Annual Research TJPS Proceeding 2013

The Thai Journal of Pharmaceutical Sciences
The Thai Journal of Pharmaceutical Sciences is an official journal of Faculty of Pharmaceutical Sciences
Chulalongkorn University. The journal published original research and review articles in the fields of
Pharmaceutical sciences.
EDITORS
Pithi Chanvorachote, Ph.D. Chulalongkorn University, Thailand
Rutt Suttisri, Ph.D. Chulalongkorn University, Thailand
Vipaporn Panapisal, Ph.D. Chulalongkorn University, Thailand
ASSOCIATE EDITORS
Kulwara Meksawan, Ph.D. Chulalongkorn University, Thailand
Varisa Pongrakhananon, Ph.D. Chulalongkorn University, Thailand
EDITORIAL ADVISORY BOARD

Prof. Lee Kirsch, Ph.D. University of Iowa, USA
Prof. Ian S. Haworth, Ph.D. University of Southern California, USA
Prof. Yon Rojanasakul, Ph.D. West Virginia University, USA
Prof. Schuyler S. Korban, Ph.D. University of Massachusetts, USA
Prof. Timothy Maher, Ph.D. MCPHS University, USA
Prof. Alexander T. Florence, Ph.D. University of London, UK
Prof. M. Jayne Lawrence, Ph.D. King’s College London, UK
Prof. Tsuneji Nagai, Ph.D. Hoshi University, Japan
Prof. Ikuo Saiki, Ph.D. University of Toyama, Japan
Prof. Masao Hattori, Ph.D. University of Toyama, Japan
Prof. Apiwat Mutirangura, MD., Ph.D. Chulalongkorn University, Thailand
Prof. Garnpimol C. Ritthidej, Ph.D. Chulalongkorn University, Thailand
Prof. Somboon Tanasupawat, Ph.D. Chulalongkorn University, Thailand
Prof. Mont Kumpugdee Vollrath, Ph.D. University of Applied Sciences, Germany
Prof. Sergio Rosales Mendoza, Ph.D. Universidad Autónoma de San Luis Potosí, México
Sudjit Luanpitpong, Ph.D. West Virginia University, USA
Todd A Streckle, Ph.D. National Institute for Occupational Safety and
Health, USA
Lying Wang, MD. Ph.D. National Institute for Occupational Safety and
Health, USA
Heinric Williams, MD. Geisinger Medical Center, USA
Rungpetch Sakulbumrungsil, Ph.D. Chulalongkorn University, Thailand
Pornchai Rojsitthisak, Ph.D. Chulalongkorn University, Thailand
Pintip Pongpech, Ph.D. Chulalongkorn University, Thailand
Pornpen Pramyothin, Ph.D. Chulalongkorn University, Thailand
Warangkana Warisnoicharoen, Ph.D. Chulalongkorn University, Thailand
Panida Laoratthaphone, Ph.D. Pan Rajdhevee Group Public Company Ltd.,
Thailand
Rossarin Tansawat, Ph.D. Chulalongkorn University, Thailand
Nithipun Suksumek, MD. Phramongkutklao college of Medicine, Thailand
Krich Rajprasit Srinakharinwirot University, Thailand
Kitiyot Yotsombut Chulalongkorn University, Thailand
Hasseri Bin Halim, Ph.D. Universiti Teknologi MARA, Malaysia
Muhummad Awais Khan, Ph.D. International Potato Center CIP, Peru
The 30th Annual Research Conference in Pharmaceutical Sciences| i

Yuepeng Han, Ph.D. Wuhan Botanical Garden of the Chinese Academy
of Sciences Moshan, China
EDITORIAL ASSISTANT
Patcharin Sithdhichankhuna Chulalongkorn University, Thailand
Arunya Khanpromma Chulalongkorn University, Thailand
Nonthalert Lertnitikul Chulalongkorn University, Thailand
All correspondence should be addressed to the Editorial Office of The Thai Journal of Pharmaceutical
Sciences. Copyright ©2006 Faculty of Pharmaceutical Sciences Chulalongkorn University. No
reproducibility is allowed without permission from the Editor.
ISSN (print version) 0125-4685
ISSN (electronic version) 1905-4637
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The 30th Annual Research Conference in Pharmaceutical Sciences| ii

Poster Sessions
Poster
Title
No.
PN-1 Highly efficient preparation of ethyl piperate from piperine isolated from Piper
nigrum
Piyapong Choochana, Thitima Lhinhatrakool and Prasan Tanguenyongwatana
PN-2 Discovery of Novel Cholinesterase Inhibitor (RKNU026) with β-Amyloid

Aggregation Inhibitory Activity
Thippatai Hiranyaekaphap, Nitipol Srimongkolpithak, Thamrong Wongchang,

Prayuth Poowaruttanawiwat, Kwanchai Rattanamanee and Ruengwit Kitbunnadaj
PN-3 Preparation of 2'-N-2,3,4-trifluorobenzoyl derivative of cytotoxic ecteinascidin 770

from the Thai tunicate Ecteinascidia thurstoni
Witaya Lowtangkitcharoen, Taksina Chuanasa, Naoki Saito and Khanit

Suwanborirux
PN-4 Extraction of oil from grape seeds with supercritical carbon dioxide
Jirawat Eiamwat, Paramee Pengpreecha, Benjaporn Tiensong, Sorada Walapa,

Kulapatr Wachirasiri and Sukit Kampruengdet
PN-5 Stingless bee honey II: Qualitative and quantitative studies on honey produced by
three stingless bee species collected from a mangosteen garden in Chantaburi
province, Thailand
Nipatha Issaro, Thorsang Weerakul, Sisipawan Machana , Phatsakorn Ornnim,

Chamipha Phanudulkitti, Tharathee Srijan, Jiratikron Laiwattanaphaisal and
Chumnan Pattarapanich
PN-6 Gene cloning and expression of a triterpene synthase from Alangium lamarckii
Nattaon Tansakul, Pimpimon Tansakul and Wanchai De-Eknamkul
PN-7 Effect of ethanol concentration on cyaniding-3-glucoside, total monomeric

anthocyanins, total phenolic content and radical scavenging properties in purple
corn (Zea mays L.) seed and cob
Penpan Srithong, Tanwarat Kajsongkram, Chuleratana Bangchonglikitkul and

Waraporn Boonsupthip
The 30th Annual Research Conference in Pharmaceutical Sciences| iii

PN-8 Antioxidant activities and phytochemicals of edible flowers
Jeerawat Sawatpipat, Vatin Phunsawat, Piyanuch Rojsanga and Pongtip Sithisarn
PN-9 Investigation on phytochemical, antioxidant and cytotoxic properties of

Momordica cochinchinensis (GAC) fruit extracted by supercritical fluid carbon
dioxide (SCF-CO2) method
Prapaipat Klungsupya, Worawan Tiatragoon, Thanchanok Muangman, Jirawat
Eiamwat, Sarunya Laovitthayanggoon , Jeerayu Thongdon-A and Srisak
Trangwacharakul
PN-10 Comparative studies on antioxidant activities, total phenolics and anthocyanin

content of four native fruits
Tanwarat Kajsongkram, Saranya Laovitthayanggoon ,Kusol Iamsub, Vilailuk

Chuennangchee, Penpan Srithong and Chuleratana Bangchonglikitkul
PN-11 Antileukemic activity and secondary metabolites of an endophytic fungus

Phomopsis sp. from Artemisia annua
Punyisa Ngankaranatikarn, Taksina Chuanasa, Nongluksna Sriubolmas and

Khanit Suwanborirux
PN-12 Detection of esterase activity converting cytotoxic renieramycin M to

jorunnamycin A in the crude enzyme of the nudibranch Jorunna funebris
Dalad Waropastrakul, Khanit Suwanborirux, Wanchai De-Eknamkul and Taksina

Chuanasa
PN-13 Comparison of HPLC and TLC densitometric methods for the quantification of
rhein in Cassia fistula pod pulp extract
Savita Chewchinda, Pongtip Sithisarn and Wandee Gritsanapan
PN-14 Use of three indigenous vegetables as potential dietary fiber sources for health food
product
Jeerayu Thongdon-A, Prapaipat Klungsupya, Thanchanok Muangman, Arkachai

Tantrawong and Srisak Trangvacharakul
PN-15 Identification of lactic acid bacteria and yeasts from fermented rice product (khao-
khab)
Somboon Tanasupawat, Wongsakorn Phongsopitanun, Wanlapa Lorliam,

Naphatrapi Luangsakul and Lawan Chatanon
PN-16 Nutritional values and TLC fingerprint of Scaphium scaphigerum fruit gel
Wichittra Phlicharoenphon, Wandee Gritsanapan and Pongtip Sithisarn
The 30th Annual Research Conference in Pharmaceutical Sciences| iv

PN-17 Quantitative analysis of epigallocatechin gallate in young and mature Assam tea
leaf extracts
Pattra Ahmadi Pirshahid, Thongchai Hemthanon , Yaowaluk Khamphan, Phatsuda

Chueboonmee and Chuleratana Banchonglikitkul
BP-1 Preliminary study of Syzygium aromaticum (L.) on analgesic activity in rats
Kanyarat Sueksakit, Krittiya Thisayakorn, Vichein Khueynok, Kanjana Sriyam,

Darunee Pahusee and Nopparat Buddhakala
BP-2 Effects of topical administration of Nymphaeae lotus flower extract on UVA-

induced photoinflammation in mice
Krittiya Thisayakorn, Ubon Rerk-am, Sinn Tangstirapakdee, Vichein Khueynok,

Kanchana Sriyam, Darunee Pahusi and Chuleratana Banchonglikitkul
BP-3 Effect of Morus alba stem extract on nitric oxide production in lipopolysaccharide-
induced RAW 264.7 macrophage cells
Nattaporn Soonthornsi, Warinkarn Hemstapat, Pongsak Utaisincharoen and

Tasana Pitaksuteepong
BP-4 Pilot study of antibacterial and anti-inflammatory activities of Indian gooseberry

(Phyllanthus emblica L.) extract against bacteria associated with pitted keratolysis
Rattanasiri Giwanon, Krittiya Thisayakorn, Pongsatorn Limsiriwong, Saowaluck

Ruengsri, Vichein Kheuynok,Kanchana Sriyam, Marudech Srisom, Darunee
Pahusee, Sawai Nakakaew, Natthachest Ketmanee, Sontaya Peungsumrong and
Chulerattana Banchonglikitkul
BP-5 Chemical profile analysis and anti-inflammatory activity of polar fraction from
soybeans (Glycine max)
Siripen Jarikasem, Amornrat Khayungarnnawee and Sarinthip Muensaen
BP-6 Aligned electrospun chitosan/poly-ε caprolactone blended scaffold: Anti-

inflammatory effect
Natthan Charernsriwilaiwat, Louise Carney, Brendan Robb, Praneet Opanasopit,

Theerasak Rojanarata and Sandra Downes
BP-7 Antibacterial activity of α-mangostin from the pericarp extract of Garcinia

mangostana L. against drug resistant bacteria
Griangsak Eumkeb, Sineewan Phitaktim and Yothin Teethaisong
The 30th Annual Research Conference in Pharmaceutical Sciences| v

BP-8 Anti-oxidative stress activity of Phikud Navakot extract in Saccharomyces
cerevisiae
Nongluksna Sriubolmas, Uthai Sotanaphun, Duangdeun Meksuriyen and Suthep

Wiyakrutta
BP-9 Radical-scavenging and cellular DNA protective activities of active fractions from
Lansium domesticum Corr. fruit
Prapaipat Klungsupya, Nava Suthepakul, Thanchanok Muangman, Sarunya

Laovitthayanggoon, Jeerayu Thongdon-A and Srisak Trangwacharakul
BP-10 Protective effect of oligomeric proanthocyanidins (OPCs) from Thai grape seeds
against H 2 O 2 -induced tight junction function disruption in Hanman CACO-2 cells
Thanchanok Muangman, Prapaipat Klungsupya, Anawat Suwanagul, Akihiro

Watari and Kiyohito Yagi
BP-11 Interaction between P-glycoprotein and Thai herbs with anti-diabetic potential
Wilasinee Dunkoksung, Nontima Vardhanabhuti, Surattana Amnuoypol and Suree
Jianmongkol
BP-12 Cytotoxic evaluation of a fruit extract from Zanthoxylum limonella in human
dermal fibroblast cell line using MTT assay
Sarunya Laovitthayanggoon, Buppachart Potduang, Natthachest Ketmanee and
Chuleratana Banchonglikitkul
BP-13 Anti-lipase activity of Quercus infectoria G. Olivier extract
Sirinan Thubthimthed, Sarunya Laovitthayanggoon, Parkpoom Siriarchavatana,

Konlawit Chaithongsri and Chuleratana Banchonglikitkul
BP-14 Acute oral toxicity test of Quercus infectoria G. Olivier extract in rats
Tuanta Sematong, Sirinan Thubthimthed, Sareeya Reungpathanapong, Sarunya

Laovitthayanggoon and Chuleratana Banchonglikitkul
BP-15 Biological activities of Cajanus cajan ethanolic extracts

Ubon Rerk-am, Bantika Kongsombat, Sinn Tangsatirapakdee and Chuleratana
Banchonglikitkul
BP-16 Synergy effect of ceftazidime with flavonoids against Streptococcus pyogenes
Supatcharee Siriwong, Pongrit Krubphachaya, Kanjana Thumanu and Griangsak

Eumkeb
BP-17 The effects of red kwao krue (Butea superba Roxb.) extract on sperm quality
and testosterone level in mice
Griangsak Eumkeb, Wanatkamol Naknarong and Kittipot Sirichaiwetchakoon
The 30th Annual Research Conference in Pharmaceutical Sciences| vi

PT-1 Development and validation of RP-HPLC method for quantitative analysis of
individual γ-oryzanol in cold pressed rice bran oil
Apirak Sakunpak, Jirapornchai Suksaeree, Laksana Charoenchai, Chaowalit

Monton, Pathamaporn Pathompak , Tossaton Charoonratana and Krisana
Kraisintu
PT-2 Quantification and validation of gallic acid content in Tri-phala extract and
effervescent tablet by HPTLC
Benjaporn Chainethvanich, Worawan Saingam and Prasan Tanguenyongwatana
PT-3 Development and validation of RP-HPLC method for determination of piperine in

Maha-Solos tablet formulation
Worawan Saingam, Chaowalit Monton, Jirapornchai Suksaeree, Natawat
Chankana, Siriporn Kittiwisut and Laksana Charoenchai
PT-4 Application of titrimetric indicator to the estimation of polyplex formation ratio

between chitosan polymer and plasmid DNA used for gene delivery formulation
Samarwadee Plianwong, Praneet Opanasopit, Tanasait Ngawhirunpat and

Theerasak Rojanarata
PT-5 Preparation of doubly crosslinked chitosan by the use of environmentally friendly

crosslinkers for enzyme immobilization
Siripran Tidjarat, Tanasait Ngawhirunpat, Praneet Opanasopit and Theerasak

Rojanarata
PT-6 Influence of chemical structures of emulsifiers on feasibility of isopropyl palmitate

lotion formation
Prapaporn Boonme, Chalida Boonthongchuay, Thanaporn Amnuaikit and

Wibul Wongpoowarak
PT-7 Effect of particle size of ultradeformable liposomes on dermal delivery of

hydrophilic compound
Worranun Rangsimawong, Praneet Opanasopit, Theerasak Rojanarata and

Tanasait Ngawhirunpat
PT-8 Development of hyaluronic acid loaded elastic liposome
Chawankorn Kasetvetin, Soravoot Rujiviphat and Waree Tiyaboonchai
PT-9 Physicochemical stability of meloxicam loaded liposome formulation: Effect of

cationic and anionic surfactant
Sureewan Duangjit, Praneet Opanasopit, Theerasak Rojanarata, and Tanasait

Ngawhirunpat
The 30th Annual Research Conference in Pharmaceutical Sciences| vii

PT-10 Meloxicam-loaded pH sensitive polymeric micelles: Solvents and entrapment
methods
Thisirak Woraphatphadung, Warayuth Sajomsang, Theerasak Rojanarata,

Tanasait Ngawhirunpat and Praneet Opanasopit
PT-11 Fabrication of meloxicam taste-masked oral fast disintegrating tablet by ion

exchange resin and cyclodextrin
Wipada Samprasit, Theerasak Rojanarata, Prasert Akkaramongkolporn, Tanasait

Ngawhirunpat and Praneet Opanasopit
PT-12 Characterization of transferrin conjugated solid lipid nanoparticles and in vitro

release pattern for targeting brain drug delivery
Su Myat Nyein Thu, Vimolmas Lipipun and Garnpimol C. Ritthidej
PT-13 Combined effect of low-frequency ultrasound and microneedles for transdermal

hydrophilic macromolecules
Suvida Laohapatarapant, Praneet Opanasopit, Theerasak Rojanarata and

Tanasait Ngawhirunpat
PT-14 Development of Garcinia mangostana extract in liquid crystal cream for topical
delivery system
Napa Bunma, Jirapan Moungjaroen and Prasan Tanguenyongwatana
PT-15 Formulation of ketoprofen matrix membrane for transdermal delivery systems
Jirapornchai Suksaeree, Wiwat Pichayakorn, Chaowalit Monton, Apirak

Sakunpak, Tun Chusut, Worawan Saingam and Fameera Madaka
PT-16 Development of capsaicin microemulsions for transdermal drug delivery
Wisuta Chairat, Praneet Opanasopit, Theerasak Rojanarata and Tanasait

Ngawhirunpat
PT-17 Preliminary formulation study of cold pressed rice bran oil mask
Jirapornchai Suksaeree, Laksana Charoenchai, Apirak Sakunpak, Chaowalit

Monton, Tun Chusut, Worawan Saingam and Pathamaporn Pathompak
PT-18 Formulation of topical preparations containing high concentration of permeation

enhancer in a treatment of onychomycosis
Phojana Komesmuneeborirak, Pornpen Werawatganone and Walaisiri Muangsiri
PT-19 Novel technique for dry powder development for intranasal delivery
Wittaya Nakachon and Garnpimol C. Ritthidej
The 30th Annual Research Conference in Pharmaceutical Sciences| viii

PT-20 Development of hair tonic comprising a mixed extract from fruits of Phyllanthus
emblica and Zanthoxylum limonella
Itsara Keeta, Nahathai Sawang and Buppachart Potduang
PT-21 Stability evaluation of a hair tonic consisting of a mixed extract from fruits of
Phyllanthus emblica and Zanthoxylum limonella
Sitthiphong Soradech, Nahathai Sawang, Buppachart Potduang, Itsara Keeta and

Arkachai Tantrawong
PT-22 Development of hair conditioning and styling gel comprising a mixed extract from
fruits of Phyllanthus emblica and Zanthoxylum limonella
Itsara Keeta, Nahathai Sawang and Buppachart Potduang
PT-23 Development of hair conditioner comprising a mixed extract from fruits of

Phyllanthus emblica and Zanthoxylum limonella
Buppachart Potduang, Itsara Keeta, Wanlapha Saisin, Suchipha

Wannaphatchaiyong and Bundit Fungsin
PT-24 Stability evaluation of a hair conditioner consisting of a mixed extract from fruits
of Phyllanthus emblica and Zanthoxylum limonella
Sitthiphong Soradech, Buppachart Potduang, Itsara Keeta, Wanlapha Saisin,

Suchipha Wannaphatchaiyong and Cholticha Niwaspragrit
PT-25 Acute skin irritation test of facial & body gel wash and moisturizing gel products
comprising a patented mixed extract from Emblica and Ma-Khwaen
Sareeya Reungpatthanaphong, Tuanta Sematong, Buppachart Potduang,

Phanukit Kunhachan, Itsara Keeta and Cholticha Niwaspragrit
PT-26 Development of shampoo comprising a mixed extract from fruits of Phyllanthus

emblica and Zanthoxylum limonella for itchy scalp
Buppachart Potduang, Itsara Keeta, Suchipha Wannaphatchaiyong, Wanlapha

Saisin and Bundit Fungsin
PT-27 Development of Jatu-Phalathika formulation in effervescent tablet dosage form
Pongsak Awaiyawanon, Anchana Sanrungmuang, Worawan Saingam and Prasan

Tanguenyongwatana
PT-28 Cosmeceutical development of oxyresveratrol from Mahad (Artocarpus lakoocha

Roxb.)
Surapol Natakankitkul, Nicha Chareonmuang, Tawan Panyakhong and Worrapon

Wangkananon
The 30th Annual Research Conference in Pharmaceutical Sciences| ix

PT-29 Particle size characteristics of gold nanoparticles
Chutima Kongsuwan and Warangkana Warisnoicharoen
PT-30 Molecular dynamics simulation of α-resorcinol crystal dissolution in water
Somwang Sae-Tang, John Kendrick, Jamshed Anwar and Jittima Chatchawalsaisin
PT-31 Miscibility study of benzocaine and poly L-lactide using solubility parameter
calculation and thermal analysis
Yada Vattanagijyingyong, Narueporn Sutanthavibul and Jittima Chatchawalsaisin
PT-32 Development of lawsone methyl ether loaded chitosan-dextran sulfate

nanoparticles; preliminary study
Pattravee Niamprem, Pharkphoom Panichayupakaranant and Waree

Tiyaboonchai
PT-33 Effect of spray drying carriers on physical properties of spray dried anti-Fee-
Mareng-Suang extract powder
Natawat Chankana, Chaowalit Monton, Worawan Saingam, Siriporn Kittiwisut,

Jirapornchai Suksaeree, Apirak Sakunpak, Krisana Kraisintu and Yupa
Tengwattanachoti
PT-34 Preliminary study of spray drying and freeze drying processes on physicochemical
properties of encapsulated rosmarinic acid
Supaporn Ketpitthaya, Suchada Sukrong, Walaisiri Muangsiri, Nontima

Vardhanabhuti, W. John Kao, Narueporn Sutanthavibul and Phanphen
Wattanaarsakit
PT-35 Fabrication and evaluation of clotrimazole-loaded PVP/HPβCD nanofiber mats for

oral candidiasis
Prasopchai Tonglairoum, Tanasait Ngawhirunpat, Theerasak Rojanarata,

Ruchadaporn Kaomongkolgit and Praneet Opanasopit
PT-36 Physicochemical properties of pseudolatex systems prepared from para rubber

sheet and preliminary drug loading for drug delivery
Wiwat Pichayakorn, Rungtiwa Waiprib, Isara Lueanpaen, Jirapornchai Suksaeree

and Wirach Taweepreda
The 30th Annual Research Conference in Pharmaceutical Sciences| x

CP-1 Time-kill study of the in vitro activity of tigecycline against carbapenem-resistant
Klebsiella pneumoniae
Maimun Halee, Wanchai Treyaprasert, Pornpan Koomanachai and Duangdao

Waywa
CP-2 Laxative effectiveness of Cassia angustifolia in Thai constipated patients
Peeranuch Mangmeesri, Kant Wongsuphasawat, Wandee Gritsanapan and Wit

Viseshsindh
CP-3 Prediction equation of glomerular filtration rate decline in patients with type 2
diabetes and preserved kidney function at Maharajnakhonsitham-marat Hospital
Pliwphai Chaidecha Sukmak, Titinun Auamnoy and Somratai Vadcharavivad
CP-4 Predicting the development of renal impairment in type 2 diabetic patients with
preserved kidney function
Suchada Kittipanyaworakun, Chaowarat Munprom, Titinun Auamnoy, Kearkiat

Praditpornsilpa and Somratai Vadcharavivad
SP-1 Production of halal herbal medicinal products in Chanae Hospital: A community

hospital in Narathiwas province, Thailand
Chaowalit Monton, Krisana Kraisintu and Natawat Chankana
PN Pharmaceutical Chemical and Natural Products

BP Biopharmaceutical Sciences
PT Pharmaceutical Technology
CP Clinical Pharmacy
SP Social Pharmacy
The 30th Annual Research Conference in Pharmaceutical Sciences| xi

Contents
Section Page
Pharmaceutical Chemical and Natural Products 1-62
Biopharmaceutical Sciences 63-123
Pharmaceutical Technology 124-264
Clinical Pharmacy 265-278
Social Pharmacy 279-281
Author Index 282-284
The 30th Annual Research Conference in Pharmaceutical Sciences| xii

Session I
Pharmaceutical Chemical and
PN 1 - 17
Natural Products
PN - 1
Thai J. Pharm. Sci. Vol. 38 (Suppl.) 2013
HIGHLY EFFICIENT PREPARATION OF ETHYL PIPERATE FROM

PIPERINE ISOLATED FROM PIPER NIGRUM
Piyapong Choochana, Thitima Lhinhatrakool, Prasan Tanguenyongwatana
Faculty of Oriental Medicine, Rangsit University, Pathumthani 12000, Thailand.
KEYWORDS: Piper nigrum, Ethyl piperate, Piperine, Piperic acid, Esterification
INTRODUCTION
Piper nigrum L. is a medicinal plant in the family Piperaceae. This plant is native to south India and
widespread in tropical region1). In Thailand, black pepper is commonly consumed as a condiment and
also used as an ingredient in traditional Thai medical preparations2). Piperine is the major alkaloid
responsible for the pungency of black pepper. The amount of piperine varies from 5-7% in white and
black pepper. Piperine has been reported to show lipid-lowering effect in drug-induced
hypercholesterolemic rats3). However, piperine was cytotoxic to cultured embryonic rat brain neurons and
caused extensive immunotoxicological effects in mice4, 5). Ethyl piperate is modified from piperine to get
rid of the toxicity of the parent compound and retain its lipid-lowering effect6). We are interested in
studying the preparation of ethyl piperate by using synthetic method for further investigation of its other
biological activities.
MATERIALS AND METHODS

Instrument and reagents Dicyclohexylcarbodiimide (DCC) and 4-dimethyl-aminopyridine (DMAP)
were purchased from Aldrich (New Jersey, USA). p-Toluene sulfonic acid was obtained from Aldrich
(New Jersey, USA). All other reagents and solvents were reagent grade and used without further
purification. TLC was performed on silica gel GF 254 plates (Merck). For column chromatography, silica
gel (Merck 230-400 mesh) was used. NMR spectra were recorded with a Bruker Avance (1H, 300 MHz)
spectrometer. Chemical shifts are reported in ppm, and coupling constants are reported in Hz. All NMR
spectra were obtained in deuterated chloroform (CDCl 3 ), DMSO-d 6 and referenced to the residual solvent
peak. Mass spectra were obtained on an Agilent GC/MS 5975C.
Plant material The dried fruits of Piper nigrum were bought from local drugstore in Nonthaburi
province, Thailand. They were identified by comparison with the specimens at the Forest Herbarium,
Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources and
Environment, Bangkok. The voucher specimen of Piper nigrum (SRU 024) was deposited at the Faculty
of Oriental Medicine, Rangsit University, Pathumthani, Thailand.
Preparation of crude extracts The dried, powdered fruits of Piper nigrum (100 g) were extracted with
95% ethanol (400 mL) at room temperature for 7 days. The extract was filtered with Whatman No.1 filter
paper and then evaporated under reduced pressure with rotary evaporator to obtain 9 g of the final crude
dark brown extract.
Isolation of piperine The crude extract (9 g) was dissolved in ethanol (30 ml). Potassium hydroxide (8.0
g) was added to the mixture and stirred until completely dissolved. Then, the mixture was filtered through
Whatman No. 1 filter paper and left overnight to freely evaporate at room temperature. After this process,
piperine precipitated in the mixture as brown solid compound. The crude product was washed with hot
water three times to remove the water-soluble residues. The solid was recrystallized with isopropanol and
kept at 15ºC for 3 days. After that, the crystals were filtered upon Whatman No. 1 filter paper to obtain
yellow solid (2.9591 g) with melting point of 129-130 ºC. UV(7) λ max 343 nm; IR(8) (KBr disc): 3010,
2942, 1633, 1581, 1433 cm-1; 1H NMR(9) (300 MHz, CDCl 3 ) δ [ppm]: 1.59 (m, 4H), 1.64 (m, 2H), 3.55
(d, 4H), 5.95 (s, 2H), 6.44 (d, J = 14.5 Hz, 1H), 6.74 (m, 3H), 6.89 (d, J = 8 Hz, 1H), 6.97 (s, 1H), 7.40
(qd, J = 15, 2 Hz, 1H). 13C NMR(10) (75 MHz, CDCl 3 ) δ [ppm]: 165.3 (C-1), 148.1 (C-3'), 148.0 (C-4'),
142.4 (C-3), 138.2 (C-5), 131.0 (C-1'), 125.3 (C-4), 122.4 (C-6'), 120.0 (C-6'), 108.4 (C-5'), 105.6 (C-2'),
101.2 (C-8), 46.8 (C-9), 43.2 (C-9), 26.6 (C-11), 25.6 (C-11), 24.62 (C-10) and MS(2) (GC/MS): M+ =
285.
Preparation of piperic acid To a round bottom flask (50 mL) containing 0.50 g of piperine, 20%
alcoholic potassium hydroxide (30 mL) was added. The mixture was refluxed at 70 ºC for 12 h and then
cooled down to room temperature. The solution was acidified with 1M HCl to pH 3.0 and then
transferred to a separatory funnel. Dichloromethane (30 mL) was added to the separator to extract the
aqueous layer. The extraction was repeated two times and the dichloromethane layers were collected,
evaporated to obtain crude piperic acid. The crude compound was recrystallized with methanol-water
The 30th Annual Research Conference in Pharmaceutical Sciences| 1

PN - 1
(8:2) to give crystals of piperic acid (0.2814 g, 57.61% yield) with melting point of 213-215 ºC. UV λ max
340 nm; IR(11) (KBr disc): 3300-2500 (broad), 1671, 1594 cm-1; 1H NMR (300 MHz, DMSO- d 6 ) δ
[ppm]: 5.92 (d, J = 15.1 Hz, 1H), 6.05 (s, 2H), 6.92-7.00 (m, 4H), 7.23 (s, 1H), 7.25-7.32 (m, 1H). 13C
NMR (75 MHz, DMSO- d 6 ): 167.8, 148.2, 148.0, 144.8, 139.9, 130.6, 124.9, 123.2, 121.2, 108.6, 105.8,
101.4 and MS (GC/MS): M+ = 218.
Preparation of ethyl piperate by acid catalyzed esterification To a round bottom flask (50 mL)
containing piperic acid (1.0003 g) in toluene (10 mL), ethanol (1.0 mL) and p-Toluene sulfonic acid (0.05
g) were added. The flask was connected with Dean-Stark Apparatus to remove the water from the
reaction. The reaction mixture was refluxed for 6 h and monitored by TLC. After 6 h, there was still
some starting material left in the reaction. The reaction mixture was evaporated by using rotary
evaporator and the residue was subjected to silica gel column chromatography. The mobile phase was
hexane (50 mL), then the mixture of hexane-ethyl acetate (7:3). The product was collected and the solvent
was evaporated to obtain pale yellow solid (0.3831 g, 38.30% yield) with melting point of 117-118 ºC.
UV λ max 342 nm,; IR (KBr disc): 1704, 1618, 1609, 1489 cm-1. 1H-NMR (300 MHz, CDCl 3 ) δ [ppm]:
1.31 (t, J = 7 Hz, 3H ), 4.22 (q, J = 7 Hz, 2H), 5.93 (d, J = 15 Hz, 1H), 5.98(s, 2H), 6.69 (dd, J = 16, 11
Hz, 1H), 6.78 (d, J = 8 Hz, 1H), 6.81 (d, J = 15 Hz, 1H), 6.91(dd, J = 1.6, 9 Hz, 1H), 6.99(d, J = 1.6 Hz,
1H), 7.41 (dd, J = 10.5, 15.3 Hz, 1H). 13C-NMR (75 MHz, CDCl 3 ): 167.1, 148.5, 148.2, 144.6, 140.1,
130.5, 124.5, 122.9, 120.4, 108.5, 105.8, 101.3, 60.2, 14.3 and MS (GC/MS): M+ = 246.
Preparation of ethyl piperate by Steglich reaction (12) To a round bottom flask (50 mL) containing
piperic acid (0.5003 g) in dry dichloromethane (15 mL), ethanol (1.0 mL), 4-dimethylaminopyridine
(DMAP) (0.22 g) and dicyclohexylcarbodiimide (DCC) (0.57 g) were added. The mixture was stirred
under nitrogen atmosphere for 24 h and the reaction was monitored by TLC. After the reaction was
complete, the reaction mixture was evaporated by using rotary evaporator and the residue was subjected
to silica gel column chromatography. The mobile phase was the mixture of hexane-ethyl acetate (7:3).
The product was collected and the solvent was evaporated to obtain pale yellow solid (0.5227 g, 92.79%
yield) with melting point of 117-118 ºC. UV λ max 342 nm,; IR (KBr disc): 1704, 1618, 1609, 1489 cm-
. H-NMR (300 MHz, CDCl 3 ) δ [ppm]: 1.31 (t, J = 7 Hz, 3H ), 4.22 (q, J = 7 Hz, 2H), 5.93 (d, J = 15
1 1
Hz, 1H), 5.98(s, 2H), 6.69 (dd, J = 16, 11 Hz, 1H), 6.78 (d, J = 8 Hz, 1H), 6.81 (d, J = 15 Hz, 1H),
6.91(dd, J = 1.6, 9 Hz, 1H), 6.99(d, J = 1.6 Hz, 1H), 7.41 (dd, J = 10.5, 15.3 Hz, 1H). 13C-NMR (75 MHz,
CDCl 3 ): 167.1, 148.5, 148.2, 144.6, 140.1, 130.5, 124.5, 122.9, 120.4, 108.5, 105.8, 101.3, 60.2, 14.3
and MS (GC/MS): M+ = 246
RESULTS AND DISCUSSION

The isolation of piperine from black pepper was uncomplicated process and the compound was obtained
at a reasonable yield of almost 3%. For the preparation of piperic acid, alkali hydrolysis with potassium
hydroxide gave enough material to go on to next step. The esterification of piperic acid with alcohol was
performed by two different methods. The first method, which was the acid catalyzed esterification, used
p-toluene sulfonic acid as a catalyst with Dean-Stark Apparatus to remove water from the reaction in
order to push the reaction equilibrium to the product side. However, this reaction need to be refluxed in
high temperature in toluene and the ethanol which was one of the reagents in the reaction could evaporate
from the reaction during the process. The reaction was therefore not complete and the product was
obtained at a poor yield. We then decided to use another reaction, the Steglich esterification, which could
be done at room temperature (Figure 1).
O O
O O
OH DCC, DMAP O CH3
O EtOH, CH2Cl 2
O
RT
Piperic acid Ethyl piperate
Figure 1 Steglich esterification reaction to prepare ethyl piperate
Steglich esterification reaction is a mild reaction which allows the conversion of sterically demanding and
acid labile substrates. This reaction used dicyclohexylcarbodiimide (DCC) and 4-dimethyl-aminopyri-
dine (DMAP) as activation agent and catalyst, respectively, to conduct more efficient esterification
reaction than the classical one. It is better for works with the highly volatile compounds because the

PN - 1
reaction can be performed at room temperature. For the synthesis of ethyl piperate with Steglich reaction,
we obtained ethyl piperate at a yield of 92.79% yield.
CONCLUSION
From our study, the isolation of piperine and the preparation of piperic acid derivatives are simple and
efficient. Ethyl piperate is obtained through Steglich esterification reaction in a high yield. This
preparation is in the first phase of projects to use ethyl piperate as UV protective agent, and also to apply
ethyl piperate in liposomes form for drug delivery study.
ACKNOWLEDGMENTS
This work was supported by Research Institute of Rangsit University, Pathumthani 12000, Thailand.
REFERENCES
1. Ahmad, N., Fazal, H., Haider, A.B., Farooq, S., Ali, M., Khan, M. 2012. Biological role of Piper
nigrum L. (Black pepper): A Review. Asian. Pac. J. Trop. Biomed., 1-10.
2. วุฒิ วุฒิธรรมเวช. สารานุกรมสมุนไพร. กรุ งเทพมหานคร:สํานักพิมพ์โอเดียนสโตร์, 2540.
3. Vijayakumar, R.S., Nalini, N. 2006. Lipid-lowering efficiency of piperine from Piper nigrum L.
in high-fat diet and antithyroid drug-induced hypercholesterolemic rats. J. Food. Biochem.,
30:405-421.
4. Unchern, S., Saito, H., Nishiyama, N. 1998. Death of cerebellar granule neurons induced by
piperine is distinct from that induced by low potassium medium. Neurochem Res., 23: 97-102.
5. Dogra, R.K., Khanna, S., Shanker, R. 2004. Immunotoxicological effects of piperine in mice.
Toxicology. 196: 229-236.
6. Borjihan, R., Wu, Y. 2005. Inventors; Inner Mongolia University, assignee. Application of
piperinic esters as lipid-lowering drugs and health care products. China patent ZL
2005101259762, 2005 Dec 1.
7. Lupina, T., Cripps, H. 1987. UV spectrophotometric determination of piperine in pepper
preparation: collaborative study. J Assoc Off Anal Chem, 70(1):112-3.
8. Ikan, R. 1991. Natural Products: A Laboratory Guide, 2nd ed. Academic Press, p236.
9. Berger, S., Sicker, D. 2009. Classic in Spectroscopy, Isolation and Structure Elucidation of
Natural Product, 1st ed. Wiley-VCH Verlag & Co. KGaA, Weinheim, p53.
10. Pretsch, E. et al. 2002. Computer-Aided Structure Elucidation, WILEY-VCH.
(http://www.sciencesoft.net/piperine/index.html)
11. Zarai, Z., Boujelbene, E., Salem, N.B., Gargouri, Y., Sayari, A. 2013. Antioxidant and
antimicrobial activities of various solvent extracts, piperine and piperic acid from Piper
nigrum. LWT-Food Science and Technology, 50:634-641.
12. Neises, B., Steglich, W. 1985. Esterification of monoethyl fumarate-DMAP as catalyst. Org
Synth, 63: 183.

PN - 2
DISCOVERY OF NOVEL CHOLINESTERASE INHIBITOR (RKNU026) WITH

β-AMYLOID AGGREGATION INHIBITORY ACTIVITY
Thippatai Hiranyaekaphap,δ Nitipol Srimongkolpithak,δ Thamrong Wongchang,δ Prayuth

Poowaruttanawiwat,ϕ Kwanchai Rattanamanee,ϕ Ruengwit Kitbunnadajδ,†
δ
Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences, Naresuan University
Phitsanulok 65000, Thailand. ϕDepartment of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Naresuan University,
Phitsanulok 65000, Thailand. †To whom correspondence should be addressed. Email: rkitbunna@hotmail.com
KEYWORDS: Alzheimer’s disease, Cholinesterase enzyme, Cholinesterase inhibitors, Beta Amyloid

aggregation, Coumarin
INTRODUCTION
Alzheimer's disease (AD) is the most common form of dementia that caused by a reduction of the
neurotransmitter, especially acetylcholine (ACh) which involves in memory and cognitive function in the
brain. ACh, however, can be broken down by acetylcholinesterase (AChE). Many studies have indicated
that butyrylcholinesterase (BuChE) activity increase continuously during progression. In addition,
inhibitors binding to the peripheral anionic site (PAS) of the enzymes have hindered the amyloid β
peptide (Aβ) formation. Such findings suggest that compound inhibiting AChE function, through an
interaction with amino acid at PAS of the enzymes, might represent a new attractive candidate in
Alzheimer’s disease (AD) therapy.1-2
In this study, coumarin was chosen as core structure because its structure is similar to dihydroindenone
ring of donepezil. Yet, coumarin and its metabolite, in particular 7-hydroxycoumarin, are known to be the
safe chemicals for human. A series of coumarin derivatives are designed and synthesized by linking of
coumarin moiety and amines with various spacer lengths. All targeted compounds will be tested for their
inhibitory activities against AChE and BuChE. Some compounds, moreover are selected for further Aβ
aggregation inhibitory activity. In order to identify binding pocket of the coumarin derivatives, a so-called
computational modelling was applied.

Experimental section Reagents were obtained from commercial suppliers and used without further
purification. Solvents used were either commercial or AR grade. Thin-layer chromatography was carried
out on Merck Kieselgel 60F254 and column chromatography was performed using Carlo erba Kieselgel
(0.063-0.200 mm). Melting points were determined on a Buchi apparatus (Buchi535) an uncorrected 1H
NMR spectra were recorded on Bruker/avance at 400 MHz using the residual undeuterated solvent peak
as reference. Mass spectrometry was performed on Applied Biosystems (API4000). Molecular docking
was performed using Accelrys Discovery studio 3.1.
Methods Compounds (1−7) were accomplished as illustrated in scheme 1. The commercially available 7-
hydroxy coumarin, 7-hydroxy-2H-chromen-2-one, was alkylated with dibromoalkanes in the presence of
potassium carbonate to afford 7-bromoalkyloxy-2H-chromen-2-one (1a−1k). Next, the 7-bromoalkyloxy-
2H-chromen-2one was treated with various amines, i.e., piperidine, diethylamine, morpholine,
methylpiperazine, isoquinoline, dimethoxy, and isoquinoline, in acetone with the presence of potassium
carbonate to give the targeted compounds (2a−2k, 3a−3c, 4a−4c, 5a−5c, 6a−6c, and 7a−7c).
Scheme 1 Synthetic pathway for coumarin derivatives.
i ii R1 R2
Br N
O O OH O O O n O O O n
1a−k (n = 2−12) 2− 7
Reagents and conditions (i) Br-(CH 2 ) n -Br, acetone, K 2 CO 3, ∆, 16 h; (ii) R 1 -NH-R 2 , acetone, K 2 CO 3, ∆.

PN - 2
Pharmacological evaluation
Cholinesterase inhibition assay The inhibitory activities of all compounds against Cholinesterases(ChEs)
were measured according to a method described previously by Ellman. The method employs 3.8 mM of
acetylthiocholine (ATC) as a substrate for ChEs. ATC was broken down to thiocholine and acetate by
ChEs. The resulting thiocholine was subsequently reacted with 3 mM of dithiobisnitrobenzoate (DTNB)
to give a 2-nitrobenzoate-5-mercaptothiocholine and a yellowish 5-thio-2-nitrobenzoate. The quantity of
yellow color which develops over time was a measurement of the activity of ChEs and can be determined
using a UV-visible spectrophotometer. Upon UV-visible radiation condition, TNB undergone excitation
and displays an absorption band with λmax of 405 nm. The positive control used in this study were
neostigmine and galantamine.3
Amyloid-beta aggregation inhibition assay Thioflavin T (Th-T) fluorescence assay was used for the
identification and quantification of amyloid (Aβ) fertilization. The mixtures of Aβ 1–42 peptide and AChE
in presence or absence of the test inhibitors were incubated for 6 hours at 37 °C. A solution of Aβ
(dissolved in DMSO and diluted sodium phosphate buffer, pH 8.0) and a solution AChE (dissolved in
sodium phosphate buffer, pH 8.0) were added. After co-incubation, the mixture was added Th-T. To
analyze co-aggregation inhibition, the fluorescence absorption and emission were measured at 450 nm
and 490 nm, respectively.4
Molecular docking Docking of the various inhibitors into the hAChE active site (PDB4EY7) was
performed using Accelrys Discovery studio 3.1. The default settings for Accelrys Discovery studio 3.1
were used. The initial structure for docking was the geometry optimized, whereas an active site of the
protein was assigned from receptor cavity. Both ligand and protein were individually prepared by
CHARMm force field. The CDOCKER algorithm optimization was then applied for the docking and
energy minimization process of the ligand-protein complex. The energies for different conformations
were calculated in term of CDOCKER energy.
RESULTS
Novel coumarin derivatives were designed and synthesized by linking of a 7-hydroxycoumarin core
structure and a piperidine with various spacer lengths (2-12 methylene unit, 2a−k). The result showed that
the optimum spacer length was 7-9 methylene units. Within the series, compounds (2f−2h) possessed the
highest inhibitory activities (Table 1). Thus, the 7-9 methylene unit spacer length was selected for the
further study. A new series of compounds was designed and synthesized by replacement of the piperidine
ring with various nitrogen-containing species. As shown in table 2, no such a compound exhibited higher
inhibitor activity over its piperidine prototype. Hence, compound 2f, RKNU026, was the most potent
cholinesterase inhibitor with IC 50 values of 0.3 and 15.8 µM against AChE and BuChE, respectively.
Docking study of RKNU026-hAChE complex indicated that the coumarin moiety indeed interacts with
Trp286 via a π-π interaction (Figure 1).
Table 1: Inhibitory of hAChE and hBuChE activities determined for the piperidine containing derivatives
O O O n
No RKNU n hAChE hBuChE No RKNU n hAChE hBuChE

IC 50 (µM) IC 50 (µM) IC 50 (µM) IC 50 (µM)
2a 021 2 3.3 56.6 2h 028 9 4.1 13.5
2b 022 3 6.0 > 100 2i 029 10 1.4 25.0
2c 023 4 1.1 29.1 2j 030 11 14.7 29.5
2d 024 5 1.3 37.0 2k 031 12 > 100 54.2
2e 025 6 4.1 16.9
2f 026 7 0.3 15.8 Neostigmine 0.8 35.1
2g 027 8 0.7 6.8 Galantamine 0.4 18.6
The half maximum inhibitory concentration (IC 50 , µM) values were measured using Ellman’s method described previously. Results
are presented as mean of at least three independent experiments.

PN - 2
Table 2: Inhibitory of hAChE and hBuChE activities determined for the coumarin derivatives bearing various nitrogen-containing
species.
O O O n
No RKNU n R hAChE hBuChE No RKNU n R hAChE hBuChE

IC 50 (µM) IC 50 (µM) IC 50 IC 50 (µM)
(µM)
2f 026 7 0.3 15.8 5a 047 7 23.8 9.1
2g 027 8 0.7 6.8 5b 048 8 7.9 5.5
N N
2h 028 9 4.1 13.5 5c 049 9 33.7 69.4
3a 059 7 0.7 > 100 6a 056 7 N 6.7 > 100
3b 060 8 N 1.6 > 100 6b 057 8 5.0 > 100
N
3c 061 9 2.6 > 100 6c 058 9 6.2 > 100
4a 050 7 O 20.9 79.7 7a 044 7 O
O
61.0 43.4
4b 051 8 4.9 28.6 7b 045 8 21.8 38.6
N
4c 052 9 16.7 > 100 7c 046 9 N 47.5 39.1
Neostigmine 0.8 35.1 Galantamine 0.4 18.6
The half maximum inhibitory concentration (IC 50 , µM) values were measured using Ellman’s method described previously. Results
are presented as mean of at least three independent experiments.
Figure 1 In silico, docking of RKNU026 into the active site of hAChE using CDOCKER algorithm on Accelrys Discovery studio
3.1. The RKNU026-AChE complex structure with the lowest energy is shown on the left panel, and the 2D diagram is depicted on
the right panel.
DISCUSSION
In this study, a novel coumarin series was designed preliminarily by linking of coumarin moiety and a
piperidine ring with various spacer lengths (2 to 12 methylene unit). The result showed that compound 2f
and 2g, with spacer length of 7 and 8 methylene unit respectively, possessed the optimum inhibitory
potency for both hAChE and hBuChE. Further study by replacement of the piperidine ring with various

PN - 2
nitrogen containing rings, i.e. dietyhlamine (3a−c), morpholine (4a−c), dietyhlamine (5a−c), 1,2,3,4-
tetrahydroisoquinoline (6a−c), and 6,7-dimethoxy-1,2,3,4 tetrahydroisoquinoline (7a−c), showed that the
bigger the ring the lower potency obtained. Compound 7a−c display the lowest potency for hAChE in
comparison with the others. In contrast to what was observed for hBuChE, compounds 3a−c and 6a−c
with a small heterocyclic ring, i.e., dietyhlamine, and dietyhlamine, respective exhibited the lowest
potency. However, in this study, compound 2f (RKNU026) was the most potent AChE and BuChE
inhibitor. The compound was further tested for Aβ aggregation inhibitory activity. In comparison with
donepezil, a well-known potent AChE inhibitor currently used for AD treatment, RKNU026 exhibited an
equal degree of Aβ aggregation inhibitory activity even with lower concentration; RKNU026 possessed
the inhibitory activity for 19% at concentration of 5 µM whereas donepezil showed the inhibitory activity
for 22% at concentration of 100 µM (Figure 2).5
In order to identify the binding interaction between RKNU026 and hAChE, computational docking was
applied. CDOCKER algorithm allowed the ligand to align flexibly in the pocket of the enzyme. The
complex structure with the lowest CDOCKER energy showed that the piperidine nitrogen of RKNU026
binds to Trp86, Tyr337, Phe338, and Ty341 via cation-π interactions. Interestingly, the coumarin ring
indeed binds to Trp286 through a π-π interaction at PAS of the enzyme. This result was in agreement to
what was reported earlier that compounds interacting to amino acid at PAS hinder the Aβ aggregation
process.
Aβ + AChE
*
RKNU026 + Aβ + AChE
Aβ + RKNU Done Aβ
Aβ
AChE 026 pezil
RKNU026 + Aβ + Aβ
+ +
AChE AChE
Figure 2 (Left panel) In situ, real time Th-T fluorescence assays of solutions of Aβ in the presence or absence of AChE and/or
RKNU026. (Right panel) The maximum Aβ aggregation in the presence and absence of inhibitor (RKNU026, 5 µM or donepezil,
100 µM), % Aβ aggregation inhibition = 100 - the maximum Aβ aggregation. *The maximum Aβ aggregation value of donepezil
was taken from reference 5.
CONCLUSION
In this study, a coumarin derivative RKNU026 (2f) was identified as novel potent cholinesterase inhibitor
with IC 50 of 0.3 µM for hAChE and 15.8 µM for hBuChE. The compound, moreover, inhibited Aβ
aggregation (19% at concentration of 5 µM). Thus, RKNU026 might be a valuable candidate in the
further development of drug for Alzheimer’s disease.
ACKNOWLEDGMENTS
The First author would like to thank Faculty of Pharmaceutical Science, Naresuan University, Phitsanulok
for some financial support and Dr. Nitra Nuengchamnong for valuable technical advice.
REFERENCES
1. Bourne, Y.; Taylor, P.; Radic, Z.; Marchot, P. Structural insights into ligand interaction at the acetylcholinesterase peripheral
anionic site. EMBO J. 2003, 22, 1-12.
2. Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V., β-Amyloid aggregation induced by human acetylcholinesterase:
inhibition studies. Biochemical Pharmacology 2003, 65 (3), 407-416.
3. Ellman, G. L.; Courtney, D. K.; Andres, V.; Featherstone, R. M. A new and rapid colorimetric determination of
acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88.
4. Biancalana, M.; Koide, S. Molecular Mechanism of Thioflavin-T Binding to Amyloid Fibrils. Biochem. Biophys. Acta. 2010,
1804, 1405-1412.
5. Mishra, N.; Sasmal, D.; Singh, K. K. Attenuation Aβ1-42-induced toxicology by a novel acetylcholinesterase inhibitor.
Neurosci. 2013, 250, 309-319.

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PREPARATION OF 2'-N-2,3,4-TRIFLUOROBENZOYL DERIVATIVE OF CYTOTOXIC

ECTEINASCIDIN 770 FROM THE THAI TUNICATE ECTEINASCIDIA THURSTONI
Witaya Lowtangkitcharoen,1 Taksina Chuanasa,1 Naoki Saito,2 and Khanit Suwanborirux1,*

1
Center for Bioactive Natural Products from Marine Organisms and Endophytic Fungi (BNPME), Department of Pharmacognosy
and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
2
Department of Pharmaceutical Chemistry, Meiji Pharmaceutical University, Tokyo 204-8588, Japan
KEYWORDS: Ecteinascidia thurstoni, 2'-N-(2,3,4-trifluorobenzoyl) ecteinascidin 770
INTRODUCTION
Ecteinascidin 743 (Et 743, Trabectedin, Yondelis®, Scheme 1), a tris-tetrahydroisoquinoline alkaloid
isolated from the Caribbean tunicate Ecteinascidia turbinata,1) has been approved by European Medicines
Agency for using in the patients with soft tissue sarcoma.2) The unique chemical structure of Et 743 is
composed of tetrahydroisoquinoline 3 subunits (A, B and C) and an α-carbinolamine functional group
responsible to its anticancer activity. Et 743 was reported to induce the DNA sequence-selective
alkylation of the guanine N 2 in the duplex DNA minor groove via iminium intermediate (Scheme 1).3)
The NMR-based studies of the DNA and Et 743 interaction have shown that the A and B-subunits and α-
carbinolamine are sequence binding recognition.4) Interestingly, the C-subunit, which is perpendicular to
the A and B-subunits, protrudes from the duplex DNA binding sites and might be related to its DNA
binding affinity.5) In addition, the α-carbinolamine is not only structurally crucial for the DNA-alkylation
but also causing the compound unstabiliy. We have succeeded in isolating a large amount of the stable
ecteinascidin 770 (Et 770, 1) from the Thai tunicate Ecteinascidia thurstoni by pretreatment with
potassium cyanide in buffer solution.6) In order to improve its biological activity, the chemical
modification of 1 has been particularly focused at the C-subunit. The first preparation of the 2′-N-acyl
derivatives of 1 was recently reported based on a three-step transformation including: a) 18,6′-O-bisallyl
protection, b) 2′-N-acylation, and c) removing of the allyl protecting group.7,8) Most of them showed
potent cytotoxicity on human solid tumor cell lines. In this presentation, preparation of 2′-N-2,3,4-
trifluorobenzoyl derivative of 1 for future cytotoxicity evaluation will be reported.
Scheme 1 Et 743 and mechanism of action

General experimental procedure Optical rotations were measured on a Perkin Elmer 341 polarimeter.
The IR spectra were obtained on a Perkin Elmer Spectrum One FT-IR spectrometer. The 1H and 13C
spectra were recorded at 500 and 125.65 MHz, respectively, on a JEOL JNM-LA-500 FT-NMR
spectrometer and at 300 and 75 MHz, respectively, on a Bruker Avance DPX-300 FT-NMR spectrometer.
The HR-FABMS spectra were recorded on a JMS-700 mass spectrometer, with a direct inlet system
operating at 70 eV. CD was obtained on a Jasco J-715 spectropolarimeter. Column chromatography was

PN - 3
performed using Silica gel 60 (No. 1.09385, particle size 0.040-0.063 mm, Merck) for flash column
chromatography, silica gel 60 (No. 1.07734, particle size 0.063-0.200 mm, Merck) for quick column
chromatography and Sephadex LH-20 (Pharmacia Biotech AB) for gel filtration chromatography.
Extraction and isolation of Et 770 The tunicate Ecteinascidia thurstoni (65 kg, wet weight) was
collected by SCUBA from Phuket Island at 1-5 m dept in May 2012. The tunicate sample was
homogenized with 20 L of phosphate buffer solution (pH 7). Potassium cyanide solution (10%) was
added to the homogenized solution to get 10 mM KCN at the final concentration, and the mixture was
stirred for 5 hours. Then the mixture was macerated with methanol (5 x 20 L). The filtered solution was
concentrated to be the aqueous emulsion, which was partitioned with EtOAc to give a dark-brown residue
of the EtOAc extract (38.87 g). The extract was subjected to quick column chromatography (Silica gel,
hexane-EtOAc 3:1 to 1:3, 100% EtOAc and methanol, respectively) and gel filtration chromatography
(Sephadex LH 20, 100% methanol). The crude 1 was separated by a silica gel flash column (CH 2 Cl 2 -
EtOAc 2:3). The precipitate of 1 was crystallized in co-solvent of CH 2 Cl 2 -methanol to give colorless
crystals of Et 770 (258.2 mg, 3.97 x 10-3 % of wet wt).
Preparation of 2′-N-(2,3,4-trifluorobenzoyl)Et 770 (Scheme 2) a) 18,6′-O-bisallyl protection: Et 770
(232.6 mg, 0.30 mmole) was dissolved in acetone (60 mL), then the solution was added K 2 CO 3 (1.04 g,
25 eq) and stirred for 5 minutes at 0˚C. Allyl bromide (522.7 μl, 20 eq) was added drop wise over 20
minutes to the vigorously stirred mixture. After the reaction suspension was stirred for 1 hour at 0˚C, the
reaction flask was further stirred at room temperature for 18 hours. Then, the reaction mixture was
filtered and concentrated in vacuo, the residue was diluted with water (50 mL) and extracted with CHCl 3
(5 x 50 mL). The combined organic layer was washed with brine (50 mL), dried with anh. Na 2 SO 4 and
concentrated in vacuo. The crude extract was separated by a silica gel flash column using elution of
gradient benzene-EtOAc 15:1 to 1:1 and 0:1 to provide 18,6′-O-bisallyl Et 770 (2) in 77.5% yield.
b) 2′-N acylation: Compound 2 (15.3 mg, 0.018 mmole) and DMAP (11 mg, 5 eq) were dissolved in
pyridine (1.5 mL), and then the solution was stirred 5 minutes at 0˚C. Then, 2,3,4-trifluorobenzoyl
chloride (114 μL, 50 eq) was slowly dropped into the stirring cold solution. After the reaction solution
was stirred for 30 minutes at 0˚C, the reaction was further stirred at room temperature until the TLC
showed disappearance of the starting material. After the solvent was removed in vacuo, the residue was
diluted with water (10 mL) and extracted with CH 2 Cl 2 (5 x 15 mL). The combined organic layer was
washed with brine (40 mL) and dried in vacuo to give a residue. This residue was purified by a silica gel
flash column using elution of gradient CH 2 Cl 2 -EtOAc 15:1 to 1:1 and 0:1 to provide 2'-N-(2,3,4-
trifluorobenzoyl)-18,6′-O-bisallyl Et 770 (3) in 80.1% yield.
c) 18,6′-O-bisallyl deprotection: Tributyltin hydride (52 μL, 16.5 eq) was added dropwise over 10
minutes to a vigorously stirred solution of 3 (11.8 mg, 0.012 mmole), (Ph 3 P) 2 PdCl 2 (5 mg, 0.6 eq), and
glacial AcOH (26 μL, 37.5 eq) in dry THF (4 mL) at 25oC, and the mixture was stirred at 25°C for 4
hours. Then, the mixture was diluted with water (10 mL), made alkaline with 5% aqueous NaHCO 3 , and
extracted with CHCl 3 (3 x 30 mL). The combined extract was washed with 5% aq. NaHCO 3 and dried in
vacuo to give a residue. This residue was purified by a silica gel flash column using elution of gradient
CH 2 Cl 2 -EtOAc 15:1 to 1:1 and 0:1 to provide 2′-N-(2,3,4-trifluorobenzoyl)Et 770 (4) in 31.5 % yield.
Spectroscopic data and physical properties of Et 770 and its derivatives Ecteinascidin 770 (1):
Spectroscopic data and physical properties see Ref. 6) 18,6'-O-bisallyl Et 770 (2): Spectroscopic data and
physical properties see Ref. 8) 2'-N-(2,3,4-trifluorobenzoyl)-18,6'-O-bisallyl Et 770 (3): Colorless
amorphous powder, [α] D 25 -56.9˚ (c 0.58); IR (KBr) 3437, 2925, 2225, 1760, 1510, 1194 cm-1; 1H
NMR (CDCl 3 , 300 MHz) δ (ppm) 7.07, 7.05, 6.52, 6.33, 6.05, 6.05, 5.96, 5.96, 5.41, 5.29, 5.20, 4.76,
4.67, 4.64, 4.46, 4.35, 4.27, 4.14, 3.72, 3.59, 3.56, 3.50, 3.50, 3.45, 2.98, 2.89, 2.49, 2.49, 2.37, 2.26,
2.12, 2.01, 1.79; 13C NMR (CDCl 3 , 75 MHz) δ (ppm) 168.8, 168.3, 164.0, 150.2, 148.8, 147.5, 147.2,
145.5, 141.4, 140.9, 134.7, 133.0, 131.2, 130.0, 127.7, 126.0, 124.6, 123.3, 123.2, 122.4, 118.1, 117.9,
116.8, 113.4, 112.8, 112.6, 112.6, 111.0, 102.1, 72.9, 70.5, 69.7, 60.9, 60.6, 60.4, 59.4, 59.4, 55.5, 55.2,
54.9, 45.5, 42.2, 41.8, 39.1, 29.3, 24.9, 20.3, 15.6, 9.8; HR-FABMS m/z 1008.3228 (MH+, calcd for
C 53 H 51 F 3 N 4 O 10 S, 1008.3227); CD Δε nm (c 10.9 μM, methanol, 22˚C) 2.9 (210), -2.4 (216), -0.6 (226),
0.8 (256). 2'-N-(2,3,4-trifluorobenzoyl)Et 770 (4): Colorless amorphous powder, [α] D 25 45.6˚ (c 0.09);

PN - 3
IR (KBr) 3436, 2925, 2854, 1759, 1629, 1462, 1196 cm-1; 1H NMR (CDCl 3 , 300 MHz) δ (ppm) 7.07,
7.05, 6.42, 6.38, 6.07, 5.97, 5.76, 5.48, 4.69, 4.62, 4.39, 4.23, 3.66, 3.65, 3.58, 3.53, 3.42, 2.98, 2.57, 2.48,
2.31, 2.21, 2.03, 1.81; HR-FABMS m/z 928.2611 (MH+, calcd for C 47 H 43 F 3 N 4 O 10 S, 928.2601); CD Δε
nm (c 19.4 μM, methanol, 22˚C) 1.8 (201), -4.2 (216), -2.3 (226), 0.7 (251).
Scheme 2 General procedure for preparation of 2'-N-(2,3,4-trifluorobenzoyl)Et 770

Preparation of 2′-N-(2,3,4-trifluorobenzoyl)Et 770 was achieved by a three-step transformation of Et 770,
including: a) 18,6′-O-bisallyl protection, b) 2′-N-acylation, and c) 18,6′-O-bisallyl deprotection (Scheme
2). The reactive 18- and 6'-OHs of 1 were protected by using allyl bromide and K 2 CO 3 to provide 2 in
77.5% yield. The 1H- and 13C-NMR spectra of 2 showed the characteristic signals similar to those of 1.
The appearance of the allyl-proton signals (around δ H 5-6) along with the disappearance of the 18-OH
(δ H 5.77) and 6′-OH (δ H 5.59) signals and the shifts of aromatic carbon signals implied the nucleophilic
substitutions at the phenolic hydroxyls by the allyl groups. The 2'-N acylation of 2 was subsequently
performed with 2,3,4-trifluorobenzoyl chloride and a catalyst DMAP to provide 3 in 80.1% yield.
The 1H- and 13C-NMR spectra of 3 showed characteristic signals similar to those of 2. The additional
signals of the corresponding two aromatic protons at δ H 7.07 and 7.05 and the additional carbonyl carbon
at δ C 168.8 implied the 2′-N substituted by a 2,3,4-trifluorobenzoyl group. For the deprotection step, the
allyl groups were converted to the OH groups by using n-Bu 3 SnH to donate hydride and using
(Ph 3 P) 2 PdCl 2 as a catalyst to provide 4 in 31.5% yield. The 1H- and 13C-NMR spectra data of 4 showed
characteristic signals similar to those of 3. The disappearance of the allyl signals along with the
appearance of the 18-OH (δ H 5.76) and 6′-OH (δ H 5.48) signals implied that the allyl groups were
completely removed and replaced by the phenolic hydroxyl protons.

PN - 3
CONCLUSION
Et 770 (1) from the Thai tunicate Ecteinascidia thurstoni is a potent cytotoxic compound. To improve its
biological activity, the chemical modification of 1 has been focused. We were able to prepare additional
2′-N-2,3,4-trifluorobenzoyl derivative of 1 via the three-step transformation. In the next step, its
cytotoxicity by measuring IC 50 values against human colon carcinoma (HCT116), lung carcinoma
(QG56) and prostate carcinoma (DU145) cell lines will be evaluated.
ACKNOWLEDGEMENTS
This research was supported by BNPME and Pharmaceutical Research Instrument Center, Faculty of
Pharmaceutical Sciences, Chulalongkorn University. This research was partially supported by Japan
Society for the Promotion of Science (JSPS) Asia/Africa Science Platform Program 2010-2012. We are
grateful to Phuket Marine Biological Center (PMBC) for supporting the SCUBA equipment.
REFERENCES
1. Rinehart, K.L., Holt, T.G., Freqeau, N.L., Stroh, J.G., Keifer, P.A., Sun, F., Li, L.H.,
Martin,D.G. 1990. Ecteinascidins 729, 743, 745, 759A, 759B and 770: potent antitumor agent
from the Caribbean tunicate Ecteinascidia turbinata. Journal of Organic Chemistry. 55: 4512-
4515.
2. European Medicines Agency. EMEA/COMP/1292/03 Rev.1. EMEA 2007. London, 30th
November 2007.
3. Pommier, Y., Kohlhagen, G., Baily, C., Waring, M., Mazumder, A., Kohn, K.W. 1996. DNA
sequence- and structure-selective alkylation of guanine N2 in the DNA minor groove by
ecteinascidin 743, a potent antitumor compound from the Caribbean tunicate Ecteinascidia
turbinata. Biochemistry. 35: 13303-13309.
4. Hurley, L.H., and Seaman, F.C. 1998. Molecular basis for the DNA sequence selectivity of
ecteinascidins 736 and 743: evidence for dominant role of direct readout via hydrogen
bonding. Journal of American Chemical Society. 120: 13028-13041.
5. Mollinedo, F., David-Cordonnier, M., Gajate, C., Olmea, O., Laine, W., Iglesia-Vicente, J.D.L.,
Perez, C., Cuevas, C., Otero,G., Manzanares, I., Bailly, C. 2005. DNA and non-DNA targets in
the mechanism of action of the antitumor drug trabectedin. Chemistry and Biology. 12: 1201-
1210.
6. Suwanborirux, K., Charunpant, K., Amnouypol, S., Pummangura, S., Kubo, A., Saito, N. 2002.
Ecteinascidins 770 and 786 from the Thai tunicate Ecteinascidia thurstoni. Journal of Natural
Products. 65(6): 935-937.
7. Puthongking, P., Patarapanich, C., Amnuoypol, S., Kubo, A., Suwanborirux, K., Saito, N. 2006.
Chemistry of ecteinascidins. Part 2: Preparation of 6'-O-acyl derivatives of stable ecteinascidin
and evaluation of cytotoxicity. Chemical and Pharmaceutical Bulletin. 54: 1010-1016.
8. Saktrakulkla, P., Toriumi, S., Tsujimoto, M., Patarapanich, C., Suwanborirux, K., Saito, N. 2011.
Chemistry of ecteinascidins. Part 3: Preparation of 2'-N-acyl derivatives of ecteinascidin 770 and
evaluation of cytotoxicity. Bioorganic and Medicinal Chemistry. 19: 4421-4436.

PN - 4
EXTRACTION OF OIL FROM GRAPE SEEDS WITH SUPERCRITICAL CARBON DIOXIDE
Jirawat Eiamwat1, Paramee Pengpreecha2, Benjaporn Tiensong2, Sorada Walapa1,

Kulapatr Wachirasiri1, and Sukit Kampruengdet1
1
Food technology department, Thailand Institute of Scientific and Technological Research, Pathum Thani 12120 Thailand
2
Industrial and Metrology and Testing Service Center, Thailand
Institute of Scientific and Technological Research, Samut Prakan 10280 Thailand
KEYWORDS: Extraction, grape seeds, supercritical carbon dioxide (SC-CO 2 )
INTRODUCTION
Grape seeds are known as a waste byproduct of juice and wine production. They contain oil between 10
and 20%, depending on grape variety, which has high amounts of unsaturated fatty acids, usually linoleic
and oleic acids ranged from 53 to 78% and 16 to 31% (Bail et al., 2008; Sabir et al., 2012). Oil from
grape seeds is also constituted with natural antioxidants, among others, tocopherols in the 50 to 520
mg/kg range that are responsible for health-benefits such as lowering cholesterol levels and alleviating
cardiovascular diseases (Bail et al., 2008; Sabir et al., 2012). Grape seed oil is a promising lipid for
human consumption due to its nutritional constituents.
The use of supercritical carbon dioxide (SC-CO 2 ) is an effective method for oil extraction. It produces
purer oil and preserves bioactive components of the oil when compared with the typical hexane extraction
and refining. This is because the low critical point (P c = 7.3 MPa, T c = 31°C) and characteristics of CO 2
that allows for organic solvent free extraction, making it safe and efficient for separation and fractionation
of thermally sensitive oils (Agostini et al., 2012). In the present study the effects of pressure (20, 30
MPa) and temperature (30 °C, 40 °C, 50 °C) were investigated on the oil yield. The quality of SC-CO 2
extracted oil in term of fatty acid content was compared to that of hexane-extracted oil.

Preparation of grape seeds sample Grape seeds of the Ribier variety were obtained from a winery in
Samut Sakhon province, Thailand. The seeds were washed and oven-dried at 55°C for 4-6 h. After that
they were ground using a miller and then sieved using the sieve screens between 10 and 100 mesh. The
ground seed sample of particle size ranging from 0.15 to 2.0 mm was stored in a sealed plastic bag at
room temperature until all extractions were carried out.
Proximal chemical analysis The proximate analysis of grape seeds sample was determined according to
AOAC (2000). The analysis included moisture, crude protein, crude fat and ash. The moisture content
was determined by drying in oven at 105°C to a constant weight. Total crude protein was determined
using the Kjeldahl method. The total fat content was determined by Soxhlet extraction with petroleum
ether (40 to 60 °C) for 4 h, and then oven-dried to dryness at 105°C for 1 h. Ash was determined by
weighing the incinerated residue obtained at 550°C for 30 min. Total available carbohydrate was
calculated as 100% minus the sum of moisture, protein, fat and ash.
SC-CO 2 extraction The SC-CO 2 extraction of oil was conducted using the Speed SFE instrument with a
300 ml extraction vessel (Applied Separations Inc., Allenton, PA, USA). The vessel was heated with an
oven controlled by a thermostat (±1°C). Liquid CO 2 was delivered into the vessel and pressurized to the
operating value (±10 bar) with a high-pressure pump (Applied Separations Inc., Allenton, PA, USA).
For each extraction, approximately 0.1 kg of ground grape seeds were loaded into the vessel, and packed
with propylene wool. SC-CO 2 was left in contact with the sample for 30 min of static extraction. Then,
dynamic extraction was performed with a CO 2 flow rate ranging about 2 L/min for 4 h. The SC-CO 2
with dissolved oil passed through a heated micrometering valve at 110°C, and was expanded to
atmospheric pressure. The oil was collected in a pre-weighed glass vial at ambient pressure and
temperature.
Solvent extraction Hexane extraction was also performed in a Soxhlet apparatus at 60°C for 4 h in cycles
of about 30 min. Hexane was removed under vacuum in a rotary evaporator at 40°C.

PN - 4
Fatty acid composition SC-CO 2 and hexane extracted oils were converted into fatty acid methyl esters
(FAME) according to AOAC (2000). In brief, 0.2 g of oil sample was dissolved in 10 mL of 1M
methanolic sodium hydroxide, refluxed at 100 °C for 15 min, and then 12 mL of 12% BF 3 and 5 mL of
n-heptane was added. After cooling, 30 mL of saturated sodium chloride was added to the mixture. The
upper n-heptane phase was transferred into a vial and injected into a GCMS-QP2010 Ultra gas
chromatrograph mass spectrometer (Shimadzu, Columbia, MD, USA) equipped with a capillary column
Cp-Sil 88 (100 m long, 0.25 mm i.d., 0.2 µm film thickness). The initial oven temperature was from
100°C heated to 240°C (3°C/min). Injector and detector temperatures were set at 225°C. The mass
spectrometer operated at ionization energy of 70 eV with a scan range of 30-320 amu. Identification of
components was carried out based on retention time and mass spectra by macthing with the NIST library.
Statistical analysis Extractions at each condition were performed in triplicate. All analysis were done in
duplicate. The mean values were calculated and subjected to analysis of variance (ANOVA) at 5% level
of significance using SPSS for Window Version 12.0 (SPSS Inc., Thailand).
RESULTS
Proximate analysis of grape seeds The results obtained showed the proximate composition of 8.0%
moisture, 7.1% crude protein, 14.3% fat, 2.6% ash and 68.0% carbohydrate. These results are in good
agreement with those reported by Elegamey et al. (2013).
Oil extraction Grape seed oil extracted with SC-CO 2 was virtually clear, light yellow-green in color,
while hexane-extracted oil was yellow-brown and turbid. Yields of SC-CO 2 extracted oils ranged from
1.7 to 5.5%, as shown in Table 1, compared to the hexane extractable oil yield of 7.1%. The highest oil
yield (5.5%), corresponding to 35.9% of the total available oil, obtained at 30 MPa, 30 °C. The oil yield
can be further improved by increasing extraction time from 5.5% to 6.9% at 8 h to achieve 44.4% oil
recovery. As seen the oil yield time profile in Figure 1, the oil was extracted rapidly in 4 h, then
gradually with the increased extraction time until approximately 8 h. Extraction time longer than 8 h
resulted in a slightly additional oil yield, representing only 1-2% of the total oil extracted. It is also
observed that the SC-CO 2 extracted oil after 8 h was from light to dark green in color, possibly that
chlorophyll was co-extracted along with the oil.
Extracted oil composition Grape seed oils obtained by SC-CO 2 under different conditions and Soxhlet
extraction contained mainly palmitic (12.8 to 15.4%), stearic (6.8 to 7.7%), oleic (26.7 to 27.5%), and
linoleic (46.0 to 50.4%) acids. The oils were rather poor in linolenic (0.5 to 0.6%) acid, as shown in
Table 2.
DISCUSSION
Although oil extraction of grape seeds with the usual solvent hexane had higher oil yield, it resulted in
the presence of impurities such as unsaponifiables and hexane residue in the crude oil (Gómez et al.,
1996), which requires further refinement. The SC-CO 2 extraction method produced the purer oil. The oil
yield obtained in this study was comparable to those of Gómez et al (1996) who extracted oil from Airen
grape seeds with SC-CO 2 , using 35 MPa, 40 °C for 3 h, obtained 6.9% oil yield, about 92% yield with
hexane extraction in 20 h. Comparing the SC-CO 2 extraction conditions, we used lower pressure and
lower temperature, but longer extraction time.

PN - 4
Table 1 Grape seed oil yield1 and recovery2 obtained with SC-CO 2 extraction under different conditions at CO 2 flow rate about 2
L/min
Pressure Temperature Oil yield1 Recovery2

(MPa) (°C) (% of total oil)
20 30 2.5b 16.4b
20 40 2.3b 15.2b
20 50 1.7a 11.3a
30 30 5.5d 35.9d
30 40 5.0c 32.5c
30 50 5.1c 32.9c
1,2
Oil yield obtained after 4 h (g oil/100 g seed, dry matter basis) is mean of duplicate;
values with the same superscript letter in a column are not significantly different (p > 0.05).
Figure 1 Cumulative oil yield from grape seeds of the Ribier variety using SC-CO 2 at 30 MPa, 30 °C and CO 2 flow rate about 2
L/min. Each point represents the mean of triplicate extractions.
Table 2 Fatty acid composition of grape seed oils obtained with SC-CO 2 under different conditions at CO 2 flow rate about 2
L/min and Soxhlet extraction
Fatty acid (% of total fatty acids)

Extraction method
C16:0 C18:0 C18:1 C18:2 C18:3
o
SC-CO 2 , 20 MPa/ 30 C 14.3 7.4 27.3 47.7 0.5
SC-CO 2 , 20 MPa/ 40 oC 15.3 7.4 27.5 46.1 0.6
SC-CO 2 , 20 MPa/ 50 oC 15.4 7.5 27.7 46.0 0.6
SC-CO 2 , 30 MPa/ 30 oC 13.1 6.8 26.7 50.4 0.5
SC-CO 2 , 30 MPa/ 40 oC 13.5 6.9 27.1 49.7 0.5
SC-CO 2 , 30 MPa/ 50 oC 13.7 7.1 27.3 48.9 0.5
Soxhlet 12.8 7.7 26.7 49.4 0.5
Values are the mean of duplicate analyses
The pressure was the most important factor. The oil yield increased (p < 0.05) by about 2-3 folds when
the pressure increased from 20 to 30 MPa at lower temperature (30 °C). This may be explained by the
increase of CO 2 density with pressure, resulting in a higher oil recovery. However, results of extraction
temperature differed from those of extraction pressure. The decreased oil yield with an increase in
temperature at constant pressure was not statistically significant. This indicates that the CO 2 density did

PN - 4
not change drastically when the temperature was increased from 30 to 50 °C. The analyzed fatty acid
composition showed high amount of unsaturated fatty acids (about 75%), indicating a good nutritional oil.
The values obtained in the present study were comparable to the results by Elegamey et al. (2013),
although wide variability in fatty acid profiles was observed with different grape varieties (Elegamey et
al., 2013; Sabir et al., 2012). The fatty acid profile of SC-CO 2 extracted oils varied slightly with pressure
and temperature, and was comparable to that of hexane-extracted oil.
CONCLUSION
SC-CO 2 extraction was used to produce oil from grape seeds and compared to Soxhlet extraction using
hexane. SC-CO 2 extraction at 30MPa, 30 °C gave the highest oil yield at 5.5% for 4 h initial extraction.
The SC-CO 2 extracted oil recovery obtained at 30MPa, 30 °C and 8 h was 44.4% of the total available
oil. The SC-CO 2 extracted and hexane-extracted oils had similar fatty acid profiles. In both cases the
content of unsaturated fatty acids in the oil was about 75%, mainly linoleic and oleic acids. Additional
characterization of tocopherols and phytosterols in grape seed oil obtained by SC-CO 2 extraction is being
investigated.
ACKNOWLEDGEMENT
The authors express their gratitude to Ministry of Science and Technology, Bangkok, Thailand for
providing research funds.
REFERENCES
1. Agostini, F., Bertussi, R.A., Agostini, G., Atti dos Santos, A.C., Rossato, M., Vanderlinde, R.,
2012. Supercritical extraction from vinification residues: fatty acids, α-tocopherol, and phenolic
compounds in the oil seeds from different varieties of grape. Scientific World Journal, 790486.
2. Bail, S., Stuebiger, G., Krist, S., Unterweger, H., Buchbauer, G., 2008. Characterisation of
various grape seed oils by volatile compounds, triacylglycerol composition, total phenols and
antioxidant capacity. Journal of Food Chemistry 108, 1122-1132.
3. Elagamey, A.A., Abdel-Wahab, M.A., Shima, M.E., Abdel-Mogib, M., 2013. Comparative study
of morphological characteristics and chemical constituents for seeds of some grape table
varieties. Journal of American Science 9, 447-454.
4. Gómez, A.M., López, C.P., De la Ossa, E.M., 1996. Recovery of grape seed oil by liquid and
supercritical carbon dioxide extraction: a comparison with conventional solvent extraction. Short
Communication. The Chemical Engineering Journal 61, 227-231.
5. Sabir, A., Unver, A., Kara, Z., 2012. The fatty acid and tocopherol constituents of the seed oil
extracted from 21 grape varieties (Vitis spp.). Journal of the Science of Food and Agriculture 92,
1982-1987.

PN - 5
STINGLESS BEE HONEY II: QUALITATIVE AND QUANTITATIVE STUDIES ON HONEY

PRODUCED BY THREE STINGLESS BEE SPECIES COLLECTED FROM A MANGOSTEEN
GARDEN IN CHANTABURI PROVINCE, THAILAND
Nipatha Issaro1, Thorsang Weerakul1, Sisipawan Machana1 , Phatsakorn Ornnim1, Chamipha

1
Phanudulkitti1, Tharathee Srijan, Jiratikron Laiwattanaphaisal1, and Chumnan Pattarapanich
1
Faculty of Pharmaceutical Sciences, Burapha University Chonburi, 12130, Thailand
KEYWORDS: Stingless bee, physicochemical parameter, Chanthaburi Province
INTRODUCTION
Stingless bee (Sb) keeping has been widely practiced in many countries such as Brazil [9], Guatemala,
Mexico, Venezuela [5] and Thailand. The bee has been raised mostly to promote flower pollination and as
the consequence, Sb honey and its propolis are the main products obtained. Interest in Sb honey is
expressed by local residents, tourist centers, gift shops and health food shops that want to promote it as
native food and the demand is expected to grow rapidly. Hence, Sb honey is economically viable bee
products that can be of significant importance to local communities and/or industry. Sb honey has long
been consumed as food due to its high proportion of monosaccharides and other minor compounds
including protein, organic acids, vitamins, flavonoids and acetylcholine [11]. In addition, honey has been
reported to possess bioactivities useful in traditional or alternative medicine. For example, honey was
reported to have wound healing [6], anti-proliferative [7], anti-bacterial [8], anti-diabetic [2], anti-
inflammatory [1], and anti-plasmodial activities [10]. In Thailand, it has been reported that the
composition and quality of Thai Sb honey differ from European ones [3].
Scant knowledges are available concerning the quality of Thai Sb honey, which is not included in the
International Standard for Honey (CODEX, 2001). Generally, several parameters (moisture content, pH,
free acidity, total acidity, nitrogen content and diastase value) are used to determine the quality of honey.
The chemical composition also dictates the grades and the market values of honey, which can be
classified according to their physiochemical parameter such as moisture content, hydroxymethylfurfural
(HMF), diastase activity and pH into Grade A (14-16% moisture content), B (17-18%), C (19-21%), or D
(>21%) [12]. In order to set the quality standard for Thai Sb honey, it is necessary to assess the quality of
available Sb honey in the markets. In addition, several factors such as botanical, geographical and insect
species variation can also affect the quality of honey. Therefore, this research work was primarily focused
on the determination of basic physicochemical properties of Thai Sb honey collected in Chanthaburi
Province, Thailand, as the baseline information for further standard establishment.
MATERIAL AND METHODS

Honey samples Three types of stingless bee honey (Trigonalaeviceps Smith, Trigona sp. and
Trigonapagdenis Schwarz) were collected from a mangosteen garden in Chanthaburi Province located in
the eastern part of Thailand. The samples were preserved at 11 to -21 °C in the dropping bottle and
analysed as soon as they arrived in the laboratory.
Physicochemical analysis The chemical analysis was performed according to the official method
prescribed by the Association of Official Analytical Chemists [4], including the determination of pH,
diastase activity, moisture content, reducing sugars content, ash content, total acidity, electrical
conductivity, HMF content and insoluble matter content. Nitrogen content was measured by the Kjeldahl
method [4].
Analysis of data The mean values of each parameter were computed and analysed by using one way
ANOVA analysis. Statistical significance was assessed as p < 0.05.
RESULTS AND DISSCUSSION

The basic parameters obtained from three types of Sb honey were shown to be similar. All samples were
relatively very slightly acidic, which likely to inhibit the growth of microorganism. Their total sugar
contents (Brix) were 82.67-84.50% and their nitrogen contents were relatively high (136.40-178.70 g/kg).
In contrast, their low ash values (0.14-0.22g/100g) indicated the cleanliness of the samples. Their mean
values of physicochemical parameters were significantly different at p < 0.05 (Table 1).

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Table 1 Physicochemical characteristics of honey samples from 3 types of stingless bee
Trigonalaeviceps Trigonapagdenis
Physicochemical parameters Trigona sp.
Smith Schwarz
pH 3.44+ 0.06 3.35+0.03 4.01+0.01
Total acidity (Eq/kg) 50.83+3.81 78.14+2.89 20.00+2.50
Diastase activity (Dn) 13.64+0.00 16.67+0.00 11.11+0.00
Electrical conductivity (mS/cm) 0.57+ 0.00 0.57+0.00 0.45+0.00
Moisture (%) 15.73+1.28 13.26+0.46 14.66+0.30
Brix (%) 82.67+1.04 84.50+0.50 83.77+0.40
Ash content (g/100g) 0.14+0.00 0.20+0.00 0.22+0.00
HMF (mg/kg) 3.32+0.00 3.18+0.00 3.97+0.00
Nitrogen (g/1kg) 178.70+0.00 173.80+0.00 136.40+0.00
Reducing sugars (%) 27.37+0.20 29.34+0.35 41.64+0.12
Insoluble matter (%) 0.03+0.00 0.11+0.00 1.75+0.00
Remark: Data are shown as the mean + S.D. and are derived from triplicate experiments
CONCLUSION
This is the first comparative study on various parameters among three types of Sb honey found in
Thailand. These findings would be basic information for establishing the standard of Sb honey and would
also be useful to the stingless bee keepers in order to improve their product.
ACKNOWLEDGMENTS
This work was supported by the Faculty of Pharmaceutical Sciences, Burapha University. Mr. Wisit
Tanooard, Mr. Samart Khrua and Mr. Wirot Khrua, as well as Thai local wisdom of Kon Chan
Channarong (stingless bee) at Chantaburi province, Thailand, are also thanked for facilities.
REFERENCES
1. Ahmad, A., Khan, R.A., Mesaik, M.A. 2009. Anti-inflammatory effect of natural honey on bovine
thrombin-induced oxidative burst in phargocytes. Phytother. Res. 23: 801-808.
2. Al-Waili, N.S. 2004. Natural honey lowers plasma glucose, C-reactive protein,homocysteine, and
blood lipids in healthy, diabetic, and hyperlipidemic subjects:comparison with dextrose and sucrose.
J. Med. Food. 7:100-107.
3. Anchalee, S., Pilanee, V., Sukantaros, T. 2009. Comparative composition of honey from Thai
stingless bee and European honeybee (Apis mellifera L.). Proceedings of 47th Kasetsart University
Annual Conference: Plants., 139-144.
4. AOAC (2005). Official methods of analysis of AOAC International. 18th ed. Maryland: AOAC
International.
5. Patricia, V., Margarita, M., Maria E.E. 2004. Quality standards for medicinal uses of Meliponinae
honey in Guatemala, Mexico and Venezuela, March Bee World.
6. Phuapradit, W, Saropala, N. 1994. Tropical application of honey in treatment of abdominal wound
disruption. Annual research abstracts and bibliography of non-formal publications. 22:56.
7. Pichichero, E., Cicconi, R., Mattei, M., Muzi, M.G., Canini, A. 2010. Acacia honey and chrysin
reduce proliferation of melanoma cells through alterations in cell cycle progression. Int. J. Oncol. 37:
973-981.
8. Sherlock, O., Dolan, A., Athman, R., Power, A., Gethin, G., Cowman, S., Humphreys, H. 2010.
Comparison of the antimicrobial activity of Ulmo honey from Chile and Manuka honey against
methicillin-resistant Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. BMC

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Complement. Altern. Med., http://biomedcentral.com/1472-6882/10/47.

9. Souza, Bruno A., Roubik, David W., Barth., Ortrud M., Heard, Tim A., Enríquez, Eunice., Carvalho,
Carlos., Villas-Bôas, Jerônimo., Marchini, Luis., Locatelli, Jean., Persano-Oddo,Livia., Almeida-
Muradian, Ligia., Bogdanov, Stefan., Vit, Patricia. 2006. Composition of stingless bee honey: setting
quality standards, Interciencia. 12(31): 867-875.
10. Jirattikarn, K., Paworn N., Atsalek, R., Pakorn, W., Chanpen, C. 2012. Preliminary Screening for
Various Bioactivities in honey and Propolis Extraction from Thai Bees. European Journal of Medical
Plants. 2(2): 74-92.
11. Wang J., Kliks M.M., Jun S., M. Jackson, Q.X. Li. 2010. Rapid analysis of glucose, fructose, sucrose
, and maltose in honeys from different geographic regions using Fourier transform infrared
spectroscopy and multivariate analysis. Journal of Food Science. 75(2) : 208–214.
12. White, J.W. and L.W. Doner. 1980. Honey composition and properties. Beekeeping in the United
States Agriculture Handbook. 335: 82-91.

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GENE CLONING AND EXPRESSION OF A TRITERPENE SYNTHASE FROM ALANGIUM

LAMARCKII
Nattaon Tansakul1, Pimpimon Tansakul2, and Wanchai De-Eknamkul1

1
Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,
Chulalongkorn University, Bangkok 10330, Thailand
2
Prince of Songkla University, Songkla 90112, Thailand
KEYWORDS: Alangium larmarckii, Triterpene synthase, Gene cloning, Gene expression
INTRODUCTION
The triterpenoids are a large group of terpenoid compounds found widely as natural products. Many
structures of tetracyclic and pentacyclic triterpenes have been discovered in higher plants and reported to
have various biological and pharmacological activities [1, 2]. However, the physiological functions of this
secondary product group of triterpenes in plants are still not clear. Triterpenes are biosynthesized by the
group of enzymes, called either as oxidosqualene cyclases (OSC), named after their common substrate, or
as triterpenoid synthases, named after their preferred products. Each of these enzymes can facilitate the
mechanism of cyclisation which comprises protonation, cyclisation, rearrangement and deprotonaton. All
of these steps occur within one enzyme to give the structure of tetracyclic or pentacyclic triterpene [3].
Due to the complex mechanism of the OSC enzymes and the diversity of the triterpene products, it is
interesting to know how each triterpene synthase produces each product specifically. Several studies have
been reported on cloning and characterization of OSCs from plant species (for review, see [4]). The data
of triterpene synthase genes from many plant species can help predicting the formation of various
triterpenes which are taken place in active sites of the enzymes.
Alangium lamarckii is a small to medium tree belonging to the family Alangiaceae. Previous study has
shown that friedelin can be found in A. lamarckii leaves [5]. Therefore, this plant is an ideal source of this
triterpene ketone. Here, we report the cDNA cloning of OSCs from A. lamarckii and heterologous
expression in a mutant yeast lacking lanosterol synthase, which is a member of OSC family.

Plant material Young leaves of Alangium lamarckii were collected from the botanical garden of the
Faculty of Pharmaceutical Sciences, Chulalongkorn University.
Cloning of OSC from A. lamarckii The leaves of A. lamarckii were ground by using liquid nitrogen in a
mortar and pestle. Total RNA was extracted by using RNeasy mini kit (Qiagen) followed by cDNA
synthesis using reverse transcriptase (Fermentas). The resulting cDNA was used as template for
amplification by PCR in a total volume of 50 µl containing the first strand cDNA 2 µl, primers (5’-
ATGTGGAGGCTGAAAGTAGCAGAAGG-3’ and 5’-TCAGAGCCTCTGGGGAGGGAACTGAA-3’),
dNTP mixture (0.2 mM), 10x buffer (5 µl) and taq DNA polymerase (Invitrogen). The PCR condition
was set for 35 cycles, at 94˚C for 30 seconds, 55˚C 40 seconds 72˚C for 3 min and final extension 72˚C
for 10 min. A 2.3-kb DNA band corresponding to the length of OSC genes was separated by agarose gel
electrophoresis. After sequencing, 5 genes from the first PCR were used as templates for the second PCR
under the same PCR condition in the solution. Primers containing yeast consensus sequence (5’-
GTACTGAACGTTHAMAMAATGTCSAGGCTGAAAGTAGCAGAAG-3’ and 5’-AAGTCAAGGGA
GGGGTCTCCGAGAGATCTATCTAG-3’) were used to get the suitable constructs for expression in
yeast following the yeast expression vector pYES2 protocol (Invitrogen). The second PCR products were
digested with NotI and XbaI, and ligated into multiple cloning site of a yeast expression vector pYES2
which was digested by the same restriction enzymes.
Functional expression in yeast and product detection The full length cDNAs were cloned into a yeast
expression vector pYES2 (Invitrogen). Forty constructs were transformed into a Sacccharomyces
cerevisiae strain GIL77 [6] by using Forzen-EZ Yeast Transformation IITM kit (ZYMO RESERCH).
The transformed cells were picked and cultured in synthetic complete medium without uracil (SC-U) at
30˚C with shaking 200 rpm, and supplemented with ergosterol, hemin, and Tween80. After 2 days, the
cells were collected and re-suspended in SC-U without glucose, supplemented with ergosterol, hemin,
Tween80 and 2% galactose, and incubated using the same condition for 1 day. Cells were collected and
re-suspended in 0.1 M potassium phosphate pH 7.0, supplemented with 3% glucose and hemin, and
incubated using the same condition for 1 day. The cells were then collected and refluxed in 20%
KOH/50% EtOH, and extracted with hexane. The obtained 40 crude extracts were checked for the

PN - 6
presence of triterpene products using thin layer chromatography (TLC). The plate was double developed
using hexane-acetone (19:1) as mobile phase with the presence of standards (β-amyrin and friedelin). The
TLC plate was visualized by spraying anisaldehyde-sulphuric acid reagent (AS).
The sample with potential product forming was chosen to scale up for detecting the structure of the
products. The obtained extract was then partial purified on a preparative TLC glass plate and double
developed using hexane-acetone (19:1) as mobile phase. The corresponding positions to the standard
triterpene mono-alcohols were scratched and eluted with acetone. The eluate was concentrated, separated
and analysed by high pressure liquid chromatography (HPLC) using a SUPER-ODS column (4.6 x 250
mm. Tosoh) eluted with 95% CH 3 CN as solvent (flow rate 1 ml/min, temperature 40 ˚C, detection UV
254 nm).

Five targeted genes from A. lamarckii cDNA were amplified by PCR. The genes consist of 2292 bp
nucleotides encoding 763 amino acids. The genes have conserved QW motif, which presumably has a
role to stabilize the protein structure, and DCTAE motif, which is responsible for substrate protonation
[7]. These motifs are conserved in the OSC family. Among the 5 genes, 4 of them showed 80% identity to
the amyrin synthase from Catharanthus roseus [8], and the other one showed 79% identity to the mixed
amyrin synthase from Olea europaea [9].
The results suggested that the genes from A. larmarckii leaves are OSC genes. Each gene was then
constructed into yeast expression vector and expressed in a mutant yeast, GIL77, lacking lanosterol
synthase. The yeast was extracted and screened on a TLC plate. Interesting samples showed a pink spot
corresponding to the standard β-amyrin and positive control pOEA [9]. The sample no. 35, which is one
of the 4 genes, showed 80% identity to amyrin synthase from C. roseus. It was scaled up to a large
volume to increase the amount of product which has the same Rf to the standard. The extract was purified
by TLC and analysed by HPLC. Only the obtained HPLC chromatogram (Fig.1) of the sample showed a
peak at the retention time of 15 min compared to the standards and negative control (empty vector
pYES2). Compared to the standards, this peak is presumably the triterpene mono-alcohol taraxasterol or
β-amyrin. This is being confirmed by using the technique of liquid chromatography mass spectrometry
(LC-MS).
mAU mAU
11.573
18.219
2 50
50
2.874
2 25
45
2 00
2.332
40
1 75
35
15.914
1 50
10.282
2.749
15.154
30
17.785
30.540
1 25
25
1 00
20
15.968
16.862
75
21.253
12.668
15
17.667
13.433
50
32.073
10
7.471
12.157
25
5
0
0
0 .0 5 .0 1 0.0 1 5.0 2 0.0 2 5.0 3 0.0 3 5.0 min 1 0.0 1 2.5 1 5.0 1 7.5 2 0.0 2 2.5 2 5.0 2 7.5 3 0.0 3 2.5 3 5.0 3 7.5 min
(A) (C)
mAU
11.455
m AU
2.322
2.347
50
18.176
2 25
45
2.874
2 00 40
1 75 35
15.866
2.745
1 50 30
10.495
25
1 25
20
1 00
15
21.127
25.795
75
19.264
3.186 3.336
10
50
11.554
7.500
5
21.208
6.224
25
0
-5
0 .0 5 .0 1 0.0 1 5.0 2 0.0 2 5.0 3 0.0 3 5.0 min
1 0.0 1 2.5 1 5.0 1 7.5 2 0.0 2 2.5 2 5.0 2 7.5 3 0.0 3 2.5 3 5.0 3 7.5 min
(B) (D)
Figure 1 HPLC chromatograms of (A) standard taraxasterol (15.86 min), (B) standard β-amyrin (15.91 min), (C) sample no. 35 and
(D) negative control pYES2.
CONCLUSION
It has been reported that friedelin is present in A. larmarckii leaves [5]. Therefore, we targeted the genes
that are involved in the formation of fridelin. However, based on the results presented, we can conclude
that the gene obtained would be involved in the formation of triterpene mono-alcohols. This suggested
that this plant has other triterpenoid compounds which have not been reported, presumably due to their

PN - 6
presence as minor compounds. However, the finding of OSC genes in this study would contribute to the
knowledge of the triterpenoid natural products in this Thai plant.
ACKNOWLEDGMENTS
The authors are grateful to Assoc. Prof. Dr. Boonchoo Sritularak and Assoc. Prof. Dr. Rutt Suttisri
(Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,
Chulalongkorn University) for authentic standards of β-amyrin and friedelin. We would also like to thank
the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences,
Prince of Songkla University, Songkla, Thailand for providing working spaces during one of the authors
(N. Tansakul)’s short visit.
REFERENCES
1. Dzubak, P., et al., Pharmacological activities of natural triterpenoids and their therapeutic
implications. Natural Product Reports, 2006. 23(3): p. 394-411.
2. Salvador, J.A.R., et al., Ursane-type pentacyclic triterpenoids as useful platforms to discover
anticancer drugs. Natural Product Reports, 2012. 29(12): p. 1463-1479.
3. K. U. Wendt, et al., Enzyme Mechanisms for Polycyclic Triterpene Formation. Angewandte
Chemie International Edition, 2000. 39: p. 2812-2833.
4. Haralampidis, K., M. Trojanowska, and A. Osbourn, Biosynthesis of Triterpenoid Saponins in
Plants, in History and Trends in Bioprocessing and Biotransformation, N.N. Dutta, et al., Editors.
2002, Springer Berlin Heidelberg. p. 31-49.
5. Gupta, N.C., B. Singh, and D.S. Bhakuni, Steroids and triterpenes from Alangium lamarckii,
Allamanda cathartica, Abrus precatorius and Holoptelea integrifolia. Phytochemistry, 1969. 8(4):
p. 791-792.
6. Kushiro, T., M. Shibuya, and Y. Ebizuka, β-Amyrin synthase. European Journal of Biochemistry,
1998. 256(1): p. 238-244. Thoma, R., et al., Insight into steroid scaffold formation from the
structure of human oxidosqualene cyclase. Nature, 2004. 432(7013): p. 118-122.
7. Thoma, R., et al., Insight into steroid scaffold formation from the structure of human
oxidosqualene cyclase. Nature, 2004. 432(7013): p. 118-122.
8. Yu, F., et al., Functional characterization of amyrin synthase involved in ursolic acid biosynthesis
in Catharanthus roseus leaf epidermis. Phytochemistry, 2013. 91(0): p. 122-127.
9. Saimaru, H., et al., Production of triterpene acids by cell suspension cultures of Olea europaea.
Chemical and Pharmaceutical Bulletin 2007. 55(5): p. 784-788.

PN - 7
EFFECT OF ETHANOL CONCENTRATION ON CYANIDIN-3-GLUCOSIDE, TOTAL

MONOMERIC ANTHOCYANINS, TOTAL PHENOLIC CONTENT AND RADICAL
SCAVENGING PROPERTIES IN PURPLE CORN (Zea mays L.) SEED AND COB.
Penpan Srithong1, Tanwarat Kajsongkram2, Chuleratana Bangchonglikitkul2 and Waraporn Boonsupthip1

1
Faculty of Agro-Industry, Kasetsart University. 50 Ngamwongwan Road, Ladyao, Chatuchak, Bangkok, Thailand . 10900
2
Pharmaceutical and Natural Products Department, Thailand Institute of Scientific and Technological Research (TISTR),
Technopolis, Klong 5, Klong Luang, Pathumthani,Thailand, 12120.
KEYWORDS: Purple corn, Anthocyanins, Solvent extraction
INTRODUCTION
Purple corn (Zea mays L.) has been extensively grown in South America, mainly in Peru and Bolivia, and
used to prepare drinks and desserts for centuries due to its high pigment content. Purple corn seed and cob
contains high concentration of anthocyanins which are an important group of flavonoid compounds.
Anthocyanins in purple corn have been used as a source of colors and phytonutrients over the last years.
Many health benefits have been associated with purple corn, including reduction of oxidation stress,
prevention of coronary heart disease, obesity and diabetics, and anticancer activity. Due to its health
benefits, we will study the preparation process of anthocyanin-rich extract as the raw material for
medical and health supplement purpose.
The aim of this study is to investigate the effect of ethanol concentration on the cyanindin-3-glucoside
content (C3G), total anthocyanins content (TAC), total phenolic contents (TPC) and free radical
scavenging (DPPH (1,1-diphenyl-2-picrylhydrazyl) method) of purple corn (Zea mays L.) seed and cob.
The ethanol concentration has been used as 90%, 70%, 50%, 30% and 0% v/v ethanol.

Plant material Purple corn seed (PCS) and cob (PCC) were obtained from Nakhonsawan Province,
Thailand. The seeds and cob were separated,and then washed, dried and ground into coarse particles.
Anthocyanin-rich extract preparation The powder of purple corn seed and cob ( 25,10 g.) were extracted
by 75 and 70 ml of aqueous ethanol by using sonicator for 10 min. The extraction were studied at the
concentration of 90%, 70%, 50%, 30%, 0%, v/v ethanol. The supernatants were filtered through a
Whatman No.1. and concentrated by using a rotary evaporator at 40 ºC under vacuum condition.
Preparation of the sample The anthocyanin extracts were dissolved in 0.01%-HCl-acidified-water and
partitioned with chloroform in separatory funnel. The upper aqueous layer, containing the anthocyanins,
was collected while the lower which is the chloroform layer was carefully discarded. Residual chloroform
was removed from the anthocyanin extract by using a rotary evaporator at 40 ºC under vacuum
condition. Extracts were taken to 50 ml in volumetric flask adjust with 0.01%-HCl-acidified-water and
kept at −80 ºC until analysis.
Determination of cyanindin-3-glucoside content in purple corn seed and cob extracts by HPLC method
Cyanindin-3-glucoside content was determinate on Waters Alliance System e2695 LC system equipped
with Waters model 2998 a photodiode-array detector and an Empower software (Waters Corporation,
USA). HPLC separation was conducted using a reversed phase 5 μm Symmetry C18 column (3.9 × 150
mm, Waters Corp.) coupled with a guard column of the same stationary phase (Waters Corp.). The mobile
phase used were A, 1% phosphoric acid/10% acetic acid/5% acetonitrile in water, and B, 100%
acetonitrile. Anthocyanins were separated by using a linear gradient from 0% to 80% A in 25 min. An
injection volume of 20 μLwith a 1mL/min of flow rate was used. Spectral information at the wavelength
of 520 nm was collected. Measurements were carried out in triplicate with the calculations being based on
a standard curve and data were presented as mg /100g dry weight. This method was modified from the
procedure described by Jing and Giusti (2009).
Determination of polyphenol and total monomeric anthocyanins content and antioxidant activity Total
anthocyanins content (TAC) was evaluated by pH-differential method (Giusti and Wrolstad, 2001). Data
were presented as mg/100g dry weight.
Total phenolics content (TPC) was determined using Folin–Ciocalteu reagent (Singleton and Rossi,1965).
Measurements were carried out with the calculations being based on a standard curve based upon gallic
acid. Results were expressed as mg gallic acid equivalents (GAE)/100g dry weight.

PN - 7
The free radical scavenging activity of the extracts was perform by using the 1,1-diphenyl-2-
picrylhydrazyl (DPPH) assay according to the procedure described by Brand-Williams et al. The results
are expressed as milligrams of Trolox equivalents (TE) per 100 g of dry sample (mgTE/100 g dry
weight).
Statistical analysis The results are expressed as means ± standard error of mean (S.E.M.). Differences in
mean values between groups were analyzed by a one-way analysis of variance (ANOVA) followed by a
post-hoc Duncan's Multiple Range test for multiple comparisons. Statistical significance was assessed as
p<0.05.

Effect of ethanol concentration on cyanindin-3-glucoside content in purple corn (Zea mays L.) seed
and cob. Cyanidin-3-glucoside content (C3G) in purple corn (Zea mays L.) seed and cob were quantified
using HPLC method. The optimum HPLC system was comprised of a C18 reverse phase column (Xterra
column, 5µm, 3.9 x 150 mm), gradient elution with 1% phosphoric acid/10% acetic acid/5% acetonitrile
in water (A) and acetonitrile (B) as mobile phase and UV detection at 520 nm. C3G content were
determined by comparison to caribration curve (Figure 1) which derived from separated injections of five
concentrations of C3G versus the peak area. Linearity was found in the concentration range between 10-
50 ppm with correlation coefficient (r2) of 0.9997. The C3G retention time of reference standard and
samples was the same at 4.88 minute (Figure 2).
The effect of ethanol concentration on C3G was investigated over the range of 0-90% v/v ethanol. The
content of the C3G in PCS and PCC extracts increased with ethanol concentration up to a maximum at
70% v/v and then decreased at 90% v/v (Figure 3). The optimal ethanol concentration for PCS and PCC
extracts were 70% v/v ethanol (p<0.05) that showed the highest C3G content (2.62 ± 0.00 and 28.38 ±
0.50 mg/ 100 g dry weight). The result of this study revealed that C3G content in PCC extract was more
than PCS extract.
Figure 1 Calibration curve of cyaniding-3-glucoside (y=76928x-33156, r2= 0.9997)
Figure 2 HPLC Chromatograms analysis of the anthocyanins (A) Referense standard of cyanidin-3-glucoside (B) PCS extracted
with 70% ethanol (C) PCC extracted with 70% ethanol Column: Xterra C 18 3.9 x 150 mm Mobile phase: 1% phosphoric acid/10%
acetic acid/5% acetonitrile in water (A) and acetonitrile (B), (gradient elution)

PN - 7
Effect of ethanol concentration on anthocyanins content in purple corn (Zea mays L.) seed and cob.
The total anthocyanin content in PCC and PCS extract could be determined using visible
spectrophotometry. Figure 4 showed anthocyanin contents in 5 ethanol concentrations varied from 0.95 ±
0.00-491.22±5.73 mg/100g dry weight. The TAC of 70% v/v ethanol extracts from PCS and PCC showed
the highest as 79.65 ± 0.26 and 491.22 ± 5.73 mg/ 100 g dry weight. The increase of the ethanol
concentration in the extraction solution up to 70% resulted to increase TAC. The TAC of extracts
significantly decrease at 90% v/v ethanol. In previous research in Benitaka cultivar grapes study, it has
similarly results which showed solvents containing 70% ethanol in water leads to extract the higher
content of total anthocyanins when comparing with 60% and 80% v/v of ethanol (Vanini, L.S. et al.,
2009)
a-e
Figure 3 Cyanidin-3-glucoside content of PCC and PCS extract. Data are presented as mean ±SEM. Point with different letters
in each figure means significant difference (p≤0.05).
Figure 4 Total anthocyanins content (TAC) (mg/100 g dry weight) of PCC and PCS extract. Data are presented as mean±SEM.
a-e
Point with different letters in each figure means significant difference (p≤0.05).
Effect of ethanol concentration on total phenolics content in purple corn (Zea mays L.) Seed And Cob.
Measurement of the TPC in PCS and PCC extracts showed that the increase of ethanol in the extraction
solution (from 0% to 70%) resulted to increase TPC of the extracts but TPC of the extracts decrease at
90% of ethanol (Figure 5). The TPC of PCS and PCC at 70% ethanol PCS was 345.07 ± 5.07 mg
GAE/100g dry weight , 1227.02 ± 5.89 mg GAE/100g dry weight ,respectively.
From the literature review, the red grape pomace study show that the solvents containing 70% and 50%
ethanol in water leads to extracting of higher content of total phenolics content, as compared to 10% and
30% ethanol, and to methanolic solutions (Monrad et, 2010).
Effect of ethanol concentration on radical scavenging properties in purple corn (Zea mays L.) seed and
cob. The DPPH radical assay has been widely used to evaluate the antioxidant activity of fruit and
vegetable extract. The method is based on the the reaction that hydrogen-donating antioxidants reduce
violet DPPH free radical to yellow DPHH-H, a non-radical form (Kumaran and karunakaran, 2005) The
reduce amount of DPPH absorption at 517 nm indicated the radical-scavenging ability of antioxidants.
Figure 6 displays DPPH radical-scavenging activity of the anthocyanins from of PCS and PCC extract.
The results show that the radical-scavenging activities of antioxidants increased with the increment of
ethanol concentrations (from 0% to 70%v/v ethanol) but decrease at 90% of ethanol. The free radical
scavenging activities of PCS and PCC at 70%v/v ethanol PCS were 567.62 ± 1.38 and 2539.02 ± 23.29
mg TE/100 g dry weight, respectively.

PN - 7
Figure 5 Total phenolics content (TPC) (mg gallic acid equivalents (GAE)/100g dry weight.) of PCS and PCC extract. Data are
presented as mean±SEM. a-e Point with different letters in each figure means significant difference (p≤0.05).
Figure 6 The free radical scavenging activity (milligrams of Trolox equivalents (TE) per 100 g of dry sample (mgTE/100 g dry
weight) of PCC and PCS extract was extracted Data are presented as mean ±SEM. a-e Point with different letters in each figure
means significant difference (p≤0.05).
CONCLUSION
From this study, 70% ethanol extraction was found to be the most effective concentration on cyaniding-3-
glucoside, anthocyanins, total phenolics content and antioxidant activity.
ACKNOWLEDGMENTS
This study was supported by Thailand Instituted of Scientific and Technological Research (TISTR),
Pathumthani 12120. We thank all TISTR colleagues who in one way or another help this project to
succeed.
REFERENCES
1. Brand-Williams W, Cuvelier ME, Berset C. 1995. Use of a free radical method to evaluate
antioxidant activity. Lebensm.- Wiss. –Technol 28: 25-30.
2. Giusti MM and Wrolstad RE. 2001. Characterization and measurement of anthocyanins by UV-
visible spectroscopy, In Current Protocals in Food Analytical Chemistry, R. E. Wrolstad, T. E.
Acree, H. An, E. A.
3. Jing P. and Giusti MM. 2007. Effects of Extraction Conditions on Improving the Yield and
Quality of an Anthocyanin-Rich Purple Corn (Zea mays L.) Color Extract. J food chemistry and
toxicology 72(7): 363-368.
4. Kumaran A, karunakaran RJ. 2005. Antioxidant and free radical scavenging activity of an
aqueous extract of Coleus aromaticus. Food Chem. 97: 109-114.
5. Monrad JK et al., 2010. Subcritical solvent extraction of anthocyanins from dried red grape
pomace, J Agric Food Chem 58(5): 2862-2868.
5. Singleton VL and Rossi JL. 1965. Colorimetry of total phenolics with phosphomolybdic-
phosphotungstic acid reagents. Am J Enol Vitic 16: 144-158.
6. Vanini LS et al., 2009. Extraction and stability of anthocyanins from the Benitaka grape cultivar
(Vitis vinifera L.). J. Food Technol 12( 3):213-219.

PN - 8
ANTIOXIDANT ACTIVITIES AND PHYTOCHEMICALS OF EDIBLE FLOWERS
Jeerawat Sawatpipat1, Vatin Phunsawat1, Piyanuch Rojsanga2 and Pongtip Sithisarn1*

1
Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, 10400, Thailand
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, Bangkok, 10400, Thailand
KEYWORDS: edible flowers, free radical scavenging activity, total phenolic, total flavonoid
INTRODUCTION
Plants are major sources of nutrition and vitamins for human life. Every parts of the plant such as leaves,
flowers, fruits, roots and rhizomes have been used as food or health supplements. Some edible flowers are
now more popularly consumed in the form of drinks, jellies, salads and main dishes. However, there are
some Thai traditional plants which their flowers are edible for a long time, but there is no report
concerning about the phytochemicals and biological activities of them. Therefore, this experiment was set
up in order to screen the free radical scavenging activities of extracts from 11 edible flowers using 1,1-
diphenyl-2-picrylhydrazyl (DPPH) assay. Then, quantitative analysis of total phenolic and total flavonoid
contents of the extracts using spectrophotometric methods was conducted. Finally, phytochemical
analysis of all flower extracts by thin layer chromatography was performed.

Plant materials Eleven edible flowers from various plants including broccoli, (Brassica oleracea),
cauliflower (B. oleracea), Chinese cabbage (B. chinensis var. parachinensis), tamarind (Tamarindus
indica), banaba (Musa sp.), rose (Rosa sp.), ixora (Ixora sp.), cowslip creeper (Telosma minor), agasta
(Sesbania grandiflora), Siamese neem tree (Azadirachta indica var. siamensis) and garlic chive (Allium
tuberosum) were purchased from local market, Bangkok, Thailand in June 2013. The samples were
cleaned and dried in hot air oven (50oC) for 6 hours and powdered with electronic mill (20 mesh sieve).
Each sample was prepared using the extraction procedures shown below.
Decoction: Dried powder of each flower sample was separately boiled (80oC) with distilled water
(plant/water ratio = 1:10 w/v) for 3 h, then filtered. The filtrate was dried upon a water bath to obtain
dried decoction extract.
Maceration: Dried powder of each flower sample was separately macerated with 95% ethanol
(plant/water ratio = 1:10 w/v) for 7 day with frequent shaking, then filtered. The filtrate was dried upon a
water bath to obtain dried ethanol extract.
Determination of free radical scavenging activity using DPPH scavenging assay The free radical
scavenging effect of flower extracts as well as standard trolox corresponding to the quenching ability of
1,1-diphenyl-2-picryl hydrazyl (DPPH) was carried out as described by Yamasaki et al.1). Each sample
was assayed in triplicate and the average percentage of inhibition at the concentration of 100 µg/ml was
calculated.
Determination of total phenolic content using Folin-Ciocalteu method Using the method modified from
Naithani et al.2), solutions of plant extracts were oxidized with Folin-Ciocalteu reagent and the reactions
were neutralized by sodium carbonate solution. The absorbance of the resulting blue colored solution was
measured at 765 nm after 60 min. Each sample was assayed in duplicate. Total phenolic content was
expressed as g gall acid equivalent in 100 g extract (g% GAE).
Determination of total flavonoid content Total flavonoid content was investigated using the method
previously described 3). Solutions of plant extracts were reacted with an equal volume of aluminium
chloride solution. Absorbances were read at 415 nm after 10 min, and flavonoid con tent was expressed as
grams quercetin equivalent in 100 g of plant extracts (g% QE).
Thin layer chromatographic fingerprints Thin layer chromatography of all flower extracts was
performed on TLC pre-coated silica gel 60 GF 254 plate, using ethyl acetate-formic acid-acetic acid-water
(137:11:11:26) as the solvent system. TLC plates were detected under UV at 254 and 366 nm, NP/PEG
and DPPH spray reagents.
RESULTS
Free radical scavenging activity: DPPH scavenging assay Comparing among all flower extracts at the
concentration of 100 µg/ml, decoction and maceration extracts from the flowers of rose, ixora and
Siamese neem tree exhibited high free radical scavenging activity. Rose flower extracts prepared from

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decoction and maceration showed the strongest free radical scavenging activity with percentages of
inhibition of 90.25 ± 0.52 and 94.00 ± 0.98 µg/ml, respectively (Table 1).
Determination of total phenolic content: Folin-Ciocalteu method As shown in Table 1, rose flower
decoction extract exhibited the highest total phenolic content of 23.03 ± 0.93 g% GAE. Other extracts
which displayed high total phenolic contents were decoction extract from the flowers of Siamese neem
tree and maceration extracts from the flowers of banana and garlic chive, with total phenolic contents of
18.05 ± 0.76, 17.73 ± 2.82 and 20.34 ± 3.92 g% GAE in the dried extracts, respectively.
Determination of total flavonoid: aluminium chloride method Rose flower decoction extract also
contained the highest amount of total flavonoids (2.60 ± 0.09 g% QE), followed by the maceration and
decoction extracts from the flowers of Siamese neem tree (Table 1). From these results, DPPH
scavenging activities of decoction extracts showed higher correlations to total phenolic and total
flavonoid contents, with coefficient determination values (R2) of 0.664 and 0.455, respectively, while
maceration extracts displayed low correlation (R2 < 0.2) between the activities and total phenolic and
flavonoid contents.
Thin layer chromatographic fingerprints As shown in Figure 1, the decoction extracts from 11 edible
flowers showed specific TLC fingerprints to NP/PEG spraying reagent. The rose and ixora flower extracts
(track numbers 8 and 10) displayed chromatographic bands corresponding to standard phenolics and
flavonoids, including quercetin and chlorogenic acid which are antioxidant compounds, suggesting their
influences on the strong in vitro antioxidant effects.
Table 1 Free radical scavenging activity, total phenolic and total flavonoid contents of extracts from 11 edible flowers
DPPH Total
Extraction Yield Total flavonoid
No. Sample scavenging phenolic
solvent (%w/w) (g% QE)
activity* (g% GAE)
1 Chinese cabbage Water 1.46 17.65 ± 1.86 5.79 ± 1.17 0.72 ± 0.13
Alcohol 0.89 13.85 ± 2.57 8.09 ± 1.47 1.29 ± 0.17
2 Banana Water 1.34 8.11 ± 1.11 2.39 ± 0.39 0.72 ± 0.17
Alcohol 0.65 Inactive 17.73 ± 2.82 0.53 ± 0.17
3 Cowslip creeper Water 3.17 13.68 ± 3.61 4.34 ± 0.03 0.96 ± 0.06
Alcohol 1.59 2.08 ± 0.66 6.83 ± 0.15 0.85 ± 0.14
4 Rose Water 3.00 90.25 ± 0.52 23.03 ± 0.93 2.60 ± 0.09
Alcohol 1.64 94.00 ± 0.98 13.46 ± 0.57 0.93 ± 0.47
5 Tamarind Water 4.70 23.06 ± 0.93 3.93 ± 0.39 0.61 ± 0.10
Alcohol 4.81 26.69 ± 5.42 7.47 ± 0.53 0.88 ± 0.01
6 Ixora Water 3.99 73.15 ± 8.41 14.41 ± 0.70 0.96 ± 0.47
Alcohol 2.41 94.24 ± 0.49 14.03 ± 1.22 0.73 ± 0.06
7 Broccoli Water 2.19 21.58 ± 0.31 5.34 ± 0.80 0.47 ± 0.08
Alcohol 1.33 1.18 ± 0.34 5.95 ± 1.80 0.28 ± 0.05
8 Cauliflower Water 3.14 20.72 ± 1.87 5.15 ± 0.44 0.63 ± 0.08
Alcohol 0.93 9.99 ± 0.61 11.83 ± 1.70 1.35 ± 0.12
9 Garlic chive Water 0.48 19.62 ± 0.61 5.76 ± 0.51 0.72 ± 0.17
Alcohol 0.53 2.27 ± 1.50 20.34 ± 3.92 1.11 ± 0.06
10 Agasta Water 2.08 21.97 ± 1.30 6.95 ± 0.39 1.10 ± 0.10
Alcohol 2.50 5.20 ± 1.52 7.68 ± 2.72 1.29 ± 0.01
11 Siamese neem tree Water 3.34 84.91 ± 0.10 18.05 ± 0.76 1.45 ± 0.03
Alcohol 1.35 29.28 ± 1.75 8.87 ± 2.20 2.26 ± 0.03
Trolox - - 29.98 ± - -
0.63**
*% inhibition at the concentration of 100 µg/ml, ** IC 50 value

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A B
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Figure 1 TLC fingerprints of decoction extracts from 11 edible flowers; 1 = standard chlorogenic acid, 2 = standard rutin, 3 =
standard quercetin, 4 = standard gallic acid, 5-15 = water extracts; 5 = Chinese cabbage, 6 = banana, 7 = cowslip creeper, 8 = rose, 9
= tamarind, 10 = ixora, 11 = broccoli, 12 = cauliflower, 13 = garlic chive, 14 = agasta, 15 = Siamese neem tree
Stationary phase: silica gel GF 245
Solvent system: ethyl acetate-formic acid-acetic acid-water (137:11:11:26)
Detection: A= UV 254 nm, B= NP/PEG spraying reagent under UV 366 nm
DISCUSSION
All extracts from edible flowers exhibited in vitro free radical scavenging activities and contained high
amounts of phenolic and flavonoid compounds. The antioxidant activities can be correlated to the
phenolic and flavonoid contents, especially in decoction extracts. This result suggested that decoction
could be the suitable extraction method to prepare health supporting products from flowers. Extracts from
rose and ixora flowers showed the bands corresponding to flavonoid and phenolic acid which could be
responsible for their strong free radical scavenging activities. Moreover, these two flowers have pink to
red color, indicating the presence of anthocyanins which could also promote antioxidant effects.
CONCLUSION
Thai edible flowers showed in vitro free radical scavenging activities, suggesting health promotion
through their consumption. Spectrophotometric and thin layer chromatographic results indicated the
presence of phenolic and flavonoid compounds. Rose and ixora flowers exhibited the strongest
antioxidant effects with high phenolic and flavonoid contents, and should therefore be studied for their
phytochemicals and other related biological activities.
ACKNOWLEDGMENTS
The authors would like to express their gratitude to the Faculty of Pharmacy, Mahidol University, for
providing financial support and research facilities.
REFERENCES
1. Yamasaki K, Hashimoto A, Kokusenya Y, Miyamoto T, Sato T. 1994. Electrochemical method
for estimating the antioxidative effects of methanol extracts of crude drugs. Chem Pharm Bull
42 (8):1663-5.
2. Naithani J, Nair S, Kakkar P. 2005. Decline in antioxidant capacity of Indian herbal teas during
storage and its relation to phenolic content. Food Res Int 39:176-81.
3. Meda A, Lamien CE, Romito M, Millogo J, Nacoulma OG: Determination of the total phenolic,
flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging
activity. Food Chem 2005; 91: 571–577.

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INVESTIGATION ON PHYTOCHEMICAL, ANTIOXIDANT AND CYTOTOXIC PROPERTIES

OF MOMORDICA COCHINCHINENSIS (GAC) FRUIT EXTRACTED
BY SUPERCRITICAL FLUID CARBON DIOXIDE (SCF-CO 2 ) METHOD
Prapaipat Klungsupya1, Worawan Tiatragoon3, Thanchanok Muangman1, Jirawat. Eiamwat2, Sarunya

Laovitthayanggoon1 , Jeerayu Thongdo-A and Srisak Trangwacharakul2
1
Department of Pharmaceuticals and Natural Products (PNPD),
2
Department of Food Technology, Thailand Institute of Scientific and Technological Research (TISTR), 35Moo 3 Techno
Polis,Klong Luang, Pathum Thani 12120, Thailand
3
Department of Pharmacology, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand
KEYWORDS: Momordica cochinchinensis, GAC, supercritical extraction, anti-oxidant, PCL, cytotoxic,

MTT
INTRODUCTION
Momordica cochinchinensis of Cucurbitaceae family has been known as “Fhuck khow” in Thailand and
called “GAC” in Vietnam. It has been consumed as indigenous vegetable and fruit. It was found that peels
and seed membranes of GAC fruit compose of high levels of β-carotene, α-tocopherol and lycopene [1].
Seed membrane of ripe GAC fruit (orange color) was reported to contain high levels of β-carotene and
lycopene at about 10 and 30 times greater than in carrots and tomatoes [2] , respectively. Though GAC is
increasing in popularity, very few information of its anti-oxidant activity was reported. Since traditional
solvent extraction has some disadvantages i.e. some residual solvent left in the extracts and matrix, and
there is always some level of environmental contamination from their use [3]. Therefore, in this study
GAC (seed and peel) extracts were prepared by using supercritical fluid carbon dioxide (SCF-CO 2 )
method. Carrots and tomatoes were also extracted in similar manner and used as reference samples. The
phytochemical components of SCF-CO 2 -GAC peel (GAC-P) and seed membrane (GAC-SM) extracts
were determined by three analytical parameters including phenolic, carotenoid and flavonoid contents.
The anti-oxidant capacity of these GAC-P and GAC-SM extracts was assessed on superoxide (O 2 -•) and
hydroxyl (OH•) radicals scavenging activity using photochemiluminescence (PCL) and deoxyribose (DR)
assays, respectively. Their cytotoxic property was evaluated by the MTT assay on TK6 human
lymphoblast and L929 mouse fibroblast cell lines.

Preparation of supercritical fluid carbon dioxide (SCF-CO 2 ) extraction The ripe fruits of GAC were
purchased from Nakhonpathom province of Thailand (Figure 1). They were washed to remove any
contaminants. The peels (P) and seed membranes (SM) were separated and dried at 50oC in hot air oven
for 48 h. Dried samples were ground and stored in the refrigerator (4oC) in light-protective container. Five
hundred grams (500 g) of air-dried peel or seed membrane were subjected to supercritical fluid CO 2
extraction (SCF-CO 2 ) under suitable conditions (data not shown regarding patent procedure). Carrots and
tomatoes were prepared and extracted by the similar manner and used as reference samples.
Phytochemical component determination
Total phenolic content (TPC) The total phenolic content of GAC-P, GAC-SM, carrot and tomato extracts
was determined using the Folin-Ciocalteu’s method [4] with a slight modification due to soluble capacity
of extracts. Gallic acid was used as the standard phenolic. The results were expressed in term of mg gallic
acid equivalents (GAE)/mg sample extract.
Total flavonoid content (TFC) The total flavonoid content of GAC-P, GAC-SM, carrot and tomato
extracts was determined using the aluminum chloride colorimetric method [5]. Rutin was used as the
standard flavonoid. The results were expressed in term of mg rutin equivalents (RE)/mg sample extract.
Total carotenoid content (TCC) The total carotenoid content of GAC-P, GAC-SM, carrot and tomato
extracts was determined using a spectrophotometer. β-carotene was used as the standard carotenoid. The
results were expressed in term of β-carotene equivalent (BE)/100 mg sample extract.
Antioxidant capacity determination
Superoxide anion radical (O 2 -•) scavenging activity by PCL assay The anti-oxidant activity of GAC-P,
GAC-SM, carrots and tomatoes was determined using photochemiluminescence (PCL) [6-7] method by
Photochem® (Analytik Jena, Germany). Its principle is based on the photo-induced auto-oxidation
inhibition of luminol by antioxidants mediated from the superoxide anion (O 2 –●) radicals. The antioxidant
evaluation was performed for both water (ACW) and lipid (ACL) soluble substance systems using the

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ascorbic acid and trolox as standard antioxidants. Antioxidant activity would be indicated by O 2 –●
reduction in the presence of test samples compared with the standard antioxidant (construction of a
calibration curve with Trolox for antioxidant capacity of ACL or ascorbic for antioxidant capacity of
ACW). The sensitivity of the photochemiluminescence (PCL) assay lies within nmol quantities of
substances. For antioxidant determination, each GAC SCF-CO 2 sample was prepared by dissolving 10
mg in 1 mL of dimethyl sulfoxide (DMSO) and filtered through 0.45 mm syringe filter. All samples were
determined in triplicate.
Hydroxyl radical (OH-•) scavenging activity by 2-deoxyribose (2-DR) degradation assay The hydroxyl
radical (OH-•) scavenging activity of GAC, carrots and tomatoes was assessed using the 2-deoxyribose (2-
DR) degradation assay (Genaro-Mattos, 2009). The 2-DR method is based on the determination of
malondialdehyde (MDA) pink chromogen which was a degraded product of 2-deoxyribose (2-DR)
damaged by OH•. Each sample fractions were prepared as previously mentioned in PCL assay except
using distilled water as solvent. Typical reactions were started by the addition of 50 µM FeCl 3 to
solutions (0.5 mL final volume) containing 5 mM 2-DR, 100 µM EDTA, 10 mM phosphate buffer (pH
7.2), 0.5 mM H 2 O 2 and various concentration of sample fractions in presence of 100 µM ascorbic acid
(reducing agent) for starting the reaction and generated OH•. Reactions were carried out for 10 min at
room temperature and stopped by the addition of 0.5 mL 2.8% trichloroacetic acid (TCA) followed by the
addition of 0.5 mL thiobarbituric acid (TBA) solution. After boiling for 15 min, solutions were allowed to
cool at room temperature. The absorbance of reaction mixture was measured to determine MDA pink
chromogen at 532 nm in micro-plate reader system (GENios Plus, TECAN®, Australia). All samples were
tested in triplicate.
Cytotoxicity determination by MTT assay Cytotoxic property of GAC-P and GAC-SM extracts was
performed on 2 cell lines including the TK6 human lymphoblasts and L929 mouse fibroblasts using the
MTT assay. The principle of MTT is based upon the quantitative colorimetric method for determining cell
proliferation. The MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium-bromide) is a yellow water
soluble tetrazolium dye that is reduced by mitochondrial dehydrogenase, in living but not dead cells, to a
purple formazan product that is insoluble in aqueous solutions. The amount of MTT-formazan formation
can be determined spectrophotometrically after being solubilized in a suitable solvent such as
dimethylsulfoxide (DMSO). Surviving cell numbers (living cells) are determined indirectly by MTT dye
reduction. Both cell lines were exposed to GAC-P and GAC-SM at different concentrations ranging from
1,000 to 5000 µg/ml for 4 h (short exposure) and 24 h (long exposure). Following the exposure times, cell
viabilities of TK6 and L929 were determined and reported as the IC 50 , which refers to the concentration
of test sample that inhibits cell growth by 50%.
% Viability = Absorbance of treated cells

Absorbance of untreated cells × 100

Phytochemical component determination
Total phenolic content (TPC) A summary of total phenolic content of extracts of GAC-P, GAC-SM,
tomatoes and carrots was demonstrated in Table 1. Among these 4 extract samples, it was found that
carrot exhibited the highest phenolic content at 0.0261 ±0.010 mg GAE/mg extract.
Table 1 Values of total phenolic content (TPC)
Total phenolic content

Samples
(mg GAE/mg extract)
GAC seed membrane (GAC-SD) 0.0156 ± 0.039
GAC peel (GAC-P) 0.0156 ± 0.039
Tomato (T) 0.0261 ± 0.010
Carrot (C) 0.0160 ± 0.032
Results were expressed as mean ± SD (n = 3).

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Total flavonoid content (TFC) A summary of total flavonoid content of four extracts including GAC-P,
GAC-SM, tomatoes and carrots was shown in Table 2. The highest flavonoid content (0.167 ± 0.005 mg
RE/mg extract) was observed in peel extract of GAC (GAC-P) sample.
Table 2 Values of total flavonoid content (TFC)
Total flavonoid content

Samples
(mg RE/mg extract)
GAC peel (GAC-P) 0.1670 ± 0.005
Tomato (T) 0.1015 ± 0.003
Carrot (C) 0.0958 ± 0.002
Total carotenoid content (TCC) The total carotenoid content was summarized in Table 3. Of this assay,
β-carotene was used as the standard carotenoid. The results were expressed in term of β-carotene
equivalent (BE)/100 mg sample extract. The result showed the highest carotenoid content value (483.12
µg ± 0.0005 µg BE/100 mg extract) was detected in an extract of GAC peel (GAC-P).
Table 3 Determination of total carotenoid content (TCT)

Samples Total carotenoid content
(µg BE/ 100 mg extract)
GAC peel (GAC-P) 483.12 ± 0.0005
Tomato (T) 354.67 ± 0.004
Carrot (C) 1.66 ± 0.016
Superoxide anion radical (O 2 -•) scavenging activity The highest antioxidant activity was found in GAC-
P for both the ACL and ACW measurement system at 1.873±0.034 nmol Trolox equiv/100 µg and at
0.166 ±0.007 nmol Ascorbic acid equiv/100 µg, respectively. The tomato sample (ACL = 1.144±0.109
nmol Trolox equiv/100 µg, ACW = 0.131±0.053 nmol Ascorbic acid equiv/100 µg) exhibited a greater
antioxidant activity over carrot (ACL = 0.412±0.099 nmol Trolox equiv/100 µg, ACW = 0.062±0.015
nmol Ascorbic acid equiv/100 µg). The PCL results also indicated that GAC-P possessed a higher
O 2 -• scavenging activity than GAC-SM. The results of ACL and ACW suggested that the O 2 -• scavenger
activity in SFE-P found both polar and non-polar phytochemical groups and the non-polar group was
a more effective antioxidant than the polar group.
Hydroxyl radical (OH-•) scavenging activity As demonstrated in Figure 1, the OH• scavenging activity of
GAC extracts exhibited a wide range of OH• scavenging activity, proved from 8.14 ± 0.31 to 55.05 ± 1.09
in the % inhibition of the 2-DR degradation. The GAC-P extract was a more effective inhibitor of the OH•
by exhibiting (55.05 ± 1.09%) than SFE-SM sample (8.14 ± 0.31%) for the inhibition of 2-DR
degradation. However, it was observed that tomato (T) extract exhibited greater antioxidant activity over
GAC-P and GAC-SM extracts in this assay. The wide range of % inhibition values among extracts was
possibly caused by their solubility in water, which was the main solvent used in the deoxyribose assay.
Figure 1 Inhibitory effect (%) of a 2-deoxyribose

degradation of GAC-SM, GAC-P, tomato (T) and
carrot (C) extracts. Results were expressed as a
means ± SD (n=3).

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Cytotoxic activity The MTT assay was carried out to examine the cytotoxicity of GAC-P and GAC-SM in
two different cell lines including TK6 human lymphoblasts and L929 mouse fibroblasts. Results of MTT
assay of L929 and TK6 displayed in Table 4. The MTT results revealed cell survival rates greater than
80% and their IC 50 > 1,000 µg/ml indicating a non-cytotoxic property of GAC peel and seed membrane
extracts.
Table 4. The growth inhibitory effect-IC 50 values (μg/ml) of GAC-SM and GAC-P extracts on the TK6 and L929 cell lines by MTT
assay after 4 and 24 h exposuretime, respectively.
Cell line Extract Treatment time (h) IC 50 (ug/ml)

4 >5,000
TK6 GAC-SM
24 >5,000
4 3,512
GAC-P
24 1,979
4 >5,000
GAC-SM
24 >5,000
L929 4 >5,000
GAC-P
24 3,162
CONCLUSION
The present study firstly revealed that Momordica cochichinensis or GAC fruit peel (SCF-P) and seed
membrane (SCF-SM) extracts displayed low to non-cytotoxic to TK6 and L929 cells, as determined by
MTT assay. In addition, the peel extract possessed higher O 2 -• and OH• scavenging activity than seed
membrane extract. For the health promoting effect, increased consumption of M
cochichinensis antioxidants in the diet of individuals is strongly recommended. Upon this purpose, further
biochemical studies may be needed to characterize its antioxidant defense characteristics or counteracting
the effects of any pro-oxidant.
ACKNOWLEDGEMENTS
This project was funded by the Ministry of Science and Technology for the Thailand Institute of
Scientific and Technological Research (TISTR).
REFERENCES
1. Kubola J, Siriamornpun S. Phytochemicals and antioxidant activity of different fruit fractions (peel,
pulp, aril and seed) of Thai gac (Momordica cochinchinensis Spreng). Food chemistry 2011; 127:
1138-45.
2. Vuong L, Chitchumroonchokchai C, Chapman M, Ishida B, King J, Failla M. High bioaccessibility
of carotenes and lycopenes in gac oil and gac fruit aril. The FASEB Journal 2003; 17 (4): abstract
456.18.
3. Reverchon E. Marrone C. Modeling and simulation of the supercritical CO2 extraction of vegetable
oils. J. Supercrit. Fluid 2001; 19: 161-75.
4. Choi, Y. M., Noh, D. O., Cho, S. Y., Suh, H. J., Kim, K. M., & Kim, J. M. (2006). Antioxidant and
antimicrobial activities of propolis from several regions of Korea. LWT - Food Science and
Technology, 39(7), 756-761.
5. Amensour, M., Sendra, E., Perez-Alvarez, J. A., Skali-Senhaji, N., Abrini, J., & Fernandez-Lopez, J.
(2010). Antioxidant activity and chemical content of methanol and ethanol extracts from leaves of
rockrose (Cistus ladaniferus). Plant Foods for Human Nutrition, 65(2), 170-178.
6. Popov IN, Lewin G. Photochemiluminescent detection of anti-radical activity; testing of lipid-
soluble antioxidants. J Biochem Biophys Methods 1996; 31: 1-8.
7. Escobar J, Cardenas G, Lissi E. Evaluation of the total content of lipid-soluble antioxidants in blood
plasma samples employing a simple chemiluminescence quenching procedure. J Biochem Biophys
Methods 1997; 35: 57-60.

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COMPARATIVE STUDIES ON ANTIOXIDANT ACTIVITIES, TOTAL PHENOLICS AND

ANTHOCYANIN CONTENT OF FOUR NATIVE FRUITS
Tanwarat Kajsongkram1, Saranya Laovitthayanggoon1 ,Kusol Iamsub1, Vilailuk Chuennangchee1,

Penpan Srithong2, Chuleratana Bangchonglikitkul1
1
Thailand Institue of Scientific and Technological Research, 35 Moo 3 Technopolis Tambon Khlong 5, Amphoe Khlong Luang,
Pathum Thani , Thailand. 12120. E-mail: tanwarat@tistr.or.th
2
Faculty of Agro-Industry, Kasetsart University. 50 Ngamwongwan Road, Ladyao, Chatuchak, Bangkok, Thailand . 10900
KEYWORDS: anthocyanin, total phenolics, antioxidant activities
INTRODUCTION
Anthocyanins are the ubiquitous water-soluble pigments found in most flowers and fruits, and are
responsible for their impressive red and blue colours. Anthocyanins have been predominantly found in
nature as glycosides of polyhydroxy and polymethoxy derivatives of 2-phenyl-benzopyryliurn or
flavylium salts (Fig. 1). Individual members are differentiated by the number of hydroxyl and methoxyl
groups of the B-ring, by the number of sugar units attached to the aglycone and their position of
attachment, and by the nature and number of aliphatic or aromatic acids attached to the sugar residues
These plant pigments exist in the form of glycosides and the most common sugar moieties are glucose,
galactose, rhamnose and arabinose. Cyanidin derivatives are the most abundant anthocyanins in plants. As
a component of the human diet, anthocyanins are known to have health promoting activities because of
their high antioxidant properties in vitro and in vivo. In recent years several studies have shown that
anthocyanins display a wide range of biological activities including antioxidant, anti-inflammatory,
antimicrobial and anti-carcinogenic. The objective of this study was to investigate antioxidant activity
(DPPH assay), total phenolics and anthocyanin content of 4 anthocyanin-rich fruits: Antidesma
thwaitesianum (Buni fruit), Syzygium cumini (Java plum), Lepisanthes fruticosa (Luna nut) and Morus
alba (Mulberry). The results would be useful as basic information for finding sources of anthocyanin-rich
raw materials with high antioxidant activities and also useful as medical and health supplement.
Figure 1 Chemical structure of anthocyanin
Materials All fresh fruits were collected from different parts of Thailand. They were graded, washed and
crushed into small pieces.
Sample preparation The fresh samples (10 g.) were extracted in 50 ml aqueous acid acetone by sonicator
for 5 min. (3 times) and filtered through a Whatman No.1 filter paper. The supernatant was collected and
partitioned with chloroform in separatory funnel. The upper aqueous layer, containing the acetone/water
mixture was collected while the chloroform/acetone layer was carefully discarded. Residual acetone and
chloroform were removed from the anthocyanin extract by using a rotary evaporator at 40 ºC under

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vacuum condition. Extracts were taken to 50 ml in volumetric flask by 0.01%-HCl-acidified-water.

Extracts were then kept at −80 ºC until further analyzed.
Determination of total monomeric anthocyanins (TAC), total phenolics (TP) and antioxidant activities
(DPPH assay)
Materials Absolute methanol, acetone, chloroform, hydrochloric acid, potassium chloride, sodium
acetate, Folin reagent and sodium carbonate were reagent grade (Lab Scan, Ireland). DPPH (2,2-diphenyl-
l-picryhydrazyl) and Trolox (hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) were purchased
from Fluka (Germany).
Apparatus UV-Vis absorbance of TAC and TP was measured on a UV-Vis spectrophotometer UV-2450
(SHIMADZU, Japan). For DPPH assay, absorbance was recorded on a Microplate reader (GENios plus,
Austria) with Magellan software.
Method The total monomeric anthocyanin content (TAC) was measured by the pH-differential method
(Giusti and Wrolstad, 2001). Anthocyanin extracts were prepared in 0.025 M potassium chloride buffer,
pH 1.0 and 0.4 M sodium acetate buffer, pH 4.5 to measure the absorbance of the colored oxonium and
the colorless hemiketal form by comparison of the absorbance value at 520 nm using spectrophotometer.
The calculated values from the pigment as cyanidin-3-glucoside, MW = 449.2 and €=26,900, were
compared and reported as the monomeric anthocyanin content.
Total phenolics (TP) were measured by the Folin–Ciocalteu (FC) method (Singleton and Rossi, 1965).
Absorbance was measured at 765 nm. TP was expressed as milligrams of gallic acid equivalent per 100 g
fresh weight.
The DPPH free radical-scavenging activity of each sample was determined (Brand-Williams et al, 1995).
Briefly, a 50% v/v of aqueous methanolic DPPH solution (607 µM) was prepared. The initial absorbance
of the DPPH was measured at 517 nm and did not change throughout the period of assay. The aliquots
(20 µl) of Trolox and each sample (with appropriate dilution if necessary) were added to 180 µl of
aqueous methanolic DPPH solution. Discoloration was measured at 517 nm after incubation for 30 min at
30 °C in the dark. Measurements were performed at least in triplicate. The percentage of DPPH (%DPPH)
was calculated as:
% DPPH = (Ac−As )× 100 /Ac
where Ac is the absorbance of the control, and As is the absorbance of the sample. IC50 values were
calculated to denote the concentration of a sample required to decrease the absorbance at 517 nm by 50%.

In this study, free radical scavenging activity, TP and total TAC of 4 native fruits were investigated and
shown in Figures 2, 3 and 4. For DPPH assay, free radical scavenging activity of Antidesma
thwaitesianum (Buni fruit), Syzygium cumini (Java plum), Lepisanthes fruticosa (Luna nut), and Morus
alba (Mulberry) was revealed as 32461.25, 4344.47, 3557.47 and 1317.20 µmoles Trolox equivalents/100
gram fresh weight, respectively. Among these four fruits, Antidesma thwaitesianum (Buni fruit) showed
the highest activity.

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Figure 2 The DPPH free radical scavenging activity of (a) Antidesma thwaitesianum (Buni fruit), (b) Syzygium cumini (Java plum),
(c) Lepisanthes fruticosa (Luna nut), (d) Morus alba (Mulberry) (in µmoles Trolox equivalents / 100 gm fresh weight
The TP differed among different types of fruits and the TP values were expressed as milligrams of gallic
acid equivalents (GAE) per gram of fresh weight. There were significant differences among these tested
fruits. The TP of Antidesma thwaitesianum (Buni fruit), Syzygium cumini (Java plum), Lepisanthes
fruticosa (Luna nut), and Morus alba (Mulberry) were found to be 816.29, 741.79, 692.63 and 296.07 mg
GAE/g fresh weight, respectively. Antidesma thwaitesianum (Buni fruit) contained the highest total
phenolics.
Figure 3 The total phenolics of (a) Antidesma thwaitesianum (Buni fruit), (b) Syzygium cumini (Java plum), (c) Lepisanthes
fruticosa (Luna nut), (d) Morus alba (Mulberry) (in mg / 100 gm fresh weight)
Total anthocyanin contents in Antidesma thwaitesianum (Buni fruit), Syzygium cumini (Java
plum), Lepisanthes fruticosa (Luna nut), and Morus alba (Mulberry) were 259.83, 285.25, 122.01 and
220.30 mg/100g fresh weight, respectively. Syzygium cumini (Java plum) had the highest anthocyanin
contents.

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Figure 4 Total anthocyanin content of (a) Antidesma thwaitesianum (Buni fruit), (b) Syzygium cumini (Java plum), (c) Lepisanthes
fruticosa (Luna nut), (d) Morus alba (Mulberry) (in mg / 100 gm fresh weight)
CONCLUSION
In this study, four fruits native to Thailand were investigated for their antioxidant activity, total phenolics
and total anthocyanin content. The result showed that Antidesma thwaitesianum (Buni fruit) contained the
highest in anthocyanins and antioxidant activity, and Syzygium cumini (Java plum) contained the highest
in total phenolics. The results would be useful as basic information in the screening for sources of
anthocyanin-rich raw materials with high antioxidant activities for medical and health supplemental
purposes.
REFERENCES
1. Brand-Williams, W., M.E. Cuvelier and C. Berset. 1995. Use of a free radical method to
evaluate antioxidant activity. Lebensm.- Wiss. –Technol. 28: 25-30.
2. Giusti, M. M. and R. E. Wrolstad. 2001. Characterization and measurement of anthocyanins by
UV-visible spectroscopy, In Current Protocals in Food Analytical Chemistry, R. E. Wrolstad, T.
E. Acree, H. An, E. A. Decker, M. H. Penner, D. S. Reid, S. J. Schwartz, C. F. Shoemaker, and
P. Sporns, eds. (NY: John Wiley &Sons, Inc.), pp. F1.2.1-F1.2.13.
3. Singleton, V.L.and Rossi JL. 1965. Colorimetry of total phenolics with phosphomolybdic-
phosphotungstic acid reagents. Am J Enol Vitic. 16: 144-158.

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ANTILEUKEMIC ACTIVITY AND SECONDARY METABOLITES OF AN ENDOPHYTIC

FUNGUS PHOMOPSIS SP. FROM ARTEMISIA ANNUA
Punyisa Ngankaranatikarn1, Taksina Chuanasa1, Nongluksna Sriubolmas2 and Khanit Suwanborirux1, *

1
Center for Bioactive Natural Products from Marine Organisms and Endophytic Fungi (BNPME), Department of Pharmacognosy
and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand.
2
School of Pharmacy, Eastern Asia University, Pathumthani 12110, Thailand.
KEYWORDS: Phomopsis sp., endophytic fungus, antileukemic activity, Artemisia annua
INTRODUCTION
Endophytic fungi are symbiotic microorganisms living within tissues of the plant hosts without causing
noticeable diseases. These microorganisms are known as rich sources of bioactive secondary
metabolites.[1] Particularly, fungi in the genus Phomopsis were commonly found in medicinal plants and
reported to produce various secondary metabolites exhibiting interesting bioactivities including
cytotoxicity,[2-4] antimicrobial activity[5] and antimycobacterial activity.[6] The metabolites with diverse
structures from Phomopsis were revealed to possess cytotoxic activity; for example, oblongolides, the
hexaketide γ-lactone, from P. oblongna and dicerandrols, the xanthone dimers, from P. longicolla
exhibiting cytotoxicity against human epidermal carcinoma (KB) and human breast cancer (BC) cell
lines,[2,3] the cadinane sesquiterpenes from P. cassiae showing significant cytotoxic activity against
human cervical cancer (HeLa) cell line.[4]
The endophytic fungus Phomopsis sp. AANN8 was isolated from the twigs of the Thai medicinal plant
Artemisia annua L. (Family Asteraceae). The crude ethyl acetate extract from a fermentation broth of this
fungus exhibited selective and strong antileukemic activity against human acute monocytic leukemia
(THP-1) cell line. Therefore, study of secondary metabolites from this endophytic fungus and their
antileukemic activity should be very interesting for anticancer drug development.

General procedures 1H-NMR (300 MHz), 13C-NMR (75 MHz), DEPT, and 2D NMR spectra were taken
on a Bruker Fourier 300 NMR spectrometer. Chemical shift values were reported with respect to the
residual solvent signals (δ H 7.26/δ C 77.0 for CDCl 3 and δ H 2.05/δ C 28.9 for acetone-d 6 ). High-resolution
mass spectra were performed on an electrospray ionization JMS-700 (JEOL) instrument with a direct inlet
system operating at 70eV. A HPLC system equipped with a Shimadzu LC20AB pump and a
LiChroSpher® RP-18 column (250 x 10 mm, 10 µm, Merck) and a SPD-20A UV/Vis detector at 210 nm
was used. TLC analysis was carried out using silica gel 60 F 254 on aluminium sheet (Merck) and spots
were visualized under UV light at 254 nm and spraying with 5%anisaldehyde in H 2 SO 4 followed by
heating. Sephadex LH20, silica gel 60 (70-230 and 230-400 mesh, Merck) were used for gel filtration,
flash and quick column chromatography, respectively.
Fungal material The fungus Phomopsis sp. AANN8 was isolated from the twigs of A. annua grown in
Kanchanaburi Province, Thailand. The twigs were washed under running sterile distilled water and then
air-dried. The cleaned twigs were cut into pieces of 5 cm in length then surface sterilized by 70% ethanol
for 1 min, 5% sodium hypochlorite solution for 5 min and sterile distilled water for three times (1 min,
each). The sterilized samples were cut into small pieces using a sterile blade and placed on a sterile water
agar plate. All samples were subsequently incubated at 25ᵒC. The hyphal tip of endophytic fungus
growing out from the plant tissue was cut by a sterile pasture pipette and transferred to a sterile potato
dextrose agar (PDA) plate. After incubation at 25ᵒC for 7 days, culture purity was determined by colony
morphology.
Fermentation and extraction The endophytic fungus was grown on PDA plates at 25ᵒC approximately
for 7 days depending on growth rate. Six pieces (1 x 1 cm2) of the grown culture were prepared and then
inoculated into a 1000 ml erlenmeyer flask containing 200 ml of yeast extract sucrose (YES) broth. After
incubation at 25ᵒC for 21 days under stationary condition, the fungal culture (total 40 l) was separated
into mycelial and filtrate parts. The filtrate broth was extracted by partition with ethyl acetate three times.
The combined ethyl acetate phase was evaporated to dryness under reduced pressure to yield the crude
ethyl acetate extract (BB1, 6.0 g).
Isolation The crude extract BB1 was isolated by antileukemic assay-guided fractionation as shown in
Scheme 1. The crude ethyl acetate extract was fractionated into five fractions (BB2-BB6) by a silica gel
quick column with gradients of hexane/dichloromethane, hexane/ethyl acetate and methanol. Fraction

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BB4 was subjected to Sephadex LH-20 gel filtration chromatography elution with methanol to provide
seven fractions (BB7-BB13). Fraction BB10 was separated by a silica gel column using 40%hexane in
ethyl acetate as a mobile phase to provide eight fractions (BB19-BB26). Fraction BB21 was purified by
semi-preparative HPLC using 30%methanol in H 2 O as a mobile phase, flow rate 3 ml/min to yield
compound 1 (9.5 mg). Fractions BB24 and BB25 were pooled and subjected to semi-preparative HPLC
using 20% methanol in H 2 O as a mobile phase, flow rate 3 ml/min to give compounds 2 (49.9 mg) and 3
(8.2 mg). Fraction BB9 was subjected to silica gel column chromatography elution with 10%acetone in
dichloromethane to provide nine fractions (BB27-BB35). Fractions BB28-30 were pooled and purified by
a semi-preparative HPLC column using 30%methanol in H 2 O as a mobile phase, flow rate 2 ml/min to
yield compound 4 (6.9 mg).
Scheme 1. Extraction and isolation of the ethyl acetate extract of Phomopsis sp. AANN8.
Compound 1: white crystals, 1H NMR (acetone-d 6 ) δ (ppm): 8.15 (1H, s, 1-OH), 7.03 (2H, d, J = 8.5 Hz, 3-H and 5-H), 6.72 (2H, d,
J = 8.5 Hz, 2-H and 6-H), 3.69 (1H, s, 2´-OH), 3.66 (2H, t, J = 7.2 Hz, 2´-H), 2.69 (2H, t, J = 7.2 Hz, 1´-H); 13C NMR (acetone-d 6 )
δ (ppm): 156.5 (s, 1-C), 131.6 (s, 4-C), 130.6 (s, 3-C and 5-C), 115.8 (s, 2-C and 6-C), 64.2 (s, 2´-C), 39.4 (s, 1´-C); HR-EIMS m/z
138.0680 (calcd for C 8 H 10 O 2 , 138.0681)
In vitro antileukemic activity assay The crude extract and fractions were evaluated for their antileukemic
activity by sulforhodamine B (SRB) colorimetric assay[7] using the human acute monocytic leukemia
(THP-1) cell line. Ellipticine was used as the positive control. The crude extract and fractions with the
final concentration of 20 µg/ml exhibiting antileukemic activity against THP-1 cell line (over 55%
inhibition) were further tested for the effective concentrations that inhibited 50% of THP-1 cell growth
(EC 50 ) and compared with ellipticine, which its EC 50 was at 19.46 µg/ml.

Compound 1 was obtained as white crystals from the culture broth of the endophyte, Phomopsis sp.
AANN8. The HR-EIMS of 1 displaying a molecular ion at m/z 138.0680 indicated its molecular formula
of C 8 H 10 O 2 with 4 degrees of unsaturation. The 1H NMR data of 1 showed two doublets of four aromatic
protons at δ H 7.03 and 6.72 with an ortho coupling constant of 8.5 Hz indicating a 1,4-disubstituted
aromatic ring and a pair of two-proton triplets of coupled methylenes at δ H 3.66 and 2.69. The 13C NMR
and DEPT spectra of 1 confirmed the 1,4-disubstituted phenyl moiety by a pair of two-carbon signals of
aromatic methane carbons at δ C 130.6 and 115.8, one oxygenated quaternary aromatic carbon at δ C 156.5
and one quaternary carbon at δ C 131.6 as well as the ethylene moiety by two methylene carbons at δ C 64.2
and 39.4. The 1H and 13C NMR data of 1 were shown in Table 1. Compound 1 was identified as 4-
hydroxyphenethyl alcohol or tyrosol by comparison of the 1H and 13C NMR data with the literature.[8]
This is the first report of tyrosol obtained from the fugus Phomopsis. Tyrosol was previously isolated
from plants, such as Acorus gramineus[8] and Olea europea[9] as well as microbes, such as Neofusicoccum
parvum[10] and Glomerella cingulate[11] The compound was reported to exhibit various bioactivities such
as antioxidant[12], anti-inflammatory[13] and neuroprotective[14].

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Table 1. 1H and 13C NMR data for 1 in acetone-d 6 (δ in ppm).
Position δ H , mult. (J in Hz) δC

1 - 156.5
2, 6 6.72, d (2H, 8.5) 115.8
3, 5 7.03, d (2H, 8.5) 130.6
4 - 131.6
1´ 2.69, t (2H, 7.2) 39.4
2´ 3.66, t (2H, 7.2) 64.2
1-OH 8.15, br -
2´-OH 3.69, br -
6 5 2'
OH
HO
1 4 1'
2 3
Tyrosol (1)
Antileukemic activity of the ethyl acetate extract (BB1) and fractions (BB2-BB6, BB8-BB10) against
THP-1 cell line was presented as percentage inhibition of THP-1 cell growth and EC 50 values in Table 2.
The extract showed more potent antileukemic activity than ellipticine. After fraction BB1 was
fractionated by a silica gel column, the obtained fraction BB4 presented higher antileukemic activity than
BB1. Unexpectedly, the antileukemic activity of fractions BB7-BB13 separated from fraction BB4
dramatically decreased. This result suggested that there might be complexing conditions in fraction BB4
influencing its antileukemic activity, for example some co-factors required for the cytotoxicity were
disappeared during the separation or several compounds would be synergistically involved in such a
bioactivity.
Table 2. Inhibition percentages of THP-1 cell growth and EC 50 values
%inhibition EC 50
Fraction
(at 20 µg/ml) (µg/ml)
Ellipticine nt 19.46
BB1 59.80 17.11
BB4 85.33 0.70
BB8 1.97 nt
BB9 0.53 nt
BB10 52.98 nt
nt = not tested
CONCLUSION
The crude ethyl acetate extract from the fermentation broth of the endophytic fungus, Phomopsis sp.
AANN8, exhibited antileukemic activity against THP-1 cell line. After separation by antileukemic assay-
guided fractionation, four pure compounds (1-4) were obtained and compound 1 was identified as tyrosol.
Further steps are structure elucidation of other three compounds and moreover, isolations are required to
obtain more compounds from fraction BB4. Once isolation and structure elucidation are achieved,
antileukemic activity of pure compounds will be evaluated.

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ACKNOWLEDGEMENTS
This project is supported by BNPME and Pharmaceutical Research Instrument Center, Faculty of
Pharmaceutical Sciences, Chulalongkorn University. The mass spectrum was measured by Professor
Naoki Saito at Meiji Pharmaceutical University. The fungal material and antileukemic activity assay was
prepared by Assistant Professor Dr. Suthep Wiyakrutta at Department of Microbiology, Faculty of
Science, Mahidol University.
REFERENCES
1. Strobel G., Daisy B., Castillo U., and Harper J. Natural products from endophytic
microorganisms. Journal of Natural Products, 2004 (67): 257-268.
2. Bunyapaiboonsri T., Yoiprommarat S., Srikitikulchai P., Srichomthong K., and Lumyong S.
Oblongolides from the endophytic fungus Phomopsis sp. BCC9789. Journal of Natural Products,
2010 (73): 55-59.
3. Clardy J., and Wagenaar M.M. Dicerandrols, New antibiotic and cytotoxic dimers produced by
the fungus Phomopsis longicolla isolated from an endangered mint. Journal of Natural Products,
2001 (64): 1006-1009.
4. Silva G.H., Teles H.L., Zanardi L.M., Young M.C.M., Eberlin M.N., Hadad R., Pfenning L.H.,
Costa-Neto C.M., Castro-Gamboa I., Bolzani V.S., and Araujo A.R. Cadinane sesquiterpenoids
of Phomopsis cassia, an endophytic fungus associated with Cassia spectabilis
(Leguminosae). Phytochemistry, 2006 (67): 1964-1969.
5. Huang Z., Cai X., Shao C., She Z., Xia X., Chen Y., Yang J., Zhou S., and Lin Y. Chemistry and
weak antimicrobial activities of phomopsins produced by mangrove endophytic fungus
Phomopsis sp. ZSU-H76. Phytochemistry, 2008 (69): 1604-1608.
6. Rukachaisirikul V., Sommart U., Phongpaichit S., Sakayaroj J., and Kirtikara K. Metabolites
from the endophytic fungus Phomopsis sp. PSU-D15. Phytochemistry, 2008 (69): 783-787.
7. Skehan P., Storeng R., Scudiero D., Monks A., McMahon J., Vistica D., Warren J.T., Bokesch
H., Kennt S., and Boyd M.R. New colorimetric cytotoxicity assay for anticancer-drug
screening. Journal of the National Cancer Institute, 1990 (82): 1107-1112.
8. Park C.H., Kim K.H., Lee I.K., Lee S.Y., Choi S.U., Lee J.H. and Lee K.R. Phenolic constituents
of Acorus gramineus. Archives of Pharmacal Research, 2011 (34): 1289-1296.
9. Capasso R., Cristinzio G., Evidente A. and Scognamiglio F. Isolation, spectroscopy and selective
phytotoxic effects of polyphenols from vegetable waste waters. Phytochemistry, 1992 (31):
4125-4128.
10. Evidente A., Punzo B., Andolfi A., Cimmino A., Melck D. and Luque J. Lipophilic phytotoxins
produced by Neofusicoccum parvum, a grapevine canker agent. Phytopathologia Mediterranea,
2010 (49): 74-79.
11. Guimaraes D.O., Borges W.S., Kawano C.Y., Riberio P.H., Goldman G.H., Nomizo A.,
Thiemann O.H., Oliva G., Lopes N.P. and Pupo M.T. Biological activities from extracts of
endophytic fungi isolated from Viguiera arenaria and Tithonia diversifolia. FEMS Immunology
and Medical Microbiology, 2008 (52): 134-144.
12. Puerta R., Dominguez E.M., Gutierrez V.R., Flavill J.A. and Hoult R.S. Effects of virgin olive
oil phenolics on scavenging of reactive nitrogen species and upon nitrergic
neurotransmission. Life Sciences, 2001 (69): 1213-1222.
13. Puerta R., Gutierrez V.R. and Hoult R.S. Inhibition of leukocyte 5- lipoxygenase by phenolics
from virgin olive oil. Biochemical Pharmacology, 1999 (57): 445-449.
14. Bu Y., Rho S., Kim J., Kim M.Y., Lee D.H., Kim S.Y., Choi H. and Kim H. Neuroprotective
effect of tyrosol on transient focal cerebral ischemia in rats. Neuroscience Letters, 2007 (414):
218-221.

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DETECTION OF ESTERASE ACTIVITY CONVERTING CYTOTOXIC RENIERAMYCIN M

TO JORUNNAMYCIN A IN THE CRUDE ENZYME
OF THE NUDIBRANCH JORUNNA FUNEBRIS
Dalad Waropastrakul1, Khanit Suwanborirux1, Wanchai De-Eknamkul1, and Taksina Chuanasa1*

1
Chulalongkorn University, Bangkok Thailand 10330
KEYWORDS: Jorunnamycin A, Renieramycin M, Esterase Activity, Jorunna funebris
INTRODUCTION
JorunnamycinA (JA) is a renieramycin member of the marine tetrahydroisoquinolinequinone alkaloids.
JA has been recently used as an important starting material to prepare various 22-O-acyl derivatives of
renieramycin M (RM) for improving its potent cytotoxicity against cancer cell lines as well as studying
structure-cytotoxicity relationships.[1] RM is a major cytotoxic component, previously isolated from a
Thai blue sponge Xestospongia sp. pretreated with potassium cyanide.[2] To chemically synthesize JA,
the angeloyl group at C-22 of RM must be removed by cleavage of the ester bond to yield a free alcohol
moiety. However, the routine acid and alkaline hydrolysis reactions were not successful to cleave this
ester bond. The chemical transformation was performed by the 3-step reaction involving hydrogenation,
hydride reduction and oxidation as shown in Figure 1.[1] Interestingly, JA was recently isolated in a good
yield from a marine nudibranch Jorunna funebris pretreated with potassium cyanide.[3] This shell-shed
gastropod mollusk is a sponge-eating animal, which specifically feeds on the sponge Xestospongia and
accumulates RM and JA in both visceral part and mantle. The absence of JA in the sponge Xestospongia,
therefore, led to a hypothesis that the reaction catalyzed by specific enzyme(s) in the nudibranch tissue
might be involved in the conversion of RM to JA. Considering the structures of these two related
alkaloids, the enzyme involved in the hydrolysis of the ester bond at C-22 is possibly an esterase type. In
this communication, our preliminary study aiming to clarify esterase activity in the crude protein extract
prepared from J. funebris is designed to support the hypothesis. The discovery of this specific enzyme
activity will be an important step for the future development to utilize the specific esterase enzyme in
replacing harmful reagents for such specific chemical reactions.
OCH3
OCH3
O CH3
OCH3 O CH3
O
H O CH3 O
H3C O 1) H2, 20%pd(OH)2/C H
N CH3 O H3C O
EtOAc H N CH3
H3C O
N 2) AlH3, THF N CH3
H3CO N
H3CO
O CN 3) O2 N RCOX, base
H3CO
O O CN
O CN O
O H (?? Esterase OH
O R
from J. funebris)
renieramycin M jorunnamycin A 22-O-acylrenieramycinM

derivatives
Figure 1 Steps in preparation of 22-O-acyl renieramycin M derivatives.

Materials The nudibranch J. funebris samples were collected in 2012 at Sichang islands, Chonburi,
Thailand. The samples were stored at -80°C until they were used for analysis of RM/JA contents and
preparation of crude enzyme extracts.
Determination of RM/JA contents in J. funebris tissues J. funebris samples were dissected to separate
the visceral and mantle parts. A pool of each part was ground into homogeneous paste under liquid
nitrogen. Each part (500 mg) was accurately weighed and pretreated with 500 µL of 10 mM KCN in 50
mM phosphate buffer (pH 7.0) for 5 h. The sample suspension was macerated with 2 mL of methanol for
24 h and subsequently adjusted to 5 mL by brine solution. The resulting mixture was further partitioned
with 5 mL of EtOAc. The EtOAc layer (1 mL) was evaporated to dryness and re-dissolved with 1 mL
methanol to yield the EtOAc extract. The EtOAc extract was subjected to HPLC (Shimadzu SPD-M20A
SIL-20A, Japan) using a LiChrospher 100 RP-18 HPLC column (4 x 125 mm, 5 μm), 20 µL of sample

PN - 12
volume, 70% MeOH in water as a mobile phase at a flow rate of 1.0 mL/min, detection with a UV
detector (Shimadzu,SDD-M10A PDA) at λ 270 nm. Retention times and UV spectra of the HPLC
chromatographic peaks responsible for RM and JA presenting in the EtOAc extract were compared with
those of the RM and JA reference standards.
Crude protein preparation Each visceral and mantle part of J. funebris samples was pooled and
accurately weighed for 1 g. Each part was homogenized with 4.0 mL of 50 mM Tris-HCl buffer (pH 8.0)
then centrifuged at 5,000g 4°C for 15 min to remove cell debris. The supernatant was added by
ammonium sulfate to bring up salt concentration to 70% and stirred at 4°C for 20 min. The mixture was
centrifuged at 15,000g, 4°C for 20 min. The pellet obtained (the 0-70% fraction) was re-dissolved in 2
mL of 50mM Tris-HCl buffer (pH 8.0) and stored at -80°C. Desalting process of the crude protein by a
PD-10 column (8.3 mL of Sephadex G-25 Medium, GE healthcare, Sweden) eluted by 50 mM Tris-HCl
buffer (pH 8.0) was performed and the crude protein was collected at 3.5-5.5 mL of the eluate.
Partial purification of the crude protein The crude protein from the visceral part of J. funebris was
partially purified by sequential ammonium sulfate precipitation. The visceral part was treated with the
process as mentioned above. The initial pellet of proteins from 20% ammonium sulfate concentration
was discarded. The remaining supernatant was sequentially precipitated by higher concentrations (40–
100%) of ammonium sulfate with 5% increment interval. The protein pellet from each precipitation was
desalted by a PD-10 column prior to the esterase activity assay, as previously described.
Total protein assay Total soluble proteins were quantified by the Bradford assay[4] using Bio-Rad protein
assay dye reagent (Bio-Rad product 500-0006) and the standard protocol for 96-well microplates. The
absorbance at 595 nm of the standard protein BSA and its corresponding concentrations (0.125–1.0
mg/mL) were plotted as the protein concentration standard curve.
Esterase activity assay of the prepared proteins The assay for esterase activity of the crude proteins and
the partially purified proteins was modified from the anti-lipase activity assay.[5] In brief, the reaction
was carried out in a 200-µL volume comprising 20 µL of RM (50 µM RM solution in 10% DMSO in
MeOH), 0.4 mg of the prepared protein, and 50 mM Tris-HCl buffer (pH 8.0) and incubated at 25°C for
90 min. Then, the reaction mixture was partitioned with200 µL of EtOAc and 100 µL of the EtOAc layer
was transferred to evaporate to dryness. The residue was dissolved with 100 µL of MeOH to give the
extract for determination of RM/JA contentsby HPLC as previously described. The enzyme activity
(U/mg) was calculated from enzyme unit (U) divided by the protein content in each fraction. Enzyme
unit (U) was defined as the amount of enzyme required to produce 1 μmol of JA per min.
RESULTS
Determination of RM/JA contents in J. funebris tissues To detect the RM and JA contents in the mantle
and the visceral parts of J. funebris, the EtOAc extracts from both tissue parts were prepared and
subjected to quantitative analysis by HPLC (Figure 2). The retention times in the HPLC chromatograms
of the references RM and JA were at 7.77 and 2.90 min, respectively and both compounds showed the
similar UV spectra with λ max at 270 nm (Figure 2A and B). The results showed that both RM and JA
were presented in the mantle and visceral parts by comparison their HPLC retention times and UV spectra
to those of RM and JA reference standards (Figure 2C and D).
Crude protein preparation and partial purification of the crude protein The crude proteins from the
mantle and the visceral tissues were prepared by precipitation with 70% ammonium sulfate to obtain
1.4%mg and 3.2%mg w/w proteins of the tissue wet weight, respectively. Partial purification of the crude
protein from the visceral part was further prepared through sequential precipitations with different
ammonium sulfate concentrations (20–100%) by 5% increasing interval (Figure 3). The proteins were
initially precipitated at the 40–45% fraction and completely precipitated at the 80–85% fraction. The 50–
55%, 55–60%, and 60–65% fractions significantly yielded higher protein contents than the others, with
0.62%mg - 0.72%mg protein of the visceral part wet weight.
Esterase activity of the protein/enzyme preparations To detect the esterase activity of the prepared
proteins, RM was used as the enzyme substrate and JA was the expected final product. HPLC was
performed to detect the presence of RM and JA from the reaction mixtures. Preliminary results showed
that RM was converted to JA by the proteins only from the visceral part (Figure 4B and C). Sequential
precipitation of the visceral proteins by ammonium sulfate showed that the protein of the 65-70% fraction
presented the highest esterase activity (Figures 3 and 4D). In addition, the neighboring fractions (55-60%,
60-65%, and 70-75%) also exhibited comparatively high activity. Reduction in the activity of the other
fractions was apparently observed (Figure 3).

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Figure 2 Detection of jorunnamycin A (JA) and renieramycin M (RM) in J. funebris tissues. The HPLC chromatograms of A) the
reference JA, B) the reference RM, C) the EtOAc extract of the mantle part of J. funebris, and D) the EtOAc extract of the visceral
part of J. funebris. The inserts show the UV spectra of JA and RM
Figure 3 Esterase activity for RM hydrolysis and total protein content of the partially purified crude proteins by sequential
ammonium sulfate precipitation.
DISCUSSION
From the results, an accumulation of RM/JA in J. funebris body suggested that these organisms certainly
contained the secondary metabolites of interest. The conversion of RM to JA was found in the crude and
partially purified enzymes from the visceral part of J. funebris. Thus, it could be inferred that the esterase
enzyme involved in converting RM to JA was extracted into both enzyme preparations. Since there was a
limitation of starting materials, we tried to achieve the suitable percentage of ammonium sulfate in our
preliminary experiment. Total proteins were precipitated at 0-70% ammonium sulfate concentration
where the activity of interest was detected. Because different proteins are precipitated in different
concentrations of ammonium sulfate[6], we expected that the protein fractions at different percentages of
ammonium sulfate would show various degrees of activity. Interestingly, the 65-70% fraction showed the
highest esterase activity whereas the 60-65% fraction possessed the largest amount of total protein
content. It suggested that the 65-70% fraction certainly contained the highest concentration of the
esterase for RM hydrolysis. The 55-75% fractions revealed a range of well-detected esterase activity,
therefore, the protein fraction precipitated by 55-75% ammonium sulfate was selected for further study.
This partially-purified enzyme from the visceral part will be used for further optimization study to
investigate effects of different parameters and substrate specificity on its esterase activity. Further studies
are required to purify and characterize the enzyme.

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Figure 4 Detection of jorunnamycin A (JA) produced from renieramycin M (RM) by esterase activity of the crude enzymes from
J. funebris tissues. The HPLC chromatograms of A) a mixture of the references JA and RM, B) and C) the EtOAC extracts of the
enzymatic reactions of the mantle and the visceral crude enzymes, respectively, and D) the EtOAC extract of the enzymatic reaction
of the 65-70% ammonium sulfate fraction of the visceral crude enzyme. The inserts show the UV spectra of JA and RM.
CONCLUSION
In this study, the esterase activity producing JA from RM of the crude enzyme prepared from the visceral
part of the nudibranch J. Funebris was revealed. The enzyme activity was demonstrated to catalyze the
hydrolysis reaction cleaving the angeloyl moiety of RM to produce JA. Finally, the 55-75% ammonium
sulfate was suitable to partially purify the enzyme with the desirable esterase activity after initial
precipitation of the visceral proteins with 55% ammonium sulfate.
ACKNOWLEDGMENT
Pharmaceutical Research Instrument Center, Faculty of Pharmaceutical Sciences, Chulalongkorn
University has been acknowledged for facilities support. Center for Bioactive Natural Products from
Marine Organisms and Endophytic Fungi (BNPME) has been supported by the Commission on Higher
Education, Thailand
REFERENCES
1. Charupant K, Daikuhara N, Saito E, Amnuoypol S, Suwanborirux K, Owa T, Saito N:
Chemistry of Renieramycins. Part 8: Synthesis and Cytotoxicity Evaluation of
Renieramycin M–jorunnamycin A Analogues. Bioorganic & medicinal chemistry 2009,
17:4548-4558.
2. Suwanborirux K, Amnuoypol S, Plubrukarn A, Pummangura S, Kubo A, Tanaka C, Saito N:
Chemistry of Renieramycins. Part 3. 1 Isolation and Structure of Stabilized Renieramycin
Type Derivatives Possessing Antitumor Activity from Thai Sponge Xestospongia Species,
Pretreated with Potassium Cyanide. Journal of natural products 2003, 66:1441-1446.
3. Charupant K, Suwanborirux K, Amnuoypol S, Saito E, Kubo A, Saito N: Jorunnamycins A—
C, New Stabilized Renieramycin-Type Bistetrahydroisoquinolines Isolated from the Thai
Nudibranch Jorunna funebris. Chemical and pharmaceutical bulletin 2007, 55:81-86.
4. Bradford MM: A Rapid and Sensitive Method for the Quantitation of Microgram Quantities
of Protein Utilizing the Principle of Protein-dye Binding. Analytical biochemistry 1976,
72:248-254.
5. McDougall GJ, Kulkarni NN, Stewart D: Berry Polyphenols Inhibit Pancreatic Lipase
Activityin vitro. Food Chemistry 2009, 115:193-199.
6. Green AA, Hughes WL: Protein Fractionation on the Basis of Solubility in Aqueous
Solutions of Salts and Organic Solvents. In Methods in enzymology. Edited by: Academic
Press; 1955:67-90.

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COMPARISON OF HPLC AND TLC DENSITOMETRIC METHODS FOR

THE QUANTIFICATION OF RHEIN IN CASSIA FISTULA POD PULP EXTRACT
Savita Chewchinda, Pongtip Sithisarn and Wandee Gritsanapan*
Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand
KEYWORDS: Cassia fistula, HPLC, rhein, TLC densitometric method
INTRODUCTION
Cassia fistlula Linn. (Fabaceae) is commonly known as “Khun” and “Ratchapruek” in Thai. C. fistula
pod pulp is used in traditional medicine as a purgative/laxative drug and also used against various
disorders such as skin diseases, diabetes and other ailments 1, 2). The major anthraquinone derivative in the
pod pulp is rhein3,4). Although high performance liquid chromatography (HPLC) is the most widely used
method for quality assessment of herbal preparations, it requires high operational cost and skilled
operator. Therefore, the purpose of this study is to compare the quantitative results obtained by validated
HPLC and TLC densitometric methods in the analysis of rhein content in the extracts of C. fistula pod
pulp. From our data, there is no significant difference (P > 0.05) between the mean content of rhein
measured by HPLC and TLC densitometric methods. Thus, TLC densitometric method could be
alternatively used as a routine analysis of rhein in C. fistula pod pulp. Moreover, TLC densitometric
method is simple, less expensive and can provide analytical data faster than HPLC.

Plant Materials Six samples of ripe pods of C. fistula were collected from Nakhon Si Thammarat
province, Thailand, in March 2010. They were identified by comparing with herbarium specimens at the
Forest Herbarium, Department of National Park, Wildlife and Plant Conservation, Ministry of Natural
Resources and Environment, Bangkok. The voucher specimens (WCF0401-6) were deposited at the
Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University. The ripe pods were cleaned
with tap water and the pod pulp (without seed) was separated and kept in a tight container at 4oC until
used.
Preparation of C. fistula Pod Pulp Extract Each sample of fresh pulp of C. fistula ripe pod (20 g) was
boiled with distilled water (200 mL) for one hour at 95-98oC and the mixture was filtered. The extraction
process was repeated until anthraquinones in the pulp were exhaustively extracted (tested by Borntrager’s
reaction). The filtrates were combined and evaporated to dryness on a boiling water bath to yield a
decoction crude extract. The yield of crude extract was recorded and the extract ratio (weight of pod pulp
: 1 g extract) was calculated.
Preparation of Sample Solution Decoction extracts of samples of C. fistula pod pulp (each 0.1 g) were
accurately weighed, separately dissolved in 60% (v/v) methanol, then adjusted to 10 mL in volumetric
flasks for both methods. Each solution was filtered through a 0.45 μm nylon membrane filter and
analyzed in triplicate.
Preparation of Standard Solution Rhein reference standard was accurately weighed and dissolved in
methanol in a volumetric flask as a stock solution (1 mg/mL). Standard working solutions of rhein were
prepared by diluting the stock solution with 60% (v/v) methanol in the range of 1.25-20 µg/mL and 25-
250 ng/spot for HPLC and TLC densitometric analysis, respectively.
HPLC Method A validated HPLC analysis5) was performed on a Shimadzu Technologies modular model
Class VP system consisting of a SCL-10A system, a UV-vis SPD-10A detector, LC-10 AD and auto
injector SIL-10A (Shimadzu, Japan). The analysis was carried out using a BDS Hypersil C18 column
(250×4.6 mm, i.d. 5 μm) (Thermo Fisher Scientific Inc., USA) with a BDS Hypersil C18 guard column
(10×4 mm, i.d. 5 μm) (Thermo Hypersil-Keystone, USA). The isocratic mobile phase was 0.5% aqueous
acetic acid solution and methanol (40:60). The total running time was 30 min and the flow rate was 1.0
mL/min. The UV detector monitored at 435 nm while the injection volume was 20 μL.
TLC Densitometric Method Thin layer chromatography was performed on an aluminum sheet of silica
gel60 F 254 (20 cm x 10 cm). Sample and standard solutions were applied on the plate as 7 mm band with
a Linomat V automatic sample spotter (Camag, Switzerland) under nitrogen flow, positioned at 10 mm
from the bottom of the plate. The mobile phase consisted of ethyl acetate-methanol-water (100:17:10,
v/v/v). The plate was developed to a distance of 8 cm in a Camag twin trough chamber. The densitometric
scanning was performed by using a TLC Scanner 3 (Camag, Switzerland) with winCATS software. The

PN - 13
wavelength of the detector was set at 435 nm. The slit dimension was 6.00 x 0.45 mm and the scanning
speed was 20 mm s-1. The sample was applied at 10 µL/spot. The used method was validated as reported
in our previous work6)
Statistical Analysis The mean values of rhein content in C. fistula pod pulp extracts determined by HPLC
and TLC densitometric methods were tested by paired t-test at 95% confident level.

The contents of rhein in C. fistula pod pulp extracts were in the range of 0.0851-0.1147% w/w. HPLC
chromatograms of the extract showed similar pattern with a major peak of rhein at a retention time of 15
minute. Regarding the quantitative analysis of rhein content by the validated TLC densitometric method,
rhein was found in the TLC chromatogram at Rf = 0.49 separated from other components (Figure 1). The
contents of rhein in the six samples of C. fistula pod pulp extracts analyzed by both methods are shown in
Table 1. The paired t-test showed no statistically significant difference (P > 0.05) between the mean
contents of rhein performed by HPLC method and TLC densitometric method. In conclusion, the
proposed TLC densitometric method could be used as an alternative method for the quantitative analysis
of rhein content in C. fistula pod pulp extract. This method showed several advantages such as simplicity,
fast data acquisition, and high efficacy.
Figure 1 3D TLC densitogram of rhein standard (track no. 4-8) and C. fistula pod pulp extract (sample#1 lot 1= track no. 1-3, lot2 =
track no.9-11, lot 3= track no. 12-14)
Table 1 The contents of rhein in 6 samples of C. fistula pod pulp extracts analyzed by HPLC and TLC densitometric methods
Sample no. Content of rhein (% w/w)*

HPLC TLC densitometry
1 0.1147± 0.004 0.1041 ± 0.006
2 0.1069 ± 0.002 0.0945 ± 0.003
3 0.0965 ± 0.001 0.0973 ± 0.004
4 0.0998 ± 0.002 0.0986 ± 0.006
5 0.0929 ± 0.003 0.1041 ± 0.004
6 0.0851 ± 0.001 0.0846 ± 0.001
average 0.0993 ± 0.002 0.0972 ± 0.004
*Expressed as mean ± SD (n = 3)

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ACKNOWLEDGEMENTS
This project is a part of a Ph.D. thesis of Mahidol University, financially supported by the Office of the
Higher Education Commission and Mahidol University under the National Research Universities
Initiative.
REFERENCES
1. Asolkar LV, Kakkar KK, Chakre OJ. 1992. Second supplement to glossary of Indian medicinal
plant with active principles. CSIR, New Delhi, 177.
2. Alam MM, Siddiqui MB, Hussian W. 1990.Treatment of diabetes through herbal drugs in rural
India. Fitoterapia 61: 240-42.
3. Sakulpanich A, Chewchinda S, Sithisarn P, Gritsanapan W. 2012. Standardization and toxicity
evaluation of Cassia fistula pod pulp extract for a source of herbal laxative drug. Phcog. J 4: 6-12.
4. Bahorun T, Neergheen VS, Aruoma OI. 2005. Phytochemical constituents of Cassia fistula. Afr
J Biotechnol 4: 1530-40.
5. Chewchinda S, Sakulpanich A, Sithisarn P, Gritsanapan W. HPLC analysis of laxative rhein
content in Cassia fistula pods of different provenances in Thailand. Thai Journal of Agricultural
Science (in press).
6. Chewchinda S, Sithisarn P, Gritsanapan W. 2012. TLC-densitometric method for chemical
stability evaluation of Cassia fistula pod pulp extract: an alternative laxative drug. J Med Plants
Res 6: 4940-44.

PN – 14
USE OF THREE THAI INDIGENOUS VEGETABLES AS POTENTIAL DIETARY FIBER

SOURCES FOR HEALTH FOOD PRODUCT
Jeerayu Thongdon-A1, Prapaipat Klungsupya1, Thanchanok Muangman1,

Arkachai Tantrawong1and Srisak Trangvacharakul2
1
Department of Pharmaceuticals and Natural Products, Thailand Institute of Scientific and Technological Research (TISTR),
35 Moo 3, Techno Polis, Klong Luang, Pathumthani 12120, Thailand
2
Department of Food Technology, Thailand Institute of Scientific and Technological Research (TISTR), 35 Moo 3, Techno Polis,
Klong Luang, Pathumthani 12120, Thailand
KEYWORDS: Polygonum odoratum, Limnophila aromatica, Gymnema inodorum, dietary fiber,

antioxidant capacity
INTRODUCTION
Nowadays, changes in life style, especially consumption behaviors, lead to an increase in patients with
non-infected chronic diseases (such as obesity, diabetes, cardiovascular diseases and cancer).
Accordingly, there has been a growing interest in health food products which help support well-being.
Recently, several Thai indigenous vegetables have gained increasing attention due to their various
bioactive substances and dietary fiber.
Among them, Polygonum odoratum Lour. (Polygonaceae), Limnophila aromatica (Lamk) Merr.
(Scrophulariaceae), and Gymnema inodorum Decne. (Asclepiadaceae) are rather well known and have
been used in traditional cuisine and medicine. Previous studies revealed the antioxidant activities of leaf
extracts derived from P. odoratum and L. aromatica, analyzed by DPPH assay. [1, 2]
G. inodorum has been well known to have therapeutic effects in curing certain diseases such as diabetes,
rheumatic arthritis and gout. It exerts hypoglycemic effect caused by triterpenoid saponins or gymnemic
acids which inhibit glucose absorption from the intestinal tract and suppress the increase in blood glucose
level in oral glucose tolerance tests in rat.[3] Moreover, its antioxidant property was also reported. [1, 3]
Hence, this study was carried out to determine dietary fiber content and antioxidant capacity of these local
vegetables to use them for health food product development emphasized on low calorie and high in
dietary fiber.
Polygonum odoratum Lour. Limnophila aromatica (Lamk) Merr.

“Phak paew” “Phak ka yaeng”
Gymnema inodorum (Lour.) Decne.

“Phak chiang da”
Figure 1 Thai indigenous vegetables used in the study

PN – 14

Plant materials and extract preparation Fresh aerial parts of P. odoratum and L. aromatica and leaves
of G. inodorum were purchased from Nakorn Pathom province in June 2012. For P. odoratum and L.
aromatica, the air-dried and ground plant samples (100 g) were extracted with 95% aqueous ethanol (300
ml x 12) at room temperature. After filtering and removing the solvent via rotary evaporator at 45°C, the
greenish brown semisolids were obtained. G. inodorum fresh leaves were chopped and blended with
distilled water (1 kg : 3 L of water). Then it was filtered and lyophilized to powder. All crude extracts
were kept at 4°C until analysis for antioxidant capacity.
Dietary fiber assay This assay quantifies the total dietary fiber (TDF) content by means of enzymatic-
gravimetric method based on the procedure of AOAC (1997) [4]. For each vegetable, quadruplicate dried-
milled samples (0.3-0.5 mm mesh) were gelatinized with heat stable α-amylase and then digested with
protease and amyloglucosidase to remove the protein and starch present in the samples. Ethanol was
added to precipitate soluble dietary fiber (SDF). After filtration, the residue was washed with ethanol and
acetone, followed by drying and weighing. Half of the samples were analyzed for protein by Kjeldahl
nitrogen analysis and the other halves were ashed (525 °C, 5 h). TDF value was the weight of the residue
less the weight of the protein and ash. The insoluble dietary fiber (IDF) contents were evaluated by the
similar method for the TDF, but the ethanolic precipitation of SDF was not undertaken. After that, SDF
value was calculated by TDF value minus IDF value.
Antioxidant capacity determination by photochemiluminescence (PCL) The antioxidant capacities of
the obtained extracts were assessed by means of PCL using PHOTOCHEM (Analytik Jena, Germany)
that provides high sensitivity and precision of the data. PCL assay was operated according to Popov and
Lewin [5] and can be carried out by two different protocols, ACW and ACL, which permit the
measurement of the antioxidant capacity of the water- and lipid-soluble components, respectively. In this
study, calibration and measurements were conducted as described in the ACL kit protocol in which
Trolox was used as the reference standard. The crude extracts were dissolved in methanol (10 mg/ml)
prior to being subjected to the assay. The results were expressed as Trolox equivalent unit (nmol) per 1 µg
of tested sample. In case of G. inodorum extract, it was subjected to ACW kit protocol in which ascorbic
acid (vitamin C) was used as the reference standard. The measurement of each sample was triplicated.

The yields of P. odoratum and L. aromatica extracts were 16.4 and 21.5 % by dry weight, respectively.
The extraction of G. inodorum leaf yielded 4.30 % by fresh weight. As shown in Table 1 and Figure 2,
TDF contents of dried powder derived from three plants varied from 34.41% - 47.51% (w/dw) which the
IDF proportions were more than 95% of the TDF values. P. odoratum showed the highest TDF content
followed by L. aromatica and G. inodorum (47.51%, 44.27% and 34.41%, respectively).
Assessments of antioxidant potency were performed via PCL assay and expressed as capacity of the
obtained extracts to counteract the superoxide anion O 2 •-, which is one of the most dangerous reactive
oxygen species to occur in human body. From the compatible procedures used, it suggested that
antioxidant capacities of the P. odoratum and L. aromatica extracts were mainly owing to the presence of
lipid-soluble components (such as carotenoids, phenolic compounds, etc.) while the active antioxidants of
G. inodorum were water-soluble compounds.
As summarized from PCL assay, L. aromatica extract was found to be more potent (1.46 nmol Trolox
equivalent /µg) than the P. odoratum extract (1.06 nmol Trolox equivalent/µg). The strong antioxidant
activities of P. odoratum and L. aromatica extracts were consistent with the result of Nanasombat and
Teckchuen (2009) [2]. Significant antioxidant property of G. inodorum extract was also demonstrated (6.50
nmol ascorbic acid equivalent /µg). Most studies indicated that the protective effect against oxidative
radicals of any botanical extracts was due to phenolic compounds.

PN – 14
Table 1 Dietary contents found in three Thai local vegetables
Plant sample % SDF (w/dw) % IDF (w/dw) % TDF (w/dw)
P. odoratum 0.95 46.56 47.51

L. aromatica 1.89 42.38 44.27
G. inodorum 0.18 34.23 34.41
Note: SDF = Soluble dietary fiber; IDF = Insoluble dietary fiber;
TDF = Total dietary fiber; dw = dry weight
Figure 2 Soluble and insoluble dietary fiber contents (% w/dry weight) of three Thai indigenous vegetables (SDF = Soluble dietary
fiber; IDF = Insoluble dietary fiber)
Three indigenous vegetables tested were composed of noticeable dietary fiber contents and antioxidant
capacities. Therefore they were chosen to utilize as raw materials for development of health food
11
products, for example, in tablet form as shown in Figure 3.
[A] [B] [C]

Figure 3 Samples of dietary fiber tablets (no coloring agent) from Limnophila aromatica [A], Polygonum odoratum [B], and
Gymnema inodorum [C]

PN – 14
CONCLUSION
P. odoratum, L. aromatica, and G. inodorum were interesting to be the alternative sources for dietary
11
fiber and antioxidant. Although G. inodorum comprises of lower dietary fiber content than P. odoratum
and L. aromatica, it exerts a prominent property i.e. glucose absorption inhibiting action that is profitable
to the consumer. Thus supplementing a balanced diet with L. aromatica, P. odoratum and G. inodorum
would be beneficial to persons who have health problems (e.g. constipation, obesity, diabetes) as well as
healthy human who are deficient in fiber intake (25-30 g/day recommended) [6]. Nevertheless, utilization
of these vegetables for developing as health food product or food supplement requires further studies such
as stability of the active components and safety assessment for long term consumption.
ACKNOWLEDGMENTS
The authors are grateful to Thailand Institute of Scientific and Technological Research (TISTR) for
financial support throughout the research project.
REFERENCES
1. Tangkanakul P., Trakoontivakorn G., Auttaviboonkul P., Niyomvit B., Wongkrajang K. 2006.
Antioxidant activity of Northern and Northeastern Thai foods containing indigenous vegetables.
Kasetsart J (Nat Sci) 40 (Suppl.): 47-58.
2. Nanasombat S., Teckchuen N. 2009. Antimicrobial, antioxidant and anticancer activities of Thai
local vegetables. J Med Pl Res. 3(5): 443-449.
3. Shimizu K., Ozeki M., Tanaka K., Itoh K., Nakajyo S., Urakawa N., Atsuchi M. 1997.
Suppression of glucose absorption by extracts from the leaves of Gymnema inodorum. J Vet Med
Sci. 59(9): 753-757.
4. Official Methods of Analysis of AOAC International, 16th Edition, Volume II, Section 45.4.07,
Method 985.29, 1997.
5. Popov I., Lewin G. 1999. Antioxidative homeostasis: characterization by means of
chemiluminescent technique. Meth Enzymol. 300: 437-456.
6. Figuerola F. et al. 2005. Fiber concentrates from apple pomace and citrus peel as potential fiber
sources for food enrichment. Food chem. 91(3): 395-401.

PN – 15
IDENTIFICATION OF LACTIC ACID BACTERIA AND YEASTS FROM

FERMENTED RICE PRODUCT (KHAO-KHAB)
Somboon Tanasupawat1, Wongsakorn Phongsopitanun1, Wanlapa Lorliam2,

Naphatrapi Luangsakul3 and Lawan Chatanon4, *
1
Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn
University, Bangkok 10330, Thailand.
2
Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand.
3
Faculty of Agricultural-industry, King’s Mongkut Institute of Technology Ladkrabang,
Bangkok 10520, Thailand.
4
Bioscience Department Thailand Institute of Scientific and Technology Research (TISTR), Technopolis,
PathumThani 12120, Thailand.
KEYWORDS: Fermented rice product, lactic acid bacteria, Lactobacillus, yeasts
INTRODUCTION
Khao-khab, a product in Utaradit province, the northern part of Thailand, is made from rice (Oryza
sativa) or glutinous rice (Oryza glutinosa). Khao-khab is comsumed as it is or the vegetables are put
on it over a steaming water pot and then wrapped its cooked and comsumed with spicy sauce. The fried
noodle was also wrapped with the sheet of khao-khab for comsumption. Acetic acid bacteria in khao-
khab, Acetobacter indonesiensis, A. lovaniensis, A. pasteurianus, A. syzygii and A. farinalis strains have
been isolated and described 1). This study, lactic acid bacteria and yeasts are isolated and identified
based on the phenotypic and genotypic characteristics.

Isolation of LAB and yeasts Twelve fermented rice products collected in Laplae district, Utaradit
province were used for the isolation. The pH of the samples was measured by a pH meter (Mettler
Toledo, USA). Ten grams of sample were homogenized with 90 ml of sterile 0.85 % NaCl solution to a
uniform suspension and then tenfold serial dilutions in 0.85 % NaCl solution were carried out. For
evaluation of LAB, for each serial dilution, 1 ml aliquots were each pipetted into a Petri dish with 20 ml
of melted isolation medium, de Man, Rogosa and Sharpe agar (MRS agar, Difco) containing 0.3 g/100 ml
CaCO 3 and incubated at 30°C for 3 days. The colonies with a surrounding clear zone were counted as
lactic acid bacteria and they were picked up for purification. For evaluation of the yeasts, the aliquots of
0.1 ml of each dilution were spread onto the surface of Yeast and Malt Extract agar plates (YM agar) and
incubated at 30°C for 3 days. The colonies were counted and their cell morphology were observed. Yeasts
were purified by the streaking technique on a YM agar plate.
Identification of LAB
Phenotypic characterization Cells and colony appearance were examined on the cells grown on MRS
agar plates after 3 days incubation. The production of gas from D-glucose, arginine hydrolysis, nitrate
reduction, slime formation and acid production from carbohydrates were determined 2,3). All the above
tests were carried out by incubating the cultures at 30 °C for 3 days. Growth at 45 °C, at different starting
pH values (4.0, 4.5, 8.0 and 8.5) and different NaCl concentrations (4, 6, 8 and 10 g/100 ml) was tested
by using half strength of MRS (MRSH) broth. Meso-diaminopimelic acid (DAP) in the cell wall
peptidoglycan was determined as described previously 4). The isomer of lactic acid produced by each
strain was analyzed enzymatically according to the method of Okada et al.5).
16S rRNA gene sequencing analysis DNA from LAB isolates was isolated from cells grown in an
MRSH broth andwas purified as reported previously 6). The 16S rRNA gene was sequenced as
previously described 7). The sequences determined (1307–1352 bases) were aligned with the selected
putative homolog sequences obtained from BLASTn searches of the Gen-homolog sequences obtained
from BLASTn searches of the Gen-Bank/EMBL/DDBJ database by employing CLUSTAL_X 8).
Identification of yeast isolates
Phenotypic characterization Cells and colony appearance of yeasts were determined from cells and
colonies grown in YM broth and on YM agar, respectively, after incubation at 30 C for 72 h. For the
spore morphology, cells were grown on YM agar, 5 g/100 ml malt agar, and acetate agar were used to
induce the sporulation of yeasts whilst the hyphae growth was also determined by using YM agar,
according to the methods of Yarrow 9). Physiological and biochemical characterization of the isolated
yeasts were determined by investigating the assimilation reactions of sugars using the ID 32C test

PN – 15
(bioMérieux, France). The isolates were grouped based on their relationships among the phenotypic
characteristics by cluster analysis. Hierarchical cluster analysis was conducted by using SPSS for
Windows version 13.0.
26S rRNA gene sequence analysis The nucleotide sequences of the D1/D2 variable domains of the 26S
rRNA encoding gene were determined by the modified methods of Kurtzman and Robnett 10). The
obtained sequences, were aligned and compared with homolog 26S rRNA gene partial sequences obtained
from the DNA databank by BLASTn homology searches.
Analytical procedures Ethanol was analysed by gas chromatography (Hewlett-Packard, HP 5890 Series;
USA) using Parapak QS (Carbowax 20 M) column and flame ionizationdetector (FID, 150 °C).

The total count of LAB in the khao-khab samples ranged from 3.2 x108 to 2.2 x1011 colonies/g sample,
whilst yeasts were from 5.9 x105 to 1.2 x108 colonies/g sample. The pH values of the samples ranged
from 3.4 to 4.0. In the identification of LAB isolates, all rod-shaped LAB isolates produced DL-lactic
acid, were identified as Lactobacillus based on morphological, physiological, and biochemical
characteristics and they were grouped based on the phenotypic characteristics by cluster analysis.
Twenty-three isolates (Group A) were heterofermentative and 7 isolates (Group B) were
homofermentative . On the basis of 16S rRNA gene sequence similarity (Table 1), the representative
isolates in Group A (FL11-1, FL18-1, FL19-1S and FL21-1) were closely related to L. fermentum (99.9-
100%) 11). Group B isolates which contained meso-diaminopimelic acid in their cell wall, including
isolates FL11-3 and FL22-2 (Group B1), isolate FL17B (Group B2), and isolate FL22-1 (Group B3),
were closely related to L. plantarum (99.9-100%), L. pentosus (100%) and L. nantensis (99.1%),
respectively 2,12). Their differential characteristics are shown in Table 2.
In Thailand, L. plantarum strains were a common species isolated from sauerkraut, pickled bean sprouts,
pickled spring onion, sweetened rice, fermented sausages 2) whilst L. pentosus was found in pickled
mustard, pickled spring onions, fermented durian, fermented tea leaves, fermented fish and fermented
Thai sausages2). In addition, L. plantarum and L. casei strains were isolated from the starter dough of
Chinese steamed buns 13).
Table 1 Group, isolate no., similarity (%) and identification of isolates.
Group Isolate no. % Similarity Identification

A FL18-1, FL11-1, 99.9-100 L. fermentum
FL19-1S, FL21-1
B1 FL11-3, FL22-2 99.9-100 L. plantarum
B2 FL17B 100 L. pentosus
B3 FL22-1 99.1 L. nantensis
For the identification of 12 yeast isolates, they were identified as Pichia kudriavzevii (3 isolates), P.
occidentalis (3 isolates), P. manshurica (2 isolates), Yarrowia lipolytica (2 isolates), and each isolate as
Yarrowia sp. and Geotrichum sp., based on the D1/D2 domain sequences of 26S rRNA gene similarity
(99-100%) (Table 3). They were grouped based on the phenotypic characteristics by cluster analysis. Six
selected yeast isolates could produce ethanol from glucose ranged from 0.132 -7.872 g/l (Table 3). Their
differential characteristics are shown in Table 4. The yeasts isolates in this study were different from
Candida tropicalis, Pichia stipitis, C. parapsilosis, Issatchenkia orientalis and Saccharomyces cerevisiae
strains that isolated from the starter dough of Chinese steamed buns 13).
CONCLUSION
In this study, 30 lactic acid bacterial isolates included Lactobacillus fermentum (23 isolates), L. plantarum
(3 isolates), L. pentosus (2 isolates) and L. nantensis (2 isolates) and 12 yeast isolates identified as
Pichia kudriavzevii (3 isolates), P. occidentalis (3 isolates), P. manshurica (2 isolates), Yarrowia
lipolytica (2 isolates), and each isolate as Yarrowia sp. and Geotrichum sp. are distributed in khao-khab
collected in Utaradit province. The LAB isolates and yeasts may play the important roles in the
fermentation of this product.

PN – 15
Table 2 Differential characteristics of Lactobacillus isolates.
Group Group Group

Characteristics Group A B1 B2 B3
(23) (3) (2) (2)
Growth at pH4.0 + + + +
pH 4.5 + (-2) + + +
pH 8.0 - (+11) + + +
pH 8.5 - (+11) + + (-1) +
Growth in
4%NaCl + (-1) + + +
6% NaCl + (-10) + + +
8% NaCl - + (-1) + -
Growth at 45 C + + + +
Arginine
hydrolysis - (+4) - w -
Gas from glucose + - - -
Acid from:
D-Amygdalin - + + +
L-Arabinose + (-11) + + -
D-Cellobiose - + + +
D-Galactose + (-4) + + +
Gluconate - (4w) + + + (-1)
Glycerol - w - -
Lactose + (-7) + + +
D-Maltose + (-1) + + +
D-Mannitol - + + -
D-Mannose + (-11) + + +
D-Melibiose + + + +
Methyl-glucoside - (+2) - - -
Raffinose + (-1) + + -
D-Ribose + (-2) + + +
Salicin - + + +
D-Sorbitol - + + -
Sucrose + (-1) + + +
D-Xylose - (+3) - (+1) - -
D-Trehalose -(+10) + + +
+, positive; w, weak positive; -, negativreaction
Numbers in parentheses indicate the number of isolates showing the reaction.
Table 3 Isolate no., ethanol (g/l), similarity (%) and identification of isolates.
Group Isolate no. Ethanol (g/l) % Similarity Identification

A1 FY19 ND 100 Pichia kudriavzevii
FY20 7.872 100 Pichia kudriavzevii
A2 FY18 3.374 99 Pichia occidentalis
FY21 ND 99 Pichia occidentalis
A3 FY11 ND 100 Pichia manshurica
FY15 0.132 98 Pichia manshurica
A4 FY14 ND 100 Pichia kudriavzevii
B FY12 0.075 100 Yarrowia lipolytica
FY13 0.055 100 Yarrowia lipolytica
FY16 ND 99 Yarrowia sp.
C FY17 0.275 100 Geotrichum sp.
D FY22 ND 100 Pichia occidentalis
ND, not determined.

PN – 15
Table 4 Differential characteristics of yeasts isolates.
Assimilation of A1 A2 A3 A4 B C D
(2) (2) (2) (1) (3) (1) (1)
Cycloheximide - - - - + + -
Erythritol - - - - + - -
Gluconate (K) - - - - -(+1) - -
N-Acetyl-Glucosamine + + - - + - -
Glucosamine + +(-1) + + + + -
Glycerol -(+1) - +(-1) + + + -
Lactic acid + - +(-1) + -(+1) - +
D-Mannitol - - - - + + -
D-Ribose - - - - +(-1) - -
D-Sorbitol - - - - -(+1) + -
L-Sorbose - + - - -(+1) + +
D-Xylose - - - - - + -
+, positive; -, negative reaction. Numbers in parentheses indicate the number of isolates showing the reaction.
REFERENCES
1. Tanasupawat S, Kommanee J, Yukphan P, Nakagawa Y, Yamada Y. 2011. Identification of
Acetobacter strains from Thai fermented rice products based on the 16S rRNA gene sequence
and 16S-23S rRNA gene internal transcribed spacer restriction analyses. J Sci Food Agric 91:
2652-2659.
2. Tanasupawat S, Ezaki T, Suzuki K, Okada S, Komagata K, Kozaki M. 1992. Characterization
and identification of Lactobacillus pentosus and Lactobacillus plantarum strains from fermented
food in Thailand. J Gen Appl Microbiol 38: 121-134.
3. Tanasupawat S, Okada S, Komagata K. 1998. Lactic acid bacteria found in Fermented fish in
Thailand. J. Gen. Appl. Microbiol. 44(3): 193-200.
4. Komagata K, Suzuki K. 1987. Lipid and cell-wall analysis in bacterial systematics. Methods
Microbiol 19:161-207.
5. Okada S, Toyoda T, Kozaki M. 1978. An easy method for the determination of the optical types
of lactic acid produced by lactic acid bacteria. Agric Biol Chem 42: 1781-1783.
6. Saito H, Miura K. 1963. Preparation of transforming deoxyribonucleic acid by phenol. Biochim
Biophys Acta 72: 619-629.
7. Tanasupawat S, Thawai C, Yukphan P, Moonmangmee D, Itoh T, Adachi O, Yamada Y. 2004.
Gluconobacter thailandicus sp. nov., an acetic acid bacterium in the alpha-Proteobacteria. J Gen
Appl Microbiol 50: 159-167.
8. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. CLUSTAL_X
windows interface: flexible strategies for multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Res 25: 4876-4882.
9. Yarrow D. 1998. Methods for the isolation, maintenance and identification of yeasts. In:
Kurtzman CP, Fell JW (eds.). The yeasts, a taxonomic study, Elsevier Science, Amsterdam.
pp.77-105.
10. Kurtzman CP, Robnett CJ. 1998. Identification and phylogeny of ascomycetous yeasts from
analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van
Leeuwenhoek73:331-371.
11. Tanasupawat S, Okada S, Suzuki K, Kozaki M, Komagata K.1993.Lactic acid bacteria,
particularly heterofermentative lactobacilli, found in fermented foods in Thailand. Bull JFCC 9:
65-78.
12. Valcheva R, Ferchichi MF, Korakli M, Ivanova I, Gänzle MG, Vogel RF, Prévost H, Onno B,
Dousset X. 2006. Lactobacillus nantensis sp. nov., isolated from French wheat sourdough. Int J
Syst Evol Microbiol 56: 587-591.
13. Luangsakul N, Keeratipibul S, Jindamorakot S, Tanasupawat S. 2009. Lactic acid bacteria and
yeasts isolated from the starter doughs for Chinese steamed buns in Thailand. LWT-Food Sci
Technol 42: 1404-1412.

PN – 16
NUTRITIONAL VALUES AND TLC FINGERPRINT OF

SCAPHIUM SCAPHIGERUM FRUIT GEL
Wichittra Phlicharoenphon, Wandee Gritsanapan, Pongtip Sithisarn*
Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand.
KEYWORD: Samrong, Scaphium scaphigerum, nutritional values, TLC fingerprint
INTRODUCTION
Samrong (Scaphium scaphigerum (G.Don) Guib. & Planch) is a tree in Sterculiaceae family. The
traditional uses of the gel from the fruits of this plant are to use as laxative and weight control agents and
for the treatments of cough and sore throat and using as expectorant1). After soaking in water,
samrong seed coats become a brown glass jelly which can be used as a bulk laxative and promote the
satiety. This gel contains polysaccharide PP-III, which the subunits are manosaccharides; galactose,
arabinose and rhamnose1-3). Even though there are a lot of beverage and dessert preparations, especially
healthy drinks for weight control from samrong fruits gel, however there is no report about the
specification and the quality control of this gel before. Therefore, this experiment was set up in order
to evaluate the nutritional values such as carbohydrate, total sugar, fat, protein, soluble and insoluble fiber
contents of samrong fruit gel and dried powders using methods according to AOAC 2005 guideline.
Investigation of phytochemical characteristic was also conducted by thin layer chromatography (TLC).

Plant material Samrong fruits were purchased from Chantaburi province, Thailand in April, 2013. The
specimen was compared with the authentic plant material of Forest Herbarium, Wildlife and Plant
Conservation Department of Thailand. Samrong fruits were prepared by cleaning and cutting upper and
lower parts of the fruits and soaked in distilled water at room temperature. Then the contaminations were
separated. The swollen samrong fruit gel was rinsed and boiled in distilled water at temperature of
100˚C for 60 minutes. The swollen samrong fruit gel was left on the sieve until the water was drained.
Samrong fruit gel was passed through the fabric filter, dried in hot air oven (65-70˚C for 8 hours) then
powdered through the sieve No. 20. The fresh gel and dried samrong gel powder were analyzed for the
nutritional values as described below.
Analysis of nutritional values Nutritional values such as carbohydrate, total sugar, fat, protein,
soluble fiber and insoluble fiber contents in samrong fruit gel and dried powder were evaluated using the
methods according to AOAC 2005 guideline4).
Preparation of samrong fruit gel Samrong gel powder (5 g) was swelled in water (1000 ml) for 16 hours
then the gel was separated. Samrong gel (5 g) was mixed with 95% ethanol (210 ml) and left at 4˚C for 12
hours then filtered. The obtained precipitate was hydrolyzed by adding 15 ml of 5% sulfuric acid solution.
The solution was heated on the water bath (90˚C) for 2 hours. The solution was cooled and neutralized
using barium carbonate. The mixture was filtered and a supernatant was used for TLC analysis.
TLC condition The hydrolyzed fraction of samrong fruit gel and the authentic monosaccharides were
analyzed by thin layer chromatography using the condition as follow;
Adsorbent: Silica gel GF 60
Solvent system: 1-propanol : ethyl acetate : water (4:0.5:0.5 v/v/v)
Detection: 10% sulfuric acid in methanol. After spraying, the TLC plate was heat at 100˚C for 10 minutes
RESULTS
As shown in Table 1, samrong fruit gel (100 g) showed the absence of any energy, total fat, carbohydrate
and total sugar with 0.25 ± 0.00 x 6.25 g of protein. This gel powder contained low soluble and insoluble
dietary fiber contents (0.57 ± 0.01and 1.26 ± 0.01 g, respectively). Samrong fruit gel dried powder (100
g) contained low energy of 335.5 ± 0.71 kcal with 28.75± 0.18 and 316.8 ± 0.49 g of protein and
carbohydrate, respectively. This gel powder showed the absence of any fat with higher amounts of soluble
and insoluble dietary fibers (5.21 ± 0.16 and 89.93 ± 0.06 g, respectively) and small amount of total sugar
(0.47 ± 0.01 g).

PN – 16
Table 1 Nutritional values of gel and gel powder of Scaphium scaphigerum fruits determined by methods according to AOAC 2005
guideline.
Nutritional values (per 100 g)

Nutritional values Samrong fruit gel Samrong fruit gel powder
Energy (kcal) Not detected 335 ± 71
Protein (Nx6.25) (g) 0.25 ± 0.00 28.75±0.18
Total fat (g) Not detected Not detected
Carbohydrate (g) Not detected 316.8 ± 0.49
Soluble dietary fiber (g) 0.57 ± 0.01 5.21 ± 0.16
Insoluble dietary fiber (g) 1.26 ± 0.01 89.93 ± 0.06
Total sugar (g) Not detected 0.47 ± 0.01
Thin layer chromatography of the hydrolyzed fraction of samrong fruit gel showed the bands at Rf
values of 0.32, 0.44 and 0.60 suggesting the presences of monosaccarides galactose, arabinose and
rhamnose, respectively (Figure 1).
Figure 1 : Thin layer chromatography fingerprint of hydrolyzed fraction of Scaphium scaphigerum fruit gel.
TLC condition The hydrolyzed fraction of samrong fruit gel and the authentic monosaccharides were
analyzed by thin layer chromatography using the condition as follow;
Adsorbent: Silica gel 60
Solvent system: 1-propanol : ethyl acetate : water (4:0.5:0.5 v/v/v)
Detection: 10% sulfuric acid in methanol. After spraying, the TLC plate was heat at 100˚C for 10 minutes
Track; 1= samrong fruit gel hydrolyzed fraction, 2 = standard glucose, 3 = standard galactose, 4 =
standard arabinose, 5 = standard rhamnose, 6 = standard mannose
DISCUSSION
The fresh samrong gel showed very low nutritional values such as no energy, fat, carbohydrate and sugar
with very low amounts of fibers. While the gel powders prepared from the fresh gel of samrong promoted
the constituent with high insoluble dietary fiber content, low amount of carbohydrate, protein, sugar and
calorie with none of fat content. Even though samrong gel in the dried powder form contained small
amount of energy, protein, sugar and carbohydrate, it contained higher amount of dietary fiber than the
fresh gel. Therefore, this dried gel powder could be used to promote the satiety as the weigh control
agent. TLC fingerprint of the hydrolyzed fraction of samrong fruit gel showed the chromatographic bands
corresponded to monosaccharides galactose, arabinose and rhamnose supporting the previous report about

PN – 16
the subunits of polysaccharides PP-III 1), the main phytochemical in samrong fruit gel. However, other
physical properties including swell volume, total acidity, total ash and loss on should be performed in the
future to qualitatively control of this samrong gel powders.
CONCLUSION
Samrong fruit gel powder promoted a high insoluble dietary fiber with low amounts of calorie and other
nutritionals suggesting the possibility to be applied as weight control agent.
ACKNOWLEDGMENTS
The authors express their gratitude to Thailand Research Fund (TRF) for financial support.
REFERENCE
1. วันดี กฤษณพันธ์. สํารอง : สมุนไพรที่สรรพคุณไม่เป็ นรองใคร. วารสารเภสัชกรรมสมาคมแห่งประเทศ
ไทยในพระบรมราชูปถัมภ์ 2550: 1/2550: 11-14
2. กิติยา ภู่เจริ ญ, พรรณทิพา สิ งห์ประชา. ผลิตภัณฑ์อาหารเสริ มเส้นใยอาหารจากผลสํารอง (โครงการพิเศษ).
กรุ งเทพมหานคร.มหาวิทยาลัยมหิ ดล; 2546
3. ชนินนั ท์ ลิมปิ ชัชวาลย์. ผลของปริ มาณโปรตีนในกัมสํารองต่อสมบัติอิมลั ชัน ปริ มาณกรดฟี นอลิก และ
ความสามารถต้านออกซิเดชัน (วิทยานิพนธ์). กรุ งเทพมหานคร. มหาวิทยาลัยเกษตรศาสตร์; 2551.
4. Official methods of analysis of AOAC International; 2005 / William Horwitz,
editor.Gaithersburg, Md.: AOAC International, c 2005. (Ref. 543/AOAC/18th ed. Appendix D.
p.9/Appendix E.p.2-3)

PN – 17
QUANTITATIVE ANALYSIS OF EPIGALLOCATECHIN GALLATE IN YOUNG AND 11
MATURE ASSAM TEA LEAF EXTRACTS
Pattra Ahmadi Pirshahid*, Thongchai Hemthanon, Yaowaluk Khamphan, Phatsuda Chueboonmee and
Chuleratana Banchonglikitkul
Pharmaceutical and Natural Products Department,Thailand Institute of Scientific and Technological Research,Technopolis, Moo 3,
Klong 5, Klong Luang, Pathum Thani 12120, Thailand.
*
Corresponding author: pattra_a@tistr.or.th (Pattra Ahmadi Pirshahid)
KEYWORDS: Epigallocatechin gallate (EGCG), Assam tea
INTRODUCTION
Assam Tea, Camellia sinensis var. assamica (Mast.) in Theaceae family, is widely distributed both in
Asia and North America. Tea has been considered as a medicinal herb and a healthy beverage since
ancient times. Nowadays, tea is one of the most consumed beverages in the world because of a major
source of flavonoids and its anti-oxidant properties. Epigallocatechin gallate (EGCG) is a phenolic
compound and a major component in tea leaves. EGCG have been reported to be an effective in health
benefits such as against cardiovascular diseases, cerebral hemorrhage and lower risk of several types of
cancer such as endometrial adenocarcinoma. Due to the health promoting of EGCG, TISTR has
developed the dietary supplement products that contained the extract of young tea leaves which was
available in market as green tea to be a part of active ingredient. The objective of this study is to analyse
the quantity of EGCG in comparison between young and mature leaves in order to substitute the mature
to the young leaves extract to lower cost of raw material by High performance liquid chromatography.
Figure 1 Chemical structure of Epigallocatechin gallate (EGCG).

Material The leaves of Assam tea (Camellia sinensis Var. assamica (Mast.)) were collected in June 2013
from Wieng Pa Pao, Chiang Rai Province, Thailand. The first 5 leaves were collected as young leaves
while the sixth up to the end of stem were collected as a mature leaves. Solvent used for extraction was
95% ethanol (commercial grade) which was obtained from The Liquor Distillery Organization. Solvents
used for Liquid Chromatography were dimethylformamide, methanol, acetic acid and deionized water.
All solvents were HPLC grade and obtained from RCI Labscan, Ireland. An authentic standard of
Epigallocatechin gallate was HPLC grade with 95.0% purity from Sigma-Aldrich, USA.
Plant material preparation The young leaves (YL) and mature leaves (ML) were separately washed with
water then dried at 45°C in oven for 20 hrs. The dried leaves were powdered for further extraction.
Extraction Ten gram of each young and mature powdered leaves of Camellia sinensis var. assamica
(Mast.) were extract with 95% ethanol by stirring at room temperature for 20 min. The solid was separate
from the mother liquor by filtration, followed by re-extraction for 4 times, the filtered solution was

PN – 17
combined, then evaporated under vacuum at 40°C to yield the crude extracts of young and mature leaves
(YL,ML).
Apparatus HPLC analysis was performed on the Water 600 controller series conncected with the Water
486 Tunable Absorbance Detector. Data analysis was performed by using Clarity chromatography
software (DataApex, Czech Republic). Separation was comprised of a C 18 reverse phase column ODS
HYPERSIL, 200x4.6 mm, 5 µm (Thermo Electron Corporation, USA). The mobile phase consisted of
dimethylformamide/methanol/acetic acid and water (gradient), 1 ml/min as a flow rate with the injection
volume of 20 µl and λmax is 278 nm.
Preparation of standard solution Epigallocatechin gallate (EGCG) was accurately weighed to 10 mg in a
volumetric flask (50 ml) and the volume adjusted to 50 ml with water. The stock solution was diluted to
create five concentrations for standard curve.
Preparation of samples One gram of each crude extract of YL and ML was dissolved with 50 ml of
water. The solution was sonicated for 15 min, filtered, then adjusted to final volume of 50 ml, and filter
through a 0.45 µm prior inject to HPLC.
Calibration curve and linearity The calibration curve was analysis of the standard epigallocatechin
gallate at five concentrations. The linearity was determined by means of linear regression analysis. The
calibration curve showed a coefficient of correlation R2 = 0.9990.
Limits of detection (LOD) and quantification (LOQ) LOD and LOQ were the concentrations that give a
signal to noise ratio equal to 3.35 and 11.17 respectively.
Figure 2 Young leaves (1) Mature leaves (2) Harvesting of tea leaves (3).

The extract of YL and ML were determination by HPLC as gradient elution with triplicate injects of
solutions and gave good resolution without any interference by other compounds in the samples under the
chromatographic condition. The retention time of EGCG was about 20.3 min. It revealed that the EGCG
content in young leaves and mature leaves are 0.73±0.05 and 2.86±0.02%, respectively.

PN – 17
Calibration Curve EGCG
20000
y = 32.552x - 327.26
15000
R2 = 0.999
Area
10000
5000
0
0 100 200 300 400 500 600
Conc.(ppm )
Figure 3 Calibrition Curve of Epigallocatechin gallate (EGCG).
Std. EGCG
young leaves extract
mature leaves extract
Figure 4 HPLC chromatogram of Standard Epigallocatechin gallate (EGCG)(1) young leaves extract (2) and mature leaves extract
(3).

PN – 17
CONCLUSION
The two different crude ethanolic extract of young and mature Assam tea leaves displayed a good
percentage of EGCG content by 0.73±0.05 and 2.86±0.02% respectively. This resulted will be value
added to mature leaves by substitution to young leaves as an active ingredients of TISTR dietary
supplement products with lower cost of raw materials.
Table 1 Percentage of EGCG in Young leaves extract and Mature leaves extract.
EGCG
Sample
(% w/w of the extract)
Young leaves extract(YL) 0.73±0.05
Mature leaves extract (ML) 2.86±0.02
REFERENCES
1. http://www.enzolifesciences.com/ALX-270-263/epigallocatechin-gallate/
2. Anderson, R.F.et al. 2001. Green tea catechins partially protect DNA from OH radical induced
strand breaks and base damage through fast chemical repair of DNA radicals. Carcinogenesis,
v.22, n.8,p. 1.189-1.193.
3. Kawai, K. et al. 2003. Epigallocatechin gallate, the main component of tea polyphenol, binds to
CD4 and interferase with gp120 binding. J. Allergy Clin. Immunol., v. 112, n. 5, p. 951-7.
4. Rijken, P.J et al. 2000. Antioxidant and Other Properties of Green and Black Tea, in: Cadenas, E;
Packer, L. Handbook of Antioxidants. 2st ed.: Marcel Dekker, New York, cap. 19, p. 371-399.
5. Sano, J. et al. 2004. Effects of Green Tea Intake on the Development of Coronary Artery Disase,
Circ J.,v. 68, p. 665-70.
6. Sato, T.; MYATA, G. 2000.The Nutraceutical Benefit. Part I: Green tea. Nutrition, v. 16, p. 315-
317.
7. Suzuki, M. et al. 2004. Protective effect for green tea catechins on cerebral ischemic damage.
Med. Sci. Monit., v. 10, n. 6, p. 166-74.
8. Drinking green tea modestly reduces breast cancer risk. J Nutr. 2009 Feb; 139(2): 310-6. Epub
2008 Dec 11.

Session 2
Biopharmaceutical Sciences BP 1 - 17
PRELIMINARY STUDY OF SYZYGIUM AROMATICUM (L.)

ON ANALGESIC ACTIVITY IN RATS
Kanyarat Sueksakit1, Krittiya Thisayakorn1, Vichein Khueynok12, Kanjana Sriyam1, Darunee Pahusee1,
and Nopparat Buddhakala
1
Pharmaceutical and Natural Product Department (PNPD), Thailand Institute of Scientific and Technological Research (TISTR),
Technopolis, Pathum Thani 12120, Thailand
BP - 1
2
Division of Biology, Faculty of Science and Technology, Ragamangala University of Technology Thanyaburi, Pathum Thani
12110, Thailand. Email: b_sueksakit@hotmail.com
KEYWORDS: Syzygium aromaticum L., Analgesic, Tail flick
INTRODUCTION
Syzygium aromaticum (Linn.) Merr.&L.M.Perry is commonly known as clove tree in the Myrtaceae
family. The clove tree is an evergreen that grows to a hight ranging from 8-12 m, used as a spice in
cuisines all over the world. In Thailand, it is widely used as a medicinal plant to treat a variety of diseases
including inflammation, tooth pain, and analgesia. Its chemical composition showed that the essential oils
mainly contained about 87.00% eugenol, 8.01% eugenol acetate, and 3.56% β-caryophyllene1. Pain, the
most common reason for patients, is an unpleasant sensory. It generally caused by inflammatory reaction
in the tissue or tissue damage and produced emotional reaction. Non steroidal anti-inflammatory drugs
(NSAIDs) are widely used in the treatment of pain, inflammation, and fever. However, these drugs has
many side effects, especially irritating the gastrointestinal tract. In nowadays, many people determine to
select medicinal plants to relief pain instead of drugs because it has less toxic effects, and can treat a
variety of diseases. Therefore, the purpose of this study was to evaluate the analgesic activity of clove oil
on tail flick method in rats. The procedures followed and applied are based on those described by Yashpal
et al. (1993)2.

Materials Male Wistar rats were purchased from the National Laboratory Animal Center, Mahidol
University, Salaya, Nakorn Pathom. Age of the rats at the beginning of study was 2 months old. All
procedures and animal care were approved by Institutional Animal Care and Use Committee of Thailand
Institute of Scientific and Technological Research. They were kept in cages at 25±2 ºC under alternate of
12 hours light/dark cycle and fed with standard pellet and tab water ad libitum. All rats were acclimatized
for 7 days before starting the experiments.
Clove oil was isolated by water steam distillation and stored in brown bottle at 4ºC. It was prepared in a
cream base (vehicle) at a various concentrations at 1%, 5%, and 10% w/w. Cream base was also used as a
vehicle. 5% w/w indomethacin in cream base were used as standard drugs for analgesic activity. The
analgesic activity was assessed by tail flick meter (Ugo Basile, Italy).
Methods Rats were randomly divided into six groups of four rats in each group. From the five groups
comprised of the control ; rats were received with cream base, standard ; 5% w/w indomethacin, and the
rest three groups ; 1%, 5% and 10% w/w clove oil, respectively. All tested materials were topically
applied to the tail within 3 cm from the tip after measured the baseline. Tail flick latency was assessed by
tail flick meter (Ugo Basile, Italy). The intensity of heat was set at 50. To prevent tissue damage, a cut off
time was set at 10 seconds. The baseline was measured 3 times for each animal and mean was used as
predrug latency. The reaction time was measured at 0, 30, 60, 90, 120, 180, and 240 minutes after
receiving drug and oil. The number (in seconds) of each rat’s tail withdrawal was calculated for the
maximal possible effect percent as follows;
% MPE = [(test latency-baseline latency)/(cut off time-baseline latency)]×100
All data were expressed as mean ± standard error. The results were analyzed for statistical significance by
one-way ANOVA. Statistical significance was defined as p<0.05.
RESULTS
Analgesic activity of Syzygium aromaticum (L.) (Clove oil) was assayed by using tail flick meter (Ugo
Basile, Italy) and results are presented in Table 1. The results showed that 5% w/w clove oil had
significantly (p<0.05) higher reaction time than the control group at 90 and 120 min and 5% w/w
indomethacin showed significantly (p<0.05) of reaction time at 120 and 180 min. The effects of clove oil

and indomethacin increased with time. Indomethacin had the highest of maximal possible effect percent
(%MPE) for 36.76% at 180 min, and 5% w/w clove oil had the highest %MPE for 18.62% at 90 min were
presented in Figure 1.
Table 1 Effect of Clove oil on tail flick method in rats
Reaction time ± S.E.

Groups
BP - 1
Baseline 0 min 30 min 60 min 90 min 120 min 180 min 240 min
Control 2.75±0.10 4.20±0.30 3.30±0.10 3.20±0.26 3.10±0.23 2.75±0.35 3.35±0.25 3.15±0.05
Indomethacin 2.41±0.04 3.33±0.24 3.97±0.29 3.78±0.48 3.75±0.40 4.28±0.40* 5.20±0.06* 3.88±0.59
Clove oil 1% 2.41±0.06 3.93±0.07 3.67±0.09 2.93±0.17 3.23±0.15 2.70±0.24 2.88±0.44 3.05±0.33
Clove oil 5% 2.59±0.06 3.90±0.05 3.57±0.38 2.97±0.27 3.97±0.22* 3.80±0.21* 3.07±0.29 4.07±0.20
Clove oil 10% 2.48±0.10 4.25±1.05 3.10±0.70 2.67±0.47 3.33±0.61 2.80±0.36 2.93±0.38 3.63±0.19
Value are represented as a Mean±S.E. (n=4 in each group). Data expressed by using one-way ANOVA.
*p<0.05 was considered as significant were compared to the control group at a specific time.
Figure 1 Maximal possible effect percent (%MPE) on tail flick method in rats
DISCUSSION
In the tail flick model of acute pain in rats, it was shown that indomethacin and 5% w/w clove oil
increased tail flick withdrawal latency. However, the standard drug (indomethacin) showed greater effect
than the clove oil did. These results are consistent with previously published data 3. The analgesic studies
revealed that clove oil exhibited potent analgesic central analgesic activity effect against thermal noxious
stimuli3. Agents such as histamine, serotonin, and bradykinin are natural pain substances liberated by
injured tissues which stimulate the nociceptive mechanism to elicit pain4. Clove oil may possibly have
acted as an analgesic agent from heat which stimulates the nociceptive mechanism.
CONCLUSION
The present study on the analgesic activity of clove oil was confirmed to have a promising role in pain
management and to select optimal concentration to the experiment study.
ACKNOWLEDGEMENTS
The authors would like to express gratitude thanks to Thailand Institute of Scientific and Technological
Research (TISTR) for their financial support.
REFERENCES
1. Alma, M.H., Ertas, M., Nitz, S. and Kollmannsberger, H. “Chemical composition and content of
essential oil from the bud of cultivated Turkish clove (Syzygium aromaticum L.),” Bioresearch,
Vol 2 (2), 2007. pp 265-269.

2. Yashpal, K., Radhakrishman, V., Coderre, T.J., Henry, J.I. “CP-96, 345, but not its stereoisomer,
CP-36, 344, blocks the nociceptive responses to intrathecally administered substance P and to
noxious thermal and chemical stimuli in the rat,” Neuroscience, Vol52, 1993, pp 1039-1047.
3. Vogal, H.G. “Drug Discovery and Evaluation Pharmacological Assays, 2nd ed,” Springer, New
York, 2002. pp 716-717.
4. Collier, H.O.J. “Analgesic. In: Evaluation of Drug Activities,” Pharmacometric, Vol 1, 1964. pp
183-203.
BP - 1

EFFECTS OF TOPICAL ADMINISTRATION OF NYMPHAEAE LOTUS FLOWER EXTRACT

ON UVA-INDUCED PHOTOINFLAMMATION IN MICE
Krittiya Thisayakorn1, Ubon Rerk-am1, Sinn Tangstirapakdee1, Vichein Khueynok1, Kanchana Sriyam1,
Darunee Pahusi1 and Chuleratana Banchonglikitkul1
1
Pharmaceutical and Natural Product Department (PNPD), Thailand
BP - 2
Institute of Scientific and Technological Research (TISTR), Technopolis, Pathum Thani 12120, Thailand.
KEYWORDS: Photoinflammation, UVA, Nymphaeae lotus Linn, Prostaglandin
INTRODUCTION
Nymphaeae lotus Linn. (water lily, NL) is an annual plant in the Nymphaceae family that grows in
freshwater. Its trunk and bulb are in the soil under water. The flowers display in a wild variety of colors
from pink, red, purple, yellow, and white. NL has a various pharmacological activities, such as
antioxidation, anti-tyrosinase, hepatoprotection, and anti-inflammation (Debnath et al., 2013). Skin
exposure to ultraviolet (UV) light produces various types of biological effects, such as
immunosuppression skin inflammation, skin cancer, and so on (Hruza and Pentland, 1993). Particularly,
skin inflammation results in the mediation of prostaglandin E 2 (PGE 2 ). An increase in PGE 2 is the sign
of UV-induced inflammation in the skin (Miller et al., 1994). Therefore, the purpose of this study was to
elevate whether NL could protect UV-induced skin inflammation in mice by determining both skin edema
and PGE 2 levels. It might be beneficial for developing as a cosmeceutical product from plants.

Materials Thirty male ICR mice were purchased from the National Laboratory Animal Center, Mahidol
University, Salaya, Nakhon Pathom. Age of the mice at the beginning of study was 2 months old. They
were acclimatized under standard conditions for 1 week with free access of food and water. All
procedures and animal care were approved by Institutional Animal Care and Use Committee of Thailand
Institute of Scientific and Technological Research.
Flower of NL was extracted with 95% w/v ethanol. It was prepared in cream base at 3% and 6% w/w.
Cream base was also used as a vehicle. PGE 2 , a primary mediator of inflammation, kit (Cayman
Chemical, USA) was used to determine PGE 2 levels in the skin.
UV box was developed in our laboratory. It was made from wood in the size of 25x30x45 cm. The cover
of the box had a small compartment (20x10x6 cm) on the top for carrying 3 UVA lamps. On/off switch
was installed outside the box. UVA lamp (8 watts; Panasonics, Switzerland) was used to employ UVA
approximately 200 mJ/cm2.
Methods
UVA-induced photoinflammation Mice were shaved into 2x2 cm on the bottom back for one day before
the experiment. On the day of experiment, mice were randomly divided into 5 groups (6 mice of each).
The shaved skin of all mice, except one group was reserved to be a sham group, was exposed to the UVA
light approximately 200 mJ/cm2 for 30 min from directly above after 1 hr of topical administration of NL
(3% and 6%, respectively) or cream base. The control group was exposed to the UVA light only, whereas
the sham group was not treatmented with either cream or UVA. 48 hr after UV exposure, all animals were
sacrificed. Exposed skin was removed. The thickness of the skin was measured at three midline sites
using a pocket thickness gage (Mitutoyo, Japan).
PGE 2 assay The method of PGE 2 assay was modified from Yoshida et al. (2006). All skin removals were
frozen in liquid nitrogen, after which were broken by crushing in a mortar on dry ice. The skins were then
homogenized in buffer containing 50 mM Tris-HCl and 0.25% sucrose (pH 8.3), and were centrifuged at
8,000 g (4°C) for 20 minutes. The supernatants were then collected and again centrifuged at 100,000 g for
20 min (4°C). The pellets were purified with SPE (C-18) according to the manufacturer’s instructions of a
PGE 2 ELISA kit.

Statistical analysis All data were expressed as mean ± standard error. Statistical analysis for comparing
treatment effects of each group of mice was done by one-way ANOVA. Comparisons among groups were
conducted using the LSD post-hoc analysis. Student’s t-test techniques were also used. Statistical
significance was defined as p<0.05.
RESULTS
UVA-induced photoinflammation The skins that were only exposed to UVA light (the control group)
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significantly increased 25% edema compared to non-exposed skins (the sham group) as shown in Figure 1
and Table 1. Pretreatment with either cream or NL was beneficial for skin photoinflammation. Cream
base and both concentrations of NL cream significantly depleted skin edema induction by UVA
irradiation (Figure 1).
70
60
a
Skin Thickness (0.01 mm)
50 b b b
Sham = no any treatments
40
Control = UVA irradiation only
30
Vehicle = Cream base + UVA irradiation
20
10
0
Sham Control Vehicle 3%NL 6%NL
Group
Figure 1 Effects of topical pretreatment with NL (3% and 5% w/w in vehicle) on UVA-induced photoinflammation. Skin thickness
was determined and expressed as mean ± standard error. a p<0.05 relative to the sham group; b p< 0.05 relative to the control group.
Table 1 Effects of topical administration of NL (3% and 5% w/w in vehicle) on UVA-induced photoinflammation. Skin edema was
shown in percents, whereas Sham = no any treatments, Control = UVA irradiation only, Vehicle = Cream base + UVA irradiation,
3%NL = 3%w/w NL + UVA irradiation, and 6%NL = 6%w/w NL + UVA irradiation
Group Skin edema (%)
Sham -
Control 25.5
Vehicle 8.8
3% w/w NL 6.7
6% w/w NL 8.8
PGE 2 assay The levels of PGE 2 in the skin samples were shown in Table 2. The skins which were only
exposed to UV irradiation (the control group) exhibited the highest amounts of PGE 2 . They were
approximately 2-folds higher (98.3 ± 33.6 pg/ml) than those seen in non UV-exposed mice (45.0 ± 9.2
pg/ml; the sham group). As displayed in Table 2, the increase in UVA-induced PGE 2 levels was
significantly diminished by pretreatment with both 3% and 5% w/w of NL. Particularly, 3% w/w NL
exhibited a significant reduction of PGE 2 levels (p<0.028).

Table 2 Effects of topical administration of NL (3% and 5% w/w in vehicle) on UVA-induced PGE 2 levels. Sham = no any
treatments, Control = UVA irradiation only, Vehicle = Cream base + UVA irradiation, 3%NL = 3%w/w NL + UVA irradiation, and
6%NL = 6%w/w NL + UVA irradiation.
Group PGE 2 levels (pg/ml) p-value p-value

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Sham 45.0 ± 9.2 1.000
Control 98.3 ± 33.6 0.067 1.000
vehicle 83.3 ± 23.7 0.177 0.591
3% w/w NL 33.2 ± 8.2 0.670 0.028
6% w/w NL 49.4 ± 11.8 0.873 0.090
DISCUSSION
Skin inflammation could be produced by UVA irradiation since the skin thickness was found
approximately twice increase compared to non-irradiated skin. Pretreatment with either cream base or NL
remarkably shielded the skin from photoinflammation. However, the amounts of PGE 2 were found a
significant decrease in only 3%w/w NL mice. Therefore, the data demonstrated that 3% w/w NL reduced
UVA-induced skin inflammation probably involved in the decrease of this primary mediator of
inflammation.
CONCLUSION
As a remarkable evidence to diminish the effect of UVA-induced photoinflammation found in 3% w/w
NL mice on both investigations of skin edema and PGE 2 level, it suggested that pretreatment with 3%
w/w NL possibly beneficiated for UV-exposed skin.
ACKNOWLEDGEMENTS
We express our appreciation to Thailand Institute of Scientific and Technological Research (TISTR) for
providing financial support.
REFERENCES
1. Debnath S, Ghosh S, Hazra B. (2013) Inhibitory effect of Nymphaea pubescens Willd. flower
extract on carrageenan-induced inflammation and CCl4-induced hepatotoxicity in rats. Food
Chem Toxicol. 59:485-91.
2. Hruza LL, Pentland AP. (1993) Mechanisms of UV-induced inflammation. J Invest Dermatol.
100:35S-41S.
3. Miller CC, Hale P, Pentland AP. (1994) Ultraviolet B injury increases prostaglandin synthesis
through a tyrosine kinase-dependent pathway. Evidence for UVB-induced epidermal growth
factor receptor activation. J Biol Chem. 269:3529-33.
4. Yoshida E, Watanabe T, Takata J, Yamazaki A, Karube Y, Kobayashi S. (2006) Topical
application of a novel, hydrophilic gamma-tocopherol derivative reduces photo-inflammation in
mice skin. J Invest Dermatol. 126:1633-40.
5. http://www.qsbg.org/database/botanic_book%20full%20option/search_detail.asp?botanic_id=16
13

EFFECT OF MORUS ALBA STEM EXTRACT ON NITRIC OXIDE PRODUCTION

IN LIPOPOLYSACCHARIDE-INDUCED RAW 264.7 MACROPHAGE CELLS
Nattaporn Soonthornsit1, Warinkarn Hemstapat2, Pongsak Utaisincharoen3 and Tasana Pitaksuteepong1

1
Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences,
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Naresuan University, Phitsanulok 65000, Thailand.
2
Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
3
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
KEYWORDS: Morus alba L., Oxyresveratrol, Macrophage 264.7 cell, Nitric oxide, Osteoarthritis
INTRODUCTION
Nitric oxide (NO) is a cellular signaling molecule known to mediate key physiologic processes in host
defense, inflammation and immunity. The role of NO in some diseases such as cancer(1) and osteoarthritis
(OA)(2) has been reported. For OA, NO has been reported to play an important role in inflammation and
pain(3,4). The significant amount of NO was found to be produced in cartilage obtained from patients with
OA(3). It was shown to mediate the expression of proinflammatory cytokines and inhibit collagen and
proteoglycan synthesis, leading to the breakdown of the extracellular matrix(2,4). It also found to induce
chondrocyte apoptosis.
Morus alba L., known as white mulberry, has been widely cultivated in Thailand under support of Queen
Sirikit. Oxyresveratrol, the bioactive compound found in M. alba extract, has shown to have the anti-
oxidant activity with IC 50 value of 3.6 ± 0.0 µM and 15.1 ± 2.3 µM determined by lipid peroxidation and
DPPH assay, respectively(5). Chung et al.(5) reported the anti-inflammatory activity of oxyresveratrol by
inhibiting the production of nitric oxide (NO) and prostaglandin (PG) E 2 , inducible nitric oxide synthase
(iNOS) expression, and NF-κB activation in the LPS-activated RAW 264.7 macrophage cells(5).
Thongsuk(6) found that the amount of oxyresveratrol in various parts of mulberry tree was different and
the ethanolic extract obtained from stems has the highest amount of oxyresveratrol compared to the
extract obtained from twigs and leaves(6). Recently, the extract prepared from the stems of M. alba was
investigated for the efficacy on pain reduction using rat model of OA which was induced in male Wistar
rats by anterior cruciate ligament transection (ACLT)(7). The pain-related behavior was determined using
hind limb weight bearing tester. The results showed that MSE significantly attenuated joint pain in dose-
dependent manner(7). The reduction on inflammation and pain may be related to various mediators. The
present study was aimed to investigate the effect of M. alba stem ethanolic extract (MSE) on NO
production in LPS-induced RAW 264.7 cells.

Plant materials and preparation of plant extract M. alba stems were obtained from the Queen Sirikit
Sericulture Center (Tak), Tak Province, Thailand. The preparation of MSE was performed as described
previously(8). Briefly, the fresh stems were chopped and dried. Then, the dried plant was extracted by
maceration technique using 80% ethanol for 2 cycles at room temperature. After filtration, the filtrate was
evaporated under reduced pressure using a rotary evaporator (BUCHI Rotavapor R-114, Switzerland) and
continued drying using a water bath (M25 LAUDA, Germany). Then, the dried powder was stored in
tight, light-protected container.
Cell culture Mouse macrophage cell line (RAW 267.4) obtained from American Type Culture Collection
(ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagles’ medium (DMEM)
(HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS) (HyClone) and 1% L-glutamine
(Biological industries, Kibbutz Beit Haemek, Israel). The cultures were incubated at 37°C under 5% CO 2
atmosphere and were subcultured every 2 days.
Cell viability assay RAW 264.7 cells were seeded in a 6-well plate at a concentration of 1.9 x 105
cell/mL. After overnight incubation, cells were treated with various tested samples in the presence of
heat-killed E. coli for 24 h. After treatment, cell viability was determined using trypan blue dye exclusion
by mixing with trypan blue dye solution (Gibco, Grand Island, NY, USA). Then, the unstained cells were
counted using hemocytometer (Reichert, Buffalo, NY, USA) under a light microscope. The percentage
cell viability was calculated by the following equation:
% Cell viability = (viable cells/total number of cells) x100

Treatment of mouse macrophage cell line RAW 264.7 cells (1.875 x 105 cell/mL) were seeded in a 6-
well plate. After overnight incubation, cells were resuspended in 1 mL of supplemented DMEM before
pretreated with various concentrations of samples for 2 h and lipopolysaccharide (LPS) was then added
and incubated for another 16 h. After that, the cell supernatants were collected to 1.5 mL microcentrifuge
tube as subjected to NO production analysis.
Determination of nitric oxide (NO) production The nitrite concentration in cell supernatant was
measured as an indicator of NO production following the Griess Reagent Kit (Molecular Probes, Inc,
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Eugene, OR). Briefly, 150 µL of each supernatant was combined with 20 µL of Griess reagent (1%
sulfanilamide in 5% phosphoric and 0.1% naphthylethylenediamine dihydrochloride in water) and
distilled water 130 µL in a 96-well plate, incubated at room temperature for 30 min. After incubation, the
absorbance of each well was determined at 548 nm using a microplate reader (Molecular Devices). The
amount of nitrite in samples was back-calculated from a sodium nitrite calibration curve (0-100 µM). The
percentage inhibition of NO production was calculated relative to control, i.e. LPS-stimulated untreated
cells. Diclofenac sodium (DCS) was used as a positive control.
Statistical analysis Data are expressed as mean ± standard deviation (S.D.) of at least three independent
experiments, which were performed in tripicate. Statistical analysis was determined using one-way
ANOVA followed by the Tukey’s test for multiple comparisons (Graphpad Prism 6.0, Graphpad Software
Inc., San Diego, USA). P-values less than or equal 0.05 were considered statistically significant.
RESULTS
Cell viability The results of the percentage cell viability are shown in Figure 1. It was shown that MSE at
concentrations of 20 and 40 μg/mL as well as oxyresveratrol at concentrations of 5 and 10 μg/mL did not
exhibit cytotoxicity to RAW 264.7 cells. The results of cell viability in all tested samples were > 90%.
Table 1 The percentage cell viability of RAW 264.7 cells following incubation with different concentrations of mulberry stem
extract (MSE) and oxyresveratrol (oxy) for 24 h
Treatments %Cell viability

Control 100.00 ± 0.00
MSE 20 μg/mL 98.05 ± 0.30
MSE 40 μg/mL 93.79 ± 1.62
oxy 5 μg/mL 99.91 ± 0.16
oxy 10 μg/mL 97.30 ± 2.58
Each value represents mean ± S.D., N=3 for all groups
Nitric oxide (NO) production LPS-stimulated murine macrophage RAW 264.7 cells model is known to
induce iNOS transcription, resulting in NO production. This cell model was therefore selected to evaluate
the effect of M. alba stem extract on the inhibition of NO production. The production of NO was
observed by measuring nitrite (NO 2 –) concentration. The results are shown in Figure 2. Diclofenac, a
positive control used in this study, was shown to have inhibitory effect on NO production. This result
is in agreement with the result of Bae et al.(9). Both MSE and oxyresveratrol at two different
concentrations significantly inhibited the production of NO in a dose-dependent manner compared with
LPS-stimulated untreated cells (control). As determined by High Pressure Liquid Chromatography
(HPLC), the percentage of oxyresveratrol in MSE used in this study was 17.87±0.61%(8). Thus, the
equivalent concentration of oxyresveratrol in MSE 20 and 40 µg/mL was 3.4 and 6.8 µg/mL,
respectively. Therefore, this study suggests that MSE possesses higher inhibitory effect of NO production
than oxyresveratrol.
DISCUSSION
MSE had no cytotoxicity to RAW 264.7 cells at the concentrations studied. It inhibited NO production
better than oxyresveratrol at an equivalent amount of oxyresveratrol. These results suggest that apart from
oxyresveratrol, the other components in MSE such as prenylated flavonoids (i.e. kuwanon E) may have
effect on the inhibition of NO production(10). In this study diclofenac was tested at the lowest
concentration, i.e. at 1 µg/mL and it was shown the strongest activities on the inhibition of NO
production. The comparable effect on the inhibition of NO production of MSE compared to diclofenac
was achieved at 40 µg/mL. However, as a bioactive natural product available in our country, it is still
valuable to be used as dietary supplement for reducing inflammation and pain in OA.

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Figure 2 The percentage inhibition on nitric oxide production in RAW 264.7 cells induced by LPS. RAW 264.7 cells (1.875 x 105
cells/mL) were treated with 100 ng/mL of LPS for 16 h after pretreating with mulberry stem extract (MSE) at concentrations of 20,
40 µg/mL, oxyresveratrol (oxy) at concentrations of 5, 10 µg/mL or diclofenac sodium (DCS) 1 µg/mL for 2 h. The cell supernatant
was then collected and the percentage inhibition on nitric oxide production was determined by Griess reaction assay. ****p<0.001
represents significant difference from LPS-stimulated untreated cells.
CONCLUSION
This study clearly demonstrates that mulberry stem ethanolic extract can inhibit NO production in LPS-
stimulated cells and has no effect on cell viability. Further investigations on the effect of MSE on the
inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 mRNA expressions are
ongoing.
ACKNOWLEDGMENTS
Financial support from the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Office of the
Higher Education Commission, Ministry of Education is grateful acknowledged. The authors would also
like to thank Naresuan University, Thailand for the research fund from the yearly budget (R2555B069)
and Queen Sirikit Sericulture Center (Tak) for plant materials.
REFERENCES
1. Xu W, Liu LZ, Loizido M, Ahmed M, Charles IG. 2002. The role of nitric oxide in cancer. Cell Res 12(5-
6): 311-20.
2. Scher JU, Pillinger MH, Abramson SB. 2007. Nitric oxide synthases and osteoarthritis. Curr Rheumatol
Rep 9: 9-15.
3. Amin AR and Abramson SB. 1998. The role of nitric oxide in articular cartilage breakdown in
osteoarthritis. Curr Opin Rheumatol 10(3):263-68.
4. Abramson SB. 2008. Nitric oxide in inflammation and pain associated with osteoarthritis. Arthritis Res
Ther 10:S2.
5. Chung KO, Kim BY, Lee MH, Kim YR, Chung HY, Park JH, et al. 2003. In-vitro and in-vivo anti-
inflammatory effect of oxyresveratrol from Morus alba L. J Pharm Pharmacol 55: 1695-700.
6. Thongsuk, P. 2007. In-vitro and clinical study of mulberry extract for skin whitening product. Master of
Sciences Naresuan University.
7. Khunakornvichaya A, Lekmeechai S, Pitaksuteepong T, Morales NP, Hemstapat W. 2012. Effect of Morus
alba L. Extract on Pain Associated with Osteoarthritis in Rats. Thai J Pharmacol 34(1): 121-25.
8. Soonthornsit N and Pitaksuteepong T. 2012. Microemulsion formulation containing mulberry stem extract.
Proceeding of the 38th Congress on Science and Technology of Thailand, 17-19 October, Empress
Convention centre, Chiang Mai, Thailand: G_G0053, pp 1-6.
9. Bae SH, Ryu YS, Hong JH, Park JC, Kim YM, Seok JH, Lee JH, Hur GM. 2001. Diclofenac inhibits IFN-
g plus lipopolysaccharide-induce iNOS gene expression via suppression of NF-kB activation in RAW 264.7
macrophages. Korean J Physiol Pharmacol 5: 521-27.
10. Yang ZG, Matsuzaki K, Takamatsu S, Kitanaka S. 2011. Inhibitory effects of constituents from Morus alba
var. multicaulis on differentiation of 3T3-L1 cells and nitric oxide production in RAW264.7 cells.
Molecules 16(7): 6010-22.

PILOT STUDY OF ANTIBACTERIAL AND ANTIINFLAMMATORY ACTIVITIES

OF INDIAN GOOSEBERRY (PHYLLANTHUS EMBLICA L. EXTRACT)
AGAINST BACTERIA ASSOCIATED WITH PITTED KERATOLYSIS
Rattanasiri Giwanon1, Krittiya Thisayakorn1, Pongsatorn Limsiriwong1, Saowaluck Ruengsri1, Vichein

Kheuynok1, Kanchana Sriyam1, Marudech Srisom1, Darunee Pahusee1, Sawai Nakakaew1, Natthachest
BP - 4
Ketmanee1, Sontaya Peungsumrong1 and Chulerattana Banchonglikitkul1
1
Pharmaceutical and Natural Product department (PNPD), Thailand Institute of Scientific and Technological Research (TISTR).
Technopolis, Pathum Thani, 12120.Thailand. E-mail address: rattanasiri@tistr.or.th
KEYWORDS: P. emblica L., K. sendentarius, pitted keratolysis, antibacterial, antiinflammatory
INTRODUCTION
Pitted keratolysis is superficial bacterial infection on the skin, especially the foot. This skin disorder is
characterized by malodour and erosions on the soles of the foot. The malodour is caused by the sulfur
byproducts of the bacteria. The causative bacteria is Kytococcus sedentarius. Phyllanthus emblica L., also
known as Indian gooseberry or Makham-pom in Thai is rich sources of bioactive constituents such as
polyphenols, gallic acids, alkaloids, tannin, saponin, flavonoids, essential oils and ellagic acids. The
ethanolic fruit extract shows pharmacological activities such as antimicrobial, anti-inflammatory,
antioxidative and antimutagenic activities. Some previous studies reported the potential antibacterial
activity of P. emblica L. against Micrococcus species. The study on antibacterial efficacies against pitted
keratolysis caused pathogens and antiinflammatory efficacies from P. emblica L. to use as an anti-foot
odor hygienic product should be accomplished. Therefore, the purpose of this study is to investigate
antibacterial potencies against bacterial associated with pitted keratolysis and antiinflammatory potencies
from P. emblica L. extract.

Plant material and Preparation of extracts 2 parts (seeds (MS) and pulp (MP)) of P. emblica L. were
collected from Nakhon Ratchasima province, Thailand. The pulps were separated from the seed and cut
into small pieces. Both parts were dried and were pulverized into fine powder by a grinder. All of these
were extracted with 95 % ethanol (w/v) for 48 h 3 times and were then evaporated. The yields of the 2
extracts (MS and MP) were 8.94 %(w/w) and 55.32 %(w/w), respectively.
Antibacterial method The antibacterial activities of the 2 extracts against 3 bacterial strains relevant to
pitted keratolysis (K. sedentarius ATCC 27573, K. sedentarius ATCC 27574 and K. sedentarius ATCC
27575) which provided from ATCC Culture Collection, were performed by agar diffusion and agar
dilution method.
The agar diffusion testing According to the Clinical and Laboratory Standards Institute (CLSI;
Document M2-A8, 2006), the diameters of inhibition zones (mm) of the 2 extracts against the 3 pitted
keratolysis associated pathogens were evaluated by agar diffusion method (20 mg/disc) using Muller
Hinton Agar (MHA). All experiments were carried out in duplicate. Student's t-Test was used for
comparison between the 2 extracts. Statistical significance was defined as p < 0.05.
The agar dilution testing According to CLSI standards (Document M7-A7, 2006), the minimum
inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) of the selected
extract were determined by agar dilution using MHA. No growth of the 3 dermatogens were observed as
MICs and MBCs. All experiments were performed in duplicate.
Antiinflammatory method (Preliminary study) The preliminary study of topical antiinflammatory
activities of the selected extract were assessed by ethyl phenylpropiolate (EPP)-induced rat ear edema
(Brattsand, et al.,1982).
Animals 15 male Wistar rats weighing 105 ± 10 g, obtaining from the National Laboratory Animal
Center, Thailand were housed under standard environmental conditions of temperature at 24±10 oC under

a 12 h dark-light cycle, and allowed free access for food and water. All animals were deprived of food
except water at least 16 h prior the experiments. The Animal Ethics Committee of TISTR approved all
experimental protocols.
Test drugs The inflammogen (EPP (Fluka Chemika,Switzerland)) was dissolved in 5 % (v/v) of acetone
(Merck, Germany). Phenylbutazone (Sigma, USA) was used as a standard anti-inflammatory drug.
Antiinflammatory testing (Preliminary study) Ear edema was induced in 15 male Wistar rats. The
BP - 4
administration of 1 % and 7.5 % (w/v) of the MP per ear was applied onto inner (10 µl/ear) and outer (10
µl/ear) of the ear 30 min prior to EPP induction. The control group received vehicle only. Ear thickness
was determined before and after treatment 30, 60 and 120 min using pocket thickness gauge after the
treatment. The anti-inflammatory activities were calculated at each time point as percent inhibition of
edema comparing with the vehicle-treated animals.
RESULTS
The 2 extracts of P. emblica L. revealed the antibacterial effects against all tested dermatogens. Both MP and
MS possessed antibacterial activities against the 3 K.sedentarius strains with their inhibition zones ranging
from 18.51-42.23 to 14.58-43.47 mm, respectively (p < 0.05). However, there was no significant
differences between these 2 extracts. Therefore, the higher yield of extraction of the MP was an index for
selecting to the further study (Table 1).
The MIC and the MBC value of the MP against the 3 K. sedentarius strains using agar dilution method was 10
mg/ml.
Both 1 % and 7.5 % (w/v) of the MP had tendency to inhibit the rat ear edema induced by EPP. (Figure 1).
Table 1 The comparison of inhibition zones between the 2 extracts from P. emblica L. (MP and MS) against the 3 K. sedentarius
strains, pitted keratolysis associated pathogens, using agar diffusion method (20 mg/disc)
Inhibition zones (diameter, mm ± SD) of

Bacterial strains
MP MS
K. sedentarius ATCC 27573 18.67 ± 0.22 14.72 ± 0.19
Figure 1 Preliminary study on anti-inflammatory activities of the MP by rat edema model
DISCUSSION
Among the tested extracts of P. emblica L, the MP possessed strong anti bacterial effect against all the 3
bacterial strains associated with pitted keratolysis. This result corresponded to some previous studies.

(Mayachiew, P and Devahastin, S., 2008, Dhale DA and Magle UP., 2011). The MP also had potent anti-
inflammatory activity in EPP-induced rat ear edema. The pulp of P. emblica L. revealed abundant
bioactive constituents such as alkaloids and phenolics. (Arunachalam M, et. al., 2011 and Rahman S, et.
al., 2009). The result suggested that the MP may potentially to be an anti-pitted keratolysis agent. For
further studies, its experimentally antiinflammatory study, active constituents identification and
toxicological effect of the MP should be conducted.
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CONCLUSIONS
The MP showed the tendency of antibacterial action against K. sedentarius, pitted keratolysis caused
pathogens and antiinflammatory action in EPP-induced rat ear edema. These findings indicate the
possibility to develop the MP as anti-pitted keratolysis agent from natural products.
ACKNOWLEDGEMENTS
We are grateful to Thailand Institute of Scientific and Technological Research (TISTR) for financial
supporting.
REFERENCES
1. Clinical and Laboratory Standards Institute (CLSI). 2006. Performance standards for antimicrobial
disk susceptibility tests: Approved Guideline . M2-A8. 9th ed. 26(1). USA;Wayne, Pa.
2. Clinical and Laboratory Standards Institute (CLSI). 2006. Method for dilution antimicrobial
susceptibility tests for bacteria that grow aerobically. Approved Guideline. M7-A7. 7th ed.
USA;Wayne, Pa.
3. Brattsand ,R., et al. 1982. Influence of 16α, 17α- acetal substitution and steroid nucleus
fluorination on the topical to systemic activity ratio of glucocorticoids. J. Steroid. Biochem.
16:779-786.
4. Mayachiew P and Devahastin S. 2008.Antimicrobial and antioxidant activities of Indian
gooseberry and galangal extracts. Food. Sci. Tech. 41:1153-1159
5. Dhale DA and Magle UP. 2011. Phytochemical screening and antibacterial activity of
Phyllanthus emblica (L.).Sci.Res.Rep. Nov;1(3): 138-142.
6. Arunachalam M, Shailja S and Sumeet KS. 2011. The anti-inflammatory potential of phenolic
compounds from Emblica officinalis L.in rat. Inflam. 19: 327-334.
7. Rahman S, Akbor M M, Howlader A and Jabbar A. 2009. Antimicrobial and cytotoxicity activity
of the alkaloids of Amlaki (Emblica officinalis). Pak.J.Bio.Sci. 12(6):1152-1155.

CHEMICAL PROFILE ANALYSIS AND ANTI-INFLAMMATORY ACTIVITY OF POLAR

FRACTION FROM SOYBEANS (GLYCINE MAX)
Siripen Jarikasem1, Amornrat Khayungarnnawee1, and Sarinthip Muensaen1
1
Pharmaceutical and Natural Products Department, Thailand Institute of Scientific and Technological Research,
BP - 5
Pathum Thani 12120, Thailand
KEYWORDS: Glycine max soybean, polar components, HPLC fingerprint, anti-inflammation
INTRODUCTION
Presently, phytochemicals from soybeans have received more attention. They are used as neutraceutical
ingredients due to their numerous health beneficial effects. Soy saponins, the main polar components,
which exhibited in vitro antioxidation and anti-inflammation (1-4), as well as cytotoxicity to several
cancer cell lines such as Hep-G2(5), Hela (6) and CaCo-2 (7-8) are among of interest. In this study, we
investigated on chemical profile of soybean’s polar fraction and its in vivo anti-inflammatory activity.
HPLC-PDA-ELSD was employed for the chemical fingerprint analysis.

Plant material: Soy beans were purchased from health food shop (Lemon green) in Bangkren District,
Bangkok, in October 2011. They were pulverized into coarse powder (20 mesh), then exhaustedly
soxhletted with hexane to give the defatted soy bean powder (DFP).
Extraction and fractionation DFP 1.1 kg were macerated with 11 L of 70% ethanol for 72 h with
occasional stirring, then filtered. After removal of ethanol under reduced pressure, 300 ml portion of
aqueous solution equivalent to 360 g of DFP was extracted with 3x200 ml of n-butanol. The combined n-
butanol solution was concentrated under reduced pressure to give 4.9 g of dried extract (BuFr).
HPLC analysis (9) BuFr was dissolved in methanol (1mg/ml) and PTFE 0.45 µm filtered, then subjected
to HPLC analysis. Confirmation of components was done by comparision of Rt and absorbance spectral
with reference standards.
Analysing condition
HPLC: Waters Pump600
Column: Nova-Pak C 18 (150 mm x 3.9 mm i.d., particle size 4 µm)
Mobile phase: 0.1% trifluoroacetic acid in water (A) and acetronitrile (B), gradient elution: 100A to 62A/
38B for 20 min, 62A/ 38B isocratic for 5 min, 62A/ 38B to 87A/ 13B for 25 min.
Flow rate: 0.8 ml/min
Inject volume: 10 µl
Detection: Photo diode array (PDA) at 205 nm and evaporative light scattering detector (ELSD) at probe
temperature 50oC, a gain of 6.0 and nebulizer nitrogen gas of 3.3±0.1 bar.
Reference standards:
Soy isoflavones: daidzin, glycitin,genistin, daidzein, glycitein, genistein (Chromadex USA)
Soy saponins: soyasaponin I (Chromadex USA)
Anti-inflammatory Test (10)

Animals Male Wistar rats were purchased from Laboratory Animal Centre, Mahidol University, Salaya,
Nakornprathom, Thailand. The animals were housed in the animal facility of Thailand Institute of
Scientific and Technological Research under standard conditions (25±2°C), 50-60% of humidity and 12
h/ 12 h light/dark cycles. The animals were kept under laboratory conditions for one week prior to the
start of the experiments. Food and water were allowed ad libitum.
Carrageenan induced paw oedema Rats weighing 80-100 g were divided in groups of six: vehicle
control (distilled water), positive control (diclofinac 50 mg/kg), test sample I (BuFr 100 mg/kg) and test

sample II (BuFr 200 mg/kg). At the beginning of the experiment, initial paw volumes were determined
using a water plethysmometer (Ugo Basil, Italy). Then, individual animal group orally received sample
accordingly. One hour after sample administration, paw oedema was induced by injection of 0.1 ml of
carrageenan (λ-carrageenan, type IV, Sigma) diluted in saline in the right hind foot pad. Paw volumes
were determined at times 1, 2, and 3 h after oedema induction. The percentage of oedema inhibition was
calculated with reference to vehicle control group.
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Analysis of Data The results are expressed as means ± S.E.M. Differences in mean values between
groups were analyzed by a one-way analysis of variance (ANOVA).
RESULTS
HPLC analysis HPLC chromatogram of BuFr detected under PDA at 205 nm (Fig. 1D) showed the
presence of isoflavones daidzin, glycitin, genistin, daidzein, glycitein and genistein at Rt of 16.09, 16.90,
20.66, 29.70, 30.58 and 32.66, respectively as confirmed by Rt of standard isoflavones (Fig. 1A). Peak
corresponding to soyasaponin I was observed at Rt 39.28 confirmed by ELSD (Fig. 1B, Fig. 1C). Among
isoflavone constituents, genistein appeared to be the main component followed by genistin and daidzein,
respectively.
Figure 1 HPLC Chromatograms of polar fraction from soybeans (BuFr) and reference standards.
(A) standard isoflavones, PDA 205 nm, (B) soyasaponin I, ELSD, (C) BuFr, ELSD, (D) BuFr, PDA 205nm.
Anti-inflammatory activity BuFr at 100 and 200 mg/kg p.o. exhibited oedema inhibitory effect at time 2
and 3 h similar to that of diclofinac 50 mg/kg (Table 1). However, the effect seemed to be less potent and
less lasting than diclofinac. Maximum effect of BuFr was observed at 2h while that of diclofinac appeared
to be not less than 3 h. Interestingly, the anti oedema effect of BuFr was dose dependent manner at time 2
and 3 h.

Table 1 Oedema inhibitory effect of polar fraction, BuFr from soybeans tested by carrageenan induced paw oedema in rat.
Samples % Oedema Oedema inhibition (%)

1h 2h 3h 1h 2h 3h
Control 31.06±2.27 63.40±7.84 76.17±7.24 - - -
Diclofenac 23.44±1.93* 20.92±3.60* 19.53±3.36* 24.00 64.00* 68.00*
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50 mg/kg
BuFr 38.66±2.24* 22.25±3.73* 23.78±3.81* 6.00 40.00* 23.00*
100 mg/kg
BuFr 25.68±3.07* 31.23±3.21* 49.15±4.07* 9.00 51.00* 41.00*
200 mg/kg
n = 6, *significant from control, P < 0.05

% Swelling = (T t - T 0 ) x 100
T0
T0 = Paw volume before induced oedema
Tt = Paw volume after sample application and induced oedema
DISCUSSION
Chemical profile of polar fraction from defatted soybeans showed the presence of the representative
phytochemicals including isoflavones and saponins. Seven components could be characterized using
HPLC-PDA-ELSD including daidzin, glycitin, genistin, daidzein, glycitein, genistein and soyasaponin I.
Structurally, there have been no UV active chromophores in saponin molecule. Thus, PDA detector at 205
nm gave a low intensity peak of soyasaponin I while the saponin selective ELSD detector gave higher
intensity peak. In considering only saponin components, ELSD is recommended.
In vivo anti-inflammatory activity was selected for our preliminary pharmacological assessment of BuFr
since many pathological disorders are associated with inflammation. BuFr at 100-200 mg/kg exhibited a
moderate anti-inflammatory activity compared to the standard anti-inflammatory drug, diclofinac at 50
mg/kg. The result is in accordance with its chemical profile and previous in vitro anti-inflammatory
activity (1-2). So BuFr is interesting to be used as a natural nutraceutical ingredient for anti-inflammation
purpose.
CONCLUSION
n-Butanol fraction from the 70% aqueous ethanolic extract from defatted soybeans comprised both
isoflavones and saponins detected by HPLC-PDA-ELSD. Seven components could be characterized from
HPLC-PDA profile at 205 nm including daidzin, glycitin, genistin, daidzein, glycitein, genistein and
soyasaponin I. The fraction given orally at 100 and 200 mg/kg showed in vivo anti-inflammatory effect
tested by carrageenan induced rat paw oedema.
ACKNOWLEDGMENTS
This work was supported grants from Thailand Institute of Scientific and Technological Research,
Ministry of Science and Technology.
REFERENCES
1. LY Z, et al. 2011. Anti-inflammatory effect of soyasaponins through suppressing nitric oxide

production in LPS-stimulated RAW 264.7 cells by attenuation of NF-κB-mediated nitric oxide
synthase expression. Bioorg Med Chem Lett. 15;2(8): 2415-8.
2. Kang JH, et al. 2005. Soybean saponins suppress the release of proinflammatory mediators by
LPS-stimulated peritoneal macrophages. Cancer Lett. 18;230(2): 219-27.

3. Lee IA, Park YJ, Joh EH and Kim DH. 2011. Soyasaponin Ab ameliorates colitis by inhibiting
the binding of lipopolysaccharide (LPS) to Toll-like receptor (TLR) 4 on macrophages. J Agric
Food Chem. 28;59(24):13165-72.
4. Lee IA, Park YJ, Yeo HK, Han MJ and Kim DH. 2010. Soyasaponin I Attenuates TNBS-
Induced Colitis in Mice by Inhibiting NF-κB Pathway. J Agric Food Chem. 58(20): 10929-34.
5. Zhang W, Popovich DG. 2008. Effect of soyasapogenol A and soyasapogenol B concentrated
extracts on HEP-G2 cell proliferation and apoptosis. J Agric Food Chem 56(8):2603-8.
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6. Xiao JX, et al. 2007. Morphological study on apoptosis Hela cells induced by soyasaponins.
Toxicol In Vitro. 21(5): 820-6.KIM HY. 2004. Antiproliferative crude soy saponin extract
modulates the expression of iKBa, protein kinase C, and cyclooxygenase-2 in human colon
cancer cells. Cancer letters 210:(1)1: 1-6.
7. Ellington AA, Berhow M and Singletary KW. 2005. Induction of macroautophagy in human
colon cancer cells by soybean B-group triterpenoid saponins. Carcinogenesis 26(1): 159-67.
8. Kang JH, et al. 2008. Soybean saponin inhibit tumor cell metastasis by modulating expressions
of MMP-2, MMP-9 and TIMP-2. Cancer letters 261:1(8): 84-92.
9. Hamburger M, Slacanin I and Hostettmann K. 1992. Acelyted saponins with molluscicidal
activity from Sapindus rarak:Unambiguous structure determination by proton nuclear magnetic
resonance and quantitative analysis. Phytochemical analysis 3:231-237.
10. Winter CA, Edwin A, Risley and Nuss GW. 1962. Carrageenan-induced edema in hind paw of
the rat as an assay for anti-inflammatory drugs. Proc. Soc. Exp. Biol. 111: 544-547.

ALIGNED ELECTROSPUN CHITOSAN/POLY ε-CAPROLACTONE BLENDED SCAFFOLD:

ANTI-INFLAMMATORY EFFECT
Natthan Charernsriwilaiwat1,2*, Louise Carney3, Brendan Robb3, Praneet Opanasopit2,

Theerasak Rojanarata2 and Sandra Downes3
1
Faculty of Pharmaceutical Sciences, Burapha University, Chonburi 20131, Thailand.
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2
Pharmaceutical Development of Green Innovations Group (PDGIG), Faculty of Pharmacy,
Silpakorn University, Nakhon Pathom 73000, Thailand.
3
School of Materials, Biomaterials Group, The University of Manchester, Manchester, M1 7HS, UK
KEYWORDS: Electrospinning, Chitosan, Poly ε-caprolactone, Tissue engineering
INTRODUCTION
Chitosan has been used to fabricate the electrospun nanofiber mats for tissue engineering. However, the
chitosan electrospun scaffold is very fragile, strongness not enough to support cell growth and
differentiation. Poly ε-caprolactone (PCL) was selected as a blending polymer due to their compensatory
characteristics such as slow degradation and good mechanical properties. The electrospun chitosan/ PCL
nanofibres has recently been investigated1-4). These studies reported the potential of chitosan/PCL
nanofibers for biomedical applications. However, the inflammatory response to chitosan/PCL nanofiber
was limit studied. Chitosan has been reported the inhibitory effect on reactive oxygen species (ROS)5) and
inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-
6 (IL-6). Therefore, the aim of this study was to investigate the effect of chitosan/PCL aligned nanofiber
scaffolds on inflammatory response to macrophage cells. The aligned nanofiber scaffolds were prepared
by electrospinning of chitosan/PCL solutions with different volume ratios. The morphology and degree of
alignment of nanofiber scaffolds were characterized using scanning electron microscope (SEM). The cell
morphology was examined by SEM. Cell proliferation was determined by DNA assay using 3T3
fibroblast cells. The effects of chitosan/PCL aligned nanofiber scaffolds on inflammatory response were
investigated by dichlorofluorecein assay using J774A.1 macrophages cells.

Materials Chitosan (degree of deacethylation = 0.85, molecular weight = 110 kDa), poly(ε-caprolactone)
(PCL) (Mn 80000), 3T3 swiss albino fibroblast cells were purchased from American type culture
collection (ATCC). J774A.1 mouse BALB/c monocyte macrophage cells were obtained from European
collection of cell cultures (ECACC). All other reagents and solvents were of analytical grade.
Preparation of Chitosan/PCL solution and electrospinning Chitosan (8% w/v) was dissolved in a
mixture of trifluoroacetic acid (TFA) and dichloromethane (DCM) (75:25 v/v). PCL (30% w/v) was
dissolved in trifluoroethanol (TFE). The 8% chitosan solution was mixed with 30% PCL solution at
volume ratio of 25/75. The solutions were taken up in a 5 ml plastic syringe connected with a 21 gauge,
stainless needle (diameter = 0.8 mm) at the nozzle. The needle was connected to the high voltage DC
power supply of a Glassman High Voltage device. The electric potential was fixed at 15 kV. The
nanofibers were collected on a rotating collector with rotation speed at 1200 rpm. The solution feed was
driven by a syringe pump, and the rate was fixed at 0.25 ml/h during spinning. The collection distance
was fixed at approximately 15 cm. The process duration was fixed at 2 h to provide fibrous meshes with
the same thickness. Prior to cell seeding the electrospun mats were cut to fit into a sterile 24 well plate
Scaffdex well insert (C00002S, Scaffdex, Tampere, Finland) to hold its at the bottom of the well plate,
and sterilized under UV light in laminar flow hood for 30 minutes on either side.
Fiber characterization The morphologies of chitosan/PCL nanofibre scaffolds were perform using
scanning electron microscope (SEM; Phenom G2 pro, Eindhoven, Netherlands). Randomly selected
areas of the fiber covered foil were cut into squares and mounted onto carbon coated stubs (Agar
Scientific) and gold sputter-coated for a total of 2 min (Edwards Sputter Coater–S150B). Fiber diameter
and angles for alignment were determined from the SEM images using the Fibermetric software (n=300,
100 fiber measurements from 3 different SEM images).
Determination of 3T3 cell proliferation For 3T3 cell viability and morphology studies cells were seeded
at 25000 cells/ml. The 3T3 fibroblast cells viability was evaluated by DNA assay at 3, 5 and 7 days after
seeding. DNA assay was performed to quantify the number of cells on the nanofibrous scaffold. At each
time point, cells were washed in PBS, covered with distilled water and frozen at - 80 °C. Cells were
frozen and thawed three times and aliquots of 50 μl (in triplicate) were moved into a clear flat bottom 96-

well plate. Fifty μl of TNE buffer (10 mM Tris, 2M NaCl, 1 mM EDTA, pH 7.4) and 100 μl of Hoechst
stain solution (stock solution: 1mg/ml Hoechst 33258 in water; dilution 1:50 in TNE buffer) were added
to each well. The number of cells was calculated base on the fluorescence at 355 nm excitation and 460
nm emission using a fluorescence plate reader. Data were expressed as cell number.
Determination of 3T3 cell morphology At day 7, cell seeded samples were fixed with 1.5%
glutaraldehyde for 30 minutes at 4ºC and rinsed in 0.1M phosphate buffer. Cellular samples were
dehydrated using a series of increasing ethanol concentration (50, 70, 90 and 100% v/v) incubations
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followed by an overnight incubation in hexamethyldisilasane (HMDS). Samples were mounted on carbon

tabs and gold sputter coated for 2 minutes. The morphology of cell attach on nanofibers scaffolds were
observed under SEM.
Determination of murine macrophage response For dichlorofluorecein assay, J774A.1 macrophages
cells were seeded at 50000 cells/ml. The inflammatory response to J774A.1 macrophages cells was
evaluated at 2, 4, 8, 24 and 48 hours after seeding. Dichlorofluorecein assay was used to determine to
peroxide released in medium when J774A.1 macrophages cells contract to different scaffolds. 2,7
Dichlorofluorescin diacetate was dissolved in fresh methanol at 2mM and incubated at room temperature
for 20 min in the dark. The cells were seeded in dilute 2mM DCF 1:100 in Hank's Balanced Salt Solution.
At each time point, transfer 100µl aliquots of cell media into the black well plates. The peroxide release
(%) was calculated base on the fluorescence at 485 excitations and 530 emissions using fluorescence plate
reader compare with control (copper discs).
Statistical analysis All experiment data were collected from triplicate samples and are expressed as the
mean ± standard deviation (S.D.). Statistically significant differences were analysed using the Student’s t-
test. The significance level was set at P < 0.05.

Electrospinning and morphology The SEM images of chitosan/PCL aligned nanofiber scaffolds with
different volume ratios are shown in Figure 1. The average diameters of PCL and 25/75 chitosan/PCL
were 519.65 ± 172.15 and 406.37 ± 98.71 nm. The diameter distribution and degree of aligment of
chitosan/PCL aligned nanofiber scaffolds are also shown in Figure 1. The average diameter was in
nanometer range and fibers were smooth. The diameters of fiber decrease when the chitosan was
incorporated into nanofiber. Chitosan is an ionic polyelectrolyte, causing a higher charge density on the
surface of the ejected jet that is formed during electrospinning. Thus, as the charge density increases, the
diameter of the final fibers becomes smaller6). The alignment of pure PCL fiber was higher than the
blended fiber. This result may be because the properties of blend polymer altered which effect to fiber
formation during electrospinning process.
Figure 1 SEM images, diameter distributions and degree of alignments of aligned nanofiber scaffolds PCL (a) and 25/75
chitosan/PCL (b).
Cell morphology and proliferation 3T3 fibroblast cells cultured on the chitosan/PCL aligned nanofiber
scaffolds for 7 days were examined with SEM. Figure 2 shows the 3T3 fibroblast cells grown on glass
control, PCL and 25/75 chitosan/PCL scaffolds. The SEM images indicated that 3T3 fibroblast were well
attached to both scaffold and showed that the cells on the glass exhibited a stellate-patterned phenotype

(Fig. 2a). Whereas, cells exhibited the fibroblastic phenotype on the chitosan/PCL aligned nanofibers,
cells gradually more elongated, presenting a spindle-shaped morphology spreading parallel to the fibers
(Fig. 2b and c). Figure 3 shows the cell number on scaffolds through DNA assay. After 5 days culture, the
cell number of cells evaluated on PCL and 25/75 chitosan/PCL aligned nanafiber scaffold was higher
compared to control (P<0.05). There were also no significant differences between both scaffolds in cell
number. The results of cell proliferation and cell number were in the same way. These indicated that the
chitosan/PCL was non toxic to 3T3 fibroblast cells and promote the cell growth.
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Figure 2 SEM images of 3T3 fibroblast cells on glass control (a), PCL and (b) 25/75 chitosan/PCL (c) aligned nanofiber scaffolds
after 7 days of cell culture.
Response to macrophage cell Dichlorofluorecein assay was used to determine to peroxide released in
medium when J774A.1 macrophages cells contract to different scaffolds. Figure 4 shows the peroxide
release (%) at each time of PCL, 25/75 chitosan/PCL aligned nanofiber scaffolds and control from
J774A.1 macrophages cells. This result shows the statistical different between peroxide level of the 25/75
chitosan/PCL with the PCL and control. The % peroxide release of the PCL and control was around
100% on the on other hand the 25/75 chitosan/PCL was around 40% and decrease to 20% in 2 day.
Chitosan has been reported the inhibitory effect on reactive oxygen species (ROS)5). These result
indicated that the chitosan in scaffolds can reduce peroxide release from J774A.1 macrophages cells.
Figure 3 Cell number of 3T3 fibroblast cells on control (), PCL Figure 4 The percentage peroxide release from J774A.1
() and 25/75 chitosan/PCL () aligned nanofiber macrophage cells with control (), PCL () and 25/75
scaffolds.*Statistically significant (P<0.05). chitosan/PCL () aligned nanofiber scaffolds.
CONCLUSION
The chitosan/PCL aligned nanofiber scaffolds were successfully prepared using the electrospinning
process. These fibers were aligned and were in the nanometer range. The chitosan/PCL aligned nanofiber
scaffolds showed well compatibility with 3T3 fibroblast cells. The scaffolds exhibited inhibitory effect on
peroxide release from J774A.1 macrophages cells. These biodegradable, biocompatible and anti-
inflammatory aligned nanofiber scaffolds have promising potential for tissue engineering application.

ACKNOWLEDGEMENTS
The authors wish to thank the Thailand Research Funds and Biomaterials group, University of
Manchester for financial support.
REFERENCES
1. Cooper A, Bhattarai N, Zhang M. 2011. Fabrication and cellular compatibility of aligned
chitosan–PCL fibers for nerve tissue regeneration. Carbohydr Polym 85:149-156.
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2. Shalumon KT, Anulekha KH, Girish CM, Prasanth R, Nair SV, Jayakumar R. 2010. Single step
electrospinning of chitosan/poly(caprolactone) nanofibers using formic acid/acetone solvent
mixture. Carbohydr Polym 80: 413-419.
3. Schueren LVD, Steyaert I, Schoenmaker BD, Clerck KD. 2012. Polycaprolactone/chitosan blend
nanofibres electrospun from an acetic acid/formic acid solvent system. Carbohydr Polym 88:
1221-1226.
4. Bhattarai N, Li Z, Gunn J, Leung M, Cooper A, Edmondson D, Veiseh O, Chen M-H, Zhang Y,
Ellenbogen RG, Zhang M. 2009. Natural-synthetic polyblend nanofibers for biomedical
applications. Adv Mater 21: 2792-2797.
5. Liu H-T, Li W-M, Xu G, Li X-Y, Bai X-F, Wei P, Yu C, Du Y-G. 2009. Chitosan
oligosaccharides attenuate hydrogen peroxide-induced stress injury in human umbilical vein
endothelial cells. Pharmacol Res 59: 167-175.
6. Charernsriwilaiwat N, Opanasopit P, Rojanarata T, Ngawhirunpat T, Supaphol P. 2010.
Preparation and characterization of chitosan-hydroxybenzotriazole/polyvinyl alcohol blend
nanofibers by the electrospinning technique. Carbohydr Polym 81: 675-680.

ANTIBACTERIAL ACTIVITY OF α-MANGOSTIN FROM THE PERICARP EXTRACT

OF GARCINIA MANGOSTANA L. AGAINST DRUG RESISTANT BACTERIA
Griangsak Eumkeb1, Sineewan Phitaktim1 and Yothin Teethaisong1*

1
School of Pharmacology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.
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KEYWORDS: Alpha-mangostin, Garcinia mangostana, Antibacterial activity, Resistant bacteria
INTRODUCTION
In the recent years, incidence of multidrug resistance in pathogenic and opportunistic bacteria has been
increasingly documented. These bacteria pose life-threatening risks to the hospitalized patients and their
care givers1). Enterobacter cloacae are significant cause of nosocomial infections, mainly causes of
pneumonia, wound infection and urinary tract infection in the most hospital. Antimicrobial resistance in
this strain has increasingly emerged, including resistant to aminopenicillins, cefazolin, and cefoxitin due
to the production of amp C β-lactamases2). Similarly, the emergence of fecal Escherichia coli isolates
exhibiting resistance to extended-spectrum cephalosporins has been reported among pigs in Spain3).
Furthermore, 19% antibiotics resistance of E. coli in hospital sewage water was resistant to ampicillin,
ceftazidime, ceftriaxone, cefotaxime, chloramphenicol, ciprofloxacin, gentamicin, nalidixic acid and
trimethoprim-sulphamethoxazole among the resistant isolates from the wells4). Staphylococcus
saprophyticus is implicated in 10-20% of urinary tract infections (UTI), usually susceptible to antibiotics
commonly prescribed for patients with UTI. However, resistance of S. saprophyticus to penicillins such
as oxacillin has also been reported5). Thus, development of novel antibacterial agents that can reverse the
resistance to β-lactam antibiotics are research objectives of far reaching importance and urgently needed.
Mangosteen (Garcinia mangostana Linn.) (GML), belonging to the family Guttifera, has been used as a
traditional medicine in Southeast Asia for the treatment of diarrhea, inflammation, and chronic ulcers6) as
well as abdominal pain, dysentery, infected wound and suppuration7). The xanthone derivertives, α-
mangostin, is a major bioactive compound found in the pericarp of the mangosteen, has been discovered
to possess antimicrobial activities against Helicobacter pylori and Methicillin-resistant Staphylococucus
aureus (MRSA), anti-inflammatory activities, inhibition of oxidative damage8, 9). However, no works
have been investigated the effect of GML extract on some drug resistant bacteria such as S.
saprophyticus, E. cloacae and E. coli. The purpose of this investigation was to examine antibacterial
activity of bioactive compounds from the pericarp of GML extract against those drug resistant bacteria,
when used alone and in combination with β-lactam antibiotics.

Isolation and purification of α-mangostin α-mangostin from pericarp of GML was isolated and purified
according to previous methods10, 11) with some modifications. The dichloromethane crude extract was
used to separate in silica gel column chromatography to yield 11 fractions. Fraction 3 was subjected to
HPLC and purified using preparative thin layer chromatography to obtain the major compound. This
compound was elucidated the chemical structure by NMR and structure spectrum data was compared with
previously reported12). The structure of α-mangostin is shown in figure 1.
Bacterial strains and antibiotics Clinical isolates of S. saprophyticus DMST 27055, E. cloacae DMST
21394 and E. coli DMST 19629 were obtained from Department of Medical Science, National Institute of
Health, Ministry of Public Health, Thailand. The reference strains S. aureus ATCC 29213 and E. coli
ATCC 25922 from the American Type Culture Collection were used as control. Oxacillin and ceftazidime
were obtained from Sigma.
Bacterial suspension standard curve In order to select bacterial suspensions with a known viable count,
the method of Liu et al13) was followed. Mueller-Hinton agar and Cation-adjusted Mueller-Hinton broth
(CAMHB) were used as medium.
Minimum inhibitory concentration determination (MIC) To determine MICs of α-mangostin and
antibiotics against these strains, the standard agar dilution method was performed. Briefly, bacterial
suspension was adjusted to approximately 1 x 108 CFU/ml. Inoculum (0.1 ml) of each stain was added to
0.9 ml MHB, plus serial dilutions of the antibacterial agents, to give approximately 1x107 CFU/ml.
Antibacterial-free tubes were use as control. Aliquots (2µl) of each inoculum were spotted on agar surface
(the final concentration was approximately 1 x 104 CFU/ml). Then, agar plates were incubated at 37 ºC
for 24 h. The MICs was defined as the lowest concentration of antibiotic at which there is no visible
growth in the triplicate spots13, 14).

Checkerboard determination Combinations of antibacterial agents were performed using checkerboard

assay in order to examine synergistic antibacterial activity of the extract and antibiotics. Cultured bacteria
and antibacterial agents were prepared and performed similar to previously described in MIC
determination. Differently, in this method, combinations of antibacterial agents plus GML extract in each
spot were employed. The interactions between antibacterial agents and these extracts were determined by
the fractional inhibitory concentration (FIC). The FIC index (FICI) was calculated and interpreted in
accordance with Odds’s described: FICI ≤ 0.5 denoting synergistic; FICI > 0.5–4.0 denoting no
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interaction; FICI > 4.0 denoting antagonism15).

Killing curve determinations (Viable counts) Viable counts for the determination of killing curve was
performed as previously described16) with slightly modifications. Inocula 5 x 106 CFU/ml were exposed to
antibacterial agents at 0.5,1,2,3,4,5,6,24 h. The cultures at each exposed time were plated on MHA in
quadruplicate. Then, incubation at 37 ºC for 24 h. Finally, these plates were allowed to count bacterial
growth.
Transmission electronmicroscopy (TEM) The combination of oxacillin plus GML extract that exhibited
synergistic effect against oxacillin-resistant S. saprophyticus was chosen to investigate primarily
mechanism of action by TEM. Oxacillin either alone or in combination with these extracts were prepared
for TEM following previously described12).
RESULTS AND DICUSSION

MIC and checkerboard determinations The MICs of CH 2 Cl 2 crude extract, Fr 3 , and α-mangostin against
clinical isolates of oxacillin-resistant S. saprophyticus (ORSS) were 50, 31 and 8 µg/ml, respectively.
Whereas, the MICs of those GML extracts against both clinical isolates of ceftazidime-resistant E. coli
and ceftazidime-resistant E. cloacae were the same value at >10,000, >10,000 and >1,024 µg/ml,
respectively (Table 1). These results indicated that the bioactive compounds exhibit higher potency
against ORSS than oxacillin alone but no effect on those of E. coli and E. cloacae, which are gram
negative bacteria and have a multi-layered and complex structure. The outer membrane can act as a
barrier to many environmental substances including antibiotics17). The results of checkerboard
determinations demonstrated that MICs of CH 2 Cl 2 crude extract, Fr 3 , and α-mangostin in combinations
with oxacillin against ORSS were dramatically decreased, exhibiting the synergistic effect at FICI 0.25,
0.138, and 0.375, respectively (Table 1). These results indicate that the bioactive compounds can reverse
the resistant strain of ORSS to its primary susceptible antibiotic.
Killing curve assay The control showed no reduction in the counts of CFU from control inoculum. The
results showed that the combination of the bioactive compounds (CH 2 Cl 2 crude extract, Fr 3 extract, and
α-mangostin) and oxacillin caused a reduction of 5 Х 105 CFU/ml of ORSS to 103 CFU/ml within 6 h and
throughout the remainder of a 24 h (Figure 2). These results provide evidence that bioactive compounds
in combination with oxacillin have synergistic activity. The results of this study seem consistent with
earlier findings that Ceftazidime at 5 µg/ml in combination with 5 µg/ml of tested flavonoids reduced the
CFU/mL of MRSA strain by 5x103 over 6 h. The reduced counts did not recover in 24 h18).
14 15
13
12 19
11 O OH
8 9 1 16
H3CO 8a 9a 18
7 2 20
17
3
6 4a
HO 10a O OH
5 4
Fig. 1 The structure of α-mangostin
TEM study Electronmicroscope investigations clearly exhibited that the combination of oxacillin with α-
mangostin from the pericarp of GML markedly showed great synergism activity against ORSS and
caused damage to ultrastructures of this strain, the majority of these cells undoubtedly exhibited cell
shape and cell envelope damage (Figure 3D). Similarly, most of these cells exposed to α-mangostin alone
were considerably smaller than those of control cells and exhibited cell envelope damage and
morphological change (Figure 3C). Whereas, cells treated with oxacillin alone seemed slightly smaller

than control cells (Figure 3B). The results of TEM in this investigation suggest that oxacillin had weaker
activity against ORSS. Whereas, biochemical compounds of GML extracts showed rather higher potency
than oxacillin against this strain. These results are consistent with previously reported that TEM clearly
showed damage to the ultrastructures of MRSA strain after exposure to the combination of galangin and
ceftazidime18). Similarly, TEM study clearly demonstrated that galangal extract caused both outer, inner
membrane damage and cytoplasm coagulation of S. aureus strain19).
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Table 1 MICs of bioactive compounds from the pericarp of GML extract when used singly and in combination with antibiotics
against drug resistant bacteria
MIC
MIC alone FIC
Strains agents Combination Outcome
(µg/ml) index
(µg/ml)
S. saprophyticus Oxacillin 128 16
DMST 27055 Crude extract 50 6.25 0.25 synergism
Oxacillin 128 16
Fr 3 31 3.875 0.138 synergism
Oxacillin 128 16
α-mangostin 8 2 0.375 synergism
E. coli DMST 19629 Ceftazidime >1,024 >1,024

Crude extract >10,000 >10,000 >2 no interaction
Ceftazidime >1,024 >1,024
Fr 3 >10,000 >10,000 >2 no interaction
α-mangostin >1,024 >1,024 >2 no interaction
E. cloacae DMST 21394 Ceftazidime >1,024 >1,024

Crude extract >10,000 >10,000 >2 no interaction
Fr 3 >10,000 >10,000 >2 no interaction

α-mangostin >1,024 >1,024 >2 no interaction
S. aureus ATCC 29213 Oxacillin 2 NT NT NT

E. coli ATCC 25922 Ceftazidime 8 NT NT NT
FICI ≤ 0.5 denoting synergistic; FICI > 0.5–4.0 denoting no interaction; FICI > 4.0 denoting antagonism; NT: not test; the
references S. aureus ATCC 29213 and E. coli ATCC 25922 were used as control strains.
1011
1010
109
108
Viable count (cfu/ml)
107
cont rol
oxacillin (32)
106 -mangost in (1.2)
Fr 3 (2.5)
crude ext ract (6.25)
105 oxacillin (8)+ -mangost in (1)
oxacillin (8)+Fr 3 (2)
oxacillin (8)+crude ext ract (3)
104
103
102
0 1 2 3 4 5 6 7 23 24 25
Time (h)
Fig. 2 The effect of oxacillin combined with bioactive compounds from the pericarp of GML extract on the clinical isolates of
oxacillin-resistant S. saprophyticus DMST 27055 (ORSS). Symbol represents: (●) control (antibacterial free); (○) oxacillin (32
µg/ml); (▼)α-mangostin (1.2 µg/ml) ;(▲) Fr3 (2.5 µg/ml); (■) crude extract (6.25 µg/ml);(■) oxaxillin (8 µg/ml)+ mangostin
(1µg/ml); (♦)oxaxillin (8 µg/ml) + Fr3 (2 µg/ml); (♦)oxaxillin (8 µg) + Fr3 (3 µg/ml). The values plotted are the means of 3
observations, and the vertical bars indicate the standard errors of the means

a A b B
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c C d D
Fig. 3 Ultrathin sections of log phase clinical isolates of oxacillin-resistant S. saprophyticus DMST 27055 (ORSS) grown in MHB:
Control (A: bar = 1µm, x4,000; a: bar=500 nm,x15,000); 32 µg/ml oxaxillin (B:bar=1µm, x4,000; b: bar=200 nm, x19,500); 1.2
µg/ml α-mangostin (C: bar= 500 nm, x9,900; c: bar 200 nm, x29,000); 8 µg/ml oxacillin plus 1 µg/ml α-mangostin (D: bar=1µm,
x5,000 ; d: bar = 200 nm, x29,000).
CONCLUSION
The bioactive compounds from the pericarp of GML, especially α-mangostin, possess a potential
antibacterial agent against clinical isolates of ORSS. Furthermore, the great synergistic activity between
this compound and oxacillin against this strain was occurred. Our findings provide evidence that these
GML extract compounds can reverse bacterial resistance to sensitive status for oxacillin. Interestingly, the
antibacterial combination approach is an interesting avenue to combat drug resistant bacteria. This study
provides evidence that the bioactive compounds from the pericarp of GML extract offer for the
development of a valuable adjunct to oxacillin against ORSS, which currently almost penicillins
resistance.
ACKNOWLEDGEMENTS
The authors are indebted and highly appreciative to the National Research Council of Thailand for
invaluable assistant in research funds support.
REFERENCES
1. Jones ME, Draghi DC, Thornsberry C, Karlowsky JA, Sahm DF, Wenzel RP. 2004. Emerging
resistance among bacterial pathogens in the intensive care unit--a European and North American
Surveillance study (2000-2002). Ann Clin Microbiol Antimicrob 3(14).
2. Zhou Q, Zhang, M., Wang, A., Xu, J., Yuan, Y.,. 2012. Eight-Year Surveillance of
Antimicrobial Resistance among Enterobacter Cloacae Isolated in the First Bethune Hospital.
Physics Procedia 33(1194-96.
3. Escudero EV, L. Teshager, T. Torres, C. Moreno, M.A. 2010. Resistance mechanisms and farm-
level distribution of fecal Escherichia coli isolates resistant to extended-spectrum cephalosporins
in pigs in Spain. Res Vet Sci 88(1):83-87.
4. Amaya E, Reyes D, Paniagua M, Calderon S, Rashid MU, Colque P, et al. 2012. Antibiotic
resistance patterns of Escherichia coli isolates from different aquatic environmental sources in
Leon, Nicaragua. Clinical microbiology and infection : the official publication of the European
Society of Clinical Microbiology and Infectious Diseases.
5. Maharat Nakhonratchasima hospital. 2012. Microbiology Report: Antibiotic Resistance Profile
and Prevalence of Isolated Organisms. Department of Clinical Pathology,Maharat Nakhon
Ratchasima hospital, Nakhon Ratchasima, Thailand

6. Suksamrarn S, Komutiban O, Ratananukul P, Chimnoi N, Lartpornmatulee N, Suksamrarn A.

2006. Cytotoxic prenylated xanthones from the young fruit of Garcinia mangostana. Chem
Pharm Bull (tokyo) 54(3):301-5.
7. Pedraza-Chaverri J, Cardenas-Rodriguez N, Orozco-Ibarra M, Perez-Rojas JM. 2008. Medicinal
properties of mangosteen (Garcinia mangostana). Food Chem Toxicol 46(10):3227-39.
8. Iikubo K, Ishikawa Y, Ando N, Umezawa K, Nishiyama S. 2002. The first direct synthesis of α-
mangostin, a potent inhibitor of the acidic sphingomyelinase. Tetrahedron Lett 43(2):291-93.
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9. Chomnawang MT, Surassmo S, Wongsariya K, Bunyapraphatsara N. 2009. Antibacterial
Activity of Thai Medicinal Plants against Methicillin-resistant Staphylococcus aureus.
Fitoterapia 80(2):102-04.
10. Chairungsrilerd N, Takeuchi K, Ohizumi Y, Nozoe S, Ohta T. 1996. Mangostanol, a phenyl
xanthone from Garcina mangostana. Phytochemistry 43(5):1099-102.
11. Sakagami Y, Iinuma M, Piyasena KG, Dharmaratne HR. 2005. Antibacterial activity of alpha-
mangostin against vancomycin resistant Enterococci (VRE) and synergism with antibiotics.
Phytomedicine 12(3):203-8.
12. Ee GC, Daud S, Taufiq-Yap YH, Ismail NH, Rahmani M. 2006. Xanthones from Garcinia
mangostana (Guttiferae). Nat Prod Res 20(12):1067-73.
13. Liu IX, Durham DG, Richards RM. 2000. Baicalin synergy with beta-lactam antibiotics against
methicillin-resistant Staphylococcus aureus and other beta-lactam-resistant strains of S. aureus. J
Pharm Pharmacol 52(3):361-6.
14. Clinical Laboratory Standards Institute. Method for dilution antimicrobial susceptibility tests for
bacteria that grow aerobically. In: Matthew AW, Franklin, R.C., William, A.C., Micheal, N.D.,
George, M.E., David W.H. Janet, F.H., Mary, J.F., Jana, M.S., Donal, E.L., Danie, J.S., Fred,
C.T., John, D.T., Melvin, P.W., & Barbara, L.Z., editor. CLSI document M7-A7. 2006. Seventh
Edition Method for dilution antimicrobial susceptibility tests for bacteria that grow aerobically.
Wayne, Pennsylvania: Clinical and Laboratory Standards Institute. p. 14-34.
15. Odds FC. 2003. Synergy, antagonism, and what the chequerboard puts between them. J
Antimicrob Chemother 52(1):1.
16. Richards RM, Xing JZ, Gregory DW, Marshall D. 1993. An electronmicroscope study of the
effect of sulphadiazine and trimethoprim on Enterobacter cloacae. J Med Microbiol 38(1):64-8.
17. Eumkeb G, Richards RME. 2004. Reversing b- Lactam Antibiotic Resistance in Gram- positive
bacteria by Some Flavonoids. Suranaree J Sci Technol 11(143-50.
18. Eumkeb G, Sakdarat S, Siriwong S. 2010. Reversing β-lactam antibiotic resistance of
Staphylococcus aureus with galangin from Alpinia officinarum Hance and synergism with
ceftazidime. Phytomedicine 18(1):40-45.
19. Oonmetta-aree J, Suzuki T, Gasaluck P, Eumkeb G. 2006. Antimicrobial properties and action of
galangal (Alpinia galanga Linn.) on Staphylococcus aureus. LWT - Food Sci Technol
39(10):1214-20.

ANTI-OXIDATIVE STRESS ACTIVITY OF PHIKUD NAVAKOT EXTRACT

IN SACCHAROMYCES CEREVISIAE
Nongluksna Sriubolmas1, Uthai Sotanaphun2, Duangdeun Meksuriyen3 and Suthep Wiyakrutta4*

1
School of Pharmacy, Eastern Asia University, Pathum Thani 12110, Thailand.
2
Department of Pharmacognosy, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom 73000, Thailand.
3
Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences,
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Chulalongkorn University, Bangkok 10330, Thailand.

4
Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
KEYWORDS: Anti-oxidant, Hydrogen peroxide, Phikud Navakot, Traditional drug, Yeast
INTRODUCTION
Phikud Navakot is a polyherbal formula in Yahom included in List of Herbal Medicinal Products A.D.
2012. Yahom is primarily used as cardiotonic agent to increase cardiac output according to Thai
traditional knowledge. Scientific evidence demonstrating biological activity of Phikud Navakot has to be
evaluated. Phikud Navakot comprises an equal weight of nine crude drugs namely Kot Soa [root of
Angelica dahurica (Fisch.) Benth. & Hook. f.], Kot Khamao [rhizome of Atractylodes lancea (Thunb.)
DC.], Kot Hua Bua (rhizome of Ligusticum chuanxiong Hort.), Kot Chiang [root of Angelica sinensis
(Oliv.) Diels], Kot Chulalumpa (aerial part of Artemisia pallens Wall. ex Besser), Kot Kradook [rhizome
of Saussurea costus (Falc.) Lipsch.], Kot Kan Prao (rhizome of Picrorhiza kurrooa Royle ex Benth.), Kot
Pung Pla (gall of Terminalia chebula Retz.), and Kot Jatamansi [root and rhizome of Nardostachys
jatamansi (D. Don) DC.]. Active compounds isolated from these herbs were found to exhibit antioxidant
activity, for example, picroliv, picroside-I and kutkoside from P. kurrooa1, and ferulic acid from root of A.
sinensis2. Oxidative stress is one contributing factor in development of several pathophysiological
conditions including cardiovascular diseases, neurodegenerative diseases, cancer and aging3. Anti-
oxidative stress activity at the cellular level of Phikud Navakot should be determined to support its
traditional use. Saccharomyces cerevisiae is a model eukaryotic organism widely used in the study of
cellular processes4,5. The present study was aimed to investigate the effect of Phikud Navakot extract
against oxidative stress induced by hydrogen peroxide in yeast model.

Preparation of Phikud Navakot extract Each crude drug was ground into coarse powder. Equal amount
(weight) of the 9 crude drugs were combined and macerated in 10 times by weight of 80% ethanol for
overnight. Extraction was done by heating at 100 ºC for 3 h and the liquid phase was separated by
filtration. The solid phase was re-extracted with the same procedure. The two filtrates were combined and
the ethanol was removed by evaporation under reduced pressure to yield the concentrate Phikud Navakot
extract (NVK).
Preparation of yeast cells S. cerevisiae ATCC 26108 was grown in yeast peptone dextrose (YPD) broth
[1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose] at 30 ºC with 160 rpm shaking for 72 h.
Yeast cells were collected by centrifugation at 3,000 g for 3 min, washed 3 times with phosphate buffer
saline (PBS) and suspended in PBS to yield 2x108 CFU/mL. The yeast suspension was incubated at 30
ºC, 160 rpm for 1 h before determining the IC 50 value induced by H 2 O 2 and anti-oxidative stress analysis.
Determination of IC 50 of hydrogen peroxide The yeast suspension was diluted in PBS to yield 1x104
CFU/mL which was further aliquoted into 2-mL screw cap microcentrifuge tubes. H 2 O 2 (100 µL) was
added to obtain the final concentrations of 0.5, 1.0, 2.0, and 3.0 mM. The tubes were loosely capped and
incubated at 30 ºC with 650 rpm shaking (Thermomixer Comfort, Eppendorf) for 1 h. The cell viability
was determined by drop plate method on YPD agar. The agar plates were incubated at 30 ºC for 48 h and
the colonies were counted. Percent cell survival was plotted against H 2 O 2 concentration and the IC 50
value was determined as the concentration of H 2 O 2 resulted in 50% survival.
Anti-oxidative stress assay The 950-µL aliquots of NVK in PBS at various concentrations were mixed
with 50 µl of the 2x108 CFU/mL yeast cell suspension in PBS pre-conditioned at 30ºC, 160 rpm for 1 h as
described above. Quercetin (0.1 mM) was used as a positive control. Sample from each tube was taken for
viable count to determine the CFU at the beginning (T 0 ) of NVK and quercetin treatments. A 200-µL
aliquot of the remaining mixture from each tube was transferred to 2-mL screw cap microcentrifuge tube
and incubated at 30 ºC, 650 rpm for 1 h. After 1 h incubation, the treated yeast cells were diluted 1,000

times in PBS and mixed with an equal volume of 2 × IC 50 H 2 O 2 and incubated at 30 ºC, 650 rpm for 1 h.
Percent cell survival was determined as described above.
Statistical Analysis Data are expressed as mean + SD. Student’s t-test was used to compare the significant
difference between two groups.
RESULTS
Determination of IC 50 value of H 2 O 2 against stationary phase S. cerevisiae There was a linear
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relationship between the killing activity (expressed as % survival of the yeast cells) and the log
concentration of H 2 O 2 as shown in Figure 1. The IC 50 value which is the concentration of H 2 O 2 that
killed half of the S. cerevisiae cells calculated from the graph was 1.6 mM.
Figure 1 Percent survival of S. cerevisiae after treated with various concentrations of H 2 O 2 for 1h. The IC 50 value calculated from
the graph is shown. Results were represented as mean ± SD from triplicate determinations.
Protective effect of NVK against H 2 O 2 -induced oxidative stress in S. cerevisiae S. cerevisiae cells were
pretreated with 5 to 1000 µg/mL of NVK (or 34 µg/mL quercetin, a known antioxidant) and then
challenged with H 2 O 2 at the IC 50 . The results are shown in Figure 2. Pretreating with 5 or 10 µg/mL of
NVK had no effect on yeast cell survival as compared with the control experiment. NVK (20 and 25
µg/mL) could increase the % survival of the yeast cells from 51% (control) to 59% and 60%, respectively.
However, these were not statistically significant different from the control. NVK (30 µg/mL) significantly
increased the survival of the yeast cells to 63%. Increasing NVK concentration to 50 µg/mL resulted in
disappearance of the protective effect. Importantly, the NVK at the concentration of 100 µg/mL and
higher concentration-dependently became toxic to the cells.
Figure 2 Percent survival of S. cerevisiae pretreated with various concentration of NVK for 1 h before treated with 1.6 mM H 2 O 2
for 1 h. Quercetin was used as a positive control. Results were expressed as mean ± SD from triplicate determinations. * indicates
statistically significant difference from the control (p < 0.05).

DISCUSSION
S. cerevisiae at stationary phase is a useful model organism for oxidative stress studies. Stationary yeast
cells and most eukaryotic multicellular organism share a number of important characteristics including
quiescent phase of cell cycle (G 0 )6. Cultivation of yeast cells in YPD broth with shaking at 30 ºC for 72 h
could reach saturation (2x108 cells/mL), suggesting that cells were in stationary phase as previously
report7. NVK (30 µg/mL could protect yeast cells from H 2 O 2 -induced oxidative stress killing, showing
anti-oxidative stress activity of NVK. However treatment of NVK at 3-fold higher concentration
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significantly decrease percent survival of yeast cells in a concentration dependent manner. Anti-oxidative
stress activity of NVK in yeast cells found in this study suggested that Phikud Navakot in Yahom might
ameliorate oxidative stress in human leading to the cardiotonic activity of Yahom.
CONCLUSION
NVK at the optimum concentration increases resistance of yeast cells against H 2 O 2 -induced oxidative
stress. Further in-depth and detailed studies, such as proteomic analysis of NVK-treated yeast cells,
should be further carried out to elucidate the target(s) and exact mechanism of this cytoprotective effect of
NVK. Nevertheless, our finding of the NVK toxicity in yeast cells implies that Phikud Navakot at high
dose might be toxic to human and should be further clarified. Special precaution of Phikud Navakot
ingestion should be taken for not taking high amount or not regularly taking for a long period.
ACKNOWLEDGMENTS
This work was financially supported by a Grant-in-Aid from the National Research Council of Thailand
(NRCT 2011-33).
REFERENCES
1. Chander R, Kapoor NK, Dhawan BN. 1992. Picroliv, picroside-I and kutkoside from Picrorhiza
kurrooa are scavengers of superoxide anions. Biochem Pharmacol 44(1):180-3.
2. Cheng CY, Ho TY, Lee EJ, Su SY, Tang NY, Hsieh CL. 2008. Ferulic acid reduces cerebral
infarct through its antioxidative and anti-inflammatory effects following transient focal cerebral
ischemia in rats. Am J Chin Med 36(6):1105-19.
3. López-Alarcón C, Denicola A. 2013. Evaluating the antioxidant capacity of natural products: a
review on chemical and cellular-based assays. Anal Chim Acta 763:1-10.
4. Petranovic D, Nielsen J. 2008. Can yeast systems biology contribute to the understanding of
human disease? Trends Biotechnol 26:584-90.
5. Műller B, Grossniklaus U. 2010. Model organisms – a historical perspective. J Proteomics 73:
2054-63.
6. Zakrajšek T, Raspor P, Jamnik P. 2011. Saccharomyces cerevisiae in the stationary phase as a
model organism - characterization at cellular and proteome level. J Proteomics. 74(12):2837-45.
7. Della Croce C, Bronzetti G, Cini M, Caltavuturo L, Poi G. 2003. Protective effect of lipoic acid
against hydrogen peroxide in yeast cells. Toxicol in Vitro 17(5-6):753-9.

RADICAL-SCAVENGING AND CELLULAR DNA PROTECTIVE ACTIVITIES OF ACTIVE

FRACTIONS FROM LANSIUM DOMESTICUM CORR. FRUIT
Prapaipat Klungsupya1, Nava Suthepakul3, Thanchanok Muangman1, Sarunya Laovitthayanggoon1,

Jeerayu Thongdon-A1 and Srisak Trangwacharakul2
1
Department of Pharmaceuticals and Natural Products (PNPD),
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2
Department of Food Technology, Thailand Institute of Scientific and Technological Research (TISTR),
35 Moo 3 Techno Polis, Klong Luang, Pathum Thani 12120, Thailand.
3
Department of Pharmacology, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand.
KEYWORDS: Lansium domesticum, Long-kong, Superoxide (O 2 -•), Hydroxyl (OH•), Hydrogen

peroxide (H 2 O 2 ), DNA damage, Comet test
INTRODUCTION
Lansium domesticum Corr belongs to the family Meliaceae. It originates from Southeast Asia and is also
cultivated in Australia, Sri Lanka, India, and Puerto Rico. Although it is planted sporadically throughout
the tropics, most of the commercial production is in Thailand, Malaysia, Indonesia, Philippines, and
Vietnam (1). L. domesticum is known under a variety of common names in different countries and
languages. In Thailand, the L. domesticum fruit, called “long-kong”, is very popular and widely
consumed. The long-kong peels were formerly medicinally used against diarrhea and intestinal spasms,
whereas the seeds were an effective remedy for fever and sickness. Previously, antimicrobial and
antimalarial properties of L. domesticum seeds were investigated (1).
Most free radical reactions involve formation of ROS, including superoxide anion radical (O 2 -•), hydroxyl
radical (OH•), peroxyl radical (ROO•) and hydrogen peroxide (H 2 O 2 ) (2). These ROS can attack healthy
cells and cause oxidative damage to DNA, lipid, protein and other bio-molecules. They can disrupt
duplication of DNA, interfere with DNA maintenance, break open the molecule or alter the structure by
reacting with the DNA bases. Lipids in cell membranes are quite prone to oxidative damage because free
radicals tend to collect in cell membranes, known as lipid peroxidation. When a cell membrane is
oxidized by an ROS, it becomes brittle and leaky. Eventually, the cell falls apart and dies (2-3). The
objective of this study was to investigate the anti-oxidative activity against DNA damage of fraction
extracted from the peels of long-kong fruits using single cell gel electrophoresis (SCGE) or comet assay
in TK6 human lymphoblast cells.

Preparation of crude extracts The fresh, mature fruits of L. domesticum Corr. purchased from Talad-Thai
market were employed. After washing, skin (peel) and seeds of the fruits were manually separated, dried
at 50ºC in a hot air oven for 48 h and finally grounded into powder using a blender. One hundred g of
dried powder of skin (SK) and seeds (SD) were extracted separately with 50% and 95% ethanol. The
extracts were then filtered and concentrated using a rotary evaporator. Four crude extracts were obtained
and named LDSD50, LDSK50, LDSD95 and LDSK95, representing parts of L. domesticum fruits and
their ethanolic extraction.
Partition of crude extracts The four ethanolic extracts (prepared as described above) were partitioned
between dichloromethane (DCM) and 50% aqueous ethanol. The obtained aqueous phase (H 2 O) was
further extracted with ethyl acetate (EA). The partition procedure yielded 12 fractions (namely; LDSK50-
DCM, LDSK50-EA, LDSK50-H2O, LDSK95-DCM, LDSK95-EA, LDSK95-H2O, LDSD50-DCM,
LDSD50-EA, LDSD50-H2O, LDSD95-DCM, LDSD95-EA and LDSD95-H2O). All twelve fractions
were concentrated at 45ºC, then kept at 4ºC and protected from light until being used.
Evaluation of free-radicals scavenging activity of fractions Twelve long-kong fractions were subjected
to photochemiluminescence (PCL) and deoxyribose assays to determine their anti-oxidant capacity on
superoxide anion radical (O 2 -•) and hydroxyl radical (OH•) scavenging activities.
Photochemiluminescence (PCL) assay In the PCL system, O 2 -• radicals were generated at about 1,000
times greater than in normal cells by optical excitation of photosensitizer substance (luminol). These O 2 -•
radicals were partially eliminated by reaction with the anti-oxidants in the test samples. Then, the
remaining O 2 -• caused the detector substance to emit luminescence. Therefore, the anti-oxidant capacities

of samples could be determined by their inhibitory effect on luminescence generation. Results of PCL
assay indicated the O 2 -•scavenging activity of 12 fractions of long-kong fruit extracts obtained from both
the lipid- and water-soluble solvent systems to possess anti-oxidant capacities (4).
Deoxyribose assay The principle of deoxyribose assay is based on the determination of malondialdehyde
(MDA) pink chromogen which is a degradation product of 2-deoxyribose (2-DR). The OH• radical,
formed in the reaction between iron (III)-EDTA and H 2 O 2 in the presence of ascorbic acid (reducing
agent), attacks the deoxyribose sugar to form products that on heating with thiobarbituric acid (TBA) at
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low pH yields a pink chromogen which can be determined using a spectrophotometer at 532 nm. Thus,
long-kong extract fractions that possessed OH• scavenging activity could inhibit the deoxyribose
degradation and yield less chromogen than the one without anti-hydroxyl radical activity (5)
Investigation on DNA protective activity in H 2 O 2 -treated TK6 cells by comet assay Subsequently, the
active fractions were assayed for their anti-oxidative activity against DNA damage by hydrogen peroxide
(H 2 O 2 ) in TK6 human lymphoblast cells (ATCC CRL-8015) using the comet assay or single cell gel
electrophoresis (SCGE) (6). The TK6 cells were pre-treated with various concentrations of long-kong
active fractions and then exposed to H 2 O 2 to induce DNA damage. The treated cells were then mixed
with low melting point (LMP) agarose and spreaded onto a microscope glass slide pre-coated with normal
melting point (NMP) agarose gel. Following the lysis treatment of cells (whole glass slides) with
detergent at high alkali salt concentration, DNA unwinding and electrophoresis were carried out at pH 13.
The comet results were quantitatively analyzed in real time under a fluorescence microscope equipped
with a camera-couple device (CCD) and connected to a computer with Comet III software. The anti-
oxidative activity of active fractions was indicated by a significant reduction in DNA damage parameters:
either the tail length (TL = distance of DNA migration from center of cell nucleus, µm) or tail moment
(TM = distance between the center of the tail and the center of the head, multiplied by the percentage of
DNA in the tail, %) values (7, 11).

Superoxide (O 2 -•) radical –scavenging activity (PCL assay) The photochemiluminescence (PCL) assay
measures the potential anti-oxidant property of L. domesticum fractions by two different protocols i.e.
ACW and ACL that indicate the anti-oxidant capacity of the water and lipid soluble components,
respectively (8-9). The anti-oxidant property is expressed in equivalent concentration units of ascorbic
acid and Trolox for water and lipid soluble systems, respectively (10). All twelve L. domesticum fractions
exhibited different degrees of the O 2 -• scavenging activity for both ACL (Figure 1, left) and ACW
(Figure 1, right) measurement systems. Results of the ACL demonstrated the anti-oxidant capacity of
these twelve fractions (at 10 µg/ml) range from 0.380 to 6.625 nmol of Trolox. Among these, LDSK50-
EA possessed the highest anti-oxidant activity (equivalent to 6.625 nmol of Trolox). Interestingly, the
anti-oxidant capacity of ACW system indicated that 50% ethanol extract of peels (LDSK50) still had high
anti-oxidant capacity. The anti-oxidant capacity of all fractions was found to be in a wide range from -
0.065 to 98.733 nmol of ascorbic acid. The highest anti-oxidant activity was found in fraction of
LDSK50-H 2 O (98.733 nmol of ascorbic acid) followed by the LDSK50-EA (54.660 nmol of ascorbic
acid).
Figure 1 The anti-oxidant capacity in lipid (ACL, Left) and water phases (ACW, Right) of twelve L. domesticum fractions by PCL
assay. Results were expressed as means ± SD (n=3). *Significant difference was detected from the lowest activity fraction of same
part-extraction (p≤0.05). **Significant difference was detected from all fractions of same part-extraction (p≤0.05).

For the lipid-soluble (ACL) system, the degree of O2-• scavenging activity for all twelve fractions (from
high to low) were as follows: LDSK50-EA (6.625 nmol) > LDSK50-H2O (1.845 nmol) > LDSK95-EA
(1.750 nmol) > LDSD50-EA (1.257 nmol) > LDSD95-EA (1.200 nmol) > LDSK95-H2O (1.195 nmol) >
LDSK50-DCM (1.028 nmol) > LDSD95-DCM (0.966 nmol) > LDSD50-DCM (0.795 nmol) > LDSD95-
H2O (0.635 nmol) > LDSD50-H2O (0.525 nmol) > LDSK95-DCM (0.380 nmol). For the water-soluble
(ACW) system, the overall anti-oxidant capacity of these fractions, ranking from high to low, are as
follows: LDSK50-H2O (98.733 nmol) > LDSK50-EA (54.660 nmol) > LDSK95-H2O (9.910 nmol) >
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LDSK95-EA (8.350 nmol) > LDSD95-EA (6.880 nmol) > LDSD50-EA (5.410 nmol) > LDSK50-DCM
(4.180 nmol) > LDSD50-H2O (2.073 nmol) > LDSD95-DCM (1.513 nmol) > LDSD95-H2O (1.105
nmol) > LDSK95-DCM (0.345 nmol) > LDSD50-DCM (-0.065 nmol).
Hydroxyl (OH•) radical scavenging activity (deoxyribose assay) Inhibitory effect of L. domesticum
fractions on 2-deoxyribose (2-DR) degradation was determined by measuring the competition between 2-
DR and sample fractions for the OH• generated from the Fe3+/ascorbate/EDTA/H2O2 system. The activity
was expressed as % inhibition for the test sample at the concentrations of 0.5, 1.0 and 2.0 mg/ml. Figure
2 demonstrated a wide range of OH• scavenging activity of these fractions from 0.50 ± 0.12 to 93.44 ±
0.84%. Fraction LDSK50-H2O, at 2,000 µg/ml, displayed maximum inhibitory effect (%I) of up to 93.44
%, equal to that of tannic acid (reference anti-oxidant) at 80 ug/ml (data not shown).
DNA protective activity on H 2 O 2 -treated TK6 cells (comet assay) Fractions LDSK50-EA and LDSK50-
H 2 O, which were most potent against O 2 -• and OH• radicals, were further studied using comet assay. TK6
cells were separately pre-treated with these fractions at 25, 50, 100 and 200 µg/ml for 24 h prior to H 2 O 2
induction. The DNA protective effect of LDSK50-H 2 O was indicated by a reduction in TL and TM
damages compared to cells treated with H 2 O 2 alone.
H2O
H2O
*
H2O H2O
*
*
*
* *
* * *
* * *
* *
Figure 2 Inhibitory effect (%) of 2-deoxyribose degradation of twelve L. domesticum fractions by deoxyribose assay. Results were
expressed as means ± SD (n=3). *Significant difference was detected from all fractions of same concentrations (0.5, 1.0 and 2.0
mg/ml) (p≤0.05).
Treatment with H 2 O 2 at 50 µM for 5 min produced DNA damage (%TM) in TK6 cells at about 10-fold
greater than in untreated cells. Figure 3 shows the DNA-protective activity of both fractions when cells
were pre-treated at 25, 50, 100 and 200 μg/ml for 24 hr prior to exposure to H 2 O 2 . The effect was
indicated by a reduction in TL and TM values in comparison to cells treated with H 2 O 2 alone (Figure 3).
Pre-treatment with LDSK50-EA could prevent DNA damage in a dose-dependent manner (Figure 4),
with the highest effect found at 200 µg/ml concentration (% DNA damage inhibition = 53.47 ± 1.99).
However, at dose greater than 200 µg/ml (up to 250 µg/ml), very little alteration in the effect was detected
but with higher cytotoxicity (data not shown). In contrast, fraction LDSK50-H 2 O exhibited only slight
anti-oxidative activity at similar concentrations. Hence, LDSK50-EA possessed anti-oxidative DNA
damage greater than LDSK50-H 2 O fraction.

*
* *
*
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Figure 3 Tail moment (TM, %) values measured in pre-treated TK6 cells with LDSK50-EA and LDSK50-H 2 O fractions followed
by H 2 O 2 damage induction by comet assay. Results were expressed as means ± SD (n = 3). *Significant difference was detected
from 50 µM H 2 O 2 treatment groups at p≤0.05 (ANOVA).
* *
*
* * * *
Figure 4 Inhibitory effect of LDSK50-EA and LDSK50-H 2 O on H 2 O 2 -induced DNA damage in TK6 cells by comet assay. Results
were expressed as means ± SD (n = 3). *Significant difference was detected from 50 µM H 2 O 2 treatment groups at p≤0.05
(ANOVA)
CONCLUSION
This study generates new and updated information on biological activity of seeds and skins (peels) of
long-kong (L domesticum Corr.) fruits that has not yet been studied before. The results on free radical
(O 2 -• and OH-•) scavenging activity and cellular DNA protective activity against H 2 O 2 will promote and
strengthen utilization of L. domesticum.
ACKNOWLEDGEMENTS
This project was carried out during 2009-2012 and funded by the Ministry of Science and Technology for
the Thailand Institute of Scientific and Technological Research (TISTR).
REFERENCES
1. Tilaar M, Wong LW, Ranti AS, Wasitaatmadja SM, and Junardy FD, (2008). Review of
Lansium domesticum Corrêa and its use in cosmetics. Bol. Latinoam. Caribe Plant. Med.
Aromaticas, 7: 183-189.
2. Halliwell, B. (1996). Antioxidants in human health and disease. Annual Review of Nutrition, 16,
33-50.
3. Ray, P. D., Huang, B.-W., & Tsuji, Y. (2012). Reactive oxygen species (ROS) homeostasis and
redox regulation in cellular signaling. Cellular Signalling, 24(5), 981-990.
4. Popov I, and Lewin G, (1999). Antioxidative homeostasis: characterization by means of
chemiluminescent technique. Methods Enzymol, 300: 437-456.
5. Genaro-Mattos, T. C., Dalvi, L. T., Oliveira, R. G., Ginani, J. S., & Hermes-Lima, M. (2009).
Reevaluation of the 2-deoxyribose assay for determination of free radical formation. Biochimica
et Biophysica Acta, 1790(12), 1636-1642.
6. Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., et al. (2000).
Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing.
Environmental and Molecular Mutagenesis, 35(3), 206-221.

7. Ostling, O., & Johanson, K. J. (1984). Microelectrophoretic study of radiation-induced DNA

damages in individual mammalian cells. Biochemical and Biophysical Research
Communications, 123(1), 291-298.
8. Popov, I., & Lewin, G. (1999). Antioxidative homeostasis: characterization by means of
chemiluminescent technique. Methods in Enzymology, 300, 437-456.
9. Popov, I., & Lewin, G. (1996). Photochemiluminescent detection of antiradical activity; IV:
testing of lipid-soluble antioxidants. Journal of Biochemical and Biophysical Methods, 31(1-2),
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1-8.
10. Besco, E., Braccioli, E., Vertuani, S., Ziosi, P., Brazzo, F., Bruni, R., et al. (2007). The use of
photochemiluminescence for the measurement of the integral antioxidant capacity of baobab
products. Food Chemistry, 102(4),1352-1356.
11. Guillamet E, Creus A, Farina M, Sabbioni E, Fortaner S, and Marcos R, (2008). DNA-damage
induction by eight metal compounds in TK6 human lymphoblastoid cells: Results obtained with
the alkaline Comet assay. Mutation Research/Genetic Toxicology and Environmental
Mutagenesis, 654, 22-28.

PROTECTIVE EFFECT OF OLIGOMERIC PROANTHOCYANIDINS (OPCs) FROM

THAI GRAPE SEEDS AGAINST H 2 O 2 -INDUCED TIGHT JUNCTION FUNCTION
DISRUPTION IN HANMAN CACO-2 CELLS
Thanchanok Muangman1, Prapaipat Klungsupya1, Anawat Suwanagul1,

Akihiro Watari2 and Kiyohito Yagi2
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1
Thailand Institute of Scientific and Technological Research (TISTR), Techno Polis, Pathum Thani 12120, Thailand.
2
Laboratory of Bio-Functional Molecular Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
KEYWORDS: Oligomeric proanthocyanidins (OPCs), Grape seed extract (GSE), Intestinal barrier,
Claudin-4, Trans-epithelium resistance (TER), Caco-2
INTRODUCTION
Oligomeric proanthocyanidins (OPCs) are polyphenolic compounds which occur abundant in red grape
seed. They take the form of oligomers or polymers of polyhydroxy flavan-3-ol units, such as (+)-catechin
and (-)-epicatechin. Many publications have been reported that OPCs is powerful antioxidant free radical
scavenger suggesting that it can be effective in reducing the risk of cancer, cardiovascular disease and the
number of chronic diseases. The extensive research both in vitro and in vivo studies shown OPCs can act
as anti-carcinogenic agent by activities include reduced proliferation, increased apoptosis, cell cycle arrest
in tumor cells.
Tight junction (TJ) is essential for the ‘‘tight’’ bonding between adjacent cells of the epithelial/endothelial
protective barriers and regulating paracellular transport across cell layers. It is also known as occluding
junctions or zonula occludens. The structure TJ is composed of integral membrane proteins such as
occluding and claudin family connecting to actin cytoskeleton through zonular occudens (ZO) proteins.
Disruption of TJ results in barrier leakage and compromises the defense mechanism of the body.
In 2008, Thailand Institute of Scientific and Technological Research (TISTR) was firstly developed
method to extract the natural OPCs from Thai red grape seeds of Vitis vinifera cv. Ribier (Pok Dum).
Furthermore, we previously reported the anti-oxidant, non-mutagenic, anti-mutagenic and anti-oxidative
DNA damage of this Thai OPCs. The current study was carried out to further investigate the protective
activity of Thai OPCs against H 2 O 2 -induced TJ function disruption and expression of claudin-4 in Caco-
2 human intestinal epithelial cells.

Extraction of oligomeric proanthocyanidins (OPCs) The natural OPCs were firstly extracted by the
Thailand Institute of Scientific and Technological Research (TISTR) from grape seeds isolated from
waste product of the winery and grape juice industry. The whole seeds were dried and subjected to
extraction using suitable ratio of ethanol/water and temperature. The chemical composition and structure
of OPCs were analyzed by high performance liquid chromatography (HPLC) and nuclear magnetic
resonance (NMR) spectroscopy. For experimentation, OPCs stock solution (3,000µg/mL) was prepared
by dissolving in distilled water and then stored at -25°C under dark condition until used.
Cell culture and maintenance The human colon adenocarcinoma Caco-2 cell line was employed. The
cells were grown in DMEM medium (Nussui pharmaceutical, Tokyo, Japan) and supplement with 10%
(v:v) of fetal bovine serum (Nichirei Biosciences, Tokyo, Japan), D-(+)-Glucose (Sigma, St.Louis, USA),
MEM non-essential amino acids (Gibgo/Invitrogen, N.Y., USA), 1% of L-Glutamine, 1% of
penicillin/streptomycin (Nacalai Tesque, Kyoto, Japan) in a humidified incubator at 37○C and 5% CO 2 .
Caco-2 cells were grown for 2-3 days to confluency of monolayers.
Determination of cytotoxicity The cytotoxic effect of MPCP#35 or Caco-2 cells was evaluated using Cell
Count Reagent SF kit (Nacalai Tesque, Kyoto, Japan). Cells were seeded in 96 well plates and cultured
for 24 h prior to treatment with various concentrations of OPCs. After treatment, the medium in each well
was replaced with 100 µl of new medium and added 10 µL of Cell Count Reagent solution. Cells were
incubated in a 5% CO 2 incubator at 37○C for 1-2 h. Cells viability was spectrophotometrically quantified
at a measuring wavelength of 450 nm (Tristar LB 941, Berthold Technologies, Japan).
Reporter gene assay MPCP#35 cells were grown for 2-3 days to confluence. For luciferase assay cells at
a density of 4 x 104 cells/ cm2 were grown on 96-well plate. Confluent MCPC#35 cells were treated with
OPCs for 24 h. Cells were washing twice by PBS after incubation time. Luciferase assay reagent (TOYO
INK, Tokyo, Japan) was added to 100 µl/ well and shake for 30 min with aluminum foil cover. The light
intensity was measure by the luminometer (TriStar LB 941, Berthold Technologies, Japan).

Measurement of trans-epithelial electrical resistance (TER) Caco-2 cells (4 x 104 cells) were seeded in
6.5 mm polycarbonate membrane inserted with 0.4 µm pore size (Corstar, Corning, USA) in culture
medium. The TER across the monolayer was measured with a chopstick-like electrode connected to a
Millicell-ERS (electrical resistance system, Millipore, USA) and the TER values were expressed as the
measured resistance in ohms (Ω) multiplied by the surface are of the Transwell membrane (cm2). CaCo-2
cells were grown until the TER was stable for 7-10 days in culture. Once stable resistance was obtain
(>200 Ω cm2), the cells were treated with protein samples. TER was measured just before adding protein
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samples and at 24 and 48 h.
RESULTS
Cell Viability OPCs had no significant effects on the viability of MPCP#35 and Caco-2 cells at the
concentration of 0.01- 0.1 mg/mL.
Effect of OPCs on claudin-4 promoter activity Luciferase reporter gene assays were performed to study
effects of OPCs on the promoter of claudin-4. The claudin-4 promoter was cloned into a PGVCL4P
vector bearing a luciferase gene for quantitative measurement of promoter activity. It was found that
OPCs at concentration of 0.2 mg/mL increased the promoter activity to 291 ± 14% expressed as
percentage of control as demonstrated in Figure 1.
Figure 1 Effect of OPCs on claudin 4-promoter activity (luciferase)
Effects of OPCs on TER values The ability of OPCs to preserve the function of tight junction structure
was tested using TER measurement. Tightness of the junction would result in high membrane electrical
resistance. The effects of OPCs on TER were measured in Caco-2 monolayers over 48 h. In comparison
with the control, OPCs (0.2 mg/mL) slightly increased the TER values at 24 h and 48h as shown in Figure
2.
Figure 2 Effects of OPCs on trans-epithelium electrical resistance (TER) values of Caco-2 cells.

DISCUSSION
A significant body of evidence has demonstrated that inflammatory bowel disease has been associated
with a disorder of the intestinal TJ barrier function [1-2]. A large number of studies have reported that
various food components provide beneficial of anti-inflammatory, anti-mutagenic effects in the intestines
and also intestinal TJ barrier functions [3-5]. OPCs are the most abundant of flavonoids found in the seed
of grape (Vitis vinifera) especially from red grape seed extract. In our study, OPCs induced claudin-4
expression at a transcriptional and protein level including of TER values improvement. Regarding results
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obtained from this study, OPCs enhanced barrier properties in Caco-2 cells used for the study of epithelial
functions. This effect ranks among a large number of beneficial effects reported for dietary
proanthocyanidins, which include suppression of carcinogenesis and prevent of cardiovascular disease.
Our present study illustrates similar results to other flavonoids such as genistein, quercetin, myricetin and
EGCG [6]. The two compounds had been reported their protective and promotive effects on intestinal TJ
barrier function. Study in colonic epithelial HT-29/B6 cells shown that genistein was completely blocked
the decreasing of TER induced by inflammatory cytokine [7]. Moreover, genistein also exhibited to
suppress decrease TER induced by enteric bacterial in Caco-2 cells [8]. The results of the two flavonols;
quercetin and myricetin demonstrated a decreased permeability of lucifer yellow and increased the TER
correlated with overexpression of claudin-4 in Caco-2 cells [9]. Furthermore, on the basis of report gene
assay, that quercetin stimulates claudin-4 promoter activity via enhanced the claudin-4 expression at a
transcriptional level in Caco-2 cells similarly as resulted in OPCs [10].
CONCLUSION
Regarding the present results, we found and concluded that OPCs enhanced tight junction (TJ) barrier
properties in Caco-2 cells via claudin-4 protein. The improvement of TJ function might represent an
important protective effect of this substance against barrier disturbance in intestinal inflammation.
However, the molecular mechanisms underlying this OPCs-mediated effect remain unclear. Thus, further
studies are suggested.
ACKNOWLEDGEMENTS
This project was funded by the Ministry of Science and Technology for the Thailand Institute of
Scientific and Technological Research (TISTR). Special thanks go to Mr.Srisak Trangvacharakul,
Director of Food Technology Department, TISTR for his kind support. Thanks to all colleagues at
Laboratory of Bio-Functional Molecular Chemistry, Graduate School of Pharmaceutical Sciences, Osaka
University, Japan.
REFERENCES
1. Sawada N, Murata M, Kikuchi K, Osanai M, Tobioka H, Kojima T, Chiba H. Tight junctions and
human diseases. Med Electron Microsc. 2003; 36: 147-56.
2. Schmitz H, Barmeyer C, Fromm M. Altered tight junction structure contributes to the impaired
epithelial barrier function in ulcerative colitis. Gastroenterology. 1999; 116: 301-9.
3. Corder R, Mullen W, Khan NQ, Marks SC, Wood EG, Carrier MJ, Crozier A. Oenology: Red wine
procyanidins and vascular health. Nature. 2006; .444: 566.
4. Seth A, Basuroy S, Sheth P, Rao RK. L-Glutamine ameliorates acetaldehyde-induced increase in
paracellular permeability in Caco-2 cell monolayer. Am J Physiol Gastrointest Liver Physiol. 2004;
287: 5107.
5. Usami M, Muraki K, Iwamoto M, Ohata A, Matsushita E, Miki A. Effect of eicosapentaenoic acid
(EPA) on tight junction permeability in intestinal monolayer cells. Clin Nutr. 2001; 20: 351-
6. Suzuki T, Hara H. Role of flavonoids in intestinal tight junction regulation. J Nutr Biochem. 2011;
7. Schmitz H, Fromm M, Bentzel CJ, Scholz P, Detjen K, Mankertz J, et al. Tumor necrosis factor-alpha
(TNFalpha) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J Cell Sci.
1999; 112 (1):137-46.
8. Wells CL, Jechorek RP, Kinneberg KM, Debol SM, Erlandsen SL. The isoflavone genistein inhibits
internalization of enteric bacteria by cultured Caco-2 and HT-29 enterocytes. J Nutr. 1999; 129: 634-
40.
9. Suzuki T, Hara H. Quercetin enhances intestinal barrier function through the assembly of zonula
occludens-2, occludin, and claudin-1 and the expression of claudin-4 in Caco-2 cells. J Nutr. 2009;
139: 965-74.
10. Amasheh M, Schlichter S, Amasheh S, Mankertz J, Zeitz M, Fromm M, et al. Quercetin enhances
epithelial barrier function and increases claudin-4 expression in Caco-2 cells. J Nutr. 2008;138: 1067-
73.

INTERACTION BETWEEN P-GLYCOPROTEIN AND THAI HERBS

WITH ANTI-DIABETIC POTENTIAL
Wilasinee Dunkoksung1, Nontima Vardhanabhuti2, Surattana Amnuoypol3 and Suree Jianmongkol4*

1
Graduate Program in Pharmacology, Faculty of Pharmaceutical sciences, Chulalongkorn University, Bangkok,
2
Thailand. Department of
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Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences,
Chulalongkorn University, Bangkok, Thailand.
3
4
Department of Pharmacology and Physiology, Faculty of Pharmaceutical sciences, Chulalongkorn University, Bangkok, Thailand.
*Corresponding author e-mail: sureejmk@yahoo.com
KEYWORDS: P-glycoprotein, Caco-2 cells, anti-diabetic herbs
INTRODUCTION
Diabetes mellitus (DM) is a chronic metabolic disease with the uncontrolled high blood glucose level. In
order to control blood sugar, current treatment plan includes diet restriction, exercise and drug therapy.
Anti-diabetic drugs control blood sugar through various mechanisms of action including increase insulin
secretion and sensitivity, and limit glucose absorption. Several herbs such as bitter cucumber or mara-
kee-nok (Momordica charantia L., Family Cucurbitaceae) and cinnamon (Cinnamomum iners Reinw.
ex Blume Family Lauraceae) have been known for their anti-diabetic action. In our preliminary study,
alcoholic extract of four Thai herbs collected from the Plant Genetic Conservation Project area under The
Royal Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn potently inhibited intestinal
alpha-glucosidase, suggesting their anti-diabetic potential. These plants include Pterospermum littorale
Craib (or Lam-pang, Family Sterculiaceae), Dialium cochinchinense Pierre (Kleng, Family Fabaceae),
Mamecylon plebejum Kurz. var. ellipsoideum Craib. (Plong-bai-ree, Family Melastomataceae) and
Thespesia populnea (L.) Soland.ex Corr. (Poe-ta-lay, Family Malvaceae).
Herb-drug interactions can affect efficacy and safety of drug therapy. Interference with the function of
P-glycoprotein (P-gp) is one of the most studied transporter-based drug interactions. P-gp is the main
energy-dependent efflux transporter protein in the ABC transporter superfamily. This drug efflux pump
can be found abundantly at the apical site of several epithelial cells and tissues such as intestine, kidney
proximal tubule and lungs1-3. Hence, interference with P-gp activity may affect the processes of
absorption, distribution and elimination, leading to an alteration of pharmacokinetic behaviors of its drug
substrates4. For example, piperine in black pepper inhibited P-gp activity, leading to an increase in the
plasma concentration of phenytoin and rifampin5. The purpose of this study was to determine whether
four herbs with anti-diabetic potential (P. littorale Craib, D. cochinchinense Pierre, M. plebejum Kurz.
var. ellipsoideum Craib, T. populnea (L.) Soland.ex Corr.) could inhibit the function of P-gp transporter,
using the in vitro model of caco-2 cells.

Plant materials and preparation of plant extract All medicinal plants were collected in January, 2011
from Chonburi Province, Thailand. Bark of P. littorale Craib (PL, 89.56 g), D. cochinchinense Pierre
(DC, 45.38 g), leaves of M. plebejum Kurz. var. ellipsoideum Craib. (MP, 16.9 g) and fruits of T.
populnea (L.) Soland.ex Corr. (TP, 149.41 g) were cut and macerated separately with 500 ml of 95%
ethanol for 5 days at room temperature, and then filtered. The filtrate was evaporated to dryness under
vacuum. The weight of each crude extract was 3.26 g for PL, 1.47 g for DC, 1.55 g for MP, and 1.71 g for
TP. All the crude extracts were kept at -20 ºC until used.
Chemicals The chemicals including verapamil (VER), vinblastine (VBL), calcein acetoxymethyl ester
(calcein-AM), Hank’s balanced salt solution (HBSS), Bradford reagent were purchased form Sigma
chemical company (St. Louis, MO, USA).
Cell cultures The Caco-2 cells (ATCC, Rockville, MD, USA), passage 96 to 112, were cultured in
DMEM supplemented with 10% FBS, 1% non-essential amino acid, 1% penicillin-streptomycin, 1%
L-glutamine and 10 nM VBL at 37°C in a humidified atmosphere of 5% CO 2 . Cells were subcultured
every 3-4 days (at approximately 70% confluency). For the uptake study, the cells were seeded onto a 24
well-plate at a density of 13,000 cells/cm2. The cells were fed with fresh VBL-containing medium every
2 days until they were used in the assay at 21 days after seeding. A day before an uptake study, the
medium was switched from VBL-containing medium to VBL-free medium.

Uptake study The Caco-2 monolayers were pretreated with the test extract for 30 minutes, followed by
addition of calcein-AM (0.4 µM). After the incubation period of 30 minutes, the cells were washed with
ice cold HBSS and lysed with 0.3N NaOH in 1% Triton X-100. The fluorescent intensity of calcein was
determined with the microplate reader at an excitation wavelength and an emission wavelength of 485nm
and 535 nm, respectively. The amounts of proteins in each sample were determined with Bradford
reagent in order to normalize the total protein in each experiment.
Data analysis The results were calculated and expressed as the percentage of the calcein retention/mg
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protein in relative to those of the control group in each experiment (100%). Data were expressed as the
mean ± SEM obtained from at least three separated experiments.
RESULTS
The expression of active P-gp in the Caco-2 monolayers after cultured for 21 days in VBL-containing
medium was at the appreciable level. The presence of verapamil (100 µM) and cyclosporine A (10 µM),
which are known inhibitors of P-gp, increased intracellular accumulation of calcein by 5 and 6.8 folds,
respectively (Figure 1).
Figure 1 Intracellaular accumulation of calcein in the VBL-resistant Caco-2 monolayers aged 21 days. The effects of verapamil
(VER) and cyclosporine A (CsA), the known P-gp inhibitors, were determined and used as our positive control. Data were
normalized per mg of proteins and expressed as the percentage of control (untreated group). Values represented the mean ± SEM
(n=5-6).
Figure 2 Effect of alcoholic extracts (MP, PL, TP, and DC) on P-gp function in the VBL-resistant Caco-2 monolayers aged 21 days.
The calcein accumulation was expressed as the percentage of control. Values represented the mean ± SEM (n=3-6).

Crude extracts of TP and DC were able to increase intracellular calcein retention in the VBL-resistant
Caco-2 cells, suggesting their inhibitory action against P-gp activity (Figure 2). At the maximal
concentration of 150 µg/ml, TP and DC increased calcein retention by 2.14 and 2.64 folds, respectively.
The inhibition was apparently concentration-dependent. Neither MP nor PL extract could inhibit P-gp
activity in our uptake assay (Figure 2). It should be noted that the maximal concentration of each crude
extract in this study was the highest soluble concentration in 1%DMSO. In addition, all the crude extracts
had no significant effects on the viability of VBL-resistant Caco-2 cells.
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DISCUSSION
In this study, the function of P-gp was assessed by measuring the intracellular calcein accumulation in the
VBL-resistant Caco-2 monolayers cultured for 21 days. The Caco-2 cell is an epithelial human colon
adenocarcinoma cell line which has been widely accepted as a model of the intestinal barrier for
predicting drug absorption6. Upon being cultured in optimum condition, the Caco-2 monolayers express
various drug transporters in the ATP binding cassette superfamily including P-gp7-8. Although the Caco-2
cells are able to express the appreciable levels of P-gp, several factors such as passage number and
subculture process can cause the variation in the number of functional active protein9. In order to increase
the expression of active P-gp, we cultured the Caco-2 cells in the presence of vinblastine (10 nM) as
described in the previously reported protocols10-12. Our VBL-resistant Caco-2 cells steadily expressed
high levels of functional active P-gp, even when the high passage numbers (96-112) of the cells were
used.
Four Thai herbs in the Plant Genetic Conservation Project area under The Royal Initiative of HRH
Princess Maha Chakri Sirindhorn were chosen for this study based on their ability to inhibit alpha-
glucosidase, which is a known drug target for diabetic control. We demonstrated that extracts of
M. plebejum and P. littorale at the highest concentration in this study were unable to alter P-gp activity.
On the other hand, extracts of D. cochinchinense and T. populnea at their maximal soluble concentration
inhibited the P-gp function in comparable potency. The inhibition could be observed at the concentration
as low as 50 µg/ml (TP) and 100 µg/ml (DC). Upon increasing the concentrations of the two extracts,
their inhibitory action increased. It was likely that certain chemical constituents in crude alcoholic
extracts of MP and DC were potent inhibitors of P-gp. Hence, concomitant use of either
D. cochinchinense or T. populnea and P-gp drug substrates might cause the problem regarding
P-gp-mediated drug interactions through the direct inhibition of this efflux pump. Further studies in this
regard are needed. In addition, isolation and identification of the compounds being P-gp inhibitors in
these two herbs should be pursued.
CONCLUSION
Crude alcoholic extracts of Thespesia populnea (TP) and Dialium cochinchinense (DC) were able to
inhibit the P-glycoprotein function.
ACKNOWLEDGMENTS
This work was supported by the Plant Genetic Conservation Project area under The Royal Initiative of
Her Royal Highness Princess Maha Chakri Sirindhorn, Thailand.
REFERENCES
1. Fojo AT, Ueda K, Slamon DJ, Poplack DG, Gottesman MM and Pastan I. 1987. Expression of a
multidrug-resistance gene in human tumors and tissues. Proc Nati Acad Sci 84: 265-9.
2. Fromm MF. 2003. Importance of P-glycoprotein for drug disposition in humans. Eur J Clin
Invest 33 (Suppl. 2): 6-9.
3. Thuerauf N and Fromm MF. 2006. The role of the transporter P-glycoprotein for disposition and
effects of centrally acting drugs and for the pathogenesis of CNS diseases. Eur Arch Psychiatry
Clin Neurosci 256(5): 281-6.
4. Fromm MF. 2004. Importance of P-glycoprotein at blood–tissue barriers. Trends Pharmacol Sci
25(8): 423-9.
5. Bhardwaj RK, Glaeser H, Becquemont L, Klotz U, Gupta SK and Fromm MF. 2002. Piperine, a
Major Constituent of Black Pepper, Inhibits Human P-glycoprotein and CYP3A4. J Pharmacol
Exp Ther 302(2): 645-50.
6. Artursson P, Palm K and Luthman K. 2001. Caco-2 monolayers in experimental and theoretical
predictions of drug transport. Adv Drug Deliv Rev 46(1-3): 27-43.

7. Taipalensuu J, Törnblom H, Lindberg G, Einarsson C, Sjöqvist F, Melhus H, et al. 2001.

Correlation of gene expression of ten drug efflux proteins of the ATP-binding cassette
transporter family in normal human jejunum and in human intestinal epithelial Caco-2 cell
monolayers. J Pharmacol Exp Ther 299(1): 164-70.
8. Le Ferrec E, Chesne C, Artusson P, Brayden D, Fabre G, Gires P, et al. 2001. In vitro models of
the intestinal barrier. The report and recommendations of ECVAM Workshop 46. European
Centre for the Validation of Alternative methods. Altern Lab Anim 29(6): 649-68.
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9. Sambuy Y, De Angelis I, Ranaldi G, Scarino ML, Stammati A and Zucco F. 2005. The Caco-2
cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-
2 cell functional characteristics. Cell Biol Toxicol 21: 1-26.
10. Shirasaka Y, Kawasaki M, Sakane T, Omatsu H, Moriya Y, Nakamura T, et al. 2006. Induction
of human P-glycoprotein in Caco-2 cells: development of a highly sensitive assay system for P-
glycoprotein-mediated drug transport. Drug Metab Pharmacokinet 21(5): 414-23.
11. Siissalo S, Laitinen L, Koljonen M, Vellonen KS, Kortejärvi H, Urtti A, et al. 2007. Effect of
cell differentiation and passage number on the expression of efflux proteins in wild type and
vinblastine-induced Caco-2 cell lines. Eur J Pharm Biopharm 67(2): 548-54.
12. Hellinger E, Bakk ML, Pócza P, Tihanyi K and Vastag M. 2010. Drug penetration model of
vinblastine-treated Caco-2 cultures. Eur J Pharm Sci 41(1): 96-106.

CYTOTOXIC EVALUATION OF A FRUIT EXTRACT FROM ZANTHOXYLUM LIMONELLA

IN HUMAN DERMAL FIBROBLAST CELL LINE USING MTT ASSAY
Sarunya Laovitthayanggoon1, Buppachart Potduang1*, Natthachest Ketmanee1

and Chuleratana Banchonglikitkul1
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1
Pharmaceuticals and Natural Products Department, Thailand Institute of Scientific and Technological Research, Technopolis,
35 Mu 3, Thanon Liab Klong 5, Klong Luang, Pathumthani 12120, Thailand. E-mail: Sarunya@tistr.or.th
KEYWORDS: Zanthoxylum limonella, Cytotoxicity, Human dermal fibroblast, MTT assay
INTRODUCTION
Zanthoxylum limonella Alston (RUTACEAE), locally called Ma-khwaen, is a deciduous tree up to 18 m
tall. It is widely distributed in the northern part of Thailand and has been used in folk medicines for
different medical purposes. Dry fruits are sold in local markets and traditionally used as spice. The bark,
root-bark and fruit contained highly effective antibacterial substances. The essential oil from Z. limonella
fruits exhibits the anti-oxidative potential. The oil contains Sabinene which is a potent bactericidal against
the multi-drug resistant bacteria 1, 2, 3. We have reported that the 60% ethanol fruit extract of Z. limonella
is effective against tested microbial including C. albicans (MIC 2.5-10 mg/ml). It is anti-oxidant (EC 50
5.94 µg/ml) and its total flavonoids is 3.61 mg rutin/g extract4. We have later found that the extract
exhibited potent anti-tyrosinase (IC 50 0.33 mg/ml) activity. The extract was proved to be a potent anti-
inflammatory active extract which was more potent than Std. Diclofenac5. The anti-inflammatory, anti-
tyrosinase and antioxidant activities make the plant valuable in cosmetics. This study was performed to
evaluate cytotoxicity of the ethanol fruit extract from Z. limonella using MTT assay that was a part of
scientific aspects.

Preparation of plant extracts The dry fruit powder of Z. limonella was separately extracted using 60%
ethanol at room temperature. The combined filtrates were evaporated under reduced pressure at 40-50ºC
to give crude ethanol extract.
Sample preparation The crude extract was weighed and dissolved in 100% dimethylsulfoxide making
stock concentration of 1 mg/ml. Filtration through a 0.2 μm filter was done before serially diluted the
sample in the culture medium of cells at a ratio of 1:2 giving 8 concentrations of 500, 400, 300, 200, 100,
50, 25 and 12.5 µg/ml.
Cell culture The human dermal fibroblast (ATCC CRL-1474 :NHFF) were grown in Dulbecco's
Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 2mM L-glutamine and
100 unit/ml penicillin and streptomycin. The cells were incubated for 72 h. at 37°C in a fully humidified,
5% CO 2 : air atmosphere.
MTT cytotoxicity test6,7 MTT assay is a tetrazolium-dye based colorimetric microtitration assay.
Metabolism competent cells are able to metabolize the tetrazolium (yellow) to formazan (blue); this color
change is measured spectrophotometrically with a plate reader. It is assumed cells that are metabolically
deficient will not survive, thus the MTT assay is also an indirect measurement of cell viability. The cells
were seeded in a 96-well plate at a density of 10,000 cells/well, and incubated for 24 hours. The sample at
various concentrations were added to the cells and incubated for 24 hours. The test samples were removed
from the cell cultures and the cells were reincubated for a further 24 hours in fresh medium and then
tested with MTT assay. Briefly, 50 µl of MTT in PBS at 5 mg/ml was added to the medium in each well
and the cells were incubated for 4 hours. Medium and MTT were then aspirated from the wells, and
formazan solubilized with 200 µL of DMSO and 25 µl of Sorensen’s Glycine buffer, pH 10.5. The optical
density was read with a microplate reader (Molecular Devices) at a wavelength of 570 nm. The average of
4 wells was used to determine the mean of each point. The data were analyzed with the SoftMax Program

(Molecular Devices) to determine the IC 50 for each toxin sample. A dose-response curve was derived
from 8 concentrations in the test range using 4 wells per concentration. Results of toxic compounds are
expressed as the concentration of sample required to kill 50% (Inhibitory concentration 50; IC 50 ) of the
cells compared to controls.

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The cytotoxicity test was shown in Table 1 and Figure 1. The treatment of NHFF cells for 24 hr with
various concentrations of Z. limonella extract at 500, 400, 300, 200, 100, 50, 25 and 12.5µg/ml. indicated
that the IC 50 value was 391.8 ± 0.01µg/ml for treatment times.
Table 1: Viability of NHFF following exposure to Z. limonella extract
Sample Concentration (µg/ml) % Viability IC50(µg/ml)

500.00 23.45 ± 0.02 391.8 ± 0.01
400.00 55.66 ± 0.05
300.00 73.70 ± 0.13
200.00 103.04 ± 0.15
Z. limonella
100.00 101.00 ± 0.02
50.00 102.00 ± 0.06
25.00 101.00 ± 0.05
12.50 102.00 ± 0.07
Figure 1 The Viability of NHFF following exposure to Z. limonella (µg/ml) extract
CONCLUSION
This assay was a modified version of conventional direct and indirect contact tests conformed to the
published standard methods (BS-EN30993-5 and ISO10993-5). The MTT assay is a tetrazolium-dye
based colorimetric microtitration assay. Metabolism competent cells are able to metabolize the
tetrazolium (yellow) to formazan (blue); this color change is measured spectrophotometrically with a
plate reader. It is assumed cells that are metabolically deficient will not survive, thus the MTT assay is
also an indirect measurement of cell viability. The cytotoxicity results showed the % survival of NHFF
cell line, at each concentration compared to control and IC 50 values, over the test concentrations of 500-
12.5 µg/ml. The results showed that The IC 50 values of Z. limonella extract was 391.8 ± 0.01 µg/ml .
ACKNOWLEDGEMENT
The author would like to gratitude thanks to Thailand Institute of Scientific and Technological Research
(TISTR) for the financial support.

REFERENCES
1. Mallikarjuna P, Uma Maheswara Rao V and Satyanarayana T. 1999. Antimicrobial activity of

Zanthoxylum limonella. Indian Drugs 36 (7): 476-478.
2. Tangjitjaroenkun J, Supabphol R and Chavasiri W. 2012. Antioxidant effect of Zanthoxylum
limonella Alston. J Medicinal Plants Res. 1407-1414.
3. Charoenying P, Laosinwattana C, Phuwiwat W, and Lomratsiri J. 2008. Biological activities of
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Zanthoxylum limonella Alston fruit extracts. KMITL Sci. J. 8(1): 12-15.
4. Ngamnon Y, Potduang B*, Fungsin B, Phasuk S, Takolpuckdee P, Rerk-am U, Kaewduang M
and Tanpanich S. 2012. Antimicrobial, antioxidant activities and total flavonoids of fruit extracts
from Ma-Khwaen (Zanthoxylum limonella). Thai J Pharm Sciences 35 (Suppl.): 36-37.
5. Khayungarnnawee A, Soradech S, Potduang B*, Sematong T, Laovitthayanggoon S, Keeta I,
Niwaspragrit C.and Tantrawong A. (2012). Anti-inflammatory activity of fruit extracts from
Phyllanthus emblica and Zanthoxylum limonella. Thai J. Pharm. Sci. 36 (Suppl.) Dec., p.38-40
6. Freshney RI. 2005. Culture of Animal Cell: A manual of Basic Technique. Wiley-Less,
NewYork, Chapter 21: pp.287-307.
7. Plumb JA, Milroy R, Kaye SB. Effects of the pH dependence of 3-(4,5- dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide-formazan absorption on chemosensitivity determined by a
novel tetrazolium based assay. Cancer Res. 1989 49:4435-4440.
8. BS-EN30993-5. 1994. Biological evaluation of medical devices, Part 5. Tests for cytotoxicity, in
vitro methods: British Standard1994
9. ISO10993-5. 2009. Biological evaluation of medical devices, Part 5. Tests for in vitro
cytotoxicity: International Standard 2009
10. Guidance Document on using In Vitro Data to Estimate In Vivo Starting Doses for Acute
Toxicity, 2001 NIH Publication No. 01-4500 available under
(http://iccvam.niehs.nih.gov/docs/acutetox_docs/guildance0801/iv_guild.pdf)

ANTI-LIPASE ACTIVITY OF QUERCUS INFECTORIA G.OLIVIER EXTRACT
Sirinan Thubthimthed1, Sarunya Laovitthayanggoon1, Parkpoom Siriarchavatana1, Konlawit

Chaithongsri1 and Chuleratana Banchonglikitkul1
1
Pharmaceutical and Natural Products Department (PNPD), Thailand Institute of Scientific and Technological Research (TISTR),
Technopolis, Pathumthani, 12120, Thailand.
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KEYWORDS: Obesity, lipase inhibitor, anti-lipase activity, Quercus infectoria, nutgall
INTRODUCTION
Obesity is the most common metabolic disease in developed nations. There are various therapeutic
approaches to treating obesity, including suppression of food intake, increased thermogenesis, accelerated
lipolysis and inhibition of lipogenesis. It has been suggested that inhibition of digestion and absorption
of dietary fat is a key factor in treating of obesity so the application of a pancreatic lipase inhibitor was
widely interesting. The only pancreatic lipase inhibitor currently appoved for longterm treatment of
obesity is Orlistat. The use of Orlistat frequently results in adverse events including flatus, oily stools,
fecal urgency or fecal incontinence, and abdominal pain (Harold E. Bays, 2004). Therefore, there is a
need for more lipase inhibitors or medicinal products that are safe and effective. Previously we screened
the anti-lipase activity of fifteen Thai herbs belonging to different families such as Compositae,
Lauraceae, Labiatae, Rutaceae and Zingiberaceae.
Qurercus infectoria (Fagaceae) is a small tree or a shrub mainly present in western Asia and Southern
Europe. The galls of Q. infectoria have been known to possess medicinal properties, such as astringent,
anti-inflammatory, antiviral, antidiabetic, larvicidal, antibacterial, antiulcerogenic and gastroprotective
activities The main constituents found in the galls of Q. infectoria are tannin (50-70%) and small amount
of free defined as water–soluble polyphenolic compounds that have theability to precipitate proteins
(Umachigia SP, et al., 2008). In this study we investigated the lipase inhibitory activity of Q. infectoria
extract by using the BALB-DTNB method (Kurooka and Kitamura, 1978) and also the levels of tannin
and phenolic contents.

Preparation of herbal extracts The galls of Q. infectoria were obtained from the local market and were
crushed to small pieces before pulverized into powder. The extraction was carried out by using 95%
ethanol in a percolator at room temperature. The extract was filtered and concentrated at 45oC under
reduced pressure in a rotary vacuum evaporator.
Determination of total tannin content The total tannin content of the Q. infectoria extract was
determined using the Folin- Ciocalteu reagent. The reaction mixture contained: 100 μl of diluted Q.
infectoria extract, 500 μl of freshly prepared diluted Folin Ciocalteu reagent and 1 ml of 20 % sodium
carbonate. Mixtures were kept in dark at ambient conditions for 1 h to complete the reaction. The
absorbance at 760 nm was measured. Gallic acid was used as standard (Concentration of 0.05, 0.1, 0.2,
0.4, 0.8 and 1.0 mg/ml of tannic acid were prepared in ethanol). The Folin-Ciocalteu reagent is sensitive
to reducing compounds including polyphenols, thereby producing a blue color upon reaction. All
determination was performed in triplicate and the total phenolic content was expressed as mg/g tannic
acid equivalents (TAE).
Determination of total phenolic content The total phenolic content of the Q. infectoria extract was
determined using the Folin- Ciocalteu reagent. The reaction mixture contained: 100 μl of diluted Q.
infectoria extract, 500 μl of freshly prepared diluted Folin Ciocalteu reagent and 1 ml of 20 % sodium
carbonate. Mixtures were kept in dark at ambient conditions for 1 h to complete the reaction. The
absorbance at 760 nm was measured. Gallic acid was used as standard (Concentration of 0.05, 0.1, 0.2,
0.4, 0.8 and 1.0 mg/ml of gallic acid were prepared in ethanol). All determination was performed in
triplicate and the total phenolic content was expressed as mg/g gallic acid equivalents (GAE).
Statistical analysis The determinations were conducted in triplicate and results were expressed as mean ±
standard error.
Preparation of pancreatic lipase enzyme The 0.2 g. of rat pancreas was mixed with 10 ml of 0.9%
normal saline, spun at 7,000 rpm/min for 10min. and then the supernatant was kept under -800C until next
experiment.
Assay for lipase inhibitory activity Solution of 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 and 1 mg/ml of
ethanolic extract of galls of Q. infectoria was prepared before enzyme assay. The positive control

(Xenical:Orlistat 120 mg) solution was prepared by dissolving in phosphate buffer saline (PBS) and
adjust to 0.5 mg/ml final concentration.
The 10 µl of each preparation were added to 1 ml of pancreatic lipase enzyme and then incubated the
reaction at room temperature for 15 min. The active extracts were determined by using a commercial
lipase assay kit (BioAssay Systems. USA) according to the manufacturer’s instructions. The activity of
the negative control was also checked with and without inhibitor. The inhibitory activity (I) was
calculated according to the following formula.
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I %= 1 - B-b x 100
A-a
Where A is the activity of the enzyme without inhibitor, a is the negative control without inhibitor, B is
the activity of the enzyme with inhibitor and b is the negative control with inhibitor.
RESULTS
The dried powder of galls of Q. infectoria yielded 88.21% (w/w) of ethanolic extract. Standard curve for
the determination of total tannin and total phenolic content was prepared by using different concentrations
of tannic acid and gallic acid, respectively. The results have been presented in table 1. The total tannin
and total phenolic contents of Q. infectoria extract were 625.83±2.08 mg of TAE/g and 655.86±2.22 mg
of GAE/g,respectively. The anti-lipase activity of various concentrations this extract were shown in
Table 2. where lipase inhibition is expressed in percentage (%). The extract at 10,50,100,200 mg/ml
concentration showed no inhibition in lipase activity while the extract at the concentration of 600-1000
mg/ml showed moderate anti-lipase activity at 20.33-35.36% inhibition.
Table 1 The total tannin and total phenolic contents of Q. infectoria extract
Total tannin Content Total Phenolic Content

(mg of TAE/g of extract) (mg of GAE/g of extract)
Ethanolic extract of Q. infectoria 625.83±2.08 655.86±2.22
The values are shown as mean±s.d. (n = 3).
Table 2 Anti-lipase activity of Q. infectoria extract
No. Concentration of % Inhibition

Q. infectoria extract
1. 10 µg/ml No Inhibition
5. 300 µg/ml 5.00
6. 400 µg/ml 18.52
7. 500 µg/ml 6.54
8. 600 µg/ml 20.33
9. 700 µg/ml 25.24
10. 800 µg/ml 27.46
11. 900 µg/ml 27.13
12. 1000 µg/ml 35.36
13. Oristat ( 5 µg/ml) 54.44
DISCUSSION
Tannins are defined as naturally occurring polyphenolic compounds of high molecular weight to form
complexes with the proteins. It has been reported that tannin has an inhibitory effect on pancreatic lipase
activity and could be applied in the treatment of obesity (Lei F. et al., 2007). In this study, ethanolic
extract of Q. infectoria had high concentration of total tannin and total phenolic contents and inhibited
lipase activity at the concentration over than 600 µg/ml. Therefore, the anti-lypase activity of Q.
infectoria extract might be due to the high amounts of total tannin. The inhibitory effect of this crude

extract is much weaker than orlistat because the crude extract contains, not only active substances, but
also non-active components.
CONCLUSION
The results of the current study showed that Q. infectoria extract has lipase inhibitory activity and has
potential for the treatment of obesity. Further studies are needed to verify the inhibitory activities in
animal models and also the safty evaluation.
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REFERENCES
1. Davidson M, Hauptman J, Digiroloamo M. 1999. Weight control and risk factor reduction in
obese subjects treated for 2 years with orlistat. JAMA. 281: 235–42.
2. Harold E. Bays,2004. Current and Investigational Antiobesity Agents and Obesity Therapeutic
Treatment Targets Obesity Research 12:1197-1211.
3. Kim J, Dae SJ, Hyojun K, and Jin SK. 2009. Anti-lipase and lipolytic activities of ursolic acid
isolated from the roots of Actinidia arguta. Arch Pharm Res 32 (7): 983-7.
4. Kurooka S and Kitamura T. 1978. Properties of serum lipase in patients with various
pancreatic diseases. Analysis by a new serum lipase assay method (the BALB-DTNB method)
in combination with gel-filtration and iso-electrofocusing techniques. J Biochem (Tokyo).
84:1459-66.
5. Sharma N,Sharma VK and Seo SY.2005 Screening of some medicinal plants for anti- lipase
activity.J Ethnopharm.97:453-6.
6. Tietz NW and Shuey DF. 1993. Lipase in serum-the elusive
enzyme:anoverview.Clin.Chem.39(5): 746-56.
7. Umachigia SP, et al., 2008, Studies on Wound Healing Properties of Quercus infectoria .Trop J
Pharm Res,7 (1): 913-919.

ACUTE ORAL TOXICITY TEST OF QUERCUS INFECTORIA G.

OLIVIER EXTRACT IN RATS
Tuanta Sematong1, Sirinan Thubthimthed1, Sareeya Reungpathanapong1,

Sarunya Laovitthayanggoon1 and Chuleratana Banchonglikitkul1
1
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Pharmaceutical and Natural Products Department, Thailand Institute of Scientific and Technological Research (TISTR)
Pathumthani 12120, Thailand.
KEYWORDS: Quercus infectoria G. Olivier, Acute oral toxicity, Rats
INTRODUCTION
Quercus infectoria G. Olivier, common name known as Ben ka nee (Thai) and belonging to the Fagaceae
family. It is indigenous to Turkey, Iran, Iraq, Kurdistan, Cyprus, East Aegean Islands, Greece, Lebanon
and Syria. The tree is occasionally cultivated for production of tanning bark and for dye production of the
wood. The main constituents found in the galls of Q. infectoria are tannin (50-70%) and small
amount of free gallic acid and ellagic acid. This plant is very useful in various parts.
The seed can be thoroughly washed in running water to remove the bitter tannins and cooked, or it can be
dried, ground into powder and used as a thickening in stews etc. The nut gall extract or powder is also
used as herbal drink or tea health purposes. Due to is very useful plant, that needs to find out more
potentials and safety information. Thus, the objective of this study is to determine the safety of 95%
ethanolic extract of Q. infectoria in rats.
Figure 1 Quercus infectoria G. Olivier : Ben ka nee
MATERIAL AND METHOD

Animals Male (250 ± 20 g) and Female (230 ± 20 g) Wistar rats were obtained from National Laboratory
Animal Centre, Mahidol University, Salaya, Nakornpathom. They were kept in cages with sterilized
wood shavings as bedding at 24 ± 2°C in 12 h light/dark cycle and feed with standard diets and tap water
ad libitum. All rats were acclimatized for 7 days prior to the experiments.
Method Acute oral toxicity test was carried out following the “Guideline No. 423: Acute oral toxicity-
Acute toxic class method of the OECD Guidelines for Testing of Chemicals (7)”. In brief, animals were
divided into five groups and each group contains five rats of both sexes. Group 1 was served as negative
control which was received 0.5% CMC or distill water in equivolume to the test group. Group 2-4 were
served as treatment groups which were received the extract at dose of 2,000, 7,500 and 15,000 mg/kg,
respectively. The rats were fasted for 16 hrs prior to dosing the test sample while drinking water was
available ad libitum, and foods were withheld for further 3-4 hrs. Any toxic signs were immediately
observed at ½, 1 and 3 hrs. The special care should be considered to animals that obviously showed toxic
signs during the first 4 hrs after dosing and observed once daily thereafter for 14 days. Body weight was

recorded weekly and at the end of the test. All survivors were euthanized by CO 2 asphyxiation and then
performed necropsy finding. The mean of body weight gain of the animals in the test groups was
calculated in comparison to the rats of the control group using Student’s t-Test (p ≤ 0.05).

As shown in Table 1 and 2. All groups of treated rats (2,000, 7,500 and 15,000) did not show any toxic
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sings through the observation period. But the number of dead rats was found at dose 7,500 and 15,000
mg/kg b.w. The body weight gain of the rats showed no difference from the control group. Necropsy
findings exhibited normal appearance and no macroscopic pathological lesions of visceral organ. Thus,
LD 50 (lethal dose) was estimated at 9,700 mg/kg.
Table 1 Summary of mortality rate and gross pathology of control and treated rats
a
Mortality rate
Treatment/Dose Gross Pathology
Male Female Total
Control group 0/5 0/5 0/10 Normal
0.5% CMC
equivolume to the treatment group
Treatment group 0/5 0/5 0/10 Normal
“Ben ka nee extract 2,000 mg/kg bw.”
a
Number of dead rats/number of rats tested
Table 2 Means of body weight gain of the control and treated rats were recorded during experimentation and at termination
*Mean of body weight gain (g)

Sex Treatment/Dose
Day 8 Day 15
Control group 46.40 ± 2.25 91.00 ± 2.42
0.5% CMC
Treatment group *58.20 ± 2.93 *79.80 ± 2.22
Male
Treatment group 36.00 ± 11.11 83.25 ± 12.46
Treatment group 52.00 ± 13.00 68.50 ± 11.50
“Ben ka nee extract 15,000 mg/kg b.w.”
Control group 18.00 ± 2.16 32.60 ± 2.40
0.5% CMC
Treatment group 19.20 ± 1.82 30.20 ± 3.48
Female “Ben ka nee extract 2,000 mg/kg bw.”
Treatment group *30.75 ± 1.97 *44.25 ± 2.46
“Ben ka nee extract 7,500 mg/kg b.w.”
“Ben ka nee extract 15,000 mg/kg b.w.” NC NC
* Data shown in the table are mean + SEM
NC = Not calculated (dead findings within 24-48h after treating with extracts)
CONCLUSION
The LD 50 of 95% ethanolic extract of Ben ka nee in rats is 9,700 mg/kg body weight. Therefore, this
study indicated that Ben ka nee extract seem to be safe in use as material source for herbal products
development. However, the repeated dose toxicity evaluation of the extract is still necessary in further
study.

ACKNOWLEDGEMENT
The author would like to express gratitude thanks to Thailand Institute of Scientific and Technological
Research for their financial support.
REFERENCES
1. G.Kaur, H. Hamid, A.Ali, MS Alam, and M. Athar, “Antiinflammatory evaluation of alcoholic
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extract of galls of Quercus infectoria,” J.Ethnopharmacol. vol.90, pp 285-92, 2004.
2. Organization for Economic Co-operation and Development. 2001. OECD Guidelines for Testing
of Chemicals, Volume 2, Section 4: Health Effects. Acute Oral Toxicity-Acute Toxic Class
Method, Test Guideline No. 423.

BIOLOGICAL ACTIVITIES OF CAJANUS CAJAN ETHANOLIC EXTRACTS
Ubon Rerk-am1, Bantika Kongsombat1, Sinn Tangsatirapakdee1 and Chuleratana Banchonglikitkul1

1
Pharmaceutical and Natural Products Department, Thailand Institute of Scientific and Technological Research.
35 Moo 3, Technopolis, Tambon Klong 5, Amphoe Klong Luang, Pathum Thani 12120, Thailand.
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KEYWORDS: Cajanus cajan (L) Millsp., Pigeon pea, Anti-oxidative activity, Anti-tyrosinase activity
INTRODUCTION
Pigeon pea (Cajanus cajan (L) Millsp) is among the dry leguminous seed which important source of
proteins in human diet food, especially in the vegetarian population. Pigeon pea has high levels of
proteins and important amino acids such as methionine, lysine and tryptophan. The Extraction of seed and
leaves are widely used in Indian and Chinese traditional folk medicine [1]. The chemical compounds of
pigeon peas compose of isoguercitrin, quercetin, quercetin-3-methyl ether and 3-hydroxy-4-prenyl-5-
methoxystilbene-2-carboxylic acid [2]. The rich content of phenolic and flavonoids contained in leaves
extracts were promising their bioactivity such as antipasmodic, anti-imflammatory, antimicrobial and
antioxidant activities [3]. Fungal endophyte MD98 extracts from leaves was produced phenolic and
flavonoids compounds which have antioxidant properties, superoxide dismutase and glutathione reductase
activities in HepG2 cell [4]. The ethanolic extract of leaves contain pinostrobin, cajanin, longstylin C,
longistylin A and cajaninstilbene, there was potent to antioxidant, antiplasmodical, anti-inflammatory and
hypocholesterolemic activities. While, the crude ethanolic extracts of roots contain genistein, genistin,
longstylin C, longistylin A and cajanol, which were antioxidant and anticancer activities [5]. The present
study was taken up to evaluate antioxidant and anti-tyrosinase activities of Cajanus cajan seed ethanolic
extract. The antioxidant is claimed that it is biologically active in protecting the body whereas the skin
collagen and elastic tissue against damaging by reactive oxygen species. Tyrosinase is known to play a
role as key enzyme for melanin biosynthesis in plants and animals. Tyrosinase inhibitors therefore can be
clinically useful for the treatment of some dermatological disorders that associate with melanin
hyperpigmentation.

Preparation of Plant Extracts The dried seed of Pigeon pea were ground into powder and extracted with
70 % ethanol at room temperature for 10 times. The combined filtrate of ethanol solution was evaporated
under reduced pressure at room temperature.
Scavenging of Diphenyl-picrylhydrazyl (DPPH) Radicals Assay The free radical scavenging activity of
ethanolic extracts was analyzed by the DPPH assay [6]. The amount of 100 µL of various concentrations
sample (20, 40, 60, 80 and 100 µg/mL) were reacted with 100 µL of 6x10-3 M DPPH ethanolic solution in
a 96-well plate, incubated at 37 oC for 30 min. The absorbance was measured at 517 nm using a UV–VIS
microplate reader. All experiments were carried out in triplicates.
Lipid Peroxidation (β-Carotene Bleaching Model) The antioxidant activity of crude extract was
measured by β-carotene bleaching model system with slight modification [7]. Emulsion I was prepared by
dissolving 10 mg of β-carotene in 10 ml of chloroform. Four milliliters of β-carotene solution, 40 mg of
linoleic acid and 400 mg of Tween 40 were mixed and removed chloroform at 50 oC under vacuum by
rotary evaporator. The emulsion was further made up to 100 ml with MillQ water. Emulsion II was
prepared same as emulsion I with out β-carotene. Test sample, 50 µl of varied concentration sample were
mixed with emulsion I. Blank sample, 50 µl of varied concentration sample were mixed with emulsion II.
Absorbance was measured at 450 nm after mixture incubation in oven at 50 oC and the reaction reading
will reccord from zero time (t=0) till 60 min.
Inhibition of tyrosinase activity Determination of tyrosinase inhibition activity was performed by the
dopachrome method using L-DOPA as the substrate and described by Iida et al [8] with slight
modifications. Briefly, 50 µL of various sample concentration, 50 µL of phosphate buffer (pH 6.8) and 50
µL of 787 units/mL of mushroom tyrosinase solution were mixed in 96-well plate. After pre-incubation at
37 oC for 10 min, 50 µL of 160 µg/mL L-Dopa was added and incubated at 37 oC for 2 min. The amount
of dopachrome was measured at 492 nm using a UV–VIS microplate reader.


The biological activity of C. cajan seed ethanolic extracts was showed in Table 1 and 2.
Table 1 Antioxidant activity (EC 50 ) of crude ethanolic extract of C. cajan seed, compared with standard.
Sample DPPH assay Lipid peroxidation
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(µg/ml) (µg/ml)
Crude ethanolic extract of C. cajan seed 44.36 25.97
Trolox 1.90 1.20
The DPPH radical was used to evaluate the free radical scavenging capacity of antioxidants extensively.
The concentration of C. cajan seed ethanolic extract to quench DPPH radical (EC 50 ) was 44.36 µg/ml.
Lipid peroxidation was estimated of capacity of crude extract to protect β-carotene color change from
yellow to colorless. The activity (EC 50 ) was 25.97 µg/ml. The results show that the antioxidant activity
of C. cajan seed ethanolic extract lower activity than Trolox, that know compound had high antioxidant
activity.
Table 2 Anti-tyrosinase activity (EC 50 ) of crude ethanolic extract of C. cajan seed, compared with standard.
Sample Anti-tyrosinase (µg/ml)

Crude ethanolic extract of C. cajan seed 267
Kojic acid 2.31
For the activity of C. cajan seed ethanolic extracts to inhibit on dopa oxidase of mushroom tyrosinase.
These extracts showed lower activity than Kojic acid, that know compound had high anti-tyrosinase
activity and used in whitening agent.
CONCLUSION
The results reveal that C. cajan seed ethanolic extracts had high antioxidant activity and slightly anti-
tyrosinase activity. Although less activity than Trolox and Kojic acid. These crude ethanolic extracts
could be potential sources of antioxidant and anti-tyrosinase activity. It would be promissing to do more
studies as skin-whitening and anti-wrinkle agents. Future biological investigations on human melanocytes
and dermal fibroblast need to further confirm, including safety evaluation.
REFERENCES
1. Olawuni, I.A., Ojukwu, M. and Eboth, B. 2012. Comparative Study on the Physio-Chemical
Properties of Pigon pea (Cajanus cajan) Flour and Protein Isolate. Intetnational Journal of
Agricultural and Food Science. 2(4). 121-126
2. Green, P.W.C, Stevenson, P.C. Simmonds, M.S.J. and Sharma, H.C. 2003. Phenolic Compounds on
the Pod-Surface of Pigeonpea, Cajanus cajan, Mediate Feeding Behavior of Helicoverpa armigera
larvae. Journal of Chemical Ecology. 29(4). 811-821
3. Zu, Y.G., Liu, X.L., Fu, Y.J., Wu, N., Kong, Y. and Wink, M. 2010. Chemical Composition of SFE-
CO 2 Extracts from Cajanus cajan (L.) Huth and their Antimicrobial Activity in vitro and in vivo.
Phytomedicine. 17. 1095-1101
4. Gao, Y., Zhao, J., Zu, Y., Fu, Y., Liang, L., Luo, M., Wang, W. and Efferth, T., 2012. Antioxidant
Properties, Superoxide Dismutase and Glutathione Eeductase Activities in HepG2 Cell with a Fungal
Endophyte Producing Apiginin from Pigeon pea (Cajanus cajan (L.) Millsp.), Food Research
International. 49. 147-152.
5. Pal, D., Mishra, P., Sachan, N. and Ghosh, A.K.. 2011. Biological Activities and Medicinal Properties
of Cajanus cajan (L) Millsp., Journal of Advanced Pharmaceutical Technology & Research. 2(4).
207-214
6. Duan, X.J, Zhang, W.W, Li, X.M, Wang, B.G. 2006. Evaluation of Antioxidant Property of Extract
and Fractions Obtained from a Red Alga, Polysiphonia urceolata. Food Chemistry. 95. 37-43.

7. R. Kawaree, R., Okonogi, S., Chowwanapoonpohn, S. and Phutdhawong, W and Kawaree, R. 2008.
Chemical Composition and Antioxidant Evaluation of Volatile Oils from Thai Medicinal Plants.
Proceedings of the International Workshop on Medicinal and Aromatic Plants. Acta Horticulturae.
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Tyrosinase Activity and Melanin Biosynthesis from Rheum officinale. Planta Med. 61. 425-428.
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SYNERGY EFFECT OF CEFTAZIDIME WITH FLAVONOIDS

AGAINST STREPTOCOCCUS PYOGENES
Supatcharee Siriwong1*, Pongrit Krubphachaya1, Kanjana Thumanu2, Griangsak Eumkeb1

1
School of Pharmacology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
2
Synchrotron Light Research Institute (Public Organization), Nakhon Ratchasima 30000, Thailand.
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KEYWORDS: Streptococcus pyogenes, FT-IR microspectroscopy, Flavonoids
INTRODUCTION
Streptococcus pyogenes is one of the most frequent pathogens of humans. S. pyogenes can infect when
defenses are compromised or when the organisms are able to penetrate the constitutive defenses. It is the
cause of many important human diseases, ranging from mild superficial skin infections to life-threatening
invasive illness. One of the most severe invasive manifestations is streptococcal toxic shock syndrome
(STSS)1). Beta-lactam antibiotics such as penicillin and amoxicillin are uniformly effective against most
strains of S. pyogenes. However, increasing antimicrobial resistance of S. pyogenes has been observed
during the last decade in Europe and worldwide2). Flavonoids constitute the largest group of plant
phenolics, accounting for over half of the eight thousand naturally occurring phenolic compound3).
Phenolic compounds and flavonoids exhibit a wide range of physiological properties such as anti-
allergenic, anti-inflammatory and antimicrobial. The antibacterial activity of flavonoids is being
increasing documented and some researchers have reported synergy between naturally occurring
flavonoids and other antibacterial agents against resistant strains of bacteria. Although the antibacterial
activity of flavonoids against various pathogens has been reported, the little is known about its activity on
S. pyogenes. FT-IR microspectroscopy technique can be employed to perform in depth molecular level
and also use to study spectral change resulting from bacterial injuries. The spectra of bacterial cells reflect
the biochemical structure and composition of the cellular constituent that include fatty acids, proteins,
polysaccharides and nucleic acids. This technique has been applied to differentiate intact microbial cells
base on the unique surface biochemistry of bacterial cell4). Therefore, this study was aimed to investigate
antibacterial effect and synergistic effect with antibiotic of flavonoids against S. pyogenes. Furthermore,
the FT-IR microspectroscopy technique was used to characterized biochemical compound on whole
bacterial cell after treatment with antibacterial compound.

Strain and growth conditions S. pyogenes ATCC 19615 was used in this study and cultivated with
aeration at 37 ºC in Brain Heart Infusion (BHI) broth. The cell culture was centrifuged at 4,000 rpm for
10 min. The cell pellets were washed with saline (0.85% NaCl), recentrifuged and resuspended in saline.
The cell was adjusted to appropriate concentration for antibiotic susceptibility testing.
Drug and chemical preparations Stock solution of ceftazidime was dissolved in distilled water and
isolated flavonoids contain luteolin, baicalein and quercetin were prepared in 5% DMSO.
Minimum Inhibitory Concentrations (MICs) determination Overnight culture (18 hr.) was adjusted to
give 5 x 105 CFU/ml in BHI broth, which plus 10% serial dilutions of the ceftazidime or isolated
flavonoid, Tubes of broth without antibacterial agent was used as the control. The lowest concentration of
each antibacterial which inhibited growth was taken to be the MIC5-6).
Checkerboard determination The culture from 18 hr was adjusted to give final concentration of 1x105
CFU/ml in BHI broth, which plus 10% serial dilutions of the ceftazidime alone and in combination with
isolated flavonoids, tubes of broth without antibacterial agent was used as the control. MICs were
determined for antibacterial combination and the isobologram was plotted. The Fractional Inhibitory
Concentration (FIC) indexes were calculated5-6).
Viability curve determination Bacterial concentration of 5 x 105 CFU/ml in BHI broth was exposed to the
ceftazidime alone or in combination with isolated flavonoids at selected concentrations an incubation
temperature at 37 ºC. Viable count was determined after contact time of 0, 0.5, 1, 2, 4 and 6 hr on BHI
agar plates in triplicate. An incubation at 37 ºC for 18-24 hr was allowed counting of growing colonies
and viability curve was plotted7).
Effect of flavonoids on cytoplasmic membrane of S. pyogenes S. pyogenes ATCC 19615 was cultured
into 100 ml BHI broth and incubated at 37 ºC for 18–24 h. After incubation, the bacterial cells were
separated by centrifugation at 10,000g for 10 min and resuspended in PBS (pH7.4). Suspensions were
adjusted to OD 600 of 2.0. Different conditions of treatment were added to the cell suspension. The

experiment was done in triplicates. Cells without ceftazidime and flavonoids were used as control. All the
samples were incubated at 37 ºC for 60 min. After treatment, the cell suspension was centrifuged at
13,400g for 15 min and OD 260 value of the supernatant was taken as a percentage of the extracellular UV-
absorbing materials released by cells8).
Bacterial whole cells preparation for FT-IR technique After treatment with ceftazidime and isolated
flavonoids. Bacterial cells were centrifuged and discard supernatant, cells were washed in saline for 2
times to remove culture media. Cell suspensions in saline were washed in sterile distilled water for 2
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times and then deposited into Mirr IR low e-microscope slides. Dried cell in desiccator was resuspended
in pure water before used9).
FT-IR microspectroscopy analysis Cell suspensions was dissolved in sterile distilled water and then
deposited into Mirr IR low e-microscope slides (Kevey slide) used as a substrate for FT-IR microscope
analysis. The samples were then desiccated under vacuum for several hours and stored in desiccators to
form films suitable before analysis. Spectra were collected on a Bruker IR spectrometer (tensor 27)
coupled to an IR microscope (Hyperion 2000) with 36x magnification. Second derivative and vector
normalize were manipulated to account for difference in sample thickness, minimize baseline variation
and allowed visual comparison. All data analysis were carried out in the spectral range from 3000-2800
cm-1and 1800-900 cm-1, which covers the mixed region of lipid, protein and polysaccharide. The original
spectra of bacterial whole cells were generated second derivatives, and subsequently vector normalized
for multivariate statistical analysis, using the Unscrambler 9.7 software (Camo, Norway) and Cluster
analysis, using the OPUS 6.5 software (German)9).
RESULTS
Minimum Inhibitory Concentrations (MICs) MIC is defined as the lowest concentration of the
antimicrobial agent in the BHI broth resulting in the complete inhibition visible growth. The MICs for the
ceftazidime, luteolin, baicalein and quercetin against S. pyogenes ATCC 19615 are shown in Table 1.
This strain was susceptible to ceftazidime, luteolin, baicalein and quercetin at MIC of 0.25 µg/ml, 128
µg/ml, >256 µg/ml and 128 µg/ml respectively.
Checkerboard assay The fraction inhibitory concentration of ceftazidime plus isolated luteolin, baicalein
and quercetin were 0.625, <0.625 and 0.531 respectively (Table 1). The results showed that ceftazidime at
concentration of 0.25 µg/ml was significantly reduced when combined with isolated flavonoids against
this strain.
Table 1 Minimum Inhibitory Concentrations (MICs), fractional inhibitory concentrations (FICs) and FIC indexes determined by
checkerboard assays of ceftazidime, luteolin, baicalein, quercetin alone and ceftazidime in combination with flavonoids against S.
pyogenes ATCC 19615
S. pyogenes ATCC 19615 MIC (µg/ml) FIC (CF + FV) (µg/ml) FIC index
Ceftazidime 0.25 - -
Luteiolin 128 0.125 + 16 0.625
Baicalein >256 0.125 + <32 <0.625
Quercetin 128 0.125 + 4 0.531

CF = Ceftazidime; FV = Flavonoids
Viability curve The synergistic activity of ceftazidime in combination with luteolin, baicalein and
quercetin were confirmed in viability curve. The combination effect of ceftazidime and these selected
flavonoids were more effective than ceftazidime alone. It can be seen in figure 1 that the viable counts of
S. pyogenes ATCC 19615 were significantly reduced in the presence of the combination of ceftazidime
and these selected flavonoids compared to ceftazidime, these selected flavonoids alone and control group.
The cells in the combination groups of either ceftazidime plus quercetin or baicalein were decreased to
104 CFU/ml at 6 hr.

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Figure 1 Viability of S. pyogenes ATCC 19615 after treatment with ceftazidime alone and in combined with flavonoids. ,
control; , ceftazidime 0.125 µg/ml; , baicalein 84 µg/ml; , quercetin 43 µg/ml; , luteolin 43 µg/ml; ,
ceftazidime 0.125 µg/ml + quercetin 4 µg/ml; , ceftazidime 0.125 µg/ml + baicalein 32 µg/ml; , ceftazidime 0.0625
µg/ml + luteolin 4 µg/ml. The bars represent the standard deviations of three replicates.
Effect of flavonoids on cytoplasmic membrane of S. pyogenes The leakage of cytoplasmic membrane

was analysed by determining the release of cell materials. Figure 2 shows that the OD 260 of bacterial cell
material after S. pyogenes ATCC 19615 suspensions were treated with either ceftazidime alone and in
combination with flaovnoids. After treatment with combination of ceftazidime and flavonoids, the OD
was increased from 0.01 in control group to 0.02 in ceftazidime alone treated group. Especially, the
ceftazidime plus either luteolin or baicalein were clearly risen up to 0.12 and 0.14 respectively. The
results suggest that combination of ceftazidime and selected flavonoids damages cytoplasmic membrane
and causes subsequent leakage of intracellular constituents.
.20
.15
OD260
.10
.05
0.00
ol Lu ue ai
ntr fta a+ +Q +B
Co Ce Ce
ft a a
Ceft Ceft
Figure 2 Measuring absorbance of the cell materials contents at 260 nm releasing from S. pyogenes ATCC 19615 after treated with
ceftazidime alone and in combination with flavonoids. (Cefta = Ceftazidime, Lu = Luteolin, Que = Quercetin, Bai = Baicalein)
FT-IR characterization studies The spectra of S. pyogenes ATCC 19615 cultivated in BHI broth are
shown in Figure 3a-b. The effect of treated group of ceftazidime and isolated flavonoids were harvested
and analyzed using FT-IR microspectroscopy. Each spectrum contains information about functional
groups arising from predominantly carbohydrate, protein and nucleic acids. In Figure 3a the band at
~2921, ~2852 cm-1 of treated groups that correspond to the C–H stretching of CH 2 vibration, were
significantly increased compared to control group. No difference was observed in band content at ~2963,
~2875 cm-1 that assigns to C–H stretching of CH 3 vibration on control and treated groups. This band
centered corresponding to stretching mode of asymmetric CH 3 , CH 2 vibration mainly due to membrane
lipids. The conformational change of protein amide I noted between 1700-1600 cm-1 can give information
of protein secondary structure such as α-helix (centered at ~1650 cm-1), β-sheet (centered at ~1635 cm-1),
β-turn (centered at ~1685 cm-1). FT-IR spectral was changed over the growth phase of S. pyogenes ATCC
19615 treatment with ceftazidime plus baicalein. The spectra show in figure 3b indicated that the α-helix,
β-sheet of amide I and amide II band were higher than all treated and control groups. In addition, the α-
helix of amide I and amide II band of ceftazidime plus baicalein was shifted after bacterial growth phase

was treated. However, the P=O symmetric stretch of nucleic acid ribose or deoxyribose moieties (~1080
cm-1) of ceftazidime plus baicalein was clearly decreased. While, ceftazidime plus quercetin treated
group revealed high content of P=O symmetric stretch of nucleic acid ribose or deoxyribose.
a b
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Figure 3a Representative 2nd derivative transformation spectra (~3000-2800 cm-1) and 3b Representative 2nd derivative
transformation spectra (~1800-900 cm-1) of S. pyogenes ATCC 19615 after treatment with ceftazidime alone and in combination
with flavonoids. The spectra were collected and averaged from 627 spectra.
DISCUSSION
The results from MICs determination indicated that the isolated flavonoids inhibited S. pyogenes ATCC
19615. These results are in substantial agreement those of Banso and Mann, they found that the
antibacterial test of the flavonoid fraction from Antiaris africana showed activity against B. subtilis, S.
pyogenes and E. coli 10). The checkerboard assay revealed that FIC indices of ceftazidime plus flavonoids
were lower than 1.0 against this strain. It has been proposed that synergy can be announced when the FIC
index < 1.0 11). Therefore, the ceftazidime plus these flavonoids exhibited synergistic effect against this
train. These synergistic effects were confirmed by killing curve effect. The measurement of UV-
absorbing materials releasing is an index of cell lysis and non selective pore formation12). After treatment
with ceftazidime and isolated flavonoids, the leakage of cytoplasmic membrane was analysed by
determining the release of cell materials such as nucleic acid which was absorbed at 260 nm. The results
indicated that the combination of ceftazidime plus flavnoids led to higher cell leakage that appeared to
lose nucleic acid, critical molecule and ions. The obtained spectral changes of the bacteria treated with
ceftazidime plus flavonoids provide evidence that the chemical composition of these cells were varied.
Our results showed significant increase in band area at ~2920 to ~2850, ~1653, ~1639 and ~1544 cm-1
which seem to represent the amounts of CH 2 of fatty acid, alpha-helix, beta-sheet of amide I and amide II,
respectively when treated with the combination of ceftazidime and flavonoids. In addition, a major
reduction in the band area of amide I and amide II was found in ceftazidime alone treated group.
Moreover, there were undoubtedly decreased in spectrum at ~1085 cm-1 of ceftazidime plus baicalein
treated group compared to others, this spectral variation suggests that phosphodiester functional group is
lower.
CONCLUSION
Our findings provide evidence that all selected flavonoids have synergistic effect with ceftazidime aginst
S. pyogenes ATCC 19615. The primary mechanism of action of ceftazidime plus these flavonoids is 1.
Disruption of the cytoplasmic membrane function of S. pyogenes ATCC 19615 which resulted in a loss of
cytoplasmic constituents and ions. 2. Alter protein synthesis and disrupt nucleic acid synthesis whereas do
not effect fat synthesis. Of the three compounds, baicalein plus ceftazidime showed good inhibited
viability curve. In conclusion, FT-IR microspectroscopy may use to determine the presence of injured
antibacterial agent that could be underestimated by conventional microbial technique.
ACKNOWLEDGMENT
The authors wish to thank the School of Pharmacology, Faculty of Science, Suranaree University of
Technology for research funds. And grateful acknowledge Synchrotron Light Research Institute (Public
Organization) Thailand to support the FT-IR microspectroscopy technique.

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phytopharmaceuticals. Phytomedicine 16: 97-110.
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antilisteria bacteriocin produced by Lactobacillus pentosus from Chinese traditional ham. Food
Control 19: 817-822.

THE EFFECTS OF RED KWAO KRUE (BUTEA SUPERBA ROXB.) EXTRACT

ON SPERM QUALITY AND TESTOSTERONE LEVEL IN MICE
Griangsak Eumkeb1, Wanatkamol Naknarong1, Kittipot Sirichaiwetchakoon1,*

1
School of Pharmacology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
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KEYWORD: Butea superba Roxb., Sperm motility, Sperm count, Reproductive system, Testosterone
INTRODUCTION
Butea superba Roxb. has been claimed to use for aphrodisiac1). Male infertility refers to the inability of a
male to achieve a pregnancy in a fertile female that low quantity or poor quality of the sperm and lack of
testosterone are the most common causes2). Approximately 30% of infertile couples experience infertility
due to male factor i.e. reduction in sperm quality and production. Clinical andrologists have therefore
tried to improve the fertility potential of the sperm cells to achieve better results in spontaneous
fertilization or IVF/ICSI3). Moreover, lower testosterone level may increase risk of Alzheimer’s disease in
older men4). Previous finding showed that sildenafil significantly increased total testosterone level, sperm
motility, sperm concentration, sperm–oocyte binding, resulting in stimulation of spermatogenesis,
promotion of the physical and functional maturation of spermatozoa, and maintaining the accessory
organs of the male reproductive tract5-6). However, the results of these studies are controversial. Some of
these studies demonstrated no significant effects of sildenafil on these parameters7). Therefore, new
generation of phytopharmaceuticals that increase spermatogenetic and androgenic effects to treat these
diseases instead of synthetic drugs alone are research objectives of far reaching importance. The objective
of this study was to investigate the effects of Butea superba extract and sildenafil on the sperm quality
and testosterone level in male mice.

Plant materials and preparation of extracts Fresh tuberous roots of Butea superba Roxb. were collected
from Chiang Rai province, Thailand. The plant specimens was authenticated and compared to herbarium
specimen (BCU 11046) at the herbarium of the Department of Botany, Faculty of Science, Chulalongkorn
University.
Extraction Dried, powdered roots of Buteasuperba Roxb. (25 kg) were extracted continuously with
ethanol by Soxhlet extractor, filtered through filter paper, and evaporated to remove the solvent. The
ethanol extract (375 g) was separated by silica gel column chromatography and high performance liquid
chromatography (HPLC). The spectral analyses, including UV, IR, LC-MS, 1H and 13C NMR were
compared with previously reported values and values obtained from standard compounds.
Animals Sixty adult male mice, aged about 130 days, weighing 30-40 g, were obtained from the Animal
Care Building, Suranaree University of Technology, Nakhon Ratchasima, Thailand. The experimental
protocol was approved in accordance with guideline for the care and use of laboratory animal by animal
care and use committee (ACUC). The animals were housed at room temperature (25 ± 0.5 ºC) on a
reverse day-night cycle.
Experimental procedures Mice were divided into 6 groups with 10 animals each. Before treatment (pre-
treatment), blood and sperm of all mice in these groups were collected for comparison to those of the
post-treatment. During treatment period, control (Con), Butea superba Roxb. crude extract (Cru),
sildenafil (Sil), fraction B (FrB), fraction C (FrC), and fraction E (FrE) mice, were fed with either 0.5 ml
of distilled water, 1, 250, 10, 40, 50, and 150 mg/kg BW/day, respectively. The animal’s weight was
recorded every day throughout 14 consecutive days. At the end of treatment period, all mice were
sacrificed and subjected to necropsy. The heart, liver, spleen, kidney, stomach and the reproductive
organs (testis, seminal vesicle and prostate glands) were examined. The blood and sperm were collected
to compare sperm motility, sperm count, haematology, testosterone level and blood chemistry with pre-
treatment in each mouse and between groups.
Statistical analysis All data are presented as the mean ± S.E.M. Significant differences between the
relative selected organ weight and body weight of control and treatment groups were analyzed by
ANOVA. The difference between pre- and post- treatment groups were calculated by paired student’s t-
test. Then, significant difference between each group was compared using ANCOVA. The Tukey HSD
post hoc test at p<0.05 and p<0.01 were considered statistically significant difference between each
group.

RESULTS
The quality and quantity of crude extract and each fraction from Butea superba The major components
of each fraction were identified and orally administered to male mice. The 1.5% (w/w) yield from dried
powder of Cru at the dose of 1,250 mg /kg BW/day consisted of 53.57 µg of genistein plus 312.95 µg of
biochanin A and 396.59 µg of unknown compound 1 (Un1). FrB (0.022% yield) at the dose of 40 mg/Kg
BW/day consisted of 1,260.00 µg of unknown compound 1 (Un1). FrC (0.030% w/w yield) at the dose of
50 mg/Kg BW/day consisted of 6.78 µg of genistein plus 31.59 µg of unknown compound 1 (Un1). Also,
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FrE (0.054% w/w yield) at the dose 150 mg/Kg BW/day consisted of 66.92 µg of biochanin A as the main
compound plus 9 unkown compounds.
Sperm motility The effects of BS extracts and sildenafil on sperm motility (%) of mice are presented in
Figure 1. The results exhibited that there were significant increases in sperm motility of all post-treated
groups compared to pre-treatment (p<0.01) except for control. Apart from this, the highest motility level
was found in the fractions C and E compared to others including sildenafil (p<0.01).
120
pre-treated level
110 post-treated level
A
ANCOVA adjusted post-treated level A
100 ** a
90
** a
B
B
** b
sperm motility (%)
80 * bc BC
C
70 d * cd
60
50
40
30
20
10
0
Con Cru Sil FrB FrC FrE
groups
Figure 1 Effects of BS extracts and sildenafil on sperm motility (%) of mice. Significant difference between pre- and post-test in
each group was compared using paired student t-test at * p <0.05, ** p <0.01. Significant difference between ANCOVA adjusted
post-treated level in each group was compared using ANCOVA and Tukey HSD post hoc test at; a p<0.05, A p<0.01.
Sperm count and morphology Figure 2 shows the effects of BS extracts and sildenafil on sperm number
(x100,000 n/ml) of mice. The results exhibited significant increase in sperm number of all post-treated
groups compared to pre-treatment (p<0.01) except for control. Also, fraction C showed the highest sperm
number. Besides, the sperm number of crude extract, sildenafil, fractions B and E treated groups were
significantly higher than control group (p<0.01). The morphology of these sperms was normal compared
with control. In fact, the result of sperm count was almost the same as that of sperm motility.
. 450
pre-treated level
post-treated level
sperm number (x 100,000 n/mL)
400
ANCOVA adjusted post-treated level
A
350 ** a
B
300 BC
** bc ** b
250 BC BC
** c **
c
200
D
150
d
100
50
0
groups
Figure 2 Effects of BS extracts and sildenafil on sperm number (x100,000 n/ml) of mice. Significant difference between pre- and
post-test in each group was compared using paired student t-test at * p <0.05, ** p <0.01. Significant difference between ANCOVA
adjusted post-treated level in each group was compared using ANCOVA and Tukey HSD post hoc test at; a p<0.05, A p<0.01.
Haematology and blood chemistry The cholesterol level of post-treated groups of fractions C, E, B and
sildenafil were significantly higher than the baseline level. The cholesterol level of fractions C, E and
sildenafil were significantly higher than those of control (p<0.05), whereas the hemoglobin level of
sildenafil and crude extract post-treated groups were significantly lower than the baseline value at p<0.01
and 0.05, respectively. Although the hemoglobin level of fraction E treated group was significantly higher
than those of control and crude extract groups (p<0.05). Obviously, only post-treated sildenafil group
exhibited significantly lower hematocrit level than baseline (p<0.01). Furthermore, the PMN level of
fractions B and C post-treated group were significantly higher than the baseline value at p<0.05 and 0.01,

respectively. However, these levels were not significantly different from that of control. Similarly, the
MCHC level of fraction C post-treated group was significantly higher than pre-treated level (p<0.05);
however, the value was not significant compared to those of control. In addition, the MPV level of crude
extract and sildenafil treated groups were significantly lower than control (p<0.01).
Histology of testis It can be seen from Figure 3 that the spermatogenesis of all treated groups yielded
more mature spermatids than that of control. These spermatogenesis processes were higher in both
spermatogenesis levels and spermatid numbers. These findings lend support to assumption that
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compounds isolated from BS can increase both sperm number and motility.
a b c
d e f
Figure 3 Micrographs of testicular (seminiferous tubules) section of mice; a = control DW 0.5 ml/kg BW/day, b = crude extract
1,250 mg/kg BW/day, c = Sildenafil 10 mg/kg BW/day, d = fraction B 40 mg/Kg BW/day, e = Fraction C 50 mg/Kg BW/day, f =
Fraction E 150 mg/kg BW/day. All groups were treated for 14 days. All micrographs displayed at X100 magnification.
Testosterone Level Figure 4 shows the effects of BS extracts and sildenafil on testosterone level of mice.
There were significant increases in testosterone level of all post-treated groups compared to pre-treatment
(p<0.01) except for the control. The highest testosterone level was observed in fraction C treated groups.
Serum testosterone level of both fractions C and E treated groups were significantly higher than other
groups (p<0.01). This level in the crude extract, fraction B and sildenafil treated groups was also
significantly higher than control (p<0.01).
1800
serum testosterone level (ng/mL)
pre-treated level
1600
post-treated level ** A
ANCOVA adjusted post-treated level
1400
** B
1200
1000
C ** C
800 ** C **
600
400
D
200
0
groups
Figure 4 Effects of BS extracts and sildenafil on testosterone level (ng/ml) of mice. Significant difference between pre- and post-
test in each group was compared using paired student t-test at * p<0.05, ** p<0.01. Significant difference between ANCOVA
adjusted post-treated level in each group was compared using ANCOVA and Tukey HSD post hoc test at; a p<0.05, A p<0.01.
Body weight There was no significant difference in the relative growth rate measured by living body
weight of male mice treated with BS extract compounds and sildenafil groups when compared to the
control (p<0.01).
Selected reproductive and vital organs The testes weights of crude extract and sildenafil treated groups
were significantly higher than those of control and other groups (p<0.01). Also, the epididymis weight of
fraction E was significantly higher than others, whereas those of the crude extract, fraction B and
sildenafil treated groups were significant lower than control (p<0.01). Furthermore, the seminal vesicle
weights of fractions B, C and E treated groups were significantly higher than the remainder of these
groups (p<0.01). The prostate gland weights were significant higher in the crude extract and sildenafil
groups (p<0.01). However, the spleen weights of fractions B, C and E treated groups were significantly
higher than control (p<0.01). On the contrary, the stomach weight of fraction E treated group was lower
than control (p<0.01).
The histopathology of the heart, liver, spleen, kidney, and stomach of all treated groups revealed normal
appearance compared to control group.

DISCUSSION
This investigation provides evidence that Butea superba extract could increase testosterone level, sperm
number and motility in mice. This extract might therefore be developed to increase testosterone, sperm
number and motility in men.
The results exhibited that there were significant increases in testosterone level of all post-treated groups
compared to pre-treatment (p<0.01) except for the control. In addition, the increase in testosterone level
of fraction C treated groups was the highest and this level of both fractions C and E treated groups were
BP - 17
significantly higher than those of other groups (p<0.01). Apart from this, the results showed significant
increase in sperm number of all post-treated groups compared to pre-treatment (p<0.01) except for
control. Fraction C also showed the highest sperm number. Besides, the sperm number of crude extract,
sildenafil, fractions B and E treated groups were significantly higher than control group (p<0.01). These
results were confirmed by sperm morphology micrographs. Similarly, there were significant increases in
sperm motility of all post-treated groups compared to pre-treatment (p<0.01) except for control. The
highest motility level was found in groups treated with fractions C and E (p<0.01). These findings
provide evidence that genistein, Un1 and biochanin A may play an important role in increasing the sperm
number, motility and testosterone level. These results support previous finding that that sildenafil-treated
mice showed significantly increased total testosterone level, as well as noteworthy ultrastructural
alterations of Leydig cells, such as a vesicular smooth endoplasmic reticulum, large vacuoles, enlarged
discontinue cristaes of mitochondria and vesicles of whorle membranes at the periphery typical of an
activated steroid-secreting cell5). Previous findings on the stimulation of Leydig cells by sildenafil can be
explained by the function of Leydig cells in producing testosterone, the male sex hormone, which
stimulates spermatogenesis, promotes the physical and functional maturation of spermatozoa, maintains
the accessory organs of the male reproductive tract, etc. Therefore, the increase in testosterone level
would result in higher sperm number and motility. Recent studies have also revealed that blood
testosterone concentrations were lower in male patients suffering from Alzheimer’s disease (AD)2,4).
These Butea superba extracts might be useful to treat AD in men in order to increase testosterone level in
these patients. Blood analysis showed that the cholesterol level of fractions C, E and sildenafil treated
groups were significantly higher than control (p<0.05). One reason could be that cholesterol, a precursor
of testosterone, is prepared to synthesize testosterone8-9).
CONCLUSION
These findings provide evidence that Butea superba extract in fractions C and E and sildenafil can
increase testosterone level, sperm number and motility of mice compared to control group. These results
can be explained by assuming that genistein, unknown compound 1 and biochanin A may play important
role. These findings may provide evidence that these BS extracts may be developed as treatment to
increase testosterone level, sperm number, sperm motility and infertility or Alzheimer’s disease in men
after safety level is investigated.
ACKNOWLEDGEMENT
We are indebted and highly appreciative to School of Pharmacology, Institute of Science, for grant
support. National Research Council of Thailand for fund. Dr. Santi Sakdarat for suggestion and Miss
Sudarat Tanpongrung for kind laboratory help.
REFERENCES
1. Soonthorn L. 1931. Herbal recipe of tuberous KwaoKrua. Chiang Mai. UppatipongPublisher :p.15.
2. Moffat SD, Zonderman AB, Metter EJ, Kawas C, Blackman MR, Harman SM, et al. 2004. Free testosterone and risk for
Alzheimer disease in older men. Neurology. 62(2):188-93.
3. Isidori AM, Pozza C, Gianfrilli D, Isidori A, 2006. Medical treatment to improve sperm quality. Reproductive
BioMedicine Online 12, 704-14.
4. Hogervorst E, Williams J, Budge M, Barnetson L, Combrinck M, Smith AD. 2001. Serum total testosterone is lower in
men with Alzheimer's disease. Neuro Endocrinol Lett 22(3):163-68.
5. Saraiva KL, Silva AK, Wanderley MI, De Araujo AA, De Souza JR, Peixoto CA. 2009. Chronic treatment with sildenafil
stimulates Leydig cell and testosterone secretion. Int J Exp Pathol. 90(4):454-62.
6. Dimitriadis F, Tsambalas S, Tsounapi P, et al., 2010. Effects of phosphodiesterase-5 inhibitors on Leydig cell secretory
function in oligoasthenospermic infertile men:a randomized trial. BJU Int. 106:1181-5.
7. Dimitriadis F, Giannakis D, Pardalidis N, et al., 2008. Effects of phosphodiesterase-5 inhibitors on sperm parameters and
fertilizing capacity. Asian J Androl. 10:115-33
8. Maqdasy S, Baptissart M, Vega A, Baron S, Lobaccaro J-MA, Volle DH. 2013. Cholesterol and male fertility: What
about orphans and adopted? Mol Cell Endocrinol. 368(1–2):30-46.
9. Midzak AS, Chen H, Papadopoulos V, Zirkin BR. 2009. Leydig cell aging and the mechanisms of reduced testosterone
synthesis. Mol Cell Endocrinol. 299(1):23-31.

Session 3
Pharmaceutical Technology PT 1 - 36
DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR QUANTITATIVE

ANALYSIS OF INDIVIDUAL γ-ORYZANOL IN COLD PRESSED RICE BRAN OIL
Apirak Sakunpak1,2*, Jirapornchai Suksaeree1,2, Laksana Charoenchai1,2, Chaowalit Monton1,2,

Pathamaporn Pathompak1,2 , Tossaton Charoonratana1,2, and Krisana Kraisintu2,3
1
Faculty of Pharmacy, Rangsit University, Pathum Thani 12000, Thailand
2
Sino-Thai Traditional Medicine Research Center, Herbal Medicinal Products Research and Development Center (Cooperation
between Rangsit University and Harbin Institute of Technology and Heilongjiang University of Traditional Chinese Medicine),
Rangsit University, Pathum Thani, 12000, Thailand
3
Faculty of Oriental Medicine, Rangsit University. Pathum Thani, 12000, Thailand
*
Corresponding author: apirak.s@rsu.ac.th (Apirak Sakunpak)
KEYWORDS: rice bran oil, oryzanol, RP-HPLC, validation
INTRODUCTION
The people of Thailand have been using the brown outer layer of the rice kernel, known as rice bran, for
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generations. Rice bran is rich in oil and frequently sold as a dietary supplement. It is a plentiful source of
many bioactive compounds, including γ-oryzanol, phytosterols, ferulic acid and phytic acid. γ-Oryzanol
(steryl ferulates) has been shown to be a major bioactive compound in rice. The four major γ-oryzanol
constituents in rice were determined to be cycloartenyl ferulate, 24-methylenecycloartanyl ferulate,
campesteryl ferulate, and β-sitosteryl ferulate. They have been previously shown to display various
biological activities including anti-inflammatory, antioxidant and anti-tumor activities [1]. The reported
values of γ-oryzanol range from 0.2 to 2.72%, depending on the method of extraction, rice variety,
weather, and area of cultivation [2, 3]. Nowadays, HPLC methods for quantitative determination of
γ-oryzanol in rice bran oil have been reported and determining the total content of γ-oryzanol in rice
products rather than on analyzing the content of individual steryl ferulate [4, 5]. Recently, the individual
steryl ferulates in conventional and organic brown rice were determined by HPLC [6]. However, long time
for analysis was required and RP-HPLC validation methods have not yet been conducted. In this study
four major of γ-oryzanol in cold pressed rice bran oil were identified by mass spectroscopy and RP-HPLC
method was developed for determination of γ-oryzanol content. The RP-HPLC method was also
validated.

Materials Solvents used for chromatography were acetonitrile (HPLC grade), methanol (HPLC grade),
isopropanol (analytical grade) and water (HPLC grade). All solvent were obtained from B&J (Korea). A
standard mixture of γ-oryzanol (analytical grade) was purchased from TCI, Tokyo, Japan. Hom-Pathum
rice bran samples were provided by a local milling company in Thailand. The rice bran samples were
passed though sieve number 20 and immediately extracted under cold press conditions.
Isolation of individual γ-oryzanol The four major of steryl ferulates were isolated from standard
γ-oryzanol mixture by HPLC. A 50 mg of γ-oryzanol mixture was dissolved in isopropanol 10 ml. An
aliquot of 30 µl was separated on HPLC (Agilent Technologies, USA) with a Poroshell 120 EC-C18
column (3.0×150 mm, 2.7µm). Elution was achieved by using a solvent mixture of acetonitrile and
methanol (60:40 v/v), with a flow rate of 0.8 ml/min. Four peak were collected (peak 1 t R 10.6 min; peak
2 t R 12.2 min; peak 3 t R 13.8; peak 4 t R 15.9 min). The isolated compounds were used as an external
standard for HPLC quantitative analysis.
Identification of individual γ-oryzanol LC-MS was carried out using Dionex Ultimate TM3000
equipped. The sample was separated at 25 ºC on a Poroshell 120 SB-C18 (2.1×150 mm, 2.7 µm) using a
mobile phase consisting of acetonitrile and methanol (60:40 v/v). Injection volume was 10 µl and the UV
detector was at 325 nm (variable wavelength detector). Individual γ-oryzanol was identified with high
capacity 3D quadrupole ion trap (Bruker Amazon SL). The mass spectrometer was equipped with an ESI
ion source. The ESI-MS spectra were acquired in negative ionization mode recorded on a mass range of

m/z 100-800. Capillary voltage was 4500 V. Drying gas temperature was set at 200 ºC with a flow rate of
7.0 L/min and nebulizing pressure was of 2 bar. Data were processed by Compass 1.3 SR2.
HPLC conditions HPLC analysis was carried out using the Agilent 1200 series equipped with an Agilent
1200 series photodiode-array detector (PDA) and autosampler. Data analysis was performed using
OpenLAB CDS EZChrom software (Agilent, USA). Separation was achieved at 25 ºC on a Poroshell 120
EC-C18, 3.0×150 mm, 2.7 µm (Agilent Technologies, USA). The mobile phase consisted of acetonitrile-
methanol (60:40 v/v) and was pumped at a flow rate of 0.8 ml/min. The injection volume was 10 µl. The
quantitation wavelength was set at 325 nm.
Preparation of standard solutions γ-Oryzanol was accurately weighed to 25 mg in a volumetric flask
(size 25 ml) and the volume was adjusted to 25 ml with isopropanol. The stock solution was serial two-
fold diluted to six concentrations and filtered through a membrane filter (0.45 µm) before HPLC analysis.
Preparation of samples Rice bran oils were accurately weighed to 25 mg in a volumetric flask (size 25
ml) and the volume was adjusted to 25 ml with isopropanol. The samples were filtered through a
membrane filter (0.45 µm) before HPLC analysis. The experiments were carried out in triplicate.
Method validation For validation of the analytical method, the guidelines of the International Conference
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on Harmonization of Technical Requirement for the Registration of Pharmaceuticals for Human Use were
followed (ICH, 2005). Calculated data were checked for their linearity, accuracy, intraday and interday
precision specificity, LOD and LOQ to validate the HPLC method.
Calibration curve and linearity The calibration curves were analysis of a mixture containing each of the
standard γ-oryzanol at six concentrations and plotting peak areas against the concentration of each
reference standard. The linearity of the detector response for the standards was determined by means of
linear regression analysis. The calibration curve should show a coefficient of correlation (R2) ≥ 0.9995.
Accuracy Rice bran oil sample solutions were fortified with three concentrations of known quantities of
the standard γ-oryzanol in order to check the accuracy of the data. Prior to fortification with standard
γ-oryzanol the background levels of standard γ-oryzanol in the rice bran oil were determined so as to
calculate actual recoveries. The amounts of γ-oryzanol were determined in triplicate and the percentage
recoveries were then calculated.
Precision Precision experiments were conducted to ascertain any intraday and inter-day variability. The
solution of one rice bran oil sample was used to check the intra-day precision. Six separate injections of
this sample were carried out on the same day. The data were used to calculate the % R.S.D. (not more
than 2%) for intraday precision. The inter-day precisions were validated by repeating the extraction
procedure on the same sample. An aliquot of each extract was then injected and quantified. This
parameter was evaluated by repeating the extraction in triplicate on three different days with a freshly
prepared mobile phase and sample. The data was used to calculate % R.S.D. (not more than 5%) for inter-
day precision.
Specificity Peak identification was carried out using authentic standards and scanning the UV spectrum of
each peak using the photodiode-array detector. The UV spectra were taken at various points of the peaks
to check peak homogeneity.
Limits of detection (LOD) and quantification (LOQ) Serial dilutions of a reference standard were carried
out with isopropanol and were then analyzed by the HPLC method. LOD and LOQ were the
concentrations that give a signal to noise ratio equal to 3 and 10, respectively.

Peak identification and assignment Peak identification and assignment in HPLC fingerprints of rice bran
oil were based on the comparison mass spectral data with published data. Four characteristic peaks were
identified as cycloartenyl ferulate (peak 1 t R 10.6 min, [M-H]- (m/z) 601.5; MS/MS (m/z) 586.5), 24-
methylcycloartenyl ferulate (peak 2, t R 12.2 min, [M-H]- (m/z) 615.6; MS/MS (m/z) 600.5), campesteryl
ferulate (peak 3, t R 13.8 min, [M-H]- (m/z) 575.5; MS/MS (m/z) 560.5), β-sitosteryl ferulate (peak 4,
t R 15.9 min, [M-H]- (m/z) 589.6; MS/MS (m/z) 574.5). The chemical structures and MS spectra of
individual γ-oryzanol are shown in figure 1 and 2, respectively.

O
O O
O O
O
HO
HO
1 2
O
O O
O O
O
HO
HO
3 4
Figure 1 Chemical structures of the steryl ferulates identified from the commercial standard γ-oryzanol mixture (1) cycloartenyl
ferulate; (2) 24-methylenecycloartanyl ferulate; (3) campesteryl ferulate; (4) and β-sitosteryl ferulate.
586.5 A 600.5 B
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615.6
601.5
575.5 C 574.5 D
560.5 589.6
Figure 2 MS/MS spectra of cycloartenyl ferulate (A), 24-methylenecycloartanyl ferulate (B), campesteryl ferulate (C), and β-
sitosteryl ferulate (D)
Validation method The optimal conditions for the simultaneous quantitative determination of
cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, campesteryl ferulate, and β-sitosteryl ferulate in
cold pressed rice bran oil were examined using an isocratic RP-HPLC system. As all four compounds
have good absorption at 325 nm, this wavelength was used for quantitation. Mixtures of acetonitrile and
methanol (60:40 v/v) were examined as the mobile phase. This mobile phase and ratio obtained a good
resolution. All four compounds were eluted within 17 min with satisfactory resolution (Figure 3).
2
1 A
3
4
Figure 3 HPLC chromatograms of standard γ-oryzanol (cycloartenyl ferulate (1), 4-methylenecycloartanyl ferulate (2), campesteryl
ferulate (3), and β-sitosteryl ferulate (4)) (A) and Hom-Pathum cold pressed rice bran oil (B)

The linearity, accuracy, intra-day and inter-day precision, specificity and limits of detection and
quantitation were determined to validate the RP-HPLC method. The calibration curve for was linear over
the concentration range 500-15.125 µg/ml. Cycloartenyl ferulate, 4-methylenecycloartanyl ferulate,
campesteryl ferulate, and β-sitosteryl ferulate exhibited linearity over the evaluated ranges with
correlation coefficients 0.9996. Both intra-day and inter-day precission were estimated by the relative
standard deviation were less than 2% and 5%, respectively. Recoveries in the range of 100.1-101.9%
were observed for all compounds. Utilising the photodiode array (PDA) makes it possible to obtain the
UV spectra. Specificity of the method was evaluated using the UV-absorption spectra produced by the
photo diode-array detector. The spectra were taken at three points of the peak for cycloartenyl ferulate,
24-methylenecycloartanyl ferulate, campesteryl ferulate, and β-sitosteryl ferulate. When it was compared
with the standard, the spectra of the peak were observed to be homogenous. Finally, it was found that the
RP-HPLC method was very sensitive for detecting cycloartenyl ferulate, 24-methylenecycloartanyl
ferulate, campesteryl ferulate with LOD and LOQ values of 0.156 and 0.312 µg/ml. The LOD and LOQ
of β-sitosteryl ferulate were 0.781 and 1.562 µg/ml, respectively. All of validated data were shown in
Table 1.
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Table 1 Validation data of RP-HPLC method
%RSD LOQ LOD

Substances Linear equations %Recovery
Intra-day Inter-day (µg/ml) (µg/ml)
R1 y = 44978x - 38615 101.15 + 1.02 0.06 0.52 0.312 0.156
R2 y = 44989x - 54136 101.97 + 1.25 0.42 0.60 0.312 0.156
R3 y = 45040x - 19571 101.35 + 1.15 1.12 4.00 0.312 0.156
R4 y = 44983x - 9316 100.10 + 1.43 0.02 0.58 1.562 0.781
R1, cycloartenyl ferulate; R2, 24-methylenecycloartanyl ferulate; R3, campesteryl ferulate; R4, β-sitosteryl ferulate
Individual γ-oryzanol content in Hom-Pathum cold pressed rice bran oil was determined using HPLC. In
the present study, the content of individual γ-oryzanol were as follows cycloartenyl ferulate
(0.53±0.08%w/w), 24-methylenecycloartanyl ferulate (0.87±0.13%w/w), campesteryl ferulate
(0.45±0.06%w/w), and β-sitosteryl ferulate (0.31±0.11%w/w). Of these four compounds,
24-methylenecycloartanyl ferulate show the highest content in Hom-Pathum cold pressed rice bran oil
(Table 2).
Table 2 Individual γ-oryzanol content in Hom-Pathum cold pressed rice bran oil
Hom-Pathum rice % Content; Mean ± SD (%w/w)

bran/Lot Number R1 R2 R3 R4 Total
1 0.45 ± 0.01 0.73± 0.02 0.35 ± 0.01 0.30 ± 0.01 1.83 ± 0.04
2 0.61 ± 0.01 0.89 ± 0.01 0.48 ± 0.01 0.43 ± 0.01 2.41 ± 0.01
3 0.61 ± 0.01 0.89 ± 0.01 0.49 ± 0.01 0.42 ± 0.01 2.40 ± 0.01
4 0.55 ± 0.00 1.07 ± 0.03 0.50 ± 0.00 0.19 ± 0.00 2.31 ± 0.05
5 0.45 ± 0.01 0.79 ± 0.01 0.43 ± 0.01 0.23 ± 0.00 1.90 ± 0.03
Average 0.53 ± 0.08 0.87 ± 0.13 0.45 ± 0.06 0.31 ± 0.11 2.17 ± 0.28
R1, cycloartenyl ferulate; R2, 24-methylenecycloartanyl ferulate; R3, campesteryl ferulate; R4, β-sitosteryl ferulate
CONCLUSIONS
A simple, specific, precise, accurate, rapid and reproducible RP-HPLC has been developed to quantify
cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, campesteryl ferulate, and β-sitosteryl ferulate in
Hom-Pathum cold pressed rice bran oil. The separation was achieved on Agilent 1260 series with
Poroshell 120 EC-C18 (3.0×150 mm, 2.7µm) column. A mixture of acetonitrile and methanol (60:40)

were use as mobile phase and was pumped at a flow rate of 0.8 ml/min. The injection volume was 10 µl.
The quantitation wavelength was set at 325 nm.
ACKNOWLEDGEMENTS
The authors wish to thank the Faculty of Pharmacy and Sino-Thai Traditional Medicine Research Center
(Cooperation between Rangsit University and Harbin Institute of Technology and Heilongjiang
University of Chinese Medicine), Rangsit University, Pathum Thani, Thailand for all chemicals and
instruments.
REFERENCES
1. Lerma-Garcia MJ, Herrero-Martinez JM, Simo-Alfonso EF, et al. (2009). Composition,
industrial processing and applications of rice bran γ-oryzanol. Food Chem 115: 389-404.
2. Butsat S, Siriamornpun S. (2010). Antioxidant capacities and phenolic compounds of the husk,
bran and endosperm of Thai rice. Food Chem 119: 606-613.
3. Zullaikah S, Melwita E, Ju YH. (2009). Isolation of γ-oryzanol from crude rice bran oil.
Bioresour Technol 100: 299-302.
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4. Ohtsubo, K., Suzuki, K., Yasui, Y, Kasumi, T, (2005). Bio-functional components in the
processed pre-germinated brown rice by a twin-screw extruder. J Food Compost Anal 18: 303-
316.
5. Lee JH, Oh SK, Cho K.M, et al. (2009). Variations in physicochemical properties of brown rice
(Oryza sativa L.) during storage. Food Sci Biotechnol 18: 1398-1403.
6. Cho JY, Lee HJ, Kim GA, et al. (2012). Quantitative analyses of individual γ-oryzanol (Steryl
Ferulates) in conventional and organic brown rice (Oryza sativa L.). J Cereal Sci 55: 337-343.

QUANTIFICATION AND VALIDATION OF GALLIC ACID CONTENT IN TRI-PHALA

EXTRACT AND EFFERVESCENT TABLET BY HPTLC
Benjaporn Chainethvanich1, Worawan Saingam2, Prasan Tanguenyongwatana1

1
Faculty of Oriental Medicine, Rangsit University, Pathumthani 12000, Thailand
2
Faculty of Pharmacy, Rangsit University, Pathumthani 12000, Thailand
KEYWORDS: Gallic acid, Tri-phala, Effervescent tablet, HPTLC, method validation
INTRODUCTION
Gallic acid is a major compound in many herbs that have been used in Traditional Thai Medicine. This
compound is known to have anti-oxidation, anti-inflammatory, antimutagenic and anticancer activities1, 2).
In Thai traditional formula, Tri-phala has been used to adjust the balance of the elements in the body,
detoxification and rejuvenation3). Tri-phala composes of three herbs which are Terminalia bellerica
Roxb., Terminalia chebula Retz, and Phyllanthus emblica L. All of them have gallic acid as an active
constituent 4-6). This research developed a high performance thin-layer chromatography (HPTLC) method
for the simultaneous quantification of gallic acid in Tri-phala extract and effervescent tablet. The tablet
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weights size was 4000 mg and composed of 25% of Triphala extract. The method was validated for
linearity, precision, accuracy, limit of detection (LOD), and limit of quantitation (LOQ).

Instruments and reagents Reagents and solvents were reagent grade and used without further
purification. Gallic acid was purchased from Aldrich (USA). Freeze Dryer was performed on EYELA
FD-1(Tokyo Rikakikai, Japan). HPTLC was performed on pre-coated HPTLC silica gel GF 254 plates cat.
No.1.05548.0001 (Merck, Germany). Spotting device was Linomat 5 automatic sample spotter (CAMAG,
Muttenz, Switzerland). TLC chamber was glass twin-trough chamber (20 × 10 cm.) (CAMAG,
Switzerland). Densitometer was TLC scanner 3 with winCATS software (CAMAG, Switzerland).
Syringe was 100 μL size (Hamilton, Bonaduz, Switzerland).
Plant materials The plant materials of Terminalia bellerica, Terminalia chebula and Phyllanthus emblica
were bought from local drugstore in Nonthaburi province, Thailand. All of them were identified by
comparison with the specimens at the Forest Herbarium, Department of National Park, Wildlife and Plant
Conservation, Ministry of Natural Resources and Environment, Bangkok. The voucher specimens of
Terminalia bellerica (SRU 021), Terminalia chebula (SRU 022), and Phyllanthus emblica (SRU 023)
were deposited at Faculty of Oriental Medicine, Rangsit University, Pathumtani, Thailand. The Tri-phala
preparation was formulated by using the equal amount of Terminalia bellerica, Terminalia chebula and
Phyllanthus emblica.
Preparation of crude extracts The Tri-phala preparation was formulated by using the equal amount of
500 g of Terminalia bellerica, Terminalia chebula and Phyllanthus emblica. Then the mixture was boiled
in water (3 L) and let the water evaporate freely to obtain 1 liter of the aqueous extract. Next, the water
was removed from the extract by using freeze dry method.
Preparation of standard solutions Stock standard solution was prepared over the range of concentrations
expressed in ng/spot by making the measurements at 6 concentrations. The standard solutions were
spotted on the TLC plate to obtain final concentrations at 440, 880, 1320, 1760, 2200 and 2640 ng/spot.
Preparation of sample solution
Tri-phala effervescent tablet composed of Tri-phala extract 25%, sodium bicarbonate, citric acid, tartaric
acid, PEG 6000, and sodium saccharin. The effervescent tablet (4000 mg) was accurately weighed and
transferred to a 100 mL beaker which filled with water (45 mL). The effervescent tablet was
disintegrated completely in a solution within 5 minutes. Then the solution was transferred to volumetric
flask 50 mL and adjusted to volume with water. The mixture was mixed well and filtered through
membrane filter 0.45 μm to give clear sample solution.
Validation of the Method The method was validated for linearity, precision, accuracy, limit of detection
(LOD) and limit of quantitation (LOQ) according to the International Conference of Harmonization
(ICH) guidelines.
(a) Linearity-- Linearity relationship between peak area and concentration of the standard solutions
was evaluated at 6 concentration levels over the range of 440-2640 ng/spot. Each concentration
was done in triplicate.

(b) Precision-- Repeatability and intermediate precision of the developed method were expressed in
term of relative standard deviation (RSD) of peak area. Intraday precision was studied by repeat
analyzing on the same day at three concentrations (440, 880 and 1320 ng/spot) of standard gallic
acid solution (n=3). Interday precision was determined on three different days at the same three
concentrations by the proposed method.
(c) Accuracy-- The accuracy of the method was evaluated by performing recovery studies by adding
known amount of the reference compound at three levels (424, 636 and 848 ng/spot) to the
effervescent tablet sample solution. Then the solutions were applied on TLC plate and conducted
chromatography. Three determinations were performed for each level of concentration and the
recoveries were calculated.
(d) Specificity--Peak purity of gallic acid was assessed by scanning the standard and sample spots.
Correlation coefficients of these spectra were calculated.
(e) LOD and LOQ-- LOD and LOQ were determined by scanning the blank (methanol) spot and
detecting the noise. A series of concentrations of gallic acid standard solution (10-100 ng/spot)
were spotted on the TLC plate. Signal-to-noise ratios of 3:1 and 10:1 were considered as LOD
and LOQ, respectively.
Chromatographic condition for quantitative analysis of gallic acid in the Tri-phala extract and
effervescent tablet sample solutions Standard and sample were applied on precoated aluminium-backed
PT - 2
HPTLC silica gel 60 GF 254 plate using Linomat 5. Each sample solution (3 μL) was applied in triplicate
as narrow bands of 6 mm length using a nitrogen aspirator. The constant application rate of 150 nL/s, 5.0
x 0.45 mm densitometer slit dimension, and 20 mm/s scanning speed were used. The mobile phase
consisted of dichloromethane : ethyl acetate : formic acid (40 : 56 : 4, v/v). Linear ascending development
was performed in a twin-trough glass chamber saturated with 50 mL of the mobile phase for a plate. The
optimized chamber saturation time for the mobile phase was 25 min at room temperature (25 ± 2 ºC). The
distance covered by the solvent font was 8 cm, which took about 20 min for each run. The spots were
scanned using the TLC scanner 3 in the reflectance-absorbance mode at 280 nm, operated by winCATS
software. The amount of gallic acid in the samples was calculated using the calibration graph.

In this study, we developed a suitable method for the analysis of gallic acid in Tri-phala extract and
effervescent tablet. Gallic acid is main chemical constituent in Terminalia bellerica, Terminalia chebula
and Phyllanthus emblica which are the major components in the extract and effervescent tablet. For the
chromatographic condition, several trials were made by using different solvent systems containing non-
polar solvents and relative polar solvent; dichloromethane : ethyl acetate : formic acid (40 : 56 : 4, v/v)
gave the best separation. The R f value of gallic acid was 0.34 ± 0.01 and separated well from other
compounds as shown in Figure 1. The UV absorption spectrums of standard and sample have been
overlaid to confirm the identity and peak purity correlation coefficient was over 0.9996 (Figure 2(a)). The
method validation was composed of linearity, accuracy, precision, LOD and LOQ. For linearity of gallic
acid, peak areas were found to have good linear relationship with the concentration with a correlation
coefficient (r2) of 0.9936 and within the concentration range of 440-2640 ng/spot (Figure 2(b)). The
correlation coefficient and regression equation are presented in Table 1. The interday and intraday
precisions of gallic acid were performed and the results showed acceptable precision at three different
concentration levels of 440 – 1320 ng/spot with RSD <2.42%. The percentage recovery at three levels of
gallic acid was 101.7, 100.7 and 98.4%, with an average of 101.4% (Table 2).
Figure 1 (a) Standard gallic acid (b) Gallic acid in Tri-phala effervescent tablet.

Figure 2 (a) The UV absorption spectrums of standard gallic acid and Tri-phala sample were overlaid from 200-400 nm.
(b) Calibration curve of standard gallic acid.
Table 1 Summary of linear regression data for calibration curve of gallic acid
Parameters Results
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Linear range 440 – 2640 ng/spot
Linear regression equation y = 3319.267 + 8.250xa
Correlation coefficient (r2) 0.9936
Limit of quantitation, ng 40
Limit of detection, ng 10
Peak purity (specificity) r (s,m) 0.9996, r (m,e) 0.9997

a
x is the amount of gallic acid in ng, y is the peak area at 280 nm
Table 2 Recovery study of gallic acid
Serial Amount added, ng Amount found, ng Recoverya,b, %

No
1 424 431.06 ± 7.51 101.70 ± 1.77
2 636 640.78 ± 14.70 100.70 ± 2.31
3 848 834.42 ± 7.54 98.40 ± 0.89
a
Expressed as mean standard deviation (SD, n=3), b Average recovery = 101.4%
The validated method was used in determination of gallic acid content in Tri-phala freeze dry extract and effervescent tablet (Table
3). The results showed that there were significantly different in the content (w/w) of gallic acid in both samples. The amount of
gallic acid in freeze dry extract ranged from 3.30 - 3.38% (w/w) in dried powder while in the effervescent tablet ranged from 1.36 –
1.88% (w/w). The freeze dry extracts showed higher amount of gallic acid than the effervescent tablets. The reason for this issue
could be subject to the preparation process to make Tri-phala effervescent tablet which used hot-air oven to dry the granule. This
process might reduce the amount of gallic acid because there is a report about gallic acid can rapidly decompose at high
temperatures between 105-150 ºC7).
Table 3 Yields of gallic acid in Tri-phala freeze dry extracts and effervescent tablets.
Samples Yield of gallic acid, % (w/w)a

Freeze dry extracts 3.30±0.03 - 3.38±0.03
Effervescent tablets 1.36±0.02 – 1.88±0.06
a
Experssed as mean standard deviation (SD, n=3)
CONCLUSION
The HPTLC method was developed and validated for qualitative and quantitative analysis of gallic acid in
Tri-phala extract and effervescent tablet. This method is simple, precise, and accurate. It can be used for
quantitative analysis of gallic acid, an active anti-oxidation component in the raw materials, extracts of
Terminalia bellerica, Terminalia chebula, and Phyllanthus emblica and Tri-phala effervescent tablet. This

study can be a good start for preparing traditional formulations into modern dosage forms which are easy
to use and take them with us in any places.
ACKNOWLEDGMENTS
This work was supported by Faculty of Oriental Medicine Graduate School Program, Rangsit University,
Pathumthani 12000, Thailand
REFERENCES
1. Bag, A., Kumar Bhattacharyya, H., Kumar Pal, N., Rajan Chattopadhyay, R. 2013. Anti-
inflammatory, anti-lipid peroxidative, antioxidant and membrane stabilizing activities of
hydroalcoholic extract of Terminalia chebula fruits. Pharm. Bio. Sep 5 [Epub ahed of print].
2. Pual, WH., Kim, SH. 2013. MAPK inhibitors augment gallic acid-induced A549 lung cancer cell
death through the enhancement of glutathione depletion. Oncol. Rep. 30(1):513-519.
3. วุฒิ วุฒิธรรมเวช. สารานุกรมสมุนไพร. กรุ งเทพมหานคร:สํานักพิมพ์โอเดียนสโตร์, 2540.
4. Pallti, F., bruni, R., Righi, D., Grandini, A., Tognolini, M., Pio Prencipe, F., Poli, F., Benvenuti,
S., Del Rio, D., Rossi, D. 2013. Metabolite profiling of polyphenols in a Terminalia chebula
Retzius ayurvedic decoction and evaluation of its chemopreventive activity. J. Ethnopharmacol.
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147(2):277-85.
5. Jadon, A., Bhadauria., Shukla, S. 2007. Protective effect of erminalia belerica Roxb. And gallic
acid against carbon tetrachloride induce damage in albino rats. J. Ethnopharmacol. 109(2):214-
8.
6. Yang, B., Kortesniemi, M., Liu, P., Karonen, M., Salminen, J.P. 2012. Analysis of hydrolysable
tannins and other phenolic compounds in emblic leafflower(Phyllanthus emblica L.) fruits by
high performance liquid chromatography-electrospray ionization mass spectrometry. J. Agri.
Food. Chem. 60(35):8672-83.
7. Boles JS, Crerar DA, Grissom G, Key TC. 1998. Aqueous thermal degradation of gallic acid.
Geochim. Cosmochim. Ac. 52(2):341-344.

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR DETERMINATION OF

PIPERINE IN MAHA-SOLOS TABLET FORMULATION
Worawan Saingam1, Chaowalit Monton1, Jirapornchai Suksaeree1, Natawat Chankana1, Siriporn

Kittiwisut1 and Laksana Charoenchai1
1
Sino-Thai Traditional Medicine Research Center (cooperation between Rangsit University, Harbin Institute of Technology, and
Heilongjiang University of Chinese Medicine), Faculty of Pharmacy, Rangsit University, Pathum Thani, Thailand, 12000
KEYWORDS: Maha-solos, Tablet, Piperine, RP-HPLC
INTRODUCTION
Anxiety disorders are prevalent and widely interspersed with sickness in the general public. There are
several categories of anxiety disorders, including generalized anxiety disorder (GAD), social phobia,
panic disorder, agoraphobia, phobias, post-traumatic stress disorder (PTSD), and obsessive compulsive
disorder (OCD). The general symptoms of anxiety disorder include obsessive thoughts, nightmares,
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problems sleeping, cold or sweaty hands and/or feet, shortness of breath, dry mouth, nausea and
dizziness that affects quality of life. These disorders are accepted as risk factors for many diseases,
especially psychiatric diseases.
The treatments of anxiety disorders currently available include tricyclic antidepressants (TCAs), selective
serotonin reuptake inhibitors (SSRIs), serotonin–noradrenergic reuptake inhibitors (SNRIs), and other
atypical antidepressant drugs such as monoamine oxidase inhibitors (MAOIs) (Nemeroff, 2007).
However, the efficacy of drugs is frequently unstable and many of them create side effects such as weight
changes, drowsiness or sedation, insomnia, agitation, apathy, fatigue, dry mouth, gastrointestinal
disturbances, headache, cognitive impairment, and sexual dysfunction (Kennedy, 2006). The alternative
possible treatment is herbal medicines. Therefore, research and development of more effective drugs
without or less side effects are required.
In Thailand herbal medicines to relieve disorders have been used for many years, and research for new
pharmacotherapy from medicinal plants has progressed significantly in the last 10 years. Moreover, a
growing number of herbal products have been introduced into treating psychiatric disorders. Clinical
studies in humans have shown that treatment with variuos Thai traditional medicines (TTM), such as
Cassod tree (or Thai copper pod), Foetid Cassia and Confederate Vine, have a therapeutic effect on
insomnia and anxiety disorders.
Maha-solos, a TTM formula, has been used more than 100 years for anxiety. These formulas are
composed of Solanum seaforthianum Andr., Glycyrrhiza glabra Linn., Amomum krervanh Pierre., Mesua
ferrea Linn., Piper nigrum Linn., Zingiber officinale Roscoe., Piper retrofractum Vahl.,
Cinnamomum verum J. Presl., Olax scandens Roxb., Nelumbo nucifera Gaertn., Dracaena
lourieri Gagnep., Tarenna hoaensis Pitard., Ligusticum sinense Oliv. cv.Chuanxiong Hort., Saussurea
lappa Clarke., Angelica sinensis (Oliv.) Diels., Dryobalanops aromatica Gaertn and sugar in the same
proportions. Among 16 medicinal plants, pepper (Piper nigrum Linn.) compose of piperine is the
medicinally use for treat many diseases such as snake venom poisoning, anti-epileptic. Maha-solos is
available in powder dosage form and is boiled in water before use. Since such side effects of this current
therapy are harmful for patients, new Maha-solos formulations have been developed to reduce side
effects. However, in case of Maha-solos is prepare by boiled, the original method of preparing herbal
medicines in the practice of TTM. The composition of herbs within a boiled solution can be changed for
the condition of the patient. However, these traditional preparations have some disadvantages, such as the
time to prepare and the administration of a large volume of unpleasant tasting medicine that can be
troublesome for patients. Therefore, the researcher is interested in developing and formulating Maha-
solos into a modern formulation and validating RP-HPLC method for determination of piperine in
formulation. This will provide a viable resolution for delivering active ingredients using a cost-effective
technology.


Materials Piperine was purchased from Sigma Chemical Co.(USA). 16 Medicinal plant: Solanum
seaforthianum Andr., Glycyrrhiza glabra Linn., Amomum krervanh Pierre., Mesua ferrea Linn., Piper
nigrum Linn., Zingiber officinale Roscoe., Piper retrofractum Vahl., Cinnamomum verum J.
Presl., Olax scandens Roxb., Nelumbo nucifera Gaertn., Dracaena lourieri Gagnep., Tarenna
hoaensis Pitard., Ligusticum sinense Oliv. cv.Chuanxiong Hort., Saussurea lappa Clarke., Angelica
sinensis (Oliv.) Diels., Dryobalanops aromatica Gaertn and sugar were purchased from Charoensuk
Osod, Nakorn pathom, Thailand. Pharmaceutical excipient: colloidal silicon dioxide, magnesium
stearate, microcrystalline cellulose, sodium starch glycolate were purchased from TTK science,
Bangkok, Thailand. The Solvent for HPLC: methanol and water (Labscan, Thailand) were also
purchased from TTK science, Bangkok, Thailand.
Quantitative analysis for determination of piperine by high performance liquid chromatography
(HPLC) Quantitative analysis of piperine was analysed using a high performance liquid chromatography
(HPLC) method, coupled to a UV detector set to 343 nm. The HPLC system consisted of a quaternary
pump system (Agilent, 1260 VL), autosampler (Agilent, 1260 TCC) and UV/VIS with diode array
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detector (Agilent, 1260 DAD VL). A reverse-phase Zorbax C-18 column 4.6 x 150 mm was eluted by
using a mixture (30:70) of methanol and water as the mobile phase with a flow rate was set to 1.0 ml/min
and the injection volume was 10 µl. Validation parameters of linearity, accuracy, and precision were
confirmed for this method.
Application to Maha-solos tablet formulation Prepared Maha-solos tablet stock solution by weighed
accurately 10 tablets and crushed with mortar and pestle to obtain fine powder. Dissolve 600 mg of
powder in 25 ml methanol and sonicated for 15 min. The solution was filtered through Whatman filter
paper no.1. Then, pipette out 2.3 ml solution from stock solution and diluted up to 10 ml with methanol.
Finally, pipette out 1 ml solution from stock solution and diluted up to 10 ml with methanol (0.55 mg/ml).
To determine the specificity of the analytical method, standard stock solution of piperine, placebo
solution and Maha-solos solution dissolve in the same solution. Both solutions were filtered through 0.45
μm Nylon filter. Equal volumes (10 μl) of each sample were injected into the chromatograph by
autosampler and peak areas were measured.
Preformulation study To estimate the suitability of mixture for formulation, a preformulation study that
checked the physical properties of the medicinal plant and excipients was required. The results of the
preformulation study provided data that necessary for formulation and manufacture of tablet.
Preformulation study, angle of repose, bulk density, tapped density, compressibility index and Hausner
ratio were determine using the appropriate techniques.
Formulation development of Maha-solos tablets
Table 1 Formula of tablet
Ingredients Weight per tablet (mg) Function

F1 F2 F3 F4
Medicinal plant 500 500 500 500 Active ingredient
Colloidal silicon dioxide 12 24 36 36 Adsorbent/Glidant
Magnesium stearate 1.5 1.5 1.5 1.5 Lubricant
Microcrystalline cellulose 86.5 74.5 62.5 38.5 Diluent
Sodium starch glycolate - - - 24 Disintegrant
Total 600 600 600 600 -
All ingredients as shown in Table 1 will be mixed and compressed by direct compression using a single
punch tablet machine to make a tablet formula. Humidity in the room will be controlled to be lower than
30%RH. Amount of piperine from tablets will be analyzed by validated RP-HPLC method.

Physical properties of Maha-solos tablets

The evaluation data composed of weight variation, thickness, hardness, friability, and disintegration.

Quantitative analysis for determination of piperine by RP-HPLC
Validation method of RP-HPLC analysis Linearity was studied by preparing standard stock solution of
piperine at different concentration levels. When the concentrations of piperine and its respective peak
areas were subjected to regression analysis by least squares method, a good linear relationship (R2 =
0.9997) was observed between the concentrations of piperine and the respective peak areas in the range
0.1-0.5 mg/ml. The regression equation was found to be y = 152988146x-293050.8667, where y is the
peak area and x is the concentration of piperine.
To ensure the accuracy and reliability of the method, recovery studies were carried out in triplicate at
three concentration levels of test concentration. The recovery of piperine was found to be in the range of
99.19-100.89 %
The intra-day precision of the assay method was evaluated by carrying out six independent assays of
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piperine test samples against qualified reference standard on same day and these studies were also
repeated on six consecutive days to determine inter-day precision. The percentage of RSD of six assay
values was calculated. Intra-day and inter-day, % RSD of piperine was found 0.63 and 0.26, respectively.
Application to Maha-solos tablet formulation The chromatogram of Maha-solos solution was compared
with chromatogram of standard and placebo. As a result, the chromatogram of placebo was not shown the
peak area at 4.026 minutes as shown in Figure 1.
Standard solution
Tablet solution
[A] [B]
Figure 1 Chromatogram of piperine in [A] standard solution and tablet solution and [B] placebo solution
Preformulation study Preformulation study, angle of repose, bulk density, tapped density, compressibility
index and Hausner ratio were showed in Table 2.
Table 2 Data of preformulaton study
Formulation F1 F2 F3 F4
Angle of repose (°) 45.51 42.27 38.66 27.47
Bulk density (g/ml) 0.43 0.48 0.34 0.35
Tapped density (g/ml) 0.50 0.50 0.42 0.53
Carr’s index (%) 14.00 4.00 19.05 33.96
Hausner ratio 1.16 1.04 1.24 1.51
Formulation development of Maha-solos tablets The formula of F1, F2, F3 and F4 contained medicinal
plant at the amount 500 mg or 83.33% by weight. Colloidal silicon dioxide was individually tested for
Maha-solos powder adsorption by mixing with excipients at the concentration of 2.0%, 4.0% and 6.0% by
weight (F1-F3). The effect of adsorbent on physical properties of Maha-solos tablets was visually
observed. It was found that colloidal silicon dioxide at the concentration of 6.0% by weight (F3) was the
most suitable adsorbent for Maha-solos powder. F1 and F2 showed poor flow and exhibited sticking with

punch and die set. F3 formulation which contained 6.0% of colloidal silicon dioxide exhibited good flow,
good compressibility and did not stick to punch and die set. Disintegretion time of F3 is 10.10±1.76 min.
Then, we added sodium starch glycolate 4.0% by weight (F4) to decrease disintegration time to 5.88±0.89
min. The physical appearance of tablet of F3 and F4 were brown, round shape and smooth surface. The
evaluation data of weight variation, thickness, hardness, friability, and disintegretion were showed in
Table 3.
Table 3 Data of physical properties
Physical properties F3 F4
Weight variation (mg) 585.99±11.48 589.40±7.35
Thickness (mm) 6.02±0.07 5.97±0.06
Hardness (kP) 7.00±1.26 7.58±1.02
Friability (%) 0.20 0.21
Disintegration (mins) 10.10±1.76 5.88±0.89
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CONCLUSIONS
The analytical RP- HPLC method was developed and validated carefully for determination an amount of
piperine. The developed method was simple and responsive which could be employed for quantity
analysis of formulation. Maha-solos tablets are prepared successfully by direct compression method.
Colloidal silicon dioxide can effectively adsorb Maha-solos powder and other excipients. The formulation
overcomes the problems associated with hygroscopic excipients like medicinal plant.
REFERENCES
1. Chandira, M., and Jayakar, B. Formulation and evaluation of herbal tablets containing Ipomoea
digitata Linn, extract. International Journal of Pharmaceutical Sciences Review and Research.
3(1)(2010): 101-10.
2. Kardani, K., Gurav, N., Solanki, B., Patel, P., and Patel, B. RP-HPLC Method Development and
Validation of Gallic acid in Polyherbal Tablet Formulation. Journal of Applied Pharmaceutical
Science. 3(5)(2013): 37-42.
3. Kennedy, S.H. A review of antidepressant treatments today. Eur europsychopharmacol.
16(2006): 619-23.
4. Kolhe, S.R., Borole, P., and Patel, U. Extraction and Evaluation of piperine from Piper nigrum,
Linn. International Journal of Applied Biology and Pharmaceutical Technology. 2(2) (2011).
5. Kondawar, M.S., Kamble, K.G., and Mali D.S. Quantitative estimation of Gallic acid and
Ascorbic acid in a marketed herbal medicine: Triphala churna by High Performance Thin Layer
Chromatography. International Journal of PharmTech Research. 3(3)(2011): 1593-9.
6. Nemeroff CB. The burden of severe depression: a review of diagnositic Challenges and
treatment alternatives. J Psychiatr Res 41(2007): 189-206.
7. Patel, M.G., Patel, V.R., and Patel, R.K. Development and Validation of Improved RP- HPLC
method for Identification and Estimation of Ellagic and Gallic acid in Triphala
churna. International Journal of PharmTech Research. 2(3)(2010): 1486-93.
8. Singh, I., and Kumar, P. Preformulation studies for direct compression suitability of cefuroxime
axetil and paracetamol: a graphical representation using SeDeM diagram. Acta Poloniae
Pharmaceutica n Drug Research. 69(1)(2012): 87-93.
9. Verzele, M., and Qureshi, S. HPLC Determination of Piperine in Pepper and in Pepper Extracts.
Chromatographia. 13(4)(1980): 241–3.
10. Wang, P., Li, L., Yang, H., and et al. Chromatographic fingerprinting and quantitative analysis
for the quality evaluation of Xinkeshu tablet. Journal of Pharmaceutical Analysis. 2(6)(2012):
422–30.

APPLICATION OF TITRIMETRIC INDICATOR TO THE ESTIMATION OF POLYPLEX

FORMATION RATIO BETWEEN CHITOSAN POLYMER AND PLASMID DNA USED FOR
GENE DELIVERY FORMULATION
Samarwadee Plianwong1, Praneet Opanasopit1, Tanasait Ngawhirunpat 1 and Theerasak Rojanarata1*

1
Silpakorn University, Nakhon Pathom 73000, Thailand
*Corresponding author: teerasak@su.ac.th, rtheerasak@yahoo.com
KEYWORDS: chitosan, chitosan pyridine, dichlorofluorescein, weight ratio, polyplex
INTRODUCTION
Gene therapy is currently a promising approach to the treatment of diseases. It is carried out by
transferring genetic materials to target cells in order to compensate for defective genes or produce
therapeutic proteins. For successful delivery and expression of exogenous genes, suitable types and
amounts of vectors are usually required1). Chitosan (CS) and its derivative ex. chitosan pyridine (CS-Py)
are commonly used as cationic polymeric carriers for gene delivery2). Once the amino groups are
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protonated, CS and CS-Py binds and condenses negatively charged DNA into smaller particles. The
optimal cationic polymers to DNA ratio has a significant correlation to the complete formation of
polyplexes and the resulting transfection efficiency1,3) since too low amounts of polymers cannot
efficiently compact DNA and neutralize the negative charge whereas a significant excess of cationic
polymers turn out to be cytotoxic4). Thus, the complete polymers/DNA complexes usually investigated
prior to in vitro and in vivo transfection experiments. For this purpose, a range of techniques have been
used to monitor the self-assembly process e.g. light scattering, the inhibition of ethidium bromide
fluorescence, zeta potential measurement5) and the most commonly used gel electrophoresis.
Nevertheless, some methods need costly specialized instruments and time- consuming. Furthermore, a
potent carcinogenic ethidium bromide6,7) is commonly employed in gel electrophoresis method. Despite
the current availability of less mutagenic alternatives for nucleic acid stains, most of them are
significantly high-priced. From these rationales, a new assay has been developed by using inexpensive
dichlorofluorescein (DCF) for the estimation of CS and CS-Py to DNA ratio at which the complete
polyplex was formed. DCF has been used as an adsorption indicator in Fajan's precipitate-forming
titration of chloride using silver nitrate as a titrant8). In analogue to this phenomenon, DNA which is
anionic like chloride ion is titrated with various amounts of cationic CS or CS-Py. When a critical amount
of CS or CS-Py is added to completely form the complex particles with nucleic acids and totally masks
the negative charge, the surface charge of the polyplexes becomes positive and DFC anions are attracted
to adsorb on the particles as counterions. In the previous study, we developed the dye adsorption based
method for determining the complete polyplex formation of polyethylenimine (PEI), the synthetic
cationic polymer with nucleic acid. The results showed the successful estimation of PEI/pDNA and
PEI/siRNA polyplex formation. In addition, the assay is also a green and low-cost alternative method for
routine task in the formulation of PEI and nucleic acids9). In this study, we developed the proposed
method for the estimation of the natural cationic polymer, CS and its derivative, CS-Py to nucleic acid
ratio at which the complete polyplex was formed.

Preparation of plasmid DNA The plasmids pEGFP-C2 (4.7 kbp) was amplified in Escherichia coli and
purified by using the commercial plasmid midi kits (Qiagen, Hilden, Germany).The quality and quantity
of purified pDNA were evaluated by the optical density at 260 nm and 280 nm and by agarose gel
electrophoresis. The purified plasmid was resuspended and kept in Tris-EDTA (TE) buffer (pH7.5).
Formulation of CS/pEGFP and CS-Py/pEGFP polyplexes The CS/pEGFP and CS-Py/pEGFP
polyplexes were prepared by adding the solution of CS or CS-Py (in 0.01 mM hydrochloric acid) to the
plasmid solution in 1.5 mL microcentrifuge tubes at the different weight ratios of 0, 0.1, 0.5, 1, 5, 10, 15
and 20. The mixtures were gently mixed and further incubated at room temperature for 30 min,
sufficiently for the complex formation. Then, the polyplex solutions were diluted with water to 30 mL.
Estimation of the ratio of complete polyplex formation
Proposed method based on DCF dye adsorption The ratio which the CS or CS-Py associated
equivalently with nucleic acid was found by using the proposed method. Five µL of 0.2 mg mL−1 DCF
solution was added into a series of polyplex solutions prepared by using varied CS or CS-Py to nucleic

acid ratios. After gently mixed, the solutions were centrifuged at 20,000 rpm for 5 min to precipitate the
polyplexes at the bottom of the reaction tubes. The pellets were washed twice by using sterile water and
briefly centrifuged to remove the unadsorbed DCF in the supernatant. The point of complete polyplex
formation was seen by the formation of pink colored smear or pellets of DCF-adsorbed polyplex. By the
other means, 30 mL of 0.01N NaOH was added to the pellets to free the adsorbed DCF from the polyplex.
Upon the exposure to UV light at 366 nm, the reaction solutions emitted green fluorescent light which
could be observed.
Spectrophotometry method The complete CS/pEGFP and CS-Py/pEGFP polyplex formation was
examined by measuring the absorbance values of unadsorbed DCF in the supernatant. The absorbance
values were measured at 504 nm by a cuvetteless drop-based NanoVuePlas spectrophotometer (GE
healthcare, UK), against a sterile water blank.
Agarose gel retardation method To compare the proposed method with the currently used assays the
association of CS or CS-Py and nucleic acid was also examined by gel retardation method, using. 0.8%
agarose gel. The electrophoresis of DNA polyplex was carried out in 1X TAE buffer at 100 V for 45 min.
Subsequently, the gel was stained with 0.5 mg mL−1 ethidium bromide. The bands were visualized and
photo graphed by a UV transilluminator using a GelDoc system.
RESULTS
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Principle of the proposed method The estimation of CS or CS-Py to nucleic acid ratio of complete
polyplex formation was developed based on the adsorption of common, safe and inexpensive dye onto the
surface of the polyplex. As illustrated in Figure 1, a series of polyplexes set up at varied weight ratios of
CS or CS-Py/nucleic acid were firstly prepared by adding the various amounts of CS or CS-Py to the
nucleic acid solutions with the fixed concentration. After the self-assembly of pDNA with cationic CS or
CS-Py (A), anionic DCF with green color was added to the solution (B). At this step, the nucleic acid
which did not completely form polyplexes because of the inadequate amount of CS or CS-Py remained
negatively charged and the adsorption of DCF on the surface of the particles would not happen. Since a
critical amount of CS or CS-Py associated equivalently to the plasmids causing the complete self-
assembly complexation, the positive charge of CS or CS-Py attracted DCF onto the particles as
counterions (C). This resulted in the appearance of pink colored smear or pellets which could be
visualized after spinning down these particles. By the other means, the adsorbed dye could be released
into the solution by the addition of sodium hydroxide solution and the green fluorescence could be
observed under UV light at 366 nm.
Figure 1 Method for the estimation of polymer to nucleic acid ratio of the complete polyplex formation based on dye adsorption.
CS or CS-Py/pDNA polyplex formulations by DCF dye adsorption method The method was applied to
the estimation of the CS or CS-Py to pEGFP ratio at which both components equivalently, giving rise to
the complete polyplex formation. As seen in Figure 2, the assembling reactions with the adequate
CS/pEGFP or CS-Py/pEGFP ratios of 5 and higher produced pink pellets or green fluorescence under
visible light or UV light, respectively whereas those comprising the sub-optimal CS/pEGFP or CS-

Py/pEGFP ratios did not showed pink pellets and no light emission. Based on this rationale, the lowest
CS/pEGFP or CS-Py/pEGFP ratio that gave pink colored pellets or green fluorescence represented the
equivalent ratio for the complete polyplex formation. In this case, both CS/pEGFP and CS-Py/pEGFP
showed the same optimal ratio which was found at 5.
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Figure 2 The estimation of CS/pEGFP ((a) and (b)) and CS-Py/pEGFP ((c) and (d)) ratio of the complete polyplex formation by
using DCF adsorption method as detected by the formation of pink pellets ((a) and (c)) or the fluorescence emission under UV light
((c) and (d)).
CS or CS-Py/pDNA polyplex formations by spectrophotometry method To understand the fundamental

aspects of the adsorption phenomenon, we have monitored the concentrations of DCF in CS/pEGFP and
CS-Py/pEGFP self-assembly reactions by measuring the absorbance values of free DCF at 504 nm. As
shown in Figure 3, the concentrations of DFC in the solutions decreased abruptly at the CS/pEGFP and
CS-Py/pEGFP ratio of 5 due to the adsorption of dye onto the polyplex particles which were formed
completely at this point.
Figure 3 Absorbance values of free DCF in the solutions at different CS/pEGFP (a) and CS-Py/pEGFP (b) weight ratios.
Agarose gel electrophoresis To compare the proposed method with the conventional assays, gel
retardation experiment confirmed that pEGFP was absolutely retained in the wells at the CS/pEGFP and
CS-Py/pEGFP weight ratios of 5 and higher (Figure 4) which were totally in agreement with the results
obtained from the proposed method.

Figure 4 Gel electrophoresis assay of CS/pEGFP (a) and CS-Py/pEGFP (b) polyplexes prepared at weight ratio of 0, 0.1, 0.5, 1, 5,
10, 15 and 20 (from left to right).
DISCUSSION
In this study, a new assay has been developed for the estimation of CS and CS-Py to pDNA ratio at which
the complete polyplexes are formed by the use of DCF dye. This method could determine by three means,
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visualization, fluorescence and spectrophotometry. From the principle of the propose method, an
inadequate amount of CS and CS-Py did not completely form polyplexes with pDNA and remained
negatively charged thus the adsorption of DCF on the surface of the particles would not happen. Once the
amount of CS and CS-Py was equivalently to the plasmids, the positive charge of the polymers attracted
DCF onto the particles and the pink smear or pellets are visible at the bottom of tubes after spinning
down. An alternative detection method, sodium hydroxide solution was added to release the adsorbed dye
into the solution and produced green fluorescence under UV light (366 nm). In both cases of CS and CS-
Py, the optimal weight ratio was found to be 5. For detection by spectrophotometry, the concentrations of
free DFC in the solutions decreased abruptly due to the adsorption of dye onto the polyplex particles
formed completely point. Compared with gel electrophoresis experiment, the complete polyplex
formation was found at the same weight ratio as the proposed method. These results suggested that the
dye adsorption-based method was a reliable alternative method for monitoring the equivalence point for
the optimal self-assembly between plasmid DNA with CS and CS-py.
CONCLUSION
Using the analog principle to the detection method for the end point of chloride titrimetry, a dye
adsorption based method was developed and applied to the estimation of the ratio representing the
complete polyplex formation between CS/pDNA and CS-Py/pDNA. The method gave comparable results
to currently used assays i.e. gel electrophoresis. However, it required no sophisticated instruments, time-
consuming, carcinogenic ethidium bromide and costly new generation of fluorescent nucleic acid staining
dyes. Moreover, the proposed method could be usually accomplished within less than 10 min. Hence, it
was a fast, facile, cost- effective and safe for operator alternative method, suited for the investigation of
the optimal CS and its derivative such as CS-Py to pDNA ratio for gene delivery.
ACKNOWLEDGMENTS
We gratefully acknowledge the financial support of this study by Thailand Research Fund (TRF) through
the Royal Golden Jubilee Ph.D. Program scholarship (PHD/0069/2553) for Samarwadee Plianwong and
by Faculty of Pharmacy, Silpakorn University, Thailand.
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1. U. Lungwitz, M. Breunig, T. Blunk, A. GÖpferich, Eur. J. Pharm. Biopharm. 60 (2005) 247.
2. S. Mao, W. Sun, T. Kissel, Adv. Drug Deliv. Rev. 62 (2010) 12.
3. T.G. Park, J.H. Jeong, S.W. Kim, Adv. Drug Deliver. Rev. 58 (2006) 467.
4. W.T. Godbey, K.K. Wu, A.G. Mikos, J. Controlled Release 60 (1999) 149.
5. A.L. Parker, D. Oupicky, P.R. Dash, L.W. Seymour, Anal. Biochem. 302 (2002) 75.
6. V.L. Singer, T.E. Lawlor, S. Yue, Mutat. Res. 439 (1999) 37.
7. M.J. Waring, J. Mol. Biol. 13 (1965) 269.
8. K. Fajans, O.Z. Hassel, Elektrochem 29 (1923) 495.
9. S. Plianwong, P. Opanasopit, T. Ngawhirunpat, T. Rojanarata. Talata. 115 (2013) 241.

PREPARATION OF DOUBLY CROSSLINKED CHITOSAN BY THE USE OF

ENVIRONMENTALLY FRIENDLY CROSSLINKERS FOR ENZYME IMMOBILIZATION
Siripran Tidjarat1, Tanasait Ngawhirunpat1, Praneet Opanasopit1 and Theerasak Rojanarata1,*
1
Silpakorn University,Nakhon Pathom 73000, Thailand (*corresponding author email: teerasak@su.ac.th)
KEYWORDS: Chitosan, Bead, Crosslink, Enzyme, Immobilization
INTRODUCTION
Chitosan, a natural polysaccharide, is an N-deacetylated derivative of chitin obtained from crustaceans,
insects and fungi. It possesses interesting features i.e. film forming ability, gelation and bioadhesive
characteristics. Moreover, it is biodegradable and biocompatible1). Because of the polymeric cationic
characteristics, chitosan can interact with negatively charged molecules or polymers such as
tripolyphosphates (TPP) leading to interpolymer linkages and the formation of beads or micro/
nanoparticles. In pharmaceutical fields, these particulate systems have been used for encapsulation of
drugs and biological substances2). In addition, they can be applied to enzyme immobilization, a practical
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approach to attach the enzyme to an inert, insoluble material in order to facilitate continuous and recycle
use of enzyme. Apart from such ionic crosslinking, chitosan which contains primary amine groups can
undergo covalent crosslinking with certain crosslinkers such as genipin. This compound is an aglycone
derived from an iridoid glycoside called geniposide present in fruit of Gardenia jasminoides3). Compared
with glutaraldehyde and many other commonly used synthetic cross-linking reagents, genipin is known as
a natural crosslinker with much lower toxicity and more environmental friendliness
D-phenylglycine aminotransferase (D-PhgAT; EC 2.6.1.72) is an enzyme firstly discovered in Thailand
from Pseudomonas stutzeri ST-2014). It catalyzes a reversible stereo-inverting transamination of D-
phenylglycine or D-4-hydroxyphenylglycine and 2-oxoglutaric acid to yield benzoylformic acid or 4-
hydroxybenzoylformic acid (HBZF) and L-glutamic acid. Currently, D-PhgAT has been applied for the
synthesis of optically pure D-phenylglycine, an intermediate for the synthesis of ampicillin5). In addition,
it has been used for the analysis of L-glutamate in foods6) and the assay of amoxicillin formulations7).
Since D-PhgAT employed for the aforedmentioned purposes were as solution or immobilized form using
expensive matrix for immobilization, the use of low cost and green carriers such as natural polymers
especially chitosan is an interesting alternative.
In this study, doubly crosslinked chitosan beads were prepared by using two safe and environmentally
friendly crosslinkers TPP and genipin and aimed to use for immobilizing D-PhgAT. The influence of
some conditions of the crosslinking reaction (pH of TPP, genipin concentration) on the resulting beads
was studied. Chitosan beads were studied in terms of size, solubility under various pH conditions and
preliminarily tested for the reaction catalyzed by immobilized enzyme.

Materials Chitosan powder (high molecular weight with the degree of deacetylation > 75%) and sodium
TPP were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Genipin was supplied by
Challenge Bioproducts Co. (Taichung, Taiwan). D-PhgAT was purified in the laboratory from
recombinant Escherichia coli expressing the cloned gene. All other chemicals were of analytical grade
from Merck (Darmstadt, Germany). Distilled water was used throughout the experiments.
Ionic crosslinkink of chitosan beads Briefly, 3% (w/v) chitosan powder was dissolved in 2% (v/v) acetic
acid. and mechanically stirred for 3 h to obtain clear chitosan solution. Sodium TPP was dissolved in
deionized water to prepare 10% (w/v) TPP aqueous solutions. The pH of TPP aqueous solutions were
adjusted from pH 8.1 (original pH value) to pH 3.0, 5.0, 7.0, 8.0, 9.0 and 11.0, respectively. The chitosan
solution was directly dropped through a syringe needle (20 G) into the solution of TPP, and the chitosan
droplets were stood in the solution for 12 hours to crosslink the gel beads. After crosslinking, the
solidified gel beads were separated and washed thoroughly with deionized water to remove residual ionic
crosslinking agents.
Covalent crosslinking of chitosan beads The previously ionic-crosslinked beads prepared using TPP at
pH 8 were immersed in genipin solution with various concentrations including 2.5, 5, 10, 25 and 50 mM.
The beads were shaken at 40 °C for 12 hours and then washed three times with distilled water and blotted
with tissue paper to remove adsorbed water on the surface.

Study of solubility of beads The solubility of wet beads was examined by shaking them in
acidic and alkali media e.g. hydrochloric acid (pH 2, 3, 5), distilled water (pH 7) and
sodium hydroxide solution (pH 9, 11, 14) at 30 °C for 12 hours.
Immobilization of D-PhgAT on crosslinked chitosan Enzyme immobilization was carried out on
crosslinked chitosan matrix which was dried at 40 °C for 12 hours and then grounded and sieved. To 100
mg of the dry grounded matrix, 1 mL of enzyme solution prepared in 20 mM Tris buffer pH 7.6 was
added. The immobilization reaction was shaken at 25 rpm, 25 oC for 1 hour. After then the enzyme
solution was removed from the reaction and the resulting enzyme-immobilized matrix was washed with
water.
Tests of the reaction catalyzed by immobilized enzyme To study the catalytic performance of D-PhgAT
immobilized on the crosslinked chitosan, transmination reaction was tested. The reaction involved the
conversion of 5 mM D-4-hydroxyphenylglycine in the presence of 10 mM 2-oxoglutaric acid at pH 8.6 to
HBZF and L-glutamic acid. After setting up the reactions by using about 100 mg of D-PhgAT-
immobilized matrix per total reaction volume of 1 mL and incubating the reactions at 37 oC for 15 mins,
the products of interest i.e. HBZF was assayed by high performance liquid chromatography (HPLC).
The chromatographic system consisted of HPLC apparatus equipped with ODS column (250 mm x 0.4
mm, 5 µm) and UV detector (340 nm for HBZF), a mobile phase containing 50 mM potassium phosphate
buffer (pH 6.8) and the flow rate set at 1.0 mL.min-1. The experiments were performed five rounds to see
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the effect of repeated use.

Properties of ionic-crosslinked chitosan beads It was found that ionic crosslinked chitosan beads were
generally round and white with the average diameter of about 2 mm (Figure 1, left). However, the use
of TPP solutions with different pH could affect some appearance of the bead and their durability under
different acid-base conditions. From the experiments, TPP solution with lower pH produced opaque
beads while more clear beads were obtained from neutral or alkaline TPP. In term of the dissolution,
beads prepared by using TPP solution at pH 3 resisted to the dissolution at all pH , but beads prepared by
using TPP solution adjusted to pH 5, 7, 8, 9 and 11 dissolved at pH 2 (Table 1). These results suggested
that the ionic crosslinking occurred at the higher extent under acidic condition because amino groups of
chitosan were positively ionized and thus crosslinked well with the negative charge of phosphate ion. As
the result, the beads prepared under acidic conditions were more durable than the beads prepared under
neutral or alkaline conditions. To further improve the durability, the ionic crosslinked beads were
subsequently subjected to covalent crosslink using genipin.
1 mm
Figure 1 Wet chitosan beads prepared by ionic crosslinking with TPP solution (left) and chitosan beads prepared by ionic
crosslinking followed by genipin-mediated covalent crosslinking (right).
Table 1 Solubility of the beads prepared via ionic crosslinking in the medium with different pH.

Table 2 Solubility of the beads prepared via ionic crosslinking, followed by covalent crosslinking in the medium with different pH.
Properties of doubly (ionic and covalent) crosslinked chitosan beads Since beads prepared by using TPP
solutions at pH 8 showed similar pH-solubility to those prepared at other pH wheras the preparation was
easier and more economical since they required minimal pH adjustment of TPP solution, this type of
beads was chosen for the subsequent covalent crosslinking. Upon the crosslink reaction with colorless
genipin solution, the beads changed the color from white to dark purple. In addition, the size of beads
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became smaller with the average diameter of about 1.5 mm (Figure 1, right). Interestingly, the resulting
beads increased their strength and durability as they did not dissolve at all pH (Table 2). However, the use
of higher concentration of genipin than 25 mM yielded brittle beads.
Catalytic performance of enzyme immobilized on crosslinked chitosan Since fine particles hold higher
surface area which is useful for enzyme immobilization and they are more practical for column packing
for the future use, doubly crosslinked chitosan beads were finely ground prior to use as enzyme carrier.
The evaluation of the catalytic performance of immobilized enzyme as determined by formation of HBZF
product revealed that both TPP-crosslinked chitosan and doubly crosslinked chitosan could adsorb the
enzyme with some extent. In general, the enzyme activity as evidenced by the amount of HBZF formed
declined upon the repeated uses of the enzyme, probably due to the release of enzyme from the carrier.
Despite the activity of enzyme immobilized on doubly crosslinked chitosan was lower that on TPP-
crosslinked chitosan in the first round of reaction, both of them were not significantly different in the
subsequent rounds. Therefore, it may imply that the covalent crosslink step which improved the pH-
stability of chitosan matrix did not drastically reduce the capability of chitosan to adsorb the enzyme as
well as the resulting catalytic performance of enzyme.
Figure 2 The formation of HBZF (mM) after round 1, 3 and 5 of the catalytic reaction using ground ionic-crosslinked chitosan
versus doubly crosslinked chitosan.
CONCLUSION
In this study, chitosan beads were prepared with ease by the use of safe and environmentally friendly
crosslinkers namely TPP and genipin. The procedures and conditions of the crosslinking method
influenced on the properties of the resulting beads. In overall, double crosslinking process could increase
the durability of the beads over the broader pH compared with those crosslinked only by TPP. Also, it did
not drastically reduce the capability of chitosan for enzyme immobilization as well as the catalytic

performance of enzyme. However, since the enzyme activity was found to decrease upon repeated uses,
some improvement strategies should be applied in the future study in order to anchor the enzyme more
tightly onto the matrix for more effecient use.
REFERENCES
1. Rinaudo M. Chitin and chitosan: Properties and applications. Prog. Polym. Sci. 31 (2006) 603–
632
2. Agnihotri SA, Mallikarjuna NN, Aminabhavi TM. Recent advances on chitosan-based micro-
and nanoparticles in drug delivery. Journal of Controlled Release 100 (2004) 5–28.
3. Muzzarelli RAA. Genipin-crosslinked chitosan hydrogels as biomedical and pharmaceutical
aids. Carbohydrate Polymers 77 (2009) 1–9.
4. Wiyakrutta S, Meevootisom V. A stereo-inverting D-phenylglycine aminotransferase
from Pseudomonas stutzeri ST-201: purification, characterization and application for D-
phenylglycine synthesis. Journal of Biotechnology 55 (1997) 193-203.
5. Rojanarata T, Isarangul D, Wiyakrutta S, Meevootisom V, Woodley JM. Controlled-release
biocatalysis for the synthesis of D-Phenylglycine. Biocatalysis and Biotransformation 22 (2004)
195-201.
6. Khampha W, Meevootisom V, Wiyakrutta S. Spectrophotometric enzymatic cycling method
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using L-glutamate dehydrogenase and D-phenylglycine aminotransferase for determination of L-
glutamate in foods. Analytica Chimica Acta 520 (2004) 133–139.
7. Rojanarata T, Opanasopit P, Ngawhirunpat T, Saehuan C,Wiyakrutta S, Meevootisom V. A
simple, sensitive and green bienzymatic UV-spectrophotometric assay of amoxicillin
formulations. Enzyme and Microbial Technology 46 (2010) 292–29.

INFLUENCE OF CHEMICAL STRUCTURES OF EMULSIFIERS ON FEASIBILITY

OF ISOPROPYL PALMITATE LOTION FORMATION
Prapaporn Boonme1,*, Chalida Boonthongchuay1, Thanaporn Amnuaikit1 and Wibul Wongpoowarak1

1
Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla, Thailand
*Corresponding author (e-mail: prapaporn@pharmacy.psu.ac.th)
KEYWORDS: Emulsion, HLB, Hydrophile-lipophile balance, Lotion
INTRODUCTION
Emulsion formulations are one of hard tasks since there are a lot of emulsifiers which can be selected. To
save time in emulsifier selection, the hydrophile-lipohile balance (HLB) system has been used for
choosing proper emulsifiers. Lipophilic compounds (oils, fats, and waxes) have an individual value called
“required HLB”. An emulsifier or blend of emulsifiers, having an HLB equal to the required HLB of the
lipophilic phase will make a more stable emulsion than emulsifiers of any other HLB values.1 Generally,
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blends of emulsifiers can provide much higher effective as emulsifiers than any single chemical
composition would be because their resulted complex interfacial films can protect the internal droplets
from coalescence, leading to more stability.1-3 Nevertheless, "right chemical type" is also as important as
"right HLB". It was reported that the properties of emulsifier mixtures, i.e. HLB and carbon number of
alkyl chain length, affected the stability of asphalt emulsions.4 Moreover, only total HLB of an emulsifier
pair was found to be not enough to formulate a physically stable coconut oil emulsions.5 Besides normal
emulsions, the stability of multiple emulsions was also depended on emulsifier chemistry.6 Therefore, it is
interesting to investigate the influence of few differences in HLB values but large differences in chemical
structures of an emulsifier in the emulsifier blends on feasibility of emulsion formation. In this study,
isopropyl palmitate (IPP) was formulated in form of lotion using four pairs of different blends of non-
ionic emulsifiers and evaluate for feasibility of emulsion formation. The required HLB for IPP is 11.5.7
Four pairs of emulsifier blends with the identical total HLB of 11.5 prepared from different components
were investigated.

Materials Fou r non-ionic emulsifiers, i.e. polyethylene [20] sorbitan monooleate (PSMO, HLB = 15.0),
polyethylene [20] sorbitan monostearate (PSMS, HLB = 14.9), sorbitan monooleate (SMO, HLB = 4.3)
and sorbitan monostearate (SMS, HLB = 4.7) were purchased from P.C. Drug Center Co., Ltd. (Bangkok,
Thailand). Isopropyl palmitate (IPP) was purchased from East Asiatic (Bangkok, Thailand). Distilled
water was used throughout the experiments. All chemicals were of pharmaceutical grade and used as
received without further purification.
Preparation of isopropyl palmitate lotions Amount of each emulsifier in each emulsifier blend was
calculated according the HLB system.1-3 Four formulations of IPP lotions containing 40% w/w IPP, 10%
w/w emulsifier blend, and 50% w/w water, as exhibited in Table 1, were prepared by an emulsification
process.5 The oil phase (IPP and an oil-soluble [low HLB] emulsifier) and the water phase (water and a
water-soluble [high HLB] emulsifier) were separately heated in a water bath to 70°C. Afterwards, the
water phase was slowly added to the oil phase with continuous stirring until the mixture was cooled to
ambient temperature.
Observation for feasibility of lotion formation All prepared samples were optically observed for their
appearance such as color and homogeneity to determine the feasibility of lotion formation. The feasibility
of lotion formation was decided from non-separated milky liquid performance of the obtained products
within 3 days after preparation, arbitrary criteria duration.

Table 1 Formulations of IPP Lotions
IPP PSMO PSMS SMO SMS Water

Formulation
(% w/w) (% w/w) (% w/w) (% w/w) (% w/w) (% w/w)
L-1 40 6.73 - 3.27 - 50
L-2 40 6.60 - - 3.40 50
L-3 40 - 6.79 3.21 - 50
L-4 40 - 6.67 - 3.33 50
RESULTS
After immediate preparation, all samples were seen as milky liquids. However, only L-4 could provide
the feasibility of lotion formation while other formulations could not as shown in Figure 1. The products
of L-1, L-2 and L-3 exhibited upward creaming and subsequently breaking within very short duration.
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Figure 1 Appearance of the investigated IPP lotions
DISCUSSION
The results indicated that only formulation L-4 could provide feasibility for physical stable IPP lotion
while other formulations (L-1, L-2, and L-3) could not. Upward creaming was seen in Formulations L-1,
L-2, and L-3 since the instable dispersed oil droplets were less dense than the water continuous phase,
resulting in a tendency to raise. Afterwards, the instable oil droplets could coalesce and phase separation
could be occurred. This physical instability also indicated an oil-in-water type of the obtained IPP lotion
which was in coincidence to HLB of emulsifier system of higher than 8.1-3
The difference in HLB value between SMO and SMS is 0.4; obviously showing that SMO is more
lipophilic.1-3 In previous study, SMO was found that it could not form strong interfacial films around
droplets of coconut oil since its very low HLB caused SMO to prefer to migrate into the oil phase rather
than to settle at the oil/water interface.5 In this study, it was also noted that SMO in formulations L-1 and
L-3 could not provide feasibility for physical stable IPP lotion. This phenomenon was due to high
lipophilicity of SMO.
The difference in HLB value between PSMO and PSMS is only 0.1; however, the stability of the obtained
IPP lotions was contrast. PSMO when combined with either SMO or SMS could not feasibly provide
physical stable IPP lotions while PSMS when combined with SMS could do. Hence, chemical structures
of emulsifiers can influence on feasibility of emulsion formation by attraction with the oil phase.1 As
illustrated in Figure 2, the unsaturated lipophilic oleate tail of PSMO could attract to IPP having saturated
bonds less than the saturated lipophilic stearate tail of PSMS.

Figure 2 Chemical structures of (a) IPP, (b) PSMO, and (c) PSMS.
It could be obviously seen in this study that the application of the HLB system could not provide the
perfect answer for emulsifier selection. Chemical compatibility resulted from chemical structure was also
a critical factor. However, the physicochemical reasons were not investigated in the current study.
In this study, it was found that proper emulsifier blend (PSMS/SMS) could provide a stable IPP lotion. It
is well-known that more the emulsion interface is strengthened by the presence of the emulsifier system
results in higher stability of the resultant emulsion. Therefore, an emulsifier blend is possible to reach a
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greater packing density of emulsifier material at the oil/water interface rather than a single material,
leading to an intrinsically more stable emulsion.1-3 To illustrate this point, the packing of PSMS/SMS at
the interface between IPP and water in order to for an oil-in-water emulsion was proposed and exhibited
in Figure 3.
Figure 3 Packing of an emulsifier blend (PSMS/SMS) at IPP/water interface.
CONCLUSION
It could be concluded that among studied emulsifier pairs (PSMO/SMO, PSMO/SMS, PSMS/SMO, and
PSMS/SMS), only the PSMS/SMS blend was suitable to emulsify IPP in water, although all pairs had the
identical total HLB value. Thus, besides HLB values, the chemical structures of emulsifiers were proved
to be another important parameter in feasibility of emulsion formation.
ACKNOWLEDGEMENTS
The authors are grateful for financial support received from Graduate School, Prince of Songkla
University, Thailand.

REFERENCES
1. ICI Americas Inc. 1980. The HLB system: a time-saving guide to emulsifier selection. Available
online: http://www.firp.ula.ve/archivos/historicos/76_Book_HLB_ICI.pdf. Accessed: January,
2013.
2. Ansel HC, Popovich NG, Allen LV Jr. 1995. Pharmaceutical Dosage Forms and Drug Delivery
Systems. 6th ed, Williams & Wilkins, Malvern, pp. 253-85.
3. Boothroyd S. 2008. Emulsions. Society of Cosmetic Science, Luton, pp. 1-52.
4. Al-Sabagh AM. 2002. The relevance HLB of surfactants on the stability of asphalt emulsion.
Colloids and Surfaces A: Physicochemical and Engineering Aspects 204: 73-83.
5. Boonme P, Maneenuan D, Channarong S. 2013. Physical stability of coconut oil lotions
formulated using hydrophile-lipophile balance system of various emulsifier pairs. International
Journal of Pharmaceutical Compounding 17(4): 347-50.
6. Schmidts T, Dobler D, Guldan AC, Paulus N, Runkel F. 2010. Multiple W/O/W emulsions -
using the required HLB for emulsifier evaluation. Colloids and Surfaces A: Physicochemical and
Engineering Aspects 372: 48-54.
7. Convergent Cosmetics. nd. Emulsions and the HLB System. Available online:
http://www.lotioncrafter.com/pdf/Emulsions_&_HLB_System.pdf. Accessed: January, 2013.
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THE EFFECT OF PARTICLE SIZE OF ULTRADEFORMABLE LIPOSOMES FOR DERMAL

DELIVERY OF HYDROPHILIC COMPOUND
Worranun Rangsimawong1, Praneet Opanasopit1, Theerasak Rojanarata1, Tanasait Ngawhirunpat1, **

1
Faculty of Pharmacy, Silpakorn Univercity, Nakron Pathom, Thailand*Corresponding author E-mail address: tanasait@su.ac.th
KEYWORDS: Vesicle size, Ultradeformable liposomes (ULs), Hydrophilic compound, Skin permeation
INTRODUCTION
Ultradeformable liposomes consist of phospholipids, an edge activator that increases deformability of the
bilayers by destabilizing them and are elastic in nature1. The influence of liposome size seems to be
important. Verma et.al. (2003) reported that the hydrophilic fluorescent compound [carboxyfluorescein
(CF)] penetration was related to the size of the liposome. The liposome with a size of 120 nm diameter
showed statistically enhanced penetration of CF into the skin as compared to large ones2. However, no
dedicated study was performed up to now to clarify the influence of vesicle size of ultradeformable
liposomes (ULs) on the skin penetration. The aim of this study was to investigate the effect of vesicle size
of ultradeformable liposomes (ULs) on the penetration of fluorescently labeled compound into the
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porcine skin. Sodium fluorescein (NaFI), a hydrophilic fluorescent compound was encapsulated into
vesicles. Liposomes were prepared by the sonication method or extruding the vesicle through different
pores sizes of membrane filter. The particle size, zeta potential, entrapment efficiency (%EE), Loading
efficiency (%LE) and in vitro skin penetration were investigated. The confocal laser scanning microscopy
(CLSM) was also used to investigate the penetration pathways of the vesicles.

Materials Phosphatidylcholine (PC) from eggs was purchased from GmbH. Cholesterol (Chol) was
purchased from Carlo Erba Reagent. Tween 20 was purchased from Ajax Finechem. NaFI was purchased
from Sigma-Aldrich.
Preparation of NaFI entrapped in liposome Ultradeformable liposomes were composed of phospholipid
(PC), cholesterol (Chol), and tween 20 as the edge activator. Liposomes were prepared by the sonication
method or extruding the vesicle through different pores sizes of membrane filter. NaFI solution was
prepared by dissolving NaFI in phosphate-buffered saline (PBS; pH 7.4). A mixture of PC and Chol in a
molar ratio of 10: 2, dissolved in chloroform/methanol (2:1, v/v), was added to a test tube, and the solvent
was evaporated with nitrogen gas. The lipid film was placed in a desiccator connected to a vacuum pump
for a minimum of 6 hours. The dried lipid film was hydrated with NaFI in PBS buffer. After, the
dispersion was sonicated in a bath for 30 minutes and then added tween 20 to the liposome dispersion.
UL50 were prepared using probe-sonicated for 30 minutes. UL100 were prepared using probe-sonicated
for 2 minutes and then extruding the vesicle through 0.22 µm membrane filter. UL 500 was prepared by
extruding the vesicle through 1.2 µm membrane filter.
Characterization of ultradeformable liposomes
Particle size and surface charge Each liposome formulation was diluted with an appropriate amount of
water and subsequently measured for size distribution and zeta potential using a Dynamic Light
Scattering (DLS) particle size analyzer (Zetasizer Nano-ZS, Malvern Instrument, Worcestershire, UK)
with a 4mW He-Ne laser at a scattering angle of 173o. All the measurements were carried out under
ambient conditions and in triplicate.
%EE and %LE The liposome dispersion (0.5 mL) was placed in an ultrafiltration tube with a molecular
weight cutoff of 3,000 Da (Microcon YM-3; Minipore, Billerica, Massachusetts, USA) and centrifuged at
4 oC at 10,000xg for 60 minutes. The filtration was discarded, and 0.25 mL of PBS was added to the
retentate before further centrifugation at 4 oC at 10,000xg for 40 minutes. The collected NaFI-loaded
liposome in the retentate were subsequently disrupted by added 0.2 mL of 0.1% (w/v) Triton X-100 and
centrifuged at 4oC at 10,000xg for 10 minutes. The NaFI content in the supernatant was determined by the
fluorescence-detection method. The drug %EE and %LE were calculated by the following equation (1
and 2)
%EE = (C L /C i ) x 100 (1)
Where C L is the concentration of NaFI-loaded in liposomal formulation, and C i is the initial concentration
of NaFI added into the liposome formulation
%LE = (D t /L t ) x 100 (2)

Where D t is the total amount of NaFI in the liposomal formulation, and L t is the total amount of PC and
Chol added into the liposomal formulation.
In vitro skin-penetration study NaFI through porcine skin was performed using Franz diffusion cells with
a penetration area of 2.31 cm2. The receiver compartment was filled with 6.5 mL of PBS (pH 7.4), stirred
with a magnetic bar at a rate of 500 rpm. The skin was mounted in the diffusion chamber of the cell, with
the stratum corneum side up. Diffusion cell were connected with a circulating water bath to maintain the
temperature at 32 oC. Each liposome (2 mL) was applied into the donor compartment. At the
predetermined times of 1, 2, 4, 6, 8, and 24 hours, 0.5 mL of receiver medium was withdrawn for analysis
by the fluorescence-detection method, and the same volume of PBS was added into the receiver
compartment to maintain a constant volume. Each sample was analyzed in triplicate.
CLSM study After the in vitro skin-penetration study at a time of 8 hours, porcine skin was visualized the
depth of NaFI penetration through the skin. The x-z sectioning confocal images were obtained from the
x20 objective lens system, equipped with an He-Ar laser (excitation wavelength 488 nm; emission
wavelength 514 nm). The piece of tissue was placed on a coverslip (22 x 50 mm) with the stratum
corneum facing up toward the microscope condenser, than an adequate amount of methyl salicylate was
added in to the piece of tissue as an immersion oil. Confocal images were illustrated as a gallery of the x-
z axis serial optical sections.
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Physicochemical characteristic of ultradeformable liposomes The particle size of ULs was in the order:
UL50 (36.0+1.03 nm) < UL100 (120.83+1.21 nm) < UL500 (451.13+8.78 nm). Sonication is one of the
popular methods to prepare unilamellar vesicles from the aqueous dispersion of phospholipids. In
addition, using difference sonication times of an ultrasonic probe system have an impact on the liposome
sizes7. All formulations showed a polydispersity index below 0.3 except for the 451 nm ULs, indicating a
narrow size distribution. The zeta potentials of all formulations were negative surface charge (-14.93 to -
15.50 mV). (Table 1)
The %EE and %LE of NaFI-loaded ULs was in the order: UL500 > UL100 > UL50. It indicated that
NaFI as a hydrophilic compound can be entrapped in the aqueous compartment (polar solute) of liposome
vesicles. Since unilamellar vesicles contain a large central aqueous compartment, they are ideally suited
for the encapsulation of water soluble agents8. In large unilamellar vesicles, UL500 showed higher NaFI-
loaded liposome vesicle than UL50 and UL100.
Table 1 Characterization parameters of the difference liposomal formulations
Formulations Particle size (nm) PDI Zeta potential %EE %LE

(mV)
UL50 36.13+1.03 0.199+0.01 -15.50+0.53 35.04+2.28 8.42+2.73
UL100 120.83+1.21 0.278+0.01 -13.20+0.56 36.79+9.00 10.17+2.89
UL500 451.13+8.78 0.367+0.01 -14.93+1.78 44.79+2.57 10.61+2.72
All data represent the mean + standard deviation (n=3).
In vitro skin-penetration study The flux of NaFI can be ranged as follows: 36 nm>138 nm> 451 nm. The
smaller size ULs of 36 nm diameter showed enhanced penetration of NaFI in the skin as compared to
larger ones. Fig.1 shows the amount of NaFI delivered from ULs of different sizes into different layers of
the skin.
Figure 1 shows in vitro cumulative amount-time profiles of NaFI in difference formulations permeated through porcine skin.
Symbols: UL 50 ( ); UL100 ( ); and UL500 ( ).

CLSM study CLSM study showed the difference in skin-penetration depth of NaFI between these three
formulations. The smaller particle size (UL50) could penetrate into the deeper skin layer (75 µm) than
UL100 (65 µm) and UL500 (55 µm).
(B) (C)
(A)
Figure 2 shows x-z axis serial images from CLSM of porcine skin treated with NaFI-loaded Rh-PE-labeled ULs at a time of 8 h:
(A) UL 50; (B) UL 100; and (C) UL 500. (20x objective lens)
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CONCLUSION
The particle sizes were 36.0+1.03 nm, 120.83+1.21 nm, and 451.13+8.78 nm and had negative surface
charge (-14.93 to -15.50 mV). The %EE and %LE of NaFI-loaded ULs was in the order: UL500 > UL100
> UL50. The flux of NaFI can be ranged as follows: UL50 > UL100 > UL500. The results correlated well
with CLSM that the smaller size of vesicles showed the deeper skin permeation of NaFI. The
transfollicular pathway also played an important role for skin permeation of ULs. The result indicated that
the NaFI penetration was related to the size of the ultradeformable liposome.
ACKNOWLEDGMENTS
The authors wish to thank the Commission of Higher Education (Thailand), the Thailand Research Funds
through the Golden Jubilee PhD Program (Grant No.PHD/0091/2554).
REFERENCES
1. Cevc G., Schätzlein A., Richardsen H. 2002. Ultradeformable lipid vesicles can penetrate the
skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM
experiments and direct size measurements. Biochim Biophy Acta, 1564(1), 21–30.
2. Verma D.D., Verma S., Blume G., Fahr A. 2003. Particle size of liposomes influences dermal
delivery of substances into skin. Int J Pharm, 258, 141-151.
3. Subongkot T., Duangit S., Rojanarata T., Opanasopit P., Ngawhirunpat T. 2012. Ultradeformable
liposomes with terpenes for delivery of hydrophilic compound. J Lipo Res, 22(3), 254-262.
4. Subongkot T., Wonglertnirant N., Songprakhon P., Rojanarata T., Opanasopit P., Ngawhirunpat
T. 2013. Visualization of ultradeformable liposomes penetration pathways and their skin
interaction by confocal laser scanning microscopy. Int J Pharm, 441, 151-161.
5. Kajimoto K, Yamamoto M, Watanabe M, Kigasawa K, Kanamura K, Harashima H, et al. 2011.
Noninvasive and persistent transfollicular drug delivery system using a combination of
liposomes and iontophoresis. Int J Pharm, 403(1–2):57–65.
6. Maheshwari RGS, Tekade RK, Sharma PA, Darwhekar G, Tyagi A, Patel RP, et al. 2012.
Ethosomes and ultradeformable liposomes for transdermal delivery of clotrimazole: A
comparative assessment. Saudi Pharm J, 20(2):161–70.
7. Silva R, Ferreira H, Little C, Cavaco-Paulo A. 2010. Effect of ultrasound parameters for
unilamellar liposome preparation. Ultrasonics Sonochemistry, 17(3), 628–32.
8. Harrington KJ, Konstantinos N, Richard GV. 2002. Liposomal targeted cytotoxic drugs for the
treatment of cancer. J Pharm Pharmacol, 54: 1573–1600.

DEVELOPMENT OF HYALURONIC ACID LOADED ELASTIC LIPOSOME
Chawankorn Kasetvetin1, Soravoot Rujiviphat1 and Waree Tiyaboonchai1

1
Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University,
Phitsanulok 65000, Thailand
KEYWORDS: hyaluronic acid, elastic liposome, entrapment efficiency
INTRODUCTION
Hyaluronic acid (HA), also called hyaluronan, is a natural glycoaminoglycan (polysaccharide) that is
mostly abundant in skin [1]. However, as skin ages, the hyaluronic acid in the skin decreases which
accounts for the loss of hydration and moisture in the skin and ultimately results in wrinkle and loss of
elasticity. As a consequence, HA is extensively utilized in cosmetic products to combat signs of aging [2].
Nevertheless, owing to its high molecular weight and charge, it is difficult to deliver HA into the skin.
To overcome this problem, elastic liposome has been developed by reversed phase evaporation technique
(REV). Elastic liposomes have been developed and evaluated as transdermal delivery systems to improve
delivery of encapsulated agent to and through skin. They are similar to conventional liposomes but with
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the incorporation of an edge activator in the lipid bilayer structure to provide elasticity. The objective of
this study was to develop and characterize the HA loaded liposome by varying the ratio of phospholipid
(HPC): cholesterol (CH): Tween80. Additionally, the entrapment efficiency of HA loaded liposome was
determined.
MATERIALS AND METODS

Materials Hyarulonic acid sodium salt from Streptococcus equi, Stain All dye (assay 95%) and uranyl
acetate dehydrate were purchased from Sigma Chemical, Capricorn, Singapore. Cholesterol from lanolin
(≥ 95.0 % GC) was purchased from Sigma-Aldrich Chemie, Buchs, Japan. Chloroform and methanol
(analytical grade) were purchased from RCI Labscan, Bangkok, Thailand. Ethanol 95% was obtained
from commercial product, TTK-Science, Thailand. Phophatidylcholine (Lipoid S100-3) was purchased
from Lipoid GmbH, Ludwigshafen, Germany and polyoxyethylene sorbitan monooleate (Tween 80) was
purchased from Thai Sanguanwat Chemical, Bangkok, Thailand.
Determination of hyaluronic acid (HA) based on Stain All dye In this experiment, HA was
determined based on Stain All by UV-visible spectrophotometer. Stain All is a dye which can bind to
glycoaminoglycans and form complexes whose optical properties are different from those of the free
dye. HA solution was prepared in deionized water with concentration ranging from 0.5-5 mg/ml. The dye
solution (0.1 mg/ml) was prepared by dissolving 2.5 mg of Stain All in 22 ml of water and 3 ml of
methanol. The HA/dye complex was detected at 640 nm [3].
Preparation of HA loaded liposome by reversed phase evaporation technique (REV) Hyaluronic acid
loaded liposome was prepared by reversed phase evaporation technique. Phosphatidylcholine (HPC) and
cholesterol (CH) were dissolved in a mixture of ethanol and chloroform (4:1). The aqueous phase,
hyaluronic acid mixed with Tween 80, was added to the oil phase and mixed. The system was sonicated
for 10 minutes in a bath type sonicator at 50°C for 10 minutes. Then, the organic solvent was removed
under reduced pressure (200 mm Hg) using a rotary evaporator (Buchi Rotovapor, USA). At this point,
the materials formed a liposomal suspension. The resulting liposomes were kept in the refrigerator
overnight at -20˚C before characterization [4].
Physicochemical characterization of the liposome Mean particle size: The mean particle size and size
distribution measurements were conducted using a ZetaPALS® (Brookhaven Instrument Co., New York,
USA). Prepared liposome formulations were diluted at least 50 times in DI water to obtain liposomal
suspension. The particle size of each sample was measured at 25ºC at a detection angle of 90º for 6
repeated measurements. All measures were performed in triplicate. Zeta potential: The zeta potential was
determined using phase analysis light scattering with ZetaPAL/90plus. Measurements were carried out at
25ºC at 14.8º to the incident light. Samples were prepared by redispersing the liposome in DI water. The
zeta potential was calculated using the electrophoretic mobility based on the Smoluchowski
approximation.
Morphology of liposome The morphology of the HA loaded liposome was characterized by transmission
electron microscopy (TEM) with an accelerating voltage of 80 kV(Phillips Tecnai12, Electron Optics,
Holland).The liposome specimen for TEM was prepared with negative staining. The prepared liposome

suspension was processed by using copper grid to adsorb liposome particles from the suspension for 5
min, then stained in uranyl acetate for 30 seconds and dried. The grid containing the liposome sample was
dried further in the air and kept in desiccators before being investigated [5].
Determination of entrapment efficiency (EE): The entrapment efficiency of the HA loaded liposome
was determined by ultracentrifugation method. Approximately 100 mg of HA loaded liposome was
suspended in 2 ml deioinzed water and subjected to centrifugation at 18,000 rpm for 30 min. After
discarding the supernatant, HA was extracted from liposome using 10 mL deionized water and sonication
for 2 min at room temperature. Then, the sample was centrifuged at 18,000 rpm for 30 min. Finally, the
supernatant was analyzed for HA content based on Stain All assay using a UV-Vis spectrophotometer at
640 nm. EE was calculated as [(Amount of HA entrapped) x 100]/(Total amount HA). The EE is reported
as the mean of three independent trials.
RESULTS
Determination of HA based on Stain All dye
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Figure 1 Standard curve of hyaluronic acid (HA) based on Stain All at 640 nm using UV-Vis spectrometer
Physiochemical characterization of elastic liposome

Table 1 Effect of HPC on the mean particle size, size distribution and zeta potential of elastic liposome
Code HPC:CH:Tween80 MS PI BI Zeta potential

(w/w) (nm±SD) (±SD) (±SD) (mV±SD)
A1 40:30:10 538 (±103) 0.18 (±0.09) 6.8 (±1.0) -33.93 (±2.28)
A2 60:30:10 616 (±34) 0.06 (±0.07) 7.8 (±0.8) -33.86 (±1.65)
A3 70:30:10 627 (±113) 0.07 (±0.06) 8.9 (±0.9) -31.85 (±4.1)
A4 80:30:10 577 (±36) 0.02 (±0.02) 8.4 (±0.9) -26.45 (±2.46)
A5 90:30:10 647 (±29) 0.01 (±0.01) 6.0 (±3.1) -27.51 (±3.30)
* MS= mean particle size, PI =polydispersity index and BI =base line index
Table 2 Effect of CH on the mean particle size, size distribution and zeta potential of elastic liposome

B1 80:0:10 N/A
B2 80:10:10 773 (±57) 0.06 (±0.05) 3.7 (±1.7) -22.05 (2.99)
B3 80:20:10 628 ( ±71) 0.007 (±0.03) 7.7 (±1.2) -22.63 (2.23)
B4 80:30:10 522 (±36) 0.016 (±0.02) 6.8 (±8.1) -25.02 (2.24)
B5 80:40:10 539 (±18) 0.005 (±0.000) 8.1 (±0.7) -24.19 (3.4)

Table 3 Effect of Tween 80 on the mean particle size, size distribution and zeta potential of elastic liposome

C1 80:40:5 527 (±102) 0.02 (±0.03) 8.7 (±0.6) -25.93 (±2.24)
C2 80:40:10 539 (±18) 0.01 (±0.00) 8.1 (±0.7) -24.19 (±3.4)
C3 80:40:20 446 (32) 0.01 (±0.00) 9.0 (±0.4) -28.93 (±5.94)
C4 80:40:30 382 (±32) 0.01 (±0.00) 9.0 (±0.4) -28.21 (±6.37)
Morphology of HA loaded liposome
(A) (B) (C)
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Figures 2-4 The transmission electron micrographs in different fields of the HA loaded liposome prepared by REV method. TEM
image (A):123000x, (B):135000x, and (C):135000x
Determination of entrapment efficiency
Table 4 The mean particle size, size distribution, zeta potential and % entrapment efficiency of HA loaded liposome
Concentration MS PI BI Zeta potential % Entrapment % drug

of HA (nm ±SD) (±SD) (±SD) (mV±SD) (±SD) content
(mg/ml)
0.2* 834 0.06 5.9 -29.59 (±1.01) 74.90 (±3.13) 71.70
(±67) (±0.07) (±1.2)
0.4* 515 0.01 8.1
(±44) (±0.00) (±0.9) -25.88 (±1.8) 78.87 (±0.89) 78.98
*n=3
DISCUSSIONS
In this study, HA loaded elastic liposome was successfully developed. The effect of HPC: CH: Tween 80
on physical properties of elastic liposome was characterized. Table 1 shows that the mean particle size,
polydispersity index and zeta potential are not significantly difference. In this experiment, increasing
HPC resulted in increased liposome formation since, as a phospholipids, it could act in the formation of
vesicle. Table 2 shows the effect of CH on physical properties of elastic liposome. The mean particle size
tended to reduce when CH was increased because the insertion of CH molecules into the lipid bilayer
helped the upper portions of the lipid hydrocarbon chains to adopt a more trans configuration, thus
decreasing the number of kinks in the bilayer and increasing the orderliness of the lipid molecules. Thus,
the inclusion of CH led to increased lateral packing density of phosphatidylcholine layers and decreased
the free volume within the hydrophobic part of the layer lipid [6]. It is well known that CH acts as
significant membrane stabilizer but also a relative vesicle formation. Thus, CH is indispensable
component for liposome formation. Formulation without CH resulted in no liposome formation. Tween
80, as a non-ionic surfactant, acts as edge activator to enhance the skin permeation [7]. The effect of
tween 80 on the particle size is similar to the effect of CH. Figures 1-3 show the TEM images of the plain
liposomes. Morphologically, these plain liposomes were nearly spherical and had multilamellar structure
characterized by multiple membrane bilayer. The candidate ratio for optimal liposome formulation was
HPC : CH : Tween 80 (80:40:30, w/w), as a blank liposome. The mean particle size was ~ 382 nm and
polydispersity index of 0.01, indicating a narrow size distribution and negatively charged at -28 mV. The

percentages of the HA entrapped into liposomes of 0.2 and 0.4 mg/ml of HA were as high as 75% and
79%, respectively.
CONCLUSION
Preparation of HA loaded elastic liposome by reversed phase evaporation has been achieved. The optimal
liposome formulation was the ratio of HPC : CH : Tween 80 = 80:40:30, w/w. The mean particle size was
382 nm with negative charge of approximately -28 mV. Cholesterol is important to vesicle formation and
can also act as membrane stabilizer (rigid membrane). The effects of CH and Tween 80 in increasing the
particle size were similar which the mean particle size tended to reduce because the increasing CH and
Tween 80 resulted in decreasing number of kinks in the bilayer and increasing the order of the lipid
molecules (condensation effect) [6]. Entrapment efficiency of HA loaded elastic liposome for 0.2 and 0.4
mg/ml of HA was nearly as high as about 75-79%. In the future, the permeation of HA loaded elastic
liposome will be carried out.
ACKNOWLEDGMENTS
The authors would like to thank the financial support from Naresuan University and Center of Excellence
for Innovation in Chemistry (PERCH-CIC).
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REFERENCES
1. Stern R, Maibach HI. Hyaluronan in skin: aspects of aging and its pharmacologic modulation.
Clinics in Dermatology. 2008; 26(2):106-22.
2. Kakehi K, Kinoshita M, Yasueda S-i. Hyaluronic acid: separation and biological implications.
Journal of Chromatography B. 2003; 797(1–2):347-55.
3. Fagnola M, Pagani MP, Maffioletti S, Tavazzi S, Papagni A. Hyaluronic acid in hydrophilic
contact lenses: Spectroscopic investigation of the content and release in solution. Contact Lens
and Anterior Eye. 2009; 32(3):108-12.
4. Zalba S, Navarro I, Trocóniz IF, Tros de Ilarduya C, Garrido MJ. Application of different
methods to formulate PEG-liposomes of oxaliplatin: Evaluation in vitro and in vivo. European
Journal of Pharmaceutics and Biopharmaceutics. 2012; 81(2):273-80.
5. Cheatanachan P, Akarachalanon P, Worawirunwong D, Dararutana P, Bangtrakunonth A,
Bunjop M, Kongmuang S. Ultrastructureal characterization of liposomes using transmission
electro microscope. 2008; 55-57:709-711.
6. Rao Z, Taguchi T. Spectroscopic studies on interactions between cholesterol-end capped
polyethylene glycol and liposome. Colloids and Surfaces B: Biointerfaces. 2012; 97(0):248-53.
7. El Maghraby GMM, Wasiams AC, Barry BW. Interactions of surfactants (edge activators) and
skin penetration enhancers with liposomes. International Journal of Pharmaceutics. 2004; 276(1–
2):143-61.

PHYSICOCHEMICAL STABILITY OF MELOXICAM LOADED LIPOSOME

FORMULATION: EFFECT OF CATIONIC AND ANIONIC SURFACTANT
Sureewan Duangjit1, Praneet Opanasopit1, Theerasak Rojanarata1, and Tanasait Ngawhirunpat1*

1
Department of Pharmaceutical Technology, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom 73000, Thailand
KEYWORDS: Transfersome, Liposome, Meloxicam, Physicochemical characteristics, Stability
INTRODUCTION
Liposomes have been developed as transdermal drug delivery carriers because there are numerous study
focusing on the use of liposomes for enhancing skin permeation of various drugs including hydrophilic
drugs (sodium fluorescein [1], carboxyfluorescein [2]), lipophilic drugs (retinoic acid [3], tretinoin [4]),
genes [5], proteins [6], and macromolecules [7]. Several studies reported that elastic vesicles were more
efficient in enhancing the transport of drugs than rigid vesicles. Accordingly, new categories of liposome
with high elasticity have been introduced. The elastic liposome mainly consists of phospholipids and
various types of penetration enhancer (e.g., edge activator, single-chain surfactant, etc.), which only a
specially designed of liposome was shown to be able to allow outstanding transdermal drug delivery
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carriers. Recently, various kinds of elastic liposomes have been developed for enhancing the skin
permeation of various drugs. The effectiveness of liposome and their physicochemical stability have been
question and not yet fully understood. For example, ethosomes are novel lipid carrier that composed of
phospholipid, water and high ethanol content (20-45%). This type of elastic liposome could enhance skin
permeation of various drugs e.g., testosterone [8], 5-fluorouracil [9] and 5-aminolevulinic acid [10]. The
high concentration of ethanol might improve the effectiveness of skin permeability. However, it might
also limit their safe and stability for transdermal delivery carriers. The intensive studies of liposomal
systems suggested that the liposome composition directly affected the physicochemical characteristics
and their stability. The physicochemical characteristics and the stability of liposome formulation might be
a major factor that should be concerned in development of novel liposome carriers. The most
effectiveness for skin delivery with instability formulation was not suitable for transdermal delivery
carriers. Meloxicam (MX), a nonsteroidal anti-inflammatory drug (NSAID) as a preferential COX-1
inhibitor. MX is often used clinically but oral and injectable administrations of MX are not appropriate
for peptic ulcers and patient compliance. Moreover, MX has no available option for transdermal delivery.
Therefore, MX is suitable for development as a transdermal delivery candidate.
The objectives of this study were to develop and investigate the effect of liposome composition on
physicochemical characteristics (i.e., vesicle size, zeta potential, elasticity and drug content) and the
stability (i.e., vesicle size, zeta potential and drug content) of liposome formulation. Three types of
liposome formulations i.e., cationic, neutral and anionic liposome were optimized for becoming optimal
transdermal delivery carrier for meloxicam. This knowledge provides the useful information for
optimizing the novel liposome formulation for enhancing skin delivery of meloxicam, especially
liposomes containing surfactant systems.

Preparation of liposome formulation Three types of liposome formulations i.e., cationic liposome (cLP),
neutral liposome (nLP) and anionic liposome (aLP) were prepared. The liposome formulations containing
phosphatidylcholine (PC), cholesterol (Chol), cationic surfactants (cetylpyridium chloride; C16), anionic
surfactants (sodium hexadecyl sulfate; A16) and meloxicam (MX) were formulated. As shown in Table 1,
liposome formulations were prepared by the sonication method. Briefly, lipid mixtures of PC, Chol,
surfactant and MX were dissolved in chloroform/methanol (2:1 v/v ratio), and the solvent was evaporated
under nitrogen gas stream. The lipid film was dried in a desiccator for 6 h to remove the remaining
solvent. The dried lipid film was hydrated with acetate buffer solution; ABS (pH 5.5). Liposomes were
subsequently sonicated for 30 min using a bath-type sonicator and then duplicated sonicated in ice-bath
using probe sonicator (Sonics Vibra CellTM, Newtown, CT, USA) for 30 min. An excess amount of lipid
composition and MX was removed by centrifugation at 4°C using 15,000 rpm for 15 min, and the
supernatant was collected. All formulations were freshly prepared or stored in airtight containers at 4 °C
prior to further studies.
Measurement of physicochemical characteristics The MX loaded liposome formulations were
characterized for vesicle size, zeta potential and MX content. Vesicle size and zeta potential of liposome

formulations were measured by photon correlation spectroscopy (Zetasizer Nano series, Malvern
Instrument, UK). Twenty µL of the liposome formulations were diluted with 1480 µL of deionized water.
All measurement samples were performed at room temperature, at least three independent samples were
taken, and the vesicle size and zeta potential were measured in triplicate (n=3).
Measurement of elasticity The elasticity value of the lipid bilayer of the vesicles was directly
proportional to J Flux × (r v /r p )2:
2
r 
-1
Elasticity value (mg·sec ·cm ) -2
= J Flux ×  V  (1)
 rP 
Where J Flux is the rate of penetration through a permeable barrier (mg·sec-1·cm-2), r v is the size of the
vesicles after extrusion (nm), and r p is the pore size of the barrier (nm) [11]. To measure J Flux , the
vesicles were extruded through a polycarbonate membrane (Nuclepore, Whatman Inc., MA, USA) with a
pore diameter of 50 nm (r p ), at a pressure of 0.5 MPa. After 5 min of extrusion, the extrudate was
weighed (J Flux ), and the average vesicle diameter after extrusion (r v ) was measured by Zetasizer Nano ZS
instrument.
Stability study of liposome formulation The MX loaded liposome formulations were kept in glass bottle
with plastic plugs and stored at 4±1 °C and 25±1 °C for 30 days. The physicochemical stability of MX
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loaded liposome formulation was evaluated. The vesicle size and zeta potential were determined using
Zetasizer Nano ZS instrument. The MX content was evaluated by measuring MX remaining in the
liposome formulation by HPLC at day 1, 7, 15, 30 and 120. The physicochemical stability of the
formulation after freshly preparation (at day1) was used as control and MX content at day 1 was also
normalized to 100%.
HPLC analysis The MX concentration in all samples was analyzed by HPLC. All samples were stored at
4 °C until analysis. The HPLC system consists of a SIL-20A autosampler, LC-20AT liquid
chromatograph and SPD-20AUV detector (Shimadzu Corporation, Kyoto, Japan). The analytical column
was YMC-Pack ODS-A (150 mm × 4.6 mm i.d., S-5, YMC Co. Ltd, Kyoto, Japan), and the mobile phase
was composed of acetate buffer solution (pH 4.6)/methanol (50:50, v/v). The flow rate was set at 0.8
ml/min, and the wavelength used was 272 nm. The calibration curve for MX was in the range of 1-50
µg/mL with a correlation coefficient of 0.999. The percent recovery was found from 99.85-100.30%, and
relative standard deviation for both intra-day and inter-day was less than 2%.
Data analysis The data are reported as the means ± standard error (S.E.) (n=3-6), and statistical analysis
of the data was carried out using one-way ANOVA followed by student’s t-test. A p-value of less than
0.05 was considered to be significant.
Table 1 The composition of different liposome formulation
Liposome component (%W/V)

Formulation ABS pH 5.5 qs to
PC Chol C16 A16 MX
cLP 0.77 0.04 0.10 - 0.07 100 mL
nLP 0.77 0.04 - - 0.07 100 mL
aLP 0.77 0.04 - 0.10 0.07 100 mL

Physicochemical characteristics of liposome The physicochemical characteristics i.e., vesicle size, zeta
potential, elasticity and MX content of liposome formulations were shown in Table 2. The addition of
cationic and anionic surfactant resulted in significant difference in physicochemical characteristics of MX
loaded liposome formulation. The vesicle size of cLP was significantly smaller than nLP. While, the
vesicle size of aLP was significantly larger than nLP. As, the effect of neutralization between anionic
drug (MX) and cationic liposome (cLP) could reduce the repulsive forces between the liposome bilayer,
these might decrease the vesicle size of liposomes. In contrast, the synergistic effect between anionic drug
(MX) and anionic liposome (aLP) might result in a large vesicle size [12]. The size distribution of all
liposome formulation was not significant difference. The zeta potential of cLP, nLP and aLP was positive,
neutral and negative charge, respectively, depended on intrinsic properties of their surfactant charge and
total net charge of liposome composition. Under the experimental condition of pH 5.5, the isoelectric
point (PI) of PC (PI = 6) was higher than pH. On the other hand, the PI of MX (PI = 2.6) was lower than
the pH. Thus, PC and MX carried the net positive charge and negative charge, respectively. The liposome

formulation consisted of neutral charge material e.g., phosphatidylcholine and cholesterol, positive charge
material e.g., cetylpyridinium chloride and negative charge material e.g., sodium hexadecyl sulfate and
meloxicam. Therefore, the total net charge of the liposome composition may affect the net charge of
liposome.
The elasticity of cLP and aLP was significantly higher than nLP. Because, the incorporation of single-
chain surfactant (edge activator) i.e., cetylpyridinium chloride (C16) and sodium hexadecyl sulfate (A16)
could increase the elasticity of liposome bilayer [13]. The cationic and anionic surfactants which have a
high radius of curvature could increase deformability of the liposome bilayer. However, these results
indicated that the same carbon chain length with different polar head group of surfactant resulted in
significant difference in elasticity value. Moreover, the MX content of cLP and aLP was significantly
higher than nLP. These results indicated that the addition of C16 and A16 surfactant might increase the
solubility of MX in liposome bilayer. Our results were consistency with the previous study [10] that the
drug content of liposome with sodium stearate (anionic surfactant) was increased.
Table 2 The physicochemical characteristics of liposome formulation after freshly preparation
Size Zeta potential Elasticity MX content

Formulation
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(nm) (mV) ( mg·sec-1·cm-2) (%)
cLP 90.60 ± 9.20 +48.30 ± 0.67 88.7 ± 0.98 68.06 ± 0.84
nLP 108.80 ± 10.60 1.30 ± 1.01 11.6 ± 1.64 26.36 ± 0.26
aLP 164.30 ± 3.20 -60.80 ± 0.51 19.2 ± 1.68 54.11 ± 0.33
Physicochemical stability of liposome Figure 1 shows the effect of cationic and anionic surfactant on the
(A) vesicle size, (B) zeta potential and (C) MX remaining of liposome formulation from day 1 to day 120
at 4 °C and 25 °C. The results indicated that no sedimentation was observed in any formulation after fresh
preparation. No sedimentation was found in any formulation, after storage at 4 °C for 30 days. The
vesicle size was not significantly different from the initial liposome formulation at day 1 (control).
However, the sedimentation was found in the some formulation (i.e., aLP and cLP), after storage at 4 °C
for 120 days. The vesicle size on day 120 had a trend to increase, and the size of liposome was larger than
control, however most of formulations was smaller than 200 nm (except; aLP). After storage at 25 °C for
30 days, no sedimentation was found in any formulations till day 30, and the vesicle size had a trend to
increase after the storage age of 15 days, however all formulations was still smaller than 200 nm. In
contrast, after storage at 25 °C for 120 days, sedimentation was found in all formulations. The vesicle
size on day 120 was significantly different from the initial formulation which was around 200-400 nm. In
addition, the size distribution of all liposome formulation was not significant difference.
The zeta potential of liposome formulation was depended on the net charge of their liposome
composition. After storage at 4 °C for 30 days, the zeta potential was slightly different from the initial
formulation; and some formulations had a trend to increase while some formulations had a trend to
decrease depended on their composition. However, after storage at 4 °C for 120 days, the zeta potential
was significantly different from the initial formulation (especially; aLP). After storage at 25 °C for 30
days, the result was similar to those found in 4 °C for 30 days. However, after storage at 25 °C for 120
days, the zeta potential was significantly different from the initial formulation, and the zeta potential was
markedly increased and higher than the initial formulation. The MX remaining in the formulation after
freshly preparation at day 1 was normalized to 100%. After storage at 4 °C for 30 days, the MX
remaining was slightly decreased but still higher than 90% of the initial formulation. However, after
storage at 4 °C for 120 days, the MX remaining of almost formulations decreased around 70-80% from
the initial formulation, while some of them (i.e., aLP) decreased around 90% from the initial formulation.
After storage at 25°C for 30 days, the MX remaining of almost formulations was slightly decreased but
still higher than 90% and 80% of the initial formulation at day 15 and day 30, respectively. However,
after storage at 25°C for 120 days, the MX remaining of all formulations decreased around 80-90% from
the initial formulation. Especially, the MX remaining in the formulation of aLP was lower than 10% of
the initial formulation.
In short term stability (on day 1 to day 30), the results suggested that the physicochemical stability in
different formulation factor was not significantly different. However, in long term stability (on day1 to
day120), the results indicated that the liposome composition i.e., A16 was a major component affecting
the physicochemical instability of aLP formulation. Moreover, the addition of Chol in liposome
formulation might be a main factor affecting the physicochemical stability of all formulations. Because,

Chol caused an increase rigidity and packing density of PC molecules, thus Chol lead to increase stability
of liposome formulation [13]. These results indicated the stability of cLP was better than nLP and aLP.
The good physicochemical stability of our liposome was at 4 °C (at least) for 30 days and was also stable
at 25 °C (at least) for 15 days. The physicochemical stability of liposome formulation was slightly or not
significantly different between the experimental temperature of 4 °C and 25 °C for 30 days, while the
physicochemical stability of liposome formulation was significantly different between the storage age of
day 1 and day 120. Therefore, the storage age was the major factor and the temperature was the minor
factor affecting the physicochemical stability of vesicle formulation. The recommended storage condition
for the vesicle formulation was 4 °C for 30 days and/or 25 °C for 15 days.
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Figure 1 The effect of cationic and anionic surfactant on the (A) vesicle size, (B) zeta potential and (C) MX remaining of liposome
formulation from day 1 to day 120 at 4 °C and 25 °C.
CONCLUSION
In this study, three types of liposome formulations were prepared for skin delivery of meloxicam. The
physicochemical characteristics of liposome were depended on types of liposome. The liposome
composition affected directly on the physicochemical characteristics of liposome formulation and their
stability. Therefore, the physicochemical characteristics and the stability of liposome formulation was the
major factor affecting the skin permeability of liposome formulations. These finding provided the useful
information for optimizing the novel liposome formulation for enhancing skin delivery of meloxicam,
especially liposomes containing cationic surfactant. The optimal formulation with good stability is used
for further transdermal delivery carrier of meloxicam.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the Thailand Research Funds through the Golden Jubilee Ph.D.
Program (Grant No. PHD/0141/2550) for financial support and thank the Faculty of Pharmacy, Silpakorn
University, Nakhon Pathom, Thailand and Department of Pharmaceutics, and Department of Drug
Delivery Research, Hoshi University, Tokyo, Japan for all facilities and support.
REFERENCES
1. Perez-Cullell, N., et al., Drug delivery, 2000(7): p. 7-13.
2. Verma, D.D., et al., Eur J Pharm Biopharm, 2003a(55): p. 271–277.
3. Montenegro, L., et al., Int J Pharm, 1996(133): p. 89-96.
4. Sinico, C., et al., J Controll Release, 2005(103): p. 123 – 136.
5. Geusens, B., et al., Eur J Pharm Sci, 2011. 43: p. 199–211.
6. Gupta, P.N., et al., Int J Pharm, 2005(293): p. 73-82.
7. Betz, G., et al., Int J Pharm, 2001(228): p. 147-159.
8. Touitou, E., et al., J Controll Release, 2000. 65: p. 403–418.
9. Zhang, Z., et al., Nanomed Nanotech Biol Med, 2012. 8(6): p. 1026–1033.

10. Fang, Y.-P., et al., Int J Pharm, 2008(356): p. 144–152.

11. Cevc, G., Handbook of Biological Physics, 1995, Elsevier Science B.V. p. 465-490.
12. Manosroi, A., et al., Int J Pharm, 2010. 392: p. 304-310.
13. Elsayed, M.M.A., et al., Int J Pharm, 2007. 332: p. 1-16.
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MELOXICAM-LOADED pH SENSITIVE POLYMERIC MICELLES:

SOLVENTS AND ENTRAPMENT METHODS
Thisirak Woraphatphadung1, Warayuth Sajomsang2, Theerasak Rojanarata1, Tanasait Ngawhirunpat1 and

Praneet Opanasopit1
1
2
National Nanotechnology Center, Thailand Science Park, Pathumthani, Thailand
KEYWORDS: Incorporation efficiency, polymeric micelles, physical entrapment
INTRODUCTION
The poorly soluble of drug is one of the important obstacles for successful and effective therapy which
frequently results in low bioavailability of the orally administered drug1,2). Polymeric micelles have been
investigated intensively as oral drug delivery applications because it is more convenient and preferred by
patient3). They are composted of amphiphilic block copolymers which consist of hydrophilic segment
(stabilize micelles) and hydrophobic segment (contain lipophilic drugs). In this way, poorly soluble drugs
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can be successfully solubilized in aqueous media4). Several techniques have been offered for the
preparation of drug-loaded micelles which the efficiency of drug loading depend on each technique such
as chemical conjugates, physical entrapments, electrostatic technique, etc5). In this study, physical
entrapment technique was selected because it was the simplest and most convenient technique and can be
divided into dialysis, O/W emulsion, dropping and evaporation. Therefore, the aim of this study was to
evaluate the effect of organic solvents and preparation methods on loading efficiency. Meloxicam (MX),
a class of non-steroidal anti-inflammatory drug with poor aqueous solubility (BCS class II), was selected
as a model drug. N-arylsuccinyl chitosan was used as synthesis pH-sensitive polymer.

Materials Meloxicam (MX) was a gift from Siam pharmaceutical, Thailand. Chitosan with 96%
deacetylation (MW 15 KDa) was purchased from OilZac Technologies Co., Ltd. (Bangkok, Thailand). 2-
Naphthaldehyde and succinic anhydride were purchased from Sigma Aldrich, USA. Dialysis bag
(CelluSep, 6000–8000 MWCO) was purchased from Menbrane Filtration Products, USA. All other
reagents and solvents were of analytical grade and used without further purification.
Synthesis of N-arylsuccinyl chitosan N-arylsuccinyl chitosan were synthesized by introducing
hydrophobic and hydrophilic moieties by reductive arylation and succinylation, respectively 6).
Preparation of micelles with and without MX
Dialysis 5 mg of N-arylsuccinyl chitosan polymer and MX (0-40 % to polymer) was dissolved in 2 ml of
dimethyl sulfoxide (DMSO) in a glass bottom. The mixture was stirred at room temperature until
completely dissolved. The mixture was then placed in a dialysis bag and dialyzed against distilled water
overnight.
O/W emulsion Micelles without MX were prepared as dialysis. For drug-loaded micelles, MX (5-40 % to
polymer) was dissolved in dichloromethane (DCM) and then was injected under constant stirring to 2 ml
of aqueous empty micellar solution. After that DCM was evaporated by overnight stirring at room
temperature. The remaining aqueous solution was centrifuged at 1000 rpm for 2 min and the supernatant
was collected.
Evaporation 5 mg of N-arylsuccinyl chitosan polymer and MX (0-40 % to polymer) was dissolved in N-
dimethylformamide (DMF) in a glass bottom. The mixture was added acetone (1/3 of DMF) and stirred at
room temperature under nitrogen gas flow until the solvent completely evaporated. 3ml of distilled water
was added, and the solution was sonicated using a probe-type sonicator (model CV 244, Sonics Vibra
CellTM, USA) in a cycle with a sonication time of 5 min and a standby time of 5 min at 80°C for 20 min.
The obtained solution was centrifuged at 1000 rpm for 2 min and the supernatant was collected.
Dropping 5 mg of N-arylsuccinyl chitosan polymer and MX (0-40 % to polymer) was dissolved in
DMSO or DMF 0.5 ml. The solution was slowly dropped into stirred water and the mixed solution was
stirred overnight. Final ratio of DMSO or DMF : water was vary as 1:5 and 1:10. The mixture was then
placed in a dialysis bag and dialyzed against distilled water overnight. After that the mixture solution was
centrifuged at 1000 rpm for 2 min and the supernatant was collected.
Incorporation efficiency MX-loaded polymeric micelles were dissolved in a mixture solution of DMSO:
H 2 O (9:1). The amount of MX-loaded into polymeric micelles was determined by UV-vis

Spectrophotometer (Model 8453E, Agilent Technologies, USA) at 365 nm. The incorporation efficiency
and loading capacity were calculated following equation(1) and (2), respectively.
(1)
(2)
Particle size The mean particle size of polymeric micelles with and without MX was determined in
triplicate at 25°C by using the Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The micelles
samples were diluted with distilled water, which was passed through a 0.22 µm membrane filter prior to
use.
In vitro release Release of MX from MX-loaded polymeric micelles was determined using a dialysis bag.
20 ml of 0.1N HCl (pH 1.2) or phosphate buffer solution pH 6.8 (PBS pH 6.8) was used as medium at
37°C under constant stirring. 1 ml of MX-loaded polymeric micelles was placed in a dialysis bag and
immersed in the medium. At certain time intervals, 1 ml aliquots of the medium were withdrawn and the
same volume of fresh medium was added. The sample solution was analyzed by UV-vis
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Spectrophotometer at 365 nm.
Statistical analysis All experimental measurements were collected in triplicate. The values are expressed
as the mean ± standard deviation (SD).

Empty polymeric micelles A novel pH responsive polymeric self-assemblies were developed based on an
introduction of hydrophobic and hydrophilic moieties into a chitosan backbone. The self-aggregates of N-
arylsuccinyl chitosan can be formed in aqueous media. The critical micelle concentration (CMC) of the
self-assembly micellar system of this polymer was determined by measuring the fluorescence intensity of
the pyrene as a fluorescent probe7). The CMC value of was found to be 0.0749 mg/ml. The particle sizes
and polydispersity index (PdI) of micelles prepared from different methods (dialysis, dropping and
evaporation) were shown in the Table1. The results revealed that different methods influenced on the
particle size of polymeric micelles. Micelles prepared by dropping technique showed the smallest particle
sizes in both DMSO and DMF solvents. The rank of particle sizes of the micelles were dropping
(84.31±0.88 nm) < dialysis (196.20±1.51nm) < evaporation (233.50±7.07 nm). The type and ratio of
solvent to water was also the important factor which affected on the particle size of polymeric micelles
prepared by dropping method. When using DMSO, the particle size of the micelles (84.31±0.88 nm) was
smaller than DMF (98.35±0.46 nm) with the similar narrow size distribution. In addition, reduction in the
ratio of organic solvent and water from 1:5 to 1:10 resulting in the increase in the particle size of the
micelles from 84.31 to 97.97 nm for DMSO and from 98.35 to 107.40 nm for DMF.
Table1 The particle sizes and polydispersity index (PdI) of polymeric micelles prepared by different methods
Method Organic solvent Ratio particle size ± S.D. (nm) PdI

(organic: water)
Dialysis DMSO - 196.20±1.51 0.061±0.031
DMF 196.10±1.15 0.081±0.012
Dropping DMSO 1:5 84.31±0.88 0.198±0.016
0.198±0.015
DMF 98.35±0.46
DMSO 1:10 97.97±0.77 0.237±0.001
0.192±0.009
DMF 107.40±1.06
DMF:acetone
Evaporation - 233.50±7.07 0.384±0.006
(3:1)
Incorporation efficiency Polymeric micelles prepared by dropping method using DMSO as organic
solvent showed a smaller particle size than DMF. Therefore, DMSO with the ratio of DMSO:water (1:5)

was selected to prepare MX-loaded polymeric micelles. For evaporation method, DMF was selected as
appropriate solvent. The effect of entrapment methods on the incorporation efficiency and loading
capacity of MX-loaded polymeric micelles was shown in the Fig.1. The X-axis represents the initial drug
used in preparation (percentage of MX to polymer), ranging from 5% to 40%, and Y-axis represents the
percentage of MX incorporated into the micelles (% CPT-loaded) (Fig 1a) and loading capacity (Fig.1b).
The results revealed that evaporation method showed the highest incorporation efficiency (25-90 %) and
loading capacity (20-65 µg/mg) when compared to other methods (less than 25 %). The loading capacity
increased ranging from 20 to 65 µg/mg with an increase in the initial MX loading from 5% to 20%. Such
a high content indicates successful incorporation of water-insoluble drug to polymeric micelles with good
water solubility. Micelle formation and drug incorporation into micelle were expected to occur
simultaneously. Interactions, mainly hydrophobic interactions, among the hydrophobic N-
phthaloylchitosan chain, MX, and solvent may be an important key to control this incorporation process.
This result was similar to the previous studies reported that the incorporation efficiency of
dexamethasone-loaded into PEGylated poly-4-(vinylpyridine) polymeric micelles was also dependent on
the incorporation methods5). Co-solvent evaporation method had percentage drug loading higher than
direct dialysis and O/W emulsion. Thus, the incorporation method affected on drug loading efficiency.
Table 2 shows the particle sizes of the polymeric micelles with and without MX. The MX-loaded
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polymeric micelles showed larger size (108 - 875 nm) than the empty micelles (84 - 233 nm). The particle
sizes of the MX-loaded micelles had tendency to increase with an increase in the weight ratio of MX to
polymer. The larger particle size of MX-loaded into polymeric micelles might be the increase of MX in
the micelles and the aggregation of micelles8).
(a) (b)
Figure1 Effect of incorporation technique and initial drug (5-40% to polymer) on (a) the incorporation efficiency and (b) loading
capacity of MX-loaded polymeric micelles; ( ) dialysis method; ( ) dropping method; ( ) evaporation method; ( ) emulsion
method.
Table 2 The particle sizes and polydispersity index (PdI) of polymeric micelles with and without MX
MX to Dialysis Dropping Evaporation O/W emulsion

polymer
Particle size Particle size Particle size Particle size
(%) PdI PdI PdI PdI
(nm) (nm) (nm) (nm)
0 196.20±1.51 0.061 84.31±0.88 0.198 233.50±7.07 0.384 - -
5 275.03±6.12 0.169 108.77±4.03 0.217 291.77±6.35 0.383 192.93±0.85 0.070
10 364.50±8.35 0.250 113.87±1.32 0.156 382.17±12.02 0.523 194.83±3.02 0.100
20 654.07±23.91 0.444 127.07±0.29 0.153 312.17±12.00 0.381 192.13±2.54 0.076
40 875.23±28.60 0.510 142.73±0.57 0.174 293.80±6.84 0.310 195.37±2.17 0.120
Micelles drug release Polymeric micelle drug delivery systems are advantageous for their wide
applicability in delivering hydrophobic drugs. pH-sensitive polymeric micelles are promising for oral drug
delivery, especially for poorly water-soluble medicines. MX (20% to polymer)-loaded polymeric

micelles prepared by evaporation method showed the highest MX loaded. Therefore, this formulation was
selected to investigate the release behavior. The in vitro release of 20% MX-loaded polymeric micelles
was measured using simulated intestinal fluid (SIF) (PBS pH 6.8) and simulated gastric fluid (SIF) (0.1 N
HCl pH 1.2) as release medium that mimic the conditions experienced in the body. The release profiles of
MX-loaded polymeric micelles in both release mediums are shown in Fig. 2. In acidic medium (0.1 N
HCl), MX released less than 50% of the MX. When the pH was changed to pH 6.8, the MX release
increased due to ionization of succinic acid moiety into N-arylsuccinyl chitosan micelles. Therefore, this
novel chitosan derivative would be the appropriate copolymer to protect poorly water-soluble drug in the
gastric fluid and release at basic pH to absorb into intestinal.
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Figure 2 Release profile of 20% MX to polymer-loaded polymeric micelles in; ( ) 0.1 N HCl pH 1.2; ( ) PBS pH 6.8
CONCLUSION
The pH responsive N-arylsuccinyl chitosan was successfully synthesized. N-arylsuccinyl chitosan could
be formed self-assembly in aqueous media and successfully incorporated poorly water-soluble drug (MX)
in hydrophobic inner core by various physical entrapment methods. Among the methods, evaporation
method and initial 20% MX to polymer showed the highest MX incorporation efficiency. The MX release
behaviors could be adjusted by pH. Therefore, this N-arylsuccinyl chitosan polymeric micelle presents
interest to develop MX carrier in oral drug delivery.
ACKNOWLEDGMENTS
The authors would like to acknowledge Commission of Higher Education (Thailand) and the Thailand
Research Funds through the Golden Jubilee Ph.D. Program and National Nanotechnology Center
(NANOTEC), Thailand.
REFERENCES
1. Aliabadi HM, Elhasi S, Mahmud A, Gulamhusein R, Mahdipoor P, Lavasanifar A. 2007. Encapsulation of
hydrophobic drugs in polymeric micelles through co-solvent evaporation: The effect of solvent composition
on micellar properties and drug loading. Int J Pharm. 329: 158–165.
2. Lu Y, Park K. 2013. Polymeric micelles and alternative nanonized delivery vehicles for poorly soluble
drugs. Int J Pharm. 453(1): 198-214.
3. Zhang Y, Chen J, Zhang G, Lu J, Yan H, Liu K. 2012. Sustained release of ibuprofen from polymeric
micelles with a high loading capacity of ibuprofen in media simulating gastrointestinal tract fluids. React
Funct Polym. 72: 359-364.
4. Yokoyama M. 2011. Clinical application of polymeric micelle carrier systems in chemotherapy and image
diagnosis of solid tumors. J Exp Clin Med. 3(4): 151-158.
5. Miller T, Colen GV, Sander B, Golas MM, Uezguen S, Weigandt M et al. 2013. Drug loading of polymeric
micelles. Pharm Res. 30: 584-595.
6. Sajomsang W, Tantayanon S, Tangpasuthadol V, Thatte M, Daly WH. 2008. Synthesis and characterization
of N-aryl chitosan derivatives. Int J Biol Macromol. 43:79-87.
7. Zhao C, Wang Y, Winnik MA, Riess G, Croucher MD, 1990. Fluorescence probe technique used to study
micelle formation inwater-soluble block co-polymer. Langmuir 6. 514–516.
8. Ngawhirunpat T, Wonglertnirant N, Opanasopit P, Ruktanonchai U, Yoksan R, Wasanasuk K et al. 2009.
Incorporation methods for cholic acid chitosan-g-mPEG self-assembly micellar system containing
camptothecin. Colloids Surf, B Biointerfaces. 74: 253-259.

FABRICATION OF MELOXICAM TASTE-MASKED ORAL FAST DISINTEGRATING

TABLET BY ION EXCHANGE RESIN AND CYCLODEXTRIN
Wipada Samprasit1, Theerasak Rojanarata1, Prasert Akkaramongkolporn1, Tanasait Ngawhirunpat1 and

Praneet Opanasopit1
1
KEYWORDS: cyclodextrin, ion exchange resins, oral disintegrating tablet, taste masking
INTRODUCTION
To overcome the problem of difficulty in swallowing tablets, the new drug delivery dosage forms known
as oral disintegrating tablets (ODTs) were developed. These tablets can be dissolved or suspended within
60 s in the mouth for easy swallowing 1). Disintegration time and taste masking are primary
considerations in the formulation of ODTs. Current taste-masking methods, including sweeteners and
flavors 2), are not sufficient for use in ODTs. Thus, new taste-masking techniques have been developed
recently, i.e. ion exchange resins and cyclodextrins 1, 3). An ion exchange resin consists of two
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components: a cross-linked polymer matrix and a functional component with bound counter ions 4).
Exchange of an ionized drug molecules with counter ions binds the drug to the resin, forming a
“resinate”. The drug resinate does not come in contact with the taste buds, and so its bitter taste is
masked. However, the controlled release of drugs from resinates can delay the onset of action 3). To
circumvent this effect, taste masking by cyclodextrins has been developed. A cyclodextrin molecule has a
lipophilic cavity that forms an inclusion complex by accepting a molecule or the nonpolar part of a
molecule into the cavity. The encapsulating the drug molecules at the molecular level reduces the
unpleasant odor and taste 5). Moreover, an inclusion complex is rapidly wetting than the free drug, and it
enhances the dissolution of the drug. Therefore, the combination of ion exchange resins and cyclodextrin
has promise for the development of ODTs for weakly water-soluble drugs with bitter taste. Meloxicam
(MX), a class of non-steroidal anti-inflammatory drugs (NSAIDs) with poor aqueous solubility and bitter
taste was selected as a model drug to formulate ODTs. Tablets containing various drug forms (free drug,
resinate, MX/HPβCD complexes, and the mixture of resinate and MX/HPβCD complexes) were prepared
and evaluated for properties that included disintegration time, taste, and dissolution profiles.

Materials Meloxicam, 2-hydroxypropyl-β-cyclodextrin (HPβCD) with molar substitution 0.6, poly
(styrene-co-divinylbenzene) sodium sulfonate (Amberlite® IRP-69), and poly(styrene codivinylbenzene)
with dimethylamine functional group in the chloride form (Dowex® 1x2-200) were purchased from Sigma
Chemical Co., USA. Microcrystalline cellulose (Comprecel® M102 D+; Mingtai chemical, Taiwan.),
spray dried lactose (Super-Tab® 11SD; DMV-fonterra excipients GmbH & Co., Germany), Crospovidone
(Kollidon®CL; BASF, Germany), mannitol (Merck, Germany), icing sugar, and magnesium stearate were
purchased and used as received. Deionized water was used in this work.
Preparation of MX resinate First, saturated MX solutions were prepared as follows. An excess amount of
MX was added into phosphate buffer solution at pH 8. The mixture was vigorously shaken on a magnetic
stirrer for 24 h. Then, the sample was filtered through a 0.45 μm membrane filter to remove any
undissolved solids. The dissolved MX was assayed at a wavelength of 362 nm by UV–Visible
spectrophotometry (NanoVueTM, GEHealthcare, UK). A MX resinate was prepared by a batch method.
The saturated MX solution were equilibrated with Dowex® 1x2-200 resin at 2:1 weight ratios of MX to
resin while stirring magnetically for 48 h. After equilibration, the resinate was collected by filtration,
washed several times with copious amounts of deionized water, and dried at 50°C overnight in a hot air
oven. The percentage of MX loading was determined by an elution method. The resinate was accurately
weighed and placed in a volumetric flask that contained 2 N potassium chloride (KCl) solution. The
mixture was stirred magnetically for 24 h and the eluted MX was assayed by spectrophotometry. The MX
loading (% w/w) of the resinate was calculated with the following equation: %MX loading = (MX EL /W) x
100% where MX EL and W are the amounts of eluted MX and resinate, respectively.
MX/HPβCD complexes Meloxicam and HPβCD were mixed at 1:1 molar ratio of meloxicam and
HPβCD that were blended in a plastic bottle for 10 min.

The mixture ratio of resinate and MX/HPβCD complexes The resinate and MX/HPβCD complexes were
mixed at 1:2 weight ratio of resinate to MX/HPβCD complexes that was blended in a plastic bottle for 10
min.
Preparation of Meloxicam ODTs Several ODTs containing equivalent doses (7.5 mg) of MX in the free
drug, resinate, MX/HPβCD complexes and the mixture ratio of resinate and MX/HPβCD complexes were
developed. The formulae of meloxicam ODTs are summarized in Table 1. All materials, except
magnesium stearate were blended in a plastic bottle for 10 min. Then, magnesium stearate was added and
materials were mixed for an additional 3 min. A portion of the mixture was accurately weighed (200 mg),
transferred to a hand hydraulic press machine (Specac P/N 15011/25011, UK), and compressed at a
constant force (29.4 kN) using stainless steel flat-face punches with a diameter of 9.53 mm.
Table 1 Composition of meloxicam ODTs
Ingredient (mg) F1 F2 F3 F4 F5 F6
Meloxicam (MX) 7.5 - - - - -
MX resinate - 15 - 5 - 5
MX/HPβCD complexes - - 7.5/29.45 5/19.65 7.5/29.45 5/19.65
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Mannitol (10%) 20 20 20 20 20 20
Icing sugar (10%) 20 20 20 20 20 20
Comprecel® M102 D+ (20%) 40 40 40 40 40 40
Kollidon®CL (5%) 10 10 10 10 10 10
Amberlite® IRP-69 (20%) - - - - 40 40
Magnesium stearate (0.5%) 1 1 1 1 1 1
Super-Tab® 11SD q.s. to 200 200 200 200 200 200
Evaluation of Meloxicam ODTs (1) The amount of MX in a tablet was determined by crushing the tablet
in a mortar. Then, the crushed powder was transferred into a 250-ml volumetric flask, and 2 N KCl, pH 8,
solution was added to adjust the volume. The mixture was stirred magnetically for 24 h. The supernatant
was filtered and assayed by UV–Visible spectrophotometry. The MX content was calculated and
expressed as % labeled amount. (2) The tablets were evaluated for uniformity of weight, diameter
hardness, and friability. Tablets were individually weighed on an analytical balance. The thickness and
diameter were measured with amicrometer (Sylvac S229, Switzerland). The hardness was measured with
a Monsanto hardness tester (Sheetal Scientific Industries, Mumbai, India). The friability was determined
with a Roche friabilator (Yeoheng Co., Thailand). (3) In vitro disintegration time. A modified method
was used to determine the disintegration time of tablets simulating the conditions similar to mouth
cavity 6). The 6 ml of artificial saliva was placed inside vessel in such way that 2 ml of artificial saliva
was below the sieve and 4 ml above the sieve. The tablet was placed on the sieve no 10 and the whole
assembly was then placed on a shaker. The time at which all the particles pass through the sieve was
taken as a disintegration time of the tablet. (4) In vivo disintegration time and taste evaluation was
performed with six healthy human volunteers. This study was approved by an Investigational Review
Board (Human Studies Ethics Committee, Faculty of Pharmacy, Silpakorn University, approval no. 24-
2553). Each volunteer randomly took a tablet and kept on their tongue, the time required for complete
disintegration of tablet was recorded as in vivo disintegration time and the bitterness after a tablet
completely disintegrated was determined immediately. The taste was evaluated and assigned a numerical
value, i.e., 0 = tasteless, 1 = slightly bitter, 2 = moderately bitter and 3 = strongly bitter, respectively. (5)
In vitro dissolution studies were performed in 0.1 N HCl for 2 h using a USP dissolution testing apparatus
II (type PTW, Pharmatest, Germany) at 37±0.5°C with a rotation speed of 50 rpm. After 2 h, the pH was
changed to pH 6.8 by the addition of 0.20 M solution of trisodium phosphate, which was equilibrated to
37°C. The MX release was measured at predetermined times; samples of medium were collected and
replaced with fresh medium, and analyzed by UV–Visible spectrophotometry.

Preparation of MX resinate MX has two dissociation constants (pKa), 1.09 and 4.18. The isoelectric
point (pI) of MX, which was computed from (pKa 1 +pKa 2 )/2 7), was 2.63. Therefore, the condition of
pH>2.63 can increase the solubility and ionization of MX into anionic forms that can be exchanged with
counter ions to form MX resinates. MX solubility increased when pH was raised from pH 3 to 8 8). The
maximum solubility was observed at pH 8, which was used to prepare MX resinate. The resinate was
successfully prepared by a batch process. After the resin was placed in the MX solution, ions from the

resin and MX solution diffused, and the dissolved MX in its ionized form (MX−) exchanged with the
counter ion (Cl−) of resin and bound to the resin via ion exchange reactions, forming the resinate
complex. The MX content was 49.69±0.35%w/w of MX to resinate, according to an elution method.
Preparation of meloxicam ODTs ODTs containing MX in various forms (free drug (F1), resinate (F2),
MX/HPβCD complexes (F3), and the mixture of resinate and MX/HPβCD complexes (F4)) were
formulated. The results showed that the MX form affected the disintegration time and hardness of the
tablets (Figure 1). The disintegration times were longer and the tablets were harder for tablets formulated
with MX/HPβCD complexes (F3) and the mixture of resinate and MX/HPβCD complexes (F4) than for
tablets formulated with the free drug and the resinate (F1 and F2). The disintegration time was prolonged
with increasing the hardness that was caused by the strengthening stronger attractive intermolecular
forces among the particles increase the hardness, hinder water penetration and prolong the disintegration
time. HPβCD contains waters of crystallization (14%) that increases binding among the particles, thus
producing stronger tablets 9). To improve the tablet disintegration times, the 20% of Amberlite® IRP-69
was selected as additional disintegrants for tablets containing MX/HPβCD complexes (F5) and the
mixtures of resinate and MX/HPβCD complexes (F6). Rapid disintegration times were occurred due to
increases in water uptake and swelling with of Amberlite® IRP-69 10).
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Figure 1 Disintegration times (bar graph) and Figure 2 Dissolution profiles of MX from tablets; (filled circle)
hardness (dot graph) of meloxicam ODTs marketed product (mobic®), (filled upright triangle) F1, (filled square)
F2, (open upright triangle) F5, and (open square) F6
Evaluation of meloxicam ODTs Tablets from F1, F2, F5, and F6 were selected and evaluated for optimal
ODTs. There were no significant differences in the weight, diameter, and hardness of these tablets, Table
2. For all tablets, the hardness was approximately 3 kg/in.2, and the friability was below the acceptable
limit (<1%), indicating that tablet hardness was high enough to withstand erosion during handling and
storage. Tablets disintegrated rapidly within 60 s, the acceptable limit for ODTs. The rapid disintegration
of tablets was due to the two types of disintegrants (Kollidon®CL and Amberlite® IRP-69). Kollidon®CL
has wicking ability that is a principal mechanism of disintegration, while Amberlite® IRP-69 has a
combination of wicking and swelling properties for disintegration. When the tablet is exposed to water,
disintegrants pull water through capillaries and the resin swelled, and the tablet disintegrated.
Table 2 Evaluation of parameters of meloxicam ODTs
diameter hardness drug content

weight (mg) friability in vitro DT (s) in vivo DT (s)
Formula (mm) (kg/inch2) (%LA)
n = 20 (%) n=6 n=6
n = 20 n = 20 n=3
F1 199.16 ± 3.27 9.61 ± 0.02 2.39 ± 0.39 0.99 52.17 ± 4.62 50.00 ± 11.98 106.51 ± 1.49
F2 202.99 ± 5.81 9.64 ± 0.05 3.00 ± 0.18 0.77 32.50 ± 11.24 42.17 ± 5.19 103.00 ± 4.62
F5 200.31 ± 3.72 9.60 ± 0.01 2.70 ± 0.39 0.62 50.67 ± 7.58 89.67 ± 15.16 100.62 ± 3.24
F6 207.43 ± 3.75 9.60 ± 0.01 2.94 ± 0.27 0.80 45.17 ± 11.13 62.67 ± 21.27 99.78 ± 3.16
The in vivo tablet disintegration times were slightly longer than the in vitro times, due to the very small
volume of human saliva that penetrated the tablet relative to the large volume of disintegration
medium 11). The MX content (% labeled amount) in all formulations were within the assay limit (90%–
110%) specified in the USP meloxicam tablet monograph. The dissolution profiles of formulated ODTs
and the marketed product (mobic®) are shown in Fig. 2. In acidic medium (0.1 N HCl), all formulae
except F2 released less than 20% of the MX due to its poor solubility at acidic pH values. When the pH
was changed to pH 6.8, the MX release dramatically increased due to its greater solubility and ionization.
The dissolution profile of the tablets containing the free drug (F1) released approximately 60% of the MX
within 6 h, which was less than the other formulae. The tablets containing resinate (F2) released
approximately 80% of the MX within 6 h, which was more than the free drug (F1) case. This finding can

be explained by the hydrophilicity of ion exchange resin. In the dissolution medium, the tablet
disintegrated, which liberated the resinate from the tablet. The ion exchange resin allowed aqueous
solutions to enter the three-dimensional resin structure and rapidly hydrate the MX resinate, thereby
enhancing the dissolution rate. The anions in the release medium displaced the loaded MX from the
resinate via ion exchange reactions, and the liberated MX was released. However, MX was not
completely released, which might be attributed to the equilibrium of the release process via the ion
exchange reaction. Tablets with MX/HPβCD complexes (F5) and the mixture of resinate and MX/HPβCD
complexes (F6) provided complete MX release within 6 h. This finding indicated that HPβCD enhanced
MX dissolution via the in situ inclusion complex with HPβCD during tablet disintegration and MX
dissolution. In the case of tablets with the mixture of resinate and MX/HPβCD complexes (F6), the MX
release was due to the combination of MX release by the MX/HPβCD complexes and MX resinate via in
situ inclusion complexation and ion exchange reactions that provided complete MX release within 6 h.
The complete MX release by F5 and F6 was similar to the release of the commercial product (Mobic®).
Table 3 presents results from the taste evaluation of ODTs with various MX forms compared to the free
drug (F1) reference. The level of bitter taste from F2 and F6 was significantly lower than F1 (p
value<0.05 analyzed from Wilcoxon signed rank test), i.e., 0.012 and 0.019 for F2 and F6, respectively.
In contrast, the bitterness level of F5 was not significantly different from F1. This finding confirmed that
PT – 11
the tablets with resinate (F2) and the mixture of resinate and MX/HPβCD complexes (F6) successfully
masked the bitter taste, while the MX/HPβCD complexes in F5 did not. However, only tablets containing
the resinate and MX/HPβCD complexes (F6) successfully masked the bitter taste of MX and enhanced
MX dissolution. Therefore, F6 was selected as the best formulation of MX ODTs.
Table 3 Taste Evaluation of Meloxicam ODTs
Bitter level*
Volunteers
F1 F2 F5 F6
1 2 1 2 1
2 1 0 1 1
3 2 1 2 0
4 2 0 1 1
5 2 0 2 0
6 2 1 2 1
*Bitterness levels: 0 = tasteless, 1 = slightly bitter, 2 = moderately bitter and 3 = strongly bitter
CONCLUSION
Taste-masked meloxicam ODTs with enhanced dissolution were successfully prepared using a
combination of ion exchange resin and cyclodextrin with the MX resinate and MX/HPβCD complexes.
This formulation demonstrated a good level of taste, rapid disintegration, and complete drug dissolution.
Thus, this tablet is advantageous for poorly water-soluble drugs with bitter taste and increases the
palatability of ODTs.
ACKNOWLEDGMENTS
The authors wish to thank the Commission of Higher Education (Thailand), the Thailand Research Funds
through the Golden Jubilee PhD Program (Grant No.PHD/0001/2553) and the Silpakorn University
Research and Development Institute for financial support (Grant No. SURDI 55/02/2555). 0
REFERENCES
1. Hirani JJ, Rathod DA, Vadalia KR. 2009. Orally disintegrating tablets: A review.Trop J Pharm
Res. 8:161-172.
2. Mohapatra A, Parikh RK, Gohel MC. 2008. Formulation, development and evaluation of patient
friendly dosage forms of metformin, Part-I: Orally disintegrating tablets. Asian J. Pharm. 2:167-
171.
3. Bhise K, Shaikh S, Bora D. 2008. Taste Mask, design and evaluation of an oral formulation
using ion exchange resin as drug carrier. AAPS PharmSciTech. 9(2):557-562.
4. Anand V, Kandarapu R, Garg S. 2001. Ion-exchange resins: carrying drug delivery forward.
Drug Discov Today. 6:905-914.

5. Loftsson T, Brewster ME. 1996. Pharmaceutical applications of cyclodextrins. 1. Drug

solubilization and stabilization, J Pharm Sci. 85:1017–1025.
6. Fu Y, Jeong SH, Park K. 2006. Preparation of fast-dissolving tablets based on mannose. ACS
Symp Ser. 924:340–351.
7. Luger P, Daneck K, Trummlitz WEG, Wagner K. 1996. Structure and physicochemical
properties of meloxicam, a new NSAID. Eur J Pharm Sci. 4:175-187.
8. Samprasit W, Rojanarata T, Akkaramongkolporn P, Ngawhirunpat T, Sila-On W, Opanasopit P.
2013. Improvement of drug loading onto ion exchange resin by cyclodextrin inclusion complex.
Drug Dev Ind Pharm. in press
9. Fenyresi E, Shirankura O, Szejtli J, Nagai T. 1984. Properties of cyclodextrin polymer as a
tableting aid. Chem Pharm Bull.32:665-669.
10. Rudnic EM, Rhodes CT, Welsh S, Bernardo P. 1982. Evaluation of the mechanism of
disintegrant action. Drug Dev Ind Pharm. 8:87–109.
11. Narazaki R, Harada T, Takami N, Kato Y, Ohwaki T. 2004. A new method for disintegration
studies of rapid disintegrating tablet. Chem Pharm Bull. 52(6):704-707.
PT – 11

CHARACTERIZATION OF TRANSFERRIN CONJUGATED SOLID LIPID NANOPARTICLES

AND IN VITRO RELEASE PATTERN FOR TARGETING BRAIN DRUG DELIVERY
Su Myat Nyein Thu1, Vimolmas Lipipun1, Garnpimol C. Ritthidej1

1
Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand.
KEYWORDS: blood brain barrier, targeted drug delivery, transferrin, conjugation, asiatic acid
INTRODUCTION
Receptor mediated transcytosis (RMT) is the most common transport mechanism to transport drugs into
the brain due to its capability to selectively uptake the macromolecules by binding the selective ligands to
its selective receptor presents on the brain capillary endothelial cells. Transferrin receptors (TfR) are
mostly abundant on the brain capillaries and the transferrin (Tf) is used as a targeting ligand binds to its
receptor (TfR) and transport drugs by RMT is becoming the interesting strategy in this study. Asiatic
Acid (AA) has been chosen as a model drug in this study due to its well known neuroprotective properties
by preventing and decreasing the amyloid β level in neurofibrillary tangles, reduce oxidative stress and
PT – 12
increase ACh synthesis in ACh deficits in cerebral cortex. Solid lipid nanoparticles (SLNs) has been
chosen as novel drug carrier in this study due to its size dependent properties, wide applications, higher
biocompatibility, higher drug loading, higher stability, low cost exipients and several other advantages.
Taken together, this study designed to focus on AA loaded SLNs conjugated with Tf targeting to the TfR
on brain capillary endothelial cells, characterize its physicochemical properties and to study its drug
release behavior in vitro.

Drugs and chemicals Asiatic Acid and human holo-transferrin, and the membrane phospholipid, 1,2-
distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) were purchased from Sigma-Aldrich, UK while
the triglycerides, trimyristin and tripalmitin were purchased from TCI, Japan. The lipophillic surfactant
Tween 80 and Span 80 were purchased from VWR International Ltd., UK. The hydrophilic surfactant,
Pluronic F127 was purchased from BASF, Mount Olive, NJ, and 1-ethyl-3-(3-dimethylaminopropyl
carbondiimide hydrochloride) (EDC) (Sigma-Aldrich, Germany) was used as zero length crosslinker. All
aqueous solution was prepared Ultrapure water (Maxima Ultra Pure Water, Elga-Prima Corp, UK) with a
resistivity greater than 18 MΩ/cm and phosphate buffered saline (PBS; pH 7.4). In addition, acetonitrile
(Sigma) and PBS (pH 7.7) were used as HPLC mobile phase.
Preparation of AA loaded SLNs AA loaded SLNs were prepared by high pressure homogenization
technique according to Soni, V., et al (2005) with the optimization results of each component by full
factorial design. The DSPE, tripalmitin and trimyristin were used as lipid phase and Span 80 was used as
lipophilic surfactatnt melted together with AA. Tween 80 and pluronic F 127 was used as hydrophilic
surfactants.
Conjugation of transferrin with AA loaded SLNs The conjugation of AA SLNs and Tf was performed
by the reported by Soni, V., et al. (2005) with slight modification. EDC was used as a zero length
crosslinking agent by crosslinking the carboxyl group of Tf with amino group presents on DSPE lipid on
the preformed SLNs via forming carbonyl amide linkage. The unbound drugs and excessive products
were removed by passing the dispersion through the sephadex G50 column.
Particle size and zetapotential measurement To measure the average size, PDI, and zetapotential of both
AA SLNs and AA Tf-SLNs, Nano-ZS zetasizer (Malvern Instruments, Malvern, UK) at 25 0C was used.
Each measurement was made in five replicates (Luo, et al., 2010).
Fourier transform infrared spectroscopy The conjugation between carboxyl group of Tf and amino
group presence on the preformed surface of SLNs were comfirmed by FT-IR using KBr pellet technique
and scanned in the range of 400 – 4000 cm-1 (Florey, 1979).
Drug Entrapment Efficiency The amount of entrapped AA in Tf conjugated and unconjugated SLNs
were determined by using the method Fry, et al. (1978). The unentrapped drug from formulations was
removed by passing the formulation through a Sephadex G-50 minicolumn and centrifuged followed by
lysing the SLNs with Triton X-100 and content of AA will be analyzed by HPLC. The encapsulation
efficiency was calculated by the following equation,

EE (%) = W entrapped drug ×100 %

W initial drug
Where W entrapped drug and W initial drug are the weight of entrapped drug in SLNs after G-50 column separation
and the initial weight of drug that added in the system, respectively.
In Vitro Drug Release Study The release of AA from the conjugated and unconjugated formulations were
studied in-vitro using dialysis membrane (Sigma Aldrich, molecular weight cut off: 12,400 Dalton,
average flat width 25 mm (1 in) in diameter, porosity 0.45 µm) with the method reported by Gupta, et al.
(2007) with slight modification. Samples were withdrawn periodically at 0.5, 1, 2, 4, 6, 8, 12, 16, 20, 24,
36 hours and replaced with same amount of fresh buffer. The amount of drug were quantified by HPLC
(Gonther, et al. 1996). The following formula was used to calculate the percentage of drug release from
the SLNs system.
Drug released (%) = Cr × Vr × 100
A
Where C r is concentration of drug in receptor compartment, V r is volume of receptor compartment, and
A is the amount of drug in donar compartment at time zero and the release mechanism was analyzed by
DD solver 1.0 add in program in Microsoft Excel (Gupta, et al. 2005).
Statistical Analysis All the mentioned experiments were repeated at least three times. Statistical analysis
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was performed using One-way ANOVA. A P-value of less than 0.05 would be considered as statistically
significant.
RESULTS
Particle size and zeta potential measurement The AA SLNs were prepared by high pressure
homogenization method and conjugated with Tf. The result obtained from Nanozetasizer showed that the
Tf conjugated nanoparticles produced the average particle size of <200 nm and the non conjugated
nanoparticles produced <180 nm in diameter. To measure the size difference upon the addition of drug
molecules, the measurement of plain nanoparticles conjugated and nonconjugated with Tf without loading
drug showed <180 nm and <160 nm respectively. Zeta potential value was found to be less negativity
after conjugation with Tf and there was no significant difference in poly dispersity index.
Fourier transform infrared spectroscopy The spectra obtained from AA loaded Tf-SLNs explained that
the intensities of lipids of CH 2 bending at 1465 cm-1 and NH 2 bending at approximately around 3310 cm-
2
after Tf conjugation and the intensities at 1729, 1736, 2916~ cm-1 IR spectra indicated the presence of
C=C, C=O and C-H bend and stretch presents in lipids and AA. The peak level at 1654.44 at AA TF-
SLNs revealed the bond formation of cabonyl group C=N and N-H bending at 1557.40 with increased and
prominent peaks after conjugation with transferrin in drug loaded SLNs while the physical mixture
showed no prominent bonding interaction when compared with AA Tf-SLNs as shown in Figure 1
(Florey, 1979).
A. Pure AA
B. AA Tf-SLNs
C. Pure Tf
Figure 1 The IR spectra of A. Pure AA, B. AA Tf-SLNs, C. Pure Tf, D. Physical mixture
Drug Entrapment Efficiency The percent of AA loaded into the plain SLNs and Tf-SLNs were analyzed
by HPLC and the result found out that the unconjugated SLNs entrapped ≤ 81% while the conjugated
nanoparticles entrapment was reduced to ≤ 70% was observed.

In Vitro Release Study The percent of drug content was analyzed by HPLC at various time interval in
vitro release study determined that the Tf conjugated AA loaded SLNs has higher drug released and
showed sustained drug release behavior when compared with unconjugated nanoparticles, as ≤ 70% and ≤
55.3 respectively as shown in Table 1 and the drug was released by Hopfenberg with Tlag model.
Table 1 The percentage of drug release at 4 different time points.
Time (hours) AA SLNs (%) AA Tf-SLNs (%)

4 ≤3 ≤5
12 ≤ 9.5 ≤ 10
24 ≤ 18.4 ≤ 22.5
36 ≤ 55.3 ≤ 70
DISSCUSSIONS
In the present study, the Tf conjugated AA loaded SLNs were prepared, characterized its physicochemical
properties and evaluated its drug release properties. The plain and AA loaded Tf conjugated and
unconjugated nanopartilces were prepared and measured its size, polydispersity and zetapotential by
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nanozetasizer as shown in. The Tf conjugated nanoparticles showed slightly bigger particle size than non-
conjugated nanoparticles in both plain and drug loaded SLNs, this could explained the conjugation of Tf
molecules on the surface of preformed SLNs and formation of Tf layer could make the particles slightly
bigger in size and zeta potential values was decreased due to the slightly negative charge on Tf layer
shield the surface of SLNs after conjugation. There was no significant change in polydispersity index
value revealed that there was no excess Tf, unentrapped drugs or others excess components dispersed in
the formulation.
From FT-IR spectrum, the intensities level in the spectra of AA Tf-SLNs at 1654.44 and 1557.40 showed
(C=N) bond bending and N-H bond stretching with the formation of peaks after conjugation with Tf
respectively when compared with physical mixture. This could confirm that the Tf was successfully
conjugated by formation of carbonyl amide linkage between Tf and amino group presents in SLNs
without interfering the physiological interactions in the preformed SLNs. Moreover, the C-H stretch and
esters bends present in AA was still maintained in the spectra revealed that the drug was successfully
loaded in the nanoparticles as shown in Figure 1.
The drug entrapment efficiency was analyzed by HPLC and the percent entrapment of Tf conjugated
SLNs was 70% which was slightly decreased then non-conjugated SLNs and this could be due to the
leaching of drug molecules on the surface of preformed SLNs when incubation during conjugation to
anchor the Tf molecules on the surface of SLNs.
The drug release of Tf conjugated SLNs were significantly higher than that of non-conjugated SLNs after
36 hour period and showed the sustained drug release behavior in vitro. The release model explained that
the drug was released by the mechanism of erosion of the erodible matrix on the surface of the molecules.
This could indicate that the peptide had dramatic impact on erosion of the polymer matrix of the drug
release profile.
CONCLUSION
In conclusion, Tf as a targeting ligand was successfully conjugated to the AA loaded SLNs due to the
formation of carbonyl amide bonding without disturbing the physicochemical properties of the SLNs. AA
was loaded in the nanoparticles without having distortion or interaction to the bonds between polymer
matrix and surfactant. High drug entrapment efficiency and sustained drug release profile were observed
in Tf conjugated SLNs with very small size with low polydispersity index. This proposed carrier could be
used as targeted and enhanced drug delivery system in order to overcome BBB limitations with Tf
targeting ability to selectively bind the Tf receptors present on brain endothelial cells.
AKNOWLEDGEMENTS
The authors express their gratitude to the Graduate School, Faculty of Pharmaceutical Sciences,
Chulalongkorn University and National Research Council of Thailand for providing research funds.
REFERENCES
1. Florey, K. 1979. Analytical Profiles of Drug Substances, Excipients and Related Methodology.
New York: Academic Press. 46-57

2. Fry, D. W., White, J. C., Goldman, I. D. 1978. Rapid separation of low molecular weight solutes
from liposomes without dilution. J.Anal. Biochem 90: 803-807.
3. Gelperina, S., Maksimenko, O., Kreuter, J., et al. 2010. Drug delivery to the brain using
surfactant-coated poly(lactide-co-glycolide) nanoparticles: Influence of the formulation
parameters. Euro. J. Pharm. and Biopharm. 74: 157–163.
4. Gupta, Y., Jain, A., Jain. S. K. 2007. Transferrin-conjugated solid lipid nanoparticles for
enhanced delivery of quinine dihydrochloride to the brain. JPP, 59: 935–940.
5. Luo, Q., Zhao, J., Zhang X. and Pan, W. 2011. Nanostructured lipid carrier (NLC) coated with
Chitosan oligosaccharides and its potential use in ocular drug delivery system. Int. J. Pharm.
403: 185-191.
6. Soni, V., Kohli, D. V., Jain, S. K., 2005. Transferrin coupled liposomes as drug delivery carriers
for brain targeting of, 5-florouracil, J. Drug Targeting, May, 13(4): 245–250.
7. Lee, K. M., Kim, I. S., Lee, Y. B., 2005. Evaluation of transferrin-polyehtylenimine conjugate
for targeted gene delivery, Arch Pharm Res Vol. 28, No 6; 722-729.
PT – 12

COMBINED EFFECT OF LOW-FREQUENCY ULTRASOUND AND MICRONEEDLES FOR

TRANSDERMAL HYDROPHILIC MACROMOLECULES
Suvida Laohapatarapant1, Praneet Opanasopit1, Theerasak Rojanarata1, Tanasait Ngawhirunpat1

1
Department of Pharmaceutical Technology, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom 73000, Thailand.
KEYWORDS: Transdermal, Low-frequency ultrasound, Microneedles
INTRODUCTION
Transdermal route offers several potential advantages over conventional routes such as avoidance of first
pass metabolism, predictable and extended duration of activity, minimizing under able side effects, utility
of short half-life drugs, improving physiological and pharmacological response, avoiding the fluctuation
in drug level, inter and intra patient valuations and most importantly. However one of the major problems
in transdermal drug delivery is the low penetration rate through the outer most layer of the skin, stratum
corneum. To date many skin penetration enhancement techniques have been developed to improve
bioavailability and increase the range of drug for topical and transdermal delivery1). Some of skin
PT – 13
penetration enhancement techniques include: heat, iontophoresis, electroporation, sonophoresis,
microneedle or magnetophoresis2, 3).
Microneedles (MNs) is one of the interesting physical enhancement method. This method can disrupt
stratum corneum barrier by creating large aqueous microchannels enough for molecules to pass through
without skin damage. Nevertheless, the length of aqueous microchannel does not reach the dermis layer,
which is filled with nerves and blood vessels. Consequently, the patient does not experience pain or
discomfort 4). Ultrasound (US), also known as phonophoresis or sonophoresis, is a technique which
involves the use of ultrasound energy to enhance skin penetration of drug. The mechanism of transdermal
skin permeation involves the disruption of the stratum corneum lipids by the formation of gaseous
cavities and heat generation, thus allowing the drug to pass through the skin. Transdermal enhancement is
particularly significant higher at low frequency regimes (20 kHz - 100 kHz) than when induced by high
frequency ultrasound (1 MHz – 16 MHz) 1 ,5). Therefore nowadays research fields of skin permeation
using the synergistic effect of ultrasound with other methods such as chemical enhancer, microneedles are
still important to be investigated. The objective of this study is to investigate the skin permeation of
hydrophilic macromolecules by using MNs, low frequency US and both of MNs and low frequency US.
Fluorescein isothiocyanate-dextrans (FITC-dextran) were used as model hydrophilic macromolecule.

Material Fluorescein isothiocyanate–dextran(FD4) (MW 3,000- 5,000) was purchased from Sigma
Aldrich (St. Louis, MO, U.S.A.) All other chemicals used in this study were analytical grade.
Skin preparation The pig skin is used in this experiment due to their similar physiological properties with
the human skin6). The pig skin is obtained from slaughterhouse at Nakhon Pathom province. The skin was
excised from pig immediately after the pig was sacrificed. The excised skin was fixed on top of a paraffin
sheet with the epidermis layer facing up. The adhering fat and other visceral debris in the skin were then
carefully removed. The underlying subcutaneous fat was gently scraped off until the skin was about 2.0-
2.5 mm thick. The preparation skin was washed, wrapped in aluminum foil, and stored at -18°C.
Preparation of microneedles array The microneedles arrays were prepared by using 9 acupuncture
needles (0.25 x 30 mm, DongBang acupuncture Inc., Boryeong, Korea). First, the silicone sheet with
thickness 2 mm, and size 15 x 15 mm was cut. Each microneedle was cut into 4 mm in length and
punctured through a silicone sheet allowing the needle tips out of a silicone sheet 1,000 µm in length. The
other end of acupuncture needle was bended to obtain a perpendicular shape in order to anchor the needle.
Then the microneedles array was fixed with an adhesive tape to ensure that the needles remained
stationary.
Low frequency ultrasound Low frequency ultrasound at 20 kHz was delivered from a commercially
available sonicator (Vibra-cell™, VCX130 PB, Sonics and Materials, Inc., Newtown, CT, USA). The
amplitude of ultrasound was set at 25%. The radiating diameter of transducer was 6.0 mm. The ultrasound
transducer was located approximately 3 mm. from surface of the skin. Continuous mode of ultrasound
application was used in this study.
Microneedles array treatment of skin The skin was punctured by using MNs array with pressure of 10 N
for 2 min. Then MNs array was removed from skin, and the skin was immediately placed on the Franz
diffusion cell.

Ultrasound treatment of skin The skin was placed on the Franz diffusion cell. Before applied ultrasound,
the donor compartment was filled with the FD4 solution. Then the pig skin was treated with with low
frequency ultrasound for 2 minutes by using continuous mode.
Microneedles array and ultrasound treatment of skin The skin was punctured by using MNs array with
pressure of 10 N for 2 min. Then MNs array was removed from skin and the skin was immediately placed
on the Franz diffusion cell. After that the low frequency ultrasound was applied on the skin for 2 minutes
by using continuous mode.
In vitro permeation studies Franz diffusion cell apparatus was used in vitro permeation studies. The
diffusion cell has an average 2.022 cm2 of diffusional area and the receptor compartment has a volume of
6 ml approximately. Before starting the experiments, the receptor compartment was filled with phosphate
buffer saline (pH 7.4) and maintained at 32 °C using a water bath. The solution in the receptor
compartment was continuously stirred at 400 rpm using magnetic stirrer in order to maintain the sink
condition during the experiments. The donor and receptor compartment were then fixed by clamper. The
top of the donor compartment was covered with parafilm in order to prevent FD4 solution loss. To
investigate the cumulative permeation profiles, 500 µl of the model drug solution in the receptor
compartment were sampled at 5, 15 and 30 minutes, 1, 2, 4, 6, 8, 12, 16, 20 and 24 h. After sampling, the
solution was immediately replaced with fresh PBS at the same volume. The samples were analyzed after
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finishing the experiments. All the tests were carried out in four experiments.
Preparation of FD4 Solution FD4 solution was prepared by dissolving FD4 in PBS pH 7.4 to obtain
concentration 2.5 μg/ml of solution. After that, the solutions were then mixed to ensure that the FD4 was
completely dissolved.
Quantitative Analysis The amount of FD4 in the solution was determined using a fluorescence
spectrophotometer (RF 5300PC; Shimadzu, Kyoto, Japan) at excitation and emission wavelengths of 495
and 515 nm, respectively7). All experiments were performed 4-6 times.

The skin permeation of FD4 by using MNs, low frequency US and both of MNs and low frequency US
was investigated. Untreated skin was also studied in this experiment for representative of passive
diffusion. After treatment skin with MNs, low frequency US and both of MNs and low frequency US at
24 hrs, the cumulative amount of FD4 permeated from skin treated with both of MNs and low frequency
US, MNs and low frequency US increased up to 8-fold (100.120 + 24.83 μg/cm2), 6-fold (74.523 + 11.47
μg/cm2) and 2.37-fold (29.373 + 4.35 μg/cm2), respectively when compared with untreated skin (passive
diffusion) (12.395 + 4.84 μg/cm2) (Table1). The permeation profiles of FD4 were shown in Figure 1. The
combination of skin penetration enhancement techniques (microneedles and low frequency ultrasound)
was significantly effective compared with microneedles alone or ultrasound alone. These results might be
caused from a combination result of disruption of stratum corneum barrier by creating large aqueous
microchannels by microneedle and disruption of the stratum corneum lipids by the formation of gaseous
cavities and heat generation by low frequency ultrasound.
Figure 1 In vitro permeation of FD4 following treatment with MNs and low frequency US (x), with MNs (▲), with low frequency
US (■) and passive diffusion (). Each point represents the mean S.E. of four experiments.
Table 1 Cumulative amount of FD4 permeated from skin after treatment with microneedles and low frequency ultrasound over 24
hrs.

CONCLUSION
In the present study, the synergistic effect with microneedles and low frequency ultrasound to transport
hydrophilic macromolecules was obtained. The skin permeation of hydrophilic macromolecules in the
combination of microneedles and low frequency ultrasound was significantly higher than microneedles
PT – 13
alone or ultrasound alone because of different mechanisms in skin transport enhancement. In addition to
increasing transdermal transport, the combination of skin penetration enhancement techniques also
increased the safety by reducing the strength of individual skin penetration enhancement technique 8).
ACKNOWLEDGMENTS
The authors thank to Faculty of Pharmacy, Silpakorn University for supporting of instrument using in this
experiment.
REFERENCES
1. Hiren JP, Darshan GT, Anand KB, Dushyant AS. 2011. Penetration enhancers for transdermal
drug delivery system: A review. IJPI’s journal. 1(2): 68- 80.
2. Patel MN, Bharadia PD, Patel MM. 2010. Skin Penetration Enhancement Techniques-Physical
Approaches. IJPAS. 1(2): 62- 72.
3. Guang Y, Kevin SW, Jie Z, Sanjay S, Bruce KG. 2010. Evaluation needle length and density of
microneedle arrays in the pretreatment of skin for transdermal drug delivery. Int J Pharm. 391: 7
-12.
4. Henry S, Devin MV, Mark AG, Mark PR. 1998. Microfabricated microneedles: A novel
approach to transdermal drug delivery. J Pharm.Sci. 87: 922 -925.
5. Ritesh K, Anil P. 2007. Modified Transdermal Technologies: Breaking the Barriers of Drug
Permeation via the Skin. Trop J Pharm Res. 6(1): 633- 644.
6. Bangtao C Jiashen W, Ciprian I. 2010. Sonophoretic enhanced microneedles array (SEMA)-
Improving the efficacy of transdermal drug delivery. Sensors and Actuators B. 145: 54- 60.
7. Yan K, Todo H, Sugibayashi K. 2010. Transdermal drug delivery by in-skin electroporation
using a microneedle array. Int J Pharm. 397: 77- 83.
8. Samir M, Joseph K. 2004. Low-frequency sonophoresis: A reviw. Advanced Drug Delivery
Reviews. 56: 589- 601.

DEVELOPMENT OF GARCINIA MANGOSTANA EXTRACT IN LIQUID CRYSTAL CREAM

FOR TOPICAL DELIVERY SYSTEM
Napa Bunma1, Jirapan Moungjaroen2, Prasan Tanguenyongwatana1, *

1
Faculty of Oriental Medicine, Rangsit University, Pathumthani 12000, Thailand
2
Numsiang International CO. LTD. 19 Sukumvit 70, Bangna, Bangkok 10260, Thailand
KEYWORDS: Garcinia mangostana, Liquid crystal, α-mangostin, HPTLC, Cream
INTRODUCTION
Garcinia mangostana L. is a medicinal plant that has long history in medicinal use in Southeast Asia for
treatment of diarrhea, skin infection and chronic wounds1). The extract of its pericarp has demonstrated
antibacterial activity against many types of microorganism2, 3). The ethanol extract of mangosteen fruit
rinds was also active against Propionibacterium acnes and Staphylococcus epidermis with MIC of 7.81
and 15.63 µg/mL, respectively4). Currently, liquid crystal systems are used to modify drug delivery
system for the delivery of topical drugs into skin5). Liquid crystals are highly anisotropic fluids that exist
as a result of long-range orientation ordering among constituent molecules. They are also three-
PT – 14
dimensional association structures that stabilize emulsions6). Our objective is to develop a liquid crystal
cream containing G. mangostana fruit rind extract to be used as a topical anti-acne product.

Instrument and reagents 1, 3-Butylene glycol was purchased from Kyowa Hakko (Tokyo, Japan).
Carbopol ultrez polymer was purchased from Lubrizol (Ohio, USA). L-Arginine was purchased from
CellMark (Balmoral Plaza, Singapore). NIKKOMULESE LC was purchased from Nikkol (Tokyo,
Japan). Cetostearyl alcohol and caprylic/capric triglyceride were purchased from Parchem (New York,
USA). The polarized light microscopy was performed on a Nikon Eclipse 50i POL (Tokyo, Japan).
Viscosity measurement was performed using a Fungilab VISCOSTAR plus viscometer (Barcelona,
Spain). All other reagents and solvents were reagent grade and used without further purification. TLC was
performed on silica gel GF 254 plates (Merck). For column chromatography, silica gel (Merck 230-400
mesh) was used. NMR spectra were recorded with a Bruker Avance (300 MHz) spectrometer. Chemical
shifts are reported in ppm, and coupling constants are reported in Hz. All NMR spectra were obtained in
deuterated chloroform (CDCl 3 ) and referenced to the residual solvent peak. Mass spectra were obtained
from an Agilent GC/MS 5975C.
Plant material The fruit rinds of G. mangostana were bought from local drugstore in Nonthaburi
province, Thailand. The material was identified by comparison with the specimen at the Forest
Herbarium, Department of National Park, Wildlife and Plant Conservation, Ministry of Natural Resources
and Environment, Bangkok. The voucher specimen of G. mangostana (SRU 026) was deposited at the
Faculty of Oriental Medicine, Rangsit University, Pathumthani, Thailand.
Preparation of crude and partially-purified extracts The dried, powdered fruit rinds of G. mangostana
(100 g) were extracted with 95% ethanol (400 mL) at room temperature for 7 days. The extract was
filtered with Whatman No.1 filter paper and then evaporated under reduced pressure to obtain 9 g of
crude dark brown extract. The crude extract (2 g) was dissolved in CH 2 Cl 2 -MeOH (8:2) (8 ml). The
mixture was then subjected to silica gel column chromatography, eluted with CH 2 Cl 2 -MeOH (8:2), to
obtain partially purified dark brown extract (1.2 g).
Isolation of α-mangostin The crude extract (1.2 g) was dissolved in 5 ml of CH 2 Cl 2 -MeOH (7:3), then
subjected to silica gel column chromatography eluted with CH 2 Cl 2 -MeOH (7:3) as the mobile phase.
After that, fractions 12-17 were collected and evaporated to obtain a yellow crystalline solid (212 mg)
with melting point of 180-182 ºC. UV 7) λ max 244, 343 nm; IR 8) (KBr disc): 3256, 2925, 1639, 1460 cm-1;
1
H NMR 9) (300 MHz, CDCl 3 ) δ [ppm]: 1.69 (s, 4H), 1.76 (s, 3H), 1.83 (s, 6H), 3.45 (d, J = 7.15 Hz,
2H), 3.81 (s, 3H), 4.09 (d, J = 6.26 Hz, 2H), 5.26 (m, 2H), 6.29 (s, 1H), 6.82 (s, 1H), 13.77 (s, 1H). 13C
NMR 9) (75 MHz, CDCl 3 ) δ [ppm]: 182.0, 161.6, 160.6, 155.8, 155.0, 154.5, 142.5, 137.0, 135.7, 132.2,
123.1, 121.4, 112.2, 108.5, 103.6, 101.6, 93.3, 62.0, 26.5, 25.8, 25.8, 21.4, 18.2, 17.9 and MS 9)
(GC/MS): M+ = 410.
Preparation of G. mangostana liquid crystal cream The G. mangostana liquid crystal cream was
prepared as o/w emulsion cream. The process started with using NIKKOMULESSE LC, cetostearyl
alcohol, caprylic/capric triglyceride and G. mangostana partially purified extract (0.1% w/w) as oil phase
adding into 1,3-butylene glycol, Carbopol ultrez, L-arginine and water at 80 ºC. The mixture was

homogenized and left to cool down to 50 ºC before DM DM Hydantoin was added and mixed together.
The product was evaluated with polarized light microscopy.
Table 1 Composition of liquid crystal cream ingredients
No. Name of chemicals % w/w
1 Water 71.4
2 1,3-Butylene glycol 5.0
3 Carbopol ultrez 0.1
4 NIKKOMULESSE LC 5.0
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5 Cetostearyl alcohol 3.0
6 Caprylic/capric triglyceride 15.0
7 L-Arginine 0.1
8 DM DM Hydantoin 0.3
9 Garcinia mangostana partially purified extract 0.1

The purification of G. mangostana crude extract with silica gel column chromatography to partially
purified extract was uncomplicated process and could dispose of undesirable components that give
unwanted color to the product. The partially purified extract of G. mangostana was standardized for α-
mangostin by using HPTLC.
For the preparation of G. mangostana liquid crystal cream, the thermotropic method was chosen to make
liquid crystals. In this formula, NIKKOMULESSE LC, which composed of cetyl alcohol, stearyl alcohol,
behenyl alcohol, phytosterol, glyceryl stearate, carprylic/capric triglyceride, hydrogenated lecithin, and
PEG-20 phytosterol, performed a major role in forming liquid crystal. After homogenized and cooled
down to room temperature, the viscous emulsion became nicely smooth, yellow cream with viscosity of
58,365 cP. We smeared a small amount of cream onto a glass slide and examined it with a polarized light
microscope (Figure 1). The first image (a) showed smooth texture of the emulsion droplets of the cream
base while the G. mangostana cream (b) showed droplets of slightly bigger size.
(a) (b)
Figure 1 Picture of cream base (a) G. mangostana cream (b) under light microscopy.
Liquid crystals are highly anisotropic fluids and their three-dimensional association structures stabilize
the emulsions. The dualism between a solid (crystal) and a flowing liquid gives rise to the term liquid
crystal. Their structure has the ability to bend/refract and reflect polarized light such that they exhibit
birefringence under the microscope 6, 10).

Birefringence
Figure 2 Liquid crystals of G. mangostana cream, when viewed under a polarized light microscope, showed the birefringence.
Figure 2 showed droplets of G. mangostana extract emulsion surrounded by a highly structured lamellar
liquid crystalline gel network acting to reduce the likelihood of particle coalescence. This
photomicrograph clearly shows the birefringence, which is the hallmark of liquid crystals surrounding
emulsion droplets.
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CONCLUSION
From our study, the formation of G. mangostana extract into liquid crystal form is easy with the help of a
specific ingredient, NIKKOMULESSE LC. The way to prove the formation of liquid crystals is also
easily done by observing the birefringence under polarized light microscope. This mixture of emulsifiers
can create a lamellar structure that responsible for delivery the active ingredients into the skin. This liquid
crystal form can enhance the absorption of active compounds through the skin. This formula can be
applied to develop other medicinal plants for cosmetic and medicinal purposes.
ACKNOWLEDGMENTS
This work was supported by Numsiang International CO. LTD, 19 Sukumvit 70, Bangna, Bangkok
10260, Thailand.
REFERENCES
1. Mahabusarakam, W., Wiriyachitra, P., Taylor, W.C. 1987. Chemical constituents of Garcinia
mangostana. J. Nat. Prod. 50:474-478.
2. Suksamram, S., Suwannapoch, N., Phakhodee, W., Thanuthiranlert, J., Ratananukul, P.,
Chimnoi, N. 2003.Antimicrobial activity of prenylated xanthones from the fruites of Garcinia
mangostana. Chem. Pharm. Bull. 51:857-859.
3. Iinuma, M., Tosa, H., Tanaka, T., Asai, F., Kobayashi, Y., Shimano, R. 1996. Antibacterial
activity of xanthones from guttiferaeous plants against methicillin-resistant Staphylococcus
aureus. J. Pharm. Pharmacol. 48: 861-865.
4. Pothitirat, W., Chomnawang., Gritsanapam, W. 2010. Anti-acne-inducing bacterial activity of
mangosteen fruit rind extracts. Med. Princ. Pract. 19(4):281-6.
5. Gosenca, M., Beŝter-Rogač, M., Gašperlin, M. 2013. Lecithin based lamellar liquid crystals as a
physiologically accepyable dermal delivery system for ascorbyl palmitate. Eur. J. Pharm. Sci.
50:114-122.
6. Wiechers, J, editor. 2009. Skin barrier: chemistry of skin delivery systems. 1st ed. Allure
Publishing Corporation; p.265.
7. Abdalrahim, F.A.A., Khalid, M.A., Mohammad, J.S., Zhari, I., Amin Malik, S.A.M. 2012.
Quantification of α-, β- and γ–mangostin in Garcinia mangostana fruit rind extracts by a reverse
phase high performance liquid chromatography. J. Med. Plants. Res. 6(2): 4526-4534.
8. Madihah, M., Bohari, M.Y., Azwam, M.L. 2013. A study on dispersion and characterization of
α–mangostin loaded pH sensitive microgel systems. Chem. Cent. J. 7:85.
9. Ly, D.H., Poul, E.H., Ole, V., Fritz, D., Hung, D.P., Lein-Hoa, D.N. 2009. Cytotoxic geranylated
xanthones and o-alkylated derivatives of α–mangostin. Chem. Pharm. Bull. 57(8): 830-834.
10. Nesseem, D.I. 2001. Formulation and evaluation of itraconazole via liquid crystal for topical
delivery system. J. Pharm. Biomed. Anal. 26(3):387-99.

FORMULATION OF KETOPROFEN MATRIX MEMBRANE

FOR TRANSDERMAL DELIVERY SYSTEMS
Jirapornchai Suksaeree1,2,*, Wiwat Pichayakorn3,4, Chaowalit Monton1,2, Apirak Sakunpak1,2, Tun

Chusut1,2, Worawan Saingam1,2, and Fameera Madaka1,2
1
Faculty of Pharmacy, Rangsit University, Pathum Thani 12000, Thailand
2
Sino-Thai Traditional Medicine Research Center (Cooperation between Rangsit University, Harbin Institute of Technology, and
Heilongjiang University of Chinese Medicine), Rangsit University, Pathum Thani 12000, Thailand
3
Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112, Thailand
4
Medical Products Innovations from Polymers in Clinical Use Research Unit, Prince of Songkla University, Hat-Yai, Songkhla
90112, Thailand *Corresponding author: jirapornchai.s@rsu.ac.th (Jirapornchai Suksaeree)
KEYWORDS: Matrix membrane, Ketoprofen patches, Drug delivery
INTRODUCTION
For matrix membrane in transdermal drug delivery systems, the drug is dispersed or dissolved in a
polymer. This membrane is attached to an adhesive layer and directly applied to the skin which can act as
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an adhesive layer by itself [1, 2]. Ketoprofen is a potent nonsteroidal anti-inflammatory drug and is
practically insoluble in water. It is commonly used for the treatment of musculoskeletal disorders such as
osteoarthritis and rheumatoid arthritis, as well as for symptoms of trauma [3]. Pseudolatex systems are
prepared in colloidal aqueous dispersions that are water-based after solvent evaporation by the
emulsification–evaporation technique which employs surfactant as stabilizers [4]. They can also be useful
in mediating drug release [5-7].
The main objective of this work was to prepare ketoprofen matrix membrane made from ethyl cellulose
(EC) and different amounts of deproteinized natural rubber latex (DNRL). Polyvinyl alcohol was used as
the surfactant and stabilizer of these systems. Glycerine was used as a skin humectant, dibutyl phthalate
as a plasticizer, and polyvinyl pyrrolidone as the channeling agent. Then, the matrix membrane for
ketoprofen transdermal patches was produced by solvent evaporation using a hot air oven. The physical
appearance and mechanical properties were characterized. Consequently, the in vitro studies of
ketoprofen were also performed.

Materials The DNRL was prepared by W. Pichayakorn’s laboratory [8-10]. It was collected from Hevea
brasiliensis: RRIM 600 clones and deproteinized by enzyme and centrifugation method. EC, ketoprofen
(98 % purity, Mw = 254.28 g/mol), polyoxyethylene-20 oleyl ether, polyvinyl pyrrolidone, glycerine,
polyvinyl alcohol (Mw = 31,000 g/mol), dibutyl phthalate were obtained from Sigma-Aldrich (USA). The
other chemicals were of analytical grade.
Preparation and physical appearance of the EC-DNRL pseudolatex systems Ketoprofen matrix
membrane was prepared by EC-DNRL pseudolatex systems composing of organic phase and aqueous
phase. The EC and dry DNRL were dissolved in 500 mL of dichloromethane by using a magnetic stirrer
until it was a clear, slightly yellow solution. After that, 6%w/w dibutyl phthalate and 2 g ketoprofen (20
mg/mL) were mixed together into this pseudolatex systems. For the aqueous phase, the 14%w/w
polyvinyl alcohol and 4%w/w polyvinyl pyrrolidone, used as surfactant and channeling agent,
respectively, were dissolved in 100 mL of water and stirred until completely dissolved. Then, 6%w/w
glycerine as the skin humectant was mixed into this solution. The two phases were mixed together by
homogenization method for 30 minutes until an emulsion-like system occurred. Dichloromethane was
then removed under vacuum by rotary evaporator with bath temperature of 40 ± 2 °C for 5 hours. Finally,
these pseudolatex systems were adjusted to 100 mL with water. Zeta potential and particle size were
measured by ZetaPALS (Brookhaven, Germany) at 25 ± 2 ºC, and presented as the effective diameter,
polydispersity index (PI), and zeta potential, respectively. The pH of the pseudolatex systems was
measured by a S220 SevenCompact™ pH/Ion pH meter (Mettler Toledo, Switzerland) at room
temperature. The pH meter was calibrated by using pH 4.0, 7.0, and 10.0 standard buffers.
Preparation and mechanical properties of ketoprofen matrix membrane Fifteen mL of EC-DNRL
pseudolatex systems preparation were poured into a Petri-dish. This was dried in a hot air oven at 70 ± 2
ºC until a complete membrane containing 4 mg/cm2 of ketoprofen was formed. Subsequently, the dry and
complete ketoprofen matrix membranes were peeled from the Petri-dish and kept in desiccators. Then, the
mechanical properties for their tensile strength (ultimate tensile strength [UTS] and elongation at break),

and the adhesion properties (T-peel strength and loop tack adhesion), were tested using the Universal
Testing Machine Model QC-508E (Cometech, Taiwan) with a 500 N loaded cell [8, 9,11].
In vitro studies The in vitro study of ketoprofen was performed using a modified Franz-type diffusion
cell with a diffusion area of 1.77 cm2. The pseudolatex-membranes were cut into 4 cm2 containing 16 mg
of ketoprofen (4 mg/cm2). The receptor compartment was filled with 12 mL of 0.5 w/v of
polyoxyethylene-20 oleyl ether in pH 7.4 isotonic phosphate buffer solution controlled with a water jacket
at 37 ± 0.5 ºC, and constantly stirred at 300 rpm with a magnetic stirrer. A 1 mL sample of isotonic
phosphate buffer solution was withdrawn at 0.5, 1, 2, 4, 6, 8, and 12 hour intervals, and an equal volume
of fresh isotonic phosphate buffer solution was then added as a replacement [12]. For the in vitro release,
the pure ketoprofen was applied to cellulose membrane (Mw cut-off 3,500 g/mol). The ketoprofen matrix
membrane was directly applied to the donor compartment. The in vitro permeation of ketoprofen
formulation was determined using pig skin and directly applied to the pig skin. The samples collected
from the in vitro study were analyzed by the HPLC system (Agilent 1260 series, USA) with an Agilent
C18 analytical column 4.6 mm × 150 mm. The mobile phases used were 0.025% v/v trifluoroacetic acid
in water (solvent A) and acetonitrile (solvent B), and they were run at a gradient of 70:30 for 5 min, then
10:90 for 8 min followed by 0:100 (solvent A:B, respectively) with a flow rate of 1 mL/min. Ketoprofen
content in the samples was determined at 255 nm. The ketoprofen content was calculated by comparing
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with the validated calibration curve. The HPLC method provided good accuracy (97.28-102.17%),
precision (0.62-1.17), and linearity in the required concentration range of 10-50 µg/mL of pure ketoprofen
in isotonic phosphate buffer solution.

Physical appearance of the EC-DNRL pseudolatex systems The DNRL material was confirmed to be a
suitably safe polymer to apply on the skin and used in this work [8, 9, 11]. Ketoprofen in its solid form could
be completely dissolved in EC-DNRL pseudolatex systems, and exhibited equally sticky emulsion. They
were yellowish pseudolatex mixtures due to the color of ketoprofen and DNRL, with good homogeneity
and smooth texture. The pH, effective diameter, polydispersity index, and zeta potential values of the
pseudolatex systems indicated their safety and ease to be applied directly on the skin. They had pH values
in range of 6.04 to 6.19 and they were safe for use on the skin with no irritation [13]. The effective
diameter, polydispersity index, and zeta potential values of ketoprofen pseudolatex mixtures were 542.23
to 787.54, 0.11 to 0.17, and -35.18 to -51.55, respectively.
Mechanical properties of ketoprofen matrix membrane The mechanical properties of ketoprofen matrix
membranes are shown in Figures 1 and 2. We found these membranes had significantly increased softness
and flexibility. They were decreased in UTS and increased percentage of elongation at break when the
DNRL was mixed into their formulation. This is directly related to the individual property of DNRL that
is very stretchy and flexible [14, 15], therefore more flexible and viscous membranes were produced. In
addition, the ketoprofen matrix membrane was improved adhesion properties that significantly increased
the T-peel and tack adhesion values. The membranes made using polyvinyl alcohol showed significantly
higher adhesive properties, i.e., T-peel strength and tack adhesion of the in situ membranes.
In vitro studies The ketoprofen pseudolatex matrix membrane was made from EC and DNRL at ratio of
1:1 was selected for in vitro studies and compared to pure ketoprofen. The pseudolatex systems could
enhance the poor water solubility of ketoprofen. These systems significantly increased ketoprofen release
profile and skin permeation profile from pseudolatex formulations (Figure 3 and 4).
CONCLUSION
We have prepared the ketoprofen matrix membrane from EC-DNRL pseudolatex systems. They had good
physical appearance and mechanical properties that were appropriate to be developed and produced
suitable ketoprofen matrix membrane. The in vitro study results showed that increased ketoprofen from
ketoprofen matrix membrane of 1:1 ratios of EC:DNRL. Thus, this work successfully prepared
ketoprofen matrix membrane for ketoprofen formulation development in skin applications.

Figure 1 The UTS and elongation at break values of ketoprofen matrix membrane (mean ± SD, n=5)
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Figure 2 The T-peel strength and tack adhesion values of ketoprofen matrix membrane (mean ± SD, n=5)
Figure 3 The in vitro release profile of ketoprofen matrix membrane at EC:DNRL=1:1 (mean ± SD, n=3)

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Figure 4 The in vitro skin permeation profile of ketoprofen matrix membrane at EC:DNRL=1:1 (mean ± SD, n=3)
ACKNOWLEDGEMENTS
The authors would like to express their gratitude to the Faculty of Pharmacy, Rangsit University, for
financial supports.
REFERENCES
1. Adrian CW. (2003). Theoretical aspects of transdermal drug delivery. In: Transdermal and Topical Drug
Deliveryed.), pp. 27-49. Pharmaceutical Press, Illinois.
2. Chien YW. (1992). Transdermal drug delivery and delivery systems. In: Novel Drug Delivery System.
(Chien YW, ed.), pp. 301-80, 2nd ed. Marcel dekker, New York.
3. Sekiya I, Morito T, Hara K, et al. (2010). Ketoprofen absorption by muscle and tendon after topical or oral
administration in patients undergoing anterior cruciate ligament reconstruction. AAPS PharmSciTech
11:154-8.
4. Sastry SV, Wilber W, Reddy IK, Khan MA. (1998). Aqueous-based polymeric dispersion: preparation and
characterization of cellulose acetate pseudolatex. Int J Pharm 165:175-89.
5. Chang R-K and Hsiao C. (1989). Eudragit RL and RS pseudolatices: Properties and performance in
pharmaceutical coating as a controlled release membrane for theophylline pellets. Drug Dev Ind Pharm
15:187-96.
6. Thioune O, Briançon S, Devissaguet JP, Fessi H. (2000). Development of a new ethylcellulose pseudolatex
for coating. Drug Dev Res 50:157-62.
7. Vyas SP, Gogoi PJ, Jain SK. (1991). Development and characterization of pseudolatex based transdermal
drug delivery system of diclofenac. Drug Dev Ind Pharm 17:1041-58.
8. Pichayakorn W, Suksaeree J, Boonme P, et al. (2012). Nicotine transdermal patches using polymeric
natural rubber as the matrix controlling system: Effect of polymer and plasticizer blends. J Membr Sci 411-
412:81-90.
9. Pichayakorn W, Suksaeree J, Boonme P, et al. (2012). Preparation of deproteinized natural rubber latex and
properties of films formed by itself and several adhesive polymer blends. Ind Eng Chem Res 51:13393-404.
10. Suksaeree J, Boonme P, Taweepreda W, et al. (2012). Characterization, in vitro release and permeation
studies of nicotine transdermal patches prepared from deproteinized natural rubber latex blends. Chem Eng
Res Des 90:906-14.
11. Suksaeree J, Charoenchai L, Monton C, et al. (2013). Preparation of a pseudolatex–membrane for
ketoprofen transdermal drug delivery systems. Ind Eng Chem Res (Just Accepted Manuscript).
12. Shah PP, Desai PR, Channer D, Singh M. (2012). Enhanced skin permeation using polyarginine modified
nanostructured lipid carriers. J Controlled Release 161:735-45.
13. Draize JH, Woodward G, Calvery OH. (1944). Method for the study of Irritation and toxicity of substance
applied topically to the skin and mucous membrane. J Pharmacol Exp Ther 82:377-90.
14. Chen Y, Peng Z, Kong LX, et al. (2008). Natural rubber nanocomposite reinforced with nano silica. Polym
Eng Sci 48:1674-7.
15. Rippel MM, Lee L-T, Leite CAP, Galembeck F. (2003). Skim and cream natural rubber particles: Colloidal
properties, coalescence and film formation. J Colloid Interface Sci 268:330-40.

DEVELOPMENT OF CAPSAICIN MICROEMULSIONS FOR

TRANSDERMAL DRUG DELIVERY
Wisuta Chairat, Praneet Opanasopit1, Theerasak Rojanarata1, Tanasait Ngawhirunpat1, *

1
Silpakorn University, Nakhon Pathom 73000, Thailand.
KEYWORDS: Microemulsions, Capsaicin, Transdermal Drug Delivery
INTRODUCTION
Capsaicin (8-methyl-N-Vanillyl-6-nonenamide) is a natural alkaloid (capsaicinoid) extracted from chili
peppers, which are plants belonging to the genus Capsicum. It is responsible for the hot pungent taste and
causes a burning sensation to the mammalian tissue. The capsaicin produces analgesia by depleting
substance P in small fiber nociceptor neurons on which Transient Receptor Potential action channel
(subfamily V), type 1 (TRPV1) is predominantly located. Capsaicin binds to the vanilloid receptor
TRPV1, which acts as a molecular integrator of chemical and physical painful stimuli1. Capsaicin is used
in topical therapy for a variety of disorders such as rheumatism, lumbago and sciatica. However,
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capsaicin has strong pungency leading to limitation in clinical use. In addition, it has very poor aqueous
solubility resulting in difficulties in the design of pharmaceutical formulation2.
Microemulsion is defined as a dispersion sytem consisting of oil, surfactant, co-surfactant and aqueous
phase, which has a single optically isotropic and thermodynamically stable liquid solution.
Microemulsion can be used to deliver drugs to patients via several routes. The topical application of
microemulsion has gained increasing interest, however the most difficult aspect of a transdermal delivery
system is to overcome the barrier of stratum corneum against foreign substances. Due to its advantages,
for example, easy making, good thermodynamically stability, enhanced drug solubility and enhanced skin
permeation3,4, microemulsion has become a potential transdermal drug delivery for both hydrophilic and
hydrophobic drugs. The aim of the study is the development of a microemulsion formulation for the
transdernal drug delivery of capsaicin using Isopropyl mistate (IPM) as oil phase, Cocamide DEA
(Comperlan KD®) as surfactant, ethanol 95% as co-surfactant and RO water as aqeous phase.

Material Capsaicin powder (synthetic 98%), Isopropyl myristate (IPM), cocamide DEA (Comperlan
KD®) were purchased from Sigma-Aldrich (St Lious, USA). All other chemicals used in this study were
analytical grade.
Construction of pseudo-ternary phase diagrams Pseudo-ternary phase diagrams were constructed using
the water titration method at ambient temperature to obtain the concentration range of the components for
microemulsion. The microemulsion system consisted of isopropyl myristate (IPM) as oil phase, cocamide
DEA (Comperlan® KD) as surfactant, ethanol 95% as co-surfactant and RO water as water phase. The
surfactant/co-surfactant weight ratio was 1:1, 2:1, 3:1 and 4:1. For each phase diagram, the mixtures of
IPM and surfactant/co-surfactant were prepared at weight ratios of 5:95, 10:90, 20:80, 30:70, 40:60,
50:50, 60:40, 70:30, 80:20, 90:10, respectively. After the microemulsion regions in the phase diagram
were identified, the microemulsion vehicles were selected and prepared at different component ratio in
order to study the effect of oil, mixture of surfactant and water ratios to the characteristic of
microemulsion.
Preparation of 0.075%w/w capsaicin-loaded microemulsion According to the microemulsion regions in
the phase diagrams, the microemulsion formulations were selected as describe in Table 1. Microemulsion
formulations were prepared by mixing surfactant mixture, IPM and water by weight ratio using magnetic
stirrer at ambient temperature. Capsaicin was accurately weighed and adjusted to weight with the
microemulsion formulations, following by stirring with magnetic stirrer at ambient temperature. The
finally concentration of capsaicin-loaded microemulsion was 0.075 % (w/w). Drug content was
determined by ultra-performance liquid chromatography (UPLC).
Microemulsion characterization The characteristics of microemulsion both before and after loading with
0.075% w/w capsaicin were studied as followed.
pH measurement The pH was determined using pH meter (Metler Toledo, Sevencompact S220). The
measurements were performed in triplicate.

Electrical conductivity measurement The conductivity of the microemulsion formulations was

determined by using conductivity meter (Metler Toledo, Sevencompact S230) at 25ºC. The measurements
were performed in triplicate.
The average particle sizes measurement The particle sizes and polydispersity index were determined by
dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern, UK) using a helium-neon gas laser with
beam wavelength 632.8nm Microemlsion were loaded into 1 cm3 disposable zeta cell. Measurement
angles were monitored at 12.8° and 175° and fixed temperature at 25°C.
Determination of capsaicin solubility in microemulsion formulation The excess amount of the capsaicin
was added in microemulsion formulations, and samples were continuously shaken for 48h at room
temperature. Then the microemulsions were centrifuged (14000rpm, 30mins) to remove the undissolved
drug. The supernatants were collected, diluted with methanol, and capsaicin concentration was
determined by ultraviolet spectrophotometer at wavelength 280nm.
Table 1 Composition of selected microemulsion formulations.
Formulati ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME ME
on No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
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Oil 35 30 25 15 10 10 20 30 40 50 60 70 20 20 20 20 20
S/Co-S
50 50 50 50 50 80 70 60 50 40 30 20 45 50 55 60 65
(3:1)
Water 15 20 25 30 40 10 10 10 10 10 10 10 35 30 25 20 15

Construction of pseudo-ternary phase diagram The phase diagrams composed of IPM as oil phase,
cocamide DEA (comperlan KD®) as surfactant, ethanol 95% as co-surfactant and RO water as water
phase. Surfactant/co-surfactant (S/Co-S) were prepared (w/w) in ratio of 1:1, 2:1, 3:1 and 4:1. RO water
was added drop-wise to the surfactant/oil mixtures using the water titration method. Phase diagrams were
constructed to visualize the microemulsion forming regions (Figure 1).
Figure 1 Pseudo-ternary phase diagrams of microemulsion composed of Isopropyl myristate (IPM), Cocamide DEA (Comperlan
KD®), Ethanol 95% and RO water.
The previous studies reported that short chain alcohol could decrease interfacial tension between oil and
water and adjust the flexibility of interfacial membrane2,3, so ethanol 95% was incorporated as co-
surfactant in this study. With the suitable ratio of the oil, mixture surfactant and water, the mixture was
changed to be transparent microemulsion, and the rest of the regions represented the turbid and
conventional emulsions based on visual inspection. The microemulsion regions were approximately 35-
45% of the phase diagrams. As S/Co-S increased, the area of the microemulsion became enlarged,
reaching a maximum at S/Co-S ratio of 3:1. The microemulsion vehicles of S/Co-S ratio 3:1 were
selected and prepared at different component ratio. The surfactant mixture at 50%w/w was used in the
formulation ME1-ME5, ME9 and ME14; the water was used at 10%w/w in formulation ME6-ME12, and
the IPM at 20%w/w was used in formulation ME7, ME13-ME17.
Formulation of 0.075%w/w capsaicin-loaded microemulsion The capsaicin content in capsaicin- loading
microemulsion formulations was between 0.0676%w/w and 0.0805%w/w.
Microemulsion characterization:
pH The pH of microemulsion system before loading capsaicin were between 9.84-10.48. The pH of
0.075%w/w capsaicin-loaded microemulsion were 9.82-10.56 (Table 2). The pH of the formulations
tended to increase when the ratio of water/surfactant mixture decreased (Figure 2).

Electrical conductivity The electrical conductivity of the microemulsion formulations before loading
capsaicin were between 0.11-271 µS/cm-1(Table 2). Water in oil microemulsions represent very low
specific conductivity (ca.10-9 – 10 -7 Ω -1cm-1)6. All formulations were o/w microemulsion except the
ME12 formulation that was w/o microemulsion. Capsaicin-loaded in the formulation did not sig nificantly
affect the conductivity of microemulsion. The conductivity of microemulsion formulations increased as
the water content increased. As the water composition is constant and the oil amount increased in the
formulations, the conductivity decreased (Figure 3).
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Figure 2 The pH of microemulsion formulations; Figure 3 The electrical conductivity of
( ) Blank ME microemulsion formulations;
( ) 0.075%w/w capsaicin-loaded ME ( ) Blank ME
( ) 0.075%w/w capsaicin-loaded ME
Particle size and polydispersity The droplet sizes of the microemulsion formulations, both before and
after loaded 0.075%w/w capsaicin, were in the nano-size range (11.57-257.2 nm). Small droplet sizes
provided increased stability against sedimentation, flocculation and coalescence7. The 0.075%w/w
capsaicin loaded in microemulsion did not significantly influence on the droplet size of microemulsion.
The polydispersity value described the homogeneity of the droplet size. The polydispersity values of
microemulsion formulations, before and after loading 0.075%w/w capsaicin, were 0.080-0.570. All
polydispersity values (except blank ME11) were smaller than 0.5, indicating that the droplet size had high
homogeneity4.
Table 2 Characterization of microemulsion formulations.
Formulation pH Conductivity (µs·cm-1) Particle size (nm) Polydispersity

NO.
Blank ME Loaded ME Blank ME Loaded ME Blank ME Loaded ME Blank ME Loaded ME
ME1 10.12±0.02 10.03±0.01 70.9±0.60 72.8±0.79 22.56±1.41 17.46±0.20 0.268±0.061 0.224±0.009
ME2 10.01±0.01 9.91±0.01 107.4±0.56 108.3±0.21 16.69±0.89 15.31±0.10 0.223±0.006 0.171±0.003
ME3 9.96±0.01 9.88±0.02 142.3±0.30 149.7±1.39 16.82±0.03 17.27±0.15 0.197±0.008 0.131±0.007
ME4 9.89±0.01 9.83±0.01 240±0.00 237±3.06 20.17±0.91 18.33±0.17 0.243±0.029 0.115±0.014
ME5 9.89±0.01 9.82±0.01 271±1.53 289±1.53 18.55±0.20 17.49±0.04 0.171±0.008 0.112±0.003
ME6 10.47±0.01 10.56±0.01 44.3±0.20 47.4±0.40 12.72±1.77 11.57±0.29 0.259±0.072 0.195±0.041
ME7 10.48±0.01 10.53±0.01 40.8±0.10 42.9±0.10 19.53±5.12 19.19±5.32 0.221±0.010 0.080±0.010
ME8 10.48±0.01 10.45±0.01 34.8±0.20 36.1±0.10 17.09±2.63 22.79±2.83 0.115±0.030 0.088±0.020
ME9 10.38±0.01 10.32±0.01 31.0±0.30 29.4±0.20 20.54±2.38 17.57±3.96 0.267±0.052 0.090±0.016
ME10 10.30±0.01 10.26±0.01 24.3±0.20 23.1±0.20 22.52±0.58 15.96±0.23 0.356±0.023 0.085±0.027
ME11 10.24±0.01 10.12±0.01 19.52±0.03 18.45±0.05 105.52±12.48 24.18±1.71 0.570±0.148 0.356±0.095
ME12 10.20±0.01 10.13±0.01 0.11±0.03 3.69±0.16 257.2±3.13 51.29±0.43 0.245±0.015 0.445±0.008
ME13 9.84±0.01 9.83±0.01 198.0±1.82 209±3.06 20.25±0.01 23.27±0.39 0.165±0.006 0.105±0.016
ME14 9.91±0.01 9.84±0.00 190.2±0.26 205±0.58 18.33±0.08 17.81±0.18 0.214±0.010 0.113±0.004
ME15 10.02±0.01 10.08±0.01 134.3±0.21 134.8±0.76 14.77±0.25 16.11±1.13 0.192±0.014 0.248±0.042
ME16 10.15±0.01 10.16±0.01 103.2±0.12 102.8±0.20 18.89±2.49 12.27±0.52 0.161±0.007 0.225±0.015
ME17 10.27±0.01 10.22±0.01 67.7±1.19 69.4±0.20 22.60±1.88 17.23±2.07 0.256±0.024 0.164±0.029

Determination of capsaicin solubility in microemulsion formulation Capsaicin solubility of

microemulsion formulations were between 94.60-231.83 mg/ml (9.46-23.18 %w/v). The capsaicin
solubility increased by increasing the mixture of surfactant and decreasing the water phase (Figure 4).
Figure 4 Capsaicin solubility in microemulsion formulation (mg/ml, %w/v).

PT – 16
CONCLUSION
The microemulsion systems for transdermal drug delivery of capsaicin were developed and characterized.
The ME systems were composed of isopropy; myristate (IPM) as oil phase, cocamide DEA (Comperlan
KD®) as surfactant, ethanol 95% as co-surfactant and RO water as aqueous phase. The mixture surfactant
at the ratio of 3:1 provided the largest microemulsion area of the phase diagram. The characterization of
microemulsion formulations showed that the oil phase, mixture of surfactant and aqueous phase affected
the properties of the microemulsion. As the amount of the ratio of water/mixture surfactant decreased, the
pH of microemulsion formulation tended to increase. When the water amount in aqueous phase was
increased, the electrical conductivity was increased, however, as the amount of oil phase increased, the
conductivity decreased. The droplet sizes of the microemulsion formulations were in nano-size range and
the polydispersity value showed the homogeneity of droplet size. Capsaicin loaded in the formulation did
not significantly influence the pH, electrical conductivity and droplet size of the microemulsions. The
capsaicin solubility in the microemulsion formulations were increased by increasing the surfactant
mixture and decreasing the aqueous phase.
ACKNOWLEDGMENTS
This study was supported by Pharmaceutical Development of Green Innovations Group (PDGIG), Faculty
of Pharmacy, Silpakorn University, Nakhon Pathom 73000, Thailand.
REFERENCES
1. Hayman, M. and P. C. A. Kam "Capsaicin: A review of its pharmacology and clinical
applications." Current Anaesthesia & Critical Care 19(5-6): 338-343.
2. Huang, Y. B., Y. H. Lin, et al. (2008). "Transdermal delivery of capsaicin derivative-sodium
nonivamide acetate using microemulsions as vehicles." International Journal of Pharmaceutics
349(1–2): 206-211.
3. Z. Pengwei, G. Wenyuan, et.al. “In vitro evaluation of topical microemulsion of capsaicin free of
surfactant” Biol. Pharm Bull. 31(12): 2316-2320.
4. Üstündağ Okur, N., Ş. Apaydın, et al. (2011). "Evaluation of skin permeation and anti-
inflammatory and analgesic effects of new naproxen microemulsion formulations." International
Journal of Pharmaceutics 416(1): 136-144.
5. Panomsuk, S. et al. “In vitro permeation of drugs through shed snake skin: Species
difference.” Electronic Proceeding of the 20th FAPA Congress, Bangkok, Thailand, 2004.
6. S.K. Mehta and Gurpreet Kaur (2011). “Microemulsions: Thermodynamic and Dynamic
Properties.” Thermodynamics, InTech, Available from:
http://www.intechopen.com/books/thermodynamics/microemulsions-thermodynamic-and-
dynamic-properties
7. Hathout, R. M., T. J. Woodman, et al. (2010). "Microemulsion formulations for the transdermal
delivery of testosterone." European Journal of Pharmaceutical Sciences 40(3): 188-196.

PRELIMINARY FORMULATION STUDY OF COLD PRESSED RICE BRAN OIL MASK
Jirapornchai Suksaeree1, *, Laksana Charoenchai1, Apirak Sakunpak1, Chaowalit Monton1, Tun Chusut1,
Worawan Saingam1 and Pathamaporn Pathompak1
1
Faculty of Pharmacy, Rangsit University, Pathum Thani 12000, THAILAND
Sino-Thai Traditional Medicine Research Center (Cooperation between Rangsit University, Harbin Institute of Technology,
and Heilongjiang University of Chinese Medicine), Rangsit University, Pathum Thani 12000, THAILAND
*
Corresponding author: jirapornchai.s@rsu.ac.th (Jirapornchai Suksaeree)
KEYWORDS: Rice bran oil, Mask, Chitosan, Eudragit
INTRODUCTION
Rice bran oil (RBO) is extracted from rice bran that obtained during milling of rice (Oryza sativa L.). It
has various components with beneficial nutritive and biological effects such as vitamin B1, B2, niacin,
carbohydrates, protein, and minerals such as iron, calcium, and phosphorous. Recently, RBO has attracted
wide interest, and is quickly growing in the health and nutritional industry due to its potential benefit to
health and several beneficial constituents [1]. However, γ-oryzanol is an important substance in RBO
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about 0.9-2.1%. It comprised of 38% monounsaturated, 37% polyunsaturated, and 25% saturated fatty
acids. The fatty acid composition is 14-22% palmitic acid, 0.9-2.5% stearic acid, 38-46% oleic acid, 33-
40% linoleic acid, and 0.2-2.9% linolenic acid, as reported in the Codex standard and other
publications [2-6]. In addition, it can reduce harmful low density lipoprotein, without reducing the good
high density lipoprotein [5, 7-9]. Moreover, γ-oryzanol has been shown to possess antioxidant, anti-
inflammatory, anti-tumor, and hypocholesterolemic activities [2]. The linoleic and linolenic acids (ω-6 and
ω-3 fatty acid, respectively) appear to reduce several health risks such as cancer, cardiovascular disease,
inflammation, developmental disorders, and cognitive aging [4, 6, 10-13]. However, compositions of RBO are
diverse mainly due to differences between varieties of rice, environmental factors, genotype, and
extraction method and conditions [3].
This preliminary research aimed to prepare the cold pressed RBO mask made from chitosan blended with
different types of eudragit including eudragit ® RL 100, eudragit ® RS 100, eudragit ® RL 30 D, and
eudragit ® RS 30 D. The formulations were examined visually and their physical properties such as pH,
viscosity, zeta potential and size were studied.

Materials Chitosan was purchased from Seafresh, Thailand. Different types of eudragit (eudragit ® RL
100, eudragit ® RL 30 D, eudragit ® RS 100, and eudragit ® RS 30 D) were gifted from Jebsen & Jessen
NutriLife (T) Ltd., Thailand. The rice bran of Hom-Pathum rice (Khaw-Hom-Pathum) was collected from
Pathum Thani province, Thailand. This sample was obtained from the mill as liquid oil prepared in the
laboratory (Figure 1). Povidone K30, glycerine, and diethyl phthalate was purchased from Sigma, USA.
Other chemicals and model drugs were pharmaceutical or analytical grade.
Preparation of cold pressed RBO mask Different formulations of cold pressed RBO mask were prepared
by pseudolatex system composing of aqueous phase and organic phase. The main polymer compositions
in these formulations were chitosan and different types of eudragit, including eudragit ® RL 100,
eudragit ® RS 100, eudragit ® RL 30 D, and eudragit ® RS 30 D. The chitosan, eudragit ® RL 30 D, and
eudragit ® RS 30 D were dissolved in aqueous phase, but eudragit ® RL 100 and eudragit ® RS 100 were
dissolved in organic phase. Firstly, the chitosan (2 g) was dissolved in distilled water (100 mL) and 0.5%
acetic acid was added. Then, chitosan solution was mixed with other ingredients which were dissolved in
aqueous phase. The cold pressed RBO was dissolved in 300 mL dichloromethane and mixed with other
ingredients which were dissolved in organic phase. Then, the aqueous phase was poured into the organic
phase under a homogenizer (IKA, Germany) at ambient temperature to obtain oil-in-water emulsions for
30 minutes, respectively. Finally, the oil-in-water emulsion was evaporated by rotary evaporator to
remove dichloromethane. The formed pseudolatex was kept in well-closed container at ambient
temperature for further evaluation. The cold pressed RBO mask formulations are presented in Table 1.

Figure 1 The process of cold pressed RBO from rice bran of Hom-Pathum rice (Khaw-Hom-Pathum)
Table 1 The ingredients of cold pressed RBO mask formulations
Formulations (g)
Ingredients
R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12
Cold pressed RBO 2 2 2 2 2 2 2 2 2 2 2 2
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Chitosan 2 2 2 2 2 2 2 2 2 2 2 2
Eudragit® RL 100 10 5 5 - - - - - - - 5 -
Eudragit® RS 100 - - - 5 - - 5 5 5 - - -
Eudragit® RL 30 D - - - - 5 - - - - 5 - -
Eudragit® RS 30 D - - - - - 5 - - - - - 5
Povidone K30 4 3 1 1 1 1 1 1 1 1 1 1
Diethyl phthalate 4 4 4 4 4 4 4 4 - - - -
Glycerine - - - - - - - - 4 4 4 4
Light mineral oil 2 2 2 2 2 2 2 2 2 2 2 2
Tween 20 - - - - - - 10 - - - - -
Tween 60 - - - - - - - 10 10 10 10 10
Tween 80 10 10 10 10 10 10 - - - - - -
Span 60 - - - - - - - 5 5 5 5
Paraben concentrate 1 1 1 1 1 1 1 1 1 1 1 1
Physical appearance and physical properties of the formulations The appearance of these formulations
were visually observed by the researcher. Zeta potential and particle size were measured by Zetasizer
(Malvern, UK). The pH value was measured using a pH meter (Mettler Toledo, Germany). Viscosity was
measured using a programmable viscometer (Brookfield, Brookfield Engineering Laboratories Inc., USA)
with a spindle LV 4.

Physical appearance of cold pressed RBO mask Their pH and viscosity are presented in Table 2. The pH
values of the formulations were in the range of 6.03-6.36. The formulations were found to be safe when
applied on the skin. Furthermore, the R9 to R12 formulas were selected to determine zeta potentials and
their particle size, as presented in Table 2. The zeta potential values of these formulations were in the
range of 10.7-35.6 mV, which expressed the mainly positive charge of chitosan. They had low
polydispersity index, indicating a relatively narrow size distribution of the particles. The zeta potential of
R11 formulas could predict the physical storage stability of cold pressed RBO mask. If cold pressed RBO
mask formulations had a good physical stability, the zeta potential values are higher than ± 30 mV [14].
However, only the R11 formula had the suitable particle size, in nanometer scale, of 198.9 nm.
The physical appearance of different cold pressed RBO mask formulations is shown in Figure 2. They
were white or slightly yellow colored. R1 and R2 formulas were very sticky cream / emulsion, so they
were not further investigated. Thus, R3 to R12 formulas were selected to test for their stability at room
temperature (Figure 2B). We found the R3 to R8 formulas to be not suitable for preparing cold pressed
RBO mask by pseudolatex systems because they were unstable. Although these formulas were initially
stabilized with emulsifiers, they were stilled inherently unstable and eventually separated (Figure 2B).
There were sedimentation and flocculation due to the assembly of the large drops and small flocci.

However, they could be reversed by agitation. Therefore, R9 to R12 formulas were suitable for future
development of cold pressed RBO mask formulation (Figure 2B).
Table 1 The pH, viscosity, zeta potential values, and particle size of cold pressed RBO mask formulations
Formulations
Physical properties
R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12
pH 6.03 6.17 6.07 6.22 6.28 6.29 6.31 6.12 6.27 6.26 6.36 6.33
Viscosity (cP0 - - 763 804 720 773 802 779 752 793 812 793
Zeta potential (mV) - - - - - - - - 33.3 10.7 35.6 18.0
Polydispersity
- - - - - - - - 0.46 0.91 0.46 0.61
index
Particle size (nm) - - - - - - - - 5418 3244 198.9 1407
PT – 17
(A) (B)
Figure 2 Physical appearance of different cold pressed RBO mask formulations (A) initial preparation and (B) after 1 month at
room temperature
CONCLUSION
We have successfully prepared cold pressed RBO mask formulations from chitosan blended with
different types of eudragit including eudragit ® RL 100, eudragit ® RS 100, eudragit ® RL 30 D, and
eudragit ® RS 30 D. The pH and viscosity values of these formulations indicated that they were safe for
direct application to the skin. The R9 to R12 formulas showed good physical properties; therefore, they
were suitable for preparing cold pressed RBO mask. Moreover, these formulations could be further
developed for cosmetic purposes.
ACKNOWLEDGEMENTS
The authors would like to express their gratitude to the Faculty of Pharmacy, Rangsit University, for
financial supports. We thank Jebsen & Jessen NutriLife Ltd.,Thailand for providing some materials in this
study.

REFERENCES
1. Kennedy G and Burlingame B. (2003). Analysis of food composition data on rice from a plant
genetic resources perspective. Food Chem. 80:589-96.
2. Aladedunye F, Przybylski R, Rudzinska M and Klensporf-Pawlik D. (2013). γ-Oryzanols of
North American wild rice (Zizania palustris). J Am Oil Chem Soc. 90:1101-9.
3. Goffman F, Pinson S and Bergman C. (2003). Genetic diversity for lipid content and fatty acid
profile in rice bran. J Am Oil Chem Soc. 80:485-90.
4. Hibbeln JR, Nieminen LR, Blasbalg TL, et al. (2006). Healthy intakes of ω−3 and ω−6 fatty
acids: estimations considering worldwide diversity. Am J Clin Nutrit. 83:S1483-93S.
5. Kahlon T, Chow I, Chiu M, et al. (1996). Cholesterol-lowering by rice bran and rice bran oil
unsaponifiable matter in hamsters. Cereal Chem 73:69–71.
6. MacLean CH, Newberry SJ and Mojica WA. (2006). Effects of omega-3 fatty acids on cancer
risk: A systematic review. JAMA. 295:403-15.
7. Nicolosi R, Austam L and Hegsted D. (1991). Rice bran oil lowers serum total and low density
lipoprotein cholesterol and apo-B levels in nonhuman primates. Atherosclerosis. 88:133–9.
8. Sugano M and Tsuji E. (1997). Rice bran oil and cholesterol metabolism. J Nutr. 127:521–3.
9. Sugano M and Tsuji E. (1996). Rice bran oil and human health. Biomed Environ Sci. 9:242–5.
PT – 17
10. Freemantle E, Vandal M, Tremblay-Mercier J, et al. (2006). Omega-3 fatty acids, energy
substrates, and brain function during aging. Prostaglandins Leukot Essent Fatty Acids. 75:213-
20.
11. Rizos EC, Ntzani EE, Bika E, et al. (2012). Association between omega-3 fatty acid
supplementation and risk of major cardiovascular disease events: A systematic review and meta-
analysis. JAMA. 308:1024-33.
12. Sala-Vila A and Calder PC. (2011). Update on the relationship of fish intake with prostate,
breast, and colorectal cancers. Crit Rev Food Sci Nutr. 51:855-71.
13. Simopoulos AP. (2002). The importance of the ratio of omega-6/omega-3 essential fatty acids.
Biomed Pharmacol. 56:365-79.
14. Riddick TM. (1968). Control of colloid stability through zeta potential. Zeta-Meter Inc., New
York.

FORMULATION OF TOPICAL PREPARATIONS CONTAINING HIGH CONCENTRATION

OF PERMEATION ENHANCER IN A TREATMENT OF ONYCHOMYCOSIS
Phojana Komesmuneeborirak1, Pornpen Werawatganone1, Walaisiri Muangsiri1

1
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Science, Chulalongkorn University,
Bangkok 10330, Thailand.
KEYWORDS: Nail, Nondermatophyte, Onychomycosis, Permeability
INTRODUCTION
Onychomycosis is fungal infection of nail plate. In Thailand, nondermatophyte molds (NDM) such as
Aspergillus spp., Fusarium spp., Scytalidium spp. are leading cause of onychomycosis.1 Standard
treatment of onychomycosis is orally administration of antifungal agents for example terbinafine,
itraconazole, or fluconazole. The nail plate is several layers of dead and compacted keratin supported by
nail bed, viable dermis with capillaries. Delivery therapeutic quantity of oral administered drugs to the
nail plate requires high oral dose and long therapeutic regimen. Therefore, treatment failure in
onychomyosis is due to low availability of drugs at the infection site (nails), high hepatotoxicity of the
PT – 18
antifungal drugs, and low patient compliance.2,3 Nail preparation containing antifungal drugs is not
commercially available in Thailand. In order to overcome the above drawbacks, the ultimate goal of this
research is to develop a topical nail formulation containing the high concentration of permeation
enhancer. The secondary objective of this research is to compare stabilities. Since the nail plate contained
water more than lipid structure, the nail formulations should be an o/w emulsion (o/w) or the hydrophilic
formula.4 In addition, the topical nail formulations must adhere to the nail plate for a reasonable time so
that permeation enhancer can facilitate drug permeation into the nail plate. Dimethylsulfoxide (DMSO) is
known to enhance the nail permeability.5 DMSO is slightly hazard to human. DMSO can cause
formulation problems since it is an organic solvent by nature. Thus, development of formulations
containing high concentration of DMSO is quite a challenge. An objective of this present work was to
develop three stable based formulations, i.e. ointment, o/w emulsion, and gel, containing DMSO to be
used as nail formulations.

Formulation of cream base The o/w emulsions were modified by varying concentration of various
ingredients as shown in Table 1.6,8 The cream base was prepared using a beaker method. Oil phase
consisted of glyceryl monostearate, cetyl alcohol, stearyl alcohol, mineral oil, and span®60 were heated to
75οC. Water phase consisted of purified water, tween®60, and DMSO were heated to 80οC. The oil phase
was added into the water phase and gently stirred until it was congealed.
Formulation of hydrophilic ointment base The hydrophilic ointment bases were prepared by varying
concentration of ingredients as shown in Table 2.7,8 In short, stearyl alcohol and white petrolatum were
fused at 75°C and the mixture was stirred until it became homogeneous. The aqueous phase composed of
sodium lauryl sulfate, propylene glycol, DMSO and purified water was heated to 75°C. The oil phase was
added to the aqueous phase. The hydrophilic ointment base was stirred gently until it was congealed.
Formulation of gel base The formulation of gel base was shown in Table 3.8 Poloxamer 407 was slowly
dispersed in cold mixture (5οC) of propylene glycol, DMSO and purified water. The mixture was gently
stirred until the gel was formed.
Physical stability evaluation Physical stability of selected preparations were evaluated by heating-cooling
method. Samples were stored in a refrigerator (Kelvinator, Australia) at 4οC for 48 hours, and then kept in
an incubator (Memmert, Thailand) at 45οC for 48 hours. The preparations were redone all steps
mentioned above for 6 cycles prior to organoleptic evaluation.
RESULTS AND DISCUSSIONS

The freshly prepared o/w cream bases containing 5-50% DMSO were opaque thick cream (Figure 1A).
After passing through 6 heating-cooling cycles, o/w cream based containing DMSO less than 30%
showed no any significant physical changes (Figure 1B). However, o/w cream based containing DMSO
more than 35% showed translucent layer at the bottom of the cream. The translucent layer was due to
high concentration of DMSO that could dissolve wax and oil in the formulation.

Table 1 The formulation of cream base
Ingredients Functions % w/w

Glyceryl monostearate Emollient, emulsifying agent 1-5
Cetyl alcohol Stiffening agent 1-5
Stearyl alcohol Stiffening agent 1-5
Mineral oil Emollient 5-10
Tween®60 Emulsifying agent 1-10
®
Span 60 Emulsifying agent 1-10
Dimethyl sulfoxide Penetration enhancer 5-50
Purified water Vehicle q.s. to 100
PT – 18
Table 2 The formulation of hydrophilic ointment base

Sodium lauryl sulfate Emulsifying agent 0.5-2.5
Propylene glycol Humectant 5-15
Stearyl alcohol Stiffening agent 1-5
White petrolatum Emollient 5-50
Purified water Vehicle q.s. to 100
Table 3 The formulation of gel base

Poloxamer 407 Gelling agent 12.5-20
Purified water Solubilizer q.s. to 100
Figure 1 Physical appearance of freshly prepared o/w cream base (A) and cream base after 6 cycles of heating-cooling cycle (B).
The numbers represent DMSO concentration presented in each formulation where CB represents cream base formulation.

The hydrophilic ointment and gel bases were formulated in the presence of 5-30% DMSO. The
hydrophilic ointments were thick opaque cream with no significant changes in physical appearance after
keeping at 4οC and 45οC for 6 cycles (the data was not shown). The obtained gels were opaque viscous
gels. The surface active properties of Poloxamer 407 gave rise to formation of air bubbles after gently
stirring. As a result, the air bubbles were entrapped inside the viscous gels. DMSO is solidified at
temperature lower than 18 °C; therefore, elimination of air bubbles by keeping the gels in a refrigerator in
order to lower the gel viscosity led to formation of DMSO crystal. However, the gels showed no
significant changes in their physical appearance after passing through 6 heating-cooling cycles.
CONCLUSION
In this present work, three based formulations; i.e. o/w emulsion, hydrophilic ointment and gel,
containing 30%DMSO were developed. All of them showed reasonable physical stability after passing
through 6 heating-cooling cycles. In the further studies, an antifungal agent will be incorporated into
these based formulations. The formulations will subject to both physical and chemical tests prior to
determination of the nail permeability.
ACKNOWLEDGEMENTS
PT – 18
The authors would like to thank the Department of Pharmaceutics and Industrial Pharmacy, Faculty of
Pharmaceutical Science, Chulalongkorn University, for providing research facilities.
REFERANCES
1. Ungpakorn, R., 2005. Mycoses in Thailand: current concerns. Japanese Journal of Medical
Mycology, 46(2), 81-86.
2. Rodgers, P. and Bassler, M., 2001. Treating onychomycosis. American Family Physician, 63(4),
663-672.
3. Welsh, O., Vera-Cabrera, L., and Welsh, E., 2010. Onychomycosis. Clinics in Dermatology,
28(2), 151-159.
4. Murdan, S., 2002. Drug delivery to the nail following topical application. International Journal of
Pharmaceutics, 236(1-2), 1-26.
5. Vejnovic, I., Simmler, L., and Betz, G., 2010. Investigation of different formulations for drug
delivery through the nail plate. International Journal of Pharmaceutics, 386(1-2), 185-194.
6. Niazi, S.K., 2009. Handbook of pharmaceutical manufacturing formulations: semisolid products.
vol.4. 2nd ed. New York: Informa Healthcare.
7. The United States pharmacopeia USP24: the national formulary NF19. 2000. Rockville, MD:
United States Pharmacopeial Convention.
8. Rowe, R.C., Sheskey. P.J., Quinn, M.E., eds. 2009. Handbook of pharmaceutical excipients. 6th
ed. London: Pharmaceutical Press.

NOVEL TECHNIQUE FOR DRY POWDER DEVELOPMENT FOR INTRANASAL DELIVERY
Wittaya Nakachon1 and Garnpimol C. Ritthidej1

1
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences,
KEYWORDS: intranasal delivery, dry powder, film casting, jet mill
INTRODUCTION
Intranasal administration has extensively gained interest in recent years for delivery of therapeutically
active drugs not only small molecules but also peptides and proteins1, 2. It can be used in delivery
therapeutic drugs for treatment local, systemic and central nervous disorders as well as for vaccination3.
Intranasal delivery also has several advantages such as non-invasive route, self and easy administration,
rapid onset of action, lack first pass hepatic metabolism, rich vascular submucosa and lymphatic system,
bypass of the blood brain barrier, and probable direct pathways to the brain1, 4, 5. However, one of the
principal drawbacks of nasal delivery system is rapidly cleared from the nose by mucociliary clearance, a
defend mechanism of upper respiratory tract to prevent the body against any noxious inhaled substances6.
PT – 19
Powder formulations can solve such disadvantage and improve bioavailability of drug, based on
deceleration of mucociliary clearance rate7. Moreover, dry powder formulations can offer essential
advantages over liquid formulations in terms of enabling higher drug payload and increasing drug product
stability8.
The aim of the present study was to develop dry powder formulation by using liquid-free film casting
technique followed by jet milling process for intranasal drug delivery. Additionally, physicochemical
evaluations including mucoadhesive property were determined.

Materials Polyvinyl caprolactam - polyvinyl acetate - polyethylene glycol graft copolymer (Soluplus®)
was a generous gift form BASF chemical company (Ludwigshafen, Germany). Eudragit® E PO was
purchased from Evonik industries (Darmstadt, Germany). Chitosan (low molecular weight, 75-85%
deacetylation) and Poly(ethylene glycol) (PEG: molecular weight 3350) were procured from Sigma-
Aldrich, Co. (St. Louis, MO, USA).
Methods
Powdered-film preparation Soluplus®, PEG, low molecular weight chitosan, and Eudragit® E PO were
mixed with appropriated ratio into 6 formulations. The ratios of mixtures of Soluplus® to chitosan or
Eudragit® E PO were ranged from 0.5 – 1.0, and amount of PEG was 20-80% of the aforementioned
polymer mixtures. The powder blends were cast on the Teflon sheets mounted on a leveled glass plate and
stored in hot air oven at 65 ˚C for 3-4 hr.
Nasal powder preparation The obtained polymeric films were coarsely ground by granulator with 30-
mesh sieve (Erweka FGS, Germany) and then pulverized by a jet mill machine (Current Jet; model CJ-10,
Nisshin Engineering Co., Ltd., Japan). The collected powders were kept in a dessicator at room
temperature until further used.
Particle morphology, size and size distribution evaluation Particle morphology was characterized by
using a scanning electron microscope (SEM; JEOL, JSM-6400 Scanning Microscope, Tokyo, Japan) and
particle size and size distribution of powder formulations were determined by a Mastersizer 2000
(Malvern Instrument, Herrenberg, Germany) equipped with Scirocco 2000 dispersion unit.
Physicochemical characterizations Differential scanning calorimeter (DSC: model DSC822e, Mettler
Toledo, Switzerland) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-
FTIR; Spectrum One FT-IR Spectrometer, PerkinElmer, USA) were performed.
In vitro evaluation of mucoadhesive property This method was adapted from Harikarnpakdee et al.
(2006). The intestine obtained from a local authorized slaughterhouse was kept in ice pack and used
within 1 hour from killing. A 5-centimeter long intestine was cut, then incised along, and cleaned by
washing with 0.9% normal saline. After that, the tissue was placed on glass Petri dish installed in the
funnel with an angle of 40˚ relative to the horizontal plane. An accurate weight of powder formulations
incorporated Brilliant Blue as indicator was applied on mucosal surface. A pH 6.0 simulated nasal fluid
(SNF) was warmed at 32±1˚C and peristaltically pumped at a rate of 5 ml/min over the mucosa tissue.
The washed was collected at 10, 20, 30, 60, 90, and 120 minutes and analyzed by UV spectrophotometer
(model UV-1800, Shimaszu, Japan). 9

RESULTS
Dry powder formulations were prepared by 3-step methods consisting of film casting, coarsed grinding,
and pulverizing. All obtained powder formulations were characterized their morphology under SEM. The
results demonstrated that all formulations had irregular shape with round edge. Their average sizes
(d(0.5)) were between 6 and 10 micron with narrow span values of 1.3 – 2.2.
DSC and ATR-FTIR were performed to elucidate physicochemical interaction among excipients used and
influence of production processes. According to DSC thermograms, formulation no. 2 both film and
powder exhibited two enderthermic peaks. The first sharp peak and the second broad peak appeared at
around 58˚C and 320˚C which may be the melting point peak of PEG and the degradation of polymers,
respectively, as shown in Figure 1. Moreover, other formulations also showed similar results. The FI-IR
spectra of physical mix, film cast, and powder of formulation no. 2 (Figure 2) demonstrated the dominant
peak of Soluplus® and PEG such as the C=O-stretching around 1732-1733 and 1632-1633 cm-1 of
Soluplus® as well as the CH-stretching around 2883-2886 and 2861 cm-1 of PEG. In addition, the
intensity of the peak appearing around 1731-1733 cm-1 in formulation no. 6 was higher than that around
1632-1634 cm-1 (data not shown), while other formulations had opposite result. This effect may be the
influence of amount of Eudragit® E PO in the formulation.
PT – 19
Film
Powder
Figure 1 DSC thermogram of film and powder of formulation no. 2.
Powder
2861
2886 1733
1633
Film
%T
2861
2886 1733
1633
Physical mix
1732
2861
1632
2883
Figure 2 FT-IR spectra of physical mix (bottom line), film (middle line), and powder (upper line) of formulation no. 2.

PT – 19
Figure 3 The percent of Brilliant Blue washed from porcine intestinal mucosa of powder formulations no.1-6.
The mucoadhesive property was determined by observing capability of powder adhering on porcine
intestinal mucosa. All powder formulations could retain on the mucosa for 60 minutes. However, it can be
seen in the figure 2 that more than75% of dye in all formulations was washed within 10 minutes. At the
first 10 minutes, although there was no significantly different among formulations, formulation no. 3-6,
containing chitosan or Eudragit® E PO, had a tendency to better adherence to the mucosa than the others.
DISCUSSION
The results of the present study may be summarized by pointing out, firstly, that powder formulations
which were prepared by liquid-free film casting technique and pulverized by jet milling process could
produce particle size with range of 6-10 micron. This size of particles is appropriate for nasal drug
delivery which requires size range about 5-120 micron8, 10, 11. In order to determine their mucoadhesion,
the formulations consisting of low molecular weight chitosan and Eudragit® E PO were likely to better
adhere to the mucosa. As there are many proposed mucoadhesive theories used to explain this phenomena
such as electronic theory, wetting theory, adsorption theory, diffusion theory, mechanical theory, and
fracture theory, but this process of mucoadhesion cannot be described by just one of these theories12. In
this case, it can be explained by assuming that electronic interaction may play a role in adhesion by
interacting between positive charge of polymer and negative charge of mucin on mucosal surface. On the
DSC and FT-IR data, there was no new peak or significant peak shift observing in the thermograms and
spectrums. The results suggested that compatibility of polymers were accepted, and the effect of
production processes to polymer interactions was scared.
CONCLUSION
According to the results of this study, dry powder formulations prepared by liquid-free film casting
technique and jet milling process showed a suitable particle size range with narrow size distribution as
well as polymer and process compatibility. Furthermore, positively charged formulations were likely to
improve mucoadhesive property. Therefore, the dry powder formulations developed in this study may be
a novel and promising candidate for intranasal delivery system. However, further study on permeation
and stability of the formulations incorporating drug should be performed.
ACKNOWLEDGMENTS
The authors wish to acknowledge the Department of Pharmaceutics and Industrial Pharmacy, Faculty of
Pharmaceutical Sciences, Chulalongkorn University for providing research facilities.

REFERENCES
1. J.J. Lochheadand R.G. Thorne. Intranasal delivery of biologics to the central nervous system.
Adv Drug Deliv Rev. 64:614-628 (2012).
2. H.R. Costantino, L. Illum, G. Brandt, P.H. Johnson, and S.C. Quay. Intranasal delivery:
Physicochemical and therapeutic aspects. Int J Pharm. 337:1-24 (2007).
3. L. Illum. Nasal drug delivery: new developments and strategies. Drug Discov Today. 7:1184-
1189 (2002).
4. K.L. Berensand T.R. Sullivan. Advances in intranasal therapeutics - Delivery of dry powder
pharmaceuticals and biologics. Touch Briefings (2007).
5. S.V. Dhuria, L.R. Hanson, and W.H. Frey. Intranasal delivery to the central nervous system:
Mechanisms and experimental considerations. J Pharm Sci. 99:1654-1673 (2010).
6. N.M. Schipper, J.C. Verhoef, and F.H.M. Merkus. The nasal mucociliary clearance: Relevance
to nasal drug delivery. Pharm Res. 8:807-814 (1991).
7. C. Callens, E. Pringels, and J.P. Remon. Influence of multiple nasal administrations of
bioadhesive powders on the insulin bioavailability. Int J Pharm. 250:415-422 (2003).
8. T. Keldmann. Advanced simplification of nasal delivery technology: Anatomy+innovative
device=added value opportunity, ONdrugDelivery Magazine, ONdrugDelivery Ltd, West
PT – 19
Sussex, 2005, pp. 4-7.
9. S. Harikarnpakdee, V. Lipipun, N. Sutanthavibul, and G. Ritthidej. Spray-dried mucoadhesive
microspheres: Preparation and transport through nasal cell monolayer. AAPS PharmSciTech.
7:E79-E88 (2006).
10. M. Copleyand P. Kippax. From actuation to deposition: Particle sizing techniques for
characterizing nasal drug delivery systems. Inhalation (2012).
11. Draft guidance for industry: Sinusitis: designing clinical development programs of non
antimicrobial drugs for treatment US. FDA; Center for Drug Evaluation and Research (CDER)
2006.
12. J.D. Smart. The basics and underlying mechanisms of mucoadhesion. Adv Drug Deliv Rev.
57:1556-1568 (2005).

DEVELOPMENT OF HAIR TONIC COMPRISING A MIXED EXTRACT FROM FRUITS OF

P. EMBLICA AND Z. LIMONELLA
Itsara Keeta1, Nahathai Sawang2 and Buppachart Potduang1,*

1
Technopolis, Klong 5, Klong Luang, Pathumthani 12120, Thailand; E-mail: buppachart@tistr.or.th
2
School of Cosmetic Science, Mae Fah Luang University, Muang, Chaing Rai 57100, Thailand
KEYWORDS: Hair tonic, Phyllanthus emblica, Zanthoxylum limonella, Anti-microbial, Antioxidant
INTRODUCTION
Hair tonic is a cosmeceutical product for use on the scalp to reduce hair loss and revitalize hair and scalp.
Marketed hair tonics claim to improve blood flow and restore small hair follicles to increase the delivery
of oxygen and nutrients for hair follicles.1 The product benefits from active natural ingredients as amino
acids, vitamins, minerals, essential fatty acids, growth factors, and antioxidants (flavonoids, glycosides,
etc.) which increase the hair cells and prevent the aging of the cells causing by the free radicals from
modern life style including pollution, chemicals, stress and UV light2.
PT – 20
Fruits of Ma-khampom (Phyllanthus emblica Linn., EUPHORBIACEAE) are consumed as fruit or in the
form of food products. The fruit extracts possess several pharmacological activities, such as anti-oxidant,
anti-tumor and anti-inflammation, attributable to its high vitamin C and polyphenolic content. The fruits
have long been used as hair growth nourishment in traditional Tibetan and Ayurvedic medicine. Recent in
vivo studies suggested P. emblica as possessing hair growth promoting activity as it is a component in
the herbal formulations that effectively enlarge the size and prolong the anagen phase of hair follicles.
However, its biological effects on follicular cells are still largely unknown3-6.
Ripe fruits of Ma-khwaen (Zanthoxylum limonella Alston, RUTACEAE) are harvested and
commercialized in local markets as a popular spice in the northern part of Thailand. Vitamin E has been
found in the seed oil. The essential oil from these fruits affects the gastrointestinal system and initiates
smooth muscle contraction by a non-specific mechanism. The oil contains sabinene, which is potently
bactericidal against the multi-drug resistant bacteria.7, 8
This research aimed to develop a hair tonic comprising a patented mixed extract from the fruits of P.
emblica and Z. limonella which are anti-oxidant, anti-inflammatory and anti-microbial9. The product also
benefits from the fruit extract of P. emblica as it is reported to promote proliferation in dermal papilla
cells of human hair follicle4. The development of hair tonic was prepared from water and alcohol as base
ingredient (water as diluents and alcohol as vehicle), together with other ingredients as humectants and
nutrients. Excipient compatibility, organoleptic characteristics and stability in accelerated conditions were
tested to select the best hair tonic formulation.

Plant material The dry, powdered fruits of P. emblica and Z. limonella were provided by the Sakaerat
Biosphere Research Center, Agricultural Technology Department of TISTR.
Preparation of a patented mixed extract Specific extracts of P. emblica and Z. limonella were separately
prepared by macerating the powdered fruits with ethanol-water and filtering through Whatman paper No.
41. The solvent was removed under reduced pressure using a rotary evaporator (Heidolph, Hei-VAP
Precision) at 45°C. An appropriate mixing ratio was used to prepare the mixed extract with anti-microbial
activity, as tested by agar diffusion assay.
Formulation of hair tonic Four formulas of hair tonic consisting of the P. emblica and Z limonella mixed
extract were formulated. They varied in the amount of ingredients in the basic formula, as shown in Table
1. The active mixed extract was dissolved in a humectant, then mixed with nutrients dissolved in purified
water. The solution of chelating agent in water was added to the previous mixture, followed by alcohol
and mixed thoroughly. Hair tonic for adults should have acidic pH.
Stability test The formulated hair tonic was tested using heating and cooling cycle, at 4°C for 24 hrs and
45°C for 24 hrs, for 6 cycles. The physical stability of samples was evaluated based on their turbidity,
precipitation and appearance.
In vitro anti-microbial assessment of the product Agar diffusion assay was performed against
Propionibacterium acnes, Staphylococcus aureus, S. epidermidis, Streptococcus pyogenes and Candida
albicans. Stock of microorganism was prepared by cultivation on agar, when microorganism reveals
good, it was separated to sterile water and adjusted to the concentration of 0.5 McFarland. Twenty

milliliters of nutrient agar was added and allowed to set in a Petri dish. The microorganism was added and
distributed evenly over the agar surface, and let dry in aseptic condition. Twenty µl of sample were added
directly onto the agar, which was then incubated at 37°C for 18-24 hrs. The clear zone of inhibition was
observed and compared to that of the hair tonic base which was used as control.
Table 1 The basic formula of Hair tonic
Ingredients Function %w/w

95%Ethanol Vehicle A
Menthol Flavor/anti-inflammatory 0.1-0.5
Propylene glycol Humectant 10-30
Glycerin Humectant B
Panthenol (Pro Vitamin B5) Moisturizer, promote blood 1-2
flow
Tocopheryl Acetate Promote blood flow 0.2-0.5
Zinc pyrithione Anti-fungal 0.2-0.5
Crude extracts Active ingredient C
PT – 20
Disodium EDTA Chelating agent q.s.
Fragrance Fragrance q.s.
Water Vehicle q.s. to 100
Preparation of sample Control: Hair tonic without crude extract of P. emblica and Z. limonella.
Sample: Hair tonic with crude extract of P. emblica and Z. limonella in varied concentrations.

Development of hair tonic Four formulas of hair tonic containing the patented mixed extract from the
fruits of P. emblica and Z. limonella were formulated. Formula 1 gave opaque brown liquid and
precipitated. Alcohol was added to formula 2, producing clearer liquid but still opaque and separated
when vitamin E was added. Zinc pyrithione was added to formula 3, resulting in turbid liquid and
precipitated. Formula 4 was formulated without vitamin E and zinc pyrithione, but more humectants were
added to eliminate problem with opaqueness. Formula 4 was stable under heating and cooling stability
test.
In vitro anti-microbial assessment of the product The agar diffusion assay of the hair tonic showed that
the products, with varied concentration of the active extract, were active against 7 strains of
Staphylococcus aureus (DMST 8013 and DMST 8840), Streptococcus pyogenes (DMST 17020),
Staphylococcus epidermidis (DMST 12228), Candida albicans (DMST 10231 and DMST 90028) and
Propionibacterium acnes (DMST 14916) as shown in Table 2.
Table 2 Anti-microbial inhibition of hair tonic with varying concentrations of active ingredient
Conc. S. aureus S. aureus S. pyogenes S. epidermidis C. albicans C. albicans P. acnes

DMST DMST DMST DMST 12228 DMST DMST DMST
8013 8840 17020 10231 90028 14916
1.5% +1.0 +1.0 +1.1 +1.2 +1.0 +0.9 +1.5
2.5% +1.0 +1.0 +1.3 +1.2 +1.0 +1.1 +1.5
3.5% +1.2 +1.6 +1.5 +1.3 +1.0 +1.2 +1.6
4.5% +1.4 +1.8 +1.8 +1.6 +1.1 +1.2 +1.8
base -/+1.0 -/+0.9 -/+1.1 +1.1 +0.9 +1.0 +1.0
+ = visible
-/+ = unclear
- = invisible
CONCLUSION
The patented mixed extract from the fruits of P. emblica and Z. limonella could be used as an active
extract for hair tonic product. The final product comprised of purified water, propylene glycol, alcohol,
glycerin, pro vitamin B5, menthol, disodium EDTA and the active extract. The P. emblica and Z.
limonella mixed extract could significantly promote the anti-microbial efficacy of the base.

ACKNOWLEDGMENTS
This study was supported by the Pharmaceutical and Natural Products Department, Thailand Institute of
Scientific and Technological Research (TISTR). We thank all TISTR colleagues who in one way or
another help this project to succeed.
REFERENCES
1. NIXODIL Tonic-Hair loss treatment. Available at http://www.nixodil.com/, accessed May 15,
2013.
2. Chuleewandevi. Tonics for stopping hair loss. Available at http://www.chuleevandevi.com/Hair-
grow-tonic/b/1596643031, accessed May 15, 2013.
3. Charoenteeraboon J, Ngamkitidechakul C, Soonthornchareonnon N, Jaijoy K and Sireeratawong
S. 2010.
4. Antioxidant activities of the standardized water extract from fruit of Phyllanthus emblica Linn.
Songklanakarin J. Sci. Technol. 32 (6), 599-604.
5. Luanpitpong S, Nimmannit U, Pongrakhananon V and Chanvorachote P. 2011. Emblica
(Phyllanthus emblica Linn.) fruit extract promotes proliferation in dermal papilla cells of human
PT – 20
hair follicle. Res J Med Plant 5: 95-100.

6. Cancer chemopreventive activity of Amla (Phyllanthus emblica): The Sustainer. J Appl Pharm
Sci 02 (01): 176-183.
7. Anthony C. Dweck FLS FRSH FRSC, Dweck Data David Mitchell, Chesham Chemicals.
Emblica officinalis [Syn: Phyllanthus Emblica] or Amla: the Ayurvedic wonder. Available at
http://www.dweckdata.com/published_papers/emblica_officinalis.pdf, accessed May 18, 2013.
8. Chaiyong S, Jatisatienr C, Dheeranupatana S and Jatisatienr A. 2007. Biological activity of
9 7
Zanthoxylum limonella (Dennst.) Alston. essential oil and its formulation. Planta Med 73: 599.
7 7

limonella Alston. J Med Plants Res 6(8): 1407-1414.
10. Potduang B*, Fungsin B, Keeta I, Ketmanee N, Intharungsri A, Soradech S, Niwaspragrit C and
Tanpanich S. 2012. Biological activities of a patented mix extarct from fruits of phyllathus
emblica and Zanthoxylum limenella. Thai J. Pharm. Sci. 36 (suppl.): 44-47.

STABILITY EVALUATION OF A HAIR TONIC CONSISTING OF A MIXED EXTRACT

FROM FRUITS OF PHYLLANTHUS EMBLICA AND ZANTHOXYLUM LIMONELLA
Sitthiphong Soradech1, Nahathai Sawang2, Buppachart Potduang1, *, Itsara Keeta1

and Arkachai Tantrawong1
1
Pharmaceutical and Natural Products Department, Thailand Institute of Scientific and Technological Research (TISTR) ,
Technopolis, Klong 5, Klong Luang, Pathumthani 12120, Thailand. E-mail: buppachart@tistr.or.th
2
School of Cosmetic Science, Mea Fah Luang University, Muangi, Chaing Rai, 57100, Thailand.
KEYWORDS: Hair tonic, Phyllanthus emblica, Zanthoxylum limonella, Physical stability
INTRODUCTION
Recently, the demand of herbal medicines is increasing rapidly due to their lack of side effects. Apart
from traditionally documented applications, some modern trials have also established the utility of herbs
in personal care products to help nourish and care for the skin. The herbal cosmeceutic products are
formulated using various permissible cosmetic ingredients to form the base in which one or more herbal
extracts are added as active ingredients. Hair tonic is a cosmeceutical product for using in the scalp to
PT – 21
reduce hair loss and revitalize hair and scalp for healthy growth1. The active natural ingredients i.e.,
amino acids, vitamins, minerals, essential fatty acids, growth factors, and antioxidants (flavonoids,
glycosides, etc.) could increase the hair cells and prevent the aging of the cells caused by the free radicals
from modern life style including pollution, chemicals, stress and UV light2-3.
Emblica (Phyllanthus emblica L., EUPHORBIACEAE), or “Ma-Kham Pom” in Thai, is a deciduous tree
found throughout India, Nepal, South China, Thailand, Indochina, Laos and Malaysia to North Australia.
The acidic fruits are eaten fresh or as condiments. The edible fruits are rich in tannins, vitamin C,
polyphenols, alkaloids, flavoniods etc. The fruits possess anti-oxidant, anti-inflammatory, anti-microbial
and tyrosinase activity. They are used for diverse internal ailments and in hair tonic in Asian medicine4- 9.
Ma-khwaen (Zanthoxylum limonella Alston, RUTACEAE) is an evergreen shrub of which non-toxic
edible fruits are harvested in northern Thailand. The fruit has many active ingredients such as alkaloids,
amides, coumarins, sterols and phenylpropanoid-lignans. The extracts of its roots, stem-barks, stems and
fruits are widely used for antibacterial, anti-inflammatory, anesthetic properties and to inhibit tyrosinase
activity 10-12.
(1a) (1b)
Figure 1 (1a) Emblica (Phyllanthus emblica) and (1b) Ma-khwaen (Zanthoxylum limonella).
The fruit extracts of Emblica and Ma-khwaen were mixed in a patented ratio to exhibit the best anti-
microbial activity. TISTR has reported that the mixed extract was anti-oxidant (EC 50 = 0.0079 µg/ml),
potently anti-inflammatory (at 1.25 mg/ear) and could reduce croton oil-induced rat ear edema to 50%
within 2 hr, which was better than diclofenac as the standard13, 14. As an alternative to commercial
products, a patented hair tonic was formulated containing the mixed extract for its anti-microbial, anti-
inflammatory and anti-oxidant benefits. In the present study, stability assessment was performed for the
hair tonic using heating and cooling method which involves cycling the product through storage
conditions of 45°C 24 hrs and 4°C 24 hrs for 6 cycles.


Biosphere Research Center, Agricultural Technology Department, Thailand Institute of Scientific and
Technological Research (TISTR).
Preparation of a patented mixed extract Specific extracts of P. emblica and Z. limonella were separately
prepared by macerating the powdered fruits with ethanol-water and filtering through Whatman paper No.
41. The solvent was removed under reduced pressure using a rotary evaporator (Heidolph, Hei-VAP
Precision) at 45 °C. An appropriate mixing ratio was used to prepare the mixed extract.
Formulation of hair tonic The hair tonic ingredients included 95% ethanol, menthol, propylene glycol,
glycerin, panthenol, tocophery acetate, zinc pyrithione, crude extracts, disodium EDTA, fragrance and
water. The active extract was dissolved in humectants and then mixed with water solution of ingredients.
Chelating agent and alcohol were then added and mixed thoroughly. The hair tonic for adults should be
acidic.
Stability testing of hair tonic The product stability in accelerated conditions was determined by heating
and cooling method (45° C, 24 hrs and 4° C, 24 hrs for 6 cycles), as shown in Figure 2. The resulted
physical and chemical properties of sample such as appearance, texture, color, odor, viscosity, pH, and
phase separation were observed.
PT – 21
Figure 2 Diagram of stability testing under heating and cooling accelerated conditions.
Analysis of sample was performed in triplicate. The appearance, odor and separation of sample were
evaluated by visual method. The viscosity of sample was evaluated by Brookfield viscometer (MLVT115,
USA). The pH of sample was evaluated by pH meter (ph 700, German. The color of sample was
investigated by color measurement (Miniscan EZ, USA).

The stability and compatibility of a suitable hair tonic formulation containing the mixed extract from
fruits of P. emblica and Z. limonella were evaluated under accelerated conditions (heating and cooling
test, at 45 °C 24 hrs and 4 °C 24 hrs for 6 cycles). No phase separation occurred and the product showed
no change in the appearance, odor and texture whereas pH, viscosity and color were slightly changed. The
pH and viscosity were changed from 4.53 to 4.63 and from 41.23 to 36.83 CPs, respectively. The color of
the products was analyzed according to three parameters including: L*, a*, b*. These values were slightly
changed from 38.65, 22.08 and 46.09 to 36.29, 24.37 and 49.11, respectively, during the 6 cycle storage
test.
Table 1 Physical and chemical changes of the hair tonic during heating and cooling test (45 °C 24 hrs and 4 °C 24 hrs, 6 cycles).

Properties 0 cycle 3 cycles 6 cycles

Appearance Brown color, clear Brown color, clear Brown color, clear
Separation None None None
Odor Good Good Good
Texture Good Good Good
pH 4.53 ± 0.01 4.47 ± 0.21 4.63 ± 0.06
Viscosity 41.23 ± 3.75 40.89 ± 2.96 36.83 ± 1.67
(CPs.)
Color
L* 38.65 ± 0.31 36.80 ± 0.66 36.29 ± 0.67
a* 22.08 ± 0.23 22.98 ± 0.73 24.37 ± 1.16
b* 46.09 ± 0.68 46.47 ± 0.83 49.11 ± 0.49
*Note L* = 100 (Reflecting diffuser, White), L* = 0 (Black)

a* = Positive a (red), Negative a (green)
b* = Positive b (yellow), Negative b (blue)
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CONCLUSION
The patented mixed extract from fruits of P. emblica and Z. limonella could be used as an active extract
for hair tonic. The patented product was stable in appearances, odor and color with no phase separation
whereas its pH, viscosity and color were slightly changed after storage under accelerated conditions (45
°C 24 hrs and 4 °C 24 hrs, 6 cycles). Stability of the sample product confirmed that it was suitable as a
hair tonic formulation with beneficial effects of the active extract.
ACKNOWLEDGMENTS
The authors express their gratitude to the Thailand Institute of Scientific and Technological Research
(TISTR) for providing research funds. We thank all TISTR colleagues who in one way or another help
this project to succeed.
REFERENCES
1. Shweta K. Gediya, Rajan B. Mistry, Urvashi K. Patel, M. Blessy and Hitesh N. 2011. Jain Herbal
Plants: Used as a cosmetics J. Nat. Prod. Plant Resour., 1 (1): 24-32
2. VEN ZEL, Hair loss treatment. Retrieved May 15, 2013, from http://www.nixodil.com/
3. Chuleewandevi. Tonics for stopping hair loss. Retrieved May 15, 2013, from
http://www.chuleevandevi.com/Hair-grow-tonic/b/1596643031
4. Kahkonen MP, Hopia AI, Vuorela HJ, Rauha JP, Pihlaja K, Kujala TS, Heinonen M. 1999.
Antioxidant activity of plant extracts containing phenolic compounds. Journal of Agricultural
andFood Chemistry 47: 3954–3962.
5. Lampronti I, Khan MTH, Bianchi N, Borgatti M and Gambar R. 2004. Inhibitory effects of
medicinalplant extracts on interactions between DNA and transcription factors involved in
inflammation. Minerva Biotechnological 16: 93–99.
6. Phyllanthus emblica L. – EUPHORBIACEAE. Available at http://www.biotik.org/laos/species/p/
phyem/phyem_en.html, accessed 3 October 2012.
7. Charoenteeraboon J, Ngamkitidechakul C, Soonthornchareonnon N, Jaijoy K and Sireeratawong S.
2010. Antioxidant activities of the standardized water extract from fruit of Phyllanthus emblica Linn.
Songklanakarin J. Sci. Technol. 32 (6), 599-604.
8. Luanpitpong S, Nimmannit U, Pongrakhananon V and Chanvorachote P. 2010. Emblica (Phyllanthus
emblica Linn.) Fruit Extract Promotes Proliferation in Dermal Papilla Cells of Human Hair Follicle.
Research Journal of Medicinal Plant, 2010
9. Singh E, Sharma S, Pareek A, Dwivedi J, Yadav S and Sharma S. 2011. Phytochemistry, traditional
uses and cancer chemopreventive activity of Amla (Phyllanthus emblica): The Sustainer. Journal of
Applied Pharmaceutical Science 02 (01): 176-183
10. Tangjitjaroenkun J, Supabphol R and Chavasiri, W. 2012. Antioxidant effect of Zanthoxylum
limonella Alston. Journal of Medicinal Plants Research: 1407-1414.
11. Charoenying P. Laosinwattana C. Phuwiwat W. and Lomratsiri J. 2008. Biological activities of
Zanthoxylum limonella Alston fruit extract. KMITL Science Journal: 8.

12. Anthony C. Dweck FLS FRSH FRSC, Dweck Data David Mitchell, Chesham Chemicals Ltd.
Emblica officinalis [Syn: Phyllanthus Emblica] or Amla: the Ayurvedic wonder. Retrieved May 18,
2013, from http://www.dweckdata.com/published_papers/emblica_officinalis.pdf
Tanpanich S. 2012. Biological activities of a patented mix extarct from fruits of Phyllathus emblica
and Zanthoxylum limenella. Thai J. Pharm. Sci. 36 (suppl.): 44-47.
14. Khayungarnnawee A, Soradech S, Potduang B*, Sematong T, Laovitthayanggoon S, Keeta I,
Niwaspragrit C and Tantrawong A. 2012. Anti-inflammatory activity of fruit extracts from
Phyllathus emblica and Zanthoxylum limenella. Thai J. Pharm. Sci. 36 (suppl): 38-40
15. Romanoski P. How to stability test a cosmetic formula. Retrieved Oct. 11, 2013, from
http://chemistscorner.com/how-to-stability-test-a-cosmetic-formula/
PT – 21

DEVELOPMENT OF HAIR CONDITIONING AND STYLING GEL COMPRISING A MIXED

EXTRACT FROM FRUITS OF P. EMBLICA AND Z. LIMONELLA
Itsara Keeta1, Nahathai Sawang2 and Buppachart Potduang1,*

1
Technopolis, Klong 5, Klong Luang, Pathumthani 12120, Thailand; E-mail: buppachart@tistr.or.th
2
School of Cosmetic Science, Mae Fah Luang University, Muang, Chaing Rai 57100, Thailand
KEYWORDS: Hair styling gel, Gelling agent, P. emblica, Z. limonella, Antioxidant
INTRODUCTION
Hair gel is a hairstyling product that is used to stiffen hair into a particular hairstyle. Many gels contain
adhesive agents which will make the hairs stick together as they dry in the configuration determined by
the styling process to give the style added strength and allow it to hold longer.1 Cationic polymers are one
of the main functional components of hair gel. The positive charges in polymer cause it to stretch, making
the gel more viscous. Hair gels resist natural protein conformations and allow hair to be styled and
textured. This is because the stretched-out polymer takes up more space than a coiled polymer and thus
PT – 22
resists the flow of solvent molecules around it. The positive charges also bind the gel to the negatively
charged amino acids on the surface of the keratin molecules in the hair. More complicated polymer
formulas exist.2, 3
The edible fruits of Ma-khampom (Phyllanthus emblica Linn., EUPHORBIACEAE) are used as an
important constituent of many Ayurvedic preparations such as chyavanprash and triphala and is regarded
as one of the best rejuvenating herbs. The fruits are globular, fleshy, smooth, and striated, of yellowish-
green color. They contain an obovate-obtusely triangular six-celled nut. Traditionally, the fruit is useful as
an astringent, cardiac tonic, diuretic, laxative, liver tonic, refrigerant, stomachic, restorative, alterative,
antipyretic, anti-inflammatory, hair tonic, and digestive medicine. It is used for a variety of ailments such
as anemia, hyperacidity, diarrhea, eye inflammation, anomalies of urine, leucorrhea, jaundice, nervine
debility, liver complaints, and cough. It is reported to have hepatoprotective, antioxidant, antimutagenic,
cytoprotective, antitumor, antifungal, antimicrobial, hypolipidemic, and antiatherosclerotic effects. The
fruit contains two hydrolysable tannins, emblicanins A and B, which have antioxidant properties; one on
hydrolysis gives gallic acid, ellagic acid and glucose, whereas the other gives ellagic acid and
glucose.4,5,6,7
Dry ripe fruits of Ma-khwaen (Zanthoxylum limonella Alston, RUTACEAE) are commercialized in local
markets in the northern part of Thailand. They have been traditionally used in food as a popular spice.
The essential oil from these fruits contains vitamin E and sabinene, which is potently bactericidal against
the multi-drug resistant bacteria.8,9
The patented mixed extract from fruits of P. emblica and Z. limonella is effective as an in vitro anti-
oxidant (EC 50 7.9 mg/ml)9. This research aimed to develop a hair conditioning and styling gel for
damaged hair comprising the mixed extract. The product would also benefit from the antioxidant effect of
the extract other than nourishing the hair.

Preparation of the mixed extract P. emblica extract and Z. limonella extract was separately prepared by
macerating the powdered fruits with ethanol-water for 3 nights, filtering through Whatman paper No.41
and rinsing with the same solvent. The solvent was removed under reduced pressure using a rotary
evaporator (Heidolph, Hei-VAP Precision) at 45 °C. The crude extracts were mixed at an appropriate
patented ratio.
Formulation of hair conditioning and styling gel Hair conditioning and styling gel consisting of the P.
emblica and Z limonella mixed extract was formulated into 5 formulas. In formulas 1 and 2, the basic
formula was varied in the type of carbopol. In the formulas 3, 4 and 5, the basic formula was varied in the
amount of carbopol and other ingredients, as shown in Table 1.

Table 1 Basic formulas of the hair conditioning and styling gel
Ingredients Function F1 F2 F3 F4 F5
%w/w %w/w %w/w %w/w %w/w
Carbopol ultrez 10 Gelling agent - 0.50 - - -
Carbopol 940 Gelling agent 0.50 - A3 A4 A5
Propylene glycol Humectant, diluent 10.00 10.00 10.00 10.00 10.00
Glycerin Humectant 8.00 8.00 X X X
Panthenol (pro vitamin B5) Moisturizer 1.00 1.00 1.00 1.00 1.00
Polyquaternium-10 Conditioning agent - - - B4 B5
Disodium EDTA Chelating agent 0.10 0.10 0.10 0.10 0.10
PEG-12 dimethicone Plasticizer 0.50 0.50 C3 C4 C5
Cyclomethicone Conditioning agent - - 0.50 0.50 0.50
Jojoba oil Conditioning agent - - 0.50 0.50 0.50
Tocopheryl Acetate Antioxidant - - 0.20 0.20 0.20
Preservative Preservative 0.50 0.50 0.50 0.50 0.50
PT – 22
Active mix extract Active ingredient D D D D D

Triethanolamine Neutralizer q.s. q.s. q.s. q.s. q.s.
Fragrance Fragrance q.s. q.s. q.s. q.s. q.s.
Water Diluent q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100 q.s. to 100
Stability test The stability of the hair conditioning and styling gel in the accelerated conditions was
assessed using heating and cooling method which defined as alternation of storage conditions from 4oC
for 24 hrs and 45°C for 24 hrs (1 cycle) for 6 cycles. The physical stability of samples was evaluated
based on turbidity, precipitation and appearances.
RESULTS
Development of hair conditioning and styling gel The hair conditioning and styling gel containing the
patented mixed extract from fruits of P. emblica and Z limonella was formulated into 5 formulas. The
appearances of each formula are shown in Table 2. All formulas were stable and did not separate under
centrifugation and stability testing.
Table 2 The appearances of hair conditioning and styling gel formulas
Formula Appearances
1 Clear, viscous and not greasy
2 Clear, not viscous and not greasy
3 More viscous than formula 1, opaque, light yellow, did not hold hair, not
sticky and not greasy
4 More viscous than formula 1 and 3, opaque, light yellow, weakly hold hair,
not sticky and not greasy
5 More viscous than formula 1, 3 and 4, opaque, light yellow, weakly hold hair,
not sticky and not greasy
CONCLUSION
for hair conditioning products which benefits from its antioxidant efficacy. The products will nourish the
hair as well as protect it from UV damage. The best formula of hair conditioning and styling gel is
formula 5, which was stable under 6 test cycles of heating and cooling.
ACKNOWLEDGMENTS

REFERENCES
1. Hairfinder, Hairstyles, Hair Care & Fashion. Conditioner & Styling Gel. Available at
http://www.hairfinder.com/hair3/styling_gel.htm, accessed May 10, 2013.
2. Feigenbaum H. The use of cationizing reagents in the preparation of conditioning polymers
for hair and skin care. SKW QUAB Chemicals. Available at
http://www.quab.com/files/Personal_Care_Article.pdf, accessed May 10, 2013.
3. Cationic polymers. Available at http://www.corporate.basf.com/basfcorp/img/stories/wip
o/haargel/Haargel_e.pdf, accessed May 10, 2013.
4. Charoenteeraboon J, Ngamkitidechakul C, Soonthornchareonnon N, Jaijoy K and
Sireeratawong S. 2010.
5. Antioxidant activities of the standardized water extract from fruit of Phyllanthus emblica
Linn. Songklanakarin J. Sci. Technol. 32 (6): 599-604.
6. Luanpitpong S, Nimmannit U, Pongrakhananon V and Chanvorachote P. 2011. Emblica
(Phyllanthus emblica Linn.) fruit extract promotes proliferation in dermal papilla cells of
human hair follicle. Res J Med Plant 5: 95-100.
7. Singh E, Sharma S, Pareek A, Dwivedi J, Yadav S and Sharma S. 2011. Phytochemistry,
traditional uses and Cancer chemopreventive activity of Amla (Phyllanthus emblica): The
PT – 22
Sustainer. J Appl Pharm Sci 02 (01): 176-183.
8. Anthony C. Dweck FLS FRSH FRSC, Dweck Data David Mitchell, Chesham Chemicals.
Emblica officinalis [Syn: Phyllanthus Emblica] or Amla: the Ayurvedic wonder. Available
at http://www.dweckdata.com/published_papers/emblica_officinalis.pdf, accessed May 18,
2013.
9. Chaiyong S, Jatisatienr C, Dheeranupatana S and Jatisatienr A. 2007. Biological activity of
9 7
Zanthoxylum limonella (Dennst.) Alston. essential oil and its formulation. Planta Med 73:
7 7
599.
limonella Alston. Journal of Medicinal Plants Research 6(8): 1407-1414.
11. Potduang B*, Fungsin B, Keeta I, Ketmanee N, Intharungsri A, Soradech S, Niwaspragrit C
and Tanpanich S. 2012. Biological activities of a patented mix extarct from fruits of
phyllathus emblica and Zanthoxylum limenella. Thai J. Pharm. Sci. 36 (suppl.): 44-47.

DEVELOPMENT OF HAIR CONDITONER COMPRISING A MIXED EXTRACT FROM

FRUITS OF PHYLLANTHUS EMBLICA AND ZANTHOXYLUM LIMONELLA
Buppachart Potduang1, *, Itsara Keeta1, Wanlapha Saisin2, Suchipha Wannaphatchaiyong2

and Bundit Fungsin3
1
Technopolis, Klong 5, Klong Luang, Pathumthani, 12120, Thailand; E-mail: buppachart@tistr.or.th
2
Department of Pharmaceutical Sciences, Prince of Songkla University, Hatyai, Songkhla, 90110, Thailand.
3
Biosciences Department, Thailand Institute of Scientific and Technological Research (TISTR), Technopolis, Klong 5, Klong Luang,
Pathumthani, 12120, Thailand.
KEYWORDS: Hair conditioner, Phyllanthus emblica, Zanthoxylum limonella, Anti-inflammation,

Antioxidation
INTRODUCTION
Hair conditioner is a hair care product that is applied after shampooing in order to condition the hair and
then rinsed out. The product is beneficial to dry or damaged hair. It works by restoring moisture, and
PT – 23
smoothing the cuticles of the hair follicles1. Ultraviolet and visible radiations are known to damage hair
involving hair color changes and protein damage. It can cause permanent damage to the cuticle, which is
the outside covering of the hair. It can penetrate into the cortex, which is the center of the hair, and do all
sorts of damage. UV breaks chemical bonds and cause dryness, rough texture, split ends, susceptibility to
breakage, unmanageability, and loss of pigmentation and luster2.
This research aimed to develop an anti-microbial, antioxidant and anti-inflammatory benefits hair
conditioner for damaged hair and nourish the scalp, comprising a patented mixed extract from fruits of
Phyllanthus emblica and Zanthoxylum limonella. The extract’s antioxidant effect will help reducing hair
damage from free radicals causing by pollutants and UV rays. Hair and scalp health could be promoted by
the anti-microbial and anti-inflammatory effects of the extract.
Phyllanthus emblica L. (EUPHORBIACEAE), or Ma-khampom, is an herbal plant commonly found in
Thailand. The edible fruit was reported as an alternative treatment of skin disorders, inflammations and
premature graying. Chemical constituents of the fruit include vitamin C, gibberellins, lupeol, kaempferol,
quercetin, emblicain A and B, punigluconin, pedunculaginn, phyllanthin, zeatin, amlaic acid, corilagin
ellagic acid, putranjivain A, digalic acid, phyllembolic acid, emblicol and galactaric acid3. The fruit also
has antioxidant activity, which provides protection against free radicals induced by UV4. We have found
that the ethanol fruit extract of P.emblica was effective as an anti-microbial (MIC 10-20 mg/ml) and anti-
oxidant (EC 50 18.7 µg/ml).
Zanthoxylum limonella Alston (RUTACEAE), or Ma-khwaen, is widely distributed in the northern part of
Thailand. Dry fruits are sold in local markets and traditionally used as spice. Its essential oil exhibits the
anti-oxidative potential. The oil contains Sabinene which is a potent bactericidal against the multi-drug
resistant bacteria 5-7. We have reported that the ethanol fruit extract of Z. limonella is effective against
tested microbial including C. albicans (MIC 2.5-10 mg/ml) and is an anti-oxidant (EC 50 5.9 µg/ml) with
total flavonoids of 3.61 mg rutin/g extract8.
The patented mixed extract from fruits of P. emblica and Z. limonella is effective as an anti-oxidant (EC 50
7.9 µg/ml) and in vitro anti-microbial (MIC 4.5 mg/ml) against Propionibacterium acne, Staphylococcus
aureus, S. epidermidis and Streptococcus pyogenes9. The mixed extract was used to develop a patented
hair conditoner for antioxidant benefits and to nourish the hair and scalp.

Plant material The dry fruits powder of P. emblica and Z. limonella were provided by the Sakaerat
Preparation of the mixed extract The water-ethanol crude extracts from dry fruits of P. emblica and Z.
limonella were mixed in a patented proportion for the best anti-microbial activity.
Formulation of hair conditoner Hair conditioners consisting of the P. emblica and Z limonella mixed
extract were formulated in 3 formulas. They were varied in amount of ingredients in the basic formula as
shown in Table 1. The cream was prepared in two phases: Phase A comprising mineral oil, emollients,
emulsifiers and thickening agents were heated at 75-80 °C. Phase B comprising water and humectants
were heated at 75-80 °C. Phase B was added into phase A and homogenized to make cream base. A water
solution of the mix extract, a solubilizer, cationic surfactant, conditioning agent and preservative was

added into the cream base while stirring, and then added perfume and stirred homogeneously with a
homogenizer. Adjust the pH range 5.0 to 8.5 with NaOH solution (5%). RP-HPLC was used for quality
control of the product.
Table 1 The basic formula of hair conditioner.
Ingredients Function %w/w

Polyquaternium-7 Cationic surfactant 1-5
Cetyl alcohol Thickener X
Stearyl alcohol Thickener X
Mineral oil Emollient 1-5
Beeswax Thickener 1-5
Polyquaternium-11 Conditioning agent 1-5
A Emulsifier 1-5
Span 80 Emulsifier 1-3
Propylene glycol Humectants 1-5
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Preservative Preservative 0.1-1
Active mix extract Active ingredient A
NaOH solution (5%) Basifying agent q.s.
Fragrance Odoring agent q.s.
Water Solubilizer q.s. to 100
Stability test Stability of the hair conditioner in accelerated conditions was assessed using heating and
cooling method which defined as cycling between storage conditions of 4 oC for 24 hrs and 45 °C 24 hrs
for 6 cycles. The physical stability of samples was evaluated on turbidity, precipitation and appearances.

A hair conditoner for antioxidant benefits was formulated in 3 formulas comprising a mixed extract from
fruits of P. emblica and Z limonella. Thickener and emulsifier was varied in Formula 1, 2 and found that
Formula 1 was more viscous and gave homogenous cream base than Formula 2. So Formula 1 was
chosen for cream base in Formula 3 in which a water solution of the mixed extract was added. Formula 3
was stable and not separate under centrifugation and stability testing.
CONCLUSION
for hair conditioner products. The best formula of hair conditioner is Formula 3 which was stable under 6
cycles of heating and cooling test. It gave soft-after feeling and moisturizing effect after used. The
product will act as a shield for harmful UV rays and protect the hair from environmental pollution as well
as benefits from the anti-microbial and anti-inflammatory effects of the mixed extract.
ACKNOWLEDGMENTS
REFERENCES
1. What is Hair Conditioner? Available at http://www.wisegeek.com/what-is-hair-conditioner.htm,
accessed December 3, 2012.
2. Beauty care. 2000. Sunlight and hot blow-drying are major causes of hair damage. Available at
http://www.beautycare.com/newspro/talk/053053590,28049.html, accessed December 3, 2012.
3. Phyllanthus emblica L. Available at http://www.globinmed.com/index.php?option=com_content
&view=article&id=79255:phyllanthus-emblica-l&catid=718:p&Itemid=150, accessed December
2, 2012.

4. Penning A. 2012. Beauty in layers: Multitasking ingredients. GCI Magazine (June). Available at
http://www.gcimagazine.com/marketstrends/segments/antiaging/156350405.html?page=3,
accessed December 2, 2012.
5. Mallikarjuna P, Maheswara U, Rao V and Satyanarayana T. 1999. Antimicrobial activity of
7. Charoenying P, Laosinwattana C, Phuwiwat W, and Lomratsiri J. 2008. Biological activities
of Zanthoxylum limonella Alston fruit extracts. KMITL Sci J 8(1): 12-15.
8. Ngamon Y, Potduang B, Fungsin B, Phasuk S, Takolpuckdee P, Rerk-am U, Kaewduang M and
Tanpanich S. 2012. Antimicrobial, antioxidant activities and total flavonoids of fruit extracts
from Ma-Khwaen (Zanthoxylum limonella). Thai J Pharm Sci 35 (January, Suppl.): 36-37.
9. Potduang B, Fungsin B, Keeta I, Ketmanee N, Intharungsri A, Soradech S, Niwaspragrit C and
Tanpanich S. 2012. Biological activities of a patented mix extarct from fruits of Phyllathus
emblica and Zanthoxylum limenella. Thai J Pharm Sci 36 (suppl): 44-47.
PT – 23

STABILITY EVALUATION OF A HAIR CONDITIONER COMPRISING A MIXED EXTRACT

FROM FRUITS OF PHYLLANTHUS EMBLICA AND ZANTHOXYLUM LIMONELLA
Sitthiphong Soradech1, Buppachart Potduang1, *, Itsara Keeta1, Wanlapha Saisin2,

Suchipha Wannaphatchaiyong2 and Cholticha Niwaspragrit3
1
Technopolis, Klong 5, Klong Luang, Pathumthani, 12120, Thailand.
2
Department of pharmaceutical sciences, Prince of Songkla University, Hatyai, Songkhla, 90110, Thailand.
3
Agricultural Technology Department, Thailand Institute of Scientific and Technological Research (TISTR), Technopolis, Klong 5,
Klong Luang, Pathumthani, 12120, Thailand.
KEYWORDS: Hair conditioner, Phyllanthus emblica, Zanthoxylum limonella, Product stability
INTRODUCTION
Hair conditioner is a hair care product that is applied after shampooing in order to condition the hair and
is then rinsed out. The product is beneficial to dry or damaged hair. It works by restoring moisture, and
smoothing the cuticles of the hair follicles1. Hair conditioner comprising of powerful antioxidants can
PT – 24
reduce UV damage to the hair including hair color changes and protein damage2.
Fruits of Ma-khampom (Phyllanthus emblica L., EUPHORBIACEAE) and Ma-khwaen (Zanthoxylum
limonella Alston, RUTACEAE) are edible and common in Thailand with various reports on their
biological activities3-11. The patented mixed fruit extract from the fruits of P. emblica and Z. limonella is
effective as an anti-oxidant (EC 50 7.9 µg/ml), in vitro anti-microbial (MIC 4.5 mg/ml against P. acne, S.
aureus, S. epidermidis and S. pyogenes) and anti-inflammatory extract12, 13. The biological activities of the
mixed extract are beneficial to various cosmeceutical products. TISTR have developed a hair conditioner
from the mixed extract to be an alternative to commercial products.
In the present study, the physical stability of the patented hair conditioner was evaluated under
accelerated conditions using heating and cooling method at 45° C 24 hrs and 4° C for 24 hrs for 6 cycles,
and then the physical and chemical properties of samples were observed.

Preparation of the mix extract The water-ethanol crude extracts from dry fruits of P. emblica and Z.
Formulation of hair conditioner The ingredients of hair conditioner included polyquaternium-7, cetyl
alcohol, stearyl alcohol, mineral oil, beeswax, polyquaternium-11, Span 80, propylene glycol,
preservative, active mixed extract, NaOH solution (5%), fragrance and water. The cream was prepared in
two phases: Oil Phase A and Water Phase B, both heated at 75-80 °C. Phase B was added into phase A
and homogenized to make cream base. A water solution of the mixed extract was added into the cream
base while stirring, and then adjusted the pH to 8.5.
Stability testing of hair conditioner The stability in accelerated conditions of sample was assessed using
heating and cooling method (45° C, 24 hrs and 4° C, 24 hrs for 6 cycles) as shown in Figure 1. The
physical and chemical stability such as appearance, texture, color, odor, viscosity, pH and phase
separation were observed. The evaluation of physical and chemical properties of sample was performed in
triplicate. The appearance, odor and separation were evaluated by visual method. The viscosity was
evaluated using a Brookfield viscometer (MLVT115, USA). The pH was evaluated using a pH meter (ph
700, German). The color of sample was investigated by color measurement (Miniscan EZ, USA).
Heating at 45 0C, o Appearance

Alternative 24 hr. Stability o Odor
Hair conditioner for 6 cycles & evaluation o Texture
Cooling at 40C , o Separation
24 hr. o pH
o Viscosity
o Color
Figure 1 Diagram of stability testing by heating and cooling method.


Stability assessment of the patented hair conditioner comprising the mixed extract from the fruits of P.
emblica and Z. limonella was performed using heating and cooling test, at 45 °C 24 hrs and 4 °C 24 hrs
for 6 cycles. The results revealed that the appearance, odor and texture of this product did not change and
no separation occurred whereas pH, viscosity and color were slightly changed after 6 cycles of storage in
accelerated conditions. This formulation showed high initial viscosity (1.67 x 104 CPs.) and the viscosity
slightly decreased during the test (from1.67 x 104 to 1.65 x 104 CPs). The pH of the product was slightly
increased from 5.13 to 5.26 due to the change in acidity of the extract. The color of the product was
investigated based on three parameters including: L*, a*, b*. These values slightly changed from 78.93,
1.57 and 9.21 to 82.74, 1.68 and 9.55, respectively.
Table 1 Physical and chemical changes of the hair conditioner during heating and cooling test (45 °C 24 hrs and 4 °C 24 hrs, 6
cycles).
Properties 0 cycle 3 cycles 6 cycles

Appearance White cream White cream White cream
Separation None None None
Odor Good volatile oil Good volatile oil Good volatile oil
PT – 24
Texture Good Good Good

pH 5.13 ± 0.01 5.23 ± 0.05 5.26 ± 0.02
Viscosity (CPs.) 1.67 x 104 ± 4.06 x 102 1.67 x 104 ± 4.30 x 102 1.65 x 104 ± 4.93 x 102
Color
L* 78.93 ± 1.25 80.33 ± 1.61 82.74 ± 1.71
a* 1.57 ± 0.02 1.62 ± 0.03 1.68 ± 0.03
b* 9.21 ± 0.06 9.38 ± 0.11 9.55 ± 0.10
*Note L* = 100 (Reflecting diffuser, White), L* = 0 (Black)

a* = Positive a (red), Negative a (green)
b* = Positive b (yellow), Negative b (blue)
CONCLUSION
The addition of patented mixed extract from the fruits of P. emblica and Z. limonella did not affect the
physical and chemical properties of the hair conditioner. The product exhibited no change in appearances,
odor and color and no separation, whereas pH, viscosity and color were slightly changed after storage for
6 cycles in accelerated conditions. The slight changes in pH, viscosity and color resulted from
temperature and humidity. Stability of the patented hair conditioner indicated that the product was
suitably formulated and could maintain its intended properties during appropriate shelf life.
ACKNOWLEDGMENTS
The authors express their gratitude to the Thailand Institute of Scientific and Technological Research
(TISTR) for providing research funds. We thank all TISTR colleagues who in one way or another help
this project to succeed.
REFERENCES
1. What is Hair Conditioner?. Available at http://www.wisegeek.com/what-is-hair-
conditioner.htm, accessed 3 December 2012.
2. Trueb RM. 2006. Pharmacologic interventions in aging hair. Clin Interv Aging. 1(2): 121–
129. Available at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2695167/, accessed 14
October 2013.
3. Kahkonen MP, Hopia AI, Vuorela HJ, Rauha JP, Pihlaja K, Kujala TS, Heinonen M. 1999.
Antioxidant activity of plant extracts containing phenolic compounds. Journal of Agricultural
andFood Chemistry 47: 3954–3962.
4. Lampronti I, Khan MTH, Bianchi N, Borgatti M and Gambar R. 2004. Inhibitory effects of
medicinalplant extracts on interactions between DNA and transcription factors involved in
inflammation. Minerva Biotechnological 16: 93–99.
5. Phyllanthus emblica L. – EUPHORBIACEAE. Available at http://www.biotik.org/laos/species/
p/phyem/phyem_en.html, accessed 3 October 2012.

6. Phyllanthus emblica L. Available at http://www.globinmed.com/index.php?

option=com_content &view=article&id=79255:phyllanthus- emblica-l&catid=718:p&
Itemid=150, accessed 2 December 2012.
7. Penning A. 2012. Beauty in layers: Multitasking ingredients. GCI Magazine, 5 pages. Available
at http://www.gcimagazine.com/marketstrends/segments/antiaging/156350405.html?page=3,
accessed 2 December 2012.
limonella Alston. J Medicinal Plants Res. 1407-1414.
Zanthoxylum limonella Alston fruit extracts. KMITL Sci. J. 8(1): 12-15.
11. Ngamon Y, Potduang B*, Fungsin B, Phasuk S, Takolpuckdee P, Rerk-am U, Kaewduang M
and Tanpanich S. 2012. Antimicrobial, antioxidant activities and total flavonoids of fruit
extracts from Ma-Khwaen (Zanthoxylum limonella). Thai J Pharm Sciences 35 (Suppl. Issue
January): 36-37.
PT – 24
Tanpanich S. 2012. Biological activities of a patented mix extarct from fruits of phyllathus
emblica and Zanthoxylum limenella. Thai J. Pharm. Sci. 36 (suppl.): 44-47.
13. Khayungarnnawee A, Soradech S, Potduang P*, Sematong T, Laovitthayanggoon S, Keeta I,
Niwaspragrit C and Tantrawong A . Anti-inflammatory activity of fruit extracts from phyllathus
emblica and Zanthoxylum limenella. Thai J. Pharm. Sci. 2012, 36 (suppl.) 38-40.

ACUTE SKIN IRRITATION TEST OF FACIAL & BODY GEL WASH AND MOISTURIZING
GEL PRODUCTS COMPRISING A PATENTED MIXED EXTRACT
FROM EMBLICA AND MA-KHWAEN
Sareeya Reungpatthanaphong1, Tuanta Sematong1, Buppachart Potduang*1, Phanukit Kunhachan1,

Itsara Keeta1and Cholticha Niwaspragrit2
1
Pharmaceuticals and Natural Products Department, Thailand Institute of Scientific and Technological Research,
Technopolis, 35 Mu 3, Thanon Liab Klong 5, Klong Luang, Pathumthani 12120, Thailand. E-mail: Buppachart@tistr.or.th
2
Agricultural Technology Department, Thailand Institute of Scientific and Technological Research,
Technopolis, 35 Mu 3, Thanon Liab Klong 5, Klong Luang, Pathumthani 12120, Thailand.
KEYWORDS: Phyllanthus emblica, Zanthoxylum limonella, Gel wash, Moisturizing gel, Skin irritation
test
INTRODUCTION
PT – 25
Emblica (Phyllanthus emblica L.; in Thai “Ma-Kham-Pom”), a euphorbiaceous plant, is widely

distributed in subtropical and tropical regions in China, India, Indonesia and Malaysia. Emblica fruit has
plentiful amounts of vitamin C and superoxide dismutase. It has been widely used in many traditional
medicines including Chinese, Tibetan and Ayurvedic medicine as anti-oxidant, anti-microbial, anti-cancer
and anti-inflammatory agents 1, 2, 3, 4).
Ma-Khwaen (Zanthoxylum limonella Alston) is found in the Northern part of Thailand. Its fruits have
been widely used in Indian traditional medicine for the treatment of cardiac, respiratory diseases, tooth
infection and against stomach infection. It has been reported to reveal antimicrobial activity 5).
In this research, the facial & body gel wash and moisturizing gel products were formulated from a
patented mixed extract of Emblica and Ma-Khwaen. The objective of this study was to evaluate and
investigate for skin irritation or contact dermatitis of these products in animal models. The skin irritation
test was conducted following the Test Guideline No.404 of the OECD Guidelines for testing of
chemical 6). The results of this study may provide adequate assurance for premarket safety evaluation.

Plant material Dry, powdered fruits of Emblica and Ma-Khwaen were provided by the Agricultural
Technology Department of TISTR.
Preparation of plant extracts Each dried powder (500 g) was macerated with different ratios of ethanol-
water. The ethanol extracts were filtered and the solvent was removed using a rotary evaporator at 45°C.
Formulations and stability study The gel products containing the patented mixed extract of Emblica and
Ma-Khwaen were formulated and studied by the Pharmaceuticals and Natural Products Department of
TISTR.
Animals Healthy adult albino New Zealand rabbits (white hybrid strain) were purchased from the
Department of Animal Science, Faculty of Agriculture, Kasetsart University. Their body weight range
was from 2 to 3 kg. They were housed individually in stainless steel cages and were fed with foods and
water ad libitum. Prior to starting the experiment, they were acclimatized to the animal room conditions
for one week.
Acute skin irritation test One day before experimentation, an area of skin approximately 10 cm × 10 cm
on the dorso-lumbar region of each rabbit (3 rabbits/sample) was clipped free of hairs. Two areas of the
shaven skin (approximately 2.5 cm × 2.5 cm) were selected. The 0.5 ml of sample on a 2.5 cm × 2.5 cm
gauze patch was moistened with water to serve as a treated patch, while 0.5 ml of distilled water on
another patch was served as a control patch. Entire trunk of the rabbit was wrapped with elastic cloth to
avoid dislocation of the patches. At the end of the 4 hr exposure period, all patches were removed and the
treated skin gently wiped with moistened cotton wool to remove any residual test material. The rabbits
were assessed for the degree of erythema and oedema evidence on each site at 1 , 24, 48 and 72 hrs after

removal of the patches. The skin reactions were independently scored by two inspectors using the
numerical scoring system as follows 5).
Skin reactions Score
Erythema (Most severely affected area graded):
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet red coloration to slight eschar formation) 4
Oedema formation (Most severely affected area graded):

No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well defined by definite raising) 2
Moderate oedema (area raised approximately 1 mm) 3
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Severe oedema (raised > 1 mm, and extending beyond the area of exposure) 4

After removal of the patches from the treated skin of each rabbit, skin reactions at 1, 24, 48 and 72 hrs
were observed. There were no erythema and oedema reactions in all rabbits treated with distilled water
(control area) which were scored as zero. The rabbit skin irritation scores for the facial & body gel wash
and moisturizing gel products are shown in Table 1.
It was found that 50% diluted facial & body gel wash product produced slight erythema of three tested
rabbits’ skin at 1 h after patch removal and the symptom disappeared within 48 h, indicating that the gel
wash could cause only insignificant and mild rash at 1 h. However, at 4 h the rabbits with single
application patch test of 100% moisturizing gel product exhibited no erythema or oedema reactions on
their skin, which could indicate that the moisturizing gel was non-irritant.
Table 1 The skin irritation scores of the rabbits treated with the facial & body gel wash and moisturizing gel products.
Scoring time (hr)

Rabbit
Treatment 1 24 48 72
No.
Erythema Oedema Erythema Oedema Erythema Oedema Erythema Oedema
1 0 0 0 0 0 0 0 0
Distilled water 2 0 0 0 0 0 0 0 0
3 0 0 0 0 0 0 0 0
1 1 0 1 0 0 0 0 0
Facial & body
gel wash 2 1 0 1 0 0 0 0 0
product
3 1 0 1 0 0 0 0 0
1 0 0 0 0 0 0 0 0
Moisturizing
2 0 0 0 0 0 0 0 0
gel products
3 0 0 0 0 0 0 0 0

CONCLUSION
Our results demonstrated that 50% diluted facial & body gel wash product from a mixed extract of
Emblica and Ma-Khwaen caused slight erythema of all tested rabbits’ skin at 1 h after patch removal and
the symptom disappeared within 48 h. This indicated that the facial & body gel wash only caused
insignificant and mild rash at 1 h. However, the 100% moisturizing gel product did not produce any skin
reactions on the tested rabbits’ skin, which could indicate that it was non-irritant. Further toxicity studies
should be conducted as safety assessment on cosmeceutical products containing the mixed extract as
active constituent.
REFERENCES
1. Verma, R.C., Gupta, A., 2004. Effect of pre-treatments on quality of solar-dried amla. Journal of
Food Engineering 65, 397–402.
2. Liu, X., M. Zhao, J. Wang, W. Luo, B. Yang and Y. Jiang. 2008. Antioxidant activity of
methanolic extract of emblica fruit (Phyllanthus emblica L.) from six regions in China. Journal
of Food Composition and Analysis. 21: 219–228.
3. Zhang, Y.J., Tanaka, T., Iwamoto, Y., Yang, C.R. and Kouno, I. 2000. Phyllaemblic acid, a
PT – 25
novel highly oxygenated norbisabolane from the roots of Phyllanthus emblica. Tetrahedron
Letters 41, 1781–1784.
4. Zhang, Y.J., Nagao, T., Tanaka, T., Yang, C.R., Okabe, H. and Kouno, I. 2004. Antiproliferative
activity of the main constituents from Phyllanthus emblica. Biological and Pharmaceutical
Bulletin 27, 251–255.
5. Charoenying, P., Laosinwattana, C., Phuwiwatand, W. and Lomratsiri, J. 2008. Biological
activities of Zanthoxylum limonella Alston fruit extracts. KMITL Sci. J. Vol.8 No.1 January –
June, 12-15.
6. Organization for Economic Co-operation and Development (2002). OECD Guidelines for
Testing of Chemicals, Volume 2, Section 4: Health Effects. Acute Dermal Irritation / Corrosion.
Test Guideline No. 404.

DEVELOPMENT OF SHAMPOO COMPRISING A MIXED EXTRACT FROM FRUITS OF

PHYLLANTHUS EMBLICA AND ZANTHOXYLUM LIMONELLA FOR ITCHY SCALP
Buppachart Potduang1, *, Itsara Keeta1, Suchipha Wannaphatchaiyong2, Wanlapha Saisin2

and Bundit Fungsin3
1
Technopolis, Klong 5, Klong Luang, Pathumthani, 12120, Thailand. E-mail: buppachart@tistr.or.th
2
Department of Pharmaceutical Sciences, Prince of Songkla University, Hatyai, Songkhla, 90110, Thailand.
3
Biosciences Department, Thailand Institute of Scientific and Technological Research (TISTR),
Technopolis, Klong 5, Klong Luang, Pathumthani, 12120, Thailand.
KEYWORDS: Shampoo, Phyllanthus emblica, Zanthoxylum limonella, Anti-microbial, Anti-

inflammation
INTRODUCTION
Shampoo is a hair care product used for the removal of oils, dirt, skin particles, environmental pollutants
and other contaminant particles that gradually build up in hair. The goal is to remove the unwanted build-
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up without stripping out so much sebum as to make hair unmanageable1.
This research aimed to develop an anti-microbial, anti-inflammatory and antioxidant benefits shampoo for
itchy scalp comprising a patented mixed extract from fruits of Phyllanthus emblica and Zanthoxylum
limonella. The anti-inflammatory and anti-microbial effects of the active extract promote itchy scalp
reduction. The antioxidant effect of the extract is beneficial for damaged hair by reducing harmful free
radicals causing by pollutants and UV rays.
Phyllanthus emblica L. (EUPHORBIACEAE), known as Ma-khampom, is an herbal plant commonly
used in Asian traditional medicine. Its edible fruits were reported to be used as an alternative treatment of
diarrhea, jaundice, skin disorders, inflammations and premature graying. The fruit extract is used as
a skin-lightening agent, benefits from its anti-tyrosinase effect. Its antioxidant activity provides protection
against free radicals induced by UV. Phenolic compounds from P. emblica exhibited anti-inflammatory
effect2-6.
Zanthoxylum limonella Alston (RUTACEAE), known as Ma-khwaen, has been extensively used in folk
medicines for different medical purposes. The edible fruit has been traditionally used as food flavor in the
northern part of Thailand. Its essential oil exhibits the anti-oxidative potential according
to Sabinene, which is a potent bactericidal against the multi-drug resistant bacteria7-9. The extract is
effective as anti-microbial (MIC 2.5-10 mg/ml) and anti-oxidant (EC 50 5.94 µg/ml) 10.
The patented mixed extract from fruits of P. emblica and Z. limonella is effective as in vitro anti-
microbial (MIC 4.5 mg/ml) against Propionibacterium acnes, Staphylococcus aureus, S. epidermidis and
Streptococcus pyogenes. It is an anti-oxidant (EC 50 7.9 µg/ml) and a potent anti-inflammatory on croton
oil-induced rat ear edema better than std. Diclofenac11-12. The mixed extract was used as an active extract
to develop a patented moisturizing shampoo for preventing and reducing dry and itchy scalp. The
shampoo formula composes of the active ingredients in an appropriate solvent. Humectants are
responsible to increase water content of the top layers of scalp, and affect the scalp moisture contain.
Surfactants wash dirt on hair and scalp. A flavoring agent was added for good smell with refresh and
relaxes feeling. A chelating agent stabilizes the product not to precipitate. The preservative is essential to
prevent product damage caused by microorganisms and to protect the product from inadvertent
contamination by the consumer during use. The mixed extract from fruits of P. emblica and Z. limonella
benefits the product for its antimicrobial and anti-inflammation.

Plant material The dry fruit powder of P. emblica and Z. limonella were provided by the Sakaerat
Preparation of the mixed extract The water-ethanol crude extracts from dry fruits of P. emblica and Z.
Formulation of shampoo Shampoo consisting of the P. emblica and Z limonella mixed extract was
formulated in 10 formulas. They were varied in amount of ingredients in the basic formula as shown in
Table 1. The active mixed extract was dissolved in a humectant with small amount of water; and then
added other surfactants, foam booster, conditioning agents, thickening agents. The chelating agent
solution in water was added to the previous mixture, added purified water and mixed thoroughly. The

preservative and fragrance were then added while stirring. 50% citric acid solution was used to adjust pH.
Shampoo for adults should have pH 5.0 – 8.5.
Stability test The formulated shampoo was tested under heating and cooling test (H&C) at 4 °̊C 24 hrs
and 45 °C 24 hrs for 6 cycles. The physical stability of samples was evaluated on turbidity, precipitation
and appearance.
In vitro anti-microbial assessment of shampoo Tissue diffusion assay was performed against P. acnes, S.
aureus, S. epidermidis, S. pyogenes and C. albicans. Stock of microorganism was prepared by cultivation
on agar, when microorganism revealed good it was separated to sterile water and adjusted to the
concentration of 0.5 McFarland. Twenty milliliters of nutrient agar was added and allowed to set in a
Petri dish. The microorganism was added and distributed evenly over the agar surface, and let dry in
aseptic condition. 20 µl of 50% shampoo solution was added directly onto 1 cm diameter tissue paper on
nutrient agar. When the time completed at 1 minute, took the tissue out of the agar cultures. Closed the lid
and incubated at 37 °C for 18-48 hrs. The clear zone of inhibition was observed compared to shampoo
base. The shampoo base was used as control.
Table 1 The basic formula of shampoo.

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Ingredients function %w/w

Sodium laureth sulfate (70%) Anionic surfactant 30-50
Coamidopropyl betaine Amphoteric surfactant 1-5
Cocamide DEA Foam boosters & Stabilizer X
A Conditioning agent 1-5
Lanolin Conditioning agent 1-5
Dimethicone Conditioning agent 1-5
PEG 400 Thickening agent X
Glycerine Humectants 1-5
Crude extracts Active ingredient X
Propylene glycol Humectants & Co-solvent 1-5
Tetrasodium EDTA Chelating agent 0-0.02
Citric acid solution (50%) Acidifying agent q.s.
B Preservative 0.1-1
Fragrance Odorant q.s.
Water Vehicle q.s. to 100
Staphylococcus aureus DMST 8013 Staphylococcus aureus DMST 8840
Streptococcus pyogene DMST 17020 Staphylococcus epidermidis DMST 12228
Figure 1 Microbial inhibition of the shampoo Formula 10 consisting of the patented mixed extract from fruits of P. emblica and Z.
limonella against 4 strains of S. aureus, S. pyogenes and S. epidermidis. The results in a Petri dish compared between shampoo base
(left) and Formula 10 shampoo (right).

Candida albicans DMST 10231 Candida albicans DMST 90028 Propionibacterium acnes DMST 14916
Figure 2 Microbial semi-inhibition of the shampoo Formula 10 consisting of the patented mixed extract from fruits of P. emblica
and Z. limonella against 3 strains of C. albicans and P. acnes. The results in a Petri dish compared between shampoo base (left) and
Formula 10 shampoo (right).

The shampoo containing the patented mixed extract from fruits of P. emblica and Z limonella was
formulated in 10 formulas. Formula 1 gave clear brown and pearlescent solution, but it was not viscous
and the opacifying agent separated. More thickening agent was added in Formula 2 and 3. Formula 2 was
thicker than Formula 1, but Formula 3 was not viscous. Viscosity adjusted with surfactant was performed
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in Formula 4 and 5. Formula 5 was thicker than 4. Viscosity and color were further adjusted in Formula 6
to 8. Formula 8 was better than Formula 6 and 7. Formula 9 was derived from Formula 8 by adding
surfactant, different conditioning agent and opacifying agent which resulted that it was not stable under
stability test. The opacifying agent was not used in Formula 10. Formula 10 was selected for their
acceptable viscosity and stability.
In vitro anti-microbial assessment of Formula 10 revealed that the shampoo was active against 4 strains of
S. aureus (DMST 8013 and DMST 8840), S. pyogenes (DMST 17020) and S. epidermidis (DMST
12228) as shown in Figure 1. It was semi-active against 3 strains of C. albicans (DMST 10231 and
DMST 90028) and P. acnes (DMST 14916) as shown in Figure 2. The anti-microbial activity was stable
after 6 cycles of heating and cooling test.
CONCLUSION
for shampoo products. The best shampoo was Formula 10 with acceptable moisten, pH and viscosity. It
comprised appropriate amount of chelating agent and solubilizer to give stable clear brown solution. An
appropriate amount of a flavoring agent was added to the Formula 10. The product benefits from the anti-
inflammatory, anti-microbial and antioxidant effects of the active extract for reducing itchy scalp as well
as harmful free radicals causing by pollutants and UV rays.
ACKNOWLEDGMENTS
REFERENCES
1. Wikipedia.Shampoo. Available at http://en.wikipedia.org/wiki/Shampoo, accessed 2 December
2012.
2. Phyllanthus emblica L. Available at http://www.globinmed.com/index.php?option=com_content
&view=article&id=79255:phyllanthus- emblica-l&catid=718:p&Itemid=150, accessed 5 October
2012.
3. Emblica. Available at http://www.drugs.com/npp/emblica.html#ref19, accessed 10 October
2012.
4. Penning A. 2012. Beauty in layers: Multitasking ingredients. GCI Magazine, 5 pages. Available
at http://www.gcimagazine.com/marketstrends/segments/antiaging/156350405.html?page=3,
accessed 7 October 2012.
5. Liu X, Cui C, Zhao M, Wang J, Luo W, Yang B and Jiang Y. 2008. Identification of phenolics in
the Fruit of emblica (Phyllanathus emblica L.) and their antioxidant activities. Food Chem 109:
909-915.
6. Muthuraman A, Sood S and Singla SK. 2011. The antiinflammatory potential of phenolic
compounds from Emblica officinalis L. in rat. Inflammopharmacology 19(6): 327-34.


Zanthoxylum limonella Alston fruit extracts. KMITL Sci J 8(1): 12-15.
10. Ngamnon Y, Potduang B, Fungsin B, Phasuk S, Takolpuckdee P, Rerk-am U, Kaewduang M and
Tanpanich S. 2012. Antimicrobial, antioxidant activities and total flavonoids of fruit extracts
from Makhwaen (Zanthoxylum limonella). Thai J Pharm Sci 35 (suppl): 36-37.
11. Potduang B, Fungsin B, Keeta I, Ketmanee N, Intharungsri A, Soradech S, Niwaspragrit C and
Tanpanich S. 2012. Biological activities of a patented mix extarct from fruits of Phyllathus
emblica and Zanthoxylum limenella. Thai J Pharm Sci 36 (suppl): 44-47.
12. Khayungarnnawee A, Soradech S, Potduang P, Sematong T, Laovitthayanggoon S, Keeta I,
Niwaspragrit C and Tantrawong A . Anti-inflammatory activity of fruit extracts from Phyllathus
emblica and Zanthoxylum limenella. Thai J Pharm Sci 2012, 36 (suppl.) 38-40.
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DEVELOPMENT OF JATU-PHALATHIKA FORMULATION IN

EFFERVESCENT TABLET DOSAGE FORM
Pongsak Awaiyawanon1, Anchana Sanrungmuang1, Worawan Saingam2 and Prasan Tanguenyongwatana1
1
Faculty of Oriental Medicine, Rangsit University, Pathum Thani, Thailand, 12000
2
Sino-Thai Traditional Medicine Research Center, Faculty of Pharmacy, Rangsit University, Pathum Thani, Thailand, 12000
KEYWORDS: Jatu-Phalathika, effervescent tablet, gallic acid
INTRODUCTION
Traditional Thai medicine (TTM) to relieve health disorders has been used for many years. It has been
widely accepted that TTM has a good potential for new pharmacotherapy from medicinal plants. Jatu-
Phalathika, a TTM formula, has been used as mild laxative, expectorant and to relieve cough. This
formula composed of equal proportions of four medicinal plants which are Terminalia bellirica (Beleric
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Myrobalan), Terminalia chebula (Chebulic Myrobolans), Phyllanthus emblica (Emblic Myrobolans) and
Terminalia sp. (Arjun). Jatu-Phalathika is prepared by boiling the plant materials with water before use.
The composition of herbs within a boiled solution can be changed depending on the condition of the
patient. This preparation has some disadvantages such as the time needed to prepare, appearance or taste
that might be considered unpleasant for some patients. Therefore, this study is interested in developing
Jatu-Phalathika into a modern formulation, the effervescent tablet. This will provide a viable solution for
delivering active ingredients using a cost-effective technology.

Materials Gallic acid was purchased from Sigma Chemical Co. (USA). All four medicinal plants:
Terminalia bellirica (Beleric Myrobalan), Terminalia chebula (Chebulic Myrobolans), Phyllanthus
emblica (Emblic Myrobolans) and Terminalia sp. (Arjun) were purchased from drug store in
Nonthaburi Province, Thailand. Pharmaceutical excipients including lactose, sodium bicarbonate, citric
acid, tartaric acid, polyethylene glycol 6000 and sodium saccharin were purchased from TTK Science,
Bangkok, Thailand. The solvents for HPLC including acetic acid, water and acetronitrile (Labscan,
Thailand) were also purchased from TTK Science, Bangkok, Thailand.
Methods
Preparation of crude extracts The Jatu-Phalathika preparation was formulated by using equal amount
(500 g) of Terminalia bellerica, Terminalia chebula, Phyllanthus emblica and Terminalia sp. (Arjun).
The mixture was then boiled in water (3 L) and left to freely evaporate in order to obtain 1 liter of the
aqueous extract. Next, the water was removed from the extract by using freeze dry method (EYELA FD-
1, Tokyo Rikakikai, Japan).
Preformulation study In order to estimate the suitability of mixture for formulation, preformulation
studies to check the physical properties of Jatu-Phalathika extract with other excipients were required.
The results of the preformulation studies provided data necessary for the formulation and manufacturing
of the effervescent tablet. Preformulation studies were performed concerning the angle of repose, bulk
density, tapped density, compressibility index and Hausner’s ratio, using appropriate techniques.
Formulation development of Jatu-Phalathika effervescent tablets All ingredients shown in Table 1 were
mixed and compressed by direct compression using a single punch tablet machine to make an
effervescent tablet following the formula. First, Jatu-Phalathika extract was absorbed by lactose, sheared
by the pestle and put through a 60-mesh sieve. The powder of Jatu-Phalathika was tray-dried at 60 °C
using a hot air oven for 1 hour. Then, the powder was mixed with sodium bicarbonate, citric acid, tartaric
acid, PEG 6000 and sodium saccharin. Humidity in the room was controlled to lower than 30%RH.
Amount of gallic acid from effervescent tablets was analyzed by validated HPLC method.

Table 1 Formula of effervescent tablets
Ingredients Weight per tablet (mg) Function

F1 F2 F3 F4
Jatu-Phalathika 250.00 250.00 250.00 250.00 Active ingredient
extract
Lactose 750.00 750.00 750.00 750.00 Absorbent/Filler
Sodium bicarbonate 500.00 489.90 479.90 469.90 Alkalizing agent
Citric acid 166.67 163.30 159.97 156.63 Acidifying agent
Tartaric acid 333.33 326.60 319.93 313.27 Acidifying agent
PEG 6000 - 20.00 40.00 60.00 Lubricant
Sodium saccharin - 0.20 0.20 0.20 Sweetening agent
Total weight (mg) 2000.00 2000.00 2000.00 2000.00 -
Physical properties of Jatu-Phalathika effervescent tablets The evaluation data composed of weight
PT – 27
variation, thickness, hardness, friability, and disintegretion.

Quantitative analysis of gallic acid by high performance liquid chromatography (HPLC) Quantitative
analysis of gallic acid was performed using high performance liquid chromatography (HPLC) method
with UV detector at 292 nm. The HPLC system consisted of a quaternary pump system (Agilent, 1260
VL), autosampler (Agilent, 1260 TCC) and UV/VIS with diode array detector (Agilent, 1260 DAD VL).
A reversed-phase Inertsil ODS3 C-18 column (4.6 x 250 mm) was eluted by using a gradient system of
acetronitrile (A) and 1.0% acetic acid (B) as the mobile phase with a flow rate of 0.8 ml/min. The
condition of mobile phase gradient elution was 10–40% (A) in 0–15 min, 40–60% (A) in 15–30 min, 60–
80% (A) in 30–40 min, and 80–100% (A) in 40–60 min. The injection volume was 5 µl. Validation
parameters of linearity, accuracy, and precision were confirmed for this method.

Preformulation study The preformulation studies to check the physical properties of the Jatu-Phalathika
extract with other excipients were shown in Table 2.
Table 2 Data of preformulaton studies
Preformulaton studies F1 F2 F3 F4
Angle of repose (°) 47.52 44.11 40.29 38.25
Bulk density (g/ml) 0.38 0.41 0.39 0.43
Tapped density (g/ml) 0.43 0.48 0.48 0.50
Carr’s Index (%) 11.63 14.58 18.75 14.00
Hausner’s Ratio 1.13 1.17 1.23 1.16
Formulation development of Jatu-Phalathika effervescent tablets The formula of F1, F2, F3 and F4
contained lactose at the amount of 37.50% by weight. Lactose could effectively absorb Jatu-Phalathika
extract in the formulation. F1, which composed of Jatu-Phalathika extract and acid-base agent, poorly
flowed and stuck to the punch and die set. Then, we added PEG 600 in F2, F3 and F4 at the amount of
1%, 2% and 3% by weight, respectively. The effervescent tablets of F2 and F3 showed a better flow than
F1 but still stuck to the punch and die set. F4 formulation, which contained 3% of PEG 6000, exhibited
good flow, good compressibility and did not stick to punch and die set. When dissolved F4 effervescent
tablet in water, it disintegrated and produced clear solution. The physical appearance of effervescent
tablet of F4 was brown, round shape with smooth surface. The evaluation data of weight variation,
thickness, hardness, friability, and disintegretion time were shown in Table 3.

Table 3 Data of physical properties
Physical properties F1 F2 F3 F4
Weight variation (mg) 2003.12±8.76 2007.15±2.25 2011.47±5.13 2006.62±5.81
Thickness (mm) 7.28±0.15 7.44±0.09 7.49±0.17 7.50±0.11
Hardness (kP) 45.23±0.02 43.56±0.11 48.25±0.13 49.68±0.05
Friability (%) 0.27 0.20 0.23 0.29
Disintegretion time (min) 3.05±0.05 3.11±0.09 3.11±0.13 3.20±0.06
Quantitative analysis of gallic acid by high performance liquid chromatography (HPLC)

Validation method of HPLC analysis Linearity was studied by preparing standard stock solution of gallic
acid at different concentration levels. When the concentrations of gallic acid and their respective peak
areas were subjected to regression analysis by least squares method, a good linear relationship (r2 =
0.9990) was observed between the concentrations of gallic acid and the respective peak areas in the range
20-200 µg/ml. The regression equation was found to be y = 13.8469x- 24.0172, where Y is the peak area
PT – 27
and x is the concentration of gallic acid.
To ensure the accuracy and reliability of the method, recovery studies were carried out in triplicate at
three concentration levels. The recovery of gallic acid was found to be in the range of 98.47-99.10 %
The intra-day precision of the assay method was evaluated by carrying out six independent assays of
gallic acid test samples against qualified reference standard on the same day and these studies were also
repeated on six consecutive days to determine inter-day precision. The percentage of RSD of six assay
values was calculated. On day 1, 2 and 3, % RSD of gallic acid was found to be 0.533, 0.701 and 0.354,
respectively.
To determine the specificity of the analytical method, standard stock solution of gallic acid, placebo
solution and Jatu-Phalathika solution dissolve in the same solution. All solutions were filtered through 0.2
μm Nylon filter. Equal volume (5 μl) of each sample was injected into the HPLC by autosampler and
diode array scanning was measured from 220-400 nm. The retention time and UV absorption spectrum of
Jatu-Phalathika solution were compared with the standard and placebo. As a result, the chromatogram of
placebo was not shown the peak area at 7.129 minutes.
CONCLUSION
Jatu-Phalathika effervescent tablets were prepared successfully by direct compression method. Lactose
can effectively absorb Jatu-Phalathika extract and other excipients. The formulation overcomes the
problems associated with hygroscopic excipients like sodium bicarbonate, citric acid and tartaric acid.
The analytical HPLC method is validated for quantitative determination of gallic acid in effervescent
tablet. This research demonstrates the new dosage form for TTM preparations to be a new way to promote
traditional medicine usage for modern lifestyle.
REFERENCES
1. วุฒิ วุฒิธรรมเวช. สารานุกรมสมุนไพร. กรุ งเทพมหานคร:สํานักพิมพ์โอเดียนสโตร์ , 2540.
2. Chandira, M., and Jayakar, B. Formulation and evaluation of herbal tablets containing Ipomoea digitata
Linn, extract. International Journal of Pharmaceutical Sciences Review and Research. 3(1)(2010): 101-110.
3. Choudhary, N., and Sekhon, B. An overview of advances in the standardization of herbal drugs. J Pharm
Educ Res. 2(2)(2011).
4. Gordon, R.E., Rosanske, T.W., Fonner, D.E., Anderson, N.R., and Banker, G.S. Granulation technology
and tablet characterization. Pharmaceutical dosage forms: tablets. 3(2)(1990): 245-348.
5. Singh, I., and Kumar, P. Preformulation studies for direct compression suitability of cefuroxime axetil and
paracetamol: a graphical representation using SeDeM diagram. Acta Poloniae Pharmaceutica n Drug
Research. 69(1)(2012): 87-93.
6. Wang, P., Li, L., Yang, H., and et al. Chromatographic fingerprinting and quantitative analysis for the
quality evaluation of Xinkeshu tablet. Journal of Pharmaceutical Analysis. 2(6)(2012): 422–430.

COSMECEUTICAL DEVELOPMENT OF OXYRESVERATROL FROM MAHAD

(ARTOCARPUS LAKOOCHA ROXB.)
Surapol Natakankitkul1, Nicha Chareonmuang1, Tawan Panyakhong1 and Worrapon Wangkananon2
1
Department of Pharmaceutical Science, Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand.
2
Mintech Laboratory Co. Ltd, 199/273 Soi Choet Wutthakat 9, Don Muang, 10210 Bangkok, Thailand.
KEYWORDS: Oxyresveratrol, Skin whitening, Mahad, Artocarpus lakoocha Roxb.
INTRODUCTION
Oxyresveratrol (Figure 1), a natural phenolic stilbene compound found in Mahad (Artocarpus lakoocha
Roxb.) heartwood extract, exhibited a potent inhibitory effect on tyrosinase enzyme which catalyzes rate-
limiting steps in biosynthesizing human melanin pigment [1,2]. In Thailand, herbal cosmetics are
becoming more popular especially for nourishing and whitening skin. Several plant extracts were found to
PT – 28
exhibit strong anti-tyrosinase activity in vitro such as those from Areca catechu L. [3], Artocarpus
incisus [4], Broussonetia spp. (paper mulberry root bark extract) [5], Glycyrrhiza glabra (licorice extract)
[6], Prunus spp. [7], and Rheum officinale [8]. Many of these extracts have been tested in vivo and
commercially developed as skin whitening agents in cosmetic preparations such as Morus alba and
licorice extracts [9,10]. However, they are quite expensive and some users may develop skin
hypersentivity especially when applying them at high concentrations. Some preliminary studies were
developed skin-whitening solution containing Mahad heartwood extract which had oxyresveratrol as the
active ingredient [11,12]. The main objective of this study was therefore to study the extraction process
and the properties of Mahad extract and measured the amount of oxyresveratrol in the extract, developed
lotion formulation from Mahad extract, clinical evaluated whitening skin and investigated the satisfaction
of using Mahad lotion in the female volunteers.
Figure 1 Oxyresveratrol chemical structure.
Figure 2 The dried wood chips of heartwood Mahad (Artocarpus lakoocha Roxb.).


Materials Oxyresveratol was donated from Youcandu Institute. Mahad (Artocarpus lakoocha Roxb.)
heartwood was received from Mintech Laboratory Factory. All other reagents were of analytical and
HPLC grade.
Methods The dried wood chips of heartwood Mahad (Figure 2) was continuous extracted with ethanol
using Soxhlet apparatus (40 mm ID extractor, with 500-mL round bottom flask) and slow alcohol
evaporation by Rotavapor (BÜCHI Labortechnik AG, Switzerland). Then the extract was identified
oxyresveratrol by TLC (Silica gel 60 GF254 plates and developing solvent of chloroform: methanol = 9:1
v/v) and UV-visible spectroscopy. The amount of oxyresveratrol in the extract was measured by HPLC.
Chromatographic analyses used a Shimadzu prominence UFLC system (Shimadzu, japan) connected with
Hypercil ODS column (4.0x250 mm, 5-micron; Agilent, USA). The mobile phase consisted of
acetonitrile and DI water with acetic acid (30:70:0.04 v/v). The flow rate of mobile phase was maintained
at 1.0 ml/min. Sample injection volume was10 µl. The UV detection for oxyresveratrol was carried out at
320 nm.
Formulation and in vivo evaluation of lotion containing Mahad extract The Mahad or A.
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lakoocha extract was formulated as an oil-in-water emulsion in 3 lotion formulations and selected the best
physical and chemical properties of the formulation. The lotions containing Mahad extracts were also
freshly prepared and used within 2 month. Additionally, the stability study; tyrosinase inhibitory activity,
HPLC, colour and odour of the formulation kept by cooling and heating cycle (4, 45 °C; 48 hr) for 6
cycles were studied.
In vivo evaluation of skin whitening activity of Mahad lotion The protocol was approved by the Ethics
Committee of the Faculty of Pharmaceutical Sciences, Chiang Mai University. Thirty female volunteers,
age between 20 and 23 years with normal healthy skin, were recruited in the study. The in vivo physical-
whitening activity was further evaluated in 30 female volunteers. The initial melanin values were taken
from both the left and right lower arms of each subject using Mexameter MX16 (Courage and Khazaka,
Germany) before application of the test lotions. The initial melanin values were tested between the left
and the right arms within each subject to make sure that the starting melanin values were the same before
product application (P > 0.05, paired t-test). The subjects were daily applied each her arm blindly with
1% w/v Mahad testing lotion and lotion base using a parallel clinical trial with self-control applied the
lotion then assessed the satisfaction of using lotion for 6 weeks. The score value of whitening was
measured each application site of each arm every two weeks using skin-colors tone band then compared
relative to the initial treated arms within the same subject using paired t-test (alpha = 0.05). All the
experimental data were expressed as means ± SD.
Figure 3 The whitening activity in average skin-colors tone score after daily application to the lower arms of female volunteers for
6 weeks with 1% Mahad lotion (sample) in comparison with lotion base (control)
Data = means ± SD (n = 30). Day42*significantly greater than control (P < 0.05) at the same week.


The results showed oxyresveratrol was the major active ingredient in the Mahad extract and the percent
yield of extraction measured by HPLC was 35.13%w/v. The stability test of formulated lotion by cooling
and heating of 6 cycles found that the test lotion was still stable with more viscous and pale color. While
Pothitirat et al. [13] reported that the whitening skin lotion containing 1.03% of Mahad extract storaged
at 4°C for 4 weeks, the antityrosinase activity, TLC, colour and odour of the lotion were not changed.
The percentage of tyrosinase inhibition of Mahad extract at concentration of 1 mg/mL was found to be
87.88±0.31 (IC 50 = 50µg/mL) [14]. The melanin-reducing efficacy was also measured using Maxameter.
The whitening activity of the selected lotion evaluated in female volunteers found that daily application
with 1% Mahad lotion to the upper arms (n = 30) of volunteers produced significant over the lotion base
after 4 weeks. From Figure 3, it can be seen that the average skin-colors tone score for whitening activity
of the lower arms treated with Mahad lotion continuously decreased with the treatment duration, i.e. from
2.84% at week 4 to 7.64% at week 6. During the same period, the values for the arms treated with lotion
base (self-control) decreased only slightly from 0.53% at week 4 to 1.06% at week 6. When paired t-test
was applied to the whitening data at various weeks, significant difference was found in the mean %
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whitening values between the arms treated with Mahad lotion and the self-control arms after 4 weeks of
application. After week 4, the significance became even more pronounced (p < 0.05) as the difference
between the two arms continued to increase until the end of the study (week 6), indicating a greater
whitening efficacy of Mahad lotion over the control. The oxyresveratrol was the most effective agent
giving the short onset of significant whitening effect after only day 28 of application (p<0.05). The effect
also increased with time with maximum whitening observed at day 42 after applied lotion significantly
(p<0.05). However, more studies are needed to confirm the clinical efficacy and safety of the Mahad
extract in larger panel of subjects using different formulations and/or in combination with other whitening
agents.
Recently, Sritularak et al. [15] have screened a large number of plants for their in vitro anti-tyrosinase
activity and found that the heartwood extract of A. lakoocha exhibited the highest activity. Purification of
the extract yielded two active components, namely oxyresveratrol and resveratrol. Comparing to other
tyrosinase inhibitors commonly used in whitening products like kojic acid and licorice extract, the readily
available and less expensive. A. lakoocha extract apparently produced a faster onset of significant
whitening effect, requiring only 2–4 weeks of application depending on the type of formulation and area
of application. The greater anti-tyrosinase activity of oxyresveratrol (tetrahydroxystilbene derivative) was
probably due to its higher number of phenolic hydroxy substituents, whereas resveratrol is a trihydroxy
analogue. It has been suggested that the number and position of phenolic hydroxy groups and the
presence of trans-olefin structure in the stilbene derivatives play an important role in their enzyme
inhibitory potencies [9,16].
The oil-in-water emulsion appeared to provide better whitening activity than the lotion base. The mild
skin whitening effect of Mahad extract could be considered a desirable attribute for use in cosmetic
preparations as its whitening activity will be gradual and more natural. However suitable UV
filters/absorber or sun-screening agents should be included in future formulations to enhance their
whitening efficacy. Regarding the side effects, no skin rashes or serious skin disorders were observed in
all subjects receiving the test lotion. Considering the low concentrations employed in this study (1%), the
extract may have a very promising potential for use as a safe, effective and economical whitening agent in
the cosmetic industry. The overall satisfactions of using Mahad lotion in the volunteers were in moderate
to good level.
CONCLUSION
The results of the studies demonstrated that the heartwood Mahad or A. lakoocha extract lotion was able
to reduce melanin formation in human volunteers have a promising potential for use as an effective and
economical skin-whitening cosmeceutical.

REFERENCES
1. Jagtap UB and Bapat VA. 2010. Artocarpus : A review of its traditional uses, phytochemistry
and pharmacology. J Ethnopharmacol 129: 142-166.
2. Chang TS. 2009. An Updated Review of Tyrosinase Inhibitors. IJMS 10: 2440-2475.
3. Lee KK and Choi JD. 1999. The effect of Areca catechu L. extract on anti-inflammatory and
antimelanogenesis. Int J Cosmet Sci 21: 275–284.
4. Shimizu K, Kondo R, Sakai K, Lee SH and Sato H. 1998. The inhibitory components
from Artocarpus incisus on melanin biosynthesis. Planta Med 64: 408–412.
5. Jang DI, Lee BG, Jeon CO. et al. 1997. Melanogenesis inhibitor from paper
mulberry. Cosmetics Toiletries 112: 59–62.
6. Yokota T, Nishio H. and Kubota Y. 1998. The inhibitory effect of glabridin from licorice
extracts on melanogenesis and inflammation. Pigment Cell Res 11: 335–361.
7. Matsuda H, Nakamura S and Kubo M. 1994. Studies of cuticle drugs from natural sources. II.
Inhibitory effects of prunus plants on melanin biosynthesis. Biol Pharm Bull 17: 1417–1420.
8. Iida K, Hase K, Shimomura K, Sudo S, Kadota S and Namba T. 1995. Potent inhibitors of
tyrosinase activity and melanin biosynthesis from Rheum officinale. Planta Med 61: 425–428.
9. Kim JH and Lee KT. 1998. Inhibitory effects of Morus alba extracts on
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melanogenesis. Cosmetics Toiletries 113: 65–70.
10. Petit L and Piérard GE. 2003. Skin-lightening products revisited. Int J Cosmet Sci 25: 169–181.
11. Zheng ZP, Cheng KV, To JT, Li H, Wang M. 2008. Isolation of tyrosinase inhibitors from
Artocarpus heterophyllus and use of its extract as antibrowning agent. Mol Nutr Food Res
52(12): 1530-1538.
12. Likhitwitayawuid K and Sritularak B. 2001. A New Dimeric Stilbene with Tyrosinase Inhibitiory
Activity from Artocarpus gomezianus. J Nat Prod 64(11): 1457-1459.
13. Pothitirat W, Pluemlamai J, Satniyom S, Leelamanitaya W, Gritsanapan W. 2010. Development
of whitening skin lotion from selected medicinal plants and its antityrosinase activity. Planta
Med 76: 174.
14. Tengamnuay P, Pengrungruangwong K, Pheansri I, Likhitwitayawuid K. 2006. Artocarpus
lakoocha heartwood extract as a novel cosmetic ingredient: evaluation of the in vitro anti-
tyrosinase and in vivo skin whitening activities. Int J Cosmet Sci 28(4): 269-276.
15. Sritularak B, De-Eknamkul W and Likhitwitayawuid K. 1998. Tyrosinase inhibitors
from Artocarpus lakoocha. Thai J Pharm Sci 22:149–155.
16. Ohguchi K, Tanaka T, Kido T. et al. 2003. Effects of hydroxystilbene derivatives on tyrosinase
activity. Biochem Biophys Res Commun 307:, 861–863.

PARTICLE SIZE CHARACTERISTICS OF GOLD NANOPARTICLES
Chutima Kongsuwan1 and Warangkana Warisnoicharoen2
1
Pharmaceutical Technology (International Program), Department of Pharmaceutics and Industrial Pharmacy,
Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
2
Department of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences,
KEYWORDS: Gold nanoparticles, Dynamic light scattering, UV-vis absorption, TEM, Stability
INTRODUCTION
Gold nanoparticles (AuNPs) are metallic nanoparticles that have attracted much attention due to the
unique optical properties, being easily visualized and detected by spectroscopic techniques [1]. AuNPs
are considered as non-toxic and are attractive to be applied in many applications. The particle size and
surface modification of AuNPs can be controlled by chemical composition [2]. The particle size of
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nanoparticles is thought to one of stability indices of the nanoparticles. Little or no change in particle size
is possibly implied the stability of the nanoparticles especially after time storage. The measurement of
nanoparticle size, however, is likely to depend upon the methodology and interpretation of the data.
Recently, some recent mentioned that the different techniques employed led to the variation in particle
sizes of the nanoparticles [3]. In this study, the citrate-stabilized gold nanoparticles (AuCt) and
polyethyleneimine-stabilized gold nanoparticles (AuPEI) were synthesized and determined for their
nanoparticle sizes and size distribution using several methods namely, UV-vis spectroscopy, transmission
electron microscopy and dynamic light scattering. The variation in size with different time intervals of
either AuCt or AuPEI was also evaluated.

Materials For nanoparticle synthesis, hydrogen tetrachloroaurate (III) trihydrate (HAuCl 4 .3H 2 O),
trisodium citrate dihydrate (Na 3 C 6 H 5 O 7 .2H 2 O) and polyethyleneimine (PEI,-(CH 2 CH 2 NH) n -, MW =
750 KDa) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Glassware were cleansed with aqua
regia (HCl:HNO 3 = 3:1, Carlo Erba, Milan, Italy) for the purpose of circumventing gold contamination,
rinsed with ultrapure water and oven dried prior to use.
Synthesis of gold nanoparticles The chemical reduction method was used for synthesis of 200 ppm
(1,015 µM) AuCt and AuPEI. The thirty-five microliters of 30% (w/w) hydrogen tetrachloroaurate
trihydrate were added to 49.5 mL ultrapure water. The sample was then heated up to 90°C in a water bath
with stirring. Then, 0.5 mL of 0.4 M trisodium citrate dihydrate was added in to the gold solution and the
mixture was continued heating until the color became dark red. The molar ratio of Au atom to trisodium
citrate used in the synthesis was 1:8. In addition, AuPEI at the same concentration of AuCt was
synthesized using polyethyleneimine as a polymeric stabilizer. The same procedure as mentioned above
was used for synthesis AuPEI except for 0.5 mL of 0.36 M PEI solution was used instead of the citrate
solution. Notably, the molar ratio of Au atom to PEI was 1 to 0.072. The synthesized AuCt and AuPEI
were stored in a light-protected container at room temperature until use.
Size measurement of gold nanoparticles The appearance and color of freshly prepared AuNPs were
visually observed prior to size measurement. The measurement of either AuCt or AuPEI was performed
using i) UV-vis spectroscopy ii) transmission electron microscopy and iii) dynamic light scattering.
UV-vis spectroscopy The optical properties of AuNPs are conquered by collective oscillations of
electrons (plasma oscillations) that are in resonance with the incident electromagnetic radiation [4]. The
surface plasmon resonance (SPR) band of AuNPs was characterized by using UV-visible
spectrophotometer model Evolution 600 (Thermo Scientific, UK). The UV quartz cuvette with a path
length of 10 nm was used and the ultrapure water was a reference standard for measurement.

The analysis of nanoparticle size was done according to the following equation [3]:
d = 3 + (7.5 * 10-5) X 4 for X < 23 (1)

d = (√X-17 – 1) for X ≥ 23 (2)
0.06
where d is the diameter of the nanoparticle (nm), when 5 ≤ d ≤ 100, and X is the maximum absorbance
wavelength (λ max ) – 500.
Transmission electron microscopy (TEM) Ten microliters of sample solution was dropped on the
carbon-coated grid (200 mesh) and dried in the air at room temperature prior to observation under TEM
model JEM-2100 (Jeol, Japan). The size distribution of AuCt and AuPEI was calculated using SemAfore
program version 5.21.
Dynamic light scattering The hydrodynamic sizes of AuCt and AuPEI were also measured by using
dynamic light scattering (DLS) method or referred as photon correlation spectroscopy (PCS) technique.
The scattered intensity of the Brownian motion of nanoparticles is recorded and calculated using Stokes-
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Einstein equation to obtain hydrodynamic diameter (d h ) [5]:
dh = kT (3)
3⋅¶⋅η⋅D
where k is the Boltzmann’s constant (1.38065 x 10-23 J⋅K-1), T is the temperature (K), η is the viscosity of
the solvent (nm-2⋅s) and D is the diffusion coefficient (nm2⋅s-1).
Then, the hydrodynamic diameter of nanoparticle was calculated by the cumulant method using the
software provided with the instrument. The width of size distribution was also reported as polydispersity
index (PdI). All PCS measurements were performed at 25°C using Zetasizer Nano ZS (Zen3600,
Malvern, UK) equipped with He-Ne laser operating at a wavelength of 633 nm and a photon detector. The
measurement was performed at a scattering angle of 173°. Prior to size measurement, AuCt and AuPEI
were diluted with ultrapure water to eliminate multiple scattering effects. The data was represented as a
mean particle diameter of three measurements.
Stability study of gold nanoparticles AuNPs were kept at room temperature with light protection. AuNPs
were determined for instability after storage for 1, 4 and 12 weeks. The change in appearance and color of
AuNPs were visually observed. The particle sizes of AuNPs were evaluated by DLS method as described
previously.
RESULTS
Particle size analysis of gold nanoparticles The SPR characteristic of AuNPs was presented as the UV
absorbance spectra. The maximum absorption wavelengths (λ max ) were found to be 522.5 ± 0.5 nm for
AuCt and 521 nm for AuPEI (Figures 1A and 2A). For AuCt, the particle size was calculated according to
equation-1 for ‘X’ value of 22 (λ = 522 nm) and equation-2 for ‘X’ value of 23 (λ = 523 nm). Thus, the
sizes of AuCt were in a range of 20.57 to 24.16 nm. Since the ‘X’ values of AuPEI were less than 23 (λ =
521 nm), the particle size of AuPEI was then calculated using equation-1. The AuPEI contained particle
sizes of 17.59 nm.
The morphology and size of the AuNPs were detected by a TEM technique. The TEM result presented
the spherical shape of either AuCt or AuPEI (Figures 1B and 2B). The average size and size distribution
of AuNPs from TEM result were further analysed. The mean particle size and of AuCt was 10.76 ± 1.26
nm (n=200) and that of AuPEI was 7.71 ± 0.53 nm (n=279). In order to obtain the hydrodynamic size of
AuNPs, the DLS technique was performed. The hydrodynamic sizes of AuCt and AuPEI were 18.37 ±
0.34 nm (n=3) and 18.59 ± 0.59 nm (n=3), respectively. The polydispersity index which was similar to

the distribution of size was 0.39 for AuCt and 0.27 for AuPEI. In comparison, the results of particle size
measurement of AuCt and AuPEI by several methods are tabulated in Table 1.
Stability study of gold nanoparticles After being kept for several time periods, AuNPS (AuCt and
AuPEI) presented rather the same physical appearance as their corresponding freshly prepared samples.
Likewise, the UV-vis absorption spectra of AuNPs after storage were in similar pattern to those of freshly
prepared samples (data not shown). The variation in size and size distribution of week-old AuNPs was
evaluated by DLS technique. The PCS results are illustrated in Figure 3.
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(A) (B)
Figure 1 UV-visible absorption spectra (A) and TEM image (inset; scale bar of 50 nm) with corresponding size distribution
histograms (B) of AuCt.
(A) (B)
Figure 2 UV-visible absorption spectra (A) and TEM image (inset; scale bar of 50 nm) with corresponding size distribution
histograms (B) of AuPEI.

Figure 3 Variation in hydrodynamic diameter (nm) (mean ± S.D., n=3) of and polydispersity index of AuCt () and AuPEI ()
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with different time storage. Solid line and dash line represent particle diameter and polydispersity index, in orderly.
Table 1 Particle size analysis of AuCt and AuPEI
AuNPs Particle size (nm) measured by different methods

i) UV spectroscopy ii) TEM iii) DLS
AuCt 20.57 ; λ = 522 nm 10.76 ± 1.26 18.37 ± 0.34
24.16 ; λ = 523 nm
AuPEI 17.59 ; λ = 521 nm 7.71 ± 0.53 18.59 ± 0.59
DISCUSSION AND CONCLUSION

In this study, gold nanoparticles stabilized by either citrate or polyethyleneimine were characterized for
particle sizes using the commonly used methods namely, UV-vis spectroscopy, transmission electron
microscopy and dynamic light scattering or so-called photon correlation spectroscopy. The results of
particle sizes obtained from UV-vis spectroscopy and DLS were considerably in the same range for AuCt
and fairly similar for AuPEI. Surprisingly, the particle sizes of AuCt and AuPEI became smaller (nearly
50% or more) when determined by the TEM analysis. Considerably, the result was not unexpected since
the stabilizers of AuNPs, citrate and polyethyleneimine were hydrophilic in nature. The hydrophilic
stabilizer can then form the layer surrounding the particles and interact with the water molecules in the
dispersion medium [6,7]. Conversely, the TEM technique allows only dehydrated form of a tested sample
to be detected, hence the interaction with the water molecules or interparticle interaction were unable to
be occurred. However, although the smaller sizes detected with the TEM results, this technique was the
only one that indicated the morphology of the AuNPs. In this study, the results were in the same trend
when the size measurement was performed by UV-vis spectroscopy and DLS. It was mentioned however
that the size analysis of UV-vis spectroscopy and DLS techniques was dependent on the assumption of
spherical shape and monodisperse non-interacting particles [8]. Notably, the DLS method provides better
advantages than UV-vis spectroscopy in term of the polydispersity index (PdI). Owing to less change in
particle sizes and lower PdI values of AuPEI after time storage, AuPEI seemed to be more stable and
narrower in size distribution than AuCt.
In conclusion, the size measurement of AuNPs should be concurrently performed using at least two
techniques. The TEM technique is inevitable when the assumption on size morphology or distribution is
required for analysis of particle sizes.

ACKNOWLEDGEMENTS
The authors wish to thank the support in part from Chulalongkorn University Graduate School Thesis
Grant for CK.
REFERENCES
1. D. Philip. Synthesis and spectroscopic characterization of gold nanoparticles, Spectrochimica
Acta Part A 71: 80-85 (2008).
2. B.K. Pong, H.I. Elim, J.X. Chong, W. Ji, B.L. Trout, and J.Y. Lee. New insights on the
nanoparticle growth mechanism in the citrate reduction of gold (III) salt: formation of the Au
nanowire intermediate and its nonlinear optical properties, J. Phys. Chem. 111: 6281-6287
(2007).
3. N.G. Khlebtsov. Determination of size and concentration of gold nanoparticles from extinction
spectra, Anal. Chem. 80: 6620-6625 (2008).
4. P. Ghosh, G.Han, M. De, C.K. Kim, and V.M. Rotello. Gold nanoparticles in delivery
applications, Adv. Drug Deliv. Rev. 60: 1307-1315 (2008).
5. T. Tadros, P. Izquierdo, J. Esquena, and C. Solans. Formation and stability of nano-emulsions,
Adv. Colloid Interface Sci. 108-109: 303-318 (2004).
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6. W. Haiss, N.T.K. Thank, J. Aveyard, and D. G. Fernig. Determination of size and concentration
of gold nanoparticles from UV-Vis spectra, Anal. Chem. 79: 4215-4221 (2007).
7. M.G. Vladimir, V. K. Alla, N.L. Yuri, and L. Roman. Photon correlation spectroscopy
investigations of proteins, Adv. Colloid Interface Sci. 105: 201-328 (2003).
8. B.N. Khlebtsov, and N.G. Khlebtsov. On the measurement of gold nanoparticle sizes by the
dynamic light scattering method, Colloid J. 73: 118-127 (2011).

MOLECULAR DYNAMICS SIMULATION OF α-RESORCINOL CRYSTAL

DISSOLUTION IN WATER
Somwang Sae-Tang1, John Kendrick2, Jamshed Anwar2,3 and Jittima Chatchawalsaisin1

1
2
The School of Life Sciences, University of Bradford, Bradford, West Yorkshire, BD7 1DP, U.K.
3
Current address: Chemical Theory & Computation, Department of Chemistry, Lancaster University, Lancaster LA1 4YB, U.K.
KEYWORDS: Dissolution, α-Resorcinol, Molecular dynamics simulation
INTRODUCTION
Chemicals including drugs are often presented in a product in their solid state, as this offers greater
stability. In pharmaceuticals the choice of crystal structure, particle morphology, and particle size can all
influence manufacturing, stability of the product, and its bioavailability. Therefore, understanding how
crystals grow and how they may be engineered to give the desired properties is important. Crystals of
polar molecules such as resorcinol and urea are particularly interesting since they can grow
asymmetrically along the polar axis and they have been used as models for investigating the effects of
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solvents1-5) and additives6). Despite much research interest, our understanding of the mechanisms of
crystal growth and dissolution in these systems is still rudimentary. Here we studied the dissolution of α-
resorcinol crystal in water at the two faces that terminate the polar axis using computer simulation, more
specifically molecular dynamics (MD) simulation.
METHODS
MD is a method which reveals the dynamic behavior of molecules in a system by calculating interaction
forces between the molecules. The specified interactions between atoms and molecules defined by a force
field are employed to calculate the resulting molecular trajectory and thus simulate the dynamical
behavior of the molecular system as a function of time.
In this study, MD simulations were performed using the DL_POLY package7) with the force field
parameters for α-resorcinol molecules8-9) and TIP4P model of water molecules10). The system consisted
of a crystal slab of α-resorcinol containing 768 molecules with the non-polar face, (011), and the polar
face, (011), exposed to 3200 water molecules (Figure 1). The crystal slab was infinite in the x and y
directions since three-dimensional periodic boundaries were applied. The simulations were performed in a
constant temperature and constant stress ensemble (NST) using the Rahman-Parrinnello approach and
coupled to a Nosé-Hoover thermostat and barostat with relaxation times of 0.1 and 1.0 ps, respectively.
The timestep was 0.002 ps and the interaction potential cutoff radius, which limits the number of
interactions between molecules, was 12 angstrom. The smooth particle mesh Ewald method was used to
calculate long range electrostatic interaction with a relative error of 10-5. The simulations were first
equilibrated for 500 ps at 3 different temperatures, 200, 250, and 300 K, with an increased mass for the
resorcinol molecules (100 fold higher) in the forcefield input. The increased mass maintained the integrity
of the crystals while allowing the water molecules to equilibrate about the exposed crystal faces. The
different temperatures were employed to generate different starting positions of the water molecules. The
equilibrated crystal slab thickness was approximately 60 angstrom, whilst the water layer was
approximately 50 angstrom. Production runs were carried out at 293, 313, and 333 K (using the 200K
equilibrated configuration). A total of three replicate simulations were carried at 333K (using equilibrated
configurations at 200, 250 and 300 K) to remove any bias resulting from the starting configuration. All
production runs were carried out for 15 ns. The production runs at 293 and 313 K were continued for
further 50 ns.
RESULTS
The molecular trajectories for each of the systems were visualized using the program Visual Molecular
Dynamics (VMD)11). Density profiles and other quantities were determined using an in-house suite of
analytical programs. The results were used to examine the dissolution of the two faces of the α-resorcinol
crystal slab in water.
Density profiles: Densities of α-resorcinol and water along the z-axis were calculated by dividing the slab
into layers of 4.9 angstrom along the z-axis. The results of average densities over the first nanosecond of
water and α-resorcinol are shown in Figure 2. The average water densities of every simulation showed
that there was a water layer on the non-polar face. The water layer on the polar face appeared only for

simulation at 293 K. At 333K the whole crystal slab completely dissolved into solution after 15 ns of
simulation (Figure 3c), while the crystal slabs simulated at 293 and 313 K gradually dissolved. After
simulating for a further 50 ns, at 293 K, one polar face layer dissolved into solution while the non-polar
face still retained its structure (Figure 3a). At 313 K, 3 layers of polar face and one layer of non-polar face
had dissolved into solution (Figure 3b). For different starting configurations at 333 K, all simulations
performed similarly as above but the rates of dissolution were faster than for simulations at 293 and 313
K.
Trajectory projections: The trajectories are useful to determine the motion of water molecules which
interact with both crystal faces and how α-resorcinol molecules dissolved into solution. The positions of
α- resorcinol and water molecules along z axis after 65 ns of simulations at 293 K and 313 K are shown in
Figures 3a and 3b, respectively. Figure 3c shows the positions of molecules after 15 ns of simulations at
333K. Clearly, the crystal dissolves faster at higher temperatures. Moreover, the crystal could not retain
its structure beyond 15 ns when the simulations were carried out at 333 K. The results for the fractional
surface
coverage present the number of α-resorcinol molecules along the z-axis of the simulation box divided by
initial number of α-resorcinol molecules on each face as a function of time. They clearly show different
dissolution rates for each face (Figure 4). At the all temperatures the polar face dissolved faster than the
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non-polar face. These profiles also confirmed the number of crystal layers which had dissolved into
solution as shown by analysis of the trajectory snapshots (Figure 3). The constant solution densities at 65
ns for simulations at 293 K (Figure 4a, d) and 313 K (Figure 4b, e) allowed us to stop continuing these
simulations.

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DISCUSSION
The polar face of α-resorcinol crystal dissolves faster than the non-polar face at all temperatures. The
adsorbed water layer could be an important factor on the dissolution of both faces as previous studies2-3).
It is clearly shown that the water layer is likely to appear on the non-polar face rather than polar face
especially at lower temperatures and that it could inhibit the dissolution of non-polar face because of the
strong binding interaction between water layer and crystal face. Temperature was also an important factor
in the dissolution of α-resorcinol. The dissolution of α-resorcinol crystal was faster at higher temperatures
since higher energy can break the binding interaction between water layer and crystal face.
CONCLUSION
The MD simulations are able to reproduce the experimental observations that the dissolution of α-
resorcinol crystal is asymmetric with it being faster at the (011) face. The cause is thought to be the
stronger adsorption of water at (011) surface but this requires confirmation through potential of mean
force (free energy) calculations for water adsorption at each of the respective surfaces.
ACKNOWLEDGEMENTS
The authors would like to thank British Council (PMI 2 Connect – Research Co-operation Award) for
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financial support.
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nucleic-acids, and organic-molecules. J Am Chem Soc 117: 5179-5197.
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crystalline phases of resorcinol. CrystEngComm 10: 437-445.
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The parameters of the TIP4P model potential. J Comput Chem 24: 973-981.
11. Humphrey W, Dalke A, Schulten K. 1996. VMD -Visual Molecular Dynamics. J Mol Graphics
14: 27-28, 33-38.

MISCIBILITY STUDY OF BENZOCAINE AND POLY L-LACTIDE USING SOLUBILITY

PARAMETER CALCULATION AND THERMAL ANALYSIS
Yada Vattanagijyingyong1, Narueporn Sutanthavibul1 and Jittima Chatchawalsaisin1

1
KEYWORDS: Benzocaine, Poly l-lactide, Miscibility, Solubility parameter, Glass transition temperature
INTRODUCTION
Solid dispersion can be classified based on physicochemical state of drug and polymeric carrier. Among
several systems of solid dispersion, solid solution is of particular interest to pharmaceutical industry
because it can overcome typical problems such as poor oral bioavailability due to poor drug solubility,
drug polymorphism, and poor chemical (solution) stability, found in drug product development. Also, the
state of the drug in solid solution can be kinetically and thermodynamically stabilized. To date, it is
feasible to manufacture solid solution through spray drying and hot melt extrusion processes. However,
PT – 31
formation of solid solution is not always possible. Evaluation of drug-polymer miscibility is often useful
in guiding successful solid solution formulation1-4). In this study, miscibility between benzocaine (BZC),
drug, and poly l-lactide (PLLA), polymeric carrier was predicted by comparison the calculated solubility
parameters of the drug and polymer and by experimental determination using differential scanning
calorimetry (DSC) and hot stage microscopy (HSM)1).

Solubility parameter calculation
Group contribution methods (GCM) GCM was employed to calculate Hansen solubility parameters of
BZC and PLLA using HSPiP program version 3. This method is based on structure fragmentation by
Hoftyzer/Van Krevelen and Hoy approaches.
Molecular dynamics (MD) simulation 3D model of BZC and a unit of l-lactic acid (LA) were obtained
from the Pubchem Substance Database. The MD simulation was carried out using Forcite molecular
mechanics and dynamics simulation module of the Materials Studio version 5.5 software package
(Accelrys, San Diego, CA, USA). The 30x30x30 Å3 simulation box, with periodic boundary condition,
containing bulk amorphous BZC of 81 molecules or PLLA of 25 unit chain length was created using
Amorphous cell tool. Atomic charges and interactions between atom and molecules were assigned with
COMPASS forcefield5). Electrostatic and van der Waals energies were calculated by Ewald summation
and atom-based summation methods, respectively. The temperature was controlled by Nose thermostat;
and the pressure was controlled by Berendsen barostat. Geometry of the system was first optimized and
then its density was corrected under NPT ensemble at 298 K and 0.0001 GPa until the density was
fluctuated around average value. After that, the simulation was carried out under NVT ensemble at 298 K
for 500 ps until the system was equilibrated. The last 400 ps configurations were used for calculating
solubility parameters. The difference between the solubility parameters of BZC and PLLA (∆δ) was used
as predictor for miscibility.
Thermal analysis
BZC (≥ 99% purity) was purchased from Sigma-Aldrich. PLLA (Naturework®PLA2003D, 0.19% of
residue lactide, 4.4% of d-isomer) was purchased from BC Polymer Co., Ltd. PLLA was cooled in liquid
nitrogen for 5-10 min before grinding in a ultra-centrifugal mill (Model ZM200, Retsch, Germany) fitted
with 0.5 mm sieve prior to use.
Differential scanning calorimetry (DSC) BZC-PLLA miscibility was evaluated using differential
scanning calorimetry (DSC Model PB822e, Mettler Toledo, Columbus, USA) under nitrogen purge at 30
ml/min. BZC, PLLA and physical blends of BZC-PLLA at 10:90, 20:80, 30:70 and 50:50 ratios were
examined by heat-cool-heat cycle. The cycle was started by heating the sample from 25 °C to 165 °C at
10 °C/min and holding it at 165 °C for 2 min, followed by cooling the melt to -60 °C at 20 °C/min and
ended with reheating the cooled melt to 165 °C at 10 °C/min. Melting temperatures (T m ) and glass
transition temperatures (T g ) were detected during heating and reheating step, respectively.
Hot stage microscopy (HSM) Thermal behavior of BZC, PLLA and their physical blends were examined
using hot stage (Model FP82HT, Mettler Toledo, Columbus, USA) and light microscope (Model Eclipse
E200, Nikon, Tokyo, Japan) connected to camera and adapters (Model EOS650D, Canon, Tokyo, Japan).
The sample placed on slide glass and covered with cover glass was fixed on the hot stage. The sample

was heated from 30 °C to 165 °C at a heating rate of 10 °C/min.
RESULTS
Solubility parameter calculation
The solubility parameter of BZC and PLLA calculated by GCM using Hoftzyer/Van Krevelen and Hoy
approaches, also MD simulation are presented in Table 1.The solubility parameter of BZC and that of
PLLA was in the range of 20.7-23.3 MPa0.5 and 17.4 - 21.3 MPa0.5, respectively. The ∆δ was quite small
and slightly varied among calculation methods.
Table 2 Solubility parameters of BZCand PLLA
Solubility parameter (δ) calculation

Method (MPa)0.5 ∆δ
BZC PLLA
Hoftyzer/Van Krevelen 20.7 17.4 3.3
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Hoy 23.3 21.3 2

MD simulation 22.6 17.4 5.2
Thermal analysis
Differential scanning calorimetry (DSC) T m peaks of BZC and PLLA were 91.83 °C and 151.49 °C,
respectively (Table 2). T m of the polymer was decreased as BZC proportion in the blends was increased
(data not shown). T g of PLLA was detected at 59.25 °C upon reheating the cool melt (Figure 1 (A)).
However, determination of T g of the drug was not possible due to recrystallization of BZC at 48.04 °C
through cooling the molten drug.
Table 3 Thermal properties from DSC
Lists BZC PLLA

T m (°C) 91.83 151.49
T g (°C) - 59.25
Figure 1 DSC thermograms of BZC:PLLA blends: (A) 0:100, (B) Figure 2 Effect of BZC on T g of drug-polymer blend.
10:90, (C) 20:80, (D) 30:70 and (E) 50:50 during reheating step of heat-
cool-heat cycle.
Single T g of BZC-PLLA blends at the ratio of 10:90, 20:80, 30:70 and 50:50 were found at 31.42 °C,
28.05 °C, 24.26 °C and -2.39 °C, respectively (Figure 1(B-E)). The relationship between the proportion of
BZC in the blends and their T g is displayed in Figure 2. For the 50: 50 blend, above its T g ,
recrystallization of amorphous BZC occurred at 48.19 °C (Figure 1(E)).

Hot stage microscopy (HSM) HSM allows thermal behaviors of the drug and polymer to be visualized. In
the presence of the drug, PLLA was melted at the temperature below its T m (Figure 3). Melting of the
polymer occurred at lower temperatures with increasing BZC proportions. Dissolution of PLLA in molten
BZC was clearly observed (Figure 3 (H, J)).
91 °C 140 °C 150 °C 160 °C
A B C D
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E F G H
I J K L
Figure 3 Hot-stage photomicrographs of BZC-PLLA blends at varied ratios: 0:100 (A-D), 10:90 (E-H) and 50:50 (I-L).
DISCUSSION
The calculated solubility parameters obtained from both methods were similar to the values reported
earlier6-7). Greenlengh has been reported that if the ∆δ was less than 7.0 MPa0.5, the blend was likely to be
miscible. If it was more than 10.0 MPa0.5, the blend tended to be immiscible2). In the present study, the ∆δ
was less than 7.0 MPa0.5, suggesting that BZC and PLLA were potentially miscible. Drug-polymer
miscibility was confirmed by exhibiting of new single T g of the blend between the individual T g of the
drug, which was reported in the literature at -31 °C8) and of the polymer, 59.25 °C. The T g values of the
blends were decreased when the proportions of the drug in the blends were increased (Figure 2). This may
be attributed to plasticizing effect of the drug like ibuprofen which was reported earlier9). Miscibility was
also confirmed by melting behavior and dissolution of the polymer in the molten drug as visualized by
HSM (Figure 3). Recrystallization of the drug during reheating the cooled melt in the DSC which was
observed for the 50:50 blend may be explained by insufficient amount of the polymer to inhibit
crystallization of the amorphous drug.
CONCLUSION
BZC and PLLA are miscible based on solubility parameter comparison and thermal analysis using DSC
and HSM. The ratio of the drug and polymer may be an essential in formation of solid solution.
ACKNOWLEDGMENTS
The authors wish to thank the Chulalongkorn University Drugs & Health Products Innovation Promotion
Center and Process Development Center for Pharmaceutical and Herbal Products for the software
packages and computer facilities. The National Metal and Materials Technology Center (MTEC) is
acknowledged for ultra-centrifugal mill.

REFERENCES
1. Forster A, Hempenstall J, Tucker I, Rades T. 2001. Selection of excipients for melt extrusion
with two poorly water-soluble drugs by solubility parameter calculation and thermal analysis. Int
J Pharm 226: 147-161.
2. Greenhalgh DJ, Williams AC, Timmins P, York P. 1999. Solubility parameters as predictors of
miscibility in solid dispersions. J Pharm Sci 88: 1182-1190.
3. Gupta J, Nunes C, Vyas S, Jonnalagadda S. 2011. Prediction of solubility parameters and
miscibility of pharmaceutical compounds by molecular dynamics simulations. J Phys Chem B
115: 2014-2023.
4. Maus M, Wagner KG, Kornherr A, Zifferere G. 2008. Molecular dynamics simulations for drug
dosage form development: thermal and solubility characteristics for hot melt extrusion. Mol
Simul 34: 1197-1207.
5. Sun H. 1998. COMPASS: an ab initio force-field optimized for condensed-phase applications
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6. Fu Y, Liao L, Yang L, Lan Y, Mei L, Liu Y, Hu S. 2013. Molecular dynamics and dissipative
particle dynamics simulations for prediction of miscibility in polyethylene
terephthalate/polylactide blends. Mol Simul 39: 415-422.
PT – 31
7. de Arenaza IM, Meaurio E, Coto B, Sarasua J-R. 2010. Molecular dynamics modelling for the
analysis and prediction of miscibility in polylactide/polyvinilphenol blends. Polymer 51: 4431-
4438.
8. Baird JA, Van Eerdenbrugh B, Taylor LS. 2010. A classification system to assess the
crystallization tendency of organic molecules from undercooled melts. J Pharm Sci 99: 3787-
3806.
9. Wu C, McGinity JW. 1999. Non-traditional plasticization of polymeric films. Int J Pharm 177:
15-27.

DEVELOPMENT OF LAWSONE METHYL ETHER LOADED CHITOSAN-DEXTRAN

SULFATE NANOPARTICLES; PRELIMINARY STUDY
Pattravee Niamprem1, Pharkphoom Panichayupakaranant2 and Waree Tiyaboonchai1*

1
Naresuan University, Phitsanulok 65000, Thailand.
2
Prince of Songkla University, Hat Yai, 90112, Thailand.
KEYWORDS: Lawsone methyl ether (LME), chitosan, dextran sulfate, nanoparticles
INTRODUCTION
Lawsone methyl ether (LME, 2-methoxy-1,4-napthoquinone), isolated from Impatiens balsamina Linn.
(Balsaminaceae), exhibited potential antifungal and antibacterial activities [1]. Both minimal inhibitory
and minimal fungicidal concentrations of LME against Candida were 1.25µg/ml [2]. In addition,
Sritrairat et al. (2011) suggested that LME might be used as alternative mouthwash in the prophylaxis of
PT – 32
oral candidiasis (OC) for denture wearers or among HIV-infected individuals or other immune-
compromised patients [3]. However, the limitation of conventional mouthwashes is their short retention
time leading to low therapeutic efficacy [4]. To overcome this problem, mucoadhesive formulations
involving micro- and nanoparticles have been developed [5]. In this study, chitosan-dextran sulfate
nanoparticles (CDNPs) were examined. Chitosan is a naturally biopolymer derived from the shells of
crustaceans such as crabs, shrimps and lobsters. It is biodegradable, biocompatible and nontoxic [6].
Dextran sulfate is a biodegradable polyanionic polymer that is widely used for pharmaceutical
applications [7]. CDNPs are formed due to the electrostatic interaction between chitosan and dextran
sulfate. The surface charge and mean particle size are adjustable by varying the concentration ratio of
both polymers and pH sensitive swelling has been reported [7, 8]. Based on these considerations, the
purpose of this study was to develop and characterize the LME loaded CDNPs. In addition, the amount of
LME released was determined using in vitro release study.

Materials Chitosan from shrimp (C, MW 30kDa with 95% deacetylation) was obtained from
Aquapremier, Inc. (Bangkok, Thailand). Dextran sulfate (D, MW 500kDa), α-Amylase from Aspergillus
oryzae and mucin from porcine stomach, Type II were purchased from Sigma-Aldrich Chemie GmbH
(Steinheim, Germany). All other chemicals and solvents were of analytical grade. De-ionized (DI) water
was used in the preparation of solutions and dispersion of CDNPs.
Preparation of LME loaded CDNPs Lawsone methyl ether was semisynthesized by methylation of
lawsone. Then, the LME loaded CDNPs were prepared by polyelectrolyte complexation as reported
earlier [5], with minor modification. LME in dimethylsulfoxide (DMSO) solution (10 mg/ml) and 0.5 ml
of 1%Tween 20 were added to 1 ml of the dextran sulfate aqueous solution. The resulting mixture was
continuously stirred at 500 rpm for 30 min. Then, the mixture of 1.67 ml of chitosan aqueous solution (pH
4) and DI water (total volume: 5 ml) was added drop-wise to the dextran sulfate mixture using
homogenization at 10,000 rpm for 15 min. Subsequently, 0.025 ml of PEG-400 was added with mixing
over a period of 15 min. CDNPs were prepared with different processing parameters to study the effect of
a number of variables on their physicochemical properties. Process parameters were varied as follows: the
polymer concentration was varied from 0.1 to 0.2%; the mass ratio of chitosan to dextran sulfate was
varied from 1:0.4 to 1:1. The ranges of these variable values were selected based on preliminary
experiments. All samples were prepared in triplicate.
Physicochemical characterization of the CDNPs: Mean particle size The mean particle size and size
distribution were measured by dynamic light scattering (DSL) using ZetaPAL/90plus (Brookhaven
Instrument, Holtsville, NY). This instrument was equipped with 35 mV HeNe laser diode operating at
632.8 nm (JDS Uniphase, San Jose, CA, USA) and a BI-200SM Goniometer with an EMI-9863
photomultiplier tube connected to a BI-9000AT digital correlator. An aliquot of nanoparticle was diluted
in DI water. The particle size of each sample was measured at 25ºC at a detection angle of 90º for 10
repeated measurements. The mean particle size and polydispersity index were obtained from the
cumulative measurements [6].

Zeta potential The zeta potential was determined using phase analysis light scattering with
ZetaPAL/90plus. Measurements were carried out at 25ºC at 14.8º to the incident light. Samples were
prepared by redispersing the CDNPs in DI water. The zeta potential was calculated using the
electrophoretic mobility based on the Smoluchowski approximation.
Determination of entrapment efficiency (EE) LME loaded CDNPs were centrifuged at 18,000 rpm for
30 min. After discarding the supernatant, LME was extracted from CDNPs using the mixture of 2%
aqueous acetic acid and methanol at a ratio of 60:40 (v/v) and applied to the HPLC system. The HPLC
system (Shimadzu, Japan) consisted of a LC20-AT pump connected to a SIL-10ADVP auto-injector, a
SPD-6AV system controller, SPD-20A UV-visible detector. LME was separated using a Gemini 5u C18
110A (5µm, 150x4.6 mm).
In viro release studies In vitro release profile of LME loaded CDNPs was determined by the shake-flask
method [5]. LME exhibits poor water soluble, thus, artificial saliva (pH 7.4) containing 0.6% (v/v) Tween
80 was used as a dissolution medium to provide sink condition. A known amount of the LME loaded
CDNPs was mixed into 5 ml of dissolution medium and shaken at 100 rpm with an orbital shaker at
37±0.5ºC. An aliquot (0.3 ml) was taken at predetermined time intervals of 5, 15, 30, 60,120 and 180 min
in triplicate. The samples were centrifuged at 18,000 rpm for 30 min and the supernatant was analyzed for
the amount of LME released using HPLC method as described above.
PT – 32
RESULTS
Physicochemical characterization of the CDNPs: Mean particle size and size distribution The effect of
the polymer concentration (0.1, 0.125, 0.15 and 0.2% chitosan and dextran sulfate) was studied by
maintaining the polymer ratio at 1:0.6. The mean particle size was increased from 301 to 411 upon an
increase of polymer concentration (Table 1). When the concentration of polymer was less than 0.1%, the
system became translucent suggesting that only a small amount of nanoparticle was obtained. In contrast,
when the concentration of polymer was more than 0.15%, particle precipitation occurred. In addition, the
effect of the mass ratio of chitosan and dextran sulfate at 1:0.4, 1:0.6, 1:0.8 and 1:1 was studied by
maintaining the polymer concentration at 0.125%. The mean particle size was increased from 317 to 428
upon an increase of mass ratio of chitosan and dextran sulfate (Table 1). When the mass ratio of chitosan
and dextran sulfate was 1:1, particle precipitation occurred.
Zeta potential Regardless of the processing parameter, the zeta potential value of all formulations
exhibited a positive charge with the same zeta potential of approximately 20 mV (Table 1).
Determination of entrapment efficiency (EE) The entrapment efficacy of LME loaded CDNPs showed
low entrapment efficacy within the range of 7-17%.
In vitro release studies The effect of polymer concentration and mass ratio of chitosan and dextran sulfate
on the drug release profile was evaluated. For all formulation tested, the LME loaded CDNPs showed
biphasic drug release pattern with a burst release, approximately 60%, within 5 min, followed by a
sustained release up to 3 h. (Figure 1 and 2). In addition, LME solution was used as a control. As
expected, approximately 90% of LME rapidly dissolved within 5 min.
DISCUSSION
To overcome the limitation of conventional mouthwash, polymeric nanoparticles are proposed as a
promising delivery system for topical oral delivery as they possess a long retention time and prolong
released characteristic. Thus, in this study, LME loaded mucoadhesive CDNPs were developed. CDNPs
were formed rapidly by phase separation induced by electrostatic interaction between the positively
charge chitosan and negatively charge dextran sulfate. In this preliminary study, the influence of polymer
concentration and mass ratio of chitosan and dextran sulfate on physicochemical properties was
evaluated. These experimental conditions resulted in LME loaded CDNPs with a mean particle size in a
range of 300-450 nm and polydispersity index of ~0.2-0.3 indicating a narrow distribution. The mean
particle size of LME loaded CDNPs was slightly larger compared with blank CDNPs, ~ 200 nm (data not
shown). The mean particle size was found to be dependent upon the polymer concentration and polymer
ratio. The mean particle size tended to increase when increasing the polymer concentration. Larger
complex nuclei may be formed when formulated with higher polymer concentration. Similarly, mean
particle size tended to increase when increasing the mass ratio of polymer concentration. In addition,

precipitation was observed when the mass of dextran sulfate was equal and/or higher than chitosan.
However, there were no significant differences in the resulting zeta potentials when varying the
processing parameters. The zeta potential of all formulations was in a range of +16 to +21 mV, suggesting
CDPNs should be adhered to the negatively charged oral cavity. Unfortunately, CDNPs showed low LME
entrapment efficacy. It might be the effect of Tween 20 that was used as an emulsifier to disperse LME in
dextran sulfate solution. Tween 20 is a non-ionic surfactant that has HLB value of 16.7 which may help
solubilize LME in aqueous medium, leading to low entrapment efficacy. Nevertheless, formulation
without Tween 20 showed LME precipitation in dextran sulfate solution resulting in no entrapment of
LME. Therefore, various types of surfactants and concentrations should be examined to overcome this
problem, and the surfactants with low HLB might be candidate.
In addition, in vitro release profile studies showed burst release of LME from nanoparticles (60% within 5
min), suggesting that LME was likely adsorbed onto the particle surface, and some were entrapped in
particles. Moreover, the mechanism of LME release from the CDNPs was analyzed using three models as
follows: zero order, first order and Higuchi equation. The correlation coefficients for the release kinetics
were calculated from the graph. All formulations were best fitted in the Higuchi equation, indicating
PT – 32
diffusion to be the predominant mechanism of LME release from particles (Figures 1B and 2B).
Table 1 The mean particle size, zeta potential and entrapment efficiency of LME loaded CDNPs
EE
Mean size Zeta potential
Formulation PI±SD
(nm±SD) (mV±SD)
(%±SD)
C-D 0.1% 301±12 0.270±0.035 19±2 17±4
C-D 0.125% 346±16 0.251±0.036 19±2 9±1
C-D 0.15% 411±10 0.211±0.015 21±1 11±0
C-D 0.2% P − − −
C:D 1:0.4 317±10 0.309±0.033 19±0 7±1
C:D 1:0.6 346±16 0.251±0.036 19±2 9±1
C:D 1:0.8 428±17 0.271±0.052 16±2 17±1
C-D 1:1 P − − −
EE = entrapment efficiency
P = precipitation which could not be measured by ZetaPAL/90plus.

B
PT – 32
Figure 1 (A) In vitro release profiles of LME loaded CDNPs prepared with a mass ratio of chitosan to dextran sulfate of 1:0.6 and
polymer concentration of (■) 0.1%, (Δ) 0.125%, and (×) 0.15% chitosan and dextran sulfate aqueous solution compared with (●)
LME solution. (B) Higuchi release model of LME loaded CDNPs.
Figure 2 (A) In vitro release profiles of LME loaded CDNPs prepared with a polymer concentration of 0.125% and a mass ratio of
chitosan and dextran sulfate of (♦) 1:0.4, (Δ) 1:0.6, and (×) 1:0.8 compared with (●) LME solution. (B) Higuchi release model of
LME loaded CDNPs.
CONCLUSION
LME loaded CDNPs prepared by electrostatic interaction between the positively and negatively charge
polymers could be successfully obtained. The prepared nanoparticles showed mean particle size of 300-
450 nm and are positively charged (+20mV), suggesting its mucoadhesive properties. In addition, the in
vitro release profile showed their ability to provide a sustained release, hence, CDNPs can reasonably be
considered as a promising oral cavity delivery system. Various type of surfactant should be further
investigated to enhance entrapment efficacy of LME incorporated.
ACKNOWLEDGMENTS
The authors would like to thank the financial support from Naresuan University.

REFERENCES
1. Farnsworth, N.R. and G.A. Cordell, A review of some biologically active compounds isolated from
plants as reported in the 1974-1975 literature. Lloydia, 1976. 39(6): p. 420-55.
2. Tripathi, R.D., H.S. Srivastava, and S.N. Dixit, A fungitoxic principle from the leaves of lawsonia
inermis lam. Experientia, 1978. 34(1): p. 51-2.
3. Sritrairat, N., et al., Antifungal activity of lawsone methyl ether in comparison with chlorhexidine.
Oral Pathology & Medicine, 2011. 40(1): p. 90-96.
4. Paderni, C., et al., Oral local drug delivery and new perspectives in oral drug formulation. Oral
Surgery, Oral Medicine, Oral Pathology and Oral Radiology, 2012. 114(3): p. e25-e34.
5. Chaiyasan, W., S.P. Srinivas, and W. Tiyaboonchai, Mucoadhesive chitosan-dextran sulfate
nanoparticles for sustained drug delivery to the ocular surface. J Ocul Pharmacol Ther, 2013. 29(2):
p. 200-7.
6. Tiyaboonchai, W. and N. Limpeanchob, Formulation and characterization of amphotericin B–
chitosan–dextran sulfate nanoparticles. International Journal of Pharmaceutics, 2007. 329(1–2): p.
142-149.
7. Anitha, A., et al., Preparation, characterization, in vitro drug release and biological studies of
curcumin loaded dextran sulphate–chitosan nanoparticles. Carbohydrate Polymers, 2011. 84(3): p.
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1158-1164.
8. Delair, T., Colloidal polyelectrolyte complexes of chitosan and dextran sulfate towards versatile
nanocarriers of bioactive molecules. European Journal of Pharmaceutics and Biopharmaceutics,
2011. 78(1): p. 10-18.

EFFECT OF SPRAY DRYING CARRIERS ON PHYSICAL PROPERTIES OF SPRAY DRIED

ANTI-FEE-MARENG-SUANG EXTRACT POWDER
Natawat Chankana1, *, Chaowalit Monton2, 3, Worawan Saingam2, 3, Siriporn Kittiwisut2, 3, Jirapornchai

Suksaeree2, 3, Apirak Sakunpak2, 3, Krisana Kraisintu1, 3, 4, and Yupa Tengwattanachoti4
1
Sun Herb Thai Chinese Manufacturing Plant, Rangsit University, Pathum Thani 12000, Thailand.
2
Faculty of Pharmacy, Rangsit University, Pathum Thani 12000, Thailand.
3
Sino-Thai Traditional Medicine Research Center (Cooperation between Rangsit University, Harbin Institute of Technology, and
Heilongjiang University of Chinese Medicine), Rangsit University, Pathum Thani 12000, Thailand.
4
Faculty of Oriental Medicine, Rangsit University, Pathum Thani, 12000, Thailand.
*
Corresponding author: natawat.c@rsu.ac.th (Natawat Chankana)
KEYWORDS: Spray dry, Physical property, Anti-Fee-Mareng-Suang, Colloidal silicon dioxide,

Maltodextrin
INTRODUCTION
Thai traditional medicine (TTM) has been used as cardiotonic agent included anti-Fee-Mareng-Suang
PT – 33
(anti-FMS); TTM formula is included in classical medical book called Tam Ra Pat Sart Songkraow, in the
chapter of Kam Pee Thip Ma La. This formula composed of 12 medicinal plants: Senna tora (L.) Roxb.,
Derris scandens Benth., Maclura cochichinensis (Lour.) Corner., Alpinia officinarum, Smilax
corbularia Kunth., Smilax glaba Roxb., Tarenna hoaensis Pitard., Dracaena lourieri Gagnep.,
Thyrsostachys siamensis Gamble., Angiopteris evecta Hoffm., Globba malaccensis Ridl., and Pinus spp.
Anti-FMS is prepared by boiling with water and ethanol, the original method for herbal medicines
preparation in the practice of TTM. The extracted composition of medicinal plants within a boiled
solution depends on boiling procedure of each person. However, this traditional preparation has some
disadvantages, such as the time to preparation and difficult to prescribed dose these can be troublesome
for patients. In pharmaceutical industry, spray drying is common procedure for change liquid to solid
especially for herbal extract solution [1]. This process is cost-effective, flexible and improving the
properties of extracted anti-FMS. It is necessary to develop new dosage forms for extracted anti-FMS
powder and/or easy to use that improving patient compliance.
The aim of this study was to evaluation the effect of type and concentration of spray drying carriers
(colloidal silicon dioxide and maltodextrin) on physical properties of spray dried anti-FMS extract
powder.

Materials The all herbs are shown in Table 1 was purchased from Charoensuk Osod, Nakorn Pathom
province, Thailand. Colloidal silicon dioxide was purchase from Changzhou Kaide Import and Export
Co.Ltd, China. Maltodextrin was purchased from TTK Sciences, Thailand. 95% Ethanol was purchased
from Samchai Chemical Co.Ltd., Thailand.
Table 1 Ingredients of anti-FMS herbal formula.
No. Scientific name Family Parts used Weight ratio

1 Senna tora (L.) Roxb. Leguminosae- Twig & leaves 5 parts
Caesalpinoidae
2 Derris scandens Benth. Leguminosae- Vine 5 parts
Papillionoidae
3 Maclura cochichinensis (Lour.) Corner. Moraceae Wood 2 parts
4 Alpinia officinarum Zingiberaceae Rhizome 2 parts
5 Smilax corbularia Kunth. Smilacaceae Rhizome 2 parts
6 Smilax glaba Roxb. Smilacaceae Rhizome 2 parts
7 Tarenna hoaensis Pitard. Rubiaceae Wood 2 parts
8 Dracaena lourieri Gagnep. Dracaenaceae Wood 2 parts
9 Thyrsostachys siamensis Gamble. Gramineae Root 2 parts
10 Angiopteris evecta Hoffm. Marattiaceae Root 2 parts
11 Globba malaccensis Ridl. Zingiberaceae Rhizome 2 parts
12 Pinus spp. Pinaceae Wood 2 parts

Extraction procedure The extraction procedure was based on original procedure since ancient time. Anti-
FMS crude drug (15 kg) was wetted with 6.5 L of 40% ethanol for 15 minutes. 10 L of water was then
added into anti-FMS crude drug and boiled for 15 minutes, for three times. All fractions of extracted anti-
FMS was filtered and combined. The combined extract was concentrated until the extract remains a half
of initial volume. The different types and concentrations of spray drying carrier: no carrier, colloidal
silicon dioxide (0.1%, 0.5%, and 1.0%), and maltodextrin (10.0% and 20.0%), was added into extracted
anti-FMS solution, the concentration of carrier was calculated on dried weight basis of anti-FMS crude
drug (Table 2).
Table 2 Percentage yield of spray dried anti-FMS herbal extract.
Formula Spray drying carrier Percentage yield (%)

F1 No carrier 1.43
F2 0.1% colloidal silicon dioxide 3.10
F5 10.0% maltodextrin 5.48
F6 20.0% maltodextrin 9.76
PT – 33
Spray drying procedure The extracted anti-FMS mixture was spray dried using factory scale spray dryer
(Model:LPG, Changzhou Kaide Import and Export Co.Ltd, China). The mixture was stirred by magnetic
stirrer and fed into spray drying chamber through a peristaltic pump with speed of 25 rpm. The inlet and
outlet air temperature were 170 °C and 80 °C, respectively. The mixture was sprayed through a nozzle
with atomizer pressure of 3 bars throughout the spray drying process. The spray dried products were
collected and weighed. Percentage yield was calculated on dried weight basis of crude drug and carrier.
The spray dried extracts were kept in desiccator until use.
Physical properties evaluation [2,3]
Angle of repose The angle of repose was investigated by the fixed funnel method. 5 g of spray dried
powder was poured into a glass funnel. The lower tip of glass funnel was 5 cm from the ground. The
height (h) and radius (r) of powder cone was measured, and then calculated using the equation (1). The
tests were performed in triplicated.
h
Tan θ =
r (1)
Where θ = angle of repose (°), h and r = height (cm) and radius (cm) of powder cone
Bulk density 2.5 g of spray dried powder was accurately weighed and gently poured into a 10 mL glass
cylinder without compacting. The volume of unsettled spray dried powder was recorded and then
calculated using the equation (2). The tests were performed in triplicated.
m
Bulk density =
V0
(2)
3
Where m = mass (g), V 0 = bulk volume (cm )
Tapped density The glass cylinder with the unsettled spray dried powder from bulk density testing, and a
tapped density tester (Erweka D-63150 Model:SVM 202, Germany) with 1,250 strokes was used for
tapped density testing. The volume of tapped spray dried powder was recorded, and then calculated using
the equation (3). The tests were performed in triplicated.
m
Tapped density =
Vf
(3)
3
Where m = mass (g), V f = final tapped volume (cm )
Compressibility index and Hausner ratio Compressibility index and Hausner ratio were calculated from
bulk volume and tapped volume. They were calculated following equation (4) and (5), respectively.
(V − Vf )
Compressibility index = 0 × 100
V0
(4)

V
Hausner ratio = 0
Vf
(5)
Moisture content The extracted and spray dried anti-FMS was tested moisture content using moisture
balance (Radwag, MAC 50/NH, Poland). The 1 g of spray dried extract was put into pan and heated up to
105 °C using standard mode, temperature rapidly increased and constant through process. Moisture
content was recorded when weight of test sample not change more than 1 mg within 120 seconds.
Moisture content was reported in unit of percentage. The tests were performed in triplicated.

Physical appearance Physical appearance of extracted and spray dried anti-FMS of various formulations
were dark-brown into light-brown depend on type and concentration of spray drying carriers (Figure 1).
Extracted and spray dried anti-FMS without carrier had dark-brown, melting when stored at ambient
condition. Nevertheless, carrier-mixed spray dried anti-FMS extract powder were less found.
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Figure 1 Physical appearance of spray dried anti-FMS herbal extract with different type and concentration of spray drying carriers
(a) no carrier (b) 0.1% colloidal silicon dioxide (c) 0.5% colloidal silicon dioxide (d) 1.0% colloidal silicon dioxide (e) 10.0%
maltodextrin (f) 20.0% maltodextrin (photographed by camera).
Percentage yield Percentage yield of spray dried extracted powder without carrier (F1) was 1.43%.
Increasing the concentration of colloidal silicon dioxide from 0.1% to 0.5% and 1.0% increased the
percentage yield that were 3.10%, 5.62%, and 6.58, respectively. In addition, when maltodextrin
concentration was increased from 10.0% to 20.0%, percentage yield increased from 5.48% to 9,76%
(Table 2).
Physical properties of spray dried anti-FMS herbal extract As results in Table 3, increasing spray drying
carrier concentration slightly decreased the angle of repose. According to the USP 33/NF28, the
flowability levels classed to 7 levels: excellent, good, fair, passable, poor, very poor and very very poor.
Spray dried extract without carrier (F1) had “passable” flowability. F2 and F5 formula revealed “fair”
flowability. F3 and F4 showed “good” flowability, and F6 showed “excellent” flowability. It mean that
spray drying carrier improve powder flowability [4,5]. Bulk density and tapped density (or bulk volume
and tapped volume) were used to calculate compressibility index and Hausner ratio. Bulk density and
tapped density of spray dried extract powder had 0.36-0.53 g/cm3 and 0.52-0.73 g/cm3, respectively.

Compressibility index and Hausner ratio had 7 levels of tabletability. F2 and F4 had “poor” tabletability.
F1, F3, and F5 formulas were “very poor” and F6 was “very, very poor” tabletability. The less value of
compressibility index and Hausner ratio means high probable of powder compression into tablets or filled
into capsules, such as F6 showed high flowability but low tabletability. The moisture content showed that
F1 and F2 formula had high moisture content, 7.00% and 6.25%, respectively. This result revealed that
colloidal silicon dioxide with concentration of 0.5% and 1.0% (F3 and F4) suitable used for spray drying
of anti-FMS extract than 0.1% (F2). Higher concentration of spray drying carrier showed lower in
moisture content of spray dried powder. Some publication use colloidal silicon dioxide up to 10% [6]. F3-
F6 had moisture content less than 5%, especially F4 had moisture content less than 3%. Moreover,
colloidal silicon dioxide had better drying performance than maltodextrin [7].
Table 3 Physical properties of spray dried anti-FMS herbal extract.
Physical properties F1 F2 F3 F4 F5 F6
Angle of repose (°) 41±4.02 38±2.24 35±3.30 34±1.53 39±3.56 27±2.04
Bulk density (g/cm3) 0.39±0.02 0.37±0.01 0.41±0.02 0.53±0.01 0.36±0.01 0.36±0.02
Tapped density (g/cm3) 0.58±0.02 0.52±0.04 0.64±0.04 0.73±0.03 0.55±0.02 0.62±0.02
Compressibility index (%) 32±1.92 31±1.45 33±1.57 29±2.54 35±0.74 41±1.76
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Hausner ratio 1.48±0.04 1.41±0.09 1.56±0.06 1.42±0.05 1.53±0.02 1.70±0.05
Moisture content (%) 7.00±0.18 6.25±0.22 3.73±0.05 2.95±0.16 4.14±0.26 4.12±0.10
Data represent as average±SD
CONCLUSION
Spray drying carriers play an important role for spray drying of anti-FMS extract. Both colloidal silicon
dioxide and maltodextrin were used as spray drying carriers. Colloidal silicon dioxide with concentration
of 0.5% and 1.0%, and maltodextrin with concentration of 10.0% and 20.0% were suitable for anti-FMS
spray drying process.
ACKNOWLEDGEMENTS
The authors are thankful to the Faculty of Pharmacy, Rangsit University for research facilities and
monetary support.
REFERENCES
1. Souza CRF, Schiavetto IA, Thomazini FCF, Oliveira WP. 2008. Processing of Rosmarinus
officinalis LINNE extract on spray and spouted bed dryers. Brazilian J Chem Eng 25(1): 59-69.
2. The United State Pharmacopeial Convention. 2010. The United State Pharmacopeia 33/the
National Formulary 28. United Book Press, Maryland.
3. Department of Medical Sciences, Ministry of Public Health. 2009. Thai Herbal Pharmacopoeia
2009 volume III, Office of National Buddishm Press, Nonthaburi.
4. Gallo L, Llabot JM, Allemandi D, Bucalá V, Piña J. 2011. Influence of spray-drying operating
conditions on Rhamnus purshiana (Cáscara sagrada) extract powder physical properties. Powder
Technol 208: 205-14.
5. Gallo L, Piña J, Bucalá V, Allemandi D, Ramírez-Rigo MV. 2013. Development of a modified-
release hydrophilic matrix system of a plant extract based on co-spray-dried powders. Powder
Technol 241:252-62.
6. Soares e Silva L, Santos da Silva L, Brumano L, et al. 2012. Preparation of dry extract of
Mikania glomerata Sprengel (Guaco) and determination of its coumarin levels by
spectrophotometry and HPLC-UV. Molecules 17: 10344-54.
7. Georgetti SR, Casagrande R, Souza CRF, Oliveira WP, Fonseca MJV. 2008. Spray drying of the
soybean extract: Effects on chemical properties and antioxidant activity. LWT 41:1521-7.

PRELIMINARY STUDY OF SPRAY DRYING AND FREEZE DRYING PROCESSES ON

PHYSICOCHEMICAL PROPERTIES OF ENCAPSULATED ROSMARINIC ACID
Supaporn Ketpitthaya1, Suchada Sukrong2, Walaisiri Muangsiri1, Nontima Vardhanabhuti1,

W. John Kao3, Narueporn Sutanthavibul1 and Phanphen Wattanaarsakit1
1
Department of Pharmaceutics and Industrial Pharmacy, 2Department of Pharmacognosy and Pharmaceutical Botany,
Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
3
Department of Biomedical Engineering, School of Pharmacy, Department of Surgery, University of Wisconsin-Madison, USA.
KEYWORDS: rosmarinic acid, encapsulation, spray drying, freeze drying, sodium alginate
INTRODUCTION
Rosmarinic acid (RA) is a polyphenol ester found in a variety of herbal plants especially in the Lamiaceae
group such as rosemary, sage, spanish sage, and oregano [1]. RA has a variety of bioactivities, especially
antioxidant and anti-inflammatory properties [1,2]. It also can break up amyloid-beta conglomerates of
Alzheimer's Disease in laboratory studies, and it has anti-viral properties with effects against Herpes
Simplex [3,4]. RA is soluble in organic solvent such as ethanol, but slightly soluble in water. Since RA is
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unstable to heat and oxidation [5], it is suitable to use as the model substance for herb extract to study of
the formulations and encapsulation process. Encapsulation is a process by which active substances are
incorporated inside particles in order to protect them from external environment or to control the release
of active substances from the microparticles [6]. Among various encapsulation techniques; coacervation,
ionic gelation, emulsion etc., spray drying (SD) and freeze drying (FD) are the two processes notably
used to support an industrial production and also to increase the stability of active substances [9-11,13].
In this study, sodium alginate (AG) was used as a biocompatible polymer to encapsulate rosmarinic acid
and to protect it from external environment [7,8,12]. Spray drying and freeze drying processes were used
and compared as encapsulation techniques. The factors of encapsulation processes and polymer
concentrations on physicochemical properties of the encapsulated RA were investigated.

Materials Rosmarinic acid (purity: 96%) and sodium alginate were purchased from Sigma-Aldrich.
Mannitol, calcium chloride dihydrate and trisodium citrate were purchased from Ajax Finechem.
Methods
Preparation of encapsulated RA microparticles The formulations of encapsulated RA prepared by spray
drying and freeze drying processes were shown in Table 1.
Table 1 Formulations and encapsulation processes of rosmarinic acid.
Spray drying Freeze drying

Blank Polymer concentration
Non-crosslinked Crosslinked Crosslinked
(without RA)
SD1-NB SD1-NC SD1-C FD1-C AG 1%
SD2-NB SD2-NC SD2-C FD2-C AG 2%
Spray drying process Non-crosslinked spray dried RA microparticles (SD-NC) were prepared by adding
75 mg of rosmarinic acid into 30 ml of polymer solution at concentrations and ratios as shown in Table 1
to make the ratio of RA polymer by weight equal to 1:4 in all formulations. The obtained solution was
then vigorously stirred by magnetic stirrer and sprayed into the chamber at the temperature of 130oC with
the spray rate of 2 ml/min. The outlet temperature was kept at 70-75 o C. Non-crosslinked spray dried
microparticles without RA (SD-NB) were also prepared as control. Crosslinked spray dried RA
microparticles (SD-C) was obtained by dispersing the non-crosslinked microparticles into 30 ml of 5%
w/v calcium chloride solution for 5 min then washed 3 times with methanol and 2 times with purified
water and was dried by freeze drying.
Freeze drying process Crosslinked freeze dried RA powders (FD-C) were prepared at the same
concentration and ratio of polymer to rosmarinic acid as in the spray drying process but with the addition
of mannitol at the concentration of 3% w/v into the solution followed by 500 µl of 5% w/v calcium

chloride as crosslinking agent. After crosslinking, freeze drying process was performed to make a matrix
of encapsulated RA and the obtained cake matrix was sieved by using mesh no. 30.
Characterization of encapsulated RA
Particle size and morphology Particle size was measured by light scattering using Malvern®
Mastersizer2000. Ethanol was used as solvent to avoid polymer swelling. Particle morphology was
observed by Scanning electron microscope (SEM).
Differential scanning calorimetry analysis (DSC) Thermal analysis by DSC was carried out to determine
the transition energy of the encapsulated samples. The samples were run at a scanning rate of 10◦ C per
min and within a temperature range of 25-250 ◦ C.
Entrapment efficiency High Pressure Liquid Chromatography (HPLC) was used to analyze the
entrapment efficiency (% EE) of the encapsulated RA, using Cosmosil®C18 column. The mobile phase
solution was prepared by using methanol and water at the ratio 35:65 with 0.1% v/v of acetic acid. The
flow rate of mobile phase was controlled to be at 1 ml/min and at 35◦C. 10 µl of the filtered solution was
injected into HPLC and equipped with UV detector at the wavelength of 320 nm. Analytical method
validation was carried out for accuracy, precision, specificity and linearity. The sample for analyzing was
prepared by dispersing 30 mg of the encapsulated RA in 5 ml of 3% w/v sodium citrate solution for 12
hours. The solution was then diluted with methanol to 50 ml and centrifuged at 3,200 rpm for 15 minutes.
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The obtained solution was filtered through 0.22 nylon syringe filter and analyzed by HPLC.
In vitro release study In vitro release study of the encapsulated RA was performed using modified Franz
diffusion cell with cellulose acetate membrane. Phosphate buffer pH 5.5 and pH 7.4 were used as donor
and receptor medium, respectively. The temperature of receiver medium was maintained constant at
32±0.5◦C. The 10 mg of encapsulated RA particles was dispersed onto the donor compartment and 2 ml
of dissolution fluid was periodically withdrawn from the receptor compartment which replaced with the
same amount of fresh medium and assayed by HPLC.
RESULTS
The particle size results from SD and FD processes were shown in Figure 1. From SD process, the sizes
of microparticles were ranged within 15 to 52 µm and were found to be decreased with the increasing of
polymer concentrations as can be seen in all formulations of SD-NC, SD-C and the control (SD-NB). The
sizes of crosslinked spray dried microparticles were found to be larger than those of non-crosslinked
spray dried microparticles. The particle sizes of FD powders which were produced by sieving the cake
matrix were found to be larger than SD particles.
The particle morphology from SEM was

shown in Figure 2. It was found that spray
drying process created spherical shape
microparticles where as freeze drying process,
after cake matrix sieving, created flatted shape
powders. Figure 3 showed the surface
appearance of FD powders. The surface of
FD2-C powder was found to be smoother than
FD1-C.
Figure 1 Particle size of non-crosslinked and crosslinked

spray dried microparticles and freeze dried powders.
Figure 2 SEM images of spray dried microparticles and freeze dried powders. (a; SD1-NC, b; SD2-NC, c; FD1-C, d; FD2-C)

The results from Figure 4 showed that the

melting endothermic peak of rosmarinic acid e f
at 170oC was seen in physical mixture with
polymer while the peak was disappeared in all
formulations through spray drying and freeze
drying process.
Figure 3 SEM images of surface of freeze dried powders

(e; FD1-C, f; FD2-C).
Figure 4 DSC thermogram of physical mixture of rosmarinic

acid with polymer (PM), encapsulated spray dried
microparticles and freeze dried powders (a; rosmarinic acid,
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b; mannitol, c; polymer AG, d; PM of RA+AG 1:1, e; SD1-

NC, f; PM of RA+mannitol 1:1, g; FD1-C).
Table 2 Entrapment efficiency of the spray dried microparticles and freeze dried powders.
Process Spray drying Freeze drying

Formulation SD1-NC SD2-NC SD1-C SD2-C FD1-C FD2-C
% EE (mean±SD) 71.97±3.04 75.59±5.49 1.71±0.08 2.13±1.01 103.9±3.76 97.71±5.85
Entrapment efficiency (% EE) results were

shown in Table 2. It was found that the
crosslinking process reduced the % EE of SD-C
significantly to lower than 5%. As well, the %
EE of the freeze drying process (FD-C) was
found to be higher than those of spray drying
process (SD-NC). The release profiles of
rosmarinic acid from the encapsulated particles
were shown in Figure 5. FD1-C showed the
fastest release profile. The profiles of both SD1-
NC and SD2-NC have similar result but SD2-NC
seem to have slightly faster release than SD1-NC
Figure 5 In vitro release profiles of SD-NC and FD-C:
at the initial phase.
(♦) SD1-NC, (■) SD2-NC, (▲) FD1-C, (x) FD2-C.
DISCUSSION
From spray drying process, using a hot gas for rapid drying, the obtained microparticles are spherical
shape and free-flowing. In order to make a stronger shell matrix, a crosslinking of microparticles was
performed by adding calcium into an alginate structure to form a stable network interaction [6,7].
However, there is also an interaction between the particles during a crosslinking process which resulted in
an agglomeration of those particles. It was also found that the crosslinking process for spray dried
microparticle affected the entrapment efficiency of rosmarinic acid. This was caused by the rosmarinic
acid loss in the process of crosslinking [8]. As for the freeze drying process, which is a drying technique
worked by freezing the samples and then reducing the pressure for water removing by sublimation into
gas, the encapsulated cake matrix was obtained. The powders obtaining from sieving step has a flatted
shape and less in free-flowing property than the particles from spray drying process. The spray dried
microparticles obtained tend to be smaller with increasing in polymer concentrations. Also the surface

roughness of particle from freeze drying was reduced. This might be caused by higher polymer
concentrations which affected the viscosity and strong structure in the solution and resulted in lesser
change in the structure of polymer surface during freeze drying process. Rosmarinic acid was
encapsulated and homogeneously dispersed in the polymer matrix as solid dispersion in amorphous state
through spray drying and freeze drying processes. The amorphous state of RA in polymer matrix might
cause this effect from the large amount of RA dissolved and lost during dispersion in crosslinking
solution. Entrapment efficiency of encapsulated RA freeze dried powders was higher than spray dried
microparticles since there was no rosmarinic acid loss in the process. From the release studies, RA was
remarkably increased at the initial phase and then sustained release from the encapsulated particles of
both processes. The result showed that higher polymer concentrations provided more sustained release
profiles. However, the smaller particle size with the higher surface area might have a possibilty to
enhance the RA dissolve rate as observed from the result of SD2-NC at initial phase. Apart from the
surface area, the more surface roughness and porosity from freeze drying process give a better
performance of release profile compared with those from spray drying process.
CONCLUSION
Rosmarinic acid, which was used as a model of natural bioactive substances in this study, can be
PT – 34
encapsulated in the particles made of sodium alginate. Sodium alginate, which is an anionic
polysaccharide with linear chain copolymer and the property of biocompatibilty, biodegradability and
nontoxicity, can be performed as a polymer for encapsulation by using spray drying and freeze drying
process. The non-crosslinked SD and crosslinked FD are suitable processes to make the encapsulated RA
particles and have a potential to be further developed for pharmaceutical applications or drug delivery
systems especially in the area of encapsulation of active substances.
ACKNOWLEDGMENTS
We thank Ratchadapiseksomphot Endowment Fund of Chulalongkorn University (RES560530157-HR)
for the funding and also thank CU.D.HIP, faculty of pharmaceutical sciences, Chulalongkorn University
for supporting lab equipments.
REFERENCES
1. Erkan N, Ayranci G, Ayranci E. Antioxidant activities of rosemary (Rosmarinus Officinalis L.) extract, blackseed
(Nigella sativa L.) essential oil, carnosic acid, rosmarinic acid and sesamol. Food Chem. 110 (2008) 76–82.
2. Swarup V, Ghosh J, Ghosh S, Saxena, Basu A. Antiviral and Anti-Inflammatory Effects of Rosmarinic Acid in
an Experimental Murine Model of Japanese Encephalitis. Antimicrob Agents Ch. 51(9) (2007) 3367-3370.
3. Iuvone T, De Filippis D, Esposito G, D'Amico A, Izzo A. The Spice Sage and Its Active Ingredient Rosmarinic
Acid Protect PC12 Cells from Amyloid-β Peptide-Induced Neurotoxicity. JPET 317(3) (2006) 1143-1149.
4. Reichling J, Nolkemper S, Stintzing FC, Schnitzler P. Impact of ethanolic lamiaceae extracts
on herpes virus infectivity in cell culture. Forsch Komplementmed. 15(6) (2008) 313-20.
5. Schwarz K. Antioxidative constituents of Rosmarinus officinalisand Salvia officinalis. III. Stability of phenolic
diterpenes of rosemary extracts under thermal stress as required for technological processes. Z. Lebensm. Unters.
Forsch. 195 (1992) 104-107.
6. Matalanis A, Jones G. O, McClements D. J. Structured biopolymer-based delivery systems for encapsulation,
protection, and release of lipophilic compounds. Food Hydrocolloid. (2011)
7. Mobus K, Siepmann J, Bodmeier R. Zinc–alginate microparticles for controlled pulmonary delivery of proteins
prepared by spray-drying. Eur J Pharm Biopharm. 81 (2012) 121–130.
8. Moebus K, Siepmann J. Bodmeier R. Novel preparation techniques for alginate–poloxamer microparticles
controlling protein release on mucosal surfaces. Eur J Pharm Sci. 45 (2012) 358–366.
9. Rochaa G. A. Favaro-Trindade C. S. Grosso C. R. F. Microencapsulation of lycopene by spray drying:
Characterization, stability and application of microcapsules. Food bioprod process. 90 (2012) 37–42.
10. Ezhilarasi P.N, Indrani D, Jena B.S, Anandharamakrishnan C. Freeze drying technique for microencapsulation of
Garcinia fruit extract and its effect on bread quality. J Food Eng. 117 (2013) 513–520.
11. Karthik P, Anandharamakrishnan C. Microencapsulation of Docosahexaenoic Acid by Spray-Freeze-Drying
Method and Comparison of its Stability with Spray-Drying and Freeze-Drying Methods. Food Bioprocess
Technol. (2013) 2780-2790.
12. Belscak-Cvitanovic A, Stojanovic R, Manojlovic V, Komes D, Cindric I. J, Nedovic V, Bugarski B.
Encapsulation of polyphenolic antioxidants from medicinal plant extracts in alginate–chitosan system enhanced
with ascorbic acid by electrostatic extrusion. Food Res Int. 44 (2011) 1094–1101.
13. Quispe-Condori S, Marleny D.A, Temelli F. Microencapsulation of flax oil with zein using spray and freeze
drying. Food Sci and Technol-LEB. 44 (2011) 1880-1887.

FABRICATION AND EVALUATION OF CLOTRIMAZOLE-LOADED PVP/HPβCD

NANOFIBER MATS FOR ORAL CANDIDIASIS
Prasopchai Tonglairoum1, Tanasait Ngawhirunpat1, Theerasak Rojanarata1, Ruchadaporn

Kaomongkolgit2 and Praneet Opanasopit1, *
1
Pharmaceutical Development of Green Innovations Group (PDGIG), Faculty of Pharmacy, Silpakorn University, Nakhon Pathom
73000, Thailand. (*corresponding author email: praneet@su.ac.th)
2
Department of Oral Diagnosis, Faculty of Dentistry, Naresuan University, Phitsanulok, Thailand.
KEYWORDS: clotrimazole, PVP, HPβCD, nanofibers, oral candidiasis
INTRODUCTION
Incidences of Oropharyngeal candidiasis (OPC) are increasing worldwide and are most common with
Candida albicans1). The clinical symptoms of oropharyngeal candidiasis can be sensations, altered taste,
and altered smell, which may affect the patient’s quality of life2). A number of effective antifungal drugs
could be used either topically or systemically for management of OPC. Clotrimazole (CZ) is a lipophilic
antimycotic drug with a broad spectrum utilized for treatment of fungal infections3-4) which is mainly used
PT – 35
in the management of superficial candidiasis. Nevertheless, it has poor aqueous solubility (0.49 µg/ml)5)
which might affect its antimycotic activity. Therefore, enhancement of solubility and release rate of the
CZ is necessary for a desirable and rapid antimycotic activity. Fast-dissolving drug delivery systems have
played an important role since their advantages, such as enhancing drug solubility, onset of action and
bioavailability6). Polyvinylpyrrolidone (PVP) is one of the polymers which can provide this feature as it
offers the possibility of ultrafast dissolution. Complexation of poorly soluble drugs with cyclodextrin is a
useful method to improve the dissolution and stability of the drugs7). Nanofibers are used in many fields
because they exhibits outstanding characteristics such as high porosity and specific surface area and very
small pore size8). Electrospinning is widely used to produce nanofibers because of simple and easy way to
control the morphology of ultrafine fibers9). In this work, CZ-loaded electrospun nanofiber mats using
HPβCD functionalized PVP as the filament-forming polymer were developed. The properties of the
electrospun nanofiber and the drug release characteristics were investigated. Moreover, the nanofiber
mats were also investigated for antifungal activity and cytotoxicity.

Material Clotrimazole, HPβCD, PVP (MW. ~1,300,000) and 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl
tetrazolium bromide (MTT), Sabouraud dextrose broth. Dimethyl sulfoxide (DMSO) were purchased
from various suppliers. The human gingival fibroblast (HGF) was obtained from the Faculty of Dentistry,
Naresuan University, Dulbecco’s modified Eagle’s medium (DMEM), trypsin-EDTA, penicillin-
streptomycin antibiotics and fetal bovine serum (FBS) were obtained from GIBCO-Invitrogen. All other
reagents and solvents were of analytical grade and used without further purification.
Electrospinning of CZ-loaded PVP/HPβCD nanofibers 8% PVP and 70 mM HPβCD were dissolved in
a solvent mixture of EtOH: H 2 O: BzOH with a volume 70:20:10. Clotrimazole (5%, 10%, 15% and
20%wt to polymer) was added into the mixture and stirred for 12 h at room temperature. The viscosity
and conductivity of these mixed solutions were determined. The electrospinning process was conducted at
25 °C with the fix applied voltage, the distance between the tip and the collector, and the feeding rate as
15 kV, 15 cm and 0.3 ml/h, respectively.
Characterization of CZ-loaded PVP/HPβCD nanofibers The morphology and diameter, chemical
structure, thermal behavior and physical status were investigated using scanning electron microscope
(SEM), Fourier Transform Infrared spectrophotometer (FT-IR), Differential scanning calorimeter (DSC)
and X-ray diffractometer (XPRD), respectively.
CZ content and In vitro release The total content of CZ in the CZ-loaded nanofiber mats and In vitro
release studies were determined in triplicate using HPLC. The In vitro release studies were modified from
the dissolution test which is official in USP (USP 35, 2011)10. Briefly, 5 mg of the CZ-loaded nanofiber
mats were placed in bottle containing 50 ml of 0.1 N HCl solution (pH 1.2) incubated at 37°C under
shaking at 150 rpm. After a given time, an aliquot of the release medium solution was analyzed.
Antifungal activity of the nanofiber mats
Susceptibility testing A broth microdilution method in accordance with the guidelines recommended by
CLSI (Clinical and Laboratory Standards Institute, 2002), using serial two-fold dilutions of CZ in
Sabouraud dextrose medium, was employed to determine susceptibility of the C. albicans and C.

dubliniensis to clotrimazole. Briefly, C. albicans and C. dubliniensis were incubated (104 CFU/ml) in 48
well plates for 48 h at 37 °C exposed to serial 2-fold dilution in the SDB culture medium of the CZ
solution (from 200 to 0.4 µg/ml).
Time-kill analysis The rate of killing of fixed inoculums of C. albicans and C. dubliniensis (usually 104
CFU/ml) is determined by incubating with agitation the test organism in SDB medium containing
different concentration of CZ form nanofiber mats, powder and lozenges (10 mg, Candinas
troche; Thailand Jan Laboratories). The aliquot of control and CZ containing medium were removed and
diluted at the 5, 15, 30, 60, 120 minute time point and then determining the survivor colony count
(CFU/ml). The kill curves are constructed by plotting the CFU/ml surviving at each time point in the
presence and absence of the nanofiber mats, CZ powder and lozenges.
Evaluation of cytotoxicity The cytotoxicities of the CZ and CZ-loaded nanofiber mats were evaluated
using an MTT cytotoxicity assay. The human gingival fibroblast cells (HGF) which obtained form
explants of gingival tissue attached to non-carious, freshly extracted third molar from three patients were
used. The HGF cells were plated in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 1% non-
essential amino acids and 0.1% penicillin-streptomycin and were distributed at a density of 10,000
cells/well in 96-well plates. The cells were grown under humidified atmosphere until confluency. The
cells were treated with CZ at various concentrations ranging from 25 to 400 µg/ml in serum-free medium
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and incubated for 24 h. For the CZ-loaded nanofiber mats, cytotoxicity test performing based on a
procedure adapted from the ISO10993-5 standard test method (indirect contact). The CZ (0-20%)-loaded
nanofibers were sterilized by UV radiation for 1 h and then immersed in a serum-free medium in an
incubator for 24 h to produce extraction media of varying concentrations. The tested extraction media at
varying concentrations were replaced and the cells were re-incubated for 2 h and 24 h. After treatment,
the serum-free medium containing CZ and the tested extraction solutions were removed. Finally, the cells
were incubated with 100 µl of an MTT-containing medium (1 mg/ml) for 4 h. The medium was removed,
the cells were rinsed with a phosphate buffer (pH 7.4), and the formazan crystals formed in the living
cells were dissolved in 100 µl DMSO per well. Relative viability (%) was calculated based on the
absorbance at 550 nm determined using a microplate reader.
Statistical analysis All experimental measurements were collected in triplicate. The values are expressed
as the mean ± standard deviation (SD). The statistical significance of the differences in each experiment
was examined using one-way analysis of variance (ANOVA), followed by a least significant difference
(LSD) post hoc test. The significance level was set at p < 0.05.

PVP/HPβCD nanofiber mats Electrospinning of PVP/HPβCD solution containing various amounts of
CZ (0, 5, 10, 15 and 20%wt CZ to polymer) were carried out from EtOH:H 2 O:BzOH (70:20:10). The
average fiber diameter of nanofiber electrospun from solution containing CZ content as 0, 5, 10, 15 and
20% wt to polymer was 665, 663, 667, 657 and 645 nm, respectively. Thus, high amount of CZ can be
incorporated in nanofiber mats without any effect on the fiber diameter.
Figure 1 The SEM image (1,000x) of the PVP/HPβCD nanofiber mats with a different CZ loading amount; a) 5, b) 10, c) 15 and d)
20%wt CZ to polymer
The FT-IR spectrum show that pure CZ powder exhibits dominant absorption peaks at 1,586.7, 1,490.7,
and 1,304.9 cm–1 corresponded to the benzene ring stretching. The band at 904.7, 823.68, and 744.59
cm–1 are assigned to the C-H stretching. The band 1,081.4 cm–1 and 1,210.1 cm–1 correspond to the
chlorobenzene and C-N stretching, respectively. The peak that was observed in the CZ powder spectrum
was also observed in spectra of the 5-20% CZ-loaded nanofiber mats. Thus, CZ was well incorporated
into the nanofiber mats.
The thermogram of CZ powder exhibits an endothermic sharp peak at 145.13 °C due to melting
temperature of CZ. The endothermic curves of the pure nanofiber mat (0% CZ) were also observed at

167.38 °C. The melting point slightly increased to 173.59, 177.12, 178.12 and 186.49 °C when the
concentration of CZ increased to 5, 10, 15 and 20%, respectively. The absence of detectable crystalline
domain, even at high content of CZ, indicates that CZ was incorporated in PVP/HPβCD nanofiber mats in
the amorphous state. These results are in accordance with XRPD result which showed that no peak is
found in the diffractograms of nanofiber mats containing various amounts of CZ.
Drug content and release studies The entrapment efficiency of CZ in the nanofiber mats was 92.60-
96.85%, which shows the excellent incorporation of CZ in the nanofiber mats. An increasing of the initial
amount of CZ resulted in an overall increase in the amount of CZ entrapped in the nanofiber mats. The
loading capacity of CZ in the nanofiber mats was increased from 0.046 to 0.161 mg/mg of nanofiber
when increasing the initial amount of CZ from 5% to 20% wt to polymer.
PT – 35
Figure 2 Release profiles of CZ from the CZ-loaded PVP/HPβCD nanofiber mats with different amounts of CZ: () 5%, () 10 %,
() 15 % and () 20 % to polymer. The data are expressed as mean ± standard deviation from three independent experiments. *
Statistically significant (P<0.05).
The release characteristics of CZ from the CZ-loaded PVP/HPβCD nanofiber mats with different
percentages of CZ are shown in Figure 2. It was observed that the CZ was rapidly released from the CZ-
loaded electrospun nanofiber mats. After 30, 60 ,60 and 120 second, all of CZ contained in the 5, 10, 15
and 20% CZ-loaded nanofiber mats were released into dissolution medium respectively. The fast release
characteristic of CZ from the nanofibers mat is due to the extremely high specific surface area and
porosity, which promoting a remarkably fast drug release, and the ability of PVP nanofiber to provide a
fast-dissolving hydrophilic environment. Moreover, the electrospinning process changes the crystalline
stage of CZ into amorphous state which facilitates dissolution of the drug in dissolution medium.
Antifungal study The MIC and MFC values of CZ are obtained from susceptibility testing using a broth
dilution assay. It is evident that CZ is strongly active against C. albicans and C. dubliniensis with the
MIC value of 12.5 and 6.25 µg/ml, respectively; besides, the MFC were 25 and 12.5 µg/ml, respectively.
Figure 3 Time kill plot of a) C. albicans and b) C. dubliniensis. (CFU/ml) versus the treatment time: () the control, () 5 mg of
20% CZ-loaded PVP/HPβCD nanofiber mats, () 20% CZ-loaded PVP/HPβCD nanofiber mats equivalent to CZ 1 mg/ml, () CZ
powder equivalent to CZ 1 mg/ml and () CZ lozenges equivalent to CZ 1 mg/ml. The data are expressed as mean ± standard
deviation from three independent experiments. * Statistically significant (P<0.05).

The antifungal activity of CZ loaded nanofiber mats against C. albicans and C. dubliniensis was tested by
counting the viable bacterial cells that rested in a Candida suspension after the contact of the nanofiber
mats with the suspension. About 104 CFU/ml of the strain was exposed to the nanofiber mat with different
CZ loading; furthermore, the CZ-loaded nanofiber mat which gives a final concentration of CZ equivalent
to 1mg/ml in Candida suspension was also tested for time kill assay in comparison to the CZ powder and
the lozenges at the same concentration of CZ. The CZ-loaded nanofibers have ability to reduce the
Candida and killed within 120 min of contact. In contrast, the pure PVP/HPβCD nanofiber did not reduce
the Candida growth. In comparison to the CZ powder and CZ lozenges, the CZ-loaded nanofiber which
gives the same final CZ concentration can significantly reduce exhibit faster activity to kill the Candida
than CZ powder and the lozenges (Figure 3) due to a very fast disintegration of the nanofiber and the
amorphous state of CZ in nanofiber which can fast and easy dissolve than the crystalline of the CZ
powder. The CZ lozenges must take time to disintegrate and follow by dissolving; thus, it also shows
slow antifungal activity. The fast antifungal activity of the CZ-loaded PVP/HPβCD nanofiber mats render
them promising for oral candidiasis application.
Cytotoxicity evaluation The cytotoxicity effect of various concentrations of CZ and the extraction
medium from 0-20% loaded nanofiber mats in HGF cells were determine as % viability (Figure 4a). A
concentration-dependent cytotoxicity of the CZ was observed for a 24-h incubation period. The IC 50
PT – 35
values, indicating 50% cell deactivation, of CZ were greater than 25 µg/ml. For the CZ-loaded nanofiber
mats, There was a significant decrease in cell viability when the HGF cells were incubated with the higher
loading amount of CZ in the nanofiber mat (0-20%) when compared to control (p<0.05).
Figure 4 The percentage cell viability in HGF cells of the CZ loaded PVP/HPβCD nanofiber mats containing 0, 5, 10, 15 and
20%wt CZ to polymer: a) incubated at () 2 h and () 24 h and b) incubated at 2 h in HGF cells derived from 3 patients: ()
patient #1, ( ) patient #2 and ( ) patient # 3. Each value represents the mean ± standard deviation of five wells. * Statistically
significant (P<0.05).
The cytotoxicity was observed after 24-h incubating with the extraction media of the nanofiber mats
containing high content of CZ (15–20 % wt. to polymer) (Figure 4a). However, the cytotoxicity of CZ
was reduced after incorporated in nanofiber mat. For the 2-h incubates the extraction media of the
nanofiber mats with different concentration of CZ, the cell viability remained similar to that of the control
cells in all concentrations. The result showed no significant difference in cytotoxicity of the CZ-loaded
nanofiber mats in HGF cells derived from three different patients as shown in Figure 4b.
CONCLUSION
The CZ-loaded PVP/HPβCD nanofiber mats were successfully prepare by electrospining using EtOH:
H 2 O: BzOH with a ratio of 70:20:10 as a solvent system. An increasing of CZ content did not affect on
the diameter of the nanofiber mats. The rapidly released CZ from the CZ-loaded electrospun nanofiber
mats were achieved. The nanofiber mats exhibit faster antifungal activity than CZ powder and CZ
lozenges. Moreover, the cytotoxicity of CZ was reduced after incorporated in nanofiber mat. Hence, these
nanofiber mats may be promising for oral candidiasis application.
ACKNOWLEDGEMENTS
The authors would like to acknowledge Commission of Higher Education (Thailand) and the Thailand

Research Funds through the Golden Jubilee Ph.D. Program (Grant No.PHD/0092/2554) and the Silpakorn
University Research and development institute for the financial support.
REFERENCES
1. Arendorf TM, Walker DM. The prevalence and intra-oral distribution of Candida albicans in
man. Arch Oral Biol. 1980; 25(1): 1-10.
2. Richart PA, Samarayake LP. Philipsen HP, Pathology and chinical correlates in oral candidiasis
and its variants: a review. Oral Dis. 2000; 6(2): 85-91
3. Ahmed MO, EI-Gibaly I, Ahmed SM. Effects of cyclodextrins on the physicochemical
properties and antimycotic activity of clotrimazole. Int J Pharm. 1998; 171: 111-121.
4. Pedersen M, Bjerregaard S, Jacobsen J, Sorensen AM. A genuine clotrimazole-cyclodextrin
inclusion complex-isolation, antimycotic activity, toxicity and an unusual dissolution rate. Int J
Pharm. 1998; 176(1): 121-131.
5. Hoogerheide JG, Wyka BE. Clotrimazole. Analytical Profiles of Drug Substances. F Klaus,
Academic Press. 1982; 11: 225-255.
6. Seager H. Drug-delivery products and the Zydis fast-dissolving dosage form. J. Pharm.
Pharmacol. 1998; 50(4): 375-382.
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7. Albers E, Muller BW. Cyclodextrin derivatives in pharmaceutics. Crit Rev Ther Drug Carrier
Syst. 1995; 12(4): 311–337.
8. Matthews JA, Wnek GE, Simpson DG, Bowlin GL. Electrospinning of collagen nanofibers.
Biomacromolecules. 2002; 3(2): 232-238.
9. Li D, Xia Y. Electrospinning of Nanofibers: Reinventing the Wheel? Adv Mater. 2004. 16(14):
1151-1170.
10. The United States Pharmacopoeia, 2011. 35 Reissue, The National Formulary 30, USP
Convention, INC. Rockville, MD.

PHYSICOCHEMICAL PROPERTIES OF PSEUDOLATEX SYSTEMS PREPARED FROM

PARA RUBBER SHEET AND PRELIMINARY DRUG LOADING FOR DRUG DELIVERY
Wiwat Pichayakorn1, 2, *, Rungtiwa Waiprib1, Isara Lueanpaen1, Jirapornchai Suksaeree1,
and Wirach Taweepreda2,3
1
2
Medical Products Innovations from Polymers in Clinical Use Research Unit, Prince of Songkla University, Songkhla 90112, Thailand.
3
Department of Materials Science and Technology, Faculty of Science, Prince of Songkla University, Songkhla 90112, Thailand.
*
Corresponding author: wiwat.p@psu.ac.th
KEYWORDS: Pseudolatex, Para rubber sheet, Drug delivery
INTRODUCTION
Applying the products onto the skin is the simplify method and much attractive for both topical and
systemic approaches in drug and cosmetic delivery. Many types of particulated systems, such as
liposomes, polymeric micelles, micro/nanoemulsions, and polymeric micro/nanoparticles have been
developed to gain the different formulations such as conventional topical products and the novel dosage
forms as reservoir-type transdermal patches or film forming polymeric solutions[1]. Some of these
PT – 36
particulated carriers can also control the release and permeation rates of active ingredients into the deeper
layers of the skin or into the blood circulation[2]. Pseudolatex system, the colloidal aqueous dispersions of
hydrophobic polymers, is one of these particulated carriers that can also be used in the same purpose. In
general, pseudolatex has been prepared from any existing thermoplastic water-insoluble polymers such as
several types of acrylate polymers[3,4], ethyl cellulose[5], and cellulose acetate[6]. Oil-in-water
emulsification is the common technique to prepare these pseudolatexes as aqueous polymeric
dispersions[3,6].
Cis 1,4-polyisoprene, the natural hydrophobic polymer obtained from the tapping process of the bark of
Para rubber tree (Hevea brasiliensis), has much interesting characteristics such as excellent elasticity and
flexibility, and ease for film forming. Our research group has successfully developed several products
from this polymer to use in transdermal drug delivery, such as matrix films[7-10], reservoir patches[11], and
film forming polymeric solution[12]. The initial raw material to prepare these products is fresh natural
rubber latex. However, this fresh latex cannot be kept in several days for commercially due to its
instability since the droplet coagulation and microbial contamination are the main stability problems.
Normally, in Thailand, this polymer is primary transformed into the Para rubber sheet for commercial.
However, there is no report to use Para rubber sheet as raw material in drug delivery. Therefore, this
study aimed to preliminary prepare the pseudolatex systems from Para rubber sheet for drug delivery and
to investigate the effects of various parameters in preparation process on properties of pseudolatexes.
Lastly, the feasibility of drug loading in these pseudolatex systems was evaluated.

Materials Para rubber sheet was kindly gifted from the farmer in Songkhla province, Thailand. The other
chemicals and model drugs were pharmaceutical or analytical grade and used without further
modification.
Preparation of drug-free pseudolatex systems Organic phase and aqueous phase were separately
prepared. A 3.5 g of Para rubber sheet was dissolved in 200 mL dichloromethane and mixed with 4%
dibutyl phthalate (DBP) and 2-8% mineral oil, and used as organic phase. In the other phase, surfactants
(5-15% Tween80 or 1-2% sodium lauryl sulfate [SLS]), thickeners (0.16-0.30% hydroxypropyl
methylcellulose [HPMC] or 0.08-0.24% methylcellulose [MC]), and preservative (2% Uniphen P-23)
were dissolved in distilled water to the final volume of 100 mL. Then, the aqueous phase was poured into
the organic phase under a high speed homogenizer (IKA, Germany) at ambient temperture to obtain oil-
in-water emulsions. The speed and time of homogenization were varied in the ranges of 12,000, 16,000,
20,000 rpm and 10, 15, 20 minutes, respectively. Finally, the oil-in-water emulsion was evaporated by
rotary evaporator to remove the dichloromethane. The formed pseudolatex was filled in the well-close
container and kept at ambient temperature for further evaluations. The pseudolatex formulations are
presented in Table 1.
Preparation of drug loaded pseudolatex systems Five model drugs were separately loaded in the selected
pseudolatex base formulation. For water-insoluble drugs, either indomethacin or lidocaine was dissolved
in the organic phase before emulsification process. For water soluble drugs, in contrast, either propranolol
HCl, lidocaine HCl, or vitamin C was dissolved in the water phase before emulsification process. Then,

both phases were further mixed and pseudolatex systems were formed according to the selected optimal
condition.
Physicochemical characterizations of pseudolatex systems The pseudolatex systems were optically
observed for their appearances and redispersibilities. Particle size was measured by a laser particle size
analyzer (Coulter, USA). Particle shape was studied under a light microscope (Olympus, Japan). The pH
value was performed by a pH meter (Mettler Toledo, Switzerland). Rhelogical behavior was measured by
a programmable rheometer (Brookfield DV-III Ultra, Brookfield Engineering Laboratories Inc., USA)
fitted with a spindle SC4-31 while set at different spindle speeds at 50-250 rpm, and the viscosity was
calculated. The protein remaining in pseudolatex systems was analyzed as total nitrogen content by
Kjeldahl method[13].
Table 1 Formulations of pseudolatex bases prepared with various parameters.
Formulations (%)
Parameters
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15
Para rubber
3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5
sheet
PT – 36
DBP 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
Mineral oil 2 4 2 4 2 4 2 2 4 6 4 4 4 4 6
Tween80 15 15 10 10 5 5 - - - - - - - - -
SLS - - - - 1 1 2 1 1 1 1 1 1 1 2
HPMC 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.30 - - - -
MC - - - - - - - - - - - 0.08 0.16 0.20 0.24
Uniphen P-23 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Effects of compositions on the properties of drug-free pseudolatex systems
Amount of Para rubber sheet In preliminary step, the Para rubber sheet could dissolve in
dichloromethane but only in a limited level that might affect the evaporation process in the last step.
Higher amount of Para rubber sheet required the higher amount of dichloromethane, then, it took more
time in preparation process and increased the production cost. The 3.5 g of Para rubber sheet could be
dissolved in 200 mL dichloromethane that be an optimum level of organic solvent used and the
appropriate time to evaporate it. Therefore, the formulations P1-P15 were then developed (Table 1). The
fixed preparation parameters at 20,000 rpm homogenization speed and 10 minutes homogenization time
were used.
Type and amount of surfactants Tween80 was firstly chosen as surfactant because it is commonly used
in oil-in-water emulsion formulations in pharmaceuticals. In this study, however, it caused the
aggregation of rubber polymer after evaporation process. Although the level of Tween80 was reduced
from 15% (P1-2) to 10% (P3-4) and 5% (P5-6), the un-appropriate products were also formed. Therefore,
SLS was then used instead of Tween80 as showed in P7-15. It was found that 1-2% SLS could be formed
the appropriate pseudolatex systems. Higher amount of SLS revealed the higher smooth dispersion.
Amount of mineral oil Mineral oil amount also affected the pseudolatex stability (P8-10). Increase
amount of mineral oil revealed more stable systems that took the longer time to phase separation occur.
Type and amount of thickeners Both HPMC and MC could stabilize the obtained pseudolatex systems.
The higher amount of thickeners revealed more stable systems. Moreover, MC resulted in the higher
product stability than HPMC formulations.
In this study, therefore, the P15 was chosen for the further studied.
Effects of preparation parameters on the properties of drug-free pseudolatex systems
Homogenization speed All the P15 formulations prepared by different homogenization speeds of 12,000,
16,000, and 20,000 rpm, and the homogenization time of 10 minutes provided the similar appearance of
pseudolatex systems. The white-color polymeric dispersions without any aggregation were obtained.
After 2 months of storage at ambient temperature, however, the separation of upper white emulsion and
lower clear slight-yellowish solution occurred that could be easily dispersed by hand-shaking. The
viscosity behaviors of all formulations revealed the Newtonian properties. The pH, percentage of
separation level after storage, particle size and viscosity before and after storage are shown in Figure 1

(top). The pH values were not different, the %separation and droplet size slightly decreased, and the
viscosity slightly increased in increasing homogenization speed. After storage, the droplet size and
viscosity also decreased.
Homogenization time All the P15 formulations prepared by different homogenization times of 10, 15,
and 20 minutes, and the homogenization speed of 20,000 rpm also provided the similar appearance of
pseudolatex systems. The results also provided the similar trends. The pH, percentage of separation level
after storage, particle size and viscosity before and after storage are shown in Figure 1 (bottom). The pH
values were not different, the %separation and droplet size slightly decreased, and the viscosity slightly
increased in increasing homogenization time. After storage, the droplet size and viscosity also decreased.
The deviation of all data at 15 minutes homogenization time was the lowest. In this study, therefore, the
optimum preparation parameters at 20,000 rpm homogenization speed and 15 minutes homogenization
time were chosen for the further studied.
PT – 36
Figure 1 Effects of (top) homogenization speed and (bottom) homogenization time on the properties of drug-free pseudolatex
systems ( ) at initial and ( ) after storage at ambient temperature for 2 months.
Effects of stabilizers on the properties of drug-free pseudolatex systems at the optimum preparation
parameters
SLS amounts The 1, 1.5, and 2% SLS were further confirmed again in the optimum preparation
parameters. The pH, percentage of separation level after storage, particle size and viscosity before and
after storage are shown in Figure 2 (top). The %separation and droplet size slightly decreased, and the pH
and viscosity slightly increased in increasing SLS amounts. After storage, the droplet size and viscosity
also decreased. The 2% SLS obtained the highest pseudolatex stability.
Mineral oil amounts The 4, 6, and 8% mineral oil were further confirmed again in the optimum
preparation parameters. The pH, percentage of separation level after storage, particle size and viscosity
before and after storage are shown in Figure 2 (bottom). The pH values were not different, but the
%separation and droplet size slightly decreased, and the viscosity significantly increased in increasing
mineral oil amounts, especially in 8% that could not determine in the same condition due to the very high
viscosity. After storage, the droplet size and viscosity also decreased. The 6% mineral oil obtained the
highest pseudolatex stability.
Therefore, the best pseudolatex formulation contained 3.5% block rubber, 0.24% MC, 6% mineral oil, 4%
DBP, 2% SLS, and 2% Uniphen P-23 using the speed and time of homogenizer as 20000 rpm and 15
minutes, respectively. The protein content of this best drug-free pseudolatex system decreased from
2.56% to 0.32. This indicated that the risk of contact allergy of this product caused by the protein
allergens could decrease. The droplet morphology under optical microscope presented as the multiple
emulsion with various size distributions (figure was not shown).

PT – 36
Figure 2 Effects of amounts of (top) SLS and (bottom) mineral oil on the properties of drug-free pseudolatex systems ( ) at
initial and ( ) after storage at ambient temperature for 2 months.
Drug-loaded pseudolatex systems

Only 2% lidocaine could be loaded into the pseudolatex system with the good physical appearances. The
white stable dispersion was obtained. The particle size of lidocaine loaded pseudolatex (26.73±11.87 µm)
was not different from the pseudolatex base (27.54±0.23 µm) but with larger size distribution. The pH
increased to 9.12±0.03 due to the basic properties of drug. The viscosity quite increased and its behavior
changes to pseudoplastic flow that the viscosity decreased when the shear force increased. Unfortunately,
the 1, 2, and 4% indomethacin and vitamin C were also loaded into the pseudolatex systems, but the
aggregation of drug and polymer droplets was found especially in the higher drug amounts due to their
acidic properties that incompatible to rubber polymer. Propranolol HCl and lidocaine HCl precipitated in
the pseudolatex systems and some polymer aggregations occurred. These might be due to the complex
incompatibility between HCl salts and SLS resulting in the lower stabilizer activity. Therefore, the
property of drug was the important factor that should be considered for loading into this pseudolatex
system.
CONCLUSION
Pseudolatex systems could be developed from Para rubber sheet. The preparation parameters in both
compositions and emulsification process directly affected the properties of pseudolatex systems. Some
drugs could be loaded into this system to form the good properties. However, many drugs could not be
applied in this system. Therefore, it should be further studied to find out the flexible condition for use this
system in widely types of drug delivery.
ACKNOWLEDGEMENTS
The authors would like to express their gratitude to the Faculty of Pharmaceutical Sciences, Prince of
Songkla University, for financial supports.
REFERENCES
1. Schröeder IZ, Franke P, Schaefer UF, et al. (2007) Delivery of ethinylestradiol from film
forming polymeric solutions across human epidermis in vitro and in vivo in pigs. J Controlled
Release 118: 196-203.
2. Barry BW (2001) Novel mechanisms and devices to enable successful transdermal drug delivery.
Eur J Pharm Sci 14: 101-114.
3. Quintanar-Guerrero D, Allémann E, Fessi H, et al. (1999) Pseudolatex preparation using a novel
emulsion–diffusion process involving direct displacement of partially water-miscible solvents by
distillation. Int J Pharm 188: 155-164.

4. Panigrahi L, Pattnaik S, Ghosal SK (2005) The effect of pH and organic ester penetration
enhancers on skin permeation kinetics of terbutaline sulfate from pseudolatex-type transdermal
delivery systems through mouse and human cadaver skins. AAPS PharmSciTech 6: 167-173.
5. Morales ME, Ruiz MA, López G, et al. (2010) Development of oral suspensions of
microparticles of ethylcellulose with tramadol. Drug Dev Ind Pharm 36: 885-892.
6. Sastry SV, Wilber W, Reddy IK, et al. (1998) Aqueous-based polymeric dispersion: preparation
and characterization of cellulose acetate pseudolatex. Int J Pharm 165: 175-189.
7. Suksaeree J, Boonme P, Taweepreda W, et al. (2012) Characterization, in vitro release and
permeation studies of nicotine transdermal patches prepared from deproteinized natural rubber
latex blends. Chem Eng Res Des 90: 906-914.
8. Pichayakorn W, Suksaeree J, Boonme P, et al. (2012) Preparation of deproteinized natural rubber
latex and properties of films formed by itself and several adhesive polymer blends. Ind Eng
Chem Res 51: 13393-13404.
9. Pichayakorn W, Suksaeree J, Boonme P, et al. (2012) Deproteinized natural rubber
latex/hydroxypropyl methylcellulose blending polymers for nicotine matrix films. Ind Eng Chem
Res 51: 8442-8452.
PT – 36
10. Pichayakorn W, Suksaeree J, Boonme P, et al. (2012) Nicotine transdermal patches using
polymeric natural rubber as the matrix controlling system: Effect of polymer and plasticizer
blends. J Membr Sci 411-412: 81-90.
11. Pichayakorn W, Suksaeree J, Boonme P, et al. (2013) Deproteinised natural rubber used as a
controlling layer membrane in reservoir-type nicotine transdermal patches. Chem Eng Res Des
91: 520-529.
12. Pichayakorn W, Suksaeree J, Boonme P, et al. (2013) Deproteinized natural rubber film forming
polymeric solutions for nicotine transdermal delivery. Pharmaceut Dev Tech 18: 1111-1121.
13. ASTM International (2001) Standard test method for rubber-nitrogen content D3533. West
Conshohocken, Pennsylvania, United States.

Session 4
Clinical Pharmacy CP 1 - 4
TIME-KILL STUDY OF THE IN VITRO ACTIVITY OF TIGECYCLINE AGAINST

CARBAPENEM-RESISTANT KLEBSIELLA PNEUMONIAE
Maimun Halee1, Wanchai Treyaprasert1, Pornpan Koomanachai2, Duangdao Waywa2

1
Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
2
Division of Infectious diseases and Tropical Medicine, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol
University, Bangkok 10700, Thailand
KEYWORDS: Tigecycline, carbapenem resistant Klebsiella pneumoniae, time-kill study
INTRODUCTION
Carbapenem-resistant Klebsiella pneumoniae (CRKP) has been disseminated worldwide. Infections caused
by CRKP have been associated with high mortality rates.(1) The prevalence of CRKP isolated from clinical
samples continues to increase globally.(2) According to a recent survey, there was high prevalence of
CRKP in clinical isolates from several hospitals in Thailand.(3) CRKP was identified as a causative agent
of pneumonia, causing death in Thai patients.(4) Tigecycline is a new glycylcycline antimicrobial agent
with broad spectrum activity. The activity of tigecycline extends to clinically relevant, susceptible and
multidrug-resistant strains of K. pneumoniae(5) including CRKP.(6) Currently, there are few reports about
the in vitro activity of tigecycline against CRKP. The aim of this study was to evaluate, by time-kill
study, the in vitro activity of tigecycline against clinical isolate of CRKP.

Drugs and microorganisms Tigecycline was provided by Pfizer Inc., Groton, CT, USA. The CRKP
clinical isolate was obtained from the Faculty of Medicine, Siriraj Hospital, Thailand.
Media preparation Cation-adjusted Mueller-Hinton Broth (CaMHB: Fluka, Bucsh, Switzerland) and
Mueller-Hinton agar (MHA: Oxoid Ltd, Basingstoke, Hamsphire, England) were prepared according to
the package inserts.
MIC determination The MIC is defined as the lowest concentration of antimicrobial agent that
CP - 1
completely inhibits the growth of the organism as detected by the unaided eye. The tests were performed
using fresh CaMHB (<12 h old)(7) by the macrodilution method.(8)
Time-kill study Time-kill study of CRKP was generated after exposure of the microbe to tigecycline in an
in vitro kinetic model. This model was used to investigate the antibacterial efficacy of constant drug
concentrations for 48 h. The model consisted of a 75 ml vented-cap tissue culture flask with a canted neck
(Corning Incorporated, NY, USA), containing 30 ml of CaMHB media incubated at 37◦C.
An aliquot of a suspension (100 µL) of initial inoculation (equivalent to 0.5 McFarland scale) was added
to the in vitro model. The model was incubated for 2 h before adding different tigecycline concentrations
to produce the log growth phase of bacterium.
Selection of the tigecycline concentrations for the test was based on their MIC values. These
concentrations were from 0.25 to 64 times of the MIC. A control experiment with bacteria and no drug
was run simultaneously. Samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 16, 24 and 48 h. Bactericidal activity
was defined as a reduction of ≥3 log 10 of total count of CFU/mL in the original inoculum. Bacteriostatic
activity was defined as maintenance of or reduction of <3 log 10 of the total CFU/mL in the original
inoculum.(9)
Bacterial quantification Bacterial counts were determined by plating 50 µL of the serial 10-fold dilutions
on MHA plates, using an adapted droplet-plate method.(8) Briefly, agar plates were divided into four
quadrants. With an automatic pipette, 5×10 µL droplets of the chosen dilution were equidistantly plated
onto one of the quadrants. A duplicate was plated onto the adjacent quadrant. Then, the plates were
incubated at 37◦C for 16-24 h before reading. The procedure was repeated at least three times per bacterial
strain and dose. Positive controls with bacteria but no drug were run simultaneously. Following
incubation, the number of CFUs was counted in each duplicate quadrant at each time-point and averaged.

RESULTS
The determined MIC values and tigecycline concentrations used in the time-kill study against CRKP are
summarized in Table 1. K. pneumoniae IF1526 was clinical isolates of CRKP. The range of tigecycline
concentrations tested in the time-kill study was from 0.25 to 64 µg/mL for CRKP.
Table 1 Tigecycline MIC values and concentrations tested in the time-kill study.
Type of organism Organism MIC (µg/mL) Tested concentrations (µg/mL)

Carbapenem-resistant K. pneumoniae IF1526 1 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64
Figure 1 Time-kill study of tigecycline against K. pneumoniae IF1526 (CRKP). Concentrations tested were 0.25×, 0.5×, 1×, 2×, 4×,
8×, 16× 32× and 64×MIC of tigecycline. Viable bacterial counts were determined over 48 h of incubation.
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The concentrations tested in time-kill study of tigecycline against K. pneumoniae IF1526 (CRKP)
covering the entire tigecycline range (minimum inhibition of bacterial growth, efficient bacterial killing
and maximum bacterial killing) are depicted in Figure 1. At concentrations of minimum inhibition of
bacterial growth (0.25×MIC, 0.5×MIC, and 1×MIC), the concentration at 1xMIC showed the inhibition of
bacterial growth but regrowth started after 8 h of incubation. As expected, efficient bacterial killing by
tigecycline (2×MIC and 4×MIC) showed its bacteriostatic activity which allowed regrowth after 12-24 h.
The concentrations of maximum bacterial killing (8×MIC, 16×MIC, and 32×MIC) showed bacteriostatic
activity without regrowth. Only concentration at 64×MIC showed a rapid bactericidal activity after 8 h
of incubation.
DISCUSSION
This is the first report on the in vitro activity of tigecycline against CRKP carried out on clinical isolate
from Thailand. In this study, we have determined MICs of tigecyline against CRKP. The MIC value was
1 µg/mL and the result was consistent with reported MIC.(6, 10) In time-kill study, tigecycline
demonstrated bacteriostatic effect against CRKP for most of the concentrations tested (2×, 4×, 8×, 16×
and 32×MIC). The result of this study confirmed that of Pournaras et al.(10) Our study has shown that
tigecycline produced bactericidal effect over 24 h with concentration at 64×MIC.
CONCLUSION
Tigecycline has good in vitro antibacterial activity against CRKP. The antimicrobial agent showed both
bacteriostatic and bactericidal activities, as well as time-dependent killing pattern.
ACKNOWLEDGMENT
This study was supported by a grant “THE 90TH ANNIVERSARY OF CHULALONGKORN
UNIVERSITY FUND (Ratchadaphiseksomphot Endowment Fund)” from Chulalongkorn University

REFERENCES
1. Gupta N, Limbago BM, Patel JB, Kallen AJ. Carbapenem-Resistant Enterobacteriaceae:
Epidemiology and Prevention. Clinical Infectious Diseases. 2011;53(1):60-7.
2. Nordmann P, Naas T, Poirel L. Global Spread of Carbapenemase-producing Enterobacteriaceae.
Emerging Infectious Diseases. 2011.
3. Trakulsomboon S, Pongparit S. Survey for carbapenemase-producing Klebsiella pneumoniae
isolated from clinical specimens in Thai hospitals. International Journal of Infectious Diseases.
2012;16, Supplement 1(0):e435.
4. Tribuddharat C, Prombhul S, Tulanont D, Aranya C, Bamrungsri N, Mekviwattanawong S. New
variant of an imipenemase, IMP-32, in a Klebsiella pneumoniae from a fatal case of Thai patient.
International Journal of Infectious Diseases. 2012;16, Supplement 1(0):e436.
5. Meagher AK, Ambrose PG, Grasela TH, Ellis-Grosse EJ. Pharmacokinetic/pharmacodynamic
profile for tigecycline-a new glycylcycline antimicrobial agent. Diagnostic Microbiology and
Infectious Disease. 2005;52(3):165-71.
6. Sader HS, Flamm RK, Jones RN. Tigecycline activity tested against antimicrobial resistant
surveillance subsets of clinical bacteria collected worldwide (2011). Diagnostic Microbiology
and Infectious Disease. 2013;76(2):217-21.
7. Petersen PJ, Jones CH, Bradford PA. In vitro antibacterial activities of tigecycline and
comparative agents by time-kill kinetic studies in fresh Mueller-Hinton broth. Diagnostic
Microbiology and Infectious Disease. 2007;59(3):347-9.
8. Treyaprasert W, Schmidt S, Rand KH, Suvanakoot U, Derendorf H.
Pharmacokinetic/pharmacodynamic modeling of in vitro activity of azithromycin against four
different bacterial strains. International Journal of Antimicrobial Agents. 2007;29(3):263-70.
9. National Committee for Clinical Laboratory Standards (1999) Methods for determining
bactericidal activity of antimicrobial agents; approved guideline. Wayne, PA: National
Committee for Clinical Laboratory Standards; 1999 (NCCLS document M62A).
10. Pournaras S, Vrioni G, Neou E, Dendrinos J, Dimitroulia E, Poulou A, et al. Activity of
tigecycline alone and in combination with colistin and meropenem against Klebsiella
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pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae strains by time–kill assay.
International Journal of Antimicrobial Agents. 2011;37(3):244-7.

LAXATIVE EFFECTIVENESS OF CASSIA ANGUSTIFOLIA IN

THAI CONSTIPATED PATIENTS
Peeranuch Mangmeesri1, 3, Kant Wongsuphasawat1, *, Wandee Gritsanapan2, * and Wit Viseshsindh3

1
School of Anti-Aging and Regenerative Medicine, Mae FahLuang University, Bangkok
2
Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok
3
Department of Surgery, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok
KEYWORDS: Cassia angustifolia, Anthraquinone, Constipation
INTRODUCTION
Cassia angustifolia Vahl., or senna, is among the most commonly used herbal laxatives and has been used
for medicinal purposes for centuries. Senna pods are more stable and produce less spasmodic pain side
effect than the leaves due to their lower content of free anthraquinones1,2). The active principle of
anthraquinone compounds such as aloe-emodin and its glycosides, physcion, rhein, rhein-8-glucoside,
sennosides A and B, which promote peristaltic movement of the large intestine, was reported to be
anthrone compound3). Although C. angustifolia has long been used for its laxative effect, no clinical
research has been performed in Thai people. The objective of this blinded, clinical controlled trial was to
test the laxative effectiveness of C. angustifolia in Thai patients with constipation.
METHODS
Patient Patients with a diagnosis of constipation who attended the outpatient clinic at Ramathibodi
Hospital, Mahidol University, Bangkok, Thailand during April to June 2013 were invited to participate in
the research project. Eligibility criteria included functional constipation as defined by Rome III criteria4).
Moreover, patients had to be older than 20 years. Patients taking concomitant medications that could
modify bowel habit were excluded. Patients with severe liver, renal or cardiac diseases, uncontrolled
diabetes mellitus or fecal incontinence, as well as pregnant women, were also excluded. The trial was
approved by the hospital’s ethics committee.
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Study drug Commercial C. angustifolia tablets containing 15 mg of total sennosides calculated as

sennoside B were used.
Instrument Daily stool chart indicated the frequency and quantity of stools, stool consistency, ease of
evacuation and the presence of other symptoms or side effects was used.
Research procedure Thirty six participants were recruited according to the eligibility criteria to join the
clinical study. All patients were assigned to five days of treatment with C. angustifolia tablets. Before entry into
the study, patients had three- to five-day period free of laxatives as a wash out period (for ethical reasons, the
maximum period without a bowel movement). Two tablets of C. angustifolia, blinded as drug “A”, were given
once daily before bedtime to each participant for 5 connective days. Patients followed their normal diet but
were asked to consume no product containing probiotics or prebiotics such as fermented milk or yogurt five
days prior to the on-study period.
Outcome measure All research participants were subjected to self-assessment report on daily stool chart,
indicating the stools frequency, quantity, stool consistency, the ease of evacuation and the presence of other
symptoms or side effects (including abdominal bloating, abdominal pain or cramps, nausea, wind or flatulence,
headache, anorexia and urgency) everyday during the research. The stool quantity can be estimated by
comparison of the stool with the chicken egg size. Stool consistency was rated using the Bristol Stool
Form (Figure 1). The stool evacuation was recorded by a scoring system (1-5 on a visual analogue scale
where 1 indicated very difficult and 5 indicated excellent results).
Statistical analysis Continuous data were shown as means and SDs. Categorical data were expressed as
percentages.
RESULTS
The results showed that most participants (48.33%) had stool frequency of 1 time/day, followed by the no
stool frequency group (26.11%), while the smallest group of participants (1.11%) had stool frequency of 3
times/day. The mean of the stool quantity was 2.19, while the mean of stool evacuation was 3.70. The stool
consistency was in type 4 (ideal stool) (27.01%). No adverse drug effect was found in most (73.33%) of the
participants.

Figure 1 Bristol Stool Form scale
Table 1 Stool frequency of the research participants
Number of
Stool frequency % of participant
participants
None 47 26.11
1 time/day 87 48.33
2 times/day 39 21.67
3 times/day 2 1.11
More than 3 times/day 5 2.78
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Total 180 100.00
Table 2 Stool consistency of the research participants
Type of stool Number of participants % of participant

Type 1 3 2.19
Type 2 28 20.43
Type 3 20 14.60
Type 4 37 27.01
Type 5 21 15.33
Type 6 19 13.87
Type 7 9 6.57
Total 137 100.00

Table 3 The adverse drug effects found in the research participants
Number of
Adverse drug effects % of participants
participants
None 132 73.33
Abdominal bloating 19 10.56
Abdominal pain or cramps 17 9.44
Nausea / vomiting 0 0
Headache 0 0
Anorexia 10 5.56
Muscular cramp 2 1.11
Total 180 100.00
DISCUSSION
Constipation is a very common problem that affects most people during their lives. The prevalence of
chronic constipation from Thai Motility Club study in Thai population is about 23.5%,5) which is very
high when compared to other functional bowel disorders. Although changing in lifestyle and diet are
suggested as the first choice of treatment for patients with constipation, those patients who still have
constipation need a laxative drug. Among the popular self-administered laxatives or drugs prescribed by
physician, C. angustifolia is one of the common laxatives used. Due to its natural origin, less toxicity,
and accessibility without a medical prescription, C. angustifolia is a popular laxative drug for constipation
treatment. However, no clinical research has been performed in Thai people. Therefore, this research was
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set up in order to use commercial C. angustifolia sold in Thailand as the laxative drug to treat Thai constipated
patients. From the results, C. angustifolia promoted moderate laxative effects in the patients. Therefore, C.
angustifolia can be considered as a laxative for the initial treatment. This study helps confirming the
laxative effectiveness of this herbal remedy and provides additional scientific support for this well-known
traditional medicinal plant.
REFERENCES
1. Gilman AG, Goodman LS, Gilman A. 1990. The Pharmacological Basis of Therapeutics, 6th ed,
Macmillan Publishing, New York.
2. Gritsanapan W. 2010. Ethnomedicinal plants popularly used in Thailand as laxative drugs. In
Chattopadhyay D. (Ed.), Research Signpost, 295-315.
3. Barnes J, Anderson LA, Phillipson JD. 2002. Herbal Medicines, 2nd ed, The Pharmaceutical
Press, London.
4. Drossman DA. 2006. Rome III: the new criteria. Chin J Dig Dis 7: 181-185.
5. Chonmaitree P, Udompunturak S, Leelakusolvong S. 2009. Efficacy and Safety of 28 days
Treatment with Sodium Phosphate Solution in Management of Chronic Constipation
(Preliminary Study). Thai J Gastroenterol 10 (1): 24-27.

PREDICTION EQUATION OF GLOMERULAR FILTRATION RATE DECLINE IN PATIENTS

WITH TYPE 2 DIABETES AND PRESERVED KIDNEY FUNCTION
AT MAHARAJNAKHONSITHAMMARAT HOSPITAL
Pliwphai Chaidecha Sukmak1, Titinun Auamnoy2, and Somratai Vadcharavivad1

1
Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences,Chulalongkorn University, Bangkok 10330, Thailand.
2
Department of Social and Administrative Pharmacy, Faculty of Pharmaceutical Sciences,
KEYWORDS: Type 2 diabetes, Preserved kidney function, Glomerular filtration rate, Prediction
INTRODUCTION
Chronic kidney disease (CKD) is one of the major complications founded in patients with type 2 diabetes
(T2DM) and is characterized by progressive deterioration in renal function (1). Early detection and
management of CKD has been suggested to prevent the major consequences including progression to
renal failure and cardiovascular complications (2). In clinical practice, the most widely used parameter for
evaluation of renal function loss is the decline in glomerular filtration rate (GFR). Some clinical factors
associated with GFR decline in T2DM patients with early-staged CKD or preserved kidney function have
been identified (3-5). However, these researches in Thai patients are limited. The aim of this study was to
determine the predictors of annual GFR decline in a cohort of Thai patients with T2DM and a baseline
GFR ≥ 60 ml/min/1.73 m2.

Study subjects The populations for this study were all T2DM patients from outpatient diabetes clinic of
Maharajnakhonsithammarat Hospital who came to see physicians during 2007-2012. One thousand
patients who met all inclusion criteria were randomly sampling by computerized method from the hospital
database. Individuals who had already been treated for T2DM with baseline GFR ≥ 60 mL/min/1.73 m2
were included in this study. Patients having non-diabetic renal diseases including glomerulonephritis,
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amputation, pregnancy, or incomplete medical records were excluded from the study. The study protocol
was reviewed for approval by the Maharajnakhonsithammarat Hospital Ethical Committee.
Measurements The retrospective medical record review were employed to obtain the associated clinical
and biochemical data. Hypertension, hyperlipidemia, and prior cardiovascular diseases including strokes,
myocardial infarction, coronary artery disease, and peripheral vascular disease, were defined by physician
diagnosis of each condition documented in the medical record. Non-fasting blood samples were obtained
for measurements of serum concentrations of creatinine, hemoglobin A1C, and lipids. Serum creatinine
was measured by using the Jaffe’s kinetic assay. Urinary albumin concentration was semi-quantified by
using a urine dipstick and classified into 2 groups thereafter including normoalbuminuria (< 30 mg/L) and
abnormal albuminuria (≥ 30 mg/L). Smoking status was recorded as current smoker and non-current
smoker. Blood pressure was measured by trained nurses using a mercury sphygmomanometer with an
appropriate cuff size at the left upper arm of patients after their resting for more than 5 minutes. All data
transferred from medical records were verified, validated and cleaned. Double visual check was
implemented as the verification method to ensure the accuracy of the data gathered. Data validation
including range check, logical check, and missing data check were as well performed to confirm that all
data were sensible. GFR was calculated by using the Thai equation recently generated by Praditpornsilpa,
et al: 375.5 × Cr(-0.848) × Age(-0.364) × 0.712 (if female) (6). According to the enzymatic method of serum
creatinine measurement used in this equation, Jaffe-measured serum creatinine in this study was
multiplied by correction factor 0f 0.906 prior to GFR calculation (7). Annual GFR decline for each patient
was calculated by the formula: GFR baseline − GFR follow-up / years of follow-up (5). Percentage of annual
GFR decline was calculated by the formula: annual GFR decline / GFR baseline × 100.
Statistical analysis Categorical data were presented as frequency and percentage while continuous data
were presented as mean and standard deviation (SD). Stepwise multiple regression analysis (MRA) was
performed to determine the prediction equation of annual GFR decline. The following variables were
considered for possible inclusion in the regression model: age, age of first diagnosis of T2DM, T2DM
duration, GFR, hemoglobin A1C, systolic blood pressure, diastolic blood pressure, serum total
cholesterol, serum LDL- cholesterol, serum HDL- cholesterol, serum triglyceride, abnormal albuminuria,
and current smoker. Four major assumptions of MRA including normality, linearity, homoscedasticity
and multicollinearity were formally inspected prior to model proceeding. Statistical analysis was

performed with SPSS 17.0 statistical package software. P values < 0.05 were considered statistically
significant.
RESULTS
Summary of study subjects Baseline characteristics of 1000 patients were shown in Table 1. All patients
had received ACEI/ARB. GFR was followed with the mean observation period of 5.3±0.1 years, ranging
from 5.0-5.6 years. In addition, there were no patients characterized by GFR increase during this
observational period.
Table 1 Baseline characteristics of T2DM patients with preserved kidney function (N=1000)
Parameter Frequency (%) Mean±SD (Range)

Age (years) - 59.4±4.4 (51/66)
Male 353 (35.3) -
Age of diabetes diagnosis (years) - 53.1±3.2 (50/65)
Duration of diabetes (years) - 6.3±3.7 (1/16)
OAD only/ insulin only/ OAD + insulin 808/120/72 (80.8/12.0/7.2) -
GFR at baseline (mL/min/1.73 m2) - 92.5±20.5 (66.7/157.2)
GFR at follow-up (mL/min/1.73 m2) - 72.1±18.8 (43.7/145.5)
GFR decline (mL/min/1.73 m2 per year) - 3.8±1.0 (1.0/6.8)
GFR decline (% per year) - 4.2±1.1 (1.1/7.6)
Abnormal albuminuria 907 (90.7) -
Hemoglobin A1C (%) - 7.8±0.8 (6.7/9.5)
Hemoglobin A1C < 7% 223 (22.3) -
Hypertension 974 (97.4) -
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Systolic blood pressure (mmHg) - 147.5±3.3 (139/153)

Diastolic blood pressure (mmHg) - 85.4±7.8 (70/99)
Hyperlipidemia 896 (89.6)
Total cholesterol (mg/dL) - 252.5 ±37.8 (172/367)
LDL-cholesterol (mg/dL) - 204.8 ±20.0 (176/237)
HDL-cholesterol (mg/dL) - 47.2 ±6.3 (36/60)
Triglyceride (mg/dL) - 229.9 ±40.4 (190/397)
Statin users 883 (88.3) -
Prior cardiovascular diseases 66 (6.6) -
Current smokers 85 (8.5) -
Abbreviations: OAD, oral antidiabetic drug
Prediction equation of annual GFR decline Stepwise MRA revealed that age and baseline GFR were the
significant predictors of the annual GFR decline with the adjusted R-square = 0.480. The regression
coefficient (B) of these two predictors was 0.141 (95% CI: 0.129-0.152) and 0.032 (95% CI: 0.030-
0.034), respectively and p<0.001 for both predictors. Furthermore, the standardized regression coefficient
(Beta) of these two predictors was 0.589 and 0.626, respectively. Thus, the prediction equation of the
annual GFR decline is presented as follows:
Annual GFR decline = 0.141 (Age) + 0.032 (baseline GFR) - 7.48

DISCUSSION
Prediction of GFR decline in patients with T2DM and preserved kidney function is of clinical importance
in order to improve the therapeutic strategies for early approach of CKD. In this long-term retrospective
study of a large cohort of patients with T2DM and preserved kidney function, clinical variables including
old age and high baseline GFR were significantly identified to be the predictors of the annual GFR
decline.
Most patients in this study cohort were characterized by old age, female predominance, poor control of
diabetes, hypertension, and hyperlipidemia, and also abnormal albuminuria. In addition, all patients had
received ACEI/ARB, the antihypertensive drug classes that provide a benefit for delaying the progression
of CKD (1-2). However, all patients were considered to be in the GFR decline stage of renal disease since
the absence of patients with increase in GFR during the period of follow-up (8).
High baseline GFR was the strongest predictor of the annual GFR decline as a result of its greatest Beta
value. From the prediction equation, it should be interpreted that the higher baseline GFR the patients
had, the faster annual GFR decline they got. Previous studies show that high baseline GFR is the
significant predictor of the annual GFR decline in T2DM patients with preserved kidney function.
Zoppini, et al. reported that baseline GFR is the independent predictor of the annual GFR decline (Beta =
-0.07, p<0.05) (3). The minus symbol of Beta reported in this study means that the higher baseline GFR
the patients have, the more decline of annual GFR they get. In addition, Babazono, et al. reported the
negative impact of baseline GFR on the annual GFR change (B = -0.060, Beta = -0.403, p<0.001) (4),
meaning that baseline GFR has a positive impact on the annual GFR decline. The cause of high GFR
leading to subsequent GFR decline is attributed to the glomerular hyperfiltration influenced by increase in
circulating nitric oxide as a resulted of hyperglycemia founded in patients with diabetes (10).
For old age, this study demonstrates a positive impact on the annual GFR decline, meaning that the older
age the patients had, the faster annual GFR decline they got. The significant association between age and
annual GFR decline in T2DM patients with preserved kidney function is reported in the previous
published data. Zoppini, et al. reported that age is the independent predictor of the annual GFR decline
(Beta = -0.07, p<0.05) (3). The minus symbol of Beta reported in this study means that the older age the
patients have, the more decline of annual GFR they get. Moreover, Meguro, et al. reported the positive
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impact of age on the annual GFR decline in patients with baseline GFR from 60 to less than 90
mL/min/1.73 m2 (Beta = 0.153, p<0.001) (5). The mechanisms involving age-related deterioration in
renal function include decrease in renal plasma flow and loss of filtration surface area with age (11).
Strengths of this study included the large number of patients in the study cohort and the long duration of
follow-up. The study also has some limitations. First, the availability of some relevant data may be
limited because of the retrospective design of this study. Second, selection bias may occur as a result of
the exclusion of individuals with incomplete medical data. Finally, some variables including albuminuria
and smoking habit were not fully quantified. Future studies with a complete quantification of these data
would be needed to further evaluate their effects on the annual GFR decline.
CONCLUSION
This study demonstrates that, in a large cohort of patients with T2DM and preserved kidney function who
were followed for at least 5 years, the annual GFR decline is strongly predicted by high GFR and old age.
ACKNOWLEDGMENTS
The authors are grateful to thank Dr. Kamol Khositrangsikun for the valuable suggestions. The authors
would also like to thank the medical record officers for their help with data collection.
REFERENCES
1. American Diabetes Association. Standards of medical care in diabetes--2013. Diabetes Care 36
(January 2013): S11-S66.
2. KDOQI Clinical Practice Guidelines and Clinical Practice Recommendations for Diabetes and
Chronic Kidney Disease. Am J Kidney Dis 49 (February 2007): S12-S154.
3. Zoppini, G.; et al. Predictors of estimated GFR decline in patients with type 2 diabetes and
preserved kidney function. Clin J Am Soc Nephrol. 7 (March 2012): 401-408.
4. Babazono, T.; et al. Lower haemoglobin level and subsequent decline in kidney function in type
2 diabetic adults without clinical albuminuria. Diabetologia. 49 (June 200): 1387-1393.
5. Meguro, S.; et al. Factors associated with the decline of kidney function differ among GFR strata
in subjects with type 2 diabetes mellitus. Int J Endocrinol. 2012 (2012): 687867.

6. Praditpornsilpa, K.; et al. The need for robust validation for MDRD-based glomerular filtration
rate estimation in various CKD populations. Nephrol Dial Transplant 26 (September 2011: 2780-
2785.
7. Stevens, L.A.; et al. Impact of creatinine calibration on performance of GFR estimating
equations in a pooled individual patient database. Am J Kidney Dis 50 (July 2007): 21-35.
8. Palmer, B.F. Screening tests for renal impairment in patients with type 2 diabetes: the what,
when, and how. Postgrad Med. 123 (January 2011): 7-14.
9. Yokoyama, H.; et al. Determinants of decline in glomerular filtration rate in nonproteinuric
subjects with or without diabetes and hypertension. Clin J Am Soc Nephrol 4 (September 2009):
1432-1440.
10. Chiarelli, F.; et al. Increased circulating nitric oxide in young patients with type 1 diabetes and
persistent microalbuminuria: relation to glomerular hyperfiltration. Diabetes. 49 (July 2000):
1258-1263.
11. Presta, P.; Lucisano, G.; Fuiano, L.; and Fuiano, G. The kidney and the elderly: why does the
risk increase? Int Urol Nephrol. 44 (April 2012): 625-632.
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PREDICTING THE DEVELOPMENT OF RENAL IMPAIRMENT IN TYPE 2 DIABETIC

PATIENTS WITH PRESERVED KIDNEY FUNCTION
Suchada Kittipanyaworakun1, Chaowarat Munprom2, Titinun Auamnoy3, Kearkiat Praditpornsilpa4 and

Somratai Vadcharavivad1
1
Department of Pharmacy Practice, Faculty of Pharmaceutical Science, Chulalongkorn University, Bangkok 10330 Thailand.
2
Department of Pharmacy, Saraburi Hospital, Saraburi, 86000 Thailand.
3
Department of Pharmacy Administration, Faculty of Pharmaceutical Science, Chulalongkorn University, Bangkok 10330 Thailand.
4
Division of Nephrology,Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok 10330 Thailand.
(Correspondence: Somratai Vadcharavivad, E-mail: somratai.r@chula.ac.th)
KEYWORDS: Type 2 diabetes mellitus, Renal impairment, Urinary albumin excretion, Glomerular
filtration rate, Predicting
INTRODUCTION
Diabetes mellitus (DM), especially type 2 diabetes, is the most common cause of end stage renal disease.
DM patients with renal impairment (GFR<60 ml/min/1.73 m2) are at greater risk of developing
cardiovascular disease leading to premature mortality1. Age, age at diabetes diagnosis, duration of
diabetes, glomerular filtration rate, HbA1C, total cholesterol, systolic blood pressure and albuminuria
have been reported to be associated with renal impairment in diabetes patients with preserved kidney
function2-7. Identification and assessment of individual risk factors for developing renal impairment may
be useful for more appropriate patient monitoring and treatment. To date, limited data has been available
on this subject regarding the Thai population. Therefore, this study aimed to develop an equation for
predicting the development of renal impairment in Thai type 2 diabetic patients.

Study design and participants This research was a retrospective cohort study. Patients were recruited
who were treated at the Outpatient Department at Saraburi Hospital during January 1, 2006-December 31,
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2007 with follow-up conducted until January 1, 2011-December 31, 2012. A total of 322 subjects with
type 2 diabetes mellitus (T2DM) according to ICD-10 identified from the hospital database aged 20 years
or older with baseline eGFR of 60 ml/min/1.73 m2 or greater and who received hypoglycemic agent for at
least 3 months were included. Subjects were excluded if they 1) were pregnant; 2) had limbs amputated;
3) had malignancy, kidney transplantation or kidney disease due to causes other than T2DM; or 4) had
incomplete variables data for analysis. This study was approved by the Ethics Committee of Saraburi
Hospital.
Parameters All parameters were established using information from the patient database and medical
records of Saraburi Hospital.
Demographic and clinical parameters: Information about age, gender, duration of diabetes, age at
diabetes diagnosis, comorbidities and blood pressure (BP) was collected. Treatment with angiotensin-
converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) was also recorded.
Biochemical parameters: Serum creatinine (Scr) was measured using Jaffe’s method in mg/dl.
Hemoglobin A1C (HbA1C) was measured using immunoturbidimetric method in percentage. Total
cholesterol (TC) was measured using enzymatic colorimetric method in mg/dl. Urine albumin was
measured using microalbumin dipstrip (Micral test-II®) in mg/l and was categorized as normal and
increased urinary albumin excretion. Increased urinary albumin excretion (UAE) was defined as urine
albumin concentration 20 mg/l or greater. These measurements were all collected at baseline.
Definition of evaluated outcome Development of renal impairment (RI) was evaluated at the end of the
study (about 5 years after baseline) which was defined as estimated GFR (eGFR) < 60 ml/min/1.73 m2 at
follow-up2. The eGFR was calculated using Thai eGFR equation8 as follows: 375.5 x Cr Enz -0.848 x age-0.364
x 0.712 (if female), where Cr Enz refers to serum creatinine measured using enzymatic method (mg/dl),
and age is in years. Since Scr in the hospital’s laboratory was measured by Jaffe’s method, values were
calibrated measuring by enzymatic method using the following equation9: Scr (enzymatic method) = Scr
(Jaffe’s method) x 0.906.
Statistical analysis Before analysis, gathered data was verified by visual checks two times and validated
by range, logical and missing data checks. The results were expressed as mean ± standard deviation for
continuous data and percentage for category data. The significant difference between groups was
analyzed by independent t-test and chi-square test. Logistic regression analysis was used to identify
significant risk factors and develop a predictive model based on renal impairment status. Age, age at

diabetes diagnosis, duration of diabetes, baseline eGFR, HbA1C, TC, systolic blood pressure and
increased urinary albumin excretion were applied as predictors in the forward procedure. All analysis was
performed with statistical package SPSS 17. Statistical significance was assessed as p<0.05.
RESULTS
Of the total of 322 patients, mean age was 59.1±8.7 years, most were female 181 (56.2%) and had
increased UAE (79.5%), and mean baseline eGFR was 86.3±18.4 ml/min/1.73 m2. The baseline
characteristic of patients between the renal impairment group and non-renal impairment group at follow-
up are shown in Tables 1 and 2. Patients who developed renal impairment at follow-up were older, more
likely to be female, be of a higher age at diagnosis, have a longer duration of diabetes, lower baseline
eGFR, higher systolic blood pressure (SBP), greater frequency of increased UAE, hypertension and use
renin-angiotensin system blockers than those who did not. Hemoglobin A1C, diastolic blood pressure,
total cholesterol, proportion of dyslipidemia and diabetes treatment were not significantly different
between the groups.
During the mean follow-up of 5.2 ± 0.3 years, 88 patients developed renal impairment. Their mean eGFR
at follow-up was 51.3 ± 6.6 ml/min/1.73 m2. Table 3 shows the results of univariate and multivariate
logistic regression analysis. In univariate analysis, older, higher age at diagnosis, longer duration of
diabetes, lower baseline eGFR, higher systolic blood pressure and increased UAE were factors
significantly associated with renal impairment at follow-up. In multivariate analysis, predictors
significantly associated with renal impairment were baseline increased UAE (OR: 5.204, p= 0.001),
baseline eGFR (OR: 0.878, p<0.001), diabetes duration (OR: 1.130, p= 0.004), baseline SBP (OR: 1.030,
p= 0.029) and age (OR: 1.054, p=0.030).
Table 1 Baseline characteristics of patients in renal impairment group and non-renal impairment group at follow-up.
Variables Non-RI (n=234) RI (n=88) All (N=322) P value

Age (years) 57.4 ± 8.6 63.6 ± 7.4 59.1 ± 8.7 <0.001*
Age at diagnosis(years) 53.2 ± 9.0 56.9 ± 8.2 54.2 ± 8.9 0.001*
CP - 4
Diabetes duration (years) 4.2 ± 3.8 6.7 ± 4.7 4.9 ± 4.2 <0.001*
eGFR (ml/min/1.73 m2) 92.0 ± 18.1 71.1 ± 6.9 86.3 ± 18.4 <0.001*
Hemoglobin A1C (%) 7.8 ± 1.4 7.7 ± 1.2 7.8 ± 1.3 0.458
Systolic blood pressure (mmHg) 135.8 ± 15.4 142.6 ± 12.3 137.7 ± 14.9 <0.001*
Diastolic blood pressure (mmHg) 80.6 ± 9.7 81.3 ± 10.1 80.8 ± 9.8 0.572
Total cholesterol (mg/dl) 208.3 ± 49.4 218.6 ± 51.3 210.6 ± 50.2 0.083
Cohort size, n=322. Data are expressed as mean ± SD. P values refer to the unpaired t test, *p<0.05
RI; renal impairment.
Table 2 Baseline characteristics of patients in renal impairment group and non-renal impairment group at follow-up.
Variables Non-RI RI (n=88) All (N=322) P value

Sex (female/male) 47.0/53.0 80.7/19.3 56.2/43.8 <0.001*
eGFR 60-89/90-120/>120 ml/min/1.73m2 48.3/44.0/7.7 98.9/1.1/0 62.1/32.3/5.6 <0.001*
Increased urinary albumin excretion 75.6 89.8 79.5 0.005*
Comorbidity
Hypertension 75.6 88.6 79.2 0.010*
Dyslipidemia 64.1 67.0 64.9 0.622
Cardiovascular disease 7.7 6.8 7.5 0.790
Treatments
Oral hypoglycemic agent(s) only 89.3 83.0 87.6 0.123
Insulin only 3.8 5.7 4.3 0.541
Insulin + Oral agent(s) 6.8 11.4 8.1 0.184
ACEI/ARB agents 63.2 79.5 68.0 0.005*
Cohort size, n=322. Data are expressed as percentages. P values refer to the chi-square test, *p<0.05
RI; renal impairment, ACEI; angiotensin-converting enzyme inhibitor, ARB; angiotensin receptor
blocker.

Table 3 Univariate and multivariate logistic regression analysis with odds ratio (95% CI) for baseline variables as the predictor for
renal impairment.
Variables Univariate P value Multivariate P value

Increased UAE (y/n) 2.827 (1.333-5.992) 0.007 5.204 (2.044-13.253) 0.001
Baseline eGFR (ml/min/1.73 m2) 0.876 (0.846-0.907) < 0.001 0.878 (0.847-0.910) < 0.001
Diabetes duration (years) 1.143 (1.078-1.212) < 0.001 1.130 (1.041-1.226) 0.004
Age (years) 1.104 (1.065-1.145) < 0.001 1.054 (1.005-1.105) 0.030
Systolic blood pressure (mmHg) 1.034 (1.015-1.053) 0.001 1.030 (1.003-1.058) 0.029
Age at diagnosis (years) 1.048 (1.019-1.079) 0.001
Total cholesterol (mg/dl) 1.004 (0.999-1.009) 0.084
Hemoglobin A1C (%) 0.937 (0.778-1.129) 0.493
Cohort size, n=322. eGFR; estimated glomerular filtration rate, UAE; urinary albumin excretion.
The final model contained 5 baseline variables as shown in equation 1 was fit well with our data (Chi-
square 6.172, p= 0.628). This model can explain variation (Nagelkerke’s R2) 58.3% indicating the model
is useful in predicting renal impairment. At the cut-off point of 0.5, the sensitivity, specificity and overall
accuracy for prediction of renal impairment were 72.72, 92.31 and 86.96%, respectively.
Probability of renal impairment = (equation 1)
Where Z = -0.027 + 0.052 x age (in years) + 0.122 x diabetes duration (in years) – 0.130 x eGFR
(in ml/min/1.73 m2) + 1.649 x increased UAE (yes=1, no=0) + 0.030 x SBP (in mmHg)
DISCUSSION
This study included 322 type 2 diabetic patients with various diabetes durations. These patients had mean
CP - 4
diabetes duration of 4.9 years at baseline. Eighty eight patients (27.3%) developed renal impairment over
a 5-year period. Modifiable risk factors of development of renal impairment include decreased eGFR,
increased UAE and high SBP. In addition, prolonged diabetes duration and old age were associated with
the development of renal impairment.
The results show that increased UAE is a major predictor for developing renal impairment. Patients with
increased UAE had a 5.2-fold increased risk of developing renal impairment. This is supported by
previous studies which showed patients with microalbuminuria and macroalbuminuria had a 1.7-2.2 and
4.3-5.8-fold increase in risk of renal impairment, respectively 4, 5, 7.
Although the association of higher baseline eGFR with an increased rate of GFR decline has been
shown10, patients with lower baseline eGFR are more likely to develop eGFR less than 60 ml/min/1.73
m2. Our findings show that each 1 ml/mim/1.73 m2 increase in baseline eGFR was associated with a
12.2% decreased risk of developing renal impairment which was corroborated by previous studies of
patients with a mean baseline eGFR of 82-94 ml/min/1.73m2 showing a 6-28% reduction of renal
impairment risk for each unit increase in eGFR5, 6.
Our results indicate that each 1 year increase in diabetes duration is associated with a 13% increased risk
of developing renal impairment. This is a comparable portion of patients developing renal impairment as
in previous studies, showing a hazard ratio of 1.02 and 1.2 after 4.6 and 6.4 years of follow-up in patients
with a mean diabetes duration of 6.3 and 12.5 years, respectively4, 5. It is contrary to other studies that
reported that diabetes duration was not significantly associated with developing renal impairment2, 6.
In agreement with previous studies, it was found that old age was associated with developing renal
impairment2, 4, 5, 7. This is compatible with the concept that kidney morphology and function can be
changed by aging and diseases including DM and HT can hasten this change11.
Our results highlight the fact that high blood pressure is a risk factor for development and progression of
renal impairment2, 5. Systolic blood pressure, not diastolic BP, in the renal impairment group was
significantly higher than non-renal impairment group.
To our knowledge, there was one other predictive model for CKD development in Thai patients.
Thakkinstian et al.12 developed a prediction score for classifying risk of developing CKD in the Thai
population from areas across Thailand (N=3,459). Four predictors were included in their final model,

which were age, kidney stones, DM and hypertension. However, only 11.9% of the participants were
diabetic, so it may be less specific for prediction of CKD in diabetic patients than our model.
The qualifications of this study were that, first, urine albumin and Scr be collected from only one
measurement. There are several factors leading to variation of these values and therefore they may not
reflect real conditions such as urine concentration and protein intake. Second, the predictive equation still
needs to be validated. However, this study was a longitudinal study and investigated the predictors in
Thai patients so that the results may be informative for monitoring and treatment, particularly in Thai
diabetic patients that have similar characteristics. Finally, in this study urine albumin was measured as 2
levels (normal and increased urinary albumin excretion) which corresponded to American Diabetes
Association guidelines 2013, which define UAE as albumin-creatinine ratio ≥ 30 mg/g, which is
comparable to albumin concentration ≥ 20 mg/l in our study13. Future studies with quantification of
albuminuria would be needed to further evaluate its effect on the development of renal impairment.
CONCLUSION
The predictive model had 5 baseline risk factors including increased UAE, eGFR, duration of diabetes,
age and SBP. Study results suggest that albuminuria, eGFR and systolic blood pressure are modifiable
risk factors and that reducing albuminuria, preserving GFR and controlling blood pressure may be
beneficial in preventing renal impairment. Furthermore, it is recommended that patients be diagnosed
with DM as soon as possible to receive appropriate monitoring and treatment.
ACKNOWLEDGMENTS
We are grateful to Mrs. Somsri Pansaksiri, programmer for Saraburi Hospital, for her assistance with the
use of the hospital’s patient database.
REFERENCES
1. Kidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group. KDIGO 2012 Clinical Practice
Guideline for the Evaluation and Management of Chronic Kidney Disease. Kidney inter. 2013;Suppl(3):1-
150.
2. Afghahi H, Cederholm J, Eliasson B, Zethelius B, Gudbjornsdottir S, Hadimeri H, et al. Risk factors for the
CP - 4
development of albuminuria and renal impairment in type 2 diabetes--the Swedish National Diabetes
Register (NDR). Nephrol Dial Transplant. 2011;26(4):1236-43.
3. Bash LD, Selvin E, Steffes M, Coresh J, Astor BC. Poor glycemic control in diabetes and the risk of
incident chronic kidney disease even in the absence of albuminuria and retinopathy: Atherosclerosis Risk in
Communities (ARIC) Study. Arch Intern Med. 2008;168(22):2440-7.
4. Luk AO, So WY, Ma RC, Kong AP, Ozaki R, Ng VS, et al. Metabolic syndrome predicts new onset of
chronic kidney disease in 5,829 patients with type 2 diabetes: a 5-year prospective analysis of the Hong
Kong Diabetes Registry. Diabetes Care. 2008;31(12):2357-61.
5. Targher G, Chonchol M, Bertolini L, Rodella S, Zenari L, Lippi G, et al. Increased risk of CKD among type
2 diabetics with nonalcoholic fatty liver disease. J Am Soc Nephrol. 2008;19(8):1564-70.
6. Zoppini G, Targher G, Chonchol M, Ortalda V, Abaterusso C, Pichiri I, et al. Serum uric acid levels and
incident chronic kidney disease in patients with type 2 diabetes and preserved kidney function. Diabetes
Care. 2012;35(1):99-104.
7. Zoppini G, Targher G, Chonchol M, Perrone F, Lippi G, Muggeo M. Higher HDL cholesterol levels are
associated with a lower incidence of chronic kidney disease in patients with type 2 diabetes. Nutr Metab
Cardiovasc Dis. 2009;19(8):580-6.
8. Praditpornsilpa K, Townamchai N, Chaiwatanarat T, Tiranathanagul K, Katawatin P, Susantitaphong P, et
al. The need for robust validation for MDRD-based glomerular filtration rate estimation in various CKD
populations. Nephrol Dial Transplant. 2011;26(9):2780-5.
9. Stevens LA, Manzi J, Levey AS, Chen J, Deysher AE, Greene T, et al. Impact of creatinine calibration on
performance of GFR estimating equations in a pooled individual patient database. Am J Kidney Dis.
2007;50(1):21-35.
10. Rossing K, Christensen PK, Hovind P, Tarnow L, Rossing P, Parving HH. Progression of nephropathy in
type 2 diabetic patients. Kidney Int. 2004;66(4):1596-605.
11. Zhou XJ, Rakheja D, Yu X, Saxena R, Vaziri ND, Silva FG. The aging kidney. Kidney Int.
2008;74(6):710-20.
12. Thakkinstian A, Ingsathit A, Chaiprasert A, Rattanasiri S, Sangthawan P, Gojaseni P, et al. A simplified
clinical prediction score of chronic kidney disease: a cross-sectional-survey study. BMC Nephrol.
2011;12:45.
13. de Zeeuw D. Albuminuria: a target for treatment of type 2 diabetic nephropathy. Semin Nephrol.
2007;27(2):172-81.

Session 5
Social Pharmacy SP 1
PRODUCTION OF HALAL HERBAL MEDICINAL PRODUCTS IN CHANAE HOSPITAL:

A COMMUNITY HOSPITAL IN NARATHIWAS PROVINCE, THAILAND
Chaowalit Monton1, 2,*, Krisana Kraisintu1, 3, 4 and Natawat Chankana4

1
Sino-Thai Traditional Medicine Research Center (Cooperation between Rangsit University, Harbin Institute of Technology and
Heilongjiang University of Chinese Medicine), Rangsit University, Pathum Thani 12000, Thailand.
2
Faculty of Pharmacy, Rangsit University, Pathum Thani 12000, Thailand.
3
Faculty of Oriental Medicine, Rangsit University, Pathum Thani, 12000, Thailand.
4
Sun Herb Thai Chinese Manufacturing Plant, Rangsit University, Pathum Thani, 12000, Thailand.
KEYWORDS: Herbal, Halal, Community hospital, Chanae hospital, Gamma irradiation
INTRODUCTION
Herbal medicines have been widely used around the world since ancient times, and are recognized for
their good therapeutic effects as they have fewer side effects than modern medicines (Ansari et al., 2012).
Furthermore, the worldwide use of herbal medicinal products has continued to increase, and nowadays a
large number of the global population use medicinal herbal products as a primitive form of health care
(Shirwaikar, 2012).
The 2012 National List of Essential Medicines of Thailand contains 71 herbal recipes. The Eleventh
National Economic and Social Development Plan of Thailand has plans to permit usage of herbal
medicines in hospitals and primary healthcare, indicating that herbal products will be more important and
used even more in the near future.
Narathiwas is a province in the southernmost area of Thailand bordering Malaysia. Chanae is a district of
Narathiwas province, and has a community hospital called Chanae Hospital following the name of the
district. Recently, Narathiwas has become an area of unrest and is targeted by terrorists. The set-up of a
herbal factory in this area would be too difficult, because of this dangerous situation. Nevertheless,
Professor Krisana Kraisintu initiated projects to set up a mini-plant in three southernmost provinces of
Thailand (Narathiwas, Pattani, and Yala) to promote herbal medicinal products using local herbs. As most
of the population in these provinces are Muslim. Muslim people must ensure that all foods (particularly
processed foods), as well as non-food items like cosmetics and pharmaceuticals, are halal. This is because
these products often contain animal by-products or other ingredients that are not permissible for Muslims
to eat or use on their bodies. It is necessary for the productions of herbal products follow halal
authentication. This article describes the procedure for the set-up of a mini-plant in an area of unrest in
Thailand, to be a model of other community hospitals in the country.
METHODOLOGY
The set-up of the mini-plant has three steps:
Initial step Three pharmacists from Rangsit University met with the director of the hospital, doctors,
pharmacists, and Thai traditional doctors of Chanae Hospital to prepare for the project at Chanae
SP – 1
Hospital. Professor Krisana Kraisintu described the importance and phases of plant set-up. The resolution
from this meeting was to train the staff to work in the mini-plant. Machines for production of the
medicines were supplied from Sri Sakorn District Public Health Office and Naradhiwas Rajanagarindra
Hospital.
(a) (b)
Figure 1 the first step of mini-plant set up (a) Professor Krisana Kraisintu met with the Chanae Hospital colleagues (b) Dislocation
of machines from Sri Sakorn District Public Health Office to Chanae Hospital by native people of Sri Sakorn district

Plant design and set-up After the mini-plant was designed, the equipment was installed. The first floor of
the plant has procedures in place regarding cleaning, baking, grinding, and sieving herbal medicines. The
second floor contains a production unit, packing unit, and quarantine area for the end products.
(a) (b)
Figure 2 the second step of mini-plant set up (a, b) Installation all machines into the building
Production Before production at Chanae Hospital, two pharmacists and one Thai traditional doctor from
the hospital visited the Sun Herb Thai Chinese Manufacturing Plant at Rangsit University to learn about
the herbal products production process. Afterwards, pharmacists from Rangsit University monitored the
trained staff of Chanae Hospital to evaluate their proficiency of production procedures.
(a) (b)
SP – 1
(c) (d)
Figure 3 Production of halal herbal products (a,b) Chanae Hospital colleagues learn about the herbal products production process at
Rangsit University (c,d) Halal herbal products production at mini-plant of Chanae Hospital

The timeline from initial phase to production phase was three months, which was extremely fast. The
principal pieces of equipment in the mini-plant were two capsule filling machines, one hot air oven, two
grinders, one sieving machine, and one tea bag packing machine.
In the initial phase, six items of herbal medicinal products were produced (Table 1), including rhizome of
turmeric (Curcuma longa L.), leaf of Asiatic pennywort (Centella asiatica Urban.), vine of jewel vine
(Derris scandens (Roxb.) Benth.), all parts above the ground of kariyat (Andrographis paniculata (Burm.
f.) Wall.ex Nees), vine of edible-stemmed vine (Cissus quadrangularis L.), and leaf of ringworm bush
(Senna alata (L.) Roxb). All herbal products were prepared as capsules. However, because of its bulky
size, there was a problem filling capsules with ringworm bushes, thus it was planned to prepare as a tea
instead. Therefore, Chanae Hospital can produce five items of herbal products.

Table 1 Herbal medicinal product items produced in Chanae Hospital
No. Herbal Indication

1 Turmeric Anti-flatulence
2 Asiatic pennywort Antipyretic, anti-apthous ulcer, treatment of internal trauma
3 Jewel vine Treatment of muscle pain, anti-inflammatory
4 Kariyat Treatment of non-infectious diarrhea, relief symptoms of
common cold
5 Edible-stemmed vine Anti-hemorrhoid
6 Ringworm bush Laxative
All herbal products that are produced at Chanae Hospital are in the official List of Herbal Medicinal
Products, and the 2012 National List of Essential Medicines of Thailand. All herbs that are in the list are
safe for appropriate for use in humans in Thailand (The Ministry of Public Health of The Kingdom of
Thailand, 2012).
Gamma irradiation is an effective procedure for sterilizing herbal products. It can destroy microorganisms
that contaminate manufactured products (Ferreira et al., 2010). Thus, all products that were produced at
Chanae Hospital were gamma irradiated before use; this step was facilitated by the Sun Herb Thai
Chinese Manufacturing Plant at Rangsit University. Therefore, Chanae Hospital is the first community
hospital in Thailand that sterilizes herbal products by gamma ray before use. Chanae Hospital is also the
first hospital plant that produces herbal medications following halal authentication. An example of
turmeric capsules is shown in Figure 4.
Figure 4 Gamma irradiated product (turmeric capsules)
CONCLUSION
The cooperation between an academic institute (Rangsit University) and community hospital (Chanae
Hospital) can promote the production of herbal medicinal products. The mini-plant was set-up on the
SP – 1
border of Thailand in an area of unrest, and five items of gamma irradiated herbal products have been
produced following halal authentication.
ACKNOWLEDGEMENTS
The authors are grateful for monetary support from Rangsit University. We would also like to thank KI
Tull for assistance with the English for this proceeding.
REFERENCES
1. Ansari SH, Islam F, Sameem M. 2012. Influence of nanotechnology on herbal drugs: A review.
J Adv Pharm Technol Res 3(3):142-6.
2. Ferreira AA, Leal AS, Ferraz VP. Machado Y, Takahashi JA. 2010. Effects of gamma radiation
on the chemistry and antibacterial activity of artichoke leaves. Bol Latinoam Caribe Plant Med
Aromat 9: 232-7.
3. Shirwaikar A. 2012. Promoting the safe use of herbal medicines (online).
(http://www.hygeiajournal.com/Downloads/2121690828Annie%20madam%20%20Editorial.
pdf, accessed 23 April 2013).
4. The Ministry of Public Health of the Kingdom of Thailand. 2012. National list of essential
medicines A.D.2012 (online). (http://www.nlem.in.th/principles/herb/measurement, accessed 30
March 2013).

AUTHOR INDEX
Akkaramongkolporn, Prasert PT-11 Hisayakorn, Krittiya BP-2
Amnuaikit, Thanaporn PT-6 Iamsub, Kusol PN-10
Amnuoypol, Surattana BP-11 Issaro, Nipatha PN-5
Anwar, Jamshed PT-30 Jarikasem, Siripen BP-5
Auamnoy, Titinun CP-3, 4 Jianmongkol, Suree BP-11
Awaiyawanon, Pongsak PT-27 Kajsongkram, Tanwarat PN-7, 10
Banchonglikitkul, Chuleratana BP-2, 4, 12-15, Kampruengdet, Sukit PN-4
PN-7, 10, 17
Boonme, Prapaporn PT-6 Kao, W. John PT-34
Boonsupthip, Waraporn PN-7 Kaomongkolgit, Ruchadaporn PT-35
Boonthongchuay, Chalida PT-6 Kasetvetin, Chawankorn PT-8
Buddhakala, Nopparat BP-1 Keeta, Itsara PT-20-26
Bunma, Napa PT-14 Kendrick, John PT-30
Carney, Louise BP-6 Ketmanee, Natthachest BP-4, 12
Chainethvanich, Benjaporn PT-2 Ketpitthaya, Supaporn PT-34
Chairat, Wisuta PT-16 Khamphan, Yaowaluk PN-17
Chaithongsri, Konlawit BP-13 Khayungarnnawee, Amornrat BP-5
Chankana, Natawat PT-3, 33, SP-1 Kheuynok, Vichein BP-1, 2, 4
Chareonmuang, Nicha PT-28 Kitbunnadaj, Ruengwit PN-2
Charernsriwilaiwat, Natthan BP-6 Kittipanyaworakun, Suchada CP-4
Charoenchai, Laksana PT-1, 3, 17 Kittiwisut, Siriporn PT-3, 33
Charoonratana, Tossaton PT-1 Klungsupya, Prapaipat BP-9-10, PN-9,
14
Chatanon, Lawan PN-15 Komesmuneeborirak, Phojana PT-18
Chatchawalsaisin, Jittima PT-30, 31 Kongsombat, Bantika BP-15
Chewchinda, Savita PN-13 Kongsuwan, Chutima PT-29
Chuanasa, Taksina PN-3, 11-12 Koomanachai, Pornpan CP-1
Chuchana, Piyapong PN-1 Kraisintu, Krisana PT-1, 33, SP-1
Chueboonmee, Phatsuda PN-17 Krubphachaya, Pongrit BP-16
Chuennangchee, Vilailuk PN-10 Kunhachan, Phanukit PT-25
Chusut, Tun PT-15, 17 Laiwattanaphaisal, Jiratikron PN-5
De-Eknamkul, Wanchai PN-6, 12 Laohapatarapant, Suvida PT-13
Downes, Sandra BP-6 Laovitthayanggoon, Sarunya BP-9, 12-14,
PN-9-10
Duangjit, Sureewan PT-9 Lhinhatrakool, Thitima PN-1
Dunkoksung, Wilasinee BP-11 Limsiriwong, Pongsatorn BP-4
Eiamwat, Jirawat PN-4, 9 Lipipun, Vimolmas PT-12
Eumkeb, Griangsak BP-7, 16-17 Lorliam, Wanlapa PN-15
Fungsin, Bundit PT-23, 26 Lowtangkitcharoen, Witaya PN-3
Giwanon, Rattanasiri BP-4 Luangsakul, Naphatrapi PN-15
Gritsanapan, Wandee CP-2, PN-13, Lueanpaen, Isara PT-36
16
Halee, Maimun CP-1 Machana, Sisipawan PN-5
Hemstapat, Warinkarn BP-3 Madaka, Fameera PT-15
Hemthanon, Thongchai PN-17 Mangmeesri, Peeranuch CP-2
Hiranyaekaphap, Thippatai PN-2 Meksuriyen, Duangdeun BP-8

Monton, Chaowalit PT-1, 3, 15, 17, Reungpathanapong, Sareeya BP-14, PT-25
33, SP-1
Moungjaroen, Jirapan PT-14 Ritthidej, Garnpimol C. PT-12, 19
Muangman, Thanchanok BP-9-10, PN-9, Robb, Brendan BP-6
14
Muangsiri, Walaisiri PT-18, 34 Rojanarata, Theerasak BP-6, PT-4-5,
7, 9-11, 13, 16,
35
Muensaen, Sarinthip BP-5 Rojsanga, Piyanuch PN-8
Munprom, Chaowarat CP-4 Ruengsri, Saowaluck BP-4
Nakachon, Wittaya PT-19 Rujiviphat, Soravoot PT-8
Nakakaew, Sawai BP-4 Sae-Tang, Somwang PT-30
Naknarong, Wanatkamol BP-17 Saingam, Worawan PT-2-3, 15, 17,
27, 33
Natakankitkul, Surapol PT-28 Saisin, Wanlapha PT-23-24, 26
Ngankaranatikarn, Punyisa PN-11 Saito, Naoki PN-3
Ngawhirunpat, Tanasait PT-4-5,7, 9-11, Sajomsang, Warayuth PT-10
13, 16, 35
Niamprem, Pattravee PT-32 Sakunpak, Apirak PT-1, 15, 17,
33
Niwaspragrit, Cholticha PT-24-25 Samprasit, Wipada PT-11
Nyein Thu, Su Myat PT-12 Sanrungmuang, Anchana PT-27
Opanasopit, Praneet BP-6, PT-4-5, Sawang, Nahathai PT-20, PT-21,
7, 9-11, 13, 16, PT-22
35
Ornnim, Phatsakorn PN-5 Sawatpipat, Jeerawat PN-8
Pahusee, Darunee BP-1, 4 Sematong, Tuanta BP-14, PT-25
Pahusi, Darunee BP-2 Siriarchavatana, Parkpoom BP-13
Panichayupakaranant, PT-32 Sirichaiwetchakoon, Kittipot BP-17
Pharkphoom
Panyakhong, Tawan PT-28 Siriwong, Supatcharee BP-16
Pathompak, Pathamaporn PT-1, 17 Sithisarn, Pongtip PN-8, 13, 16
Pattarapanich, Chumnan PN-5 Sitthiphong Soradech PT-21
Pengpreecha, Paramee PN-4 Soonthornsi, Nattaporn BP-3
Peungsumrong, Sontaya BP-4 Soradech, Sitthiphong PT-24
Phanudulkitti, Chamipha PN-5 Sotanaphun, Uthai BP-8
Phitaktim, Sineewan BP-7 Srijan, Tharathee PN-5
Phlicharoenphon, Wichittra PN-16 Srimongkolpithak, Nitipol PN-2
Phongsopitanun, Wongsakorn PN-15 Srisom, Marudech BP-4
Phunsawat, Vatin PN-8 Srithong, Penpan PN-7, 10
Pichayakorn, Wiwat PT-15, 36 Sriubolmas, Nongluksna BP-8, PN-11
Pirshahid, Pattra A. PN-17 Sriyam, Kanjana BP-1-2, 4
Pitaksuteepong, Tasana BP-3 Sueksakit, Kanyarat BP-1
Plianwong, Samarwadee PT-4 Sukmak, Pliwphai C. CP-3
Poowaruttanawiwat, Prayuth PN-2 Sukrong, Suchada PT-34
Potduang, Buppachart BP-12, PT-20- Suksaeree, Jirapornchai PT-1, 3, 15, 17,
26 33, 36
Praditpornsilpa, Kearkiat CP-4 Sutanthavibul, Narueporn PT-31, 34
Rangsimawong, Worranun PT-7 Suthepakul, Nava BP-9
Rattanamanee, Kwanchai PN-2 Suwanagul, Anawat BP-10
Rerk-am, Ubon BP-2, 15 Suwanborirux, Khanit PN-3, 11-12

Tanasupawat, Somboon PN-15 Vardhanabhuti, Nontima BP-11, PT-34
Tangsatirapakdee, Sinn BP-2, 15 Vattanagijyingyong, Yada PT-31
Tanguenyongwatana, Prasan PN-1-2, 14, 27 Viseshsindh, Wit CP-2
Tansakul, Nattaon PN-6 Wachirasiri, Kulapatr PN-4
Tansakul, Pimpimon PN-6 Waiprib, Rungtiwa PT-36
Tantrawong, Arkachai PN-14, PT-21 Walapa, Sorada PN-4
Taweepreda, Wirach PT-36 Wangkananon, Worrapon PT-28
Teethaisong, Yothin BP-7 Wannaphatchaiyong, Suchipha PT-23-24, 26
Tengwattanachoti, Yupa PT-33 Warisnoicharoen, Warangkana PT-29
Thisayakorn, Krittiya BP-1, 4 Waropastrakul, Dalad PN-12
Thongdon-A, Jeerayu BP-9, PN-9, 14 Watari, Akihiro BP-10
Thubthimthed, Sirinan BP-13-14 Wattanaarsakit, Phanphen PT-34
Thumanu, Kanjana BP-16 Waywa, Duangdao CP-1
Tiatragoon, Worawan PN-9 Weerakul, Thorsang PN-5
Tidjarat, Siripran PT-5 Werawatganone, Pornpen PT-18
Tiensong, Benjaporn PN-4 Wiyakrutta, Suthep BP-8
Tiyaboonchai, Waree PT-8, 32 Wongchang, Thamrong PN-2
Tonglairoum, Prasopchai PT-35 Wongpoowarak, Wibul PT-6
Trangvacharakul, Srisak BP-9, PN-9, 14 Wongsuphasawat, Kant CP-2
Treyaprasert, Wanchai CP-1 Woraphatphadung, Thisirak PT-10
Utaisincharoen, Pongsak BP-3 Yagi, Kiyohito BP-10
Vadcharavivad, Somratai CP-3-4

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The Thai Journal of Pharmaceutical Sciences

EDITORIAL ADVISORY BOARD

The 30th Annual Research Conference in Pharmaceutical Sciences| i

The 30th Annual Research Conference in Pharmaceutical Sciences| ii

Piyapong Choochana, Thitima Lhinhatrakool and Prasan Tanguenyongwatana

PN-2 Discovery of Novel Cholinesterase Inhibitor (RKNU026) with β-Amyloid

Thippatai Hiranyaekaphap, Nitipol Srimongkolpithak, Thamrong Wongchang,

PN-3 Preparation of 2'-N-2,3,4-trifluorobenzoyl derivative of cytotoxic ecteinascidin 770

Witaya Lowtangkitcharoen, Taksina Chuanasa, Naoki Saito and Khanit

Jirawat Eiamwat, Paramee Pengpreecha, Benjaporn Tiensong, Sorada Walapa,

Nipatha Issaro, Thorsang Weerakul, Sisipawan Machana , Phatsakorn Ornnim,

Nattaon Tansakul, Pimpimon Tansakul and Wanchai De-Eknamkul

PN-7 Effect of ethanol concentration on cyaniding-3-glucoside, total monomeric

Penpan Srithong, Tanwarat Kajsongkram, Chuleratana Bangchonglikitkul and

The 30th Annual Research Conference in Pharmaceutical Sciences| iii

Jeerawat Sawatpipat, Vatin Phunsawat, Piyanuch Rojsanga and Pongtip Sithisarn

PN-9 Investigation on phytochemical, antioxidant and cytotoxic properties of

PN-10 Comparative studies on antioxidant activities, total phenolics and anthocyanin

Tanwarat Kajsongkram, Saranya Laovitthayanggoon ,Kusol Iamsub, Vilailuk

PN-11 Antileukemic activity and secondary metabolites of an endophytic fungus

Punyisa Ngankaranatikarn, Taksina Chuanasa, Nongluksna Sriubolmas and

PN-12 Detection of esterase activity converting cytotoxic renieramycin M to

Dalad Waropastrakul, Khanit Suwanborirux, Wanchai De-Eknamkul and Taksina

Savita Chewchinda, Pongtip Sithisarn and Wandee Gritsanapan

Jeerayu Thongdon-A, Prapaipat Klungsupya, Thanchanok Muangman, Arkachai

Somboon Tanasupawat, Wongsakorn Phongsopitanun, Wanlapa Lorliam,

Wichittra Phlicharoenphon, Wandee Gritsanapan and Pongtip Sithisarn

The 30th Annual Research Conference in Pharmaceutical Sciences| iv

Pattra Ahmadi Pirshahid, Thongchai Hemthanon , Yaowaluk Khamphan, Phatsuda

BP-1 Preliminary study of Syzygium aromaticum (L.) on analgesic activity in rats

Kanyarat Sueksakit, Krittiya Thisayakorn, Vichein Khueynok, Kanjana Sriyam,

BP-2 Effects of topical administration of Nymphaeae lotus flower extract on UVA-

Krittiya Thisayakorn, Ubon Rerk-am, Sinn Tangstirapakdee, Vichein Khueynok,

Nattaporn Soonthornsi, Warinkarn Hemstapat, Pongsak Utaisincharoen and

BP-4 Pilot study of antibacterial and anti-inflammatory activities of Indian gooseberry

Rattanasiri Giwanon, Krittiya Thisayakorn, Pongsatorn Limsiriwong, Saowaluck

Siripen Jarikasem, Amornrat Khayungarnnawee and Sarinthip Muensaen

BP-6 Aligned electrospun chitosan/poly-ε caprolactone blended scaffold: Anti-

Natthan Charernsriwilaiwat, Louise Carney, Brendan Robb, Praneet Opanasopit,

BP-7 Antibacterial activity of α-mangostin from the pericarp extract of Garcinia

Griangsak Eumkeb, Sineewan Phitaktim and Yothin Teethaisong

The 30th Annual Research Conference in Pharmaceutical Sciences| v

Nongluksna Sriubolmas, Uthai Sotanaphun, Duangdeun Meksuriyen and Suthep

Prapaipat Klungsupya, Nava Suthepakul, Thanchanok Muangman, Sarunya

Thanchanok Muangman, Prapaipat Klungsupya, Anawat Suwanagul, Akihiro

BP-13 Anti-lipase activity of Quercus infectoria G. Olivier extract

Sirinan Thubthimthed, Sarunya Laovitthayanggoon, Parkpoom Siriarchavatana,

Tuanta Sematong, Sirinan Thubthimthed, Sareeya Reungpathanapong, Sarunya

BP-15 Biological activities of Cajanus cajan ethanolic extracts

BP-16 Synergy effect of ceftazidime with flavonoids against Streptococcus pyogenes

Supatcharee Siriwong, Pongrit Krubphachaya, Kanjana Thumanu and Griangsak

Griangsak Eumkeb, Wanatkamol Naknarong and Kittipot Sirichaiwetchakoon

The 30th Annual Research Conference in Pharmaceutical Sciences| vi

Apirak Sakunpak, Jirapornchai Suksaeree, Laksana Charoenchai, Chaowalit

Benjaporn Chainethvanich, Worawan Saingam and Prasan Tanguenyongwatana

PT-3 Development and validation of RP-HPLC method for determination of piperine in

PT-4 Application of titrimetric indicator to the estimation of polyplex formation ratio

Samarwadee Plianwong, Praneet Opanasopit, Tanasait Ngawhirunpat and

PT-5 Preparation of doubly crosslinked chitosan by the use of environmentally friendly

Siripran Tidjarat, Tanasait Ngawhirunpat, Praneet Opanasopit and Theerasak

PT-6 Influence of chemical structures of emulsifiers on feasibility of isopropyl palmitate

Prapaporn Boonme, Chalida Boonthongchuay, Thanaporn Amnuaikit and

PT-7 Effect of particle size of ultradeformable liposomes on dermal delivery of

Worranun Rangsimawong, Praneet Opanasopit, Theerasak Rojanarata and