Division of Physical and Analytical Chemistry, Faculty of Pharmaceutical Sciences, Nihon Pharmaceutical University.
Chem Pharm Bull (Tokyo). 2021;69(1):67-71. doi: 10.1248/cpb.c20-00568.
We examined the method of oxidative hemolysis for assessment of antioxidant activity of various compounds, especially lipophilic compounds. 2,2'-Azobis(amidinopropane) dihydrochloride (AAPH) was used as the source of free radicals for the oxidative hemolysis of horse erythrocytes. We found that absorbance at 540 nm is not appropriate for monitoring AAPH-induced hemolysis. Instead, we should use absorbance at 523 nm (an isosbestic point), because AAPH oxidizes the oxygenated hemoglobin to methemoglobin and absorbance at 540 nm does not correctly reflect the amount of released hemoglobin by AAPH-induced hemolysis. The corrected method of AAPH-induced hemolysis was applicable to assess the antioxidant activity of various hydrophilic compounds such as ascorbic acid, (-)-epicatechin, and edaravone. For the assessment of antioxidant activity of lipophilic compounds, we need appropriate dispersing agents for these lipophilic compounds. Among several agents tested, 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC) liposome at a concentration of 0.34 mM was found to be useful. Exogenous α-tocopherol incorporated using DMPC liposome as a dispersing agent was shown to protect erythrocytes from AAPH-induced hemolysis in a concentration-dependent manner.
我们研究了氧化溶血法来评估各种化合物的抗氧化活性，特别是脂溶性化合物。2,2'-偶氮双（脒基丙烷）二盐酸盐（AAPH）被用作氧化马红细胞溶血的自由基源。我们发现，540nm 处的吸光度不适于监测 AAPH 诱导的溶血。相反，我们应该使用 523nm 处的吸光度（等色点），因为 AAPH 将氧合血红蛋白氧化为高铁血红蛋白，而 540nm 处的吸光度不能正确反映 AAPH 诱导的溶血释放的血红蛋白量。经修正的 AAPH 诱导溶血法适用于评估抗坏血酸、(-)-表儿茶素和依达拉奉等各种亲水性化合物的抗氧化活性。对于脂溶性化合物的抗氧化活性评估，我们需要这些脂溶性化合物的适当分散剂。在测试的几种试剂中，发现浓度为 0.34mM 的 1,2-二肉豆蔻酰-sn-甘油-3-磷酸胆碱（DMPC）脂质体是有用的。用 DMPC 脂质体作为分散剂掺入的外源性α-生育酚能够以浓度依赖的方式保护红细胞免受 AAPH 诱导的溶血。
