Determination of Total Phenol Content

The total phenol content (TPC) was determined through Folin–Ciocalteau’s method, using gallic acid as standard (Sigma-Aldrich, St. Louis, MO, USA). Briefly, 0.125 mL of sample (properly diluted with water to obtain an absorbance value within the linear range of the spectrophotometer) underwent an addition of: 0.5 mL of distilled water, 0.125 mL of Folin–Ciocalteau’s (Sigma-Aldrich, St. Louis, MO, USA) reagent and 1.25 mL of an aqueous solution of Na₂CO₃ 7.5% (w/v%), bringing the final volume to 3 mL with water. After mixing, the samples were kept in the dark for 90 min. After the reaction period, the absorbance was measured at 760 nm using a V-730 UV-visible/NIR spectrophotometer operated by Spectra Manager^™ Suite (Jasco Inc., Easton, MD, USA). Each sample was analyzed in triplicate, and the concentration of total polyphenols was calculated in terms of gallic acid equivalents (GAE) [14 (link)].