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1、密 级： 学校代码：10075 分类号： 学 号：20091032 理学硕士学位论文 胀果甘草愈伤组织多糖的合成调控和生 物活性分析 学位申请人：雷琪瑶 指 导 教 师 ： 梁玉玲 教授 学 位 类 别 ： 理学硕士 学 科 专 业 ： 植物学 授 予 单 位 ： 河北大学 答 辩 日 期 ： 二一二年六月 Classified Index: CODE:10075 U.D.C: NO:20091032 A Dissertation for the Masters Degree of Science Regulation on synthesis of polysaccharide in Gly
2、cyrrhiza inflata Bat. callus and biological activity analysis Candidate: Lei Qiyao Supervisor: Prof. Liang Yuling Academic Degree Applied for: Master of Science Specialty: Botany University: Hebei University Date of Oral Examination: June, 2012 摘 要 I 摘 要 豆科植物甘草（Glycyrrhiza spp.）是我国重要的传统药材之一，多生长在干旱、半
3、 干旱的荒漠草原、沙漠边缘和黄土丘陵地带，含有三萜皂苷类、黄酮类及多糖类等化合 物。甘草多糖（Glycyrrhiza polysaccharide）作为甘草中的一种重要的生物活性成分，可 以控制和调控机体细胞的分裂分化和生长衰老， 具有多种免疫增强功能以及较强的抗肿 瘤抗病毒作用。 甘草的现代化学及生物活性的研究重点大多集中于甘草酸和黄酮类化合 物， 对甘草多糖的研究报道较少。 本文以甘草资源三大品种之一的胀果甘草 （Glycyrrhiza inflata Bat.）为研究对象，在 MS 基本培养基的基础上，通过调整培养基成分对胀果甘 草愈伤组织细胞的生长及多糖合成进行调控，达到了增加其产物甘
4、草多糖的含量的目 的，并利用正交试验法找出了超声波提取愈伤组织中甘草多糖的最优条件。所得到甘草 多糖经过除蛋白、半透膜透析、层析柱洗脱等纯化流程去除了绝大部分的杂质，最后对 达到一定纯度后的甘草多糖与相同条件提取纯化的天然甘草药材中甘草多糖的抗氧化 活性以及抗肿瘤活性进行了比较分析。 主要试验结果如下： 1. 当采用液料比 70:1，超声时间 30min，超声温度 90，超声功率 80W 时甘草子 叶愈伤组织材料的多糖提取率达最大值 7.72%，甘草多糖含量为 86.80%；当提取条件 为液料比 40:1，超声时间 20min，超声温度 80，超声功率 80W 时，甘草下胚轴愈伤 组织材料的多
5、糖提取率达最大值 7.49%，甘草多糖含量为 76.71%。而相同条件下甘草 药材的多糖提取率仅仅为 5.26%，甘草多糖含量仅为 58.37%，明显低于从甘草愈伤组 织中提取的多糖含量。 2MS 基本培养基附加蔗糖最利于甘草愈伤组织生长，6,7-V 基本培养基虽利于甘 草多糖的合成但不利于愈伤组织生长从而影响多糖产量； 综合愈伤组织的生长和多糖合 成两方面考虑培养基中 PO43浓度确定为 1.25 mmol/L、Ca2+浓度选择 2.99 mmol/L 时， 有利于甘草愈伤组织生长及多糖的生物合成 3 经过 Sevag 法、 流水透析、 二乙基氨基纤维素 （DEAE-52） 、 葡聚糖凝胶
6、（Sephadex G-75）柱层析等技术对甘草多糖进一步纯化后，愈伤组织中的甘草多糖的纯度可达 92.69%，明显高于天然甘草药材中的多糖纯度 86.42%。 摘 要 II 4试验材料甘草愈伤组织与天然甘草药材中的甘草多糖均具有一定的抗氧化活性 和抗肿瘤活性， 且愈伤组织中的甘草多糖对于清除自由基和对肿瘤细胞的抑制作用略比 天然甘草药材明显。 目前甘草多糖的获得大多取材于天然甘草药材， 但由于药物资源的过度开采而造成 稀缺， 本试验利用诱导甘草愈伤组织的方法从中提取甘草多糖并对组织培养甘草细胞的 多糖组分、生物活性以及与天然甘草药材多糖的异同做了初步的研究。试验结果表明， 通过对胀果甘草愈伤
7、组织的生长及多糖合成调控能够提高甘草多糖的含量， 通过组织培 养方法获得的甘草多糖具有一定得抗氧化活性和抗肿瘤活性， 且与天然甘草药材中的甘 草多糖活性相当，为甘草多糖的规模化生产开辟了新的途径。 关键词 胀果甘草 愈伤组织 甘草多糖 合成调控 生物活性 Abstract III Abstract As one of the most crucial Chinese traditional medicine, the radices and rhizomes of Glycyrrhiza spp. containing triterpene glycosides, compounds such
8、 as flavonoids and polysaccharides, which grows in arid and semi-arid desert steppe, desert margins and loess hilly area. As an important biological activity of components in Glycyrrhiza spp, Glycyrrhiza polysaccharides (GPS) can regulate the bodys cell division or differentiation and growth or agin
9、g, as well as have a variety of immune function and the role of strong anti-tumor virus resistance. Modern researches of chemical and biological activity of Glycyrrhiza spp. are mostly focused on Glycyrrhizic acid and flavonoids, but lack of the research reports of glycyrrhiza polysaccharides. This
10、paper to one of the three major varieties of licorice resources of Glycyrrhiza inflate Bat. for the study, MS medium on the basis of, by adjustment medium of nutrition components on expansion fruit licorice more injury organization cell of growth for regulation, achieved the purpose which has increa
11、sed GPS in its product. And using orthogonal experimental method to find out the ultrasonic extraction of callus tissue of glycyrrhiza polysaccharide in optimal conditions. By protein clearing, and semi-permeable membrane dialysis, and chromatography column washing, most of the impurities has been r
12、emoved from GPS. Finaly, we compared the GPS that extracted from Callus with GPS in the same conditions of extraction and purification of natural licorice medicinal materials which have anti-oxidant and anti-tumor activity. Results show that: a.Among the tested basal medium, MS with the addition of
13、sucrose was the best for callus growth, 6,7-V was most suitable for glycyrrhiza polysaccharide production. The optimal concentration of PO43 and Ca2+ of medium was 1.25 mmol/L and 2.99 mmol/L for biomass accumulation of callus and bio-synthesis of glycyrrhiza polysaccharide. The higher concentration
14、 of phosphate and Ca2+ were benefit of the production of glycyrrhiza polysaccharide, but not the growth of callus. b.The extraction rate of polysaccharide from Glycyrrhiza inflata callus induced from Abstract IV cotyledon reached the maximum content of 7.72% and when the liquid-solid ratio was 70:1,
15、 ultrasonic power with 80W , 90 and 30 min, content of Glycyrrhiza polysaccharide was 86.7980%. Extraction rate of polysaccharide in Glycyrrhiza inflata callus from hypocotyl reached the maximum content of 7.49%,and when the liquid-solid ratio was 40:1,after ultrasound with 80W , 80and 20 min, conte
16、nt of Glycyrrhiza polysaccharide was 76.7110%. The extraction rate of polysaccharide from medicinal materials was only 5.26%, and the of Glycyrrhiza polysaccharide was only 58.374%, which is significantly lower than the polysaccharide from Glycyrrhiza inflata callus. c.We use the method of Sevag flowing water dialysis DEAE-52 and Sephadex G-75 to the further purify GPS. The content of
