Research Article ISSN: 0974-6943

Kadiyala Gopi et al. / Journal of Pharmacy Research 2011,4(4),1010-1012

Available online through www.jpronline.info

The neutralization effect of methanol extract of Andrographis paniculata on Indian cobra Naja naja snake venom
Kadiyala Gopi, Kadali Renu, Mohan Raj, Dinesh Kumar and Muthuvelan B* School of Bio sciences and Technology, VIT University, Vellore - 632 014,Tamil Nadu, India Received on: 05-12-2010; Revised on: 14-01-2011; Accepted on:09-03-2011

ABSTRACT
The present study reports the antivenom properties of Indian medicinal plant Andrographis paniculata (Acanthaceae), commonly referred as Nilavembu to reduce the effect of snake venom of Indian cobra Naja naja. Shade dried stem and leaf parts of the Andrographis paniculata were extracted with different solvents on the basis of polarity nature such as petroleum ether (polarity 0), ethyl acetate (4.4), methanol (5.1), and water (9.0). All the four extracts were tested for their antivenom activities through in vivo experiments. Among those, methanol extract of Andrographis paniculata has shown significant inhibition on neurotoxic symptoms caused by the venom (450 g/ kg b.w) and prolonged the survival time of mice (22 2 g) maximum up to 14.44 0.55 h compared to other extracts. This in vivo screened active methanol extract was further tested for direct inhibitory activity on Naja naja snake venom major enzymes like; acetyl cholinesterase, hyaluronidase, ATPase, protease, and hemolytic activities in vitro. In these experiments, the venom was preincubated with different concentrations of Andrographis paniculata methanol extract at 37 C for 1h before adding to the reaction mixture in vitro. The results confirmed that, the methanol extract of Andrographis paniculata possesses potent snake venom inhibitors. Phytochemical screening for active components of methanol extracts are still under investigation. Key words:Andrographis paniculata, Naja naja, neutralization.

INTRODUCTION
Snake bites constitute a health problem in many tropical and subtropical countries, with an estimated 2.5 million peoples envenomed each year on a global basis [8, 33] and in India alone number of deaths have been estimated between 10,000- 15,000 per annum [14]. The snakes responsible for such high death rate are cobra; saw scaled viper, Russells viper and common Krait [7]. A careful study of the literature shows that cobra and viper are the two important categories of snakes in India which are subjecting maximum deaths [5]. The Indian cobra Naja naja, family Elapidae poses a dangerous public health problem in numerous tropical and subtropical countries like India [24] and the different components of N. naja venom from India are need to be well characterized [1, 4 and 25]. The venom consists, proteins and enzymes like; acetyl cholinesterase, a post synaptic neurotoxin [24], hyaluronidase, a spreading factor [11] ATPase [19], protease [12] and alkaline phosphatases [27]. All these enzymes cause severe damage to a victim who leads to paralysis, cardiac arrest and finally death. Envenomations due to snake bites are commonly treated by parenteral administration of horse or sheep-derived polyclonal antivenoms aimed at neutralization of toxins [30]. It does not provide enough protection against venom- induced hemorrhage, necrosis, and it often produce adverse hypersensitivity reactions [29]. However, despite the wide spread success of this therapy, it is still important to search for different venom inhibitors, either synthetic or natural, that could inhibit or substitute for the action of antivenins. In rural areas traditional herbal medicine is readily available to treat snake bites. Application of the plant or its sap on to the bite area, chewing leaves and bark or drinking plant extracts or decoctions are some procedure intended to act against snake venom activity. Andrographis paniculata Ness (Acanthaceae) commonly referred as Nilavembu is the most important plant among those plants which have been used by the tribal to treat victims of snakebites in rural areas of southern India especially in Andhra Pradesh and Tamilnadu [29]. However, in most cases the efficacy of this traditional treatment regimen is unproven and this is of particular importance especially in local areas where antivenin is not available readily. Therefore, the present study has been initiated to investigate the snake venom (Naja naja) neutralization by medicinal plant Andrographis paniculata. 2. MATERIALS AND METHODS 2.1 Collection of medicinal plant The present investigation was carried out by conducting surveys and using questionnaire with tribal people and irulars living in Irularpatti, Vallimalai, Alamalrangapuram and Ammorupalli in Vellore district of Tamilanadu and also from the vaidyars, tribal and rural people living in and around the Southern part of Chittoor district, Andhra Pradesh, India. Our main aim was to collect the oral information about the medicinal plants used by irulars and tribal for snakebites and to select the most effective and popularly used plant among them for the present study. Among all plants, Andrographis paniculata was taken for the present study which was identified by plant taxonomist Dr.K.Madhava Chetty, Assistant Professor, Dept. of Botany, Sri Venkateswara University, Tirupati. 2.2 Extraction of plant Five hundred grams of the powdered shade dried plant material was dissolved in 1lit (w/v) petroleum ether and allowed for overnight at room temperature. The suspension was then filtered using muslin cloth, the residue obtained was resuspended in freshly taken petroleum ether solvent (1:2 ratios). The process was repeated for one more day with same solvent. Then the residue was completely air dried and the same process was adapted with different solvents like ethyl acetate, methanol and finally with water (polar ranges started from 0.0, 4.4, 5.1 and 9.0 respectively). Solvents from the extracts were removed by using rotary evaporator and obtained crude extracts were stored in tightly stopper bottles at 4 C for further studies. 2.3 Animals Cross breed albino mice (CBA) of both sexes with average weighing 22 2 g each were used as test animal. The animals, which were obtained from the laboratory animal facility of the Karigiri (CMC) hospital Tamilnadu, India, were housed in stainless steel cages at room temperature of 28-32 C under a light period of 16-18 h daily. They were fed on standard commercial feed. The experiments were carried out in the same lab in accordance with the institutional guidelines for animal care and use. 2.4 Snake venom & chemicals Lyophilized snake venom ( Naja naja) purchased from Irula snake catchers Industrial cooperative society limited. Chennai, India. Snake venom (mg/ml) was dissolved in 0.9% saline and centrifuged at 2500 rpm for 10 min. The supernatant of the venom was taken for the study. Actyle choline, ATP, and Hyaluronic acid were purchased from Sigma Aldrich (US). P- Nitro phenyl phosphate, p- nitrophenol and alkaline phosphatase were purchased from Sisco Research Laboratory, Mumbai India. All other reagents were analytical grades. 2.5 Lethal potency and symptoms/signs of neurotoxicity LD50 was determined according to the method of Meier and Theakston [22]. Five groups of 20 mice were injected intraperitonially (i.p) with Naja naja venom in 0.3 ml of saline with the doses ranging from 0.1-0.7 mg/kg body weight of the mice and control group has received only saline. Survival time of each animal in each set was recorded for 24 h. LD50 was calculated by the comparison of doses injected, with the observed survival time within 24 h. The effect of all the plant extracts on the lethal potency was studied by pre-incubating the venom with different extracts at 37 C for 1 h and injected intraperitonially into four groups of 20 mice and fifth group served as control which received only saline (0.9% NaCl). 2.6. Enzymatic inhibition studies with active methanol extract 2.6.1 Acetylecholinesterase activity Acetyl cholinesterase inhibition assay was carried out according to the modified method of Ellman et al., [9]. 200 l of venom (1 mg/ml) was preincubated (1 h) with different concentrations of in vivo directed active methanol extract and supernatant was added to the assay mixture which consists of 100 l of 75 mM acetylcholine idodate in 1 ml of phosphate buffer. The activity was was measured by taking the absorbance at 412 nm. Venom without plant extract was considered as control or 100% activity. 2.6.2 Proteolytic activity Proteolytic activity was determined according to the method of Satake et al., [32]. Using 2% casein as substrate in 0.02 M Tris-HCl buffer (pH 8.5). Venom 200 l (1 mg/ml) and different dilutions of plant extract were pre- incubated with 1 ml of substrate for 2 h at 37 C. The undigested casein was precipitated by the addition of 1.5 ml of 0.44 M trichloroacetic acid (TCA) and centrifuged. The digested casein in the supernatant was determined using Folin ciocalteus reagent. Venom without plant extract was considered as control or 100% activity. 2.6.3 Hyaluronidase activity Hyaluronidase activity was assayed according to the method of Ressig et al., [28]. Venom (1 mg /ml) 200 l was pre incubated with different amounts of plant extract for 30 minutes. The mixture was then added to 50 g of hyaluronic acid in 0.2 M sodium acetate buffer pH 5.0 containing 0.15 M NaCl, at 37 C. The change in absorbance was monitored at 585 nm. The venom and reaction mixture without plant extract was referred as control or 100% activity.

*Corresponding author.
B. Muthuvelan, Professor School of Bio sciences and Technology, VIT University, Vellore - 632 014 Tamil Nadu, India Tel.: 91-416-2243091 Ext. 2556 Fax: 91-416-2243092 E-mail:muthusurya@yahoo.com

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2.6.4 ATPase activity ATPase activity was measured according to the modified method of Manjunath Kini and Veerabasappa [19]. Naja naja venom 400 l ( 1 mg/ml ) was pre-incubated with different concentrations of active methanol extract of Andrographis paniculata (1 mg/ml) for 30 M with minutes.To the reaction,1 ml of assay mixture (750 l of 0.1 M Tris pH 7.5,100 l of 0.1MgCl2, 50 l of 0.1 M ATP,and 100 l of BSA) was added with gentle shaking at 37 C and stopped at a certain time (1 h) by adding 1 ml of SDS solution. The inorganic phosphate formed was determined by phosphate determination method by taking 400 l of sample along 600 l of TCA and incubated separately for 10 min at 37 C followed by centrifugation at 1500 rpm for 10 min. About 500 l of supernatant was added together with 500 l of ferrous sulphateammonium molybdate reagent and the absorbance was measured at 820 nm with in 2 h for every 10 minutes of intervals. Reaction mixture without plant sample was referred as control or 100 % activity. Inhibition reaction was calculated in terms of percentage (100%). Values represent mean S.E of three experiments. 2.6.5 Hemolytic activity Hemolytic activity was measured according to the method of Nelson et al., [26]. Naja naja venom 200 l (1 mg/1 ml) was pre-incubated with different amounts of active methanol extract of the Andrographis paniculata for 30 minutes. To the reaction mixture RBC with PBS was added and incubated for 45 minutes at room temperature followed by centrifugation at 5000 rpm per 10 minutes. Supernatant was taken for absorbance at 540 nm. Reaction mixture without plant sample was referred as control. 2.6.6 Statistical Analysis All the data were subjected to t-test and the data with p < 0.01 & 0.05 were considered as significant for further analysis. Further, the results are represented in the form of mean S.D/ S. E. 3. RESULTS 3.1 Plants used for the treatment of snakebites Overall, from our survey in the rural south India, the following plants are considered and recommended by vaidyars as a traditional medicine for snakebites; Acalypa indica, Achyranthes aspera, Andrographis paniculata, Andrographis lineata, Euphorbia hirta, Leucas aspera, and Mimosa pudica. Among these plants, decotion obtained from leaves of Andrographis paniculata is recorded as most effective herbal medicine in the treatment of cobra (Naja naja) bites. 3.2 Lethal potency and symptoms/signs of neurotoxicity LD50 evaluated for the Naja naja crude venom is 450 g/kg body weight of the mice. The administration of preincubated mixture of venom and petroleum ether extract of plant (1:500 w/ w) resulted in over 3.15 0.05 h survival times while the venom preincubated with ethyl acetate extract 1:500 w/w showed increase up to 7.27 0.26 h in the survival period. The survival period recorded for venom preincubated with methanol extract had shown significant prolongation on survival time up to 14.44 0.05 h (Table 1), comparing to all other extracts.
Table. (1). Survival time of mice injected with crude snake venom pre-incubated with different extracts of Andrographis paniculata at different concentrations, on 450 g/kg b.w of venom.
Sample Venom alone Petroleum ether extract Ethyl acetate extract Methanolic extract Aqueous extract Normal saline Venom: Plant ratio (w/w) 1:00 1:250 1:500 1:250 1:500 1:250 1:500 1:250 1:500 Nill Survival time (h) 1.300.06 3.080.05 3.150.05 6.130.05 7.270.26 10.200.06 14.440.55 8.210.03 9.010.27 > 24 h

3.4 Inhibition of protease Activity To assess the in vitro antagonism of protease, the venom degrade the substrate (casein) into peptide precipitation could be observed at 600 nm. In this experiment lane-1 has considered to be 100% of protease activity or zero percentage inhibition of protease. Maximum of protease inhibition (55.9%) was occurred at 1:20 w/w concentration of venom and methanol extract of plant respectively (Fig. 2). From the results it was observed that increased amount of plant extract could increase in the inhibition of protease of Indian cobra venom (Naja naja) protease activity.

Fig. (2). Inhibition of proteolytic activity of Naja naja venom by methanolic extract of Andrographis paniculata. Venom sample (100 g) without plant extract was considered as 100% protease activity or 0 % inhibition of protease.

3.5 Inhibition of hyaluronidase activity In this assay when different amounts of plant extract were pre-incubated with venom (200 g) and the reaction was observed by calorimetrically at 585 nm for every 10 minutes intervals at 585 nm. The results have shown highest inhibition up to 59.4 % of hyaluronidase inhibition at 1:20 w/w dilutions of methanol extract of the plant. Venom sample without plant extract was referred as a control or 100 % inhibition (Fig. 3).

Fig. (3). Inhibition of hyaluronidase of Naja naja venom by active methanolic extract of Andrographis paniculata. (1) Venom sample (100 g) without plant extracts (control, 100 % activity).

3.6 ATPase inhibition ATPase inhibition was calibrated by liberation of inorganic phosphate with of positive control of venom (400 g) and substrate as ATP (10 m). Different concentrations of venom and substrate were used for this reaction. When the amount of venom was increased to 350 g and 400 g, the reaction was observed the kinetics. The Same concentration of venom 400 g with different amounts of active methanol extract of the plant (1:5 to 1:20 w/w) was pre-

3.3 Inhibition of Acetyl cholinesterase activity The methanol extract of the plant was taken in different dilutions starting from 1:5 w/w ratios to 1:20 w/w ratio with triplicate experiments. Starting (1:5 w/w venom and plant extract) dilution has shown least inhibition while maximum inhibition (51.43%) has been recorded in 1:20 dilution. The activity was calculated in terms of percentage of inhibition compared to venom pre-incubated with different amounts of plant extract and venom with substrate. The enzyme reaction was observed for every 10 minutes intervals at 412 nm (Fig. 1). Acetyl cholinesterase activity of the venom was considered as 100%.
Fig. (4). Effect of methanolic extract of Andrographis paniculata on Indian cobra venom ATPase: Venom sample in absence of plant extract was considered as 100% activity. Values represent mean S.E of three experiments.

incubated for the reaction. Maximum inhibition up to 26.28 % (Fig. 4) has been noted at highest amount of plant concentration. 3.7 Inhibition of hemolytic activity Hemolytic activity of Indian cobra (Naja naja) in absence of Andrographis paniculata was considered as 100% activity. In in vitro the venom was preincubated for 1 h with different amounts of active methanol extract of Andrographis paniculata to observe hemolytic inhibition. The extract has shown significant inhibition up to 90.08% on venom hemolytic activity

Fig. (1). Inhibition of acetyl cholinesterase by increased amounts of active methanolic extract of Andrographis paniculata; (1) venom (1 mg/ml) with acetylcholine idodate substrate (control, 100% activity).

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at 1:20 w/w concentration. Increased plant concentration could probably show complete neutralization on venom hemolytic activity (Fig. 5). the plant and further study is required to evaluate the active lead molecules of this extract against venom toxicity. ACKNOWLEDGEMENTS We would like to thank the management of VIT University, Vellore for providing us their premises for carrying out this research work. Our heart felt thanks goes to Directors, SBST & SSH, and N. S. Karthikeyan CSIR-SRF, of VIT University for their valuable suggestions and support. REFERENCES
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(Ed.), Enzymes from Snake Venom. Alaken, Colorado 1998, 689-703. Houghton PJ, Osibogan IM. Flowering plants used against snake bite. J. Ethnopharmacol., 1993,39, 1-29. Hutt MJ, Houghton PJ. A survey from the literature of plants used to treat scorpion stings. J. Ethnopharmacol., 1998, 60, 97-110. Liang GD, Theakston RDG, Leite RP, de-Silva D, Warrell WA, Bias G. Comparison of the potency of three Brazilian Bothrops antivenoms using in vitro rodent and in vitro assays. Toxicon., 1992,30, 1219. Manjunath Kini R, Veerabasappa Gowda T. Studies on snake venom Enzymes: Part I-Purification of ATPase, A Toxic component of Naja naja Venom and its inhibition by Potassium Gymnemate . Indian J. Biochem. Biophys., 1981, 19, 152-154. Manoj L, Dinesh K, Dharma Ram C, Sanjeev S, Pravesh J. A Case of Saw Scale Viper Snakebite Presenting as Pleuro-pericardial Haemorrhage. JIACM., 2002, 3 (4), 392-4. Markland FS. Snake venoms and the hemostatic system. Toxicon 1998, 36: 1749-1800. Meier J, Theakston RG. 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Fig. (5). Inhibition of hemolytic activity of Indian cobra (Naja naja) venom by Andrographis paniculata: Venom without plant extract referred as 100% hemolytic activity.

4. 5.

4. DISCUSSION AND CONCLUSION Plant extracts have been traditionally used as a folk medicine in the treatment of snakebite envenomations all over the world. This is of particular importance especially in local areas where antivenin is not available readily. As a part of it this study has been carried out to understand the scientific basis for the traditional application of Andrographis paniculata to use as snake bite remedy in our region. Aqueous paste and decoction obtained from the leaves of Andrographis paniculata are widely used for snake bites by indigenous people of Southern India [3]. Snake venoms are reported as complex mixtures of different classes of proteins and peptides such proteases, hyaluronidase, esterase, ATPases, which plays a specific role in the envenomation process acting by different mechanisms [6, 21, and 33]. It has also been suggested that the small dose of venom, as typically injected in humans, leads to continuous activation of fibrinogen, producing a fragile fibrin more susceptible to lyses than ordinary fibrin, leading to bleeding manifestations. Vascular endothelial damage caused by the haemorrhagin present in the venom also contributes to bleeding manifestations [20]. Antiserum are being the only therapeutic agent development from animal source used for snake bites and they are time consuming and expensive. Although, use of plants against the effects of snakes bite has been long recognized, more scientific attention has been given since last 20 years [31]. As a part of it, this study has been carried out to understand the scientific basis for the traditional application of Andrographis paniculata (Acanthaceae) to use as snake bite remedy in our region. Earlier reports state that, aqueous paste and decoction obtained from the stem and leaves of Andrographis paniculata are widely used for snake bites by indigenous people of Southern India [2, 36]. An important point of this work was the manner in which the neutralizing effects of plant extracts against snake venoms were evaluated. Neutralization studies can be performed by adopting two basic strategies: (1) Pre-incubation of venom and antivenin prior to testing and (2) Injecting venom and anti-venom separately to experimental mice [14, 11]. In our study, we have used pre-incubation method for screening the active plant extracts against venom. Mice when injected with Naja naja venom in absence of the extract had shown severe neurotoxic symptoms such as paralysis of hind limbs, lacrimation, and urination. The survival time of animal was 1.3 0.06 h followed by injection of lethal dose (450 g/kg, w/w) of Naja naja. The methanol extract of the plant significantly reduced the neurotoxic symptoms caused by venom and extended the survival time of the mice to a greater extent up to 14 0.55 h (Table. 1). The inhibition of Naja naja venom enzymes by increased amounts of methanol extract of Andrographis paniculata was very effective, when the extract was previously mixed with venom. There was a significant neutralization of acetylcholine esterase (Fig. 1), protease (Fig. 2) hyaluronidase (Fig. 3), and hemorrhagic (Fig. 5) activities in vitro and of lethality in vivo experiments by methanol extract which may be the main reason of using, aqueous extract and decoctions of Andrographis paniculata as immediate remedy for snake bites in rural areas. Hyaluronidases in venoms are referred to as spreading factors because they facilitate toxin diffusion into the tissue of the victims by catalyzing the hydrolysis of the glycosamino glycan in the connective tissues, thus contributing to systemic, toxication [34, 11]. The N. naja venom has been found to have a higher hyaluronidase activity when compared to russelii venom [10]. The methanol extract of the plant might contain some active compounds like triterpenoids and phenol compounds which are major constituents of antisnake venom plants [23, 34]. The most likely mechanism for antisnake venom activities by plant extracts could be due to the binding of venom proteins with active neutralizing compounds like; terpenoids and polyphenolic substances of the extracts. Some compounds from plants used for inflammation were known to inhibit enzymes of snake venoms [16, 17] by binding with the venom enzymes, thereby making them unable to act on receptors [17]. Based on the results obtained, Andrographis paniculata seems to have very potent anti-snake venom compounds. In conclusion, our study on the different extracts of Andrographis paniculata showed that the methanol extract of the plant contains active compounds to inhibit the symptoms and toxicity caused by Indian cobra (Naja naja) venom enzymes, in comparison with the other extracts of

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.

Source of support: Nil, Conflict of interest: None Declared

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