Integrated affinity ultrafiltration-based multidimensional screening of acetylcholinesterase inhibitors from industrially cultivated Gastrodia elata

Efficient discovery of bioactive compounds from medicinal industrial crops requires robust and objective screening strategies capable of integrating biochemical activity with preliminary biological validation. Gastrodia elata is an important medicinal crop widely used as an industrial raw material for natural product extraction, yet systematic identification of its acetylcholinesterase (AChE)-active constituents remains limited. In this study, an integrated multidimensional screening platform was established to identify and prioritize AChE inhibitors from unprocessed industrially cultivated G. elata . The workflow combined affinity ultrafiltration–LC screening, molecular docking, molecular dynamics simulations, enzyme inhibition kinetics, preparative HSCCC separation, preliminary cellular validation, and network pharmacology analysis. Affinity ultrafiltration coupled with LC analysis identified parishin A and parishin C as the prioritized AChE-binding constituents, exhibiting high binding degree and recovery values. Molecular docking and molecular dynamics simulations further demonstrated stable interactions. Enzyme kinetic analysis confirmed that parishins A and C acted as mixed-type AChE inhibitors, with IC50 values of 0.0145 and 0.0248 mM, respectively. HSCCC separation enabled efficient isolation of the six active constituents with purities above 98%. To provide preliminary biological validation beyond enzyme-level activity, the potential protective effects of parishins A and C were evaluated in injured PC12 cells. Both compounds significantly improved cell viability in a dose-dependent manner, reduced apoptotic nuclear morphology, and were associated with alterations in apoptosis - and oxidative stress-related proteins, including Bax/Bcl-2, cleaved caspase-3, p-AKT/AKT, Nrf2, and HO-1. These findings suggest that the prioritized compounds possess preliminary neuroprotective relevance in addition to AChE inhibitory activity. Network pharmacology analysis further indicated potential associations with Alzheimer’s disease–related signaling pathways. This study establishes an integrated and industrially applicable workflow for the discovery and prioritization of natural AChE inhibitors from medicinal crops and identifies parishins A and C as promising bioactive constituents of G. elata for further neuroprotective investigation.