Antifungal Effects And Mechanisms Of Propolis On Aspergillus Fumigates In Vitro

Propolis is a resinous material that is collected by Apis mellifera bees from buds,leaves,bark,which mixed with secreted bees wax.It has special aromatic smell.Propolis has some pharmacological activities,such as anti-inammatory,antitumor,antioxidant and antifungal activity.Recently,with the deterioration of the ecological environment and the using of new type medical materials,fungal infection rate is increasing gradually.The most effective agents are chemical synthesis which is toxic for organisms.A large amount of literature suggests that the ethanolic extract of propolis has some antifungal activity.In this paper,we studied the antifungal mechanisms of propolis on Aspergillus fumigates so as to provide material foundation and theoretical data for exploiting the promising new drugs.The main contents and academic contributions of this thesis are summarized as follows:1.The study of antibacterial activity of propolis on Aspergillus fumigatus:Total phenolic acid and flavonoid content detected by Al(NO3)3-NaNO2-NaOH and Folin-Ciocalteu colorimetric method in ethanolic extracts of propolis samples were 220.8±16.98 mg/g and 223.5±2.98 mg/g,respectively.The experiment was divided into five groups:blank control group(sterile water treated A.fumigates),EEP group,solvent group(0.7%ethanol),negative control(without A.fumigates and drugs)and positive control group(itraconazole).Then,EEP diluted into different concentrations(10,20,30,40,50,100,150,200 ?g/mL),and used to deal with A.fumigatus,the results showed that there was a dose-dependent relationship between propolis and bacteriostatic ring size.Next,the minimum inhibitory concentrations(MIC)of propolis were determined by agar dilution method,A.fumigatus spores of weights and respiratory rate were significantly lower than blank control group when exposure to MIC50 of propolis with incubation for 3 d..Unltrastructure of spore was seriously damaged.Measurement of ATP content found that the ATP content of EEP group was significantly lower than blank control group.2.iTRAQ-based proteomics analysis of ethanolic extracts of propolis with A.fumigatus:A total of 285207 spectra were generated from the experiment used iTRAQ technology to analysis A.fumigatus protein relative quantitative in EEP group and blank control group.Mascot identified a total of 26350 spectra matched to known spectra,25617 spectra matched to unique peptides,9766 peptides,9573 unique peptides,and 2730 proteins.LC-ESI-MS/MS identified a total of 284 differentially expressed proteins when EEP group compared with blank control group;161 proteins were up-regulated and 123 were down-regulated.GO and KEGG functional enrichment analysis of these candidate proteins were performed to reveal the underlying mechanism,results suggested that these proteins mainly participated in liposome inorganic ion transport,energy metabolism and carbon metabolism,signal transduction,indicating that the compound in propolis interfered A.fumigatus normal physiological and biochemical processes in cell.3.Real time-PCR analysis the effect of propolis on A.fumigatus gene:KEGG analysis showed that phosphatidylinositol signaling pathway was different.Semi-quantitative interpretation analysied the gene expression on the pathway,the expression level of PKC,PLC1,PI4K mRNA were different.So,Real time-PCR analysied the three genes,then these proteins expression were compared.The results showed that the three genes expression were down-regulated,compared with the control group,PKC mRNA and protein expression levels were the same,but the expression of PLC1 and PI4K levels were not consistent.