在傳統醫學上，燕窩具有止咳化痰、補中益氣、治虛勞和增強免疫力的效果，而實質的功效還是需要去探討及研究。燕窩富有高單位之黏多醣(mucin)和唾液酸。而唾液酸又普遍存在於動物的唾液、黏膜等部分，也是病毒感染宿主重要的關鍵之一。前人的研究已發現，燕窩具有抗流感病毒之功效。本研究利用膠體管柱分析將帶有唾液酸的胜肽分離出來，做進一步的抗病毒測試，以瞭解其抗病毒之功效。實驗首先利用SDS-PAGE、凱氏氮含量測定以及醣類指紋圖譜進行鑑偽分析，以確保燕窩樣品為正品。利用pronase將燕窩中的蛋白質水解成短胜肽，經由膠體層析(gel filtration)將不同大小片段區分開，進行唾液酸含量、凝集素分析以及病毒結合性測試。在凝集素分析中得知，燕窩唾液酸主要以α2,3鍵結。高效能陰離子交換色層分析(HPAEC)分析出燕窩的唾液酸以Neu5Ac為主體。在病毒結合分析中，發現從燕窩分離出的物質可以有效和病毒H5N1及H1N1結合。此實驗結果顯示，燕窩對於能辨認唾液酸α2,3鍵結之流感病毒有結合能力，也代表其可以達到抗流感之功效。在核磁共振(NMR)分析中證實燕窩中含有類似於唾液酸的4,8-anhydro-N-acetylneuraminic acid，其是否影響燕窩與病毒間的結合，相關需做進一步的探討。另外檢測出燕窩中含有微量的Tn結構，因此燕窩也可能含有抗癌效果。此結果對於往後燕窩保健功能及流感病毒防治研究有很大的幫助。

Bird’s nest is the saliva from the Colloclia that have a lot of mucin and sialic acid. However the sialic acid is an important intermediate of influenza virus infection. In 2006, Guo et al was confirmed the Inhibitory effect of bird’s nest extract on influenza virus infection. But the anti-virus component has not been isolated and purified. First, the bird's nest used in this study was proved to be a pure bird’s nest by SDS-PAGE, sialic acid content and protein content. Qualification analysis by SDS-PAGE showed sample possessed three bands at about 50 kDa, 100 kDa and 150 kDa, by Warren assay showed sample possessed a sialic acid content about 10% of dry weight, by Kjeldahl method showed sample possessed a protein content about 50% of dry weight, and by fluorescence analysis in 365 nm showed sample emitted a blue-white light. According to the criteria on previous studies, the sample was considered to be a real and pure bird’s nest. Subsequently, the sample was applied to pronase digestion and then separated by Bio-gel P2 for size exclusion chromatography. All fractions were performed carbohydrate analysis including sialic acid. In lectin blotting, Maackia amurensis agglutinin (MAA) lectin can interact with the fraction that suggest the structure of sialic acid is sialic acid-2,3 galactose in bird’s nest. And carbohydrate membrane array to demonstrate the influenza virus of H5N1 and H1N1 can bind the fractions that suggest the antivirus effect of bird’s nest. These fractions will be further analyzed by neutralization assay to study their antivirus effects in vitro.

論文審定書 i
誌謝 ii
中文摘要 iii
Abstract iv
圖次 viii
表次 x
第一章 背景介紹 1
1.1 燕窩 1
1.1.1 燕窩的來源 1
1.1.2 燕窩的分類 1
1.1.3 燕窩的傳統療效 2
1.1.4 燕窩的加工處理及商品 3
1.1.5 燕窩的成分分析與研究 4
1.1.6 燕窩功效的科學研究 5
1.2 唾液酸 6
1.3 流感病毒 10
1.3.1 流感病毒與唾液酸的辨認 12
1.3.2 流感病毒感染途徑 13
1.3.3 流感防治與治療 15
1.4 實驗目的 17
1.5 實驗架構 17
第二章 材料與方法 18
2.1 SDS-PAGE樣品製備 18
2.2 SDS-PAGE 18
2.3 鹽酸水解燕窩唾液酸 19
2.4 Trifluoroacetic acid (TFA)水解燕窩 19
2.5 燕窩蛋白質水解 19
2.6 Warren assay 20
2.7 蛋白質含量分析(凱氏氮) 22
2.8 燕窩螢光分析 23
2.9 膠體層析(gel filtration) 23
2.10 高效能陰離子交換色層分析(High performance anion exchange chromatography, HPAEC) 23
2.10.1 唾液酸分析 24
2.10.2 GalNAc分析 25
2.11 醣晶片(carbohydrate membrane array)製備 26
2.12 醣晶片凝集素測試 27
2.13 醣晶片抗體測試 28
2.14 醣晶片病毒測試 28
第三章 結果與討論 30
3.1 燕窩的鑑偽 30
3.1.1 以SDS-PAGE分析正品燕窩之特徵色帶 30
3.1.2 燕窩螢光反應的分析 31
3.1.3 燕窩粗蛋白含量分析 31
3.1.4 燕窩中唾液酸含量分析 32
3.1.5 HPAEC分析燕窩中的唾液酸 33
3.1.6 唾液酸/乙醯半乳糖胺 (Neu5Ac/GalNAc) 比值分析 33
3.2 抗病毒成分的分離純化 34
3.2.1 蛋白酶水解燕窩樣品 34
3.2.2 膠體層析 35
3.3 燕窩與燕窩水解胜肽的抗病毒效果 36
3.3.1 燕窩水解胜肽的純化分液之抗病毒效果分析 36
3.3.2 燕窩水解胜肽分液的唾液酸及GalNAc含量分析 37
3.3.3 凝集素對燕窩水解胜肽分液中的唾液酸鍵結結構分析 38
3.3.4 燕窩分液與流感病毒的結合分析 38
3.3.5 燕窩水解胜肽上的唾液酸對於與凝集素和流感病毒結合力的探討 39
3.3.6 核磁共振分析燕窩中的唾液酸 40
3.4 燕窩水解胜肽分液中的STn及Tn含量分析 41
第四章 結論 42
參考文獻 83
附錄 91

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