A Experimental Studies On The Adme/tox Characteristics And Liver Pharmacodynamic & Toxic Mechanism Of Xiao Chai Hu Tang

In this paper, Caco-2 cell model, primary hepatocyte model and their combined model for ADME/Toxity test were established and evaluated respectively. The combined model of Caco-2 cell and primary hepatocyte(CMCH) was used to test ADME/Tox, and the liver pharmacodynamic & toxic mechanism of Xiao Chai Hu Tang.1. In this study,the Caco-2 cell mono layer model was established.The Caco-2 cell was cultivated in Millicell plate inserts for 21 days in standard procedure.Caco-2 cell model was evaluated by morphology feature using inverted microscope.The compactification and integrality of the mono layer cell was evaluated by transepithelial eleetrical resistance (TEER). The results were the same as the model established befor in laboratory. So, this model could be used for intestinal epithelial permeability as a valuable model.2. The primary hepatocyte model was established based on the two-step perfusion of Seglen's.The viability was between 70% and 93% detected by Trypan blue dye exclusion method. Then, the primary heaptocyte was cultured adherently.24 hour later, the cell changed in the shape and the cultureal supernatant was used to judge the excretion function by testing the ALB and BUN. The results showed that when the ALB reached a high amount at the third day, the BUN attained the best status. So, the third day was decided as the drug administration time.3. The Caco-2 cell model and the primary hepatocyte model were combined together then estabilished an ADME/Tox combined system(CMCH) to research the absorption and metabolism process of EXCHT in vitro. EXCHT was added in AP of Caco-2, and 2 hour later, the sample in BL was used to investigate the variation in chemical composition of EXCHT which had acted on the combined model for 2 hours. In this paper, an analytical method based on high performance liquid chromatography (HPLC) had been established to determine the metoblism constitutents of xiao chai hu decoction.The conditions were as follows:column-Symmetry ShieldTM RP18 (5.0μm,4.6mm×250mm Column);mobile phase-Acctonitrile-0.01% H3PO4 in gradient elution; detection wavelength-270nm; flow rate-0.45mL/min;column temperature-30℃. The accuracy, precision, sensitivity, specificity and linearity of this method were satisfied with the requirements.The baicalin, Wogonoside, Wogonin in the sample could be tested at the same time. The results showed that the three contents in the combined model group were as follows:2.60±0.11μg,2.03±0.11μg, 0.65±0.05μg as well, the two step model group were as follows:2.37±0.37, 2.72±0.53,0.44±0.09μg.Both baicalin and Wogonoside in the two groups were more than compared group's,however,the Wogonin was belower.WGN could not be testd in the standard model group but with baicalin 2.62±0.30μg.WGS in the two step model group was more than which in the combined model group,p=0.011(p<0.05),but WGN was less than which in the combined model group,p=0.001(p<0.01)。4. As the EXCHT was acted on the combined model group and the two step model group respectively for 2 hours,the sample in BL was used to test AST and LDH.We found that,the AST and LDH of the combined model group were higher than the compared group(P<0.01).The LDH in the two step model group was lower than the compared group,p=0.000(p<0.01),but with the same AST.The LDH of standard model group was higher than the compared group but with the same AST(P>0.05). We consider that the hepatocyte can be protected by EXCHT in the two step model group and standard model group, but it can be damaged in the CMCH.