| Plant-derived essential oils are a new type of antifungal agent.However,its antifungal mechanism still stays on the surface layer.In this paper,studies of fungal inhibition mechanism of essential oils were carried out.The results showed that trans-cinnamaldehyde could inhibit the growth of mycelium by destroying the cell membrane.At the same time,it also disrupted mitochondrial function and triggered mitochondria-mediated apoptosis.In addition,as a new high-sensitivity fingerprint analysis method,Raman spectroscopy has been applied to the analysis of the antifungal mechanism of essential oils.Researches on antifungal mechanism help to develop better natural antifungal agents.The specific results are as follows:First,trans-cinnamaldehyde exerts an effect of inhibiting mycelium growth through a membrane damage mechanism.Trans-cinnamaldehyde changed the permeability of cell membranes leading to the leakage of cellular contents such as nucleic acids,proteins,potassium ions and the like.Cell membrane could be stained red by propidium iodide which indicated that the integrity of cell membrane was reduced from 100.0±1%lowest to 12.1±3%.At the same time,scanning electron microscopy showed that the surface of the hyphae after the treatment of trans-cinnamaldehyde showed uneven wrinkles,suggesting the morphology of the cells had changed.Second,trans-cinnamaldehyde destroyed the normal function of the mitochondria and triggered mitochondria-mediated apoptosis.The tricarboxylic acid cycle was deregulated by changing the enzyme activity of the tricarboxylic acid cycle.The specific evidence was that the content of citric acid first increased and then decreased,the activity of succinate dehydrogenase decreased from 107.8±2.2 U to 52.7±5.3 U,and the activity of malate dehydrogenase increased from 4.8±0.5 U up to 15.2±0.9 U.The electrons in the respiratory chain in the mitochondria leaked a lot.The level of reactive oxygen species in cells increased sharply,and the redox level was out of regulation.Trans-cinnamaldehyde caused the mitochondrial membrane potential collapse by disrupting the mitochondrial membrane integrity,leading to cytochrome c leakage,and the leakage of cytochrome c triggered the activation of apoptotic protease,which was characterized by an increase in the activity of Caspase-3 which was from3.6±0.6 U up to 8.8±1.1 U,followed by cascade amplification,then cell died.Finally,Raman spectroscopy method with high sensitivity and fingerprint information was introduced into the research process of essential oil's antifungal mechanism.Raman spectroscopy and surface-enhanced Raman spectroscopy could play a good diagnostic role in fast detection of internal and external changes in the process of essential oil's inhibitory effect.Surface-enhanced Raman spectroscopy was used to detect the leakage of cellular proteins,nucleic acids and lipids.The characteristic intensity of protein which was at 1244 cm-1 in the extracellular supernatant increased,and the characteristic intensity of nucleic acid-734 cm-1?1330 cm-1?and 1338 cm-1-increased.In addition,the intensity of the characteristic peak representing lipid at 1466 cm-1 also increased.When confocal micro-Raman spectroscopy was used to detect the cytochrome c in mitochondria,it was found that the characteristic intensity index I750/I2944 of cytochrome c decreased,suggesting a decrease in the amount of cytochrome c in mitochondria.In addition,the Raman intensity could establish a strong correlation with the classical analysis method,such as OD260,leakage of protein,leakage of cytochrome c,and the correlation coefficient was above 0.9.In conclusion,the above results indicated that trans-cinnamaldehyde exerts an excellent antifungal effect by inhibiting the mycelial growth of Penicillium italicum via disrupting the cell membrane and by activating mitochondria-mediated apoptosis.Raman spectroscopy and surface-enhanced Raman spectroscopy could be well applied to substance changes characterization of the antifungal process of essential oils. |
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