Use of electrochemically-induced oxidation as an evaluation method for low molecular weight antioxidants

Free radical and antioxidant biochemistry has been the subject of increased interest and controversy. The recognition that free radicals are related to aging and various pathologies has lead to an increasing effort to discover natural and synthetic low molecular weight antioxidants that may strengthen natural defenses against free radicals. This research investigated a novel approach employing electrochemically-induced oxidation as a convenient and controllable tool to accelerate naturally occurring free radical reactions for the evaluation of antioxidant compounds.; An Antioxidant Evaluation Procedure (AEP) for water-soluble antioxidants was developed by using n-propyl gallic acid (GA) and Trolox as reference antioxidants. The rate constant of oxidation that occurs in the presence of a reference antioxidant was compared with that which occurs in its absence as a measure of antioxidant or prooxidant capacity. Five amino acids, tryptophan, methionine, cysteine, tyrosine, and histidine, and four Met-containing peptides, Met-Phe. Phe-Met, Met-Leu-Phe and Met-Enkephalin were evaluated, from which Met-Leu-Phe was selected as the substrate for the AEP because Met-Leu-Phe behavior provided greater sensitivity based on kinetic evaluations.; Using the AEP, the order of antioxidant effects was found to be, GA > ascorbic acid (AA) > Trolox. Prooxidant effects for these antioxidants were observed under some of the conditions used. GA was found to synergize the antioxidant activity of Trolox and AA under some conditions. Competitive kinetics approach has been employed and a term “Relative Antioxidant Capacity” (RAC) was defined to provide a quantitative measure for the relative antioxidant capacity (relative to Trolox). RAC was calculated to be 2.71 for GA and 1.15 for AA. The degradation of both Met-Leu-Phe and the antioxidants were studied by LC-MS and the results suggest that a combination of reactive oxygen species was produced and that hydroxyl free radical was the major radical responsible for the oxidation.; Water-insoluble antioxidants, α-tocopherol and L-ascorbyl-6-palmitate, were further evaluated using a previously designed approach. The protective effect of L-ascorbyl-6-palmitate on linoleic acid was less than that of α-tocopherol at low concentrations. The oxidation products of α-tocopherol were identified to be α-tocopherolquinone and α-tocopherolhydroquinone.