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研究生:林彥君
研究生(外文):Yen-Chun Lin
論文名稱:北蟲草子實體微波萃取物基因毒性之安全性評估
論文名稱(外文):Genotoxicity Analysis of Microwave-Extract of Cordyceps militaris by Ames Test and In Vitro Mouse Lymphoma tk Assay
指導教授:孫芳君陳小玲陳小玲引用關係
指導教授(外文):Fang-Chun SunHsiao-Ling Chen
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
中文關鍵詞:北蟲草微波萃取安姆測試鼷鼠淋巴瘤 tk+/- 分析
外文關鍵詞:Cordyceps militarisMicrowave ExtractionAmes TestMouse Lymphoma Assay
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北蟲草(Cordyceps militaris)原名北冬蟲夏草,又名蛹蟲草,與冬蟲夏草(Cordyceps sinensis)同為蟲草屬,經各方研究發現北蟲草與冬蟲夏草其醫用價值類似,可抗腫瘤、真菌、消炎及增加免疫力。因北蟲草價格便宜,所以市場佔有率已漸漸取代傳統冬蟲夏草。目前市面上有關探求北蟲草基因毒性資料甚少,且往往僅著重於北蟲草與冬蟲夏草活性比較。本研究目的為檢測北蟲草微波萃取物細胞毒性及基因毒性。樣品為子實體水萃取物及基座乙醇萃取物。本研究選用安姆測試 (Ames Test) 及鼷鼠淋巴瘤 tk+/- 分析法作為安全性評估平台。本研究 Ames Test 為平板混合試驗,並選用5株 Samonella typhimurium (TA97a、TA98、TA100、TA102及TA1535) 做為測試菌株。鼷鼠淋巴瘤細胞突變試驗則以 L5178Y tk+/- 細胞接種於96-well 作為測試方法。於 Ames Test 之毒性測試發現,北蟲草萃取物具有明顯抑制細菌生長。當北蟲草子實體水萃取物濃度為5 mg/plate 時,TA100 及 TA98 存活率百分分別為41.1% 和52.7%。而北蟲草基座乙醇萃取物濃度為0.63 mg/plate時,TA100及TA98存活率百分率分別為40.8%及49.1%。基因毒性試驗結果顯示,北蟲草經微波萃取處理萃取物,無論是否經 S9 mix 誘導,均不具有微生物及體外哺乳類細胞基因突變毒性。本試驗亦針對北蟲草萃取物進行抑癌測試,以 XTT 作為細胞存活指示劑,萃取液分別處理CHO-K1 (中國倉鼠細胞) 及 MB-MDA231 (乳癌細胞),結果發現北蟲草萃取物均有明顯毒殺 MB-MDA231 的能力,子實體水萃取物及基座乙醇萃取物IC50 分別為29 μg/ml及27 μg/ml。但對正常細胞不具有毒殺力。
Cordyceps militaris, belongs to the Dong-Chong-Xia-Cao group in Chinese herbs, possesses pharmacolugcal activities similar to Cordyceps sinensis. Several studies have been demosetrated that C. militaris has multiple biologacal actions, including anti-cancer, anti-infection, anti-inflammatory and immunological stimulating activities. In recent years, C. militaris is more popular and cheaper than C. sinensis on the market. In this study the cytotoxicity and mutagenicity of extracts from C. militaris were determined in the Ames test and in vitro mouse lymphoma tk assay. The Ames test was conducted by the plate incorporation method using five strains of Samonella typhimurium, TA97a, TA98, TA100, TA102 and TA1535. The L5178Y tk+/- cell was used for detecting and assessing the mutability of extracts from C. militaris by microwell method. In the cytotixicity test, water extracts and 50% ethanol extracts form C. militaris significantly inhibited the growth of S. typhimurium. When the concentration of water extract of fruiting body from C. militaris was 5 mg/plate, the survival rate was 41.1% and 52.7% in TA100 and TA98, respectively. However, when the concentration of 50% ethanol extracts of C. militaris locus was 0.63 mg/plate, the survival rate of TA100 and TA98 were 40.8% and 49.1%, respectively. In the mutagenesis analysis, the results show that whether the water extract and enthanol extract of fruiting body from C. militaris were treated with the S9 mix or not, no significant was shown. In addition, the inhibition of cell proliferantion was assessed by XTT. The results showed that the water and ethanol extracts of fruiting body from C. militaris effectively inhibited the proliferation of breast cancer (MB-MDA231 cell) with the range of 50% inhibited concentration (IC50) of 29μg/ml and 27μg/ml, respectively.
目錄

封面內頁
簽名頁
授權書                         iii
中文摘要                        iv
英文摘要                        vi
誌謝                        vii
目錄                         viii
圖目錄                        xiii
表目錄                       xiv

1. 前言                       1
2. 文獻回顧                       2
2.1 北蟲草                     2
2.2 蟲草活性成份                 4
2.2.1 蟲草素 (Cordycepin)             4
2.2.2 蟲草多醣 (Cordyceps polysaccharide)    5
2.2.3 蟲草酸 (D-Mannitol)              5
2.2.4 硒 (selenium)                 5
3. 微波萃取                       7
3.1 微波萃取背景                   7
3.2 微波萃取特色                   7
4. 基因毒性相關法規                   9
4.1 《健康食品管理法》                9
5. Ames Test                      12
5.1 背景                       12
5.2 食品常見致癌物            12
5.3 Histidine 生合成路徑               13
5.4 本研究 S. typhimurium 差異           15
6. 鼷鼠淋巴瘤 tk+/- 分析法               17
6.1 鼷鼠淋巴瘤 tk+/- 分析法試驗背景         17
7. 材料方法                      19
7.1 Ames Test                    19
7.1.1 藥劑                    19
7.1.2 藥品配法                  20
7.1.3 製備樣品                  23
7.1.4 菌株活化                  23
7.1.5 菌株保存                  24
7.1.6 平板培養                  24
7.1.7 菌株表現型確認               24
7.1.7.1 Histidine 篩檢          25
7.1.7.2 rfa mutation 檢測            25
7.1.7.3 uvrB mutation 檢測          25
7.1.7.4 R質體檢測            26
7.1.8 經各正對照組確認菌株            27
7.1.9 毒性試驗                  28
7.1.10 致突變測試                28
7.2 鼷鼠淋巴瘤 tk+/- 分析法              30
7.2.1 藥品                    30
7.2.2 藥品配置                  31
7.2.3 製備樣品                  32
7.2.4 細胞培養                  32
7.2.5 L5178Y tk-/- 基因型刪除         33
7.2.6 基因毒性測試                33
7.2.6.1樣品處理及細胞誘導          32
7.2.6.2 細胞毒性測試 (Cytotoxicity)       34
7.2.6.3 細胞存活測試 (Survival test)       34
7.2.6.4 Expression period            35
7.2.6.5 Viability test             35
7.2.6.6 TFT test              36
7.3 北蟲草抑癌測試                  37
7.3.1 藥品                    37
7.3.2 細胞培養                 38
7.3.3 細胞生長抑制率測試 (XTT assay)       38
7.4 統計分析                     39
8. 結果與討論                      39
8.1 Ames test 結果                 41
8.1.1 菌株確認                 41
8.1.2 毒性測試                 42
8.1.3 致突變測試                 44
8.2 鼷鼠淋巴瘤 tk+/- 分析結果             50
8.2.1 細胞毒性評估                50
8.2.2 生長毒性評估                50
8.2.3 基因毒性評估                55
8.3 抑制乳癌細胞測試                 58
9. 結論                         60
參考文獻                        63

圖目錄

圖2.2.1. Cordycepin 化學結構圖               4
圖5.3.1. Histidine 主要生成路徑              14
圖7.1.1. 致突變測試流程圖                 30
圖7.3.1. 細胞生長抑制率測試 (XTT assay) 流程圖      39
圖8.1.1. 北蟲草子實體水萃取物在無 S9 mix 誘導下之 Ames Test 結果     47
圖8.1.2. 北蟲草子實體水萃取物在有 S9 mix 誘導下之 Ames Test 結果     47
圖8.1.3. 北蟲草基座50%乙醇萃取物在無 S9 mix 誘導下之 Ames Test 結果   49
圖8.1.4. 北蟲草基座50%乙醇萃取物在有 S9 mix 誘導下之 Ames Test 結果   49
圖8.2.1. 北蟲草萃取物在無 S9 mix 誘導下之細胞24小時生長抑制結果      53
圖8.2.2. 北蟲草萃取物在有 S9 mix 誘導下之細胞24小時生長抑制結果      53
圖8.2.3. 北蟲草萃取物在無 S9 mix 誘導下之細胞14天生長抑制結果       54
圖8.2.4. 北蟲草萃取物在有 S9 mix 誘導下之細胞14天生長抑制結果        54
圖8.2.5. 北蟲草子實體水萃取物在無 S9 mix 誘導下致細胞基因突變檢測      56
圖8.2.6. 北蟲草子實體水萃取物在有 S9 mix 誘導下致細胞基因突變檢測     56
圖8.2.7. 北蟲草基座50%乙醇萃取物在無 S9 mix 誘導下致細基因傷害檢測     57
圖8.2.8. 北蟲草基座50%乙醇萃取物在有 S9 mix 誘導下致細胞基因傷害檢測    57
圖8.3.1. 北蟲草子實體水萃取物抑制 MB-MDA231 細胞生長檢測          59
圖8.3.2. 北蟲草基座50%乙醇萃取物抑制 MB-MDA231 細胞生長檢測        59

表目錄

表2.1.1. 北蟲草與冬蟲夏草成分分析比較           3
表4.1.1. 健康食品分類                  11
表5.4.1. Ames Test 菌株基因型突變差異           16
表7.1.1. Ames Test 菌株在 uvrB mutation 檢測條件    26
表7.1.2. Ames Test 所使用正對照組              28
表8.1.1 Ames Test 菌株表現型確認結果            41
表8.1.2 Ames Test 菌株經各正對組確認結果         42
表8.1.3. 北蟲草子實體水萃取物對 TA100 及 TA98 毒性測試 43
表8.1.4. 北蟲草基座50%乙醇萃取物對 TA100 及 TA98 毒性測試         44
表8.1.5. 北蟲草子實體水萃取物之 Ames Test 結果      46
表8.1.6. 北蟲草基座50%乙醇萃取物之 Ames Test 結果    48
表8.2.1. 北蟲草萃取物對細胞24~48小時及14天生長抑制結果  52
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