【作者】 李珊珊；

【导师】 周义发；

【作者基本信息】 东北师范大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 蜂花粉是蜜蜂采集花粉后，加入少量的分泌物和花蜜，加工成的扁圆形花粉团。蜂花粉具有多种生物活性，例如提高机体免疫力、抗肿瘤、抗病毒、抗氧化和美容养颜等。多糖是蜂花粉的重要活性成分，本论文研究了六种蜂花粉果胶的结构以及抑制流感病毒神经氨酸酶的活性。主要包括；蜂花粉中I型聚鼠李半乳糖醛酸（RG-I）和聚半乳糖醛酸（HG）果胶的精细结构比较研究；蜂花粉果胶结构及其抑制流感病毒神经氨酸酶的活性；以荷花蜂花粉RG-I果胶（WNPP-2-RG）为例分析抗病毒活性的可能机制；对WNPP-2-RG的精细结构进行了分析，探讨了其结构与免疫活性的关系。果胶在自然界中含量丰富，结构复杂，活性多样。本论文的实验结果将为研究果胶结构与活性的关系奠定基础，促进果胶类多糖的进一步应用。论文选取荷花（Nelumbo nucifera）、虞美人（Papaver rhoeas）、玫瑰（Rosa rugosa）、荞麦（Fagopyrum esculentum）、五味子（Schisandra chinensis）和油菜（Brassicacampestris）六种蜂花粉为原料，通过系统地分级与纯化，得到六种蜂花粉RG-I和六种HG型果胶。结构分析表明，玫瑰蜂花粉RG-I型果胶只含有I型阿拉伯半乳聚糖（AG-I）侧链，其余五中RG-I型果胶都同时含有I型和II型阿拉伯半乳聚糖（AG-II）侧链；鼠李糖（Rha）与半乳糖醛酸（GalA）比例约为1:1，不含或仅含少量HG结构域；Rha和GalA构成的主链结构域含量在16%~50%之间；半乳糖和阿拉伯糖的总含量多数在70%~75%之间；油菜蜂花粉的RG-I型果胶除含有上述结构外，还含有葡聚糖和木聚糖侧链；RG-I型果胶分子量多在3.8×10~5Da左右。六种蜂花粉HG型果胶都含有较多的GalA；少量的RG-I和大量的HG结构域构成核心结构；侧链存在一定的差异，荷花蜂花粉HG果胶同时含有AG-I和AG-II两种类型的侧链，虞美人和玫瑰蜂花粉HG果胶含有1,5-arabinan和1,3-galactan侧链，五味子蜂花粉HG果胶含有1,5-arabinan和1,4-galactan侧链，荞麦蜂花粉HG果胶含有1,4-xylan侧链；虞美人和油菜蜂花粉HG型果胶中含有20%左右的葡聚糖；分子量在8×103~3.6×10~4Da之间。为了分析果胶的构效关系，对六种RG-I和六种HG果胶抑制流感病毒神经氨酸酶的活性进行了研究。神经氨酸酶是流感病毒发挥活性的关键酶，抑制其活性可达到抗流感病毒的作用，因此该结果可用于探讨果胶结构与抗流感病毒活性的关系。利用本实验室建立的生物发光法和经典的底物荧光法，对果胶级分地抑制流感病毒H1N1和H5N1神经氨酸酶的活性进行了研究。结果表明，富含HG结构域的果胶级分抑制活性明显高于RG-I级分。其中，来源于虞美人和油菜蜂花粉中的HG型果胶对流感病毒神经氨酸酶抑制活性高于其它HG型果胶，对两种酶的IC50均在1mg/mL以下。根据各个级分的结构特征可以推测，HG结构域是具有抑制流感病毒神经氨酸酶活性的主要活性单元，而葡萄糖侧链的存在会大大促进这种抑制作用。RG-I果胶没有直接抑制流感病毒神经氨酸酶的活性，可能是通过其它途径达到抗流感病毒效果。选择荷花蜂花粉RG-I级分WNPP-2-RG为代表，研究结果表明，它能够显著促进免疫指标，免疫调节作用可能是其发挥抗流感病毒活性的途径之一。对WNPP-2-RG的精细结构进行了鉴定。它以RG-I结构域为核心，AG-I和AG-II为侧链，分子量为3.8×10~5Da。侧链中1,6-Gal构成骨架结构，末端主要为Gal、少量Glc和Ara。为了进一步研究果胶结构与活性的关系，对WNPP-2-RG结构与免疫活性的关系进行了分析。通过部分酸水解除去侧链中的Ara，剩余核心结构为RG-I和AG-II结构域。体外免疫活性结果表明，侧链中的Ara的除去和Gal的暴露，对淋巴细胞增殖作用影响不大，但能够显著促进巨噬细胞的吞噬作用，降低NO释放能力。将WNPP-2-RG与人参RG-I型果胶（WGPA-2-RG）的结构和活性进行了对比研究。结果表明，侧链的类型、糖残基连接方式、甲酯化度、乙酰化度和分子量等不同，对活性的影响也不同。更多还原

【Abstract】 Bee pollen is the male gametophyte of gymnosperm and angiosperm, which containsmost of the nutrients afford to plant growth and development. It is well known as a naturalnutrition and healthy food especially in improving human’s immunity. Bee pollen had manyactivities, such as antitumor, antivirus and immunobiological activities. Polysaccharides arethe main active components in the bee pollen. In this paper, we analyzed the structure ofpectic polysaccharides from six types of bee pollen and their antivirus activity, including thestructure comparision of type I rhamnogalacturonan (RG-I) and homogalacturonan (HG); therelationship of structure and inhibitory activity on H1N1and H5N1neuraminidase; the fluvirus inhibitory mechanism of RG-I pectin; the fine structure of RG-I pectin from bee pollenof Nelumbo nucifera (WNPP-2-RG) and its structure-activity relationship. The results of thispaper can offer some datas about structure-activity relationship of pectins, improved theapplication of pectin.In this paper, we choosed six kinds of bee pollens, including Nelumbo nucifera,Papaver rhoeas, Rosa rugosa, Fagopyrum esculentum, Schisandra chinensis and Brassicacampestris, for studying the structure features and structure-activity relationship of pectins.Six RG-I and six HG pectins were obtained by a combination of DEAE-Cellulose andSepharose CL-6B colume. Structure analysis showed six RG-I pectins from bee pollencontained RG-I core region, both type I arabinogalactan (AG-I) and type II arabinogalactan(AG-II) sidechains except RG-I pectin from Rosa Rugosa which only contained AG-Isidechains; the ratio of rhamnose (Rha) and galacturonic acid (GalA) was nearly1:1,contained little HG region; RG-I mainchain contents was about16%~50%; total contents ofgalactose (Gal) and arabinose (Ara) were among70%~75%; the RG-I pectin from Brassicacampestris was contained glucan and xylan sidechains; the molecular weight of RG-Ifractions is about3.8×10~5Da. HG pectins from six kinds of bee pollen contains largeamounts of GalA; HG and RG-I region composed the core structure, ratio of Ara and Galwas nearly1:1; HG pectin from Nelumbo nucifera contained both AG-I and AG-IIsidechains, HG pectin from Papaver rhoeas and Rosa rugosa contained1,5-arabinan and1,3-galactan sidechains, HG pectin from Schisandra chinensis contained1,5-arabinan and1,4-galactan sidechains, HG pectin from Fagopyrum esculentum contained1,4-xylansidechains; HG pectin Papaver rhoeas and Brassica campestris contained glucose (Glc,about20%); the molecular weight was among8×103~3.6×10~4Da. The inhibitory activity of RG-I and HG fractions on H1N1and H5N1neuraminidaseswas studied by bioluminescent assay and typical fluorescent method. The results indicatedHG fractions had better inhibition activity than RG-I fractions, especially the HG fracionsfrom Papaver rhoeas and Brassica campestris, IC50were below1mg/mL. According to thestructure features of each fraction, we can concluded HG region was the important region oninhibitory activity of H1N1and H5N1neuraminidases, the Glc sidechains can improve thisactivity.The immunological activity of RG-I pectin (WNPP-2-RG) from bee pollen of Nelumbonucifera was analysed, it can improve the immunological activities, suggested RG-I mighthave antivirus activity through immunological way. Structure analysis showed WNPP-2-RGhas an RG-I core region, both AG-I and AG-II sidechains.1,6-Gal composed of thebackbone of sidechains, branched at O-3or O-4. Most of the Gal residues were determinedto be close to the molecular core, some Gal and Ara on the surface of the molecules. Theimmunological activitiy of WNPP-2-RG was related to its side chains. Ara residues affectedits macrophage phagocytosis and NO production, but no effect on lymphocyte proliferation.The comparation of structure-immunological activity between WNPP-2-RG and ginsengRG-I fraction WGPA-2-RG was performed. Ara residues in different RG-I pectins haddifferent effects on their activities.更多还原