藤梨根多糖的分离提取及生物活性分析

论文编号:SP103  论文字数:10419,页数:27

摘要：藤梨根（Radix Actinidiae Argutae）作为一种常用抗肿瘤中药,在临床医药方面具有很好的功效。近几年来,随着国内外对藤梨根研究的逐渐深入,人们陆续从藤梨根中提取出多种天然成分,其中不乏活性很好的化合物。因此中药藤梨根的抗肿瘤作用越来越受到国内外研究者的重视,具有不错的发展潜力和市场前景。
 藤梨根多糖是藤梨根中的重要抗肿瘤成分，具有显著的生物学活性。但是,目前关于藤梨根多糖的分离纯化及抗肿瘤活性分析的研究报道甚少。我们经过沸水提取，乙醇沉淀，Sevag＋酶解法脱蛋白，DEAE-Sepharose Fast Flow离子交换层析，Sephadex G-100凝胶过滤层析得到纯化的藤梨根多糖Ⅱa、Ⅱb（APSⅡa、APSⅡb）。通过正交试验筛选出最佳浸提条件组合为料液比1：20、浸提温度100℃、提取时间4.0hr。试验表明用乙醇沉淀多糖，乙醇的倍数是决定沉淀率的主要因素。通过实验，确定3倍体积乙醇为最佳沉淀倍数。
 最后在体外通过MTT法研究分离出的藤梨根多糖组分对癌细胞的凋亡影响。结果显示，组分APSⅡb对肺腺癌细胞SPC-A1有抑制作用，并且表现出一定的剂量依赖性。而组分APSⅠ和APSⅡa虽然对肺腺癌细胞有一定的抑制作用，但抑制作用并未随着用药量的增加而增加，因此认为其不具备抗肿瘤活性。藤梨根多糖APSⅡb能明显抑制癌细胞增殖，促进细胞凋亡。其具体的抗肿瘤机制有待进一步的研究。
关键词：藤梨根；多糖；分离纯化；MTT；抗肿瘤

Abstract:The Radix Actinidiae Argutae is a kind of in common use Chinese herbal medicine, having good effects in the anti-tumor clinical medicine. In the past years, various natural components have been extracted from Radix Actinidiae Argutae. Followed by the further studies, more and more concerns have been put into the development potential and market prospects of Radix Actinidiae Argutae.
 The polysaccharides of Radix Actinidiae Argutae (APS) are kinds of mainly effective components of Radix Actinidiae Argutae. However, there is few report focused on the isolation、purifiation or biological activity analysis of APS. In this research, we extracted the polysaccharides with hot water and precipitated by alcohol. Protein was removed by Sevag and enzymolysis method. Then the products were further purified with column chromatography on DEAE-Sepharose Fast Flow and Sephadex G-100 orderly,fractionized as 3 fractions: APSⅠ、APSⅡa and APSⅡb. The optimal extracting conditions were concluded from a orthogonal experiment as material and liquid are at ratio of 1:20,extrating at 100℃ for 4 hours. The experiment indicated that the alcohol addition is the determintive factor of polysaccharide precipitation. From the results, we found 3 times volume of extract is the best alcohol addition.
 Finally, we demonstrated the apoptotic effects of APS by MTT. The effects of APSⅠ、APSⅡa and APSⅡb on the lung cancer cells were studied in vitro. The results indicated that the APSⅡb had significant inhibtion of lung cancer cell proliferation alone at a series of concentrations. What’s more, the inhibtion ratio raise when the concentration increase. However, APSⅠand APSⅡa do not have those obvious effects. It was concluded that APSⅡb was a kind of polysaccharide which had the anti-tumor function.

Keyword：Radix Actinidiae Argutae, polysaccharides, isolation, purifiation, MTT, anti-tumor

目 录
中文摘要.Ⅰ
英文摘要…………………………………………………………………….……Ⅱ
目录……………………………………………………………………………….Ⅴ
绪论……………………………………………………………………………1
2. 实验部分……………………………………………………….………………3
 2.1 实验材料与仪器………………………………………….……………3
 2.1.1 实验材料…………………………………………………………3
 2.1.2 实验试剂…………………………………………………………3
 2.1.3 实验仪器…………………………………………………………3
 2.2 实验方法………………………………………………….……………4
 2.2.1 藤梨根多糖的提取……………………………………….……….4
 2.2.1.1 藤梨根粗多糖的提取……………………………………….4
 2.2.1.1.1 粗多糖提取流程………………………………………4
 2.2.1.1.2 藤梨根多糖提取条件的优化…………………………5
 2.2.1.2 标准葡萄糖曲线制作及糖含量测定……………………….7
 2.2.1.3 脱蛋白……………………………………………………….7
 2.2.1.4 粗多糖的脱色……………………………………………….8
 2.2.2 藤梨根多糖的纯化…………………………………………………8
 2.2.2.1 DEAE-Sepharose Fast Flow 阴离子交换层析………….….8
 2.2.2.2 Sephadex G-100凝胶过滤层析……………………………9
 2.2.3 生物活性分析…………………………………………………….…9
 2.2.3.1 癌细胞种类…………………………………………………9
 2.2.3.2 对癌细胞抑制影响的测定……………………………….….9
3.结果与讨论………………………………………………….……….………….10
 3.1 藤梨根多糖的提取…………………………………………………….10
 3.1.1 藤梨根粗多糖的提取……………………….……………….…10
 3.1.2 多糖提取条件的确定…………………….………………….10
 3.1.2.1 单因素实验…….10
 3.1.2.2 正交实验…….14
 3.1.3 标准葡萄糖曲线制作及糖含量测定……15
 3.1.4 脱蛋白方法比较……16
 3.2 藤梨根多糖的纯化…….17
 3.2.1 DEAE-Sepharose Fast Flow 阴离子交换层析……17
 3.2.2 Sephadex G-100凝胶过滤层析……18
 3.3 生物活性分析……20
4. 总结与展望……20
 4.1 藤梨根多糖的提取……21
 4.2 藤梨根多糖的纯化……21
 4.3 藤梨根多糖的生物活性分析……22
致谢…….……24
参考文献…….……25
中英文对照…….……27