S2 IKD minat Farmako 2018

DRUG DISCOVERY FROM

NATURAL RESOURCES

Mae Sri Hartati Wahyuningsih

Department of Pharmacology and therapy
Faculty Of Medicine, Public Health and Nursing
Universitas Gadjah Mada
 Specific instructional objectives
1. To know definition and types of Herbal Medicine
2. To know the development of traditional Medicine to be a
modern drug.
3. To know how to get active compound from natural
resources.
4. To know some example of research funding
THE USE OF NATURAL MATERIALS

Natural material

Cosmetics Food

DRUGS
Traditional
medicine
Food supplement

+Vit, Amino. acid

& mineral
 DEVELOPMENT OF TRADITIONAL MEDICINE
TO BE A MODERN DRUG

Flora, Animals Marine lives JAMU

Microorganisms
Traditional Medicine Standardized
herbal medicine
Single or
mixture FITOFARMAKA
Standardized, preclinic
Bioassay guided
&clinic testing
active compounds
isolation
Dose v/s
ACTIVE respons CHEMOPREVENTIVE
COMPOUND CHEMOPROTECTIVE
Synthetic, Derivatives,
Analogs synthetic ,
Isolation, preclinic &
clinic testing. etc

DRUGS
PENGELOMPOKAN DAN PENANDAAN OBAT HERBAL
INDONESIA

DATA EMPIRIK JAMU

TURUN MENURUN
SWA
PENGOBATAN

HERBAL
DATA PRA KLINIK TERSTANDAR
(Bahan Uji Terstandar)

DATA KLINIK FITOFARMAKA

(Bahan Uji terstandar)
 PENGELOMPOKAN OBAT HERBAL INDONESIA

JAMU OBAT HERBAL FITOFARMAKA

TERSTANDAR
1. Disediakan secara 1. Terbuat dari ekstrak 1. Terbuat dari ekstrak,
tradisional (pil, dan dapat disejajarkan
serbuk seduh dsb) dengan obat modern
2. Dasar pengalaman 2. Dasar penelitian ilmiah 2. Dasar penelitian ilmiah

3. Tanaman penyusun (5- 3. Tanaman penyusun 3. Tanaman penyusun

10) atau lebih maksimum 5 (lima) maksimum 5 (lima)
4. Aman 4. Aman 4. Aman
5. Pembuktian secara 5. Pembuktian secara 5. Pembuktian secara
empiris
ilmiah dengan data ilmiah dengan data klinik
praklinik
6. Memenuhi persyaratan 6. Memenuhi persyaratan 6. Memenuhi persyaratan
mutu yg berlaku mutu yg berlaku mutu yg berlaku

7. Bahan baku belum 7. Bahan baku 7. Bahan baku terstandari

terstandarisasi terstandarisasi (FI, MMI) sasi (FI, MMI)
 Riset untuk memastikan
Khasiat dan keamanan

Quality Safety
CPOTB
Efficacy

Budidaya Standardisasi MUTU SIMPLISIA

Riset
 Definisi Fitofarmaka

KEP. Ka. B.POM. RI., No: HK.00.05.4.2411., th 2004

Tentang
PENGELOMPOKAN DAN PENANDAAN OBAT BAHAN ALAM
INDONESIA.

 Fitofarmaka merupakan bentuk obat tradisional

dari bahan alam yang dapat disejajarkan dengan
obat modern karena proses pembuatannya yang
telah terstandar, ditunjang dengan bukti ilmiah
sampai dengan uji klinik pada manusia.
PEDOMAN FITOFARMAKA
Kep. Men. Kes.RI. (761/92)

PRIORITAS PEMILIHAN
1. Bahan baku relatif mudah diperoleh
2. Didasarkan pada pola penyakit di Indonesia
3. Perkiraan manfaat terhadap penyakit tertentu cukup besar
4. Memiliki rasio resiko dan kegunaan yang menguntungkan
penderita
5. Merupakan satu-satunya alternatif pengobatan
 TAHAP PENGEMBANGAN FITOFARMAKA

1. Seleksi bahan tanaman

2. Pengujian farmakologi (in vivo)
- Penapisan aktivitas (belum ada petunjuk aktivitas)
- Langsung pemastian khasiat (ada petunjuk)
3. Pengujian toksisitas (akut, subakut, kronik, spesifik)
- Spesifik (Toksik pada janin, mutagenisitas, karsinogen)
4. Pengujian farmakodinamika (in vitro & in vivo) (Preklinik ??)
5. Pengembangan sediaan (formulasi)
6. Penapisan fitokimia dan standarisasi sediaan
7. Pengujian klinik  ??
  UJI PRAKLINIK DAN KLINIK
Kep. Men. Kes. RI. (56/2000)

UJI PRAKLINIK:
1. Uji toksikologi (keamanan & spektrum efek toksik)
- Umum (akut, subakut/subkronis, kronis)
- Khusus (teratogenik, mutagenik, karsinogenik)
2. Uji farmakodinamik ( khasiat)
 Hasil Uji Praklinik:
Indikasi awal
Perkiraan dosis efektif
Perkiraan batas aman

Hasil Uji Klinik:

Fase I : Menegaskan keamanan & profil
farmakokinetik obat pd manusia sehat (farklin)
 Tolerabilitas dan perkiraa dosis
Fase II : Menegaskan kemanjuran & keamanan pd
penderita skala sedang (100-200)
 Kemanjuran & keamanan
Fase III : Menegaskan kemanjuran & keamanan pd
penderita skala besar (200-1000)
 Manfaat klinis lebih absolut
Bandingkan manfaat dan resiko
Fase IV :Menegaskan keamanan obat (Survei pasca pasar)
 Resiko penggunaan
LEGALITAS
Undang-undang No. 29 tahun 2004 tentang Praktik Kedokteran,
dokter/dokter gigi dalam memberikan pelayanan kesehatan harus
memenuhi standar pelayanan medis, yang pada prinsipnya harus
memenuhi kaidah praktik kedokteran berbasis bukti (evidence based
medicine)

Peraturan Menteri Kesehatan No. 03/MENKES/PER/I/2010

tentang Saintifikasi Jamu.

Saintifikasi Jamu adalah pembuktian ilmiah jamu melalui penelitian

berbasis pelayanan kesehatan.

Salah satu tujuannya adalah memberikan landasan ilmiah (evidenced

based) penggunaan jamu secara empirik melalui penelitian berbasis
pelayanan yang dilakukan di sarana pelayanan kesehatan, dalam hal ini
klinik pelayanan jamu/dokter praktik jamu
 Hortus Medicus clinic is saintifikasi Herbal Clinic Type A,
Implementation saintifikasi Herb.
 Materials used in the form of crude material that have proven efficacy
and safety through preclinical trials.
 Supporting staff human resources
8 doctors
2 person pharmacist
3 assistant pharmacist
1 person healthcare analyst (laboratory)
1 nurse and 1 medical record
 Diagnosis is implemented by incorporating the conventional
diagnosis results of laboratory analysis of medical records and also is
developed with qualitative data to assess the healthy aspect.
Clinic location:
Balai Besar Penelitian dan Pengembangan Tanaman Obat dan Obat Tradisional Jl.
Raya Lawu No. 11 Tawangmangu
Karanganyar, Jawa Tengah
Telp. 0271-697010
 TAHAPAN UJI UNTUK PENGEMBANGAN OBAT HERBAL
Inventarisasi  Observasi  Seleksi

UJI PRAKLINIK OBAT HERBAL

Kel. I Kel. II Kel. III Kel. IV

Aman (+) Aman (+) Aman (-) Aman (-)
Khasiat (+) Khasiat (-) Khasiat (+) Khasiat (-)

Terus beredar Boleh beredar Tidak dipakai sampai Dilarang beredar

(jalur formal) (jalur non formal) penelitian lanjut dan dipakai
Isolasi
Standardisasi ISOLAT
Tek. Farmasi
Uji Klinik
Uji Klinik Uji Klinik
Bermanfaat
Bermanfaat Bermanfaat

Obat Jadi
PELAYANAN
KESEHATAN
ACTIVE COMPOUND
ISOLATION
 ACTIVE COMPOUND ISOLATION
(Active substance and standardization marker)

 Phytochemical approach
- Takes a long time, costly

 Bioassay Guided Isolation approach

(each –stage monitoring of activity testing)
- Fast and in expensive
Phytochemical approach

TLC of Sixteen top plants BPOM

Bioassay Guided Isolation Approach
Heksan : EtOAc
(3 : 1, v/v)
Material

Extraction Crude extracts

Inactive
CHCl3 MeOH Bioactivity screening extracts 1:1,v/
v

TLC
Fractionation Active
analysis
extracts
Heksan : EtOAc
(3 : 1, v/v)

F1
F2
F3 Pure
Cytotoxicity Active Isolation &
F4 bioactive
screening fractions Purification
F5 compounds
F1 F2 F3 F4 F5 P
Identification
Inactive (UV, IR,MS,NMR)
fractions
Chemical structure
 ACTIVE COMPOUND IDENTIFICATION

Determining the chemical structure of isolated compound

Chemical structure is used:

- To find out physical property & chemical compound
- To find out activity estimate
- To find out activity mechanism
- To serve as identity in the standardization of natural resouces
 Manners:
 Spectroscopic UV, IR, MS dan NMR
 Crystalography
 Derivation
 Others
MAJOR TYPES OF SECONDARY METABOLITES

PHENOLIC COMPOUNDS (Aromatik)

- Flavonoids, Anthraquinones etc.
TERPENOID COMPOUNDS (Isoprene)
- Eugenol, Carotenoids, Taxol, Oleanolic acid etc.
ALKALOIDS (Mengandung atom N, nitrogen)
- Vincristine, quinine, camptothecine etc.
POLYPHENOLIC COMPOUNDS (banyak aromatik)
-EGCG (Epi Gallo Catechin gallate) etc.
POLYKETIDE COMPOUNDS (dari asam asetat)
- Discodermolide etc.
BIOLOGICALLY ACTIVE POLYPHENOLIC COMPOUNDS
OH OH

OH OH OH

HO O O
HO
OH
O OH

O
HO O
OH OH OH

(-)-Epigallocatechin 3-O-gallate OH
OH
EGCG
OH OH Theaflavin

Green tea OH
Black & green tea
HO
Stimulant, diuretics, antioxidant,
HO preventive effect on dental caries
O OH
OH
OH Antimutagenic invitro, prevention
HO
on cancer cells proliferation, inhibit
HO
HO O
OH
lipid peroxidation

OH
But little data on bioavailability of
Theasinensin C
these polyphenols
OH
Black tea
Biosintesis Terpenoid IPP=Isopentenil pirofosfat
GPP = Geranyl pyrophosphate
FPP = Farnesyl pyrophosphate
GGPP = Geranyl geranyl pp
Squalene = 30 carbons or 6 isoprene
MVA IPP DMAPP
Asam mevalonat Dimetil Alil pirofosfat

GPP C10
O-PP
SESQUITERPENOIDS
+ IPP
MONOTERPENOIDS
FPP C15
O-PP

+ IPP
FPP
GGPP
O-PP

Squalene
DITERPENOIDS

TRITERPENOIDS + STEROLS
 OPP
BIOLOGICALLY ACTIVE OPP

TERPENOID COMPOUNDS DMAP IPP

Building blocks

β-Carotene (Carotenoids)

Daucus carota Degraded to retinol (Vitamin A), preventive

O actions against degenerative dissorders, non
toxic coloring agent.
O
O O O
OH
Taxol promotes the assembly of tubulin
NH O dimers into microtubules, which
OH stabilizes by inhibiting
HO
O O O depolymerization

O
O
For ovarian tumors that do not
Taxol
respond to cis-Sisplatin, recently
reported for metastatic breast cancers
Taxus brevifolia (yew tree) 0.01%
unresponsive to anthracycline etc.
Paclitaxel, Docetaxel (derivative)
 BIOLOGICALLY ACTIVE ALKALOIDS
•Alkaloid structures are varies, biogenetically most of
alkaloids derived from amino acids (–N- atom
contributors). Alkaloids are compounds having –N- in
their molecule structures. Therefore alkaloids react
alkalis due to a pair of free electron on –N- atom. Most
of biologically active Secondary Metabolites are
alkaloids
OH
Antimitotics, they bind
to tubulin and prevent
R = -CH3, Vinblastine the formation of the
N R = -CHO, Vincristine microtubules that
responsible for the
N formation of the mitotic
H N
H spindle
H 3CO
O Treatment of cells with
OH these alkaloids leads to an
CH 3 accumulation of cells in
H 3CO N O the M and G2 phases, and
H the effect is lethal in the S
Catharanthus roseusR O
phase.
(Tapak doro) O OCH 3
Lead compounds from marine chemistry
N
H
N
H Anti malaria, comparable to artemisinin. A
OH collaborative research between U of Miss-
UGM-MMV Switzerland. Manzamine-A active
N

H
N
against chloroquine resistent plasmodium.

Manzamine-A
(Acanthostrongilophora sponge.)

Facts:
Manzamine-A structure is complex. Very
expensive collection cost. However, the sponge
grows very fast and manzamine-A is also
produced by microorganism symbiotic to sponge-
---- FERMENTATION is possible
 DETERMINING COMPOUND
POTENCY/PRODUCT

Compared to drugs (used clinically)

Preclinical testing in vivo:
- General toxicity testing
(acute, sub acute, chronic)
- Special toxicity testing
(carcinogenicity, teratogenicity)
  More compounds needed
`
To decrease the number of animals used for trials
and samples.
In vitro testing :
Enzyme activity testing
Anticancer testing (cell line)
Antimicrobe testing
Antiinflammation testing.

Determining action mechanism

(Prediction of mechanism action tract )

 Apoptosic stimulation
 Proliferation inhibition
 Angiogenesis inhibition
 Legitimation and formality


 The Republic of Indonesia’s Supervisory Agency Chief’s
Decree on Food and Drug (BPOM-RI)>> Indonesia
 Food and Drug Administration (FDA)>> Amerika Serikat

Manner:
• Documen of preclinic and clinic:
According to indication, efficacy and safety (quality control)
• Proven as effective and useful as safe medicine legal drug (trade mark)

 Incooperation with industries

- HAKI Facility
(Intelectual wealth right facility)
EXAMPLES OF RESEARCH
FINDINGS
 Catharanthus roseus

Taxus brevifolia

Nerium indicum M. Jalapa L P. macrocarpa

O
HO
6
1'
6' Podofilotoksin
5
1 5'
4'
2 2'
4
HO 3 3' OCH3
OH 6'' O
5'' 1''
O
HO
4''
2'' OH Phalerin ( 1)
T. diversifolia OH
3''
 BIOASSAY GUIDED ISOLATION
(I)

Nerium indicum Mill.

Nama daerah : Kembang Mentega, Jure
Familia : Apocynaceae

Dilaporkan sebagai antikanker secara tradisional

(Hartwel, 1982)
 Tumbuh subur sepanjang tahun di
Indonesia sebagai tanaman hias.
 Serbuk daun kering
CHCl3 ISOLASI SENYAWA
Sari CHCl3 Sisa (tak aktif)
Eter sentrifus (5oC,10’)

Sari tak larut Sari larut

Partisi dengan 3 corong pisah
n-heksan:MeOH:H2O (28:18:4 v/v)

Lapisan Atas Lapisan bawah Vakum cair kromatografi

F1 F2 F3 F4 F5 F6
BST 400 g/ml BST 400 g/ml
(72%) (100%) Preparatif KLT

NiO1 Ni-(2,3) Ni-C

(Oleandrin)
NiO2 NiO3 Preparatif KLT

NiO2A NiO2B NiO2C NiO2D

Uji Sitotoksik
Senyawa paling aktif
Identifikasi
Penelusuran
UV IR MS NMR MEKANISME KERJA
 n-heksana : etil asetat (3 : 1 Heksan : Eter (1 : 1, v/v) EtOAc
v/v)
100 % Etil asetat 100%

1 2 3 4 5 6 7 F5 1 2 3 4 5
1 2 3 4 5 6

KLT kromatogram hasil ekstraksi, partisi, fraksinasi dan isolasi

daun Nerium indicum Mill. (Fase diam : silika gel GF254 )
Dipandu dengan Bioassay
 A B C D

Heksan : Eter (1 : 3 v/v), PE : Eter (1 : 1 v/v) PE : Etil asetat (1 : 1 v/v)

CHCl3 : Eter (1 : 1 v/v)
3x

1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6

Kromatogram senyawa NiO1, NiO2A-D dan

NiO3
Keterangan:

1. NiO1 4. NiO2C
2. NiO2A 5. NiO2D
3. NiO2B 6. NiO3
Fase diam : Silika gel GF254
Nilai IC50(ng/ml) 6 senyawa hasil isolasi dari daun Nerium indicum Mill.
(konsentrasi tertinggi 12500 ng/ml)

Cell Test Compounds

Lines
NiO1 NiO2A NiO2B NiO2C NiO2D NiO3 Dox Cpt

EVSA-T 9.2 >12500 74.1 36.6 17.5 195.6 9.2 246

MCF-7 5.1 8339 16.4 9.7 11.5 99.3 6.2 610.9
T47-D 887 >12500 11109 >12500 >12500 >12500 -- --
A498 12.0 11882 11.2 7.4 22.7 124.8 91.9 1048
H226 12.7 7989 286.2 137.1 25.6 259.2 133 1518
IGROV 8.2 >12500 42.6 19.2 9.8 71.2 22.9 161.2
M19 9.0 >12500 32.2 12.5 12.1 89.3 15.6 239.5
WiDR 8.0 >12500 35.0 14.9 13.9 120.7 14.2 537.7
HeLa 4.64 3470 14.17 >12500 4.83 29.769 8.9 --
Vero 325.38 >12500 >12500 >12500 >12500 >12500 -- --
Catatan : A498=Renal, EVSA-T=Breast, H226=Lung, IGROV=Ovarian, HeLa=Serviks,
MCF-7=Breast, Vero= Normal sel, M19=Melanoma, WiDR=Colon, T47-D= Breast,
Dox=Doksorubisin, Cpt=Cisplatinum
 C-O-C

CH

OH C=O

Spektra UV (NiO2D) Spektra IR

CH3-C=O

H-19
-OCH3

5’-CH3
H-18
H-1’
-OCH3
CH3-C=O

C-21

H-
C-18

21
C-17
C-22

C-19

H-22H-16
C-1’
C-23

Spektra IR
C-20

CDCl3

Spektra 13C-NMR Spektra 1H-NMR

 O
21 
O
23
 
18 20 22
12
17
11
19 13
16 O
1 9 14
2
10 8 15 O
OH
3 5 7
O 4 6
H
1'

5' O 2'

HO
3' (C32H48O9)
4'
H 3CO

Struktur kimia 5α-oleandrin (NiO2D)

Western Blotting
Control 5-oleandrin

0 2 6 10 15 24 2 6 10 15 24 hrs

Bcl-2 A. 26 kDa

Bax B. 23 kDa

β-actin C.

Ekspresi protein Bcl-2 (A), Bax (B) dan β-actin (C) sel HeLa
setelah pemberian 5-oleandrin kadar 3,88x10-4mM inkubasi
selama periodik waktu
 C-23

C-20

C-23
C-17
CH3-C=O
C-20 C-16
264

C-22

C-22 C-1’

C-1’
C-3
C-21 C-5

Spektra 13C-NMR
C-17 -OCH3
Spektra UV (NiO2C)
-OCH3
C-13

C-18

C-19 C-19
C-18
5’-CH3

CDCl3
OH

H-22
CH

H-21
C=O

-O-CH3

Spektra 1H-NMR
Spektra IR

5’-CH3
C-O-C

H-18
H-19
 O
21 
O
23
 
18 20 22
12
17
11
19 13
16
1 9 14
2 15
10 8
OH
3 5 7
O 4 6
H
1'

5' O 2'

HO 4'
H 3CO
3'
(C30H44O7)

Struktur kimia 16,17-dehidrodeasetil-5-oleandrin.

PUBLIKASI
1. Wahyuningsih, M.S.H., Wahyuono, S, and Wayan, T.A. 2000, Efek Sitotoksik Oleandrin,
Senyawa Bioaktif Hasil Isolasi dari daun Nerium indicum Mill. Terhadap Sel Mieloma.
Berkala Ilmu Kedokteran, 32(4),235-241.
2. Wahyuningsih, M.S.H., Wahyuono, S, and Wayan, T.A., 2000, Isolasi dan Identifikasi
Senyawa Bioaktif dari daun Nerium indicum Mill. Majalah Farmasi Indonesia,
11(2),86-95
3. Prasetyawati C., Donatus, IA., Wahyuningsih MSH., Wahyuono, S., 2004. Sub chronic
toxicity asssay of aqueous extract of the leaves of Nerium indicum Mill. on male white
mice (Ratus norvegicus) strain wistar, Indonesian Journal of Pharmacy, 15(1), 13-19
4. Wahyuningsih, M.S.H., Mubarika S., RLH Bolhuis., K. Nooter., Ganjar, I. G and
Wahyuono S., 2004, Cytotoxicity of oleandrin isolated from the leaves of Nerium
indicum Mill. on several human cancer cell lines. Indonesian Journal of Pharmacy,
15(2), 96-103
5. Wahyuningsih MSH., Mubarika S., Ganjar IG., and Wahyuono S., 2005, Deteksi
mekanisme antikanker oleandrin hasil isolasi dari daun Nerium indicum Mill. terhadap
sel MCF-7 menggunakan fluorescein isothiocyanate-annexin V dan gel agarose,
Journal of Traditional Medicine, 10(33) 21-26.
6. Wahyuningsih MSH., Mubarika S., Ganjar IG., Hamann, M.T., and Wahyuono S, 2006
Identification of Cardenolide compounds as selective anticancer isolated from Nerium
indicum Mill. Leaves and its cytotoxic effect. Journal of Traditional Medicine,
11(37).13-19.
PUBLIKASI
7. Wahyuningsih MSH., Mubarika S., Gandjar IG., Wahyuono S., Boersma AWM.,
Nooter K., 2008, Detection of apoptosis mechanism on renal cancer cell treated by
16,17-dehydrodeacetyl-5α-oleandrin compound isolated from Nerium indicum Mill.
Leaves, Indonesian Journal of Pharmacy, 19 (4), 178-184.
8. Wahyuningsih MSH., Mubarika S., Bolhuis RLH., Nooter K., Oostrum RG.,
Wahyuono S., Gandjar IG., 2008, Selectivity of Compounds Isolated From The
Leaves Of Nerium indicum Mill. On Various Human Cancer Cell Lines, The Medical
Journal Of Malaysia, Suppl. A, 63, 24-25.
9. Wahyuningsih MSH.,Mubarika S., Mark T. Hamann, Gandjar IG., dan Wahyuono S.,
2008, Structure identification of potential compound as selective renal anticancer
isolated from Nerium indicum Mill. Leaves, Indonesian Journal of Pharmacy, 19 (2),
57-64.
10. Wahyuningsih MSH., Mubarika S., Ganjar IG., Wahyuono S.,Takeya T., 2017, 5α-
Oleandrin reduce Bcl-2 protein and increase Bax protein expression on Hela
cervical cancer cell, Univ Med, 36(2): 102-109.
11. Muhammad F., Yuliani FS., Wahyuningsih MSH, 2017,Aktivitas Antifibrotik Ekstrak
Klorofom Nerium Indicum dalam Menghambat Proliferasi Fibroblas Keloid dengan
MTT Assay, Prosiding Seminar Nasional “Peran Herbal untuk mencegah Proses
Degenerasi, Auditorium Fakultas Kedokteran UGM, 22 April 2017, ISBN: 978-602-
50277-0-3: p.46-50.
 BIOASSAY GUIDED ISOLATION
(II)

MAHKOTA DEWA
[Phaleria macrocarpa (Scheff.). Boerl.]

Nama daerah : Makuto Dewo

Familia : Thymelaeaceae
Dilaporkan sebagai antikanker secara
tradisional (Anonim, 2002), seduhan daun untuk
kanker hati (Syariefa, 2001)

 Tumbuh subur sepanjang tahun di

Indonesia
Procedure
Powdered, dried leaves
Macerated CHCl3

CHCl3 Ext. Material

MeOH Ext. Material

Bioassay Guided Fractionation (BST)

Active fraction
Bioassay Guided isolation
Chemical
Active cmpd. LC50 (BST)
Structure

Cytotoxicity and selectivity (IC50) assays on: Other mechanisms..?

1. Cancer cells in vitro
2. Normal cells in vitro
 MS spektra : mol. Weight (m/z 422),
HR-EIMS : C20H22O10

UV IR

MS
13C-NMR
1H-NMR

O
6 6'
HO 5 1'
5'
1
4'
2 2'
4
HO 3 3' OCH 3

O Phalerin
O
H 1''
HO LC50 = 1.5 x 10-1 mM
5'' OH H
H 6''
2'' (BST)
4'' H IC50 = 1,9 x 10-1 mM
3'' [Myeloma cells (NS-1)]
HO HO H
Cytotoxic activity of Phalerin on several cancer cells lines

No. Dose Cells death (%)

(ug/ml)
A498 Raji Vero
1 500 50,90 23,65 14,79
2 250 38,39 19,28 13,99
3 83,33 22,74 18,51 8,49
4 27,78 14,69 15,68 7,96
5 9,26 10,27 15,16 6,08
6 3,087 4,16 13,62 2,11
7 1,028 0,49 11,05 0,45
8 0,34 0 3,8 0

Note: A498 = Kidney cancer cells

Raji = Lymphoma cancer cells
Vero = Normal cells
Number of latex consumed by 100 of macrophages

No Concentration (g/ml) R1 R2 R3 Average

1 Control (medium) 285 270 258 271

2 Control (solvent, DMSO) 253 249 251 251
3 Phalerin 1,85 324 367 358 350
4 Phalerin 5,56 321 352 350 341
5 Phalerin 16,67 442 374 420 412
6 Phalerin 50 406 433 425 421
7 Phalerin 100 460 390 416 422
Increasing Number Of Latex Consumed By 100 Macrophages

No. Tested samples (μg/ml) Increasing ratio (%)

1 1.85 39.44
2 5.56 35.86
3 16.67 64.14
4 50 67.73
5 100 68.13
 A B C

(A). Makrofag sebelum diinkubasi control (100x)

(B). Makrofag setelah dicuci RPMI dan distimuli lateks (400x)
(C). Makrofag setelah pemberian phalerin 50 g/ml
 NO 2
ANTIRADICAL BIOASSAY .
(DPPH)
..
O 2N N N
..

NO 2

No. Samples R-1 R-2 R-3 Average

(µg/ml) (%) (%) (%) (%)

1 Phalerin 1.85 16.27 17.02 21.63 18.31

2 Phalerin 5.56 24.09 24.20 18.84 22.38

3 Phalerin 16.67 29.12 21.31 24.41 24.95

4 Phalerin 50 34.80 31.05 28.80 31.55

5 Phalerin 75 32.65 35.33 30.94 32.97

6 Phalerin 250 36.17 31.26 34.37 33.93

7 Phalerin 500 41.11 34.37 42.61 39.36

8 Quersetin 20 88.54 88.01 88.97 88.51

ANGIOGENESIS
Hasil pengamatan makroskopik pembentukan pembuluh darah dengan
sampel pada berbagai kadar dari kecil hingga besar
PUBLIKASI
1. Wahyuono S., Wahyuningsih MSH., Mubarika S., Sudarmanto BA., Setiadi J. and Ganjar IG.,
2005, Penetapan kadar Phalerin dalam buah Mahkota Dewa Phaleria macrocarpa (Scheff).
Boerl secara KLT-Densitometri. Journal of Traditional Medicine, 10(32).10-13.
2. Wahyuningsih MSH., Mubarika S., Artama WT., Ganjar I G and Wahyuono S., 2005,
Sitotoksisitas Phalerin hasil isolasi dari daun Mahkota Dewa Phaleria macrocarpa (Scheff).
Boerl terhadap berbagai sel kanker manusia in Vitro. Journal of Traditional Medicine,
10(32).5-9.
3. Wahyuningsih MSH., Mubarika S., Ganjar I G., Hamann MT., Rao, K.V. and Wahyuono S., 2005,
Phalerin, A New Benzophenoic glucoside isolated from the methanol extract of Mahkota Dewa
(Phaleria macrocarpa (Scheff). Boerl. Leaves. Indonesian Journal of Pharmacy, 16(1).51-57
4. Wijanarko H., Wahyuningsih MSH., Mubarika S., Ganjar I G and Wahyuono S., 2005, Aktivitas
Phalerin hasil isolasi dari daun Mahkota Dewa Phaleria macrocarpa (Scheff). Boerl sebagai
pemacu fagositosis makrofag in Vitro. Journal of Traditional Medicine, 10(33) 11-15.
5. Wijanarko H., Wahyuningsih MSH., Mubarika S., Ganjar IG and Wahyuono S, 2006, Antiradical
activity of Phalerin isolated from the leaves of Mahkota Dewa (P. macrocarpa (Scheff). Boerl),
Journal of Traditional Medicine, 11(35).16-20.
6. Wahyuningsih MSH., Mubarika S., Wahyuono S., 2008, Effect Of Phalerin Isolated From
Phaleria macrocarpa (Scheff) Boerl. Leaves on EVSA-T and P53 Protein Expression in Vitro,
Journal of Traditional Medicines, 13(44), 83-89.
 BIOASSAY GUIDED ISOLATION
(III)

Tithonia diversifolia (Hemsley) A. Gray

Nama daerah : Kembang bulan
Familia : Asteraceae

 Tumbuh subur sepanjang tahun di

Indonesia
Heksan : EtOAc
Heksan : EtOAc
(3 : 1, v/v)
Serbuk daun kering (500 gram) (3 : 1, v/v)

CHCl3

Ext CHCl3 Residu

IC50=16,61g/ml MeOH

n-Heksan Ext MeOH

IC50=1006,99 µg/ml CHCl3 MeOH
Insol Hk Sol

Sari Larut Sari tak larut

IC50.325,331g/ml) IC50. 3,078 g/ml)
Heksan : EtOAc
Vakum cair liq kromatografi
(3 : 1, v/v)

F1 F2 F3 F4 F5
32,17g/ml 11, 42 g/ml 6,22 g/ml 94,31 g/ml 41,74 g/ml

KLT Preparatif

A B C
146.89 g/ml 9,77 g/ml 136.58 g/ml

Identifikasi dan mekanisme kerja

F1 F2 F3 F4 F5 P
 KLT Isolat teraktif fase diam (silika gel GF254 ) dan fase
gerak {WB: EtOAc(1:1; v/v) (A), dan (3:1,v/v (B)}
Deteksi : UV 254 dan 366 nm
Nilai IC50 Isolat A, B, dan C kembang bulan pada sel HeLa

160 146.886
136.579
140

120

100

80

60 47.074

40

20

0
Isolat atas (A) Isolat tengah (B) Isolat bawah ©

IC50 (ug/mL)

PEMURNIAN
 Indeks Selektivitas Isolat Kembang Bulan
pada berbagai sel kanker

69.02
70

60

50

40.53
40

30
22.59

20 16.16 16.55

11.46
9.77 8.59
10 4.13 4.7
2.5 2.44 3.522
1.79 0.996
0.59
0
HeLa WiDR Myeloma RAJI MCF7 T47D M19 EVSA-T

IC50 (ug/mL) Selektivitas

Spektra UV (B) Spektra IR (B)

Spektra 13C-NMR Spektra 1H-NMR

Molekuler model dari Tagitinin C
(C19H24O6) BM=348
 A B

C D

Ekspresi caspase 9 sel WIDR inkubasi 6 jam (A). Kontrol, (B).

Pemberian Tagitinin C dosis ½ IC50, (C).Dosis IC50, (D).
Dosis 2xIC50 (Perbesaran 400x)
PUBLIKASI
1. Mahardika, AB., Wahyuono S., Wahyuningsih, MSH., 2016, Sitotoxicity of
Compound isolated from the leaves of Tithonia diversifolia (Hemsley) A.Gray)
against T47D, MCF-7 and EVSA-T cells, Journal of Pharmaceutics, Inpress
2. Ranti I., Wahyuningsih MSH., Wirohadidjojo YW., 2016, The Antifibrotic Effect of
Isolate Tagitinin C from Tithonia diversifolia (Hemsley) A. Gray on Keloid Fibroblast
Cell, Pan African Medical Journal, Inpress
3. Wahyuningsih, MSH., Wijayanti MA., Budiyanto A., Muhammad Hanafi, 2015,
Isolation and Identification of Potential Cytotoxic Compound from Tithonia
diversifolia (Hemsley) A. Gray Leaves, Int J Pharm Pharm Sci. 7 (6), 298-301.
4. Wahyuningsih, MSH., Wirohadidjojo, YW., Hidayat, R., Sadid, A., 2015, Antifibrotic
Effect of Standardized Ethanol Extract of Tithonia diversifolia (Hemsley) A. Gray on
Keloid Fibroblasts, Int J Pharmacognosy and Phytochemical Research, 7(4); 642-647
5. Syarif, RA., Wahyuningsih MSH., Mustofa, Ngatidjan, 2014, Inhibitory Activity of
Purified Extract of Tithonia diversifolia (Hemsley) A.Gray) Leaves on Plasmodium
falciparum Growth and Heme Polymerization, Journal of the Indonesian Medical
Association (J Indon Med Assoc), 64 (5), 228-233.
PUBLIKASI

6. Mardihusodo HR., Wahyuningsih MSH., Astuti I., 2013, The effect of active
compound isolated from the leaves of T. diversifolia (Hemsley) A. Gray on
cell cycle and angiogenesis of WiDr cell line, J Med Sci, 45 (3), September
7. Mahardika, AB., Wahyuono S., Wahyuningsih, MSH., 2016, Sitotoxicity of
Compound Wahyuningsih MSH., Syarif RA., Suharmi S., Murini Tri., Saputra F.,
Adiguno Suryo W., 2013, Selectivity of Purified Extract from the leaves of
Tithonia diversifolia (Hemsley) A.Gray) against Hela Cells, Trad. Med. J., 18(1),
22-28
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