Green synthesis of iron (Fe) nanoparticles using Plumeria obtusa extract as a reducing and stabilizing agent: Antimicrobial, antioxidant and biocompatibility studies

2.1 Reagents/chemicals

The chemicals/reagents used of analytical grade, including ferrous acetate (Fe(C₂H₃O₂)₂), deionized water, dimethyl sulfoxide (DMSO), streptomycin, sabouraud dextrose, Fluconazole, resazurin indicator, DPPH, ascorbic acid (AA) (C₆H₈O₆), ferric chloride hexahydrate (FeCl₃: 6H₂O), butylated hydroxyl toluene (BHT), sodium nitroprusside, fresh human blood, ethylene diamine tetra acetate (EDTA), phosphate-buffer saline (PBS), Triton X-100, MTT, Doxorubicin and Cisplatin, Hela cells and AU565 cell lines, DMEM reagent, bovine serum, penicillin. The Escherichia coli and Bacillus subtilis, Aspergillus niger and Schizophyllum commune pure cultures were obtained from Biochemistry Laboratory, University of Agriculture Faisalabad.

2.2 Extract preparation

The P. obtuse fresh leaves were taken University of Agriculture Faisalabad, Pakistan (Botanical Garden). The leaves were properly rinsed using water for cleaning purpose. A 20 g fresh leaves of P. obtusa were used into 100 mL of deionized water to prepare extract. The mixture was stirred continuously and heated at 80 °C for ∼60 min. The resulting solution was cooled down, filtered and extract was preserved at 4 °C for further use in bio-fabrication of FeNPs (Halanayake et al., 2021).

2.3 Bio-fabrication of FeNPs

The FeNPs were fabricated by adding 50 mL of 3 mM solution of ferrous acetate (Fe(C₂H₃O₂)₂) into 100 mL of extract of P. obtusa. The contents was heated at 70 °C for 2 h with continuous stirring, while keeping the pH of solution at 9.0 using NH₄OH solution. Then, it was cooled down, centrifuged (3000 rpm) for 20 min. The FeNPs were rinsed with deionized water many times to eliminate residues of P. obtusa leaves extract residues and ions. The resulting powdered was incubated for 2 h at 75 °C and the calcination of FeNPs was performed at 600 °C (Iqbal et al., 2020), which were further subjected to characterization and bioactivity evaluation. The biosynthesis study plan is shown in Fig. 1.

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Fig. 1

Schematic presentation of biosynthesis of FeNPs using P. obtusa extract and applications.

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2.4 Characterization of FeNPs

The biosynthesized FeNPs were subjected to different characterization techniques UV/Vis spectroscopy (UV-4000 spectrophotometer, Germany), FTIR (Alpha, Bruker, Germany) using the KBr method (0.002 g sample was grinded with 0.98 g KBr). The XRD of FeNPs was investigated using X-ray diffractometer (Bruker Germany, BRUKE-D8-ADVANCE) at current voltage of 40 mA and Cu Kα radiations source energized at 45 kV (λ = 0.15418 nm) in a diffraction angle range of 2θ from 10° to 80°. The SEM (NOVAFEISEM-450) and TEM (JEOL 2000 FX) for morphological analysis of the FeNPs using at voltage of 10–15 kV and analysis was carried out using Image J software for SEM. The UV/Vis spectra were observed in the range of 200–800 nm wavelength. FTIR analysis was performed in range of 500–4500 cm⁻¹ to characterize potential bioactive responsible for the reduction of metal ions to atom. The average crystalline size of NPs was recorded using crystallite size calculator (Scherrer formula) (Bihani et al., 2021; Halanayake et al., 2021).

2.5 Antimicrobial activity evaluation

2.6 Antioxidant activity evaluation

To elucidate free radical scavenging potential of FeNPs, the spectrophotometric method was used according to protocol reported elsewhere (Hamelian et al., 2018). For this purpose, DPPH (2.4 mg) was added into 25 mL CH₂OH and vortexed properly. Later on, FeNPs with different concentrations ranging from 25 to 500 μg/mL were utilized to explore their free radical quenching capacity. The standards used in this assay were ascorbic acid (AA) (positive control) and DMSO (negative control). The 200 μL reaction mixture containing 20 μL FeNPs and 180 μL reagent solution was laden into 96-well Eliza plate and incubated for 1 h in dark. After that the absorbance was monitored at 517 nm via reader (microplate). The % scavenging of DPPH was determined using following equation.

The total reducing power ability of FeNPs was performed using K₃[Fe(CN)₆] according to method (Abbasi et al., 2019) with slight modification. The DMSO and ascorbic acid (AA) served as negative and positive controls. A 200 μL of FeNPs were taken in different concentration ranging from 25 to 250 μg/mL. To each solution of FeNPs, 5 mL of phosphate buffer (6.6 pH) with 0.2 M concentration and 2.5 mL K₃[Fe(CN)₆] (1%) were added and incubated the resulting solution for 30 min. After 30 min, 2.5 mL solution of C₂HCl₃O₂ (10%) was poured and then, centrifuged (3000 rpm) for 15 min. The upper layered 2.5 mL solution was mixed with 0.5 mL FeCl₃ (0.1%) and 2.5 mL water (deionized) and then, absorbance is monitored (630 nm). The reducing power is presented in terms of μg ascorbic acid equivalent per mg (μg AAE/mg) of NPs.

The hydrogen peroxide (H₂O₂) radical scavenging activity was evaluated utilizing FeNPs (25–500 μg/mL) and ascorbic acid as standard. A 200 μL FeNPs were mixed with 5 mM H₂O₂ solution and incubated for half an hour at room temperature and Eliza reader was employed for absorbance analysis at 610 nm. To compare the antioxidant capacity of FeNPs, the DMSO (negative control), while AA was employed as a positive control. The results were determined as μg AAE/mg of FeNPs by following the protocol (Khalil et al., 2017).

Nitric oxide (NO) scavenging potential of FeNPs was monitored taking different concentration (25–250 μg/mL) and butylated hydroxyl toluene (BHT) used as standard. Each concentration of FeNPs was mixed with equal volume of 20 mM freshly prepared sodium nitroprusside and the resultant solution incubated for 90 min at 25 °C. The optical density was recorded at 546 nm and radical scavenging activity was calculated (Sangaonkar and Pawar, 2018).

2.7 Biocompatibility and cytotoxicity evaluation

The biocompatibility assay of synthesized FeNPs was performed against human RBCs. The haemolytic activity was conducted using human RBCs (Abbasi et al., 2019). For this assay, 1 mL fresh human blood was taken into tube containing EDTA and subsequently, centrifuged for 15 min at 13,000 rpm. After that, the supernatant was discarded and pallets were washed thrice with phosphate-buffer saline (PBS). The suspensions of erythrocytes were prepared by adding 9.8 mL to PBS 200 μL erythrocytes. Then, 100 μL of erythrocytes suspension was mixed to different concentrations of FeNPs, incubated for 1 h/35 °C and centrifuged for 10 min at 10,000 rpm. The supernatants were shifted to 96-wells plate and the hemoglobin released was analyzed at 540 nm. PBS and Triton X-100 served as negative and positive controls. The hemolysis (%) was estimated by employing the Eq. (2).

In addition, the human macrophage was also utilized to test biocompatibility of FeNPs by MTT assay. This assay is relayed on colorimetric detection using 96 well flat bottom micro plate integrated with cultured cells (100 μL/well) having 5 × 10⁴ cells/mL concentration. Medium was replaced with 200 μL fresh medium after overnight incubation and to this, 200 μL of FeNPs with different concentrations (30–100 μg/mL) were added and the resulting medium again incubated for 2 days. Insoluble crystals of formazan were formed after addition of 200 μL of MTT (0.5 mg/mL) into this medium, which was kept at 37 °C for 4 h. Multichannel well plate used to remove excess bulk medium without disturbing formazan crystals. These NPs were dissolved by addition of 100 μL DMSO in each well, which further 15–20 min agitated followed by immediate measurement of absorbance at 570 nm with 650 nm reference wavelength. Doxorubicin and Cisplatin were used as standards, whereas positive control (without sample) served as 100% and 50% inhibition in growth (IC₅₀) of cell lines caused by specific concentration termed as cytotoxicity (Rolim et al., 2019). The percent inhibition was determined as shown in Eq. (3).

Hela cells and AU565 breast carcinoma cells were individually employed to assess cytotoxicity of synthesized FeNPs. The DMEM (Dulbecco’s Modified Eagle Medium) having pH 7.2 was implemented for cells growth along with fetal bovine serum (5%), 100 IU/mL penicillin and 100 μg/mL streptomycin taken in 75 cm² which further incubated with 5% CO₂ atmosphere at room temperature (Nadhe et al., 2020).

2.8 Statistical analysis

The standard and means deviation (±S.E) of the three replicates was calculated. Statistical Package for Social Science Version 20.0 and Microsoft Office Excel Version 2010 was used for analysis. The ANOVA method followed by Duncan's multiple range test at P < 0.05 was applied to analyze the significant differences of the experimental data (Li et al., 2020).

3.1 Characterization

The fabrication of FeNPs from ferrous acetate (Fe(C₂H₃O₂)₂) using leaves extract of P. obtuse was performed. The Fe(C₂H₃O₂)₂/P. obtusa solution color changed gradually at 70 °C to reddish black from dark brown, which is a signal of FeNPs formation. The color change was the preliminary confirmation for the bioreduction of Fe ions this, which was also confirmed by estimating the absorbance and the vibration of surface plasmon (Iqbal et al., 2020). The fabrication of FeNPs in an aqueous media was elucidated via UV/Vis spectroscopy by scanning the solution in 200–800 nm range. The maximum absorbance (λ_max) was observed 331 nm (Fig. 2) confirming the synthesis of FeNPs that was within range of surface plasmon resonance of Fe, which is consistent with previous reports (Iqbal et al., 2020). The additional peaks appeared in UV/Vis spectra are due to biomolecules present in the extract, these peaks are due to π/π* transitions confirming the occurrence of aromatic compounds in the extract.

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Fig. 2

UV/Visible spectra of P. obtusa extract and FeNPs.

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The FT-IR analysis was employed for the analysis of possible biomolecules and functional molecules responsible for biosynthesis. Fig. 3 indicates FTIR spectra of P. obtusa and biosynthesized FeNPs, respectively. The broad band was analyzed corresponding to the phenol functional group, N-H/C-H/O-H stretching bonding of amines, amides, —COOH, C-N groups was observed between 3503 and 2019 cm⁻¹ range. The presence of carbonyl functional groups was attributed to small peak at 1769 cm⁻¹, while a strong peak was seen at 1620 cm⁻¹ due to stretching of C⚌C. There were other peaks particularly observed at 1481, 1308, 1182 and 663 (cm⁻¹), which are related to the aliphatic ether. However, in case of FeNPs. The bands appearing in spectra of FeNPs at 3556–3254, 2165, 1655, 1434, 1307, 1174 (cm⁻¹) are due to the O—H, C—H, C—N, C—O and S⚌O of aromatic ester vibrations. The band at 621 cm⁻¹ showed the existence of Fe—O bond vibration. There was remarkable shifting of peaks in plant mediated FeNPs, thus suggesting participation of respective functional groups in reduction process and FeNPs formation. FT-IR studies revealed that synthesized FeNPs possessed biomolecules of P. obtusa extract. The distinct and significant IR peaks of these biomolecules were present in the IR spectra of synthesized FeNPs. As the Fe⁺³ reduced upon interaction of ferric acetate with leaves extract of P. obtusa. Previous reports also revealed the presence of bioactive compounds, i.e., quercetin, gallic acid, kaempferol, chrysophanol, naringenin, etc.) in plant extracts (Yadav and Fulekar, 2018; Al Banna et al., 2020; Awwad and Amer, 2020), which are responsible for reduction metal ions to metal stable atom. These biomolecules bind to metal ions surface and play substantial role in reducing and stabilizing the NPs. The bioactive molecules on NPs were responsible for the biological activities of NPs (Al-Fa'ouri et al., 2021; Amer and Awwad, 2021; Shammout and Awwad, 2021).

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Fig. 3

FTIR spectra of FeNPs and P. obtusa extract.

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The XRD analysis was performed to study structure of biosynthesized FeNPs. The Bragg peaks observed in P. obtusa mediated FeNPs were in agreement to pure phase of FeNPs that agree with JCPD: 95-905-1447. The FeNPs distinct XRD diffraction peaks were indexed at 2θ values of 10.46°, 15.8°, 29.22°, 35.14° and 40.59°, which representing (1 1 0), (2 0 0), (2 0 1), (2 1 1), (0 2 0) and (2 2 2) crystallographic planes, respectively (Fig. 4). The sharp XRD peaks showed highly crystalline nature of FeNPs. Scherer's approximation was used to calculate average size of FeNPs and it was found to be 50 nm. Fig. 5 depicts the SEM images of FeNPs of spherical shape. The FeNPs were also characterized using TEM in order to further elucidate morphology and size of NPs. The spherical shape of NPs was observed from TEM images as given in Fig. 6. These spectroscopic analysis are in closer agreement with previous reported (Khalil et al., 2017; Abbasi et al., 2019; Li et al., 2020).

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Fig. 4

XRD pattern of FeNPs synthesized using P. obtusa extract.

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Fig. 5

SEM analysis of FeNPs synthesized using P. obtusa extract.

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Fig. 6

TEM analysis of FeNPs synthesized using P. obtusa extract.

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3.2 Antimicrobial activity

Traditional medications available in pharmaceutical industries play imperative part to treat different microbial ailments, but are associated to problems, i.e., resistance against antibiotics. Hence, researchers are exploring the alternative ways to design alternate materials in order to combat against antibiotic resistance as well as to minimize the risks of spread of diseases (Patil Shriniwas, 2017; Abbasi et al., 2019). Thus, the advances in nano-biotechnology have opened the avenues for fabrication of advanced materials (metal NPs) with the special antimicrobial potential for biomedical applications.

The antibacterial activities of FeNPs and aqueous leave extract were executed by using bacterial strains viz. E. coli and B. subtilis and response are depicted in Fig. 7(a and b) and Fig. 8a. The antibacterial activity of P. obtusa leaves extract and different concentrations FeNPs was evaluated by disc diffusion method. The zone of inhibition (maximum) was observed at 100 μg/mL, 18 ± 0.03 mm and 16 ± 0.03 mm for extract of P. obtusa against E. coli and B. subtilis, respectively. Whereas, the highest inhibition zone in case of FeNPs (at 100 μg/mL) was 29 ± 0.03 mm against E. coli and 24 ± 0.04 mm against B. subtilis. At 100 μg/mL concentration, the standard (streptomycin) exhibited the zone of inhibition 24 ± 0.06 mm against E. coli and 22 ± 0.05 mm against B. subtilis. The FeNPs showed promising (P < 0.01) activity in comparison to standard. The bactericidal activity of FeNPs was due to the cations (Fe⁺²) formation, which get inclined to the bacterial cell-wall having negative charge owing to electrostatic attractions. The FeNPs then further penetrate inside the bacteria thus inhibiting its growth. The NPs in the cytoplasm interact with biomolecules (-S- containing enzymes and proteins) as well as DNA and preclude them from the normal functioning inside the cell. Thus, resulting in complete destruction of bacterial cell (Khan et al., 2017; Abbasi et al., 2019; Iqbal et al., 2020). Besides, previous studies revealed that generation of ROS (reactive oxygen species) is the base of damage (oxidative) in bacterial cells for the NPs-based toxicity. Moreover, the defects (on the surface) of NPs also might account for cells injury and antibacterial growth inhibition. Furthermore, the bioactive on the exterior of FeNPs from the extract used for stabilization and capping of NPs played significant antibacterial potential role (Abbasi et al., 2019; Ibrahim et al., 2021). The dose-dependent antibacterial activities of the present study are in agreement studies reported earlier [31,32].

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Fig. 7

Antimicrobial activity, zone of inhibition (mm), (a, b) against E. coli, B. subtilis and (c, d) against S. commune and A. niger.

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Fig. 8

(a) Antibacterial, (b) antifungal, (c) hemolytic activity, (d) biocompatibility against macrophages, (e) in vitro cytotoxicity exhibited by green synthesized FeNPs at different concentrations.

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The antifungal activities were assessed for both P. obtusa extract and FeNPs in 20–100 μg/mL and the findings are displayed in Figs. 7(c, d) and 8b. In case of P. obtuse extract, the obtained zones of inhibition were 14 ± 0.02 mm against A. niger and 17 ± 0.02 mm against S. commune. The FeNPs showed 24 ± 0.02 mm zones of inhibition against A. niger and 28 ± 0.03 mm against S. commune at 100 μg/mL concentration. The zone of inhibition exhibited by standard (fluconazole) against A. niger was 21 ± 0.05 mm, whereas it was 26 ± 0.4 mm against S. commune. A significantly antifungal activity (P < 0.005) was shown by FeNPs versus standard. The FeNPs furnished a dose-dependent activity and was enhanced markedly as the concentration of the NPs increased. The FeNPs inhibition activity was due to its small particle size. The smaller sized FeNPs could penetrate in the lipid membrane easily consequently blocked the binding sites of fungal enzymes thus inhibited mycelial growth. The damage of cytoplasm by deteriorating the structure of fungus due to apoptosis. The previous reports suggested that the FeNPs interference with spores and hyphae of fungus, the generation of ROS also led to inhibition in growth of fungus (Abbasi et al., 2019, Iqbal et al., 2020; Wang et al., 2021). The antifungal characteristic exhibited by green synthesized metal NPs are very important candidate for the biological applications. These findings are in accordance with the earlier observations (Yadav and Fulekar, 2018; Abbasi et al., 2019; Ibrahim et al., 2021).

The MICs values were observed as 125 ± 0.11, 250 ± 0.09, 250 ± 0.13 and 125 ± 0.08 for P. obtusa and 62.5 ± 0.9, 31.25 ± 0.07, 62.5 ± 0.11 and 62.5 ± 0.12 μg/mL in case of FeNPs against E. coli, B. subtilis, A. niger and S. commune microbial strains, respectively. The MICs values of 63.4 ± 0.1, 32.31 ± 0.09, 61.95 ± 0.13 and 62.15 ± 0.08 (μg/mL) were observed against E. coli, B. subtilis, A. niger and S. commune, respectively for standard drugs (streptomycin for bacterial strain and Fluconazole for fungal strain). All the results were statistically significant with probability value of P < 0.005. The MIC is defined as the visible inhibition of microorganism growth as an antimicrobial agent is added under definite conditions. The less MIC value indicates the higher activity of the NPs employed as antimicrobial agent. Lower values of MICs was observed in case of P. obtusa mediated FeNPs as compared to P. obtusa against these strains. The difference in MIC value of FeNPs and extract might be due to smaller size of NPs beside the bio-molecules nature in different plant extracts utilized for the formulation of FeNPs (Khan et al., 2017; Abbasi et al., 2019; Iqbal et al., 2020; Hasanin, 2021).

3.3 Cytotoxicity and biocompatibility

The toxicological and biocompatible nature of FeNPs was assessed against human macrophages and RBCs. According to the standard guidelines set by American Society, the biological substances are hemolytic if exposing the RBCs and its hemolysis greater than 5%, 2–5% are slightly hemolytic, while those having <2% are non-cytotoxic. The % hemolysis is the release of hemoglobin after rupturing of RBCs on treating with FeNPs. If tested NPs is hemolytic, it will rupture the RBCs and results in release of hemoglobin. The hemolytic assay of FeNPs was done against RBCs (human) in the range of 20–100 μg/mL as depicted in Fig. 8c. The P. obtusa showed 1.8 ± 0.01 and FeNPs 2.1 ± 0.01% hemolysis at 100 μg/mL and below this concentration no lysis was observed for both extract and NPs. The 100% lysis was examined for positive control (Triton-X-100), however 0% was observed for negative control (PBS). The presence of phyto-constituents in plant extract also plays a significant in biosafety of the NPs. Up to 2.1%, the RBCs lysis is due to the action of biomolecules that maintain RBCs membrane integrity by inhibiting oxidation phenomenon. In addition to this, the plasma antiradical activity also enhanced (Jamdagni et al., 2018; Abou-Yousef et al., 2021). This biocompatibility assessment is pivotal for understanding the cytotoxic behavior of NPs. Our studies concluded that biosynthesized FeNPs at low concentrations are non-hemolytic against RBCs, thus confirming their safe nature and biocompatible nature. Our findings are also consistent with earlier reports (Iqbal et al., 2019) who determined the hemolysis of RBCs using metal NPs synthesized using plant extract. There was no apparent lysis seen in RBCs on exposure of these metal NPs at low concentrations.

The MTT cytotoxic assay was performed to confirm the biosafety of FeNPs against human macrophages. The results of MTT assay showed inhibition response was dose-dependent. It was inferred from findings that macrophages response was higher on treating with FeNPs higher concentration, while cytotoxicity reduced at lower concentration. The results depicted in Fig. 8d revealed that 31.32 ± 0.04 and 21 ± 0.0.3% inhibition of P. obtusa and FeNPs in macrophages growth at 200 μg/mL concentration thus confirming the FeNPs non-toxic behavior (P < 0.01). The macrophage mechanism of FeNPs was to scavenge the ROS produced from external sources by its reduction. The FeNPs at low concentration were non-toxic to macrophages and human erythrocytes except its quantity enhanced beyond the threshold limit (Prach et al., 2013; Abou-Yousef et al., 2021).

The FeNPs were also analyzed against the AU565 and HeLa cell lines, the cell viability decreased as the concentration increased from 30 to 100 μg/mL. More cell decline observed in AU565 (82.03 ± 0.08 to 23.65 ± 0.065 %) with IC₅₀ of 47.51 ± 0.08 μg/mL in comparison to the HeLa cell lines, in which the analyzed cell viability ranged from 88.61 ± 0.06 to 33.34 ± 0.06 % (IC₅₀ 55.36 ± 0.07 μg/mL) (Fig. 8e). The cells retained intact and shown 100% cell viability without any treatment served as control. The HeLa cell lines results compared to Cisplatin (standard), which showed 97.15 ± 0.09% decline in viability of cells and another standard Doxorubicin employed for AU565 cell lines with 98.80 ± 0.09% cell viability was declined. The higher FeNPs concentration enhanced the cell viability loss in both cases. AU565 and HeLa cell lines show decreased cell viability at higher concentration, but the extent was observed to be different in the two cell lines. This difference indicates the more resistant nature of HeLa cell lines under similar conditions as compared to AU565. The FeNPs IC₅₀ value was recorded to be 371.3 μg/mL. The findings of biocompatibility, i.e., hemolytic (P < 0.01), microphages (P < 0.01), cell lines (P < 0.01) showed significant values at 100 μg/mL concentration of FeNPs. Similarly, (Rajan et al., 2017; Dacrory et al., 2021) studied the cytotoxicity of NPs from E. cardamomum extract against HeLa cell lines and an increased activity shown was observed in concentration dependent manner.

3.4 Antioxidant activities

The reactive oxygen species (ROS) in living organism cause membrane damage via lipid oxidation resulting in various ailments. Plant mediated metal NPs have potential to scavenge these ROS. Since P. obtusa leaf extract mediated FeNPs was used in present study as strong capping and reducing agent. It is assumed, that some flavonoids quench ROS on the surface of FeNPs (Khan et al., 2017; Abbasi et al., 2019; Iqbal et al., 2020; Hasanin 2021). The antioxidant activities like total reducing power ability DPPHH₂O₂ and NO of P. obtusa mediated FeNPs were evaluated at different concentrations. The findings of DPPH scavenging for different concentration of FeNPs as % inhibition values at concentration 25–500 μg/mL are presented in Fig. 9a. The results revealed that DPPH radical scavenging potential values as 56.34 ± 0.03% for P. obtuse, while 70.23 ± 0.02% for FeNPs at concentration 500 μg/mL, respectively. The radical scavenging potential of ascorbic acid (standard) determined was 66 ± 0.02%. It was found that % inhibition increases by increasing the concentration. The FeNPs showed significant scavenging activity and results were comparable to the standard. It was deduced that free radicals such as DPPH has central nitrogen having ability to accept hydrogen radical or electron and some antioxidants compound in extract and NPs might involve in reducing the free radicals. The purple-colored DPPH solution turned yellow during the reduction of DPPH radical to non-radical form. The absorption for this reduction was determined at 517 nm which indicated strong antioxidant potential (Abbasi et al., 2019; Dacrory et al., 2021).

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Fig. 9

(a) DPPH, (b) TRPA, (c) H₂O₂, (d) NO radical scavenging activities at different concentrations of extract and FeNPs.

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The antioxidant potential of leaves extract and FeNPs in concentration range of 25–250 μg/mL was investigated using total reducing power ability (TRPA). The reducing power ability values observed in case of P. obtusa extract was 67.46 ± 0.03 μg AAE/mg, these values were 76.65 ± 0.02 μg AAE/mg in case of FeNPs at 250 μg/mL. The findings revealed that P. obtusa mediated FeNPs showed strong antioxidant activity in concentration dependent way (Fig. 9b). The mechanism for TRPA activity was the involvement of the reduction in antioxidant potentials of FeNPs by providing H-atom that quench the free radical chain. When compared to plant extract, the activity was significant of biosynthesized NPs. Ferrous ions (Fe²⁺) are obtained from the ferricyanide complex (Fe³⁺) during this assay as evident from the change in color from yellow to bluish green (Abbasi et al., 2019).

The inhibition of H₂O₂ radical scavenging responses have been shown in Fig. 9c for the concentration range of 25–500 μg/mL. The radical scavenging was enhanced in a dose dependent manner, i.e., maximum radical quenching was observed at 500 μg/mL that was 56.82 ± 0.02% for P. obtusa extract and 67.34 ± 0.02% in case FeNPs. The values of H₂O₂ radical scavenging for standard (ascorbic acid) was 74 ± 0.02%. The phenomenon of aging and other similar disorder are associated with the generation of free radicals including H₂O₂, which disturb the numerous energy producing systems. The NPs have strong ability to scavenge these radicals and exhibited antioxidant potential, which increased with concentration (Patil Shriniwas, 2017; Abu-Elghait et al., 2021).

The NO radical scavenging activity was evaluated at concentrations (25–250 μg/mL), P. obtusa showed inhibition of 67.08 ± 0.03%, whereas FeNPs showed 74.43 ± 0.02% at 250 μg/mL concentration (Fig. 9d). The BHT (butylated hydrogen toluene) was employed as a standard to test NO scavenging potential, which showed 72.3 ± 0.02% NO radical scavenging activity. The biosynthesized FeNPs exhibited strong scavenging ability. The NO is involved in many biological functions including neuro transmission, antimicrobial, anticancer properties and smooth muscle relaxation in addition to regulation of blood pressure (Khan et al., 2017). The reaction of NO with superoxide produces peroxynitrite anion, which result in oxidation of lipids and DNA fragmentation responsible for oxidative damage. The NO regularization should be managed to avoid its harmful effects and scavenging through NPs quite significant in this regard (Patil Shriniwas, 2017; Abu-Elghait et al., 2021). The FeNPs exhibited significant free radical scavenging potential for assays performed viz. TRPA (P < 0.01), DPPH (P < 0.005), H₂O₂ (P < 0.05) and NO (P < 0.001) for DPPH and H₂O₂ the concentration was 500 μg/mL and 250 μg/mL for TRPA and NO. The FeNPs proved to be promising for biomedical applications since the DPPH, TRPA, H2O2 and NO showed the potential activity and also previous findings are in line for NPs biosynthesized using Callistemon viminalis, Rhamnus virgata and Fagonia indica (Khan et al., 2017). Also green route for the fabrication NP is eco-benign and facile versus other physicochemical approaches (Remya et al., 2017; Al Banna et al., 2020; Awwad et al., 2020; Fatima et al., 2020; Amer and Awwad, 2021; Shahid et al., 2021).