Hydrothermal green synthesis of silver nanoparticles using Pelargonium/Geranium leaf extract and evaluation of their antifungal activity

2.1 Materials

Pelargonium/Geranium was purchased from a local market in Tabriz, Iran. Silver salt (AgNO₃) was purchased from Dr. Mojallali (Dr. Mojallali Chemical Complex Co., Tehran, Iran). Standard solution of AgNPs (with particle size of 10 nm and concentration of 1000 ppm) was obtained from Tecnan-Nanomat (Navarra, Spain).

Aspergillus flavus (PTCC 5004) and Aspergillus terreus (PTCC 5021) were purchased from microbial Persian type culture collection (PTCC, Tehran, Iran). Potato dextrose agar (PDA) as culture medium was provided by Oxoid Ltd. (Hampshire, UK).

2.2 Preparation of the Pelargonium/Geranium extract

The leaves of Pelargonium/Geranium were washed thoroughly two to three times with tap water, followed by washing with double-distilled water to remove any impurity, and then shade-dried for 1 week to completely remove the moisture. The resulting dried leaves were powdered using a domestic miller (MX-GX1521; Panasonic, Tokyo, Japan). Finally, 1 g of the powdered leaf was boiled for 5 min in 100 ml of deionized (DI) water and then filtered through Whatman no. 1 filter paper. The filtered Pelargonium/Geranium extract was stored in the refrigerator at 4°C.

2.3 Synthesis of AgNPs using Pelargonium/Geranium leaf extract

According to the literature, silver nitrate solution (1 mm) was prepared by dissolving 0.017 g of its powder in 100 ml of DI water [4], [18], [19]. Different amounts of AgNO₃ solution (9.5–20.5 ml) were mixed with 0.1 ml of Pelargonium/Geranium leaf extract with different concentrations (0.400–1.100 g/100 ml) and then the mixture solutions were kept in autoclave at 15 psi pressure and 121°C temperature for 15 min.

2.4 Analysis

2.5 Experimental design and statistical analysis

Response surface methodology (RSM) using a central composite design (CCD) with two independent variables (synthesis parameters), namely Pelargonium/Geranium leaf extract concentration (PLEC) (0.4–1.1 g/100 ml) (X₁) and amount of 1 mm AgNO₃ solution (9.5–20.5 ml) (X₂), was applied to determine the least mean λ_max (Y₁) and the highest color (Y₂) of the AgNP solution.

As compared to other experimentation methodologies, which were established on classical one-variable-a-time optimization, RSM has several advantages including creating numerous valuable data using a small run of experiments and the ability of estimating the interactions of independent variables with the response variables. Therefore, it is a proper method that can be used to optimize the main independent parameters of the process to obtain a product with desired attributes [29]. The selected synthesis parameters were studied at five different levels, i.e. central point (X₁: 0.750 g, X₂: 15.000 ml), level –1 (X₁: 0.400 g, X₂: 15.000 ml), level 1 (X₁: 1.100 g, X₂: 15.000 ml), level –α (X₁: 0.502 g, X₂: 11.110 ml), and level α (X₁: 0.997gr, X₂: 18.889 ml). In order to estimate the pure error, a central point was repeated for five times [27]. Therefore, 13 experimental runs, including four factorial points (levels ±1), four star points (levels ±α), and five central points were generated using the software Minitab v.16 statistical package (Minitab Inc., PA, USA) (Table 2). All experiments were carried out throughout 1 day by using one block. A second-order polynomial equation [Eq. (2)] was used to correlate the mean λ_max (Y₁) and the highest color (Y₂) of the AgNP solution to the studied synthesis variables.

where Y is the response variable; A₀ is a constant; A₁, A₂ correspond to the linear terms; A₁₁, A₂₂ represent the quadratic terms; and A₁₂ indicates the interaction terms. The suitability of the model was studied accounting for the coefficient of determination (R²) and the adjusted coefficient of determination (R²-adj). Analysis of variance was also carried out to provide the significance determinations of the resulting models in terms of p-value and F-ratio. High values of the F-ratio and small p-values (<0.05) were considered as statistically significant. Based on the fitted polynomial equations, three-dimensional surface plots and two-dimensional contour plots were designed to better visualize the independent variable interactions [30]. In order to obtain the optimum levels of PLEC and the amount of AgNO₃ solution with the desired response variables, numerical multiple response and graphical optimizations were used [31]. In fact, optimal values for the synthesis parameters were obtained by estimating the resulting surface plots with limitations on the responses of a minimum value for the λ_max (AgNP size) as well as a maximum value of the AgNP solution color. For verification of the validity of the statistical experimental approaches, three additional approval tests were performed at obtained optimum synthesis conditions.

3.1 Characterization of the Pelargonium/Geranium leaf extract

FT-IR measurement was performed to identify the responsible biomolecules in the Pelargonium/Geranium leaf extract for reduction and stability of the bioreduced AgNPs. In the FT-IR spectrum (Figure 1), several absorption peaks were centered at 616.94, 1637.52, 2043.77, and 3452.89 cm⁻¹, which were in the region range of 400–4000 cm⁻¹. The widest spectrum absorption was observed at 3452.89 cm⁻¹, which is attributed to the stretching vibrations of OH (hydroxyl groups) responsible for reducing the Ag⁺ ions to atoms. Furthermore, the suppressed bands at 1637.52 cm⁻¹, stretching vibration of C=C (alkane groups), are responsible for stabilizing the NPs. The spectrum absorption at 616.94 cm⁻¹ corresponded to the ring and skeletal modes of the main components.

Figure 1:

FT-IR spectrum of Pelargonium/Geranium leaf extract.

GC-MS analysis (Table 1) was also used to identify the active ingredients in the leaf extracts that were responsible for the synthesis of AgNPs. It was found that there were approximately 13 active compounds recorded within 32 min of retention time. However, there were eight prominent compounds found at different peaks of the chromatogram. These included tannins, flavonoids, phenolic acid, cinnamic acid, coumarin, monoterpenes, and sesquiterpenes, which may influence the reduction process and the stability of the synthesized AgNPs. The obtained results were in agreement with the results of the FT-IR analysis. These findings show that the main reducing (flavonoids, phenolic acid, and cinnamic acid) and stabilizing (monoterpenes and sesquiterpenes) compounds of the leaf extract had OH and C=C groups in their chemical structures, respectively.

Table 1:

Main reducing and stabilizing compounds of Pelargonium/Geranium leaf extract.

Name/molecular formula	Structural formula	Retention time (min)	Main fragments
Tannin, C76H52O46		35	1701.2
Flavonoids		18.2	302, 268, 191.62
		17.1	289, 254.74, 178.87
Sesquiterpenes	Three isoprenea units, sesquiterpenes may be linear (acyclic) or contain rings	15.3	204
Phenolic acid, C9H10O4		14.6	182.17, 137, 135
Cinnamic acid, C9H8O2		12	148.16, 103
Coumarin, C9H6O2		11.6	146.14, 130
Monoterpenes	Two isoprene units, monoterpenes may be linear (acyclic) or contain rings	10.8	136

^aIsoprene:

The mean pH of leaf extracts was about 5.15, indicating that Pelargonium/Geranium leaf extract is an acidic solution. The reason for this can be traced back to Table 1, which shows that extracts contain large amounts of phenolic and cinnamic acids. The AgNO₃ solution was colorless (Figure 2A), whereas the leaf extract was light yellow (Figure 2B).

Figure 2:

Color and appearance of the AgNO₃ solution (A), Pelargonium/Geranium leaf extract (B), and synthesized AgNPs using Pelargonium/Geranium leaf extract (C).

3.2 Fitting the response surface models

According to the design of the experiments, second-order polynomial models were fitted using the response variables obtained from the experimental runs (Table 2). The predictable regression coefficients and the corresponding significance of regressions for final reduced models are given in Table 3. In the final reduced models, insignificant effects were removed. However, the non-significant main term of the studied independent variables could not be removed from the model, if either their quadratic or interaction effects were significant (p<0.05) (Tables 3 and 4) [31]. The F-ratio and p-values of the all terms in the obtained final reduced models, evaluating their effectiveness, are also shown in Table 4.

It should be noted that the resulting models were significant only in the defined ranges for the studied independent variables, and they may not be used to predict the responses out of these ranges [32], [33].

As the overall model performance could be manifested in coefficients of determinations, the resulting rather high values for R² and R²-adj verified the fitness of the proposed models. Moreover, the achieved insignificant lack of fits for the proposed final models confirm their sufficient fitness to the synthesis parameter effects (Table 3). As clearly observed in Table 4, the synthesis parameters have significant (p<0.05) effects on all the studied characteristics of synthesized AgNPs.

3.2.1 λ_max

The λ_max of the obtained AgNPs ranged from 402 to 408 nm (Table 2). In all cases, λ_max was obtained in a favorable range for AgNPs using UV-Vis analysis [34].

The results also demonstrated that the synthesis variables had significant (p<0.05) effects on the λ_max (Y₁) variations (Table 4). In fact, the particle size of the synthesized AgNPs can be manifested in the λ_max of the AgNPs. It is well known that the longer wavelengths in absorption spectra of metal NPs are correlated to their bigger size [34]. The AgNP size changes could also be explained as a function of PLEC and the amount of AgNO₃ solution. As shown in Figure 3A, at low and constant PLEC, λ_max increases by increasing the amount of AgNO₃ in the mixture reaction, resulting in the production of bigger-sized AgNPs. This can be explained by the fact that at a low PLEC, the alkene groups of monoterpenes and sesquiterpenes had weak binding ability with Ag⁺. This behavior creates a layer around the AgNPs that acts as a capping agent to decrease AgNP agglomeration and increase their stability in the medium. The obtained results are in agreement with the findings of Zhang et al., who investigated the amount of AgNO₃ solution on λ_max and size of NPs. Based on their study, by increasing the AgNO₃ volume, the surface plasmon peak of AgNPs trended to red-shift and the particle size was increased [35].

Figure 3:

Surface (A) and contour (B) plots for λ_max of the synthesized AgNP solution as a function of the amount of AgNO₃ (ml) and PLEC (g/100 ml).

On the other hand, at high and constant PLEC, by increasing the amount of the AgNO₃ solution, the size of AgNPs was decreased (Figure 3A). Due to the presence of high concentration of stabilizing agents in the extract, upon the addition of low amounts of AgNO₃ solution, silver ions caused repulsion and capping agents had strong binding with AgNPs. As shown in Figure 3A, by increasing the PLEC at constant high and low amounts of AgNO₃ solution, the size of AgNPs were constant and increased, respectively. At high amounts of AgNO₃ by increasing the PLEC, due to the presence of more stable compounds, the λ_max and NP size remained constant. The obtained results also indicated a significant (p<0.05) interaction between the amount of AgNO₃ solution and the PLEC on changing AgNP size (Table 4).

As clearly observed in Figure 3B, the minimum achievable λ_max (405 nm) could be at low amounts of AgNO₃ solution and PLEC. The individual optimum conditions indicated that the maximum (Y₁=405 nm) was predicted at PLEC of 0.62 g/100 ml and 9.8 ml of AgNO₃ solution.

3.2.2 Color of AgNP solution

Based on the results, the synthesis variables had a significant (p<0.05) effect on the color of the AgNP solution (Y₂) variations (Table 4). Thus, the AgNP solution color changes could also be explained as a function of PLEC and the amount of AgNO₃. As shown in Figure 4, at low and constant amounts of PLEC, by increasing the amount of AgNO₃ the intensity of the mixture color decreased, denoting the production of a smaller number of AgNPs in the medium. The result can be explained by the fact that in the small amounts of dried powder, reducing agents can react with Ag⁺ completely. In fact, after complete reduction, by increasing the amount of AgNO₃ solution, the amount of silver ions increased while the number of synthesized AgNPs was constant, and their concentration decreased due to an increase in the volume of the AgNP solution. On the other hand, at high and constant PLEC, increasing the amount of AgNO₃ had no significant effect on the color of mixture (Figure 4A). Due to high concentration of reducing agents in the extract, small amounts of AgNO₃ solution rapidly reduced the free silver ions to form AgNPs, which, in turn, changed the color of the mixture solution from yellow to dark gray (Figure 2C). The presence of surface plasmon vibration bands due to the formation of AgNPs could change the color of mixture to dark gray [23]. By increasing the amount of AgNO₃ solution, the number of synthesized AgNPs was increased. However, due to the high intensity of mixture color, these changes could not be visualized. As clearly observed in Figure 4A, at constant low and high amounts of AgNO₃ solution, by increasing the concentration of PLEC, the color of mixture was constant (at low concentrations) and increased (at high concentrations), respectively. At low amount of AgNO₃, by increasing the PLEC concentration, all of the silver ions were sharply reduced to AgNPs and the color of the solution turned into dark gray. Due to low concentration of silver ions, further increase in the concentration of PLEC did not result in any significant (p<0.05) effect on the concentration of the formed AgNPs and the color of mixture. At high amounts of AgNO₃ solution, an increase in the concentration of PLEC could lead to increase in the concentration of synthesized AgNPs and color intensity of the solution. Bindhu and Umadevi also indicated that by increasing the concentration of dried hemp extract, the concentration of synthesized AgNPs increased, which was observable by the color change of the solution [36].

Figure 4:

Surface (A) and contour (B) plots for AgNP solution color as function of the amount of AgNO₃ (ml) and PLEC (g/100 ml).

The obtained results also indicated a significant (p<0.05) synergistic effect of the amount of AgNO₃ solution and the concentration of PLEC on the AgNP solution color (Table 4).

As shown in Figure 4B, the maximum AgNP concentration of 100.73 ppm and color intensity of 1.98 IU could be achieved at high and low concentrations of PLEC and the amount of AgNO₃ solution, respectively. The individual optimum conditions showed that the maximum color was predicted using 0.62 g/100 ml of PLEC of and 9.8 ml of AgNO₃ solution.

3.3 Optimization of processing parameters for the synthesized AgNPs

The optimum conditions for AgNP synthesis would be achieved when the process resulted in the formation of the smallest mean particle size (λ_max) with the highest number of AgNPs (color) in the solution. Graphical optimization based on an overlaid contour plot was used to find the optimum region for synthesis parameters to produce AgNPs with the minimum particle size and the maximum AgNP solution color (Figure 5). The white area in Figure 5 indicates the desired PLEC and AgNO₃ solution levels to obtain the optimum AgNPs. The quartiles of AgNP solution color were considered as their accepted higher levels. The first quartile of the mean particle size was also selected as its desired low level. As shown in Figure 5, the most desirable AgNPs were obtained from the synthesis conditions with AgNO₃ solution and PLEC of less than 16 ml and 0.9 g/100 ml, respectively.

Figure 5:

Overlaid contour plot of λ_max and color of AgNP solution with acceptable levels as a function of the amount of AgNO₃ (ml) and PLEC (g/100 ml).

Numerical multiple optimizations were also used to find the optimum levels of the studied variables. The results also suggested that the synthesis conditions with AgNO₃ solution of 9.8 ml and PLEC of 0.62 g/100 ml would give the most desirable AgNPs with λ_max and mixture solution color of 405 nm and 1.98 IU, respectively. Furthermore, the overall closeness between the predicted and experimental values of the responses could be concluded from the p-values of t-test analysis between them (1.00 for both responses). Moreover, three AgNP solutions were prepared according to the recommended optimal levels by numerical multiple optimization and were characterized in terms of studied physicochemical properties. The measured experimental values for the λ_max and AgNP solution color for these three AgNP solution samples were 405±2.33 nm and 1.98±0.6 IU, respectively. The insignificant differences found between the predicted and experimental values of the optimum suggested sample was reconfirmed by the adequacy of the final reduced models fitted by RSM.

3.4 Physico-chemical characteristics of synthesized AgNPs at optimum synthesis conditions

By synthesis of the AgNPs at obtained optimum conditions, the colorless reaction mixture was turned into dark gray solution (Figure 2C). The color change occurred due to the presence of the active molecules in the leaf extract, which could reduce the silver metal ions into AgNPs. Formation of AgNPs from 1 mm solution of AgNO₃ could be evaluated by using UV-Vis spectral analysis. Metal NPs have free electrons, the combined vibration of which in resonance with the light wave gives rise to an SPR absorption band [37]. As clearly observed in Figure 6A, an SPR spectrum for AgNPs was achieved at 405 nm.

Figure 6:

SPR spectrum (A) and particle size distribution (B) of the synthesized AgNPs at obtained optimum synthesis conditions.

The average particle size and the PDI, as a relative width of the size distribution, were determined using DLS measurements. DLS could also be used to determine the particle size distribution of the synthesized NPs in the suspension. At the optimum synthesis condition, the particle size of AgNPs ranged between 22 and 36 nm, with an average size of 29 nm. The PDI value of the synthesized AgNPs was 0.413 at obtained optimum synthesis condition, and its particle size distribution is shown in Figure 6B.

The ζ potential value of +0.6 mV for synthesized AgNPs at the optimum conditions showed that the capping biomolecules around the AgNPs are primarily composed of positively charged groups that are also responsible for the moderate stability of the NP [38]. TEM analysis was performed to evaluate the shape and microstructure of the synthesized AgNPs. A representative TEM image of the synthesized AgNPs is shown in Figure 7. As clearly observed, the synthesized NPs were dispersed with spherical structures. In fact, spherical NPs were more abundant than other shapes of NPs.

Figure 7:

TEM image of the synthesized AgNPs at obtained optimum conditions.

3.5 Antifungal activity of synthesized AgNPs at optimum synthesis conditions

The effects of synthesized AgNPs on mycelial growth of Aspergillus flavus and Aspergillus terreus during an incubation period of 7 days are shown in Figure 8A and B, respectively. As shown in Figure 8, the growth of A. flavus and A. terreus mycelia were significantly (p<0.05) inhibited by the presence of AgNPs as compared to the control plates and those amended with only Pelargonium/Geranium leaf extract. This result indicated that the synthesized AgNPs had high antifungal activity, and this was in line with the findings of Shankar et al., who indicated that AgNPs synthesized with Pelargonium/Geranium extract ranged in size from 16 to 40 nm and had a strong detrimental effect on the fungus Fusarium oxysporum [39]. According to Kim et al., AgNPs affect fungal cells by attacking their membranes and then disrupting the membrane potential [40]. Lamsal et al., with the aid of microscopic observations, indicated that the synthesized NPs caused antimicrobial effects not only on fungal hyphae but also on conidial germination [41]. It also seems that the high antifungal properties of AgNPs could be related to their chelating character and capability to modify the concentrations of ions existing in the growth media (e.g. Ca²⁺), essential minerals, and trace elements, which are all important for microbial growth especially for filamentous fungal growth.

Figure 8:

Antifungal activity of the synthesized AgNPs at obtained optimum conditions against Aspergillus flavus (A) and Aspergillus terreus (B). Data are the mean value of three replicates (each replicate contains four plates).