Calorimetric adaptation of the inhibited autoxidation method to determine the activity of individual antioxidants and natural extracts

This work aims to determine the antioxidants activity of individual antioxidants and natural extracts by isothermal calorimetry. The proposed method is an adaptation of the inhibited autoxidation method. It consists of the monitoring of the heat-flow signal evolved during the autoxidation of styrene, in the presence of 2,2 '-azobis(isobutyronitrile) as radical initiator, with individual antioxidants or plant extracts as inhibitors, and under isothermal conditions (303 K). The resulting calorimetric traces were transformed in conversion fractions over time. The antioxidant activity was expressed with the initial slope of the inhibited period of styrene autoxidation. Also, the induction period observed at the onset of the uninhibited styrene autoxidation leaded the stoichiometry number of the reaction between antioxidants and free radicals. The approach was very simple, and it allowed to rank individual antioxidants (alpha-tocopherol > > gallic acid = quercetin > syringic acid > > synaptic acid > 4-hydroxybenzoic acid) and plant extracts (Olea europea > Melissa officinalis > Fraxinus excelsior > > Papaver rhoeas), based on their ability to decrease the conversion rate of styrene autoxidation. The results on plant extracts were critically compared with those from the DPPH assay. Differently to the many antioxidant assays widely used nowadays, the proposed approach has the merit to test the activity of micromolar quantities of antioxidants to inhibit the autoxidation of a large excess of an oxidizable substrate, whose oxidation is induced by a constant rate of free radicals formation under well controlled and reproducible experimental conditions.

Automated summary

This study used isothermal calorimetry to determine the antioxidants activity of individual antioxidants and natural extracts. The proposed method monitored the heat-flow signal during the autoxidation process and evaluated the ability of antioxidants to inhibit the oxidation reaction. The results allowed for ranking of antioxidants and plant extracts, and were compared with other antioxidant assays.