【作者】 李宁；

【导师】 刘方；

【作者基本信息】 南开大学 ， 微生物学， 2013， 博士

【摘要】 天然抗氧化成分因具有高效低毒和多重抗氧化活性而备受研究者的关注。菇类作为一种重要的天然资源是抗氧化活性成分的主要来源之一本研究利用分子印迹技术,以天然抗氧化活性成分为模板分子,采用本体聚合法合成分子印迹聚合物,从致孔剂、功能单体以及交联剂等方面对分子印迹聚合物的制备过程进行优化,并将制备的分子印迹聚合物作为固相萃取的吸附材料,从菇类提取物中分离富集天然抗氧化成分。分子印迹聚合物的制备和应用主要从以下三方面进行：(1)以咖啡酸苯乙酯为模板分子,4-乙烯吡啶为功能单体,乙二醇二甲基丙烯酸酯为交联剂,四氢呋喃和异辛烷的混合溶液作为致孔剂合成分子印迹聚合物。实验考察了分子印迹聚合物对模板分子咖啡酸苯乙酯和结构类似物咖啡酸的选择性,并通过吸附动力学和吸附等温线对聚合物进行表征。将咖啡酸苯乙酯分子印迹聚合物作为固相萃取的吸附材料,从25种菇类中分离富集咖啡酸苯乙酯和咖啡酸。实验结果表明,从7种菇类中检测到咖啡酸的存在,含量在0.004-0.340μg g-1(dw)之间,其中裂褶菌、巴氏蘑菇、杏鲍菇、黄伞以及鲍鱼菇为首次报道分离到咖啡酸。此外,与商品化C18柱相对比,分子印迹柱对目标物表现出较好的亲和性和选择性。(2)以没食子酸为模板分子,4-乙烯吡啶为功能单体,乙二醇二甲基丙烯酸酯为交联剂,四氢呋喃和异辛烷的混合溶液作为致孔剂合成分子印迹聚合物。本实验比较了模板分子没食子酸和4种结构类似物(焦性没食子酸、间苯二酚、原儿茶酸以及3,5-二羟基甲苯)在分子印迹柱上的吸附能力,并对聚合物的吸附性能进行表征。没食子酸分子印迹聚合物对于模板分子没食子酸表现出选择性高、传质速度快等特点。25mg L-1的没食子酸在50mg分子印迹物中静态吸附120min后,基本达到平衡。将没食子酸分子印迹柱应用于25种菇类粗提物的分离纯化,并从9种菇类中检测到没食子酸,含量在0.044-0.528μg-1(dw)之间,其中从鹿茸菌中分离得到了没食子酸的结构类似物原儿茶酸。本实验对没食子酸分子印迹柱与商品化C18柱对菇类粗提物的分离效果进行了比较,结果表明,分子印迹柱能够特异性的富集没食子酸,而C18柱对菇类粗提物的吸附能力较差。(3)以硬毛素为模板分子、丙烯酰胺和乙二醇二甲基丙烯酸酯作为功能单体和交联剂、四氢呋喃和异辛烷的混合溶液作为致孔剂制备得到的分子印迹聚合物对模板分子硬毛素表现出较强的选择性,对硬毛素的吸附能力明显强于结构类似物hispolon和inotilone。同样,通过吸附动力学模型和吸附等温模型对分子印迹聚合物的吸附性能进行了评价。以此分子印迹物作为固相萃取吸附填料对8种菇类提取物进行分离富集,从3种菇类中检测到硬毛素,其中桑黄中硬毛素的含量最高,为8.44μg g-1(dw)。与商品化C18柱相比,以分子印迹聚合物作为吸附材料的固相萃取柱对目标物表现出特异的吸附能力,从而起到富集目标物、纯化样品的目的。为了进一步评价所合成的分子印迹聚合物对目标活性成分的分离富集能力,本研究检测了菇类粗提物在经过分子印迹柱萃取后,样品的抑制红细胞溶血活性、抑制脂质过氧活性以及超氧阴离子清除活性的变化情况,从抗氧化活性的角度考察分子印迹柱的分离富集效果。实验结果表明,大多数含有目标活性成分的菇类粗提物在经过分子印迹柱萃取分离后,其抗氧化活性有所提高,说明菇类粗提物中的杂质成分被去除的同时活性成分得到富集。氧化应激引起的细胞氧化损伤而造成的细胞凋亡在疾病的发生和发展中所起到的作用越来越受到研究人员的关注。因此,对活性氧造成机体氧化损伤的机制以及抗氧化活性成分作用途径的研究尤为重要。本研究利用H202诱导小鼠脾细胞氧化损伤构建细胞氧化应激模型,以从美味牛肝菌、鹿茸菌和桑黄中分离得到的活性成分Be、Rf和Pi作为实验对象,评价天然抗氧化成分对氧化损伤细胞的保护作用。H202的暴露会导致小鼠脾细胞存活率的下降,并且与H202呈现剂量和时间依赖性关系。通过流式细胞仪分析发现,H202能够明显提高细胞内活性氧水平。Be、Rf和Pi能够显著抑制H202诱导的小鼠脾细胞氧化损伤,使细胞存活率从63.7%提高到73.7-77.7%,并且与活性成分的浓度呈现剂量依赖性关系,同时三种活性成分的预保护可以抑制H202诱导的胞内活性氧的产生。为了进一步研究活性成分的作用途径,本研究从线粒体依赖的信号转导途径和死亡受体介导的信号转导途径为切入点,探讨天然活性成分发挥其抗氧化活性的作用机制。研究发现,H202诱导小鼠脾细胞氧化损伤主要是通过线粒体依赖的信号转导途径实现的,并未激活死亡受体介导的凋亡通路中的Fas/FasL途径。天然活性成分对H202诱导小鼠脾细胞氧化损伤的保护作用与其对细胞线粒体的保护作用密切相关。Be、Rf和Pi都能不同程度的提高因H202造成的细胞线粒体膜电位降低,恢复线粒体正常生理功能。同时,也抑制了线粒体内促凋亡因子细胞色素C的释放。活性成分还能通过提高抗凋亡蛋白Bcl-2mRNA和蛋白表达量,抑制Caspase-3、Caspase-9激活的方式阻断活性氧介导的细胞氧化损伤。抑制细胞凋亡,从而起到抗氧化作用。本研究将分子印迹技术应用于菇类中天然抗氧化成分的分离纯化,并对得到的天然活性成分的体外抗氧化活性以及抗细胞氧化应激机制进行探讨,推断抗氧化成分对细胞的保护机制,为建立一套快速、高效的从菇类资源中分离纯化活性成分的分离筛选体系奠定理论基础,从而实现菇类资源中抗氧化成分的开发和利用。更多还原

【Abstract】 Natural products with antioxidant activity have been paid much attention by many researchers because of its efficiency and low toxicity. Mushroom, as an important natural resource, is one of the main sources of antioxidant components.In this study, a method to separate and enrich the active compounds from mushrooms using molecularly imprinted technology (MIT) is reported. The molecularly imprinted polymers (MIPs) were prepared by bulk polymerization and the antioxidants were the template. The effects of porogenic solvents, functional monomers and cross linkers on the MIPs were investigated. MIPs coupled with solid phase extraction (SPE) provided an approach for separation and enrichment active compounds from mushrooms. The synthesis and application of the natural active compound-MIPs consist of three segments:(1) The MIPs were synthesized using caffeic acid phenethyl ester (CAPE) as the template,4-vinyl pyridine (4-vp) as functional monomer, ethylene glycol dimethacrylate (EDMA) as cross linker and a mixture of tetrahydrofuran (THF) and isooctane as porogenic solvent. The selectivty, the sorption kinetic and the adsorption isotherm were investigated. MIP as the adsorbent phase for SPE (MISPE) was used to separate and enrich CAPE and its analog caffeic acid (CA) from25species of mushrooms. CA was detected in7out of the25mushroom species and the concentration was range from0.004to0.340μg g-1(dw). This is the first report describing the isolation of CA from Schizophyllum commune, Agaricus blazei, Pleurotus eryngii, P. adiposa, and P. cystidiosus. In comparison with C18-SPE, MISPE displayed high selectivity and good affinity for the target compounds.(2) The MIPs was synthesized by gallic acid (GA) as the template,4-vp as functional monomer, EDMA as cross-linker and a mixture of THF and isooctane as porogenic solvent. The selectivity of the MIPs was evaluated by GA and its structure analogs (pyrogallic acid,1,3-dihydroxybenzene, protocatechuic acid,3,5-Dihydroxytoluene) and its adsorption behavior was investigated in detail. The results showed that the MIPs exhibited good selective ability, offered a faster kinetics for the adsorption and desorption of GA. The adsorption equilibrium was almost reached within120min at GA concentration of25mg L-1in50mg MIPs. GA was detected in9out of the25mushroom species and the concentration was in range of0.044-0.528μg g-1(dw). The structure analog protocatechuic acid (PA) was also detected in Ramaria formosa. Comparison with C18-SPE, the separation of GA from the extract of mushrooms with MISPE demonstrated high affinity and good recognition. The C18-SPE exhibited much lower extraction yields to the extract of R. formosa.(3) The MIPs were prepared by hispidin as the template, acrylamide (AA) as functional monomer, EDMA as cross-linker and a mixture of THF and isooctane as porogenic solvent. Comparing with the two analogs of hispidin, hispolon and inotilone, the template hispidin exhibited good affinity to the MIPs. The adsorption behaviors were evaluated by the sorption kinetic and the adsorption isotherm. The prepared MIPs were employed as an adsorbent phase for convenient direct separation and enrichment hispidin from8species mushrooms. Hispidin was detected in3out of the8mushroom species and the highest concentration of hispidin was detected in Phellinus igniarius,8.44μg g-1(dw). Comparison with commercial C18-SPE, MISPE displayed high selectivity and good affinity for hispidin for extract of P. igniarius and most of interfering components present in the extract of the mushroom were removed using MISPE column.The antioxidant activity of the extracts after using MIPs was evaluated by inhibitions of erythrocyte hemolysis and lipid peroxidation, the scavenging activity of free radical. The extracts contained target compounds after perconcentration using the MIPs exhibited higher inhibition activity than those before using MIPs. It indicated that the extracts of the mushrooms after extracted by MIPs have been clean up and the active ingredients have been enriched.More and more researchers pay attention to the occurrence and development of the disease caused by oxidative stress-induced cell oxidant damage. Therefore, the mechanism of the oxidative damage caused by reactive oxygen species (ROS) and the antioxidant pathway of the active compounds are particularly important. The antioxidants Be, Rf and Pi, which was extracted from Boletus edulis, Ramaria formosa and Phellinus igniarius by MIPs, used to assess the efficacy against hydrogen peroxide (H2O2)-induced mouse splenocytes oxidative stress. The H2O2-induced cell death in dose-and time-dependent was more pronounced. The extracts of the mushrooms showed moderate protective effects against H2O2induced cell death. The cell viability increased from63.9to73.7-77.7%and the protective effects of the active compounds were dose dependent. Intracellular ROS levels in the cells was enhanced after H2O2treatment but effectively suppressed in the cells pretreated with the extracts. In order to study the antioxidant mechanism of the active compounds, the mitochondria-dependent signal transduction pathway and the death receptor-mediated signal transduction pathway were studied. The oxidant damage induced by H2O2seems to be through the mitochondrial dependent signaling pathway, did not activate Fas/FasL pathway in the death receptor-mediated apoptosis pathway. The protective effect of the active compounds to H2O2-induced oxidative damage of mouse splenocytes is closely related its protective effect on mitochondria. Be, Rf and Pi could significantly increased the disruption of mitochondrial membrane potential caused by H2O2, restored the mitochondrial physiological function and inhibited the release of cytochrome c from the mitochondria into the cytosol. The pretreatment with the extracts significantly increased the mRNA and protein level of Bcl-2and inhibited caspase-3and caspase-9cleavage and block H2O2induced cell apoptosis.In the present researches, MIT was applied to the separation and purification of the antioxidant compounds form mushrooms. In order to study the antioxidant mechanism, the antioxidant activity in vitro and the protection effect of the antioxidant compounds H2O2against induced oxidate damage were discussed. MIT provided a rapid and effective approach for separation and enrichment active compounds from mushrooms, which is the basis to the development of mushroom resources.更多还原