TPC was estimated by the Folin–Ciocalteu method (Zill-e-Huma
et al., 2009) using a kit (SEPPAL (Isitec-lab), France) especially suitable
for food products. This kit includes: Reagent A (modified Folin–
Ciocalteu reagent), reagent B (alkaline buffer) and a gallic
acid solution (3 g/L). A small volume (20 lL) of H2O (blank), gallic
acid solution (standard) or the extract (sample) was mixed with
(2 ml) of reagent A. After 1 min, 1 ml of reagent B was added.
The mixtures were allowed to stand for 30 min in the dark at room
temperature. Then, their absorbance was measured at 760 nm with
a diode-array Hewlett–Packard 8453 spectrophotometer. TPC were
calculated by using the following formula: