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Objective: To investigate the metabolic stability, the main CYP450 enzymes phenotypes and metabolites of Diosbulbin B based on in vitro metabolism model. Methods: For metabolic stability study, UPLC-MS/MS was used to detect the remaining Diosbulbin B content in the incubation solution after being incubated with human and rat liver microsomes, respectively. Ten recombinant human CYP450 enzymes (1A1, 1A2, 1B1, 2A13, 2A6, 2B6, 2D6, 2C9, 2C19, 3A4) were used for identifying the metabolic enzyme phenotypes of Diosbulbin B. Moreover, the major metabolic enzyme phenotype for the metabolism of Diosbulbin B was confirmed and verified by the rat isolated hepatic perfusion model. The metabolites of Diosbulbin B in human and rat liver microsomes were determined by LC-MS/MS. Results: The metabolic percentage of Diosbulbin B in human and rat liver microsomes were 37% and 59%, respectively. Its half-lives t1/2 in human and rat liver microsomes were 97.4 and 52.3 min, respectively. The intrinsic clearance rates CLint in human and rat livers were 8.23 and 23.9 mL/(min•kg), and liver clearance CLh in human and rat livers were 5.89 and 16.8 mL/(min•kg). It can be found that the metabolic rate of Diosbulbin B in rat liver microsomes was faster than in human liver microsomes. There were five CYP enzymes, including 3A4, 2C19, 2C9, 1A13 and 1A1, related to the metabolism of Diosbulbin B, especially CYP3A4. The hepatic perfusion experimental results showed that the metabolism of Diosbulbin B was inhibited by ketoconazole, and the inhibitory effect was enhanced along with the increasing dosage of ketoconazole, which confirmed that CYP3A4 played an important role in metabolism of Diosbulbin B. There was one metabolite (M1) of Diosbulbin B has been found in both human and rat liver microsomes incubation. Conclusion: The metabolic rate of Diosbulbin B in rat liver microsomes was faster than human liver microsomes. The CYP3A4 plays a leading role in the metabolism of Diosbulbin B. And a demethylated metabolite of Diosbulbin B was appeared in both human and rat liver microsomes incubation.
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OBJECTIVE To characterize pharmacokinetics and tissue distribution of diosbulbin B (DIOB)in rats,and compare exposure and elimination of DIOB in plasma and tissues after ig adminis-tration of compound DIOB and ethanol extract of Dioscorea bulbifera L.(EEDB),respectively.METHODS Liquid chromatography coupled with tandem mass spectrometry method was developed and validated for quantitative determination of DIOB in plasma and tissue samples. The sensitivity, matrix effect, accuracy and precision of the method were determined. After a single ig administration of DIOB 1.3 mg·kg-1and EEDB 1.0 g·kg-1, respectively, plasma and tissues samples were collected separately from male SD rats at prescheduled time points.The drug-protein binding of DIOB in plasma and tissues was measured using Rapid Equilibrium Dialysis Assay. The concentration-time profiles of DIOB were analyzed using WinNonLin 6.4 to obtain pharmacokinetic parameters. RESULTS Rapid absorption and elimination of DIOB were observed in rats.The plasma maximum concentration(cmax),peak time(tmax) and eliminate half life(t1/2)of DIOB in rats receiving DIOB or EEDB were 312±67 and(131±84)μg·L-1(P<0.01), 0.12 ± 0.07 and (0.23 ± 0.17) h, and 1.17 ± 0.28 and (2.34 ± 0.83) h (P<0.05). DIOB was rapidly distributed in most tissues,with the AUC(0-24 h)of DIOB in lungs,liver and kidneys for DIOB and EEDB groups were 4527.0±557.7,183.0±51.1 and(64.4±22.4)ng·h·g-1,and 6507.9±424.3(P<0.01),467.5±202.7(P<0.05) and (238.6 ± 70.0) ng·h·g-1(P<0.05), respectively. The protein binding rate of DIOB was 38.7%-43.6%,61.3%-66.9% and 61.4%-64.7% in plasma,livers and lungs,respectively.The ratio of free concen-trations between tissue and plasma for lungs was more than 48,indicating the specific distribution of DIOB in lungs.CONCLUSION There is difference in pharmacokinetics and tissue distribution of DIOB in rats after taking compound DIOB and EEDB.Among the tissues detected,lungs are the major target of DIOB with the highest exposure level.These characterizations should be considered in pharmacological and toxicological studies of DIOB and relevant herbs.
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Rhizoma Dioscoreae Bulbiferae is a traditional Chinese medicine with hepatotoxicity, but the metabolic profile of fatty acids has not been identified in the rats with liver injury. In this project, a gas chromatography-mass spectrometry method was applied to simultaneous quantification of 16 non-esterified fatty acids (NEFA) and esterified fatty acids (EFA) in the serum of control, ethanol extraction of Rhizoma Dioscoreae Bulbiferae (ethanol extraction, ET) and diosbulbin B (DB)-treated rats. Meanwhile, the change of fatty acid metabolic profile of liver injured rats was analyzed by principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). The results of NEFA concentration indicated that the serum concen-trations of palmitic acid (C16:0), stearic acid (C18:0), palmitoleic acid (C16:1n7), oleic acid (C18:1n9), vaccenic acid (C18:1n7), linoleic acid (C18:2n6), linolenic acid (C18:3n3), eicosatrienoic acid (C20:3n6), arachidonic acid (C20:4n6) and docosahexaenoic acid (C22:6n3) in DB-treated rats decreased significantly, while that of C18:2n6 and C20:3n6 obviously increased and that of C20:4n6 and C22:6n3 noticeably dropped in ET-treated rats when compared with control. Furthermore, the results of EFA concentration illus-trated that the serum concentrations of C16:0, C18:0, C20:4n6, C22:6n3 and eicosapentaenoic acid (C20:5n3) in two toxic groups were remarkably decreased when compared with control. The fatty acid meta-bolic profiles of the two toxic groups exhibited significant difference from the normal levels, and the degree of deviation of ET group was higher than that of DB group. More importantly, the results of PLS-DA showed that C20:4n6 and C22:6n3 were important indicators of the hepatotoxicity induced by ET and DB, and the serum concentrations of the two fatty acids had good correlation with the levels of alanine aminotransferase, aspartate aminotransferase and total bilirubin using Pearson's correlation analysis and canonical correlation analysis (CCA). Therefore, C20:4n6 and C22:6n3 were identified as potential biomarkers of ET and DB-induced liver injury. The project can provide a foundation for furture investigation of molecular mechanism of hepato-toxicity caused by Rhizoma Dioscoreae Bulbiferae.
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Objective To establish a HPLC method for determining and comparing the contents of diosbulbin B in Dioscorea bulbifera L. from different regions. Methods The medicine powders were extracted twice by water, then the water extracts were combined and detected by HPLC at 35℃,with Kromasil-C18 column (4. 6 mm×250 mm,5μm) and a mobile phase of acetonitrile-water-glacial-acetic acid (34660. 1). The flow rate was 1. 0 mL·min-1 and the detection wavelength was 210 nm. Results The linear range of diosbulbin B was 26. 0-260. 0 μg·mL-1(r=0. 999 9). The average content of diosbulbin B in Dioscorea bulbifera L. from different areas was that of Hebei>Guangxi>Jiangsu>Sichuan>Zhejiang. Conclusion The method is simple,quick,accurate and suitable for the determination of diosbulbin B in Dioscorea bulbifera L. from different regions.