In vitro and In vivo Anti-Arthritic and Anti-Inflammatory Activity of Bungarus Fasciatus Venom

In the Indian traditional medicinal system Ayurveda and Unani, the use of snake venom has been mentioned to treat various diseases including arthritis. This prompted us to investigate the anti-arthritic and anti-inflammatory activity of banded krait venom (BFV). In the present study, in vitro anti-arthritic activity of BFV was evaluated by bovine serum protein denaturation and egg albumin denaturation and in vivo antiarthritic activity was evaluated by collagenase induced arthritis in male albino Wistar rats. In vitro anti-inflammatory activity was evaluated by RBC membrane stabilization and in vivo anti-inflammatory activity was evaluated by histamine/ formalin induced paw edema in male albino Swiss mice. BFV treatment significantly inhibited bovine serum protein and egg albumin denaturation. In collagenase induced arthritis model, BFV treatment significantly reduced serum proinflammatory cytokines TNF-α, IL-1β and IL-17, significantly increased percentage stabilization of RBC membrane and significantly decreased histamine/ formalin induced paw edema. The findings of the present study showed that Bungarus fasciatus venom possesses significant in vitro and in vivo antiarthritic and anti-inflammatory activities and may serve as a natural biomedicinal remedial agent against inflammatory conditions. Copyright: © 2015 Ghosh S, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Citation: Ghosh S, Saha K, Dasgupta SC, Gomes A. In vitro and In vivo Anti-Arthritic and Anti-Inflammatory Activity of Bungarus Fasciatus Venom. J Toxins. 2015;2(1): 5. J Toxins 2(1): 5 (2015) Page 02 ISSN: 2328-1723 bovine serum protein denaturation method [18] and egg albumin denaturation method [18]. Bovine serum albumin (BSA) denaturation method: Test solution (0.5 ml): 0.5% w/v aqueous solution of BSA (0.45 ml) and test solution (0.05 ml) of different concentrations were used. Test control solution (0.5 ml): 0.5% w/v aqueous solution of BSA (0.45 ml) and distilled water (0.05 ml) were used. Product control (0.5 ml): 0.45 ml distilled water and test solution (0.05 ml) of different concentrations were used. Standard solution (0.5 ml): 0.5% w/v aqueous solution of BSA (0.45 ml) and diclofenac sodium (0.05 ml) of different concentrations were used. Test solution (0.05 ml) of different concentrations (2, 5, 10, 20, 30 and 60 μg/ml) and standard drug diclofenac sodium (0.05 ml) of different concentrations (2, 5, 10, 20, 30 and 60 μg/ml) were mixed with 0.5% w/v aqueous solution of BSA (0.45 ml). Then the samples were incubated at 37 oC for 20 min followed by incubation at 57 oC for 3 min. 2.5 ml of phosphate buffer (pH 6.3) was added to all the above samples after cooling. UV-Visible spectrophotometer (Analab) was used to measure the absorbance at 255 nm. The control represents 100% protein denaturation. The percentage inhibition of protein denaturation was calculated by the following formula: Percentage inhibition = 100 – [{(optical density of test solution – optical density of product control)/ optical density of test control}/ × 100]. Egg albumin denaturation method: 0.2 ml of fresh hen’s egg albumin, 2.8 ml of phosphate buffered saline (pH 6.4) and test solution of different concentrations (2, 5, 10, 20, 30 and 60 μg/ml) or standard drug diclofenac sodium (0.05 ml) of different concentrations (2, 5, 10, 20, 30 and 60 μg/ml) were mixed to form a reaction mixture of 5 ml. Double distilled water of same volume served as control. The samples were incubated at 37±2 oC in a BOD incubator for 15 min followed by heating at 70 oC for 5 min. UV-Visible spectrophotometer (Analab) was used to measure the absorbance at 255 nm. The percentage inhibition of protein denaturation was calculated by the following formula: Percentage inhibition = 100 × [absorbance of test sample/ absorbance of control – 1] In vivo anti-arthritic activity In vivo anti-arthritic activity of BFV was evaluated using collagenase induced osteoarthritis model [19]. Collagenase induced arthritis model: Induction of osteoarthritis was done in adult Wistar male albino rats (120±10 gm) by intraarticular injection of 20 μl bacterial collagenase (5 CDU) solution in the right knee joint. Same amount of saline was injected in the left knee joint. After treatment of 14 days, animals were sacrificed and serum pro inflammatory cytokines were measured. Treatment schedule: Animals were randomised in 4 groups (n=6). Group 1: Sham control, Group 2: Osteoarthritic control, Group 3: Standard drug, Indomethacin treated (0.25 mg.kg-1, p.o. × 5 days, alternatively), Group 4: BFV treated (0.3 mg.kg-1, i.p. × 14 days) Measurement of serum cytokines: Serum pro inflammatory cytokines TNF-α, IL-1β and IL-17 were measured using ELISA kits (R & D Systems, USA). In vitro anti-inflammatory activity In vitro anti-inflammatory activity of BFV was evaluated using RBC membrane stabilization method [20]. RBC membrane stabilization method: Goat red blood cells were used for the study of membrane stability. Goat blood was collected and mixed with 3.8% tri-sodium citrate solution and centrifuged at 3000 rpm. After washing the packed cells in isosaline, 10% suspension was made. 4.5 ml reaction mixture was prepared with 0.5 ml of 10% RBC suspension, 1 ml of phosphate buffer (pH 7.4), 2 ml of hyposaline (0.25% NaCl) and 1 ml of test solution of different concentrations (2, 5, 10, 20, 30 and 60 μg/ml). The samples were then incubated at 37 oC for 30 min followed by centrifugation for 20 min at 3000 rpm. Supernatant solution was tested for haemoglobin content at 560 nm using a UV-Visible spectrophotometer (Analab). In vivo anti-inflammatory activity In vivo anti-inflammatory activity of BFV was evaluated using histamine induced paw edema model [21] and formalin induced paw edema model [22]. Histamine induced paw edema model: Histamine induced paw edema was analyzed in mice model. Mice were randomised into three groups (n=6). Gr.1: Histamine control, Gr.2: Standard drug, Indomethacin treated (10 mg.kg-1, i.p.), Gr.3: BFV treated (1.25 mg.kg-1, i.p.). The initial volume of right hind paw of mice was measured using digital caliper (Mitutoyo, Japan). Then, 0.02 ml of 1% histamine was injected subcutaneously into the subplantar region of right hind paw. The volume of right hind paw was measured at 1, 2, 3 hour after histamine injection for determining the volume of paw edema. Standard drug, Indomethacin and BFV were given one hour before histamine injection. Formalin induced paw edema model: Formalin induced paw edema was analyzed in mice model. Mice were randomised into three groups (n=6). Gr.1: Formalin control, Gr.2: Standard drug, Indomethacin treated (10 mg.kg-1, i.p.), Gr.3: BFV treated (1.25 mg.kg-1, i.p.). The initial volume of right hind paw of mice was measured using digital caliper (Mitutoyo, Japan). Then, 0.02 ml of 2% formalin was injected subcutaneously into the subplantar region of right hind paw. The volume of right hind paw was measured at 1, 2, 3 hour after formalin injection for determining the volume of paw edema. Standard drug, Indomethacin and BFV were given one hour before formalin injection. Statistical analysis Data expressed as mean ± SEM (n=6). One way ANOVA was carried out for determination of significant differences between groups. *p<0.05 was considered to be statistically significant (Gr.1 vs Gr.2) and **p<0.05 was considered to be statistically significant (Gr.2 vs Gr.3/Gr.4). Results In vitro anti-arthritic activity Effect of BFV on bovine serum albumin (BSA) denaturation: Citation: Ghosh S, Saha K, Dasgupta SC, Gomes A. In vitro and In vivo Anti-Arthritic and Anti-Inflammatory Activity of Bungarus Fasciatus Venom. J Toxins. 2015;2(1): 5. J Toxins 2(1): 5 (2015) Page 03 ISSN: 2328-1723 BFV at a concentration of 2, 5, 10, 20 and 30 μg/ml showed inhibition of BSA denaturation by 3.06%, 4.36%, 4.97%, 9.39% and 20.86% respectively whereas standard drug, diclofenac sodium at a concentration of 2, 5, 10, 20 and 30 μg/ml showed inhibition of BSA denaturation by 2.92%, 5.69%, 9.79%, 13.05% and 20.29% respectively (Figure 1). Effect of BFV on egg albumin denaturation: BFV at a concentration of 2, 5, 10, 20 and 30 μg/ml showed inhibition of egg albumin denaturation by 12.06%, 17.25%, 21.81%, 26.59% and 31.15% respectively whereas standard drug, diclofenac sodium at a concentration of 2, 5, 10, 20 and 30 μg/ml showed inhibition of egg albumin denaturation by 5.67%, 7.11%, 12.46%, 17.33% and 21.65% respectively (Table 1). In vivo anti-arthritic activity Effect of BFV on serum proinflammatory cytokines: BFV significantly reduced serum pro inflammatory cytokines TNF-α, IL1β and IL-17 by 42.17%, 58.14% and 26.91% respectively; whereas standard drug, Indomethacin showed a reduction of TNF-α, IL-1β and IL-17 by 46.83%, 57.07% and 45.81% respectively when compared to arthritic control rats (Figure 2). In vitro anti-inflammatory activity Effect of BFV on RBC membrane stabilization: BFV at a concentration of 2, 5, 10, 20 and 30 μg/ml showed stabilization of RBC in hypotonic solution by 25.98%, 32.66%, 40.43%, 56.40 % and 60.29% respectively whereas standard drug, diclofenac sodium at a concentration of 2, 5, 10, 20 and 30 μg/ml showed stabilization of RBC in hypotonic solution by 22.67%, 29.84%, 45.52%, 52.59% and 56.66% respectively (Figure 3). In vivo anti-inflammatory activity Effect of BFV on histamine induced paw edema: BFV showed significant reduction of histamine induced paw edema by 65.85% whereas standard drug, Indomethacin showed significant reduction of histamine induced paw edema by 70.73% when compared to histamine control group (Table 2). Effect of BFV on formalin induced paw edema: BFV showed significant reduction of formalin induced paw edema by 34.23% whereas standard drug, Indomethacin showed significant reduction of formalin in