ANTI-WRINKLE ACTIVITY OF P. STROBILACEA 215 at 96°C for 3 min, then 25 cycles of 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min, and fi nally extension at 72°C for 10 min. The fi nal products were detected with 2% aga- rose gel. The gel was photographed, and the intensity of the stained PCR fragments was quantifi ed from the photographs by a densitometric analysis with Gel Doc 2000 (Bio- Rad Laboratories, Segrate, Milan, Italy). EGCG ((-)epigallocatechin-3-gallate) was used as a positive control. ASSAY OF TYPE I COLLAGEN MRNA BY RT-PCR The human fi broblast cells were seeded at a density of 1 × 106 cells/dish in a 60-mm cell culture dish incubated for one day at 37°C in 5% CO2. The culture medium was replaced with a serum-free medium, and the samples were added to each dish. After 24 h culture, total RNA were isolated with TRIzol reagent and the isolated total RNA were measured at 260 nm. RT-PCR of type I collagen and β-actin were performed using an all-in-one RT-PCR kit (Superbio, Korea) with 1 μg of RNA. The sequences of the primers were as follows: 5′-CTGGCAAAGAAGGCCGCAAA-3′ (sense) and 5′-CTCACCACGATCA- CCACTCT-3′ (anti-sense) for type I collagen mRNA 5′-GAGACCTTCAACACCCC- AGCC-3′ (sense) and 5′-GGCCATCTCTTGCTCGAAGTC-3′ (anti-sense) for β-actin. Type I collagen mRNA RT-PCR reactions involved reverse transcription at 50°C for 30 min, denaturing at 96°C for 3 min, then 22 cycles of 94°C for 1 min, 62°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 reac- tions involved reverse transcription at 50°C for 30 min, denaturing at 96°C for 3 min, then 25 cycles of 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min, and fi nally exten- sion at 72°C for 10 min. The PCR products were identifi ed by electrophoresis on 2% agarose gel and EtBR (Ethidium bromide, Sigma) staining. The intensity of the stained PCR fragments was also quantifi ed from the photographs by a densitometric analysis with Gel Doc 2000 as above. ETHICS This study was conducted according to the guidelines laid down in the Declaration of Helsinki of 1975 as amended in 2000, and all procedures involving human subjects were approved by the Ellead Skin Research Center Institutional Review Board. The volunteers were clearly and precisely informed of the particular objectives and protocol of the study, and of the foreseeable risks involved in the in vivo clinical trial. Written informed consent was obtained from all subjects. IN VIVO CLINICAL TRIAL Twenty fi ve subjects (34∼49-year-old females in good general health) were recruited for this clinical study on a formulation containing P. strobilacea fruit extract (the test cream contained 0.2% P. strobilacea fruit extract and the placebo did not). This double-blind, placebo-controlled, left-right randomized clinical study was carried out during a 12- week period in order to assess the test and placebo formulations. We measured in this clinical test the effi cacy of the formulation in terms of its anti-wrinkling effect on the skin

JOURNAL OF COSMETIC SCIENCE 216 through a visual evaluation conducted by dermatologists, photometric evaluation, the manufacturing of skin replicas, and image analysis with a Skin-Visiometer SV 600 (Cour- age & Khazaka, Germany). A cutaneous examination of the crow’s feet area was con- ducted by two dermatologists via a double-blind method. Twenty-fi ve healthy volunteers with dry to very dry skin visited the Ellead Skin Research Center (Republic of Korea) at 0, 4, 8, and 12 weeks, and cutaneous readings were taken according to a photodamage score of 0 to 7 (0, none 1, none/mild 2, mild 3, mild/moderate 4, moderate 5, moder- ate/severe 6, severe 7, very severe) (18). We analyzed wrinkling on a monitor by using the 3-Dimensional Skin System program, measuring the number of wrinkle peaks and the depth of each wrinkle. The measuring principle of the Skin-Visiometer SV 600 is based on light transmission through a very thin replica comprising blue-dyed two-com- ponent silicone the light absorption of which is known. The roughness parameters inves- tigated were R1 (depth of roughness), R2 (maximum roughness), R3 (mean depth of roughness), R4 (smoothness depth), and R5 (arithmetic average roughness). STATISTICS In determining the signifi cance of the data, an independent t-test was used to confi rm whether the difference was statistically signifi cant (5%). MS EXCEL 2003 software was used for the statistical analysis. RESULTS AND DISCUSSION FREE-RADICAL SCAVENGING ACTIVITY It has been reported that free radicals induced by ultraviolet light or oxidative stress ac- celerate skin aging (19). Therefore, assays of free-radical scavenging capacity were carried out by the DPPH method. The free-radical scavenging capacity of P. strobilacea fruit extract and its fractions was measured at each concentration (1–10 μg/ml), the results being shown in Table I The free-radical scavenging capacity is expressed as SC50, the con- centration needed to reduce 50% of the DPPH radical. The crude P. strobilacea fruit extract and all the fractions except the hexane fractions (SC50 10 μg/ml) showed high free-radi- cal scavenging capacity (SC50 10 μg/ml). Among these, the EtOAc fraction and the BuOH fraction had the highest free-radical scavenging activity (the SC50 were 4.9 and 4.8 μg/ml, respectively) compared to BHT (di-t-butyl hydroxyl toluene SC50 = 25.4 μg/ml), which was used as a positive control. Ellagic acid especially showed good free-radical scav- enging capacity (SC50 = 2.5 μg/ml), while ellagic-4-O-xyropyranoside did not have DPPH radical scavenging activity. Ellagic acid and ellagic-4-O-xyropyranoside isolated from the n-BuOH fraction are known as effective components of P. strobilacea (12). Ellagic acid is comprised of four rings of poly phenol, commonly found as a precursor form of ellagic acid, ellagitannin. It is a vegetable phenol found in grapes, strawberries, pomegranates, strawberry trees, peanuts, and green tea and has antioxidant, antiviral, antimutation, and anti-tumor effects. A recent report showed that ellagic acid suppressed the growth of breast, gullet, skin, colon, prostate, and pancreatic cancer cells (13). Ellagic acid is a primary constituent of several tannin-bearing plants that produce the category of tannins known as gallotannins, which give rise to ellagic acid and gallic acid upon hydrolysis by water.