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論文名稱: | 台灣蜆(Corbicula fluminea)與一條根(Glycine tomentalle)組合物對肝細胞、大鼠及吳郭魚肝臟抗氧化酵素活性之影響
| Effects of freshwater clam (Corbicula fluminea) and I-Tiao-Gung (Glycine tomentella) extracts on the antioxidant enzymes activity of clone-9 rat hepatocyte cultures, rat and tilapia liver.
研究生: | 謝欣瑜 Hsin Yu Hsieh
指導教授: | 邱思魁博士 Tze-Kuei Chiou, Ph. D.
| 孫寶年博士 Bonnie Sun Pan, Ph. D.
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: | M95320067
畢業學年度: | 96
語文別: | 中文
關鍵字: | 台灣蜆 Corbicula fluminea
| 一條根 Glycine tomentalle
| 抗氧化酵素 antioxidant enzymes
全文影像連結: | | (本論文20110729對校內公開)
| 論文全文(pdf)

論文頁數: | 102
摘要: | 本論文以台灣蜆與一條根組合物評估其對肝臟抗氧化酵素活性之影響。台灣蜆 (Freshwater clam, Corbicula fluminea) 熱水萃取物具抗氧化、保肝及抗癌的效果，水解物可降血壓。一條根 (Glycine tomentalle) 95% 乙醇萃取物(GTE)具抗氧化性、抗發炎及抗環境緊迫之功效，兩者組合可能成為多功效之保健食品。本研究以肝細胞 (clone-9)、大鼠與吳郭魚為試驗模式，分別給予化學性急性、慢性及物理性急性緊迫，以抗氧化酵素活性為指標，探討組合物對細胞與動物受到緊迫時的影響，以評估組合產品之功效。
| 花蓮(H-)、彰化地區(C-) 之台灣蜆熱水萃取物 (FC-HW) 及其乙醇區分 (FC-HW-E) 分別與GTE組合，以FC-HW 1.42~6.11 mg/mL與GTE 0.07~0.28 mg/mL混合清除DPPH自由基具相乘性，清除率達60%；FC-HW與GTE濃度比為20：1，DPPH自由基清除率最大；FC-HW-E 0.47~2.35 mg/mL與GTE 0.07~0.14 mg/mL混合清除DPPH自由基具相乘性，清除率達65%，FC-HW-E與GTE濃度比為8：1，DPPH自由基清除率最大，以清除率較高的組合進行細胞、大鼠與吳郭魚的實驗。細胞模式中，單一萃取物或組合物與clone-9培養48 h，細胞存活率皆大於80%。單一萃取物或組合物與細胞培養3或6 h後，以10 mM CCl4刺激2 h，FC與GTE皆可降低肝細胞GPT活性，組合物降低GPT活性的能力較單一萃取物強，但組合物與單一萃取物與細胞培養12 h，降低GOT之效果無差異；在CCl4損傷的細胞，SOD活性較低，蜆萃取物可提高受損細胞的SOD活性，H-FC-HW或H-FC-HW-E與GTE組合物提高抗氧化酵素活性的能力較單一萃取物強。
| 大鼠餵食蜆肉乙醇區分(C-FC-E)、蜆肉乙酸乙酯區分(C-FC-EA)、GTE 及 C-FC-E/GTE 2 週後，同時以每週三次腹腔注射DMN誘導肝纖維化，持續4週後，負控制組 GPT、GOT活性受 DMN 損傷而上升，餵食 C-FC-E 及組合物後 GPT 顯著降低，效果較 silymarin 強，C-FC-EA 可降低 GOT；慢性 DMN 損傷的大鼠肝臟抗氧化酵素活性上升，餵食蜆肉脂溶性萃取物後，抗氧化酵素活性較負對照組低且趨於控制組，餵食組合物的抗氧化酵素活性低於單一萃取物且趨於控制組；餵食蜆肉脂溶性萃取物大鼠肝臟TBARS含量亦顯著降低。
| 吳郭魚動物實驗分兩部份。實驗一，吳郭魚 (70.52 ± 8.84 g) 飼料內添加 2% H-FC-HW，再分別加入0%、0.08%、0.16% GTE，餵食12週後，各組HSI (hepatosomatic index)、SOD、GPx、GR 及 G6PDH 的活性與控制組沒有顯著差異，僅餵食組合物組 CAT 的活性，隨著 GTE 含量的增加顯著提高。實驗二，吳郭魚 (82.19 ± 12.43 g) 餵食飼料添加 2% H-FC-HW 或 2% C-FC-HW，再加入 0%、0.1% GTE，餵食 4 週後，受急速降溫至 12℃ 緊迫 3 h，各組肝體比無顯著差異；抗氧化酵素 SOD、CAT 和 GPx 等的活性，以餵食蜆的組別高於控制組並顯著降低吳郭魚肝臟 TBARS，餵食 H-FC-HW/GTE組合物提升肝臟抗氧化酵素的活性、降低 TBARS的能力較 H-FC-HW 強，餵食C-FC-HW/GTE提升SOD、CAT 和 GPx 等的活性較C-FC-HW強，但此兩組吳郭魚肝臟 TBASR 值無顯著差異。
| FC/GTE組合物可提升急性CCl4損傷肝細胞與低溫緊迫吳郭魚的抗氧化性，減少氧化壓力，以花蓮產蜆 (H-FC) 與GTE的相乘效果較為顯著；蜆肉脂溶性萃取物可降低慢性DMN損傷大鼠肝臟的抗氧化活性及TBARS量，由細胞及大鼠模式顯示，組合物對抗氧化酵素活性的影響具有相乘效果。
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| The purpose of this thesis effects of freshwater clam (Corbicula fluminea) and I-Tiao-Gung (Glycine tomentella) extracts on the antioxidant enzymes activity. The hot water extract of freshwater clam (FC-HW) has antioxidant, hepatoprotective and anticancer effects. Clam muscle protein hydrolysates inhibited angiotensin I converting enzyme. The 95% ethanol extract of I-Tiao-Gung (GTE) showed antioxidative, anti-inflammatory and anti- stress effects. Combination of FC-HW and GTE can be multi-functional health foods. The purpose of this study was to elucidate the combined effects of FC-HW and GTE on the antioxidant enzymes activities using rat and it’s hepatocyte and tilapia as 3 test model .
| The FC cultured in Hualien (H) and in Changhua (C) were extracted with hot water to make FC-HW, which at concentration 1.42~6.11 mg/mL was mixed with GTE at concentration 0.07~0.28 mg/mL. The composite produced synergistic effect on DPPH scavenging activity being 60%. FC-HW and GTE at concentration ratio 20/1 had the highest DPPH scavenging activity. The FC-HW was extracted with 80% ethanol. Either H-FC-HW-E or C-FC-HW-E was mixed with GTE at concentration ratio 8/1 had the highest DPPH scavenging activity, which was 65%.
| Clone-9 liver cell treated with FC-HW, FC-HW-E, GTE and FC/GTE had not toxicity. Cell viability was ＞80%. Cell pretreated with different extract or composite for 3, 6 or 12 hours, then induced oxidative damage by 10 mM CCl4 for 2 hours, FC, GTE and FC/GTE significantly decreased GPT and GOT activities, but were not significanly different between treatments with FC or GTE. Cells induced oxidative damage by 10 mM CCl4 for 2 hours decreased SOD activity. Cells treated with FC for 6 hours followed by the same oxidative damage significantly increased SOD activity. The composite of H-FC-HW/GTE and H-FC-HW-E/GTE increased SOD activity more than FC or GTE alone.
| Rat fed diet containing ethanolic or ethyl acetate extract of freshwater clam (C-FC-E, C-FC-EA), GTE and C-FC-E/GTE for 2 weeks, then added with Dimethylnitrosamine (DMN) in diet for 4 weeks to induce hepatic fibrosis and increased GOT and GPT activity as the negative control group. Rat fed diet containing C-FC-E and C-FC-E/GTE significantly decreased GPT activity, more effective than diet containing silymarin as a positive control. Rat fed diet containing C-FC-EA decreased GOT activity. DMN increased antioxidant enzyme activity and TBARS in rat liver in the negative control group. Rat fed diet containing C-FC-E, C-FC-EA or C-FC-E/GTE significantly decreased liver antioxidant enzyme activity and TBARS of those in the control group. Intake of C-FC-E/GTE was more effective to decrease antioxidant enzyme activities than single treatment of either FC or GTE.
| Tilapia (70.52 ± 8.84 g) fed diets containing 2% H-FC-HW in combination with 0%, 0.08% or 0.16% GTE cultured for 12 weeks (Experiment 1) using biomarkers of HSI, SOD, GPx, GR and G6PDH activities showed no significant difference from those of the control group. CAT activity increased in the GTE-containing group. Tilapia (82.19 ± 12.43 g) fed diet as control or diets containing 2% H-FC-HW or 2% C-FC-HW in combination with 0% or 0.1% GTE cultured for 4 weeks (Experiment 2) at 24℃, then instantaneously exposed to 12℃ of water temperature for 3 hours. HSI of the 4 sample groups were not significantly different from that in control group. The liver antioxidant enzyme activities of SOD, CAT and GPx were higher and TBARS was lower in tilapia fed with FC-HW diet than those of the control group. Tilapia fed 2% H-FC-HW/GTE was more effective to increase liver antioxidant enzyme activities and lower TBARS than those of the FC group. Tilapia intaked C-FC-HW/GTE had higher SOD, CAT and GPx activities in liver than those in FC group but TBARS were not significantly different between both groups.
| FC/GTE combined increased antioxidation and decreased oxidative stress of CCl4-injured hepatocytes and anti-cold activity in cultured stress activity in cultured tilapia. H-FC/GTE combined showed synergistic effect in cell culture and tilapia and rat. Rat fed diet containing C-FC-E, C-FC-EA, GTE or C-FC-E/GTE decreased DMN injury by decreases in antioxidant enzyme activities and TBARS.
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論文目次: | 摘要  1
| 圖目錄  1
| 表目錄  2
| 縮寫表  3
| 壹、前言  4
| 貳、文獻整理  7
| 一、蜆及一條根之生理活性    7
| (一) 蜆    7
| (二) 一條根    9
| (三) 交互作用    11
| 二、自由基與 ROS 對生物體之傷害    14
| (一) 自由基與ROS    14
| (二) 氧化作用之傷害    15
| 三、抗氧化防禦系統    16
| (一) 非酵素性抗氧化物    16
| (二) 抗氧化酵素    18
| 1. 超氧化物歧化酵素 (Superoxide dismutase, SOD)    18
| 2. 過氧化氫酵素 (Catalase)    19
| 3. 麩胱甘肽過氧化酵素 (Glutathione peroxidase, GPx)    19
| 4. 麩胱甘肽還原酵素 (Glutathione reductase, GR)    19
| 5. 葡萄糖六磷酸去氫酵素(Glucose 6 phosphate dehydrogenase，G6PDH)    20
| 四、研究模式    21
| (一) 肝細胞模式    21
| (二) 吳郭魚動物模式    21
| 參、材料與方法  23
| 一、材料    23
| 二、藥品試劑    24
| 三、使用儀器    25
| 四、方法    26
| (一) 材料製備    26
| (二) 大鼠肝細胞模式    30
| (三) 大鼠動物模式    33
| (四) 吳郭魚模式    34
| 五、分析項目    36
| (ㄧ) 蛋白質濃度測定    36
| (二) 脂質過氧化測定    36
| (三) 肝功能指標之測定    37
| (四) 抗氧化酵素活性之測定    38
| (五) 細胞存活率 (MTT) 測定    41
| 六、統計分析    42
| 肆、結果討論  43
| 一、台灣蜆與一條根組合物清除DPPH自由基之能力    43
| (一) 花蓮產台灣蜆與一條根萃取物之組合產品    43
| (二) 彰化產台灣蜆與一條根萃取物之組合產品    47
| 二、組合樣品對四氯化碳損傷肝細胞之影響    52
| (一) 四氯化碳劑量對肝細胞損傷之影響－以 MTT 為指標    52
| (二) 組合樣品濃度對肝細胞損傷之影響－以 MTT 為指標    56
| (三) 細胞肝功能指標之變化    58
| (四)組合樣品對肝細胞抗氧化酵素之影響    63
| 三、組合樣品對大鼠肝臟之影響    67
| (一) 血液肝指數之變化    67
| (二) 脂質過氧化之變化    69
| (三) 抗氧化酵素活性之變化    70
| 四、組合樣品對吳郭魚肝臟之影響    74
| (一) 吳郭魚餵食後之肝體比    74
| (二) 肝臟脂質過氧化之變化    76
| (三) 肝臟抗氧化酵素活性之變化    79
| 五、台灣蜆與一條根組合物之相乘性    83
| 伍、結論  85
| 陸、參考文獻  87
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2009 年 11 月 22 日 23:15