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.

THE ANTI-WRINKLE AND ANTI-MELANOGENIC EFFECTS 279 protein after induction by TNF-α was determined by immunoblotting, p-coumaric acid was shown to be highly potent in the suppression of MMP-1 expression (Figure 5B). This result implies that p-coumaric acid has an inhibitory effect on MMP-1 protein expression and Figure 4. Ef fect of SSE and f SSE on the expression levels of MMP-1, -2, and -3 protein. HDF-N cells were treated with different doses of SSE and f SSE in the presence TNF-α (10 ng/ml) for 24 h. Then, equal amounts of cell lysate were subjected to immunoblotting (n = 3). (A) Representative image of immunoblots. (B) Densitomet- ric analysis of immunoblots. The expression level of protein is expressed relative to the level of β-actin. * and # indicate p 0.05 versus untreated control and TNF-α treated group, respectively. SSE 50% ethanol extract of S. bicolor L. stalk, f SSE A. oryzae NK- fermented form of SSE, MMP matrix metalloproteinase, TNF-α tumor ne- crosis factor-α.