COMT In The Disposition Of Luteolin And Its Regioselectivity Of Methylation

Catechol-O-methyltransferase (COMT, EC2.1.1.6) enzyme catalyzes the transfer of a methyl group from Sadenosyl-L-methionine (AdoMet) to one of the hydroxyls in a catechol substrate. The substrates of COMT in mammals include catecholamines and hormones, such as dopamine, norepinephrine, epinephrine, catecholestrogens.Flavonoids are widely distributed in the nature, which possess a variety of pharmacological activities, such as anti-ancer, anti-xidant, anti-inflammatory. Lots of epidemiologic studies showed, flavonoid intake was associated with reduced cancer risk. Therefore, many flavonoids are potential to be developed into drugs or functional foods. Due to its phenol hydroxyls, they are good substrates of UDP-glucuronosyltransferases and sulfotransferases. And some catechol containing flavonoids are also substrates of COMT. The present study was aimed to study the role of COMT in the disposition of luteolin (3’,4’,5,7-tetrahydroxyflavone) from Chrysanthemum morifolium extract(CME) in vitro and in vivo and its regioselectivity of methylation of luteolin by COMT.1. Mechanism responsible for the much lower exposure of luteolin than apigenin when orally administration of Chrysanthemum morifolium extract to ratsLuteolin (3’,4’,5,7-tetrahydroxyflavone) and apigenin (4’,5,7-trihydroxyflavone) are two common flavones and major bioactive components in Chrysanthemum morifolium extract(CME). Although CME contains approximate luteolin (6.5%, w/w) and apigenin (5.4%, w/w), luteolin showed a much lower exposure than apigenin when CME was orally administered to rats. The aim of the present study is to elucidate the mechanisms that caused the pharmacokinetics difference between luteolin and apigenin in rats. The results of in situ rat intestinal single-pass perfusion model showed that the permeability of luteolin (ka,7.96min-1and Peff,4.87cm/min) was about50%of that of apigenin (ka,18.5min-1and Peff,10.8cm/min), which agreed with that oral bioavailability of luteolin (30.4%) from CME was significantly lower than that of apigenin (51.1%). On the other hand, luteolin was much more unstable than apigenin during the incubation with primary rat hepatocytes and rat tissue homogenates, and a unknown methylated metabolite of luteolin was detected after incubation. In conclusion, Rat intestinal absorption and tissue metabolism caused luteolin a lower exposure than apigenin after orally giving CME to rats. Methylation might be responsible for the disposition of luteolin in vivo.2. Contribution of Catechol-O-methyl transferase on the disposition of luteolin in ratsMethylated metabolite of luteolin was detected after incubation of luteolin with rat tissue homogenates and primary rat hepatocytes, which might cause the lower exposure in rat in vivo. But there is lack of information about the structure of the methyated metabolite and metabolic enzyme. So the present study was to identify the methylated metabolite and its enzyme and to evaluate its role in disposition of luteolin. In the study, two methylated metabolites, M1(meta-O-methylation, chrysoeriol) and M2(para-O-methylation, diosmetin), were identified in hydrolyzed urine after i.v. injection of luteolin to rats and Catechol-O-methyl transferase(COMT) was identified to be responsible for methyaltion of luteolin by using a specific COMT inhibitor, entacapone. Methylation of luteolin was studied in vitro with rat tissue homogenates (liver, kidney, small intestine, lung and erythrocyte) and the liver displayed the highest COMT activity, followed by the kidney, small intestine, lung and erythrocyte. Enzyme kinetic studies of the formation of M1and M2in the rat tissue homogenates were conducted, the Km for the formation of M1and M2were similar in liver and kidney (3.382-3.567μM) but different in other tissues. Regioselective methylation of luteolin was observed in the study. In vitro, there was a preference for the formation of M2over Ml in all the tissues investigated. In contrast, the concentrations of M1were significantly higher than M2in both plasma and urine when intravenous administration of6.5mg/kg luteolin to rats. To evaluate the role of methylation in pharmacokinetics of luteolin in vivo, plasma pharmacokinetics and urinary excretion were studied in rats. The COMT inhibitor, entacapone, significantly decreased the formation of M1and M2and increased the concentration of luteolin in plasma and urine. This study reveals the important role of COMT in pharmacokinetics of luteolin.3. Role of COMT in the pharmacokinetics of Chrysanthemum morifolium extract(CME) in ratsThe research had proved that COMT mediated methylation of luteolin played an important role in disposition of luteolin in vivo. We hypothesized that COMT also played a role when Chrysanthemum morifolium extract(CME) was given to rats and might cause a lower exposure of luteolin than apigenin in rats. To confirm it, an HPLC-UV method for the simultaneous determination of plasma concentrations of luteolin, apigenin, chrysoeriol and diosmetin was established, and applied to pharmacokinetics study of apigenin, luteolin chrysoeriol and diosmetin when CME was orally given to rats with or without co-giving a COMT inhibitor, entacapone. The concentrations of luteolin were significantly increased after co-administration of entacapone, and those of of chrysoeriol were dramtically decreased, which revealed the COMT might play a role in the disposition of luteolin in rats after dosing of CME. In conclusion, the method was sensitive and accurate for simultaneous determination of luteolin, apigenin, chrysoeriol and diosmetin, and the pharmacokinetic study gave us more information of CME in vivo.4. Expression, purification and identification of recombinant human COMT*1and COMT*2in Escherichia coli.The important role of COMT in pharmacokinetics of luteolin and regioselectivity of methylation in rats were revealed. But there was lack of model to study the COMT mediated methylation in human. Recombinant human COMT is very useful tool to study substates and inhibitors of human COMT. Due to widely distribution of soluble hCOMT(S-COMT) in the body and that hCOMT108V/M is an important SNP, the present study was to estabalish a recombinant S-hCOMT*1(108V) and S-hCOMT*2(108M) in Escherichia coli..Method:1) Plasmid pET-28a(+)-hCOMT*1and pET-28a(+)-hCOMT*2were obtained through gene technology.2) Plasmid pET-28a(+)-hCOMT*1and pET-28a(+)-hCOMT*2were transformed into Escherichia coli.Rosseta(DE3), which coulod expressed the objective protein.3) The hCOMT*1and hCOMT*2were purified by simply affinity purification.4) COMT activity were identified with typical COMT substrate (quercetin) and hCOMT*2showed significantly more unstable than hCOMT*2. Results:Escherichia coli. Rosseta(DE3) pET-28a(+)-hCOMT*1and pET-28a(+)-hCOMT*2were estabolished and relatively pure(>65%) hCOMT*1and hCOMT*2after purification. And recombinant human COMT protein could be utilized in the further study of COMT.5. Methylation of Luteolin Human in vitro and in vivoCOMT can transfer a methyl group to one of hydroxyls in catechol ring, in which there was a preference of meta-methylation in the most known COMT substrates, such as dopamine(meta/para:4.3) and levodopa (meta/para:15.4). Though catechol containing flavonoids are good substrates of COMT, there was lack of information on their regioselectivity of methylation. The present study aims to elucidate the regioselectivity of methylation of luteolin by human COMT, and determine its meta/para methylated metabolite ratios in vitro and in vivo. Recombinant human COMT was used to study the formation kinetics of methylation of luteolin; human liver microsomes was used to evaluate the stability of the meta-MeO-and para-MeO-luteolin, while a series of typical inhibitors of CYPs were utilized to identify the subtype of CYPs contributing to further metabolism of meta-MeO-and para-MeO-luteolin. The methylated metabolites of luteolin in human urine(0-8h) collected after oral administration of1mg/kg luteolin were analyzed with HPLC after hydrolysis treatment. Vmax values(pmol mg protein-1min-1) for formation of meta-MeO-luteolin(2735±313), para-MeO-luteolin(4972±558) Km values(μM) of meta-MeO-luteolin(1.889±0.5994),para-MeO-luteolin(4972±558.2) The ratio of meta-MeO/paraMeO of CLint(Vmax/Km) were0.62. Methylated metabolites were also detected in human urine after dosing of luteolin, but the ratio of the meta-to para-mathylated metabolite was1.98±0.5. Human liver microsomes was preferential to metabolize para-methylated luteolin over meta-methylated luteolin. Furthermore, CYP1A2and CYP3A4, preferentially metabolized the para-MeO-luteolin, which partially resulted in different ratios of meta/para methylated metabolites between in vivo and in vitro. Our study gave new information on regioselectivity of methylaiton of flavonoids by human COMT, which deeply explained the metabolism pathway of luteolin and other catechol containing compounds.