Phytotoxic potential of Ranunculus testiculatus aqueous extract: physiological responses and molecular docking/putative ligand interactions with barley α-amylase

Exploiting allelopathic plants and elucidating their mechanisms of action are essential for sustainable weed management and the discovery of natural bioherbicides. This study investigated the allelopathic potency of Ranunculus testiculatus aqueous extract on seed germination, seedling biochemistry, and α-amylase activity, supported by hypothesis-generating molecular docking. Dose–response bioassays were conducted on Hordeum vulgare var. Sardari to determine the concentration causing 50% inhibition of germination (IC50 = 236 mg L⁻¹), which was subsequently applied to a panel of 38 crop and weed species. Germination was suppressed in all tested species, with weeds being markedly more susceptible (91.5% mean inhibition) than crops (64.8%). Tetrazolium staining revealed a progressive loss of metabolic activity from the aleurone–endosperm interface toward the embryo axis. Antioxidant enzyme activities increased at the lowest concentration and declined at higher concentrations, declining in parallel with the membrane stability index. α-Amylase activity declined dose-dependently, reaching a 72% reduction relative to the control. GC-MS profiling identified putative secondary metabolites. After rigorous exclusion of analytical artifacts, compounds such as 2,3-dihydrobenzofuran (ΔG = − 6.5 kcal mol⁻¹) and 2,4-di-tert-butylphenol (ΔG = − 6.1 kcal mol⁻¹) were predicted to dock within the substrate-binding cleft, engaging the catalytic nucleophile Asp179 and neighboring residues His288 and Arg177 through hydrogen-bond and hydrophobic contacts. Benchmarking against the standard inhibitor acarbose (ΔG = − 7.2 kcal mol⁻¹) provided a reference for comparing their predicted docking scores. Ranunculus testiculatus aqueous extract exhibited phytotoxic activity consistent with a dual-mode action: oxidative stress responses associated with membrane damage, and reduced α-amylase activity potentially associated with specific phenolic and heterocyclic constituents. The apparent differential sensitivity toward weeds, and the identification of candidate α-amylase ligands through in silico prediction, highlight this species as a promising natural template for developing bioherbicides and candidate carbohydrate-hydrolyzing enzyme inhibitors. However, experimental validation using purified enzymes and authentic compounds is required before any agricultural application can be considered.