Bioactivity-Guided Isolation and Molecular Docking of Potential Anti Naja nigricollis Venom Agent from Sclerocarya birrea Root-Bark Extracts

Background and Objective: Snakebite envenomation remains a major public health challenge in Sub-Saharan Africa, where limited access to effective antivenom persists. This study aimed to isolate, characterize, and evaluate bioactive antivenom constituents from Sclerocarya birrea root-bark against Naja nigricollis venom. Materials and Methods: Methanol extract of Sclerocarya birrea root-bark was sequentially fractionated using solvents of increasing polarity. Lyophilized Naja nigricollis venom obtained from Ahmadu Bello University, Zaria, Nigeria, was used for biological assays. Antivenom activity of the crude extract and fractions was evaluated in vivo against venom-induced lethality in albino rats, while in vitro phospholipase A2 (PLA2) inhibitory activity was assessed. Bioactivity-guided chromatography of the most active fraction was performed, followed by chemical characterization using GC-MS, FTIR, and UV-Vis spectroscopy. Molecular docking of the identified compound with venom PLA₂ was conducted using AutoDock 4.0. Data were expressed as Mean±SEM and analyzed using one-way ANOVA, with Duncan’s Multiple Range Test at p<0.05. Results: The crude methanolic extract significantly (p<0.05) prolonged survival time in Naja nigricollis-envenomed rats in a dose-dependent manner. Among the solvent fractions, the ethyl acetate fraction (EAF) exhibited the most significant (p<0.05) in vivo and in vitro antivenom activities. Bioassay-guided chromatographic separation of EAF yielded 30 pooled chromatographic fractions (CPFs), of which CPF-25 showed the highest antivenom potency, achieving 92.1% phospholipase A2 inhibition. GC-MS analysis of CPF-25 identified nine major constituents, predominantly hexadecanoic acid (57.83%), erucic acid (12.46%), di-n-octyl phthalate (7.85%), 13-octadecenal (4.09%), and 9,12-octadecadienoyl chloride (6.18%). FTIR spectra revealed peaks at 3381 cm^-1 (O-H), 1780 cm¹ (C = O) and 1641 cm^-1 (C = C), while UV-Visible spectroscopy exhibited absorption bands between 242.5-310.5 nm consistent with conjugated unsaturated systems. Molecular docking demonstrated strong binding affinity of 9,12-octadecadienoyl chloride to venom phospholipase A₂, with a binding energy of -6.29 kj/mol. Conclusion: Sclerocarya birrea root-bark contains bioactive compounds with antivenom potential, supporting its ethnomedicinal relevance and possible application as a plant-based antivenom adjunct.