Green Synthesis of Silver Nanoparticles Using Juniperus procera Extract: Their Characterization, and Biological Activity

: Plant extract-based green synthesis of metal nanoparticles (NPs) has become a popular approach in the ﬁeld of nanotechnology. In this present investigation, silver nanoparticles were prepared by an efﬁcient and facile approach using Juniperus procera extract as a bioreducing and stabilizing agent. The as-synthesized silver nanoparticles (JP-AgNPs) were characterized by several characterization techniques such as UV–Vis, XRD, FT-IR, HR-TEM, and EDX analysis. The XRD analysis evidently conﬁrms that the as-synthesized Ag nanoparticles (NPs) from Juniperus procera plant extract (JP-AgNPs) are crystalline in nature. FT-IR analysis conﬁrms that the plant extract plays a dual role as a bioreducing and capping agent, while HR-TEM revealed the spherical morphology of as-synthesized JP-AgNPs with the size of ~23 nm. Furthermore, the synthesized JP-AgNPs were evaluated for antibacterial properties against several bacterial and fungal strains such as Staphylococcus aureus (ATCC 12228), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 4753), Cryptococcus neoformans (ATCC 16620), and Candida albicans (ATCC 885-653). The JP-AgNPs displayed an efﬁcient mean zone of inhibition (MZI) at 50.00 µ L for bacterial associated with fungal pathogens than the plant extract. Mainly, MZI values against microbial pathogens were as follows; E. coli (17.17 ± 0.72 mm), P. mirabilis (14.80 ± 0.17 mm), and C. albicans (14.30 ± 0.60 mm), whereas JP-AgNPs showed moderate activity against P. aeruginosa (11.50 ± 0.29 mm) and C. neoformans (9.83 ± 0.44 mm). Notably, the tested JP-AgNPs have displayed almost similar antimicrobial activities with that of standard antimicrobial drugs, such as streptomycin and nystatin. The enhanced antimicrobial activity of JP-AgNPs can be ascribed to the quality of resultant NPs including, uniform size, shape, and aqueous colloidal stability of nanoparticles. band evidently the accomplishment of reaction. The UV–Vis spectrum obtained to display the absorption in the visible range from to 460 nm, with a sharp intense peak appearing at 424 nm (Figure 1a,b) which conﬁrms the formation of JP-AgNPs, additional absorption peaks at 260 nm is also observed, which belongs to the phytomolecules from J. procera extract (JP) remaining on the surface of JP-AgNPs as capping agents, which is conﬁrmed by the UV–Vis analysis of an extract of J. procera (JP).


Introduction
Nanotechnology is a prominent field in material science [1], which effectively combines various disciplines of basic sciences including engineering [2], physics [3], chemistry [4], and biological sciences [5]. Principal to the advancement of this field is the development of novel approaches for the efficient and facile preparation of nanoparticles (NPs). NPs offer remarkable physico-chemical characteristics when compare to their bulk counterparts, which have been successfully utilized for various advanced applications [6]. So far, NPs have been produced through various approaches involving a variety of physical, chemical and biological processes. Generally, most of the physical, chemical and hybrid approaches are expensive and hazardous in nature due to the involvement of sophisticated instruments and toxic materials including solvents, reducing agents and precursors etc. [7]. On the other hand, NPs preparation using green approaches is less expensive, eco-friendly and easy to apply. Hence, these approaches have become popular and are increasingly employed for the eco-friendly preparation of metallic NPs. To date, the green synthesis of NPs has been attempted using a variety of natural materials like microorganisms, biomolecules and plant extracts etc. [8]. Although a significant number of studies have been published in the literature on the green synthesis of NPs using biomolecules, microorganisms and plant extracts, plant extracts are more favored over other natural materials [7,9,10]. Since plant extract-based approaches are inexpensive, efficient, and does not involve a cumbersome process of the isolation of the biomolecules and storing the microbial cultures etc. [11]. Moreover, plants based synthesis of nanomaterials does not require expensive reagents, high temperatures and pressure etc.
Among various materials, NPs developed from noble metals have received significant recognition over the past few decades, as they can be utilized in sensors, biomedical devices and various other advanced applications [8,[12][13][14][15]. Out of a variety of metallic NPs, silver NPs have received immense popularity owing to their extraordinary characteristics, comprising of excellent electrical conductivity, high chemical stability, and superior antibacterial activity [16]. Therefore, the synthesis of silver nanoparticles has been attempted using a number of methods and still is an evolving field. Chemically synthesized Ag NPs are of high quality, however, they are mostly not suitable for biomedical application due to the chances of contamination of resulting NPs with the hazardous chemical used during their synthesis. Since the biocompatibility of nanomaterials is of utmost importance when they are considered for biological applications. The green synthesis of Ag NPs using plant extracts is an attractive option, which is less likely to contaminate the final products. [17]. Due to this, several studies have demonstrated the successful preparation of Ag NPs using the extracts of various plants, such as Origanum vulgare L. [16], Pulicaria glutinosa [17], Cordia myxa [18], and Salvadora persica [7]. In addition to the whole plant extracts, other components of the plants like seeds, roots, leaves, fruits, ad stems have been also been applied to prepare diverse metallic nanoparticles. Since these parts are known to contain a wide range of phytochemicals in their extracts, which can effectively play the role of stabilizing and bioreducing agents for the synthesis of nanoparticles [18,19]. In this context, Juniperus procera extract, which is known to consist of a variety of polyphenolic phytoconstituents will be a potential candidate to be explored for the preparation of NPs. However, this has been rarely investigated for the synthesis of silver nanoparticles. Juniperus procera plant belongs to the Cupressaceae family, and phytomolecules of this plant are known to possess excellent antioxidant, antimicrobial, and anticancer activities. Several literature reports stated that more than 65 species allied to Juniperus are spreading all over the world. Analyses performed on J. procera extracts revealed the occurrence of several constituents that might replicate its pharmacological properties [20]. Although Juniperus procera has been used prior for the preparation of Ag NPs, only its leaves' extract is known to produce NPs [21]. Besides, the resultant Ag NPs were not of very good quality due to the large size and wide size distribution of particles between 30-90 nm. To the best of our knowledge, the extract of the whole ariel parts of the plant has never been used for this purpose.
Herein, in the present study, a biosynthesis approach was adopted for the synthesis of silver nanoparticles using J. procera extract. The as-synthesized silver nanoparticles (JP-AgNPs) were characterized by several characterization techniques such as UV-Vis, XRD, FT-IR, TEM, and EDX analysis. Furthermore, the synthesized silver nanoparticles were investigated for biological activity using various bacterial and fungal strains (Scheme 1).

Materials
The aerial parts of J. procera are abundantly available in the Al-Baha region of Saudi Arabia and were collected in the month of April-2020 After the collection of plant material it was firstly identified in the herbarium division by a plant taxonomist at King Saud University, Riyadh. A voucher specimen (HZKJP-20) of the J. procera is maintained with us. AgNO3 (99.8%, Sigma Aldrich, St. Louis, MO, USA), and other chemicals and solvents exploited in this work were purchased from Sigma Aldrich.

Preparation of Plant Extract for the Synthesis of Ag NPs
To start with, fresh aerial parts of the J. procera were first dried at room temperature and ground into small pieces. About 1 kg of J. procera aerial parts was refluxed for ~3 h on a heating mantle at 80 °C. After three hours of reflux, the solution was allowed to cool down and filtered. The water extract obtained was dried completely on a rotary evaporator to get the dark brownish color extract. The resultant extract was stored at room temperature and was used for the synthesis of silver nanoparticles. 1.0 g extract from aerial parts of the J. procera was dissolved in 10 mL of DI water and was used for the NPs synthesis.

Preparation of Silver Nanoparticles Using J. procera (JP-AgNPs)
The synthesis of silver nanoparticles (JP-AgNPs) was carried out by adding 1 mL from the stock solution of the aerial part extract of the J. procera to 50 mL of 0.5 mM of AgNO3 aqueous solution in a 250 mL flask. The flask with the resultant solution is attached with a magnetic stir bar and fixed on a condenser. The reaction was continued for ~120 min at 90 ± 5 °C. While stirring, the reaction color transformed from light green to dark brown color, and afterward, no further color transformation is perceived until the end of the reaction. To eliminate any prospect of the existence of unbound residual phytomolecules on the surface of the green as-synthesized JP-AgNPs, it is re-dispersed in double distilled water via sonication for 20 min after centrifugation, this procedure is continued three times to isolate pure JP-AgNPs. Afterward, the attained reaction product is washed three times with double distilled water, and then the obtained product was dried at 70 °C for 12 h in an oven. Finally, a black powder is achieved. Scheme 1. Graphical representation of silver nanoparticles (JP-AgNPs) synthesis using J. procera extract.

Materials
The aerial parts of J. procera are abundantly available in the Al-Baha region of Saudi Arabia and were collected in the month of April-2020 After the collection of plant material it was firstly identified in the herbarium division by a plant taxonomist at King Saud University, Riyadh. A voucher specimen (HZKJP-20) of the J. procera is maintained with us. AgNO 3 (99.8%, Sigma Aldrich, St. Louis, MO, USA), and other chemicals and solvents exploited in this work were purchased from Sigma Aldrich.

Preparation of Plant Extract for the Synthesis of Ag NPs
To start with, fresh aerial parts of the J. procera were first dried at room temperature and ground into small pieces. About 1 kg of J. procera aerial parts was refluxed for~3 h on a heating mantle at 80 • C. After three hours of reflux, the solution was allowed to cool down and filtered. The water extract obtained was dried completely on a rotary evaporator to get the dark brownish color extract. The resultant extract was stored at room temperature and was used for the synthesis of silver nanoparticles. 1.0 g extract from aerial parts of the J. procera was dissolved in 10 mL of DI water and was used for the NPs synthesis.

Preparation of Silver Nanoparticles Using J. procera (JP-AgNPs)
The synthesis of silver nanoparticles (JP-AgNPs) was carried out by adding 1 mL from the stock solution of the aerial part extract of the J. procera to 50 mL of 0.5 mM of AgNO 3 aqueous solution in a 250 mL flask. The flask with the resultant solution is attached with a magnetic stir bar and fixed on a condenser. The reaction was continued for~120 min at 90 ± 5 • C. While stirring, the reaction color transformed from light green to dark brown color, and afterward, no further color transformation is perceived until the end of the reaction. To eliminate any prospect of the existence of unbound residual phytomolecules on the surface of the green as-synthesized JP-AgNPs, it is re-dispersed in double distilled water via sonication for 20 min after centrifugation, this procedure is continued three times to isolate pure JP-AgNPs. Afterward, the attained reaction product is washed three times with double distilled water, and then the obtained product was dried at 70 • C for 12 h in an oven. Finally, a black powder is achieved.

Preparation of Plant Extracts and JP-AgNPs for Biological Activity
Juniperus procera, sect. Sabina is commonly known as African-cedar. It is a coniferous tree native of Africa and the Arabian Peninsula. In order to prepare the samples for biologi- cal studies, one milligram of extracted plant powder (JP) and one mg of biosynthesized silver nanoparticle (JP-AgNPs) were separately dissolved in one mL of dimethyl sulfoxide (DMSO) each. In this case, the final concentrations of both the samples remained 1 mg/mL.

Antimicrobial Activity of JP-AgNPs
The antibacterial and antifungal activity was performed as per method agar well diffusion method described by Bonev et al. (2008) [22]. Selected bacterial and fungal strains used in the antimicrobial assessment were procured from the Microbiology Lab, King Khalid University Hospital, Riyadh, Saudi Arabia. A list of microbial pathogens namely, Staphylococcus aureus (ATCC 12228), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 4753), Cryptococcus neoformans (ATCC 16620), and Candida albicans (ATCC 885-653). Concisely, Sabouraud dextrose agar (SDA) for C. albicans and C. neoformans and Mueller-Hinton for bacterial strains was poured into Petri plates. The agar plates were swabbed with broth cultures standardized with 0.5 McFarland standard (1.0 × 10 8 CFU/mL). six mm well containing known concentrations of plant extract (JP), JP-AgNPs, and standard drug (10 µg)) were loaded and then incubated at 37 • C for 24 h. Well loaded only with DMSO consider as a negative control. The streptomycin and nystatin were kept as positive controls for bacterial and fungal pathogens, respectively. The mean zone of inhibition (MZI) in millimeters (mm) was determined for each microbial pathogen in later incubation periods. All experiments were conducted in triplicate. The JP-AgNPs with MZI values more than or equal to 12.00 mm were selected for minimum inhibitory concentration (MIC) assays.
For MIC assay [23], a stock solution (mg/mL) of test JP-AgNPs was dissolved in DMSO. Further, a doubling dilution of test JP-AgNPs was performed to achieve concentrations ranging from 0.5 to >64.00 µg/mL whereas, streptomycin and nystatin (0.5 to 15.00 µg/mL). Test JP-AgNPs at various concentrations were then added to culture medium in a 96-well titer plate and different microbial strains were inoculated at 1 × 10 8 CFU/mL concentration. Muller Hinton broth (MHB) medium was employed for both bacterial and fungal pathogens. The titer plate was incubated at 37 • C for 24 h and then observed for the growth of the tested pathogens. Streptomycin and nystatin were used as standard antibacterial and antifungal drugs, respectively. The lowest concentration of the test JP-AgNPs where the well remained clear, indicating that the bacterial or fungal growth was completely inhibited and that the least concentration was considered as MIC value.

Characterization
UV-Visible analysis performed on lambda 35, Perkin Elmer, Waltham, MA, USA. Fourier transform infrared analyses were performed on a 1000 FT-IR instrument, Perkin-Elmer Waltham, MA, USA. The FT-IR samples were measured under the resolution of 4 cm −1 . The X-ray powder diffraction analysis of the as-prepared silver nanoparticles was performed on D2-Phaser, Bruker, Germany. Transmission electron microscopy (TEM) analysis was performed using JEM-1101, Jeol, Japan. Scanning electron microscopy (SEM) and energy-dispersive X-ray analysis was performed using JSM 7600F instrument, JEOL, Tokyo, Japan. The images were captured under an operational voltage of 5 kV and a working distance of 4.5 mm over different magnifications.

UV-Visible Analysis
The silver nanoparticles (JP-AgNPs) formation is monitored by UV-Vis analysis, which is carried out by collecting the reaction mixture from time to time. However, the obvious color transformation in the reaction mixture from light green to dark brown also pointed out the formation of JP-AgNPs. The UV-Vis analysis of the reaction mixture revealed at the start of the reaction, the nucleation process was in progress rapidly, which ensued in the fast development of JP-AgNPs for the first 1 h. After two hours, the nucleation process is found to slow down and no further transformation in the intensity of the absorption band was noticed, which evidently specified the accomplishment of reaction. The UV-Vis spectrum obtained to display the absorption in the visible range from 370 to 460 nm, with a sharp intense peak appearing at 424 nm (Figure 1a,b) which confirms the formation of JP-AgNPs, additional absorption peaks at 260 nm is also observed, which belongs to the phytomolecules from J. procera extract (JP) remaining on the surface of JP-AgNPs as capping agents, which is confirmed by the UV-Vis analysis of an extract of J. procera (JP).
pointed out the formation of JP-AgNPs. The UV-Vis analysis of the reaction mixture revealed at the start of the reaction, the nucleation process was in progress rapidly, which ensued in the fast development of JP-AgNPs for the first 1 h. After two hours, the nucleation process is found to slow down and no further transformation in the intensity of the absorption band was noticed, which evidently specified the accomplishment of reaction. The UV-Vis spectrum obtained to display the absorption in the visible range from 370 to 460 nm, with a sharp intense peak appearing at 424 nm (Figure 1a,b) which confirms the formation of JP-AgNPs, additional absorption peaks at 260 nm is also observed, which belongs to the phytomolecules from J. procera extract (JP) remaining on the surface of JP-AgNPs as capping agents, which is confirmed by the UV-Vis analysis of an extract of J. procera (JP).

FT-IR Analysis
FT-IR analysis was performed to ascertain the possible biomolecules in the J. procera extract. FT-IR spectra of plant extract and as-synthesized JP-AgNPs are displayed in Figure 3. The phytomolecules analysis of J. procera extract discloses the existence of flavonoids, alkaloids, steroids, rosins, saponins, etc., [24]. In the plant extract, the peaks are noticed at 535, 617, 1063, 1267, 1429, 1603, and 3419 cm −1 . After reaction with AgNO 3 , the peaks are shifted slightly, such as 533, 619, 1078, 1273, 1431, 1620, and 3421 cm −1 . The strong intense peaks at 1429 cm −1 correspond to C-N stretching, which may belong to the proteins in the plant extract [25]. The strong band at 1063 cm −1 may correspond to the ether linkages of flavanones [26]. The existing phenolic groups in the plant extract possibly contributing to the reduction are appeared in the~1325-1040 and~1455-1600 cm −1 regions [27]. For example, phenols are typically identified by the characteristic broad band representing OH stretch roughly between 3400 to 3500 cm −1 , as appeared in this case at 3410 cm −1 . There is a bend at~1380 cm −1 , which may correspond to the O-H bending of both phenol and carboxylic acid. Between the wave numbers 1300 cm −1 and 1000 cm −1 , the largest peak is at 1247 cm −1 and this peak is assigned as the C-O stretch of phenol. Whereas, another sharp peak at 1058 cm −1 can be representative of C-O stretching of alcohols. Besides, a sharp band at 1620 cm −1 is due to the C=C aromatic ring stretching, pointing towards the existence of the aromatic group. All these peaks strongly indicate the presence of polyphenolic contents in the plant extract. Therefore, the rich polyphenolic and flavonoic contents present in the plant extract can be potentially responsible for reducing and stabilizing the resultant Ag NPs. Indeed, this is further confirmed by an elaborated study on the extraction of extracts from different parts of the J. procera plant in a variety of solvents [28]. The study revealed that ethanolic, methanolic extracts of leaves demonstrate high concentrations of total phenolic and total flavonoid content. The majority of these peaks are also existing in the FT-IR spectrum of JP-AgNPs with some marginal shifts. Consequently, the occurrence of these peaks in the FT-IR spectrum of JP-AgNPs is evidently directed towards the effective dual role of the aqueous plant extract of J. procera, both as a reducing and stabilizing agent.   The term 'D' is the Crystal size, 'λ' corresponds to X-ray wavelength, 'k' is a dimensionless shape factor, 'β' is the line broadening at half the maximum intensity, and 'θ' is Bragg's angle. The average crystallite size of JP-AgNPs is found to be~23.7 nm (Table 1).

TEM and EDX Analysis
The as-synthesized size and morphology of JP-AgNPs were investigated b transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscop analysis ( Figure 4). Figure 4a displays the spherical morphology of the JP-AgNPs AgNPs are well distributed. Additionally, the particle size distribution graph w mated by using Image J software ( Figure 5), the particle size distribution graph o AgNPs displays the average particle size is in the range of 10-30 nm. Whereas, in a study, the leaves extract of JP has produced large sized NPs with wide size dist between 30-90 nm [21]. The relatively high quality of NPs obtained in our stud due to the presence of large contents of polyphenols and flavonoids in the whole of aerial parts of J. Procera when compare to the leave extract. Furthermore, Figur hibits the energy-dispersive X-ray analysis of the synthesized silver nanoparticle recommends the existence of silver as the main element. Generally, the silver na cles display a typically strong signal at 3 keV, due to surface plasmon resonan profile showed a strong signal for silver along with some other signal peaks whic have initiated from the biomolecules that are bound to the surface of JP-AgNP senting the reduction of silver ions to elemental silver. The other peak of Cu in th an artifact of the Cu grid on which the sample was coated. There were no othe

TEM and EDX Analysis
The as-synthesized size and morphology of JP-AgNPs were investigated by using transmission electron microscopy (TEM) and energy-dispersive X-ray spectroscopy (EDX) analysis ( Figure 4). Figure 4a displays the spherical morphology of the JP-AgNPs. The JP-AgNPs are well distributed. Additionally, the particle size distribution graph was estimated by using Image J software (Figure 5), the particle size distribution graph of the JP-AgNPs displays the average particle size is in the range of 10-30 nm. Whereas, in an earlier study, the leaves extract of JP has produced large sized NPs with wide size distribution between 30-90 nm [21]. The relatively high quality of NPs obtained in our study can be due to the presence of large contents of polyphenols and flavonoids in the whole extract of aerial parts of J. Procera when compare to the leave extract. Furthermore, Figure 4b exhibits the energy-dispersive X-ray analysis of the synthesized silver nanoparticles, which recommends the existence of silver as the main element. Generally, the silver nanoparticles display a typically strong signal at 3 keV, due to surface plasmon resonance. EDX profile showed a strong signal for silver along with some other signal peaks which might have initiated from the biomolecules that are bound to the surface of JP-AgNPs, representing the reduction of silver ions to elemental silver. The other peak of Cu in the EDX is an artifact of the Cu grid on which the sample was coated. There were no other signal peaks noticed for silver. This authorizes the thorough reduction of silver compounds to JP-AgNPs as revealed in the EDX spectrum.

Possible Mechanism of Reduction by Phytomolecules
The antioxidant properties of phytoconstituents consisting of flavonoids, polyphenols are generally caused by their redox properties [29]. Due to this, the polyphenolic compounds function as strong reductants, hydrogen donors, singlet oxygen quenchers [30]. It is believed that these compounds generate reactive hydrogen during the process of tautomerization (conversion from enol-form to the keto-form), which facilitates the reduction of metal ions [31]. Apart from this, other internal oxidations including ketone to carboxylic acid reactions are also known to promote the reduction of metal ions to metal NPs. Therefore, in the case of J. Procera, we believe that Ag NPs were probably formed in three different steps similar to the process that occur during polyol based synthesis of NPs [32]. Initially, Ag seeds are generated due to the reduction of metal ions by virtue of the redox properties of polyphenols and flavonoids, this process is referred to as the induction phase. Subsequently, the small Ag seeds ultimately grow into larger aggregates during the growth process. Finally, in the termination phase, the surface of the Ag aggregates are stabilized by the surface-bound phytoconstituents, which also define the final morphology and size of NPs.

Possible Mechanism of Reduction by Phytomolecules
The antioxidant properties of phytoconstituents consisting of flavonoids, polyphenols are generally caused by their redox properties [29]. Due to this, the polyphenolic compounds function as strong reductants, hydrogen donors, singlet oxygen quenchers [30]. It is believed that these compounds generate reactive hydrogen during the process of tautomerization (conversion from enol-form to the keto-form), which facilitates the reduction of metal ions [31]. Apart from this, other internal oxidations including ketone to carboxylic acid reactions are also known to promote the reduction of metal ions to metal NPs. Therefore, in the case of J. Procera, we believe that Ag NPs were probably formed in three different steps similar to the process that occur during polyol based synthesis of NPs [32]. Initially, Ag seeds are generated due to the reduction of metal ions by virtue of the redox properties of polyphenols and flavonoids, this process is referred to as the induction phase. Subsequently, the small Ag seeds ultimately grow into larger aggregates during the growth process. Finally, in the termination phase, the surface of the Ag aggregates are stabilized by the surface-bound phytoconstituents, which also define the final morphology and size of NPs.

Possible Mechanism of Reduction by Phytomolecules
The antioxidant properties of phytoconstituents consisting of flavonoids, polyphenols are generally caused by their redox properties [29]. Due to this, the polyphenolic compounds function as strong reductants, hydrogen donors, singlet oxygen quenchers [30]. It is believed that these compounds generate reactive hydrogen during the process of tautomerization (conversion from enol-form to the keto-form), which facilitates the reduction of metal ions [31]. Apart from this, other internal oxidations including ketone to carboxylic acid reactions are also known to promote the reduction of metal ions to metal NPs. Therefore, in the case of J. Procera, we believe that Ag NPs were probably formed in three different steps similar to the process that occur during polyol based synthesis of NPs [32]. Initially, Ag seeds are generated due to the reduction of metal ions by virtue of the redox properties of polyphenols and flavonoids, this process is referred to as the induction phase. Subsequently, the small Ag seeds ultimately grow into larger aggregates during the growth process. Finally, in the termination phase, the surface of the Ag aggregates are stabilized by the surface-bound phytoconstituents, which also define the final morphology and size of NPs.

Biological Activity
In the recent past, antimicrobial resistance is a common problem around the globe, more often microbial pathogens are showing resistance against standard drugs in medicine to treat dreadful diseases. However, various chemical and green synthesized nanoparticles are explored in every part of the world and scientific communities extensively investigate the biological potency of such promising nanoparticles. In this lieu, biosynthesized JP-AgNPs were evaluated for their antimicrobial activities.
Overall, these results showed substantial antimicrobial activities against a panel of tested microbial pathogens (Table 2 and Figure 6). The JP-AgNPs exhibited efficient MZI values at 50.00 µL against bacterial pathogens, especially compared to the fungal pathogens. The MZI range between 8.00 ± 0.28 mm and 17.17 ± 0.72 mm. Particularly, MZI values against microbial pathogens were as follows; E. coli (17.17 ± 0.72 mm), P. mirabilis (14.80 ± 0.17 mm) and C. albicans (14.30 ± 0.60 mm), whereas JP-AgNPs showed moderate activity against P. aeruginosa (11.50 ± 0.29 mm) and C. neoformans (9.83 ± 0.44 mm). In the case of S. aureus (8.00 ± 0.28), it exhibited much less activity compared to all the microbial pathogens. Overall antimicrobial activity of plant extract was moderate when compared with JP-AgNPs, and its range was between 6.50 ± 0.10 and 11.25 ± 0.50 mm. Thus, the tested JP-AgNPs was found to hold as same antimicrobial activities when compared to standard antimicrobial drugs, such as streptomycin and nystatin (Table 2 and Figure 7). # a . significant at the level of p < 0.05 in comparison with S. aureus; b . significant at the level of p < 0.05 in comparison with E. coli; c . significant at the level of p < 0.05 in comparison with P. aeruginosa; d . significant at the level of p < 0.05 in comparison with P. mirabilis; e . significant at the level of p < 0.05 in comparison with C. albicans; f . significant at the level of p < 0.05 in comparison with C. neoformans; ns -non-significant; $ -statistically nonsignificant; * = 50 µL of the solution from the stock solution of 1 mg/mL was used, which is equivalent to 50 µg of the powder.
In addition, the assessment of prospective antimicrobials was determined by MIC for the JP-AgNPs with MZI values above or equal to 12.00 mm. The results displayed in Table 3, showed that the tested JP-AgNPs possessed significant antimicrobial activities with minimum (16.00 µg/mL) and maximum (>64.00 µg/mL) MIC value. The JP-AgNPs showed a significant MIC value against E. coli and C. albicans (16.00 µg/mL) followed by P. mirabilis and P. aeruginosa (32.00 µg/mL). This antimicrobial activity incident was described among different strains variation and JP-AgNPs stabilization. Comparatively, MIC values of tested JP-AgNPs were moderately lower than commercially available antimicrobial drugs tested in this study. However, enhanced antimicrobial activity of JP-AgNPs due to synthesis with uniform shape, size, and particle size distribution and also colloidal stability of nanoparticles interacted with the cell wall of microbial pathogens [33,34].
C. neoformans ATCC 16620 9.50 ± 0.25 9.80 ± 0.44 ns,b,ns,d,e 16.00 7.93-11.73 # a . significant at the level of p < 0.05 in comparison with S. aureus; b . significant at the level of p < 0.05 in comparison with E. coli; c . significant at the level of p < 0.05 in comparison with P. aeruginosa; d . significant at the level of p < 0.05 in comparison with P. mirabilis; e . significant at the level of p < 0.05 in comparison with C. albicans; f . significant at the level of p < 0.05 in comparison with C. neoformans; ns -non-significant; $ -statistically non-significant; * = 50 µ L of the solution from the stock solution of 1 mg/mL was used, which is equivalent to 50 µ g of the powder.

Conclusions
In the present study, a novel approach for the synthesis of silver nanoparticles from J. procera plant extract was given. The as-synthesized JP-AgNPs are characterized by various characterization techniques such as UV-Vis, XRD, FT-IR, TEM, and EDX analysis. The as-synthesized JP-AgNPs were spherically shaped with the size of ~20 nm. The UV-  It is widely believed that the antimicrobial activity of nanoparticles usually occur due to the breaking of cell walls, and leakages in the cell membranes [35]. In this case, also the JP-AgNPs were possibly clinging to the bacteria cell wall and liberated silver ions into the bacteria cell by the process of diffusion, which ultimately disrupts the cell wall and causes the death of the bacteria.
In addition, this massive antimicrobial activity is mainly due to the formation of reactive oxygen species (ROS) inside the pathogens [22]. Moreover, the development of the electron spin effect by JP-AgNPs drives the accumulation of free radicals. The free radicals readily interact with the cell membrane of pathogens and create pores resulting in the death of microbial pathogens [36,37]. Reports also demonstrate that JP-AgNPs disrupt the cellular metabolism by which control DNA replication and ATP synthesis [38].

Conclusions
In the present study, a novel approach for the synthesis of silver nanoparticles from J. procera plant extract was given. The as-synthesized JP-AgNPs are characterized by various characterization techniques such as UV-Vis, XRD, FT-IR, TEM, and EDX analysis. The as-synthesized JP-AgNPs were spherically shaped with the size of~20 nm. The UV-Vis spectral analysis confirmed the formation of JP-AgNPs. Potential biomolecules responsible for reducing and stabilizing the silver nanoparticles are indicated by FT-IR measurements. Furthermore, the synthesized JP-AgNPs were evaluated for antibacterial properties against several bacterial and fungal strains such as S. aureus (ATCC 12228), E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), P. mirabilis (ATCC 4753), C. neoformans (ATCC 16620), and C. albicans (ATCC 885-653). The tested JP-AgNPs were found to possess almost similar antimicrobial activities to that of standard antimicrobial drugs, such as streptomycin and nystatin. The JP-AgNPs have revealed antibacterial against a wide range of pathogenic bacteria which recognized their application in biomedicine. Furthermore, it is concluded that the synthesis of AgNPs using J. procera extract is an inexpensive, easy and eco-friendly approach that eliminates the application of toxic reagents which may cause environmental damage.