J. Cosmet. Sci., 66, 1–13 (January/February 2015) 1 Antioxidant properties of fermented mango leaf extracts ANNA PARK, TAEKYU KU, and ILSOU YOO, Department of Advanced Organic Materials Engineering, Faculty of Engineering and Applied Science, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea. Accepted for publication January 11, 2015. Synopsis Antioxidant properties of mango (Mangifera indica) leaves were evaluated. Hydroalcoholic leaf extracts that were lyophilized were subsequently fermented with either Lactobacillus casei or effective microorganisms (EM) such as probiotic bacteria and/or other anaerobic organisms. Antioxidant properties were measured as a func- tion of the mango leaf extract concentration in the fermentation broth. Tests for radical scavenging using the 1,1-diphenyl-2-picrylhydrazyl radical showed higher antioxidant ac- tivity for Lactobacillus- and EM-fermented mango leaf extracts than for the synthetic antioxidant butylated hydroxytoluene. Antioxidant activity generally increased with increasing fermented extract concentration as did the fermented extracts’ polyphenol and fl avonoid contents. Fermented extracts reduced reactive oxygen species generation by lipopolysaccharide in RAW 264.7 cells when measured via fl uorescence of dichlorodihydrofl uorescein acetate treated cells using fl ow cytometry. RAW 264.7 cells also showed a concentration-dependent cytotoxic effect of the fermented extracts using the 3-(4,5-dimethylthialol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Inhibition of mushroom tyrosinase activity as well as nitrite scavenging by the fermented extracts increased as fermented extract concentrations increased. Tyrosinase activity was assayed with 3,4-dihydroxyphenylalanine as substrate. Nitrite scavenging was assessed via measurement of inhibition of chromophore production from nitrite –naphthylamine–sulfanilic acid mixtures. The antioxidant properties of fermented mango leaf extracts suggest the fermented extracts may be useful in developing health food and fermentation-based beauty products. INTRODUCTION Among the components of mango, mangiferin and catechol oxidase are recognized for their colon cleansing effects and capacity to enhance resistance against pathogens and diseases. Mango also contains high levels of tryptophan, a precursor of the neurotransmit- ter serotonin (1). Lupeol, a triterpene present in mango, is known as an effective inhibitor of cellular growth in both prostate and skin cancers (1). Extensive research efforts have focused on the fermentation of natural bioactive substances using microorganisms. Microbial fermentation of natural substances is known to enhance Address all correspondence to Anna Park at dranna@hanmail.net.

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