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詳目顯示區塊

第 10 筆
| (以作者名查詢海洋大學圖書館館藏系統)
| (以作者名查詢全國圖書書目資訊網)
| (以作者名查詢全國博碩士系統)
論文名稱: | 吳郭魚為動物模式評估一條根降血脂、降膽固醇功能及抗發炎與抗環境緊迫之功能
| Development of tilapia as an animal model for evaluation of I-Tiao-Gung (Glycine tomentella) on hyperlipidemia, hypercholesterolemia, inflammation and environmental stress
研究生: | 陳翠瑤 Tsui-Yao Chen
指導教授: | 孫寶年 Bonnie Sun Pan
學位類別: | 博士
學校名稱: | 國立臺灣海洋大學
系所名稱: | 食品科學系
學號: | D90320004
畢業學年度: | 94
語文別: | 中文
關鍵字: | 一條根 I-Tiao-Gung, Glycine tomentella
| 吳郭魚 tilapia
| 抗發炎 anti-inflammation
| 降血脂 hypolipidemia
| 降膽固醇 hypocholesterolemia

全文影像連結:

論文全文(pdf)

論文頁數: | 116
摘要: | 摘 要
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| 金門產闊葉大豆屬一條根 (Glycine tomentella Hayata) 民間用於治療風濕、關節炎、筋骨酸痛，但其藥理作用未明。本研究先以體外試驗 (in vitro) 探討一條根之抗氧化及抗發炎作用，再以吳郭魚飼養試驗 (in vivo)，探討一條根之生理活性，建立以吳郭魚為模式動物初步篩選保健食品之功效。
| 一條根 95% 乙醇萃取物 (GT-E) 抑制亞麻油酸自氧化之 IC50 為 4.6 μg/mL，抗氧化力是水溶性抗氧化劑 trolox (IC50=14.2 μg/mL) 的 2.3 倍。清除 DPPH 的 EC50 為 18.4 μg/mL，與 trolox 相似 (17.1 μg/mL)。GT-E 抑制誘發型環氧合酶 (cyclooxygenase-2；COX-2) 的 IC50 為 42.0 μg/mL，較 indomethacin 高 (0.61 μg/mL)，抑制 5-脂氧合酶 (5-lipoxygenase； 5-LOX) 的 IC50 為 0.40 μg/mL，較 nordihydroguaiaretic acid (NDGA) 的低 (2.3 μg/mL)。
| 以商用水產飼料 (蛋白質 53.0%，脂質 7.4%，灰分11.4%、粗纖維 1%，碳水化合物 21.4%，熱量 3194 kcal/kg) 餵食體重 185.0±19.3 g 雜交種吳郭魚 (Oreochromis niloticus × O. aureaus)  1.6 % body mass/day ，8 週後吳郭魚出現高血脂症及高膽固醇血症，血漿總三酸油酯為 1218.6±79.2 mg/dL，總膽固醇為740.3±276.3 mg/dL，低密度脂蛋白-膽固醇 (low density lipoprotein-cholesterol ; LDL-C) 392.1±56.3 mg/dL，另以相同條件飼養體重約 60 g 的莫三比克吳郭魚 (Oreochromis mossambicus) ，餵食 2% body mass/day 8 週後，血漿三酸甘油酯變為原來的 10.6倍，總膽固醇為 4.5 倍，LDL-C 為5.9 倍，與餵食 1% body mass/day  12 週後，均發生高血脂症及高膽固醇血症。
| 餵食體重 59.5± 9.5 g 雄性純種莫三比克吳郭魚 (Oreochromis mossambicus) 成魚，實驗組16尾，在飼料中添加1% GT-E 及控制組16 尾，餵食1.0% body mass/day 12 週後，GT-E 組較控制組之血漿三酸甘油酯低41.9%，膽固醇低30.4%、LDL-C也低 36.1%，均具有顯著差異 (p<0.05)。GT-E組血漿總抗氧化力 (total antioxidant status ; TAS) 顯著高於控制組 (p<0.05)，其 LDL 粒子內的α-tocopherol 含量為 13.3±3.0 α-tocopherol molecule/LDL ，顯著高於控制組的 8.8±2.5 α-tocopherol molecule/LDL (p<0.05)。
| 以銅離子誘導LDL氧化之 in vitro 試驗，GT-E (3.2 μg/mL) 延長 LDL 氧化遲滯期的時間接近 trolox 0.5 μg/mL 的效果。離體 (ex vivo) 實驗， GT-E 組較控制組的 LDL 氧化遲滯期長，氧化速率較慢，其LDL 脂肪酸組成中，單元不飽和脂肪酸較控制組高、多元不飽和脂肪酸則較低，餵食 GT-E吳郭魚可保護 LDL 延緩氧化，並改變 LDL 脂肪酸的組成。
| 吳郭魚在氯化銨環境緊迫下血漿之 LOX 活性上升，攝食 GT-E 可抑制 LOX 活性。以 40 尾雄性雜交種吳郭魚成魚，分別餵食添加GT-E 飼料之實驗組 (20 尾)、與控制組 ( 20 尾) 12 個月後，曝露於 100 ppm 氯化銨水中，GT-E組與控制組的血比容積、血糖、AST、ALT 均無顯著變化，且兩組之間無顯著差異，但兩組的滲透壓在曝露氯化銨12 及 24 小時均較未曝露者低。在曝露至12 小時兩組吳郭魚的血小板5-LOX 活性明顯增加，控制組的活性顯著高於 GT-E 組 (p<0.05)，其 12-LOX 及 15-LOX 活性也上升，GT-E 組的 12-LOX 及15-LOX則無明顯增加 (p>0.05)，吳郭魚攝取GT-E 後在環境緊迫時，較能維持正常生理狀況。
| 整體而言，GT-E 可抑制 LDL 氧化、 LOXs 及COX-2 的活性，應具有抗發炎及抗動脈粥狀硬化的功效。GT-E 在ex vivo 及 iv vivo 實驗中，對高膽固醇血症及高血脂症的吳郭魚有降血脂、保護血漿中 LDL，延緩氧化的功效。由吳郭魚會發生高血脂症，其脂蛋白形態、脂肪酸代謝與膽固醇合成及利用類似哺乳動物，吳郭魚可做為評估降血脂的實驗動物，初步篩選具有降血脂功效的保健食品。
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| Abstract
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| I-Tiao-Gung (Glycine tomentella Hayata) has been wildly cultured in Kinmen and used as a folk medicine for treatment of rheumatism, gout, arthritis, and ache of physique indicative of its anti-inflammatory activities. But its biological activities and pharmacological functions have not been studied systematically. The purpose of this study was to elucidate the long-claimed anti-inflammatory activity of I-Tiao-Gung by in vitro test and to assess its potential effects on atherosclerosis, using tilapia as an alternative animal to evaluate the in vivo effects of I-Tiao-Gung.
| The 95% ethanol extract of I-Tiao-Gung (GT-E) showed antioxidant activity on hemoglobin-catalyzed oxidation of linoleic acid. The IC50 value was 4.6 μg/mL lower than that of trolox (IC50=14.2 μg/mL). GT-E had the ablility to scavenge 1,1-diphenyl-2- picrylhydrazyl radicals (DPPH). Its EC50 value was 18.4 μg/mL similar to trolox (17.1 μg/mL). GT-E inhibited tilapia thrombocyte (the nucleated platelet) 5-, 12-, 15-lipoxygenase (LOX). The IC50 values were 0.43, 0.72 and 0.42 μg/mL respectively, while those of nordihydroguaiaretic acid (NDGA) on 5-, 12-, 15-LOX were 2.3, 1.6, and 1.7 μg/mL respectively. The IC50 on cyclooxygenase-2 (COX-2) inhibition by GT-E was 42.0 μg/mL while that for indomethacin (as a positive control) was 0.61 μg/mL. GT-E showed antioxidant activity by prolonging the lag phase (△Tlag) of human LDL oxidation induced by Cu2+ in a dose-dependent manner. The △Tlag of LDL added with 3.2 μg/mL GT-E was similar to that with trolox at 2.0 μM (ca. 0.5 μg/mL ).
| Hybrid tilapia (Oreochromis niloticus × O. aureaus), body mass 185.0±19.3, were fed daily with a commercial fish feed (protein  53.0%, lipid 7.4%, ash 11.4%, crude fiber 1%, carbohydrate 21.4%, total energy 3194 kcal/kg) at 1.6% body mass for 8 weeks. The plasma total triacylglycerol increased to 1218.6±79.2 mg/dL, total cholesterol increased to 740.3±276.3 mg/dL, and low density lipoprotein (LDL-C) increased to 392.1±56.3 mg/dL, respectively. Hyperlipidemia and hypercholesterolemia were found in fish. Mossambicus tilapia (Oreochromis mossambicus) (n=32), body mass 60±6.4 g, cultured with the same condition, fed daily 2% body mass for 8 weeks, and 1% body mass for 12 weeks were found hyperlipidemia and hypercholesterolemia. Matured male mossambicus tilapia  (body mass 59.5±9.5, n=32) were randomly divided into two groups and fed with feed containing 1% (w/w) GT-E for 12 weeks. Total triacylglycerol of the GT-E group was lower than that of the control group by 41.9%, total cholesterol was lower by 30.4%, LDL-C was lower by 36.1%. Plasma total antioxidant status (TAS) of the GT-E group was higher than that of the control group by 79%. GT-E fed group had longer lag phase of Cu2+-induced LDL oxidation and retained significantly more α-tocopherol (13.3±3.0 α-tocopherol molecule/LDL) in LDL particles than the control group  (8.8±2.5 α-tocopherol molecule/LDL). LDL from the GT-E group had more monounsaturated fatty acids and less polyunsaturated fatty acids than the control, indicating that GT-E may affect fatty acids metabolism. These evidences demonstrated that GT-E had hypolipidemic and hypocholesterolemic effects.
| Using tilapia exposed to ammonium chloride to evaluate the fuction of GT-E on anti-environmental stress. Hybrid male tilapia (n=40) weighed 185 g were randomly divided into two groups and fed with feed either containing 1% (w/w) GT-E or control diet for 12 months than exposed to 100 ppm ammonium chloride for up to 24 hours. Both groups decreased in osmosis measured at 12 and 24 hours. 5-LOX activity increased when exposed for 12 hours. Thrombocyte 12- and 15-LOX increased in the control group but not in the GT-E group. Intake of GT-E seemed to retard the changes in tilapia exposed to stress as abnormal concentration of ammonium chloride.
| The prevention of LDL oxidation and the dual inhibition of LOXs and COX-2 are indicative of the possible roles of GT-E on anti-atherosclerosis and anti-inflammation. Since GT-E inhibited LDL oxidation of tilapia in vitro, ex vivo and in vivo, GT-E had hypolipidemic and hypocholesterolemic effects in tilapia similar to that previously reported in hamster. Tilapia can serve as a surrogate animal model for prescreening hypocholesterol and anti-atherosclerosis effects of nutraceuticals and health foods.
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論文目次: | 目  錄
| 中文摘要……………………………………………………………......….I
| 英文摘要….…..………………………………….…………………….....IV
| 英文縮寫對照...…………………………………..……………………XVII
| 一、前言    1
| 二、文獻回顧    5
| 1. 一條根生理活性    5
| 1.1  一條根來源    5
| 1.2 一條根的成分與藥理作用    5
| 1.3  一條根之藥理活性    5
| 1.4 闊葉大豆 (金門產一條根) 的毒性試驗    6
| 2. 動脈粥狀硬化發生機制    7
| 2.1  LDL氧化與發炎作用    12
| 2.2 血小板的活化    14
| 2.2.1 血小板的 LDL receptor    14
| 2.2.2 血小板TXA2 作用與濃度變化    15
| 2.2.3 血小板細胞內Ca 2+ 濃度 (［Ca2+］) 變化    15
| 2.3  LDL對血小板凝集之影響    16
| 2.3.1活化Rho kinase    16
| 2.3.2 增加 fibronectin 的表現    16
| 2.4 蛋白酶與粥狀動脈硬化    17
| 3. 脂氧合酶與動脈粥狀硬化的關係    18
| 3.1  5-LOX 與發炎反應    18
| 3.2  巨噬細胞 12/15-LOX 的表現    19
| 3.3  巨噬細胞 LOX 對 LDL的氧化作用    21
| 3.4  LOX 抑制劑之作用    21
| 4. 多酚類化合物預防動脈粥狀硬化之研究    22
| 4.1 多酚類化合物的分類與來源    22
| 4.2 抗氧化及抗發炎能力    23
| 4.3 降低血漿總膽固醇及 LDL-C 之能力    24
| 4.4 保護內皮細胞 (endothelium；EC) 的功能    25
| 4.5 抗發炎及抑制血管新生的功能    26
| 5. 應用於動脈粥狀硬化研究之動物模式    26
| 5.1 兔子    27
| 5.2 基因轉殖小鼠    28
| 6. 魚類生化學變化    29
| 6.1 環境緊迫的生理變化    29
| 6.2 魚類動物實驗    30
| 7. 魚類動脈粥狀硬化的研究    31
| 7.1 循環系統    31
| 7.2 鮭魚、鱒魚動脈粥狀硬化    32
| 8.1 吳郭魚生長條件    33
| 8.1.1 溫度    33
| 8.1.2 水質    33
| 8.1.3 營養需求    33
| 8.2 吳郭魚血液生化特性    34
| 三、材料與方法    35
| 1. 材料    35
| 1.1 一條根95 %乙醇萃取物 (GT-E) 之製備    35
| 1.2 人的血漿    36
| 1.3 吳郭魚血漿    36
| 1.4 吳郭魚餵食試驗 (一).    36
| 1.5 吳郭魚餵食試驗 (二)    37
| 1.6 吳郭魚餵食試驗 (三)    37
| 1.7 吳郭魚氯化銨毒性試驗    37
| 2. 方法    38
| 2.1 一條根 95% 乙醇萃取物抗氧化能力分析    38
| 2.1.1 抑制亞麻油酸自氧化之能力    38
| 2.1.2 清除 DPPH 自由基之能力    38
| 2.1.3 一條根抑制 LDL 氧化之能力    39
| 2.1.3.1 血漿 LDL 的分離    39
| 2.1.3.2 電泳檢測 LDL 分離效果    39
| 2.1.3.3  LDL 透析    40
| 2.1.3.4  抑制 Cu2+ 誘導LDL 氧化之能力    40
| 2.2 一條根 95% 乙醇萃取物異黃酮含量    40
| 2.3 一條根95% 乙醇萃取物抑制COX-2 之能力    41
| 2.4血球個數    42
| 2.4.1 紅血球數 (red blood cell count；RBC)    42
| 2.4.2. 白血球數 (white blood cell count；WBC)    42
| 2.4.3 血色素 (hemoglobin, HGB)    42
| 2.4.4血比容積 (hematocrit；Hct)    42
| 2.5 血漿生化數值分析    43
| 2.5.1 血糖 (plasma glucose)    43
| 2.5.2 血漿膽固醇 (plasma cholesterol)    43
| 2.5.3  plasma HDL-C    43
| 2.5.4  plasma LDL-C    44
| 2.5.5  plasma total antioxidant capacity (TAC)    44
| 2.5.6 三酸甘油脂 (triacylglycerol；TG)    45
| 2.5.7 血小板脂氧合酶活性測定    45
| 2.5.7.1. 血液脂氧合酶粗酵素液製備    45
| 2.5.7.2. 蛋白質定量    45
| 2.5.7.3. 高效能液相層析法    46
| 2.5.8  ALT (alanine aminotransferase ；EC 2.6.1.2)    46
| 2.5.9  AST (aspartate aminotransferase ；EC 2.6.1.1)    47
| 2.5.10  LDL α-tocopherol 含量    47
| 2.5.11  LDL 脂肪酸組成分析    48
| 2.6 統計分析    49
| 四、結果    50
| 1 GT-E抗氧化能力    50
| 1.1 抑制血紅素催化亞麻油酸自氧化及清除 DPPH    50
| 1.2  GT-E抑制 cyclooxygenase-2、5-lipoxygenase    50
| 1.3  GT-E 抑制 12-，15-lipoxygenase    51
| 2.1 吳郭魚及健康男性血漿LDL 之分離    52
| 2.2  Cu2+ 誘導吳郭魚LDL 氧化之條件    52
| 2.3  GT-E 抑制人與吳郭魚血漿 LDL 氧化之能力    55
| 2.3.1. 延長氧化遲滯期    55
| 2.3.2 降低 propagation phase 之氧化速率    57
| 3  GT-E 之異黃酮含量    60
| 4. 餵食一條根飼料對吳郭魚之影響    62
| 4.1 雜交吳郭魚 (Oreochromis. niloticus × O. aureus) 之高血脂症    62
| 4.2  GT-E對雜交種吳郭魚降血脂之功效    63
| 4.3 莫三比克吳郭魚 (Oreochromis mossambicus) 之高血脂症    64
| 4.4  GT-E 對莫三比克吳郭魚降血脂之功效    67
| 4.5  GT-E 組投餵控制組飼料    68
| 4.6  GT-E 抑制LDL氧化的ex vivo試驗    68
| 4.7  GT-E 對吳郭魚 LDL 脂肪酸組成的影響    70
| 5. 餵食GT-E對魚類抗發炎抗緊迫之影響    72
| 5.1  LOX 活性    72
| 5.2 血比容    74
| 5.3血糖    74
| 5.4 滲透壓    75
| 5.5  肝功能    75
| 五、討論    77
| 1. 一條根的抗發炎功效    77
| 2. 抗環境緊迫功效    78
| 3 一條根降血脂功效    80
| 4  降膽固醇功效    81
| 5. 一條根預防動脈粥狀硬化功效    83
| 6. 吳郭魚動物模式試驗之可行性    85
| 六、結論    88
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| 表目錄
| 表 1-1、美國國家膽固醇教育計畫 (NCEP) 高血脂之分類    8
| 表 3-1、一條根 95% 乙醇萃取物抑制亞麻油酸及清除自由基的能力    50
| 表3-2、一條根 95% 乙醇萃取物與indomethancin 抑制 cyclooxygenase-2 及 NDGA 抑制吳郭魚血小板脂氧合酶活性之差異    51
| 表 3-3、GT-E 濃度改變銅離子誘導人體 LDL氧化之氧化速率及共軛雙烯最大生成量之變化    59
| 表 3-4、GT-E 濃度改變銅離子誘導吳郭魚 LDL氧化之氧化速率及共軛雙烯最大生成量之變化    60
| 表 3-5、餵食吳郭魚添加 GT-E 飼料 8 週後體重、血球數及血比容    62
| 表 3-6、飼料餵食量對吳郭魚血漿中血糖、總膽固醇、三酸甘酸酯的影響    63
| 表 3-7、餵食量對吳郭魚飼養八週後體重及血漿生化指標之影響    65
| 表 3-8、實驗動物發生高血脂症之誘導及其血漿三酸甘油酯、膽固醇的含量與 LDL 氧化遲滯期    66
| 表 3-9、飼料添加GT-E 餵食吳郭魚 12 週後血糖、三酸甘油酯及膽固醇含量的變化    67
| 表 3-10、吳郭魚餵食 GT-E 飼料6 個月後，再以控制組飼料餵食二個月血糖、血漿總膽固醇、三酸甘油酯含量    68
| 表 3-11、吳郭魚餵食GT-E 飼料12 週後血漿總抗氧化力、LDL α-tocopherol 含量與銅離子誘導LDL 氧化參數之變化    70
| 表3-12、吳郭魚餵食一條根血漿 LDL 脂肪酸組成    71
| 表3-13、 吳郭魚餵食一條根曝露於 100 ppm 氯化銨血小板之脂氧合酶活性的變化    73
| 表 3-14、吳郭魚餵食一條根曝露於 100 ppm 氯化銨血比容的變化    74
| 表 3-15、吳郭魚餵食一條根曝露於 100 ppm 氯化銨血糖和血漿滲透壓的變化    75
| 表3-16、吳郭魚餵食一條根曝露於 100 ppm 氯化銨肝功能指數的變化    76
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| 圖目錄
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| 圖 1-1、動脈粥狀硬化形成過程的「損傷反應假說」    10
| 圖 1-2、動脈粥狀硬化形成之氧化修飾圖。    12
| 圖 2-1、闊葉大豆屬Glycine tomentella 一條根    35
| 圖 2-2 莫三比克吳郭魚    36
| 圖 3-1、人及吳郭魚血漿脂蛋白在 agarose 膠片電泳上的分佈    52
| 圖 3-2、 Cu2+ 誘導健康人禁食後血漿 LDL 氧化之 A 232 nm 隨時間之變化。    53
| 圖 3-3、 Cu2+ 誘導吳郭魚LDL 氧化隨時間變化之 A 232 nm 。    55
| 圖 3-4、GT-E 抑制銅離子誘導 LDL 氧化之效果    56
| 圖 3-6、GT-E 濃度對延長銅離子誘導血漿 LDL 氧化遲滯期之影響    58
| 圖 3-7、 RP-HPLC 圖譜 (A) 異黃酮標準品 (B) GT-E    61
| 圖 3-8、吳郭魚餵食 GT-E 血漿 LDL 受銅離子誘導氧化的變化    69
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2009 年 11 月 22 日 23:31