| The polysaccharides were isolated sequently from Enteromorpha intestinalis by hot water, diluted alkali solution. Purification, immunostimulation and antitumor activities and the structure of these polysaccharides were studied in this paper. The relationship between the structure and activities of the polysaccharides was also discussed.First, the air dried algae was extracted with 95% ethanol at 60°C for 2 h to remove pigment and micromolecular substance. Then the residues were extracted with distilled water at 90°C for 2 h, the solid-liquid ratio was 1:20, the progress was repeated twice. After combination, concentration and lyophillization, we obtained the crude polysaccharide WE. The water unextractable solid was washed, dried and extracted with 0.5 M NaOH solution which contained 0.3% (w/w) KBH4 at room temperature for 2 h, and the green slurry was filtered through line cloth. The suspension was neutralized with hydrochloric acid (1 M) and filtered. The supernatant containing water-soluble polysaccharide was dialyzed, concentrated, lyophilized and then dried, yielding the crude polysaccharide AE.The water-extracted polysaccharide WE was fractionated by frozen-thawed, deproteinated by Sevag method, and purified by anion-exchange chromatography on a column of DEAE-Sepharose CL-6B and gel filtration chromatography on a column of Sepharose CL-6B. Then two fractions were obtained and named as WEA (the neutral polysaccharide) and WEB ( the acid polysaccharide), respectively. WEA and WEB appeared as single symmetrical peak on HPLC, and the average molecular weights were 72.03kDa and 60.12kDa which were caculated through calculation from dextran standard. The alkali-extracted polysaccharide AE was fractionated by frozen-thawed, deproteinated by Sevag method, and purified ultrafiltration and gel filtration chromatography on a column of Sepharose CL-6B. Then two fractions were obtained and named as DAEA and DAEB, respectively. DAEA and DAEB appeared as single symmetrical peak on HPLC, and the average molecular weights were 18.21kDa and 46.80kDa.GC analysis showed that WE was composed of Rha (58.00%), Glc (20.50%), Xyl (13.54%), Gal (2.27%), Man (2.26%), and small content of glucuronic acid (3.43%); WEA was composed of Glc (55.24%), Rha (23.76%), Xyl (17.11%), Man (2.21%) and Gal (1.67%); the acid fraction WEB was composed of Rha (68.81%), Xyl (9.40%), Gal (4.89%) and Glucuronic acid (16.89%), the sulfate content of WEB was 19.98%. AE was composed of Rha (15.93%), Man (18.04%), Gal (13.09%), Glc (11.96%), Ara (10.94%), Rib (8.89%), Xyl (5.29%), and glucuronic acid (15.86%); DAEA consisted of Glc (33.69%), Rha (11.44%), Gal (9.64%), Man (7.92%), Ara (7.58%) and Xyl (2.79%); DAEB consisted of Rha (69.20%), Xyl (12.90%), Glc (8.20%), Gal (7.40%) and small content of glucuronic acid (2.30%), the sulfate content of WEB was 9.6%.WE, WEA, WEB, AE, DAEA and DAEB was cultivated with lymphocyte cells or peritoneal macrophages. Among the six polysaccharides, WEA, WEB and DAEB could induce lymphoctes proliferation, increase the production of TNF-αin macrophages, and dose-dependently stimulate macrophages to produce NO through the up-regulation of inducible NO synthase (iNOS) activity. The activity of WEB was better than that of WEA and WE. After desulfation, the immunostimulation activities of the polysaccharide WEB was strongly decreased, or even completely disappeared. It was indicated the sulfate group was indispensable for immunostimulation of WEB. Among the alkali-extracted polysaccharides, DAEB showed the better immunostimulation activity.At the dose of 100, 200, and 400mg/kg, WEA, WEB and DAEB could inhibit tumor growth in S180 tumor-bearing mice, WEB had better activity than WEA and DAEB, the maximal tumor inhibition rate was 74.70% at the does of 400 mg/kg. Meanwhile, they increased in the relative spleen and thymus weight and expression of tumor necrosis factor-alpha (TNF-α) in serum.However, WE, WEA, WEB, AE, DAEA and DAEB at the concentration up to 800μg/ml did not affect the growth of S180, Hela and HepG2 tumor cells. These results indicated that the tumor inhibitory activity of the three fractions was not due to their cytotoxicities on tumor cells, but may be related to the stimulation of immune system, which has a great significance in therapeutics of the tumor growth.On the basis of the methylation analysis applied for native polysaccharide, desulfated polysaccharide and desulfated-reduced polysaccharide, partial acid hydrolysis, periodate oxidation and 13C NMR analysis suggested that WEB was mainly contained (1→4)-linked-rhamnose, and other linkages as (1→2), (1→2, 4) and (1→3)-rhamnose, and with a branch at the O-2 position every five (1→4)-linked-rhamnose residues. It also had nonreducing terminal Xyl and Gal; amount of (1→4)-linked-GlcA, and small content of (1→)-linked-GlcA. The O-3 of (1→4)-linked-rhamnose was the sites of sulfation. DAEB was branched and contained principally terminal, (1→4), (1→2,4)-linked-rhamnose; terminal, (1→3)-linked-xylose, (1→4)-linked-glucose, and (1→3)-linked-galactose. Desulfaction of DAEB demonstrated that the O-3 of rhamnose and O-2 of xylose were the sites of sulfation.
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