槲皮素-替米考星混合物纳米粒的制备及对金黄色葡萄球菌小菌落变异株抗菌活性研究

doi:10.16431/j.cnki.1671-7236.2022.04.029

摘要/Abstract

摘要： 【目的】制备一种适用于治疗由金黄色葡萄球菌小菌落变异株(Staphylococcus aureus small colony variants,SASCVs)引起奶牛乳腺炎感染的槲皮素-替米考星混合物纳米粒,评价其对SASCVs菌株的抗菌活性。【方法】通过对槲皮素(0.05、0.10、0.15和0.20 g)和替米考星原料药用量(0.2、0.3、0.4和0.5 g)及转速(500、1 000、1 500 r/min)、反应时间(0.5、1.0、2.0 h)、反应温度(25、50、75 ℃)、溶剂浓度(5.0%、7.5%、10.0%)进行考察,以外观性状为指标,筛选槲皮素-替米考星混合物纳米粒最优配方和制备条件;以微观形态、沉降体积比、再分散性、粒径和Zeta电位为指标,对制备的槲皮素-替米考星混合物纳米粒表征;通过琼脂扩散法、微量肉汤稀释法和体外杀菌曲线,对比槲皮素-替米考星混合物纳米粒和市售替米考星溶液对SASCVs菌株的抗菌活性,评价槲皮素-替米考星混合物纳米粒的优势。【结果】当替米考星为0.4 g、槲皮素为0.1 g、溶剂为20 mL 7.5%的聚乙二醇溶液,在转速为1 000 r/min、反应时间为1 h、反应温度为50 ℃时,所制备的槲皮素-替米考星混合物纳米粒最优,其溶液外观性状显示颜色清亮,无沉淀和絮状物;微观形态显示该混合物纳米粒分布均匀,颗粒大小一致;沉降体积比为1,再分散性良好,平均粒径为741.9 nm,Zeta电位为-7.69 mV。替米考星标准品、槲皮素-替米考星混合物纳米粒和市售替米考星溶液对SASCVs菌株的抑菌圈直径分别为2.54、1.51和1.38 cm,对SASCVs菌株的最低抑菌浓度分别为1、1和2 μg/mL;体外杀菌曲线表明,槲皮素-替米考星混合物纳米粒对SASCVs菌株表现出明显的浓度依赖性。【结论】本研究所制备的槲皮素-替米考星混合物纳米粒对SASCVs菌株具有较强杀菌能力,可为治疗由SASCVs菌株引起的奶牛乳腺炎提供基础研究资料。

关键词: 槲皮素; 替米考星; 混合物纳米粒; 金黄色葡萄球菌小菌落变异株; 抗菌活性

Abstract: 【Objective】 Quercetin-tilmicosin mixture nanoparticles was prepared for the treatment of Staphylococcus aureus small colony variants (SASCVs) infection,and its antibacterial activity against SASCVs strain was evaluated.【Method】 The dosage of quercetin (0.05,0.10,0.15 and 0.20 g) and tilmicosin API (0.2,0.3,0.4 and 0.5 g),rotating speed (500,1 000 and 1 500 r/min),reaction time (0.5,1.0 and 2.0 h),reaction temperature (25,50 and 75 ℃) and solvent concentration (5.0%,7.5% and 10.0%) were determined to screen the optimal formula and preparation conditions of quercetin-tilmicosin mixture nanoparticles.The prepared quercetin-tilmicosin mixture nanoparticles were characterized by micro-morphology,sedimentation volume ratio,re-dispersibility,particle size and Zeta potential.The antibacterial activities of quercetin-tilmicosin mixture nanoparticles and commercial tilmicosin solution against SASCVs strains were compared by agar diffusion method,micro broth dilution method and in vitro sterilization curve,and the advantages of quercetin-tilmicosin mixture nanoparticles were evaluated.【Result】 Quercetin-tilmicosin mixture nanoparticles were the best when tilmicosin was 0.4 g,quercetin was 0.1 g,solvent was 20 mL of 7.5% polyethylene glycol solution,rotating speed was 1 000 r/min,reaction time was 1 h and reaction temperature was 50 ℃,respectively.Its appearance properties of formulation showed no precipitate and floccule,and the color was clear.Its micro-morphology showed that the mixture nanoparticles were evenly distributed and the particle size was the same.The sedimentation volume ratio was 1,and the re-dispersibility was good.The average particle size was 741.9 nm;Zeta potential was -7.69 mV.The inhibition zone diameters of tilmicosin standard,quercetin-tilmicosin mixture nanoparticles and commercial tilmicosin solution were 2.54,1.51 and 1.38 cm,respectively,and the minimum inhibitory concentrations were 1,1 and 2 μg/mL,respectively.The in vitro killing curves showed that quercetin-tilmicosin mixture nanoparticles against SASCVs strains showed obvious concentration-dependent.【Conclusion】 The quercetin-tilmicosin mixture nanoparticles prepared in this study had strong bactericidal ability against SASCVs strains,and could provide basic research data for the treatment of dairy cow mastitis caused by SASCVs strain.

Key words: quercetin; tilmicosin; mixture nanoparticles; Staphylococcus aureus small colony variants; antibacterial activity

中图分类号:

S859.7

刘金环, 张珊玲, 宋玮, 罗万和, 陈伟. 槲皮素-替米考星混合物纳米粒的制备及对金黄色葡萄球菌小菌落变异株抗菌活性研究[J]. 中国畜牧兽医, 2022, 49(4): 1470-1478.

LIU Jinhuan, ZHANG Shanling, SONG Wei, LUO Wanhe, CHEN Wei. Preparation of Quercetin-tilmicosin Mixture Nanoparticles and Its Antibacterial Activity Against Small Colony Variant Strains of Staphylococcus aureus[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1470-1478.

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