CN116921688A - 基于桃叶提取液制备纳米银的方法及其在抑菌中的应用 - Google Patents
基于桃叶提取液制备纳米银的方法及其在抑菌中的应用 Download PDFInfo
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Abstract
本发明公开了基于桃叶提取液制备纳米银的方法及其在抑菌中的应用。基于桃叶提取液制备纳米银的方法包括以下步骤:将桃叶提取液与银离子溶液混合,反应生成纳米银。合成的纳米银颗粒可抑制Hafnia菌的生长,最小抑菌浓度为64μg/mL。P‑AgNPs对哈夫尼亚菌生物被膜、AHLs、蛋白酶、群集和泳动等毒力因子具有抑制作用,为开发具有抑制AHL型QS的Ag纳米颗粒型抑制剂提供了一定的理论基础,也为纳米银在水产品养殖和贮藏运输过程中的应用提供了一定的研究基础。
Description
技术领域
本发明涉及纳米材料的制备领域,具体涉及一种基于桃叶提取液制备纳米银的方法及其在抑菌中的应用。
背景技术
银纳米颗粒(silver nanoparticles,AgNPs)是指粒径为纳米级别(1-100nm)的单质银颗粒,具有优良的表面活性、催化特性、导电性以及抑菌特性,能抑制包括G+菌和G-菌的多种细菌,其抑菌机制主要为破坏细菌的细胞膜和呼吸链以干扰DNA的复制等。传统的银纳米颗粒的合成方法包括物理和化学合成法。其中物理合成法直接将单质银分散成纳米银,在合成过程中具有成本高、危险性高的缺点;化学合成法成核速率难以控制并且需加入还原剂和稳定剂,容易对环境造成污染。而植物提取物因为具有多种活性物质,利用其合成AgNPs时,AgNPs被活性物质自然修饰,因此合成的AgNPs具有良好的安全性和较好的生物兼容性,并且对环境污染小。Faizan等人利用印度藏茴香(Carum copticum)种子提取物合成的Ag@CC-NPs对铜绿假单胞菌、紫色杆菌、粘质沙雷氏菌等都具有良好的抑菌能力(DOI:10.1016/j.micpath.2020.104172)。
哈夫尼亚菌属(Hafnia sp.)为革兰氏阴性杆菌,在自然界中普遍存在。主要为真空包装和冷藏肉制品中常见的一种腐败菌,相关研究表明其致腐行为受到以N-酰基-L-高丝氨酸内酯(N-acyl-L-homoserine lactones,AHLs)作为信号分子的群体感应(quorumsensing,QS)系统调控,包括生物被膜形成、蛋白酶和胞外多糖等导致腐败的毒力因子的产生都会受到QS系统影响。在传统的水产养殖和水产贮藏的过程中常添加各类抗生素来达到抑菌的目的,但长而久之的使用则会导致耐药菌的产生并且残留的抗生素也不利于消费者的身体健康。而通过影响QS来减弱细菌毒力的策略会在一定程度上避免耐药性的产生,目前已经有相关研究表明AgNPs可以抑制一些腐败或致病菌的QS,但是没有AgNPs对哈夫尼亚菌QS系统影响的相关研究,这表明AgNPs这种广谱抗菌剂在水产品的养殖和贮藏运输过程中具有一定应用潜力。
桃(Peach)为蔷薇科植物,原产于中国并在亚洲各国广泛种植,其植物部分已被证明具有多种药用价值,例如其花具有治疗黄疸的功效、果实具有降低胆固醇和维持血压等效果,而其叶子中含有的酸类、酚类等成分也具有清热解毒、消炎杀虫等能力。
发明内容
本发明的第一个目的是提供一种基于桃叶提取液制备纳米银的方法,包括以下步骤:将桃叶提取液与银离子溶液混合,反应生成纳米银。
优选地,所述的桃叶提取液是将桃叶洗净后剪碎,加入水蒸煮至溶液变为黄绿色后,抽滤后获得桃叶提取液。
优选地,所述的银离子溶液是AgNO3溶液。
优选地,所述的反应条件为60℃避光进行水热法反应6h。
优选地,具体步骤为:
(1)将采摘后的桃叶叶片洗净剪碎后,称取5.0g加入150mL蒸馏水于60℃蒸煮至溶液变为黄绿色,抽滤即得桃叶提取液;
(2)量取30mL的桃叶提取液与10mL 10mM的AgNO3混匀后,在避光条件下60℃进行水热法合成6h,待溶液变为黑褐色终止,12000r/min、4℃离心45min弃上清,沉淀用10mL无菌水洗涤3次后冻干。
本发明的第二个目的是提供上述方法制备的纳米银。
本发明的第三个目的是提供上述纳米银在制备抑制哈夫尼亚菌药物中的应用。
优选地,所述的纳米银的抑菌浓度为64μg/mL。
优选地,所述的哈夫尼亚菌为哈夫尼亚菌HA1或HA2。
优选地,所述的抑制哈夫尼亚菌为抑制哈夫尼亚菌生物被膜形成、抑制哈夫尼亚菌产AHLs、抑制哈夫尼亚菌产胞外蛋白酶、抑制哈夫尼亚菌产胞外多糖、抑制哈夫尼亚菌群集能力、以及抑制哈夫尼亚菌泳动能力中的至少一种。
本发明的优点:
本发明拟采用桃叶提取液作为还原剂,采用水热法与AgNO3合成桃叶-AgNPs(P-AgNPs),桃叶中所含有的生物活性成分不仅容易获取,而且在AgNPs的生物绿色合成过程中,使所得的AgNPs被这些成分自然修饰以降低对合成环境的污染并具有更好的生物相容性。
合成的纳米银颗粒可抑制Hafnia菌的生长,最小抑菌浓度为64μg/mL。
在sub-MIC下P-AgNPs对哈夫尼亚菌生物被膜、AHLs、蛋白酶、群集和泳动等毒力因子具有抑制作用,为开发具有抑制AHL型QS的Ag纳米颗粒型抑制剂提供了一定的理论基础,也为纳米银在水产品养殖和贮藏运输过程中的应用提供了一定的研究基础。
附图说明
图1是利用桃叶提取液制备的P-AgNPs(A)及其紫外-可见吸收光谱图(B)。其中(A)图中左边试管为桃叶提取液在60℃与AgNO3进行水热反应合成0h的混合液,右边试管为桃叶提取液在60℃与AgNO3进行水热反应合成6h后溶液颜色由棕黄变为黑褐色。
图2是P-AgNPs的动态光散射(DLS)图和Zeta电位图。
图3是P-AgNPs的X射线衍射(XRD)图。
图4是P-AgNPs的傅里叶变换红外光谱(FTIR)图。
图5是P-AgNPs的扫描电镜(SEM)和X射线能谱(EDM)分析图。
图6是P-AgNPs对哈夫尼亚菌HA1、HA2的抑菌圈(A)、以及在不同浓度P-AgNPs对哈夫尼亚菌HA1、HA2的抑制效果(B),a:桃叶提取液;b:无菌水;c:P-AgNPs;d:AgNO3。
图7是结晶紫法测定P-AgNPs对哈夫尼亚菌HA1和HA2生物被膜的抑制效果(A)、以及在1/2MIC(32μg/mL)浓度的P-AgNPs下光学显微镜(B)和扫描电镜(C)下的HA1和HA2生物被膜形态。
图8是P-AgNPs对哈夫尼亚菌HA1和HA2产生AHLs的抑制效果。
图9是P-AgNPs对哈夫尼亚菌HA2胞外蛋白酶(A)和哈夫尼亚菌HA1、HA2胞外多糖的影响(B)。
图10是P-AgNPs对哈夫尼亚菌HA1和HA2群集(A)和泳动(B)的抑制效果。
具体实施方式
以下实施例是对本发明的进一步说明,而不是本发明的限制。
实施例1:桃叶提取液的制备
将采摘后的桃(红花碧桃Amygdalus persica L.var.persica f.rubro-plenaSchneid.)叶叶片洗净剪碎后,称取5.0g加入150mL蒸馏水于60℃蒸煮至溶液变为黄绿色后,抽滤后作为桃叶提取液并于4℃冰箱保存。
实施例2:P-AgNPs的合成及表征
量取30mL的按实施例1的方法制备的桃叶提取液与10mL 10mM的AgNO3混匀后,在避光条件于60℃进行水热法合成6h,使得溶液变为黑褐色即为终止,于12000r/min、4℃下离心45min弃上清;沉淀用10mL无菌水洗涤,于12000r/min、4℃下离心45min弃上清,重复3次后冻干保存,即为桃叶-AgNPs(P-AgNPs)。
对合成的P-AgNPs为初步测定其有无AgNPs形成,将冻干后的30mgAgNPs溶解在30mL蒸馏水中,在波长300~600nm、扫描频率1.0Hz条件下进行紫外-可见吸收光谱扫描有无AgNPs的吸收特征峰出现,在25℃、入射波长633nm、散射角90°条件下利用动态光散射(Dynamic Light Scattering,DLS)技术测定测定P-AgNPs的粒径分布,在25℃、入射波长633nm、散射角90°、μ=0.8872cP、RI=0.135条件下测定Zeta电位以判断P-AgNPs的稳定性;为测定P-AgNPs的结晶程度和元素成分,将冻干后的P-AgNPs粉末20mg在管电压40kV、管电流30mA、扫描速度5°/min、2θ=20°~80°条件下获得其X射线衍射(XRD)图谱并结合德拜-谢乐(Debye-Scherer)公式:式中D为晶体的尺寸、K为谢乐常数(0.9)、λ为X射线波长(0.15406nm)、β为衍射峰半高宽度、θ为布拉格衍射角,以测定P-AgNPs的平均粒径尺寸;为测定P-AgNPs中有无桃叶提取液的活性成分作为还原剂和封端剂,将冻干后的P-AgNPs粉末20mg使用溴化钾压片法在500~4000cm-1范围内扫描,以得到P-AgNPs的傅里叶变换红外光谱(FTIR)图;为测定P-AgNPs的形态样貌和元素组成,将冻干后的P-AgNPs粉末20mg在1.0~2.0kV、10mA条件下进行扫描电镜(SEM)观察并结合X射线能谱仪(EDS)进行元素分析。
采用MDI Jade 6软件和Omnic软件分别对P-AgNPs的X射线衍射图谱和傅里叶变换红外光谱图平滑一次并去除背景噪声后,采用Excel、Origin 2018软件进行数据处理和作图,采用SPSS 26软件进行显著性差异分析。
结果如下:
(1)桃叶提取液在60℃与AgNO3进行水热反应合成6h后溶液颜色由棕黄变为黑褐色,表明可能有AgNPs的形成(图1A)。后通过300~600nm的UV-vis分析发现在447nm处有吸收共振峰的形成,在380~460nm之间的表面等离子共振峰是AgNPs特有的吸收共振峰,单一峰表明有AgNPs的形成(图1B)。
(2)采用动态光散射(Dynamic Light Scattering,DLS)中的数量分布测定P-AgNPs的粒径小于100nm,且平均粒径为90.06±1.80nm(图2A),其平均多分散性指数(Polydispersity index,PDI)为0.228,之后通过Zeta电位测定发现P-AgNPs的电位为-21.83±1.33mV(图2B)。
(3)通过20°~80°的XRD扫描发现P-AgNPs在2θ为38.06°、44.22°、64.38°、77.34°出现了Ag的衍射峰(PDF#87-0597)以分别对应其(111)、(200)、(220)、(311)的晶体结构,表明桃叶提取液成功合成了面心立方结构的AgNPs,且采用德拜-谢乐(Debye-Scherer)公式: 式中:D为晶体的尺寸、K为谢乐常数(0.9)、λ为X射线波长(0.15406nm)、β为衍射峰半高宽度、θ为布拉格衍射角,初步计算了D-AgNPs的平均晶体尺寸为36.3nm(图3)。
(4)通过500~4000cm-1的FTIR扫描发现P-AgNPs在3411.94cm-1处可能出现了O-H拉伸振动,在2958.58cm-1、2918.30cm-1处可能出现了C-H弯曲振动,在1735.32cm-1处可能出现了C=O的伸缩振动,在1635.36cm-1处可能出现了N-H弯曲振动,在1453.50cm-1处可能出现了C=C骨架振动,在650~1350cm-1的指纹区的1243.96cm-1、1039.31cm-1处可能出现了C-O或N-H的伸缩振动,在602.36cm-1处可能出现了有机卤化物C-X的伸缩振动(图4)。
(5)通过SEM观察桃叶提取液可以将AgNO3还原并形成球形AgNPs(图5A),进一步通过EDS能谱分析发现P-AgNPs主要由Ag、Cl、C、O元素组成,且Ag元素的质量比已经超过50%(图5B)。
实施例3:P-AgNPs对哈夫尼亚菌的抑菌能力
为测定P-AgNPs对哈夫尼亚菌的抑菌能力,将0.05g合成的P-AgNPs分散于10mL无菌水中,得到P-AgNPs溶液。并将过夜活化后的哈夫尼亚菌菌株HA1和HA2按1%(v/v)接种于经牛津杯打孔的LB营养琼脂中,孔中各加入200μL的桃叶提取液、10mMAgNO3或200μL P-AgNPs溶液,28℃培养12h后观察有无抑菌圈的形成,以200μL的无菌水为空白对照,每组3个平行。将有抑菌能力的P-AgNPs采用微量肉汤稀释法确定其最小抑菌浓度(MIC)。
经牛津杯打孔法测定P-AgNPs对哈夫尼亚菌HA1和HA2均有抑菌圈的形成(图6A),之后通过96孔板微量肉汤稀释法测定P-AgNPs对于哈夫尼亚菌HA1和HA2的MIC为64μg/mL(图6B)。
实施例4:P-AgNPs对哈夫尼亚菌毒力因子的抑制作用
(1)P-AgNPs对哈夫尼亚菌生物被膜的抑制作用
为测定P-AgNPs对哈夫尼亚菌生物被膜的抑制作用,将培养至OD595nm为0.5左右的哈夫尼亚菌菌株HA1和HA2按1%(v/v)接种于含200μL LB肉汤的96孔板中并添加1/128~1/2MIC(1/128MIC、1/32MIC、1/16MIC、1/8MIC、1/4MIC和1/2MIC,下同)浓度的P-AgNPs28℃培养24h后测定OD595nm时的OD值,吸去菌液并用PBS缓冲液洗涤3次,风干后用200μL 0.1%结晶紫溶液染色15min后吸去结晶紫溶液并用PBS缓冲液洗涤至无紫色出现,风干后用200μL33%冰醋酸溶解结晶紫染液15min并测定OD595nm时的OD值以测定P-AgNPs对生物被膜的抑制作用,每组6个平行,以不加P-AgNPs为空白对照,并以公式:ODP-AgNPs-ODControl)/ODControl计算P-AgNPs抑制哈夫尼亚菌生物被膜形成的抑制率。将培养至OD595nm为0.5左右菌株HA1、HA2按1%(v/v)接种量接种于预置无菌盖玻片、含有终浓度为1/128~1/2MICAgNPs或不含有AgNPs的4mLLB肉汤的6孔板中培养24h,取出盖玻片用生理盐水冲洗除去浮游细菌后用甲醇固定15~20min,再用0.1%结晶紫染液染色15~20min,清洗并自然风干后于光镜下观察生物被膜形态的变化。将培养至OD595nm为0.5左右HA1、HA2按1%(v/v)接种量接种于预置无菌锌片、含有终浓度为1/128~1/2MICAgNPs或不含有AgNPs的2mLLB肉汤的24孔板中培养24h,取出锌片用生理盐水冲洗除去浮游细菌后转入新的无菌孔板中加入4℃预冷的2.5%戊二醛固定液固定2~4h,再用50%、60%、70%、80%、90%、100%的无水乙醇进行梯度洗脱,自然风干后喷金于扫描电镜下观察生物被膜形态变化。
通过结晶紫染色法测定1/128~1/2MIC(0.5~32μg/mL)浓度下P-AgNPs对哈夫尼亚菌HA1和HA2生物被膜形成的抑制作用,发现P-AgNPs在终浓度1/8~1/2MIC(8~32μg/mL)时对哈夫尼亚菌HA1和HA2生物被膜的形成均有部分抑制作用(图7A),且P-AgNPs在1/8MIC(8μg/mL)时对HA1和HA2形成生物被膜的抑制率分别为18.28%和14.89%,在1/4MIC(16μg/mL)时对HA1和HA2形成生物被膜的抑制率分别为32.26%和25.53%,在1/2MIC(32μg/mL)时对HA1和HA2形成生物被膜的抑制率分别为37.63%和51.06%。
通过光学显微镜和扫描电镜观察哈夫尼亚菌HA1和HA2生物被膜的微观结构,发现在1/2MIC(32μg/mL)时P-AgNPs相较于不添加P-AgNPs的空白对照组可以明显的抑制生物被膜形成,在扫描电镜下空白对照组生物被膜的微观结构更为致密、杂乱,而经P-AgNPs处理后则只产生小部分的生物被膜且可以观察到作为载体的锌片背景(图7B、图7C),但在1/128~1/16MIC(0.5~4μg/mL)的这种MIC低浓度时P-AgNPs反而会促进哈夫尼亚菌生物被膜的形成(图7A)。
(2)P-AgNPs对哈夫尼亚菌产AHLs的抑制作用
为测定P-AgNPs对哈夫尼亚菌HA1和HA2产AHLs能力的抑制作用,将培养至OD595nm为0.5左右的HA1和HA2按1%(v/v)接种于含1/128~1/2MIC浓度的P-AgNPs的10mL LB肉汤中28℃培养24h后,于8000r/min、4℃下离心2min,取200μL上清液注入经牛津杯打孔的含有紫色色杆菌CV026的LB营养琼脂中,培养24h测定产紫圈的直径以测定P-AgNPs对AHLs的抑制作用,每组3个平行,以不加P-AgNPs为空白对照。并以(DControl-DP-AgNPs)/DControl计算P-AgNPs对哈夫尼亚菌HA1和HA2产AHLs的抑制率,其中D为形成的变色圈直径。
通过测定1/128~1/2MIC(0.5~32μg/mL)浓度下P-AgNPs对哈夫尼亚菌HA1和HA2产生AHLs的抑制作用,发现P-AgNPs随着sub-MIC浓度的增加,即在1/128~1/8MIC(0.5~8μg/mL)浓度时P-AgNPs对哈夫尼亚菌HA1和HA2产生AHLs的抑制作用呈浓度依赖趋势,而在1/2MIC(32μg/mL)浓度时P-AgNPs只能抑制哈夫尼亚菌HA1产生AHLs,对哈夫尼亚菌HA2产生AHLs的能力则没有抑制效果,因此P-AgNPs分别在1/2MIC(32μg/mL)和1/8MIC(8μg/mL)时对哈夫尼亚菌HA1和HA2产AHLs能力的抑制效果最好,且最大抑制率分别为28.67%和7.36%(图8)。
(3)P-AgNPs对哈夫尼亚菌产胞外蛋白酶和胞外多糖的抑制情况分析
为测定P-AgNPs对哈夫尼亚菌HA2产胞外蛋白酶能力的抑制作用,将培养至OD595nm为0.5左右的菌株HA2按1%(v/v)接种于含1/128~1/2MIC浓度的P-AgNPs的10mL LB肉汤中28℃培养24h后,于8000r/min、4℃下离心2min后取上清作为粗酶液,采用酪蛋白为底物并以福林酚法测定P-AgNPs对哈夫尼亚菌HA2胞外蛋白酶活力的抑制作用,即将2mL0.5%酪蛋白溶液于28℃水浴5min后加入预热的1mL上清液(粗酶液)常温反应10min后,加入10%三氯乙酸溶液3mL终止反应15min后于12000r/min、28℃下离心10min,取1mL上清液和0.55mol/LNa2CO3溶液5mL混匀后加入福林酚试剂1mL,显色15min后测定OD680nm值。每组3个平行,以水代替粗酶液作为空白对照,以y=0.0097x+0.0994(R2=0.9992)为酪氨酸标准方程,式中x为酪氨酸含量(μg/mL),y为680nm处吸光值。哈夫尼亚菌HA2的蛋白酶活力定义为在28℃时,1mL粗酶液1min水解酪蛋白产生1μg酪氨酸为1个酶活力单位,并根据以下公式计算出粗蛋白酶液的酶活力。酶活力(U/mL)=(OD680nm×K×V)/(t×N);式中,OD680nm为样品的OD680nm值,K为标准曲线OD680nm为1时酪氨酸的含量(μg),t为酶促反应10min的时间(min),V为酶促反应6mL的总体积(mL),N为粗酶液的稀释倍数1。
为测定P-AgNPs对哈夫尼亚菌HA1和HA2产胞外多糖能力的抑制作用,将培养至OD595nm为0.5左右的菌株HA1和HA2按1%(v/v)接种于含1/128~1/2MIC浓度的P-AgNPs的10mL LB肉汤中28℃培养24h后,于8000r/min、4℃下离心2min后,上清液加入10%三氯乙酸(w/w)并静置12h后于8000r/min、4℃下离心30min以除去蛋白,取上清液加入3倍体积95%乙醇于4℃静置24h后于8000r/min、4℃下离心30min以醇沉多糖,沉淀用10mL ddH2O溶解并用苯酚-硫酸法测定胞外多糖含量,即先在冰水浴中加入0.5mL 6%苯酚,充分振荡混匀后逐滴加入3mL浓硫酸后于沸水浴中反应20min后于凉水中静置10min并在OD490nm处测定吸光度值,以未加入多糖为空白对照,以y=0.0103x+0.0075(R2=0.9996)为葡萄糖标准方程,式中x为葡萄糖含量(μg/mL),y为490nm处吸光值。
通过福林酚法测定1/128~1/2MIC(0.5~32μg/mL)浓度下P-AgNPs对哈夫尼亚菌HA2蛋白酶活性的抑制作用,发现P-AgNPs在1/128~1/4MIC(0.5~16μg/mL)浓度下随着浓度的增加也会增强对哈夫尼亚菌HA2胞外蛋白酶活力的抑制效果,且在1/4MIC(16μg/mL)时有最大抑制率为27.86%,但在1/4~1/2MIC(16~32μg/mL)浓度下P-AgNPs反而会对哈夫尼亚菌HA2胞外蛋白酶活力的抑制作用呈现出下降趋势(图9A)。
通过苯酚-硫酸法测定1/128~1/2MIC(0.5~32μg/mL)浓度下P-AgNPs对哈夫尼亚菌产胞外多糖的抑制作用,发现P-AgNPs对哈夫尼亚菌产胞外多糖的抑制同其对生物被膜的抑制趋势相似,即在1/128~1/8MIC(0.5~8μg/mL)浓度时呈现促进作用,在1/4~1/2MIC(16~32μg/mL)浓度时才能呈现抑制作用,且在1/2MIC(32μg/mL)浓度时对哈夫尼亚菌HA1和HA2产胞外多糖的能力有最大抑制率并分别为29.20%和30.03%(图9B)。
(4)P-AgNPs对哈夫尼亚菌群集和泳动能力的抑制作用
将培养至OD595 nm为0.5左右的哈夫尼亚菌菌株HA1和HA2吸取4μL菌液接种于含1/128~1/2MIC P-AgNPs的群集培养基(NaCl 5g/L、琼脂6g/L、葡萄糖5g/L、蛋白胨10g/L,余量为水)和泳动培养基(NaCl 5g/L、琼脂3g/L、胰蛋白胨10g/L,余量为水)中心,待其风干后于28℃培养24h并观察其迁移直径以验证P-AgNPs对哈夫尼亚菌群集和泳动能力的抑制作用,每组3个平行,以不加P-AgNPs为空白对照。
通过测定1/128~1/2MIC(0.5~32μg/mL)浓度下P-AgNPs对哈夫尼亚菌HA1和HA2的群集和泳动抑制能力,发现P-AgNPs对于哈夫尼亚菌HA1和HA2的群集能力均有抑制作用且均表现出随着sub-MIC浓度的增加抑制作用增强的趋势,其中对于哈夫尼亚菌HA2群集能力的抑制作用最为明显,即P-AgNPs在1/8MIC(8μg/mL)时即可抑制哈夫尼亚菌HA2的群集能力,而对于哈夫尼亚菌HA1则需P-AgNPs在1/4MIC(16μg/mL)时才能抑制其群集能力(图10A);在P-AgNPs对于哈夫尼亚菌HA1和HA2泳动能力的抑制方面,发现P-AgNPs也表现出随着sub-MIC浓度的增加抑制作用增强的趋势,且在1/8MIC(8μg/mL)时即可抑制哈夫尼亚菌HA1和HA2的泳动能力(图10B)。
Claims (10)
1.一种基于桃叶提取液制备纳米银的方法,其特征在于,包括以下步骤:将桃叶提取液与银离子溶液混合,反应生成纳米银。
2.根据权利要求1所述的方法,其特征在于,所述的桃叶提取液是将桃叶洗净后剪碎,加入水蒸煮至溶液变为黄绿色后,抽滤后获得桃叶提取液。
3.根据权利要求1所述的方法,其特征在于,所述的银离子溶液是AgNO3溶液。
4.根据权利要求1所述的方法,其特征在于,所述的反应条件为60℃避光进行水热法反应6h。
5.根据权利要求1所述的方法,其特征在于,具体步骤为:
(1)将采摘后的桃叶叶片洗净剪碎后,称取5.0g加入150mL蒸馏水于60℃蒸煮至溶液变为黄绿色,抽滤即得桃叶提取液;
(2)量取30mL的桃叶提取液与10mL10mM的AgNO3混匀后,在避光条件下60℃进行水热法合成6h,待溶液变为黑褐色终止,12000r/min、4℃离心45min弃上清,沉淀用10mL无菌水洗涤3次后冻干。
6.权利要求1-5任一所述的方法制备的纳米银。
7.权利要求6所述的纳米银在制备抑制哈夫尼亚菌药物中的应用。
8.根据权利要求7所述的应用,其特征在于,所述的纳米银的抑菌浓度为64μg/mL。
9.根据权利要求7所述的应用,其特征在于,所述的哈夫尼亚菌为哈夫尼亚菌HA1或HA2。
10.根据权利要求7所述的应用,其特征在于,所述的抑制哈夫尼亚菌为抑制哈夫尼亚菌生物被膜形成、抑制哈夫尼亚菌产AHLs、抑制哈夫尼亚菌产胞外蛋白酶、抑制哈夫尼亚菌产胞外多糖、抑制哈夫尼亚菌群集能力、以及抑制哈夫尼亚菌泳动能力中的至少一种。
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