藤梨根多糖的分離提取及生物活性分析

食品科學論文編號:SP103  論文字數:10419,頁數:27

目 錄
中文摘要.Ⅰ
英文摘要…………………………………………………………………….……Ⅱ
目錄……………………………………………………………………………….Ⅴ
緒論……………………………………………………………………………1
2. 實驗部分……………………………………………………….………………3
 2.1 實驗材料與儀器………………………………………….……………3
 2.1.1 實驗材料…………………………………………………………3
 2.1.2 實驗試劑…………………………………………………………3
 2.1.3 實驗儀器…………………………………………………………3
 2.2 實驗方法………………………………………………….……………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 藤梨根多糖提取條件的優化…………………………5
 2.2.1.2 標準葡萄糖曲線制作及糖含量測定……………………….7
 2.2.1.3 脫蛋白……………………………………………………….7
 2.2.1.4 粗多糖的脫色……………………………………………….8
 2.2.2 藤梨根多糖的純化…………………………………………………8
 2.2.2.1 DEAE-Sepharose Fast Flow 陰離子交換層析………….….8
 2.2.2.2 Sephadex G-100凝膠過濾層析……………………………9
 2.2.3 生物活性分析…………………………………………………….…9
 2.2.3.1 癌細胞種類…………………………………………………9
 2.2.3.2 對癌細胞抑制影響的測定……………………………….….9
3.結果與討論………………………………………………….……….………….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 多糖提取條件的確定…………………….………………….10
 3.1.2.1 單因素實驗…….10
 3.1.2.2 正交實驗…….14
 3.1.3 標準葡萄糖曲線制作及糖含量測定……15
 3.1.4 脫蛋白方法比較……16
 3.2 藤梨根多糖的純化…….17
 3.2.1 DEAE-Sepharose Fast Flow 陰離子交換層析……17
 3.2.2 Sephadex G-100凝膠過濾層析……18
 3.3 生物活性分析……20
4. 總結與展望……20
 4.1 藤梨根多糖的提取……21
 4.2 藤梨根多糖的純化……21
 4.3 藤梨根多糖的生物活性分析……22
致謝…….……24
參考文獻…….……25
中英文對照…….……27

摘要：藤梨根（Radix Actinidiae Argutae）作為一種常用抗腫瘤中藥,在臨床醫藥方面具有很好的功效。近幾年來,隨著國內外對藤梨根研究的逐漸深入,人們陸續從藤梨根中提取出多種天然成分,其中不乏活性很好的化合物。因此中藥藤梨根的抗腫瘤作用越來越受到國內外研究者的重視,具有不錯的發展潛力和市場前景。
 藤梨根多糖是藤梨根中的重要抗腫瘤成分，具有顯著的生物學活性。但是,目前關于藤梨根多糖的分離純化及抗腫瘤活性分析的研究報道甚少。我們經過沸水提取，乙醇沉淀，Sevag＋酶解法脫蛋白，DEAE-Sepharose Fast Flow離子交換層析，Sephadex G-100凝膠過濾層析得到純化的藤梨根多糖Ⅱa、Ⅱb（APSⅡa、APSⅡb）。通過正交試驗篩選出最佳浸提條件組合為料液比1：20、浸提溫度100℃、提取時間4.0hr。試驗表明用乙醇沉淀多糖，乙醇的倍數是決定沉淀率的主要因素。通過實驗，確定3倍體積乙醇為最佳沉淀倍數。
 最后在體外通過MTT法研究分離出的藤梨根多糖組分對癌細胞的凋亡影響。結果顯示，組分APSⅡb對肺腺癌細胞SPC-A1有抑制作用，并且表現出一定的劑量依賴性。而組分APSⅠ和APSⅡa雖然對肺腺癌細胞有一定的抑制作用，但抑制作用并未隨著用藥量的增加而增加，因此認為其不具備抗腫瘤活性。藤梨根多糖APSⅡb能明顯抑制癌細胞增殖，促進細胞凋亡。其具體的抗腫瘤機制有待進一步的研究。
關鍵詞：藤梨根；多糖；分離純化；MTT；抗腫瘤

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