青蒿素与过氧化物酶作用的荧光研究

目的:研究青蒿素与过氧化物酶间的作用关系,建立青蒿素荧光检测方法。方法:以吡罗红B为指示剂,用荧光降低法研究青蒿素与辣根过氧化物酶的相互作用。结果:青蒿素与辣根过氧化物酶之间的催化反应米氏常数Km为2.72×10-5mol/L,最大反应速度vmax为1.74×10-4mol/(L·s),催化常数Kcat为24.27s-1。反应作用位点分别是青蒿素内过氧基团与酶中心离子Fe。该反应可用于对黄花蒿药材中青蒿素含量的测定,测定工作曲线为c=0.1684?F-0.8143(cartemisinin单位为1.0×10-7mol/L),相关系数r=0.9965,3σ检出限为3.58×10-8mol/L。结论:青蒿素与辣根过氧化物酶之间的作用具有酶与底物反应的特征,该特征反应可用于对黄花蒿药材中青蒿素含量的测定。

Studies on Fluorescence Investigation of the Interaction of Artemisinin with Peroxidase

Objective： To explore the interaction between artemisinin and peroxidase（horseradish peroxidase, HRP） and establish quantification method for determination of artemisinin in Chinese medicinal herb Artemisia annua L. Methods： Pyronine B was used as an indicator to study on the interaction between artemisinin and HRP by fluorescence detection. Results： The Michaelis-Menten parameters of the reaction including Km, vmax and Kcat, were 2.72×10^-5 mol/L, 1.74×10^-4 mol/（L · s）, 24.27 s^-1 respectively. The action sites were endperoxide moiety of artemisinin and the central metal ion of enzyme. Under optimal conditions, fluorescence intensity change of pyronine B was proportional to the artemisinin concentration. The linear regression equation was c = 0.168 4 △F=0.814 3 （the concentration measurement unit of artemisinin is 1.0 ×10^-7 mol/L） and the detection limit （3 σ） was 3.58×10^-8 mol/L. Conclusion： The interaction between HRP and artemisinin was in an enzyme substrate model. This method could be applied to determine the concentration of artemisinin in A. annua.