ARECA CATECHU L. EXTRACT 357 lOO 80 6o 4o 20 o o 200 400 600 800 lOOO Concentration ( pg/m I) Figure 2. Dose-dependent inhibition of hyaluronidase activities. The effects of CC-516 (I) and Gyhyrrhiza uralensis (O) on enzyme activities are indicated as % inhibition compared to the control. Significance of the data is P 0.01. IC5o values are 416 pg/ml (I) and 330 pg/ml (O), respectively. gests that CC-516 might reduce the inflammation in the skin induced by various inducers of inflammation. Because CC-516 also showed inhibitory activity against de- layed hypersensitivity, CC-516 may be a useful agent to treat various skin problems. Previously, several groups, including our groups, demonstrated that flavonoids showed anti-inflammatory activity and anti-allergic activity (29). These results indicate that CC-516 may be, at least in a part, the compound contributing the antioxidative activity in vitro and the topical anti-inflammatory activity in vivo. Table II Mouse Ear Edema Inhibition of CC-516 Dose Thickness increased [mm] Thickness increased [mm] Groups (mg/ear) (% inhibition) • (% inhibition) 2 Control -- 0.25 _+ 0.03 0.16 _+ 0.03 Prednisolone 0.5 0.16 _+ 0.02 (36.0) NT Indomethacin 0.5 NT 0.08 _+ 0.02 (50.0) 5% CC-516 3 x 50 0.19 ñ 0.02 (24.0) 0.14 _+ 0.01 (12.5) 1 Croton-oil-induced ear edema. 2 Arachidonic-acid-induced ear edema. NT: not tested. P 0.05, significantly different from control (n = 5).

358 JOURNAL OF COSMETIC SCIENCE Table III Inhibition of Delayed Hypersensitivity by CC-516 Dose Groups (mg/ear) Thickness increased [mm] (% inhibition) Control -- Prednisolone 7 x 0.1 5% CC-516 7 x 50 0.22 ñ 0.04 0.05 ñ 0.04 (77.3) 0.15 _+ 0.03 (31.8) P 0.001, significantly different from control (n = 10). Table IV IC5o values of Tyrosinase Inhibitors for Mushroom Tyrosinase Materials IC5o (mg/ml) Arbutin 6.52 Kojic acid 0.37 Licorice extracts 2.87 CC-516 0.48 Table V The Effect of CC-516 on Melanin Contents of B-16 Melanoma Cells (n = 3) CC-516 Melanin content Inhibition of (mg/ml) (pg/1 x 10 6 cell) melanogenesis (%) Control 0.051 ñ 0.002 0.00 ñ 4.0 0.5 0.026 ñ 0.005 49.1 ñ 9.8 1.0 0.015 ñ 0.003 70.6 ñ 6.1 2.0 0.007 ñ 0.003 86.3 ñ 7.1 Melanogenesis is a series of oxidative polymerization reactions starting from tyrosine and activated by oxidative stress caused by UV. Tyrosinase plays an important role in melanogenesis. Dopaquinone, an intermediate of melanogenesis, is unstable and con- verted to dopachrome by tyrosinase or autoxidation, and melanin can be formed through the subsequent polymerization reaction. Therefore, inhibition of melanogenesis can be achieved by antioxidation and inhibition of tyrosinase. In fact, kojic acid (29) and arbutin (18), known as tyrosinase inhibitors, have been used in cosmetics for skin whitening. In this study, the effect of CC-516 on melanogenesis was examined using an in vitro enzyme assay and cell culture method. At first, inhibition of tyrosinase-catalyzed do- pachrome formation was examined (Table IV). CC-516 (IC5o: 0.48 mg/ml) showed stronger inhibition than arbutin (IC5o: 6.52 mg/ml) but was weaker than kojic acid (IC5o: 0.37 mg/ml). We also examined melanogenesis in B-16 melanoma cells. The results with various concentrations of CC-516 are summarized on Table V. Melanin contents are expressed as A4oo/106 cells. CC-516 also showed a strong effect on cultured melanoma cells. The IC5o value of CC-516 is about 0.5 mg/ml. From this result, it is deduced that CC-516 may inhibit cellular pigmentation more effectively than arbutin by its antioxidative activity against oxidation of the unstable intermediates of melanin as well as by direct inhibition of tyrosinase. Its inhibitory activity against cellular