CN115869350B - 黑骨藤提取物在制备亚胺培南抗铜绿假单胞菌增敏剂中的应用 - Google Patents
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Abstract
本申请公开了中草药技术领域中一种黑骨藤提取物在制备亚胺培南抗铜绿假单胞菌增敏剂中的应用,具体是将黑骨藤与有机溶剂混合后提取得到黑骨藤提取物,亚胺培南按照不同临床菌株的1/4MIC和1/8MIC分别联合黑骨藤提取物1024μg/mL和512μg/mL四种配伍,结果说明黑骨藤提取物可以增加铜绿假单胞菌对亚胺培南的敏感性。
Description
技术领域
本发明涉及医药技术领域,具体涉及一种黑骨藤提取物在制备亚胺培南抗铜绿假单胞菌增敏剂中的应用。
背景技术
铜绿假单胞菌(Pseudomonas aeruginosa,PA)是目前临床上最常见的引起严重院内获得性感染的条件致病菌之一。在机体抵抗力下降或免疫功能受损或是侵入性操作时(如留置尿管、呼吸机辅助通气)易引起各种感染,如泌尿系感染、呼吸道感染、脑膜炎、烧伤感染等,其中以下呼吸道感染最为常见,包括支气管扩张合并感染、慢性阻塞性肺疾病合并感染、呼吸机相关性肺炎等,且主要分布于重症监护病房。
抗生素的广泛使用甚至于滥用导致多重耐药铜绿假单胞菌(Multidrug-resistant Pseudomonas aeruginosa,MDRPA)的出现,给人们的安全带来巨大的威胁。目前,碳青霉烯类抗生素是治疗MDRPA的临床一线药物,也是治疗革兰阴性杆菌严重感染的最后一道防线。然而,随着碳青霉烯类抗菌药物(亚胺培南、美罗培南等)的不断使用,碳青霉烯耐药的铜绿假单胞菌(Carbapenem-resistant Pseudomonas aeruginosa,CRPA)的不断出现,使临床上对CRPA感染彻底陷入了无药可用的尴尬境地。最新的CHINET中国细菌耐药监测结果显示(2020年),CRPA感染所致的死亡率排名上升至第三位,已经成为感染死亡率上长最快的耐药菌之一。因此,在全球对新抗菌药物的开发陷入低谷期的时刻,“间接抗菌策略”,即找到具有提高现有抗菌药物作用的药物(抗菌增敏剂),延缓耐药菌的传播迫在眉睫。
苗药黑骨藤苗药民族名称为锐松怪(贵州铜仁)、嘎巴又赊(贵州黔东南),又称黑骨头、滇杠柳等,是马钱科蓬莱葛属植物黑骨藤的根或全株。主要分布于贵州、云南等地,药用历史悠久,在《贵州本草》、《贵州草药》等医药著作中均有记载。有祛风湿,通经络,消炎解毒之功效。主治风湿痹痛,跌仆损伤,闭经,乳痈,骨折,口腔炎,胃痛,消化不良,疟疾。在现有技术的报道中,并未有黑骨藤协同亚胺培南抗菌增效的报道。
发明内容
本发明针对现有技术的不足,研究黑骨藤提取物在铜绿假单胞菌标准菌株与临床分离菌中与多种抗生素的抗菌增效作用,尤其是可以显著提高铜绿假单胞菌对碳青霉烯类抗生素亚胺培南敏感性的生物学作用。
本发明的目的之一是提供一种黑骨藤提取物在制备亚胺培南抗铜绿假单胞菌增敏剂中的应用。
进一步的,所述铜绿假单胞菌为多重耐药铜绿假单胞菌。
进一步的,所述铜绿假单胞菌为碳青霉烯耐药的铜绿假单胞菌。
进一步的,所述黑骨藤为马钱科蓬莱葛属植物干燥的根或全株。
进一步的,所述黑骨藤提取物为将黑骨藤与适当比例的有机溶剂混合,提取数次,合并提取液,回收溶剂后得黑骨藤提取物。
进一步的,所述有机溶剂为乙醇。
进一步的,所述提取方法包括加热回流提取、闪式提取、超声提取或渗漉提取。
本发明还有一个目的是提供一种抗菌组合物,主要由亚胺培南和黑骨藤提取物混合而成。对碳青霉烯耐药的铜绿假单胞菌具有较好的抑菌效果。
进一步的,当所述亚胺培南的量为不同临床菌株的1/8MIC~1/4MIC。亚胺培南的用药浓度更低,抗菌敏感性更强。
本发明的工作原理及有益效果:亚胺培南属于碳青霉烯类抗生素,碳青霉烯类抗生素是抗菌谱最广的一类β-内酰胺类抗生素,抗菌活性强,对铜绿假单胞外膜的透过性大。其作用机制是抑制胞壁粘肽合成酶,即青霉素结合蛋白(PBPs),从而阻碍细胞壁粘肽合成,使细菌胞壁缺损,菌体膨胀致使细菌胞浆渗透压改变和细胞溶解而杀灭细菌。
在技术中,黑骨藤提取物在体外实验中明显增强亚胺培南对于铜绿假单胞菌的抑制作用。
申请人利用黑骨藤提取物联合不同抗生素对多种细菌进行筛选实验,在进行大量筛选实验后发现黑骨藤提取物可显著增强亚胺培南对铜绿假单胞菌的抗菌作用,即黑骨藤提取物对亚胺培南具有抗菌增效作用,关于其抗菌增效作用机制需要进一步的研究。
具体实施方式
下面通过具体实施方式进一步详细说明:
在本实施方案中首先参考2020年美国临床和实验室标准协会(Clinical andLaboratory Standards Institute,CLSI)药物敏感标准判断,选择质控菌株:铜绿假单胞菌ATCC27853,临床分离菌株来源于贵州遵义地区。
实施例1:
黑骨藤提取物对铜绿假单胞菌质控菌株ATCC27853与临床分离株最低抑菌浓度(MIC)的测定。
使用小型打粉机将黑骨藤根药材打碎,过筛,用75%乙醇浸泡过夜(w/w=1:10),超声波提取3次,每次30min,抽滤,合并滤液并浓缩得黑骨藤粗提物。
采用微孔二倍稀释法,调整细菌浓度为1×106CFU/mL,接种于96孔无菌培养板内,加入各药物至含有细菌培养孔内,根据溶解度依次倍比稀释,置37℃培养箱孵育18~24h,读取阳性和阴性对照孔,阴性对照孔清亮,阳性对照孔混浊。黑骨藤提取物对细菌的MIC为抑制细菌肉眼可见生长的最低药物浓度。此次实验结果表明黑骨藤提取物单独使用不能抑制细菌生长,结果示于表1。
表1黑骨藤提取物对铜绿假单胞菌质控菌株与19株临床分离菌株的MIC
实施例2:亚胺培南对铜绿假单胞菌质控菌株ATCC27853与临床分离菌株的最低抑菌浓度测定。
采用微孔二倍稀释法,调整细菌浓度为1×106CFU/mL,接种于96孔无菌培养板内,加入各药物至含有细菌培养孔内,根据溶解度依次倍比稀释,置37℃培养箱孵育18~24h,读取阳性和阴性对照孔,阴性对照孔清亮,阳性对照孔混浊。药物对细菌的MIC为抑制细菌肉眼可见生长的最低药物浓度。此次实验观察了亚胺培南对铜绿假单胞菌质控菌株ATCC27853和19株临床分离菌株的抑菌效力。根据实验室标准协会(Clinical andLaboratory Standards Institute,CLSI)药物敏感标准,当MIC值≤2μg/mL时,即判断铜绿假单胞菌对亚胺培南敏感;当MIC值≥8μg/mL时,即判断铜绿假单胞菌对亚胺培南耐药。根据表2结果可知,只有铜绿假单胞菌标准株及临床株1、4、5、6、7、11和15对亚胺培南敏感;铜绿假单胞菌临床株3、8、12和14在敏感与耐药的临界值之间,其它8株临床分离株都有耐药性(耐药菌比例超过40%),特别是PA17有着极强的耐药性(MIC≥512μg/mL)。
表2亚胺培南对铜绿假单胞菌质控菌株和19株临床分离菌株的MIC
实施例3:黑骨藤提取物与碳青霉烯类抗生素亚胺培南联合应用时对铜绿假单胞菌临床菌株MIC影响。
采用棋盘式微孔稀释法,调整细菌浓度为1×106CFU/mL,接种于96孔无菌培养板内,将不同浓度黑骨藤提取物(1024μg/mL、512μg/mL)分别与1/4MIC、1/8MIC亚胺培南配伍,观察配伍后的药物对铜绿假单胞菌临床分离菌株MIC的影响。
此次实验结果表明黑骨藤提取物单独使用虽然不能抑制细菌生长,但在1024μg/mL或者512μg/mL血人参提取物与抗生素亚胺培南联合应用时,可使亚胺培南的MIC降低8倍,说明黑骨藤提取物可以增加亚胺培南对铜绿假单胞菌的敏感性。结果见表3。
表3黑骨藤提取物联合亚胺培南对铜绿假单胞菌MIC的影响
注:√代表有效,本实验中配合设计分别为亚胺培南对不同临床菌株的1/4MIC和1/8MIC分别联合黑骨藤提取物1024μg/mL和512μg/mL四种配伍,其中除菌种PA03在512μg/mL黑骨藤提取物和1/4MIC亚胺培南配伍下有效,512μg/mL黑骨藤提取物和1/8MIC亚胺培南的配伍无效,其余各个菌种的配伍均有效。以上实验说明黑骨藤提取物可以显著增加亚胺培南对铜绿假单胞菌临床菌株的敏感性。
Claims (5)
1.一种黑骨藤乙醇提取物在制备亚胺培南抗铜绿假单胞菌增敏剂中的应用。
2.根据权利要求1所述的应用,其特征在于:所述铜绿假单胞菌为碳青霉烯耐药的铜绿假单胞菌。
3.根据权利要求2所述的应用,其特征在于:所述黑骨藤为马钱科蓬莱葛属植物干燥的根或全株。
4.根据权利要求3所述的应用,其特征在于:所述黑骨藤乙醇提取物为将黑骨藤与适当比例的乙醇混合,提取数次,合并提取液,回收溶剂后得到。
5.根据权利要求4所述的应用,其特征在于:所述提取方法包括加热回流提取、闪式提取、超声提取或渗漉提取。
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