| Extraction, isolation, purification and structural identification of polysaccharide from Spirulina (Arthrospira) platensis of alkaline lakes in Erdos Plateau and Chad Lake in Africa were studied, and also comparatively studied the mechanism of antitumor, activity of antibacteria and antitumor of the polysaccharide. The aim of this paper is to establish the basis of theoretical and applied research on developing polysaccharide of S. (A.) platensis of alkaline lakes in Erdos Plateau and its products, screening new antitumor drugs, natural alternatives to antibiotics and immunostimulant. The thesis is divided into four parts:(1)Polysaccharide from S. (A.) platensis of two ecogeographic species were extracted with the traditional water extraction and alcohol precipitation method, and purified preliminarily with the trichloroacetic acid and active carbon method. The two sorts of Polysaccharide were further purified with SephadexG-100 gel column chromatography, ESP of polysaccharide from S. (A.) platensis of alkaline lakes in Erdos Plateau and FSP of polysaccharide from S. (A.) platensis of Chad Lake in Africa were achieved.ESP and FSP were homogeneous component, which have been proved with Paper chromatography, UV spectrum, specific rotation and SephadexG-100 gel column chromatography methods. ESP was milky white flocculent crystal, FSP was light yellow flocculent crystal after freeze-drying by low temperature, and they were both soluble in water. The Polysaccharide′content of ESP and FSP were 89.75% and 62.87%, which have been determined with Sulfuric acid-phenol method. Non-polysaccharide components were mainly ash and protein in FSP and ESP, which have been verified too. ESP and FSP were polysaccharide combined protein possibly that were conjectured.The molecular weight and structure of ESP and FSP were studied with SephadexG-150 gel column chromatography, TLC, HPLC, IR and NMR methods on the basis of above studies. The results showed that the molecular weight of ESP and FSP were 77625 Dr and 85114 Dr, the connection pattern of single sugar in ESP were mainly [α-Glc(1→4)-]n, including fewα-Glc(1→6)-,α-Fuc(1→2)-,β-Xyl(1→3), -α-Rha as ended glycosylation, The monosaccharide of constituting ESP′were mainlyα-pyranose, largest in glucose, followed by rhamnose and few in arabinose, xylose, galactose, mannose and fructose. The connection pattern of single sugar in FSP wereα-Glc(1→6)- andα-Xyl(1→2), -β-Xyl, -β-Gal and -α-Rha were ended glycosylation, the monosaccharide of constituting FSP were mainlyα- orβ- type glucopyranose, followed by rhamnose and few xylose, mannose, galactose and arabinose.(2)The antibacterial function of ESP and FSP were studied in vitro with cup-plate method. The results showed that ESP and FSP had bactericidal activity, but inhibitory effect to bacteria was related to concentration of polysaccharide and species of bacteria. Inhibition of ESP was better significant to Shigella dysenteriae in the concentration of 20 mg/mL and 40 mg/mL, and the diameter of bacteriostasis circle were 14.17 millimeters and 12.16 millimeters. FSP had bactericidal activity to Shigella dysenteriae, Pseudomonas aeruginosa and Proteus vulgaris in the concentration of 20 mg/mL, the diameters of the inhibition zone were 12.13 millimeters, 9.10 millimeters and 9.07 millimeters.(3)The inhibitory effect of ESP and FSP to S180 ascites tumor in mice has been studied with the animal model of S180 ascites tumor. And then the effect of ESP and FSP on proliferation of spleen lymphocyte cell and the concentration of immune cell factors in serum were detected with MTT and ELISA methods, the molecular mechanism of immune enhancement of ESP and FSP were studied preliminarily to tumor-bearing mice. The results showed that ESP and FSP could improve the survival quality of tumor-bearing mice, inhibit the growth of S180 transplanted in mice, reduce the scope of tumor invasion in the body, lengthening survival time of tumor-bearing mice. Effect of the medium dose of ESP and FSP was most significantly on the survival time of tumor-bearing mice, the lengthening rate of life were 50.67% and 52.00%. ESP and FSP could reduce injury of organ in tumor-bearing mice, raising the thymus and spleen index, thus increasing immune function, enhance the levels of IL-2, TNF-αand IFN-γin serum of tumor-bearing mice when inhibit growth of tumor. ESP and FSP could promote the proliferation of spleen lymphocyte cell induced by ConA In vitro. The influence of medium dose of ESP and FSP to cell factors, the proliferation of spleen lymphocyte and immune organ index were most significantly. The above results show that ESP and FSP of appropriate dose can inhibit S180 ascites tumor significantly, enhance T cell-driven immune function, improve the immune regulate function of body. Immune regulate effect may be one of the mechanisms of antitumor.(4)The effect of ESP and FSP on growth of sarcoma 180, mouse leukemia cells L1210, human chronic myelogenous leukemia K562 and mouse lymphoma cells YAC-1 had been studied with MTT methods, meanwhile effect of ESP and FSP on cell cycle and cell apoptosis of S180 have been studied too with flow cytometry. The results showed that ESP and FSP of different dose all had inhabitation to different degrees on above tumor cells in vitro. But, only the medium doses of ESP and FSP had a inhibition rate more than 30%, reached 35.88% and 33.98%. The medium dose of ESP and FSP all had the more obviously inhibition to the cell proliferation on above tumor cells, but the inhibition effect were significantly lower than CTX group to the S180, YAC-1 and K562 (P<0.05). Comparison with Astragalus sugar group, only the medium dose of ESP and FSP to K562, the medium dose of FSP to YAC-1 had more significant inhabitation than Astragalus sugar group (P<0.05).The results of FCM showed that, ESP and FSP could cause cell cycle changes, block S180 cells in the G1 phase, induce apoptosis, and the effect of the medium dose of ESP and FSP were most significant (P<0.05) comparison with each other.
|
PDF Full Text Request