【作者】 於将林；

【导师】 李学刚； 李宗澧；

【作者基本信息】 西南大学 ， 药用资源化学， 2007， 硕士

【摘要】 异黄酮类化合物是植物次生代谢的产物，在常见植物中，发现含有异黄酮的植物主要是大豆，葛根，红车轴草等。其中，研究最多最深入的是对豆科植物的大豆中的异黄酮的研究。纯大豆异黄酮的熔点大都在100℃以上，呈黄白色粉末状，有轻微苦涩味，易溶于丙酮、乙醇、甲醇、乙酸乙酯等极性溶剂中，微溶于水。异黄酮具有多种生理功能，其具有抗肿瘤作用、弱雌激素和抗雌激素作用、对心血管作用、对骨代谢的作用，另外还具有抗氧化作用、提高非特异性免疫功能及抗炎等作用。因此，目前对其各种生理活性功能的较非常活跃。目前，从植物中提取异黄酮的工艺路线主要是：提取—分离—纯化—测定。提取方法主要有溶剂法、超声波法等，而以传统的溶剂法为主。分离蛋白质则主要有等电点法、离心法、冷冻—等电点—离心法、乙醇沉淀法、超滤法等方法，纯化则主要有溶剂萃取法、超滤法、树脂吸附法和柱层析法等，测定的方法则有紫外分光光度法(UV)、薄层色谱法(TLC)、高效液相色谱法(HPLC)、气相色谱法(GC)、高效液相色谱一质谱法(HPLC-MS)等。本论文通过选择资料最常用的溶剂法对菜籽饼中异黄酮进行提取，并通过薄层色谱及高效液相色谱证明菜籽饼中是否含有异黄酮及含有的种类及含量。在此基础上，以高效液相色谱为标准方法，比较不同的紫外波长(254nm、259nm、260nm、262nm、三波长)下紫外比色的准确性及可靠性，选择一种快速简便的测定异黄酮的方法；通过比较不同的提取条件(溶剂、浓度、温度、时间、料液比)，对提取工艺技术进行优化；通过比较等电点法、离心法、冷冻—等电点—离心法等几种方法，选择一种最佳的分离蛋白的工艺技术；通过比较乙酸乙酯萃取法和树脂吸附法，选择最佳的纯化方法；并对所提纯的异黄酮进行活性研究。其主要实验结果如下：(1)本文发现油菜饼中含有异黄酮类化合物为染料木素(genistin)，油菜饼中染料木素的含量范围为0.100％—0.290％。(2)对异黄酮快速测定方法的研究表明：用紫外比色法测定油菜饼中的异黄酮的含量时，其最佳的比色波长为259nm，其校正系数为1.67，不能用三波长紫外比色法测定油菜饼粗提取物中的异黄酮含量。(3)通过比较不同溶剂(甲醇、乙醇)、不同的提取时间(1h、2h、3h)、不同的提取次数(1、2、3)、不同的提取浓度(50％、60％、70％、80％)、不同的料液比(1：5、1：10、1：15、1：20)、不同的提取温度(50℃、60℃、70℃、80℃)的异黄酮得率及杂质含量，发现用溶剂法提取油菜饼中异黄酮类物质的最佳工艺条件为：用乙醇做为溶剂，在浓度为60％的情况下，温度为60℃，料液比为1：15，提取时间为3h，提取2次。这与其他研究者的结果较为一致。(4)通过等电点沉淀法、离心法、冷冻—等电点—离心法三种方法的比较，认为最佳的分离蛋白的方法为：先在0℃下冷冻24h，再用盐酸调节pH值约在3—4之间，后在2000r／min条件下，离心20min。(5)乙酸乙酯萃取法、大孔树脂吸附法两种方法比较表明：用大孔树脂吸附法纯化提取物的效果较用乙酸乙酯萃取法为佳，纯化后的干物质中染料木素的含量最高可达77.7％。并且提出了异黄酮的的提取技术路线为：乙醇提取——冷冻—等电点—离心法分离蛋白质——大孔树脂纯化浓缩——测定。(6)对所提取菜籽饼的异黄酮的抗氧化活性能力的研究表明：染料木素能显著降低脂类物质的过氧化物(POV)和硫代巴比妥酸(TBA)值，对脂类物质的自动氧化具有明显的抑制作用。更多还原

【Abstract】 Isoflavones are the plant secondary metabolic products, they contain isoflavones which are mainly soybeans, Gegen and Trifolium pratense in the common plants. Within this total, the largest and most in-depth study is the soybean isoflavone research in legumes. The melting point of most of pure isoflavones is above 100℃, with the yellow-white colour, and it tastes bitterness slightly. The isoflavones can dissolved in acetone, Ethanol, methanol, ethyl acetate and other polar solvents, but they cann’t dissolved in petroleum ether, n-hexane and other non-polar solvents. Isoflavones have a variety of physiological functions,which are anti-tumor effect, the weak estrogen and anti-estrogen effect, the cardiovascular effect, the bone metabolism effect, and it also has anti-oxidation, enhancing non-specific immune function and anti-inflammatory effects. So it’s a hot research in the physiological function of isoflavones.At the present time, the main routes of extracting isoflavones from plants is that: extraction—separating protein—purification—determination. The main extraction is using solvent, ultrasonic method.The main separating protein is isoelectric point method, centrifugation and freeze-isoelectr -ic point-centrifugation, ethanol precipitation, ultrafiltration method and so on. Purification mainly contains solvent extraction, ultrafiltration, resin adsorption and column chromatography. the meth -ods of Determination are UV spectrophotometry (UV), thin layer chromatography (TLC), High performance liquid chromatography (HPLC), gas chromatography (GC), HPLC a mass spectrum -etry (HPLC-MS)..In this paper, it chooses the most commonly method for rapeseed’s isoflavones extraction by solvent.usingTLC and HPLC, we can make sure that it has isoflavones or not,and then make clear the type and the content of isoflavones in Rapeseed meal. On this basis, by using high-performance liquid chromatography method as a standard, comparing the accuracy and reliability of different UV wavelength (254nm, 259nm,260nm,262nm, three wave -length) ,we can choose a simplier and rapider method of determining isoflavones; by comparing different extraction condition (solvent, concentration, temperature, time, weight ratio,number) ,we can also select the optimalitical extracting condition;by comparing the isoelectric point method, centri -fugation, freeze-isoelectric point-centrifugation, we can make clearly of the best protein separation condition; by comparing solvent extraction and resin adsorption, we can also choose the best puri -fication methods; and then we do a text between the standard sample and the purified isoflavone for physiological activity.The main results are as follows:(1) In this paper, we found that Rapeseed Meal does contain genistin which is one of the isoflavones by extraction of rapeseed meal. and the content range of genistein is 0.100%-0.290%. (2) Rapid determination of isoflavones Research shows : Using UV to Determinate the isoflav -one’s content which extracted from the rapeseed meal is feasible, angd its best colorimetric wavele -ngth is 259nm, the calibration coefficient is 1.67. 3 wavelength can not be used to determinated the isoflavone’s content.(3) By comparing the rate of isoflavone and impurities under different solvents (methanol, ethanol), the extraction time (1,2 and 3 hours), different numbers(1,2,3) and different tweight ratio (1:5,1:10. 1:15,1:20), the different temperature (50℃, 60℃, 70℃, 80℃) ,it conclude the optimizatial condition: 60%ethanol, the temperature is 60℃, and weight ratio is 1:15, the extraction time is 3 hours, extraction 2nd,.(4) By using isoelectric point precipitation, centrifugation and freeze—isoelectric point—centrifugation, it conclude the best method for protein separation : chilled for 24 hours, after HC1, make sure pH between at 3-4. Then at 2000r/min, centrifugal 20min..(5) Two method of parification which is ethyl acetate extraction, macroporous resin adsorption method results showed that: using macroporous resin adsorption purified isoflavones better than extraction with ethyl acetate. using macroporous resin adsorption,the content of genistin in dry matter is up to 77%. And the extraction line of isoflavones is that: Ethanol extraction—Freeze-isoelectric point-Centrifugal Separation of protein—macroporous resin extract—Determination.(6) The isoflavone’s anti-oxidant capacity from rapeseed meal showed : Genistin can signify -cantly reduce the lipid substance’s POV and TBA value.更多还原