JOURNAL OF COSMETIC SCIENCE 276 mobile phase was used under binary linear gradient conditions as follows: 0–15 min, 10–30% B 15–30 min, 30–60% B 30–35 min, 60–100% B 35–40 min, 100% B 40–41 min, 100–10% B 41–55 min 10% B. The fl ow rate was 1.0 ml/min and the injection volume was 10 μl. SSE (10 mg/ml), f SSE 10 mg/ml), and p- coumaric acid (1,000 μg/ml) were dissolved in methanol using an appropriate dilution. Peaks were identifi ed by com- paring their retention time and UV-vis spectra with p-coumaric acid and were quanti- tated using the corresponding p-coumaric acid curve. STATISTICAL ANALYSIS Data were expressed as means ± SEM. Statistical analyses were performed using the Sigma Plot 12.0 software program. One-way analysis of variance was used to identify signifi - cance ( p 0.05). When signifi cant, specifi c differences between groups were identifi ed using Tukey’s multiple-comparison tests. RESULTS DETERMINATION OF OPTIMAL ETOH EXTRACTION OF SSE AND ITS CYTOTOXICITY To fi nd the optimal EtOH extraction of S. bicolor L. stalks containing high MMP-1 sup- pressing potential, each EtOH fraction of SSE (50 μg/ml) was used to treat NIH-3T3 cells for 24 h. The expression levels of MMP-1 were then determined by immunoblotting. As shown in Figure 1, a 50% EtOH extraction of S. bicolor L. stalk showed the highest suppression of collagen-degrading enzyme MMP-1 expression. In a subsequent experi- ment, SSE lyophilized from a 50% EtOH extraction of S. bicolor L. stalk was used. When the cytotoxicities of SSE and f SSE were assessed, there was no signifi cant cytotoxicity in HDF-N cells treated with up to 100 μg/ml for 24 h (Figure 2). SSE and f SSE resulted in cell cytotoxicity above 200 μg/ml, but the cytotoxicity of f SSE was less than that of its original extract, SSE. These fi ndings suggest that both SSE and f SSE could be produced as effective active ingredients with no associated cytotoxicity when used at concentrations up to 100 μg/ml. MEASUREMENT OF SSE AND FSSE ANTIOXIDANT ACTIVITY Oxidative-stress promotes MMP activity and melanin synthesis, which are causal factors in the induction of skin aging (15). Here, the antioxidant activities of SSE and f SSE were determined using an ORAC assay. As shown in Table I, both SSE and f SSE showed re- markable antioxidant scavenging activity. The antioxidant capacity of SSE at 50 μg/mL was similar to that of Trolox, and f SSE showed higher antioxidant scavenging activity even in lower concentrations compared with SSE. TYROSINASE INHIBITORY EFFECT OF SSE AND FSSE The antioxidant scavenging activities of SSE and f SSE were demonstrated by the ORAC assay (Table I). Skin pigmentation is caused by melanin production mediated by tyrosinase,

THE ANTI-WRINKLE AND ANTI-MELANOGENIC EFFECTS 277 however, its inhibition is one scheme used to inhibit the pigmentation process (12). Thus, the level of anti-tyrosinase activity, another determinant in skin whitening, was determined using purifi ed mushroom tyrosinase. Arbutin (100 μg/ml) was used as a pos- itive control for tyrosinase inhibition. The antityrosinase activity of SSE was evident at a dose of 50 μg/ml, but f SSE showed inhibitory effects at lower concentrations than SSE (Figure 3). This result suggests that the fermentation process enriched the content of ac- tive compounds involved in tyrosinase inhibition. Figure 2 . Cell cytotoxicities of SSE and f SSE. HDF-N cells were treated with different doses of SSE and f SSE for 24 hr. Then cell cytotoxicity was determined by MTT reduction assay. Data are the mean ± SEM (n = 3). * and ** indicate p 0.05 and p 0.01 versus untreated control. SSE 50% ethanol extract of S. bicolor L. stalk, f SSE A. oryzae NK- fermented form of SSE, MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Table I Antioxidant Effects of SSE and f SSE μg/ml SSE f SSE 3.125 0.32 ± 0.02 0.35 ± 0.02 6.25 0.37 ± 0.01 0.55 ± 0.02 12.5 0.66 ± 0.07 0.79 ± 0.02 25 0.85 ± 003 0.98 ± 0.01 50 1.02 ± 0.03 1.09 ± 0.00 100 1.11 ± 0.01 1.12 ± 0.00 200 1.33 ± 0.03 1.29 ± 0.01 Antioxidant capacity was determined using an oxygen radical absorbance capacity (ORAC) assay. Trolox (6.25 μg/ml) was used as a positive control and set at 1.0. Data are the mean ± SEM (n=3) and expressed as an index of Trolox. SSE 50% ethanol extract of S. bicolor L. stalk, f SSE A. oryzae NK- fermented form of SSE.