FUCUS EXTRACT: COSMETIC TREATMENT FOR UNDER-EYE DARK CIRCLES 105 HO occurs as three isozymes: an inducible heme oxygenase-1 (HO-1) and constitutively expressed heme oxygenase-2 (HO-2) and heme oxygenase-3 (HO-3) (7). HO-1 can be induced by variety of stimuli, which indicate that HO-1 plays a vital role in maintaining cellular homeostasis in addition to heme degradation (8). THE PROTECTIVE ROLES OF HO-1 HO-1 mediated protection of cells and tissues is supported by several animal models of oxidative injury and acute infl ammation (9,10). In these models, HO-1 elevation confers potent resistance to stress, cell injury, and lipopolysaccharide-induced death. Blocking of HO activity abrogated cytoprotection, resulting in severe tissue damage. In addition, increased HO-1 expression levels have been clinically demonstrated to resolve of a wide variety of infl ammatory conditions (11). HO-1 activity forms a feedback loop by attenu- ating leukocyte adhesion and migration and by promoting the resolution of infl ammatory responses. Overexpression of HO-1 in endothelial cells and fi broblasts protected these cells from TNF-α-mediated apoptosis (12). HO-1 also plays a role in the regulation of angiogenesis, and overexpression of HO-1 has accelerated cutaneous wound healing in mice (13). THE PROTECTIVE EFFECT OF HO-1 IS ALSO THROUGH ITS PRODUCTS: (i) CO inhibits proliferation of vascular smooth muscle cells, platelet aggregation, and protects against apoptosis. In addition, CO inhibits proinfl ammatory genes while aug- menting anti-infl ammatory cytokine production (14,15). (ii) HO-1 induction is accompanied by increased ferritin synthesis. A recent study dem- onstrated that ferritin plays a central role in cellular antioxidant defense and cytoprotec- tion (16). The elevated level of ferritin would result in increased resistance to iron-mediated oxidative stress (17). (iii) It was shown in vitro that at micromolar concentrations, both biliverdin and bilirubin are effi cient free-radical scavengers (5). In liposomes, bilirubin suppressed oxidation even more effectively than α-tocopherol or vitamin E, which is regarded as an excellent anti- oxidant. These results indicate that bilirubin is a very good antioxidant and provides potent protection against oxidative injury and infl ammation (18). HO-1 and its products: CO, ferritin, and bilirubin all working together, play an essential role in antioxidation, anti-infl ammation, and cytoprotection. COLLAGEN AND WRINKLES Collagen, a group of naturally occurring fi brous proteins, is a major component of the extracellular matrix (ECM) that supports tissues. It is also the main component of fascia and skin (19), providing them with strength and elasticity. With aging, the amount of collagen production in the skin decreases, which results in the formation of fi ne lines and wrinkles (20). This is especially evident around the eye area.

JOURNAL OF COSMETIC SCIENCE 106 Collagen is composed of a triple helix of amino acid chains and is produced and assembled by fi broblast cells through a series of steps. By identifying active cosmetic ingredients that can stimulate collagen production, the appearance of fi ne lines and wrinkles can be minimized. MATERIALS AND METHODS REAGENT AND CHEMICALS Human IL-1β and IL-8 ELISA kit were from RnD systems (RnD system, Minneapolis, MN) Mouse anti-HO-1 antibody was from BD Biosciences (San Jose, CA) Mouse anti-β actin was from Sigma (Sigma, St Louis, MO) Goat anti-mouse HRP antibody from Santa Cruz Biotechnology, Inc. (Dallas, TX) SV 96 total RNA isolation kit from Promega (San Luis Obispo, CA) Probes of HO-1 and GUSB from Life Technologies (Grand Island, NY) 2,2-diphenyl-1-picrylhydrazyl (DPPH), Trolox, RIPA buffer, and a cocktail of pro- tease inhibitors are from Sigma. All medium including Dulbecco’s modified Eagle’s me- dium (DMEM), medium 154, and media supplement are from Invitrogen (Life Technologies). BCA assay kit from Pierce (Thermo Fisher Scientifi c Inc. Rockford, IL). CELL CULTURES AND TREATMENT PROTOCOL Normal human dermal fi broblasts (NHDF) were obtained from Invitrogen (Life Tech- nologies) and were subcultured in DMEM medium supplemented with fetal bovine se- rum (FBS 10%), penicillin (100 U/ml) and streptomycin (100 μg/ml), and incubated at 37°C in a humidifi ed incubator with 5% CO2. Normal human epidermal keratinocyte (NHEK) were also obtained from Invitrogen and were subcultured in medium 154 with supplement kit at 37°C with 5% CO2. After confl uence, cells were seeded in 6-well or 96-well plates and incubated for two more days before treatment. Tested products were diluted into respective treatment medium to different concentrations, and then were added to the cells. RNA ISOLATION AND RT-PCR ON HO-1 Cells were grown to confl uence and then were treated with tested product in their respec- tive medium for 24 h at 37°C with 5% CO2. After treatment, cells were rinsed once and total RNA was extracted from the cells using Promega SV 96 total RNA isolation kit. Two hundred nanograms of total RNA was used for reverse transcription with M-MLV and oligo (dT) primers. Real time PCR was performed with probe sets specifi c for human HO-1 (Hs01110250_m1). These reactions were run in duplex, with probe sets for GUSB, a housekeeping gene for data normalization. The cycling conditions for the RT-PCR are as following: 50°C for 2 min, 95°C for 10 min, and then 40 cycles of 95°C for 15 s, 60°C for 60 s. Data were analyzed with ΔCt in Excel.