CN111773270A - 一种抑制铜绿假单胞菌生长及其生物膜形成的苦楝果提取物的制备方法和应用 - Google Patents
一种抑制铜绿假单胞菌生长及其生物膜形成的苦楝果提取物的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种抑制铜绿假单胞菌生长及其生物膜形成的苦楝果提取物的制备方法和应用。经试验证明，本发明的苦楝果提取物在含量为0.5‑1.0g/mL能有效抑制铜绿假单胞菌浮游菌的生长及其生物膜的形成，在含量为0.25‑0.5g/mL对铜绿假单胞菌浮游菌的抑制效果较差，但能较好抑制铜绿假单胞菌生物膜的形成。本发明的原材料天然环保，抑菌抑膜效果显著，有益于人体健康。苦楝果提取物可应用于制备清洗压疮、糖尿病足溃疡、下肢动静脉溃疡等慢性伤口、疥疮、头癣、湿疮、湿疹痛痒等的药物、食品保鲜防腐剂或日化产品抗菌剂。
Description
技术领域
本发明属于医药、日化、植物和微生物领域，具体涉及一种抑制铜绿假单胞菌生长及其生物膜形成的苦楝果提取物的制备方法和应用。
背景技术
化学杀菌剂长期大量以及不正当使用使得很多微生物对其产生了不同程度的抗药性，与此同时，化学杀菌剂的残留也会对人类健康造成威胁，因此，化学杀菌剂在很多领域受限受禁，这促使科研人员从现有的具优异杀菌功能的天然植物和中草药中挖掘更多的活性成分。特别是从黄花蒿中提取到有效抑制疟疾的青蒿素的屠呦呦教授获得诺贝尔生理学或医学奖，以及在新型冠状病毒防治过程中，中草药制剂和疗法多次出现在诊疗方案中，这些必定会将我国的天然产物抑菌抑病毒研究和开发利用推向新的高潮。
而截止目前，科研人员已发现全世界约1600种以上的植物含有杀菌、杀虫、抗病毒功效物质，其中研究较多的是楝科植物，主要包括苦楝、川楝和印楝。苦楝在我国黄河以南各省区较常见。诸多文献报道了苦楝对多种微生物有抑菌、杀菌、驱虫、杀虫、抗病毒、抗肿瘤等多重活性。有学者研究发现苦楝树乙醇提取液对黑曲霉、绿色木霉有很好的抑菌活性，苦楝浸剂对黄色毛癣菌、同心性毛癣菌、许兰氏黄藓菌、奥杜盎氏小芽孢癣菌、铁锈色小芽孢癣菌、羊毛状小芽孢癣菌、红色皮肤癣菌、星形奴卡氏菌等均有一定的抑菌效果。
细菌生物膜(Bacterial biofilm,BF)是细菌的一种生存形式，有数据表明在自然界中高达95％的细菌都是以细菌生物膜的形式存在，它是一种附着于生物或非生物材料表面，被自身分泌的胞外大分子包裹并具有一定三维结构特点的细菌群体。由于生物膜的形成，从而导致细菌抗药性的产生，有研究表明，细菌生物膜的抗药性比其在浮游状态高10～1000倍。细菌生物膜的形成还会导致很多的危害，如对附着的材料表面产生腐蚀作用、污染医疗器械和引发人类疾病等。慢性伤口细菌生物膜就是细菌附于伤口床，与自身分泌的细胞外基质成分相互融合形成的一种膜状组织，慢性伤口因持续时间长、影响因素多而易发感染甚至形成难以处理的细菌生物膜，增加了治疗难度，影响愈合效果。临床多见于压疮、糖尿病足溃疡、下肢动静脉溃疡等慢性伤口。
铜绿假单胞菌(Pseudomonas aeruginosa,PA)俗称绿脓杆菌，是一系列严重化脓性感染，如支气管扩张、慢性支气管炎和囊状肺纤维化等基础疾病继发感染的重要致病菌，铜绿假单胞菌极易在体内外形成生物膜。该菌引起的很多感染发生在衰弱或免疫受损的住院病人，是重症监护室感染的第二位最常见的病原菌。患代谢性疾病、血液病和恶性肿瘤的患者，以及术后或某些治疗后的患者极易感染本菌，常引发术后伤口感染，烧伤伤口也易感染铜绿色假单胞菌，也可引起褥疮、脓肿、化脓性中耳炎等，引起的感染病灶可导致菌血症和败血症，可造成死亡。因此需要筛选抑制铜绿假单胞菌生长的药物或者方法，从而减少该菌的危害性。很多的研究已经证实PA对多种抗生素等杀菌药物产生了抗药性，如果能从天然植物提取物中发现对PA有杀灭效果的活性成分，并加以开发利用，将极大拓展防治该菌的药物谱。抗生素治疗细菌生物膜历来存在争议，有文献报道，抗生素虽然可杀灭链球菌，但却促进铜绿假单胞菌和沙雷菌等其他细菌形成生物膜，这些细菌定植于伤口的深层，抗生素难以发挥作用，导致细菌持续存在从而延缓伤口的愈合。疾病也能影响细菌生物膜生存与否，如糖尿病患者伤口中的链球菌是非糖尿病的63倍。
发明内容
本发明的第一个目的是提供一种抑制铜绿假单胞菌生长及其生物膜形成的苦楝果提取物的制备方法，包括以下步骤：将苦楝果清洗后烘干至恒重，粉碎，得到苦楝果粉；往苦楝果粉中加入水，采用超声辅助水提法进行提取，得到提取液，将提取液离心，取上清液，将上清液浓缩，得到苦楝果提取物。
优选，所述的制备方法，包括以下步骤：将苦楝果清洗后烘干至恒重，用高速粉碎机粉碎至颗粒直径≤1mm，得到苦楝果粉；将苦楝果粉按100g/L的比例与蒸馏水混合，室温下超声提取30min，然后加热煮沸提取30min，再次超声提取30min，过滤，得到提取液；往滤渣中加入蒸馏水，煮沸提取30min，过滤，得到提取液，合并两次提取液，5000r/min离心10min，取上清液，将上清液减压抽滤，真空浓缩，灭菌，得到苦楝果提取物。
上述制备方法中的超声提取的频率为40kHz，功率为100W。
本发明的第二个目的是提供一种由上述的制备方法制备得到的苦楝果提取物。
所述的苦楝果提取物的含量为0.25～1.0g/mL。
本发明的第三个目的是提供由上述的制备方法制备得到的苦楝果提取物在抑制铜绿假单胞菌生长及其生物膜形成中的应用。
本发明的苦楝果提取物可用于制备清洗压疮、糖尿病足溃疡、下肢动静脉溃疡、疥疮、头癣、湿疮、湿疹痛痒的药物、食品保鲜防腐剂或日化产品抗菌剂。
本发明根据苦楝果中活性成分特性，配置特定的提取方法，提高了活性成分的提取率，提高苦楝果提取物对铜绿假单胞菌的生长及其生物膜形成的抑制效果。
本发明的原材料源于植物，天然环保，抑菌效果显著，有益于人体健康，可应用于制备清洗伤口的药物、食品保鲜防腐剂或日化产品抗菌剂，应用范围和前景广阔。
附图说明
图1为苦楝果提取物对铜绿假单胞菌的抑菌效果。(A)为不同浓度的苦楝果提取物培养铜绿假单胞菌24h对细菌浮游菌的抑制效果(每个实验浓度8个平行，n＝8)，(B)为不同浓度的苦楝果提取物培养铜绿假单胞菌24h对细菌生物膜形成的抑制效果(每个实验浓度8个平行，n＝8)。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整的描述，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。以下实施例是对本发明的进一步说明，而不是对本发明的限制。
以下实施例中的测试菌株为铜绿假单胞菌(P.aeruginosa ATCC 9027)时，测试方法为：用该实施例中营养琼脂培养基(NA)斜面培养基将测试菌株于37℃培养1d，然后用PBS清洗，稀释到OD600＝1.0配制成菌悬液工作液，同时以无菌蒸馏水作为空白对照。按照梯度稀释的方法在培养体系中加入一定浓度的苦楝果提取物，吸取150μL到96孔细胞培养板中(每个实验浓度8个平行，n＝8)，然后将培养板放入37℃恒温培养箱静置培养24小时，最后将96孔板取出，用全波长酶标仪测定菌液的在600nm处的吸光度值(OD600)，与空白对照的吸光度值比较，以此评价苦楝果提取物对细菌浮游菌的抑制效果；然后，弃浮游菌并轻轻清洗板孔，晾干，在每个板孔内加入200μL 1％(v/w)结晶紫染色液，静置染色30分钟，弃结晶紫染色液，再用无菌水洗板孔，室温晾干后，用200μL 95％的乙醇脱色30min，利用全波长酶标仪测定其在595nm处的吸光度值(OD595)，与空白对照的吸光度值比较，以此评价苦楝果提取物对细菌生物膜形成的抑制效果。
在本说明书中使用以下缩写：ATCC是美国模式培养物集存库(American TypeCulture Collection)的简写，PA是铜绿假单胞菌，NA是营养琼脂培养基，PBS是磷酸缓冲盐溶液(phosphate buffer saline)，温度的单位是摄氏度(℃)，min＝分钟，d＝天，mL＝毫升，μL＝微升，mg＝毫克，g＝克，OD600指的是溶液在600nm波长处的吸光值，OD595指的是溶液在595nm波长处的吸光值。
实施例1
将购买的苦楝果用蒸馏水清洗干净以除去表面尘土，沥干水分放入烘箱，65℃烘干至恒重，用高速粉碎机粉碎至颗粒直径≤1mm，得到苦楝果粉，用密封袋保存备用。称取100g苦楝果粉置于1000mL蒸馏水中，室温下采用超声仪(频率40kHz，功率100W)超声作用30min，然后加热煮沸30min，再次置于超声仪中作用30min，用200目纱布过滤得到提取液，将滤渣加入到500mL蒸馏水中煮沸30min，用200目纱布过滤得到提取液，合并两次提取液，高速离心分离(5000r/min)10min，取上清液。将上清液先用400目尼龙滤布减压抽滤，再用0.45μm滤膜减压抽滤。并在旋转蒸发仪上保持真空度-0.1～-0.08MPa，减压浓缩至100mL，最终得到浓度约为1.0g/mL的浓缩液。浓缩液经121℃灭菌20min后，得到苦楝果提取物，于4℃冰箱内保存备用。
用铜绿假单胞菌(P.aeruginosa ATCC 9027)作为测试菌株，以1.0g/mL的苦楝果提取物按照上述测试方法范例中的方法进行测试，用96孔细胞培养板在37℃静置培养菌株24h，测得的苦楝果提取物对PA的OD600为0.1190，空白对照的OD600为1.1444，与空白对照相比，OD600下降了89.60％。同时，用结晶紫染色法和全波长酶标仪测定菌株在1.0g/mL苦楝果提取物条件下的细菌生物膜形成能力，测得的OD595均值为：0.0951，空白对照的OD595为2.0813，与空白对照相比，OD595下降了95.43％。由此可见，使用本发明所提供的苦楝果提取物培养铜绿假单胞菌(P.aeruginosa ATCC 9027)，除了可以有效抑制该菌浮游菌的生长以外，还可以有效的抑制其细菌生物膜的形成。
实施例2
按照实施例1的方法制备苦楝果提取物。将1.0g/mL苦楝果提取物用蒸馏水稀释至浓度为0.5g/mL，备用。
用铜绿假单胞菌(P.aeruginosa ATCC 9027)作为测试菌株，以0.5g/mL的苦楝果提取物按照上述测试方法范例中的方法进行测试，用96孔细胞培养板在37℃静置培养菌株24h，测得的苦楝果提取物对PA的OD600为0.3869，空白对照的OD600为1.1444，与空白对照相比，OD600下降了66.19％。同时，用结晶紫染色法和全波长酶标仪测定菌株在0.5g/mL苦楝果提取物条件下的细菌生物膜形成能力，测得的OD595均值为0.2560，空白对照的OD595为2.0813，与空白对照相比，OD595下降了87.70％。由此可见，使用本发明所提供的苦楝果提取物培养铜绿假单胞菌(P.aeruginosa ATCC 9027)，除了可以有效抑制该菌浮游菌的生长以外，还可以有效的抑制其细菌生物膜的形成。
实施例3
按照实施例1的方法制备苦楝果提取物。将1.0g/mL苦楝果提取物用蒸馏水稀释至浓度为0.33g/mL，备用。
用铜绿假单胞菌(P.aeruginosa ATCC 9027)作为测试菌株，以0.33g/mL的苦楝果提取物按照上述测试方法范例中的方法进行测试，用96孔细胞培养板在37℃静置培养菌株24h，测得的苦楝果提取物对PA的OD600为0.7845，空白对照的OD600为1.1444，与空白对照相比，OD600下降了31.45％。同时，用结晶紫染色法和全波长酶标仪测定菌株在0.33g/mL苦楝果提取物条件下的细菌生物膜形成能力，测得的OD595均值为0.5610，空白对照的OD595为2.0813，与空白对照相比，OD595下降了73.05％。由此可见，使用本发明所提供的苦楝果提取物在浓度为0.33g/mL时抑制铜绿假单胞菌(P.aeruginosa ATCC 9027)浮游菌的生长较弱，但能有效抑制铜绿假单胞菌生物膜的形成。
实施例4
按照实施例1的方法制备苦楝果提取物。将1.0g/mL苦楝果提取物用蒸馏水稀释至浓度为0.25g/mL，备用。
用铜绿假单胞菌(P.aeruginosa ATCC 9027)作为测试菌株，以0.25g/mL的苦楝果提取物按照上述测试方法范例中的方法进行测试，用96孔细胞培养板在37℃静置培养菌株24h，测得的苦楝果提取物对PA的OD600为0.9323，空白对照的OD600为1.1444，与空白对照相比，OD600下降了18.53％。同时，用结晶紫染色法和全波长酶标仪测定菌株在0.25g/mL苦楝果提取物条件下的细菌生物膜形成能力，测得的OD595均值为0.8370，空白对照的OD595为2.0813，与空白对照相比，OD595下降了59.78％。由此可见，使用本发明所提供的苦楝果提取物在浓度为0.25g/mL时抑制铜绿假单胞菌(P.aeruginosa ATCC 9027)浮游菌的生长较差，但能有效抑制铜绿假单胞菌生物膜的形成。
上述实施例1-4的苦楝果提取物(浓度分别为1.0g/mL、0.5g/mL、0.33g/mL、0.25g/mL)对铜绿假单胞菌的抑菌效果见图1。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.一种抑制铜绿假单胞菌生长及其生物膜形成的苦楝果提取物的制备方法，其特征在于，包括以下步骤：将苦楝果清洗后烘干至恒重，粉碎，得到苦楝果粉；往苦楝果粉中加入水，采用超声辅助水提法进行提取，得到提取液，将提取液离心，取上清液，将上清液浓缩，得到苦楝果提取物。
2.根据权利要求1所述的制备方法，其特征在于，包括以下步骤：将苦楝果清洗后烘干至恒重，用高速粉碎机粉碎至颗粒直径≤1mm，得到苦楝果粉；将苦楝果粉按100g/L的比例与蒸馏水混合，室温下超声提取30min，然后加热煮沸提取30min，再次超声提取30min，过滤，得到提取液；往滤渣中加入蒸馏水，煮沸提取30min，过滤，得到提取液，合并两次提取液，5000r/min离心10min，取上清液，将上清液减压抽滤，真空浓缩，灭菌，得到苦楝果提取物。
3.根据权利要求2所述的制备方法，其特征在于，所述的超声提取的频率为40kHz，功率为100W。
4.权利要求1-3任一所述的制备方法制备得到的苦楝果提取物。
5.根据权利要求4所述的苦楝果提取物，其特征在于，所述的苦楝果提取物的含量为0.25～1.0g/mL。
6.权利要求1-3任一所述的制备方法制备得到的苦楝果提取物在抑制铜绿假单胞菌生长及其生物膜形成中的应用。
7.权利要求1-3任一所述的制备方法制备得到的苦楝果提取物在制备清洗压疮、糖尿病足溃疡、下肢动静脉溃疡、疥疮、头癣、湿疮、湿疹痛痒的药物、食品保鲜防腐剂或日化产品抗菌剂中的应用。
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Application publication date: 20201016 |