JOURNAL OF COSMETIC SCIENCE 108 were disrupted with dedicated lysis solution. This solution allows the disruption of cell membranes without solubilizing the deposited matrix. The deposited collagen I was de- tected by primary antibody anti-collagen I (Interchim, Montlucon, France) and with the secondary antibody anti-IgG coupled to a DELFIA® Europium (Perkin Elmer, Courta- boeuf, France) probe. Europium-related fl uorescence (λexc, 340 and λem, 615 nm), propor- tional to the quantity of deposited collagen was measured. STATISTICAL ANALYSIS The statistical analysis (two-tailed) for in vitro studies were performed using Student’s t-test and analysis of variance on the multiple comparisons with a threshold of signifi - cance set to 5% ( p 0.05). RESULTS AND DISCUSSION EFFECT OF FUCUS EXTRACT ON HO-1 STIMULATION IN VITRO Skin responds to environmental assault and endogenous factors by changing the vascular integrity, leading to the blood leakage and heme accumulation in the surrounding tissue space and causing dark circles. In this study, experiments were performed to evaluate the effect of Fucus extract in the HO-1 stimulation activity in cultured cells. The HO-1 stimulation effi cacy of Fucus extract is evidenced fi rst at the gene level by RT- PCR (Figure 3). For initial screening, both fi broblasts and keratinocytes were treated with 5% of the Fucus extract in their respective medium for 24 h at 37°C with 5% CO2. Total RNA was isolated, cDNA was synthesized and used as template, and a probe set specifi c to HO-1 gene was used for this reaction. The HO-1 mRNA expression profi le was compared to non-treated control, and the results from both cell types are described in Table I. Figure 3. Fucus extract stimulates heme oxygenase-1 gene expression in keratinocytes- (Log base is 2, 1log = 2 fold) Mean ± SD on 3 assays – Paired Student’s t- test - **: p 0.01- ***: p 0.001 compared to control.

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.