【作者】 李绮丽；

【导师】 吴卫国；

【作者基本信息】 湖南农业大学 ， 园艺产品采后科学与技术， 2013， 博士

【摘要】 莲子是睡莲科水生草本植物莲(Nelumbo nucifera Gaertn.)的成熟种子,在我国及东南亚作为传统已开拓的食材和中药材已为人所熟知。莲子的加工通常要经过干燥、分级、去壳、除皮、去芯等工序。在除皮过程中产生的莲子皮(约占莲子重量的15%)目前只是用于动物饲料,甚至是作为废弃物处理掉,造成了资源的极大浪费。近年来随着莲子种植规模的增大,其产量迅速增加,使得莲子皮的产量也随之增加。莲子皮富含丰富的原花青素,而原花青素已被证实具有抗氧化,抗肿瘤,抗心血管疾病,抗衰老等多种功能活性,且无毒性,是一种极具开发潜力的天然活性物质。本研究就是以莲子皮为原料,提取分离低聚原花青素,并对其进行结构鉴定,同时对抗氧化机理进行探讨。研究结果对于提高莲子皮的综合利用,提升莲子加工的附加值,推动莲子产业的健康发展具有积极意义。全文主要研究内容及结果如下：1、香草醛/盐酸比色法测定莲子皮原花青素方法的研究在香草醛/盐酸比色法测定葡萄和高粱中原花青素含量研究的基础上,对香草醛/盐酸比色法测定莲子皮原花青素含量的具体方法进行了研究。探讨了盐酸浓度、香草醛浓度、反应时间、反应温度、光照等因素对原花青素与香草醛显色反应的影响规律后,确定了香草醛/盐酸比色法测定莲子皮原花青素含量的最佳条件为：取0.5m1待测液,加入3m1的4%香草醛甲醇溶液,1.5m1浓盐酸,30℃水浴20min,500nm下测定吸光度值A,以儿茶素为标样绘制标准曲线Y=1.3476X+0.0166, R2=0.9996,计算待测液中原花青素的含量。结果表明,优化后的香草醛/盐酸法用于测定莲子皮中原花青素含量,其稳定性和重现性好,回收率高。2、运用丙酮溶液提取莲子皮原花青素研究利用原花青素在丙酮溶液中具有较高溶解的特性,采用丙酮溶液提取莲子皮中原花青素。本部分研究在分别探讨丙酮浓度,温度,pH值,时间,料液比等因素对莲子皮原花青素得率影响规律的基础上,运用均匀设计和支持向量机(Uniform Design and Support Vector Regression,UD-SVR)的方法对丙酮溶液提取莲子皮原花青素的技术参数优化。研究结果显示：经过两轮均匀设计试验,确定了料液比,丙酮浓度,pH值对原花青素得率的影响大于温度和时间,且原花青素提取优化的工艺条件为：丙酮浓度(v/v)为67%,料液比1：57,pH值2.7,提取温度为37℃,提取时间为90min。在此最佳条件下,莲子皮原花青素的得率为5.87%。3、莲子皮低聚原花青素的分级分离研究丙酮溶液提取获得的原花青素为不同聚合度原花青素的混合物,为了得到不同聚合度级别的原花青素,利用不同聚合度级别的原花青素极性和有机溶剂中溶解度的差异,采用乙酸乙酯溶剂萃取法与柱层析相结合的方法对丙酮溶液提取的原花青素进行分级分离。丙酮溶液提取的原花青素经乙酸乙酯溶液萃取分级,可获得萃取于乙酸乙酯相的低聚原花青素；低聚原花青素经AB-8柱大孔树脂层析分离,选择60%丙酮溶液洗脱得大孔树脂纯化物(PMR)。PMR采用聚酰胺柱做进一步分级分离,优化聚酰胺柱层析条件(上样浓度为0.48mg/ml,上样流速为16BV/h,上样量在8BV,洗脱液流速为24BV/h)后,用浓度分别为10%,30%,50%,70%的丙酮溶液依次分级洗脱,分别得到丙酮洗脱物：M1、M2、M3、M4四个洗脱组分,四个洗脱组分的原花青素回收率分别为：12.32%、19.84%、54.41%、2.30%,四个洗脱组分的原花青素纯度分别为：78.47%、84.61%、96.26%、34.15%。4、莲子皮低聚原花青素组分鉴定分析经乙酸乙酯初提的低聚原花青素组分经AB-8柱大孔树脂层析分离的大孔树脂纯化物(PMR)通过近红外光谱扫描(IR)显示：图谱表现出明显的原花青素特征骨架振动,表明其主要成分是由儿茶素或表儿茶素单元构成的原花青定聚合物。对低聚原花青素大孔树脂纯化物(PMR),聚酰胺分离组分M1,M2和M3四种级分的平均聚合度进行分析。结果显示：大孔树脂纯化物(PMR),聚酰胺分离组分M1,M2和M3等四种级分的平均聚合度分别2.12,1.41,1.62和1.84。聚酰胺分离组分M1,M2和M3三种低聚原花青素组分经液质联用(RP-HPLC-ESI-MS)检测共得出9种原花青素单体和低聚体成分(F1-F9),其中包括三种黄烷单体F1(m/z305),F4(m/z289)和F7(m/z289),四种原花青定二聚体的同分异构体F2,F3, F6, F8(m/z577)和两种原花青定四聚体的同分异构体F5和F9(m/z1153)。5、莲子皮低聚原花青素分离组分的空间结构鉴定通过对1H-NMR及13C-NMR的分析得到化合物F4确定为儿茶素单体(+)-catechin,化合物F2确定为二聚体-(-)表儿茶素-(4p→8)-+(-)儿茶素结构,即原花青素B1,化合物F6确定为二聚体-(-)表儿茶素-(4p→8)-(-)-表儿茶素结构,即原花青素B2。另通过标准品的对比分析,确定化合物F7为表儿茶素单体(-)-epicatechin,化合物F1为桔儿茶素(+)-gallocatechin,化合物F3为二聚体+(-)儿茶素-(4a→8)--(-)表儿茶素,即原花青素B4。6、莲子皮低聚原花青素的抗氧化机理研究对莲子皮低聚原花青素大孔树脂纯化物PMR,以及儿茶素单体和VC的抗氧化性进行了比较研究。结果显示：莲子皮低聚原花青素PMR的总抗氧化能力,亚油酸体系的氧化抑制能力,以及清除自由基DPPH, OH-和02-的能力都要高过VC,但都略低于儿茶素。其中,抗氧化剂对自由基DPPH的清除能力最强,羟基自由基次之,超氧阴离子自由基的清除能力最弱。另外,通过细胞试验结果发现,PMR可以有效的激活Nrf2-ARE信号通道,促进Nrf2的解离,从而加强了具有抗氧化抗炎抗肿瘤的保护基因如HO-1的转录,其中PMR在50μg/ml的浓度下激活能力最为显著。更多还原

【Abstract】 Lotus seeds are the seeds of plants in the genus Nelumbo. Xiangtan is the main area to plant Lotus seeds in China. In recent years, with the developing of planting scale, the yield of lotus seeds is increasing rapidly and annual yield can arrived at one hundred thousand tons. Due to the lotus seeds are normally sold de-peeled in the supermarket, the lotus seed peel depeeled with the machine become the by-product in the process of lotus seeds and they are either thrown or used for feeding livestock. In fact, the recent research showed the lotus seed peel contained high concentration proanthocyanidins. Hence, recovering proanthocyanidins from the lotus seed peel and applying in humans’ daily life will be a meaningful and effective way to make wealth from waste. The main research results were shown as follows:1Vanillin assay was carried out in HC1medium to determine the content of proanthocyanidins in the lotus seed peel.Several parameters affecting the precision and accuracy of vanillin assay including HC1concentration, vanillin concentration, reaction time, reaction temperature, sunlight were studied. The results showed that the proper reaction conditions were as the following:the medium comprised of0.5ml of sample,3ml of4%vanillin solution in methanol, and1.5ml of concentrated HC1in methanol, and the reaction was carried out at30℃for20min (measured at500nm). Assessment of the method by statistics proved its high stability, reproduction quality, and recovery.2Based on the uniform design and support vector regression, UD-SVR, a novel experimental design and analysis approach was applied. It was used to optimize the extraction process including five independent variables for the proanthocyanidins from the lotus seed peel. The optimization results by UD-SVR showed the proanthocyanidins yield increased from2.9%in the initial scheme and3.98%in the optimal scheme of single factor to5.87%after two rounds of uniform design and testing of36schemes. The optimal scheme was solid:liquid of1:57, acetone concentration of67%, pH value of2.7, temperature of37℃and extration time of90mins. 3In order to obtain the high purity proanthocyanidins, ethyl acetate solvent extraction combining with macroporous resin AB-8was used to purify the extraction firstly. Then polyamide chromatograph was optimized to further purify. The results showed that the suitable velocity of loading, the concentration and capacity of sample loading and the velocity of flow are16BV/h,0.48mg/ml,8BV and24BV/h, respectively. Four different acetone concentration10%,30%,50%and70%were used to elute the polyamide. The four fractions M1、M2、M3、M4were obtained, and the rocovery rate were12.32%、19.84%、54.41%、2.30%, respectively. The purity were78.47%、84.61%、96.26%、34.15%, respectively。4After eluting with10%,30%and50%acetone solvent, M1, M2, M3were obtained and further analyzed by infrared spectru, RP-HPLC and RP-HPLC-MS/MS. In conclusion, infrared spectrum confirmed that the procyanidins, consisted of catechin units, was the mainly construction in the extraction of proanthocyanidins from the lotus seed peel. Compounds were further-confirmed by RP-HPLC and RP-HPLC-MS/MS. Through the above mentioned methods, the basic components of proanthocyanidins had been concertained including the monomer flavanoid ([M-H]" m/z289) and ([M-H]" m/z305), four kinds of isomeric compounds of procyanidin dimers ([M-H]-m/z577) and two kinds of tetramers ([M-H]-m/z1153). Moreover, the MS/MS spectrum showed fragmentation of dimers all include m/z of407and289, one of tetramers include m/z of1001,865,695,575and287, and the other include865,739,575,423and287.5Compounds F2, F4and F6were further identified by NMR:compound F4was (+)-catechin, compound F2was epicatechin-(4β→8)-catechin (procyanidin B1) and compound F6was epicatechin-(4β→8)-epicatechin (procyanidin B2). In addition, compared with the standard sample, other compounds were comfirmed including F1of (+)-gallocatechin, F3of catechin-(4α→8)-epicatechin (procyanidin B4) and F7of (-)-epicatechin.6The research on the antioxidate capacity of PMR, catechin and VC showed: Firstly, total antioxidate capacity, the antioxidative capacity on linolic acid and scavenging capacity on DPPH, O2-and OH-radicals of proanthocyanidins PMR were all higher than VC, but lower than catechin. Secondly, all the antioxidants have higher scavenging capacity on DPPH·system than OH-and O2-. In addition, PMR was found to active Nrf2-ARE signal pathway and result in activation of Nrf2through its dissociation from Keapl. The expression of cytoprotective target genes like antioxidant proteins HO-1is then transactivated in response to the stress.更多还原