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研究生:簡淑娟
研究生(外文):Shu-Chuan Chien
論文名稱:人工固體栽培樟芝成分抽離暨自由基清除抗變異活性探討
論文名稱(外文):Active components isolation and their antimutagenic activities from Solid-state cultivated Taiwanofungus camphoratus via hydroxy radical scavenging effect
指導教授:洪清霖洪清霖引用關係
指導教授(外文):Ching-Lin Hong
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:公共衛生學系
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:126
中文關鍵詞:樟芝抗變異安姆氏生物檢定法
外文關鍵詞:Taiwanofugus camphoratus、Antimutagenic、Ames test
相關次數:
  • 被引用被引用:6
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  • 下載下載:142
  • 收藏至我的研究室書目清單書目收藏:1
摘要
樟芝為台灣本土獨特菌種,價格昂貴,民間傳為解毒、保肝、抗癌之機能性食品即食療補助用品;產學界多數以液體深層發酵培養為主,但無法令其自行合成具有作用的三萜類 (Triterpenoids)。固體栽培是新開發出來的栽培技術;運用此法培養出的樟芝,已有報告指出其成分與野生樟芝較為相似。
本研究利用固體栽培樟芝子實體作為研究材料,探討其抗突變之作用機轉。將樟芝粉末分別經乙醇和冷水萃取,其乙醇萃取液再經由分配萃取,初步分離成正己烷層、乙酸乙酯層、甲醇層等三部分,而水萃取液則經透析後再行酒精沈澱探討其多醣的生物活性。
本研究以安姆氏試驗法之Salmonella typhimurium TA98及TA100突變株,發現無論加或不加老鼠肝臟均質物S9 mix活化酶,其均呈現陰性反應,顯示樟芝對微生物不具致突變性。此外以TA102進行基因毒性試驗及自由基抗變異活性分析;結果發現樟芝不具基因毒性且其乙醇粗萃取物及水萃取物對清除過氧化氫自由基之抗變異效果達統計上顯著抑制效果。而進一步分離所得的三個部分,對清除過氧化氫自由基抗變異效果依序為:甲醇層>乙酸乙酯層>正己烷層。
因此再利用中壓液態層析分離抽取甲醇層,依序得出三個部分。抗變異能力測試結果,Fraction3的效果最佳,其SR50為0.005mg/ml,即具有較佳的抗變異效果。綜合以上實驗結果,顯示樟芝不具基因毒性及致突變性,此外還可藉由去變異(Desmutation)及抗變異(Antimutation)達到清除自由基之功效。
Abstract
Taiwanofugus camphoratus is an unique fungal species in Taiwan which is rare and expensive. It has been used as a functional food for drug intoxication, hepatoprotection and anti-tumor. This study used the fruiting body of T. camphoratus obtained from solid stage to further explore its anti-tumor mechanism. Fruiting body was extracted by ethanol and cold water. Ethanol extracts were further partitioned using n-Hexane, ethyl acetate, and methanol fraction. Cold water extracts were eluted through dialysis membrane (M.W. 3000) and precipitated in ethanol which were then used to analyze the bioactivation of T. camphoratus. This study also use the Ames test to evaluate the mutagenesis of T. camphorates. The Salmonella typhimurium TA98, and TA100 mutant strains were used. Either alone or in the presence of S9 mix, T. camphoratus extracts were not mutagenic. The genotoxicity and antimutagenic effects of T. camphoratus extracts were examined using S. typhimurium strain TA102. Our results showed that T. camphoratus extracts were non-genotoxic as well as antimutagenic for TA102, which were pretreated with H2O2-generated ROS in the Ames test. The H2O2 scavenging effect of the T. camphoratus ethanol extracts was highest is the methanol layer, followed by Ethyl acetate layer and n-Hexane layer. Therefore we further isolated three fractions seriesly from the methanol layer by Medium Pressure Liquid Chromatography. Through the antimutgenic assay, Fraction 3 had the highest antimutagenic ability (SR50:0.005mg/ml). In summary, we found that T. camphoratus is antigenotoxic and antimutagenic, which also has the ability of desmutation and antimutation to scavenge free radicals.
【目錄】

第一章前言 2
第一節文獻探討 2
第二節 研究背景與動機  2
第三節 研究目的 2
第二章 研究基礎之引用原理 2
第一節 樟芝的藥理活性 2
第二節 樟芝固體栽培 2
第三節 自由基與抗氧化物 2
第四節 安姆式沙門菌試驗 2
第三章 材料與方法 2
第一節 實驗材料與儀器 2
第二節 實驗方法 2
第四章 結果與討論 2
第一節 樟芝有效成分之分離 2
第二節 安氏試驗(Ames test)特性檢視 2
第三節 樟芝抗氧化致突變性試驗結果 2
第五章 結論 2
第一節 樟芝有效成分化學萃離 2
第二節 樟芝有效成分抗羥基自由基引變作用 2
第三節 討論 2
參考文獻 2
圖表 2
附錄 2

【表目錄】
Table 1. Extraction Yield of Ethanol Extracts from Taiwanofungus camphoratus Fruiting Body powder……………………………………………………....63
Table 2. Genotype marker / culture-check test of Salmonella typhimurium TA98 、TA100 and TA102 …………………………………..……………….……..64
Table 3. Mutagenicity of Ethanol extracts from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA98 with and without S9 mixture………65
Table 4. Mutagenicity of Methanol extracts from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA98 with and without S9 mixture………..66
Table 5. Mutagenicity of cold water extracts from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA98 with and without S9 mixture………..67
Table 6. Mutagenicity of Fraction 1 extracts from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA98 with and without S9 mixture………..68
Table 7. Mutagenicity of Fraction 2 extracts from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA98 with and without S9 mixture………..69
Table 8. Mutagenicity of Fraction 3 extracts from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA98 with and without S9 mixture………..70
Table 9. Mutagenicity of Lycopene on S. typhimurium TA98 with and without S9 mixture…………………………………………………………………...…..71
Table 10. Mutagenicity of Ethanol extracts from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA100 with and without S9 mixture…..…..72
Table 11. Mutagenicity of Methanol extracts from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA100 with and without S9 mixture………73
Table 12.Mutagenicity of cold water extracts from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA100 with and without S9 mixture………..74
Table 13.Mutagenicity of Fraction 1 extracts from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA100 with and without S9 mixture………..75
Table 14.Mutagenicity of Fraction 2 extracts from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA100 with and without S9 mixture………..76
Table 15. Mutagenicity of Fraction 3 extracts from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA100 with and without S9 mixture………………………………………………………………………...77
Table 16. Mutagenicity of Lycopene on S. typhimurium TA100 with and without S9 mixture………………………………………………………………………...78
Table 17 Genotoxicity check of cold water extract from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA102……………………..79
Table 18 Genotoxicity check of Fraction 1 from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA102……………………………………….80
Table 19. Genotoxicity check of Fraction 2 from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA102……………………………………….81
Table 20.Genotoxicity check of Fraction 3 from the fruiting body of Taiwanofungus camphoratus on S. typhimurium TA102……………………………………….82
Table 21.Genotoxicity check of Lycopene on S. typhimurium TA102……………….83
Table 22.Hydrogen peroxide induced mutagenicity on S.typhimurium TA102 diminished from radical scavenging activities of Taiwanofungus camphoratus Ethanol extracts………………………………………………………………..84
Table 23. Hydrogen peroxide induced mutagenicity on S.typhimurium TA102. diminished from radical scavenging activition of each separated layer separated from ethanol extracts Taiwanofungus camphorates…………………………...85
Table 24.Hydrogen peroxide induced mutagenicity on S.typhimurium TA102 diminished from the radical scavenging activities of Taiwanofungus camphoratus Methanol residual extracts………………………………………86
Table 25. Hydrogen peroxide induced mutagenicity on S.typhimurium TA102 diminished from the radical scavenging activities of Taiwanofungus camphoratus cold water extracts………………………………………………87
Table 26. Hydrogen peroxide induced mutagenicity on S.typhimurium TA102 diminished from the radical scavenging activities of Taiwanofungus camphoratus Fraction 1 extracts………………………………………………88
Table 27. Hydrogen peroxide induced mutagenicity on S.typhimurium TA102 diminished from the radical scavenging activities of Taiwanofungus camphoratus Fraction 2 extracts………………………………………………89
Table 28. Hydrogen peroxide induced mutagenicity on S.typhimurium TA102 diminished from the radical scavenging activities of Taiwanofungus camphoratus Fraction 3 extracts…………..…………………………………..90
Table 29. Hydrogen peroxide induced mutagenicity on S.typhimurium TA102 diminished from the radical scavenging activities of Lycopene………………91
Table 30.Antimutagenic Effects of Taiwanofungus camphoratus cold water extracts on S.typhimurium TA102 against the oxidative damage from H2O2 , under the antimutation model……………………………………………………………92
Table 31.Antimutagenic Effects of Taiwanofungus camphoratus methanol extracts Fraction 1 on S.typhimurium TA102 against the oxidative damage from H2O2 , under the antimutation model…………………………………………………93
Table 32. Antimutagenic Effects of Taiwanofungus camphoratus methanol extracts Fraction 3 on S.typhimurium TA102 against the oxidative damage from H2O2 , under the antimutation model…………………………………………………94
【圖目錄】
Figure.1 Schematic flowchart in the sample extraction and separation of Taiwanofungus camphorates…………………………………………………95
Figure 2. Elution profile of ethanol extraction of Taiwanofungus camphoratus fruiting body from the high performance liquid chromatography(HPLC)………...….96
Figure 3. Elution profile of methanol residual layer extraction of Taiwanofungus camphoratus fruiting body from the Medium performance liquid chromatography(MPLC)……………………………………………...……...97
Figure 4. Elution profile of methanol residual layer extraction of Taiwanofungus camphoratus fruiting body from the high performance liquid chromatography(HPLC)…………………………………………….………..98
Figure 5. Elution profile of Fraction 1 layer extraction of Taiwanofungus camphoratus fruiting body from the liquid chromatography/mass spectrometry………......99
Figure 6. Elution profile of Fraction 2 layer extraction of Taiwanofungus camphoratus fruiting body from the liquid chromatography/mass spectrometry ….…….100
Figure 7. Elution profile of Fraction 2 layer extraction of Taiwanofungus camphoratus fruiting body from the liquid chromatography/mass spectrometry…….…...101
Figure 8. Suppression of Hydrogen peroxide mutagenicity via scavenging activities by Taiwanofungus camphoratus Ethanol extract…………………………...102
Figure 9. Suppression of Hydrogen peroxide mutagenicity via scavenging activities by each separated layer from the ethanol extracts of Taiwanofugus camphorates…………………………………………………………………103
Figure 10. Suppression of Hydrogen peroxide mutagenicity via scavenging activities of Taiwanofungus camphoratus Methanol extract……………………….....104
Figure 11. Suppression of Hydrogen peroxide mutagenicity via scavenging activities of Taiwanofugus camphoratus cold water extract…………………………..105
Figure 12. Antimutagenic effect via Hydrogen peroxide scavenging activity of methanol extract Fraction 1…………………………………………………106
Figure 13. Antimutagenic effect via Hydrogen peroxide scavenging activity of methanol extract Fraction 2…………………………………..……………..107
Figure 14. Antimutagenic effect via Hydrogen peroxide scavenging activity of methanol extract Fraction 3…………………………………………...…….108
Figure 15 .Antimutagenic Effects of Taiwanofungus camphoratus cold water extracts on S.typhimurium TA102 against the oxidative damage from H2O2 , under the antimutation model………………………………………………………….109
Figure 16. Antimutagenic Effects of Taiwanofungus camphoratus methanol extract Fraction 1 on S.typhimurium TA102 against the oxidative damage from H2O2 , under the antimutation model……………………………………………….110
Figure 17. Antimutagenic Effects of Taiwanofungus camphoratus methanol extract Fraction 3 on S.typhimurium TA102 against the oxidative damage from H2O2 , under the antimutation model…………………………………………….…111
Figure 18. Antimutagenic effect via Hydrogen peroxide scavenging activitiy of Lycopene , under the antimutation model…………………………………..112
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