| Currently, the severest challenges for the modernization of Traditional Chinese Medicine (TCM) are the unclear of pharmacodynamic materials and action mechanism, and the unreliable quality control methods. For this reason, taking Lindera aggregata (Wu-yao) and Phyhalis alkekengi (Chinese lantern) as the research carriers, this dissertation accordingly focused on the investigation of pharmacodynamic materials, quality control methods and related pharmacokinetics of the two TCMs, by combination of multiple research modes and modern instrument analytical techniques. Ultimately, the pharmacodynamic materials of Lindera aggregata and Phyhalis alkekengi for hepatoprotective and anti-inflammatory activities, respectively, were clarified; the reliable quality control methods of the two TCM were established, and the main pharmacokinetics features of Phyhalis alkekengi were revealed. The main innovation and academic contributions of this dissertation are presented as follows:1.13compounds were isolated from95%ethanol extract of Lindera aggregata that collected from Taizhou, Zhejiang province, by using modern chromatographic techniques. Structures of the13compounds were identified by physico-chemical properties and modern spectral techniques (including UV, IR,1H NMR,13C NMR, HMQC, HMBC, NOESY, COSY and HRMS). They were Linderagalactone B (1), Linderagalactone D (2), Linderagalactone E (3),3-eudesmene-1β,11-diol (4), Linderagalactone A (5), Linderagalactone C (6), Hydroxylindestenolide (7), Strychnistenolide (8),8-hydroxyisogermafurenolide (9), Atractylenolide Ⅲ (10), Linderalactone (11), Linderane (12) and Neolinderalactone (13). They were all sesquiterpene lactones except for4, and1,2,3,5and6were new compounds. Among them,3,7,11and12showed highly hepatoprotective activity against H2O2-induced oxidative damages on HepG2cells. This research clarified the pharmacodynamic materials of Lindera aggregata for hepatoprotective, and found some new compounds. These results provided the theoretical basis and scientific reference for quality control, new drug development and application of Lindera aggregata.2. Based on the established HPLC fingerprint of Phyhalis alkekengi (Chinese lantern) preparations (CLP), analysis and comparison were carried out among the HPLC profiles of rat plasma (samples obtained after intragastric administration of CLP), CLP and blank plasma, about twenty compounds of CLP were found absorbed into blood. Then, directed by the drug contented plasma’s fingerprint, the isolation, purification and identification of the absorbed constituents were performed,9compounds were isolated and indentified, and7of them belonged to physalins. Finally, through in vitro anti-inflammatory mode, combining with literature researchs, physalins were determined to be the pharmacodynamic materials of CLP for anti-inflammatory activity. This research is very important for establishing quality evaluation methods and exploring pharmacokinetic properties of Phyhalis alkekengi.3. UPLC-MS/MS methods were established to evaluate the quality of Lindera aggregata and Phyhalis alkekengi through simultaneous quantitation of5sesquiterpene lactones and5physalins, respectively. The results showed that the developed methods had good linearity, precision, accuracy and sensitivity for the determination of the active compounds. Finally, these simple, rapid and reliable methods were successfully applied to determine the sesquiterpene lactones and physalins in Lindera aggregata and Phyhalis alkekengi samples from different areas of China. Otherwise, this study found that4,7-didehydrophysalin B was instable under room temperature, and its3transformation products were detected and identified for the first time. This study laid a solid foundation for the establishment of comprehensive quality control methods of Lindera aggregata and Phyhalis alkekengi.4. Chromatographic fingerprinting analysis methods of Lindera aggregata and Phyhalis alkekengL were established using UPLC-DAD-MS/MS, respectively. MS fragmentation behavior and UV characteristics of sesquiterpene lactones and physalins were also investigated. According to the UPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by MS/MS spectra,11fingerprint peaks were identified from Lindera aggregata (including3alkaloids and8sesquiterpene lactones), and19fingerprint peaks were identified from Phyhalis alkekengi (14of them belonged to physalins). Finally, the established fingerprinting methods were applied to evaluate the quality of13Lindera aggregata and31Phyhalis alkekengi samples through similarity and PCA. The proposed methods provide a scientific technical platform for the quality control of TCM and the rapidly study of chemical components of TCM.5. An UPLC-MS/MS method was developed for the quantification of3major active ingredients (physalin D, physalin G and4,7-didehydroneophysalin B) of CLP in rat plasma. The developed method provided a simple sample preparation and a stable analytical result for the determination of these physalins in plasma samples. This method was successfully applied to the determination of the3major components of Phyhalis alkekengi in rat plasma after intragastric or intravenous administration. In addition, the metabolism of4,7-didehydroneophysalin B in liver microsomes was investigated. Eight metabolites were identified by MS/MS data, and metabolic pathways of4,7-didehydroneophysalin B were deduced. |
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