Effects Of Inhibitors On Tyrosinase And Their Regulation On Melanogenesis

Tyrosinase (EC 1.14.18.1) is a kind of redoxidase containing copper, widely distributed in nature. It is a key enzyme involved in the melanin biosynthesis. Its inhibitors are usuallyused in whitening cosmetic.Firstly, screening the tyrosinase inhibitors and studying their inhibitory mechanism have been conducted. In view of the relationship between structure and activity (SAR) of tyrosinase inhibitors, two series of compounds containing sulfur were designed and synthesized. The inhibitory effects and mechanism of these synthetical compounds had been elucidated. Then, some tyrosinase inhibitors were screened out through studying their inhibitory capacities on prepared cell-free tyrosinase from B16. Furthermore, through UV-Vis spectrum and DPPH method, the inhibitory mechanism of thiols on mushroom tyrosinase and the anti-oxidant activity of compounds were investigated, respectively. In the end, the B16 cell viability, tyrosinase activity, melanin content and mRNA and protein levels of tyrosinase, TRP-1, TRP-2 were assayed in presence of different concentrations of thiosemicarbazones. The contents and results were as follows:The effects of alky-substituted benzaldehydes, hydroxy-substituted benzaldehydes, halide-substituted benzaldehydes, 4-vinylbenzaldehyde, 4-vinylbenzoic acid and alkoxy-substituted benzoic acids on mushroom tyrosinase were studied. They can all inhibit the tyrosinase activity. And it is concluded that the inhibitory ability of benzaldehydes is apparently stronger than that of benzoic acids. According to the inhibitory mechanism of alky-substituted benzaldehydes and alkoxy-substituted benzoic acid, it is proved that the substituted group on 4-position and the certain length of carbon chain are benefit for the inhibitory capacity of inhibitors.The inhibitory effects and mechanism of cysteine, cysteine derivatives and thioureas on mushroom tyrosinase were investigated. Cysteine and its derivatives are all irreversible inhibitors, while thioureas were all reversible inhibitors on mushroom tyrosinase. Moreover, thiourea and N-aminothiourea are noncompetitive inhibitor, and allythiourea and phenylthiourea both are Mixed-I type inhibitor. Then, according to the kinetics of substrate, the inhibitory kinetics constants of phenylthiourea on mushroom tyrosinase were determined.The design and synthesis of two kinds of tyrosinase inhibitors containing sulfur were done. They were cysteine schiff bases and thiosemicarbazones. The inhibitory mechanisms of the synthetical compounds on mushroom tyrosinase were elucidated, too. It is studied that they are all potent reversible inhibitors of tyrosinase. With the changing of substituted group on benzene ring in compounds, they appear different inhibitory type.The inhibitory mechanisms of thiols on mushroom tyrosinase were investigated through UV-Vis spectrum. It is indicated that cysteine and four kinds of cysteine schiff bases can react with the oxidant product of L-DOPA by tyrosinase or NaIO₄ forming colorless compounds in order to reduce the quantity of the oxidant product.The anti-oxidant activity of compounds were assayed though DPPH method. Except for 3,4-dihydroxybenzaldehyde, other benzaldehydes can't scavenge free radical. They are of free radical scavenging activity in presence of high concetration of thioureas. And, different thiosemicarbazones appear different ability of scavenging free radicalArbutin, vitamins (vitamin B₁, C, K₁), benzoic acids, benzaldehydes, thioureas and thiosemicarbazones all can inhibit the activity of cell-free tyrosinase in B16. Among them, allythiourea, phenylthiourea and thiosemicarbazones are all potent this enzyme inhibitors.Kojic acid as positive control, the cell viability, tyrosinase activity, melanin content and the mRNA level of tyrosinase in B16 by thiosemicarbazones were studied. According that, 4-(dimethylamino)-benzaldehyde can more effectively inhibit melaningenesis than others. But, these four compounds can't apparently affect the mRNA levels of tyrosinase, TRP-1, TRP-2. While, 4-(dimethylamino)-benzaldehyde can dramatically reduce the protein levels of tyrosinase, TRP-1, TRP-2.In a word, the synthesis and inhibitory mechanism of new potent tyrosinase inhibitors were done in this study. Furthermore, the inhibition of melaningenesis by these compounds were investigated, which provided a foundation to use for whiten cosmetic.