Iron nanoparticles (INPs) due to their unique physicochemical and biological characteristics are one of the most applied nanostructures in the science and technology. These nanoparticles are now employed in a variety of applications ranging from environmental remediation to biomedicine and pharmaceutical sciences [1, 2, 3, 4, 5, 6, 7, 8, 9, 10]. Over the last decade, several chemical and physical techniques were developed for the synthesis of INPs. Due to intrinsic limitations and problems of these techniques, several attempts have been made to find out a sustainable approach for the synthesis of INPs [11,12]. Green synthesis has emerged as a promising approach in this regard. Due to employing natural compounds from plants or microorganisms, this method has significant advantages over the physical and chemical procedures [13, 14, 15, 16, 17, 18]. However, there are still some difficulties with employing microorganisms for the synthesis of nanostructures [19,20].
Thanks to plant mediated green synthesis, fabrication of nanostructures is now possible in a facile, economic, and environmental friendly manner without any elaborate and complicated procedures [6,16,17,21, 22,23]. Plant extract mediated synthesis owing to simple processing and cheap raw materials provides a suitable opportunity for biosynthesis of nontoxic and biocompatible nanoparticles . Plant extracts contain phytochemicals such as polyphenols, flavonoids, reducing sugars, proteins, carbohydrates, nitrogen bases, and amino acids that can act as reducing and capping agents for converting metal ions to metal nanoparticles and stabilizing them [6,16,17,22,23]. So far, variety of plants such as green tea, Syzygium cumini, Eucalyptus, Cupressus sempervirens, Hordeum vulgare, stinging nettle (Urtica dioica), Mediterranean cypress (Cupressus sempervirens), and Rumex acetosa have been used to produce INPs [1,6,8,11,16,21,25,26]. However, there are limited data available about the effective factors in the green synthesis of nanoparticles and the optimal reaction conditions [22,23,25,27,28]. Meanwhile, knowledge about the effective parameters in the reaction process and optimal reaction condition is the critical point to achieve higher productivity. These data can be achieved successfully by using a design of experiment software which less employed for the plant mediated synthesis of nanoparticles.
Pinus eldarica (also known as Pinus brutia var. eldarica, calabrian pine, mondell pine, and Eldarian pine) is a very famous tree of the Pinaceae family with evergreen foliage. It height can be about 9-25 m and usually found with brown or green cones. Pine tree adapted to survive in wide range of climates, so can be found in many regions of world. Furthermore, it is native to Iran, Iraq, Armenia, Azerbaijan, and Georgia. This tree has gained medical applications for treating hyperlipidemia, atherosclerosis, nerve malfunction, neuralgic disorders and rheumatism. The needle leaves of the tree are source of natural compounds such as polyphenols, antioxidants, proanthocyanidins, luteins, and beta-carotene that are key elements in the green synthesis of metal nanoparticles [29, 30, 31, 32]. However, bark extract of this tree was just used for the green synthesis of silver nanoparticles . Therefore, in the present study the potential application of pine tree needles extract for production of INPs were investigated. The effective factors in the synthesis reaction and optimal reaction condition for highest rate of nanoparticles production were also demonstrated by using design of experiments (DoE) and response surface methodology (RSM), respectively [33, 34, 35].
2 Materials and methods
Pinus eldarica needle leaves were collected from garden campus of the Fasa university of Medical sciences (Fasa, Fars province, Iran). Ferric chloride (FeCl3·6H2O) was purchased from Merck Chemicals (Darmstadt, Hessen, Germany). Millipore water (Millipore Corp., Bedford, MA, USA, conductivity range of 0.055-0.294 μS/cm) was used for all the experiments.
2.2 Leaf extract preparation
Needle leaves were rinsed with deionized water in order to remove possible dusts and mud particles. The needles were dried at room temperature under dust free condition for about two weeks and then powdered with household miller and passed through a particular mesh sieve. Five gram of leaves powder was mixed with 100 mL deionized water in a 250 mL round bottom flask [36,37]. The mixture was heated up to boiling by using a heater mantel under reflux. After 15 min boiling resultant extract was cooled to room temperature and filtered through a Whatman filter paper (Reeve angel, Grade 201) to remove the sludge. The filtrate was centrifuged (5000 rpm, 12 min) to remove fine leaf particles. A clear supernatant was obtained and used for further experiments.
2.3 Experimental design
The MODDE software version 9 (Umetrics, Sweden) as a tool for statistical design of experiment was employed with two predominant stages in order to optimize the factors involved in synthesis process for maximizing the production of INPs. First stage was carried out to specify the important factors with significant effects on the amount of synthesized nanoparticles. A fractional factorial design was utilized to screen the effect of four variables namely plant extract quantity, iron precursor concentration, reaction temperature, and reaction time.
Second stage was carried out to achieve an empirical model to specify the optimum concentrations of selected efficient factors. In this step, central composite face (CCF) design and RSM were used to optimize the effective factors selected from screening stage. The variables and their range of values are depicted in Table 1.
2.4 Synthesis of INPs
For INPs fabrication, leaf extract (9 mL) and deionized water (750 μL) were added to a 50 mL round bottom flask and the mixture was stirred vigorously at room temperature. Consequently, 250 μL iron precursor (FeCl3·6H2O, 1 M) was added to the flask and reaction was followed for 30 min at room temperature. The reaction product was centrifuged and resulting black precipitate was washed with deionized water for three times to remove any unreacted solutes and phytochemicals. Finally, the black pellet was oven dried at 50°C for 48 h.
2.5 Characterization of INPs
Physicochemical properties of the synthesized nanoparticles through optimum experiment were evaluated by material analysis techniques. Transmission electron microscope (TEM, Zeiss, EM900, HT-100 KV) studies were carried out to identify the morphology and size of the prepared particles [38, 39, 40]. Analyses were done without any sample preparation. A drop of INPs suspension was dripped on a cupper grid and dried at room temperature. Particle size analysis was conducted by using an image analysis software (ImageJ version 1.47v, developed by NIH, http://imagejnihgov/ij/) Fourier-transform infrared (FTIR) spectroscopy (Perkin Elmer Spectrum One) analysis was done using KBr pellets for characterizing the synthesized INPs and also for understanding the existence of surface functional groups on the nanoparticles. The IR absorption analysis was done from 4500 cm-1 to 400 cm-1 [13,41]. The crystallographic analysis of INPs was performed by X-ray powder diffractometer (Siemens D5000). Resulting XRD pattern was evaluated by X’Pert High Score version 1.0d (PAN analytical B.V., Almelo, the Netherlands) [42,43]. The magnetic properties of nanoparticles and values of magnetic parameters such as saturation magnetization (Ms), coercive force (Hc) and magnetic remanence (Mr) were characterized using
vibrating sample magnetometer (VSM) (American-Lake Shore Cryotronics company, 7407 Model) with increasing magnetic field up to 19 kOe and field sweeping from −19 to +19 kOe [44,45]. Thermo gravimetric analysis (TGA) and derivative thermo gravimetric (DTG) analyses were done by a TGA, 209 F3 Tarsus. This technique was done to determine the thermal stability, presence, and the quantification of organic compounds from Pinus eldarica leaf extract in the final INPs product. TGA thermo grams were recorded for 10.8 mg of powder sample at a heating rate of 10°C/min in the temperature range of 30 to 600°C under air atmosphere. Energy-dispersive X-ray spectrometer (EDS) (Rontec analyser, Germany) was employed to determine the elemental composition of nanoparticles.
3 Results and discussion
3.1 Effective parameters in the synthesis reaction
Reaction time, iron precursor concentration, leaf extract quantity, and reaction temperature were selected to investigate their effect on INPs production. DoE was carried out based on these four parameters and the amount of prepared nanoparticles was chosen as the response . The results of initial screening for effective parameters in the synthesis reaction are depicted in Table 1. Table 2 illustrates the statistical analysis and variables coefficient in order to evaluate the main effects of single parameters and their interactions on the weight
of produced nanoparticles . These results indicate that two factors namely iron precursor concentration and amount of leaf extract had significant positive effect on nanoparticles production (P-value less than 0.05 indicates that the factor is significant). These findings are in close agreement with previous report for the synthesis of iron nanoparticles using green tea extract. Where, FeCl3-to-green tea extract ratio found to be the effective parameter in the amount of nanoparticles formation .
3.2 Optimization of nanoparticles production
Determination of optimum amount for iron precursor and leaf extract to enhance the INPs production was carried out by using the central composite face (CCF) design as shown in Table 3. The ANOVA test (Table 4) indicates a good fitness of the model, because of the high F value of 160.421 and a very low probability value. The linear regression coefficient R2 = 0.994 and the adjusted
determination coefficient R2 of 0.988 for the model demonstrate the accuracy of the model.
The response surface plots are shown in Figure 1. Analysis of the plots demonstrates that the maximum INPs is achievable when the final concentration of iron precursor and leaf extract are set as 25-45 mM and 8.5-9 mL, respectively. As can be seen in Figure 1, formation of nanoparticles decreases when the values of ferric chloride and leaf extract are set to be more than 50 mM and less than 8.5 mL, respectively. The highest productivity was predicted by the model to be 0.015 g per reaction (1.5 mg per mL of the reaction mixture) with optimum values of ferric chloride (25 mM) and leaf extract (9 mL). Consequently, the validation experiment was carried out under the optimized condition which resulted in the production of 0.015 g mL-1 INPs.
Although vast investigations have been done in regards to green synthesis of nanoparticles, there are very rare investigations for the optimization of nanoparticles fabrication . Therefore, in the present study, the effects of reaction parameters on the fabrication of INPs were investigated and the reaction condition was optimized to achieve highest amount of nanoparticles in a constant reaction volume. Reaction condition has an immense influence on the physicochemical characteristics of the green synthesized nanoparticles [17,22,23]. So, investigations must be done to determine the impacts of synthesis conditions and involved factors on the reactivity, morphology, and other properties of the synthesized nanoparticles. For instance, the influences of various parameters such as the ratio of iron precursor (Fe2+) and leaf extract (tea extract), reaction temperature, and reaction pH were systematically investigated and have been reported by Lanlan Huang . It was revealed that the reactivity of the prepared nanoparticles was highly depended to the synthesis conditions and the reduction rate is kinetically dominated by these factors.
3.3 Characterization of INPs
Physicochemical properties of the prepared INPs under optimal reaction condition were evaluated. Figure 2a is illustrating TEM micrograph of the produced nanoparticles and corresponding size distribution histogram is depicted in Figure 2b The obtained particles were dominantly spherical in shape with 8-34 nm in diameter with the mean particle size of 18 nm. Green synthesized INPs with similar particle size distribution were also reported by using a red wine, pomegranate, mulberry, and cherry leaf extract [12,48]. Based on the TEM micrographs, INPs were heavily surrounded by biological components from leaf extract (Figure S1), therefore, the resulting structure is a microstructure which composed of INPs and phytochemical compounds. Similar phenomenon is reported for the green synthesis of metal nanoparticles using a variety of plant extracts [16,23,49,50]. In some cases it has been confirmed that this biologic matrix is mainly composed of carbohydrates from leaf extract [23,49].
FTIR analysis was conducted to determine the chemical components which were responsible for stabilizing and capping of INPs. FTIR spectrum of the prepared INPs is presented in Figure 3. The OH groups induce a broad indicative peak which appeared at 3420.97 cm−1 . The carbonyl group stretching vibration can be seen at 1616.05 cm−1 and the peak at 1069.05 cm–1 is due to C–O bonds . The peaks of aliphatic C–H appeared around 2918 cm–1 [38, 39, 40]. The FTIR results indicate that prepared INPs are capped with biologic compounds which were derived from Pinus eldarica leaf extract [13,22,51]. These finding are in agreement with TEM micrograph which shows that INPs are surrounded by a biologic matrix. The Fe-O characteristic peaks of iron oxide nanoparticles commonly appear at about 640 cm−1 and 450 cm–1 [42,43, 44 52,53]. These bonds were not observed in the FTIR spectrum of the prepared particles, which is
indicative of zero-valent INPs. Similar spectra were also reported for chemically or plant mediated synthesized zero-valent INPs [16,54]. Oxidation of the exposed iron atom to iron oxide/hydroxide results in a core-shell structure that Fe0 forming the core which is surrounded by an iron oxide shell [11,55,56]. The thin iron oxide shell lead to the appearance of Fe-O absorption bonds in the FTIR spectra, but in lower intensity than that usually observed for iron oxide nanoparticles . Absence of these peaks in FTIR spectra of the prepared particles indicated the absence of iron oxide shell. Organic capping from leaf extract seems to protect the surface of zero-valent iron atoms from oxidation.
The XRD pattern of the INPs is presented in Figure 4. XRD analysis confirmed that no distinctive peaks were present on the spectra. This indicated that the INPs were not crystalline in nature and were amorphous structures. Fabrication of amorphous zero-valent INPs was also reported by using leaf extract of different plants such as eucalyptus, mulberry, pomegranate, and cherry . The broad shoulder peak from 20° to 30° of 2θ values was proposed to be due to the presence of organic component from leaf extract which are responsible for capping and stabilizing nanoparticles . Some researchers also considered a tinny peak appearing at around 2θ of 44-45° as indicative peak for zero-valent INPs [11,58,59]. Production of other amorphous nanostructures was also reported for the green synthesized INPs. For instance, green synthesis of INPs by using extracts of Eucalyptus tereticornis, Melaleuca nesophila, and Rosemarinus officinalis was reported to result in amorphous iron-polyphenol nanoparticles [4,60].
TGA and DTG curves of the produced INPs are provided in Figure 5, demonstrating a significant weight loss processes. The initial weight loss below 263.7°C should be attributed to the evaporation of residual and adsorbed water. The weight loss above 263.7°C corresponds to the decomposition of the capping materials [16,61]. Decomposition of organic materials makes the main peaks at 373.9°C and 415.9°C in the DTG curve [16,62]. There was no weight loss at above 440°C. TGA analysis showed that the weight percentage of biologic coating in the product is about 74.43% . Since now, the weight percentage of organic compounds in the green synthesized nanoparticles was reported to be up to 60% . So, pine tree needles can be introduced as a biologic source for reducing and capping agents to provide nanoparticles with heavy coating. This unique feature can be a great advantage for future applications.
The magnetization curve as a function of the applied magnetic field at room temperature is shown in Figure 6. The sample showed no hysteresis and the magnetization curve was completely reversible that exhibits a superparamagnetic behavior of nanoparticles. The saturation magnetization value of the synthesized particles was found to be 33 memu/g. The very low saturation value is possibly due to the diamagnetic properties of biologic capping material and amorphous state of the prepared INPs . Previous investigations have indicated that increase in the intensity of biological coating resulted in significant reduction of saturation magnetization [52,53]. Also, Santra et al., reported that the magnetization of the uncoated magnetic nanoparticles (1.3 emu/g) is higher than coated nanoparticles (0.5 emu/g) . In the other experiment, the saturation magnetization values of the iron nanoparticles which were synthesized by using Plectranthus amboinicus leaf extract was reported to be 1.25 emu/g . Low saturation magnetization value of the prepared nanoparticles in contrast to previous report can be in agreement with TGA results which indicate a heavy biologic coating.
Localized elemental information of the iron nanoparticles was determined by EDS and results are depicted as Figure 7. There are intense peaks of C, O, Ca, and Fe in the spectrum and quantitative data indicated that the atomic percentages of the prepared material were 53.08% C, 44.76% O, 0.30% Ca, and 1.87% Fe. The high percentage of carbon and oxygen elements originated mainly from the polyphenol groups and other C, O-containing compounds in Pinus eldarica extract. Also, Ca element is attributed to the plant extract. As can be observed from the TEM graph, the produced iron nanoparticles were heavily surrounded by biologic compounds from leaf extract, which is accountable for the low iron content in the sample. These results are in agreement with previous findings for the green synthesized INPs [57,58,66]. But, in comparison, the low Fe weight percent is due to heavy biological coating that is provided by Pinus eldarica extract . This value (1.87%) for iron content is one of the least atomic percentage that already were reported.
Phytochemical compounds from pine tree (Pinus eldarica) needles were successfully utilized as a natural source of reducing and capping agents for the green synthesis of zero valent INPs. Analysis based on the statistical design of experiment indicated that quantity of leaf extract and concentration of iron precursor are the most effective parameters in the green synthesis reaction. Reaction time and temperature showed no significant effects on the nanoparticles fabrication. The prepared particles were intensely surrounded by biologic compounds from leaf extract. Extraordinary capability of Pinus eldarica extract for heavily entrapment of nanoparticles can be introduced as a unique property for the green synthesis of nanoparticles. These findings highlighted the interesting potential of the pine tree needles for application in industrial scale green synthesis of INPs.
This study was financially supported by a grant (grant No. 96006) from the Fasa University of Medical Sciences.
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About the article
Published Online: 2019-08-18
Published in Print: 2019-01-28
Citation Information: Green Processing and Synthesis, Volume 8, Issue 1, Pages 846–855, ISSN (Online) 2191-9550, ISSN (Print) 2191-9542, DOI: https://doi.org/10.1515/gps-2019-0055.
© 2019 Kheshtzar et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 Public License. BY 4.0