Quantification of Total Phenolic Content

The total phenolic content was established by using the Folin–Ciocalteu reagent following a little modified method [48 (link)]. The sub-extracts, extracts were evaporated and then dissolved with 500 μL of 70% methanol (HPLC-Gradient grade, Sigma–Aldrich, Darmstadt, Germany VWR chemicals) at 70 °C. The mixtures were centrifuged at 3500 g for 10 min, and the supernatants were collected in separate tubes. The pellets were re-extracted under identical conditions. Supernatants were combined and used for total phenolic assay. For the total phenolic assay, 20 μL of extract was dissolved into 2 mL of distilled water. Two hundred microliters of dissolved extract were mixed with 1 mL of Folin–Ciocalteu reagent (previously diluted tenfold with distilled water) and kept at 25 °C for 3–8 min; 0.8 mL of sodium bicarbonate (75 g L⁻¹) solution was added to the mixture. After 60 min at 25 °C, absorbance was measured at 765 nm. The results were expressed as gallic acid equivalents. The measurement of total phenolics was done under absorbance at 765 nm with the use of a Jenway UV/Vis 6405 spectrophotometer (Jenway, Chelmsford, UK). The total phenolic contents were determined from the linear equation of a standard curve prepared with gallic acid. The results were calculated as mg of gallic acid equivalent per g dry weight of extract.