Establishment Of Immobilized Capillary AChE Reactor-based Online Ligand Fishing Platform And Its Application In Natural Products Screening

Natural products are attracting increasing attention all over the world,due to their long historical clinical practice and appealing therapeutic efficacy.Moreover,natural products possess high chemical scaffold diversity and can be considered as a huge and invaluable source of bioactive compounds for discovering promising new drugs.However,because of the chemical complexity of TCMs,it is neither easy to identify bioactive constituents nor to elucidate their pharmacological mechanism.The conventional bioassay-guided fractionation approach has been a mainstream method for discovering bioactive compounds from natural products.Unfortunately,the isolation procedures central to this approach are usually labor-intensive,time-consuming and costly.Therefore,in order to develop a direct and reliable method for discovering lead compounds from natural products,a comprehensive two-dimensional online ligand fishing platform was developed using immobilized capillary enzyme reactors in combination with liquid chromatography-mass spectrometry.In the first chapter,the importance of natural products in the field of drug discovery and the screening strategies of active compounds from natural products were reviewed.In addition,the related researches on immobilized enzyme technology and acetylcholinesterase were also introduced.On the basis of the sumarization,the research ideas and novelties of this research were put forward.In the second chapter,a polymerization mixture composed of the functional monomers?GMA and EDMA?,ternary porogens?1,4-butanediol,1-propanol and H₂O?and initiator AIBN was employed to prepare poly?GMA-co-EDMA?monolith,and further the poly?GMA-co-EDMA?monolith was used as support for immobilizing acetylcholinesterase to prepare enzyme reactor.During this process,the compositions of the polymerization mixture and enzyme concentration as well as immobilization time were systematically optimized.Subsequent characterization and evaluation of the optimized immobilized acetylcholinesterase reactor were performed.In the third chapter,based on the affinity chromatography theory,the immobilized acetylcholinesterase reactor was used as an affinity solid-phase extraction column to reintegrate with commercial instrument components to build an online ligand fishing platform.The kinetic parameters of the immobilized acetylcholinesterase reactor and the important debugging parameters of the online ligand fishing platform were investigated.In the fourth chapter,the immobilized acetylcholinesterase reactor was used to evaluate the overall enzyme inhibitory activity of five plant extracts,and the extract of Corydalis yanhusuo exhibits the highest enzyme inhibitory activity.Then the constructed online ligand fishing platform was applied to fish bioactive compounds from Corydalis yanhusuo extract.Inhibitory activities of the online found compounds were further confirmed by using a standard plate reader based enzymatic assay.In the fifth chapter,all the research contents were summarized,and on this basis,the future research work was prospected.