桉叶提取物抑制黄嘌呤氧化酶活性成分的分离纯化及其动力学研究

Inhibition effects on xanthine oxidase and kinetics research of extracts from eucalyptus leaves

为了对尾巨桉属广林9号品种桉叶提取物进行体外抑制黄嘌呤氧化酶(XOD)活性研究,并对其主要活性成分进行分离纯化、结构鉴定及抑制动力学研究。采用高效液相色谱法(HPLC)检测酶反应生成尿酸的量,以甲醇-K2HPO4缓冲液(50 mmol/L,pH值7.5)(5∶95)为流动相,检测波长为290 nm,测定了桉叶的不同溶剂提取物及其萃取组分对XOD活性的抑制作用,确定了最佳提取条件,并采用Diaion HP-20大孔树脂、反相硅胶柱层析法和制备液相色谱仪进行分离纯化。确定桉叶中最高活性单体为鞣花酸、槲皮素-3-O-葡萄糖苷酸和五没食子酰葡萄糖,测得其IC50分别为22.51、32.96、31.75μg/mL;并通过酶抑制动力学试验比较了他们的抑制类型和Ki值,得出鞣花酸的Ki值最小、为1.048μg/mL,可知其抑制黄嘌呤氧化酶活性效果在3种活性单体中最优。

Purified the anti-hyperuricemic components from Guanglin No.9 eucalyptus leaves by screening xanthine oxidase inhibitory activity in vitro and identified their structures. The inhibitory effects of extracts from eucalyptus leaves on xanthine oxidase （XOD） were measured by HPLC analysis. The mobile phases consisted of methanol-K2HPO4 buffer liquid （50mmol/L, pH 7.5, 5：95） and detection wavelength was 290 nm. The inhibitory effects on XOD of different extracts from eucalyptus leaves were determined to confirm the optimal extraction conditions, and used Diaion HP-20 macroporous resin, ODS and preparation HPLC to purify the extracts from eucalyptus leaves. The most active compounds, which were ellagic acid, Quercetin-3-O-glucuronide and Pentagalloyl glucose, were isolated with ICes 22.51, 32.96, 31.75 μg/mL, respectively. The inhibitory types and Kis of these three compounds were also studied through kinetics research, as the Ki of ellagic acid was minimum, 1.048 μg/mL, so the inhibitory effect on XOD of the ellagic acid was the best among these three compounds.