Methods used to evaluate the peroxyl (ROO.) radical scavenging capacities of four common antioxidants

Different methods that are used to measure peroxyl radical (ROO) scavenging capacity were used in model systems containing four common antioxidant compounds: ascorbic acid, cysteine, gallic acid and Trolox, a water-soluble vitamin E analogue. Five tests were performed, with differences in the method of peroxyl radical generation (lipoxygenase, azo-compound thermal degradation and spontaneous oxidation from a natural source of fatty acids) and in the addition of probes such as linoleic acid, crocin, fluorescein and electron paramagnetic resonance (EPR) by spin-trapping with a-phenyl-N-tert-butylnitrone. Significant differences were found in antioxidant reactivity, evaluated by dose–effect semi-logarithmic plots and EC50 values, and a possible explanation for these differences was explored. In assays in which ROO was generated by the azo-compound or spontaneous oxidation, gallic acid was the most powerful antioxidant. When lipoxygenase was used to produce ROO, ascorbic acid had the highest scavenging index, while cysteine and gallic acid had low levels of activity. Another difference was identified regarding the molecular probe: ascorbic acid and Trolox were the least reactive in the EPR system and azo-bis system with fluorescein, while in assays with crocin, these compounds had intermediate activities. Ascorbic acid and Trolox were the best antioxidants in an enzymatic-linoleic acid system, with a reciprocal ratio of 6:1, while adding crocin to the same system changed the reciprocal ratio to 2:1. A literature examination indicated that out of the antioxidant compounds tested, cysteine has the highest bond dissociation enthalpy, and this compound showed, as expected, low scavenging activity in three of the five tests in this study.