臺灣博碩士論文加值系統

樟芝（Antrodia camphorata）為台灣特有種之食藥用菇，本研究以A . camphorata菌種所生產之固體栽培及液體發酵培養的菌絲體，經乙醇萃取後其萃取液再經由分配萃取，初步分離為n-Hexane layer、Ethyl acetate(EA) layer、Ethanol(EtOH) layer等三部分，經進一步進行生物活性之分析後，再針對具有活性的部分進行管柱色層層析分離。而經乙醇萃取後之殘渣再以熱水萃取經凍乾得熱水萃取物，經進一步進行透析或酒精沉澱探討其多醣分子量及其生物活性。
樟芝之固體栽培及液體發酵菌絲體乙醇萃取物，對Escherichia coli、 Salmonella choleraesuis subsp. choleraesuis、Staphylococcus aureus subsp. aureus、Pseudomonas aeruginosa、Proteus vulgaris、Pseudomonas glumae、Listeria monocttogenes、Aspergillus niger、Penicillium citrinum、Fusarium moniliforme皆有抑制生長或抑制產孢之能力。固體栽培菌絲體乙醇萃取物對DPPH• (1,1-Diphenyl-2-picryl-hydrazyl)之清除能力遠大於液體發酵菌絲體乙醇萃取物，而不論固體栽培或液體發酵菌絲體乙醇萃取物，其對DPPH•之清除能力，在反應三十分鐘後(0.4mg/mL)，EA layer效果最佳，EtOH layer次之，n-Hexane layer較差。而固體栽培或液體發菌絲體熱水萃取物(10mg/mL)則以ABTS測定總抗氧化力，以固體栽培菌絲體熱水萃取物較佳。固體栽培菌絲體乙醇萃取液(1mg/mL)，對子宮頸癌(HeLa)、乳癌(MCF-7)、胃癌(AGS)等細胞具有抑制生長之能力，而液體發酵菌絲體乙醇萃取液(1mg/mL)則對乳癌(MCF-7)細胞、胃癌(AGS)細胞有較好的抑制生長能力。在初步分離之三部分(n-Hexane layer、EA layer、EtOH layer)，對抑制癌細胞之生長效果，n-Hexane layer與EA layer較優於EtOH layer。固體栽培與液體發酵之熱水萃取物(5mg/mL)，對於子宮頸癌與胃癌細胞有較佳抑制能力。而固態酒精沉澱物則對子宮頸癌、乳癌、胃癌及肝癌之抑制能力較液體發酵酒精沉澱物佳。
在多醣體成分之測定，不論以透析(透析膜M.W.3000)或酒精沈澱法分析，液態發酵之多醣含量及濃度皆優於固體栽培菌絲體。而經由膠體過濾層析(GPC)分析得知，二者皆有分子量百萬以上之多醣體，且透析後之分子量遠比酒精沈澱的分子量大。
綜合本研究之結果，樟芝固體栽培及液體發酵之菌絲體乙醇萃取物和熱水萃取物在抗菌能力、抗氧化能力以及細胞株之抗癌試驗上，皆有良好之效果。

Chang-Zhi, namely Antrodia camphorata, is an endemic species in Taiwan, which was used for food and medicine. This study we used mycelia obtained by solid stage and liquid fermentation for further experiments. Mycelia were extracted by ethanol and following partitions were extracted by n-Hexane, ethyl acetate (EA), and ethanol (EtOH) formation.
Ethanol extractions from Antrodia camphorata mycelia produced by solid and liquid stage fermentation were further used in the tests of antibiosis, antioxidant, and antitumor activities. The following microorganisms, such as Escherichia coli, Salmonella choleraesuis subsp. Choleraesuis, Staphylococcus aureus subsp.aureus, Pseudomonas aeruginosa, Proteus vulgaris, Pseudomonas glumae, Listeria monocttogenes, Aspergillus niger, Penicillium citrinum, and Fusarium moniliforme, were investigated for antibiotic examination by using double layers plate method. Except for Pe. citrinum, both extractions of mycelia (0.1g/mL) fermented by solid and liquid stage fermentation evidently contained antibiosis activity to inhibit the growth or sporulation of upper microorganisms.
Scavenging effect of DPPH• (1,1-Diphenyl-2-picryl-hydrazyl) by ethanol extracts of solid stage fermentation was identically better more than that of liquid stage fermentation. Reaction after 30 minutes the scavenging effect on DPPH• comparison with ethanol extraction of solid and liquid stage fermentation (0.4mg/mL) showed that the activity of extraction is EA layer > EtOH layer > n-Hexan layer. Meanwhile, the total antioxidant status of hot water extracts by solid stage fermentation was better than that of by liquid stage fermentation.
Both ethanol extractions (1mg/mL) indicated the ability of antitumor activity to cervical cancer cell (HeLa), breast cancer cell (MCF-7), and gastric cancer cell (AGS) was acceptable. On n-Hexane, ethyl acetate (EA), and ethanol (EtOH) formation, the inhibition antitumor cells comparison with solid and liquid stage fermentation proved that n-Hexane and EA layer was better than EtOH layer.
Mycelia hot water extracts (5mg/mL) by solid and liquid stage fermentation inhibited the HeLa and AGS tumor cell viabilities. On the ethanol precipitate (5mg/mL) obtained from solid stage fermentation in antitumor HeLa, MCF-7 AGS, and Hep G2 cells was better than that from liquid stage fermentation.
The polysaccharides of hot water extractions from A. camphorata mycelia eluted through dialysis membrane (M.W. 3000) or precipitated in ethanol indicated that liquid stage fermentation was always higher than those by solid stage fermentation. The molecular weight of polysaccharides of solid and liquid stage fermentations eluted through gel filtration chromatograph (GPC) was over 106 Da, which was higher than that of by alcohol precipitate method.
Evidently, the functional activities of ethanol extractions and hot water extracts of A. camphorata mycelia in antibiotic, antioxidant, and antitumor were proved from both of solid and liquid stage fermentations.

目　　次
中文摘要……………………………………………………………………………….Ⅰ
英文摘要………………………………………………………………………….........Ⅲ
誌謝………………………………………………………………………….................Ⅴ
目次………………………………………………………………………….................Ⅵ
表目錄……………………………………………………………………….................Ⅸ
圖目錄………………………………………………………………………….............Ⅹ
第一章 緒論………………………………………………………………………….…1
1.1 研究動機………………………………………………………………………....1
1.2 樟芝之介紹…………………………………………………………………..…..3
1.3 菇類之機能性………………………………………………………………..…..4
1.3.1 第一級功能……………………………………………………………….....4
1.3.2 第二級功能……………………………………………………………….....4
1.3.3 第三級功能……………………………………………………………….....5
1.4 文獻回顧…………………………………………………….…………….……..6
1.4.1 樟芝之成分分析………………………….…………..……………………..6
1.4.1.1 一般營養成分之分析……………….…………...……………………..6
1.4.1.2 微量金屬含量分析……………….…………..…….…………………..6
1.4.2 樟芝之成分分離………………………….………………..………………..7
1.4.3 樟芝之生物功能性………………….…………………..………………....13
1.4.3.1 抗氧化能力…….……………….…………………..………………....13
1.4.3.2 肝功能試驗…….……………….…………………..………………....13
1.4.3.3 降血脂功能…….……………….…………………..………………....14
1.4.3.4 安全性評估…….……………….…………………..………………....14
第二章 材料與方法……………………………………………………………….....16
2.1 實驗材料……………………………………………………………..………....16
2.1.1 樣品及來源……………………………………………….……..………....16
2.1.2 儀器設備………………………………………………….……..………....16
2.2 實驗方法…………………………………………………….………….……....18
2.2.1 樣品萃取與分離………………………….……………….……………….18
2.2.1.1 試劑及設備……………………….….……………….……………….18
2.2.1.2 方法………………………….……………….………………….…….18
2.2.1.2.1 乙醇萃取物之分離…….……………….………………….……..18
2.2.1.2.2 熱水萃取物之分離…….……………….…………….…….…….18
2.2.2 抗菌活性試驗………………..…………………………………….……....21
2.2.2.1 試劑及設備……………..…………………………………….…….....21
2.2.2.2 方法……………..……………….…………………………….……....21
2.2.3 抗氧化能力測定…………………………….……….…………….……....22
2.2.3.1 試劑及設備……………..…………………………………….…….....22
2.2.3.2 方法……………..……………….…………………………….……....22
2.2.3.2.1 總抗氧化力之測定………….…………………………….……...22
2.2.3.2.2 清除DPPH•能力之測定…….…………………………….……...23
2.2.4 抑制腫瘤細胞成長試驗……………………..…………………….……....23
2.2.4.1 試劑及設備……………………..…………………….………….…....24
2.2.4.2 動物細胞株……………………..…………………….………….…....24
2.2.4.3 方法……………………..…………………….………………….…....24
第三章 結果與討論……………………………………………………….………....25
3.1 樟芝之成分分離………………………………………….….………………....25
3.1.1 乙醇萃取物之初步分離…………………………….……..……….……....25
3.1.2 固態栽培菌絲體乙醇萃取物層析分離…………………...………….……25
3.1.2.1 EA layer層析分離…………………………….……..………………....25
3.1.2.2 n-Hexane layer層析分離…………………………….……..……….….25
3.1.3 液態發酵菌絲體乙醇萃取物層析分離………………………………...….31
3.1.3.1 EA layer層析分離…………………………….……..………………....31
3.1.3.2 n-Hexane layer層析分離…………………………….……..……….….31
3.1.4 野生樟芝子實體之分析……………………. ………………………...…...31
3.1.5 多醣之萃取分離…………………………………………………….…..….31
3.2 乙醇萃取物之生物活性探討……………………………………………...……38
3.2.1 抗菌活性……………………………...………………………………….....38
3.2.1.1 乙醇萃取物之抗菌能力………...…………….…………………….....38
3.2.1.2 初步分離之抗菌能力………...…………….……………………….....38
3.2.1.3 Fractions之抗菌能力………...…………….………………….…….....39
3.2.2 抗氧化能力測定…………………………...…………………………….....51
3.2.2.1 乙醇萃取物之清除DPPH•能力……...…………………………….....51
3.2.2.2 初步分離之清除DPPH•能力……...……………………………….....51
3.2.2.3 Fractions之清除DPPH•能力……...…………………..…………….....51
3.2.3 抑制腫瘤細胞成長試驗……………………………...…………………….56
3.2.3.1 乙醇萃取物之抑制腫瘤細胞成長能力………...…………….……….56
3.2.3.2 初步分離之抑制腫瘤細胞成長能力………...………………….…….56
3.2.3.3 Fractions之抑制腫瘤細胞成長能力………...………………...……….56
3.3 熱水萃取物之生物活性探討…………………..……………………………….71
3.3.1 抗菌活性……………………………….…..……………………………….71
3.3.2 抗氧化能力………………………….…………..………………………….71
3.3.2.1 總抗氧化力…………………….……….…..………………………….71
3.3.2.2 清除DPPH•能力…………………….…………..…………………….71
3.3.3 抑制腫瘤細胞成長試驗………………………...………………………….72
第四章 結論………………………………………………………………………….83
第五章 參考文獻…………………………………………………………………….85
表目錄
表一、抗菌活性測試菌株……………………………………………………………...21
表二、樟芝菌絲體粉末乙醇萃取物之初步分離萃取率……………………………...29
表三、樟芝菌絲體粉末熱水萃取率………………………………………………...…34
表四、樟芝熱水萃取物之聚合物含量………………………………………………...35
表五、樟芝菌絲體中多醣體之含量………………………………………………...…35
表六、固態栽培與液態發酵菌絲體乙醇萃取物之抗菌活性………………...………41
表七、固態栽培菌絲體乙醇萃取物初步區分之抗菌活性…………………………...48
表八、液態發酵菌絲體乙醇萃取物初步區分之抗菌活性…………………………...49
表九、固態栽培菌絲體乙醇萃取之分離物(fraction)之抗菌能力……………………50
表十、液態發酵菌絲體乙醇萃取之Hexane layer分離物(fraction)之抗菌能力……..46
表十一、液態發酵菌絲體乙醇萃取之EA layer分離物(fraction)之抗菌能力…….…47
表十二、乙醇萃取物清除DPPH•能力相對對照組之相對作用濃度………………...54
表十三、乙醇萃取分離物(fractions)清除DPPH•能力………………………………..55
表十四、固態栽培與液態發酵菌絲體熱水萃取物之抗菌活性……………………...73
表十五、熱水萃取物清除DPPH•能力………………………………………………...76
圖目錄
圖一、樟芝之單孢子壁………………………………………...…………….………....3
圖二、楊書威由樟芝分離得到九個成分…………………………………….…………9
圖三、高曉薇由樟芝分離得到三個成分…………………………………………….....9
圖四、程一華分離得到十個成分………………………………………………….......10
圖五、吳德鵬分離得到十二個成分…………………………………………………...11
圖六、黃惟敏分離得到之四個新化合物……………………………………………...12
圖七、樟芝樣品萃取與分離流程圖…………………………………………………...20
圖八、固態栽培菌絲體乙醇萃取液初步分離之HPLC層析圖……………………...27
圖九、液態發酵菌絲體乙醇萃取液初步分離之HPLC層析圖………….…………...28
圖十、固態栽培菌絲體EA layer層析分離流程圖…………………………………...30
圖十一、固態栽培菌絲體Hexane layer層析分離流程圖………….............................30
圖十二、液態發酵菌絲體EA layer層析分離流程圖………………………………...33
圖十三、液態發酵菌絲體Hexane layer層析分離流程圖…………………………....33
圖十四、野生樟芝乙醇萃取液之HPLC層析圖……………………………………...32
圖十五、膠體過濾層析(GFC)分子量之分佈圖…………………………………….....36
圖十六、膠體過濾層析(GFC)標準品LogM.W.—R.T.(min)之標準曲線………...…...37
圖十七、固態栽培與液態發酵菌絲體乙醇萃取物之抗菌活性……….......................42
圖十八、固態栽培菌絲體乙醇萃取物初步區分之抗菌活性 ……………………….44
圖十九、液態發酵菌絲體乙醇萃取物初步區分之抗菌活性………………………...46
圖二十、乙醇萃取分離物(fraction)之抗菌測試………………………………………47
圖二十一、固態栽培菌絲體乙醇萃取物之清除DPPH•能力…………………..…....52
圖二十二、液態發酵菌絲體乙醇萃取物之清除DPPH•能力………………………..53
圖二十三、固態栽培菌絲體乙醇萃取物抑制腫瘤細胞生長能力…………………..58
圖二十四、液態發酵菌絲體乙醇萃取物抑制腫瘤細胞生長能力………………..…59
圖二十五、固態栽培菌絲體Hexane layer抑制腫瘤細胞生長能力…………………60
圖二十六、液態發酵菌絲體Hexane layer抑制腫瘤細胞生長能力…………….……61
圖二十七、固態栽培菌絲體EA layer抑制腫瘤細胞生長能力……………..………..62
圖二十八、液態發酵菌絲體EA layer抑制腫瘤細胞生長能力……………....………63
圖二十九、固態栽培菌絲體EtOH layer抑制腫瘤細胞生長能力……………………64
圖三十、液態發酵菌絲體EtOH layer抑制腫瘤細胞生長能力………………………65
圖三十一、固態栽培乙醇分離物抑制腫瘤細胞生長能力…………………..……….66
圖三十二、固態栽培純化物抑制腫瘤細胞生長能力…………………….…….….…67
圖三十三、液態發酵Hexane layer之分離物抑制腫瘤細胞生長能力…………….…68
圖三十四 液態發酵EA layer之分離物抑制腫瘤細胞生長能力……………………69
圖三十五、液態發酵純化物抑制腫瘤細胞生長能力………………………………...70
圖三十六、固態栽培與液態發酵菌絲體熱水萃取物之抗菌活性…………………...74
圖三十七、固態栽培與液態發酵菌絲體熱水萃取物之總抗氧化力………………...75
圖三十八、固態栽培熱水萃取物之抑制腫瘤細胞生長能力………………………...77
圖三十九、液態栽培熱水萃取物之抑制腫瘤細胞生長能力………………………...78
圖四十、固態栽培熱水萃取透析物之抑制腫瘤細胞生長能力…………………...…79
圖四十一、液態栽培熱水萃取透析物之抑制腫瘤細胞生長能力……………..…....80
圖四十二、固態栽培熱水萃取酒精沈澱物之抑制腫瘤細胞生長能力……….….....81
圖四十三、液態栽培熱水萃取酒精沈澱物之抑制腫瘤細胞生長能力………….….82

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