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.

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