【作者】 明建；

【导师】 陈宗道； 桂明英；

【作者基本信息】 西南大学 ， 农产品加工及贮藏工程， 2008， 博士

【摘要】 本文以云南特有食药用菌天麻[Gastrodia elata Blume]、野生羊肚菌[Morchellaesculenta（L.）Pers.]子实体为原料,利用多糖分离纯化技术、现代医学分析技术、降血脂功能食品的评价及检测方法和仪器分析技术对两种食药用菌活性多糖进行了系统研究,主要获得如下结果:（1）通过对天麻和羊肚菌多糖分离提取的工艺条件研究发现,天麻粗多糖提取的最佳工艺条件为:温度50℃、浸提时间3h、水料比10:1、乙醇浓度80%,在该条件下水提天麻粗多糖得率约为9.67%（干重）。羊肚菌粗多糖提取的最佳工艺条件:为浸提温度85℃、浸提时间2h、水料比50:1、乙醇浓度80%,在该条件下羊肚菌粗多糖得率约为4.96%（干重）。（2）通过对天麻和羊肚菌多糖分离纯化的系统研究发现,两种食药用菌多糖分别经水浸提、减压浓缩、Sevag法脱蛋白、透析、乙醇沉淀、DEAE-52纤维素柱层析和Sephadex G-100柱层析等一系列分离纯化过程,分别得到天麻多糖组分PGEB-3-H和羊肚菌多糖组分PMEP-1,经高效液相色谱（HPLC）鉴定为均一多糖,其得率分别为0.824%（干重）和1.58%（干重）。经凝胶层析测定,天麻多糖（PGEB-3-H）和羊肚菌多糖（PMEP-1）的相对分子质量分别为28 800和43 600。经过理化性质鉴定分析发现两种菌类食物活性多糖中都不含游离单糖、糖醛酸、蛋白质、多酚类物质等,均为非淀粉类中性纯粹多糖。（3）首次利用多糖化学结构分析的化学方法（单糖组成分析、甲基化分析、高碘酸氧化、Smith降解等）和现代仪器分析方法(气相色谱、红外光谱、GC-MS、¹H和¹³C核磁共振等)对天麻多糖（PEGB-3-H）和羊肚菌多糖（PMEP-1）进行全面结构特征分析,结果发现天麻多糖（PEGB-3-H）是α-1,4-连接为主链有少量α-1,6-连接分支的吡喃葡聚糖。羊肚菌多糖（PMEP-1）是以α-1,4-葡萄糖残基、α-1,6-半乳糖残基、α-1,2-甘露糖残基及α-1,5-阿拉伯糖残基为主要连接,同时含有α-1,2,6-甘露糖分支、α-1,4,6-葡萄糖分支和β-1,2,6-半乳糖分支的高度分支杂多糖。（4）通过选用与血脂代谢及肥胖、肿瘤细胞增殖相关的PPARs和A549模型对天麻多糖（PGEB-3-H）及羊肚菌多糖（PMEP-1）的生物活性进行高通量药物筛选（High-Throughput Screening,HTS）,结果发现PGEB-3-H和PMEP-1对PPARδ模型有一定的激活作用,但作用很弱,效果不显著;对PPARα模型没有激活作用;可见,两种多糖具有一定的生物活性,但比对照阳性药物（2-Bro:2-溴十六烷酸;WY:WY14643）的活性要弱得多。两种多糖在体外对肿瘤细胞A549均无明显的抑制作用。（5）对天麻多糖（PGEB-3-H）和羊肚菌多糖（PMEP-1）降血脂的功能进行动物实验。结果发现,所用的高脂饲料在实验周期内能使大鼠血清中总胆固醇（TC）、甘油三酯（TG）和低密度脂蛋白胆固醇（LDL-C）显著升高（P＜0.05）,高密度脂蛋白胆固醇（HDL-C）显著下降（P＜0.05）。两种多糖的各试验组(低剂量100mg·kg^-1·d^-1、中剂量200mg·kg^-1·d^-1、高剂量400mg·kg^-1·d^-1)大鼠血清中TG显著地低于高脂模型组（P＜0.05）。两种多糖对TG影响具有明显的剂量效应关系,剂量越高,TG降低幅度越大。在饲用高脂饲料的同时,给予高、中剂量PGEB-3-H和高剂量PMEP-1能使大鼠血清TG维持在正常水平,能达到与正常对照组无明显差异（P＞0.05）的水平。在各实验组中,只有低剂量PGEB-3-H组大鼠血清中TC明显低于高脂对照组,但仍然显著地高于正常对照组:只有低、中剂量PMEP-1组大鼠血清中LDL-C明显低于高脂对照组,但仍然显著地高于正常对照组;只有中剂量PGEB-3-H组大鼠血清中HDL-C明显高于高脂对照组,且与正常对照组无明显差异。PMEP-1三个实验组大鼠血清中HDL-C虽然不是显著地高于高脂对照组,但已达到与正常对照组无显著差异的水平。由此可见,实验用两种多糖对大鼠高脂血症的预防主要是通过对TG的影响实现的,两种多糖对预防高脂血症的形成有积极意义。（6）对天麻多糖（PGEB-3-H）改善学习记忆力的功能进行了研究。Morris水迷宫测试结果显示,东莨菪碱记忆损伤模型组（MC）小鼠找到平台的时间显著长于生理盐水组（NC）和阳性对照组（PC）。PGEB-3-H三个剂量组在大多数训练测试中,小鼠找到找到平台的时间短于MC组,且高剂量组与模型组之间的差异达到了显著水平（P＜0.05,P＜0.01）。由多糖对小鼠脑内乙酰胆碱（Ach）含量影响发现,高剂量PGEB-3-H组小鼠脑内乙酰胆碱（Ach）含量明显高于生理盐水组和阳性对照组(20 g·kg^-1·d^-1天麻全粉),而中、低剂量组与生理盐水组之间无明显差异（P＞0.05）。可见,天麻多糖（PGEB-3-H）对改善记忆能力有一定的作用,其作用机理可能是通过提高脑内乙酰胆碱（Ach）含量。本论文的创新之处在于:（1）利用云南产天麻和野生羊肚菌为原料,以开发现代保健食品为目的,成功地分离纯化获得了两种活性多糖,并对其理化性质进行了系统研究。（2）采用GC、IR、GC-MS、NMR等先进的化学仪器方法研究了天麻多糖（PGEB-3-H）和羊肚菌多糖（PMEP-1）的一级结构特征,明确地提出两种多糖的结构特征并对天麻多糖的可能重复单元的高级结构进行了模拟计算。（3）尝试运用高通量药物筛选（HTS）技术研究了天麻多糖（PEGB-3-H）和羊肚菌多糖（PMEP-1）的生物活性。（4）首次研究并确证了天麻多糖是天麻改善学习记忆能力的重要活性物质之一,也为天麻益智功能的物质填补了新的内容。（5）研究发现了天麻多糖和羊肚菌多糖具有预防高血脂形成的作用,尤其是对甘油三酯的影响。本文研究方法规范可靠,所得结论完全可以有效地应用于现代保健食品及多糖药物的生产指导中。更多还原

【Abstract】 Two polysaccharides were isolated and purified from the fruiting body of Gastrodia elata Blume and wild Morchella esculenta（L.）Pers,edible-medicinal fungi grown in Yunnan.Then,anticaner ability,blood lipids reducing activity and congitive activity of two polysaccharides were investigated by high-throughput screening and animal experiments in vitro or in vivo.The following results were obtained.（1） The optimal parameters for extraction of two crude polysaccharides were achieved.Highest recovery（9.76%db） of crude polysaccharide from Gastrodia elata Blume could be obtained with liquid-material ratio 10:1,50℃,3 h and precipitating alcohol concentration 80%.When the pruiting body of Morchella esculenta（L.）Pers. extracted with liquid-material ratio 50:1,85℃for 2 h,and precipitated with 80%,the highest recovery of crude polysaccharide（4.96%,db） was gained.（2） Two HPLC proved purified polysaccharides,PGEB-3-H from Gastrodia elata Blume and PMEP-1 from Morchella esculenta（L.）Pers.,were obtained from two crude polysaccharides successicvely purified by Sevag deprotein,dialysis,DEAE-52 column and Sephadex G-100 column.The recovery of two purified polysaccharides were 0.824%and 1.58%respectively.The molecular weight of PGEB-3-H and PMEP-1 were determined with column chromography as 28800 and 43600 respectively. Physico-chemical characteristics analysis indicated that two polysaccharides do not contain monosaccharose,uronic acid,protein and polyphenols.Therefore,it can be concluded that they are non-starch neutral pure polysaccharides. （3） Using chemical methods（methylation reaction,periodate oxidation,Smith degradation） and instrument analysis(IR,GC-MS,¹H-NMR and ¹³C-NMR),the structural characterics of PGEB-3-H and PMEP-1 were investigated.It was found that PGEB-3-H was mainly composed by glucose,and it had aα-1,4-linked glucan main chain occasionally branched withα-1,6 glycosidic linkage PMEP-1 was identified as a high-ramified heteropolysaccharide,whose main chain was a mixing linkage ofα-1,4-glucose,α-1,6-galactose,α-1,2-mannose andα-1,5-arabose,and its side chain attached atα-1,2,6-mannose,α-1,4,6-glucose andα-1,2,6-galactose.（4） The possible bologival activities of PGEB-3-H and PMEP-1 were tested with blood lipids matablism and obesity related PPARs and cancer related A549 models by high-throughput screening（HTS）.The result showed PGEB-3-H and PMEP-1 had slight activation effects on PPARδmodel but they are not significantly differed from control ones.No effects were obsereved of testing polysaccharides on PPARαmodel. Then,it can be concluded on the basis of this experiment that the tested two polysaccharides exerted a slight bioacticity,which were evry low as compared to positive controls,2-Bro and WY14643.The high-throughput screening proved that neither PGEB-3-H nor PMEP-1 has obvious inhibitory effect on tumour cell A549 in vitro.（5） The effet of PGEB-3-H and PMEP-1 on reducing serum blood lipids of rats was studied,and the results indicated that the high-fat diet useded in this experiment could successfully induced formation of hyperlipemia（increase in TC,TG and LDL-C, decrease in HDL-C） in rats,and the model of rat was properly bulited.Low,medium and high dose of PGEB-3-H and PMEP-1 could significantly（P＜0.05） reduce TG in serum of rats.The higher the dose,the stronger function in reducing blood lipids was founded.It was very interesting that serum TG level was maintained at normal level when rats fed with high-fat diet as well as medium or high dose of tested polysaccharides.However,for TC in serum,marked reduction,but still higher than normal level,was only observed with low dose of PGEB-3-H when compared to high-fat diet control.Low or medium dose of PMEP-1 could significantly（P＜0.05） reduce LDL-C when compared to high-fat diet control.However,medium dose of PGEB-3-H could significantly（P＜0.05） increase total cholesterin HDL-C to normal level when compared to high-fat diet control.Although,no significant difference was observed between PMEP-1 groups and high-fat diet control in HDL-C,the level of HDL-C in PMEP-1 groups incereased to a higher level which caoul not be significantly differed from normal control.So PGEB-3-H and PMEP-1 could significantly reduce the blood lipids（or inhibit the formation of hyperlipemia） of rats,and they have stronger effecs on TG than TC.（6）The congitive activity of PGEB-3-H was conducted in this paper.Morris water maze showed that scopolamine treated mice need loner time to corss maze than noral and positive control.PGEB-3-H had a positive effect scopolamine treated mice to corss maze and high dose of PGEB-3-H could significantly shorten the crossing time.At the same time,it was founded that high dose of PGEB-3-H could significantly could significantly increase（P＜0.05） acetylcholine（Ach） content in mice cerebra,which even higher than that of positive control(20 g·kg^-1·d^-1 Gastrodia elata Blume powder).So,it can be concluded that PGEB-3-H could enhance congitive ability and its mechanism may be regarded as increasing Ach content.The creative aspects of this paper were summarized into five points as following. （1） In order to make basis for 3^rd generation healthy foods,two purified polysaccharides were firstly and successfully obtained from edible-medicinal fungi Gastrodia elata Blume and wild Morchella esculenta（L.）Pers.,and the physico-chemical characteristics were also systematically studied.（2） The primary structure of PGEB-3-H and PMEP-1 was researched firstly by chemical and instrument methods such as gas chromagraphy, IR,GS-MS and NMR,the structural character of PMEP-1 and the repeating unit of PGEB-3-H were firstly identified.On this basis,3D structure of PGEB-3-H was therotical modelled with MM2 programme.（3） High-throughput screening（HTS） technique was subjected for possible boiactivity of PGEB-3-H and PMEP-1.（4） A polysaccharide,PGEB-3-H,was firstly proved as a new substance member for congitive function of Gastrodia elata Blume besides gastrodine,which incoporated new infromation into the studies in congitive activity of Gastrodia elata Blume.（5） PGEB-3-H and PMEP-1 was approved as hepful drugs to act against the formation of hyperlipidemia,especially TG in serum,when a high fat diet was consumed.In this paper,the research methods are normative and reliable,and the conclusion can be effectively used for production of the 3^rd generation healthy foods.更多还原