Melanin is essential for skin protection against UV-light, but the over-production of melanin causes serious skin disorders such as freckles, discoloration, and pigmented age spots.1) Melanogenesis is carried out in the melanocytes, which are mainly located in the basal layer of the epidermis.2)The degree of melanization is a major factor for skin color in animal bodies. Melanin biosynthesis within the melanocytes is controlled by several enzymes. In the melanin biosynthesis pathway, tyrosinase catalyzes the first rate-determining step, and is considered to be the key enzyme in melanin production.3,4) Therefore, searching for potent tyrosinase inhibitors is valuable in the development of skin whitening agents.Cinnamic acid is one of the major components of Cinnamomum cassia BLUME.5) This compound is reported to have various biological activities such as anti-oxidant, antiinflammatory, and anti-cancer properties.6-9) Moreover, Lee 10) and Masamoto et al. recently reported the inhibitory effect of cinnamic acid on tyrosinase activity. 11) However, its effects on melanocytes and animal skin were not yet reported.This study examined the depigmenting ability of cinnamic acid using melan-a cells and brown guinea pigs; and its effects on the expression of melanin biosynthesis-related enzymes were examined. Furthermore, cinnamic acid's UV-protecting effect and cell extracted-tyrosinase inhibitory effect were investigated.
MATERIALS AND METHODS
MaterialsMushroom tyrosinase, L-dopa, kojic acid, and cinnamic acid were purchased from Sigma-Aldrich Co. (U.S.A). FBS, RPMI, and PS were purchased from Gibco BRL (U.S.A).Tyrosinase Extraction The melan-a cells were disrupted by resuspension in a tyrosinase buffer (80 mM PO 4 bufferϩ1% Triton-X 100ϩ100 mg/ml of PMSF), which was followed by sonication in an ice bath. After centrifugation at 12500 rpm for 15 min, the supernatant was used for the enzyme assay; 150 mg of the proteins were required for each reaction.
12)Measuring Tyrosinase Activity Tyrosinase activity was measured by its dopa oxidase activity, using a slightly modified version of the method reported by Shono and Toda.
13)Each concentration (1 mM, 500 mM, 100 mM, 10 mM) of the test substance was dissolved in MeOH. Next, 120 ml of Ldopa (5 mM, dissolved in a 67 mM phosphate buffer, pH 6.8), and 40 ml of either the same buffer or the test sample, were added to a 96-well microplate; then 40 ml of tyrosinase was added. The amount of dopachrome in the reaction mixture was measured after incubation at 37°C for 30 min. Based on the optical density at 490 nm (OD 490), the inhibitory activity of the sample was expressed as the concentration that inhibited 50% of the enzyme activity (IC 50 ). Kojic acid was used as the reference.Cell Line and Culture Procedures The melan-a cells were cultured in RPMI 1640 medium under 10% FBS and 200 nm phobol 12-myristate 13-acetate (TPA) conditions. To a 100p tissue culture dish, 10 ml of medium was added and then seeded with 5ϫ10 5 cells. If the cells were confluently grown after 3 to 4 d at ...
