【作者】 张华；

【导师】 王振宇；

【作者基本信息】 哈尔滨工业大学 ， 化学工程与技术， 2013， 博士

【摘要】 随着科学技术的发展，由于空间技术和核技术的应用，辐射伤害不仅见于战时，已渗透到各个领域，辐射产生大量自由基能引发机体氧化应激反应，现代医学认为炎症、肿瘤、衰老、血液病、以及心、肝、肺、皮肤等近百余种相关疾病的发生机理与体内自由基产生过多而得不到及时有效清除有着密切的关系，由于合成抗氧化剂存在稳定性差、作用时间短、毒副作用、不适合长期服用等问题，发掘新型天然抗氧化剂已成为食品科学和航天医学的研究重点。本课题采用化学修饰方法制备黑木耳中性多糖片段硫酸酯（Sulfate of neutralAuricularia auricular polysaccharides, SNAAP），采用清除体外自由基进行活性跟踪，经细胞模型确证，SNAAP对氧化应激具有很好的防护作用，进一步建立氧化应激动物模型，从细胞、分子水平系统研究SNAAP辐射防护作用，旨在促进黑木耳及其多糖资源的深度开发利用。选用氯磺酸-N, N二甲基甲酰胺（CSA-DMF）作为酯化试剂，采用单因素实验，以硫酸酯化试剂（CSA：DMF）体积比为1：6；酯化温度为50℃，反应时间为3h，进行非选择性修饰制备木耳中性多糖片段硫酸酯：得率为51.89±1.36%，离子色谱法测定其硫酸根含量为31.00±2.79%，计算硫酸基取代度为0.78±0.11；经气相色谱法分析该工艺下制备SNAAP产物中甲酰胺和N, N-二甲基甲酰胺溶剂残留量分别为80.1±7.5μg/L和64.1±4.8μg/L，均符合人用药品注册技术规范国际协调会（ICH）和中国药典（2000版）限量标准。采用活性跟踪法，研究了SNAAP的体外抗氧化活性，当受试样品浓度为2.0mg/mL时，SNAAP对超氧自由基清除率为95.71%，而黑木耳中性多糖片段清除率仅为3.75%，表明硫酸酯化能显著提高黑木耳中性多糖片段对超氧自由基的清除能力，筛选得到SNAAP对超氧自由基清除活性最强。红外光谱解析SNAAP在1249cm^-1（νS=O）和820cm^-1（νC-O-S）处有-OSO₃的S=O伸缩振动和C-O-SO₃基团的伸缩振动特征吸收峰，说明合成产物SNAAP有硫酯键。且硫酸基位于SNAAP中单糖残基平伏位置，并在C₆位上发生取代；紫外光谱显示SNAAP未出现新的特征吸收峰，证明该酯化方法是较为温和的硫酸化修饰手段。原子力显微镜表征SNAAP分子中具有三维空间网状结构，相对分子量为6.53×10⁶Da，且与黑木耳中性多糖片段相比，经过硫酸化，SNAAP的分子量分布变窄，分子量较为均一和集中。SNAAP单糖组成分别由D-核糖、D-甘露糖、L-鼠李糖、L-阿拉伯糖、D-木糖、D-葡萄糖、D-半乳糖组成，各单糖之间摩尔比例为1.0：7.0：1.0：1.0：4.0：10：2.0。MTT法评价了SNAAP不具有细胞毒性作用，以X射线辐射诱导人外周血为氧化应激模型，采用血常规分析，筛选得到SNAAP对氧化应激防护作用最佳，进一步验证实验研究发现SNAAP中剂量组（60μg/mL）可以将辐射受损的白细胞数维持至对照组水平，减少辐射引起的畸变白细胞数，具有辐射防护功效。构建γ辐射诱导的氧化应激动物模型，研究发现SNAAP各剂量组能够直接清除辐射产生的自由基，可有效的促进造血系统恢复和免疫细胞增殖和生长，提高机体的免疫系统功能；能够显著提高小鼠各脏器中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）和乳酸脱氢酶（LDH）活性，增加还原型谷胱甘肽（GSH）的含量，降低髓过氧化物酶（MPO）活性，降低丙二醛（MDA）水平，证实SNAAP能够有效激活抗氧化酶系，抑制氧化酶系，抑制脂质过氧化，减轻细胞膜损伤，减少骨髓微核和染色体畸变率；揭示SNAAP抗辐射机制与自由基清除、抗氧化酶系水平、细胞增殖、谷胱甘肽水平、脂质氧化、细胞周期停滞等因素有关。因此，SNAAP对辐射诱导氧化应激防护作用这一研究对航天、军事及核辐射接触人员的健康和安全具有十分重要理论意义和实际应用价值。更多还原

【Abstract】 As technology develops, human beings enjoy life more convenient than everbefore. Yet, at the same time they are suffering ever greater oxidative stress fromfree radicals caused by a variety of genres of radiation. It is believed that inmodern iatrology more than one hundred human diseases are associated withoxidative stress and that synthesized antioxidants are not suitable for long-termuse due to their instability and toxicity. Therefore, the investigation anddevelopment of natural antioxidants has been the focus for researchers in the foodscience and aerospace medicine disciplines.In this research, specific chemical methods were applied to synthesize asulfate of neutral Auricularia auricular polysaccharide fragments （SNAAP）together with neutral Auricularia auricular polysaccharide. Their antioxidant andradio-protective effects were assessed by in vitro radical scavenging experiments,oxidative stress cells models and oxidative stress animal models. Correspondingly,the radio-protective effect of SNAAP was systematically examined in levels ofanimal cells and molecules. Therefore, this research promotes exploitation ofAuricularia auricular and its polysaccharides. The main content of this study isdescribed as follows.NAAP was prepared through deproteinization, decolorization, purificationand dialysis. The chemical structure analysis of NAAP and its sulfated derivative,SNAAP, revealed the sulfation of the NAAP was successfully achieved.Experiments verified the neutral Auricularia auricular polysaccharide fragmentswere sulfated under a rigorous sulfation using chlorosulfonic acid （DMF volumeratio of1:6） at temperature of50℃, for3hours, then treated by theChloro-sulfonic acid-N, N dimethyl formamide method. With this procedure,SNAAP was produced at a yield of51.89±1.36%with a degree of substitution（DS） of0.78±0.11. The residual amount of formamide and N, N dimethylformamide in the SNAAP was determined by gas chromatography, to be80.1±7.5μg/L and64.1±4.8μg/L, within the limit by ICH and Chinese Pharmacopoeia（2000）, signifying the safe use of SNAAP in cell and animal experiments.The in vitro antioxidant capacity of the SNAAP was systematically evaluated.The results confirmed that the SNAAP had better superoxide radical scavengingability than the non-sulfated polysaccharides fragments. At a sample concentrationof2.0mg/mL for SNAAP a superoxide radical scavenging rate of95.71%wasobtained; the respective values of the neutral Auricularia auricular polysaccharide fragments was only3.75%. The super scavenging ability of SNAAP was due tosulfate groups. The FTIR analysis established that the SNAAP had specificabsorbing peaks of-OSO₃and C-O-SO₃at both1249cm^-1（νS=O） and820cm^-1（νC-O-S）. A UV spectrum analysis did not find any new absorbing peaks, whichsignified that this sulfating method was mild and that the SNAAP possesses athree dimensional network structure with a molecular mass of6.53×106.Compared with NAAP fragments, the molecular mass of the SNAAP increaseddue to introduction of sulfates, consisting of D-ribose, D-mannose, L-rhamnose,L-arabinose, D-xylose, D-glucose, D-galactose, with a mole ratio of1.0：7.0：1.0：1.0：4.0：10.0：2.0.MTT analysis certified that the SNAAP was not cell toxic. X-ray inducedhuman peripheral blood was used as an oxidative stress model. Routine bloodexamination showed that SNAAP possessed the best radio-protective effect, inaccordance with the findings that a mid-level dose of SNAAP （60μg/mL） cansustain the leukocyte level of irradiated rats at the same level as control rats and toreduce the amount of aberrant leukocytes induced by irradiation.In addition, animal models with γ-ray induced oxidative stress weremechanistically built to further explore the radiation-protective effect of SNAAP.The improvements of the immune system, are caused by direct scavenge of freeradicals in vitro by SNAAP, enhance by hematopoiesis and promote by the growthand proliferation of the immunocytes. Furthermore, SNAAP can improve theexpression of many important enzymes involved in oxidative stress response suchas superoxide dismutase, catalase, and lactic dehydrogenase and at the same timereduce the expression of marrow peroxidase. Likewise, SNAAP can increase theamount of reduced glutathione and reduce the amount of malondialdehyde intested organs. Consequently, SNAAP can activate the anti-oxidase system, inhibitthe oxidase system, alleviate lipid peroxidation, reduce damage to cell membrane,and decrease the mutation of the micronucleus rate and of chromosomesaberrations.The results revealed radioprotection mechanisms of SNAAP as radicalscavenging, anti-oxidases expression, cell proliferation, GSH level, lipid oxidation,cell cycle arrest, DNA-protein binding and chromosome folding. Therefore,SNAAP will be a potential new radio-protector, humans working with radiationexposure.更多还原