DPPH自由基清除活性的光度微量滴定模型及应用

传统的DPPH自由基清除活性评价方法以半数清除浓度EC₅₀为评价指标，但EC₅₀随DPPH初始加入量增加而增加，随分析体积增加而减小，因此，不同条件EC₅₀值不具有可比性。提出以DPPH与抗氧化剂相互反应的化学计量数比(R)作为评价DPPH清除活性的指标，该指标只与DPPH与抗氧化剂相互反应的化学计量关系有关，与DPPH初始加入量和分析体积等因素无关，解决了EC₅₀可比性差的问题。提出了测定化学计量数比(R)的光度微量滴定法，建立了利用滴定过程吸光度差(ΔA)与抗氧化剂加入量之间的滴定方程计算R值、以R计算EC₅₀的光度微量滴定模型，并利用芦丁对模型进行验证。结果：芦丁与DPPH反应R值在1.817～1.846之间，当DPPH加入量为1.12×10-7，2.24×10-7，4.48×10-7和6.72×10-7 mol时，分别计算得EC₅₀值分别为1.196×10-3，2.392×10-3，4.819×10-3和7.292×10-3 mg·mL-1。在此基础上，基于文献报道的芦丁清除DPPH条件，利用得到的芦丁R值计算出相应EC₅₀，结果与文献报道EC₅₀值相当。方法可比性好，样品消耗量明显降低，简单、成本低，结果可靠，为自由基清除活性评价提出了一种新的思路。

Photometric Micro-Titration Model of DPPH Radicals Scavenging Activity and Its Application

In the present paper，the stoichiometric ratio (R) for the interreaction of DPPH radicals with the antoxidant was employed as a evaluation index for DPPH radicals scavenging activity of antioxidants. This evaluation index was related only with the stoichiometric relationship between DPPH radicals and the antioxidant, not the relationship with the initial DPPH amount and the volume of sample，which could offer a solution for the problem of poor comparability of EC₅₀ under different conditions. A novel photometric micro-titration method was proposed for the determination of the stoichiometric ratio (R) for the interreaction of DPPH radicals with the antoxidant. The titration equation was established based on the absorbance difference (ΔA) of DPPH radicals in the titration process and the added amount of antoxidant. The stoichiometric ratio (R) for the reaction of DPPH radicals with the addition amount of antoxidant was determined by the titration equation obtained, while, the DPPH median elimination concentration (EC₅₀) of antoxidant can be calculated by the stoichiometric ratio (R). The above photometric micro-titration model was verified using rutin as DPPH radicals scavenger. As experiment results, the stoichiometric ratio (R) of DPPH radicals to rutin was determined to be in the range of 1.817～1.846. The calculated value of EC₅₀ was 1.196×10-3, 2.392×10-3, 4.819×10-3 and 7.292×10-3 mg·mL-1 for 1.12×10-7, 2.24×10-7, 4.48×10-7 and 6.72×10-7 mol of the addition amount of DPPH radicals, respectively. The proposed method has better precision and reliability with smaller amount of sample than conventional method. While, the obtained stoichiometric ratio value (R) of rutin was employed to calculate the rutin median elimination concentration for DPPH (EC₅₀) according to the conditions as reported in the literatures, and the calculated results were consistent with that reported in the literatures.