Experimental and computational evaluation of bioactive secondary metabolites from Trewia nudiflora stem bark.

Trewia nudiflora L. (Euphorbiaceae) stem bark is traditionally used in Ayurvedic medicine for treating pain and gastrointestinal disorders, yet its bioactive constituents and underlying mechanisms remain unvalidated. This study aimed to isolate bioactive secondary metabolites from the methanol stem bark extract (MeOH-extract) of T. nudiflora and systematically evaluate its antidiarrheal, analgesic, thrombolytic, and membrane-stabilizing activities. Secondary metabolites were isolated via column chromatography and structurally characterized using 1H and 13C NMR. In vivo antidiarrheal (castor oil-induced), central (tail-immersion), and peripheral (acetic acid writhing) analgesic profiles were evaluated in Swiss-albino mice. In vitro thrombolytic and membrane-stabilizing assays were complemented by in silico molecular docking and ADMET profiling against five validated protein targets. Stigmasterol and (5α)-gitoxigenin 3-(α-L-rhamnopyranoside) were isolated as known constituents, providing chemotaxonomic confirmation, while the novelty lies in their integrated pharmacological and in silico characterization. The MeOH extract significantly reduced diarrheal feces by up to 60.46% (400 mg/kg), prolonged tail-immersion latency by 217.65%, and inhibited abdominal writhing by up to 65.39%. The extract showed 23.10% clot lysis and 79.14% hemolysis inhibition, while stigmasterol and gitoxigenin preferentially targeted μ-opioid (-5.856 kcal/mol) and EP2 receptors (-5.649 kcal/mol), respectively. ADMET profiling confirmed favorable oral absorption for stigmasterol (HIA = 96.4%, log BB = 0.80, LD₅₀ = 890 mg/kg) and flagged gitoxigenin's narrow cardenolide safety margin (Toxicity Class II; LD₅₀ = 41 mg/kg). These findings provide phytochemical and mechanistic support for the ethnomedicinal claims of T. nudiflora and identify both compounds as pharmacological leads warranting further investigation.