基于肝药酶代谢的金铃子散复方配伍研究
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摘要
研究背景
“辨证论治”是中医药认识和治疗疾病独特的思维方法,中药复方配伍是体现中医理论和“辨证论治”思想的载体。揭示中药复方配伍规律的科学内涵,一直是中药复方研究领域亟待解决的关键问题；探究中药配伍规律,对阐明中医“辨证论治”理论的科学内涵和中药复方的作用机制有着重要意义。中药复方配伍,从化学本质上来讲是不同中药的多组份间构成一个系统的、复杂的相互作用体系,这些相互作用的组份群是复方整体效应的重要物质基础。这些成分群可能直接作用于机体产生作用,也可能进入机体之后与机体代谢系统相互作用后产生药理作用。如果说中药化学解决的是中药复方起效的物质基础,中药药理学解决的是中药复方发生作用的结果,那么药代动力学就是联系起中药化学和中药药理学的一个纽带,它通过研究复方成分群在体内的动态变化规律,揭示复方如何相互作用产生药效作用。所以药代动力学的兴起和发展为研究中药复方物质基础、配伍规律和作用机理开辟了新的研究思路和方法以及必要的手段。运用药代动力学研究方法定性、定量地分析中药复方配伍后化学成分在体内的吸收、分布、代谢、排泄的动态变化规律,能够真实的反映中药的体内过程,阐明中药配伍后发挥药效的物质基础,是药效配伍研究方法的强有力补充。所以,探讨中药复方成分群与机体代谢系统之间的相互作用是阐述配伍规律的有效途径之一。
中药复方最常见、最经典的用药方式是口服汤剂,方剂中的药效物质群必须经过胃肠道的吸收进入体内才能发挥药理效应。肠道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之一,因此,从肠道吸收的角度研究方剂配伍时的吸收动力学变化,可以阐释方剂配伍的科学内涵。
药物在机体内的生物转化主要由肝细胞内滑面内质网上的肝药酶催化,在肝脏中参与药物代谢的代谢酶中以P450酶最为重要,人类肝微粒体中参与药物代谢的P450酶比较简单,主要有CYP1A、CYP2C、CYP2D、CYP2E和CYP3A五大类,由P450酶催化的Ⅰ相反应是其体内代谢转化的关键性步骤,因为这一步反应常常是药物从体内消除的限速步骤,它可以影响到药物的许多重要的药动学特性。
中药复方数以万计,为了体现研究的代表性和科学性,本课题通过遴选临床疗效确切、应用较为广泛的金铃子散为研究对象：金铃子散由川楝子和延胡索等量配比而成,首见于刘完素《素问·病机气宜保命集》为“疗肝郁化火而致的胸胁、腹脘、疝气痛、痛经”等痛症的代表方剂。方子虽小,但却不失配伍原则,有明确的君臣佐使关系,方中用川楝子疏肝气,泄肝火,为君药,延胡索行气活血,为臣使药,二药相配,气行血畅,疼痛自止,功效卓著。基于小复方或药对的配伍研究对于揭示中药复方配伍规律有着先导和示范意义。
1实验目的
本研究以经典小复方金铃子散为范例,基于药代动力学的研究思路,从药物吸收、代谢两个主要环节着手,利用正交设计的方法,一方面,从体内和体外两个层面探索金铃子散不同配比方对肝药酶P450活性的影响(诱导或抑制作用)；另一方面,提出并建立适合中药复杂系统研究的药物吸收-代谢模型,探讨金铃子散不同配比方吸收行为、吸收-代谢行为与复方配伍间的内在规律。从中药复方与肝药酶代谢相互作用角度探讨中药复方配伍规律及其科学内涵。
2实验方法和结果
2.1金铃子散及不同配比方对CYPlA2活性影响的研究
方法：采用L9(34)正交设计表,将金铃子散中的川楝子和延胡索作为两因素,每个因素设3个不同剂量作为实验的三水平,研究金铃子散9个不同配比及其相应的单味药物在体外和体内对CYP1A2活性的影响。体外实验以肝微粒体为研究工具,采用单一探针非拉西丁观察不同配伍的金铃子散对CYP亚型(CYP1A2)的半数抑制浓度(IC50)。体内实验采用探针药物法,先以金铃子散对大鼠进行预处理,再给予探针药(非拉西丁)后,应用微透析采样技术直接在肝脏采集样本,测定非拉西丁和对乙酰氨基酚的体内药动学参数,考察金铃子散不同配比方对酶(CYP1A2)活性的影响。实验结果进行正交-t检验和方差分析统计。
结果：川楝子、延胡索单味提取物和配比方1-9对肝药酶CYP1A2的半数抑制浓度(IC50)分别为：0.0252±0.0052、0.0121±0.0079、0.0919±0.0150、0.0719±0.0053、0.0282±0.0045、0.0754±0.0155、0.0628±0.0033、0.0919±0.0150、01976±0.0273、0.1591±0.0081、0.1311±0.0085g·L-1,对CYP1A2活性无显著抑制作用。体内实验,药代参数显示：金铃子散不同配比方均能诱导CYP1A2活性,诱导作用强弱顺序是：诱导剂组(方3)>方4(方5)>方9(方6,方1,方2,方8,川楝子,延胡索组)>方7>正常对照组。
2.2金铃子散及不同配比方对CYP3A4活性影响的研究
方法：采用L9(34)正交设计表,将金铃子散中的川楝子和延胡索作为两因素,每个因素设3个不同剂量作为实验的三水平,研究金铃子散9个不同配比及其相应的单味药物在体外和体内对CYP3A4活性的影响。体外实验以肝微粒体为研究工具,采用单一探针睾酮观察不同配伍的金铃子散对CYP亚型(CYP3A4)的半数抑制浓度(IC50)。体内实验采用探针药物法,先以金铃子散对大鼠进行预处理,再给予探针药(睾酮)后,应用微透析采样技术直接在肝脏采集样本,测定其体内药动学参数,考察不同配比方对酶(CYP3A4)活性的影响。实验结果进行正交-t检验和方差分析。
结果：川楝子、延胡索单味提取物和配伍方1-9体外对肝药酶CYP3A4的IC50值分别为2.59±0.33,0.87±0.30,1.14±0.20,1.00±0.13,1.19±0.10,2.33±0.15,1.39±0.19,1.14±0.20,1.29±0.14,1.43±0.32,1.49±0.28mg·L-1：体内实验结果显示：与正常对照组比较,金铃子散不同配比方、川楝子和延胡索单味提取物处理组大鼠CYP3A4酶活性均降低,金铃子散配比方抑制CYP3A4酶活性的强弱顺序：方1(方4)>方7>方2(方5)>方8>方3>方9。
2.3Cocktail探针药物法观察金铃子散及不同配比方对CYPs活性的影响
方法：采用L9(34)正交设计表,将金铃子散中的川楝子和延胡索作为两因素,每个因素设3个不同剂量作为实验的三水平,研究金铃子散9个不同配比及其相应的单味药体外对CYPs活性的影响。实验以肝微粒体为研究工具,采用多探针药物非拉西丁(PHE)、香豆素(COM)、甲苯磺丁脲(TOL)、奥美拉唑(OME)、睾酮(TST)为工具,观察金铃子散的不同配伍对CYP亚型(CYP1A2,CYP2A6,CYP2C9,CYP2C19和CYP3A4)的半数抑制浓度(IC50)。实验结果进行正交-t检验和方差分析。
结果：Cocktail探针法川楝子、延胡索单味提取物和配伍方1-9体外对肝药酶CYP1A2的IC50值分别为0.029±0004,0.015±0.006,0.096±0.015,0.077±0.003,0.033±0.005,0.079±0.016,0.068±0.009,0.096±0.015,0.194±0.023,0.156±0.048,0.111±0.012g·L-1；对肝药酶CYP2A6的IC50值分别为0.051±0.003,0.059±0.009,0.129±0.013,0.068±0.004,0.064±0.007,0.070±0.013,0.079±0.008,0.129±0.013,0.054±0.010,0.060±0.013,0.091±0.010g.L-1；对肝药酶CYP2C9的IC50值分别为0.149±0.020,0.125±0.013,0.067±0.005,0.059±0.004,0.064±0.005,0.077±0.003,0.061±0.006,0.067±0.005,0.059±0.004,0.064±0.003,0.077±0.003g·L--1；对肝药酶CYP2C19的IC50值分别为0.056±0.010,0.091±0.008,0.104±0.010,0.105±0.008,0.091±0.010,0.070±0.006,0.062±0.008,0.104±0.010,0.094±0.010,0.085±±0.009,0.069±±0.005g.L"1；对肝药酶CYP3A4的IC50值分别为：0.0031±0.0003,0.0010±0.0004,0.0014±0.0002,0.0010±0.0003,0.0019±0.0001,0.0025±0.0005,0.0016±0.0009,0.0014±0.0002,0.0019±0.0004,0.0023±0.0003,0.0017±0.0008g·L1。Cocktail探针法对CYP1A2和CYP3A4的IC50与单一探针法数值上虽然不等同,但是结果的生物学意义一致。
2.4吸收-代谢模型的建立和验证
方法：中药复杂体系的体内过程(ADME/Tox)重点和核心是吸收和代谢环节,建立了吸收-代谢模型对于中药药效物质、复方配伍研究意义重大。针对药物体内过程的特点建立中药代谢研究的体外模型,将药物的肠吸收模型和肝微粒体代谢模型结合起来,体外模拟药物的吸收-代谢过程,在外翻肠囊的基础上,向肠囊外浴槽内注入肝微粒体CYPs底物,30min后取样,向肠囊内加入肝微粒体及还原型辅酶Ⅱ,30min、60min后取样,HPLC分析和测定吸收样品中探针药物的原型及吸收-代谢样品中原型物、代谢物。
结果：单一探针吸收-代谢验证结果显示吸收液样品中能检测到探针药物原型、吸收-代谢样品中能同时检测到原型物、代谢产物；多探针吸收-代谢也验证上述结论,该模型可用于药物的吸收和代谢过程研究。
2.5基于吸收-代谢模型的金铃子散及其配伍研究
方法：建立适合中药研究的吸收-代谢模型,将药物的肠吸收研究模型和肝微粒体代谢模型结合起来；向肠囊外浴槽内注入不同配比的金铃子散,30min后取样,加入肝微粒体及还原型辅酶Ⅱ,30min、60min后取样,HPLC定性和定量分析吸收样品中金铃子散可吸收组份及吸收-代谢样品中可吸收-代谢组份,同时采用LC-MS进行验证。
结果：金铃子散提取物中15个主要组份中的10个组份可被小肠吸收,经A-M模型代谢后出现一个新的组份峰；金铃子散吸收组份比与配伍相关,川楝子是影响主要组份吸收比(Ar)和吸收-代谢比(Mr)的主要因素,延胡索次之,方1和方9主要组份吸收比最大,吸收-代谢比的顺序：方5>方6(方1)>方9>方4>方3>方7>方2>方8。
3结论
从中药复方对肝药酶的影响而言,金铃子散配比方对肝药酶CYP1A2活性有诱导作用,延胡索是诱导作用的主要因素；对CYP3A4酶的活性有抑制作用,配伍之后复方抑制作用显著增强,二者配伍显示出协同抑制作用；Cocktail探针药物法体外实验显示对CYP2A6、CYP2C9、CYP2C19无显著抑制作用。
从代谢酶对复方作用的角度而言,建立了适合中药复杂体系研究的吸收-代谢模型并用于金铃子散复方配伍研究,金铃子散配伍可吸收组份比与方剂配伍相关,方1(川楝子：延胡索比为1：1)和方9(川楝子：延胡索比为4：3)主要组份的吸收比最大,方5、方6(方1)主要组份吸收-代谢比最大；同时从复方药代(吸收-代谢差异)层面也能阐释方剂配伍理论的内涵：君药(川楝子)是影响全方吸收、吸收-代谢的主要因素,臣使药(延胡索)是处于从属地位的因素。
Background
The "syndrome differentiation and treatment" is unique understanding thought method which is recognition and treatment of the disease of the traditional Chinese medicine (TCM). Complex prescription compatibility of TCM is to reflect the characteristics and essence of TCM theory and "syndrome differentiation and treatment" thinking. Revealing the scientific connotation of complex prescription compatibility law of TCM is always the key problems expected to solve in TCM compound research field. It is important to explore compatibility law of TCM for clarifying the scientific connotation of "syndrome differentiation and differentiation"(TCM) theory and the mechanism of action of complex prescription of TCM. Essentially, the compatibility of traditional Chinese complex prescription constitutes a systematic and complex interaction system of multi-component Chinese medicine. These ingredients group, which may have pharmacological effects directly or after the interaction with the body's metabolic system, is an important material basis of therapeutic effects of traditional Chinese complex prescription. If what Chinese medicine chemical solves is the material basis of complex prescription (TCM) and the things that Pharmacology resolved are the result of complex prescription (TCM), then Pharmacokinetics, which is through the research on the dynamic change regularity in vivo of compound composition to reveal compound how to produce therapeutic effects, is a key link between the TCM chemistry and pharmacology. So the rise and development of Pharmacokinetic opened up new ideas, methods and provided necessary means for the research on the material basis of TCM compound, compatibility law and mechanism of action. With the using of Pharmacokinetic methods, after complex prescription compatibility, the qualitative and quantitative analysis of chemical composition's dynamic change regulation of absorption, distribution, metabolism, excretion in vivo can reflect the true process of TCM in the body, really clarify the material basis of efficacy after complex prescription compatibility, and be a strong complement of research on relation between efficacy and compatibility. Therefore, it is one of the most effective ways for describing compatibility law to explore the interaction between the group of complex prescription and the metabolic system of the body.
To take decoction orally is the most common and classic administration method of complex prescription. Only the substance groups which can express efficacy in the prescription go through the gastrointestinal tract and be absorbed into the body, can they play a pharmacological effect. Intestinal absorption is one of key factors in determining drug bioavailability and the therapeutic impact of drugs. Therefore, the research on change of absorption dynamics from a view of intestinal absorption can explain the scientific law of prescription compatibility.
The biotransformation of drugs in the body is mainly by the hepatic drug enzyme catalyst, which is on the slip endoplasmic of liver cells. Cytochrome P450is the most important in all enzymes that take part in drug metabolism in liver. Liver microsomal cytochrome P450enzymes in human body that take part in drug metabolism are relatively simple, mainly includes five categories:CYP1A, CYP2C, CYP2D, CYP2E, and CYP3A4. I reaction through the P450enzyme catalytic is a critical step of metabolic transformation in vivo. This reaction is often rate-limiting step in drug elimination from the body, so it can affect many important drug pharmacokinetic properties.
There are tens of thousands of Chinese medicine compound. But in order to reflect the research's features of representativeness and science, after selection, this topic selected JinLingZi powder as object, which has definitely clinical efficacy and is widely used. JinLingZi Powder is composed of Toosendan fruit and the same amount of Rhizoma Corydalis. Jinlingzi powder consists of Toosendan fruit and Rhizoma Corydalis with equal amount into ratio. It was firstly recorded in the "SuWen·BingJiQiYiBaoMingJi", written by LiuWansu. And it is the representative prescription of pain diseases caused by liver depression of the fire, such as hypochondrium pain, bellyache, hernia pain and dysmenorrhea. This prescription may be small, but it did not lose the principle of compatibility at all. In this prescription, Toosendan fruit, as the Monarch, can disperse liver stagnation and clear away the liver fire, Rhizoma Corydalis can promote Qi and activate blood, so it is the Minister and the Guide. The foregoing herbs being coordinative work, the Qi and the blood can move swimmingly and then the pain can stopped. The research based the compatibility of the small complex prescription and the herb couple have pioneering and demonstrative effect in clarifying the regulation of prescription compatibility in TCM.
1Objective
In this study, took the classic small compound Jinlingzi Powder as an example, Pharmacokinetics research ideas and orthogonal experimental design were applied to explore the effects of Jinlingzi powder formula and the compatibility on CYP enzyme activity in vitro and in vivo. On the other hand, absorption-metabolic model was proposed and established for the study of traditional Chinese medicine complex systems.The absorption-metabolic model was used to searching for the internal disciplines between the compability and the Pharmacokinetics. From the interaction point of view of traditional Chinese medicine and liver drug metabolic enzymes to explore the compatibility law of traditional Chinese medicine and its scientific connotation.
2Methods and Results
2.1The effects of Jinlingzi powder formula and the compatibility on CYP1A2
Method:L9(34) orthogonal design table was used. Put the Toosendan fruit and Rhizoma Corydalis of Jinlingzi powder as the two factors, each factor has three different doses as three levels of the experiment, which is to study the effects of nine different compatibility and corresponding single herbs in vitro and in vivo on enzymatic activity of CYP1A2employs. In vitro test, rat liver microsome was applied to observe the50%inhibitory concentration of Jinlingzi powder on CYP subtypes (CYP1A2) by using a single probe phenacetin. In vivo experiment by the means of specific subtype probe drug, rats were administered with medicine, then injected probe drug phenacetin. Pharmacokinetic parameters in vivo were determined so as to examine the effects of different formulas on the activity of enzyme (CYP1A2). The statistics is carried out by orthogonal-t test and variance analysis.
Result:The half maximal inhibitory concentrations (IC50) of the extract of Toosendan fruit, Rhizoma Corydalis and Jinlingzi Powder(formula.1-9) on the enzymatic activity of CYP1A2were:0.0252±0.0052,0.0121±0.0079,0.0919±0.0150,0.0719±0.0053,0.0282±0.0045,0.0754±0.0155,0.0628±0.0033,0.0919±0.0150,0.1976±0.0273,0.1591±0.0081,0.1311±0.0085g·L-1g·L-1, respectively. No significant inhibition CYP4501A2enzyme activity. In vivo test, pharmacokinetic parameters showed that the CYP1A2enzyme activity was inhibited by the different compatibility of Jinglingizi powder. The inhibited intensity of Jinglingizi powder followed the order: induction group (formula3)>formula4(formula5)>formula9(formula6, formula1, formula2, formula8, Toosendan fruit, Rhizoma Corydalis group)>formula7> normal group.
2.2The effects of Jinlingzi powder formula and the compatibility on CYP3A4
Method:L9(34) orthogonal design table was used. Put the Toosendan fruit and Rhizoma Corydalis of Jinlingzi powder as the two factors, each factor has three different doses as three levels of the experiment, which is to study the effects of nine different compatibility and corresponding single herbs in vitro and in vivo on enzymatic activity of CYP sub-typed(CYP3A4)employs. In vitro test, rat liver microsome was applied to observe the50%inhibitory concentration of Jinlingzi powder on CYP3A4by using a single probe testosterone. In vivo experiment by the means of specific subtype probe drug, rats were administered with Jinglingzi Powder, then injected probe drug testosterone. Pharmacokinetic parameters in vivo were determined so as to examine the effects of different formulas on the activity of enzyme (CYP3A4). The statistics is carried out by orthogonal-t test and variance analysis.
Result:The half maximal inhibitory concentrations (IC50) of the extract of Toosendan fruit, Rhizoma Corydalis and Jinlingzi Powder(formula.1-9) on the enzymatic activity of CYP3A4were2.59+0.33,0.87±0.30,1.14±0.20,1.00±0.13,1.19±0.10,2.33±0.15,1.39±0.19,1.14±0.20,1.29±0.14,1.43±0.32,1.49±0.28mg·L-1mg·L-1, respectively. In vivo test, the CYP3A4enzyme activity was inhibited by the different compatibility of Jinlingizi powder, compared with the normal control group. The inhibited intensity of Jinlingzi powder followed the order:formula1(formula4)> formula7> formula2(formula5)> formula8>formula3> formula9.
2.3The effects of Jinlingzi Powder formula and the compatibility on CYPs by Cocktail probe substrates approaches
Method:L9(34) orthogonal design table was used. Put the Toosendan fruit and Rhizoma Corydalis of Jinlingzi powder as the two factors, each factor has three different doses as three levels of the experiment, which is to study the effects of nine compatibility and corresponding single herbs in vitro and in vivo on enzymatic activity of CYPs employs.Rat liver microsome was applied to observe the50%inhibitory concentration of Jinlingzi powder on CYP sub-typed(CYP1A2, CYP2A6, CYP2C9, CYP2C19and CYP3A4) by the means of probe phenacetin (PHE), coumarin (COM), tolbutamide (TOL), omeprazole(OME), testosterone (TST). The statistics is carried out by orthogonal-t test and variance analysis.
Result:The half maximal inhibitory concentrations (IC50) of the extract of Toosendan fruit, Rhizoma Corydalis and Jinlingzi Powder(formula.1-9) on the CYP1A2:0.029±0.004,0.015±0.006,0.096±0.015,0.077±0.003,0.033±0.005,0.079±0.016,0.068±0.009,0.096±0.015,0.194±0.023,0.156±0.048,0.111±0.012g·L-1, respectively; on the CYP2A6:0.051±0.003,0.059±0.009,0.129±0.013,0.068±0.004,0.064±0.007,0.070±0.013,0.079±0.008,0.129±0.013,0.054±0.010,0.060±0.013,0.091±0.010g·L-1, respectively; on the CYP2C9:0.149±0.020,0.125±0.013,0.067±0.005,0.059±0.004,0.064±0.005,0.077±0.003,0.061±0.006,0.067±0.005,0.059±0.004,0.064±0.003,0.077±0.003g·L-1, respectively; on the CYP2C19:0.056±0.010,0.091±0.008,0.104±0.010,0.105±0.008,0.091±0.010,0.070±0.006,0.062±0.008,0.104±0.010,0.094±0.010,0.085±0.009,0.069±0.005g·L-1; on the CYP3A4:0.0031±0.0003,0.0010±0.0004,0.0014±0.0002,0.0010±0.0003,0.0019±0.0001,0.0025±0.0005,0.0016±0.0009,0.0014±0.0002,0.0019±0.0004,0.0023±0.0003,0.0017±0.0008g·L-1。IC50value of CYP1A2and CYP3A4by Cocktail probe method is not equivalent to that by individual probe method, but the biological significance of the results are consistent.
2.4Modeling and verification of Absorption-Metabolism model
Method:Absorption and metabolism was the key and core to ADME in vivo of TCM complex system. It is important to make a suitable absorption-metabolic model for TCM, which can contact the model of intestinal absorption with liver microsomal metabolic for TCM efficacy material research. Aiming at the characteristics of drug in the body, the establishment of Chinese medicine in vitro model must simplify and grasp the key link. Put the drug intestinal absorption model and the liver microsomal metabolism model together and then simulate the process of drug absorption and metabolism in vivo. Rat liver microsome CYPs was injected to gut sac outside bath slot based on everted gut sac. Then, rat liver microsome and NADPH would be injected after30min. The amount of components which can be absorbed from the prototype and the metabolin of probe drugs was analysed and detected by HPLC after30and60min.
Result:A single probe absorption-metabolism verification results show that the prototype and the metabolin of probe drugs can be detected in absorbing sample simultaneously. It can also be verified in multi-probe absorption-metabolism, which can be used in study of drug absorption and metabolism process.
2.5Study on the Compatibility of Jinlingzi powder formula based on the Absorption-Metabolism model
Method:The absorption-metabolic model suitable for TCM research was made to contact the model of intestinal absorption with liver microsomal metabolism model. Different prescriptions of Jinlingzi powder were injected to gut sac outside bath slot, and took samples after30min, then, rat liver microsome and NADPH would be injected into the everted gut sac. The amount of different components which can be absorbed from absorption-metabolic and the metabolic sample of Jinlingzi powder was analysed and detected by HPLC after30and60min, and meanwhile detected by LC-MS.
Result:About16main components which were derived from Jinlingzi Powder,10of them can be absorbed by intestinal tract. A new component peak was shown after the A-M metabolism model. The absorption ingredient proportion of Jinlingzi Powder was related to compatibility. Toosendan fruit is the major factor in influence on main group absorption ratio (Ar) and absorption-metabolic ratio (Mr), and Rhizoma Corydalis's influence followed it. The main group absorption ratios of formula1and formula9are maximum than others. The order of absorptio-metabolism ratio:formula5> formula6(formula1)> formula9> formula4> formula3> formula7> formula2> formula8.
3Conclusions
From the perspective of complex prescription of TCM's impact on hepatic enzyme, the different compatibility of Jinlingizi powder can induce the CYP1A2activity, and the intensity of this induction is associated with the compatibility of Jinlingzi powder, it can also inhibit the activity of CYP3A4. Synergistic inhibitory effect was shown on compatibility between Toosendan fruit and Rhizoma Corydalis. The experiment in vitro by Cocktail indicates that it has no significant effect on CYP2A6、CYP2C9、CYP2C19.
From the perspective of the metabolic enzyme's impact on complex prescription, the absorption ingredient proportion of Jinlingzi Powder was related to its compatibility. The main absorption ratios of formula1and formula9are maximum in all groups. The main absorption-metabolic ratios of formula5and formula6(formula1) are maximum. From the perspective of compound pharmacokinetics (the difference between absorption and metabolism), the connotation of prescription compatibility theory can also be explained meanwhile, which is that as the Monarch herb, Toosendan fruit plays a leading role factor in absorption, absorption-metabolism of complex prescription, while Rhizoma Corydalis,as the Minister herb, plays a subordinate role in it.
“辨证论治”是中医药认识和治疗疾病独特的思维方法,中药复方配伍是体现中医理论和“辨证论治”思想的载体。揭示中药复方配伍规律的科学内涵,一直是中药复方研究领域亟待解决的关键问题；探究中药配伍规律,对阐明中医“辨证论治”理论的科学内涵和中药复方的作用机制有着重要意义。中药复方配伍,从化学本质上来讲是不同中药的多组份间构成一个系统的、复杂的相互作用体系,这些相互作用的组份群是复方整体效应的重要物质基础。这些成分群可能直接作用于机体产生作用,也可能进入机体之后与机体代谢系统相互作用后产生药理作用。如果说中药化学解决的是中药复方起效的物质基础,中药药理学解决的是中药复方发生作用的结果,那么药代动力学就是联系起中药化学和中药药理学的一个纽带,它通过研究复方成分群在体内的动态变化规律,揭示复方如何相互作用产生药效作用。所以药代动力学的兴起和发展为研究中药复方物质基础、配伍规律和作用机理开辟了新的研究思路和方法以及必要的手段。运用药代动力学研究方法定性、定量地分析中药复方配伍后化学成分在体内的吸收、分布、代谢、排泄的动态变化规律,能够真实的反映中药的体内过程,阐明中药配伍后发挥药效的物质基础,是药效配伍研究方法的强有力补充。所以,探讨中药复方成分群与机体代谢系统之间的相互作用是阐述配伍规律的有效途径之一。
中药复方最常见、最经典的用药方式是口服汤剂,方剂中的药效物质群必须经过胃肠道的吸收进入体内才能发挥药理效应。肠道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之一,因此,从肠道吸收的角度研究方剂配伍时的吸收动力学变化,可以阐释方剂配伍的科学内涵。
药物在机体内的生物转化主要由肝细胞内滑面内质网上的肝药酶催化,在肝脏中参与药物代谢的代谢酶中以P450酶最为重要,人类肝微粒体中参与药物代谢的P450酶比较简单,主要有CYP1A、CYP2C、CYP2D、CYP2E和CYP3A五大类,由P450酶催化的Ⅰ相反应是其体内代谢转化的关键性步骤,因为这一步反应常常是药物从体内消除的限速步骤,它可以影响到药物的许多重要的药动学特性。
中药复方数以万计,为了体现研究的代表性和科学性,本课题通过遴选临床疗效确切、应用较为广泛的金铃子散为研究对象：金铃子散由川楝子和延胡索等量配比而成,首见于刘完素《素问·病机气宜保命集》为“疗肝郁化火而致的胸胁、腹脘、疝气痛、痛经”等痛症的代表方剂。方子虽小,但却不失配伍原则,有明确的君臣佐使关系,方中用川楝子疏肝气,泄肝火,为君药,延胡索行气活血,为臣使药,二药相配,气行血畅,疼痛自止,功效卓著。基于小复方或药对的配伍研究对于揭示中药复方配伍规律有着先导和示范意义。
1实验目的
本研究以经典小复方金铃子散为范例,基于药代动力学的研究思路,从药物吸收、代谢两个主要环节着手,利用正交设计的方法,一方面,从体内和体外两个层面探索金铃子散不同配比方对肝药酶P450活性的影响(诱导或抑制作用)；另一方面,提出并建立适合中药复杂系统研究的药物吸收-代谢模型,探讨金铃子散不同配比方吸收行为、吸收-代谢行为与复方配伍间的内在规律。从中药复方与肝药酶代谢相互作用角度探讨中药复方配伍规律及其科学内涵。
2实验方法和结果
2.1金铃子散及不同配比方对CYPlA2活性影响的研究
方法：采用L9(34)正交设计表,将金铃子散中的川楝子和延胡索作为两因素,每个因素设3个不同剂量作为实验的三水平,研究金铃子散9个不同配比及其相应的单味药物在体外和体内对CYP1A2活性的影响。体外实验以肝微粒体为研究工具,采用单一探针非拉西丁观察不同配伍的金铃子散对CYP亚型(CYP1A2)的半数抑制浓度(IC50)。体内实验采用探针药物法,先以金铃子散对大鼠进行预处理,再给予探针药(非拉西丁)后,应用微透析采样技术直接在肝脏采集样本,测定非拉西丁和对乙酰氨基酚的体内药动学参数,考察金铃子散不同配比方对酶(CYP1A2)活性的影响。实验结果进行正交-t检验和方差分析统计。
结果：川楝子、延胡索单味提取物和配比方1-9对肝药酶CYP1A2的半数抑制浓度(IC50)分别为：0.0252±0.0052、0.0121±0.0079、0.0919±0.0150、0.0719±0.0053、0.0282±0.0045、0.0754±0.0155、0.0628±0.0033、0.0919±0.0150、01976±0.0273、0.1591±0.0081、0.1311±0.0085g·L-1,对CYP1A2活性无显著抑制作用。体内实验,药代参数显示：金铃子散不同配比方均能诱导CYP1A2活性,诱导作用强弱顺序是：诱导剂组(方3)>方4(方5)>方9(方6,方1,方2,方8,川楝子,延胡索组)>方7>正常对照组。
2.2金铃子散及不同配比方对CYP3A4活性影响的研究
方法：采用L9(34)正交设计表,将金铃子散中的川楝子和延胡索作为两因素,每个因素设3个不同剂量作为实验的三水平,研究金铃子散9个不同配比及其相应的单味药物在体外和体内对CYP3A4活性的影响。体外实验以肝微粒体为研究工具,采用单一探针睾酮观察不同配伍的金铃子散对CYP亚型(CYP3A4)的半数抑制浓度(IC50)。体内实验采用探针药物法,先以金铃子散对大鼠进行预处理,再给予探针药(睾酮)后,应用微透析采样技术直接在肝脏采集样本,测定其体内药动学参数,考察不同配比方对酶(CYP3A4)活性的影响。实验结果进行正交-t检验和方差分析。
结果：川楝子、延胡索单味提取物和配伍方1-9体外对肝药酶CYP3A4的IC50值分别为2.59±0.33,0.87±0.30,1.14±0.20,1.00±0.13,1.19±0.10,2.33±0.15,1.39±0.19,1.14±0.20,1.29±0.14,1.43±0.32,1.49±0.28mg·L-1：体内实验结果显示：与正常对照组比较,金铃子散不同配比方、川楝子和延胡索单味提取物处理组大鼠CYP3A4酶活性均降低,金铃子散配比方抑制CYP3A4酶活性的强弱顺序：方1(方4)>方7>方2(方5)>方8>方3>方9。
2.3Cocktail探针药物法观察金铃子散及不同配比方对CYPs活性的影响
方法：采用L9(34)正交设计表,将金铃子散中的川楝子和延胡索作为两因素,每个因素设3个不同剂量作为实验的三水平,研究金铃子散9个不同配比及其相应的单味药体外对CYPs活性的影响。实验以肝微粒体为研究工具,采用多探针药物非拉西丁(PHE)、香豆素(COM)、甲苯磺丁脲(TOL)、奥美拉唑(OME)、睾酮(TST)为工具,观察金铃子散的不同配伍对CYP亚型(CYP1A2,CYP2A6,CYP2C9,CYP2C19和CYP3A4)的半数抑制浓度(IC50)。实验结果进行正交-t检验和方差分析。
结果：Cocktail探针法川楝子、延胡索单味提取物和配伍方1-9体外对肝药酶CYP1A2的IC50值分别为0.029±0004,0.015±0.006,0.096±0.015,0.077±0.003,0.033±0.005,0.079±0.016,0.068±0.009,0.096±0.015,0.194±0.023,0.156±0.048,0.111±0.012g·L-1；对肝药酶CYP2A6的IC50值分别为0.051±0.003,0.059±0.009,0.129±0.013,0.068±0.004,0.064±0.007,0.070±0.013,0.079±0.008,0.129±0.013,0.054±0.010,0.060±0.013,0.091±0.010g.L-1；对肝药酶CYP2C9的IC50值分别为0.149±0.020,0.125±0.013,0.067±0.005,0.059±0.004,0.064±0.005,0.077±0.003,0.061±0.006,0.067±0.005,0.059±0.004,0.064±0.003,0.077±0.003g·L--1；对肝药酶CYP2C19的IC50值分别为0.056±0.010,0.091±0.008,0.104±0.010,0.105±0.008,0.091±0.010,0.070±0.006,0.062±0.008,0.104±0.010,0.094±0.010,0.085±±0.009,0.069±±0.005g.L"1；对肝药酶CYP3A4的IC50值分别为：0.0031±0.0003,0.0010±0.0004,0.0014±0.0002,0.0010±0.0003,0.0019±0.0001,0.0025±0.0005,0.0016±0.0009,0.0014±0.0002,0.0019±0.0004,0.0023±0.0003,0.0017±0.0008g·L1。Cocktail探针法对CYP1A2和CYP3A4的IC50与单一探针法数值上虽然不等同,但是结果的生物学意义一致。
2.4吸收-代谢模型的建立和验证
方法：中药复杂体系的体内过程(ADME/Tox)重点和核心是吸收和代谢环节,建立了吸收-代谢模型对于中药药效物质、复方配伍研究意义重大。针对药物体内过程的特点建立中药代谢研究的体外模型,将药物的肠吸收模型和肝微粒体代谢模型结合起来,体外模拟药物的吸收-代谢过程,在外翻肠囊的基础上,向肠囊外浴槽内注入肝微粒体CYPs底物,30min后取样,向肠囊内加入肝微粒体及还原型辅酶Ⅱ,30min、60min后取样,HPLC分析和测定吸收样品中探针药物的原型及吸收-代谢样品中原型物、代谢物。
结果：单一探针吸收-代谢验证结果显示吸收液样品中能检测到探针药物原型、吸收-代谢样品中能同时检测到原型物、代谢产物；多探针吸收-代谢也验证上述结论,该模型可用于药物的吸收和代谢过程研究。
2.5基于吸收-代谢模型的金铃子散及其配伍研究
方法：建立适合中药研究的吸收-代谢模型,将药物的肠吸收研究模型和肝微粒体代谢模型结合起来；向肠囊外浴槽内注入不同配比的金铃子散,30min后取样,加入肝微粒体及还原型辅酶Ⅱ,30min、60min后取样,HPLC定性和定量分析吸收样品中金铃子散可吸收组份及吸收-代谢样品中可吸收-代谢组份,同时采用LC-MS进行验证。
结果：金铃子散提取物中15个主要组份中的10个组份可被小肠吸收,经A-M模型代谢后出现一个新的组份峰；金铃子散吸收组份比与配伍相关,川楝子是影响主要组份吸收比(Ar)和吸收-代谢比(Mr)的主要因素,延胡索次之,方1和方9主要组份吸收比最大,吸收-代谢比的顺序：方5>方6(方1)>方9>方4>方3>方7>方2>方8。
3结论
从中药复方对肝药酶的影响而言,金铃子散配比方对肝药酶CYP1A2活性有诱导作用,延胡索是诱导作用的主要因素；对CYP3A4酶的活性有抑制作用,配伍之后复方抑制作用显著增强,二者配伍显示出协同抑制作用；Cocktail探针药物法体外实验显示对CYP2A6、CYP2C9、CYP2C19无显著抑制作用。
从代谢酶对复方作用的角度而言,建立了适合中药复杂体系研究的吸收-代谢模型并用于金铃子散复方配伍研究,金铃子散配伍可吸收组份比与方剂配伍相关,方1(川楝子：延胡索比为1：1)和方9(川楝子：延胡索比为4：3)主要组份的吸收比最大,方5、方6(方1)主要组份吸收-代谢比最大；同时从复方药代(吸收-代谢差异)层面也能阐释方剂配伍理论的内涵：君药(川楝子)是影响全方吸收、吸收-代谢的主要因素,臣使药(延胡索)是处于从属地位的因素。
Background
The "syndrome differentiation and treatment" is unique understanding thought method which is recognition and treatment of the disease of the traditional Chinese medicine (TCM). Complex prescription compatibility of TCM is to reflect the characteristics and essence of TCM theory and "syndrome differentiation and treatment" thinking. Revealing the scientific connotation of complex prescription compatibility law of TCM is always the key problems expected to solve in TCM compound research field. It is important to explore compatibility law of TCM for clarifying the scientific connotation of "syndrome differentiation and differentiation"(TCM) theory and the mechanism of action of complex prescription of TCM. Essentially, the compatibility of traditional Chinese complex prescription constitutes a systematic and complex interaction system of multi-component Chinese medicine. These ingredients group, which may have pharmacological effects directly or after the interaction with the body's metabolic system, is an important material basis of therapeutic effects of traditional Chinese complex prescription. If what Chinese medicine chemical solves is the material basis of complex prescription (TCM) and the things that Pharmacology resolved are the result of complex prescription (TCM), then Pharmacokinetics, which is through the research on the dynamic change regularity in vivo of compound composition to reveal compound how to produce therapeutic effects, is a key link between the TCM chemistry and pharmacology. So the rise and development of Pharmacokinetic opened up new ideas, methods and provided necessary means for the research on the material basis of TCM compound, compatibility law and mechanism of action. With the using of Pharmacokinetic methods, after complex prescription compatibility, the qualitative and quantitative analysis of chemical composition's dynamic change regulation of absorption, distribution, metabolism, excretion in vivo can reflect the true process of TCM in the body, really clarify the material basis of efficacy after complex prescription compatibility, and be a strong complement of research on relation between efficacy and compatibility. Therefore, it is one of the most effective ways for describing compatibility law to explore the interaction between the group of complex prescription and the metabolic system of the body.
To take decoction orally is the most common and classic administration method of complex prescription. Only the substance groups which can express efficacy in the prescription go through the gastrointestinal tract and be absorbed into the body, can they play a pharmacological effect. Intestinal absorption is one of key factors in determining drug bioavailability and the therapeutic impact of drugs. Therefore, the research on change of absorption dynamics from a view of intestinal absorption can explain the scientific law of prescription compatibility.
The biotransformation of drugs in the body is mainly by the hepatic drug enzyme catalyst, which is on the slip endoplasmic of liver cells. Cytochrome P450is the most important in all enzymes that take part in drug metabolism in liver. Liver microsomal cytochrome P450enzymes in human body that take part in drug metabolism are relatively simple, mainly includes five categories:CYP1A, CYP2C, CYP2D, CYP2E, and CYP3A4. I reaction through the P450enzyme catalytic is a critical step of metabolic transformation in vivo. This reaction is often rate-limiting step in drug elimination from the body, so it can affect many important drug pharmacokinetic properties.
There are tens of thousands of Chinese medicine compound. But in order to reflect the research's features of representativeness and science, after selection, this topic selected JinLingZi powder as object, which has definitely clinical efficacy and is widely used. JinLingZi Powder is composed of Toosendan fruit and the same amount of Rhizoma Corydalis. Jinlingzi powder consists of Toosendan fruit and Rhizoma Corydalis with equal amount into ratio. It was firstly recorded in the "SuWen·BingJiQiYiBaoMingJi", written by LiuWansu. And it is the representative prescription of pain diseases caused by liver depression of the fire, such as hypochondrium pain, bellyache, hernia pain and dysmenorrhea. This prescription may be small, but it did not lose the principle of compatibility at all. In this prescription, Toosendan fruit, as the Monarch, can disperse liver stagnation and clear away the liver fire, Rhizoma Corydalis can promote Qi and activate blood, so it is the Minister and the Guide. The foregoing herbs being coordinative work, the Qi and the blood can move swimmingly and then the pain can stopped. The research based the compatibility of the small complex prescription and the herb couple have pioneering and demonstrative effect in clarifying the regulation of prescription compatibility in TCM.
1Objective
In this study, took the classic small compound Jinlingzi Powder as an example, Pharmacokinetics research ideas and orthogonal experimental design were applied to explore the effects of Jinlingzi powder formula and the compatibility on CYP enzyme activity in vitro and in vivo. On the other hand, absorption-metabolic model was proposed and established for the study of traditional Chinese medicine complex systems.The absorption-metabolic model was used to searching for the internal disciplines between the compability and the Pharmacokinetics. From the interaction point of view of traditional Chinese medicine and liver drug metabolic enzymes to explore the compatibility law of traditional Chinese medicine and its scientific connotation.
2Methods and Results
2.1The effects of Jinlingzi powder formula and the compatibility on CYP1A2
Method:L9(34) orthogonal design table was used. Put the Toosendan fruit and Rhizoma Corydalis of Jinlingzi powder as the two factors, each factor has three different doses as three levels of the experiment, which is to study the effects of nine different compatibility and corresponding single herbs in vitro and in vivo on enzymatic activity of CYP1A2employs. In vitro test, rat liver microsome was applied to observe the50%inhibitory concentration of Jinlingzi powder on CYP subtypes (CYP1A2) by using a single probe phenacetin. In vivo experiment by the means of specific subtype probe drug, rats were administered with medicine, then injected probe drug phenacetin. Pharmacokinetic parameters in vivo were determined so as to examine the effects of different formulas on the activity of enzyme (CYP1A2). The statistics is carried out by orthogonal-t test and variance analysis.
Result:The half maximal inhibitory concentrations (IC50) of the extract of Toosendan fruit, Rhizoma Corydalis and Jinlingzi Powder(formula.1-9) on the enzymatic activity of CYP1A2were:0.0252±0.0052,0.0121±0.0079,0.0919±0.0150,0.0719±0.0053,0.0282±0.0045,0.0754±0.0155,0.0628±0.0033,0.0919±0.0150,0.1976±0.0273,0.1591±0.0081,0.1311±0.0085g·L-1g·L-1, respectively. No significant inhibition CYP4501A2enzyme activity. In vivo test, pharmacokinetic parameters showed that the CYP1A2enzyme activity was inhibited by the different compatibility of Jinglingizi powder. The inhibited intensity of Jinglingizi powder followed the order: induction group (formula3)>formula4(formula5)>formula9(formula6, formula1, formula2, formula8, Toosendan fruit, Rhizoma Corydalis group)>formula7> normal group.
2.2The effects of Jinlingzi powder formula and the compatibility on CYP3A4
Method:L9(34) orthogonal design table was used. Put the Toosendan fruit and Rhizoma Corydalis of Jinlingzi powder as the two factors, each factor has three different doses as three levels of the experiment, which is to study the effects of nine different compatibility and corresponding single herbs in vitro and in vivo on enzymatic activity of CYP sub-typed(CYP3A4)employs. In vitro test, rat liver microsome was applied to observe the50%inhibitory concentration of Jinlingzi powder on CYP3A4by using a single probe testosterone. In vivo experiment by the means of specific subtype probe drug, rats were administered with Jinglingzi Powder, then injected probe drug testosterone. Pharmacokinetic parameters in vivo were determined so as to examine the effects of different formulas on the activity of enzyme (CYP3A4). The statistics is carried out by orthogonal-t test and variance analysis.
Result:The half maximal inhibitory concentrations (IC50) of the extract of Toosendan fruit, Rhizoma Corydalis and Jinlingzi Powder(formula.1-9) on the enzymatic activity of CYP3A4were2.59+0.33,0.87±0.30,1.14±0.20,1.00±0.13,1.19±0.10,2.33±0.15,1.39±0.19,1.14±0.20,1.29±0.14,1.43±0.32,1.49±0.28mg·L-1mg·L-1, respectively. In vivo test, the CYP3A4enzyme activity was inhibited by the different compatibility of Jinlingizi powder, compared with the normal control group. The inhibited intensity of Jinlingzi powder followed the order:formula1(formula4)> formula7> formula2(formula5)> formula8>formula3> formula9.
2.3The effects of Jinlingzi Powder formula and the compatibility on CYPs by Cocktail probe substrates approaches
Method:L9(34) orthogonal design table was used. Put the Toosendan fruit and Rhizoma Corydalis of Jinlingzi powder as the two factors, each factor has three different doses as three levels of the experiment, which is to study the effects of nine compatibility and corresponding single herbs in vitro and in vivo on enzymatic activity of CYPs employs.Rat liver microsome was applied to observe the50%inhibitory concentration of Jinlingzi powder on CYP sub-typed(CYP1A2, CYP2A6, CYP2C9, CYP2C19and CYP3A4) by the means of probe phenacetin (PHE), coumarin (COM), tolbutamide (TOL), omeprazole(OME), testosterone (TST). The statistics is carried out by orthogonal-t test and variance analysis.
Result:The half maximal inhibitory concentrations (IC50) of the extract of Toosendan fruit, Rhizoma Corydalis and Jinlingzi Powder(formula.1-9) on the CYP1A2:0.029±0.004,0.015±0.006,0.096±0.015,0.077±0.003,0.033±0.005,0.079±0.016,0.068±0.009,0.096±0.015,0.194±0.023,0.156±0.048,0.111±0.012g·L-1, respectively; on the CYP2A6:0.051±0.003,0.059±0.009,0.129±0.013,0.068±0.004,0.064±0.007,0.070±0.013,0.079±0.008,0.129±0.013,0.054±0.010,0.060±0.013,0.091±0.010g·L-1, respectively; on the CYP2C9:0.149±0.020,0.125±0.013,0.067±0.005,0.059±0.004,0.064±0.005,0.077±0.003,0.061±0.006,0.067±0.005,0.059±0.004,0.064±0.003,0.077±0.003g·L-1, respectively; on the CYP2C19:0.056±0.010,0.091±0.008,0.104±0.010,0.105±0.008,0.091±0.010,0.070±0.006,0.062±0.008,0.104±0.010,0.094±0.010,0.085±0.009,0.069±0.005g·L-1; on the CYP3A4:0.0031±0.0003,0.0010±0.0004,0.0014±0.0002,0.0010±0.0003,0.0019±0.0001,0.0025±0.0005,0.0016±0.0009,0.0014±0.0002,0.0019±0.0004,0.0023±0.0003,0.0017±0.0008g·L-1。IC50value of CYP1A2and CYP3A4by Cocktail probe method is not equivalent to that by individual probe method, but the biological significance of the results are consistent.
2.4Modeling and verification of Absorption-Metabolism model
Method:Absorption and metabolism was the key and core to ADME in vivo of TCM complex system. It is important to make a suitable absorption-metabolic model for TCM, which can contact the model of intestinal absorption with liver microsomal metabolic for TCM efficacy material research. Aiming at the characteristics of drug in the body, the establishment of Chinese medicine in vitro model must simplify and grasp the key link. Put the drug intestinal absorption model and the liver microsomal metabolism model together and then simulate the process of drug absorption and metabolism in vivo. Rat liver microsome CYPs was injected to gut sac outside bath slot based on everted gut sac. Then, rat liver microsome and NADPH would be injected after30min. The amount of components which can be absorbed from the prototype and the metabolin of probe drugs was analysed and detected by HPLC after30and60min.
Result:A single probe absorption-metabolism verification results show that the prototype and the metabolin of probe drugs can be detected in absorbing sample simultaneously. It can also be verified in multi-probe absorption-metabolism, which can be used in study of drug absorption and metabolism process.
2.5Study on the Compatibility of Jinlingzi powder formula based on the Absorption-Metabolism model
Method:The absorption-metabolic model suitable for TCM research was made to contact the model of intestinal absorption with liver microsomal metabolism model. Different prescriptions of Jinlingzi powder were injected to gut sac outside bath slot, and took samples after30min, then, rat liver microsome and NADPH would be injected into the everted gut sac. The amount of different components which can be absorbed from absorption-metabolic and the metabolic sample of Jinlingzi powder was analysed and detected by HPLC after30and60min, and meanwhile detected by LC-MS.
Result:About16main components which were derived from Jinlingzi Powder,10of them can be absorbed by intestinal tract. A new component peak was shown after the A-M metabolism model. The absorption ingredient proportion of Jinlingzi Powder was related to compatibility. Toosendan fruit is the major factor in influence on main group absorption ratio (Ar) and absorption-metabolic ratio (Mr), and Rhizoma Corydalis's influence followed it. The main group absorption ratios of formula1and formula9are maximum than others. The order of absorptio-metabolism ratio:formula5> formula6(formula1)> formula9> formula4> formula3> formula7> formula2> formula8.
3Conclusions
From the perspective of complex prescription of TCM's impact on hepatic enzyme, the different compatibility of Jinlingizi powder can induce the CYP1A2activity, and the intensity of this induction is associated with the compatibility of Jinlingzi powder, it can also inhibit the activity of CYP3A4. Synergistic inhibitory effect was shown on compatibility between Toosendan fruit and Rhizoma Corydalis. The experiment in vitro by Cocktail indicates that it has no significant effect on CYP2A6、CYP2C9、CYP2C19.
From the perspective of the metabolic enzyme's impact on complex prescription, the absorption ingredient proportion of Jinlingzi Powder was related to its compatibility. The main absorption ratios of formula1and formula9are maximum in all groups. The main absorption-metabolic ratios of formula5and formula6(formula1) are maximum. From the perspective of compound pharmacokinetics (the difference between absorption and metabolism), the connotation of prescription compatibility theory can also be explained meanwhile, which is that as the Monarch herb, Toosendan fruit plays a leading role factor in absorption, absorption-metabolism of complex prescription, while Rhizoma Corydalis,as the Minister herb, plays a subordinate role in it.
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