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Plant-mediated biosynthesis of silver nanoparticles using Prosopis farcta extract and its antibacterial properties
Accepted Manuscript Plant-mediated biosynthesis of silver nanoparticles using Prosopis farcta extract and its antibacterial properties Abdolhossein Miri, Mina Sarani, Mahere Rezazade Bazaz, Majid Darroudi PII: DOI: Reference:
S1386-1425(15)00034-7 http://dx.doi.org/10.1016/j.saa.2015.01.024 SAA 13192
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
5 December 2014 11 January 2015 14 January 2015
Please cite this article as: A. Miri, M. Sarani, M.R. Bazaz, M. Darroudi, Plant-mediated biosynthesis of silver nanoparticles using Prosopis farcta extract and its antibacterial properties, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.01.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Plant-mediated biosynthesis of silver nanoparticles using Prosopis farcta extract and its antibacterial properties Abdolhossein Miria,b, Mina Sarania, Mahere Rezazade Bazazc,d , Majid Darroudie,f,* a
Zabol Medicinal Plants Research Center, Zabol University of Medical Sciences, P.O.Box,3333-669699, Zabol, Iran b Department of Pharmacognosy, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran c Neurogenic Inflammation Research Centre, School of Medicine, Mashhad University of Medical Sciences, Mashhad 9177948564, Iran d Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran e Nuclear Medicine Research Center, Mashhad University of Medical Sciences, Mashhad, Iran f Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Abstract “Green” synthesis of metal nanoparticles has become a promising synthetic strategy in nanoscience and nanotechnology in recent years. In this work, silver nanoparticles (Ag-NPs) were synthesized from extract of Prosopis farcta at room temperature. Formation of Ag-NPs at 1mM concentration of AgNO3 gave spherical shape nanoparticles with mean diameter about 10.8 nm. The formation of nanoparticle was confirmed by the surface Plasmon resonance (SPR) band illustrated in UV-vis spectrophotometer. The morphology and size of the Ag-NPs were determined using high magnification transmission electron microscopy (TEM). The crystalline structure of obtained nanoparticles was investigated using the powder X-ray diffraction (PXRD) pattern. In addition, these green synthesized Ag-NPs were found to show higher antibacterial activity against multi drug resistant clinical isolates. Key words: Silver nanoparticles; green synthesis; Prosopis fracta; TEM; PXRD
*
Corresponding author:
M. Darroudi E-mail: [email protected] & [email protected] Tel.: +98-513-8002286 & Fax: +98-513-8002287
1
1. Introduction Nowadays, nanoparticles have been improved as a result of truly important recent advances by applying “green” chemistry rules to nanotechnology and materials sciences [1]. Silver nanoparticles (Ag-NPs) are one of the most important commercialized nanoparticles. Ag-NPs have attracted considerable attention during the past few years because of their potential applications in different fields such as biomedicine [2, 3], biosensor [4], and oxidative catalysis [5]. Different types of Ag-NPs are used in over 200 consumer products. As an antibacterial, antimicrobial, and antiviral agent, Ag-NPs are employed in medical devices, cosmetics, home water purification systems, textiles, and household appliances [6]. Many techniques of synthesizing Ag-NPs have been investigated. Some of them are wet chemical reduction [7], electrochemical [8], γ-ray irradiation [9], UV-irradiation [10], photochemical reduction [11], ultrasonic assisted [12], microwave [13], and laser ablation [14, 15]. Synthesis of Ag-NPs via different “green” chemico-physical conditions as well as the biosynthetic methods by numerous microorganisms has been extremely researched. When Ag-NPs are chemically synthesized, three main components are needed: (1) silver salt (e.g., AgNO3), (2) reducing agent (e.g., NaBH4), and (3) stabilizing or capping agent (e.g., polyvinyl alcohol) for controlling the growth and size of the nanoparticles and prevent them from aggregating [16]. In biosynthesis of Ag-NPs, the reducing and stabilizing agents are replaced by existence molecules in living organisms which these molecules (bio-reducing and bio-stabilizing agents) can be present in different biological systems such as bacteria, fungi, yeasts, algae, or plants [17, 18]. The exact mechanism of biological reduction in conversion of Ag+ to Ag0 is not completely understood but normally enzymatic reduction can be distinguished from non-enzymatic reduction. In enzymatic reduction, silver cations can be trapped and reduced by negatively charged carboxylated groups of enzymes
2
in the cell wall [19]. On the other hand, the non-enzymatic reduction of Ag+ is based on the chemical reduction by reducing and stabilizing agents are produced by microorganisms or plants [20, 21] such as proteins which cap the Ag-NPs and prevent them from aggregation [22]. Large scale production generating inexpensive Ag-NPs is needed. This can only be achieved when the biological production system is chosen in function of the yield. Hence, the true challenge for biogenic Ag is finding the balance between scalability, price, and applicability. Then, a more scalable, available, and low cost route to fabrication Ag-NPs would be through the plantmediated synthetic approach. Some microorganisms, such as bacteria, yeasts, and viruses, in the living environment are often pathogenic and cause severe infections in human beings. On the other hand, in recent years, most of these pathogenic microorganisms are resistant to antimicrobial agents [23-25]. Consequently, the development and modification of novel antimicrobial agents (e.g., natural and inorganic based antimicrobial substances) are necessary due to increasing demand of alternative treatments [26, 27]. Among inorganic antimicrobial agents, Ag-NPs have been most widely used due to its unique chemo-physical properties. Several approaches reported antimicrobial activity of Ag-NPs and its mechanisms in Gram positive and Gram negative bacteria [28-30]. Therefore, it was of great interest to take advantage and explore a novel “green” template for biosynthesizing AgNPs as new antimicrobial agent. Herein, a novel approach for the biosynthesis of Ag-NPs as a green chemistry method using extract of Prosopis farcta (P. farcta) at room temperature has been reported. The reduction of Ag+ in aqueous solution exhibits colloidal Ag-NPs with particle mean diameter about 10.8 nm. In addition these bio-synthesized Ag-NPs were found to show higher antibacterial activity against different multi drug resistant bacteria.
3
2. Materials and methods 2.1. Materials Silver nitrate is purchased from Sigma-Aldrich chemicals for this study. Prosopis farcta (P. farcta) leaves were collected from Zabol, Sistan and baluchestan province, IRAN. The P. farcta leaves were cleaned with double distilled water and shade-dried for a week at ambient temperature and further P. farcta leaves were ground to powder and stored for further study. All glassware’s are washed with HNO3 and distilled water and dried in oven.
2.2. Preparation of leaf extract In a typical reaction process, P. farcta leaf extract was prepared by taking 5.0 g of dry leaf powder with 50 ml of distilled and the extract was placed in orbital shaker for 4 h and then extract was filtered through Whatman filter paper No. 1. The clearly filtered, light brown extract was stored in refrigerator at 4⁰C. The extract is used as reducing agent as well as stabilizing agent.
2.3. Biosynthesis of Ag-NPs For the synthesis of Ag-NPs, 5 ml of the extract was added dropwise to 95 ml of silver nitrate (1 mM) solution and the solution was placed in orbital shaker at room temperature for 1 h. The color of solution change involved in the formation of Ag-NPs from light brown to dark brown as shown in Fig. 1. The final solution containing Ag-NPs was centrifuged at about 10,000 rpm for 15 min. The obtained precipitation was kept into petri plates and left in the oven for drying at about 60⁰C for 24 h. The dried Ag-NPs were scrapped out for the further study.
4
2.4. Antibacterial activity Antibacterial effect of Ag-NPs was evaluated using disk diffusion method against common Gram positive bacteria, i.e., Staphylococcus aureus (PTCC 1431), Bacillus subtilis (PTCC 1420) and Gram-negative bacteria, i.e., Escherichia coli (PTCC 1399), Pseudomonas aeruginosa (PTCC 1074) according to standard methods (NCCLS 2000). Overnight culture of bacterial suspension was adjusted to 0.5 McFarland equivalents to 1.5×108 colony-forming units/ml. Dried surface of Muller–Hinton agar (MHA) inoculated with above bacterial suspension by streaking the swab three times and then 6 mm discs impregnated with 15 µl of Ag-NPs solution were placed on inoculated agars [31]. Besides discs impregnated with aqueous extract solution (0.1% v/v) considered as negative control. All plates were incubated at 37⁰C for 24 h. Gentamicin and Streptomycin were used as antibacterial standards against all pathogens. The zones of inhibition were measured and numbers reported in average (Table 1). Experiments were performed in triplicate.
2.5. Characterization of Ag-NPs The biosynthesized Ag-NPs in a solution was confirmed by measuring the UV-vis spectrum of the solution (1:4 diluted) of the reaction mixture. UV-vis spectrum was recorded on double beam spectrophotometer (Shimadzu, model UV-1800) from 300 to 900 nm at a resolution of 0.5 nm. The double distilled water containing the extract was used as a blank. The Ag-NPs synthesized with 5% leaf extracts and 1 mM silver nitrate solution were characterized with the help of transmission electron microscopy (TEM, model LEO 440i). The observed reflection planes corresponding to fcc Ag-NPs in PXRD diffraction pattern (Philips, X’pert, Cu Kα radiations (k = 1.5418 Å)).
5
3. Results and discussion 3.1. UV–vis spectral studies As shown in Fig. 1, incubation of culture supernatant with silver nitrate illustrated a color change from light brown to brown whereas no color change was observed in culture supernatant without silver nitrate. The appearance of a brownish color in silver nitrate-treated culture supernatant suggested the formation of Ag-NPs [32, 33], which is a part of confirmation to support the preparation of Ag-NPs. Further confirmation of synthesis of Ag-NPs was examined by UV-vis spectrophotometer. Light wavelengths in the range of 300–800 nm are normally used for characterizing different metallic nanoparticles in the nanoscale [34, 35]. It is distinguished that Ag-NPs exhibit brownish colors, depending on the concentration and the size of nanoparticles; the colors arise due to the excitation of surface Plasmon resonance (SPR) of the Ag-NPs [36]. The appearance of the brownish color was due to the excitation of the SPR, typical of Ag-NPs having λmax values in the visible range of 400–500 nm [37]. In the UV–vis absorption spectrum, a strong SPR peak, located at about 433 nm, was observed due to formation of Ag-NPs using the culture supernatant of P. farcta (Fig. 2). 3.2. PXRD analysis The crystalline structure of synthesized Ag-NPs was investigated by PXRD analysis. Fig. 3 illustrated that the PXRD pattern of the as prepared Ag-NPs. The diffraction peaks were detected in the 2θ angles in a range of 35–80°, which can be indexed to the (111), (200), (220), and (311). 6
Accordingly, it confirms that the biosynthesized Ag-NPs have faced centered cubic (fcc) crystalline structure. The obtained result was matched with the Joint Committee on Powder Diffraction Standards (JCPDS) file No. 04-0783. The average crystallite size of biosynthesized Ag-NPs was estimated from the full width half maximum (FWHM) of (111) reflection as shown in Fig. 3 using Scherrer formula as presented in Eq. 1 [38]: (1)
D = 0.9λ/βCosθ
where D is the crystallite size, λ is the wavelength of the X–ray source (0.1541 nm), β is the FWHM, θ is the angle of diffraction. It was obtained that the average diameter of the Ag-NPs crystal was about 8.5 nm. This result was confirmed with calculated mean diameter of nanoparticles from the TEM analysis (Fig. 4). Some unassigned intense diffraction peaks, might be related to the crystallization of bioorganic phases that attach on the surface of the nanoparticles. Because of the biomass residue, other crystallographic impurities such as AgCl were also observed in the XRD profile [39-41]. 3.3. The morphology and size of Ag-NPs analysis by TEM The morphology and size of biosynthesized Ag-NPs were evaluated by TEM. A representative TEM micrograph recorded from the aqueous Ag-NPs deposited on carbon coated copper grids is shown in Fig. 4. The typical monodispersed spherical Ag-NPs apparently can be distinguished in the TEM micrograph. From the TEM micrograph and size distribution analysis (Fig. 4) it is observed that diameter of biosynthesized Ag-NPs are about 10.8 nm and approximately all the Ag-NPs are homogeneous in nature.
7
3.4. Antibacterial activity of biosynthesized Ag-NPs Disk diffusion is a distinguished and accustomed method to determine how is toxicity effect of a material in solution against to bacterial colonies [42]. In this work, we are interest to know the antibacterial activity of biosynthesized Ag-NPs against Gram positive and Gram negative bacteria. Fig. 5 illustrates the inhibition zone at around the disks due to the diffusion of Ag-NPs. Compared with the control, the diameters of inhibition zones increased for all the test pathogens. As shown in Fig. 5, the biosynthesized Ag-NPs could inhibit four different pathogenic bacteria, including Staphylococcus aureus, Bacillus Subtilis, Pseudomonas aeruginosa, and Escherichia coli as previously reported [43, 44]. Inhibition zone values represented as averages in Table 1 and the assays were performed in triplicate.
S1386-1425(15)00034-7 http://dx.doi.org/10.1016/j.saa.2015.01.024 SAA 13192
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
5 December 2014 11 January 2015 14 January 2015
Please cite this article as: A. Miri, M. Sarani, M.R. Bazaz, M. Darroudi, Plant-mediated biosynthesis of silver nanoparticles using Prosopis farcta extract and its antibacterial properties, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.01.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Plant-mediated biosynthesis of silver nanoparticles using Prosopis farcta extract and its antibacterial properties Abdolhossein Miria,b, Mina Sarania, Mahere Rezazade Bazazc,d , Majid Darroudie,f,* a
Zabol Medicinal Plants Research Center, Zabol University of Medical Sciences, P.O.Box,3333-669699, Zabol, Iran b Department of Pharmacognosy, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran c Neurogenic Inflammation Research Centre, School of Medicine, Mashhad University of Medical Sciences, Mashhad 9177948564, Iran d Department of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran e Nuclear Medicine Research Center, Mashhad University of Medical Sciences, Mashhad, Iran f Department of Modern Sciences and Technologies, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Abstract “Green” synthesis of metal nanoparticles has become a promising synthetic strategy in nanoscience and nanotechnology in recent years. In this work, silver nanoparticles (Ag-NPs) were synthesized from extract of Prosopis farcta at room temperature. Formation of Ag-NPs at 1mM concentration of AgNO3 gave spherical shape nanoparticles with mean diameter about 10.8 nm. The formation of nanoparticle was confirmed by the surface Plasmon resonance (SPR) band illustrated in UV-vis spectrophotometer. The morphology and size of the Ag-NPs were determined using high magnification transmission electron microscopy (TEM). The crystalline structure of obtained nanoparticles was investigated using the powder X-ray diffraction (PXRD) pattern. In addition, these green synthesized Ag-NPs were found to show higher antibacterial activity against multi drug resistant clinical isolates. Key words: Silver nanoparticles; green synthesis; Prosopis fracta; TEM; PXRD
*
Corresponding author:
M. Darroudi E-mail: [email protected] & [email protected] Tel.: +98-513-8002286 & Fax: +98-513-8002287
1
1. Introduction Nowadays, nanoparticles have been improved as a result of truly important recent advances by applying “green” chemistry rules to nanotechnology and materials sciences [1]. Silver nanoparticles (Ag-NPs) are one of the most important commercialized nanoparticles. Ag-NPs have attracted considerable attention during the past few years because of their potential applications in different fields such as biomedicine [2, 3], biosensor [4], and oxidative catalysis [5]. Different types of Ag-NPs are used in over 200 consumer products. As an antibacterial, antimicrobial, and antiviral agent, Ag-NPs are employed in medical devices, cosmetics, home water purification systems, textiles, and household appliances [6]. Many techniques of synthesizing Ag-NPs have been investigated. Some of them are wet chemical reduction [7], electrochemical [8], γ-ray irradiation [9], UV-irradiation [10], photochemical reduction [11], ultrasonic assisted [12], microwave [13], and laser ablation [14, 15]. Synthesis of Ag-NPs via different “green” chemico-physical conditions as well as the biosynthetic methods by numerous microorganisms has been extremely researched. When Ag-NPs are chemically synthesized, three main components are needed: (1) silver salt (e.g., AgNO3), (2) reducing agent (e.g., NaBH4), and (3) stabilizing or capping agent (e.g., polyvinyl alcohol) for controlling the growth and size of the nanoparticles and prevent them from aggregating [16]. In biosynthesis of Ag-NPs, the reducing and stabilizing agents are replaced by existence molecules in living organisms which these molecules (bio-reducing and bio-stabilizing agents) can be present in different biological systems such as bacteria, fungi, yeasts, algae, or plants [17, 18]. The exact mechanism of biological reduction in conversion of Ag+ to Ag0 is not completely understood but normally enzymatic reduction can be distinguished from non-enzymatic reduction. In enzymatic reduction, silver cations can be trapped and reduced by negatively charged carboxylated groups of enzymes
2
in the cell wall [19]. On the other hand, the non-enzymatic reduction of Ag+ is based on the chemical reduction by reducing and stabilizing agents are produced by microorganisms or plants [20, 21] such as proteins which cap the Ag-NPs and prevent them from aggregation [22]. Large scale production generating inexpensive Ag-NPs is needed. This can only be achieved when the biological production system is chosen in function of the yield. Hence, the true challenge for biogenic Ag is finding the balance between scalability, price, and applicability. Then, a more scalable, available, and low cost route to fabrication Ag-NPs would be through the plantmediated synthetic approach. Some microorganisms, such as bacteria, yeasts, and viruses, in the living environment are often pathogenic and cause severe infections in human beings. On the other hand, in recent years, most of these pathogenic microorganisms are resistant to antimicrobial agents [23-25]. Consequently, the development and modification of novel antimicrobial agents (e.g., natural and inorganic based antimicrobial substances) are necessary due to increasing demand of alternative treatments [26, 27]. Among inorganic antimicrobial agents, Ag-NPs have been most widely used due to its unique chemo-physical properties. Several approaches reported antimicrobial activity of Ag-NPs and its mechanisms in Gram positive and Gram negative bacteria [28-30]. Therefore, it was of great interest to take advantage and explore a novel “green” template for biosynthesizing AgNPs as new antimicrobial agent. Herein, a novel approach for the biosynthesis of Ag-NPs as a green chemistry method using extract of Prosopis farcta (P. farcta) at room temperature has been reported. The reduction of Ag+ in aqueous solution exhibits colloidal Ag-NPs with particle mean diameter about 10.8 nm. In addition these bio-synthesized Ag-NPs were found to show higher antibacterial activity against different multi drug resistant bacteria.
3
2. Materials and methods 2.1. Materials Silver nitrate is purchased from Sigma-Aldrich chemicals for this study. Prosopis farcta (P. farcta) leaves were collected from Zabol, Sistan and baluchestan province, IRAN. The P. farcta leaves were cleaned with double distilled water and shade-dried for a week at ambient temperature and further P. farcta leaves were ground to powder and stored for further study. All glassware’s are washed with HNO3 and distilled water and dried in oven.
2.2. Preparation of leaf extract In a typical reaction process, P. farcta leaf extract was prepared by taking 5.0 g of dry leaf powder with 50 ml of distilled and the extract was placed in orbital shaker for 4 h and then extract was filtered through Whatman filter paper No. 1. The clearly filtered, light brown extract was stored in refrigerator at 4⁰C. The extract is used as reducing agent as well as stabilizing agent.
2.3. Biosynthesis of Ag-NPs For the synthesis of Ag-NPs, 5 ml of the extract was added dropwise to 95 ml of silver nitrate (1 mM) solution and the solution was placed in orbital shaker at room temperature for 1 h. The color of solution change involved in the formation of Ag-NPs from light brown to dark brown as shown in Fig. 1. The final solution containing Ag-NPs was centrifuged at about 10,000 rpm for 15 min. The obtained precipitation was kept into petri plates and left in the oven for drying at about 60⁰C for 24 h. The dried Ag-NPs were scrapped out for the further study.
4
2.4. Antibacterial activity Antibacterial effect of Ag-NPs was evaluated using disk diffusion method against common Gram positive bacteria, i.e., Staphylococcus aureus (PTCC 1431), Bacillus subtilis (PTCC 1420) and Gram-negative bacteria, i.e., Escherichia coli (PTCC 1399), Pseudomonas aeruginosa (PTCC 1074) according to standard methods (NCCLS 2000). Overnight culture of bacterial suspension was adjusted to 0.5 McFarland equivalents to 1.5×108 colony-forming units/ml. Dried surface of Muller–Hinton agar (MHA) inoculated with above bacterial suspension by streaking the swab three times and then 6 mm discs impregnated with 15 µl of Ag-NPs solution were placed on inoculated agars [31]. Besides discs impregnated with aqueous extract solution (0.1% v/v) considered as negative control. All plates were incubated at 37⁰C for 24 h. Gentamicin and Streptomycin were used as antibacterial standards against all pathogens. The zones of inhibition were measured and numbers reported in average (Table 1). Experiments were performed in triplicate.
2.5. Characterization of Ag-NPs The biosynthesized Ag-NPs in a solution was confirmed by measuring the UV-vis spectrum of the solution (1:4 diluted) of the reaction mixture. UV-vis spectrum was recorded on double beam spectrophotometer (Shimadzu, model UV-1800) from 300 to 900 nm at a resolution of 0.5 nm. The double distilled water containing the extract was used as a blank. The Ag-NPs synthesized with 5% leaf extracts and 1 mM silver nitrate solution were characterized with the help of transmission electron microscopy (TEM, model LEO 440i). The observed reflection planes corresponding to fcc Ag-NPs in PXRD diffraction pattern (Philips, X’pert, Cu Kα radiations (k = 1.5418 Å)).
5
3. Results and discussion 3.1. UV–vis spectral studies As shown in Fig. 1, incubation of culture supernatant with silver nitrate illustrated a color change from light brown to brown whereas no color change was observed in culture supernatant without silver nitrate. The appearance of a brownish color in silver nitrate-treated culture supernatant suggested the formation of Ag-NPs [32, 33], which is a part of confirmation to support the preparation of Ag-NPs.
Accordingly, it confirms that the biosynthesized Ag-NPs have faced centered cubic (fcc) crystalline structure. The obtained result was matched with the Joint Committee on Powder Diffraction Standards (JCPDS) file No. 04-0783. The average crystallite size of biosynthesized Ag-NPs was estimated from the full width half maximum (FWHM) of (111) reflection as shown in Fig. 3 using Scherrer formula as presented in Eq. 1 [38]: (1)
D = 0.9λ/βCosθ
where D is the crystallite size, λ is the wavelength of the X–ray source (0.1541 nm), β is the FWHM, θ is the angle of diffraction. It was obtained that the average diameter of the Ag-NPs crystal was about 8.5 nm. This result was confirmed with calculated mean diameter of nanoparticles from the TEM analysis (Fig. 4). Some unassigned intense diffraction peaks, might be related to the crystallization of bioorganic phases that attach on the surface of the nanoparticles. Because of the biomass residue, other crystallographic impurities such as AgCl were also observed in the XRD profile [39-41].
7
3.4. Antibacterial activity of biosynthesized Ag-NPs Disk diffusion is a distinguished and accustomed method to determine how is toxicity effect of a material in solution against to bacterial colonies [42]. In this work, we are interest to know the antibacterial activity of biosynthesized Ag-NPs against Gram positive and Gram negative bacteria. Fig. 5 illustrates the inhibition zone at around the disks due to the diffusion of Ag-NPs. Compared with the control, the diameters of inhibition zones increased for all the test pathogens. As shown in Fig. 5, the biosynthesized Ag-NPs could inhibit four different pathogenic bacteria, including Staphylococcus aureus, Bacillus Subtilis, Pseudomonas aeruginosa, and Escherichia coli as previously reported [43, 44]. Inhibition zone values represented as averages in Table 1 and the assays were performed in triplicate.