Plackett-Burman design in the biosynthesis of silver nanoparticles with Mutisia acuminatta (Chinchircoma) and preliminary evaluation of its antibacterial activity

2.1 Preparation of the total extract and obtaining the methanol fraction (MF)

The leaves of the Peruvian medicinal plant Mutisia acuminatta (chinchircoma) were washed, sorted, dried, pulverized, and sieved through a Tyler #100 mesh, yielding 60.00 g of powdered plant material. The powder was degreased with 300 mL of petroleum ether, shaken until homogenization, and subjected to ultrasound treatment in a bath (Branson model CPX5800H) under the following conditions: 25°C, 10 min, 40 kHz, and moderate power. The degreased sample was then filtered using Whatman fast passage paper and dried at room temperature. To prepare the extract, 58.1734 g of the dried defatted sample was added to 290 mL of 80% methanol and sonicated. The extract was centrifuged at 5000 RPM for 5 min in a swing-rotor centrifuge (Eppendorf), and the supernatant was collected for further fractionation by chromatography. Gradient column chromatography (CC) was performed using the following solvents as the mobile phase: petroleum ether, ethyl acetate, and methanol, in order of increasing polarity. The fractions obtained were monitored by thin-layer chromatography (TLC), and the fractions with the highest polarity were selected.

2.2 Identification of flavonoids by UHPLC - MS

To prepare the samples, 100 μL of each solution was taken and diluted with methanol in a ratio of 1:10. The resulting mixture was then sonicated for 5 minutes and filtered through a 0.25 μm PVDF disc filter into an HPLC vial. The samples were analyzed using a UHPLC chromatograph model Dionex Ultimate 3000 UHPLC system (Thermo Scientific), equipped with a Luna Omega C18 100 Å, Phenomenex column (150 x 2.1 mm, 1.6 μm) kept at 40 °C, and an injection volume of 2 μL. A gradient elution system was used with eluent A (1% HCOOH in H2O) and eluent B (1% HCOOH in ACN) at a flow rate of 0.2 mL/min. The coupled mass spectrometer model Q Exactive Plus (Thermo Scientific) was used to detect the compounds. Full MS scan parameters included a range of 130-1600 m/z, a resolution of 70,000, micro scans of 1, an automatic gain control (AGC target) of 1 × 10⁶, and a maximum IT of 100 ms.

2.3 Biosynthesis of silver nanoparticles

The biosynthesis of silver nanoparticles was carried out using different volumes (100-150 μL) of the FM methanolic fraction solution, which were added to the AgNO₃ precursor salt (1 mM), under the same synthesis conditions as described earlier.¹ Each experimental trial was developed according to the Plackett and Burman factorial design. For biosynthesis, 15 mL of AgNO3 salt (1 mM) was used as a precursor, and 5 mL of 20% EMT from the plant species (Mutisia acuminatta) was added. This solution was previously alkalinized by adding drops of NaOH (0.1 N) until a basic pH of 8-9 was obtained, using a multiparameter (Thermo Scientific model Star PRO “Orion”). The other synthesis conditions were as follows: temperature 45-48°C, agitation speed 600 rpm, and a synthesis time of 90 min. Agitation was carried out using a magnetic plate with a temperature sensor (Thermo Scientific model “Super Nuova +”). All solutions and dilutions were prepared using deionized water with a conductivity of 0.050 μS, supplied by the water purification system (Thermo Scientific, model “Smart 2 Pure”).

2.4 Characterization of biosynthetized AgNPs

2.4.1 Ultraviolet-Visible Spectrophotometry (UV-VIS)

The UV-VIS spectrophotometer (Thermo Scientific brand, model Evolution 220) was used to read and monitor the biosynthesized AgNPs. Each reading was taken from a 1000 uL sample, which was diluted with an 80% methanolic solution in a 1:2 ratio for each sample analyzed. Spectral scanning was performed between 350 nm - 750 nm to determine the maximum peak absorbance as a function of wavelength, corresponding to the surface plasmon resonance (SPR) of the AgNPs, in accordance with the Plackett and Burman design (PBD). The spectrophotometric readings were taken using a quartz cuvette with a step width of 1 cm. The maximum absorbance was determined at a wavelength of 411 nm, and the target used for each spectral sweep was an 80% methanolic solution. Thermo INSIGHT software was used for spectral processing.

2.4.2 Fourier transform infrared spectrophotometry (FT-IR)

The FT-IR spectrophotometric analysis was performed using a Thermo Scientific Nicole iS50 Analytical model equipped with “OMNIC Spectra” software for data analysis. Aliquots of the 20% ETM, FM, and AgNPs samples were individually placed on the diamond ATR lens for each reading. Prior to each measurement, a spectral background (BG) was recorded in transmittance (T) mode within the mid-IR range (4000 cm^-1 to 400 cm^-1). The diamond ATR accessory was cleaned with Kimtech absorbent cloths and 98% absolute ethyl alcohol to prevent cross-contamination between samples. A final BG spectral background was taken after each data collection.

2.4.3 Dynamic light scattering (DLS)

The hydrodynamic size of the biosynthesized AgNPs was measured using dynamic light scattering (DLS) with a Malvern Zetasizer Nano ZSP instrument. Prior to analysis, the samples were filtered using a 10 mL syringe and a 0.22 μm pore size filter. For each measurement, 1.5 mL of the filtered sample was added to a polypropylene cuvette. The instrument software required several input parameters for the analysis, including the refractive index (IR) of silver (1.330), the dispersant medium (methanol), and the dynamic viscosity (Cp) of the dispersant medium (0.587 for 20°C). The software generated a histogram plot of the size distribution, which showed the percentage of scattered light intensity (%) as a function of the hydrodynamic particle diameter distribution (d.nm).

2.4.4 Transmission electron microscopy (TEM)

The biosynthesized NPsAg were analyzed using an atomic resolution TEM (Thermo Scientific, model Talos F200i) with a working voltage of 20 to 200 kV and a line resolution of less than 0.10 nm. For analysis, the sample was supported on a MESH 300 CU Grid (TEM). Prior to analysis, the AgNPs sample was centrifuged at 5000 RPM using a tilting angle rotor centrifuge, and the solid phase obtained (precipitate) was separated from the liquid phase. This solid phase was then washed with pure methanol three times, and a 10 uL sample was taken and supported on the MESH 300 CU Grid. The sample was dried for 48 hours in a chamber with a relative humidity (RH) of 30% and a temperature of 25°C. To ensure that the AgNPs were not conglomerated or in a state of structural aggregation at the bond level, an ultrasound pretreatment (BRANSON 5800) was performed for approximately 15 minutes.

2.5 Experimental design

2.5.1 Software

Minitab 19 software was used for assay development, Plackett-Burman experimental design (PBD), and analysis of variance (ANOVA). The software was also used to generate 3D response surface models and 2D contour plots, perform correlation analysis of variables, and visualize the interaction of results.

2.5.2 Plackett - Burman design (PBD)

The Plackett-Burman experimental design (PBD)³⁸ was used to investigate the influence of six factors on the absorbance (411nm) as the dependent variable for the biosynthesis of silver nanoparticles. The six factors included were stirring speed (X1, RPM), potential hydrogen (X2, pH), synthesis temperature (X3, °C), synthesis time (X4, min), volume of methanolic fraction (X5, μL), and concentration of AgNO₃ (X6, mM). Twelve factorial tests and three tests with PBD center points were conducted to determine the most significant factors, as presented in Table 1 and Table 2 of the experimental trials. The polynomial regression model shown in equation 1 was used to analyze the data:

In addition, the effects of each variable were determined, including the degrees of freedom (df), the Sum of Squares (SC), the Mean of the Sum of Squares (MC), and the calculation of the Fo statistic. A Fisher’s test was also conducted using the F statistic according to tables at the 95% significance level (p-value < 0.05) with 1 and 2 degrees of freedom, respectively. The F statistic was used to evaluate the significance of the biosynthesis variables, which were considered significant for Fo > F (tables). The statistical software Minitab 19 Ink was used for assay development, PBD, ANOVA, analysis of 3D response surface models and 2D contour plots, correlation analysis of variables, and graphical interaction of results.

2.5.3 Antibacterial evaluation

2.5.3.1 Zone of inhibition method and sensitivity analysis

Certified strains of Staphylococcus aureus ATCC^® 25923 (Epower) and Escherichia coli ATCC 33876 were obtained from Sigma Aldrich. All glassware was sterilized in a unidirectional laminar flow chamber at 80°C for 15 min in a Pasteur oven prior to use. The culture medium (Mueller Hinton) was prepared by dissolving 34 g per 1000 mL of distilled water under agitation and constant boiling, following the manufacturer’s specifications (Merck KGaA). The medium was then sterilized using an LA autoclave (Biogenics brand, model AVDA 20-D) at 121°C for 15 min and 15 lb of pressure. Inoculum preparation followed the laboratory’s recommendations for acquiring Escherichia coli and Staphylococcus aureus strains, with the lyophilized strains reactivated in previously sterile 0.1% peptone water. To do this, 0.1 g of peptone (Liofilchem brand) was weighed and diluted in 100 mL of double distilled water and used as a diluent for the lyophilized strains. Sterile swabs were moistened with the inoculum, distributed and sown in test tubes with slant agar, covered, and incubated at 37°C. Approximately 15 mL of the Mueller Hinton agar medium was then poured into each sterile petri dish and allowed to cool before use.

3.1 Preparation of the total methanolic extract (EMT) and obtaining the methanolic fraction (FM)

The defatted plant sample showed a very intense dark green color after drying at room temperature, and its dry weight was recorded using an analytical balance, yielding a total of 58.1730 g, which was used to prepare a 20% total methanolic extract (TME). The TME obtained had an intense dark brownish-green color. The yield of the crude extract from the methanolic extraction was 70%, which was prepared using ultrasound under the following conditions: 35°C, 45 min, 40 kHz, and moderate power. The FM was then obtained in a series of test tubes using column chromatography (LC) eluates with varying concentrations and polarities of solvents used as the mobile phase. The eluates obtained were grouped based on their characteristics observed in the TLC plates, which showed a variable intensity of coloration ranging from brown to light yellow. The chromatographic plates (TLC) were developed up to 85% of the total height in a horizontal glass chamber (10 cm × 15 cm) with the medium saturated with the mobile phase for 30 min at room temperature and 60% relative humidity. The TLC plates were then dried with airflow and developed with UV light. Figure 1 shows the TLC plate of tubes 31-40 corresponding to the methanolic fraction.

Figure 1. TLC (thin layer chromatography) plate of the methanolic fraction.

3.2 Metabolite identification by High-Performance Liquid Chromatography coupled to mass spectrophotometry (HPLC - MS)

Figure 2A, 2B, and 2C show the total ionic current chromatograms (TIC) of samples obtained from Mutisia acuminata leaves, including the total methanolic extract at 20% (EMT), the methanolic fraction (FM), and the biosynthesized silver nanoparticles (AgNPs - FM).⁷³ The number of signals and their intensity in the chromatograms varied according to the concentration and structural characteristics of the molecules present in the samples. The total methanolic extract (Figure 2A) showed a higher number of signals in the chromatogram, detecting derivatives of caffeic acid, coumarins, flavonoids, and lignans in the zone between 0-15 minutes. After 15 minutes, many unknown compounds were detected, corresponding to new nitrogenous derivatives of coumarins. The FM (Figure 2B) exhibited a similar profile to the EMT, where several of the compounds detected in the EMT were not observed, especially in the zone above 15 minutes. Finally, in Figure 2C (AgNPs - FM), the number of detected signals was very low, detecting a disaccharide, coumarins, and a complex formed between silver and the solvent AgCH₃CN+ at t = 1.95 min. The isomeric compounds at m/z 430 (t = 15.27 min and t = 15.56 min) were the most intense signals in this sample, whose structures could not be identified. There is a high possibility that these are new coumarin-derived compounds.

Figure 2. A) Chromatogram of the total methanolic extract at 20% (EMT 20%) of Mutisia acuminata. B) Chromatogram of the methanolic fraction (FM) of Mutisia acuminata. and C) Chromatogram of the biosynthesized silver nanoparticles (AgNPs-FM) of Mutisia acuminata.

3.3 UV-VIS Spectrophotometry

Figure 3A displays the UV-Visible spectral scan of the 20% EMT of Mutisia acuminata obtained from 400 nm to 750 nm wavelength range. The main absorption peak with a maximum absorbance of around 650-680 nm and a secondary peak at around 470 nm corresponds to the phytochemical components of the TME, including chlorophyll. These specific UV-VIS absorbance peaks are directly related to the time, extraction method, and concentration of the methanolic extract obtained. Additionally, in Figure 3A, the UV-VIS spectrum of the FM obtained by LC is shown, where no peak can be observed between 400-420 nm, corresponding to the RPS of the AgNPs. This finding confirms that the starting EMT and the FM obtained do not contain the characteristic peak of the silver nanoparticle.

Figure 3. A) UV-VIS spectra of the 20% total methanolic extract (TME) of Mutisia acumintta and B) UV - VIS spectra of the biosynthesized AgNPs showing the absorbance at different time intervals.

3.4 FT-IR Spectroscopy

The FT-IR absorption spectra of AgNPs obtained by green synthesis were studied in the range of 400 cm^-1 to 4000 cm^-1 wavenumber in transmittance mode. In Figure 4A, 4B and 4C, we can identify a broad band around 3000 cm^-1 and 3400 cm^-1 approximately due to the N-H stretching vibration of the NH₂ group and the O-H group of the stretching vibration overlap attributed to the water and phenolic compounds present. Figure 4A, which corresponds to the EMT at 20%, defined transmission peaks were observed at: 3316.19; 2947.28; 2834.93; 1654.52; 1449.23; 1406.69, 1111.46; 1015.61 and 543.82 cm^-1. Figure 4B, which corresponds to FM, peaks with lower absorption were observed at: 3616.16; 2972.89; 1452.06; 1026.15; 879.92; 648.59 cm^-1, where the disappearance of some IR peaks concerning the total methanolic extract (TME) is appreciated. Figure 4C shows the FTIR spectrum of the biosynthesized AgNPs, bands and spectral peaks of higher intensity and the differences concerning the FTIR spectra of Figure 4A and 4B, where the formation of a peak at 1637.00 cm^-1 was observed producing an increase in intensity in the band between 1990.32 and 2500.00 cm^-1, which reveals the possible biomolecules of Mutisia acuminata extract responsible for reducing silver ions (Ag⁺) and their interaction with the AgNPs.³⁹ Moreover, notoriously, the absence of several peaks in Figure 4A and 4B corresponding to EMT and FM, respectively, was observed.

Figure 4. FT-IR spectra.

A) Total Methanolic Extract at 20% (EMT); B) Methanolic fraction, C) AgNPs biosynthesized with methanolic fraction.

3.5 Dynamic Light Scattering (DLS)

Figure 5 shows the DLS results for the size of the AgNPs obtained in colloidal suspension, which was biosynthesized with the FM of Mutisia acuminata. The results show the size distribution of the silver nanoparticles using a monomodal histogram plot with moderate dispersity, presenting hydrodynamic sizes between 20-180 nm approximately and with a polydispersity index (PDI) of 0.260, the average hydrodynamic size (Z- average) was 45.91 nm. The maximum reported size distribution of AgNPs as a function of % intensity was 60.87 nm ± 24.95 nm, as observed in the histogram in Figure 5.

Figure 5. Hydrodynamic size distribution of AgNPs synthesized from the FM of Mutisia acuminatta#.

3.6 Transmission Electron Microscopy (TEM)

Figure 6A shows the results by transmission electron microscopy (TEM), where the biosynthesized AgNPs present a quasi-spherical morphology with agglomerates, the agglomeration observed in Figure 6A could be due to the drying process before analysis by microscopy.⁴⁰ The size range observed in Figure 6A was between 30 to 60 nm; these results illustrate the formation of AgNPs used as a reducing agent in the phytoconstituents of Mutisia acuminata FM. Figure 6A also shows the biomolecular coating on the surface layer of AgNPs, which could be responsible for the stability of the obtained AgNPs.³⁹ Figure 6B, from the TEM micrograph, shows the regular distribution pattern of silver atoms forming the crystal lattice of the obtained nanoparticles, which present a distance between 260-270 pm in the observed interatomic plane. These results could correspond to the interplanar (111) separation of the planes in the cubic crystal structure centered on the faces of the silver atomic arrangement.⁴¹

Figure 6. A) TEM micrographs of Mutisia acuminatta biosynthesized AgNPs, the nanoscale image of the obtained shape of the AgNPs. B) TEM image of silver with atomic resolution and crystallinity pattern.

3.7 Experimental design and statistical analysis

3.7.1 Level of influence of the factors according to the Plackett-Burman Design (PBD)

The Plackett-Burman design (PBD) helps to recognize significant variables with fewer experiments³⁸; the variables were tested at two levels (high and low) indicated by (+1) and (-1), respectively, as shown in Table 1. The PBD design configuration was developed with Minitab 19 software, which predetermined 15 experimental tests of the factorial design, plus 3 central tests, to evaluate the curvature of the design, as presented in Table 2. According to Asfaram et al.⁴² in Table 1, the number of total variables identified for the process of synthesis of AgNPs bi-synthesized Ag with the methanolic fraction (AgNPs-FM) of Mutissia acuminatta is summarized, where the independent variables identified by PBD were as follows; i.e., K (number of factors) equal to 6. Likewise, the independent variables (Xi) are also categorized here with their respective denomination and physical units; the upper (+1), lower (-1) and central (0) levels are also presented for each of the variables on the coded scale, and the dependent variable (Y) is also denoted as the response variable. The results (Y) were obtained by spectral scanning using the UV-VIS spectrophotometer, determining the maximum absorbance of the spectrum at a fixed wavelength of 411 nm.

The experimental design was developed to reduce the number of experimental runs.^38,42,43 As analyzed in Table 1 and Table 2, the concentration effects of stirring speed X₁ (RPM), potential hydrogen X₂ (pH), synthesis temperature X₃ (°C), synthesis time X₄ (min), the volume of the methanolic fraction X₅ (μL) and the concentration of AgNO₃ X₆ (mM), on the absorbance (411 nm), were investigated empirically using the Plackett-Burman design developed⁴⁴ in Table 2.

3.7.2 Analysis of Variance (ANOVA) and significance effect

Table 3 presents the ANOVA and significance analysis of the variables; the statistical significance of the model was evaluated and estimated by the F-value (5.40) and the p-value (p < 0.020). The results suggest that the listed factors had remarkable influences on the absorbance (411 nm) recorded, except for the pH of synthesis (X₂) and AgNO₃ con-centration (X₆), due to their low p-value (p-value = ∼0.002 and ∼0.027 < 0.05) respectively. Therefore, these two factors had significance in Mutisia acuminata AgNPs biosynthesis, having some impact on the response variable (Y). Therefore, Table 3 shows the results identifying 2 factors that significantly influence the response variable Y (Abs 411 nm). The development of the significance test with the F-statistic and the calculations developed in Table 3 was performed at 95% (p < 0.05), with 1 and 7 degrees of freedom, respectively, for the development of the ANOVA. The standard plot of the effects (Figure 7) and the Pareto diagram (Figure 8) are presented, which indicate that of the total of 6 variables evaluated: the hydrogen potential (pH) (X₂) and the AgNO₃ concentration (mM) (X₆) were significant variables of the AgNPs biosynthesis process, with a probability α = 0.05. The results of the Plackett-Burman design of experiments are shown as a standardized Pareto plot in Figure 8. The graphs show the p-values of the independent variables, which were considered to have a significant effect when the values were <0.05, with 95% confidence.

Figure 7. Normal plot of standardized effects.

Figure 8. Pareto diagram of standardized effects.

3.7.3 Polynomial regression

The multiple linear regression analysis and the Plackett-Burman factorial design allowed the design of a statistical polynomial model representative of the experimental data. ANOVA determined the significant effect for each variable, and the model’s coefficients were found from this. The significance of the variables evaluated was detected with the T-value and p-values (p < 0.05). The regressions were represented by the corresponding polynomial equations 2 and 3 for the coded and uncoded parameters, respectively:

3.7.4 Adjustment of the polynomial regression model

Figure 9 represents the fit model of the experimental data and the data estimated from the polynomial regression equation (equation 2), finding that the value of the correlation coefficient (R²) to be 84.40%, which is a value closely related to the adjusted coefficient of determination (R² adj.) with a value of 83.20%, showing the acceptability of the empirical data. Therefore, Figure 9 shows the adjusted model of the data obtained, where the scattered points in the graph correlate with fit to the trend line, thus presenting a high adjusted correlation coefficient (Adjusted R² = 83.20%).

Figure 9. Model fit of experimental data (ABS 41 nm) and estimated data (Fit 1).

3.7.5 Contour plots and surface plots of the polynomial model

Figure 10 shows the set of 3D surface plots and 2D contour plots of the obtained polynomial model; these plots show the hyperplanes and contours that were generated by the model for Absorbance (411 nm) as a function of the evaluated variables X₁ (RPM), X₂ (pH), X₃ (°C), X₄ (min), X₅ (μL) and X₆ (mM). Each graph was generated by Minitab 19 statistical analysis software. Surface and contour Figure 10A, B, C and D combine two independent parameters for response prediction (Abs 411nm), as described below: Figure 10A shows the surface and contour plots of Absorbance (411 nm) as a function of stirring speed (RPM) and pH. Figure 10B shows the surface and contour plots of Absorbance (411 nm) as a function of stirring speed (RPM) and temperature (°C). Figure 10C shows Absorbance’s surface and contour plots (411 nm) as a function of pH and temperature (°C). Figure 10D also shows the surface and contour plots of Absorbance (411 nm) as a function of stirring speed (RPM) and AgNO₃ concentration (mM). Graphs A, B, C and D are response surfaces that present slight curvatures detected by the central points of the PBD. Also, the maximum extremes located for this region of points can be appreciated as effective responses of the polynomial prediction model about the parameters of greater significance than the ANOVA determined.

Figure 10. 3D response surface and 2D contour plots of the polynomial regression model for ABS (411 nm) as a function of (A) stirring speed (RPM) vs. pH, (B) stirring speed (RPM) vs. temperature, (C) pH vs. temperature, (D) stirring speed (RPM) vs. AgNO₃.

3.8 Antibacterial activity

Table 4 shows the results obtained by the inhibition halos method for the sensitivity analysis of the samples evaluated against the bacterial strains Escherichia coli ATCC 33876 and Staphylococcus aureus ATCC 25923. According to the data, the most significant antibacterial activity was against Escherichia coli ATCC 33876, showing inhibition halos (mm) of greater diameter concerning the Staphylococcus aureus ATCC 25923 strain. According to the data in Table 4, the most significant inhibition halos recorded were given by the following samples: (1) AgNPs - FM, (2) EMT (5%), (3) FM and followed by (4) MetOH, highlighting the inhibition effect of biosynthesized silver nanoparticles (AgNPs - FM). According to Table 4, the nanoparticles synthesized with the methanolic fraction (AgNPs - FM) presented higher antibacterial activity against Escherichia coli ATCC 33876, with the following average inhibition halos for each cultured plate: 5.167 ± 0.289 mm; 5.333 ± 0.289 mm and 5.167 ± 0.289 mm respectively. However, the NPsAg-FM against Staphylococcus aureus ATCC 25923 presented a smaller average inhibition halo: 3.667 ± 0.289 mm; 4.000 ± 1.000 mm; 3.833 ± 0.289 mm.

On the other hand, Figure 11 shows the categorical graphical representation of the antibacterial effect, where it is clearly distinguished that AgNPs – FM and EMT (5%) show a high sensitivity against both strains Escherichia coli ATCC 33876 and Staphylococcus aureus ATCC 25923, concerning the other substances evaluated (FM and MetOH).

Figure 11. Representation of antibacterial effect against certified strains of Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 33876: (a) MetOH, (b) AgNPs - FM, (c) FM and (d) EMT (5%).

Figures 12 and 13 below show the images of the agar well assay in the Petri dishes, with the respective media and bacteriological strains and the different substances evaluated: (a) EMT (5%), (b) FM, (c) AgNPs-FM and (d) Methanol, against Escherichia coli ATCC 33876 and Staphylococcus aureus ATCC 25923 respectively, where the formation of the inhibition halos of the medium against the bacterial strains after 48 hours of incubation at 35°C can be observed. The formation of halos of greater average distance for both bacterial strains is given for biosynthesized silver nanoparticles (AgNPs-FM), followed by EMT (5%), presented halos of inhibition with high magnitude of measurement (mm), concerning the other substances as presented in Table 4. The substances that had little sensitivity effect against both bacterial strains were: FM followed by pure methanol with little noticeable inhibition halos and low measurement magnitudes (bacteriostatic), as shown in Table 4.

Figure 12. Agar well assays for the evaluation of antibacterial activity against Staphylococcus aureus ATCC 25923: (A) EMT (5%), (B) FM, (C) AgNPs-FM and (D) Methanol (blank).

Figure 13. Agar well assays for the evaluation of antibacterial activity against Escherichia coli ATCC 33876: (A) EMT (5%), (B) FM, (C) AgNPs-FM and (D) Methanol (blank).