Quantification of Total Phenolic Content

A solution of methanol:acetic acid 99%:1% v/v was added to 10 g of a sample in a 2:1 v/w ratio (28 (link)). Samples were perfectly pounded and left for 24 h in the dark at 4°C. Then, they were centrifuged at 4 °C at 11,600 g (Biofuge, Beckman, Cassina de Pecchi, Italy) for 10 min. The removed, filtered, and dried supernatant was then resuspended in 10 ml of 80% ethanol, and ethyl acetate was added. This last step was repeated three times. The organic fraction obtained was then treated with sodium sulphate anhydrous, filtered, and dried again. The residue was resuspended in 1 ml of 50% methanol and stored at −26°C until the analysis. The colorimetric analysis of total phenolics followed the method of Singleton and Rossi (29 (link)) with Folin–Ciocalteu phenol as a reagent. The absorbance at λ = 760 nm was determined at room temperature with a Cary UV/Vis spectrophotometer (Varian, Palo Alto, CA, USA). Quantification was based on a standard curve created using gallic acid (y = 0.497 x + 0.0239 R² = 0.9908). The results are expressed as mg of gallic acid equivalents (GAE)/100 g of fresh weight (FW) of the product ± SD.