Rapid Screening Of Bioactive Compounds From Traditional Chinese Medicines By Chemical Biology Approaches Combined With High Resolution Mass Spectrometry

Elucidating the bioactive constituents in traditional Chinese medicine(TCM)plays a key role in the modernization of TCM,which could help to interpret the mechanism of action,discover new drugs,and develop standards for quality control.Therefore,rapid screening of bioactive compounds from TCM is very important.The focus of this dissertation is the development of new chemical biology approaches for rapid discovering bioactive compounds from TCM.The main works and academic contributions of this dissertation are included as follow:1.HPLC-ESI-Q-TOF-MS/MS method was applied for comprehensive analysis of the chemical constituents in Yangxinshi Tablet(YXST).A total of 127 compounds were identified or tentatively deduced by comparing their retention times and MS spectra with those of authentic standards or literature data.To further prove the antioxidant activity of YXST,its free radical scavenging capacity was assessed by 1,1-diphenyl-2-picrylhydrazyl(DPPH)spectrophotometric assay and the antioxidants in YXST were rapidly screened by DPPH-HPLC experiment.Especially,salvianolic acid A and salvianolic acid B showed excellent DPPH scavenging activities with the EC50 of 151.9 and 275.6 ?g/mL,respectively,which were stronger than that of L-ascorbic acid(positive control)with the EC50 of 297.1 ?g/mL.Additionally,these two most potent antioxidants were detectable in rat plasma after oral administration,indicating salvianolic acid A and salvianolic acid B may be the bioactive compounds for antioxidation in vivo.2.A novel strategy for screening brain-penetrable antioxidants from natural products was developed by blood-brain barrier specific permeability assay and subsequent recognition with DPPH probe.2,6,4'-Trihydroxy-4-methoxybenzophenone(ZM)in ethyl acetate extract of Rhizoma Anemarrhenae was found to be an active compound.ZM showed moderate DPPH radical scavenging activity,which was lower than VC.However,the blood-brain barrier permeability potential of ZM was very well,which made it a good candidate for cerebral protection study.3.A simple and efficient in vitro method based on ultrafiltration LC-MS and molecular modeling has been developed to study the human serum albumin(HSA)binding of the compounds in DHIL Seven major components including protocatechuic aldehyde,p-coumaric acid,salvianolic acid D,rosmarinic acid,salvianolic acid E,lithospermic acid and salvianolic acid B were identified as HSA ligands.Considering the drug-HSA binding property of the compounds in DHI may change during drug combination therapy,competitive binding assay was carried out to evaluate the influence of aspirin on the DHI-HSA binding.Experimental results revealed that the salvianolic acids in DHI had stronger binding ability to HSA than sodium salicylate.To further verify the results above,molecular modeling and probe displacement assay were conducted to investigate the optimum binding site and binding affinity of the ligands on HSA.Our findings suggested that the established method could be a powerful tool to study the drug-HSA binding property of Chinese medicine injections.4.A novel immobilized fusion protein affinity chromatography(IFPAC)was developed to achieve rapid screening of PPARy ligands from TCM.First,functional human PPAR7 ligand binding domain(hPPAR,yLBD)was bacterially produced by fusion to glutathione S-transferase(GST).The unpurified GST-hPPARyLBD was directly applied to a 96-well filter plate prepacked with glutathione sepharose.Due to the strong affinity between GST and glutathione,the fusion protein could selectively attach to the glutathione matrix with an oriented immobilization,which was rapidly purified under non-denaturing conditions.Experimental results indicated that the prepared 96-affinity column array exhibited excellent selectivity and sensitivity for fishing novel interacting compounds.The proposed approach was applied in the high-throughput screening of PPARy ligands from natural products,followed by rapid characterization of active compounds using HPLC-ESI-Q-TOF-MS/MS.Isochlorogenic acid A in Dendranthema indicum flowers was found to be a PPARy ligand.Our findings suggested the IFPAC could be a powerful tool for drug discovery from natural products.