Isolation, Purification And Structure Analysis Of Water-Soluble Polysaccharides From Mycelium Of Xylaria Nigripes

Xylaria nigripes belongs to Xylaria,Xylariaceae, Xylariales,Ascomycotina and is also called "wulingshen, jizongdan" in China.It is a rare and expensive medicinal fungi with nourishing effect of replenishing qi,tonifying kidney, strengthening the spleen and eliminating dampness,sedation and relieving uneasiness of body and mind and so on.The latest results showed that it also could improved immunity function and adjusted endocrine function.Its crude polysaccharides could enhanced macrophage phagocytic activity.The fermented mycelium of Xylaria nigripes had the similar pharmacological activity and clinic curative effect to that of its fruiting body and had no toxic and other side effects.In this paper, isolation,purification and structure analysis of water-soluble polysaccharides from mycelium of Xylaria nigripes was investigated,in order to provide an academic basis for the pharmacological function and clinic application of Xylaria nigripes.The results were as follows:Crude water-soluble polysaccharides were isolated from its fermented mycelium by hot water extraction and precipitated by ethanol.The polysaccharide XNW-1 and XNW-2 were obtained by successive ethanol fraction,deproteination and gel column chromatography.XNW-1 and XNW-2 were both of white substances and rather difficult to be soluble in cold water than in hot water and no infusibility in organic solvents such as alcohols and ketones.Homogeneity and molecular weight of XNW-1 and XNW-2 were determined by UV spectrum detection and Sephadex G-100 column chromatography.The aqueous solution of XNW-1 and XNW-2 did not change the colour when added iodine solution,which indicated both of them were non-starch polysaccarides,in other words,it showed XNW-1 and XNW-2 did not contain consecutiveα—(1,4)glycosidic bonds with no-branches.Composition analysis revealed XNW-1 and XNW-2 were made up of only glucose.The total sugar content of XNW-1 and XNW-2 were 96 %and 94%.The results showed that XNW-1 was a homogeneous polysaccharide with a molecular weight 9.0×10⁵D by mean of SephoroseCL-6B column chromatography.The more purified polysaccharide(XNW-1)was carried out further structural analysis.The structure of XNW-1 was elucidated by infrared spectroscopy(IR)analysis,methylation analysis,¹H nuclear magnetic resonance(¹H NMR)analysis and ¹³C nuclear magnetic resonance(¹³C NMR)analysis.These analyses indicated that XNW-1 has the following structure:XNW-1 is mainly made up ofαconfiguration glycosidic,Its basic structure is composed of major(1→4)linked glucose(82%),other linkages including (1→4,6)glucose(12%),(1→6)glucose(1%)and terminal(1→)glucose(5%).XNW-1 is a kind of lowly branched structure.In vitro antitumor activities was investigated by MTT methods.The results revealed that XNW-1 had no direct killing action for MCF-7 cells(P＞0.05).