Determination And Pharmacokinetics Study On Ingredients In A Traditional Chinese Medicine Formula Danggui-beimu-kushen-pill

Objective： Analyse seven components (AWS, OMT, OSP, MT, SP, JS, YS) in rats’ plasmapharmacokinetic behavior、tissue distribution and feces、urine excretion, after orally administeredDanggui-Beimu-Kushen-Pill. The pharmacokinetic parameters of compositions provide theoreticalreference for clinical medication.Method:(1) Crude drugs were used70%ethanol reflux extraction three times, RRLC-MS/MSmethod were used to quantitative the extract ingredients from herbs.(2) Blood samples werecollected from the retinal venous plexus at0,5,15,30,45,60,90,120,180,36,480,720and1080min, after oral administration(3×1.35g/kg), respectively.100μl plasma which obtained aftercentrifugation was precipitated by2times methanol. Nitrogen was used to dry supernatant under40℃then reconstitution. RRLC-MS/MS method was used to quantitative plasma componentconcentration at each time point.(3) Took the tissues and organs at0,15,30,90and240min afteradministered prescription to normal rats. Homogenized it with5times volumes ice bath ultra-purewater. Took100μl homogenate which obtained after centrifugation for precipitation treatment by3times acetonitrile methanol. Nitrogen was used to dry supernatant under40℃then reconstitution.RRLC-MS/MS method were used to quantitative each component in tissues at each time point.(4)Metabolic cages were used to collecte feces、urine at0-2h,2-4h,4-8h,8-12h,12-24h after orallyadministered Danggui-Beimu-Kushen-Pill to normal rats. Took100μl urine after homogenization,extracted it with4times ethyl acetate. RRLC-MS/MS method were used to quantitative eachcomponent in urine samples at each time point. DAS software was used to calculate pharmacokineticparameters of each excretory. Fecal samples were dried for24h under45℃and homogenized itwith8times volumes of (w: v)50%methanol, extracted it with four times ethyl acetate.RRLC-MS/MS method were used to quantitative each component in fecal samples at each time point.Combining fecal dry weight with the dosage to convert the total fecal excretion rate and the relationbetween cumulative excretion rate and time. Result:(1)The content of AWS in Angelica sinensis Radix (Danggui) was0.1053mg/g, OMT, OSP,MT and SP in Sophora flavescen Radix (Kushen) were14.896mg/g、3.716mg/g、1.648mg/g and0.542mg/g respectively, JS and YS in Thunberg Fritillary Bulbus(Beimu) were0.488mg/g and0.323mg/g respectively.(2) OMT, OSP, MT, SP, JS and YS showed two-compartment models andAWS showed one-compartment model in rats. Tmax of MT was around2h and Cmax was1121.10±175.58ug/L, t1/2α and t1/2β were88.39±52.20min and143.00±50.36min respectively,CL/F was0.006±0.002L/min/kg, showed slow absorption and slow elimination. SP pharmacokineticbehavior in the normal rat plasma was similar to MT. Both things distributed more in the plasma.Pharmacokinetic behavior of OMT and OSP were similar in rats and Tmax was around30min, V1/Fof both OMT and OSP were more than100, Presumably both things distributed to some organs orlarge range of organization centralizely. Tmax of AWS was4.5±1.225min. After6h AWS in plasmawas less than limit of quantification (0.4ng/ml), showed fast absorption and fast elimination. JSpharmacokinetic behavior in the normal rat plasma was similar to YS and both weretwo-compartment models and showed slow absorption and slow elimination.(3) OMT and OSPdistributed more in rat stomach and intestinal tissues and followed by the kidneys and lungs. MT andSP distributed more in the intestines and followed by the Kidneys and livers. JS and YS distributedmore in the Stomachs and intestines and followed by the Livers and lungs. AWS distributed more inthe Stomachs and intestines and followed by the Kidneys. Each component of the total excretionrates of urinary excretion were OMT:(0.089±0.073)%, OSP:(0.456±0.301)%, MT:(14.214±11.258)%, SP:(7.703±3.485)%, JS:(0.454±0.262)%, YS:(2.389±1.106)%, AWS:(3.801±1.451)%respectively, after administration24h. Each component of the total excretion ratesof fecal excretion were OMT:(0.005±0.002)%, OSP:(0.015±0.006)%, MT:(0.339±0.182)%, SP:(0.851±0.498)%, JS:(0.21±0.149)%, YS:(0.177±0.114)%, AWS:(2.256±1.531).Conclution: This study is the first time to establish RRLC-MS/MS simultaneous determinationmethod of seven compositions of biological samples in rats after orally administeredDanggui-Beimu-Kushen-Pill. Clarifies the plasma pharmacokinetic parameters of each component,tissue distribution and feces, urine excretion. provide experimental basses for pharmacological andpharmacodynamic study and theoretical reference for clinical medicine ofDanggui-Beimu-Kushen-Pill.