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論文名稱: | 高硫酸基含量青海菜寡醣之製備及生理活性探討
| Studies on the Prduction of Oversulfated Oligosaccharides from Monostroma nitidum and Their Biological Activities
研究生: | 鄭蓉芳 Jung-Fang Cheng
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: | 49942019
畢業學年度: | 100
語文別: | 中文
關鍵字: | 膠體過濾層析 GPC
| 硫酸基 sulfate
| 抗凝血 anticoagulant
全文影像連結: | | (本論文20150730對校內公開)
| 論文全文(pdf)

論文頁數: | 94
摘要: | 本研究目的在於探討青海菜熱萃液經纖維素酶及 Pseudomonas vesicularis MA103 及 Aeromonas salmonicida MAEF108 所產之酵素水解後，區分不同聚合度的青海菜寡醣，並利用氯磺酸-二甲基甲醯胺法 (Chlorsulfonic acid-N,N-dimethylformamide method) 提升其硫酸基含量，並探討其抗氧化性、人血漿抗凝血及抗血小板凝集、及抗病毒之生理活性變化。青海菜經由熱萃、纖維素酶及 P. vesicularis MA103 與 A. salmonicida MAEF108 海藻多醣誘導粗酵素液降解後，其總醣量分別由 2.05 mg/mL 增加至 19.35 mg/mL 與 20.34 mg/mL，而還原醣含量分別由 1.98 mg/mL 提升為 18.67 mg/mL 與 18.76 mg/mL，經超過濾系統 (UF) 區分出小於 3 kDa 分子量之寡醣混合液 (Mon-UF) 後，再以 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) 分析與分離後，依其聚合度 (Degree of polymerization，DP) 分別命名為 Mon-C7、Mon-C4、和 Mon-C2/3。利用氯磺酸-二甲基甲醯胺法提高青海菜寡醣之硫酸基含量，結果Mon-UF、Mon-C7、Mon-C4、和 Mon-C2/3 之硫酸基含量從 0.15%-1.95% 提升至 20.04%-22.73%。4 組高硫酸基含量青海菜寡醣分別命名為 Mon-UF-DMF、Mon-C7-DMF、Mon-C4-DMF、及 Mon-C2/3-DMF。4 組青海菜寡醣與 4 組高硫酸基含量青海菜寡醣，對於 DPPH 自由基清除能力及亞鐵離子螯合能力皆未呈現活性，但在還原力方面，除了每 1000 μg Mon-C4 具相當於 3.49 μg 維生素 C 之還原力外，其餘皆未能檢出。在人血漿之抗凝血實驗中，Mon-C7-DMF 20 mg/mL 之活化部分凝血活酶時間 (Activated partial thromboplastin time, APTT) APTT 為 77 sec，比臨床檢驗 APTT 正常值之 30-50 sec 長，凝集穩定後平均 OD660nm 吸光值為 0.450，比未添加抗凝血劑之對照組之吸光值降低 16%，凝血酶原時間 (Prothrombin time, PT) 實驗中，4 組青海菜寡醣與 4 組高硫酸基含量青海菜寡醣，皆未顯示延長 PT 之活性，當高硫酸基含量青海菜寡醣於 20 mg/mL 各管下，則呈現降低 PT 試驗中凝血程度之表現。於抗血小板凝集實驗中，5 mg/mL Mon-UF-DMF 與 20 mg/mL Mon-UF-DMF 皆有助降低志願者 A 及志願者 B 之血小板凝集反應，分別比低硫酸基含量之 Mon-UF (5 mg/mL、20 mg/mL) 所呈現的凝集百分比分別降低了 13.48%-15% (志願者 A) 及 20%-24.97% (志願者 B)，Mon-C7-DMF 於志願者 A 及志願者 B 的試驗中，皆有助於抑制血小板凝集程度，分別可以降低血小板凝集程度約 4.94%-7.10% (志願者 A) 及 13.59%-15.23% (志願者 B)。抗病毒試驗，抑制日本腦炎病毒吸附 BHK-21 細胞反應，結果顯示，高硫酸基含量青海菜寡醣實驗組具有較高的細胞存活率 (p ＜ 0.05)，而於抑制日本腦炎病毒繁殖反應，則與對照組相較，未能呈現具差異性的細胞存活率。推斷高硫酸基含量青海菜寡醣仍無法抑制病毒的繁殖，但可能具有干擾日本腦炎病毒附著於宿主細胞表面的吸附反應。高硫酸基含量青海菜寡醣顯示較佳的抗凝血及抗血小板凝集活性，故未來具有發展為預防血栓栓塞疾病相關保健食品之潛力。
| The aim of this study is to investigate M. nitidum via hot-water-extract, and cellulase, and then crude polysaccharides (PS) degrading enzymes from Pseudomonas vesicularis MA103 and Aeromonas salmonicida MAEF108 (induced with M. nitidum PS) for enzyme hydrolysis to separate oligosaccharides (OS) with different degree of polymerization (DP), and using Chlorsulfonic acid- N, N-dimethylformamide (DMF) method to increase sulfate contents of these OS, and then examine the physiological activity effects of their antioxidant activity, anticoagulant and anti-platelet activity of human plasma, and anti-viral activity. After M. nitidum was extracted by hot-water to obtain PS, added the cellulase hydrolysis as well as the crude polysaccharides (PS) degrading enzymes from P. vesicularis MA103 and A. salmonicida MAEF108 (induced with M. nitidum PS) for enzyme hydrolysis. Total sugar content of M. nitidum PS increased from 2.05 mg/mL to 19.35 mg/mL, and 20.34 mg/mL. Reducing sugar content of M. nitidum PS increased from 1.98 mg/mL to 18.67 mg/mL, and 18.76 mg/mL, respectively. Oligosaccharides hydrolysates with molecular mass < 3 kDa were collected by Ultra-filtration system, then though Sephadex G-10 gel filtration chromatography (GPC) and high performance liquid chromatography (HPLC) to obtain nine peaks. The 4-7 peaks are named as the Mon-C7, Mon-C4, and Mon-C2/3, respectively according to their degree of polymerization. After using chlorsulfonic acid- N,N-dimethylformamide (DMF) method to increase sulfate contents of these OS, the sulfate content of Mon-UF, Mon-C7, Mon-C4, and Mon-C2/3 increased from 0.15%-1.95% to 20.04%-22.73%, and designated as Mon-UF-DMF, Mon-C7-DMF, Mon-C4-DMF, and Mon-C2/3-DMF. In the evaluation of the antioxidation effect of M. nitidum OS and oversulfated OS, DPPH free radical scavenging and the ability of chelating on Fe (II) ion of OS and oversulfated OS did not exist significant activity. However, the reducing power could not found except that Mon-C4 existed 3.49 μg/mL of vitamin C in reducing power. In the experiment on anticoagulant activities of human plasma, activated partial thromboplastin time (APTT) of Mon-C7-DMF (20 mg/mL) was 77 sec, which is longer than that of clinical examination normal value of APTT 30-50 sec. The average absorbance value of OD660nm was 0.450, about 16% lower than the blank. In the experiment on prothrombin time (PT) analysis, M. nitidum OS and oversulfated OS were no obvious delayed in PT, but there were a tendency for reduction on Coagulation degree while oversulfated OS concentration was 20 mg/mL. In platelet aggregation analysis, 5 mg/mL and 20 mg/mL of Mon-UF-DMF,  will help to reduce the platelet aggregation response of volunteer A and volunteer B, respectively, compared with Mon-UF (5 mg/mL and 20 mg/mL) showing the percentage of agglutination was decreased by 13.48%-15% (volunteer A) and 20%-24.97% (volunteer B). The Mon-C7-DMF helped volunteer A and volunteer B to inhibit platelet aggregation level. The reduction of platelet aggregation level of volunteer A and volunteer B were about 4.94%-7.10% 13.59%-15.23%, respectively. In anti-virus test, inhibition of Japanese encephalitis virus (JEV) adsorption BHK-21 cells response. The results showed that in the prevention experiment, M. nitidum oversulfated OS performed higher survival rate of host cell. In contrast, inhibition of JEV propagation reaction had not significant cell survival rate. The aforementioned outcome means that M. nitidum oversulfated OS cannot inhibit the propagation of the virus, but it may interfere with the JEV to attach to the BHK-21 cells surface. After comparing M. nitidum OS and oversulfated OS showed better effect on the anticoagulant and anti-platelet agglutination experiments than OS. The results can serve as the basis for the development of a novel health food to prevent thromboembolic disease in the future.
論文目次: | 目錄
|
| 目錄    i
| 表目錄    v
| 圖目錄    vi
| 附錄目錄    viii
| 中文摘要    ix
| 英文摘要    xi
| 壹、前言    1
| 貳、文獻整理    3
| 一、青海菜 (Monostroma nitidum) 之利用與研究    3
| 1-1. 簡介    3
| 1-2. 利用與研究    3
| 1-3. 組成    3
| 二、海藻硫酸多醣之生理活性    4
| 2-1. 抗凝血性    4
| 2-2. 抗氧化性    5
| 2-3. 抗病毒活性    6
| 2-4. 抗癌活性    7
| 2-5.    抑制血管收縮素轉化酶 (Angiotensin converting enzyme, ACE) 抗癌活性    7
| 2-6. 其他的活性    8
| 三、止血反應與血栓形成    8
| 3-1. 止血反應    8
| 3-2. 血管壁    9
| 3-3. 血小板    9
| 3-4. 凝固因子    10
| 3-4-1. 內在途徑    11
| 3-4-2. 外在途徑    11
| 3-4-3. 最終的共同途徑    12
| 3-4-4. 凝血酶的正回饋機制    12
| 3-5. 抗凝血系統    12
| 3-5-1. 抗凝血蛋白酶 III    12
| 3-5-2. 蛋白質 C 與其輔因子蛋白質S    13
| 3-5-3. 組織因子路徑抑制劑    13
| 3-6. 纖維蛋白溶解系統    13
| 四、臨床抗凝血藥物簡介    14
| 五、自由基的形成與功用    15
| 叁、實驗設計    17
| 肆、材料方法    18
| 一、實驗材料    18
| 1. 原料    18
| 2. 試驗菌株    18
| 3. 試驗藥品    18
| 4. 儀器設備    21
| 5. 試驗細胞株    22
| 6. 培養基的組成    22
| 7. 青海菜熱萃多醣誘導酵素反應基質    23
| 二、實驗方法    24
| 1. 菌株之保存與活化    24
| 1-1. 菌株之保存    24
| 1-2. 菌株之活化    24
| 2. 青海菜粉末之一般成分分析    24
| 2-1. 水分含量    24
| 2-2. 灰分含量    25
| 2-3. 粗蛋白含量    25
| 2-4. 粗脂肪含量    25
| 3.    A. salmonicida MAEF108或 P. vesicularis MA103
| 經青海菜熱萃多醣誘導所產之青海菜水解酵素    26
| 3-1. 青海菜熱萃取多醣之製備    26
| 3-2. 菌株活化與誘導    26
| 3-3. 青海菜熱萃多醣誘導粗酵素液之製備    26
| 3-4. 青海菜熱萃多醣誘導酵素之活性測定    27
| 4. 青海菜寡醣之分離與純化    27
| 4-1. 青海菜寡醣水解物之製備    27
| 4-2. 超過濾 (Ultra filtration, UF)    27
| 4-3.膠體過濾層析 (Gel permeation chromatography, GPC)    28
| 5. 青海菜寡醣利用氯磺酸-二甲基甲醯胺法高度硫酸化    28
| 6. 青海菜寡醣與高度硫酸化之成分分析    28
| 6-1. 總醣含量測定    28
| 6-2. 還原醣含量測定    29
| 6-3. 總酚含量測定    29
| 6-4. 硫酸酯含量測定    29
| 7. 抗氧化測定    30
| 7-1. 清除 DPPH 自由基能力測定    30
| 7-2. 亞鐵離子螯合能力測定    30
| 7-3. 還原力之測定    31
| 8. 抗血小板凝集測定    31
| 8-1. 人類富含血小板血漿之製備    31
| 8-2. 血小板凝集的測定    32
| 9. 抗凝血測定    32
| 9-1. 活化部分凝血酶原時間    32
| 9-2. 前凝血酶原時間    33
| 10. 抗病毒活性之測定    33
| 10-1. 細胞株培養及繼代方法    33
| 10-2. 細胞毒性試驗 (MTT assay)    33
| 10-3. 抑制病毒吸附宿主細胞及抑制病毒繁殖反應    34
| 伍、結果與討論    36
| 陸、結論    46
| 柒、參考文獻    48
| 捌、附錄    88
|
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2012 年 11 月 19 日 8:51