CN114392253B - 大蒜辣素在制备金属β-内酰胺酶抑制剂中的用途 - Google Patents
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Abstract
本发明公开了天然提取物大蒜辣素的一种新用途，可作为金属β‑内酰胺酶(NDM)抑制剂，用于碳青霉烯耐药菌感染的治疗。本申请大蒜辣素作为金属β‑内酰胺酶抑制剂，用于防止金属β‑内酰胺酶对β‑内酰胺类抗生素的水解，对该类抗生素起保护作用。在美罗培南等碳青霉烯类抗生素的各类制剂中添加大蒜辣素，以抑制产金属β‑内酰胺酶的细菌对β‑内酰胺类抗生素的水解，从而提高β‑内酰胺类抗生素的稳定性，维持抗生素的疗效，具有良好的制药应用前景。
Description
技术领域
本发明属于医药技术领域，特别是大蒜辣素在制备金属β-内酰胺酶抑制剂中的用途。
背景技术
耐药细菌已成为人类健康的主要威胁和全球面临的医学难题，尤其碳青霉烯类耐药肠杆菌(CRE)在全球范围内的广泛传播给临床治疗带来巨大挑战。美国疾病控制预防中心(CDC)在其最新的18种抗生素耐药威胁报告中，将CRE列为三大紧急公共健康威胁之一。CRE感染导致患者出现发烧、尿路感染、肺炎、败血症等症状，死亡率可高达70％。值得警惕的是，碳青霉烯类抗生素虽未被批准应用于兽医临床，但CRE却已经出现在了动物源性菌株及其所在环境之中，不仅给动物细菌性疾病防控带来困难，而且为动物源性食品安全敲响警钟。
碳青霉烯类耐药肠杆菌(CRE)耐药机制主要为细菌产生碳青霉烯酶，该酶不同于其他β-酰胺酶，能够水解除单环β-内酰胺环类抗生素外的其他所有β-内酰胺类抗生素，其中包括结构更稳定、抗菌谱更广的碳青霉烯类抗生素。β-内酰胺酶根据ambler分类法可以分为A、B、D类，其中A类和D类称为丝氨酸-β-内酰胺酶，属于非金属酶；B类称为金属-β-内酰胺酶(MBLs)，其依赖于金属锌离子而产生催化活性。MBLs主要包括新德里金属β-内酰胺酶(NDM)、维罗纳整合子编码的金属β-内酰胺酶(VIM)和亚胺培南金属β-内酰胺酶(IMP)酶等，其中NDM在世界范围内最为常见。携带NDM的菌株又被称为“超级细菌”，自从2010年被报道以来，引起全球关注。
NDM于2009年首次在印度新德里一名尿路感染患者分离的肺炎克雷伯菌中发现，被命名为NDM-1。随后NDM在40多种细菌中检测到，并在全球七十余个国家广泛传播。目前已发现NDM-1的突变体已多达25种，且突变体的水解活性不断增强。我国在人群、动物和环境中主要流行的是NDM-5和NDM-9。NDM具有典型αβ/βα的三明治结构，其水解机制为位于活性中心的两个Zn(Ⅱ)与水或氢氧根结合形成亲核试剂，攻击β-内酰胺环上的羰基碳导致其环结构被破坏，最终导致该类抗生素失效。目前临床应用的β-内酰胺酶抑制剂克拉维酸、舒巴坦、他唑巴坦等只对丝氨酸-β-内酰胺酶有显著抑制作用，但对金属β-内酰胺酶无抑制活性，目前也尚未有NDM抑制剂上市。产金属β-内酰胺酶菌株的出现，极大地限制了碳青霉烯类抗生素的使用，针对超级细菌的最后一道防线药物储备几乎面临枯竭。筛选特异性酶抑制剂以控制其对抗生素的灭活是抗耐药菌药物研发的主要策略之一，因此寻找各类新型NDM抑制剂显得尤为迫切与重要。
大蒜是药食两用的植物，已有数千年的应用历史。大蒜中含有多种化学成分，主要包括含硫有机化合物和皂苷类，而含硫有机化合物是其重要的活性物质。1944年Cavallito等发现大蒜切碎后的活性成分具有显著抗菌效果。进一步研究表明该类活性成分为大蒜辣素(Allicin)，化学名为二丙烯基硫代亚磺酸酯，结构式如下：
完整的大蒜中不存在大蒜辣素，而是大蒜在被切开或碾碎后，导致大蒜细胞被破坏，液泡中的蒜酶释放，进而与细胞质中的蒜氨酸发生催化裂解反应而产生。我们已有的研究结果证实大蒜辣素对产NDM的肠杆菌科细菌有较好的抗菌活性，且发现其对NDM酶活性有显著的抑制作用。
发明内容
发明目的：针对上述现有技术，本申请提供了大蒜辣素在制备金属β-内酰胺酶抑制剂中的用途。
技术方案：本申请公开了大蒜辣素在制备金属β-内酰胺酶抑制剂中的用途。
本申请公开了大蒜辣素在制备治疗碳青霉烯耐药菌感染的药物中的用途。
本申请公开了一种抗菌药物组合物，其包括碳青霉烯类抗生素和大蒜辣素，大蒜辣素抑制抑制金属β-内酰胺酶的活性，提高碳青霉烯类抗生素的抗菌活性。
其中，所述大蒜辣素的浓度应大于或等于16μg/mL。
所述碳青霉烯类抗生素优选美罗培南，亚胺培南等其它药物也适用。
本申请公开了大蒜辣素联合β-内酰胺类抗生素在制备碳青霉烯耐药菌抑菌剂中的应用。
本申请公开了大蒜辣素联合碳青霉烯类抗生素在制备碳青霉烯耐药菌抑菌剂中的应用。
本申请公开了大蒜辣素联合美罗培南在制备碳青霉烯耐药菌抑菌剂中的应用。所述大蒜辣素可抑制金属β-内酰胺酶，可与碳青霉烯类抗生素联用治疗产酶菌引起的感染，所述大蒜辣素的用量大于或等于16μg/mL。
有益效果：本申请公开了大蒜辣素的一种新用途，即作为金属β-内酰胺酶抑制剂，用于保护碳青霉烯类抗生素免受金属β-内酰胺酶的水解，保持该类抗生素的抗菌作用。在美罗培南等碳青霉烯类抗生素的各类制剂中添加大蒜辣素，以抑制产金属β-内酰胺酶的微生物对碳青霉烯类抗生素的水解，从而提高碳青霉烯类抗生素的稳定性，维持抗生素的疗效，具有良好的制药应用前景。体外联合抑菌实验表明大蒜辣素与美罗培南有联合抑制NDM-5阳性肠杆菌的作用，其联合抑菌指数FICI为0.375。通过构建pET-28a/NDM-5载体，成功表达了具有活性的NDM-5金属β-内酰胺酶，测定了大蒜辣素对NDM-5的半数抑制浓度(IC50)为18.63±1.39μM，测定了大蒜辣素对NDM-5的抑制方式为非竞争性抑制，抑制常数Ki为17.69μM。本发明发现了大蒜辣素对NDM-5的抑制作用，为临床上治疗碳青霉烯类耐药菌引起的感染性疾病提供了新选择。
附图说明
图1是NDM-5水解美罗培南的米氏曲线；
图2是大蒜辣素对NDM-5的抑制曲线；
图3是大蒜辣素对NDM-5抑制反应动力学曲线。
具体实施方式
下面结合具体实施例对本申请技术方案作出详细说明。
其中大蒜辣素可从大蒜中提取，美罗培南购于上海阿拉丁生化科技股份有限公司，NDM-5蛋白为本实验室通过构建pET-28a/NDM-5载体表达而获得。
实施例1微量肉汤稀释棋盘法测定大蒜辣素与美罗培南对NDM阳性菌株的协同作用具体实施方法如下：
将冻存于-80℃的NDM阳性的大肠杆菌接种于LB琼脂平板上，置于37℃恒温培养箱中静置培养，第二日挑取单菌落接种于LB肉汤中，37℃过夜振荡培养。将增菌液按1:100接种于新鲜LB肉汤中，37℃振荡培养复壮2h。再以1:1000接种于新鲜MH肉汤中。
配制大蒜辣素母液：精密称取159.7mg蒜粉(其有效含量为2.56mg)，溶于10mL超纯水中，置于涡旋仪上振荡10min，8000rpm离心5min后用直径0.22μm无菌滤头过滤上清即为浓度为2560μg/mL的母液。用超纯水倍比稀释母液后得到梯度浓度分别为8、16、32、64、128、256μg/mL大蒜辣素的初始工作液。
配制美罗培南母液：精密称取2.56mg美罗培南溶于10mL的超纯水，然后用直径0.22μm无菌滤头过滤药液，得到浓度为2560μg/mL的母液。用灭菌单蒸水倍比稀释母液后得到梯度浓度分别为8、16、32、64、128、256μg/mL初始工作液。
采用棋盘法测定大蒜辣素和美罗培南的联合抑菌作用。于无菌96孔板中7×7棋盘范围内每孔加入100μL细菌培养液。1～6横行每孔依次加入50μL不同梯度的美罗培南溶液，1～6纵列每孔依次加入50μL不同梯度的大蒜辣素溶液。第7行与第7列则加入100μL的超纯水以得到单药MIC。加样完毕后置于37℃恒温培养箱中培养18-20h后判读结果。FICI的计算公式为：(A药联用时的MIC/A药单用时的MIC)+(B药联用时MIC/B药单用时的MIC)。判读标准如下：FICI≤0.5为协同作用；>0.5～1为相加作用；>1～2为无关作用；>2为拮抗作用。实验结果表明大蒜辣素与美罗培南的联合抑菌指数FICI＝0.375，此结果表明二者有协同抑菌作用。
实施例2半数抑制浓度(IC50)的测定
大蒜辣素对NDM-5的抑制活性检测。首先证实了表达的NDM水解底物美罗培南的活性。反应于96孔板中进行。测定方法如下：不同浓度(10、20、40、50、60、80、120、160、180、200、300μM)的美罗培南溶液加入到96孔板的1-11孔中，然后每孔加入100μL制备好的NDM-5(使反应终浓度为20nM)，第12孔为无酶对照。将样品混匀后置于酶标仪中检测OD300的变化，每隔1min检测一次，共检测30min。以OD300值与美罗培南浓度作线性回归，求得OD300值下降量(ΔOD300)所对应的美罗培南浓度下降量(Δs)，以此求得线性范围内反应速度(v＝Δs/t)，根据美罗培南的浓度和其对应的反应速度，采用GraphPad Prism 9拟合米氏反应曲线。图1结果显示本实验室表达纯化的NDM-5具有水解美罗培南的活性，其米氏常数(Km)和最大反应速率(Vmax)分别为50.54μM和0.1035μM/s。
证实NDM酶活性后，将纯化的NDM-5蛋白稀释至20nM，与不同浓度的大蒜辣素溶液在30℃孵育30min，同时设置不加大蒜辣素的对照组。加入100μL终浓度为100μM的美罗培南溶液后立刻放入酶标仪中检测30min内吸光度OD300的变化，由于美罗培南的β-内酰胺环在OD300处有吸光度，因此美罗培南吸光度的变化可以反映其水解程度。计算处理组的反应速率(Vi)，未处理的对照组反应速率(V0)，通过公式1-Vi/V0计算酶活抑制率，采用软件GraphPad Prism 9拟合出大蒜辣素对NDM-5的抑制曲线并求出半数抑制浓度(IC50)。实验重复3次。结果如表1和图2所示：大蒜辣素对NDM-5有明显的抑制作用，其IC50为(18.63±1.39)μM。
表1.大蒜辣素对NDM-5的抑制率测定结果
实施例3大蒜辣素对NDM-5抑制反应动力学及抑制类型分析
将NDM-5蛋白稀释至终浓度20nM，分别与终浓度为0、20、40μM的大蒜辣素溶液在30℃下孵育30min。加入终浓度10、20、40、60、80μM的美罗培南启动反应，立刻通过酶标仪检测吸光度OD300的变化，每隔1min检测一次，共检测30min。求出相应的反应速率，按Lineweaver-Burk双倒数作图法作图，求出抑制常数Ki。抑制双倒数曲线如图3所示：结果显示反应速率Vmax随抑制剂浓度增加而变小，双倒数曲线相交于横轴，为典型的非竞争性抑制动力学曲线，表明大蒜辣素为NDM-5的非竞争性抑制剂，其抑制常数Ki为17.69μM。
Claims (1)
1.大蒜辣素联合美罗培南在制备治疗碳青霉烯耐药菌引起感染的药物中的用途，所述碳青霉烯耐药菌为NDM阳性的大肠杆菌。
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| 大蒜的主要化学成分及其药理作用研究进展;严常开；等;《中国新药杂志》;20040830;第13卷(第8期);688-691页 * |
| 大蒜素联合美罗培南对耐碳青霉烯类抗生素的鲍曼不动杆菌的体外杀菌研究;于亮；等;《中华临床医师杂志(电子版)》;20120315;第6卷(第6期);1452-1457页 * |
| 大蒜辣素药理作用研究进展;易小翠 等;《宜春学院学报》;20190930;第41卷(第9期);19-22页 * |
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