| Assay Method Information | |
| | In Vitro Tyrosinase Inhibition Assay |
| Description: | Tyrosinase inhibition assays were performed in a 96-well microplate format using the SpectraMax 340 microplate reader (Molecular Devices, CA) according to the method developed by Hearing [Khan et al., Pure Appl. Chem., 79:2277-2295]. First the samples were screened for the o-diphenolase inhibitory activity of mushroom tyrosinase (Lyophilised, ≥2000 units/mg, Sigma, Montana, USA) using L-3,4-dihydroxyphenylalanine (L-DOPA) (Sigma) as substrate. All the active samples from the preliminary screening were subjected to half maximal inhibitory concentration (IC50) studies. The samples were dissolved in methanol/DMSO to a final concentration of 0.5%. At higher concentrations like 3.3-6.7% DMSO shows a dose dependent inhibitory activity against the enzyme tyrosinase [Yu et al., J. Agric. Food Chem., 51:2344-2347]. So the final concentration for DMSO at 0.5% should be comparatively safe. Thirty units of mushroom tyrosinase (28 nM) were preincubated with the test compounds in 50 nM Na-phosphate buffer (pH 6.8) for 10 min at 25°C. Then the L-DOPA (0.5 mM) was added to the reaction mixture and the reaction was monitored by measuring the change in absorbance at 475 nm (at 37°C) due to the formation of the DOPA chrome for 10 min. |
| Affinity data for this assay | |
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