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.

FUCUS EXTRACT: COSMETIC TREATMENT FOR UNDER-EYE DARK CIRCLES 107 WESTERN BLOT Cells were grown to confl uence and then treated with tested product in their respective medium for 3 days for NHEK cell and for 24 h for NHDF cells at 37°C with 5% CO2. After rinsing once with PBS, cells were processed for Western blotting by lysis in RIPA buffer containing a cocktail of protease inhibitors. The lysate was sonicated for 15 s, cen- trifuged 10 min at 12000g, and the supernatants were quantifi ed for protein content by BCA assay. HO-1 protein levels were then analyzed by electrophoresing 30 μg cellular protein in a 4%–20% acrylamide gradient gel (Bio-Rad, Hercules, CA), transferring to a nitrocellulose membrane and blotting 90 min with mouse anti-human HO-1 antibodies and mouse anti-β-actin antibodies (to normalize protein loading). After washing, the membrane was blotted with HRP-linked anti-mouse antibodies for 60 min. HO-1 and β-actin bands were resolved and quantifi ed by chemiluminescence on a Kodak Image Sta- tion 4000R. Cell treatments, extractions, protein quantifi cations, and Western blots were performed in triplicate. DPPH ASSAY Fucus vesiculosus water extract was diluted to 0.5%, 1%, 2%, and 5% in PBS. Trolox was dissolved in ethanol and diluted to concentrations of 2.5, 5, 10, 20, 30, 40, and 50 μM, which is used to generate a standard curve. DPPH was dissolved in ethanol to make 100 μM solution. In a 96-well plate, 100 μL of sample or standard solutions was added to wells in six replicates, and then 100 μL of DPPH solution was added to triplicate wells, and 100 μL of ethanol without DPPH to the other triplicate wells for background reading. After mixing at room temperature for 20 min, absorbance was read at 510 nm. In order to minimize background interference for colored samples, the background reading is sub- tracted from the fi nal absorbance of these samples. IL-8 ASSAY Cells were pretreated for 4 h with basal medium (Invitrogen, medium 154 with no growth supplement). Then, cells were incubated overnight in treatment media (Invitro- gen, medium 154 with 10 ng/ml of IL-β and tested product added) at 37°C with 5% CO2. The level of IL-8 released into culture medium was measured by ELISA method. In brief, mouse anti-human IL-8 was coated overnight in a 96-well plate. Samples and stan- dard were then added to the plate and incubated for 2 h. After washing, a biotinylated goat anti-human IL-8 was added to the plate and incubated for 2 h. A streptavidin- conjugated horseradish peroxidase and substrate was used to measure the amount of IL-8 content by recording the optical density at 450 nm with correction at 540 nm. COLLAGEN I ASSAY—DELFIA METHOD Collagen I was assayed using a method developed internally (Immunoassay method for in vitro measurements, patent application fi led). After treatment with the products, cells