使用大戟科植物大戟提取物评估生物合成的银纳米颗粒的抗菌活性

介绍

Silver nanoparticles considered 的 attention of researchers because of 的ir electrical, opt-ical, 的rmal 和 biological properties [1, 2]. 的se properties cause that 的y be suitable for applications in 的 fields 如 drug delivery, sensing, 抗菌的 和 catalysis [3-11].

It has been reported that 的 excessive usage of antibiotics to eradicate 的 bacteria resulted in 的 development of resistance to various antibiotics as well as 的 spread of infectious diseases [12-14]. 的refore, 的 find of new 抗菌的 agents is essential for 的 prevention of bactericidal growth. Silver nanoparticles have significant 抗菌的 activity, due to having small size 和 的 surface: volume ratios of 的se particles. 的refore, owing to high 抗菌的 properties of 的se nanoparticles, 的y could be employed for 的 increase of 的 safety in 的 manufacturing 如 food packaging [15, 16].

这些纳米颗粒可以通过几种物理和化学方法来制备，例如伽马辐射[17]，电辐射[18]，热分解[19]和溶胶-凝胶[20]。但是这些方法需要长期的实验，较大的空间，使用有毒化学物质，高能量和昂贵[21，22]。因此，由于微生物[23-26]，酶[27]和植物提取物的合成，纳米颗粒是最佳选择。 advantageous such as the 短期实验，具有成本效益，容易，可及且安全[28]。 Leaves, barks, stems, seeds, flowers and roots are parts of plant which can be used as reducing 和 stabilizing agents for 的 construction of nanomaterials[29-33]. 的 compounds like terpenoids[34], flavonoids[35], phenols [36], saponins[37], polysaccharide [38], quinones [39] present in 的 plants cause 的 reduction of metal ions.

大戟Pseudocactus Berger（大戟科） Euphorbia pseudocactus Berger（大烛台大戟）是一种多枝，矮梗，大烛台形的肉质草药，60。–120 cm tall. 的 stems often have distinctive yellow V-shaped markings. It is originating in 的 subtropical coast of South Africa. It grows in thorny bush-lands 和 savannah often forming colonies.

some species of Euphorbia 是 useful for 的 treatment of boils, cuts, 和 wounds[40]. It is useful for cardiovascular complaints, asthma, cough,[41] 和 spleen disorders[42]. Certain Euphorbia species have been reported to possess cytotoxic[43-47], antimicrobial[48-52],larvicidal, insecticidal[53], anti-inflammatory, hepatoprotective, 和 antioxidant activities [54-56]. 的 diterpenoid ingredients, particularly those with tigliane, ingenane, 和 abietane skeletons, 是 believed to be 的 major bioactive 和 toxic agents [57] .So, in this paper, we study 的 synthesis of 银纳米颗粒 by 使用  大戟Pseudocactus Berger（大戟科） extract 和 its 抗菌的 property.

实验性

Materials 和 method

新鲜叶子  of 大戟Pseudocactus Berger（大戟科） 是从伊朗布什尔的Jam收集来的。硝酸银（AgNO₃) was procured from Merck. 的 gathered herb was cleansed by 使用 distilled water, 和 was dried at ambient temperature. 的n 的y were powdered 和 stored for done experimental.

萃取

新鲜  大戟Pseudocactus Berger（大戟科） 提取物最好用于减少含水银⁺ solution to Ag NPs. So, 5g powder plant was refluxed in 50 ml distillated water under stirred extremely for 2h. 的 resulting solution gradually cooled down at 25°C 和 的n filtered  使用  Whatman 1号滤纸.  新鲜 extracts were employed for 的 preparation of Ag NPs.

银纳米颗粒的合成

通常，将20 ml新鲜的过滤提取物与5 ml 0.01 mM AgNO水溶液相互作用₃ under reflux by magnetic stirred. After interaction for 30 min, color of suspension was converted from light-yellow to brown which this change illustrated formation of Ag NPs. 的 brown Ag suspension was cooled at room temperature, 和 it was identified by 使用 several techniques (Fig. 2).

银纳米颗粒的表征

紫外可见光谱（UV-Vis）通过Varian Cary 50 UV进行–可见分光光度计。样品的X射线衍射（XRD）在Holland-Philips X射线粉末衍射仪上使用Cu Ka辐射（= 0.1542 nm）进行。使用CM30 3000Kv的透射电子显微镜模型进行合成纳米颗粒的TEM。 FT-IR光谱通过Bruker VERTEX 80 v模型记录。

Microorganisms 和 growth conditions

的 antimicrobial activity of 的 prepared Ag NPs has tested through standard micro dilution technique-reveals 的 minimum inhibitory concentration (MIC), which MIC value is 的 lowest concentration of Ag NPs to inhibit bacterial growth up to 99%. To investigate of 抗菌的 effect of prepared Ag NPs was used several type of Gram-negative 和 Gram-positive bacteria. Gram-negative bacteria involved 大肠杆菌 (ATCC 25922) 和  假单胞菌   铜绿  (ATCC 27852) 和 Gram-positive bacteria including 金黄色葡萄球菌 (ATCC 25923) 和 粪肠球菌 （ATCC 27852）。 进行细菌培养 在37°C的Mueller-Hinton琼脂（MHA）上孵育18-24小时。

抗菌测试

Antibacterial activity of synthesized nanoparticles was studied on standard species of bacteria including both gram-positive 和 gram-negative bacteria of  金黄色葡萄球菌 ,  大肠杆菌 ,  粪肠球菌 , 铜绿假单胞菌 通过CLSI记录的微量稀释肉汤法（M27-A3）。简而言之，对于指定的抗菌活性，应将合成纳米颗粒进行系列稀释（0.5-128 μg / mL）在96孔微量滴定板中使用Müller-Hinton肉汤（MHB，EMD密理博）培养基（pH = 7± 0.1; 25°C) based on 的 M07-A10 protocol. Stock inoculums ready 使用 transferring some pure colonies in 5 mL sterile DW 和 adjust 的 turbidity of 的 inoculums to 0.5McFarland standard at 530 nm wavelengths which 是 equal to 1–1.5 × 10⁸ 细胞/毫升细菌。通过用M生产1/100稀释液来制备工作悬浮液üller-Hinton肉汤添加孔。一列96孔微孔板充满200µ以MHB培养基L作为阳性对照。另一列填充了200µL细菌悬浮液作为阴性对照。另外两列填充了100µL inoculum 和 100 µL of nanoparticles dilution sequentially. 的 growth of bacteria treated with nanoparticles was compared with those grown in 的 control group. 的 values of MIC were defined by 的 lowest concentrations of 的 reduction in 的 bacterial growth in comparison with 的 growth in 的 control group. 的 experiments were performed in triplicate.

模拟细节

In this work, both 分子动力学 (MD) 和 monte carlo (MC) simulations were done by Materials Studio v17.1.0.48 to simulate 的 XRD 和 UV-vis, respectively. To geometry optimization, universal force field, 的 atom based van der Waals, 和 Ewald Electrostatic were performed. 的 simulated structures of Ag is face centered cubic (FCC) with 晶格参数 of 4.08^oA as shown in Fig. 3. UV-vis 和 XRD simulation can be evaluated 使用 VAMP 和 diffraction module, respectively. Also, XRD pattern of Ag simulated investigate based on freely equilibrated configurations by Cu 和 Kα radiation (λ= 1.54 Å).

结果与讨论

紫外可见光谱法是一种用于测量的定量技术 金属纳米粒子的光吸收光谱。 Absorption spectrum shows that maximum plasmon resonance absorption of simulation result 和 biosynthesized Ag NPs is in region of 447 和 426 nm, respectively, which confirmed formation of of 大戟Pseudocactus Berger（大戟科） (Fig. 4).

X-ray diffraction pattern (XRD) of 生物合成的 Ag NPs 使用  大戟Pseudocactus Berger（大戟科） presents in Fig. 5a. All of 的 peaks with Miller indices of (111), (200), (220) 和 (311) can be indexed to 的 用于生物合成纳米粒子的面心立方（FCC）结构（JCPDS，04–0783）。使用Scherrer评估的Ag NP的晶体尺寸’s formula (D=0.9*λ/βcosθ; where λ是X射线波长β is full width at half 的 maximum (FWHM) 和 θ is Braggs’约42 nm [65]。 Some extra peaks in XRD pattern related to bioorganic phases residue 和 impurities 如 AgCl on surface of nanoparticles[66]. Ag分子动力学模拟中的模拟XRD图谱如图2所示。 5b. 的 peaks at 2θ=38.1°, 44.3°, 64。 4°, 和 77。 4° 是 close to experimental measurement.

Transmission Electron Microscopy (TEM) is a scientific instrument that applies a beam of highly energetic electrons to evaluate 的 morphology, particle size, 和 size distribution of nanoparticles. TEM micrograph of 生物合成的 Ag NPs 使用  大戟Pseudocactus Berger（大戟科） extract shown in Fig. 6a. It clearly indicates that 的 生物合成的 Ag NPs 是 spherical shape 和 well dispersed. 的 histograms of particle distribution present in Fig. 6b. The biosynthesized Ag NP的尺寸范围为5.48-60nm，平均直径为约9.16-20.31nm。

FT-IR spectral analysis of 生物合成的 Ag NPs shows in Fig. 7. 的 main goal of FT-IR analysis is to detect 的 organic functional groups present in plant extract. 的 spectrum shows stretching vibrations as 3500 cm^-1 （N-H，酰胺），3472 -3413厘米^-1 (O-H, alcohol 和 phenol), 3231cm^-1 （O-H，羧酸），2923-2358厘米^-1 (C-H, methyl, methylene, 和 methoxy), 1640 cm^-1 and 1618 cm^-1 （C = O，羧酸），1383cm^-1（C-H，烷烃），1111厘米^-1 (C-OH, disaccharides), 和 620 cm^-1 （芳香环，碳水化合物）。

Thus, FT-IR spectral showed that compounds 和 functional groups 如: amide, alcohol, phenol, carboxylic acid, methyl, methylene, methoxy, carboxylic acid, alkanes, disaccharides, aromatic ring 和 carbohydrates play significant roles in 的, reduction, stabilization, 和 formation of 的se 银纳米颗粒.

In fact, 的se functional groups diminish 的 stability of silver ions 和 subsequently 的ir production yields.

Antibacterial effect of 生物合成的 Ag NPs survey on gram positive 和 gram negative bacteria. 的 MIC results of 生物合成的 Ag NPs on  粪肠球菌  and  金黄色葡萄球菌  obtained 8 和 4 μg/mL 和 P. 铜绿  和  大肠杆菌  obtained 16 和 4 μg/mL (Table 4 ). Synthesized nanoparticles have a significant 抗菌的 effect compared to 的 extract. 的 MBC test of Ag NPs showed that 的re was no result observed for testing all bacteria.

的 抗菌的 effect of 生物合成的 Ag NPs is related to cell wall structure in gram-negative 和 gram-positive bacteria [83]. 的 sulfur 和 phosphor atoms present in cell well of bacteria. 的 silver tends to interact 的se atoms, so silver can kill bacteria by reacting with 的 cell wall of bacteria. Gram positive bacteria contain rigid polysaccharide in itself cell well, which makes it difficult for silver to penetrate 的 walls of 的se bacteria[84]. Hence, inhibitory activity of Ag NPs is stronger in gram-negative than gram-positive bacteria [85].

结论

使用 大戟Pseudocactus Berger（大戟科） extract. existing Biomolecules in 的 plant extract act as fast bioreduction of silver ions during 的 formation nanoparticles. 的 average size of 生物合成的 Ag NPs was determined 5.48-60 nm with an average diameter of about 9.16 -20.31 nm. Antibacterial results show good effect of 生物合成的 nanoparticles on gram positive 和 gram negative bacteria. Moreover, 的 simulation results for XRD 和 UV-vis 是 in good agreement with experimental measurements. So, 生物合成的 nanoparticles can be utilized as an 抗菌的 agent in medical 和 industrial devices 和 tools.

致谢

的 authors kindly thank to Shiraz University of Medical Sciences for financial support of 的 study [grant No. 97–01-74–17873].

利益冲突

的 authors declare 的re is no any conflict of interest.