Mechanistic Investigation of Acetylcholinesterase Inhibition by Asiatic acid: An Integrated Experimental and Computational Study

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder in which cholinergic dysfunction remains an important therapeutic target. Centella asiatica remains a major source of neuroactive phytoconstituents, but the molecular basis of its acetylcholinesterase (AChE) inhibition remains incompletely understood. In this study, a bioactivity-guided fractionation strategy was combined with computational analyses to characterize Asiatic acid (AA) from the active hydroalcoholic extract as an AChE inhibitor. AA showed the strongest hAChE inhibition (IC50 = 45.50 ± 1.31 μM) and competitive inhibition kinetics with a Ki of 8.64 μM. Molecular docking followed by 500 ns molecular dynamics simulations demonstrated stable accommodation of AA within the AChE catalytic site, with limited ligand conformational fluctuation and preservation of the binding orientation. Interaction analysis identified Glu202, Arg296, Phe295, Phe297, Tyr337, Tyr341, His447, and Leu76 as key contributors to ligand stabilization through complementary electrostatic, hydrogen-bonding, hydrophobic, and aromatic interactions. Per-residue MM/GBSA decomposition further supported the major energetic contributions of Glu202 and Arg296, while the overall binding free energy was favorable (−61.26 kcal/mol). Well-tempered metadynamics revealed a stepwise dissociation pathway involving four metastable states, indicating progressive reorganization of protein–ligand interactions during unbinding. DFT analysis further demonstrated spin-dependent frontier orbital characteristics, electronic delocalization, and distinct electrostatic potential regions that may support complementary interactions with AChE. Altogether, the integrated experimental and computational findings provide mechanistic insight into AA-mediated AChE inhibition and establish a structural and energetic framework for further investigation of AA and related pentacyclic triterpenoids as potential leads for AD drug development.