Studies On Chemometries And Immobilized Enzyme Based Approaches For Rapid Discovery Of Hioactive Compounds From Traditional Chinese Medicine

Screening bioactive compounds from traditional Chinese medicine (TCM) is a crucial part in elucidating the chemical composition of TCM. It not only has great significance in the modernization of TCM but also is important for discovering new drugs. In this thesis, we developed two novel approaches for rapid discovery of bioactive compounds from TCM based on traditional bio-guided assay strategy. An approach based on partial least squares (PLS) modeling and sorting was proposed. Through integrated use of liquid chromatography-mass spectrometry and chemometric analysis, cardioprotective and anti-inflammatory constituents from a clinical used Chinese medicine Tongmai Yangxin pills (TMYXP) were identified. Moreover, lipase-adsorbed halloysite nanotubes were fabricated and were successfully applied in screening potential lipase inhibitors from the extract of Magnoliae Cortex. The results provided new approachs for rapid screening of bioactive compounds from TCM.The main contents of the thesis included:1. H2O2 oxidative damage H9c2 rat cardiac myocytes was used to screen cardioprotective constituents from TMYXP. We found five components with moderate cardioprotective effects among 18 components of TMYXP. Their EC50 ranged from 7.332 to 17.44μg/mL, with good dose-effect relationships. By use of LC-MS technology 11 probable cardioprotective compounds were putatively identified from the five components.2. An approach for rapid discovery of bioactive compounds from TCM based on PLS modeling and sorting was proposed, which could analyze relevant LC-MS and activity evalution data, to identify rapidly potential active compounds. Based on the approach, LPS-stimulated RAW264.7 cells were used to screen anti-inflammatory constituents from TMYXP. Five components were discovered with strong anti-inflammatory effects. Their IC50 ranged from 6.702 to 32.31μg/mL, with good dose-dependent manners. PLS regression modeling was established to analyze the correlation between compounds and bioactivities, and the 82 compounds were ranked according to their correlation coefficients. The validation experiments were conducted to verify the anti-inflammatory activities of top ranking compounds. The results indicated that six compounds among seven top-rank compounds were found to exert dose-dependent anti-inflammatory activities. Our findings suggested the proposed method could be used to rapidly figure out active compounds from mixture.3. A rapid and easy approach was proposed to fabricate lipase-adsorbed nanotube reactor (LANR) through electrostatic interaction. HNTs immobilized enzyme could be adopted to screen lipase inhibitors with orlistat as experimental group and schisandrin as contrast, and successfully applied in Magnoliae Cortex. Four neolignans compounds from the extract of Magnoliae Cortex were found to be potent ligands of lipase. We also isolated two of them and confirmed their structures by NMR. Their lipase inhibitory activities were verified; with IC50 were 213.03μM and 96.96μM respectively. This new type of lipase inhibitors from Magnoliae cortex was reported for the first time. The structure-activity relationship of those compounds was further investigated by molecule docking. The results indicated the type of compounds could combine with key amino acids of lipase active domain; occupying enzyme catalytic sites could be their action mechanism of inhibiting lipase activity.