JOURNAL OF COSMETIC SCIENCE 400 concentrations of emblica (0, 0.01, 0.1, 0.5, 1, and 2 mg/ml) for 24 hours. The cell lysates were analyzed by Western blot analysis using a specifi c antibody for type I pro-collagen protein. Consistent with the immunocytochemistry assay, our Western blot results showed that emblica extracts dramatically increased intracellular type I pro-collagen in mouse fi - broblast cells. Approximately 6.87-fold induction of type I pro-collagen was observed at the treatment concentration of 0.1 mg/ml emblica (Figure 3). As the dose was in- creased to 0.5 mg/ml and 1 mg/ml, the pro-collagen level appeared to decrease and the level was signifi cantly decreased compared to the untreated control at the concentra- tion of 2 mg/ml. Since asiaticoside has been recently reported to possess a collagen-enhancing effect (12), we used the same concentrations of asiaticoside (0.1 mg/ml and 0.5 mg/ml) for compari- son. Our results indicated that emblica extract dramatically up-regulated the type I pro- collagen level to 7.55-fold compared to the untreated control (Figure 4), whereas only 1.01- and 2.57-fold inductions were found in 0.1 mg/ml and 0.5 mg/ml of asiaticoside treatment, respectively. EFFECTS OF EMBLICA EXTRACT ON COLLAGENASE ACTIVITY Collagenase is a metalloproteinase with an active site zinc ion that is important in fa- cilitating interaction with an inhibitor (13). Quantifi cation of the anti-collagenase activ- ity of emblica extracts was determined by using an EnzChek® gelatinase/collagenase assay kit. As shown in Figure 5, we found that emblica extract signifi cantly inhibited collagenase activity in a dose-dependent manner. At the concentration of 0.5 mg/ml, emblica extract showed collagenase inhibition of 72.11 ± 5.95%, and the inhibition activity slightly in- creased up to 78.67 ± 3.51% when the concentration was extended to 1 mg/ml. Figure 2. Effect of emblica extracts on type I pro-collagen level in mouse fi broblast cells. Cells were treated with various concentrations of emblica extract (0, 0.01, 0.1, 0.5, 1, and 2 mg/ml) for 24 hours. Type I pro- collagen protein was determined by immunocytochemistry assay and fl ow cytometry. Data are shown in terms of relative fl uorescence intensity to untreated control. The experiments were performed independently in triplicate, and the number of cells was 100,000 cells in each sample. The data are represented as mean ± S.D. and were analyzed by the Student’s t-test. *Signifi cant difference, p 0.05, compared to untreated control.

P. EMBLICA EXTRACT AND PRO-COLLAGEN SYNTHESIS 401 Figure 3. Effect of emblica extract on type I pro-collagen expression level. Cells were treated with various concentrations of emblica extract (0, 0.01, 0.1, 0.5, 1, and 2 mg/ml) for 24 hours, and cell lysates were sub- jected to Western blot analysis detection with specifi c antibody for type I pro-collagen protein. All Western blot results were quantifi ed using analyst/PC densitometry software. Mean densitometry data from three inde- pendent experiments were normalized to result in cells in the control. The data are represented as mean ± SD and were analyzed by the Student’s t-test. *Signifi cant difference, p 0.05, compared to untreated control. Figure 4. Effect of emblica extract and asiaticoside on type I pro-collagen expression level. Cells were treated with 0.1 mg/ml emblica extract or 0.1 mg/ml and 0.5 mg/ml asiaticoside for 24 hours, and cell lysates were subjected to Western blot analysis detection with specifi c antibody for type I pro-collagen protein. All Western blot results were quantifi ed using analyst/PC densitometry software. Mean densitometry data from three inde- pendent experiments were normalized to result in cells in the control. The data are represented as mean ± SD and were analyzed by the Student’s t-test. *Signifi cant difference, p 0.05, compared to untreated control.