Synergistic bacteriostatic properties of 4-hydroxycoumarin-Ag composite
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摘要:
抗生素的滥用导致大量耐药菌的出现，这对卫生健康和社会经济造成严重威胁，因此，迫切需要开发新型抗菌剂来解决细菌耐药问题。当今现有的抗菌剂大致可分为有机抗菌剂和无机抗菌剂，有机抗菌剂因易产生耐药性而限制了其发展，无机抗菌剂Ag NPs因其具有抑菌活性强、杀菌持久、广谱抑菌、且不产生耐药等特性被应用于生物和医用材料等领域。研究表明，Ag NPs粒径越小，比表面积越大，抑菌活性越强，但粒径越小，表面能越高，Ag NPs越易团聚，从而降低其抑菌活性。且目前相关研究多集中于复合材料的基础抑菌活性，而对于两者的结合方式和初步的抑菌机制探究的较少。本研究以软硬酸碱理论为基础，将4-羟基香豆素和Ag NPs配位制备4-羟基香豆素-Ag复合抑菌材料，以革兰氏阴性菌E.coli、革兰氏阳性菌S.aureus和耐药菌T-Salmonella为模型菌研究协同抑菌活性，并讨论其抑菌机制。所制备得到的复合材料不仅具备单独无机抗菌剂和有机抗菌剂的杀菌特性，且协同作用可明显提高其抑菌性能。本论文旨在得到性能更加优异的抗菌材料，并探讨其抑菌机制，其不仅可以解决细菌耐药性问题，还可以为抗生素的改性和研发新型抗生素提供实验依据。 4-羟基香豆素-Ag复合材料抑菌机制图 Abstract: The abuse of antibiotics leads to the emergence of a large number of drug-resistant bacteria, which poses a serious threat to health and social economy. Therefore, it is urgent to develop new antibacterial agents to solve the problem of bacterial drug resistance.In this paper, silver trifluoroacetate (CF3COOAg) as raw material, using chemical reduction method to prepare nano-silver (Ag NPs) particles, and ultrasonic assisted coordination with 4-hydroxycoumarin to prepare 4-hydroxycoumarin-Ag NPs new composite antibacterial material. Transmission electron microscopy (TEM), X-ray diffrotometer (XRD), X-ray photoelectron spectroscopy (XPS), UV-visible absorption spectroscopy (UV-Vis), infrared spectroscopy (FTIR), Zeta potential and theoretical calculation were used for characterization. Gram-negative bacteria E.coli, Gram-positive bacteria S. aureus and drug-resistant bacteria T-Salmonella were used as model bacteria to study the synergistic bacteriostatic activity and bacteriostatic mechanism of 4-hydroxycoumarin-Ag.Compared with Ag NPs, the antibacterial efficiency of the composite material for E.coli, S. aureus and T-Salmonella has been improved 62.5%, 37.5% and 44.4% respectively. When the concentration of the composite mat- erial was 150 μg/mL, the bacteriostatic rate of the tested bacteria could reach 99.9% within 60min.The inhibition mechanism showed that the composite material could significantly destroy the bacterial cell wall and enter the bacterial interior to inhibit the bacterial respiratory system.The material not only has the unique bactericidal properties of inorganic antibacterial agent and organic antibacterial agent, but also has stronger antibacterial activity and can solve the problem of bacterial resistance, which can provide scientific basis for the modification of antibiotics and the development of new antibacterial agent.-
Key words:
- Synergistic antibacterial /
- Ag NPs /
- 4-hydroxycoumarin /
- antibacterial mechanism /
- coordination
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图 1 Ag NPs以及 4-羟基香豆素-Ag复合材料表征(a、b)Ag NPs的透射电镜图像和粒径分布图；(c)Ag NPs和复合材料的XRD谱图；(d)Ag NPs的XPS谱图；(e)Ag NPs、4-羟基香豆素以及复合材料的UV-Vis吸收光谱；(f)Ag NPs、4-羟基香豆素以及复合材料的FT-IR光谱；(g)Ag NPs与4-羟基香豆素结合理论计算分析图
Figure 1. Ag NPs and 4-hydroxycoumarin-Ag composite characterization (a, b) Transmission electron microscope image and particle size distribution map of Ag NPs; (c) XRD spectra of Ag NPs and composite materials; (d) XPS spectra of Ag NPs; (e) UV-Vis absorption spectra of Ag NPs, 4-hydroxycoumarin and composite materials; (f) FT-IR spectra of Ag NPs, 4-hydroxycoumarin and composite materials; (g) Theoretical calculation and analysis diagram of Ag NPs combined with 4-hydroxycoumarin
图 2 不同材料对E.coli 、S.aureus 和T-Salmonella的滤纸片扩散照片(注：A、B、C、D分别对应溶剂二甲基亚砜、Ag NPs、4-羟基香豆素以及复合材料。图中(a1-a4)代表浓度为35.5、75、150、300 μg/mL的不同抑菌材料(4-羟基香豆素负载量为5%)对E.coli的抑菌结果照片。(b1-b4)、(c1-c4)为S.aureus以及T-Salmonella的抑菌结果照片。(d)、(e)、(f)分别为E.coli、S.aureus、T-Salmonella的抑菌圈直径随浓度变化曲线)
Figure 2. Different materials for E.coli、S.aureus and T-Salmonella filter paper spread photos (Note: A, B, C and D correspond to dimethyl sulfoxide, Ag NPs, 4-hydroxycoumarin and composite materials respectively. In the figure, (a1-a4) represents the bacteriostatic results of E.coli of different bacteriostatic materials(The loading of 4-hydroxycouma- rin was 5%)with concentrations of 35.5, 75, 150 and 300 μg/mL.(b1-b4) and (c1-c4) show the antibacterial results against S.aure- us and T-Salmonella. (d)、(e)and (f) show E.coli, S.aureus and T-Salmonella bacteriostatic rings diameter changes with concentr- ation, respectively)
图 3 4-羟基香豆素-Ag复合材料菌落计数照片E.coli(a)、S.aureus(b)以及T-Salmonella(c)的菌落计数分布图。(d)为复合材料的时间-杀菌曲线图。(e)图为三种测试菌的在不同时间的抑菌率比较图
Figure 3. Photo of colony count of 4-hydroxycoumarin-Ag composite E.coli(a), S.aureus(b) and T-Salmonella(c) colony count diagrams. Figure (d) is the time-germicidal curve of the composite. Figure (e) shows the comparison of the bacteriostatic rates of the three tested bacteria at different times
图 4 4-羟基香豆素-Ag复合材料与E.coli、S.aureus以及T-Salmonella作用不同时间的Zeta电位值图
Figure 4. The Zeta potential diagrams of the 4-hydroxycoumarin-Ag composite have different reactions with E.coli, S.aureus and T-Salmonella for different times
图 5 4-羟基香豆素-Ag复合材料对E.coli (b)、S.aureus(d)及T-Salmonella(f)的PI染色照片。(a、c、e)为对应的纯菌对照效果图
Figure 5. The 4-hydroxycoumarin-Ag composite has been able to color PI photos of E.coli (b), S.aureus (d) and T-Salmonella (f). (a), (c) and (e)are the corresponding pure bacteria control renderings
图 6 4-羟基香豆素-Ag复合材料对E.coli(a)、S.aureus(b)、T-Salmonella(c)的微量热分析以及复合材料毒理性实验结果分析(d)
Figure 6. Microthermal analysis of E.coli (a), S.aureus (b) and T-Salmonella (c) for 4-hydroxycoumarin-Ag composite and analysis of toxicity test results of composites (d)
图 7 4-羟基香豆素-Ag复合材料抑菌机制图
Figure 7. Diagram of antibacterial mechanism of 4-hydroxycoumarin-Ag composite
表 1 溶剂、Ag NPs、4-羟基香豆素、4-羟基香豆素-Ag对E.coli 、S.aureus 和T-Salmonella的抑菌圈尺寸
Table 1. The size of bacteriostasis circles for E.coli, S.aureus and T-Salmonella of solvent, Ag NPs, 4-hydroxycoumarin and 4-hydroxycoumarin -Ag
Bacterial Concentration/ (μg·mL−1) Inhibition zones/cm DMSO Ag 4-hydroxycoumarin 4-hydroxycoumarin-Ag E. coli 35.5 0.6 0.6 0.6 0.7 75 0.6 0.7 0.6 1.0 150 0.6 0.9 0.65 1.5 300 0.6 1.2 0.7 1.95 S. aureus 35.5 0.6 0.6 0.6 0.65 75 0.6 0.65 0.6 0.9 150 0.6 0.75 0.65 1.25 300 0.6 1.2 0.85 1.62 T-Salmonella 35.5 0.6 0.6 0.6 0.6 75 0.6 0.65 0.65 0.8 150 0.6 0.7 0.65 1.1 300 0.6 0.9 0.7 1.3 
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