Anti-Venom Activity of Albizia Lebbeck Bark Extracts Against Naja Kaouthia Snakevenom

“Anti-venom activity of Albizia lebbeck bark extracts against Naja Kaouthia snakevenom”

SYNOPSIS FOR

M. PHARM DISSERTATION

SUBMITTED TO

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

BENGALOORU, KARNATAKA

SUBMITTED BY

KIRAN SINGH J

I M. PHARM

DEPARTMENT OF PHARMACOLOGY

P.E.SCOLLEGE OF PHARMACY

BENGALOORU – 560050

(2008-2009)

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES

BENGALOORU, KARNATAKA.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECT FOR

DISSERTATION

1.0 / NAME AND ADDRESS OF THE CANDIDATE. / KIRAN SINGH J
PERMANENT ADDRESS
No. 670, 12thMain, 1st Block, 3rd Stage, Basaveshwaranagar, Bangalore-560079.
LOCAL ADDRESS
SAME AS ABOVE
2.0 / NAME OF THE INSTITUTION. / P.E.S.COLLEGE OF PHARMACY
Hanumanthanagar, 50 feet road, B.S.K 1st stage, Bengalooru – 50,
3.0 / COURSE OF STUDY AND SUBJECT. / M.PHARM.
PHARMACOLOGY.
4.0 / DATE OF ADMISSION TO COURSE. / 31st May, 2008
5.0 / TITLE OF THE TOPIC:
“Anti-venom activity of Albizia lebbeck bark extracts against Naja Kaouthia snakevenom”
6.0 / 6.1. NEED FOR THE STUDY:
Snakebite is a global problem, especially in the tropical countries like India. In Asia, as a whole, there may be up to four million snakebites every year, of which almost 50% are envenomed. It is estimated that annual snakebite mortality in Indian subcontinent is more than 25000.
The Indian monocled cobra Naja kaouthia Family Elapidaeis prevalent in the entire eastern and north-eastern parts of the country and is responsible for a large number of snakebite case.1
Polyvalent antivenom or antiserum is the only therapeutic agent available throughout the world. Majordrawback of serum therapy is its prohibitive cost and chance that victims are often away from medical care when bitten. Generally antivenom serum scarce commodity in quality and supply to rural areas, moreover they require ideal storage condition which may not be possible in rural area. Apart from these it also causes hypersensitivity
In ancient Indian system of ayurveda, there are many plants recommended for using in snakebite therapy. Some of these are popularly used by snake charmers of India for treating snakebite patients, but without any scientific validation. Therefore, this type of treatment remains questionable and needs thorough scientific investigation.
The flowersof Albizia lebbeck are used for snake-bite. All parts of the plant are recommended for the treatment of snake-bite (Ayurveda)2.The use of the plant against the effect of snake bite long been recognized even in modern times. More than 700 plants have been reported as used in folk medicine in the world for snake bite. Many people living in rural area are highly exposed to snake bite, unfortunately antivenom and medical resources are limited in such areas. So patient cannot seek adequate and rapid attention for envenomation.
Therefore, in the present investigation, an effort has been given to evaluate the neutralization capacity of Albizia lebbeckbark extracts against lethality, myotoxicity, coagulation activity and some toxic enzymes of Naja Kaouthia venom.
6.2REVIEW OF THE LITERATURE:
Methanolic extract of Albizia lebbeckbark extract when administered showed Antispermatogenic, Anti-androgenic activities in male albino rats3.The ethanolic extracts of leaves of Albizzia lebbeck exhibited anticonvulsant activity4. This plant is also used in management of helminthes of animals5. An inhibition of the proliferationof murine leukemia and human mouth epidermalcarcinoma cell lines by Albizia lebbeck oil is also seen6 .Oral administration of saponins isolated from Albizia lebbeck bark to male ratsbrought about a significant decrease in the weights of testes, epididymides, seminal vesicle and ventral prostate.Albizia lebbeck reduced the fertilityof male rats by 100 %7.
Three main saponins named albiziasaponins A, B and C were isolated from the barks of Albizia lebbeck. Their structures were established through spectral analyses as acacic acid lactone 3-O-β-D-xylopyranosyl-(1→2)-α-L-arabinapyranosyl-(1→6)- β-D-glucopyranoside, 3-O-β-D- glucopyranosyl-(1→2)-O-[α-L-arabinapyranosyl-(1→6)]- β-D-glucopyranoside and 3-O-β-D- xylopyranosyl-(1→2)-α-L-arabinapyranosyl-(1→6) -O-[ β-D- glucopyranosyl-(1→2)]- β-D-glucopyranoside8.
Two new tri-O-glycoside flavonols: kaempferol and quercetin 3-O-α-rhamnopyranosyl(1→6)β-glucopyranosyl(1→6)]- β- galactopyranosides, were identified from the leaves of Albizia lebbeck. Structures were established by conventional methods of analysis and confirmed by ESI-MS, H and C-NMR spectral analysis9.
Some of the scientific reports on plant based preparations or agents have antivenom property are given here under:
The isolation and purification of lupeol acetate from the methanolic root extract of Indian medicinal plant Hemicesmus indicus which could neutralize venom induced action of Doboia russellii and Naja Kaouthia on animals. Lupeol acetate neutralized Naja Kaouthia venom induced lethality, cardiotoxicity, neurotoxicity and respiratory changes in experimental animals10.
Plant polyphenols from the aqueous extracts of Pentace burmanica, Pithecellobium dulce, Areca catechu and Quercus infectoria were tested for their inhibitory acticities against Naja Kaouthia venom by in vitro neutralization method. The anti-venom activites of these plant polyphenols by selectively blocking the nicotinic acetylcholine receptor and non-selectively by precipitation of the venom proteins were suggested11.
Weak neurotoxins from Naja Kaouthiasnake venom have demonstrated their ability to interact with both muscle type and neuronal α 7 nicotinic acetylcholine receptors. It was found that i.v. injection of WTX induced a dose-dependent decrease in BP and an increase in HR in rats and mice, the rats being more sensitive to WTX. Blockade of either nAChRs or mAChRs affected WTX action differently in rats and mice, thus reflecting interspecies differenced in haemocynamic regulation12.
The methanolic root extracts of Vitex negundo Linn. and Emblica officinalis reported to posses significant antagonistic activity against Vipera russellii and Naja kaouthia venom induced lethal activity both in vitro and in vivo studies. The plant significantly neutralizes the lethality, haemorrhage, coagulant, defibrinogenating and inflammatory activity induced by venom13.
The aqueous and alcoholic extracts of dried roots of Mimosa pudica reported to have significant inhibitory effect of lethality, myotoxicity and enzymatic activity namely PL-A2, Protease against Naja kaouthia1.
PLANT PROFILE14
PLANT NAME:Albizia lebbeck
FAMILY: Mimosaceae
VERNACULAR NAME:Eng: Siris tree; Hin: Siris, Siris; Kan:Begemara; Mal: Nenmenivaka, Vaka; San: Sirisah, Bhandi; Tam: Vakai, Siridam; Tel: Dirisana.
DISCRIPTION:A medium to large sized unarmed deciduous tree about 20 m in height with an umbrella-shaped crown and grey to dark brown rough irregularly cracked bark; leaves abruptly bipinnate, main rachis with a large gland above the base and one below the upper-most pair of pinnae, pinnae 2-4 pairs, leaflets 5-9 airs with glands between their bases, the lateral ones elliptic-oblong, the two terminal ovate-oblong, all unequal sided; flowers white, fragrant, in globose umbellate heads; fruits long, characteristic pods, bluntly pointed, tin, pale yellow, smooth, shiny, reticulately veined above the seed; seeds 4-12, pale brown, ellipsid, oblong, compressed.
PART USED: Bark, flower, seeds.
HABITATE: Throughout India, also cultivated.
CHEMICAL CONSTITUTION:The bark yield tannins of condensed type, viz. D-catechin, isomers of leucocyanidin and melacacidin and a new leucoantho-cyanidin, lebbecacidin. It also gives friedelin and β-sitosterol. Seeds gave crude protein, calcium, phosphorus, iron, niacin, and ascorbic acid, amino acid composition of the protein is: arginine, histidine, leucine & isoleucine lysine, methionine, phenylalanine, threonine, tyrosine, and valine. The flowers contain lupeol, α-and β-amyrin and a pigment similar to crocetin.
MEDICINAL USES:The bark is astringent, bitter, acrid, sweet, mildly thermogenic, expectorant, aphrodisiac, anti-inflammatory, anodyne, cephalic, ophthalmic, depurative, restorative and tonic. It is useful in vitiated conditions of pitta and kapha, cough and catarrh, asthma, enlarged cervical glands, ophthalmopathy, nyctalopia, strengthening gums, scrofula, skin eruptions, leprosy, leucoderma, sprains, wounds, ulcers, neuralgia, inflammations, erysipelas, diarrhoea and all types of poisoning. It is also used as a dentifrice in odontopathy. The flowers are useful in chronic cough and bronchitis. The seeds are useful in inflammations. Scrofula, skin diseases, leprosy, leucoderma, chronic catarrh, seminal weakness, ophthalmopathy and poisoning.
The flowers are used for snake-bite. All parts of the plant are recommended for the treatment of snake-bite (Ayurveda)2.
6.3OBJECTIVE OF THE STUDY:
# Preparation of bark extracts using various solvents and their Preliminary phytochemical
evaluation.
# Evaluation of LD50 of venom as per OECD guidelines.
# Evaluation of acute oral toxicity of bark extracts in mice.
# Evaluation of neutralization of lethality, both in-vivo and in-vitro (in mice) against venom.
# Evaluation for the neutralization activity of bark extract against myotoxicity induced Naja
Kaouthia venom in mice.
# Evaluation of Neutralization activity of Phospholipase A2 of venom by Extract.
# In-vivo and In-vitro evaluation of Anticoagulant activity of bark extract against Naja
Kaouthia induced coagulation in mice
# To evaluate the anti-inflammatory activity of bark extracts against Naja Kaouthia induce
inflammation in rat paw.
7.0 /

MATERIAL AND METHODS:

7.1 SOURCE OF DATA :
The plant material will be procured from authenticated suppliers. Whole experiment is planned to generate data from laboratories studies. Experiment will be performed as described in the standard bibliography, may be obtained from standard journals and text books available within the college or from other pharmacy colleges or from libraries of National Institutes or through internets from industry.

7.2 METHODS OF COLLECTION DATA( including sampling procedure if any):
The whole study is divided into following phases.
I. Collection of plant material.
a) The roots of Albizia lebbeck will be procured from Authenticated supplier
from Bangalore and it will be Authenticated by Taxonomist, Dept. of
Botany, BangaloreUniversity, Bangalore.
b) Lyophilized venom will be procured from the authenticated Supplier; Calcutta
SnakePark, Hindustan park Road, Calcutta.
Preparation of extracts.
Dried powderbark material will be successively extracted with water and alcohol (70%) in a soxhlet apparatus for 72 hours. The residue obtained after extraction is dried. The residue mass obtained is evaporated and reduce temperature to dryness. A% yield of all the two extracts will be determined.
Preliminary Phytochemical investigation.
Preliminary phytochemical investigation will be done as described in Practical Pharmacognosy- Techniques and Experiments 15.
Acute oral toxicity of plant extract16.

Female Swiss albino mice (18-20 g) are individually identified and allowed to acclimate to the laboratory condition for 7 days before the start of the study. Only one mouse receives single dose at a particular time. First animal receives a dose of 175 mg/kg and is observed for any toxicity signs, survival or death up to 48 hrs.. If the first animal died or appeared moribund, the second animal receives a lower dose (55mg/kg). The dose progression or reduction factor is 3.2 times of the previous dose.If no mortality is observed in the first animal then the second animalreceives a higher dose (55 mg/kg). Dosing of the next animal iscontinued depending on the outcome of the previously dose for a fixed time interval (48 hours). The test is stopped when one ofthe stopping criteria is observed.

5 reversals occur in any 6 consecutive animals tested.
3 consecutive animals died at one dose level.
Survived animals are observed for long-term outcomes for a period of 14 days. The acute oral toxicity values are calculated using AOT 425 software (Environmental Protection Agency, USA) based on the short term (48 hours) and long term out come (14 days).
Determination of MLD of venom (LD50).
Since this is continuation of Dissertation work from Previous year using same Naja Kaouthia venom.From unpublished dissertation work from Mr. Bhupendra Patel, Dept of Pharmacology. The LD50 of Naja Kaouthia in mice was found to be 0.6 μg/gm of mice. Same dose will be used for present work17.
II. PHARMACOLOGICAL EVALUATION.
Animals: Male albino mice of body weight 18-20 g and Rats having weight of 180-200 g will be selected for all the experiments. Animals will be kept in our animal house at an ambient temperature of 25C and 45-55% relative humidity with a 12/h dark: 12 hour light cycle. Animals will have free access to food and water.
Evaluation of Antivenom Activity of root extract of Albizia lebbeck.

1: Neutralization of lethality18

Animal used:- Albino Mice

Sex :- Either Sex
Weight :- 18-20 g
Number of animal in each group:- 6
Swiss Albino mice (n = 6) weighing (18 – 20 gm) were used to find out neutralization of lethality caused by Envenomation. The animals are administered either 1.5,2,3 MLD (LD50) of Naja Kaouthia venom (i.p). Immediately after envenomation mice will receive required dose of bark extract of Albizia lebbeck / Phosphate Buffer / Polyvalent antivenom for competitive purpose. Animals are observed for 48hrs and time for death will be recorded.
Group I / Control (Phosphate Buffer)
Group II / Venom+ Aq. Extract of A lebbeckdose level I
Group III / Venom+ Aq. Extract of A lebbeck dose level II
Group IV / Venom+ Alc. Extract of A lebbeck dose level I
Group V / Venom+ Alc. Extract of A lebbeckdose level II
Group VI / Venom + Polyvalent Antivenom
2: Neutralization of myotoxicity of venom19

Animal used:- Albino Mice

Sex :- Either sex
Weight :- 18-20 g
Number of animal in each group:- 6
Swiss Albino mice will be injected intramuscularly (i.m.) in the right gastrocnemius with 2.5μg of venom in 50μl of PBS and control animals will receive Phosphate buffer are bled 1, 3, 6, 12 and 24h later through the ophthalmic plexus under light ether anesthesia. Sera will be collected after cold centrifugation (10500 rpm) for Creatine Kinase activity in serum is determined by Auto span Diagnostic Kit Bangalore analysed in semi auto analyzer. One unit corresponds to the amount of enzyme that hydrolyses 1μm of creatine per min at 25°C. Enzyme activity is expressed in U/l.
Group I / Control (Phosphate buffer)
Group II / Venom
Group III / Venom+ Aq. Extract of A lebbeck dose level I
Group IV / Venom+ Aq. Extract of A lebbeck dose level II
Group V / Venom+ Alc. Extract of A lebbeck dose level I
Group VI / Venom+ Alc. Extract of A lebbeck dose level II
3 : Neutralisation of Phospholipase A2 activity of venom20
It is carried out by turbidimetric method using egg yolk suspension as substrate. A fresh egg yolk suspension is prepared in 0.9% NaCl this suspension is diluted in 0.9% NaCl insuch way that the absorbance at 925nm will be 1.2. A varying concentration of Naja Kaouthia venom / venom + varying conc. Of various root extracts is incubated with 2ml of this egg yolk suspension at 37 0 C for 20 mins. The reaction is stopped by adding 3ml ice cold 0.9% NaCl and O.D is measured at 925 nm against 0.9% NaCl as Blank. 1unit of activity is defined as the amount of enzyme which produces a decrease in 1milli unit of absorbance at 925nm/min.
4: Anti-coagulant activity21
Various amounts of venom, dissolved in 100 μl of PBS, will be added to 200 μl of human plasma incubated at 37 °C, and the clotting times recorded. The minimum coagulant dose (MCD) is the amount of venom inducing coagulation of plasma in 60 s.
5: Determination of anti-inflammatory activity of Albizia lebbeck22

Animal used:- Albino rat

Sex :- Male
Weight :- 180-200 g
Number of animal in each group:- 6
The minimum oedematic dose (MOD) of venom/ carrageenan is defined as the least amount of venom / carrageenan which, when injected into male albino mice, produced inflammation (oedema) in the paw. Non fasted male albino rats (180–200 g) were treated with different dose of venom/ carrageenan and plant extracts. Varying doses of the plant extracts, various amount of venom/carrageenan (in 0.01 ml) were injected (intraplanter) route. Control animals will be injected with Phosphate Buffer / Saline. The oedamtogenic response is evaluated by using screw gauge at varying intervals of 15, 30, 60, 120 and 180 mins of injection.
The percentage inhibition was calculated by following formula.

Following formula VD = Volume of paw odema at 0 min
VT = Maximum Volume of paw odema after time.
Group I / Normal (Saline / phosphate buffer)
Group II / Naja Kaouthia venom
Group III / carrageenan
Group IV / Carrageenan + Aq. Extract of A lebbeck dose level I
Group V / Carrageenan + Aq. Extract of A lebbeck dose level II
Group VI / Venom + Alc. Extract of A lebbeck dose level I
Group VII / Venom + Alc. Extract of A lebbeck dose level II
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