DPPH method
The antiradical activity of the oils of the seven olive varieties (IM < 2, 2012/2013 crop season) were measured using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) following the procedure reported by Prior, Wu, and Schaich (2005). The assays were performed in a Hitachi U-2900 spectrophotometer (Tokio, Japan), using PS cuvettes.
To a 60 μM methanolic solution of DPPH (HPLC-grade, 1.17 ml) was added the methanolic solution of the extract (30 μl, 7 different concentrations), or pure methanol as the control. The corresponding mixtures were kept in the darkness at rt for 30 min and then, the absorbance was measured at 515 nm against a blank (MeOH). Plotting the values of DPPH remaining vs. extract concentration allows a straight line from which the EC50 (the concentration of the antioxidant required to reduce the concentration of the DPPH to 50% of its initial value) is calculated. All measurements were carried out in triplicate. The remaining DPPH concentration was calculated using the expression: