ANTIOXIDANT PROPERTIES OF FERMENTED MANGO LEAF EXTRACTS 3 10 ml 50% Ethanol) was prepared by heating the solution on Ho t plate stirrer (PC -4200, Co rning Inc., Corning, NY) for 4 hours at 80°C. The extract vapor was condensed and returned to extract liquid using a Re fl ux condenser (Labpia, Seoul, Korea) to control the rate of heating and to prevent loss of extract through evaporation. The extracted leaves were cleared of impurities using fi lter paper (Advantec No. 2, Tokyo, Japan) and a rotary evaporator (Rotary Evaporator N-1000SW, Eyela, Tokyo, Japan). Mango leaf extracts were kept in a deep freezer (−62°C) (DE8525, Ilshin Lab Co. Ltd., Dongduchon, Korea) for a day, lyophilized at 0 mTorr in a vacuum between −70° and −60°C (FD5508, Ilshin Lab Co. Ltd., Dongduchon, Korea), and air-sealed until analysis. PREPARATION OF MANGO LEAF FERMENTATION EXTRACT To prepare mango leaf lactobacillus fermentation extract (MLFE), we inoculated Lactoba- cillus casei (KCTC2180, Sahmyook University, Seoul, Korea) in a 55 mg/ml sterile MRS broth and incubated the culture at 37°C for 1 day to be used as the main culture. Differ- ent amounts of mango leaf extracts were each added to 10 ml of distilled water. Ap- proximately 0.1 ml of the seed culture with Lactobacillus casei concentration of 6.2 × 10 colony forming units (cfu)/ml was added, mixed thoroughly, and maintained in an incu- bator (Wonil Incu1, Wonil Tech., Jeonju, Korea) set at 37°C for 2 days to obtain a fer- mentation broth. To prepare mango leaf effective microorganism fermentation extract (MEFE), we obtained an EM Activity Solution (EverMiracle, Jeonju University, Jeonju, Korea). EM Activi ty Solutions (5%) and sugar (molasses, 5%) were combined in distilled water (90%) and fermented (or activated) at 37°C for 7 days in a sealed container. Upon ensuring the acti- vated EM solution’s pH is under 3.5, we added 0.1 ml of seed culture with lactobacillus concentration of 6.2 × 10 cfu/ml to 10 ml of distilled water containing different amounts of mango leaf extracts to obtain fermentation broth of varying concentrations for anti- oxidant activity tests. CELL CULTURE The cell line utilized in this study was RAW 264.7 (mouse leukemic monocyte macro- phage cell line (KCLB 40071)) obtained from Korean Cell Line Bank (Seoul, Korea). The cells were cultured at 37°C in a 5% CO2 incubator (Thermo Scientifi c, Waltham, MA) using Dulbecco’s modifi ed Eagle’s medium (Gibco® DMEM, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco® FBS, Life Technologies) and 1% antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin) (11). ANTIOXIDANT ACTIVITY TESTING Polyphenol measurement. The amount of polyphenol per gram of extract was assessed using the Folin–Denis me thod (12). Approximately 100 μl of the extract and 1 ml of 2% Na2CO3 were mixed and reacted for 2 minutes at room temperature. After the reaction, 50% Folin–Ciocalteu’s phenol reagent (100 μl) was added, vortexed, and incubated for

JOURNAL OF COSMETIC SCIENCE 4 30 min at room temperature. Subsequently, the absorbance of the mixture was measured using the UV/VIS spectrophotometer (OPTIZEN 2020UV plus, Mecasys Co., Daejeon, Korea) at a wavelength of 750 nm. Using Tannic acid (Sigma-Aldrich Korea Ltd, Youngjin-city, Korea) as a standard substance, the replicates were tested three times for the analysis. Flavonoid measurement. The fl avonoids content per gram of extract was measured using the colorimetric method in the presence of diethylene glycol (13). Approximately 100 μl of the extract, 100 μl of 1 N NaOH, and 1 ml of diethylene glycol were combined using a vortex mixer and incubated for 1 h in a 30°C water bath. Following the reaction, the absorbance of the mixture was measured using a UV/VIS spectrophotometer at a wave- length of 420 nm. Catechin (Sigma-Aldrich Korea Ltd) was used as a standard substance for three repeat examinations. DPPH radical scavenging ac tivity measurement. The DPPH radical scavenging activity was measured using a modifi ed version of the method described by Blois (14). First, we pro- duced 500 mM of DPPH using 0.1 M Tris base-HCl buffer (Tris buffer) at pH 7.4 and methanol. Then, a sample aliquot of 100 μl, 400 μl of 0.1 M Tris buffer and 500 μl of 500 mM DPPH were combined and vortexed. The mixture was reacted for 30 min in the dark at room temperature and the absorbance of the mixture was analyzed using a UV/VIS spec- trometer at 517 nm in three replicate runs. The artifi cial antioxidant butylated hydroxytoluene (BHT) was used as a control and the DPPH radical scavenging activity was expressed as electron donation abilit y (EDA) percent- age, a percentage based on the differences in absorbance of DPPH solutions between absence (A_initial) and presence (A_fi nal) of antioxidant samples (BHT, MLFE, or MEFE) (15): EDA (%) = (A_initial – A_fi nal/A_initial) × 100%. Cell viability estimation using MTT assay. Cell viability was measured using the methodol- ogy of Mosmann (16). RAW 264.7 cells were seeded onto 96-well plates at a density of 3 × 105 cells/well and cultured at 37°C in a 5% CO2 incubator. The cells were cultured for another 24 h after treatment using various concentrations of the MLFE and MEFE extracts. After culturing, 100 μl of MTT solution was placed into each well, cultured for 4 hours, and the formazan precipitate was then dissolved in 100 μl of dimethylsulfoxide after the removal of the supernatant. An ELISA microplate reader (E-max, Molecular Device, Sunnyvale, CA) was used to measure the absorbance at 570 nm in three replicate viability assays. Cell viability is expressed in percentage using the following formula (17): Cell viability (%) = (A−B)/A × 100% A: Absorbance of untreated RAW 264.7 cells at 570 nm B: Absorbance of extract sample at 570 nm. ROS production measurement. RAW 264.7 cells (4 × 105 cells) were seeded in a glass bottom dish (MatTek Corp., Ashland, MA) and incubated for 24 h. Cells were treated with a various amounts of the MLFE or MEFE for 10 hours and incubated for 20 h with 1 ml of 1 μg/ml LPS (Sigma-Aldrich Korea Ltd: E. Coli 0111:B4, L3024). Then, cells were treated with 10 μM of dichlorofl uorescein-diacetate (DCFH-DA, Sigma-Aldrich) for 0.5 h and harvested with trypsin/EDTA (Gibco) for three separate fl ow cytometric analyses.