CN109432103B - 齐墩果酸在制备β内酰胺酶抑制剂中的医用用途 - Google Patents

齐墩果酸在制备β内酰胺酶抑制剂中的医用用途 Download PDF

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CN109432103B
CN109432103B CN201811365552.7A CN201811365552A CN109432103B CN 109432103 B CN109432103 B CN 109432103B CN 201811365552 A CN201811365552 A CN 201811365552A CN 109432103 B CN109432103 B CN 109432103B
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邓旭明
周永林
王建锋
郭岩
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Abstract

本发明提供一种齐墩果酸在制备β内酰胺酶抑制剂中的医用用途，通过β‑内酰胺酶活性检测试验、微量肉汤稀释棋盘法、时间‑杀菌曲线试验验证了齐墩果酸能够抑制β‑内酰胺酶的活性并恢复青霉素等β‑内酰胺类抗生素对β‑内酰胺酶阳性菌（包括金黄色葡萄球菌、大肠杆菌和肺炎克雷伯氏菌）的抗菌活性。因此齐墩果酸作为β‑内酰胺酶抑制剂可以与抗生素联用，增加对耐药菌使用药物的选择性，对开发抗β‑内酰胺酶菌感染新药有着重要意义。

Description

齐墩果酸在制备β内酰胺酶抑制剂中的医用用途
技术领域
本发明公开了齐墩果酸的一种新的用途，进一步讲涉及齐墩果酸在制备β-内酰胺酶抑制剂中的医用用途，属于医学制药技术领域。
背景技术
细菌耐药性的问题已经成为全球的一个关注热点，其中以耐β-内酰胺类抗生素的金黄色葡萄球菌、大肠杆菌和肺炎克雷伯氏菌较为严重。耐药菌感染导致β-内酰胺类抗生素的治疗失败和较高的死亡率，给治疗院内感染、特别是治疗ICU感染带来了更大的压力。
β-内酰胺酶抑制剂在市场上已流通，但是该类抑制剂亦属于抗生素衍生物，本身具有一定的抗菌活性，通过使用时极易产生耐药性。齐墩果酸属于中药活性成分，对所检测的几种致病菌无抗菌活性。因此，开发齐墩果酸作为新型β-内酰胺酶抑制剂具有十分重要的意义。齐墩果酸为存在于女贞子、齐墩果和罗锅地等多种植物中。齐墩果酸有抗炎、抗肿瘤和增强免疫力的作用。目前大量研究表明齐墩果酸在抗病毒感染的治疗和防护中有较好的作用。
发明内容
本发明提供了一种齐墩果酸在制备β-内酰胺酶抑制剂中的医用用途，公开了齐墩果酸能够抑制β-内酰胺酶的活性，恢复β-内酰胺类抗生素对携带β-内酰胺酶金黄色葡萄球菌、大肠杆菌和肺炎克雷伯氏菌的杀菌活性。
本发明所述的齐墩果酸，其分子式：C30H48O3，分子量：456.70；
本发明通过β-内酰胺酶活性检测、棋盘法最小抑菌浓度和时间-杀菌曲线法验证齐墩果酸能够抑制β内酰胺酶的活性并恢复β-内酰胺类抗生素对β内酰胺类抗生素耐药的金黄色葡萄球菌、大肠杆菌和肺炎克雷伯氏菌抗菌活性。
本发明的积极效果在于：
提供了齐墩果酸在制备β-内酰胺酶抑制剂中的新医用用途，公开了齐墩果酸能够抑制β内酰胺酶的活性，恢复青霉素对携带β-内酰胺酶金黄色葡萄球菌、大肠杆菌和肺炎克雷伯氏菌的杀菌活性。
附图说明
图1为本发明检测不同浓度齐墩果酸对主要β-内酰胺酶的活性影响结果；
图2为本发明齐墩果酸联合β-内酰胺类抗生素对携带β-内酰胺酶金黄色葡萄球菌、大肠杆菌的时间-杀菌曲线。
具体实施方式
通过以下实施例进一步举例描述本发明，并不以任何方式限制本发明，在不背离本发明的技术解决方案的前提下，对本发明所作的本领域普通技术人员容易实现的任何改动或改变都将落入本发明的权利要求范围之内。
实施例1
齐墩果酸作为β内酰胺酶抑制剂用于药学上可接受的任何载体。
实施例2
齐墩果酸作为β内酰胺酶抑制剂用于制备治疗感染性疾病的药物。
实施例3
齐墩果酸作为β内酰胺酶抑制剂用于治疗细菌引起的感染性疾病。
试验例1
β-内酰胺酶活性鉴定
过夜培养表达β-内酰胺酶的金葡菌和携带NDM-1的大肠杆菌，备用。取无菌试管3只分别加入0 μg/mL、8 μg/mL和32μg/mL齐墩果酸，之后每管内均加入10 μL过夜培养的金葡菌和携带NDM-1的大肠杆菌菌液，于37 ℃恒温摇床中，200rpm培养3 h，12000g，4 ℃离心5 min后分别取上清50μL与无菌96孔板中，加入25 μL头孢硝噻吩作为底物，加入PBS(磷酸盐缓冲液)125 μL，于37 ℃恒温培养箱中静止孵育1 h左右，通过观察颜色变化和492 nm出吸光值来判定β-内酰胺酶活性。结果见表1和附图1。
表 1 共培养后齐墩果酸对β-内酰胺酶活性抑制效果
Figure 833370DEST_PATH_IMAGE001
同时，构建携带不同耐药酶的工程菌，表达活性耐药酶。检测耐药酶活性并确定一个最佳的耐药酶浓度，加入PBS(磷酸盐缓冲液)174 μL与无菌96孔板中，并加入活性耐药酶1 ul震荡孵育30 min，然后加入25 μL头孢硝噻吩作为底物，于37 ℃恒温培养箱中静止孵育1 h左右，通过观察颜色变化和492 nm出吸光值来判定β-内酰胺酶活性。结果见表2。
表2 共孵育后齐墩果酸对β-内酰胺酶活性抑制效果
Figure 647743DEST_PATH_IMAGE002
结论：齐墩果酸与金黄色葡萄球菌USA300共培养后可降低分泌到上清中的β-内酰胺酶的活性。同时，纯化的β-内酰胺酶与齐墩果酸共同孵育，可降低β-内酰胺酶的活性。
试验例2 最小抑菌浓度试验
于96孔无菌微孔板内按棋盘法进行齐墩果酸、青霉素单用及二者联用抗产β内酰胺酶金葡菌的抑菌活性实验，确定二者单独应用及联合应用MIC值，计算部分抑菌浓度指数(FIC)。 FIC=MIC(青霉素联合)/MIC(青霉素单用)+MIC(齐墩果酸联合)/MIC(齐墩果酸单用)，结果见表3：
表 3 齐墩果酸联用青霉素对表达β内酰胺酶金黄色葡萄球菌的MIC及FIC值
Figure 407888DEST_PATH_IMAGE003
结论：齐墩果酸单独应用不具备抑菌效果，与青霉素联用能够降低青霉素对β-内酰胺酶阳性金葡菌MIC值8倍，FIC数值表明二者具有协同作用。
试验例3 时间-杀菌曲线试验
取表达β内酰胺酶的金葡菌过夜培养物，调整至5×107 CFUs/mL，备用。取无菌试管3组（无抗生素对照组、4 μg/mL青霉素组及32 μg/mL齐墩果酸联用4 μg/mL青霉素组），每组标记为1、3、5、7 和9h，所有试管内均加入1 mL高压灭菌的LB培养基，之后，每管内均加入10 μL调整后的菌液，使每个试管内的菌液浓度为5×105 CFUs/mL。其中4 μg/mL青霉素组及32 μg/mL齐墩果酸联用4 μg/mL青霉素组分别加入对应用量的抗生素及抑制剂，混匀之后立即将无抗生素对照组的菌液进行涂板计数，作为0 h的菌落数。之后，每1、3、5、7及9 h分别取对应试管内的菌液，涂板计数，绘制时间-杀菌曲线（图2）。
结论：齐墩果酸联用青霉素能够在7h后杀死表达β内酰胺酶金葡菌USA300。

Claims (1)

1.齐墩果酸在制备用于恢复青霉素对β-内酰胺酶阳性的金葡菌的体外抗菌活性制剂中的用途。
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CN113750084B (zh) * 2021-09-12 2024-07-26 东北农业大学 酞磺醋胺作为ndm-1抑制剂或抗生素保护剂的新用途
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