Title

探討離子液體(ILs)的致突變性與其對微生物與細胞的毒性影響之研究

Translated Titles

Mutagenicity and toxicity of ionic liquids on microorganisms and cells

DOI

10.6845/NCHU.2015.00234

Authors

黃祖駿

Key Words

離子液體 ; 微生物毒性 ; 細胞毒性 ; 致回復突變性 ; 抑菌 圈 ; MTT assay ; 發炎反應 ; Microbial toxicity ; cytotoxicity ; mutagenicity ; inhibition zone ; MTT assay ; inflammation

PublicationName

中興大學食品暨應用生物科技學系所學位論文

Volume or Term/Year and Month of Publication

2015年

Academic Degree Category

碩士

Advisor

方繼

Content Language

繁體中文

Chinese Abstract

離子液體(ionic liquids, ILs)乃由陰離子及陽離子所組成之有機化合物,具有非揮發性、較有機溶劑更佳的溶解能力以及較廣之液體溫度範圍等特殊性質,故可取代一般具有揮發性有機溶劑的使用,被視為一種新穎的綠色溶劑(green solvent)。近年來,離子液體被廣泛地應用於各種領域,如電化學、有機合成、萃取、生物技術等;然而,離子液體的潛在毒性在生物技術領域之應用為一重要課題。 本研究針對五種不同類型離子液體(1-ethyl-3-methylimidazolium ethylsulfate ([EMIM][EtSO4]), 1-butyl-3- methylimidazolium acetate ([BMIM][OAc]), trihexyl(tetradecyl) phosphonium decanoate (THTDPD), choline hydroxide ([Ch][OH])和cholin acetate ([Ch][OAc])和Mix-ILs (由親水性的[EMIM][EtSO4]和親油性的THTDPD混合配製的多元離子液體)的致回復突變性和對於微生物和細胞之毒性進行探討。 微生物毒性試驗中,[BMIM][OAc]對於四株食品中常見細菌(Escherichia coli BCRC 11634、Staphylococcus aureus BCRC 12154、Salmonella enterica BCRC 10747、Listeria monocytogenes BCRC 15330)產生之抑菌圈分別為23.94 mm、30.49 mm、31.10 mm和31.78 mm; 就黴菌而言, Aspergillus japonicus CY6-1和Aspergillus carneus M34所產生的抑菌圈分別為14.34 mm和18.75 mm,皆較四株細菌所產生之抑菌圈為小,此結果顯示黴菌較細菌對於離子液體更具抗性。Mix-ILs對於A. japonicus CY6-1和A. carneus M34相較於單一離子液體[EMIM][EtSO4]或THTDPD而言,Mix-ILs對黴菌的毒性增強,具有協同作用。Cholin-based ILs ([Ch][OH]和[Ch][OAc])與imidazolium-based ILs ([EMIM][EtSO4]和[BMIM][OAc]) 相較之下,Cholin-based ILs對於微生物形成的抑菌圈和MIC、MBC值都較高,此結果說明[Ch][OH]和[Ch][OAc]對於微生物的潛在毒性較低。 細胞毒性試驗中,[BMIM][OAc]離子液體對三株癌細胞[(胃癌細胞 (AGS)、肝癌細胞 (HepG2)、腸癌細胞 (Caco-2) ]和小鼠巨噬細胞(Raw 264.7)在培養48小時後其存活率最低,細胞存活率在高濃度1,000 g/ml分別為26.4%、13.7%、37.5%和14.1%,顯示[BMIM][OAc]具有細胞毒性。Mix-ILs對於AGS胃癌細胞、Caco-2腸癌細胞和Raw 264.7小鼠巨噬細胞作用24小時後相較於單一離子液體[EMIM][EtSO4]和THTDPD對於AGS胃癌細胞其細胞存活率來的低,顯示Mix-ILs具有協同作用(synergism),即相較於單子液體[EMIM][EtSO4]和THTDPD其毒性具有增強的現象。不同類型離子液體對於Raw264.7小鼠巨噬細胞產生發炎反應的影響。實驗結果顯示不同類型離子液體在不同濃度下(100-1000 g/ml)與Raw264.7小鼠巨噬細胞分別作用24小時和48小時後,與控制組相比並無顯著性差異,顯示本實驗所使用的離子液體並不會與Raw264.7小鼠巨噬細胞表面的受體結合而引發發炎反應,說明本實驗所使用不同類型離子液體並無促發炎反應的效果。 台灣衛生福利部食品藥物管理署規定基因毒性測試必須使用五株鼠傷寒沙門氏菌 (Salmonella typhimurium TA97、TA98、TA100、TA102和TA1535)進行測試。Salmonella typhimuriumTA102和TA1535在基因型試驗測試中與文獻不符,說明菌株已經呈現弱化的現象,最後選用另外三株鼠傷寒沙門氏菌(Salmonella typhimurium TA97、TA98和TA100)進行測試。致回復突變能力試驗結果顯示本實驗所使用不同類型的離子液體對於Salmonella typhimurium TA97、TA98和TA100皆不具有致突變性,說明本實驗使用的離子液體不具有基因毒性。 以上結果得知不同類型五種不同類型離子液體對於微生物和細胞的毒性皆不同,且Cholin-based ILs對於微生物和細胞的潛在毒性較低,具有廣泛應用於生物技術領域之潛力。

English Abstract

Ionic liquids (ILs) are solvent formed by an anion and a cation and usually liquid state at room temperature. Compared with conventional organic solvents, ILs possesses an array of advantageous properties, such as non-volatility, high solvent capacity and high thermal stability, etc. Currently, they are regarded as promising “green solvent”. In recent years, ionic liquids are widely used in various fields, such as organic synthesis, extraction, biotechnology, etc. However, the potential toxicity of ionic liquids is an important issue in the industry of biotechnology. Five different types of ionic liquids, including 1-ethyl-3- methylimidazolium ethylsulfate ([EMIM][EtSO4]), 1-butyl-3- methylimidazolium acetate ([BMIM][OAc]), trihexyl(tetradecyl) phosphonium decanoate (THTDPD), choline hydroxide ([Ch][OH]), cholin acetate ([Ch][OAc]) and Mix-ILs ([EMIM][EtSO4] and THTDPD, dissolved in deionized (DI) water, were used in this study to evaluate the mutagenicity and toxicity of ionic liquids on microorganisms and cells. The microbial toxicity results showed that the inhibition zones of the four strains of bacteria exist commonly in food (Escherichia coli BCRC 11634、Staphylococcus aureus BCRC 12154、Salmonella enterica BCRC 10747、Listeria monocytogenes BCRC 15330) treated with [BMIM][OAc] were 23.94 mm, 30.49 mm, 31.10 mm and 31.78 mm, respectively. However, the inhibition zones of the tested molds (Aspergillus japonicus CY6-1 and Aspergillus carneus M34) treated with [BMIM][OAc] were 14.34 mm and 18.75 mm, respectively, indicating that mold is more resistant to ionic liquids compared with bacteria. The toxicity of Mix-ILs on A. japonicus CY6-1 and A. carneus M34 showed the synergistic effect compared with the single ILs, [EMIM][EtSO4] and THTDPD. On the other hand, the toxicity of choline-based ionic liquids, including [CH[OAc] and [Ch][OH] showed the lower toxicity on the tested microorganism compared with imidazolium-based ionic liquids ([EMIM][EtSO4] and [BMIM][OAc]) used in the test. In the cytotoxicity tests, [BMIM][OAc] showed the highest toxicity on three cancer cells [(gastric adenocarcinoma cell line (AGS), human hepatoblastoma cell line (HepG2), human colon adenocarcinoma (Caco-2)] and mouse leukaemic monocyte macrophage cell line (Raw 264.7). After 48 hours incubation, the cells viability of AGS, HepG2, Caco-2 and Raw 264.7 at 1,000 g/ml concentration treated with [BMIM][OAc] were 26.4%, 13.7%, 37.5% and 14.1%, respectively. The toxicity of Mix-ILs on AGS, HepG2, and Raw 264.7 cells after 24 hours incubation also showed the synergistic effect compared with the toxicity of single ILs, [EMIM][EtSO4] and THTDPD. In addition, different types of ionic liquids in various concentrations (100 g/ml-1,000 g/ml) showed no significant difference in NO production compared with the control group after the incubation of 24 hours and 48 hours, respectively. According to the regulations of The Food and Drug Admistration, the five strains of Salmonella typhimurium (Salmonella typhimurium tester strains TA97、TA98、TA100、TA102 and TA1535) would be chosen for the testing of genotoxicity test. In our study, the genotype test of Salmonella typhimurium TA102 and TA1535 showed the negative result compared with literature, indicating that the attenuation of the tester strains Salmonella typhimurium TA102 and TA1535. Thus, the Ames test employing histidine mutants of the Salmonella typhimurium tester strains TA97, TA98 and TA100 were used to examine the mutagenicity of the different types of ionic liquids in the further test. No mutagenic activity was observed for either test strains at all used doses of ionic liquids, with or without S9 activation. The above results showed that the different types of ionic liquids have the significant toxicity on microorganisms and cells. In summary, the choline-based ionic liquids have the potential to be widely used in the industry of biotechnology due to its lower toxicity compared with imidazolium-based ionic liquids.

Topic Category 農業暨自然資源學院 > 食品暨應用生物科技學系所
生物農學 > 生物科學
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