Investigating the Possibility of Green Synthesis of Silver Nanoparticles Using Vaccinium Arctostaphlyos Extract and Evaluating Its Antibacterial Properties

Hindawi BioMed Research International Volume 2021, Article ID 5572252, 13 pages https://doi.org/10.1155/2021/5572252

Research Article Investigating the Possibility of Green Synthesis of Silver Nanoparticles Using Vaccinium arctostaphlyos Extract and Evaluating Its Antibacterial Properties

Sedighe Khodadadi ,1 Nafiseh Mahdinezhad ,1 Bahman Fazeli-Nasab ,2,3 Mohammad Javad Heidari ,4 Baratali Fakheri ,1 and Abdolhossein Miri 5

1Department of Biotechnology and Plant Breeding, Faculty of Agriculture, University of Zabol, Zabol, Iran 2Research Department of Agronomy and Plant Breeding, Agricultural Research Institute, University of Zabol, Zabol, Iran 3Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran 4Faculty of Pharmacy, Cyprus International University, Lefkosa, Turkish Republic of Northern Cyprus, Lefkosa, Cyprus 5Department of Pharmacognosy, School of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran

Correspondence should be addressed to Bahman Fazeli-Nasab; [email protected]

Received 25 January 2021; Revised 17 February 2021; Accepted 17 April 2021; Published 3 May 2021

Academic Editor: Nowsheen Shameem

Copyright © 2021 Sedighe Khodadadi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Objective. Vaccinium genus plants have medicinal value, of which Vaccinium arctostaphylos (Caucasian whortleberry or Qare-Qat in the local language) is the only available species in Iran. Public tendency to use herbal remedies and natural products such as synthesized nanoparticles is increasing due to the proof of the destructive side effects of chemical drugs. Nanosilver products have been effective against more than 650 microbe types. This study was aimed at assessing the possibility of green synthesis of silver nanoparticles using Vaccinium arctostaphylos aqueous extract and at evaluating its antibacterial properties, as well. Materials and Methods. In order to synthesize silver nanoparticles, different volumes of Vaccinium arctostaphylos aqueous extract (3, 5, 10, 15, and 30 ml) were assessed with different silver nitrate solution concentrations (0.5, 1, 3, 5, and 10 mM) and different reaction time durations (1, 3, 5, 10, and 20 minutes) at room temperature using a rotary shaker with a speed of 150 rpm. Ultraviolet-visible (UV-Vis) spectroscopy, X-ray diffraction analysis (XRD), Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM) were carried out. The antibacterial activity of the aqueous extract and the synthesized nanoparticles was evaluated, as well. Results. Silver nanoparticle formation process was confirmed with XRD analysis, transmission electron microscopy (TEM), and FTIR spectroscopy. The UV-Vis spectroscopy of silver colloidal nanoparticles showed a surface plasmon resonance peak at 443 nm under optimal conditions (3 ml aqueous extract volume, 1 mM silver nitrate solution concentration, and 3 min reaction time under sunlight exposure). The reduction of silver ions to silver nanoparticles in solution was confirmed, as well. Based on X-ray diffraction analysis, the size of silver nanoparticles was in the range of 7-16 nm. TEM images showed an even distribution of silver nanoparticles, with a spherical shape. FTIR spectroscopy demonstrated the presence of different functional groups of oxygenated compounds such as carboxyl, hydroxyl, and nitrogenous groups. The antibacterial properties of the synthesized nanoparticles were confirmed. Conclusion. The synthesized nanoparticles showed more antibacterial properties against gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) than gram-negative ones (Escherichia coli and Salmonella enteritidis).

1. Introduction aﬀect more than 650 microbe types [1–3]. Increasing the surface area of silver nanoparticles makes one gram of silver Strong antibacterial activity is the main objective for the nanoparticles enough to kill bacteria on a surface area of one development of nanosilver products as it has been shown to hundred square meters [4]. There are several ways to reduce 2 BioMed Research International

Ag+ to Ag0, including the use of gamma rays, ultraviolet World Health Organization reports, up to 80% of the world’s light, heating, electrochemical reduction, and reducing che- population, especially those of developing countries and poor micals such as sodium borohydride (NaBH4). Although and remote areas, use medicinal plants. On the other hand, physicochemical methods may have flourishing and well- medicinal plants are part of natural resources and a lot of known net production, however, they are usually expensive, countries have such a resource, though the type, number, time-consuming, and potentially hazardous to the environ- and variety of plant species vary based on the conditions ment [5]. and geographical location of each region [19]. Unfortunately, many chemical methods of nanoparticle The medicinal genus Vaccinium from Ericales order and synthesis have disadvantages such as the use of hazardous the Ericaceae family are popular plants whose medicinal, chemicals and the use of materials with low recyclability nutritional, and industrial value is being increased every and high energy consumption [6]. Rising the environmental day. There are 100 species of this genus worldwide of which concerns over the past decade and the exploration for envi- Vaccinium arctostaphylos is the only available species in Iran ronmentally friendly synthetic methods have always directed that has been reported in 6 regions [18]. scientists toward biosynthesis which is both economically The most important metabolites in Vaccinium arctosta- viable and biologically ecofriendly. Biosynthesis and the use phylos leaves and fruits are phenols, especially cyanosides, of plants and microorganisms such as bacteria, yeasts, fungi, which have antioxidant properties. Vaccinium arctostaphylos etc., are among the most widely used methods which have fruits contain 30% sugar, 15.5% protein, and 1.5% fat, with several advantages over chemical and physical methods branches containing hexadecanoic acid, tis-pyrene, beta- including environmental compatibility, application of non- ionone, and sandracupimaradinin. In Iranian traditional toxic and nondestructive environmental materials, the lack medicine, brewed fruit is recommended for lowering blood of need for temperature, pressure, energy and high-dose che- sugar and blood pressure [19]. micals, and also nanoparticle production on a large scale [7– Vaccinium arctostaphylos contains proanthocyanidins 9]. The use of nontoxic chemicals, harmless solvents, and which inhibit the adhesion and subsequent proliferation of recyclable materials has convinced us to apply biological bacteria, especially E. coli and Helicobacter pylori, in the epi- principles in nanoparticle biosynthesis [10]. thelium of the urinary tract and gastric mucosa, respectively. Plant chemicals including terpenoids, flavonoids, Although anthocyanins are found in many other similar ketones, aldehydes, amides, and carboxylic acids are directly fruits, it has shown that the anthocyanidin compounds in involved in ion reduction and silver nanoparticle production. Vaccinium arctostaphylos have the ability to block bacteria, Flavones, organic acids, quinones, and water-soluble phyto- as well [19]. chemicals are responsible for the immediate reduction of sil- Vaccinium arctostaphylos due to its anticyanocidal com- ver ions [11]. pounds helps to form stronger capillaries and reduce platelet Electrostatic attraction between negative charge of bacte- adhesion [19]. Vaccinium genus and Vaccinium arctostaphy- rial cell and positive charge of nanoparticles [12], Ag + bind- los as its only medicinal species in Iran has economic value. ing to protein functional groups and their denaturation [13] The antibacterial properties of silver nanoparticles have silver interference with biological macromolecules such as led to the expansion of its applications in the textile, paint, enzymes and DNA through electron emission mechanism ceramic, pharmaceutical, agricultural, livestock, food packag- [6, 14], and the fact that DNA loses its replication ability ing, and cosmetic industries. On the other hand, due to the [15] determine the antibacterial activity of nanoparticles. unique properties of nanoparticles, the use of appropriate Biosynthesis of silver nanoparticles using sesame seed production methods in order to achieve nanoparticles with extract and assessment of the shape of nanoparticles using optimal properties, lower cost, and environmental protection TEM images showed spherical particles with an average size is one of the important challenges in the field of nanotechnol- of 14 nm and silver ion to silver nanoparticle conversion per- ogy. Therefore, based on what was said, the aim of this centage of about 61.99% [16]. The dimension of silver nano- research was to synthesize silver nanoparticles using Vacci- particles produced using Cynodan dactylon extract was nium arctostaphylos, to identify important factors involved estimated to be 30-50 nm in the SEM images and 10-70 nm in the synthesis of more stable and suitable nanoparticles, in XRD spectroscopy [17]. Biosynthesis of silver nanoparti- and also to investigate the antibacterial effect of the synthe- cles using Salvia spinosa seed extract and assessment of the sized nanoparticles. shape of nanoparticles using FESEM images showed spherical particles. The nature of particles was crystalline based 2. Materials and Methods on XRD spectroscopy. The dimension of silver nanoparticles was estimated to be 19-125 nm based on FESEM imaging, 2.1. Plant and Extract Preparation. Vaccinium arctostaphylos and the diameter of growth inhibition zone against B. subtilis, fruit was collected in the summer of 2020 from West Azer- B. vallismortis, and E. coli was 15 mm, 16 mm, and 12 mm, baijan Province, Iran, and subsequently verified by the respectively [18]. botany department, Mohaghegh Ardabili University, Iran Public tendency to use herbal remedies and natural prod- (Figure 1). Collected fruits were dried in the dark. The extrac- ucts such as synthesized nanoparticles is increasing due to tion was carried out by the maceration method (soaking in the proof of the destructive side effects of chemical drugs. water solvent). 100 ml of double-distilled water was added More than 60% of Germans and Belgians and 74% of British to 10 g of dried sample and subsequently placed on a rotary people prefer natural herbal remedies. Moreover, based on shaker (Pars Azma, Iran) at a speed of 150 rpm for 24 hours. BioMed Research International 3

with a copper target emitting Cu Ka line with a wavelength of 1.54 Å and Bragg angle of 30° ≤ 2θ ≤ 80. Silver nanoparticles formed sharp peaks at 38°,44°,67°, and 78° which shows the crystal structure of the characteristic peaks correspond- ing to (111), (200), (220), and (311) planes, respectively [22]. If the peaks in these four angles are quite sharp and clear, it indicates a favorable interaction between the mineral nanoparticles and the extract, as the nanoparticles get stabi- lized, giving a characteristic sharp peak. Otherwise, the peaks are weak [23]. On the other hand, the nanoparticle Figure 1: Morphological characteristics of leaves and fruit of Vaccinium arctostaphylos L. size cannot be obtained directly from the peaks, but its limits can be obtained through the Debye-Scherrer equation [24] as follows: Following extraction, the extract was filtered using Whatman No. 1 filter paper. Dehydration was performed by anhydrous λ L K Ɵ ð Þ sodium sulfate. For microbial analysis, the extracts were dis- = β Cos , 2 solved in one ml of DMSO, with a dilution rate of 20 mg/μl. To prevent extract decomposition by light and heat, a dark glass container was used to store the extract. Following where β is the width of the peak (full width half maximum), collection, the resulting extract was stored in the above- λ is the X-ray wavelength at 1.54178 Å (15.4178 nm), L is the mentioned container in the refrigerator [20]. mean diameter of nanoparticles, θ is the angle between radi- ation and particle plane (diffraction angle here is 40.01), and 2.2. Nanoparticle Synthesis. In order to synthesize silver K is a constant which is considered 1 for cubic structures but nanoparticles, different volumes of Vaccinium arctostaphylos is 0.9 for silver. aqueous extract (3, 5, 10, 15, and 30 ml) were assessed with Nanoparticle size was obtained by averaging the values different silver nitrate solution concentrations (0.5, 1, 3, 5, obtained from three characteristic silver peaks (in the range and 10 mM) and different time durations (1, 3, 5, 10, and of 38°,44°, and 67°). That is, first, the particle size for each 20 minutes) at room temperature using a rotary shaker with sample is calculated at three characteristic silver peaks in a speed of 150 rpm. Finally, Ag2O was obtained from the the above-mentioned range. Then, the approximate size of mentioned treatments according to the following reaction: the nanoparticles is obtained by getting averages.

− − 2Ag+ + 2NO +2Na+ + 2OH → Ag O + 2NaNO +H O 2.5. FTIR Spectroscopy. For FTIR spectroscopy, 25 ml of 3 2 3 2 1 mM silver nitrate solution (Merck, Germany) was added ð Þ 1 to 5 ml of seed extract and kept at 20°C for 24 hours. After completion of the reaction, the sample was centrifuged for Ag2O precipitate was isolated and washed three times 30 minutes at 5000 rpm and then the supernatant was with distilled water. The resulting precipitate was dried at removed. This was repeated three times to ensure. After cen- ° 50 C for 2 hours. Thus, brown Ag2O was obtained. Color trifugation, the sample was dried and the powder was used change to brown and opacity was considered as the first sign for FTIR analysis using the PerkinElmer FTIR spectrometer of nanoparticle synthesis. Finally, the absorption spectra of (GX Model, USA). A similar process was conducted for the all samples were measured at 300 to 800 nm [21]. All experi- extract. ments were carried out in triplicate. 2.6. Scanning Electron Microscopy (SEM). In order to specify 2.3. UV-Vis Spectroscopy. In order to verify the presence of the size and morphology of the nanoparticles produced using silver nanoparticles, the absorption spectra of the synthesized electron microscopy, the reaction mixture was centrifuged nanoparticles were taken using the PerkinElmer spectropho- three times for 15 minutes at 12,000 rpm. Then, a few drops tometer (UV/Visible-Lambda 45 model, USA). For this pur- of the resulting precipitate were dried on a piece of aluminum pose, the treated extract was sampled at different times and foil at room temperature, and subsequently, the image was the absorption spectra were immediately measured in the taken by electron microscopy (Philips SEM, CMC-300 KV wavelength range of 420 to 450 nm, before the sample got model, Netherlands). dried. This wavelength range corresponds to the silver ele- ment, showing fluctuation in the absorption spectrum 2.7. Assessment of the Antibacterial Activity. Antibacterial ff depending on the size and shape of these particles. e ect of silver nanoparticles against gram-positive bacteria including Staphylococcus aureus (ATCC25923) and Bacillus 2.4. XRD Spectroscopy. XRD spectroscopy was used to inves- subtilis (ATCC9372) and gram-negative bacteria including E. tigate the presence of nanoparticles synthesized by fruit coli (ATCC25922) and Salmonella enteritidis (ATCC13311) extract. Actually, this method investigates the stepwise for- was inspected. The bacterial strains were obtained from mation of bioreduced nanoparticles. The crystal structure of Zabol Medical Science University. the synthesized nanoparticles was examined using an X-ray To evaluate the antibacterial activity by the disk diffusion diffractometer (APD2000 model, Italy), with an X-ray tube method [25], a microbial suspension was prepared from a 4 BioMed Research International single bacterial colony according to the 0.5 McFarland stan- dards. The bacterial strains were cultured separately on a nutrient agar medium. Eight discs including different concentrations of silver nitrate solution (0.5, 1, 3, 5, and 10 mM), negative control (aqueous extract of the plant), AgNO3(aq) and positive controls (gentamicin and streptomycin antibi- (a) μ (b) otics) were placed on the surface of each plate. 20 l of each 3 min sample was poured onto each disk. The plates were then incubated in an incubator at 37°C. After 24 hours, the inhibition zone was observed. Measurement of the inhibition diameter zone was carried out at least 3 times in each exper- iment following which the mean was calculated, as well. All concentrations of silver nanoparticles were used to specify the minimum inhibitory concentration (MIC). In this Figure 2: Color change of fruit extract (a) before and (b) after test, two positive controls (streptomycin and gentamicin adding silver nitrate solution. antibiotics) and one negative control (extract) were used. To determine the minimum bactericidal concentration of Vaccinium arctostaphylos aqueous extract and time expo- (MBC), all turbidity-free wells were cultured on Müller- sure of 3 minutes (Figure 3(c)). It was observed that with Hinton agar media and subsequently placed in an incubator increasing AgNO3 concentration, the color of the solution at 37° for 24 hours. The lowest concentration at which the gets browner. bacteria did not grow was reported as the minimum bactericidal concentration. 3.5. Assessment of the Effect of Reaction Time on Nanoparticle Synthesis. The effect of reaction time (1, 3, 5, 10, 20, and 30 3. Results minutes) on silver nanoparticle synthesis was assessed in the presence of 1 mM silver nitrate solution and 3 ml of Vac- 3.1. Biosynthesis of Silver Nanoparticles. The fresh aqueous cinium arctostaphylos aqueous extract (Figure 3(d)). It was extract of Vaccinium arctostaphylos fruit was pink which observed that with increasing the reaction time, there is an changed to dark red after adding 0.001 M silver nitrate solu- opportunity for the reduction phenomenon to be more com- tion and exposure to sunlight for 3 minutes (Figure 2). plete, increasing the number of nanoparticles formed and converting more Ag+ into Ag0. Moreover, the solution color ff ff 3.2. UV-Vis Spectroscopy. Assessment of the e ect of di erent got darker, and as a result, the maximum wavelength parameters including silver nitrate solution concentration, increased with time. Due to the instability of the formed sil- aqueous extract volume, reaction time, and the presence of ver nanoparticles, an optimum time is required to complete sunlight catalyst on the formation of silver nanoparticles the reaction, which was obtained as 3 minutes in the present indicated the optimal conditions for nanoparticle synthesis study. as 1 mM silver nitrate solution, 3 ml of aqueous extract, and 3-minute sunlight exposure. Considering that UV-Vis spec- 3.6. Assessment of the Effect of Sunlight on Nanoparticle troscopy is an important method for determining the forma- Synthesis. Assessment of the effect of sunlight on nanoparti- tion and stability of metal nanoparticles in aqueous solution cle synthesis in the presence of 1 mM silver nitrate solution [26], the formation and stability of silver nanoparticles were and 3 ml of Vaccinium arctostaphylos aqueous extract verified by the UV-Vis spectrum. Therefore, the appearance showed that in the absence of light, the reaction is not com- of a suitable peak at 443 nm indicated the formation of nano- plete, but when the sample is exposed to sunlight during particles [27]. As silver nanoparticles have adsorption at the the autumn and winter days, the reaction gets completed range of 400 to 500 nm, the adsorption band of surface plas- quickly, reducing silver particles (Figure 4). mon resonance intensification at 443 nm was observed for the synthesized silver nanoparticles (Figure 3(a)). 3.7. FTIR Spectroscopy. FTIR spectroscopy was used to identify the active and functional reducing groups of silver ions. 3.3. Assessment of the Effect of Different Extract Volumes on The results of the analysis on Vaccinium arctostaphylos fruit Nanoparticle Synthesis. The effect of different volumes of extract in the presence of silver nitrate solution showed two Vaccinium arctostaphylos aqueous extract (3, 5, 10, 15, and peaks in 1638 cm-1 and 3449 cm-1, confirming the presence 30 ml) on nanoparticle synthesis was investigated in the pres- of oxygenated compounds such as carboxyl, hydroxyl, and ence of 1 mM silver nitrate solution, sunlight catalyst, and nitrogenous groups (Figure 5). time exposure of 3 minutes. Based on the results, the optimal volume of 3 ml was selected to follow the experiments 3.8. XRD Spectroscopy. XRD spectroscopy of the synthesized (Figure 3(b)). silver nanoparticles showed the presence of four sharp peaks at 38°,44°,67°, and 78°. The diameter of silver nanoparticles 3.4. Assessment of the Effect of Changes in Silver Nitrate Salt was measured as 11.94 nm in the range of 7-16 nm (Figure 6). Solution Concentration on Nanoparticle Synthesis. The effect ff of di erent AgNO3 concentrations (0.5, 1, 3, 5, and 10 mM) 3.9. TEM Analysis. TEM analysis of the synthesized silver on silver nanoparticle synthesis was investigated using 3 ml nanoparticles showed that the particles are nanoscale in size BioMed Research International 5

1 (E) 1 (D) 0.9 0.9 (C) (B) 0.8 0.8 0.7 0.7 (A) 0.6 0.6 0.5 0.5

Absorption 0.4

Absorption 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0 0 320 520 720 320 420 520 620 720 Wavelength (nm) Wavelength (nm)

1 mM 3cc 3 min (A) 3 cc (D) 15 cc (B) 5 cc (E) 30 cc (C) 10 cc (a) (b) 1 2.5 (D) 0.9 (F) (E) 0.8 (C) 2 0.7 (E)

0.6 (B) 1.5 0.5 (D) (A) Absorption

0.4 Absorption 1 (C) 0.3 (B) 0.2 0.5 0.1 (A)

0 0 320 420 520 620 720 320 420 520 620 720 Wavelength (nm) Wavelength (nm)

(A) 0/5 mM (D) 5 mM (A) 1 min nor (D) 10 min nor (B) 1 mM (E) 10 mM (B) 3 min nor (E) 20 min nor (C) 3 mM (C) 5 min nor (F) 30 min nor (c) (d)

Figure 3: (a) UV-Vis spectrum under optimal conditions; (b) UV-Vis spectra of silver nanoparticles using different volumes of Vaccinium arctostaphylos aqueous extract (3, 5, 10, 15, and 30 ml), in the presence of 1 mM silver nitrate solution, sunlight catalyst, and time ff ff exposure of 3 minutes; (c) the e ect of di erent AgNO3 concentrations on silver nanoparticle synthesis using 3 ml of Vaccinium arctostaphylos aqueous extract; (d) the effect of reaction time on nanoparticle synthesis using Vaccinium arctostaphylos aqueous extract.

and almost spherical in shape. The size of the synthesized of growth inhibitory zone of E. coli, B. subtilis, and S. aureus nanoparticles under optimal conditions was measured as (p <0:01) (Table 1). While no inhibition zone was observed 21 nm (Figure 7). for Salmonella bacterial species, however, the appearance of inhibition zone around the disks for the other three bacterial 3.10. Assessment of the Antibacterial Activity of Silver species verified the antibacterial effect of synthesized silver Nanoparticles. Antibacterial effect of silver nanoparticles nanoparticles. An increment of the concentration of synthe- against gram-positive bacteria including Staphylococcus sized silver nanoparticles increased inhibition zone diameter, aureus and Bacillus subtilis and gram-negative bacteria as well (Figures 8 and 9). Investigating the minimum inhibi- including E. coli and Salmonella enteritidis was inspected. tory concentration (MIC) showed more antibacterial activity The green synthesized silver nanoparticles using Vaccinium of silver nanoparticles against Staphylococcus aureus as arctostaphylos extract had different effects on the diameter compared to Escherichia coli and Bacillus subtilis bacterial 6 BioMed Research International

2.0 Sunlight (~1000 mW/cm2)

Sample 1.5 Light source

1.0 Transmittance (%) 0.5 13.3 mW/cm2

0.0 200 300 400 500 600 700 800 900 Wavelength (nm)

0.8

0.6 (A) Absorption 0.4

0.2

0 320 520 720 −0.2 (B) Wavelength (nm)

(B) 3 min b nor (A) 3 min nor

Figure 4: The eﬀect of the presence and absence of 3-minute sunlight exposure on nanoparticle synthesis using Vaccinium arctostaphylos aqueous extract.

3449.38 1637.98 4. Discussion

1.4 The appearance of dark red color in the biosynthesis of silver 1.2 nanoparticles following reaction with silver ions is an obvi- 1.0 ous indicator of the reduction of metal ions and the formation of silver nanoparticles which can be due to the changes 0.8 in the surface plasmon resonance of metal nanostructures Absorbance urits 0.6 [18]. This initial color change which specifies the production of a colloidal suspension of silver nanoparticles [28] can be a 0.4 good tool to detect the formation of silver nanoparticles in 3500 3000 2500 2000 1500 1000 500 the reaction mixture. Herbal chemical compounds act as -1 Wavenumber cm the reducing and stabilizing agent to reduce silver ions. ff Figure 5: FTIR spectrum of silver nanoparticles synthesized under The e ect of light on the production of silver nanoparti- optimal conditions. cles varies depending on their size and shape. As the concentration of silver ions increases, the adsorption rate also increases. This is because with increasing the amount of strains, increasing with rising the concentration and number metal ions, more ions are reduced and consequently more of nanoparticles (Table 2). Assessment of the minimum bac- nanoparticles will be synthesized [29–31]. tericidal concentration (MBC) showed more antibacterial Assessment of the effect of different extract volumes on activity of silver nanoparticles against Staphylococcus aureus nanoparticle synthesis showed that in smaller plant extract as compared to Escherichia coli and Bacillus subtilis bacterial volumes, the intensity of peaks is lower which can be due to strains (Table 2). decreased amount of reducing agents and thus decreased rate BioMed Research International 7

Counts Assessment of the intensity of solution discoloration due 4000 910B-634-440 111 to the changes in the concentration of silver nitrate solution indicates an increase in the concentration of silver nanopar- 3000 ticles in the solution [35] which is due to the growth of par-

311 ticle size [21]. Spectroscopy analysis results showed that as 2000 200 220 the concentration of silver nitrate in the solution increases, the particle size and consequently peak intensity increase, as well (Figure 5), while a further increase in silver ion con- 1000 centration leads to the opposite trend [36]. Increasing silver nitrate solution concentration increases silver nanoparticle 0 synthesis and consequently nanoparticle size and maximum 40 50 60 70 Position [°2Teta] (Copper (Cu)) absorption rate [21]. The reaction time is effective in the synthesis and stability Figure 6: XRD spectrum of silver nanoparticles synthesized under of nanoparticles, because if the synthesis process is not com- optimal conditions. plete, the production of nanoparticles will increase over time. Moreover, time is the most significant factor in the verifica- tion of the stability of synthesized nanoparticles, because if no increase in the adsorption rate of silver nanoparticles is observed over time, it specifies that the resulting nanoparticles are absolutely stable over time [37]. In FTIR spectroscopy, oxygenated compounds present in the alcoholic and phenolic substances of the extract act as one of the active stabilizing agents to reduce silver nanoparticles [33]. Although the exact mechanism of nanoparticle formation during green synthesis is not yet known, it seems that terpenoids, sugars, phenols, and other plant 100 nm extract compounds can be used in the synthesis of metal Figure nanoparticles [38]. 7: TEM analysis of silver nanoparticles synthesized under -1 -1 optimal conditions. Adsorption in 1638 cm and 3449 cm in FTIR analysis corresponds to the affinity of the C-O-H and C = O groups of Table the extract compounds, the tensile vibrations of the alkyl 1: Diameter variance analysis of inhibitory zone of green group, and the C-C bond to a benzene ring. This attribution synthesis of silver nanoparticles using Vaccinium arctostaphylos indicates the presence of the CHO group which can be the extract against different bacterial agents. main agent to reduce Ag+ to Ag0 and being converted to MS CO after reaction. S.O.V df E. coli B. subtilis S. aureus XRD analysis of silver nanoparticles synthesized in ∗∗ ∗∗ ∗∗ Source 6 113.857 147.969 141.714 Euphorbia hirta extract showed the presence of silver nanoparticles with a diameter of 15-50 nm, but the SEM image Error 14 0.857 1 0.857 confirmed them as 40-50 nm. The results of UV-Vis spec- Total 20 troscopy also showed the absorption peak at 425 nm [39]. It ∗∗Significant at the level of one percent. has shown that Acalypha indica leaf extract can synthesize silver nanoparticles with a diameter of 20-30 nm and has of Ag+ ion to Ag0reduction. Furthermore, with increasing the been effective in inactivating Escherichia coli and Vibrio chol- concentration of the extract, the intensity of the peaks and era bacterial strains [40]. the adsorption rate will be increased which can be due to Green synthesis of silver nanoparticle using aqueous the increase in the number of silver nanoparticles and their extract of Fumaria parviflora and study of its antibacterial agglomeration. Such changes in the adsorption rate may and antioxidant properties was achieved. The synthesized sil- indicate the changes 1in nanoparticle size [32]. In addition, ver nanoparticles showed the most absorbance at 430 nm and assessment of the optimal concentration among different had a spherical shape with an average size of 10-50 nm. The extract concentrations has shown that increasing the extract synthesized nanoparticles had had antioxidant properties μ concentration will augment nanoparticle synthesis. On the with IC50 of 21 g/ml. The results of the antibacterial studies other hand, increasing the concentration of silver nitrate showed that the synthesized silver nanoparticles had had solution has increased nanoparticle synthesis rate, as well more antibacterial activity against the gram-positive bacteria [33]. In general, when the volume of the extract increases of Staphylococcus aureus than the gram-negative bacteria of under constant conditions, more organic material surrounds Escherichia coli [41]. In this research, the synthesized nano- the nanoparticles. Therefore, the particles settle down, and in particles showed more antibacterial properties against very large quantities, the nanoparticles come out of the gram-positive bacteria than gram-negative, and also, the size nanostate which makes the characteristic peak of these nano- of silver nanoparticles was in the range of 7-16 nm that these particles no longer visible in the desired range [34]. results were similar to the related research. 8 BioMed Research International

30 A 25 A A B 20 B B BC DE CD 15 E C CC

zone (mm) DD 10 D D DE E 5 Te diameter of growth inhibitor E F 0 S GM 5 mM 3 mM 1 mM 10 mM 0.5 mM arctostaphylos arctostaphylos arctostaphylos arctostaphylos arctostaphylos V. V. V. V. V. Source E.Coli B.Subtilis S.Aureus

Figure 8: Inhibition zone diameter of different concentrations of silver nitrate solution synthesized in Vaccinium arctostaphylos aqueous extract. Similar letters indicate no significant difference.

S. aureus

E. coli

B. subtilis

Control Gentamicin Streptomycin A B C D E

Figure 9: Disc diﬀusion test using 5 diﬀerent concentrations of silver nitrate solution ((a) 10 mM; (b) 5 mM; (c) 3 mM; (d) 1 mM; (e) 0.5 mM). The aqueous extract of the plant was considered as the negative control, and gentamicin and streptomycin antibiotics were considered as the positive control.

Table 2: MBC and MIC test analysis against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis bacterial strains.

E. coli B. subtilis S. aureus Cases under review MIC (μg/ml) MBC (μg/ml) MIC (μg/ml) MBC (μg/ml) MIC (μg/ml) MBC (μg/ml) Extract ——————

AgNO3 100 ppm 200 ppm 100 ppm 200 ppm 12.5 ppm 25 ppm

Green synthesis and antimicrobial eﬀects of silver nano- was about 5-40 nm [42]. In this research, the size of silver particles were investigated using Orange blossom extract, and nanoparticles was in the range of 7-16 nm, and also, the it was concluded that the formation of silver bioparticles in shape was the spherical particles that the results are the same. the range of 400 to 450 nm was demonstrated using a spectro- Although the shape of silver nanoparticles synthesized in photometer (UV). The size and morphology of these nanopar- the present study is very similar to those of other plants, but ticles were determined by a passing electron microscope, and their size in this study is smaller than the size of 61 to 117 nm the shape of the spherical particles and their average size which is reported in a number of studies [43]. BioMed Research International 9

At low concentrations of silver nitrate solution, larger solution concentration, and 3 min reaction time under sun- nanoparticles are synthesized due to the slower nucleation light exposure) that the optimal conditions are the same of nanoparticles. At high concentrations, the final nanoparti- with related research. cles become smaller due to faster nucleation and increasing The major property of silver nanoparticles is their anti- the number of nuclei in the reaction solution. The size and bacterial activity which is used in wound dressing, skin shape of nanoparticles play a significant role in many of their wound ointments, disinfectants, and medical instrument pharmaceutical applications [44]. coatings. These nanoparticles shorten the duration of the The green synthesis of silver nanoparticles was investi- wound healing process by reducing the activity of metallo- gated using Allium paradoxum plant extract and its antimi- proteases and boosting the apoptosis of neutrophils, making crobial activity. It was concluded that the best concentration the appearance of scars more natural. In addition, they will be for the synthesis of silver nanoparticles was 5 mM silver effective in better collagen orientation and enhance the nitrate over a period of 30 minutes [45]. However, in the mechanical strength of the tissues. Assessment of wound present study, the best concentration was 1 mM silver nitrate dressings has shown that silver usage for similar applications and exposed to sunlight for 3 minutes, which indicates the does not cause toxicity or adverse effects on human cells. greater power of Vaccinium arctostaphylos plant in the green Recent studies have also demonstrated that the use of silver synthesis of silver particles. nanoparticles in skin ointments causes silver penetration into Silver has long been known for its antibacterial proper- the wound bed, absorption by the epidermal cells of the ties. In fact, silver nanoparticles exhibit such properties wound margin, accumulation in the wound debris, and even- against aerobic and anaerobic bacteria due to the release of tually transfer to the peripheral circulatory system which silver ions. Binding of these particles to sulfur-containing helps the wound healing process and prevent infection [54]. proteins on the surface of bacterial membranes allows the In this study, the antimicrobial activity of Vaccinium arc- entry and subsequent changes in the morphology and respi- tostaphylos aqueous extract and its synthesized silver nanopar- ratory chain of bacteria which finally leads to the death of the ticles were evaluated on four bacterial strains. The results foreign agent by affecting the cell death process [46]. showed that the nanoparticles in concentrations used had The effect of nanoparticles on the cells of living organ- stronger antimicrobial effects than the extract itself which isms depends on their diameter, size, and shape [47]. Larger was concentration dependent in both the extract and the nanoparticles are more toxic due to their easier absorption nanoparticle solution. While silver nanoparticles had more and larger surface area. Furthermore, the release rate of sil- antimicrobial activity against gram-positive bacteria, gram- ver ion is increased as the size of silver nanoparticles negative bacteria showed resistance to silver nanoparticles. decreases. Therefore, the toxicity of silver nanoparticles To date, different reports have been presented about the depends on their size and the amount of silver ion release greater effect of silver nanoparticles on gram-negative bacte- rate [48]. However, other factors such as crystallinity, stabi- ria [3, 55–57] or gram-positive bacteria [29, 58–60] and even lizing reagents, and environmental factors including pH, the the lack of difference between gram-positive and gram- presence of ligands, and divalent cations in solution and negative ones [61]. macromolecules have also been considered significant [49]. Some reports have attributed the difference in the In general, silver nanoparticles, due to their smaller size, response of gram-positive and gram-negative bacteria to sil- have a greater contact surface with the environment and ver nanoparticles to the difference in their cell wall structure microorganisms. This increases their biological and chemi- [48, 62], and some reports have pointed to the e ffect of silver cal activities and consequently their impact on cell mem- nanoparticles on the structure of the bacterial cell membrane branes [50–52]. At low concentrations, due to the small and the reduction of cell leakage and ultimately the bacterial number of nanoparticles, the degree of interaction of the death [58]. However, the effect of silver nanoparticles on cell nanoparticles with the bacterial cell membrane is less and membrane structure and reduction of cell leakage (extracel- thus the inhibitory effect of nanoparticles is low and the lular vesicle production) seems to be greater. However, if diameter of the inhibition zone is small. For this reason, a the antibacterial effect of silver nanoparticles is related to balanced concentration of nanoparticles is always desirable the peptidoglycan layer (a specific feature of the membranes to inhibit the bacterial growth [25]. of bacterial species which is not observed in mammalian The antimicrobial effect and effective parameters on the cells), the use of silver nanoparticles as an antibacterial agent stability of silver nanoparticles synthesized using Mentha will be easier and more specified. pulegium were investigated, and it was concluded that the All domains of life on Earth, including gram-negative distribution of nanoparticles with spherical shapes was and gram-positive bacteria as well as eukaryotic organisms, almost uniform. The study of the effect of different variables actively secrete nanometer-sized vesicles (conserved intercel- in the synthesis of these nanoparticles has shown that the lular evolutionary components). These structures are round, volume of the extract is 3 ml, the concentration of silver bilayer containing biologically active substances such as pro- nitrate salt is 1 mM, and the reaction time of 10 minutes in teins, lipids, nucleic acids, and metabolites that can act as sunlight is the best conditions to achieve maximum stability secretory systems for the secretion of toxins, enzymes, and of silver nanoparticles [53]. In the present research, the UV- pathogens in bacteria. On the other hand, these structures Vis spectroscopy of silver colloidal nanoparticles showed a can increase the survival rate in bacteria. Resistance to surface plasmon resonance peak at 443 nm under optimal antibacterial compounds can take place by releasing these conditions (3 ml aqueous extract volume, 1 mM silver nitrate vesicles at all stages of the bacterial growth. Actually, the 10 BioMed Research International production of these vesicles can augment the survival of bac- UV-Vis spectroscopy and transmission electron microscopy. teria when attacked by phages. These structures can remove The silver nanoparticles obtained at the reaction time of 3 antibacterial agents such as supplements and antibiotics, as minutes had an average size of 12 nanometers and an almost well [63]. It has even been reported that S. aureus extracellu- spherical shape. Synthesized silver nanoparticles showed lar vesicles contain beta-lactamase which causes the survival more antibacterial activity against gram-positive bacteria of ampicillin-sensitive gram-positive and gram-negative bac- than gram-negative bacteria, especially against Staphylococ- teria in the presence of ampicillin [64–66] [60–62]. Evidence cus aureus. also suggests that the secretion of extracellular vesicles is a In general, our results showed that Vaccinium arctosta- completely conserved evolutionary process in gram-positive phylos aqueous extract has a good potential to produce silver bacteria [64, 67] [60, 63]. Therefore, the higher sensitivity nanoparticles. Both the extract and the synthesized nanopar- of gram-positive bacteria as compared to gram-negatives in ticles have significant biological potential. In case additional this study may be due to the role of silver nanoparticles in studies are carried out and side effects are assessed and the further reducing the production of extracellular vesicles in conditions are optimized, they can be used in food, pharma- gram-positive bacteria. ceutical, and agricultural industries and in the near future as In general, the mechanism of antimicrobial activity by sil- natural antioxidant supplements with more efficacy. ver nanoparticles is related to their ability to cross cell membranes and how they affect cellular components such as Data Availability DNA, proteins, and others which ultimately disrupt DNA replication and the activity of proteins and bacterial enzymes, No data were used to support this study. leading to bacterial death [33]. The most common mechanism for the antibacterial effect Conflicts of Interest of silver nanoparticles is the ionic release of silver and the inactivation of thiol groups in enzymes, which inactivate bac- The authors declare no conflicts of interest. terial enzymes. The released silver ions inhibit bacterial DNA replication, damage cell cytoplasm, reduce adenosine tri- References phosphate (ATP) levels, and eventually kill bacterial cells. Increasing the surface to volume ratio of nanoparticles [1] M. Khaksar, S. Vasileiadis, R. Sekine et al., “Chemical charac- increases the binding level of nanoparticles to the bacterial terisation, antibacterial activity, and (nano) silver transforma- cell. This action increases the release of silver ions to the bac- tion of commercial personal care products exposed to household greywater,” Environmental Science: Nano, vol. 6, teria and thus improves the antibacterial effect of silver [68]. no. 10, pp. 3027-3028, 2019. Nanotechnology is an attractive field of research for [2] A. Lateef, S. A. Ojo, and J. A. Elegbede, “The emerging roles of human beings due to the production of nanoparticles in dif- arthropods and their metabolites in the green synthesis of ferent sizes, shapes, chemical compositions and distributions, metallic nanoparticles,” Nanotechnology Reviews, vol. 5, and its many applications. The fabrication, manipulation, no. 6, pp. 601–622, 2016. and use of metal nanoparticles are of great importance due [3] M. Singh, S. Singh, S. Prasad, and I. Gambhir, “Nanotechnol- to their reduced dimensions and consequently their unique ogy in medicine and antibacterial effect of silver nanoparti- thermal, optical, and electronic properties [69]. cles,” Digest Journal of Nanomaterials and Biostructures, Today, the synthesis of silver nanoparticles is very com- vol. 3, no. 3, pp. 115–122, 2008. mon due to their many applications in various fields. The [4] M. Gericke and A. Pinches, “Biological synthesis of metal synthesis of these nanoparticles is done through physical, nanoparticles,” Hydrometallurgy, vol. 83, no. 1-4, pp. 132– chemical, and biological methods. The biological method is 140, 2006. preferable to the other two methods due to its compatibility [5] M. N. Nadagouda, G. Hoag, J. Collins, and R. S. Varma, with the environment and its low cost. Green nanoparticle “Green synthesis of Au nanostructures at room temperature production is a nature-friendly method that uses natural sol- using biodegradable plant surfactants,” Crystal Growth & vents [70]. Design, vol. 9, no. 11, pp. 4979–4983, 2009. Past studies in the field of nanoparticles show that due to [6] H. Rasouli, J. Popović-Djordjević, R. Z. Sayyed, S. Zarayneh, the lack of a logical model of the interaction of these particles M. Jafari, and B. Fazeli-Nasab, “Nanoparticles: a new threat to ” with biological molecules, so far from trial and error methods, crop plants and soil rhizobia?, in Sustainable Agriculture ff Reviews 41: Nanotechnology for Plant Growth and Development, the properties of a nanoparticle and its e ects on the reaction – medium and molecules are present. They found out in that S. Hayat, J. Pichtel, M. Faizan, and Q. Fariduddin, Eds., pp. 201 214, Springer International Publishing, Cham, 2020. environment. Due to the entry of these particles into plants [7] E. Castro-Longoria, A. R. Vilchis-Nestor, and M. Avalos- and animals, and especially their increasing use in products “ ff Borja, Biosynthesis of silver, gold and bimetallic nanoparticles used by humans, it seems necessary to study the e ects of using the filamentous fungus Neurospora crassa,” Colloids and these particles on molecules and biological hosts [71]. Surfaces B: Biointerfaces, vol. 83, no. 1, pp. 42–48, 2011. [8] M. B. Ahmad, M. Y. Tay, K. Shameli, M. Z. Hussein, and J. J. 5. Conclusion Lim, “Green synthesis and characterization of silver/chitosan/- polyethylene glycol nanocomposites without any reducing The biosynthesis of silver nanoparticles by Vaccinium arctos- agent,” International Journal of Molecular Sciences, vol. 12, taphylos aqueous extract was determined and confirmed by no. 8, pp. 4872–4884, 2011. BioMed Research International 11

[9] B. Fazeli-Nasab and R. Sayyed, “Plant growth-promoting rhi- improved antibacterial activity,” Colloids and Surfaces B: zobacteria and salinity stress: a journey into the soil,” in Plant Biointerfaces, vol. 103, pp. 9–14, 2013. Growth Promoting Rhizobacteria for Sustainable Stress Man- [24] P. C. Dey, S. Sarkar, and R. Das, “X-ray diffraction study of the – agement, pp. 21 34, Springer, 2019. elastic properties of jagged spherical CdS nanocrystals,” Mate- [10] Y. Kim, V. R. Babu, D. T. Thangadurai et al., “Synthesis, char- rials Science-Poland, vol. 38, no. 2, pp. 271–278, 2020. acterization, and antibacterial applications of novel copoly- [25] J.-J. Hwang and T.-W. Ma, “Preparation, morphology, and ” meric silver nanocomposite hydrogels, Bulletin of the antibacterial properties of polyacrylonitrile/montmorillonite/- – Korean Chemical Society, vol. 32, no. 2, pp. 553 558, 2011. silver nanocomposites,” Materials Chemistry and Physics, [11] V. K. Sharma, R. A. Yngard, and Y. Lin, “Silver nanoparti- vol. 136, no. 2-3, pp. 613–623, 2012. ” cles: green synthesis and their antimicrobial activities, [26] S. Naraginti and A. Sivakumar, “Eco-friendly synthesis of sil- Advances in Colloid and Interface Science, vol. 145, no. 1- ver and gold nanoparticles with enhanced bactericidal activity – 2, pp. 83 96, 2009. and study of silver catalyzed reduction of 4-nitrophenol,” Spec- [12] I. Sondi and B. Salopek-Sondi, “Silver nanoparticles as antimi- trochimica Acta Part A: Molecular and Biomolecular Spectros- crobial agent: a case study on E. coli as a model for gram- copy, vol. 128, pp. 357–362, 2014. ” negative bacteria, Journal of Colloid and Interface Science, [27] R. Amooaghaie, M. R. Saeri, and M. Azizi, “Synthesis, charac- – vol. 275, no. 1, pp. 177 182, 2004. terization and biocompatibility of silver nanoparticles synthe- [13] S. Mandal, S. K. Arumugam, R. Pasricha, and M. Sastry, “Silver sized from _Nigella sativa_ leaf extract in comparison with nanoparticles of variable morphology synthesized in aqueous chemical silver nanoparticles,” Ecotoxicology and Environmen- foams as novel templates,” Bulletin of Materials Science, tal Safety, vol. 120, pp. 400–408, 2015. – vol. 28, no. 5, pp. 503 510, 2005. [28] S. P. Reise and N. G. Waller, “Item response theory and clinical [14] S. Shaikh, N. Nazam, S. M. D. Rizvi et al., “Mechanistic insights measurement,” Annual Review of Clinical Psychology, vol. 5, into the antimicrobial actions of metallic nanoparticles and no. 1, pp. 27–48, 2009. ” their implications for multidrug resistance, International [29] A. Kaur, S. Preet, V. Kumar, R. Kumar, and R. Kumar, “Syner- Journal of Molecular Sciences, vol. 20, no. 10, p. 2468, 2019. getic effect of vancomycin loaded silver nanoparticles for [15] S. A. Anuj, H. P. Gajera, D. G. Hirpara, and B. A. Golakiya, enhanced antibacterial activity,” Colloids and Surfaces B: “Bacterial membrane destabilization with cationic particles Biointerfaces, vol. 176, pp. 62–69, 2019. ffl of nano-silver to combat e ux-mediated antibiotic resis- [30] S. Francis, S. Joseph, E. P. Koshy, and B. Mathew, “Microwave ” tance in gram-negative bacteria, Life Sciences, vol. 230, assisted green synthesis of silver nanoparticles using leaf – pp. 178 187, 2019. extract of Elephantopus scaber and its environmental and bio- [16] T. Mohasseli and S. P. Sayedi, “Green synthesis and character- logical applications,” Artificial cells, nanomedicine, and bio- ization of silver nanoparticles using aqueous extract of Sesa- technology, vol. 46, no. 4, pp. 795–804, 2018. ” mum indicum seeds, Modares Journal of Biotechnology, [31] V. Swetha, S. Lavanya, G. Sabeena, E. Pushpalaksmi, S. J. Jen- – vol. 6, no. 1, pp. 10 20, 2015. son, and G. Annadurai, “Synthesis and characterization of sil- [17] S. Supraja, S. M. Ali, N. Chakravarthy et al., “Green synthesis ver nanoparticles from Ashyranthus aspera extract for of silver nanoparticles from Cynodon dactylon leaf extract,” antimicrobial activity studies,” Journal of Applied Sciences International Journal of ChemTech Research, vol. 5, no. 1, and Environmental Management, vol. 24, no. 7, pp. 1161– pp. 271–277, 2013. 1167, 2020. [18] S. Pirtarighat, M. Ghannadnia, and S. Baghshahi, “Green syn- [32] A. Tripathy, A. M. Raichur, N. Chandrasekaran, T. Prathna, thesis of silver nanoparticles using the plant extract of Salvia and A. Mukherjee, “Process variables in biomimetic synthesis spinosa grown in vitro and their antibacterial activity assess- of silver nanoparticles by aqueous extract of Azadirachta ment,” Journal of Nanostructure in Chemistry, vol. 9, no. 1, indica (Neem) leaves,” Journal of Nanoparticle Research, pp. 1–9, 2019. vol. 12, no. 1, pp. 237–246, 2010. [19] T. Hasanloo, M. Jafarkhani Kermani, Y. Dalvand, and [33] R. Karamian and J. Kamalnejade, “Green synthesis of silver S. Rezazadeh, “A complete review on the genus Vaccinium nanoparticles using aqueous seed extract of Cuminum cymi- and Iranian Ghareghat,” Journal of Medicinal Plants, vol. 18, num L. and evaluation of their biological activities,” Scientific no. 72, pp. 46–65, 2019. Journal of Ilam University of Medical Sciences, vol. 26, no. 5, [20] M. A. Ferhat, B. Y. Meklati, J. Smadja, and F. Chemat, “An pp. 128–141, 2019. improved microwave Clevenger apparatus for distillation of [34] A. Miri, M. Sarani, M. R. Bazaz, and M. Darroudi, “Plant- essential oils from orange peel,” Journal of Chromatography mediated biosynthesis of silver nanoparticles using Prosopis A, vol. 1112, no. 1-2, pp. 121–126, 2006. farcta extract and its antibacterial properties,” Spectrochimica [21] A. Miri, N. Mahdinejad, O. Ebrahimy, M. Khatami, and Acta Part A: Molecular and Biomolecular Spectroscopy, M. Sarani, “Zinc oxide nanoparticles: biosynthesis, characteri- vol. 141, pp. 287–291, 2015. zation, antifungal and cytotoxic activity,” Materials Science [35] S. Moshahary and P. Mishra, “Synthesis of silver nanoparticles and Engineering: C, vol. 104, article 109981, 2019. (AgNPs) using culinary banana peel extract for the detection [22] M. S. Tamboli, M. V. Kulkarni, R. H. Patil, W. N. Gade, S. C. of melamine in milk,” Journal of Food Science and Technology, Navale, and B. B. Kale, “Nanowires of silver-polyaniline nano- vol. 58, pp. 797–804, 2020. composite synthesized via _in situ_ polymerization and its [36] S. M. Pourmortazavi, M. Taghdiri, V. Makari, and M. Rahimi- ” novel functionality as an antibacterial agent, Colloids and Sur- Nasrabadi, “Procedure optimization for green synthesis of sil- – faces B: Biointerfaces, vol. 92, pp. 35 41, 2012. ver nanoparticles by aqueous extract of _Eucalyptus oleosa_,” [23] P. Boomi, H. G. Prabu, and J. Mathiyarasu, “Synthesis and Spectrochimica Acta Part A: Molecular and Biomolecular Spec- characterization of polyaniline/Ag-Pt nanocomposite for troscopy, vol. 136, pp. 1249–1254, 2015. 12 BioMed Research International

[37] K. P. Kumar, W. Paul, and C. P. Sharma, “Green synthesis of resistant bacteria,” Journal of Applied Microbiology, vol. 112, gold nanoparticles with Zingiber officinale extract: characteri- no. 5, pp. 841–852, 2012. zation and blood compatibility,” Process Biochemistry, vol. 46, [52] N. Durán, M. Durán, M. B. De Jesus, A. B. Seabra, W. J. Fávaro, – no. 10, pp. 2007 2013, 2011. and G. Nakazato, “Silver nanoparticles: a new view on mecha- [38] J. Y. Song, E.-Y. Kwon, and B. S. Kim, “Biological synthesis of nistic aspects on antimicrobial activity,” Nanomedicine: Nano- platinum nanoparticles using Diopyros kaki leaf extract,” Bio- technology, Biology and Medicine, vol. 12, no. 3, pp. 789–799, process and Biosystems Engineering, vol. 11, no. 1, p. 159, 2010. 2016. [39] E. Elumalai, T. Prasad, J. Hemachandran, S. V. Therasa, [53] J. P. Mohamadzade, H. Azizi, M. Khatami, and H. Yaghobi, T. Thirumalai, and E. David, “Extracellular synthesis of silver “Optimization of effective parameters on the suspension dura- nanoparticles using leaves of Euphorbia hirta and their anti- bility of green synthesis silver nanoparticles and evaluation bacterial activities,” Journal of Pharmaceutical Sciences and their antimicrobial effect,” Journal of Torbat Heydariyeh Uni- Research, vol. 2, no. 9, pp. 549–554, 2010. versity of Medical Sciences, vol. 8, no. 1, pp. 36–47, 2020. [40] C. Krishnaraj, E. Jagan, S. Rajasekar, P. Selvakumar, [54] G. G. Walmsley, A. McArdle, R. Tevlin et al., “Nanotechnology P. Kalaichelvan, and N. Mohan, “Synthesis of silver nanoparti- in bone tissue engineering,” Nanomedicine: Nanotechnology, cles using Acalypha indicas leaf extracts and its antibacterial Biology and Medicine, vol. 11, no. 5, pp. 1253–1263, 2015. ” activity against water borne pathogens, Colloids and Surfaces [55] E. Pazos-Ortiz, J. H. Roque-Ruiz, E. A. Hinojos-Márquez et al., – B: Biointerfaces, vol. 76, no. 1, pp. 50 56, 2010. “Dose-dependent antimicrobial activity of silver nanoparticles [41] B. Dousti, F. Nabipour, and A. Hajiamraei, “Green synthesis of on polycaprolactone fibers against gram-positive and gram- silver nanoparticle using aqueous extract of Fumaria parvi- negative bacteria,” Journal of Nanomaterials, vol. 2017, Article flora and study of its antibacterial and antioxidant properties,” ID 4752314, 9 pages, 2017. – Razi Journal of Medical Sciences, vol. 26, no. 6, pp. 105 117, [56] A. Al-Sharqi, K. Apun, M. Vincent, D. Kanakaraju, and L. M. 2019. Bilung, “Enhancement of the antibacterial efficiency of silver [42] S. Heydarzadeh and H. Yaghoubi, “Green synthesis and anti- nanoparticles against gram-positive and gram-negative bacte- bacterial effect of silver nanoparticles by using extract of Citrus ria using blue laser light,” International Journal of Photoe- aurantium,” Razi Journal of Medical Sciences, vol. 24, no. 157, nergy, vol. 2019, Article ID 2528490, 12 pages, 2019. – pp. 15 24, 2017. [57] S. Gurunathan, J. W. Han, D.-N. Kwon, and J.-H. Kim, [43] P. Teirlinck and F. Petersen, “The nature of gold-iodide “Enhanced antibacterial and anti-biofilm activities of silver adsorption onto coconut-shell carbon,” Minerals Engineering, nanoparticles against gram-negative and gram-positive bacte- vol. 9, no. 9, pp. 923–930, 1996. ria,” Nanoscale Research Letters, vol. 9, no. 1, pp. 1–17, 2014. [44] M. Gnanadesigan, M. Anand, S. Ravikumar et al., “Antibacte- [58] J. Li, K. Rong, H. Zhao, F. Li, Z. Lu, and R. Chen, “Highly selec- rial potential of biosynthesised silver nanoparticles using Avi- tive antibacterial activities of silver nanoparticles against Bacil- cennia marina mangrove plant,” Applied Nanoscience, vol. 2, lus subtilis,” Journal of Nanoscience and Nanotechnology, no. 2, pp. 143–147, 2012. vol. 13, no. 10, pp. 6806–6813, 2013. [45] T. Ranjbar, F. Sadeghian, H. R. Goli, M. Ahanjan, and M. A. [59] A. K. Suresh, D. A. Pelletier, W. Wang et al., “Silver nanocrys- Ebrahimzadeh, “Green synthesis of silver nanoparticles with tallites: biofabrication using Shewanella oneidensis, and an Allium paradoxum extract and evaluation of their antibacterial evaluation of their comparative toxicity on gram-negative activities,” Journal of Mazandaran University of Medical Sci- and gram-positive bacteria,” Environmental Science & Tech- ences, vol. 29, no. 182, pp. 1–11, 2020. nology, vol. 44, no. 13, pp. 5210–5215, 2010. [46] F. Ahmadi, S. Abolghasemi, N. Parhizgari, and F. Moradpour, [60] M. Premanathan, K. Karthikeyan, K. Jeyasubramanian, and “Effect of silver nanoparticles on common bacteria in hospital G. Manivannan, “Selective toxicity of ZnO nanoparticles surfaces,” Jundishapur Journal of Microbiology, vol. 6, no. 3, toward gram-positive bacteria and cancer cells by apoptosis p. 21, 2013. through lipid peroxidation,” Nanomedicine: Nanotechnology, – [47] M. D. Scherer, J. C. V. Sposito, W. F. Falco et al., “Cytotoxic Biology and Medicine, vol. 7, no. 2, pp. 184 192, 2011. and genotoxic effects of silver nanoparticles on meristematic [61] A. Abbaszadegan, Y. Ghahramani, A. Gholami et al., “The cells of Allium cepa roots: A close analysis of particle size effect of charge at the surface of silver nanoparticles on antimi- dependence,” Science of the Total Environment, vol. 660, crobial activity against gram-positive and gram-negative bac- pp. 459–467, 2019. teria: a preliminary study,” Journal of Nanomaterials, [48] D. P. Tamboli and D. S. Lee, “Mechanistic antimicrobial vol. 2015, Article ID 720654, 8 pages, 2015. approach of extracellularly synthesized silver nanoparticles [62] A. Russell and W. Hugo, “7 antimicrobial activity and action of against gram positive and gram negative bacteria,” Journal of silver,” in Progress in medicinal chemistry, pp. 351–370, Else- Hazardous Materials, vol. 260, pp. 878–884, 2013. vier, 1994. [49] L. Kvítek, A. Panáček, J. Soukupova et al., “Effect of surfactants [63] M. Ebrahimi Vargoorani, M. H. Modarressi, M. Sheikhpour, and polymers on stability and antibacterial activity of silver and S. D. Siadat, “Medical function of bacterial extracellular nanoparticles (NPs),” The Journal of Physical Chemistry C, vesicles,” Iranian Journal of Medical Microbiology, vol. 12, vol. 112, no. 15, pp. 5825–5834, 2008. no. 3, pp. 140–159, 2018. [50] M. Bindhu and M. Umadevi, “Antibacterial and catalytic activ- [64] E. Y. Lee, D. Y. Choi, D. K. Kim et al., “Gram-positive bacteria ities of green synthesized silver nanoparticles,” Spectrochimica produce membrane vesicles: proteomics-based characteriza- Acta Part A: Molecular and Biomolecular Spectroscopy, tion of Staphylococcus aureus-derived membrane vesicles,” vol. 135, pp. 373–378, 2015. Proteomics, vol. 9, no. 24, pp. 5425–5436, 2009. [51] M. K. Rai, S. Deshmukh, A. Ingle, and A. Gade, “Silver nano- [65] L. Karthik, G. Kumar, T. Keswani, A. Bhattacharyya, S. S. particles: the powerful nanoweapon against multidrug- Chandar, and K. V. B. Rao, “Protease inhibitors from marine BioMed Research International 13

actinobacteria as a potential source for antimalarial com- pound,” PloS One, vol. 9, no. 3, article e90972, 2014. [66] J. Lee, E.-Y. Lee, S.-H. Kim et al., “Staphylococcus aureus extracellular vesicles carry biologically active β-lactamase,” Antimicrobial Agents and Chemotherapy, vol. 57, no. 6, pp. 2589–2595, 2013. [67] A. Olaya-Abril, R. Prados-Rosales, M. J. McConnell et al., “Characterization of protective extracellular membrane- derived vesicles produced by Streptococcus pneumoniae,” Jour- nal of Proteomics, vol. 106, pp. 46–60, 2014. [68] F. Hajmohammadi, “Biosynthesis of silver nanoparticles and the comparison of their antibacterial activity against Bacillus cereus and Serratia marcescens,” Journal of Cellular and Molec- ular Research (Iranian Journal of Biology), vol. 32, no. 1, pp. 16–32, 2019. [69] G. Arya, R. M. Kumari, N. Gupta, A. Kumar, R. Chandra, and S. Nimesh, “Green synthesis of silver nanoparticles using Pro- sopis juliﬂora bark extract: reaction optimization, antimicrobial and catalytic activities,” Artiﬁcial cells, nanomedicine, and biotechnology, vol. 46, no. 5, pp. 985–993, 2018. [70] B. Sharma, I. Singh, S. Bajar, S. Gupta, H. Gautam, and P. Kumar, “Biogenic silver nanoparticles: evaluation of their biological and catalytic potential,” Indian Journal of Microbiol- ogy, vol. 60, no. 4, pp. 468–474, 2020. [71] H. Veisi, S. Azizi, and P. Mohammadi, “Green synthesis of the silver nanoparticles mediated by _Thymbra spicata_ extract and its application as a heterogeneous and recyclable nanoca- talyst for catalytic reduction of a variety of dyes in water,” Jour- nal of Cleaner Production, vol. 170, pp. 1536–1543, 2018.