CN105663244A - 一种具有细菌群体感应淬灭活性的无花果叶提取物及用途 - Google Patents
一种具有细菌群体感应淬灭活性的无花果叶提取物及用途 Download PDFInfo
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Abstract
本发明提供了一种具有细菌群体感应淬灭活性的无花果叶提取物及用途。具体涉及无花果叶粗提物的制备、细菌群体感应淬灭活性的筛选及在细菌群体感应抑制剂制备方面的应用。本发明首次报道无花果叶的提取物具有淬灭细菌群体感应的活性。由不同极性的有机溶剂提取制备的无花果叶粗提物对紫色杆菌和铜绿假单胞菌群体感应系统具有抑制作用，其中二氯甲烷提取物对群体感应系统的淬灭活性最强，该粗提物能够显著降低群体感应介导的紫色杆菌紫色素的产量以及铜绿假单胞菌的群集运动，减弱致病菌的耐药性，可用于制备新型细菌群体感应抑制剂，因此在细菌感染性疾病防治方面具有广阔的应用前景。
Description
技术领域
本发明属于植物有效成分开发与利用领域，具体涉及一种具有细菌群体感应淬灭活性的无花果叶提取物的制备、活性筛选及在细菌群体感应抑制剂制备方面的用途。
背景技术
随着抗生素大量的使用和滥用，临床上出现了广泛的耐药菌株，使得细菌耐药性成为越来越严重的世界性难题。传统的抗菌药物由于产生生存压力很容易使细菌产生耐药性，因此，现有的抗菌药物已不足以解决细菌性感染疾病问题。目前研究抗菌的另一种策略是抗毒力策略，即在不抑制细菌生长的情况下，直接抑制细菌相关毒力因子的表达，降低或者失去对宿主的伤害能力。这种策略不杀死或者抑制细菌的生长，因此对细菌没有生存压力，理论上不会产生耐药性。近些年来研究表明细菌毒力因子的表达很大程度上都是由群体感应系统调控的，因此通过抑制细菌群体感应，能够降低细菌毒力因子的产生，抑制细菌群体感应调控的致病行为，不易诱导耐药突变。
群体感应（QuorumSensing，QS）是广泛存在于细菌间的一种信息交流现象。细菌可以通过生产、识别、感应一种或多种可溶性的化学信号分子，即自诱导物质（AutoInducer,AI）来实现相互间信息的传递和交流。20世纪60年代在一种海洋费氏弧菌（Vibriofischeri）中发现了群体感应现象，但是直到1994年细菌群体感应这一概念才被提出（J.Bacteriol.,1994,176:269-275）。当一个特定环境中细菌的数量急剧增加时，其所分泌的信号分子的浓度也会相应的升高，当达到一定的阈值时细菌与细菌之间就会通过这种信号分子来调控基因的表达，比如生物发光、生物被膜形成、抗生素产生、毒素产生、生产孢子等。
细菌群体感应信号分子主要分为四类：第一类是革兰氏阴性菌由酰基高丝氨酸内酯（Acyl-homoserineLactones，AHLs）介导的群体感应系统，其信号分子为AHL；第二类是革兰氏阳性菌由自诱导肽（AutoinducingPeptide，AIP）介导的群体感应系统，其信号分子为氨基酸或者短肽；第三类是在革兰氏阴性菌和阳性菌中都存在的一类细菌群体感应系统，其信号分子为呋喃硼酸二酯（AI-2）；第四是其它类信号分子，包括一些喹诺酮类化合物，如Holden等发现的假单胞菌中的次级代谢产物2-庚基-3-羟基-4-喹诺酮(thePseudomonasquinolonesignal,PQS)，可以引起调控假单胞菌的毒性因子基因的表达；研究人员还发现二烃基间苯二酚类化合物能够代替AHLs来介导PauR参与的QS系统。另外，某些酯类、二酮呱嗪类化合物和脂肪酸等都可被用作QS系统信号分子。
无花果(Ficuscaricalinn)，隶属于桑科榕属，一种落叶小乔木开花植物，主要生长在热带和温带。中医上主要用于健胃清肠，消肿解毒和食欲不振等。由于无花果的栽培历史悠久和重要的药用价值，植物化学家对无花果的叶、根、果实和分泌的乳液都进行了研究，无花果叶中的主要成分为黄酮类、香豆素类、甾醇类、萜类，具有细胞毒、光敏剂、抗肿瘤、抗原虫药、化学防御、抗炎等作用；根茎中的主要成分是萜类和甾醇类，具有降血脂、遮光剂等作用；果实中的主要成分是花色苷类物质，具有抗氧化和自由基清除作用；乳液中的主要成分是萜类物质，具有降血脂的作用。除此以外，文献中还报道了抗真菌活性、抗血管生成、退热、抑菌活性、免疫调节、保肝药、降胆固醇、止痉挛、驱虫等作用。但是，至今还没有文献报道其提取物的群体感应抑制活性。本发明首次报道了无花果叶提取物具有细菌群体感应抑制的活性。
发明内容
本发明提供了一种具有细菌群体感应淬灭活性的无花果叶提取物及用途。
本发明提供了一种具有细菌群体感应淬灭活性的无花果叶提取物在细菌群体感应抑制剂制备方面的用途。
本发明提供了一种具有细菌群体感应淬灭活性的无花果叶提取物的制备方法按照以下步骤进行：
室温下自然晾干的无花果叶，粉碎成沫，称取1g原材料置于三角摇瓶，加入20ml有机溶剂，超声30min，再置于28℃下摇床48h震荡提取，然后过滤得滤液和滤渣；滤渣再用同比列有机溶剂依照上述方法提取两遍，共得3次滤液，合并，真空浓缩，反相硅胶C18预柱处理后获得无花果叶提取物粗浸膏。
本发明包含以下有益效果：
本发明得到的无花果叶提取物在有效浓度范围内能显著地抑制紫色杆菌和铜绿假单胞菌QS系统的作用。其中二氯甲烷提取物的淬灭活性最强。在0~0.1mg/ml范围内，该二氯甲烷提取物不抑制紫色杆菌CV026的菌体生长，但能显著地降低由QS系统介导的紫色杆菌紫色素的产生，且随着浓度的增加，降低紫色杆菌紫色素产量的作用越明显，呈浓度依赖性，在加入0.1mg/ml的提取物时抑制率达到80%；在加入0.8mg/ml二氯甲烷提取物时能显著地减弱铜绿假单胞菌的群集运动，可用于防治紫色杆菌和铜绿假单胞菌所引起的疾病感染研究。
附图说明
图1为无花果叶提取物的QS淬灭活性筛选图。
图2为不同含量的无花果叶提取物对紫色杆菌CV026菌体生长影响的测定图。
图3为不同含量的无花果叶提取物对紫色杆菌紫色素产量影响的测定图。
图4为无花果叶提取物对铜绿假单胞菌PA01群集运动（swarming）影响的测定图。
具体实施方式
实施方式一：一种具有细菌群体感应淬灭活性的无花果叶提取物的制备方法
室温下自然晾干的无花果叶，粉碎成沫，称取1g原材料置于三角摇瓶，加入20ml二氯甲烷溶剂，超声30min，再置于28℃下摇床48h震荡提取。过滤得滤液与滤渣；滤渣再用20ml二氯甲烷溶剂依照上述方法提取两遍，共得3次滤液，合并，真空浓缩，反相硅胶C18预柱处理后获得无花果叶提取物粗浸膏。
实施方式二：本实施方式二与实施方式一不同的是：采用的提取试剂为甲醇，其它步骤与实施方式一相同。
实施方式三：本实施方式三与实施方式一或二不同的是：采用的提取试剂为正己烷，其它步骤与实施方式一相同。
实施方式四：本实施方式四与实施方式一至三不同的是：采用的提取试剂为50%丙酮（v/v），其它步骤与实施方式一相同。
实施方式五：无花果叶提取物抑制紫色杆菌群体感应活性的测试
1实验样品及实验方法
以上述制备方法获得的粗浸膏用DMSO配置浓度为50mg/ml的母液，并依次稀释为20mg/ml、10mg/ml、5mg/ml、2mg/ml、1mg/ml的样品溶液。
活性筛选所用培养基为LB培养基（蛋白胨10g/L；酵母提取物5g/L；NaCl10g/L;去离子水1L，121℃，灭菌20分钟），其固体培养基加入1.5%的琼脂。紫色杆菌的代谢产物紫色素是受群体感应系统严格调控的（Microbiology,1997,143(12):3703-3711），紫色杆菌CV026为信号分子缺失株，自身不能产生信号分子，只有在加入外源性的信号分子己酰高丝氨酸内酯C6-HSL后，才能启动群体感应调控的紫色素产生，当受到群体感应抑制因子的干扰时，则能抑制紫色素的产生，在琼脂板上表现出浑浊但不透明的晕圈。
紫色杆菌QS抑制剂平板筛选方法：取15ml热融化的LB固体培养基，待冷却至35℃左右，加入200μl培养24h的紫色杆菌CV026菌液、50μl信号分子C6-HSL（10μmol/ml）及20μl卡那霉素（50mg/ml），轻轻混匀，倒入平板中，待凝固后，用打孔器打孔，将无花果叶提取物加入孔中（20μl），并置于28℃恒温培养箱中培养48h，观察现象。若无花果叶提取物中含有细菌QS抑制因子，则能够抑制紫色杆菌紫色素的产生，在琼脂平板上表现出浑浊但不透明的晕圈。
紫色杆菌QS抑制剂摇瓶筛选方法：用LB液体培养基将培养至对数期的紫色杆菌CV026菌液稀释至OD600nm=0.1，每瓶10ml；分别加入20μl不同浓度的无花果叶提取物，50μl信号分子和20μl的卡那霉素，置入恒温摇床（28℃、160rmp）培养24h；DMSO溶剂做空白对照。培养结束后，4000rmp离心30min，将上清液吸出，用1mlDMSO重新溶解，再离心（4000rmp，30min），将上清液分别倒入已标记好的离心管中，然后分别依次取200μl上清液加入到96孔微板中，用酶标仪测585nm的吸光度A585，以表征紫色杆菌紫色素的产量；第二次离心获得的沉淀，各用1ml的无菌水重新悬浮菌体，然后依次取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。
2实验结果
紫色杆菌QS抑制剂平板筛选结果表明：c(50μg)、d(100μg)、e(200μg)、f(500μg)的无花果叶二氯甲烷提取物均能抑制紫色杆菌紫色素产生，且随着浓度的增加抑制效果增强（图1）。
紫色杆菌QS抑制剂摇瓶筛选：以无花果叶提取物浓度为横坐标，以600nm处的光吸收度为纵坐标，绘制紫色杆菌CV026在无花果叶提取物作用24h下的菌体生长情况（图2），无花果叶提取物在0~0.1mg/ml浓度范围内对紫色杆菌CV026的生长不产生影响；同时以585nm处的光吸收度为纵坐标，绘制紫色杆菌CV026在无花果叶提取物作用24h下的紫色杆菌紫色素的产量（图3），实验证明无花果叶提取物在有效浓度范围内能显著的降低紫色杆菌紫色素的产量，且随着浓度的增加，降低紫色杆菌紫色素产量的作用越明显，呈浓度依赖性，其中无花果叶二氯甲烷提取物抑制作用最好，在加入0.1mg/ml的提取物时抑制率达到80%。
实施方式六：无花果叶提取物抑制铜绿假单胞菌swarming活性的测试
1实验样品及实验方法
样品溶液的配制与实施方式五中步骤1的实验样品及实验方法一致。
群集运动（swarming）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。铜绿假单胞菌是医院感染的重要机会致病菌和耐药菌之一，其生物被膜、绿脓菌素、群集运动、弹性蛋白酶等毒力因子的表达均受到QS系统的调控。铜绿假单胞菌的群集运动是一种细菌群体在半固体培养基表面快速、同步转移的复杂型运动（J.Bacteriol,1999,181(13):4133-4136）。
群集运动抑制活性的筛选方法：取4.8ml热融化的swarming培养基，待冷却至30℃左右，加入200μl不同浓度的无花果叶提取物，轻轻混匀，倒入平板中，待凝固后，用打孔器打孔，将1μl过夜培养的铜绿假单胞菌PA01菌液加入孔中，37℃恒温培养24h，观察现象。若无花果叶提取物中含有细菌QS抑制因子，则能够抑制QS介导的毒力因子的表达，在琼脂平板上表现出群集运动减弱。
2实验结果
筛选结果表明：20mg/ml的无花果叶二氯甲烷提取物能显著地抑制铜绿假单胞菌的群集运动（图4）。
Claims (5)
1.一种具有细菌群体感应淬灭活性的无花果叶提取物及用途，其特征在于具有细菌群体感应淬灭活性的无花果叶提取物在制备细菌群体感应抑制剂方面的用途。
2.根据权利要求1所述的一种具有细菌群体感应淬灭活性的无花果叶提取物及用途，其特征在于所述的具有细菌群体感应淬灭活性的无花果叶提取物的制备方法按照以下步骤进行：
室温下自然晾干的无花果叶，粉碎成沫，称取1g原材料置于三角摇瓶，加入20ml有机溶剂，超声30min，再置于28℃下摇床48h震荡提取，然后过滤得滤液和滤渣；滤渣再用同比列有机溶剂依照上述方法提取两遍，共得3次滤液，合并，真空浓缩，反相硅胶C18预柱处理后获得无花果叶提取物粗浸膏。
3.根据权利要求2所述的具有细菌群体感应淬灭活性的无花果叶提取物的制备方式，其特征在于溶剂提取法选用石油醚、二氯甲烷、三氯甲烷、乙酸乙酯、甲醇、50%丙酮（v/v）等多种有机溶剂。
4.根据权利要求1所述的一种具有细菌群体感应淬灭活性的无花果叶提取物及用途，其特征在于该提取物在0~0.1mg/ml有效浓度范围内不抑制紫色杆菌CV026（ChromobacteriumviolaceumCV026）的菌体生长，而能显著地降低由群体感应系统介导的紫色杆菌素的合成，可用于制备群体感应抑制剂防治紫色杆菌所引起的疾病感染。
5.根据权利要求1所述的一种具有细菌群体感应淬灭活性的无花果叶提取物及用途，其特征在于该提取物在0~0.8mg/ml浓度范围内能显著地降低由群体感应系统介导的铜绿假单胞菌PA01(PseudomonasaeruginosaPA01)的群集运动（swarming），可用于制备群体感应抑制剂防治铜绿假单胞菌所引起的疾病感染。
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