植物甾醇及其酯化衍生物的分析测试研究

【作者】 沙鸥；

【导师】 许文林；

【作者基本信息】 扬州大学 ， 物理化学， 2004， 硕士

【摘要】 植物甾醇是一种重要的天然活性物质，主要来源于植物油精炼下脚料，特别是脱臭馏出物中。由于其特有的生理功能及物化性质，被广泛地应用于医药、食品及化工行业。本文选择了植物甾醇及其酯化衍生物的分析测试为研究体系，分别采用红外光谱法、紫外可见分光光度法，差示扫描热分析法和反相高效液相色谱法对植物甾醇及其酯化衍生物进行了表征和分析测定，旨在建立一种简单、快速、准确的分析测试方法，为植物甾醇的分离与精制过程研究和植物甾醇酯化反应过程研究提供分析测试的理论依据和技术参数。 1、植物甾醇及其酯化衍生物的紫外可见分光光度法分析测定 利用3-羟基甾族化合物与Deniges磺基醋酸汞试剂发生显色反应、产生紫外吸收的原理，测定了混合植物甾醇中豆甾醇、β-谷甾醇与Deniges磺基醋酸汞络合物的紫外光谱，探讨了显色反应时间、显色剂用量及浓硫酸与冰醋酸配比对紫外吸收光谱的影响。结果表明，在一定浓度范围内，甾醇络合物的吸光度A与其浓度成线性关系，并利用紫外光谱法测定了混合植物甾醇中豆甾醇和β-谷甾醇的含量，为混合植物甾醇中单组分的测定提供了一种准确可信的分析测试方法，采用该法测定结果与用液相色谱法测定结果相近。同时采用紫外分光光度法对甾醇酯化衍生物的固体样品进行了分析测试，为液相色谱紫外检测提供了检测波长和各物质在该检测波长下的摩尔吸光系数。 2、植物甾醇及其酯化衍生物的高效液相色谱法分析测试 (1)植物甾醇中豆甾醇和β-谷甾醇的高效液相色谱法分析测定 应用反相高效液相色谱对混合植物甾醇中豆甾醇和β-谷甾醇的含量以及重结晶过程中获得的植物甾醇样品进行了分析测定。实验采用的色谱柱为Hypersil ODS反相柱(4.6×150mm，5μm)，流动相为色谱纯甲醇，紫外检测波长为210nm，恒溶剂洗脱，混合植物甾醇在12分钟内得到了很好的分离。实验结果表明，豆甾醇、扬州大学硕士学位毕业论文尽谷幽醇在色谱上均有响应并达到基线分离;采用面积归一法和外标法均可为植物幽醇中豆幽醇、刀一谷幽醇的含量进行分析测定，为植物幽醇重结晶过程中单组分含量的测定建立了一种快速准确的定量分析方法。 (2)植物幽醇乙酞化过程中的高效液相色谱法分析测试 应用反相高效液相色谱对植物幽醇乙酞化过程中取样样品和乙酞化产物进行了分析测定。实验采用HyPersil ODs反相柱(4.6 x 1 50~，5“m)为色谱分析柱，色谱纯甲醇为流动相，紫外检测波长为21OIun，恒溶剂洗脱的操作条件。实验结果表明，采用该法可对植物幽醇及其乙酞化物同时直接进样分析，克服了气相色谱法不能直接对出醇及其乙酞化物同时分析、侧定植物幽醇样品时需衍生化预处理的缺点;该法测定植物幽醇及其乙酸酷的响应时间小于25min，且各种出醇及其乙酸酷的分离效果较好;采用反相高效液相色谱法能够快速准确地对植物幽醇乙酸化反应进行的程度及乙酞化产物中豆出醇乙酸醋、刀一谷凿醇乙酸醋的含量进行分析测定。(3)植物幽醇唬拍酸醉单酷表征及玻拍酸配单酷化过程中的高效液相色谱法分析测试 分别采用反相高效液相色谱法、红外光谱法、紫外可见分光光度法及差示扫描热分析法对植物幽醇中豆幽醇、刀一谷幽醇与玻拍酸醉反应生成的醋化产物进行了分析表征并对豆幽醇、刀一谷出醇分别与玻泊酸醉反应生成幽醇玻拍酸单醋过程进行了分析测定。分析测定结果表明，在实验条件下豆幽醇、尽谷幽醇分别与玻拍酸醉反应生成产物为幽醇唬拍酸单醋;豆幽醇玻拍酸单酷和尽谷幽醇玻拍酸单酷的熔点分别为154.8℃和131.3℃左右:豆幽醇、刀一谷幽醇及其相应唬拍酸单醋在色谱上均有响应并达到基线分离;采用面积归一法能够快速准确地对植物幽醇唬拍酸配醋化反应进行的程度及酷化产物的含量进行分析测定。 本研究的创新之处体现在:(1)由于植物幽醇结构的特殊性，将红外光谱法和紫外分光光度法用于混和植物幽醇中单组分的定性与定量分析，为单组分幽醇含量的测定提供了准确可行的分析测试方法，通过该法可以完成对植物街醇不同要求的分析。(2)由于本实验体系中植物幽醇及其醋化产物结构的特殊性，在液沙鸥;植物幽醇及其醋化衍生物的分析测试相色谱定量分析上可应用面积归一化法对混合植物幽醇及其酷化产物中各组分含量进行分析测定。采用紫外检测器对待分析样经色谱柱分离后的单组分进行检测，在色谱设定操作条件下，待测物在检测波长下具有近似相等的摩尔吸光系数，公式X，%一(A‘/艺A，)‘100%成立，且实验结果与理论推导一致，该理论为反应与结晶分离相结合分离精制植物幽醇的研究提供了一种快速准确的分析测定方法。(3)应用反相高效液相色谱法、红外光谱法、紫外可见分光光度法及差示扫描热分析法对植物幽醇与唬拍酸醉反应生成唬拍酸单醋进行了研究和表征，使植物幽醇的乳化性通过对幽核上经基基团进行化学改性而得到改善，获得具有良好体内抗癌活性的植物幽醇墟拍酸醋化衍生物。更多还原

【Abstract】 Phytosterols are an important group of naturally occurring substances derived from waste oil of refining vegetable oil, especially from deoderizer distillate. Since phytosterol are known to have a wide range of biological activility and physical properties, they are useful steroids for food industry, pharmaceutical trade and chemical industry. In this research phytosterols and phytosterol esterified derivatives were characterized, analyzed and deteriminated by the UV-vis spectral method, FT-IR method, DSC method and HPLC method respectively. The experimental result could offer the theoretic basic and the technological parameter for the study of recrystallization process and esterification process of phytosterol.1. Determination of the content of yff-sitosterol and stigmasterol in phytosterols by UV -vis spectral methodThe UV-vis spectra of the complex compound of -sitosterol or stigmasterol with Deniges reagent was measured. The effects of the time for color development reaction, the quantity of colored indicator, and the volume ratio of H2SO4 and HAC on the UV-vis spectra were studied. The experimental results show that the UV spectra of these two complex compounds are different, and there exists a linear relationship between absorption A and -sitosterol or stigmasterol content in a certain content range. There is a good agreement of the results obtained by UV-vis spectral and HPLC methods. The solid sample of phytosterols and their esters were also studied by the UV-vis spectral method. The detection wavelength for the analysis of HPLC and the molar extinction coefficient were offered by the UV-vis spectral method.2. The analysis and determination of phytosterols and their esterified derivatives by HPLC method(1) HPLC analysis of stigmasterol and -sitosterol in phytosterolsStigmasterol and yft-Sitosterol, as well as mixed phytosterols purified by recrystallization, were analyzed by HPLC. A reverse-phase column Hypersil ODS (4.6 X150mm,5 u m) was used for chromatographic separation under isocratic conditions with methanol as the mobile phase at flow-rate of 0.7 mL/min. The detection wavelength was set at 210nm. The experimental results indicated that Stigmasterol and. -sitosterol in phytosterols could be separated well within 12 minutes. External standard method and peak area normalization method could be used to determinate the contents of -sitosterol and Stigmasterol accurately and efficiently.(2) HPLC determination of phytosterols and their acetatesDetermination of phytosterol acetates and phytosterols in the esterification process of phytosterols were studied by a RP-HPLC method. Chromatographic separation was carried out using a Hypersil ODS column under isocratic conditions with methanol as the motile phase at a flow-rate of 0.7 ml/min. The operative wavelength was set at 210nm and phytosterol acetates could be separated well within 25 minutes. With this method phytosterols and phytosterol acetates can be analyzed directly at the same time while phytosterols must go through the derivatization pretreatment when GC method was used. The experimental results showed that the phytosterols and their acetates in the esterification process could be analyzed accurately and quickly by the peak area normalization method.(3) Characterization, analysis and determination in the reaction process of phytosterol with succinic anhydride by RP-HPLCThe reaction products were characterized by DSC, FT-IR, UV-vis and RP-HPLC in the reaction process of Stigmasterol, -sitosterol with succinic anhydride. It was confirmed that Stigmasterol hemisuccinate and -sitosterol hemisuccinate were the reaction products and the melt points were 154.8 C and 131.3 C accordingly under the experimental conditions. And RP-HPLC was studied to analyze and determinate the content of phytosterols and their esterified products in the reaction process ofstigmasterol and -sitosterol with succinic anhydride accordingly. Chromatographic separation was carried out using a Hypersil ODS column under isocratic conditions with methanol as t更多还原