FUCUS EXTRACT: COSMETIC TREATMENT FOR UNDER-EYE DARK CIRCLES 109 Results were further confi rmed by a dose-dependence study in keratinocyte as shown in Figure 3. At concentrations from 0.5% to 2%, Fucus extract positively stimulated HO-1 gene expres- sion in keratinocyte culture. At 1%, Fucus extract stimulated HO-1 to 2.2 log, which is more than 4-fold of control at 2%, the stimulation went to about 10-fold of control ( p 0.001). The HO-1 stimulation activity of Fucus extract at protein level analyzed by Western blot is presented in Figure 4 for keratinocyte and in Figure 5 for fi broblast cells. Fucus extract at 1% and 2% concentrations induced HO-1 protein in keratinocyte sig- nifi cantly in a dose-dependent manner (Figure 4). Quantitative densitometric analysis of protein band intensities showed that at 1%, Fucus extract induced HO-1 protein about 6-fold of control, at 2%, it induced HO-1 protein about 30-fold of control. The p values of paired Student’s t-test for both concentrations are less than 0.001 when compared to control, which is statistically signifi cant. In fi broblast cells (Figure 5), HO-1 protein was induced by Fucus extract at both 1% and 2% concentrations too. When the analyzed protein band intensities were compared to control for a paired Student’s t-test, the p values for both concentrations are less than 0.01, which is statistically signifi cant. The induced HO-1 protein content in fi broblast cells reached maximum levels when treated with 1% of Fucus extract. These results clearly show that Fucus extract is capable of inducing HO-1 in both keratinocyte and fi broblast cells, suggesting Fucus extract could have a benefi cial Table 1 Fucus extract up-regulated HO-1 mRNA expression in both fi broblast and keratinocyte cells Cell type NHDF NHEK HO-1 log2 fold change relative to control 1.6 5.8 HO-1 fold change relative to control 3 55 Figure 4. Fucus extract increased HO-1 content in keratinocyte by western blot. Mean ± SD on 3 assays – Paired Student’s t test - ***: p 0.001.

JOURNAL OF COSMETIC SCIENCE 110 effect in reducing dark circles around the eyes caused by blood leakage and heme ac- cumulation. EFFECT OF FUCUS EXTRACT ON ANTIOXIDATION BY DPPH ASSAY Antioxidants help to prevent and repair damage to your body’s tissue by slowing or pre- venting the effect of free radicals, and help to protect your skin from the damaging effects of the sun (21). DPPH is a common abbreviation for the organic chemical compound 2,2-diphenyl-1- picrylhydrazyl. It is a dark-colored crystalline powder composed of stable free-radical molecules. It is used in a common assay for determining the antioxidant capacity of active cosmetic ingredients. Figure 5. Fucus extract increased HO-1 content in fi broblast by western blot. Mean ± SD on 3 assays – Paired Student’s t test -**: p 0.01 - ***: p 0.001. Figure 6. Fucus extract: Anti-oxidation effect Mean + SD of triplicate assays - Paired Student’s t test - ***: p 0.001.