ABSTRACT
The mass spectrometry-based metabolomics method was used to systematically investigate the formation of celastrol metabolites,and the effect of celastrol on endogenous metabolites. The mice plasma,urine and feces samples were collected after oral administration of celastrol. Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry( UPLC-QTOF-MS) was applied to analyze the exogenous metabolites of celastrol and its altered endogenous metabolites. Mass defect filtering was adopted to screen for the exogenous metabolites of celastrol. Multivariate statistical analysis was used to identify the endogenous metabolites affected by celastrol. Celastrol and its eight metabolites were detected in urine and feces of mice,and 5 metabolites of them were reported for the first time. The hydroxylated metabolites were observed in the metabolism of both human liver microsomes and mouse liver microsomes. Further recombinant enzyme experiments revealed CYP3 A4 was the major metabolic enzyme involved in the formation of hydroxylated metabolites. Urinary metabolomics revealed that celastrol can affect the excretion of intestinal bacteria-related endogenous metabolites,including hippuric acid,phenylacetylglycine,5-hydroxyindoleacetic acid,urocanic acid,cinnamoylglycine,phenylproplonylglycine and xanthurenic acid. These results are helpful to elucidate the metabolism and disposition of celastrol in vivo,and its mechanism of action.
Subject(s)
Animals , Humans , Mice , Chromatography, High Pressure Liquid , Mass Spectrometry , Metabolomics , Microsomes, Liver , Metabolism , Triterpenes , Metabolism , PharmacokineticsABSTRACT
An ultra performance liquid chromatography tandem quadrupole time-of-flight mass spectrometric method was developed for rapid analysis of glycerophospholipids in RAW264.7 macrophage. The modified Bligh-Dyer was applied to extract glycerophospholipids from RAW264.7 macrophage. The target compounds, detected by mass spectrometry in ESI+ and ESI- mode, were separated by gradient elution with mobile phase (A) water (containing 10mmol·L-1 ammonium acetate and 0.25% acetic acid) and (B) acetonitrile/isopropanol (1:1) (containing 10mmol·L-1 ammonium acetate and 0.25% acetic acid). A total of 82 glycerophospholipids including 57 phosphatidylcholines (PCs), 21 phosphatidylethanolamines (PEs), three phosphatidylglycerols (PGs) and one phosphatidylinositol (PI) were deduced. The UHPLC-QTOF/MS method is rapid, simple and credible for targeting analysis of glycerophospholipids of RAW264.7 macrophage.