JOURNAL OF COSMETIC SCIENCE 396 Skin aging is a process involving an alteration of type I collagen, the major component of dermis. An increase in type I collagen degradation and a decrease in its regeneration are considered major causes of wrinkle formation (3). Type I collagen is originally synthe- sized from intracellular type I pro-collagen containing propeptide extensions at both ends of the molecule (4,5). The pro-collagen is secreted to the extracellular matrix where the propeptides are removed and the collagen molecules aggregate to form the fi bril (5,6). The matrix metalloproteinases (MMPs) are a large family of zinc-dependent endopro- teases degrading all extracellular matrix proteins (ECMs) including collagen. MMP-1, interstitial collagenase, mediates type I and type III collagen degradation (7). Several fac- tors promoting this degradation process have been identifi ed, including ultraviolet (UV) radiation and reactive oxygen species (ROS) (8). Due to their potent antioxidant activity, emblica extracts appear to have promise as effectors for anti-aging actives. The objective of the present study is to investigate the novel pharmacological activities of emblica extract, namely type I collagen promoting and anti-collagenase activities. MATERIALS AND METHODS MATERIALS Dulbecco’s Modifi ed Eagle’s medium (DMEM), L-glutamine, fetal bovine serum (FBS), penicillin/streptomycin, and phosphate-buffered saline were obtained from Gibco-BRL (Gaithersburg, MD). Formaldehyde, triton X-100, glycerol, sodium formate, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoside (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO). Pro-collagen type I rabbit polyclonal antibody, horseradish peroxidase-coupled isotype-specifi c secondary antibod- ies, and FITC-coupled secondary antibodies were obtained from Santa Cruz Biotechnol- ogy (Santa Cruz, CA). A protease inhibitior mixture was obtained from Roche Molecular Biochemicals (Switzerland). A chemiluminescence detection system was obtained from Amersham Biosciences (Piscataway, NJ), and a Western blot system was obtained from Bio-Rad (Hercules, CA). An EnzChek® gelatinase/collagenase assay kit was purchased from Molecular Probes®, Invitrogen (Carlsbad, CA). ISOLATION OF MOUSE EMBRYONIC FIBROBLASTS Isolation of fi broblast cells from mouse embryo was carried out according to a previously described method (Bradley, Baylor College of Medicine, Waco, TX). Briefl y, mouse uterus was dissected from 13- or 14-post coitum pregnancy female mice. Each embryo was separated and washed with phosphate buffer solution. Then the embryo was forced through a syringe that was fi tted with an 18G 11/2" needle into Dulbecco’s Modifi ed Eagle’s medium (DMEM). Primary fi broblasts were the only cells that attached and proliferated after fi ve days incubation at 37°C. Cells were cultured in DMEM containing 10% fetal bovine serum, 2 mmol/l L-glutamine, and 100 units/ml of penicillin/strepto- mycin in a 5% CO2 environment at 37°C. The cells from passages 2–5 were used for the experiments.

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