SCREENING METHOD FOR TYROSINASE INHIBITORS 521 (50 μM), 3-(4-hydroxyphenyl)lactic acid (70 μM), p-methoxycinnamic acid (120 μM), cinnamic acid (200 μM), caffeic acid (250 μM), m-coumaric acid (270 μM), o-coumaric acid (300 μM), 3-(4-hydroxyphenyl)pyruvic acid (700 μM), ferulic acid (750 μM), and 3-phenyllactic acid (1000 μM). The results indicate that the single phenolic hydroxyl group at the para position and the double bond on the side chain are critical for TYR enzyme inhibition. Additional hydroxyl or methoxy groups on the phenyl moiety and the hydroxyl and carbonyl groups on the side chain appear to have negative effects on TYR enzyme inhibition. The results support that p-coumaric acid has an optimal structure for the inhibition of TYR activity. As expected, the IC50 value of p-coumaric acid against TYR activity in HEK293 cell extracts was identical to the value previously determined using HEM extracts (3 μM) and very different from the value against mushroom TYR (300 μM) (11). Because p-coumaric acid, 3-(4-hydroxyphenyl)propionic acid and 3-(4-hydroxyphe- nyl)lactic acid appeared to be strong inhibitors of human TYR, their effects on cel- lular melanogenesis were compared in HEMs. In this experiment, cells were pretreated with a test compound and then treated with L-tyrosine to stimulate melanogenesis (22). As shown in Figure 3A,B, treatment of HEMs with L-tyrosine decreased cell vi- ability by 7.5% and increased intracellular melanin content by 68%. Among the three test compounds, p-coumaric acid inhibited the cellular melanin synthesis most effec- tively without signifi cant cytotoxicity (Figure 3A,B). The other two compounds had no signifi cant effects on cellular melanogenesis. Thus the inhibitory effects of these phenylpropanoids on cellular melanogenesis correlate well with their inhibitory ef- fects on TYR activity in vitro, supporting the usefulness of the TYR inhibitor screen- ing method developed in this study. These results also support a great potential of Figure 3. Effects of p-coumaric acid, 3-(4-hydroxyphenyl)propionic acid and 3-(4-hydroxyphenyl)lactic acid on melanin synthesis in HEMs. HEMs were pretreated with the vehicle or p-coumaric acid (Comp. 1), 3-(4-hydroxyphenyl)propionic acid (Comp. 2), or 3-(4-hydroxyphenyl)lactic acid (Comp. 3) at 100 μM and then stimulated with 1.0 mM L-tyrosine. Cell viability (A) and intracellular melanin content (B) are pre- sented as percent of vehicle control without L-tyrosine treatment (means ± SE, n=4). Data not sharing the same letters are signifi cantly different from each other.

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