白芷促进P-gp介导活性成分吸收的物质基础及机理研究

P-glycoprotein (P-gp), an ATP-dependent ef?ux protein, its efflux effect on drug transport was a major impact that induced many active ingredients low bioavailability. The results of our previous study showed that Radix Angelicae Dahuricae could improve transport of the drugs affected by P-gp. So we speculated that "there are active ingredients that could improve the transport of drugs affected by P-gp in Radix Angelicae Dahuricae, and the mechanism of the active ingredients might be relate to reduce the express of P-gp,and/or block the efflux function of P-gp". P-gp plays an important role in regulating the intestinal absorption of Paeoniflorin, Paclitaxel, Vinblastine, so the three drugs were selected in this topic. Based on the hypothesis, using Caco-2 cell monolayer model and in situ single pass intestine perfusion model, combined of pharmacokinetic studies, by means of the separation and purification technology, structural identification technology of natural organic compounds to trace the active substance of Radix Angelicae Dahuricae that improve the transport of "Paeoniflorin", "Paclitaxel", "Vinblastine". Using MDCK-MDR1cell model, by the technology of FQ-RT-PCR , protein electrophoresis and SDS-PAGE to study the effect of active substance of Radix Angelicae Dahuricae on the express of P-gp, P-gp drug binding site, the ATPase activity, the cell membrane fluidity. On the basis of the study to demonstrate the material base and the mechanism of Radix Angelicae Dahuricae on promoting drugs transport that affected by P-gp. The research will lay a foundation for development of new absorption enhancer, and will provide new ideas for screening of absorption enhancer from traditional Chinese medicine.

P-gp外排是导致药物口服生物利用度低的重要因素，课题组前一个国基金研究结果表明，白芷具有促进药物肠道转运的作用，且这些被促进转运的药物大多受P-gp的外排。因此，我们推测"白芷中具有促进P-gp介导活性成分吸收的活性物质，其作用机理可能与抑制P-gp的表达和/或功能有关"。为此，本课题以肠道转运受P-gp外排活性成分"芍药苷""紫杉醇""长春花碱"为探针药，采用Caco-2细胞、在体肠灌流模型，结合体内药代动力学研究，借助天然有机化合物分离纯化和结构鉴定技术，追踪白芷促进"探针药"肠道转运的活性物质；运用MDCK-MDR1细胞膜型，采用荧光定量PCR、蛋白电泳、SDS-聚丙烯酰氨凝胶电泳等技术，研究白芷活性物质对P-gp表达和功能的影响，以阐明白芷促进P-gp介导活性成分吸收的物质基础和作用机理，为白芷活性成分开发新型辅料-吸收促进剂奠定基础，同时提供以中药为原料筛选吸收促进剂的新思路。

P-gp外排是导致药物口服生物利用度低的重要因素，课题组前国基金研究结果表明，白芷具有促进药物肠道转运的作用，且这些被促进转运的药物大多受P-gp的外排。因此，在本课题中我们提出假说“白芷中具有促进P-gp介导活性成分吸收的活性物质，其作用机理可能与抑制P-gp的表达和/或功能有关”。本课题以肠道转运受P-gp外排活性成分“芍药苷”“多烯紫杉醇”“长春新碱”为探针药，首先根据文献分离10种呋喃型香豆素，即氧化前胡素、佛手柑内酯、珊瑚菜素、欧前胡素、异欧前胡素、白当归脑、异补骨脂素、东莨菪内酯、伞形花内酯、白当归素。采用Caco-2及MDCK-MDR1细胞考察各香豆素成分对探针药物肠道转运的影响。研究结果表明欧前胡素、异欧前胡素、氧化前胡素能显著促进多烯紫杉醇、长春新碱的肠道转运；但对芍药苷的肠道转运无促进作用。我们又增加了一个新的探针药物葛根素，考察了欧前胡素、异欧前胡素、氧化前胡素对葛根素肠道转运的影响，发现欧前胡素、异欧前胡素、氧化前胡素能显著促进葛根素透过Caco-2单层细胞转运。.根据细胞模型的研究结果，进一步结合在体肠灌流模型，药代动力学验证欧前胡素、异欧前胡素及氧化前胡素促进探针药物肠道转运的结果。研究结果表明欧前胡素、异欧前胡素、氧化前胡素能增加长春新碱、多烯紫杉醇的肠道转运、提高长春新碱、多烯紫杉醇在大鼠体内的生物利用度。.为了研究欧前胡素、异欧前胡素及氧化前胡素促进P-gp介导药物肠道转运的机制研究，开展了白芷中欧前胡素、异欧前胡素、氧化前胡素、佛手柑内酯对ATP酶活性的影响；对细胞膜流动性的影响；对Rh123透过MDCK-MDR1细胞肠道转运的影响；对P-gp蛋白表达的影响以及对P-gp mRNA的影响。研究结果表明欧前胡素、及氧化前胡素为非浓度依赖性的ATP酶诱导剂；异欧前胡素为浓度依赖的ATP酶双向调节子，其可能通过干扰ATP酶活性而阻断了P-gp的功能。三者均能降低细胞膜的流动性，抑制药物的外排。欧前胡素、氧化前胡素具有增强Rh123的细胞摄取作用。欧前胡素、异欧前胡素、氧化前胡素均能不同程度的降低P-gp的表达。欧前胡素（1µg/ml、5µg/ml、10µg/ml）、氧化前胡素（1µg/ml、5µg/ml、10µg/ml）均可降低P-gp mRNA的表达，异欧前胡素（10µg/ml）具有降低P-gp mRNA的表达。