[Analysis and evaluation of bioactive constituents from different parts of Epimedium brevicornum].

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.

Qualitative Analysis and Componential Differences of Chemical Constituents in Lysimachiae Herba from Different Habitats (Sichuan Basin) by UFLC-Triple TOF-MS/MS.

Lysimachiae Herba (LH), called Jinqiancao in Chinese, is an authentic medical herb in Sichuan Province often used in the prescription of traditional Chinese medicine (TCM). However, in recent years, there has been a lack of comprehensive research on its chemical components. In addition, the landform of Sichuan Province varies greatly from east to west and the terrain is complex and diverse, which has an important influence on the chemical constituents in LH. In this study, ultrafast liquid chromatography coupled with triple-quadrupole time-of-flight tandem mass spectrometry (UFLC-triple TOF-MS/MS) was used to analyze the samples of LH from eight different habitats in Sichuan Basin. The constituents were identified according to the precise molecular weight, the fragment ions of each chromatographic peak and the retention time of the compound obtained by high-resolution mass spectrometry, combined with software database searches, standard comparisons and the related literature. Differential chemical constituents were screened using partial least squares discriminant analysis (PLS-DA) and t-tests. The results showed that a total of 46 constituents were identified and inferred, including flavonoids, phenolic acids, amino acids, tannins, fatty acids and coumarins; the fragmentation pathways of the main constituents were preliminarily deduced. According to the variable importance in projection (VIP) and p-values, four common differential constituents were screened out, 2-O-galloylgalactaric acid, quercetin 3-O-xylosyl-rutinoside, nicotiflorin and kaempferol 3-rutinosyl 7-O-alpha-l-rhamnoside. This study provides basic information for the establishment of a comprehensive quality evaluation system for LH.

Quality Evaluation of Tetrastigmae Radix from Two Different Habitats Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis.

Tetrastigmae Radix, also known as Sanyeqing (SYQ) in Chinese, is an important traditional Chinese medicine with a long history. Tetrastigma hemsleyanum Diels et Gilg mainly grows in the south of the Yangtze River and is widely distributed. The content of bioactive constituents in SYQ varies greatly in different habitats, and there are obvious differences in the content of bioactive constituents between southwestern SYQ (WS) and southeastern SYQ (ES). To distinguish and evaluate the quality of ES and WS, an analytical method based on ultrafast performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry (UFLC-QTRAP-MS/MS) was established for the simultaneous determination of 60 constituents including 25 flavonoids, 9 phenolic acids, 15 amino acids, and 11 nucleosides in 47 samples from ES and WS. In addition, orthogonal partial least squares discriminant analysis (OPLS-DA), t-test, and gray correlation analysis (GRA) were used to discriminate and evaluate the ES and WS samples based on the contents of 60 constituents. The results showed that there were significant differences in the bioactive constituents between ES and WS, and ES was superior to WS in terms of quality evaluation. This study not only provides basic information for differentiating ES and WS but also provides a new perspective for the comprehensive evaluation and quality control of SYQ from two different habitats.

[Analysis and evaluation of dynamic accumulation of multiple active constituents in Taxilli Herba in Guangxi].

A method based on ultra-high performance liquid chromatography with triple quadrupole/linear ion trap mass spectrometry(UHPLC-QTRAP-MS/MS) was developed for the simultaneous determination of 33 active constituents, including flavonoids, organic acids, nucleosides, and amino acids in Taxilli Herba to analyze and evaluate the dynamic accumulation of their multiple active constituents. The separation was performed at 30 ℃ on an XBridge~® C_(18) column(4.6 mm×100 mm, 3.5 µm) with gradient elution using 0.1% formic acid aqueous solution-methanol as the mobile phase at a flow rate of 0.5 mL·min~(-1), and the injection volume was 2 µL. The constituents were ionized in the electrospray ionization source(ESI) and quantitated by the multiple reaction monitoring(MRM) mode. The entropy weight TOPSIS method was used to objectively assign weights to the target constituents and rank them according to their relative closeness coefficient(C_i) to construct a multi-index comprehensive evaluation model of Taxilli Herba. The results showed that the concentrations and peak areas of 33 target constituents had good linearity in their respective linear ranges, and the correlation coefficients(r) were not less than 0.999 0. The RSD of precision, reproducibility, and stability were not higher than 4.7%. The average recoveries were 98.03%-101.3% with RSD less than 4.0%. There were differences in the content of 33 active constituents in Taxilli Herba at different harvest periods. The overall quality of Taxilli Herba harvested from mid-February to early March was better, which was consistent with the traditional harvest period. This study provides basic information for revealing the rule of dynamic accumulation of multiple active constituents in Taxilli Herba and determining the suitable harvesting period. Meanwhile, it also provides a new methodological reference for the comprehensive evaluation of the intrinsic quality of Taxilli Herba.

Comparison of Multiple Bioactive Constituents in the Corolla and Other Parts of Abelmoschus manihot.

Abelmoschus manihot (L.) Medic (AM), called Huangshukui in Chinese, is a widely used medicinal plant. Each part of AM has medicinal value, including Abelmoschi Radix (AR), Abelmoschi Herba (AH), Abelmoschi Folium (AF), Abelmoschi Corolla (AC), and Abelmoschi Semen (AS). However, only AC is documented in the Chinese Pharmacopoeia. In order to investigate whether there is any difference between AC and the other parts of AM, an analytical method based on ultra-fast performance liquid chromatography coupled with triple quadrupole-linear ion trap mass spectrometry (UFLC-QTRAP-MS/MS) was established for the simultaneous determination of 35 constituents in different parts of AM. Moreover, principal components analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were applied to classify and evaluate the different parts of AM based on the content of the 35 constituents. The total contents of the 35 constituents in AC were significantly higher than in the other parts of AM and the results revealed significant differences between AC and the other parts of AM. Eight constituents were remarkably related to the sample classifications. This research does not just provide the basic information for revealing the distribution patterns in different parts of AM from the same origin, but also complements some of the scientific data for the comprehensive quality evaluation of AC.

Qualitative Analysis and Componential Differences of Chemical Constituents in Taxilli Herba from Different Hosts by UFLC-Triple TOF-MS/MS.

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-ß-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.

Quality Evaluation of Taxilli Herba from Different Hosts Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis.

Taxilli Herba (TAXH) is an important traditional Chinese medicine with a long history, dating from the Eastern Han Dynasty to the present times. However, the active constituents in it that parasitize different hosts vary, affecting its clinical efficacy. Given the complexity of the host origins, evaluating the quality of TAXH is critical to ensure the safety and effectiveness of clinical medication. In the present study, a quantitative method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established, which simultaneously determined the content of 33 active constituents, including 12 flavonoids, 4 organic acids, 12 amino acids, and 5 nucleosides in 45 samples. Orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to classify and distinguish between TAXH and its adulterants, Tolypanthi Herba (TOLH). A hierarchical clustering analysis (HCA) was conducted combined with a heatmap to visually observe the distribution regularity of 33 constituents in each sample. Furthermore, gray relational analysis (GRA) was applied to evaluate the quality of samples to get the optimal host. The results demonstrated that TAXH excelled TOLH in quality as a whole. The quality of TAXH parasitizing Morus alba was also better, while those that were parasitic on Cinnamomum camphora and Glyptostrobus pensilis had relatively poor quality. This study may provide comprehensive information that is necessary for quality control and supply a scientific basis for further exploring the quality formation mechanism of TAXH.

[Simultaneous determination of multiple bioactive constituents in Abelmoschi Corolla by UFLC-QTRAP-MS/MS].

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 38 active components in Abelmoschi Corolla, including flavonoids, organic acids, nucleosides and amino acids, so as to investigate the effects of different harvesting and processing methods on multi-active components in Abelmoschi Corolla. The chromatographic separation was performed on a XBridg®C_(18) column(4.6 mm×100 mm, 3.5 µm) with(0.1% formic acid water) methanol-acetonitrile(1∶1) as the mobile phase for gradient elution at 30 ℃. The flow rate was 0.5 mL·min~(-1). The components were detected in a multiple-reaction monitoring(MRM) mode. The gray relational analysis(GRA) was used to comprehensively evaluate the multiple active components of Abelmoschi Corolla at different harvesting times and drying temperatures. The results showed that 38 components had a good linearity with correlation coefficients all above 0.999 0. The method featured a good precision, repeatability and stability with the relative stan-dard deviations(RSDs) of less than 5.0%. Recoveries ranged from 98.06% to 104.4% with RSD between 0.22% and 4.9%. The results of GRA indicated that a better quality in the samples collected on September 9 th. Samples dried at 90 ℃ had a better quality. The established method is accurate and reliable, and can be used to assess the internal quality of Abelmoschi Corolla. This study can provide basic materials for determining appropriate harvesting time and processing method of Abelmoschi Corolla.

Distribution Patterns for Bioactive Constituents in Pericarp, Stalk and Seed of Forsythiae Fructus.

Forsythiae Fructus (FF) is a widely used folk medicine in China, Japan, and Korea. The distribution of bioactive constituents throughout the fruit segments has rarely been addressed, although mounting evidence suggests that plant secondary metabolites are synthesized and distributed regularly. The phytochemical profiles of three segments of FF (pericarp, stalk and seed) were firstly revealed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantitative analysis of twenty-one bioactive constituents, including three phenylethanoid glycosides, five lignans, eight flavonoids, and five phenolic acids to explore the spatial distribution of bioactive constituents. Furthermore, the hierarchical clustering analysis (HCA) and one-way analysis of variance (one-way ANOVA) were conducted to visualize and verify the distribution regularity of twenty-one analytes among three segments. The results showed that phytochemical profiles of the three segments were similar, i.e., phenylethanoid glycosides covering the most part were the predominant compounds, followed by lignans, flavonoids and phenolic acids. Nevertheless, the abundance of twenty-one bioactive constituents among three segments was different. Specifically, phenylethanoid glycosides were highly expressed in the seed; lignans were primarily enriched in the stalk; flavonoids were largely concentrated in the pericarp, while the contents of phenolic acids showed no much difference among various segments. The research improves our understanding of distribution patterns for bioactive constituents in FF, and also complements some scientific data for further exploring the quality formation mechanism of FF.

A Method to Study the Distribution Patterns for Metabolites in Xylem and Phloem of Spatholobi Caulis.

Spatholobi Caulis (SC), the vine stem of Spatholobus suberectus Dunn, is a widely used traditional Chinese medicine (TCM) for the treatment of blood stasis syndrome and related diseases. Xylem and phloem are the main structures of SC and the color of xylem in SC is red brown or brown while the phloem with resin secretions is reddish brown to dark brown. They are alternately arranged in a plurality of concentric or eccentric rings. In order to investigate the distribution patterns of metabolites in xylem and phloem of SC, an analytical method based on UFLC-QTRAP-MS/MS was established for simultaneous determination of 22 constituents including four flavanols, nine isoflavones, two flavonols, two dihydroflavones, one flavanonol, one chalcone, one pterocarpan, one anthocyanidin and one phenolic acid in the samples (xylem and phloem) from Laos. Furthermore, according to the contents of 22 constituents, heat map, principal components analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and t-test were used to evaluate the samples and discover the differences between xylem and phloem of SC. The results indicated that the measured ingredients in xylem and phloem were significantly different. To be specific, the contents of flavonoids in xylem were higher than that in phloem, while the content of protocatechuic acid showed a contrary tendency. This study will not only reveal the distribution patterns of metabolites in xylem and phloem of SC but also facilitate further study on their quality formation.