P. EMBLICA EXTRACT AND PRO-COLLAGEN SYNTHESIS 397 EXTRACTION AND SAMPLE PREPARATION Emblica fresh fruits were freed from foreign matter like dust or other organic matter. The cleaned raw material was chopped up to reduce its size. The raw material was then agi- tated in water. The liquid part was separated and then converted to powder form by spray drying. Various samples of emblica extract were prepared by dissolving the dry emblica extract powder in deionized water to the indicated concentrations. CELL VIABILITY ASSAYS Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Cells in 96-well plates were incubated with 500 μg/ ml of MTT for four hours at 37°C. The intensity of the MTT product was measured at 550 nm using a microplate reader. The relative percentage of cell survival was calculated by dividing the absorbance of treated cells by that of the control in each experiment. WESTERN BLOT ANALYSIS After specifi c treatments, cells were incubated in lysis buffer containing 20 mmol/l Tris- HCl (pH 7.5), 1% Triton X-100, 150 mmol/l NaCl, 10% glycerol, 1 mmol/l Na3VO4, 50 mmol/l NaF, 100 mmol/l phenylmethylsulfonyl fl uoride, and a commercial protease inhibitor mixture (Roche Molecular Biochemicals) for 20 minutes on ice. After insoluble debris was pelleted by centrifugation at 14,000g for 15 minutes at 4°C, the supernatants were collected and the protein content was determined using the Bradford method (Bio- Rad Laboratories, Hercules, CA). Proteins (40 μg) were resolved under denaturing condi- tions by SDS-PAGE (10%) and transferred onto nitrocellulose membranes (Bio-Rad). The transferred membranes were blocked for one hour in 5% nonfat dry milk in TBST [25 mmol/l Tris-HCl (pH 7.4), 125 mmol/l NaCl, 0.05% Tween 20] and incubated with the appropriate primary antibodies at 4°C overnight. Membranes were washed twice with TBST for ten minutes and incubated with horseradish peroxidase-coupled isotype- specifi c secondary antibodies for one hour at room temperature. The immune complexes were detected by an enhanced chemiluminescence detection system (Amersham Biosci- ences) and quantifi ed using analyst/PC densitometry software (Bio-Rad). Mean densi- tometry data from independent experiments were normalized to control results. The data were presented as the mean ± SD and analyzed by the Student’s t-test. IMMUNOCYTOCHEMISTRY After specifi c treatment, cells in six-well plates were rinsed once with cold PBS and fi xed for three minutes with a fi xing reagent containing 4% formaldehyde in phosphate-buffered saline (PBS) solution, pH 7.4. After removal of the fi xing reagent, cells were washed twice with TBS buffer (pH 7.4) and incubated with permeabilizing solution (1% Triton X-100 in PBS) at room temperature for fi ve minutes. After washing with TBS buffer, the cells were blocked with blocking buffer (2.5% FBS in TBS) for 30 minutes at room

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