【作者】 国旭丹；

【导师】 王敏；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2013， 博士

【摘要】 苦荞为国际粮农组织公认的优秀药食兼用作物，其保健功能自中国历代古医书就多有阐述。现代医学研究也证明苦荞具有抗氧化、抗炎、降血糖、降血脂、降血压、抗肿瘤等多种药理活性。专家预测，到2020年全球范围内，在人类历史上心血管疾病（CVD）将首次成为首位的致死原因。CVD的共同病理基础是代谢紊乱。“代谢综合征”（MS）是以胰岛素抵抗（IR）为特征的一类共同发生发展、相互紧密联系的代谢紊乱的总称，它不是一种单一的疾病，而是一系列病理生理现象的统一体，如肥胖、Ⅱ型糖尿病、高血脂、高血压（或冠心病）等，亦称为胰岛素抵抗综合征。胰岛素抵抗实际上是机体长期存在并呈进行性发展的一个亚健康状态，而对防治胰岛素抵抗强调的是早期干预和长期干预，这正符合功能性食品的应用特征。苦荞在食品及医药领域已广受关注，多酚成分是其功能性开发的基础。本研究以苦荞多酚为研究对象，分析苦荞的多酚抗氧化组分的存在形式与影响因素，并采用活性追踪法分离主要抗氧化物质，同步研究苦荞主要抗氧化物质槲皮素及其体内代谢物槲皮素-3-O-葡萄糖醛酸改善内皮IR活性，明确其在体内代谢转化过程中改善血管内膜IR的活性形式、作用效果与作用机理。本研究将苦荞的功能性推向了一个新的高度。本文以苦荞麦为实验材料，以苦荞黄酮和酚酸为研究对象，将植物化学、现代色谱分析、分子生物学和药理学等学科有机结合，以抗氧化活性为切入点，以自由基、活性物质与功能关系及IR理论为指导，采用HPLC分析、植物化学分离纯化NMR鉴定技术及Elisa和Western blot相结合的研究方法和技术，研究苦荞多酚和抗氧化性的内在和外在影响因素，并活性追踪分离纯化鉴定其主要抗氧化物质，探讨主要抗氧化物质槲皮素及体内代谢物槲皮素-3-O-葡萄糖醛酸改善IR状态下血管内膜功能的效应及作用机制，以期为苦荞预防代谢综合征提供理论依据。主要研究结果为：（1）苦荞籽的绝大部分（76%-95%）酚物质以游离形式存在，游离酚是其发挥抗氧化性的主要贡献者，游离酚和结合酚的分布研究有利于理解食用苦荞对健康的益处。（2）苦荞籽除富含芦丁外，含有的酚酸包括p-羟基苯甲酸、阿魏酸、原儿茶酸、p-香豆酸、没食子酸、咖啡酸、香草酸、紫丁香酸和绿原酸，其中原儿茶酸和阿魏酸是苦荞籽的主要酚酸。（3）苦荞品种和生长环境及其它们的交互作用影响着苦荞酚物质的含量和抗氧化性。环境参数如高海拔有助于提高苦荞芦丁和酚酸含量。该结果提示通过优化特定苦荞品种的生长条件便能培育出富含天然抗氧化成分的苦荞存在可能。（4）苦荞的酚含量、组成及其抗氧化性受组织结构影响。原儿茶酸是苦荞壳（>40目）的主要酚酸，p-羟基苯甲酸是苦荞麸（40-80目）和细粉（<80目）的主要酚酸。苦荞壳、麸皮、细粉的总酚含量排序为：麸>壳>细粉。清除DPPH自由基和ABTS自由基能力的排序为：麸>壳>细粉。总还原力的排序为：壳>麸>细粉。该结果暗示苦荞籽壳和麸皮，（常被弃去的部分）的多酚等抗氧化功能性物质高于细粉（常被食用部分）。如果将壳和麸皮超微粉碎后添加到其它粮谷食品中，将极大的提高其粮谷食品的功能价值。因此，苦荞壳和麸皮极有潜力作为功能食品原料以提高苦荞的附加值。（5）苦荞麸皮乙醇粗提物乙酸乙酯萃取相的总酚和总黄酮含量及抗氧化性均显著高于正丁醇相和水相，说明乙酸乙酯是富集苦荞黄酮粗提物的良好溶剂。（6）采用活性追踪法分离的苦荞麸皮的主要抗氧化物质为槲皮素、p-羟基苯甲酸和胡萝卜苷，在这三种物质中槲皮素含量最高，p-羟基苯甲酸首次从苦荞样品中分离出来。该结果有助于理解苦荞的保健功能，将会促进苦荞在保健食品和医药领域的利用。（7）在人脐静脉内皮细胞（HUVECs）中，苦荞麸皮主要抗氧化物质槲皮素和其代谢物槲皮素-3-O-葡萄糖醛酸能显著抑制棕榈酸（PA）引起的活性氧（ROS）的过量产生并能恢复下降的线粒体膜电位，从而表现出抑制氧化应激的活性；能有效抑制PA诱导的IKKβ和NF-κB的激活，降低炎性因子IL-6和TNF-α蛋白的产生，从而表现出针对IKKβ/NF-κB途径的抗炎活性。（8） PA通过炎性反应和氧化应激阻碍了胰岛素在HUVECs内沿PI3K信号通路的转导。而槲皮素和槲皮素-3-O-葡萄糖醛酸能显著有效抑制PA刺激的IRS-1丝氨酸磷酸化的增加，恢复胰岛素作用下IRS-1酪氨酸磷酸化及PI3K信号下游Akt和eNOS的磷酸化活化，进而促进NO的释放，从而表现出对PI3K/Akt信号通路转导的调控作用，即改善内皮功能紊乱的作用。（9）槲皮素和槲皮素-3-O-葡萄糖醛酸改善血管内膜IR的信号途径为ROS/IKKβ/IRS-1/PI3K/Akt/eNOS/NO。该结果明确了苦荞对IR状态下血管内膜功能的调节效应、作用特点和关键靶点，勾勒出苦荞干预血管内膜IR的分子机制。该结果提示槲皮素和体内代谢物槲皮素-3-O-葡萄糖醛酸对IR状态下内皮功能紊乱具有一定的保护作用。本研究同时考察槲皮素和其代谢物对内皮细胞胰岛素敏感性影响的结果为槲皮素预防或治疗与IR有关的内皮功能紊乱提供了有力的理论依据，而且对预防和治疗MS具有重要的理论意义。本研究从活性物质与功能关系和分子信号转导途经方面为苦荞预防或治疗MS提供了强有力的理论依据，并将苦荞的功能性推向了一个新的高度。更多还原

【Abstract】 Tartary buckwheat (Fagopyrum tataricum), as a unique food medicine dual-use cerealcrop, which health benefits have been reported in many China traditional medicine books.And modern medical researches have also shown its many pharmacological activities such asantiinflammation, antioxidant, antidiabetes, antihyperlipidemia, antihypertension andantitumor et al. By the year2020, reportedly, cardiovascular disease (CVD) will become thefirst cause of death in the first time in the global scope. Metabolic disorder is a commonpathological basis of CVD. Metabolic syndrome (MS) is the general term that describes anumber of metabolic disorders, and characterized by insulin resistance (IR). MS is not asingle disease, but in fact it is a unity of a series of pathologic physiology phenomenon, suchas obesity, type2diabetes, hyperlipidemia, hypertension and coronary heart disease et al.Therefore, MS is also referred to as insulin resistance syndrome. As a matter of fact, IR is asub-health state of a body with gradually development. Early and long-term prevention aregood ways to prevent IR, and it is in keeping with application characteristics of functionalfood. People have been paying more attention on the application of tartary buckwheat in foodand medical fields. Tartary buckwheat is rich in polyphenols with a wide array of bioactivitiesimplicated in cardiovascular protection, and polyphenols are a group important functionalsubstance in tartary buckwheat. This study aims to establish the link between the polyphenolantioxidants and their metabolites of ameliorating insulin resistance and further elucidateunderlying mechanism. Determination of the functional substances and verification of theirbeneficial regulation of insulin sensitivity should contribute to further applications of tartarybuckwheat in the management of metabolic syndrome and diabetes insulin resistanceinvolved.This research incorporated plant organic chemistry, modern chromatography analysisand molecular biology. And the composition, antioxidant activity and regulation of insulinaction of flavonoids and phenolic acids of tartary buckwheat were researched using HPLC,NMR, Elisa and Western blot methods, and free radicals and anti-oxidation, structure-activityand insulin resistance theories. The objectives of present study were the following:(1) toanalysis the influencing factors of phenolics and antioxidant activity of tartary buckwheat;(2)to look for the pure antioxidant substance;(3) to investigate the molecular mechanism ameliorating insulin resistance of pure antioxidant substance (quercetin) and its metabolite(quercetin-3-O-glucuronide). The results will provide a theoretical basis for the application oftartary buckwheat in the prevention and treatment of MS.The main conclusions of this study are as follows:(1) The majority (76%-95%) of phenolic compounds of tartary buckwheat was presentin the free form and the distinction between free and bound phenolics helps to understand thepotential benefit of tartary buckwheat consumption.(2) Phenolic acids in tartary buckwheat seed included p-hydroxybenzoic, ferulic,protocatechuic, p-coumaric, gallic, caffeic, vanillic, syringic and chlorogenic acids. Andp-hydroxybenzoic, protocatechuic and ferulic acids were the prominent phenolic acids intartary buckwheat seed.(3) The variety and growing environment and the interaction between variety andenvironment may contribute more to individual antioxidant properties and phenolics oftartary buckwheat. Environmental parameter such as higher altitudes may also have anincreasing effect on rutin and phenolic acids content of tartary buckwheat. The results alsoshow the possibility of increasing the content of natural antioxidants by optimizing thegrowing conditions of a selected variety.(4) The milling fractions of tartary buckwheat influenced their phenolics and antioxidantproperties. Protocatechuic acid was the dominant phenolic acid of the hull. p-Hydroxybenzoicacid was the dominant phenolic acid of bran and light flour. The orders of total phenoliccontent and DPPH· and ABTS·+scavenging activities of milling fractions were bran> hull> light flour. The orders of total reducing power of milling fractions was hull> bran> lightflour. The results indicate that the antioxidants including phenolic acids are concentrated inthe outer layers of tartary buckwheat. These results suggest that the use of bran fractionationto reduce the particle size can help in developing grain-based products with increasednutritional and functional potential. Therefore, hull and fine bran have the potential to be usedas functional food ingredients and may increase the added value of tartary buckwheat.(5) The ethyl acetate fraction of tartary buckwheat bran ethanol extract had higherphenolic and flavonoid contents and showed stronger antioxidant activity than n-butanol andaqueous fractions. Therefore, ethyl acetate is excellent solvent concentrated tartarybuckwheat flavonoids.(6) Quercetin, p-hydroxybenzoic acid and daucosterol were identified as the mainantioxidant constituents in the ethanol extracts of tartary buckwheat bran following abioguided chromatographic strategy. p-Hydroxybenzoic acid was isolated from buckwheatgrain for the first time. The result will contribute to the understanding of the health benefits and the novel utilization of tartary buckwheat bran in the health food and medical industries,improving the potential to add value of tartary buckwheat.(7) Quercetin and quercetin-3-O-glucuronide inhibited ROS overproduction andeffectively restored Δψm, demonstrating their chemorpotection of mitochondrial functionthrough antioxidative actions. Also, quercetin and quercetin-3-O-glucuronide inhibitedROS-associated inflammation by inhibition of IL--6and TNF-α production with suppressionof IKKβ/NF-κB phosphorylation.(8) Inflammation and ROS impaired insulin PI3K signaling and reducedinsulin-mediated NO production. Quercetin and quercetin-3-O-glucuronide facilitated PI3Ksignaling by positive regulation of serine/tyrosine phosphorylation of IRS-1and restorationof downstream Akt/eNOS activation, leading to an increased insulin-mediated NO level.(9) Quercetin and quercetin-3-O-glucuronide inhibited ROS-associated inflammation byblocking IKKβ/NF-κB activation and effectively facilitated insulin signaling transductionalong IRS-1/Akt/eNOS pathway in the endothelium. Because quercetin is rapidlymetabolized during absorption, by methylation, glucuronidation and sulfation, parallelinvestigation of effects of quercetin and its metabolite on insulin sensitivity in theendothelium should be beneficial for us to get more information about quercetin in themanagement of endothelial dysfunction involved in insulin resistance.In this study, the results provided evidences of application of tartary buckwheat inprevention and treatment of MS in terms of structure effect model and signal transductionpathways. Meanwhile, the functionality of tartary buckwheat would be promoted to a newhigh.更多还原