2.4. Determination of total phenolic content

The Folin Ciocalteu (FC) method with certain modifications was used to determine the total phenolic content of the five different extracts [[28], [29], [30]]. Gallic acid was used as a standard for this assay. Extract solutions for the assay were prepared in methanol or water depending on their solubility with a concentration of 10 mg mL⁻¹ and gallic acid solutions were prepared in methanol with different concentrations (15.625, 31.25, 62.5, 125, 250, 500 and 1000 μg mL⁻¹). In a 96-well microplate, 20 μL of the extract/standard solution was mixed with 100 μL of FC reagent and incubated for 5 min. After the incubation period, to each well containing the mixture, 80 μL of 7.5% NaHCO₃ solution was added and incubated for an additional 30 min at room temperature. The absorbance was measured at wavelength 765 nm. A standard gallic acid calibration curve was used to calculate the total phenolic content of each extract. The results were shown in terms of mg gallic acid equivalent per gm of extract.