ANTIOXIDANTS IN MUNG BEAN SPROUTS AND SAFETY FOR COSMETIC USE 213 Biotechnology Co., Ltd., Beijing, China) FRAP work liquid (Beyotime Institute of Bio- technology, Shanghai, China) Chick embryos (Merial Vital Laboratory Animal Technology Co.. Ltd., Beijing, China) Ferrozine (3-(2-pyridyl)-5, 6-diphenyl-1,2,4-triazine-4′,4″- disulfonic acid sodium salt), AR (Sigma-Aldrich, Poznań, Poland) Ferrous sulfate (FeSO4·7H2O), AR (Beyotime Institute of Biotechnology, Shanghai, China) Ethylene diamine tetraacetic acid (EDTA), AR (Beijing Chemical Reagent Company, Beijing, China). Other reagents were bought from Beijing Chemical works. SAMPLE PREPARATION Whole, unbroken mung beans without insect damage or mildew were chosen as the ex- perimental raw materials. Each group consisted of 12 g mung bean seeds, which were budded. The mung bean seeds were washed in water and disinfected by 0.1% sodium hypochlorite solution. Three-times-deionized water (W/V) was put in 26 constant- temperature incubators to soak the mung bean seeds for 16 h, until the mung bean shells burst to bud. To sprout the mung beans, they were put in petri dishes (Φ = 150 mm) that had been sterilized by 75% alcohol, with two layers of fi lter paper as a germination bed (14). The germinating seeds were kept moist with sterile water and incubated in the in- cubator without light at 26°C. Assays were performed daily for the next 8 days. EXTRACTI ON AND ANALYSIS OF COMPOSITION Every 4 g of sprout pieces (1.0 g, 0–8 days, respectively) with 20 ml water was kept at 50°C for 2 h and centrifuged at 5000 rpm for 5 min. The supernatant of the sample was used for the analysis of composition. Concentrations of polyphenols in the extracts of mung bean sprouts were measured by the Folin–Ciocalteu colorimetry (14). The Folin–Ciocalteu reagent was prepared by diluting the commercial reagent concentrate in a 1:4 ratio with water. The supernatant of the sample (0.3 ml), the Folin–Ciocalteu reagent (1.0 ml), 20% Na2CO3 (3.0 ml), and water (5.7 ml) were added in order and then kept in the dark for 2 h. Then the samples were Table I Results of Human Skin Patch Test Group Time (h) No change (0) Light erythema (1) Erythema, infi ltration, papules (2) Edematous erythema or papules (3) Signifi cant erythema with pimples or blisters (4) Negative control 0.5 32 0 0 0 0 24 32 0 0 0 0 48 32 0 0 0 0 Mung bean Sprouts (10 mg/ml) 0.5 32 0 0 0 0 24 32 0 0 0 0 48 32 0 0 0 0

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) ).