JOURNAL OF COSMETIC SCIENCE 398 temperature and further incubated with pro-collagen type I rabbit polyclonal antibody (Santa Cruz) for one hour at room temperature. After washing with TBST buffer for ten minutes, the cells were incubated with secondary antibody (FITC-coupled anti-rabbit, Santa Cruz) for one hour with gentle rocking at room temperature. They were then washed, trypsinized, resuspended in PBS (1 × 106/ml), and immediately analyzed by fl ow cy- tometry using an excitation wavelength at 488 nm and an emission wavelength at 520 ± 20 nm (FACSort, Becton Dickinson, Rutherford, NJ) with CellQuest software (Becton Dickinson). ANTI-COLLAGENASE ASSAY Collagenase inhibitory activity was performed using the EnzChek® gelatinase/collagenase assay kit (E-12055) (Molecular Probes) that was used in the previous study (9). The em- blica extract was diluted in 1X reaction buffer. The diluted collagenase inhibitor was added to each well of a 96-well plate, and 1,10-phenanthroline served as a control in- hibitor. DQ gelatin solution was added. Then 100 μl of the diluted enzyme or 100 μl of 1X reaction buffer (blank) was added to the sample wells preloaded with substrate and inhibitor. The samples were incubated at room temperature and protected from light for two hours. The fl uorescence intensity was measured by a fl uorescence microplate reader set for excitation at 485 nm and emission detection at 535 nm (Molecular Probes, prod- uct information, 2001). The increase in fl uorescence is proportional to its proteolytic ac- tivity. Therefore, the decrease in fl uorescence compared with the enzyme activity alone was observed to assay for a potential gelatinase/collagenase inhibitor. The percent inhibi- tion of collagenase reaction was calculated as follows: % Collagenase inhibition = [(A B) (C D)] *100 A B - - - - where A is the fl uorescence after incubation without the test sample (control) B is the fl uorescence after incubation without the test sample and enzyme (blank of A) C is the fl uorescence after incubation with the test sample and D is the fl uorescence after incuba- tion with the test sample, but without the enzyme (blank of C). RESULTS EFFECT OF EMBLICA EXTRACT ON PRIMARY MOUSE FIBROBLAST VIABILITY Collagen fi ber is primarily synthesized by fi broblasts as a pro-collagen protein, which is secreted and further processed to be a collagen fi ber in the extracellular matrix (10,11). Among collagens, type I is the most abundant. It comprises between 85% and 90% of the total collagen in skin (4). To investigate the effect of emblica extract on collagen syn- thesis, we fi rst characterized cell viability response to emblica treatment in primary 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). Cell viability was determined after 24 hours incubation by MTT assay. The viability of cells was determined by measuring the optical density (OD) of formazan formation at wavelength 570–620 nm. Treatment of the cells with emblica

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.