Evaluation Of The Effects Of Plyphyllin ? On Four Cytochrome P450 Isoforms In Rats

Objective:To study the influence of Paris saponin ?(PS ?),one of the main active extracts from rhizoma Paridis Saponins(RPS),on different cytochrome P450 isoforms(CYP1A2?CYP2C11?CYP3A1?CYP2D1)in rats.The purpose is to provide the referred information and theoretical basis for further research and development on it,to more reasonable application of PS ? in clinical practice.Methods:(1)Phenacetin,tolbutamide,midazolam,and dextromethorphan as the probe drug for CYP1A2,CYP2C11,CYP3A1,and CYP2D1,respectively.Male sprague-dawley(SD)rats as the research object,development and validation of a Cocktail approach by LC-MS/MS for measuring the concentration of phenacetin(PHE),tolbutamide(TB),midazolam(MDZ),and dextromethorphan(DM)in rats plasma.(2)Male SD rats as the research object,the single groups were given a single dose of 1%CMC-Na solution or different concentration of PS ?.After one hour,5 ml/kg of probe cocktail solution,containing PHE(15.0 mg/kg),TB(3.0 mg/kg),MDZ(10.0 mg/kg)and DM(15.0 mg/kg)in CMC-Na solution,was administered orally to all rats in each group.Heparinised blood samples were then collected pre-dose(0 h)and 5 min,10 min,20 min,40 min,1 h,2 h,3 h,4 h,6 h,8 h,12 h,24 h,36 h and 48 h after dosing and immediately centrifuged at 10,000 rpm at room temperature for 5 min to obtain 100 ?L rat plasma samples.These were stored at-80 ? until HPLC-MS/MS analysis.The pharmacokinetic parameters of each probe drug were calculated by the DAS 3.0 software with a noncompartmental model.The significance of the difference between the group means was assessed by SPSS software(Version 22).(3)Male SD rats as the research object,the multiple groups were given multiple dose of 1%CMC-Na solution or different concentration of PS? for seven days.Then the SD rats were fasted overnight but with access to water ad libitum.On the eleventh day,5 ml/kg of probe cocktail solution,containing PHE(15.0 mg/kg),TB(3.0 mg/kg),MDZ(10.0 mg/kg)and DM(15.0 mg/kg)in CMC-Na solution,was administered orally to all rats in each group.Heparinised blood samples were then collected pre-dose(0 h)and 5 min,10 min,20 min,40 min,1 h,2 h,3 h,4 h,6 h,8 h,12 h,24 h,36 h and 48 h after dosing and immediately centrifuged at 10,000 rpm at room temperature for 5 min to obtain 100 ?L rat plasma samples.These were stored at-80 ? until HPLC-MS/MS analysis.The pharmacokinetic parameters of each probe drug were calculated by the DAS 3.0 software with a noncompartmental model.The significance of the difference between the group means was assessed by SPSS software(Version 22).Results:(1)The results showed that the Cocktail approach,measuring the concentration of PHE,TB,MDZ and DM in rats plasma,exhibited good specificity,good sensitivity and has a satisfactory precision,accuracy and reproducibility.The validated results of method meet the regulation,and all data indicated that the Cocktail approach could be used to determine the probes plasma concentration in rats.(2)Compared the pharmacokinetic parameters of each probe drug in different treatment groups with the control group(1%CMC-Na solution)to evaluate the effects of PS ? on CYP1A2?CYP2C11?CYP3A1 and CYP2D1 in single dose groups.The pharmacokinetics parameters results indicated:the AUC(0-?)?CL?t1/2?MRT(0-?)?Cmax of PHE?DM?MDZ were no significant difference between the different treatment groups and control group;the pharmacokinetic parameters(AUC(0-?)?t1/2?MRT(0-?))of TB were increased significantly compared to those of the corresponding control group in the high dose of PS ? group,CL of TB was decreased significantly.(3)Compared the pharmacokinetic parameters of each probe drug in different treatment groups with the control group(1%CMC-Na solution)to evaluate the effects of PS? on CYP1A2?CYP2C11?CYP3A1 and CYP2D1 in multiple dose groups.The data showed:the AUC(0-?)?CL?t1/2?MRT(0-?)?Cmax of DM?MDZ were no significant difference between the different treatment groups and control group;AUC(0-?)?t1/2?MRT(0-?))of PHE were decreased significantly compared to those of the corresponding control group in high dose treatment groups,CL of TB was increased significantly;The pharmacokinetic parameters(AUC(0-?),t1/2,MRT(0-?))of TB were increased significantly compared to those of the corresponding control group in different treatment groups,CL of TB was decreased significantly.Conclusions:Development and validation of a Cocktail approach method for quantification of probes(PHE,TB,MDZ,and DM)in rat plasma.For the first time obtained the effects of PS ? on CYPs(CYP1A2?CYP2C11?CYP3A1 and CYP2D1).According our results,the CYP2C11 activity could be significantly inhibited by PS VII after a single dose or multiple doses of PS ? administrations in rats.Multiple dose of PS ? could weakly induce the activity of CYP1A2.