A modified total oxyradical scavenging capacity assay for antioxidants in plant tissues

The total oxyradical scavenging capacity (TOSC) assay of Winston et al. [Free Radical Biol. Med. 24 (1998) 480] was modified through the development and incorporation of a standard curve, yielding a relatively simple but reliable method to quantify the total water-soluble antioxidant capacity (TAC) of plant tissues. The basis of the TOSC assay is the oxidation of alpha-keto-gamma-methiolbutyric acid (KMBA) by 2,2-azobis-amidinopropane (ABAP) with the evolution of ethylene as the quantifiable end product. A standard curve was established by using a serial dilution (0-100 muM) of the antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) which scavenges peroxyl radicals formed from the thermal homolysis of ABAP, thereby reducing the rate of ethylene evolution. Regression analysis of the standard curve yielded a second-degree polynomial (R-2 = 0.9975) that was used to predict the TAC (muMoles Trolox equivalents) of plant tissues. The precision and utility of the method was tested by determining TAC of skin tissue from 'Delicious' and 'Empire' apples (Malus domestica Borkh.) at various maturities, following cold storage, and after treatment with 1-methylcyclopropene (1-MCP). The TAC of 'Delicious' apples was twofold greater than that of 'Empire' apples, and remained relatively constant for up to 60 days in cold storage, then significantly decreased thereafter. This decrease during the latter stages of cold storage may be related to the development of superficial scald in this cultivar. 'Delicious' and 'Empire' fruit treated with 1-MCP had also a significantly higher TAC at various maturities after 120 days of cold storage. This is the first study to show that a treatment with 1-MCP can potentially maintain some intrinsic functional food quality parameters (i.e. antioxidant levels) in stored fruit. The establishment of a standard curve for use, in conjunction with the TOSC assay, resulted in a precise and highly reproducible predictor of TAC in apple fruit, while also permitting a rapid turnover of tissue samples for analysis.