| BackgroundThe "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.1ObjectiveIn 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 Results2.1The effects of Jinlingzi powder formula and the compatibility on CYP1A2Method: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 CYP3A4Method: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 approachesMethod: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 modelMethod: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 modelMethod: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> formula8.3ConclusionsFrom 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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