Folin-Ciocalteu Assay for Total Phenols

The total phenol content was determined by the Folin–Ciocâlteu method with minor modifications [71 (link)]. Thus, a quantity of 0.5 mL was taken from each extract prepared as described above, over which 1.25 mL of Folin–Ciocâlteu reagent (Sigma-Aldrich Chemie GmbH, Munich, Germany) was added, diluted 1:10 with distilled water. The samples were incubated for 5 min at room temperature. After incubation, 1 mL of Na₂CO₃ (Geyer GmbH, Renningen, Germany; 60 g/L aqueous solution) was added. The samples were incubated for 30 min at 50 °C, using the thermostat (INB500, Memmert GmbH, Schwabach, Germany). The absorbance was read at 750 nm using a UV–VIS spectrophotometer (Specord 205; Analytik Jena AG, Jena, Germany). As a control, we used 70% ethanol (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The calibration curve was obtained with gallic acid (2.5–250 μg/mL). Results were expressed in mg GAE per kg dry matter (d.m.). All determinations were made in triplicate.