Effects Of Seven Kinds Coumarin On The Hepatic Microsome CYP3A4Activity In Rat

ObjectiveBy vitro incubation reaction system,to study the effect of six furancoumarin compounds （psoralen, xanthotoxol, xanthotoxin, imperatorin， isop-impinellin）and a simple coumarin osthole on hepatic microsome CYP4503A4（Cytochrome P4503A4）with MDZ as probe activity in rats,calculate halfmaximal （50%） inhibitory concentration （IC） of a substance IC50, and comparethe strength of the compound effect of these compounds. In vitro metabolismexperiments results based on, preliminary exploration to Total coumarrins fromRadix Angelicae Dahuricae （TCD） nifedipine （by CYP3A4metabolism） in ratsin vivo pharmacokinetic parameters.MethodsFirst,Four adult male Wistar rats, phenobarbital-induced precipitation ofcalcium Preparation of rat liver microsomes and determination of the totalprotein content of rat liver microsomes, CYP total and cytochrome b5（cytb5）,Second,The500μL incubated in vitro cultivation of the system, added to each ofsix kinds of6,7-Furanocoumarins compounds and a simple the coumarin thatosthole or positive control drug chloramphenicol concentration of1.0,2.0,4.0,8.0,16.0and32.0μmol· mL^-1, and create a blank control group; tube CYP3A4probe drug midazolam （midazolam） after the first pre-incubation5min and thenincubated for15min at37°C shaking water bath, ice， The cooling bath thereaction was terminated. Liquid chromatography the final concentration of thethe probe drug metabolites in vitro incubation reaction system1-OH-MDZ, theoperation of the experiment was repeated five times and calculate the IC50evaluation of these seven coumarin compounds on rat liver microsomesCYP3A4activity.Third,Take30healthy male Wistar rats were randomly dividedinto three groups, Angelica total coumarin groups were ig given coumarin25,50 mg/kg once daily for3consecutive days; the control group was given the sameamountsaline. The last time ig1h after each group ig nifedipine10mg/(2.5mL·kg^-1), nifedipine orally to the end of the start timing,5,15,30min and1,1.5,2,3,4,56,8h, blood was collected from the tail0.5mL, blood sampleswere placed in a plastic tube prior to adding sodium heparin,4000rpmcentrifuged for15min, isolated plasma nifedipine was measured at differenttime points by reversed-phase high performance liquid plasma concentration,the pharmacokinetic parameters were calculated based on the determination ofsample data in rats nifedipine.ResultsFirst,Phenobarbital induced rat liver microsomes CYP total0.55n mol·mg^-1Protein, and the protein content20.64mg·g^-1Live Wet the content of cytb50.38nmol mg^-1Protein. Phenobarbital induced rat liver microsomal protein thanblank an increase of22.4%.Second,Six concentrations of positive control drugchloramphenicol were strongly inhibited the generation of1-OH-MDZ rate anddose-dependent inhibition （P<0.01）. Each coumarin compounds on CYP3A4showing different degrees of inhibition and dose-dependent inhibition was alsofound., CYP3A4IC50values (μmol·L^-1) were psoralen （6.74）, xanthotoxol（7.45）,imperatorin（9.63）, isoimperatorin（16.48）, xanthotoxin（67.34）, isopimpinellin（75.69）, osthole （89.56）, chloram-phenicol （0.64）.Seven coumarin compoundshave different degree of CYP3A4inhibition, the positive control drugchloramphenicol inhibits the strongest and dose-dependent inhibition （P<0.01）.Third, Three groups nifedipine main pharmacokinetic parameters: control groupKa (0.26h^-1)、Ke (0.15h^-1)、t1/2α（0.93h）、 t1/2β（2.42h）、Cmax(114.24μg·L^-1)、Tmax（1.52h）, Vd(1.32L·kg^-1)、 CL (0.24L·h^-1)、AUC (868.44μg·h·L^-1); low dosegroup Ka (0.32h^-1)、Ke (0.09h^-1)、t1/2α（1.29h）、t1/2β（2.85h）、Cmax(132.48μg·L^-1)、Tmax（1.83h）、Vd(1.35L·kg^-1), CL (0.19L·h^-1)、AUC (1127.61μg·h·L^-1)；high dosegroup, Ka (0.37h^-1)、Ke (0.04h^-1)、t1/2α（1.85h）、t1/2β（3.65h）、Cmax(149.75μg·L^-1)、Tmax（2.04h）、Vd(1.41L·kg^-1)、CL (0.16L·h^-1)、AUC (1481.69μg·h·L^-1). The twogroups in to give Angelica total coumarin, nifedipine peak plasma concentration,the area under the concen-tration curve was significantly higher than the singledrug group that Angelica total coumarin components significantly affect theplasma concentration of nifedipine and a dose-dependent inhibition. The elimination rate constant Ke and a clearance CL compared with the controlgroup also had a significantly lower （P<0.05）.ConclusionsFirst,Preparation of rat liver microsomal CYP total significantly improvephenobarbital induced by CaCl2precipitation, can fully meet the needs of the invitro drug metabolism studies.Second,Was moderate inhibition of rat livermicrosomal CYP3A4activity, the psoralen xanthotoxol imperatorin,Othercoumarin compounds inhibitory effect is not obvious, IC50<100μmol·L^-1. TheCYP3A4inhibition approximate order: the psoralen≥xanthotoxol> imperatorin> isoimperatorin> xanthotoxol>xanthotoxin>osthole.Third,The pharmacokineticparameters in rats Angelica total coumarin component can inhibit themetabolism of nifedipine, change of nifedipine, the K reduce, t1/2extended, AUCincreased, CL decreased.