Green synthesis of Ag–Fe₂O₃ nanocomposite using Adiantum venustum D. Don. extract and evaluation of its antimicrobial and antioxidant properties

Abstract

Green synthesis of metal oxide nanoparticles and nanocomposites has emerged as an eco-friendly alternative to conventional nanomaterial fabrication methods due to reduced chemical toxicity and improved biological compatibility. In the present study, Adiantum venustum D. Don extract was used for the green synthesis of an Ag–Fe₂O₃ nanocomposite, followed by physicochemical characterization and evaluation of antimicrobial and antioxidant activities. Synthesized nanocomposite was analyzed by Fourier transform infrared (FTIR), X-ray diffraction (XRD), Scanning electron microscopy and energy dispersive X-ray (SEM-EDX), Dynamic light scattering (DLS) and Zeta potential analysis. FTIR analysis confirmed the involvement of plant-derived functional groups in the reduction and stabilization of the nanocomposite, and XRD analysis confirmed the formation of crystalline α-Fe₂O₃ and the presence of diffraction peaks related to Ag. The SEM analysis revealed predominantly spherical nanoparticles with limited aggregation, while the EDX analysis confirmed the presence of Fe, O, and Ag. The synthesized nanocomposite showed concentration-dependent antibacterial properties against S. aureus (33.00 ± 0.50 mm) and E. coli (21.00 ± 0.60 mm) at a concentration of 1.0 mg/mL. MIC values were 0.25 mg/mL for S. aureus and 1.50 mg/mL for E. coli. Furthermore, antifungal activity against Fusarium oxysporum and high antioxidant activity in DPPH and FRAP assays were also observed. The improved biological activity of the Ag–Fe₂O₃ nanocomposite can be attributed to the synergistic effect of the incorporation of silver, the reactivity of the Fe₂O₃ surface, generation of reactive oxygen species, and phytochemical-assisted stabilization of the surface. The results indicate that the green synthesized Ag–Fe₂O₃ nanocomposite has potential biomedical applications.

Clinical trial Not applicable

Introduction

In recent years, nanotechnology has gained prominence in material science, focusing on the design and manipulation of particles with dimensions below 100 nm [1]; [2]. Nanomaterials, ranging from 1 to 100 nm, find applications in diverse fields such as medicine [3, 4, 5, 6], food packaging [7], optics, electronics [8, 9], agriculture [10], environmental remediation [11], and wastewater treatment [12, 13]. Metal and metal oxide NPs have potential applications in antidiabetic therapy [14], biosensing systems [15, 16], anticancer activity [17] anti-inflammatory [18] and in antioxidant applications, and antibacterial formulations. NPs exhibit unique optical, chemical, electrical, and photoelectrochemical properties, making them significant for various biological applications [19]; [20]. Their precision in treating challenging diseases is noteworthy. NPs exhibit effective antimicrobial properties by causing DNA damage, protein breakdown and cell wall degradation [21].

Traditional synthesis methods are limited in terms of biocompatibility, toxicity, and environmental impact [22]. The biological sources, however, provide a means of biocompatible, non-toxic, and eco-friendly NP synthesis [23]. Biological sources such as plants, algae, fungi, and bacteria have been utilized for NP synthesis [24], with a growing preference for plant extracts combined with specific acids or metal salts such as manganese (Mn) [25], platinum (Pt) [26], gold (Au) [27], silver (Ag), copper (Cu) [28], cobalt (Co) [29], titanium (Ti) [30], zinc (Zn) [31], and Iron (Fe) [32]. Such combinations offer an effective platform for the in vivo synthesis of NPs as well as a bio-reactive platform [14]. Plants are particularly suitable for this purpose because of their wide variety of phytochemicals, including flavonoids, terpenoids, saponins, amino acids, proteins, reducing sugars, carbohydrates, and nitrogenous bases [33, 34, 35].

Iron oxide (α -Fe₂O₃) is a widely recognized metallic NPs with biocompatibility and magnetic properties. These features make it suitable for disease management and treatment of infections [36]. Fe₂O₃NPs have a broad spectrum of applications, including catalysts, dyes, antibiotic degradation, and heavy metal adsorption. Their antimicrobial activity against disease causing bacteria and fungi has been extensively studied [37]. In microbial systems, Fe₂O₃NPs bind with cell membrane, induce oxidative stress, and disrupt cellular functions, thereby exhibiting antibacterial and antifungal properties against pathogenic microorganisms. Incorporation of silver into Fe₂O₃ may further improve antimicrobial efficiency due to strong silver–microbial interactions and broad-spectrum activity. Therefore, green-synthesized Ag–Fe₂O₃ nanocomposite represents an eco-friendly platform with potential antimicrobial and antioxidant applications [38].

Adiantum venustum D. Don, with a rich history in ethnomedicine, is used to treat snake and scorpion stings. Its fronds possess astringent, diuretic, and expectorant properties, while its rhizomes aid in healing wounds and minor injuries [39]. A decoction of the leaves is effective in treating cough, stomach pain, and urinary issues. The fronds are traditionally used as an expectorant and to alleviate dry cough, and they are employed in the treatment of various ailments, including chest inflammation, biliousness, diarrhea, common cold, digestive disorders, vomiting, diuresis, and menstrual irregularities. They are also traditionally used for managing fever, ophthalmia, headache, piles, jaundice, urinary ailments, and wounds, and are reported to have tonic and antitumor uses [40].

Despite its long ethnomedicinal history, A. venustum has not been sufficiently explored as a biological source for the biosynthesis of Ag–Fe₂O₃ nanocomposites or for evaluating nanocomposite -mediated antimicrobial activity. Therefore, the present study aimed to develop an environmentally friendly and cost-effective approach for producing Ag–Fe₂O₃ nanocomposite using A. venustum extract. The synthesized nanocomposite was characterized using FTIR, XRD, SEM, EDX, DLS, and zeta potential analyses to confirm its functional groups, crystalline structure, morphology, elemental composition, particle-size distribution, and colloidal stability. In addition, its antimicrobial and antioxidant activities were evaluated to determine its potential biological applications. This study contributes to the understanding of A. venustum-mediated nanocomposite synthesis and its possible role in pathogen control and biomedical applications.

Materials and methods

Materials and collection of plant sample

All chemicals and reagents used in this study were of analytical grade and used without further purification. Ferric chloride hexahydrate (FeCl₃·6 H₂O, ≥ 98% purity) and silver nitrate (AgNO₃, ≥ 99% purity) were purchased from Sigma-Aldrich (USA). Methanol, potassium ferricyanide, ferric chloride, trichloroacetic acid (TCA), sodium phosphate buffer components, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were obtained from Merck (Germany). Ascorbic acid used as the positive antioxidant standard was procured from Sigma-Aldrich (USA). Sabouraud Dextrose Agar (SDA) and Mueller–Hinton Agar (MHA) media used for antifungal and antibacterial assays were purchased from Oxoid Ltd. (UK). Deionized water was used throughout the experimental procedures.

Fresh Adiantum venustum D. Don. plants were collected from Kafir Banda (34°58′53″ N, 72°47′50″ E; 2519 m a.s.l (meters above sea level) in District Shangla, Khyber Pakhtunkhwa, Pakistan. This species typically grows on rocks and forest floors. The species is not protected, and not listed as endangered. Therefore, no specific permissions or licenses were required for its collection. All procedures complied with relevant institutional, national, and international guidelines and legislation. The collected specimens were carefully identified using standard literature and previous fern studies in Pakistan. To ensure authenticity, the specimens were compared with authenticated herbarium samples from the National Herbarium of Pakistan (NARC), the Herbarium of Pakistan (ISL) at Quaid-i-Azam University (QAU), and the Botanical Science Division of the Pakistan Museum of Natural History (PMNH) in Islamabad. Plant names and families were verified using The Plant List (TPL) and International Plant Name Index (IPNI). The plant species was identified by Syed Nasar Shah through a comparison with herbarium specimens housed at the Herbarium of Pakistan (ISL), Quaid-i-Azam University, and the National Herbarium of Pakistan (PARC), Islamabad. Voucher specimens were assigned the voucher number SNS-22 and deposited in both herbaria for future reference.

Extract preparation of A. venustum

The leaves were washed with sterile distilled water to remove any detritus and then dried in the shade for 7 days. The cleaned and dried A. venustum leaves were crushed into a fine powder using a mortar and pestle. To prepare the extract, 10 g of leaf powder was combined with 100 mL of distilled water and kept at room temperature for 36 h. The mixture was boiled for 5 min and then incubated in a water bath at 80 °C for 30 min. The mixture was then cooled and passed through a muslin cloth to eliminate any solid residues. The resulting liquid was filtered using Whatman filter paper no. 42 and used for the synthesis of the nanocomposite synthesis [41].

Biosynthesis of Ag-Fe₂O₃ nanocomposite

The synthesis of Ag–Fe₂O₃ nanocomposite was carried out through a sequential green-mediated process following the protocol of Al-Zahrani et al. [42] with few changes. Ferric chloride hexahydrate was used as the iron precursor, while A. venustum extract acted as a natural reducing and stabilizing agent because of its phytochemical constituents. During the initial reaction, Fe³⁺ ions interacted with the plant extract under heating and stirring, leading to nucleation and formation of iron oxide precursor particles, as indicated by the color change from light green to brownish-black, as shown in Fig. 1. Silver nitrate was then introduced as the Ag⁺ source. The phytochemicals present in the extract facilitated partial reduction of Ag⁺ and promoted the anchoring or deposition of silver species onto the Fe₂O₃ nanocomposite surface. Subsequent centrifugation, drying, and calcination at 400 °C promoted crystallization of the α-Fe₂O₃ phase and formation of the Ag–Fe₂O₃ nanocomposite. The Fe: Ag precursor ratio was maintained at approximately 10:1. The final reaction mixture showed a pH of approximately 8.2, as measured after mixing the plant extract with the metal precursors; however, no external base or pH-adjusting agent was added during synthesis.

Fig. 1.

Schematic representation of the green synthesis of Ag-Fe₂O₃ nanocomposites using A. venustum extract

Characterization of Ag-Fe₂O₃ nanocomposite

The nanocomposite was thoroughly validated at the Central Resource Laboratory, Department of Physical Chemistry, University of Peshawar, using a range of analytical techniques. Powder X-ray diffraction (XRD) (JDX-3532, JEOL, Tokyo, Japan, ) was used to analyze the crystalline structure of the nanocomposite. Fourier-transform infrared spectroscopy (FTIR) (Shimadzu FTIR 8400 Spectrometer) was used to identify and evaluate the functional groups in the samples. Scanning electron microscopy (SEM) was used to investigate the surface characteristics and determine the average particle sizes. Moreover, energy-dispersive X-ray (EDX) analysis coupled with SEM was conducted to determine the elemental composition of the nanocomposite. The hydrodynamic shell size was measured using a zeta sizer analyzer. Statistical analyses were conducted using SPSS software (version 16).

Antifungal assay of Ag-Fe₂O₃ nanocomposite

Antimicrobial activity assessment was conducted at the Microbiology Department of QAU, Islamabad. In vitro mycelial growth measurements were performed according to a previously described protocol [43]. An antifungal assay to evaluate fungal inhibition was conducted using Sabouraud Dextrose Agar (SDA) plates incubated at 32 ± 2 °C. The experimental setup included control plates and plates containing Ag-Fe₂O₃ nanocomposite at various concentrations (1.0, 0.5, and 0.25 mg/mL). A 5 mm piece of a three-day-old pure fungal culture was placed at the center of each Petri dish for in vitro analysis. On the 8th day, the impact of the nanocomposite was evaluated by computing the average of the two perpendicular radii (in mm) of each fungal colony.

Antibacterial assay of Ag-Fe₂O₃ nanocomposite

The Well Diffusion Method was used to perform the antibacterial evaluation of the green-synthesized Ag-Fe₂O₃ nanocomposite. The bacterial strains tested were S. aureus and E. coli, which are classified as gram-positive and gram-negative bacteria, respectively. Bacterial cultures were obtained from the Microbiology Laboratory at QAU in Islamabad, Pakistan. Mueller–Hinton agar was prepared, sterilized by autoclaving at 121 °C for 20 min, and poured into Petri dishes after cooling. Bacterial strains were inoculated onto agar surfaces. The well diffusion method was used with different concentrations of Ag-Fe₂O₃ nanocomposite (1.0, 0.75, and 0.5 mg/mL), along with 10 µL of positive control (ciprofloxacin), which were loaded onto the bacterial cultures. The antibacterial activity was determined by measuring the zone of inhibition after incubation at 37 °C for 24 h [44, 45].

The minimum inhibitory concentration (MIC) of Ag-Fe₂O₃ nanocomposite against S. aureus and E. coli was determined using the broth microdilution method. Different nanocomposite concentrations (0.25, 0.50, 1.00, 1.50, and 2.00 mg/mL) were prepared in sterile nutrient broth and inoculated with 100 µL freshly prepared bacterial cultures. The inoculated tubes were incubated at 37 °C for 24 h. The MIC value was recorded as the lowest concentration of Ag-Fe₂O₃ nanocomposite showing no visible bacterial growth after incubation [46]. All experiments were performed in triplicate, and results were expressed as mean ± SD.

Antioxidant activity

According to the procedures outlined by [47]; [48] with few changes, a mixture of 2 mL of methanol and 1 mL of a methanolic solution containing 0.1 mM DPPH free radicals was prepared using a range of Ag-Fe₂O₃ nanocomposite concentrations, from 50 to 250 µg/mL. After thorough mixing, the mixture was incubated at ambient temperature in the dark for 30 min. The absorbance was then measured at 517 nm using a Shimadzu UV-1800 spectrophotometer [49]. Methanol containing DPPH solution without nanocomposite was used as the blank/negative control, whereas ascorbic acid served as the positive reference standard. The following equation was used to evaluate the DPPH antioxidant potential (Eq. 1):

1

Where Ac517 is the absorbance of the control and As517 is the absorbance of the sample.

The FRAP test, based on the Ferric Reducing Antioxidant Power, was performed using the approach outlined by [50], with a few changes. Different quantities of Ag-Fe₂O₃ nanocomposite (varying from 50 to 250 µg/mL) were combined with 2.5 mL of a sodium phosphate buffer (PBS) solution at a pH of 6.6 and 2.5 mL of a potassium ferricyanide solution with a concentration of 1%. The solution was incubated at 50 °C for 20 min. After incubation, a solution of trichloroacetic acid with a concentration of 10% weight/volume was introduced. The solution was then centrifuged for 10 min at 3000 rpm. Deionized water (2.5 mL) and a 0.1% ferric chloride solution were added to the liquid phase. Utilizing a Shimadzu UV-1800 spectrophotometer, the absorbance was measured at 700 nm. A reagent mixture without nanocomposite was used as the blank control, while ascorbic acid was employed as the positive reference standard. The antioxidant potential, as indicated by the FRAP experiment, was estimated using the following equation (Eq. 2):

2

The antioxidant capacity was quantified using an ascorbic acid calibration curve. The concentration of antioxidant activity was calculated using the linear regression equation x = y/0.0026 (R² = 0.9673), where x represents the antioxidant capacity expressed as µg ascorbic acid equivalents per mL (µg AAE/mL) and y is the measured absorbance at the selected wavelength. The coefficient of determination (R² = 0.9673) indicates good linearity of the calibration curve over the tested concentration range.

Statistical analysis

All biological experiments, including antimicrobial and antioxidant assays, were performed in triplicates (n = 3). Data are expressed as mean ± standard deviation (SD). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test using SPSS (version 16), with p-values < 0.05 considered significant.

Results

Characterization of nanocomposite

FT-IR of biosynthesized Ag-Fe₂O₃ nanocomposite

FTIR analysis was performed to identify the functional groups involved in the reduction, capping, and stabilization of the biosynthesized Ag–Fe₂O₃ nanocomposite as shown in Fig. 2. The broad absorption band at approximately 3390.35 cm⁻¹ corresponds to O–H stretching vibrations, indicating the presence of hydroxyl groups from phenolic and alcoholic compounds in A. venustum extract. The band at 2922.11 cm⁻¹ is attributed to C–H stretching vibrations of aliphatic compounds, while the peak at 1738.15 cm⁻¹ indicates C = O stretching of carbonyl/ester groups. The absorption band at 1639.81 cm⁻¹ may be assigned to amide C = O stretching or N–H bending vibrations, suggesting the involvement of proteinaceous or amine-containing phytochemicals. Peaks around 1381.65, 1215.41, 1108.41, and 1042.39 cm⁻¹ correspond to C–N, C–O, and C–O–C stretching vibrations, confirming the contribution of plant-derived metabolites as natural reducing and stabilizing agents. The low-frequency bands at 667.12, 533.40, and 512.99 cm⁻¹ are associated with Fe–O and Ag–O/metal–oxygen vibrations, confirming the formation of Ag–Fe₂O₃ nanocomposite. Overall, the FTIR spectrum supports the role of A. venustum phytochemicals in nanocomposite biosynthesis and surface stabilization.

Fig. 2.

FT-IR spectra of the Ag-Fe₂O₃ nanocomposite synthesized using A. venustum extract

XRD of biosynthesized Ag-Fe₂O₃ nanocomposite

The X-ray diffraction (XRD) pattern of the biosynthesized Ag–Fe₂O₃ nanocomposite confirmed the crystalline nature of the synthesized nanocomposite (Fig. 3). The diffraction peaks observed at 2θ values of approximately 24.1°, 33.1°, 35.6°, 49.5°, and 54.1° were indexed to the (012), (104), (110), (024), and (116) planes of rhombohedral α-Fe₂O₃, respectively, in good agreement with the standard hematite phase (JCPDS card no. 33–0664). In addition, characteristic diffraction peaks detected at approximately 38.1°, 44.3°, 64.4°, and 77.4° corresponded to the (111), (200), (220), and (311) planes of face-centered cubic (FCC) metallic silver, respectively, matching JCPDS card no. 04-0783. The strong diffraction intensity and sharp peaks indicated good crystallinity of the synthesized nanocomposite. The coexistence of characteristic reflections of both α-Fe₂O₃ and metallic Ag confirmed the successful formation of Ag–Fe₂O₃ nanocomposite. The average crystallite size calculated using the Scherrer equation was approximately 40.3 nm.

Fig. 3.

XRD pattern of biosynthesized Ag-Fe₂O₃ nanocomposite through A. venustum extract

Energy-dispersive X-ray analysis (EDX) and scanning electron microscopy (SEM) of biosynthesized Ag-Fe₂O₃ nanocomposite

SEM images depicted the surface morphology of green-synthesized Ag-Fe₂O₃ nanocomposite (Fig. 4 (A), revealing spherical shapes with an average size of 54.50 ± 1.69 nm, as illustrated in Fig. 4 (B). Particle size was estimated from SEM micrographs by measuring the external diameter of clearly visible and well-separated nanocomposite particles using the calibrated SEM scale bar and image-analysis software (ImageJ^®). The obtained values were used to generate the particle-size histogram, and the average particle size was reported as mean ± SD. The elemental composition of the synthesized green nanocomposite is illustrated in Fig. 4 (C), showing weighted percentages of iron, oxygen, and silver in A. venustum extract-fabricated Ag-Fe₂O₃ nanocomposite as 75.2%, 19.94%, and 2.90%, respectively. The identification of iron and oxygen peaks validated the formation of iron oxide in our samples, demonstrating results similar to those of the EDX analysis, highlighting the presence of Fe, Ag, and O peaks.

Fig. 4.

(A) SEM micrograph of synthesized Ag-Fe₂O₃ nanocomposite using A. venustum extract, (B) Histogram of the biosynthesized Ag-Fe₂O₃ nanocomposite, (C) EDX spectra of synthesized Ag-Fe₂O₃ nanocomposite using A. venustum extract, (D) DLS zeta sizer analysis Ag-Fe₂O₃ nanocomposite using A. venustum extract

DLS analysis of biosynthesized Ag-Fe₂O₃ nanocomposite

Dynamic light scattering (DLS) analysis of Ag-Fe₂O₃ nanocomposite synthesized using A. venustum extract showed a sharp size distribution peak at approximately 69.67 nm, indicating a narrow and uniform particle size distribution, as shown in Fig. 4 (D). The green synthesis approach is environmentally friendly and uses a natural plant extract to reduce and stabilize the nanostructures, avoiding the use of toxic chemicals. These green methods have been highlighted in recent studies employing different plant extracts and the resulting NPs have shown interesting biomedical and environmental applications due to their biocompatibility and low environmental impact [51].

The zeta potential distribution of the biosynthesized Ag-Fe₂O₃ nanocomposite, as seen in Fig. 5, is positively charged with a peak of 43.62 mV which indicates good colloidal stability of the nanocomposite in suspension. A high zeta potential indicates excellent colloidal stability, which is crucial for its efficient use. Green synthesis, which involves the use of plant extracts or other biological methods, offers an environmentally friendly manufacturing process, avoiding the use of harmful chemicals. NPs provide a great degree of versatility for medical, environmental remediation, and industrial processes owing to the combination of silver antibacterial activities and iron oxide magnetic capabilities. The zeta potential is important for ensuring the stability, uniform dispersion, and efficacy of NPs in many applications.

Fig. 5.

Zeta potential distribution of the green-synthesized Ag–Fe₂O₃ nanocomposite

Antimicrobial activity

Antifungal assay of biosynthesized Ag-Fe₂O₃ nanocomposite

Table 1; Fig. 6 present the percentage inhibition of Fusarium oxysporum by Ag-Fe₂O₃ nanocomposite at different concentrations (1.0, 0.5, and 0.25 mg/mL). The highest inhibition rate of 70.34% was observed at a concentration of 1.0 mg/mL. This was followed by an inhibition of 58.94% at a concentration of 0.5 mg/mL. The lowest growth inhibition percentage (36%) was recorded at a concentration of 0.25 mg/mL of Ag-Fe₂O₃ nanocomposite.

Table 1.

Percentage growth inhibition of F. oxysporum was determined after in vitro treatment with Ag-Fe₂O₃ nanocomposite synthesized using an extract from A. venustum

Treatments	F. oxysporum (% inhibition)
Ag-Fe₂O₃ nanocomposite concentration (mg/mL)
0.25	36.50 ± 2.11
0.5	58.94 ± 3.27
1.0	70.34 ± 2.84

Values are expressed as mean ± SD of triplicate measurements (n = 3)

Fig. 6.

Growth inhibition of F. oxysporum by Ag-Fe₂O₃ nanocomposite at various concentrations: (A)1.0, (B) 0.5, (C) 0.25, (D) = 0

In Vitro antibacterial assay of biosynthesized Ag-Fe₂O₃ nanocomposite

Table 2; Fig. 7 demonstrate the dose-dependent antibacterial efficacy of the green-synthesized Ag-Fe₂O₃ nanocomposite. One-way ANOVA revealed highly significant differences (p < 0.0001) across all treatments for both S. aureus (F = 667) and E. coli (F = 177). For S. aureus, Tukey HSD analysis showed that while the positive control (36.7 mm) was the most effective, there was no significant difference between the 0.5 mg/mL and 1.0 mg/mL concentrations (both falling into Group B), suggesting a plateau in antibacterial activity at these doses. In contrast, for E. coli, all four treatment groups were significantly different from one another (p < 0.05), with the 1.0 mg/mL concentration (21.0 mm) showing the highest nanocomposite -mediated inhibition, although still significantly lower than the ciprofloxacin control (22.97 mm).

Table 2.

Zone of inhibition (mm) and MIC of bacterial strains treated with different concentrations of Ag-Fe₂O₃ nanocomposite (Mean ± SD, n = 3)

Ag-Fe₂O₃ nanocomposite (mg/mL)	Zone of inhibition (mm) S. aureus	Zone of inhibition (mm) E. coli
0.25	22.00 ± 0.42c	13.97 ± 0.51d
0.5	32.00 ± 0.60b	17.97 ± 0.46c
1.0	33.00 ± 0.50b	21.00 ± 0.60b
C+	36.70 ± 0.60a	22.97 ± 0.45a
Estimated MIC	0.25 mg/mL	1.50 mg/mL

Note: Means followed by different superscript letters (a-d) within a column are significantly different from each other (p < 0.05) according to Tukey’s HSD test

Fig. 7.

Growth inhibition (a) S. aureus and (b) E. coli by Ag-Fe₂O₃ NPs at different concentrations; (A) positive control (C⁺), (B) Ag-Fe₂O₃ nanocomposite concentration (1.0 mg/mL), (C) Ag-Fe₂O₃ nanocomposite concentration (0.5 mg/mL), (D) Ag-Fe₂O₃ nanocomposite concentration (0.25 mg/mL)

The MIC analysis demonstrated concentration-dependent antibacterial activity of Ag–Fe₂O₃ nanocomposite against both tested bacterial strains. Based on the broth microdilution assay, the MIC value against S. aureus was observed at 0.25 mg/mL, indicating high sensitivity of the Gram-positive bacterium to the synthesized nanocomposite. In contrast, E. coli showed comparatively lower sensitivity, with an MIC value of 1.50 mg/mL as shown in Table 2. The greater susceptibility of S. aureus may be associated with the absence of an outer membrane barrier, which facilitates nanocomposite interaction with the bacterial cell wall. These findings support the enhanced antibacterial potential of Ag–Fe₂O₃ nanocomposite against Gram-positive bacteria.

DPPH assay of biosynthesized Ag-Fe₂O₃ nanocomposite

The DPPH assay is commonly used to measure the antioxidant potential of various substances. DPPH, a stable free radical, reacts with hydrogen or electrons from the sample, resulting in a color change from purple to yellow as diphenyl picrylhydrazine is formed. The antioxidant activity of the biosynthesized Ag-Fe₂O₃ nanocomposite and ascorbic acid was assessed using a Shimadzu UV-1800 spectrophotometer, as shown in Fig. 8 (A). Significant free-radical scavenging activity was observed at all tested concentrations, with the highest activity (70.00%) at a concentration of 250 µg/mL for the biosynthesized Ag-Fe₂O₃ nanocomposite.

Fig. 8.

Antioxidant activity of the biosynthesized Ag-Fe₂O₃ nanocomposite (A) DPPH assay, (B) FRAP assay

FRAP assay biosynthesized Ag-Fe₂O₃ nanocomposite

The ferric-reducing antioxidant power (FRAP) assay was performed to evaluate the antioxidant activity of the Ag-Fe₂O₃ nanocomposite. Ascorbic acid, a potent secondary antioxidant, was used as the reference standard owing to its ability to neutralize free radicals and terminate free-radical chain reactions. The hydroxyl groups in vitamin C contribute to its strong antioxidant properties through its polyhydroxyl structure and free radical neutralization capability. In the assay, the potassium ferrocyanide (K₃Fe(CN)₆) complex was precipitated and removed using trichloroacetic acid (TCA). The addition of FeCl₃ resulted in the formation of a green to blue complex known as “Berlin Blue,” indicating the reduction potential of the sample. The ability to reduce ferric (Fe³⁺) ions to ferrous (Fe⁺²) ions is a key measure of antioxidant activity. Figure 8 (B) shows that the inhibition ranged from 47.77 to 81.11 µg AAE/mL for biosynthesized Ag-Fe₂O₃ nanocomposite, volumes between 50 and 250 µg/mL, compared to an ascorbic acid standard curve. The synthesized nanocomposite demonstrated considerable antioxidant capacity, which may be attributed to its ability to stabilize radicals through mechanisms such as direct proton transfer, electron transfer, hydrogen atom transfer, and successive proton loss and electron transfer.

Discussion

Plant-mediated NPs synthesis has received considerable attention as an eco-friendly alternative to chemical and physical synthesis methods [52]. Fern extracts represent promising biological reducing and stabilizing agents due to their rich phytochemical composition, although their use in nanomaterial synthesis remains relatively unexplored than higher plants. Previous studies have reported fern-mediated synthesis of Ag and Au NPs with antibacterial, antioxidant, mosquitocidal, anticancer, antidiabetic, and dye-remediation potential. In this context, the present study expands fern-based nanotechnology by using A. venustum extract for green synthesis of Ag–Fe₂O₃ nanocomposite and evaluating its physicochemical, antimicrobial, and antioxidant properties.

FTIR analysis supported the involvement of plant-derived functional groups in nanocomposite formation and stabilization. Absorption bands corresponding broadly to hydroxyl, carbonyl/amide, C–O, C–N, and metal–oxygen vibrations indicate that organic residues from A. venustum extract remained associated with the nanocomposite surface. These biomolecules are likely to contribute to metal ion reduction during biosynthesis and subsequently act as capping agents that limit uncontrolled growth and aggregation. Similar roles of aldehydes, carboxylic acids, flavonoid-like compounds, ketones, terpenoid-like compounds, amides, and phenolic constituents in metal ion reduction and nanocomposite stabilization have been reported previously [51]; [53, 54]. However, FTIR evidence should be interpreted as functional-group evidence rather than direct identification of exact phytochemical compounds. Therefore, specific compounds responsible for reduction and capping cannot be confirmed without HPLC, GC-MS, LC-MS, or NMR analysis.

The XRD pattern confirmed crystalline Ag–Fe₂O₃ nanocomposite formation. Reflections at approximately 24.1°, 33.1°, 35.6°, 49.5°, and 54.1° were assigned to rhombohedral α-Fe₂O₃ planes, matching hematite phase data [55]. Additional reflections at approximately 38.1°, 44.3°, 64.4°, and 77.4° corresponded to metallic Ag planes, supporting the presence of silver within the nanocomposite system [56]; [57]. Therefore, the present results support the formation of an Ag–Fe₂O₃ nanocomposite rather than definitive lattice-level silver doping, which would require additional evidence from advanced techniques such as XPS, HRTEM-SAED, Rietveld refinement, or elemental mapping. The calculated crystallite size of 40.30 nm indicates nanoscale crystalline domains, while absence of major impurity peaks supports successful formation of the intended nanocomposite. The functional properties of iron-based nanomaterials are strongly influenced by their crystal structure [58].

SEM analysis showed predominantly spherical nanoparticles with limited aggregation. Such aggregation is commonly observed in iron oxide-based nanocomposite due to high surface energy, magnetic interactions, and drying effects during SEM sample preparation. Differences among XRD, SEM, and DLS sizes are therefore expected. The differences among the particle sizes obtained from XRD, SEM, and DLS are expected because each technique measures a different physical property of the material. XRD determines the average crystallite size of the crystalline domains, whereas SEM provides the dry-state particle morphology and particle size from the solid sample surface. In contrast, DLS measures the hydrodynamic diameter of particles dispersed in colloidal suspension, which includes the particle core together with the surrounding solvation layer, surface-bound phytochemical capping molecules, and any small agglomerates present in suspension [59]. Consequently, DLS typically yields larger particle sizes than XRD and SEM, consistent with the phytochemical capping and colloidal behavior of the synthesized Ag–Fe₂O₃ nanocomposite [60, 61].

EDX analysis confirmed the elemental presence of Fe, O, and Ag, supporting Ag–Fe₂O₃ nanocomposite formation. Although Ag percentage was relatively low, its detection by EDX, together with Ag-related XRD peaks, confirms the presence of silver in the synthesized material. Low Ag content may reflect the Fe: Ag precursor ratio, surface-level distribution of Ag species, and partial dispersion of silver over the Fe₂O₃ matrix. The positive zeta potential value of + 43.62 mV indicates excellent colloidal stability. Such stability may result from adsorption of protonated plant-derived molecules on the nanocomposite surface, which can provide electrostatic repulsion and steric protection against aggregation. Zeta potential values between ± 40 and ± 60 mV are generally considered highly stable, supporting good dispersion behavior of the synthesized NPs [62].

The antimicrobial activity of Ag–Fe₂O₃ nanocomposite may be explained through multiple interacting mechanisms [63]. Its nanoscale size and high surface area allow close contact with microbial cell walls and membranes, leading to membrane disruption, altered permeability, and leakage of intracellular components [64]. Silver species may further contribute through Ag⁺ release, interaction with thiol-containing proteins, enzyme inhibition, and interference with DNA replication and respiratory pathways [65, 66]. Fe₂O₃ may also contribute to oxidative stress through reactive oxygen species generation under suitable conditions. These combined mechanisms may explain the dose-dependent inhibition observed against S. aureus, E. coli, and F. oxysporum. Similar antimicrobial effects have been reported for plant-mediated iron oxide NPs against pathogenic bacteria and fungi [67]; [68].

The higher sensitivity of S. aureus compared with E. coli may be linked to structural differences between Gram-positive and Gram-negative bacterial cell wall structure. Gram-negative bacteria possess an outer membrane that restrict NPs penetration and reduce susceptibility, whereas Gram-positive bacteria have a thick peptidoglycan layer but lack an outer membrane barrier [69, 70]. This difference may allow stronger interactions between the NPs and bacterial cells in S. aureus. Antifungal activity against F. oxysporum may be attributed to nanocomposite interaction with fungal cell walls, oxidative stress induction, and disruption of membrane integrity [71, 72]. However, these mechanisms were not directly investigated in the present work and therefore remain proposed explanations requiring further experimental verification. These effects suggest that Ag–Fe₂O₃ nanocomposite may offer broader antimicrobial action than plant extract alone. Although the present study demonstrated the antimicrobial activity of the synthesized Ag–Fe₂O₃ nanocomposite, the antimicrobial activity of the A. venustum extract alone was not evaluated under identical experimental conditions. Therefore, the individual contribution of the plant extract to the overall antimicrobial performance could not be directly assessed. Future studies should include comparative evaluations of the crude A. venustum extract, Fe₂O₃ nanoparticles, Ag nanoparticles, and the Ag–Fe₂O₃ nanocomposite to better distinguish their individual and synergistic antimicrobial effects.

Antioxidant activity observed in DPPH and FRAP assays may be associated with surface-bound plant-derived functional groups and redox-active NPs surfaces. Hydroxyl, carbonyl, amide, C–O, and C–N groups identified through FTIR may participate in electron donation, hydrogen transfer, and radical stabilization. The Fe₂O₃ component may also contribute through redox behavior, while Ag and phytochemical capping molecules may enhance surface reactivity. However, antioxidant activity cannot be assigned to specific flavonoids, terpenoids, or phenolics without compound-level analysis. Therefore, the activity is best interpreted as a combined effect of Ag–Fe₂O₃ nanocomposite structure and A. venustum-derived surface functional groups. The antioxidant activity of Ag–Fe₂O₃ nanocomposite was supported by DPPH and FRAP responses.

Compared with crude plant extract, Ag–Fe₂O₃ nanocomposite offers several potential advantages, including higher surface reactivity, improved stability, nanoscale interaction with microbial cells, and possible synergy between Ag, Fe₂O₃, and phytochemical capping molecules [73]. The nanocomposite form may also protect or immobilize bioactive plant-derived groups on the surface, allowing prolonged interaction with microbial or radical species. Nevertheless, future studies should directly compare nanocomposite with crude extract, Fe₂O₃-only NPs, and Ag-only NPs to define individual and synergistic contributions. Further characterization studies, including elemental mapping, XPS, ICP-OES, and comprehensive phytochemical profiling, are warranted to provide deeper insights into the elemental composition, surface chemistry, and phytochemical contributions of the synthesized nanocomposite. In addition, future studies should include in vivo and toxicological evaluations to assess its biological safety, biocompatibility, and therapeutic potential. Cytotoxicity studies and comparative evaluation with the plant extract alone are also recommended to better establish the biological efficacy and safety of the nanocomposite before its potential biomedical applications can be confidently considered.

Conclusion

This study demonstrates the successful green synthesis of the Ag–Fe₂O₃ nanocomposite using Adiantum venustum extract as a biological reducing and stabilizing source. FTIR, XRD, SEM-EDX, DLS, and zeta potential analyses confirmed the formation of a crystalline, stable, and phytochemical-capped nanocomposite. The antimicrobial and antioxidant findings indicate that the synthesized Ag–Fe₂O₃ nanocomposite showed strong potential for pathogen control, antioxidant applications, and the development of eco-friendly antimicrobial materials. Its biological activity is linked to the combined contribution of Ag, Fe₂O₃, and surface-bound plant-derived functional groups. Future studies should focus on cytotoxicity assessment, comparison with crude A. venustum extract, testing against broader microbial strains, and advanced analyses such as HPLC, GC-MS, LC-MS, XPS, and elemental mapping to identify active phytochemicals and confirm silver distribution. Overall, A. venustum represents a promising green resource for developing biologically active Ag–Fe₂O₃ nanocomposites.

Author contributions

Conceptualization: AN, NA. Data Curation: MI, AN. Formal Analysis: NA, SNS. Validation: MNK, SNS. Visualization: MNK, MI. Investigation: WS, MI. Supervision: NA, MI. Project Administration: WS, MNK. Resources: SAZ, MSA. Methodology: AN, SNS. Software: JAM, NAC, AV. Writing-Original draft: SNS, NA. Writing – Review and Editing; SAZ, MSA, JAM, Funding Acquisition: NAC, AV.

Funding

Not applicable .

Data availability

All experimental supporting data and procedures are available within this article.

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Conflict of interest

The authors declare that they have no conflicts of interest to disclose.

Competing interests

The authors declare no competing interests.