CN117427146A - 桑黄酮和粘菌素在制备抑制鲍曼不动杆菌感染的药物中的应用 - Google Patents
桑黄酮和粘菌素在制备抑制鲍曼不动杆菌感染的药物中的应用 Download PDFInfo
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Abstract
桑黄酮和粘菌素在制备抑制鲍曼不动杆菌感染的药物中的应用,属于生物医药技术领域。本发明提供了桑黄酮和粘菌素联合使用在制备抑制鲍曼不动杆菌感染的药物中的应用。所述鲍曼不动杆菌包括粘菌素耐药鲍曼不动杆菌或敏感鲍曼不动杆菌。本发明提供了桑黄酮能够协同粘菌素发挥抗鲍曼不动杆菌的作用,从而治疗粘菌素耐药鲍曼不动杆菌引发的感染。当桑黄酮与粘菌素采用一定浓度联用时,所有的粘菌素耐药鲍曼不动杆菌在2h内被杀死,证明桑黄酮和粘菌素存在协同效应。
Description
技术领域
本发明属于生物医药技术领域,具体涉及桑黄酮和粘菌素在制备抑制鲍曼不动杆菌感染的药物中的应用。
背景技术
鲍曼不动杆菌(Acinetobacter baumannii,Ab)是一种非发酵革兰氏阴性的机会致病菌,易引起免疫力低下人群的感染,是医院感染暴发的重要病原菌之一,通常会引起呼吸机相关性肺炎(VAP)、菌血症、脑膜炎、心内膜炎以及泌尿道和皮肤感染等。
鲍曼不动杆菌临床主要使用抗生素为碳青霉烯类和内酰胺酶抑制剂,而随着近年来耐碳青霉烯鲍曼检出率的增高和新药开发的缺乏,迫使临床医生使用传统抗生素多粘菌素(多粘菌素B和粘菌素)作为MDRAB治疗的最后一道防线。多粘菌素于20世纪40年代首次被发现,是一种天然多碱基环状脂肽家族。多粘菌素B和粘菌素在20世纪50年代末首次被引入临床实践。但是,粘菌素在临床使用上具有显著的剂量限制性肾毒性,其治疗窗口非常狭窄,其次,急性毒性严重限制了静脉给药的最大剂量。目前世界范围内已出现多例粘菌素耐药的鲍曼不动杆菌,因此,迫切需要寻找新的治疗策略以提高疗效和减轻毒性。
发明内容
针对上述现有技术中存在的问题,本发明的目的在于设计提供桑黄酮和粘菌素在制备抑制鲍曼不动杆菌感染的药物中的应用,桑黄酮能够抑制多粘菌素耐药鲍曼不动杆菌引起的感染。
为了实现上述目的,本发明提供以下技术方案:
一方面,本发明提供了桑黄酮和粘菌素联合使用在制备抑制鲍曼不动杆菌感染的药物中的应用。
通过桑黄酮对鲍曼不动杆菌生物膜和胞内ATP水平等的研究,发现桑黄酮能显著抑制鲍曼不动杆菌生物膜的形成,降低胞内ATP水平,从而能够减少鲍曼不动杆菌的聚集,减轻药物扩散的障碍,扰乱细菌内稳态,最终增强粘菌素的抗菌效果。桑黄酮能够显著降低粘菌素耐药鲍曼不动杆菌对粘菌素的MIC值,协同粘菌素发挥抗鲍曼不动杆菌的作用,从而治疗粘菌素耐药鲍曼不动杆菌引发的感染。
所述的应用,所述鲍曼不动杆菌为粘菌素耐药鲍曼不动杆菌或敏感鲍曼不动杆菌。
所述的应用,所述桑黄酮和粘菌素联合使用降低鲍曼不动杆菌生物膜形成能力。
一种抑制鲍曼不动杆菌感染的药物组合物,所述药物组合物的组分包含独立包装的桑黄酮和粘菌素。
所述的药物组合物,所述桑黄酮的质量浓度为4~32μg/mL,所述粘菌素的质量浓度为0.5~16μg/mL。
所述的药物组合物,所述粘菌素的质量浓度为0.5-8μg/mL。
所述的药物组合物,所述药物组合物的剂型选自注射剂、软膏剂或片剂中的一种。
任一项所述的抑制鲍曼不动杆菌感染的药物组合物的制备方法,分别制备桑黄酮溶液和粘菌素溶液,与药学上可接受成分混合均匀后获得药物组合物。
所述的制备方法,所述桑黄酮溶液的溶剂为二甲基亚砜。
所述的制备方法,所述粘菌素溶液的溶剂为ddH2O。
与现有技术相比,本发明具有以下有益效果:
本发明提供了桑黄酮和粘菌素在制备抑制鲍曼不动杆菌感染的药物中的应用,特别是在抑制粘菌素耐药鲍曼不动杆菌上也具有良好的效果。桑黄酮与粘菌素在抑制鲍曼不动杆菌上具有协同作用,从而降低治疗鲍曼不动杆菌感染时使用的粘菌素浓度。当桑黄酮与粘菌素采用其1/4MIC浓度联用时,耐粘菌素鲍曼不动杆菌在2h内被完全杀死,证明桑黄酮和粘菌素存在协同效应,这极大的降低了粘菌素使用剂量,提高了治疗的安全性。
附图说明
图1为桑黄酮和粘菌素对粘菌素耐药鲍曼不动杆菌ATCC19606R、AB13R、AB18R和粘菌素敏感鲍曼不动杆菌ATCC19606的棋盘实验,FIC<0.5表示有协同作用;
图2为桑黄酮和/或粘菌素对粘菌素耐药鲍曼不动杆菌的时间杀菌曲线,其中:A为菌株ATCC19606R通过不同的药物浓度单独或联用处理后在24h内的细菌数量变化趋势;B为菌株AB13R通过不同的药物处理后在24h内的细菌数量变化趋势;
图3为桑黄酮和/或粘菌素对形成生物膜能力较强的ATCC19606R的抑制生物膜形成的作用,其中:A为浓度梯度增高的桑黄酮单独作用对生物膜的抑制效果;B为桑黄酮联合粘菌素对鲍曼不动杆菌生物膜的抑制作用;
图4为桑黄酮和/或粘菌素联合用药的安全性评估,其中:A为绵羊红细胞进行溶血性实验,B为通过CCK8法测定细胞毒性。
具体实施方式
下面将结合附图和实施例,对本发明中的技术方案进行清楚、完整地描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明桑黄酮购于MCE公司,粘菌素购于Selleck公司。
实施例1:棋盘试验检测桑黄酮与粘菌素的协同作用
将来源不同的鲍曼不动杆菌临床分离株AB13、AB18和标准菌株ATCC19606诱导不同程度的粘菌素耐药,并将产生的耐药菌株命名为:AB13R、AB18R和ATCC19606R。其中,AB13、AB18、ATCC19606、AB13R、AB18R和ATCC19606R保藏于扬州大学病原微生物学实验室。桑黄酮溶剂为DMSO,粘菌素溶剂为ddH2O,配置成储存液后用MH肉汤培养基稀释成所需浓度(27g/1000mL蒸馏水)。
用棋盘试验检测桑黄酮与粘菌素的协同作用:在96孔板中,每列倍增桑黄酮浓度(0~32μg/mL),每行倍增粘菌素(0~512μg/mL)。每孔接种100μL测试菌株悬液(5×105CFU/mL),终体积为200μL,然后在37℃下孵育24h。结果见图1,可以看出桑黄酮降低AB13R MIC值4~64倍,降低AB18RMIC值64倍,降低ATCC19606RMIC值16~64倍,降低ATCC19606MIC值2~4倍。由此可见,不同浓度的桑黄酮均显著降低了粘菌素耐药鲍曼不动杆菌对粘菌素的MIC值,也可降低粘菌素敏感鲍曼不动杆菌对粘菌素的MIC值。提示桑黄酮能协同粘菌素发挥抗菌作用,从而治疗粘菌素耐药鲍曼不动杆菌引发的感染或降低治疗粘菌素敏感鲍曼不动杆菌粘菌素的使用剂量。
实施例2:时间杀菌曲线检测桑黄酮与粘菌素的协同效果
为了进一步证明桑黄酮与粘菌素的协同作用,对上述证明有效的2株粘菌素耐药鲍曼不动杆菌进行时间杀菌曲线试验。分别向5×108CFU/mL的AB13R和5×106CFU/mL的ATCC19606R接种物中加入不同药物,37℃摇床培养,每隔2h取样进行平板计数,12h后每隔4h取样进行平板计数。本试验中选取的药物浓度根据前面结果中的MIC浓度所设置,分别选取MIC,1/2MIC,1/4MIC浓度进行单独和联合处理。结果见图2,可以看出24h内单独用药组细菌生长和对照组无显著差异。然而当桑黄酮与粘菌素联用时,所有细菌在2h内被杀死,证明桑黄酮与粘菌素存在协同效应。
实施例3:桑黄酮与粘菌素联合使用降低鲍曼不动杆菌生物膜形成能力
生物膜形成是细菌抵抗抗生素的一种重要方式,细菌可以在生物膜中生长并逃逸机体免疫应答,从而阻碍抗菌药物发挥作用,本试验选取形成生物膜能力较强的ATCC19606R菌株。向96孔板接种1×106CFU的菌液,加入不同浓度的药物处理,在37℃静置培养48h,通过结晶紫染色测定OD590值来定量生物膜形成。结果见图3,可以看出桑黄酮单独使用时便能抑制其生物膜的形成并呈浓度依赖性,当桑黄酮与粘菌素联用时更是显著降低了鲍曼不动杆菌的生物膜形成能力。
实施例4:桑黄酮与粘菌素联合使用安全性评价
药物的安全性是决定该药物是否具有研发和应用价值的重要因素之一,通过绵羊红细胞溶血性试验和CCK8细胞毒性实验初步评估桑黄酮与粘菌素联合使用的安全性。首先取新鲜的8%的绵羊血红细胞,PBS洗涤3次后用粘菌素和桑黄酮单独或联合等方式处理,以等体积的0.2%Triton X-100为阳性对照,PBS为阴性对照,37℃孵育1小时,用酶标仪测定576nm处血红蛋白的吸光度。结果见图4A,所使用的药物组合浓度中溶血率都低于5%,说明桑黄酮与粘菌素在体外有效的联合用药浓度之内不会出现显著的溶血现象。
用人类肺泡基底上皮细胞A549细胞进行CCK-8细胞毒性实验,检测粘菌素联合桑黄酮对哺乳动物细胞的细胞毒性作用。首先将细胞以约2×103个/孔接种于96孔板中,培养16~24h,然后用不同浓度的桑黄酮和粘菌素单独或联合处理细胞24h,只加药物和培养基的孔作为空白对照。培养24h后,每孔加入10μL的CCK8溶液,37℃孵育2h,检测450nm处的吸光度。结果如图4B所示,当粘菌素使用浓度为16μg/mL以内时,桑黄酮不会增加粘菌素对上皮细胞A549的细胞毒性。这些结果表明,在体外抑菌有效的最高浓度内桑黄酮与粘菌素具备一定的安全性。
Claims (10)
1.桑黄酮和粘菌素联合使用在制备抑制鲍曼不动杆菌感染的药物中的应用。
2.如权利要求1所述的应用,其特征在于,所述鲍曼不动杆菌为粘菌素耐药鲍曼不动杆菌或敏感鲍曼不动杆菌。
3.如权利要求1所述的应用,其特征在于,所述桑黄酮和粘菌素联合使用降低鲍曼不动杆菌生物膜形成能力。
4.一种抑制鲍曼不动杆菌感染的药物组合物,其特征在于,所述药物组合物的组分包含独立包装的桑黄酮和粘菌素。
5.如权利要求4所述的药物组合物,其特征在于,所述桑黄酮的质量浓度为4~32μg/mL,所述粘菌素的质量浓度为0.5~16μg/mL。
6.如权利要求4所述的药物组合物,其特征在于,所述粘菌素的质量浓度为0.5-8μg/mL。
7.如权利要求4所述的药物组合物,其特征在于,所述药物组合物的剂型选自注射剂、软膏剂或片剂中的一种。
8.如权利要求4-7任一项所述的抑制鲍曼不动杆菌感染的药物组合物的制备方法,其特征在于,分别制备桑黄酮溶液和粘菌素溶液,与药学上可接受成分混合均匀后获得药物组合物。
9.如权利要求8所述的制备方法,其特征在于,所述桑黄酮溶液的溶剂为二甲基亚砜。
10.如权利要求8所述的制备方法,其特征在于,所述粘菌素溶液的溶剂为ddH2O。
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