4.4.3. Gallic Acid-Equivalent Antioxidant Activity through DPPH Assay

A stock solution of DPPH radical 1 mM in EtOH was freshly prepared and used within 5 h. A stock solution of gallic acid 0.2 mM in EtOH was prepared. The calibration curves were built using a standard solution of gallic acid. The gallic acid standard solution with different linear increasing volumes (10–100 µL) and a known volume (100 μL) of the extract was added to a fixed volume of the DPPH solution (100 µL). After 15 min of incubation in the dark at room temperature, the EPR spectra were recorded. The antioxidant activity was plotted through the decay percentage of the area of the DPPH signal versus increasing concentrations of gallic acid standard solution. The area of the EPR spectra was calculated through the double integral of the DPPH signal.

The decay percentage for the plotting refers to the Formula (3) where A₀ is the area of the DPPH signal without the addition of the antioxidant or extract, A_S is the area the DPPH signal after the addition of scavenger agent such as antioxidant gallic acid or the extract.

The decay area percentage expressed in gallic acid equivalent was obtained through the calibration curve built with the standard solution (R² = 0.928) and reporting the area of the DPPH EPR signal after the addition of the extract. The extract was diluted a hundred times in respect to the stock solution. The measurements were repeated in triplicate and the antioxidant activity of the extract was expressed as mole/g of acid gallic equivalent.