Study On Screening Method For Trypsin Inhibitors From Natural Products Based On Monolithic Affinity Chromatography

Natural products with clear clinical efficacy and abundant resources,are an important source for the development of lead compounds and active scaffolds.However,due to the complex composition and diverse structure of natural products and low content of active ingredients,screening bioactive compounds from natural product still faces enormous challenges.Affinity chromatography screening method,based on the specific affinity between the biological target and its ligands,can directly separate the active ingredient from the complex system,and then combine with modern chromatographic techniques for the rapid analysis and identification of active compounds.Organic polymer monolith is widely used in affinity chromatography because of its simple preparation,easy surface modification,good biocompatibility and easy miniaturization.In this work,organic polymer monolith was used as support material for the preparation of affinity chromatography screening model.In order to build a reasonable and effective affinity screening model,two different biological target affinity columns were prepared and used as affinity extraction columns for the screening of active ingredients in natural products by combining with HPLC-MS.In the first chapter,the recent development of bio-affinity chromatography in natural products screening was summarized,with a particular emphasis on the reported bio-affinity stationary phases and chromatographic supports as well as the applications of bio-affinity chromatography coupled with GC-MS or LC-MS.At the same time,the research ideas and innovations of this research were put forward.In the second chapter,a polymerization mixture composed of the functional monomers(GMA),crosslinker(PEGDA),porogens(1-propanol and lauryl alcohol)and initiator(azobisisobutyronitrile)was used to prepare poly(GMA-co-PEGDA)monolith,and then,trypsin were covalently bonded on to the surface of poly(GMA-coPEGDA)monolith to prepare immobilized trypsin reactor.The compositions of the polymerization mixture and immobilization conditions were systematically optimized.Subsequent characterization and evaluation of the optimized immobilized trypsin reactor were performed.The results show that the immobilized trypsin reactor has good physical and chemical properties and enzymatic activity.In the third chapter,based on the affinity chromatography theory,the immobilized trypsin reactor was applied to screen trypsin inhibitors from natural products.Firstly,the kinetic parameters of immobilized trypsin were investigated and the inhibitory activities of 15 compounds were determined to assess its ligand affinity.Twelve compounds including fisetin,catechin and myricetin showed inhibitory activities.Subsequently,the immobilized trypsin reactor was used as solid phase extraction column and combined with HPLC-MS for screening trypsin inhibitors from Scutellaria baicalensis Georgi extract.Inhibitory activities of the binders scutellarin,baicalin and wogonoside were further confirmed by using a standard plate reader based enzymatic assay.In the fourth chapter,the poly(GMA-co-PEGDA)monolith was used as the carrier for LasR protein immobilization.LasR protein was immobilized onto the surface of poly(GMA-co-PEGDA)monolith via amino and carboxyl groups of protein,respectively.Subsequent characterization and evaluation of the immobilized LasR protein affinity columns were performed.The effect of immobilization methods on LasR activity was investigated by determining the retention of positive drug on two LasR protein affinity columns.The results showed that immobilization via carboxyl group is an effective method for preparing LasR protein affinity column.In the fifth chapter,all the research contents were summarized,and the future research work was prospected.