Determination of Total Polyphenol Content

Total polyphenol content was measured following the procedure reported in Costanzo et al. [23 (link)]. An aliquot (274 μL) of a suitable diluted methanol sample was added to 274 μL of the Folin–Ciocalteu reagent. The mixture was shaken and allowed to stand for 3 min, before adding 1.452 mL of 700 mM sodium carbonate (Na₂CO₃) solution. Samples were incubated for 2 h in darkness. Then, the absorbance was measured at 765 nm by a spectrophotometer (UV-VIS Cary 100, Agilent Technologies, Palo Alto, CA, USA). The total polyphenol content was calculated using gallic acid standard curve and expressed as mg Gallic Acid Equivalents (GAE) g⁻¹ FW.