【作者】 冉军舰；

【导师】 樊明涛；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2013， 博士

【摘要】 我国是世界上苹果种植面积和产量最大的国家，陕西是中国主要的苹果产区，苹果浓缩汁产量居全国前列，苹果中的多酚类物质是重要的活性物质，目前已有部分研究，主要集中在多酚的提取以及部分功能特性的研究，对其中各个单体酚的了解较少，比如各单体酚的生物活性等。本文采用超声波辅助纤维素酶法提取苹果多酚，比较了苹果不同部位（果皮、果肉、果核）中的酚类化合物；并建立了一种高效液相色谱联合二极管阵列检测器的方法能够同时检测苹果中10种酚类化合物；利用高效液相色谱测定了苹果不同品种和不同时期的多酚变化；用大孔树脂对苹果多酚粗提物进行纯化；采用不同的抗氧化活性评价方法评价了不同苹果品种和不同多酚的抗氧化能力；对苹果多酚的抗癌性进行了研究。取得如下结果：1.对超声波辅助条件下，纤维素酶联合柠檬酸提取苹果皮中多酚的工艺进行了优化，目的是为了提高提取率。优化的提取条件如下：提取时间37min,提取温度37℃,加酶量2500U/g，在优化工艺条件下，总多酚、绿原酸、根皮苷和金丝桃苷提取量达到最大，并且试验值和其预测值高度一致。2.建立了高效液相色谱联合二极管阵列检测器同时检测苹果粗提液中的10种多酚类物质的方法，通过试验得到这10种标准品的标准曲线，线性关系良好，决定系数均大于0.99，测定准确度时回收率的范围在96.09%到103.61%之间，考察了液相条件的稳定性，测定精确度时日内和日间的变异性RSD<2%，在日内和日间的稳定性可以接受。对建立的液相方法进行了应用，选取嘎啦、红星、乔纳金、富士、秦冠作为试验样品，对苹果中的10种多酚进行了检测，发现此方法稳定，可以用于多种苹果多酚的同时检测。3.研究了苹果不同部位的多酚含量，发现苹果皮中多酚的含量最高，果肉中的多酚含量最低；对苹果不同时期的多酚变化进行了检测，发现在苹果的生长过程中，多酚的含量随着时间的变化而有明显的降低；不同品种的品质差别主要在于金丝桃苷、根皮苷、鞣花酸、绿原酸等含量的变化。4.通过六种不同树脂对苹果多酚的吸附率和解吸率的测定，最终确定NKA-9树脂为纯化苹果多酚的最佳树脂，它对苹果多酚的吸附量较大，解吸效果也较好。优化条件为：上样液浓度为1.46mg/mL，pH为原始多酚值，进样流速为1.0mL/min，洗脱流速0.5mL/min，此条件下苹果多酚的纯度由原来的0.67%提高到41.65%，纯度提高了62倍。5.不同苹果品种的乙醇提取液均具有一定的抗氧化活性，有清除DPPH·、NO2-·、O2-·和OH-·的能力，可考虑作为天然抗氧化剂使用。测定了苹果中的多酚类物质不同单体酚的抗氧化活性，发现绿原酸、金丝桃苷、表儿茶素能力较强。6.本实验以人体结肠癌细胞Caco2和肝癌细胞HepG2为试验材料，体外观察苹果多酚的抗肿瘤作用。MTT法测定的结果显示苹果多酚抑制Caco2和HepG2细胞的最适浓度在1mg/mL-64mg/mL这个范围内。随后选取苹果多酚中含量较高的几种多酚分别作用于两种癌细胞，通过观察发现，表儿茶素、根皮苷和绿原酸的抑制作用明显强于其他多酚。更多还原

【Abstract】 China is the largest producer of apple in the world. Shaanxi is one of the mainproduction areas, planting area, annual production and the export of apple concentrated juicelead the nation. Apple polyphenols have demonstrated to have potential beneficial effects onhealth. There have been some studies on the polyphenols; the most concentrated point is onthe functional activity of apple polyphenols, but the reported materials on the singlepolyphenol in apple and biological activity of each polyphenol and their antioxidant activityare very little.In the research, cellulase enzymolysis-citric acid extraction with ultrasonic-assistedtechnology was applied for polyphenols extraction from apple peel, the polyphenols ofsample in different tissues of apple were studied; A liquid chromatography with a diode arraydetector (DAD) method to simultaneously detect ten major polyphenols in apples has beenestablished; the change of polyphenols content in different periods and varieties of apple wereanalyzed; The changes of different apple varieties and different growth periods weredetermined; Resin adsorption was empolyed for purification of apple polyphenols; theantioxidant activity of different varieties of apples and different polyphenols evaluated bydifferent antioxidant activity method; Their antiproliferative activity evaluated by two cancercells.The main results are as follows.1. Cellulase enzymolysis-citric acid Extraction with Ultrasonic-assisted technology wasapplied for polyphenols extraction from apple peel, and response surface methodology wasused to optimize the conditions of polyphenols extraction. Efficiency of extraction wasoptimised by measuring total polyphenol content and three individual polyphenol. Theoptimal extraction conditions of polyphenols were determined as follows: Extraction time of37min, extraction temperature of37℃, enzyme amount2500U/g which was agreed closelywith the predicted value.2. This study developed a liquid chromatography with a diode array detector (DAD)method to simultaneously detect ten major polyphenols in apples. The linearity of thecalibration curves for each polyphenol in the desired concentration range was found to begood (r2>0.99). The method is accurate and precise with recoveries ranging from96.09%to 103.61%and both intra-day and inter-day variabilities (RSD<2%). The chromatographicparameters, such as flow rate, mobile phase pH and column temperature, were evaluated forrobustness and determined to be acceptable. The applicability of the proposed method wasdemonstrated by analyzing these10polyphenols in real samples such as Gala, Starkrimzon,Jonagold, Fuji and Qinguan from Baishui (Shaanxi, China).3. The content of polyphenols in different tissues were determined, there were thehighest content of polyphnols in apple peel, the lowest in apple pulp; The changes ofcompositon of polyphenols in different growth periods were analyzed. The results showedthat the proportions of all polyphenols were decreased during fruit development. The maindifferences of all apple varieties were the changes of the content of hyperoside, phloridzin,ellagic acid, chlorogenic acid.4. By studying the characteristics of adsorbing apple polyphenols with six resin, NKA-9was selected, the optimum conditions for separating and purifying apple polyphenol wereobtained. The results showed that when the content of apple polyphnols of test solution is1.46mg/mL, pH is original value，the moving speed is1.0mL/min and the eluting speed is0.5mL/min，the purity of apple polyohenols can be up to41.65%, it shows that the purity ofpolyphenol advance64times.5. The antioxidant activities of extracts from different apple varieties were studied,Nitroso Radical scavenging activity, DPPH· radical scavenging activity, hydroxyl radical(·OH) scavenging activity and Oxygen Radical scavenging activity. The chlorogenic acid,hyperoside, epicatechin show the stronger antioxidant power than those polyphenols.However, the antioxidant activities in vivo need to be further research.6. The antiproliferative effect of apple polyphnols against Caco2colon cancer cells andHepG2cells in vitro were analyzed. The results showed that the ranges of adequatefconcentrations were1mg/mL-64mg/mL. The effcets of antiproliferative of chlorogenic acid,phloridzin and epicatechin were better than other polyphenols.更多还原