Separation And Purification Of Cuttlebone Polysaccharides & Research On Its Biological Activities

This paper includes two main parts: 1. Extraction and separation of Cuttlebone polysaccharides& Purification of its active component CPS-1;2. Effect of Cuttlebone polysaccharide CPS-1 on experimental ulcerative colitis in mice& other biological activities of CPS-1.Research background: As we know, many rare Chinese traditional medicines come from ocean. But so far, most of research regarding halobios' polysaccharides put their focus on alga polysaccharides. It hasn't been reported yet regarding any research on active polysaccharides from animal parenchyma. Cuttlebone, also called OsSepiae. is a Chinese traditional medicine. It is widely used in clinical treatment on various ulcers, and has very good therapy effect. As scraps in food processing, only small part of them are recycled then used as medicine.Research methods and results: Buy cuttlebones from Zhejiang province, crash into powder, then sunk in 95% ethanol to remove lipid. Use hot-water to extract crude polysaccharides from cuttlebone powder, use ethanol to precipitate the crude polysaccharides from water-solution. Re-dissolve the crude polysaccharides in water: remove the useless protein by using Phenol-Chloroform method. Then do concentration. Remove the low molecular weight substances and salts fromconcentrated solution by using dialysis for 24 hours. Get the crude polysaccharides solid after freeze drying. The total sugar contents of crude polysaccharides were determined. Crude polysaccharides are separated into water-eluted part and salt-eluted part by using ion-exchange chromatography on DEAE Sepharose Fast Flow. Concentrate the water-eluted part and then freeze drying. Re-dissolve the water-eluted part and then get one purified cuttlebone polysaccharide-CPW-2 by gel filtration on Sephacryl S-300. Concentrate the salt-eluted part and then freeze drying. Get purified cuttlebone polysaccharide-CPS-1 by gel filtration on Sephacryl S-300 and ion-exchange chromatography on Sepharose CL-6B. HPLC was used to determine the purity of refined CPS-1 and CPW-2 and their molecular weight by polysaccharides standard curve. Determined the monosaccharide composition of CPS-1 by GC analysis. Initially determine the structure of CPS-1 by UV atlas, 1R atlas, NMR-C atlas, periodic acid oxidation and methylation analysis.We use Dextran sodium sulfate (DSS)-induced method to establish mice UC model and use SMP+TMP as control. Then we primarily observed the protective activity of Cuttlebone Polysaccharide CPS-1 on experimental ulcerative colitis (UC) in mice by evaluating the indexes including clinic symptoms, body weight changes, and pathological changes in partial intestines mucous membrane. Then separately determine the quantity of some cytokines which are related to endothelial cell proliferation and inflammation in mice blood of each team by using EL1SA kits. After statistic calculation and comparison of experiment data, we could find that body weight of mice in curative team weren't distinctly decreased compared with negative control team. The general conditions of mice in curative team are good. Quantity of EGF and PDGF of curative team obviously increased, by contrast, the quantity of TNF-a decreased(P<0.05). Cuttlebone polysaccharide CPS-1 can obviously increasethe quantity of EGF and PDGF of experimental ulcerative colitis mice. It can accelerate the concrescence and repair of ulcer. It also can decrease the expression of TNF- a , which can abate the seriousness of inflammation.Research meaning: Firstly extract the biological active polysaccharide from cuttlebone solid, and get the homogeneous cuttlebone polysaccharide by several purification steps. Optimized the extraction conditions. We established techno-platform for extraction and purification of active polysaccharides from oceanic Chinese traditional medicine. The result of this research also provides us the experimental basis for further development regarding natural sugar drug used for ulcerative colitis treatment.