JOURNAL OF COSMETIC SCIENCE 2 their bioavailability owing to the preservation of nutritional components during fermen- tation. In addition, microorganisms secrete various hydrolyt ic enzymes and release bioac- tive substances bonded to intracellular components (2). Koreans traditionally consume high amounts of fermented food such as kimchi, miso, and soy sauce. With the recent unveiling of physiological activity of fermented foods, intensive research has been conducted to use such foods as health-promoting functional materials. Broadly speaking, fermentation can be regarded as the microorganism-mediated production of useful bioma terials. Microorganisms assimilate nutrients and contribute to the maintenance of life and growth, as well as generate energy through their enzymatic degradation of nutrients. Foolad et al. (3) reported that children with allergic skin diseases could benefi t from the intake of food supplements containing probiotic compo- nents, which have preventive and inhibitory effects against diseases Lactobacillus rhamnosus GG, a strain of L. rhamnosus, showed a substantial long-term preventative effect. The in vivo formation of reactive oxygen species ( ROS), oxygen-derived highly reactive metabolic substances such as hydrogen peroxide, superoxide anion, hydroxyl radical, and oxygen-free radicals, which inevitably occur during aerobic metabolism, generate toxic- ity that can disrupt normal physiolog y (3). ROS provoke damage to multiple cellular organelles and metabolic processes, thereby being involved in aging, cancer, cardiovascu- lar diseases, and infl ammation. Such free radicals are usually removed or suppre ssed in vivo by antioxidants. It has been reported that natural antioxidants such as vitamin C, carotenoids and phenolic compounds (i.e., fl avonoids, tannins, cumminoids, etc.) are highly effective in inhibiting aging in vivo or preventing atherosclerosis, infl ammation, degenerative diseases, and cancer (4–7). Against the backdrop of recent intensive research on improving fermentation-induced physiological functionality, this study aimed to evaluate the antioxidative properties of fermented hot-water extracts of mango leaves. Spectrophotometry was used to estimate the total polyphenol and fl avonoid contents in mango leaf extracts fermented with either lactic acid bacteria or other effective microorganisms (EM). EM refers to the combination of probiotic and/or anaerobic microbes in commercial agricultural products, pharmaceu- ticals, and nutritional supplements based on the trademarked product, EM-1® Microbial Inoculant (8,9). The antioxidant levels were also studied using various tests including 1,1-diphenyl-2-picrylhydrazyl (DPPH)-mediated electron-donating activity, nitrite- scavenging activity, ROS production, and cytotoxicity via the MTT colorimetric assay. The MTT assay uses the tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, along with a NAD(P)H-dependent cellular oxidoreduc- tase enzyme to assess cell viability (10). MATERIALS AND METHODS SAMPLES AND EXTRACTION Mango leaves were harvested in September from a mango farm in Jeju-do, Korea, dried at room temperature (25°C) for 2 weeks with room relative humidity of 40–50% , placed into a grinder, and subjected to extraction. The dried leaves were accurately weighed us- ing an analytical balance. Subsequently, the 10-fold extract (1 gram dried mango leaf in

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