Study On The Quality Control Of Panax Notoginseng And Related Preparations Based On Chemical Characterization And Data Mining

Chemical characterization of the complex traditional Chinese medicine(TCM)systems contributes to illuminating the medicinal material basis and improving the quality control systems,and thus promoting the standardization and internationalization of TCMs.However,the TCM components are diversified in types,complex in structures and varied in contents,and it is urgent to solve the problems of how to efficiently and sensitively characterize the active components of TCM.The dissertation is designed to develop new profiling methods in two aspects,including chromatographic separation and data mining,based on UHPLC-HRMS platform,integrating intelligent and automatic software,taking P.notoginseng and its related preparations as research objects.A strategy named “offline two dimensional LC separation-extended library screening” was proposed and applied to the target characterization of the ginsenosides from the total saponins of P.notoginseng and the leaves of P.notoginseng.The preparations of the total saponins from different manufacturers were compared and the new ginsenosides were targetedly isolated according to the characterization results.1)The offline comprehensive 2D LC system combing the orthogonal HILIC and reversed phase mechanisms was constructed.The theoretical peak capacity and orthogonality was 8976 and 0.84.The characteristic data was acquired in fast DDA mode by tandem QTOF high resolution MS,and 148 ginsenosides were identified in Xueshuantong injection.The study provided a reference method for the exposures and separations of minor and trace components,illuminated the chemical material basis of Xueshuantong injection and provided the basis for its quality control.2)Nontargeted plant metabolomics methods were adopted to systematically compare 130 batches of Xueshuantong and Xuesaitong injections,based on the aforementioned identification research of the total saponins.The results showed that no obvious differences were observed between Xueshuantong and Xuesaitong,or between different dosage forms,but obvious differences were observed between Xueshuantong from Wuzhou and the total saponins from other manufacturers.Twenty marker compounds were selected by OPLS-DA and identified by combining the identification results of the total saponins,and the main difference was that the components close to ginsenoside Rd in Xueshuantong were significantly lower than the products from other manufacturers.In addition,different roots of P.notoginseng were compared in the same way,and 15 maker compounds between the main roots and rhizomes were selected and identified.Correlation analysis of two groups of markers showed that the differences between the total saponin preparations were not due to the different parts of P.notoginseng.The research established the foundation for the construction of quality control system for the total saponins of P.notoginseng.3)An “offline 2D LC separation-extended library screening” strategy to systematically characterize the ginsenosides in the leaves of P.notoginseng was proposed based on the established offline 2D LC system.On the basis of UNIFI platform,the data acquired by 2D LC QTOF MS was interpreted by matching the library.In view of that the matching results of the library screening were limited by the compound items in the library,we proposed the predicted metabolite method to expand the library.Modification groups,including malonyl group,glucose,rhamnose and xylose and up to two steps of modification were selected to expand the library for 14 times,and the matched components with the new library were 2.56 times of that with the primary library.Finally,945 ginsenosides were identified in the leaves of P.notoginseng,and among which,662 ginsenosides were speculated to be the new ones.The expanded library screening method based on UNIFI greatly simplified the identification procedure,and by integrating the offline 2D LC method,it made a breakthrough in the number of the identified components from a single TCM herb.At the same time,the research established a foundation for the discovery of novel ginsenosides.4)LC-MS guided isolation of the leaves of P.notoginseng was conducted to confirm the structures of the above potential novel ginsenosides.Various separation techniques and spectroscopic methods were used in combination,and at last 19 novel ginsenosides were isolated and identified,including three nordammarane triterpenoid saponins(notoginsenosides L1-L3),one 3-OH acetylated ginsenoside(notoginsenoside L4),eight 3-OH malonylated ginsenosides(notoginsenosides L5-L12)and seven C17-side chain varied ginsenosides(notoginsenosides L13-L19).Among them,eight 3-OH malonylated ginsenosides were found abundant in the leaves and the flowers,but rare in the roots of P.notoginseng.Therefore,the eight 3-OH malonylated ginsenosides may be potential quality markers to differentiate the acrial parts from underground parts of P.notoginseng.In conclusion,we established methods to targetedly characterize the ginsenosides,systematically analyzed the components in P.notoginseng and its related preparations,compared the preparations of the total saponins from different manufacturers,and isolated the potential new ginsenosides from the leaves of P.notoginseng,thus laid a foundation for the quality control research.At the same time,it also provided new strategies and methods for the chemical material basis research of the complex TCM systems.