Green Synthesis And Characterization Of Iron Nanoparticles Synthesized From Aqueous Leaf Extract Of Vitex Leucoxylon And Its Biomedical Applications

Abstract: The cold extraction method was used to obtain the aqueous extract of Vitex leucoxylon leaves in a ratio of 1:10. Iron nanoparticles (FeNPs) were synthesized using aqueous leaf extract of V. leucoxylon as a reducing agent. The phytomedicine approach was used to make FeNPs by mixing 1 mL of plant extract with 1 mM of ferric sulfate. Scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), Ultraviolet–visible spectroscopy (UV-Vis), and energy-dispersive X-ray spectroscopy were used to examine the synthesized FeNPs. The reducing reaction was shown by a change in the color of the solution, and the formation of black color confirms that FeNPs have been formed. The greatest absorption peak (max) was found at 395 nm in UV-Vis spectral analysis. The FTIR spectra of V. leucoxylon aqueous leaf extract showed shifts in some peaks, namely 923.96 cm−1 and 1709.89 cm−1, with functional groups carboxylic acids, unsaturated aldehydes, and ketones, which were lacking in the FTIR spectra of FeNPs and are responsible for FeNPs formation. FeNPs with diameters between 45 and 100 nm were observed in SEM images. The creation of FeNPs was confirmed by EDX, which shows a strong signal in the metallic iron region at 6–8 Kev. XRD revealed a crystalline nature and an average diameter of 136.43 nm. Antioxidant, anti-inflammatory, cytotoxic, and wound healing in vitro tests reported significant activity of the FeNPs. The cumulative findings of the present study indicate that the green synthesis of FeNPs boosts its biological activity and may serve as a possible dermal wound-healing agent and cytotoxic agent against cancer. Future study is needed on the identifification of mechanisms involved in the synthesis of FeNPs by V. leucoxylon and its biomedical applications.

Keywords: Vitex leucoxylon; iron nanoparticles; SEM; wound healing; cytotoxic 

CISTANCHE BENEFITS FOR ANTIOXIDANT, ANTI-INFLAMMATORY, CYTOTOXIC, AND WOUND HEALING 

1. Introduction 

The application of nanotechnology in science and technology to manufacture new materials at the nanoscale level is a rapidly growing field [1]. Nanotechnology deals with manufacturing materials at the atomic level to gain distinctive properties, which can be manipulated for preferred applications. This field is rapidly growing with its applications in science and technology to manufacture new materials at the nanoscale level [2]. Various industrial sectors have embraced nanotechnology in recent years due to its applications in the fields of electronic storage systems [3], biotechnology [4], magnetic separation and preconcentration of target analytes, targeted drug delivery [5], and vehicles for gene and drug delivery [3,5,6]. Consequently, these particles have the potential to make a substantial impact on society as a result of the wide range of applications for which they can be used. Nanoparticles (NPs) are masses of particles that have a size of less than one hundred nanometers and are regarded to be key structural masses in the field of nanotechnology. The NPs’ higher activity is both their defining characteristic and the quality for which they are most notable [7]. Organic and inorganic NPs are the two primary classifications that can be applied to nanoparticles. Inorganic nanoparticles may include magnetic NPs, noble metal NPs (such as gold and silver), and semiconductor NPs (such as titanium dioxide and zinc oxide). Organic nanoparticles may include carbon NPs. Inorganic nanoparticles are attracting huge attention because they offer superior material properties along with functional versatility. They have been investigated as possible tools for medical imaging as well as for the treatment of diseases due to the size characteristics they possess [8]. The diverse chemical, physical, and biological properties of NPs are heavily influenced by a variety of parameters, including nanoparticle size and morphology, as well as surface coating, which are normally determined during the synthesis of nanoparticles. The loss of intended biological activity is caused by a decrease in colloidal stability. pH, ionic strength, and a slew of proteins that interact with AgNPs under relevant circumstances all have an impact on colloidal stability. As a result, proper synthesis technique selection is critical for obtaining the desired particle characteristics for specifific applications [9]. 

Currently, there is a wide variety of NPs that can be synthesized using a variety of physical, chemical, biological, and hybrid processes. According to the findings of a large number of research studies, the physical and chemical processes for producing nanoparticles include the use of organic solvents, hazardous compounds, signifificant amounts of energy, and stabilizing agents that are not biodegradable [10]. Therefore, in the field of green nanotechnology, the synthesis of NPs using naturally available materials such as plant extracts, various microorganisms, their metabolites, and a few natural humic substances [11,12] as reducing and capping agents is becoming increasingly popular. Synthesis methods that are friendly to the environment have been used to produce a wide variety of metallic nanoparticles, including silver, gold, iron, copper, and zinc. The development of easy and environmentally acceptable methods for the synthesis of NPs is one of the key focuses of nanotechnology. Biomaterials such as microorganisms and plant extracts can be utilized in the process of preparing a wide variety of NPs [13,14]. However, because some organisms are pathogens, it is dangerous to handle them. In order to thrive, microorganisms need to be maintained in culture and subjected to carefully controlled conditions, including temperature, pH, and other parameters. Because it eliminates the laborious process of maintaining the microbial culture, the synthesis of NPs using plant parts can sometimes prove to be more advantageous than other biological processes [15]. As a result, it has garnered a lot of attention due to its inherent characteristics, which include the utilization of natural resources, rapidity, eco-friendliness, and benignancy. These alluring characteristics are absolutely necessary for use in medical applications. The nanoparticles produced by green synthesis have a size that is well-defifined and under control, they are free of pollutants, and the method is simple to scale up. These are some of the additional benefits of green synthesis [16]. The biological activity of the synthesized nanoparticles is largely determined and fine-tuned by the green materials utilized for the stability and reduction of metal ions. One of the ideal properties of the NPs should be that it must have an outstanding capacity to discriminate between potential targets (pathogens) and mammalian (host) cells [17].

Because of this, the aqueous extract of V. leucoxylon leaves was investigated for its potential to facilitate the formation of iron nanoparticles (FeNPs) in the current research. Iron is one of the elements that can be found in the greatest abundance on Earth. Recently, it has come to be recognized as a new class of important NPs due to the fact that it possesses a variety of unique properties, including high coerciveness and superparamagnetism. Catalysis, electronic devices, information storage, sensors, drug-delivery technology, biomedicine, magnetic recording devices, and environmental cleanup are just some of the many intriguing applications that have made use of FeNPs [18]. Moreover, according to a number of studies, FeNPs may be created from various plant extracts. These plant extracts include Eucalyptus globulus leaf [19], pomegranate leaf [20], and banana peel ash [21]. The plant V. leucoxylon, which was used in the current study, is a member of the family Verbenaceae. It is also known as the five-leaved chaste tree (Kannada: Sengeni, Holenekki, Hollalakki) and can be found in the region along river banks in evergreen and semi-evergreen forests and moist deciduous forests along streams. It can reach a height of up to 20 m and is classifified as a modest to large deciduous tree. Along the length of India’s Western Ghats forests, it can be found in large numbers. V. leucoxylon leaf extract infusion has been reported to possess a wide variety of pharmacological activities such as anti-inflflammatory, antioxidant, antipsychotic, antidepressant, antiparkinsonian, and antihyperlipidemic activities [22]. Natural products have, throughout history and particularly in folk medicine, been utilized for the treatment of a wide variety of ailments and illnesses. This practice dates back to ancient times. The methods of natural product chemistry that have been around for a long time have made it possible to find a huge variety of bioactive secondary metabolites that come from terrestrial and marine sources. A signifificant number of these naturally occurring substances are now being considered for use as potential pharmaceuticals [23]. 

There are a great number of naturally occurring chemicals and nutrients that have yet to be uncovered that are useful to humanity. As a consequence of this, there is an immediate demand for research and development of innovative therapeutic options that may be utilized successfully in therapeutic interventions while creating a minimum amount of adverse effects. 

The currently suggested green synthesis process for FeNPs is distinct and cost-effective. In the current study, an attempt was made to create nanoparticles at room temperature without the use of any chemicals or physical techniques. The Vitex leucoxylon plant was chosen in an effort to synthesize iron nanoparticles, and comprehensive systematic in vitro models were carried out to assess the potency of iron nanoparticles. Only a limited number of research studies on the concept of nanoparticles and their biomedical applications have been published on this plant. As a consequence, we decided that it would be beneficial to carry out this research with the following goals in mind: screening for phytochemicals and measuring the number of secondary metabolites in V. leucoxylon; green synthesis and characterization of FeNPs from V. leucoxylon; comparative study of antioxidant and antiinflflammatory effects of V. leucoxylon and its FeNPs in vitro; in vitro cytotoxic activity of aqueous leaf extract of V. leucoxylon and its FeNPs against skin cancer, lung cancer, and oral cancer; in vitro wound-healing activity of aqueous leaf extract of V. leucoxylon and its synthetic FeNPs by scratch assay.

2. Materials and Methods

2.1. Collection of Plant Material 

In the month of March 2022, fresh leaves of V. leucoxylon were picked from the Anshi forest area of the Western Ghats in the Uttar Kannada District of the state of Karnataka in India. The leaves were identifified and authenticated by Dr. Kotresha K., Taxonomist, Department of Botany, Karnataka Science College, Dharwad; Karnataka, by referring to the voucher specimen deposited in the Department of Botany, Karnataka Science College, Dharwad, Karnataka. After being collected, the fresh plant leaf material was washed under running tap water, sun-dried, and then ground into a coarse powder using a mechanical grinder. The powder was kept in containers that were sealed at room temperature so that it could be used later in the process of crude solvent extraction.

2.2. Preparation of Plant Extract 

Using a Soxhlet device, 25 g of powdered leaves was extracted for 48 h with 250 mL of distilled water. The aqueous extract was concentrated further using a roto-evaporator, and was then dried in desiccators before being kept in an enclosed bottle at 4 ◦C until use. For the synthesis of FeNPs, the aqueous extract was utilized as a reducing and stabilizing agent. 

2.3. Solvents and Reagents 

All of the employed chemicals and solvents were of analytical quality and were purchased from Hi-media (Hubli, India). 

2.4. Phytochemical Analysis

Following the procedure described by Deepti et al. (2012), the crude aqueous leaf extract V. leucoxylon was qualitatively tested for the presence of various phytochemical constituents such as flavonoids, alkaloids, phenols, glycosides, sterols, lignins, saponins, anthraquinones, tannins, and reducing sugars [24].

2.5. Synthesis of Iron Nanoparticles 

First, 1 mL of V. leucoxylon aqueous leaf extract was added to 10 mL of 0.05 mM FeSO4 aqueous solution, and the mixture was shaken. At room temperature and in a dark environment, the complete reaction mixture process was carried out. The oxidation/reduction reaction was clearly visible after the colorless reaction mixture had been incubated and reacted for the required amount of time [25]. In order to remove any traces of aqueous extract from the freshly synthesized FeNPs, which were allowed to dry in powder after centrifugation at 10,000 rpm for ten 10 min during the desired reaction period, the aqueous mixture containing FeNPs was centrifuged a second time and redispersed in double-distilled water and dried [26]. 

2.6. Characterization of FeNPs 

Several methods, such as Ultraviolet–visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy, and energy dispersive X-ray spectroscopy, X-ray Diffraction (XRD), particle size analyzer, and zeta potential, were utilized in order to characterize the FeNPs. 

2.6.1. UV–Visible-Spectroscopy-Based Analysis

 First, 1 mL aliquot of colloidal FeNPs solution in quartz cuvettes was evaluated using UV–visible spectroscopy (U-3310, Hitachi, Tokyo, Japan), using distilled water as a reference and 0.05 mM FeSO4 as a blank, to validate the reduction of the ferric ions in the colloidal solution [27]. 

2.6.2. FTIR-Based Analysis 

The function groups (groups) that were bound on the iron surface and were involved in the synthesis of FeNPs were identified using FTIR spectroscopy (S700, Nicolet, MA, USA), [28]. After 72 h of incubation, the FeNPs were isolated by repeated centrifugation (3–4 times) of the reaction mixtures at 10,000 rpm for 15 min. The supernatant was replaced by deionized water and the pellet was stored as powder. After being dried, the FeNPs were put through an FTIR analysis using the potassium bromide pelleting process at a ratio of 1:100. 

2.6.3. Scanning-Electron-Microscopy-Based Analysis

Scanning electron microscopy (JSM-IT 500, Jeol, Boston, MA, USA), was used to examine the nanoparticles and establish their surface shape. Substrates were prepared on a clean 5 mm × 5 mm Si substrate cleaved from a 100 mm diameter wafer. The substrate was allowed to react for 2 h to 6 h and the sample was prepared by centrifuging a colloidal solution at 10,000 rpm for 5 min. The pellet was dried after being recentrifuged many times,after which it was redispersed in deionized water and the procedure was repeated. Finally, the dry pellet was obtained, which was further subjected to the structural characterization by SEM analysis as per the procedure described by National Institute of Standards and Technology, NIST-2007 [29]. 

2.6.4. Energy Dispersive X-ray 

Following drying on a carbon-coated copper grid, the reduced FeNPs were analyzed using EDX (JSM-IT 500, Jeol, Boston, MA, USA), which also allowed the elemental composition to be determined. 

2.6.5. Zeta Potential 

Observations of NPs The zeta potential is a useful tool for gaining additional insights on the stability of the colloidal NPs. The amplitude of the zeta potential provides a hint as to the possible stability of the colloid. According to Meléndrez et al. (2010), particles are regarded as being stable if their zeta potential values are either more positive than +30 mV or more negative than 30 mV [30]. This fact should be taken into consideration. The laser zeta meter was utilized in order to acquire readings of the surface zeta potentials (Malvern zeta seizer 2000, Malvern, UK). The liquid samples of the nanoparticles, totaling 5 milliliters, were diluted with 50 milliliters of double-distilled water, and 2 mm per square meter of sodium chloride was used as the suspending electrolyte solution. After that, the pH was modifified until it reached the desired level. The samples were agitated for a total of 30 min. After shaking the container, the pH at equilibrium was noted, and the zeta potential of the metallic particles was determined. For the purpose of determining the surface potential of the FeNPs, zeta potential was utilized. In each instance, the stated value was the average of the results of three individual measurements. When the values of the zeta potential ranged from higher than +30 mV to lower than 30 mV, the criteria for the stability of NPs were determined [31]. 

2.6.6. Particle Size Analyzer

In order to determine the sample’s particle size, PSA testing was performed on it after it had been lyophilized and then dispersed using an ultrasonicator (SZ-100, Horiba, Kyoto, Japan). 

2.6.7. X-ray Diffraction Analysis (XRD) Analysis

The synthesized iron nanoparticles from aqueous leaf extract of V. leucoxylon were subjected to XRD analysis (Smart Lab SE, Rigaku, Tokyo, Japan)to determine the nature as well as the average size of the nanoparticles.

2.7. Determination of Antioxidant Activity by Using In Vitro Methods

2.7.1. Ferric-Ion-Reducing Antioxidant Power Assay (FRAP) 

According to Oyaizu (1986), with a minor modification, ferric ions’ reducing power was assessed [32]. For 30 min at 50 ◦C, 2.5 mL of 20 mM phosphate buffer and 2.5 mL of 1% potassium ferricyanide were added to 2.5 mL of V. leucoxylon leaf extract and its synthesized FeNPs blended with the combination. Following the incubation period, the mixture was supplemented with 2.5 mL of 10% w/v trichloroacetic acid and 0.5 mL of 0.1% w/w ferric chloride before being incubated for an additional 10 min. Finally, a UV-V spectrophotometer was used to detect the absorbance at 700 nm. As a standard, ascorbic acid was utilized. Each sample was tested three times. 

2.7.2. Hydrogen Peroxide Scavenging Assay 

Based on the ability of V. leucoxylon aqueous leaf extract and its synthetic FeNPs to scavenge hydrogen peroxide, the antioxidant activity of these compounds was evaluated. First, 0.6 mL of phosphate buffer (pH—7.4) containing 4 mM H2O2 was added to 0.5 mL of standard ascorbic acid at a known concentration, as well as tubes containing plant extracts at various concentrations ranging from 100 µL to 500 µL (pH—7.4). Using a phosphate buffer and hydrogen-peroxide-free blank solution, we assessed the solution’s absorbance at 230 nm after 10 min. Phosphate buffer was used to create the control instead of the sample or standard [33]. Each sample was tested three times. The formula approach was used to calculate the inhibition percentage.

2.7.3. DPPH Free-Radical-Scavenging 

Assay V. leucoxylon leaf extract and synthesized FeNPs were tested for their ability to scavenge free radicals using DPPH radical as a reagent [34]. Samples were combined with DPPH radical solution (60 M) in ethanol (100 µL) at different concentrations (w/v). A UV-Vis spectrophotometer was used to measure the absorbance of the mixture at 517 nm after 30 min of incubation in the dark at room temperature. Ascorbic acid was employed as a standard for the experiment. The following equation was used to determine each sample’s DPPH scavenging activity:

where Ac represents the absorbance of the control reaction, which is performed by mixing 100 L of ethanol with 100 L of the DPPH solution, and At represents the absorbance of the test sample. Experiments were carried out in triplets. The IC50 value was computed for each sample. A higher level of free radical activity was indicated by the reaction mixture having a lower absorbance.

2.7.4. Phosphomolybdenum (PM) Assay

Total antioxidant activity was determined using the Prieto et al., 1999, standard technique. Each test tube containing 3 mL of distilled water and 1 mL of molybdate reagent solution received an aqueous leaf extract of V. leucoxylon and its FeNPs in varied concentrations ranging from 100 µL to 500 µL. These tubes were incubated for 90 min at 95 ◦C. The absorbance of the reaction mixture was measured at 695 nm after these tubes were adjusted to room temperature for 20–30 min after incubation. The reference standard was ascorbic acid [35]. 

2.8. Evaluation of In Vitro Anti-Inflflammatory Activity 

The anti-inflflammatory effect of V. leucoxylon aqueous leaf extract and its generated FeNPs was assessed using the protein denaturation method outlined by Elias et al., 1988, with slight modifications [36]. As a standard drug, diclofenac sodium was utilized. A reaction mixture comprising 2 mL of known concentration of manufactured FeNPs (100 g/mL) with standard diclofenac sodium (100 g/mL) and 2.8 mL of phosphate-buffered saline (pH 6.4) was mixed with 2 mL of fresh hen’s egg albumin (1 mM) and incubated at 27 ± 1 ◦C for 15 min. Denaturation was induced by putting the reaction mixture in a water bath at 70 ◦C for 10 min. After cooling, the absorbance at 660 nm was measured using doubledistilled water as a blank. Each test was carried out three times. The following formula was used to compute the % inhibition of protein denaturation: 

where, At = absorbance of test sample; Ac = absorbance of control.

2.9. Determination of Cytotoxic and Anticancer Activity of Iron Nanoparticles Using MTT Assay

The effect of V. leucoxylon and its synthesized FeNPs on the viability of non-cancerous fibroblast cells L292 and its anticancer activity on skin cancer (A375), lung cancer (A549), and oral cancer (KB-3-1) was evaluated using the standard MTT assay, according to Carmichael et al., (1987) [37]. All the cell lines were obtained from the National Centre for Cell Science (NCCS), Pune, India. Percentage inhibition of cell growth (IC50) values were derived using dose-response curves for each cell line, and the following formula was used to compute the percentage growth inhibition. The conversion of MTT to a purple formazan product by healthy cells’ mitochondrial dehydrogenase is the basis of this experiment [38]. 

2.10. In Vitro Wound-Healing Study by Using Scratch Assay Test

The spreading and migratory capabilities of L292 cell line cells caused by samples with known concentrations of plant extract and iron nanoparticles were examined in the current investigation [39]. Animal cell culture plates with DMEM media supplemented with 10% FBS and 2% Pen-Strep antibiotic (Darmstadt, Germany) were used to start the cell culture process. A sterile plastic pipette tip was used to scratch the monolayer confluent of cells after they had grown to roughly 50,000 cells per mL. PBS solution was used to remove any unwanted cell debris. As a negative control, untreated cells were used, whereas standard ascorbic acid was used as a positive control for polymer samples of known concentration. For the next 24 h, the cells were kept at 37 ◦C with 5% CO2. For the examination of relative cell migration and wound closure, the scratched cell layers were incubated and imaged at intervals ranging from 0 h to 6 h to 12 h and 24 h. MagVision Software’s measurement (X64, 2016, Magnus, New Delhi, India) calibration at 4× resolution was used to quantify the gap distance. In order to determine the wound closure and migration rate, the formula shown below was used: 

with respect to the following: A0h = wound area measured immediately after scratching; ATh = wound area measured after h hours; Rm = migration rate (µm/h); Wf = initial wound width (µm); and T = migration time (hour).

2.11. Statistical Analysis 

The data are presented as the mean standard deviation and standard error, and each experiment was carried out three times. SPSS software version 20 was used to perform a one-way analysis of variance (ANOVA) on the differences in mean scores that existed between the groups.

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