【作者】 肖卫民；

【导师】 杨光伟；

【作者基本信息】 西南农业大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 葛根是一种传统中草药，是豆科葛属植物野葛(Pueraria lobata(willd) ohwi)或粉葛(Pueraria thomsonii Beueh)的根。 作为一种传统药，葛根具有独特的功效。传统中约学认为葛根味甘辛、性平，有清热解肌、生津止渴、醒酒解脾、透疹、升阳止泻之功效。现代药理研究表明，葛根制剂可以调节循环系统、解除痉挛、降低血糖，抗氧化、抑制肿瘤、提高免疫、改善记忆等，对高血压、冠心病、心绞痛、脑血栓、突发性耳聋等疾病有特殊疗效。 葛根的药理活性成分主要是其含有的异黄酮类物质。目前，从葛根提取物中分离出的异黄酮有20余种，主要活性成分是葛根素、黄豆苷元和黄豆苷。其中，葛根素是葛属植物的特有成分，含量最高；黄豆苷元有“植物雌激素”之称。在植物界中，异黄酮有“生物雌激素”之称，而黄豆苷的结构与哺乳动物的雌激素最相似。 目前，中草药提取的现代化技术有超临界萃取(Supercritical Fluid Extraction，SFE)、超声波提取(The Ultrasonic withdraws the technique)、半仿生提取法(Semi-bionic Extraction method，SEB)、微波辅助萃取(Microwave-assisted Extraction，MAE)等。 微波辅助萃取(MAE)是八十年代中期发展起来的一种新技术，它利用极性分子在微波场中瞬时极化、超速转动和离子导电而使物质由内向外迅速至热，同时还伴有其它效应。微波辅助萃取在环保、食品、医药和生化等领域，已经得到广泛应用。近几年，微波辅助萃取在提取中草药成分方面的研究日益受到重视。 大孔树脂吸附柱层析是二十世纪六十年代发展起来的一种分离纯化技术，具有快速简便、分离效果好等特点，从八十年代开始应用于中草药成分的分离纯化，越来越受到重视。 本文以水为溶剂，以甘葛根为原料，利用微波辅助萃取葛根异黄酮。首先，进行单因素的研究，考察微波作用档次和葛根取材部位对葛根总异黄酮提取的影响，结果是两者对葛根总异黄酮的提取的影响显著，确定微波萃取的初步条件：在微波作用时间为45s时，选用微波高火档和有葛根粉末粗纤维的原料中，葛根异黄酮的提取率最高。其次本文以葛根粉末的重西南农业人学硕「学位论文摘要旦口目妞口巴口.口口口口目目里国量(mg)与水的体积(ml)的比例(称为固液比，w二v)、微波作用时间以及乙醇与浓缩液的体积比(V:v)二个因素为编码因子，以葛根总异黄酮的提取率(Y%)为考察对象，利用通用旋转组合设计葛根总异黄酮的微波辅助萃取和乙醇初步纯化试验，建立了数学模烈，回归方程为 Y=43.98一4.0477X一+l.138XZ+0，0786X3+6.9275X一XZ一9.5025 XI X3+2.94 XZ X3一4 .1 o57x一2一9.0一97 x22一0305 x32 经统计分析，方程的拟合不理想，原因是多方面的，试验精度、选择因素和模型的选择都是进一步考虑的问题。 本文对葛根总异黄酮粗提物进行大孔树脂的吸附层析处理，以乙醇不连续梯度进行洗脱，对吸附时间、洗脱剂流速以及层析柱的使用次数等影响因素进行研究，葛根总异黄酮的纯度和提取率为考察对象，结果是:在吸附时间分别为0.5、1、3、5、10h时，30%乙醇洗脱，吸附0.5h时的洗脱效果最好，纯度为39.02%，比样品液提高了47倍，而收率以30%乙醇洗脱，吸附3h时最高，为76.84%，总异黄酮的纯度约14%;在洗脱剂流速研究中，洗脱速度为!ml/min，乙醇浓度为30%，葛根总异黄酮的纯度最高;大孔树脂使用次数对葛根总异黄酮的提取影响明显，新柱的效果为佳，30%乙醇洗脱的纯度最好。三个研究因素的影响明显，确定的人孔树脂层析条件为:吸附时间0.5h，乙醇流速lml/min，层析柱使用一次后再生。 葛根异黄酮提取物在大孔树脂纯化后，进行了反相液相色谱的鉴定。对两个样品进行分析，样品l是在吸附0.5h，流速lmllmin，新活化的吸附柱，30%乙醇洗脱的条件卜分离得到，样品2是流速lml/min，吸附5h时，95%乙醇的洗脱分离所得。结果是:样品1中葛根素含量为25.36%，样品2中葛根素含量极低。 微波辅助萃取葛根异黄酮的研究有不少报道，但是都是以野葛根为材料，乙醇为提取介质，采用正交设计、通用旋转设计或均匀设计进行研究。本文以栽培品种甘葛为原料，以水为溶媒，采用通用旋转设计进行微波辅助提取葛根异黄酮研究，对提取影响因素进行了探讨，建立了数学模型。结果表明，采用微波提取甘葛的葛根异黄酮是可行的，但是需要进一步提高提取率。建立理想的提取模型。 研究显示，AB一8型大孔吸附树脂是分离纯化葛根异黄酮的一种较理想的树脂，本文的研究进一步证明这一结论。然而，以往研究都是以乙醇的单一浓度洗脱，以提取率为考察因素进行研究。本文以乙醇的不连续梯度洗脱，以葛根异黄酮的纯度为考察因素进行研究。结果显示，对从甘葛中提取的异黄酮，AB一8型大孔吸附树脂分离纯化效果良好，杂质少。 本研究为从甘葛中提取的异黄酮的工业生产奠定了良好的基础，具有应用价值。关键词:葛根总异黄酮微波大孔树脂更多还原

【Abstract】 Radix Puerariae (RP), a traditional Chinese herb, also named Lobed Kudzuvine Root, is the root of Pueraia labata(willd) ohwi or Pueraria thomsonii Beueh which belongs to the Leguminosae family.As a Chinese Herbal Medicine (CHM), Kudzuvine Root has special traits: Traditional Chinese Medicine Description(TCM) classifies it as a sweet and pungent herb with a mild property. It is spasmolytic, antipyretic, secretory, and anti-diarrheal. Modern pharmacological research shows that extract of isoflavone from Radix puerariae has beneficial effects on several diseases such as hypertension, coronary heart disease, angina, cerebral thrombus and paroxysmal deaf thanks to its function of regulation of blood circulation system, ant iconvulsant, lowing blood, antioxidants, anti-cancer, enhancing immune system and improving memory.Until now, more than 20 kinds of active principles which belong to isoflavones, have been isolated from the extract of the rhizome of Puerariae lobata, the major three of which are daidzin, daidzein and puerarin, in which puerarin is a specific material which mainly give rise to the contribution to the pharmacology of radix puerariae. Isoflavones are naturally occurring plant chemicals belonging to the "phytoestrogen" class; in which daidzein is named estrogen in plant because its chemical structure is similar to mammalian estrogen at the most.Today, many modern technologies are used in Chinese herbal medicine industry such as Supercritical Fluid Extraction (SFE), The Ultrasonic technique, Semi-bionic Extraction method (SBE), Microwave-assisted Extraction (MAE).The technology of microwave assisted-extraction (MAE) was developed in the middle of 1980s. Material in the microwave field is rapidly heating from inner part to outer in that its polar molecules are polarized promptly and roting in hypervelocity, and then become dipolar ion current. MAE are widely applied in research area such as environment analysis, food industry, medical analysis andbiochemistry. Recent years MAE has been paid more attention as an effective method in extraction bioactive ingradients from Traditional Chinese herbs.Microporous resin chromatography was invented in 1960s, which is an effective and covenient method with many advantages. Microporous resin chromatography technology has been employed in preparation of bioactive ingredients from Traditional Chinese herbs since 1980s and become an more and more important method of purification in Traditional Chinese Medicine industry.This research demonstrates a processing technique to extract total isoflavones from the rhizome of Pueraria thomsonii Beueh by means of microwave and to purifiy extracted isoflavone preparations using microporous resin absorbing chromatography. The method of rotation-general combination is employed in the extraction experiment of total isoflavones. Extract bioactive ingradients from puerariae radix was verified by reverse phase HPLC. The primary results are shown as follows:1. Studied experimental conditions of total isoflavones extraction from mixture of RP powder and water by means of microwave, in which microwave power, heating time and selection of organs from RP are factors selected in the research. The findings are that applying highest level of microwave power can get higher extract of total isoflavones from the rhizome of Pueraria, and the raw fibre from RP is the favorite material being to suffering processing.2. Established the mathematical model of total isoflavones extraction using quadratic rotation-general combination method, in which the ratio of weight of RP powder to water volumn (named the solid to liquid, W/V, mg/ml), heating time (s) and the volumn ratio of ethonal to extracted solution (V/V) are factors of design. But the mathematical model is not fit very well.3. Determined conditions of microporous resin absorbing chromatography for raw extract of RP isoflavones: absorbing time 0.5h, eluant velocity 1ml/min, and regeneration after working one time immediately.4. Verified ingredients of extract of RP isoflavones by reverse pha更多还原