辣椒素的香草环在p450的催化下的代谢产生的活性中间体.ppt

Reactive Intermediates Produced from the Metabolism of theVanilloid Ring of Capsaicinoids by P450 Enzymes Contents Introduction Materials and methods Results Discussion Conclusion Introduction Capsaicinoids are the pungent compounds in hot peppers.Capsaicin (trans)represents 60?70% of all capsaicinoids in natural sources. Introduction Pharmacodynamics studies Several studies have demonstrated that capsaicinoids may be chemopreventive. Paradoxically, capsaicinoids were also reported to act as Cocarcinogens，and increased rates of liver and stomach cancer. These results are consistent with previous reports of genotoxicity for capsaicinoids following S9 bioactivation. Introduction objectives Characterized the formation of reactive intermediates from capsaicinoids by P450 enzymes. Characterized the way of the GSH addition to the intermediates . . Materials and methods Materials and methods HRP HRP is a glycoprotein, because its content is very high in horseradish, so named. It’s composed of several isozymes, based on iron porphyrins, molecular weight is 40000, isoelectric point is PH3 ~ 9, hydrogen peroxide can catalytic the polymerization reactions of phenol and phenylamine and their substituendums when H2O2 existed. Usually used as organization chemical tracers in optical and electron microscope. Materials and methods Metabolic Incubations (P450) Incubations (250 μL) in phosphatebuffered saline, pH 7.2, contained ～50 pmol P450 (human liver microsomes) , 2.5 mM NADPH, 2.5 mM GSH, capsaicin, typically at 25 or 100μM. Reactions were performed at 37 °C,5min ,and P450 turnover was initiated by the addition of NADPH (2.5 mM). Materials and methods Metabolic Incubations (HRP) Samples were prepared in 50 mM sodium acetate, pH 5.5, at room temperature and initiated by the addition of 500 μM H2O2. Reactions using HRP were processed as described above. incubation time was 10 min. For the determination of Km and the rel