Discovery and validation of COX2 as a target of flavonoids in Apocyni Veneti Folium: Implications for the treatment of liver injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Apocyni Veneti Folium (AVF), a popular traditional Chinese medicine (TCM), is known for its effects in soothing the liver and nerves and eliminating heat and water. It is relevant from an ethnopharmacological perspective. Pharmacological research has confirmed its benefits on antihypertension, antihyperlipidemia, antidepression, liver protection, immune system boosting, antiaging, and diabetic vascular lesions. Previous studies have shown that flavonoids, the active ingredients, have a hepatoprotective effect. However, the exact mechanism has not been clarified. AIM OF THE STUDY: This study aimed to identify the active flavonoids in AVF and their corresponding targets for liver injury. Multiple methods were introduced to confirm the targets. MATERIAL AND METHODS: AVF compounds were analyzed using liquid chromatography-mass spectrometry (LC-MS). Then, network pharmacology was utilized to screen potential hepatoprotection targets of the compounds. An enzyme activity assay was performed to determine the effect of the compounds on the targets. Biolayer interferometry (BLI) was applied to confirm the direct interaction between the compounds and the targets. RESULTS: A total of 71 compounds were identified by LC-MS and 19 compounds and 112 shared targets were screened using network pharmacology. These common targets were primarily involved in the TNF signaling pathway, cancer pathways, hepatitis B, drug responses, and negative regulation of the apoptotic process. Flavonoids were the primary pharmacological substance basis of AVF. The cyclooxygenase 2 (COX2) protein was one of the direct targets of flavonoids in AVF. The enzyme activity assay and BLI-based intermolecular interactions demonstrated that the compounds astragalin, isoquercitrin, and hyperoside exhibited stronger inhibition of enzyme activity and a higher affinity with COX2 compared to epigallocatechin, quercetin, and catechin. CONCLUSIONS: COX2 was preliminarily identified as a target of flavonoids, and the mechanism of the hepatoprotective effect of AVF might be linked to flavonoids inhibiting the activity of COX2. The findings can establish the foundation for future research on the traditional hepatoprotective effect of AVF on the liver and for clinical studies on liver disorders.

A study for quality evaluation of Lysimachiae herba from different origins based on fingerprint-activity relationship modeling and multi-component content determination.

ETHNOPHARMACOLOGICAL RELEVANCE: Lysimachiae Herba (LH), called Jinqiancao in Chinese, is a commonly used traditional Chinese medicine in clinical practice. Doctors in the Qing Dynasty recorded that it tastes bitter, sour, and slightly cold, and it belongs to the liver, gallbladder, kidney, and bladder meridians. It has the effects of removing dampness and jaundice, eliminating gallstones, and reducing blood stasis. Because of its potent pharmacological effects, it is extensively utilized in the treatment of hepatobiliary and urinary system stones, jaundice, hepatitis, and cholecystitis. Although LH is included in "Sichuan authentic Chinese herbal medicine records", the quality of it from different origins still lacks reliable evaluation methods, which is difficult to reflect the high quality of LH from Sichuan. AIMS OF THE STUDY: This study aimed to establish a fingerprint-activity relationship model between the fingerprint of LH and its protective effect on cholestatic liver injury, and to evaluate the quality of LH from Sichuan and Guizhou by multivariate statistical analysis. MATERIALS AND METHODS: 20 batches of LH samples were collected from Sichuan and Guizhou. Characteristic fingerprints of samples were established by UHPLC-Triple TOF-MS/MS and the chemical pattern recognition analysis was carried out by HCA. Then, a rat model of cholestatic liver injury was established by intragastric administration of ANIT. Combined with the common peak information of fingerprint and pharmacodynamic index results, GCA and BCA were used to screen the efficacy markers. Finally, based on UHPLC-QTRAP-MS/MS, the content of efficacy markers was simultaneously determined, and the overall quality of LH from two origins was evaluated by PCA and TOPSIS. RESULTS: In the fingerprint of 20 batches of LH, 15 common peaks were identified in the negative ion mode, and the similarity was between 0.887 and 0.981. Pharmacological results showed that, compared with the control group, the content of AST, ALT, ALP, TBA, TBIL, and MDA in serum increased, and the content of GSH and SOD activity decreased after 48 h of ANIT administration. In addition, compared to the model group, different doses of LH from the two origins could decrease the serum levels of AST, ALT, ALP, TBA, TBIL, and MDA, raise the levels of GSH and SOD activity, reduce the infiltration range of inflammatory cells, and improve the cholestatic liver injury in rats. Among them, the pharmacodynamic indices of the SCHD group were significantly better. GCA and BCA showed that a total of 7 constituents related to the efficacy were screened, which were proanthocyanidin B1, ferulic acid, hyperoside, astragalin, nicotiflorin, afzelin, and kaempferol. Besides, the content of 7 active constituents in samples from Sichuan was higher than that from Guizhou, indicating that the quality of samples from Sichuan may be better, consistent with the result of the pharmacological experiment. CONCLUSION: The quality and efficacy of LH from different origins were stable, and all of them had protective effects on cholestatic liver injury in rats. The method established in this study is accurate and reliable, and it can be used to comprehensively evaluate the internal quality of LH.

[Quality evaluation of Lysimachiae Herba from different habitats based on simultaneous determination of multiple bioactive constituents combined with multivariate statistical analysis].

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.

[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.

Metabolite Profiling and Transcriptome Analysis Explain the Difference in Accumulation of Bioactive Constituents in Taxilli Herba from Two Hosts.

Taxilli Herba (TH) is a semi-parasitic herb and the host is a key factor affecting its quality. Flavonoids are the main bioactive constituents in TH. However, studies on the difference in accumulation of flavonoids in TH from different hosts are vacant. In this study, integrated transcriptomic and metabolomic analyses were performed on TH from Morus alba L. (SS) and Liquidambar formosana Hance (FXS) to investigate the relationship between the regulation of gene expression and the accumulation of bioactive constituents. The results showed that a total of 3319 differentially expressed genes (DEGs) were screened in transcriptomic analysis, including 1726 up-regulated genes and 1547 down-regulated genes. In addition, 81 compounds were identified using ultra-fast performance liquid chromatography coupled with triple quadrupole-time of flight ion trap tandem mass spectrometry (UFLC-Triple TOF-MS/MS) analysis, and the relative contents of flavonol aglycones and glycosides were higher in TH from SS group than those from the FXS group. A putative biosynthesis network of flavonoids was created, combined with structural genes, and the expression patterns of genes were mostly consistent with the variation of bioactive constituents. It was noteworthy that the UDP-glycosyltransferase genes might participate in downstream flavonoid glycosides synthesis. The findings of this work will provide a new way to understand the quality formation of TH from the aspects of metabolite changes and molecular mechanism.

Decreased Sirt3 contributes to cyclic production of reactive oxygen species and islet ß-cell apoptosis in high glucose conditions.

BACKGROUND: Reactive oxygen species (ROS) plays a vital role in the apoptosis of islet ß-cells in type 2 diabetes mellitus (T2DM). Sirt3 (Sirtuin 3, a deacetylase) and FoxO1 (a transcription factor) might be involved in ROS production. This study was to investigate mechanism of ROS production and ß-cell apoptosis in T2DM. METHODS: Oxidative stress and apoptosis in islets of db/db mice and high glucose cultured ß-cells were observed by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay and western blotting. Then, H2O2 was used to ascertain the effect of ROS on the expression of Sirt3. Meanwhile, FoxO1, antioxidant enzymes - catalase (CAT) and manganese superoxide dismutase (MnSOD) and ß-cell apoptosis were also determined by western blotting. Finally, Sirt3 was knocked down to evaluate the effect on oxidative stress and apoptosis of ß-cells. RESULTS: Under high glucose environment, enhanced ROS made a decrease of Sirt3 expression, which increased acetylation of FoxO1, thus reduced the expression of its target proteins -MnSOD and CAT, and further significantly increased ROS levels. Increased ROS finally led to the apoptosis of ß-cells. CONCLUSION: Down-regulation of Sirt3 plays an important role in the cyclic production of ROS and ß-cell apoptosis. Targeting Sirt3 may be favorable for the treatment of T2DM.

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.

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.

[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.

Comparative transcriptome analysis reveals variations of bioactive constituents in Lonicera japonica flowers under salt stress.

Lonicera japonica flowers (LJF) is a traditional Chinese medicine packed with phenols constituents and widely used in the treatments of various diseases throughout the world. However, there is still very little known on how LJF identifies and resists salt stress. Here in, we systematically investigated the effect of salt on the phenotypic, metabolite, and transcriptomic in LJF. During long term stress (35 days), 1055 differential expression genes (DEGs) involved in the biosynthesis of secondary metabolites were screened through transcriptome analysis, among which the candidate genes and pathways involved in phenols biosynthesis were highlighted; and performed by phylogenetic tree analysis and multiple nucleotide sequence alignment. Ninety compounds were identified and their relative levels were compared between the control and stressed groups based on the LC-MS analysis, Putative biosynthesis networks of phenolic acid and flavonoid were con-structed with structural DEGs. Strikingly, the expression patterns of structural DEGs were mostly consistent with the variations of phenols under salt stress. Notably, the upregulation of UDP-glycosyl transferases under salt stress indicated post-modification of glycosyl transferases may participate in downstream flavonoids synthesis. This study reveals the relationships of the gene regulation and the phenols biosynthesis in LJF under salt stress, paving the way for the use of gene-specific expression to improve the yield of biocomponent.

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.

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.

Metabolite Profiling and Transcriptome Analysis Explains Difference in Accumulation of Bioactive Constituents in Licorice (Glycyrrhiza uralensis) Under Salt Stress.

Salinity stress significantly affects the contents of bioactive constituents in licorice Glycyrrhiza uralensis. To elucidate the molecular mechanism underlying the difference in the accumulation of these constituents under sodium chloride (NaCl, salt) stress, licorice seedlings were treated with NaCl and then subjected to an integrated transcriptomic and metabolite profiling analysis. The transcriptomic analysis results identified 3,664 differentially expressed genes (DEGs) including transcription factor family MYB and basic helix-loop-helix (bHLH). Most DEGs were involved in flavonoid and terpenoid biosynthesis pathways. In addition, 121 compounds including a triterpenoid and five classes of flavonoids (isoflavone, flavone, flavanone, isoflavan, and chalcone) were identified, and their relative levels were compared between the stressed and control groups using data from the ultrafast liquid chromatography (UFLC)-triple quadrupole-time of flight-tandem mass spectrometry (TOF-MS/MS) analysis. Putative biosynthesis networks of the flavonoids and triterpenoids were created and combined with structural DEGs such as phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase [4CL], cinnamate 4-hydroxylase [C4H], chalcone synthase [CHS], chalcone-flavanone isomerase [CHI], and flavonoid-3',5' hydroxylase (F3',5'H) for flavonoids, and CYP88D6 and CYP72A154 for glycyrrhizin biosynthesis. Notably, significant upregulation of UDP-glycosyltransferase genes (UGT) in salt-stressed licorice indicated that postmodification of glycosyltransferase may participate in downstream biosynthesis of flavonoid glycosides and triterpenoid saponins. Accordingly, the expression trend of the DEGs is positively correlated with the accumulation of glycosides. Our study findings indicate that key DEGs and crucial UGT genes co-regulate flavonoid and saponin biosynthesis in licorice under salt stress.

Quercetin protects islet ß-cells from oxidation-induced apoptosis via Sirt3 in T2DM.

OBJECTIVES: Sirt3 may regulate ROS production and might be involved in ß-cell apoptosis, which plays an important role in the progression of type 2 diabetes mellitus (T2DM). Quercetin is a potent anti-oxidative bioflavonoid, but its effects on T2DM remain to be explored. This study aimed to investigate the effects of quercetin on ß-cell apoptosis and explore its mechanisms. MATERIALS AND METHODS: The effects of quercetin were conducted on db/db mice and INS1 cells. Fasting blood glucose was determined by the colorimetric method, serum insulin was measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, Sirt3 in INS1 cells was knocked down by plasmid transfection. The antioxidant proteins (SOD2 and CAT), apoptosis proteins (cleaved Caspase-3, Bax, and BCL-2), and Sirt3 protein in pancreases and INS1 cells were determined by western blotting. RESULTS: When INS1 cells and diabetic mice were treated with quercetin, the levels of SOD2, CAT, and Sirt3 proteins were increased, the levels of cleaved Caspase-3 and the ratio of Bax to BCL-2 were decreased at different degrees, along with reduced blood glucose levels and elevated insulin levels in diabetic mice. When Sirt3 was knocked down in INS1 cells, increase of two antioxidants and decrease of cell apoptosis generated by quercetin could not occur. CONCLUSION: Quercetin protected islet ß-cells from oxidation-induced apoptosis via Sirt3 in T2DM, which would be beneficial to develop new strategies for preventing ß-cell failure in T2DM.

[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.

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.

Variations in morphology, physiology, and multiple bioactive constituents of Lonicerae Japonicae Flos under salt stress.

Lonicerae Japonicae Flos (LJF) is an important traditional Chinese medicine for the treatment of various ailments and plays a vital role in improving global human health. However, as unable to escape from adversity, the quality of sessile organisms is dramatically affected by salt stress. To systematically explore the quality formation of LJF in morphology, physiology, and bioactive constituents' response to multiple levels of salt stress, UFLC-QTRAP-MS/MS and multivariate statistical analysis were performed. Lonicera japonica Thunb. was planted in pots and placed in the field, then harvested after 35 days under salt stress. Indexes of growth, photosynthetic pigments, osmolytes, lipid peroxidation, and antioxidant enzymes were identified to evaluate the salt tolerance in LJF under different salt stresses (0, 100, 200, and 300 mM NaCl). Then, the total accumulation and dynamic variation of 47 bioactive constituents were quantitated. Finally, Partial least squares discrimination analysis and gray relational analysis were performed to systematically cluster, distinguish, and evaluate the samples, respectively. The results showed that 100 mM NaCl induced growth, photosynthetic, antioxidant activities, osmolytes, lipid peroxidation, and multiple bioactive constituents in LJF, which possessed the best quality. Additionally, a positive correlation was found between the accumulation of phenolic acids with antioxidant enzyme activity under salt stress, further confirming that phenolic acids could reduce oxidative damage. This study provides insight into the quality formation and valuable information to improve the LJF medicinal value under salt stress.

Quality evaluation of Lonicerae Japonicae Flos and Lonicerae Flos based on simultaneous determination of multiple bioactive constituents combined with multivariate statistical analysis.

INTRODUCTION: Lonicerae Japonicae Flos (LJF) and Lonicerae Flos (LF) belong to different genera of Caprifoliaceae. They have been historically utilised as herbal medicine to treat various diseases. However, the comprehensive assessment of them still remains a challenge. OBJECTIVE: To develop a comprehensive method of ultra-fast liquid chromatography-tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) coupled with multivariate statistical analysis for the quality evaluation and reveal differential components of LJF and LF. METHODOLOGY: A validated UFLC-QTRAP-MS/MS method was established for simultaneous determination of 50 constituents, including 12 organic acids, 12 flavonoids, 6 iridoids, 3 saponins, 13 amino acids and 4 nucleosides. The obtained data were employed to multivariate statistical analysis. Principal component anlysis (PCA) and partial least squares determinant analysis (PLS-DA) were performed to classify and reveal differential components of samples; grey relational analysis (GRA) was introduced to assess the samples according to the contents of 50 constituents by calculating the relative correlation degree of each sample. RESULTS: Fifty constituents were simultaneously determined of LJF and LF. Based on obtained data, PCA and PLS-DA were easy to distinguish samples and the classification of the samples was related to 11 chemical constituents. GRA implied the quality of LJF was better, and that the flower buds were superior to the flowers. Moreover, organic acids are the main components of samples. CONCLUSION: This study not only established a method of simultaneous determination of multiple bioactive constituents in LJF and LF, but provided comprehensive information on the quality control of them. The developed method is conducive to distinguish orthologues or paralogues of them, and supply the support for "heterologous effects".

Qualitative and quantitative analysis of the major constituents in Spatholobi Caulis by UFLC-Triple TOF-MS/MS and UFLC-QTRAP-MS/MS.

There have been few comprehensive studies on the holistic chemical composition of Spatholobi Caulis (SC) and consequently, the information is lacking for the in-depth study of the major constituents. SC is a kind of widely used traditional Chinese medicine with its xylem and phloem alternately arranged in 3-10 rings, but the relationship of phloem ring number and the quality remains unclear. In this study, the characterization of the major constituents in SC was analyzed by ultra-fast liquid chromatography coupled with triple quadrupole-time of flight tandem mass spectrometry (UFLC-Triple TOF-MS/MS), and the content of 19 flavonoids in SC with different phloem ring numbers was simultaneously determined by ultra-fast liquid chromatography coupled with triple quadrupole-linear ion trap tandem mass spectrometry (UFLC-QTRAP-MS/MS). Correlation analysis was performed to evaluate the quality of SC with different phloem ring numbers according to the content of 19 flavonoids. Results showed that 50 constituents in SC were identified and the fragmentation pathways of different types of compounds were preliminarily deduced by the fragmentation behavior of the 50 constituents. In addition, the content of flavonoids increased with phloem ring number, which demonstrated that the content of flavonoids in SC was positively correlated with the number of phloem rings. Our research will contribute to the variety identification and quality evaluation of SC, and provide a scientific basis for evaluating the quality of medicinal materials based on its appearance and characteristics.

Metabolomics characterizes the metabolic changes of Lonicerae Japonicae Flos under different salt stresses.

Salt stress affects the metabolic homeostasis of medicinal plants. However, medicinal plants are sessile organisms that cannot escape from salt stress. They acclimatize themselves to the stress by reprogramming their metabolic pathways. Lonicerae Japonicae Flos (LJF) with strong antioxidant activity is commonly used in traditional Chinese medicine, tea, and beverage. Nevertheless, the variation of integrated metabolites in LJF under different salt stresses remains unclear. In this study, High Performance Liquid Chromatography tandem triple time-of-flight mass spectrometry (HPLC- triple TOF-MS/MS) coupled with multivariate statistical analysis was applied to comparatively investigate the metabolites changes in LJF under different salt stress (0, 100, 200, 300 mM NaCl). Total 47 differential metabolites were screened from 79 metabolites identified in LJF under different salt stress. Low salt-treated group (100 mM NaCl) appeared to be the best group in terms of relative contents (peak areas) of the wide variety in bioactive components. Additionally, the phenylpropanoid pathway, monoterpenoid biosynthesis, glycolysis, TCA cycle, and alkaloid biosynthesis were disturbed in all salt-stress LJF. The results showed that LJF metabolisms were dramatically induced under salt stress and the quality of LJF was better under low salt stress. The study provides novel insights into the quality assessment of LJF under salt stress and a beneficial framework of knowledge applied to improvement the medicinal value of LJF.