沈立言臺灣大學:食品科技研究所胡芳晴Hu, Fang-ChingFang-ChingHu2010-05-112018-06-292010-05-112018-06-292009U0001-2107200918024300http://ntur.lib.ntu.edu.tw//handle/246246/182221根據行政院衛生署在2008年的資料統計中可見慢性肝疾病及肝硬化為國人的十大死因之一，而在十大癌症死因中，肝癌更位居第二位，而肝病所造成的併發症也造成極大的醫療支出，因此在台灣肝病的預防及治療可說是當務之急。肝臟是解毒代謝的重要器官，相對而言，國人因病毒、藥物或酒精毒性引起的肝損傷比率很高。乙醯胺基苯酚(acetaminophen, APAP)是臨床上常用的鎮痛解熱劑，每日建議量為1~4克，當一次服用大量時會導致肝臟損害，而其中毒機轉主要是因肝臟的Phase I代謝途徑中經CYP450氧化酶會產生有毒的中間產物N-acetyl-p-benzoquinoemine (NAPQI)，使肝貯存之glutathione (GSH)含量下降，並造成脂質過氧化而引起肝臟傷害。本研究欲建立APAP誘導SD大鼠肝損傷之模式，結果顯示以管餵(oral gavage)方式給予大鼠高劑量(3.5 g/kg bw)仍難以誘導，並與先前本實驗室所建立之APAP誘導Balb/c小鼠肝損傷模式加以比較評估後，由病理組織切片中發現小鼠模式較符合人體的病理進展，因此小鼠模式是較適合之動物模式。七層塔(Ocimum gratissimum L.)是台灣特有的民間草藥，有解毒、利尿、消炎的功能，並有抗脂質過氧化及抗氧化之功效，先前研究中發現，以適當濃度七層塔精油處理大白鼠初代肝細胞，能有效提升GSH相關之抗氧化與解毒代謝系統，因此本實驗藉由已建立之APAP誘導小鼠肝損傷之模式，評估七層塔精油之護肝功效與相關代謝途徑及探討可能的護肝有效成分。急性(600 mg APAP/kg bw, 腹腔注射後四小時犧牲)及慢性(400 mg APAP/kg bw, 腹腔注射每週兩次共四週)實驗結果顯示，以APAP誘導小鼠肝損傷會顯著提升血清中aspartate transferase (AST)及alanine transferase (ALT)值，同時嚴重耗損肝臟貯存之GSH含量，並造成肝臟抗氧化酵素glutathione peroxidase (GPx)、glutathione reductase (GRd)、superoxide dismutase (SOD)及catalase (CAT)活性顯著降低(p < 0.05)，也使肝臟解毒酵素glutathione S-transferase (GST)活性下降及cytochrome P450 2E1 (CYP2E1)氧化酶(oxidase)活性上升。而以管餵方式給予小鼠N-acetyl-L-cysteine (NAC)則能有效恢復APAP所造成之肝細胞傷害，且管餵給予2 mg/kg bw七層塔精油也有恢復酵素活性之趨勢。而管餵給予七層塔精油之主成分丁香酚(eugenol)則有類似於七層塔精油的效果，但效果較不顯著，推測七層塔中含有其他可與eugenol有護肝之加成效果的成分。此結果指出，七層塔精油可能具有發展為保健植物產品的潛力。According to the 2008 report of Department of Health of Taiwan, liver cancer is the second of ten leading cancer deaths, and chronic liver disease and cirrhosis is the seventh of ten leading causes. Therefore hepatoprotective issue is very important in Taiwan. Acetaminophen (APAP) is a mild nonnarcotic, analgesic and antipyretic agent, and is effective and safe when consumed as recommended (1-4 g per day). However, APAP overdose will undergo oxidative conversion by Phase I reactions to the toxic metabolite N-acetyl-p-benzoquinoemine (NAPQI) and may increase the risk of hepatotoxicity. Ocimum gratissimum L. (OG; tree-basil) is an aromatic plant and has been used as a folk medicine in the treatment of hepatitis and cirrhosis in Taiwan. In this study, the applicable animal model by the comparison of SD rats and Balb/c mice was evaluated. Moreover, the hepatoprotective effects of OG essential oil at various concentrations on the serum and liver enzymes, hepatic inflammation, and pathological changes in APAP-intoxicated Balb/c mice were investigated. The experimental results showed that in acute liver injury, OG oil treatment decreased 600 mg/kg bw APAP-induced liver injury. The chronic liver injury results indicated that administration of 400 mg APAP/kg bw increased the aspartate transferase (AST) and alanine transferase (ALT) levels in mice serum along with the enhancement of hepatic lipid peroxidation. APAP also reduced antioxidant enzyme activities (GPx, GRd, SOD and CAT) and glutathione (GSH) level in the liver. Treatment with the 2 mg OG oil/kg bw significantly reversed these changes (p < 0.05). Besides, histopathological examination also revealed the protective nature of OG oil against APAP-induced necrotic change in the liver. The major compound of OG oil is eugenol. However, the treatment with eugenol had less hepatoprotective effects on APAP-induced liver damage compared with OG oil. The results suggest that the OG oil protects hepatic tissues against oxidative damages and can be used as an effective protector against APAP-induced hepatotoxicity.目錄謝....................................................................................................................................i文摘要.........................................................................................................................Ⅰ文摘要.........................................................................................................................III錄.................................................................................................................................V次.................................................................................................................................IX次..................................................................................................................................X一章、前言.....................................................................................................................1二章、文獻回顧.............................................................................................................2 第一節、肝臟介紹.....................................................................................................2 一、生理構造.....................................................................................................2 二、肝臟形態學.................................................................................................2 三、肝臟的功能.................................................................................................5 四、肝臟解毒系統.............................................................................................6 五、肝損傷機制.................................................................................................7 第二節、活性氧化物介紹.........................................................................................9 一、種類及其對體內造成的影響.....................................................................9 二、粒線體抗氧化防禦系統...........................................................................11 第三節、免疫系統介紹...........................................................................................12 一、免疫系統簡介...........................................................................................12 二、發炎反應簡介...........................................................................................13 三、細胞激素之功能.......................................................................................14 第四節、乙醯胺基苯酚(acetaminophen, APAP)介紹.............................................16 一、基本介紹...................................................................................................16 二、肝臟中之代謝途徑...................................................................................16 三、N-acetyl-p-benzoquinoemine (NAPQI)造成肝臟毒性之機制.................18 四、APAP誘導肝損傷的危險因子.................................................................19 第五節、乙醯半胱胺酸(N-acetyl-L-cysteine, NAC)介紹......................................20 一、基本介紹...................................................................................................20 二、生化及功能...............................................................................................20 三、給予方式及其毒性與副作用...................................................................21 四、劑量及使用方法.......................................................................................21 第六節、七層塔(Ocimum gratissimum L.)介紹......................................................22 一、精油簡介...................................................................................................22 二、七層塔基本介紹.......................................................................................24 三、七層塔精油之重要活性成分...................................................................25 四、七層塔的重要生理活性...........................................................................25 五、七層塔精油之安全性...............................................................................27三章、乙醯胺基苯酚誘導Sprague-Dawley大鼠急性及慢性肝損傷之模式建立.....................................................................................................................................29 第一節、實驗目的...................................................................................................29 第二節、急性及慢性實驗架構...............................................................................30 一、建立APAP誘導Sprague-Dawley大鼠急性肝損傷動物模式................30 二、建立APAP誘導Sprague-Dawley大鼠慢性肝損傷動物模式................31 第三節、實驗材料...................................................................................................32 一、乙醯胺基苯酚(acetaminophen, APAP) ....................................................32 二、乙醯半胱胺酸(N-acetyl-L-cysteine, NAC) .............................................32 三、其他實驗藥品及儀器...............................................................................32 第四節、實驗方法...................................................................................................34 一、急性期實驗動物.......................................................................................34 二、急性期實驗設計.......................................................................................34 三、慢性期實驗動物.......................................................................................35 四、慢性期實驗設計.......................................................................................35 五、分析方法...................................................................................................37 六、統計分析...................................................................................................41 第五節、結果...........................................................................................................42 一、急性期結果...............................................................................................42 二、慢性期結果...............................................................................................44 第六節、討論...........................................................................................................47四章、七層塔對乙醯胺基苯酚誘導Balb/c小鼠急性及慢性肝損傷之護肝功效....49 第一節、實驗目的...................................................................................................49 第二節、急性及慢性實驗架構...............................................................................50 一、七層塔精油對於APAP誘導Balb/c小鼠急性肝損傷之護肝功效.........50 二、七層塔精油對於APAP誘導Balb/c小鼠慢性肝損傷之護肝功效.........51 第三節、實驗材料...................................................................................................52 一、七層塔.......................................................................................................52 二、丁香酚(eugenol)及乙醯半胱胺酸(N-acetyl-L-cysteine, NAC) ..............53 三、其他實驗藥品及儀器...............................................................................53 第四節、實驗方法...................................................................................................56 一、急性期實驗動物.......................................................................................56 二、急性期實驗設計.......................................................................................56 三、慢性期實驗動物.......................................................................................58 四、慢性期實驗設計.......................................................................................58 五、分析方法...................................................................................................60 六、統計分析...................................................................................................65 第五節、結果...........................................................................................................66 一、七層塔精油萃出率及密度.......................................................................66 二、急性期結果...............................................................................................66 三、氣相層析儀分析七層塔精油中之成分丁香酚(eugenol).......................69 四、慢性期結果...............................................................................................69 第六節、討論...........................................................................................................73 一、七層塔精油對於APAP誘導Balb/c小鼠急性肝損傷之護肝功效.........73 二、七層塔精油對於APAP誘導Balb/c小鼠慢性肝損傷之護肝功效.........75五章、結論...................................................................................................................78六章、參考文獻...........................................................................................................79驗結果圖表.................................................................................................................91錄...............................................................................................................................120次一 肝臟前視圖.............................................................................................................2二 肝臟組織功能單位之概念圖.................................................................................3三 肝腺胞(hepatic acinus)之概念圖............................................................................4四 藥物代謝.................................................................................................................7五 藥物誘導肝損傷之機制.........................................................................................8六 粒線體中自由基的生成過程及影響...................................................................10七 外來抗原引起之免疫反應...................................................................................13八 活化之庫佛細胞(Kupffer cell)產生自由基簡圖.................................................14九 發炎引起之體內媒介物所扮演的角色...............................................................15十 肝細胞中由CYP2E1所導致的毒性....................................................................17十一 APAP在人體中之代謝.....................................................................................19十二 精油中成分之化學結構...................................................................................23十三 以水蒸氣蒸餾法萃取七層塔精油裝置圖.......................................................53十四 給予健康成人每日四克之APAP於血清中AST及ALT值影響......................76十五 急性期處理不同劑量APAP對Sprague-Dawley大鼠肝臟病理組織變化之影響.......................................................................................................................93十六 慢性期處理不同劑量APAP對Sprague-Dawley大鼠肝臟病理組織變化之影響.......................................................................................................................98十七 於APAP誘導小鼠急性肝損傷實驗模式下餵食七層塔精油對小鼠肝臟病理組織變化之影響.............................................................................................108十八 丁香酚標準品及七層塔精油揮發性成分之氣相層析圖.............................109十九 於APAP誘導小鼠慢性肝損傷實驗模式下連續餵食七層塔精油五週對小鼠肝臟病理組織變化之影響.............................................................................119次一 肝損傷機制.............................................................................................................9二 抗氧化防禦機轉...................................................................................................12三 乙醯胺基苯酚之代謝物在不同物種間代謝之差異...........................................47四 急性期處理不同劑量APAP對Sprague-Dawley大鼠血液生化值之影響........91五 急性期處理不同劑量APAP對Sprague-Dawley大鼠肝臟抗氧化狀態之影 響...........................................................................................................................92六 慢性期處理不同劑量APAP對Sprague-Dawley大鼠肝重與體重百分比之影 響...........................................................................................................................94七 慢性期處理不同劑量APAP對Sprague-Dawley大鼠血清AST值之影響........95八 慢性期處理不同劑量APAP對Sprague-Dawley大鼠血清ALT值之影響........96九 慢性期處理不同劑量APAP對Sprague-Dawley大鼠肝臟組織之抗氧化狀態影響...........................................................................................................................97十 於APAP誘導小鼠急性肝損傷實驗模式下餵食七層塔精油對小鼠存活率之影響...........................................................................................................................99十一 於APAP誘導小鼠急性肝損傷實驗模式下餵食七層塔精油對小鼠體重及肝腎脾相對重量之影響.....................................................................................100十二 於APAP誘導小鼠急性肝損傷實驗模式下餵食七層塔精油對小鼠血清AST及ALT值之影響.............................................................................................101十三 於APAP誘導小鼠急性肝損傷實驗模式下餵食七層塔精油對小鼠肝臟促發炎細胞激素分泌量之影響.............................................................................102十四 於APAP誘導小鼠急性肝損傷實驗模式下餵食七層塔精油對小鼠肝臟抗氧化狀態之影響.................................................................................................103十五 於APAP誘導小鼠急性肝損傷實驗模式下餵食七層塔精油對小鼠肝臟GSH及GSSG含量之影響......................................................................................104十六 於APAP誘導小鼠急性肝損傷實驗模式下餵食七層塔精油對小鼠肝臟GRd、GPx、CAT及SOD酵素比活性之影響...........................................................105十七 於APAP誘導小鼠急性肝損傷實驗模式下餵食七層塔精油對小鼠肝臟GST及CYP2E1酵素比活性之影響......................................................................106十八 於APAP誘導小鼠急性肝損傷實驗模式下連續餵食七層塔精油對小鼠肝臟肝細胞萎縮及肝臟肝細胞發炎之影響.........................................................107十九 於APAP誘導小鼠慢性肝損傷實驗模式下連續餵食七層塔精油五週對小鼠存活率之影響.................................................................................................110二十 於APAP誘導小鼠慢性肝損傷實驗模式下連續餵食七層塔精油五週對小鼠體重及肝腎脾相對重量之影響.....................................................................111二十一 於APAP誘導小鼠慢性肝損傷實驗模式下連續餵食七層塔精油五週對小鼠血清AST值之影響.................................................................................112二十二 於APAP誘導小鼠慢性肝損傷實驗模式下連續餵食七層塔精油五週對小鼠血清ALT值之影響.................................................................................113二十三 於APAP誘導小鼠慢性肝損傷實驗模式下連續餵食七層塔精油五週對小鼠肝臟抗氧化狀態之影響.........................................................................114二十四 於APAP誘導小鼠慢性肝損傷實驗模式下連續餵食七層塔精油五週對小鼠肝臟GSH及GSSG含量之影響.............................................................115二十五 於APAP誘導小鼠慢性肝損傷實驗模式下連續餵食七層塔精油五週對小鼠肝臟GRd、GPx、CAT及SOD酵素比活性之影響.................................116二十六 於APAP誘導小鼠慢性肝損傷實驗模式下連續餵食七層塔精油五週對小鼠肝臟GST及CYP2E1酵素比活性之影響.............................................117二十七 於APAP誘導小鼠慢性肝損傷實驗模式下連續餵食七層塔精油五週對小鼠肝臟表面凹凸不平程度、充血、肝細胞萎縮及肝臟肝細胞發炎之影響.................................................................................................................118application/pdf19276632 bytesapplication/pdfen-US乙醯胺基苯酚動物模式七層塔精油抗氧化酵素丁香酚肝損傷acetaminophenanimal modelOcimum gratissimum L. essential oilantioxidant enzymeseugenolliver damage[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182221/1/ntu-98-R96641030-1.pdf