Refining,Structure Elucidation And Antitumor Activity In Vitro Of Polysaccharides From Lentinus Edodes Of Fangxian

Lentinus edodes has been valued by humankinds as an edible and medical fungus. A number of bioactive moleculars have been identified, inculuding lentinan which is the best known and most potent substance. As an immunomodulator, Lentinan have an important development and research value because of its small toxicity and good curative effect. In recent years, many researches indicated that some polysaccharides isolated from L. edodes exhibited effects on anti-virus, anti-tumor, regulating the immune system, lowering blood sugar, antioxidant and other functions. Two single molecular weight polysaccharide preparations, LNT₁ and LNT₂, were isolated from the fruiting bodies of Lentinus edodes (Fangxian.), by extracted with sodium hydroxide, bleached with hydrogen peroxide, fractionated and purified with ultrafiltration. The structure, sugar chains conformation and antitumor activity in vitro are discussed.Part one The extraction and purification of LNT₁ and LNT₂The crude polysaccharide was extracted by sodium hydroxide solution, The optimal alkali extraction factors were temperature 25℃, sodium hydroxide solubility 5% and time 24 h by orthogonal tests. Refined polysaccharides, LNT₁ and LNT₂, were obtained from the crude polysaccharides, by fractionation and purification methods with hydrogen peroxide and ultrafiltration. The purity of LNT₁ and LNT₂ were comfirmed by HPLC and UV. The molecular weights (LNT₁:Mw=605.4 kDa,LNT₂:Mw=185.2 kDa) and the sugar content (LNT₁:95.61%,LNT₂:94.99%) were measured by HPLC and phenol-sulfuric acid method respectively. The chemical structures of LNT₁ and LNT₂ were studied by Acid hydrolysis,Periodate oxidation,Smith degradation,Methylation analysis,1D and 2D NMR (DQF-COSY, HSQC, and HMBC) experiments. The chain conformation of LNT₁ and LNT₂ were evaluated by Congo Red Test. Analysis of monosaccharide composition showed that LNT₁ and LNT₂ only contain D-glucose. Periodate oxidation-Smith degradation Test showed that LNT₁ and LNT₂ contain (1-3) or (1-3-6), (1-6) or terminal sugar resides. Congo Red Test showed that LNT₁ and LNT₂ exsits as triple-helix in distilled water and a helix-coil transition may occur when NaOH concentration is higher than 0.20M. Results from FT-IR showed that LNT₁ and LNT₂ wereβ-D-Glycopyranoside polysaccharides. On the basis of Periodate oxidation,Methylation analysis,1D and 2D NMR (DQF-COSY, HSQC, and HMBC) experiments the polysaccharides (LNT₁ and LNT₂) were shown to consist of repeating units with the following structure.The cytotoxicity assay of LNT₁ and LNT₂ on H-22 tumor cells were evaluated in vitro using MTT assay. The growth of H-22 tumor cells could be inhibited by LNT₁ and LNT₂ at a concentration as low as 12.5μg/mL, and the inhibition ratios are 24.38% and 27.52%, respectively, and a dose-dependent manner was observed up to 800μg/mL. LNT₁ and LNT₂ exhibited strongest inhibition ratio against H-22 tumor cells at the concentration of 800 μg/mL, reached 52.31% and 52.62%, respectively. So LNT₁ and LNT₂ have the value to further study.