【作者】 王军辉；

【导师】 罗建平；

【作者基本信息】 合肥工业大学 ， 农产品加工及贮藏工程， 2011， 博士

【摘要】 金钗石斛（Dendrobium nobile Lindl.）为兰科（Orchidaceae）石斛属（Dendrobium Sw.）多年生附生草本植物,是中国药典（2010版）收载的名贵中药。金钗石斛化学成分的研究主要为生物碱、萜类、木脂素、酚类等小分子化合物,对其多糖类组分的研究较少。为了了解金钗石斛多糖的化学结构及生物活性,本文对金钗石斛多糖进行了提取、分离纯化、结构表征、糖链构象和抗白内障活性的研究。金钗石斛干燥的茎脱色脱脂后经热水浸提获得金钗石斛水提粗多糖DNP-W,DNP-W进一步用DEAE-纤维素和葡聚糖凝胶进行分离纯化,获得7个均一多糖组分DNP-W1A、DNP-W1B、DNP-W2、DNP-W3、DNP-W4、DNP-W5和DNP-W6。采用化学（甲基化分析、高碘酸氧化-Smith降解反应、部分酸水解）与波谱学（UV、IR、GC、GC-MS、ESI-MS、NMR）结合的手段,对各均一多糖的一级结构进行了表征。结果表明DNP-W1A是由Man、Glc和Gal组成的半乳葡甘聚糖,主链由α-（1→6）-linked Manp和α-（1→3）-linked Manp组成,Glcp和Galp残基支链分别通过α-（1→6）-linked Manp的3位和2位连接在主链上。DNP-W1B是一个阿拉伯半乳葡聚糖,拥有[→4)-β-Glcp-（1→6）-β-Glcp-（1→]二糖单位的主链结构,在β-（1→6）-linked Glcp的4位形成分支,支链由Galp和Arap残基组成。DNP-W2是一个部分乙酰化的半乳甘露葡聚糖,主链由β-（1→4）-linked Glcp、β-（1→6）-linked Glcp及β-（1→4）-linked Manp组成,其中部分β-（1→4）-linked Glcp及β-（1→4）-linked Manp的6位连接有末端Galp。DNP-W3为阿拉伯半乳聚糖,主链由β-（1→3）-linked Galp组成,在其4位连接Arap和Rhap残基支链。DNP-W4是一个主链由β-（1→4）-Glcp、β-（1→6）-linked Glcp及β-（1→6）-linked Galp组成的酸性杂多糖。DNP-W5和DNP-W6为果胶类多糖,由[→4）-α-GalAp-（1→2）-α-Rhap-(1→]二糖重复单位构成主链,DNP-W5主链GalAp的3位和鼠李糖的4位存在分支,侧链包括半乳糖区域和葡萄甘露糖区域和木糖链。DNP-W6主链Rhap的4位存在分支,支链区包括半乳糖区域和葡萄甘露糖区域。抗白内障活性试验表明,金钗石斛水提粗多糖及各均一多糖组分都对链脲佐菌素（STZ）诱导的糖尿病性白内障有一定的延缓作用,但各多糖处理组之间差异较大,以粗多糖与DNP-W1A处理组的效果最好,呈剂量依赖性。实验结束时,粗多糖与DNP-W1A处理组大鼠晶体混浊程度明显低于模型及其它多糖处理组,体重增加幅度在150-190%之间,血糖下降了25%以上,晶体各清除氧自由基酶活性及GSH含量显著高于STZ模型组,膜脂过氧化指标MDA与H₂O₂、蛋白质氧化损伤产物、糖基化终产物等含量显著低于STZ模型组。结合金钗石斛多糖抗白内障活性试验的生化指标测定及各均一多糖的结构分析,金钗石斛多糖抗白内障活性与其减轻大鼠晶体氧化损伤及抑制糖基化终产物的形成有关,其活性作用的化学结构基础可能与多糖中甘露糖含量及其连接方式有关。进一步采用扫描电镜（SEM）、X-射线、差示扫描量热仪（DSC）、尺寸排除色谱-激光光散射（SEC-LLS）和原子力显微镜（AFM）等方法对抗白内障活性多糖DNP-W1A的溶液行为进行了探讨。SEM、X-射线及DSC分析表明DNP-W1A不具有完整单晶结构,其玻璃化转变温度为85℃。AFM显示DNP-W1A糖链分子间互相缠绕,形成大小不一的无规线团与岛屿状结构。SEC-LLS结果表明DNP-W1A在溶液中主要以半柔顺链的无规线团存在。更多还原

【Abstract】 Dendrobium nobile Lindl. （Chinese name“Jin-Chai-Shi-Hu”） is a species of Orchidaceae with distribution in southeast Asia, southwest and south of China and has been recorded in Chinese Pharmacopoeia （2010 Edition）. The chemical structures of several low molecular weight compounds from D. nobile have been elucidated, such as alkaloids, bibenzyls, stilbenoids, glycosides, sesquiterpenes, fluorenones and phenanthrenes. In contrast, the polysaccharides of D. nobile were much less investigated, even though polysaccharides have been proved to be one of major active constituents of Dendrobium plants. In order to investigate the chemical structural features, solution behavior and biological activities of the polysaccharides obtained from D. nobile, the isolation, purification, structural elucidation, solution behavior and anti-cataract tests of polysaccharides from D. nobile were carried out in this paper.The hot-water extracts from the dried stems of D. nobile were precipitated with 4 volume ethanol to give the crude polysaccharide DNP-W. Further, DNP-W was fractionated on DEAE–Cellulose anion-exchange column and purified by gel filrtaiton chromatography to obtain seven homogeneous polysaccharide fractions: DNP-W1A, DNP-W1B, DNP-W2, DNP-W3, DNP-W4, DNP-W5 and DNP-W6. The structural features of seven homogeneous polysaccharides were revealed by a combination of chemical and instrumental analysis, including IR, GC, GC-MS, ESI-MS, methylation analysis, periodate oxidation, partial acid hydrolysis, and NMR. The results showed that DNP-W1A had the backbone consisting of 1,6-linked and 1,3-linkedα-D Manp residues with branches at O-3 and O-2 of 1,6-linkedα-D-Manp residues. The branches were composed of terminalβ-D-Glcp residues, 1,4-linkedβ-D-Glcp residues, 1,6-linkedα-D-Galp residues, and terminalα-D-Galp residues. DNP-W1B possessed a backbone of a disaccharide of [→4)-β-Glcp-（1→6）-β-Glcp-（1→], with 33% branches at O-4 of （1→6）-linkedβ-D-Glcp residues. The side chains contained arabinosyl and galactosyl residues. DNP-W2 was a 2-O-acetyl galactomannoglucan and had a backbone consisting of （1→4）-linkedβ-D-Glcp, （1→6）-linkedβ-D-Glcp, and （1→4）-linkedβ-D-Manp residues, with branches at O-6 of （1→4）-linkedβ-D-Glcp andβ-D-Manp residues. The branches were composed ofα-D-galp residues. The acetyl groups were substituted at O-2 of （1→4）-linked Manp residues. DNP-W3 was a rhamnoarabinogalactan and had the backbone consisting of 1,3-linkedβ-D-Galp residues with branches at O-4. The branches were composed of 1-linkedβ-L-Arap and 1,4-linkeα-L-Rhap residues. DNP-W4 was a complex heteropolysaccharide and possessed a backbone compoesd of （1→4）-linkedβ-D-Glcp, （1→6）-linkedβ-D-Glcp, and （1→6）-linkedβ-D-Galp residues, with substitutes at O-4/6 of Glcp residues and O-3 of Galp residues. The branches composed of terminal Manp residues, （1→6）-linkedβ-D-Manp, （1→3）-linkedβ-D-Glcp,β-D-Glcp,β-D-Galp, （1→4）-linkedα-D-GalAp, （1→2）-linkedα-L-Rhap, and Xylp residues. DNP-W5 and DNP-W6 were pectic polysaccharides with the backbone of a disaccharide of [→4）-α-GalAp-（1→2）-α-Rhap-(1→]. The side chains of DNP-W5 contained galactosyl, mannosyl, glucosyl, and xylosyl residues substituted at O-4 of the Rhap and O-3 of the GalpA residues. The side chains of DNP-W6 were attached at the O-4 of Rhap residues, including terminal Manp,β-（1→4）-linked Glcp residues,β-（1→6）-linked Manp residues, terminal Galp,β-（1→3） andβ-（1→6）-linked Galp （galactan） residues.The anti-cataract activities of polysaccharide fractions from the stems of D. nobile were assessed through streptozotocin（STZ）-induced cataract model in Sprague-Dawley （SD） rats. The results indicated that all the polysaccharide fractions from D. nobile exhibited anti-cataract activities to some extent. Among tested polysaccharide samples, DNP-W and DNP-W1A presented higher anti-cataract activities and there was a dose–response relationship between concentration of polysaccharides and anti-cataract activities. According to DNP-W and DNP-W1A treatment groups, at the end of experiment, the level of lenticular opacification from rats was much lighter than that of other polysaccharide treatment groups, the added value of body weight was between 150 and 190%, and the blood sugar decreased over 25%. The mean activities of the antioxidant enzymes and GSH content were significantly higher in lenses of rats from DNP-W and DNP-W1A treatment groups than in lenses of rats from model group. Conversely, the mean levels of MDA, H₂O₂, advanced glycation end products and protein carbonyl content in lenses was significantly lower in DNP-W and DNP-W1A group rats than in model group rat lenses. Together with the results of biochemical indicator in anti-cataract activity experiments and structural characteristics of seven homogeneous polysaccharide fractions, the biochemical mechanism of protective effect in rat lenses appeared to occur by maintaining the antioxidant defense system and decreasing protein glycation. And the chemical structure basis for anti-cataract activities is related to mannose content and its glycosyl linkage types.The conformation and solution behavior of DNP-W1A were investigated with scanning electron microscope （SEM）, X-ray, differential scanning calorimetry （DSC）, size-exclusion chromatography-Laser light scattering （SEC-LLS）, atomic force microscopy （AFM） and Congo-red reaction. The results of SEM,X-ray and DSC indicated that the physical structure state of DNP-W1A was noncrystal, and its vitrification temperature was 85℃. The AFM observation showed that the molecular chain morphology of DNP-W1A was different size islands and random coils in solution. DNP-W1A was proved to be the hemi-flexible with the technique of SEC-LLS, Congo-red reaction and intrinsic viseosity, and the conformation could be changed by temperature.更多还原