虎杖不同溶剂提取物对黄嘌呤氧化酶的抑制作用与酶动力学研究

Study on Inhibition and Enzyme Kinetics of Different Solvent Extractions From Polygonum cuspidatum on Xanthine Oxidase

目的:研究虎杖不同溶剂提取物对黄嘌呤氧化酶的抑制作用与酶动力学。方法:质量浓度分别为200、100、50μg/ml的虎杖总提取物、石油醚提取物、乙酸乙酯提取物、正丁醇提取物、水提取物与120μl黄嘌呤氧化酶(0.02 U/ml)作用于240μl黄嘌呤溶液(300μmol/L),采用高效液相色谱(HPLC)法测定黄嘌呤氧化酶-黄嘌呤反应体系尿酸生成量,计算抑制率。以40、20、10、5μg/ml虎杖乙酸乙酯提取物与100、80、60、40μg/ml虎杖正丁醇提取物作用于240μl黄嘌呤溶液,采用HPLC法测定尿酸生成量。酶动力学研究采用双倒数作图法确定有效溶剂提取物的抑制类型,采用二次作图法求药物对游离酶的抑制常数(K)i、药物对酶底物络合物的抑制常数(Kis)。结果:质量浓度为200、100、50μg/ml时,虎杖乙酸乙酯、正丁醇提取物对黄嘌呤氧化酶的抑制率均大于50%;虎杖乙酸乙酯提取物的Ki和Kis分别为14.46、61.82μg/ml,虎杖正丁醇提取物的Ki和Kis分别为82.97、148.93μg/ml。结论:虎杖乙酸乙酯、正丁醇提取物对黄嘌呤氧化酶具有抑制作用,二者均为混合型抑制,对游离酶的抑制作用均强于对酶-底物复合物的抑制作用。该结论为虎杖治疗痛风的新药开发和抑制黄嘌呤氧化酶作用有效成分研究提供了依据。

OBJECTIVE：To study the inhibition and Enzyme kinetics of different solvent extractions from Polygonum cuspida- turn on xanthine oxidase. METHODS： Total extractions, ethyl acetate extractions, n-butanol extractions, water extractions from P. cuspidaturn with concentrations of 200, 100, 50 μg/ml＋120 μl xanthine oxidase （0.02 U/ml） were respectively acted on 240 μl xanthine solution （300 μmol/L）. HPLC was employed to test the formation amount of uric acid in the xanthine oxidase-xanthine re- action system and calculate the inhibition rate. 40, 20, 10, 5 μg/ml ethyl acetate extractions from P cuspidaturn and 100, 80, 60, 40 μg/ml n-butanol extractions from P cuspidaturn were acted on 240 μl xanthine solution. Uric acid content was determined by HPLC. The inhibitory type of effective solvent extractions was determined by double reciprocal plot and K1 and Kis were obtained by quadratic mapping in the enzyme kinetics experiment. RESULTS： The inhibition rates of ethyl acetate and n-butanol extractions from P. cuspidaturn on xanthine oxidase were more than 50% with different concentrations （200, 100, 50 μg/ml）. The K1 and Kis of petroleum ether and ethyl acetate extractions from P. cuspidaturn on free enzyme were 14.46, 61.82 μg/ml respectively and the K1 and Kis of n-butanol from P cuspidaturn were 82.97, 148.93 μg/ml. CONCLUSIONS： The ethyl acetate and butanol extractions from P. cuspidatum showed significant inhibitory activities with mixed inhibitions. The inhibitory effects of both on free enzyme were stronger than those on enzyme-substrate complex. The study provides supporting ideas for the development of P. cuspidaturn on treatment of gout and the study of active ingredients inhibiting xanthine oxidase.