| Rhizoma alismatis (RA), the dried rhizome of Alisma orientale (Sam.) Juz. and a famous traditional Chinese medicine that has been widely used for removing dampness and promoting diuresis in clinic, was selected and investigated for its potential toxicological mechanism, toxic section and components with the application of metabonomic approach and by the in vitro cytotoxicity test. Pharmacokinetics and the in vitro metabolism of alisol A were also studied. The results provide a meaningful scientific basis for the elucidation of the pathogenesis and prevention and diagnosis of renal injury induced by RA, and for reasonable safe medication.1. UPLC-MS based metabonomic approaches for analysis of urine and serum samples from rats were developed and successfully applied to the study on the metabolic changes in urine and serum from rats treated with RA. Caulis aristolochiae manshuriensis was selected as a positive control medicine. Serum biochemical indicator and histopathology were tested to evaluate renal function. The potential mechanism of RA-induced nephrotoxicity is also discussed. The values of Scr/BW and BUN of RA-treated group were significantly higher than those of normal control group. Obviously pathological changes were observed in kidneys from RA-treated group. The differences in the urine (and serum) metabolic profiles of the control and treated rats were clearly distinguishable with principal components analysis (PCA)of the chromatographic data. 10 and 6 correlative bio markers were obtained in the urine and serum loading plots. According to the physiologic function of these biomarkers, it is supposed that mechanism of RA-induced renal injury may refer to a series of metabolic disturbance,such as phospholipids metabolism, amino acid metabolism, sphingosine metabolism and enterobacteria metabolism, and may be concerned with dysfunction of liver and sausage and oxidative stress.2. The nephrotoxicity of chloroform layer, ethyl acetate layer, n-butanol layer, and water layer extracted from RA water extract were investigated to find out the toxic section of RA by combination metabonomic approach with serum biochemistry and histopathology analysis.The results of serum biochemical and histopathology indicated that chloroform layer may be the nephrotoxic section of RA. Rats treated with chloroform layer were away from normal control group, and the distance was longer than others to normal control group. Most of the.biomarkers obtained were the same as the ones obtained from renal injury induced by RA water extract. It is reveled that chloroform layer may be the nephrotoxic section of RA from the metabonomic aspect. It is also indicated that metabonomic technology could be an effective approach for clinical diagnosis and mechanism research.An HPLC-IT/MSn method was developed for screening and identification of the main chemical constituents in chloroform layer of RA water extract. Chromatographic peaks of 17 were detected, 10 of which were identified as alisol A, alisol A 23-acetate, alisol A 24-acetate,16-oxo-alisol A, 13,17-epoxy-alisol A, alisol C, alisol C 23-acetate, alisol L, alisol M and alismaketone C 23-acetate.3. The cytotoxic effect of alisol A on renal epithelial cell (HKC cells) was investigated using aristolochic acid (a known nephrotoxic substance) as a positive control. The cytotoxic effect of alisol A was found to be time and concentration dependent. The IC50 values of alisol A in HKC cells following incubation for 24, 48, and 72 h, were significantly lower than those of aristolochic acid, respectively, which indicated that alisol A has considerable cytotoxic effect on the HKC cells. Based on the results obtained from the fluorescence microscope, low concentration of alisol A induced apoptosis, while high concentrarion induced neorobiosis to cell death. The results in this study suggest that alisol A may be one of the nephrotoxic chemical constituents in RA.4. An LC-MS method was developed and validated for the simultaneous determination of alisol A and alisol A 24-acetate in rat plasma, and was successfully applied to the study on the pharmacokinetics of them in rat plasma. Both of the concentration-time curves of alisol A and alisol A 24-acetate in rat plasma after oral administration of Rhizoma Alismatis extract showed significant double-peak phenomenon. The tnax was 9.7 ± 0.5 h and 9.2 ± 1.0 h, Cmax was 476.1 ± 188.6 ng·ml-1 and 233.1 ± 55.4 ng·ml-1,t1/2 was 3.1 ± 0.6 h and 2.9 ± 0.9 h,AUC(0-24) was 2945 ± 1024 ng·h·ml-1 and 1820 ± 464 ng·h·ml-1,AUC(0-∞) was 3453 ± 1623 ng·h·ml-1 and 1874 ± 450 ng h mL-1,respectively for alisol A and alisol A 24-acetate5. An LC-MS method was developed and successfully applied to the study on the enzyme kinetics of alisol A in RLM and HLM incubation systems, and employed for semi-quantitative determination of each metabolite of alisol A. The metabolites of alisol A in RLM, HLM and human recombinant CYP3A4 enzyme incubation systems were identified by HPLC-Q-TOF/MS. A total of 3 and 6 oxidative metabolites were identified in RLM and HLM incubation systems, respectively. Chemical inhibitors of CYP450 and individual human recombinant CYP450 enzyme were used to identify the CYP450 isozymes involved in the formation of each metabolite of alisol A. The result indicated that the formation of each metabolite of alisol A was mainly catalyzed by CYP3A4 enzyme. |
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