Inhibition of Toxic Effects of Viper and Cobra Venom by Indian Medicinal Plants

The mortality rate from snakebites in West Bengal is very high and most of the deaths are caused by the Daboia russelli and Naja naja envenomation. Twenty-three plants from the seventeen families were collected from the traditional healers and explored for the first time for antisnake venom activity. In our previous report, the methanolic root extract of the Indian medicinal plants Pluchea indica, Hemidesmus indicus, Vitex negundo and Emblica officinalis significantly neutralized the Viper and Cobra venom-induced pathophysiological changes [1] [2]. In the present study, we explored four plant extracts (Curcuma aromatica, Aristolochia indica, Androgrphis paniculata and Curcuma zeodaria) for the antisnake venom activity. The plant extracts significantly antagonized Daboia russelli, Echis carinatus, Ophiophagus hannah and Naja kaouthia venom-induced lethal activity both in in vitro and in vivo studies. Daboia russellii venom-induced haemorrhage, coagulant, defibrinogenating and PLA2 activity were significantly neutralized by the extracts. No precipitating bands were observed between the plant extract and venom. This observation confirmed the role of active constituents of plants and plant materials involved in snake venom inhibition. Further studies are going on in our laboratory for the identification of active molecules as well as their mechanism of venom inhibition.


Introduction
Every year more than 100,000 people die due to snakebite worldwide.In India morality rate is very high, 40,000 -50,000 die annually [3] [4].Daboia russelli, Echis carinatus, Naja kaouthia and Naja naja are the common snakes found throughout India and a large number of deaths occur due to envenomation by these snakes.The antiserum is the only therapeutic agent available for the treatment of snakebite and does not provide enough protection against venom inducing haemorrhage, necrosis, nephrotoxicity and often produces hypersensitive reactions [5]- [7].Antiserum development in animal is time consuming, expensive and requires ideal storage condition.Monovalent antiserum is not available and the health center is usually far and few in number.To overcome these drawbacks, there is a great need to search, to develop new affordable and suitable antidote against snakebite.
The World Health Organization estimates that 80% of the world's population depends on traditional medicine for their primary health care needs.As long as man can remember, plants/plant materials have been used worldwide in traditional medicine for the treatment of different diseases.It is estimated that even today approximately seventy percent of the world population rely on medicinal plants as their primary source of medicines [8].Till today many of the drugs used are derived from natural products or are dependent upon natural products for the development.In the recent discoveries of the analgesic, anti-arthritis, antimicrobial and anticancer agent (taxol) indicate the continuing importance of plant species in drug discovery.Currently, a large number of plants and plant materials are being screened for pharmacological activities especially those used in traditional/folk medicine against different diseases.Over the years, many attempts have been made for the development of snake venom antagonists especially from plant sources in spite of the existence of antiserum.Many Indian medicinal plants/plant materials are recommended for the treatment of snakebite [2] [9] [10] and some are tested, but so far no systematic analysis has been done.In our earlier studies, it was showed that four of these plants (Table 1), successfully neutralized the venom-induced pathophysiological changes [1] [2].The present investigation attempts to identify those species, which are used in folk medicine in West Bengal, India and are worthy of further investigation as leads for drug development in management of snake bite.

Venom
Lyophilized snake venom of Daboia russelli, Echis carinatus, Ophiophagus hannah and Naja kaouthia were obtained from Calcutta snake park, Kolkata, India and were preserved in a desiccator at 4˚C for further use.It was dissolved in 0.9% saline and centrifuged at 2000 rpm for 10 min.The solution was used as venom and was stored at 4˚C until further use.Venom concentration was expressed in terms of dry weight.

Plant Extracts
Plants/plant materials were obtained from different places of West Bengal after being indicated by traditional healer and then they were identified by Dr. N. Paria, Department of Botany, University of Calcutta, West Bengal.Some of these plants were also purchased commercially from M/s United chemical and Allied products, Kolkata, and processed in the following manner.
After drying and crushing individual sample of the relevant parts of each plant species, was extracted first by refluxing with petroleum ether (50˚C -60˚C, for 72 h) and then by methanol (60˚C -80˚C for 72 h).Methanol extract was concentrated in vaccuo and kept in a desiccator at room temperature for further use.Before use, it was dissolved in saline and centrifuged at 2000 rpm for 10 min at room temperature.The solution was used for further investigation and kept at 4˚C.The plant extracts were expressed in terms of dry weight.

Snake Venom Antiserum
Lyophilized polyvalent snake venom antiserum (as reference serum) was obtained from Bengal chemicals and pharmaceuticals Private Ltd.Kolkata.Before use antiserum was dissolved in 0.9% saline.

Animals
Male albino mice of body weight about 18 -20 g were used in this study after acclimatization to the laboratory condition.The study plan was approved by the Animal Ethics Committee of University of Calcutta, Kolkata; India.All animals were cared for and handled in accordance with the guideline of the ethical committee.

Inhibition of Venom Lethal Effect
The toxicity of snake venom was assessed by injection of different concentration of venom in 0.2 ml physiological saline into tail vein of male albino mice 18 -20 gm [1].To assess the in vitro antagonism, various concentrations of venom (1 -300 LD 50 ) were mixed with a fixed amount of plant extract (2 mg), the mixture incubated at 37˚C for 1h, and centrifuged at 2000 rpm for 10 min.The supernatant was injected intravenously into male albino mice (18 -20 g), ten mice per dose.The median lethal dose (LD 50 ) was calculated 24 h after injection of the venom-extract mixture.Lethal toxicity was also assessed by subcutaneously injection of various doses of venom.The neutralizing potency of each plant extract was assessed by s.c injection of venom (1 -5 LD 50 ) into groups of ten mice followed by immediate injection of fixed dose of plant extract (5 mg) orally (p.o.).

Inhibition of Venom Haemorrhagic Activity
The minimum haemorrhagic dose (MHD) of venom (defined as the least amount of venom which when injected intradermaly (i.d) into mice results in a haemorrhagic lesion of 10 mm diameter 24 h later was measured [11].
Neutralization of the haemorrhagic activity was estimated by mixing a fixed amount of plant extract (2 mg) with different amounts of venom (1 -300 MHD).The plant extract-venom mixture was incubated at 37˚C for 1h, centrifuged at 2000 rpm × 10 min, and 0.1 ml of supernatant injected (i.d).The haemorrhagic lesion was estimated after 24 h.To assess the antihaemorrhagic activity of venom in vivo, various amount of venom (1 -6 MHD) were injected (i.d) followed immediately by the plant extract (5 mg, p.o.) and the haemorrhagic lesion measured after 24 h.

Inhibition of Venom Coagulant Activity
The minimum clotting dose of plasma (MCDP) was determined as described by Alam et al. [1].Neutralization of this activity was estimated by mixing different amount of venom (1 -200 MCD), with a fixed amount of plant extract (100 µg), incubating for 1 h at 37˚C.Different concentration of incubate were added to the experimental tube in place of 0.1 ml physiological saline, and the clotting time was recorded.

Inhibition of Venom Serum Inhibitory Activity
Inhibition of snake venom serum activity was measured in order to examine if the decreased clotting of V. russellii venom on goat plasma was due to venom inhibition in serum.Goat blood was collected without anticoagulant, incubated in glass tubes for 2 h at 37˚C and then centrifuged at 2000 rpm for 15 min.Pooled sera were kept frozen at −20˚C.Equal volumes of Tyrode-Hepes buffer (137 mM Nacl; 2.7 mM KCl; 12 mM NaHC0 3 , 0.42 mM NaH 2 Po 4 , 10 mM Hepes; pH 7.4) or goat serum was incubated with venom solution of different concentration for 5 min at 37˚C.Aliquots of 50 µl were then added to 200 µl of goat serum (maintained for 2 min at 37˚C) and the clotting time recorded.MCDS were then calculated.Neutralization of serum inhibitory activity was estimated by mixing different amount of venom with fixed amount of plant extract, incubating for 1 h at 37˚C and centrifuged.Supernatant was then added to the experimental group and the clotting time was observed.

Inhibition of Venom Defibrinogenating Activity
Minimum defibrinogenating dose (MDD) of V. russellii venom is defined as the minimum amount of venom which when injected i.v into mice causes' incoagulable blood 1 h later [1].Neutralization of this activity was estimated by mixing different amount of venom with fixed amounts of plant extract, incubating at 37˚C for 1 h, and centrifuge at 3000 rpm at 5 minutes.The supernatant was injected i.v into albino mice (18 -20 g) as described above.For in vivo studies, the MDD of venom was injected i.v followed by the plant extract (p.o.) and the nature of the blood observed after 1 h.The neutralization ability of plant extracts was expressed as effective dose 50 (ED 50 ) defined as the amount of plant extract at which the venom induced (lethal and haemorrhagic) action was reduced by 50%.The effective dose (ED) was defined as the amount of plant extract at which the venom induced MCDP and MDD activities were effectively neutralized.
For carrying out the experiment, 2.0 ml of egg yolk suspension and 0.2 ml of test material (venom or venom compound) were mixed in the test tube and incubated at 37˚C for 1 h.After incubation the time required for coagulation of egg yolk was recorded by placing the test tube containing test materials on the boiling water bath.A blank was run with normal saline instead of test material.One unit enzyme activity was defined as the amount of venom, which increased the coagulation time of the egg yolk control by one minute.
Neutralization of the enzyme activity was estimated on the different amount of plant extract mixed with the different amount of viper venom.The venom-extract mixture was incubated at 37˚C for 1 h.Centrifuged at 2000 rpm for 10 min, supernatant was tested in a total of 0.2 ml for the enzyme neutralization activity.

Immunological Interaction of Venom and Plant Extracts
To assess the immunological interaction of venom and plant extract, immunogel diffusion [12] and immunogel electrophoresis [13] were performed.

Statistical Analysis
All results are expressed as Mean ± SE.The significance of the difference between mean was determined by student's t test.All assays were performed ten times.
Through collection from different rural places of West Bengal and identification, twenty three plants/plant materials of seventeen families were examined for anti-snake venom activity.Eight plant materials were found to be active against snake bite (Table 2).In the present paper only four plants those are not reported earlier were taken for anti-snake venom activity.The plant extracts (up to 1 mg/kg, i.v) did not produce lethal effect.
The ED 50 of the plant extracts of C. aromatica, A. indica, A. paniculata and C. zeodaria were found to be 0.21 mg, 0.22 mg, 0.31 mg, 0.53 mg in vitro and 2.6 mg, 2.7 mg, 2.8 mg, 2.8 mg respectively in in vivo against Daboia russelli venom (Table 2).

Coagulant Activity
The effect of Daboia russellii venom on coagulant activity and their neutralization by plant extract were examined as estimated by plasma recalcification time.The normal value (0.9% saline + calcium induced) of minimum clotting dose of plasma (MCDP) was found to be 25 ± 0.17 seconds and venom + calcium induced MCDP was found to be 15 ± 0.20 second.
The extracts (0.2 mg each) were incubated with different amount of venom (1 -75 µg) and tested for coagulant activity.The venom induced plasma recalcification were antagonized effectively by the plant extracts (Table 5).

Serum Inhibitory Activity
Daboia russellii venom was taken for the serum inhibitory activity.The normal value (calcium-induced) of minimum clotting dose of serum (MCDS) was found to be 25 ± 0.20 seconds.The plant extracts (0.2 mg each) incubated with different amount of venom (1 -75 µg), antagonized serum inhibitory activity significantly (Table 5).

Defibrinogenating Activity
The methanolic root extract of the plants effectively antagonized the viper venom induced defibrinogenating activity.In in vitro study, the plant extracts (2 mg) gave protection up to 20 MDD against venom-induced defibrinogenation.In in vivo study, venom-induced defibrinogenation was antagonised by the plant extracts.The effective dose of C. aromatica, A. indica, A. paniculata and C. zeodaria was found to be 0.32 mg, 0.34 mg, 0.43 mg, 0.44 mg respectively in in vitro and 2.4 mg, 2.6 mg, 2.5 mg, 2.5 mg respectively in in vivo of Daboia russellii venom (Table 6).The fold of protection was always higher in in vitro studies.

Phospholipase Activity
Phospholipase A 2 activity of Daboia russellii venom was assessed by egg yolk coagulation method.One unit of Daboia russellii venom activity was found to be 2 μg which increased the coagulation time by one minute (control 0.9% saline, coagulation time was found to be 45 ± 1.16 seconds).
The extracts (C.aromatica, A. indica, A. paniculata and C. zeodaria: 0.2 mg each) were incubated with different amount of viper venom (2 -100 μg) and tested for phospholipase A 2 activity.The venom PLA 2 was effectively neutralized by the plant extracts.The Curcuma aromatic extract (0.2 mg) significantly neutralized the venom (80 μg) induced PLA 2 activity.The degree of protection was found to be higher in Curcuma aromatic.The effective dose to be 18 μg, 20 μg, 20 μg and 20 μg by C. aromatica, A. indica, A. paniculata and C. zeodaria respectively (Table 7).Effective dose of plant extract which neutralized venom dose of 10 units of PLA2 activity.Results are expressed as mean of ten observations.1 unit = one unit of enzyme activity of Daboia russelli venom was defined as the amount of venom which increased the coagulation time of egg yolk by one minute.

Immunological Interaction of Snake Venom and Plant Extracts
Immunogel diffusion and immunogel eclectophoresis of snake venom of Daboia russelli, Echis carinatus, Ophiophagus hannah and Naja kaouthia were produced precipitating band with polyvalent antisnake venom antiserum.However venom antigen and plant extract did not produced any precipitating band.

Discussion
More than hundreds of plants have been used in folk medicine throughout the world for snakebites [14] [15].Till date few plants/plant materials have been evaluated in well controlled assays and about forty of them have been found to be effective against Cobra and Viper envenomation [2] [16].In our earlier studies we found that the methanolic root extracts of H. indicus, P. indica, E. officinalis and V. negundo possessed potent anti-snake venom activity [1] [2].These plant extracts effectively neutralized the snake venom inducing lethal, haemorrhagic, coagulant and anti-coagulant activity both in vivo and in vitro.An organic acid, 2-hydroxy-4-methoxy benzoic acid was isolated, purified from the Indian medicinal plant Hemidesmus indicus root extract, which effectively antagonized the Daboia russelli venom inducing pathophysiological changes both in vitro and in vivo.
The mechanism of venom inhibition by the compound was due to free radical formation inhibition through lipoxygenase and cyclo-oxygenase pathway [17].
In the present work, four plants out of twenty three plant species were reported for the first time in the Indian context.The plants/plant materials were collected from the traditional healers used in snakebite in the eastern parts of India and were identified by the botanist.In the present investigation the four new plant species which were found active against snake venom were taken for further study.The four plant extracts Aristolochia indica, Andrographis paniculata, Curcuma aromatica and Curcuma zeodaria were effective against snake-venom of Cobra (Naja kaouthia and Ophiophagus hannah) and Viper (Daboia russelli and Echis carinatus) venom both in vivo and in vitro.The maximum fold of protection was found by the methanolic root extract of Curcuma aromatica both in Viper and Cobra venom.The Daboia russellii venom which induced haemorrhagic, coagulant and anticoagulant activities (both in vitro and in vivo) were significantly neutralized by all the four plant extracts.
Of all the plant compounds reported against snake envenomation, the mechanism of action was somehow similar.Some of the plant constituents like coumarin, polyphenols, flavonoids, cinnamic acid derivatives, hydroxyl benzoic acid derivatives, tannins and aristolochic acid have the protein binding and enzyme inhibiting properties [18].This is due to their affinity to peptides, proteins (enzymes and other toxic components) and metal binding capacity present in the compounds.The PLA 2 enzyme is present in large quantity in almost all the snake venom and has been found to be neutralized by several plant compounds [17] [19] [20].
Curcumin present in Curcuma aromatica may be involved in venom inactivation process.Another unsaturated ketone of turmeric, ar-turmerone was shown to inhibit the lethal action of rattle snake venom [21] [22].Aristolochia species are famous for snakebite remedies.The aristolochic acid effectively neutralized the viper venom-induced edema [23].This compound may act through enzyme association and cause significant change in the secondary structure of the snake venom protein.The other chemical compounds present in the plant can be held responsible for the neutralizing effect of snake venom actions [24].Many compounds identified from plants having different chemical structure were reported to be capable of interacting with peptides and proteins (enzyme) of snake venom.The mechanism of action of the plant extracts/plant compounds are still not clear [25], and they may be attributed to the blocking of receptors-structure prone to chemical attack, and may block the active site of the snake venom.Other mechanism of action of the plant compounds are inhibition of metalloproteinase present in the snake venom.This is due to the metal chellator substances present in the plant extracts/plant compounds.The presence of benzoic acid derivatives and phenolic compounds seem to act through LOX, COX and free radical formation inhibiting systems [17].This is one of the possible mechanisms of action of venom inhibition.The above evidence may provide some clues of mechanism of venom inhibitions.In our laboratory further work is going on for the identification of pure compound(s) from the plants/plant extracts and their mechanism of venom inhibition.

Table 1 .
Medicinal plants used by traditional healer against snake bites were tested in laboratory against snake venoms in experimental rodents.

Table 2 .
Neutralization of lethal action of Daboia russellii and Echis carinatus venom by plant extracts in male albino mice.
* Defined as the effective dose of plant extract which neutralized venom dose of 2 LD50 in vitro/2LD50 in vivo.Results are expressed as mean of ten observations.** Duration of deaths time following venom exposure: Daboia russellii venom (1 Minimum lethal dose) = 13.24 ± 0.30 hours; Echis carinatus venom (1 Minimum lethal dose) = 12.20 ± 0.30 hours.

Table 4 )
. In in vivo study, venom (1 -2 MHD) injected (i.d) into mice followed by plant extracts (5 mg, p.o.), gave protection against venom induced haemorrhagic activity.The degree of protection in in vivo was less than that of in vitro.Effective dose of C. aromatica, A. indica, A. paniculata and C. zeodaria were found to be 0.25 mg, 0.26 mg, 0.35 mg, 0.34 mg in vitro and 2.5 mg, 2.6 mg, 2.5 mg, 2.7 mg respectively in vivo against Daboia russellii venom (Table4).

Table 3 .
Neutralization of lethal action of Naja kaouthia and Ophiophagus hannah venom by plant extracts in male albino mice.
* Defined as the effective dose of plant extract which neutralized venom dose of 2 LD50 in vitro/2LD50 in vivo.Results are expressed as mean of ten observations.** Duration of deaths time following venom exposure: Ophiophagus hanah venom (1 Minimum lethal dose) = 6.24 ± 0.20 hours; Naja kaouthia venom (1 Minimum lethal dose) = 10.68 ± 0.30 hours.

Table 4 .
Neutralization of haemorrhagic action of Daboia russellii and Echis carinatus venom by plant extracts in male albino mice.
Defined as the effective dose (ED) of plant extract which neutralized venom dose of 25 MHD in vitro and 1 MHD in vivo.Results are expressed as mean of ten observations.

Table 5 .
Neutralization of coagulant activity of Daboia russellii venom by the plant extracts.

Table 6 .
Neutralization of defibrinogenating activity of Daboia russellii venom by plant extracts in male albino mice.Defined as the effective dose of plant extract which neutralized venom dose of 3 MDD in vitro and 1 MDD in vivo.Results are expressed as mean of 10 observations.MDD minimum defibrinogenating.Dose = 2.5 µg. *

Table 7 .
Neutralization of Phospholipase A2 activity of Daboiarusselli venom by plant extracts.