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  • 學位論文

以流式細胞儀及系統生物學分析山胡椒超臨界流體萃取物之抑菌機制

Use Flow Cytometry and Systemic Biology Approach to Analyze the Bacterial Inactivation Mechanism of Litsea cubea

指導教授 : 徐源泰
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摘要


山胡椒Litsea cubeba,為台灣原住民常用之食用香料,添加於飲食中增加風味並且延長保存時間,其抑菌及抗氧化效果具有研究潛力。本實驗使用蒸餾萃取法與超臨界流體萃取法萃取山胡椒中之物質。結果顯示,超臨界流體萃取物 DPPH 自由基清除率 31.51%;總酚類含量達 22.17 mg of GAE/g ,高於其他市售精油。精油與超臨界流體萃取物之最低抑菌濃度皆為 312.5 ppm,低於食品防腐劑。利用流式細胞儀比較二種山胡椒萃取物與四種已知機制之抗生素Penicillin、Tetracycline、Rafipincin、Trimethoprim 在五種濃度下抑制 Bacillus subtilis ATCC 6633、Escherichia coli ATCC 8739 之螢光變化,三種螢光染劑分別為 Propidium iodide (PI)、Bis (1,3-dibarbituric acid)-trimethine oxanol (BOX)、Carboxyfluorescein diacetate (cFDA) 。顯示二種山胡椒萃取物之抑菌方法為破壞細胞外壁,改變通透性與電位來抑制細菌生長,但較無法明顯分辨從內部抑制之原因。進一步利用氣相層析質譜儀分析二萃取物組成,山胡椒精油與超臨界流體萃取物中可分析出30與44種物質;二者共同組成比例佔精油之92.17% ,僅佔超臨界流體萃取物 73.94%,顯示後者能夠取得更多種化合物,這些化合物大部分屬於揮發性較低之長碳鏈有機物;精油組成分與前人研究類似。以 STITCH 3.1 模擬分析;發現山胡椒精油之成分與 B. subtilis 及 E. coli 內部多種蛋白相關聯,影響細胞之氧氣運輸感知與催化、DNA損傷偵測與移除、胺基酸與乳酸之代謝等;山胡椒超臨界流體萃取物則與脂肪酸之生合成與代謝相關,此結果與流式細胞儀觀察相符合。結合流式細胞儀比對與 STITCH 等創新方法可以快速掌握複雜組成物之植物萃取物功用,嘗試解決植物組成複雜且不易比較之問題;未來應用此方法可減少探索成本,增加尋找有效物質之機會。

並列摘要


Litsea cubeba is a plant frequently used as spice by Taiwanese aborigines as flavoring agent or food preservatives. Recently, its noticeable antibiotic and antioxidant properties have attracted much attention for scientific research. In this study, we employed steam distillation and supercritical fluid extraction techniques to extract compounds from L. cubeba. The supercritical extract of L. cubeba has a DPPH clearance rate of 31.51% and a total phenol content up to 22.17mg GAE/g, a value higher than other essential oil on the market. The minimum concentration of both essential oil and supercritical extract of L. cubeba capable of inhibiting cell growth is 312.5ppm. Flow cytometer is used to measure the effectiveness of L. cubeba extracts on the inhibition of Bacillus subtilis ATCC 6633 and Escherichia coli ATCC8739, the results were then compared in together with Penicillin, Tetracycline, Rafapincin, and Trimethoprim, at 5 different concentrations. Associated fluorescent signal changes were detected by fluorescence-based cell counter. The three fluorescent molecules used in this study were Propidium iodide (PI), Bis (1,3-dibarbituric acid)-trimethine oxanol (BOX), and Carboxyfluorescein diacetate (cFDA). Experiments have confirmed that both extracts of L. cubeba may disrupt the integrity of cell wall and further on change cell permeability and electrical potential, thereby inhibiting cell growth. Yet, it is unclear how such growth inhibition occurs at the molecular level. Further analysis of L. cubeba extracts by Gas-Chromatography-Mass Spectrophotometer, followed by simulation analysis using STITCH 3.1, has identified 30 and 44 compounds in L. cubeba essential oil and supercritical fluid, respectively. The common components of both extracts account for 92.17% of essential oil but only 73.94% of supercritical fluid extract, clearly demonstrating that the later method extracts far more compounds than the former. Based on possible interactions between of the chemical components of L. cubeba and endogenous proteins in B. subtilis and E. coli, it can be postulated that L. cubeba affects cell’s oxygen transport and sensing, enzyme catalysis and electron transfer, DNA damage detection and removal, and metabolism of amino acids and lactic acid. Supercritical extract of L. cubeba was found closely associated with both the synthesis and metabolism of fatty acids and lipids. Further application of flow cytometer, it is now workable for rapid identification of useful chemical compounds in plants.

參考文獻


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