ARECA CATECHU L. EXTRACT 355 HUMAN SKIN IRRITATION TEST We studied the potential of the CC-516 to irritate human skin in 50 healthy female volunteers using a 48-hr closed patch. No skin irritation occurred after application in 50 volunteers. RESULTS AND DISCUSSION We previously screened the inhibitory effect on elastase of methanolic extracts of 150 medicinal plants. The Areca catechu extract showed high inhibitory effects comparable to reference compounds (7). For inhibitory effects of several solvent extracts on elastase the ethanolic extract showed the highest effect, and thus CC-516 was used as the sample in this study. We investigated the effect of CC-516 on the anti-inflammatory and anti-melanogenesis activity by various methods. Table I shows the antioxidative activity of CC-516 and activities of reference compounds such as dl-tx-tocopherol, L-ascorbic acid, and BHT. Figure 1 gives a good dose-response relationship between antioxidative activity. BHT appeared to be the most potent inhibitor, with an IC5o value of 1.5 lag/ml, while other reference compounds, dl-tx-tocopherol and L-ascorbic acid, showed IC5o values of 33.6 lag/ml and 219 lag/ml, respectively. The IC5o value of CC-516 was 45.4 lag/ml. Natural anti-oxidants are usually phenolic or polyphenoic compounds, and these com- pounds include tocopherol, flavonoid, and cinnamic acid derivatives (20). It is known that there are two types of antioxidants (21). The first type of antioxidant inhibits the formation of free radicals that may initiate oxidation. The second type of antioxidant inhibits the free-radical chain propagation reactions. Therefore, some plant extracts may act at the initiation stage of peroxidation, interfering with Fenton's reaction, thus breaking the chain reaction. L-ascorbic acid is one of the most potent scavengers. IC5o values of ascorbic acid, dl-tx-tocopherol, and BHT were found to be 29.7 lag/ml, 33.5 lag/ml and 37.2 lag/ml, respectively. On the other hand, the IC5o value of CC-516 (IC5o: 10.2 lng/ml) showed much lower activity than the well-known reference compounds. Free-radical damage to biosystems is one of the major processes that contribute to degenerative diseases like cancer and aging (22). Detailed free-radical mechanisms and their quantitative contributions are still not clear. Despite these uncertainties, it is clear that free-radical scavengers may inhibit endogeneous, metabolically driven, oxidative DNA damage, as well as mutations and aging caused by exogeneous agents (23-25). Most plants have phenolic compounds such as tannins and flavonoids that may contrib- Table I Inhibition of Lipid Peroxidation by the CC-516 Measured by Fenton's Reagent Linoleate System and Free-Radical Scavenging Activity of CC-516 in a 1.1-diphenyL2-picryl hydrazine (DPPH) Free-Radical Generating System Components IC50 (lag/ml) IC50 (lag/ml) BHT 1.5 37.2 d14x-Tocopherol 33.6 33.5 Ascorbic acid 219.0 29.7 CC-516 45.4 10.2

356 JOURNAL OF COSMETIC SCIENCE lOO 80- 60 40 2O 0 20 40 60 80 100 Concentration(pg/ml) Figure 1. Antioxidative activities of CC-516 in response to CC-516 concentrations. ute to their antioxidative activities. Masaki (26) reported that Paeo,ia suffruticosa has a strong scavenging activity against DPPH radicals, and he isolated galloyl glucose as an active compound. In the anti-hyaluronidase activity assay, CC-516 exhibited greater than 65% and 78% of inhibition at concentrations of 0.5 mg/ml and 1 mg/ml, respectively. As shown in Figure 2, CC-516 with 0.1-1 mg/ml as the final concentration exhibited 10% to 78% inhibition. Anti-hyaluronidase activity of Glycyrrhi•a •rale,sis as a control exhibited 10-84% of inhibition at a concentration of 0.1-1 mg/ml. IC5o values of Glycyrrhixa urale,sis and CC-516 were found to be 330 ug/ml and 416 Fg/ml, respectively. CC-516 contains phenolic compounds such as flavonoids and tannins (27), and thus it could be speculated that the active constituents of CC-516 and Glycyrrhixa urale,sis, which have inhibitory effects on hyaluronidase activity, are phenolic compounds. Tables II and Ill represent the topical anti-inflammatory activity of CC-516. Although the activity was found to be weak compared to the potent activity of the reference compounds, prednisolone and indomethacin, CC-516 showed anti-edematous activity as well as anti-hypersensitivity. The mouse ear edema assay is a frequently used animal model for topical application of the various compounds (28). In croton-oil-induced ear edema, 12-o-tetradecanoylphorbol-13-acetate (TPA) is suggested to produce edema, and leukotriene B 4 i'S found in ear area. In arachidonic-acid-induced ear edema, eicosanoids such as prostaglandins and leukotrienes are involved in the inflammation. CC-516 did show anti-inflammatory activity against both inflammatory mediators, which sug-