Walnut leaf extract-based green synthesis of selenium nanoparticles via microwave irradiation and their characteristics assessment

Selenium nanoparticles (Se NPs) have predominant characteristics compared to that in their bulk usage due to their high surface-to-volume ratio. The walnut (Juglans regia L.) leaf extract containing different bioactive compounds that act as reductant and stabilizing agents has been used for the green synthesis of the Se NPs. Influences of two synthetic variables, namely, the amount of selenium salt solution ranging from 15 to 25 mL and the amount of walnut leaf extract ranging from 1 to 5 mL, on broad emission peak (λmax) and absorbance of colloidal solutions having Se NPs were evaluated via the response surface methodology. Obtained results indicated that using microwave radiation (800W for 4min) and 5mL of walnut leaf extract and 15 mL of selenium salt solution, Se NPs with λmax, absorbance, particle size, polydispersity index, and zeta potential values of 375 nm, 3.65% absorbance unit (a.u.), 208 nm, 0.206, and −24.7 mV were synthesized, which had high bactericidal activity toward Escherichia coli and Staphylococcus aureus. The transmission electron microscopy analysis also indicated that spherical and monodispersed Se NPs with a mean particle size of 150 nm were formed using the walnut leaf extract.


Introduction
Selenium, a nonmetal trace element, has gained more attention in pharmaceuticals and medicine areas due to its unique properties such as lower toxicity, higher antioxidant activity, anticarcinogenic, muscle functioning, antimicrobial activities, and progressive effect on thyroid metabolism ). Selenium is found in the body of humans and animals in the form of seleno proteins, which plays a key role in enzymatic reactions and the synthesis of seleno enzymes such as glutathione peroxidase and thioredoxin reductase (Fairweather-Tait et al. 2011).
Selenium in nanoscale has predominant characteristics compared to that in its bulk usage. These distinctive properties are related to the high surface-tovolume ratio and surface energy of the selenium nanoparticles (Se NPs) (Prasad et al. 2013). Green fabrication of metal and metal oxide nanoparticles (NPs) using different parts of plants (e.g., flower, leaf, stem and root) is a novel branch of nanobiotechnology. Infact, natural existed bioreductants and stabilizers in the plans, such as carbohydrates, alkaloids, proteins and enzymes, phenolic acids, flavonoids and polyphenols have key roles in the synthesis of NPs (Mohammadlou et al. 2016;Eshghi et al. 2018). However, compared to the physicochemical synthesis methods to fabricate inorganic NPs, green processes are time consuming, which can be fixed by fast heating methods such as hydrothermal and microwave radiation (Torabfam and Jafarizadeh-Malmiri 2017; Ghanbari et al. 2018). In fact, moving of the metal NPs is accelerated in the microwave electric field because they carry electrical charges (Eskandari-Nojedehi et al. 2016).
Walnut belongs to Juglandaceae family (Juglans genus), and Persian walnut (Juglans regia L.) is one of its more famous members (Hashemi-Seyede and Rafati 2015; Eshghi et al. 2018). It is believed that its leaf is the main source of healthcare bioactive components that are widely used in the treatment of several diseases such as antihemorrhoidal, antidiarrheic, antimicrobial (i.e., antifungal and antibacterial), and antiscrofulous (Pereira et al. 2007). The main bioactive compounds of the walnut leaf contain important phenolic components such as flavonoids and naphthoquinones. 3-Caffeoylquinic, 3-p-coumaroylquinic, and 4-p-coumaroylquinic acids are the major hydroxycinnamic acids that existed in the walnut leaf, and its main flavonoids are quercetin 3-galactoside, quercetin 3-arabinoside, quercetin 3-xyloside, quercetin 3-rhamnoside, quercetin 3-pentoside, and kaempferol 3-pentoside derivatives Due to the advantages of response surface methodology (RSM) over classical one-variable-at-a-time optimization, such as the generation of large amounts of information from a small number of experiments and the possibility of evaluating the interaction effect between the variables on the response, it is a suitable procedure to assess the relationships between the synthesized variables and the response variables of the formed NPs (Anarjan et al. 2014). RSM has several benefits compared to other statistical methods to reduce the number of experiment runs with adequate replications at the center point Therefore, the main objectives of the present study were to (i) assess the potent uses of the walnut leaf extract in the green synthesis of Se NPs, (ii) model the fabrication process of Se NPs and optimize the synthesized parameters, namely, amounts of selenium salt solution and walnut leaf extract, based on RSM, to form Se NPs with minimum λ max , particle size and polydispersity index (PDI), and maximum absorbance and zeta potential values, and (iii) measure the physicochemical and antibacterial activities of the fabricated Se NPs using the obtained optimum values of the synthesized parameters under microwave radiation.

Materials
Walnut leaves were collected from a local garden in Tabriz, Iran. Na 2 SeO 3 , as selenium salt, was obtained from Merck Company (Darmstadt, Germany). Escherichia coli (PTCC 1270) and Staphylococcus aureus (PTCC 1112), as indexes of Gram negative, were obtained from microbial Persian Type Culture Collection (PTCC, Tehran, Iran). Nutrient agar, as culture media, was provided by Biolife Co. (Milan, Italy).

Walnut leaf extraction and Se NP formation
Walnut leaves were washed with tap water, dried, and powdered using domestic miller. One gram of the prepared powder was then added into 100 mL of the boiling water for 5 min. Finally, the mixture solution was cooled and filtered using Whatman No. 1 filter paper and subjected into a vacuum-Buchner funnel, and the filtrate was then kept in a bottle at dark (Fardsadegh and Jafarizadeh-Malmiri 2019). The previous study indicated that 10

Physicochemical analysis
The important functional groups in the walnut leaf were detected via Fourier transform infrared (FT-IR) spectroscopy using a Bruker Tensor 27 spectrometer (Bruker Co., Karlsruhe, Germany) in the 4,000-400 cm −1 region and using KBr pellets (Eshghi et al. 2018). The formation of Se NPs could be easily confirmed using a Jenway UV-Vis spectrophotometer 6705 (Cole-Parmer Co., Staffordshire, UK) due to the formation of NPs surface plasmon resonance (SPR) signal that can be revealed as a broad emission peak (λ max ) in the wavelength ranging from 270 to 400 nm Absorbance (% a.u.) of the samples at λ max could be related to the fabricated Se NP concentration. A dynamic light scattering (DLS) analyzer (Zetasizer Nano ZS; Malvern instruments, Worcestershire, UK) was employed to evaluate the PDI, particle size, particle size distribution, and zeta potential of the Se NPs. Transmission electron microscopy (TEM CM120, Philips, Amsterdam, Netherlands) with an acceleration voltage of 120 kV was employed to assess the morphology of the synthesized NPs (Ahmadi et al. 2019).

Antibacterial assay
The bactericidal activity of the made Se NPs toward both S. aureus and E. coli, Gram-positive and Gram-negative bacterial strains, respectively, was estimated via the well diffusion method as described by Eshghi et al. (2018). In fact, the bacterial species were inoculated on an NA media plate (90 mL in diameter) for 18-24 h at 37°C. A three-to five-well isolated colonies of the same morphological type were mixed in 10-15 mL of sterile normal saline solution. The bacterial suspension density was adjusted to 0.5 McFarland standard. This is equivalent to 1.5 × 10 8 colony forming units of bacteria in 1 mL of prepared inoculums. A total of 0.1 mL of that amount was spread on the surface of the solid NA plates, and a hole, with a diameter of 5 mm, was made in the solid agar. Then, 10 µL of the produced Se NPs solution was poured into the well and incubated at 37°C for 24 h. The antibacterial activity of the synthesized silver NPs was correlated with the diameter of the clear zone around the holes.

Design of experiments, optimization, and statistical analysis
Design of experiments, based on the central composite design (CCD), and RSM have been employed to assess the influences of the synthesized variables, including amounts of walnut leaf extract (X 1 , 1-5 mL) and amounts of Na 2 SeO 3 solution (X 2 , 15-25 mL), on the absorbance value (Y, % a.u.) of the samples having made Se NPs. According to CCD, 13 experiments were performed (Table 1), with all these runs completed at 1 day (one block)   They also found that proteins and enzymes existed in the walnut leaf extract by observing the amide group (1637.48 cm −1 ), as shown in Figure 1, that was related to these biomolecules. In fact, proteins could excellently participate in the formation of Se NPs as a stabilizing agent.

Se NP synthesis by the walnut leaf extract
Literature have shown that the made Se NPs had λ max at UV-Vis wavelength ranging from 270 to 400 nm due to their SPR. SPR also caused color changes from yellow (before exposure into the microwave) to pale red (after accomplishing the Se NP synthesis) in the solution mixture containing walnut leaf extract and selenium salt. Figure 2 shows the color changes in the samples without and with Se NPs. As presented in Table 1

Model generation
According to the experimental values obtained for different runs, a second-order model was generated to show absorbance variation of colloidal solutions containing fabricated Se NPs as a function of the synthetic parameters, namely, amounts of the selenium salt and walnut leaf extract. Higher values of the absorbance can be directly interrelated to the concentration of fabricated Se NPs in the colloidal solution. Table 2 presents regression coefficients for all terms of the fitted equation with its R 2 (0.9957), R 2 -adj (0.9903), and lack-of-fit p-value (0.075). The higher values for R 2 and R 2 -adj and

Effects of selenium salt and walnut leaf extract on formation of Se NPs
The effects of two independent and synthetic factors on the response variable (absorbance of the colloidal solutions containing fabricated Se NPs) are exhibited in Figure 3. As could be observed in this figure, at lower amounts of selenium salt, by increasing the concentration of walnut leaf extract, the absorbance increased. It seems that at lower amounts of selenium salt by increasing the amounts of walnut leaf extract, the concentration of bioreductants, presented in the extract, increased, which can reduce more amounts of the selenium ions into the Se NPs reported that by increasing the amounts of the Aloe vera leaf extract and mushroom extract, at lower amounts of silver and gold salts, the concentration of the formed silver and gold NPs increased, respectively. The obtained results also illustrated that at higher amounts of selenium salt, by increasing the amounts of walnut leaf extract, insignificant changes were observed in the absorbance of the colloidal solutions. This result revealed that the interaction of two synthetic parameters had a significant (p < 0.05) effect on the absorbance of the colloidal solutions having made Se NPs. This result was in line with the statistical analysis ( Table 3), which illustrated that the interaction term had a significant effect on the absorbance. To better visualize the optimum amounts of the selenium salt and walnut leaf extract, for the formation of Se NPs with higher absorbance (concentration), a two-dimensional contour plot for absorbance of the colloidal solutions containing Se NPs as a function of the synthetic parameters is shown in Figure 4. As this figure indicates, high values for the absorbance of the solutions were achieved at maximum and minimum amounts of the walnut leaf extract and selenium salt, respectively. Therefore, optimum synthetic conditions for the formation of Se NPs with high concentration would be obtained using 5 mL of walnut leaf extract and 15 mL of selenium salt. The generated model was predicted 3.65% a.u. for the absorbance of the colloidal solution prepared under these optimized conditions. Using the achieved optimized synthetic conditions, three additional experiments were accomplished, and the statistical analysis revealed an insignificant difference between the experimental and predicted values of the absorbance. The obtained result indicated the suitability of the fitted model to predict the absorbance value of the colloidal solutions containing formed Se NPs, which are prepared in the defined ranges for the amounts of walnut leaf extract and selenium salt. Fardsadegh et al.

Physicochemical characteristics of the formed Se NPs using optimal synthesized conditions
Se NPs were fabricated under obtained optimum processing conditions and their physicochemical attributes were evaluated. The UV-Vis spectrum of the made Se NPs using optimal conditions is presented in Figure 5. As this figure shows, the obtained λ max for the solution containing Se NPs was achieved at 375 nm. The obtained data by DLS also revealed that the formed Se NPs under   The TEM image of the formed Se NPs revealed that spherical and monodispersed Se NPs with a mean particle size of 150 nm were synthesized using the walnut leaf extract (Figure 7).

Antibacterial activity of the formed Se NPs using optimum synthesized conditions
The bactericidal activity of the fabricated Se NPs using optimal synthetic conditions toward S. aureus (A) and E. coli (B), with the diameter of created clear zones, is shown in Figure 8.    Gram-positive bacteria. The achieved results were consistent with the finding of Fardsadegh et al. (2019). They also reported that the bactericidal activity of the fabricated Se NPs using the Pelargonium zonale leaf extract on the S. aureus was higher than that on E. coli.

Conclusions
The main achieved results of the present work revealed the high green synthetic potential of the walnut leaf extract to fabricate Se NPs with high concentration and antibacterial activity and suitable physicochemical characteristics. Furthermore, microwave radiation effectively accelerated the fabrication of Se NPs within minimum processing time. On the other hand, RSM could be excellently modeled, optimized, and predicted the fabrication process parameters to form Se NPs with minimum particle size and PDI, and maximum stability, as manifested in the high zeta potential value. Synthesized Se NPs using the walnut leaf extract can be utilized in food and pharmaceutical supplements. However, the developed green fabrication technique in the present study can be easily employed in the synthesis of other useful metal and metal oxide NPs.

Conflict of interest:
The authors declare no conflict of interest.