【作者】 王璐；

【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】 电离辐射能够诱导机体产生氧化应激反应，破坏机体的内环境稳态，造成机体的抗氧化防御系统、免疫系统及胃肠功能等的严重损伤，甚至导致遗传突变及引发癌症。因此，寻找能够降低氧化损伤、提升免疫活性，且高效无毒的天然辐射防护物质成为研究热点。本论文以山荆子多酚（Polyphenols from Malus baccata (Linn.) Borkh.，MBP）作为研究对象，采用不同溶剂进行分离并获得抗氧化活性最优组分丙酮提取物MBP-3，对其分离及纯化工艺进行研究，并对活性最强的梯度40%乙醇洗脱物MBP-3b进行组成分析及结构鉴定；系统的研究了MBP-3b对辐射诱导机体氧化损伤的防护作用途径及作用机制。采用四种不同极性的溶剂（水、乙醇、丙酮和乙酸乙酯）分别提取获得山荆子多酚MBP-1~4四个组分，对多酚类化合物含量进行分析，并在体外建立不同抗氧化体系，研究MBP的体外抗氧化活性和脂质过氧化抑制活性。结果表明山荆子丙酮提取物MBP-3的总酚、黄酮和花色苷含量最高；四种MBP均能不同程度的清除非生理性和生理性自由基，并具有显著的总还原能力及脂质过氧化抑制能力，其中MBP-3的抗氧化活性最为显著，说明MBP-3具有更有效的供电子及供氢能力，能够终止自由基链式反应、降低氧化应激从而抑制脂质过氧化的发生。采用超声辅助提取方法分离山荆子多酚，通过响应面法优化得到山荆子多酚最佳提取工艺为：丙酮作为提取溶液，液料比13：1，提取温度47℃和超声功率330W，在最优条件下所得山荆子多酚含量为1170.45±28.15mg GAE/100g；大孔树脂NKA-9层析纯化MBP-3最优条件为上样体积1/5BV，上样流速1.5BV/h，洗脱流速2.0BV/h，动态梯度洗脱下40%（v/v）乙醇洗脱物MBP-3b总酚含量最高，且抗氧化活性最强。采用UPLC-Q-TOF-MS，结合UV-Vis及FT-IR光谱对MBP-3b进行组成分析和结构鉴定，结果发现MBP-3b主要由十种多酚化合物组成，分别为飞燕草素3,5-二葡萄糖苷、矢车菊-3-葡萄糖苷、绿原酸、原花青素C1、槲皮素-3-半乳糖苷、槲皮素-3-葡萄糖苷、槲皮素-3-木糖苷/阿拉伯糖苷、根皮素-2-木糖葡萄糖苷、槲皮素-3-鼠李糖苷和根皮苷，相对质量百分含量分别为3.60%、9.29%、11.96%、1.64%、18.74%、6.63%、4.75%、7.16%、4.32%和18.24%。在体外建立60Coγ辐射诱导脾细胞和外周血白细胞氧化损伤模型，通过对辐射后脾细胞的存活率、脾细胞内氧化还原状态、外周血白细胞形态、脾细胞DNA的损伤以及脾细胞内ROS的含量进行分析，结果发现MBP-3b能够显著的降低辐射后外周血中异形白细胞的数量，并能通过促进脾细胞内抗氧化酶SOD、CAT的活性，降低细胞内脂质过氧化产物MDA的产生，从而有效的抑制胞内ROS的生成，降低细胞的氧化应激反应的进程而抑制了DNA的损伤，提高了辐射条件下脾细胞存活率。建立60Coγ辐射诱导小鼠氧化损伤模型，通过分析辐射后小鼠DNA损伤、免疫功能、抗氧化防御系统活性来研究MBP-3b对小鼠的辐射防护作用途径，结果发现，MBP-3b能够通过提高机体免疫功能，促进机体酶促（SOD、CAT、GSH-Px及GST）及非酶促（T-AOC、GSH）抗氧化防御系统功能，降低辐射后机体的脂质过氧化发生，从而有效抑制辐射后小鼠骨髓微核及染色体畸变的发生，对辐射诱导的小鼠机体氧化损伤具有防护作用。通过对辐射条件下小鼠小肠及脾脏形态学及细胞凋亡进行研究，结果表明，MBP-3b能够缓解电离辐射诱导的小鼠小肠及脾脏组织病变，抑制免疫细胞的大量凋亡；通过免疫组化方法对小鼠脾细胞线粒体凋亡信号途径相关蛋白表达进行研究，结果表明MBP-3b能够有效抑制促凋亡Bax蛋白表达，促进抗凋亡蛋白Bcl-2的表达，从而抑制了细胞色素c的释放及Caspases-3活性亚基的表达，对辐射引起的脾细胞凋亡具有明显的抑制作用。本课题研究表明山荆子多酚MBP-3b作为一种抗氧化组分，能够有效的促进机体酶促及非酶抗氧化防御系统功能，抑制60Co γ辐射诱导的活性氧的过量产生，调控线粒体凋亡信号途径相关蛋白表达，对辐射诱导的机体氧化损伤具有防护作用。本研究对于进一步揭示多酚类化合物的辐射防护作用机制、开发天然辐射防护物质具有理论和现实意义。更多还原

【Abstract】 Ionizing radiation could induce oxidative stress and cause the serious damage of hematopoietic system and gastrointestinal function in the body, or even lead to genetic mutation and carcinogenic. Therefore, looking for natural and non-toxic radioprotective substances which could effectively reduce oxidative damage, enhance immune activity become a hot topic. This study selected polyphenols from Malus baccata (Linn.) Borkh.(MBP) as the research object, obtained the best antioxidant component-actone extract MBP-3by separating of MBP extracts using different solvent, optimized the separation and purification processes of MBP-3and characterized the structure of40%(v/v) ethanol eluate MBP-3b, which possessed the best antioxidant activity; systematically studied the protective effect of MBP-3b on oxidative damage of organism induced by radiation and revealed the mechanism of radioprotective effect.MBP-1~4were obtained respectively by extracting using four kinds of solvents with different polarity and analyzed the contents of polyphenols. Established of different antioxidant system in vitro, the free radical scavenging activity, inhibition of lipid peroxidation and reducing power of MBP were systematically researched. The results showed that MBP-3possessed the most abundant of total polyphenols, flavonoids and anthocynins. All MBPs could scavenge physiological and non-physiological free radicals in different degree. MBP also had significant reducing power and could effectively inhibit lipid peroxidation of rat liver, and. The biological activity of MBP-3was better than the others. This implied that MBP-3possess the better ability of supplying electron and hydrogen, which could terminate free radical chain reaction and inhibite oxidative stress, as well as inhibited lipid peroxidation.Ultrasonic-assisted extraction process was used to separate MBP, the optimized extraction conditions obtained by response surface methodology were as follows: acetone as the extraction solvent, liquid/material was13:1, extraction temperature47℃and ultrasonic power330W, and the content of polyphenols was reached to1170.45mg GAE/100g. The best purification parameters of MBP-3on NKA-9chromatography resins were as follows: loading sample volume1/5BV, loading flow rate1.5BV/h, elution flow rate2.0BV/h and40%(v/v) ethanol eluate-MBP-3b possessed the highest content of total phenolic with the best antioxidant activity.MBP-3b constituents were analyzed and identified by UPLC-Q-TOF-MS, UV-Vis and FT-IR spectrums The results indicated that MBP-3b was composed of ten kinds of phenolic compounds, including delphinidin-3,5-diglucoside, cyanidin-3-glucoside, chlorogenic acid, proanthocyanidins C1, quercetin-3-galactoside, quercetin-3-glucoside, quercetin-3-xyloside/arabinoside, phloretin-2-xyloseglucoside, quercetin-3-rhamnoside and phlorizin, their relative quality percents were respectively3.60%,9.29%,11.96%,1.64%,18.74%,6.63%,4.75%,7.16%,4.32%and18.24%.The oxidative damage model of splenocytes and peripheral blood leukocytes induced by60Co γ radiation was established in vitro. The survival of splenocytes, redox state in splenocytes, peripheral blood leukocyte morphology, DNA damage of splenocytes and contents of ROS in splenocytes after radiation were analyzed. The results indicated that MBP-3b could effectively reduce the amount of damaged leukocytes, increase the activity of antioxidant enzymes SOD and CAT in splenocytes, and accordingly reduce the contents of lipid peroxidation products MDA. Thereby, MBP-3b inhibited the generation of ROS, then lowered the process of cellular oxidative stress and inhibited DNA damage, thus increased the survival of splenocytes after radiation.The oxidative damage model of mice induced by60Coγ radiation was established. By analyzing DNA damage, immune activity and activity of antioxidant defense system to investigate the radioprotective effect of MBP-3b. The results found that MBP-3b could promote immune function, enhance the enzymatic (SOD, CAT, GSH-Px and GST) and non-enzymatic (T-AOC and GSH) antioxidant defense system and lower lipid peroxidation after radiation, then effectively inhibit the micronucleus and chromosome aberrations. These indicated that MBP-3b possessed radioprotective effect on oxidative damage of mice.The morphology and apoptosis of small intestine and spleen of mice after radiation were studied. The results suggested that MBP-3b could alleviate the lesions of spleen and small intestinal tissues, and reduce a large number of immune cells apoptosis induced by radiation. The related proteins of apoptosis in mitochondrial pathway were determined using immunohistochemical methods. The results showed that MBP-3b could significantly inhibit the expression of pro-apoptosis protein Bax, promote the expression of anti-apoptosis protein Bcl-2, thereby inhibited the release of Cytochrome c and the expression of activity subunit of Caspase-3, and then effectively protect the splenocytes away from oxidative damage induced by radiation.The research indicated that MBP-3b possessed significant antioxidant activities and could promote the function of enzymatic and non-enzymatic antioxidant system, reduce the excessive production of ROS induced by60Co γ radiation. It could also regulate the expression of associated protein in mitochondrial apoptotic signal pathway. Accordingly, MBP-3b showed protective effect on oxidative damage of organism. The present study possesses theoretical and practical implication to revealing radioprotective mechanism of polyphenols and exploiting natural raidioprotectors.更多还原