Inhibition of Naja naja venom enzymes by the methanolic extract of Leucas aspera and its chemical profile by GC–MS

Purpose: The present investigation was aimed at evaluating the anti-ophidian properties of ethnomedicinal herb Leucas aspera against Indian cobra, Naja naja venom enzymes. Methods: Methanolic extract of Leucas aspera was evaluated, in vitro, for its ability to inhibit the major enzyme activities of Naja naja venom including protease, phospholipase A2, hyaluronidase and hemolytic factors. The type of phytochemicals present in the extract was analyzed. Also, the major phytoconstituents in the extract was determined by gas chromatography–mass spectrometry (GC–MS). Results: Venom protease and hyaluronidase activities (two isoforms) were completely (100%) neutralized by the L. aspera methanolic extract at ratio of 1:50 w/w (venom: plant extract) and venom hemolytic activity was also completely neutralized at a ratio of 1:80 w/w by the plant extract. However, the extract failed to neutralize phospholipase A2 activity even at the highest concentration used. Phytochemical analysis revealed the presence of alkaloids, acidic compounds, flavonoids, steroids and cardiac glycosides in the extract. GC–MS analysis indicated that a total of 14 compounds were present in the extract. The major bioactive constituents were found to be 6-octadecenoic acid (32.47%), n-hexadecanoic acid (25.97%), and 17-octadecen-14-yn-1-ol (14.22%) along with the minor constituents, sitosterol (2.45%) and stigmasterol (2%), which was previously reported to exhibit antivenom activity. Conclusion: The results obtained demonstrate for the first time that the methanolic extract of Leucas aspera possesses anti-venom activity and could be considered as a potential source for the anti-ophidian metabolites

Quercetin-3-O-rhamnoside from Euphorbia hirta protects against snake Venom induced toxicity

Background The plant Euphorbia hirta is widely used against snake envenomations in rural areas and it was proved to be effective in animal models. Therefore, the scientific validation of its phytoconstituents for their antiophidian activity is aimed in the present study. Methods E. hirta extract was subjected to bioactivity guided fractionation and the fractions that inhibited different enzyme activities of Naja naja venom in vitro was structurally characterized using UV, FT-IR, LC–MS and NMR spectroscopy. Edema, hemorrhage and lethality inhibition activity of the compound were studied in mice model. In addition, molecular docking and molecular dynamic simulations were also performed in silico. Results The bioactive fraction was identified as Quercetin-3-O-α-rhamnoside (QR, 448.38 Da). In vitro experiments indicated that protease, phospholipase-A2, hemolytic activity and hemorrhage inducing activity of the venom were inhibited completely at a ratio of 1:20 (venom: QR) w/w. At the same concentration, the edema ratio was drastically reduced from 187 to 107. Significant inhibition (93) of hyaluronidase activity was also observed at a slightly higher concentration of QR (1:50). Further, in in vivo analysis, QR significantly prolonged the survival time of mice injected with snake venom. Conclusion For the first time Quercetin-3-O-α-rhamnoside, isolated from E. hirta, has been shown to exhibit anti-snake venom activity against Naja naja venom induced toxicity. General significance Exploring such multifunctional lead molecules with anti-venom activity would help in developing complementary medicine for snakebite treatments especially in rural areas where anti-snake venom is not readily available