| Lunasin is a kind of soybean bioactive peptide,which process biological activities such as anti-oxidation,anti-inflammation,anti-cancer and reducing cholesterol in vivo.However,it was difficult to realize the industrial production and commercial utilization of Lunasin due to its low content(about 0.5 ? 1.2 mg/g)and complexity of extraction and purification.In this paper,we studied the influencing factors controlling accumulation of Lunasin in germinated soybean,the functional activity of Lunasin and the heterogenous expression of Lunasin peptide in millet in order to lay a foundation for the application of Lunasin.Firstly,the effects of germination temperature,germination time and Na Cl concentration on Lunasin accumulation in soybean germination were studied.The content of Lunasin in bean sprouts was determined by Western blot and enzyme-linked immunosorbent assay(ELLISA).The results showed that the optimum conditions for Lunasin accumulation in soybean germination under Na Cl stress were as follows:Na Cl concentration 50 m M,germination time 6h,germination temperature 25?.Under these conditions,the Lunasin content in soybean sprouts was 2.34 mg/g.Secondly,the biological activity of Lunasin peptide was studied.(1)The antioxidant activity of the purified Lunasin was evaluated by ABTS and DPPH free radical scavenging experiments.The results showed that the free radical scavenging activities of non-sprouting group,water treatment group and salt treatment group were all dose-dependent in the range of 0.125?1 mg/m L.The free radical scavenging activity of salt treatment group was significantly higher than that of water treatment group and non-sprouting group.(2)In the determination of NO of mouse RAW264.7macrophages,salt treatment group,water treatment group and non-sprouting group significantly inhibited the accumulation of NO in culture medium with dose-dependent.When the concentration was 2 mg/m L,the inhibition rate of salt treatment group(70.2%)was significantly higher than that of water treatment group(58.47%)and ungerminated group(51.97%).In addition,Lunasin could inhibit the expression of NOS,IL-1,IL-6 and TNF-? induced by lipopolysaccharide,and the inhibitory effect was as follows: salt treatment group > water treatment group.(3)The cytotoxicity and anti-proliferation effect of Lunasin on human breast cancer MDA-MB-231 cells were determined.The results of cytotoxicity test showed that non-germination group,water treatment group and salt treatment had no cytotoxic effect on MDA-MB-231 cells inthe concentration range of 0.5 mg/m L.In the anti-proliferation experiment,non-germination group,water treatment group and salt treatment group had higher inhibitory effect on the proliferation of MDA-MB-231 cells.At the concentration of 2mg/m L,the inhibition rate of cell proliferation in salt treatment group(69%)was significantly higher than that in water treatment group(52%)and ungerminated group(37%).Finally,heterogenous Lunasin peptide was expressed using millet as plant expression system.Besides,its biological activity was characterized.The transgenic plants of Lunasin millet were obtained by Agrobacterium tumefaciens-mediated transformation.Western-blot results showed that Lunasin peptide was successfully expressed in millet.The free radical scavenging activity of ABTS and DPPH showed that the free radical scavenging ability of transgenic type(Lunasin-enriched fraction from trans-Lunasin millet)was significantly higher than that of wild type(peptide-enriched fraction from wild type rice).The result of Lunasin lipid-lowering activity showed that transgenic type could better inhibit the differentiation of 3T3-L1 preadipocytes and reduce the accumulation of lipid in the process of differentiation compared with the wild type.At the same time,the inhibition rate of transgenic type on human breast cancer MDA-MB-231 cells was also higher than that of wild type. |
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