Development and validation of a chemiluminogenic method for the evaluation of antioxidant activity of hydrophilic and hydrophobic antioxidants Original Research Article

A sensitive and simple procedure is described for evaluating the antioxidant activity of 21 known hydrophilic and hydrophobic compounds by using the chemiluminogenic reaction of lucigenin with hydrogen peroxide. The method was validated for linearity, limit of detection, precision, additivity and ruggedness and compared to the DPPH method. Furthermore, the IC50 values of all compounds were calculated and compared by both methods and results are discussed on the basis of structure–antioxidant activity relationship. Among hydrophilic antioxidants, phenolic compounds with adjacent hydroxyl groups (gallic acid, caffeic acid, pyrocatechol, (±)-catechin, oleuropein) were found to be efficient antioxidants in chemiluminescence method with IC50 values at the sub-micromolar range, while phenolic compounds without adjacent hydroxyl groups (p-coumaric acid, BHT, BHA, resorcinol) exhibited weak activity with IC50 values > 10 μM. α-Tocopherol was the most efficient hydrophobic antioxidant (IC50 = 6.7 μM) while oleic and linoleic acids were found to be very poor antioxidants (IC50 > 300 μM). Finally the method was applied to the estimation of antioxidant activity of complex samples such as olive oils. To the best of our knowledge, this is the first time that total antioxidant activity can be determined directly in olive oil and not in aqueous extracts.