ANTI-SKIN-AGING BENEFITS OF EXOPOLYMERS FROM AUREOBASIDIUM PULLULANS 289 mixed with 80 μl of diluted E-AP-SM2001 (12.5, 25, 50, 100, 200, and 400 μg/ml) or oleanolic acid (6.25, 12.5, 25, 50, 100, and 200 μg/ml) as a standard under exactly the same experimental conditions. Then 100 μl of diluted MMP-1 (0.2 U/ml Sigma) was added to each well and the plate was incubated at room temperature for 1–2 h protected from light. Fluorescence was measured at an excitation maximum of 495 nm and an emission maximum of 515 nm. All the dilutions were made with reaction buffer contain- ing 0.5 M Tris-HCl, 1.5 M NaCl, 50 mM CaCl2, and 2 mM sodium azide (pH 7.6). The control used for this experiment was buffer with the substrate and the inhibitors but without MMP-1. The MMP-1 inhibitory activity of each sample was calculated using equation (6): MMP-1 inhibitory activity (%) = 100 − [(ODs/ODc) × 100], where ODs and ODc are, respectively, the absorbances of the experimental sample and the vehicle- treated control at 515 nm. The results are reported in terms of IC50 (the concentration at which the percentage inhibition of MMP-1 activity was 50%). TYROSINASE INHIBITION ASSAY Tyrosinase inhibition was assayed according to the method of Masamoto (28). Briefl y, aliquots (0.05 ml) of E-AP-SM2001 (12.5, 25, 50, 100, 200, and 400 μg/ml) were mixed with 0.5 ml of L -DOPA (Sigma) solution (1.25 mM) and 0.9 ml of sodium acetate buffer solution (0.05 M, pH 6.8), and preincubated at 25°C for 10 min. Then, 0.05 ml of an aqueous solution of mushroom tyrosinase (333 U/mL Sigma) was added to the mixture. This solution was immediately monitored for the formation of dopachrome by measuring the linear increase in OD at 475 nm with a UV/Vis spectrophotometer, and the tyrosinase inhibitory activity of each sample was calculated using equation (7): Tyrosinase inhibi- tory activity (%) = 100 − [(ODs/ODc) × 100], where ODs and ODc are, respectively, the absorbances of the experimental sample and the vehicle-treated control at 475 nm. The results are reported in terms of IC50 (the concentration at which the percentage inhibition of tyrosinase activity was 50%). Kojic acid (1.25, 2.5, 5, 10, 20, and 40 μg/ml) was used as a standard under exactly the same experimental conditions. MELANIN FORMATION TEST IN B16/F10 MELANOMA CELLS B16F10 murine melanoma cells (CRL-6475) were purchased from the American Type Culture Collection (ATCC Manassas, VA). The cells were cultured in Dulbecco’s modi- fi ed Eagle’s medium (DMEM Sigma-Aldrich) containing 2 mM L -glutamine (Sigma), supplemented with 10% fetal bovine serum (Gibco, Irvington, NJ), 100 U/ml penicillin (Sigma) and 100 μg/ml streptomycin (Sigma), in culture fl asks in a CO2 incubator with a humidifi ed atmosphere containing 5% CO2 in air at 37°C. The culture medium was changed every 2 days. The cells were harvested by trypsinization when they were about 70% confl uent, counted with a hemocytometer and seeded at appropriate numbers into wells of cell culture plates for further experiments. Melanin content was measured as described previously (29) with slight modifi cations. The B16F10 melanoma cells were seeded (2 × 105 cells/well) in 3 ml of medium in 6-well culture plates and incubated overnight to allow them to adhere. The cells were exposed to various concentrations of E-AP-SM2001 (50, 100, 200, 400, 800, and 1,600 μg/ml) for 72 h in the presence or absence of 100 nM α-MSH (Sigma). At the end of the treatment, the cells were washed

JOURNAL OF COSMETIC SCIENCE 290 with PBS and lysed with 800 μl of 1 N NaOH (Merck, Darmstadt, Germany) containing 10% DMSO (Sigma) for 1 h at 80°C. The absorbance at 400 nm was measured using a microplate reader. The inhibitory activity of each sample against melanin production was calculated using equation (8): Inhibitory activity (%) = 100 − [(ODs/ODc) × 100], where ODs ODc are, respectively, the absorbances of the experimental sample and α-MSH-treated control at 400 nm. The results are reported in terms of IC50 (the concentration at which the percentage inhibition of melanin production was 50%). Kojic acid (25, 50, 100, 200, 400, and 800 μg/ml) was used as a standard under exactly the same experimental conditions. IN VIVO SKIN MOISTURIZING MEASUREMENT The assay for skin moisturizing measurement was carried out according to the method reported previously by Hou et al (30). In this study, 192 male ICR mice (6-weeks old on receipt SLC, Shizuoka, Japan) were used after 7 days of acclimatization. Animals were housed four per polycarbonate cage in a temperature-controlled (20–25°C) and humid- ity-controlled (40–45%) room. The light:dark cycle was 12 h:12 h and normal rodent pellet diet and water were supplied freely during acclimatization. After acclimatization, mice were divided into four groups for each time point (30 min and 1, 2, 4, 8, and 24 h eight mice per time point) based on body weight. Then, 100 μl of vehicle (distilled water) or E-AP-SM2001 was directly applied to shaved dorsal skin. A gel mask pack (area 2 × 3 cm) containing E-AP-SM2001 or SFF (area 2 × 3 cm) was patched on the shaved dorsal skin. Then, 30 min after application of test materials, all remaining samples were elimi- nated using cotton balls. Subsequently, 0.5, 1, 2, 4, 8, and 24 h after the end of exposure to the test materials, 2 × 3-cm skin samples were removed and skin water content (%) was measured using an automated moisture balance analyzer (MB23 Ohaus, Parsippany, NJ). In addition, percentage change compared to the vehicle control was calculated to help determine the effi cacy of test materials using equation (9): Change compared with vehicle control (%) = {[(Data for test material-treated group − Data for vehicle-treated control)/Data for vehicle-treated control] × 100}. STATISTICAL ANALYSES All in vitro data are expressed as the mean ± S.D. of fi ve independent experiments, and skin water content was calculated as the mean ± S.D. of eight mouse skins at each time point. Multiple comparison tests for different dose groups were conducted. Variance ho- mogeneity was examined using the Levene test (31). If the Levene test indicated no sig- nifi cant deviations from variance homogeneity, the obtained data were analyzed by one-way ANOVA followed by a least-signifi cant differences (LSD) multicomparison test to determine which pairs in the group comparison were signifi cantly different. If the Leven test showed signifi cant deviation from variance homogeneity, a nonparametric comparison test, the Kruskal–Wallis H test, was conducted. When a signifi cant differ- ence was observed in the Kruskal–Wallis H test, the Mann–Whitney U (MW) test was conducted to identify the specifi c pairs in the group comparison that were signifi cantly different. IC50 values for each in vitro assay were calculated by probit methods. Statistical analyses were conducted using SPSS for Windows (release 14.0K SPSS, Chicago, IL) (32).