Ding_2025_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1261_124657

Acetylcholinesterase (AChE) is widely concerned as a hotspot target for the treatment of Alzheimer's disease (AD). This study aimed to screen AChE inhibitors from walnut kernel (WK) through the techniques combined of bio-affinity ultrafiltration, AChE-functionalized magnetic nanoparticles and UPLC-Q-Exactive Orbitrap MS. The results showed that WK extract improved acetylcholine level and reduced AChE level in the hippocampus and cortex of AD mice with kidney yang deficiency. AChE inhibition experiment showed WK extract has the best activity with IC(50) values at 1.310 mg/mL. Bio-affinity ultrafiltration combined with UPLC-Q-Exactive Orbitrap MS screened out 3 active compounds, including ellagic acid 4-O-xyloside, ellagic acid (EA) and glansreginin A. In order to avoid false positive results, AChE-functionalized magnetic nanoparticles combined with HPLC was used to ligand fish again, and EA was fished. And the IC(50) value of EA on AChE was 1.486 mg/mL. Molecular docking showed EA had the calculated binding energies at -8.468 kJ/moL with AChE, and could interact with the amino acid residues of ASP 74, TYR 133 and GLU 202 via hydrogen bonds and had the pi-pi bonds with TRP 86. The developed ligand fishing method is simple and efficient for screening potential AChE inhibitors from medicinal plant.