CN115778939B - Dt-10在制备抗真菌药物和日用品中的应用 - Google Patents
Dt-10在制备抗真菌药物和日用品中的应用 Download PDFInfo
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Abstract
本发明公开了DT‑10在制备抗真菌药物和日用品中的应用。DT‑10对氟康唑耐药和敏感白色念珠菌都具有抑制活性，最小抑菌浓度(MIC)为3.125‑12.5μg/mL。作用机理研究表明，DT‑10能够结合细胞膜磷脂，进而增强白色念珠菌SC5314细胞膜渗透性，同时降低细胞膜流动性。该作用机制使DT‑10能够迅速杀菌，在16×MIC浓度下，处理20min时能够将活性菌量控制在检测线以下，故不易产生耐药性。另外，DT‑10对新生隐球菌和烟曲霉也具有较强抑制活性。鉴于DT‑10低细胞毒性和强抗真菌活性，可用于制备预防或治疗真菌感染的药物，也可用于制备抗真菌日用品。
Description
技术领域：
本发明属于天然产物结构优化物领域，具体涉及天然产物木豆素C结构优化物DT-10在制备抗真菌药物或抗真菌日用品中的应用。
背景技术：
近年来，免疫力低下人群数量不断增加，这也导致侵袭性真菌感染越发严重。真菌感染行动基金组织(GAFFI)统计数据显示，每年有超过3亿人患上严重的真菌感染疾病，其中大约有200万人会因为真菌感染而死亡。其中，念珠菌属、隐球菌属和曲霉属是三大主要致病菌属，它们能够侵袭免疫力低下人群，例如器官移植、肿瘤化疗、人类免疫缺陷病毒(HIV)感染患者，成为导致患者死亡的真正原因。
目前，临床上用于治疗真菌感染的药物种类较少，主要有三大类，包括唑类、多烯类和棘白菌素类。这三大类抗真菌药物不仅抗菌谱窄，而且副作用强，很难满足临床真菌治疗需求。更糟糕的是，在本就缺乏抗真菌药物的情况下，真菌耐药问题日益突出，给真菌治疗带来难度。因此，抗真菌药物的研发迫在眉睫。
天然产物是新药开发的重要来源。DT-10是天然产物木豆素C(Longistylin C，LGC)异戊烯基环化产物，其结构优化物DT-10的结构式如下所示：
公开于专利CN102276433A中。但是本专利之前未发现任何与DT-10抗真菌的研究报道。
发明内容：
本发明的目的是提供化合物DT-10在制备抗真菌药物或抗真菌日用品中的应用。
本发明通过实验表明，DT-10不仅能够抑制氟康唑敏感白色念珠菌SC5314和临床分离菌株11D、11E以及11F，而且对氟康唑耐药白色念珠菌ATCC10231、CMCC(F)98001、BNCC186382、BNCC337321和临床分离菌株CA632、CWQ1、CWQ2也具有较强的抑制活性，其最小抑菌浓度MIC为3.125-12.5μg/mL。作用机理研究表明，DT-10通过结合细胞膜磷脂，增加膜渗透性，降低膜流动性。同时，在16×MIC浓度下，DT-10在20min内能够将活性菌量降低至检测线以下，故不易产生耐药性。另外，DT-10对氟康唑耐药新生隐球菌BNCC339771的MIC＝3.125μg/mL，对烟曲霉BNCC 340016的MIC＝12.5μg/mL。
因此，本发明的第一个目的是提供化合物DT-10在制备抗真菌药物中的应用；
所述的化合物DT-10的结构式如下所示：
本发明的第二个目的是提供一种抗真菌感染的药物，其含有化合物DT-10作为活性成分。
优选，所述的药物以化合物DT-10为活性成分和其他药物赋形剂或载体制成。
优选，所述的真菌包括但不限于白色念珠菌、新生隐球菌和烟曲霉感染。
优选，所述的药物可以是颗粒剂、胶囊剂、片剂、散剂、滴丸剂、缓释剂或注射剂中的任意一种或多种。
本发明第三个目的是提供一种防止真菌感染或污染的日用品，其含有化合物DT-10作为活性成分。
优选，所述的真菌包括但不限于白色念珠菌、新生隐球菌和烟曲霉感染。
优选，所述的日用品包括但不限于包括但不限于衣物消毒液、洗手消毒液、化妆品、洗发水、阴道洗液或漱口水。
化合物DT-10是天然产物木豆素C结构优化物，具有比LGC更低的细胞毒性(ShanYan，et al.Chinese Journal of Natural Medicines.2015)。进一步研究发现，DT-10对白色念珠菌、新生隐球菌和烟曲霉具有较强抑制活性，可用于制备抗真菌的药物或抗真菌日用品。
附图说明：
图1是DT-10结合细胞膜磷脂活性图，注：PG，phosphatidylglycerol，磷脂酰甘油；PE，phosphatidylethanolamine，磷脂酰乙醇胺；CL，cardiolipin，心磷脂。
图2是DT-10增大细胞膜渗透性图。
图3是DT-10降低细胞膜流动性图。
图4是DT-10和LGC溶血活性测定图，注：T，代表Triton-X-100。
图5是DT-10杀菌时间图。
具体实施方式：
下面通过实施例进一步描述本发明，但本发明并不局限于这些实施方式，任何在本实施例基本精神层面的改进或代替，仍属于本发明权利要求所保护的范围。
实施例1：DT-10抑菌活性测定
1、仪器和设备
96孔细胞培养板(康宁)、生物安全柜、恒温振荡器、恒温培养箱、万分之一天平。
2、菌株
白色念珠菌(Candida albicans)标准菌株SC5314由安徽中医药大学邵菁教授惠赠，BNCC菌株购买于北纳生物-河南省工业微生物菌种工程技术研究中心，白色念珠菌临床分离菌株CA632、CWQ1、CWQ2、11D、11E、11F由山东大学常文强副教授惠赠，其他菌种为实验室保存菌株。
3、试剂配制
(1)DT-10母液和工作液配制：称取化合物DT-10粉末10mg，溶解于1mL DMSO(麦克林公司)，即得到10mg/mL DMSO母液，-20℃保存。取母液40μL，加入80μL DMSO，即得到4mg/mL工作液。
(2)抗生素母液配制：氟康唑(Fluconazole)、两性霉素B(Amphotericin B)和制霉菌素(Nystatin)母液和工作液配制方法同DT-10。
(3)RPMI-1640培养基：称取RPMI-1640粉末10.4g和MOPS粉末34.53g，加入1L灭菌去离子水，完全溶解后用NaOH调节pH至7.0。用0.22μm的无菌滤膜过滤除菌，4℃保存。
(4)YPD固体培养基：称取蛋白胨20g、葡萄糖20g、酵母提取物10g、琼脂粉20g，加入1L去离子水，高温高压灭菌。
(5)YPD液体培养基：称取蛋白胨20g、葡萄糖20g、酵母提取物10g，加入1L去离子水，高温高压灭菌。
(6)PDA固体培养基：马铃薯200g切成小块，加入800mL去离子水煮沸20min，纱布过滤，称取葡萄糖20g和琼脂15～20g，加入到滤液中，去离子水定容至1L，高温高压灭菌。
4、实验方法
(1)白色念珠菌和新生隐球菌测试方法：
将白色念珠菌和新生隐球菌接种于YPD固体培养基，30℃，培养2d。挑取菌落于YPD液体培养基，30℃，200rpm，培养至指数生长后期。取该菌液10μL再次接种于1mL YPD液体培养基，30℃，200rpm，培养至指数生长期。血细胞计数板计数，用RPMI-1640培养基调整菌液浓度为1×104CFU/mL。96孔细胞培养板第一行加入195μL菌液和5μL待测化合物工作液，其余孔各加入100μL菌液，取第一行孔中的液体100μL于第二行孔中，依次二倍梯度稀释。将96孔细胞培养板置于37℃恒温培养箱中，白色念珠菌24h后观察最小抑菌浓度MIC值，新生隐球菌48h后观察MIC值。肉眼观察每孔菌生长情况，以不生长菌落孔对应的浓度为最小抑菌浓度。
(2)烟曲霉测试方法：
将烟曲霉接种到PDA培养基，置于30℃培养5d，用PRMI-1640培养基冲洗平板，获得孢子悬浮液，血细胞计数板计数，将孢子悬浮液浓度调整为1×105CFU/mL。化合物对烟曲霉的抑制活性测定方法同白色念珠菌。
5、实验结果
表1
如表1所示，DT-10对新生隐球菌、烟曲霉和白色念珠菌都具有较强的杀菌活性，其MIC值为3.125-12.5μg/mL。其中，新生隐球菌BNCC 339771、烟曲霉BNCC 340016、白色念珠菌(ATCC10231、CMCC(F)98001、BNCC 186382和BNCC 337321)以及白色念珠菌临床分离菌株(CA632、CWQ1和CWQ2)皆为氟康唑(FCZ)耐药菌株，DT-10对这些菌株都有抑制活性。
实施例2：DT-10结合细胞膜磷脂
1、试剂
细胞膜磷脂成分磷脂酰甘油(Sigma-Aldrich,841188P)和磷脂酰乙醇胺(Sigma-Aldrich,840027P)溶解于甲醇，心磷脂(Sigma-Aldrich，C1649)为5mg/mL乙醇溶液，麦角甾醇(ALFA，AB0565)溶解于乙醇。
2、实验方法
使用棋盘肉汤稀释法探究不同细胞膜成分对DT-10和两性霉素B抗菌活性的影响。在96孔板中加入50μL RPMI-1640培养基，第一行前10个孔中加入相应浓度的DT-10或两性霉素B，第一行后两个孔中加入待研究细胞成分，依次向下倍比稀释至第八行。在第一列各孔中加入对应浓度待研究细胞成分，依次从左到右倍比稀释至第8列，室温孵育3h。随后，第10和12列加入50μL RPMI-1640培养基，其他列全部加入50μL稀释好的白色念珠菌SC5314菌液。96孔板置于37℃培养18h后测定OD600吸光度。
3、实验结果
两性霉素B为多烯类抗真菌药物，它能够结合真菌细胞膜上的麦角甾醇，进而改变真菌细胞膜的通透性。如图1所示，体系中加入麦角甾醇能够明显降低两性霉素B抑菌活性，而细胞膜磷脂(心磷脂、磷脂酰乙醇胺和磷脂酰甘油)对其活性没有显著影响。与两性霉素B相反，细胞膜磷脂能够明显降低DT-10抑菌活性，而麦角甾醇对其影响不明显。
实施例3：DT-10增大细胞膜渗透性
1、实验方法
稀释好的白色念珠菌SC5314菌液分别加入终浓度1/2×MIC的DT-10或氟康唑，阴性对照加入等体积DMSO。同时加入终浓度为80μg/mL的DAPI和10μM的SYTOX-Green，37℃摇菌5、15、30、60min取样，PBS缓冲液清洗后分别测定激发波长364nm/发射波长454nm和激发波长488nm/发射波长538nm处的荧光强度。
2、实验结果
氟康唑(FCZ)能够特异性干扰真菌细胞色素P450，进而抑制细胞膜上麦角固醇的合成并杀死真菌细胞。如图2所示，与阴性对照DMSO组相比，随着氟康唑处理时间延长，细胞中荧光强度增加不明显，而DT-10处理5min便可导致荧光强度显著增加，说明DT-10能够破坏细胞膜，增加膜渗透性。
实施例4：DT-10降低细胞膜流动性
1、实验方法
稀释好的白色念珠菌SC5314菌液加入终浓度2μmol/L的Laurdan染液(DMSO溶解)，37℃摇菌10min后菌液加入终浓度1/2×、1×、2×MIC的DT-10，加入终浓度50mM的苯甲醇(Benzyl alcohol)为阳性对照，加入等体积DMSO为阴性对照。处理40min后PBS缓冲液清洗，酶标仪分别测定激发波长350nm/发射波长435nm和激发波长350nm/发射波长500nm处的荧光强度，计算广义偏振度GP值，GP＝(435nm荧光强度-500nm荧光强度)/(435nm荧光强度+500nm荧光强度)。GP值为-1和+1分别代表最高和最低膜流动性。
2、实验结果
如图3所示，DT-10能够明显降低细胞膜流动性，而且随着DT-10浓度增加，流动性降低越明显。
实施例5：DT-10和LGC溶血活性测定
1、实验方法
无菌脱纤维羊血PBS清洗三次后加入二倍梯度稀释的DT-10或LGC，37℃孵育1h，2500rpm离心，吸取上清测定543nm吸光度。等体积DMSO为阴性对照，0.2％Triton-X-100为阳性对照。每个样品3个技术重复，实验进行3次生物学重复。
2、实验结果：
如图4所示，在浓度为256μg/mL(白色念珠菌MIC值的40倍)时，DT-10溶血活性可忽略，而LGC却表现出明显增高的溶血活性。
实施例6：杀菌时间测定
1、实验方法
调整好的白色念珠菌SC5314菌液分别加入终浓度为8×和16×MIC的DT-10、制霉菌素(NST)、两性霉素B(AMP)、氟康唑(FCZ)和5-氟胞嘧啶(5-F)，加入等体积的DMSO为阴性对照。37℃摇菌处理20min后分别10倍梯度稀释进行菌落平板计数。
2、实验结果
两性霉素B和制霉菌素能够结合细胞膜上的麦角甾醇，增强细胞膜渗透性，进而将细胞杀死；氟康唑通过干扰真菌细胞色素P450抑制麦角甾醇的合成,从而起到灭杀真菌细胞的作用；5-氟胞嘧啶通过干扰DNA和蛋白质合成来发挥抗真菌作用。通过作用方式可知，两性霉素B和制霉菌素能够直接杀菌，因此在该两种药物处理下细胞快速死亡。而氟康唑和5-氟胞嘧啶通过抑制细胞成分合成方式导致细胞死亡，因此在这两种药物处理下细胞死亡速度相对较慢。如图5所示，DT-10表现出比制霉菌素和两性霉素B更快的杀菌速度，在16×MIC处理浓度下，20min内DT-10能够将活性菌量降低至检测线以下。
Claims (3)
1.化合物DT-10在制备抗真菌药物中的应用，所述的真菌为白色念珠菌、新生隐球菌或烟曲霉；
所述的化合物DT-10的结构式如下所示：
。
2.根据权利要求1所述的应用，其特征在于，所述的药物以化合物DT-10为活性成分和其他药物赋形剂或载体制成。
3.根据权利要求1所述的应用，其特征在于，所述的药物是颗粒剂、胶囊剂、片剂、散剂、滴丸剂、缓释剂或注射剂中的任意一种。
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