3.4. Folin-Ciocalteu Assay

The total phenolic content (TPC) of the samples was determined using the Folin-Ciocalteu method as described previously with slight modifications [28]. Briefly, 200 μL aliquots of the sample, a standard, and a blank solution were each mixed with 1 mL Folin-Ciocalteu reagent (diluted 1:10 from stock solution) in a semi-microcuvette. Between 30 s and 8 min later, 800 μL 7.5% Na₂CO₃ solution was added at the same time to each mixture and incubated for 2 h at room temperature. The extinction was then measured at λ 760 nm using a spectrophotometer (Jasco, Groß-Umstadt, Germany). The TPC was calculated with a calibration curve constructed using gallic acid solutions. Each sample was measured in triplicates, and the results were expressed as grams of gallic acid equivalents per 100 g extract (g GAE/100 g).