Tyrosinase inhibition activity was determined as described by Momtaz et al.
[35 (link)], with L-3,4-dihydroxyphenylalanine (L-DOPA, Sigma) and tyrosine as substrates. Samples were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 20 mg/ml, and further diluted in potassium phosphate buffer (50 mM, pH 6.5) to 600 μg/ml. Assays were carried out in a 96-well micro-titre plate and a Multiskan FC plate reader (Thermo scientific technologies, China) was used. All the steps in the assay were conducted at room temperature. In triplicate, each prepared sample (70 μl) was mixed with 30 μl of tyrosinase (333 Units/ml in phosphate buffer, pH 6.5). After 5 min incubation, 110 μl of substrate (2 mM L -tyrosine or 12 mM L-DOPA) was added to the reaction mixtures and incubated further for 30 min. The final concentration of the extract was between 2.6 – 333.3 μg/ml. Arbutin (1.04 – 133.33 μg/ml) was used as a positive control while a blank test was used as each sample that had all the components except L-tyrosine or L-DOPA. Results were compared with a control consisting of DMSO instead of the test sample. Absorbance values of the wells were then determined at 492 nm. The percentage tyrosinase inhibition was calculated as follows:
where Acontrol is the absorbance of DMSO and Asample is the absorbance of the test reaction mixture containing extract or arbutin. The IC50 values of extracts and arbutin were calculated.
[35 (link)], with L-3,4-dihydroxyphenylalanine (L-DOPA, Sigma) and tyrosine as substrates. Samples were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 20 mg/ml, and further diluted in potassium phosphate buffer (50 mM, pH 6.5) to 600 μg/ml. Assays were carried out in a 96-well micro-titre plate and a Multiskan FC plate reader (Thermo scientific technologies, China) was used. All the steps in the assay were conducted at room temperature. In triplicate, each prepared sample (70 μl) was mixed with 30 μl of tyrosinase (333 Units/ml in phosphate buffer, pH 6.5). After 5 min incubation, 110 μl of substrate (2 mM L -tyrosine or 12 mM L-DOPA) was added to the reaction mixtures and incubated further for 30 min. The final concentration of the extract was between 2.6 – 333.3 μg/ml. Arbutin (1.04 – 133.33 μg/ml) was used as a positive control while a blank test was used as each sample that had all the components except L-tyrosine or L-DOPA. Results were compared with a control consisting of DMSO instead of the test sample. Absorbance values of the wells were then determined at 492 nm. The percentage tyrosinase inhibition was calculated as follows:
where Acontrol is the absorbance of DMSO and Asample is the absorbance of the test reaction mixture containing extract or arbutin. The IC50 values of extracts and arbutin were calculated.
Free full text: Click here
