Preparation of nanoemulsion from polysaccharides of Grifola frondosa and evaluation of its inhibition effect on H9 subtype avian influenza virus and common bacteria
To develop the nanoemulsions from polysaccharide of Grifola frondosa(GFP)and assess its physicochemical and morphological characteristics,as well as their potential to inhibit avian influenza viruses(AIV)and three common bacteria,we employed a low-energy emulsification method to prepare stable GFP nanoemulsions,which were then subjected to various treatments to evaluate their stability.Morphological characteristics were observed by transmission electron microscopy(TEM)and scanning electron microscopy(SEM),which particle size and polydispersity index(PDI)were determined with a laser particle size analyzer.The Zeta potential was measured using potentiometry.The results showed that the GFP nanoemulsion presented a clear and transparent appearance,and it remained stable without any signs of layering or emulsion breakdown after centrifugation at 8000r/min for 20 minutes or being left room temperature for 7 days.Electron microscope observation shows that the presence of uniformly distributed spherical particles,enveloped by a grid-like gel matrix.The particle size of GFP nanoemulsion ranges from 10nm to 100nm,with an average particle size of 24.12nm,a PDI of 0.096,and a Zeta potential of-18.6±4.3 mV,all of which are indicative of good stability.The GFP nanoemulsion and a hand wash-free gel disinfectant were tested for their inhibitory effects on the H9 subtype AIV NJ02 strain in MDCK cells.The virus inhibition rate were calculated for each treatment,and it was observed that the GFP nanoemulsion demonstrated a significantly higher virus inhibition rate compared to the hand wash-free gel at concentrations above 40µL/mL(P＜0.01).Notably,at a concentration of 80μL/mL,the GFP nanoemulsion was able to completely inhibit virus growth without any cytotoxic effects.Further,the GFP nanoemulsion was tested at various dilutions,along with GFP solution,hand wash-free gel,and 75％ethanol,against the AIV NJ02 strain.The copy number of the viral gene was quantified using fluorescence quantitative PCR(qPCR),and the inhibition rate of each drug on the virus was calculated.The results showed that the virus inhibition rate was significantly higher for the 10-fold diluted drugs compared to the 100-fold diluted ones,with the 10-fold diluted GFP nanoemulsion outperforming the hand wash-free gel,GFP stock solution and 75％ethanol(P＜0.01).The antibacterial effects of GFP nanoemulsion,GFP stock solution,and hand wash free gel on three common bacteria in poultry houses were tested using the drug-sensitive paper method.The results of paper diffusion method showed that GFP nano emulsion had no obvious inhibitory effect on Staphylococcus aureus,strong antibacterial effect on Bacillus subtilis and Escherichia coli.However,the overall antibacterial effect of GFP nano emulsion is slightly worse than that of hand wash-free gel.The above results indicate that the GFP nanoemulsion prepared in this study exhibited stable properties,a potent inhibitory effect on AIV cultured in vitro,and a certain inhibitory effect on common poultry bacteria.It was found to be safe and non-toxic,making it a promising candidate for use as a safe and efficient nanodisinfectant for both livestock and humans.This research provides a solid foundation for the development of novel antiviral herbal nanodisinfectants.
polysaccharides of Grifola frondosa(GFP)nanoemulsionsH9 subtype avian influenza virusantibacterial effect
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