【摘要】：植物甾醇因具有降低膽固醇吸收的功效，近年來備受關注，許多國家相繼開發(fā)了種類繁多的富含植物甾醇的功能性食品。然而，由于其化學結(jié)構(gòu)上含有雙鍵，植物甾醇和膽固醇都容易受到外界各種因素（如空氣、光、熱、酶等）的影響，在生產(chǎn)和儲存過程可能發(fā)生氧化，產(chǎn)生大量氧化物。已有研究表明，膽固醇氧化物與一系列有害的與磷脂代謝和細胞功能相關的人類疾病有關，并顯示出細胞毒性、致突變和致癌等性質(zhì)。植物甾醇可以經(jīng)歷與膽固醇同樣的氧化過程，生成植物甾醇氧化物，因此植物甾醇氧化物很有可能對人體健康產(chǎn)生不利影響。 目前對于這類物質(zhì)的生理活性研究卻很少，主要原因是由于目前尚無商業(yè)化的植物甾醇氧化物標準品，因此對于植物甾醇氧化物的生理效應和安全性尚無定論。 本研究以膽固醇、豆甾醇和混合植物甾醇為實驗材料，采用薄層色譜法和高效液相色譜法分析制備了植物甾醇單體和熱氧化物，采用化學合成法合成了兩類主要的甾醇氧化物，對氧化物及中間產(chǎn)物的色譜行為和化學結(jié)構(gòu)性質(zhì)進行了研究，并對氧化物的細胞毒性效應進行了初探。 高效液相色譜法是分析植物甾醇及其氧化物的主要方法之一。在本實驗中，通過對液相色譜柱和流動相的試驗，獲得了最佳分析條件：色譜柱為Agilent EclipseXDB-C8柱，流動相為乙腈/異丙醇=9:1(v/v)的混合溶劑，流速為1.0mL/min，紫外檢測波長208nm。該分析條件不僅能夠有效地分離植物甾醇混合物，，而且還能成功分離后續(xù)合成的氧化產(chǎn)物，且對各類被分析物的保留時間較短，可在化學合成過程中監(jiān)測反應進度。 為了獲得足量生理活性研究所需的植物甾醇氧化物，本研究首先分別從高純度的膽固醇和豆甾醇單體出發(fā)，化學合成了結(jié)構(gòu)為7-keto-和7β-OH-膽固醇氧化物，以及7-keto-和7β-OH-豆甾醇氧化物；然后，以一種混合植物甾醇（Generol95R）為起始物，合成了7-keto-和7β-OH-混合植物甾醇氧化物。根據(jù)薄層色譜（TLC）和高效液相色譜（HPLC）的分析結(jié)果可以發(fā)現(xiàn)，植物甾醇氧化物及其中間產(chǎn)物的保留值與膽固醇和豆甾醇同結(jié)構(gòu)的化合物相同，且洗脫順序具有相似的規(guī)律，這表明起始混合植物甾醇中的各單體以相同的速率參與了化學反應。 基于前述的分析型HPLC對7-keto-和7β-OH-植物甾醇氧化物的分析結(jié)果，我們采用同型號的半制備高效液相色譜（semi-preparative HPLC）對植物甾醇氧化物進行了分離制備，經(jīng)過循環(huán)進樣并回收餾分，獲得了四種高純度的植物甾醇氧化物單體，分別為：7-keto-菜油甾醇、7-keto-谷甾醇、7β-OH-菜油甾醇和7β-OH-谷甾醇。對這些氧化物進行了結(jié)構(gòu)表征，其中7-keto-谷甾醇和7β-OH-谷甾醇的核磁和質(zhì)譜數(shù)據(jù)均與文獻報道一致，而7-keto-菜油甾醇和7β-OH-菜油甾醇的相關結(jié)構(gòu)表征數(shù)據(jù)是首次報道。本研究提供了一種利用混合植物甾醇獲得其氧化物單體的新方法。 利用獲得的植物甾醇氧化物對人類小腸癌細胞HIC進行了初步的細胞毒性效應研究，結(jié)果表明植物甾醇氧化物具有和膽固醇氧化物相似的毒性效應，但是其毒性效應要遠低于膽固醇氧化物。
[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,
【學位授予單位】：安徽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：TQ460.1;R329.2

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