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.

JOURNAL OF COSMETIC SCIENCE 278 SUPPRESSION OF MMP-1, -2, AND -3 BY SSE AND FSSE The suppressive effects of SSE and f SSE on MMP-1, -2, and -3 protein expression levels were determined by immunoblotting. Here, TNF-α (10 ng/ml) was used as an inducer of MMP expression in HDF-N cells (16,17). As shown in Figure 4, the protein expression levels of MMP-1, -2, and -3 were signifi cantly increased in the presence of TNF-α. SSE at concen- trations more than 100 μg/ml signifi cantly suppressed the expression of MMP-1, -2, and -3. However, f SSE was more potent than SSE because f SSE showed suppressive effects beginning at 50 μg/ml. This result implies that fermentation of SSE with A. oryzae NK increased the components associated with inhibiting the protein expression of MMP-1, -2, and -3. This result suggests that SSE and f SSE could have potential as antiwrinkle agents for use in cosmeceutical products. IDENTIFICATION OF P-COURMARIC ACID IN SSE AND FSSE USING HPLC AND ITS SUPPRESSIVE EFFECT ON EXPRESSION OF MMP-1 PROTEIN To identify components increased in f SSE relative to SSE, HPLC was performed on SSE and f SSE. As shown in Figure 5A, the amount of p-coumaric acid in f SSE was 1.5-times higher than that in SSE. When the suppressive effect of p-coumaric acid on expression of MMP-1 Figure 3. Tyrosinase inhibitory effect of SSE and f SSE. Antityrosinase activity was determined using a colo- rimetric method using purifi ed mushroom tyrosinase and L-tyrosine at the indicated doses of SSE and f SSE. Data are % of untreated control as mean ± SEM (n = 3). * and ** indicate p 0.05 and p 0.01 versus un- treated control, respectively. SSE 50% ethanol extract of S. bicolor L. stalk, f SSE A. oryzae NK-fermented form of SSE.