【作者】 王雅；

【导师】 樊明涛；

【作者基本信息】 西北农林科技大学 ， 食品科学与工程， 2012， 博士

【摘要】 沙枣（Elaeagnus Angustifolia L.）为胡颓子属植物，在我国西北干旱地区生长面积较大，果实富含蛋白质、糖类、鞣质、黄酮、矿物质及多种微量元素等，具有止泻、利尿、降血脂、降血压、降血糖等生理功能。本文以甘肃武威地区腾格里沙漠生长的沙枣为试验材料，研究了提取溶剂对沙枣提取物得率和抗氧化活性的影响；沙枣生长期内多酚、黄酮和原花青素及抗氧化活性的动态变化；成熟沙枣不同部位多酚、黄酮和原花青素含量及抗氧化活性；沙枣多酚、黄酮和原花青素的提取工艺、大孔吸附树脂对沙枣多酚的吸附和解吸能力；沙枣提取物的体内外抗肿瘤作用及沙枣中活性物质的分离鉴定及其抗氧化活性，主要研究结果如下：（1）在其它条件相同的情况时，50%乙醇和50%丙酮提取的沙枣提取物得率最高，且其提取物的抗氧化活性较强。以乙醇-水和丙酮-水提取体系提取得到的沙枣提取物总得率与其抗氧化活性之间呈现显著的相关性，提取物的得率越高，其抗氧化活性越强；（2）沙枣果实在生长期内重量、体积及果皮和果肉占果实的质量分数逐渐增大，果核占果实的质量分数逐渐降低，在生长后期趋于平稳。沙枣果实多酚、黄酮和原花青素在幼果中含量最高，随着果实发育，多酚含量下降，黄酮和原花青素含量呈现先上升后下降又小幅上升的趋势。沙枣果实提取物的抗氧化活性在生长期内呈现先下降，到生长后期又有所回升的趋势；（3）沙枣果实在生长期多酚、黄酮及原花青素的含量与提取物的抗氧化活性之间存在显著的相关性，尤其与原花青素的含量相关性最高。沙枣多酚、黄酮和原花青素的含量越高，抗氧化活性越强。还原力、DPPH清除能力与油脂氧化稳定性测定相互之间的相关性均极显著（P＜0.01），说明这三种抗氧化活性检测方法可用于沙枣提取物抗氧化活性的研究；（4）成熟沙枣果实中，多酚、黄酮和原花青素在不同部位分布不同。果肉部位的含量显著高于果皮和果核。另外，部位不同，提取物的抗氧化活性不同，沙枣果肉提取物的抗氧化活性显著高于果核和果皮；（5）在单因素试验基础上，对影响沙枣多酚、黄酮和原花青素提取效果的乙醇浓度、超声时间、料液比和超声功率进行响应面设计，建立了沙枣多酚、原花青素和黄酮提取因素的回归方程，这些方程能在试验范围内较好地反映沙枣多酚、黄酮及原花青素的提取效果。优化得到沙枣多酚的工艺参数为：超声功率309.35W，超声时间9.71min，料液比1:11.40，乙醇浓度48.10%，在此条件下沙枣多酚最大响应值为8.58%；沙枣原花青素的最优工艺参数为：超声功率278.86W，超声时间10.28min，料液比1:11.77，乙醇浓度49.36%，在此条件下沙枣原花青素最大响应值为4.53%；黄酮提取的最佳工艺参数：超声功率293.18W，超声时间8.04min，料液比1:20.20，乙醇浓度63.84%，在此条件下沙枣黄酮最大响应值为5.37%；（6）在供试的6种大孔吸附树脂中，NKA-9对沙枣多酚吸附解吸有较好的作用。当上柱液体积为25.12mL，沙枣多酚浓度为1.00mg/mL，上柱流速为1.0mL/min时，NKA-9树脂对沙枣多酚的吸附效果最好。当用60%乙醇为洗脱剂，洗脱液体积为18.84mL，洗脱流速为1.0mL/min时，沙枣多酚的动态解吸效果最好。经NKA-9大孔树脂纯化，沙枣多酚的含量是纯化前的4.8倍，提高了其抗氧化活性和对体外培养的人肝癌细胞HepG2的抑制效果；（7）沙枣提取物对体外培养的人肝癌HepG2细胞增殖有显著的抑制作用，在培养的前48h内，抑制效果与浓度有明显的依赖关系，提取物的浓度越大，抑制效果越好；体内试验表明，沙枣提取物对肝癌H22有显著的抑制作用，且可以提高机体的免疫功能；通过肿瘤组织的病理学切片观察，沙枣提取物实验组肿瘤组织中可见到明显的肿瘤细胞坏死区域，细胞排列较为疏松，存在凋亡小体，异常形态细胞居多，细胞核崩解现象严重。在急性毒性试验中，在最大剂量27.4g/kg并未表现出毒性；（8）采用大孔树脂纯化、反复硅胶柱层析、半制备液相色谱等手段对沙枣果实中的化合物进行分离，得到其中的10个化合物。通过化合物的MP、UV、MS、1H-NMR、13C-NMR、DEPT、2D-NMR等数据鉴定了其中的8个化合物，其中7个为黄酮类化合物，1个氨基酸。化合物4(槲皮素-3，4’-O-β-D-葡萄糖苷)、7(槲皮素-3-O-β-D-半乳糖-4’-O-β-D-葡萄糖苷)和化合物8(异鼠李素-3-O-β-D-半乳糖-4’-O-β-D-葡萄糖苷)为首次从该属植物中分离得到。化合物3（槲皮素）、4、6（异鼠李素-3-O-β-D-吡喃半乳糖甙）、7、8均有一定的抗氧化活性，其抗氧化活性强弱顺序是：化合物3＞化合物4＞化合物7＞化合物6＞化合物8。更多还原

【Abstract】 Elaeagnus Angustifolia L.(Elaeagnus), is widely distributed in Northwest China. Its fruitis rich in many effective components including protein, carbohydrates, tannin, flavonoid,minerals and trace elements which have antidiarrheal, diuresis and hypoglycemic effects, andcan lower blood pressure and reduce blood lipid. In this paper, the fruits of E. angustifoliacollected from the Tengger Desert in Wuwei, were used as test material for studying theextraction rate and antioxidant activity of these extractions from different extraction solvent;The dynamic changes for content of polyphenol, flavonoid and proanthocyanidins, and theantioxidant activities during the tested growing period were evaluated; The content ofpolyphenol, flavonoid and proanthocyanidins, and antioxidant activity in different parts ofripe fruits were also determined; The extraction processes for polyphenol, flavonoid andproanthocyanidins were optimized; Adsorption and desorption capability of polyphenol bymacroporous resin were determined; In addition, the isolation and identification, antioxidantactivity of chemical constituents were also studied. The major findings were as follows:（1） In same conditions, the yield of extracts from the fruits of Elaeagnus AngustifoliaL.(EFEA) by50%ethanol and50%acetone was the highest, which possessed the strongestantioxidant activities. The EFEA obtained from the solvent systems of ethanol-water andacetone-water showed significant correlations with their antioxidant activities. The moreextraction rate of the extracts had, the more high antioxidant activities of the extractsdisplayed.（2） The weight and volume of the whole fruit increase in early growth period. Inaddition, the mass fraction of peel and fresh quickly increase in early period, the mass fractionof stone decrease in early period, and steadily grow closer to maturity.The content ofpolyphenol, flavonoid and proanthocyanidins obviously changes in the tested growing stage,and young fruit has high one; With the fruit constantly growing, the content of polyphenolgradually decrease. On the other hand, the content of flavonoid and proanthocyanidinsinitially increase, then decrease, and finally slightly increase. As far antioxidant activities, thefruit showed this trend of decreasing at firstly and increasing later in the growth.（3） There are significant correlations between the content of polyphenol, flavonoidand proanthocyanidins in the growth stage with the antioxidant activities of extracts, especially between proanthocyanidins and antioxidant activities. The antioxidant activitiesimprove when the content of EFEA increase. Moreover, there are correlations among thesedetermination methods of DPPH scavenging, reducing power and oxidation stability of lard.The result show these three methods can be used for determination the antioxidant activitiesof EFEA.（4）In different part of ripe fruit,the distribution of polyphenol,flavonoid andproanthocyanidins is different. The contents of these chemical components in the fresh arehigher than one in fruit stone and peel. In addtion, antioxidant activities in the fresh is alsohigher than in fruit stone and peel.（5） Response surface experiments based on single factor experiment were designed toresearch the impact of ethanol concentration、ultrasonic time、solid-liquid ratio、ultrasonicpower on yield of polyphenol, flavonoid and proanthocyanidins. Regression equations ofpolyphenol,flavonoid and proanthocyanidins were established. These equations couldeffectively predict the effect of polyphenol, flavonoid and proanthocyanidins yield inexperiment range. The conditions for polyphenol extraction were shown as ultrasonic power309.35W, ultrasonic time9.71min, solid-liquid ratio1:11.40, ethanol concentration48.10%,and the yield of polyphenol were8.58%under these conditions. The conditions forproanthocyanidin extraction were shown as ultrasonic power278.86W, ultrasonic time10.28min, solid-liquid ratio1:11.77, ethanol concentration49.36%, and the yield ofproanthocyanidin was4.53%; The conditions for flavonoid extraction were shown asultrasonic power293.18W, ultrasonic time8.04min, solid-liquid ratio1:20.20, ethanolconcentration63.84%, and the yield of flavonoid was5.37%.（6）In the six tested macroporous resins, the NKA-9was the most desirable adsorptionand desorption material for polyphenol of Elaeagnus Angustifolia L.. When25.12mL of1mg/mL sample was flowed through NKA-9resin column at a speed of1.0mL/min, theadsorption reached dynamic equilibrium. The adsorbed polyphenol could be completelyeluted with18.84mL of60%ethanol at1mL/min. After the purification,the content ofpolyphenol was4.8times of untreated one, and antioxidant activities and inhibition on HepG2tumor cells were obviously improved.（7） The EFEA significantly inhibited the proliferation of Human hepatoma cellsHepG2in vitro test. During first48h of culture stage, the inhibitory effect was in adoes-dependent manner. The study of the tumor-bearing mice model suggested that theextracts had significantly inhibitory effect on hepatoma cells H22, and improve immunity inmice. By pathology slice of tumor tissue, it could be found that there were obvious necroticareas of tumor cells from EFEA group, and cells grow in a more loose state; there were some apoptotic bodies and a lot of abnormal morphologic cells; The nucleus were seriously brokendown. In the acute toxicity test, the highest does of27.4g/kg did not show toxicity to mice.（8） Ten compounds were isolated by macroporous resin, silica column chromatoprap-hy, Sephadex LH-20and semi-prepared HPLC, in which eight compounds were identified byMP, UV, IR, MS,1H-NMR,13C-NMR, DEPT,2D-NMR, including seven flavonoids and aamino acid. Among these, compounds4(Quercetin3,4’-O-β-D-diglucoside),7(Quercetin3-O-β-D-Galactopyranoside-4’-O-β-D-glucopy-ranoside) and8(isorhamnetin3-O-β-D-Galactopy-ranoside-4’-O-β-D-glucopyranoside) were isolated for the first time from Elacagnus. Thecompound3、compound4、compound6、compound7and compound8had antioxidant activities,which sequence was compounds3＞compound4＞compound7＞compound6＞compound8.更多还原