Bioactivity assays

Measurement of antioxidant activity was carried out with DPPH assays, following the methodology originally proposed by Villano et al. [36], with some modifications. In brief, the DPPH (200 μmol) reagent was dissolved in ethanol and maintained for 20 min at 60–70 °C until the solution absorbance reached 1.0 ± 0.02 at 515 nm, as measured by a spectrophotometer (Thermo Electron, Spectronic Genesys 6, Madison, WI, USA). The resulting solution was kept stable for the next 16 h and stored at 4 °C. The assays were performed by adding 180 μL of the DPPH solution to the plant sample extracts (20 μL, 1 mg mL^− 1), and the resulting mixture was incubated for 20 min at 37 °C in the dark. The reaction absorbance was measured at 515 nm. Results were expressed as Trolox equivalent activity concentrations (mM), and as the mean value of the three analytical replicates.

Total phenol content assays were performed in two steps. First, the reaction mixture, containing 20 μL of plant sample extract in 80% methanol (1 mg mL^− 1) and 100 μL of 0.2 N Folin-Ciocalteu’s phenol reagent, was incubated for 5 min in the dark. Then, 80 μL of 7.5% Na₂CO³ was added, and the resulting reaction mixture was incubated for 60 min. Finally, absorbance was measured at 750 nm. Assay results were expressed in terms of gallic acid equivalent of the activity (μg mL^− 1), and as the mean value of three analytical replicates.

For total flavonoid content assays, reaction mixtures contained 20 μL of plant sample extract in 80% methanol (1 mg mL^− 1), 20 μL of 0.1 N NaOH, and 160 μL of 90% diethylene glycol. The reaction mixture was incubated for 60 min and the resulting absorbance was recorded at 405 nm. Results were expressed as naringin equivalent activity concentrations (μg mL^− 1). The data were presented as the mean of three analytical replicates.

Mushroom tyrosinase inhibitory activity was determined using the following method. A reaction mixture was prepared with 125 μL of 0.1 M sodium phosphate buffer (pH 6.5), 5 μL of plant sample extract in 80% methanol (10 mg mL^− 1), 30 μL of mushroom tyrosinase (1000 unit mL^− 1), and 40 μL of 1.5 mM L-tyrosine, and was added to 96-well plates. The reaction mixture was incubated at 37 °C for 20 min and absorbance was measured at 490 nm. The data were presented as the mean value of three analytical replicates.