Chemical Composition, Antibacterial Activity And Related Mechanism Of The Essential Oil From The Leaves Of Juniperus Rigida Sieb. Et Zucc.

Juniperus rigida Sieb. et Zucc. belongs to Cupressaceae and it is an evergreen coniferous shrub occurring in North, Northeast of China, Korea and Japan, which is a small tree with a strong central axis. The branches and leaves of J. rigida have been used in Tibetan Medicine and Traditional Mongolian Medicine for the treatment of brucellosis, skin disease and rheumatic arthritis. However, the chemical composition, antibacterial activity and related mechanism of the essential oil from the leaves of J. rigida have not been reported. The aim of this study was to evaluate antibacterial potential of essential oils from J. rigida leaves against Klebsiella pneumonia and to examine its possible related mechanisms. The study was undertaken in order to scientifically validate the traditional use of J. rigida. The main obtained results are as follows:1. The essential oil was extracted from the leaves of J. rigida by supercritical CO2 fluid extraction technology, and the extraction rate of the essential oil was 0.8%. Chemical composition of the essential oil was analyzed by gas chromatography-mass spectrometry(GC-MS). 62 components were identified from the essential oil. Caryophyllene(13.11%), α-Caryophyllene(11.72%), Caryophyllene oxide(10.30%), Germacrene D(6.57%) and Linalool(6.31%) were found to be the major components.2. The antibacterial activity was evaluated against 10 bacteria by the paper disc diffusion method. The results showed that the essential oil had certain antibacterial activity on all of the tested bacteria including both Gram-positive and Gram-negative bacteria. The DIZ of the essential oil of J. rigida varied from 8.05-16.00 mm. The minimum inhibitory concentration(MIC) and minimum bactericide concentration(MBC) values of the essential oil were estimated by agar dilution method. The results showed that the essential oil had certain antibacterial activity on all of the tested bacteria, The MIC and MBC values for tested bacterial strains were in the range of 3.125-12.50 mg/mL. All these bacteria, the essential oil performed both MIC and MBC of 3.125 mg/mL against K. pneumoniae, which indicated that the essential oil was an effective bacterial inhibitor and bactericide against K. pneumoniae.3. The antibacterial mechanism was evaluated by growth curve, the integrity of cell membrane, the SDS-PAGE of protein patterns and scanning electron microscope(SEM). The growth curve assay was used to investigate the bactericidal effects of the essential oil. The results indicated that different incubation time and concentration of the essential oil of J. rigida had great impacts on antibacterial activities. The increase in proteins, 260 nm absorbing materials of bacterial cells suspension indicated that the cytoplasmic membranes were broken by the essential oil. The SDS-PAGE of bacterial proteins demonstrated that the essential oil could damage bacterial cells through the destruction of cellular proteins. Scanning electron microscopy(SEM) showed that the essential oil damaged the morphology of cell wall and membrane. These results indicated that the essential oil of J. rigida has great potential antibacterial activities against K. pneumonia. Further phytochemical and pharmacological studies are required for proper scientific validation of the folk use of this plant species.