黑柄炭角菌菌丝体水溶性多糖的分离纯化与结构鉴定
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摘要
黑柄炭角菌[Xylaria nigripes(KI.)Sacc.]属于子囊菌亚门(Ascomycotina)、炭角菌目Xylariales、炭角菌科Xylariaceae、炭角菌属Xylaria,又名乌灵参,鸡枞蛋,是一种名贵稀有的药用真菌,具有补气、固肾、健脾除湿、镇静安神等功效。新近研究发现该菌能够提高机体免疫功能和调节内分泌代谢平衡,其粗多糖能提高巨噬细胞吞噬功能。其发酵菌丝体在化学成分、药理和临床方面和天然黑柄炭角菌基本一致,且无任何毒副作用。本文拟对黑柄炭角菌发酵菌丝体的水溶性多糖进行分离纯化,确定其结构特征,以期为其药理作用和临床应用等方面提供理论基础。研究结果如下:
黑柄炭角菌菌丝体经热水煮提、乙醇沉淀得到粗多糖,经乙醇分级、蛋白酶法和Sevag法联合脱蛋白、凝胶柱层析方法纯化得到两种水溶性多糖XNW-1和XNW-2。XNW-1和XNW-2均为白色粉末,都较难溶于冷水,易溶于热水,均易被醇、酮等有机溶剂析出。经紫外扫描检测和Sephadex G-100分析表明XNW-1和XNW-2为均一组分;XNW-1和XNW-2遇碘液不变色,说明均为非淀粉性多糖,即不含多个连续无分支的α—(1→4)键型;单糖组成分析表明两者均只含葡萄糖。XNW-1和XNW-2的糖含量分别为96%和94%。经SephoroseCL-6B柱色谱测得XNW-1的相对分子质量为9.0×10~5。并对纯度高的XNW-1进行了进一步的结构分析。
XNW-1经IR、甲基化分析、~1H NMR、~(13)C NMR分析表明:XNW-1由α型糖苷键构成;主要连接方式为(1→4)葡萄糖(82%),构成主链的核心;其它连接方式包括(1→4,6)葡萄糖(12%),(1→6)葡萄糖(1%),末端(1→)连接葡萄糖(5%);末端糖基所占比重不大,说明XNW-1为低分支结构。
溴化四唑蓝法初步测定了XNW-1体外抗肿瘤活性。体外活性研究实验表明:XNW-1在体外抗肿瘤实验中对MCF-7细胞无直接的杀伤作用(P＞0.05)。
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 9.0×10~5D 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 ~(13)C nuclear magnetic resonance(~(13)C 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).
黑柄炭角菌菌丝体经热水煮提、乙醇沉淀得到粗多糖,经乙醇分级、蛋白酶法和Sevag法联合脱蛋白、凝胶柱层析方法纯化得到两种水溶性多糖XNW-1和XNW-2。XNW-1和XNW-2均为白色粉末,都较难溶于冷水,易溶于热水,均易被醇、酮等有机溶剂析出。经紫外扫描检测和Sephadex G-100分析表明XNW-1和XNW-2为均一组分;XNW-1和XNW-2遇碘液不变色,说明均为非淀粉性多糖,即不含多个连续无分支的α—(1→4)键型;单糖组成分析表明两者均只含葡萄糖。XNW-1和XNW-2的糖含量分别为96%和94%。经SephoroseCL-6B柱色谱测得XNW-1的相对分子质量为9.0×10~5。并对纯度高的XNW-1进行了进一步的结构分析。
XNW-1经IR、甲基化分析、~1H NMR、~(13)C NMR分析表明:XNW-1由α型糖苷键构成;主要连接方式为(1→4)葡萄糖(82%),构成主链的核心;其它连接方式包括(1→4,6)葡萄糖(12%),(1→6)葡萄糖(1%),末端(1→)连接葡萄糖(5%);末端糖基所占比重不大,说明XNW-1为低分支结构。
溴化四唑蓝法初步测定了XNW-1体外抗肿瘤活性。体外活性研究实验表明:XNW-1在体外抗肿瘤实验中对MCF-7细胞无直接的杀伤作用(P＞0.05)。
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 9.0×10~5D 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 ~(13)C nuclear magnetic resonance(~(13)C 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).
引文
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