UC Berkeley

The effect of various naturally occurring and structurally related chemical compounds on mushroom tyrosinase and B16-F10 melanoma cells were examined. For each natural product or related compound, the detailed mechanism of regulatory effect on tyrosinase-catalyzed melanin synthesis was elucidated, and occasionally, the optimal mechanism of cytotoxicity on B16 melanoma cells exhibited by chemical compounds are evaluated. Arbutin (hydroquinone-o-β-D-glucopyranoside), a well-known depigmenting reagent, was oxidized by tyrosinase to the corresponding o-quinone with an extremely slow rate, and this reactive metabolite caused melanocytotoxicity, resulting in the antimelanogenic effect. Another monophenol derivative, thymol (5-methyl-2-isopropylphenol), on the other hand, did not act as either a substrate or an inhibitor, but it acted as a redox inhibitor, due to its prooxidative property, to disrupt the melanin formation. This prooxidant effect also triggered the prooxidative-related toxicity on melanoma cells. In the case of cardols (5-alkylresorcinol), naturally occurring resorcinolic lipid, hydrophilic head, and hydrophobic tail concept was applied to the mechanism of both tyrosinase inhibition and cytotoxicity. More specifically, resorcinol moiety quickly and reversibly bound to binuclear copper of tyrosinase, and then, hydrophobic tail portion slowly and irreversibly interacted with the hydrophobic portions proximate to the active site of the enzyme. However, this inhibitory mechanism was only observed when the hydrophobic alkyl chain was long enough to interact with the hydrophobic portion of the enzyme. In the case of cellular effect, cardol (C15:3) preferentially acted as a surfactant to disrupt the function of cellular membrane while cardol (C5:0) produced prooxidant-related toxicity. Alkyl-3,5-dihydroxybenzoate and 3,5-dihydroxyphenyl alkanoate with various lengths of alkyl chain were examined to clarify the effect of the lengths of alkyl chain on inhibitory and toxic effect. In the cases of both alkyl-3,5-dihydroxybenzoate and 3,5-dihydroxyphenyl alkanoate, basically, both compounds with alkyl chain longer than C9 caused a two-step inactivation on mushroom tyrosinase. In addition, alkyl-3,5-dihydroxybenzoate acted as a surfactant to cause cytotoxicity while 3,5-dihydroxyphenyl alkanoate were decomposed in the growth medium. Alkyl-3,5-dihydroxybenzoate, interestingly and importantly, inhibited melanogenesis without affecting any cell growth, which is due to the combined effect of the two-step tyrosinase inactivation and surfactant activity. Finally, the effect of polyphenolic compounds, resveratrol and luteolin, were subjects to elucidate their effect on tyrosinase and B16 melanoma cells. In both cases, tyrosinase oxidized them to the corresponding o-quinone. However, in the case of resveratrol (trans-3,5,4'-trihydroxystilbene), the corresponding o-quinone irreversibly interacted with tyrosinase, indicating that kcat type (suicide) inhibition was the mechanism of inhibition. Resveratrol did not show any toxicity up to 200 μM, and at 200 μM, melanogenesis was suppressed with the addition of resveratrol. Luteolin (2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-chromenone), on the other hand, the corresponding o-quinone did not inhibit tyrosinase but did act as a redox cycler, which oxidized leukodopachrome to dopachrome when luteolin was coexisted L-DOPA and tyrosinase. In the case of luteolin, luteolin o-quinone was also an active principle for the toxicity. Through the investigation, the biological significance of each molecule was observed. Based on the dynamic perspective and the biological significance, the possible utilization of these chemicals was also discussed.