Screening acetylcholinesterase inhibitors from traditional Chinese medicines by paper-immobilized enzyme combined with capillary electrophoresis analysis

Abstract

Discovering acetylcholinesterase (AChE) inhibitors is one of the important ways to develop new drugs for the treatment of Alzheimer's disease. In this work, a simple strategy was developed for screening AChE inhibitors from traditional Chinese medicines (TCMs) by capillary electrophoresis (CE) analysis combined with enzymatic assay, in which immobilized AChE was employed. For AChE immobilization, cellulose filter paper (CFP) was used as the carrier material and physically coated with chitosan owing to moderate viscosity of chitosan to be introduced into amino groups, and then AChE was covalently bonded to the amino-functionalized CFP through a Schiff base reaction using glutaraldehyde (GA) as a cross-linking agent. The CFP-immobilized AChE exhibited enhanced endurance to pH and temperature, improved storage stability, excellent repeatability and reusability. More remarkably, CFP-immobilized AChE can be instantly separated from enzyme reaction mixture thus greatly simplifying the operational process. For immobilized AChE, the Michaelis-Menten constant, inhibition constant and IC₅₀ were determined using huperzine A as a model inhibitor. Finally, CFP-immobilized AChE was applied to inhibitor screening from 17 TCMs, among which Chebulae Fructus (ripe fruits of Terminalia chebula) exhibited the strongest inhibitory effect on AChE. The positive results indicated that such a screening strategy may open up a new avenue to discover active components from TCMs.

Keywords: Acetylcholinesterase inhibitor; Capillary electrophoresis; Inhibitor screening; Paper-Immobilized enzyme; Traditional Chinese medicine.