植物甾醇氧化物的制备及其细胞毒性的初探
发布时间：2018-11-05 06:50
【摘要】：植物甾醇因具有降低胆固醇吸收的功效，近年来备受关注，许多国家相继开发了种类繁多的富含植物甾醇的功能性食品。然而，由于其化学结构上含有双键，植物甾醇和胆固醇都容易受到外界各种因素（如空气、光、热、酶等）的影响，在生产和储存过程可能发生氧化，产生大量氧化物。已有研究表明，胆固醇氧化物与一系列有害的与磷脂代谢和细胞功能相关的人类疾病有关，并显示出细胞毒性、致突变和致癌等性质。植物甾醇可以经历与胆固醇同样的氧化过程，生成植物甾醇氧化物，因此植物甾醇氧化物很有可能对人体健康产生不利影响。 目前对于这类物质的生理活性研究却很少，主要原因是由于目前尚无商业化的植物甾醇氧化物标准品，因此对于植物甾醇氧化物的生理效应和安全性尚无定论。 本研究以胆固醇、豆甾醇和混合植物甾醇为实验材料，采用薄层色谱法和高效液相色谱法分析制备了植物甾醇单体和热氧化物，采用化学合成法合成了两类主要的甾醇氧化物，对氧化物及中间产物的色谱行为和化学结构性质进行了研究，并对氧化物的细胞毒性效应进行了初探。 高效液相色谱法是分析植物甾醇及其氧化物的主要方法之一。在本实验中，通过对液相色谱柱和流动相的试验，获得了最佳分析条件：色谱柱为Agilent EclipseXDB-C8柱，流动相为乙腈/异丙醇=9:1(v/v)的混合溶剂，流速为1.0mL/min，紫外检测波长208nm。该分析条件不仅能够有效地分离植物甾醇混合物，，而且还能成功分离后续合成的氧化产物，且对各类被分析物的保留时间较短，可在化学合成过程中监测反应进度。 为了获得足量生理活性研究所需的植物甾醇氧化物，本研究首先分别从高纯度的胆固醇和豆甾醇单体出发，化学合成了结构为7-keto-和7β-OH-胆固醇氧化物，以及7-keto-和7β-OH-豆甾醇氧化物；然后，以一种混合植物甾醇（Generol95R）为起始物，合成了7-keto-和7β-OH-混合植物甾醇氧化物。根据薄层色谱（TLC）和高效液相色谱（HPLC）的分析结果可以发现，植物甾醇氧化物及其中间产物的保留值与胆固醇和豆甾醇同结构的化合物相同，且洗脱顺序具有相似的规律，这表明起始混合植物甾醇中的各单体以相同的速率参与了化学反应。 基于前述的分析型HPLC对7-keto-和7β-OH-植物甾醇氧化物的分析结果，我们采用同型号的半制备高效液相色谱（semi-preparative HPLC）对植物甾醇氧化物进行了分离制备，经过循环进样并回收馏分，获得了四种高纯度的植物甾醇氧化物单体，分别为：7-keto-菜油甾醇、7-keto-谷甾醇、7β-OH-菜油甾醇和7β-OH-谷甾醇。对这些氧化物进行了结构表征，其中7-keto-谷甾醇和7β-OH-谷甾醇的核磁和质谱数据均与文献报道一致，而7-keto-菜油甾醇和7β-OH-菜油甾醇的相关结构表征数据是首次报道。本研究提供了一种利用混合植物甾醇获得其氧化物单体的新方法。 利用获得的植物甾醇氧化物对人类小肠癌细胞HIC进行了初步的细胞毒性效应研究，结果表明植物甾醇氧化物具有和胆固醇氧化物相似的毒性效应，但是其毒性效应要远低于胆固醇氧化物。
[Abstract]:Because of its effect of reducing the absorption of cholesterol, plants have been attracted much attention in recent years. Many countries have developed a wide variety of functional foods rich in vegetable oil. However, since the chemical structure contains double bonds, both phytohormones and cholesterol are susceptible to external factors such as air, light, heat, enzymes, and the like, and oxidation may occur in the production and storage processes to produce a large amount of oxide. Studies have shown that cholesterol oxides are associated with a series of harmful human diseases associated with phospholipid metabolism and cellular function, and show the properties of cytotoxicity, mutation, and carcinogenicity. The plant carbon dioxide can undergo the same oxidation process as cholesterol to generate plant oxygenic oxide, so it is very likely that plant parasitic oxide may have adverse effects on human health. At present, there is little research on the physiological activities of these substances, mainly due to the fact that there is no commercial plant oxycarbide standard at present, so there is no physiological effect and safety for phytochrome oxide. In this study, the experimental materials were prepared by TLC and high performance liquid chromatography. Two kinds of main compounds were synthesized by chemical synthesis. The chromatographic behavior and chemical structure properties of oxides and intermediate products were studied. The cytotoxicity of oxides was also studied. A preliminary study was carried out. High performance liquid chromatography (HPLC) was used to analyze plant growth rate and its oxidation. In this experiment, the optimal analysis conditions were obtained by testing liquid chromatography column and mobile phase: column was Agilent ElipseXDB-C8 column, mobile phase was acetonitrile/ isopropanol = 9: 1 (v/ v) mixed solvent, flow rate was 1. 0mL/ min, UV detection. The test wavelength is 208nm. The analysis condition not only can effectively separate the plant oil mixture, but also can successfully separate the oxidation products of subsequent synthesis, and the retention time of various analytes can be relatively short, and can be chemically synthesized. In order to obtain a sufficient amount of phytochrome oxide needed for the Institute of Biological Activities, the study first proceeds from high-purity cholesterol and soy-derived monomers, chemically synthesizing 7-keto-and 7-keto-OH-cholesterol oxides, as well as 7-keto-and 7-and 7-keto-and 7-keto-OH-cholesterol oxides, respectively. A mixture of 7-keto-and 7-keto-O was synthesized using a mixed plant extract (Generol95R) as the starter. H-mixed plant oxytetracycline. According to the analysis results of thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC), it can be found that the retention value of phytochrome oxide and its intermediate product is the same as that of the structure of cholesterol and beans, and The elution sequence has a similar pattern, indicating that the monomers in the initial mixed plant have a phase The same rate was involved in the chemical reaction. Based on the analysis results of the aforementioned analytical HPLC on 7-keto-and 7-keto-OH-plants, we used the same model semi-preparative high performance liquid chromatography (CEi-prep HPLC) to separate the phytochrome oxide. The preparation method comprises the following steps of: circulating a sample and recovering a fraction, and obtaining four high-purity phytochrome oxide monomers, namely, 7-keto-rapeseed oil, 7-keto-glutamic acid, 7-keto-OH-The results showed that the nuclear magnetic and mass spectra of 7-keto-cereal oil and 7-keto-OH-glutamate were consistent with the literature, while 7-keto-rapeseed oil and 7-keto-OH-rapeseed oil were all consistent with the literature. The related structural characterization data is first reported. This study provides a hybrid plant A new method for obtaining the oxide monomer of the human intestinal cancer cell is carried out by using the obtained plant oxytetracycline oxide, and the results show that the plant oxytetracycline has a similar toxicity effect to the cholesterol oxide,
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：TQ460.1;R329.2
[Abstract]:Because of its effect of reducing the absorption of cholesterol, plants have been attracted much attention in recent years. Many countries have developed a wide variety of functional foods rich in vegetable oil. However, since the chemical structure contains double bonds, both phytohormones and cholesterol are susceptible to external factors such as air, light, heat, enzymes, and the like, and oxidation may occur in the production and storage processes to produce a large amount of oxide. Studies have shown that cholesterol oxides are associated with a series of harmful human diseases associated with phospholipid metabolism and cellular function, and show the properties of cytotoxicity, mutation, and carcinogenicity. The plant carbon dioxide can undergo the same oxidation process as cholesterol to generate plant oxygenic oxide, so it is very likely that plant parasitic oxide may have adverse effects on human health. At present, there is little research on the physiological activities of these substances, mainly due to the fact that there is no commercial plant oxycarbide standard at present, so there is no physiological effect and safety for phytochrome oxide. In this study, the experimental materials were prepared by TLC and high performance liquid chromatography. Two kinds of main compounds were synthesized by chemical synthesis. The chromatographic behavior and chemical structure properties of oxides and intermediate products were studied. The cytotoxicity of oxides was also studied. A preliminary study was carried out. High performance liquid chromatography (HPLC) was used to analyze plant growth rate and its oxidation. In this experiment, the optimal analysis conditions were obtained by testing liquid chromatography column and mobile phase: column was Agilent ElipseXDB-C8 column, mobile phase was acetonitrile/ isopropanol = 9: 1 (v/ v) mixed solvent, flow rate was 1. 0mL/ min, UV detection. The test wavelength is 208nm. The analysis condition not only can effectively separate the plant oil mixture, but also can successfully separate the oxidation products of subsequent synthesis, and the retention time of various analytes can be relatively short, and can be chemically synthesized. In order to obtain a sufficient amount of phytochrome oxide needed for the Institute of Biological Activities, the study first proceeds from high-purity cholesterol and soy-derived monomers, chemically synthesizing 7-keto-and 7-keto-OH-cholesterol oxides, as well as 7-keto-and 7-and 7-keto-and 7-keto-OH-cholesterol oxides, respectively. A mixture of 7-keto-and 7-keto-O was synthesized using a mixed plant extract (Generol95R) as the starter. H-mixed plant oxytetracycline. According to the analysis results of thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC), it can be found that the retention value of phytochrome oxide and its intermediate product is the same as that of the structure of cholesterol and beans, and The elution sequence has a similar pattern, indicating that the monomers in the initial mixed plant have a phase The same rate was involved in the chemical reaction. Based on the analysis results of the aforementioned analytical HPLC on 7-keto-and 7-keto-OH-plants, we used the same model semi-preparative high performance liquid chromatography (CEi-prep HPLC) to separate the phytochrome oxide. The preparation method comprises the following steps of: circulating a sample and recovering a fraction, and obtaining four high-purity phytochrome oxide monomers, namely, 7-keto-rapeseed oil, 7-keto-glutamic acid, 7-keto-OH-The results showed that the nuclear magnetic and mass spectra of 7-keto-cereal oil and 7-keto-OH-glutamate were consistent with the literature, while 7-keto-rapeseed oil and 7-keto-OH-rapeseed oil were all consistent with the literature. The related structural characterization data is first reported. This study provides a hybrid plant A new method for obtaining the oxide monomer of the human intestinal cancer cell is carried out by using the obtained plant oxytetracycline oxide, and the results show that the plant oxytetracycline has a similar toxicity effect to the cholesterol oxide,
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：TQ460.1;R329.2
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