氟苯尼考纳米乳制备及其抑菌效果观察
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摘要
为制备氟苯尼考纳米乳并了解其抑菌效果,本试验进行了处方筛选、物理性质考察、抑菌圈试验,通过测定氟苯尼考在多种油相中的溶解度,确定最佳油相;再以乳化剂-助乳化剂(Smix)体积比Kv值及Smix-油相体积比为考察指标,结合伪三元相图筛选最佳处方组成;利用染色法鉴别纳米乳类型,通过透射电镜观察纳米乳微观形态;通过激光粒度分析仪测定粒度分布、Zeta电位;采用离心及长期试验考察其稳定性;经抑菌圈试验观察氟苯尼考纳米乳对金黄色葡萄球菌、大肠埃希菌、微杆菌、枯草芽孢杆菌的体外抑菌效果。结果显示,橄榄油为最佳油相,EL-40为最佳乳化剂,丙三醇为最佳助乳化剂,优选的Smix体积比Kv值为2.0,Smix-油相体积比为8∶2。氟苯尼考纳米乳最佳处方组成为:氟苯尼考120mg,N,N-二甲基甲酰胺0.1mL,橄榄油2mL,EL-40 5.7mL,丙三醇2.3mL,水6mL。氟苯尼考纳米乳为水包油(O/W)型,外观呈球形,大小均匀无黏连,平均粒径28nm,粒径呈正态分布,Zeta电位为-0.454mV,经离心试验及长期试验样品稳定,对4种常见菌体外抑菌效果强于同浓度氟苯尼考溶液。本试验结果表明,氟苯尼考纳米乳制备方法简单、可行,且纳米乳稳定,抑菌效果良好,在畜牧养殖中具有潜在应用价值。
In order to successfully prepare florfenicol nanoemulsion and investigate its bacteriostatic effect,the prescription screening,physical properties investigation and bacteriostatic activity test were conducted.The oil phase,emulsifier and coemulsifier were optimized by determining the solubility of florfenicol in oil phases,emulsifiers and coemulsifier,respectively.The optimum formula was designed based on Kv of emulsifier-coemulsifier(Smix)and Smix-oil phase volume ratio which was selected using pseudo-ternary phase diagrm.Florfenicol nanoemulsion was distinguished by staining method,and the configuration of florfenicol nanoemulsion were measured by TEM,the particle size and Zeta potential were measured by laser particle size analyzer.The stability was investigated by a centrifugation and a long-term test.The antibacterial effect of florfenicol nanoemulsion on Staphylococcus aureus,Escherichia coli,Microbacterium and Bacillus subtilis was studied by a bacteriostatic activity test.The results showed that olive oil was the optimized oil phase,EL-40 was the optimized emulsifier,glycerin was the optimized coemulsifier,the optimized ratio of Kv was 2.0,and the volume ratio of Smix-oil was 8∶2.The optimum formula of florfeni-col nanoemulsion was the composition of fluorobenicol(120 mg),N,N-dimethylformamide(0.1 mL),olive oil(2 mL),EL-40(5.7 mL),propanol(2.3 mL),and distilled water(6 mL).The uniform size and no adhesion droplet shape of florfenicol nanoemulsion was O/W,with the average diameter of 28 nm and Zeta potential of-0.454 mV,maintained to be stable up to six months.The florfenicol nanoemulsion performanced a better antibacterial effect than the same concentration of fluorphenicol solution for four bacterias.The results demonstrated that the preparation method of florfenicol nanoemulsion was simple,feasible,and showed high stability and good bacteriostasis.The study proved that the florfenicol nanoemulsion had potential application value in the animal husbandry and aquaculture.
In order to successfully prepare florfenicol nanoemulsion and investigate its bacteriostatic effect,the prescription screening,physical properties investigation and bacteriostatic activity test were conducted.The oil phase,emulsifier and coemulsifier were optimized by determining the solubility of florfenicol in oil phases,emulsifiers and coemulsifier,respectively.The optimum formula was designed based on Kv of emulsifier-coemulsifier(Smix)and Smix-oil phase volume ratio which was selected using pseudo-ternary phase diagrm.Florfenicol nanoemulsion was distinguished by staining method,and the configuration of florfenicol nanoemulsion were measured by TEM,the particle size and Zeta potential were measured by laser particle size analyzer.The stability was investigated by a centrifugation and a long-term test.The antibacterial effect of florfenicol nanoemulsion on Staphylococcus aureus,Escherichia coli,Microbacterium and Bacillus subtilis was studied by a bacteriostatic activity test.The results showed that olive oil was the optimized oil phase,EL-40 was the optimized emulsifier,glycerin was the optimized coemulsifier,the optimized ratio of Kv was 2.0,and the volume ratio of Smix-oil was 8∶2.The optimum formula of florfeni-col nanoemulsion was the composition of fluorobenicol(120 mg),N,N-dimethylformamide(0.1 mL),olive oil(2 mL),EL-40(5.7 mL),propanol(2.3 mL),and distilled water(6 mL).The uniform size and no adhesion droplet shape of florfenicol nanoemulsion was O/W,with the average diameter of 28 nm and Zeta potential of-0.454 mV,maintained to be stable up to six months.The florfenicol nanoemulsion performanced a better antibacterial effect than the same concentration of fluorphenicol solution for four bacterias.The results demonstrated that the preparation method of florfenicol nanoemulsion was simple,feasible,and showed high stability and good bacteriostasis.The study proved that the florfenicol nanoemulsion had potential application value in the animal husbandry and aquaculture.
引文
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[2]张东超,杨宁宁,林静,等.猪传染性胸膜肺炎放线杆菌的分离鉴定及药敏试验[J].中国畜牧兽医,2016,43(6):1604-1609.ZHANG D C,YANG N N,LIN J,et al.Isolation,identification and drug sensitive tests of porcine contagious Actinobacillus pleuropneumoniae[J].China Animal Husbandry&Veterinary Medicine,2016,43(6):1604-1609.(in Chinese)
[3] GRAHAM R,PALMER D,PRATT B,et al.In vitro activity of florphenicol[J].European Journal of Clinical Microbiology and Infectious Diseases,1998,7(5):691-694.
[4] VARMA K,ADAMS P,POWERS T,et al.Pharmacokinetics of florfenicol in veal calves[J].Journal of Veterinary Pharmacology and Therapeutics,1986,9(4):412-425.
[5] ZHAO Q,WANG Y,WANG S L,et al.Prevalence and abundance of florfenicol and linezolid resistance genes in soils adjacent to swine feedlots[J].Scientific Reports,2016,6:32192.
[6]吉姆·E·里维耶尔,马克·G·帕皮奇著.操继跃,刘雅红译.兽医药理学与治疗学[M].第9版.北京:中国农业出版社,2012.RIVIERE J E,PAPICH M G(Editor).CAO J Y,LIU Y H(Translator).Veterinary Pharmacology&Therapeutics[M].9th Edition.Beijing:China Agriculture Press,2012.(in Chinese)
[7]杨鹏.氟苯尼考琥珀酸钠的合成与药理作用初探[D].重庆:西南大学,2008.YANG P.Sysnthesis of florfenicol sodium succinate and research on its pharmacological effect[D].Chongqing:Southwest University,2008.(in Chinese)
[8]刘畅,周俊杰,符华林,等.尿素/PVPk30联合增加氟苯尼考溶解度的研究[J].西北农林科技大学学报(自然科学版),2017,45(8):29-35.LIU C,ZHOU J J,FU H L,et al.Increasing florfenicol solubility by combined urea and PVPk30[J].Journal of Northwest A&F University(Natural Science Edition),2017,45(8):29-35.(in Chinese)
[9]陈建平,赵玲,苏健裕,等.氟苯尼考—羟丙基-β-环糊精包合物的制备及其结构表征[J].应用基础与工程科学学报,2013,21(1):63-70.CHEN J P,ZHAO L,SU J Y,et al.Preparation and structure identification of inclusion complex of florfenicol with hydroxypropyl-β-cyclodextrin[J].Journal of Basic Science and Engineering,2013,21(1):63-70.(in Chinese)
[10]杨宝亭,王庚南,黄康东,等.氟苯尼考—羟丙基-β-环糊精包合物的研制[J].中国畜牧医,2017,44(6):1854-1860.YANG B T,WANG G N,HUANG K D,et al.Preparation and identificaton of florfenicol-hydroxypropyl-β-cyclodextrin[J].China Animal Husbandry&Veterinary Medicine,2017,44(6):1854-1860.(in Chinese)
[11] KHATRI P,SHAO J.Mechanism and structural factors of lipid and surfactant in the formation of selfemulsified nanoemulsion[J].Journal of Pharmaceutical Sciences,2018,107(8):2198-2207.
[12] KOROLEVA M,NAGOVITSINA T,YURTOV E.Nanoemulsions stabilized by non-ionic surfactants:Stability and degradation mechanisms[J].Physical Chemistry Chemical Physics Pccp,2018,20(15):10369-10377.
[13]许晶,李洋洋,金花,等.纳米乳在食品工业中应用[J].东北农业大学学报,2017,48(5):89-96.XU J,LI Y Y,JIN H,et al.Application of nanoemulsion in food industry[J].Journal of Northeast Agricultural University,2017,48(5):89-96.(in Chinese)
[14] EL-SHERBINY M,ELDOSOKY M,EL-SHAFEY M,et al.Vitamin D nanoemulsion enhances hapatoprotective effect of conventional vitamin D in rats fed with a high-fat diet[J].Chemico-biological Interactions,2018,288:65-75.
[15] ALKHATIB M H,ALSHEHRI W S,ABDU F B.In vivo evaluation of the anticancer activity of the gemcitabine and doxorubicin combined in a nanoemulsion[J].Journal of Pharmacy and Bioallied Sciences,2018,10(1):35-42.
[16]丁志英,张越,刁亚茹,等.口服纳米载药系统研究进展[J].中国生化药物杂志,2016,36(6):30-32.DING Z Y,ZHANG Y,DIAO Y R,et al.Progress in oral drug-loaded nano-system[J].Chinese Journal of Biochemical and Pharmaceuticals,2016,36(6):30-32.(in Chinese)
[17]高娴,欧阳五庆,李梦云,等.托氟沙星纳米乳的制备及其急性毒性研究[J].畜牧兽医学报,2015,46(11):2069-2077.GAO X,OUYANG W Q,LI M Y,et al.Study on the preparation and acute toxicity of tosufloxacin nanoemulsion[J].Acta Veterinaria et Zootechnica Sinica,2015,46(11):2069-2077.(in Chinese)
[18]丁丹,盛小莉,梁孔贤,等.伊维菌素纳米乳透皮制剂的研究[J].中国畜牧兽医,2015,42(2):401-407.DING D,SHENG X L,LIANG K X,et al.Study on ivermectin nanoemulsion for transdermal drug delivery[J].China Animal Husbandry&Veterinary Medicine,2015,42(2):401-407.(in Chinese)
[19]姚静,周建平,杨宇欣,等.微乳对难溶性药物增溶机理的研究[J].中国药科大学学报,2004,35(6):14-17.YAO J,ZHOU J P,YANG Y X,et al.Study on the mechanism of lipophilic drugs incorporation into microemulision system[J].Journal of China Pharmaceutical University,2004,35(6):14-17.(in Chinese)
[20] RUCKENSTEIN E,CHI J C.Stability of microemulsions[J].Journal of the Chemical Society Faraday Transactions Molecular&Chemical Physics,1975,71(2):1690-1707.
[21]刘安刚,李引乾,孙娇,等.氟苯尼考纳米乳的制备及品质评价[J].西北农业学报,2011,20(5):44-49.LIU A G,LI Y Q,SUN J,et al.Preparation and quality evaluation on florfenicol nanoemulsion[J].Acta Agriculturae Boreali-occidentalis Sinica,2011,20(5):44-49.(in Chinese)
[22]朱术会,王成森,王加才,等.氟苯尼考纳米乳的制备及性状研究[J].黑龙江畜牧兽医,2018,3:209-211.ZHU S H,WANG C S,WANG J C,et al.Preparation and characterization of florfenicol nanoemulsion[J].Heilongjiang Animal Science and Veterinary Medicine,2018,3:209-211.(in Chinese)
[23] KUMARI S,KUMARASWAMY R V,CHOUDHARY R C,et al.Thymol nanoemulsion exhibits potential antibacterial activity against bacterial pustule disease and growth promotory effect on soybean[J].Scientific Reports,2018,8:6650.
[24] NAJAFI-TAHE R,GHAEMI B,KHARAZI S,et al.Promising antibacterial effects of silver nanoparticleloaded tea tree oil nanoemulsion:A synergistic combination against resistance threat[J].Aaps Pharmsci tech,2018,19(3):1133-1140.
[2]张东超,杨宁宁,林静,等.猪传染性胸膜肺炎放线杆菌的分离鉴定及药敏试验[J].中国畜牧兽医,2016,43(6):1604-1609.ZHANG D C,YANG N N,LIN J,et al.Isolation,identification and drug sensitive tests of porcine contagious Actinobacillus pleuropneumoniae[J].China Animal Husbandry&Veterinary Medicine,2016,43(6):1604-1609.(in Chinese)
[3] GRAHAM R,PALMER D,PRATT B,et al.In vitro activity of florphenicol[J].European Journal of Clinical Microbiology and Infectious Diseases,1998,7(5):691-694.
[4] VARMA K,ADAMS P,POWERS T,et al.Pharmacokinetics of florfenicol in veal calves[J].Journal of Veterinary Pharmacology and Therapeutics,1986,9(4):412-425.
[5] ZHAO Q,WANG Y,WANG S L,et al.Prevalence and abundance of florfenicol and linezolid resistance genes in soils adjacent to swine feedlots[J].Scientific Reports,2016,6:32192.
[6]吉姆·E·里维耶尔,马克·G·帕皮奇著.操继跃,刘雅红译.兽医药理学与治疗学[M].第9版.北京:中国农业出版社,2012.RIVIERE J E,PAPICH M G(Editor).CAO J Y,LIU Y H(Translator).Veterinary Pharmacology&Therapeutics[M].9th Edition.Beijing:China Agriculture Press,2012.(in Chinese)
[7]杨鹏.氟苯尼考琥珀酸钠的合成与药理作用初探[D].重庆:西南大学,2008.YANG P.Sysnthesis of florfenicol sodium succinate and research on its pharmacological effect[D].Chongqing:Southwest University,2008.(in Chinese)
[8]刘畅,周俊杰,符华林,等.尿素/PVPk30联合增加氟苯尼考溶解度的研究[J].西北农林科技大学学报(自然科学版),2017,45(8):29-35.LIU C,ZHOU J J,FU H L,et al.Increasing florfenicol solubility by combined urea and PVPk30[J].Journal of Northwest A&F University(Natural Science Edition),2017,45(8):29-35.(in Chinese)
[9]陈建平,赵玲,苏健裕,等.氟苯尼考—羟丙基-β-环糊精包合物的制备及其结构表征[J].应用基础与工程科学学报,2013,21(1):63-70.CHEN J P,ZHAO L,SU J Y,et al.Preparation and structure identification of inclusion complex of florfenicol with hydroxypropyl-β-cyclodextrin[J].Journal of Basic Science and Engineering,2013,21(1):63-70.(in Chinese)
[10]杨宝亭,王庚南,黄康东,等.氟苯尼考—羟丙基-β-环糊精包合物的研制[J].中国畜牧医,2017,44(6):1854-1860.YANG B T,WANG G N,HUANG K D,et al.Preparation and identificaton of florfenicol-hydroxypropyl-β-cyclodextrin[J].China Animal Husbandry&Veterinary Medicine,2017,44(6):1854-1860.(in Chinese)
[11] KHATRI P,SHAO J.Mechanism and structural factors of lipid and surfactant in the formation of selfemulsified nanoemulsion[J].Journal of Pharmaceutical Sciences,2018,107(8):2198-2207.
[12] KOROLEVA M,NAGOVITSINA T,YURTOV E.Nanoemulsions stabilized by non-ionic surfactants:Stability and degradation mechanisms[J].Physical Chemistry Chemical Physics Pccp,2018,20(15):10369-10377.
[13]许晶,李洋洋,金花,等.纳米乳在食品工业中应用[J].东北农业大学学报,2017,48(5):89-96.XU J,LI Y Y,JIN H,et al.Application of nanoemulsion in food industry[J].Journal of Northeast Agricultural University,2017,48(5):89-96.(in Chinese)
[14] EL-SHERBINY M,ELDOSOKY M,EL-SHAFEY M,et al.Vitamin D nanoemulsion enhances hapatoprotective effect of conventional vitamin D in rats fed with a high-fat diet[J].Chemico-biological Interactions,2018,288:65-75.
[15] ALKHATIB M H,ALSHEHRI W S,ABDU F B.In vivo evaluation of the anticancer activity of the gemcitabine and doxorubicin combined in a nanoemulsion[J].Journal of Pharmacy and Bioallied Sciences,2018,10(1):35-42.
[16]丁志英,张越,刁亚茹,等.口服纳米载药系统研究进展[J].中国生化药物杂志,2016,36(6):30-32.DING Z Y,ZHANG Y,DIAO Y R,et al.Progress in oral drug-loaded nano-system[J].Chinese Journal of Biochemical and Pharmaceuticals,2016,36(6):30-32.(in Chinese)
[17]高娴,欧阳五庆,李梦云,等.托氟沙星纳米乳的制备及其急性毒性研究[J].畜牧兽医学报,2015,46(11):2069-2077.GAO X,OUYANG W Q,LI M Y,et al.Study on the preparation and acute toxicity of tosufloxacin nanoemulsion[J].Acta Veterinaria et Zootechnica Sinica,2015,46(11):2069-2077.(in Chinese)
[18]丁丹,盛小莉,梁孔贤,等.伊维菌素纳米乳透皮制剂的研究[J].中国畜牧兽医,2015,42(2):401-407.DING D,SHENG X L,LIANG K X,et al.Study on ivermectin nanoemulsion for transdermal drug delivery[J].China Animal Husbandry&Veterinary Medicine,2015,42(2):401-407.(in Chinese)
[19]姚静,周建平,杨宇欣,等.微乳对难溶性药物增溶机理的研究[J].中国药科大学学报,2004,35(6):14-17.YAO J,ZHOU J P,YANG Y X,et al.Study on the mechanism of lipophilic drugs incorporation into microemulision system[J].Journal of China Pharmaceutical University,2004,35(6):14-17.(in Chinese)
[20] RUCKENSTEIN E,CHI J C.Stability of microemulsions[J].Journal of the Chemical Society Faraday Transactions Molecular&Chemical Physics,1975,71(2):1690-1707.
[21]刘安刚,李引乾,孙娇,等.氟苯尼考纳米乳的制备及品质评价[J].西北农业学报,2011,20(5):44-49.LIU A G,LI Y Q,SUN J,et al.Preparation and quality evaluation on florfenicol nanoemulsion[J].Acta Agriculturae Boreali-occidentalis Sinica,2011,20(5):44-49.(in Chinese)
[22]朱术会,王成森,王加才,等.氟苯尼考纳米乳的制备及性状研究[J].黑龙江畜牧兽医,2018,3:209-211.ZHU S H,WANG C S,WANG J C,et al.Preparation and characterization of florfenicol nanoemulsion[J].Heilongjiang Animal Science and Veterinary Medicine,2018,3:209-211.(in Chinese)
[23] KUMARI S,KUMARASWAMY R V,CHOUDHARY R C,et al.Thymol nanoemulsion exhibits potential antibacterial activity against bacterial pustule disease and growth promotory effect on soybean[J].Scientific Reports,2018,8:6650.
[24] NAJAFI-TAHE R,GHAEMI B,KHARAZI S,et al.Promising antibacterial effects of silver nanoparticleloaded tea tree oil nanoemulsion:A synergistic combination against resistance threat[J].Aaps Pharmsci tech,2018,19(3):1133-1140.