Extraction And Separation Of Essential Oil From Peppermint And Study On Antibacterial Activity Of L-menthol PLGA

The process optimization of extraction of peppermint oil by steam distillation and supercritical CO₂ extraction was explored.In the orthogonal experiment,the optimum process of extraction of peppermint oil by steam distillation was 1:6,the distillation time was 6h,the immersion time was 1h,the yield of mint essential oil was about 2.20%.The extraction time of the mint essential oil yield is more significant.The results of orthogonal test show that the optimal extraction rate of peppermint oil is about 2.93%when the extraction pressure is 14MPa,the extraction temperature is 60℃and the extraction time is 1.5h.The extraction pressure of the mint essential oil yield is more significant.There are 25 kinds of known components were identified in the supernatotic CO₂ extraction method,and 16 known components were identified by the steam distillation method.Ingredients containing more than 1%of the composition of the mint essential oil in the two methods are:1,8-eucalyptol,limonene,L-menthone,menthylfuran,neo-menthol,L-menthol,carvone.In the experiment of separating L-menthol from peppermint oil,we chose silica gel column chromatography as the separation method,select the ratio of petroleum ether:ethyl acetate=80:1 as the eluent,and use 5%vanillin vanacate solution As a color developing solution for silica gel column chromatography.Among the thin layer chromatography（TLC）,the ratio of petroleum ether:ethyl acetate=8:1 was used as the developing agent.The results of thin layer chromatography showed that the L-menthol fraction was well separated.The results of freeze crystallization and GC-MS showed that the purity of L-menthol was 96.985%.We use PLGA on the L-menthol molecules wrapped modified to make it better play a bacteriostatic effect.Scanning electron microscopy（SEM）indicates that PLGA has been coated with L-menthol to modify the advantages of L-menthol nanomaterials.In the L-menthol PLGA against common pathogen inhibition experiments,we screened its resistance to E.coli and S.aureus have a strong inhibitory effect.The results of CFU showed that the antibacterial activity of L-menthol PLGA was higher than that of L-menthol.The MIC results showed that the MIC of L-menthol PLGA to E.coli was 3.7±0.02μg/mL and the MIC of S.aureus was 5.2±0.04μg/mL.The inhibitory effect of L-menthol PLGA on E.coli was even higher than that of the control group.LIVE/DEAD experiments showed that the lethal rate of L-menthol PLGA to bacterial cells was quite high,and the proportion of dead cells to total cells was increasing with the increase of the concentration of the dosing group.With the increase in the concentration of dosing group,the survival rate of bacterial cells is getting lower and lower.According to the results of scanning electron microscopy,we can conclude that L-menthol PLGA nanoparticles have certain damage to E.coli and S.aureus,which will cause the cell membrane to fold,rupture,and even with the inclusions.It can be seen that the antibacterial mechanism of L-menthol PLGA nanoparticles may be the destruction of bacterial cell membrane to achieve bacteriostatic effect.Ultra-thin section experiments show that L-menthol PLGA nanoparticles can enter the bacterial cells,destruction of bacterial cell walls and cell membrane,so as to achieve the purpose of killing bacteria.β-galactosidase experiments showed that with the L-menthol PLGA concentration increased,the greater the damage to E.coli membrane structure.With the increase of drug concentration,the destruction of E.coli membrane structure will be more and more;and after the nano-modified L-menthol on E.coli membrane structure damage degree will be significantly improved.