【作者】 卫平；

【导师】 罗佳波；

【作者基本信息】 南方医科大学 ， 中药学， 2014， 博士

【副题名】麻黄甘草及麻黄桂枝药对血浆药动学、组织分布及排泄的研究

【摘要】 研究背景与意义药对,又称“对药”,即两味药的配伍应用,是历代医家经过临床验证,把配伍后疗效最佳的药物固定下来而形成的,是中药配伍中最小的固定单位。药对是连接单味中药与方剂的桥梁,是方剂配伍的精华与核心所在,具备了方剂的基本主治功能,又体现了方剂的整体疗效。中药复方临床疗效的发挥,在很大程度上取决于中药的配伍,而药对是介于单味药与复方方剂之间的配伍单元,与按“君臣佐使”原则组成的中药复方方剂有着不可分割的联系,既具有复方配伍的特性,又具备药味少、化学成分相对简单,便于开展现代医药学研究的特点。因此以药对研究为切入点,阐明其配伍机制,寻求发挥最佳药理效应的配伍比例,有利于探索与发掘药对的基本功用。开展药对配伍规律的研究,能进一步揭示药对配伍的客观规律与科学内涵,促进中药配伍应用理论的进步；能有效地指导临床遣方用药,既有利于更好地应用已有的药对,亦有利于针对疾病谱的变化和病证的发展而创制新的药对；特别是在新药研究中以有效成分或有效部分进行配伍研究越来越多的今天,以传统配伍的普遍规律为指导的新的配伍形式,对促进中药新产品的研究及发展具有重要的现实意义。近几年国外将含麻黄的制剂用作运动与减肥的食品补充剂,引发了一系列的副作用事件尤其是心血管系统的毒副作用,美国已出台法案禁用含麻黄制剂。流行病学研究己证明与长期使用麻黄有关,而麻黄及麻黄制剂的毒副作用主要与其中的麻黄类生物碱成分有关。含麻黄的中药方剂在临床使用已经有2000多年的历史,其疗效显著,国内对其副作用的报道也较少。从国外含麻黄制剂的用量来看,都远远低于含麻黄中药方剂的临床用量,但后者的毒副作用事件却远远的少于前者。究其原因,除了临床用法方面的差异,中药方剂配伍的精髓不可忽视。本文为国家自然科学基金重点项目—麻黄类药对组成规律基础研究的部分研究内容。麻黄-甘草及麻黄-桂枝药对是中医临床常用的药对,在很多经典方剂中都有应用,如麻黄汤、大青龙汤等。麻黄与甘草伍用,可缓和麻黄燥烈之性,增强麻黄发汗散水和止咳平喘之功；而麻黄与桂枝伍用则发汗解表,善治风寒感冒,恶寒发热,头身疼痛之表实证,为辛温解表之重剂。在实际的临床应用中,麻黄-甘草及麻黄-桂枝药对针对不同的主治证候则采用不同的配伍比例。研究目的中药配伍的主要目的是增效减毒,药对组成规律的研究从增强药理作用和降低毒副作用的入手,是两个比较可行的研究角度。我们认为无论增效或减毒均是成分间相互作用的结果,即效应成分相互影响而引起的体内动态过程改变的综合效果。在前期不同配伍配比麻黄-甘草及麻黄-桂枝药对化学成分、药理药效及代谢组学的基础上,本研究以麻黄-甘草及麻黄-桂枝药对为研究对象,运用现代技术手段,从该两药对不同比例配伍前后的血药-浓度时间曲线及药动学参数、组织分布和排泄特征方面对麻黄-甘草及麻黄-桂枝药对进行较为全面和系统的研究,通过比较分析该两药对不同比例配伍后主要效应成分血浆动力学、组织分布和排泄方面的差异,探讨其量效、时效及毒效间的关系,进一步验证及深化麻黄-甘草及麻黄桂枝药对“相使”、“相须”的配伍关系,揭示麻黄-甘草及麻黄-桂枝药对“增效减毒”物质基础的体内过程与作用机制,据此为中医临床遣方用药、创制新药对及开发新药提供理论和实验依据。研究方法《金匮要略》中甘草麻黄汤组成为：“甘草二两,麻黄四两(去节)”。本课题选取甘草麻黄汤中的药对—麻黄-甘草(12：6)为研究对象,依据药对文献报道的配比,上下成倍浮动,设计成配比：12：3,12：6和12：12三个配伍组。《伤寒论》中麻黄汤组成为：“麻黄三两(去节),桂枝二两(去皮),甘草一两(炙),杏仁七十个(去皮尖)。”本课题选取麻黄汤中的药对—麻黄-桂枝(3：2)为另一研究对象,依据药对文献报道的配比,上下成倍浮动,设计成配比：3：1,3：2和3：4三个配伍组。1.麻黄-甘草及麻黄-桂枝药对中麻黄、甘草、桂枝主要效应成分血浆代谢动力学研究分别建立大鼠血浆中麻黄主要效应成分麻黄碱(E)、伪麻黄碱(PE)、去甲基麻黄碱(NME)、去甲基伪麻黄碱(NMP)及甲基麻黄碱(ME),甘草主要效应成分甘草酸(GLY)、甘草次酸(GA)及甘草苷(LQ),桂枝主要效应成分桂皮酸(CA)、桂皮醇(CAL)及香豆素(CN)的UPLC-MS/MS检测方法。SD大鼠分别灌胃给药麻黄、麻黄-甘草药对(12：3、12：6、12：12,w/w)和甘草(3)、甘草(6)、甘草(12)水煎液；麻黄、麻黄-桂枝药对(3：1、3：2、3：4,w/w)和桂枝(2)水煎液后,采用建立的方法检测血浆中麻黄、甘草及桂枝主要效应成分的含量并对其血药浓度-时间曲线及主要药动学参数进行比较,探讨配伍对麻黄、甘草及桂枝效应成分血浆药动学行为的影响,分析麻黄与甘草、麻黄与桂枝的药物相互作用。2.麻黄-甘草及麻黄-桂枝药对麻黄主要效应成分组织分布研究建立SD大鼠组织样品中麻黄主要效应成分E、PE、NME、NMP及ME的UPLC-MS/MS检测方法。SD大鼠灌胃给药麻黄、麻黄-甘草药对(12：6)和麻黄、麻黄-桂枝药对(3：2)水煎液后,根据血浆药动学实验,分别在吸收相(0.25h)、分布相(1.5h)和消除相(3h)选择一个时间点进行采样。采用建立的方法检测上述3个时间点SD大鼠组织样品中麻黄主要效应成分的含量,并对其在各组织的分布及蓄积情况进行比较分析。3.麻黄-甘草及麻黄-桂枝药对麻黄主要效应成分经尿及粪便的排泄研究建立大鼠尿液及粪便中麻黄主要效应成分E、PE、NME、NMP及ME的UPLC-MS/MS检测方法。SD大鼠灌胃给药麻黄、麻黄-甘草药对(12：6)和麻黄、麻黄-桂枝药对(3：2)水煎液后,收集0～2h、2～6h、6～12h、12～24h、24～36h、36-48h及48-72h共7个时间段的尿液和粪便,采用建立的方法检测尿液及粪便样品中麻黄主要效应成分的含量,并对其排泄情况进行比较分析。4.统计学处理运用DAS3.0软件(上海博佳医药科技有限公司,中国上海),采用非房室模型计算麻黄(甘草,桂枝)主要效应成分的药代动力学参数；运用SPSS13.0软件进行统计分析。计量资料以均数±标准差(x±s)表示；多组之间主要药动学参数的均数比较采用单向方差分析(One-way ANOVA),其中各配伍组与麻黄组相比,采用Dunnett法；两组之间均数的比较采用独立样本的t检验；组织分布数据采用重复测量数据的方差分析进行分析(p<0.05,具有统计学意义)。实验结果1.麻黄-甘草及麻黄-桂枝药对不同配伍配比主要效应成分血浆药动学的研究本部分成功建立了大鼠血浆样品中NME、NMP、E、PE和ME, GLY、GA和LQ, CA、CAL及CN的UPLC-MS/MS检测方法。本方法准确度高,重现性和稳定性良好,提取回收率和基质效应符合生物样品的检测要求,各分析物的分离良好,无内源性物质及可能的代谢物的干扰。在麻黄、甘草单煎液及麻黄-甘草药对不同配比水煎液的大鼠血浆药动学的研究中,麻黄类生物碱及甘草效应成分的药动学参数均存在差异。麻黄组、麻黄-甘草(12：3)组、麻黄-甘草(12：6)组和麻黄-甘草(12：12)组中NME的AUC0-t、MRT0-t、Tmax、t1/2z、CLz/F; NMP的AUC0-t、MRT0-t、Tmax、t1/2z、VzF、 CLz/F; E的AUC0-t、MRT0-t、CLz/F; PE的AUC0-t、MRT0-t、t1/2z、Vz/F、CLz/F和ME的AUC0-t、CLz/F均具有显著性差异(均p<0.05)。其中与麻黄组相比,麻黄-甘草(12：3)组降低了NME和NMP的MRT0-t,降低了PE的t1/2z和Vz/F(p<0.05)；麻黄-甘草(12：6)组增大了NME、E和PE的AUCo-t(均p<0.05),降低了E和PE的CLz/F(均p<0.05),降低了PE的Vz/F(<0.05)；麻黄-甘草(12：12)组则增大了的E和PE的MRT0-t、ME的CLz/F(均p<0.05)。与甘草(3)相比,麻黄-甘草(12：3)降低了LQ的AUC(0-t)和Cmax,而Vz/F和CLz/F则增加了(均p<0.05)；降低了GLY的AUV0-t、MRT(0-t)、Tmax的值,增加了CLz/F的值(均p<0.05)；降低了GA的AUCo-t和Cmax,增加了GA的MRT0-t、Tmax、 t1/2z、Vz/F和CLz/F(均p<0.05)。与甘草(6)相比,麻黄-甘草(12：6)组中LQ的AUCo-t(减小)、MRTo-t(增大)、Cmax(减小)、t1/2z(增大)、Vz./F(增大)和CLz/F(增大)均存在统计学的差异(均p<0.05)；降低了GLY的AUC0-t、Tmax、Cmax的值,而MRT0-t、t1/2z、V,/F和CLz/F则增加了(均p<0.05)；降低了GA的AUCo-t和Cmax,增加了GA的MRT0-t、Tmax、t1/2z、Vz/F的值(均p<0.05)。与甘草(12)相比,麻黄-甘草(12：12)降低了LQ的AUCo-t和Cmax,增加了其MRT0-t、Vz/F和CLz/F的值(均p<0.05)；增大了GLY的AUCo-t和Cmax降低了其CLz/F的值(均p<0.05)；增大了GA的MRT0-t、Tmax、t1/2z,而减小了其CLz/F的值(均p<0.05)。在麻黄、桂枝单煎液及麻黄-桂枝药对不同配比水煎液的大鼠血浆药动学研究中,麻黄类生物碱及桂枝有效成分的药动学参数也均存在一定的差异。麻黄组、麻黄-桂枝(3：1)组、麻黄-甘草(3：2)组和麻黄-甘草(3：4)组中NIMIE的MRT0-t、Cmax、t1/2z、Vz/F; NMP的AUC0-t、MRT0-t、Cmax、t1/2z、Vz/F、CLz/F;E的AUC0-t、MRT0-t、Tmax、Cmax、t1/2z、Vz/F、CLz/F; PE的MRT0-t、Tmax、 Cmax、t1/2z、Vz/F和ME的MRT0-t、Cmax、t1/2z、Vz/F均具有显著性差异(均p<0.05)。其中与麻黄组相比,麻黄-桂枝(3：1)组降低了NME、E、PE、ME的MRT0-t、t1/2z和Vz/F(均p<0.05),增加了E的Tmax (p<0.05);麻黄-桂枝(3：2)组降低了NME、E、PE和ME的MRTo-t(均p<0.05),降低了NME、E、ME的t1/2z和Vz/F(均p<0.05),降低了NMP的CLz/F(p<0.05),而增大了NME、NMP、E、PE和ME的Cmax(均p<0.05),增大了NMP的AUCo-t(p<0.05)；麻黄-桂枝(3：4)组同样降低了NME、NMP、E、PE、ME的MRT0-t、t1/2;和Vz/F(均p<0.05),降低了NMP的CLz/F(p<0.05),而增加了NME、NMP、E、PE和ME的Cmax(均p<0.05),增大了NMP的AUCo-t和E、PE的Tmax(均p<0.05)。与桂枝(2)相比,麻黄-桂枝(3：2)增加了CN的AUC0-t和MRTot,降低了其t1/2z、Vz/F和CLz/F的值(均p<0.05)；增加了CAL的AUCo-t和MRTo-t,而减小了其Vz/F和CLz/F的值(均p<0.05)；同样也增加了CA的AUCo-t和MRTo-t,降低了其t1/2z、Vz/F和CLz/F的值(均p<0.05)。2.麻黄-甘草及麻黄-桂枝药对麻黄主要效应成分在大鼠体内的组织分布研究与麻黄单煎液相比,麻黄和甘草以12：6(w/w)配伍后,心、脑、肝、脾、肺、肾中NME的含量均随时间的变化而呈现由增加到降低的变化(分组与时间交互作用的p<0.05),其中均增加了给药后0.25h时上述组织中NME的含量(均p<0.05),1.5h时心组织中NME的含量仍高于麻黄组(p<0.05),到3h时均降低了NME在脑、心、肝、肺和肾中的含量(均p<0.05)；麻黄与甘草配伍后,脑和心组织中NMP的含量随时间的变化而变化(分组与时间交互作用的p<0.05),在给药后0.25h时降低了NMP在心中的含量,1.5及3h时降低了NMP在脑和心脏中的含量(均p<0.05)；麻黄与甘草配伍后,心、脑、肝、脾、肺、肾中E的含量均随时间的变化而呈现由增加到减少的趋势(分组与时间交互作用的p<0.05),在给药后0.25及1.5h时均可增加上述组织中E的含量(均p<0.05),到3h时降低了E在脑、心和脾组织中分布(均p<0.05)；麻黄与甘草配伍后,脑、心、脾和肾组织中的PE的含量均随时间的变化而变化(分组与时间交互作用的p<0.05),在给药后0.25、1.5及3h均增加了PE在心、肺、肝、脾和肾中的含量(p<0.05),脑组织中PE变化由增加(0.25及1.5h,p<0.05)到降低(3h,p<0.05)；麻黄与甘草配伍后,心、肺、肝和肾组织中的ME的含量均随时间的变化而变化(分组与时间交互作用的p<0.05),其中,在给药0.25及1.5h时均降低了ME在心组织中的含量(p<0.05),3h时均降低了其在脑、脾、肾组织中的含量(p<0.05),增加了在肺组织的含量(0.25及1.5h,p<0.05),3h时又降低了其在肺组织的含量(p<0.05)。与麻黄单煎液相比,麻黄-桂枝(3：2)配伍后,心、肺、肝、脾和肾中NME的含量均随时间的变化而变化(分组与时间交互作用的p<0.05),在给药0.25及1.5h后降低了NME在脑、肝中的含量(均p<0.05),3h增加了其在脑中的含量,3h时降低了NME在心组织中含量,0.25h时降低了其在脾组织中的含量,1.5及3h均增加了其在肾组织中的含量(均p<0.05)；麻黄与桂枝配伍后,各组织中NMP的含量均随时间的变化而变化(分组与时间交互作用的p<0.05),在给药0.25及3h时均增加了NMP在肺组织中的含量,1.5及3h时增加了其在脑组织中的含量,三个时间点均增加了其在脾中的含量,1.5h时增加了其在心、肝、肾中的含量(均p<0.05)；麻黄与桂枝配伍后,脑、肺、脾和肾中E的含量均随时间的变化而变化(分组与时间交互作用的p<0.05),在给药0.25及3h后降低了心组织中E的含量,脑组织中E的含量由降低(1.5h)到增加(3h),肺组织中E的含量呈现由降低(0.25h)到增加(1.5h)再降低(3h)的趋势,增加了E在脾(0.25h)和肾(3h)的含量(均p<0.05)；麻黄与桂枝配伍后,PE在各组织中分布与E相似；麻黄与桂枝配伍后,心、肺和肾中ME的含量均随时间的变化而变化(分组与时间交互作用的p<0.05),在给药3h时增加了ME在脑中的含量,ME在心组织中的分布由增加(1.5h)到降低(3h),增加了其在肺和肾(0.25h)的含量(均p<0.05)。3.麻黄-甘草及麻黄-桂枝药对麻黄主要效应成分经大鼠尿液及粪便的排泄研究与麻黄单煎液相比,麻黄与甘草配伍后降低了粪便中E的累积排泄量(t=3.430,p=0.009),降低了尿液中ME的累积排泄量(t=3.955,p=0.004)。麻黄类生物碱经粪便排泄的趋势比尿液较缓。麻黄单煎液中的NME、NMP、E、PE和ME经尿液和粪便总的排泄量分别占给药量的12.69%、13.56%、3.48%、3.65%和3.26%；而麻黄-甘草(12：6)中的NME、NMP、E、PE和ME经尿液和粪便总的排泄量则分别占给药量的12.28%、14.52%、3.29%、3.21%和2.27%。与麻黄单煎液相比,麻黄与桂枝配伍后可增加尿液中NME (t=2.462,p=0.039)、NMP (t=3.187,p=0.013)和E(t=2.703,p=0.027)的累积排泄量,降低了粪便中E的累积排泄量(t=2.905,p=0.020)；麻黄单煎液中的NME、NMP、E、PE和ME经尿液和粪便总的排泄量分别占给药量的11.34%、14.76%、6.35%、4.54%和8.08%；而麻黄-桂枝(3：2)中的NME、NMP、E,PE和ME经尿液和粪便总的排泄量则分别占给药量的15.01%、18.07%、5.89%、4.35%和7.35%。结论1.血浆药动学的研究结果表明,与麻黄、甘草单煎液相比,麻黄-甘草药对3个配比水煎液中麻黄类生物碱及甘草效应成分的药动学参数均存在差异。其中,麻黄-甘草(12：6)组可显著提高麻黄类生物碱(NME、E和PE)的生物利用程度,延缓了E和PE从体内的清除。因此,麻黄与甘草以12：6(w/w)配伍后,甘草可增强麻黄的药理作用,这与“甘草麻黄汤”中的配比相一致,验证了麻黄甘草“相使”配伍思想的科学性；也与本课题组前期发现麻黄-甘草(12：6)组具有较好的抗炎、利尿作用的结果相佐证。各配伍组中甘草效应成分甘草酸的生物利用度和在体内的存在时间随着甘草量的增加,表现出由抑制到促进的影响；另各配伍组中甘草苷及甘草次酸的生物利用度也均降低,且它们在体内的平均滞留时间都比各单味甘草组有所延长,推断麻黄与甘草配伍,甘草对麻黄的解毒作用可能与甘草酸、甘草次酸、甘草苷在体内的平均滞留时间的延长有关,其中甘草酸为甘草解毒的主要成分。与麻黄、桂枝单煎液相比,麻黄-桂枝药对3个配比水煎液中麻黄类生物碱及桂枝主要效应成分的药动学参数也存在一定的差异。麻黄与桂枝以3：2或3：4(w/w)配伍后,麻黄类生物碱的生物利用度均得到了显著提高,并且3个配比组均降低了麻黄类生物碱在大鼠体内的蓄积；另桂枝与麻黄以3：2配伍后,也显著提高桂枝主要效应成分的生物利用。因此,麻黄与桂枝配伍以3：2配伍后,两者的药理作用均得到了增强,两者之间具有协同作用；另麻黄与桂枝配伍后,均可降低麻黄类生物碱在体内的蓄积,降低了其毒副作用,这与本课题组前期发现麻黄-桂枝(3：2)组增效减毒作用明确,麻黄-桂枝3个配伍组毒性拮抗作用明确、解热药效实验存在一定的协同作用的结果相吻合。结果佐证了麻黄桂枝“相须”配伍的科学内涵。2.麻黄-甘草及麻黄-桂枝药对中麻黄类生物碱组织分布的研究。与麻黄组相比,麻黄与甘草(12：6)配伍后,可促进NME、E和PE在心、脑、肺、肝、脾、肾中的吸收和分布,使它们迅速发挥药理作用,这与血浆药动学研究部分相吻合；加快了NME、E、PE和ME从脑中消除,减少了它们在脑组织中的蓄积；抑制了NMP在心和脑组织中的吸收和分布,加快了其从心和脑组织中消除。另麻黄与甘草(12：6)配伍后加快了NME从心、肝、脾、肺、肾中消除；加快了E从心和脾组织中消除；加快了ME从肺、肝、脾、肾中消除,减少了它们在组织中的蓄积。因此麻黄与甘草(12：6)配伍后,可促进NME、E和PE的吸收和分布,增强麻黄的药理作用；另也可不同程度地降低麻黄类生物碱在各研究组织中的蓄积,达到减毒的目的,这与前期麻黄-甘草药对药理毒理研究的结果相一致。与麻黄组相比,麻黄-桂枝(3：2)组抑制了E和PE在心、肺和脾中的吸收,抑制了NME和NMP在脾中的吸收,促进了NMP和ME在肺中的吸收；增加了E和PE在肺中的分布,增加了NME在肺和肾中的分布,增加了NMP在脑、心、肝、脾和肾组织中的分布,增加了其在肝组织中的分布,减少了NME、E和PE在脑中的分布；延缓了5个麻黄类生物碱从脑中的消除,一定程度上起到增加药理效应的作用；另麻黄与桂枝配伍后,加快了E和PE在心和肺组织的消除,也加快了NME和ME在心组织中的消除,降低了它们在心组织中的蓄积,起到减毒的作用。这也与前期麻黄-桂枝药对药理毒理研究的结果相一致。3.麻黄-甘草及麻黄-桂枝药对中麻黄类生物碱经尿及粪便的排泄研究。大鼠灌胃给药麻黄、麻黄-甘草(12：6)和麻黄、麻黄-桂枝(3：2)水煎液后,麻黄类生物碱以原形从尿及粪便中累积排泄量的比例极低。提示麻黄类生物碱在体内可能存在着一定的生物转化。不管是麻黄与甘草配伍,还是麻黄与桂枝配伍,对麻黄类生物碱经尿及粪便的累积排泄率都没有太显著的影响。更多还原

【Abstract】 Background and SignificanceHerbal couples, also called herbal pairs, which as the basic composition units of Chinese herbal formulas have special clinical significance in traditional Chinese medicine (TCM), and are much simpler than other complex formulas without altering their basic therapeutic features. Herbal couples is the bridge of the single Chinese herb and TCM formula, is the essence and core of the prescription compatibility, have the basic function of the attending of prescription, and also reflects the overall efficacy of the TCM formula.The clinical efficacy of Chinese herbs largely depends on the compatibility of medicine. The herbal couples, as the compatibility unit between single herb and Chinese herbal formulas, and have the inseparable connection of Chinese herbal formulas, which composed according to the principle of "monarch, minister, assistant and guide". The herbal couples have less herbal medicines, and chemical composition is relatively simple, which not only have the features of formula, but also easy to carry out the characteristics of the modern medical science research. So research on the herbal couples, as a starting point, may be beneficial to elucidate its mechanism of actions of formulas, seek the best the ratio of compatibility, and explore the basic effects, and function expansion. Carrying out research on the compatibility lawof herbal couples, is of great significance to reveal the scientific contents of formulas compatibility, mine the theory of TCM compatibility, and expand the clinical ideas of the treatment, as well as provide clues to the basis for in-depth study of herbal compatibility law, and also the basis for the formation of the new formulas. Especially with the increasing of compatibility using active ingredients or effective parts, applying the traditional compatibility theory to guide the new form of compatibility, is of practical significance to promote the new products of traditional Chinese medicine for research and innovation.In recent years, foreign agents use preparation which containing ephedra as food supplements of exercise and weight-loss, which lead to a series of adverse events, especially the side effects of the cardiovascular system, the United States has introduced legislation to disabled the preparation which containing ephedra. Epidemiological studies have proven this associated with long-term use of ephedra, and the side effects of ephedra and its preparations mainly associated with the ephedra alkaloids. Containing ephedra prescriptions of TCM have been used for2000years in clinical, the curative effects of that is distinct, and fewer side effects have reported in domestic. The dosage of foreign preparation which containing ephedra are far below the TCM prescriptions, but the side effects of the event are far less than the former. Investigate its reason, in addition to the differences in clinical use, the essence of compatibility of TCM cannot be ignored.This study is the part of basic research on the composition of the law of Herba Ephedrae herbal couples, funded by the National Science Foundation. Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herb couples (also called Mahuang-Gancao and Mahuang-Guizhi herb-couple in Chinese) are commonly used in TCM clinical, which used in many classical prescriptions, such as Mahuang decoction and Daqinglong decoction. The combination of Mahuang and Gancao can moderated the drying chamber of Mahuang, and strengthen the sweating aproll, cough and asthma of Mahuang. While the combination of Mahuang and Guizhi can sweating nourish, and have good treatment on chill cold, bad cold fever, headache and body pain. In the actual clinical application, using the different ratio of Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herb couples for different syndromes, is a question worth considering concering the connotation of compatibility.ObjectivesTo improve effect and decrease toxicity is the main purpose of the compatibility of TCM, study on prescription-formulating principle of herb-couples from enhancing pharmacological efficacy and reduce side effects are two feasible platforms. We think no matter synergy or attenuated is the result of interaction between components, the comprehensive effect which is the effect of a dynamic process in vivo of the composition influence each other. The object of this topic is Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herb couples, using the modern technology, have a more comprehensive and systematic study on the blood drug concentration time curve, pharmacokinetic parameters, tissue distribution and excretion of these two herbal couples in different proportion and compatibility of their bioactive compounds. By explore the changes in the pharmacokinetic parameters, tissue distribution and excretion, it will not only further to validate the compatibility-relationship between mahuang and gancao (also mahuang and guizhi) and also play a role in finding the best compatibility proportion. Furthermore, by finding out the relationship of dose-efficiency, time-efficiency and toxic-efficiency, this will to bring into further validation and depth of the compatibility relationship of "mutual-assistance","mutual promotion", to reveal the intracorporal process of material basis and action mechanism of improve effect and decrease toxicity of Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herb couples. That can provide the experimental basis for the clinical treatment of TCM, development of new drug-couples and drugs.ContentsTo clarify the compatibility rules of Traditional Chinese herb-couple Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi with different compatibility ratio, we compared the changes of active ingredients in plasma pharmacokinetics, tissue distribution and excretion. Mainly containing:1. Study on the plasma pharmacokinetics of Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herbal couples.Develop and validate the ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to determination the content of norpseudoephedrine (NMP), norephedrine (NME), ephedrine (E), pseudoephedrine (PE) and methylephedrine (ME) in rat plasma, which are the major active ingredients of Mahuang; the content of glycyrrhizic acid (GLY), glycyrrhetinic acid (GA) and liquiritin (LQ) in rat plasma, which are the major active ingredients of Gancao; the content of coumarin (CN), cinnamic alcohol (CAL) and cinnamic acid (CA) in rat plasma, which are the major active ingredients of Guizhi. Using the developed method to determined the major active ingredients content of Mahuang (also Gancao and Guizhi) in rat plasma after orally administered Mahung, Mahuang-Gancao herb-couple (ratios12:3,12:6,12:12,w/w) and Mahuang, Mahuang-Guizhi herb-couple (atios3:1, 3:2,3:4,w/w) extracts (also after orally administered the Gancao (3), Gancao (6), Gancao (12) and Guizhi (2) extracts). Compare the changes of blood drug concentration-time curve and the main pharmacokinetic parameters of the main bioactive ingredients of Mahuang (also Gancao and Guizhi). Discuss the influence of compatibility for the plasma pharmacokinetic behavior of Mahuang, Gancao and Guizhi, analysis the drug interaction of Mahuang and Gancao (also Mahuang and Guizhi).2. Study on the tissue distribution of Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herbal couplesDevelop and validate the ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to determinate the content of norpseudoephedrine (NMP), norephedrine (NME), ephedrine (E), pseudoephedrine (PE) and methylephedrine (ME) in rat tissues, which are the major active ingredients of Mahuang. Using the developed method to determined the major active ingredients content of mahuang in rat tissues after orally administered Mahung, Mahuang-Gancao herb-couple (ratio12:6,w/w) and Mahuang, Mahuang-Guizhi herb-couple (atio3:2,w/w) extracts. Compare the changes of tissue distribution, and discussed the accumulation of the ephedrine alkaloids in each tissue.3. Study on the excretion from urine and feces of Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herbal couplesDevelop and validate the ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method to determinate the content of norpseudoephedrine (NMP), norephedrine (NME), ephedrine (E), pseudoephedrine (PE) and methylephedrine (ME) in rat urine and feces, which are the major active ingredients of Mhuang. Using the developed method to determined the major active ingredients content of mahuang in rat urine and feces samples after orally administered Mahuang, Mahuang-Gancao herb-couple (ratio12:6, w/w) and Mahuang, Mahuang-Guizhi herb-couple (ratio3:2, w/w) extracts. Compared and analysis the changes of its excretion.4. Statistial analysisThe pharmacokinetic parameters of the major active compound of mahuang (also gancao and guizhi) were calculated by the non-compartmental analysis of plasma concentration vs. time data using Drug and Statistics (DAS)3.0software (BioGuider Co., Shanghai, China). The differences of samples in different groups were conducted with univariate statistical analysis using software of SPSS16.0. The measurement data was presented as mean±SD (x±s); multiple groups means were compared with One-way ANOVA, among them, using the Dunnett method to compared the three compatibility groups and Mahuang group; two groups means were compared with indepent-samples T tese; the analysis of tissue distribution using the variance of repeated measurement data. A value of p<0.05was considered statistically significant.Results1. Study on pharmacokinetics of the major component in rat plasma after oral Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herbal couples in different proportionWe have established UPLC-MS/MS method to determine the norephedrine, norpseudoephedrine, ephedrine, pseudoephedrine, methyleephedrine; glycyrrhizic acid, glycyrrhetinic acid, liquiritin; and cinnamic acid, cinnamic alcohol, coumarin in rat plasma, which pass the methodological study with high accuracy, good reproducibility and stability, extraction recovery and matrix effects in biological samples meet the testing requirements. Under these UPLC method, separation of each analyte is good, there have no endogenous interfering substances and possible metabolites, so the method is rapid, sensitive and accurate.The pharmacokinetic parameters of ephedra alkaloids and the active ingredients of Gancao were different after oral administration of Mahuang, Gancao and Mahuang-Gancao herb-couple. The AUC0-t, MRT0-t, Tmax, t1/2z, CLz/F of NME; the AUCo-t, MRTo-t, Tmax, ti/2z, Vz/F, CLz/F of NMP; the AUC0-t, MRT0-t, CLz/F of E; the AUCo-t, MRTo-t, ti/2z, Vz/F, CLz/F of PE; and the AUC0-t, CLz/F of ME in Mahung group, Mahuang-Gancao (12:3) group, Mahuang-Gancao (12:6) group and Mahuang-Gancao (12:12) group have significant difference(p<0.05). Compared with the Mahuang group, Mahuang-Gancao (12:3) group reduced the MRT0-t of NME and NMP, reduced the t1/2z and Vz/F of PE (p<0.05); Mahuang-Gancao (12:6) group increased the AUC0-t of NME, E and PE(all p<0.05), reduced the CLz/F of E and PE(all p<0.05), reduced the Vz/F of PE(p<0.05); Mahuang-Gancao (12:12) group also increased the MRT0-t of E and PE, the CLz/F of ME(all p<0.05).Compared with Gancao (3) group, Mahuang-Gancao (12:3) reduced the AUC0-t and Cmax of LQ, but increased its Vz/F and CLz/F value (all p<0.05); It can reduced the AUC0-t, MRT0-t and Tmax of GLY, but increased its Vz/F value (all p<0.05); reduced the AUC0-t and Cmax of GA, increased its MRT0-t, Tmax, tmz, Vz/F and CLz/F value (all p<0.05).Compared with Gancao (6) group, Mahuang-Gancao(12:6) group reduced the AUC0-t and Cmax of LQ, but increased its MRT0-t, t1/2z, Vz/F and CLz/F value (all p<0.05); reduced the AUC0-t, Tmax and Cmax of GLY, increased its MRT0-t, tmz, Vz/F and CLz/F value(all p<0.05); reduced the AUC0-t and Cmax of GA, but increased its MRT0-t, Tmax, t1/2z and Vz/F value(all p<0.05).Compared with Gancao (12) group, Mahuang-Gancao(12:12) group reduced the AUC0-t and Cmax of LQ, but increased its MRT0-t, Vz/F and CLz/F value(all p<0.05); increased the AUC0-t and Cmax of GLY, and reduced its CLz/F value(all p<0.05); reduced the CLz/F of GA, but increased its MRT0.t, Tmax and t1/2z value(all p<0.05).The phanrmacokinetic parameters of ephedra alkaloids and the active ingredient of Guizhi (CA, CN and CAL) were different after oral administration of Mahuang, Guizhi and Mahuang-Guizhi herb-couple. The MRT0-t, Cmax, t1/2z, Vz/F of NME, the AUC0-t, MRT0-t, Cmax, t1/2z Vz/F, CLz/F of NMP, the AUC0-t, MRT0-t, Tmax, Cmax, t1/2z, Vz/F, CLz/F of E, the MRT0-t, Tmax, Cmax, t1/2z, Vz/F of PE, and the MRT0-t, Cmax, t1/2Z, Vz/F of ME in Mahung group, Mahuang-Guizhi (3:1) group, Mahuang-Guizhi (3:2) group and Mahuang-Guizhi (3:4) group have significant difference(p<0.05). Compared with the Mahuang group, Mahuang-Guizhi (3:1) group reduced the t1/2z, MRT0-t and Vz/F of NME, E, PE and ME(all p<0.05), increased the Tmax of E (p=0.001); Mahuang-Guizhi (3:2) group reduced the MRT0-t of NME, E, PE and ME(all p<0.05), the t1/2z and Vz/F of NME, E and ME(all p<0.05), the CLz/F of NMP (p<0.05), but increased the Cmax of the five ephedra alkaloids, the AUC0-t of NMP(p<0.05); Mahuang-Guizhi(3:4) group also reduced the t1/2Z, MRT0-t and Vz/F of the five ephedra alkaloids(all p<0.05), the CLz/F of NMP(p<0.05); but increased the Cmax of five ephedra alkaloids(all p<0.05), the AUC0-t of NMP, the Tmax of E and PE (all p<0.05).Compared with the Guizhi (2) group, Mahuang-Guizhi(3:2) group increased the AUC0-t and MRT0-t of CN, but reduced its t1/2z, Vz/F and CLz/F value(all p<0.05); increased the AUC0-t and MRT0-t of CAL, but reduced its Vz/F and CLz/F value(all p<0.05); also increased the AUC0-t and MRT0-t of CA, reduced the t1/2Z, Vz/F and CLz/F of CA(all p<0.05).2. Study on tissue distribution of the major component of mahuang in rat after oral Ephedra sinica-Glycyrrhiza uralensis and Ephedra sinica-Ramulus Cinnamomi herbal-couple decoction.Compared with the Mahuang decoction, after compatibility of Mahuang and Gancao(12:6), the concentration of NME in heart, brain, liver, spleen, lung and kidney are changed along with time(The p of group interaction with time0.05), at0.25h after oral administration, the concentration of NME in six tissue were all increased (p<0.05), at1.5h the concentration of NME in heart is still higher than Mahuang group, at3h the concentration of NME in brain、heart、liver、lung and kidney are reduced(p<0.05); after compatibility of Mahuang and Gancao, the concentration of NMP in brain and heart are changed along with time, at0.25h after oral administration the concentration of NMP are reduced, at1.5and3h the concentration of NMP in brain and heart are reduced (all p<0.05); after compatibility of Mahuang and Gancao, the concentration of E in heart, brain, liver, spleen, lung and kidney are changed along with time(from increase to reduce), at0.25and1.5h after oral administration, the concentration of E in tissues are increased(all p<0.05), and at3h, the concentration of E in brain, heart and spleen are reduced(all p<0.05); after compatibility of Mahuang and Gancao, the concentration of PE in heart, brain, spleen and kidney are changed along with time, at0.25,1.5and3h, the concentration of PE in heart, lung, liver, spleen and kidney are increased(all p<0.05), the concentration of PE in brain changes from increased (0.25and1.5h, p<0.05) to reduced (3h, p<0.05); after compatibility of Mahuang and Gancao, the concentration of ME in heart, lung, liver and kidney are changed along with time, at0.25and1.5h after oral administration, the concentration of ME in heart are reduced(p<0.05), the concentration of ME in brain, spleen and kidney are reduced at3h, the concentration of ME in lung are increased at0.25and1.5h, and reduce at3h(all p<0.05).Compared with the Mahuang decoction, after compatibility of Mahuang and Guizhi(3:2), the concentration of NME in heart, lung, liver, spleen and kidney are changed along with time(The p of group interaction with time<0.05), the concentration of NME in brain and liver are reduced at0.25and1.5h after oral administration,the concentration of NME in brain are increased at3h, the concentration of NME in heart are reduced at3h, the concentration of NME in spleen are reduced at0.25h, the concentration of NME in kidney are increased at1.5and3h(all p<0.05); after compatibility of Mahuang and Guizhi, the concentration of NMP in brain, heart, lung, liver, spleen and kidney are changed along with time, the concentration of NMP in lung are increased at0.25and3h, the concentration of NMP in brain are also increased at1.5and3h, the concentration of NMP in spleen are increased at three time, the concentration of NMP in heart, liver and kidney are increased at1.5h(all p<0.05); after compatibility of Mahuang and Guizhi, the concentration of E in brain, lung, spleen and kidney are changed along with time, the concentration of E in heart are reduced at0.25and3h, the concentration of E are changed from reduced(1.5h) to increase(3h), the concentration of E are from reduced(0.25h) to increase(1.5h), and reduced at3h, the concentration of E in spleen(0.25h) and kidney(3h) are increased; after compatibility of Mahuang and Guizhi, the concentration of PE in tissues are similar with E; after compatibility of Mahuang and Guizhi, the concentration of ME in heart, lung and kidney are changed along with time, the concentration of ME in brain are increased at3h, and the concentration of ME in heart are changed from increased (1.5h) to reduced (3h), the concentration of ME in lung and kidney(0.25h) are incresed(all p<0.05).3. Study on the urine and feces excretion of the bioactive ingredients of mahuang in rat after oral Herba Ephedrae-Radix Glycyrrhizae and Herba Ephedrae-Ramulus Cinnamomi herb-couple decoction.Compared with the Mahuang decoction, Mahuang-Gancao herb-couple can reduced the cumulative feces excretion of E (t=3.430, p=0.009), reduced the cumulative urinary excretion of ME (t=3.955, p=0.004). Five ephedra alkaloids in feces excretion were more moderate than urine. In Mahuang decoction, the urine and fecal excretion of NME, NMP, E, PE and ME accounted for a total dose was12.69%,13.56%,3.48%,3.65%and3.26%, respectively; while it was12.28%,14.52%,3.29%,3.21%and2.27%of NME, NMP, E, PE and ME in Mahuang-Gancao(12:6) group.Compared with the Mahuang decoction, Mahuang-Guizhi herb-couple increased the cumulative urinary excretion of NME (t=2.462, p=0.039), NMP (t=3.187, p=0.013) and E (t=2.703, p=0.027), reduced the cumulative feces excretion of E (t=2.905, p=0.020); In Mahuang decoction, the urine and fecal excretion of NME, NMP, E, PE and ME accounted for a total dose was11.34%,14.76%,6.35%,4.54%and8.08%, respectively; while it was15.01%,18.07%,5.89%,4.35%and7.35%of NME, NMP, E, PE and ME in Mahuang-Guizhi(3:2) group.Conclusion1. The results of the plasma pharmacokinetic showed that:After oral administration of Mahuang, Gancao and Mahuang-Gancao herb-couple decoction in rat, the pharmacokinetic parameters of ephedra alkaloids and the active ingredients of Gancao were different. Mahuang-Gancao (12:6) group can improve the degree of bioavailability of ephedra alkaloids, reaching synergistic effect. Therefore, Gancao can strengthen the pharmacological effects of Mahuang. The ratio of this is consistent with "GanMahuang-Tang", and this is also verified the science thought of the compatibility of "mutual-assistance" of Mahuang and Gancao. Also compared the the active ingredient of Gancao between Gancao and Mahuang-Gancao herb-couple decoction, with the increasing amount of Gancao, the bioavailability and existence time in vivo of glycyrrhizic acid are changed from inhibiting to promoting; the bioavailability of liquiritin and glycyrrhetinic acid in all Mahuang-Gancao groups are also reduced, compared with all Gancao group, the average residence time of them are extended.Study on the plasma pharmacokinetic of Mahuang, Guizhi and Mahuang-Guizhi herb-couple in rat, the pharmacokinetic parameters of ephedra alkaloids and the active ingredients of Guizhi are different. After the combined use of them, the bioavailability of the each ingredient has been improved, and the accumulation of ephedra alkaloids in rat was reduced. When Mahuang combination with Guizhi, the pharmacological effects of them all enhanced, and there are have synergism between them; Guizhi also can reduced the accumulation of ephedra alkaloids in vivo, can reduced the side effects. The results of this confirmed the scientific connotation of "mutual promotion" of Mahuang-Guizhi.2. Studies on the tissue distribution of ephedra alkaloids before and after drug compatibility. Compared with Mahuang group, Mahuang-Gancao (12:6) group can promoted the absorption and distribution of NME, E and PE in heart, brain, lung, liver, spleen and kidney, make them play a role of pharmacological quickly. Accelerated the elimination of NME, E, PE and ME from brain, reduced their accumulation in brain. Inhibited the absorption and distribution of NMP in the heart and brain, accelerated its elimination from the heart and brain, and reach the role of attenuated. After combined the Mauang with Gancao, it can accelerated the elimination of NME from heart, liver, spleen and kidney, accelerated the elimination of E from heart and spleen, and accelerated the elimination of ME from lung, liver, spleen and kidney, reduced the accumulation of them in the tissues, also have the effect of attenuated.Compared with Mahuang group, Mahuang-Guizhi group can inhibited the absorption of E and PE in heart, lung and spleen, inhibited the absorption of NME and NMP in spleen, and promoted the absorption of NMP and ME in lung; increased the distribution of E and PE in lung, increased the distribution of NME in lung and kidney, increased the distribution of NMP in brain, heart, liver, spleen and kidney, and reduced the distribution of NME, E and PE in brain; delayed the elimination of five ephedra alkaloids from brain, at a certain extent, have an effect of increasing pharmacological; Mahuang-Guizhi group can accelerated the elimination of E and PE from heart and lung, also accelerated the elimination of NME and ME from heart, reduced the accumulation of them in heart.3. The excretion study of ephedra alkaloids before and after two drugs matched in the urine and feces. After rat oral administration of Mahuang, Mahuang-Gancao (12:6) and Mahuang-Guizhi (3:2) decoction, the proportion of cumulative excretion in urine and feces of ephedra alkaloids from the original drug was very low. It showed ephedra alkaloids might exist some biotransformation in the body. Whether Mahuang and Gancao matched, or Mahuang and Guizhi matched, it had litter significant impact on cumulative urinary and feces excretion rate of ephedra alkaloids.更多还原