Study On Antibacterial Activity And Mechanism Of Phloretin

The antimicrobial activity of phloretin has been studied and variable results have been reported on different microbial species. The reports showed that phloretin inhibited the growth of a number of Gram-positive and Gram-negative bacteria including Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium et al. and plant pathogenic fungi including Phytophthora capsici, Alternaria panax, M. grisea et al. However, its antibacterial mechanism is not yet fully understood. In this paper, Lister monocytogenes CMCC 54004 was selected as the model organism to elaborate the antibacterial mode of action of phloretin. The potential antibacterial mechanisms were determined by permeability and integrity of cell membrane, ATP content, key metabolic enzymes activity, intracellular protein content as well as nucleic acid morphological character. The results could provide a theoretical basis for the developing of phloretin into high-performance and low-toxicity antibacterial drugs or preservative. The main results were as follows:(1) Phloretin exhibited remarkable antibacterial activity against Listeria monocytogenes and had a better inhibitory effect with increased concentrations. Phloretin at final concentrations from 100 to 200 ?g/mL could completely inhibit the growth of L. monocytogenes up to 48h and showed significantly bactericidal activity.(2) Scanning electron microscopy, transmission electron microscopy, determination of K+concentration and leakage of intracellular macromolecules (nuclear acid and protein) were employed to study the effect of phloretin on the integrity of cell membrane. The experiments showed that phloretin increased membrane permeability, but no leakage of intracellular macromolecules implied that phloretin induced a minor damage to the cell membrane and not damaged the integrity of the cell membrane.(3) Phloretin influenced the energetic metabolism of L.monocytogenes by reducing cellular ATP synthesis and decreasing the total enzymatic activity of key metabolic enzymes, but the decrease of the specific activity of key metabolic enzymes was not obvious. Compared to the control, ATP content decreased by 21.92%,28.45%,40.69%, and 74.80% after addition of phloretin at different concentrations from 25 to 200 ?g/mL, respectively. Statistical analysis revealed that no significant difference in enzyme activity per microgram protein (P>0.05) was found between treated and untreated groups for the specific activities of hexokinase (HK) and isocitrate dehydrogenase (IDH).(4) Phloretin treatment induced intracellular DNA aggregation, thereby affecting cellular protein concentration. The remarkable electron-light region in the center of the cells treated with phloretin suggested that intracellular structure was destroyed. The agarose gel electrophoresis results indicated that phloretin had no ability to cause DNA cleavage. So it is inferred that the electron-light region was caused by DNA aggregation. Intracellular protein concentration was measured according to Bradford and SDS-PAGE and the results showed that phloretin could significantly reduce the synthesis of protein, with the protein concentrations decreasing by 83.75% after 200 ?g/mL phloretin treatment.From these results, phloretin had strong antibacterial activity on Listeria monocytogenes and its antibacterial mechanism was decreasing the intracellular protein content and energy level resulting from DNA aggregation. Subsequently, natural death occurred in virtue of cell metabolic disturbance.