【作者】 龙文静；

【导师】 刘仲华； 张盛；

【作者基本信息】 湖南农业大学 ， 营养与食品卫生， 2011， 硕士

【摘要】 咖啡作为世界消费量最大的饮品,其营养价值一直备受瞩目。早在19世纪国外就对咖啡豆中的功能成分展开了研究,随着科技的进步,研究方法和技术的不断革新,咖啡豆中的营养素成分及非营养素成分已逐渐明了,相应的保健功能及药理学作用也得到了进一步证实。尤其是,咖啡豆中所含的大量非营养素成分——多酚类化合物己成为目前国际天然药物学领域的关注重点,这些物质在先导物发现和创新药物研制中扮演了越来越重要的角色。云南、海南、广西及广东是我国咖啡豆种植的主产区,且资源十分丰富。因此,建立标准化和规范化的咖啡多酚提制体系,有效开发和合理利用我国咖啡豆资源,改变仅靠原料出口的贸易模式,具有非常重要的经济价值和现实意义。天然产物的开发利用关键在于先进的提取分离技术与手段以及准确、高效、快速的分析方法。本研究建立了咖啡豆中多酚类物质的高效液相色谱(HPLC)分析方法；确定了咖啡多酚的最优提取工艺参数,进一步研究了聚酰胺树脂初步分离纯化咖啡多酚的最优条件,并利用HPLC技术对咖啡多酚的具体组成进行了分析；评价了咖啡豆多酚体外抗氧化活性,结果如下：建立了同时测定咖啡豆中6种多酚类化合物(咖啡酸、3-咖啡酰奎尼酸、4-咖啡酰奎尼酸、5-咖啡酰奎尼酸、3,5-二咖啡酰奎尼酸、4,5-二咖啡酰奎尼酸)的反相高效液相色谱测定方法。采用Kromasil C18柱(4.6 mm×200 mm,5μm),以乙腈和0.1%甲酸水溶液为流动相进行梯度洗脱,二极管阵列检测器检测,45 min内可对6种目标物进行同时检测,且各化合物都能达到基线分离。经测定,该方法的平均回收率为90.76%-104.73%,相对标准偏差为0.7%-3.9%。该法简便、快速、灵敏度高,适用于咖啡豆中6种酚酸类化合物的同时分析。此外,试验还利用HPLC法比较了云南四个产区咖啡豆原料中多酚含量,结果显示保山产区咖啡豆的总多酚含量最高。通过单因素试验研究了提取溶剂、提取方法(温度)、提取时间及次数对咖啡多酚提取效果的影响,得到咖啡多酚的最佳提取工艺：用70%乙醇水溶液,按液料比1：7,80℃下回流提取两次,每次提取2h。从7种大孔树脂中筛选出最佳树脂—聚酰胺,研究其初步纯化咖啡多酚的适宜条件。结果表明：聚酰胺树脂纯化咖啡多酚的最佳吸附和解吸条件为上样液浓度4.44 mg/ml左右,重复过柱1次,最佳洗脱剂为80%乙醇,洗脱剂用量约为3倍的柱体积,上样及洗脱流速均为2 BV/h。多酚含量由14.43%提高到53.32%,得率达到78.21%。分别采用紫外分光光度计和HPLC对咖啡多酚初步纯化物进行分析,前者测得总多酚含量为53.32%,后者检测6种多酚类物质的总含量为47.55%,表明咖啡多酚主要为绿原酸及其衍生物。通过5种不同的体系(清除过氧化氢、羟基自由基、超氧阴离子自由基、DPPH自由基能力及还原能力的测定)研究了咖啡多酚的抗氧化性能。结果表明,咖啡多酚具有一定的自由基清除能力和还原Fe3+的能力,且自由基清除率和还原Fe3+的能力与样品浓度之间存在显著相关性。更多还原

【Abstract】 As the largest consumption of drinks in the world, coffee’s nutritional value has much more attracted people’eyes. Early in the 19th century, the abroad began to study the function of components of coffee beans. With the advances in technology, innovation in research methods and technology, the nutrient composition of coffee beans and non-nutrient components has been gradually clear, corresponding health function and pharmacological effect has been further confirmed. In particular, a large number of non-nutrient components—polyphenols in the coffee beans has become the focus in international natural medicine field, these substances play an increasingly important role in the leading compound discovery and the innovation of drugs development. Yunnan, Hainan, Guangxi and Guangdong is China’s main coffee growing areas, and very rich in resources. Therefore, the establishment of the standard and regular distilling system of coffee polyphenols, in the effective development and rational utilization of the coffee resources, change the export trade mode of raw materials alone, has the very important economic value and practical significance.The key of the exploitation and use of natural products is the advanced extraction and separation technologies, and the accurate, efficient and fast method of analysis. This study established the high performance liquid chromatography (HPLC) analytical methods of the polyphenols in the coffee beans, identified the optimal extraction parameters of the polyphenols from the coffee, and further studyed the optimal conditions that the polyamide resin separated and purified the polyphenol of the coffee, analyzed the specific composition of polyphenols of the coffee by using the HPLC techniques, evaluated the in vitro antioxidant activity of polyphenols in coffee beans, the results were as follows:A method of reversed-phase high performance liquid chromatography coupled with diode array detector (HPLC-DAD) was established for the simultaneous determination of 6 phenolic acids (Caffeic acid,3-O-Caffeoylquinic acid,4-O-Caffeoylquinic acid,5-O-Caffeoylquinic acid, 3,5-O-Dicaffeoylquinic acid,4,5-O-Dicaffeoylquinic acid) in green coffee beans. Separation was achieved using a Kromasil C18 column (4.6 mm×200 mm,5μm), under the gradient elution with the mobile phase of acetonitrile and 0.1%(v/v) formic acid/water. The 6 phenolic acids were well separated within 45 min. The recoveries from 90.76% to 104.73% with the relative standard deviation between 0.7%-3.9%.The method is simple, rapid and highly sensitive, suitable for the simultaneous determination of 6 phenolic acids and quality control of coffee beans. In addition, using the HPLC method the test also compared the content of coffee polyphenol in four areas of Yunnan, the result showed that the highest content of total polyphenols is in Baoshan coffee.The effects on yield of extraction solution, extraction method (temperature), extraction time, and extraction times were researched. The optimization technics parameters of the extraction of coffee polyphenols were as follows:coffee was extracted at 80℃in 70% ethanol twice, with 1:7 as ratio of solid/liquid,extraction 2h every time.7 kinds of macroporous resins were compared for the separation of polyphenol of coffee, Polyamide resin exhibited superior adsorption and regeneration properties. The optimum conditions for separating and purifying polyphenol of coffee were obtained. The results showed that the separation optimization technics parameters with Polyamide resin were as follows:the sample concentration is 4.44mg/ml,the moving speed is 2BV/h, adsorbing twice,and eluting volume is 3BV with 80% ethanol as eluting solvent. The purity of polyphenol of coffee can be improved from 14.43% up to 53.32% and the recovery rate is 78.21%. UV spectrophotometer and HPLC were used to analyze Preliminary purified coffee polyphenols. The former measured the total polyphenol content was 53.32%, the latter measured total content of 6 kinds of the polyphenols was 47.55%, the results indicated that the main Phenolic substances in coffee are caffeyolquinic acid (CQA) and its derivatives.Through five kinds of detection system (scavenging hydrogen peroxide, hydroxyl radical, superoxide anion radical, DPPH radical and reducing power determination), the experiment studied the antioxidant properties of coffee polyphenols. The result showed that coffee polyphenols have certain abilities of scavenging free radieals and reducing Fe3+, and Significant correlation exists between the clearance rate of the free radieals, the abilities of reducing Fe3+ and the Sample concentration.更多还原