2.6 Total phenolic content (TPC) and Total flavonoid content (TFC)
2.6.1 Total phenolic content (TPC)
Using Ainsworth and Gillepsie’s (2007) Folin-Ciocalteu procedure, the TPC of the flours and cookie extracts was evaluated. A solution of 10 µL extract and 50 µL Folin-Ciocalteu phenol reagent (Sigma Aldrich, Germany) was added to 500 µL of distilled water. After letting it stand in the dark for three minutes, 200 µL of 20% (g/v) Na2CO3 (Sigma Aldrich, Germany) was added, followed by the addition of 245 µL of distilled water. The mixture (300 µL) was precisely pipetted into a 96-well microplate, covered with aluminum foil, and then incubated in darkness for 30 min. The plate’s absorbance was then measured using a micro plate reader (iMark, Biorad, South Africa) at 750 nm. The standard utilized was garlic acid (Sigma Aldrich, Germany), and the results were reported as mg GAE/g sample.
2.6.2 Total flavonoid content (TFC)
The TFC was determined using the method proposed by Ar-Farsi and Lee (2008), involving the mixing of 30 µL of the extract with 20 µL of 36 mM NaNO2. About 20 µL of 94 mM AlCl3 was then added. A micro plate reader (iMark, Biorad, South Africa) was used to measure the mixture’s absorbance at 450 nm. Using catechin (Sigma Aldrich, Germany) as a reference, the results were reported as mg of catechin equivalents per g of the sample (mg CE/g sample).