Antimicrobial Activities And Mechanisms Of Punicalagin Against Staphylococcus Aureus

Staphylococcus aureus is a common pathogen.The growth of S.aureus in foods presents a potential public health hazard.Synthetic preservatives have been used for years to extend shelf life of food products,but the effects of some preservatives on human health are frequently questioned.With increasing demand for natural food products by consumers,the search for natural antimicrobials,especially plant-derived antimicrobial compounds,has gained increasing attentions in recent years.It is of great significance to explore safe and effective antimicrobials for the prevention and control of S.aureus or other microbial contamination.Pomegranate is a medicinal and edible plant resource.It is nutritious and has a variety of health care effects.Pomegranate peel as the by-product of pomegranate processing industry shows great value in food,medicine and other fields for its rich polyphenols.Punicalagin,one of the main active compounds in pomegranate peel,has been reported to possess many properties,including antioxidant,anti-inflammatory,anticancer and antiviral activities.It also exhibited inhibitory effects against various food-borne pathogens.The main purpose of this study was to isolate and purify punicalagin from pomegranate peel,and to reveal its inhibitory effects against S.aureus.The mechanisms of action were explained by four aspects,including the ultrastructure of bacteria,expression of virulence factors,biofilm formation and proteome.The findings of this study will provide theoretical basis for the development and utilization of punicalagin.The results are as follows:(1)Punicalagin was purified from pomegranate peel extract by Amberlite XAD-16 macroporous resin.Gradient concentrations of methanol were used as elution solutions.The content of punicalagin increased first and then decreased with the increasing concentration of methanol.The highest content was 79.6%.Punicalagin exhibited good inhibitory effects against S.aureus.The diameter of the inhibition zone increased with the concentration of the extract.(2)The minimum inhibitory concentration of punicalagin on S.aureus was 0.25 mg/mL.After the treatment of lethal concentrations of punicalagin,the cell membrane potential of S.aureus was decreased.It means that punicalagin caused membrane hyperpolarization.Besides,the release of potassium into the extracellular was promoted by punicalagin.Results from scanning electron microscopy showed that punicalagin bound to the surface of the bacteria to form a rough polymer layer.The higher the concentration of punicalagin was,the thicker the polymer layer became.Thus the cells were damaged and led to death.A larger size of bacteria was seen after the treatment of punicalagin under the transmission electron microscope.The polymer around the cells was visible as well.(3)Punicalagin reduced the expression of ?-hemolysin,plasma coagulase and total enterotoxins at subinhibitory concentrations.The transcription of sea(enterotoxin gene),hla(hemolysin gene)and agrA(regulatory gene)were inhibited significantly after the treatment of punicalagin at the concentration of 1/8×MIC.(4)The formation of biofilm was inhibited by punicalagin at the concentrations that did not inhibit the growth of S.aureus.The inhibitory effect on the biofilm was enhanced and the number of bacteria in the biofilm decreased with the increase of the concentration of punicalagin.Scanning electron microscopy and laser confocal microscopy intuitively showed that the formation of S.aureus biofilm was inhibited.The changed surface morphology of the bacteria treated with punicalagin was observed under the scanning electron microscope.After treated with punicalagin at the concentrations of 1/32 and 1/16×MIC,the expression of fib,ebps was inhibited while the expression of icaA and cidA increased.There was no significant effect on the expression of agrA,fnbA,clfA,clfB,sarA,eno and sigB.(5)A total of 1026 proteins were detected by tandem mass tag combined with high performance liquid chromatography and mass spectrometry.There were 423 proteins showing significant changes in expression between the punicalagin-treated and the control group(P <0.05).Among those proteins,251 proteins were down-regulated while 172 proteins were up-regulated.Most of the proteins were cytoplasmic protein(77%),followed by cytoplasmic membrane protein(7%).Cell wall and extracellular proteins accounted for 2%,respectively.Many metabolic pathways were affected by punicalagin,especially purine metabolism,pyrimidine metabolism and tricarboxylic acid circulation pathway.The expression of key enzymes was inhibited,leading to the suppression of the pathways,thereby inhibiting the growth and proliferation of bacteria.