Gastrodia elata, a traditional Chinese medicinal herb, is known for its glycoside-rich extracts with anti-inflammatory and anti-Alzheimer's disease (AD) effects. Acetylcholinesterase (AChE), a key enzyme involved in AD, is a major therapeutic target, with AChE inhibitors playing a significant role in managing neurodegenerative diseases. However, the specific active components responsible for the AChE inhibitory effects in G. elata remain unclear, hindering further research. This study aimed to identify and optimize the active compounds in G. elata for their potential as AChE inhibitors. Seven potential AChE inhibitors were rapidly screened from the G. elata extract using affinity ultrafiltration liquid chromatography-mass spectrometry (AUF-LC-MS). To optimize the extraction process, the response surface method combined with Euclidean distance and NSGA-III was applied, and then, gastrodin, parishins A, B, C, D, G, and K, with purities of 98.82%, 98.23%, 98.16%, 98.78%, 98.25%, 99.11%, and 98.84%, respectively, using the ternary counter current chromatography via the solvent systems of n-butanol-ethyl acetate-methanol-water at volume ratio of 3.0:4.5:2.0:5.0, n-butanol-ethyl acetate-methanol-water at volume ratio of 3.0:5.0:2.5:4.5, and n-butanol-ethyl acetate-methanol-water at volume ratio of 2.5:4.5:2.5:4.5. Molecular docking and dynamics simulations indicated that these components could bind tightly to AChE, forming stable complexes. Enzyme kinetics assays showed that all seven compounds exhibited reversible inhibition of AChE. Specifically, gastrodin and parishins B, D, G, and K were identified as mixed-competitive inhibitors, parishin A as an uncompetitive inhibitor, and parishin C as a non-competitive inhibitor. The inhibition rates followed the order: parishin G > gastrodin > parishin B > parishin K > parishin D. These findings suggest that the active components of Gastrodia elata have significant potential as AChE inhibitors, providing a basis for the development of natural AD therapies.

Gastrodia elata, Acetylcholinesterase, Multi-Objective Extraction Optimization, Quaternity Method Screening, High-Speed Countercurrent Chromatography