|Place of Conferral||北京|
|Keyword||金丝桃苷 异槲皮苷 分子印迹 苯硼酸 固相萃取|
中文摘要异槲皮苷和金丝桃苷属于黄酮类化合中黄酮醇类的苷，二者都具有多种的生理活性，广泛存在植物中。异槲皮苷和金丝桃苷的分子结构非常类似，仅仅是黄酮苷的糖基为差向异构体（葡萄糖和半乳糖），极性非常近似，因此异槲皮苷和金丝桃苷的分离纯化以及制备的提取分离，成为其开发应用的关键。分子印迹技术（MIT）是基于生物蛋白抗原抗体的“锁钥原理”发展起来，用于人为制备特异性选择性吸附材料-分子印迹聚合物（MIP）的技术。为解决分子印迹本体合成法存在操作复杂等诸多缺点，分子印迹整体柱应运而生。本文工作以异槲皮苷为模板，4-乙烯基吡啶为单体、二甲基丙烯酸乙二醇酯为交联剂、离子液/二甲亚砜/N’N-二甲基甲酰胺为三元致孔剂，偶氮二异丁腈为引发剂，混合后装入不锈钢柱管中，密封水浴60℃反应18 h，得异槲皮苷印迹整体柱。最优的合成条件是模板/单体/交联剂为1:5:20；绿色溶剂-离子液体的体积占有率为63.2–69.3%；得到最高的印迹因子为2.97。本文把异槲皮苷印迹材料和固相萃取技术结合，通过优化萃取条件，异槲皮苷印迹聚合物结合通过实验，我们得到最优化的条件为：甲醇-水混合溶液(70 : 30，v/v)作为上样试剂；甲醇-水(20 : 80, v/v) 和甲醇-水(25 : 75, v/v)作为淋洗试剂；乙腈-水混合溶液(30 : 70 v/v)为洗脱试剂；聚合物的用量为1.9g。得到的最高异槲皮苷、金丝桃苷和紫云英苷的回收率分别为87.78%, 93.26% 和 83.25%。 硼酸亲和材料可以特异性吸附顺二羟基化合物, 经过长期的探索，硼酸亲和色谱（BAC）随后获得的巨大的发展。合成的填料有以下几个特点，即可以共价键吸附含顺式二醇的化合物，通过pH调节与目标化合物的结合/释放，动力学快并且可以与质谱联用，但是硼酸亲和整体柱的制备依然是一个挑战。本文以4-乙烯基苯硼酸为聚合单体，二甲基丙烯酸乙二醇酯为交联剂、四氢呋喃/甲苯为二元致孔剂，聚苯乙烯为线性大分子致孔剂，偶氮二异丁腈为引发剂，混合后装入不锈钢柱管中，密封水浴55℃反应24 h，得苯硼酸亲和整体柱。最优的合成条件:单体/交联剂为7:3，聚苯乙烯的浓度为40 mg/mL，制备了通透性良好的苯硼酸亲和整体柱，对金丝桃苷和异槲皮苷最大的选择因子为16.7。苯硼酸材料结合固相萃取技术优化固相萃取条件，最优的处理条件：甲醇为上样试剂，甲醇和甲醇/水(3:7, v/v)为淋洗试剂，甲醇/水(3:7, pH = 4.0, v/v)作为洗脱试剂，用于分离金丝桃苷和异槲皮苷的混标，得到了高纯度的异槲皮苷和金丝桃苷(>99.96%)，回收率分别为83.7%和78.6%；用于棉花花总黄酮中，材料对棉花花总黄酮中的金丝桃苷和三叶豆苷表现出了很好的吸附分离效果。
AbstractIsoquercitrin (ISO) and hyperoside (HYP), the important flavonoids with vivo and vitro pharmaceutical and physiological activity, are enriched in various plants. Flavonoid glycosides sugar-based (glucose and galactose) of ISO and HYP are epimer which show very similar chemical structure and polarity. Therefore, it is still great scientific key for ISO and HYP purification from crude extract and separation of the two compounds in separation science.Molecular imprinting technique based on the “key-lock”principle of antigen and antibody in biological protein create the special selectively affinity polymer material-molecular imprinting polymer (MIP). To overcome the defects of bulk synthesis method, just as complex operation, molecular imprinting monolith has been developed sharply recent years. Thus in our work, imprinted monoliths were prepared by the following process: a mixture with isoquercitrin as template, 4-vinylpyridine as the functional monomer, ethylene glycol dimethacrylate as the cross-linking agent, 1-butyl-3-methylimidazoliumtetrafluoroborate/N’N-dimethylformamide/dimethyl sulfoxide as porogen, azobisisobutyronitrile as initiator was injected into njected into a stainless steel column (100 mm × 4.6 mm), then was sealed and submerged in a 60 ℃ water bath for 18h. Through the comparing research, the best protocol template/monomer/cross-linking is 1:5:20; The percentage of the green solvent (ionic liquid, IL) in the total volume of porogenic solvent account 63.2–69.3% and the highest imprinting factor (IF) was 2.97. The optimal MIPs were used as solid-phase extraction (SPE) sorbents for purification of ISO, hyperoside, and astragalin and a SPE protocol was optimized. It was found that the most suitable solvents for loading, washing and elution steps were methanol/water (70 : 30, v/v), methanol/water (20 : 80, v/v) and acetonitrile/water (30 : 70, v/v), respectively. The highest recovery rate of ISO, hyperoside, andastragalin was 87.78%, 93.26% and 83.25%, respectively, from the crude extract of cotton flowers. Boronate affinity materials have emerged as important media of molecular recognition and selective separation for cis-diol-containing compounds. Dependent on the traits, kinds of function monomer, combining with cis-diol-containing compounds in reversible covalent coordinated by pH, boronate affinity chromatography (BAC) show fast kinetic processes and also easy combined to mass spectrum (MS). In our expectation, we tried to exploit the phenylboronic acid monolithic column for separating ISO and HYP in preparation scale. However, the synthesis of benphylboronic acid monolith is still great challenge because of too much column pressure. In our work, the monolithic polymer was made using 4-vinylphenylboronic acid (4-VPBA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as crosslinker monomer, and a mixture of PS solution in tetrahydrofuran, the linear macromolecular porogen, and toluene as porogen. The monolith with good permeability and selectivity factor (highest 16.7) was achieved with monomer/cross-linking (7:3) and PS solution in tetrahy-drofuran (40 mg/mL). After optimization of the SPE protocol, it was found that when pure methanol was used as rinsing solvent to wash ISO and its analogs, the most of ingredients were still retained in the SPE column, which can be rinsed by methanol/water (3:7, v/v) further. In non-competitive SPE experiments, the high purify HYP (99.96%) and ISO (100%) were obtained with recovery of 83.7% and 78.6%, respectively. The poly(VPBA-co-EDMA) polymer combined with SPE was also applied to extract cis-diol flavonoid glycosides from the crude extraction of cotton flower. The boronate-affinity materials for SPE can remove the most of ISO and the interfering compounds, leading to the high recovery of hyperoside and trifolin.
|李相杰. 异槲皮苷印迹整体柱及苯硼酸亲和整体柱的制备及应用[D]. 北京. 中国科学院大学,2016.|
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