【作者】 赵海田；

【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， 2012， 博士

【摘要】 电离辐射能诱导机体生成活性氧自由基，导致细胞和组织发生氧化应激反应，造成机体神经、造血、消化和免疫系统发生急性或慢性损伤，因此，筛选和评价具有优良抗氧化、抗辐射活性的天然产物成分已成为医学、生物学和食品科学研究的热点。本论文以蓝靛果花色苷（Anthocyanin from Lonicera caerulea var. edu-lis，ALC）为研究对象，对ALC分离纯化工艺进行了优化，并对ALC进行了结构表征；对ALC抗氧化、抗辐射活性和对辐射诱导氧化伤害防护作用及机制进行了系统的研究。主要研究结果如下：采用超声波辅助提取法分离提取了ALC，通过响应面法优化得到ALC最佳提取工艺条件：提取时间97min，料液比1:16.35，乙醇浓度47%；单次提取率为86.53%，反复提取3次，总提取率达94.70%。大孔树脂层析法纯化ALC最优条件：采用AB-8型大孔树脂，50%乙醇（HCl调pH值至3.0）为洗脱剂，径高比1:15，洗脱流速1.5BV/h，洗脱剂用量4.0BV，所得ALC色价为79.68与粗提物（色价3.58）相比，纯化倍数为22.26。采用高效液相色谱-二极管阵列检测器（HPLC-DAD）、高效液相色谱-电喷雾离子阱质谱（HPLC-ESI-IT-MS）和高分辨质谱（Q-TOF-MS）技术，结合紫外-可见光谱（UV-VIS）、红外光谱（IR）分析技术对ALC进行了结构表征。从ALC中发现8种花色苷，分别是矢车菊素-3-槐糖-5-葡萄糖苷、矢车菊素-3,5-二葡萄糖苷、矢车菊素-3（阿魏酰）-葡萄糖苷、矢车菊素-3-葡萄糖苷、矢车菊素-3-芸香苷、天竺葵素-3-葡萄糖苷、芍药素-3-葡萄糖苷和芍药素-3-芸香糖苷，相对质量百分含量分别为1.12%、3.71%、1.38%、72.42%、9.62%、2.30%、7.10%和2.34%。系统性研究了ALC的体外抗氧化活性作用，清除生理自由基、非生理自由基、总还原能力和抑制脂质过氧化能力。结果表明，ALC具有很强的传递氢原子能力，可以通过提供氢原子直接清除羟基自由基（·OH）或终止自由基的链反应，从而防止自由基诱导氧化损伤的发生。同时，ALC具有较高的抗氧化活性和还原能力，对于脂质过氧化具有一定的抑制作用，并呈良好的剂量效应关系。建立X了射线和60Coγ辐射诱导细胞损伤模型，结果表明，ALC可以拮抗电离辐射损伤作用，提高辐射损伤小鼠脾淋巴细胞活力，降低小鼠脾淋巴细胞DNA的辐射损伤程度。建立60Coγ辐射诱导氧化损伤动物模型，对ALC的辐射防护作用进行研究。结果表明，ALC能减轻辐射造成小鼠免疫器官的损伤，显著增加小鼠各脏器中超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）活性，提高还原型谷胱甘肽(GSH)含量，降低丙二醛（MDA）含量，有效激活抗氧化酶系，减少脂质过氧化，减轻细胞膜的氧化损伤，降低小鼠外周血淋巴细胞异形和骨髓细胞微核率。通过蛋白免疫印迹技术（Western blot）对小鼠肝脏组织MAPK信号通路和线粒体介导的细胞凋亡信号通路主要相关蛋白表达进行了研究。结果表明，ALC对辐射致小鼠肝脏组织细胞蛋白表达具有显著的调控作用。ALC防护辐射诱导机体氧化损伤的作用机制是：ALC能诱导小鼠肝脏组织细胞中Bcl-2表达上调，阻抗辐射引起Bax表达升高，诱导HSP70表达升高，阻抗辐射诱导的p-JNK1/2和p-p38表达升高，从而降低活化caspase-3表达，减少细胞凋亡。ALC能够改善由电离辐射诱导的氧化损伤，降低活性氧自由基的产生，调节细胞氧化还原和凋亡信号转导，抑制线粒体凋亡信号途径，对辐射诱导氧化伤害具有显著的防护作用。本研究结果对于揭示辐射诱导氧化损伤防护机制及开发研制新型抗辐射药物具有重要的理论价值和实际意义。更多还原

【Abstract】 Ionizing radiation can induce acute or chronic damage in the nervous,hematopoietic, digestive and immune systems through reactive oxygen species (ROS)caused oxidative stress in cells and tissue. Hence screening and evaluation antioxidantand radiation protective activity components from natural product resources havebecome a hot spot in the study of biology, medicine and food science. In this thesis,separation, purification and identification of anthocyanin in Lonicera.caeruleavar.edulis (ALC) were researched; antioxidant activity and protective effect onradiation-induced oxidative damage of ALC were also investigated. The main results areas follows:The optimum extraction conditions for ALC were47%ethanol,97min extractiontime, and the ratio of material to solvent of1:16.35. The optimum purificationconditions for ALC were AB-8macroporous resin,4.0BVs ethanol (50%, pH=3.0)eluant under1.5BV/h flow rate, and the ratio of diameter to height of1:15. Then we got22.26purification multiple than crude extracts and79.68color value ALC.ALC constituents were analyzed and identified by HPLC-DAD, HPLC-ESI-MS, Q-TOF-MS, UV-VIS and IR. The results show that the main constituents ofALC contained eight anthocyanins: cyaniding-3-sophoroside-5-glucoside, cyanidin-3,5-diglucoside, cyaniding-3-(feruloyl)-glucoside, cyaniding-3-glucoside, cyanidin-3-rutinoside, pelargonidin-3-glucoside, peonidin-3-glucoside and peonidin-3-rutinoside.Their mass percent contents were1.12%,3.71%,1.38%,72.42%,9.62%,2.30%,7.10%and2.34%, respectively.Antioxidant activities of ALC were studied in vitro, such as removal physiologicaland non-physiological free radicals, total reducing capacity and anti lipid peroxidationactivity. The results show that ALC has high scavenging activity on hydroxide radical(·OH). The main mechanism is that ALC is an active hydrogen atom donor, which canbreak down radical chain reactions through hydrogen atom transfer. ALC also showsignificant total reducing capacity and inhibition activity on lipid peroxidation with adose-dependent manner.X-ray or60Coγ radiation induced mouse spleen cell damage model was establishedto test and verify ALC’s significant protective effect on radiation induced damagethrough enhancing cell viability and reducing DNA damage induced by ionizingradiation.Then, oxidative damage animal model induced by60Coγ radiation was establishedto investigate the protective effect in vivo. The results show that ALC can significantlyreduce mouse immune organs oxidative damage through enhancing antioxidant enzymes activities such as superoxide dismutase (SOD) and glutathione peroxidase(GSH-Px). ALC can ameliorate glutathione (GSH) and reduce malondialdehyde (MDA)level in organs and serum of mouse to decrease lipid peroxidation and protect cellmembrane. ALC also can inhibit generation of atypical lymphocytes and bone marrowmicronuclei induced by radiation.Regulating effect of ALC on apoptosis and MAPK path related proteins expressstatus were analyzed through western blot assay. The results demonstrate that theprotective effect mechanism of ALC on oxidative damage induced by radiation was thatALC can increase expression of Bcl-2and HSP70, block radiation-induced up regulatedof Bax, phosphorylated JNK1/2and p38, inhibit the release of cytochrome c, depressthe activities of caspase-3, and thus inhibit cell apoptosis.These results demonstrate that ALC has effective antioxidant and antiradiationactivities through regulating cell oxidoreduction and apoptosis signal transduction. Thepresent study has important theoretical and practical significance to revealingmechanism of preventing radiation induced oxidative damage and developing newradioprotectors.更多还原