A. INCISUS EXTRACT AND WRINKLE REDUCTION 315 suspension was transferred from the 75-cm2 fl ask into 12-well plate at a density of 1×105 cells/well of DMEM with FBS and antibiotics. The cells were incubated at 37°C for 24 h in a humid atmosphere containing 5% CO2. There were two groups of cultures: (i) serum-free DMEM + DMSO (0.1%, control group) and (ii) serum-free DMEM + extract (treated group). The cells continued to be incubated at 37°C with 5% CO2 for 72 h. Type I procollagen and MMP-1 assay. In this study, the procollagen assay used an antibody against the C-terminal propeptide region that is part of the transcribed collagen protein that is then proteolytically cleaved after secretion. Thus, this assay is a measure of newly synthesized collagen. After incubation with the extract for 72 h, the cell-free supernatant was collected, stored at −80°C, and then assayed later. The amount of type I procollagen was measured by using a commercial human procollagen type-I C-peptide EIA kit (Takara Bio Inc., Shiga, Japan). The same samples were also assayed for MMP-1 (interstitial collagenase) by using a commercial human MMP-1 ELISA kit (RayBiotech, Inc., Geor- gia, USA). The levels of type I procollagen and MMP-1 were normalized against a stan- dard dose-response curve based on the absorption at the wavelength of 450 nm using a Labsystems Multiskan RC 96-well microplate reader. The determinations were performed in triplicate. EFFECT OF THE EXTRACT ON CONTRACTION OF A FIBROBLAST-EMBEDDED COLLAGEN LATTICE Fibroblast-embedded collagen lattice preparation. A three-dimensional collagen lattice was prepared according to the previous studies (11,26) with modifi cation. Fibroblasts from nonwrinkled and wrinkled skin were collected from the same explants as mentioned above. Cells at passage 7 were used to prepare the fi broblast-embedded lattice. Briefl y, the disk-shaped matrix consisted of 1.98 ml of concentrated DMEM 1.96 X (GIBCOTM, In- vitrogenTM Life Technologies, California) containing 50 μg/ml of the extract (treated group) or 0.1% of DMSO (control group), 1.5 ml of rat tail type I collagen (Institut de Biotechnologies Jacques Boy, Reims, France), 0.25 ml of 0.1 N NaOH (Prolabo, Fon- tenay-Sous-Bois, France), 0.17 ml of 7.5% NaHCO3 (PanTM Biotech GmbH), 0.50 ml of FBS (PanTM Biotech GmbH), and 0.5 ml of cell suspension (8 × 105 cells/ml). Matrices were prepared in 60-mm Petri dishes (Falcon bacteriological dishes, Elvetec Services, Clemont-Ferrand, France) and then placed in a 37°C incubator in a humid atmosphere containing 5% CO2. Matrix disks were prepared in triplicate for each group and experi- ments were performed in duplicate. Contraction capacity determination. The contraction capacity of the fi broblast-embedded lat- tice was visually determined from the lattice diameter. To measure matrix diameters, they were placed on a transparent metric ruler on a dark background. The matrix diameters were measured over the seven-day culture period. STATISTICAL ANALYSIS All quantitative data reported here are expressed as means of the samples for each treat- ment. Student’s unpaired t-test was used for comparison between the two groups. P 0.05 was considered signifi cant.
JOURNAL OF COSMETIC SCIENCE 316 RESULTS AND DISCUSSION THE APPEARANCE AND ARTOCARPIN CONTENT OF THE EXTRACT Extraction of dietyl ether provided a yellow solid powder. The HPLC chromatograms of the artocarpin standard and the artocarpin contained in the extract are shown in Figure 1A and 1B, respectively. The amount of artocarpin contained in the extract was 44.5 ± 0.1% (w/w). This fi nding coincided with our previous study indicating 45.2% (w/w) of artocarpin in the ether extract (20). EFFECTS OF THE EXTRACT ON THE VIABILITY AND PROLIFERATION OF HUMAN FIBROBLASTS To investigate the effect of the extract on cell viability, the fi broblasts were treated with various concentrations (0.5–50 μg/ml) of the extract for 24, 48, or 72 h. As shown in Figure 2A, the extract did not affect the viability of nonwrinkled-skin fi broblasts. Figure 1. Chromatograms of (A) 0.2 mg/ml purifi ed artocarpin (standard) and (B) 1.0 mg/ml A. incisus extract.
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