Figure 1. Effect of cubebin on melanin amount and cell proliferation in B16 cells. The cells were treated with various concentrations of cubebin for 72 h. (a) The melanin amount was determined. (b) The cell viability was determined by the trypan blue exclusion test. Melanin amount and cell proliferation were expressed as percentage values. Each percentage value in the treated cells was calculated with respect to that in the control cells. Data represent means ± SD of two different experiments each carried out in duplicate. ^*Significantly different from the control cells (^*p < 0.05, ^**p < 0.01).

Figure 2. Effect of cubebin on tyrosinase activity in B16 cells. The cells were treated with 10 μM cubebin for the indicated times. (a) Melanin amount was determined. (b) Tyrosinase activity was determined by measuring the formation of dopachrome. Melanin amount and tyrosinase activity were expressed as percentage values. Each percentage value of tyrosinase activity in the treated cells was calculated with respect to that in the pre-treated cells. Data represent means ± SD of two different experiments each carried out in duplicate. ^*Significantly different from that in the pre-treated cells (^*p < 0.05, ^**p < 0.01).

Figure 3. Effects of cubebin on tyrosinase and tyrosinase mRNA expressions in B16 cells. The cells were treated with 10 μM cubebin for the indicated times. (a) The levels of tyrosinase expression were determined by Western blot analysis using the mouse anti-tyrosinase antibody. (b) The levels of tyrosinase mRNA were determined by RT-PCR analysis.

Figure 4. Effect of cubebin on phosphorylation of p38 MAPK in B16 cells. The cells were treated with 10 μM cubebin for the indicated times. Phosphorylation of p38 MAPK, ERK 1/2, and p70 S6K1 was assessed by Western blot analysis with using the mouse anti-tyrosinase antibody for phosphorylated forms of p38 MAPK, ERK 1/2, and p70 S6K1.

Figure 5. Effect of cubebin with SB203580, a selective inhibitor of p38 MAPK, on tyrosinase mRNA expression in B16 cells. The cells were treated with 10 μM cubebin for 12 h with or without 10 μM SB203580. The level of tyrosinase mRNA was determined by RT-PCR analysis.

Figure 6. A proposed scheme showing the activation mechanism of cubebin on melanogenesis.