Research Of Separation Purification And Activity Screening For Homogenous Components In Traditional Chinese Medicine

The material basis research of traditional Chinese medicine (TCM), quality evaluation and activity screening based on TCM resources were the important parts of modern TCM. In this study, piper longum L. and lobelia chinensis Lour. were selected as research subjects and two-dimensional chromatography was used as the core technology. Combined with the development of chromatographic separation materials and separation purification technologies, systemic separation methods including pretreatment, one-dimensional efficient preparation and two-dimensional orthogonal separation were developed. As a result, homogenous components, activity screening components and compounds were obtained. Furthermore, activity evaluations of material resources above were carried out. Separation purification, quality evaluation and activity screening of TCM were researched systematically.Alkaloid homogenous component was prepared from piper longum L. using saponification reaction and traditional extraction method. A two dimension (2D)-normal phase liquid chromatography (NPLC)/reversion phase chromatography (RPLC) method was constructed and its orthogonality reached to 58.3%. Then, this method was enlarged to preparation level and 20 screening components and 28 compounds were obtained. A 2D-ultra-performance supercritical fluid chromatography/ultra-performance liquid chromatography method with good orthogonality was developed and 340 peaks were identified in alkaloid homogenous component, enriching the 2D analysis methods.Quality evaluation was carried out for the alkaloid homogenous component. Eighteen compounds prepared from alkaloid homogenous component were used for accurate qualitative analysis and twenty-five minor compounds were identified by LC-MS, making the material basis of the alkaloid homogenous component relatively clear. Multi-index quantitative analysis was carried out using four reference compounds and the content of them reached to 16.47%. Furthermore, the neuro protective effect of alkaloid homogenous component and activity screening components were evaluated preliminarily on SH-SY5Y and PC-12 cell models which were induced by 6-OHDA and H2O2. Three possible active components were found and in-depth research need to be done in the future.Using the difference in polarity between alkaloids and flavonoids, and combing traditional extraction method with solid phase extraction method, alkaloid and flavonoid homogenous components were prepared from lobelia chinensis Lour. respectively. A preparative 2D-hydrophilic interaction liquid chromatography (HILIC)/RPLC method with good orthogonality was developed for the purification of flavonoids. Then,20 flavonoid homogenous components and 10 compounds were obtained. What’s more, eight compounds were obtained firstly. A mix-mode/RPLC method was researched preliminarily for the analysis of alkaloid homogenous component. In this study, two homogenous components were prepared from single TCM. It was beneficial for sample simplification. In-depth research could be carried out according to their own characteristics.The regulation and control of flavonoid homogenous components and compounds for GPR35 was investigated using new cellular label-free integrative pharmacology (CLIP) technology. Ten activity screening components and five new compounds could active GPR35 receptor. The activity of these compounds on GPR35 receptor was verified by agonism, desensitization and antagonism analysis. They were beneficial to explain the acting mechanism of anti-inflammatory and analgesic. Then, the toxicity for the growth of Zebrafish embryonic and the effect for the growth of vascular system were evaluated based on transgenic zebrafish model. Two compounds, which inhibited the development of ISV and DLAV obviously, were founded and this inhibition effect was related to the inhibition of cell proliferation or cell migration. Further investigations of its acting mechanism would to be done.Taking homogenous components as the core, systemic separation purification, quality evaluation and activity screening researches were carried out. The preparation of homogenous components, which aimed to enrich a certain ingredient, was thecourse of discarding the dross and selecting the essential. Two dimensional analysis and preparation based on homogenous components were the deep research course of the material basis. All fractions and compounds, which were characterized by the stability of preparation method and the good quality control, were essential resources of new medicine development. In this study, the systemic separation methods and researches of the crucial issues were the necessary technical indemnifications for the work above. Furthermore, it benefited for the understanding of material base in TCM and promoting the level of quality control.