INHIBITORY EFFECTS OF GERANIC ACID 355 Geranic acid suppressed only 8.3% of viable cells, but this compound reduced melanin production by 43.9% at 500 μM. By way of contrast, geraniol and citronellol evidenced high cell toxicity at above 50 μM, and citronellic acid did not inhibit melanin production at any concentration. INHIBITORY EFFECTS ON TYROSINASE ACTIVITY For many years, mushroom tyrosinase has been studied for its use in cosmetics as well as in food industries (25). The inhibitory effects of geranic acid derivatives against the dopa- oxidase activity of tyrosinase are shown in Figure 2. According to our results, geranic acid signifi cantly inhibited tyrosinase activity with a 77% inhibition at 500 μM. Citronellic acid and geraniol also evidenced tyrosinase-inhibitory activity in a dose-dependent man- ner, but to a lesser degree than was noted with geranic acid. The IC50 of geranic acid, citronellic acid, and geraniol were measured as 195.4, 645.9, and 950.4 μM, respectively. CHANGES OF INTRACELLULAR LEVEL OF TYROSINASE-RELATED PROTEINS Based on the results of our depigmenting activity measurements, the regulatory effects of geranic acid on the expression of melanin generation-related protein in Melan-a cells were assessed via Western immunoblotting analysis. Both tyrosinase and dopachrome tau- tomerase (TRP-2) are melanin generation-related enzymes, and perform a central role in the melanogenesis pathway. Tyrosinase catalyzes the hydroxylation of L -tyrosine to L - Dopa and the oxidation of L -Dopa to o-dopaquinone in the fi rst two melanogenetic steps. The yield of o-dopaquinone is spontaneously converted to dopachrome, which is pro- cessed either into 5,6-dihydroxyindole (DHI) or 5,6-dihydroxyindole-2-carboxylic acid Figure 2. The inhibitory effects of geranic acid derivatives against the dopa-oxidase activity of tyrosinase. Kojic acid was utilized as a positive control. Each value is expressed as the mean ± S.E. of three experiments. The inhibitory effects of 500 μM citronellol could not be determined because of its absorbance at 490 nm.

JOURNAL OF COSMETIC SCIENCE 356 (DHICA). TRP-2 catalyzes the tautomerization of dopachrome to DHICA, and the fu- ture oxidation of DHICA by DHICA oxidase (TRP-1) gives rise to the DHICA-eumelanin (26,27). As is shown in Figure 3, treatment with 500 μM of geranic acid for 3 days resulted in a signifi cant reduction in the generation of intracellular tyrosinase. However, the level of intracellular TRP-2 was not altered by geranic acid treatment. Therefore, geranic acid acts specifi cally to reduce the expression of tyrosinase. DISCUSSION Based on previous reports of the tyrosinase-inhibitory effects of geranic acid (11), the inhibitory activity of geranic acid and three geranic acid derivatives on the biosyn- thesis of melanin pigment in Melan-a cells was evaluated. According to our results, geranic acid inhibited relative melanin contents by 25.6% as compared to the cell viability level at 500 μM. Additionally, geranic acid evidenced inhibitory effects on tyrosinase activity and intracellular expression. However, the inhibitory activities of geranic acid derivatives on melanin formation were found to be weaker than that of geranic acid. Geraniol, an alcohol analog of geranic acid, evidenced more profound cell toxicity than was observed with geranic acid. Citronellol and citronellic acid evidenced no signifi cant melanin synthesis-inhibitory activity at any tested concentration. Geranic acid is a colorless to pale yellow clear oily liquid, with a typical fl avor. It is fre- quently used as a perfuming agent in the cosmetics industry. Although the tyrosinase- inhibitory activity of geranic acid has been recently reported, its depigmenting properties in melanocytes were fi rst reported, to the best of our knowledge, in this study. Overall, these results indicated that geranic acid may prove useful not only as a perfuming agent but also as a skin depigmentation agent. Although Melan-a cells are commonly used in such studies, experiments using normal human melanocytes would be an appropriate next step. Figure 3. Effects of geranic acid on tyrosinase and dopachrome tautomerase expression in Melan-a cells. The Melan-a cells were treated with geranic acid for 3 days. TRP-2 (tyrosinase-related protein-2) dopachrome tautomerase.