ANTI-SKIN-AGING BENEFITS OF EXOPOLYMERS FROM AUREOBASIDIUM PULLULANS 287 α-(1,4)-(1,6)-glucan (8%), glucose (37.7%), galactose (0.8%), mannose (1.5%), protein (3.1%), and ash (7.2%). The standard references ascorbic acid, oleanolic acid, and kojic acid were purchased from Sigma (St. Louis, MO), and a facial treatment mask containing Saccharomycopsis ferment fi ltrate (SFF) (P&G, Japan) was obtained from a local cosmetics shop. DPPH RADICAL SCAVENGING ACTIVITY TEST The assay for free radical scavenging capacity was carried out according to the method reported previously by Blois et al. (22). The DPPH radical shows a deep violet color due to its unpaired electron, and radical scavenging capacity can be followed spectrophotometrically by the loss of absorbance at 525 nm. Briefl y, 0.2 mM DPPH (Sigma, Steinheim, Germany) in a 95% etha- nol solution (1 ml) was added to a sample of the stock (2 ml). Each sample solution was diluted with distilled water to fi nal E-AP-SM2001 concentrations of 12.5, 25, 50, 100, 200, and 400 μg/ml or fi nal ascorbic acid concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg/ml, and the samples were then agitated. The optical density (OD) at 525 nm was measured after 10 min with a UV/V is spectrophotometer (Beckman, Munich, Germany). The free radical scavenging activity of each sample was calculated using equation (1): DPPH radical scavenging activity (%) = 100 − [(ODs/ODc) × 100], where ODs and ODc are, respectively, the absorbances of the experimental sample and the vehicle-treated con- trol at 525 nm. The results are reported in terms of IC50 (the concentration needed to reduce 50% of DPPH). Ascorbic acid, a representative antioxidant, was used as a control. SOD-LIKE ACTIVITY TEST The assay for free radical scavenging capacity was carried out according to the method reported previously by Marklund and Marklund (23). Each sample solution was diluted with distilled water to fi nal E-AP-SM2001concentrations of 12.5, 25, 50, 100, 200, and 400 μg/ml or fi nal ascorbic acid concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg/ml, and the samples (0.2 ml) were then agitated with Tris-HCl buffer [50 mM Tris (hydroxy- methyl)aminomethane (Sigma, St. Louis, MO) and 10 mM EDTA (Sigma), pH 8.5] (3 ml) and 7.2 mM pyrogallol (Merck, Rahway, NJ) (0.2 ml) for 10 min at 25°C. After agita- tion, the reactions were stopped by adding 1N HCl (Daejung, Siheung-si, Korea). Among reacted solutions, the oxidized pyrogallol was detected by measuring the absorbance at 420 nm after 10 min with a UV/V is spectrophotometer (Beckman). The SOD-like activ- ity of each sample was calculated using equation (2): SOD-like activity (%) = 100 − [(ODs/ODc) × 100], where ODs and ODc are, respectively, the absorbances of the experimental sample and the vehicle-treated control at 420 nm. The results are reported in terms of IC50 (the concentration needed to reduce pyrogallol oxidation by 50%). Ascorbic acid, a representative antioxidant, was used as a control. HYALURONIDASE INHIBITION ASSAY The assay was performed according to a method reported previously (24). Hyaluronidase reacts with the substrate hyaluronic acid to release N-acetyl glucosamine. In the presence of an inhibitor, the release of N-acetyl glucosamine, which is monitored by measuring the

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