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Background and Purpose: Hepatic mitochondrial pyruvate carrier (MPC) transports pyruvate into mitochondria. This study investigated the involvement of MPC1 in hepatic glucagon response, in order to identify a possible pharmacological intervention.Experimental Approach: The correlation between hepatic glucagon response and MPC1 induction was investigated in fasted mice and primary hepatocytes. The effects of ginsenoside Rb1 on MPC1 function were observed.Key Results: Glucagon challenge raised blood glucose with hepatic MPC1 induction, and inhibition of MPC induction coincided with a reduced rise in blood glucose. cAMP-responsive element-binding protein (CREB) knockdown blocked glucagon-induced MPC1 expression, while CREB overexpression increased MPC1 expression. Luciferase reporter, chromatin immunoprecipitation assay, and promoter mutation confirmed that CREB increased MPC1 transcription through gene promoter induction. CREB regulated transcription co-activator 2 nuclear translocation was also required for CREB to promote MPC1 induction. Glucagon shifted mitochondrial pyruvate towards carboxylation for gluconeogenesis via the opposite regulation of pyruvate dehydrogenase and carboxylase with respect to MPC1 induction. MPC1 induction was necessary for glucagon to promote pyruvate-driven hepatic glucose production (HGP), but glucagon failed to influence HGP from other gluconeogenic substrates routed into the tricarboxylic acid cycle, independent of MPC. Rb1 blocked cAMP signalling by inhibiting AC activity and deactivated CREB by dephosphorylation, possibly contributing to inhibiting MPC1 induction to reduce HGP.Conclusions and Implications: CREB transcriptionally up-regulates MPC1 to provide pyruvate for gluconeogenesis. Rb1 reduced cAMP formation which consequently reduced CREB-mediated MPC1 induction and thereby might contribute to limiting pyruvate-dependent HGP. These results suggest a therapeutic strategy to reduce hyperglycaemia in diabetes. [ABSTRACT FROM AUTHOR]

SUMMARY: Salvianolic acids (SalAs), a group of secondary metabolites in Salvia miltiorrhiza, are widely used for treating cerebrovascular diseases. Their biosynthesis is modulated by a variety of abiotic factors, including ultraviolet‐B (UV‐B) irradiation; however, the underlying mechanisms remain largely unknown. Here, an integrated metabolomic, proteomic, and transcriptomic approach coupled with transgenic analyses was employed to dissect the mechanisms underlying UV‐B irradiation‐induced SalA biosynthesis. Results of metabolomics showed that 28 metabolites, including 12 SalAs, were elevated in leaves of UV‐B‐treated S. miltiorrhiza. Meanwhile, the contents of several phytohormones, including jasmonic acid and salicylic acid, which positively modulate the biosynthesis of SalAs, also increased in UV‐B‐treated S. miltiorrhiza. Consistently, 20 core biosynthetic enzymes and numerous transcription factors that are involved in SalA biosynthesis were elevated in treated samples as indicated by a comprehensive proteomic analysis. Correlation and gene expression analyses demonstrated that the NAC1 gene, encoding a NAC transcriptional factor, was positively involved in UV‐B‐induced SalA biosynthesis. Accordingly, overexpression and RNA interference of NAC1 increased and decreased SalA contents, respectively, through regulation of key biosynthetic enzymes. Furthermore, ChIP‐qPCR and Dual‐LUC assays showed that NAC1 could directly bind to the CATGTG and CATGTC motifs present in the promoters of the SalA biosynthesis‐related genes PAL3 and TAT3, respectively, and activate their expression. Our results collectively demonstrate that NAC1 plays a crucial role in UV‐B irradiation‐induced SalA biosynthesis. Taken together, our findings provide mechanistic insights into the UV‐B‐induced SalA biosynthesis in S. miltiorrhiza, and shed light on a great potential for the development of SalA‐abundant varieties through genetic engineering. [ABSTRACT FROM AUTHOR]

Oplopanax horridus, widely distributed in North America, is an herbal medicine traditionally used by Pacific indigenous peoples for various medical conditions. After oral ingestion, constituents in O. horridus extract (OhE) could be converted to their metabolites by the enteric microbiome before absorption. In this study, in order to mimic gut environment, the OhE was biotransformed using the enteric microbiome of healthy human subjects. For accurate and reliable data collection with optimized approaches in sample preparation and analytical conditions, ultra‐performance liquid chromatography and quadrupole time‐of‐flight mass spectrometry were used to characterize parent constituents and their metabolites. In the extract, 20 parent compounds were identified including polyynes, sesquiterpenes, monoterpeondids, phenylpropanoids and phenolic acids. After the biotransformation, a total of 78 metabolites were identified, of which 37 belonged to polyynes metabolites. The common biotransformation pathways are hydroxylation, acetylization, methylation and demethylation. Based on the pathway distributions, the metabolism signature of OhE has been explored. The metabolism pathways of OhE compounds are dependent on their structural classifications and hydrophilic/hydrophobic properties. In summary, with comprehensive analysis, we systematically investigated human microbiome‐derived OhE metabolites. The enteric microbial metabolism signature provides novel information for future effective use of O. horridus. [ABSTRACT FROM AUTHOR]

Background & Aims: Nonalcoholic fatty liver disease encompasses isolated steatosis or nonalcoholic fatty liver and nonalcoholic steatohepatitis (NASH). NASH develops from isolated steatosis with obscure driving forces. We aim to identify key factors promoting this transition. Methods: Following 21‐week of high‐fat diet feeding, obese mice were classified into two groups termed as isolated steatosis and NASH based on hematoxylin‐eosin staining of liver histology. The integrated multi‐omics analysis of lipidome, transcriptome and gut microbiome were performed in mice with isolated steatosis and NASH, and confirmed in human samples. Results: Livers in mice with NASH lost most lipids, and the transcriptional landscape was also changed dramatically in mice with NASH in relative to mice with isolated steatosis. Plasma lipidome analysis demonstrated a very clear difference between these two groups of mice, which was partially recapitulated in serum of patients with isolated steatosis and NASH. The microbiota composition revealed that Bacteroides genus and Bacteroides uniformis species decreased while Mucispirillum genus and Mucispirillum schaedleri species increased largely in mice with NASH. More importantly, we found that Bacteroides uniformis correlated positively with triglycerides (TGs) and negatively with free fatty acids (FFAs) and PE(18:1/20:4), while Mucispirillum schaedleri correlated positively with FFAs, LysoPC(20:3), LysoPC(20:4) and DG(16:1/18:2). Mechanically, administration of Bacteroides uniformis increased specific TGs, and decreased hepatic injury and inflammation in diet‐induced mice. Conclusions: Overall, through multi‐omics integration, we identified a microbiota‐lipid axis promoting the initiation of NASH from isolated steatosis, which might provide a novel perspective on NASH pathogenesis and treatment. [ABSTRACT FROM AUTHOR]

Background and Purpose: Hypoxic activation of hypoxia-inducible factor 1α (HIF-1α) and fibrosis in adipose tissue contribute to adipose dysfunction. This study was designed to investigate the effects of metformin and resveratrol on the regulation of HIF-1α and fibrosis in hypoxic adipose tissue.Experimental Approach: Mice were fed a high-fat diet to induce hypoxia and fibrosis in adipose tissue; adipose tissue incubated in vitro in 1% O2 showed a similar change. The effects of metformin and resveratrol on hypoxia, HIF-1α accumulation, endoplasmic reticulum stress and gene expressions of extracellular matrix components and pro-inflammatory cytokines were examined.Key Results: Oral administration of metformin or resveratrol prevented hypoxia and reduced HIF-1α accumulation with dephosphorylation of inositol-requiring enzyme 1α and eukaryotic initiation factor 2α, indicative of suppression of hypoxic HIF-1α activation and endoplasmic reticulum stress. Metformin and resveratrol down-regulated gene expressions of Col3α, Col6α, elastin and lysyl oxidase and thereby reduced collagen deposition in adipose tissue. The increased gene expressions of TNF-α, IL-6, monocyte chemoattractant protein 1 and F4/80 were also down-regulated by metformin and resveratrol. Metformin and resveratrol had similar effects in adipose tissue exposed to 1% O2 . Metformin reduced ATP production and prevented the reduction in oxygen tension in 3T3-L1 cells, suggesting that it prevented hypoxia by limiting oxygen consumption, whereas resveratrol reduced HIF-1α accumulation by promoting its proteasomal degradation via the regulation of AMPK/SIRT1.Conclusion and Implications: Hypoxia and fibrosis are early causes of adipose dysfunction in obesity. Both metformin and resveratrol effectively inhibited HIF-1α activation-induced fibrosis and inflammation in adipose tissue, although by different mechanisms. [ABSTRACT FROM AUTHOR]

After ingestion of ginseng, the bioavailability of its parent compounds is low and enteric microbiota plays an important role in parent compound biotransformation to their metabolites. Diet type can influence the enteric microbiota profile. When human subjects on different diets ingest ginseng, their different gut microbiota profiles may influence the metabolism of ginseng parent compounds. In this study, the effects of different diet type on gut microbiota metabolism of American ginseng saponins were investigated. We recruited six healthy adults who regularly consumed different diet types. These subjects received 7 days' oral American ginseng, and their biological samples were collected for LC-Q-TOF-MS analysis. We observed significant ginsenoside Rb1 (a major parent compound) and compound K (a major active metabolite) level differences in the samples from the subjects consuming different diets. Subjects on an Asian diet had much higher Rb1 levels but much lower compound K levels compared with those on a Western diet. Since compound K possesses much better cancer chemoprevention potential, our data suggested that consumers on a Western diet should obtain better cancer prevention effects with American ginseng intake compared with those on an Asian diet. Ginseng compound levels could be enhanced or reduced via gut microbiota manipulation for clinical utility. [ABSTRACT FROM AUTHOR]

A copper-catalyzed remote C−H amination of 8-aminoquinoline scaffolds on the C5 position is described. The protocol employs commercially available N-fluorobenzenesulfonimide (NFSI) as the amination reagent and shows broad substrate scope, providing various 5-aminated quinolines in moderate to excellent yields under mild conditions. These reactions feature complete regioselectivity, operational simplicity, and high efficiency. [ABSTRACT FROM AUTHOR]

Circulating microRNAs (miRNAs) have been used as promising diagnostic biomarkers for esophageal squamous cell carcinoma (ESCC). We performed miRNA expression profiling using quantitative reverse transcription polymerase chain reaction (qRT-PCR) based Exiqon panels from three ESCC pools and one normal control (NC) pool samples. Using qRT-PCR, identified serum miRNAs were further confirmed in training (32 ESCC vs. 32 NCs) and testing stages (108 ESCC vs. 96 NCs). Consequently, five serum miRNAs (miR-20b-5p, miR-28-3p, miR-192-5p, miR-223-3p, and miR-296-5p) were significantly overexpressed in ESCC compared with NCs. The diagnostic value of the 5-miRNA signature was validated by an external cohort (60 ESCC vs. 60 NCs). The areas under the receiver operating characteristic curve (ROC) of the 5-miRNA signature were 0.753, 0.763, and 0.966 for the training, testing, and the external validation stages, respectively. The expression levels of the miRNAs were also determined in tissues, arterial serum, and exosomes. MiR-20b-5p, miR-28-3p, and miR-192-5p were significantly upregulated in ESCC tissues, while miR-296-5p was overexpressed in ESCC serum exosomes. In conclusion, we identified a 5-miRNA signature in serum for the detection of ESCC. [ABSTRACT FROM AUTHOR]

The emergence of direct analysis in real time (DART) ion source provides the great possibility for rapid analysis of hazardous substance in drugs. DART mass spectrometry (DART-MS) enabled the conducting of a fast and non-contact analysis of various samples, including solid or liquid ones, without complex sample preparation or chromatographic separation. In this study, a modified DART-quadrupole time-of-flight mass spectrometry (DART-QTOF-MS) method was developed for identification and determination of 5-hydroxymethylfurfural (5-HMF) in Shenfu (SF) injection. The quantitative transfer of sample solution was introduced to the glass tips of DIP-it sampler at a fixed volume, which significantly increases the repeatability and accuracy of analytical results. The protonated ion of dibutyl phthalate in the atmosphere was used as the reference mass for TOF-MS recalibration during the data acquisition for constant high accuracy mass measurements. Finally, the developed DART-MS method was used to determine 5-HMF in seven batches of SF injection, and the contents of 5-HMF were not higher than 100 µg/mL. The results obtained were further confirmed by an ultra-high performance liquid chromatography combined with triple quadrupole mass spectrometer (UHPLC-QQQ-MS). The overall results demonstrated that the DART-QTOF-MS method could be applied as an alternative technique for rapid monitoring 5-HMF in herbal medicine injection. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A mild and metal-free method to functionalize aliphatic C−H bonds adjacent to a nitrogen atom through radical translocation, oxidation to an iminium intermediate, and nucleophilic trapping with alcohols is described. The transformation proceeds efficiently with edible ascorbic acid (vitamin C) as the radical initiator and readily available tert-butyl nitrite as the nitrosating reagent. Notably, any transition-metal or additive is not required. The current method provides simple, efficient, and practical access to valuable α-alkoxybenzamides in moderate to good yields. [ABSTRACT FROM AUTHOR]

The pharmacokinetics of 5-fluorouracil (5-FU) in combination with or without American ginseng (seven-consecutive days oral dose) in rats were evaluated using liquid chromatography-electrospray ionization-mass spectrometry (LC-MS). Chromatographic separation was performed on a reverse LC column within a total run time of 6.5 min, which allowed for a relatively quick analysis. The limit of quantification for 5-FU was 15 ng/mL and this method was linear over 15-50,000 ng/mL. This method supported stabilizing determination of the plasma concentration of 5-FU over a period of 24 h. Precision both interday and intraday (coefficient of variation) was within 14% and accuracy (relative error) ranged from −5 to 14%. In view of the observed pharmacokinetic parameters, including maximum concentration, time to maximum concentration, area under the concentration-time curve (AUC), mean residence time, elimination half-life and clearance, our results showed no significant differences in all of the pharmacokinetic parameters between the ginseng co-treated group and 5-FU alone group. Some increase in AUC was observed in 5-FU plus ginseng group; however, the difference did not reach statistical significance compared with 5-FU alone. It appeared that American ginseng administration did not significantly alter the kinetics of 5-FU. More studies are still needed to confirm our results. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Panaxadiol (PD) is a purified sapogenin of ginseng saponins, which exhibits anticancer activity. Epigallocatechin gallate (EGCG), a major catechin in green tea, is a strong botanical antioxidant. In this study, we investigated the possible synergistic anticancer effects of PD and EGCG on human colorectal cancer cells and explored the potential role of apoptosis in the synergistic activities. Effects of selected compounds on HCT-116 and SW-480 human colorectal cancer cells were evaluated by a modified trichrome stain cell proliferation analysis. Cell cycle distribution and apoptotic effects were analyzed by flow cytometry after staining with PI/RNase or annexin V/PI. Cell growth was suppressed after treatment with PD (10 and 20 µ m) for 48 h. When PD (10 and 20 µ m) was combined with EGCG (10, 20, and 30 µ m), significantly enhanced antiproliferative effects were observed in both cell lines. Combining 20 µ m of PD with 20 and 30 µ m of EGCG significantly decreased S-phase fractions of cells. In the apoptotic assay, the combination of PD and EGCG significantly increased the percentage of apoptotic cells compared with PD alone ( p < 0.01). The synergistic apoptotic effects were also supported by docking analysis, which demonstrated that PD and EGCG bound in two different sites of the annexin V protein. Data from this study suggested that apoptosis might play an important role in the EGCG-enhanced antiproliferative effects of PD on human colorectal cancer cells. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

ABSTRACT Herbal formulations are complex natural mixtures. Researchers usually tend to focus more on analysis of nonvolatile components but pay less attention to volatile compounds. In this study, an analytical strategy combining two approaches was established for comprehensive analysis of herbal formulations. Guizhi Fuling capsule (GFC), a drug approved by the FDA to enter phase II clinical trial for treatment of primary dysmenorrhea, was taken as a case for analysis. Gas chromatography-mass spectrometry (GC-MS) with automated mass spectral deconvolution and identification system (AMDIS) led to rapid identification of 48 volatile components including four acetophenones, three fatty acid esters, 13 phenylpropanoids and 19 sesquiterpenes. Most of them were found from Guizhi. The volatile oils of Guizhi have been proved to exhibit many pharmacological activities. This is helpful in understanding the pharmacological mechanism of GFC. Furthermore, AMDIS turned out to be efficient and reliable for analysis of complex herbal formulations. Rapid-resolution liquid chromatography (RRLC) coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS/MS) allowed the identification of 70 nonvolatile components including six acetophenones, 12 galloyl glucoses, 31 monoterpene glycosides, three phenols and 12 triterpene acids. Fragmentation behaviors of assigned components, especially triterpene acids, which are hard to identify by low-resolution MS, were first investigated by TOF MS/MS. Characteristic ions and typical loss of assigned triterpene acids were summarized. Combinatorial use of GC-MS-AMDIS and RRLC-ESI-Q-TOF MS/MS could be of great help in global qualitative analysis of GFC, as well as other herbal products. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Rapid resolution liquid chromatography (RRLC) coupled with diode array detection (DAD) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) method was applied to the mass spectral study of a series of naturally occurring iridoid glycosides and phenylpropanoid glycosides in Radix Scrophulariae, which provides higher speed and increased sensitivity without loss of resolution. With dynamic adjustment as the key role of the fragmentor voltage and confirmed with authentic standards, valuable structural information regarding the nature of both the glycoside skeletons was thus obtained. Most compositions were found to possess organic acid moiety such as cinnamoyl, caffeoyl and ferulyol. Besides extensive fragmentation of the carbohydrate moiety, losses of the hydroxyl and glucose residue units showed in the spectra, permitting the exploration of the skeleton and the identity of substituents in the molecule. Ten major iridoid glycosides and 10 phenylpropanoid glycosides were identified or tentatively characterized based on their retention times, UV and TOF MS data. The major fragmentation pathways of PGs in Radix Scrophulariae obtained through the MS data was schemed systematically for the first time, which provides a reference for other PGs derivatives. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Asperosaponin VI (also named akebia saponin D) is a typical bioactive triterpenoid saponin isolated from the rhizome of Dipsacus asper Wall (Dipsacaceae). In this work, a sensitive high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) assay has been established for determination of asperosaponin VI in rat plasma. With losartan as the internal standard (IS), plasma samples were prepared by protein precipitation with methanol. Chromatographic separation was performed on a C18 column with a mobile phase of 10 m m ammonium acetate buffer containing 0.05% formic acid-methanol (32 : 68, v/v). The analysis was performed on an ESI in the selected ion monitoring mode using target ions at m/z 951.4 for asperosaponin VI and m/z 423.2 for the IS. The calibration curve was linear over the range 3-1000 ng/mL and the lower limit of quantification was 3.0 ng/mL. The intra- and inter-assay variability values were less than 9.5 and 7.8%, respectively. The accuracies determined at the concentrations of 3.0, 100.0, 300.0 and 1000 ng/mL for asperosaponin VI were within ±15.0%. The validated method was successfully applied to a pharmacokinetic study in rats after oral administration of asperosaponin VI. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Buyang Huanwu Decoction (BYHYD) is a classical traditional Chinese medicinal prescription that has been widely used for treating cerebrovascular illnesses for hundreds of years. In this study, a comprehensive analytical method has been developed for quantitative analysis of the major constituents in BYHWD. This method was based on high-performance liquid chromatography coupled to a diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) on a common reverse-phase C18 column. HPLC coupled with on-line time-of-flight mass spectrometry (HPLC-TOF/MS) was additionally adopted to provide further validation for the constituents. It was found that 0.3% aqueous formic acid and acetonitrile was the optimum mobile phase for gradient elution. This method, which showed excellent precision and accuracy, was successfully applied to quantify the bioactive constituents in six BYHWD products. The validated HPLC-DAD-ELSD method, together with the HPLC-TOF/MS analysis, provided a new basis for assessing the quality of traditional Chinese medicinal prescription consisting of many bioactive components. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Buyang Huanwu Decoction (BYHWD), is a well-known traditional Chinese preparation consisting of Radix Astragali, Radix Angelicae Sinensis, Rhizoma Ligustici Chuanxiong, Radix paeoniae Rubra, Flos Carthami, Semen Persicae and Lumbricus. An ultra-fast high-performance liquid chromatography (HPLC) method coupled with diode array detection (DAD) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF/MS) has been developed for rapid separation and structural identification of constituents in BYHWD. Using an ultra-fast HPLC system with short columns (4.6 × 50 mm, 1.8 µm), the total analysis time for this complex prescription is less than 30 min. With various fragmentor voltages in TOF/MS, accurate mass measurements (less than 5 ppm error) for molecular ions and characteristic fragment ions could represent reliable identification criteria for these compounds. Fifty-four major constituents from BYHWD sample, including four C-glycosyl quinochalcones, four flavonoid O-glycosides, sixteen isoflavones, six monoterpene glycosides, eight saponins, four organic acids and five amino acids, were identified or tentatively characterized based on their retention times, DAD and TOF/MS data. All the compounds were further assigned in the seven individual crude drugs. In conclusion, the ultra-fast HPLC with DAD-TOF/MS is a highly useful and efficient technique to separate and identify constituents in complex matrices of herbal medicines or preparations. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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A cell extraction coupled with high-performance liquid chromatography (HPLC) analysis has been developed to screen the potential bioactive components in combined prescription of Danggui Buxue decoction. The method was validated by using HL-7702 cells, RAW 264.7 cells and Caco-2 cell extraction. According to the hypothesis that, when cells are incubated together with the extract of traditional Chinese medicines (TCMs), the potential bioactive compounds in the extract should selectively combine with the cells, cell-combining compounds were analyzed by HPLC-diode array-evaporative light scattering detectors. Their structures were elucidated in comparison with available reference compounds, and further confirmed by HPLC-electrospray ionization time-of-flight mass spectrometry with accurate mass measurement. The results demonstrated that nine compounds were combined with the HL-7702 cells, seven with RAW 264.7 cells, and thirteen with the Caco-2 cell line. In view of the two key steps of drugs action, absorption by intestinal epithelium cells and interaction with target cells, this rapid and reliable method could be utilized to predict the bioactive constituents in TCMs, and it was in agreement with the characteristics of combined prescriptions of TCMs as multi-components, multi-target sites and multi-channel actions. Copyright © 2007 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A novel method, using high-performance liquid chromatography combined with principal component analysis, was developed for the quality evaluation of danshen through simultaneous determination of seven components, namely danshensu, protocatechuic acid, protocatechuic aldehyde, salvianolic acid B, tanshinone I, cryptotanshinone and tanshinone IIA. These seven components were simultaneously separated on a Zorbax SB-C18 column. The mobile phase consisted of 0.05% phosphoric acid water and methanol:acetonitrile (1:1) with a gradient elution, and the detection wavelength was set at 280 nm. Thirty samples of danshen and its substitutes from different sources were investigated by the established method. The results showed that the content of each analyte varied considerably in different danshen samples. Among the seven components tested, salvianolic acid B, tanshinone IIA, cryptotanshinone, tanshinone I, danshensu and protocatechuic aldehyde were proved suitable and representative as chemical markers for the quality control of danshen except for protocatechuic acid. Moreover, principal component analysis was used for the similarity evaluation of different samples, and it could be straightforward and reliable to differentiate danshen samples of different origins. In conclusion, simultaneous quantification of multiple components by high-performance liquid chromatography combined with principal component analysis would be a better strategy for the quality evaluation of danshen. Copyright © 2007 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]