| Traditional Chinese medicine (TCM) was created with the birth of Chinese civilization which dates back four to five thousand years ago when Emperor Yan ruled China. Although TCM has used the processed crude multi-component natural products to treat different symptoms in accordance with a unique theory, understanding the principle of treatment of TCM has been an enormous challenge to modern scientists. Modernization of TCM has been proclaimed and put in practice for several decades, but such a road is still difficult in nowadays. Simple isolation and characterization of one or several active components in TCM and assaying their corresponding bioactivity can not reveal its real efficacy basis because the efficacy of TCM is resulted from multiple components at multiple targets. Therefore, the goal of this thesis is to propose several strategies to achieve qualitative identification of all secondary metabolites (including both major and trace ones) in TCM. This will provide clear indication for further pharmacodynamical research, quality control and safety evaluation of TCMs.Besides the traditional phytochemical research method, the on-line identification method, which is based on the modern hyphenated techniques, has been used as an important tool for phytochemistry research of TCMs. By the traditional phytochemical research method, chemical structure elucidation of the isolated components can be completely conducted. However, this process is time-consuming, laborious and costly, and it is usually hard to implement the investigation of the trace or labile constituents in TCMs. In comparison, the on-line identification process is usually very simple and rapid, and it is suitable for analyzing trace and labile ones in complicated matrix. However, structure information afforded by this method is usually limited. Thus, the general thinking of this thesis is to achieve global and accurate qualitative analysis of the active compounds in TCMs based on the mutual complementarity of traditional phytochemical isolation techniques and on-line identification techniques. Two feasible schemes, named as 'Scheme 1'and'Scheme 2'respectively, were established based on the actual situation. Three flavonoids-rich TCM samples, i.e. the roots of Scutellaria bacalensis Georgi, the stems and leaves of Scutellaria bacalensis Georgi and Dracocephalum tanguticum Maxim, were used as model plants to testify the feasibility of the proposal described above. The details are summerized as follow.1. Based on the phenomenon that hydrophilic acetonitrile can be separated from water by adding a hydrophobic solvent, a novel phase transition extraction (PTE) method for extraction and isolation of flavonoids in TCMs has been proposed. This method integrates the extraction process and the isolation process into one simple step. The flavonoid aglycones and flavonoid glycosides in TCMs can be completely separated by this simple method. The application of this method in a systemic phytochemical isolation study will greatly simplify the downstream isolation process.2. As guided by'Scheme 1', global and accurate qualitative analysis of the flavonoids in the roots of Scutellaria bacalensis Georgi was conducted. Step 1, HPLC-UV-MSn was used to analyse the methanol extract of the roots of S. baicalensis.31 components were detected and all were assigned to be flavonoids. Step 2, a systemic isolation study was conducted based on the SIPTE method proposed above.18 major components were successfully isolated and their structures were unambiguously elucidated by their spectroscopic and spectrometric data. Step 3, The UV pattern and MS fragmentation characteristics of the eighteen known flavonoids were studied in detail. The rules summarized provided valuable indications for the subsequent on-line identification process. Step 4, by interpreting both the MS and the UV data in detail, other thirteen minor flavonoids in the roots of S. baicalensis were on-line identified successfully. This study has testified the feasibility of'Strategy 1'proposed. Furthermore, the global qualitative result is quite significative for quality control, safety evaluation and pharmacodynamical research of the roots of S. baicalensis and its related products.3. As guided by'Scheme 2', global and accurate qualitative analysis of the flavonoids in the stems and leaves of S. bacalensis was conducted. Step 1, HPLC-UV-MSn analysis was also performed.21 flavonoids were detected and most of them were found to be the unique constituents for the stems and leaves of S. baicalensis. Step 2, On-line identification were performed at first. The UV pattern and MS fragmentation rules summarized above still provided valuable indications for the on-line identification study here.17 components were successfully on-line identified. These on-line results were further confirmed by comparing with corresponding literature data. Step 3, object isolation of the rest four flavonones, which were failed to be on-line identified, were conducted. One of them is a new compound. This study has testified the feasibility of 'Scheme 2'proposed. The global qualitative result is also significative for quality control, safety evaluation of the stems and leaves of S. baicalensis and its related products.4. To conduct a global qualitative analysis of the flavonoids in Dracocephalum tanguticum Maxim under guidance of'Scheme 1', i.e. Step 1, HPLC-UV-MSn analysis; Step 2, systemic isolation; Step 3, study and conclusion of UV and MS rules; Step 4, on-line identification of unisolated components.8 flavone glycosides were detected. Six of them were isolated while the rest two were on-line identified. All of these eight flavone glycosides were the first time reported from D. tanguticum.5. A novel MSn method used for exact hydroxylation position characterization of flavonoid glycones has been proposed. In classical MSn studies of flavonoids, product ions (i,jA+/- and i,jB+/-) produced by the cleavage of C-ring including retro Diels-Alder (RDA) fragmentations, are indicative for the distribution of hydroxyl substituents between the A- and B-ring, but the exact substitution position could not be determined. In the present work, characteristic A-and B-ring related product ions, such as 1,3A+,1,3B+ and 0,2B+ ions, obtained from hydroxylated flavonoid aglycones in positive ESI ion mode, were further fragmented via collisionally activated dissociation (CAD). The obtained fragmentation patterns of the selected ions are structural informative for the detailed hydroxylation patterns of the corresponding A- or B-ring. This new method is more convenient and can provide more information than conventional ones.
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