戊己丸不同配伍的肠吸收特征及与P-gp相互作用研究

【作者】 董宇；

【导师】 朱晓新； 姜廷良；

【作者基本信息】 中国中医科学院 ， 中西医结合基础， 2008， 博士

【摘要】 1实验目的中药复方配伍是中医用药的一大特色和优势,了解其配伍规律对临床辨证施治有重要的指导意义。中药复方多以口服形式入药,药物在肠道中的吸收是决定药物生物利用度和影响药物发挥治疗作用的关键因素。本研究以戊己丸为例,利用正交设计的方法,从肠吸收角度,通过监测中药复方组成中药的代表成份小檗碱（Ber）、巴马汀（Pal）、吴茱萸碱（Evo）、吴茱萸次碱（Rut）、芍药苷（Peo）,以其数、量、相互比例为着眼点,研究戊己丸不同比例的配伍肠道吸收过程中的动态变化规律,以及组成药物的贡献度、贡献形式等,并以数学方程和图解对其进行描述,借以探讨中药复方的配伍原理以及科学性。2实验方法和结果2.1戊己丸组方及其配伍的肠外翻吸收研究方法:采用L₉（3⁴）正交设计表,将戊己丸组方中的黄连、制吴茱萸、炒白芍作为3因素,各因素的3个不同剂量作为实验的水平数,研究戊己丸9个不同配比及其相应的单味药物在肠道中的吸收。采用肠外翻生物模型,以戊己丸中代表成份Ber、Pal、Evo、Rut、Peo为检测对象,用HPLC检测五个成分在单味药中的经时吸收量,用LC-MS同时检测戊己丸配伍中五个代表成份的变化。通过单因素方差分析（ANOVA）、正交设计的方差分析及归一化加权综合评分法对实验结果进行统计分析。结果:黄连提取物中Ber和Pal在肠道吸收过程中为零级吸收,其R²＞0.9,在不同剂量时Kα显示两者为被动扩散,各肠段吸收总趋势为空肠＞回肠＞结肠;制吴茱萸提取物中Evo和Rut在肠道中为零级吸收,R²＞0.9,Evo在小肠各段、Rut在空肠、回肠中均表现为主动转运,Rut在结肠中Kα随剂量增加而增加,表现为被动扩散形式,低剂量时结肠吸收少于空肠、回肠,高剂量时结肠多于空肠、回肠;Peo吸收为零级吸收,R²＞0.9,吸收形式以被动扩散为主,不同肠段的吸收为空肠＞回肠＞结肠。用LC-MS结合快速分离色谱柱,建立同时测定戊己丸中5个性质不同化合物的分析检测方法,实现了在较短时间内（t＜3.5min）同时分离目标检测成分;同时对方中5个化学成分均采用ESI正离子模式选择性离子检测法,排除了其他杂质的干扰,方法操作简单、快速、灵敏度高、特异性强、重现性好。正交设计研究显示戊己丸中5个代表成份在不同配伍比例时在肠道的吸收动力学不同。在空肠、回肠、结肠中5个代表成分的最佳吸收组合分别为,Ber（12:6:3、12:6:3、12:2:3）,Pal（12:1:12、12:1:12、12:2:12）,Evo（6:6:3、6:6:3、6:6:3）,Rut（12:6:12、12:6:3、12:6:3）,Peo（6:2:6、6:2:6、6:6:6）,相同成分在肠道不同部位的吸收趋势大致相同,但不同成分之间的吸收趋势变化较大。用多指标正交实验分析方法进行方剂配伍比例的优选,首先将分散的数据归一化,使其具有可比性。然后针对指标间的重要性差异给出各指标的权重,本实验依据戊己丸组方中的君臣佐使,确定Ber和Pal的权重为6,Evo、Rut和Peo的权重为4,给出综合评分进行正交统计。结果为黄连在各肠段吸收贡献度最大,确定了其君药地位,制吴茱萸和炒白芍次之,为臣药,其吸收最优配伍比例为黄连:制吴茱萸:炒白芍=6:3:6。2.2戊己丸单方及优选配伍组方在Caco-2细胞中的吸收研究方法:利用Caco-2细胞类似小肠上皮细胞的特性,研究中药提取物及其配伍在细胞中的摄取和转运特征。摄取实验将细胞种于24孔板,加药1h后细胞匀浆,测定细胞内药物含量;转运实验将细胞接种到Millicell小室膜上,待细胞具有极性、分化完成、完整性好后进行转运实验,实验时分别将药物加入AP或BL侧,检测另一端小室内的药物吸收/外排量的变化。结果:细胞生长21天碱性磷酸酶活力AP侧为BL侧的2倍以上（P＜0.05）,说明细胞具有极性,分化完成;荧光素钠Papp值在4.4×10⁷cm·sec^-1,2 h其透过率在0.7%,TEER值达到600Ω·cm²（7-8 d）,证明细胞完整性较好,可以用于转运实验。Caco-2细胞实验进一步证明了Ber、Pal、Peo以被动转运为主,P_b-a/P_a-b比例均在1左右,Evo、Rut以主动转运为主要转运机制,Evo的P_b-a/P_a-b比例＜0.5,Rut的P_b-a/P_a-b比例＜0.8;配伍6:1:6时Ber、Pal、Evo的摄取和外排均有所增加,Ber吸收减少,Rut摄取、吸收和外排均不同程度降低。配伍12:2:3和12:1:12时各代表成份吸收变化趋势大致相似,但在12:1:12配伍中Ber吸收增加、Evo细胞摄取量增多,这可能是引起两个配伍组产生相同药效的同时各有侧重点的原因。2.3戊己丸单方与P-gp相互作用研究方法:Ver抑制P-gp实验,采用肠外翻法,在肠外加入含有或不含有Ver（100μM）的测试药液,比较Ver对戊己丸组成单方中成分的吸收变化影响;Rho123外排实验,大鼠按实验分组分别给药5天,用肠外翻模型研究对Rho123的外排改变情况。结果:Ber、Pal、Evo在加入Ver后,在肠道各段吸收没有明显改变,Pal在加入Ver后吸收略有增加,尤其以结肠和回肠吸收增加较多,Peo在回肠中吸收比不加Ver时有显著增加（P＜0.05）,空肠和结肠改变不明显。对Rho123外排影响,提前服用Ver后,Rho123的外排显著减少（P＜0.01）,说明Ver抑制了P-gp,黄连组在空肠中对Rho123的外排也显著减少（P＜0.05）,回肠中外排有减少趋势。炒白芍组在回肠中Rho123外排显著增加（P＜0.01）,其余各组对Rho123外排没有明显影响。2.4戊己丸单方及不同配伍对MDR1-mRNA表达的影响方法:在Caco-2细胞中加入含药培养基培养2d,收集细胞后用Trizol一步法提取总RNA,逆转录合成cDNA,RT-PCR对MDR1-mRNA进行定量分析。结果:配伍6:1:6、12:2:3、12:1:12以及相同剂量黄连提取物单味药物均可以降低MDR1-mRNA的表达,制吴茱萸提取物及炒白芍提取物则可以诱导MDR1-mRNA表达,使其表达量增加。3结论本实验利用肠外翻模型和Caco-2细胞模型,研究戊己丸组方中单味药物和不同配伍下的代表成份吸收变化情况,结果显示戊己丸中代表成份在肠道中的吸收机制不同,不同配伍比例时吸收变化较大,经统计学分析最终得出黄连:制吴茱萸:炒白芍=6:3:6为最佳吸收配比,同时证明了黄连在方中的贡献度最大,确定其君药地位,制吴茱萸和炒白芍次之,为臣药。通过研究戊己丸单方与配伍对P-gp的相互作用,证明戊己丸单方与不同配比可能通过诱导/降低MDR1-mRNA的表达,产生药物间的相互作用,从而引起方中成份吸收的改变。更多还原

【Abstract】 1 ObjectiveThe compatibility of prescription in traditional Chinese medicine（TCM） is the special feature and superiority of Chinese Materia Medica,and it is the significance guidance for clinical prescription to understand the rule of compatibility.The oral administration is the main form of Chinese Herbs,and the intestinal absorption is one of the important factors to contribute the bioavailability and the pharmaco- dynamic.According to orthogonal design, Wuji Pill is used as a case to investigate the absorption of the represents compounds in intestinal.Some representative constituents such as berberine,palmatine,evodiamine, rutacarpine and paeoniflorin are monitored.Three index including difference number, difference dose and difference proportion of Wuji Pill are used to describe their dynamic variety regulation and calculate the contribution degree and modality of the prescripted durgs. Some mathematics equations and illustrations are presented to describe all the above results. At last the principle and scientification of the compatibility of prescription in Chinese Materia Medica is discussed.2 Method and Result2.1 Research the absorption of Wuji Pill in the single herbs and difference compatibility in the everted gut sacsMethod:The experiment employs the L₉（3⁴） orthogonal design,which the extracts of Rhizoma Coptidis,Evodiae Fructus and Radix Paeoniae Alba are used as three factors and difference dosage as three levels.The absorbance of nine compatibility and corresponding single herbs are studied by the means of the everted gut scas.Five representative composition of Wuji Pill,Ber、Pal、Evo、Rut and Peo,were detected by HPLC（in the single herbs） and LC-MS（in the compatibility of Wuji Pill）.The statistics is carried out by One-way ANOVA, the generallinear model and normalization synthetic weighted mark method.Resuk:The absorption of Ber and Pal in intestines is the zero order rate process,R²＞0.9. The Ka of Ber and Pal display the passive absorption,and the absorption trend in different intestines is jejunum＞ileum＞colon.The absorption of Evo and Pal conforms to the zero order rate process,R²＞0.9.The mechanism of absorption of Evo is the active transport in various intestines,and Rut is the active transport in the jejunum and the ileum,and belongs to the positive transport in the colon.The absorption trend is that in low dose level,the colon is lower than the jejunum and ileum,while higher in higher dose.The absorption of Peo is the zero order rate process,R²＞0.9.The Peo display the passive absorption,and the absorption trend in different intestine is jejunum＞ileum＞colon.We established the method of simultaneous determination 5 variety property compounds in Wuji Pill using LC-MS with tacho-isolation colum,and a good separation is obtained between the object composition in 3.5 min.To remove the interference from other materials, the mass scan patterns are all ESI poison mode in 5 compounds.The advantage of our method is simple,fast,sensitivity,specificity and good reproducibility.The absorption dynamics of 5 compounds are distinct in Wuji Pill with the difference compatibility in orthogonal design.In the jejunum,ileum and colon,the optimal absorption compatibility of 5 compounds are as follows:Ber（12:6:3、12:6:3、12:2:3）,Pal（12:1:12、12:1:12、12:2:12）,Evo（6:6:3、6:6:3、6:6:3）,Rut（12:6:12、12:6:3、12:6:3）,Peo（6:2:6、6:2:6、6:6:6）.It is showed that the same compounds have the similar absorption trend in different intestine,and the different compounds are various in the absorption.The optimal proportion of the prescription compatibility is obtained by employing the multicriteria orthogonal analysis.We first normalize the scattered data to make it comparability,and calculate the index-weight based on monarch,minister,assistant and guide of Wuji Pill.The index-weight of Ber and Pal are six, and Evo、Rut and Peo are four,and then calculate synthetic score to statistic.The result shows the extract of Rhizoma Coptidis made the greatest contribution in different intestine that determine it monarch,and the extract of Evodiae Fructus and that of Radix Paeoniae Alba are lower that make it minister.The optimization of the compatibility is 6:3:6（extractive Rhizoma Coptidis / extractive Evodiae Fructus / extractive Radix Paeoniae Alba）.2.2 Research the absorption of Wuji Pill in single herbs and the optimization compatibility in the Caco-2 cell monolayersMethod:the absorption of Caco-2 cell monolayers is similar with the small intestine epithelium,which can investigate the uptake and transport of extract of Chinese herbs and their compatibility.In the uptake experiment,The Caco-2 cells are seeded 24 pore plates,and add the drugs.One hour later,the cells are homogenated and the compounds content in cells are determined.In the transport experiment,the Caco-2 cells are seeded the Millicell,and the cells complete the differentiation and can be used to transport.Add drugs in AP or BL,and detect the receptor compounds.Result:The alkaline phosphatase in AP is over double BL at 21 day（P＜0.05）,which shows the cells already come into being polarity,and complete the differentiation.The Papp of the fluorescein sodium is 4.4×10^-7 cm·sec^-1,and the transmissivity is 0.7%in two hours. TEER is over 600Ω·cm²（7-8 d） that means the good integration and could use for transport experiment.The experiment of Caco-2 cells verified the passive absorption of Ber、Pal、Peo,and the P_b-a/P_a-b is about 1.Evo and Rut is the positive transport,and the P_b-a/P_a-b of Evo is lower than 0.5,the P_b-a/P_a-b of Rut is lower than 0.8.The uptake and excretion of Ber、Pal、Evo in 6:1:6 are increase,and the absorption of Ber is decrease,the uptake、absorption and excretion of Rut are all depress in 6:1:6.Changes of the compatibility in 12:2:3 and 12:1:12 are similar, but the absorption of Ber and the uptake of Evo in 12:1:12 are increase,which can produce the particular theraoeutic based on the same pharmacodynamic. 2.3 Research the interaction of the single prescription in Wuji Pill with P-glycoproteinMethod:In the inhibition study with P-gp,the drugs with or without inhibitor verapamil （100μM） are added to the mucosal medium in the everted gut seas,and the represent absorption,in the single prescription in Wuji Pill,are observed to determine the interaction with verapamil.In the excretion study with Rho123,rats are treated with drugs for 5 days,the Rho 123（100μM） is introduced into the everted gut scas,and observate the excretion.Result:There is no obviously change in Ber、Pal and Evo after adding verapamil,and increase of Pal in the colon and ileum,while significantly increase of Peo at the ileum（P＜0.05）.Rho123 is the substrate of P-gp.Ver,the inhibitor of P-gp,can significantly decrease the excretion of Rho123（P＜0.01）.The extract of Rhizoma Coptidis reduce the excretion of Rho123 in jejunum（P＜0.05）,and the extract of Radix Paeoniae Alba significantly increase the excretion of Rho123 in ileum（P＜0.01）.There are no obvious changes in the other groups.2.4 Research the effect to MDR1-mRNA with Wuji Pill in the single herbs and the optimization compatibility in the Caco-2 cell monolayersMethod:Caco-2 cells are cultivated in medium with drugs for 2 days.After collecting cells,RNA is isolated by the method of Trizol one-step,reverse transcription synthesis cDNA, and quantitative assay the MDR1-mRNA by RT-PCR.Result:The groups of the extract Rhizoma Coptidis and their compatibility（6:1:6、12:2:3、12:1:12） could reduce the expression of MDR1-mRNA.The extract of Radix Paeoniae Alba could promote the expression of MDR1-mRNA.The extract of Evodiae Fructus in lower dose could induce the expression of MDR1-mRNA,while higher dosage is no entrainment.3 ConclusionThe experiment is to research the intestinal absorption of the represents in the single herbs and the difference compatibility of Wuji Pill in the everted gut sacs and Caco-2 cells monolayers,which to elucidate the regularity and scientification of the compatability of prescription.The mechanism of absorption of the represents in Wuji Pill is different,and they have a great change in the different compatibility.The optimal compatibility in absorption is 6:3:6（extract of Rhizoma Coptidis / extract of Evodiae Fructus / extract of Radix Paeoniae Alba）,and the extract of Rhizoma Coptidis make the greatest contribution which is the monarch,and the extract of Evodiae Fructus and the extract of Radix Paeoniae Alba are a bit lower that make them minister.The single drugs and the compatibility of Wuji Pill make the increasing or reducing the expression of MDR1-mRNA that produce the interaction of the different compounds in the intestinal absorption.更多还原