# Antioxidant and Antibacterial Activities of Silver Nanoparticles Biosynthesized byMoringa oleifera through Response Surface Methodology

Authors: A. B. Abeer Mohammed; Amr Mohamed; Noura El-Ahmady El-Naggar; Hoda Mahrous; Ghada M. Nasr; Asmaa Abdella; Rasha H. Ahmed; Sibel Irmak; Mohamed S. A. Elsayed; Samy Selim; Amr Elkelish; Dalal Hussien M. Alkhalifah; Wael N. Hozzein; Abdallah S. Ali
Journal: Journal of Nanomaterials
 (2022)

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## Abstract

The research highlights the environmentally sustainable biosynthesis of silver nanoparticles from fresh leaves of the herbal medicinal plantMoringa oleifera. They may have been used as anti-inflammatory, anticancer, and antimicrobial agents. M. oleifera extract both reduces and stabilizes silver nanoparticles (AgNPs). Optimum factors needed for AgNP biosynthesis were studied using a central composite design (CCD) matrix. Ultraviolet-visible (UV–Vis) absorption spectroscopy, transmission electron microscopy, and Fourier-transform infrared spectroscopy were used to confirm and characterize the synthesized AgNPs. The biogenic AgNPs demonstrated substantial antibacterial potential against the pathogenic strains Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Bacillus subtilis. The antioxidant activity of biosynthesized AgNPs with M. oleifera extract increased from 11.96% when the concentration of the extract was 4 mg/mL to 63.79% at a plant concentration of 20 mg/mL. This research provides an easy and cost-effective technique for the production of stable nanoparticles, with an evaluation of their bioactivity.


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## Body

## 1. Introduction

Nanoparticles (NPs) can be generated by various chemical, biological, and physical methods [1, 2]. Biological approaches are preferred because they are environmentally friendly [3], competitive [4], and more economical than other approaches [5]. NPs can be created using microorganisms or medicinal plants [6]. Green synthesis is performed by environmentally friendly materials such as herbs and bacteria for NP biosynthesis [7]. This increases the therapeutic value of the NPs produced because the plant phytocompounds provide antioxidant and antibacterial activities for the synthesized silver nanoparticles (AgNPs) [8]. The potential uses of plant materials in the green synthesis of NPs eliminate the need for additional procedures, such as intracellular purification steps for the synthesized compounds and the cultivation of microbial cell cultures [9].Silver has long been recognized as an antimicrobial agent, and AgNPs are nontoxic to eukaryotes such as humans; despite this, AgNPs are extremely poisonous to prokaryotic cells such as bacteria [10]. Researchers are studying their applications in nanomedicine because of their antimicrobial [11], antiplatelet [12], anticancer [13], and wound healing properties [14]. The high surface area-to-volume ratios of AgNPs ensure high reactivity in biomedical research [15]. Various studies have been documented on plant extracts used for AgNP synthesis, such as Papaver somniferum [16], Bauhinia variegata L. [17], Hevea brasiliensis [18], Aloe vera [19], and Acacia farnesiana [20]. In these studies, phytocompound extracts were used as alternative precursors for reducing or capping the silver from silver nitrate during the biosynthesis of AgNPs [16].Moringa oleifera is widely disseminated and used throughout the world’s tropical and subtropical regions, and it is native to India and Africa [21]. It has been used for centuries because of its health benefits and medicinal properties. M. oleifera leaves contain a variety of beneficial chemicals. These are classified into carotenoids, vitamins, glucosinolates, polyphenols, flavonoids, phenolic acids, oxalates, isothiocyanates, alkaloids, phytates, saponins, and tannins [22]. The medicinal significance of M. oleifera is associated with its high antioxidant activity, as well as its substantial antibacterial activity [23]. M. oleifera has been reported to synthesize natural bioactive compounds such as flavonoids and terpenoids. These compounds have been discovered to be potential antimicrobials against pathogenic microorganisms [24–27]. Various studies have documented the biosynthesis of AgNPs from M. oleifera and assessed their many potential uses, such as antimicrobial agents [27–35], their sensing properties [27], water detoxification abilities [31], and their anticancer potential [33].In the current study,M. oleifera was used as a low-cost and environmentally acceptable precursor for bioreduction, biostabilization, and biocapping of silver in the biosynthesis of AgNPs with antibacterial activity. Green chemistry principles were designed for the biosynthesis of AgNPs. The biosynthesis parameters were optimized using a central composite design (CCD) with response surface methodology. The antibacterial and antioxidant activities of biosynthesized AgNPs were assessed against the pathogenic human bacteria Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Bacillus subtilis.


## 2. Results and Discussion

### 2.1. Evaluation of AgNP Biosynthesis UsingM. oleifera Leaf Extract and UV-Visible Spectral Analysis

Silver nanoparticles were biosynthesized using an extract ofM. oleifera. leaves mixed with 50 mL when added to an aqueous solution of silver nitrate (1 mM). The reaction mixture was maintained in complete darkness. The color changed from pale brown to dark brown, indicating that AgNPs were formed. A dark brown coloration of the solution following the reaction with the Ag+ ions is an obvious indicator of metal ion reduction and the formation of AgNPs. Due to the excitation of silver nanoparticles’ surface plasmon vibrations, AgNPs exhibit a light yellow to brown coloration [36]. The authors in many previous studies apply a variety of AgNO3 concentrations, varying from 0.1 mM to 1 M, in the biosynthesis of AgNPs. For the biosynthesis of AgNPs, the most frequently used concentration of AgNO3 is 1 mM when an aqueous extract of Plectranthus amboinicus leaves is used [37]. Sadeghi and Gholamhoseinpoor [38] synthesized AgNPs from a methanol-extracted aqueous filtrate of Ziziphora tenuior leaves using 0.1 mM AgNO3. On the other contrary, AgNP production required 500 mM of AgNO3 mixed with a vacuoevaporated methanol extract of Viburnum lantana leaves [39], while Veerasamy et al. [40] employed 5 mM of AgNO3 in the synthesis of AgNPs using aqueous filtrate of Garcinia mangostana leaves as a reducing agent.Scanning UV–visible spectroscopy in the 200–800 nm region was used to confirm the biosynthesis of AgNPs. The surface plasmon resonance (SPR) spectroscopy of silver nanoparticles synthesized using extractMoringa oleifera leaves confirmed the presence of AgNPs via a prominent absorption band at 460 nm. This result was consistent with Sarkar et al.’s [41] finding. The absorption spectrum of SPR is greatly influenced by stabilizing molecules, particle size, and particles adsorbing to the surface. When the size of silver nanoparticles in aqueous solution increases, the SPR peak changes to longer wavelengths [42].


### 2.2. Biosynthesis Optimization of AgNPs

The RSM is a strategy that is based on the fundamental concepts of statistics as well as random assignment and validation. It is a highly successful statistical strategy that contributes to the simplification of the optimization of a large number of variables in order to anticipate the optimal processing conditions with the fewest possible experiments and to explain the individual and the mutual interactions of independent variables throughout a range of possible values on the response [43]. Table 1 displays the experimental results of the CCD matrix of the main variables, as well as related biosynthesized AgNPs (μg/mL). The CCD experiments showed a large variance in AgNP biosynthesis; this variance reflected the importance of the optimization process in AgNP biosynthesis. There was a broad variation in AgNP biosynthesis ranging from 0.227 to 3.068 μg/mL across the various CCD trials. The difference underlined the significance of the optimization process in achieving a higher biosynthesis of AgNPs. Table 2 shows the results of the statistical analysis using multiple regression and variance analysis (ANOVA) for CCD results of AgNP biosynthesis using M. oleifera leaf extract.Table 1
The experimental and predicted values of silver nanoparticle biosynthesis withMoringa oleifera leaf extract were designed by central composite design.
RunInitial pH level (X1)Temperature (X2)Incubation time (X3)Plant extract volume (X4)AgNPs (μg/mL)CodedActualCodedActual (°C)CodedActual (hr)CodedActual (mL)ExperimentalPredicted1110180-16-112.0041.9802-16180110-111.0691.0833080600801.51.491.3504110180110122.0882.0435080600801.51.2491.35060806008-20.51.9732.0477-16-140-16120.6030.80880806021201.52.0022.032908-2200801.50.7720.81710110-140-16121.9641.76111080600801.51.3181.35012-240600801.50.2270.11713-16-140110121.05910.89414110-140-16-111.2721.27815110-140110-112.9692.8191608060-2401.50.9030.993170821000801.50.8520.927182120600801.52.692.92019080600822.51.7041.74920-16-140-16-110.3610.21721-16180-16120.8420.80422110-140110122.6462.71823-16-140110-110.7190.88724110180110-113.0682.93225080600801.51.3051.35026080600801.51.2451.35027080600801.51.4921.35028-16180-16-111.0041.00129-16180110120.240.30230110180-16121.7751.675Table 2
ANOVA and regression coefficients for CCD results of AgNP biosynthesis usingM. oleifera leaf extract.
Source of varianceSum of squaresDfMean squareF-valueP valueConfidence levelCoefficient estimateStandard error95% CI low95% CI highRank (%)Model16.77141.2047.71<0.000199.99Linear effectsX1 (initial pH level)11.78111.78469.25<0.000199.990.700.030.630.7732.492X2 (temperature)0.0210.020.720.410758.930.030.03-0.040.101.269X3 (incubation time)1.6211.6264.44<0.000199.990.260.030.190.3312.040X4 (plant extract volume)0.1310.135.300.036196.39-0.070.03-0.14-0.013.453Interaction effectsX1X20.0110.010.270.611438.86-0.020.04-0.100.060.953X1X30.7610.7630.13<0.000199.990.220.040.130.3010.084X1X40.0110.010.460.506749.33-0.030.04-0.110.061.250X2X30.3510.3513.780.002199.79-0.150.04-0.23-0.066.820X2X40.6210.6224.720.000299.98-0.200.04-0.28-0.119.133X3X40.3410.3413.570.002299.78-0.150.04-0.23-0.066.768Quadratic effectsX120.0510.051.940.184081.600.040.03-0.020.111.954X220.3910.3915.600.001399.87-0.120.03-0.18-0.065.541X320.0510.051.800.199280.080.040.03-0.020.111.885X420.5210.5220.550.000499.960.140.030.070.206.360Error effectsLack of fit0.31100.032.440.1684Pure error0.0650.01Std. Dev.0.16R-squared0.9780Mean1.43AdjR-squared0.9575C.V. (%)11.08PredR-squared0.8896PRESS1.89Adeq precision25.1233The coefficients’ signs (positive or negative effect on the response) and statistical significance were used to interpret the data. Interactions between two variables can be antagonistic (negative coefficient) or synergistic (positive coefficient) (positive coefficient) [44]. The estimated coefficients with negative values (Table 2) imply an antagonistic relationship between variables and the biosynthesis of AgNPs, while positive coefficients imply a synergistic interaction between variables and the biosynthesis of AgNPs. If the value is close to zero, it indicates that the process variable has low to no influence on AgNP biosynthesis. The contribution percentages for the various variables are shown in Table 2.To determine the significance of each coefficient,P values were used. This is necessary for understanding the behavior of independent variables and their mutual interactions [36]. The factors with a confidence level greater than 95% (.) were considered significant. Thus, the process factors with P<0.05 and confidencelevel>95 percent) were judged to have a significant effect on silver nanoparticle production [45]. The most important factors influencing AgNP biosynthesis were pH (P<0.0001), time of incubation (P <0.0001), and plant extract volume (P=0.0361). The quadratic effects of the temperature and the plant extract volume were also found to be significant with P values of 0.0013 and 0.0004, respectively. It was also noticed that the interaction effects between pH×incubationtime, temperature×incubationtime, temperature×theplantextractvolume, and incubationtime×theplantextractvolume were also significant with probability values of P<0.0001, P=0.0021, P=0.0002, and P=0.0022, respectively (Table 2).The determination coefficient (R2) values indicate the extent to which the experimental factors and their interactions may contribute for the observed response values’ variability. The R2 value is always in the range of 0 to 1. When the R2 value is close to 1, the model is more robust and accurate in predicting the response [46]. In this case, an R2 of 0.9780 suggested that 97.80% of the total data variance is attributable to the tested variables (Table 2). Additionally, the Adj R2 and predicted R2 values are 0.9575 and 0.8896. The model’s significance was confirmed by the model’s exceptionally high Adj R2 value (Table 2). The predicted R2 value is very close to the adjusted R2 value. This demonstrated a high degree of consistency between predicted and actual values for AgNP biosynthesis. “Adeq Precision” is a signal-to-noise ratio meter. A ratio greater than four is preferred. A value of 25.1233 for “Adeq Precision” shows an adequate signal-to-noise ratio. Additionally, in Table 2, the value of PRESS is 1.89; standard deviation value is 0.16, and mean value is 1.43 and a very low value of C.V. (11.08) showing that this experimental performance was accurate and fit.Table3 summarizes the fit summary results obtained when linear, 2FI, and quadratic models were used to determine the optimal polynomial model for the experimental data on AgNP biosynthesis. The fit summary results indicated that the quadratic model of AgNP biosynthesis is highly significant, having an extremely low P value < 0.0001. Lack of fit test for AgNP biosynthesis with F‐value=2.44 and P value = 0.1684 is nonsignificant. The model summary statistics for the quadratic model of AgNP biosynthesis (Table 3) revealed a low standard deviation of 0.16, a high adjusted R2 of 0.9575, and a predicted R2 of 0.8896.Table 3
Fit summary for experimental results of CCD for optimization of AgNP biosynthesis usingM. oleifera leaf extract.
Model summary statisticsSourceSDR2Adjusted R2PredictedR2PRESSLinear0.380.79030.75680.67535.572FI0.280.91180.86530.80733.30Quadratic0.160.97800.95750.88961.89Lack of fit testsSourceSSDfMSF-valuePvalueLinear3.53200.176513.780.00432FI1.45140.10358.080.0153Quadratic0.31100.03132.440.1684Pure error0.0650.0128Sequential model sum of squaresSourceSSDfMSF valueP valueLinear vs. mean13.5543.3923.56<0.00012FI vs. linear2.0860.354.360.0062Quadratic vs. 2FI1.1440.2811.310.0002Residual0.1770.02MS: mean square; SS: sum of squares; SD: standard deviation; 2FI: two-factor interaction; PRESS: sum of squares of prediction error; Df: degree of freedom.


### 2.3. Three-Dimensional Surface Plots

To show the factors’ interactions and the optimal value of each variable required for maximum silver nanoparticle biosynthesis, three-dimensional response surface curves were generated to understand the factors’ interaction effects. Each graph demonstrates the effect of two factors on the biosynthesis of AgNPs, with the other two variables remaining constant at their central value [44]. Figures 1(a)–1(f) depict the three-dimensional responses for the four variables by plotting AgNP biosynthesis on the z-axis against pairs of process factors while keeping the value of the other two factors at their central value constant. They made it easier to understand the presence and magnitude of interdependencies between variables and to establish each parameter’s optimum levels for maximum output of nanoparticle synthesis.Figure 1
Three-dimensional plots showing the mutual effects of selected variables on AgNP biosynthesis withM. oleifera leaf extract.
(a)(b)(c)(d)(e)(f)The three-dimensional plots in Figure1(a) demonstrate that AgNP biosynthesis depended on the pH (X1) and temperature (X2) effects while the incubation time (X3) and plant extract volume (X4) were maintained at a zero level (8 h and 1.5 mL, respectively). Figure 1(b) shows a response surface plot for pH (X1) against incubation time (X3) while maintaining the temperature (X2) and extract volume (X4) at zero level (60°C and 1.5 mL, respectively). Figure 1(c) shows a response surface plot for the extract volume (X4) and pH (X1) while the incubation time (X3) and temperature (X2) were kept fixed at a zero level (8 hours and 60°C, respectively). Figure 1(d) shows a response surface plot for the incubation time (X3) and temperature (X2) while the pH (X1) and extract volume (X4) were kept fixed at a zero level (8.0 and 1.5 mL, respectively). Figure 1(e) shows a response surface plot for the extract volume (X4) and temperature (X2) while the incubation time (X3) and the pH (X1) were kept fixed at a zero level (8 h and 8.0, respectively). Figure 1(f) shows a response surface plot for the extract volume (X4) and time (X3) while the pH (X1) and temperature (X2) were kept fixed at a zero level (8.0 and 60°C, respectively). It was found from the plots that the maximum biosynthesis of AgNPs was achieved at a pH of 10, incubation temperature of 80°C for 10 hours, and a volume of extract of 1 mL.The AgNP process mechanism is well known to depend on the temperature and pH because they may influence the AgNP net charge and functional group [47]. Mokgweetsi et al. investigated the optimum conditions for the biosynthesis of AgNPs using M. oleifera plant parts (flowers, leaves, and stem barks). They found the optimum conditions to be 40 min of reaction time at 85°C using leaves; the reaction time was increased to 60 min with flowers and stem barks [48].The particle size and morphological shape of the biologically synthesized NPs were affected by the pH, and the pH had a serious impact on the electrical charges of the capping agents and biomolecules, leading to a change in their ability to attach by reducing of metal ions [49]. Raising the temperature increases the production of AgNPs because new active adsorption sites form. Nevertheless, at temperatures above 50°C, metal biosorption can decrease owing to physical damage to the biosorbent surface or the destruction of certain active binding sites due to poor adaptive forces on the sites between the biosorbent surface and the metal ions [50].


### 2.4. Model Validation

The normal probability plot of the residuals is depicted in Figure2(a). The residuals are regularly distributed and near to the diagonal line, showing that the model is well-fitting the data. It is essential to use the normal probability plot to determine whether the residuals have a normal distribution, in which case the points will follow a straight line [51]. The Box–Cox graph is depicted in Figure 2(b). Lambda’s optimal value is within the maximum and minimum confidence ranges. There was no need for data transformations. The blue line is situated in the middle of the two red ones; this indicates that the model fits the experimental data well. The plot of predicted values vs. residuals of AgNP biosynthesis (Figure 2(c)) shows the residuals scattered uniformly and randomly parallel to the diagonal line, confirming the model’s adequacy. Actual versus predicted silver nanoparticle biosynthesis is depicted in Figure 2(d). The presence of points close to the diagonal line indicates a significant correlation between the silver nanoparticle biosynthesis predicted by the model and the actual results.Figure 2
(a) Normal probability plot of internally studentized residuals, (b) Box-Cox plot of model transformation, (c) plot of internally studentized residuals versus predicted values, and (d) plot of predicted versus actual of AgNP biosynthesis by usingM. oleifera leaf extract as determined by second-order polynomial equation.
(a)(b)(c)(d)


### 2.5. Characterization of Biosynthesized AgNPs

#### 2.5.1. Color Change and UV–Vis Spectroscopy

Biofabrication of AgNPs can be confirmed using the chromatic shift of the substratum reaction as a visual marker. Nanometals display conspicuous spectral characteristics by surface plasmon resonance (SPR) based on the mutual vibration of free electrons and light wave resonance affected by the sizes and shapes of each of the synthesized NPs. This property is controlled by physical properties such as the form of the particles as to the size and shape and the local chemical effect [52, 53].Figure3 shows the development of 450 nm-based SPR AgNPs. This is in line with the findings of Bonigala et al. [54], who reported that the SPR zone of AgNPs appeared exclusively within the range of ~400 to 500 nm. The change in color from yellow to brown suggested that the Ag+ ion had been biotransformed into Ag0, becoming an AgNP [24]. The color shift in metal NPs was caused by surface plasmon vibration excitation [55].Figure 3
UV–visible spectrum recorded as a function of reaction time for an aqueous solution of 10−3 M AgNO3 and Moringa oleifera leaf extract.On the contrary, the adsorption peak of the SPR of AgNPs was reported at different locations at 415 to 426 nm [48], 430 to 440 nm [24], and 440 to 450 nm [25] from leaf extracts of M. oleifera in different conditions.


#### 2.5.2. Transmission Electron Microscopy Imaging

Transmission electron microscopy (TEM) can provide details about the bulk content from a very low to a higher magnification [56]. TEM images demonstrated clearly that particles had a spherical shape and that their sizes ranged from 15.22 to 29.45 nm (Figure 4). Furthermore, the biosynthesized NPs were distributed uniformly without morphological abnormalities or aggregation [57]. The biofunctional components derived from nature (alkaloids, terpenes, fatty acids, and amino acids) were effective as stabilizing agents for preventing NP aggregation [58]. TEM imaging confirmed the green synthesis of AgNPs by M. oleifera aqueous extract [25]. A similar observation was made by Ghosh et al. [26], Prasad and Elumalai [24], and Moodley et al. [25] that the synthesized AgNPs had a spherical shape when using M. oleifera leaf extracts. Rodríguez- Félix et al. synthesized the AgNPs using safflower with the same characteristics (spherical shape, crystalline structure) [59]. Bapat et al. reported that the bioactivity of AgNPs depends on the morphological, charges, and surface properties of the AgNPs, in addition to the synergistic effects of AgNPs and cellular response and pH [60].Figure 4
TEM image of AgNPs biosynthesized withM. oleifera leaf extract.


#### 2.5.3. Fourier-Transform Infrared Spectroscopy Studies

Fourier-transform infrared (FTIR) spectroscopy studies of AgNPs were made to find out the possible compounds responsible for the efficient stabilization and capping of AgNPs synthesized withM. oleifera extract [61]. The FTIR spectra of stabilized AgNPs are shown in Figure 5. The spectral analysis found that the functional groups were responsible for reducing and stabilizing AgNPs while they were converted to their oxidized derivatives.Figure 5
FTIR spectrum of aqueous extract of AgNPs synthesized withM. oleifera leaf extract.The stretching vibrations of the O–H functional groups in the structure (phenolic, alcoholic, and/or carboxylic acid compounds), as well as amine groups that might be overlapped with the O-H peak, were correlated to the broadband visible at 3000 to3500 cm−1 [25, 31]. The infrared (IR) bands at 1631 to 1650 cm−1 were stretch vibrations of –C=O belonging to the amide II band (mainly from in-plane N-H bending), which is one of the major bands in the IR spectra of proteins. In fact, the peak at 1631 to 1650 cm−1 attributed to both C=C stretching and amide (N-H) stretching could be involved in the protein stabilization of NPs [25, 31, 62]. The peak at 2000 to 2100 cm−1 was attributed to C-H bending in aromatic compounds such as flavonoids that existed in M. oleifera extract [20, 27]. The broadband at 680 to 500 cm−1 attributed to the aliphatic chain’s bending region existed in the extract compounds [47]. A similar interpretation was reported by Mehwish et al. [31], Moodley et al. [25], and Bindhu et al. [27], who showed functional groups with slight peak shifts were presented in AgNPs, and this indicated the reduction and stabilization of AgNPs by the extract of the M. oleifera leaf.Infrared data indicated that the protein in the extract ofM. oleifera was involved in the coating and biosynthesis of AgNPs [62]. The protein covering AgNPs prevents agglomeration [63]. According to reports, free amide groups in the protein molecules can interact with AgNPs [64]. The groups of carbonyl and free amine in the protein residues have a stronger potential to bind AgNPs to prevent the agglomeration and stabilization of AgNPs [65].


### 2.6. X-Ray Diffraction (XRD)

The crystallinity of green synthesized AgNP-based leaf extract ofMoringa oleifera was confirmed by the XRD (Figure 6). The XRD diffractogram at 2 θ values identified the main integrated peaks at 27.79°, 32.08°, 45.88°, 56.88, and 66.50°; these bands were similar to the reported findings in a previous investigation on the crystalline nature of AgNPs [66].Figure 6
XRD diffractogram of AgNPs synthesized withM. oleifera leaf extract.


### 2.7. Antimicrobial Activity of AgNPs

AgNP biosynthesized by theM. oleifera extract were investigated for antimicrobial effectiveness against various strains of pathogenic bacteria (E. coli, K. pneumoniae, S. aureus, and B. subtilis). The results reported in Table 4 show that AgNPs exhibited a zone of inhibition for all test species, either the Gram-negative (G-) strain (E. coli and K. pneumoniae) or Gram-positive (G+) strain (S. aureus and B. subtilis). The Gram-negative strain (E. coli and K. pneumoniae) is the most resistant bacterial species against the AgNPs synthesized in the current study (Figure 7). It displayed the smallest inhibition zone (17 and 11 mm, respectively) and the highest minimum inhibitory concentration (MIC) (3.6 and 5.8 μg/mL, respectively). However, S. aureus was the most prone, with a maximum inhibition zone of 19 mm and the lowest MIC of 2.9 μg/mL. The high sensitivity of (G+) bacteria may be attributed to the relative permeability of the outer membrane in contrast to (G-) bacteria [67]. Several mechanisms for understanding the antimicrobial activity of biosynthesized AgNPs have been postulated. Feng et al. [68] reported that AgNP antibacterial activity of Ag+ ions is attributed to their ability to bind to various elements of bacterial cells, such as the cytoplasm and DNA molecules, which flow out of a wounded cell wall. The bacterial inactivation process of AgNPs can be caused by the peroxidation of membrane lipids and protein inactivation in the plasma membrane and cell wall, which weaken the structural integrity of the membrane, resulting in transport disorders and potassium leakage [69]. Similar findings were reported by Bindhu et al. [27], Prasad and Elumalai [24], and Ghosh et al. [26], who investigated the antimicrobial activity of AgNPs synthesized by the extract of M. oleifera against different pathogenic bacteria such as Pseudomonas aeruginosa, Salmonella paratyphi, B. subtilis, K. pneumoniae, and E. coli. They found that the most resistant strains were Gram-negative strains, especially K. pneumoniae, of the tested strains.Table 4
Antibacterial activity of biosynthesized AgNPs fromM. oleifera extract against bacterial pathogen strains.
MicroorganismZOI∗ (mm)MIC∗∗ (μg mL −1)Escherichia coli17±0.33.6±0.034Klebsiella pneumonia11±0.25.8±0.023Staphylococcus aureus19±0.62.9±0.05Bacillus subtilis15±0.54±0.03∗ZOI: zone of inhibition diameter (mm); ∗∗MIC: minimal inhibitory concentrations (μg mL −1).Figure 7
Effect of biosynthesized AgNPs fromM. oleifera extract against bacterial pathogen strains.In the bacteria cell, AgNPs can interact with the signaling pathway during bacterial growth by regulating tyrosine phosphorylation on the substrates of the polypeptide that are important for cell proliferation [70]. Sondi and Salopek-Sondi investigated the mechanism of AgNP penetration into E. coli and found that AgNPs led to the formation of pits in the plasma membrane that could be responsible for major increases in the permeability of the cell membrane, affecting transport through the cell membrane [71].Furthermore, metal-induced free radicals such as reactive oxygen species (ROS) cause oxidative stress which can harm DNA, mitochondria, and bacterial membranes. The cell finally dies as a result of this [72]. Another possible effect of antibacterial activity by AgNPs is due to losing the ability for DNA replication and cellular synthesis of proteins after Ag+ treatment [68]. Bindhu et al. [27], Prasad et al. [24], Moodley et al. [25], and Ghosh et al. [26] investigated the antimicrobial effect of AgNPs using M. oleifera extract. The combination of the antibacterial properties of AgNPs and M. oleifera produced an excellent antimicrobial agent against pathogenic microorganisms such as (K. pneumoniae, S. aureus, E. coli, P. aeruginosa, and Candida albicans).


### 2.8. Antioxidant Activity of AgNPs

The antioxidant potential of biosynthesized AgNPs is due to their high phenolic content, which was calculated by the DPPH radical scavenging assay [73]. The DPPH radical was scavenged by M. oleifera extracts in a dose-dependent technique (Table 5). The scavenging potency was increased if the concentration of M. oleifera extracts was increased. Based on statistical analysis, the highest percentage of scavenging (63.79%) was discovered at concentrations of 20 mg/mL, followed by concentrations of 16 mg/mL (31.89%) and 12 mg/mL (16.95%) (P<0.05), whereas a concentration of 4 mg/mL showed the lowest scavenging capacity with a median value of 11.96% (P<0.05).Table 5
Radical scavenging activity of AgNPs synthesized withM. oleifera leaf extracts at different concentrations.
Concentration of plant extract (mg/mL)% inhibition411.96±0.0586.98±0.0.031216.95±0.051631.89±0.12063.79±0.09Capped AgNPs were proved to have potent free radical scavenger activity. Antioxidants prevent oxidation by neutralizing the production of free radicals. The antioxidant activity of capped AgNPs is possibly due to the natural phytocompounds in theM. oleifera extract, which neutralize the free radicals produced [74]. It could also be due to the functional groups on the surface of AgNPs [75]. Some studies reported that the phenolic compounds in plant extracts are the main response for the antioxidant and antimicrobial activity of the NPs [76, 77].


## 2.1. Evaluation of AgNP Biosynthesis UsingM. oleifera Leaf Extract and UV-Visible Spectral Analysis

Silver nanoparticles were biosynthesized using an extract ofM. oleifera. leaves mixed with 50 mL when added to an aqueous solution of silver nitrate (1 mM). The reaction mixture was maintained in complete darkness. The color changed from pale brown to dark brown, indicating that AgNPs were formed. A dark brown coloration of the solution following the reaction with the Ag+ ions is an obvious indicator of metal ion reduction and the formation of AgNPs. Due to the excitation of silver nanoparticles’ surface plasmon vibrations, AgNPs exhibit a light yellow to brown coloration [36]. The authors in many previous studies apply a variety of AgNO3 concentrations, varying from 0.1 mM to 1 M, in the biosynthesis of AgNPs. For the biosynthesis of AgNPs, the most frequently used concentration of AgNO3 is 1 mM when an aqueous extract of Plectranthus amboinicus leaves is used [37]. Sadeghi and Gholamhoseinpoor [38] synthesized AgNPs from a methanol-extracted aqueous filtrate of Ziziphora tenuior leaves using 0.1 mM AgNO3. On the other contrary, AgNP production required 500 mM of AgNO3 mixed with a vacuoevaporated methanol extract of Viburnum lantana leaves [39], while Veerasamy et al. [40] employed 5 mM of AgNO3 in the synthesis of AgNPs using aqueous filtrate of Garcinia mangostana leaves as a reducing agent.Scanning UV–visible spectroscopy in the 200–800 nm region was used to confirm the biosynthesis of AgNPs. The surface plasmon resonance (SPR) spectroscopy of silver nanoparticles synthesized using extractMoringa oleifera leaves confirmed the presence of AgNPs via a prominent absorption band at 460 nm. This result was consistent with Sarkar et al.’s [41] finding. The absorption spectrum of SPR is greatly influenced by stabilizing molecules, particle size, and particles adsorbing to the surface. When the size of silver nanoparticles in aqueous solution increases, the SPR peak changes to longer wavelengths [42].


## 2.2. Biosynthesis Optimization of AgNPs

The RSM is a strategy that is based on the fundamental concepts of statistics as well as random assignment and validation. It is a highly successful statistical strategy that contributes to the simplification of the optimization of a large number of variables in order to anticipate the optimal processing conditions with the fewest possible experiments and to explain the individual and the mutual interactions of independent variables throughout a range of possible values on the response [43]. Table 1 displays the experimental results of the CCD matrix of the main variables, as well as related biosynthesized AgNPs (μg/mL). The CCD experiments showed a large variance in AgNP biosynthesis; this variance reflected the importance of the optimization process in AgNP biosynthesis. There was a broad variation in AgNP biosynthesis ranging from 0.227 to 3.068 μg/mL across the various CCD trials. The difference underlined the significance of the optimization process in achieving a higher biosynthesis of AgNPs. Table 2 shows the results of the statistical analysis using multiple regression and variance analysis (ANOVA) for CCD results of AgNP biosynthesis using M. oleifera leaf extract.Table 1
The experimental and predicted values of silver nanoparticle biosynthesis withMoringa oleifera leaf extract were designed by central composite design.
RunInitial pH level (X1)Temperature (X2)Incubation time (X3)Plant extract volume (X4)AgNPs (μg/mL)CodedActualCodedActual (°C)CodedActual (hr)CodedActual (mL)ExperimentalPredicted1110180-16-112.0041.9802-16180110-111.0691.0833080600801.51.491.3504110180110122.0882.0435080600801.51.2491.35060806008-20.51.9732.0477-16-140-16120.6030.80880806021201.52.0022.032908-2200801.50.7720.81710110-140-16121.9641.76111080600801.51.3181.35012-240600801.50.2270.11713-16-140110121.05910.89414110-140-16-111.2721.27815110-140110-112.9692.8191608060-2401.50.9030.993170821000801.50.8520.927182120600801.52.692.92019080600822.51.7041.74920-16-140-16-110.3610.21721-16180-16120.8420.80422110-140110122.6462.71823-16-140110-110.7190.88724110180110-113.0682.93225080600801.51.3051.35026080600801.51.2451.35027080600801.51.4921.35028-16180-16-111.0041.00129-16180110120.240.30230110180-16121.7751.675Table 2
ANOVA and regression coefficients for CCD results of AgNP biosynthesis usingM. oleifera leaf extract.
Source of varianceSum of squaresDfMean squareF-valueP valueConfidence levelCoefficient estimateStandard error95% CI low95% CI highRank (%)Model16.77141.2047.71<0.000199.99Linear effectsX1 (initial pH level)11.78111.78469.25<0.000199.990.700.030.630.7732.492X2 (temperature)0.0210.020.720.410758.930.030.03-0.040.101.269X3 (incubation time)1.6211.6264.44<0.000199.990.260.030.190.3312.040X4 (plant extract volume)0.1310.135.300.036196.39-0.070.03-0.14-0.013.453Interaction effectsX1X20.0110.010.270.611438.86-0.020.04-0.100.060.953X1X30.7610.7630.13<0.000199.990.220.040.130.3010.084X1X40.0110.010.460.506749.33-0.030.04-0.110.061.250X2X30.3510.3513.780.002199.79-0.150.04-0.23-0.066.820X2X40.6210.6224.720.000299.98-0.200.04-0.28-0.119.133X3X40.3410.3413.570.002299.78-0.150.04-0.23-0.066.768Quadratic effectsX120.0510.051.940.184081.600.040.03-0.020.111.954X220.3910.3915.600.001399.87-0.120.03-0.18-0.065.541X320.0510.051.800.199280.080.040.03-0.020.111.885X420.5210.5220.550.000499.960.140.030.070.206.360Error effectsLack of fit0.31100.032.440.1684Pure error0.0650.01Std. Dev.0.16R-squared0.9780Mean1.43AdjR-squared0.9575C.V. (%)11.08PredR-squared0.8896PRESS1.89Adeq precision25.1233The coefficients’ signs (positive or negative effect on the response) and statistical significance were used to interpret the data. Interactions between two variables can be antagonistic (negative coefficient) or synergistic (positive coefficient) (positive coefficient) [44]. The estimated coefficients with negative values (Table 2) imply an antagonistic relationship between variables and the biosynthesis of AgNPs, while positive coefficients imply a synergistic interaction between variables and the biosynthesis of AgNPs. If the value is close to zero, it indicates that the process variable has low to no influence on AgNP biosynthesis. The contribution percentages for the various variables are shown in Table 2.To determine the significance of each coefficient,P values were used. This is necessary for understanding the behavior of independent variables and their mutual interactions [36]. The factors with a confidence level greater than 95% (.) were considered significant. Thus, the process factors with P<0.05 and confidencelevel>95 percent) were judged to have a significant effect on silver nanoparticle production [45]. The most important factors influencing AgNP biosynthesis were pH (P<0.0001), time of incubation (P <0.0001), and plant extract volume (P=0.0361). The quadratic effects of the temperature and the plant extract volume were also found to be significant with P values of 0.0013 and 0.0004, respectively. It was also noticed that the interaction effects between pH×incubationtime, temperature×incubationtime, temperature×theplantextractvolume, and incubationtime×theplantextractvolume were also significant with probability values of P<0.0001, P=0.0021, P=0.0002, and P=0.0022, respectively (Table 2).The determination coefficient (R2) values indicate the extent to which the experimental factors and their interactions may contribute for the observed response values’ variability. The R2 value is always in the range of 0 to 1. When the R2 value is close to 1, the model is more robust and accurate in predicting the response [46]. In this case, an R2 of 0.9780 suggested that 97.80% of the total data variance is attributable to the tested variables (Table 2). Additionally, the Adj R2 and predicted R2 values are 0.9575 and 0.8896. The model’s significance was confirmed by the model’s exceptionally high Adj R2 value (Table 2). The predicted R2 value is very close to the adjusted R2 value. This demonstrated a high degree of consistency between predicted and actual values for AgNP biosynthesis. “Adeq Precision” is a signal-to-noise ratio meter. A ratio greater than four is preferred. A value of 25.1233 for “Adeq Precision” shows an adequate signal-to-noise ratio. Additionally, in Table 2, the value of PRESS is 1.89; standard deviation value is 0.16, and mean value is 1.43 and a very low value of C.V. (11.08) showing that this experimental performance was accurate and fit.Table3 summarizes the fit summary results obtained when linear, 2FI, and quadratic models were used to determine the optimal polynomial model for the experimental data on AgNP biosynthesis. The fit summary results indicated that the quadratic model of AgNP biosynthesis is highly significant, having an extremely low P value < 0.0001. Lack of fit test for AgNP biosynthesis with F‐value=2.44 and P value = 0.1684 is nonsignificant. The model summary statistics for the quadratic model of AgNP biosynthesis (Table 3) revealed a low standard deviation of 0.16, a high adjusted R2 of 0.9575, and a predicted R2 of 0.8896.Table 3
Fit summary for experimental results of CCD for optimization of AgNP biosynthesis usingM. oleifera leaf extract.
Model summary statisticsSourceSDR2Adjusted R2PredictedR2PRESSLinear0.380.79030.75680.67535.572FI0.280.91180.86530.80733.30Quadratic0.160.97800.95750.88961.89Lack of fit testsSourceSSDfMSF-valuePvalueLinear3.53200.176513.780.00432FI1.45140.10358.080.0153Quadratic0.31100.03132.440.1684Pure error0.0650.0128Sequential model sum of squaresSourceSSDfMSF valueP valueLinear vs. mean13.5543.3923.56<0.00012FI vs. linear2.0860.354.360.0062Quadratic vs. 2FI1.1440.2811.310.0002Residual0.1770.02MS: mean square; SS: sum of squares; SD: standard deviation; 2FI: two-factor interaction; PRESS: sum of squares of prediction error; Df: degree of freedom.


## 2.3. Three-Dimensional Surface Plots

To show the factors’ interactions and the optimal value of each variable required for maximum silver nanoparticle biosynthesis, three-dimensional response surface curves were generated to understand the factors’ interaction effects. Each graph demonstrates the effect of two factors on the biosynthesis of AgNPs, with the other two variables remaining constant at their central value [44]. Figures 1(a)–1(f) depict the three-dimensional responses for the four variables by plotting AgNP biosynthesis on the z-axis against pairs of process factors while keeping the value of the other two factors at their central value constant. They made it easier to understand the presence and magnitude of interdependencies between variables and to establish each parameter’s optimum levels for maximum output of nanoparticle synthesis.Figure 1
Three-dimensional plots showing the mutual effects of selected variables on AgNP biosynthesis withM. oleifera leaf extract.
(a)(b)(c)(d)(e)(f)The three-dimensional plots in Figure1(a) demonstrate that AgNP biosynthesis depended on the pH (X1) and temperature (X2) effects while the incubation time (X3) and plant extract volume (X4) were maintained at a zero level (8 h and 1.5 mL, respectively). Figure 1(b) shows a response surface plot for pH (X1) against incubation time (X3) while maintaining the temperature (X2) and extract volume (X4) at zero level (60°C and 1.5 mL, respectively). Figure 1(c) shows a response surface plot for the extract volume (X4) and pH (X1) while the incubation time (X3) and temperature (X2) were kept fixed at a zero level (8 hours and 60°C, respectively). Figure 1(d) shows a response surface plot for the incubation time (X3) and temperature (X2) while the pH (X1) and extract volume (X4) were kept fixed at a zero level (8.0 and 1.5 mL, respectively). Figure 1(e) shows a response surface plot for the extract volume (X4) and temperature (X2) while the incubation time (X3) and the pH (X1) were kept fixed at a zero level (8 h and 8.0, respectively). Figure 1(f) shows a response surface plot for the extract volume (X4) and time (X3) while the pH (X1) and temperature (X2) were kept fixed at a zero level (8.0 and 60°C, respectively). It was found from the plots that the maximum biosynthesis of AgNPs was achieved at a pH of 10, incubation temperature of 80°C for 10 hours, and a volume of extract of 1 mL.The AgNP process mechanism is well known to depend on the temperature and pH because they may influence the AgNP net charge and functional group [47]. Mokgweetsi et al. investigated the optimum conditions for the biosynthesis of AgNPs using M. oleifera plant parts (flowers, leaves, and stem barks). They found the optimum conditions to be 40 min of reaction time at 85°C using leaves; the reaction time was increased to 60 min with flowers and stem barks [48].The particle size and morphological shape of the biologically synthesized NPs were affected by the pH, and the pH had a serious impact on the electrical charges of the capping agents and biomolecules, leading to a change in their ability to attach by reducing of metal ions [49]. Raising the temperature increases the production of AgNPs because new active adsorption sites form. Nevertheless, at temperatures above 50°C, metal biosorption can decrease owing to physical damage to the biosorbent surface or the destruction of certain active binding sites due to poor adaptive forces on the sites between the biosorbent surface and the metal ions [50].


## 2.4. Model Validation

The normal probability plot of the residuals is depicted in Figure2(a). The residuals are regularly distributed and near to the diagonal line, showing that the model is well-fitting the data. It is essential to use the normal probability plot to determine whether the residuals have a normal distribution, in which case the points will follow a straight line [51]. The Box–Cox graph is depicted in Figure 2(b). Lambda’s optimal value is within the maximum and minimum confidence ranges. There was no need for data transformations. The blue line is situated in the middle of the two red ones; this indicates that the model fits the experimental data well. The plot of predicted values vs. residuals of AgNP biosynthesis (Figure 2(c)) shows the residuals scattered uniformly and randomly parallel to the diagonal line, confirming the model’s adequacy. Actual versus predicted silver nanoparticle biosynthesis is depicted in Figure 2(d). The presence of points close to the diagonal line indicates a significant correlation between the silver nanoparticle biosynthesis predicted by the model and the actual results.Figure 2
(a) Normal probability plot of internally studentized residuals, (b) Box-Cox plot of model transformation, (c) plot of internally studentized residuals versus predicted values, and (d) plot of predicted versus actual of AgNP biosynthesis by usingM. oleifera leaf extract as determined by second-order polynomial equation.
(a)(b)(c)(d)


## 2.5. Characterization of Biosynthesized AgNPs

### 2.5.1. Color Change and UV–Vis Spectroscopy

Biofabrication of AgNPs can be confirmed using the chromatic shift of the substratum reaction as a visual marker. Nanometals display conspicuous spectral characteristics by surface plasmon resonance (SPR) based on the mutual vibration of free electrons and light wave resonance affected by the sizes and shapes of each of the synthesized NPs. This property is controlled by physical properties such as the form of the particles as to the size and shape and the local chemical effect [52, 53].Figure3 shows the development of 450 nm-based SPR AgNPs. This is in line with the findings of Bonigala et al. [54], who reported that the SPR zone of AgNPs appeared exclusively within the range of ~400 to 500 nm. The change in color from yellow to brown suggested that the Ag+ ion had been biotransformed into Ag0, becoming an AgNP [24]. The color shift in metal NPs was caused by surface plasmon vibration excitation [55].Figure 3
UV–visible spectrum recorded as a function of reaction time for an aqueous solution of 10−3 M AgNO3 and Moringa oleifera leaf extract.On the contrary, the adsorption peak of the SPR of AgNPs was reported at different locations at 415 to 426 nm [48], 430 to 440 nm [24], and 440 to 450 nm [25] from leaf extracts of M. oleifera in different conditions.


### 2.5.2. Transmission Electron Microscopy Imaging

Transmission electron microscopy (TEM) can provide details about the bulk content from a very low to a higher magnification [56]. TEM images demonstrated clearly that particles had a spherical shape and that their sizes ranged from 15.22 to 29.45 nm (Figure 4). Furthermore, the biosynthesized NPs were distributed uniformly without morphological abnormalities or aggregation [57]. The biofunctional components derived from nature (alkaloids, terpenes, fatty acids, and amino acids) were effective as stabilizing agents for preventing NP aggregation [58]. TEM imaging confirmed the green synthesis of AgNPs by M. oleifera aqueous extract [25]. A similar observation was made by Ghosh et al. [26], Prasad and Elumalai [24], and Moodley et al. [25] that the synthesized AgNPs had a spherical shape when using M. oleifera leaf extracts. Rodríguez- Félix et al. synthesized the AgNPs using safflower with the same characteristics (spherical shape, crystalline structure) [59]. Bapat et al. reported that the bioactivity of AgNPs depends on the morphological, charges, and surface properties of the AgNPs, in addition to the synergistic effects of AgNPs and cellular response and pH [60].Figure 4
TEM image of AgNPs biosynthesized withM. oleifera leaf extract.


### 2.5.3. Fourier-Transform Infrared Spectroscopy Studies

Fourier-transform infrared (FTIR) spectroscopy studies of AgNPs were made to find out the possible compounds responsible for the efficient stabilization and capping of AgNPs synthesized withM. oleifera extract [61]. The FTIR spectra of stabilized AgNPs are shown in Figure 5. The spectral analysis found that the functional groups were responsible for reducing and stabilizing AgNPs while they were converted to their oxidized derivatives.Figure 5
FTIR spectrum of aqueous extract of AgNPs synthesized withM. oleifera leaf extract.The stretching vibrations of the O–H functional groups in the structure (phenolic, alcoholic, and/or carboxylic acid compounds), as well as amine groups that might be overlapped with the O-H peak, were correlated to the broadband visible at 3000 to3500 cm−1 [25, 31]. The infrared (IR) bands at 1631 to 1650 cm−1 were stretch vibrations of –C=O belonging to the amide II band (mainly from in-plane N-H bending), which is one of the major bands in the IR spectra of proteins. In fact, the peak at 1631 to 1650 cm−1 attributed to both C=C stretching and amide (N-H) stretching could be involved in the protein stabilization of NPs [25, 31, 62]. The peak at 2000 to 2100 cm−1 was attributed to C-H bending in aromatic compounds such as flavonoids that existed in M. oleifera extract [20, 27]. The broadband at 680 to 500 cm−1 attributed to the aliphatic chain’s bending region existed in the extract compounds [47]. A similar interpretation was reported by Mehwish et al. [31], Moodley et al. [25], and Bindhu et al. [27], who showed functional groups with slight peak shifts were presented in AgNPs, and this indicated the reduction and stabilization of AgNPs by the extract of the M. oleifera leaf.Infrared data indicated that the protein in the extract ofM. oleifera was involved in the coating and biosynthesis of AgNPs [62]. The protein covering AgNPs prevents agglomeration [63]. According to reports, free amide groups in the protein molecules can interact with AgNPs [64]. The groups of carbonyl and free amine in the protein residues have a stronger potential to bind AgNPs to prevent the agglomeration and stabilization of AgNPs [65].


## 2.5.1. Color Change and UV–Vis Spectroscopy

Biofabrication of AgNPs can be confirmed using the chromatic shift of the substratum reaction as a visual marker. Nanometals display conspicuous spectral characteristics by surface plasmon resonance (SPR) based on the mutual vibration of free electrons and light wave resonance affected by the sizes and shapes of each of the synthesized NPs. This property is controlled by physical properties such as the form of the particles as to the size and shape and the local chemical effect [52, 53].Figure3 shows the development of 450 nm-based SPR AgNPs. This is in line with the findings of Bonigala et al. [54], who reported that the SPR zone of AgNPs appeared exclusively within the range of ~400 to 500 nm. The change in color from yellow to brown suggested that the Ag+ ion had been biotransformed into Ag0, becoming an AgNP [24]. The color shift in metal NPs was caused by surface plasmon vibration excitation [55].Figure 3
UV–visible spectrum recorded as a function of reaction time for an aqueous solution of 10−3 M AgNO3 and Moringa oleifera leaf extract.On the contrary, the adsorption peak of the SPR of AgNPs was reported at different locations at 415 to 426 nm [48], 430 to 440 nm [24], and 440 to 450 nm [25] from leaf extracts of M. oleifera in different conditions.


## 2.5.2. Transmission Electron Microscopy Imaging

Transmission electron microscopy (TEM) can provide details about the bulk content from a very low to a higher magnification [56]. TEM images demonstrated clearly that particles had a spherical shape and that their sizes ranged from 15.22 to 29.45 nm (Figure 4). Furthermore, the biosynthesized NPs were distributed uniformly without morphological abnormalities or aggregation [57]. The biofunctional components derived from nature (alkaloids, terpenes, fatty acids, and amino acids) were effective as stabilizing agents for preventing NP aggregation [58]. TEM imaging confirmed the green synthesis of AgNPs by M. oleifera aqueous extract [25]. A similar observation was made by Ghosh et al. [26], Prasad and Elumalai [24], and Moodley et al. [25] that the synthesized AgNPs had a spherical shape when using M. oleifera leaf extracts. Rodríguez- Félix et al. synthesized the AgNPs using safflower with the same characteristics (spherical shape, crystalline structure) [59]. Bapat et al. reported that the bioactivity of AgNPs depends on the morphological, charges, and surface properties of the AgNPs, in addition to the synergistic effects of AgNPs and cellular response and pH [60].Figure 4
TEM image of AgNPs biosynthesized withM. oleifera leaf extract.


## 2.5.3. Fourier-Transform Infrared Spectroscopy Studies

Fourier-transform infrared (FTIR) spectroscopy studies of AgNPs were made to find out the possible compounds responsible for the efficient stabilization and capping of AgNPs synthesized withM. oleifera extract [61]. The FTIR spectra of stabilized AgNPs are shown in Figure 5. The spectral analysis found that the functional groups were responsible for reducing and stabilizing AgNPs while they were converted to their oxidized derivatives.Figure 5
FTIR spectrum of aqueous extract of AgNPs synthesized withM. oleifera leaf extract.The stretching vibrations of the O–H functional groups in the structure (phenolic, alcoholic, and/or carboxylic acid compounds), as well as amine groups that might be overlapped with the O-H peak, were correlated to the broadband visible at 3000 to3500 cm−1 [25, 31]. The infrared (IR) bands at 1631 to 1650 cm−1 were stretch vibrations of –C=O belonging to the amide II band (mainly from in-plane N-H bending), which is one of the major bands in the IR spectra of proteins. In fact, the peak at 1631 to 1650 cm−1 attributed to both C=C stretching and amide (N-H) stretching could be involved in the protein stabilization of NPs [25, 31, 62]. The peak at 2000 to 2100 cm−1 was attributed to C-H bending in aromatic compounds such as flavonoids that existed in M. oleifera extract [20, 27]. The broadband at 680 to 500 cm−1 attributed to the aliphatic chain’s bending region existed in the extract compounds [47]. A similar interpretation was reported by Mehwish et al. [31], Moodley et al. [25], and Bindhu et al. [27], who showed functional groups with slight peak shifts were presented in AgNPs, and this indicated the reduction and stabilization of AgNPs by the extract of the M. oleifera leaf.Infrared data indicated that the protein in the extract ofM. oleifera was involved in the coating and biosynthesis of AgNPs [62]. The protein covering AgNPs prevents agglomeration [63]. According to reports, free amide groups in the protein molecules can interact with AgNPs [64]. The groups of carbonyl and free amine in the protein residues have a stronger potential to bind AgNPs to prevent the agglomeration and stabilization of AgNPs [65].


## 2.6. X-Ray Diffraction (XRD)

The crystallinity of green synthesized AgNP-based leaf extract ofMoringa oleifera was confirmed by the XRD (Figure 6). The XRD diffractogram at 2 θ values identified the main integrated peaks at 27.79°, 32.08°, 45.88°, 56.88, and 66.50°; these bands were similar to the reported findings in a previous investigation on the crystalline nature of AgNPs [66].Figure 6
XRD diffractogram of AgNPs synthesized withM. oleifera leaf extract.


## 2.7. Antimicrobial Activity of AgNPs

AgNP biosynthesized by theM. oleifera extract were investigated for antimicrobial effectiveness against various strains of pathogenic bacteria (E. coli, K. pneumoniae, S. aureus, and B. subtilis). The results reported in Table 4 show that AgNPs exhibited a zone of inhibition for all test species, either the Gram-negative (G-) strain (E. coli and K. pneumoniae) or Gram-positive (G+) strain (S. aureus and B. subtilis). The Gram-negative strain (E. coli and K. pneumoniae) is the most resistant bacterial species against the AgNPs synthesized in the current study (Figure 7). It displayed the smallest inhibition zone (17 and 11 mm, respectively) and the highest minimum inhibitory concentration (MIC) (3.6 and 5.8 μg/mL, respectively). However, S. aureus was the most prone, with a maximum inhibition zone of 19 mm and the lowest MIC of 2.9 μg/mL. The high sensitivity of (G+) bacteria may be attributed to the relative permeability of the outer membrane in contrast to (G-) bacteria [67]. Several mechanisms for understanding the antimicrobial activity of biosynthesized AgNPs have been postulated. Feng et al. [68] reported that AgNP antibacterial activity of Ag+ ions is attributed to their ability to bind to various elements of bacterial cells, such as the cytoplasm and DNA molecules, which flow out of a wounded cell wall. The bacterial inactivation process of AgNPs can be caused by the peroxidation of membrane lipids and protein inactivation in the plasma membrane and cell wall, which weaken the structural integrity of the membrane, resulting in transport disorders and potassium leakage [69]. Similar findings were reported by Bindhu et al. [27], Prasad and Elumalai [24], and Ghosh et al. [26], who investigated the antimicrobial activity of AgNPs synthesized by the extract of M. oleifera against different pathogenic bacteria such as Pseudomonas aeruginosa, Salmonella paratyphi, B. subtilis, K. pneumoniae, and E. coli. They found that the most resistant strains were Gram-negative strains, especially K. pneumoniae, of the tested strains.Table 4
Antibacterial activity of biosynthesized AgNPs fromM. oleifera extract against bacterial pathogen strains.
MicroorganismZOI∗ (mm)MIC∗∗ (μg mL −1)Escherichia coli17±0.33.6±0.034Klebsiella pneumonia11±0.25.8±0.023Staphylococcus aureus19±0.62.9±0.05Bacillus subtilis15±0.54±0.03∗ZOI: zone of inhibition diameter (mm); ∗∗MIC: minimal inhibitory concentrations (μg mL −1).Figure 7
Effect of biosynthesized AgNPs fromM. oleifera extract against bacterial pathogen strains.In the bacteria cell, AgNPs can interact with the signaling pathway during bacterial growth by regulating tyrosine phosphorylation on the substrates of the polypeptide that are important for cell proliferation [70]. Sondi and Salopek-Sondi investigated the mechanism of AgNP penetration into E. coli and found that AgNPs led to the formation of pits in the plasma membrane that could be responsible for major increases in the permeability of the cell membrane, affecting transport through the cell membrane [71].Furthermore, metal-induced free radicals such as reactive oxygen species (ROS) cause oxidative stress which can harm DNA, mitochondria, and bacterial membranes. The cell finally dies as a result of this [72]. Another possible effect of antibacterial activity by AgNPs is due to losing the ability for DNA replication and cellular synthesis of proteins after Ag+ treatment [68]. Bindhu et al. [27], Prasad et al. [24], Moodley et al. [25], and Ghosh et al. [26] investigated the antimicrobial effect of AgNPs using M. oleifera extract. The combination of the antibacterial properties of AgNPs and M. oleifera produced an excellent antimicrobial agent against pathogenic microorganisms such as (K. pneumoniae, S. aureus, E. coli, P. aeruginosa, and Candida albicans).


## 2.8. Antioxidant Activity of AgNPs

The antioxidant potential of biosynthesized AgNPs is due to their high phenolic content, which was calculated by the DPPH radical scavenging assay [73]. The DPPH radical was scavenged by M. oleifera extracts in a dose-dependent technique (Table 5). The scavenging potency was increased if the concentration of M. oleifera extracts was increased. Based on statistical analysis, the highest percentage of scavenging (63.79%) was discovered at concentrations of 20 mg/mL, followed by concentrations of 16 mg/mL (31.89%) and 12 mg/mL (16.95%) (P<0.05), whereas a concentration of 4 mg/mL showed the lowest scavenging capacity with a median value of 11.96% (P<0.05).Table 5
Radical scavenging activity of AgNPs synthesized withM. oleifera leaf extracts at different concentrations.
Concentration of plant extract (mg/mL)% inhibition411.96±0.0586.98±0.0.031216.95±0.051631.89±0.12063.79±0.09Capped AgNPs were proved to have potent free radical scavenger activity. Antioxidants prevent oxidation by neutralizing the production of free radicals. The antioxidant activity of capped AgNPs is possibly due to the natural phytocompounds in theM. oleifera extract, which neutralize the free radicals produced [74]. It could also be due to the functional groups on the surface of AgNPs [75]. Some studies reported that the phenolic compounds in plant extracts are the main response for the antioxidant and antimicrobial activity of the NPs [76, 77].


## 3. Materials and Methods

### 3.1. Moringa Oleifera Plant Collection and Preparation of Leaf Extract

FreshM. oleifera leaves were gathered from the local market in Giza, Egypt; cleaned in distilled water; and air-dried. The leaf material was kept in a freeze dryer for 72 h to remove all moisture and then stored at −16°C until use. Extrication processes were prepared by homogenization of 10 g of an M. oleifera leaf in 50 mL of distilled water, and the final volume was set at 100 mL. The flasks were coated with foil and incubated at 25°C for 24 h with shaking at 150 rpm. Then, the extracts were filtered by Whatman No. 1 filter paper (Whatman International Ltd, Maidstone, England).


### 3.2. Synthesis of AgNPs

Five mL extracts ofM. oleifera leaves were added subsequently to 50 mL of the aqueous solution of AgNO3 (1.0 mM). The suspension was stirred for 20 min with a magnetic stirrer and held at 25°C for 48 h in dark. The color changed from light brown to dark brown, which indicated the end of the reaction. The nanoparticles were recovered after synthesis by centrifugation at 15,000 rpm for 15 minutes, rinsed thoroughly with sterile distilled water, and dried overnight. The characterization tests and analysis were performed on this dry sample. A 50 mL aliquot containing 5 mL of distilled water was treated and utilized as a negative control in the experiments [11]. All experiments and analyses were performed in triplicate.


### 3.3. Biosynthesis Optimization of AgNPs via the Central Composite Design Matrix

The design matrix of central composite (CCD) is a surface-response technique used to evaluate the optimum rates of the major independent variables for increasing the AgNP biosynthesis. In the central composite model, four factors with the highest degrees of confidence were identified for further optimization (Table1). These factors were pH (X1, 6–10), temperature (X2, 20–80°C), incubation time (X3), and plant extract volume (X4), all experimental factors were evaluated in the absence of light and are presented in Table 1.The central composite design matrix was created with 30 separate tests and 3 center points using Design-Expert software (Ver. 7). The significant variables were evaluated and coded at 3 levels (-1, 0, and +1) for the levels of low, middle, and high, respectively. To define and compare the relation between the AgNP biosynthesis (Y) and the relevant four factors, as well as to predict the optimal factor rates, the equation for polynomial was as follows:
(1)Y=β0+ΣiβiXi+βΣiiβiiXii+ΣijβijXiXj,where the code of Y is AgNP biosynthesis, β0 is the regression coefficients, βi is the linear coefficient, Xi is the independent variable rates, βii is the quadratic coefficients, and βij is the interaction coefficients. Trials were conducted in three replicates, with reported intermediate results.


### 3.4. Characterization and Analysis of AgNPs

The characterization and analysis of the biosynthesis of AgNPs were analyzed using a spectrophotometer (UV-Vis Vision Technology V3.20, ATI Unicam-5625) within 200-900 nm at the Faculty of Science, Mansoura University, Egypt. The biofunctional groups ofMoringa oleifera extract involved in the bioreduction of silver nanoparticles were determined by a Fourier transform infrared (FT-IR) (Thermo-Fisher Nicolet IS10, USA) in the region of 4000–500 cm–1 at a resolution of 1 cm−1. The transmission of electron microscopy (TEM) (JEOL JSM-6510/v, Japan) was applied to characterize the morphological shape and particle size of the biosynthesized AgNPs. The crystallinity nature of biosynthesized AgNPs was studied using a D8-Advance X-ray diffractometer (Bruker, Germany); the analysis was performed at 40 kv, mA, scanning rate at 6°/min, and Cu Kα radiation; the 2θ scanning ranged between 20 and 80° at 0.02 step size.


### 3.5. Microorganism

The experiments employed four normal microbial strains:Escherichia coli (ATCC-35218), Klebsiella pneumoniae (ATCC-700603), Staphylococcus aureus (ATCC-25923), and Bacillus subtilis (ATCC-27853). The bacterial cultures were cultivated and tested in media (Difco, Sparks, MD) for soy broth trypticase (TSB) and soy agar trypticase (TSA).


### 3.6. Paper Disc Diffusion Assay

Cell suspension (100μL) was evenly distributed on TSA for disk diffusion assay. The inoculated agar was examined with impregnated discs of filter paper (Whatman No. 41, 6 mm diameter) with 25 μL plant extract at a concentration of 10 mg/mL. The antibacterial control agent was chloramphenicol/ketoconazole at a dosage of 10 mg/mL. Incubation of inoculated plate was incubated at 37°C/24 h. The diameter zone of the inhibition (ZOI) was then correctly determined, and the mean of their triplicates was estimated [78].


### 3.7. Determination of Minimal Inhibitory Concentration (MIC)

To assess minimum inhibitory concentrations (MIC), each of theMoringa oleifera extracts was tested against examined bacterial strains as a quantitative assay [79]. As the predictor of bacterial production, p-iodonitro-tetrazolium violet (INT) was used in the method of broth’s microdilution. Serial concentrations of Moringa oleifera extracts (50 μL of each well) were distributed on the plates of TSA and incubated for 24 h/37°C.


### 3.8. Antioxidant Activity of Silver Nanoparticles

The antioxidant activity of AgNPs was assayed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) to determine the scavenging function of free radicals [80]. In this assay, the removal of the H-atom from the antioxidant compound leads to reducing the color of DPPH from violet to a pale-yellow. The assay reaction mixture contained 1 mL of AgNPs at various concentrations (10, 20, 30, 40, and 50 mg/mL) and 1 mL methanolic solution (0.1 mM) of DPPH and incubated under extreme shaking at 37°C for 0.5 hours. The absorbance of the mixture was measured at 517 nm, and the positive control was ascorbic acid. The higher scavenging activity of free radical was related to more decrease in absorbance, which was calculated using the following formula:
(2)DPPHscavengingactivity%=100×A0−A1A0,where A0 is the absorbances of the control and A1 is the sample absorbances. The results are presented as the average of three replicate analyses, with the main values as well as the standard deviation (SD) provided.


### 3.9. Statistic Data Analysis

The design of the experiments and statistical analysis was done using Design-Expert software (Ver.7), and the 3D surface plots were drawn using Statistica® 8.0 (StatSoft Inc., Tulsa, USA).


## 3.1. Moringa Oleifera Plant Collection and Preparation of Leaf Extract

FreshM. oleifera leaves were gathered from the local market in Giza, Egypt; cleaned in distilled water; and air-dried. The leaf material was kept in a freeze dryer for 72 h to remove all moisture and then stored at −16°C until use. Extrication processes were prepared by homogenization of 10 g of an M. oleifera leaf in 50 mL of distilled water, and the final volume was set at 100 mL. The flasks were coated with foil and incubated at 25°C for 24 h with shaking at 150 rpm. Then, the extracts were filtered by Whatman No. 1 filter paper (Whatman International Ltd, Maidstone, England).


## 3.2. Synthesis of AgNPs

Five mL extracts ofM. oleifera leaves were added subsequently to 50 mL of the aqueous solution of AgNO3 (1.0 mM). The suspension was stirred for 20 min with a magnetic stirrer and held at 25°C for 48 h in dark. The color changed from light brown to dark brown, which indicated the end of the reaction. The nanoparticles were recovered after synthesis by centrifugation at 15,000 rpm for 15 minutes, rinsed thoroughly with sterile distilled water, and dried overnight. The characterization tests and analysis were performed on this dry sample. A 50 mL aliquot containing 5 mL of distilled water was treated and utilized as a negative control in the experiments [11]. All experiments and analyses were performed in triplicate.


## 3.3. Biosynthesis Optimization of AgNPs via the Central Composite Design Matrix

The design matrix of central composite (CCD) is a surface-response technique used to evaluate the optimum rates of the major independent variables for increasing the AgNP biosynthesis. In the central composite model, four factors with the highest degrees of confidence were identified for further optimization (Table1). These factors were pH (X1, 6–10), temperature (X2, 20–80°C), incubation time (X3), and plant extract volume (X4), all experimental factors were evaluated in the absence of light and are presented in Table 1.The central composite design matrix was created with 30 separate tests and 3 center points using Design-Expert software (Ver. 7). The significant variables were evaluated and coded at 3 levels (-1, 0, and +1) for the levels of low, middle, and high, respectively. To define and compare the relation between the AgNP biosynthesis (Y) and the relevant four factors, as well as to predict the optimal factor rates, the equation for polynomial was as follows:
(1)Y=β0+ΣiβiXi+βΣiiβiiXii+ΣijβijXiXj,where the code of Y is AgNP biosynthesis, β0 is the regression coefficients, βi is the linear coefficient, Xi is the independent variable rates, βii is the quadratic coefficients, and βij is the interaction coefficients. Trials were conducted in three replicates, with reported intermediate results.


## 3.4. Characterization and Analysis of AgNPs

The characterization and analysis of the biosynthesis of AgNPs were analyzed using a spectrophotometer (UV-Vis Vision Technology V3.20, ATI Unicam-5625) within 200-900 nm at the Faculty of Science, Mansoura University, Egypt. The biofunctional groups ofMoringa oleifera extract involved in the bioreduction of silver nanoparticles were determined by a Fourier transform infrared (FT-IR) (Thermo-Fisher Nicolet IS10, USA) in the region of 4000–500 cm–1 at a resolution of 1 cm−1. The transmission of electron microscopy (TEM) (JEOL JSM-6510/v, Japan) was applied to characterize the morphological shape and particle size of the biosynthesized AgNPs. The crystallinity nature of biosynthesized AgNPs was studied using a D8-Advance X-ray diffractometer (Bruker, Germany); the analysis was performed at 40 kv, mA, scanning rate at 6°/min, and Cu Kα radiation; the 2θ scanning ranged between 20 and 80° at 0.02 step size.


## 3.5. Microorganism

The experiments employed four normal microbial strains:Escherichia coli (ATCC-35218), Klebsiella pneumoniae (ATCC-700603), Staphylococcus aureus (ATCC-25923), and Bacillus subtilis (ATCC-27853). The bacterial cultures were cultivated and tested in media (Difco, Sparks, MD) for soy broth trypticase (TSB) and soy agar trypticase (TSA).


## 3.6. Paper Disc Diffusion Assay

Cell suspension (100μL) was evenly distributed on TSA for disk diffusion assay. The inoculated agar was examined with impregnated discs of filter paper (Whatman No. 41, 6 mm diameter) with 25 μL plant extract at a concentration of 10 mg/mL. The antibacterial control agent was chloramphenicol/ketoconazole at a dosage of 10 mg/mL. Incubation of inoculated plate was incubated at 37°C/24 h. The diameter zone of the inhibition (ZOI) was then correctly determined, and the mean of their triplicates was estimated [78].


## 3.7. Determination of Minimal Inhibitory Concentration (MIC)

To assess minimum inhibitory concentrations (MIC), each of theMoringa oleifera extracts was tested against examined bacterial strains as a quantitative assay [79]. As the predictor of bacterial production, p-iodonitro-tetrazolium violet (INT) was used in the method of broth’s microdilution. Serial concentrations of Moringa oleifera extracts (50 μL of each well) were distributed on the plates of TSA and incubated for 24 h/37°C.


## 3.8. Antioxidant Activity of Silver Nanoparticles

The antioxidant activity of AgNPs was assayed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) to determine the scavenging function of free radicals [80]. In this assay, the removal of the H-atom from the antioxidant compound leads to reducing the color of DPPH from violet to a pale-yellow. The assay reaction mixture contained 1 mL of AgNPs at various concentrations (10, 20, 30, 40, and 50 mg/mL) and 1 mL methanolic solution (0.1 mM) of DPPH and incubated under extreme shaking at 37°C for 0.5 hours. The absorbance of the mixture was measured at 517 nm, and the positive control was ascorbic acid. The higher scavenging activity of free radical was related to more decrease in absorbance, which was calculated using the following formula:
(2)DPPHscavengingactivity%=100×A0−A1A0,where A0 is the absorbances of the control and A1 is the sample absorbances. The results are presented as the average of three replicate analyses, with the main values as well as the standard deviation (SD) provided.


## 3.9. Statistic Data Analysis

The design of the experiments and statistical analysis was done using Design-Expert software (Ver.7), and the 3D surface plots were drawn using Statistica® 8.0 (StatSoft Inc., Tulsa, USA).


## 4. Conclusion

The findings of this study suggest that AgNPs can be biofabricated from an aqueous extract ofM. oleifera. The leaves of M. oleifera showed potent antioxidant and antibacterial activities. M. oleifera showed good potential for synthesizing AgNPs through rapid reduction of Ag+ ions. The synthesized AgNPs had a spherical shape and high efficiency against some pathogenic microorganisms. They have potential uses as antioxidants for medical applications. A recommended approach for biosynthesis is using a green procedure for the biosynthesis of AgNPs because of their eco-friendly nature.



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*Source: 9984308-2022-04-20.xml*