食品科學(xué)論文編號(hào):SP103  論文字?jǐn)?shù):10419,頁(yè)數(shù):27

目 錄
中文摘要.Ⅰ
英文摘要…………………………………………………………………….……Ⅱ
目錄……………………………………………………………………………….Ⅴ
緒論……………………………………………………………………………1
2. 實(shí)驗(yàn)部分……………………………………………………….………………3
 2.1 實(shí)驗(yàn)材料與儀器………………………………………….……………3
 2.1.1 實(shí)驗(yàn)材料…………………………………………………………3
 2.1.2 實(shí)驗(yàn)試劑…………………………………………………………3
 2.1.3 實(shí)驗(yàn)儀器…………………………………………………………3
 2.2 實(shí)驗(yàn)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(yōu)化…………………………5
 2.2.1.2 標(biāo)準(zhǔn)葡萄糖曲線制作及糖含量測(cè)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(guò)濾層析……………………………9
 2.2.3 生物活性分析…………………………………………………….…9
 2.2.3.1 癌細(xì)胞種類…………………………………………………9
 2.2.3.2 對(duì)癌細(xì)胞抑制影響的測(cè)定……………………………….….9
3.結(jié)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(shí)驗(yàn)…….10
 3.1.2.2 正交實(shí)驗(yàn)…….14
 3.1.3 標(biāo)準(zhǔn)葡萄糖曲線制作及糖含量測(cè)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(guò)濾層析……18
 3.3 生物活性分析……20
4. 總結(jié)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(xiàn)…….……25
中英文對(duì)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(yī)藥方面具有很好的功效。近幾年來(lái),隨著國(guó)內(nèi)外對(duì)藤梨根研究的逐漸深入,人們陸續(xù)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(lái)越受到國(guó)內(nèi)外研究者的重視,具有不錯(cuò)的發(fā)展?jié)摿�和市�?chǎng)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(xué)活性。但是,目前關(guān)于藤梨根多糖的分離純化及抗腫瘤活性分析的研究報(bào)道甚少。我們經(jīng)過(guò)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(guò)濾層析得到純化的藤梨根多糖Ⅱa、Ⅱb（APSⅡa、APSⅡb）。通過(guò)正交試驗(yàn)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(shí)間4.0hr。試驗(yàn)表明用乙醇沉淀多糖，乙醇的倍數(shù)是決定沉淀率的主要因素。通過(guò)實(shí)驗(yàn)，確定3倍體積乙醇為最佳沉淀倍數(shù)。
 最后在體外通過(guò)MTT法研究分離出的藤梨根多糖組分對(duì)癌細(xì)胞的凋亡影響。結(jié)果顯示，組分APSⅡb對(duì)肺腺癌細(xì)胞SPC-A1有抑制作用，并且表現(xiàn)出一定的劑量依賴性。而組分APSⅠ和APSⅡa雖然對(duì)肺腺癌細(xì)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(rèn)為其不具備抗腫瘤活性。藤梨根多糖APSⅡb能明顯抑制癌細(xì)胞增殖，促進(jìn)細(xì)胞凋亡。其具體的抗腫瘤機(jī)制有待進(jìn)一步的研究。
關(guān)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