PROTECTION FROM PHOTOAGING BY P. EMBLICA EXTRACT 53 observed by immunocytochemistry that there was signifi cant damage to the procollagen (precursor of collagen) synthesized inside the cell and signifi cant damage to the collagen synthesized (from the exocytosed procollagen) and deposited on the extracellular matrix (ECM) of the cell. There was a 64% reduction in fl uorescence intensity due to UVB- induced collagen damage and a 43% reduction in fl uorescence intensity due to UVB- induced procollagen damage as compared to the respective unexposed control cells. Thereafter, the effect of emblica extract on UVB-induced collagen damage was deter- mined using the CosmoBio human type 1 collagen estimation ELISA kit, which is spe- cifi c for type 1 collagen estimation. Normal human dermal fi broblasts were grown for 24 h and then treated with varying concentrations of emblica extract and exposed to UVB ir- radiation of 50 mJ/cm2. After exposure, the medium was replaced with fresh medium and incubated for 24 h in a CO2 incubator. After incubation, the collagen content in the ECM of the cells was determined by ELISA. Usually collagen is also secreted in the culture me- dium however, under our experimental conditions, signifi cant collagen was not detected in the culture medium. Hence, collagen was estimated in the ECM of the cells by ELISA. The collagen estimated in non-irradiated cells was 3.23 μg/ml. The collagen estimated in untreated UVB-irradiated cells was 0.285 μg/ml, whereas the collagen detected in em- blica-treated UVB-irradiated cells was 2.72 μg/ml and the collagen detected in ascorbic acid-treated UVB-irradiated cells was 1.05 μg/ml (Figure 2). Treatment with emblica extract signifi cantly provided protection from collagen damage in normal human dermal fi broblasts in a dose-dependent manner (at concentrations ranging from 0.125 to 0.5 mg/ml), with the maximum response of 9.5 ± 0.28-fold protection from collagen damage at a concentration of 0.5 mg/ml. Since ascorbic acid is reported to provide UV protection, we used ascorbic acid for comparison. Our results indicated that ascorbic acid showed only 3.7 ± 0.07-fold protection from collagen damage at a concentration of 0.5 mg/ml. ROS INHIBITION OF EMBLICA EXTRACT Reactive oxygen species (ROS) are generated by a variety of sources from the environment (e.g., photo-oxidations and emissions) and normal cellular functions (e.g., mitochondrial metabolism and neutrophil activation). ROS include free radicals (e.g., superoxide and Figure 1. Effect of emblica extract on cell viability. Normal human dermal fi broblasts were treated with various concentrations (0, 0.125. 0.25, 0.5, 1, and 2 mg/ml) of emblica extract for 24 h. Cell viability was analyzed by NRU assay. The data are represented as percentage of cell viability compared with the untreated control. The experiments were performed independently in triplicate, and the number of cells was 50,000 cells in each sample. The data are represented as mean ± S.D. and were analyzed by Student's t-test. *Signifi cant difference, p 0.05, compared to untreated control.

JOURNAL OF COSMETIC SCIENCE 54 hydroxyl radicals), nonradical oxygen species (e.g., hydrogen peroxide and peroxynitrite), and reactive lipids and carbohydrates (e.g., ketoaldehydes and hydroxynonenal) (14). ROS is the main cause for photoaging. As shown in Figure 3, we found that emblica ex- tract signifi cantly inhibited ROS induced by UVB exposure in normal human dermal fi broblasts in a dose-dependent manner. Untreated UVB-irradiated cells showed 84 ± 1.4% induction in ROS as compared to the non-irradiated cells. Emblica extract treat- ment lowered the induction of ROS in UVB-irradiated cells in a dose-dependent manner, with maximal reduction of ROS induced to 15 ± 4% at a concentration of 0.5 mg/ml, while ascorbic acid reduced the induction in ROS to 64 ± 2% at a concentration of 0.5 mg/ml (Figure 3). DISCUSSION UV irradiation is a major environmental factor responsible for a high incidence of premature skin aging, referred to as photoaging, as well as skin cancer and melanoma. UV irradiation Figure 2. Effect of emblica extract and ascorbic acid on UVB-induced collagen damage. Normal human dermal fi broblasts were treated with various concentrations (0, 0.125. 0.25, and 0.5 mg/ml) of emblica ex- tract and 0.5 mg/ml of ascorbic acid and irradiated with a UVB dosage of 50 mJ/cm2 . Followed by irradiation and 24-h incubation, the collagen content was estimated using the CosmoBio human collagen type 1 ELISA kit. 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 Student's t-test. **Signifi cant difference, p 0.05, compared to untreated UVB-irradiated cells. Figure 3. Effect of emblica extract and ascorbic acid on UVB-induced ROS generation. Normal human dermal fi broblasts were treated with various concentrations (0, 0.125. 0.25, and 0.5 mg/ml) of emblica ex- tract and 0.5 mg/ml of ascorbic acid and irradiated with a UVB dosage of 50 mJ/cm2. Followed by irradiation and 24-h incubation, the ROS induction was estimated. The data are represented as percentage of ROS induction compared with the non-irradiated control cells. 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 Student's t-test. **Signifi cant difference, p 0.05, compared to untreated UVB-irradiated cells.