臺灣博碩士論文加值系統

牛樟芝為生長於台灣特有種牛樟樹（Cinnamomum kanehirae Hayata）上獨有之真菌，近來，人們越來越重視從天然產品中尋找藥物，牛樟芝的藥理效用逐漸受世人認可，諸如腫瘤抑制、增強免疫等，而其保健使用與治療效果必須築基於毒性劑量觀念之上。本研究以微生物回復突變性試驗法(mammalian microsome reversion assay, Ames test)針對牛樟芝萃取純化次級代謝產物-倍半萜內酯Antrocin進行致突變及抗致突變性試驗，希冀了解牛樟芝之重要成份Antrocin在使用上之安全劑量範圍及可能治療癌症效果。
本實驗以鼠傷寒沙門氏菌TA98和TA100菌株分為兩部分來測試：第一組為致突變試驗，進行安全性評估討論；第二組為抗致突變性試驗，進行功能性評估討論。在安全性評估中，Antrocin試驗最高劑量100μg/ plate對這兩個菌株的菌落數量沒有致突變作用。因此，在此劑量下同時進行Antrocin的功能性評估抗致突變性測試。
在抗致突變性試驗中，Antrocin對TA100菌株的抑制率較低，在不經肝臟酵素系統中試驗只能達到24.3％的抑制率，低於本實驗室以前對各種方法培養的樟芝提取物的研究；Antrocin對TA98菌株在不經肝臟酵素系統中試驗的抑制率為33.9％，高於本實驗室以前對各種方法培養的樟芝提取物的研究，而當加入肝臟酵素系統後，抑制率可提高至66％，具高度抗致突變能力，顯示Antrocin在抗致突變之治癌效果評估需經體內肝臟酵素之代謝才能產生。

Antrodia cinnamomea, a heart-rot wood decay fungus of stout camphor (Cinnamomum kanehirae Hayata), is an endemic fungal species in Taiwan. Recently, people are paying more and more attention to search medicines from natural products. It has been reported that many chemical components of A. cinnamomea carry functional properties like tumor suppression, immunological enhancement. However the application of these components for medical cure has to found on the knowledge of the toxic dose of these components. This research studied the mutagenicity and anti-mutagenicity of Antrocin, an extract of A. cinnamomea, via mammalian microsome reversion assay (Ames test) to find the safe dose range and to verify the anti-cancer property of Antrocin and its derived pure compounds.
The TA98 and TA100 strains of Salmonella typhimurium were used to conduct Ames test. Up to 100 µg/plate of Antrocin gave no suppression effect to the number of colony multiplied of these two strains. The mutagenicity test assessing the security of Antrocin and the anti-mutagenicity test assessing the function of Antrocin were therefore tested up to this dose of 100 µg/plate. No mutagenic potential was found in the security assessment for both of TA98 and TA 100 strains. For anti-mutagenicity assessment, the inhibition rate to TA100 strain exhibited by Antrocin was weak, only up to 24.3%, if S9 was not added, lower than previous studies of this laboratory on row extract of Antrodia cinnamomea cultivated from various methods. However the inhibition rate to TA98 strain was 33.9%, higher than previous studies of this laboratory on row extract of Antrodia cinnamomea, and approximately doubled, 66%, while the liver activating enzyme S9 is present.

摘要 I
ABSTRACT III
致謝 V
目錄 VII
圖目錄 XI
表目錄 XII
第一章、 前言 1
1.1 研究緣起 1
1.2 研究目的 3
1.3 研究架構 3
第二章、 文獻回顧 5
2.1 天然產物之代謝物與生物活性 5
2.2 真菌代謝產物及生物活性 6
2.3 牛樟芝介紹 9
2.4 牛樟芝活性成分研究 13
2.4.1 多醣體 13
2.4.2 萜類 14
2.4.3 三萜類 15
2.4.4 倍半萜類 16
2.5 癌症與飲食 18
2.6 抗致突變作用機制 19
2.7 健康食品認證 20
2.7.1 健康食品安全性評估 20
2.7.2 健康食品查驗登記雙軌制 21
2.8 藥品非臨床安全性試驗 24
2.9 安姆氏測試法 26
第三章、 材料與方法 29
3.1 試驗材料 29
3.1.1 牛樟芝(YMT1002)倍半萜內酯Antrocin 29
3.1.2 試驗菌株 30
3.1.3 培養基及溶液配置 33
3.1.4 供試藥品 36
3.1.5 供試器材 37
3.2 試驗方法 38
3.2.1 固態培養 38
3.2.2 液態培養 38
3.2.3 菌性測定 40
(一) 組胺酸需求特性測定 40
(二) rfa突變型測定 40
(三) ∆uvrB突變型測定 40
(四) R質體測定 40
3.2.4 Antrocin致突變試驗 41
3.2.5 Antrocin抗致突變試驗 41
3.3 結果判定與品保 43
3.3.1致變異結果分析參考對照 43
3.3.3抗致突變率計算 45
3.3.4數據分析 45
第四章、 結果與討論 46
4.1 S. typhimurium TA98與TA100菌株基因型態試驗 46
4.1.1 組胺酸需求特性測定 46
4.1.2 rfa突變型測定 46
4.1.3 ∆uvrB突變型測定 47
4.1.4 R質體測定 47
4.2 牛樟芝萃取物Antrocin之致變異性評估 48
4.2.1 致突變試驗 48
4.2.2 抗致突變試驗 50
4.3 Antrocin與牛樟芝四種不同培養方式萃取物治癌性比較 52
第五章、 結論 57
第六章、 建議 58
參考文獻 60
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圖目錄
圖一、研究架構圖 4
圖二、牛樟芝 11
圖三、Antrocin的化學結構式 29
圖四、框構位變異示意圖 32
圖五、鹼基對換變異示意圖 32
圖六、致突變試驗 42
圖七、抗致突變試驗 42
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表目錄
表一、沙門氏菌株DNA序列特異性 31
表二、S. typhimurium TA98與TA100菌株基因型態試驗結果 47
表三、牛樟芝萃取物Antrocin在有或無S9混合物下對鼠傷寒桿菌TA98菌株之致突變性 49
表四、牛樟芝萃取物Antrocin在無S9混合物下對鼠傷寒桿菌TA100菌株之致突變性 49
表五、抗致突變活性區間 50
表六、牛樟芝萃取物Antrocin對4NQO(-S9)在鼠傷寒沙門桿菌TA98及TA100之抗致突變性上之影響 51
表七、牛樟芝萃取物Antrocin對2-AF (+S9)在鼠傷寒沙門桿菌TA98之抗致突變性上之影響 51
表八、牛樟芝萃取物Antrocin與牛樟芝四種不同培養方式萃取物對4NQO (-S9)在鼠傷寒沙門桿菌TA98之抗致突變性上之影響 54
表九、牛樟芝萃取物Antrocin與牛樟芝四種不同培養方式萃取物對4NQO (-S9)在鼠傷寒沙門桿菌TA100之抗致突變試驗 55
表十、牛樟芝萃取物Antrocin與牛樟芝四種不同培養方式萃取物對2-AF (+S9)在鼠傷寒沙門桿菌TA98之抗致突變試驗 56

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