【作者】 茆广华；

【导师】 吴向阳；

【作者基本信息】 江苏大学 ， 食品科学， 2014， 博士

【摘要】 硒(Se)是人体必需的微量元素,是重要含硒酶(谷胱甘肽过氧化物酶和硫氧还蛋白还原酶等)的必需组分,具有提高机体免疫能力、抗肿瘤、缓解体内重金属蓄积、抗衰老和防止心血管疾病等活性。人体缺硒会导致免疫能力下降、癌症、克山病和大骨节病等的发生,危害人体健康,但日常膳食不能满足人体对硒的最低需要,因此硒的营养补充尤为重要。研究表明,食用菌具有很好的富硒能力,但由于栽培基质缺硒,导致食用菌中的含硒量低,因此采用人工富硒方式增加栽培基质硒含量或菌面喷施无机硒,是获得富硒食用菌子实体行之有效的方法。灰树花(Grifola frondosa, Maitake)隶属担子菌纲,多孔菌目,多孔菌科,树花属,是一种药食两用真菌,多糖是其主要活性物质,具有增强免疫、抗肿瘤、抗氧化等活性,灰树花子实体富硒后,其硒多糖兼具硒和多糖的活性,具有重要的研究价值和应用前景,而目前关于灰树花子实体富硒及其硒多糖的研究鲜见报道。本文通过富硒栽培,获得富硒灰树花子实体,研究其中硒的主要赋存形式；采用DEAE-52和Sephacryl S-400对富硒灰树花子实体粗硒多糖进行分离和纯化,获得富硒灰树花子实体硒多糖组分(Se-GP11),并采用HPLC、GC、GC-MS、 NMR、AFM、TEM及CD等现代分析手段对其进行全面结构分析；采用Heps荷瘤小鼠模型,研究Se-GP11的体内抗肿瘤活性、对5-氟尿嘧啶(5-Fu)的减毒作用及其免疫机制,为富硒灰树花子实体及其硒多糖在食品和医药领域中的应用提供依据,该研究对人类健康和社会发展具有重要意义。本文主要研究内容如下：(1)灰树花子实体富硒及其硒的赋存形式研究。建立了富硒灰树花子实体中硒含量的氢化物-原子荧光光谱测定方法,最佳检测条件为：灯电流80mA、负高压280V、载流盐酸浓度10%、硼氢化钠浓度1.0%；采用菌面和拌料施硒两种种植方式对灰树花进行富硒研究,结果表明菌面喷施亚硒西酸钠5.785mg/段和袋料拌施57.143mg/段时,其富硒的效果较好,产量分别为28和24g/段,有机硒含量为11.98和20.36μg/g,分别占总硒的87.19%和61.51%,说明菌面喷施硒的富硒效果优于袋料拌硒,其有机硒含量是未富硒灰树花子实体的98.81倍,因此富硒灰树花子实体粉可以作为硒营养强化剂；研究了富硒灰树花子实体中硒的赋存形式,结果表明硒多糖和硒蛋白是富硒灰树花子实体中硒的主要赋存形式,分别占有机硒的13.68%和57.52%。(2)富硒灰树花子实体粉及其粗硒多糖的抗肿瘤活性和经口急性毒性研究。采用Heps和Lewis荷瘤小鼠模型,比较了富硒灰树花子实体粉及其粗硒多糖与未富硒灰树花子实体粉的体内抗肿瘤活性,结果表明,三者对Heps荷瘤小鼠的效果优于Lewis荷瘤小鼠,其中粗硒多糖的抑瘤活性最好,抑瘤率达20.63%(环磷酰胺(CTX)阳性对照组为36.26%),其次为富硒灰树花子实体粉,未富硒灰树花子实体粉最低,仅为1.92%；小鼠经口急性毒性研究表明,富硒灰树花子实体粉及其粗硒多糖属于实际无毒级,其经口LD50均大于15.1g/kg b.w..(3)富硒灰树花子实体硒多糖的提取、分离纯化及结构鉴定。采用响应面分析法(RSM)研究富硒灰树花子实体粗硒多糖的提取工艺,最佳提取工艺条件为：提取温度100℃、提取时间2.6h、料液比1：34.7,该工艺条件下富硒灰树花子实体粗硒多糖得率为15.37%,粗多糖含量为48.68%；采用DEAE-52, Sephacryl S-400对富硒前后灰树花子实体粗多糖进行分离纯化,两者皆获得了3个多糖组分,除硒含量外,其多糖含量、分子量和单糖组成均无显著差异,其中富硒及未富硒灰树花多糖水洗纯化硒多糖组分Se-GP11和多糖组分GP11的量较多；红外分析表明Se-GP11和GP11表现为相同的特征吸收峰,富硒后使得吡喃糖环上的3个特征峰发生了红移,因此硒可能是以Se-H键的形式存在于Se-GP11的支链上；通过甲基化、GC-MS、1H NMR、13C NMR、1H-1H COSY谱、HSQC谱和HMBC谱等分析手段对Se-GP11的一级化学结构进行解析,确定其一级结构的重复单元为：采用CD、激光粒度测定、AFM和TEM等方法对Se-GP11的构象及分子形貌进行研究。CD谱研究表明,温度、离子强度、pH和络合物等环境条件改变均影响其立体构象及非对称性；激光粒度测定结果表明,Se-GP11粒度分布较均匀,粒径为690nm,分散度为0.208；AFM观察发现,温度改变了Se-GP11形貌,常温下Se-GP11多糖分子链纵横交错,形成立体多层网状,加热处理冷却后,Se-GP11清晰的链结构发生了变化,有形成不规则聚集体的趋势；TEM观察发现Se-GP11在水溶液中聚结成网络状。(4) Se-GP11的抗肿瘤活性及其对5-Fu的减毒作用研究。采用Heps荷瘤小鼠模型,考察Se-GP11的体内抗肿瘤活性及其对5-Fu的减毒作用。结果表明,Se-GP11活性显著高于GP11,在剂量为54mg/kg时,Se-GP11的抑制率达64.38%,接近5-Fu阳性对照组的抑瘤率(68.58%)；Se-GP11可显著提高荷瘤小鼠胸腺指数、血清中IL-2和TNF-α水平,外周血白细胞的数量,肝脏的SOD活力,可使血清中AST水平恢复正常,降低肝脏中MDA水平；对5-Fu的减毒研究表明,Se-GP11效果显著好于GP11, Se-GP11可明显对抗5-Fu所引起的小鼠免疫器官指数、外周血白细胞数量的减少和机体TNF-α、IL-2水平的降低,此外,Se-GP11对5-Fu所引起的肝损伤具有一定的改善作用。可见,Se-GP11具有较好的抗肿瘤和对5-Fu的减毒作用,效果优于GP11且对机体无毒。(5) Se-GP11抗肿瘤免疫机制研究。以腹腔巨噬细胞RAW264.7为研究对象,研究了Se-GP11对腹腔巨噬细胞的免疫调节机制。结果表明,Se-GP11可显著提高RAW264.7中性红吞噬能力,效果优于GP11; MTT实验结果表明,1000μg/mL的Se-GP11对RAW264.7细胞无明显的毒性作用；经Se-GP11诱导的RAW264.7细胞及其上清液均可显著抑制HepG-2细胞的增殖且Se-GP11的活性高于GP11; Se-GP11可显著提高RAW264.7细胞释放NO, TNF-a和IL-1β的能力且显著高于GP11;Western blot和RT-PCR法研究表明,Se-GP11可显著上调iNOS蛋白、iNOS和TNF-α mRNA的表达,且其表达量显著高于GP11,说明Se-GP11的免疫调节功能显著高于GP11；抗体阻断结果表明明,TLR-4和TLR-2抗体可显著阻断Se-GP11诱导RAW264.7细胞分泌NO和TNF-α。表明Se-GP11通过受体TLR-4和TLR-2激活巨噬细胞,促进细胞因子蛋白和基因的表达,从而提高机体的免疫功能,是其免疫激活机理之一。更多还原

【Abstract】 Selenium (Se) is an essential element and important part of Se-contained enzymes (glutathione peroxidase, thioredoxin redutase and so on). It has many bio-activities, such as improving of immunity, anti-oxidant, anti-virus and antagonistic accumulation of heavy metals and so on. Deficiency of Se endangers human health and leads to various clinical consequence, including Keshan disease, Kashin Beck disease, cardiovascular disease and diabetes. It was reported that edible fungus has good Se accumulation ability, whereas, the Se content is very low due to lack of Se in the culture medium. Therefore, the artifical way to increase Se in plant medium or spraying Se at the growth stage of fungus is an effective method to gain Se-enriched fungus. Grifola frondosa (Basidiomycetes, Aphyllophorales, Polyporaceae) is a kind of edible and medicinal mushroom. Polysaccharide is the main bioactive compound with diverse biological activities, such as enhancing immunity, anti-tumor and anti-oxidant and so on. The Se polysaccharide from Se-enriched G. frondosa (Se-GF) has both the activities of Se and polysaccharide. It has important research value and application prospect. To our best knowledge, there is little study on Se accumulation of G. frondosa and it Se-polysaccharide (Se-GP). In this study, the optimal method for G. frondosa accumulation of Se was obtained and the Se distribution in Se-GF was studied. Three homogenous Se-GPs polysaccharides (Se-GP11, Se-GP22and Se-GP33) from Se-GF were obtained via DEAE-52and Sephacryl S-400purification and the structure of Se-GP11was comprehensively analyzed by GC-MS, NMR, CD, AFM and other advanced analytical methods. The anti-tumor activity, toxicity attenuation effect to5-Fluorouracil (5-Fu) and immunomodulatory mechanism of Se-GP11were investaged systematically. This study will provide the basis for the application of Se-GF and Se-GP in the fields of food and medicine. It has important significance for human health and social development. The main researches content are given bellow:(1) To study on the G. frondosa accumulation of Se and the distribution Se. The hydride generation atomic fluorescence method for Se test was established and the optimal condition is lamp current (80mA), minus high voltage (280V), hydrochloride concentration (10%) and sodium borohydride concentration (1%). Spraying Se (5.785mg/kg) at the growth stage of G. frondosa is the effective way to obtain Se-GF. The organic Se content of Se-GF11.98μg/g, which is98.81times of G. frondosa, account for87.19%of the total Se. Therefore, Se-enriched G. frondosa can used as an Se nutrition fortifier. Se-polysaccharide and Se-protein are two main form of Se in Se-GF, accounted for13.68%and57.52%, respectively.(2) To study the anti-tumor activity and acute oral toxicity of Se-GF and its Se-GP. Heps and Lewis tumor bearing mice were applied to study the anti-tumor activity of Se-GF, Se-GP and G. frondosa (GF). The results indicated that all of them given better effect on Heps than Lewis. The Se-GP show best activity and the inhibition rate reach to20.63%(Cyclophosphamide(CTX) is36.26%), followed by Se-GF, GF(only1.92%) is the lowest. Both Se-GF and Se-GP belong to actually non-toxic grade and the oral LD50is greater than15.1g/kg b.w.(3) Isolation, purification and characterization of Se-GP were studied. Se-GP was obtained from Se-GF using response surface method (RSM) method and compared to G. frondosa polysaccharide (GP) for their preliminary characterization. Under optimal condition, the yield of Se-GP is15.37%. Three homogenous Se-GPs (Se-GP11, Se-GP22and Se-GP33) and GPs (GP11, GP22and GP33) were obtained via DEAE-52and Sephacryl S-400purification. Their molecular weight, polysaccharide content and IR spectrum were not obviously different except for the Se content. To clarify the structure of Se-GP11by methylation, GC-MS, NMR. The predicted primary structure of Se-GP11was given bellow: The advanced structure of Se-GP11was studied by CD, AFM, TEM and so on. CD spetrum of Se-GP11changed significantly with the change of external condition (temperature, pH value and ion intensity etc). The laser particle study of Se-GP11distribute uniformly with particle diameter690nm and polydisoersity0.208. The results of AFM indicate that the polysaccharide molecules chain of Se-GP11is arranged in a crisscross pattern at room temperature. However, after heat treatment, the clear chain structure of Se-GP11changed and has irregular aggregates trend. The result of SEM revealed that the Se-GP11in water solution coalesce into network.(4) The HepG-2mouse tumor model is established to study the anti-tumor activity, attenuation to5-Fu on tumor bearing mice of Se-GP11. The activity of Se-GP11is signigicantly higher than GP11. The inhibition rate of Se-GP11was64.38%at the dose of54mg/kg, which is near to5-Fu (68.58%). Se-GP11increased the thymus index, raised the production of TNF-a and IL-2in serum, ameliorated the5-Fu induced toxicity effect on body weight loss, regained changes in hepatic biochemical and hematological parameters, and protected the effect against5-Fu induced oxidation damage.(5) To study the immunomodulatory mechanism of Se-GP11. Compared to the control group, the phagocytic activity was significantly enhanced in Se-GP11-treated murine macrophage RAW264.7cells, and higher than that of GP11. When RAW264.7cells were treated with Se-GP11, cytotoxic activity against HepG-2was significantly induced, and higher than that of GP11. Moreover, Se-GP11increase the level of TNF-α and IL-1β and the production of NO in dose-dependent manner, and higher than that of GP11. In addition, western blot and RT-PCR analysis suggest Se-GP11promoted the expression of iNOS protein, iNOS and TNF-a mRNA. The expression is significantly higher than that of GP11, indicating the immune function of Se-GP11is better than GP11. TLR-2/4is a possible receptor for Se-GP11-or GP11-mediated macrophage activation for function blocking antibodies to TLR-2/4, markely suppressed Se-GP11-or GP11-mediated TNF-a and NO production. The data suggest that stimulating macrophage via TLR-2/4, promoting the expression of cytokine protein and gene, and then improving the immune function is one of the immune activation mechanisms of Se-GP11.更多还原