Studies On The Identification And Quality Analysis Of Gentiana Macrophylla Radix

Gentiana macrophylla Radix is a Traditional Chinese medicine（TCM）with efficacy of dispelling wind-dampness and relieving pain,mainly distributed in Qinghai,Ningxia,Shaanxi,Shanxi,Inner Mongolia,Yunnan and other places in China,with complicated sources and numerous base origins.As the demand for Gentiana macrophylla Radix herbs has increased greatly,the wild resources have been destructively mined,the market is confused,different medicinal parts are mixed,and many local customary species not included in the pharmacopoeia are used instead of Gentiana macrophylla Radix,which seriously affects the quality of herbs and directly affects the clinical efficacy of Chinese medicine.Using modern analytical technology for multi-index quantitative detection and fingerprint research is an important means of the TCM quality control,which is widely used to evaluate the quality,identify the authenticity and source and so on.Capillary electrophoresis（CE）is a fast,low-cost,low sample and solvent consumption method.Different compounds can be separated by changing the composition of background electrolyte and operation mode.In the field of TCM analysis,CE fingerprinting has been widely used in the quality evaluation of TCM.Near Infrared（NIR）spectroscopy has the advantages of rapid,non-destructive,green and simple operation.It is an important means to identify the species and origin of TCM and to quickly detect the content of active ingredients by extracting spectral information and establishing qualitative and quantitative models through chemometrics.In this thesis,the quality control method of TCM is studied by taking Gentiana macrophylla Radix as an example,the capillary electrophoresis analysis method of Gentiana macrophylla Radix is established based on AQbD（Analytical Quality by Design）concept,and the qualitative and quantitative quality control model of NIR spectra of Gentiana macrophylla Radix is developed;and combining the advantages of spectroscopy and chromatography,we proposed a NIR spectroscopy and CE fingerprinting conversion strategy for rapid evaluation of the overall quality of TCM.The specific studies are as follows:1.A preliminary study on the analytical method of CE in Gentianae Macrophyllae Radix based on AQbD concept.Four iridoid compounds in Gentianae Macrophyllae Radix were analyzed by capillary electrophoresis photodiode array detector.The peak area RSD（n=3）and prepeak resolution of the four active ingredients were taken as critical method attributes（CMAs）.The critical method parameters（CMPs）were filtered out by the partial analysis design test combined with Pareto analysis as buffer concentration and pH,SDS concentration,column temperature and voltage.Then,a central composite design was used for method optimization,and the response model between CMPs and CMAs was established and the design space was constructed to obtain the optimal separation conditions:51 mmol·L^-1borax solution,pH=9.47,40 mmol·L^-1SDS as background electrolyte,separation voltage of 20 k V,and column temperature of 23℃.Finally,the validation experiments were carried out,and the results show that the test results were close to the predicted value of the model,and the method was rapid and reliable for the quantitative analysis of the four iridoids in Gentianae Macrophyllae Radix,providing a reference for the application of AQbD concept in the analysis of TCM by CE.2.Qualitative and quantitative study of Gentianae Macrophyllae Radix based on near infrared spectroscopy and chemometrics.The standard deviation spectrum was used to optimize the near-infrared acquisition conditions,and the near-infrared spectra of 196 samples of Gentianae Macrophyllae Radix were collected by second-order derivatives combined with multiple scattering correction to establish an orthogonal partial least squares discriminant analysis（OPLS-DA）model.According to the 3D score chart,Gentiana macrophylla Pall.,Gentiana crassicaulis Duthie ex Burk.,Gentiana.dahurica Fisch.and the mixture can be clearly distinguished,and the verification accuracy of the model for all samples were 100%.Subsequently,the partial least squares regression（PLSR）quantitative models of gentiopicrin,loganic acid and quercitric acid in Gentianae Macrophyllae Radix were established.Four pretreatment methods and their combinations and four variable screening methods were compared,and the model performance was evaluated in terms of root mean square error（RMSE）and correlation coefficient.The results showed that the root mean square error of prediction for all three components was less than 0.70%（w/w）and the R_p value was greater than 0.850.The qualitative and quantitative models established in this study were accurate and reliable,which can reflect the overall quality information of Gentianae Macrophyllae Radix.It can be used for the identification and the determination of effective components for the Gentianae Macrophyllae Radix,which contribute to provide reference for the quality control of other TCM.3.Preliminary study on multivariate signal conversion of NIR spectroscopy and CE chromatography fingerprint based on chemometrics.The CE fingerprint method of Gentianae Macrophyllae Radix was established and validated.NIR spectroscopy were converted to CE fingerprint profiles by PLSR algorithm.The study compared four pre-processing methods and their combinations and four variable screening methods,and established a PLSR conversion model,the R_p values greater than 0.900 and RMSEP less than 0.350 m AU,model prediction performance was satisfactory.The conversion performance of the model was evaluated by similarity analysis using the vectorial angle cosine method,and the results showed that 36.2%of the sample model conversion similarity was between 0.85 and 0.95,and 62.1%of the sample conversion spectra were similar to the real spectra more than 0.95,and the model conversion effect was satisfactory.The PLSR algorithm can initially realize the conversion of multivariate signals between NIR spectroscopy and CE chromatography.The established method can greatly shorten the analysis time and fully exploit the poorly interpretable NIR spectra data,which has application value in the field of TCM analysis.