【作者】 辜明；

【导师】 张玉； 王凯平；

【作者基本信息】 华中科技大学 ， 药剂学， 2009， 硕士

【摘要】 香菇多糖是香菇中最重要的一类生物活性物质,作为一种免疫促进剂,在20世纪60年代已引起人们广泛的研究兴趣。现代研究表明:香菇多糖具有抗病毒、抗肿瘤,调节免疫力、降血糖及抗氧化等功能。LT1是本实验室从香菇子实体中提取得到的一种单一分子量多糖,经结构研究发现LT1是一种未报道的新型香菇多糖,其分子量Mw=642 kDa,经检验符合国家香菇多糖注射剂原料药的质量标准的要求。本论文对LT1的提取分离方法、化学结构和糖链构象进行了较为系统的研究,内容主要包括香菇多糖LT1的分离纯化、LT1的质量控制研究、LT1的化学结构研究和糖链构象研究四部分。第一部分香菇多糖LT1提取纯化研究对香菇多糖的提取纯化工艺进行研究,改进了传统的提取纯化方法,以更少的有机试剂以及纯化步骤提取出香菇多糖LT1,经高效液相色谱证实LT1为一种单一分子量多糖,其分子量Mw=642 kDa。Molish实验说明LT1为糖类物质,KI-I2实验表明LT1不是淀粉类物质。第二部分香菇多糖LT1的质量控制研究依据国家香菇多糖注射剂原料药质量标准WS1-320(X-263)-2004Z,对香菇多糖LT1进行质量控制研究。采用傅立叶红外光谱、紫外分光光度计、高效液相色谱等方法,考察LT1的光谱特征、糖含量、糖链特征等物理化学性质。傅立叶红外光谱分析结果表明香菇多糖LT1中含有α、β构型单糖;经硫酸-苯酚法测得LT1的糖含量达到98.7%(以葡萄糖计);表征糖链特征的高碘酸氧化实验测得氧化前后的吸光度之差为0.19。以上测试结果均符合国家质量标准WS1-320(X-263)-2004Z的要求,说明香菇多糖LT1符合国家香菇多糖注射剂原料药的要求。第三部分香菇多糖LT1的化学结构研究对香菇多糖LT1的化学结构进行研究。毛细管粘度法测定香菇多糖LT1的粘度性质。粘度分析结果显示,LT1显示与一般高分子化合物不同的粘度性质,其比浓粘度和增比粘度均随浓度增加而减小。综合运用气相色谱-质谱联用技术、傅立叶红外光谱、糖类甲基化分析、高碘酸氧化实验、Smith降解实验、核磁共振波谱等方法,考察LT1的光谱特征、单糖组成和糖链连接等化学结构性质。单糖组成GC分析结果表明香菇多糖LT1仅由D-glucose组成;傅立叶红外光谱显示LT1为典型的多糖类物质,含有α和β构型两种D-glucose;高碘酸氧化和Smith降解表明LT1含有1-3,1-4,1-6连接的糖残基;甲基化实验证明了上述结果,从甲基化实验GC图谱上可知:还原末端,1-3连接糖残基,1-4连接糖残基和1-4-6连接糖残基的分子比例约为1:1:2:0.6;由于LT1分子量较大,水溶性较差,故对其部分酸水解产物进行核磁共振实验(1H、13C、DQF-COSY、HSQC、HMBC),结合上述化学以及波谱学实验,可以推测LT1分子可能为如下结构:由以上结构可知,香菇多糖LT1是一种从香菇子实体中提取出的新型单一分子量多糖。第四部分香菇多糖LT1的糖链构象研究相关研究表明,多糖的药理活性与其糖链的三螺旋结构有密切联系。多糖在不同溶液中其分子会呈现不同的构象,同时多糖分子的构象改变往往伴随着一些理化参数如旋光度等的变化。通过测定LT1在不同浓度NaOH溶液中的旋光度和刚果红实验证明,LT1分子在0.1M NaOH中以三螺旋结构存在,在NaOH浓度大于0.20M时可能会发生构象的转变。从LT1的原子力显微镜(AFM)图谱中可以发现LT1分子在水溶液中存在着线性和分支结构,经过测量其厚度为2.20nm左右,与文献报道的多糖三螺旋糖链的厚度相似。可以由此推断LT1分子在水溶液中由三条糖链螺旋组成,并在此基础上形成线性或分支结构。综上所述,从香菇子实体中提取的多糖LT1达到高效液相色谱纯,由α构型和β构型吡喃葡萄糖组成。其化学结构可能为:以1, 4连接的α构型吡喃葡萄糖和1, 3连接的β构型吡喃葡萄糖构成其主链,主链中1, 4连接的葡萄糖C6位上含有支点(每4个D-葡萄糖含有一个支点),其侧链为1个α-D-1-连接的葡萄糖残基末端。LT1糖链构象研究相关实验表明:LT1分子在水溶液中呈现三螺旋结构,当溶解于浓度高于0.20M的NaOH溶液中时,可能发生构象的转变,由三螺旋变为随机缠绕的单链状结构。更多还原

【Abstract】 Polysaccharides from Lentinus edodes is one of the most important biomacromolecules. It attracts much attention since 1960 for its immunomodulating effects. In recent years, many researches indicated that some polysaccharides isolated from L. edodes exhibited effects on anti-virus, anti-tumor, regulating the immune system, lowering blood sugar, antioxidant and other functions. LT1 was a novel polysaccharide isolated from L. edodes with molecular weight of 642 kDa and we found it reach the criteria of National Drug Standards WS1-320(X-263)-2004Z. This paper summarizes the results of chemical structure and chain conformation studies on LT1, mainly including the extraction and purification of LT1; the quality control of LT1; the chemical structure of LT1and the chain conformation of LT1.PART ONE THE EXTRACTION AND PURIFICATION OF LT1The methods for extraction and purification of polysaccharides from L. edodes were studied, so as to improve the conditions of isolating single molecular weight polysaccharides from L. edodes. Then a novel polysaccharide LT1(Mw=642 kDa) was obtained. The viscosity properties of LT1 were studied with capillary viscometric measurements. The viscometric measurements show moleculer association of LT1 in aqueous solutions. The results of molish test and KI-I2 test indicated LT1 is different from the structure of amylum.PART TWO STUDY ON QUALITY CONTROL OF LT1Quality control of LT1 was studied by national lentinan quality standard ( NO. WS1-320(X-263)-2004Z). FT-IR, UV-VIS and HPLC were performed to quantify its total carbohydrate content and characterized its chemical structure. FT-IR showed LT1 contains bothαandβtype residues. Total carbohydrate content quantified by the phenol–sulfuric acid method as 98.7%. The difference of absorption before and after smith-degradation was 0.19. All the results indicated LT1 confirming the national lentinan quality standard ( NO. WS1-320(X-263)-2004Z ). PART THREE THE CHEMICAL STRUCTURE OF LT1The chemical structure of LT1 was studied. Gas chromatography-mass spectrometry (GC-MS), Fourier transform infrared spectroscopy (FT-IR), methylation analysis, HIO4 oxidation-Smith degradation, 1D and 2D NMR (DQF-COSY, HSQC, and HMBC) experiments were performed to characterize the chemical structure of LT1. The GC analysis result shows that LT1 only contained D-glucose. The FT-IR result shows that LT1 was consisted of bothαandβtype glucose. Based on the results of methylation analysis, HIO4 oxidation-Smith degradation and NMR spectra, a proposed chemical structure of LT1 was established:PART FOUR THE CHAIN CONFORMATION OF LT1The order–disorder transitions of a helix are often accompanied by some changes, which can be monitored by optical rotation. The NaOH weight fraction (WNaOH) dependence of [α]25D for LT1 in water–NaOH mixtures at 25℃were measured. Congo Red test was used to determine the chain conformation of LT1 in 0.1M NaOH solution. Atomic Force microscopy(AFM) was used to observe the LT1 molecule in aqueous solution and measure the thickness of it. [α]25D sharply decreases when WNaOH increases from 0.20M to 0.30M indicating that the helix-coil conformation transition of LT1 may carry out when NaOH concentration was higher than 0.20M. Compared with a pure Congo Red solution, Congo Red-glucan complex exhibiting a large red shift inλmax indicates that the existence of triple helix of LT1 dissolved in NaOH solution(0.1M).From the AFM image of the LT1 sample in distilled water, multiple molecules of thick rope-like structures can be observed. The thickness of the lentinan measured by AFM is about 2.20 nm averaged over hundreds of molecules which is approximately to the triple-strand thickness of a lentinan molecule with 1.73nm measured by X-ray diffraction . Some linear and several branched structures were also observed in image.Based on all the studies in this paper, we can conclude that LT1 is a novel polysaccharide isolated from L. edodes. It has a linear structure with a backbone composed ofα-1,4-linked,β-1,4-linked andβ-1,3-linked glucopyranosyl units and slightly branched at C-6 ofβ-1,4-linked glucopyranosyl by side chains, mainly as a single glucosyl stubs. Studies on chain conformation of LT1 show that LT1 exsits as triple-helix in distilled water and a helix-coil transition may occur when NaOH concentration is higher than 0.20M.更多还原