Separation And Purification Of Polysaccharide From The Brown Seaweed Sargassum Graminifolium And Its Effects On Biological Activity

With the improvement of living standards, people’s excessive intakes of energy nowadays result in body fat--obesity. Zimmet Paul, one famous diabetes specialist,once said, "Obesity is spreading throughout the world like epidemic disease”. In United States, medical expenses caused by obesity are added up to hundreds of billions of dollars. As a super nation with 13.7 billion people, the number of obese patients is increasing rapidly. It can be seen that the cost of medical expenses which is brought about by obesity will be astronomical. So the treatment of obesity and studies of related drugs should be given more attentions.China possesses over 10,000 kilometers of mainland coastlines, vast sea areas,abundant ocean resources. In Recent years, both domestic and overseas’ s scholars have turned to ocean looking for source of raw materials; “drug from the sea” is no longer a slogan. Seaweed, as a producer of ocean resources, it plays an important role as a member of marine biological resources. According to statistics, there are over one thousand seaweeds around the globe, and they contain various bio-active substances,making it needless to worry about the abundance of raw materials. Sargassum graminifolium is a kind of seaweed distributed in southeast coastal areas of China which is also a traditional ocean drug. Sargassum graminifolium possesses many bio-active substances such as polysaccharides, unsaturated fatty acids, polyp Henols etc, extracting those bio-active compounds from them as drugs to treat obesity and related diseases has great values both on theory and practice.This study discusses the separation and purification of polysaccharides from Sargassum graminifolium, and investigates the regulation of intestinal flora,immunoregulation and hypolipidemic effect of these polysaccharides in obese rats.In order to optimize the extraction technology of Sargassum graminifolium, we used both single-factor experiments and orthogonal designed method to determined optimum conditions of microwave extraction. In our experiments we selected three factors:microwave power, extraction temperature and extraction time; and determined three levels of each factor by the single-factor experiment: microwave power: 300 W, 450 W,600W; extraction temperature: 50℃, 55℃, 60℃; extraction time: 10 min, 12 min, 14 min.As a result, the optimum microwave extraction conditions of SGP were: At microwave power 450 W, extraction temperature 60℃, extraction time 14 min. Using these conditions, the yield of SGP was 10.83%, extraction rate of SGP was 4.94%, the purity of SGP was 45.62%.To purify the SGP crude extracts, we went through deproteinization, decolorization and removal of small molecules methods. The single-deproteinization ratio was 47.4%,polysaccharide loss ratio was 13.7%, and the quintic deproteinization ratio 96.3% using Sevage method, however its SGP loss ratio was also as high as 48.3%. In TCA precipitation method, protein removal rate was 88.5% and polysaccharide loss rate was16.7%. Compared the deproteinization ratio and SGP loss ratio, TCA precipitation method was better in SGP deprivation. Second, the results of SGP decoloration tests showed that hydrogen peroxide has better effects than active carbon in decoloration.This test was designed like this: Use dose of activated carbon powder 2g/m L, 4g/m L,6g/m L, 8g/m L, 10g/m L, 12g/m L and mass fraction of hydrogen peroxide 5%, 6%, 7%,8%, 9%, 10% at the conditions of p H=6, 40℃ to decoloured for 3 h respectively. The results showed that optimum dose of active carbon was 10g/ml, the decoloration rate was 41.7% under this condition, and SGP loss rate was 14.6%. While the optimum mass fraction of hydrogen peroxide was 8%, the decoloration rate was 73.9%, and SGP loss rate was 12.9% under this condition. Thus hydrogen peroxide showed better decoloration effects. Finally, we used microfiltration membrane to ultrafiltered trichloroacetic acid, oligosaccharide, small molecules and other impurities, the SGP loss rate was 7.21% during this process.In the last part of this study, we evaluate SGP activity through animal models of obese mouse. High-fat-induced obese rats model were built by high-fat diet. Then SGPs were treated to mouse by intragastric administration, blood samples were drawn from orbit, tested the level of TC, TG, HDL and LDL in plasma. The rats were killed andanatomized immediately. The spleen and thymus were taken for organ index tests in sterile condition, and was then used to culture lymp Hocytes by MTT method, tested its conversion rate. Collected the contents in small intestinal to make suspension and diluted in gradient, then it was inoculated into selection culture medium of Escherichia coli, Lactobacillus and Bifidobacterium separately. The result showed that the concentrations of TC, TG and LDL from plasma in the treatment groups were lower than those in the blank group, but the concentration of HDL was higher than that in the treatment group. The number of Escherichia coli from treatment group was 3.79%-13.83% lower than the blank group. Compared with the blank group, the number of lactobacillus and bifidobacterium came from treatment group increased 10.07%-14.47% and 4.34%- 14.06% respectively. TC, TG and LDL concentrations have positive correlations with the number of intestinal bacteria, and the negative correlation between the number of Lactobacillus and the number of bacteria in intestines, HDL has the reverse effect. It can be concluded that SGP can suppress the growth of Escherichia coli in the intestinal of high- fat- diet induced obese mice; on the other hand SGP can promote the growth of lactic acid bacteria and Bacillus. The number of intestinal flora is also related to blood lipid biochemical indicators.