2.4. Stoichio-Kinetic Modelling of DPPH• Assay

The decay of DPPH• was measured at its maximum absorbance at 515 nm with a Cary 60 UV-VIS spectrophotometer (Agilent Technology, Santa Clara, CA, USA). A 100 µM DPPH• solution in methanol usually had maximum absorbance at 1.1 ± 0.4 (ε in methanol = 10,870 ± 200 M⁻¹cm⁻¹) [9]. The following procedure was used to determine the value of the rate constants and stoichiometric factor (n) for all phenols and extracts. First, 800 µL of a 125 µM DPPH• working solution (final concentration 100 µM) was transferred into a quartz cuvette, while 200 µL of the antioxidant solutions was injected with a syringe to prevent the loss of data during the initial seconds of the reaction. Different concentrations of antioxidants (10, 20, 50, 100, 200, 400 and 800 µM) reacted with 100 µM DPPH•, and the absorbance decay was measured from the beginning to 3 min for gallic and ascorbic acid; to 5 min for caffeic acid, Trolox and sinapic acid; to 10 min for chlorogenic acid and to 15 min for ferulic acid. The concentration of the DPPH• was deduced from the Beer–Lambert law, and a concentration versus time graph was generated. To calculate the rate constants and the stoichiometry of the reaction, different models were created in the software Copasi, and the best fitting is reported in Section 3.1 (see below).