LAPORAN AWAL PRAKTIKUM

KIMIA BAHAN ALAM FARMASI

ISOLASI ALKALOID DARI BUAH LADA HITAM
(Piper nigrum L)

OLEH:

NAMA : ALFATHRI YUNEDI

NO BP : 1811013018
SHIFT /KLP : 1 (SENIN) / 4
HARI/TGL : SELASA/12 MEI 2020
REKAN KERJA : 1. MAISUNNA BUNGA ULI 1811011046
2. SUCI WULANSARI 1811012040
3. YULMA HERDALINA 1811012052

LABORATORIUM KIMIA BAHAN ALAM FARMASI

FAKULTAS FARMASI
UNIVERSITAS ANDALAS
PADANG
2020
 BAB I
PENDAHULUAN
I.1 Tujuan

1. Mengetahui dan mempraktekan cara mengisolasi golongan senyawa alkaloid.

2. Mengetahui cara mengidentifikasi senyawa alkaloid hasil isolasi.

I.2 Manfaat

1. Praktikan dapat mengetahui tinjauan botani, kandungan kimia, kegunaan

tradisional, bioaktivitas, dan metode ekstraksi dari Piper nigrum L.
2. Praktikan dapat memahami teori tentang isolasi senyawa alkaloid (piperin)
dari Piper nigrum L.
3. Praktikan memahami bioaktivitas senyawa piperin
 BAB II
TINJAUAN PUSTAKA

2.1 Tinjauan Botani

Gambar 1: (Piper nigrum L)

2.1.1 Klasifikasi

Kingdom : Plantae
Divisi : Spermatophyta
Sub divisi : Angiospermae
Kelas : Dicotyledoneae
Ordo : Piperales
Family : Piperaceae
Genus : Piper
Spesies : Piper nigrum L [1]

2.1.2 Morfologi

Tanaman lada hitam memiliki bentuk seperti tanaman kayu memanjat. Akar lada
hitam merupakan akar tunggang, namun jika berkembang biak secara penyerakan
maka berakar serabut.Ukurannya kecil-kecil dan tidak panjang sebagaimana akar
tunggang biasanya.Sesuai dengan jenisnya,akar tanaman ini dibedakan menjadi dua
yakni akar lekat dan akar tanah.Akar lekat adalah akar yang tumbuh pada setiap ruas
buku yang ada di permukaan tanah, dan mempunyai panjang rata-rata 2,5-3,5 cm.
Dalam satu ruas buku bisa tumbuh sebanyak 10-15 helai akar. Kemudian akar tanah
adalah akar yang tumbuh pada batang tanaman lada yang berada di dalam
tanah.Batang lada berbentuk agak pipih dan beruas-ruas dengan panjang ruas 4-7cm,
panjang batang mencapai 15cm. Daun pada tanaman ini berupa daun tunggal, dengan
panjang 12-18cm dan lebar 3 cm dengan panjang tangkai 4 cm. Daun tumbuhan
Piper nigrum L berbentuk bulat dengan ujung daun meruncing, pertulangan daun
curvinervis ukuran daunnya mencapai 12-18cm lebar 5-10cm dan tumbuh berselang-
seling.Warna daun dewasa hijau, permukaan daun halus dan mengkilap. Bunganya
merupakan bunga majemuk dan berlokasi terminal. Buahnya berbentuk bundar secara
botani dikenal sebagai buah berbiji. Buah matang bewarna merah.[2]

2.1.3 Sinonim

Nama daerah: Lada (aceh), pedes(sunda),merica(jawa),lada kecik

(Bengkulu),marica(makasar),rica jawa (ternate), dan malita iodawa (gorontalo).[3]
Nama luar negri : Black pepper(inggris), hu zhiau(cina).[3]

2.1.4 Habitat dan sebaran

Tanaman lada hitam(Piper nigrum L) merupakan tanaman tropis yang membutuhkan
curah hujan dan kelembapan yang cukup. Lada hitam tumbuh dengan baik pada
daerah dengan ketinggian 1500 m diatas permukaan laut. Suhu yang di kehendaki
antara 10-400C. Lada hitam dapat tumbuh subur pada tanah yang memiliki pH 4,5-
6,5. Tanaman lada hitam berasal dari wilayah india dan tersebar di daerah pantai
Malabar, Malaya , jawa, dan Thailand. [3]
2.2 Kandungan Kimia
Penapisan fitokimia buah Piper nigrum L menunjukan bahwa buah ini megandung
4% alkaloid,piperine,pipene,piperamid dan piperamin, yang merupakan senyawa
yang paling aktif di isolasi dari Piper nigrum L yang memiliki potensi untuk berbagai
aktivitas biologis. [4]
Piperin terdiri dari empat isomer yaitu piperin,isopiperin,isochavicine,dan chavicine.
Piperamid adalah metabolit sekunder yang ada di bagian luar lada dan biji lada
hitam.[4]
Berbagai peneliti telah mengisolasi senyawa dari spesies ini termasuk senyawa
fenolik, berbagai turunan dari lignin, terpen, kalkon, alkaloid, dan steroid.
Brachyamide B, dihydropiperland, kelompok benzamide (2E,4E) N-ecosadienoyl
pereridine, N- trans –feroloyitryamine, N-formylpiperidine, guineensine,
pentadienoyl, N-isobutyldeca dienamide, isobutyl – eicosadienamide, isobutyl
eicosatrienamide, isobutyl octadienamide, piperamide, piperamine, piperettine,
piperamine, piperin, piperolein B trichostachine, sarmentosine tricholein retro
fractamide A.[5]
Struktur
Piperine (1-[5-(1,3-Benzodiosol-5-yil-1-oxo-2,4-pentadienyl])

Gambar 2: Struktrur piperin

2.3 Kegunaan Tradisional

 Di Jawa penggunaan tradisional lada hitam dicampur dengan jahe dan madu,
digunakan sebagai obat kuat sesudah melahirkan. Secara eksternal digunakan untuk
kulit sebagai counter-irritant, bisa juga digunakan sebagai tapal pada perut yang
mulas atau sakit. Untuk rematik, sakit kepala dan dioleskan. Piper nigrum
direkomendasikan untuk berbagai pengobatan secara eksternal (dioleskan)3 .

 Di China lada dipercaya memiliki aktivitas insektisida terhadap nyamuk dan lalat,
anti bakteri, anti jamur, membantu menghilangkan rasa sakit rematik, kedinginan,flu,
pilek,nyeri. Sebagai obat eksternal digunakan untuk rubefacient sebagai aplikasi local
untuk sakit radang tenggorokan dan beberapa gangguan kulit, antimutagenik5 .

 Di Eropa lada banyak digunakan untuk tambahan masakan. Daya tarik lada sebagai
bahan masakan karena adanya zat piperin. Lada diketahui berkhasiat dalam
menambah nafsu makan, memperbaiki sistem pencernaan, menambah cita rasa
makanan, meluruhkan keringat, meningkatkan sekresi lambung, meluruhkan flatus,
mengurangi rasa mual, menigkatkan suhu tubuh, serta sebagaistimulan dan anti
bakteri. Sementara itu lada hitam dipercaya dapat digunakan untuk mengobati
konstipasi, diare, sakit telinga, gangren, penyakit jantung, hernia, suara serak,
gangguan pencernaan, gigitan serangga, kesulitan tidur, linu sendi, gangguan hati,
paru, bisul dalam mulut dan sakit gigi [7]

2.4 Bioaktivitas

2.4.1 Ekstrak

1. Aktivitas Antimikroba
Di antara beberapa ekstrak pelarut (air dingin, air panas dan methanol) dari buah
Piper nigrum L ditemukan bahwa ekstrak air dingin memiliki zona maksimum
terhadap e-coli 1/4/23 mm, sedangkan ekstrak air panas menunjukan zona maksimum
inhibisi terhadap S. thypi dan S.aureus 1/4/22 mm. Selain itu ekstrak dari methanol
menunjukan penghambatan tertinggu terhadap e-coli, S.thypi, dan S.aureus dengan
1/4
/21mm.[8]
2. Aktivitas Antioksidan
Ekstrak biji Piper nigrum L memiliki aktivitas pembilasan yang lebih tinggi dari
pada ekstrak etanol terhadap opph, anion superoksida, hydrogen peroksida dan
aktivits antioksidan total berdasarkan metode tiosianat, sedangkan yang terakhir
menunjukan daya pereduksi besi yang lebih tinggi. Kekuatan antioksidan daun Piper
nigrum L dinilai menggunakan sejumlah tes . Diantara tiga ekstrak pelarut (etil
asetat,aseton dan air). Ekstrak etil asetat menunjukan efek OPPH, ABTS dan
Superperoksida tertinggi, sementara ekstrak aseton menunjukan penghambatan
tertinggi terhadap hydrogen peroksida, nitrat oksida dan paling efektif dalam uji
fosfomolibdenum.[9]

2.4.2 Senyawa Metabolit Sekunder

1. Aktivitas antimikroba
Di dalam Piper nigrum L terdapat dua senyawa fenolik yaitu 3,4-dihydroxyphenyl
etanol glukosida (A) dan 3,4-dihydroxy-6(N-ethylamino) benzamide (B). Ditemukan
menghambat pertumbuhan pathogen bawaan makanan, termasuk S.aureus,B.cereus,
E-coli dan S.thypi (dengan pengecualian senyawa A untuk bakteri yang terakhir)
secara umum senyawa A lebih efektif dari pada senyawa B tetapi kurang efektif
terhadap kontrol positif 4-methylcatechol. Selain itu kombinasi piperin dengan
ciprofloxacin secara signifinikan mengurangi MIC dan konsentasi pencegahan mutasi
ciprofloxacin terhadap S.aureus termasuk strain yang resisten metisilin. Kehadiran
piperine menghasilkan peningkatan akumulasi dan penurunan penghabisan etidium
bromide pada tipe luas dan strain mutan (cipi-1) sehingga menunjukan perannya
dalam penghambatan pompa penghabisan bakteri. Selain itu ditemukan bahwa asam
piperik menunjukan aktivitas anti bakteri yang lebih tinggi dari piperin (MIC) dalam
kisaran 312,5-625 mg/ml terhadap banyak bakteri gram positif dan gram negatif.[10]

2. Aktivitas Antioksidan
Senyawa Fenolik dari Piper nigrum L termasuk 3,4-dihydroxyphenol ethanol
glucoside glucoside . 3,4-dihydroxy -6- (N-ethylamino) benzamide dan glucoside
asam fenolik ditemukan untuk mencari radikal DPPH dengan nilai EC50 masing
masing 0,76 , 0,27, dan o,12 mg/ml. Senyawa Piper nigrum L lain N-
transferuloyiltryramine juga menunjukan aktivitias DPPH. Senyawa yang tersisolasi
seperti piperin dan asam piperat juga menunjukan aktivitas DPPH dan antioksidan
yang lebih tinggi dalam uji fosfomolibdenum di bandingkan dengan ekstrak etanol
buah beri. Piperin adalah DPPH yang lebih kuat dibandingkan asam piperik,
Sementara asam piperik menunjukan aktivitas produksi molibdenum yang lebih
tingggi. [11]

2.5 Metode Ekstraksi

Metode ekstraksi yang digunakan yaitu maserasi. Maserasi merupakan jenis ekstraksi
sederhana karena pengerjaannya hanya di lakukan dengan cara merendam bahan
simplesia dalam cairan penyari. Cairan penyari akan menembus dinding sel dan
masuk ke dalam rongga sel yang mangandung zat aktif. Zat aktif akan larut dan
adanya perbedaan konsentrasi antara larutan zat aktif di dalam sel dengan luar sel ,
maka zat aktif (zat terlarut) ditarik keluar. Peristiwa tersebut terjadi berulang kali
hingga terjadi keseimbangan konsentrasi antara larutan diluar dan di dalam sel.[12]
Metode maserasi digunakan untuk penyarian simplesia yang mengandunng zat aktif
yang mudah larut, mudah mengembang dalam cairan penyari , tidak mengandung
benzoin, tiraks dan lilin. Keuntungan dari metode ini adalah cara pengerjaan dan
peralatan yang digunakan sederhana dan mudah di lakukan. Pada umumnya maserasi
dilakukan dengan merendam sampel dalam pelarut yang sesuai selama tiga hari pada
suhu ruang.[12]
 BAB III
PROSEDUR PERCOBAAN

3.1 Alat dan Bahan

Alat:

 Corong
 Botol 100 ml
 Vial
 Pipet tetes
 Seperangkat alat rotary evaporator
 Chamber
 Pentotol
 Spatel

Bahan :

 Buah lada hitam 20 g

 Methanol
 Kalium hidroksida
 Etil asetat
  Kapas / kertas saring
 Plat KLT
 Larutan dragendorf

3.2 Cara kerja

Buah lada hitam kering

Grinder & timbang 20 g

Simplesia halus lada hitam

Maserasi dgn methanol (3-5 hari) dan saring

Ekstrak methanol lada hitam

Diuapkan menggunakan rotary evapotaror

Ekstak kental lada hitam

+KOH 10% (10 ml) saring, diamkan

Kristal
Rekristalisasi dgn etil asetat dan lakukan

Pendesakan dgn n-heksan

Krsital murni piperin

 Uji KLT
fase diam : silica gel 60 F 254
Fase gerak : n-heksan : etil asetat(2:3)
panjang gelombang 254nm

DAFTAR PUSTAKA

[1] Tjitrosoepomo, G . Taksonomi Tumbuhan (Spermatophyta). Yogyakarta: UGM

press. 2010

[2] Sarjani, TM, Mawardi, Pandia, Wulandari. Identifikasi Morfologi dan Anatomi
Tipe Stomata Famili Piperaceae di Kota Langsa. 2017:1(2):182-191

[3] Munawaroh dan Yuzammi. The Diversity and Conservation of Piper (

Piperaceae) in Bukit Barisan Selatan National Park, Lampung Province. Media
konservasi.2017:22(2):118-128

[4] Ahmad, N. Biological Role of Piper nigrum L. 2012:2(1):945-953

[5] Gorgani, L. Piperine The Bioactive Compound of Black Papper:From Isolation to

Medical Formulation .2017: 16:124-140

[6] Marta, J. Antibacterial Activity of Piperine Extracted from Piper nigrum Againts
e-coli dan bacillus subtilis. Europan Journal of Biomedical and Pharmaceuticals
Sciences.2016 Mei : 3(6): 497-500

[7] Agoes, Azwar. Tanaman Obat Indonesia. Jakarta : Salemba Medika. (2010)
[8] Hartati, Pagarra. Differences of Ethanol Extract and Ethyl Acetate of Piper leaf
against Anti Microbial Activity.2017 November : 7(2) : 1-7

[9] Insanu, M, Martiani. Comparison of Antioxidant acrivities from Four Spesies of

Piper. 2017: 7(2): 305-312

[10] Hikal. Antibacterial Activity of Piperine and Black Pepper oil. 2018 December:
15 (4) : 877-880

[11] Andrade, Regina. Antioxidant Activity of Black Pepper Oil Obtained

Supercritical CO2.2014 : 14(2) : 1-5

[12] Najib, A. Ekstraksi Senyawa Bahan Alam. Yogyakarta: Deepublish.2018

 REVIEW JURNAL

Judul : Evaluation of Aristolochia indica L. and Piper nigrum L. methanol

extract against centipede Scolopendra moristans L. using Wistar albino rats
and screening of bioactive compounds by high pressure liquid
chromatography: a polyherbal formulation
Journal : journal homepage: www.elsevier.com/locate/biopha
Tahun : 2018
Penulis : D. Sivaraja., S. Shanmugamb., M. Rajana., S.P Sasidharana., S.
Sathyanarayanana., K. Muniyandia ., P. Thangaraja,., A.A de Souza Araújob
Reviewer : Alfathri Yunedi

Metode Penelitian
1. Identifikasi tanaman dan kelabang dan estimasi protein racun kandungan
A. indica dan P. nigrum dikumpulkan dari Dharapuram dan Ooty, Tamil Nadu, India
selama bulan Oktober dan Desember (2014), masing-masing. Kemudian tanaman itu
diidentifikasi oleh Botanical Survey of India, Coimbatore (Ref. No: BSI / SRC / 5/23 /
2014-15 / Tech.1253 dan 1457 masing-masing). Kelabang dikumpulkan dari
Dharapuram dan diidentifikasi oleh Survei Zoologi India, Kerala. Scolopendra
morsitans L. (no. Reg: ZSI / WGRC / IR / INV / 4152). Selanjutnya, racun
dikumpulkan menggunakan alat listrik dengan menstimulasi kelenjar racun dan
disimpan pada suhu −20 ° C untuk penggunaan lebih lanjut. Kandungan protein racun
itu diukur menggunakan metode Lowry.

2. Ekstraksi dan analisis fitokimia

100 g bubuk kulit akar A. indica (RBAI), biji P. nigrum (SPN) dan keduanya dalam
kombinasi rasio 1: 1 (CAIPN) diekstraksi menggunakan Alat soxhlet dengan metanol
(400 mL). Ekstrak dipekatkan dengan rotary vacuum evaporator (Equitron, India),
udara kering, dan ditimbang. Persentase pemulihan ekstrak dihitung menggunakan
formula: Berat ekstrak × 100 / Berat sampel. Total fenolat, tanin dan kandungan
flavonoid ekstrak (masing-masing 200 dan 400 μL) ditentukan pada 725 dan 510 nm
menggunakan spektrofotometer (UV-1800, Shizmadzu) dan hasilnya dinyatakan
sebagai Gallic setara asam (GAE), setara asam Tannat (TAE) dan Rutin ekuivalen (RE)
masing-masing.

3.Uji antioksidan in vitro

Aktivitas antioksidan dari ekstrak ditentukan menggunakan DPPH • aktivitas
pemulungan, diukur pada 517 nm dan hasilnya adalah dinyatakan dalam IC50 (μg /
mL); Uji fosfomolibdenum dan hasilnya dinyatakan dalam mg ekstrak ekivalen asam
askorbat (AAE) / g; Uji peroksidasi lipid digunakan untuk mengukur% penghambatan
peroksidasi pada 532 nm. Semua absorbansi sampel diukur menggunakan
spektrofotometri UV-vis

4. Analisis kromatografi cair tekanan tinggi (HPLC)

Ekstrak CAIPN dilarutkan dalam metanol (20 mg / 100 mL) dan disonikasi selama 30
menit dan disaring melalui filter membran 0,45 μm (PTFE). Senyawa fitok standar juga
disiapkan dengan cara yang sama. Analisis dilakukan dengan menggunakan degasser
DGU-20A3, dua LC-20CE pompa, injektor otomatis SIL-20A HT, oven kolom CTO-
20A, detektor array fotodioda (DAD) SPDM20Avp dan sistem CBM-20A pengontrol
(Shimadzu Co., Kyoto, Jepang). Pemisahan kromatografi dilakukan menggunakan
Phenomenex luna C18 analitik kolom 4,6 × 250 mm (ukuran partikel 5 μm). Volume
injeksi adalah 20 μL dan laju aliran 1 mL / menit. Fase seluler terdiri dari gradien asam
asetat A, air dan 1% (v / v) (sistem Milli-Q, Millipore, Bedford, USA) dan B, methanol.
Detektor dipasang pada 340 nm untuk mendapatkan kromatogram. Akhirnya, puncak
sampel adalah dibandingkan dengan standar waktu retensi puncak dan kuantifikasi
selesai.
Hasil
1. Ekstrak persentase pemulihan dan konten fitokimia
Persentase pemulihan ekstrak berada di urutan sebagai: CAIPN (16,4%)> RBAI
(7,8%)> SPN (6,01%). Ekstrak CAIPN menunjukkan kandungan fenolat, tanin, dan
flavonoid total yang lebih tinggi daripada RBAI dan Ekstrak SPN dan signifikan pada
p <0,05.
2. Antioksidan in vitro dan pemblokiran proteolisis
Ekstrak CAIPN menunjukkan aktivitas mengurangi molibdenum yang baik,
penghambatan peroksidasi lipid dan aktivitas pembersihan radikal DPPH dari RBAI
dan SPN dengan signifikansi p <0,05. Kandungan protein dari racunnya adalah 2,72
μg setara BSA / mL. Memblokir racun proteolitik Aktivitas dengan ekstrak CAIPN
sangat luar biasa dibandingkan dengan dua sampel lainnya signifikansi (p <0,05)
3. Analisis HPLC
Ekstrak metanol CAIPN mengungkapkan keberadaan dua utama puncak pada waktu
retensi yang berbeda. Puncak 1 (RT: 11.827 mnt), Puncak 2 (RT: 16,401 mnt)
diidentifikasi sebagai asam ferulat (15,04 ± 0,09 ug / mg) dan quercetin (35,30 ± 0,30
μg / mg) .

Kesimpulan
Tikus yang diberi perlakuan CAIPN memiliki tingkat elektrolit yang signifikan
properti dan mempertahankan biomarker lipid dan darah, hati dan ginjal (tanpa
hipersensitif) dengan dosis yang lebih rendah. Dengan demikian itu menunjukkan S.
potensi penetral racun morsitans dari ekstrak CAIPN lebih luar biasa daripada ekstrak
metanol RBAI dan SPN. Hasil penelitian ini mengkonfirmasi manfaat terapeutik lebih
tinggi dalam polyherbal daripada terapi tanaman tunggal. Selanjutnya, isolasi
antivenomic senyawa dari ekstrak CAIPN sedang dalam proses yang membantu untuk
merumuskan obat antivenomik ecofriendly baru yang efektif untuk kesejahteraan
manusia.
 Biomedicine & Pharmacotherapy 97 (2018) 1603–1612

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Original article

Evaluation of Aristolochia indica L. and Piper nigrum L. methanol extract T

against centipede Scolopendra moristans L. using Wistar albino rats and
screening of bioactive compounds by high pressure liquid chromatography:
a polyherbal formulation
Dhivya Sivaraja, Saravanan Shanmugamb, Murugan Rajana, Sreeja Puthanpura Sasidharana,
⁎
Saikumar Sathyanarayanana, Kasipandi Muniyandia, Parimelazhagan Thangaraja, ,
b
Adriano Antunes de Souza Araújo
a
Bioprospecting Laboratory, Department of Botany, Bharathiar University, Coimbatore-46, Tamil Nadu, India
b
Department of Pharmacy, Federal University of Sergipe, Cristovao, Sergipe, CEP, 49100-000, Sao Cristovao, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: The present study was aimed to explore the anti-venom activity of Aristolochia indica and Piper nigrum plants
Scolopendra moristans against the centipede (Scolopendra moristans) envenomation in animal model. In vtiro phytochemical, antioxidant
Hypersensitivity and blocking of proteolysis were carried out by using standard spectrophotometric methods. In vivo anti-venom
Aristolochia indica activity of methanol extracts was determined using Wistar albino rats after fixing lethal and effective doses. The
Piper nigrum
electrolytes, lipid, liver, kidney, hematological parameters were analyzed and histopathology of skin and liver
Polyherbalism
were also examined. Anti-skin cancer by MTT method and HPLC analysis were also carried out. The CAIPN
Anti-venom
extract showed higher total phenolics (150.65 ± 0.08 mg GAE/g extract) and flavonoids (158.97 ± 0.93 mg
RE/g extract) content. Further, the same extract revealed the higher molybdenum reducing, inhibition of lipid
peroxidation (80.08 ± 0.22%), DPPH radical scavenging (3.05 μg/mL), and blocking of proteolysis activities
(96.45 ± 0.04%). The parameters like hypersensitivity, electrolytes, lipids, blood components, liver and kidney
marker of the CAIPN methanol extract (200 mg/kg) treated envenomated rats was remarkable and same as in the
normal animals. Such status was also achieved by RBAI and SPN at 600 mg/kg. The histopathological scoring of
skin and liver confirmed the venom neutralizing activity of CAIPN. Also, the CAIPN methanol extract was no-
table in anti-skin cancer activity (208 μg/mL). The presence of the ferulic acid (04 ± 0.09 μg/mg) and quercetin
(35.30 ± 0.30 μg/mg) like compounds was confirmed by HPLC analysis. Hence, the present investigation re-
sults conclude that the CAIPN was significant in their action and this polyherbal formulation could be considered
as a new source for the pharmaceutical industries to develop a new effective, ecofriendly anti-venom drug.

1. Introduction
Centipede (Class: Chilopoda) is the venomous terrestrial arthropods
especially, Scolopendromorpha (order) is the largest and most dan-
gerous organism. Its envenomation case reports were recorded with the
effects including rhabdomyolysis, acute renal failure, intracranial he-
morrhage, pruritus, skin disorders, fever and chills [1,2], acute myo-
cardial infarction [3], tachypnea, palpitation and hypotension [4].
These effects are due to the complexity of venom components like
metalloprotease, hyaluronidase, phospholipase A2, CAP (CRiSP (cy-
steine rich proteins), Allergen (Ag-5), and Pathogenesis-related (PR-1))
proteins, cardiotoxin, cytotoxin, myotoxin, neurotoxin, histamine,
serotonin, ion channel modulators, etc. that are responsible for the
hypersensitive allergic reaction [5]. Furthermore, the enzyme hyalur-
onidase is also expressed highly in cancer cells, helps in rapid multi-
plication [6]. In this connection, its envenomation is may be considered
as risk factor for cancer. In Allopathy medicine system, drugs like
steroids, antihistamine, analgesic, anti-inflammatory, Tetanus toxoid
(TT) injection are prescribed to overcome the hypersensitive effects but
this medication aid for temporary suppression of effects alone which is
the major problem (unpublished data). In point of chemotherapy above
said medicine is under ambiguity.
The traditional healing system Ayurveda was highlighted the poly-
herbalism and its therapeutic benefits in “Sarangdhar Samhita” [7]. The

⁎
Corresponding author at: Department of Botany, Bharathiar University, Coimbatore 641 046, India.
E-mail address: drparimel@gmail.com (P. Thangaraj).

https://doi.org/10.1016/j.biopha.2017.11.114
Received 27 September 2017; Received in revised form 19 November 2017; Accepted 20 November 2017
0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
D. Sivaraj et al. Biomedicine & Pharmacotherapy 97 (2018) 1603–1612

Table 1
The phytochemicals content, antioxidant and blocking of total proteolytic activity of RBAI, SPN and CAIPN.

Samples Total phenolics(mg Tannins (mg TAE/g Flavonoids(mg RE/g Phosphomoly-bdenum Lipid peroxidation DPPH radical Inhibition of
GAE/g extract) extract) extract) assay(mg AAE/g extract) (%) scavenging Proteolytic activity
activity (IC50) (%)

RBAI 54.27 ± 0.77c 21.31 ± 0.77c 76.46 ± 0.87b 76.36 ± 1.24c 74.54 ± 0.28 b
35.92 50.09 ± 0.05 c

SPN 102.02 ± 2.48b 52.38 ± 2.50b 79.65 ± 0.43 b 130.82 ± 1.24 b

69.10 ± 0.12 c
12.91 54.34 ± 0.01 b

CAIPN 150.65 ± 0.08a 72.43 ± 0.16a 158.97 ± 0.93 a 172.21 ± 3.02 a

80.08 ± 0.22 a
3.05 96.45 ± 0.04 a

GAE – Gallic Acid Equivalents; TAE = Tannic acid Equivalents; RE – Rutin Equivalents; Values are mean of triplicate determination (n = 3) ± SD; statistically significant at p < 0.05
where a > b > c in each column. Except DPPH radical scavenging activity column.

Fig. 1. Lethal dose of venom and effective dose of extracts of RBAI (Root bark of Aristolochia indica); SPN (Seed of Piper nigrum) and CAIPN (Combination of Aristolochia indica and Piper
nigrum). (a). Lethal dose (LD) of venom; (b) Effective dose (ED) of RBAI (Root bark of Aristolochia indica); (c) Effective dose (ED) of SPN(Seed of Piper nigrum); (d) Effective dose (ED) of
CAIPN (Combination of Aristolochia indica and Piper nigrum).

Aristolochiaceae and Piperaceae family medicinal plants Aristolochia
indica and Piper nigrum respectively are used as good sources of anti-
venom by the traditional healing practitioners [8]. Both plants are used
to treat skin diseases [9,10]. Furthermore, the scientific articles re-
ported that the presence of chemical constituents like ishwarone, aris-
tolochene, ishwarol, aristolinidquinone and aristolochic acid with the
properties including anti-inflammatory, anti-phospholipase A2, anti-
hemorrhagic activity, enzyme inhibitory and anti-snake venom in A.
indica [11]. The phytochemicals like 4-nerolidylcatechol, piperine, tri-
cholein, piperamide, piperamine were reported in P. nigrum with the
antimicrobial, anticancer, immunomodulatory, antioxidant, analgesic
and antihypertensive activities [12,13]. Both plants were studied sci-
entifically for their many pharmacological activities separately. Still
now, there is no scientific report on anti-centipede venom activity of
polyherbal formulation using this both plants. Hence, the present study
aimed to investigate the effectiveness of polyherbal formulation to heal
Scolopendra morsitans envenomation effects and also to provide the
scientific validation.
2. Materials and methods
2.1. Chemicals
Chemicals used in this study were analytical and HPLC grade,
purchased from Himedia, Mumbai, and Sigma-Aldrich, Bangalore.

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Fig. 2. Hypersentive behaviour of envenomated rats. (a) Itching (b)Writhing (c) Paw licking (d) Hemolytic lesion after envenomaiton. Group 2: 0.6% CMC (p.o.); Group 3: Standard drug
“Medrol” (295 mg/kg p.o.); Group 4: Methanol extract of Root bark ofAristolochia indica (RBAI: 200 mg/kg p.o.); Group 5: Methanol extract of Root bark of Aristolochia indica (RBAI:
400 mg/kg p.o.); Group 6: Methanol extract of Root bark of Aristolochia indica (RBAI:600 mg/kg p.o.); Group 7: Methanol extract of Seed of Piper nigrum (SPN:200 mg/kg p.o.); Group 8:
Methanol extract of Seed of Piper nigrum (SPN:400 mg/kg p.o.); Group 9: Methanol extract of Seed of Piper nigrum (SPN:600 mg/kg p.o.); Group10: Methanol extract of Combination of
Aristolochia indica and Piper nigrum (CAIPN:100 mg/kg p.o.); Group11: Methanol extract of Combination of Aristolochia indica and Piper nigrum (CAIPN:200 mg/kg p.o.); Group12:
Methanol extract of Combination of Aristolochia indica and Piper nigrum (CAIPN:300 mg/kg p.o.). Values (Mean ± SEM) showing significance (< control ANOVA, *p < 0.001) are
compared to envenomated vechile control.

2.2. Identification of plants and centipede and estimation of venom protein using the electrical instrument by stimulating the venom gland [14]
content and stored at −20 °C for further use. The protein content of venom was
measured using Lowry’s method [15].
A. indica and P. nigrum were collected from Dharapuram and Ooty,
Tamil Nadu, India during the month of October and December (2014),
2.3. Extraction and phytochemical analysis
respectively. Then the plant was identified by Botanical Survey of India,
Coimbatore (Ref. No: BSI/SRC/5/23/2014-15/Tech.1253 and 1457
The 100 g powder of root bark of A. indica (RBAI), seed of P. nigrum
respectively). The centipede was collected from Dharapuram and
(SPN) and both in combination of 1:1 ratio (CAIPN) was extracted using
identified by Zoological Survey of India, Kerala. Scolopendra morsitans
Soxhlet apparatus with methanol (400 mL). The extracts were con-
L. (Reg. no: ZSI/WGRC/IR/INV/4152). Further, venom was collected
centrated by rotary vacuum evaporator (Equitron, India), air dried, and

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Table 2
Lipids and electrolytes level in the envenomated rats.

Group Cholesterol (mg/dl) Triglycerides (mg/ HDL Cholesterol LDL Cholesterol VLDL Cholesterol Sodium (mEq/I) Potassium (mEq/I) Calcium (mEq/I)
dl) (mg/dl) (mg/dl) (mg/dl)

1 65.54 ± 0.59 100.89 ± 0.70 53.36 ± 0.33 13.63 ± 0.14 20.17 ± 0.14 142.55 ± 1.24 5.1 ± 0.43 9.93 ± 0.04
2 29.54 ± 0.66* 19.05 ± 0.05* 19.30 ± 0.39* 3.08 ± 0.01* 3.81 ± 0.01* 98.87 ± 0.18* 7.02 ± 0.06* 3.68 ± 0.51*
3 34.90 ± 0.01* 36.73 ± 0.26* 23.57 ± 0.16* 5.52 ± 0.40* 7.34 ± 0.05* 121.65 ± 2.45* 6.82 ± 0.80* 5.33 ± 0.55*
4 34.90 ± 0.01* 36.73 ± 0.26* 23.57 ± 0.16* 5.52 ± 0.40* 7.34 ± 0.05* 102.25 ± 1.48* 8.23 ± 0.58 4.06 ± 0.04*
5 55.01 ± 0.02* 78.27 ± 0.32* 36.37 ± 2.15* 12.89 ± 0.01 15.65 ± 0.06* 112.65 ± 1.07* 6.42 ± 0.05 6.33 ± 0.55*
6 64.96 ± 0.69 99.86 ± 0.91 99.86 ± 0.91 13.56 ± 0.09 19.97 ± 0.18 141.88 ± 0.18 4.86 ± 0.05 9.30 ± 0.50
7 29.09 ± 0.01* 29.73 ± 0.26* 21.23 ± 0.67* 21.09 ± 0.67* 5.94 ± 0.05* 98.58 ± 1.45* 8.07 ± 0.02* 3.96 ± 0.04*
8 52.34 ± 0.55* 52.34 ± 0.55* 68.27 ± 0.32* 31.70 ± 0.16* 51.09 ± 0.75* 103.65 ± 1.54* 7.23 ± 0.06* 5.96 ± 0.03*
9 65.16 ± 0.56 100.97 ± 1.56 53.76 ± 0.71 43.76 ± 0.71* 59.03 ± 0.70 141.55 ± 0.97 4.76 ± 0.66 9.59 ± 0.53
10 39.75 ± 1.14* 70.73 ± 0.86* 37.57 ± 1.14* 6.51 ± 1.94* 14.14 ± 0.17* 102.92 ± 2.57* 6.89 ± 0.005* 6.96 ± 0.04*
11 64.34 ± 1.50 99.74 ± 0.70 53.37 ± 1.39 13.68 ± 1.46 19.94 ± 0.14 140.65 ± 0.56 4.89 ± 0.44 9.33 ± 0.55
12 65.16 ± 0.56 99.92 ± 0.09 53.76 ± 0.71 12.99 ± 0.01 19.98 ± 0.01 142.55 ± 0.76 5.29 ± 0.44 9.86 ± 0.05

Group 1 (Normal); Group 2 (vehicle control); Group 3 (300 mg/kg Medrol); Group 4 (200 mg/kg RBAI extract); Group 5 (400 mg/kg RBAI extract); Group 6 (600 mg/kg RBAI extract);
Group 7 (200 mg/kg SPN extract); Group 8 (400 mg/kg SPN extract); Group 9 (600 mg/kg SPN extract); Group 10 (100 mg/kg CAIPN extract); Group 11 (200 mg/kg CAIPN extract);
Group 12 (300 mg/kg CAIPN extract).Values (Mean ± SEM) showing significance (2 sided post hoc ANOVA, *p < 0.001) are compared to normal control. Hence, the values which are
not sigificant are close to the values of normal control.

Table 3
Hematological parameters of envenomated rats.

Group Neutrophils (%) Lymphocytes (%) Eosinophils (%) Monocytes (%) Basophils (%) Platelet count (103/ μL) RBC count (Millions/μL)

1 22.24 ± 0.18 51.48 ± 0.61 0.12 ± 0.01 1.36 ± 0.23 0.55 ± 0.01 1010 ± 23 8.92 ± 1.12
2 45.3 ± 0.06* 90.71 ± 0.53* 5.86 ± 0.05* 5.45 ± 0.40* 1.07 ± 0.01* 1375.33 ± 4.61* 4.81 ± 0.121*
3 14.65 ± 0.57* 78.47 ± 0.40* 0.08 ± 0.00* 3.63 ± 0.08* 0.20 ± 0.03* 1171 ± 6.08* 8.92 ± 0.01*
4 37.90 ± 0.10* 78.63 ± 0.58* 4.86 ± 0.05* 4.08 ± 0.00* 0.96 ± 0.03* 1175.33 ± 4.61* 5.52 ± 0.23*
5 29.6 ± 0.22* 68.74 ± 0.13* 3.63 ± 0.53* 3.63 ± 0.23* 0.61 ± 0.00 1266 ± 10.01* 6.04 ± 0.01*
6 19.66 ± 0.32 51.59 ± 0.52 0.12 ± 0.00 1.60 ± 0.20 0.52 ± 0.02 1006.33 ± 3.60 8.64 ± 0.58
7 35.23 ± 0.58* 87.1 ± 1.47* 7.59 ± 0.44* 6.51 ± 0.45* 4.96 ± 0.03* 1373.33 ± 1.16* 3.44 ± 0.15*
8 28.26 ± 0.76* 79.41 ± 0.57* 4.96 ± 0.05* 4.45 ± 0.47* 2.85 ± 0.032* 1264.33 ± .01* 6.04 ± 0.01*
9 20.65 ± 0.03 50.44 ± 0.77 0.12 ± 0.07 1.3 ± 0.12 0.53 ± 0.01 1016.5 ± 0.70 8.72 ± 0.07
10 26.23 ± 0.58* 69.1 ± 0.26* 1.59 ± 0.44* 3.81 ± 1.01* 2.96 ± 0.03* 1128.66 ± 1.01* 5.83 ± 0.08*
11 24.26 ± 0.76 53.74 ± 1.09 0.20 ± 0.01 1.62 ± 0.22 0.61 ± 0.00 1017.33 ± 1.52 8.11 ± 0.06
12 22.55 ± 1.76 51.54 ± 0.06 0.125 ± 0.00 1.2 ± 0.01 0.54 ± 0.02 1010.5 ± 2.12 8.87 ± 0.14

Group 1 (Normal); Group 2 (vehicle control); Group 3 (300 mg/kg Medrol); Group 4 (200 mg/kg RBAI extract); Group 5 (400 mg/kg RBAI extract); Group 6 (600 mg/kg RBAI extract);
Group 7 (200 mg/kg SPN extract); Group 8 (400 mg/kg SPN extract); Group 9 (600 mg/kg SPN extract); Group 10 (100 mg/kg CAIPN extract); Group 11 (200 mg/kg CAIPN extract);
Group 12 (300 mg/kg CAIPN extract). Values (Mean ± SEM) showing significance (2 sided post hoc ANOVA, *p < 0.001) are compared to normal control. Hence, the values which are
not significant are close to the values of normal control.

Table 4
The biochemical parameters of liver and Kidney.

Group Total Bilirubin (mg/dl) Total proteins (mg/dl) AST (U/L) ALT (U/L) AL P (U/L) GGTP (U/L) Urea (mg/dl) Creatinine (mg/dl)

1 0.11 ± 0.01 7.02 ± 0.06 149.47 ± 6.41 25.88 ± 0.99 90.27 ± 1.05 22.67 ± 0.2 17.72 ± 0.71 0.45 ± 0.04
2 0.80 ± 0.27* 2.03 ± 0.05* 244.43 ± 1.25* 70.56 ± 0.49* 139.52 ± 1.08* 36.44 ± 0.67* 32.75 ± 1.06* 0.04 ± 0.05*
3 0.34 ± 0.00* 3.02 ± 0.02* 211.69 ± 0.63* 50.22 ± 6.02* 101.34 ± 1.52* 32.51 ± 0.32* 30.56 ± 0.49* 0.95 ± 0.05*
4 0.31 ± 0.01* 2.73 ± 0.51* 211.48 ± 0.99* 60.26 ± 1.64* 125.28 ± 4.31* 35.02 ± 0.56* 29.08 ± 0.45* 1.07 ± 0.07*
5 0.09 ± 0.01 4.98 ± 0.07* 198.41 ± 1.05* 48.58 ± 0.85* 104.58 ± 4.98* 29.87 ± 0.10 20.47 ± 0.09* 0.63 ± 0.06
6 0.10 ± 0.01 7.10 ± 0.09 144.73 ± 0.84 25.85 ± 1.15 93.22 ± 2.58 22.67 ± 0.19 14.07 ± 1.81 0.41 ± 0.07
7 0.41 ± 0.02* 1.32 ± 0.01* 305.90 ± 0.01* 120.10 ± 0.0* 223.56 ± 0.49* 1.47 ± 0.40* 31.74 ± 0.23* 0.78 ± 0.05*
8 0.063 ± 0.00 4.27 ± 0.05* 248.41 ± 1.05* 68.58 ± 0.85* 124.58 ± 1.98* 32.87 ± 0.10* 25.47 ± 0.09* 0.67 ± 0.017*
9 0.09 ± 0.00 6.96 ± 0.02 142.32 ± 2.18 24.82 ± 3.04 91.89 ± 0.155 22.11 ± 0.94 18.62 ± 0.37 0.44 ± 0.02
10 0.40 ± 0.02* 1.37 ± 0.01* 303.90 ± 0.01* 118.10 ± 0.05* 221.56 ± 0.49* 1.46 ± 0.40* 28.08 ± 0.92* 0.68 ± 0.00*
11 0.11 ± 0.01 6.27 ± 0.05 147.53 ± 1.90 25.91 ± 1.90 90.92 ± 0.94 22.21 ± 0.66 17.14 ± 1.23 0.57 ± 0.01
12 0.11 ± 0.01 6.96 ± 0.02 142.32 ± 2.18 24.85 ± 3.04 91.89 ± 0.155 22.11 ± 0.94 18.12 ± 1.08 0.45 ± 0.02

Group 1 (Normal); Group 2 (vehicle control); Group 3 (300 mg/kg Medrol); Group 4 (200 mg/kg RBAI extract); Group 5 (400 mg/kg RBAI extract); Group 6 (600 mg/kg RBAI extract);
Group 7 (200 mg/kg SPN extract); Group 8 (400 mg/kg SPN extract); Group 9 (600 mg/kg SPN extract); Group 10 (100 mg/kg CAIPN extract); Group 11 (200 mg/kg CAIPN extract);
Group 12 (300 mg/kg CAIPN extract). Values (Mean ± SEM) showing significance (2 sided post hoc ANOVA, *p < 0.001) are compared to normal control. Hence, the values which are
not sigificant are close to the values of normal control.

weighed. The extract recovery percentage was calculated using the
formula: Weight of extract × 100/ Weight of sample. The total phe-
nolics, tannins and flavonoids content of extracts (200 and 400 μL re-
spectively) were determined at 725 and 510 nm using spectro-
photometer (UV-1800, Shizmadzu) and results were expressed as Gallic
acid equivalents (GAE), Tannic acid equivalents (TAE) and Rutin
equivalents (RE) respectively [16].
2.4. In vitro antioxidant assay
The antioxidant activity of the extracts was determined using the
DPPH• scavenging activity, measured at 517 nm and the results were
expressed in IC50 (μg/mL); Phosphomolybdenum assay and the results
were expressed in mg of ascorbic acid equivalents (AAE)/g extract;
Lipid peroxidation assay was employed to quantify the % of inhibition

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in peroxidation at 532 nm. All sample absorbance was measured using
the UV–vis spectrophotometer [16].
2.5. In vitro blocking of the total proteolytic activity
The blocking of the proteolytic activity of venom by the extracts was
determined at 280 nm based on Kunitz [17] method. Blocking of total
proteolytic activity (%) = 1 − (Absorbance of casein − Absorbance of
casein exposed to venom plus sample)/(Absorbance of casein −
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Biomedicine & Pharmacotherapy 97 (2018) 1603–1612
Fig. 3. (a) Histopathology of skin. A: Group
1(Normal); B: Group 2 (vehicle control); C: Group 3
(standard drug “Medrol”); D: Group 4 (600 mg/kg
methanol extract of Root bark of Aristolochia indica
(RBAI)); E: Group 5 (600 mg/kg methanol extract of
Seed of Piper nigrum (SPN)); F: Group 6 (200 mg/kg
methanol extract of Combination of Aristolochia in-
dica and Piper nigrum (CAIPN)). (b) Histopathology
of liver. A: Group 1 (Normal); B: Group 2 (vehicle
control); C: Group 3 (standard drug “Medrol”);
D:Group 4 (600 mg/kg methanol extract of Root bark
of Aristolochia indica (RBAI)); E: Group 5 (600 mg/kg
methanol extract of Seed of Piper nigrum (SPN)); F:
Group 6 (200 mg/kg methanol extract of
Combination of Aristolochia indica and Piper nigrum
(CAIPN)).
Absorbance of casein exposed to venom plus buffer).
2.6. In vivo studies
2.6.1. Ethical clearance and animal management
All the animal experiments were subject to the scrutiny of
Institutional Animal Ethics Committee for the ethical clearance (NCP/
IAEC/2015-16-06). Swiss albino mice (25–30 g) and Wistar albino rats
of either sex (120–180 g) were housed in a polypropylene cage at 26 °C
D. Sivaraj et al. Biomedicine & Pharmacotherapy 97 (2018) 1603–1612

Table 5
The histopathological scoring of skin and liver tissue section (n = 3, P < 0.001).

Groups/Doses (w/w) Organ Leucocytes infiltration Congestion Inflammation Collagen damage Necrosis Hyperplasia

Epidermal cells Kupffer cells

Normal control Skin – 1± 00 1± 00 1± 00 1± 00 1 ± 00 –

Liver 1 ± 00 1± 00 1± 00 1± 00 1± 00 – 1 ± 00
Vechilce control Skin – 3± 00* 3± 00* 3± 00* 3± 00* 3 ± 00 *
–
Liver 3. ± 00* 3± 00* 3± 00* 2.99 ± 01* 3± 00* – 3 ± 00*
Medrol (295 mg/kg) Skin – 2.97 ± 03* 2.89 ± 11* 3± 00* 2.44 ± 06* 3 ± 00* –
Liver 3 ± 00* 3± 00* 3± 00* 3± 00* 3± 00* – 3 ± 00*
RBAI (600mg/kg) Skin – 1.85 ± 15* 1.84 ± 16* 2± 00* 1.88 ± 12* 2 ± 00* –
Liver 2 ± 00* 2± 00* 2± 00* 1.95 ± 05* 2± 00* – 2 ± 00*
SPN (600mg/kg) Skin – 2.95 ± 05* 2.82 ± 18* 3± 00* 3± 00* 2 ± 00* –
Liver 2 ± 00* 2± 00* 1.98 ± 02* 2± 00* 2± 00* – 3 ± 00*
CAIPN (200mg/kg) Skin – 1± 00 1± 00 1± 00 1± 00 1 ± 00 –
Liver 1 ± 00 1± 00 1± 00 1± 00 1± 00 – 1 ± 00

Scoring calculation: No abnormality:1, Moderate:2, Extensive:3. Values (Mean ± SEM) showing significance (2 sided post hoc ANOVA, *p < 0.001) are compared to normal control.
Hence, the values which are not sigificant are close to the values of normal control.

in clean environment and fed with standard chow and a clean drinking
water for 7days for their acclimatization.
2.6.2. Acute toxicity
The acute toxicity study was performed using Swiss albino mice
(n = 6) as per the guidelines of organization for economic co-operation
for development (OECD), Test No. 423 [18]. The animals were ad-
ministered orally with methanol extracts of RBAI, SPN, and CAIPN at a
dose of 500, 1000 and 2000 mg/kg body weight and the animals were
observed for 24 h (2 h for morbidity and up to 24 h for mortality) to
determined a toxic dose.
2.6.3. Determination of median lethal dose (LD50) of venom and effective
dose (ED30, 50 and 100) of drug and extracts
The LD50 (dose required to kill half number of the rats in test group
at specific duration) of S. morsitans venom was determined based on
[19] by intradermal (i.d.) administration of different concentrations
(10–200 μL) of venom to respective groups of rats (?? = 6). The LD50 of
venom was determined from the occurrence of death within 24 h of
venom injection by probit analysis [20]. The ED30, 50 and 100 (a dose that
produce a desired therapeutic response) of RBAI, SPN and CAIPN ex-
tract was determined according to method of Arley [21]. The animals
(n = 6) were pretreated (3 days) with respective extracts (100 to 1000
mg/kg) and the ED of standard drug Medrol (Methylprednisolone) de-
termined using the formula, Animal dose (mg/kg) = Human equivalent
dose (mg/kg) x conversion factor [22]. On the 4th day, LD50 of venom
was injected to animals and effective dose of extracts was determined
from the occurrence of death within 24 h of venom injection by probit
analysis [20].
2.6.4. Antivenom activity
The rats were divided into twelve groups (n = 6) randomly and
pretreated for 3 days with their respective drug dissolved in 0.6%
Carboxymethyl cellulose (CMC). Group 1(Normal rats, without en-
venomation and drug/extract administration); Group 2 (0.6% CMC
p.o.); Group 3 (Standard drug “Medrol” 295 mg/kg p.o.); Group 4–6
(RBAI 200, 400 and 600 mg/kg p.o.); Group 7–9 (SPN 200, 400 and
600 mg/kg p.o.) and Group10-12 (CAIPN 100, 200 and 300 mg/kg
p.o.).
On the fourth day, the animals were envenomated with 100 μL via
intradermal injection and observed up to the 10th day of study for
hypersensitive activity like itching, writhing, paw licking, hemolysis,
edema and shivering for 2 h. The pathological affect of venom on skin,
liver, kidney and blood was studied. Blood was drawn from the CAIPN
(5th day), RBAI, SPN, Medrol, vehicle and normal (10th day) group
animals based on reduction hypersensitivity by puncturing retro-orbital
plexus under diethyl ether anesthesia and collected into the with an-
ticoagulant, ethylenediamine tetra acetic acid (EDTA) and without
anticoagulant bottles. The serum was separated by centrifugation at
1000 g for 10 min to analyze the electrolytes (Atomic absorbance
spectrophotometry), lipid profile (Lipid profile analyzer), liver and
kidney function test (Span Diagnostics Limited kit, India). The antic-
oagulated blood was used to measure the hematological parameter
(Hematology analyzer, ABX-Micro- S-60). On the 10th day, the abrasion
and lacerations portion of skin induced by hypersensitivity and liver
were dissected and stored in 10% formalin.
2.6.4.1. Histopathological scoring analysis. The histopathological
analysis was performed by cutting 2 mm sections of the tissue using
the microtome and staining with hematoxylin and eosin. The samples
were evaluated for necrosis, pyknosis, congestion, collagen damage,
haemorrhage, kupffer cells and epidermal hyperplasia, inflammatory
and leucocytes cell infiltration using a semi quantitative scale [23]. The
variations were graded like No abnormality (Score1); Moderate (Score
2) abnormality affecting 25% of samples; Extensive (Score3)
abnormality affecting more than 75% of samples.
2.7. Anti-skin cancer activity of CAIPN
Anti-skin cancer activity of CAIPN was evaluated using the epi-
dermoid carcinoma A 431 cell line by MTT method and results was
expressed in inhibitory concentration of 50% cells (IC50) Camptothecin
was used as standard [24].
2.8. High-pressure liquid chromatography (HPLC) analysis
The CAIPN extract was dissolved in methanol (20 mg/100 mL) and
sonicated for 30 min and filtered through 0.45 μm membrane filter
(PTFE). The standard phytocompounds also prepared in same way. The
analysis was performed using degasser DGU-20A3, two LC-20CE
pumps, a SIL-20A HT auto-injector, CTO-20A column oven,
SPDM20Avp photodiode array detector (DAD) and a CBM-20A system
controller (Shimadzu Co., Kyoto, Japan). The chromatographic se-
paration was performed using a Phenomenex luna C18 analytical
column 4.6 × 250 mm (5 μm particle size). The injection volume was
20 μL and the flow rate 1 mL/min. The mobile phase consisted of the
gradient of A, water and 1% (v/v) acetic acid (Milli-Q system,
Millipore, Bedford, USA) and B, methanol. The detector was set at
340 nm to get the chromatograms. Finally, the sample peaks were
compared with standards peak retention time and quantification was
done.

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Fig. 4. (a) Anticancer activity of CAIPN methanol

extract. MCAIPN:Methanol extract of CAIPN (b)
CAIPN methanol extract treated A 431 Cell viability
percentage. MCAIPN: Methanol extract of CAIPN.

2.9. Statistical analysis statistically significant at p < 0.05 and p < 0.001.

The results were expressed as Mean ± Standard Deviation (SD)/ 3. Results

Standard Error Mean (SEM). The data were statistically analyzed using
SPSS version 20.0 by means of one way ANOVA followed by Duncan’s 3.1. Extract recovery percentage and phytochemical contents
test for phytochemical, in vitro antioxidant and antivenom studies and
Dunnett’s t-test for in vivo studies. Mean values were considered The extract recovery percentage lies in order as: CAIPN

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Fig. 5. The HPLC analysis of CAIPN methanol extract. (a) CAIPN methanol extract (b)
Ferulic acid (c) Quercetin.
(16.4%) > RBAI (7.8%) > SPN (6.01%). CAIPN extract showed
higher content of total phenolics, tannins and flavonoids than RBAI and
SPN extract and are significant at p < 0.05 (Table 1).
3.2. In vitro antioxidant and blocking of proteolysis
The CAIPN extract showed good molybdenum reducing activity,
inhibition of lipid peroxidation and DPPH radical scavenging activity
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Biomedicine & Pharmacotherapy 97 (2018) 1603–1612
than RBAI and SPN with significance p < 0.05. The protein content of
venom was 2.72 μg BSA equivalents/mL. Blocking of venom proteolytic
activity by CAIPN extract was remarkable than other two samples with
the significance (p < 0.05) and are presented in Table 1.
3.3. In vivo animals studies
3.3.1. Acute toxicity
The RABI, SPN, and CAIPN extract were subjected to acute toxicity
study on albino mice, monitored for 24 h after administration of ex-
tracts at 500, 1000 and 2000 mg/kg doses. The parameters like alert-
ness, grooming, touch and pain response, tremors, righting reflex,
gripping strength, urination, salivation etc., were normal and there was
no mortality up to 2000 mg/kg. Hence, all were found to be nontoxic.
3.3.2. Lethal dose of venom and effective dose of drug
LD50 of venom was 100 μL (Fig. 1a). The ED30, 50 and 100 of RBAI
(200, 400, 600 mg/kg), SPN (200, 400, 600 mg/kg), CAIPN (100, 200,
300 mg/kg) and the probit analysis are present in (Fig. 1b–d respec-
tively). The ED50 of Medrol was 295 mg/kg.
3.3.3. Antivenom activity
3.3.3.1. Hypersensitivity. The hypersensitive behaviour of vehicle and
Medrol treated animals was higher than the extract treated animals.
CAIPN (200 mg/kg) treated animals were normal in condition (from 5th
day) than the RBAI, and SPN extract treated rats. The edema and
shivering also observed for one hour after envenomation expect the
CAIPN (200 and 300 mg/kg) treated animals. Abrasion and lacerations
were developed in animals due to the itching and hemolysis. The
hypersensitive behaviours of rats (itching, writhing, paw licking and
hemolysis) are present in Fig. 2(a–d) and were significant at p < 0.001.
3.3.3.2. Electrolytes and lipid profile. Hyponatremia, hyperkalemia, and
hypocalcemia were noted in envenomated vehicle group than normal
rats. CAIPN was remarkable at 200 mg/kg in electrolytes range than the
RBAI and SPN treated animals at 600 mg/kg and with significant at
p < 0.001. The electrolytes were not significant in Medrol treated
animals and the observed data are expressed in Table 2. CAIPN
(200 mg/kg) treated rats were significant at p < 0.001 in
maintaining cholesterol, triglycerides, HDL, LDL, VLDL in normal
range and which was accomplished by RBAI at 600mg/kg. But SPN
was not significant for LDL even at 600 mg/kg. The Medrol treated
animals was found with unstable lipid content and also not significant
at p < 0.001 (Table 2).
3.3.3.3. Hematological and biochemical parameter. CAIPN (200 mg/kg)
was effective in boosting WBC (neutrophils, lymphocytes, eosinophils,
monocytes, basophils), platelet and RBC count and are significant at
p < 0.001, same status was registered in 600 mg/kg RBAI and SPN
treated rats and the Medrol treated rats were not significant (Table 3).
RBAI and SPN (600 mg/kg) where appreciable in both urea and
creatinine level but comparably lower dose 200 mg/kg of CAIPN was
significant in protecting kidney. The activity of RBAI and SPN (600 mg/
kg) was notable since, bilirubin, AST, ALT, ALP, and GGTP were
maintained in the normal range but fortunately lower dose (200 mg/
kg) of CAIPN was significant than the both extracts and the results were
significant at p < 0.001. The Medrol treated animals liver and kidney
marker was not significant at p < 0.001 (Table 4).
3.3.3.4. Histopathology. The histopathological of skin (Fig. 3a) and
pathological scoring (Table 5) showed abnormal architecture
including the presence of congestion, inflammatory cells, collagen
damage, necrosis, epidermal hyperplasia with score 3 in the vehicle
and medrol treated rats. The cellular architecture of 600 mg/kg RBAI
and SPN treated rats was not worse as in vehicle rats and were observed
with score 2. CAIPN extract (200 mg/kg) treated rats skin cells were in
D. Sivaraj et al.
the normal skin cellular architecture with granulation tissue, and other
dermal cells and score was 1.
Histopathological examination of the liver and its scoring (Fig. 3b
and Table 5) reveals that in vehicle and medrol rats experience a high
level of liver injury with the noticeable changes like central vein con-
gestion, kupffer cells hyperplasia, nuclear pyknosis, cellular in-
flammation, necrosis, and leucocytes infiltration with score 3. The liver
cellular arrangement of RBAI and SPN treated rats (600 mg/kg) were
observed with same changes in score 2, but the CAIPN extract (200 mg/
kg) treated rat liver were observed with similar architecture to normal
rat (score1).
3.4. Anti-skin cancer activity of CAIPN extract
The CAIPN extract showed dose-dependent inhibition activity
against A431, a skin cancer cell line and the 50% growth inhibition
concentration (IC50) of the extract was 208 μg/mL. The interaction of
methanol extract on the skin cancer cells is represented in Fig. 4a. The
cell viability at 450 μg/mL was 37.69% and represented in Fig. 4b. The
activity of the extract was not equivalent to the camptothecin at 80 μM.
3.5. HPLC analysis
The methanol extract of CAIPN revealed the presence of two major
peaks at different retention times. Peak 1 (RT: 11.827 min), Peak 2 (RT:
16.401 min) was identified as ferulic acid (15.04 ± 0.09 μg/mg) and
quercetin (35.30 ± 0.30 μg/mg) like compounds respectively and are
represented in (Fig. 5a–c).
4. Discussion
The Medrol is trade name of steroid tablet contain
Methylprednisolone as a main component which acts as anti-in-
flammatory, anti-allergen which also include in prescription to treat
envenomation. Fortunately, its side effects on the liver, heart, immunity
etc. are well known. Considering this and incompetence of the drug in
healing the envenomation effects the present study step forward to find
the new effective polyherbal antivenom drug using the medicinal plants
A. indica and P. nigrum.
Soxhlet method aids to extract the thermostable compounds like
polyphenolics including flavonoids a potential anti-venomic compound.
This thermostable nature helps to increase the absorption and bioa-
vailability that enhance the therapeutic value [25].
The venom components stimulates reactive free radical generation
within cells and cause the oxidative stress, affects the mitochondria and
macromolecules lipids, proteins and DNA which results in apoptosis
[26]. To overcome this, antioxidant ability of drug is considered and
hence the present study confirmed that CAIPN methanol extract was
significant in their radical scavenging activity and this was due to their
polyphenolic contents. Furthermore, it also found significant in
blocking of the proteolytic activity of venom and that might be due to
the bioactive compounds phenolics that denatures the metalloprotease,
phospholipase A2, and other components, responsible for the in-
flammation by releasing the arachidonic acid and also by preventing
the lysis of erythrocytes [27,28]. The phenolics could form hydrogen
bonds with the three histidine residues and could chelate the Zn2+
atom of the metalloprotease and also it bind to the active sties of
phospholipase A2 and other components which could potentially result
in inhibition of the venom enzymatic activities [29].
The acute toxicity results confirmed that methanol extract of all
three samples were found safe up to dose 2000 mg/kg. The animal
model results confirmed that CAIPN methanol extract was potential
over the RBAI and SPN extract in reducing hypersensitivity, main-
taining electrolytes and biomarkers of blood, liver, and kidney which
assured the prevalence of peaceful internal environment. It was evident
that plant extract was effective against the hypersensitive allergic
1611
Biomedicine & Pharmacotherapy 97 (2018) 1603–1612
effects induced by envenomation.
The rats without itching showed that CAIPN extract acts as anti-
neurotoxin that protects the terminal parts of the motor axons and
boutons from susceptibility and thereby it prevents self-injury to skin
[30]. Transport of electrolytes is regulated by ion channels. The pre-
sence of normal electrolytes range in CAIPN extract treated rats con-
firmed its potential action against the ion channel modulators, μ-
SLPTX-Ssm1a (Na), κ-SLPTX-Ssm1a, κ-SLPTX-Ssm2a and κ-SLPTX-
Ssm3a (K) and ω-SLPTX-Ssm1a and ω-SLPTX-Ssm2a (Ca) [5]. Thereby,
it could completely reduce the osmotic imbalance and oxidative stress
and that could inhibit inflammation. This was the evidence that the
polyphenols and flavonoids found in CAIPN extract act synergistically
as competitive blocker to the respective receptor site [31,32].
The lipid content is essential for proper functioning of cells and
membranes. The remarkable action of CAIPN extract in maintaining
lipids indicates that it is able to inactivate the phospholipase A2 and
other lipid destroying venom components. Particularly, sesquiterpenes
and aristolochic acid in A. indica has the anti-phospholipase A2 and
anti-inflammatory property [33,34]. Likewise, piperine and 4-ner-
olidylcatechol in P. nigrum act as antivenom, anti-myotoxicity and anti-
phospholipase A2 [13,35]. Phospholipase A2 induces hemolysis of RBC
and liberates lysolecithin and mediated the oxidative stress [36]. He-
matological parameters also present in appropriate range and this
confirmed the anti-phospholipase A2 activity of CAIPN methanol ex-
tract. The kidney and liver biomarker also again ensured the venom
denaturing potential of CAIPN methanol extract. The histopathological
scoring analysis confirmed that CAIPN extract was significant at
p < 0.001 in recuperating of liver and skin cells, incredible ability to
counteract the 100 μL of venom protein at 200 mg/kg.
The appreciable anti-skin cancer potential of methanol extract was
due to the tannin, a potent inhibitor of hyaluronidase which breaks the
hyaluronic acid that found majorly in skin [5,28,37,38]. The presence
of the quercetin and ferulic acid compounds in methanol extract con-
firmed by HPLC analysis has the property like hepatic-renal protectant,
anti-inflammatory, anticancer, antioxidant and skin care agent [39,40].
On the whole, as discussed above the antivenomic compounds in
CAIPN might act synergistically on specific competitive receptor as
blocker to inhibit venom action that helps to eliminate the root cause of
the both known and unknown effects as stated by Ayurveda [7,41].
Eventually, the present findings confirmed that polyherbal extract has
the ability in curing S. morsitans envenomation effects.
5. Conclusion
The CAIPN treated rats were with the significant level of electrolytes
property and maintain the lipid and blood, liver and kidney biomarker
(without hypersensitivity) at a lower dose. Thus it indicates the S.
morsitans venom neutralizing potential of CAIPN extract was remark-
able than the RBAI and SPN methanol extracts. The present study re-
sults confirmed the therapeutic benefits were higher in polyherbal than
the single plant therapy. Furthermore, the isolation of antivenomic
compounds from CAIPN extract is in process which helps to formulate
the new effective ecofriendly antivenomic drug for the welfare of
human beings.
Conflicts of interest
None.
Acknowledgements
We humbly thank Mr. P.M. Sureshan, Zoological Survey of India,
Kozhikode, Kerala for his indeed support in identification of centipedes;
Dr. Sengottuvelu and Mrs. Lalitha, Nandha College of Pharmacy, Erode
for their assistance during pharmacology study.
D. Sivaraj et al.
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