天麻有效成分积累规律及天麻多糖结构研究
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摘要
天麻(Gastrodia elata BL.)为兰科真菌营养型多年生草本植物,药用其干燥块茎。天麻素、天麻多糖和蛋白质是天麻有效成分。天麻多糖有抗氧化、清除体内自由基、延缓衰老等重要作用,可开发利用的价值很大。
但目前有关天麻多糖研究还很少,主要由于天麻多糖还没有成熟的纯化方法,且天麻多糖的结构还不清楚。了解天麻主要有效成分的积累分布规律,在此基础上探讨天麻多糖适宜的纯化方法,分析天麻多糖的组成及结构,为天麻用药和有效成分提取过程中选择天麻原料提供依据,为天麻多糖的药理作用研究提供结构基础。
本文通过对天麻有效成分在不同生长期和天麻块茎不同部位的积累分布进行研究:筛选天麻多糖提取纯化方法,得到3种纯化的天麻多糖,并对其中含量最多的一种天麻多糖TM1进行了结构分析。主要研究结果如下:
1.天麻多糖和蛋白质在生长期180d时含量最高,天麻素在240d天时含量最高。考虑到天麻有效成分含量和天麻产量,生产中天麻最佳的采收时间为240d。
2.对天麻块茎不同部位来说,天麻多糖和天麻素在皮部和茎芽部含量最高,天麻蛋白质在髓部和茎芽部含量最高。
3.采用10%乙醇为提取溶剂、50℃恒温水浴锅回流提取2h,重复3次,95%沉淀,可以获得较好的天麻多糖提取效果。
4.DEAE纤维素阴离子交换层析柱分离纯化得到三种天麻多糖:TM1(白色结晶状固体)、TM2(淡黄色结晶状固体)、TM3(白色粉末状固体)。经理化性质鉴定,天麻多糖TM1为非淀粉类多糖,没有还原性。
5.纯度分析表明天麻多糖TM1是分子量均一的多糖,分子量范围为2.02×10~5,可作为结构分析使用。
6.经紫外光谱鉴定天麻多糖TM1是糖蛋白,采用纸色谱和高压电泳以及柱色谱分析结果表明天麻多糖TM1满足结构分析的要求。天麻多糖TM1经过完全酸水解衍生化后,用气相色谱分析,结果表明该多糖是由L-鼠李糖、L-岩藻糖、D-木糖、D-甘露糖、D-葡萄糖以摩尔比0.22:14.80:0.36:39.00:66.52构成。通过红外光谱、核磁共振、质谱及氨基酸分析等研究,可以初步确定天麻多糖TM1的主体鲒构为α(1-6)-D-Glc构成,含有少量β-构型,侧链分支点在C-4位,以α(1-4)糖苷键或者β(1-4)糖苷键与Glc相连,侧链较少。糖蛋白由11种氨基酸组成,以N-型连接于主链上。
Gastrodia elata BL.whose nutrition comes from epiphyte is a kind of perennially herbaceous plant, belonging to Orchidaceae.It's tuber was used as chinese traditional medicine.The Gastrodia elata BL. polysaccharide,Gastrodia and protein are the mainly effective ingredients in Gastrodia elata BL. Gastrodia elata BL.polysaccharide plays an important role on antioxidantion,clearing away oxidized free radical and delaying caducity.So,it is valuble to study Gastrodia elata BL.polysaceharide
Few studies on Gastrodia elata BL.polysaccharide were reported.There was not a full-blown method to purify Gastrodia elata BL.polysaccharide and it's chemical structure hasn't been known clearly.
Realizing the rules of the mainly effective ingredient's accumulation and distribution in Gastrodia elata BL.to explore the best purification method and analyse the structures of Gastrodia elata BL. polysaccharide.The results could be provided theory basis for choosing Gastrodia elata BL.in medicine,extracting effective ingredients in Gastrodia elata BL.and studying the pharmacological effect on Gastrodia elata BL.polysaccharide.
Study on accumulation and distribution of effective ingredient during different growth phases and different parts of Gastrodia elata BL.Filtrating the method of purifying Gastrodia elata BL. polysaccharide to got three polysaccharides TM1,TM2 and TM3 and analysed the chemical structure of TMI whose content is most.The results as follows:
1.The content of polysaccharide and protein extracted from Gastrodia elata BL.were the highest on the growth phase of 180 days and Gastrodia was the highest on the growth phase of 240 days.In order to gain the highest content of effective ingredients and biomass of Gastrodia elata BL.,we choosed 240 days as optimal picking time.
2.In different parts of Gastrodia elata BL.,the maximum contents of Gastrodia and polysaccharide were in the hull and bud of Gastrodia elata BL.and the maximum protein were in marrow and bud.
3.The optimum extraction condition was three times(2h/times,50℃)with 10%alcohol;the 95% alcohol sedimentation could get better extraction effect on polysaccharide extracted from Gastrodia elata BL.
4.After DEAE-Cellulose ion change column chromatography,there were three polysaccharides TM1(white,crystal,solid),TM2(yellow,crystal,solid),and TM3(white,powder,solid).After property appraisal,TM1 was a kind of polysaccharide which don't belong to amylum and had no deoxidization.
5.Molecular weight of polysaccharide TM1 extracted from Gastrodia elata BL.was hornogeneousand, about 2.02×10~(-5)and could use for construction measurement research.
6.After the appraisal of UV spectrum,TM1 contained litttle protein and satisfied the demand of structure analysis through PC,electrophoresis and pole chromatogram.After TMI were completely hydrolyzed by acid,analysis through Gas Chromatography indicated that TM1 were mainly made up of L-Rhamnose,L-Fucose,D-Xylopyranose,D-Mannitose and D-Glc by 0.22:14.80:0.36:39.00:66.521inmolar.Analyzed by Infrared Ray(IR),Nuclear Magnetic Resonance (NMR)Mass Spectrum(MS),and Aminophenol,the structure of TMI is chiefly linked by a-glycoside bonds and a fewβ-glycoside bonds,while the backbone of TM1 is mainly linked by 1-6 glycoside bonds and a few branch is linked by 1-4 linkage.The mueoprotein of TM1 are mainly made up of 11 kinds ofaminophenol,the mucoprotein and the backbone of TM1 is linked by N-glycosidic linkage.
但目前有关天麻多糖研究还很少,主要由于天麻多糖还没有成熟的纯化方法,且天麻多糖的结构还不清楚。了解天麻主要有效成分的积累分布规律,在此基础上探讨天麻多糖适宜的纯化方法,分析天麻多糖的组成及结构,为天麻用药和有效成分提取过程中选择天麻原料提供依据,为天麻多糖的药理作用研究提供结构基础。
本文通过对天麻有效成分在不同生长期和天麻块茎不同部位的积累分布进行研究:筛选天麻多糖提取纯化方法,得到3种纯化的天麻多糖,并对其中含量最多的一种天麻多糖TM1进行了结构分析。主要研究结果如下:
1.天麻多糖和蛋白质在生长期180d时含量最高,天麻素在240d天时含量最高。考虑到天麻有效成分含量和天麻产量,生产中天麻最佳的采收时间为240d。
2.对天麻块茎不同部位来说,天麻多糖和天麻素在皮部和茎芽部含量最高,天麻蛋白质在髓部和茎芽部含量最高。
3.采用10%乙醇为提取溶剂、50℃恒温水浴锅回流提取2h,重复3次,95%沉淀,可以获得较好的天麻多糖提取效果。
4.DEAE纤维素阴离子交换层析柱分离纯化得到三种天麻多糖:TM1(白色结晶状固体)、TM2(淡黄色结晶状固体)、TM3(白色粉末状固体)。经理化性质鉴定,天麻多糖TM1为非淀粉类多糖,没有还原性。
5.纯度分析表明天麻多糖TM1是分子量均一的多糖,分子量范围为2.02×10~5,可作为结构分析使用。
6.经紫外光谱鉴定天麻多糖TM1是糖蛋白,采用纸色谱和高压电泳以及柱色谱分析结果表明天麻多糖TM1满足结构分析的要求。天麻多糖TM1经过完全酸水解衍生化后,用气相色谱分析,结果表明该多糖是由L-鼠李糖、L-岩藻糖、D-木糖、D-甘露糖、D-葡萄糖以摩尔比0.22:14.80:0.36:39.00:66.52构成。通过红外光谱、核磁共振、质谱及氨基酸分析等研究,可以初步确定天麻多糖TM1的主体鲒构为α(1-6)-D-Glc构成,含有少量β-构型,侧链分支点在C-4位,以α(1-4)糖苷键或者β(1-4)糖苷键与Glc相连,侧链较少。糖蛋白由11种氨基酸组成,以N-型连接于主链上。
Gastrodia elata BL.whose nutrition comes from epiphyte is a kind of perennially herbaceous plant, belonging to Orchidaceae.It's tuber was used as chinese traditional medicine.The Gastrodia elata BL. polysaccharide,Gastrodia and protein are the mainly effective ingredients in Gastrodia elata BL. Gastrodia elata BL.polysaccharide plays an important role on antioxidantion,clearing away oxidized free radical and delaying caducity.So,it is valuble to study Gastrodia elata BL.polysaceharide
Few studies on Gastrodia elata BL.polysaccharide were reported.There was not a full-blown method to purify Gastrodia elata BL.polysaccharide and it's chemical structure hasn't been known clearly.
Realizing the rules of the mainly effective ingredient's accumulation and distribution in Gastrodia elata BL.to explore the best purification method and analyse the structures of Gastrodia elata BL. polysaccharide.The results could be provided theory basis for choosing Gastrodia elata BL.in medicine,extracting effective ingredients in Gastrodia elata BL.and studying the pharmacological effect on Gastrodia elata BL.polysaccharide.
Study on accumulation and distribution of effective ingredient during different growth phases and different parts of Gastrodia elata BL.Filtrating the method of purifying Gastrodia elata BL. polysaccharide to got three polysaccharides TM1,TM2 and TM3 and analysed the chemical structure of TMI whose content is most.The results as follows:
1.The content of polysaccharide and protein extracted from Gastrodia elata BL.were the highest on the growth phase of 180 days and Gastrodia was the highest on the growth phase of 240 days.In order to gain the highest content of effective ingredients and biomass of Gastrodia elata BL.,we choosed 240 days as optimal picking time.
2.In different parts of Gastrodia elata BL.,the maximum contents of Gastrodia and polysaccharide were in the hull and bud of Gastrodia elata BL.and the maximum protein were in marrow and bud.
3.The optimum extraction condition was three times(2h/times,50℃)with 10%alcohol;the 95% alcohol sedimentation could get better extraction effect on polysaccharide extracted from Gastrodia elata BL.
4.After DEAE-Cellulose ion change column chromatography,there were three polysaccharides TM1(white,crystal,solid),TM2(yellow,crystal,solid),and TM3(white,powder,solid).After property appraisal,TM1 was a kind of polysaccharide which don't belong to amylum and had no deoxidization.
5.Molecular weight of polysaccharide TM1 extracted from Gastrodia elata BL.was hornogeneousand, about 2.02×10~(-5)and could use for construction measurement research.
6.After the appraisal of UV spectrum,TM1 contained litttle protein and satisfied the demand of structure analysis through PC,electrophoresis and pole chromatogram.After TMI were completely hydrolyzed by acid,analysis through Gas Chromatography indicated that TM1 were mainly made up of L-Rhamnose,L-Fucose,D-Xylopyranose,D-Mannitose and D-Glc by 0.22:14.80:0.36:39.00:66.521inmolar.Analyzed by Infrared Ray(IR),Nuclear Magnetic Resonance (NMR)Mass Spectrum(MS),and Aminophenol,the structure of TMI is chiefly linked by a-glycoside bonds and a fewβ-glycoside bonds,while the backbone of TM1 is mainly linked by 1-6 glycoside bonds and a few branch is linked by 1-4 linkage.The mueoprotein of TM1 are mainly made up of 11 kinds ofaminophenol,the mucoprotein and the backbone of TM1 is linked by N-glycosidic linkage.
引文
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