Simultaneous determination of bioactive constituents in Danggui Buxue Tang for quality control by HPLC coupled with a diode array detector,an evaporative light scattering detector and mass spectrometry

Danggui Buxue Tang (DBT), a classical traditional Chinese formula comprising Radix Angelicae Sinensis (RAS) and Radix Astragali (RA), has been widely used to treat menopausal irregularity in Chinese women for nearly 800 years. In this study, a comprehensive analytical method of simultaneously determining the main types of bioactive constituents, eighteen in all from the formula, involving flavonoids, saponins, organic acid and some volatile compounds, was developed. This method was based on HPLC coupled to a diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) on a common reverse-phase C(18) column. Liquid chromatography coupled with on-line electrospray ionization mass spectrometry (LC-ESI-MS) was also used to further validate and analyze 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 good precision and accuracy, was successfully used to quantify the bioactive constituents in six products. As a result, the validated HPLC method, together with the LC-ESI-MS analysis, provided a new basis for assessing the quality of traditional Chinese medicinal compound preparations (TCMCPs) consisting of many bioactive components.     

Study on the dynamic metabolic characteristic of main active ingredients in Danggui Buxue Decoction by liquid chromatography-tandem mass spectrometry based on in situ sequential metabolism strategy

Danggui Buxue Decoction is a classic formula for replenishing qi and nourishing blood. Despite its widespread use, its dynamic metabolism involved remains unclear. Based on the sequential metabolism strategy, blood samples from different metabolic sites were obtained via in situ closed intestine ring integrated with a jugular venous continuous blood supply technique. An ultra-high-performance liquid chromatography-linear triple quadruple-Orbitrap-tandem mass spectrometry method was developed for the identification of prototypes and metabolites in rat plasma. The dynamic absorption and metabolic landscape of flavonoids, saponins, and phthalides were characterized. Flavonoids could be deglycosylated, deacetylated, demethylated, dehydroxylated, and glucuronicated in the gut and then absorbed for further metabolism. Jejunum is an important metabolic site for saponins biotransformation. Saponins that are substituted by Acetyl groups tend to lose their acetyl groups and convert to Astragaloside IV in the jejunum. Phthalides could be hydroxylated and glucuronidated in the gut and then absorbed for further metabolism. Seven components serve as crucial joints in the metabolic network and are potential candidates for the quality control of Danggui Buxue Decoction. The sequential metabolism strategy described in this study could be useful for characterizing the metabolic pathways of Chinese medicine and natural products in the digestive system.     

Metabolic infrastructure of pregnant women with methylenetetrahydrofolate reductase polymorphisms: A metabolomic analysis

The aim of this study was to demonstrate the altered metabolic infrastructure of pregnant women with methylenetetrahydrofolate reductase (MTHFR) polymorphisms at first trimester and during delivery. Eight singleton pregnant women with MTHFR polymorphisms were compared with 10 normal pregnant women. Maternal blood samples were obtained twice during their pregnancy period (between the 11th and 14th gestational weeks and during delivery). Metabolomic analysis was performed using GC–MS. The GC–MS based metabolomic profile helped identify 95 metabolites in the plasma samples. In the MTHFR group, the levels of 1-monohexadecanoylglycerol, pyrophosphate, benzoin, and linoleic acid significantly decreased (P ˂ 0.05 for all), whereas the levels of glyceric acid, l -tryptophan, l -alanine, l -proline, norvaline, l -threonine, and myo-inositol significantly increased (P ˂ 0.01 for the first two metabolites, P ˂ 0.05 for the others) at 11–14 gestational weeks. Conversely, the levels of benzoin, 1-monohexadecanoylglycerol, pyruvic acid, l -proline, phosphoric acid, epsilon-caprolactam, and pipecolic acid significantly decreased in the MTHFR group, whereas metabolites such as hexadecanoic acid and 2-hydroxybutyric acid increased significantly in the study group during delivery. An impaired energy metabolism pathway, vitamin B complex disorders, tendency for metabolic acidosis (oxidative stress), and the need for cell/tissue support seem prevalent in pregnancies with MTHFR polymorphisms.     

UPLC–QTOF/MS‐based metabolomics reveals the mechanism of chronic unpredictable mild stress‐induced hypertension in rats

Hypertension is a common chronic disease, and it is the strongest risk factor for cardiovascular disease. Recently, the number of patients with hypertension‐related complications has increased significantly, adding a heavy burden to the public health system. It is known that chronic stress plays an important role in the pathogenesis of cardiovascular diseases such as hypertension and stroke. However, the impact of hypertension on the dysfunctions induced by chronic stress remains poorly understood. In this study, using LC–MS‐based metabolomics, we established a chronic stress model to demonstrate the mechanisms of stress‐induced hypertension. We found that 30 metabolites in chronically stressed rats were changed; of these metabolites, seven had been upregulated, and 23 had been downregulated, including amino acids, phospholipids, carnitines and fatty acids, many of which are involved in amino acid metabolism, cell membrane injury, ATP supply and inflammation. These metabolites are engaged in dysregulated pathways and will provide a targeted approach to study the mechanism of stress‐induced hypertension.     

Urinary metabolomic study of systemic lupus erythematosus based on gas chromatography/mass spectrometry

Systemic lupus erythematosus (SLE) is an autoimmune disease with heterogeneous organ and system manifestations. In this study, urinary metabolic alterations related to SLE were investigated by performing gas chromatography/mass spectrometry (GC/MS) based metabolomics and multivariate statistical analysis. Patients with SLE and healthy controls could be clearly differentiated in view of the metabolic abnormity in urine. Among 70 identified endogenous metabolites, 23 metabolites were dramatically increased in SLE patients, which involved in several key metabolic pathways including energy metabolism, nucleotide metabolism, oxidative stress and gut‐microbiome‐derived metabolism. This noninvasive and GC/MS‐based metabolomic technique is a promising and potent strategy for identifying novel biomarkers and understanding pathogenesis of SLE. Copyright © 2016 John Wiley & Sons, Ltd.     

Glucose challenge metabolomics implicates the change of organic acid profiles in hyperlipidemic subjects

Hyperlipidemia (HLP) is a major risk factor of diabetes and cardiovascular disease. Here, we applied gas chromatography–mass spectrometry to study differences in postprandial organic acid profiles in healthy and HLP subjects. In fasting status, six intermediates of the tricarboxylic acid cycle showed significant differences in HLP and healthy controls (P < 0.05). The percentage changes of 17 metabolites including three intermediates of the tricarboxylic acid cycle were significantly different during the oral glucose tolerance test. Postprandial changes in ethylmalonic acid and pimelic acid were negatively associated with HOMA-IR (homeostasis model assessment of insulin resistance; all P < 0.05) in the HLP group. Postprandial metabolism of organic acid profiles revealed energy metabolism perturbations in HLP. Our findings provide new insights into the complex physiological regulation of HLP postprandial metabolism.     

Metabolomics study of cured tobacco using liquid chromatography with mass spectrometry: Method development and its application in investigating the chemical differences of tobacco from three growing regions

Cured tobacco is an important plant material. Component studies are a big challenge for its significantly diverse chemical properties and vastly different concentrations. In this work, liquid chromatography with quadrupole time‐of‐flight mass spectrometry was used to perform a metabolomics study of cured tobacco owing to its efficient separation and detection of semipolar metabolites. A solvent of methanol/water (8:2, v/v) and 30 min of ultrasound time were found to be optimal to perform extraction. 95, 92, and 93% of metabolite features had within 20% of coefficient of variation for repeatability, intraday and interday precision analysis, respectively, indicating a good stability of the method developed. 113 metabolites were identified in cured tobacco based on accurate mass, retention time, and MS/MS fragments. The developed method was applied to a metabolomics study of cured tobacco from three growing regions. Forty three metabolites were found to be contributed to the classification. It is shown that the developed method can be applied to metabolomics analysis of plant materials.     

Comprehensive investigation of mechanism and effective ingredients of Fangji Huangqi Tang by serum pharmacochemistry and network pharmacology

Fangji Huangqi Tang (FHT), has been reported to show effects on nephrotic syndrome, but its mechanism of action and bioactive components have not yet been determined. In this study, a method using UPLC–HRMS/MS was established for the detection and identification of the chemical constituents and metabolites absorbed into the blood. Absorbed components in serum were then used for the network pharmacology analysis to deduce the mechanism and effective components. A total of 86 compounds were identified or tentatively characterized. Based on the same instrumental conditions, 85 compounds were found in rat serum after oral administration of FHT, including 22 prototypes and 63 metabolites. Network pharmacology analysis showed that absorbed components, such as (3R)-2′,3′,4′,7-tetrahydroxyisoflavan, astrapterocarpan, cycloastragenol, 7,2′-dihydroxy-3′,4′-dimethoxyisoflavan, astragaloside IV, astrapterocarpan glucoside and glycyrrhetinic acid, could be responsible for the pharmacological activity of nephrotic syndrome by regulating the VEGF signaling pathway, focal adhesion and MAPK signaling pathway. Furthermore, the pathway-target network showed that the MAPK1, AKT2 and CDC42 were involved in the signal pathways above. This study provides a scientific basis for the mechanism and effective ingredients of FHT.     

Urinary metabolite profiling provides potential differentiation to explore the mechanisms of adjuvant‐induced arthritis in rats

To explore the pathogenesis of rheumatoid arthritis (RA) from the perspective of metabolomics, gas chromatography time‐of‐flight mass spectrometry (GC‐TOF/MS) technology was used to observe changes in the metabolic profiles of urine output from rats with adjuvant‐induced arthritis (AA). Sprague–Dawley rats were randomly divided into a control group and an experimental group, with eight in each. Rats in the experimental group were induced by intracutaneous innoculation of 0.1 mL Freund's complete adjuvant to right paws. On day 20 after immunization, the metabolic profiles between rat control and experimental groups were compared by combining GC‐TOF/MS technology with multivariate statistical approaches, including principal component analysis, partial least squares discriminant analysis and orthogonal projections to latent structures–discriminant analysis. Nine potential biomarkers were identified, including 2,2‐dimethylsuccinic acid, tartronic acid, dehydroshikimic acid, hippuric acid, adenine, phenaceturic acid, l ‐dopa, 1,4‐dihydroxy‐2‐naphthoic acid and melibiose. The findings indicate that the rats with AA are disturbed in metabolism of purine, amino acid, fat and energy. This study also demonstrates that the dysfunction in a range of biosynthetic and catabolic pathways, which leads to increased oxygen free radicals and inflammation, could cause underlying pathogenesis of RA. Copyright © 2016 John Wiley & Sons, Ltd.     

Metabolomics study on promoting blood circulation and ameliorating blood stasis: Investigating the mechanism of Angelica sinensis and its processed products

Angelica sinensis (Danggui, DG) parched with alcohol (Jiu Danggui, JDG) and charred DG are the main processed products of DG, which are used to treat blood stasis syndrome (BSS). However, their therapeutic effect and mechanisms are still unclear. Based on an acute rat BSS model, the intervention effects of DG and its processed products (DGPPs) were evaluated by the hemorheology and coagulation function parameters. Meanwhile, plasma and urine metabolites were detected and analyzed by liquid chromatography coupled to quadrupole time‐of‐flight mass spectrometry and multivariate statistical analysis method. The results of hemorheology, coagulation function parameters and metabolomics all showed that the BSS model was successfully established, DGPPs intervention could significantly relieve rats BSS and the therapeutic effect of JDG was best. Moreover, 23 differential metabolites (14 in plasma and nine in urine) were identified that were closely related to the BSS, involving seven potential target metabolic pathways. DGPP intervention showed different degrees of reverse effect on these metabolites. JDG was the most effective owing to extensive regulation effect on differential metabolites. This study provides a reference for understanding the pathological mechanism of BSS and the mechanism of DGPPs, which lays a theoretical foundation for the rational use of DGPPs in clinical practice.     

Metabolomics analysis of the therapeutic mechanism of Semen Descurainiae Oil on hyperlipidemia rats using 1H‐NMR and LC–MS

Semen descurainiae oil (SDO) is an important traditional Chinese medicine that was recently discovered to have the function of reducing blood lipids. Metabolomics analyses of plasma, liver and kidney in rats were performed using ¹H‐NMR and LC–MS to illuminate the lower blood lipid concentration effect of SDO, and niacin was considered as the active control. The measure of total cholesterol (TC) and low‐density‐lipoprotein cholesterol (LDL‐C) in plasma showed that SDO treatment decreased significantly the content of TC and LDL‐C. An orthogonal partial least squares–discriminant analysis approach was applied to identify the different metabolic profiles of plasma, liver and kidney in rats and to detect related potential biomarkers. The results suggested that the metabolic profiles of the control group and hyperlipidemia group showed significant difference and the SDO and niacin group had effective anti‐hyperlipidemia function. The biomarkers primarily concern lipid metabolism, amino acid metabolism and glycometabolism, and the change in biomarkers indicated that hyperlipidemia could cause the unbalance of these metabolic pathways in vivo. SDO reduced blood lipids by repairing amino acid and lipid metabolism.     

Nontargeted urine metabolomics analysis of the protective and therapeutic effects of Citri Reticulatae Chachiensis Pericarpium on high-fat feed-induced hyperlipidemia in rats

In this study, we focused on studying the changes in urine metabolites in hyperlipidemic rats using ultra-performance liquid chromatography coupled with quadrupole time-of-fight mass spectrometry (UPLC–Q-TOF/MS) and metabolomics, as well as the effect of Citri Reticulatae Chachiensis Pericarpium (CRCP) on hyperlipidemia. These urine samples were examined by UPLC–Q-TOF/MS to obtain MS data. The MS data were analyzed by principal component analysis and partial least squares-discriminant analysis to identify the differential metabolites. CRCP reduced the body weight and levels of triglycerides, total cholesterol and low-density lipoprotein cholesterol and abnormally decreased high-density lipoprotein cholesterol in hyperlipidemic rats, which were significantly raised by a high-fat diet. Twenty-seven potential biomarkers were identified within the complex sample matrix of urine. Fourteen biomarkers increased in the hyperlipidemia rats compared with normal rats. Meanwhile, 13 biomarkers decreased. CRCP reversed abnormal changes in biomarkers, including 5-l -glutamyl-taurine, 5-aminopentanoic acid, cis-4-octenedioic acid and 2-octenedioic acid. These biomarkers show that hyperlipidemia is related to the metabolic pathways of taurine and hypotaurine metabolism, fatty acid biosynthesis , and arginine and proline metabolism . CRCP mainly prevents hyperlipidemia by intervening in these metabolic pathways.     

Overall metabolic network analysis of urine in hyperlipidemic rats treated with Bidens bipinnata L.

Hyperlipidemia has been highlighted as one of the most prominent and global chronic conditions nowadays. Bidens bipinnata L. (BBL), a folk medicine in contemporary China, has efficacy in the treatment of hyperlipidemia (HLP) in China. Although some physiological and pathological function parameters of hyperlipidemia have been investigated, little information about the changes in small metabolites in biofluids has been reported. In the present study, global metabolic profiling with high-performance liquid chromatography–linear ion trap/Orbitrap high-resolution mass spectrometry (HPLC–LTQ/Orbitrap MS) combined with a pattern recognition method was performed to discover the underlying lipid-regulating mechanisms of BBL on hyperlipidemic rats induced by high-fat diet (HFD). The total of four metabolites, up- or down-regulated (p < 0.05 or 0.01), were identified and contributed to the progression of hyperlipidemia. These promising identified biomarkers underpin the metabolic pathway, including glyoxylate and dicarboxylate metabolism, the TCA cycle, sphingolipid metabolism and purine metabolism. They are disturbed in hyperlipidemic rats, and are identified using pathway analysis with MetPA. The altered metabolite indices could be regulated closer to normal levels after BBL intervention. The results demonstrated that urinary metabolomics is a powerful tool in the clinical diagnosis and treatment of hyperlipidemia to provide information on changes in metabolite pathways.     

Metabonomics study on the effect of Siwu Decoction for blood deficiency syndrome in rats using UPLC–Q/TOF–MS analysis

Siwu decoction (SWD), a traditional Chinese medicinal formula with over 1000 years of clinical history, is widely used for gynecological disease, especially blood deficiency syndrome, which is similar to anemia in modern medicine. In view of metabonomics being useful approach to investigate the potential mechanisms of action from the point of view of systems biology, in this study an ultra‐performance liquid chromatography–quadrupole‐time of flight mass spectrometry method was employed for a holistic evaluation of SWD on a blood‐deficiency rat model induced by N‐acetylphenylhydrazine and cyclophosphamide via plasma metabonomics study. Routine blood examination results showed that SWD could significantly improve the declining hemogram indices. Meanwhile, the plasma metabonomics profiles in different groups were analyzed and differentiating metabolites were primarily visualized through chemometric analysis. Seven biomarkers were identified in plasma samples of blood‐deficiency rat model compared with the normal group. Five main metabolism pathways were suggested using the Kyoto Encyclopedia of Genes and Genomes Pathway Analysis and Pathway Activity Profiling algorithm analysis. This indicated that SWD played a therapeu role in blood deficiency by regulating the aberrant endogenous metabolites. To sum up, this study provides clear evidence that a metabonomics study could serve as a useful tool to elucidate the systematic therapeutic profiles and mechanisms for blood deficiency syndrome of Chinese herbal medicines.     

基于代谢组学技术的快速蒸发电离质谱与智能手术刀联用实时鉴别果汁掺伪方法研究

近年来,果汁掺伪问题日益突出.作为一种新型的实时鉴别方法,智能手术刀(IKnife)与快速蒸发电离质谱(REIMS)联用技术无需样品前处理即可解决这一问题.该文采用代谢组学指纹识别技术建立橙汁、苹果汁和葡萄汁的实时鉴别方法,并对其掺假水平进行预测.主成分分析-线性判别分析联用法建立的判别模型能够区分不同类型的果汁,排除...