| 1. Application of metabonomics in evaluations of drug toxicity and disease diagnosisNMR-or LC-based metabonomics is to understand the changes of ednognous small molecules, analyze the differences between metabolic profiles with the aid of bioinformatics under different physiological or pathological conditions, and then discover biomarkers, provide a whole picture of organism in specific time points and conditions. In this study, H NMR-based metabonomics is applied for evaluation of drug toxicity and disease diagnosis.1.1. NMR-based metabonomics on drug toxicity evaluationNMR-based metabonomics approach has been proved to be an efficient technique to investigate the biochemical effects of Cimicidol-3-O-pβ-D-xyloside (CX) isolated from Cimicifugae rhizomes that have been used controversially in clinic. This work is to access the toxicological effects of CX after oral administration (50 mg/kg/day) over a 7-day period in female SD rats using metabonomic analyses of 1H NMR spectra of urine, serum and liver tissue extracts. The metabolic signature of 1H NMR based urinalysis of daily samples displayed an increment in the levels of taurine, trimethylamine-N-oxide (TMAO), betaine and acetate. Elevated serum levels of creatinine, glucose, alanine, TMAO and betaine and lower level of lactate were observed. Metabolic profiling on aqueous soluble extracts of liver showed simultaneously the increases of succinate, glycogen, choline, glycerophosphorylcholine (GPC), TMAO and betaine levels and reduction in valine, glucose, lactate levels. Nevertheless, no changes in any metabonomic level were found in lipid soluble extracts of liver. These findings indicate that CX has a slight toxicity in liver and kidney via disturbance in metabolisms of energy and amino acids. The present study provides reasonable explanation for clinical hepatotoxicity of Cimicifugae extract.1.2. NMR-based metabonomics on disease diagnosisThe present NMR-based metabonomic approach combining with DFA provided a rapid and sensitive method to differentiate malignant pleural effusions from benign ones. Total 12 small molecules (9 are newly discovered in pleural effusions) were determined with the one shot:H NMR measurement in the effusions. In the malignant pleural effusions of training group, the mean values of valine, lactate and alanine were significantly higher, while, the levels of acetoacetate, trimethylamine-N-oxide (TMAO)/betaine, α-andβ-glucose were markedly lower compared with those in benign ones. In the DFA model, the best discrimination (P< 0.001) was achieved when lactate, acetoacetate, TMAO/betaine and a-glucose were considered together as biomarkers. Accuracy was 95.8% and 97.0% for training group and validation group, respectively. The small molecules in the pleural effusions might serve as useful biomarkers for diagnosis or prognosis of malignancy of pleural effusions. The present study provided a rapid and sensitive method to predict the malignancy of pleural effusions.2. pharmacokinetic studies on anti-osteoporosis drugPharmacokinetics is an essential part of pharmacology. In the process of development of drug, it is getting important on the drugs preclinical studies and clinical research. And liquid chromatography-mass spectrometry (LC-MS) is commonly used analytical tools in biomedical research, which combines the separation of liquid chromatography and the identification of mass. In this study, two LC-MS methods were developed and validated for determination of two anti-osteoporosis drug in plasma and their application to pharmacokinetics2.1. pharmacokinetics of Cimicidol-3-O-p-D-xyloside in miceA highly sensitive and specific liquid chromatography-ESI-mass spectrometric (LC-ESI-MS/MS) method was established for investigating the pharmacokinetics of Cimicidol-3-O-p-D-xyloside in mice, using Cimicidanol as an internal standard (IS). Sample preparation was performed by liquid-liquid extraction with acetic ether and the separations were achieved using a C18 column. A triple quadrupole mass spectrometer equipped with an electrospray ionization interface was operated in the ion positive mode. The calibration curve was linear in the range of 5-20000 ng/ml. The lower limit of quantification (LLOQ) was 5 ng/ml. The RSD%s of inter-and intra-day were met the methodological requirements. The mice were given a single dose at 25 mg/kg, its pharmacokinetic models were two-compartment mode and three-compartment model for oral administration and intravenous injection, respectively. The relative bioavailability of CX was 24.8% by calculation.2.2. Pharmacokinetics of QOA-8a in vitro and in vivoA HPLC was employed to determine the enzyme kinetics of QOA-8a in vitro metabolic system using the rat and human liver microsomes. The calibration curve was linear in the range of 1-100 μM. The RSD%s of inter-and intra-day were met the methodological requirements. The enzyme kinetics parameters of QOA-8a were calculated and the results showed QOA-8a had a rapid metabolic process in rat and human liver microsomes. And a metabolite of QOA-8a was determined.A LC-MS/MS method was established for investigating the pharmacokinetics of QOA-8a in rats. Sample preparation was performed by liquid-liquid extraction with acetic ether and the separations were achieved using a C18 column. A triple quadrupole mass spectrometer equipped with an electrospray ionization interface was operated in the ion positive mode. The calibration curve was linear in the range of 1-1000 ng/ml and 5-1000 ng/ml for QOA-8a and GA, respectively. The RSD%s of inter-and intra-day were met the methodological requirements. The mice were given a single dose at 1 mg/kg, its pharmacokinetic models were two-compartment mode for oral administration and intravenous injection. The relative bioavailability of CX was 32.6% by calculation. |
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