**2. Results**

#### *2.1. Formation of Ag NPs*

During the synthesis of Ag NPs, the color of the mixture solution containing silver salt and walnut leaf extract changed from colorless into brown and dark. In fact, the synthesized Ag NPs, due to their surface plasmon resonance (SPR), changed the color of the mixture solution and this color change verified the formation of Ag NPs using walnut leaf extract and microwave irradiation (Figure 1). As clearly observed in Table 1, λmax of the synthesized Ag NPs was varied 424–429 nm, which was in a favorable range for Ag NPs [8,10]. The particle size of the synthesized Ag NPs could be correlated with their broad emission peaks (λmax), where longer wavelengths in λmax of Ag NPs were associated with bigger size [9]. This indicated that *J. regia* leaf extract successfully reduced silver ions and formed Ag NPs.

**Figure 1.** Color and appearance changes during synthesis of silver nanoparticles (Ag NPs) using *J. regia* leaf extract. *J. regia* leaf extract containing silver nitrate before ( **A**) and after (**B**) exposure to microwave irradiation.

Figure 2 shows the Fourier transform-infrared (FT-IR) spectrum of *J. regia* leaf extract which was in the region range of 400–4000 cm<sup>−</sup>1. The IR spectrum of leaf extract absorption bands at 3454.87 and 2072.86 cm<sup>−</sup><sup>1</sup> represent the phenolic–OH and N=C groups. Absorption bands at 1637.48 cm<sup>−</sup><sup>1</sup> are related to the amino group, bending vibrations, and the –OH group. The obtained results indicated that the phenolic compounds and proteins were the two main components of the *J. regia* leaf extract which had key roles in the formation of the stabilized Ag NPs [11,12].


**Table 1.** Experimental runs according to the central composite design (CCD) and response variables for synthesis of Ag NPs.

Exp, Experimental values of studied responses; Pre, Predicted values of studied responses. \*, Out of range.

**Figure 2.** Fourier transform-infrared (FT-IR) spectrum of *J. regia* leaf extract.

#### *2.2. Models Generation and Synthesis Conditions Optimization*

Based on experimental runs, the values of broad emission peaks (λmax, nm) and concentration (ppm) for the fabricated Ag NPs were achieved (Table 1) and according to these obtained data, models were created. Table 2 shows coefficients of the model terms and models accuracy based on R square and its adjusted (R2, R2-adj) and lack of fit. The high values of R<sup>2</sup> (>77.80) and R2-adj (>85.61) and lack of fit (*p* > 0.05) verified higher suitability of the generated models to predict the synthesis response parameters. *p*-Values of the generated model terms are also described in Table 3. As can be

seen, the interaction of the amounts of leaf extract and silver salt solution had significant (*p* < 0.05) effects on λmax and concentration of the formed Ag NPs.


**Table 2.** Regression coefficients, R2, R2-adj, and probability values for the fitted models.

β0 is a constant, β1, β11 and β12 are the linear, quadratic, and interaction coefficients of the quadratic polynomialequation, respectively.

**Table 3.** *p*-Values of the regression coefficients in the obtained models.


Figure 3A,B indicates the effects of the amount of *J. regia* leaf extract and amount of AgNO3 on the λmax of the synthesized Ag NPs. As clearly observed in Figure 3, the minimum λmax (particle size) was obtained at both minimum amount of *J. regia* leaf extract and of AgNO3 solution and the maximum amount of *J. regia* leaf extract and of AgNO3 solution. The experimental value of the concentration for the synthesized Ag NPs ranged 19–80 ppm (Table 1). The effects of the amount of *J. regia* leaf extract andof AgNO3 on the concentration of the fabricated Ag NPs are shown in Figure 4A,B.

As clearly observed in Figure 4, the maximum concentration of the synthesized Ag NPs was obtained using the highest amount of the *J. regia* leaf extract. The obtained results were in line with the findings of other research [8–10]. They found that by increasing the amount of plant extract, the concentration of the bioreductants increased in the extract, which in turn increased the concentration of the formed Ag NPs.

In the synthesis of Ag NPs, the main objective is formation of NPs with desirable physico-chemical properties, including minimum particle size (λmax) and maximum concentration. This synthesis process is known as optimized procedure. According to the generated models for the synthesis of Ag NPs, an overlaid contour plot, as graphical optimization (Figure 5), was plotted to better visualize of optimum area (white colored zone).

The result of a numerical optimization also demonstrated that by using 0.1 mL of *J. regia* leaf extract and 15 mL of AgNO3, Ag NPs with minimum λmax of 421 nm and maximum concentration of 135.64 ppm were produced. A verification test using optimum synthesis parameters also indicated insignificant (*p* > 0.05) differences between the values of predicted and experimental of λmax and concentration of the fabricated Ag NPs and verified the suitability of the models.

**Figure 3.** Response surface (**A**) and contour plots (**B**) for λmax of the synthesized Ag NP solution as function of the amount of *J. regia* leaf extract and amount of AgNO3 solution.

**Figure 4.** Response surface (**A**) and contour plots (**B**) for the concentration of the synthesized Ag NP solution as function of amount of *J. regia* leaf extract and amount of AgNO3 solution.

**Figure 5.** Overlaid contour plot of Ag NPs λmax and concentration with acceptable levels as a function of amount of *J. regia* leaf extract and amount of AgNO3 solution.

#### *2.3. Physico-Chemical Characteristics of the Synthesized Ag NPs at Obtained Optimum Conditions*

Formation of Ag NPs using *J. regia* leaf extract at obtained optimum conditions was confirmed by changes in the color of the mixture solution. Dynamic light scattering (DLS) analysis also indicated that the synthesized Ag NPs had particle size, polydispersity index (PDI), and zeta potential values of 168 nm, 0.419 and −15.6 mV, respectively. The particle size distributions (PSD) of the sample are also shown in Figure 6.

**Figure 6.** Particle size distribution of synthesized Ag NPs at obtained optimum synthesis conditions using *J. regia* leaf extract.

#### *2.4. Antibacterial Activity*

The antibacterial activity of synthesized Ag NPs on the growth of Gram-positive (*S. aureus*) and Gram-negative (*E. coli)* bacteria during incubation indicated that the diameter of the clear zone for synthesized Ag NPs in the plate containing *S. aureus* and *E. coli* were 16 and 10 mm, respectively (Figure 7). The results also indicated that the mean diameter of formed clear zone around the Ampicillin disc in the plates containing *S. aureus* and *E. coli* were 35 and 33 mm, respectively.

**Figure 7.** Created zones of inhibition with *S. aureus* (**A**) and *E. coli* (**B**) incubated at 37 ◦C for 24 h for synthesized Ag NPs using *J. regia* leaf extract.

The obtained results indicated that the fabricated Ag NPs had higher antibacterial activity against Gram-positive bacteria compared to the Gram-negative bacteria. The obtained results were in agreemen<sup>t</sup> with findings of Ahmadi et al., Mohammadlu et al., and Torabfam and Jafarizadeh-Malmiri [8–10]. The main reason behind the bactericidal activity of the Ag NPs against the bacteria strains is related to their effects on the permeability of the cell wall and membrane. In fact, the released silver ions (Ag+) from the Ag NPs were attached into the anionic groups of the cell wall, such as polycyclic aromatic hydrocarbon and teichoic acids. They also formed polyelectrolyte complexes, which limited the transference of nutrients and provided metabolites into and outside the cell [9]. Furthermore, caffeoylquinic acid is the main phenylpropanoids in *J. regia* leaf extract, which has various bioactivities such as antioxidant, antibacterial, anticancer, antihistamic, and other biological effects. The direct antimicrobial activity of caffeoylquinic acid implies an array of possibilities, including effects in the cell envelope [13].

#### **3. Materials and Methods**
