沈立言Sheen, Lee-Yan臺灣大學:食品科技研究所陳與國Chen, Yu-KuoYu-KuoChen2010-05-112018-06-292010-05-112018-06-292009U0001-2707200901434900http://ntur.lib.ntu.edu.tw//handle/246246/182229靈芝 (Ganoderma lucidum) 自古以來即為著名的藥用真菌，且在亞洲地區被當成中草藥來使用已有數千年之久。許多證據顯示，靈芝具有抗過敏、抗致突變、抗病毒、抗發炎、抗癌及降血糖等功效。另外，也有研究指出豆科植物黑豆 (Glycine max (L.) Merr.) 與黃耆 (Astragalus membranaceus) 含有許多具保健功效之活性成分，但到目前為止尚無將豆科植物當作基質發酵靈芝之研究。因此本研究以豆科植物 (黑豆與黃耆) 作為基質發酵所得之靈芝為試驗樣品，並以體外及體內試驗探討靈芝菌絲體乙醇萃取物 (EMG) 對人類肝癌細胞株Hep3B之抑制效果。此外，進一步純化EMG中之活性成分並測定其抑制肝癌活性。體外實驗方面，以200 L發酵槽將黑豆與黃耆作為基質發酵靈芝12天，所得之菌絲體的乙醇粗萃物 (EMG) 可有效抑制人類肝癌細胞株Hep3B之細胞存活率，並且具有劑量與時間效應。EMG對Hep3B細胞在24、48及72小時的處理下之IC50分別為156.8、89.8及70.1 μg/mL。另外，EMG增加Hep3B細胞週期中sub-G1的比率，且可誘導其產生DNA片段化與染色質的濃縮與斷裂，並藉由提高Hep3B細胞caspase-3、caspase-8及caspase-9的活性誘導細胞凋亡的發生。由此可知EMG可藉由誘導Hep3B細胞產生細胞凋亡而達到抑制其生長之目的。活體實驗方面，預先給予裸鼠不同劑量之EMG (20、40及200 mg/kg bw) 兩週後，將Hep3B細胞 (1.5×107 cells/mouse) 接種於裸鼠身上，接種後繼續給予九週的EMG。結果顯示，40或200 mg/kg bw之EMG可以有效地抑制裸鼠身上腫瘤之體積與重量 (與控制組相比)，其對腫瘤體積之抑制率分別為48及50% (p<0.05)，而對腫瘤重量之抑制率則分別為60及61% (p<0.05)，且在腫瘤組織切片方面可以觀察出細胞凋亡的現象。除此之外，在實驗結束時各組之體重彼此間均無顯著差異，且由血液樣品分析結果顯示，處理組與控制組之間的AST及ALT值同樣沒有顯著性差異，顯示EMG的給予對裸鼠並不會產生不良之影響。成分鑑定的預實驗方面，將靈芝菌絲體乙醇粗萃物以管柱層析的方式進行區分，獲得29個區分物，並以MTT分析方法探討此些區分物對Hep3B細胞存活率之影響。結果顯示，第8個與第13個區分物 (PF8及PF13) 對於Hep3B細胞有較好的抑制生長效果，其IC50分別為15.7 and 21.8 μg/mL。而在細胞週期的結果中，PF8及PF13可造成Hep3B細胞sub-G1比率的增加。此外，經PF8及PF13處理過之Hep3B細胞可觀察到DNA片段化的現象產生。顯示PF8及PF13可誘導Hep3B走向細胞凋亡。但是由於PF8及PF13的量太少，因此無法進一步純化與鑑定。第二次的區分實驗中，總共獲得7個區分物，以第5個與第6個區分物 (F5及F6) 有較好的抑制肝癌活性，其中F6的量 (約2.1 g) 較為適合進行接下來的抑癌試驗及成分鑑定。故進一步利用高效能液相層析法將F6分離純化，並以NMR鑑定其化合物結構，從其中分離出兩種化合物，分別為5α,8α-epidioxy-22E-ergosta- 6,9(11),22-trien-3β-ol (又稱為9,11-dehydroergosterol peroxide, 9(11)-DHEP) 及5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol (又稱為ergosterol peroxide, EP)。在抑癌活性方面，9(11)-DHEP以及EP對於Hep3B細胞之存活率皆具有抑制效果，且在20 μg/mL的處理濃度之下可誘導其產生DNA片段化。推測9(11)-DHEP以及EP可能為EMG抑制肝癌的活性來源。體而言，以豆科植物作為基質發酵所得之靈芝菌絲體乙醇萃取物 (EMG)，不管在體外或是體內實驗方面，皆具有抑制人類肝癌細胞株Hep3B生長之活性，推測EMG的抑癌活性來自其促進Hep3B細胞產生凋亡之能力，顯示EMG具有預防癌症之潛力，而EMG的抑癌活性則可能來自於9(11)-DHEP以及EP。Ganoderma lucidum is a famous medicinal fungus that has been used as an herbal medicine in Asian regions for thousands of years. Several lines of evidence indicate that G. lucidum possess anti-allergic, anti-mutagenic, anti-viral, anti-inflammatory, antitumour and hypoglycaemic properties. On the other hand, Glycine max (L.) Merr. and Astragalus membranaceus have been reported that contain several important components with various physiological and biological activities. However, the liver protective function of fermentation mycelia of G. lucidum cultivated in the medium containing leguminous plants (G. max (L.) Merr. and A. membranaceus) has not been studied yet. The objective of our study was to investigate the anti-cancer effect of ethanolic extract of mycelia of G. lucidum (EMG) cultivated in the medium containing leguminous plants on human hepatocellular carcinoma cells (Hep3B) in vitro and in vivo assays. Furthermore, the active components of the fraction of EMG and their anti-hepatoma activity were determined.he anti-proliferation effect was observed in vitro growth of Hep3B cell lines after incubation with ethanolic extract of mycelia of G. lucidum (EMG) cultivated in the medium containing G. max (L.) Merr. and A. membranaceus using 200 L fermentor for 12 days. The results indicated that EMG inhibited cell viability in a dose- and time-dependent manner, and the IC50 treated with EMG for 24, 48 and 72 hr were 156.8, 89.9 and 70.1 μg/mL, respectively. EMG inhibited the growth of Hep3B cells via the sub-G1 peak rise in cell cycle analysis, DNA ladder, and increasing of caspase-3, caspase-8 and caspase-9 activities. These results suggested that EMG might suppress Hep3B cells growth through apoptosis in vitro.n vivo, we studied the anti-tumor activity of EMG in Hep3B tumor-bearing nude mice model. EMG was administered for 2 wk before inoculation of Hep3B cells and was continued for 9 wk. The results indicated that oral administration of 40 and 200 mg/kg bw/day of EMG significantly reduced the tumor volume and weight in xenografted nude mice; tumor volume was inhibited by 48 and 50% (p<0.05); tumor weight was inhibited by 60 and 61% (p<0.05), respectively. Immunohistochemistry showed that apoptosis is visualized by detecting cleaved caspase-3. Furthermore, there was no significant difference in body weights among the various groups at the end of the study. The AST and ALT values showed no significant difference between treatment groups and control group. The results implied that EMG feeding to mice did not appear to induce any adverse effects.n the pre-experiment of separation and identification of active components from EMG, the EMG was separated further by gel filtration chromatography to obtain 29 fractions. The effects of various fractions on cell viability of Hep 3B cells were analyzed by MTT assay. The results showed that fraction 8 (PF8) and 13 (PF13) had higher growth inhibition on Hep3B cells. The IC50 values of PF8 and PF13 for Hep3B cells were 15.7 and 21.8 μg/mL, respectively. Cell cycle analysis showed that the growth inhibition effect was associated with sub-G1 peak rising. Moreover, DNA ladder was observed from Hep3B cells treated by PF8 and PF13. Our results indicate that the fractions (PF8 and PF13) of EMG could inhibit growth of Hep3B cells through apoptosis. But the amounts of PF8 and PF13 were too few to identify.urthermore, EMG was fractionated into seven fractions (F1-F7). We found that F5 and F6 had higher growth inhibitory effects on Hep 3B cells than the other fractions, and F6 possessed enough amounts (about 2.1 g) to carry out a more detailed study. The F6 was separated further by HPLC to obtain two compounds, and was identified as 5α,8α-epidioxy-22E-ergosta-6,9(11),22-trien-3β-ol (9,11-dehydro- ergosterol peroxide, 9(11)-DHEP) and 5α,8α-epidioxy-22E-ergosta-6,22-dien-3β-ol (ergosterol peroxide, EP) by NMR. 9(11)-DHEP and EP showed growth inhibition effect on Hep3B cells and DNA ladder was observed with 20 μg/mL of treatment. Thus, it was suggested that 9(11)-DHEP and EP should be the major active compounds in EMG to induce apoptosis of hepatoma cells.verall, these finding demonstrated that EMG could suppress Hep3B cells growth through apoptosis in vitro and exhibit anti-tumor effects in vivo model, and suggested that it has a useful potency in cancer prevention. The anti-cancer activity of EMG might attribute to 9(11)-DHEP and EP.摘 要 Ibstract III 錄 VI 次 X 次 XI、前言 1、文獻回顧 2一節 癌症 2、癌症之簡介 2、肝癌之簡介 3二節 細胞週期與細胞凋亡 7、細胞週期 7、細胞凋亡 8三節 靈芝 13、靈芝之簡介 13、靈芝之栽培方式 14、靈芝之生理功效 16、靈芝之活性成分 19四節 豆科基質 25、黑豆之簡介 25、黃耆之簡介 27五節 異種移植 29、裸鼠之特性 29、人類肝癌細胞株Hep3B之特性 30amp;#21442;、研究目的與實驗架構 45一節 研究目的 45二節 實驗架構 46、整體架構 46、靈芝菌絲體乙醇萃取物 (EMG) 抑制肝癌細胞株活性與機制探討 47、以動物腫瘤模式探討靈芝菌絲體乙醇萃取物 (EMG) 之抑癌活性 48、靈芝菌絲體乙醇萃取物 (EMG) 區分物抑制肝癌細胞株活性探討與成分鑑定 49、實驗材料與方法 51一節 實驗材料與儀器設備 51、實驗材料 51、化學藥品與試劑 51、儀器設備 54、實驗細胞株 56、動物來源與管理 56二節 實驗方法 56、樣品之前處理與製備 56、矽膠管柱層析 57、人類肝癌細胞株 (Hep3B) 之解凍、繼代培養及保存 59、細胞存活率分析 (MTT assay) 60、細胞形態觀察 62、細胞週期分析 62、DNA片段化測定 62、細胞核形態觀察 63、Caspases活性之分析 63、動物試驗 64一、統計分析 65、結果與討論 66一節 靈芝樣品之決定 66、樣品之發酵條件 66、樣品之抑制肝癌細胞活性 (以lovastatin協同處理) 66二節 靈芝菌絲體乙醇萃取物 (EMG) 對人類肝癌細胞株Hep3B之影響 68、EMG對細胞生存率之影響 68、EMG對細胞形態之影響 69、EMG對細胞週期之影響 69、EMG對DNA片段化及細胞核形態之影響 70、EMG對caspase-3、-8及-9活性之影響 71、EMG對粒線體膜電位之影響 72三節 以動物腫瘤模式探討靈芝菌絲體乙醇萃取物 (EMG) 之抑癌活性 73、腫瘤動物模式之建立 73、EMG對人類肝癌細胞異種移植裸鼠腫瘤生長之影響 74、EMG對腫瘤組織形態與caspase-3表現之影響 75、肝功能指數與其他血液生化值 76四節 靈芝菌絲體乙醇萃取物 (EMG) 區分物之抑制Hep3B細胞活性與成分鑑定 77、預備區分實驗之結果 77、正式區分實驗之結果 78、EMG區分物對細胞生存率之影響 78、EMG區分物F6對細胞週期之影響 78、EMG區分物F6對DNA片段化之影響 79、EMG活性成分之結構分析與鑑定 79、活性成分對細胞生存率之影響 84、活性成分對DNA片段化之影響 84、活性成分對細胞核形態之影響 84、結論 86、參考文獻 87一 細胞週期及其相對應之Cyclin-CDK complex 31二 G. lucidum之三萜類成分與生理功效 32三 以豆科植物作為基質發酵所得靈芝之發酵條件 100四 不同發酵條件所得靈芝菌絲體之乙醇萃出率 100五 靈芝菌絲體乙醇萃取物在以lovastatin協同處理48小時之下對Hep3B細胞存活率之影響 101六 試驗結束時裸鼠之血液生化值 102七 預實驗所得區分物之產率 103八 第二次區分實驗所得區分物之產率 104一 多步驟性之致癌機轉 33二 癌細胞之能力 34三 誘發肝癌之相關機制 35四 細胞週期及其相關調控分子 36五 以電子顯微鏡觀察到之細胞形態 (A)一般細胞 (B)凋亡細胞 (C)壞死細胞 37六 細胞凋亡之死亡受體、粒線體及內質網路徑 38七 粒線體路徑 39八 Bcl-2家族成員 40九 人類caspase之結構與功能 41十 死亡受體路徑 42十一 靈芝 (Ganoderma lucidum) 子實體之型態 43十二 從G. lucidum子實體中發現之三萜類化合物之化學結構 44十三 靈芝菌絲體乙醇萃取物 (EMG) 對Hep3B細胞存活率之影響 105十四 不同濃度之EMG對Hep3B細胞形態之影響 (處理48小時) 106十五 EMG誘導Hep3B細胞週期sub-G1 peak的產生 107十六 EMG對Hep3B細胞 (A) DNA片段化 (B) 細胞核形態之影響 108十七 EMG對Hep3B細胞內caspase-3活性之影響 109十八 EMG對Hep3B細胞內caspase-8活性之影響 110十九 EMG對Hep3B細胞內caspase-9活性之影響 111二十 EMG對Hep3B細胞內粒線體膜電位之影響 112二十一 接種Hep3B細胞2-3週後 (實驗期第28-35天) 控制組裸鼠之外觀。 113二十二 試驗期間給予EMG對各組裸鼠體重之影響 114二十三 EMG對裸鼠腫瘤體積之影響 115二十四 試驗結束時腫瘤在裸鼠身上之生長情形 116二十五 EMG對裸鼠腫瘤重量之影響 117二十六 EMG對腫瘤組織形態與cleaved caspase-3表現之影響 118二十七 預實驗所得區分物對Hep3B細胞存活率之影響 119二十八 PF8誘導Hep3B細胞週期sub-G1 peak的產生 120二十九 PF13誘導Hep3B細胞週期sub-G1 peak的產生 121三十 預實驗所得區分物對Hep3B細胞DNA片段化之影響 122三十一 第二次區分實驗之TLC結果 123三十二 區分物對Hep3B細胞存活率之影響 124三十三 F6誘導Hep3B細胞週期sub-G1 peak的產生 125三十四 F6對Hep3B細胞DNA片段化之影響 126三十五 化合物1之1H-NMR光譜圖 127三十六 化合物1之紅外線光譜圖 127三十七 化合物2之1H-NMR光譜圖 128三十八 化合物2之紅外線光譜圖 128三十九 活性成分對Hep3B細胞存活率之影響 129四十 活性成分對Hep3B細胞DNA片段化之影響 130四十一 活性成分對Hep3B細胞細胞核形態之影響 131application/pdf2806017 bytesapplication/pdfen-US靈芝抑制肝癌活性細胞凋亡ergosterol peroxideGanoderma lucidumanti-hepatoma activityapoptosis[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182229/1/ntu-98-D92641002-1.pdf