Breinholt Vibeke M, Rasmussen Salka E, Brøsen Kim, Friedberg Thomas H
Institute of Food Safety and Nutrition, Division of Biochemical and Molecular Toxicology, The Danish Veterinary and Food Administration, Mørkhøj Bygade 19, 2860 Søborg, Denmark.
Pharmacol Toxicol. 2003 Jul;93(1):14-22. doi: 10.1034/j.1600-0773.2003.930102.x.
Recombinant cytochrome P450 (CYP) 1A2, 3A4, 2C9 or 2D6 enzymes obtained from Escherichia coli and human liver microsomes samples were used to investigate the ability of human CYP enzymes to metabolize the two dietary flavonoids, genistein and tangeretin. Analysis of the metabolic profile from incubations with genistein and human liver microsomes revealed the production of five different metabolites, of which three were obtained in sufficient amounts to allow a more detailed elucidation of the structure. One of these metabolites was identified as orobol, the 3'-hydroxylated metabolite of genistein. The remaining two metabolites were also hydroxylated metabolites as evidenced by LC/MS. Orobol was the only metabolite formed after incubation with CYP1A2. The two major product peaks after incubation of tangeretin with human microsomes were identical with 4'-hydroxy-5,6,7,8-tetramethoxyflavone and 5,6-dihydroxy-4',7,8-trimethoxyflavone, previously identified in rat urine in our laboratory. By comparison with UV spectra and LC/MS fragmentation patterns of previously obtained standards, the remaining metabolites eluting after 14, 17 and 20 min. were found to be demethylated at the 4',7-, 4',6-positions or hydroxylated at the 3'- and demethylated at the 4'-positions, respectively. Metabolism of tangeretin by recombinant CYP1A2, 3A4, 2D6 and 2C9 resulted in metabolic profiles that qualitatively were identical to those observed in the human microsomes. Inclusion of the CYP1A2 inhibitor fluvoxamine in the incubation mixture with human liver microsomes resulted in potent inhibition of tangeretin and genistein metabolism. Other isozymes-selective CYP inhibitors had only minor effects on tangeretin or genistein metabolism. Overall the presented observations suggest major involvement of CYP1A2 in the hepatic metabolism of these two flavonoids.
从大肠杆菌和人肝微粒体样品中获得的重组细胞色素P450（CYP）1A2、3A4、2C9或2D6酶，用于研究人CYP酶代谢两种膳食黄酮（染料木黄酮和陈皮素）的能力。对染料木黄酮与人肝微粒体孵育的代谢谱分析显示产生了五种不同的代谢物，其中三种产量充足，足以对其结构进行更详细的阐释。其中一种代谢物被鉴定为奥洛波尔，即染料木黄酮的3'-羟基化代谢物。其余两种代谢物也是羟基化代谢物，液相色谱/质谱（LC/MS）证明了这一点。奥洛波尔是与CYP1A2孵育后形成的唯一代谢物。陈皮素与人微粒体孵育后的两个主要产物峰与4'-羟基-5,6,7,8-四甲氧基黄酮和5,6-二羟基-4',7,8-三甲氧基黄酮相同，这两种物质先前已在我们实验室的大鼠尿液中鉴定出。通过与先前获得的标准品的紫外光谱和LC/MS裂解模式进行比较，发现分别在14、17和20分钟后洗脱的其余代谢物在4',7-、4',6-位去甲基化，或在3'-位羟基化并在4'-位去甲基化。重组CYP1A2、3A4、2D6和2C9对陈皮素的代谢产生的代谢谱在定性上与在人微粒体中观察到的代谢谱相同。在与人肝微粒体的孵育混合物中加入CYP1A2抑制剂氟伏沙明，可有效抑制陈皮素和染料木黄酮的代谢。其他同工酶选择性CYP抑制剂对陈皮素或染料木黄酮的代谢只有轻微影响。总体而言，所呈现的观察结果表明CYP1A2在这两种黄酮的肝脏代谢中起主要作用。
