UPLC-MS/MS测定吴茱萸汤活性组分在硝酸甘油致偏头痛大鼠血浆和脑组织的药代动力学

UPLC-MS/MS determination of active components from Wuzhuyu Decoction in plasma and brain tissues of nitroglycerin-induced migraine rats

建立硝酸甘油致偏头痛大鼠血浆和脑组织中吴茱萸汤中吴茱萸次碱(Ru)、吴茱萸碱(Ev)、吴茱萸苦素(Rv)、柠檬苦素(Li)、人参皂苷Rb1(Rb1)的UPLC-MS/MS分析方法,并开展药代动力学研究。选用雄性SD大鼠,多剂量灌胃(ig)未经组分优化(UNOS)和经组分优化的吴茱萸汤样品(OS),在硝酸甘油造模后不同时间点采集血液与脑干样品,采用UPLC-MS/MS测定血浆和脑干中5种成分的浓度,以Phoenix WinNonlin 5.2.1药动学程序软件对血浆数据进行非房室模型拟合,计算药动学参数。经方法学考证,6种成分的峰面积与其在血样及脑组织中的浓度线性关系良好(r>0.9947),日内、日间准确度、精密度,基质效应、稳定性等均符合体内药物分析的要求,建立的方法可用于大鼠血浆及脑组织中相关成分的药动学研究。比较2种不同吴茱萸汤组活性成分的血浆药动学参数,发现OS组中的Rb1的Tmax,Cmax,AUC0-24 h,AUC0-∞均升高;Ev的t1/2,Tmax升高,但其Cmax,AUC0-24 h,AUC0-∞降低;Ru的t1/2升高,Cmax,AUC0-24 h,AUC0-∞降低。比较2种汤剂中各成分的脑组织分布,则在OS中含量高的正向作用成分,如给药后30 min,2 h时的Ru,30 min时的Ev,30 min,2 h时的Rb1,均显著低于UNOS组;而在UNOS中含量高的负向作用成分,如给药后30 min,2 h,12 h的吴茱萸苦素(Rv)显著高于OS组,部分阐释了优化吴茱萸汤疗效好于未优化吴茱萸汤的机制。

A sensitive and specific ultra-performance liquid chromatography-mass spectrometry(UPLC-MS/MS)method was deve-loped for analysis of rutaecarpine(Ru),evodiamine(Ev),rutaevine(Rv),limonin(Li),ginsendside Rb1(Rb1),ginsendside Re(Re)in rat plasma and brain tissues of nitroglycerin-induced migraine rats.Male healthy Sprague-Dawley(SD)rats were orally given multiple dose of optimized(OS)and un-optimized Wuzhuyu Decoction(UNOS),and their blood samples and brainstem were collected at different time points after injection of nitroglycerin(10 mg·kg-1)into the frontal region.The drug concentrations of the 6 analytes in plasma and brainstem were determined by UPLC-MS/MS method.Subsequently,the main pharmacokinetics parameters of plasma were calculated by using Phoenix WinNolin 5.2.1 software.The methodological test showed that all of analytes in both plasma and brainstem homogenate exhibited a good linearity within the concentration range(r>0.9947).The intra-day and inter-day accuracy,precision,matrix effect,stability of the investigated components meet the requirements for biopharmaceutical analysis.The developed method was successfully applied in pharmacokinetic studies on abovementioned ingredients in rat plasma and brain stem.The plasma pharmacokinetic parameters of active ingredients in two different Wuzhuyu Decoction group were compared,it was found that Rb1 had higher t1/2,Tmax,Cmax,AUC0-24 h and AUC0-∞in OS group.Meanwhile,Ev had higher t1/2 and Tmax but lower Cmax,AUC0-24 h and AUC0-∞,Ru has higher t1/2 but lower Cmax,AUC0-24 h and AUC0-∞in OS group.The brain tissue distribution of each component were compared between the two groups,the component with higher content in OS,such as Ru at 30 min and 2 h after administration,Ev at 30 min,Rb1 at 30 min and Rb1 at 2 h after administration have lower brain tissue distribution than those in UNOS group,while the component with higher content in UNOS,such as Rv at 30 min,2 h and 12 h after administration had higher brain tissue distribution than those in OS group.