FRAP Assay

In FRAP assays, the reduction of Fe3+-TPTZ to blue-colored Fe2+-TPTZ complex was monitored by the method described by Damiano et al., (2017) with slight modifications [40]. The FRAP reagen<sup>t</sup> was prepared by mixing ten volumes of acetate buffer (300 mM, pH 3.6), one volume of TPTZ solution (10 mM TPTZ in 40 mM HCl) and one volume of FeCl3 solution (20 mM FeCl3·6H2O in 40 mM HCl). Reaction mixture (25 μL sample and 175 μL FRAP reagent) was incubated in the dark for 30 min at room temperature and the absorbance of each solution was measured at 593 nm using a SPECTROstar Nano Multi-Detection Microplate Reader with 96-well plates (BMG Labtech, Ortenberg, Germany). A TroloxTM calibration curve (0.01–0.10 mg/mL) was plotted as a function of blue-colored Fe2+-TPTZ complex formation, and the results were expressed as milligrams of trolox equivalents (TE) per milligram of extract (mg TE/mg extract).

## 3.5.2. Tyrosinase Inhibitory Activity

Tyrosinase inhibitory activity of each sample was determined by the method previously described by Likhitwitayawuid and Sritularak, (2001) and Masuda *et al*., (2005) [41,42] using a SPECTROstar Nano Multi-Detection Microplate Reader with 96-well plates (BMG Labtech, Ortenberg, Germany). Samples were dissolved in water containing 5% DMSO; for each sample four wells were designated as A, B, C, D; each one contained the reaction mixture (200 μL) as follows: (A) 120 μL of 0.66 M phosphate buffer solution (pH = 6.8) (PBS) and 40 μL of mushroom tyrosinase in PBS (46 U/mL) (Tyr), (B) 160 μL of PBS, (C) 80 μL of PBS, 40 μL of Tyr, and 40 μL of sample, and (D) 120 μL of PBS and 40 μL of sample. The plate was then incubated at room temperature for 10 min; after incubation, 40 μL of 2.5 mM L-DOPA in PBS solution were added in each well and the mixtures were incubated again at room temperature for 20 min. The absorbance of each well was measured at 475 nm, and the inhibition percentage of the tyrosinase activity was calculated by the following equation, using as positive control a kojic acid solution (0.10 mg/mL):

$$\%I = \frac{(A-B) - (C-D)}{(A-B)} \times 100\tag{1}$$

The results were also expressed as mg kojic acid equivalents per gram of dry weight extract (mg KAE/g extract) using a calibration curve between 0.01–0.10 mg kojic acid per milliliter of solution.
