Acetylcholinesterase-inhibiting flavonoids from Eupatorium adenophorum: structure–activity relationship, binding mechanisms, and in vivo insecticidal evaluation

The increasing risks associated with pesticide toxicity, resistance, and ecological disruption underscore the urgent need for sustainable, multi-target insecticides. In this study, 18 flavonoids were isolated from Eupatorium adenophorum Spreng and evaluated for their acetylcholinesterase (AChE) inhibition and insecticidal activity. Compounds 8 , 9 , 12 , and 14 strongly inhibited AChE, with compound 12 demonstrating the highest potency (IC₅₀ = 10.13 ± 0.45 μ g/mL for Caenorhabditis elegans and 5.61 ± 0.14 μ g/mL for Myzus persicae ). Molecular docking studies confirmed the stable binding of compound 12 with AChE, whereas enzyme kinetics revealed competitive, mixed, and noncompetitive inhibition modes for compounds 1 , 9 , and 12 , respectively. Fluorescence quenching and thermodynamic analyses indicated that static, spontaneous binding was driven by hydrogen bonding and hydrophobic interactions. In vivo assays revealed LC₅₀ values of compound 12 are 134.3 μ g/mL for C. elegans and 162.9 μ g/mL for M. persicae . Structure–activity relationship analysis suggested that hydroxylation, glycosylation, and acylation patterns significantly influenced AChE inhibition. These findings provide a mechanistic basis for the development of environmentally friendly pesticides derived from invasive plants. • Eighteen flavonoids from Eupatorium adenophorum were systematically isolated and characterized. • Caffeoylated flavonol glycosides showed strong AChE inhibition and in vivo insecticidal activity. • Kinetic, thermodynamic, and docking analyses revealed distinct mechanisms of AChE inhibition. • The 6″- O -caffeoylation of the A-ring was identified as a key structural determinant of potency. • This study provides a molecular-to-organism basis for developing insecticidal leads from invasive plants.