Establishments Of Two Platforms For ?-glucosidase Inhibitors Screening And Their Application In Natural Products

For drug discovery,natural products be considered as main source of new lead compounds.Although modern analytical techniques which are including high performance liquid phase chromatography(HPLC),liquid chromatography–high resolution mass spectrum(LC-HRMS),can achieve rapid identification and acquisition of structure information of constituents in natural products,simultaneous accessing to the bioactivity characterization of the ingredients remains a challenge.It is necessary to develop some fast and effective methods to screen bioactivity compounds from natural products.Conventional screening method requires multi-step isolation and purification,fractions collection and bioactivity assessments,which is time consuming and laborious,and some active ingredients may easily be ignored and lost.To overcome the shortcomings presented in traditional screening methods,some new screening methods appeared,mainly divided into two categories: based on bioactivity and based on affinity between protein and ligand.In this article,two screening platforms representing different screening strategies were set up,one is at-line post-column bioassay another is immobilized enzyme for ligand fishing.Both screening platforms were used to screening ?-glucosidase inhibitors from natural products.The first chapter introduces the importance of natural products in the field of drug research and the relevant screening strategies of active ingredients from natural products were reviewed.In addition,related research on diabetes and ?-glucosidase were also introduced.At the same time,the research ideas and innovations of this thesis were presented.In the second chapter,the HPLC circuit was retrofitted,and the post-column pipeline was connected to an automatic collection device to develop an at-line nanofractionation thus allowing post-column bioassay for ?-glucosidase inhibitors screening.Optimizing the concentration of the enzymatic reaction to establish a miniaturized ?-glucosidase bioassay in 384-well format;selecting appropriate inhibitors to determine the limit of detection of the screening platform;and the feasibility of the screening platform was verified by a mixed model including positive and negative drugs,and the tested results indicated that the screening platform has been successfully established and can be used for screening ?-glucosidase inhibitors from complex samples.In the third chapter,?-glucosidase was immobilized on the surface of carboxy magnetic beads to prepared ?-glucosidase reactors.The amount of magnetic beads for immobilization was optimized,the amount of magnetic beads prepared the best active enzyme reactors was selected as the optimal dosage.At the same time,the fluorescent characterization of the prepared ?-glucosidase reactors indicated that the ?-glucosidase was successfully immobilized on the surface of magnetic beads.Based on the protein-ligand affinity,the prepared ?-glucosidase reactors were used for ligand fishing.The incubation time and elution conditions during the ligand fishing process were optimized.Finally,a mixed model consisting of positive and negative drugs is used to verify the feasibility of ?-glucosidase reactors for ligand fishing,and the results indicated that the ?-glucosidase reactors has the potential for fishing out active ingredients from natural products.In the fourth chapter,ten ?-glucosidase inhibitors were screened out from Green Tea and Ginkgo Biloba L.by using at-line post-column bioassay,while five ?-glucosidase inhibitors were screened out from Green Tea by using the ?-glucosidase reactors.By comparing the screening results of Green Tea obtained respectively from at-line post-column bioassay and ligand fishing to further verifying the reliability and accuracy of these two screening platforms for bioactives screening.In the fifth chapter,all the research contents were summarized,the future research works were prospected.