Synthesis and characterization of novel silver nanoparticles using Chamaemelum nobile extract for antibacterial application

The present study reports green synthesis of silver nanoparticles (AgNPs) at room temperature using aqueous Chamaemelum nobile extract for the first time. The effect of silver nitrate concentration, quantity of the plant extract and the reaction time on particle size was optimized and studied by UV–Vis spectroscopy and dynamic light scattering. The appearance of brownish color with λmax of 422 nm confirmed the formation of AgNPs. Synthesized nanoparticles were further characterized by Fourier transform infrared spectroscopy, x-ray diffraction and transmission electron microscopy. In addition, antimicrobial activity of the AgNPs against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Bacillus subtilis was evaluated based on the inhibition zone using the disc-diffusion assay and measurement of minimal inhibition concentration and minimal bactericidal concentration by standard microdilution method. In conclusion, synthesis of nanoparticle with aqueous Chamaemelum nobile extract is simple, rapid, environmentally benign and inexpensive. Moreover, these synthesized nanoparticles exhibit significant antibacterial activity.


Introduction
The field of nanotechnology is one of the emerging areas of research in modern materials science, especially in biotechnology [1]. Nowadays, noble metal nanoparticles are being used in a broad range of application in the fields of biology, medicine, physics, chemistry and material science [2,3]. Among nanostructured noble metals the most important commercialized nanoparticles are silver nanoparticles (AgNPs). AgNPs have attracted considerable attention for their application in molecular imaging [4], drug delivery [5], photonics [6,7], micro-electronics [8], biomedicine [9,10] and biosensors [11]. Different physical and chemical techniques have been adapted throughout the world to develop AgNPs. In recent years, attention has been focused on the use of environ mentally benign materials like plant extracts, bacteria and fungi for the synthesis of nanoparticle and green synthesis of metal nanoparticles has received considerable attention because of its numerous benefits over other chemical and physical methods [12,13]. Green synthesis of nanoparticles is a simple and reliable method which creates nanoparticle with good stability and appropriate dimensions. Plant-mediated nanoparticles synthesis is preferred because it is possible to produce large scale nanoparticles with a negligible cost; it is environmental friendly and, at the same time, safe for human therapeutic use [14]. Although synthesis of AgNPs by plant extracts has already been reported with different plants such as Terminalia cuneata [15], Erythrina indica lam [16], Calotropis gigantea L. [17], Pistacia atlantica [18], Cocos nucifera [19], Alternanthera dentate [20], Croton sparsiflorus morong [21], Tribulus terrestris [22], Desmodium gangeticum [23] and Prosopis farcta [24] there is still a lot of attention paid to this field because of the diversity and the high potential of plants in producing nanoparticles with different shapes [18].
In the last three decades, there are many new antibiotics that have been produced by pharmacological industries. However, the resistance to these drugs has also increased in microorganisms. The problem of microbial resistance is growing and the outlook for the use of antimicrobial drugs in the future is still uncertain [25,26]. Due to the increasing bacterial resistance to classic antibiotics, the development and modification of novel antimicrobial agents (e.g. natural and inorganic based antimicrobial substances) are necessary [27]. In the recent years, studies on AgNPs have developed because of their inhibitory effect on bacteria [28,29]. Antimicrobial properties of AgNPs triggered the use of these nanoparticles in different fields of medicine, various industries, animal husbandry, packaging, cosmetics, health and military [30,31]. Therefore, development of new AgNPs by green synthesis with active antimicrobial effect is favorable. Herein, the principal objective of this study was to achieve a novel approach for biosynthesis of AgNPs as a green chemistry method using aqueous extract of Chamaemelum nobile (Roman chamomile) at room temperature. Characterization of the synthesized nanoparticles was evaluated using UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), x-ray diffraction (XRD), transmission electron microscope (TEM) and dynamic light scattering (DLS). Furthermore, antibacterial activity of synthesized AgNPs was assessed against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Bacillus subtilis.

Preparation of the plant extract
Chamaemelum nobile (C. nobile) was obtained from Shiraz (Fars, Southern Iran). To prepare C. nobile aqueous extract, 5.0 g of dry plant powder was crushed into 50 ml of sterile distilled water and placed in orbital shaker for 6 h then the extract was filtered through whatman no.1 filter paper. The clearly filtered, light yellow extract was stored in refrigerator at 4 °C. The extract was used as reducing agent as well as stabilizing agent for producing AgNPs.

Biosynthesis of AgNPs
In order to prepare AgNPs, 5 ml of plant extract was added to 95 ml (1 mM) aqueous solution of AgNO 3 and it was incubated at room temperature. The reduction of metal ions was initially confirmed by visual inspection of color change from light brown to dark brown then by spectrophotometric absorption at 200-800 nm. Various reaction parameters including quantity of plant extract, concentration of silver nitrate solution and reaction time were optimized to enhance the yield of nanoparticle at room temperature (24 °C). Spectrophotometric absorption and DLS measurements were recorded for each of the trials during the reaction time (table 1).
To remove un-reacted extract from the final solution, the reaction medium containing AgNPs was centrifuged at 10 000 rpm for 15 min. The obtained precipitation was freezedried and used for further studies.

Characterization of nanoparticles
As mentioned above, the formation of AgNPs were monitored by UV-vis absorbance. Absorbance for synthesized AgNPs was taken in the range of 200 to 800 nm using HACH-DR5000 spectrophotometer. In order to estimate the average size of nanoparticles a Microtrac Nanotrac wave particle size analyzer (Microtrac, USA) was used. TEM measurements were taken on TEM instrument (Philips CM-10 electron microscope) operated at an accelerating voltage at 100 keV. Samples were prepared by drop coating of colloidal solution into formvar/ carbon coated TEM grids. To identify the functional groups of synthesized nanoparticles FTIR (Perkin Elmer) spectroscopy was performed. For XRD analysis, freeze-dried nanoparticles were coated on XRD grid and the spectra were recorded using XRD instrument operating at 40 kV and a current of 40 mA μg ml −1 , 1.9 μg ml −1 , 0.9 μg mll −1 and 0.4 μg ml −1 in a final volume of 100 μl. From the bacterial suspension containing 10 6 CFU ml −1 bacteria, 10 μl was inoculated in wells. Moreover, the bacterial suspension (without any drug) was added to three wells and served as growth control. A multi detection microplate reader (BioTek's PowerWave XS2, USA) at 600 nm was used to measure optical densities in 24 h at 37 °C and the optical density (OD) for each well was recorded every 2 h spontaneously. The MIC was defined as the lowest concentration of the compounds to inhibit the growth of microorganisms. Minimal bactericidal concentration (MBC) was determined by plotting 5 μl samples from clear wells into nutrient agar plates. The MBC was the concentration at which there was no microbial growth. All experiments were performed in triplicate and results were reported in terms of mean ± SD.

Disc-diffusion assay.
The antimicrobial activity of biosynthesized Ag-NPs was also determined by disc diffusion method [35]. Afterward, 100 μl of the suspension containing 10 6 CFU ml −1 of the test microorganisms were swabbed uniformly on nutrient agar. The discs with 6 mm diameter were impregnated with 30 μl of AgNPs at the concentration of 39 μg ml −1 for E. coli and S. typhimurium, 156.2 μg ml −1 for S. aureus and 78 μg ml −1 for B. subtilis (the concentrations were quintuple the MIC value). Quintuple MIC values for aqueous solution of AgNO 3 was also used as the final concentration for discs that served as negative control. In addition, tetracycline (30 μg/disc) was used for comparison. The inoculated plates were incubated at 37 °C for 24 h and inhibition zones were measured in terms of the diameter (in millimeters) and compared with C. nobile extract, aqueous solution of AgNO 3 and tetracycline. Each assay in the experiment was performed in triplicate.

UV-Vis spectroscopic study
As shown in figure 1, the appearance of brownish color in the reaction mixture indicates the formation of AgNPs. The change in color of the solution was due to the surface plasmon resonance (SPR) and reduction of silver ions by aqueous extract of C. nobile. Furthermore, the UV-Vis absorption spectrum of AgNPs in the range of 200-800 nm at the optimum condition revealed a peak at 414 nm in 2 h and 422 nm in 24 h (figure 2). This strong SPR peak confirms the formation of AgNPs. The color of the solution started to change within 20 min by the addition of aqueous plant extract and after 24 h of incubation there was no significant change, neither in color nor in the SPR peak. Moreover, AgNO 3 solution which was used as control showed no color change.

Optimization of different parameters
For the synthesis of AgNPs at room temperature, different parameters were optimized including quantity of plant extract, concentration of silver nitrate and reaction time. The different parameters and their average sizes measured by DLS are shown in table 1. Researches have reported that changes in the composition of the reaction mixture will control the properties of nanoparticles [35]. In this case, different amount of extract from various plants has been used to obtain the best morphology and size of nanoparticles. In the present study, increasing the amount of plant led to an increase in peak absorbance in UV/Vis spectrum. In addition, larger quantities of extract reduced the particle size. This finding is in agreement with the researches done by Ibrahim [35], Iravani and Zolfaghari [30]. Moreover, increasing the AgNO 3 concentration in the reaction mixture from 0.5 to 1 mmol l −1 led to AgNPs with larger size. Meanwhile, enhancement of the reaction time from 8 to 24 h caused reduction in the size of AgNPs. These results are in agreement with the earlier investigations conducted by Gavade et al [36], Krishnaraj et al [37] and Pourmortazavi et al [38]. Therefore, to produce the smallest size of AgNPs by C. nobile aqueous extract at room temperature, the optimum condition is as following: 0.5 mmol l −1 silver ion concentration, reaction time of 24 h, and 1 ml plant extract (trial number 9, table 1).   Adv. Nat. Sci.: Nanosci. Nanotechnol. 8 (2017) 025004 C. nobile extract. As it is shown in figure 3(b), these peaks are missing in the FTIR spectra of the AgNPs indicating that the O-H groups were one of the groups responsible for synthesis of AgNPs. Furthermore, the IR spectra of AgNPs showed an absorption band at 1375 cm −1 , corresponding to the NO 3 stretching which is due to the residue of silver nitrate. The peak at 1058 cm −1 was attributed to the C-O group which is shifted to 1053 cm −1 after the reduction of silver. The band at the peak observed at 1617 and 675 cm −1 is assigned to bounded amine groups (-NH) of leaf extract. 675 cm −1 peak also shifted to 668 cm −1 after the reduction of silver. It is well known that biological components interact with metal salts and mediate reduction process with these functional groups [39]. Comparison of the FTIR spectra of AgNPs with C. nobile aqueous extract revealed the presence of functional groups such as amines, carboxylic acids, aldehydes and alcohols which are responsible for reducing and capping the silver ions [40].

XRD analysis
The XRD spectrum was recorded to confirm the crystalline structure of synthesized AgNP (figure 4). The diffraction peaks were observed at 38.1°, 44.2°, 64.4° and 77.4° in the 2θ range. The peaks can be indexed to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) reflections of face centered cubic structure of metallic silver which suitably matched the standard diffraction data with those reported for silver by joint committee on powder diffraction standards (JCPDS) file no: 040783. The average estimated crystallite size of the synthesized AgNPs at optimum condition was in the range of 24 nm. These results were consistent with the sizes of AgNPs obtained from TEM analysis.

TEM analysis
To characterize the shape, surface morphology and size of synthesized AgNPs, TEM was used. TEM micrographs recorded from the drop coated TEM grid of the synthesized nanoparticles at optimum condition showed spherical shape AgNPs with the average size of 24.2 ± 3.1 nm (figure 5). These outcomes were consistent with the sizes of AgNPs obtained from the XRD.

DLS analysis
To analyze the particle size distribution in different trials DLS was used (Microtrac, USA). Particle size measurement was done for all trials in order to choose the primary optimum size of nanoparticles (table 1). Based on the results, the mean size of AgNPs at optimum condition was recorded 47.3 nm and the range of nanoparticles was from 39 to 78.5 nm ( figure 6). As expected, the DLS measured size is slightly larger than the TEM size. As it has been mentioned previously, TEM sizes were similar to XRD measurements, while the DLS sizes were significantly larger than both (table 1). The differences possibly reflect the fact that TEM only measures a number based size distribution of the physical size and does not include any capping agent, while DLS measures the hydrodynamic diameter, which is the diameter of the particle, plus ions or molecules that are attached to the surface and moves with the AgNPs in solution [41]. These ions or other associated molecules make the particle appear larger to the instrument in comparison to TEM. Hence, the hydrodynamic diameter is always greater than the size estimated by TEM [42]. Nevertheless, many  studies proposed the importance of hydrodynamic diameter for understanding and optimizing the size of nanoparticles and their performance in biological assays [41][42][43][44].

Antibacterial effect of silver nanoparticles
AgNPs have recently received a great deal of attention and concern due to their antibacterial activity [45]. In the present study, the biologically synthesized AgNPs showed an excellent antimicrobial activity against test microorganisms. Table 2 shows the result of antimicrobial activity for biosynthesized AgNPs, aqueous solution of AgNO 3 and aqueous extract of C. nobile and tetracycline by disc diffusion method. Clear zone around the disc was considered as the inhibitory effect. As it is shown in figure 7, AgNPs had respectable inhibition zone against all strains. The zone of inhibition of AgNPs for E. coli, S. typhimurium, S. aureus and B. subtilis was 15.1 ± 0.2, 14.3 ± 0.3, 13.0 ± 0.1 and 14.3 ± 0.2 mm, respectively. In general, AgNPs showed more anti-bactericidal activity compared with the AgNO 3 solution, the inhibition zone diameter of AgNO 3 solution was 8.1 ± 0.1, 7.6 ± 0.4, 8.3 ± 0.2 and 8.0 ± 0.1 mm for E. coli, S. typhimurium, S. aureus and B. subtilis, respectively. It should be pointed out that the C. nobile extract showed no activity towards any of the organisms.
Moreover, the antimicrobial properties of synthesized silver nanoparticles were determined by means of minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC). MIC was recorded as the lowest concentration at    In the case of AgNO 3 for gram negative bacteria E. coli and S. typhimurium the MBC was determined 15.6 μg ml −1 and 31.2 μg ml −1 while for gram positive bacteria S. aureus the MBC was 125 μg ml −1 and for the B. subtilis the value was 62.5 μg ml −1 . The least MIC and MBC value of AgNPs than AgNO 3 may be due to the smaller size of the nanoparticles [46].
In general, gram negative bacteria E. coli and S. typhimurium showed better antimicrobial activity when compared to gram positive bacteria S. aureus and B. subtilis. These results agree with those presented by researchers which have reported that gram positive bacteria are less susceptible to the antimicrobial activity of silver [47][48][49][50]. Grigor'eva et al [51] investigated the cell responses to AgNPs for S. aureus and S. typhimurium. In this study they reported that overall damage of S. aureus culture was somewhat lower than that of the culture of S. typhimurium incubated with AgNPs during the same time. Goswami et al [52] reported larger zone of inhibition and lower MBC and MIC values for E. coli in comparison with B. subtilis. In studies of Martinez-Castanon et al [53] the lower MIC values for E. coli were compared against S. aureus. Furthermore, Kim et al [49] researches showed that AgNPs inhibit E. coli at the low concentration, whereas the growth-inhibitory effects on S. aureus were mild. Evidently, these differences in gram  positive and gram negative bacteria are based on structural features of cell envelope in these microorganisms [48,51]. The cellular wall for gram positive strains is wider than the cellular wall for gram negative strains [54]. Thick cell wall of gram positive bacteria is because of their multiple layers of peptidoglycan compared to the cell wall of gram negative bacteria. Several layers of peptidoglycans and molecules of teichoic acids or lipoteichoic acids make a strong negative charge on the cell wall of gram positive bacteria which may contribute to sequestration of free silver ions. Therefore, the outer membrane of gram-positive bacteria may allow less silver to reach the cytoplasmic membrane than gram negative bacteria [47,48,55]. Consequently, gram negative bacteria are more susceptible.

Conclusion
Green synthesis is an ecofriendly and cost effective procedure for large scale production of nanoparticles. In the present study, silver nanoparticles were synthesized using C. nobile aqueous extract. By changing the quantity of plant extract, silver ion concentration and reaction time at room temperature the size of nanoparticles was varied. At optimum condition the average diameter of AgNPs was 24 nm. The development of nanoparticles was monitored by UV-Vis spectroscopy and the primary particle size was measured by DLS. Characterization of nanoparticle at optimum condition was evaluated by TEM analysis and XRD measurements. FTIR analysis confirmed the bioreduction of Ag + ions to AgNPs by various functional groups. Moreover, the synthesized nanoparticles exhibit a pronounced antibacterial activity against different microorganisms.