ANTI-WRINKLE ACTIVITY OF P. STROBILACEA 213 EXTRACTION AND FRACTIONATION P. strobilacea fruits were collected from Jeju island and authenticated by Dr. C. S. Kim, the director of Halla Arboretum in Mt. Halla National Park. The collected fruits were dried in the shade at room temperature and stored in a dark, cold room until needed. The dried whole fruits (180 g) were extracted twice with 70% (v/v) ethanol (20 times as much as the weight of the dried fruits) for 24 h at room temperature. The extract of the fruits was fi ltered through fi lter paper (Whatman, No. 5) and then evaporated at 60°C. The fi ltrate was evaporated to produce the crude P. strobilacea fruit extract as a powder (49.5 g, 27.5% yield). The crude P. strobilacea fruit extract (20 g) was dissolved in distilled water at a concen- tration of 2% (w/w), and the aqueous suspension was successively extracted with n-hexane, ethyl acetate, and n-BuOH. Each fraction was evaporated to dryness to give a residue [The yields were 0.72 g (3.6%) of the hexane fraction 1.66 g (8.3%) of the EtOAc fraction 3.0 g (15.1%) of the n-BuOH fraction and 14.1 g of the H2O fraction (70.5%).] The n-BuOH fraction (1.0 g) was chromatographed on octadecylsilyl-silica gel (ODS, Capcell Pak C18 UG120, 5 μm, 20 × 250 mm, Shiseido Co., Ltd. Tokyo) by preparative HPLC with isocratic eluent (CH3CN:H2O=45:55) at a fl ux of 14.0 ml/ min. Two major compounds on prep-HPLC, ellagic acid and ellagic-4-O-xyropyrano- side, were isolated (64 mg and 22 mg, respectively) (mp 254–258°C (dec.)) and identi- fi ed by 1 H-NMR and 13 C-NMR, the results of which were in agreement with previously published data (12). FREE-RADICAL SCAVENGING ACTIVITY TEST The assay for free-radical scavenging capacity was carried out according to the method that has been reported previously by Blois (16). The DPPH (1,2-diphenyl-2-picrylhydra- zyl) 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. In brief, a 0.2 mM DPPH 95% ethanolic solution (1 ml) was added to a sample of the stock (2 ml). Each sample solution was diluted with a 70% ethanolic solution to fi nal concen- trations of 10, 5, and 1 μg/ml, and the samples were then agitated. The optical density at 525 nm was measured after 10 min with a UV/Vis spectrophotometer. The free-radical scavenging activity of each sample was calculated according to the following formula: DPPH radical scavenging activity (%) = [1– (ODs – ODb)/ODc] × 100 where ODs is the absorbance of the experimental sample, ODb is the absorbance of the blank, and ODc is the absorbance of the control at 525 nm. The results are reported in terms of SC50 (the concentration needed to reduce 50% of DPPH). BHT (di-t-butyl hy- droxy toluene), a representative antioxidant, was used as a control. ELASTASE INHIBITION ACTIVITY The elastase activity was evaluated according to the method previously reported by Kraunsoe et al. (17). In order to evaluate the inhibition of elastase activity, the amount of

JOURNAL OF COSMETIC SCIENCE 214 released p-nitroaniline, which was hydrolyzed from the substrate, N-succinyl-Ala-Ala- Ala-p-nitroanilide, by elastase, was read with a maximum absorbance at 410 nm. In brief, 1.015 mM of N-succinyl-Ala-Ala-Ala-p-nitroanilide was prepared in a 0.1232 M Tris- HCl buffer (pH 8.0), and this solution (1300 μl) was added to the stock sample (100 μl). Each sample solution was diluted to fi nal concentrations of 100, 50, and 10 μg/ml. The solutions were vortexed and preincubated for 10 min at 25°C before 100 μl of the elastase (pancreatic, Type IV, E0258, Sigma, St. Louis, MO, 0.0375 unit/ml) was added. After vortexing, each solution was placed in a water bath for 10 min at 25°C, and the absor- bance was measured at 410 nm. CYTOTOXICITY ASSAY IN A MONOLAYER CULTURE Evaluation of cytotoxicity was performed by the 3-[4,5-dimethylthiazol]-2,5-diphe- nyltetrazolium bromide (MTT) assay. The human fi broblast cells were seeded in 24-well plates at a density of 1 × 105 cells per well and cultured at 37°C in 5% CO2. After one day, a culture medium (Iscove’s modifi ed Dulbeco’s medium, IMDM) was replaced with a serum-free medium and the cells were incubated in a CO2 incubator at 37°C in the pres- ence of samples for 24 h, before being treated with 100 μl of 2.5 mg/ml MTT solution. The cells were then incubated at 37°C for an additional 4 h. The medium containing MTT was discarded, and MTT formazan that had been produced by the live cells was extracted with 1 ml of DMSO. The absorbance was read at 570 nm, with a reference wavelength of 650 nm. The cell viability was calculated as follows: Cell viability (%) = (OD570(sample)/OD570(control)) × 100 where OD570(sample) is the absorbance of the treated cells at 570 nm and OD570(control) is the absorbance of the negative control at 570 nm (non-treated cells). ASSAY OF MMP-1 EXPRESSION BY RT-PCR Human fi broblasts were cultured with IMDM + 10% FBS, 50 U/ml of penicillin, and 50 μg/ml of streptomycin at 37°C in 5% CO2 the medium was changed every two or three days. When the cells had reached confl uence, they were separated by treatment with a 0.25% trypsin–0.03% EDTA (ethylenediamine tetraacetic acid) solution. The cells were seeded into a 60-mm dish at a density of 1 × 106 cells/dish and cultured for one day at 37°C in 5% CO2. The cultured cells were exposed to UVA (6 J/m2), and a fresh medium without FBS was added to the cells, which were then treated with samples for 24 h. Total RNA was isolated from the cells with TRIzol (Invitrogen, USA) according to the instruc- tions of the manufacturer. First-strand cDNA synthesis was performed by using random hexamers. The sequences of the primers were as follows: 5′-TGGGAGCAAACA- CATCTGA-3′ (sense) and 5′-ATCACTTCTCCCCGAATCGT-3′ (anti-sense) for MMP-1 5′-GAGACCTTCAACACCCCAGCC-3′ (sense) and 5′-GGCCATCTCTTGCTCGA- AGTC-3′ (anti-sense) for β-actin. MMP-1 RT-PCR reactions involved reverse transcrip- tion at 50°C for 30 min, denaturing at 96°C for 3 min, then 22 cycles of 94°C for 1 min, 48°C for 1 min, and 72°C for 1 min, and fi nally extension at 72°C for 10 min. The β-actin RT-PCR reactions involved reverse transcription at 50°C for 30 min, denaturing