The previously described method of [49 (link)], using L-DOPA (5 mM; Sigma Aldrich) was used for performing tyrosinase assay. L-DOPA diphenolase substrate was mixed with 10 μL of test sample along with sodium phosphate buffer (50 mM, pH 6.8). The final volume of the reaction mixture was raised to 200 μL by adding 0.2 mg/mL of mushroom tyrosinase solution (Sigma Aldrich). The extraction solvent replacing the tested sample was used as control. Microplate reader (BioTek ELX800; BioTek Instruments) was used to trace the reaction processes at 475 nm. Relative to corresponding control tyrosinase effect was expressed as percent inhibition.
Mushroom tyrosinase solution
Manufactured by Merck Group
Sourced in United States, Australia
Mushroom tyrosinase solution is a laboratory reagent containing the enzyme tyrosinase extracted from mushrooms. Tyrosinase is an oxidoreductase enzyme that catalyzes the conversion of monophenols to o-diphenols and the oxidation of o-diphenols to o-quinones.
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Lab products found in correlation
8 protocols using mushroom tyrosinase solution
1
Tyrosinase Inhibition Assay Using L-DOPA
2
Tyrosinase Inhibition Assay Protocol
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The tyrosinase inhibition assay was measured as described by Neely et al. (2009) [42 (link)]. Each 1 mL assay contained a final concentration of 100 mM sodium phosphate (pH 6.5) and 2 mM L-DOPA. Finally, 0.2 mg/mL of mushroom tyrosinase solution (Sigma-Aldrich) was added to the mixture. Control, with an equal amount of extraction solvent replacing the extract, was routinely carried out. Reaction processes were traced by using a microplate reader (BioTek ELX800; BioTek Instruments Inc., Winooski, VT, USA) at a wavelength of 475 nm. The tyrosinase inhibitory effect was expressed as a % of inhibition relative to the corresponding control for each extract. Standard compound concentrations applied in a study were 100 µM and the concentration of tested extracts was 50 µg/mL. The experiments were repeated three times and the average results with standard deviation values were given in Figure 3 B.
3
Extraction and Characterization of Resveratrol and Viniferin
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E-resveratrol and other standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). E-ε-viniferin was purified from grape canes as previously described [40 (link)]. Mushroom tyrosinase solution and L-DOPA were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was obtained from a Millipore Milli-Q water purification system (Merck Millipore, city, Germany).
4
Tyrosinase Inhibition Assay Protocol
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A tyrosinase assay was performed following the technique described by Chai and his research team [48 (link)]. In brief, the L-DOPA (5 mM; Sigma-Aldrich) was employed to be diphenolase’s substrate, then mixed in sodium phosphate buffer (50 mM, pH 6.8) and 10 μL of the samples. After that, the mushroom tyrosinase solution (Sigma Aldrich) 0.2 mg/mL was put into that mixture for a final volume 200 μL. This enzymatic reaction was measured at wavelength 475 nm using the microplate reader (BMG labtech, Victoria, Australia). Tyrosinase’s inhibitory result was reported as % inhibition relative to the control. The specific tyrosinase inhibitor was kojic acid (10 μM).
5
Tyrosinase Inhibitory Assay Protocol
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Method of Chai et al. (2018) was used for the determination of tyrosinase assay [80 (link)]. In brief, L-DOPA (5 mM; Sigma Aldrich) was used as diphenolase substrate and mixed in sodium phosphate buffer (50 mM, pH 6.8) with 10 µL of I. rugosus extract. Finally, 0.2 mg/mL of mushroom tyrosinase solution (Sigma Aldrich) was added to this mixture to make a final volume of 200 µL. Control, with an equal amount of extraction solvent replacing the extract, was routinely carried out. The reaction processes were traced by using a microplate reader (BioTek ELX800; BioTek Instruments) at a wavelength of 475 nm. The tyrosinase inhibitory effect was expressed as a % of inhibition relative to the corresponding control for each extract.
6
Tyrosinase Inhibitory Assay for Plant Extracts
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Tyrosinase inhibitory assay was carried out as described by Chai et al. (2018) (link). Briefly, the diphenolase substrate l- DOPA (5 mM; Sigma Aldrich) was mixed in sodium phosphate buffer (50 mM, pH 6.8) with 10 µl of L. usitatissimum extract after which, 0.2 mg.ml−1 of mushroom tyrosinase solution (Sigma Aldrich) was added to the reaction mixture to make a final volume of 200 µl. A control experiment was performed in parallel using an equal amount of extraction solvent. The absorbance of the reaction was measured using an absorbance microplate reader (BioTek ELX800; BioTek Instruments) at 475 nm. All the experiments were performed in triplicate, and the hyaluronidase inhibitory action was expressed as a percentage of inhibition relative to the control for each extract. Kojic acid (10 µM) was used as the specific inhibitor of tyrosinase leading to an inhibition of 51.2 ± 0.9%.
7
Tyrosinase Inhibition Assay Protocol
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The tyrosinase assay was conducted following the method described Chai et al. [53 (link)]. Briefly, l -DOPA (5 mM; Sigma Aldrich) was used as the diphenolase substrate, and then mixed in sodium phosphate buffer (50 mM, pH 6.8) with 10 µL of the extract. Lastly, 0.2 mg/mL of mushroom tyrosinase solution (Sigma Aldrich) was added to this mixture, in order to reach the final volume of 200 µL. Control tests, using an equal amount of extraction solvent to replace the extract sample, were routinely carried out. The reaction processes were detected using a microplate reader (BioTek ELX800; BioTek Instruments) at a wavelength of 475 nm. Tyrosinase’s inhibitory effect was expressed as the percent of inhibition relative to the control. Kojic acid (10 µM) was used as the specific inhibitor of tyrosinase.
8
Tyrosinase Inhibition Assay Protocol
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The inhibitory activity of tyrosinase was conducted according to the procedure of Hanh et al., (2017) with some minor modifications. The experiment was conducted with a 96-well plate, each initially containing 70 µl of the liverwort extract made in potassium phosphate buffer (50 mM and pH 6.8) containing 5% DMSO, 30 µl mushroom tyrosinase solution (Sigma T3824-25KU) 124.61 U ml -1 . The mixture was incubated at 32-34 o C for 15 min. Then, continue to add 110 µl of L-tyrosine substrate 2 mM and incubate for another 20 min. The product of the reaction was measured for absorbance at 490 nm. The blank sample is enzyme-free and the negative control is a non-extracts sample. Fraction extracts were prepared at 2 mg ml -1 and positive control was kojic acid at 200 µg ml -1 .
The percentage of inhibition is calculated by the formula:
With:
As: mixture absorbance of test sample Asb: mixture absorbance of test blank sample Ac: mixture absorbance of negative control Acb: mixture absorbance of negative blank control
The percentage of inhibition is calculated by the formula:
With:
As: mixture absorbance of test sample Asb: mixture absorbance of test blank sample Ac: mixture absorbance of negative control Acb: mixture absorbance of negative blank control
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