| Spirulina is a kind of new resource food which is rich in protein, carbohydrates, vitamins, minerals, unsaturated fatty acids, trace elements and other nutrients. Spirulina polysaccharide is a natural active substance with the effects of anti-tumor, anti-oxidation, anti-radiation, decreasing blood sugar, antivirus, and improving the gastrointestinal function. This paper used spirulina powder as raw material, and identified the extraction and purification technology of spirulina polysaccharide. Also we studied the inhibitory effect of spirulina polysaccharide on Caco-2 during simulated intestinal barrier oxidative damage of biological activity, and analyze the structure of polysaccharide with high hypoglycemic activity. The main results are as follows:(1) Extract SPS with freezing and thawing techniques and ultrasonic treatment with hot water. The technological conditions are optimized through orthogonal test based on single factor. The single factor experiment results showed that the optimization time of ultrasonic, extraction temperature, solid-liquid ratio, extraction time and extraction times were respectively: 15 min, 90℃, 1.5h, 1: 25 and 2. After verification experiment, the extraction rate of SP was approximately(13.17 ± 0.49)%.(2) Remove the impurities in SP by sevage treatment and get the optimization condition with high extraction rate of protein and low SP comsumption by single factor and orthogonal screening analysis. Thecondition were as follows: chloroform: n-butanol = 5: 1, removing liquids: Raw Sugar solution = 3: 1, Remove protein 6 times, shaking time 25 min. After verification experiment, the protein removal rate was approximately(63.24 ± 0.43)%, and the polysaccharide retention rate was approximately(82.12 ± 0.51)%.(3) PSP was get after the optimized extraction technology and protein technology,and three different polysaccharide PSP-1, PSP-2 and PSP-3 was get after the PSP ultrafiltration centrifugal separation and purification technology.Then PSP-3 was separated by gel permeation chromatography and get PSP-3-1 and PSP-3-2, The molecular weight of the PSP-3-1 the high concentration of the polysaccharide, with the purity of 99% or more. The molecular weight of which is 623.02 k Da, by Gpc detection.(4) The inhibotory effect of PSP-1, PSP-2 and PSP-3 on colon adenocarcinoma cells(Caco-2 cells) was detected by MTT assay and the optimized composition and concentration was determined. The result showed that the three components all have some specific effects on Caco-2, but the effect of PSP-1 and PSP-2 was invalid and unstable, whereas the inhibition effect of PSP-3 remained stable and obvious, and have a dose-effect relationship. The best concentration of PSP-3 inhibing Caco-2 growth was 5.00 mg / m L, while IC50 was 0.63 mg / m L.(5) Caco-2 cells stimulate intestinal barrier simulation model was established to study the prevention effect of PSP-1, PSP-2 and PSP-3 on intestinal barrier oxidative damage of Caco-2 cells. The results showed that 0.1mg / m L of PSP-3 had the best effect. The products of PSP-3 after separation and purification, PSP-3-1 and PSP-3-2, and the positive control group of glutamine on intestinal Caco-2 cells during simulated barrier prevent oxidative damage was studied, which showed that 0.1mg / m L of PSP-3-1-glutamine is better than the control group, indicating that PSP-3-1 can prevent Caco-2 cells simulated intestinal barrier oxidative damage.(6) IR, GC analysis showed that Spirulina contains polysaccharidecharacteristic infrared absorption is a polysaccharide; monosaccharide composition was determined by GC results are: D- ribose: L- rhamnose: L- arabinose: L- fucose: D- glucose = 1:0.70:1.03:2.1:6.59,after periodate oxidation analysis results containing type 1 → 2,1 → 4 glycosidic linkages, which may contain 1 → 3 glycosidic bond type, almost no 1 → 6 glycosidic bond. NMR analysis showed that PSP-3-1 sugar chain containing α, β two kinds of glycosidic configuration, comprising galactosamine, indicating PSP is a polysaccharide conjugates containing protein. PSP-3-1 through atomic force microscopy analysis showed the existence of two forms, one is a branched chain forms, one is a straight-chain form, when the solubility increases, spontaneously gathered into a network structure,where in the linear polysaccharide molecule width of 34.132 nm, a height of 819.169 pm... |
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