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
JOURNAL OF COSMETIC SCIENCE 52 overnight at 4°C with continuous shaking. After incubation, the samples were centri- fuged at 10,000g for 10 min and the supernatant was neutralized with neutralization solution containing 200 mM of Tris and 150 mM of NaCl. The assay sample and the biotinylated anti-collagen antibody solution were mixed well in the ratio of 1:9. Then 50 μl of each of the samples was added to the wells of the collagen-coated microplate and incu- bated for 1 h at 28°C with moderate shaking. After washing with wash buffer, 50 μl of Avidin-horseradish peroxidase conjugate was added to each well and incubated for 1 h at 28°C with moderate shaking. After washing the wells, 50 μl of 3,3′,5,5′-tetramethylben- zidine (TMB) substrate was added and incubated for 15 min at 28°C without shaking. After incubation, 50 μl of a stop solution was added and the optical density was measured at 450 nm within 10 min. The assay is a competitive enzyme immunoassay and the opti- cal density is proportional to the collagen content. The culture medium of the cells was also analyzed in a similar manner to estimate the collagen content. The assay was per- formed using a human collagen type I ELISA kit purchased from CosmoBio (Carlsbad, USA) as per the kit data sheet. ROS INHIBITION ASSAY ROS was estimated using DCFH-DA dye (12,13). After specifi c treatment, the cells were incubated with 0.002% DCFH-DA dye for 1 h at 37°C. The fl uorescence intensity was measured by a fl uorescence microplate reader set for excitation at 485 nm and emission detection at 520 nm. The increase in fl uorescence is proportional to the ROS induced. The percentage of ROS induced is calculated with respect to the fl uorescence intensity of non-irradiated control cells: % ROS enhanced = {[100/A] * B} 100 where A is the fl uorescence of non-irradiated cells (control) and B is the fl uorescence of UVB-irradiated cells with and without sample treatment. RESULTS EFFECT OF EMBLICA EXTRACT ON NORMAL HUMAN DERMAL FIBROBLAST VIABILITY Cells were treated with varying concentrations of emblica extract (0.125, 0.25, 0.5, 1, and 2 mg/ml). Cell viability was determined after 24-h incubation by NRU assay. Treatment of cells with emblica extract did not have signifi cant effect on cell viability at a concen- tration of 0.125–0.5 mg/ml. At higher concentrations, emblica extract had a toxic effect, as indicated by the decrease in cell viability to 67% and 42% at treated doses of 1 mg/ml and 2 mg/ml, respectively (Figure 1). EFFECT OF EMBLICA EXTRACT ON UV-INDUCED COLLAGEN DAMAGE IN NORMAL HUMAN DERMAL FIBROBLAST CELLS When 24-h cultures of normal human dermal fi broblasts were irradiated with 10, 20, 30, 50, and 100 mJ/cm2 of UVB irradiation, a 50% inhibition in cell viability and a maximal enhancement in ROS generation up to 53% was observed at 50 mJ/cm2. It was also
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