Kojic acid

All chemicals were of an analytical grade and were used without further purification. A phosphate buffer solution (0.1 M, pH 6.8), prepared from sodium phosphate dibasic and sodium dihydrogen phosphate, was used as the supporting electrolyte for all measurements. Tyrosinase (EC 1.14.18.1, from mushroom), catechol, L-tyrosine, Bovine Serum Albumin (BSA), Glutaraldehyde (GA), benzoic acid and sodium azide (NaN₃) were purchased from Sigma-Aldrich (Burlington, MA, USA), whereas kojic acid was purchased from Alfa-Aesar (Tewksbury, MA, USA). The carbon black paste electrode was prepared by mixing 50% (w/w) carbon black powder N220 (Ravenna, Italy) with 50% (w/w) mineral oil (from Fluka, Buchs, Switzerland).
All electrochemical measurements were carried out using a PalmSens Potentiostat electrochemical analyzer provided by PalmSens BV (Utrecht, The Netherlands), controlled by PSTrace 4.0 software. A conventional three-electrode system in an electrochemical cell of 5 mL volume, containing the carbon black paste electrode (CBPE) as a working electrode, a platinum electrode as a counter electrode, and an Ag/AgCl electrode as a reference electrode.
An automated microplate reader (Biotek) was used to measure the absorbance of free enzyme at 490 nm, and the data were evaluated with Gen5 software. OriginPro8 was used as data analysis software.

The mushroom tyrosinase inhibitory activity was determined by a spectrophotometric method using a microplate reader, Synergy HT (Biotek), based on Kamkaen et al. [29 ] with modifications. The procedure was followed by the mixture of 150 μL of 1.5 mmol/L of L-tyrosine solution (phosphate buffer 0.05 M, pH 6.8) with 40 μL of test sample or pure solvent of the sample (blank, control), followed by a volume of 10 μL of 0.2 mg/mL of mushroom tyrosinase (T3824-250KU, Sigma-Aldrich) in phosphate buffer (0.05 M, pH 6.8).
Tyrosinase-driven conversion of L-Tyr to dopachrome was followed by an increase in the absorbance of the samples at 475 nm for 25 minutes. All tested samples were incubated at 25 °C during the process of data acquisition. Kojic acid (purity 99%, Alfa Aesar) was used as a positive control. The points in the linear range of the absorbance versus time plots were applied to calculate the slopes, directly proportional to tyrosinase activity. Then, the values of tyrosinase inhibition, expressed as the percent of the activity of the test samples versus the control experiment (pure solvent), were calculated for all samples according to the following equation: $\mathrm{Tyrosinase}\mathrm{activity}(\mathrm{TA}\%)=\frac{Slope of sample}{Slope of blank }\times 100$