Study on Cholinesterase Inhibitory Activities and AChE Inhibition Mechanism of Gastrodin Compounds Based on Molecular Docking and Kinetic Simulation.

Alzheimer's disease (AD) is characterized by cholinergic neurotransmission dysfunction, and inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) represents a key therapeutic strategy. Gastrodin exhibits broad biological activity. In vitro enzyme inhibition assays, enzyme kinetic analysis, fluorescence spectroscopy, and molecular docking simulations validated the inhibitory effects of gastrodin and its analogues on acetylcholinesterase. All compounds exhibited concentration-dependent inhibition curve inhibition against AChE, with compound 3 demonstrating significant inhibitory activity, yielding an AChE IC50 value of 25.64 ± 4.59 µM. The inhibitory activity against AChE was ranked as follows: 2-hydroxymethylphenyl-β-D-glucopyranoside (compound 3) > 4-hydroxymethylphenyl-β-D-glucopyranoside (compound 2) > 4-methoxyphenyl-β-D-glucopyranoside (compound 4) > phenyl-β-D-glucopyranoside (compound 1). Enzyme kinetics revealed that compounds 1-4 exhibit reversible mixed-site inhibition mechanisms, simultaneously binding to both the catalytic active site (CAS) and peripheral anion site (PAS) of acetylcholinesterase. Fluorescence spectroscopy indicated that compounds 1-3, except compound 4, form stable complexes with acetylcholinesterase via static quenching. Molecular docking studies revealed multiple bond interactions between the compounds and AChE, with compound 3 exhibiting the lowest binding free energy. Subsequent investigation of the compounds' inhibitory activity against BChE demonstrated that they also possess significant inhibitory effects on BChE. This study elucidates the inhibitory mechanism of gastrodin-derived compounds against AChE and their structure-activity relationship, providing a theoretical basis for developing anti-Alzheimer's disease drug candidates based on these compounds.