“Antivenom activity of Amaranthus spinosusroot extracts against Naja Kaouthia snakevenom”
SYNOPSIS FOR
M. PHARM DISSERTATION
SUBMITTED TO
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES
BENGALOORU, KARNATAKA
SUBMITTED BY
MAKARAND GOVIND DESHMUKH
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. / MAKARAND GOVIND DESHMUKHLOCAL ADDRESS
#317, 10th Main, Bank Colony, Srinivas nagar, Bengalooru-50.
PERMANENT ADDRESS
I-2, Mantri Market, Hadapsar, Pune-411028.
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. / 30th MAY, 2008
5.0 / TITLE OF THE TOPIC:
“Antivenom activity of Amaranthus spinosusroot extracts against Naja Kaouthia snakevenom”
6.0 / 6.1. NEED FOR THE STUDY:
Snakebite is a global problem, especially in the tropical countries likeIndia. 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 case1.
Polyvalent antivenom or antiserum is the only therapeutic agent available through out the world. Majordrawback of antiserum 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 investigation2.
It has been mentioned that whole plant or roots ofAmaranthus spinosus are used in snakebite3. Roots ofAmaranthus spinosusare popularly used against cobra bite by snake charmers and rural parts of India4.
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 can not seek
adequate and rapid attention for envenomation.
Therefore, in the present investigation, an effort has been given to evaluate the neutralization capacity of Amaranthus spinosusextracts against lethality, myotoxicity, coagulation activity and some toxic enzymes of N. Kaouthia venom.
6.2REVIEW OF THE LITERATURE:
Amaranthus spinosus plant used by Paliyar tribal’s in Theni district of Tamil Nadu, India. For Stomachache5.The hepatoprotective and antioxidant activity of 50% ethanolic extract of whole plant of Amaranthus spinosus was evaluated against carbon tetrachloride (CCl4) induced hepatic damage in rats6.It also shows antimalerial activity7. Inventory of local knowledge regarding Amaranthus spinosus plant of Churu district in the Thar Desert, India, they use whole plant or roots for boils, toothache, snake bites and piles.Amaranthus spinosus water extract directly stimulates proliferation of B lymphocytes in vitro8. This plant also shows good activity like Antidiabetic, anti-hyperlipidemic and spermatogenic effects9.
Some of the scientific reports on plant based preparations or agents having antivenom property are given here under:
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.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 venom10.
Extracts of Eclipta prostratediminish the lethality, myotoxicity of crotalus durissus terrificus venom11,Aristolochia shimadai neutralizes lethality of crotalid venom12,Diodia scandens delays the death due to Echis carnatus venom13, and Andrographis paniculata showed life prolonging effect against Naja Naja venom14.
PLANT PROFILE
PLANT NAME:Amaranthus spinosus
FAMILY:Amaranthaceae
SYNONYMS:Pigweed species, spiny amaranthus.
VERNACULAR NAME:Cholai (hindi), Kanata notya (Bengali), Kanta-mi-dant(Gujarati), kante math(Marathi), mullak-kirai (Tamil), mullatota- kura (Telgu), mulludantina soppu (Kannada)15
DISCRIPTION:An erect, glabrous, spinosus herb, varying in colour from green to red or purple, 30-60 cm in height with grooved branches and sharp divaricate spines in the leaf axils, leaves simple, alternate, ovate, lanceolate or oblong, entire, glabrous above, main nerves numerous, conspicuous below, flowers small, sessile, yellowish white or pale green, numerous, in dense axiallary clusters and in terminal or interrupted spikes; fruits ovoid capsules, membranous, circumscissile about the middle16.
PART USED: Whole Plant, Root.
CHEMICAL CONSTITUTION:Higher alkanes and their methyl derivatives, higher aliphatic alcohols, acid and esters, amino acids, β- sitosterol, stigmasterol, campesterol, cholesterol, α- spinasterol, α- spinasterol octacosanoate, glycosides of α- spinasterol and oleanolic acid have been reported in the plant16.
MEDICINAL USES:Amaranthus spinosus has long been used in India as a medicine.Hepatoprotective activity, Antioxidant activity, Galactogenic, laxative, emollient, spasmolytic, diuretic. Pollen extract—used for allergic asthma and allergic rhinitis. Root—used in menorrhoea. Anti-diabetic, anti-hyperlipidemic and spermatogenic.
The plant is cooling, laxative diuretic, and anemia. The roots are thermogenic and haemostatic.
6.3OBJECTIVE OF THE STUDY:
# Preparation of root extracts using various solvents and their Preliminary phytochemical
evaluation.
# Evaluation of LD50 of venom as per OECD guidelines.
# Evaluation of acute oral toxicity of root extracts in mice.
# Evaluation of neutralization of lethality, both in vivo and in vitro (in mice) against venom.
# Evaluation for the neutralization activity of root 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 root Extract against Naja
Kaouthia induced coagulation in mice
# To evaluate the anti-inflammatory activity of root 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 Amaranthus spinosus 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 powder of root material 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 by Practical Pharmacognosy- Techniques and Experiments17.
Acute oral toxicity of plant extract18.
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)19.
Since this is continuation of Dissertation work from Previous year using same Naja Kauthia 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 work.
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 25C 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 Amaranthus spinosus
1: Neutralization of lethality20
Animal used:- Albino Mice
Sex :- Either SexWeight :- 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 root extract of Amaranthus spinosus / 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 dose level I
Group III / Venom+ Aq. Extract dose level II
Group IV / Venom+ Alc. Extract dose level I
Group V / Venom+ Alc. Extract dose level II
Group VI / Venom + Polyvalent Antivenom
2: Neutralization of myotoxicity of venom21
Animal used:- Albino Mice
Sex :- Either SexWeight :- 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.spinosus dose level I
Group IV / Venom+ Aq. Extract of A.spinosus dose level II
Group V / Venom+ Alc. Extract of A.spinosus dose dose level I
Group VI / Venom+ Alc. Extract of A.spinosus dose dose level II
3 : Neutralization of Phospholipase A2 activity of venom22
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 activity23
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 Amaranthus spinosus24,10.
Animal used:- Albino rat
Sex :- MaleWeight :- 180-200 g
Number of animal in each group:- 6
The minimum oedematic dose (MOD) of venom/carrageenanis 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. Root Extract dose level I
Group V / Carrageenan + Aq. Root Extract dose level II
Group VI / Venom + Alc. Root Extract dose level I
Group VII / Venom + Alc. Root Extract dose level II
8.0 / REFERENCES :
- Mahanta M, MukharjeeAK. Neutralisation of lethality, myotoxicity and toxic enzymes of Naja kaouthia venom by Mimosa pudica root extracts. J. Ethnopharmacol. 2002;75(1):55-60.
- Chopra RN, Nayar SL, Chopra IC. Glossary of Indian Medicinal Plants. New
- Houghton PJ, Osibogun IM, Flowering plants used against snakebite.J.Ethnopharmacol. 1993;39:1-29.
- Praveen, Upadhyay B, Roy S, Kumar A. Traditional uses of medicinal plants among therural communities of Churu district in the Thar Desert, India. J Ethnopharmacol. 2007; 113:387-99.
- S. Ignacimuthu , Ayyanar M, Sankarasivaraman K.Ethnobotanical study ofmedicinalplants used by Paliyar tribals in Theni district of Tamil Nadu, India. Fitoterapia. 2008;79:562-8.
- Zeashan H, Amresh G, singh S, Rao CV. Hepatoprotective activity of Amaranthus
- Hilou A, Nacoulma OG, GuiguemdeTR.In vivo antimalerial activities of extracts from Amaranthus spinosus L. and Boerhaavia erecta L. in mice.J Ethnopharmacol. 2006; 103:236-40.
- Lin B-F, Chiang B-L, Lin J-Y.Amaranthus spinosus water extract directly stimulates proliferation of B lymphocytes in vitro. International Immunopharmacology. 2005;5:711-22.
- Sangameswaran B, Jayakar B. Anti-diabetic, anti-hyperlipidemic and spermatogenic effects of Amaranthus spinosus Linn. on streptozotocin-induced diabetic rats.J Nat Med. 2008;62:79-82.
- Alam MI, Gomes A. Snake venom neutralization by Indian medicinal plants (Vitexnegundo and Emblica officinalis) root extracts. J Ethnopharmacol 2003;86(1):75-80.
- Mors WB, Do Nascimento MC, Parente JP, Da silve MH,Melo PA, Suarez Kurtz G.
- Tsai LH, Liu HJ, Yang CP, Chang C. Inactivation of Formosan snake venoms in vivo by crude extracts of Aristolochia radix. J Formosan Med Assoc 1975;74:352-360.
- Mittal GC, Aguwa CN, Ezeira VU, Akubue PI. Preliminary pharmacological studies on antivenom action of Diodia scandensleaves. Nig J Pharmac 1981; 12: 432-6.
- NazimudeenSK, Ramaswamy S, Kameswaran L. Effect of Andrographis paniculataonsnake venom induced death and its mechanism. Ind J Pharm Sci 1978;40:132-3.
16. A Handbook of Medicinal Plants, A complete Source Book. Prajapati, Purohit, Sharma,
Kumar, 37.
17. Khandelwals KR. Practical Pharmacognosy- Techniques and Experiments. Pune: Nirali
Prakashan; 1996.
18. 423 Acute Oral toxicity - Acute Toxic Class Method (Updated Guideline, adopted
20thDecember 2001)
- 35k.
19. Bhupendra P, Mukund H. Unpublished work :Neutrilisation of Naja Kaouthia
venom byAqeous and Alcoholic extract of Tinospora cordifoliaDisseretation work
Rajiv GandhiUniversity of Health Science, Bangalore; Jan 2007.
20. Fernando C, Gilbert DL, Adriana S, Jose MG. Intramuscular administration of
antivenoms in experimental envenomation by Bothrops asper: comparison between Fab
andIgG. Toxicon.2003;41(1):71-9.
21. Moraes FV, Sousa-e-silva MC, Barbaro KC, Leitao MA, Furtado MF. Biological
andimmunochemical characterization of Micrurus altirostris venom and serum
neutralizationof its toxic activities. Toxicon 2003;41(1):71-9.
22. Kyoko UC, David TV, Eladio FS. Pharmacological characterization and
neutralization of the venoms used in the production of Bothropic antivenom in Brazil.
Toxicon2002;40(5):501-9.
23. Viviana, Ermila R, Charlotte LO, Gustavo R, José MG. Preclinical assessment of the
ability of polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced in Costa
Rica to neutralize the venoms of North American snakes. Toxicon 2003;41(7):851-60.
24. Trebien HA, Calixto JB. Pharmacological evaluation of rat paw oedema induced by
Bothrops jararacavenom. Agent and Action. 1989; 26: 292–300.
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12. /
Signature of the candidate:
(Makarand G Deshmukh)
Remarks of the guide:
Name And Designation of:
11.1 Guide Mr. Mukund Handral
Asst. Professor
Dept., Pharmacology
PES college of pharmacy
Bangalore-560050
11.2 Signature
11.3 Co-Guide
NOT APPLICABLE
11.4 Signature
11.5 Head of the department
Dr. K. Nandakumar
Asst. Professor & HOD
Dept., Pharmacology
PES college of pharmacy
Bangalore-560050
11.6 Signature
12.1 Remarks of the Principal :
12.2 Signature of the principle:
Prof. Dr. S. Mohan
Principal
PES college of pharmacy
Bangalore-560050