ANTIOXIDANT PROPERTIES OF FERMENTED MANGO LEAF EXTRACTS 5 DCFH-DA is a non-fl uorescent material that can permeate cell membranes. Within cells, it hydrolyzes to dichlorodihydrofl uorescein (DCFH), which in the presence of ROS is oxi- dized to fl uorescent dichlorofl uorescein (DCF), a high-performance fl uorescent substance. To quantify the fl uorescent cells, fl ow cytometry was also performed using a Flow Cytom- etry Caliber (Becton Dickinson, Franklin Lakes, NJ) with CellQuest Pro software. The percentage of cells in positive events was calculated as the events within the gate divided by total number of events then subtracting percentage of the control sample (untreated cells) (18). Tyrosinase inhibitory activity estimation. Tyrosinase inhibition activity was assessed using the modifi ed protocol of Masamoto et al. (19). To det ermine in vitro mushroom tyrosinase inhibition activity, 0.3 ml of 2.5 mM 3,4 dihydroxyphenylalanine (L-DOPA), 0.05 ml of the mango extract, and 1.5 ml of 0.1 M phosphate buffer solution (pH 6.8) were com- bined using a vortex mixer and pre-incubated at 25°C. Appro ximately 0.05 ml of mushroom tyrosinase (1.380 units/ml, Sigma-Aldrich) was placed into the vortex mixer and allowed to react for 2 min at 25°C. The absorbance was then measured using a UV/VIS spectrophotometer at a wavelength of 475 nm. The fol- lowing equation was used in the calculations for the percentage of tyrosinase inhibition: Tyrosinase inhibition (%) = [(A−B)/A] × 100% A = Difference in absorbance between 0.5 and 1 minutes in the reactant without the sample B = Difference in absorbance between 0.5 and 1 minutes in the reactant with the sample using 0.05 ml of 1380 units/ml mushroom tyrosinase. Nitrite-scavenging ability assessment. Nitrite scavenging was assessed using a modifi ed ver- sion of Gray and Dugan’s methodology ( 20). We combined 0.1 ml of 1 mM NaNO2 and 0.3 ml of sample MLFE and MEFE, respectively. Then, 0.2 M citrate buffer-HCl (pH 2.5) was added to produce the fi nal volume of 1 ml. The components were combined using a vortex mixer and incubated in a water bath at 37°C for 1 h, after which Griess’ reagents [30% acetic acid solution dissolved 1% sulfa- nilic acid and 1% naphthylamine = 1:1 (v/v)] and 3 ml of 2% acetic acid solution were added. After incubating for 15 min at room temperature, the absorbance of the mixture was measured using a UV/VIS spectrophotometer at a wavelength of 520 nm in three repeat examinations. The nitrite scavenging activity (%) was calculated using the follow- ing formula (21): Nitrite scavenging (%) = [1−(A−B)/C] × 100% A: Absorbance of test sample at 520 nm B: Absorbance of sample blank with H20 instead of NaNO2 at 520 nm C: Absorbance of control (H20) without test sample at 520 nm. STATISTICAL ANALYSIS This study is conducted to test the null hypothesis of equality of antioxidant activities for the MLFE and MEFE. The antioxidant activities at each concentration were expressed as

JOURNAL OF COSMETIC SCIENCE 6 mean ± S.D. The Student t-tests were carried out to test the difference in means with the signifi cance level of p 0.05 using SPSS 20.0 (IBM SPSS Statistics for Windows, Version 20.0, Armonk, NY). RESULTS AND DISCUSSION POLYPHENOL AND FLAVONOID CONTENTS Polyphenols affect the skin in a variety of ways and neutralize harmful oxidative species, thus acting as strong antioxidants (22). Polyphenols do not stimulate th e skin but suppress melanin production and, therefore, whiten the skin. This cosmetic effect may be due to a relationship between the amount of polyphenol used and the whitening effect (23). As outlined in Table I, the polyphenol content increased as the MLFE concentration in- creased from 7.43 ± 0.09 mg/g at 2 mg/ml to 30.58 mg/g ± 0.05 at 50 mg/ml. In the MEFE, the polyphenol content increased from 8.51 ± 0.05 mg/g at 2 mg/ml to 35.21 ± 0.04 mg/g at 50 mg/ml. The statistically signifi cant increases in polyphenol contents were detected for the MEFE in comparison to the MLFE for all concentration levels ex- cept at 10 mg/ml. However, a dose-dependent increase in polyphenol content was ob- served in both the MLFE and the MEFE. Fermentation-dependent fl avonoid contents are outlined in Table II. The fl avonoid con- tent in the MLFE increased from 1.35 ± 0.07 mg/g at 0.4 mg/ml to 8.89 ± 0.01 mg/g at 10 mg/ml. In addition, the fl avonoid content in the MEFE increased from 1.91 ± 0.02 mg/g at 0.4 mg/ml to 9.80 ± 0.05 mg/g at 10 mg/ml. These results confi rm that the fl avonoid content increased with increasing the MLFE and MEFE concentrations. The MEFE again yielded statistically higher concentration of fl avonoid at all concentra- tion levels except at 50 mg/ml where the MLFE yielded a higher fl avonoid content than the MEFE. These results are consistent with the previous study (24). In our study, we estimated that the change in intensity of mango leaf fermentation concentration infl uenced the fl avonoid con- tent, thus confi rming the antioxidative property of the mango leaf fermentation extracts (25). DPPH RADICAL SCAVENGING ACTIVITY DPPH radical scavenging activity allows the measurement of antioxidant activity based on EDA. Free radicals within the body react with proteins and other substances to Table I Polyphenols in the MLFE and the MEFE (n = 3) MLFE concentration (mg/ml) Polyphenol concentration (mg/g) t p-Value MLFE MEFE 0.4 3.38 ± 0.05 5.84 ± 0.02 −78.642 0.000 2 7.43 ± 0.09 8.51 ± 0.05 −18.262 0.000 10 22.52 ± 0.07 17.91 ± 0.06 86.552 0.000 50 30.58 ± 0.05 35.21 ± 0.04 −127.735 0.000