| Phytochemical diversity is the fundamental substance of therapeutic effect of traditional Chinese medicine(TCM) and important materials for complementing the search for new drugs. The therapeutic effects of TCM are commonly attributed to synergistic effect of abundant metabolites rather than a single compound. Additionally, the environment and genotype have great influence on accumulation and distribution of phytochemical ingredients, which indicated that single compound could not reflect the holistic quality of TCM. Chemical fingerprints, the well-established tools for quality evaluation, are focused on integrating complex chemical characterization of biological samples. Integrated with with chemometrics, it was widely used for diverse applications such as quality control, differentiation of geographical region, and activity screening.In this study, ultra-fast liquid chromatography tandem mass spectrometry(UFLC-MS/MS) and Fourier transform mid-infrared spectroscopy(FT-MIR) combined with chemometrics were used for screening the marker metabolites and monitoring quality variations of Gentiana rigescens Franch. ex Hemsl. Meanwhlie, comparative analysis was performed on G. rigescens and other Gentianaceae species. The results provided reasonable foundation for exploitation and utilization of the selected Gentianaceae species.This dissertation is composed of four sections. Sections 1 including chapter 2 and charpter 3 is about the development of methods for analysis of characteristic ingredients. Section 2 including chapter 4 and charpter 5 is about the exploration of similarities and differences between G. rigescens and other Gentianaceae species. The comparative analysis of G. rigescens and other Gentianaceae species have been reported in section 3(chapter 5-7). Sections 4 is a review about the 593 chemical constituents of 60 Gentiana species reported between 1960 and 2013(152 references).(1) Gentisides A–K, natural products isolated from G. rigescens with an alkyl 2, 3-dihydroxybenzoate nucleus possessing varying alkyl chain lengths and different termini on the alkyl chains(isobutyl or isopropyl groups), were potent inducers of neurite outgrowth on PC12 cells. A simple, sensitive, selective and reliable UPLC-MS/MS method based on multiple reaction monitoring(MRM) acquisition mode was developed and validated for the simultaneous determination and detection of four neu ritogenic compounds(gentisides A, B, J and K) isolated from G. rigescens. In total of nine gentisides were detected in a precursor ion scan by four dignose ions(m/z 153, 135, 109 and 91). The results showed that gentisides mainly distribute in root and the contents of four gentisides were inconsistent in samples from different geographical regions.(2) A rapid ultra-fast liquid chromatography ultraviolet method was developed for simultaneous determination of four major iridoid glycosides in G. rigescens. Response surface methodology based on the Box-Behnken design was applied to optimizing the extraction conditions of iridoid glycosides. The optimal extraction conditions were as follows: methanol concentration, 80%; the ratio of solid material to liquid, 1:70(g/m L); and extraction time, 32 minutes. The yield of the four iridoid glycosides under the optimal process was found to be 63.08 mg/g, which was consistent with the predicted yield. In addition, the total content of four iridoid glycosides of 50 cultivated samples from Lincang, Yunnan, China, was within the range of 33.6-113.26 mg/g.(3) UFLC-UV-MS/MS and FT-MIR fingerprints with chemometrics could effectively evaluate the quality of G. rigescens under different processed approaches(raw, wine-, vinegar- and salt water-processed). According to the high similarity value of fingerprints, different processing approaches were likely to result in insignificant changes in chemical constituents. However, the contents of the four iridoid glycosides and gentisides decrease upon different processing approaches. Wine-processed samples displayed the highest content of gentiside and salt water-processed approaches could lead to significant decrease in content of metabolites. Combined with PLS-DA and VIPs, eight compounds were selected as potential marker metabolites for quality control of samples under different processed approaches.(4) Fingerprint based on FT-MIR spectroscopy and UFLC-UV-MS/MS were established and complement each other for monitoring the metabolite changes. Different samples were subjected to qualitative and quantitative analysis. The results suggested that metabolites in different parts showed chemical diversity. The contents of iridoid glycosides vary largely among samples from different geographical regions. Moreover, metabolites displayed potential competitive relationship during different growth stages.(5) Four traditional ethnomedicines derived from Gentiana species were subjected to fingerprint analysis. The chromatographic fingerprints analysis combined with principal component analysis showed that the similarity values of the four species were <0.788 while the similarity values of G. scabra and G. rigescens were >0.993, which provided reasonable foundation for utilization and clinical application of the four ethnomedicines.(6) Gentiana rhodantha Franch. ex Hemsl. collected from eleven geographical sites in Yunnan, Guizhong and Guangxi province, were subjected to fingerprint analysis. Combined with PCA, PLS-DA and VIPs principle, 64 samples according to their geographical origins could be classified, and nine compounds including two new compounds identified by mass spectrometry could be regarded as characteristic compounds for discriminating samples from different geographical origins.(7) This charpter systematically characterized the variations of holistic metabolome and characteristic metabolites(iridoid glycosides and phenols) associated with nine Gentianaceae species from Western China using FT-IR, LC-MS/MS and multivariate statistical analysis. The results indicated that the holistic metabolomics in genera Gentiana and Swertia were largely different while significant diversity on the biosynthetic pathway of iridoid glycosides were also observed in these species. PCA based on targeted metabolic profiling showed a clear separation according to infrageneric classifications of genus Gentiana. However, the accumulation of the six major metabolites seems to depend on geographical regions in Sect. Monopodiae and Sect. Cruciata. Furthermore, a possible biosynthetic pathway on iridoid glycosides of Gentianaceae species was also disscussed. |
PDF Full Text Request