The mixtures were homogenized at 4��C using an ultrasonic cell disruptor (Sonics Vibra Cell http://www.selleckchem.com/products/mek162.html VCX105, Sonics Vibra Cell VCX105, Branson, MO, USA). The solution was then centrifuged at 12,000 rpm at 4��C for 15 minutes. The supernatants were collected for oxidative biomarker analysis. The activities of glutathione (GSH) and superoxide dismutase (SOD), and the levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA) in the liver extracts were examined. All commercial colorimetric assay kits were purchased from Beyotime Biotech Ltd and the assays were performed according to manufacturers�� instructions. Protein concentrations were determined using the bicinchoninic acid method.
Clinical chemistry and hematology analysis Standard spectrophotometric methods of a Cobas Integra 400 plus Automatic Biochemistry Analyzer (Roche) were used for the measurement of the following serum parameters: alanine aminotransferase, AST, alkaline phosphatase, total bile acid blood urea nitrogen, cholesterol, triglyceride, uric acid creatine kinase, high-density lipoprotein, and low-density lipoprotein. Hematological parameters consisting of white blood cell count (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MONO), erythrocytes, hemoglobin, and platelet count were determined using a hematological autoanalyzer (Coulter T540 hematology system; Beckman Coulter, Inc). The TNF-�� level in sera was measured using an ELISA kit. 1H NMR spectroscopic analysis of liver tissue extracts Samples of liver tissue (250 mg) were homogenized in 2 mL of 50% acetonitrile in an ice water bath.
The homogenates were centrifuged at 5070 g for 15 minutes at 4��C. The supernatants were removed and lyophilized before being reconstituted in 500 ��L of D2O containing 1 mM3-(trimethylsilyl)[2,2,3,3�C2H4] propionate. 1H NMR spectra were acquired for each sample at 500 MHz on a Bruker Avance III spectrometer (Bruker Corporation, Billerica, MA, USA) at ambient probe temperature. To explore the metabolic information embedded in NMR spectra, all 1D FIDs were multiplied by an exponential function of a 0.3 Hz line-broadening factor prior to Fourier transformation. The chemical shifts were referenced to the methyl group of sodium trimethylsilylpropionate (TSP) at �� 0.000. The integrals were normalized by the sum of the spectral intensity to compensate for differences in sample concentrations.
PLS-DA was applied for the classification of NMR data. Statistical analysis Data were Drug_discovery expressed as the mean �� standard deviation. Statistical analyses were performed using SPSS software (v 12.0; IBM Corporation, Armonk, NY, USA), and statistical comparisons were analyzed using the t-test and one-way ANOVA followed by Tukey��s Honestly Significant Difference (HSD) post hoc test. Differences were considered statistically significant when P < 0.05.