3.3.2. Total Ortho-Diphenolic Content

The ortho-diphenolic content was measured as described by Arnow [49]. Briefly, 100 μL of extract were diluted to 400 μL with deionized water. Then, 400 μL of 0.5 M HCl (solution A), 400 μL of 1.45 M NaNO₂ and 0.4 M Na₂MoO₄ (solution B), and 400 μL of 1 M NaOH (solution C) were added in sequence. The resulting mixture was immediately read at 500 nm using a blank containing 400 μL of deionized water. The quantification was carried out by a calibration curve obtained with increasing quantities of a standard solution of caffeic acid (range 5–50 μg). The results were expressed as mg CAE (Caffeic Acid Equivalents)/g DE, and subsequently converted, through a conversion factor, to mg GAE in order to compare the amount of ortho-diphenols with the total phenolic content. One milligram of CAE corresponded to 0.86 mg GAE.