ANTIOXIDANT PROPERTIES OF FERMENTED MANGO LEAF EXTRACTS 7 accelerate aging, prompting researchers to explore natural substances that could neutral- ize free radicals (26). The antioxidative effects of variou s concentrations of the MLFE and the MEFE were evaluated by estimating the level of DPPH radical scavenging activity, as manifested in its EDA percentage. Figure 1 illustrates the increase in DPPH radical scavenging activity as EDA percentage increase from 33% at 2 mg/ml to 69% at 50 mg/ml for the MLFE and from 61% at 2 mg/ml to 72% at 50 mg/ml for the MEFE, thus demonstrat- ing that EM fermentation is more effective than lactobacillus fermentation at lower concentration levels but equivalent at higher levels (i.e., 50 mg/ml). The MLFE dem- onstrated signifi cantly higher antioxidative properties compared to the synthetic anti- oxidant, BHT, at all concentrations except for10 mg/ml (EDA 45%). Compared to BHT, the MEFE showed statistically higher EDA level for all levels of mango leaf fer- mentation extract concentrations. The exact mechanism of antioxidant action by the mango leaf extract is unknown, although previous reports have suggested that the material itself could promote cell proliferation or impart a protective effect from exter- nal stimulants (27–29). Table II Flavonoids in the MLFE and the MEFE (n = 3) MLFE concentration (mg/ml) Flavonoid concentration (mg/g) t p-Value MLFE MEFE 0.4 1.35±0.07 1.91 ± 0.02 −13.382 0.000 2 3.62 ± 0.02 5.84 ± 0.01 −171.00 0.000 10 8.89 ± 0.01 9.80 ± 0.05 −31.691 0.000 50 20.91 ± 0.04 16.74 ± 0.03 144.72 0.000 Figure 1. DPPH radical scavenging ability measured using percentage of EDA of the MLFE and the MEFE at different concentration levels (n = 3, mean ± S.D.).The synthetic antioxidant, B HT was used as control. P-values of t-tests comparing mean BHT and MLFE or MEFE are noted above.

JOURNAL OF COSMETIC SCIENCE 8 CELL VIABILITY ESTIMATION USING MTT ASSAY Using the MTT assay procedures performed in previous studies (30,31), this study evalu- ated cytotoxic effects of mango leaf fermented extracts. Figures 2 and 3 show the viabili- ties of RAW 264.7 cells for mango leaf fermentation extracts measured using the MTT assay. Compared to the control, the cell viability decreased from 97.70%, 93.26% to 85.82% as the MEFE concentrations increased from 0.5, 1.25 to 2.5 mg/ml (Figure 2). In the case of MLFE, cell viability measures were 102.78%, 94.88%, and 85.90% at concentrations of 0.5, 1.25, and 2.5 mg/ml, respectively, which clearly demonstrates a dose-dependent cytotoxicity effect (Figure 3). At 0.5 mg/ml, the MEFE and the MLFE both demonstrated similar safety profi le to control. The higher concentrations of fer- mented extracts (1.25 and 2.5 mg/ml) showed signifi cant increase in cytotoxicity ex- pressed in decreasing EDA% in contrast to control. REACTIVE OXYGEN SPECIES Macrophages produce excessive amounts of ROS (32,33). Figures 4 and 5 show the antioxidant effects of the MLFE and the MEFE evaluated by estimating the degree of ROS generation in LPS-stimulated macrophages using fl ow cytometry. Untreated cells showed weak DCF fl uorescence, indicating the minimal level of ROS generation. LPS treatment induced a large amount ROS, evidenced by the signifi cant rightward shift of DCF fl uorescence. Both MLFE and MEFE showed a concentration-dependent response. MLFE showed marginal inhibitory effect on ROS generation at a concentration of 0.125 mg/ml (Figure 4). However, the MEFE showed signifi cant inhibitory effects of ROS generation, evidenced by the remarkable leftward shift of DCF fl uorescence. At a concentration of 0.05 mg/ml, the MEFE showed the strongest antioxidative effects (Figure 5). The results show EM fermentation enhances the antioxidative effects of mango leaf extract. ESTIMATION OF TYROSINASE INHIBITORY ACTIVITY Tyrosinase inhibition may play an important role in the cosmetic industry and in the development of skin-whitening products (34). Tyrosinase is an enzyme involved in Figure 2. Effects of mango leaf extracts MLFE vs. Control on RAW 264.7 cell viability measured by MTT assay. Data expressed as mean ± S.D. (n = 3).