牛膝（Radix Achyranthis Bidentatae）多糖的提取纯化工艺及抗衰老活性成分研究

【作者】 邓君；

【导师】 谈锋；

【作者基本信息】 西南师范大学 ， 植物学， 2001， 硕士

【摘要】 中药牛膝（Radix Achyranthis Bidentatae）是苋科植物牛膝（Achyranthesbidentata Bl.）的干燥根，它具有活血、通经、补肝肾、强筋骨之功用。在祖国医学中，牛膝被用于治疗多种疑难杂症，而且现代医学临床也证实牛膝对很多疾病有确切疗效。又因其原植物牛膝分布广泛，资源丰富，很有开发价值。 牛膝含有多种次生代谢产物，如齐墩果酸及其皂甙、蒽醌类化合物、黄酮类化合物、生物碱、蜕皮素等，它们都具有生理活性。牛膝还含有较高比例的可溶性多糖。牛膝多糖有显著的抗癌、抗放射、抗肝炎、抗衰老等功效，而且具有双向免疫调节作用。牛膝多糖又有分子量小，水溶性好，毒性低的优点，不但可以口服，还可以注射，是一种很有前景的治疗用多糖药物。但有关牛膝多糖种类、结构和药理作用的研究不多，关于其结构及药理作用关系的报道更是鲜见，对牛膝多糖的提取、分离、纯化方法也是各持己见。本文的主要研究目的是提取、分离、纯化牛膝多糖，进而研究其部分性质，以及比较怀牛膝（Achyranthes bidentata Bl. var. bidentata forma bidentata）和本地（北碚）产红牛膝（Achyranthes bidentata Bl. var. bidentata forma rubra）的多糖含量、多糖组分及各组分比例，然后以果蝇作动物模型研究分离所得的各种怀牛膝多糖的抗衰老作用。 一、牛膝质量的外观标准 牛膝视其粗细衡量质量，越粗者，多糖含量越高。φ＞0.9cm的牛膝中可溶性多糖的平均含量比φ＜0.3cm者高32.36％。 二、牛膝多糖提取条件的研究 用有机溶剂预处理牛膝可提高多糖的收率和纯度，尤以80％乙醇的除杂效果极显著，而且仅用80％乙醇预提取材料即可，不必再加极性更弱的有机溶剂脱脂。 用纤维素酶和菠萝蛋白酶预处理牛膝，然后用热水浸提，可显著提高多糖浸出率，复合酶系在pH6.5时作用最强，酶浓度对提高牛膝多糖浸出率也有极显著的正向影响。 影响牛膝多糖提取的因素有提取时间、温度、用水量、提取次数和pH值。提取时间以提取液中多糖浓度达到基本稳定为度，用连续取样法确定。不同温度下所需提取时间不等，室温渗漉需60—70小时，60℃浸泡耗时24小时，煮沸提取仅需沸腾1.5小时。提取温度、加水量和提取次数都影响牛膝多糖收率。温度越高，收率越高，且在常压和自然pH条件下，从室温到沸腾，提取温度的升高仅改变所得牛膝多糖中不同分子量范围的组分浸出的比例，而不会 改变各组分的分子量和荷电情况。加水量为材料的80倍且分三次提取时，牛 膝多糖的收率在97％以上，再增加加水量和提取次数，收率的增幅也不大。40 倍水分三次提取，多糖收率即达90％左右，而能耗大为降低，是一种较经济的 选择。提取温度还影响粗多糖纯度，二者呈正相关。用 linol·L－们CI于 4 oC 和用lind叫们。OH于儿流下提取，发现牛膝中酸溶性多糖和碱溶性多糖含量 都很低。 三、分离及纯化方法的研究 在常压条件下，不同的浓缩温度所制得牛膝多糖在 Septladex G—100和 DEAE一SePharose fast一flow柱上的层书行为相同，即浓缩温度的爿高并不 影响牛膝多糖中各组分的比例以及各组分的分子量和荷电，情况。所以提取液的 浓缩可直接于常压下沸腾。提取液中的杂蛋白的去除，用TCA法比用Sevag法 和鞍酸法的效果好，在沉降蛋白质的同时可除去大量色素。多糖的脱盐用 SePhadex G—50比透析效果较好，透析的时耗长，多糖损失大，且易受微生 物污染，Sephadex G——50脱盐无这些弱点。单糖、低聚糖、贰类等杂质可于 醇析步骤除去。醇析时应调 PH至 10，方可沉淀完全。沉淀用 80％乙醇洗涤几 次，然后依次用90％、95％和无水乙醇脱水，60oC真空干燥，所得粗多糖疏松， 颜色浅。 先后用 Sephadex G一100 和 DEAE一epharose fast一flow 柱层析可将牛 膝多糖分成四个组分，分别命名为ABPS、ABPS 11、ABPS Ill、ABPS IV。 四、牛膝多糖的部分性质 用 SePhadex G—100柱层析测定各组分分子量，用薄层层析展开各组分 的酸水解液，测得ABPS分子量为50．359kD，凯氏定氮法证实其中含氮元素， 可能是蛋白多糖，多糖部分由葡萄糖、半乳糖、鼠李糖、阿拉伯糖和半乳糖酸 组成。ABPS分子量为 1．770kD，由葡萄糖和甘露糖组成。ABPS分于量 为4．352kD，由葡萄糖、果糖和葡萄糖酸组成。ABPS IV分子量为 2．323kD， 由葡萄糖、半乳糖、葡萄糖酸和半乳糖酸组成。ABPS 11、ABPS Ill和 AB陀 IV 都不含氮元素。更多还原

【Abstract】 Radix Achyranthis bidentatae, a traditional Chinese medicine in common use is the dried root of Achyranthes bidentata Bl., a herbaceous perennial in Amaranthaceae. It can invigorate the circulation of blood, stimulate the menstrual flow, build the liver and the kidney and strengthen the physique. In Chinese traditional medical science, Radix Aehyranthis bidentatae is used to treat difficult and complicated cases. Modern clinical medicine has been proving its exact curative effect against many illnesses. Furthermore, Achyranthes bidentata is widely distributed , it is a rich resource and worth to be developed. Radix Achyranthis bidentatae contains many kinds of secondary metabolic products, such as oleanic acid and its saponin, anthraquinone, alkaloid, flavonoid and so on. All of them have physiological effects. Polysaccharides抯 proportion in Radix Achyranthis bidentatae is rather high. Achyranthes bidentata polysaccharides (ABPS) has functions of anticancer, antiradiation, antihepatitis and antiage. ABPS have such advantages as small molecular weight, good solubility and slight toxicity. Not only can it be taken orally, but also be injected, so it is a promising curative polysaccharides medicine. But the studies on the categories, structure and pharmacological functions of ABPS are short, and reports about the relationship between its struture and its pharmacological functions are even less. Methods of extracting, isolating and purifying ABPS are far from satisfaction and have not been confirmed thoroughly. The main goal of our research is to find a good technique of extracting, isolating and purifying ABPS, to study some nature of ABPS, to compare the polysaccharides content in roots of A. 53 bidentata B1. var. bidentata forma bidentata grown in Henan provincy and that in roots of A. bidentata Bl. var. bidentata forma rubra grown in Beibei, Chongqing, China, and compare the two polysaccharides? constituents. and the ratioes between these constituens. At last, we studied the antisenile activity of all the constituents of ABPS on Drosophila me]a-nogas ter. 1. The Quality Standard of Radix Achyranthis bidentatae in Outer Appearance The quality of Radix Achyranthis bidentatae can be judged from its thickness. The thicker, the more it contains polysaccharides. The Radix Achyranthis bidentatae with diameter over 0.9 cm contains polysaccharides 32. 36 percent more than that with diameter under 0. 3 cm. 2. Studies on the Conditions of Extracting Polysaccharides from Radix Achyranthis bidentatae Pretreating Radix Achyranthis bidentatae with organic solvent, particularly with 80 percent ethanol, can increase purity and rate of recovery of polysaccharides Predrawing raw material only with 80 percent ethanol has significant effect of removing impurity, it is unnecessary to use other organic solvent with weaker polar solvent in addition. Drawing Radix Achyranthis bidentatae pretreated by compound enzymes composed of cellulase and proteinase from pineapple, the rate of recovery of ABPS can rise significantly. The compound enzymes system is very sensitive to pH value. The optimum pH value is 6. 5. The rate of recovery of ABPS ascends markedly along with increasing concentration of the enzymes. The treating temperature should be about 35C, and the treating time, 60 mm or so. The factors influencing the extractio更多还原