Proteomic Characterization of Trimeresurus insularis Venom and Hemotoxic Neutralization by Azadirachta indica Extract

Background and objectives: Trimeresurus insularis is a medically important pit viper endemic to the Lesser Sunda Islands, Indonesia. Envenomation typically causes local hemorrhage, edema, and necrosis, attributed to snake venom metalloproteinases (SVMPs), serine proteases (SVSPs), and phospholipases A2 (PLA2). This study aimed to characterize the venom proteome of T. insularis using LC-HRMS and to assess the in vivo neutralizing potential of aqueous extracts from six medicinal plants (Ricinus communis, Nerium oleander, Azadirachta indica, Abrus precatorius, Cyanthillium cinereum, and Ageratum conyzoides). Methods: Venom proteins were profiled using LC-HRMS. Mice received intradermal injections of venom at 2 the minimum hemorrhagic dose (MHD; 2-8.4 µg) pre-incubated with plant extracts for 30 min at 28 °C (venom extract neutralization screen). Hemorrhagic lesion diameter was measured, and histopathological analysis of skin tissue was performed. Results: Proteomic profiling of venom from a single adult male T. insularis collected in Boyolali, Central Java identified 38 proteins across 11 major families, with SVSPs and SVMPs as the dominant toxin classes, along with PLA2s, L-amino acid oxidases (LAAOs), and snaclecs. Among the extracts tested, only A. indica bark extract significantly reduced hemorrhagic lesion diameter in a dose-dependent manner. Histology indicated reductions in inflammatory infiltrates and erythrocyte extravasation at intermediate extract doses, but higher doses were ineffective or exacerbated tissue responses. Collagen degradation was moderately alleviated but not statistically significant. Conclusion: These findings highlight the potential of A. indica bark extract as a plant-based adjunct for treating T. insularis envenomation and support further investigation into its active constituents and mechanisms. © 2026, Iranian Society of Pharmacognosy. All rights reserved.