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研究生:鍾采穎
研究生(外文):Tsai-Ying Chung
論文名稱:香椿水萃取物對細菌之抗致突變性及對哺乳動物腫瘤細胞之毒性作用
論文名稱(外文):Antimutagenicity in bacteria and cytotoxicity in mammalial cancer cells by water extracts of Toona sinensis Roemor
指導教授:葉貞吟葉貞吟引用關係鍾雲琴
指導教授(外文):Jan-Ying YehYun-Chin Chung
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2005/07/
畢業學年度:93
語文別:中文
論文頁數:100
中文關鍵詞:細胞毒性抗致突變性香椿
外文關鍵詞:cytotoxicityantimutagenicityToona sinensis Roemor
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香椿(Toona sinensis Roemor)為藥食兩用的植物,其葉、果實、樹汁與樹皮都是常見的中醫藥材,雖然香椿為民間常用之藥用保健植物,但其真正的生理功效有待更完整的科學研究證實之。本研究擬探討香椿葉水相萃取物對微生物的抗致突變效果,與對哺乳動物腫瘤細胞活性之影響。本論文以沙門氏菌回復突變測試法(即Ames assay)進行抗致突變試驗,測試菌株包括Salmonellae typhimurium TA97、TA98、TA1535、TA100、TA102;此外,以大鼠神經膠瘤細胞(Rat glioma)C6細胞與人類結腸腺癌細胞(Human colon adenocarcinoma)Caco-2 細胞為細胞模式,利用MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay測試細胞活性 ,評估香椿葉水相萃取物對腫瘤細胞株生長的影響。結果發現,不論是否添加S9 mix,香椿葉水相萃取物於測定濃度範圍內,對所有測試菌株無致突變效果。香椿葉水相萃取物於添加S9 mix時,對須代謝酵素活化之突變劑(2-aminofluorene及2-anthramine)所誘發TA100及TA1535的突變有明顯的抑制作用,且分別於1.25 mg/plate及1.50 mg/plate即可抑制90%以上之突變;但未添加S9 mix,對TA100及TA1535則不具抗突變效果。此外,香椿葉水相萃取物3.0 mg/plate時不論是否添加S9 mix,對TA97及TA98之抗突變效果超過50%;而對TA102之突變不具抑制作用。細胞毒性試驗結果顯示,香椿葉水相萃取物對於C6細胞毒性之影響較其對Caco-2明顯。於濃度3.12~25.00 mg/ml範圍內對C6細胞毒性具有明顯抑制效果(90 ~ 92 %);而對Caco-2細胞之活性影響較不顯著,於測試濃度範圍(0.09 ~25.00 mg/ml)最高抑制作用只有約81 %。綜合以上結果可知,香椿葉水相萃取物對微生物並無基因毒性產生,於代謝活化酵素(S9 mix)存在下具抑制點突變(例如對S. typhimurium TA100及TA1535具抗致突變)能力,對腫癌細胞株活性之影響程度亦隨細胞種類而異。本結果發現香椿可能對特定種類之突變及腫瘤細胞具抑制作用,但是其作用機制仍待探討。
Toona sinensis Roemor, an edible plant, is also used as Chinese medicine. Not only its leaves, fruit, sap and cortex are used commonly for medication. Further scientific study is needed to confirm the biofunctions of Toona sinensis Roemor. The aims of this study were to evaluate the antimutagenicity in bacteria and cytotoxicity in mammalian cancer cells by water extracts of Toona sinensis Roemor leaves. In this study, the bacterial antimutagenicity for water extract of Toona sinensis Roemor leaves was determined by Salmonellae revertant assay (Ames assay) and the tester stains included Salmonellae typhimurium TA97, TA98, TA1535 TA100, and TA102. The effect for water extract of Toona sinensis Roemor leaves on mammalial cancer cells was detectived by MTT 〔3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide〕assay using rat C6 glial cell and human Caco-2 colon adenocarcinoma cell. The results showed that the tested extract had no mutagenicity effect toward all tester strains. When the S9 mix was added, the extract exhibited significant antimutagenicity against TA100 and TA1535. At concentrations of 1.25 mg/plate and 1.5 mg/plate, more than 90% inhibition wsa achieved on mutagenicities induced by metabolic enzymes-activated mutagens (2-aminofluorene and 2-anthramine ). However, the extract was deficient in antimutagenicity on TA97 and TA1535 if S9 mix was not present. Among the tested concentrations, with or without adding S9 mix, the highest capability of antimutagenicity was about 50% inhibition toward TA97 and TA98 and no inhibition toward TA102. Results of cellular activities showed that the water extract of Toona sinensis Roemor leaves was more effective on C6 than Caco-2 cell. Up to 90 % inhibition was obtained when C6 cells were treated with 3.12~25.0 mg/ml of extract, but only 81% inhibition obtained in Caco-2 cell. The results of this study showed that the water extract of Toona sinensis Roemor leaves had no microbial mutagenicity, but showed inhibitory ability on point mutation (antimutagenicity on TA 100 and TA 1535) and cytotoxicity in cancer cells. These results demonstrated that the water extract of Toona sinensis Roemor leaves may inhibit certain type of mutation in bacteria and be cytotoxic to cancer cells. However, the inhibitory mechanism of the water extract of Toona sinensis Roemor leaves needs further investigation.
目錄
頁次
中文摘要………………………………………………………… 3
英文摘要………………………………………………………… 5
表一、基因毒性測試系統……………………………………… 17
表二、Salmonella tester strains之DNA序列與基因型……… 23
表三、S9與TA98於含氮鹼基異構物之致突變結果………… 26
表四、Ames test測試菌株對於不同測試物質之回復突變菌落數………………………………………………………………… 27
表五、國際對抗致突變作用結果的解釋……………………… 32
表六、良性腫瘤與惡性腫瘤的區別…………………………… 33
表七、細胞毒性試驗測定方法………………………………… 38
表八、MTT assay方法………………………………………… 47
表九、香椿葉水相萃取物對Salmonella typhimurium TA97,
TA98與TA1535之毒性試驗……………………………………… 79
表十、香椿葉水相萃取物對Salmonella typhimurium TA100與
TA102之毒性試驗………………………………………………… 80
表十一、香椿葉水相萃取物對Salmonella typhimurium TA97,
TA98與 TA1535之致突變試驗…………………………………… 81
表十二、香椿葉水相萃取物對Salmonella typhimurium TA100
與TA102之致突變試驗…………………………………………… 82
表十三、香椿葉水相萃取物對Salmonella typhimurium TA97
、TA98與 TA1535之抗致突變試驗……………………………… 83
表十四、香椿葉水相萃取物對Salmonella typhimurium TA100
與TA102之抗致突變試驗………………………………………… 84
圖一、香椿……………………………………………………… 10
圖二、Ames assay試驗流程…………………………………… 19
圖三、利用染色法測定細胞毒性之操作過程………………… 41
圖四、MTT及其經由succinate-dehydrogenase還原後所形成
之formazan產物構造…………………………………………… 43
圖五、MTT assay中的細胞生長與吸光值線性關係…………… 44
圖六、主要抗致突變食品成分之作用模式…………………… 55
圖七、香椿葉水相萃取物對C6細胞與Caco-2細胞之毒性…… 85
第一章 緒論…………………………………………………… 7
第二章 文獻回顧……………………………………………… 8
第一節 香椿簡介……………………………………
A.營養價值……………………………… 11
B.生理功能……………………………… 11
C.成分分析……………………………… 14
第二節 抗致突變之評估方法………………………… 15
A.致突變試驗…………………………… 15
B.測試菌株之特性……………………… 20
C.藥物轉化酵素S9……………………… 24
D.試驗方法與結果判定………………… 28
E.應用…………………………………… 28
F.抗致突變試驗………………………… 31
第三節 抗腫瘤之評估方法…………………………… 32
A.MTT assay……………………………… 42
B.Neutral red uptake frame………… 50
C.Sulforthodamine B(SRB)………… 50
D.動物腫瘤細胞簡介………………………51
第四節 天然物及食品之抗致突變及抗腫瘤效果…… 53
A.天然物之抗致突變及抗腫瘤效果………53
B.食品之抗致突變及抗腫瘤效果…………54
第三章 材料與方法…………………………………………… 59
第一節 抗致突變性之評估…………………………… 59
A.藥品配製…………………………………59
B.實驗方法…………………………………64
第二節 動物腫瘤細胞活性評估……………………… 67
A.藥品配製……………………………… 67
B.實驗方法……………………………… 68
C.統計分析……………………………… 70
第四章 結果與討論…………………………………………… 71
第一節 香椿水萃物之抗致突變性…………………… 71
A.毒性試驗……………………………… 71
B.致突變性試驗………………………… 72
C.抗致突變試驗………………………… 73
第二節 香椿水萃物動物腫瘤細胞活性之抑制作用… 76
第五章 結論…………………………………………………… 78
參考文獻 ………………………………………………………… 86
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