【作者】 刘刚；

【导师】 胡先明；

【作者基本信息】 武汉大学 ， 有机化学， 2010， 博士

【摘要】 真菌已经对人类的生活产生了几千年的影响,无论是作为食物来源,还是作为药品,真菌的营养价值和药用价值早已被世界所认可。研究表明,许多真菌都具有抗肿瘤、增强机体免疫功能、降糖、降脂、降压和防治冠心病等多种生物活性。松茸(Tricholoma matsutake Sing.)是世界上稀有名贵的野生食用菌,分布在非洲、美洲、欧洲和亚洲的许多地区,除了味道鲜美,其提取物具有增强免疫、抗肿瘤、降低胆固醇,抗氧化等生物活性。本文研究了松茸的化学组成,并对其多糖的分离纯化、理化性质、结构表征及体外抗肿瘤活性进行了初步研究。在第一部分中,通过常规的分析方法测定了松茸的基本化学组成,包括粗蛋白、粗纤维、粗脂肪、碳水化合物、灰分和矿物元素；对松茸中氨基酸和脂肪酸的种类和含量进行了研究,系统地评价了其营养价值。结果显示,在松茸中粗蛋白(20.3%),粗纤维(29.10%)和碳水化合物(36.67%)的含量丰富；粗脂肪含量较低(5.04%),但是其中油酸和亚油酸含量超过脂肪酸的75%。松茸中必需氨基酸的含量占总氨基酸的34.65%,与FAO/WHO提出的理想蛋白模式接近；第一限制氨基酸为蛋氨酸；其氨基酸比值系数分高达80.16。此外松茸中还含有丰富的矿物质。研究结果表明,松茸是一种对人类健康具有特殊贡献的良好的保健营养植物。第二部分对多糖的提取、分离、纯化工艺进行了优化。实验通过苯酚-硫酸法检测多糖含量,以考马斯亮蓝法检测蛋白质含量。采用传统的水提醇沉法,以多糖得率为指标,通过单因素实验和正交实验,考察了提取时间、提取温度、提取次数以及液料比等工艺参数；以多糖醇沉得率为考察指标,优化了醇沉工艺；以多糖损失率和脱蛋白率为考察指标,优化了脱蛋白工艺；以脱色前后溶液颜色的改变和多糖损失率为考察指标,优化了脱色工艺。实验还通过D101大孔吸附树酯和Sephadex G-100凝胶滤过色谱对松茸多糖进行了分离纯化,建立了松茸多糖的最佳提取、分离和纯化工艺,最终得到3个松茸多糖MTS-1、MTS-2、MTS-3。第三部分研究了松茸多糖的理化性质和结构表征。实验采用间羟联苯法和硫酸钡浊度法测定了3个多糖的糖醛酸含量及硫酸根含量；通过自动旋光仪和元素分析仪测定了多糖的比旋光度和C、H元素组成比例；运用高效凝胶渗透色谱(HPGPC)对多糖的纯度和分子量进行了分析和测定；多糖经酸水解、三甲基硅醚衍生化后,通过气相色谱(GC)分析了其中的单糖组成；结合紫外-可见光谱(UV-VIS)、傅立叶红外光谱(FTIR)、电喷雾飞行时间质谱(ESI-TOF-MS)、一维及二维核磁共振光谱(NMR)等数据分析了多糖的结构表征。分析结果表明MTS-1、MTS-2、MTS-3为纯多糖,平均分子量分别为5939、180357、232372。MTS-1主要由葡萄糖组成,含有少量的木糖和半乳糖,葡萄糖、木糖、半乳糖的摩尔比为12.89：1.20：1。MTS-2同样是由葡聚糖、木糖和半乳糖组成,摩尔比为16.77：1.63：1；结合波谱数据综合分析,推断MTS-2是以α-D-葡萄糖通过1→6糖苷键连接的葡聚糖为主链,含有少量的α-D-木糖和β-D-半乳糖,可能位于侧链,也可能杂合于主链中。MTS-3是由葡萄糖聚合而成的均多糖,结合波谱数据综合分析,确定MTS-3为β-D-葡萄糖通过1→6连接聚合而成的直链葡聚糖,聚合度约为36～37,其结构式如下。第四部分采用四甲基偶氮唑盐(MTT)法结合显微镜下细胞形态学观察的方法,研究了松茸多糖的体外抗肿瘤活性。实验考察了不同浓度的松茸多糖作用不同时间对人舌癌细胞Tca8113、人肝癌细胞HepG-2、人胰腺癌细胞HS766T、人宫颈癌细胞Hela、人乳腺癌细胞MCF-7的增殖抑制作用。结果表明,松茸多糖在低浓度时对HepG-2、HS766T的生长影响较小,但对Tca8113、Hela;和MCF-7的抑制作用明显,随着浓度升高对5种肿瘤细胞的抑制作用都随之增强,并呈现出一定的量效关系,在100μg·mL-1作用72h对上述5种肿瘤细胞最大抑制率分别为67.71%,74.89%,83.83%,75.57%,76.15%。从细胞形态学上观察,松茸多糖对上述5种肿瘤细胞均能引起不同程度的细胞凋亡,其作用机理有待进一步深入研究。整个实验研究了松茸基本化学组成,多糖的提取分离纯化工艺优化,多糖的理化性质和结构表征,还对松茸多糖的抗肿瘤活性进行了初步研究。本论文对松茸系统的研究,为该稀有珍贵的食药用真菌的进一步开发利用提供了理论依据。更多还原

【Abstract】 Fungi have influenced human life for thousands of years, either as a direct food source or as a medicine. The nutritive and medicinal values of mushrooms have long been recognized all over the world. Many researches showed that fungi have several biological activities such as anticancer, boost in the immune system, hypoglycemic action, lipid-lowering, anti-hypertension and reduce in coronary heart disease.Tricholoma matsutake is a kind of rare, edible and medicinal fungus in the world. It can be found in many regions of Africa, America, Europe and Asia. It is a valuable species throughout the world, exhibiting a characteristic delicate flavour as well as several biological activities, such as lower cholesterol, anti-oxidant, immuno-modulating and antitumor effects in humans.In this paper, the chemical composition of T. matsutake was analyzed. Besides, the separation and purification, physicochemical properties, structure characterization and anti-tumor activity of the polysaccharides from T. matsutake were investigatedIn the first part, the chemical compositions of T. matsutake were determined by routine laboratory assays, including crude protein, crude fibre, crude fat, carbohydrate, ash and mineral elements. Moreover, the amino acid and fatty acid profiles of proteins and lipids were examined to establish a preliminary guide for assessing the relative nutritive qualities and provide a basis for taping the nutritive potential of the natural resource. The analysis results indicated that the wild edible mushroom contained rich sources of crude protein (20.3%), crude fibre (29.10%) and carbohydrate (36.67%) but little fat (5.04%) in which oleic and linoleic acids accounted for more than 75%. In general, the analysis revealed that T. matsutake had rich sources of protein and fibre and had low amounts of fat. The essential amino acid accounted for 34.65% of the total amino acid which was similar to the ideal protein requirements suggested by the FAO/WHO. The first limited amino acid was identified as methionine and the score of ratio coefficient of amino acid of its protein was 80.16. Additionally, the mushroom had been found to contain good amounts of minerals. The results showed that T. matsutake was a good nutritional plant for their unique contributions to health.The second part was the preparation technology of the polysaccharides. In this section, the contents of polysaccharides and protein were determined by the reaction with phenol in the presence of sulfuric acid and the coomasssie brilliant blue method, respectively. The polysaccharides were extracted with water extraction and alcohol precipitate (WEA). The effects of several parameters such as temperature, time, liquid-solid ratio and frequency on the extraction of the polysaccharides were observed by the yield determination of the polysaccharides with odd factor experiment and orthogonal experiment. The optimal technology for precipitation with ethanol was confirmed by the yield determination of the polysaccharides. The optimal technology for deproteinizing was obtained by comparing the effects of different methods on the loss of polysaccharides and protein. The parameter of decolorizing technology was optimized by the determination of the polysaccharides and the color comparison. Moreover, D101 macroporous resin column chromatography and sephadex G-100 gel filtration chromatography were employed to obtain the reasonable preparation technology of the polysaccharides. Finally three kinds of polysaccharides MTS-1, MTS-2 and MTS-3 were obtained from T. matsutake.In the third part, the physicochemical properties and structure characterization of polysaccharides were investigated. The glucuronic acid content and SO42" content were determined by means of m-hydroxydiphenyl colorimetry and turbidimetry method, respectively. The specific rotation and C, H compositions were determined by automatic polarimeter and elementary analyzer. The molecular weight and purity of polysaccharides were analyzed by high performance gel permeation chromatography (HPGPC). After completely acid-hydro lyzed and trimethylsilyl derivatized, component analysis of monosaccharides in polysaccharides were analyzed by gas chromatography (GC). Besides, the structural investigation of polysaccharides were synthesized and characterized by ultraviolet-visible spectrophotometry (UV-VIS), Fourier transform infrared spectrophotometry (FTIR), one and two-dimensional nuclear magnetic resonance spectroscopy (NMR) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS).The results showed that the homogeneity of these three polysaccharides was confirmed, their molecular weights were 5939,180357,232372, respectively. Polysaccharide MTS-1 from T. matsutake contain glucose, xylose and galactose in a molar ratio:Glu:Xyl:Gal=12.89:1.20:1. Polysaccharide MTS-2 was confirmed to contain the same monosaccharides as MTS-1, but the molar ratio of monosaccharides was 16.77:1.63:1. The main backbone of the MTS-2 was found to be composed of a-D-glucose linked by 1→6 glucoside bond, along with small amounts of a-D-xylose and P-D-galactose could be in the side chain. MTS-3 was identified to be composed of glucose units. According to the comprehensive analysis, the backbone of MTS-3 was consisted of 36-37 units (3-D-glucoses combined by 1→6 glycosidic linkage and the structure of MTS-3 was as follows.In the forth part the anti-tumor activity was assayed by MTT method together with morphological observation under inversed light microscope. The cytotoxic activities of different concentrations and different time of MTS in vitro were investigate against human tongue carcinoma cells Tca8113, human liver cancer cells HepG-2, human pancreatic cancer cells HS766T, human cervical cancer cells Hela and human breast cancer cells MCF-7. The results showed that the low concentration of polysaccharides from T. matsutake weakly inhibited the growth of HepG-2 and HS766T but obviously affected the growth of Tca8113, Hela and MCF-7. It showed potential antiproliferative activity with dose-dependent and time-dependent relation in vitro. Maximum inhibition ratios of MTS under the 100μg·mL-1 and 72h to Tca8113, HepG-2, HS766T, Hela and MCF-7 were 67.71%,74.89%,83.83%,75.57%,76.15%, repectively. From the cell morphology observation, polysaccharides of T. matsutake can give rise to different degrees of apoptosis on the five tumor cells mentioned above, the mechanism needs further study.In summary, this study investigated not only the basic chemical composition of T. matsutake, but the extraction and purification, physicochemical properties, structural characterization and the anti-tumor activity of mushroom polysaccharides. These systematic studies of the rare and valuable of edible-medicinal fungi provide a theoretical basis for the further development of T. matsutake.更多还原