One-pot facile green synthesis of biocidal silver nanoparticles

The plant root extract mediated green synthesis method produces monodispersed spherical shape silver nanoparticles (AgNPs) with a size range of 15–30 nm as analyzed by atomic force and transmission electron microscopy. The material showed potent antibacterial and antifungal properties. Synthesized AgNPs display a characteristic surface plasmon resonance peak at 420 nm in UV–Vis spectroscopy. X-ray diffractometer analysis revealed the crystalline and face-centered cubic geometry of in situ prepared AgNPs. Agar well diffusion and a colony forming unit assay demonstrated the potent biocidal activity of AgNPs against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, Pseudomonas diminuta and Mycobacterium smegmatis. Intriguingly, the phytosynthesized AgNPs exhibited activity against pathogenic fungi, namely Trichophyton rubrum, Aspergillus versicolor and Candida albicans. Scanning electron microscopy observations indicated morphological changes in the bacterial cells incubated with silver nanoparticles. The genomic DNA isolated from the bacteria was incubated with an increasing concentration of AgNPs and the replication fidelity of 16S rDNA was observed by performing 18 and 35 cycles PCR. The replication efficiency of small (600 bp) and large (1500 bp) DNA fragments in the presence of AgNPs were compromised in a dose-dependent manner. The results suggest that the Thalictrum foliolosum root extract mediated synthesis of AgNPs could be used as a promising antimicrobial agent against clinical pathogens.


Introduction
The current research trend in the field of nanotechnology is increasing due to attractive and diversified biomedical application of metallic nanomaterials having a variable size raging from 1-100 nm. Synthesis of silver nanoparticles (AgNPs) is of much interest to the scientific community because of its wide range of applications. These AgNPs are being successfully used in cancer diagnosis and treatment as well [1,2]. Generally, nanoparticles are prepared by a variety of chemical and physical methods which are quite expensive and potentially hazardous to the environment due to the presence of residual chemicals that are responsible for various biological risks [3]. The development of biologically-inspired experimental processes or greener approaches for the syntheses of nanoparticles is evolving into an important branch of nanotechnology. The microbial method of AgNPs synthesis requires the elaborate process of culture maintenance, purification and the adherence of bacteria or fungi on the nanoparticle surface, which raises the potential risk to human and environmental health [4][5][6]. Plant mediated syntheses of nanoparticles provides selective advancement over microbial methods as they are simple, one step, cost-effective, eco-friendly and relatively reproducible and often result in more stable materials [7]. A large number of plants have been reported in the literature for biosynthesis

Experimental section 2.1. Preparation of aqueous root extracts and synthesis of AgNPs
The roots of TF were freshly collected and the powdered samples were boiled in a 250 ml glass beaker for 10 min. The filtrate obtained was used for the in situ reduction of silver ions at physiological pH and room temperature. In a simple reaction procedure, 5 ml of TF root extract was added to 95 ml of 1 × 10 −3 M aqueous AgNO 3 solution.

Characterization of AgNPs
The phytosynthesized AgNPs obtained by TF root extract were centrifuged at 15 000 rpm for 10 min to get rid of any uncoordinated biological materials [15]. UV-Vis absorption spectrum was measured using a Multiscan GO spectrophotometer (Thermo Scientific). The pellet was re-suspended in a small amount of sterilized double distilled water and then a small amount of suspension was sprayed on a glass slide to make thin film. The thin film was kept in a hot air oven to dry and then the thin film was used for the energy-dispersive x-ray spectroscopy (EDX) analysis equipped with a scanning electron microscope (SEM-JEOL JSM 6390LV, Japan) and x-ray diffractometer (XRD). Fourier transform infrared (FTIR, Perkin Elmer, Model: Spectrum 100) spectra for TF root extracts alone and AgNPs was recorded five times per scan in the frequency region 4000-400 cm −1 at room temperature to identify characteristic peaks and their functional groups involved in the reduction of Ag + ions by KBr pellet method. Photoluminescence (PL) spectra were recorded in Vrian (Perkin Elmer, Model: LS55) spectrofluorimeter using 90°illumination. Based on the excitation maximum (340 nm), an emission scan was carried out in the range of 500-600 nm. The morphological and topographical analysis of the materials was carried out by transmission electron microscopy (FEI Technai G2 F205-TWIN TEM) and atomic force microscopy (Multiview 2000 (NSOM/SPM). The crystallinity of the nanoparticles was characterized using an x-ray diffractometer (Rigaku, Model-Miniflex) operated at a voltage of 40 kV and a current of 30 mA with Cu Kα radiation in θ-2θ configurations. The crystallinity of the particle was calculated from the width of the XRD peaks by assuming that they were free from non-uniform strains and using the Scherrer formula, D = 0.94 λ/β cos θ, where D is the average crystallite domain size perpendicular to the reflecting planes, λ is the x-ray wavelength, β is the full width at half maximum band, θ is the diffraction angle [16].

In vitro anti-oxidant activity
The total phenolic content of the fractions was determined by the Folin-Ciocalteu method with slight modification [17]. The aqueous root extract and AgNPs (0.5 ml; 1 mg ml −1 ) were mixed with Folin-Ciocalteu reagent (2.5 ml, diluted 1:10 with distilled water) for 5 min; aqueous Na 2 CO 3 (2.5 ml, 75 g l −1 ) was then added to the mixture. The mixture was allowed to stand at 25°C for 30 min and the phenolic content was determined by spectrophotometry at 750 nm. The standard curve was prepared with 12.5, 25, 50, 100 and 200 μg ml −1 solutions of gallic acid (GA) in methanol. The total phenolic content was expressed as GA equivalents (mg GA g/ dry weight).

Antibacterial well diffusion assay
The antibacterial and antifungal activity of AgNPs was determined by the agar-well diffusion method against both gram-negative, gram-positive and fungal strains [18]. The microbial cultures were grown in an appropriate media and centrifuged at 5000 rpm for 5 min. Then the pellet was washed with 1X PBS and the 100 μl of the suspended culture was spread uniformly on agar plates and the plates were incubated at 4°C for 2 h for proper diffusion of the materials. The aqueous root extracts, AgNPs alone and in combination with standard antibiotics were loaded into the wells. The anti-microbial activity was determined by measuring the diameter (mm) of the zone of growth clearance after 24 h.

In vitro time killing assay
To determine the antibacterial activity of the AgNPs, the overnight grown cultures were centrifuged at 5000 rpm for 5 min, washed with 1X PBS, and the pellet was suspended in LB medium. Finally the optical density of the sample was adjusted to 0.2 at 600 nm. Bacteria grown in the presence of the medium alone (control) and AgNPs (5 μM) were incubated with 2−3 × 10 7 CFU (colony forming units)/ml bacteria in 50 ml LB medium in triplicates. The number of CFUs were analyzed by harvesting bacteria at different time points by plating serial dilutions on LB plates, and surviving colonies were enumerated after 24 h for all bacterial strains except M. smegmatis, in which the colonies were enumerated after 72 h on 7H10 plates.
2.6. Bacterial cell morphology using scanning electron microscopy SEM was performed to examine the morphological changes of two representative strains, namely S. aureus and E. coli, after incubation with AgNPs. The control and AgNPs treated bacterial cells were fixed with 2% glutaraldehyde overnight on glass coverslips. The fixed samples were then dehydrated for 30 min in a graded series of ethanol and observed by SEM.

Changes in the membrane permeability
The membrane permeability assay was examined spectrophotometrically using the protocol described previously [19]. In this study, we have chosen representative strains from gram-positive (S. aureus) and negative (E. coli) bacteria to examine the integrity of the cell membrane. Briefly, the bacterial cells grown overnight with continuous shaking in nutrient broth at 37°C were harvested and washed with 10 mM EDTA followed by two washes with distilled water. The final absorbance of the re-suspended bacterial cells was adjusted to 0.2 at A 600 . AgNPs and aqueous extracts (AE) were added to the bacterial suspension after incubation for 30 min at room temperature. At regular intervals of 2 h, aliquots of the samples were taken, centrifuged and the supernatants were analyzed for the presence of UV absorbing material by taking absorbance at UV 260 and UV 280 .

Determination of biofilm activity using crystal violet assay
We have chosen a sterile 96-well polystyrene micro titer plate (hydrophobic) as a model system to study the bacterial adhesion as reported previously [15]. Overnight grown bacterial cultures were diluted 1:100 in fresh LB medium. Different concentrations of AgNPs were added to the 96-well micro-titer plate and incubated at 37°C for 48 h. Thereafter the medium was removed and the wells were thoroughly washed with 1X PBS and 100 μl of 0.1% (w/v) crystal violet (CV) was added and incubated for 20 min. The CV was removed and washed thoroughly with 1X PBS. For quantification of the attached cells, the CV was solubilized in absolute ethanol and the absorbance was measured at 570 nm. Reduction of the biofilm was correlated with the cells grown in the absence of AgNPs in the medium as described previously.
2.9. Effect of AgNPs on in vitro DNA replication fidelity using PCR In this work, we mechanistically investigated the effects of AgNPs on in vitro DNA replication of small (600 bp) and large (1500 bp) DNA fragments using polymerase chain reactions (PCR). Briefly, the full length (5493 kb) of the plasmid pET-22(b + ) containing the rotavirus NSP5 gene (600 bp) was used as a template during PCR with primers (P5F 5′-ATCTAGGATCCGATGTCTCTCAGCATTGACG-3′ and P5R 5′-ATGTACTCGAGGCCAAATCTTCAATCAATTG-3′). PCR amplifications were performed in 30 μl reaction volumes with 2.5U Taq DNA polymerase, 200 μM each dNTP, and 0. 2 μM each primer and increasing concentration of AgNPs. The PCRs began with a denaturation step at 94°C for 5 min, and 18 and 35 cycles of amplification were performed using the following conditions: 40 s at 94°C; 30 s at 60°C; 1 min at 72°C. PCR amplification of 16 s rDNA was performed using genomic DNA isolated from E. coli (MTCC 40) with primer pairs (16 F 5′-ACATAGGATCCAGCGAACGCTGGCGGCAG-3′ and 16 R 5′-ATCTGAAGCTTTTAGCAGGTTCCCCTACGGCTA-3′). The 16S rDNA PCR consisted of an initial denaturation at 94°C for 5 min, followed by 18 cycles of 40 s at 94°C, 30 s at 55°C and 1.6 min at 72°C, and a final extension of 10 min at 72°C.
2.10. Evaluation of biocompatibility of AgNPs using macrophages cell line (RAW 246.7) and Cucumis sativus seed germination experiment In this study, we used the macrophage cell line RAW 246.7 to determine the biocompatible nature of AgNPs. Briefly, RAW 264.7 in DMEM was grown in a 96-well plate at 37°C and 5% CO 2 in the presence or absence of different concentrations of AgNPs. The cell viability was determined after 24 h incubation by treating cells with MTT solution for 4 h at 37°C in a cell culture incubator. In metabolically active cells, MTT was reduced to an insoluble dark purple formazan by mitochondrial dehydrogenases. The formazan crystals were dissolved in a denaturing buffer (SDS, HCl and isopropanol) and the absorbance was read at 570 nm in an ELISA reader. The total number of viable cells relative to the viable cells in the untreated control was calculated [17].
The germination experiment was conducted using C. sativus seeds surface sterilized with 10% sodium hypochlorite and then soaked in double distilled water and AgNPs suspensions separately for 2 h. To examine which process (seed soaking or incubation after the soaking) primarily retarded the various developmental stages of the seedlings, the following four treatments were given in respective petri dishes (90 mm) containing one piece of sterile filter paper with 5 ml each of AgNPs, sterile water (positive control) and TF extract alone (negative control): (a) both seed soaking and incubation were done in double distilled water; (b) both seed soaking and incubation were done in AgNPs suspension; (c) seeds soaked in double distilled water were incubated with AgNPs suspension; (d) seeds soaked in AgNPs suspension were incubated with double distilled water. Seven seeds were placed in each plate at a distance of 1 cm or larger. The above experimental treatments were allowed to germinate for 7 days until roots emerged and the seed germination rate and radical length was measured [20].

Synthesis and characterization of AgNPs
The development of an inexpensive, reliable and eco-friendly method will help to facilitate the increasing application of noble nanoparticles that are beneficial for mankind. The yellow color of the mixture of silver nitrate and TF root extract changed rapidly to a dark brown suspended mixture after 1 h of incubation at room temperature. The formation of AgNPs was observed by the appearance of a silver surface plasmon resonance (SPR) peak at 420 nm in UV-Vis spectroscopy ( figure 1(a)).
This typical SPR peak is similar to the plasmon vibrations of the AgNPs prepared by chemical reductions [21]. Similar observations of rapid biosynthesis of stable silver, gold and bi-metallic Ag/Au core-shell nanoparticles with 20 g of Azadirachta indica extract has been reported earlier [14]. The EDX spectrum of synthesized AgNPs clearly indicated the reduction of silver nitrate to AgNPs ( figure 1(b)). The average crystalline size of AgNPs was found to be 12.95 nm and the XRD pattern demonstrated four peaks indexed to the (111) and (200), (220), and (311) Bragg reflections, which may be indicative of the face-centered cubic structure of AgNPs [15], and the AgNPs formed were essentially pure and crystalline in nature (figure 1(c)). The putative biomolecules responsible for the green synthesis of AgNPs were identified by FTIR ( figure 1(d)). The aqueous root extract of TF (black line) showed distinct peaks at 3393, 2937, 1644, 1412, 1027, and 602 cm −1 . Similarly, in the IR spectrum of AgNPs (red line), the peaks were observed at 3435, 2929, 1634, 1384, 1061, and 616 cm −1 . Comparing the IR spectra of the root extract and AgNPs, the relative shift in the distribution of peak was observed at 3393, 2937, 1644, and 1027 cm −1 , corresponding to carbonyl stretch (C=O, 1634 cm −1 ), carboxylic acids (O-H, 2937 cm −1 ) and polyphenols (O-H, 3435 cm −1 ) functional group. The aqueous root extract of TF showed a good antioxidant activity (62.7%, figure S1(a)) as compared to the positive control-L-ascorbic acid (86.6%) using the modified DPPH scavenging assay [22]. However, the phenolic content of biosynthesized AgNPs and the extract alone as measured by the Folin-Ciocalteu method ( figure S1(b), see inset) demonstrated the attachment of lower amounts (29.8 ± 1.0 mg GA g −1 ) of phenolic compounds in AgNPs as compared to the TF root extract alone (59.6 ± 0.5 mg GA g −1 ). The proposed mechanism for the reduction and stabilization of AgNPs might be due to the presence of polyphenols, flavonoids and other active compounds present in the root extract of TF, as schematically represented in figure 1(e). The presence of flavanoids, polyphenolic and terpenoids might be responsible for the reduction and capping of silver ions and the results correlate with the total phenolic content and IR spectral analysis (figures 1(d) and S1(b)). Polyphenolic compounds are known to possess metal chelating and reducing properties leading to the formation of metallic nanoparticles [23,24]. AgNPs are reported to exhibit visible PL, which is shown in figure 1(e). The AgNP showed the presence of a visible PL band centered at 440 nm when excited at 300 nm wavelength and the luminescence coud be due to the presence of antioxidants present in the plant extract. The nanoparticles synthesized using olive leaf extract are also reported to be luminescent with an emission band at 425 nm and the observed PL property of AgNPs might have the potential for use in photoelectronic devices [25]. AFM and TEM micrographs revealed the diameter of AgNPs in the range of 15-30 nm (figures 2(a)-(d)). The biosynthesized nanoparticles were monodispersed, roughly spherical and the agglomeration of a few AgNPs were visible as observed through AFM ( figure 2(e)). The AFM micrograph shows that the particle size is bigger than those of the TEM measurements. This discrepancy could be due to the tendency of AgNPs to form an aggregate on the surface during deposition for analysis by AFM. Also, the shape of the AFM probe tip may cause misleading cross sectional views of the sample [26]. Similar to our present findings, a rapid synthesis of spherical shaped AgNPs with a dimension of 50-100 nm have been reported using the leaf extracts of Azadirachta indica, Acalypha indica, A. dentate and A. indicum [8,9,13,14].

Anti-bacterial and anti-fungal activity of AgNPs
The antibacterial and antifungal effects of biosynthesized AgNPs was investigated using an agar well diffusion assay. Prior to the antimicrobial studies, the AgNPs were thoroughly washed and centrifuged to remove any unreduced Ag + ions. Both gram-positive bacteria (B. subtilis and S. aureus) and gram-negative bacteria (E. coli, K. pneumoniae and P. diminuta) showed a maximum zone of growth inhibition (table 1). Interestingly, the activity of synthesized AgNPs against clinically isolated pathogenic fungi, T. rubrum, A. versicolor and C. albicans exhibited remarkable antifungal activity with a growth inhibition diameter measuring 15-30 nm (table 1). The moderate antibacterial property of the aqueous root extract of TF observed in this study might be due to the presence of antioxidant molecules (see figure S1(a)) and other secondary metabolites. However, the aqueous root extract of TF mediated biosynthesized AgNPs could increase the antibacterial and antifungal activities of active biomolecules adsorbed on AgNPs (table 1). Plants constitute an important source of numerous secondary metabolites (viz. alkaloids, tannins, flavonoids, phenolic compounds, phenolic glycosides) having bacteriostatic and bactericidal effects. Most of these secondary metabolites, other than possessing antibacterial and antifungal potential, can also act as potent antioxidants [27][28][29]. However, the biosynthesized AgNPs exhibited a negligible synergistic activity with the combination of standard antibiotics ampicillin, gentamicin, nystatin and clotrimazole (table 1). Figures 3(a) Table 1. Comparison of anti-microbial activity of crude AE, AgNPs alone and in combination with antibiotics against human pathogenic bacteria and fungi using agar-well diffusion assay. AE: aqueous extract; AgNPs: silver nanoparticles; ± standard deviation; ND: not determined. growth inhibition of bacteria (E. coli and B. subtilis) and fungi (C. albicans, A. versicolor, and T. rubrum) using AE, AgNPs alone and in combination with antibiotics by the agar-well diffusion method. The moderate synergistic effect of AgNPs with antibiotics observed in this study could be due to the lack of appropriate experimental conditions favorable for proper bonding reaction between the antibiotic and nanosilver. The antibiotic molecules contain many active groups such as hydroxyl and amido groups, which react easily with nanosilver by chelation [30]. Our results also correlate with the work previously done by some researchers where the moderate or minute increase in the inhibition zone of AgNPs against the bacteria was observed in the presence of antibiotics [31][32][33]. The present observation that biosynthesized AgNPs could inhibit the growth of dermatophytes correlates well with the research findings published previously [34,35]. Intriguingly, the M. smegmatis cell wall is rich in lipids such as mycolic acids, which limit the binding and the permeability of drug molecules. However, the present observation of the activity of AgNPs against M. smegmatis corroborate with the AgNPs synthesized using gallic acid as a capping agent [36]. The bactericidal efficiency of biosynthesized AgNPs was further confirmed by performing a CFU assay against E. coli, K. pneumoniae, P. diminuta, B. subtilis, S. aureus, M. smegmatis, and C. albicans (figures 4(a)-(g)).

Diameter of mean zone of inhibition (mm)
The results of the time kinetic curves revealed that AgNPs (5 μM) could significantly kill E. coli, P. diminuta and B. subtilis within the first 30 min of exposure to AgNPs. In contrast, the biocidal effect of AgNPs against K. pneumoniae, S. aureus, M. smegmatis, and C. albicans were observed after 2 h of incubation with AgNPs ( figures 4(b), (e), (f) and (g)). The potent bactericidal activity of AgNPs may be attributed to the strong interactions between AgNPs and the macromolecules such as proteins and the DNA of bacteria [8,9,37,38].

Induction of membrane damage and morphological changes in cells treated with AgNPs
SEM microscopy of two representative bacterial strains (E. coli MTCC 40 and S. aureus MTCC 3160) were carried out to observe changes in the surface morphologies of the bacterial cells after treatments with subinhibitory concentration of the AgNPs (5 μM) for 4 h. Native E. coli and S. aureus cells showed normal morphologies without any treatment (figures 5(a) and (b)).
Upon treatment with AgNPs, the bacterial cells underwent considerable morphological alterations as compared to the control. The results of the SEM micrographs indicated extensive damage to the cell wall, which could result in leakage of the UV absorbing material, mainly nucleic acid and protein. It can be seen that AgNPs treatment promotes the release of extracellular UV-absorbing materials, such as DNA and protein, over a period of 8 h (figures 5(c) and (d)). The results implied that the AgNPs induced the loss of UV absorbing materials from E. coli and S. aureus, probably through alteration of the membrane structure [19]. The CV assay revealed the A negative control with 1% DMSO did not inhibit the capacity of the bacteria to form biofilm on a polystyrene plate. The antimicrobial properties of AgNPs depend on the size and type of the capping agent used [6]. The exact mechanisms of antimicrobial properties of AgNPs are well debated and the investigations are still ongoing to understand the antibactericidal action. The positive charge on the Ag ions is suggested for antimicrobial activities of AgNPs. The high bactericidal activity might be attributed to the silver cations released from the AgNPs that probably interact with the cell wall of the bacteria, leading to increased membrane permeability [12,37,38]. The findings of a recent study revealed that AgNPs can simultaneously induce apoptosis in E. coli and inhibit new DNA synthesis in the cells in a positive concentration-dependent manner [39]. Other mechanisms involving the interaction of silver molecules with biological macromolecules, such as enzymes and DNA through an electron-release mechanism or free radical production, have been proposed [40,41].
3.4. Role of AgNPs in suppression of DNA replication fidelity using PCR Nanosilver is increasingly used in the food industry and biomedical applications. Several studies have implicated the potential toxicity of nanosilver through the modulation of phosphorous and sulphur containing macromolecules [40][41][42]. However, there is limited information on whether or how AgNPs cause changes in genetic materials. In this study, the replication fidelity of small (600 bp) and large (1500 bp) DNA fragments was quantified when nanosilver particles were present in PCRs. The results showed that the replication fidelity of a small gene was compromised by a nanosilver particle in a dose-dependent manner (  . This observation corrolates with the hypothesis that surface interactions between metallic nanoparticles and Taq DNA polymerase play an essential role and that BSA displaces Taq DNA polymerase from the silver surface. Gold nanoparticles were recently reported to reduce the formation of nonspecific products in PCR by hypothesized mechanisms, including adsorption of DNA and heat-transfer enhancement [43]. It was also reported that gold nanoparticles suppress the amplification of longer products while favoring amplification of shorter products, independent of specificity [44]. In contrast, we In concordance with observations from other research work, our experimental results imply that the nanoparticles nonspecifically adsorb polymerase, thus effectively reducing DNA polymerase concentration, leading to the suppression of amplicon size [42].

Biocompatibility of synthesized AgNPs
Despite their potent antimicrobial activity and wide biological applications, the use of AgNPs as a therapeutic agent is limited due to their potential toxic effect against mammalian cells. In this study, we tested the cytotoxicity of a plant extract that stabilized AgNPs against a macrophage cell line by the MTT assay, which relies on the fact that metabolically active cells reduce MTT to purple formazan. Untreated cells as well as cells exposed to different concentrations of the AgNPs for 24 h were subjected to the MTT assay for cell viability determination. Hence, the intensity of the dye read at 570 nm was directly proportional to the number of viable cells. AgNPs exerted no significant cytotoxic effect on mouse macrophage cell line RAW264.7 at the bactericidal concentration (5 μM), indicating that the phytochemicals present in the aqueous TF root extract provide a nontoxic capping on the AgNPs ( figure 8). However, treatment with AgNPs at higher doses (10, 312, and 625 μM) resulted in approximately 10%-40% reduction in cell viability.
It is reported in the literature that different researchers showed cytotoxic effects of metallic nanoparticle preparation on mammalian cells. Gold nanoparticles showed no significant toxicity in HeLa cells [45,46], whereas significant size-dependent toxicity was observed in fibroblasts and melanoma cells [47]. Further, it was shown that AgNPs that did not contain any capping or stabilizers exhibited a significant toxic effect on mouse macrophage J774.A1 [48], whereas starch-stabilized AgNPs had no effect on cancer cell lines U251 and fibroblasts IMR-90 [49]. In our study, seeds showing an emergence of radicle or cotyledon were recorded as being germinated to assess the phytotoxicity of AgNPs (table S1). As can be seen from table S1, the germination rate of seeds treated with AgNPs were not much affected (71.4%) as compared to the double distilled water treated seed controls (85.7%). At the gross phenotype level, AgNPs treated seedlings had shorter roots than the controls. The observation of the present study on phytotoxicity requires further research work to understand the interaction between metal-based nanoparticles and seed coats thereby modulating the phyto-physiology.

Conclusions
The AgNPs synthesized using the aqueous root extract of TF is an efficient and environmentally-friendly process. AFM and TEM micrographs showed that the synthesized AgNPs were of a monodispersed spherical shape with a size range of 15-30 nm. The synthesized AgNPs revealed potent biocidal activity against bacteria and dermatophytes used in this study. The biocompatible AgNPs could be used as an antimicrobial agent in the field of biomedicine.