| Selenium （Se） accumulation in germinated brown rice and the purification of selenoproteins and their antioxidant and antitumor activities were studied in this paper. Mainly in brown rice cultivars selection for Se enrichment and the optimization of culture conditions, effects of Se on the changes of physical activities and the mobilization of major nutrients of brown rice during germination, Se accumulation in tissues and protein fractions of Se-enriched brown rice, the purification and identification of Se-containing proteins extracted from Se-enriched brown rice and their antioxidant and antitumor activities in vitro. The major results were summarized as follows:1. Total and protein-bound Se contents were singnificantly increased by adding sodium selenite or sodium selenate. The concentrations of selenite or selenate less than 60μmol/L had no significant effets （P> 0.05） on the gemination rates and length of sprouts. However, they were significantly inhibited （P<0.05） when the concentrations increase further. During germination, selenite and selenate had different metabolic pathways in brown rice. Total Se content in Se-enriched brown rice were Michaelis-Menten trend （R2=0.974, P<0.01） increase when cultivated with selenate, however, a linear trend （R2=0.985, P<0.01） increase was observed when cultivated with selenite. Selenite was easily transformed into selenoproteins compared to selenate, and it was suitable for the producing of Se-enriched brown rice. Significant differences （P<0.01） were found among cultivars with respect to the capacities for Se accumulation. In the selection of ten cultivars brown rice, Z8 and X57 showed a stronger ability than the other cultivars. Germination temperature, germination time and selenite concentrations significantly affected Se accululation and transformation in brown rice. A Box-Behnken experiment design and response surface method was used to optimize the germination conditions of brown rice based on the single factor experiments. In the range of temperature of 20-30℃, germination time of 2-4 d and the selenite concentrations of 30-90μmpl/L, the optimum conditions were temperature 26℃, germination time 4 d and selenite concentrations of 52μmpl/L. The total Se content of Se-enriched brown rice was 9.19μg/g under the optimum conditions, and the protein-bound Se occupied 54.21%.2. Effects of Se on the changes of physical activities and the mobilization of major nutrients of brown rice during germinating were studied. Selenite had no significant influence on the respiration rates of brown rice when the concertrations were lower than 60μmol/L. However, when cultivated with 60μmol/L selenite, the respiration tares of Z8 and X57 decreased by 19.7% and 22.6%, respectively. In the range of 0-90μmol/L, selenite had no sinnificant influences on the loss of dry matter of germinated brown rice. There was a corresponding relationship between reducing sugars and starch contets in germinated brown rice, and they were significantly affected by the selenite concentrations. When the concentration of selenite was higher than 90μmol/L, it inhibited the starch degradation, and consequently the contents of reducing sugars were decreased. In Z8 and X57, starch contents were increased by 9.53% and 8.26% respectively, while the contents of reducing sugars were decreased by 13.47% and 19.37% respectively when 90μmol/L selenite was added during germination. However, starch and reducing sugar contents were not significantly affected （P> 0.05） when the concentrations of selenite was in the range of 10-60μmol/L. Tere was a negative correlation between soluble protein contents and selnite concentrations. In Z8 and X57, the soluble protein contents decreased by 19.38% and 16.72% respectively when they were cultivated with 90μmol/L selenite. Contents of free amino acids had largest value when the concentration of selenite was 10μmol/L, in Z8 and X57, they increased by 12.80% and 14.82% respectively, while cultivated with 90μmol/L selenite, they were decreased by 9.02% and 12.72%, respectively. Germinated brown rice contains a variety of protease isozymes, low concentrations of selenite can significantly improve the protease activitives, while they were inhibted at high selnite concentrations. Changes of GSH-Px activitives had the same regularity of proteases.3. Distribution of Se in Se-enriched brown rice and protein fractions was nonuniform. It decreased remarkably from bran layers to endosperm, and the sprouts had the highest Se concentration. About 40-50% of Se accumulated in the tissues of sprouts, bran layers and outer endosperm, which accounted only 10-15% of weight of Se-enriched brown rice. Changes of albumin, globulin, prolamin and glutelin were related to germination time and concentrations of selenite. Except prolamin, all the other three protein fractions decreased significantly after germination, but the degradation of albumin and globulin earlier than glutenin. When selenite concentrations ranged of 0-60μmol/L, it had no significant effects on the contents of prolamin and glutelin, while it showed a promoting effect on albumin and globulin degradation at low concentration. The four protein fractions were combined with different levels of Se, total and prtotein-bound Se contents in the order of albumin> globulin> prolamin> glutelin. However, the ratio of protein-bound Se to total Se showed different trend. Albumin had the highest contents of total and protein-bound Se among the four protein fractions, however, it showed the lowest ratio of protein-bound Se to total Se and prolamin had the highest value, through the lowest total and protein-bound Se contents were observed in it. Protein molecular weights of Se-enriched brown rice mainly distributed in the range of 13.6-121.4 kDa, and Se was able to combine in various molecular weight proteins. However, their capacities of combinding Se were negatively correlated with their molecular weights. Total of 84.3% of Se was distributed in the proteins whose molecular weights were not higher than 36.3 kDa.4. Sephadex G-100 filtration and DEAE-Sephcrose-FF ion exchange chromatography were used for separation and purification selenoproteins extracted from Se-enriched brown rice. The purified proteins with molecular weights of 14.3-15.2 kDa showed a single band analyzed by SDS-PAGE. Amino acids compositions of the purified proteins were determined by HPLC. The results showed that the Glu was predominate, accounting for 17.97-21.49% of total amino acids, followed by Leu, while contents of Cys, His and Met were less than 1.5%. Compared with protein amino acids, contents of SeCys2 and SeMet were accounting for 0.091-0.012μg/100μg and 0.015-0.017μg/100μg, respectively. However, the Se-MSC was not detected. The MALDI-TOF-MS peptide mass fingerprint identification showed that these three groups of proteins were hypothetical protein LOCOs12g27080, hypothetical protein OsJ19801 and Os01g0348900, whose moleculars weights were 5.11,14.93 and 15.16 kDa, respectively. All these proteins were from japonica rice （Oryza sativa, japonica cultivar-group）, consistent samples properties.5. The reducing power, hydroxyl radical scavenging activities, superoxide radical scavenging activities, DPPH free radical scavenging activities and the effects on proliferations of human hepatocarcinoma HepG2 cell lines and human gastric cancer MGC-823 cell lines of selenoproteins extracted from Se-enriched brown rice were studied. Selenoptoteins of F1, F4 and F5 extracted from Se-enriched brown rice showed strong antioxidant activities. F1 had higher hydroxyl radical scavenging ability than F4 and F5, while F5 had the highest superoxide radical and DPPH radical scavenging capacities. These three selenoproteins showed significant inhibitions on the proliferations of human hepatocarcinoma HepG2 cell lines and human gastric cancer MGC-823 cell lines, in which F4 had stronger inhibition than F1 and F5. With the increase of added concentrations, these three selenoproteins showed higher inhibitions. Morphological observation showed that these three selenoproteins were able to promote cells of human hepatocarcinoma HepG2 and gastric cancer MGC-823 apoptosis. |
