JOURNAL OF COSMETIC SCIENCE 286 INTRODUCTION Skin aging is characterized by clinical signs including wrinkles, irregular dryness, dyspigmen- tation, sallowness, deep furrows or severe atrophy, dehydration, telangiectases, premalignant lesions, laxity and a leathery appearance of skin (1). Skin aging is a complex biological process involving intrinsic factors (genetic factors, hormonal status, and metabolic reactions, such as oxidative stress) and extrinsic factors (chronic light exposure, pollution, ionizing radiation, chemicals, and toxins). A combination of these factors causes physiological alterations and progressive changes in each skin layer, and concomitant changes in skin appearance (2). Oxidative stress caused by reactive oxygen species (ROS) plays a pivotal role in the process of skin aging at the cellular level (3,4). ROS can block the formation of collagen, disrupt cellular renewal cycles, damage DNA, and stimulate the release of proinfl ammatory mediators (cyto- kines), which cause infl ammatory skin diseases (5–8). Additionally, ROS causes the depletion of antioxidant enzymes and destroy the cytoprotective defense mechanism by weakening an- tioxidant systems, thus rendering the skin susceptible to oxidative injury (9–11). To prevent and reduce skin aging, many people have used functional cosmetics that have a potent skin protective pharmacological effect (antiaging, whitening, antiwrinkle, mois- turizing, and skin protective effects) (12). Currently, there are various available ingredi- ents for functional cosmetics in the market. However, they have a number of limitations they are too expensive and have side effects, and their exact pharmacological mechanisms are not fully understood (13). Because of these factors, various investigations have con- tinuously attempted to search for affordable and effective functional ingredients, with fewer side effects, especially from natural sources (12,14). Purifi ed exopolymers from Aureobasidium pullulans SM2001 (E-AP-SM2001) comprise mostly β-1,3/1,6-glucan and other organic materials [amino acids, mono- or di-unsaturated fatty acids (linoleic and linolenic acids) and fi brous polysaccharides] (15). Recently, our team demonstrated that E-AP-SM2001.shows antiosteoporotic (16), anti-infl ammatory (17,18), and immunomodulatory effects (19). This fi nding prompted us to examine the protective effects of E-AP-SM2001 against skin aging in vitro and in vivo. The murine B16F10 cell line was used in this study, as it can produce melanin in response to α-melanocyte stimulating hormone (α-MSH) activation (20,21). The antioxidant effects of E-AP-SM2001 were determined by DPPH assay and by measuring superoxide dismutase (SOD)-like activity. Antiwrinkle effects were evaluated through the inhi- bition of hyaluronidase, elastase, collagenase, and matrix metalloproteinase (MMP)-1, because there is much evidence of close correlation between wrinkle formation and the loss of elasticity, collagenase, and MMP-1. Whitening effects were measured by tyrosinase inhibition assay and by measuring melanin formation in B16/F10 melanoma cells. To assess the skin moisturizing effects of E-AP-SM2001, skin water content was measured in Imprinting Control Region mice. METHODS AND MATERIALS CHEMICALS The solution and a viscous mask pack containing E-AP-SM2001 were supplied by Ari-Med Therapeutics (Daegu, Korea). Based on a previously reported analysis (15), the exopolymers of E-AP-SM2001 are known to consist of β-1,3/1,6-glucan (17%), β-1,4-glucan (18%),

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