CN106117232B - 一种细菌群体感应抑制剂及其应用 - Google Patents
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Abstract
本发明涉及一种细菌群体感应抑制剂的发现及其应用。本发明从无花果叶中分离鉴定得到两种香豆素类化合物即补骨脂素和佛手柑内酯。经实验首次证实,该类化合物具有抑制紫色色杆菌和铜绿假单胞菌群体感应系统的活性,不会引起致病菌产生生存压力,进而产生耐药性,是一种细菌群体感应抑制化合物。本发明具有原料易得、制备方法易行、无耐药性、无毒害作用的优点,因此该香豆素类化合物有开发成细菌群体感应抑制先导化合物的潜力。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种细菌群体感应抑制剂及其应用。
背景技术
传统上认为细菌是一种非社会性的生物个体。近年来的研究发现,细菌可以通过分泌、识别、感应一种或多种可溶性的化学信号分子,即自诱导剂(Autoinducer,AI)来实现相互间信息的传递和交流,被称为细菌群体感应(Quorum sensing,QS)现象。细菌QS系统参与许多生物学功能的调控,包括产生毒素、形成生物膜、生产孢子、产生抗生素、生物发光等。近年来,细菌QS系统成为药物研究者关注的焦点。以细菌QS系统为靶标筛选的抗菌药物,与传统抗生素的作用机制完全不同,它不杀死细菌或抑制单个细菌的生长,只抑制细菌QS调控的致病行为,不易诱导耐药突变。因此,利用QS作为控制感染的靶标筛选高效的QS系统干扰药物有望成为解决耐药细菌感染的有效途径之一。
香豆素类化合物是多产于无花果等中草药的一种活性苯丙素类化合物。采用有机溶剂萃取法获得的无花果叶提取物具有显著地抑制紫色杆菌QS系统调控的紫色素的生成。对其活性成分详细研究发现香豆素类化合物补骨脂素和佛手柑内酯化合物能够强烈抑制紫色色杆菌和铜绿假单胞菌QS系统的活性,目前尚未见该类化合物具有抑制紫色色杆菌和铜绿假单胞菌QS活性的报道,因此市场上也尚未见到与此相关的药物。
发明内容
本发明旨在提供一类新型的具有抑制紫色色杆菌和铜绿假单胞菌QS系统活性的化合物,其结构式为:
其结构特征是:此结构为香豆素类化合物,其中,R1、R2为氢、羟基、氧甲基、氨基或酰氧基。
本发明优选的结构中R1=R2=H或R1=H,R2=OCH3。
本发明所涉及的化合物可通过中草药植物提取、微生物发酵培养来获取含有香豆素类化合物的粗浸膏,然后从粗浸膏中采用硅胶柱层析、Sephadex LH20凝胶柱层析、制备HPLC等方法分离纯化得到,或者是通过人工合成的方法得到。
本发明中细菌QS抑制剂补骨脂素和佛手柑内酯化合物能显著地降低由QS系统介导的紫色素合成,可用于防治紫色色杆菌所引起的疾病感染。
本发明中细菌QS抑制剂补骨脂素和佛手柑内酯化合物能显著地降低由铜绿假单胞菌QS系统介导的群集运动、绿脓菌素的合成及生物被膜的形成,可用于防治铜绿假单胞菌所引起的疾病感染。
本发明的目的在于提供一种细菌QS抑制剂。补骨脂素和佛手柑内酯在多种植物中广泛存在,并且含量较高,此外还可以通过人工合成的方式获得。因此,本发明具有制备原料易获得、制备方法简单、无耐药性、无毒害作用等优点。
附图说明
图1为不同浓度补骨脂素对紫色色杆菌菌体及紫色素产量的影响;
图2为不同浓度佛手柑内酯对紫色色杆菌菌体及紫色素产量的影响;
图3为40μg/ml补骨脂素和160μg/ml佛手柑内酯对铜绿假单胞菌群集运动的影响;
图4为不同浓度补骨脂素对铜绿假单胞菌绿脓菌素产量的影响;
图5为不同浓度佛手柑内酯对铜绿假单胞菌绿脓菌素产量的影响;
图6为不同浓度补骨脂素对铜绿假单胞菌生物被膜产量的影响;
图7为不同浓度佛手柑内酯对铜绿假单胞菌生物被膜产量的影响。
具体实施方式
实施例1:中药无花果叶中补骨脂素和佛手柑内酯的纯化及结构鉴定
甲醇粗浸膏制备方法:称取1g原材料加入50ml甲醇溶液萃取,超声30分钟,再置于摇床48小时震荡提取。过滤得滤液与滤渣。渣体再用20ml甲醇溶剂依照上述方法提取两遍,共得3次滤液,合并,真空浓缩得粗浸膏。
化合物的分离纯化:上述粗浸膏用适量硅胶H拌样后,采用减压硅胶柱层析柱进行分离,使用不同比例的洗脱剂石油醚、石油醚:二氯甲烷(1:5)、石油醚:二氯甲烷(1:1)、二氯甲烷、二氯甲烷:甲醇(100:1)、二氯甲烷:甲醇(50:1)、二氯甲烷:甲醇(10:1)、二氯甲烷:甲醇(1:1)、甲醇分别冲洗硅胶柱,每个洗脱组分以3个柱体积进行洗脱。收集石油醚:二氯甲烷(1:1)与二氯甲烷洗脱的组分,减压浓缩后采用Sephadex LH20凝胶柱层析进行二次分离,甲醇为洗脱液。以紫色杆菌为指示菌株,检测各收集组分的群体感应抑制活性,合并活性流份,减压浓缩后用高效液相制备系统以甲醇:水=60:40比例进行纯化,得到单体化合物补骨脂素和佛手柑内酯。
化合物的结构鉴定:综合利用质谱和核磁共振技术对化合物进行结构鉴定。补骨脂素阳离子质谱在187处给出[M+H]+峰,209处给出[M+Na]+峰,提示化合物的分子量为186。佛手柑内酯阳离子质谱在217处给出[M+H]+峰,239处给出[M+Na]+峰,提示化合物的分子量为216。结合一维核磁数据,推测化合物的分子式分别为C11H6O3、C12H8O4。查阅文献,比对核磁数据发现两者的1H-NMR、13C-NMR及DEPT谱与文献一致,因此确定化合物为补骨脂素和佛手柑内酯。
两个化合物的核磁数据如表1所示:
表1.化合物补骨脂素和佛手柑内酯的1H-NMR和13C-NMR数据(CDCl3)
(600MHz and 150MHz,TMS,δppm)
实施例2:化合物补骨脂素与佛手柑内酯抑制紫色色杆菌QS活性的分析
紫色杆菌筛选模型及其方法:过夜培养的紫色色杆菌CV026菌液用LB液体培养基稀释至OD600nm=0.1,每瓶10ml;分别加入20μl不同浓度的化合物补骨脂素和佛手柑内酯,50μl信号分子和20μl的卡那霉素,放入摇床28℃、160rmp培养18h;DMSO溶剂做空白对照。培养结束后,6000rmp离心10min,将上清液吸出,用1ml DMSO重新溶解,再离心(6000rmp,10min),将上清液分别倒入已标记好的离心管中,分别依次取200μl上清液加入到96孔微板中,用酶标仪测585nm的吸光度A585,以表征紫色色杆菌紫色素的产量;第二次离心获得的沉淀,加入1ml的无菌水重新悬浮菌体,依次取200μl菌体悬浮液加入到96孔微孔板中,用酶标仪测600nm的吸光度OD600,以表征菌体生长密度的变化。
实验结果:补骨脂素在0-40μg/ml浓度范围内对紫色色杆菌CV026的生长不产生影响但是能够显著的降低紫色素的产量,且随着浓度的增加,降低紫色素产量的作用越明显,呈浓度依赖性,其中在加入40μg/ml时抑制率达到70%(图1)。当浓度大于40μg/ml时,对紫色色杆菌的生长呈现明显的抑制作用,因而起到杀菌作用。佛手柑内酯在0-160μg/ml浓度范围内对紫色杆菌CV026的生长不产生影响并且能够降低紫色素的产量,且随着浓度的增加,降低紫色素产量的作用越明显,但是不如补骨脂素作用明显,在加入160μg/ml时抑制率仅达到40%(图2)。
实施例3:化合物补骨脂素与佛手柑内酯抑制铜绿假单胞菌swarming活性的分析
群集运动(swarming)筛选方法:取14.8ml热融化的swarming软琼脂培养基(葡萄糖10g;琼脂粉5g;蛋白胨5g;酵母浸粉2g;溶于1L去离子水,121℃,灭菌20min),待冷却至30℃左右,分别向其中加入200μl补骨脂素和佛手柑内酯化合物,终浓度分别为40μg/ml和160μg/ml。倒入平板中,待凝固后,用打孔器打孔,将2μl过夜培养的铜绿假单胞菌PA01(OD600nm=0.1)菌液加入孔中,37℃恒温培养24h,观察现象。
实验结果:40μg/ml补骨脂素和160μg/ml佛手柑内酯能够显著的抑制铜绿假单胞菌PA01的群集运动(图3),说明补骨脂素和佛手柑内酯可作为铜绿假单胞菌QS系统的抑制因子。
实施例4:化合物补骨脂素与佛手柑内酯抑制铜绿假单胞菌绿脓菌素合成的分析
绿脓菌素筛选方法:过夜培养的铜绿假单胞菌稀释至OD600nm=0.1,分别加入不同浓度化合物,DMSO作为对照,37℃震荡培养24小时。培养结束后,分别取2ml上清液加入1ml氯仿震荡萃取,留下层有机相,再加入0.3ml盐酸溶液(0.2M)震荡萃取,取上层水相200μl加入到96孔微孔板中,用酶标仪测520nm的吸光度,以表征绿脓菌素的含量。
实验结果:补骨脂素和佛手柑内酯均能抑制铜绿假单胞菌绿脓菌素的合成,40μg/ml时补骨脂素的抑制率为55%(图4),160μg/ml时佛手柑内酯的抑制率为48%(图5)。
实施例5:化合物补骨脂素与佛手柑内酯抑制铜绿假单胞菌生物被膜形成的分析
生物被膜测试方法:过夜培养的铜绿假单胞菌PA01用LB培养基稀释至OD600nm=0.1,分别加入不同浓度的补骨脂素和佛手柑内酯,DMSO做空白对照,37℃静置培养24小时。PBS缓冲液洗去浮游的菌体,用1%结晶紫染液染色10分钟。然后再用PBS溶液洗去多余的染液,空气中干燥30分钟,再加入30%乙酸溶液,590nm处测其吸光度,以表征生物被膜的形成。
实验结果:补骨脂素和佛手柑内酯均能抑制铜绿假单胞菌生物被膜的形成,补骨脂素的抑制效果较明显,在40μg/ml时其抑制率为58%(图6),而佛手柑内酯在160μg/ml时的抑制率仅为20%(图7)。
Claims (2)
1.化合物补骨脂素在5-40μg/ml浓度范围内制备紫色色杆菌群体感应抑制剂的应用。
2.化合物佛手柑内酯在20-160μg/ml浓度范围内制备紫色色杆菌群体感应抑制剂的应用。
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