| Objective:1.Establish and validate the L-02liver cell model and Caco-2cell model, and establish their combined model for ADME/Toxity test on these two basis.2. Use the combined model of Caco-2cell and L-02liver cell model to test the ADME(absorption、distribution、metabolism、elimination) and toxicity,as well as the mechanism of toxicity of Fructus Evodiae.Method:1. In this study, the Caco-2cell monolayer model was established according to the pre-established methods in our laboratory. The compactification and integrality of the monolayer cell was evaluated by observing the cell morphology and determining the transmembrane resistance (TEER).2.The L-02liver cell model was established based on the regulatory culture method. The viability was detected by the Cell morphological,the cell’s survival rate (Trypan blue staining method) and the contant of the ALB and BUN in the liver cell culture supernatant, determining the L-02liver cell’s excretion function to judge the reliability of the L-02liver cell model.3. The L-02liver cell model and the Caco-2cell model were combined together then estabilished an ADME/Tox combined system to study the absorption, metabolism and toxicity of Fructus Evodiae (WZY) in vitro. WZY was added in AP of the joint model, then collecting the the metabolic samples. After the absorption and metabolism,WZY’s composition changes in ingredients was determined by LC/MS. The WZY metabolites hepatotoxicity was evaluated by Biochemical, ELISA detection.Results:1.Caco-2cell model:The results show that the Caco-2cell monolayer tightness and integrity were the same as the laboratory pre-established. So, the model could be used for drug transport processes in vitro study.2. L-02liver cell model:L-02liver cells cultured by conventional, after24hours, the liver cells have been adherent. cultured3days’L-02liver cells showed that Liver cells are connected into pieces. The results showed that the cell survival rate of95%-98%, The BUN attained the best status in the third and fourth days. The liver cells secrete albumin Function reached a peaked at the third day. So, the third day was decided as the drug administration time.3. Remove available Caco-2cells (Millicell film), inoculated into6-well plates of the L-02liver cells, That was the Caco-2/L-02liver cells combined model. In this paper, an analytical method based on LC/MS had been successfully established to determine the metoblism constitutents of WZY. The results showed that this method’s accuracy, precision, specificity and quantitative linear range are required. The method is simple, rapid, accurate and reliable.The evodiamine (EVO), rutaecarpine (RUT) in the sample could be tested at the same time. WZY was absorbeded and metabolized by joint model. Joint model groupe:the content of evodiamine (EVO) ingredients in1h,2h,24h:2.53±1.60μg,4.57±0.03μg,15.86±0.06μg; the content of rutaecarpine (RUT):3.56±1.49μg,5.87±0.09μg,18.70±0.21μg. Caco-2cell model:the content of evodiamine (EVO) ingredients in1h,2h,24h:3.55±0.47μg,5.11±0.02μg,21.82±0.76μg; the content of rutaecarpine (RUT):4.63±0.15μg,8.31±0.14μg,22.00±0.85μg. L-02liver cell model:the content of evodiamine (EVO) ingredients in24h:79.85±0.15μg; the content of rutaecarpine (RUT):57.96±0.11μg. WZY absorption and metabolism by the joint model2h after24hours, including the EVO, RUT two ingredients caco-2cells were lower than the model group (p<0.01). WZY was absorbeded and metabolized by joint model for2and24h later, the content of (evodiamine) EVO and (rutaecarpine)RUT were lower than the caco-2cells model group (p<0.01). The Biochemical measurement results showed that:The joint model group, AST (18.67±1.53) was Higher than the control group①(14.00±1.41).L-02liver cells group, AST (20.11±1.10), ALT (16.33±3.21), ALP (6.00±1.00) were Higher than the control group②AST (13.00±3.00), ALT (8.33±2.52), ALP (3.33±1.15). The ELISA assay results showed that:The joint model group, CYP1A1(7.68±0.39), CYP2C9(5.30±0.74) were Higher than the control group①CYP1A1(6.70±0.03), CYP2C9(4.56±0.30). L-02liver cells group,CYP1A1(3.96±0.11), CYP2C9(2.90±0.30) were Higher than the control group②CYP1A1(3.53±0.19), CYP2C9(2.42±0.03). Conclusion:1. Evodiamine (EVO), rutaecarpine (RUT) were all well absorbed in Caco-2cell model.2.Evodiamine (EVO),rutaecarpine (RUT) were metabolized mainly to prototype way.3. Water extracts of nearly ripe fruit of Evodia rutaecarpa (WZY), and its metabolites evodiamine (EVO), rutaecarpine (RUT), which has some damaged on liver cells.Its mechanism of action may be related to the induction of CYP1A1, CYP2C9. |
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