| Objective: To investigate the effects of aqueous extract of Semen Cassiae(AESC)on CYP450 enzyme in rat liver by using UPLC-QTOF-MS and metabolomics related methods.By analyzing the difference of endogenous metabolites and the characteristics of the metabolism process of Aurantio-obtusin in vivo.We can provide experimental evidence for the rational clinical use of Semen Cassiae.Methods:(1)The microsomes of rat liver were prepared after oral administration with AESC for two weeks.Then the substrate metabolites were detected of by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry(UPLC-MS/MS).Meanwhile the mRNA and protein expressions were detected by RT-PCR and Western blot.(2)The urine was collected with metabolic cages.The urine metabolic profiling was analysed by using UPLC-QTOF-MS,based on which the PCA and OPLS-DA models were established for metabonomic analysis.Potential biomarkers were screened by using variable importance in the projection(VIP)and T test.(3)Sprague-Dawley(SD)rats were given Aurantio-obtusin(500 mg·kg-1)respectively.Plasma,bile,urine and feces were collected before and after oral administration.All samples were discovered using Waters Metabolynx XS software,the potential metabolites were screened out based on well-defined representative structure with mass defect filter(MDF)technology.Then,their structures were further identified based on MS/MS fragmentation and retention time.Results:(1)The result of this experiment was that AESC obviously induced the enzyme activities of CYP1A2,CYP2B1,CYP2C11,CYP2D2,CYP2E1 and CYP3A1.Low dose of AESC significantly reduced the activity of CYP2D2,but it was induced the activity of CYP2D2 by middle dose and high dose of AESC.These subtypes were a dose-dependent increase except CYP3A1.The mRNA levels of CYP1A2,CYP2C11,CYP2D2 and CYP2E1 were also induced in rats treated with AESC,but with no significant effect in CYP2B1 and CYP3A1 mRNA expressions.The protein levels of CYP2C11 and CYP2E1 were also induced in rats treated with AESC,but did not statistically differ from control.(2)ALT was significantly increased in the middle dosage treatment group,but with no significant effect in low and high dosage treatment group.AST,SOD,ALB and GST did not statistically differ from control.The liver samples from the treatment group did not show pathological changes in liver tissue samples from any of the groups.The relative contents of uric acid?proline betaine?glycine and taurine in the AESC 5 and 15g·kg-1groups were significantly lower than those in the control group,but there was no significant difference between AESC 1.5g·kg-1group and control group.The relative contents of 1,7-Dimethylguanosine in the AESC 1.5 and 15g·kg-1groups were significantly lower than those in the control group,but there was no significant change between AESC 5g·kg-1group and control group.The relative contents of citric acid in the AESC 15g·kg-1group were significantly lower than those in the control group,the relative contents of citric acid in the AESC 1.5 g·kg-1group were significantly higher than those in the control group,but there was no significant change between AESC 5 g·kg-1group and control group.(3)The metabolic transformation pathways of Aurantio-obtusin in rats included demethylation,hydroxylation,methylation,O-glucuronide conjugation and O-sulphate conjugation.Conclusion:(1)The enzyme activity of CYP450,particularly CYP2C11 and 2E1 subtypes,were induced or inhibited by AESC to varing degrees.(2)6 endogenous metabolites like 1,7-Dimethylguanosine?Proline betaine?Glycine?Uric acid?Taurine and Citric acid have been found,through analysis metabolism pathway of each biomarkers,Semen Cassiae's mechanism of pharmacological and toxicity effects may be relate to taurine,purine,amino acid and energy metabolism.(3)Among the 6metabolites,the sulfonated product and glucuronidated conjugates(M2,3,4)were major metabolites of Aurantio-obtusin in rats,M2 and M4 in plasma were in the peak for a short time and the absorption is better. |
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