乙酰异戊酰泰乐菌素纳米乳的制备及药效评价
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摘要
目的：制备乙酰异戊酰泰乐菌素纳米乳（Acetylisovaleryltylosin Nanoemulsion，AIV-NE）,并对其质量、安全性和药效进行评价。
方法：（1）根据AIV在油相和助表面活性剂中的溶解度和伪三元相图，对油相、表面活性剂、助表面活性剂、K_m值进行筛选，制备纳米乳；用染色法，鉴别其结构类型；用透射电镜和粒度分析仪，考察AIV-NE的形态和粒径分布；通过高速离心试验、加速试验和长期试验考察其稳定性；（2）建立了AIV-NE中AIV含量测定的紫外分光光度法；（3）通过小鼠急性毒性试验考察AIV-NE的安全性；（4）通过体外抑菌试验评价AIV-NE对5种常见病原菌的抗菌活性；（5）对健康雏鸡进行攻毒，使其感染金黄色葡萄球菌，将动物分为AIV-NE高、中、低剂量组、AIV原料药对照组、替米考星溶液对照组、感染对照组和健康对照组，考察各给药组对患病鸡的治疗有效率和治愈率，对纳米乳的体内药效进行评价。
结果：（1）AIV-NE各组分的质量分数为：吐温-8024%、无水乙醇12%、乙酸乙酯4%、 AIV1%、蒸馏水59%。该纳米乳为澄清透明、稳定均一、水包油（O/W）型的黄色乳液；乳滴呈圆球形，分布均匀；平均粒径为11.1nm，多分散系数为0.057；稳定性良好。（2）AIV在5～40μg·mL~(-1)浓度范围内线性关系良好；平均回收率为（99.71±0.853）%，相对标准偏差（RSD）为0.855%；5次重复测量的吸收度值为0.1708，RSD为0.878%；日内精密度RSD为0.065%，日间精密度RSD为0.087%。该分析方法专属性良好，回收率、重复性和精密度均符合要求。（3）AIV-NE的半数致死量（LD50）为2425mg/kg，LD50的95%可信限为：2204～2707mg/kg，属于低毒级药物。（4）AIV-NE对大肠杆菌、金黄色葡萄球菌、沙门氏菌、巴氏杆菌和无乳链球菌的最小抑菌浓度（MIC）分别为16、2、8、4、8μg/mL，均小于AIV溶液和替米考星溶液组，且差异极显著（P<0.01）。（5）AIV-NE高、中、低剂量组的治愈率分别为96.7%、93.3%、76.7%，有效率分别为100%、96.7%、83.3%；AIV原料药对照组的治愈率为66.7%，有效率为76.7%；替米考星溶液对照组的治愈率为60%，有效率为66.7%。统计结果显示：AIV-NE高剂量组的治疗有效数显著高于AIV-NE低剂量组（P<0.05），极显著高于AIV溶液组和TMS溶液组（P<0.01）；AIV-NE中剂量组治疗有效数显著高于AIV溶液组（P<0.05），极显著高于TMS溶液组（P<0.01）；AIV-NE高剂量组治疗治愈数显著高于AIV-NE低剂量组（P<0.05），极显著高于AIV溶液组和TMS溶液组（P<0.01）；AIV-NE中剂量组治疗治愈数显著高于AIV溶液组（P<0.05），极显著高于TMS溶液组（P<0.01）；AIV-NE高、中剂量组鸡的平均增重均极显著高于AIV溶液组、 TMS溶液组和感染鸡对照组（P<0.01）。
结论：制备出的AIV-NE澄清透明、稳定性良好，口服安全性高，属于低毒药物，与其原料药和同类药物替米考星相比，具有更强的抑菌和杀菌活性，对人工感染金黄色葡萄球菌病的雏鸡的治疗效果明显优于其原料药和对照药物替米考星。
Objective: We prepared Acetylisovaleryltylosin Nanoemulsion(AIV-NE) and studied itsstability, safety and therapeutic efficacy.
Method:(1)We determined the oil and cosurfactant according to the solubility of Acetyl-isovaleryltylosin in them, screed the surfactant and K_mby studying the pseudoternary phasediagram. The structure type, shape and particle diameter distribution of AIV-NE wereinvestigated by staining method, transmission electron microscope and laser particle sizeanalysator respectively. Its stability was verified through high speed centrifuge, acceleratedtest and long term experiment.(2)The analytical method of AIV was established throughultraviolet-visible spectrophotometer.(3)We evaluated the safety and antibacterial activitythrough acute toxicity test and broth dilution method respectively.(4)The high, middle andlow dose of AIV-NE, AIV and Tilmicosin solution was respectively dosed by drinking freelyto the chicken which were infected by Staphylococcus and the therapeutic efficacy in vivowas investigated.
Result:(1) The mass fraction of the components in AIV-NE was Tween-8024%, ethanol12%, ethyl acetate4%, Acetylisovaleryltylosin1%, distilled water59%. AIV-NE was yellow,transparent, homogeneous and oil-in-water nanoemulsion. The shape of the nanoemulsiondrop was spherical and the average partical size was11.1nm with a polydispersity index of0.057. AIV-NE was stable after high speed centrifuge, accelerated test and long term exper-iment.(2)The result showed that the linearity of AIV was fine in the range of5～40μg·mL~(-1),and the average recovery, relative standard deviation(RSD), RSD of the with-in-day precisionand the day-to-day precision were（99.71±0.853）%,0.855%,0.065%,0.087%, respectively.The analytical method of ultraviolet spectrophotometry (UV) had high recovery rate, goodrepetitiveness and precision which could offer a good method for determining the content ofAIV.(3)The median lethal dose(LD50) and95%confidence limit of AIV-NE were2425mg/kgand2204～2707mg/kg respectively. So its toxicity is low and it is safe by oral administration.(4)Antibacterial experiments in vitro showed that the MIC of AIV-NE on Enteropathog-enicE.coli, Staphylococcus, Salmonella, Pasteurella, Streptococcus agalactiae were16、2、8、4、8μg/mL respectively, its antibacterial effect was better than AIV and Tilmicosin solution.(5)The cure rate of AIV-NE in high, middle and low dose group was96.7%,93.3%and76.7%respectively, and effective rate was100%,96.7%and83.3%respectively. The cure rate and effective rate of AIV and Tilmicosin solution was66.7%、76.7%and60%、66.7%respectiv-ely.The effective number and cure number of AIV-NE high dose group has significant differe-nce with AIV-NE low dose group(P<0.05), and has extremely significant difference with AIVgroup and Tilmicosin group(P<0.01);and the AIV-NE middle dose group has significant diff-erence with AIV group(P<0.05),and has extremely significant difference with Tilmicosingroup (P<0.01). The average weight gained of high and middle group both has extremely sig-nificant difference with AIV and Tilmicosin group(P<0.01).
Conclusion: The prepared AIV-NE has good stability, safety and better therapeutic effi-cacy, which would have wide prospect in clinical application.
方法：（1）根据AIV在油相和助表面活性剂中的溶解度和伪三元相图，对油相、表面活性剂、助表面活性剂、K_m值进行筛选，制备纳米乳；用染色法，鉴别其结构类型；用透射电镜和粒度分析仪，考察AIV-NE的形态和粒径分布；通过高速离心试验、加速试验和长期试验考察其稳定性；（2）建立了AIV-NE中AIV含量测定的紫外分光光度法；（3）通过小鼠急性毒性试验考察AIV-NE的安全性；（4）通过体外抑菌试验评价AIV-NE对5种常见病原菌的抗菌活性；（5）对健康雏鸡进行攻毒，使其感染金黄色葡萄球菌，将动物分为AIV-NE高、中、低剂量组、AIV原料药对照组、替米考星溶液对照组、感染对照组和健康对照组，考察各给药组对患病鸡的治疗有效率和治愈率，对纳米乳的体内药效进行评价。
结果：（1）AIV-NE各组分的质量分数为：吐温-8024%、无水乙醇12%、乙酸乙酯4%、 AIV1%、蒸馏水59%。该纳米乳为澄清透明、稳定均一、水包油（O/W）型的黄色乳液；乳滴呈圆球形，分布均匀；平均粒径为11.1nm，多分散系数为0.057；稳定性良好。（2）AIV在5～40μg·mL~(-1)浓度范围内线性关系良好；平均回收率为（99.71±0.853）%，相对标准偏差（RSD）为0.855%；5次重复测量的吸收度值为0.1708，RSD为0.878%；日内精密度RSD为0.065%，日间精密度RSD为0.087%。该分析方法专属性良好，回收率、重复性和精密度均符合要求。（3）AIV-NE的半数致死量（LD50）为2425mg/kg，LD50的95%可信限为：2204～2707mg/kg，属于低毒级药物。（4）AIV-NE对大肠杆菌、金黄色葡萄球菌、沙门氏菌、巴氏杆菌和无乳链球菌的最小抑菌浓度（MIC）分别为16、2、8、4、8μg/mL，均小于AIV溶液和替米考星溶液组，且差异极显著（P<0.01）。（5）AIV-NE高、中、低剂量组的治愈率分别为96.7%、93.3%、76.7%，有效率分别为100%、96.7%、83.3%；AIV原料药对照组的治愈率为66.7%，有效率为76.7%；替米考星溶液对照组的治愈率为60%，有效率为66.7%。统计结果显示：AIV-NE高剂量组的治疗有效数显著高于AIV-NE低剂量组（P<0.05），极显著高于AIV溶液组和TMS溶液组（P<0.01）；AIV-NE中剂量组治疗有效数显著高于AIV溶液组（P<0.05），极显著高于TMS溶液组（P<0.01）；AIV-NE高剂量组治疗治愈数显著高于AIV-NE低剂量组（P<0.05），极显著高于AIV溶液组和TMS溶液组（P<0.01）；AIV-NE中剂量组治疗治愈数显著高于AIV溶液组（P<0.05），极显著高于TMS溶液组（P<0.01）；AIV-NE高、中剂量组鸡的平均增重均极显著高于AIV溶液组、 TMS溶液组和感染鸡对照组（P<0.01）。
结论：制备出的AIV-NE澄清透明、稳定性良好，口服安全性高，属于低毒药物，与其原料药和同类药物替米考星相比，具有更强的抑菌和杀菌活性，对人工感染金黄色葡萄球菌病的雏鸡的治疗效果明显优于其原料药和对照药物替米考星。
Objective: We prepared Acetylisovaleryltylosin Nanoemulsion(AIV-NE) and studied itsstability, safety and therapeutic efficacy.
Method:(1)We determined the oil and cosurfactant according to the solubility of Acetyl-isovaleryltylosin in them, screed the surfactant and K_mby studying the pseudoternary phasediagram. The structure type, shape and particle diameter distribution of AIV-NE wereinvestigated by staining method, transmission electron microscope and laser particle sizeanalysator respectively. Its stability was verified through high speed centrifuge, acceleratedtest and long term experiment.(2)The analytical method of AIV was established throughultraviolet-visible spectrophotometer.(3)We evaluated the safety and antibacterial activitythrough acute toxicity test and broth dilution method respectively.(4)The high, middle andlow dose of AIV-NE, AIV and Tilmicosin solution was respectively dosed by drinking freelyto the chicken which were infected by Staphylococcus and the therapeutic efficacy in vivowas investigated.
Result:(1) The mass fraction of the components in AIV-NE was Tween-8024%, ethanol12%, ethyl acetate4%, Acetylisovaleryltylosin1%, distilled water59%. AIV-NE was yellow,transparent, homogeneous and oil-in-water nanoemulsion. The shape of the nanoemulsiondrop was spherical and the average partical size was11.1nm with a polydispersity index of0.057. AIV-NE was stable after high speed centrifuge, accelerated test and long term exper-iment.(2)The result showed that the linearity of AIV was fine in the range of5～40μg·mL~(-1),and the average recovery, relative standard deviation(RSD), RSD of the with-in-day precisionand the day-to-day precision were（99.71±0.853）%,0.855%,0.065%,0.087%, respectively.The analytical method of ultraviolet spectrophotometry (UV) had high recovery rate, goodrepetitiveness and precision which could offer a good method for determining the content ofAIV.(3)The median lethal dose(LD50) and95%confidence limit of AIV-NE were2425mg/kgand2204～2707mg/kg respectively. So its toxicity is low and it is safe by oral administration.(4)Antibacterial experiments in vitro showed that the MIC of AIV-NE on Enteropathog-enicE.coli, Staphylococcus, Salmonella, Pasteurella, Streptococcus agalactiae were16、2、8、4、8μg/mL respectively, its antibacterial effect was better than AIV and Tilmicosin solution.(5)The cure rate of AIV-NE in high, middle and low dose group was96.7%,93.3%and76.7%respectively, and effective rate was100%,96.7%and83.3%respectively. The cure rate and effective rate of AIV and Tilmicosin solution was66.7%、76.7%and60%、66.7%respectiv-ely.The effective number and cure number of AIV-NE high dose group has significant differe-nce with AIV-NE low dose group(P<0.05), and has extremely significant difference with AIVgroup and Tilmicosin group(P<0.01);and the AIV-NE middle dose group has significant diff-erence with AIV group(P<0.05),and has extremely significant difference with Tilmicosingroup (P<0.01). The average weight gained of high and middle group both has extremely sig-nificant difference with AIV and Tilmicosin group(P<0.01).
Conclusion: The prepared AIV-NE has good stability, safety and better therapeutic effi-cacy, which would have wide prospect in clinical application.
引文
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