An integrated strategy by absorbed component characterization, pharmacokinetics, and activity evaluation for identification of potential nephroprotective substances in Zhu-Ling decoction.

An efficient strategy for the identification of potential nephroprotective substances in Zhu-Ling decoction has been established with the integration of absorbed components characterization, pharmacokinetics, and activity evaluation. A qualitative method was developed to characterize the chemical constituents absorbed components in vivo of Zhu-Ling decoction by using ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. A quantitative method was established and validated for the simultaneous determination of eight compounds in rat plasma by using ultra-performance liquid chromatography-triple quadruple tandem mass spectrometry. Finally, the nephroprotective activities of absorbed components with high exposure were assessed by cell survival rate, superoxide dismutase, and malondialdehyde activities in hydrogen peroxide-induced Vero cells. As a result, 111 compounds in Zhu-Ling decoction and 36 absorbed components were identified in rat plasma and urine, and poricoic acid A, poricoic acid B, alisol A, 16-oxo-alisol A, and dehydro-tumulosic acid had high exposure levels in rat plasma. Finally, poricoic acid B, poricoic acid A, 16-oxo-alisol A, and dehydro-tumulosic acid showed remarkable nephroprotective activity against Vero cells damage induced by hydrogen peroxide. Besides, superoxide dismutase and malondialdehyde activities were obviously regulated in hydrogen peroxide-induced Vero cells by treatment with the four compounds mentioned above. Therefore, these four compounds were considered to be effective substances of Zhu-Ling decoction due to their relatively high exposure in vivo and biological activity. This study provided a chemical basis for the action mechanism of Zhu-Ling decoction in the treatment of chronic kidney diseases.

Chemical and metabolic profiling of Codonopsis Radix extract with an integrated strategy using ultra-high-performance liquid chromatography coupled with mass spectrometry.

Codonopsis radix was commonly used as food materials or herbal medicines in many countries. However, the comprehensive analysis of chemical constituents, and in vivo xenobiotics of Codonopsis radix remain unclear. In the present study, an integrated strategy with feature-based molecular networking using ultra-high-performance liquid chromatography coupled with mass spectrometry was established to systematically screen the chemical constituents and the in vivo xenobiotics of Codonopsis radix. A step-by-step manner based on a composition database, visual structure classification, discriminant ions, and metabolite software prediction was proposed to overcome the complexities due to the similar structure of chemical constituents and metabolites of Codonopsis radix. As a result, 103 compounds were tentatively characterized, 20 of which were identified by reference standards. Besides, a total of 50 xenobiotics were detected in vivo, including 26 prototypes and 24 metabolites, while the metabolic features of the pyrrolidine alkaloids were elucidated for the first time. The metabolism reactions of pyrrolidine alkaloids and sesquiterpene lactones included oxidation, methylation, hydration, hydrogenation, demethylation, glucuronidation, and sulfation. This study provided a generally applicable approach to the comprehensive investigation of the chemical and metabolic profile of traditional Chinese medicine and offered reasonable guidelines for further screening of quality control indicators and pharmacodynamics mechanism of Codonopsis radix.

Simultaneous determination of multiple constituents of Qi-Lin pill by UPLC-MS/MS: Applications to pharmacokinetics and testicular tissue distribution in rats.

Qi-Lin pill (QLP) is an effective traditional Chinese medicine prescription (TCMP) that has been used for the treatment of the oligoasthenozoospermia in China. Recently, some articles described the pharmacological effects of QLP and multiple ingredients in QLP contribute to its effects. However, the pharmacokinetic and target tissue distribution data of QLP are still unknown. In the present study, according to the Bioanalytical Method Validation Guidance of FDA, a sensitive and selective UPLC-MS/MS method was developed and validated for simultaneous determination of multiple constituents in rat plasma and testicular tissue, including morusimic acid A, codonopyrridium B, magnoflorine, emodin, 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG), ecliptasaponin A, paeoniflorin, albiflorin, gallic acid, danshensu, salvianolic acid A, catechin, isosinensetin, nobiletin, formononetin, calycosin, icariside II, icariin and epimedin C. For 19 analytes, the LLOQs reached 0.01-4 ng/mL. And all calibration curves showed favorable linearity (r ≥ 0.9903) in linear ranges. The intra-day and inter-day precision (relative standard deviation) for all analytes was less than 14.92 %, and the accuracies (as relative error) were in the range of - 6.44 % to 6.22 %. Extraction recoveries and matrix effects of analytes and IS were acceptable. All analytes were stable during the assay and storage in plasma samples. The method was successfully applied for the pharmacokinetics and testis distribution of multiple chemical constituents in QLP after a single oral dose. As a result, high exposure of danshensu, gallic acid, paeoniflorin and albiflorin were observed in rat plasma and testicular tissue. Among the flavonoids, isosinensetin and nobiletin had high exposure in testicular tissue. Moreover, alleviation of progesterone reduction was evaluated in H2O2-induced R2C leydig cells, and danshensu, gallic acid, paeoniflorin, albiflorin and nobiletin showed potent activity. Therefore, these five components were considered to be the effective components of QLP due to their relatively high exposure in vivo and biological activity. This finding also provided relevant information on action mechanism of QLP in the treatment of oligoasthenozoospermia.

Pharmacokinetics, hepatic disposition, and heart tissue distribution of 14 compounds in rat after oral administration of Qi-Li-Qiang-Xin capsule via ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry.

In the present study, a specific and sensitive approach using ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry was developed and validated for the quantitative analysis of 14 constituents in rat plasma, liver, and heart. The method was fully validated and successfully applied to pharmacokinetic, hepatic disposition, and heart tissue distribution studies of 14 compounds after the oral administration of Qi-Li-Qiang-Xin capsule. Ginsenoside Rb1, alisol A, astragaloside IV, and periplocymarin were found to be highly exposed in rat plasma, while toxic components such as hypaconitine, mesaconitine, and periplocin had low circulation levels in vivo. Moreover, sinapine thiocyanate, neoline, formononetin, calycosin, and alisol A exhibited significant liver first-pass effects. Notably, high levels of alisol A, periplocymarin, benzoylmesaconine, and benzoylhypaconine were observed in the heart. Based on high exposure and appropriate pharmacokinetic features in the systemic plasma and heart, astragaloside IV, ginsenoside Rb1, periplocymarin, benzoylmesaconine, benzoylhypaconine, and alisol A can be considered as the main potentially effective components. Ultimately, the results provide relevant information for discovery of effective substances, as well as further anti-heart failure action mechanism investigations of Qi-Li-Qiang-Xin capsule.

[Study on metabolic dynamics,metabolic enzyme phenotype and species difference of hepatic and intestinal microsome of psoralidin].

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 µL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 µL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 µL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 µL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 µL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 µL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 µL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 µL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 µL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 µL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 µL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 µL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 µL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.

Effects of Xian-Ling-Gu-Bao capsule on the gut microbiota in ovariectomized rats: Metabolism and modulation.

Xian-Ling-Gu-Bao capsule (XLGB) has been proven to prevent and treat osteoporosis. However, as a long-term oral formula, XLGB's effects on the metabolic capacity, structure and function of gut microbiota have yet to be elucidated in ovariectomized (OVX) rats. Our objectives were to evaluate the capacity of gut microbiota for metabolizing XLGB ingredients and to assess the effect of this prescription on gut microbiota. Herein, an integrated analysis that combined ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and ultrahigh-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-TQD-MS) was conducted to determine the metabolic capacity of gut microbiota. The effects of XLGB on gut microbiota were explored by metagenomic sequencing in OVX rats. Fecal samples from each group were collected after intragastric administration for three months. In total, 64 biotransformation products were fully characterized with rat gut microbiota from the OVX group and the XLGB group. The deglycosylation reaction was the main biotransformation pathway in core structures in the group that was incubated with XLGB. Compared with the OVX group, different biotransformation products and pathways of the XLGB group after incubation for 2 h and 8 h were described. After three months of feeding with XLGB, the domesticated gut microbiota was conducive to the production of active absorbed components via deglycosylation, such as icaritin, psoralen and isopsoralen. Comparisons of the gut microbiota of the OVX and XLGB groups showed differences in the relative abundances of the two dominant bacterial divisions, namely, Firmicutes and Bacteroidetes. The proportion of Firmicutes was significantly lower and that of Bacteroidetes was significantly higher in the XLGB group. This result demonstrated that XLGB could provide a basis for the treatment of osteoporosis by regulating lipid and bile acid metabolism. In addition, the increase in Lactobacillus, Bacteroides and Prevotella could be an important factor that led to easier production of active absorbed aglycones in the XLGB group. Our observation provided further evidence of the importance of gut microbiota in the metabolism and potential activity of XLGB.

Discovery of potential Q-marker of traditional Chinese medicine based on plant metabolomics and network pharmacology: Periplocae Cortex as an example.

BACKGROUND: Quality control exerted great importance on the clinical application of drugs for ensuring effectiveness and safety. Due to chemical complexity, diversity among different producing areas and harvest seasons, as well as unintentionally mixed with non-medicinal parts, the current quality standards of traditional Chinese medicine (TCM) still faced challenges in evaluating the overall chemical consistency. PURPOSE: We aimed to develop a new strategy to discover potential quality marker (Q-marker) of TCM by integrating plant metabolomics and network pharmacology, using Periplocae Cortex (GP, the dried root bark of Periploca sepium Bge.) as an example. METHODS: First, plant metabolomics analysis was performed by UPLC/Q-TOF MS in 89 batches of samples to discover chemical markers to distinguish medicinal parts (GP) and non-medicinal parts (the dried stem bark of Periploca sepium Bge. (JP)), harvest seasons and producing region of Periplocae Cortex. Second, network pharmacology was applied to explore the initial linkages among chemical constituents, targets and diseases. Last, potential Q-marker were selected by integrating analysis of plant metabolomics and network pharmacology, and the quantification method of Q-marker was developed by using UPLC-TQ-MS. RESULTS: The chemical profiling of GP and JP was investigated. Fifteen distinguishing features were designated as core chemical markers to distinguish GP and JP. Besides, the content of 4-methoxybenzaldehyde-2-O-ß-d-xylopyranosyl-(1→6)-ß-d-glucopyranoside could be used to identify Periplocae Cortex harvested in spring-autumn or summer. Meanwhile, a total of 15 components targeted rheumatoid arthritis were screened out based on network pharmacology. Taking absorbed constituents into consideration, 23 constituents were selected as potential Q-marker. A simultaneous quantification method (together with 11 semi-quantitative analysis) was developed and applied to the analysis of 20 batches of commercial Periplocae Cortex on the market. The PLS-DA model was successfully developed to distinguish GP and JP samples. In addition, the artificially mixed GP sample, which contained no less than 10% of the adulterant (JP), could also be correctly identified. CONCLUSION: Our results indicated that 9 ingredients could be considered as Q-marker of Periplocae Cortex. This study has also demonstrated that the plant metabolomics and network pharmacology could be used as an effective approach for discovering Q-marker of TCM to fulfill the evaluation of overall chemical consistency among samples from different producing areas, harvest seasons, and even those commercial crude drugs, which might be mixed with a small amount of non-medicinal parts.

Discovery of anti-flu substances and mechanism of Shuang-Huang-Lian water extract based on serum pharmaco-chemistry and network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuang-Huang-Lian preparation has captured wide attention since its clinical applications for the successful treatment of upper respiratory tract infection. However, its functional basis under actual therapeutic dose in vivo was still unrevealed. AIM OF THE STUDY: This study aimed to reveal the anti-flu substances and mechanism of Shuang-Huang-Lian water extract (SHL) on H1N1 infected mouse model by a strategy based on serum pharmaco-chemistry under actual therapeutic dose and network pharmacology. MATERIALS AND METHODS: H1N1 infected mouse model was employed for evaluation of the anti-flu effects of SHL. A simultaneous quantification method was developed by UPLC-TQ-XS MS coupled switch-ions mode and applied to characterize the pharmacokinetics of the multiple components of SHL under actual therapeutic dose. The potential active ingredients were screened out based on their pharmacokinetic parameters. And then, a compound mixture of these active candidates was re-evaluated for the anti-flu activity on H1N1 infected mouse model. Furthermore, the anti-flu mechanism of SHL was also predicted by network pharmacology coupled with the experimental result. RESULTS: SHL significantly increased the survival rate and prolonged survival days on H1N1 infected mice at a dosage of 20 g crude drug/kg/day by reversing the increased lung index, down-regulating the inflammatory cytokines (TNF-α, IL-1ß, IL-6) and inhibiting the release of IFN-ß in bronchoalveolar lavage fluids (BALF). Concomitantly, the pharmacokinetic parameters of fourteen quantified and twenty-one semi-quantified constituents of SHL were characterized. And then, five compounds (baicalin, sweroside, chlorogenic acid, forsythoside A and phillyrin), which displayed satisfactory pharmacokinetic features, were considered as potential active ingredients. Thus, a mixture of these five ingredients was administered to H1N1-infected mice at a dose of 4.24 mg/kg/day. As a result, the therapeutical effects of the mixture were similar to SHL in terms of survival rate, lung index and the release of cytokines (TNF-α, IL-1ß and IL-6) in BALF. Moreover, network pharmacology analysis indicated that the TNF-signal pathways might play a role in the anti-flu mechanism of SHL. CONCLUSIONS: A mixture of five compounds (baicalin, sweroside, chlorogenic acid, forsythoside A and phillyrin) were the anti-flu substances of SHL. The strategy based on serum pharmaco-chemistry under actual therapeutic dose provided a new sight on exploring in vivo effective substances of TCM.

Characterization of lignans in Forsythiae Fructus and their metabolites in rats by ultra-performance liquid chromatography coupled time-of-flight mass spectrometry.

OBJECTIVES: This study was designed to profile the chemical information of Forsythiae Fructus (FF) and investigate the in-vivo FF-related xenobiotics, especially for lignans. METHODS: Rats were oral administrated of FF and pinoresinol-4-O-glucoside, respectively. Blood and urine samples were collected after ingestion, and xenobiotics was profiled by an UPLC/Qtof MS method. KEY FINDINGS: A total of 19 lignans were identified or tentatively characterized in FF, and 63 lignan-related xenobiotics were found in rat plasma and urine after ingestion of FF. It was found that lignans could be transformed into metabolites by furan ring opening, hydrogenation, demethylation, dehydration and phase II reactions (sulfation and glucuronidation). The whole metabolic behaviour of bisepoxylignan was revealed by evaluating the metabolism of pinoresinol-4-O-glucoside in vivo. It was found that the configuration of C-8/C-8' was retained after furan ring opening and metabolic reactions always occurred at position of C-3/C-4/C-5 or C-3'/C-4'/C-5'. Additionally, other types components in FF and in vivo were also characterized. CONCLUSIONS: This work revealed the in-vivo metabolism of FF, and reported the characteristic metabolic reactions of lignans for the first time. It was also provided the foundation for the further investigation on pharmacodynamic components of FF or TCMs containing FF.

Identification and characterization of chemical constituents in Qi-Lin pills and their metabolites in rat bio-samples after oral administration using ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

Qi-Lin pill (QLP), a traditional Chinese medicine prescription (TCMP), composed of fifteen herbal medicines, has been widely used for the treatment of male infertility. However, an in-depth understanding of the chemical constituents of QLP and its in vivo metabolic study is lacking. In this study, a method using ultra-performance liquid chromatography coupled with quadruple time-of-flight mass spectrometry (UPLC/Q-TOF-MS) was established for comprehensive analysis of chemical constituents of QLP and their metabolites in plasma, urine, bile and feces after gastric perfusion. The method guaranteed the fast discovery of representative structural fragment information and provided efficient structure clues for identification based on data from MSE mode. As a result, a total of 202 constituents were unambiguously identified or tentatively characterized. In addition, a total of 203 QLP-related xenobiotics were characterized, including 41 (22 prototypes and 19 metabolites) in plasma, 144 (47 prototypes and 97 metabolites) in urine, 50 (27 prototypes and 23 metabolites) in bile and 68 (51 prototypes and 17 metabolites) in feces. The metabolism reactions included phase I reactions (demethylation, hydroxylation, deglycosylation, deoxygenation, hydrogenation, dehydration, oxidation and hydrolysis) and phase II reactions (methylation, conjugation with glucuronide and sulfate). This was the first comprehensive investigation on chemical constituents and metabolic profiles of QLP in vivo, and the results provided chemical foundation for further research on effective substances and action mechanism of QLP.

Simultaneous Quantitative Analysis of Multiple Biotransformation Products of Xian-Ling-Gu-Bao, a Traditional Chinese Medicine Prescription, with Rat Intestinal Microflora by Ultra-Performance Liquid Chromatography Tandem Triple Quadrupole Mass Spectrometry.

Xian-Ling-Gu-Bao (XLGB), a famous traditional Chinese medicine prescription consisted of six herbal medicines, was used for prevention and treatment of osteoporosis in China. As an oral formulation, the multiple components contained in XLGB were inevitably biotransformed by the intestinal microflora before absorption via the gastrointestinal tract. However, the dynamic profiles of biotransformation products of XLGB remain unknown. In this paper, a rapid and sensitive ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry method was developed for the simultaneous quantitative analysis of multiple biotransformation products of XLGB with rat intestinal microflora. For 10 selected quantitative compounds, all calibration curves revealed good linearity (r2 > 0.99) within the sampling ranges considered. The whole intra- and inter-day precisions (as relative standard deviation) of all analytes were <13.5%, and the accuracies (as relative error) were in the range from -11.3 to 11.2%. The lower limits of quantification were 20, 10, 5, 20, 2, 2, 2, 5, 2 and 2 ng/mL for sweroside, timosaponin BII, epimedin C, asperosaponin VI, psoralen, isobavachin, icariside II, timosaponin AIII, isobavachalcone and icaritin, respectively. The matrix effects, extraction recoveries and stabilities were all satisfactory. Meanwhile, dynamic profiles of 21 additional biotransformation products were also monitored by their area-time curves. The analytical method was successfully applied to describe dynamic profiles of 31 biotransformation products of XLGB and the recipes with removal of a definite composed herbal medicine (Anemarrhenae Rhizoma or Rehmanniae Radix).

Characterization of chemical components of Periplocae Cortex and their metabolites in rats using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Periplocae Cortex, named Xiang-Jia-Pi in China, has been widely used to treat autoimmune diseases, especially rheumatoid arthritis. However, the in vivo substances of Periplocae Cortex remain unknown yet. In this study, an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was used for profiling the chemical components and related metabolites of Periplocae Cortex. A total of 98 constituents were identified or tentatively characterized in Periplocae Cortex: 42 C21 steroidal glycosides, 10 cardiac glycosides, 23 organic acids, 4 aldehydes, 7 triterpenes, and 12 other types. Among them, 18 components were unambiguously identified by comparison with reference standards. In addition, 176 related xenobiotics (34 prototypes and 142 metabolites) were screened out and characterized in rats' biosamples (plasma, urine, bile, and feces) after the oral administration of Periplocae Cortex. Moreover, the metabolic fate of periplocoside S-4a, a C21 steroidal glycoside, was proposed for the first time. In summary, phase II reactions (methylation, glucuronidation, and sulfation), phase I reactions (hydrolysis reactions, oxygenation, and reduction), and their combinations were the predominant metabolic reactions of Periplocae Cortex in rat. It is the first report to reveal the in vivo substances and metabolism feature of Periplocae Cortex. This study also provided meaningful information for further pharmacodynamics study of Periplocae Cortex, as well as its quality control research.

Fuziline alleviates isoproterenol-induced myocardial injury by inhibiting ROS-triggered endoplasmic reticulum stress via PERK/eIF2α/ATF4/Chop pathway.

Fuziline, an aminoalcohol-diterpenoid alkaloid derived from Aconiti lateralis radix preparata, has been reported to have a cardioprotective activity in vitro. However, the potential mechanism of fuziline on myocardial protection remains unknown. In this study, we aimed to explore the efficacy and mechanism of fuziline on isoproterenol (ISO)-induced myocardial injury in vitro and in vivo. As a result, fuziline effectively increased cell viability and alleviated ISO-induced apoptosis. Meanwhile, fuziline significantly decreased the production of ROS, maintained mitochondrial membrane potential (MMP) and blocked the release of cytochrome C, suggesting that fuziline could play the cardioprotective role through restoring the mitochondrial function. Fuziline also could suppress ISO-induced endoplasmic reticulum (ER) stress via the PERK/eIF2α/ATF4/Chop pathway. In addition, using ROS scavenger NAC could decrease ISO-induced apoptosis and block ISO-induced ER stress, while PERK inhibitor GSK2606414 did not reduce the production of ROS, indicating that excess production of ROS induced by ISO triggered ER stress. And fuziline protected against ISO-induced myocardial injury by inhibiting ROS-triggered ER stress. Furthermore, fuziline effectively improved cardiac function on ISO-induced myocardial injury in rats. Western blot analysis also showed that fuziline reduced ER stress-induced apoptosis in vivo. Above these results demonstrated that fuziline could reduce ISO-induced myocardial injury in vitro and in vivo by inhibiting ROS-triggered ER stress via the PERK/eIF2α/ATF4/Chop pathway.

Modulation of hepatic lipidome by rhodioloside in high-fat diet fed apolipoprotein E knockout mice.

BACKGROUND: Rhodioloside is a glucoside of tyrosol isolated from Rhodiola rosea. However, its regulating effect on hepatic dyslipidemia of atherogenic mice has rarely been studied. PURPOSE: The specific aims of current study included to clarify lipidomic perturbation in liver tissues of apolipoprotein E deficient (apoE-/-) mice fed with high-fat diet, and to examine the effects of rhodioloside against atherosclerosis and dyslipidemia. STUDY DESIGN: The comparisons of hepatic lipidome were executed between wide type (WT) mice fed with normal diet (NDC) and apoE-/- mice fed with high-fat diet (Model), WT mice fed with high-fat diet (HFDC) versus the model mice, as well as the model mice versus rhodioloside-treated atherosclerotic mice. METHODS: Ultra high performance liquid chromatography coupled with a Q exactive hybrid quadrupole-orbitrap mass spectrometry (UPLC-MS/MS) was employed to provide an unbiased and simultaneous measurement of individual lipid species in liver tissues. RESULTS: Multivariate statistical analysis derived from LC-MS spectra revealed that high-fat diet and apoE deficiency caused a series of disturbances on glyerolipid metabolism, glycerophospholipid metabolism and sphingolipid metabolism. Rhodioloside administration showed atheroprotective effects on the apoE-/- mice with regulating the levels of 1 phosphatidylcholine, 2 phosphatidylserines, 5 alkyldiacylglycerols and 3 alkenyldiacylglycerols back to normal. In particular, PC (4:0/15:0) was positively associated with high-density lipoprotein cholesterol in blood, both of which could be ameliorated by rhodioloside. CONCLUSION: Our results identified the abnormal hepatic lipids in atherosclerosis progression that could efficiently improved by rhodioloside. These lipids contributed to biological understanding of atherogenic dyslipidemia in liver and could also served as sensitive indicators for drug target screening.

Simultaneous determination of multiple components in rat plasma and pharmacokinetic studies at a pharmacodynamic dose of Xian-Ling-Gu-Bao capsule by UPLC-MS/MS.

Xian-Ling-Gu-Bao capsule (XLGB) is an effective traditional Chinese medicine prescription (TCMP) that is used for the prevention and treatment of osteoporosis in China. A rapid, simple, efficient and stable method based on UPLC-MS/MS technology was developed for simultaneous determination of multiple components of XLGB in rat plasma. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI). For twenty-one selected quantitative prototypes, all calibration curves showed favourable linearity (r>0.9932) in linear ranges. The lower limits of quantification (LLOQs) were 2 ng/mL for psoralen (PL), 2.5 ng/mL for asperosaponin VI (AS), 1 ng/mL for isopsoralen (IPS) and sweroside (SW), 0.5 ng/mL for magnoflorine (MA), bavachinin (BVN), tanshinone IIA (TA), timosaponin BII (TBII) and icaritin (ICT), 0.1 ng/mL for epimedin B (EB) and epimedin C (EC), 0.05 ng/mL for icariin (IC), isobavachalcone (IBC), psoralidin (PD), bavachin (BV), bavachalcone (BC), epimedin A (EA) and isobavachin (IBV), 0.02 ng/mL for neobavaisoflavone (NEO) and icariside I (ICI) and 0.01 ng/mL for icariside II (ICII). The intra-day and inter-day (low, medium, high) precision (relative standard deviation) for all analytes was less than 8.63%, and the accuracies (as relative error) were in the range of -12.45% to 8.91%. Extraction recoveries and matrix effects of analytes and IS were acceptable. All analytes were stable during the assay and storage in plasma samples. The validated method was successfully applied to the pharmacokinetics (PK) studies of the twenty-one prototypes at pharmacodynamic doses (0.3 and 1 g/kg/day). In addition, dynamic profiles of 28 metabolites (phase II conjugates: 23 glucuronide conjugates, 2 sulfate conjugates and 3 glucuronide or sulfate conjugates) were also monitored by their area/IS area-time curves. As a result, coumarins, prenylated flavonoids from Psoraleae Fructus, alkaloids and prenylated flavonol glycosides from Epimedii Herba, and iridoid glycosides, triterpenoid saponins from Dipsaci Asperoidis Radix were considered to be the key effective substances of XLGB due to their high exposure and appropriate pharmacokinetic features. This is the first report to reveal pharmacodynamic ingredients by a reversed pharmacodynamic (PD) - pharmacokinetics (PK) study.

Phenolic acids and their glycosides from the rhizomes of Cimicifuga dahurica.

Phytochemical study on rhizomes of Cimicifuga dahurica resulted in the isolation of nine new neolignan and phenylpropanoid glycosides, cimicifugasides A-E (1, 2, 7-9), cimicifugamides B-D (3-5), shomaside G (6) along with four known compounds (10-13). Their structures were identified by extensive spectroscopic analyses (1D-, 2D-NMR, MS, CD, IR, UV) and chemical methods. Their anti-inflammatory potentials were evaluated by measuring their effects on PGE2 production of LPS-stimulated RAW264.7 cells, and compounds 12 and 13 showed moderate anti-inflammatory activities.

Discovery of cardio-protective constituents of Gualou Xiebai Decoction, a classical traditional Chinese medicinal formula.

BACKGROUD: Finding effective compounds of TCMs has always been the basis for achieving marker-based quality control which is currently most widely used quality control strategy. Gualou Xiebai Decoction (GLXB), a classical TCM formula, is recorded and proven as a therapy for curing coronary heart disease but the effective constituents are unidentified and the substantial basis of the therapeutic effects is not clear. PURPOSE: The present research is an investigation on the chemistry of this formula aiming at finding and precisely identifying effective compounds. STUDY DESIGN AND METHODS: This research started with screening for effective fractions of GLXB by rat myocardial infarction model and H9c2 cell hypoxia/reoxygenation model, then compounds in effective fractions were isolated and identified by phytochemical and spectroscopic methods. The cardio-protective activities of the compounds were tested in vitro and one of the effective compounds was taken as example to investigate the mechanisms. RESULTS: The water-insoluble parts of GLXB were identified as effective parts in both in vitro and in vivo experiments. Systematic isolation of compounds in the effective fractions resulted in the isolation of 34 compounds including 7 new compounds, whereas 8 compounds were effective in protecting H9c2 cells against hypoxia/reoxygenation injury. One of the effective compounds, macrostemonoside P (MP) possibly exerted its effect by activating RISK pathway and attenuating apoptosis. CONCLUSION: An array of effective constituents of GLXB were discovered, and discovery of these compounds contributed to elucidating the substantial basis for the therapeutic effects of this formula, and provides fundaments for establishing Q-markers for further reliable quality control of GLXB.

Systematic screening and characterization of Qi-Li-Qiang-Xin capsule-related xenobiotics in rats by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Qi-Li-Qiang-Xin capsule (QLQX), a well-known traditional Chinese medicine prescription (TCMP), is consisted of eleven commonly used herbal medicines, has been widely used for the treatment of chronic heart failure (CHF). However, the absorbed components and related metabolites after oral administration of QLQX are still remaining unknown. In the present work, a reliable and effective method using ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) was established to identify QLQX-related xenobiotics in rats. Based on a representative structure based homologous xenobiotics identification (RSBHXI) strategy, a total of eleven compounds (salvianolic acid B, formononetin, benzoylmesaconine, alisol A, sinapine thiocyanate, naringin, tanshinone IIA, ginsenoside Rg1, ginsenoside Rb1, astragaloside IV and periplocin), bearing different chemical core structures, were selected and investigated for their metabolism in vivo. And then, comprehensive metabolic profiles of the holistic multi-ingredients in QLQX were achieved. As a result, a total of 121 QLQX-related xenobiotics (47 prototypes and 74 metabolites) were identified or tentatively characterized, among them eight prototypes (mesaconine, hypaconine, songorine, fuziline, neoline, talatizamine formononetin, neocryptotanshinone) and two metabolites (calycosin-gluA, formononetin-guA) were relatively the main existing xenobiotics exposed in blood. All absorbed prototype constituents were mainly from six composed herbal medicines (Aconiti lateralis radix, Astragali radix, Ginseng radix, Alismatis rhizoma, Salvia miltiorrhiza radix, Periploca cortex). The main metabolic reactions were methylation, hydrogenation, hydroxylation, oxidization, sulfation and glucuronidation. This is the first study on in vivo metabolism of QLQX. These results enabled us to focus on several high exposure ingredients in the discovery of effective substances of QLQX, however further pharmacokinetic study on these QLQX-related xenobiotics are needed to be carried out.

A combination of representative compounds, metabolism platform and diagnostic extraction strategy for characterization of metabolites of Shuang-Huang-Lian oral liquid in vivo by ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry.

Traditional Chinese medicines (TCMs) usually contained a large number of chemical components, which could be transformed into more complex metabolites in vivo. In this work, a "Representative compounds-Metabolism platform-Diagnostic extraction" strategy (RMD strategy) was proposed for comprehensively identification or characterization of xenobiotics in rat after oral administration of TCMs. Shuang-Huang-Lian oral liquid (SHL), a well-known traditional Chinese medicine preparation, was used as an example. The metabolic pathways of six representative compounds, bearing five different core structures in SHL, were elucidated and their metabolic reactions were employed for exploring metabolites of homologous components in metabolism platform. Meanwhile, diagnostic ions extraction were also used for screening more structural analogues in biofluids. All this work was completed by ultra-performance liquid chromatography coupled electrospray ionization quadruple time-of-flight mass spectrometry (UPLC/Qtof MS) and UNIFI metabolism platform. As a result, a total of 254 xenobiotics were identified or tentatively characterized in rat plasma and urine after oral administration of SHL and six representative compounds. The metabolism reaction included phase I reaction (hydroxylation, hydrolysis reaction, deglycosylation, hydrogenation, demethylation, dehydroxylation and ring opening reaction) and phase II reaction (glucuronidation, sulfation and methylation). This research provided useful information for further study of the pharmacology and mechanism of SHL in vivo. It also demonstrated that RMD strategy was an efficient approach for facilitate screening-out and rapid identification of xenobiotics in biological samples after oral administration of TCMs.

Biotransformation and metabolic profile of Xian-Ling-Gu-Bao capsule, a traditional Chinese medicine prescription, with rat intestinal microflora by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry analysis.

Xian-Ling-Gu-Bao capsule (XLGB), a well-known traditional Chinese medicine prescription, has been used for the prevention and treatment of osteoporosis. The safety and efficacy of XLGB have been confirmed based on the principle of evidence-based medicine. XLGB is usually administered orally, after which its multiple components are brought into contact with intestinal microflora in the alimentary tract and biotransformed. However, investigations on the comprehensive metabolic profile of XLGB are absent. In this study, 12 representative compounds bearing different typical structures (including iridoid glycosides, prenylated flavonol glycosides, prenylated flavonoids, triterpenoid saponins, steroidal saponins, coumarins and monoterpene phenols) were selected and then investigated for their biotransformation in rat intestinal microflora. In addition, the metabolic profile of XLGB in rat intestinal microflora was investigated by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. As a result, a total of 87 biotransformation components were identified from incubated solutions of 12 representative compounds and XLGB, which underwent 16 metabolic reactions (including deglycosylation, glycosylation, dehydrogenation, hydrogenation, oxidation, epoxidation, hydroxylation, dehydration, hydration, hydrolysis, methylation, isomerization, cyclization, pyrolysis reaction, amino acid conjugation and nucleophilic addition reaction with NH3 ). This demonstrated that the deglycosylation reaction by cleavage of the sugar moieties is the main metabolic pathway of a variety of glycosides, including prenylated flavonol glycosides, coumarin glycosides, iridoid glycosides and saponins. In addition, compared with the biotransformation of 12 representative compounds, a different biotransformed fate was observed in the XLGB incubated samples of rat intestinal microflora. It is worth noting that the amino acid conjugation was first discovered in the metabolism of prenylated flavonol glycosides in rat intestinal microflora.