Evaluation of hepatic protection of Agaricus blazei-based herbal extract against CCl4-induced oxidative liver injury

本研究為利用衛生署公告之護肝功能動物模式，探討味全公司研發之養甘露樣品，是否具有對於四氯化碳誘導大鼠肝損傷保護作用。本實驗分為六組，分別為正常控制組 (未經四氯化碳處理)、四氯化碳處理負對照組 (僅投予四氯化碳之處理組)、Silymarin處理正對照組 (四氯化碳+Silymarin處理組) 及1X、2X、5X劑量實驗組 (四氯化碳+740、1480、3700 mg/kg bw/day養甘露樣品處理組)。實驗結果顯示，經連續餵食八週養甘露樣品後，三種劑量之養甘露樣品皆可以增加肝臟中抗氧化物質glutathione (GSH) 的含量並且使大鼠肝臟中脂質過氧化情況改善以及肝臟生化功能指標酵素丙胺酸轉胺酶 (alanine aminotransferase, ALT)、天門冬胺酸轉胺酶 (aspartate aminotransferase, AST) 數值顯著的減少，表示應具有護肝之功能 (p < 0.05)。在肝臟抗氧化及解毒代謝酵素活性方面，2X、5X劑量養甘露樣品處理組之麩胱甘肽過氧化酶 (glutathione peroxidase, GPx1)、麩胱甘肽還原酶 (glutathione reductase, GRd)、麩胱甘肽硫轉移酶 (glutathione S-transferase, GST)、超氧化物歧化酶 (superoxide dismutase, SOD) 酵素活性均顯著高於四氯化碳處理負對照組 (p < 0.05)。在四氯化碳投予後之第八週血清中三酸甘油酯 (triglyceride, TG) 值方面，三種劑量養甘露樣品處理實驗組之TG均較四氯化碳處理負對照組低且達顯著性差異 (p < 0.05)。此外，肝臟組織病理學觀察方面，5X劑量養甘露樣品可使肝細胞發炎壞死、脂肪變性及肝臟纖維化等現象都有顯著的改善 (p < 0.05)。由以上實驗結果推測養甘露樣品對於保護肝臟應具有正面的效果。Mushrooms have been used as an important source of nutrition and/or therapy throughout the world since ancient times. Agaricus blazei polysaccharides has been reported to induced with immunomodulatory and antitumor activities. This study was designed to evaluate the effects of Agaricus blazei-based herbal extract on CCl4-induced chronic hepatotoxicity in rats. Male Wistar rats (210 ±10 g) were divided into six groups in this study, including normal control (not treated with CCl4) and groups treated with CCl4, i.e. negative control (only treated with CCl4), positive control (treated with CCl4 and Silymarin), and three experimental groups (treated with CCl4 and low, medium or high dose of Agaricus blazei-based herbal extract, i.e. 0.74, 1.48, or 3.7g/kg bw/day Agaricus blazei-based herbal extract, respectively). On the other hand, rats were administrated with Agaricus blazei-based herbal extract for one week before the administration of CCl4. Rats were treated with Agaricus blazei-based herbal extract once a day until the eighth week of CCl4 treatment. Blood samples were collected for assaying serum biological parameters. The livers were excised for evaluating peroxidation products and antioxidant substances, as well as the activities of antioxidant enzymes. Pathological histology was also performed. The results showed that the Agaricus blazei-based herbal extract significantly (p<0.05) lowered the serum levels of hepatic enzyme markers (alanine aminotransferase, aspartate aminotransferase), inhibited inflammation, necrosis, fatty degeneration and fibrosis. Also reduced the content of the peroxidation product malondialdehyde, and the elevated the content of reduced form of glutathione (GSH). Treatment of rats with Agaricus blazei-based herbal extract could increase the activities of glutathione peroxidase (GPx1), glutathione reductase (GRd) and glutathione S-transferase (GST) in liver when compared with CCl4-treated group (p<0.05). Therefore, the results of this study show that Agaricus blazei-based herbal extract can be proposed to protect the liver against CCl4-induced oxidative damage in rats, and the hepatoprotective effect might be correlated with its antioxidant and free radical scavenger effects.目錄.................................................................................................................................Ⅰ 中文摘要.........................................................................................................................Ⅴ 英文摘要.........................................................................................................................Ⅵ 表次.................................................................................................................................Ⅷ 圖次.................................................................................................................................Ⅹ 第一章 前言...................................................................................................................1 第二章 文獻整理...........................................................................................................3 第一節 肝臟之生理功能及肝臟疾病...................................................................3 一、肝臟的生理結構與功能.............................................................................3 二、肝臟損傷的機制.........................................................................................6 三、四氯化碳誘導肝損傷機制.........................................................................8 四、護肝成分介紹...........................................................................................12 第二節 自由基與氧化.........................................................................................14 一、自由基及活性氧的定義...........................................................................14 二、脂質過氧化物的產生...............................................................................14 三、氧化狀態失調與疾病...............................................................................17 四、生物體內抗氧化防禦機制之介紹...........................................................17 第三節 巴西洋菇的相關研究資料.....................................................................21 一、簡介...........................................................................................................21 二、生理機能...................................................................................................21 第四節 羅漢果的相關研究資料.........................................................................24 一、簡介...........................................................................................................24 二、生理機能...................................................................................................24 第五節 枸杞子的相關研究資料.........................................................................26 一、簡介...........................................................................................................26 二、生理機能...................................................................................................26 第六節 大棗的相關研究資料.............................................................................28 一、簡介...........................................................................................................28 二、生理機能...................................................................................................28 第七節 黃耆的相關研究資料.............................................................................30 一、簡介.........................................................................................................30 二、生理機能...................................................................................................30 第三章 研究目的與實驗架構.....................................................................................33 一、急性動物實驗架構...................................................................................33 二、慢性動物實驗架構...................................................................................34 第四章 材料與方法.....................................................................................................35 第一節 材料...........................................................................................................35 一、實驗材料...................................................................................................35 二、實驗動物...................................................................................................36 三、實驗藥品...................................................................................................36 四、實驗儀器...................................................................................................37 五、四氯化碳誘導大鼠急性肝損傷實驗模式...............................................38 六、四氯化碳誘導大鼠慢性肝損傷實驗模式...............................................38 第二節 方法...........................................................................................................39 一、肝功能生化指數的檢測...........................................................................39 二、肝臟與血漿脂質過氧化之分析...............................................................39 三、肝臟麩胱甘肽濃度之分析.......................................................................40 四、麩胱甘肽過氧化酶活性之分析...............................................................42 五、麩胱甘肽還原酶活性之分析...................................................................42 六、麩胱甘肽硫轉移酶活性之分析...............................................................42 七、超氧化物歧化酶之測定...........................................................................43 八、過氧化氫酶之測定...................................................................................43 九、蛋白質濃度之測定...................................................................................43 十、組織病理學的觀察...................................................................................44 十一、統計方法...............................................................................................45 第五章 結果.................................................................................................................48 第一節 四氯化碳誘導大鼠急性肝損傷實驗.......................................................48 一、平均體重變化...........................................................................................48 二、臟器相對重量...........................................................................................48 三、肝臟與血漿中脂質過氧化.......................................................................48 四、肝臟中抗氧化物質麩胱甘肽含量...........................................................49 五、血清中ALT、AST值.................................................................................49 六、肝臟抗氧化與解毒代謝酵素活性...........................................................50 七、肝臟與血清中三酸甘油酯與總膽固醇含量...........................................50 第二節 四氯化碳誘導大鼠慢性肝損傷實驗.......................................................51 一、平均體重變化...........................................................................................51 二、臟器相對重量...........................................................................................51 三、肝臟與血漿中脂質過氧化.......................................................................52 四、肝臟中抗氧化物質麩胱甘肽含量...........................................................52 五、血清中ALT、AST值.................................................................................52 六、肝臟抗氧化與解毒代謝酵素活性...........................................................54 七、肝臟與血清中三酸甘油酯與總膽固醇含量...........................................55 八、肝臟組織病理切片觀察...........................................................................56 第六章 討論.................................................................................................................59 第七章 結論.................................................................................................................64 第八章 圖表.................................................................................................................65 第九章 參考文獻.........................................................................................................89 附錄...............................................................................................................................10