Zheng Lin, Lin Lianzhu, Su Guowan, Zhao Qiangzhong, Zhao Mouming
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510641, China; Guangdong Food Green Processing and Nutrition Regulation Technologies Research Center, Guangzhou 511458, China.
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510641, China; Guangdong Food Green Processing and Nutrition Regulation Technologies Research Center, Guangzhou 511458, China; State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510641, China.
Food Res Int. 2015 Oct;76(Pt 3):359-365. doi: 10.1016/j.foodres.2015.06.045. Epub 2015 Jul 21.
DPPH assay is widely used to evaluate the radical scavenging activities of peptides. Effects of pH and buffers on the stability of DPPH• and its reduced product (DPPHH) in the ethanol-buffer solution were investigated in this study and the reactivity of DPPH• towards several dipeptides was compared to that of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•) and the peroxyl radicals in oxygen radical absorbance capacity (ORAC) assay. Results showed that the deprotonation of DPPHH under basic condition could interfere with the spectrophotometric measurement at 515-525nm. It was suggested that the reaction mixture be maintained at a final pH range of 5.0-6.5 in 1:1 ethanol-acetate/citrate buffer medium when evaluating the activities of peptides. Additionally, among tested dipeptides, only Cys-containing dipeptides displayed DPPH• scavenging activity with 0.14-0.28μmol TE (Trolox equivalent)/μmol, while Tyr/Trp-containing dipeptides with high reactivity towards ABTS• and peroxyl radicals were inert to DPPH• with TE values less than 0.02.
DPPH 法被广泛用于评估肽的自由基清除活性。本研究考察了 pH 和缓冲液对乙醇 - 缓冲溶液中 DPPH•及其还原产物（DPPHH）稳定性的影响，并将 DPPH•与几种二肽的反应活性与 2,2 - 联氮 - 双 -（3 - 乙基苯并噻唑啉 - 6 - 磺酸）（ABTS•）以及氧自由基吸收能力（ORAC）测定中的过氧自由基进行了比较。结果表明，在碱性条件下 DPPHH 的去质子化会干扰 515 - 525nm 处的分光光度测量。建议在评估肽的活性时，反应混合物在 1:1 乙醇 - 醋酸盐/柠檬酸盐缓冲介质中的最终 pH 范围保持在 5.0 - 6.5。此外，在所测试的二肽中，只有含半胱氨酸的二肽表现出 DPPH•清除活性，其清除能力为 0.14 - 0.28μmol TE（Trolox 当量）/μmol，而对 ABTS•和过氧自由基具有高反应活性的含酪氨酸/色氨酸的二肽对 DPPH•呈惰性，TE 值小于 0.02。
