INHIBITORY EFFECTS OF N-FERULOYLDOPAMINE 141 mushroom tyrosinase. Comparable results have previously been reported, notably with p-coumaric acid (23). Such a difference between the two models might be explained by the different structural features of human and mushroom tyrosinases. Although both ty- rosinases exhibit high homology in their active sites, human tyrosinase is a monomeric protein while mushroom tyrosinase is mostly tetrameric. Moreover, regulation of mush- room tyrosinase differs signifi cantly in several respects from mammalian tyrosinase, nota- bly with regard to post-translational modifi cations (4). It is also worth noting that qRT-PCR results showed no effect of N-feruloyldopamine on tyrosinase gene expression in normal human melanocytes (data not shown). Figure 4. Total melanin content of murine melanoma B16-F10 cells. Mean ± SD for n = 6. **Statistically signifi cant vs. untreated control, p 0.01. Figure 5. Radical-scavenging activity of N-feruloyldopamine. DPPH assay. Mean ± SD, n = 6. *,**Statisti- cally signifi cant vs. untreated control, p 0.05 and p 0.01, respectively.

JOURNAL OF COSMETIC SCIENCE 142 N-feruloyldopamine was next shown to signifi cantly decrease total melanin content in B-16-F10 murine cells. Over 30% inhibition was observed when N-feruloyldopamine was used at 10 μM. Using linear regression, IC 50 in that model system was evaluated at 27.1 μM (% inhibition = −1.4758 × [N-feruloyldopamine] + 90.03 R² = 0.9045). Taken together, the results of human tyrosinase, mushroom tyrosinase, and melanin synthesis in B16 cells support the idea that tyrosinase inhibition by N-feruloyldopamine may be specifi c to mammalian-type tyrosinases. To investigate other mechanisms that may contribute to a melanogenesis inhibitory effect of N-feruloyldopamine, radical-scavenging properties as well as melanosome maturation gene regulation were evaluated. Indeed, one of the reactions catalyzed by tyrosinase is the oxidation of L -DOPA to dopaquinone using copper and molecular oxygen. Some antioxi- dants can thus interfere with the oxidation reaction. Moreover, other antioxidants such as vitamin C act as hypopigmenting agents by reducing intermediates of melanin polymers (24,25). By stimulating the synthesis of α-MSH in keratinocytes, ultraviolet-induced reactive oxygen species (ROS) also stimulate melanin production (26). Reducing ROS generation can thus help minimize melanin production. N-feruloyldopamine showed sig- nifi cant antioxidant effi ciency with around 70% of radical-scavenging activity for con- centrations equal to or higher than 30 μM. Using qRT-PCR methods, the effect of N-feruloyldopamine on Pmel17, MART-1, and Prot P—the best-described melanosome maturation genes—was evaluated. Despite poor inhibition of MART-1 and Protein P gene expressions, N-feruloyldopamine sig- nifi cantly decreased Pmel17 gene expression when used at 100 μM on cultured NHEM. Under physiological conditions, only mature melanosomes are transferred from mela- nocytes to the surrounding keratinocytes. Hence, factors that regulate melanosome maturation are expected to reduce skin pigmentation (27). In that respect, Pmel17 Figure 6. Effect of N-feruloyldopamine on Pmel17 mRNA level. qRT-PCR. Mean ± SD, n = 7. *Statisti- cally signifi cant vs. untreated control, p 0.05.