Study On The Mechanism Of α-terpineol Against Escherichia Coli

At room temperature, a-terpineol (relative density of0.9337) is a colorless, viscous liquid and low melting point transparent crystal, which can be applied to the flavors preparation, deodorant, in medicine, plastic, soap, printing ink, solvent of glass ware. In plant families, a-terpineol, a monoterpene, is widespread contained in the Cinnamomum camphora, Melaleuca alternefolia, E. grandis×E. urophylla, Cinnamomum longepaniculatum and so on, which the content of a-terpineol were11.20%、3.93%、11.76%and15.43%respectively. Minimal inhibitory concentration (MIC) were determined by the same ratio dilution method with broth medium and minimal bactericidal concentration (MBC) by SDA. Permeability change experiment of cell wall and membrane, nucleic acid synthesis inhibitory experiment, SDS-polyacrylamide gel electrophoresis and endotoxin inhibitory experiment were study in the study. The main results are as follows1. The study on the antibacterial activities.to determine the antibacterial activities of a-terpineol (98%),1,8-cineole (99.5%), terpinene-4-alcohol (99%), y-terpinene (99%) which is the main chemical constituents of Cinnamomum longepaniculatum leaf essential oils. Minimal nhibitory concentration (MIC) were determined by the same ratio dilution method with broth medium and minimal bactericidal concentration (MBC) by SDA. Then, draw the time-kill curve by using flat colonies countting. In result, the values of MIC and MBC which a-terpineol,1,8-cineole, terpinene-4-alcohol, y-terpinene against on Escherichia coli (CMCC(B)44102) were 0.78μL/mL and0.78μL/mL,6.25μL/mL and6.25μL/mL,1.56μL/mL and1.56μL/mL,>50μL/mL and>50μL/mL. Time-kill curves of show the the strongest antibacterial activity and time-concentration dependent of a-terpineol against the Escherichia coli.2. The study on the cell structure and function of Escherichia coli.Measuring the effect on cell wall and membrane permeability of a-terpineol on Escherichia coli, through determining the activity of ALP and β-galactosidase; Escherichia coli were treated with a-terpineol (MIC) and observed under a transmission electron microscope. In result, After120min, ALP activity increased gradually and maintained at about14; After180min, β-galactosidase activity increased sharply and maintained at about30; Escherichia coli exposed to MIC levels of the a-terpineol show decreased cell size, irregular cell shape, cell wall and cell membrane were ruptured, nucleus cytoplasm was reduced and nuclear area gathered aside. Results suggest that antibacterial mechanism of a-terpineol against Escherichia coli was realized through influence the membrane function and destroying the Escherichia coli structure.3. The study on the cellular proteins and DNA.nucleic acid synthesis inhibitory experiment and SDS-polyacrylamide gel electrophoresis were designed to study the effect of cellular soluble proteins and DNA. In results, at the level of0.5MIC, DNA fluorescence intensity become strengthened and soluble protein bands become dark than the the control group; at the level of1MIC, DNA fluorescence intensity become weakened and soluble protein bands become light than the the control group after16h; at the level of2MIC, DNA fluorescence intensity become weakened and soluble protein bands become light obviously than the the control group. Results suggest that a-terpineol have the fuction of inhibited the synthesis of nucleic acid and proteins.4. The study on the endotoxin (ET) of Escherichia coliThe degradation effects of a-terpineol on endotoxin were detected by quantitative chromogenic modification of the limulus amebocyte lysate test in vitro; Study the effection of a-terpineol on Escherichia coli endotoxin through the determination of rabbit anal temperature method. In results, When the concentration of α-terpineol between the0.1-0.39μ L/mL, Escherichia coli endotoxin decomposition rate have no changes; Between the0.78-3.9μ L/mL, Escherichia coli endotoxin decomposition rate rise obviously. In the negative control and experimental group, the rabbit temperature were no more than0.6℃and the temperature sum of three rabbits were less than1.4℃; In the positive control group, the three rabbits temperature were all more than0.6℃and the temperature sum of three rabbits were more than1.4℃. Results suggest that α-terpineol have the fuction of anti-endotoxin activity.