Wuhong Li

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.

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.

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.

Identification of multiple components in Guanxinning injection using hydrophilic interaction liquid chromatography/time-of-flight mass spectrometry and reversed-phase liquid chromatography/time-of-flight mass spectrometry

An approach for the identification of multiple components in traditional Chinese medicine injections (TCMIs) using a combination of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) coupled with time-of-flight mass spectrometry (TOFMS) was developed for the quality control of Guanxinning injection (GXNI), a widely used TCMI, composed of Salvia miltiorrhiza and Ligusticum Chuanxiong. A total of 50 compounds from five compound classes, including saccharides, amino acids, organic acids, phenolic acids and phthalides, were identified or tentatively characterized on the basis of accurate mass measurements and subsequent TOFMS product ions. Six groups of isomers of phenolic acids and saccharides were tentatively distinguished. It was observed that the ESI-TOFMS fragmentation behavior of phthalides was different in negative and positive ion mode, and the fragmentation pathways were tentatively elucidated using structurally-relevant product ions. Several highly polar constituents were characterized for the first time from GXNI by HILIC/TOFMS. In addition, all the constituents identified from GXNI were further assigned in the two individual crude drugs. The integrated strategy has provided a powerful approach for the separation and identification of the multiple components in GXNI, and it has also assisted in the establishment of methods for the comprehensive safety and quality evaluation of TCMIs.

Comparative normal/failing rat myocardium cell membrane chromatographic analysis system for screening specific components that counteract doxorubicin-induced heart failure from Acontium carmichaeli.

Cell membrane chromatography (CMC) derived from pathological tissues is ideal for screening specific components acting on specific diseases from complex medicines owing to the maximum simulation of in vivo drug-receptor interactions. However, there are no pathological tissue-derived CMC models that have ever been developed, as well as no visualized affinity comparison of potential active components between normal and pathological CMC columns. In this study, a novel comparative normal/failing rat myocardium CMC analysis system based on online column selection and comprehensive two-dimensional (2D) chromatography/monolithic column/time-of-flight mass spectrometry was developed for parallel comparison of the chromatographic behaviors on both normal and pathological CMC columns, as well as rapid screening of the specific therapeutic agents that counteract doxorubicin (DOX)-induced heart failure from Acontium carmichaeli (Fuzi). In total, 16 potential active alkaloid components with similar structures in Fuzi were retained on both normal and failing myocardium CMC models. Most of them had obvious decreases of affinities on failing myocardium CMC compared with normal CMC model except for four components, talatizamine (TALA), 14-acetyl-TALA, hetisine, and 14-benzoylneoline. One compound TALA with the highest affinity was isolated for further in vitro pharmacodynamic validation and target identification to validate the screen results. Voltage-dependent K+ channel was confirmed as a binding target of TALA and 14-acetyl-TALA with high affinities. The online high throughput comparative CMC analysis method is suitable for screening specific active components from herbal medicines by increasing the specificity of screened results and can also be applied to other biological chromatography models.

Computer-aided molecular modeling study of enantioseparation of iodiconazole and structurally related triadimenol analogues by capillary electrophoresis: Chiral recognition mechanism and mathematical model for predicting chiral separation

Chiral separation of iodiconazole, a new antifungal drug, and 12 new structurally related triadimenol analogues had been developed by capillary electrophoresis (CE) using hydroxypropyl-γ-cyclodextrin (HP-γ-CD) as the chiral selector. The effect of structural features of analytes on Δt and R(s) was studied under the optimum separation conditions. Using molecular docking technique and binding energy calculations, the inclusion process between HP-γ-CD and enantiomers was investigated and chiral recognition mechanisms were discussed. The results suggest that hydrogen bonding between fluorine at position 4 of the phenyl group beside the chiral carbon and the hydroxyl group on the HP-γ-CD rim and face to face π-π interactions between two phenyl rings highly contributed to the enantiorecognition process between HP-γ-CD and iodiconazole. The N-methyl group beside chiral carbon also played an important role in enantiomeric separation. Additionally, the big difference in binding energy (ΔΔE) highly contributed to good separation in the presence of HP-γ-CD chiral selector, which may be a helpful initial guide for chiral selector selection and predicting the result of enantioseparation. Furthermore, the new mathematical equation established based on the results of molecular mechanics calculations exhibited good capability in predicting chiral separation of these triadimenol analogues using HP-γ-CD mediated CE.

Screening and analysis of aconitum alkaloids and their metabolites in rat urine after oral administration of aconite roots extract using LC-TOFMS-based metabolomics.

Aconite roots are popularly used in herbal medicines in China. Many cases of accidental and intentional intoxication with this plant have been reported; some of these are fatal because the toxicity of aconitum is very high. It is thus important to detect and identify aconitum alkaloids in biofluids. In this work, an improved method employing LC-TOFMS with multivariate data analysis was developed for screening and analysis of major aconitum alkaloids and their metabolites in rat urine following oral administration of aconite roots extract. Thirty-four signals highlighted by multivariate statistical analyses including 24 parent components and 10 metabolites were screened out and further identified by adjustment of the fragmentor voltage to produce structure-relevant fragment ions. It is helpful for studying aconite roots in toxicology, pharmacology and forensic medicine. This work also confirmed that the metabolomic approach provides effective tools for screening multiple absorbed and metabolic components of Chinese herbal medicines in vivo.

Metabolic Profiling Provides a System Understanding of Hypothyroidism in Rats and Its Application

Background Hypothyroidism is a chronic condition of endocrine disorder and its precise molecular mechanism remains obscure. In spite of certain efficacy of thyroid hormone replacement therapy in treating hypothyroidism, it often results in other side effects because of its over-replacement, so it is still urgent to discover new modes of treatment for hypothyroidism. Sini decoction (SND) is a well-known formula of Traditional Chinese Medicine (TCM) and is considered as efficient agents against hypothyroidism. However, its holistic effect assessment and mechanistic understanding are still lacking due to its complex components. Methodology/Principal Findings A urinary metabonomic method based on ultra performance liquid chromatography coupled to mass spectrometry was employed to explore global metabolic characters of hypothyroidism. Three typical hypothyroidism models (methimazole-, propylthiouracil- and thyroidectomy-induced hypothyroidism) were applied to elucidate the molecular mechanism of hypothyroidism. 17, 21, 19 potential biomarkers were identified with these three hypothyroidism models respectively, primarily involved in energy metabolism, amino acid metabolism, sphingolipid metabolism and purine metabolism. In order to avert the interference of drug interaction between the antithyroid drugs and SND, the thyroidectomy-induced hypothyroidism model was further used to systematically assess the therapeutic efficacy of SND on hypothyroidism. A time-dependent recovery tendency was observed in SND-treated group from the beginning of model to the end of treatment, suggesting that SND exerted a recovery effect on hypothyroidism in a time-dependent manner through partially regulating the perturbed metabolic pathways. Conclusions/Significance Our results showed that the metabonomic approach is instrumental to understand the pathophysiology of hypothyroidism and offers a valuable tool for systematically studying the therapeutic effects of SND on hypothyroidism.

Serum metabonomics coupled with Ingenuity Pathway Analysis characterizes metabolic perturbations in response to hypothyroidism induced by propylthiouracil in rats

A serum metabonomic profiling method based on ultra-performance liquid chromatography/time-of-flight mass spectrometry (UHPLC/TOF-MS) was applied to investigate the metabolic changes in hypothyroid rats induced by propylthiouracil (PTU). With Significance Analysis of Microarray (SAM) for classification and selection of biomarkers, 13 potential biomarkers in rat serum were screened out. Furthermore, Ingenuity Pathway Analysis (IPA) was introduced to deeply analyze unique pathways of hypothyroidism that were primarily involved in sphingolipid metabolism, fatty acid transportation, phospholipid metabolism and phenylalanine metabolism. Our results demonstrated that the metabonomic approach integrating with IPA was a promising tool for providing a novel methodological clue to systemically dissect the underlying molecular mechanism of hypothyroidism.

Simultaneous Determination of Oleanolic and Ursolic Acids in Rat Plasma by HPLC-MS: Application to a Pharmacokinetic Study After Oral Administration of Different Combinations of QingGanSanJie Decoction Extracts.

A liquid chromatography-mass spectrometry method has been developed and validated for rapid simultaneous determination of the oleanolic and ursolic acid contents in rat plasma with betulinic acid as the internal standard (IS). The plasma samples were prepared by a liquid-liquid extraction procedure. Chromatographic separation was performed with a Chromasil-C18 column (250 mm × 4.6 mm, i.d. 5 μm) with methanol-water as mobile phase at 1 mL/min. The detection was accomplished under selected-ion-monitoring mode with a negative electrospray ionization interface. Linear calibration curves were obtained between the range of 0.86-421.2 and 0.94-462.0 ng/mL for oleanolic and ursolic acids, with lower limits of quantification at 0.43 and 0.47 ng/mL, respectively. The extraction recovery exceeded 70% in plasma. The intra- and interday precision values were <9.8% with the accuracy as -7.0 to 9.9% at three different QC levels in both cases. The pharmacokinetic behaviors of oral dosage of QingGanSanJie decoctions were then studied in rats following the developed approach. The t1/2 values of the oleanolic and ursolic acids after oral administration of the monarch medicine extract were significantly different (P < 0.05) from other prescription extracts containing different herb pieces with different compatibilities. Bimodal phenomena appeared in every concentration-time curve for the oleanolic and ursolic acids at 3-8 h after administration. The minister, assistant and guide medicines in the formula could prolong the metabolism of the oleanolic and ursolic acids in vivo, providing an experimental basis for the slow onset and long action of the Traditional Chinese Medicine compound.