β-Carotene assay revisited. application to characterize and quantify antioxidant and prooxidant activities in a microplate.

The β-carotene bleaching assay, a common method for evaluating antioxidant activity, has been widely criticized due to its low reproducibility, problematic quantification, complex reagent preparation, and interference of different factors (temperature, pH, solvents, and metals). In this work we have examined the effects of these factors and developed a highly reproducible procedure for microplate assay, evaluated the critical points of the method, and proposed a kinetic model for quantifying both antioxidant and prooxidant activities. The application of these tools produced very consistent results, which provide robust and meaningful criteria to compare in detail the characteristics of several well-known commercial antioxidants, as well as several predictable prooxidants, and can be easily applied to natural extracts, food samples, and many other type of compounds. As an example, we have tested a set of commercial antioxidants and some typical lipophilic prooxidants. The activity of the tested antioxidants decreased in the following order: ethoxyquin ≫ α-tocopherol > butylhydroxyanisole > butylhydroxytoluene ≫ propyl gallate. On the other hand, hemoglobin and Fe(2+), Fe(3+), Co(2+), and Cu(2+) showed a strong prooxidant effect, and the activity was null in Cd(2+), Ni(2+), and Sr(2+), slightly antioxidant in Mg(2+), and strongly antioxidant in Zn(2+) and Mn(2+).