Isolation And Identification Of Metabolites Of Bioactive Xanthones Isolated From Halenia Elliptica D. Don By High Performance Liquid Chromatography Coupled To Ion Trap Time-of-flight Mass Spectrometry

Halenia elliptica D. Don, belonged to the Gentianaceae family and also called "Tibetan artemisia capillaris", is widely distributed in Tibetan altiplano. The whole plant of Halenia elliptica is used as folk medicine, which characterizes by bitter in taste and cold in nature. As Halenia elliptica has the function of clearing heat, eliminating dampness, calming the liver and cholagogic action, it is a Tibetan medicinal herb used for the treatment of hepatitis and gastritis in system of Tibetan medicine.Xanthones are active components in Halenia elliptica D. Don. There were eight xanthones isolated, purified and identified from Halenia elliptica D. Don by silica gel column chromatography and Sephadex LH-20chromatography, which were seven xanthone aglycone and one xanthone glycoside, including1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1),1-hydroxy-2,3,4,7-tetramethoxy-xanthone (HM-2),1-hydroxy-2,3,4,5-tetramethoxy-xanthone (HM-3),1,7-dihydroxy-2,3,4,5-tetramethoxy-xanthone (HM-4),1,5-dihydroxy-2,3-dimethoxy-xanthone (HM-5),1,7-dihydroxy-2,3-dimethoxy-xanthone (HM-7),1,8-dihydroxy-2,3-dimethoxy xanthone (HM-8) and1-O-[β-D-xylopyranosyl-(1-6)-β-D glucopyranosyl]-2,3,5-trimethoxy-xanthone (HM-6). And the main active xanthones extracted from Halenia elliptica were determined by HPLC-DAD method.In the present investigation, both the in vitro metabolism of three xanthones, HM-3, HM-4and HM-5, and the in vivo metabolism of another xanthone, HM-1, had been studied. After their metabolites were identified, the metabolic pathways had been deduced subsequently.The metabolisms of three xanthones HM-3, HM-4and HM-5had been studied in rat liver microsomes in vitro. High performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LC-ESI-IT-TOF) was applied for identification of metabolites of these three xanthones mentioned above and1H NMR and13C NMR was used to elucidate the major metabolite of HM-5, HM-5B. The structures of eight metabolites were identified and five of them had not been reported before. It was indicated from metabolic pathways of HM-3, HM-4and HM-5that in vitro metabolic transformation of these xanthones occurred mainly at4-,2-,5-carbonic positions on their structures of parent drugs.The in vivo metabolism of1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1), an active ingredient from Halenia elliptica D. Don used as a Tibetan medicinal herb, was explored in SD rats. Phase Ⅰ and phase Ⅱ metabolites of HM-1in bile samples were detected after intravenous administration by LC-ESI-IT-TOF and the major metabolic pathway of HM-1was estimated. LC-ESI-IT-TOF was applied for on-line analysis of bile samples of rats without or with enzymatic hydrolysis. There were four metabolites of HM-1, of which Phase Ⅰ metabolites were HM-5and phase Ⅱ metabolites were two glucuronide conjugates of HM-5and one sulfate conjugate of HM-5. Meanwhile, two glucuronide conjugates of HM-1were also found out. HM-1was metabolized into HM-5by demethylation, a type of phase Ⅰ reaction, in rats in vivo. Simultaneously, parent drug HM-1or its metabolite HM-5was transformed into glucuronide conjugates or sulfate conjugate. Glucuronide conjugates were predominate among phase Ⅱ metabolites.Taken together, the results showed that the in vitro and in vivo metabolisms of structurally similar xanthones would be characteristic. Demethylation was observed in phase Ⅰ. In addition, conjugation with glucuronic acid (GlcA) and sulfatation occurred in phase Ⅱ.