| Litsea cubeba(Lour.)Pers.is one of the characteristic woody plant resources in China,which essential oil from its peel has a good commercial value.In order to develop and apply the bioactivity of Litsea cubeba essential oil(LCEO)better and to expand its market value,In this study,the anti-inflammatory activity of LCEO was investigated at the cellular,genetic and protein levels using RAW264.7 cells as the anti-inflammatory model,and the main antiinflammatory component was identified.Secondly,the antibacterial activity of citral,the main component of LCEO,was investigated by using Aspergillus niger(A.niger)as the test bacterium,and the antibacterial mechanism was investigated by measuring the relevant indicators on the intracellular,cell wall and cell membrane.Finally,ultrasonic emulsification was used to prepare the nano-emulsion of LCEO,which physicochemical properties were analyzed.The antifungal effect of LECO’s nano-emulsion was dynamically monitored by hyperspectral imaging technology.The main research results are as follows,(1)The effect of RAW264.7 cells on the secretion of NO and TNF-α was determined,which confirmed the good anti-inflammatory activity of organic LCEO.The GC-MS results showed that the main component of LCEO was citral accounting for 62.61%.After the intervention with LCEO and commeasurable citral,their effects on the expression of i NOS,COX-2 and TNF-α at the m RNA level in the inflammatory response were evaluated using Real Time Quantitative PCR(RT-q PCR),where the expression in the citral group was significantly lower than that in the LCEO group.The effect on the expression of i NOS and COX-2 proteins was examined using Western Blot,where the expression in the citral group was also significantly lower than that in the LCEO group,indicating that citral as the main anti-inflammatory component in LCEO.(2)The anti-A.niger activity mechanism of citral was studied,and the minimum inhibitory concentration of citral was determined to be 50 μg/m L.The effects of citral on the intracellular and apoptotic indicators of A.niger were determined,including reactive oxygen species(ROS),nitrate reductase(NR),nitric oxide synthase(NOS)and cysteine protease-9(Caspase-9).Compared to untreated A.niger,ROS content significantly increased after 50 μg/m L of citral intervention,and the NR,NOS and Caspase-9 enzyme activities also improved significantly.The results of Annexin V-FITC/PI showed that treatment with 50 μg/m L of citral led to the massive apoptosis of A.niger,the increase of extracellular protein,nucleic acid and alkaline phosphatase(AKP)content in the medium indicated that citral disrupted the cell membrane and cell wall structure,resulting in cell apoptosis due to the leakage of contents.(3)Ultrasonic emulsification was used to prepare the LCEO nanoemulsion,which physicochemical properties and antifungal effects were evaluated.Results showed that the mean size of LCEO nanoemulsion was 119.8 nm,the polydispersity factor was 0.216,and the encapsulation efficiency was 75.9%.As observed in transmission electron microscopy,the morphology of nanoemulsions was close to spherical shape with uniform dispersion.Compared to LCEO,the water solubility and slow-release properties were significantly improved and it has a good storage stability at room temperature and under low temperature.Considering the good anti-A.niger activity of LCEO,a non-destructive fast method was developed using hyperspectral imaging to dynamically monitor the growth of A.niger,LCEO nanoemulsion was proved to have a good slow-release effect,which can prolong the anti-A.niger efficacy of LCEO. |
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