【作者】 于丽；

【导师】 周义发； 台桂花；

【作者基本信息】 东北师范大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 人参(Panax ginseng C. A. Meyer)作为名贵中药在我国已有4000多年的应用历史。人参中含有多种有效成分,如人参皂甙、多肽、挥发油、寡糖和人参多糖等。人参多糖主要是由人参淀粉和人参果胶两部分组成。人参淀粉是淀粉样葡聚糖,来源于造粉体;人参果胶为含有半乳糖醛酸(GalA)的一大类多糖分子,来源于人参根细胞壁。研究表明人参果胶具有调节免疫、抗辐射、抑制肿瘤细胞迁移、促进细胞凋亡等活性。但目前对人参果胶在细胞壁中的分布情况以及精细结构缺少全面系统的研究。本论文通过免疫组化的方法,利用抗体作为探针对植物细胞壁多糖的分布进行了研究。然后提取分离人参多糖,在本实验室前期研究的基础上,进一步利用酶水解、离子交换和凝胶层析等组合方法制备人参果胶分子的各种结构域,研究其分子的精细结构。研究结果如下:利用细胞壁多糖抗体,采用免疫组化的方法,结合碱处理和酶作用研究了结晶化纤维素、半纤维素、果胶和蛋白聚糖这些细胞壁多糖或蛋白多糖在人参根中的分布和含量。人参根初级细胞壁多糖组成为:结晶化的纤维素、木葡聚糖、甘露聚糖、I型聚鼠李半乳糖醛酸(RG-I)型果胶结构域、阿拉伯半乳聚糖蛋白(AGP)以及高度甲酯化的同聚半乳糖醛酸(HG)结构域。次级细胞壁主要含有木聚糖、extensin以及未甲酯化和乙酰化的HG型果胶。AGP蛋白聚糖分布比较广泛,分布于人参根中的三个部位:淀粉粒表面,初级细胞壁以及树脂道中。经研究证明在人参根中HG能够掩盖一些多糖的抗原表位,这种现象广泛存在于植物体中。HG能够掩盖半纤维素的抗原表位证明了HG和半纤维素之间存在某种方式的连接;HG能够掩盖阿拉伯聚糖和AGP糖基的抗原表位,但是不能掩盖半乳聚糖的抗原表位,进一步表明了在植物体中果胶分子组建成为高级结构时HG和半乳聚糖装配于高级结构的表面,而阿拉伯聚糖和阿拉伯半乳聚糖包裹于高级结构的内部。根据本实验室系统分级人参多糖的方法,制备出人参多糖的五个级分WGPA-H、WGPA-1、WGPA-2、WGPA-3和WGPA-4,再根据分子量分布用凝胶层析将WGPA-1～WGPA-4进一步分级得到WGPA-1-HG~WGPA-4-HG四个HG结构域和WGPA-1-RG~WGPA-4-RG四个RG结构域。为制备非HG型结构域,将人参果胶WGPA用果胶酶水解除去其中的HG结构域,然后采用DEAE-Sepharose Fast Flow离子交换柱,以0、0.07、0.16、0.22和0.3 M的NaCl水溶液分步洗脱,得到相应的五个级分WGPA-UD1、WGPA-UD2、WGPA-UD3、WGPA-UD4和WGPA-UD5。根据分子量大小的不同,用凝胶柱层析将WGPA-UD2~WGPA-UD5进一步纯化得到RG-I-1、RG-I-2、RG-I-3A、RG-I-3B和RG-I-4五个RG-I级分以及AG1、AG2两个AG级分和HM-HG一个HG级分。通过酶学方法、酶联免疫吸附法、高效液相色谱、13C-核磁共振(13C NMR)分析测定人参果胶多糖各级分的结构特征。结果表明WGPA-1-HG和WGPA-2-HG主要含有甲酯化和乙酰化的HG,另外还有少量的RG-I型果胶,其侧链为II型阿拉伯半乳聚糖(AG II)。WGPA-3-HG是甲酯化非常高的HG,WGPA-4-HG是未酯化的HG型果胶,在这两个级分中也存在短的AG II。RG-I-2、RG-I-3A、RG-I-3B和RG-I-4都是带有RG-I型和聚GalA片段的结构域的多糖分子。RG-I-2和RG-I-3B的分子量较小,为5 Kd,GalA的含量远远高于鼠李糖(Rha)含量。通过NMR结果显示RG-I-2和RG-I-3B的甲酯化和乙酰化度比较高,说明这两个级分中含有长的甲酯化或者乙酰化的聚GalA片段。这两个级份的侧链比较复杂,有阿拉伯半乳聚糖。RG-I-3A和RG-I-4的分子量大于50 Kd,在这两个级分中Rha/GalA的比例分别为0.34和0.64,它们的乙酰化度近于100%,说明这两个级分中都含有高乙酰化的聚GalA片段,其侧链为阿拉伯半乳聚糖,半乳聚糖和少量的阿拉伯聚糖。应用细胞壁抗体检测这些果胶中含有的抗原表位,发现在树脂道中结合的抗体与这些RG-I果胶结构域都有很强的结合,而在树脂道中没有结合的抗体也不能与这些RG-I果胶结构域结合,由此推测人参水提果胶多糖来源于人参根的树脂道以及组成树脂道的上皮细胞。RG-I-3A和RG-I-4两个级分经过各种与果胶相关的酶进一步水解或者部分酸水解处理之后,再采用13C NMR和酶联免疫吸附法分析得到的多糖分子,以便探讨RG-I-3A和RG-I-4的精细结构。结果表明RG-I-4具有较长的高度乙酰化的聚GalA片段。RG-I-4的主链以α-(1→4)–GalpA构成的HG型结构域作为“平滑区”,以[→4)-α-GalpA-(1→2)-α-Rhap-(1→]构成的RG-I型结构域连接较多侧链形成的“毛发区”。RG-I-3A的主链由两部分组成,四糖结构域[→4)-α-GalpA-(1→4)-α-GalpA(1→4)-α-GalpA-(1→2)-α-Rhap-(1→]n以及二糖结构域[→4)-α-GalpA-(1→2)-α-Rhap-(1→]n。RG-I-3A和RG-I-4的GalA全部在O-2或者O-3被乙酰基取代。由13C NMR和酶联免疫吸附法的结果也推测RG-I-3A和RG-I-4的侧链相似,侧链多且较短,RG-I-3A的侧链取代度为71%,RG-I-4的取代度为67%,侧链有以下种形式:[α-1,5-Araf]5-7,[α-1,5-Araf]1-4,含量较低,更多的Ara处于非还原末端;少量的[β-1,4-Galp]4-6,80%以上的[β-1,4-Galp]1-4;非常少量的[β-1,3/1,6-Galp]1-2,其中[β-1,3/1,6-Galp]1-2通过Rha的C-4连接到主链。更多还原

【Abstract】 Panax ginseng C. A. Meyer (P. ginseng) has been used in China as traditional medicine for more than 4,000 years. P. ginseng contains many active components including ginsenosides, ginseng peptide, oliogosaccharides and ginseng polysaccharides. Ginseng polysaccharides are composed of starch-like glucans and ginseng pectic polysaccharides. Ginseng pectic polysaccharides are one of the three main components of ginseng root cell walls. Ginseng pectic polysaccharides have a wide range of pharmacological and therapeutically actions. They act on the central nervous system, cardiovascular system and endocrine secretion, promote immune function and metabolism, possess bio-modulation action and also have anti-stress and anti-ageing activities. However, little is known about the distribution of ginseng pectin polysaccharides in cell walls and their fine structures. We have undertaken an immunochemical study of ginseng polysaccharides. Antibodies directed to cell wall polysaccharide epitopes are important tools for the immunochemical analysis of polysaccharides in situ and they can also be useful to identify structural features present in isolated polysaccharide fractions. A combination of enzyme treatment, anion-exchange and size-exclusion chromatographies are used to fractionate different pectic polysaccharide domains; then the structures of the isolated pectic polysaccharide domains were studied by enzymolysis, ELISA, chemical methods and instrumental analysis.Here we report a study of the use of the cell wall antibodies in immunofluorescence procedures to determine the distribution and contents of cellulose, hemicelluloses, pectic polysaccharides and protoglycan in ginseng roots. Primary cell wall polysaccharides in ginseng roots are made of crystalline cellulose, xyloglucan, mannan, rhamonogalacturonan I (RG-I), protoglycans and high-methyl homogalacturonan (HG). Secondary cell wall is mainly composed of xylan, extensin and high-acetylated HG. Arabinogalactan protein (AGP) glycan is present on the surface of starch granules, in secretory cavities and in primary cell walls, which indicate diverse functions of AGP in ginseng roots. The results also showed that HG masking the epitopes of hemicelluloses and pectic polysaccharides in situ is widespread phenomenon in the primary ginseng cell walls. HG could mask the epitopes of hemicelluloses indicates that there are linkages between HG and hemicelluloses although we could not deduce if it is covalent linkages or not. HG could mask the epitopes of arabinan and AGP glycans but not galactan indicate that when the polymers assemble into cell walls, HG and galactan are present on the surface of cell wall, while arabinan and AGP glycans are wrapped inside.In order to study the structure characteristics of water-extracted pectic polysaccharides, ginseng pectin were fractionated systematically and detailedly according to the previous study in our lab. WGPA-H, WGPA-1, WGPA-2, WGPA-3 and WGPA-4 were made according to the method in our lab. Then WGPA-1~WGPA-4 were separated by size-exclusion chromatography to obtain four HG fractions and four RG fractions. In order to get non-HG pectic polysaccharides, ginseng pectin WGPA was hydrolyzed by endo-polygalacturonase to remove the un-esterified HG domain, then the hydrolysate was separated by a combination of anion-exchange and size-exclusion chromatography to get five RG-I fractions (RG-I-1, RG-I-2, RG-I-3A, RG-I-3B and RG-I-4), two AG fractions (AG1 and AG2) and one HG fraction (HM-HG).The structural features of isolated pectic fractions were elucidated using enzymolysis, HPLC, ELISA and 13C NMR. Analytical results showed that WGPA-1-HG and WGPA-2-HG were composed of a linearα-1,4-linked D-GalA with different degree of methyl-esterification and acetylation, and a small amount of neutral sugars that existed in these fractions might constitute RG-I domains with type II arabinogalactan linked to HG domains. Both WGPA-3-HG and WGPA-4-HG contain un-esterified and methyl-esterified HG. RG-I-2, RG-I-3A, RG-I-3B and RG-I-4 contain the characteristic monosaccharide compositions of RG-I which are GalA, Rha, Gal and Ara. Fractions RG-I-2 and RG-I-3B were two similar fractions, which had small molecular weights (around 5 Kd) and low ratios of Rha/GalA (around 0.25). RG-I-2 and RG-I-3B contained highly esterified HG segments and the the side chains were very complexed with type I and type II arabinogalactan (AG) as side chains. RG-I-3A and RG-I-4 are high molecular weight fractions that have similar compositions including typical RG-I monosaccharides and NMR peaks indicating high acetylation. Results showed that these two fractions contained highly esterified HG segments and the side chains were AG. Immunochemical analysis was processed on the structure features of the isolated ginseng pectic fractions which showed some antibodies bound strongly on these fractions. The striking feature of the in situ localization of the epitopes (that were observed to be abundant in the isolated ginseng fractions) is their predominant occurrence in the regions of cortical secretory cavities of the roots. The other cell wall components such as hemicelluloses that were not detected in the isolated fractions were not abundantly detected at these cavities. So the water-extracted pectic polysaccharides originated from secretory cavities.Pectin related enzyme hydrolysis, partial acid hydrolysis, 13C NMR and ELISA were used to study the fine structures of RG-I-3A and RG-I-4. The results showed that the backbone of RG-I-3A consists of two segments: the four residues repeats [→4)-α-GalpA-(1→4)-α-GalpA(1→4)-α-GalpA-(1→2)-α-Rhap-(1→]n and two residues repeats [→4)-α-GalpA-(1→2)-α-Rhap-(1→]n. The backbone of RG-I-4 consists of the―smooth region‖ofα-(1→4)–GalpA and [→4)-α-GalpA-(1→2)-α-Rhap-(1→]n as the―hairy region‖. The side chains of RG-I-3A and RG-I-4 are short and concentrated. All the side chains are attached to the O-4 of Rha in the backbone. 71% of Rha in RG- I-3A and 67% of Rha in RG-I-4 contained side chains. There are several types of side chains: small amount of [α-1,5-Araf]5-7; [α-1,5-Araf]1-4, most Ara is on the non-reduce terminate; less [β-1,4-Galp]4-6, more than 80% [β-1,4-Galp]1-4; less [β-1,3/1,6-Galp]1-2 which linked to the C-4 of Rha.更多还原