Studies On Quality Evaluation Of Panacis Japonici Rhizoma

Panacis Japonici Rhizoma(Zhu-Jie-Shen in Chinese),is derived from the dried rhizome of Panax japonicus C.A.Mey.(Araliaceae),which is used to treat hemoptysis and sputum for cough,flutter injury and weakness after illness with the medicinal properties of scattering stasis detumescence,arresting bleeding,relieving pain and replenishing deficiency[1].Panacis Japonici Rhizoma mainly produce in Hubei,Yunnan,Sichuan and Guizhou in China.Among them,the wild and cultivated resources are rich in Enshi,Hubei.Modern studies show that it mainly contains saponins,sugars,amino acids and volatile oil,and saponins are the main active ingredients[2].The harvest time and processing method specified in the 2015 edition of the Chinese Pharmacopoeia is "excavated in the autumn,and dried after removing the main root and outer skin",while the places of origin are generally dried in the sun or instrument after washing.Pharmacopoeia regulation is more general,and the quality of it on the actual market varies due to various factors(such as,habitats,harvest time and processing methods).How to ensure the quality and quality uniformity has become the critical problem in the production process of Panacis Japonici Rhizoma.Therefore,it is necessary to explore the quality formation of Panacis Japonici Rhizoma from the source of medicinal materials production,systematically analyze the influence of these factors(habitats,harvest time,processing methods,and etc.)on the chemical constituents and comprehensively evaluate its quality.In this study,the simultaneous determination of the content of saponins,amino acids and nucleosides in Panacis Japonici Rhizoma were analyzed by ultra-fast liquid chromatography coupled with triple quadrupole-linear ion trap tandem mass spectrometry(UFLC-QTRAP-MS/MS)method,Panacis Japonici Rhizoma samples of different habitats,harvesting time and growth years,processing methods,cultivated and wild types,different characters and didderent parts(roots and rhizomes)of Panax japonicus samples were adopted for comparative analysis and comprehensive evaluation;an efficient and sensitive method based on ultra-fast liquid chromatography coupled with triple quadrupole-time of flight tandem mass spectrometry(UFLC-Triple TOF-MS/MS)was established to identify saponins in Panacis Japonici Rhizoma,further,Panacis Japonici Rhizoma samples of cultivated and wild types,different characters,and roots and rhizomes of Panax japonicus samples were analyzed and compared for difference in saponins.This study is mainly divided into the following parts:1.Literature Research By the systematic review of the literature related to Panacis Japonici Rhizoma at home and abroad,carried on the existing research literature research to summarize.2.Quanlitative analysis of the saponins in Panacis Japonici Rhizoma.UFLC-Triple TOF-MS/MS was adopted to the identification and analysis of saponins in Panacis Japonici Rhizoma.Based on the accurate molecular mass information of the quasi-molecular and fragment ions provided by high-resolution mass spectrometry,combined with standard reference data and related literature data,53 saponins were finally identified,including glycosides of protopanaxadiol(PPD),glycosides of protopanaxatriol(PPT),glycosides of oleanolic acid(OA)and glycosides of ocotillone(OC),and fragmentation ways of 53 saponins were preliminarily deduced.3.Quantitative analysis of multiple-index constituents in Panacis Japonici Rhizoma.An accurate and reliable method for the simultaneous determination of saponins,amino acids and nucleosides(33 constituents)in Panacis Japonici Rhizoma from different habitats,Panacis Japonici Rhizoma of different harvesting time and growth years and processed by different methods by UFLC-QTRAP-MS/MS method was established.The result showed that the content of saponins was highest in three kinds of constituents,followed by amino acids and nucleosides.Nevertheless,the content of 33 constituents in Panacis Japonici Rhizoma from different habitats was different,the total content of saponins in Puer(Yunan,cultivated)and Wenshan(Yunan,cultivated)was higher,while the total content of amino acids and nucleosides was both higher in Enshi(Hubei,cultivated);The result of one-way analysis of variance(ANOVA)and t-test showed that the content of 33 constituents among different samples demonstrated significant differences,then,the ginsenoside Ro/Re ratios were found to be different when compared across samples from different harvest time,the result of curve fitting showed that the relationship between the content of amino acids and nucleosides with growth years was consistent with specific curves,but the saponins was not all in line;the grey relational analysis(GRA)result showed that the comprehensive quality of the processing method,which used 50?microwave drying for the intact rhizome without peeling,was better.4.Comparative analysis of constituents in cultivated and wild Panacis Japonici Rhizoma.The content of saponins,amino acids and nucleosides in cultivated and wild Panacis Japonici Rhizoma was determined and analyzed by UFLC-QTRAP-MS/MS method.The differences of saponins in cultivated and wild Panacis Japonici Rhizoma were measured and analyzed by UFLC-Triple TOF-MS/MS method,combined with multivariate statistical method.The determination result showed that the content of some constituents of the cultivated was higher than the wild samples according to 33 constituents except OC(pseudoginsenoside F11)and amino acids;the result of analysis on differences of saponins showed that cultivated and wild samples could be distinguished by principal component analysis(PCA)and the partial least squares discrimination analysis(PLS-DA),19 different constituents were screened out and 6 of them were identified according to PLS-DA and t-test.5.Comparative analysis of constituents in Panacis Japonici Rhizoma of different characters.The content of saponins,amino acids and nucleosides in Panacis Japonici Rhizoma of different characters(long,globular,mixed type of rhizomes)was determined and analyzed by UFLC-QTRAP-MS/MS method.UFLC-Triple TOF-MS/MS combined with multivariate statistical method was also adopted to analyze the differences of saponins in different characters.The determination result showed that the content of 33 constituents and various constituents in the long type was higher;the result of analysis on differences of saponins showed that three kinds of rhizomes couldn't be clearly distinguished by PCA,but they could be separated by PLS-DA.Every two groups(long type VS globular type,long type VS mixed type and globular type VS mixed type)could been could be distinguished by Orthogonal partial least squares discrimination analysis(OPLS-DA),screened out 9,8 and 10 different constituents,identified 2,2 and 2 of them by OPLS-DA and t-test,respectively.6.Comparative analysis of constituents in roots and rhizomes of Panax japonicus.The content of saponins,amino acids and nucleosides in roots and rhizomes of Panax japonicus was determined and analyzed by UFLC-QTRAP-MS/MS method,combined with hierarchical clustering(HCA),nonlinear mapping and PCA.The differences of saponins in roots and rhizomes were also measured and analyzed by UFLC-Triple TOF-MS/MS method,combined with multivariate statistical method.The determination result showed that the roots were similar to rhizomes according to the content of 33 constituents;the result of analysis on differences of saponins showed that there was no significant difference between roots and rhizomes by PCA,but they could be be distinguished by PLS-DA,further analysis of PLS-DA and t-test filtered out 1 different constituent and identified it.