Eco-benign phytochemically-functionalized Ag-core Fe-shell bimetallic nanoparticles for targeted antioxidant and anti-inflammatory drug delivery

Abstract

The etiology of several degenerative diseases is linked to oxidative stress and inflammations. This study presents an eco-benign phytochemically-functionalized Ag-core Fe-shell (Ag@Fe) bimetallic nanoparticles (NPs) for targeted antioxidant and anti-inflammatory drug delivery. The NPs were synthesized, and modified with phytochemicals from the aqueous leaf extract of Costus lucanusianus; subsequently, characterized using advance microscopes and spectroscopes. Atomic force microscope revealed the grain size of 4.54 nm with the total projected area of 95.65% confirming the surface modification of the nanoparticles, and the energy dispersive spectroscope confirmed the strong presences of Ag and Fe. At 500 µg/mL, the NPs and ascorbic acid (standard) demonstrated similar (p < 0.05) antioxidant properties by reducing oxidative stress through the effective scavenging of hydroxyl, and hydrogen peroxide radicals. Similarly, at 500 µg/mL, the anti-inflammatory performance of the NPs, and acetyl salicylic acid (standard) through the inhibition of protein denaturation, heat-induced haemolysis, and scavenging of nitric oxide radicals as 52.90 ± 6.65 and 78.26 ± 4.35; 70.83 ± 2.90 and 82.52 ± 8.43; 56.93 ± 5.18 and 74.65 ± 2.43 respectively; were statistically (p < 0.05) similar; thus, revealing the antioxidant and anti-inflammatory potency of the NPs. The dual functionality of these nanoparticles highlights their potential for therapeutic applications, particularly in managing oxidative stress-related and inflammatory conditions.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13065-025-01640-w.

Introduction

Oxidative stress is the imbalance between the production of free radicals (reactive oxygen and nitrogen species) and antioxidants, in favor of the free radical production. Oxidative stress and chronic inflammation have been significantly linked to the etiology of various diseases, including cardiovascular disorders, cancer, diabetes, and neurological diseases. The brain is particularly vulnerable to oxidative stress due to its high oxygen demand, and metabolic rate. Consequently, neurological disorders like Alzheimer’s, Parkinson’s, multiple sclerosis, and ischemic stroke have been associated with oxidative stress. Increased production of reactive oxygen species (ROS) including superoxide radicals, hydroxyl radicals, and hydrogen peroxide, can damage vital biomolecules in the body, such as proteins, lipids, and nucleic acids. This damage activates pathways that trigger an inflammatory response through the activation of nuclear factor-kappa B (NF- κB), and inflammasome [1–4].

Inflammation is characterized by the activation of various signaling pathways, including toll-like receptors, leading to elevated levels of nitric oxides, inflammatory cytokines, and chemokines. Increased oxidative stress is associated to the primary source of chronic inflammation [5]. The inflammatory route starts with the activation of the immune cells and the release of cytokines, which recruit and activates more immune cells, thus intensifying the inflammatory response. This activity generates ROS, leading to oxidative stress that can damage cells if not counteracted by antioxidants. Persistent inflammation can lead to chronic inflammation, tissue damage, fibrosis, and imbalances in the immune system, potentially causing autoimmune diseases and immunosuppression [5, 6]. Antioxidants and anti-inflammatory agents counteract the effects of oxidative stress.

Different antioxidants and anti-inflammatory compounds have been widely used to mitigate oxidative stress and inflammation in various pathological conditions. Antioxidants are molecules that neutralize free radicals (reactive oxygen & nitrogen species) and prevent oxidative damage. They can be natural or synthetic. The natural antioxidants are derived either from dietary sources, plants, or endogenous systems. Examples of these natural antioxidants include ascorbic acid (vitamin C), Vitamin E (tocopherols & tocotrienols), polyphenols, and glutathione; while the synthetic antioxidants include compounds like butylated hydroxy toluene (BHT) and butylated hydroxy anisole (BHA). Anti-inflammatory agents are used to reduce inflammation by either inhibiting pro-inflammatory pathways or mediators. They can be classified as steroidal or non-steroidal agents. The non-steroidal anti-inflammatory drugs (NSAIDs) include Ibuprofen, Aspirin, and Naproxen; while the corticosteroids (Steroidal anti-inflammatory drugs) include prednisone and dexamethasone. Some of the anti-inflammatory agents from plants sources include curcumin (derive from turmeric), omega-3 fatty acids (from fish oil), and gingerol (from ginger) [4, 7, 8].

Conventional antioxidants, and anti-inflammatory drugs have exhibited potentials in controlling these disorders, nevertheless, their clinical uses are frequently constrained by challenges such as inadequate stability, restricted bioavailability, and possible toxicity at certain doses [9]. Advancement in nanotechnology, such as the development of multifunctional nanocomposites, offers promising solutions to overcome these challenges by enhancing stability, targeted efficiency, and minimized side effects. This approach holds potential for the next generation of therapeutics agents to combat oxidative stress and inflammation effectively.

Plant-derived chemicals possessing antioxidant and anti-inflammatory properties can be integrated with nanoparticles to create nanostructured frameworks. These frameworks may enhance the efficacy of plant-extracted products, mitigate their possible adverse effects, and promote their biological activities. The bioavailability of bioactive compounds following oral administration can be enhanced using nano-encapsulation for targeted delivery. Nano-encapsulation facilitates precise release of the bioactive compounds and prevents their degradation during processing and storage [9–11]. Silver and iron nanoparticles have considerable antioxidants, anti-inflammatory, and antibacterial properties [12] among the diverse array of nanomaterials. The integration of these two nanomaterials to form nanocomposites, and their functionalization with plant chemicals presents a possibility to attain synergistic therapeutic benefits, while mitigating the limitations inherent to individual nanoparticles, boost their biocompatibility, and improve their bioactivity. This formation arises from the necessity for more efficacious antioxidant and anti-inflammatory agents.

Although the antioxidant and the anti-inflammatory properties of silver and iron nanoparticles have been widely reported [12, 13], there is paucity of report on their combination and functionalization with plant chemicals for antioxidant and anti-inflammatory capabilities. Much of the studies on silver-iron NPs, and Costus lucanusianus [14] emphasized their antimicrobial application.

This study’s innovation is the strategic integration of silver and iron nanoparticles into NPs, and the modification of the NPs with plant chemicals for enhanced therapeutics properties. The study sought to produce a new category of NPs that can significantly overcome the limitations of the existing therapeutics in effective treatments of illnesses associated with oxidative stress and inflammation. The aqueous leaf extract of Costus lucanusianus was utilized to synthesize and functionalize the Ag@Fe NPs due to its safety and rich phytochemical composition [15], and advanced microscopes and spectroscopes were employed to assess the physico-chemical properties of the NPs. In vitro biological assays were also utilized to evaluate their antioxidants and anti-inflammatory efficiencies.

Materials and methods

Sample collection and preparation

Fresh leaves of Costus lucanesianus were collected at the Federal University of Technology Owerri and subsequently identified by Mr. Francis Farawa from the Department of Forestry and Wildlife in the School of Agriculture and Agricultural Technology. The sample was deposited at the Department of Forestry and Wild Lives Herbarium, Federal University of Technology Owerri, with the voucher number FUTO/SAAT/FWT/040018. Subsequently, the leaves were carefully plucked out, thoroughly washed under a running distilled water, allowed to completely drain out water and then exposed under the shield for two weeks to dry.

Five grams of the dry leaves were ground, dissolved in 200 mL of deionized water, and heated for 30 min at 80 ℃. Subsequent to heating, the solution was filtered and evaporated to dryness to obtain the crude extract, which was stored at – 4 ℃ for further analysis.

Synthesis of Ag@Fe NPs

A 0.1 g of the previously obtained crude leaf extract was dissolved in 150 mL of deionized water to form a filtrate. Then, 2 g of iron (III) chloride hexahydrate (FeCl₃·6H₂O) and 1.38 g of silver nitrate (AgNO₃) were dissolved in the filtrate to form a solution. The pH of the solution was stabilized at 7.4 through dropwise addition of 0.1 molar solution of sodium hydroxide. Subsequently, the resultant solution was placed on a magnetic stirrer, and heated for 30 min at 80 ℃ while continuously stirred. Afterwards, the solution was allowed to cool for 1 h while continuously stirred to avert agglomeration, then followed by centrifugation at 1790 × g for 5 min. The colloidal solution of the particles was heated to dryness at the temperature of 400 ℃ for 30 min.

Functionalization of Ag@Fe NPs

The bimetallic nanoparticles were formed through co-synthetic approach, in which the bulk precursors, FeCl₃·6H₂O and AgNO_3, were dissolved simultaneously in the filtrate for co-reduction of their ions by the plant chemicals.

1

2

The functionalization of the Ag/Fe NPs followed the method described by Iheme et al. [16] with slight modification. The colloidal solution of the nanoparticles was mixed, in the ratio of 1:1, with the previously produced aqueous leaf extract, and the mixture was allowed to age for 24 h to ensure proper attachment of the plant chemicals to the nanoparticles’ surfaces. Afterwards, the solution was centrifuged at 1790 xg to separate the modified Ag/Fe NPs from the free plant chemicals. Subsequently, the solution was washed with distilled water three times to remove unbound bioactive compounds and impurities; then gradually heated at low temperature of 70 °C for 1 h to remove residual moisture, followed by rapid calcination at 100 °C for 30 min to enhance the crystalline structures of the nanoparticles. The particles were then stored for characterization. The hybridization of the phytochemicals on the surfaces of the nanocomposites were determined with Fourier infrared spectrophotometer (FTIR).

Instrumental analysis of the nanoparticles

The possible phytochemicals represented by their functional groups that capped the Ag/Fe NPs were verified using FTIR instruments. During the measurements, the FTIR spectrum was obtained within the frequency range of 4,000–600 cm^− 1. The crystallinity of the Ag/Fe NPs was determined using the Rigaku D/Max-lllC X-ray diffractometer developed by Rigaku Int. Corp. Tokyo, Japan, and set to produce diffractions at a scanning rate of 20/min in the range of 2 to 500 at room temperature with CuKa radiation set at 40 kV and 20 mA. The possible size was predicted by the Scherrer equation: , where k represents the shape factor (ranging from 0.62 to 2.08, taken at 0.59 intervals), L signifies the size of the nanocrystalline material, θ is the diffraction angle, λ represents the wavelength, and β corresponds to the peak width at half maximum (FWHM) determined from the diffraction pattern at a 2θ angle of 20 degrees. The purity and energy content of the Ag/Fe NPs were determined using an energy dispersive x-ray spectroscope (EDX) instrument. In determining the microstructure of the Ag/Fe NPs, the precise morphological characteristics of the Ag/Fe NPs were examined using a scanning electron microscope (SEM) with specific details (JEOL JSM-6380 LV, operating at 20 Kv). The sample was evenly spread onto a SEM holder, followed by a gold (Au) coating to enhance its conductivity. Furthermore, a High-Resolution transmission electron microscope (TEM), specifically the Tecnai G2 Stwin model operating at 200 kV, was used to determine the particle diameter of the Ag/Fe NPs. The powdered sample was sonicated, deposited onto carbon-coated copper grids, and examined under the microscope. The composites underwent gas chromatography coupled with mass spectrometry (GC-MS) to ascertain their volatile phytochemical makeup. The GC-MS utilized was the Thermo GC-TRACE Ultra Ver. 5.0, equipped with the thermal MS DSQ IL from Thermal Scientific Co. The GC-MS experimental parameters were as follows: column length: 30 m, inner diameter: 0.25 mm, and film thickness: 0.25 μm. The flow rate of the mobile phase (carrier gas: helium) was established at 1 mL/min. The temperature protocol for the gas chromatography apparatus commenced at 40 ℃ and was elevated to 150 ℃ for a duration of 10 min. The mass spectra of the composites were studied, and the findings were compared to the National Institute of Standards and Technology (NIST) database. The mass spectra of the known constituents in the NIST Library were juxtaposed with the composite spectrum. The relative proportion (%) of each component was calculated by comparing the average peak area to the overall area. The atomic force microscope (AFM) measurements were conducted using a Veeco Nanoman V, which features a precise three-axis scanner functioning under closed-loop control (hybrid XYZ-scanner with a range of 90 μm × 90 μm × 8 μm). All measurements were conducted in air utilizing tapping mode and OTESPA-R3 probes. The cantilever resonance frequency is 300 kHz, and the nominal radius of curvature of the tip is approximately 7 nm. The cantilever’s nominal rigidity is 42 N/m. The amplitude of tip oscillation for all measurements was approximately 40 nm. The amplitude setpoint was established at a high level, close to the free amplitude (80%), to mitigate excessive interactions with the sample and subsequent nanoparticle displacements. The pixel size was established at 5.0 nm for all AFM measurements. The scanning velocity is 4 μm/s, with fixed PID settings of 0.8 for integral gain and 10 for proportional gain. A Genesys UV-VIS Spectrophotometer, model Prove 30, with running software version ISW 1.5.1, was employed for conducting UV-vis spectroscopy. The absorption spectra were recorded within the wavelength range of 200 nm to 900 nm.

Determination of band gap energy (Eg)

The band gap energy (Eg) is the energy difference between the valence band and the conduction band in a material. It influences the interaction of materials with light and electricity.

The band gap energy of the silver doped iron nanoparticles was determined using the equation:

3

Where h is the planck’s constant (6.626 × 10^− 34 Js), c is the speed of light (3.0 × 10⁸ m/s), and λ is the maximum light absorption wavelength of the nanoparticles.

Antioxidant assay

Nitric oxide scavenging activity

Nitric oxide (NO) is produced in biological tissues by specific digestive enzymes called nitric oxide synthases. These enzymes convert arginine to citrulline via a five-electron oxidative process, resulting in the creation of NO. The chemical, sodium nitroprusside undergoes decomposition in an aqueous solution with a pH of 7.2, resulting in the formation of stable compounds namely nitrate and nitrite. The amounts of these products may be measured using Griess reagent, as previously described by [17, 18]. A solution containing 2 mL of sodium nitroprusside with a concentration of 10 mM was dissolved in 0.5 mL of phosphate buffer saline with a pH of 7.4. This solution was then mixed with 0.5 mL of the Ag/Fe NPs at different concentrations ranging 100 to 1000 µg/mL. The solution is thereafter placed in an incubator at a temperature of 250 ℃. After a 150 min incubation period, 0.5 mL of the incubated solution was removed and combined with 0.5 mL of Griess reagent, consisting of 1.0 mL of 4-Aminobenzenesulfonic acid reagent (0.33% concentration in 20% glacial acetic acid) and 1 mL of naphthyl-ethylenediamine dichloride (0.1%w/v concentration) at room temperature. Subsequently, the mixture was left to incubate at ambient temperature for 30 min, after which the absorbance of the solution was assessed spectrophotometrically at a wavelength of 546 nm. The nitric oxide radical was determined using:

4

where A₀ equals the initial absorbance and A₁ represents the final absorbance after reaction has occurred using Griess reagent.

Hydrogen peroxide scavenging assay

The capacity of Ag/Fe NPs to remove hydrogen peroxide was determined using the Rush et al. [19], approach. A 0.1 mL portion of the Ag/Fe NPs (ranging from 25 to 450 µg/mL) was put into Eppendorf tubes. The volume of the Ag/Fe NPs was then adjusted to 0.4 mL by adding 50 Mm phosphate buffer (pH 7.4). Next 0.6 mL of a 40 Mm hydrogen peroxide solution was added to each tube. The reaction mixture was vigorously mixed using a vortex mixer. After a reaction period of 10 min, the absorbance of the mixture was determined at a wavelength of 230 nm. Ascorbic acid served as the positive control. The capacity of the Ag/Fe NPs to remove hydrogen peroxide was determined using:

5

where the variables A0 and A1 represent the absorbance of the control and sample respectively.

Superoxide radical scavenging activity

Applying the approach of Robak & Gryglewski [20], , the superoxide anion scavenging activities was measured with slight modification.

A 3.0 mL of Tris-HCl buffer (16 mM, pH 8.0) containing 0.5 mL of nitro-blue tetrazolium (NBT) (0.3 mm), 0.5 mL of NADH (0.936 mM) solution, 1.0 mL of the nanocomposites at different concentrations (100, 200, 500, 100 µg/mL) and 0.5 mL Tris-HCl buffer (16 mM, pH 8.0) was used to generate superoxide anion radicals. The reaction was activated by adding 0.5 mL of phenazine methosulfate (PMS) solution (0.12 mM) to the mixture. It was then incubated at 250℃ for 5 min, and the absorbance at 560 nm is measured in comparison to a blank sample. The % inhibition was determined using:

6

where, A0 is absorbance of the control and AS is absorbance of the sample.

Hydroxyl radical scavenging activity

The hydroxyl radicals scavenging capacity of Ag@Fe NPs was tested using the Kunchandy and Rao [21] approach with slight modification.

The reaction mixture (1.0 ml) contains 100 µl of 2-deoxy-dribose (28 mM in 20 Mm KH₂P0₄-KOH buffer, pH 7.4), different concentrations of the nanocomposites (100, 200, 500 and 1000 µL), EDTA (1.04 mM) and 200 µM FeCl₃ (1:1 v/v), 100 µL of H₂O₂ (1.0 mM) and 100L of ascornbic acid (1.0 mM). The mixture was then incubated at 37℃ for an hour. Following this, one milliltre of thiobarbituric acid (1%) and 1.0 m L of trichloroacetic acid (2.8%) were added and then incubated for 20 min at 100℃. Absorbance was measured at 532 nm following cooling, in comparison to a blank sample. The inhibition percentage is calculated as follows:

7

where A0 represents the absorbance of the control (without nanocomposites), and A1 represents the absorbance of the reaction mixture containing the nanocomposites.

β-carotene Linoleic acid method/conjugated diene assay

This is among the quickest methods for antioxidant screening. It operates on the premise that oxygenated water produces Reactive Oxidative Species (ROS), which oxidizes unsaturated fatty acids like linoleic acid. Discoloration results from the oxidation of beta-carotene, which was initiated by the products of this oxidation process. The activity is measured at 434 nm and the degree of discoloration is reduced by antioxidants. As stated by [22], the process entails mixing 200 mg of an emulsified tween-80 mixture with 25 µL of linoleic acid and 0.5 mg of β-carotene in 1 mL of chloroform. The chloroform was evaporated at 400℃, 100 mL of oxygen-saturated distilled water was progressively added to the residue, and the mixture was quickly swirled to produce a stable emulsion. Four (4) millilitres of the mixture were put into test tubes containing 200 µL of NPs made in methanol at final concentrations (100, 200, 400, and 1000 µg/mL). As soon as the emulsified solution was put into the tubes, the zero-time absorbance at 470 nm was measured. For two hours, the tubes were incubated at 500℃. Ascorbic acid served as the standard. Antioxidant activity was calculated as the percentage of inhibition (%) relative to the control using the formula below:

8

whereas Ac represented the initial absorbance of the negative control, and Ac120 represented the absorbance of the negative control at 120 min, As represented the initial absorbance, and As120 represented the absorbance of sample at 120 min.

Anti-inflammatory activity

Inhibition of protein denaturation

With a slight modification, the methods of Sakat et al., [23] and Mizushima and Kobayashi [24] were used to assess the inhibition of protein denaturation. One hundred microliter (100 µL) of NPs was mixed with 500 µL of 1% bovine serum albumin. After 10 min at room temperature, this combination was heated for 20 min at 51℃. The resultant mixture was cooled to room temperature, and the absorbance at 660 nm was measured. Acetyl salicylic acid served as the standard. The following was used to determine the % inhibition of protein denaturation during the triplicate experiment:

9

where A1 is the absorbance of the nanocomposites, A2 is the absorbance of the control and A0 is the absorbance of the positive control.

Membrane stabilization

Preparation of red blood cell (RBC) suspension

Human anticoagulated blood was obtained from the Federal University of Technology Owerri Medical Center, with the informed consent of the donors, and handled according to strict protocol. Subsequently, the samples were centrifuged for 10 min at 1790 xg. Three saline washes were performed on the resultant solution. The RBC layer was taken and diluted with phosphate buffer saline (PBS) to achieve 10%v/v [24].

Heat induced hemolysis

To each concentration (100, 200, 500, 1000 µL) of the nanocomposites, 100 µL of 10% RBC was added. The mixture was centrifuged at 1587 xg for 10 min at room temperature, after which. the resultant solution was heated to 56℃ for 30 min. Absorbance was read at 560 nm after collecting the supernatant. Acetyl-salicylic acid served as the positive control. The percentage of membrane stabilization was computed as computed:

10

where A1 is the absorbance of the sample, A2 is the absorbance of the product control and A0 is the absorbance of the positive control.

Statistical analysis

The one-way analysis of variance ANOVA was employed to evaluate the obtained data, with significant differences identified at p < 0.05. The mean values and standard deviation were utilized to represent the results. The Statistical package for Social Science version 18 was used to conduct this analysis.

Result and discussion

Phytochemical composition of Ag@Fe NPs

The result of GC-MS revealed the phytochemical characterization of the nanoparticles (Table 1). Accordingly, the nanoparticles composed of eleven volatile plant chemicals, prominent among which are Cis-13-Octadecanoic acid, methyl ester (28.17%), Cis-vaccenic acid (17.19%), Hexadecanoic acid, methyl ester (16.49%), and n-Hexadecanoic acid (15.40%). These and the other phytochemicals may have contributed significantly to the anti-inflammatory, and the antioxidant properties of the nanoparticles. Cis-13-Octadecanoic acid, methyl ester, Cis-vaccenic acid, Hexadecanoic acid, methyl ester, and n-Hexadecanoic acid are fatty acids and their derivatives. Some of them have been reported to exhibit antioxidant and anti-inflammatory properties. Cis-vaccenic acid is an omega-7 monounsaturated fatty acid, which has been reported to poses anti-inflammatory properties by modulating inflammatory pathways, such as reducing the production of pro-inflammatory cytokines. However, its antioxidant activity is less well-documented. Hexadecanoic acid, methyl ester (methyl palmitate) is the methyl ester of palmitic acid. While palmitic acid is often associated with pro-inflammatory effects, its esterified form (methyl palmitate) has shown some antioxidant and anti-inflammatory properties, particularly, in reducing oxidative stress and inflammation in cellular models. N-Hexadecanoic acid (Palmitic acid) is a saturated fatty acid. It is generally considered pro-inflammatory and is not known for significant antioxidant activity. However, in some contexts, it may play a role in cellular signaling pathways that indirectly influence inflammation and oxidative stress. 2,6-octadienal, 3,7-dimethyl-(Citral) is well-known for its significant antioxidant and anti-inflammatory properties. Citral is reported to work by scavenging free radicals and inhibiting pro-inflammatory mediators like cytokines and enzymes like COX-2 and LOX. Oleic acid is a monounsaturated omega-9 fatty acid widely recognized for its anti-inflammatory and antioxidant properties. It reduces inflammation by inhibiting the production of pro-inflammatory cytokines like TNF-α, IL-1β, IL-6, IL-8 and IL-17, and modulates oxidative stress by enhancing cellular antioxidant defenses. They achieve this by modulation of NF-KB signaling, a pathway that regulates cytokine production. They promote lipid raft alterations reducing immune cell activation and finally enhance the production of anti-inflammatory cytokines, like IL-10 [25–27]. The anti-inflammatory activity of oleic acid could also result from its contribution to the synthesis of leukotriene A3, which is a potent inhibitor of pro-inflammatory leukotriene B4 and probably reduces the sensitivity of response to TNF-α [6, 28–31].

Table 1.

Volatile phytochemical composition of the nanocomposites

S/N	RT	Compd	Conc. (%)
1	7.08	10-Undecen-4-one, 2,2,6,6-tetramethyl-	2.92
2	7.69	2,6-Octadienal, 3,7-dimethyl-, €	4.22
3	16.99	Hexadecanoic acid, Methyl ester	16.49
4	17.64	n-Hexadecanoic acid	15.40
5	18.81	Cis-13-Octadecanoic acid, Methyl ester	28.17
6	18.97	Fumaric acid, Pen-4-en-2-yl tridecyl ester	0.93
7	19.05	Heptadecanoic acid, 16-Methyl-, Methyl ester	6.00
8	19.45	Cis-Vaccenic acid	17.19
9	19.65	Oleic acid	6.02
10	19.70	12-Methyl-E, E-2, 13-Octadecadien-1-Ol	2.80
11	19.80	1 H-indene, 2-butyl-5-hexyloctahydro-	0.33

Microscopic analysis of Ag@Fe nanocomposites

The UV-visible spectrum of the functionalized(fFA)- and the non-functionalized-silver doped iron nanoparticles (FA) offered insight into the optical characteristics of the nanoparticles (Fig. 1a). The absorption peak located between 350 and 380 nm is indicative of silver doped iron nanoparticles. This observation corresponds with the findings of [32], who reported the absorption of patterns of silver modified hematites. The absorption variations between the functionalized and the non-functionalized silver doped iron nanoparticles indicates the presence of phytochemicals in the functionalized nanoparticles. The presence of the plant chemicals slightly impacted on the optical properties of the NPs hence, the changes in the absorbance patterns between the FA and fFA. Using the Tauc’s equation, the band gap energy of the nanoparticles was determined as 3.27 eV. With 3.27 eV, these nanoparticles can absorb UV light, potentially leading to electron excitation and the generation of electron-hole pairs. These charge carriers can interact with reactive oxygen species (ROS), neutralizing them and reducing oxidative stress. Similarly, Ag-Fe NPs may have inhibited the production of pro-inflammatory cytokines such as TNF-α and IL-1β by reducing ROS-mediated activation of nuclear factor-kappa B (NF-κB), which is a key transcription factor in inflammation.

Fig. 1.

a and b represent the UV vis plot of Fe-Ag nanoparticles, and the FTIR spectral of the Ag-Fe nanoparticles respectively

The FTIR peaks were observed in the range of 450 to 1500 cm^− 1 (Fig. 1b). Prominent peaks were noted at 467.34946 cm^− 1 and 592.93676 cm^− 1. The peak at 467.34946 cm^− 1 indicates Ag-O bond stretching vibration, while the peaks at 592.93676 cm^− 1 reflects Fe-O vibrations in the octahedral and tetrahedral positions, typically in magnetite (Fe₃O₄) or hematite (Fe₂O₃).

The extensive peaks observed between 100 and 2500 cm^− 1 reflects the functional groups of the plant chemicals that capped the nanoparticles. The functional groups of ethers and esters are associated with C-O stretching around 1050–1250 cm^− 1. Meanwhile, the C = C stretching representing aromatic or alkene associates around 1450–1600 cm^− 1, while the symmetric and asymmetric stretching of nitro compounds (NO₂) vibrate around 1350 –530 cm^− 1 (Supplementary Table 2). The FTIR spectra of the Ag-Fe NPs aligns with that of Padilla-Cruz et al. (2021), with slight variations. The variations may have resulted from the synthetic conditions adopted during the process of synthesis.

The SEM images revealed a dense aggregation of Fe-Ag NPs with primarily trigonal morphologies (Fig. 2b). The particles were measured at the range of 50 to 100 nm using ImageJ software, confirming a certain level of agglomeration. The particle size distribution seemed reasonably uniform, while some big aggregates were evident. These observations align with Vijayaraghavan [33] who reported the rhombohedral structure of the silver doped iron nanoparticles. The structure and size of nanoparticles are influenced by the conditions of synthesis. Also, the observed minor aggregates may result from the drying process during sample preparation for SEM imagining or the interactions of the two metal nanoparticles, Fe and Ag.

Fig. 2.

Micrograph of Fe-Ag nanoparticles showing a, b and c as AFM, SEM, TEM and EDX images of Fe-Ag nanoparticles respectively

Silver doped iron oxide NPs are also shown in Fig. 2a and c as AFM and TEM images respectively. In addition to offering crucial information of the nanoparticles’ shapes, the images clearly depict the topographical, morphological, and size characteristics of the particles. Under 200 nm magnification, the TEM picture displays the morphology and internal structure of the Ag-FeO NPs, which have a trigonal morphology with varying diameters. The AFM revealed the grain size of 4.54 nm with the total projected area (rel.) of 95.65% (Supplementary Table 1).

The EDX spectra exhibited significant peaks for silver (Ag) and iron (Fe), while at the same time, revealing less significant peaks for oxygen (O), carbon (C), and sulfur (S), thus, confirming the composition of Ag-Fe NPs (Fig. 2d). The minor peaks revealing the presences of carbon, oxygen, and sulfur may have resulted from the functional plants chemicals. The comparative peak strengths of Ag and Fe align with the formation of Ag-Fe NPs. The absence of other metal peaks indicates the purity of the nanoparticles, thus, attributing the aftermaths antioxidants and anti-inflammatory effects to the functionalized Ag-Fe NPs. The EDX results correspond with Padilla-Cruz [34], for the synthesis, and prominent dominations of Ag and Fe peaks.

The synthesized silver doped iron oxide nanoparticles’ X-ray diffractogram (Fig. 3) shows two differential peaks at 2θ values of 30.00◦ and 40.00◦. The average spacing (d-spacing) between the planes of the NPs atoms at the two differential peaks of 30.00◦ and 40.00◦ are 2.98 Å and 2.25 Å. A d-spacing range of 2.25–2.98 Å correspond to larger, more exposed crystal planes, which can improve adsorption of reactive oxygen species. This d-spacing suggest favorable arrangements of the nanoparticles atoms that could influence their antioxidant and their anti-inflammatory potentials. The XRD result revealed the synthesized Ag@Fe NPs as amorphous. This implies that the NPs have larger surface area and more active sites compared to their crystalline counterparts. This property allows for more contact points with free radicals, and enhance their free radical-scavenging potential. More so, in their disorganized amorphous nature, the synthesized Ag@Fe have enhanced solubility, allowing them to disperse effectively, and reaching sites of oxidative stress for antioxidant and anti-inflammatory functions. This finding is contrary to the findings of Idris and Roy; Dimulescu et al. [35, 36] who reported crystalline structure of Ag@Fe NPs. The variations in the structure of the NPs may have resulted from the synthesis approach.

Fig. 3.

XRD spectra of the Ag-Fe NPs

Mechanism of antioxidant and Anti-inflammatory properties of Ag-Fe NPs

Oxidative stress is the key driver of many diseases, including neurodegenerative disorders, cardiovascular diseases, diabetes, and cancer [37–41]. The inflammatory process is a response to tissue lesions, infections, and tumors. Degenerative diseases like cancer, autoimmune disorder, and metabolic diseases, result from the failure to repair inflammatory tissue damages [42–44].

When nanoparticles are synthesized using plant extracts, their surfaces become coated with phytochemicals like flavonoids and polyphenols. These compounds are potent antioxidants that can effectively scavenge free radicals by donating electrons, neutralizing them before they can cause cellular damage [45–47].

Engineering of nanoparticles with specific coatings, can cause them to mimic the function of natural antioxidant. This allows them to catalyze reactions that break down ROS into less harmful molecules, providing a long-lasting defense against oxidative stress [48–51]. The nanoparticles themselves can act as a stable platform, protecting the delicate plant-based antioxidants from degradation and enhancing their bioavailability and effectiveness.

Silver nanoparticles have been reported to poses anti-inflammatory properties. The anti-inflammatory mechanism of Ag NPs begins with their binding and penetration into cell membrane, which is stimulated by inflammatory process [43]. Within the cells, Ag⁺ ions are released and consequently, reactive oxygen species (ROS) are generated. These ROS have been implicated in suppressing the NF-κB transcription factor pathway, thus reducing the production of cytokines IL-1, IL-1-α, IL-1-β, IL-6, IL-9, IL-12, IL-18, IFN- γ, and TNF-α. The ROS are eventually inactivated by intracellular gluthathione to minimize cytotoxicity of healthy cells [52–54]. Silver nanoparticles have also been reported to exacerbate inflammation by selectively inhibiting the cyclooxygenase-2 (COX-2) enzyme through the induction of structural change that inhibit the production of prostaglandins from arachidonic acid [54, 55].

Similarly, the antioxidants properties of FeO NPs are linked to their method of synthesis; whereas, green synthesized FeO NPs have been reported to have antioxidant properties [56] (Iheme et al. 2024b), the same cannot be said of the NPs synthesized through chemical or physical approach. The biological properties result from the plant chemicals on the surfaces of the NPs. Consequently, FeO NPs can act as oxidants, antioxidants, pro-inflammatory or anti-inflammatory agent, depending on the phytochemical composition of their surfaces. The primary mechanism of the oxidant nature of FeO NPs is the fenton reaction, in which H₂O₂ is converted to hydroxyl radicals (.OH) in the presence of iron ions [57, 58] (Eq. 11).

11

Conversely, FeO NPs can as well act as antioxidants, especially when they are richly capped with antioxidant phytochemicals [56]. This unique property is associated with “green synthesis” of the nanoparticles. In this instance, the nanoparticles serve as a stable carrier, enhancing the antioxidant effects, as well as protecting the phytochemicals from degradation. Certain NPs, like magnetite (Fe₃O₄), have been reported to mimic the natural antioxidant enzymes, like superoxide dismutase or catalase, by converting ROS into less toxic molecules [48, 56].

The doping of iron with silver, and the functionalization of the NPs with plant chemicals is aimed at producing multifunctional nanoparticles that combines the desirable properties of each component for enhanced antioxidant and anti-inflammatory properties.

Zero-valent iron nanoparticles are reactive and prone to rapid oxidation and aggregation. Doping them with silver promotes their stability, reduce their aggregation, and enhance their shelf life [59]. Functionalizing the bimetallic nanoparticles with plant chemicals provides additional layer of functionality and safety for biological applications [60]. Given that Ag and Fe can be pro-oxidants; antioxidant-rich plant chemicals on their surfaces can neutralize the pro-oxidant properties of the metal core shell, while promote their biocompatibility, and reduce their toxicity in biological systems. Ag@Fe NPs, in this case, serves as carrier for the phytochemicals and a synergistic amplification platform for antioxidant activity.

The combination of the nanoparticles and the plant chemicals can provide a more potent antioxidant effect than either component alone. Plant-based antioxidants function by donating electrons to neutralize free radicals. When these compounds are on the surface of nanoparticles, they can interact with and scavenge free radicals more readily, given the large surface area-to-volume ratio of the nanoparticles.

The oxidant properties of the core nanoparticles shell are not completely eliminated. In certain conditions, such as a highly acidic environment that might degrade the plant-based capes, the nanoparticles could still release Ag⁺ and Fe³⁺ ions, leading to localized oxidative stress. This pro-oxidant effect is sometimes intentionally used in biomedical applications to target and destroy cancer cells. However, in most physiological conditions, particularly when the nanoparticles are well-stabilized by phytochemicals, the antioxidant properties dominate [61].

Many of the phytochemicals associated with the synthesized Ag@Fe NPs have been linked to anti-inflammatory and antioxidant activities. For instance, Cis-vaccenic acid which accounted for the 17.19% composition of the NPs, have been reported to have anti-inflammatory effects by modulating immune response, and have also demonstrated antioxidant properties [62, 63]; Oleic acid has also been reported to have anti-inflammatory and antioxidant properties [64], while, n-Hexadecanoic acid (Palmitic acid) and its derivative, Hexadecanoic acid, methyl ester, are associated with pro-inflammatory effects [65]. Given that the NPs were capped with these antioxidant- and anti-inflammatory- rich compounds (Table 1), may have accounted for their antioxidant and anti-inflammatory properties.

The potential of Ag-Fe NPs to scavenge a wide spectrum of reactive oxygen species (ROS) were studied (Supplementary Table 3). The nanoparticles were tested for hydrogen peroxide, hydroxyl, nitric oxide and beta carotene-linoleic acid radicals, and the ability of the Ag-Fe NPs to scavenge these different reactive species indicates that these nanocomposites exhibit broad-spectrum antioxidant properties. This suggests that Ag-Fe NPs can neutralize reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are implicated in oxidative stress and inflammation.

The hydrogen peroxide generates hydroxyl radicals in cells, which, while not highly reactive, can occasionally be damaging to the cells [66]. This free radical and others are scavenged through reduction reaction. The reducing power of a substance correlates with its antioxidant activity; therefore, compounds with significant reducing capacity can demonstrate strong free radical scavenging activity [67]. In this study, the reductive potential of Ag-Fe NPs was demonstrated in a concentration-dependent manner. Figure 4a demonstrated a noteworthy representation of percentage inhibition of hydrogen peroxide radical by the silver-doped iron nanoparticle, with the highest percentage inhibition (p < 0.05) observed at 1000 µg/mL. The percentage H₂O₂ scavenged by Ag-Fe NPs at 500 and 1000 µg/mL were statistically (p < 0.05) similar to ascorbic acid (standard) at 500 µg/mL. This implies that the Ag-Fe NPs possess strong hydrogen radical scavenging activity, as they effectively neutralize H₂O₂ radicals, similar to ascorbic acid in a dose-dependent manner. This suggests that Ag-Fe NPS could be used as an alternative antioxidant agent. The statistical analysis suggests that there is no significant (p < 0.05) difference between the scavenging ability of the Ag-Fe NPs and ascorbic acid at these concentrations. This corresponds with the findings of [16]. The scavenging potential of the nanoparticles may be attributed to the nanoparticles’ narrow band gap energy (Eg), which reflects the gap between the conduction and the valence shells of the nanoparticles.

Fig. 4.

Scavenging (%) inhibition of Ag-Fe NPs with (A–D) showing hydrogen peroxide, hydroxyl, and nitric oxide radicals respectively. Bars with different alphabets are statistically significant (p < 0.05)

Nitric oxide (NO) is a reactive free radical generated by phagocytes and endothelial cells, leading to the formation of more reactive species such as peroxynitrite, which can breakdown to produce hydroxyl radicals [33]. In this study, the concentration of hydroxyl (Fig. 4b) and nitric oxide (Fig. 4c) radicals were markedly decreased at the 500 µg/mL of Ag-Fe NPs similar to 500 µg/mL of ascorbic acid, thus suggesting that the nanoparticles exhibited the same performance as the standard in scavenging the OH and NO radicals. The hydroxyl radical scavenging activity indicates that Ag-Fe NPs can neutralize highly reactive and damaging species formed in the Fenton reaction [57, 58]. Given that nitric oxide is integral to the etiology of inflammation, this may substantiate the anti-inflammatory property of the NPs. Nitric oxide scavenging suggests that the nanoparticles may interact with RNS, reducing nitric oxide’s potential to form peroxynitrite [16, 68, 69], a damaging compound. The nanoparticles’ similarity to ascorbic acid at 500 µg/mL underscores their relevance as effective alternatives to conventional antioxidants. The nitric oxide radical scavenging activity of Ag-Fe NPs further implies potential anti-inflammatory effects, as nitric oxide plays a key role in inflammatory signaling. Scavenging of NO helps to keep its concentration at physiological level thereby maintaining vascular homeostasis. This may have good implications for employing Ag-Fe NPs for suppression of leukocyte adhesion to endothelial cells, reducing excessive immune cell infiltration. Ag-Fe NPs may also be applicable in limiting platelet aggregation and thrombosis. Generally, the scavenging of nitric oxide by Ag-Fe NPs have far reaching therapeutic potentials.

Superoxide anion radicals are oxygen-centered radicals produced by the transfer of a single electron and are crucial in the formation of other reactive oxygen species (ROS) such as hydroxyl radicals, hydrogen peroxide, and singlet oxygen in biological systems, leading to lipid peroxidation [70]. Superoxide anion radicals are regarded as precursors to reactive free radicals that can interact with biological molecules, potentially causing tissue damage [71]. Superoxide pose a threat to cellular integrity due to their capacity to oxidize DNA and proteins [20]. This study demonstrates the concentration-dependent effectiveness of Ag-Fe NPs in scavenging superoxide anion radicals (Fig. 5a). There is no significant (p < 0.05) difference between the nanoparticles at 1000 µg/mL and ascorbic acid at 500 µg/mL. This implies that at 1000 µg/mL, Ag-Fe NPs were able to scavenge as much superoxide radicals as ascorbic acid (the standard drug) at 500 µg/mL. This finding aligns with [13] who reported dose-dependent effectiveness of Ag@Fe bimetallic nanoparticles in scavenging of free radicals.

Fig. 5.

Scavenging (%) inhibition of Ag-Fe NPs with (A, B), showing superoxide anion and β-carotene-linoleic acid radicals respectively. Bars with different alphabets are statistically significant (p < 0.05)

The β-carotene-linoleic acid assay is widely used to evaluate the antioxidant activity of compounds, particularly in biological or natural product research. The antioxidant properties of the NPs were assessed to determine their ability to inhibit the oxidation of the β-carotene [72, 73]. The β-carotenes-linoleic acid assay (Fig. 5b) indicated that at 1000 µg/mL, Ag@Fe can inhibit lipid peroxidation, which is critical for protecting cellular membranes and other lipophilic structures from oxidative damage. These findings suggest that Ag-Fe NPs have significant potential as versatile antioxidants capable of neutralizing multiple types of free radicals. This opens opportunities to their use in medical application, subject to further research into their mechanisms, and safety profile.

The stabilization of red blood cell membranes was investigated to elucidate the mechanism underlying the anti-inflammatory effects of Ag-Fe NPs (Supplementary Table 4). The nanoparticles successfully inhibited the heat induced hemolysis (Fig. 6a). At 500 µg/mL, the nanoparticles demonstrated a remarkable membrane stabilizing effect comparable to acetyl salicylic acid at the same concentration. Considering the similarity between the red blood cells and the lysosomal membrane, the nanoparticles may potentially impede the release of lysosomal contents from neutrophils at the site of inflammation [74]. Inhibition of heat-induced hemolysis by Ag-Fe NPs suggests potential benefits due to its ability to stabilize cell membranes, reduce oxidative stress, reduce hemolysis-driven inflammation and inhibit inflammation-driven damage. It is known that free hemoglobin and heme from lysed RBCs generate reactive oxygen species (ROS), contributing to tissue injury [75]. The protection conferred by the Ag-Fe NPs on RBCs from hemolysis could also reduce ROS production, leading to lower inflammation and tissue protection in diseases such as atherosclerosis (where oxidized lipids trigger chronic inflammation) and neuroinflammatory disorders (where iron from hemolysis can exacerbate brain inflammation).

Fig. 6.

Anti-inflammatory properties of Ag-Fe NPs, with (A, B) representing Heat-induced hemolysis and Protein denaturation respectively. Bars with different alphabets are statistically significant (p < 0.05)

The primary cause of inflammation is protein denaturation [76]. In this study, the potentials of the Ag-Fe NPs to inhibit protein denaturation was investigated. Silver-doped iron nanoparticles were found to inhibit the denaturation of albumin through heat. On heating, Ag@Fe NPs containing the reaction mixture had no cloudy appearance and precipitation, while the egg albumin without the NPs on heating turned into white precipitation. The inhibition increased with the increase in the concentration of Ag@Fe NPs with the maximum value of 52.90 ± 6.65 obtained at the concentration of 500 µg/mL. At this concentration, there was no statistically (p < 0.05) difference between the inhibition by the NPs and the reference drug, acetyl salicylic acid, which showed 78.26 ± 4.35 inhibition (Fig. 6b). This implies that at 500 µg/mL, Ag-Fe NPs are able to serve as alternative to acetyl salicylic acid in prevention of inflammation.

Implication of findings

The eco-friendly synthesis of the Ag@Fe reduces the environmental footprint of the nanoparticles, making the approach attractive for scalable and sustainable nanomedicine development.

The nanoparticles were functionalized with phytochemicals that impart their antioxidant and anti-inflammatory therapeutic properties, thus, improving the nanoparticles’ compatibility with biological systems.

Since the bimetallic structure resulted from the combination of the unique properties of silver, iron nanoparticles and plant chemicals; the synergistic effects among silver, iron nanoparticles and the phytochemicals would enhance the nanoparticles’ overall performance in antioxidant and anti-inflammatory activities.

The dual antioxidant and anti-inflammatory properties suggest that these nanoparticles could be integrated into therapies for diseases involving oxidative stress and inflammation, such as cancers, autoimmune disorders, and aging-related conditions.

Since the Ag@Fe nanoparticles have the ability to reduce inflammation by modulating inflammatory responses, possibly by inhibiting inflammatory mediators, they can be potentially applied in managing inflammatory conditions such as arthritis, asthma, inflammatory bowel disease, or used in topical formulations for wound healing or skin-related inflammatory disorders.

Conclusion

This study adopted an environmentally-friendly approach to synthesize silver-doped iron nanoparticles using leaf extract from Costus lucanusianus. Using UV-vis, FTIR, SEM, XRD, EDX. and TEM, the Ag@Fe NPs were characterized, and the synthesis of the Ag@Fe NPs were successful as evidenced by the XRD pattern, SEM, EDX, and TEM images which also validated the particle’s crystalline nature, trigonal and polydispersed morphology. Ag@Fe NPs, at 500 to 1000 µg/mL, exhibited significant (p < 0.05) antioxidant and anti-inflammatory properties comparable to the standard drugs. The narrow band gap energy of 3.27 eV, has been attributed to these enhanced functionalities. The implication of the findings suggest that Ag@Fe NPs can be applied to the treatment of many illnesses associated with reactive oxygen diseases. To determine the safety level of nanomaterials, toxicity study is recommended, and in vivo studies are recommended to confirm the nanoparticles’ reported in vitro antioxidant capabilities. Also recommended is the mechanistic insight into the molecular pathways involved in the observed antioxidant and anti-inflammatory effects.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualized and designed the experiment: CIIData curation: MAMFormal analysis: BKIInvestigation: PNAMethodology: AUNProject Administration: CJOSoftware: OOIResources: CEESupervision: NACValidation: VCSUVisualization: CKOWriting-Original draft: MAM and BKIWriting-review and editing: COU and CIIAll the authors read the manuscript and approved of it.

Funding

Not applicable.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study conformed to the principles of the Declaration of Helsinki, the Nigerian National Code of Health Research Ethics (2016), and the National Health Act (2014). The ethical approval was sought and obtained from the Research Ethics Committee, School of Biological Sciences, Federal University of Technology Owerri, with the approval Reference no as FUTO/SOBS/REC/2024/0015. Written informed consent was obtained from healthy, consenting human adult volunteers aged 18–50 years before blood collection, with the option of retaining the right to withdraw at any time without penalty.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.