Study On Metabolism Of The Main Active Components Of Total Coumarins Of Cnidii Monnieri In Rats And Human Liver Microsomes

Background:Cnidii was the dry and ripe fruit of Cnidium monnier (L．) Cuss. that was annualherb. Many researches certificated that the active components of Cnidii wasCoumarins,which were known as Total Coumarins of Cnidii Monnieri (TCCM). Asosthole, imperatorin is two highest active compounds of TCCM, often served as thequality control indicators of evaluating quality of different sources of Cnidiummedicinal and proprietary Chinese medicines containing cnidii. This study was toidentify which isoforms of CYP450were responsible for Ost and Imp metabolism inrats and human liver microsomes.To provide experimental basis for futher researchof the metabolism of Ost Imp in vivo and clinical rational drug using.Objectives:To study the metabolism of Chinese medicine Cnidium effective monomerOsthole(Ost) and Imperation(Imp) in vitro by liver microsomes, exploring theenzyme kinetics and metabolic pathways. In order to get more knowledge of totalcoumarins of Cnidium and more in depth study in pharmacokinetics to providetheoretical and experimental basis for clinical.Methods:1,To set up the HPLC analyse method of Ost and Imp.2,To investigated the metabolic characteristics of Imp from incubation time,substrate concentration and Liver microsomal concentration.3,CYP3A4inhibitor Ketoconazole(KET), CYP2D6inhibitor Qunidine (Qui),CYP2C8inhibitor trimethoprim (TMP), CYP2C9inhibitor sulfaphenazole (Sul),CYP1A2inhibitor α-Naphthoflavone (α-NF) were used to investigate the effects onthe metabolism of Imp.4, To investigated the metabolic characteristics of Ost from incubationtime,substrate concentration and Liver microsomal concentration.5,To study these inhibitors inciuding CYP3A4inhibitor Ketoconazole(KET), CYP2D6inhibitor Qunidine (Qui), CYP2C8inhibitor trimethoprim (TMP), CYP2C9inhibitor sulfaphenazole (Sul), CYP1A2inhibitor α-Naphthoflavone (α-NF) used toincubation with Ost could lead to effects on metabolism.Results1,Establishing the HPLC analyse method of Ost and Imp.2,When the Liver microsomal incubation solution maintained at37℃in ashaking bath of120rpm/min for0～30min, the disappeared rate of Imp all linearlyincreased.So the choice of incubation was30min. To determine the concentration ofliver microsomes were o.2mg/ml. The metabolism of Imp in Liver microsomalincubation solution showed the characteristic of enzyme kinetics: In the concentrationrange of0.8-12.8μg/ml, the disappeared rate of Imp in Liver microsomal incubationsolution all linearly increase, when the concentration above25.6μg/ml, the metabol-ism of Imp showed saturation.According Eadie-Hofstee to count the enzyme kineticparameters of Imp. In rat liver microsomes: V_max=31.50±1.32nmol/min/pmol,K_m=18.34±1.07uM, Cl_int=1.72±0.11mL/min/pmol;in human liver microsomes:V_max=41.37±5.12nmol/min/pmol, K_m=14.64±1.91μM, Cl_int=2.83±2.83mL/min/pmol.3,At low,medium and high concentrations in rat liver microsomes, α-NF andTMP can inhibit the metabolism of Imp,the metaboli rate of Imp compared with thecontrol group have significant(P＜0.5﹚,and with the increasing concentration ofinhibitor,suppression role is strengthened;In human liver microsomes,in addition tothe above,at medium concentration,Qui can significant inhibit the metabolism.4,When the Liver microsomal incubation solution maintained at37℃in ashaking bath of120rpm/min for0～20min, the disappeared rate of Ost all linearlyincreased.So the choice of incubation was20min. To determine the concentration ofliver microsomes were o.2mg/ml. The metabolism of Ost in Liver microsomalincubation solution showed the characteristic of enzyme kinetics: In the concentrationrange of1.6-19.2μg/ml, the disappeared rate of Ost in Liver microsomal incubationsolution all linearly increase, when the concentration above38.4ug/ml, themetabolism of Ost showed saturation.According Eadie-Hofstee to count the enzymekinetic parameters of Ost. In rat liver microsomes: V_max=6.58±0.76nmol/min/pmol,K_m=51.17±6.32uM, Cl_int=7.76±1.01mL/min/pmol in human liver microsomes: V_max=4.36±0.37nmol/min/pmol, K_m=36.49±4.21uM, Cl_int=8.35±1.21mL/min/pmol.5,At low,medium and high concentrations in rat liver microsomes, KET caninhibit the metabolism of Ost,the metaboli rate of Ost compared with the controlgroup have significant(P＜0.5﹚,and with the increasing concentration ofinhibitor,suppression role is strengthened;In human liver microsomes,at lowconcentration,KET and Qui can inhibit the metabolism of Ost. Only KET cansignificant inhibit the metabolism at mediu concentration. And at highconcentration,KET,Qui and Sul can inhibit the metabolism of Ost.Conclusions:1,We have established the sensitive HPLC analyse method of Ost and Imp inLiver microsomes.2,The metabolism of Imp and Ost in rat and human Liver microsomalincubation solution showed the characteristic of enzyme kinetics.3,CYP1A, CYP2C mediate the metabolism of Imp in rat liver microsomes,CYP1A2, CYP2C8, CYP2D6involve in the metabolism of Imp in humanmicrosomes. CYP3A mediate the metabolism of Ost in rat liver microsomes,CYP3A4, CYP2D6involve in the metabolism of Ost in human microsomes.