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目的应用体外肝细胞模型研究中草药银杏叶提取物的代谢途径,即细胞色素P450酶(CYP450)药物代谢酶系对银杏叶拮抗血小板聚集效应的影响。方法制备人超低温冷冻肝细胞,通过与银杏叶提取物预孵育,评估肝脏CYP450药物代谢酶系(CYP1A2、CYP286、CYP2C19、CYP2E1、CYP3A4)对银杏叶水提取物拮抗血小板活化因子(PAF)激活的血小板聚集作用。富含血小板血清(PRP)和少含血小板血清(PPP)与不同质量浓度(25、50、100、200和1000μg·L~(-1))PAF孵育,建立PAF激活血小板聚集的体外模型。银杏叶水提物与人超低温冷冻复苏肝细胞预孵育后,与PRP和PAF培养,观察银杏叶水提物的体外效应(抗PFA血小板聚集激活作用)是否受人肝细胞代谢的影响。CYP450药物代谢酶的特定抑制物伊曲康唑(CYP3A4)、α-萘黄酮(CYP1A2)、奥芬那君(CYP286)、奥美拉唑(CYP2C19)、4-甲基吡唑(CYP2E1)与人肝细胞预孵育后、与银杏叶水提物和PRP和PAF孵育,评估银杏叶水提物的体外效应与何种CYP450药物代谢同工酶有关。结果PAF激活血小板聚集作用遵循米-曼氏动力学方程,其K_m为98μ·L~(-1)。银杏叶水提物抑制PAF的血小板聚集激活作用,其半数抑制剂量为33μg·L~(-1)。人肝细胞与银杏叶水提物预孵育后,其体外效应(抗PAF血小板聚集激活作用)增强30%,差异有统计学意义(P<0.05)。人肝细胞与细胞色素CYP450同工酶CYP286、CYP2C19、CYP2E1、CYP3A4抑制剂预孵育后,银杏叶水提物的体外效应不受影响。人肝细胞与CYP1A2抑制剂预孵育后,人肝细胞对银杏叶水提物体外效应的增强作用基本消失,差异有统计学意义(P<0.05)。结论银杏叶水提物在体外能抑制PAF的血小板聚集作用,人肝细胞能显著增强这一体外效应,CYP450药物代谢酶CYP1A2可能参与银杏叶的这一体外效应的代谢活化。
Objective To study the effect of cytochrome P450 (CYP450) drug metabolizing enzymes on the antagonistic effect of Ginkgo biloba leaves platelet aggregation by using the in vitro hepatocyte model to study the metabolic pathway of Chinese herbal medicine Ginkgo biloba extract. Methods Human cryopreserved hepatocytes were prepared and pre-incubated with Ginkgo biloba extract to evaluate the effects of CYP1A2, CYP286, CYP2C19, CYP2E1, and CYP3A4 on antagonistic platelet activating factor (PAF) activation of Ginkgo biloba extract. Platelet aggregation. Platelet-rich serum (PRP) and platelet-free serum (PPP) were incubated with PAF at different concentrations (25, 50, 100, 200, and 1000 μg·L -1 ) to establish an in vitro model of PAF-activated platelet aggregation. Ginkgo biloba water extract was pre-incubated with human cryopreserved cryopreserved hepatocytes and cultured with PRP and PAF to observe whether the effect of ginkgo leaf aqueous extract in vitro (anti-PFA platelet aggregation activation) was affected by human hepatocyte metabolism. Specific inhibitors of CYP450 drug metabolizing enzymes Itraconazole (CYP3A4), α-Naphthoflavone (CYP1A2), Orphenadine (CYP286), Omeprazole (CYP2C19), 4-methylpyrazole (CYP2E1) and After pre-incubation of human hepatocytes and incubating with Ginkgo biloba extract and PRP and PAF, the in vitro effects of Ginkgo biloba extract were assessed in relation to what CYP450 drug metabolism isoenzymes. Results The platelet aggregation induced by PAF followed the Miemann’s kinetic equation, and the K_m was 98μ·L -1 . Ginkgo biloba extract inhibited platelet aggregation and activation of PAF, and its half inhibitor amount was 33 μg·L~(-1). After pre-incubation of human liver cells with ginkgo leaf aqueous extract, its in vitro effect (anti-PAF platelet aggregation and activation) was enhanced by 30%, and the difference was statistically significant (P<0.05). Preincubation of human hepatocytes with cytochrome CYP450 isozymes CYP286, CYP2C19, CYP2E1, and CYP3A4 inhibitors did not affect the in vitro effects of Ginkgo biloba extract. After pre-incubation of human hepatocytes with CYP1A2 inhibitors, the enhancement effect of human hepatocytes on the external effects of ginkgo leaf water extraction disappeared, and the difference was statistically significant (P<0.05). Conclusion Ginkgo biloba extract can inhibit PAF platelet aggregation in vitro, and human hepatocytes can significantly enhance this in vitro effect. CYP450 drug metabolism enzyme CYP1A2 may be involved in the metabolic activation of Ginkgo biloba in vitro.