Determination of total phenolic and flavonoid content

The total phenolic content (expressed as TPC) was determined by the Folin-ciocalteau method reported in literature with slight modifications²⁵. The standard calibration curve was plotted using gallic acid at the concentrations of 0.02–0.1 mg/mL. The assay was done by mixing 0.75 mL of 10-fold diluted Folin-Ciocalteau reagent and 100µL of each solvent extract in a test tube. The mixture was allowed to stand at room temperature for 5 min, after which 0.75 mL of 6% (w/v) sodium carbonate (Na₂CO3) solution was added. The mixture was homogenized and allowed to stand at room temperature for 90 min. Mixture turned blue on adding 6% (w/v) Na₂CO3 solution indicating the presence of phenolics. Optical density of the sample was measured at 725 nm in triplicates using a Spcetrumlab 752S UV-VIS Spectrophotometer and the standard curve was drawn. The total phenolic contents were expressed as mg gallic acid equivalent (GAE) / g dry weight.

Colorimetric aluminium chloride method was used for total flavonoid content determination^26,27. Briefly, 0.5 mL solution of each extracts in methanol was separately mixed with 1.5 mL of methanol, 0.1 mL of 10% aluminium chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water and left at room temperature for 30 min. The absorbance of the reaction mixture was measured at 415 nm using Spcetrumlab 752S UV-VIS Spectrophotometer. The calibration curve was prepared using quercetin solutions at 6.25 to 100 µg/mL in methanol. Determination was done in triplicates. Total flavonoid contents were expressed as milligrams of quercetin equivalent (QE) / g dry weight.