CN116617361A - 百里香酚在制备mcr-1酶抑制剂中的应用 - Google Patents
百里香酚在制备mcr-1酶抑制剂中的应用 Download PDFInfo
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Abstract
本发明涉及百里香酚在制备MCR‑1抑制剂中的应用，并通过棋盘法、时间‑杀菌曲线法、体外酶活性试验、荧光光谱法、细胞保护性试验证明百里香酚能恢复黏菌素对MCR‑1阳性肠杆菌(主要包括大肠杆菌、肺炎克雷伯菌、沙门氏菌)的抗菌活性。进一步通过建立雏鸡肠道感染模型证明百里香酚口服制剂联用黏菌素对能够产生MCR‑1的肠杆菌造成的感染具有良好的治疗效果，同时通过毒性实验证明百里香酚药物浓度在治疗剂量内无明显毒性。因此百里香酚作为MCR‑1抑制剂可以与黏菌素联用，恢复黏菌素应对超级耐药菌的活性,对开发抗MCR‑1阳性肠杆菌感染的新型给药方案具有重要意义。
Description
技术领域
本发明公开了百里香酚的一种新的用途，具体为百里香酚在制备MCR-1酶抑剂中的医用用途，属于医学制药技术领域。
背景技术
产碳青霉烯酶的革兰氏阴性肠杆菌引发了严重的院内感染，特别是ICU重症感染，几乎对临床一线抗生素全部耐药，死亡率极高。细菌多重耐药危机迫使沉寂多年的“老药”黏菌素重新回归临床，通过破坏细菌细胞膜的完整性呈现快速的杀菌效果，对该类耐药菌体外敏感性高达90％。然而随着黏菌素抗性基因MCR-1的发现及在全球迅速传播，应对上述重症感染的“最后一道”防线岌岌可危，MCR-1阳性菌株引发的感染性疾病将无药可用。截至目前，尚无有效的MCR-1抑制剂产品应用于临床。基于此，进一步研发新的安全有效的MCR-1抑制剂并应用于临床具有十分重要的意义。
百里香酚亦称为麝香草酚，是百里香(一种食用和药用植物)中提取的一种十分有价值的活性成分，亦可通过化学合成途径获取，具有多种药理活性，包括抗氧化、抗炎和抗血脂活性，在有效剂量范围内未见毒副作用报道，但目前国内外未见百里香酚在制备MCR-1抑制剂中的相关研究。
发明内容
本发明提供了一种百里香酚在制备MCR-1酶抑制剂中的医用用途，公开了百里香酚能够抑制MCR-1酶的活性，恢复黏菌素对MCR-1阳性革兰氏阴性菌的杀菌活性。
本发明所述的百里香酚，其分子式：C10H14O，分子量：150.22；
本发明所述的黏菌素包括多黏菌素B和黏菌素的硫酸盐，其分子式分别为：C55H96N16O13·H2SO4和C52H100N16O17S；分子量分别为：1287.53和1253.51。
本发明通过棋盘法、时间-杀菌曲线法、体外酶活性试验、荧光光谱法验证百里香酚能够抑制MCR-1酶的活性，恢复黏菌素对MCR-1阳性肠杆菌的抗菌活性，试验所用肠杆菌主要包括MCR-1阳性大肠杆菌、肺炎克雷伯菌和沙门氏菌。通过细胞实验证明百里香酚协同黏菌素能有效保护细胞免受细菌的侵袭感染。进一步通过建立小鸡肠道感染模型证明百里香酚联用黏菌素对携带MCR-1的肠杆菌造成的肠道感染具有良好的治疗效果。
本发明的积极意义在于：提供了百里香酚在制备MCR-1酶抑制剂中的新医用用途，公开了百里香酚能够抑制MCR-1酶的活性，恢复黏菌素对MCR-1肠杆菌的杀菌活性。在进一步的体内治疗试验中，百里香酚联用黏菌素对MCR-1阳性肠杆菌引起的肠道感染等感染疾病具有良好的治疗效果，具有广泛的医用用途。
附图说明
图1为本发明百里香酚联合黏菌素对MCR-1阳性沙门氏菌的时间-杀菌曲线；
图2为本发明百里香酚抑制MCR-1体外酶活性TLC色谱图；
图3为本发明百里香酚与MCR-1蛋白互作荧光光谱图；
图4为本发明百里香酚与黏菌素协同对细胞的保护试验；
图5为本发明百里香酚口服制剂联合黏菌素对感染雏鸡的治疗实验。
具体实施方式
进一步通过下述实施例描述本发明，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
百里香酚作为MCR-1酶抑制剂用于可供临床药用的任何制剂形式。
实施例2
百里香酚作为MCR-1酶抑制剂用于制备治疗感染性疾病的药物。
实施例3
百里香酚作为MCR-1酶抑制剂用于治疗细菌引起的感染性疾病，特别是MCR-1阳性肠杆菌引起的感染。
试验例1
最小抑菌浓度试验
于96孔无菌微孔板内按棋盘法进行百里香酚、黏菌素单用及二者联用抗产MCR-1大肠杆菌、肺炎克雷伯菌和沙门氏菌的抑菌活性实验，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(联用)/MIC(多粘菌素)+MIC(联用)/MIC(百里香酚)，结果见表1：
表1百里香酚联用多粘菌素对MCR-1阳性肠杆菌分离株的MIC及FIC值
结论：百里香酚单独应用对MCR-1阳性菌株不具备抑菌效果，与黏菌素联用能够降低黏菌素对MCR-1阳性大肠杆菌的MIC值16倍，对MCR-1阳性沙门氏菌的MIC值8倍，对MCR-1阳性肺炎克雷伯菌MIC值32倍，FICI数值进一步表明二者具有协同作用。
试验例2
时间-杀菌曲线试验
挑取MCR-1阳性沙门氏菌分离株单菌落于无菌LB液体培养基中过夜培养，取无菌试管加入2mL高压灭菌的LB培养基，加入适量过夜培养的菌液至终浓度为5×105CFUs/mL，按下述处理组(无抗生素对照组、2μg/mL黏菌素组，64μg/mL百里香酚，64μg/mL百里香酚联用2μg/mL黏菌素组)加入黏菌素和百里香酚适量，每组标记为1、3、5、7、9、24h，充分涡旋后，立即将无抗生素对照组的菌液进行涂板计数，作为0h的菌落数。随后按标记的时间点，分别取对应试管内的菌液进行涂板计数，绘制时间-杀菌曲线，结果见图1。
结论：与空白对照组，单独给药组相比，百里香酚联合黏菌素24h内表现了显著的杀菌效果。
试验3
体外酶活性试验
MCR-1蛋白与不同浓度的百里香酚(Thymol)(0，16，32μg/mL)在缓冲液中37℃共孵育过夜，点样于TF254薄层版中，于展开剂(乙酸乙酯/甲醇/水)中展开，观察底物展开行为变化,结果见图2。
结论：从薄层色谱中，明显观测到了NBD-gly-3-PEA加入MCR-1蛋白后，产生了NBD-gly前移的荧光斑点，MCR-1体外活性较好。而柚皮素的加入可明显抑制MCR-1转移磷酸乙醇胺的活性，尤其百里香酚浓度达到32μg/mL时，MCR-1活性最低，产物NBD-gly的荧光斑点最弱。
试验4
荧光光谱检测
将百里香酚(Thymol)(0、2、4、8、16、32μg/mL)与MCR-1蛋白(0.3mg/mL)混合，室温孵育10min，用荧光光谱仪在200-600nm范围内扫描，激发波长为280nm，记录MCR-1蛋白荧光光谱行为的变化，结果见图3。
结论：百里香酚随着浓度的增加，明显降低了MCR-1蛋白的荧光强度，说明药物通过猝灭蛋白内源性荧光的机制与蛋白发生了相互作用。
试验5
细胞保护试验
收集百里香酚(32μg/mL)、黏菌素(1μg/mL)及联合处理后的菌悬液，PBS洗涤3次，用DMEM重悬至4.0×106CFUs/mL。前一天将HeLa细胞按2×104个/孔接种，细胞/细菌悬液孵育6小时。DMEM和0.2％ Triton X-100分别为阴性和阳性对照。按照LIVE/DEADTM活力/细胞毒性试剂盒的说明进行后续试验操作，荧光显微镜显示荧光图像，结果见图4。
结论：与模型组、单独药物治疗组相比，联合用药后，细胞的存活率明显增强，荧光强度与阴性对照组相当，说明百里香酚可有效增强黏菌素的杀菌作用，保护细胞免受细菌的侵袭。
试验6
雏鸡肠道感染体内治疗试验
1日龄雏鸡(约35g)，灌胃MCR-1阳性沙门氏菌菌悬液(1×109CFUs/只)，建立雏鸡肠道感染模型。
保护率试验
雏鸡接种MCR-1阳性沙门氏菌后，立即灌胃100μL百里香酚口服溶液(以百里香酚计20mg/kg)，黏菌素10mg/kg，以及二者联合给药，每8h给药一次，一共给药三次。阳性对照组给与100μL的水溶液，不给予任何药物进行治疗；另单独设立未感染雏鸡的空白溶剂对照组，给予100μL的口服液空白溶剂，评价所用辅料的安全性。随后分别记录雏鸡感染MCR-1阳性沙门氏菌7天后的死亡率(每隔12小时记录一次)，结果见附图5。
结论：经百里香酚口服液联用黏菌素治疗后，显著降低雏鸡肠道感染的的死亡率，而百里香酚或黏菌素单独治疗组不具有显著的保护效果。
Claims (4)
1.百里香酚与黏菌素联合在制备治疗MCR-1阳性的肠杆菌引起的感染性疾病药物中的用途，其特征在于百里香酚可显著抑制MCR-1蛋白体外转移磷酸乙醇胺的活性。
2.根据权利要求1，百里香酚通过与MCR-1蛋白结合，影响其蛋白结构进而影响其体外活性。
3.根据权利要求1或2，百里香酚作为MCR-1抑制剂，能够恢复黏菌素对MCR-1阳性肠杆菌的杀菌活性。
4.根据权力要求1，百里香酚与黏菌素联合应用的制剂形式可以为胃肠道给药、非胃肠道给药的任何制剂形式。
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