Biosynthesis, characterization and antimicrobial activities assessment of fabricated selenium nanoparticles using Pelargonium zonale leaf extract

2.1 Materials

Pelargonium zonale leaves and sodium selenite (Na₂SeO₃) (purity >99%), as a natural source of bioreducing and stabilizing agents, and a selenium precursor, were provided from the local market (Tabriz, Iran) and Merck Company (Merck Co., Darmstadt, Germany), respectively. Escherichia coli (PPTCC 1270) and Staphylococcus aureus (PTCC 1112) were purchased from the microbial Persian type culture collection (PTCC, Tehran, Iran). Nutrient agar and potato dextrose agar (PDA) were purchased from Himedia (Himedia Co., Mumbai, India) and Oxoid (Oxoid Ltd., Hampshire, UK), respectively. Colletotrichum coccodes and Penicillium digitatum were isolated from local spoiled tomatoes and oranges, respectively.

2.2 Pelargonium leaf extract preparation

In order to prepare the PLE, the prepared leaves of Pelargonium zonale of similar size, shape and color, were washed and shade-dried during a week at ambient conditions (temperature of 30°C and relative humidity of 27%). The dried leaves were then powdered by domestic miller (MX-GX1521; Panasonic, Tokyo, Japan) and 2 g of the prepared powder was added into the 100 ml of boiling deionized double distilled water for 5 min. PLE was finally obtained by filtration of the boiled solution through Whatman No. 40 filter paper.

2.3 Se NPs synthesis using PLE

Previous studies which completed the synthesis of Se NPs utilized 10 mm Na₂SeO₃ as selenium solution [5, 19, 20]. Therefore, 0.263 g of the selenium salt was dissolved in 100 ml of deionized double distilled water to obtain 10 mm colorless selenium solution and several defined amounts of Na₂SeO₃ solution (15–65 ml) and PLE (0.5–2.5 ml) were mixed and then exposed to the microwave radiation using a domestic microwave oven (MG2312w, LG Co., Seoul, South Korea) for 4 min and at constant power of 800 W.

2.4 PLE characteristics assay

A Fourier transform-infrared (FT-IR) instrument (Shimadzu 8400S spectrometer, Kyoto, Japan) coupled with KBr pellets (4000–400 cm⁻¹ region) was utilized to monitor and detect the functional groups of PLE. These groups are related to the main presented reducing and stabilizing bioactive compounds in the PLE. The pH of PLE was measured using a pH meter (DELTA 320; Delta, Shanghai, China).

2.5 Assessment of the fabricated Se NPs characteristics

UV-Vis spectrophotometry (Jenway UV-Vis spectrophotometer 6705, Stone, UK) was used to confirm the formation of the Se NPs based on their surface plasmon resonance (SPR) signal. Due to the SPR signal of the NPs, the mixture solution containing the formed Se NPs indicated the broad absorption peak (λ_max) in the wavelength ranged 270–350 nm [19, [20]. The measured absorbance of the solution containing the formed NPs could be correlated to the concentration of the synthesized Se NPs. By contrast, the higher absorbance unit (% a.u.) of the mixture solution revealed that Se NPs with higher concentrations were synthesized and vice versa 15]. Furthermore, by formation of the Se NPs, the color of the mixture solution containing selenium ions converted from pale yellow into dark red for the solution containing the formed Se NPs [19, 21, 22].

Particle size, polydispersity index (PDI), particle size distribution and zeta potential values of the synthesized Se NPs were evaluated and recorded by a dynamic light scattering (DLS) analyzer (Malvern instruments, Zetasizer Nano ZS, Worcestershire, UK). For the morphology studies of the synthesized Se NPs, transmission electron microscopy (TEM CM120, Philips, Amsterdam, Netherlands) with an acceleration voltage of 120 kV, was used. The exact value of the fabricated Se NPs was also calculated and determined by TEM.

2.6 Antimicrobial activities of the synthesized Se NPs

Antibacterial activities of the synthesized Se NPs against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria were evaluated using the well diffusion method described by Torabfam and Jafarizadeh-Malmiri [23]. The antibacterial assay was performed in incubation conditions of the 37°C for 24 h.

The antifungal activities of the fabricated Se NPs were evaluated against C. coccodes and P. digitatum using an inhibition in radial mycelia growth method described by Mohammadlou et al. [15]. The antifungal assay was completed in incubation conditions of 26°C during 7 days. In order to isolate the studied fungi strains, pieces of the spoiled tomato and orange peels, which were inoculated with the C. coccodes and P. digitatum, respectively, were separated and put into the center of the plates into which PDA was poured. The provided plates were then incubated at 26°C for 1 week and after that a 5 mm disc (in diameter) of the growth fungi were provided to place on the surface of the plates containing PDA and synthesized Se NPs, PDA and PLE, and PDA as a control sample.

2.7 Statistical analysis

Central composite design with five replications of the center point, and response surface methodology (RSM) based on a second-order polynomial model, were used to design experiments and evaluate the effects of the synthesis parameters, namely amounts of PLE (X₁, ml) and Na₂SeO₃ solution (X₂, ml), on the λ_max (nm) and absorbance (% a.u.) values of the solutions containing formed Se NPs [16, [24]. As compared to other statistical techniques, RSM has several advantages including decreasing the experimental runs, and costs and evaluation of the interaction effects of the independent variables on the response variables 25, [26]. Coefficient of determination (R²), adjusting the coefficient of determination (R²-adj) and lack of fit (p-value) were evaluated for the suitability of the generated models. In addition, the significance determination of the resulted models was studied using analysis of variance in term of p-value at the 95% confidence interval for the level of the significance. Three-dimensional surface plots and two-dimensional contour plots were established to well observation of the interaction of the synthesis parameters and the optimum region of the response variables, respectively 15].

Based on the synthesis of Se NPs with minimum λ_max (nm) and maximum absorbance (% a.u.), a graphical optimization using an overlaid contour plot was obtained and a numerical optimization was carried out to achieve the strict optimum levels of the selected fabrication variables in the defined ranges. Validity of the generated models based on RSM to predict the optimum synthesis conditions, was evaluated using three additional approval tests for the synthesized Se NPs at obtained optimum conditions [27, 28]. The experimental design, data analysis optimization and verification procedures were performed using Minitab software (Minitab Inc., v. 16, PA, USA).

3.1 Specification of the PLE

The main functional groups existing in the PLE are shown in Figure 1. As can be seen in the FT-IR spectra of the PLE (Figure 1), there were four highlighted peaks centered at 3452.5 cm⁻¹, 2066 cm⁻¹, 1636.5 cm⁻¹ and 662.8 cm⁻¹. The two main peaks were detected at 3452.5 cm⁻¹ and 1636.5 cm⁻¹, which were related to the stretching vibrations of hydroxyl and amide groups, respectively. Hydroxyl groups have a key role in the reduction of selenium ions to their element and can be found in the structure of the tannins, flavonoids, phenolic acid, cinnamic acid, coumarin, monoterpenes, and sesquiterpenes which are presented in the PLE [15]. By contrast, amide groups have a main role in the stabilizing of the formed Se NPs, and these exist in the structure of the proteins and enzymes of the PLE [15, 16]. The pH of the provided PLE was approximately 5.5, indicating that the PLE is an acidic solution.

Figure 1:

Fourier transform-infrared (FT-IR) spectrum of Pelargonium zonale leaves extract (PLE).

3.2 Evaluation of the fitted models as a function of the amounts of PLE and Na₂SeO₃ solution

According to the design of experiments, and achieved experimental values for the λ_max (nm) and absorbance (% a.u.) (Table 1), the second polynomial models based on Eq. (1) were generated as a function of the linear (X_i), quadratic (X_ii) and interaction (X_ij) of the amounts of PLE (X₁, ml) and Na₂SeO₃ solution (X₂, ml). Y is the response variable:

The regression coefficients of the generated models with their R², R²-adj and p-values of the lack of fit are given in Table 2. As can be seen in this table, the higher values of the R² (>0.88) R²-adj (>0.80) and high p-values of the lack of fit (p>0.05) confirmed the adequate fitness of the generated models. Table 3 presents the p-values of all the terms of the models. As can be observed in this table, the interaction of the synthesis parameters had non-significant effects on the λ_max (nm) of the solution containing Se NPs. By contrast, the quadratic effect of the amounts of PLE (X₁₁, ml) had insignificant effects on the absorbance of the resulting solutions.

The λ_max of the solutions containing Se NPs was changed from 319 nm to 324 nm. It is noted that the particle size of the synthesized Se NPs can be exhibited in the λ_max of them. It is identified that the higher λ_max values for the NPs are interrelated to their larger size [29]. The effects of the synthesis parameters on the λ_max of the synthesized Se NPs are shown in Figure 2A and B. As can be detected in Figure 2A, at any constant amount of PLE, by increasing the amount of Na₂SeO₃ solution the λ_max were increased. The same trend was also observed for increasing the amount of PLE at any constant amount of the Na₂SeO₃ solution. This indicated that the interaction of the synthesis variables had non-significant effects on the λ_max of the solution containing Se NPs. The obtained result was reconfirmed by the obtained p-value>0.05 of the synthesis parameters interaction in Table 3. As can be seen in Figure 2B, minimum λ_max of the solution containing Se NPs could be obtained at the lower amounts of the PLE and Na₂SeO₃ solution.

Figure 2:

Surface plot (A) and contour plot (B) for λ_max, and surface plot (C) and contour plot (D) for absorbance of the synthesized selenium nanoparticles (Se NPs) as a function of amounts of Na₂SeO₃ solution (ml) and Pelargonium zonale leaves extract (PLE) (ml).

As can be seen in Table 1, the absorbance (% a.u.) values of the solutions containing formed Se NPs varied from 1.654 to 35.58 (% a.u.). It is noted that the concentration of the synthesized Se NPs can be manifested in the absorbance of their solutions. It is identified that the higher absorbance for the NPs is correlated to their higher concentrations [29]. The effects of the studied fabrication variables on the absorbance of the solutions are indicated in Figure 2C and D. As clearly detected in Figure 2C, at a constant amount of PLE, by increasing the amount of the Na₂SeO₃ solution the absorbance was decreased. This trend was observed throughout any amount of the PLE. At constant low and high amounts of the Na₂SeO₃ solution, by increasing the amount of PLE, different patterns were observed. The obtained results indicated that the fabrication parameters interaction had a significant (p<0.05) effect on the absorbance value of the solution containing fabricated Se NPs, which was in agreement with the obtained p-value for the interaction of the synthesis parameters (Table 3). Figure 2D, revealed that the maximum values for the absorbance of the solution containing Se NPs were obtained at higher amounts of PLE and lower amounts of the Na₂SeO₃ solution. The achieved result can be described by the point that, at high amounts of PLE, the concentrations of the bioreductant compounds existing in the PLE are high and these reducing agents can easily and rapidly reduce the presented selenium in the solution and convert them into their NPs, which caused an increase in the concentration of the synthesized Se NPs. The attained results were in line with findings of Mohammadlou et al. [15] and Eskandari et al. [17, 30]. Those indicated that in synthesis of the silver and gold NPs using PLE and mushroom extract, by increasing the amounts of the extracts, the concentrations of the fabricated NPs drastically increased.

3.3 Optimization and validation procedures to obtain optimum levels of the synthesis parameters

The optimum synthesis conditions for fabrication of the inorganic NPs obtained when the process leads to synthesis of the NPs with minimum particle size and higher concentration [18, 24] (Figure 3A) indicates the optimum area for the synthesis parameters to produce Se NPs with lower λ_max (nm) and higher absorbance (% a.u.). Numerical multiple optimization also indicated that the strict optimum values of selected fabrication parameters would be attained using 1.48 ml PLE and 15 ml of Na₂SeO₃ solution; the λ_max and absorbance of the solution containing Se NPs were 319 (nm) and 35.33 (% a.u.), respectively. The verification tests for the synthesis of the Se NPs at obtained and predicted optimum conditions resulted in Se NPs with λ_max and absorbance of 319±1 (nm) and 34.6±2 (% a.u.), respectively.

Figure 3:

Overlaid contour plot of λ_max, absorbance with acceptable levels as a function of amount of Na₂SeO₃ (ml) and Pelargonium zonale leaves extract (PLE) (ml) (A), UV-Vis spectra (B), particle size distribution (C) and transmission electron microscopy (TEM) image (D) of the synthesized selenium nanoparticles (Se NPs) at obtained optimum conditions.

3.4 Characteristics of the fabricated Se NPs at obtained optimum synthesis conditions

Particle size, PDI and zeta potential values for the formed Se NPs using DLS at obtained optimum conditions were 136 nm, 0.321 and −24.6 mV, respectively. Mixture solutions containing NPs with zeta potential above +20 mV or below −20 mV are physically stable [31]. High value of the particle size by DLS is related to the diameter of the synthesized Se NPs and the thick layer of stabilizing compounds which surrounded the Se NPs [32]. The PDI value of the synthesized Se NPs at attained optimum fabrication conditions revealed that the fabricated NPs were moderately polydisperse. The UV-Vis spectra of the formed Se NPs are presented in Figure 3B. As evidently detected in this figure, the formed Se NPs at achieved optimum conditions had λ_max of 319 nm. Particle size distribution of the fabricated Se NPs is also shown in Figure 3C.

A typical TEM image gained from the formed Se NPs solution at achieved optimum conditions is displayed in Figure 3D. The TEM image of the Se NPs synthesized revealed the existence of approximately spherical moderately polydisperse NPs with a particle size range of 40–60 nm. The obtained results were in agreement with findings of other investigators on green synthesis of Se NPs [6, 7, 33].

3.5 Antimicrobial activities of the fabricated Se NPs at obtained optimum synthesis conditions

The antibacterial activities of the synthesized Se NPs as compared to the PLE and Na₂SeO₃ solution on the growth of Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria are presented in Figure 4A and B, respectively. As can be seen in these figures, fabricated Se NPs indicated weak antibacterial activities against both bacteria strains. However, the diameter of the inhibition zones around the S. aureus (10 mm) was higher than that around E. coli (9 mm). The obtained results indicated that the PLE and Na₂SeO₃ solution had non bactericidal activities toward the studied bacteria strains. The obtained results were in line with findings of other studies which indicated moderate antibacterial activities of the Se NPs toward both Gram-positive and Gram-negative bacteria species [34, 35].

Figure 4:

Created zones of inhibition for Pelargonium zonale leaves extract (PLE) (1), Na₂SeO₃ (2) and selenium nanoparticles (Se NPs) (3) on the inoculated plates with Escherichia coli (A) and Staphylococcus aureus (B) incubated at 37°C for 24 h. Antifungal activity of the synthesized Se NPs at obtained optimum conditions against Penicillium digitatum (C) and Colletotrichum coccodes (D). Data are the mean value of three replicates.

The influences of the fabricated Se NPs and PLE on mycelial growth (mm) of P. digitatum and C. coccodes during the incubation period (7 days) are indicated in Figure 4C and D, respectively. As clearly detected in these figures, the synthesized Se NPs had high antifungal activities against both selected fungi strains. However, the fungicidal activity against P. digitatum was higher than against C. coccodes (Figure 4C and D). The results were in line with the findings of other researches which also revealed higher antifungal activities of the Se NPs [36, 37]. The attained results also specified that the PLE had low antifungal activities toward both studied and selected fungi.