金银花总黄酮脂质体的制备及对MRSA的抑制作用  

Preparation of total flavonoid-liposomes of Lonicerae Japonicae flos and their inhibitory effect on methicillin-resistant Staphylococcus aureus

作　　者：熊瑞 倪睿 刘恒旭 张世鹏 王璐 来小丹 XIONG Rui;NI Rui;LIU Hengxu;ZHANG Shipeng;WANG Lu;LAI Xiaodan(Department of Pharmacy,Jiangbei Campus of the First Affiliated Hospital of Army Medical University/No.958 th Hospital of PLA Army,Chongqing 400020;Department of Pharmacy,Army Medical Center of PLA/Daping Hospital,Army Medical University,Chongqing,400042,China)

机构地区：[1]陆军军医大学第一附属医院江北院区(陆军第九五八医院)药剂科,重庆400020 [2]陆军特色医学中心(大坪医院)药剂科,重庆400042

出　　处：《陆军军医大学学报》2024年第24期2755-2764,共10页Journal of Army Medical University

基　　金：重庆市科卫联合医学科研项目(2022MSXM187)。

摘　　要：目的制备金银花总黄酮(Lonicerae Japonicae Flos total flavonoids,LJFTF)脂质体,并评价其对耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)的抑制作用。方法采用乙醇注入法制备LJFTF脂质体(LJFTF liposome,LJFTFL),以磷脂浓度、磷脂与胆固醇质量比(脂胆比)、LJFTF质量浓度为考察因素,以包封率、载药量为评价指标,通过星点设计-效应面法优化制备工艺。通过观察性状、利用激光粒度仪测定粒径、多分散指数(Polymer dispersity index,PDI)和Zeta电位对LJFTFL进行表征,并通过高效液相色谱法测定其体外释放率。采用平板法和细菌活死染色实验观察LJFTFL对MRSA的抗菌活性,采用结晶紫染色观察LJFTFL对MRSA生物膜的抑制作用。结果经过优化和验证确定LJFTFL的最佳处方为:磷脂浓度35.00 mg/mL,脂胆比15.00,LJFTF浓度5.74 mg/mL;制得的LJFTFL呈淡黄色乳状液,测得其平均包封率为86.37%(RSD=0.37%,n=3),平均载药量为11.69%(RSD=0.09%,n=3),粒径为(173.60±1.07)nm,PDI值为0.15±0.05,Zeta电位为(-4.86±0.60)mV;游离的LJFTF在4 h内释放完全,LJFTFL在体外12 h的累计释放率为(59.44±3.58)%,在30 h的累计释放量为(63.58±5.78)%。平板法和细菌活死染色实验表明,LJFTFL可显著抑制MRSA生长并促进其死亡;结晶紫染色结果显示,LJFTFL可显著抑制MRSA生物膜形成,且LJFTFL干预组与同浓度游离的LJFTF干预组相比差异具有统计学意义(P<0.001)。结论本研究成功制得LJFTFL并对其处方工艺进行了优化,LJFTFL在体外对MRSA呈显著的抗菌增效活性。Objective To prepare the liposomes of total flavonoids from Lonicerae Japonicae flos(LJFTF)and evaluate their inhibitory effect on methicillin-resistant Staphylococcus aureus(MRSA).Methods LJFTF liposomes were prepared by ethanol injection.Then the preparation process was optimized by star point design-effect surface method,with the concentrations of phospholipid,mass ratio of phospholipid to cholesterol and mass concentration of LJFTF as the influencing factors,and the encapsulation rate and drug-loading rate as evaluation indicators.The obtained liposomes were characterized by observing their properties,measuring particle size,polymer dispersity index(PDI)and Zeta potential through laser particle size analyzer.High performance liquid chromatography(HPLC)was used to determine the release rate of the liposomes.The antibacterial activity of the liposomes against MRSA was observed by plate method and live/dead staining,and the inhibitory effect of the liposomes on MRSA biofilm was observed by crystal violet staining.Results After optimization and verification,the best preparation process was as follows:phospholipid concentration 35.0 mg/mL,phospholipid to cholesterol mass ratio 15.0,and LJFTF concentration 5.7 mg/mL.The prepared liposomes were light yellow emulsion,with an average encapsulation rate of 86.37%(RSD=0.37%,n=3),and an average drug-loading rate of 11.69%(RSD=0.09%,n=3),a particle size of 173.60±1.07 nm,a PDI value of 0.15±0.05,and a Zeta potential of-4.86±0.60 mV.The free LJFTF was completely released within 4 h from the dialysis bag,and the in vitro cumulative release of LJFTF was(59.44±3.58)%at 12 h and(63.58±5.78)%at 30 h.Plate and live/dead staining showed that the prepared liposomes significantly inhibited the growth and promoted the death of MRSA strain.Crystal violet staining displayed that the liposomes significantly inhibited the formation of MRSA biofilm(P<0.001),with significant difference in comparison with same dose of LJFTF(P<0.001).Conclusion LJFTF liposomes are successfully prepared

关 键 词：金银花总黄酮 脂质体 星点设计-效应面法 耐甲氧西林金黄色葡萄球菌 

分 类 号：R285.5[医药卫生—中药学] R378.11[医药卫生—中医学] R944.9