| Wheat is one of the most widely used food crops of the global acreage, the people main focus on the use of its seed all the time, while ignoring the comprehensive utilization of the natural resources of wheat seedlings, wheat seedlings is rich in superoxide dismutase, SOD has the function of scavenging superoxide radicals(·O2-), It can be effective in preventing the toxic effects of superoxide radicals organisms.In this study, the wheat seedlings as raw material, the extraction and purification technology of SOD were studied. And the use of extraction, purification of the SOD on Vivo and in vitro antioxidant activity.The main results are as follows:1. In this research, SOD,an antioxidant component from wheat seedlings was extracted by the phosphate buffer, and measured activity of the SOD through adjacent benzene three phenolic autoxidation method. The SOD activity (U·mL-1) as an index, material-liquid ratio, phosphate buffer solution pH value, extraction time, the temperature of heat treatment, heat treatment time of 5 process parameters were Examined. Finally, the extraction process was optimized by response surface analysis(RSA). The optimal extraction process of superoxide dismutase are phosphate buffer pH value 8.0; extraction time 1h; the heat treatment temperature 70℃; the heat treatment time 15min. And under these conditions, the activity of the SOD is up to 580.3U/mL.2. SOD wsa purifid by sulfate fractional precipitation, dialysis, DEAE-52 cellulose column chromatography and SephadexG-75 molecular sieve column chromatography, Coomassie brilliant blue G-250 method to measure protein content. Prepared dry powder 1.53 mg. The specific activity of the enzyme is 457.8 U/mg-Pr. Recovery of activity is 55.1%, the purify multiple times is 28.3. the resultd of Native-PAGE and SDS-PAGE both show a band, which indicated that the purity to the electrophoretically pure. Its physicochemical properties analysis showed that the peak of ultraviolet absorption of SOD from wheat seedlings is at 262 nm. Its molecular weight is 32.1 KD, It is very sensitive to KCN, H2O2, no-sensitive to the chloroform-ethanol. It is illustrated that the category of wheat seedlings is Cu/Zn-SOD.3. Antioxidant activity in vitro of SOD were studied by various antioxidant assays including hydroxyl radical scavenging capacity, DPPH radical scavenging capacity and reducing power. The results showed that the scavenging capacity of hydroxyl radical:the antioxidant activities were in the order:SOD enzyme solution of wheat seedings> 1mg/mL Vc> wheat seedings Juice>SOD Crude enzyme solution of wheat seedings> 1mg/mL Rutin; To the scavenging capacity of DPPH radical:SOD enzyme solution of wheat seedings, 1mg/mL Vc and lmg/mL Rutin were Equal in ability, But were greater than wheat seedings Juice and SOD Crude enzyme solution of wheat seedings; To the reducing power: 1mg/mL Vc>wheat seedings Juice>SOD enzyme solution of wheat seedings>SOD Crude enzyme solution of wheat seedings> 1mg/mL Rutin.4. Antioxidant activity In vivo were studied by various antioxidant assays, including content of MDA, activity of GSH-Px and CAT in mouse liver tissue. The results showed that:MDA content in mouse liver tissue has a significant difference between the high dose group and normal control group. But no significant difference compared with the positive control group. GSH-Px activity in the mouse liver tissue has a significant difference between the high dose group and normal control group, and there still has a significant difference between the high dose group and positive control group; CAT activity in the mouse liver tissue has a significant difference between the high dose group and normal control group, and still has a significant difference between the high dose group and positive control group all of results shows that taking a certain dose of the test substance can reduce the content of MDA, increase the activity of GSH-Px and CATThereby improving the antioxidant capacity. |
PDF Full Text Request