The Interaction Of Compound Inhibitors On Tyrosinase

Modern research of traditional Chinese medicine has proved that either compound or single herb medicine is basically based on a variety of chemical constituents in different biological macromolecules target in different ways, and finally realizes the overall regulation effectiveness people tend to focus on specific pharmacological study, ignoring the interaction of a plurality of components on biological macromolecules. By studying interactions of complex inhibitor on tyrosinase and discussing the interaction mechanism, this paper can also provide a theoretical basis for the compound inhibitors in terms of whitening effect.Tyrosinase(EC.1.14.18.1) is a key enzyme in the human body, animals and plants. The metabolite of tyrosinase, melanin, not only determines skin color degree, but also is the key enzyme in the browning of fruits and vegetables and in molting process of insects. The current researches on the mechanism of inhibition of tyrosinase activity are mainly focused on inhibitory effect of single inhibitor, while research and application of compound tyrosinase inhibitors are less. In order to achieve the best tyrosinase inhibitory effect, researchers began to try to use a variety of compound tyrosinase inhibitors. But the interaction between the various inhibitors is not necessarily the desired synergistic effect. It may also be a simple additive effect or even antagonistic effect. So research of mechanism and application of composite tyrosinase inhibitors is of great significance in cosmetics industry, pharmaceutical industry, food preservation and pest control, and so on. Azelaic acid and 4-hexyl resorcinol are currently the common tyrosinase inhibitors in cosmetics. Cinnamic acid is antimicrobial, and is a potential cosmetic additive. This paper takes the azelaic acid, cinnamic acid and 4- hexylresorcinol these three inhibitors as representatives to study the inhibition of composite inhibitors on tyrosinase. Azelaic acid, also known as azaleas acid, naturally present in azaleas, Chinese angelica, Codonopsis, yellow daisy, hibiscus bark and Birch. Cinnamic acid, also known as β- cinnamic acid, 3-phenyl-2-acrylic acid, is isolated from cinnamon bark or benzoic acid. Azelaic acid and 4-hexyl resorcinol are commonly used in cosmetic whitening tyrosinase inhibitors added ingredients, and cinnamic acid has antiseptic antibacterial effect and whitening effect. Research and results of single and mutual action of these three ingredients on tyrosinase are as follows:The effects on the activity of tyrosinase when using azelaic acid, cinnamic acid and 4-hexylresorcinol these three inhibitors as single and combined inhibitors were studied. Graphic analysis of Lineweaver-Burk showed that azelaic acid and 4- hexylresorcinol were competitive inhibitors, and that cinnamic acid was the non-competitive inhibitor. Lineweaver-Burk line of the compound of two competitive inhibitors(azelaic acid and 4-hexylresorcinol) was similar with single inhibitor’s, and they all intersected at the same point on the longitudinal axis. The lines of non-competitive inhibitors compounds intersected at different coordinate points in the second quadrant. Concentration of half inhibition IC50 to tyrosinase of azelaic acid, 4-hexyl resorcinol and cinnamic acid, alone and compound, were measured. Results displayed a dose dependence of three inhibitors to tyrosinase, concentration of half inhibition IC50 of azelaic acid, cinnamic acid and 4-hexyl resorcinol were 8.61mmol/L, 0.953mmol/L, 1.70μmol/L, respectively. 4-hexylresorcinol had the strongest inhibition, cinnamic acid was the second and azelaic acid was the weakest. Utilized the equivalent linear method and the median effect principle of complex function effect to judge the effect of two different inhibitors compound. The results showed that compound of azelaic acid, cinnamic acid and 4-hexylresorcinol can cause mutual synergy inhibition effect.The breed of B16 melanoma cells of mice was used to screen inhibitors and study cell toxicity. The experiment studied the function of single and compound inhibitors on the proliferation of B16 melanoma cells. By using the CDI and Q value method to assess the results, we found that by using different method, the interaction effect may be different.Using grayscale as an indicator, effect of inhibition of azelaic acid, cinnamic acid, 4-hexyl resorcinol in yam slice browning were studied. The results showed that, these three kinds of tyrosinase inhibitors had a certain inhibition in yam slice browning. Inhibition effect of compound inhibitors increased than single inhibitor, but there was no significant synergistic effect.The structure changes of enzyme before and after binding with inhibitors were analyzed by using fluorescence spectrometry. Results showed that the Stern-Volmer lines of azelaic acid and cinnamic acid had good liner relationship. Fluorescence quenching of cinnamic acid to tyrosinase was static quenching effect, and that of azelaic acid was dynamic quenching effect. Binding capacity of cinnamic acid with tyrosinase was stronger than that of azelaic acid, and this may be one reason that activity inhibition of cinnamic acid was stronger than azelaic acid. With azelaic acid, fluorescence quenching of cinnamic acid had a more significant change, and the binding constant and binding sites significantly increased, displaying a promotion of binding of cinnamic acid and tyrosinase by azelaic acid.