Quality Evaluation Of Panax Notoginseng (Burk.) F.H.Chen Using Supercritical Fluid Chromatography And Chemical Pattern Recognition

Panax notoginseng（Burk.）F.H.Chen is a precious Traditional Chinese medicine（TCM）in China,it has occupied an important position in the field of TCM and health care products.It is particularly important for the quality control and evaluation of Panax notoginseng to fully understand its chemical composition,determine the key active components,and clarify the interaction mechanism between components.However,there are many kinds of compounds in Panax notoginseng which are complicated,large differences in structures,these problems have brought great challenges to the analysis of chemical components in Panax notoginseng and their quality evaluation.At present,the analysis and quality evaluation methods of Panax notoginseng are mainly liquid chromatography（LC）,such as reversed-phase chromatography,hydrophilic chromatography,and ion-exchange chromatography.Although LC can effectively elute most of the chemical components in Panax notoginseng,it has disadvantages such as long analysis time,large consumption of organic solvents,poor elution effect of highly polar substances,and long equilibration time.In order to make up for the insufficiency of LC and achieve the comprehensive analysis of Panax notoginseng,a more efficient and fast analytical technology that can complement LC is required.Supercritical Fluid Chromatography（SFC）is a chromatographic technique that uses supercritical fluid as the mobile phase and separates substances according to the different distribution coefficients of solutes in the two phases.It has the advantages of fast analysis speed,high separation efficiency and environmental friendliness.In recent years,SFC has been widely used in many fields such as TCM analysis,environmental monitoring,food analysis and so on.In this study,the Panax notoginseng was taken as the object,and the SFC combined with pattern recognition method was applied to study the quality evaluation of Panax notoginseng.Firstly,the influence of various parameters of SFC combined with Evaporative Light-scattering Detector（ELSD）on the signal was investigated,which provided a guide for the development of SFC-ELSD method for saponins in Panax notoginseng.Similarity analysis was conducted to evaluate different samples of Panax notoginseng,which proved the feasibility of SFC-ELSD for the analysis of Panax notoginseng.Secondly,an efficient and fast SFC-MS（Supercritical Fluid Chromatography-Mass Spectrometry,SFC-MS）method combined with chemical pattern recognition was developed for the quality evaluation of traditional Chinese medicine Panax notoginseng.The SFC-MS combined with the PLS-DA model was successfully implemented.Different harvest periods and different parts of Panax notoginseng samples were distinguished,and potential characteristic components were obtained which could be used as indicators of Panax notoginseng quality.Finally,the accuracy of the model was verified by in vitro pharmacological activity tests.The hemostatic activity of Panax notoginseng combined with statistical methods was selected to deeply excavate the correlation between the index components of Panax notoginseng and the classical anticoagulant activity,and the material basis of blood efficacy was discussed.Chapter Ⅰ:The mainly components of Panax notoginseng and the quality evaluation method commonly used are systematically introduced,and the shortcomings of exisiting methods are summarized.Then the application of SFC in quality evaluation of TCM is comprehensively introduced.At last,the research route and innovations of this paper is proposedChapter Ⅱ:The influence of parameters of the SFC-ELSD on the detector signal were investigated by the response surface design（RSM）.First,the significant factors were screened by the Plackett-Burman design,and the Box-Behnken design combined with the RSM method was used to investigate the relationship between significant parameters.The best method was optimized and used in the quality evaluation of Panax notoginseng combined with fingerprints.This study shows that SFC-ELSD can be used as a suitable method for the analysis of saponins in Panax notoginseng,in addition,it provides an optimization guide for the development of SFC-ELSD analysis methods for saponins.Chapter Ⅲ:A SFC-MS method combined with chemical pattern recognition（CPR）was established for the quality evaluation of Panax notoginseng.Firstly,the chromatographic conditions for saponin analysis were obtained by screening the stationary phase,modifiers and additives,then the method was further optimized by Do E,and the feasibility of the method was verified by methodological indicators.By combining CPR methods such as HCA,PCA and PLS-DA,a classification model was established based on the peak area and content of 12 saponin compounds to evaluate the quality of different parts and different collecting time of Panax notoginseng.The results show that the PLS-DA model based on peak area can accurately distinguish samples of Panax notoginseng in different collecting time and different parts,then the characteristic components（ginsenoside Rg₂,ginsenoside Rg₁,ginsenoside Rb₁） which obtained by feature extraction can be used as potential quality marker of Panax notoginseng,and SFC-MS combined with PLS-DA method shows good application prospects in the field of quality evaluation of Panax notoginseng.Chapter Ⅳ:In order to validate the scientificity of the classification model and characteristic components,the classical in vitro hemostatic activity of Panax notoginseng was selected and the in vitro activity evaluation method was used to determine the effects of extracts from different parts and different collecting time of Panax notoginsengon the prothrombin time,thrombin time and activated partial thrombin time of blank human plasma.The correlation between characteristiccompoundscontents and anticoagulant activity was analyzed.The results showed that only different collecting time of Panax notoginseng had significant differences in thrombin time,and there was a significant correlation between the three characteristic components and the thrombin time,which further proved that the characteristic components screened out can be used as potential quality markers of Panax notoginseng..Chapter Ⅴ:The content of this paper was summarized,based on this,and the shortcomings of this research was discussed.Thenthe development of SFC and it’s application prospect in the flied of quality evaluation of TCM are prospected.