Synthesis of Biogenic Silver Nanoparticles (AgCl-NPs) Using a Pulicaria vulgaris Gaertn. Aerial Part Extract and Their Application as Antibacterial, Antifungal and Antioxidant Agents.

In this study, very simple and fast one-step synthesis of biogenic silver chloride nanoparticles (AgCl-NPs) using a Pulicaria vulgaris Gaertn. aerial part extract from an aqueous solution of silver nitrate at room temperature is proposed. The proceedings of the reaction were investigated by UV-Vis spectroscopy. AgCl-NPs were characterized using X-ray diffraction spectroscopy (XRD), Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). Antibacterial and antifungal activities of these nanoparticles were evaluated by disk diffusion and microdilution methods against Staphylococcus aureus, Escherichia coli, Candida albicans, and C. glabrata. In addition, the antioxidant activity of the synthesized AgCl-NPs was determined by the DPPH radical scavenging assay. The antimicrobial test confirmed the bactericidal activity of biosynthesized AgCl-NPs against Gram-positive and Gram-negative bacteria. They also exhibited good antifungal activities with minimum inhibitory concentration (MIC) values ranging from 40 to 60 µg/mL against Candida glabrata and Candida albicans, respectively. In addition, biosynthesized AgCl-NPs were established to have remarkable antioxidant activity. All this pointed out that the proposed new biosynthesis approach resulted in production of AgCl-NPs with convenient biomedical applications.


Introduction
Nanotechnology is the most fascinating field of study in modern material sciences [1]. Nanoparticles (NPs) show a particle size ranging from 1 to 100 nanometers (nm) [2]. Because of the large surface area to volume ratios, nanoscale materials have unique and superior physicochemical characteristics in comparison to their bulk structures [3]. Most of studied NPs are metallic because their syntheses are easier. In addition, monometallic or bimetallic NPs have a wide range of applications; i.e., from detectors, chemical sensors, and catalysts, to surface coating agents, electronic components, photographic components, and pharmaceutical products [4,5]. Metallic NPs can be synthesized using different methods; i.e., chemical, physical, and biological. Conventional physical and chemical methods normally include expensive physical and chemical processes that often use substrates or are accompanied by byproducts, which pose cytotoxicity, environmental toxicity, and carcinogenicity [6]. Bio-based synthesis, facilitated by the use of enzymes, microorganisms, and cell or plant extracts to reduce dissolved metal ions, is considered as an inexpensive, simple, nontoxic, and environment-friendly approach to the production of well characterized, highly stable, range of 200-800 nm were acquired with an UV-Vis spectrophotometer (UV-1800 240 V, Shimadzu Corporation, Kyoto, Japan).
The presence of a variety of functional groups from the plant extract, which could contribute to synthesis of biogenic AgCl-NPs, was evaluated by FTIR spectroscopy. A mixture of dried AgCl-NPs and KBr was ground into a fine powder and pressed into a pellet. Using a Nicolet 800 FTIR spectrometer (Nicolet, Madison, USA), absorption spectra of AgCl-NPs included in pellets were acquired at wavelengths ranging from 4000 to 400 cm −1 at resolution of 4 cm −1 , as described by Das et al. [24].
Crystalline behavior of synthesized biogenic AgCl-NPs was evaluated by X-ray diffraction (XRD), using a Cu-Kα radiation source (λ = 1.54 Å). Diffractograms were scanned in the region of 2θ from 10 • to 80 • at a rate of 0.026 • /min, using an X-ray diffractometer (Siemens D5000, Munich, Germany). The Debye-Scherer equation was applied to determine the average crystallite size of biogenic AgCl-NPs, as described by Bagherzade et al. [25].
The average sizes and shapes of synthesized biogenic AgCl-NPs were assessed using a Philips GM-30 transmission electron microscope (Hillsboro, OR, USA), operated at 120 kV with 2.5 Å resolution, as recommended by a TEM instrument supplier. One drop of an AgCl-NP-containing solution was put onto a Cu grid and evaporated to dryness under an infrared lamp. Digimizer software (version 4.1.1.0, MedCalc Software, Ostend, Belgium) was used for determination of particles size distribution based on TEM images.

Disc Diffusion Method
Antimicrobial and antifungal activities of AgCl-NPs were evaluated using the disc diffusion method as described by National Committee for Clinical Laboratory Standards (NCCLS) [26]. Accordingly, 100 µL of 0.5 McFarland standard suspensions of tested microorganisms, which contained 1.5 × 10 8 CFU/mL of bacteria or 1.5 × 10 6 CFU/mL of fungal strains, were applied in these tests. Mueller-Hinton (Merck KGaA, Darmstadt, Germany) and Sabouraud dextrose agars were used to inoculate tested bacterial and fungal strains. Different concentrations of biogenic AgCl-NPs (20 and 40 µg/mL), dispersed in sterile distill water, were used in these experiments. Solutions of amoxicillin (20 and 40 µg/mL) and fluconazole (20 and 40 µg/mL), both purchased from Farabi pharmaceutical Company, Iran, were also used in combination with AgCl-NPs for antibacterial and antifungal activity, respectively. Sterilized paper discs (6 mm in diameter) were impregnated with 10 µL of above mentioned solutions, and allowed to dry at room temperature. After that, disks were placed on Petri dishes, and inoculated by tested microorganisms. Discs treated with DMSO were used as negative controls. All Petri dishes were incubated at 37 • C for 24 h (bacterial strains) or 48 h (fungal strains). Susceptibility of the tested microorganisms to synthesized biogenic AgCl-NPs was evaluated by measuring appropriate diameters of resulting inhibition zones (in mm).

Determination of Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration (MIC) was measured by using the microdilution assay, as explained by the Clinical and Laboratory Standards Institute (CLSI) [27]. In case of antibacterial activity, serial dilutions of a AgCl-NP bulk solution (at 20, 40, 60, 80, 100, and 120 µg/mL) were made in a 96-well microtiter plate, using the Muller-Hinton broth medium. To evaluate antifungal activity, serial dilutions of the above mentioned AgCl-NP solution (at 20, 40, 60, 80, 100, and 120 µg/mL) were made, using the RPMI-1640 medium (Merck KGaA, Darmstadt, Germany) supplemented with 3-morpholinopropane-1-sulfonic acid (MOPS) (Sigma, St. Louis, MO, USA). In total, 100 µL of 0.5 McFarland standard suspensions of the aforementioned microorganisms were then added to wells of microtiter plates and incubated at 37 • C for 24 or 48 h for bacterial and fungal strains, respectively. Media with inoculums but without AgCl-NPs were intended as negative controls, while amoxicillin and fluconazole were used as positive controls for bacteria and fungi, respectively. MIC values reflected the lowest concentrations of biogenic AgCl-NPs that led to no visible growth of the microorganisms.

Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC)
The minimum bactericidal concentration (MBC) and the minimum fungicidal concentration (MFC) were assessed according to CLSI [27]. In short, 100 µL aliquots of media from wells containing bacteria and fungi, in which growth was not visible, were sub-cultured on Mueller-Hinton agar and Sabouraud dextrose agar plates. These plates were incubated for 24 h at 37 • C. The lowest concentrations of AgCl-NPs that showed no bacterial and fungal growth were considered as MBC and MFC values, respectively.

Antioxidant Properties of the Biosynthesized AgCl-NPs
DPPH Radical Scavenging Activity 2,2-diphenyl-1-picrylhydrazyl (DPPH) from Merck (Merck KGaA, Darmstadt, Germany) was used to measure free radical scavenging potential of biosynthesized AgCl-NPs, as described by Brand-Williams et al. [28] with slight modifications. Methanolic solutions of AgCl-NPs at various concentrations (20,40,60,80, 100, and 120 µg/mL) were prepared. Methanolic solutions of butylated hydroxytoluene (BHT) at the same concentrations were also prepared and used as standards. One milliliter aliquots of the abovementioned solutions were added to 2 mL of a methanolic solution of DPPH (1 mmol/L), and thoroughly vortexed. Resulting mixtures were incubated in dark for 30 min at room temperature. After that, the absorbance value of each mixture was measured at 517 nm. The DPPH absorbance value was considered as a control. Free radical scavenging activity of AgCl-NPs or BHT solutions was expressed as the inhibition percentage that was calculated by the following equation: DPPH radical scavenging activity (%) = 100 × (A c −A s )/A c , where A c is absorbance of the control (containing solvent and DPPH), A s -absorbance measured for solutions of AgCl-NPs or BHT.

Statistical Analysis
Data were analyzed using the statistical software package SPSS version 11.5 (IBM Corporation, Armonk, NY, USA). The one-way analysis of variance test (ANOVA) and the Duncan's multiple range test were applied to investigate differences between various groups. All results were expressed as mean values ± standard deviations (SDs).

Visual Confirmation of Green Synthesis of AgCl-NPs
Differently concentrated (1%, 2%, 4%, and 6%) ethanolic extracts of aerial parts of P. vulgaris were used for synthesis of biogenic AgCl-NPs. It was established that only in case of the addition of the 6% (v/v) extract to a 1 mmol/L AgNO 3 solution, would bioreduction and biosynthesis processes take place, and the colorless AgNO 3 solution became dark brown with time ( Figure 1). This is likely attributable to the completion of the synthesis process, and the formation of biogenic AgCl-NPs [11]. It was previously reported that P. vulgaris contains alkaloids, tannins, flavonoids, and phenolic compounds [29] that could be responsible for the bioreduction of Ag(I) ions into AgCl-NPs as well as their capping in Nanomaterials 2020, 10, 638 5 of 17 the solution [30]. The green synthesis mechanism of AgCl-NPs is not clear yet, but it seems that secondary metabolites of P. vulgaris played an important role in their formation and stabilization. These compounds, containing hydroxyl and ketonic groups, likely bound Ag(I) ions-which was necessary to reduce them to form appropriate nuclei [31]. During the development phase, AgCl nuclei were grown to form spherical AgCl-NPs, which were additionally stabilized by mentioned plant metabolites [31]. take place, and the colorless AgNO3 solution became dark brown with time ( Figure 1). This is likely attributable to the completion of the synthesis process, and the formation of biogenic AgCl-NPs [11]. It was previously reported that P. vulgaris contains alkaloids, tannins, flavonoids, and phenolic compounds [29] that could be responsible for the bioreduction of Ag(I) ions into AgCl-NPs as well as their capping in the solution [30]. The green synthesis mechanism of AgCl-NPs is not clear yet, but it seems that secondary metabolites of P. vulgaris played an important role in their formation and stabilization. These compounds, containing hydroxyl and ketonic groups, likely bound Ag(I) ions-which was necessary to reduce them to form appropriate nuclei [31]. During the development phase, AgCl nuclei were grown to form spherical AgCl-NPs, which were additionally stabilized by mentioned plant metabolites [31].

UV-visible Spectroscopy
UV-Vis spectra of resulting reaction mixtures were measured at 15, 30, 60, 90, 150, and 210 min after mixing the AgNO3 solution and the ethanolic 6% (v/v) plant extract ( Figure 1). According to these spectra, intensity of the localized surface plasmon resonance (LSPR) absorption band was increased with time and its maximum was observed at approximately 460 nm, which corresponded to formation of spherical AgCl-NPs [32,33]. UV-VIS spectra of reaction mixtures were also acquired at times higher than 210 min. These spectra overlapped with the spectra acquired at 210 min that pointed out that the end of reaction was reached.

FTIR Spectroscopy
FTIR analysis was carried out to identify groups of compounds present in the extract that were linked to biosynthesized nanoparticles ( Figure 2A). C-Cl stretching vibrations were observed at 639 cm −1 and corresponded to alkyl halides [34]. Absorption bands at 1610 cm −1 were assigned to C=C and carbonyl (C=O) stretching vibrations of amide groups (amide I/II), relating to functional groups take place, and the colorless AgNO3 solution became dark brown with time ( Figure 1). This is likely attributable to the completion of the synthesis process, and the formation of biogenic AgCl-NPs [11]. It was previously reported that P. vulgaris contains alkaloids, tannins, flavonoids, and phenolic compounds [29] that could be responsible for the bioreduction of Ag(I) ions into AgCl-NPs as well as their capping in the solution [30]. The green synthesis mechanism of AgCl-NPs is not clear yet, but it seems that secondary metabolites of P. vulgaris played an important role in their formation and stabilization. These compounds, containing hydroxyl and ketonic groups, likely bound Ag(I) ions-which was necessary to reduce them to form appropriate nuclei [31]. During the development phase, AgCl nuclei were grown to form spherical AgCl-NPs, which were additionally stabilized by mentioned plant metabolites [31].

UV-visible Spectroscopy
UV-Vis spectra of resulting reaction mixtures were measured at 15, 30, 60, 90, 150, and 210 min after mixing the AgNO3 solution and the ethanolic 6% (v/v) plant extract ( Figure 1). According to these spectra, intensity of the localized surface plasmon resonance (LSPR) absorption band was increased with time and its maximum was observed at approximately 460 nm, which corresponded to formation of spherical AgCl-NPs [32,33]. UV-VIS spectra of reaction mixtures were also acquired at times higher than 210 min. These spectra overlapped with the spectra acquired at 210 min that pointed out that the end of reaction was reached.

FTIR Spectroscopy
FTIR analysis was carried out to identify groups of compounds present in the extract that were linked to biosynthesized nanoparticles ( Figure 2A). C-Cl stretching vibrations were observed at 639 cm −1 and corresponded to alkyl halides [34]. Absorption bands at 1610 cm −1 were assigned to C=C and carbonyl (C=O) stretching vibrations of amide groups (amide I/II), relating to functional groups 15, Nanomaterials 2020, 10, x FOR PEER REVIEW 5 of 17 take place, and the colorless AgNO3 solution became dark brown with time ( Figure 1). This is likely attributable to the completion of the synthesis process, and the formation of biogenic AgCl-NPs [11]. It was previously reported that P. vulgaris contains alkaloids, tannins, flavonoids, and phenolic compounds [29] that could be responsible for the bioreduction of Ag(I) ions into AgCl-NPs as well as their capping in the solution [30]. The green synthesis mechanism of AgCl-NPs is not clear yet, but it seems that secondary metabolites of P. vulgaris played an important role in their formation and stabilization. These compounds, containing hydroxyl and ketonic groups, likely bound Ag(I) ions-which was necessary to reduce them to form appropriate nuclei [31]. During the development phase, AgCl nuclei were grown to form spherical AgCl-NPs, which were additionally stabilized by mentioned plant metabolites [31].

UV-visible Spectroscopy
UV-Vis spectra of resulting reaction mixtures were measured at 15, 30, 60, 90, 150, and 210 min after mixing the AgNO3 solution and the ethanolic 6% (v/v) plant extract ( Figure 1). According to these spectra, intensity of the localized surface plasmon resonance (LSPR) absorption band was increased with time and its maximum was observed at approximately 460 nm, which corresponded to formation of spherical AgCl-NPs [32,33]. UV-VIS spectra of reaction mixtures were also acquired at times higher than 210 min. These spectra overlapped with the spectra acquired at 210 min that pointed out that the end of reaction was reached.

FTIR Spectroscopy
FTIR analysis was carried out to identify groups of compounds present in the extract that were linked to biosynthesized nanoparticles ( Figure 2A). C-Cl stretching vibrations were observed at 639 cm −1 and corresponded to alkyl halides [34]. Absorption bands at 1610 cm −1 were assigned to C=C and carbonyl (C=O) stretching vibrations of amide groups (amide I/II), relating to functional groups 30, Nanomaterials 2020, 10, x FOR PEER REVIEW 5 of 17 take place, and the colorless AgNO3 solution became dark brown with time ( Figure 1). This is likely attributable to the completion of the synthesis process, and the formation of biogenic AgCl-NPs [11]. It was previously reported that P. vulgaris contains alkaloids, tannins, flavonoids, and phenolic compounds [29] that could be responsible for the bioreduction of Ag(I) ions into AgCl-NPs as well as their capping in the solution [30]. The green synthesis mechanism of AgCl-NPs is not clear yet, but it seems that secondary metabolites of P. vulgaris played an important role in their formation and stabilization. These compounds, containing hydroxyl and ketonic groups, likely bound Ag(I) ions-which was necessary to reduce them to form appropriate nuclei [31]. During the development phase, AgCl nuclei were grown to form spherical AgCl-NPs, which were additionally stabilized by mentioned plant metabolites [31].

UV-visible Spectroscopy
UV-Vis spectra of resulting reaction mixtures were measured at 15, 30, 60, 90, 150, and 210 min after mixing the AgNO3 solution and the ethanolic 6% (v/v) plant extract ( Figure 1). According to these spectra, intensity of the localized surface plasmon resonance (LSPR) absorption band was increased with time and its maximum was observed at approximately 460 nm, which corresponded to formation of spherical AgCl-NPs [32,33]. UV-VIS spectra of reaction mixtures were also acquired at times higher than 210 min. These spectra overlapped with the spectra acquired at 210 min that pointed out that the end of reaction was reached.

FTIR Spectroscopy
FTIR analysis was carried out to identify groups of compounds present in the extract that were linked to biosynthesized nanoparticles ( Figure 2A). C-Cl stretching vibrations were observed at 639 cm −1 and corresponded to alkyl halides [34]. Absorption bands at 1610 cm −1 were assigned to C=C and carbonyl (C=O) stretching vibrations of amide groups (amide I/II), relating to functional groups 60, Nanomaterials 2020, 10, x FOR PEER REVIEW 5 of 17 take place, and the colorless AgNO3 solution became dark brown with time ( Figure 1). This is likely attributable to the completion of the synthesis process, and the formation of biogenic AgCl-NPs [11]. It was previously reported that P. vulgaris contains alkaloids, tannins, flavonoids, and phenolic compounds [29] that could be responsible for the bioreduction of Ag(I) ions into AgCl-NPs as well as their capping in the solution [30]. The green synthesis mechanism of AgCl-NPs is not clear yet, but it seems that secondary metabolites of P. vulgaris played an important role in their formation and stabilization. These compounds, containing hydroxyl and ketonic groups, likely bound Ag(I) ions-which was necessary to reduce them to form appropriate nuclei [31]. During the development phase, AgCl nuclei were grown to form spherical AgCl-NPs, which were additionally stabilized by mentioned plant metabolites [31].

UV-visible Spectroscopy
UV-Vis spectra of resulting reaction mixtures were measured at 15, 30, 60, 90, 150, and 210 min after mixing the AgNO3 solution and the ethanolic 6% (v/v) plant extract (Figure 1). According to these spectra, intensity of the localized surface plasmon resonance (LSPR) absorption band was increased with time and its maximum was observed at approximately 460 nm, which corresponded to formation of spherical AgCl-NPs [32,33]. UV-VIS spectra of reaction mixtures were also acquired at times higher than 210 min. These spectra overlapped with the spectra acquired at 210 min that pointed out that the end of reaction was reached.

FTIR Spectroscopy
FTIR analysis was carried out to identify groups of compounds present in the extract that were linked to biosynthesized nanoparticles (Figure 2A). C-Cl stretching vibrations were observed at 639 cm −1 and corresponded to alkyl halides [34]. Absorption bands at 1610 cm −1 were assigned to C=C and carbonyl (C=O) stretching vibrations of amide groups (amide I/II), relating to functional groups 90, Nanomaterials 2020, 10, x FOR PEER REVIEW 5 of 17 take place, and the colorless AgNO3 solution became dark brown with time ( Figure 1). This is likely attributable to the completion of the synthesis process, and the formation of biogenic AgCl-NPs [11]. It was previously reported that P. vulgaris contains alkaloids, tannins, flavonoids, and phenolic compounds [29] that could be responsible for the bioreduction of Ag(I) ions into AgCl-NPs as well as their capping in the solution [30]. The green synthesis mechanism of AgCl-NPs is not clear yet, but it seems that secondary metabolites of P. vulgaris played an important role in their formation and stabilization. These compounds, containing hydroxyl and ketonic groups, likely bound Ag(I) ions-which was necessary to reduce them to form appropriate nuclei [31]. During the development phase, AgCl nuclei were grown to form spherical AgCl-NPs, which were additionally stabilized by mentioned plant metabolites [31].

UV-visible Spectroscopy
UV-Vis spectra of resulting reaction mixtures were measured at 15, 30, 60, 90, 150, and 210 min after mixing the AgNO3 solution and the ethanolic 6% (v/v) plant extract (Figure 1). According to these spectra, intensity of the localized surface plasmon resonance (LSPR) absorption band was increased with time and its maximum was observed at approximately 460 nm, which corresponded to formation of spherical AgCl-NPs [32,33]. UV-VIS spectra of reaction mixtures were also acquired at times higher than 210 min. These spectra overlapped with the spectra acquired at 210 min that pointed out that the end of reaction was reached.

FTIR Spectroscopy
FTIR analysis was carried out to identify groups of compounds present in the extract that were linked to biosynthesized nanoparticles (Figure 2A). C-Cl stretching vibrations were observed at 639 cm −1 and corresponded to alkyl halides [34]. Absorption bands at 1610 cm −1 were assigned to C=C and carbonyl (C=O) stretching vibrations of amide groups (amide I/II), relating to functional groups 150 and Nanomaterials 2020, 10, x FOR PEER REVIEW 5 of 17 take place, and the colorless AgNO3 solution became dark brown with time ( Figure 1). This is likely attributable to the completion of the synthesis process, and the formation of biogenic AgCl-NPs [11]. It was previously reported that P. vulgaris contains alkaloids, tannins, flavonoids, and phenolic compounds [29] that could be responsible for the bioreduction of Ag(I) ions into AgCl-NPs as well as their capping in the solution [30]. The green synthesis mechanism of AgCl-NPs is not clear yet, but it seems that secondary metabolites of P. vulgaris played an important role in their formation and stabilization. These compounds, containing hydroxyl and ketonic groups, likely bound Ag(I) ions-which was necessary to reduce them to form appropriate nuclei [31]. During the development phase, AgCl nuclei were grown to form spherical AgCl-NPs, which were additionally stabilized by mentioned plant metabolites [31].

UV-visible Spectroscopy
UV-Vis spectra of resulting reaction mixtures were measured at 15, 30, 60, 90, 150, and 210 min after mixing the AgNO3 solution and the ethanolic 6% (v/v) plant extract (Figure 1). According to these spectra, intensity of the localized surface plasmon resonance (LSPR) absorption band was increased with time and its maximum was observed at approximately 460 nm, which corresponded to formation of spherical AgCl-NPs [32,33]. UV-VIS spectra of reaction mixtures were also acquired at times higher than 210 min. These spectra overlapped with the spectra acquired at 210 min that pointed out that the end of reaction was reached.

FTIR Spectroscopy
FTIR analysis was carried out to identify groups of compounds present in the extract that were linked to biosynthesized nanoparticles (Figure 2A). C-Cl stretching vibrations were observed at 639 cm −1 and corresponded to alkyl halides [34]. Absorption bands at 1610 cm −1 were assigned to C=C and carbonyl (C=O) stretching vibrations of amide groups (amide I/II), relating to functional groups 210 min). Additionally, color changes of reaction mixtures are shown.

UV-Visible Spectroscopy
UV-Vis spectra of resulting reaction mixtures were measured at 15, 30, 60, 90, 150, and 210 min after mixing the AgNO 3 solution and the ethanolic 6% (v/v) plant extract (Figure 1). According to these spectra, intensity of the localized surface plasmon resonance (LSPR) absorption band was increased with time and its maximum was observed at approximately 460 nm, which corresponded to formation of spherical AgCl-NPs [32,33]. UV-VIS spectra of reaction mixtures were also acquired at times higher than 210 min. These spectra overlapped with the spectra acquired at 210 min that pointed out that the end of reaction was reached.

FTIR Spectroscopy
FTIR analysis was carried out to identify groups of compounds present in the extract that were linked to biosynthesized nanoparticles (Figure 2A). C-Cl stretching vibrations were observed at 639 cm −1 and corresponded to alkyl halides [34]. Absorption bands at 1610 cm −1 were assigned to C=C and carbonyl (C=O) stretching vibrations of amide groups (amide I/II), relating to functional groups of proteins and peptides, deformations of aromatic rings, and C=O stretching vibrations related to flavonoids [35]. C-H stretching vibrations were found at 2930 cm −1 , and were characteristic of aromatic components [25]. The absorption band at 3432 cm −1 corresponded to OH functional groups in phenolic components and alcohols with strong hydrogen bonds [25]. All these FTIR absorption peaks verified that synthesized AgCl-NPs were capped with P. vulgaris metabolites, likely including phenolic compounds and flavonoids. These compounds stabilized their structure and likely added novel properties to biosynthesized nanoparticles. phenolic compounds and flavonoids. These compounds stabilized their structure and likely added novel properties to biosynthesized nanoparticles.

Transmission Electron Microscopy (TEM)
TEM analysis ( Figure 3A,B) clearly confirmed that the shape of biosynthesized AgCl-NPs was spherical, while their size was ranged from 14.3 to 50.7 nm with the average of 28.6 ± 9.0 nm-that being the same as the particle size measured based on the XRD pattern.

Transmission Electron Microscopy (TEM)
TEM analysis ( Figure 3A and B) clearly confirmed that the shape of biosynthesized AgCl-NPs was spherical, while their size was ranged from 14.3 to 50.7 nm with the average of 28.6 ± 9.0 nmthat being the same as the particle size measured based on the XRD pattern. (A)

Disc Diffusion Method
Results of the inhibition zone test of biosynthesized AgCl-NPs against studied bacterial and fungal pathogens are shown in Figures. 4, 5, and 6. They clearly showed that biogenic AgCl-NPs had significant antibacterial and antifungal properties. AgCl-NPs indicated higher inhibition activity against the Gram-positive S. aureus strain than against the Gram-negative E. coli strain. Greater susceptibility of Gram-positive bacterial strains as compared to Gram-negative bacterial strains to action of AgCl-NPs could be related to differences in membrane permeability of these microorganisms [36]. For all tested bacteria and fungi, 20 or 40 µ g/mL solutions of synthesized biogenic AgCl-NPs alone were established to have appropriate antibacterial and antifungal activities. Results obtained in the present work using the disc diffusion method for bacterial and fungal strains could suggest that AgCl-NPs caused physical changes in the structural integrity of the membranes of the studied pathogens, resulting in their permeability. This could be responsible for leakage of cellular contents and cell death, as was also observed in [37,38]. The mechanism of antimicrobial activity of synthesized biogenic AgCl-NPs could also be related to the release of Ag + ions and their effect on studied bacterial and fungal strains [39].

Disc Diffusion Method
Results of the inhibition zone test of biosynthesized AgCl-NPs against studied bacterial and fungal pathogens are shown in Figure 4, Figure 5, and Figure 6. They clearly showed that biogenic AgCl-NPs had significant antibacterial and antifungal properties. AgCl-NPs indicated higher inhibition activity against the Gram-positive S. aureus strain than against the Gram-negative E. coli strain. Greater susceptibility of Gram-positive bacterial strains as compared to Gram-negative bacterial strains to action of AgCl-NPs could be related to differences in membrane permeability of these microorganisms [36]. For all tested bacteria and fungi, 20 or 40 µg/mL solutions of synthesized biogenic AgCl-NPs alone were established to have appropriate antibacterial and antifungal activities. Results obtained in the present work using the disc diffusion method for bacterial and fungal strains could suggest that AgCl-NPs caused physical changes in the structural integrity of the membranes of the studied pathogens, resulting in their permeability. This could be responsible for leakage of cellular contents and cell death, as was also observed in [37,38]. The mechanism of antimicrobial activity of synthesized biogenic AgCl-NPs could also be related to the release of Ag + ions and their effect on studied bacterial and fungal strains [39].     Interestingly, a combination of biosynthesized AgCl-NPs with amoxicillin or fluconazole was found to have synergetic effects on their antibacterial and antifungal activities, respectively. Since bacterial cell membranes include hydrophobic structures, they likely prevent the passage of antibiotics. In case of their combination with the AgCl-NP nanostructures used, due to their small particle size, they could enter target cells and enhance the death of pathogens [40]. A similar synergistic effect of biosynthesized AgCl-NPs in combination with antibiotics and fungicides was previously reported by Patra and Baek [13].

Determination of MIC, MBC, and MFC Values
Biosynthesized AgCl-NPs showed the best antibacterial activity against all tested bacteria strains with MIC values ranging from 60 to 80 µg/mL (see Figure 7A). MBC values of these AgCl-NPs for S. aureus and E. coli were 80 and 100 µg/mL, respectively. It was assumed that AgCl-NPs could effectively bind to bacteria biomolecules, and posing high cytotoxicity, limit their activities, and lead to their death [41]. MIC values for tested C. glabrata and C. albicans strains were 40-60 µg/mL, respectively. MFC values of these AgCl-NPs were 80 and 100 µg/mL for C. glabrata and C. albicans, respectively ( Figure 7B). These results were in agreement with Salati et al. [42], who investigated biological synthesis of AgNPs by a mango plant extract, and their anti-Candida effects. They reported that biogenic AgNPs were most effective against C. glabrata as compared to C. albicans. Nanomaterials 2020, 10, x FOR PEER REVIEW 12 of 17 Interestingly, a combination of biosynthesized AgCl-NPs with amoxicillin or fluconazole was found to have synergetic effects on their antibacterial and antifungal activities, respectively. Since bacterial cell membranes include hydrophobic structures, they likely prevent the passage of antibiotics. In case of their combination with the AgCl-NP nanostructures used, due to their small particle size, they could enter target cells and enhance the death of pathogens [40]. A similar synergistic effect of biosynthesized AgCl-NPs in combination with antibiotics and fungicides was previously reported by Patra and Baek [13].

Determination of MIC, MBC, and MFC Values
Biosynthesized AgCl-NPs showed the best antibacterial activity against all tested bacteria strains with MIC values ranging from 60 to 80 µ g/mL (see Figure 7A). MBC values of these AgCl-NPs for S. aureus and E. coli were 80 and 100 µ g/mL, respectively. It was assumed that AgCl-NPs could effectively bind to bacteria biomolecules, and posing high cytotoxicity, limit their activities, and lead to their death [41]. MIC values for tested C. glabrata and C. albicans strains were 40-60 µ g/mL, respectively. MFC values of these AgCl-NPs were 80 and 100 µ g/mL for C. glabrata and C. albicans, respectively ( Figure 7B). These results were in agreement with Salati et al. [42], who investigated biological synthesis of AgNPs by a mango plant extract, and their anti-Candida effects. They reported that biogenic AgNPs were most effective against C. glabrata as compared to C. albicans. (A)

Antioxidant Properties of Biosynthesized AgCl-NPs
Free radical scavenging activity of biosynthesized AgCl-NPs was evaluated by the DPPH radical scavenging assay ( Figure 8). They had a remarkable scavenging potential, but their radical scavenging was lower when compared to BHT. The observed effect of synthesized biogenic AgCl-NPs on DPPH radicals was likely related to their hydrogen donation activity [43]. It has been reported that the P. vulgaris extract contains a high number of polyphenolic compounds [17]. These compounds have strong antioxidants, which help to protect cells from oxidative damage by free radicals. Phenolic compounds entrapped on the surface of AgCl-NPs could enhance their antioxidant activity, having the vital ability to scavenge free radicals and up-regulate certain metal chelation reactions [44]. We suggest that antioxidant properties of AgCl-NPs could be due to the simultaneous activity of AgCl-NPs as a catalyst and polyphenolic compounds on their surfaces as antioxidant agents [45].

Antioxidant Properties of Biosynthesized AgCl-NPs
Free radical scavenging activity of biosynthesized AgCl-NPs was evaluated by the DPPH radical scavenging assay (Figure 8). They had a remarkable scavenging potential, but their radical scavenging was lower when compared to BHT. The observed effect of synthesized biogenic AgCl-NPs on DPPH radicals was likely related to their hydrogen donation activity [43]. It has been reported that the P. vulgaris extract contains a high number of polyphenolic compounds [17]. These compounds have strong antioxidants, which help to protect cells from oxidative damage by free radicals. Phenolic compounds entrapped on the surface of AgCl-NPs could enhance their antioxidant activity, having the vital ability to scavenge free radicals and up-regulate certain metal chelation reactions [44]. We suggest that antioxidant properties of AgCl-NPs could be due to the simultaneous activity of AgCl-NPs as a catalyst and polyphenolic compounds on their surfaces as antioxidant agents [45].

Conclusion
The current study demonstrated the application of ethanolic extracts of Pulicaria vulgaris Gaertn. aerial parts for one-stage synthesis of AgCl-NPs in a cost-effective, simple, and environmentally friendly way at room temperature. Results showed that the P. vulgaris extract contained organic compounds such that functional groups possibly can act as both reducing and stabilizing agents in the biosynthesis of AgCl-NPs. Biosynthesized AgCl-NPs revealed the LSPR absorption band at 460 nm, and had the average particle size of 28.6 nm, as measured based on TEM images and the XRD pattern. They showed significant antibacterial, antifungal, and antioxidant activates, and had synergetic effects in combination with a conventional antibiotic and a fungicide, amoxicillin and fluconazole, against the tested bacterial and fungal pathogens. These convenient properties of our biogenic AgCl-NPs mean that they could successfully be used as appropriate biocidal agents in biomedical and food industry applications.

Conclusions
The current study demonstrated the application of ethanolic extracts of Pulicaria vulgaris Gaertn. aerial parts for one-stage synthesis of AgCl-NPs in a cost-effective, simple, and environmentally friendly way at room temperature. Results showed that the P. vulgaris extract contained organic compounds such that functional groups possibly can act as both reducing and stabilizing agents in the biosynthesis of AgCl-NPs. Biosynthesized AgCl-NPs revealed the LSPR absorption band at 460 nm, and had the average particle size of 28.6 nm, as measured based on TEM images and the XRD pattern. They showed significant antibacterial, antifungal, and antioxidant activates, and had synergetic effects in combination with a conventional antibiotic and a fungicide, amoxicillin and fluconazole, against the tested bacterial and fungal pathogens. These convenient properties of our biogenic AgCl-NPs mean that they could successfully be used as appropriate biocidal agents in biomedical and food industry applications.