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Affinity Chromatography Separation, Purification And Monosaccharide Components Determination Of Camellia OleiferaAbel Seed Oligosaccharide Ⅰ

Posted on:2013-01-09Degree:MasterType:Thesis
Country:ChinaCandidate:H L WangFull Text:PDF
GTID:2230330395481540Subject:Biochemistry and Molecular Biology
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This paper studies the isolation and purification of oil tea seed polysaccharide I, the selection of the traditional methods of extraction and affinity chromatography method are compared. Traditional methods use50℃soaks exacting temperature,1:3ratio of solid to liquid, and use the alcohol precipitation, Sevag method to take off the protein from the coarse product of oil tea seed polysaccharide.Then through the DEAE-cellulose column, SephadexG-100column chromatography further purification get three types of oil tea seed polysaccharide (CPS Ⅰ, CPS Ⅱ, CPS Ⅲ). There’s no obvious absorption in the aqueous solution of sample oil tea seed polysaccharide ultraviolet-visible260nm (nucleic acids) and280nm (proteins), but in the190nm to210nm there’s polysaccharide characteristics between absorption peaks. Affinity chromatography has high specificity. This paper using the method of principle that phosphoric acid perssad and metal ion groups can chelating, and the research results from Jun Tao-" oil tea seed polysaccharide Ⅰ has phosphoric acid groups " to treat with the specificity of the high affinity chromatography processing. According to the principle of affinity chromatography column preparation, this paper selects various metal ion (Cu2+、Fe3+、Mg2+、Ca2+) to modify nickel agarose gel column. Finally we find when agarose gel take Cu2+the specificity adsorption ability of gel to the oil tea seed polysaccharide Ⅰ can achieve maximum. Then use different concentration and pH of phosphate buffer to chromatography column for elution. The phosphate concentration are1/5mol/L and1/15mol/L, the pH are6.0,6.5,7.0,7.5,8.0. The final determination of run in the best condition is pH6.0, the concentration of phosphate buffer is0.2mol/L. By comparison, the traditional method extraction efficiency is higher, but the purity is not high; And through modifing nickel agarose gel column, the affinity chromatography method has high specificity in the process of oil tea seed polysaccharide Ⅰ purification, which can get higher purity of the object, and the latter can also remove some of the protein when adsorbing the target polysaccharides.Through the capillary electrophoresis ways to get the monosaccharide components of oil tea seed polysaccharide Ⅰ, Ⅱ. First hydrolyze the CPS Ⅰ, CPS Ⅱ with acid, getting the hydrolysate. Then using PMP to its hydrolysate on the treatment of derivatization, with borax-sodium hydroxide of pH10.5as embarkation buffer. To do the capillary electrophoresis in the condition of25℃、15.0kV, compared with the standard of electrophoresis, concluded the monosaccharide components of these two kinds of polysaccharide. According to the relationship between the peak area and the concentration in the monosaccharide capillary electrophoresis, this paper further making the standard curves of sugar xylose, half of lactose, rat Lee, and concluded the molar ratio of xylose and half of lactose of CPS Ⅰ, and the molar ratio of xylose、 half of lactose and rat Lee of CPS Ⅱ.Giving corresponding oil tea seed polysaccharide Ⅰ intraperitoneal injection to different groups of hyperlipidemia mice, determining the index of the mice’s blood fat to verify its has certain lowering blood lipid. In this paper, we using the high, medium and low doses of CPS Ⅰ solution to hyperlipidemia mice intraperitoneal injection, then seven days for fast venous blood sampling. The index shows that the high dose of CPS I solution have certain Hypolipidemic effects.
Keywords/Search Tags:Oil Tea Seed, Polysaccharide, Chromatography, Capillary Electrophoresis, Hypolipidemic effects
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