Quantifying Total Phenolic Content

The total phenolic content was determined through the Folin–Ciocalteau reagent [33 (link)]. Briefly, a solution of the test extracts (20 μL), 10% Folin–Ciocalteau reagent (40 μL), and 7.35% sodium carbonate (140 μL) were added to a well of a 96-well plate. The blank comprised deionized water (20 μL), 10% Folin–Ciocalteau reagent (40 μL), and 7.35% sodium carbonate (140 μL). The mixture reacted in the dark for 1 h, and then absorbance was measured at 765 nm using a Thermo Scientific Varioscan LUX microplate reader (Thermo Fischer Scientific, Waltham, MA, USA). The absorbance values were converted to total phenolic content using a calibration curve previously constructed with a gallic acid standard (Sigma-Aldrich, St. Louis, MO, USA). Thus, the total phenolic contents were expressed as mg gallic acid equivalents per gram of seed powder (mg eq gallic acid/100 g dsp). Determinations were performed by using three biological and three technical replicates. The analytical method performance was monitored for each analysis batch, involving five QC samples spiked with 10% gallic acid to assess the method response variations (CV < 5%).