| The quality control of traditional Chinese medicine and ethnic medicine is closely related to their safety and effectiveness in clinical use. Due to the complex chemical compositions combined with the limitations of instrumental analysis conditions, research ideas and methods, the current quality control methods are not effective for controlling the quality of traditional Chinese medicine and ethnic medicine. At present, in order to establish a suitable quality control method that not only reflects the integrity and systematization, but also has the features of "safety, effectiveness, science, advance and practicality", we must summarize and change the research ideas and methods of quality control for traditional Chinese medicine and ethnic medicine.Traditional Chinese medicine and ethnic medicine mostly originates from various species, which leads to the difficulty for distinguishing the identity and evaluating the quality of crude drugs. Species diversity also seriously hampers the quality control, quality standards development and clinical rational use of medicinal material. Therefore, it is very important to strengthen the research of species identification, quality control and quality evaluation for traditional Chinese medicine and ethnic medicine from various species by using modern science and technology. These studies are necessary to heighten the technological level of traditional Chinese medicine and ethnic medicine, and promote the process of their modernization and industrialization.Purposes:In this paper, Rhizoma Coptidis and "Zangyinchen", two broadly used multi-species medicinal plants, were used as the research objects. The aims of this study were to evaluate the differences of the overall metabolites for the two medicinal materials from various species, and to find out the characteristic metabolic markers by means of1H NMR-based metabolomics. These studies can provide not only a theoretical basis for the species identification, quality control and clinical rational use of Rhizoma Coptidis and "Zangyinchen", but also a new research ideas and methods for the quality control and/or quality evaluation of traditional Chinese medicine and ethnic medicine. Methods:1. A1H NMR-based metabolomics protocol coupled with multivariate statistical analysis (e.g., principal component analysis and partial least squares discriminant analysis) was applied to establish the metabolic fingerprinting of Rhizoma Coptidis, to evaluate the differences of the three species (Coptis chinensis, C. deltoidea and C. teeta) for their primary and secondary metabolites, and to identify potential metabolic markers that could be responsible for the discrimination of the three species.2. A rapid, simple and accurate quantitative nuclear magnetic renonance (qNMR) method was developed for simultaneous determination of berberine, jatrorrhizine, epiberberine, coptisine, palmatine and columbamine in Rhizoma Coptidis. Moreover, paired t-test was performed to compare the quantitative data obtained by the’H NMR with those obtained through a HPLC-DAD method.3. A1H NMR-based metabolomics method coupled with multivariate statistical analysis (e.g., partial least squares discriminant analysis and hierarchical cluster analysis) was applied to simultaneously analyze the overall metabolites of "Zangyinchen", and to study the phylogenetic relationship of the five Swertia species (Swertia chirayita, S. mussotii, S. franchetiana, S. tetraptera and S. elata) and their differences in the primary and secondary metabolites.Results:1. Approximately16metabolites were identified in the methanol extract of Rhizoma Coptidis including berberine, palmatine, coptisine, epiberberine, columbamine, jateorrhizine, magnoflorine, chlorogenic acid, ferulic acid, sucrose, a-glucose, β-glucose, alanine, unsaturated fatty acids, saturated fatty acids and sterols.By combining PCA, PLS-DA and ANOVA results, we found that the40samples of Rhizoma Coptidis could be clearly classified into three groups according to different species, respectively. Palmatine, coptisine, epiberberine, columbamine and fatty acids contents were highest in C. chinensis, whereas C. deltoidea and C. teeta showed the highest levels of sucrose and chlorogenic acid, respectively.2. A quantitative1H NMR method having good linearity, precision, repeatability and accuracy was successfully applied for simultaneous determination of six protoberberine alkaloids in Rhizoma Coptidis. The average recoveries were between96.93%and102.39%with RSD values of less than2.33%for all the six compounds. The results of the paired t-test showed that there are good agreements between the1H NMR and HPLC-DAD methods for their quantitative results.3. A broad range of metabolites belonging to different chemical types were detected from "Zangyinchen" by means of1H NMR spectroscopy, including (1) xanthones:mangiferin,1,8-dihydroxy-3,5-dimethoxy xanthone,1,8-dihydroxy-3,7-dimethoxy xanthone,1-hydroxy-3,7,8-trimethoxy xanthone,1,5,8-trihydroxy-3-methoxy xanthone,7-O-[a-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyl]-1,8-dihydroxy-3-methoxy xanthone;(2) iridoid glycosides:swertiamarin, sweroside, gentiopicrin, loganic acid;(3) pentacyclic triterpenoids:oleanolic acid;(4) flavonoids: isoorientin;(5) amino acids:alanine, valine;(5) sugars:sucrose, a-glucose, β-glucose;(6) others: gallic acid, choline, formic acid, acetic acid, succinic acid, saturated fatty acids, unsaturated fatty acids, sterols.By combining PCA, PLS-DA and ANOVA results, we found that all samples of "Zangyinchen" could be clearly classified into five groups according to different species, respectively. Oleanolic acid, mangiferin, fatty acids and valine contents were highest in S. chirayita, whereas S. tetraptera showed the highest levels of gentiopicroside and choline, S. mussotii showed the highest levels of loganic acid and β-glucose, S. franchetiana showed the highest level of swertiamarin.Conclusions:1. Metabolites of C. chinensis, C. deltoidea and C. teeta have significant differences, and so it was important to show solicitude for their bioequivalence in clinical use. In addition, the critical markers responsible for the discrimination of different Rhizoma Coptidis species were found to be palmatine, coptisine, epiberberine, jateorrhizine, columbamine, berberine, sucrose, fatty acids and chlorogenic acid. Among them, epiberberine and sucrose are the most important metabolic markers as their contents have significant differences between any two of the three species. 2. The qNMR method is simple, fast, accurate and reproducible, which can be applied for the quality control and/or quality evaluation of Rhizoma Coptidis or other herbs containing alkaloids.3. Metabolites of S. chirayita, S. mussotii, S. franchetiana, S. tetraptera and S. elata have significant differences, thus they should not be together as the original plant source of the "Zangyinchen". The critical markers responsible for the discrimination of different "Zangyinchen" species were found to be oleanolic acid, mangiferin, swertiamarin, gentiopicroside, loganic acid, fatty acids, valine, choline and sugars. These metabolites can be effective as the indicators for the species identification and quality control of "Zangyinchen". In addition, from the chemotaxonomic point of view, S. chirayita has the farthest phylogenetic relationship with the other four Swertia. whereas S. mussotii has a closer phylogenetic relationship with S. tetraptera and S. franchetiana has a closer phylogenetic relationship with S. elata.Innovations:1. In this study, by integration of multidisciplinary theories and techniques, we applied the1H NMR-based metabolomic technologies and analysis methods to study the species identification, quality evaluation and chemical taxonomy of multi-species traditional Chinese medicine and ethnic medicine from the perspective of the overall metabolome.2. This paper has established the metabolic fingerprinting of Rhizoma Coptidis. In addition, by the combination of chemometric methods, we studied the difference of primary and secondary metabolites of the three species (C. chinensis, C. deltoidea and C. teeta) from the metabolome level for the first time.3. Quantitative1H NMR (qNMR) method was first developed for simultaneous determination of berberine, jatrorrhizine, epiberberine, coptisine, palmatine and columbamine in Rhizoma Coptidis, which provides a new mean for the quality control and/or quality evaluation of Rhizoma Coptidis. 4. The overall metabolites present in "Zangyinchen" were simultaneously detected for the first time including xanthones and their glycosides, pentacyclic triterpenoids, iridoid glycosides, flavonoids, sugars, amino acids and organic acids. Among them, sucrose, choline, formic acid, acetic acid and succinic acid were reported for the first time in "Zangyinchen"... |
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