PurposeThe present investigation was aimed at evaluating the anti-ophidian properties of ethnomedicinal herb Leucas aspera against Indian cobra, Naja naja venom enzymes.MethodsMethanolic 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).ResultsVenom 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.ConclusionThe 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.
The present study reports the formulation of soy protein nanoparticles and its conjugation to antivenom. The conditions for nanoparticle formation were optimised by considering particle size, count rate, stability and zeta potential. The smallest particle size of 70.9 ± 0.9 nm with a zeta potential of -28.0 ± 1.4 mV was obtained at pH 6.2, with NaOH 5.4 % and 28 μg/mg glutaraldehyde. The nanoparticle was conjugated with antisnake venom immunoglobulins (F(ab')2 fragments) using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide. TEM analysis indicated the increased size of particle to 600 nm after conjugation to antivenom. Further, in vitro studies indicated that conjugated antibodies inhibited the activity of protease, phospholipase and hyaluronidase enzymes of Bungarus caeruleus venom more efficiently than the free antivenom. This is the first report on the use of protein nanoparticles for conjugating snake venom antibodies and their implications for neutralising snake venom enzymes with increased efficiency.
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