At-line Nanofractionation Screening Platform With Sample Simplification And Enrichment Strategy Based On Magnetic Beads Ligand Fishing

Natural products are major source of leading compounds in drug discovery.To effectively screen active compounds from complex matrix remains an interesting topic.However,traditional screening methods suffer from high consumption,long development cycles and difficult operations.As the evolutions of basic analytical instruments such as High performance liquid chromatography(HPLC),high resolution mass spectrometry(HRMS)and Nuclear magnetic resonance(NMR),many new active screening methods have been developed and applied.In this study,in order to overcome disadvantages of traditional screening methods,one of those new methods,at-line nanofractionation screening platform,was established for pancreatic lipase inhibitors screening.Moreover,to overcome the disadvantages of at-line nanofractionation screening platform,a tool for sample simplification and enrichment using magnetic beads based ligand fishing was established.The whole system was finally applied to screen pancreatic lipase inhibitors from green tea extracts.In the first chapter,new advanced HPLC based approaches in natural products screening,including pre-column,on-column and post-column screening methods were summarized.Their advantages,disadvantages and prospect were also discussed.What’s more,information about pancreatic lipase such as pancreatic lipase and obesity,pancreatic lipase inhibitors screening from natural products,bioassays for pancreatic lipase was briefly introduced.In the second chapter,at-line nanofractionation screening platform was established through instrumentations based on HPLC and automatic sampler system.Bioassay for pancreatic lipase and optimizations of screening platform were set up.A mixture model of pancreatic lipase inhibitors and negative control was made up to demonstrate feasibility of at-line nanofractionation screening platform.Green tea extracts was chosen to demonstrate the screening ability of at-line nanofractionation screening platform in complex matrix from natural products.In the third chapter,a tool for sample simplification and enrichment was set up using commercial carboxyl magnetic beads based ligand fishing.A preparation method of pancreatic lipase magnetic beads was built up.Conditions of preparation of pancreatic lipase and elution were optimized.The feasibility of pancreatic lipase magnetic beads as a sample simplification and enrichment tool was demonstrated by mixture models of pancreatic lipase inhibitors and negative control and natural products of green tea extracts.In the fourth chapter,pancreatic lipase magnetic beads and at-line nanofractionation screening platform were combined to implement active compounds screening from mixture models and green tea extracts after sample simplification and enrichment.Finally,in the fifth chapter,study was systematically summarized,and further works was prospected.