A method based on ultra-high performance liquid chromatography coupled with triple quadrupole linear ion trap-tandem mass spectrometry(UHPLC-QTRAP-MS/MS) was developed for the simultaneous determination of 41 bioactive constituents of flavonoids, organic acids, nucleosides, and amino acids in Lysimachiae Herba. The content of multiple bioactive constituents was compared among the samples from different habitats. The chromatographic separation was performed in a Waters XBridge®C_(18) column(4.6 mm×100 mm, 3.5 μm) at 30 ℃. The gradient elution was performed with 0.4% methanol(A)-formic acid water(B) as the mobile phase at a flow rate of 0.8 mL·min~(-1), and the multiple-reaction monitoring(MRM) mode was adopted. According to the content of 41 constituents, hierarchical cluster analysis(HCA), orthogonal partial least squares discriminant analysis(OPLS-DA), and gray relational analysis(GRA) were perfomed to comprehensively evaluate the samples from different habitats. The results showed that the 41 constituents exhibited good linear relationship within the tested concentration ranges, with the correlation coefficients(r) greater than 0.999 4. The method featured good precision, repeatability, and stability with the relative standard deviations(RSDs) less than 5.0%. The average recoveries of the 41 constituents ranged from 98.06% to 101.9%, with the RSDs of 0.62%-4.6%. HCA and OPLS-DA separated 48 batches of Lysimachiae Herba samples from different habitats into three categories: the producing areas in Sichuan and Chongqing, the producing areas in Jiangsu, Zhejiang, and Jiangxi, and the producing areas in Guizhou. The content of 41 constituents varied among the Lysimachiae Herba samples from different habitats. The GRA results revealed that the Lysimachiae Herba sample from Nanchong City, Sichuan Province had the best comprehensive quality. The method developed in this study was accurate and reliable and thus can be used for comprehensive evaluation of Lysimachiae Herba quality and provide basic information for the selection of habitats.
The demand for licorice and its natural product derivatives in domestic and foreign market is considerably huge. The core production areas of licorice are covered with salinity and drought land in northwestern China. Studies have shown that suitable environmental stress can promote the accumulation of glycyrrhizin and liquiritin to improve its quality as medicinal materials. However, there are few reports on other bioactive constituents of licorice, not to mention their dynamic accumulation under stressed conditions. To explore the quality formation of licorice from the perspective of salt influence, a reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC–MS/MS) was established for simultaneous determination of sixteen bioactive constituents, including triterpenoids, flavonoids, chalcones and their glycosides. Physiological experiments were performed to investigate salt tolerance of licorice under different salinity treatments. The expressions of crucial genes (bAS and CHS), key enzymes of triterpenoid and flavonoid synthesis, were also tested by qRT-PCR. Our study found that 50 mM NaCl treatment (low stress) was the most favorable to promote the accumulation of bioactive constituents in the long term, without harming the plants. Flavonoid accumulation of non-stressed and low-stressed groups became different in the initial synthesis stage, and glycosyltransferases may have great influence on their downstream synthesis. Furthermore, bAS and CHS also showed higher levels in low-stressed licorice at harvest time. This work provides valuable information on dynamic variations in multiple bioactive constituents in licorice treated by salt and insight into its quality formation under stressed conditions.
A comprehensive analytical method based on ultra-fast liquid chromatography coupled with triple quadrupole/linear ion trap tandem mass spectrometry(UFLC-QTRAP-MS/MS) was established for simultaneous determination of the content of 45 bioactive constituents including flavonoids, alkaloids, amino acids, phenolic acids, and nucleosides in Epimedium brevicornum. The multiple bioactive constituents in leaves, petioles, stems and rhizomes of E. brevicornum were analyzed. The gradient elution was performed at 30 ℃ in an XBridge~® C_(18) column(4.6 mm×100 mm, 3.5 μm) with 0.4% formic acid aqueous solution-acetonitrile as the mobile phase at a flow rate of 0.8 mL·min~(-1). Single factor experiment and response surface methodology were employed to optimize the extraction conditions. Multivariate statistical analyses including systematic cluster analysis(SCA), principal component analysis(PCA), partial least squares discriminant analysis(PLS-DA), and one-way analysis of variance(One-way ANOVA) were carried out to classify the samples from different parts and identify different constituents. Grey relation analysis(GRA) and entropy weight-TOPSIS analysis were performed to build a multi-index comprehensive evaluation model for different parts of E. brevicornum. The results showed that there was a good relationship between the mass concentrations of 45 constituents and the corresponding peak areas, with the correlation coefficients(r) not less than 0.999 0. The precision, repeatability, and stability of the established method were good for all the target constituents in this study, with the relative standard deviations(RSDs) less than 5.0%(0.62%-4.9%) and the average recovery of 94.51%-105.7%. The above results indicated that the bioactive constituents varied in different parts of E. brevicornum, and the overall quality followed the trend of leaves > petioles > rhizomes > stems. This study verified the rationality of the Chinese Pharmacopoeia(2020 edition) stipulating that the medicinal part of E. brevicornum is the leaf. Moreover, our study indicated that the rhizome had the potential for medicinal development. The established method was accurate and reliable, which can be used to comprehensive evaluate and control the quality of E. brevicornum. This study provides data reference for clarifying the medicinal parts and rationally utilizing the resources of E. brevicornum.
Tetrastigmae Radix (TR) is the tuber of Tetrastigma hemsleyanum Diels et Gilg, which is frequently used in Chinese folklore medicine as a therapeutic food. TR is becoming more popular because of its superior pharmacological properties, including anticancer and antioxidant properties. However, wild-TR (WTR) resources are few in the market, and cultivated-TR (CTR) is progressively replacing WTR as the primary source of commercial herbs. Furthermore, because of its diverse growing environment, CTR may differ from WTR in terms of characteristics, metabolites, and pharmacological actions. There are currently fewer investigations on the two ecotypes, and their distinctions are unclear. As a result, this study used UFLC-Triple TOF-MS/MS to undertake untargeted metabolomics analysis of CTR and WTR, and then used statistical analysis to screen for differential accumulate metabolites (DAMs). KEGG enrichment analysis was performed on the DAMs, and the active pharmaceutical DAMs in the enrichment pathway were measured using UFLC-QTRAP-MS/MS. The results reveal that 152 metabolites of 14 different categories were discovered, with flavonoids and lipids accounting for the majority of the 35 DAMs tested by OPLS-DA. Some lipids were up-regulated in the CTR, whereas flavonoids were up-regulated in the WTR; and 777 pairs of DAM metabolites exhibited significant connection based on Pearson correlation analysis. The mainly enriched pathways were flavone and flavonol biosynthesis, the TCA cycle, galactose metabolism, and others. KEGG enrichment analysis revealed four active pharmaceutical metabolites in the enriched pathway, and demonstrated that their concentrations were greater in WTR than in CTR. Overall, this work used a comprehensive metabolomic analysis to provide a theoretical foundation for resource identification, diet formulation, and pharmacological studies of CTR and WTR.
Forsythiae Fructus (FF, Lianqiao in Chinese) is the dried fruit of Forsythia suspensa (Thunb.) Vahl. As one of the commonly used bulk medicinal materials, FF has been used as a heat-clearing and detoxifying traditional Chinese medicine for the treatment of infectious diseases. Epicarp spots are inherent traits of the FF. Previous reports have suggested that it might be related to the quality of FF, while the accurate correlation between them remains ambiguous. In this study, 32 groups of FF were selected from the same batch, and the information of epicarp spots was converted into spot area index (SAI). Then, the overall quality of FF was evaluated by principal component analysis (PCA) based on the content of 21 major bioactive ingredients. Subsequently, the Pearson correlation analysis was conducted to explore the correlation between SAI and the overall quality of FF. Ingredients related to SAI were identified. The results indicated that a significant negative correlation was shown between the SAI and the overall quality of FF. Six bioactive ingredients, including forsythoside B, p-coumaric acid, galuteolin, hesperidin, baicalin and kaempferol, were negatively correlated with SAI. This research will provide scientific information on the relationship between epicarp spots and the overall quality of FF and facilitate the general quality assessment of FF in a rapid and convenient way.
Taxilli Herba (TH) is a well-known traditional Chinese medicine (TCM) with a wide range of clinical application. However, there is a lack of comprehensive research on its chemical composition in recent years. At the same time, Taxillus chinensis (DC) Danser is a semi parasitic plant with abundant hosts, and its chemical constituents varies due to hosts. In this study, the characterization of chemical constituents in TH was analyzed by ultra-fast liquid chromatography coupled with triple quadrupole-time of flight tandem mass spectrometry (UFLC-Triple TOF-MS/MS). Moreover, partial least squares discriminant analysis (PLS-DA) was applied to reveal the differential constituents in TH from different hosts based on the qualitative information of the chemical constituents. Results showed that 73 constituents in TH were identified or tentatively presumed, including flavonoids, phenolic acids and glycosides, and others; meanwhile, the fragmentation pathways of different types of compounds were preliminarily deduced by the fragmentation behavior of the major constituents. In addition, 23 differential characteristic constituents were screened based on variable importance in projection (VIP) and p-value. Among them, quercetin 3-O-β-D-glucuronide, quercitrin and hyperoside were common differential constituents. Our research will contribute to comprehensive evaluation and intrinsic quality control of TH, and provide a scientific basis for the variety identification of medicinal materials from different hosts.
Background: Licorice is an herbal medicine applied extensively worldwide, and most of the licorice for clinical consumption is provided by Glycyrrhiza uralensis Fisch. Evidence suggests that there is a significant difference in the metabolite composition of licorice from different ecotypes. Objective: To better understand the proteomic changes and molecular mechanisms of metabolite formation in wild and cultivated Glycyrrhiza uralensis Fisch. Methods: Firstly, we established a proteome database by annotating protein sequences according to the genomic and transcriptomic data of G. uralensis. Then, iTRAQ and LC-MS/MS were applied to detect significant protein changes between cultivated and wild G. uralensis. A total of 2751 validated proteins were obtained with high confidence, and 333 were differentially expressed. Differentially expressed proteins were identified and analysed by GO, KEGG, and STRING for network and pathway enrichment. Ultimately, we combined the iTRAQ results with our previous investigation on metabolites to understand the molecular mechanisms underlying metabolite accumulation. Results: The results showed that differentially expressed proteins were mainly involved in the anabolism of carbohydrates and important amino acids that participate in primary metabolism and secondary metabolite synthesis. Another important pathway is the synthesis of flavonoids, which are generally accepted as important bioactive constituents of G. uralensis, and the accumulation of flavonoids in different synthesis stages in two ecotypes of G. uralensis was diverse. Therefore, the differentially abundant proteins in wild and cultivated G. uralensis possibly resulted in differences in medicinal compounds. Conclusion: Our study will provide novel clues for revealing the molecular mechanism of secondary metabolite synthesis as well as quality formation in wild and cultivated G. uralensis.
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