ARECA CATECHU L. EXTRACT 353 of Fugita et al. (13). The sample solution (2 ml) was added to 2 ml of 60 pM 1.1- diphenyl-2-picryl hydrazine (DPPH) ethanolic solution and kept at room temperature for 30 min. The absorbance was measured at 520 nm. ANTI-INFLAMMATORY INHIBITION OF HYALURONIDASE Hyaluronidase activity was determined spectrophotometically by measuring the amount of N-acetylglucosamine formed from sodium hyaluronate (14). Fifty microliters of bo- vine hyaluronidase (7,900 units/ml) dissolved in 0.1 M acetate buffer (pH 3.5) was mixed with 100 pl of a designated concentration of CC-516 dissolved in 5% DMSO, and then incubated in a water bath at 37øC for 20 min. The control group was treated with 100 pl of 5% DMSO instead of the CC-516. One hundred microliters of 12.5 mM calcium chloride was added to the reaction mixture, and then the mixture was incubated in a water bath at 37øC for 20 min. This Ca 2+ activated hyaluronidase was treated with 250 pl of sodium hyaluronate (1.2 mg/ml) dissolved in 0.1 M acetate buffer (pH 3.5), and then incubated in a water bath at 37øC for 40 min. One hundred microliters of 0.4 N sodium hydroxide and 100 pl of 0.4 M potassium borate were added to the reaction mixture, and then incubated in a boiling water bath for 3 min. After cooling to room temperature, 3 ml of dimethylaminobenzaldehyde solution (4 g of p-dimethylamino- benzaldehyde dissolved in 350 ml of 100% acetic acid and 50 ml of 10 N hydrochloric acid) was added to the reaction mixture, and then incubated in a water bath at 37øC for 20 min. Optical density at 585 nm of the reaction mixture was measured by using a spectrophotometer. The percentage of inhibition was calculated as: Inhibition (%) = [(ODc-ODs)/OD c] x 100 where ODe is the OD at 585 nm of the control, and ODs is the OD at 585 nm of the sample. ANTI-INFLAMMATORY ACTIVITY For measuring the topical anti-inflammatory activity, the mouse ear edema assay was employed. According to the modified method of Tonnel et al. (15), preparations of the CC-516 were topically applied to the right ears of mice (18-22 g) three times at 3-hr intervals. Thirty minutes after the final treatment of the test compounds, 2.5% croton oil or 2% arachidonic acid dissolved in acetone (25 lal/ear) was applied topically to the ears of the mice, and the ear thickness was measured 5 hr after croton oil treatment or 1 hr after arachidonic acid treatment. Percent inhibition of ear edema was calculated by comparison with the control group having the vehicle and anti-inflammatory only. Inhibitory activity against delayed hypersensitivity was measured according to the method of Tarayre et al. (16). Briefly, 3% picryl chloride (acetone) was applied to the abdomen of mice (18-22 g). One week later, 3% picryl chloride was applied to the ears of the mice, and ear thickness was measured 24 hr after the picryl chloride solution treatment. Preparations of the test compounds were applied to the ears of the mice daily for 7 days starting from day 0. The differences between the ear thickness of the extract- treated group and the control group treated with picryl chloride and vehicle only were regarded as an indication of inhibitory activity.

354 JOURNAL OF COSMETIC SCIENCE INHIBITION OF TYROSINASE Tyrosinase activity is generally determined by spectrophotometry. The procedure fol- lowed that described by Vanny et aL (17). The reaction mixture consisted of 0.05 M phosphate buffer (pH 6.8, 2.3 ml), 1.5 mM L-tyrosine solution (0.4 ml), and 2,000 U/ml mushroom tyrosinase (Sigma), in 0.05 M phosphate buffer (pH 6.8, 0.1 ml). A sample solution (0.2 ml) was added to the reaction mixture and incubated at 37øC for 10 min. The optical density at 475 nm was measured by a spectrophotometer (Beckman). The inhibitory activity of the sample was expressed as the concentration of inhibitor (IC5o) at which it inhibits 50% of the enzyme activity. The percent inhibition of tyrosinase reaction was calculated as follows: Inhibition (%) = [(A-B)/A] x 100 where A is absorbance at 475 nm without a test sample after incubation, and B is absorbance at 475 nm with a test sample after incubation. INHIBITION OF MELANIN SYNTHESIS IN B-16 MELANOMA CELLS We examined the inhibition of melanogenesis in B-16 melanoma cells by CC-516 using the modified method of Maeda and Fukuda (18). B-16 melanoma cells (ATCC CRL 6323) were seeded into 60-mm petri dishes at a density of 5 x 105 cells per dish. After the cells were cultured at 37øC in Dulbecco's Modified Eagle's Medium (DMEM) containing 4.5 g/1 of glucose, 10% fetal bovine serum (FBS), and 1% antibiotic- antimycotic (Gibco, BRL), the medium was replaced with a fresh medium containing various concentrations of chemicals. Then the cells were cultured for 2 days and the medium was replaced with fresh medium, further incubated for a day. Then cells were harvested with a cell scraper, counted with a haemacytometer, and collected by cen- trifugation. Melanin was extracted and measured according to the method of Maeda and Fukuda with some modifications (18). Briefly, cell pellets were resuspended in 1 ml of distilled water, frozen at -20øC, and thawed at 37 øC. This freezing-thawing process was performed three times. Perchloric acid was added to the cell suspensions at a final concentration of 0.5 N. The tubes were set on ice for 10 min and centrifuged at 15,000 g for 5 min. The pellets were extracted with 0.5 N perchloric acid two times, with cold ethanol/ether (3:1) two times, and with ether. The resulting pellets were dried in air, and 1 ml of 1 N NaOH was added to each tube. The tubes were incubated in a boiling water bath for 10 min to dissolve the pellets. Melanin contents were measured by reading the absorbance at 400 nm, expressed as A4oo/106 cells. CYTOTOXICITY TEST OF CC-516 Human fibroblasts were seeded in a 96-well plate at a density of 104 cells/well, supple- mented with 0.2 ml of Eagle's minimal essential medium (EMEM) containing 2% FBS, and incubated for 24 hours. After sample addition, the cells were incubated for another 24 hr, and the survival and proliferation of cells were evaluated by MTT assay (19). One-tenth milliliter of MTT solution was added to each well and incubated for 3 hr. After removing the media, 0.5 ml of DMSO was added, and formed formazan was measured by absorbance at 570 nm using an ELISA reader.