脂氧化酶活性体外测定方法研究

Studies on Determination Methods of Lipoxygenase Activity in vitro

目的:研究脂氧化酶(LOX)活性体外测定方法。方法:以亚油酸为底物,3ml反应液在25℃和pH8.0条件下反应5分钟,加5ml甲醇终止反应。采用高效液相色谱法检测亚油酸,Agilent Eclipse XDB-C8(150mm×4.6mm,5μm)色谱柱;以乙腈-0.1%磷酸溶液(80∶20)为流动相,检测波长205nm。根据反应后亚油酸减少的量计算酶活性。采用文献报道的UV法和本文首创的HPLC法,对比测定了中药土连翘体外抑制LOX活性。结果:亚油酸浓度在5.296～264.8μg/ml范围内呈线性关系,r=0.9998,检测方法精密度RSD为1.74%,测定方法重复性RSD为2.43%。土连翘体外抑制LOX活性IC50为235μg药材/ml。与传统UV法比较,HPLC法专属性更好,更准确可靠。结论:我们创立的测定方法快速、简便、专属性强、准确、重复性好。

Objective：To research the determination methods of lipoxygenase（LOX） activity in vitro.Methods：The enzyme reaction was carried out at pH 8.0 at 25 °C in 3.0 ml volume for 5 min using linoleic acid as the substrate,and the reaction was ended by adding 5.0 ml of methanol.linoleic acidwas determined by RP-HPLC,with Agilent Eclipse XDB-C8（150mm×4.6mm,5μm）,acetonitrile-0.1% phosphoric acid in water（80：20） as a mobile phase,the detective wavelength was 205 nm.The enzyme activity was calculated according to the reduced amount of linoleic acid after reaction.The inhibitory potency of Hpericum henryi on LOX activity were determined in vitro by UV and HPLC comparatively.Results：The linear range of linoleic acid was 5.296～264.8μg/ml,r=0.9998,and the detection precision RSD was 1.74%,the method′s repeatability RSD was 2.43%.Determining by HPLC,Hpericum henryi exhibited a dose-dependent inhibition on the LOX activity with an IC50 value of 235μg herbs/ml.Compared with the UV,HPLC method is greater specificity,more accurate and reliable.Conclusion：The HPLC method is rapid,simple,specific,accurate,reproducible.