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.

INHIBITION OF MELANOGENESIS BY P. ACIDULA EXTRACT 47 EFFECT OF PAE ON TYROSINASE PROTEIN AND ITS mRNA LEVELS IN B16 CELLS To study the inhibitory effect of PAE on melanogenesis in more detail, we conducted western blot and qRT-PCR analyses to verify levels of tyrosinase protein and its mRNA in the PAE-treated cells. The results are shown in Figure 4 (western blot) and Figure 5 (qRT-PCR). After stimulation by IBMX, both tyrosinase protein (Figure 4) and its mRNA (Figure 5) were signifi cantly increased. Moreover, the increased levels of both tyrosinase protein and its mRNA in the IBMX-stimulated cells were down-regulated by PAE treatments. These results reasonably explain the previous fi nding wherein both cellular tyrosinase activity (Figure 3) and melanin content (Figure 2) were decreased in the PAE-treated cells due to the inhibitory effect on tyrosinase gene expression by the extract in the IBMX-stimulated mouse melanoma B16 cells. CONCLUSION Our results clearly demonstrate that PAE is an effective melanogenesis inhibitor that functions via down-regulation of tyrosinase expression. These results indicate that PAE may be useful in the treatment of skin hyperpigmentation. ACKNOWLEDGMENTS This research was fi nancially supported by a grant (NSC 99-2622-E-024-001-CC3) from the National Scientifi c Council of Taiwan. Figure 5. Effect of PAE on the expression of the tyrosinase gene. B16 cells were seeded in six-well plates for 1 d and treated with or without 100 μM of IBMX with or without the test drug for 6 h after which total RNA isolation and then reverse transcription was carried out to obtain cDNAs that were used for quantitative real- time PCR. Relative mRNA expression was calculated by the ΔΔCt method, where ΔCt is the value obtained by subtracting the Ct value of GAPDH mRNA from the Ct value of the tyrosinase mRNA and ΔΔCt is the value obtained by subtracting the ΔCt value of a reaction from the ΔCt value of the control with IBMX stim- ulation. Specifi cally, relative mRNA expression is expressed as 2-ΔΔCt. Results represent the mean ± SD of three independent experiments. *Statistically signifi cant (p 0.05) difference between the control and the treated cells.