| Gentiana Radix et Rhizoma(Long-Dan)with Gentiopicroside that belong to iridoids as the main substance is a common traditional medicine used in Chinese clinical medicine for treating damp-heat jaundice,liver-fire headache,convulsive convulsions and digestive system diseases.It has been recorded in Chinese Pharmacopoeia(2020edition),as it derived from the roots and rhizomes of four botanical origins of Gentiana,which are Gentiana triflora Pall.?Gentiana manshurica Kitag.?Gentiana scabra Bge.and Gentiana rigescens Franch are the authentic sources.However,the chemical differences of these four species of Gentian are still unclear with no systematic characterization and chemical comparison research.The quality control was primarily focus on qualitative and quantitative of Gentiopicroside,the effective quality control of Long-Dan was severely restricted by single index.To resolve these problems,this dissertation is proposed to establish a new strategy for systematically characterize research of the major substance“iridoid group”in multiple botanical origins of Long-Dan by ultra-high performance liquid chromatography tandem linear ion-trap Orbitrap mass spectrometry,UPLC/LTQ-Orbitrap analytical platform,and to develop a new method for multi-index simultaneously quantitative measure.1?Study on fingerprint of multiple botanical origins Long-Dan88 batches botanical samples from three Gentiana species and 30 batches commercial samples were collected and authenticated.Based on the UPLC analysis platform with 88 batches botanical drug samples,the fingerprint spectrum quality analysis method of multiple botanical origins of Long-Dan was established.The method was validated with applicable stability,specificity precision and repeatability.Finally 30batches commercial samples were evaluated and identified by similarity evaluation,principal component analysis and orthogonal partial least squares method.2?Study on systematic profiling of iridoid in Long-DanAccording to the literatures and databases retrieve,an in-house library of isolated129 iridoids structure with NMR information from Gentiana family was constructed based on Progenesis QI SDF studio and Excel.In the negative mode,10 iridoids standards of MS~n fragment rule was consistently studied and summarized by UPLC/LTQ-Orbitrap MS.The characterization analysis of iridoid in Long-Dan was investigated by polygonal mass defect filter method combined multidimensional data acquiring mode.Firstly,a polygonal MDF algorithm was established based on 129iridoid in-house library to screen out potential iridoid from total ion list using Excel“IF”language.In order to get high-selectivity trigger precursor ion list MS~n fragment,various data acquiring methods had been compared,consequently,the multi-dimensional data acquiring method combined with time-segment and mass-segment mode was used in the fellow research.Then,a strategy of expanding in-house library using predicted metabolite method was constructed to infer potential iridoid structure.Finally,433iridoids were identified in three species of Gentiana,there are 259 in G.scabra,224 in G.manshurica,279 in G.rigescens.3?Based on untargeted metabolomics combined multivariate statistical analysis for screening of chemical markers between G.scabra and G.rigescensIn this part,52 batches G.scabra and 33 batches G.rigescens were systematically compared using untargeted metabolomics with univariate analysis,orthogonal partial least squares discriminant analysis and random forest for screening out the chemical markers which could be applied to accurately distinguish such two species of Gentiana.Firstly,the identification and characterization of chemical components of two species was performed using ultra-high-performance liquid chromatography coupled with LTQ-Orbitrap mass spectrometry.Subsequently,the chemical marker of two species were screened based on metabolomics and multivariate statistical analysis.Totally,87chemical constituents were characterized and identified in G.scabra Bge.(65 chemical constituents)and G.rigescens Franch.(51 chemical constituents),and 29 common chemical constituents were found.Thereafter,11 differential metabolic characteristics which could be used to differentiate two species were acquired with orthogonal partial least squares discriminant analysis(OPLS-DA)and random forest(RF)iterative modeling.Finally 7 characteristics identified as(+)syringaresinol,lutonarin,trifloroside,4-O-?-D-glu-trifloroside,4''-O-?-D-glucopyranosy1-6'-O-(4-O-?-D-glucaffeoyl)linearroside,Macrophylloside A and scabraside were selected as chemical markers for the recognition of two Gentiana species.It was implied that the results could be used to distinguish the Long-Dan derived from different botanical sources.4?Simultaneously determine 9 iridoid constituents in Long-Dan by QAMS methodAn UPLC method in which Gentiopicroside was used as internal standard was elaborated to calculate the relative correction factors between 6-glu-Loganic acid?Loganic acid?Secologanoside?6-glu-Gentiopicroside?Swertiamarin?Sweroside?Rindoside and Trifloroside,meantime,the contents of these iridoid constituents were quantitated.The methodology of 9 iridoid constituents was validated by accuracy,precision,stability,linear range and the applicability of the relative correction factor.The results were acceptable.The quantitative analysis of multi-components of nine iridoid substance in Long-Dan could be achieved by way of the proposed method.At the meantime,the contents of these 9 indexes were calculated by external standard method.Then,compared the results of these methods by standard method difference(SMD%).The outcome showed that there is no significant difference between the result of ESM and QAMS.The developed QAMS method could be applied to quantitate the contents of 9 iridoid constituents in Long-Dan without using any other reference standards in addition to Gentiopicroside.Finally,according to the result of content 9 iridoid constituents,the quality of 30 batches commercial Long-Dan samples were comprehensively evaluated using multi-index decision analysis which is technique for order preference by similarity to ideal solution(TOPSIS).In conclusion the study mentioned above,the innovation of this dissertation are inducted three points:A new targeted profiling strategy for iridoid in Long-Dan was proposed,which is more effective and sensitive.It provided a new reference for the characterization of targeted components in the complex system of traditional chines medicine(TCM).Based on untargeted botanical metabolomics research and multivariate statistical analysis,the 11 chemical marker between G.scabra and G.rigescens were found,which provided a new method for the identification of botanical origin of TCM and a scientific basis for evaluating the rationality of the concurrent use of these two botanical origins of Gentiana Radix et Rhizome.Based on the UPLC platform,an elaborated QAMS method was developed for 9iridoid constituents'quantitative analysis,and on the basis of the results,a comprehensive evaluation of commercial Long-Dan samples was implemented,which constructed a foundation for establishment of a scientific and reasonable standard of Gentiana radix et rhizome quality control. |
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