The Study Of The Mechanism Of Compatibility Of Wuji Pill By The Perfused Intestine-Liver Preparation

1 ObjectiveThe Chinese medicine theory is broad and profound, the traditional Chinese medicine compound prescription is the main form of traditional Chinese medicine clinical practice, allocated proportion in the compound prescription between the taste is the Chinese medi ci ne theory essence i s. Thi s experi ment takes the tool medi ci ne by the Wuji pill, uses the orthogonal test design the method, With the aid of perfuse intestine-liver preparation, inspect the dynamic change of Ber?Pal?Evo?Rut and Peo in perfusate and bile, to study the mechanism of blending across the absorption, metabolism, excretion angles. Inspect the influence of Wuji pill to exsomatizeintestines, exsomatize colon smooth muscle cell, form PK-PD modle, common explain the rationality of Wuji pill.2 Method and Result2.1 The optimization of perfuse intestine-liver preparation IM ethod:The rat i s di vi ded 8 groups accordi ng to the perfusate formul a Observes each group of perfusates pH, the lactic dehydrogenase (LDH), alanin amino transferase (ALT) activeness, as well as the D/W of intestine and liver, thus quite different perfusate to liver function, liver cell damage as well as tissue edema degree influence.Result:The 8th group of perfusate (does not add red blood cell, dexamethasone, norepinephrine) pH, LDH, ALT with the 1st group of perfusate (to add red blood cell, dexamethasone, noradrenalin group) to compare the non-significance difference; The 8th group of perfusate D/W of intestine and liver compare the non-significance difference with the normal control group.Conclusion:The red blood cell, the dexamethasone, norepinephrine to fill the class model functi on regardi ng the mai ntenanceintesti nes liver to be not remarkable affect.2.2 The optimization of perfuse intestine-liver preparation IIMethod:The rat is divided to 4 groups according to the perfusate formula, Observes each group of perfusate returns-ratio, the lactic dehydrogenase (LDH), the alanin amino transf erase (ALT) activeness, as well as the D/W of intestine and liver, to compare the liver function, liver cell damage as well as tissue edema degree.Result:The 2nd group of perfusate (K-R buffer solution,1%BSA) the returns-ratio is highest; LDH, the ALT activeness is lowest; The D/W of liver compared to compare with the normal control group, has the significance difference (P<0.01), the intestinal D/W compared to the normal control group, non-significance difference.Conclusion:K-H buffer solution will be replaced with K-R buffer solution, whi ch can be appl ied to the next step experiment. 2.3 Research of PK of Wuji pill in perfusate of circle perfused intestine-liver preparationMethod:Uses L9(34) orthogonal design table, Berberine, Evodia, white peony root is regarded 3 factors, and each factor has 3 different dose levels. To inspect the bioavai I ability and Ka. Statistical analysis through single factor analysis of variance (ANOVA), the orthogonal design range analysis, analysis of variance.Result:The Opti mal rati o to enhances the bi oavailability of Ber?Pal?Evo?Rut and Peo is Berberine:Evodia:white peony=3:1:3?3:1:3?3:1:6?3:1:3?3:1:6?The Optimal ratio to speed the asorption is Berberine: Evodia:white peony=3:1:3?3:1:3?3:1:12?3:1:6?3:1:6?2.4 Research of PD of Wuji pill in perfusate of circle perfused intestine-liver preparationMethod:Observe the influence of perfusate to isolated guinea pig intestine which is contracted by Ach.Result:Ach contracted isolated guinea pig intestine significantly(P<0.01).All showed the effection of anti-Ach, and have significance but Berberine,3rd?9rd party. DAS3.0 analysis parameters of Berberine> Evodia> white peony root. So the contribution to effection is Berberine>Evodia>white peony root. The King position of Berberi ne i s confirmed.2.5 Research of PK of Wuji pill in perfusate of once-through perfused intestine-liver preparationMethod:Uses L9(34) orthogonal design table, Berberine, Evodia, white peony root is regarded 3 factors, and each factor has 3 different dose levels. To inspect the bioavailability.AUC, Liver extraction rate and Extraction rate of total liver.Statistical analysis through single factor analysis of variance (ANOVA), the orthogonal design rangeanalysis, anal ysi s of vari ance.Result:The Optimal ratio to enhances the absorption and liver extraction of Ber is Berberine:Evodia:whitepeony=12:1:12,3:6:3; The Optimal ratio to enhances the absorption and liver extraction of Pal is Berberine:Evodia:white peony=6:1:12,3:2:3; The Optimal ratio to enhances the absorption and liver extraction of Evo is Berberine:Evodia:white peony=12:6:6,12:1:12:The Optimal ratio to enhances the absorption and liver extraction of Rut is Berberine: Evodia:white peony=12:6:6,6:6:1; The Optimal ratio to enhances the absorption and liver extraction of Peo is Berberine:Evodia:white peony=6:2:12,3:2:6.2.6 Research of PD of Wuji pill in perfusate of once-through perfused intestine-liver preparationMethod:Observe the influence of perfusate to colonic smooth muscle cells which is contracted by Ach.Result:Ach contracted colonic smooth muscle cells significantly(P<0.01).All showed the effection of anti-Ach, but some group have no significance. DAS3.0 analysis parameters of Berberine>Evodia>white peony root. So the contribution to effection is Berberine>Evodia>white peony root. The King position of Berberine is conf i rmed.2.7 PK-PD link model Method:Fitting the result of PK and PD by WinonlinResult:Wuji pill PK-PD link model is a kind of inhibition model. That means the higher the concentration, the lower shrinkage, This is consistent with the experimental results. By PK-PD equation E=EmaxX (1-(Ce/(Ce+EC50)))?The goodness of fit of 1?2?3?6?9group is better2.8 Research of excretion of bile of Wuji pill Method:Intubate to bile, tap the bile after duodenum administration at different time same as the time point of once-through perfused intestine-liver preparation. detected by LC-MS. Inspect the excrete ration of Ber?Pal?Evo?Rut and Peo in bile,AUC. Statistical analysis through single factor analysis of variance (ANOVA), the orthogonal design range analysis, analysis of variance.Result:The Optimal ratio to promote the excretion of Ber?Pal?Evo?Rut and Peo in bileis Berberine:Evodia:white peony=12:1:6; 12:1:6; 3:6:3; 6:6: 3,respectively.3 ConclusionThis experiment is to study the diversification of the absorption, metabolism, excretion of the representative components in Wuji pill in different compatibility. the representative components showed different absorption, metabolism, excretion in different compatibilities. And Wuji pill have the effectiveness of anti-Ach was verified, Coptis showed greatest influence on this Pharmacology.