Green Synthesis of Iron Nanoparticles from Spinach Leaf and Banana Peel Aqueous Extracts and Evaluation of Antibacterial Potential

Noida Institute of Engineering and Technology, Greater Noida, 201306 Uttar Pradesh, India Young Scientist of UPCST Scheme, Department of Biotechnology, Noida Institute of Engineering and Technology, Greater Noida, 201306 Uttar Pradesh, India Chemical and Biochemical Processing Division, ICAR-Central Institute for Research on Cotton Technology, Mumbai 400019, India Division of Physiology, Biochemistry and Post-Harvest Technology, ICAR-Central Plantation Crops Research Institute (CPCRI), Kasaragod, 671 124 Kerala, India Department of Biology, Faculty of Science, Sivas Cumhuriyet University, 58140 Sivas, Turkey Beekeeping Development Application and Research Center, Sivas Cumhuriyet University, 58140 Sivas, Turkey Facultad de Medicina, Universidad del Azuay, Cuenca, Ecuador


Introduction
Nanotechnology is the manipulation of matter through chemical and/or physical processes to produce materials with specific properties that can be used in multifaceted applications [1]. A nanoparticle (NP) is a microscopic particle with at least one dimension smaller than 100 nm in diameter [2,3]. NPs have unique optical, thermal, electrical, chemical, and physical properties [4,5], and as a result, they have a wide range of applications in medicine, chemistry, the environment, energy, agriculture, information and communication, heavy industry, and consumer goods [6][7][8]. Attrition and pyrolysis, for example, are traditional NP synthesis methods with drawbacks such as defective surface formation, low production rate, and high manufacturing costs with high energy requirements [9]. Toxic chemicals are used in chemical synthesis methods (e.g., chemical reduction and sol-gel technique), as well as the formation of hazardous by-products and contamination from precursor chemicals [10,11]. As a result, there is a growing need to develop clean, nontoxic, and environmentally friendly NP synthesis procedures. Biological synthesis protocols have several distinct advantages over traditional physical and chemical methods: (i) nontoxic chemicals are not used, making this a clean and environmentally friendly method [12][13][14][15]; (ii) the active biological component, such as an enzyme, acts as a reducing and capping agent, lowering the synthesis process' overall cost [15]; (iii) even in large-scale production, small NPs can be produced [16]; and (iv) external experimental conditions such as high energy and high pressure are unnecessary, resulting in significant energy savings [17]. For NP synthesis, a wide range of biological resources such as microorganisms (bacteria, yeast, fungi, algae, and viruses) and plants can be used [18]. Plantmediated biological synthesis of NPs has only recently gained importance [19], whereas microbe-based protocols have been developed from the combined research efforts of several authors. Plant extracts, as opposed to microbes, reduce metal ions in a shorter time. NPs can be made in a matter of minutes or hours, depending on the plant type and phytochemical concentration, whereas microorganismbased methods take longer [20]. The main disadvantage of microbe-mediated NP synthesis is the requirement for aseptic conditions, which requires trained personnel and raises the scaling-up cost [21]. Plants are preferred biological resources over microbes for all of these reasons and the ease of finding them in nature. Magnetic NPs have risen to prominence as a new class of important nanoparticles with unique properties such as superparamagnetism and high coercivity. When made with traditional methods, these NPs have some drawbacks. At such tiny levels, conventional rules of physics and chemistry become dormant and inactive. Iron NPs are submicrometer particles of iron metal. NPs are highly reactive because of their large surface area. Iron NPs retain the magnetic properties of the metal iron. Due to its large surface area, iron NPs find their application in medical imaging, MRI [22], and targeted drug delivery [23] and help in treating several neurodegenerative diseases such as Alzheimer's disease. Iron NPs are also used to scour organic pollutants in groundwater. Conventionally, NPs were synthesized by physical [24] and chemical [25] methods.
The green synthesis of iron NPs from plant extract is under exploitation and a better process than culturing cells of microorganisms. The green synthesis of iron NPs has gained interest for its nontoxicity [26] and is inexpensive to synthesize. It is an ecofriendly method as it does not use any harmful chemicals. Iron NPs are very efficient and promising to reform many environmental issues. In this research, spinach leaf extract and banana peel are considered for the production of iron NPs. Spinacia oleracea, commonly known as spinach, is a leafy green plant that belongs to the family of Amaranthaceae and is a superfood as it has lots of nutrients that are beneficial to humans [27]. It is an excellent source of iron and contains 0.81 g of iron. It also provides vitamins, proteins, and minerals. Banana is the most stand-out fruit in the world, and it belongs to the family Musaceae; banana peels are the outer covering of the banana and are also known as banana skin. It forms 18-30% of the whole banana [28]. Banana peels have diverse compounds which contain phenol that can act both as reductant and coating agents and can be used in the green synthesis approach [28]. It also includes a huge quantity of phenols that helps in the production of metallic NPs. As banana peels are not consumed and are thrown in the garbage as waste in heaps, it becomes necessary to find their applications to be used for the betterment of the environment. Promoting plant extracts over synthetic and chemical methods is very admirable as it gives way to green technology. It does not require any usage of high pressure or high temperature. The objective of this research paper is a green synthesis of iron NPs using spinach leaves and banana peel and their use as a potential antimicrobial agent against pathogenic bacteria as well as evaluating their toxicity in vivo on Drosophila melanogaster fruit fly.

Materials and Methods
2.1. Materials. Iron (III) chloride (ferrous chloride) was acquired from Central Drug House (P) Ltd. CDH, which was obtained and used without further decontamination. The ultrapurified and distilled deionized water (DDW) was obtained from the Institute's molecular biology laboratory. M. acuminata (banana) were collected from dumping sites, and Spinacia oleracea (spinach) was obtained from a local market.

Preparation of Aqueous
Extract. The spinach leaf extract (SLE) and banana peel extract (BPE) were used to make aqueous extracts. Spinach leaves were collected and thoroughly cleaned with distilled water to ensure that they were free of dirt. Using a mortar and pestle, about 20 g of fresh leaves were weighed and crushed. To make the plant extract, 20 g of leaves were boiled in 100 mL deionized water for 20 minutes and magnetically stirred at 60°C and then filtered through filter paper. The extract was kept in the fridge for later use. About 100 g of fresh banana peels was collected, washed thoroughly with distilled water, cut into fine pieces, and vacuum dried for the banana peel extract. The dry banana peel powder (8.3 g) was extracted with 125 mL DDW and stirred for 30 minutes at 60°C. Before filtering, the concentrate was allowed to cool. The samples were centrifuged for 30 minutes at 8000 rpm after extraction. Before use, the supernatant was collected, filtered, and stored at -20°C [29].
2.3. Biosynthesis of Iron Nanoparticles (FeNPs). SLE-FeNPs were made by dissolving the FeCl 3 in 40 mL of deionized water; a 0.1 M FeCl 3 solution was created. FeNPs were synthesized by mixing a 2 : 1 volume ratio of 0.1 M FeCl 3 solution with aqueous SLE at 60°C for 30 minutes with continuous magnetic stirring. As the Fe +3 ions decrease, the black color appears. The reaction mixture was prepared in 2 : 1 ratios, and the maximum absorption wavelengths of FeNPs in the UV/visible region were recorded. The BPE-FeNPs were made using a 2 : 1 volume ratio of 0.1 M FeCl 3 solution and aqueous BPE (supernatant at ambient temperature). After that, the mixture was hand-shaken for 1 minute and allowed to sit at room temperature for 1 hour. After 10 minutes, the solution color changed from transparent yellow to dark black, which was noted and recorded. The supernatant was poured out after centrifuging the mixture at 8000 rpm for 30 minutes. The black paste was redispersed in ethanol to remove any remaining biological molecules before being washed with ultrapurified water. To completely purify the NPs, the centrifugation and redispersion in ethanol and ultrapurified water process were repeated three times. The light black paste was then oven-dried overnight at 60°C, packed, and stored for characterization.

Characterization of Biosynthesized Iron Nanoparticles
2.4.1. UV-Vis Analysis. The changes in the reaction mixtures were observed visually and recorded. The sample was then spectrophotometrically examined in the UV-Vis range. The wavelength was scanned at 1 nm intervals from 200 to 700 nm. The instrument was turned on and given ten minutes to initialize. The sample solutions were placed in a quartz cuvette (1 cm), and the baseline was adjusted using ultrapurified water as the baseline. In the wavelength range of 200 to 700 nm, the spectra of extract, FeNPs, and ferric chloride solutions were obtained.

FTIR
Analysis. An FTIR system was used to make the measurements. The extract functional groups that may have played a role in the synthesis of iron microstructures were identified using FTIR scans of iron NPs and both extracts. The transmission mode range of 400-4000 cm -1 was used for the FTIR analysis.

Transmission Electron Microscopy (TEM).
A TEM microscope with a 200 keV acceleration voltage was used to create the TEM images. The FeNP sample solutions were sonicated for 20 seconds in analytical grade methanol before being mounted on a carbon-coated copper grid with a mesh size of 200. The cleaned Teflon block was placed face-up on the treated grid, and 10 L of the NP sample solution was poured onto the grid and dried under an infrared lamp before the grid was loaded onto the microscope for measure-ments. The average particle size was calculated and recorded using FeNP samples randomly selected from TEM images.

XRD
Analysis. An X-ray diffractometer was used to examine crystalline metallic iron NPs. The instrument used Cu Kα radiation at 45 kV and a monochromatic filter with a 20-80-degree wavelength range. Before being stacked in the cubes of the XRD equipment, the biosynthesized FeNPs were thoroughly dried to powder form.
2.5. Antibacterial Potential. The antibacterial potential of biogenic FeNPs derived from BPE and SLE was tested using the agar well diffusion method against two bacterial strains: Bacillus subtilis (MTTC 1133) and Escherichia coli (MTTC 62) obtained from the Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India, and the zone of inhibition was measured in millimeters using the agar well diffusion method described by Thiyagarajan et al. [7,[30][31][32]. Antibiotics such as chloramphenicol and gentamicin were used as positive controls in the study.
2.6. Impact of SLE-and BPE-Derived FeNPs on Drosophila melanogaster. Drosophila melanogaster was chosen in this investigation to identify if SLE-and BPE-derived green FeNPs had any toxic effects on life cycle traits. D. melanogaster were grown at 25°C on cornmeal, sucrose, agar-agar, dry yeast, live yeast, propionic acid, and sodium benzoate in a normal cornmeal feeding medium [7,33]. After being combined in distilled water, all food components were boiled in appropriate cooked semisolid medium. The SLE-and BPEderived green FeNP-treated food media were given to D. melanogaster at 10 mg/L concentrations, whereas the control sample was given only normal food medium as diet. Fecundity (the capacity to lay eggs), hatchability (the ability to bring forth from the egg), viability (larvae matured into adult fly), and development time (the time it takes for eggs to grow into adults) were all examined in D. melanogaster life cycle traits. In this investigation, ten replicates were carried out.
2.7. Statistical Analysis. The data in this paper were analysed using Statistica 7.0, an advanced analytics software system.

Results and Discussion
3.1. Visual Inspection of the Formation of the Iron Nanoparticles. The FeCl 3 solution and the aqueous extract solution from SLE and BPE were used to synthesize FeNPs, as shown in Figure 1. The color of the mixture (FeCl 3 +SLE) changed from light green to dark blackish-brown and (FeCl 3 +PBE) transparent yellow to dark black in 10 minutes at 60°C, demonstrating the synthesis of FeNPs from SLE and BPE (Figure 1). The surface plasmon vibrations of the FeNPs were excited, resulting in a dark color.

UV-Vis
Analysis. The UV-Vis spectrophotometric method was used to examine the NPs first. Visual and UVvisible spectroscopy was used to monitor the formation of iron NPs. The wavelength was scanned from 200 to 700 nm, and the spectra obtained two peaks at 240 and  [34]. Eucalyptus leaf extract was utilized to synthesize FeNPs; similar results on visual observations and a peak around 276 nm have been reported [35]. The optimal conditions for synthesizing iron NPs in this study were pH 5.0 and a temperature of 90°C when 10 mg of BPEs were mixed with 1 mM of FeCl 3 [35].
The peak denoting O-H stretching in phenol was decreased (phenolic compound was used for the synthesis of FeNPs).
3.3. FTIR Analysis. Different functional groups on BPE and SLE were shown using FTIR spectroscopy, and their role in iron NP synthesis was predicted (    Journal of Nanomaterials (v) A strong peak at 540 was attributed to nanoparticle vibrations confirming the production of nanoparticles (vi) A strong peak at 460 was attributed to nanoparticle vibrations confirming the production of nanoparticles 5 Journal of Nanomaterials decrease in peak intensity in the FeNP spectra indicated that these groups might be involved in the NP synthesis process. The intensity of the nitriles stretched at 2131 cm -1 in the BPE decreased, indicating that nitrile groups in the BPE concentrate may be involved in FeNP capping. The presence of the C-H stretch of aromatics at 685 cm -1 in the BPE spectrum and the absence of the same peak in the FeNP spectrum indicated that it was involved in NP synthesis. The strong peak at 639 cm -1 was attributed to FeNP vibrations, which further confirmed the NP synthesis and absence in the extract FTIR of BPE. When these findings were compared to prior studies [36,37], it was determined that FTIR from banana peel extract and FeNPs produced identical results. The possible biomolecules responsible for reducing FeNPs and capping of the bioreduced FeNPs synthesized by the SLE were identified using FTIR analysis. In the extract FTIR of SLE, a strong and prominent peak with O-H stretching in phenolic chemicals was found at 3418 cm -1 . In the FTIR of FeNPs, the shift towards higher frequencies indicated that polyphenol compounds in the extract may be connected to the O-H group in the reduction process. O-H stretching vibrations were also detected at 2917 cm -1 in the extract FTIR of SLE, and this O-H stretching vibration of phenol groups was responsible for the synthesis and stabilization of FeNPs. There was a stretching vibration of C=O groups in ketones, aldehydes, and carboxylic acids, and a strong peak was present at 1620 cm -1 in the extract, representing a higher amount of the carbonyl group compound, and this peak was extremely weak, only indicating the existence of a very little amount of the carbonyl compound in FTIR of FeNPs. The extract had a weak absorption peak at 1438 cm -1 , which could be responsible for the bioreduction of ferric chloride into FeNPs, and the extract works as a capping material. In the FTIR of FeNPs, absorption bands at 540 cm -1 and 460 cm -1 were found, which correspond to the Fe stretches in ferrous chloride and were attributed to nanoparticle vibrations, indicating the formation of nanoparticles. The SLE extracts did not include these two peaks.
According to FTIR analysis, the bioreduction of ferric chloride into iron NPs is due to the reduction of the spinach leaf extract by capping material [38,39]. When these findings were compared to previous reports [40][41][42], it was discovered that the hydroxyl, carboxyl, and amide groups in BPE might be involved in the NP synthesis process.

Transmission Electron Microscopy (TEM).
The transmission electron microscopy (TEM) method investigated the synthesized nanoparticles to determine their morphology and size [43]. The results are shown in Figure 3. According to TEM micrographs, the biologically synthesized iron NPs (SLE and BPE) are granular and dispersed throughout the grid. The particles are nanoscale, ranging from 20 to 50 nm for BPE-derived FeNPs to 10 to 70 nm for SLE-derived FeNPs. Using different plant extracts, similar iron NP results were obtained [44][45][46][47][48][49][50]. Despite being much larger than the 50 nm for FeNPs synthesized from tea extracts [34,[51][52][53] and Couroupita guianensis (cannonball tree), the 17 nm NPs were obtained [54], and this size range was more similar in size to NPs synthesized from Damask rose, with average sizes of 100 nm; Thymus vulgaris (garden thyme); and Urtica dioica (nettle leaf) [55]. Figure 4 shows that the synthesized FeNPs (BPE and SLE) were amorphous, with a weak characteristic peak of iron, indicating that the iron NPs were noncrystalline. In BPEderived FeNPs, crystal planes of (110), (200), and (112) were responsible for the intense peaks observed at Bragg reflection 2θ = 18:15°, 24.64°, and 35.78°, whereas in SLE-derived FeNPs, only one clear crystal plane of (112) and some fused crystal planes were responsible for the intense peaks observed at Bragg reflection 2θ = 47:55° (Figure 4). To confirm the presence of FeNPs, the Miller indices were used. Scherrer's formula was used to calculate the crystallite sizes: D = Kλ/β cos θ, where D is the crystallite size, K is a constant of 0.94, λ is the X-ray wavelength, β is the half-width  Journal of Nanomaterials of the most intense peak, and θ is the most intense peak's Bragg angle. The crystallite size was calculated using this equation to be 2.44 nm. The unassigned peaks on the XRD pattern could indicate the crystallization of the extract's bionatural phase. This is due to some banana peel extracts being adsorbed onto the formed iron NPs, which act as capping and stabilizing agents, preventing Fe from oxidation. The XRD results for Tridax procumbens leaf extract, used to make pure Fe 3 O 4 with a spinal structure, are quite similar [56]. FeNPs produced by reduction with green tea [57] and sorghum bran [29] extracts were also found to be amorphous, according to recent reports.

X-Ray Diffractometer Analysis (XRD). The XRD analysis in
3.6. Antibacterial Potential. The antibacterial potential of biogenic iron NPs synthesized from BPE and SLE extracts against B. subtilis and E. coli was investigated in this study. FeNPs are an excellent material for use as antibacterial agents against pathogenic bacterial species, according to the findings. Similar results were also reported for photosynthesis silver and gold NPs [53,[58][59][60][61][62], zinc NPs [63,64], iron NPs [65,66], and selenium NPs [67]. Even though NPs are widely used as antimicrobials, little is known about their mechanism. Antimicrobial mechanisms include interfering with cell wall synthesis, inhibiting protein synthesis, interfering with nucleic acid synthesis, and inhibiting a metabolic pathway [35,68,69]. It is also reported that these NPs penetrate the microbial membrane and create leakage of electrolytes, ultimately causing cell death. Nanomaterials can increase cell membrane permeability within the bacterial cell, interfere with DNA replication, denature bacterial proteins, and release silver ions [70].

In Vivo Toxicity Evaluation of SLE-and BPE-Derived
FeNPs on Drosophila melanogaster. The purpose of this study was to investigate the impact of SLE-and BPEderived FeNPs on Drosophila melanogaster life history characteristics as in vivo. For contrast, FeNPs derived from SLE and BPE were mixed in food medium of Drosophila melanogaster and some flies were breaded on without nanoparticlemixed food media as a regular diet (control sample). FeNPs were fed to flies at 10 mg/L concentrations up to the 1st generation, and 7-day-old flies from the 2nd generation were chosen for this investigation. Fecundity was measured by  7 Journal of Nanomaterials counting the eggs laid by 7-day-old flies within the 24th hour. The fecundity/fly data clearly demonstrates that the SLE-and BPE-derived FeNPs and control sample had no significant variation in fecundity. The control flies lay 63:70 ± 0:67 eggs/fly/day on average, whereas SLE-and BPE-derived FeNP-fed flies laid 62:98 ± 2:78 and 62:99 ± 2:41 eggs/fly/day, respectively ( Figure 5). The obtained results are suggesting that there is no significant difference between normal food and SLE-and BPE-derived FeNP food fed by flies. For hatchability testing, we placed 100 eggs of normal food and FeNP-fed flies in a line pattern on an ethanol-soaked black paper strip (contamination free) on the slant surface of food medium (10 × 10 eggs/vial). Furthermore, the eggs of normal food-fed flies hatched 100% of the time, but those of the SLE-and BPE-derived FeNPs hatched 99% of the time ( Figure 6). These findings again confirm the nontoxic nature of SLE-and BPE-derived FeNPs. Furthermore, the viability (larvae developed into adult fly) of both types of food medium-fed flies was tested. The viability results showed that 100% of adult's emergence in both normal-and FeNP-fed flies (Figure 7). These findings again confirm the nontoxicity of nanoparticles. In continuation, we further investigate any effect of nanoparticles on the duration of development times of D. melanogaster fly eggs to adults in hours at 25°C. The time it took for con-trol flies to grow eggs to adulthood was 251 hours, whereas SLE-and BPE-derived FeNP-treated flies take one hour extra, i.e., 252 hours ( Figure 8). This finding again confirms the nontoxic nature of FeNPs. Similar findings were found by other researchers [71][72][73].

Conclusion
In this research, spinach leaf extract and banana peel are considered for the production of iron NPs. The synthesized NPs were confirmed using visual screening, UV-Vis analysis, FTIR analysis, and XRD analysis. FeNPs were also investigated using size and morphology using TEM. The synthesized green NPs showed antimicrobial activity against the two most common food-borne bacteria, i.e., B. subtilis and E. coli. Green FeNP impact as in vivo toxicity on D. melanogaster was evaluated on fecundity, hatchability, viability, and DOD traits after feeding FeNP concentration of 10 mg/L and compared to the normal diet-fed flies (control sample). After evaluating all of the life history traits, we concluded that green-synthesized FeNPs are nontoxic in nature. Overall, FeNPs show a considerable antibacterial potential and might be utilized as antibacterial agents against pathogenic bacteria while maintaining nontoxicity to humans.    Figure 5: Pattern of egg laying capability/fly/day in D. melanogaster fed with SLE-and PBE-derived FeNPs and normal diet food (control sample). 8 Journal of Nanomaterials

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.