Shuping Wang

Metabolomic analysis of biochemical changes in the plasma and urine of collagen-induced arthritis in rats after treatment with Huang-Lian-Jie-Du-Tang

ETHNOPHARMACOLOGICAL RELEVANCE Huang-Lian-Jie-Du-Tang (HLJDT oren-gedoku-to in Japanese), a classical traditional Chinese medicine (TCM) formula, is well known for the treatment of inflammatory-related diseases such as gastritis, dermatitis, and ulcerative colitis. Our previous studies have indicated that HLJDT has therapeutic potential in rheumatoid arthritis treatment. To investigate the therapeutic mechanism of a traditional Chinese medicine formula Huang-Lian-Jie-Du-Tang (HLJDT oren-gedoku-to in Japanese) and its constituents combination for collagen-induced arthritis in rats using a metabolomics approach. MATERIALS AND METHODS Rats were divided into 9 groups, and drugs were administered from on the day after the onset of arthritis (day 12) until day 31 of the experiment once daily continuously. Urine and plasma were analyzed by reversed-phase liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS). Partial least-squares discriminate analysis (PLS-DA) models were built to evaluate the therapeutic effects of HLJDT and its constituents combination. 15 identified CIA biomarkers were investigated to explain its therapeutic mechanism. RESULTS Administration of HLJDT and its constituents combination in CIA rats not only significantly reduced arthritic scores and serum levels of IL-1β but also improved histopathologic changes in joint architecture. Urinary and plasma metabolic profiling revealed that perturbation of energy metabolism, lipid metabolism, oxidative injury and some amino acids metabolism occurred in collagen-induced arthritis (CIA). Our results also indicated that the disturbed urinary levels of succinic acid, citric acid, creatine, uridine, pantothenic acid, carnitine, phenylacetylglycine, allantoin and plasma levels of phenylpyruvic acid in model rats were gradually restored to normal after administration of HLJDT. The treatment of constituents combination of HLJDT group was able to restore to normal the disturbed urinary levels of citric acid, creatine, pantothenic acid, carnitine, pantothenic acid, phenylacetylglycine and plasma levels of uric acid, L-histidine, and l-phenylalanine in model rats. CONCLUSIONS Our study indicates that HLJDT and its constituents combination treatment can ameliorate CIA through partially regulating the perturbed energy metabolism. Our work demonstrated that metabonomics-based approach is a promising new tool to evaluate the therapeutic effects and mechanism of complex TCM prescriptions.

Plasma pharmacochemistry based approach to screening potential bioactive components in Huang-Lian-Jie-Du-Tang using high performance liquid chromatography coupled with mass spectrometric detection

ETHNOPHARMACOLOGICAL RELEVANCE Huang-Lian-Jie-Du-Tang (HLJDT), a classic prescription of traditional Chinese medicine (TCM), has been used in clinical over 1700 years for the treatment of gastrointestinal disorders, cardiovascular diseases and Alzheimer disease. But the active components of HLJDT were ambiguous, which seriously restricted its clinical application. MATERIALS AND METHODS The methodology of plasma pharmacochemistry was applied to screen the bioactive components in HLJDT. A reliable LC/MS system was established for detecting the prototype compounds and metabolites in dosed plasma after oral administration of HLJDT. By comparative analysis of the chemical profiles of HLJDT extracts, blank plasma and dosed plasma, potential bioactive compounds in HLJDT may be discovered. RESULTS By comparing the retention time, MS and MS/MS spectra with those of reference standard and literature data, 30 components including 22 prototype compounds and 8 metabolites from HLJDT were discovered as potential bioactive components in rat plasma. CONCLUSIONS A reliable and effective method was established to screen the potential bioactive components in the formula of HLJDT, which provided useful information for the further study of action mechanism of HLJDT.

Biomarkers in the early period of acute myocardial infarction in rat serum and protective effects of Shexiang Baoxin Pill using a metabolomic method.

ETHNOPHARMACOLOGICAL RELEVANCE To identify the biomarkers in early period of acute myocardial infarction (AMI) in rat serum and reveal the effective mechanism of a Traditional Chinese Medicine (TCM) named Shexiang Baoxin Pill (SBP). MATERIAL AND METHOD A metabolomic approach using reversed-phase liquid chromatography/quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) was developed. RESULTS Fourteen biomarkers in the early period of acute myocardial infarction (AMI) in rat serum were identified. These biomarkers include 5-methylcytosine, cystathionine ketimine, 2-oxoadipic acid, thymidine, epinephrine, homocystine, uric acid, 12(S)-hydroperoxyeicosatetraenoic acid (12s-HPETE), 11-dehydrocorticosterone, 12(S)-hydroxyeicosatetraenoic acid (12s-HETE), deoxycorticosterone, corticosterone, aldosterone and cortisol. Through pathway analysis of these biomarkers, inflammation, hypertrophy and oxidative injury were considered the most relevant pathological changes in early period of AMI. CONCLUSION Identification of AMI biomarkers not only supplied a systematic view of the progression of AMI in the early period but also provided the theoretical basis for the prevention or treatment of AMI. The results demonstrated that SBP pretreatment could offer protective effects for AMI through regulating the pathway of steroid hormone biosynthesis.

Structural Characterization and Identification of Major Constituents in Jitai Tablets by High-Performance Liquid Chromatography/Diode-Array Detection Coupled with Electrospray Ionization Tandem Mass Spectrometry

In the present study a universally applicable HPLC-DAD/ESI-MS/MS method was developed for carrying out the comprehensive characterization of Jitai tablets (JTT). Based on the ESI-MSn fragmentation patterns of the reference standards, a total of 101 components were identified or tentatively characterized by comparing their retention times, UV and MS spectra with those of reference standards or through the matching of empirical information with those of published components in the in-house library. The characteristic fragmentation pattern of alkaloids, phenolic acids, tanshinones, flavonoid glycosides, cyanogenic glycosides, ginsenosides, 2-(2-phenylethyl) chromones, phthalides and gingerol-related compounds were tentatively elucidated using structurally-relevant product ions. It was observed that neutral losses of C9H10O3 and C9H8O2 were the characteristic product ions of scopola alkaloids. Neutral fragment mandelonitrile was the characteristic ion of cyanogenic glycosides. To our knowledge, tropylium ion and C4H2O unit were the characteristic ions of 2-(2-phenylethyl) chromone, which resulted from the Retro-Diels-Alder (RDA) cleavage of the C ring. The results indicated that the developed analysis method could be employed as a rapid, effective technique for structural characterization of chemical constituents in TCM. This work is expected to provide comprehensive information for the quality evaluation and pharmacokinetic studies of JTT.

The serum metabolomic study of intervention effects of the traditional Chinese medicine Shexiang Baoxin Pill and a multi-component medicine polypill in the treatment of myocardial infarction in rats.

A metabolomic approach based on liquid chromatography coupled with quadrupole time-of-flight detector (LC-Q-TOF/MS) was developed to investigate the therapeutic mechanism of a traditional Chinese medicine (TCM) formula Shexiang Baoxin Pill (SBP) and a multi-component medicine polypill (consisting of simvastatin (Sim), atenolol (Ate), ramipril (Ram), hydrochlorthiazide (Hyd) and aspirin (Asp), named as SARHA). Twenty-seven biomarkers were identified in the serum of MI rats. Thirteen related pathways and 4 main pathological processes including oxidative injury, energy metabolism dysfunction, amino acid metabolism dysfunction and inflammation are involved in MI development. Our study revealed that SBP showed better therapeutic effectiveness than the polypill on MI through regulation of the energy metabolism dysfunction, oxidative injury and inflammation. The combination agent polypill had only certain therapeutic effects on inhibiting oxidative injury and inflammation induced by MI. The reverse effect of the polypill on biomarkers related to MI was much better than mono-therapy groups.

Metabolomic Study of Collagen-Induced Arthritis in Rats and the Interventional Effects of Huang-Lian-Jie-Du-Tang, a Traditional Chinese Medicine

Huang-Lian-Jie-Du-Tang (HLJDT) is a traditional Chinese medicine (TCM) with anti-inflammatory activity. The present study used a metabolomic approach based on LC-Q-TOF-MS to profile rheumatoid-arthritis- (RA-) related metabolic changes and to investigate the interventional mechanisms of HLJDT in collagen-induced arthritis rats. Forty male Wistar rats were randomly divided into five groups: (1) a model group, (2) a normal control group, (3) a dexamethasone group, (4) a HLJDT group, and (5) a group that received 13 components of HLJDT. Plasma samples were collected 8, 15, and 22 days after the rats were injected with bovine type II collagen. By combining variable importance in the projection values with partial least squares discriminant analysis, 18 potential biomarkers were identified in the plasma samples. The biomarkers were primarily involved in glycerophospholipid metabolism, fatty acid metabolism, tryptophan metabolism, linoleic acid metabolism, phenylalanine metabolism, purine metabolism, arachidonic acid metabolism, and bile acid biosynthesis. Using the potential biomarkers as a screening index, the results suggest that HLJDT can potentially reverse the process of RA by partially regulating fatty acid oxidation and arachidonic acid metabolism. This study demonstrates that a metabolomic strategy is useful for identifying potential RA biomarkers and investigating the underlying mechanisms of a TCM in RA treatment.

Pharmacokinetics and tissue distribution of five bufadienolides from the Shexiang Baoxin pill following oral administration to mice

ETHNOPHARMACOLOGICAL RELEVANCE Shexiang Baoxin Pill (SBP) is a well-known composite formula of traditional Chinese medicine (TCM), widely used to treat cardiovascular diseases such as angina pectoris and myocardial infarction. Bufadienolides are major active compounds of Venenum Bufonis, which is one of the seven materiamedicas that comprise the Shexiang Baoxin Pill. Previous pharmacokinetics studies of bufadienolides have typically used a single medicinal material delivered to rats. In this study, we have chosen the mouse, a more proper animal model than the rat, to investigate the in vivo pharmacokinetics and tissue distribution of bufadienolides from the Shexiang Baoxin Pill. MATERIALS AND METHODS The concentrations of bufadienolides in plasma and tissues were identified using high performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS/MS). The samples were prepared by liquid-liquid extraction with ethyl acetate, and the separation of bufadienolides was achieved using an ACQUITY HSS T3 column by gradient elution using water (containing 0.1% formic acid) and acetonitrile as the mobile phase at a flow rate of 0.3 mL/min. The pharmacokinetic parameters were determined using non-compartmental analysis. RESULTS The results showed that the five bufadienolides were rapidly absorbed and distributed into the body. The pharmacokinetic curve showed double peaks after oral administration. The major tissue depots for resibufogenin, bufalin, and bufotalin in mice were the intestines, lung and kidney, whereas the major tissue depots of gamabufotalin and arenobufagin were the intestines, liver and kidney. CONCLUSION The information gained from this research provides a meaningful insight for the clinical applications of the Shexiang Baoxin Pill.

Screening and analysis of the multiple absorbed bioactive components and metabolites in rat plasma after oral administration of Jitai tablets by high-performance liquid chromatography/diode-array detection coupled with electrospray ionization tandem mass spectrometry.

Based on the serum pharmacochemistry technique and high-performance liquid chromatography/diode-array detection (HPLC/DAD) coupled with electrospray tandem mass spectrometry (HPLC/ESI-MS/MS), a method for screening and analysis of the multiple absorbed bioactive components and metabolites of Jitai tablets (JTT) in orally dosed rat plasma was developed. Plasma was treated by methanol precipitation prior to liquid chromatography, and the separation was carried out on a Symmetry C(18) column, with a linear gradient (0.1% formic acid/water/acetonitrile). Mass spectra were acquired in negative and positive ion modes, respectively. As a result, 26 bioactive components originated from JTT and 5 metabolites were tentatively identified in orally dosed rat plasma by comparing their retention times and MS spectra with those of authentic standards and literature data. It is concluded that an effective and reliable analytical method was set up for screening the bioactive components of Chinese herbal medicine, which provided a meaningful basis for further pharmacology and active mechanism research of JTT.

The antidepressant-like effect of bacopaside I: possible involvement of the oxidative stress system and the noradrenergic system.

In the present study, the antidepressant-like effect of bacopaside I, a saponin compound present in the Bacopa monniera plant, was evaluated by behavioral and neurochemical methods. Bacopaside I (50, 15 and 5 mg/kg) was given to mice via oral gavage for 7 successive days. The treatment significantly decreased the immobility time in mouse models of despair tests, but it did not influence locomotor activity. Neurochemical assays suggested that treatment by bacopaside I (50, 15 and 5 mg/kg) improved brain antioxidant activity to varying degrees after the behavioral despair test. Bacopaside I (15 and 5 mg/kg) significantly reversed reserpine-induced depressive-like behaviors, including low temperature and ptosis. Conversely, bacopaside I did not affect either brain MAO-A or MAO-B activity after the behavioral despair test in mice. Additionally, 5-hydroxytryptophan (a precursor of 5-serotonin) was not involved in the antidepressant-like effect of bacopaside I. These findings indicated that the antidepressant-like effect of bacopaside I might be related to both antioxidant activation and noradrenergic activation, although the exact mechanism remains to be further elucidated.

Simultaneous determination of six hydrophilic components in rat plasma after oral administration of Jitai tablet by liquid chromatography–electrospray ionization–tandem mass spectrometry: Application to a pharmacokinetic study

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated for the simultaneous determination of amygdalin (ADL), danshensu (DSS), ferulic acid (FA), hydroxysafflor yellow A (HSYA), salvianolic acid A (SAA) and salvianolic acid B (SAB) in rat plasma. Plasma samples were pretreated by protein precipitation with acetonitrile. LC separation was performed on a Zorbax Eclipse Plus C18 column (3.0mm×100mm I.D, 1.8μm) with gradient elution using a mobile phase consisting of acetonitrile-0.1% formic acid in water at a flow rate of 0.3mL/min. ESI-MS spectra was acquired in negative ion multiple reaction monitoring mode. The mass transition ion-pair was followed as m/z 456.0→323.1, m/z 197.3→178.8, m/z 193.0→133.9, m/z 611.1→325.2, m/z 493.0→295.0, and m/z 717.0→519.0 for ADL, DSS, FA, HSYA, SAA and SAB, respectively. All analytes showed good linearity over a wide concentration range (r>0.99). The lower limit of quantification was 7ng/mL, 2ng/mL, 4ng/mL, 1ng/mL, 2ng/mL, and 4ng/mL for ADL, DSS, FA, HSYA, SAA and SAB, respectively. The mean recovery of the analytes ranged from 86.29% to 93.16%. The intra- and inter-day precisions were in the range of 1.50-9.98% and the accuracies were between 91.17% and 99.46%. The validated method was successfully applied to a pharmacokinetic study of the six hydrophilic components in rat plasma after oral administration of Jitai tablet.