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

• Main Authors: Chun-Hao Yang, 楊淳皓
• Other Authors: Yuan-Tay Shyu
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/pgdyhs

碩士 === 國立臺灣大學 === 園藝暨景觀學系 === 101 === 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.