Quantifying Hydroxyl Radical Scavenging Capacity

In vitro generation of the hydroxyl radical was carried out using the Fenton reaction involving Fe²⁺-EDTA/H₂O₂, during which peroxide decomposition to OH^● occurs [77 (link)]. These radicals react with terephthalic acid to form 2-hydroxyterephthalate acid, which has a high fluorescence. The reaction mixture consisted of 100 μL of the different extracts diluted in water, 300 μL of a 1 × 10⁻⁴ M sodium terephthalate solution, 2420 μL of phosphate buffer (0.2 M and pH 7.4), 90 μL of an EDTA (ethylenediaminetetraacetic acid disodium salt dihydrate) solution (1 × 10⁻² M), and 90 μL of Fe²⁺ (1 × 10⁻² M). The kinetics of the reaction was followed for 6 min in a LS-55 fluorometer (Perkin-Elmer, Waltham, MA, USA) at an excitation λ of 326 nm and an emission λ of 432 nm, with slits of 10 nm, without attenuation [78 (link)]. The results were presented as the ability to scavenge hydroxyl radicals, using Equation (4).
$$\% Hydroxyl radical activity=\left( \frac{A_2-A_0 }{A_1-A_0 }\right)\times 100,$$
where A₀ is the absorbance of the blank, A₁ is the absorbance of the control (without sample), and A₂ is the absorbance of the samples.