INTRODUCTION:

Indian cobra (Naja Naja) Family -Elapidae also known as Asian cobra is a species of the genus Naja found in the Indian subcontinent and causes the most snakebites in India. The Indian cobra is native to the Indian subcontinent which includes present day Nepal, Pakistan, India, Bangladesh, and Sri Lanka. It can be found in plains, jungles, open fields and the regions heavily populated by people. Its distribution ranges from sea-level up to 2,000 metres (6,600 ft) above sea-level. This species normally feed on rodents, toads, frogs, birds and other snakes. Its diet of rats leads it to areas inhabited by humans including farms and outskirts of urban areas. The Indian cobra's venom mainly contains a powerful post-synaptic neurotoxin and cardiotoxin^4,5. The venom acts on the synaptic gaps of the nerves, thereby paralyzing muscles, and in severe bites leading to respiratory failure or cardiac arrest. The venom components include enzymes such as procoagulant enzymes, presynaptic and post synaptic neurotoxins, necrotic toxins, phospholipases B,C,D, Hydrolases, cardio toxic substance pyrophosphatase¹, hyaluronidase that cause lysis and increase the spread of the venom. Envenomation symptoms may manifest between 15 minutes and 2 hours following the bite. In mice, the SC LD50 range for this species is 0.45 mg/kg – 0.80 mg/kg. The average venom yield per bite is between 169 and 250 mg. Though it is responsible for many bites, only a small percentage is fatal if proper medical treatment and anti-venom are given.

In India, it is conservatively estimated that up to 20,000 people die annually from snakebites. Morbidity is also significant. The most effective method for treating snake bite is through antivenom which is prepared from the venom of the snake. The first anti venom was prepared by Albert calmette, a French scientist of Pasteur Institute in 1895 against Indian cobra NajaNaja. Antivenom binds to neutralises the venom, stopping further damage but do not reverse the damages already happened. Other alternative therapy involves the usage of folk and traditional medicines in snake bites. Various medicinal plants and their compounds reported against snake venom activity. Many Indian herbs have been used for the treatment of snake bites. An ethno botanical survey of folk plants used in snake bites in southern parts of Tamil Nadu reports the use of 72 medicinal plants in snake bites. But they also possess their individual toxicity and they have no scientific validation.

Orthosiphon stamineus known as Java tea has traditionally been used in Java for the treatment of hypertension and diabetes. It has also been used in folk medicine for bladder and kidney disorders, gallstones, gout and rheumatism .Java tea is stated to have diuretic properties. Different parts of this plant have been reported to exhibit several medicinal properties like antidiabetic⁸ anti-inflammatory⁹ diuretic^3,6 hepatoprotective and nephroprotective activities.

No study has reported the protective activity of this plant against Naja naja snake venom. Hence present investigation has been designed to evaluate the effect of leaf extract of Orthosiphon stamineus against Naja naja snake venom..

MATERIALS AND METHODS:

Plant Material

The leaves of Orthosiphon stamineus were collected from siddha research institute, Arumbakkam, Chennai. The plant was identified and authenticated by Botanical Survey of India, Coimbatore. The material was dried in shade; they were powdered and extracted with methanol. The extract was evaporated under low pressure by using Buchi type evaporators.

Preliminary Phytochemical Screening

The methanol extract of Orthosiphon stamineus ( OSE) was subjected to preliminary phytochemical screening for their presence or absence of active Phytochemical constituents by the various methods such as alkaloids test, carbohydrates, steroids, proteins, tannins, phenols, flavanoids, gums and mucilage, glycosides, saponins and test for terpenes

Animals:

Swiss albino mice weighing between 20-25g were obtained from R.V.S. College of Pharmaceutical Sciences, Sulur, Coimbatore, Tamilnadu. They were maintained at standard housing conditions and fed with commercial diet and provided with water ad libitum during the experiment. The Institutional animal ethics committee (Reg No.1012/c/06/ CPCSEA) permitted the study.

Acute Oral Toxicity study:

Acute toxicity was carried by up and down/stair case method as per OECD guidelines. The extract was orally administered to different groups of rats at the doses of 50, 300, 1000 and 2000mg/kg body weight respectively.⁷ Animals were observed for 48 hours to study the general behaviour of animals sign of discomfort and nervous manifestation. The extract was devoid of mortality of animals at the dose of 2000 mg/kg body weight.Hence1/5th and 1/10th of the dose selected for the screening of Anti-Snake venom activity.

Snake venom

Snake venom of Naja Naja was obtained from The Irula Snake-Catchers Industrial Cooperative Society (ISCICS), Chennai and were preserved at 2 to 8°C. The Naja naja snake venom was dissolved in 0.9% (w/v) saline, centrifuged and the supernatant was used whenever required. The venom concentration was expressed in terms of dry weight (mg/ml) of the stock venom.

In vivo anti- snake venom activity

Twenty for adult Swiss albino mice of both sexes were divided into four groups of six mice each. Oral administration of extract 5 min prior to the injection of venom.¹⁰ The control group was injected with only venom (lethal dose 61mcg/20 g of the mice , i.p), while the other groups were treated separately with venom, after 5 min of oral administration of anti snake venom serum (10mg/kg) and methanolic extracts (200, 400mg/kg), respectively. The mice were observed for 24 hours for the number of mice which were survived.

RESULTS AND DISCUSSION:

The preliminary phytochemical investigation report indicates that the methanol extract of Orthosiphon stamineus found to contains alkaloids, flavanoids, phenolic compounds, glycosides, tannins, proteins and carbohydrates.

Administration of methanol extracts of Orthosiphon stamineus orally to male Wistar rats produced no observable side effects up to 2000 mg/kg body weight even after 48 h of observation.

The LD 50 value of the Naja Naja venom was already reported in the literature 61mcg/20 g of the mice.

Table -1

In vivo Anti-snake venom activity of the methanolic extract of the leaves of Orthosiphon stamineus

S. No	Treatment	Dose (mg/kg)	No of animals Survived	% Survival
1	Control	---	1/6	16.67
2	Snake Venom Antiserum	10	6/6	100
3	OSE 200 mg/kg	200	4/6	66.67
4	OSE 400 mg/kg	400	5/6	83.33

The methanol extract was screened for in-vivo anti snake venom activity. The extract at 400mg/kg increased the percentage survival which was comparable to that of standard anti venom serum. It was observed that the survival of the mice increased progressively with increasing the dose of the extract in a dose dependant manner. α-Cobratoxin is a substance of the venom of Naja Naja. It is a nicotinic acetylcholine receptor (nAChR) antagonist which binds antagonistically and slowly reversible to muscle-type and neuronal type nAChRs². This bond will block the receptor’s ability to bind acetylcholine and thereby inhibits the ion flow through the postsynaptic membrane, which will lead to paralysation. The probable mechanism of preventing the neurotoxic effect by Orthosiphon stamineus may be by interfering with the acetylcholine receptor sites by antagonising the actions of the neurotoxic substances in the venom at the acetylcholine receptor sites.

CONCLUSION:

The methanolic extract showed significant inhibitory activity was used for in vivo anti-snake venom activity. It showed significant anti-snake venom activity at the dose level of 400mg/kg body weight which was comparable with that of the standard. Thus, the present study has confirmed the ethnomedical use of the plant for the treatment of snake bite. It is hoped that subsequent fractionation of the extract to obtain the pure active compound will enhance its anti-snake venom potential.

ACKNOWLEDGEMENT:

Authors are thankful to the management of R.V.S. College of Pharmaceutical Sciences for providing all the necessary facilities to carry out this work.

REFERENCES:

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7. Ghosh MN. Fundamentals of Experimental Pharmacology 1984.

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Received on 14.02.2014 Modified on 25.03.2014

Res. J. Pharmacology & P’dynamics. 6(3): July- Sept. 2014; Page 126-128