JOURNAL OF COSMETIC SCIENCE 288 absorbance at 600 nm, is reduced. The inhibitory activity of E-AP-SM2001 (12.5, 25, 50, 100, 200, and 400 μg/ml) was compared with that of oleanolic acid (6.25, 12.5, 25, 50, 100, and 200 μg/ml) as a standard under exactly the same experimental conditions. The 600 nm value of intact undigested hyaluronic acid was set at 100%. The OD at 600 nm was measured after 15 min with a 96-well microplate reader (Tecan Sunrise, Männedorf, Switzerland) and the hyaluronidase inhibitory activity of each sample was calculated using equation (3): Hy- aluronidase inhibitory activity (%) = 100 − {[(ODs + ODc)/ODc] × 100}, where ODs and ODc are, respectively, the absorbances of the experimental sample and the vehicle-treated control at 600 nm. The results are reported in terms of IC50 (the concentration at which the percentage inhibition of hyaluronidase activity was 50%). ELASTASE INHIBITION ASSAY The elastase inhibition assay was performed by measuring the release of p-nitroaniline due to proteolysis of N-succinyl-(Ala)3-p-nitroanilide by human leucocyte elastase (Sigma) (25) in the presence or absence of 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. The absorbance was measured at 410 nm with a 96-well microplate reader and the elastase inhibitory activity of each sample was calcu- lated using equation (4): Elastase inhibitory activity (%) = 100 − [(ODs/ODc) × 100], where ODs and ODc are, respectively, the absorbances of the experimental sample and the vehicle-treated control at 410 nm. The results are reported in terms of IC50 (the concen- tration at which the percentage inhibition of elastase activity was 50%). COLLAGENASE INHIBITION ASSAY The collagenase inhibition assay was performed according to the method reported previ- ously by Niemann (26). Accordingly, 0.15 ml of collagenase (1 mg/ml Sigma) was added to mixed solutions consisting of 0.25 ml of 2 mM 4-phenylazobenzyloxycarbonyl- pro-leu-gly-pro-d-ar (Sigma) and 0.1 ml of E-AP-SM2001 (12.5, 25, 50, 100, 200, and 400 μg/ml) in 0.1 M Tris-HCl buffer (pH 7.5) and then reacted for 20 min at 37°C. After that, the reactions were stopped by adding 0.5 ml of 6% citric acid (Daejung). The ab- sorbance was measured at 320 nm with a UV/Vis spectrophotometer after addition of 1.5 ml of ethyl acetate (Sigma) and the collagenase inhibitory activity of each sample was calculated using equation (5): Collagenase inhibitory activity (%) = 100 − [(ODs/ODc) × 100], where ODs and ODc are, respectively, the absorbances of the experimental sample and the vehicle-treated control at 320 nm. The results are reported in terms of IC50 (the concentration at which the percentage inhibition of collagenase activity was 50%). Olea- nolic acid (6.25, 12.5, 25, 50, 100, and 200 μg/ml) was used as a standard under exactly the same experimental conditions. MMP-1 INHIBITION ASSAY The assay was performed using a fl uorescence microplate according to a previous report (27) with slight modifi cations. Briefl y, 20 μl of type I collagen (substrate Sigma) was

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