A. INCISUS EXTRACT AND WRINKLE REDUCTION 317 Figure 2. Effects of A. incisus extract on viability of fi broblasts (A) from nonwrinkled skin and (B) from wrinkled skin. Fibroblasts were treated with 0.1% DMSO or the extract at concentrations in the range of 0.5–50 μg/ml for 24, 48, and 72 h. Results are expressed as percentage of cell viability as compared to un- treated cells for which the optical density was adjusted to 100%. Each bar represents mean ± S.D. of triplicate study *p 0.05 and **p 0.01 denote signifi cant differences when compared to untreated cells (Student’s t-test).

JOURNAL OF COSMETIC SCIENCE 318 However, a signifi cant increase in percent viability as compared with the untreated cells was found in the wrinkled-skin fi broblasts treated with the extract at concentrations of 10 μg/ml ( p 0.0.5), 20 μg/ml ( p 0.05), and 50 μg/ml ( p 0.01) for 24 h (Figure 2B). An increase in the incubation time to 48 or 72 h resulted in signifi cant increases in cell viability after treatment with the extract at a lower concentration (5 μg/ml, p 0.05). This effect might be due to increased fi broblasts numbers, and so this was tested by counting cell numbers. Cells were treated with the extract at concentrations of 20 μg/ml or 50 μg/ml for 1, 3, 6, or 10 days and directly counted by using the trypan blue exclusion method. Focusing on untreated cells, the proliferation of cells from wrinkled skin was lower than that of cells from nonwrinkled skin, particularly during the fi rst three days of the study. Treatment with 50 μg/ml of extract resulted in a higher proliferation of the cells from wrinkled skin during the incubation period as compared with the untreated cells ( p 0.05), as shown in Figure 3B. Such a phenomenon was not observed in the fi broblasts from nonwrinkled skin (Figure 3A). The morphology of the fi broblasts did not change compared to that of the untreated cells (Figure 4A–D). The fi broblasts still retained the typical spindle-shape after treatment with the extract at the highest concentration used in this study (50 μg/ml). Figure 3. Effects of A. incisus extract on proliferation of fi broblasts (A) from nonwrinkled skin and (B) from wrinkled skin. Fibroblasts were treated with 0.1% DMSO or the extract at concentrations of 20 μg/ml or 50 μg/ml for 1, 3, 6, or 10 days. Results are expressed as the number of cells. Each point represents mean ± S.D. of triplicate study *p 0.05 and **p 0.01 denote signifi cant differences when compared to untreated cells (Student’s t-test).