Experimental validation for mechanisms of Qizhiweitong particles against Chronic Non-atrophic gastritis based on metabolomics and network pharmacology.

Qizhiweitong particles (QZWT), a classic Chinese herbal prescription derived from the Sinisan decoction in Shang Han Za Bing Lun, has definitive clinical efficacy in treating Chronic Non-atrophic Gastritis (CNG) in China. However, its mechanism of action at the metabolic level remains unclear. The aim of this study was to explore the mechanisms of QZWT against CNG based on non-targeted metabolomics combined with network pharmacology and experimentally validated by enzyme linked immunosorbent assays (ELISA). First, CNG model rats were established by free drinking ammonia water combined with starvation and satiety disorder for 12 weeks. Taking gastric tissue as the object, ultra-high performance liquid chromatography tandem mass spectrometry based metabolomics and network pharmacology were conducted to identify the key compounds, core targets and pathways that mediate the effects of QZWT against CNG. Furthermore, the targets from network pharmacology and the metabolites from metabolomics were jointly analyzed to select crucial metabolism pathways by MetaScape. Finally, the key metabolic enzymes and metabolites were experimentally validated by ELISA. The results indicated that there were 29 differential metabolites were identified and considered to be metabolic biomarkers of QZWT in the treatment of CNG. Among them, 8 of the differential metabolites showed a significant reduction in the content of QZWT groups. Arachidonic acid (AA) metabolic and glycerophospholipid (GP) metabolic are the most crucial metabolic pathways for QZWT to treat CNG. QZWT regulated AA and GP metabolism by synergetic reducing the level of AA, Phospholipid acid and Lysophosphatidic acid and inhibiting the enzyme activity of prostaglandin endoperoxide synthase 1 and prostaglandin endoperoxide synthase 2. And a compound-reaction-enzyme-gene network of mechanism for QZWT against CNG was established. In conclusion, this study reveals the complicated mechanisms of QZWT against CNG. Our work presents a novel strategy to identify the potential mechanisms of pharmacological effects derived from a compound prescription of TCM.

Computational method for rapid screening of the metabolites of Pulsatilla chinensis in rats using UHPLC-Q-TOF/MS combined with mass spectrum-based orthogonal projection.

Screening metabolites in vivo can be challenging due to the complexity of traditional Chinese medicine (TCM) and the ambiguous intracorporal process. To resolve this problem, we established the mass spectrum-based orthogonal projection (MSOP) method to differentiate prototype compounds from metabolites in vivo and applied it to the study of metabolites of Pulsatilla chinensis (PC). Initially, the validity and feasibility of the MSOP method were verified by using the ultra- high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) data of reference solution. Then, the MSOP method was applied to screen the metabolites of PC. A total of 63 metabolites were identified in vivo (urine, feces, bile, and plasma samples) and in vitro (intestinal bacteria biological sample). The results indicated that the main metabolic pathways of pentacyclic triterpenoids were demethylation, oxidation, dehydration, sulfation, and glucuronidation reactions. This study contributes to developing an integrated strategy based on chemometrics to characterize and classify the metabolism feature of pentacyclic triterpenoids of PC. This will support the scientific and rational application of PC in the clinic. The MSOP method based on the orthogonality of MS signals was used to differentiate the prototype compounds from metabolites in vivo. The method provides scientific and reliable support for fully understanding the metabolic fate of TCM.

Development of trisiloxane surfactant vesicles ultrasonic extraction method combined with ultrahigh-performance liquid chromatography tandem mass spectrometry for the rapid differentiation of Bupleuri Radix based on metabolomics.

INTRODUCTION: Due to the variety, chemical composition and complex structure, the quality control of Bupleuri Radix (BR) is a challenging task. There are still many trace compounds in BR that are difficult to extract and detect. OBJECTIVE: To develop an innovative method of trisiloxane surfactant vesicles ultrasonic extraction (TSVUE) combined with ultrahigh-performance liquid chromatography tandem mass spectrometry for the identification from Bupleurum chinense DC. (BC) to Bupleurum scorzonerifolium Willd (BS) based on metabolomics. METHODS: Based on extraction effect for BR, five different types of surfactants vesicles were prepared and compared. Then, a single-factor test and a response surface methodology study were adopted to obtain the optimal conditions for the surfactant vesicles ultrasonic extraction method. Finally, a non-targeted metabolomics method with information dependent acquisition mode was performed to analyse differential metabolites in BC and BS. RESULTS: Sugar-based surfactant containing trisiloxane [N-3-propyl-methyltrisiloxane-N-glucoheptonamne (Si(3)N-GHA)] displayed higher extraction efficiency compared to other types of surfactants when it comes to being used in pretreatment methods. And a TSVUE method was established and optimised. In total, 131 constituents were identified in two BR herbs, of which 35 were unreported, and 11 were characterised as chemical markers. CONCLUSIONS: This method provides promising perspectives for rapidly identifying trace compounds in complex systems of traditional Chinese medicine (TCM), as well as for laying the foundation in the identification of similar herbs from the same species. Meanwhile, these findings serve as a promising application of trisiloxane surfactant vesicles in the extraction field of TCM.

An integrated strategy to explore the wine-processed mechanism of Corni Fructus on chronic renal failure based on metabolomics, network analysis and bioinformatics approaches.

OBJECTIVES: Corni Fructus is one of the most famous traditional Chinese medicines (TCMs) for the treatment of various chronic kidney diseases. Wine-processed Corni Fructus (WCF) is the main processed form of Crude Corni Fructus (CCF). In this study, potential mechanisms of action of CCF and WCF on chronic renal failure (CRF) model were developed to explore wine-processed mechanism of Corni Fructus. METHODS: An integrated strategy combining metabolomics, network analysis and bioinformatics analysis has been established to investigate the therapeutic mechanisms of WCF and CCF in rats with CRF. KEY FINDINGS: The histopathological results showed that both WCF and CCF improved kidney injury and dysfunction of CRF rats, but WCF was more effective than CCF. Metabolic pathway analysis indicated that 24 metabolites and 5 major disturbed pathways associated with CCF, while WCF regulated 27 metabolites and 2 metabolic pathways. Bioinformatic analysis and network analysis revealed that 8 genes and 7 genes were regulated by CCF and WCF on CRF rats, respectively. The quantitative real-time polymerase chain reaction experiments verified the regulatory ability of CCF and WCF on the expression of 4 genes. CONCLUSIONS: An integrated strategy combined metabolomics, network analysis and bioinformatics was established to provide valuable holistic insight to explore the processing mechanism of TCMs.

Integrative strategy for quality control of Radix Bupleuri based on non-targeted metabolomic profiling and molecular networking.

Quality control of Radix Bupleuri (RB) can be challenging due to the complexity of origin, the similar morphological characteristics, and the diversity of the multiple components. In this study, an integrated strategy for extensive identification of metabolites in plants based on multiple data processing methods was proposed to distinguish four commercially available RB species. First, the pre-processed mass spectrometry data was uploaded to Global Natural Products Social Molecular Networking (GNPS) for spectral library search and molecular network analysis, which can effectively differentiate isomers and reduce molecular redundancy. Second, the possible cleavage mode was summarized from the characteristic MS/MS fragment ions of saikoside standard, and then the possible structure of saikoside in the sample was deduced according to the cleavage patterns. Third, collected all kinds of RB components reported in the literature and matched the information in the samples to obtain more comprehensive information about metabolites. Finally, chemical markers were found employing chemometrics. This strategy not only increases the variety and number of identified components, but also improves the accuracy of the data. Based on this strategy, a total of 132 components were identified from different species of RB, and 14 chemical constituents were considered to be potential chemical markers to distinguish four kinds of RB. Among them, saikogenin a, hydroxy-saikosaponin a, hydroxy-saikosaponin d, and rutinum were of great significance for identification. The method proposed in this study not only successfully identified and distinguished four species of RB, but also laid a good theoretical foundation for regulating the RB market. This strategy provides promising perspectives in the accurate analysis of the ingredients of traditional Chinese medicine.

Integrative analyses of widely targeted metabolomic profiling and derivatization-based LC-MS/MS reveals metabolic changes of Zingiberis Rhizoma and its processed products.

Zingiberis Rhizoma (ZR) has nutritional value and application potentiality, while Zingiberis Rhizoma Praeparatum (ZRP) and Carbonised Ginger (CG) are two main processed products of ZR based on different methods. Here, we performed a widely targeted metabolomics method with Sequential Windowed Acquisition of all Theoretical fragment ions (SWATH) mode to analyze differential metabolites in ZR, ZRP and CG. Additionally, the chemical derivatization was applied to characterize different submetabolomes and improve the separation effect and MS response of metabolites. In total, 369 metabolites were identified and divided into 14 categories, 104 of which were differential metabolites. Our results suggest that carbohydrates, nucleotides, organic acids, vitamins, lipids, indoles, alkaloids, and terpenes contributed to a downward trend after processing, but the maximum content of flavanones, phenylpropanes and polyphenols appeared in ZRP, and that of alcohols appeared in CG. These findings serve as promising perspectives for developing functional food in ZR, ZRP and CG.

Quality control of Zingiberis Rhizoma and its processed products by UHPLC-Q-TOF/MS-based non-targeted metabonomics combining with SIBDV method.

Zingiberis Rhizoma (ZR) is a homologous plant with pungent tastes and aromas, which has unique nutritional value and tremendous application potentiality. Zingiberis Rhizoma Praeparatum (ZRP) and Carbonised Ginger (CG) are processed products of ZR through different processing methods, and they are commonly used ingredients in food supplements. This study used ZR, ZRP and CG from different batches to further understand composition differences after processing. Additionally, we performed non-targeted metabolomics-based profiling of gingerols by ultra-high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) in combination with multivariate analysis and compounds identification. In which, we developed a comprehensive SWATH-IDA bi-directionally verified (SIBDV) method integrating the advantages of Sequential Windowed Acquisition of all Theoretical fragment ions (SWATHTM) and traditional information-dependent acquisition (IDA) mode for characterization of gingerols. Potential chemical markers were selected by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) of chemometrics methods. After that, the threshold variable importance in projection (VIP) value and P value were employed to screen the valuable MS features for discriminating ZR, ZRP and CG. In total, 59 gingerols in the different samples were structurally identified. Results allowed the selection of 33 gingerols, which are nominated as novel markers for materials authentication in ZR, ZRP and CG. The analysis of the study showed that the content of gingerols showed a downward trend after processing, but shogaols and gingerone compounds had an upward trend, resulting in differences in application and pharmacodynamic efficacy. These findings provide promising perspectives in the quality control of ZR, ZRP and CG, as well as for laying the foundation in food design and development.

Miniaturized solid-phase extraction using a mesoporous molecular sieve SBA-15 as sorbent for the determination of triterpenoid saponins from Pulsatilla chinensis by ultrahigh-performance liquid chromatography-charged aerosol detection.

A simple, sensitive and efficient solid-phase extraction method, combined with ultrahigh-performance liquid chromatography-charged aerosol detection, was applied to the pre-concentration and determination of four triterpenoid saponins from Pulsatilla chinensis (P. chinensis) ultrasound extract samples. Mesoporous molecular sieve Santa Barbara Amorphous 15 (SBA-15) displayed higher selectivity compared to Mobil Composition of Matter 41 (MCM-41), NH2-SBA-15 and mesoporous carbon when it comes to being used in pretreatment methods. It was applied as an effective sorbent in the SPE for the enrichment of the target analytes. Additionally, several key experimental parameters including the kinds of sorbents, the amount of SBA-15, the elute pH and types of elution solvent were investigated in detail. Under the optimized conditions, the satisfactory linearity (r2 ≥ 0.9940) was acquired and the limits of detection reached 0.461-0.976 µg/mL for the target analytes. The recoveries ranged from 95.1%-103.2%. The experimental results showed that SBA-15 was a candidate material for the purification and concentration of target triterpenoid saponins from complex P. chinensis samples. The study provided theoretical support for the application of mesoporous materials in the field of drug separation and provided references for the extraction and determination of trace compounds in the complex systems of traditional Chinese medicine.

Integrated strategy for accurately screening biomarkers based on metabolomics coupled with network pharmacology.

Screening diagnostic biomarkers can be challenging due to the complexity of traditional Chinese medicine (TCM) and ambiguous pharmacological mechanisms. In this study, we reported an integrated strategy for accurately screening diagnostic biomarkers based on metabolomics coupled with network pharmacology. First, a feasible pharmacological model was established through systems pharmacology and based on metabolomics-based techniques to explore diagnostic biomarkers. While the components satisfying the q-value < 0.05, fold change (FC) ≥ 1.2 or FC ≤ 0.8, coefficient of variance (CV) ≤ 30%(QC) and the variable importance in the project (VIP) value > 1 are considered to be diagnostic biomarkers. Second, the ingredients were retained only when oral bioavailability (OB), Caco-2 permeability, drug half-life, TPSA and drug likeness (DL) satisfied the criteria (OB ≥ 40%; Caco-2 ≥ -0.4; HL ≥ 4 h; TPSA˂140; DL ≥ 0.18) suggested by the TCMSP database. Moreover, ingredients that exhibit extensive biological activity in TCM are also retained. Third, the effect targets of TCM were screened using the TCMSP database, Swiss Target Prediction and STICH online software. Disease targets were gathered from the therapeutic target database (TTD), PharmGkb and TCMSP database. Hub genes were screened by potential protein-protein interaction (PPI) network pharmacology analysis. Finally, a metabolic network pathway is established between the diagnostic biomarker and the hub gene. In the network analysis of metabolic pathways, most of the genes involved in this pathway are the second-step-obtained hub genes, which can explain the accuracy of the identified biomarkers. The proposed integrated strategy was successfully applied to explore the mechanism of action of Pulsatilla decoction (PD) in the treatment of acute ulcerative colitis (UC). Based on this integrated strategy, 23 potential biomarkers of acute UC treated with PD were identified. In conclusion, the integrated strategy provides novel insights into network pharmacology and metabolomics as effective tools to illuminate the mechanism of action of TCM.

A high-throughput metabolomics approach for the comprehensive differentiation of four Pulsatilla Adans herbs combined with a nontargeted bidirectional screen for rapid identification of triterpenoid saponins.

Pulsatilla Adans (PSA) herbs (Ranunculaceae) have been widely used in traditional medicine in China and other countries. However, the authentication and quality control of PSA herbs have always been a challenging task due to their similar morphological characteristics and the diversity of the multiple components that exist in the complicated matrix. Herein, a novel integrated strategy combining UHPLC/Q-Orbitrap-MS techniques with chemometrics analysis is proposed for the discrimination of PSA materials. We developed a comprehensive method integrating a nontargeted bidirectionally screened (NTBDS) MS data set and a targeted extraction peak area analysis for the characterization of triterpenoid saponins of PSA from different species. After that, partial least-squares discriminant analysis (PLS-DA) was performed on the obtained MS data set and the parameter variable importance for the projection (VIP) value and P value were employed to screen the valuable MS features to discriminate PSA from different species. In addition, the receiver operating characteristic (ROC) curve is used to verify the reliability of MS features. Finally, heatmap visualization was employed to clarify the distribution of the identified triterpenoid saponins, and four medicinal species of PSA were successfully differentiated. Additionally, 34 constituents were reported in PSAs for the first time, 81 triterpenoid saponins were identified as differential components, and 12 chemical ingredients were characterized as potential chemical markers to differentiate the four officinal PSA herbs. This is the first time that the differences in different PSA herbs have been observed systematically at the chemical level. The results suggested that using the identified characteristic components as chemical markers to identify different PSA herbs was effective and viable. This method provides promising perspectives in the analysis and identification of the ingredients of Chinese herbal medicines, and the identification of similar herbs from the same species.

Metabolites identification of (+)-usnic acid in vivo by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

(+)-usnic acid (UA) is an active natural phenolic acid ingredient originating from Chinese traditional Tibetan herb. Usnea acid is expected to become a new agent for anticancer and remarkable antitumor. To reveal its metabolic profile, metabolites identification of UA in vivo was studied using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) in this present study. The chromatographic separation was performed on a C18 column with a mobile phase consisted of methanol and water with a flow rate of 0.4 ml/min. The mass spectral analysis conducted in a negative electrospray ionization mode combined with information-dependent acquirement technology (IDA) was used to trace all the potential UA metabolites. Several sensitive and specific multiple data-mining techniques especially key product ions (KPIs) filter were applied to hunt and identify metabolites rapidly. As a result, a total of 36 metabolites were detected after oral administration of UA, including 33, 8 and 16 in rat urine, plasma and bile, respectively. These results showed that the probable metabolite pathways of UA were oxidation, reduction, dihydroxylation, glycine conjugation, glucuronide conjugation, N-acetylcysteine conjugation and methylation. It is the first time to elucidate the profile of UA in vivo. These results not only provided the basis of UA pharmacological properties, but also gave the guidance in clinical medication. Moreover, the analysis strategy and methodology proposed in this paper could be widely used in characterization of other phenolic acids metabolites.

Study on the metabolites of betulinic acid in vivo and in vitro by ultra high performance liquid chromatography with time-of-flight mass spectrometry.

Betulinic acid is a triterpenoid organic acid with remarkable antitumor properties and is naturally present in many fruits, condiments and traditional Chinese medicines. Currently, a strategy was developed for the identification of metabolites following the in vivo and in vitro biotransformation of Betulinic acid with rat intestinal bacteria utilizing ultra high performance liquid chromatography with time-of-flight mass spectrometry with polymeric solid-phase extraction. As a result, 46 metabolites were structurally characterized. The results demonstrated that Betulinic acid is universally metabolized in vivo and in vitro, and Betulinic acid could undergo general metabolic reactions, including oxidation, methylation, desaturation, loss of O and loss of CH2 . Additionally, the main metabolic pathways in vivo and in vitro were determined by calculating the relative content of each metabolite. This is the first study of Betulinic acid metabolism in vivo, whose results provide novel and useful data for better understanding of the safety and efficacy of Betulinic acid.

UPLC-Q-TOF-MS/MS-guided dereplication of Pulsatilla chinensis to identify triterpenoid saponins.

INTRODUCTION: Triterpenoid saponins are the major bioactive constituents of Pulsatilla chinensis, playing an important role in various biological activities such as anti-tumour, cognition-enhancing, anti-biosis, anti-inflammatory, hypoglycemic and immunological adjuvant. OBJECTIVE: To establish a systematic strategy based on ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) for the efficient characterisation and identification of triterpenoid saponins in crude extracts from Pulsatilla chinensis. METHODOLOGY: In this work, the strategy includes two aspects: (1) positive mode: by target screening, we can deduce the aglycone type and the composition of sugar moiety according to the fragment ions; untargeted screening includes four steps, find unknown, formula finder, ChemSpider search and MS/MS identification; (2) negative mode: according to the MS/MS spectra, the composition of sugar chain bonded to C-28 is inferred reasonably. The extract of Pulsatilla chinensis was separated within 60 min on a C18 column and eluted with methanol and water both containing 0.1% formic acid. RESULTS: As a result, a total of 22 triterpenoid saponins (11 pairs of isomers) with four aglycone skeletons were tentatively identified or elucidated in crude extracts from Pulsatilla chinensis based on their retention times, the mass spectrometric fragmentation patterns, and MS and MS/MS data. CONCLUSION: This study provides an efficient analysis strategy to rapidly identify the triterpenoid saponins in Pulsatilla species even in traditional Chinese medicines.

UHPLC-Q-TOF-MS/MS based screening and identification of the metabolites in vivo after oral administration of betulin.

Betulin is an active natural pentacyclic triterpene ingredient with valuable anti-cancer and anti-HIV efficacies. In the present study, an efficient approach was developed to screening and identification of metabolites and to assess the metabolic profiles of betulin in vivo using UHPLC-Q-TOF-MS/MS system based on multiple mass defect filter data acquisition (MMDF) and multiple data processing techniques. Based on the proposed method, 56 phase I and 6 phase II metabolites were detected in rats after oral administration of betulin. The main biotransformation routes of betulin were identified as demethylation, dehydroxylation, deoxidization, dehydration. Conjugation with sulfate, taurine, cysteine and N-acetylcysteine groups produced 6 phase II metabolites. This study not only provided useful information for further study of the pharmacology and mechanism of betulin in vivo, but also provided essential data for further pharmaceutical studies of other pentacyclic triterpenes.

UHPLC-Q-TOF-MS/MS-oriented characteristic components dataset and multivariate statistical techniques for the holistic quality control of Usnea.

The holistic quality evaluation of Traditional Chinese Medicine (TCM) is confronted with significant challenges due to its extreme chemical complexity. In this study, a sensitive strategy based on ultra-high-performance liquid chromatography-triple/time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) and chemometric analysis was established and validated for the qualitative and semi-quantitative analyses of characteristic components in Usnea. First, three mass spectrometry fragmentation patterns of phenolic acid standards were studied and summarized. Then, an extract of this herb was analyzed by the full-scan MS spectra and identified by extracted ion chromatography (XIC). Based on the abovementioned methods, a total of 38 compounds (8 dibenzofurans, 11 didepsides, 13 depsidones, and 6 mono-substituted phenyl rings) were identified. Subsequently, the qualities of Usnea samples from different regions were evaluated by the semi-quantitative analysis based on their relative peak areas. Furthermore, principal component analysis (PCA) was performed to compare the Usnea herbs and to find possible diagnostic chemical components. This novel and powerful strategy could provide a potential approach for the holistic quality control of TCM.

Discrimination and Chemical Phylogenetic Study of Four Pulsatilla Herbs Using UPLC-ESI-MS/MS Combined with Hierarchical Cluster Analysis.

A tissue-smashing based ultra-rapid extraction coupled with ultra-performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) method was developed to determine 10 major triterpenoid saponins from Pulsatilla herbs. Compound 4 was characterized as betulinic acid glycoside 3-O-α-arabinopyranosyl-28-O-ß-glucopyranosyl-23-hydroxy with HR-ESI-MS, 1H-NMR and 13C-NMR experiment. The MS spectra result showed that the ionization of compound 4 was more efficient in the positive mode. Meanwhile, the ions at m/z 789.6 and m/z 627.5 were selected as precursor and product ion for the determination, respectively. The chromatographic separation was carried out on a Phenomenex Kinetex C18 column using a gradient mobile phase system composed of 0.1% formic acid both in methanol and water at a flow rate of 0.4 mL/min. The detection was performed by multiple reaction monitoring mode, using electrospray ionization in the positive and negative mode. The total run time was 6 min. The calibration curves possessed good linearity with all coefficients higher than 0.9987. The intra- and interday precisions were no more than 4.9%, and the average recoveries were from 97.6% to 103.4% with RSD <4.7%. Moreover, hierarchical cluster analysis was performed to compare and discriminate the Pulsatilla herbs based on the quantitative data. The hierarchical cluster analysis results demonstrated that Pulsatilla chinensis, Pulsatilla cernua, Pulsatilla dahurica, Pulsatilla turczainovii samples could be easily discriminated from each other based on the contents of triterpenoid saponins and the established method is feasible for quality control of Pulsatilla herbs.

Simultaneous determination of 12 active components in the roots of Pulsatilla chinensis using tissue-smashing extraction with liquid chromatography and mass spectrometry.

Ultra high performance liquid chromatography coupled with mass spectrometry and combining a tissue-smashing extraction technique was developed for the simultaneous quantitative analysis of 12 compounds in the roots of Pulsatilla chinensis. Among them, compound 6 was characterized and accurately quantified in this herb for the first time. The parameters of extraction condition were simultaneously optimized with a Box-Behnken design and Derringer's function. The optimized conditions were as follows: sample quantity of 0.5 g, ethanol concentration of 70%, and extraction time of 200 s. Multiple-reaction monitoring scanning was employed for the quantification between positive and negative mode in a single run of 6 min. Full validation of the method was carried out, and the results indicated that the method was rapid, specific, and reliable. The developed method was successfully applied to quantify the 12 compounds in 33 batches of P. chinensis from different provinces. Moreover, the principal component analysis was performed to compare the P. chinensis collected from different provinces of China based on quantitative data and the results indicated that the content of compounds could be used to differentiate the origins of P. chinensis. These results demonstrated that this method is feasible and reliable for the quality control of P. chinensis.

Dietary vitamin C deficiency depressed the gill physical barriers and immune barriers referring to Nrf2, apoptosis, MLCK, NF-κB and TOR signaling in grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare.

This study explored the effects of vitamin C on the physical barriers and immune barriers, and relative mRNA levels of signaling molecules in the gill of grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) (1) increased reactive oxygen species, malondialdehyde and protein carbonyl (PC) contents (P < 0.05), decreased the copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities and mRNA levels (P < 0.05), and glutathione and vitamin C contents (P < 0.05), down-regulated NF-E2-related factor 2 mRNA level (P < 0.05), and up-regulated Kelch-like ECH-associating protein (Keap) 1a (rather than Keap1b) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency induced oxidative injury in fish gill; (2) up-regulated caspase-3, -7, -8, -9, Fas ligand, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1 mRNA levels (P < 0.05), and down-regulated inhibitor of apoptosis protein and B-cell lymphoma-2 (rather than myeloid cell leukemia-1) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated cell apoptosis in fish gill; (3) up-regulated pore-forming TJs Claudin-12, 15a, -15b, and related signaling molecules myosin light chain kinase, p38 mitogen-activated protein kinase (rather than c-Jun N-terminal kinases) mRNA levels (P < 0.05), and down-regulated barrier-forming TJs Occludin, zonula occludens (ZO) 1, ZO-2, Claudin-c, -3c, -7a, -7b mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency disrupted tight junctional complexes in fish gill; (4) decreased lysozyme and acid phosphatase (ACP) activities, and complement 3 (C3), C4 and IgM contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, Hepcidin, ß-defensin mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency decrease fish gill immune function; (5) down-regulated the mRNA levels of anti-inflammatory cytokines-related factors interleukin 10 (IL-10), IL-11, transforming growth factor (TGF) ß1, TGF-ß2, inhibitor of κBa and eIF4E-binding protein 1 (4E-BP1) (rather than 4E-BP2) (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors interferon γ2, IL-1ß, IL-6, IL-8, IL-12 P35, IL-12 P40, nuclear factor κB (NF-κB) p65 (rather than NF-κB p52), IκB kinases (IKK) (only IKKα and IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated fish gill inflammation. In conclusion, vitamin C deficiency disrupted physical barriers and immune barriers, and regulated relative mRNA levels of signaling molecules in fish gill. The vitamin C requirement for against gill rot morbidity of grass carp (264-1031 g) was estimated to be 156.0 mg/kg diet. In addition, based on the gill biochemical indices (antioxidant indices MDA, PC and vitamin C contents, and immune indices LA and ACP activity) the vitamin C requirements for grass carp (264-1031 g) were estimated to be 116.8, 156.6, 110.8, 57.8 and 134.9 mg/kg diet, respectively.

Simultaneous determination of nine coumarins in rat plasma by HPLC-MS/MS for pharmacokinetics studies following oral administration of Fraxini Cortex extract.

A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the simultaneous determination of nine coumarins including aesculin, aesculetin, fraxin, fraxetin, scopoletin, isoscopoletin, 6-hydroxy-7,8-dimethoxy coumarin, 8-hydroxy-6,7-dimethoxy coumarin and umbelliferone in rat plasma using nodakenin as the internal standard (IS). The plasma samples were pretreated by a one-step direct protein precipitation with methanol. The chromatographic separation was carried out on a C18 column with a gradient mobile phase consisting of methanol and water (containing 0.05% acetic acid). All analytes and IS were quantitated through electrospray ionization in negative ion multiple reaction monitoring (MRM) mode. This method was fully validated in terms of the sensitivity, specificity, accuracy, precision (intra- and inter-day), matrix effect, recovery as well as the stability of the analyte under various conditions, and the results satisfied the requirements of biological sample measurement. The validated method was successfully applied to pharmacokinetic study of the nine coumarins in rat plasma after oral administration of Fraxini Cortex aqueous extract, among which the pharmacokinetics of four coumarins including fraxetin, isoscopoletin, 6-hydroxy-7,8-dimethoxy coumarin and 8-hydroxy-6,7-dimethoxy coumarin were studied for the first time.

UPLC-QTOF-MS/MS based screening and identification of the metabolites in rat bile after oral administration of imperatorin.

The detection of drug metabolites, particularly for minor metabolites, continues to be a challenge owing to the complexity of biological samples. Imperatorin is an active natural furocoumarin ingredient originating from many traditional Chinese herbal medicines. In the present study, the metabolites of imperatorin after oral administration were qualitatively investigated, and possible metabolic pathways of it were subsequently proposed. Bile samples were collected after oral administration and pretreated by the application of Waters Ostro. The QTOF-MS/MS data was acquired using ultra high performance liquid chromatography coupled to quadrupole time flight spectrometry (UPLC-QTOF-MS). Based on this analytical strategy, 32 metabolites (23 phase I and 9 phase II metabolites) were identified in rat bile. The results demonstrated that C5H8 could be easily eliminated from imperatorin forming the metabolite M1. It also indicated that imperatorin and M1 underwent extensive metabolic reactions including oxidation, hydrolysis, methylation, glucuronide conjugation, C2H5NO2S conjugation and C3H5NO2S conjugation. This is the first study of imperatorin metabolism in bile samples. The proposed metabolic pathways in this research will provide essential data for further pharmaceutical studies of other linear-type furocoumarins.