Acetylcholinesterase Inhibitor Screening Based On Paper-immobilized Enzyme And Spectrum-effect Relationship Study On Terminalia Chebula Fruits

Natural products and traditional Chinese medicines（TCMs）have always been the source of new drug discovery,and enzymes are important targets for drug screening.And lots of enzyme inhibitor drugs have been available for the treatment of related diseases such as Alzheimer’s disease,cancer,high blood pressure.Therefore,the establishment of a rapid and simple method to screen enzyme inhibitors from TCMs has attracted extensive attention.In this dissertation,acetylcholinesterase,a therapeutic target for Alzheimer’s disease,was used as the research object.A simple strategy was developed for screening ACh E inhibitors from TCMs by capillary electrophoresis（CE）analysis combined with enzymatic assay,in which paper-immobilized ACh E was employed.And spectrum-effect relationship study of inhibition on ACh E,antioxidant compounds of Terminalia chebula fruits was also carried out.This dissertation consists of the following parts:（1）Paper-immobilized enzyme was prepared and applied to screening acetylcholinesterase inhibitors from traditional Chinese medicines combined with capillary electrophoresis analysis.Cellulose filter paper（CFP）was used as the carrier material.Chitosan,as an amino-functionalization agent,has a certain viscosity and thus could be physically coated on the surface of CFP to introduce amino groups.And then ACh E was covalently bonded to the amino-functionalized CFP through a Schiff base reaction using glutaraldehyde（GA）as a cross-linking agent.Combined with capillary electrophoresis（CE）,the enzymatic kinetics and inhibition kinetics and performance of immobilized ACh E were studied,and further immobilized ACh E was applied to enzyme inhibitor screening from 17 TCMs.The CFP immobilized ACh E exhibited endurance to p H and temperature,improved storage stability,excellent repeatability and reusability.Among 17 kinds of water extracts of TCMs,Chebulae Fructus（ripe fruits of Terminalia chebula）exhibited the strongest inhibitory effect on ACh E.In this work,a simple strategy was developed for screening ACh E inhibitors from TCMs by CE analysis combined with enzymatic assay,in which paper-immobilized ACh E was employed,which may open up a new avenue to discover active components from TCMs.（2）Spectrum-effect relationship study was performed for screening ACh E inhibitory and antioxidant components from Terminalia chebula fruits.Eighteen samples were used as the research object.The water extract of Terminalia chebula fruits was separated by macroporous resin to obtain six eluate fractions,and then used for the evaluation of ACh E inhibition and antioxidant activities.The results showed that 30%ethanol eluate fraction exhibited the strongest inhibitory effect on ACh E and DPPH free radical scavenging activity.Further,IC₅₀（the concentration needed for50%ACh E inhibition or 50%DPPH scavenging percentage）were determined for30%ethanol eluate fraction of eighteen batches Terminalia chebula fruits.And their chromatographic fingerprints were established by UPLC.Fifteen common peaks were selected by similarity analysis,from which twelve common peaks were further screened out by principal component analysis.The peak areas of the twelve common peaks were taken as independent variables,and the IC₅₀values of eighteen batches of samples were used as dependent variables to established spectrum-effect relationship model by chemometrics methods,including grey relational analysis,partial least square and multiple linear regression.Three UPLC peaks with strongest inhibitory effect on ACh E（Peak 3,2 and 15）and five UPLC peaks with strongest antioxidant activity（Peak 1,2,5,12 and 14）were screened out.Peak 3,14 and 15 were preliminarily identified as gallic acid,chebulagic acid,and corilagin,respectively,by comparing the retention time and ultraviolet spectrum of peaks with those of standard compounds.Using UPLC-MS/MS technology,16 compounds were identified from30%ethanol eluate fraction of Terminalia chebula fruits through mass spectrometry fragment ion,fragmentation pattern and reference data.In this study,the chromatographic fingerprint analysis and activity evaluation of Terminalia chebula fruits were carried out and the correlation between chemical composition and pharmacodynamics was explored.The obtained result provided a reference for clarifying the pharmacodynamic substance basis of Terminalia chebula fruits.