Studies On Antioxidation Activitity And Liposomes Preparation Technology Of Squalene

Squalene, a typical biological active substance, has many physiological functions, for example, anti-oxidant, anti-aging, anti-fatigue, anti-tumor and other functions. However, squalene which contains six double bonds is very unstable and easily oxidized in air. To solve this problem, the liposomes preparing technology was applied. In this study, squalene as the research object was analysed on its antioxidant properties and automatic oxidation ability. The liposomes of squalene were preparated to prevent the oxidation by thin-film evaporation-freeze drying method and ether injection method. After its preparation methods were optimized, the stability of liposomes was assessed. Specific research contents and results are as follows:1. The anti-oxidation effect of squalene was evaluated by measuring the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and protein carbonyl content and hydrogen peroxide content of the pig skin slurry.The results showed that squalene could remarkably improve SOD, GSH-Px and CAT activity of the pig skin. It also significantly reduced protein carbonyl groups and protein hydroperoxides content of the pig skin, and the effect of squalene was better than vitamin E. All data indirectly displayed squalene had anti-oxidation effect on the skin.2. Utilizing differential scanning calorimeter the self-acting combustion curve of squalene and vitamin E were determined. Squalene was easier occurring automatic oxidation than vitamin E, and the curve displayed squalene existed evident variation from120℃to340℃.3. Using thin-film evaporation-ultrasonic method, thin-film evaporation-freeze drying method and the ether injection method for the preparation of liposomes, we found that the liposomes had higher embedding rate by thin-film evaporation-freeze drying method and ether injection method than thin-film evaporation-ultrasonic process, so we choose thin-film evaporation-freeze arefaction method and the ether injection method for test.4. Through the single factor experiment of thin-film evaporation-freeze drying technology, we found that the quality ratio of lecithin and cholesterol, squalene content, water bath temperature and concentration of trehalose had a deep influence on envelopment rate of squalene liposomes. As the quality ratio of lecithin and cholesterol increased gradually, the embedding rate also increased gradually; along with increasing the amount of squalene, the embedding rate had a clear increasing trend; when water bath temperature was50℃, the embedding rate of squalene liposome was highest; the embedding rate augmented in the wake of adding content of trehalose. By further orthogonal optimization test on process conditions of thin-film evaporation-freeze drying method, we found influence factors of the preparation process and arranged the primary and secondary order as follows:quality ratio of lecithin and cholesterol (A)> squalene content (B)> water bath temperature(D)> trehalose concentration (C). The optimum technological combination was A3B3C3D2.5. Through the single factor experiment of ether injection method,we found that quality ratio lecithin and cholesterol, squalene content, PEG2000concentration and magnetic stirring speed were essential to envelopment rate of squalene liposomes. As the quality ratio of lecithin and cholesterol increased gradually, the embedding rate also increased gradually; along with increasing the amount of squalene, the embedding rate had a clear increasing trend, and the embedding rate were generally higher; when magnetic stirring speed was120r/min, the embedding rate of squalene liposome was great; the embedding ratio added with PEG2000concentration rising. Further orthogonal optimization test about process conditions of the ether injection method was analyzed, and results showed that the primary and the secondary order of influence factors during preparing process, magnetic stirring rate (D)> PEG2000content (C)> lecithin and cholesterol quality ratio (A)> squalene content (B).The optimum combination was A2B3C3D2.6. By testing the stability of squalene liposomes under the optimum preparation conditions, we found that the embedding rate of liposomes was92.85%and93.57%on average through thin-film evaporation-freeze drying method and the ether injection method preparation. They reflected that experiment was feasible. In thin-film evaporation-freeze-drying process, phase transition temperature of squalene liposomes was approximately115℃, the peak was relatively stable and the particle size was1299nm. As the quality ratio of lecithin and cholesterol increased gradually, the particle size also had a gradually decreasing trend. In the ether injection method, phase conversion temperature of squalene liposomes was about118℃, the peak was relatively stable and the particle size was1287nm, they belonged to the big monolayer liposome. Particle size was influenced by magnetic stirring speed, we found that the particle size of liposomes was most ideal when the magnetic force stirring speed was 120r/min. The pH changes of liposomes were smaller by thin-film evaporation-freeze drying than by the ether injection method. MDA content of liposomes by the ether injection method were significantly higher than thin-film evaporation-freeze drying method.