P. EMBLICA EXTRACT AND PRO-COLLAGEN SYNTHESIS 399 extract at the concentration of 0.01 mg/ml caused a signifi cant increase in cell viability over the control level, (Figure 1). At the treatment concentration of 0.1–0.5 mg/ml, em- blica extract did not have a signifi cant effect on cell viability. Emblica extract at high concentrations caused a toxic effect on the cells, as indicated by cell viability that ap- proached 71% and 51% at the treated doses of 1 mg/ml and 2 mg/ml, respectively. EFFECTS OF EMBLICA EXTRACT ON TYPE I PRO-COLLAGEN EXPRESSION To determine the effect of emblica extract on pro-collagen expression, mouse fi broblast cells were treated with various concentrations for 24 hours at 37°C. The type I pro-collagen protein level was determined using immunocytochemistry based on the principle of specifi c protein–antibody complex. After treatment, cells were fi xed, permeabilized, and incubated with specifi c anti-type I pro-collagen antibody followed by chemiluminescence FITC secondary antibody. The fl uorescence intensity correlating to the type I pro-collagen level was detected by fl ow cytometry. Treatment with emblica extract signifi cantly increased the type I pro-collagen level in a dose-dependent manner (at a concentration ranging from 0.01 to 1 mg/ml), with the maximum response at a concentration of 0.1 mg/ml (1.65 ± 0.085-fold). However, fur- ther increasing emblica concentrations (0.5–1 mg/ml) slightly decreased pro-collagen levels compared to the maximal response, but levels were signifi cantly increased com- pared to the control (Figure 2). Due to its cytotoxic effect, treatment of emblica extract at 2 mg/ml signifi cantly decreased the type I pro-collagen level. In order to confi rm the effect of emblica extract on the type I pro-collagen level, Western blot analysis for type I pro-collagen was performed. Cells were treated with various Figure 1. Effect of emblica extracts on cell viability. Various concentrations (0, 0.01, 0.1, 0.5, 1, and 2 mg/ ml) of emblica extract were incubated with mouse fi broblast cells for 24 hours. Cell viability was analyzed by MTT assay. The data are presented as percentage of cell viability compared with untreated control. The ex- periments were performed independently in triplicate, and the number of cells was 20,000 cells in each sample. The data are represented as mean ± S.D. and were analyzed by the Student’s t-test. *Signifi cant dif- ference, p 0.05, compared to untreated control.

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.