Evaluation of Daboia russelii Venom Induced Biochemical Changes in Calotropis gigantea (L). R.Br Treated Mice

Abstract

Objective To study the venom neutralization ability and prophylactic effect of Calotropis gigantea methanolic root extract (CGMR) against Daboia russelii envenomation using in vitro, in silico, and in vivo methods. Background Snake venom is composite, that contains neurotoxins, disintegrins, hemotoxins, and proteases in complex with phospholipase A2 [PLA2] enzymes. Hence toxicity of venom is mainly attributed to PLA2 enzymes or their protein complexes per se. Roots of the plant, Calotropis gigantea are used extensively as a phyto-antidote by tribal communities to treat snake bite victims. However the antivenom property of root has not been comprehensively studied so far. This study scientifically reasserted the use of Calotropis Gigantea R.Br (root extract) in neutralization of D.russelii venom, alongside investigated the protection rendered by the extract. Materials and Methods Phytochemicals of Calotropis gigantea methanolic root (CGMR) extract were fractionated into hexane (non-polar fraction) and methanol (polar fraction) based on polarity. The in vitro PLA2 inhibitory action of crude extract and both fractions was determined using biochemical assay. Since significant PLA2 inhibition was observed in non-polar fraction, it was subjected to GC/MS analysis. The compounds obtained through GC/MS analysis were virtually docked to PLA2 macromolecule by protein-ligand binding simulation programs. The acute and sub acute toxicity of the extract was determined using guidelines 423 and 407 respectively in mice. The LD50 dose of Daboia russelii snake venom (DRSV) was determined using standard protocol. The in vivo neutralization ability and the in vitro neutralization (pre-incubated) LD50 of venom was determined to be 11µg/dose and the % survival of group receiving only venom was 50%. High dose of CGMR effectively neutralized LD50 dose of venom in both in vivo (%survival = 67.67%) and in vitro neutralization experiment (% survival =100%). Neutralization of venom was better in in vitro neutralization (pre-incubation). The histopathological analysis of organs necropsied from CGMR pre-treated animals demonstrated significant tolerance against venom. Conclusion Together these results indicate that CGMR has significant venom neutralizing potential as traditionally claimed. Further the extract showed significant venom neutralization ability and protection effects against venom insult. The high venom neutralization ability may be due to PLA2 inhibitors present in the extract.