Enzymes involved in melanin synthesis or degradation of the extracellular matrix play an important role in skin ageing. Many of these enzymes are present in the top layers of the skin, where they can be influenced by the application of cosmetic products with active ingredients. Many cosmetic products contain enzyme inhibitors as active ingredients, with the idea to affect the condition and appearance of the skin. The development of simple and robust assays for the determination of enzyme activity is crucial for the detection of potential new enzyme inhibitors.
In this study, we optimised the activity assay for two enzymes involved in skin ageing, tyrosinase and hyaluronidase. We also tried to optimise the assay for matrix metalloproteinase-1 (MMP-1), but were unable to obtain adequate measurements. In all cases, we chose the spectrophotometric assay as the baseline as it has a simple procedure and allows easy signal detection even with a large number of samples. First, the activity of the individual enzymes was measured in the absence of the inhibitor to determine the concentration of the enzyme giving a signal of the appropriate intensity. These tests were then repeated in the presence of a known enzyme inhibitor to prove its effectiveness. In the tyrosinase activity assay, different concentrations of enzyme, tetramethylbenzidine (TMB) dye and Ag$^+$ ions were tested. Based on these measurements, an optimised procedure was developed and validated by measuring the inhibitory effect of kojic acid, a known tyrosinase inhibitor. For the hyaluronidase activity test, different incubation times and concentrations of the carbocyanine dye »Stains-all« and of hyaluronic acid, which acts as a substrate in the reaction, were tested. Validation of the optimised procedure was carried out by measuring the inhibitory effect of apigenin, which inhibits a wide range of hyaluronidases. Using these assays, it is possible to determine the activity of new inhibitors and to compare their efficacy with already known inhibitors of these enzymes. For the MMP-1 activity assay, fluorescently labelled gelatin was used as a substrate, but unexpectedly, no increase in fluorescence was detected. To optimise this assay, we would probably need to consider other substrates and perform additional tests.
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