Studies On The Chemical Constituents And Analysis Methods For Several Traditional Chinese Medicines

This dissertation consisted of four sections. The first two sections elaborated the phytochemical investigation of the rhizomes Gymnadenia conopsea R. Br., method development for rapid identifying and qutifying the chemical condtituent of this tibetant medicine, and the chemical fingerprint analysis of rhizomes of G. conopsea, Salviae miltiorrhiza and P. notoginseng. The third section studied the fragmentation mechanism of six alkaloids from Zanthoxylum nitidium and method development for rapid identifying varieties of alkaloids from the extract of this herbal medicine. The fourth section reviewed HPLC-MS method in drug metabolism studies.The first section consisted of chapters 1, 2 and 3. Chapter 1 elaborated the phytochemical investigation of G conopsea. Four succinate derivative esters were isolated from the methanol extract of the rhizomes of G. conopsea through repeated column chromatography on normal and reversed phase silica gel, their structures were determined by ESI-MS, 1D and 2D NMR evidence. They were firstly discovered from this species. In chapter 2, a high-performance liquid chromatography-diode array detection (HPLC-DAD) method has been firstly developed for quantitation of two characteristic constituents, adenosine and 4-hydroxybenzyl alcohol, from the extract of rhizomes of G conopsea. All 10 samples of G. conopsea contained different amount of adenosine and 4-hydroxybenzyl alcohol. Adenosine and the 4-hydroxybenzyl alcohol can be applied in identification and quality control for the roots of G. conopsea. In chapter 3, a high-performance liquid chromatography-diode array detection-tandem mass spectrometry (HPLC-DAD-MS~n) method has been firstly developed for chemical fingerprint analysis of rhizomes of G. conopsea and rapid identification of major compounds in the fingerprints. Comparing the UV and MS spectra with those of authentic compounds, seven main peaks in the fingerprints were identified as adenosine, 4-hydroxybenzyl alcohol, 4-hydroxybenzyl aldehyde, dactylorhin B, loroglossin, dactylorhin A and militarine. The Computer Aided Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (CASES) was employed to evaluate the similarities of 10 samples of the rhizomes of G. conopsea collected from Sichuan, Qinghai and Hebei provinces and Tibet autonomous region of China, and Nepal. These samples from different sources had similar chemical fingerprints to each other.The second section consisted of chapters 4, 5, 6 and 7. In chapter 4, both the characteristic spectra of liposoluble tanshinones and aqueous-soluble salvianolic acids were established by the electrospray ionization mass spectrometry (ESI-MS) technique and the differences between standard and crude rhizomes of S. miltiorrhiza from 5 sources were analyzed. The law of electrospray ion trap mass (ESI-ITMS) of typical tanshinones and salvianolic acids is studied.The analysis of the chemical constituent of rhizomes of S. miltiorrhiza by liquid chromatography coupled with mass spectrum (LC/MS) technique was established, and the distances among standard herb and crude herb from 5 sources were calculated by clustering analysis. According the DAD spectra and MS~2 data, 9 tanshinones could be speculated. In chapter 5, the character spectra of total saponins in P. notoginseng extracts were established by ESI ITMS and selective ion monitoring (SIM) technology. The law of notoginsenosides by ESI-MS~2 was studied. In chapter 6, the characteristic spectra of Compound Danshen Tablet established and compared by ESI-MS and HPLC/DAD/MS, 6 known tanshinones and 3 saponins were speculated. In chapter 7, the advantage and disadvantage of the strategy, using the ESI-ITMS and LC/MS techniques for study of characteristic spetra of danshen and Compound Danshen Tablet, were summerized.The third section (chapter 8) studied the fragmentation mechanism of six alkaloids, dihydronitidine, dihydrochelerythrine, 8-acetonyl dihydronitidine, 8-acetonyldrochelerythrine, nitidine and l,3-bis(8-dihydronitidinyl)-acetone, by ESI-MS~n. Tandem mass spectrometry experiments indicated that different substitution sites of the methoxyl groups at C-9 and C-10 or at C-10 and C-11 determined the different abundances of the MS~2 fragmentation ions using the same collision energy. According to the different abundances of MS~2 product ions, positional isomeric benzo[c] phenanthridine alkaloids can be differentiated. Moreover, ten constituents in the crude alkaloids extract from the roots of Z. nitidium were rapidly identified by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS~n), through comparing the retention times and ESI-MS~n spectra with the authentic standards.The fourth section (chapter 9) is a review on HPLC-MS method development in drug metabolism studies.