The Activities And Action Mechanisms Of Clove And Rosemary Essential Oils Against Propionibacterium Acnes And The Preparation Of Liniment

In order to solve the problems of high incidence of acne,the short half life and severe side effects of anti-acne drugs in the clinic,in this study,clove and rosemary essential oils were used as major materials for the preparation of plant essential oil Liniment.It possessed high efficacy,no cytotoxicity and no stimulative side effect,and it could provide scientific evidence for the application of plant essential oils to prevent acne.The compositions of clove and rosemary essential oils were analysed by GC-MS.Flow cytometry,atomic force microscopy and protein electrophoretic technique were used to investigate the changes of Propionibacterium acnes before and after treatment with essential oils,the action mechanisms of clove and rosemary essential oils against Propionibacterium acnes were discussed.The prescription screening,preparation process and quality control specification of Liniment were studied,and its antimicrobial and anti-inflammation activities and stimulative test on skin were also investigated.The results were as follows:1.The GC-MS results showed that the main compositions of clove essential oil were euqenol andβ-caryophyllene,which relative contents were 68.52%and 19.00%,the main compositions of rosemary essential oil were 1,8-cineole,α-pinene,camphor and camphene, and which relative contents were 27.23%,19.43%,14.26%and 11.52%,respectively.2.The agar diffusion method,micro-broth dilution method and time-kill curves were used to investigated the antimicrobial activities of clove and rosemary essential oils.The results indicated that clove and rosemary essential oils exhibited broad-spectrum antimicrobial activity. The clove essential oil possessed the best antimicrobial activity against Aspergillus nigar and its MIC value was 0.062%v/v,however exhibited the poorest antibacterial activity against Pseudomonas aerμginosa,and its MIC value was 0.500%v/v.The rosemary essential oil exhibited better antimicrobial activity against other strains except Pseudomonas aerμginosa and Aspergillus nigar.Antimicrobial activities of mixture of the two essential oils in different ratio indicated their additive,synergistic or antagonistic effects against individual microorganism tests.The time-kill curves showed clearly bactericidal and fungicidal processes of different concentration's essential oil at different time.The two essential oils also exhibited better antibacterial activity against Propionibacterium acnes,and inhibition diameters were 24.0±0.5mm and 18.0±1.0mm,MIC values were 0.31mg/mL(0.031%v/v) and 0.56mg/mL(0.062%v/v).The time-kill curves of clove and rosemary essential oils against Propionibacterium acnes showed the bactericidal effect in concentration-dependent and time-dependent manner. 3.Flow cytometry,atomic force microscopy and SDS-PAGE techaniques were firstly used to investigate the action mechanisms of clove and rosemary essential oils against Propionibacterium acnes from the fluorescence intensity of cell membrane,bacterial morphology and proteins changes.The results of flow cytometry showed that fluorescence intensity of bacteria after stained with PI became stronger with increasing concentrations,cell membrane of bacteria were damaged gradually.Atomic force microscopy detected morphological changes of Propionibacterium acnes before and after treatment with essential oils at nanometer level.With the concentration and time increasing,the damage degree of bacterial body became stronger,cell wall and membrane were damaged,the bacterial body collapsed and cytoplasm overflew,the dimension of bacteria reduced.The results of SDS-PAGE showed the molecular mechanisms of essential oils against Propionibacterium acnes, clove essential oil could be play a role through inhibiting the two special proteins(18kDa and 33kDa),however,rosemary essential oil may be inhibit 56.8kDa and 67kDa proteins to prevent acne developing.4.The clove and rosemary essential oils were firstly used as main materials for anti-acne Liniment,the preparation process and quality control specification of Liniment were investigated,and its pharmacodynamics was primarily evaluated.The parameters were optimized by orthogonal experiment and synthetical consider from external quality and forming film time,they were as following,basic prescription:PVA-124 5g,tween-80 4ml, ethanol absolute 10ml,glycerol 4ml,then clove essential oil 1 mL and rosemary essential oil 0.5 mL were added and prepared to the Liniment according to the method of pharmaceutics. The quality specification of the Liniment were established by qualitative and quantitative analysis,and the tests of precision,reproducibility and stability were analysed.The pharmacodynamics of the Liniment mainly included antimicrobial,anti-inflammation activity and stimulus test on skin.The results indicated that the Liniment possessed better antibacterial activity against Propionibacterium acnes,and could effectively inhibite ear dwelling of mice, moreover,the Liniment and matrix have no stimulus on normal and injured skin.