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論文名稱: | 攝食蜆萃取物對吳郭魚血脂與 LDL 脂肪酸組成之影響
| Effects of clam extracts fed to tilapia on blood lipid level and LDL fatty acid composition
研究生: | 顏維良 Wei-Liang Yan
指導教授: | 孫寶年 Bonnie Sun 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: | M94320016
畢業學年度: | 95
語文別: | 中文
關鍵字: | 蜆 Freshwater clam
| 血脂 blood lipid
| 低密度脂蛋白 low density lipoprotein
| 有效區分 active fraction
全文點閱次數: | 12

全文影像連結:

論文全文(pdf)

論文頁數: | 91
摘要: | 吳郭魚餵食等熱量 (3.97 ± 0.04 kcal/g) 等蛋白質含量 (48.29 ± 1.03 %) 之飼料 8 週後，攝食台灣蜆組與日本蜆組之血漿總膽固醇濃度分別為 272.3 ± 64.2 與 271.8 ± 86.2 mg/dL，皆顯著低於控制組 (401.5 ± 133.2 mg/dL)，台灣蜆組與日本蜆組之 LDL 膽固醇濃度亦顯著較低於控制組，而兩種萃取物之間無顯著差異。對於三酸甘油酯與高密度脂蛋白膽固醇濃度的影響，三組無顯著差異。LDL 之總抗氧化力以台灣蜆組 0.279 ± 0.004 mM Trolox equivalent 最高，其次為日本蜆組 0.255 ± 0.004 顯著高於控制組 0.214 ± 0.007。台灣蜆組 LDL 之 α-tocopherol 含量為 11.59 ± 1.17 molecule/LDL，日本蜆組為 11.37 ± 0.31 顯著高於控制組 8.81 ± 0.50。吳郭魚攝食蜆萃取物後 LDL 中 C16:0、C18:1、SFA 與 MUFA 的含量增加，而 C18:2 含量與 PUFA/(SFA+MUFA) 的比例下降。分離吳郭魚血漿 LDL，並以銅離子誘導做 ex vivo 試驗，LDL 氧化過程分為遲滯期 (Lag phase)、連鎖反應期 (propagation phase) 與終止期 (termination phase)，吳郭魚 LDL 並無顯著之氧化遲滯期，連鎖反應期因氧化速率 (Δ A 232 nm /min) 不同而分為兩段，第一段氧化速率三組無顯差異，第二段氧化速率則以台灣蜆組顯著最低為6.66 ± 0.36 × 10-3 Δ A 232 nm/min，其次為日本蜆組 7.29 ± 0.09 皆明顯低於控制組 (8.53 ± 0.16)，達終止期時，氧化程度 (max. Δ A 232 nm) 與控制組相較皆顯著下降。
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| 取人血漿低密度脂蛋白，以銅離子誘導 in vitro 氧化試驗，添加兩種萃取物皆可延長 LDL 氧化遲滯期，且與添加濃度成正相關，而氧化程度則無顯著差異。在添加濃度為 0.32 mg/ml 時，台灣蜆與日本蜆萃取物延長遲滯期之效果無顯著差異。兩種萃取物皆具抑制 15-LOX 之效果，IC50 分別為 201 與 121 μg/mL。
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| 台灣蜆萃取物經過 5 kDa molecular weight cut-off 膜過濾與 Sephadex G-25 管柱層析所得抑制 15-LOX 效果最強區分物 (fraction C) 之胜肽含量為 145.84 ± 1.13 mg/g，含醣 0.82 %，分子量約介於 2.1~2.4 kDa，可能為含醣基的胜肽。
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| 此外，以 GC-MS 分析台灣蜆萃取物脂質部分中的植物固醇類，初步確定含有 campesterol 與 stigmasterol，此結構與從文蛤中分離出可誘導肝癌細胞死亡之固醇類化合物 (Pan et al., 2006) 類似。
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| Tilapia were fed with feeds of isocalorie (3972 ± 38 kcal/kg) and equal protein content (48.29 ± 1.03 %) for 8 weeks. The plasma total cholesterol concentrations of groups fed freshwater clam extract (FC) and Japanese clam extract (JC) were 272.3 ± 64.2 and 271.8 ± 86.2 mg/dL, respectively. LDL total antioxidant capacity (TAC) of FC and JC as 0.279 ± 0.004 and 0.255 ± 0.004 mM Trolox equivalent, respectively. LDL α-tocopherol contents in FC and JC were 11.59 ± 1.17 and 11.37 ± 0.31 molecule/LDL, respectively while that of control was 8.81 ± 0.50. The LDL fatty acids composition of tilapia fed clam extract were higher in saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) and lower in polyunsaturated fatty acid/(SFA+MUFA) ratio. Tilapia LDL were oxidized by Cu2+ as an ex vivo model. There were 3 phases during LDL oxidation, lag phase, propagation phase and termination phase. However, tilapia did not noticeable lag phase, while the propagation phase was divided into two stages because of the different oxidative rate. The second oxidative rate was the lowest in FC group (6.66 ± 0.36 × 10-3 Δ A 232 nm /min), followed by the JC group (7.29 ± 0.09), both were significantly lower than the control (8.53 ± 0.16). When reaching termination phase, the max. Δ A 232 nm were significantly lower than the control.
| Human LDL oxidation was induced by Cu2+ as an in vitro model. Both clam extracts prolonged the lag phase in a dose-dependent manner. The effect of FC was better than JC. Both clam extracts inhibited the 15-LOX activity. The IC50 of FC and JC were 201 and 121 μg/mL, respectively.
| The active fraction obtained from freshwater clam extract membrane filtration and Sephadex G-25 gel filtration had molecular weight between 2.1 to 2.4 kDa that inhibited 15-LOX most effectively. It contained 145.84 ± 1.13 mg/g peptide and 0.82% carbohydrate suggesting a glycopeptide.
| Campesterol and stigmasterol were also found in the lipid fraction of freshwater clam extract tentative identified with GC-MS.
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論文目次: | 中文摘要    I
| 英文摘要    III
| 目錄    i
| 圖目錄    iv
| 表目錄    v
| 壹、前言    1
| 貳、文獻回顧    3
| 1. 蜆類    3
| 2. 貝類萃取物之生理活性    3
| 2.1 抗氧化活性    3
| 2.2 調節免疫    4
| 2.3 降血壓功效    5
| 2.4 保護肝臟    5
| 2.5 抗發炎作用    6
| 3. 藻類萃取物之生理功能    7
| 3.1 抗氧化性    7
| 3.2 降血脂功效    8
| 4. 高脂血症    10
| 5. 低密度脂蛋白    10
| 6. 脂氧合酶與 LDL 氧化    11
| 7. 脂肪酸合成    12
| 8. 脂肪酸延長作用    12
| 9. 脂肪酸去飽和作用    14
| 9.1 Delta-9 desaturase    16
| 9.2 Delta-6 desaturase    18
| 參、實驗架構    20
| 肆、材料方法：    21
| 一、材料    21
| 1. 蜆萃取物    21
| 二、方法    21
| 1. 動物試驗    21
| 1.1. 吳郭魚 (Tilapia, Oreochromis mossambicus)    21
| 1.2.. 實驗飼料製備    22
| 1.2.1. 飼料脂肪酸分析    23
| 1.3. 採血    26
| 1.4. 血漿生化數值測定    26
| 1.4.1. 三酸甘油酯    26
| 1.4.2. 總膽固醇    27
| 1.4.3. HDL 膽固醇    28
| 1.4.4. LDL 膽固醇計算    29
| 1.5. 吳郭魚血漿 LDL 分離    29
| 1.6. 血漿與 LDL 總抗氧化力    30
| 1.7. LDL α-tocopherol測定    31
| 1.8. Ex vivo LDL 氧化試驗    32
| 1.9. LDL 氧化遲滯期之計算    33
| 2. In vitro LDL 氧化試驗    33
| 2.1 LDL 分離    33
| 2.2. 透析    34
| 2.3. 銅離子誘導 LDL 氧化    34
| 3. 蜆萃取物對脂氧合酶活性之影響    35
| 3.1. 抑制黃豆脂氧合酶活性試驗    35
| 4. 台灣蜆萃取物抑制脂氧合酶成分之區分    36
| 4.1. 5 kDa 分子量區分    36
| 4.2. Sephadex G-25 膠體過濾    36
| 4.3 醣含量測定    37
| 4.4 胜肽含量測定    37
| 4.5 區分物純度鑑定    38
| 4.6 LC-MS 分析    38
| 5. 台灣蜆萃取物植物固醇分析    39
| 5.1 皂化    39
| 5.2 GC-MS 分析    39
| 6. 統計分析    40
| 伍、結果討論    41
| 一、攝食蜆萃取物對吳郭魚之影響    41
| 1. 體重與體長    41
| 2. 血脂濃度    41
| 3. 血漿與 LDL 總抗氧化力    44
| 4. LDL 中 α-tocopherol 含量    46
| 5. LDL脂肪酸組成    47
| 二、In vitro 蜆萃取物抑制 LDL 氧化能力    55
| 1. 吳郭魚 LDL    55
| 2. 人 LDL    58
| 三、蜆萃取物抑制脂氧合酶之有效成分    63
| 1. 蜆萃取物對 15-LOX 之抑制效果    63
| 2. 分子量 5 kDa 區分    64
| 3. Sephadex G-25 膠體過濾    65
| 4. 蜆萃取區分物之 RP-HPLC 分離    68
| 5. LC/MS    70
| 四、蜆萃取物之植物固醇類    71
| 柒、參考文獻    77
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2009 年 11 月 22 日 23:18