Pyrex

The inhibitory activity against tyrosinase of root, stem, and leaf extracts of C. latifolia was determined colorimetrically based on the modification of previous methods [30 (link), 31 (link)]. The reaction mixture in a 96-well microplate (Iwaki Pyrex) consisted of 80 µL of phosphate buffer (50 mM, pH 6.5), 40 µL of extract solution (1–500 µg/mL), 40 µL of L-DOPA solution (4 mM), and 40 µL of mushroom tyrosinase solution (75 U/mL). The solution mixture was shaken for 60 seconds and incubated for 30 minutes at 25°C. The absorbance was measured using a microplate reader (Promega GloMax) at 490 nm. The blank solution in phosphate buffer was prepared in the same way as the sample solution. Control samples and blanks were made without the addition of the tyrosinase enzyme. The following equation was used to calculate the percentage of tyrosinase inhibition: $\begin{array}{c}\text{inhibition}\left(\%\right)=\frac{\left(A-B\right)-\left(C-D\right)}{\left(A-B\right)}\times 100\%,\end{array}$ where A is the absorbance of the blank solution with enzymes (blank), B is the absorbance of the blank solution without enzymes (blank control), C is the absorbance of the sample solution with enzymes (sample), and D is the absorbance of the sample solution without enzymes (control sample). The IC₅₀ value was calculated using a linear regression equation generated from plots of sample concentration versus percentage of inhibition (% inhibition).