Folin ciocalteau reagent

TPC were determined by using the Folin-Ciocalteau reagent (VWR) (Singleton et al., 1999) (link).
Briefly, 100 µL of the extract were mixed with 2.8 mL of water and subsequently with 100 µl of the Folin-Ciocalteau reagent. After that, 2 mL of sodium carbonate 7.5 % (w/v) were added to the mixture. Absorbance was measured at 750 nm after 60 min of reaction (spectrophotometer V-750, Jasco, Japan). A calibration curve was prepared with standard solutions of gallic acid and results
were expressed as mg gallic acid equivalent (GAE)/gDOL.
The total flavonoids content (TFC) was determined following the procedure described by Chang et al. (Chang et al., 2002) . Briefly, 0.5 mL of the OL extract were mixed with 2.8 mL of distilled water and 0.1 mL of AlCl3 (10%, w/v). Finally, 0.1 mL of CH₃ COOK (1M) and 1.5 mL of ethanol were added. Absorbance at 415 nm was measured after 30 minutes in darkness (spectrophotometer V-750, Jasco, Japan). A quercetin standard curve in ethanol was determined and results were expressed as mg Quercetin Equivalent (QE)/gDOL.
To determine the TPC and TFC in the freeze-dried extract (FDE), a solution of 1 mg/mL of the FDE in 80 % ethanol was used for the analytical procedures.