沈立言臺灣大學:食品科技研究所蘇正元Su, Zheng-YuanZheng-YuanSu2010-05-112018-06-292010-05-112018-06-292009U0001-2704200917133700http://ntur.lib.ntu.edu.tw//handle/246246/182227肝臟為人體中執行代謝、解毒與抗氧化系統之重要器官，但國人肝臟疾病罹患率甚高且為死亡的主要原因，因此肝臟之保健在台灣是一個重要的課題。而近年來已有許多研究證實真菌類(如靈芝和巴西洋菇)與豆科植物(如黑豆和黃耆)中，具有多種機能性成分能調節人體生理機能。但是，目前仍少有對添加豆科植物(黑豆和黃耆)作為靈芝或巴西洋菇發酵基質之研究。本研究之目的乃在探討「靈芝-豆科」或「巴西洋菇-豆科」發酵產物之抑制肝癌活性與護肝功能，並分析可能活性成分及其抑制肝癌活性之相關性，以及深入探討抑制肝癌之作用機轉，期能作為保健食品開發之重要參考。 本研究首先針對不同發酵條件之「靈芝-豆科」和「巴西洋菇-豆科」小量(5公升發酵槽)發酵產物中，篩選具最佳抑制肝癌Hep 3B細胞活性之樣品，再探討此樣品對大鼠正常初代肝細胞(ex vivo)與四氯化碳誘導大鼠初代肝細胞損傷(ex vivo)之影響。最佳的抑制肝癌細胞活性之發酵產物為：發酵基質含50 g/L黑豆和20 g/L黃耆，且於24℃發酵溫度、0.75 vvm通氣量下發酵11天之靈芝菌絲體乙醇萃取物(GL-3-mE) (IC50值為26.6 μg/mL)，以及發酵基質不含豆科植物，且於28℃發酵溫度、0.05 vvm通氣量下發酵45天之巴西洋菇菌絲體乙醇萃取物(AB-9-mE) (IC50值為14.3 μg/mL)。此外，當GL-3-mE或AB-9-mE於200 μg/mL處理濃度以下對大鼠正常初代肝細胞均無不良影響，而10或100 μg/mL處理濃度可保護四氯化碳所誘導初代肝細胞之損傷。 進一步針對不同發酵條件之「巴西洋菇-豆科」大量(500公升發酵槽)發酵產物，篩選具最佳抑制肝癌Hep 3B和Hep G2細胞活性之樣品結果顯示，二階段發酵(起始發酵基質含20 g/L黃豆，再於第6天添加含10 g/L黑豆之發酵基質)且發酵17天後所得之巴西洋菇發酵產物，其乙醇萃取物[AB(GF3)-pE]最能降低人類肝癌Hep 3B (IC50值為161.1 μg/mL)和Hep G2 (IC50值為86.9 μg/mL)細胞存活率。矽膠管柱層析法與薄層層析法進行AB(GF3)-pE之區分試驗後，低極性沖提液(正己烷/乙酸乙酯 = 97:3和19:1，v/v)所得之區分物AB(GF3)-pE-F3具最佳抑制肝癌Hep 3B和Hep G2細胞生長之能力，IC50值分別為3.7和2.2 μg/mL。使用RP-HPLC分離與製備AB(GF3)-pE-F3內有效抑癌成分，並經UV、IR、Mass、13C-NMR和1H-NMR鑑定出2個化合物，blazeispirol A和blazeispirol C，其含量會隨著發酵天數增加而增加，且與肝癌細胞存活率有極高的負相關性。 於探討「巴西洋菇-豆科」發酵產物抑癌機轉方面，blazeispirol A會減少人類肝癌Hep 3B細胞內Bcl-2磷酸化和Bcl-xL表現量，及增加Bax表現量，結果導致粒線體膜電位下降，進而活化caspase-9、caspase-3、poly(ADP-ribose)polymerase (PARP)，另有HtrA2/Omi和AIF自粒線體內釋出至細胞質中，最後引起DNA片段化，故其能同時誘發caspase依賴型與非依賴型細胞凋亡。研究結果亦顯示，blazeispirol A是AB(GF3)-pE和AB(GF3)-pE-F3促進人類肝癌Hep 3B細胞凋亡的主要貢獻成分。 進一步探討AB(GF3)-pE對大鼠正常初代肝細胞(ex vivo)、四氯化碳誘導大鼠初代肝細胞損傷(ex vivo)，以及四氯化碳誘導大鼠肝損傷(短期動物試驗)之影響。結果顯示AB(GF3)-pE於處理濃度200 μg/mL以下時對大鼠正常初代肝細胞均無不良影響，而於10和100 μg/mL處理濃度下對四氯化碳誘導大鼠初代肝細胞損傷具有保護作用。於四氯化碳誘導大鼠肝損傷短期實驗中，餵食AB(GF3)-pE可減少四氯化碳所造成的肝細胞壞死與空泡化等損傷現象，且顯著增加肝臟中還原態麩胱甘肽(glutathione)含量與麩胱甘肽過氧化酶(GPx, glutathione peroxidase)、麩胱甘肽還原酶(GRd, glutathione reductase)和麩胱甘肽硫轉移酶(GST, glutathione S-transferase)等活性(P < 0.05)。Liver is the major organ in human being for metabolism, detoxification and antioxidation. However, the annual report of the Department of Health, Executive Yuan, Taiwan, indicated chronic liver disease and liver cancer are the leading causes of death in 2007. Recently, many researches have reported that fungi, such as Ganoderma lucidum (GL) and Agaricus blazei (AB), and leguminous plants, such as black soybean and Astragalus membranaceus, contained many active compounds which can manipulate biological activities, including antitumor activity, induction of apoptosis, and hepato-protection etc. However, there is almost no research on the fermentation products of GL and AB cultivated in the medium containing leguminous plants. The objectives of this project were to investigate the anti-hepatoma activity and liver protective function of the fermentation products of GL and AB cultivated in the medium containing black soybean and/or A. membranaceus; to evaluate the potential active compunds and their anti-hepatoma activities; and to study their anti-hepatoma mechanisms. The study may provide the important information for the development of health food in the near future.irstly, the anti-hepatoma activity and liver protective function of the fermentation products (5 L fermenter) of GL and AB cultivated in the medium containing black soybean and/or A. membranaceus were investigated. Human hepatoma Hep 3B cells and primary rat hepatocytes were used as experimental models. The results indicated that the ethanolic extracts of mycelia from the fermentation products with best anti-hepatoma activity were GL-3-mE (medium containing 50 g/L black soybean and 20 g/L A. membranaceus, 24℃, 0.75 vvm, 11 days) (IC50 26.6 μg/mL) and AB-9-m-E (medium without leguminous plants, 28℃, 0.05 vvm, 45 days) (IC50 14.3 μg/mL). GL-3-mE or AB-9-mE at 10 and 100 μg/mL reduced the CCl4-induced damage in primary rat hepatocytes, while neither extracts at a dose as high as 200 μg/mL caused any effect on the growth of the primary rat hepatocytes. Furthermore, the anti-hepatoma activity of the fermentation products (500 L fermenter) of AB with different fermentation conditions was investigated. The ethanolic extract of AB(GF3)-pE inhibited the growth of Hep 3B and G2 cells (IC50 161.1 and 86.9 μg/mL, respectively) most efficiently. Its fermentation condition was two stages: the initial medium contained 20 g/L soybean, and the inoculum was transferred to new medium containing 10 g/L black soybean on day 6. The total fermentation period was 17 days. AB(BS)-pE was further separated by silica gel column chromatography and eluted with n-hexane/ethyl acetate/methanol gradient solvent system into 21 fractions. Fraction 3 [AB(BS)-pE-F3], eluted with n-hexane/ethyl acetate (97:3 and 19:1, v/v), was the most active fraction to inhibit the proliferation of Hep 3B and G2 cells (IC50 3.6 and 1.9 μg/mL, respectively). Two active compounds from AB(BS)-pE-F3 were seperated by RP-HPLC and identified by UV, IR, EIMS, and 1H and 13C NMR to be blazeispirols A and C, and they were increased after feeding the medium containing black soybean at day 6 and reached the maximum at day 12. The contents of blazeispirols A and C were highly negatively correlated with Hep 3B and G2 cell viabilities. It showed that blazeispirol A inhibited the growth of Hep 3B cells by induction of apoptois. Decrease in phosphor-Bcl-2 and Bcl-xL proteins, increase in Bax protein, disruption of mitochondrial membrane potential, activation of caspases-3 and -9 and poly(ADP-ribose)polymerase (PARP), and DNA fragmentation were involved with blazeispirol A treatment. Additionally, HtrA2/Omi and AIF were also released from mitochondria to cytosol. The results indicated that blazeispirol A can induce caspase-dependent and -independent apoptosis in Hep 3B cells. It also suggested that blazeispirol A is the major contributor in AB(GF3)-pE and AB(GF3)-pE-F3 to induce apoptosis in human hepatoma Hep 3B cells. The effects of AB(GF3)-pE on normal primary rat hepatocytes ex vivo, primary rat hepatocytes damaged by carbon tetrachloride ex vivo, and carbon tetrachloride-induced liver damage in rats in vivo were investigated. The results showed that 10 and 100 μg/mL of AB(GF3)-pE reduced the CCl4-induced damage in primary rat hepatocytes, while 200 μg/mL didn’t cause any effect on the growth of normal primary rat hepatocytes. In addition, AB(GF3)-pE reduced the inflammation and necrosis of hepatocytes, and up-regulated glutathione peroxidase (GPx)、glutathione reductase (GRd), and glutathione S-transferase (GST) to preserve the level of reduced glutathione in the liver.中文摘要................................................................................................................... Ibstract.................................................................................................................... III錄........................................................................................................................... VI次........................................................................................................................... XII次........................................................................................................................... XV一章 前言............................................................................................................. 1二章 文獻整理..................................................................................................... 2 第一節 靈芝......................................................................................................... 2 一、簡介............................................................................................................. 2 二、生理機能..................................................................................................... 3 第二節 巴西洋菇................................................................................................. 8 一、簡介............................................................................................................. 8 二、生理機能..................................................................................................... 8 第三節 黑豆......................................................................................................... 12 一、簡介............................................................................................................. 12 二、生理機能..................................................................................................... 12 第四節 黃耆......................................................................................................... 15 一、簡介............................................................................................................. 15 二、生理機能..................................................................................................... 15 第五節 肝臟......................................................................................................... 18 一、肝臟之簡介................................................................................................. 18 二、肝臟病變..................................................................................................... 18 三、護肝功能之評估方法................................................................................. 19 四、肝癌............................................................................................................. 20 第六節 細胞生長與死亡..................................................................................... 22 一、細胞週期..................................................................................................... 22 二、細胞計畫性死亡......................................................................................... 24三章 研究目的與實驗架構................................................................................. 28 第一節 研究目的................................................................................................. 28 第二節 實驗架構................................................................................................. 29 一、總實驗架構................................................................................................. 29 二、實驗流程..................................................................................................... 31 第一部分 「靈芝-豆科」小量(5公升發酵槽)發酵產物對肝癌細胞生長與大鼠初代肝細胞之影響........................................................ 31 第二部分 「巴西洋菇-豆科」小量(5公升發酵槽)發酵產物對肝癌細胞生長與大鼠初代肝細胞之影響................................................ 31 第三部分 「巴西洋菇-豆科」大量(500公升發酵槽)發酵產物抑制肝癌細胞活性及其有效成分之分離與鑑定.................................... 32 第四部分 不同發酵天數之「巴西洋菇-豆科」大量(500公升發酵槽)發酵產物抑制肝癌細胞活性及其有效成分之變化................ 33 第五部分 「巴西洋菇-豆科」大量(500公升發酵槽)發酵產物抑制肝癌細胞生長之作用機轉................................................................ 34 第六部分 「巴西洋菇-豆科」大量(500公升發酵槽)發酵產物對大鼠初代肝細胞與四氯化碳誘導大鼠肝損傷短期試驗之影響........ 35四章 材料與方法................................................................................................. 37 第一節 實驗材料與細胞株................................................................................. 37 一、實驗材料..................................................................................................... 37 二、實驗細胞株................................................................................................. 38 第二節 實驗藥品................................................................................................. 38 一、藥品與溶劑................................................................................................. 38 二、檢測套組..................................................................................................... 41 第三節 儀器設備................................................................................................. 42 一、實驗材料前處理與樣品製備之儀器設備................................................. 42 二、細胞培養之儀器設備................................................................................. 43 三、樣品分析之儀器設備................................................................................. 44、 結構鑑定之儀器設備............................................................................... 45 第四節 實驗方法................................................................................................. 46 第一部分 「靈芝-豆科」小量(5公升發酵槽)發酵產物對肝癌細胞生長與大鼠初代肝細胞之影響.................................................................. 46 (一) 樣品前處理.......................................................................................... 46 (二) 人類肝癌Hep 3B細胞株之培養與保存............................................. 46 (三) 發酵產物抑制人類肝癌Hep 3B細胞活性之評估............................. 48 (四) 可能三萜類化合物之分析………………………………………...... 49 (五) 發酵產物對大鼠正常初代肝細胞之影響.......................................... 50 (六) 發酵產物對四氯化碳誘導大鼠初代肝細胞損傷之影響.................. 57 (七) 統計分析.............................................................................................. 58 第二部分 「巴西洋菇-豆科」小量(5公升發酵槽)發酵產物對肝癌細胞生長與大鼠初代肝細胞之影響.......................................................... 59 (一) 樣品前處理.......................................................................................... 59 (二) 發酵產物抑制人類肝癌Hep 3B細胞活性之評估............................. 59 (三) 發酵產物對大鼠正常初代肝細胞之影響.......................................... 60 (四) 發酵產物對四氯化碳誘導大鼠初代肝細胞損傷之影響.................. 60 (五) 統計分析.............................................................................................. 60 第三部分 「巴西洋菇-豆科」大量(500公升發酵槽)發酵產物抑制肝癌細胞活性及其有效成分之分離與鑑定.............................................. 60 (一) 樣品前處理.......................................................................................... 60 (二) 人類肝癌Hep G2細胞株之培養與保存............................................. 61 (三) 發酵產物抑制人類肝癌Hep 3B和Hep G2細胞活性之評估........... 61 (四) 發酵產物之區分試驗.......................................................................... 62 (五) 有效抑癌化合物之分離...................................................................... 63 (六) 有效抑癌成分之鑑定.......................................................................... 64 (七) 樣品中有效抑癌化合物含量之分析.................................................. 64 (八) 統計分析.............................................................................................. 65 第四部分 不同發酵天數之「巴西洋菇-豆科」大量(500公升發酵槽)發酵產物抑制肝癌細胞活性及其有效成分之變化.............................. 65 (一) 樣品前處理.......................................................................................... 65 (二) 發酵產物抑制人類肝癌Hep 3B和Hep G2細胞活性之評估........... 65 (三) 樣品中有效抑癌化合物含量之分析.................................................. 66 (四) 統計分析.............................................................................................. 66 第五部分 「巴西洋菇-豆科」大量(500公升發酵槽)發酵產物抑制肝癌細胞生長之作用機轉.......................................................................... 66 (一) 樣品抑制Hep 3B細胞生長之形態觀察............................................. 66 (二) 抑制肝癌Hep 3B細胞生長之作用機轉............................................. 66 (三) 樣品抑制經z-VAD-fmk前處理Hep 3B細胞生長之影響................. 68 (四) 特定蛋白質表現之測定...................................................................... 69 (五) 統計分析.............................................................................................. 71 第六部分 「巴西洋菇-豆科」大量(500公升發酵槽)發酵產物對大鼠初代肝細胞與四氯化碳誘導大鼠肝損傷短期試驗之影響.................. 72 (一) 發酵產物對大鼠正常初代肝細胞之影響.......................................... 72 (二) 發酵產物對四氯化碳誘導大鼠初代肝細胞損傷之影響.................. 72 (三) 發酵產物對四氯化碳誘導大鼠肝損傷短期試驗之影響.................. 72 (四) 統計分析.............................................................................................. 77五章 結果與討論................................................................................................. 78 第一部分 「靈芝-豆科」小量(5公升發酵槽)發酵產物對肝癌細胞生長與大鼠初代肝細胞之影響......................................................................... 78 (一) 發酵產物抑制人類肝癌Hep 3B細胞活性之評估................................. 78 (二) 發酵產物中可能三萜類化合物之分析……………………....……...... 79 (三) 發酵產物對大鼠正常初代肝細胞之影響.............................................. 79 (四) 發酵產物對四氯化碳誘導大鼠初代肝細胞損傷之影響...................... 80 (五) 討論.......................................................................................................... 81 第二部分 「巴西洋菇-豆科」小量(5公升發酵槽)發酵產物對肝癌細胞生長與大鼠初代肝細胞之影響................................................................. 92 (一) 發酵產物抑制人類肝癌Hep 3B細胞活性之評估................................. 92 (二) 發酵產物對大鼠正常初代肝細胞之影響.............................................. 92 (三) 發酵產物對四氯化碳誘導大鼠初代肝細胞損傷之影響...................... 93 (四) 討論.......................................................................................................... 95 第三部分 「巴西洋菇-豆科」大量(500公升發酵槽)發酵產物抑制肝癌細胞活性及其有效成分之分離與鑑定..................................................... 104 (一) 發酵產物抑制人類肝癌Hep 3B和Hep G2細胞活性之評估............... 104 (二) 發酵產物之區分試驗.............................................................................. 105 (三) 區分物抑制人類肝癌Hep 3B和Hep G2細胞活性之評估................... 105 (四) 區分物中有效抑癌化合物之分離.......................................................... 106 (五) 化合物抑制人類肝癌Hep 3B和Hep G2細胞活性之評估................... 106 (六) 有效抑癌成分之鑑定.............................................................................. 107 (七) 大量發酵產物(500公升發酵槽)中有效抑癌化合物含量之分析及其與抑制肝癌細胞活性之相關性............................................................... 108 (八) 討論.......................................................................................................... 109 第四部分 不同發酵天數之「巴西洋菇-豆科」大量(500公升發酵槽)發酵產物抑制肝癌細胞活性及其有效成分之變化..................................... 135 (一) 發酵產物抑制人類肝癌Hep 3B和Hep G2細胞活性之評估............... 135 (二) 發酵產物中有效抑癌化合物含量之分析及其與抑制肝癌細胞活性之相關性................................................................................................... 135 (三) 討論.......................................................................................................... 136 第五部分 「巴西洋菇-豆科」大量(500公升發酵槽)發酵產物抑制肝癌細胞生長之作用機轉................................................................................. 141 (一) AB(GF3)-pE與AB(GF3)-pE-F3影響人類肝癌Hep 3B細胞生長之情形............................................................................................................... 141 (二) AB(GF3)-pE與AB(GF3)-pE-F3導致人類肝癌Hep 3B細胞死亡之作用機轉....................................................................................................... 141 (三) Blazeispirol A影響人類肝癌Hep 3B細胞生長之情形.......................... 144 (四) Blazeispirol A影響人類肝癌Hep 3B細胞生長之作用機轉.................. 144 (五) 討論.......................................................................................................... 149 第六部分 「巴西洋菇-豆科」大量(500公升發酵槽)發酵產物對大鼠初代肝細胞與四氯化碳誘導大鼠肝損傷短期試驗之影響......................... 169 (一) 發酵產物對大鼠正常初代肝細胞之影響.............................................. 169 (二) 發酵產物對四氯化碳誘導大鼠初代肝細胞損傷之影響...................... 169 (三) 發酵產物對四氯化碳誘導大鼠肝損傷短期試驗之影響...................... 170 (四) 討論.......................................................................................................... 172六章 綜合討論..................................................................................................... 186 第一節 發酵產物之生理活性............................................................................. 186 一、抑癌作用..................................................................................................... 186 二、護肝功效..................................................................................................... 187 第二節 發酵產物之安全性................................................................................. 188七章 結論............................................................................................................. 189八章 參考文獻..................................................................................................... 191九章 附錄............................................................................................................. 212application/pdf4988325 bytesapplication/pdfen-US靈芝巴西洋菇黑豆黃耆抗肝癌活性護肝Ganoderma lucidumAgaricus blazeiblack soybeanAstragalus membranaceusanti-hepatoma activityliver protection[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182227/1/ntu-98-D91641007-1.pdf