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論文名稱: | 台灣蜆 (Corbicula fluminea) 萃取物抗氧化成分之鑑定與一條根 (Glycine tomentella)組合物對降血脂之影響
| Identification of antioxidant activity component from clam extracts (Corbicula fluminea) and effect of I-Tiao Gung (Glycine tomentella) combination on hyperlipidemia
研究生: | 蕭欣宜 Sin-Yi Hsiao
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: | M95320066
畢業學年度: | 96
語文別: | 中文
關鍵字: | 台灣蜆 clam extracts
| 一條根 I-Tiao Gung
| 組合 combination
| 抗氧化 antioxidant
| 降血脂
全文影像連結: | | (本論文20100730對校內公開)
| 論文全文(pdf)

論文頁數: | 73
摘要: | 台灣蜆 (Freshwater clam, Corbicula fluminea) 之熱水萃取物具有抑制15-LOX活性、延長 LDL 氧化遲滯期之功效，並與劑量呈正相關。一條根萃取物可抑制COX-2及5-LOX的活性、與亞麻油酸自氧化作用並可清除DPPH自由基。合併使用兩種以上抗氧化物，可能相乘作用。本研究即以蜆熱水萃取物 (FC-HW) 為基礎，與一條根乙醇萃取物 (GTE) 組合，並以吳郭魚為模式動物，探討組合物抗氧化、降血脂、降膽固醇、抑制LDL氧化之功效。
| 吳郭魚 (70.5 ± 8.8 g) 餵食等熱量 (3756.8 ± 39.2 kcal/kg) 等蛋白含量 (59.8± 0.6%) 之飼料，分別添加2% FC-HW 與0、0.08 或0.16% GTE之飼料共四組，餵食12週，以飼料中含2% FC-HW組具降低血漿三酸甘油酯、總膽固醇、LDL膽固醇，提高HDL膽固醇，增加血漿及LDL總抗氧化力之功效。隨飼料中GTE添加量增加，降血脂功效及總抗氧化力較2% FC-HW組下降。吳郭魚攝食FC-HW 與GTE組合物後，肝臟中脂質含量減少，飽和脂肪酸 (SFA) 及單元不飽和脂肪酸 (MUFA)增加。吳郭魚血漿LDL離體後極易自氧化，以銅離子誘導之ex vivo氧化試驗，無氧化遲滯期，連鎖反應期具兩段氧化速率 (△A234 nm / min)，兩段氧化速率皆以2% FC-HW與0.08% GTE 組顯著最低。
| 取人血漿LDL以銅離子誘導之in vitro氧化試驗，添加FC-HW或GTE皆可延長LDL氧化遲滯期，並功效與劑量呈正相關。FC-HW與GTE組合時，在FC-HW 濃度高於160 μg/mL與GTE 濃度高於9 μg/mL時開始具有相乘作用，並以FC-HW 240 μg/mL與GTE 9 μg/mL 相乘作用較強。
| FC-HW 與GTE皆能抑制15-LOX活性，IC50 分別為170.79 μg/mL 及9.21 μg/mL。分別取FC-HW 與 GTE低於IC50之濃度組合，當FC-HW 濃度高於80 μg/mL與GTE 濃度高於6 μg/mL 對15-LOX抑制效果大於50%。
| FC-HW以膜過濾，得分子量小於5 kDa區分 (FC-HW-2) 其延緩LDL氧化遲滯期及降低LDL氧化速率較FC-HW有效。再以Sephadex G-25管柱分離，得5個次區分物，其中分子量200~300 Da之次區分物抑制15-LOX活性效果最強，inhibitory efficiency ratio (IER) 為2548.9 %/mg/mL。
| 取蜆肉乙酸乙酯及蜆熱水萃取物乙酸乙酯萃取，其中較親脂溶性之區分，以 GC-MS初步鑑定，有膽固醇 (Cholesterol) 及膽固醇衍生物，尚有植物固醇類麥角固醇 (Ergosterol) 衍生物、豆甾醇 (Stigmasterol) 及谷甾醇 (Sitosterol)，可能與蜆熱水萃取物降血脂、降膽固醇之功效有關。
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| The hot water extract of freshwater clam (Corbicula fluminea) (FC-HW) has inhibitory effect against 15-lipoxygenase (LOX) activities, protecting low-density lipoprotein (LDL) from oxidation in a dose dependent maner. The 95% ethanol extract I-Tiao-Gung (Glycine tomentella Hayata) (GTE) has inhibitory effect on 5-LOX, COX-2, hemoglobin-catalyzed oxidation of linoleic acid and scavenging 1,1-diphrnyl-2-picrylhydrazyl radical (DPPH). Combination of two antioxidants may have synergistic effect. The purpose of this study was using an animal model to elucidate the combination effect of FC-HW and GTE on antioxidative hypolipidemic, hypocholesterolemic activities, and prolongation of lag phase of LDL oxidation and inhibitory 15-LOX activity.
| Tilapia (70.5 ± 8.8 g) were fed isocalorie (3756.8 ± 39.2 kcal/kg) and feeds with protein content (59.8± 0.6%) containing 2% (w/w) FC-HW combined with 0, 0.08 or 0.16% (w/w) GTE and cultured for test groups for 12 weeks. The group fed with 2% FC-HW was hyperlipidemia, hypercholesterolemia, increasing high density lipoprotein cholesterol (HDL-C) and total antioxidant capacity (TAC) of plasma or LDL cholesterol. Tilapia fed with feeds containing FC-HW and GTE reduced liver lipid content, increased saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) in liver lipid. Tilapia LDL was oxidized in vitro by Cu2+-induction. Tilapia LDL oxidation was used as an ex vivo model. Tilapia LDL did not have noticeable lag phase, in oxidation while the propagation phase was divided into two stages because of the different oxidative rates (△A234 nm / min). The 2% FC-HW and 0.08% GTE composite yielded oxidation rater the lowest both at the first and the second propagation stage significantly lower than those in the control group.
| Cu2+-induced human LDL oxidation was used as an in vitro model. Both FC-HW and GTE prolonged the lag phase of LDL oxidation in a dose dependent maner. FC-HW and GTE composite had synergistic antioxidative effect as FC-HW was ≧ 160 μg/mL and GTE ≧ 9 μg/mL. The composite of FC-HW 240 μg/mL and GTE 9 μg/mL had the best synergistic effect.
| Both FC-HW and GTE inhibited the 15-LOX activity of which the IC50 was 170.79 μg/mL and 9.21 μg/mL, respectively. Combined the FC-HW and GTE lowered their IC50 on 15-LOX activity.
| The active fraction of FC-HW had molecular weight＜5000 Da (FC-HW-2) which prolonged lag phase of human LDL oxidation more effective than FC-HW. FC-HW-2 fractionated by Sephadex G-25 gel filtration got 5 sub-fractions, of which the molecular weight between 300 ~ 400 Da had the strongest inhibitory efficiency against 15-LOX activity. It inhibitory efficiency ratio (IER) was 2548.9 %/mg/mL.
| The lipophilic fractions of FC or FC-HW extracted with EA, was tentatively identified with GC/MS using authentic standards as references. Cholesterol, cholesterol derivatives, phytosterols including ergosterol derivatives, stigmasterol and sitosterol were found in FC and FC-HW. The phytosterols may be responsible for the hypocholesterolemic effects.
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論文目次: | 目錄
| 目錄    I
| 圖目錄    IV
| 表目錄    V
| 中文摘要    VII
| 英文摘要    IX
| 縮寫表    VII
| 壹、前言    1
| 貳、文獻整理    3
| 一、相乘作用 (Synergistic action)    3
| 二、蜆    4
| 三、蜆萃取物之生理活性    4
| 1. 抗氧化    4
| 2. 降血脂    5
| 3. 降血壓    5
| 4. 保肝    6
| 5. 抗腫瘤    6
| 四、一條根及其萃取物之生理活性    6
| 五、高血脂症    7
| 1. 低密度脂蛋白 (low-density lipoprotein, LDL)    8
| 2. 脂氧合酶 (lipoxygenase, LOX) 與LDL氧化    9
| 參、實驗架構    10
| 一、台灣蜆及一條根萃取物之組合物對吳郭魚之影響    10
| 二、台灣蜆及一條根萃取物組合抗氧化之功效    10
| 三、台灣蜆熱水萃取物之初步分離    11
| 肆、材料與方法    12
| 一、材料    12
| 二、方法    13
| 1. 吳郭魚養殖試驗    13
| 2. 採血    14
| 3. 肝臟及心臟組織切取    14
| 4. 血漿一般生化指標    14
| 4.1. 血糖 (glucose)    14
| 4.2. 三酸甘油酯 (triacylglycerol, TG)    15
| 4.3. 膽固醇 (cholesterol)    15
| 4.4. 血漿HDL-C與LDL-C    16
| 5. 血漿總抗氧化力 (Total Antioxidant Capacity, TAC)    17
| 6. 脂質萃取及脂肪酸組成分析    18
| 7. 抑制Cu2+ 誘導 LDL 氧化    19
| 7.1. 血漿LDL分離    19
| 7.2. LDL的透析    20
| 7.3. Cu2+ 誘導LDL 氧化    21
| 8. 冷光法測定15-脂氧合酶活性    22
| 9. 蜆萃取物抗氧化成分之區分    23
| 9.1. 濾膜過濾    23
| 9.2. Sephadex G-25 膠體過濾    23
| 9.3. 胜肽含量測定    24
| 9.4. Reverse-phase HPLC 分析    24
| 10. 台灣蜆萃取物植物固醇分析    25
| 10.1. 油溶性物質萃取    25
| 10.2. 皂化    25
| 10.3. 衍生化    25
| 10.4. 衍生化物質之薄層層析 (TLC)    25
| 10.5. GC/MS 分析    26
| 伍、結果與討論    27
| 一、攝食台灣蜆及一條根萃取物之組合物對吳郭魚之影響    27
| 1. 成長速率    27
| 2. 血脂濃度    27
| 3. 脂蛋白膽固醇濃度    30
| 4. 低密度脂蛋白    31
| 5. 血漿及LDL總抗氧化力    34
| 6. 肝臟脂肪酸組成    36
| 二、台灣蜆及一條根萃取物組合之功效    42
| 1. 抑制15-LOX活性    42
| 2. 延緩LDL氧化    45
| 三、台灣蜆熱水萃取物之分離    51
| 1. Sephadex G-25膠體過濾    51
| 2.    延緩LDL氧化    52
| 3. 抑制15-LOX 活性    55
| 4. 蜆萃取物之RP-HPLC分離    56
| 四、台灣蜆之固醇類    58
| 陸、結論    62
| 柒、參考文獻    64
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| 圖目錄
|
| 圖一、吳郭魚攝食台灣蜆萃取物 (FC-HW) 與一條根萃取物 (GTE) 之 組合12週對LDL ex vivo氧化之影響    33
| 圖二、魚類高度不飽和脂肪酸生合成途徑    38
| 圖三、台灣蜆萃取物 (FC-HW) 與一條根萃取物 (GTE) 之組合物與15-LOX抑制率之關係    44
| 圖四、台灣蜆萃取物 (FC-HW) 與一條根萃取物 (GTE) 組合比例對人LDL離體氧化之影響    49
| 圖五、台灣蜆萃取物 (FC-HW)、一條根萃取物 (GTE) 與人LDL 離體氧化遲滯期之關係    50
| 圖六、台灣蜆萃取物分子量小於5 kDa (FC-HW-2) 之 Sephadex G-25層析圖譜    52
| 圖七、台灣蜆萃取物FC-HW-2-D及E 之 RP-HPLC 圖譜    56
| 圖八、蜆肉及蜆熱水萃取物脂質區分中非皂化物之TLC薄層層析圖....................58
| 圖九、台灣蜆肉脂質區分中非皂化物之GC/MS圖譜    59
| 圖十、台灣蜆熱水萃取物脂質區分中非皂化物物之GC/MS圖譜    60
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| 表目錄
| 表一、飼料配方    13
| 表二、吳郭魚攝食台灣蜆萃取物 (FC-HW) 與一條根萃取物 (GTE) 之組合物十二週後體重、體長、肝體比、心體比及比成長率之變化    28
| 表三、吳郭魚攝食台灣蜆萃取物 (FC-HW) 與一條根萃取物 (GTE) 之組合物十二週後血糖、血脂變化(1)    29
| 表四、吳郭魚攝食台灣蜆萃取物 (FC-HW) 與一條根萃取物 (GTE) 之組合物十二週後血漿中HDL膽固醇及LDL膽固醇之含量    30
| 表五、吳郭魚攝食台灣蜆萃取物 (FC-HW) 與一條根萃取物 (GTE) 之組合12週對LDL ex vivo氧化之影響    32
| 表六、吳郭魚攝食台灣蜆萃取物 (FC-HW) 與一條根萃取物 (GTE) 之組合物十二週後血漿與LDL總抗氧化力 (TAC) 之影響    35
| 表七、吳郭魚攝食蜆萃取物與一條根萃取物組合之飼料12週後肝臟體比與肝臟萃油率之比較    36
| 表八、實驗飼料之脂肪酸組成    40
| 表九、吳郭魚攝食蜆萃取物與一條根萃取物組合之飼料12週後肝臟脂肪酸組成    41
| 表十、台灣蜆萃取物 (FC-HW) 及一條根萃取物 (GTE) 對15-LOX之半抑制濃度 (IC50)    42
| 表十一、台灣蜆萃取物 (FCHW) 與一條根萃取物 (GTE) 之組合物對15-LOX之抑制率    43
| 表十二、蜆萃取物 (FC-HW)與一條根萃取物 (GTE) 對人LDL in vitro氧化遲滯期、氧化速率及最大氧化程度之影響    46
| 表十三、蜆萃取物 (FC-HW) 與一條根萃取物 (GTE) 的組合對人LDL in vitro氧化遲滯期、氧化速率及最大氧化程度之影響    47
| 表十四、蜆萃取物 (FC-HW) 經Sephadex G-25膠體過濾之區分物對對人LDL in vitro氧化遲滯期、氧化速率及最大氧化程度之影響    54
| 表十五、台灣蜆萃取物分子量小於5 kDa 之區分物 (FC-HW-2-A~E) 對15- LOX 之抑制效果    55
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2009 年 11 月 22 日 23:11