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論文名稱: | 海藻寡醣經分離與提高硫酸基含量後之生理活性探討
| Study on the Isolation and Oversulfation of Marine Algal Oligosaccharides and Evaluation on Their Biological Activities
研究生: | 林季磊 Chi-Lei Lin
指導教授: | 潘崇良 Chorng-Liang Pan
學位類別: | 碩士
學校名稱: | 國立臺灣海洋大學
系所名稱: | 食品科學系
學號: | M98320047
畢業學年度: | 99
語文別: | 中文
關鍵字: | 寡醣 Oligosaccharides
| 高度硫酸化 Oversulfation
| 抗凝血 Anticoagulant
| 血小板凝集 Platelet aggregation
全文影像連結: | | (本論文20140729開放校外瀏覽)
| 論文全文(pdf)

論文頁數: | 158
摘要: | 本研究目的在於探討龍鬚菜、馬尾藻、及石蓴熱萃液經酵素水解後區分不同聚合度的寡醣，利用氯磺酸-二甲基甲醯胺法 (Chlorsulfonic acid-N,N-dimethylformamide method)，提升其硫酸基含量，並比較兔血漿及人血漿抗凝血及抗血小板凝集等生理活性之變化。龍鬚菜、馬尾藻、及石蓴於 121oC 熱萃 40 min後以 3% 纖維素酶水解 6 hr，以及 2 U/mL 海藻熱萃多醣粉末誘導 Pseudomonas vesicularis MA103 及Aeromonas salmonicida MAEF108所產之酵素水解 24 hr後，其全醣量分別為 51.21 mg/mL、 31.89 mg/mL 、及 26.83 mg/mL，而還原醣含量為 22.01 mg/mL、 23.01 mg/mL 、及 23.22 mg/mL，利用超過濾系統區分出小於 3 kDa 分子量之寡醣，經過 Sephadex G-10 膠體過濾層析 (Gel permeation chromatography, GPC) 及高效能液相層析儀 (High performance liquid chromatograph, HPLC) 可初步分離出 8 種不同聚合度的單一寡醣，龍鬚菜 Gra-I、 Gra-II、及 Gra-III，馬尾藻 Sar-I、 Sar-II、及 Sar-III，石蓴 Ulva-I 和 Ulva-II。龍鬚菜、馬尾藻、及石蓴經區分後之單一寡醣利用氯磺酸-二甲基甲醯胺法於 25oC 下反應 4 小時，結果顯示個別寡醣之硫酸基含量 Gra-I 、 Gra-II、及 Gra-III 從 ND-4.72% 提升至 14.33%-28.17%，Ulva-I 與Ulva-II 從ND-3.18% 提升至 20.22%-26.25%， Sar-I、 Sar-II 、及 Sar-III 從1.75%-3.12% 提升至 12.71%-28.86%，而 Heparin (69.2 U/mL) 之硫酸基含量為 26.15%，且經硫酸基添加後之寡醣於 HPLC 分析下會將洗堤時間提前，推測為寡醣接上更多硫酸基造成之結果，在 FT-IR 分析下可以發現於 1,250 cm-1 及 820 cm-1 有明顯之吸收波峰，推測為硫酸基添加所造成之結果。在寡醣提高硫酸基含量前後的抗氧化活性變化觀察中，經提高硫酸基之寡醣對於 DPPH 自由基清除能力無明顯活性，但在螯合亞鐵離子活性方面皆能提高活性，Gra-II-DMF、Sar-II-DMF、及 Ulva-I-DMF 之清除能力分別為 89.46%、88.91%、及 86.88%。在兔血漿之抗凝血實驗中，Heparin 10 g/mL 之活化部分凝血活酶時間 (Activated partial thromboplastin time, APTT) 延遲至352 sec，而 20 mg/mL 的 Gra-II DMF、 Sar-II DMF、及 Ulva-II DMF 可分別延遲至 440 sec、 495 sec 、及 297 sec，凝血酶原時間 (Prothrombin, PT) 實驗中，經添加硫酸基之寡醣皆無顯示延遲 PT 之活性，但會隨著提高硫酸基含量及寡醣濃度而降低凝血程度。經提高硫酸基含量的寡醣於人血漿之 APTT 與 PT 實驗中較無明顯之抗凝血效果。於抗血小板凝集實驗中，20 mg/mL 的 Gra-II DMF、Sar-I DMF、Sar-II DMF、與 Sar-III DMF 較相對應未添加硫酸基寡醣所呈現的凝集百分比分別降低了 4.2%、8.5%、8.5%、與 5.6%，且皆與相對之控制組有顯著差異。經提高硫酸基含量之龍鬚菜寡醣 Gra-II DMF、馬尾藻寡醣 Sar-I DMF、 Sar-II DMF、及 Sar-III DMF 在抗凝血及抗血小板凝集實驗中與未提高硫酸基之寡醣相比較，皆顯示較佳的活性，未來可能有做為預防血栓性心血管疾病食品之發展潛力。
論文目次: | 目錄            i
| 表目錄            v
| 圖目錄            vi
| 附錄目錄    ix
| 中文摘要    x
| 英文摘要 (Abstract)    xii
| 壹、前言    1
| 貳、文獻整理    3
| 一、海藻之來源與應用    3
| 1-1. 龍鬚菜    3
| 1-2. 馬尾藻    4
| 1-3. 石蓴    4
| 二、洋菜酶對洋菜水解的作用機制    5
| 三、海藻寡醣    6
| 3-1. 海藻寡醣之萃取方法    6
| 3-2. 含硫酸酯寡醣之生理活性    7
| 3-2-1. 抗凝血性及抗血小板凝集活性    7
| 3-2-2. 抗病毒    8
| 3-2-3. 其他生理活性    9
| 四、醣類的高度硫酸化    10
| 五、海藻寡糖組成成分之鑑定    11
| 5-1. 高效能液相層析法 (High-performance liquid
| chromatography, HPLC)    11
| 5-2. 傅立葉轉換紅外線光譜儀(Fourier-transform
| infrared spectroscopy, FTIR)    12
|
| 六、凝血機制    13
| 6-1. 內在途徑    14
| 6-2. 外在途徑    15
| 6-3. 共同途徑    15
| 6-4. 凝血作用的測試    16
| 七、血小板    16
| 7-1. 血小板構造及其生理功能    16
| 7-2. 血小板凝集作用(Aggregation)    17
| 参、實驗設計    18
| 肆、材料方法    19
| 一、實驗材料    19
| 1-1. 原料    19
| 1-2. 試驗菌株    19
| 1-3. 試驗藥品    19
| 1-4. 儀器設備    21
| 1-5. 培養基組成    22
| 1-5-1. P. Vesicularis MA 103 多醣降解酵素誘導
| 培養基    22
| 1-5-2. A. salmonicida MAEF108多醣降解酵素誘導
| 培養基    22
| 1-6. 海藻熱萃多醣誘導酵素反應基質    23
| 1-7. DNS 溶液    23
| 二、實驗方法    23
| 2-1. 菌株之保存與活化    23
| 2-1-1. 菌株之保存    23
| 2-1-2. 菌株之活化    23
| 2-2. 海藻之一般成分分析    24
| 2-2-1. 水分含量    24
| 2-2-2. 灰分含量    24
| 2-2-3. 粗蛋白含量    24
| 2-2-4. 粗脂肪含量    24
| 2-2-5. 粗纖維含量    25
| 2-3.    P. vesicularis MA103 或 A. salmonicida
| MAEF108 經個別海藻熱萃多醣誘導所產之
| 水解酵素與其活性測定    25
| 2-3-1. 個別海藻熱萃多糖之製備    25
| 2-3-2. 菌株活化與誘導    25
| 2-3-3. 個別海藻熱萃多醣誘導粗酵素之製備    26
| 2-3-4. 個別海藻熱萃多醣誘導酵素的活性測定    26
| 2-4. 個別海藻寡醣之分離與純化    27
| 2-4-1. 海藻寡醣水解物之製備    27
| 2-4-2. 超過濾(Molecular weight cut off, MWCO)    27
| 2-4-3.    GPC 膠體過濾層析    27
| 2-5. 高度硫酸化    27
| 2-5-1. 氯磺酸–二甲基甲醯胺法    27
| 2-6. 海藻單一寡糖與硫酸化海藻單一寡糖之成分分析    28
| 2-6-1. 還原糖含量    28
| 2-6-2. 總醣含量    28
| 2-6-3. 硫酸基含量測定    28
| 2-6-4. 高效能液相層析法 (High-performance liquid
| chromatography, HPLC)    29
| 2-6-5. 傅立葉轉換紅外線光譜法 (Fourier transform
| infrared spectrometer, FT-IR)    30
| 2-7.      抗氧化活性測定    30
| 2-7-1. 清除 DPPH 自由基能力之測定    30
| 2-7-2. 亞鐵離子螯合能力之測定    30
| 2-8.      抗凝血生理活性    31
| 2-8-1. 凝血酶原時間 (Prothrombin time, PT)    31
| 2-8-2. 活化部份凝血活酶時間 (Activited partial
| thromboplastin time, APTT)    31
| 2-9.      抗血小板凝集 (Platelet aggregation) 生理活性    31
| 2-9-1. 富含血小板血漿之製備    31
| 2-9-2. 血小板凝集測定 (Platelet aggregation)    32
| 2-10. 統計分析    32
| 伍、結果與討論    33
| 陸、結論    47
| 柒、參考文獻    49
|
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