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.

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.

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.

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.

^*Plant possess anti-snake venom activity as reported earlier (Alam and Gomes, 2003; Alam et al., 1996).

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 LD₅₀) 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 (LD₅₀) 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 LD₅₀) into groups of ten mice followed by immediate injection of fixed dose of plant extract (5 mg) orally (p.o.).

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.

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 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 NaHC0₃, 0.42 mM NaH₂Po₄, 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 ml were then added to 200 ml 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.

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 (ED₅₀) 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 PLA₂ activity experiments, egg yolk (obtained from commercially available eggs), 0.5% EDTA, 50 mM Tris HCl buffer (pH 7.5), 2% sodium chloride, 1% calcium chloride and 0.9% sodium chloride from Sigma except otherwise stated were used.

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.

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

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.

In in vitro study, viper venom (1 - 50 LD₅₀) was incubated with plant extracts (2 mg) and gave protection against venom-induced lethality. In in vivo study, viper venom (1 - 6 LD₅₀) was injected s.c into male albino mice followed by the plant extracts (5 mg/mouse, p.o.). Daboia russellii, Echis carinatus, Ophiophagus hannah and Naja kaouthia venom induced lethality was significantly antagonized by the plant extracts (Table 2 & Table 3). The ED₅₀ 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).

In in vitro study, venom (1 - 10 MHD) incubated with extract (2 mg) and injected (i.d) into mice, gave protection against venom induced haemorrhagic activity (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 (Table 4).

^*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.

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.

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 mg) and tested for coagulant activity. The venom induced plasma recalcification were antagonized effectively by the plant extracts (Table 5).

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 mg), antagonized serum inhibitory activity significantly (Table 5).

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 A₂ 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 A₂ activity. The venom PLA₂ was effectively neutralized by the plant extracts. The Curcuma aromatic extract (0.2 mg) significantly neutralized the venom (80 μg) induced PLA₂ 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).

^*Defined as the effecting dose of plant extract which neutralized venom dose of 15 MCDP/15 MCDS. Results are expressed as mean of 10 observations. MCDP = minimum clotting dose plasma; MCDS = Minimum clotting dose serum.

^*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 mg.

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.

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.