ANTI-WRINKLE ACTIVITY OF P. STROBILACEA 219 ASSAY OF COLLAGEN TYPE I mRNA BY RT-PCR Collagen fi ber is the main component of the extracellular matrix (ECM), as the represen- tative connective tissue that comprises about 90% of the dermis. Therefore, collagen has a direct infl uence on skin tension. To evaluate the amount of collagen type I synthesis that occurred upon exposure to the extract, collagen type I mRNA was quantitatively mea- sured by RT-PCR. P. strobilacea fruit extract increased the expression of collagen type I mRNA about 30%, irrespective of concentrations of 25–50 μg/ml. Ellagic acid isolated from the P. strobilacea fruit extract especially increased the expression of collagen type I mRNA in a dose-dependent manner (up to 41.3% at 1 μg/ml), comparable to that of ascorbic acid (up to 39.5% at 500 μM), as shown in Figure 3. IN VIVO CLINICAL TRIAL We measured the effi cacy of the anti-wrinkling effect on the skin in this clinical test through a visual evaluation by dermatologists, photometric evaluation, the manufactur- ing of skin replicas, and image analysis using the Skin-Visiometer SV 600. The cutaneous evaluation was performed on volunteers during scheduled visits (0, 4, 8 and 12 weeks). The cutaneous readings were based on a photodamage score of 0 to 7 (0, none 1, none/ mild 2, mild 3, mild/moderate 4, moderate 5, moderate/severe 6, severe 7, very se- vere) and evaluated by two dermatologists (18). The results show that the difference between the test group and the placebo group was not signifi cant until four and eight weeks after the treatment, but that there was a signifi cant difference 12 weeks after the Figure 2. Expression of MMP-1 mRNA in human fi broblasts (ATCC, CRL-2076) by RT-PCR: (a) Control (b) UVA 6J (c) UVA 6J + 10 μg/ml EGCG, (-)epigallocatechin-3-gallate (d) UVA 6J + 10 μg/ml P. strobi- lacea fruit extract (e) UVA 6J + 20 μg/ml P. strobilacea fruit extract (f) UVA 6J + 50 μg/ml P. strobilacea fruit extract (g) UVA 6J + 0.25 μg/ml ellagic acid (h) UVA 6J + 0.5 μg/ml ellagic acid (i) UVA 6J + 1.0 μg/ml ellagic acid.

JOURNAL OF COSMETIC SCIENCE 220 Figure 3. Expression of collagen type I mRNA in human fi broblast cells: (a) Control (b) 100 μM vitamin C (c) 250 μM vitamin C (d) 500 μM vitamin C (e) 10 μg/ml P. strobilacea fruit extract (f ) 20 μg/ml P. strobilacea fruit extract (g) 50 μg/ml P. strobilacea fruit extract (h) 0.25 μg/ml ellagic acid (i) 0.5 μg/ml el- lagic acid (j) 1.0 μg/ml ellagic acid. The data are expressed as the mean values (± standard deviation) of three experiments. Figure 4. Weekly comparison of photodamage scores in the crow’s feet area after the use of the formulation containing P. strobilacea fruit extract for 12 weeks (*p 0.05). The visual evaluation was conducted by two dermatologists via a double-blind method. In any case of the readings of the two doctors not agreeing, the lower score for the anti-wrinkle activity was selected for analytical purposes. treatment (Figure 4). In an image analysis of skin replicas by the Skin-Visiometer SV 600, among the roughness parameters already mentioned, skin roughness R1 is the dis- tance between the basic and reference profi le, referred to a given reference length L. R3,