FUCUS EXTRACT: COSMETIC TREATMENT FOR UNDER-EYE DARK CIRCLES 111 Trolox is a water-soluble form of vitamin E. It is commonly used as a benchmark for an- tioxidant capacity evaluation, especially for water extracts like Fucus extract. As analyzed by the DPPH assay, Fucus extract showed dose-dependent antioxidation ac- tivity at concentrations from 0.5% to 5%. As shown in Figure 6, 1% Fucus extract is equivalent to about 50 μM Trolox in its antioxidation capacity. When compared to a negative control using a paired Student’s t-test, the p values for all concentrations tested are less than 0.001, which is statistically signifi cant. EFFECT OF FUCUS EXTRACT ON ANTI-INFLAMMATION IN KERATINOCYTES (IN VITRO TEST) Interleukin-8 (IL-8) is a chemokine produced by macrophages and other cell types such as epithelial cells. This chemokine is one of the major mediators of the infl ammatory re- sponse. Under normal growth conditions, a very low level of IL-8 cytokine is detected in the medium of normal keratinocyte cultures. But when cells were treated with IL-1β, the IL-8 level secreted to media is elevated. The anti-infl ammation property of Fucus extract was evaluated by IL-8 ELISA under the treatment of IL-1β (Figure 7). Control is the treatment without Fucus extract added. As shown in Figure 7, the IL-8 level induced by IL-1β was reduced by the Fucus extract treatment in a dose-dependent manner at concentrations from 0.5% to 2%. At 1%, Fucus extract reduced IL-8 level to about 40% of control, and at 2%, to about 25% of control. Since IL-8 is often associated with infl ammation, reduction of chemokine IL-8 is a posi- tive indicator of anti-infl ammation effi cacy. EFFECT OF FUCUS EXTRACT ON COLLAGEN I IN FIBROBLAST CELLS (IN VITRO TEST) In this experiment, we evaluate the capacity for Fucus extract to stimulate collagen I synthesis in cultured human fi broblast cells. The assay shown below measures collagen I deposited in the ECM as opposed to secreted collagen. Figure 7. Fucus extract decreases IL-8 content compared to control Mean ± SEM on 3 assays in triplicate - Student’s t test - *: p0.05 - ***: p0.001.

JOURNAL OF COSMETIC SCIENCE 112 As shown in Figure 8, Fucus extract increased collagen I in the ECM in a dose-dependent way. The increases were signifi cant at all concentrations tested (0.01% to 0.5%). At 0.01%, 0.05%, 0.1%, and 0.5%, Fucus extract enhanced collagen synthesis to 1.3-, 1.6-, 2.5-, and 3.7-fold of control, respectively. Vitamin C, the positive control increased col- lagen signifi cantly to 1.8-fold of control. At 0.1% and above, Fucus extract increased collagen more than vitamin C. Vitamin C at 50 μM, a commonly used bench mark in collagen I stimulation assays, was used as a positive control. CONCLUSION This study demonstrated that Fucus extract is a novel product with quadruple in vitro effi cacy. Fucus extract stimulates in vitro the production of HO-1 enzyme. In addition, Fucus extract was found to be an anti-infl ammatory, antioxidative, and collagen boosting ingredient. Ingredients with such properties are likely to aid in alleviating the appearance of dark circles around the eye area. Additional in vivo studies are necessary to confi rm our hypothesis that Fucus extract could aid in periorbital rejuvenation. REFERENCES (1) S. Eberlin, M. Del Carmen Velazquez Pereda, G. de Campos Dieamant, C. Nogueira, R. M. Werka, M. L.de Souza Queiroz, Effect of a Brazilian herbal compound as a cosmetic eyecare for periorbital hy- perchromia (“Dark circles”), J. Cosmet. Dermatol., 8, 127–135 (2009). (2) S. M. Sadrzadeh, E. Graf, S. S. Panter, P. E. Hallaway, J. W. Eaton, Hemoglobin. A biologic fenton re- agent, J. Biol. Chem., 259, 14354–14356 (1984). (3) A. Pamplona, A. Ferreira, J. Balla, V. Jeney, G. Balla, S. Epiphanio, A. Chora, C. D. Rodrigues, I. P. Gre- goire, M. Cunha-Rodrigues, S. Portugal, M. P. Soares, and M. M. Mota, Heme oxygenase-1 and carbon monoxide suppress the pathogenesis of experimental cerebral malaria, Nat. Med., 13, 703–710 (2007). (4) S. W. Ryter A. M. K. Choi, Heme oxygenase-1/Carbon Monoxide: From metabolism to molecular therapy, Am. J. Respir. Cell Mol. Biol., 41, 251–260 (2009). (5) R. Stocker, Y. Yamamoto, A. F. McDonagh, A. N. Glazer, B. N. Ames, Bilirubin is an antioxidant of possible physiological importance, Science, 235, 1043–1046 (1987). (6) D. E. Baranano, M. Rao, C.D. Ferris, S. H. Snyder, Biliverdin reductase: A major physiologic cytopro- tectant, Proc. Natl. Acad. Sci. U.S.A., 99, 16093–16098 (2002). Figure 8. Fucus extract increase the collagen I on fi broblast cells. Mean ± SD on 3 assays in triplicate – Student’s t test - *: p 0.05 - ***: p 0.001.