Mechanisms Of Vapor-phase Antibacterial Action Of Essential Oil From Cinnamomum Camphora Var.Inaloofera Fujita

The aim of this project is the evaluation of vapor-phase antibacterial activity of essential oil from Cinnamomum camphora var. linaloofera Fujita (EOL) and its mechanism of bactericidal action against Escherichia coli ATCC25922. The mode of action of EOL was determined by SEM, TEM, FT-IR spectroscopy, fluorescence spectra, crystal violet assay, electric conductivity and byproducts of lipid peroxidation. The composition of EOL and the atmosphere generated by EOL were determined by GC/MS, and SPME-GC/MS respectively.The major antibacterial chemicals in EOL atmosphere were found and the active centres were analysed by quantum chemical technology.The vapor-phase antibacterial activity of EOL against6respiratory pathogens (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus. Streptococcus pneumoniae and Hemolytic streptococcus) of12essential oils were compared. The antibacterial activities were determined by solid diffusion and vapour diffusion methods. The antibacterial tests indicate that EOL showed the most significant comprehensive antibacterial ability as tea tree oil among all test oil, while its vapor-phase activities were more effective. E. coli, S. aureus, S. pneumoniae and H. streptococcus were sensitive to vapor-phase EOL, the minimum bactericidal concentration(MBC) was250μl/L. In contrast. P. aeruginosa was the most insensitive bacaterial.After treated by vapor-phase EOL, the microstructure of E. coli appeared significant variations. The SEM micrographs of E. coli showed distorted and shrink cell with rough surface. TEM photomicrographs show the distorted cell shape while the cell wall appeared normal as in control. The plasma lemma was distorted and notable structural disorganisation was seen within the cytoplasm. Furthermore, pyknotic nuclei cytoplasm electronic density increased. Some bacteria cell walls dissolved and broken completely, cytoplasm material leakage till even the "hole" appeared, the cell has severe deformation shrinks which and uneven shape was observed.The alternation of membrane permeability was detected by crystal violet assay and electric conductivity, the result show that vapor-phase EOL had a high-performance bactericidal activity, which could cidal E. coli within30min at the MIC level (200μl/L). The EOL atomosphere may destroy the membrane structure and altered membrane permeability, csused the uptake of dry crystal violet increase and subsequent leakage of intracellular constituents. This changes kill E. coli at a certain extent.The bactericidal effect of EOL caused changes in the spectral features of bacterial ester functional groups of lipids, structural proteins and nucleic acids. Fluorescence spectrum and FTIR spectrum showed that EOL altered the secondary and tertiary structure of membrane. Fluorescence intensity of E. coli increased after the vapor-phase treatment, which indicates the alternation of membrane tertiary structure. Fluorescence intensity was positively correlated in certain concentration. The appropriate EOL process could cause the spreading of membrane protein molecules. However membrane proteins would be curled and folded beyond certain concentration. The secondary structure change as a-helix decreased and β-fold increased, which appeared vapor-phase EOL made peptide chains of a-helix stretch into linear structure due to alternation of the molecular hydrogen bond orientation. However, the vapor-phase EOL could not change a-helix structure to (3-fold infinitude. From lipid peroxidation assay, vapor-phase EOL could not lead to lipid peroxidation of E. coli. indicate that lipid peroxidation in membrane was not the cidal effect factor.The major constituents of the EOL including linalool(35.17%), camphor(15.36%),1,8-eucalyptol(13.73%), while atmosphere was linalool(69.94%), camphor(10.90%). safrole(8.24%), nerlidol (10.92%). Only linalool show antibacterial effect within the concentration3000μl/L, indicated that the EOL antibacterial ability mainly came from linalool.Quantum chemical analysis of linalool, camphor and nerlidol found that linalool held the largest rail energy, the strongest antibacterial activity, which show the most significant antimicrobial activity. Furthermore, we found that electron-cloud densities is strongest in O atom of linalool molecule. Fukui indices of linalool indicate the significant electrophilic and radical attack activity. Therefore, the hydroxyl in linalool is the antibacterial activity center in the electronic transfer process which play the role of providing electronic.