类黄酮分子印迹聚合物的合成及其在柑橘中识别性能的研究

发布时间：2018-08-20 08:31

【摘要】：分子印迹技术(Molecularly imprinted technique,MIT)是制备对目标分子有特异性识别能力的分子印迹聚合物(Molecularly imprinted polymer,MIP)的实验室技术。分子印迹聚合物具有高特异性、高选择性、高强度等特点,而且制备过程简单、成本低、可重复利用、耐受高温、高压、酸碱和有机溶剂。目前主要应用于目标分子的分离识别、对映体的分离、临床药物分析、催化剂和吸附剂等方面。已在生物工程、食品工业、临床医学、环境监测等行业形成产业化的应用,尤其分子印迹固相萃取(MISPE)技术在食品营养功能成分的分离、富集和纯化等方面有广泛的应用。本研究以柑橘中具有营养价值的类黄酮(FLDS)为研究对象,进行了类黄酮分子印迹聚合物的制备以及一系列相关的研究。本文综述了柑橘中类黄酮的分离富集技术、分子印迹聚合物的制备、分子印迹聚合物的表征以及分子印迹固相萃取技术。本研究利用分子印迹技术的原理,以单一类黄酮为模板分子,优化了制备条件,得到具有类特异性吸附、高选择性的分子印迹聚合物微球,采用紫外光谱(UV)、傅里叶红外光谱(FTIR)、扫描电镜(SEM)等手段对聚合物进行表征。用聚合物微球做填充材料制备了分子印迹固相萃取柱,利用其类特异性吸附的特点来分离和富集结构类似的一类物质,实现了柑橘复杂基质中类黄酮的高效分离与净化。本研究的主要成果和结论:1.采用分子印迹技术,以橙皮素(HET)为模板分子,2-乙烯基吡啶(2-VP)为功能单体,乙二醇二甲基丙烯酸酯(EGDMA)为交联剂,乙腈为致孔剂,沉淀聚合法制备了具有类特异性识别能力的橙皮素分子印迹聚合物。用紫外光谱确定了功能单体的种类、模板分子与功能单体的配比。分别测定了不同交联剂用量和致孔剂用量下制备的印迹聚合物的静态吸附性能,以此确定了交联剂用量和致孔剂的用量。实验结果表明,当模板分子、功能单体、交联剂的比例为1:6:20时,致孔剂乙腈用量为50 m L时,得到的分子印迹聚合物具有很好的吸附性能。在此基础上用傅里叶红外(FTIR)、扫描电镜(SEM)等手段对分子印迹聚合物进行表征。通过静态吸附性能和Scatchard分析,研究了分子印迹聚合物的平衡吸附性能,试验结果表明MIP有两类不同类型的结合位点:特异性结合位点和非特异性结合位点。MIP所对应的K_(d1)=0.243μmol/m L,Q_(max1)=143.61μmol/g;K_(d2)=3.88μmol/m L,Q_(max2)=5.29μmol/g。而NIP线性关系较好,说明NIP只有一种非特异性结合位点,其所对应的K_d=0.58μmol/m L,Q_(max)=30.86μmol/g。2.实验评价了橙皮素分子印迹聚合物的选择性,实验结果表明印迹聚合物对四种黄烷酮印迹因子分别为1.40,1.39,1.59和2.89。以橙皮素分子印迹聚合物为填充料制备了分子印迹固相萃取小柱,筛选和优化了固相萃取条件,建立了基于MISPE技术快速分离和富集四种黄烷酮(FLES)的高效液相色谱方法,研究了分子印迹聚合物固相萃取柱的实用性,实现了枳实中柚皮苷(NGIN)、橙皮苷(HES)、柚皮素(NGEN)、橙皮素(HET)的分离和富集,该方法在2~120 mg/L浓度范围内具有良好的线性关系(R~2≥0.9991),检出限(LOD)范围0.2~0.5 mg/L。分子印迹固相萃取柱(MISPE)对四种黄烷酮的提取率大小顺序为:NGENHETNGINHES,检测出枳实中四种黄烷酮的含量分别为0.075 mg/g(NGIN),7.55 mg/g(HES),0.49mg/g(NGEN),0.29 mg/g(HET),说明MIP对四种黄烷酮有较好的识别能力和类特异性吸附性能,可以很好地应用于枳实中四种黄烷酮的分离与富集。3.以桔皮素为模板分子,丙烯酰胺(AM)为功能单体,乙二醇二甲基丙烯酸酯(EGDMA)为交联剂,乙腈为致孔剂,沉淀聚合法制备了桔皮素分子印迹聚合物,并优化了合成条件。采用傅里叶红外光谱(FTIR)和扫描电镜(SEM)等手段对分子印迹聚合物进行表征。通过静态吸附性能实验来测定聚合物对三种多甲氧基黄酮的吸附性能,结果表明MIP对甜橙黄酮(SNT)、川皮苷(NBT)和桔皮素(TNG)具有较好的吸附性能,最大吸附量分别为6.67μmol/g,6.34μmol/g,7.19μmol/g。以伞形花内酯(7-HC)为参照,评价了MIP对三种多甲氧基黄酮(PMFS)的选择性,结果显示MIP对甜橙黄酮、川皮苷、桔皮素和伞形花内酯的印迹因子IF分别为1.56、1.83、1.46、0.96。将印迹聚合物微球作为色谱填料制备分子印迹固相萃取柱,用于分离和富集柑橘果皮中三种多甲氧基黄酮,评价了分子印迹固相柱的实用性和选择性。建立了甜橙黄酮、川皮苷、桔皮素的分子印迹聚合物-高效液相色谱检测方法,实现了柑橘中三种多甲氧基黄酮的快速分离和高效富集,该方法在2~120 mg/L浓度范围内具有良好的线性关系,相关系数(R~2)皆为0.9999,检出限为0.2~0.5 mg/L,RSD≤1.9%。该固相萃取柱对三种多甲氧基黄酮检测含量分别为0.49 mg/g(SNT),1.13 mg/g(NBT),0.82 mg/g(TNG),说明该固相萃取柱对三种多甲氧基黄酮具有较好的识别能力和选择性,分离效果良好,有效去除了样品中杂质的干扰,大大提高了柑橘果皮中的三种多甲氧基黄酮的萃取效率。
[Abstract]:Molecularly imprinted technology (MIT) is a laboratory technology for the preparation of molecularly imprinted polymer (MIP) with specific recognition ability to target molecules. Utilization, tolerance to high temperatures, high pressures, acids, bases and organic solvents. Currently, it is mainly used in the separation and identification of target molecules, enantiomer separation, clinical drug analysis, catalysts and adsorbents. It has been industrialized in bioengineering, food industry, clinical medicine, environmental monitoring and other industries, especially molecular imprinting solid phase extraction (MISPE) technology. Atractylodes molecularly imprinted polymers of flavonoids (FLDS) with nutritional value in citrus were prepared and a series of related studies were carried out. The separation and enrichment techniques and molecular imprinting of flavonoids in citrus were reviewed in this paper. The preparation of imprinted polymers, characterization of molecularly imprinted polymers and molecular imprinting solid phase extraction (MISPE) were studied. Based on the principle of molecularly imprinted technology, single flavonoid was used as template molecule to optimize the preparation conditions. Molecularly imprinted polymer microspheres with class-specific adsorption and high selectivity were prepared. Molecularly imprinted solid phase extraction column was prepared by using polymer microspheres as filling materials. A class of substances with similar structure were separated and enriched by using their specific adsorption characteristics. The high efficiency separation and purification of flavonoids in Citrus complex matrix was achieved. The main results and conclusions are as follows: 1. Hesperidin-like molecular imprinted polymers with specific recognition ability were prepared by molecular imprinting technique, using hesperidin (HET) as template molecule, 2-vinylpyridine (2-VP) as functional monomer, ethylene glycol dimethacrylate (EGDMA) as crosslinking agent and acetonitrile as porogen. The types of functional monomers and the ratio of template molecules to functional monomers were determined. The static adsorption properties of the imprinted polymers were measured under different dosages of cross-linking agents and porogens, and the dosages of cross-linking agents and porogens were determined. Molecularly imprinted polymers with 50 m L acetonitrile were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The equilibrium adsorption properties of the molecularly imprinted polymers were studied by static adsorption and Catchard analysis. The experimental results were presented. MIP has two different types of binding sites: specific binding sites and nonspecific binding sites. The corresponding K_ (d1) = 0.243 micromol/m L, Q_ (max1) = 143.61 micromol/g, K_ (d2) = 3.88 micromol/m L, Q_ (max2) = 5.29 micromol/g. However, NIP has a good linear relationship, indicating that there is only one non-specific binding site, and the corresponding K_d = 0.58 micromol/m, Q_ (d2) = 3.88 micromol/g. _ (max) = 30.86 micromol / g.2. The selectivity of hesperidin molecularly imprinted polymers was evaluated. The results showed that the imprinted polymers had 1.40, 1.39, 1.59 and 2.89 flavanone imprinting factors, respectively. A high performance liquid chromatography (HPLC) method based on MISPE for rapid separation and enrichment of four flavanones (FLES) was established. The practicability of molecularly imprinted polymer solid phase extraction column was studied. The separation and enrichment of naringin (NGIN), hesperidin (HES), naringin (NGEN) and hesperidin (HET) in Fructus Aurantii Immaturus were realized. The method had good performance in the range of 2-120 mg/L. The linear relationship (R~2 < 0.9991) and the detection limit (LOD) ranged from 0.2 mg/L to 0.5 mg/L. The order of extraction rates of four flavanones from Fructus Aurantii by molecularly imprinted solid phase extraction column (MISPE) was NGENHE TNGINHES. The contents of four flavanones in Fructus Aurantii were 0.075 mg/g (NGIN), 7.55 mg/g (HES), 0.49 mg/g (NGEN) and 0.29 mg/g (HET), respectively. Hesperidin molecularly imprinted polycondensation was prepared by precipitation polymerization using hesperidin as template molecule, acrylamide (AM) as functional monomer, ethylene glycol Dimethylacrylate (EGDMA) as crosslinking agent and acetonitrile as porogen. The molecularly imprinted polymers were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The adsorption properties of the polymers for three polymethoxy flavonoids were determined by static adsorption experiments. The results showed that MIP had better adsorption properties for SNT, NBT and TNG. The maximum adsorption capacity of MIP was 6.67, 6.34 and 7.19 micromol/g, respectively. The selectivity of MIP to three polymethoxy flavonoids (PMFS) was evaluated with umbelliferol (7-HC). The results showed that the imprinting factors IF of MIP to orange flavonoids, kaempferide, hesperidin and umbelliferolide were 1.56, 1.83, 1.46, 0.96, respectively. Compound microspheres were used as chromatographic fillers to prepare molecularly imprinted solid phase extraction column for separation and enrichment of three polymethoxy flavonoids from citrus peel. The practicability and selectivity of molecularly imprinted solid phase column were evaluated. Rapid separation and high-efficiency enrichment of polymethoxy flavonoids showed that the method had a good linear relationship in the range of 2-120 mg/L. The correlation coefficients (R~2) were 0.9999, the detection limits were 0.2-0.5 mg/L and RSD was less than 1.9%. The detection contents of three polymethoxy flavonoids were 0.49 mg/g (SNT), 1.13 mg/g (NBT) and 0.82 mg/g (TNG), respectively. The results showed that the solid phase extraction column had good recognition ability and selectivity for three polymethoxy flavonoids, and the separation effect was good. The interference of impurities in the sample was effectively removed, and the extraction efficiency of three polymethoxy flavonoids in citrus peel was greatly improved.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O631.3;TS255.1

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