JOURNAL OF COSMETIC SCIENCE 50 Ultraviolet-B (UVB) 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. The majority of UV energy is absorbed by unidentifi ed photosensitizers in the cells that are postulated to generate reactive oxygen species (ROS). UV irradiation in- creases ROS production via PKCdelta signaling in primary murine fi broblasts (3). UVB irradiation has been demonstrated to produce ROS in the cells and skin, which induces the synthesis of matrix metalloproteinases (MMPs), causing skin photoaging (4). Photo- aging is a process involving alteration of type I collagen, the major component of dermis on exposure to UV radiation. Long-term exposure to UV radiation induces various cutaneous changes that differ because of physiological aging, including structural destruction of dermal collagen fi ber bundles, which comprise the major component of the dermis (5). Wrinkling, a representative change in skin surface associated with photoaging, is often seen at the corners of the eyes and in the space between the eyebrows (5). Type I collagen degra- dation is the major cause of wrinkle formation. Several factors promoting this degra dation process have been identifi ed, including UV radiation and ROS (6). Intracellular and extra- cellular oxidative stress initiated by ROS advance skin aging, which is characterized by wrinkles and atypical pigmentation. Because UV enhances ROS generation in cells, skin aging is usually discussed in relation to UV exposure. The use of antioxidants is an effec- tive approach to prevent symptoms related to photo-induced aging of the skin (7). Strat- egies to prevent or at least minimize ROS-induced photoaging and intrinsic aging of the skin necessarily include protection against UV irradiation and antioxidant homeostasis (8). The level of procollagen type I protein in photoaged skin is lower than that in naturally aged skin. The level of matrix metalloproteinase-1 protein and the activity of matrix metalloproteinase-2 were higher in the dermis of photoaged skin than in naturally aged skin (9). Emblica extract has type I pro-collagen promoting and anti-collagenase activity that is attributed to its anti-aging potential (10). Emblica fruit had been claimed to be a rich source of ascorbic acid, and its high antioxidant potential was earlier attributed to the presence of ascorbic acid. Also, it has become obvious that the high ascorbic acid content could contribute to the anti-aging and anti-photoaging properties of emblica extract. However, recent studies have shown only trace amounts of ascorbic acid in em- blica fruits (2). Therefore, we studied the comparative UVB protection effi cacy of ascorbic acid and emblica extract. To investigate the effect of emblica extract on UVB-induced collagen damage, we initially studied the cell viability response to emblica treatment in normal human dermal fi broblast cells. In continuation with earlier studies on emblica extract, the objective of the present study was to investigate the protective effect of em- blica extract in comparison to ascorbic acid against UVB-induced ROS and the collagen damage that results in photoaging. MATERIALS AND METHODS MATERIALS Normal human dermal fi broblasts (from adult donors) and its ready-to-use growth medium were obtained from PromoCell GmbH, Heidelberg, Germany. A neutral red stain (NR) was procured from Himedia Laboratories, Mumbai, India. A human collagen type I ELISA kit was purchased from Cosmo Bio (Carlsbad, USA). Procollagen type-I goat

PROTECTION FROM PHOTOAGING BY P. EMBLICA EXTRACT 51 polyclonal antibody, collagen type-I goat polyclonal antibody, and FITC-coupled donkey anti-goat IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A Bio-Sun [R] Vilber Lourmat stimulator fi tted with a UVB irradiation source composed of T20.M 312-nm Vilber Lourmat tubes, low-pressure mercury vapor tubes, hot cathodes, and a Vilber Lourmat RMX-365/312 radiometer with a microprocessor programmable in en- ergy with time was procured from Marne la Vallee, France and was used as the source of the UVB irradiation. EXTRACTION AND SAMPLE PREPARATION The fresh fruits of P. emblica were procured locally from Bangalore, India, and freed from dust or other organic matter. The fruits were cut into small pieces and macerated with cold water at room temperature for 2 h with continuous stirring. The fi ltered juice was freeze-dried to obtain the dry powder form. Various samples of emblica extract were pre- pared by dissolving the dry emblica extract powder in deionized water at the indicated concentrations. CELL VIABILITY ASSAY Cell viability was determined by a neutral red uptake (NRU) colorimetric assay. Cells in 96-well plates were incubated with 30 μg/ml of neutral red prepared in a pre-warmed culture medium for 3 h at 37°C. The excess dye was then washed off with phosphate buffer saline. The dye was extracted in a 100 μl/well of developer solution consisting of 25 ml of water, 24.5 ml of ethanol, and 0.5 ml of glacial acetic acid at room temperature for 20 min. The optical density was measured at 492 nm using a microplate reader. The relative percentage of cell survival was calculated by dividing the absorbance of the treated cells by that of the control in each experiment. IMMUNOCYTOCHEMISTRY Twenty-four hour cultures of normal human dermal fi broblasts in a six-well plate, with or without exposure to UVB irradiation of 50 mJ/cm2, were fi xed and incubated with pro-collagen type-I goat polyclonal antibody or collagen type-I goat polyclonal antibody for 1 h at room temperature. After being washed with TBS, the cells were incubated with secondary antibody (FITC-coupled donkey anti-goat IgG) for 1 h with gentle rocking at room temperature. The cells were then washed, trypsinized, and resuspended in PBS. Two hundred microliters of each sample was transferred to a 96-well black microplate and the fl uorescence intensity was measured by a fl uorescence microplate reader set for excitation at 485 nm and emission detection at 535 nm (10,11). ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) After specifi c treatment of the cells with the sample and UVB irradiation of 50 mJ/cm2, the culture medium was removed and the cells were detached using a cell scraper. The cells were then treated with 0.1 mg/ml of pepsin in 50 mM of acetic acid and incubated