Hong-Mei Jia

Qualitative and quantitative characterization of chemical constituents in Xin-Ke-Shu preparations by liquid chromatography coupled with a LTQ Orbitrap mass spectrometer.

Xin-Ke-Shu (XKS), a traditional Chinese medicine (TCM) preparation containing five herbal medicines, has been commonly used for the treatment of coronary heart disease in China. However, the chemical constituents in XKS have not been clarified yet. In order to quickly define the chemical profiles and control the quality of XKS preparations, liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap (LC-LTQ-Orbitrap) mass spectrometry was applied for simultaneous identification and quantification of multi-constituent. A total of 51 compounds, including phenolic acids, isoflavone-C-glycosides, isoflavone-O-glycosides, flavonoids, and triterpenoid saponins, were identified or tentatively deduced on the base of their retention behaviors, MS and MS(n) data, or by comparing with reference substances and literatures. In addition, an optimized LC-ESI-MS method was established for quantitative determination of 15 marker compounds in XKS preparations from 7 independent pharmaceutical companies. The validation of the method, including spike recoveries, linearity, sensitivity (LOD and LOQ), precision, and repeatability, was carried out and demonstrated to be satisfied the requirements of quantitative analysis. This is the first report on the comprehensive determination of chemical constituents in XKS preparations by LC-LTQ-Orbitrap mass spectrometry. The results suggested that the established methods would be a powerful and reliable analytical tool for the characterization of multi-constituent in complex chemical system and quality control of TCM preparations.

Metabolic pathways involved in Xin-Ke-Shu protecting against myocardial infarction in rats using ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Xin-Ke-Shu (XKS) is a patent drug used for coronary heart diseases in China. This study evaluated the protective effect of XKS against isoproterenol (ISO)-induced myocardial infarction (MI). For its underlying mechanism in rats with MI, a metabonomic approach was developed using ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/QTOF-MS). Plasma metabolites were profiled in MI rats, pretreated orally with or without XKS. Two genres of metabolic biomarkers were used to elucidate the pharmacological action of XKS: pathological biomarkers and pharmaco biomarkers. Fifteen metabolites significantly varying between MI rats and normal rats were characterized as potential pathological biomarkers related to MI, including L-acetylcarnitine (1), L-isoleucyl-L-proline (2), tyramine (3), isobutyryl-L-carnitine (4), phytosphingosine (5), sphinganine (6), L-palmitoylcarnitine (7), lysoPC(18:0) (8), uric acid (9), L-tryptophan (10), lysoPC(18:2) (11), lysoPC(16:0) (12), docosahexaenoic acid (13), arachidonic acid (14) and linoleic acid (15). Among them, eight (1-6, 9 and 10) were first reported as pathological biomarkers related to ISO-induced MI, which mainly involved into fatty acid β-oxidation pathway, sphingolipid metabolism, proteolysis, tryptophan metabolism and purine metabolism. The metabolites significantly varying between MI rats with and without XKS pretreatment were considered as pharmaco biomarkers. A total of 17 pharmaco biomarkers were recognized, including 15 pathological biomarkers (1-15), hexanoylcarnitine (16) and tetradecanoylcarnitine (17). The results suggested that pretreatment of XKS protected metabolic perturbations in rats with MI, major via lipid pathways, amino acid metabolism and purine metabolism, which also provided a promising approach for evaluating the pharmacodynamics and mechanism of traditional Chinese medicines (TCM) formulas.

UPLC-Q/TOF MS standardized Chinese formula Xin-Ke-Shu for the treatment of atherosclerosis in a rabbit model

Xin-Ke-Shu (XKS), a patent traditional Chinese medicine (TCM) preparation, has been commonly used for the treatment of coronary heart disease in China. In order to understand its mechanism of action, a metabonomic approach based on ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS) was utilized to profile the plasma metabolic fingerprints of atherosclerosis (AS) rabbits with and without XKS treatment. The metabolic profile of model group clearly separated from normal, and that of XKS group was closer to the control group. Metabolites with significant changes during atherosclerosis were characterized as potential biomarkers related to the development of atherosclerosis by using orthogonal partial least-squares-discriminate analysis (OPLS-DA). Twenty potential biomarkers, including l-acetylcarnitine (1), propionylcarnitine (2), unknown (3), phytosphingosine (4), glycoursodeoxycholic acid (5), LPC(14:0) (6), sphinganine (7), LPC(20:5) (8), LPC(16:1) (9), LPC(18:2) (10), LPC(18:3) (11), LPC(22:5) (12), LPC(16:0) (13), LPC(18:1) (14), LPC(22:4) (15), LPC(17:0) (16), LPC(20:2) (17), elaidic carnitine (18), LPC(18:0) (19) and LPC(20:1) (20), were identified by their accurate mass and MS(E) spectra. The derivations of those biomarkers can be regulated by administration of XKS, which suggested that the intervention effect of XKS against AS may involve in regulating the lipid perturbation including fatty acid β-oxidation pathway, sphingolipid metabolism, glycerophospholipid metabolism and bile acid biosynthesis. This study indicated that the UPLC-Q/TOF MS-based metabonomics not only gave a systematic view of the pathomechanism of AS, but also provided a powerful tool to study the efficacy and mechanism of complex TCM prescriptions.

Variations in gut microbiota and fecal metabolic phenotype associated with depression by 16S rRNA gene sequencing and LC/MS-based metabolomics

&NA; As a prevalent, life‐threatening and highly recurrent psychiatric illness, depression is characterized by a wide range of pathological changes; however, its etiology remains incompletely understood. Accumulating evidence supports that gut microbiota affects not only gastrointestinal physiology but also central nervous system (CNS) function and behavior through the microbiota‐gut‐brain axis. To assess the impact of gut microbiota on fecal metabolic phenotype in depressive conditions, an integrated approach of 16S rRNA gene sequencing combined with ultra high‐performance liquid chromatography‐mass spectrometry (UHPLC–MS) based metabolomics was performed in chronic variable stress (CVS)‐induced depression rat model. Interestingly, depression led to significant gut microbiota changes, at the phylum and genus levels in rats treated with CVS compared to controls. The relative abundances of the bacterial genera Marvinbryantia, Corynebacterium, Psychrobacter, Christensenella, Lactobacillus, Peptostreptococcaceae incertae sedis, Anaerovorax, Clostridiales incertae sedis and Coprococcus were significantly decreased, whereas Candidatus Arthromitus and Oscillibacter were markedly increased in model rats compared with normal controls. Meanwhile, distinct changes in fecal metabolic phenotype of depressive rats were also found, including lower levels of amino acids, and fatty acids, and higher amounts of bile acids, hypoxanthine and stercobilins. Moreover, there were substantial associations of perturbed gut microbiota genera with the altered fecal metabolites, especially compounds involved in the metabolism of tryptophan and bile acids. These results showed that the gut microbiota was altered in association with fecal metabolism in depressive conditions. These findings suggest that the 16S rRNA gene sequencing and LC–MS based metabolomics approach can be further applied to assess pathogenesis of depression. Graphical abstract Depression not only perturbs the gut microbiota at abundance level but that it also alters the fecal metabolic phenotype. In addition, there was a strong correlation between gut microbiota and fecal metabolites. Figure. No caption available. HighlightsA combined 16S rRNA gene sequencing and MS‐based metabolomics has been established.Depression led to significant changes of gut microbiota at phylum and genus levels.Depression led to significant changes of fecal metabolic phenotype.Strong correlation of gut microbiota, fecal metabolites, and catecholamine levels.

Aberrant purine metabolism in allergic asthma revealed by plasma metabolomics

Asthma is a disease characterized by chronic relapsing airways, and its etiology remains incompletely understood. To better understand the metabolic phenotypes of asthma, we investigated a plasma metabolic signature associated with allergic asthma in ovalbumin (OVA)-sensitized mice by using ultra high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Sixteen metabolites were characterized as potential pathological biomarkers related to asthma. Among them, 6 (dodecanoic acid (P1), myristic acid (P2), phytosphingosine (P3), sphinganine (P4), inosine (P13) and taurocholic acid (P15)) were first reported to have potential relevance in the pathogenesis of experimental asthma. The identified potential biomarkers were involved in 6 metabolic pathways and achieved the most entire metabolome contributing to the formation of allergic asthma. Purine metabolism was the most prominently influenced in OVA-induced asthma mice according to the metabolic pathway analysis (MetPA), suggesting that significantly changes in inflammatory responses in the pathophysiologic process of asthma. The metabolites of purine metabolism, especially uric acid (P12) and inosine (P13), may denote their potential as targeted biomarkers related to experimental asthma. The decreased plasma uric acid (P12) suggested that inflammation responses of allergic asthma inhibited the activity of xanthine oxidase in purine metabolism, and manifested the severity of asthma exacerbation. The increased level of inosine (P13) suggests that inflammatory cells induce adenosine triphosphate (ATP) breakdown, resulting in excessive expression of adenosine deaminase (ADA) in the formation of allergic asthma. These findings provided a novel perspective on the metabolites signatures related to allergic asthma, which provided us with new insights into the pathogenesis of asthma, and the discovery of targets for clinical diagnosis and treatment.

Integration of 1H NMR and UPLC-Q-TOF/MS for a Comprehensive Urinary Metabonomics Study on a Rat Model of Depression Induced by Chronic Unpredictable Mild Stress

Depression is a type of complex psychiatric disorder with long-term, recurrent bouts, and its etiology remains largely unknown. Here, an integrated approach utilizing 1H NMR and UPLC-Q-TOF/MS together was firstly used for a comprehensive urinary metabonomics study on chronic unpredictable mild stress (CUMS) treated rats. More than twenty-nine metabolic pathways were disturbed after CUMS treatment and thirty-six potential biomarkers were identified by using two complementary analytical technologies. Among the identified biomarkers, nineteen (10, 11, 16, 17, 21–25, and 27–36) were firstly reported as potential biomarkers of CUMS-induced depression. Obviously, this paper presented a comprehensive map of the metabolic pathways perturbed by CUMS and expanded on the multitude of potential biomarkers that have been previously reported in the CUMS model. Four metabolic pathways, including valine, leucine and isoleucine biosynthesis; phenylalanine, tyrosine and tryptophan biosynthesis; tryptophan metabolism; synthesis and degradation of ketone bodies had the deepest influence in the pathophysiologic process of depression. Fifteen potential biomarkers (1–2, 4–6, 15, 18, 20–23, 27, 32, 35–36) involved in the above four metabolic pathways might become the screening criteria in clinical diagnosis and predict the development of depression. Moreover, the results of Western blot analysis of aromatic L-amino acid decarboxylase (DDC) and indoleamine 2, 3-dioxygenase (IDO) in the hippocampus of CUMS-treated rats indicated that depletion of 5-HT and tryptophan, production of 5-MT and altered expression of DDC and IDO together played a key role in the initiation and progression of depression. In addition, none of the potential biomarkers were detected by NMR and LC-MS simultaneously which indicated the complementary of the two kinds of detection technologies. Therefore, the integration of 1H NMR and UPLC-Q-TOF/MS in metabonomics study provided an approach to identify the comprehensive potential depression-related biomarkers and helpful in further understanding the underlying molecular mechanisms of depression through the disturbance of metabolic pathways.

Chronic unpredictive mild stress leads to altered hepatic metabolic profile and gene expression

Depression is a complex disease characterized by a series of pathological changes. Research on depression is mainly focused on the changes in brain, but not on liver. Therefore, we initially explored the metabolic profiles of hepatic extracts from rats treated with chronic unpredictive mild stress (CUMS) by UPLC-Q-TOF/MS. Using multivariate statistical analysis, a total of 26 altered metabolites distinguishing CUMS-induced depression from normal control were identified. Using two-stage receiver operating characteristic (ROC) analysis, 18 metabolites were recognized as potential biomarkers related to CUMS-induced depression via 12 metabolic pathways. Subsequently, we detected the mRNA expressions levels of apoptosis-associated genes such as Bax and Bcl-2 and four key enzymes including Pla2g15, Pnpla6, Baat and Gad1 involved in phospholipid and primary bile acid biosynthesis in liver tissues of CUMS rats by real-time qRT-PCR assay. The expression levels of Bax, Bcl-2, Pla2g15, Pnpla6 and Gad1 mRNA were 1.43,1.68, 1.74, 1.67 and 1.42-fold higher, and those of Baat, Bax/Bcl-2 ratio mRNA were 0.83, 0.85-fold lower in CUMS rats compared with normal control. Results of liver-targeted metabonomics and mRNA expression demonstrated that CUMS-induced depression leads to variations in hepatic metabolic profile and gene expression, and ultimately results in liver injury.

Identification of the Chemical Constituents in Aqueous Extract of Zhi-Qiao and Evaluation of Its Antidepressant Effect

The immature fruit of Citrus aurantium L. (Zhi-Qiao, ZQ) has been used as a traditional medicine in China. Our previous study has shown that ZQ decoction may contribute to the antidepressant-like action of Chaihu-Shu-Gan-San. However, there are no reports on the chemical constituents of ZQ aqueous extract or its anti-depression effects. Firstly, this research reported the on-line identification of the chemical constituents in the aqueous extract of ZQ by coupling ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC-Q-TOF/MS). A total of 31 chemical constituents were identified in ZQ aqueous extract, including one tannic acid, five flavones, 13 flavanones, one limonoid, three coumarins, three cyclic peptides, and five polymethoxylated flavonoids. The antidepressant effect of ZQ aqueous extract was evaluated in vivo and the results indicated that the mice immobility time during the forced swimming test and the tail suspension test were significantly reduced with ZQ treatment. MTT assays showed both ZQ aqueous extract and its major constituents (naringin, hesperidin, neohesperidin, and nobiletin) had neuroprotective effect on corticosterone-induced neurotoxicity in PC12 cells. The in vivo and in vitro results suggest that ZQ has an antidepressant effect.

Standardized Chinese Formula Xin-Ke-Shu inhibits the myocardium Ca 2+ overloading and metabolic alternations in isoproterenol-induced myocardial infarction rats

Xin-Ke-Shu (XKS) is a traditional Chinese patent medicine used for treatment of coronary heart diseases in China. However, its mechanism of action is still unclear. In this paper, the mediation of XKS on the isoproterenol (ISO)-induced myocardial infarction (MI) rat were evaluated based on a tissue-targeted metabonomics in vitro/vivo. The result indicated that twelve metabolic pathways were involved in the therapeutic effect of XKS in vivo, where seven pathways were associated with the Ca2+ overloading mechanism. In agreement with regulation on metabolic variations, XKS markedly reversed the over-expressions of three involved proteins including phospholipase A2 IIA (PLA2 IIA), calcium/calmodulin-dependent protein kinase II (CaMK II) and Pro-Caspase-3. The metabolic regulations of XKS on H9c2 cell also partially confirmed its metabolic effect. These metabolic characteristics in vitro/vivo and western blotting analysis suggested that XKS protected from MI metabolic perturbation major via inhibition of Ca2+ overloading mechanism. Furthermore, 11 active ingredients of XKS exerted steady affinity with the three proteins through the molecular docking study. Our findings indicate that the metabonomics in vitro/vivo combined with western blotting analysis offers the opportunity to gain insight into the comprehensive efficacy of TCMs on the whole metabolic network.

Simultaneous HPLC Determination of Costunolide and Dehydrocostuslactonein Xin-ke-shu Preparations

The objective of this study is to establish a quantitative method for simultaneous determination of costunolide and dehydrocostuslactone in Xin-ke-shu preparations. The HPLC quantitative analysis was established on a Hypersil BDS column (100� 4.6 mm, 2.4 μm, Thermo) with UV detection at 225 nm. The mobile phase was water and methanol (30:70, v/v), and used at a flow rate at 0.5 mL·min - 1 . The established method showed a good linearity (R 2 > 0.9999) over the investigated concentration ranges (0.07-2.80 μg), good inter-day and intra-day precisions (less than 3%) and good recoveries (from 99.82% to 99.98%) for both target compounds. The method was found to be suitable for simultaneous determination of costunolide and dehydrocostuslactone in Xin-ke-shu preparations and can be used as a comprehensive approach to the previous reported quality control of Xin-ke-shu preparations.