Interaction effects on cytochrome P450 both in vitro and in vivo studies by two major bioactive xanthones from Halenia elliptica D. Don.

The major components, 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) and 1,5-dihydroxy-2,3-dimethoxy-xanthone (HM-5) isolated from Halenia elliptica D. Don (Gentianaceae), could cause vasodilatation in rat coronary artery with different mechanisms. In this work, high-performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMS-IT-TOF) was used to clarify the metabolic pathways, and CYP450 isoform involvement of HM-1 and HM-5 were also studied in rat. At the same time, in vivo inhibition effects of HM-1 and ethyl acetate extracts from origin herb were studied. Three metabolites of HM-5 were found in rat liver microsomes (RLMs); demethylation and hydroxylation were the major phase I metabolic reactions for HM-5. Multiple CYP450s were involved in metabolism of HM-1 and HM-5. The inhibition study showed that HM-5 inhibited Cyp1a2, 2c6 and 2d2 in RLMs. HM-1 inhibited activities of Cyp1a2, Cyp2c6 and Cyp3a2. In vivo experiment demonstrated that both HM-1 and ethyl acetate extracts could inhibit Cyp3a2 in rats. In conclusion, the metabolism of xanthones from the origin herb involved multiple CYP450 isoforms; in vitro, metabolism of HM-5 was similar to that of its parent drug HM-1, but their inhibition effects upon CYP450s were different; in vivo, Cyp3a2 could be inhibited by HM-1 and ethyl acetate extracts.

Biotransformation and in vitro metabolic profile of bioactive extracts from a traditional Miao-nationality herbal medicine, Polygonum capitatum.

Polygonum capitatum Buch.-Ham.ex D. Don, a traditional Miao-nationality herbal medicine, has been widely used in the treatment of various urologic disorders. Recent pharmacological studies demonstrated that a pure compound, FR429, isolated from the ethanol extracts of P. capitatum could selectively inhibit the growth of four hepatocellular carcinoma (HCC) cell lines in a dose-dependent manner. Thus, P. capitatum probably exhibits potential antitumor activity. However, there is very little information on the metabolism of substances present in P. capitatum extracts. In this study, gallic acid, quercetrin, ethanol extracts and ethyl acetate fraction of ethnolic extract (EtOAc fraction) of P. capitatum were cultured anaerobically with rat intestinal bacteria. A highly sensitive and selective liquid chromatography electrospray ionization-ion trap-time of fight mass spectrometry (LC/MSn-IT-TOF) technique was employed to identify and characterize the resulting metabolites. A total of 22 metabolites (M1-M22), including tannins, phenolic acids and flavonoids, were detected and characterized. The overall results demonstrated that the intestinal bacteria played an important role in the metabolism of P. capitatum, and the main metabolic pathways were hydrolysis, reduction and oxidation reactions. Our results provided a basis for the estimation of the metabolic transformation of P. capitatum in vivo.

In vitro identification of cytochrome P450 isoforms responsible for the metabolism of 1-hydroxyl-2,3,5-trimethoxy-xanthone purified from Halenia elliptica D. Don.

1-Hydroxyl-2,3,5-trimethoxyxanthone (HM-1) is one of the main constituents extracted from Halenia elliptica D. Don, which is a traditionally used Tibetan medicinal plant. The aim of this study was to illustrate the proposed metabolic pathways of HM-1 and identify which cytochrome P450 (CYP450) isoforms involved in its metabolism by using pooled human liver microsomes (HLMs) and recombinant CYP450 isoforms with selective chemical inhibitors. Metabolites were identified by high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMS(n)-ESI-IT-TOF) and nuclear magnetic resonance spectroscopy (hydrogen-1 NMR and carbon-13 NMR). Three metabolites (M1-M3) were identified, which demonstrated that demethylation and hydroxylation were the major Phase I metabolic reactions for HM-1 in HLMs. The structure of another metabolite (M4) was still unclear. The enzymatic kinetics of M1 (K(m)=23.19±14.20 µM) and M2 (Km=32.06±17.09 µM) exhibited substrate inhibition; whereas, the formation of M3 (K(m)=5.73±0.70 µM) and M4 (K(m)=16.43±5.12 µM) displayed Michaelis-Menten kinetics. The intrinsic clearance (V(max)/K(m)) of M3 was highest among these metabolites, suggesting that M3 was the major metabolite of HM-1. Moreover, CYP3A4 and CYP2C8 were the primary CYP450 isoform responsible for the metabolism of HM-1. CYP1A2, CYP2A6, CYP2B6, CYP2C9 and CYP2C19 were also involved in HM-1 metabolism, especially in the formation of M3. This study finally provides evidence of substrate inhibition and metabolism-based drug-drug interaction for the medicinal preparations containing HM-1 used in clinic.

Identification of metabolites of FR429, a potential antitumor ellagitannin, transformed by rat intestinal bacteria in vitro, based on liquid chromatography-ion trap-time of flight mass spectrometry analysis.

FR429 is an ellagitannin with a potential antitumor activity, isolated and purified from Polygonum capitatum Buch.-Ham.ex D.Don, which is a traditional Miao-nationality herbal medicine in Guizhou and Yunnan of China. Our preliminary result of pharmacology study has indicated that the antitumor activity of FR429. However, the metabolism of FR429 has not been reported yet. In this study, LC-ion trap-time of flight mass spectrometry (LC-IT-TOF/MS) was used to characterize unpredictable metabolites of FR429 biotransformed by intestinal bacteria in vitro. Total thirteen metabolites were detected and characterized via comparisons of their accurate molecular masses and fragment ions of each MS(n) stage with those of the parent drug, and four of them were also elucidated by NMR. The results demonstrated that FR429 could be transformed by intestinal bacteria in vitro, mainly via hydrolysis and reduction reaction. This work provided a basis for the further study on the biotansformation of FR429 in vivo.

Enzyme kinetic and molecular docking studies on the metabolic interactions of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from Halenia elliptica D. Don, with model probe substrates of human cytochrome P450 enzymes.

Halenia elliptica D. Don is a Tibetan herb and medicinal preparations containing Halenia elliptica have been commonly used for the treatment of hepatitis B virus infection in China. The metabolism of 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) to its metabolites is mediated through cytochrome P450 enzymes. This study aimed to investigate the herb-drug interaction potential of HM-1 by studying its effects on the metabolism of model probe substrates of five major CYP450 isoforms in human liver microsomes. HM-1 showed moderate inhibitory effects on CYP1A2 (IC50 = 1.06 µM) and CYP2C9 (IC50 = 3.89 µM), minimal inhibition on CYP3A4 (IC20 = 11.94 µM), but no inhibition on model CYP2D6 (dextromethorphan) and CYP2E1 (chlorzoxazone) probe substrates. Inhibition kinetic studies showed that the K(i) values of HM-1 on CYP1A2, CYP2C9 and CYP3A4 were 5.12 µM, 2.00 µM and 95.03 µM, respectively. HM-1 competitively inhibited testosterone 6ß-hydroxylation (CYP3A4) but displayed mixed type inhibitions for phenacetin O-deethylation (CYP1A2) and tolbutamide 4-hydroxylation (CYP2C9). Molecular docking study confirmed the inhibition modes of HM-1 on these human CYP isoforms.

In vitro study on metabolite profiles of bioactive xanthones isolated from Halenia elliptica D. Don by high performance liquid chromatography coupled to ion trap time-of-flight mass spectrometry.

The metabolisms of five xanthones isolated from a Tibetan medicinal herb Halenia elliptica D. Don, including 1-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) and 1,5-dihydroxy-2,3-dimethoxy-xanthone (HM-5), were 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 five xanthones mentioned above and (1)H NMR was used to elucidate the major metabolites. The structures of thirteen metabolites were identified and seven of them had not been reported before. Moreover, xanthone isomers herein could be distinguished by difference of fragmentation behaviors with increase of stages or relative abundances. The results indicated that in vitro metabolic transformation of HM-1, HM-2, HM-3, HM-4 and HM-5 occurred mainly at 2-, 4-, 5-, 7-carbonic positions on their structures of parent drugs. The metabolites could be new vasoactive substances. This work will provide a basis for study on the structure-activity relationships of these xanthones and their derivatives from Tibetan herbal in the next work.