摘要
原花色素为多羟基酚类化合物,衍生于黄烷类化合物,属生物类黄酮。它具有独特的化学和药理活性,可在许多领域中获得广泛的应用。对其进行研究开发具有十分重要的现实意义。
研究工作主要分4个方面:(1) 比较70%乙醇、70%甲醇和70%丙酮的原花色素提取率,比较60%乙醇、70%乙醇、80%乙醇的原花色素提取率,对温度、酸碱性、提取时间、料液比对原花色素提取率的影响进行研究;(2) 采用冷却沉淀、盐析结合溶剂分级法对原花色素进行初级分离,对产品进行高压液相色谱和质谱分析并测定原花色素含量,考查加盐量、pH值对低聚原花色素获得率的影响,研究溶剂直接分级法低聚原花色素的获得率;(3) 对大孔吸附树脂AB-8和葡聚糖凝胶LH-20进行原花色素的静态吸附试验、静态解吸试验、动态解吸试验,对大孔吸附树脂AB-8进行原花色素的动态吸附试验;(4) 试验对葡萄籽原花色素的还原能力、对亚油酸过氧化的抑制作用、对羟自由基的清除作用、对超氧阴离子的清除作用、对亚硝酸根离子的清除作用进行测定,并与VC,BHT进行比较。
研究结果表明:
(1) 原花色素溶剂提取采用1:4的料液比,粉碎10目左右,70%的乙醇或丙酮室温酸性环境下浸提8~10 h的条件较为适宜。此路线具有提取率高,易过滤等特点。
(2) 冷却沉淀、盐析结合溶剂分级法对原花色素进行初级分离可得四种产品。产品1和产品2为高聚原花色素,产品3和产品4为寡聚和低聚原花色素的混合物,产品3具有良好的水溶性。对于本方法,每100ml液体加入6.0gNaCl进行盐析是较为适宜的。pH=3.5较有利于乙酸乙酯对低聚原花色素的萃取。有机溶剂直接分级法由于叶绿素的影响效果不如冷却沉淀、盐析结合溶剂分级法。冷却沉淀、盐析结合溶剂分级法具有方法简便,成本低廉,原花色素获得率较高,产品无毒无害,低聚原花色素产品纯度高,水溶性好等特点。
(3) SephadexLH-20和AB-8对原花色素均具有较强的吸附能力,在1小时内均快速吸附水溶液中的原花色素,1小时后基本达到吸附平衡,其中LH-20无论吸附量还是吸附速率均优于AB-8。较低的流速有利于原花色素的充分吸附和解吸,但太低的流速不利于生产,对于本试验所用AB-8大孔吸附树脂层析柱,1.5ml/min的上样和洗脱速率是较适宜的。产品3可能主要为原花色素二聚体和三聚体的混合物。AB-8对原花色素的分离效果不如SephadexLH-20。
(4) 一定浓度范围内,葡萄籽提取物的还原能力随着浓度的增加而增强。当浓度低于
lm岁ml时,葡萄籽提取物对亚油酸的过氧化表现出一定的诱发效应,随着浓度的增加,葡萄
籽原花色素对亚油酸过氧化的抑制作用也逐渐增强。葡萄籽原花色素对轻自由基的清除作用
随着浓度的增加而增强,当浓度为600mg/L时,对轻自由基的清除率可达%.8%。在低浓度
条件下,葡萄籽原花色素对邻苯三酚自氧化的抑制作用较弱,在高浓度条件下具有显著的抑
制作用。在光照核黄素体系中,不同浓度的葡萄籽原花色素对超氧阴离子均具有显著的清除
作用。明显优于同浓度的V。。在低浓度条件下,葡萄籽原花色素对亚硝酸根离子的清除作用
较弱,但随着浓度的增大,葡萄籽原花色素对亚硝酸根离子的清除作用也明显增强。原花色
素抗氧化性能优于vc和BHT,是一种优良的纯天然抗氧化剂。
The proanthocyanidin , a polyphenol compound of many hydroxy, derives from the flavan compound. It is a kind of the living creature flavonoid.The proanthocyanidin has special chemical and pharmacological activity,and can be applied diffusely in many fields.So it is of great significance to research and empolder on it.
Research work can be divided into four aspects. (1),Compare the proanthocyanidin extration rate of 70% ethanol,70% carbinol,and 70% acetone; compare the proanthocyanidin extration rate of 60% ethanol,70% ethanol,and 80% ethanol;do research on the effect on the proanthocyanidin extration rate by temperature, acidity, marination time, solution cubage /weight etc. (2), To separate the proanthocyanidin primarily ,adopt the following separation method : cooling off to precipitate,combination of solvent extraction and salting out; analyse the product by means of HPLC and MS; mensurate the content of the proanthocuanidin;examine the effect on the acquisition rate of OPC by the added quantity of salt and pH;do rasearch on the OPC's acquisition rate get by the method of solvent-immediacy-separation. (3),Do proanthocuanidin static absorption experiments > static desorption experiments dynamic desorption experiments on the big-bore-adsorbing resin AB-8 and dextrose gel Sephadex LH-20;do proanthocuanidin dynamic absorp
tion experiments on the big-bore-adsorbing resin AB-8. (4),Test the deoxidization capacity of the extractions of the grape seeds and the inhibitory effects on the hyoxidation of linoleic acid ,and the effects of scavenging hydroxyl and ultra-oxygenic anion free radicals,and compare with Vc ,BHT. The research results indicates that:
(1) It is feasible that the proanthocuanidin solvent selects 1:4 rate of the weight of grape seeds to the cubage of the extraction solvent, and takes 8-10 hours to extract proanthocuanidin in the environment of 70% ethanol or acetone and room temperature;This route has the characteristic of higher extraction rate and easier filtration,etc.
(2) The separation method of the proanthocuanidin( cooling off to precipitate,combination of solvent extraction and salting out) to separate the proanthocyanidin primarity,will produce four kinds of extractions;For this mothod ,it is feasible to add 6.0g NaCl to per 100ml solvent;It is of avail to ethyl acetate to extract the OPC when pH=3.5;Owing to the effect of chlorophyl,the outcome of the method of organic-solvent-immediacy-separation is not better than the other mothod (cooling off to precipitate, combination of solvent extraction and salting out);The latter operates simply ,has little cost and the characteristic of higher extraction rate;The extractions of this method is not poisonous and harmful and NO 3 product has the characteristic of better water-solubility, and higher purity of the extractions ,etc.
(3) Both the SephadexLH-20 and AB-8 have comparatively strong absorption capacity to the proanthocuanidin;In less than an hour ,both can absorb the proanthocuanidin in water solution rapidly and can reach the absorption balance on the whole after an hour;After all, whether the absorption quantity or the absorption rate, the SephadexLH-20 is better than the AB-8; A comparatively low velocity of flow is propitious to the enough absorption and desorption of the proanthocuanidin,but a too low velocity of flow is not propitious to manufacture;As to the column used ia the experiment , a 1.5ml/min rate of elution and providing the proanthocuanidin water solution is comparatively appropriate;NO 3 product may chiefly be the compound made up of dimers and trimer;The extraction effect of the AB-8 is not better than the SephadexLH-20.
(4) In a certain range of concentration, the deoxidization capacity of the extractions from grape seeds enhances with the increasing of the concentration; When the concentration is lower than 1.0mg/ml, the extractions from grape seeds shows the induced effect to the hyoxidation of linoleic acid; With the increasing of the concentration, the inhibitory effect of the proanthocuanidin from grape seeds
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