| Halenia elliptica, D. Don is commonly used as a Tibetan medicinal herb in the treatment of hepatitis and cholecystitis. The preliminary results indicated that the major components, xanthones1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) and1,5-dihydroxy-2,3-dimethoxy-xanthone (HM-5) could cause vasodilatation in rat coronary artery pre-contracted. HM-5, which is also the major metabolite of HM-1in vitro and in vivo, exhibited endothelium-independent mechanism in rat coronary artery, other than endothelium-dependent of HM-1. We have illustrated the metabolic characteristic on CYP450of HM-1in our previous studies; therefore it is necessary to study the metabolic characteristic on CYP450of HM-5, with the similar activity but different mechanism from HM-1, in order to find out the intrinsic relationship between these two compounds.Using rat liver microsomes (RLMs), pooled human liver microsomes (HLMs) and cDNA expressed recombinant CYP450(rCYP450) isoforms, combining high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC/MSn-IT-TOF), the metabolic pathways, CYP450isoforms involved and inhibition types of HM-5were studied. At the same time, in vivo inhibition effects of HM-1and xanthones ethyl acetate extracts from origin herb were studied. Computer aided molecular docking strategy (Docking) was used to predict the inhibition types and explain the inhibition mechanism. In addtion, biotransformation of three xanthone compounds HM-1, HM-2and HM-3in vivo were studied and the metabolic characteristic of HM-1in RLMs CYP450system was studied, to assitly prove the experiments.The results showed that, twenty-five metabolites were detected after intravenous administration and their structure could be determined, metabolic pathways of HM-1, HM-2and HM-3in vivo were proposed; six metabolites of HM-1were found in RLMs, and four of them were the metabolites of HM-1in HLMs; three metabolites of HM-5were found in RLMs, and two of them were the metabolites of HM-5in HLMs; all three metabolites were detected in the CYP450metabolism of HM-1as well; demethylation and hydroxylation were the major Phase I metabolic reactions for HM-5and HM-1in RLMs and HLMs. CYP1A2,2C6/2C11,2D2,2E1and3A2were studied in RLMs and results indicated that most of the isoforms were involved in the metabolism of HM-5; combining HLMs and rCYP450studies, CYP1A2,2B6,2C8,2C9,2C19,2D6and3A4were involved in metabolism of HM-5in human in vitro. The inhibition study presented that in RLMs, HM-1inhibited CYP1A2,2C6/2C11and3A2with IC50values were34.99μM,246.37μM and14.06μM (IC20) respectively; HM-5inhibited CYP1A2,2C6/2C11and2D2with IC50values were16.54μM,14.50μM (IC20) and20.91μM (IC20) respectively. In HLMs, HM-1and HM-5were verified to be time-independent in inhibiting of CYP3A4, and HM-5showed mixed mode in inhibiting CYP1A2,2D6,3A4, with Ki values were23.13μM,15.19μM and10.23μM respectively. These inhibition mechanisms could also be explained by Docking. The data of in vivo study demonstrated that both HM-1(i.v.) and xanthones ethyl acetate extract (p.o.) could inhibit CYP3A2in rats.In conclusion, the metabolism of xanthones from the origin herb was multi-CYP450isoforms involved; in vitro, metabolism of HM-5was similar to its metabolic precursor HM-1in the metabolites, but different from it in inhibition effect and inhibition mode; in vivo, CYP3A2was accessible to be inhibited by HM-1and xanthones ethyl acetate extract. This study could provide evidence for metabolism study on CYP450of xanthones. |
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