【作者】 宋岩；

【导师】 于国萍；

【作者基本信息】 东北农业大学 ， 食品科学， 2013， 硕士

【摘要】 我国黑豆资源十分丰富，东北地区和华北各地均有分布。黑豆含有的一些容易被体内所吸收营养物质，还可以促进其它营养物质的吸收、改善新陈代谢以及疾病预防和保健作用。现在随着消费者的关注，越来越多的研究者开始从植物中提取天然产物来代替人工合成的物质。花色苷就是存在植物中很好的一种天然产物，其化学性质稳定，着色力强、色泽鲜艳、价格低廉可以应用在食品、健品和生物制药领域，因此越来越为人们所接受。本试验以黑豆为原料，通过脱脂成为脱脂黑豆粉，在研究超声波辅助提取及纯化，并对纯化黑豆花色苷后进行体内生物活性研究。在提取工艺的研究中，首先脱脂溶剂的选择，以花色苷含量和总酚含量为指标，考察不同有机脱脂溶剂对黑豆花色苷的影响，确定石油醚为最好的脱脂溶剂，提取时以花色苷含量为指标，采用单因素验设计对超声波辅助提取法进行了试验条件的优化，实验表明最佳的提取条件：料液比1∶30，超声时间为30min，超声温度50℃，超声功率280W，此时花色苷质量分数1.42mg/g.在纯化工艺研究中，采用柱层析法，选择AB-8大孔树脂为柱填料，对黑豆花色苷的粗提液进行分离纯化，得到纯度较高的花色苷样品。通过单因素实验，确定AB-8纯化工艺条件的最佳条件为：AB-8大孔树脂以约为0.1mg/mL的花色苷粗提液为上样液浓度，40min为吸附平衡时间，70min为解吸时间，吸附流速2.0mL/min，70%的乙醇溶液作为洗脱液，洗脱流速2.0mL/min，此条件下实验数据表明，纯化后比纯化前花色苷质量分数提高约3倍。在黑豆花色苷的体内动物试验中，建立衰老模型小鼠，选取Ve作为对照，考察纯化后黑豆花色苷对衰老小鼠的保护作用。实验过程中，检测全血中的T-AOC和血清、肝脏和脑组织中SOD、GSH-Px、MDA活性和蛋白质羰基含量，试验结果：纯化后黑豆花色苷灌胃的小鼠与衰老模型组小鼠比较，体内T-AOC增加，SOD和GSH-Px活性升高，MDA和蛋白质羰基含量明显下降，并且黑豆花色苷的中剂量组与VE的结果相近，之间没有差异显著性，高剂量组优于VE组，表明黑豆花色苷提取物的抗衰老作用明显，是一种高效天然的抗氧化剂。在体外抗氧化实验研究中，进一步研究纯化后黑豆花色苷对DPPH和ABTS清楚自由基的能力、还原能力、清楚超氧阴离子的能力。并与同质量浓度VC进行对照实验表明，DPPH清除能力相接近，ABTS清楚自由基与还原能力略低于VC，但清楚超氧阴离子自由基的能力明显高于VC，证实了黑豆花色苷具有较强的抗氧化作用。更多还原

【Abstract】 Black beans are very rich in Northern China and Northeast areas.In recent years, nutritivesubstances rich in black soybean are absorbed easy by the bodies, and promoting other nutritivesubstances absorbed by bodies and improving metabolism and enhancing anti-diseases and healthprotection effect.There has been an increased interest in the development of food cororants fromnatural sources as alternatives to synthenic.Anthocyain are a group of plant pigments that arewidely distributed in nature, anthocyanins in plants are chemical stability,safe, bright colorful, lowprice substances and are possible for use in food, nutraceutical and pharmaceutical preparations.Therefore, more and more people accepted its function.In this paper, black soybean were used as raw material, extraction with ultrasound assistanceand purification methods of black soybean anthocyanins wear studied,then purified black soybeananthocyanins were studied by the analysis of activities in vitro and in vivo. First degreasing solventwas selected, the anthocyanins content and total phenol content in extraction liquids were as theindex, the impact of different solvent was examined, and petroleum ether was as the best degreasingsolvent. An optimum procedure for the extraction of anthocyanins from black soybean was obtained,with the anthocyanins content as the index. Results show that material/liquid ratio,1:30; extractionduration,30min; ultrasonic temperature50℃; ultrasonic power,280W. Under this condition, thecontent of anthocyanin was1.42mg/g.In the separation and purification process, column chromatography provides a method ofseparating anthocyanins, followed by AB-8macroporous as the column packing. The crude extractsof anthocyanins were isolated and purified to obtain samples of high purity. According to theresearch of AB-8macroporous resin purification process, the optimum purification conditions:adsorption equilibrium time40min, desorption time70min,0.1mg/mL of the sample solutionconcentration, adsorption flow rate2.0mL/min,70%ethanol solution as eluent, desorption flow rate2.0mL/min, under this condition, purity of anthocyanin contents was about three times greater thanthat of non-purified.The effect of black soybean anthocyanins in vivo was evaluated,by establishing mice agingmodel, Ve was used as the positive control, the T-AOC,SOD and GSH-Px, MDA and protein carbonyl in serum, liver and brain were used as indicators to investigate the protect effect ofpurified black soybean anthocyanins to aging mice. The results showed that T-AOC, SOD andGSH-Px activities in the purified black soybean anthocyanin gavage group were increasedobviously than model group, while the MDA and carbonyl content of protein were decreasedsignificantly. In middle doses purified anthocyanin group, the result was similar with Ve group, andthe high group was superior to Ve group. The test showed that the anti-aging effect of black soybeanextract was obvious, so black soybean anthocyanin was a highly effective natural antioxidant.In vitro antioxidant experiments, black soybean anthocyanin the scavenging rate of the DPPHand ABTS, reducing power, superoxide anion scavenging activity were determined in order tocharacterization its antioxidant capacity. The results showed that purified black soybeananthocyanins had excellent antioxidant, the scavenging rate of DPPH was similar with VC, butABTS scavenging rate, reducing power was lower than VC, so they may be belong to slowantioxidants. The scavenging activity superoxide anion of purified black soybean anthocyanins wasstronger than VC. The results showed that the antioxidant capacity of black soybean anthocyaninwas highly effective.更多还原