JOURNAL OF COSMETIC SCIENCE 214 shaken thoroughly and the absorbance was measured at 765 nm using a microplate reader. The standard curve was constructed using gallic acid. Total proteins were measured using aluminum nitrate colorimetric assay developed by Qu et al. (15), in which 1.0 ml of extracts of mung bean sprouts were mixed with 5.0 ml of the confi gured Coomassie Brilliant Blue G-250 for 10 min. Then the samples were mixed and the absorbance was measured at 595 nm using a microplate reader. The stan- dard curve was constructed using bovine serum albumin. ANTIOXIDANT ACTIVITY Scavengin g of DPPH radicals. DPPH radical scavenging rates in the extracts were measured by the DPPH method according to Li et al. (16). Seed pieces (1.0 g, 0–8 days, respectively) with 5.0 ml water were extracted by ultrasound for 30 min and centrifuged at 5,000 rpm for 5.0 min. For each sample, an aliquot of 0.5 ml at different concentrations was added to 1.0 ml DPPH solution. Methanol was used as a blank solution. Ascorbic acid was used as the positive control. The decrease in absorbance was measured at 517 nm. Total antioxidant capacity assay. The total antioxidant capac ity of the extracts was mea- sured by the FRAP method. We analyzed our results using a similar method to that used by Wang et al. (17). In 96-well plates of each detection hole, we added 180 μl FRAP working liquid. In the blank control hole we added 5 μl distilled water. We then added 5 μl of FeSO4 standard solution at concentrations of 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mM to the standard curve detection hole. We took 5 μl cultivated bean sprout samples ex- tracted from 0 to 8 days after germination and added these to the sample detection hole in proper sequence, each group of three parallels, in addition to 0.15–1.5 mM Trolox reagent as the positive control. Gently shaking it, we then incubated it at 37°C for about 5–7 min, and then the decrease in absorbance was measured at 593 nm. Finally, according to the standard curve, we calculated the total antioxidant capacity of the sample. Chelation abilities of ferrous ion. Fe(II) chelation activity of water extract from mung beans and their sprouts (V. radiata) was examined using ferrozine (18). Solutions (0.1 ml) were mixed with 0.1 mL of 1.0 mM FeSO4·7H2O, and then 0.1 ml of 5 mM ferrozine and 2. 7 ml deionized water were added. Then the reaction mixture was left for 10 min at room temperature and absorbance at 562 nm was measured. The positive control was prepared in the same way, but EDTA was added instead of the sample extract. Deionized water was selected as a blank control. The percentage of Fe(II) bound was calculated according to the following formula: chelat- ing capability = [(Acontrol − Asample)/Acontrol] × 100%. SAFETY DETERMINATION Red blood cell test. Potential irritation by mung bean sprout extracts (200 mg/ml) was de- tected using the RBC test according to the method of Xue et al. (19–20). 0.4% SDS and PBS were used as positive control and negative control, respectively. The hemolysis ratio was given by the equation H (%) = 100% × (OD530 nm (sample) − OD530 nm (negative control) )/ (OD530 nm (positive control) − OD530 nm (negative control) ).

ANTIOXIDANTS IN MUNG BEAN SPROUTS AND SAFETY FOR COSMETIC USE 215 Chicken chorioallantoic membrane assay. Potential irritation of mung bean sprout extracts was detected using the chicken CAM assay according to the methods of Wang et al. (21) and Bi et al. (22). 0.4% SDS and 0.9% normal saline were used as positive control and negative control, respectively. Morphological changes were observed and data were ana- lyzed using SPSS 17.0 software. Human skin patch test. According to the method of Zheng et al. (23), skin toxicity of mung bean sprout extracts (200 mg/ml) was detected with the human skin patch test. Thirty- two subjects (23 females and 9 males, 20–30 years old) were chosen extracts were applied on their arms for 24 h. Then results were classifi ed into fi ve grades according to the pro- cedure set out in Hygienic Standard for Cosmetics (24). STATISTICAL METHODS The analyses of the data were done using the IBM SPSS Statistics v17.0 statistical pack- age (IBM Corporation, New York, NY). The experimental data were subjected to χ2 tests, with p 0.05 as a signifi cant difference. CONCLUSIONS Mung bean sprout extracts have good antioxidant property. Compared with mung bean seeds, the antioxidant capacity of sprout extracts has improved signifi cantly after bud- ding for 4 days. Later, the antioxidant capacity shows a trend of fl uctuations. After bud- ding for 8 days, the DPPH removal effect is the best. Mung bean sprout extracts have remarkable Fe(II) chelation activity. The effectiveness of the chelation of the iron ions tested remains at high level during the beginning of the germination (0–4 days). Safety tests show that extracts of mung bean sprouts from 0 to 8 days after germination are safe and nonirritating to human skin. Therefore, mung bean sprout extracts can be used as potential antioxidant additive to cosmetics for a wide range of applications. ACKNOWLEDGMENT This article was supported by Beijing Municipal Education Commission (KM200910011001). REFERENCES (1) S. Wang, J. Lin, M. Ye, T. Ng, P. Rao, and X. Ye, Isolation and characteri zation of a novel mung bean protease inhibitor with antipathogenic and anti-proliferative activities, J. Peptides, 27, 3129–3136 (2006). (2) A. Mubarak, Nutritional composition and antinutritional factors of mung bean seeds (Phaseolus aureus) as affected by some home traditional processes, J. Food Chem., 89, 489–495 (2005). (3) F. Lai, Q. Wen, L. Li, H. Wu, and X. Li, Antioxidant activities o f water-soluble polysaccharide extracted from mung bean (Vigna radiata L.) hull with ultra-sonic assisted treatment, J. Carbohydr. Polym., 81, 323–329 (2010). (4) Y. Wang, Study on the dynamic chan ge of total fl avonoids content and its antioxidant function in ger- minative mung bean. Master’s thesis, J. Jilin. Agr. Univ. (2011).