| Normal cell metabolism results in a continuous generation of reactive oxygen species. When the cell is in a normal state, the intracellular antioxidant system plays a role in scavenging excessive free radicals, and the cell is in an state of oxidative equilibrium. But when the body is aging, in a state of pathological or oxidative stress, the active oxygen scavenging ability of the body declines, and thus the chemical structure of normal tissue cell is destruced, and morphology and function of the tissue is damaged. Superoxide dismutase (SOD) is an internal antioxidant manufactured by the body as the first line of defense against free radical damage. Researchers find there is a very close relationship between SOD and diseases, such as cancer. In the paper, we use mushroom as the raw materials and purify SOD by ultrasonic disruption, ammonium sulfate salt precipitation, ion exchange chromatography and gel filtration chromatography. purity of SOD was detected by SDS-PAGE and HPLC, and the type of SOD was identified by UV, MALDI-TOF-TOF MS, ICP-MS. In addition, single factor and orthogonal experiment optimized the factors that influenced SOD liposomes preparation, and the biological activity of SOD liposomes was measured.SOD is purified from the fruiting bodies of Pleurotus eryngii, the HPLC result shows that there is only a peak in 16.5min.The SOD is 177 times puried, the activity is 3900U/mg and the recovery rate is 13.0%. In the ultrasonic extraction process, ultrasonic power, ultrasonic time, and chromatography order were optimized. The final choice of the ultrasonic power is 160W, and the ultrasonic time is 9min. Result of MALDI-TOF-TOF MS showed that the molecular weight of SOD is 31503 and the subunit of the enzyme has a molecular weight of 15790. ICP-MS showed the enzyme contains copper and zinc but not manganese. The UV spectrum showed a maximum absorption at 258nm. The results of SOD activity inhibition experiment showed that the SOD can be inhibited by hydrogen peroxide and sodium azide, but not sensitive to SDS. All of these evidence shows the purified SOD should be Cu/Zn-SOD. Entrapment efficiency and particle size was selected as evaluation factors.The influence of hospholipids and cholesterol ratio, extraction time, pH value and water phase volume to preparation of liposome was studied. Based on it, preparation conditions of SOD liposomes was optimized by orthogonal method. The ultimate condition of SOD liposome preparation is selected as follows:PC:CH=1:1, pH value is 8.2, ultrasonic time is 8min and water phase volume is 3mL. The stability result shows that SOD liposome leaks less than 20% in 2 months.SOD liposomes were prepared and used as a drug。The ability of SOD liposome to inhibit liver cancer cells is tested, and unencapsulated SOD was used as control of the SOD liposome. The results shows that, SOD liposomes Inhibit the growth of hepG2 and L02 well, but unencapsulated SOD shows no inhibition. In contrast, there is no difference between unencapsulated resveratrol and resveratrol Liposomes. Three is an increase of intracellular SOD activity and decrease of ROS level, indicating that SOD may inhibited the growth of liver cancer cells by eliminate intracellular ROS. Unencapsulated SOD may not able to enter the cell, as a result of that, it is not able to inhibit tumor cell growth.The results of this study shows that, the SOD separated and purified from fruiting body of Pleurotus eryngii, which is a rare edible and medicinal mushroom fungus, was used to prepare SOD liposomes. After treated with SOD liposomes, the growth of tumor cell was significantly inhibited. |
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