INHIBITION OF MELANOGENESIS BY P. ACIDULA EXTRACT 45 control in the present study. As shown in Figure 1, the result indicated that 50 μg/ml PAE does not exert signifi cant cytotoxicity to mouse B16 melanoma cells. In order to avoid the cytotoxic effect of PAE, we used a PAE concentration of up to 50 μg/ml in the depig- menting experiments of the present study. EFFECT OF PAE ON MELANOGENESIS AND TYROSINASE ACTIVITY IN B16 CELLS To study the anti-melanogenic effect of PAE in B16 cells, the melanin content of the cells in each treatment was determined. The results are presented in Figure 2. The melanin content of B16 cells increased considerably after stimulation by IBMX. Thereafter, PAE Figure 1. Effect of PAE on B16 cell viability. The cells were seeded in 24-well plates for 1 d and then treated with various dosages of PAE (100–3.125 mg/ml) for 2 d. The cell viability was then examined by the MTT assay, as described in the Experimental section. Figure 2. Effect of PAE on melanogenesis in B16 cells. The cells were cultivated for 1 d and then stimulated with 100 μM of IBMX for 2 d in the absence or presence of PAE. The melanin content of the cells was determined using spectrometry, as described in the Experimental section.

JOURNAL OF COSMETIC SCIENCE 46 treatment resulted in a signifi cant decrease in the melanin content of IBMX-stimulated B16 cells. In addition, the melanogenic inhibition of PAE showed a dose-dependent manner with an IC50 value of 33.5 μg/ml. When the inhibitory potency of PAE and ar- butin were compared, PAE exhibited a stronger activity than that of arbutin. Because tyrosinase plays an important role in melanogenesis, we determined the effect of PAE on tyrosinase activity. After PAE treatments, B16 cells were lysed to obtain cellular tyrosinase. We measured the enzyme activity by using l-dopa as an enzyme substrate. The result is shown in Figure 3. The cellular tyrosinase activity was signifi cantly in- creased after stimulation by IBMX, while PAE treatments would signifi cantly inhibit the stimulated cellular tyrosinase activity. Hence, the result suggested that PAE re- duced melanogenesis of IBMX-stimulated B16 cells via down-regulated cellular tyrosi- nase activity. Figure 3. Effect of PAE on cellular tyrosinase activity of B16 cells. The cells were cultivated for 1 d and stimulated with 100 μM of IBMX in the absence or presence of various dosages of PAE or arbutin for 2 d. Cellular tyrosinase activity in the cells was determined using spectrometry as described in the Experimental section. Figure 4. Effect of PAE on amounts of tyrosinase protein. Cells were inoculated in 24-well plates for 1 d and then stimulated by 100 μM of IBMX with or without the test drug. The cells were harvested and the total protein was analyzed by western blot as described in the Experimental section. The band intensity of tyrosi- nase was normalized by that of β-actin, and the normalized band intensity in the IBMX-stimulated control was recalculated to be 1.