| The dissertation was focused on chemical modification of polysaccharides extracted fromthe submerged Ganoderma lucidum fermentation broth. In order to enhance the antitumor andimmunomodulating activities of Ganoderma lucidum polysaccharides, a new method, usingsulfamide acid as the agent for sulfation modification on polysaccharides, was developed inthis paper. The esterification conditions of polysaccharides using sulfamide acid and SO3-Pyrcomplex to treat the polysaccharides were studied, the effects of the polysaccharides sulfatewere evaluated. The effects of polysaccharides sulfation on antitumor activity were evaluated,and the relationships between the chemical structure of G. lucidum polysaccharides sulfateand its antitumor activity was also studied.Mycelia from submerged fermentation of G. lucidum were crashed by a high pressurehomogenizer, and then intracellular polysaccharides from the mycelia were initially extractedin hot water. The optimal extraction condition was determined by response surface analysisand listed below: extraction temperature, 95°C;extraction time, 3.5h;the weight ratio ofmycelia and water, 1:2. A maximum intracellular polysaccharides yield of 11.56 mg/g drymycelia was achieved under this condition.Alkaline extraction was used for extracting the residual mycelia of G. lucidun, and theoptimal extraction condition was determined as listed below: extraction temperature, 65°C;extraction time, 4h;the ratio of extracting substances to extractant (NaOH), 1:4;NaOHconcentration, 0.5mol/L. The yield of the water soluble polysaccharides, treated by theabove mentioned method, reached 93.5mg/g dry mycelia.The fermentation broth was concentrated by ultrafiltration method, to further improvethe effectiveness of the extraction process and the quality of the final product. Theultrafiltration condition was determined as follows: the cut-off molecular weight, 10000;operating pressure, 0.2 Mpa;diluting the fermentation broth with hot water to double originalvolume. Compared with the traditional vacuum concentration method, higher yield andcontent of polysaccharides could be obtained by the ultrafiltration concentration method.The later method could also improve the quality of polysaccharides, and the HFR scavengingratio of polysaccharides was higher than that of the vacuum concentration method.Based on the view of high-throughput screening and the ideas of combinatorial chemistry,method of using sulfamic acid or SO3-Pyr complex as the reaction agent was adopted torefine the extracellular G.lucidum polysaccharides and to replace extensive method of usingchlorosulfonic acid-pyridine. The factors influencing on the sulfation were studied in details.The optimum sulfamic acid conditions were determined as follows: the molar ratio ofsulfamic acid to sugar, 1:2.5;sulfation temperature, 90°C;sulfation time, 2h;the ratio ofpolysaccharides weight to solvent volume (g:ml), 1:50;the DS of the derivative,1.46. Theoptimal condition of using SO3-Pyr complex method was: the molar ratio of sulfamic acid tosugar, 1:2;sulfation temperature 80°C;sulfation time, 2h;the ratio of polysaccharides weightto solvent volume (g:ml), 1:40;and the DS of the derivative, 1.53.The factors influencing on sulfation of intercellular polysaccharides were also studied.The results showed that the molar ratio of sulfamic acid to sugar unit, reaction temperature,reaction time, and the ratio of polysaccharides weight to solvent volume (g:ml) largelyeffected the sulfation modification of the intercellular polysaccharides. The optimal sulfationmodification condition was determined as below: the molar ratio of sulfamic acid to sugar, 1:3;sulfation temperature 80°C;sulfation time, 6h;the ratio between polysaccharides weight tosolvent volume (g:ml), 1:60;the DS of the derivative, 0.91. When using this optimalcondition for sulfating the alkaline extracted water-soluble ganoderma polysaccharide, the DSof its derivative was 0.71. The results of agarose electrophoresis showed that intercellularganoderma polysaccharides and alkaline extracted water-soluble ganoderma polysaccharidecould be deeply stained by toluidine blue. IR spectrum of these two kind of polysaccharidesshowed that characteristic absorptions of sulfate ester bond appeared at 1260cm-1 and 810cm-1, respectively.The components of the alkaline extracted water-soluble ganoderma polysaccharide wereidentified by GC. Rhamnose, arabinose, mannose, glucose,galactose were detected withmolar ratio of 48:51:10:25:14.The antitumour activity of G. lucidum and its sulfate derivatives were tested in vitro andin vivo.Their inhibition to the cell lines in vitro was tested by MTT method,and the resultsshowed that the G. lucidum polysaccharides could not inhibit the proliferation of KB,SGC-7901, BEL-7402, HePG-2, MCF-7 and SHZ under the tested ranges, but the inhibitionof its sulfate derivatives significantly increased, to about 34% at the dosage of 500μg/ml.The inhibitory effect of GLP-Sa was better than GLP-Sp, at the dosage of 500μg/ml. Theinhibitory effect of GLP-Sa over HePG-2, BEL-7402, and SHZ reached 74.79%, 55.46%, and85.88%. The inhibitory effect of GLP-Sp over HePG-2, BEL-7402, and SHZ also went to thelevels of 53.26%, 62.59%, 68.4%, respectively. The result showed that SO3-group play animportant role in influencing the inhibition of the GLP sulfate on the tested cancer cell lines.GLP-Sa and its purified components had no inhibition to the normal human hepatic cellline(L02), but could effectively suppress the proliferation of HePS in vivo, at the dosage of0.5mg/kg.d. The inhibitory effects of GLP-Sa and its purified components over theproliferation of HePS in vivo were 55.5%, 44.1% respectively, and the immunity over theproliferation of HePS in vivo could also be promoted at certain degree.AbstractThe physicochemical properties of S-GLP were preliminary investigated,the structure ofS-GLP was elucidated by glycosyl residual composition analyses, IR spectroscopy, UVspectroscopy and NMR spectroscopy. The sugar components were rhamnose, arabinose,mannose, glucose and galactose, and their molar ratio was 9:3:2:4:48. The sugar componentswere rhamnose,arabinose, mannose, glucose and galactose,and the ratio was 9:3:2:4:48.Themain chain was estimated to be α–D-Glc(1→6) ,and α-D-Gal,α-D-Glc,α-D-Man,rhamnose and arabinose would be in the side chain.The SO3-group would be sulfated at C-3,C-4,C-6 of the sugar unit. Sulfation modification caused the changes in the primary structureand the sophisticated structure of G. lucidum polysaccharides. The S-GLP conformation wastriple helix structure, whereas the parent G.lucidum polysaccharide's conformation was singlehelical before sulfation, suggesting that the conformation of polysaccharides became moreordered, which was also confirmed by x-ray diffraction. This might be one of the reasons forthe improvement of the antitumor activity of the modified G.lucidum polysaccharides.
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