CN114406281A - 一种植物多酚还原制备金纳米颗粒的方法及应用 - Google Patents
一种植物多酚还原制备金纳米颗粒的方法及应用 Download PDFInfo
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Abstract
本发明公开了一种植物多酚还原制备金纳米颗粒的方法及应用,属于生物医药材料技术领域。上述方法包括以下步骤:(1)加热煮沸质量分数为0.01‑0.05%的HAuCl4水溶液,得到HAuCl4溶液;(2)将植物多酚溶解成质量浓度为1‑10mg/mL的溶液,过滤、煮沸,得到植物多酚溶液;(3)将所述植物多酚溶液滴加到所述HAuCl4溶液中,煮沸15min后,转移到常温搅拌至室温,得到植物多酚还原的金纳米颗粒。本发明通过利用植物多酚还原Au3+形成金纳米颗粒,该方法简单易操作,并且所制备的金纳米颗粒可以有效的杀灭多种耐药细菌。
Description
技术领域
本发明涉及生物医药材料领域,特别是涉及一种植物多酚还原制备金纳米颗粒的方法及应用。
背景技术
细菌感染是人类健康长期以来的威胁。抗生素的发现对抗细菌感染带来了革命性的胜利。然而,抗生素的滥用加速了细菌耐药性的产生,使得抗生素失效且加深了细菌感染治疗的难度。耐药菌株与诸多疾病相关,包括骨髓炎、呼吸道感染、手术相关和植入体相关感染以及伤口感染。目前临床感染的金黄色葡萄球菌中,有60%以上的是耐甲氧西林的金黄色葡萄球菌,其造成的患者死亡率要比感染普通金黄色葡萄球菌感染高出64%;70%以上的绿脓杆菌存在多重耐药问题,对环丙沙星、头孢他啶、亚胺培南和阿米卡星等药物表现出严重的耐药性。因此,杀灭耐药型细菌对人类健康至关重要。
引起细菌耐药性问题的原因主要包括两个方面:一个是细菌生物膜的形成,另一个是在抗生素选择压力下细菌的自我进化。生物膜中的胞外基质、低pH和高酶活性可以保护细菌免受抗生素、宿主免疫防御和外部严酷的物理或化学环境的影响。研究表明,生物膜中细菌的耐药性是其浮游细菌的10~1000倍。另外,抗生素杀菌的过程会促进细菌自然选择和进化。获得耐药性的细菌需要新型抗生素进行杀灭,然而抗生素更新的速度远远小于细菌产生耐药性的速度,最新研制的抗生素开发于30年前。因此新型抗生素或抗菌材料亟待开发。
纳米材料由于其小的尺寸可以渗透进入生物膜,且一些纳米材料具有产生活性氧自由基的能力,可通过非抗生素的方式杀灭细菌,为抗多重耐药性细菌的杀灭提供了可能。
发明内容
本发明的目的是提供一种植物多酚还原制备金纳米颗粒的方法及应用,以解决上述现有技术存在的问题,通过利用植物多酚还原Au3+形成金纳米颗粒,该方法简单易操作,并且所制备的金纳米颗粒可以有效的杀灭多种耐药细菌。
为实现上述目的,本发明提供了如下方案:
本发明提供一种植物多酚还原制备金纳米颗粒的方法,包括以下步骤:
(1)加热煮沸质量分数为0.01-0.05%的HAuCl4水溶液,得到HAuCl4溶液;
(2)将植物多酚溶解成质量浓度为1-10mg/mL的溶液,过滤、煮沸,得到植物多酚溶液;
(3)将所述植物多酚溶液滴加到所述HAuCl4溶液中,煮沸15min后,常温搅拌至室温,得到植物多酚还原的金纳米颗粒。
优选的是,步骤(1)中,加热温度为130℃。
优选的是,步骤(2)中,所述植物多酚为鞣花酸、单宁酸、表没食子儿茶素没食子酸酯、没食子酸、熊果苷中任意一种。
优选的是,步骤(3)中,所述植物多酚:所述HAuCl4溶液的体积比为(11:10)-(11:20)。
本发明还提供一种所述的方法制备的金纳米颗粒。
本发明还提供所述的金纳米颗粒在非诊断和治疗目的的杀灭耐药细菌中的应用。
本发明还提供所述的金纳米颗粒在制备杀灭耐药细菌的药物中的应用。
优选的是,所述耐药细菌包括粪肠球菌属,金黄色葡萄球菌属,克雷伯菌属,鲍曼不动杆菌属,绿脓杆菌属和大肠杆菌属的细菌。
本发明公开了以下技术效果:
本发明以植物多酚作为还原剂,将Au3+还原成金纳米颗粒(Au NPs),构筑具有广谱杀菌性和抗多种耐药性细菌的金纳米颗粒的制备平台,该方法原料易得,制备过程简单快速,形成的纳米颗粒均一稳定,能够高效杀灭多种耐药性细菌,这为开发新的抗耐药细菌的药物奠定了基础,为耐药细菌的防治提供了新的方法。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为实施例1不同多酚还原得到的Au NPs透射电镜(TEM)照片;(a)TA-AuNPs;(b)EGCG-AuNPs;(c)EA-AuNPs;(d)GA-AuNPs;(e)Arb-AuNPs;
图2为传统涂布法表征实施例1不同多酚(浓度为3.12μg/mL)还原的Au NPs的对金黄色葡萄球菌的杀灭效果;
图3为传统涂布法表征实施例1不同浓度的EA-Au NPs对耐药菌的杀灭效果;(a)Acinetobacter baumanii;(b)Staphylococcus aureus;(c)Klebsiella pneumoniae;(d)Pseudomonas aeruginosa;(e)Enterobacter species;(f)Enterococcus faeciu;
图4为生物发光法表征不同浓度的EA-Au NPs对生物膜的消灭效果。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1植物多酚还原制备金纳米颗粒
(1)在100mL圆底烧瓶中加入HAuCl4(47.5mL,0.01wt%),130℃条件下加热煮沸待用;
(2)分别将不同的多酚(鞣花酸(Ellagic acid,EA)、单宁酸(Tannic acid,TA)、表没食子儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)、没食子酸(Gallic acid,GA)、熊果苷(Arbutin,Arb)溶解在超纯水中得到2mg/mL的溶液,并且进行过滤处理,煮沸待用;
(3)分别取上述2.75mL不同的多酚滴加到煮沸的47.5mL HAuCl4溶液中,煮沸15分钟后转移到常温搅拌至室温即得到不同多酚还原的Au NPs。
实施例2植物多酚还原制备金纳米颗粒
(1)在100mL圆底烧瓶中加入HAuCl4(30.5mL,0.03%),130℃条件下加热煮沸待用;
(2)分别将不同的多酚(鞣花酸(Ellagic acid,EA)、单宁酸(Tannic acid,TA)、表没食子儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)、没食子酸(Gallic acid,GA)、熊果苷(Arbutin,Arb)溶解在超纯水中得到5mg/mL的溶液,并且进行过滤处理,煮沸待用;
(3)分别取上述30.5mL不同的多酚滴加到煮沸的30.5mL HAuCl4溶液中,煮沸15分钟后转移到常温搅拌至室温即得到不同多酚还原的Au NPs。
实施例3植物多酚还原制备金纳米颗粒
(1)在100mL圆底烧瓶中加入HAuCl4(20.0mL,0.05%),130℃条件下加热煮沸待用;
(2)分别将不同的多酚(鞣花酸(Ellagic acid,EA)、单宁酸(Tannic acid,TA)、表没食子儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)、没食子酸(Gallic acid,GA)、熊果苷(Arbutin,Arb)溶解在超纯水中得到10mg/mL的溶液,并且进行过滤处理,煮沸待用;
(3)分别取上述10.0mL不同的多酚滴加到煮沸的20.0mL HAuCl4溶液中,煮沸15分钟后转移到常温搅拌至室温即得到不同多酚还原的Au NPs。
为了验证本发明制备的金纳米颗粒的效果,采用上述实施例1制备的金纳米颗粒进行了如下试验。
(1)将上述实施例1制备的Au NPs滴加到铜网上,静置24h并烘干,进行TEM测试。结果如图1所示,图中可见,经过不同植物多酚还原的HAuCl4溶液均显示出规则的纳米颗粒结构。
(2)利用实施例1制备的不同的Au NPs评价其对金黄色葡萄球菌杀菌效果,具体操作如下:
a:将金黄色葡萄球菌(S.aureus Xen36)离心分离后重悬在无菌PBS中,浓度为2×107CFU/mL;
b:将制备的五种金纳米材料进行过滤膜处理,然后用无菌PBS进行稀释,稀释浓度为6.25μg/mL;
c:将稀释好的材料与细菌溶液按照体积比1:1混合,1h后以涂布的方法评价杀菌效果。
结果如图2所示,由鞣花酸(EA)还原制备的Au NPs的杀菌效果最好,在浓度为3.12μg/mL时可以将细菌全部杀灭。
(3)利用实施例1制备的不同的Au NPs评价其对耐药菌杀灭效果,其操作步骤如下:
a:将耐药菌(包括“ESKAPE”(粪肠球菌属Enterococcus faeciu,金黄色葡萄球菌属Staphylococcus aureus,克雷伯菌属Klebsiella pneumoniae,鲍曼不动杆菌属Acinetobacter baumanii,绿脓杆菌属Pseudomonas aeruginosa和大肠杆菌属Enterobacter species))分离后重悬在无菌PBS中,浓度为2×107CFU/mL。
b:将EA-Au NPs进行过滤膜处理,然后用无菌PBS进行稀释,稀释不同浓度(3.12、2.00、1.00、0.50和0.25μg/mL),最后将稀释好的材料与细菌溶液混合,1h后以梯度稀释并涂布的方法评价杀菌效果。
结果如图3所示,EA-Au NPs的浓度为3.12μg/mL时,可以将六种“ESKAPE”耐药菌全部杀灭。
(4)利用实施例1制备的不同的Au NPs评价其对生生物膜消灭效果,具体操作步骤如下:
a:将金黄色葡萄球菌(S.aureus Xen36)分离后重悬在无菌PBS中,浓度为1×108CFU/mL,然后取100μL细菌溶液置于96孔板中,放入37℃细菌恒温培养箱中静置培养1h。培养结束后将菌液吸出来,随后加入200μL TSB培养基培养24h,即得到生物膜。
b:将EA-Au NPs进行过滤膜处理,然后用无菌PBS进行稀释,稀释不同浓度(50.00、25.00、12.50、6.25、3.12、1.56μg/mL),最后将稀释好的材料加入到生物膜中,1h后测量细菌生物发光的强度。
结果如图4所示,EA-Au NPs对生物膜具有非常明显的消灭效果,并且随着EA-AuNPs的浓度增加消灭生物膜现象越明显。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (8)
1.一种植物多酚还原制备金纳米颗粒的方法,其特征在于,包括以下步骤:
(1)加热煮沸质量分数为0.01-0.05%的HAuCl4水溶液,得到HAuCl4溶液;
(2)将植物多酚溶解成质量浓度为1-10mg/mL的溶液,过滤、煮沸,得到植物多酚溶液;
(3)将所述植物多酚溶液滴加到所述HAuCl4溶液中,煮沸15min后,常温搅拌至室温,得到植物多酚还原的金纳米颗粒。
2.如权利要求1所述的方法,其特征在于,步骤(1)中,加热温度为130℃。
3.如权利要求1所述的方法,其特征在于,步骤(2)中,所述植物多酚为鞣花酸、单宁酸、表没食子儿茶素没食子酸酯、没食子酸、熊果苷中任意一种。
4.如权利要求1所述的方法,其特征在于,步骤(3)中,所述植物多酚:所述HAuCl4溶液的体积比为(11:10)-(11:20)。
5.一种如权利要求1-4任一项所述的方法制备的金纳米颗粒。
6.如权利要求5所述的金纳米颗粒在非诊断和治疗目的的杀灭耐药细菌中的应用。
7.如权利要求5所述的金纳米颗粒在制备杀灭耐药细菌的药物中的应用。
8.如权利要求6或7所述的应用,其特征在于,所述耐药细菌包括粪肠球菌属,金黄色葡萄球菌属,克雷伯菌属,鲍曼不动杆菌属,绿脓杆菌属和大肠杆菌属的细菌。
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