j. Cosmet. sci., 49, 351-359 (November/December 1998) Areca catechu L. extract. II. Effects on inflammation and melanogenesis KUN KOOK LEE and JUNG DO CHOI, R&D Center, Coreana Cosmetics Co. Ltd., 204-1 Jeongchon-ri, Cheonan-si, Chungnam 333-830 (K. K. Lee), and Department of Biochemistry, College of Natural Science, Chungbuk National University, Cheong/u, Chungbuk 361-763 •. D. Choi), South Korea. Accepted for publication October 15, 1998. Synopsis Ethanolic extract (CC-516) from Areca catechu L. was prepared, and its various biological activities were evaluated. CC-516 showed potent anti-oxidative, free-radical scavenging, and anti-hyaluronidase activity. The anti-oxidative effect of CC-516 (IC5o: 45.4 t•g/ml) was lower than that of butylated hydroxytoluene (IC5o: 5 t•g/ml), but similar to that of tocopherol and higher than that of ascorbic acid. Especially, CC-516 exhibited relatively high free-radical scavenging activity (IC5o: 10.2 t•g/ml) compared to that of the control. CC-516 effectively inhibited hyaluronidase activity (IC5o: 416 t•g/ml) and showed inhibition in vivo on delayed hypersensitivity as well as croton-oil-induced ear edema in mice when it was topically applied. These results strongly suggest that CC-516 may reduce immunoregulatory/inflammatory skin trouble. Also, from the results, we have elucidated that CC-516 showed anti-allergic and anti-cytotoxicity activity. The whitening effect of CC-516 is shown by the inhibition of mushroom tyrosinase activity with an IC5o value of 0.48 mg/ml and of melanin synthesis in B 16 melanoma cells. This study indicates that CC-516 is effective on anti-inflammatory activity/anti-melanogenesis, and can be used as a new agent for cosmetics. INTRODUCTION Plant sources have been evaluated for developing natural antioxidants and melanogenesis inhibitors that may be involved in anti-aging and skin whitening (1). Many endoge- neous plant compounds have been reported to retard the oxidation process in their natural environment (2). Natural antioxidants occur in all higher plants and in all parts of the plant. Typical compounds that possess antioxidative activities include tocopherols, fiavonoids, cinnamic acid derivatives, phosphates, and polyfunctional organic acids. Recent studies indicate that the compounds with antioxidative and free-radical scav- enging activities can inhibit mutagenesis and carcinogenesis in addition to retardation of aging (1-3). High-molecular-weight hyaluronic acid has an important role in the regulation of scarless repair in fetal wound healing by markedly diminishing the in- fiammatory response (4). However, degradation products of hyaluronic acid lead to increased inflammation, angiogenesis, fibrosis, and collagen deposition in wound heal- 351

352 JOURNAL OF COSMETIC SCIENCE ing. Recently the harmfulness of ultraviolet (UV) radiation is increasing due to destruc- tion of the ozone layer. Excessive exposure to UV radiation causes postinflammatory pigmentation (4-5). Pigmentary disorders are caused by various factors, including in- flammation, the imbalance of hormones, and genetic disorder (6). Melanins play a critical role in the absorption of free radicals and melanogenesis in the skin in a kind of process that produces photoprotective agents against the damaging effect of UV. Many cosmetic and pharmaceutical companies have tried to find an inhibitor of melanogenesis. The regulation of cellular pigmentation can be controlled at many different stages of melanogenesis. Especially, tyrosinase inhibitors and antioxidants can be used for inhi- bition of cellular pigmentation since the melanin-producing process involves enzymatic and nonenzymatic oxidation reactions. Plant extracts having such biological activities may be a good choice for cosmetic purposes. We previously screened the inhibitory effects of 150 medicinal plants on elastase activ- ity, and examined their anti-inflammatory effects. The Areca catechu methanolic extract showed a high inhibitory effect on elastase and an anti-inflammatory effect, compared to reference compounds (7), and we selected the Areca catechu extract as a candidate for new anti-aging and anti-inflammatory agents. Areca catechu L. is widely cultivated, especially in southern Asia, and its seed is used as a chewing material anthelmintic and also as kompo-traditional medicine. Preparations containing Areca are also used as digestive medicines since Areca promotes the secretion of saliva (8-9). Areca catechu L. contains a number of chemical components, such as alkaloids, tannins, flavonoids, and fatty acids (10,11). To clarity the anti-aging mecha- nism of Areca catechu L. extract against aging, we have studied the anti-inflammatory effect in vitro and in vivo. Furthermore, the skin-whitening efficacy of the Areca catechu extract was also examined by inhibition of mushroom tyrosinase and inhibition of melanogenesis on B-16 melanoma cells. The anti-aging effect of Areca catechu L. extract was evaluated by measuring anti-oxidative activity, the free-radical scavenging effect, and inhibition of hyaluronidase in vitro, and anti-inflammatory effect/inhibition of de- layed hypersensitivity in vivo. The safety of CC-516 was evaluated by cytotoxicity on human fibroblasts and skin irritation testing. MATERIAL AND METHODS ANTIOXIDATIVE ACTIVITY A lipid peroxidation system was induced by Fenton's reagent. Each test sample (0.1 ml) and ethyl linoleate (10 pl) were added to an incubation medium (4.89 ml) containing 2% sodium dodecyl sulfate, 1 pM ferrous chloride, and 0.5 pM hydrogen peroxide. The known synthetic antioxidant, butylated hydroxytoluene (BHT) was used as a reference compound. The incubation medium was kept at 55 øC for 16 hr. Each reaction mixture (0.2 ml) was transferred into a test tube, followed by addition of 4% BHT (50 pl) to prevent further oxidation. Antioxidative activity of the sample was measured using a thiobarbituric acid (TBA) assay according to the method of Ohkawa eta/. (12). FREE-RADICAL SCAVENGING ACTIVITY Scavenging effect against free-radical generation was measured following the procedure