Yifeng Chai

Metabonomic Profiles Delineate the Effect of Traditional Chinese Medicine Sini Decoction on Myocardial Infarction in Rats

Background In spite of great advances in target-oriented Western medicine for treating myocardial infarction (MI), it is still a leading cause of death in a worldwide epidemic. In contrast to Western medicine, Traditional Chinese medicine (TCM) uses a holistic and synergistic approach to restore the balance of Yin-Yang of body energy so the bodys normal function can be restored. Sini decoction (SND) is a well-known formula of TCM which has been used to treat MI for many years. However, its holistic activity evaluation and mechanistic understanding are still lacking due to its complex components. Methodology/Principal Findings A urinary metabonomic method based on nuclear magnetic resonance and ultra high-performance liquid chromatography coupled to mass spectrometry was developed to characterize MI-related metabolic profiles and delineate the effect of SND on MI. With Elastic Net for classification and selection of biomarkers, nineteen potential biomarkers in rat urine were screened out, primarily related to myocardial energy metabolism, including the glycolysis, citrate cycle, amino acid metabolism, purine metabolism and pyrimidine metabolism. With the altered metabolism pathways as possible drug targets, we systematically analyze the therapeutic effect of SND, which demonstrated that SND administration could provide satisfactory effect on MI through partially regulating the perturbed myocardial energy metabolism. Conclusions/Significance Our results showed that metabonomic approach offers a useful tool to identify MI-related biomarkers and provides a new methodological cue for systematically dissecting the underlying efficacies and mechanisms of TCM in treating MI.

Comparative pharmacokinetics of baicalin and wogonoside by liquid chromatography–mass spectrometry after oral administration of Xiaochaihu Tang and Radix scutellariae extract to rats

The aim of this study was to compare the pharmacokinetics of baicalin and wogonoside in rats following oral administration of Xiaochaihu Tang (Minor Radix Bupleuri Decoction) and Radix scutellariae extract. Thus, a specific LC-MS method was developed and validated for the determination of these flavonoids in the plasma of rats after oral administration Xiaochaihu Tang and Radix scutellariae extract. Chromatographic separation was performed on a Zorbax SB C(18) column (150mmx4.6mm, i.d.: 5microm) with 0.1% formic acid in water and acetonitrile by linear gradient elution. Baicalin, wogonoside and carbamazepine (internal standard, I.S.) were detected in select-ion-monitoring (SIM) mode with a positive electrospray ionization (ESI) interface. The following ions: m/z 447 for baicalin, m/z 461 for wogonoside and m/z 237 for the I.S. were used for quantitative determination. The calibration curves were linear over the concentration ranges from 0.1231 to 6.156microg mL(-1) for baicalin and 0.08832 to 4.416microg mL(-1) for wogonoside. The lower limit of detection (LLOD) based on a signal-to-noise ratio of 2 was 0.06155microg mL(-1) for baicalin and 0.04416microg mL(-1) for wogonoside. Intra-day and inter-day precisions (RSD%) were within 10% and accuracy (RE%) ranged from -6.4 to 4.4%. The extraction recovery at three QC concentrations ranged from 74.7 to 86.0% for baicalin and from 71.3 to 83.7% for wogonoside. The plasma concentrations of baicalin and wogonoside in rats at designated time periods after oral administration were successfully determined using the validated method, pharmacokinetic parameters were estimated by a non-compartment model. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t(1/2) of baicalin was 3.60+/-0.90 and 5.64+/-1.67, the C(max1) was 1.64+/-0.99 and 5.66+/-2.02, the t(max1) was 0.13+/-0.05 and 0.20+/-0.07, the C(max2) was 2.43+/-0.46 and 3.18+/-1.66, and the t(max2) were 6.40+/-1.67 and 5.66+/-2.02, respectively. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t(1/2) of wogonoside was 4.97+/-1.68 and 7.71+/-1.55, the C(max1) was 1.39+/-0.83 and 1.45+/-0.37, the t(max1) was 0.21+/-0.20 and 0.17+/-0.01, the C(max2) was 1.90+/-0.55 and 1.42+/-0.70, and the t(max2) was 5.60+/-1.67 and 5.20+/-1.79, respectively. A significant difference (p<0.05) was observed for t(1/2), and the elimination of baicalin and wogonoside in Xiaochaihu Tang was increased.

Candida albicans cells lacking CaMCA1-encoded metacaspase show resistance to oxidative stress-induced death and change in energy metabolism

Candida albicans, an opportunistic pathogen, can undergo programmed cell death upon various stimuli, including oxidative stress. In this study, we showed that deletion of CaMCA1, a homologue of Saccharomyces cerevisiae metacaspase YCA1, could both attenuated oxidative stress-induced cell death and caspase activation. Compared to wild-type strain, Camca1Delta mutant showed higher accumulation of trehalose and transcription of the genes related to trehalose biosynthesis (TPS2 and TPS3) under the condition of oxidative stress. Furthermore, lower intracellular ATP concentration and mitochondrial membrane potential, less endogenous reactive oxygen species (ROS) generation were observed in Camca1Delta mutant. Our results suggest that CaMCA1 might mediate the sensitiveness to oxidative stress by affecting energy metabolism in C. albicans.

Determination of synthetic drugs used to adulterate botanical dietary supplements using QTRAP LC-MS/MS

Adulteration of botanical dietary supplements with prohibited synthetic drugs has become a serious problem. In this paper, a method for testing synthetic drugs used to adulterate botanical dietary supplements was developed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) coupled with a linearity ion-trap system in the multiple reaction monitoring (MRM) plus enhanced product ion (EPI) mode. Twenty-three drugs exhibiting various pharmacological effects, comprising blood pressure and lipid-lowering agents, sedative drugs, anti-diabetic drugs, weight-reducing agents and aphrodisiac compounds, were studied. For all drugs, a single transition was monitored using protonated molecules as precursor ions. EPI spectra were stored in a library and recognized by library searching. Several undeclared drugs were identified in herbal remedies, e.g., glibenclamide, sibutramine hydrochloride and sildenafil. Overall, 35 positive samples were found out of a total of 105 botanical dietary supplements tested. The method was selective, sensitive, rapid, high-throughput and reliable.

Comparative pharmacokinetic study of paeoniflorin after oral administration of pure paeoniflorin, extract of Cortex Moutan and Shuang-Dan prescription to rats

Shuang-Dan (SD) is a traditional Chinese prescription containing Cortex Moutan and Radix Salviae miltiorrhizae and commonly used for treating cardiovascular disease. Paeoniflorin is a main effective ingredient of Cortex Moutan and the pharmacokinetic differences of paeoniflorin following oral administration of pure paeoniflorin, Cortex Moutan extract and SD decoction to rats were studied with approximately the same dose of 30mg/kg paeoniflorin. At different time points (5, 10, 15, 30, 45, 60, 90, 120, 150, 210, 270, 360, 450min), plasma concentration of paeoniflorin was determined using a simple and rapid HPLC-MS method. Unpaired students t-test was used for the statistical comparison. A bimodal phenomenon was observed in the plasma profile after oral administration of Cortex Moutan extract. Statistically significant increase (P<0.05) in pharmacokinetic parameters of paeoniflorin including AUC(0-t), AUC(0-infinity) and MRT were obtained after oral administration of Cortex Moutan or SD decoction comparing with pure paeoniflorin. The investigation showed that among all calculated parameters, AUC(0-t), AUC(0-infinity), MRT, k(e) and T(1/2), there were no significant differences between the two decoctions. The results indicated that the reason which delay the elimination of paeoniflorin and enhance its bioavailability might be some ingredients in Cortex Moutan extract.

Comprehensive two-dimensional HepG2/cell membrane chromatography/monolithic column/time-of-flight mass spectrometry system for screening anti-tumor components from herbal medicines.

Cell membrane chromatography (CMC) is a biological affinity chromatographic method using specific cell membrane as stationary phase. It has been proved to be a practical tool for investigating binding interactions between drugs and membrane receptors. In this study, a novel comprehensive two-dimensional (2D) chromatography approach was established for screening anti-tumor components from herbal medicines (HMs). HepG2/CMC model was first developed and applied as the first dimensional column. Using an automatic ten-port switching valve equipped with two sample loops, the fractions of the first-dimension were introduced in the second-dimension consists of a monolithic column and a time-of-flight mass spectrometry (TOFMS) with high resolving ability. Based on the stability, selectivity and suitability assays of the HepG2/CMC/monolithic column/TOFMS system, berberine (BBR) and tetrahydropalmatine (THP) from Cortex phellodendri amurensis, oxymatrine and matrine from Radix sophorae flavescentis were screened and identified as potential active components. The competitive displacement assay suggested that the four components could act on epidermal growth factor receptor region on the HepG2 cell membrane in similar manner of gefitinib. Furthermore, their inhibiting effects on cell proliferation in vitro were also confirmed and, BBR and THP showed concentration dependently inhibitory ability on HepG2 cell proliferation (p<0.05). The result demonstrated that the proposed comprehensive 2D HepG2/CMC/monolithic column/TOFMS system has the advantages of strong recognition and rapid analysis abilities for the total screening procedure, which will be selectable and practical in drug discovery from complex HM samples and can also be applied to other biochromatography models.

Analysis of phenolic and triterpenoid compounds in licorice and rat plasma by high-performance liquid chromatography diode-array detection, time-of-flight mass spectrometry and quadrupole ion trap mass spectrometry

High-performance liquid chromatography with diode-array detection (HPLC/DAD), time-of-flight mass spectrometry (HPLC/TOFMS) and quadrupole ion trap mass spectrometry (HPLC/QITMS) were used for separation and identification of several compounds in licorice and rat plasma after oral administration of the herbal extract. Three phenolic compounds and one triterpenoid in licorice extract were unambiguously identified by comparing with the standard compounds. A formula database of known compounds in licorice was established, against which the other 42 compounds were identified effectively based on the accurate extract masses and formulae acquired by HPLC/TOFMS. In order to differentiate the isomers, tandem mass spectrometry was also used. The deduced fragmentation behaviors in QITMS were used to distinguish seven groups of isomers in licorice. By means of the three detectors, 46 compounds in licorice were identified. After oral administration of the extract, 25 compounds in rat plasma were detected and identified by comparing and contrasting the compounds measured in licorice with those in the plasma samples by HPLC/TOFMS. It is concluded that a rapid and effective method based on three analytical techniques was established, which is useful for identification of multiple compounds in licorice in vitro and in vivo. The result should be very useful for the quality control and curative mechanism study of licorice.

Potential Biomarkers in Mouse Myocardium of Doxorubicin-Induced Cardiomyopathy: A Metabonomic Method and Its Application

Background Doxorubicin (DOX) is one of the most potent antitumor agents available; however, its clinical use is limited because of the risk of severe cardiotoxicity. Though numerous studies have ascribed DOX cardiomyopathy to specific cellular pathways, the precise mechanism remains obscure. Sini decoction (SND) is a well-known formula of Traditional Chinese Medicine (TCM) and is considered as efficient agents against DOX-induced cardiomyopathy. However, its action mechanisms are not well known due to its complex components. Methodology/Principal Findings A tissue-targeted metabonomic method using gas chromatography–mass spectrometry was developed to characterize the metabolic profile of DOX-induced cardiomyopathy in mice. With Elastic Net for classification and selection of biomarkers, twenty-four metabolites corresponding to DOX-induced cardiomyopathy were screened out, primarily involving glycolysis, lipid metabolism, citrate cycle, and some amino acids metabolism. With these altered metabolic pathways as possible drug targets, we systematically analyzed the protective effect of TCM SND, which showed that SND administration could provide satisfactory effect on DOX-induced cardiomyopathy through partially regulating the perturbed metabolic pathways. Conclusions/Significance The results of the present study not only gave rise to a systematic view of the development of DOX-induced cardiomyopathy but also provided the theoretical basis to prevent or modify expected damage.

NMR and LC/MS-based global metabolomics to identify serum biomarkers differentiating hepatocellular carcinoma from liver cirrhosis

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. However, current biomarkers that discriminate HCC from liver cirrhosis (LC) are important but are limited. More reliable biomarkers for HCC diagnosis are therefore needed. Serum from HCC patients, LC patients and healthy volunteers were analyzed using NMR and LC/MS‐based approach in conjunction with random forest (RF) analysis to discriminate their serum metabolic profiles. Thirty‐two potential biomarkers have been identified, and the feasibility of using these biomarkers for the diagnosis of HCC was evaluated, where 100% sensitivity was achieved in detecting HCC patients even with AFP values lower than 20 ng/mL. The metabolic alterations induced by HCC showed perturbations in synthesis of ketone bodies, citrate cycle, phospholipid metabolism, sphingolipid metabolism, fatty acid oxidation, amino acid catabolism and bile acid metabolism in HCC patients. Our results suggested that these potential biomarkers identified appeared to have diagnostic and/or prognostic values for HCC, which deserve to be further investigated. In addition, it also suggested that RF is a classification algorithm well suited for selection of biologically relevant features in metabolomics.

Molecular modeling study of chiral separation and recognition mechanism of β-adrenergic antagonists by capillary electrophoresis.

Chiral separations of five β-adrenergic antagonists (propranolol, esmolol, atenolol, metoprolol, and bisoprolol) were studied by capillary electrophoresis using six cyclodextrins (CDs) as the chiral selectors. Carboxymethylated-β-cyclodextrin (CM-β-CD) exhibited a higher enantioselectivity power compared to the other tested CDs. The influences of the concentration of CM-β-CD, buffer pH, buffer concentration, temperature, and applied voltage were investigated. The good chiral separation of five β-adrenergic antagonists was achieved using 50 mM Tris buffer at pH 4.0 containing 8 mM CM-β-CD with an applied voltage of 24 kV at 20 °C. In order to understand possible chiral recognition mechanisms of these racemates with CM-β-CD, host-guest binding procedures of CM-β-CD and these racemates were studied using the molecular docking software Autodock. The binding free energy was calculated using the Autodock semi-empirical binding free energy function. The results showed that the phenyl or naphthyl ring inserted in the hydrophobic cavity of CM-β-CD and the side chain was found to point out of the cyclodextrin rim. Hydrogen bonding between CM-β-CD and these racemates played an important role in the process of enantionseparation and a model of the hydrogen bonding interaction positions was constructed. The difference in hydrogen bonding formed with the –OH next to the chiral center of the analytes may help to increase chiral discrimination and gave rise to a bigger separation factor. In addition, the longer side chain in the hydrophobic phenyl ring of the enantiomer was not beneficial for enantioseparation and the chiral selectivity factor was found to correspond to the difference in binding free energy.