| Objective: Nitazoxanide nanoemulsion (NTZ-NE) was prepared. The quality, stability,safety, and in vitro and clinical effectiveness of the drug were evaluated, which provided anexperimental basis for NTZ-NE in the veterinary clinical application.Methods:(1) Oil, surfactant, cosurfactant and cosolvent were decided according to thedrug loading, stability and safety of the nanoemulsion. Then the optimum formula wasselected on the basis of the pseudo-ternary phase diagrams. After preparation, the drug’sstructure type was identified by means of staining and dilution. Its shape was observed byTEM, and the particle size distribution, mean diameter, PDI and Zeta potential were measuredby the Laser Particle Size Analyzer.The stability of the nanoemulsion was tested as well.(2)The drug content standard curve was established by means of ultraviolet-visiblespectrophotometry, with which the drug content was calculated.(3) The safety property of theNTZ-NE was evaluated by the acute toxicity test.(4) To determine the minimal inhibitoryconcentration (MIC) and the minimal bactericidal concentration (MBC) of the NTZ-NE, invitro bacteriostatic test was carried out with common pathogens in clinicalpractice(Salmonella pullorum, Pathogenic Escherichia coli O78, Staphylococcus aureus,Streptococcus agalactiae, and Acinetobacter).(5) The pathologic model of Salmonellapullorum was made according to the result of the in vitro bacteriostatic test, by which thedrug’s clinical efficacy of Salmonella pullorum was evaluated.Results:(1) The proper components were selected with the oil of cinnamaldehyde, thesurfactant of EL-40, the cosurfactant of1,2-propanediol and the cosolvent of DMSO. Massratio of surfactant to cosurfactant was6:1. The optimum formula was cinnamaldehyde6.0%,EL-4030.0%,1,2-propanediol5.0%, DMSO0.3%, NTZ0.45%and water58.25%. Thenanoemulsion (O/W) was yellow and transparent with good fluidity, which can be infinitelydiluted by distilled water. Nanoemulsion droplets were spherical with a size distribution of5to30nm, a mean diameter of13.8nm, a PDI of0.155and Zeta potential of-14.7mV. Itremained stable after high-speed centrifugation test (4000r/min) and sealed storage attemperature-4℃,20℃,37℃and60℃.(2) The NTZ-NE’s maximum absorption wavelengthwas355nm, with good linear relationship among concentration of0-100μg/mL. The standardcurve equation was y=0.0503x＋0.0345, and R2=0.9998.(3) The NTZ-NE’s MIC for Escherichia coli O78,Salmonella pullorum,Acinetobacter, Streptococcus agalactiae andStaphylococcus aureus were2μg/mL,1μg/mL,8μg/mL,16μg/mL and8μg/mL,respectively, and MBC were2μg/mL,1μg/mL,8μg/mL,32μg/mL,16μg/mL,respectively.(4) The LD50of NTZ-NE for chicken was5391mg/kg, and95%confidenceinterval was5387~5395mg/kg, which demonstrated that the new drug was nontoxic.(5)According to the results of the drug’s effectiveness test, the protection rates of NTZ-NE highdose group, middle dose group, low dose group, nitazoxanide crude drug group, infectioncontrol group and blank control group were87%,83%,70%,63%,47%and97%,respectively, and relative weight gaining rates were90%,86%,79%,72%,56%and100%,respectively. The data showed that middle dose (0.2%) of the nanoemulsion could be appliedas clinical dose.Conclusion: The prepared NTZ-NE meets the clinical requirements with good stabilityand safety. It has significant in vitro antibacterial effects to Escherichia coli O78,Salmonellapullorum,Acinetobacter, Streptococcus agalactiae and Staphylococcus aureus.Withrecommended dose, the mortality of chickens with pullorum disease can reduce by36%, andbody weight can increase by30%. |
