ANTI-WRINKLE ACTIVITY OF P. STROBILACEA 217 INHIBITION OF ELASTASE ACTIVITY Elastin is the main component of the elastic fi bers of the connective tissue and tendons. In the skin, the elastic fi bers, together with the collagenous fi bers, form a network under the epidermis. Elastase is able to attack all major connective tissue matrix proteins, in- cluding elastin, collagen, proteoglycans, and keratins. Because elastic fi ber is easily de- composed by elastase secretion and activation caused by exposure to UV light or ROS (reactive oxygen species), an approach that inhibits elastase activity could also be applied as a useful method to protect against skin aging (20). Table II shows the results of the inhibition of elastase activity. The n-BuOH fraction of P. strobilacea fruit extract showed the highest elastase inhibition activity (IC50 = 35.1 μg/ml). It was over two times higher in effect compared to oleanolic acid (IC50 = 83.8 μg/ml), which was used as a positive control. The P. strobilacea fruit extract also showed signifi cantly high DPPH radical scavenging activity, as mentioned above. Therefore, this result suggested that a P. strobilacea fruit extract would have potential as an anti-wrinkle agent for use in cosmetic products. CYTOTOXICITY ASSAY IN A MONOLAYER CULTURE In order to evaluate the cytotoxicity of the P. strobilacea fruit extract and ellagic acid in vitro, samples were prepared at various concentrations and used to treat human fi broblasts (ATCC, CRL-2076). The results of this evaluation are shown in Figure 1. The P. strobila- cea fruit extract showed no cytotoxicity compared to that of the positive control, up to the effective concentration for anti-wrinkle activity (less than 50 μg/ml). These fi ndings sug- gest that P. strobilacea fruit extract could be used as an effective and safe active ingredient without any associated cytotoxicity. INHIBITION OF MMP-1 mRNA EXPRESSION In order to evaluate the inhibition activity of the degradation of collagen fi bers in the skin, we investigated the reduction of MMP-1 expression by P. strobilacea fruit extract Table I Free-Radical Scavenging Activity of P. strobilacea Fruit Extract Sample Fraction DPPH radical scavenging activity (%) SC50b (μg/ml) 1 μg/ml 5 μg/ml 10 μg/ml P. strobilacea fruit extract 70% EtOH 10.9 50.1 82.2 5.0 Hexane fr. 2.9 7.8 23 10 EtOAc fr. 13.8 51.2 80.7 4.9 BuOH fr. 11.1 52.0 80.7 4.8 H2O fr. 11.4 48.4 78.6 6.1 Ellagic acid 34.7 81.3 87.8 2.5 BHT a 82.5 (100 μg/ml) 64.1 (50 μg/ml) 33.1 25.4 a BHT (di-t-butyl hydroxyl toluene). b SC50 indicates the concentration (μg/ml) at which the percentage inhibition of the DPPH radical scavenging activity was 50%.

JOURNAL OF COSMETIC SCIENCE 218 and ellagic acid by using the RT-PCR method. EGCG was used as a positive control because its activities are well known to have an inhibitory effect on collagenase and stromelysin mRNA expression induced by IL-1β (21) and to have a protective effect against skin damage caused by UV rays (22). The MMP-1 expression assay on human fi broblasts was carried out with a Gel Doc 2000 image analyzer (Bio-Rad). The P. strobi- lacea fruit extract reduced the expression of MMP-1 (by up to about 76% at 50 μg/ml), as shown in Figure 2. The P. strobilacea fruit extract inhibited the expression of MMP-1 from 17.3% to 76.0% at concentrations of 25–50 μg/ml in a dose-dependent manner. But ellagic acid isolated from P. strobilacea showed only 15% of inhibition at its maxi- mum concentration (1μg/ml). Figure 1. Cytotoxicity assay of P. strobilacea fruit extract, ellagic acid, and ellagic acid 4-O-xylopyranoside in human fi broblast cells: (a) Control (b) 10 μg/ml P. strobilacea fruit extract (c) 20 μg/ml P. strobilacea fruit extract (d) 50 μg/ml P. strobilacea fruit extract (e) 0.25 μg/ml ellagic acid (f) 0.5 μg/ml ellagic acid (g) 1.0 μg/ml ellagic acid (h) 0.25 μg/ml ellagic acid 4-O-xylopyranoside (i) 0.5 μg/ml ellagic acid 4-O-xylopyranoside (j) 1.0 μg/ml ellagic acid 4-O-xylopyranoside. The data are expressed as mean values (± standard deviations) of fi ve experiments. Table II Elastase Inhibition Activity of P. strobilacea Fruit Extract Sample Fraction Elastase inhibition activity (%) IC50a (μg/ml) 100 μg/ml 50 μg/ml 10 μg/ml P. strobilacea fruit extract 70% EtOH 72.8 63.7 12.1 37.9 Hexane fr. — — — — EtOAc fr. 74.3 69.2 7.8 35.1 BuOH fr. 82.2 67.5 6.1 37.0 H2O fr. 56.2 27.6 4.6 89.2 Ellagic acid 6.2 (1 μg/ml) 57.3 (5 μg/ml) 80.6 4.6 Oleanolic acid 57.7 35.2 9.9 83.8 a IC50 indicates the concentration (μg/ml) at which the percentage inhibition of elastase activity was 50%.