CAJ | 학술논문

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、CYP2B6、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1 000 μg·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(CYP2B6)、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Km为98 μg·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33 μg·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CYP2B6、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.
목적 응용체외간세포모형연구중초약은행협제취물적대사도경,즉세포색소P450매(CYP450)약물대사매계대은행협길항혈소판취집효응적영향.방법 제비인초저온냉동간세포,통과여은행협제취물예부육,평고간장CYP450약물대사매계(CYP1A2、CYP2B6、CYP2C19、CYP2E1、CYP3A4)대은행협수제취물길항혈소판활화인자(PAF)격활적혈소판취집작용.부함혈소판혈청(PRP)화소함혈소판혈청(PPP)여불동질량농도(25、50、100、200화1 000 μg·L-1)PAF부육,건립PAF격활혈소판취집적체외모형.은행협수제물여인초저온냉동복소간세포예부육후,여PRP화PAF배양,관찰은행협수제물적체외효응(항PFA혈소판취집격활작용)시부수인간세포대사적영향.CYP450약물대사매적특정억제물이곡강서(CYP3A4)、α-내황동(CYP1A2)、오분나군(CYP2B6)、오미랍서(CYP2C19)、4-갑기필서(CYP2E1)여인간세포예부육후、여은행협수제물화PRP화PAF부육,평고은행협수제물적체외효응여하충CYP450약물대사동공매유관.결과 PAF격활혈소판취집작용준순미-만씨동역학방정,기Km위98 μg·L-1.은행협수제물억제PAF적혈소판취집격활작용,기반수억제제량위33 μg·L-1.인간세포여은행협수제물예부육후,기체외효응(항PAF혈소판취집격활작용)증강30%,차이유통계학의의(P＜0.05).인간세포여세포색소CYP450동공매CYP2B6、CYP2C19、CYP2E1、CYP3A4억제제예부육후,은행협수제물적체외효응불수영향.인간세포여CYP1A2억제제예부육후,인간세포대은행협수제물체외효응적증강작용기본소실,차이유통계학의의(P＜0.05).결론 은행협수제물재체외능억제PAF적혈소판취집작용,인간세포능현저증강저일체외효응,CYP450약물대사매CYP1A2가능삼여은행협적저일체외효응적대사활화.
AIM To estimate the effects of cytochrome P450 isozymes on efficacy of Ginkgo biloba leaf extract(GBE)-inhibition of platelet aggregation by using cryopreserved human primary hepatocytes (HPHs) in vitro. METHODS Plasma with rich platelets (PRP), plasma with poor platelets (PPP), and HPHs were prepared from donation. Platelet L-1. Effect of GBE on PAF induced platelet aggregation was estimated by preincubaiton of GBE before the addition of PAF. The effects of first pass metabolism on the efficacy of the GBE were investigated by preincubation of HPHs with GBE. The metabolism pathways involved for GBE were identified by preincubation of HPHs with selective inhibitors of CYP1A2 (α-naphthoflavon), CYP2B6 (orphenadrine hydrochlorid), CYP2C19 (omeprazole), CYP2E1 (4-methylpyrazole), and CYP3A4 (itraconazole). RESULTS PAF induced platelet aggregation followed Michaelis-Menten kinetics 30% enhancement over the control. These effects of GBE were reduced significantly by selectively inhibiting CYP1A2 but CYP2B6, 2C19, 2E1 and 3A4. CONCLUSION The inhibitive effects of GBE on PAF induced platelet aggregation are regulated by CYP1A2. The study implies an application for identifying the metabolism pathway of herb medicines in vitro.