2.7.6. Determination of Total Phenolic Content (TPC)

TPC was performed according to the Folin–Ciocalteu method described by de Araujo et al. [18]. Briefly, 300 μL of sample was mixed with 125 μL of Folin–Ciocalteu reagent and 1825 μL of DW. After the mixture was vortexed for 5 min at ambient temperature, 250 μL of sodium carbonate solution (20%, *w*/*v*) was added and it was vortexed for another 5 min. Then, the mixture was placed in a water bath at 40 ◦C for 30 min. The samples were then centrifuged at 10,000 rpm for 10 min, and the absorbance of the upper phase was measured at 765 nm by a spectrophotometer (Ultrospec 2000; Scinteck, Cambridge, UK). The results were expressed as mg gallic acid per g sample using the following formula [18]:

$$\mathbf{C} = c \frac{V}{\mathbf{m}} \tag{5}$$

where *C* is the total phenolic content (mg GAE/g dry extract), *c* is the concentration of gallic acid obtained from the calibration curve (mg/mL), *V* is the volume of extract (mL), and m is the mass of the extract (g).
