Photometric Micro-Titration Model of DPPH Radicals Scavenging Activity and Its Application
GAO Yun-tao1, 2, WEI Wei1, 2, YE Li-qing1, 2, LI Xiao-fen1,2, LIU Ping1, 2, ZHANG Hong-jiao1, 2, YANG Lu1, 2, YU Jiao-jiao1, 2, CHA Jia-wei1, 2
1. School of Chemistry and Biotechnology，Yunnan Minzu University，Kunming 650500，China 2. Key Laboratory of Ethnic Medicine Resource Chemistry，State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu University，Kunming 650500，China
Abstract：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.
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