Biofabrication of Fe3O4 Nanoparticles from Spirogyra hyalina and Ajuga bracteosa and Their Antibacterial Applications

Iron oxide nanoparticles (NPs) have attracted substantial interest due to their superparamagnetic features, biocompatibility, and nontoxicity. The latest progress in the biological production of Fe3O4 NPs by green methods has improved their quality and biological applications significantly. In this study, the fabrication of iron oxide NPs from Spirogyra hyalina and Ajuga bracteosa was conducted via an easy, environmentally friendly, and cost-effective process. The fabricated Fe3O4 NPs were characterized using various analytical methods to study their unique properties. UV-Vis absorption peaks were observed in algal and plant-based Fe3O4 NPs at 289 nm and 306 nm, respectively. Fourier transform infrared (FTIR) spectroscopy analyzed diverse bioactive phytochemicals present in algal and plant extracts that functioned as stabilizing and capping agents in the fabrication of algal and plant-based Fe3O4 NPs. X-ray diffraction of NPs revealed the crystalline nature of both biofabricated Fe3O4 NPs and their small size. Scanning electron microscopy (SEM) revealed that algae and plant-based Fe3O4 NPs are spherical and rod-shaped, averaging 52 nm and 75 nm in size. Energy dispersive X-ray spectroscopy showed that the green-synthesized Fe3O4 NPs require a high mass percentage of iron and oxygen to ensure their synthesis. The fabricated plant-based Fe3O4 NPs exhibited stronger antioxidant properties than algal-based Fe3O4 NPs. The algal-based NPs showed efficient antibacterial potential against E. coli, while the plant-based Fe3O4 NPs displayed a higher zone of inhibition against S. aureus. Moreover, plant-based Fe3O4 NPs exhibited superior scavenging and antibacterial potential compared to the algal-based Fe3O4 NPs. This might be due to the greater number of phytochemicals in plants that surround the NPs during their green fabrication. Hence, the capping of bioactive agents over iron oxide NPs improves antibacterial applications.

This paper reports the biofabrication of Fe 3 O 4 NPs by exploiting the biomolecules of Spirogyra hyalina and Ajuga bracteosa as bioreductants. Moreover, the capping of diverse phytochemicals on Fe 3 O 4 NPs may enhance the bioactivities of these biofabricated NPs. The green-fabricated Fe 3 O 4 NPs were characterized using different analytical methods to determine their unique properties. Furthermore, the present study also explored the biomedicinal potential of green-synthesized Fe 3 O 4 NPs and algal and plant extracts by accessing their antibacterial and antioxidant activities. The outcomes revealed the enhanced biological potential of Fe 3 O 4 NPs, which might be because the capping of bioactive agents around NPs is present. Nevertheless, further research will be needed to understand the chemistry of metallic oxide NPs.

Characterization of Iron Oxide NPs
The Fe 3 O 4 NPs were biofabricated using the algal and plant extracts of S. hyalina and A. bracteosa. In the case of algal-based Fe 3 O 4 NPs, the color change from yellow to reddishbrown was observed. The instantaneous dark brownish color change from light yellow confirmed the fabrication of plant-based Fe 3 O 4 NPs. The color change was attributed to surface plasmon resonance and the interaction of biomolecules with metal ions. These biosynthesized NPs were analyzed via various analytical techniques.

UV-Vis Analysis of Fe 3 O 4 NPs
UV-visible spectroscopy was used to monitor and approve the fabrication of Fe 3 O 4 NPs. The absorption peaks of biofabricated Fe 3 O 4 NPs were observed because surface plasmon vibrations reached the excitation stage. The figure shows that algae and plant extracts had no distinct peaks. In the case of algal-based Fe 3 O 4 NPs, the sharp peak was displayed at 289 nm. While in the case of plant-based Fe 3 O 4 NPs, the peak value for absorbance was observed at 306 nm shown in Figure 1. Many studies revealed the maximal absorbance at 250, 291, 297, 300, 328, and 330 nm for Fe 3 O 4 NPs synthesized from algae and plants, including Petalonia fascia [40], Ficus carica [41], Mimosa pudica [42], Moringa oleifera [31], Bauhinia tomentosa [43], and Phyllanthus niruri [44], which supported these findings. These variations of maximal absorbance were attributed to the capping of biomolecules on NPs. These biomolecules affect the size of NPs through bioreduction, which also affects maximal absorbance [30,31].

FTIR Analysis of Fe 3 O 4 NPs
FTIR spectroscopy was performed to determine the functional groups of various biomolecules interacting with the fabricated Fe 3 O 4 NPs. The FTIR spectra revealed the active biomolecules present in the algal and plant extracts that had played an essential role in reducing the precursor salt during the fabrication of iron oxide NPs and maintaining their stability. In the FTIR spectrum of the algal extract, shown in Figure 2, the prominent peak detected at 3313 cm −1 corresponded to the stretch of the O-H group of either alcohol or algal phenolics. Kale et al. also reported the presence of an O-H bond close to this position [37]. The peaks at 2924 cm −1 and 2852 cm −1 indicate the stretching vibration of a C-H bond (alkane) [45]. The C=C (alkene) stretching vibration was observed at 1641 cm −1 as a stretched vibration form. The peak at 1033 cm −1 was attributed to the stretching vibration of the primary amine C-N [34]. The FTIR spectrum of algal-based iron oxide NPs revealed a peak at 3373 cm −1 representing the O-H stretching vibration of alcohol or algal phenolics [35]. The peaks at 2932 cm −1 and 2852 cm −1 refer to the C-H stretched vibrations of an alkane. The peak at 2355 cm −1 was attributed to the absorption of atmospheric CO 2 , and the peak at 1646 cm −1 was assigned to the C=C or alkene stretching vibration [33]. The dominant peak at 674 cm −1 was attributed to the Fe-O in iron oxide, confirming the fabrication of NPs. The current result strongly agrees with the study reported by Aisida et al. [46].

FTIR Analysis of Fe3O4 NPs
FTIR spectroscopy was performed to determine the functional groups of various biomolecules interacting with the fabricated Fe3O4 NPs. The FTIR spectra revealed the active biomolecules present in the algal and plant extracts that had played an essential role in reducing the precursor salt during the fabrication of iron oxide NPs and maintaining their stability. In the FTIR spectrum of the algal extract, shown in Figure 2, the prominent peak detected at 3313 cm −1 corresponded to the stretch of the O-H group of either alcohol or algal phenolics. Kale et al. also reported the presence of an O-H bond close to this position [37]. The peaks at 2924 cm −1 and 2852 cm −1 indicate the stretching vibration of a C-H bond (alkane) [45]. The C=C (alkene) stretching vibration was observed at 1641 cm −1 as a stretched vibration form. The peak at 1033 cm −1 was attributed to the stretching vibration of the primary amine C-N [34]. The FTIR spectrum of algal-based iron oxide NPs revealed a peak at 3373 cm −1 representing the O-H stretching vibration of alcohol or algal phenolics [35]. The peaks at 2932 cm −1 and 2852 cm −1 refer to the C-H stretched vibrations of an alkane. The peak at 2355 cm −1 was attributed to the absorption of atmospheric CO2, and the peak at 1646 cm −1 was assigned to the C=C or alkene stretching vibration [33]. The dominant peak at 674 cm −1 was attributed to the Fe-O in iron oxide, confirming the fabrication of NPs. The current result strongly agrees with the study reported by Aisida et al. [46].
The FTIR spectrum of the plant extract revealed a peak at 3305 cm −1 , confirming the presence of the O-H bond in a stretched state for alcohol or plant phenolics [37]. The absorption peaks at 2918 cm −1 and 2854 cm −1 denote the C-H bond of the alkane in the mode of stretched vibrations [45]. The peak at 1609 cm −1 might be due to the C=C bond of an alkene in the stretched state [33]. In contrast, the absorption peak at 1020 cm −1 is probably due to the C-N bond of the amine in a stretched form [34]. The FTIR spectrum of plant- corresponds to the C=C bond of an alkene in its stretched state [33]. The peak at 622 cm −1 is the typical peak of the Fe-O of iron oxide, confirming the fabrication of iron oxide NPs, and it is consistent with the findings reported by Liu et al. [47]. A. bracteosa has been used to incorporate diverse bioactive agents, such as ergosterol, diterpenoids, glycosides, phytoecdysones, and flavonols that assist the present FTIR spectra [38]. Many studies disclosed the existence of essential bioactive molecules in Fe3O4 NPs and their binding to the NPs [39].  Figure 3a. The XRD peaks observed for the algalbased iron oxide NPs were similar to the standard magnetite XRD pattern in the ICCD file (00-003-0863), confirming the cubic structure of the crystalline system. The mean crystal- The FTIR spectrum of the plant extract revealed a peak at 3305 cm −1 , confirming the presence of the O-H bond in a stretched state for alcohol or plant phenolics [37]. The absorption peaks at 2918 cm −1 and 2854 cm −1 denote the C-H bond of the alkane in the mode of stretched vibrations [45]. The peak at 1609 cm −1 might be due to the C=C bond of an alkene in the stretched state [33]. In contrast, the absorption peak at 1020 cm −1 is probably due to the C-N bond of the amine in a stretched form [34]. The FTIR spectrum of plant-based iron oxide NPs suggests that there are diverse phytochemicals that play a vital role in the fabrication of iron oxide NPs. The peak at 3312 cm −1 was attributed to the O-H functional group of alcohol or plant phenolics. These findings are correlated with the study reported by Qasim et al. [45]. The absorption peaks at 2926 cm −1 and 2852 cm −1 were assigned to the stretching vibration of the alkane C-H group [36]. The peak at 2362 cm −1 was attributed to the absorption of CO 2 in the sample. At 1635 cm −1 , the absorption band corresponds to the C=C bond of an alkene in its stretched state [33]. The peak at 622 cm −1 is the typical peak of the Fe-O of iron oxide, confirming the fabrication of iron oxide NPs, and it is consistent with the findings reported by Liu et al. [47]. A. bracteosa has been used to incorporate diverse bioactive agents, such as ergosterol, diterpenoids, glycosides, phytoecdysones, and flavonols that assist the present FTIR spectra [38]. Many studies disclosed the existence of essential bioactive molecules in Fe 3 O 4 NPs and their binding to the NPs [39].

X-ray Diffraction of Fe 3 O 4 NPs
The XRD analysis of algal-based   Figure 4a,b show the morphology of fabricated algal-based Fe 3 O 4 NPs. As shown in the SEM image, the synthesized NPs were spherical and spindle-shaped. The mean size of biosynthesized NPs was 52 nm (35-80 nm). A few clusters of agglomerates were also observed, probably due to the accretion of bioactive reductive agents in algal extract or the magnetic tendency of Fe 3 O 4 NPs [40]. Figure 4d-e shows the morphology of plant-based NPs. The fabricated Fe 3 O 4 NPs were spherical and rod-shaped. The mean size of the NPs was 75 nm (45-100 nm). Some of the NPs were in an aggregated form. The aggregation of NPs might be due to the accumulation of diverse reductive agents in plant extracts or the magnetic tendency of biofabricated Fe 3 O 4 NPs. SEM confirmed the fabrication of spindle-shaped Fe 3 O 4 NPs via forced hydrolysis with a size range of 60-250 nm [49]. Another study reported the fabrication of spherical-shaped Fe 3 O 4 NPs from Perilla frutescens, approximately 50 nm in size, which is also consistent with the present findings [50]. Previous research reported that the size of the Fe 3 O 4 NPs from the peel extract of plantains and the seed extract of pomegranates were 30-50 nm and 25-55 nm, respectively [51,52].  Figure 4a,b show the morphology of fabricated algal-based Fe3O4 NPs. As shown in the SEM image, the synthesized NPs were spherical and spindle-shaped. The mean size of biosynthesized NPs was 52 nm (35-80 nm). A few clusters of agglomerates were also observed, probably due to the accretion of bioactive reductive agents in algal extract or the magnetic tendency of Fe3O4 NPs [40]. Figure 4d-e shows the morphology of plantbased NPs. The fabricated Fe3O4 NPs were spherical and rod-shaped. The mean size of the NPs was 75 nm (45-100 nm). Some of the NPs were in an aggregated form. The aggregation of NPs might be due to the accumulation of diverse reductive agents in plant extracts or the magnetic tendency of biofabricated Fe3O4 NPs. SEM confirmed the fabrication of spindle-shaped Fe3O4 NPs via forced hydrolysis with a size range of 60-250 nm [49]. Another study reported the fabrication of spherical-shaped Fe3O4 NPs from Perilla frutescens, approximately 50 nm in size, which is also consistent with the present findings [50]. Previous research reported that the size of the Fe3O4 NPs from the peel extract of plantains and the seed extract of pomegranates were 30-50 nm and 25-55 nm, respectively [51,52].

EDX Analysis of Fe 3 O 4 NPs
The EDX spectrum in Figure 5a revealed the intense peaks of iron at 6.4 keV and oxygen at 0.6 keV that affirmed the formation of Fe 3 O 4 NPs. Their mass percentages were 36.27% and 38.47%, respectively. The chlorine peak was also observed in the EDS graph with a 14.33% mass. It was determined from Figure 5b that the EDX spectrum of plantbased Fe 3 O 4 NPs were comprised of significant iron peaks at 6.4 keV and oxygen peaks at 0.6 keV, with mass percentages of 51% and 29%, respectively, confirming the fabrication of Fe 3 O 4 NPs. Figure 5 shows other elements, such as Cl and Cr. The Cl peak was attributed to the ferric chloride salt precursor utilized to fabricate Fe 3 O 4 NPs [21]. The chromium peak might be due to the sputtering of Fe 3 O 4 NPs before SEM analysis. Sputter coating prevents specimen charging and increases the signal-to-noise ratio to produce better quality images [53]. The EDX analysis of the currently biofabricated Fe 3 O 4 NPs is also consistent with other studies [54,55]. According to other studies, the expected values from EDX are closely related to the current findings [43].
to the ferric chloride salt precursor utilized to fabricate Fe3O4 NPs [21]. The chromium peak might be due to the sputtering of Fe3O4 NPs before SEM analysis. Sputter coating prevents specimen charging and increases the signal-to-noise ratio to produce better quality images [53]. The EDX analysis of the currently biofabricated Fe3O4 NPs is also consistent with other studies [54,55]. According to other studies, the expected values from EDX are closely related to the current findings [43].

In Vitro Studies
Different biological activity measures, such as the antibacterial and antioxidant properties of various fractions and Fe3O4 NPs, were used to demonstrate that these NPs performed better than algae and plant fractions.

Antibacterial Activity
The antibacterial activity of the AE, PE, and Fe3O4 NPs was investigated for both gram-positive (B. pumilus and S. aureus) and gram-negative (E. coli and P. aeruginosa) bacteria. Based on the zone of inhibition produced, both algal and plant-based Fe3O4 NPs showed remarkable antibacterial activity compared to the algal and plant extracts against both types of investigated bacteria shown in Figures 6 and 7. Overall, the antibacterial graphs shown in Figure 8 that plant-based Fe3O4 NPs had strong antibacterial potency compared to that of algal-based Fe3O4 NPs against the various types of gram-positive and gram-negative bacterial strains. This result might be due to the higher number of phytochemicals found in plant extracts compared to algal extracts.

In Vitro Studies
Different biological activity measures, such as the antibacterial and antioxidant properties of various fractions and Fe 3 O 4 NPs, were used to demonstrate that these NPs performed better than algae and plant fractions.     In many studies, the same results of antibacterial potency were revealed by Fe3O4 NPs biofabricated from extracts of Carcia papaya [55], Sida cordifolia [56], Purpureocillium platinum [57], and Phoenix dactylifera [58] against gram-positive and gram-negative bacterial strains. These metallic oxide NPs may decrease the expression of an antibiotic-resistant gene in an antibiotic-resistant bacterium [59]. Iron oxide NPs have an aptitude for adsorption and penetration into bacterial biofilms because of their distinct physicochemical characteristics, such as hydrophobicity, surface charge, and large surface area [60]. Moreover, the respective NPs attach directly to the cell walls of various microorganisms and destroy themselves efficiently [58].

Antioxidant Activity
The scavenging activity of biofabricated Fe3O4 NPs was compared with algae and plant extracts determined by DPPH assay. The methanolic extract of S. hyalina and the aqueous extract of A. bracteosa showed IC50 values of 21 ± 1.23 µg/mL and 15.8 ± 1.3 µg/mL, respectively. Both algal-and plant-based Fe3O4 NPs showed remarkable scavenging potential (IC50 = 16.1 ± 0.74 µg/mL and 11.6 ± 0.76 µg/mL) compared to that of algal and plant extracts. A low IC50 value shows that the substance has a higher antioxidant potential.    In many studies, the same results of antibacterial potency were revealed by Fe3O4 NPs biofabricated from extracts of Carcia papaya [55], Sida cordifolia [56], Purpureocillium platinum [57], and Phoenix dactylifera [58] against gram-positive and gram-negative bacterial strains. These metallic oxide NPs may decrease the expression of an antibiotic-resistant gene in an antibiotic-resistant bacterium [59]. Iron oxide NPs have an aptitude for adsorption and penetration into bacterial biofilms because of their distinct physicochemical characteristics, such as hydrophobicity, surface charge, and large surface area [60]. Moreover, the respective NPs attach directly to the cell walls of various microorganisms and destroy themselves efficiently [58].

Antioxidant Activity
The scavenging activity of biofabricated Fe3O4 NPs was compared with algae and plant extracts determined by DPPH assay. The methanolic extract of S. hyalina and the aqueous extract of A. bracteosa showed IC50 values of 21 ± 1.23 µg/mL and 15.8 ± 1.3 µg/mL, respectively. Both algal-and plant-based Fe3O4 NPs showed remarkable scavenging potential (IC50 = 16.1 ± 0.74 µg/mL and 11.6 ± 0.76 µg/mL) compared to that of algal and plant extracts. A low IC50 value shows that the substance has a higher antioxidant potential. In many studies, the same results of antibacterial potency were revealed by Fe 3 O 4 NPs biofabricated from extracts of Carcia papaya [55], Sida cordifolia [56], Purpureocillium platinum [57], and Phoenix dactylifera [58] against gram-positive and gram-negative bacterial strains. These metallic oxide NPs may decrease the expression of an antibiotic-resistant gene in an antibiotic-resistant bacterium [59]. Iron oxide NPs have an aptitude for adsorption and penetration into bacterial biofilms because of their distinct physicochemical characteristics, such as hydrophobicity, surface charge, and large surface area [60]. Moreover, the respective NPs attach directly to the cell walls of various microorganisms and destroy themselves efficiently [58].

Antioxidant Activity
The scavenging activity of biofabricated Fe 3 O 4 NPs was compared with algae and plant extracts determined by DPPH assay. The methanolic extract of S. hyalina and the aqueous extract of A. bracteosa showed IC 50 values of 21 ± 1.23 µg/mL and 15.8 ± 1.3 µg/mL, respectively. Both algal-and plant-based Fe 3 O 4 NPs showed remarkable scavenging potential (IC 50 = 16.1 ± 0.74 µg/mL and 11.6 ± 0.76 µg/mL) compared to that of algal and plant extracts. A low IC 50 value shows that the substance has a higher antioxidant potential. Plant-based Fe 3 O 4 NPs outperformed the algal-based Fe 3 O 4 NPs in terms of antioxidant potential. This might be due to the increased activity of phenolic molecules after the fabrication of Fe 3 O 4 NPs shown in Figure 9. Phenolic active agents found in plant extracts play a crucial role in producing Fe 3 O 4 NPs due to their high antioxidant activity and ability to act as reducing agents [47].
small-sized NPs may be amenable to the best scavenging activity by acting as a stabil agent [61]. The pharmacological features of bioactive agents accelerated the therap efficacy of NPs with enhanced biological applications [53]. Another study investigate scavenging effect of Fe3O4 NPs using the DPPH method and revealed the best antiox potential that supports these findings [17].

Preparation of Algal and Plant Extract
Ajuga bracteosa Wall ex Benth and Spirogyra hyalina were collected from Azad K mir, Pakistan, at coordinates 34.22° N 73.28° E in April 2022. A taxonomist of the De ment of Biotechnology, Mirpur University of Science and Technology Mirpur, Azad K mir, Pakistan, identified the plant and algae. The voucher specimens (MUST MUST1403) were deposited at the Herbarium of the Department of Biotechnology.
Spirogyra hyalina was collected from the freshwater region of Jatlan Head, di Mirpur, Azad Kashmir, Pakistan. The material was washed with distilled water to rem unwanted particles and placed in a dark area to maintain dryness. For the metha algal extract, the material was soaked in methanol for 72 h by keeping it in a shaker. a specified period, the methanolic algae extract was obtained by concentrating it in a uum via a rotary evaporator. Subsequently, 10 g of algae was dissolved in 200 mL o tilled water and then filtered via Whatman filter paper No. 1 before being stored in frigerator for later use. The herbaceous plant, Ajuga bracteosa, was gathered from Sam District Bhimber, Azad Kashmir, Pakistan. The fresh plant parts were washed with tilled water to remove the undesirable particles. These were dried in the shade. The w plant parts were minced into a fine powder, and 10 g of the minced powder was disso in 200 mL of distilled water. The suspension was placed on a hot plate at 60 °C fo hour with constant stirring. After the specified period, it was filtered through Wha filter paper No. 1 before being stored in a refrigerator for later use. Previous studies reported the green fabrication of iron oxide NPs using the methanolic and aqueous tions of algal and plant extracts [56][57][58]. Plant-based Fe 3 O 4 NPs exhibited better antioxidant potency than various oxidants that might be linked to plentiful bioactive agents [37]. The phytochemicals capped on the small-sized NPs may be amenable to the best scavenging activity by acting as a stabilizing agent [61]. The pharmacological features of bioactive agents accelerated the therapeutic efficacy of NPs with enhanced biological applications [53]. Another study investigated the scavenging effect of Fe 3 O 4 NPs using the DPPH method and revealed the best antioxidant potential that supports these findings [17].

Preparation of Algal and Plant Extract
Ajuga bracteosa Wall ex Benth and Spirogyra hyalina were collected from Azad Kashmir, Pakistan, at coordinates 34.22 • N 73.28 • E in April 2022. A taxonomist of the Department of Biotechnology, Mirpur University of Science and Technology Mirpur, Azad Kashmir, Pakistan, identified the plant and algae. The voucher specimens (MUST1402-MUST1403) were deposited at the Herbarium of the Department of Biotechnology.
Spirogyra hyalina was collected from the freshwater region of Jatlan Head, district Mirpur, Azad Kashmir, Pakistan. The material was washed with distilled water to remove unwanted particles and placed in a dark area to maintain dryness. For the methanolic algal extract, the material was soaked in methanol for 72 h by keeping it in a shaker. After a specified period, the methanolic algae extract was obtained by concentrating it in a vacuum via a rotary evaporator. Subsequently, 10 g of algae was dissolved in 200 mL of distilled water and then filtered via Whatman filter paper No. 1 before being stored in a refrigerator for later use. The herbaceous plant, Ajuga bracteosa, was gathered from Samahni, District Bhimber, Azad Kashmir, Pakistan. The fresh plant parts were washed with distilled water to remove the undesirable particles. These were dried in the shade. The whole plant parts were minced into a fine powder, and 10 g of the minced powder was dissolved in 200 mL of distilled water. The suspension was placed on a hot plate at 60 • C for one hour with constant stirring. After the specified period, it was filtered through Whatman filter paper No. 1 before being stored in a refrigerator for later use. Previous studies also reported the green fabrication of iron oxide NPs using the methanolic and aqueous solutions of algal and plant extracts [56][57][58].

Green Biofabrication of Algal-and Plant-Based Iron Oxide NPs
Ferric chloride hexahydrate was used as a salt precursor to fabricate IONPs. An amount of 1.35 g (0.1M) of FeCl 3 .6H 2 O was dissolved in 50 mL of distilled water in a bottom flask and heated for 20 min at 70 • C with mild stirring using a hot plate. Subsequently, 50 mL of the algal extract was added dropwise to the iron solution and heated to 60 • C for 30 min. The reddish-brown color that appeared indicates the formation of iron oxide NPs. The resulting product was centrifuged to remove impurities by washing two times with distilled water and three times with ethanol at 6000 rpm for 20 min. The final product was dried and stored for future use. In the case of the fabrication of iron oxide NPs from Ajuga bracteosa, the same salt precursor was applied shown in Figure 10. A 0.1 M FeCl 3 .6H 2 O solution in 100 mL of distilled water was prepared in a bottom flask and heated for 20 min at 70 • C with continuous mild stirring using a hot plate. Thirty milliliters of the plant extract were added dropwise to the iron solution and heated to 60 • C for 30 min. The dark brownish color indicated the formation of iron oxide NPs. The rest of the experiment was the same.

Green Biofabrication of Algal-and Plant-based Iron Oxide NPs
Ferric chloride hexahydrate was used as a salt precursor to fabricate IONPs. An amount of 1.35 g (0.1M) of FeCl3.6H2O was dissolved in 50 mL of distilled water in a bottom flask and heated for 20 min at 70 °C with mild stirring using a hot plate. Subsequently, 50 mL of the algal extract was added dropwise to the iron solution and heated to 60 °C for 30 min. The reddish-brown color that appeared indicates the formation of iron oxide NPs. The resulting product was centrifuged to remove impurities by washing two times with distilled water and three times with ethanol at 6000 rpm for 20 min. The final product was dried and stored for future use. In the case of the fabrication of iron oxide NPs from Ajuga bracteosa, the same salt precursor was applied shown in Figure 10. A 0.1 M FeCl3.6H2O solution in 100 mL of distilled water was prepared in a bottom flask and heated for 20 min at 70 °C with continuous mild stirring using a hot plate. Thirty milliliters of the plant extract were added dropwise to the iron solution and heated to 60 °C for 30 min. The dark brownish color indicated the formation of iron oxide NPs. The rest of the experiment was the same.

Characterization of Fe3O4 NPs
The fabricated Fe3O4 NPs were characterized using various analytical techniques to determine their size, shape, functional groups, crystallographic structure, and elemental analysis. The characterization techniques include UV-Vis spectroscopy, scanning electron microscopy (SEM), Fourier transform infrared (FTIR, spectrum 65, Mirpur, Azad Jamu and Kashmir), X-ray diffraction (XRD), and energy dispersive X-ray spectroscopy (EDX). The optical qualities of fabricated Fe3O4 NPs were investigated and approved by utilizing a double-beam UV-Vis spectrophotometer (Optizen, 3220) with a spectral range of 200-500 nm. The FTIR spectra were used to determine bioactive agents amenable to fabricated NPs and their stabilization in the range of 4000-500 cm −1 . The XRD (Quaid-i-Azam University Islamabad, Pakistan) pattern of the synthesized NPs was revealed at 40 kV and 40 mA using CuKα radiation (1.5406) from 10-80° 2θ at 2°/step. The morphology and size of NPs were determined using the SEM technique (Institute of Space and Technology, Islamabad, Pakistan) operating at 10 kV. The EDX technique was used to determine the elemental composition of synthesized NPs, which was estimated by employing the EDX detector connected to the SEM instrument.

Characterization of Fe 3 O 4 NPs
The fabricated Fe 3 O 4 NPs were characterized using various analytical techniques to determine their size, shape, functional groups, crystallographic structure, and elemental analysis. The characterization techniques include UV-Vis spectroscopy, scanning electron microscopy (SEM), Fourier transform infrared (FTIR, spectrum 65, Mirpur, Azad Jamu and Kashmir), X-ray diffraction (XRD), and energy dispersive X-ray spectroscopy (EDX). The optical qualities of fabricated Fe 3 O 4 NPs were investigated and approved by utilizing a double-beam UV-Vis spectrophotometer (Optizen, 3220) with a spectral range of 200-500 nm. The FTIR spectra were used to determine bioactive agents amenable to fabricated NPs and their stabilization in the range of 4000-500 cm −1 . The XRD (Quaid-i-Azam University, Islamabad, Pakistan) pattern of the synthesized NPs was revealed at 40 kV and 40 mA using CuKα radiation (1.5406) from 10-80 • 2θ at 2 • /step. The morphology and size of NPs were determined using the SEM technique (Institute of Space and Technology, Islamabad, Pakistan) operating at 10 kV. The EDX technique was used to determine the elemental composition of synthesized NPs, which was estimated by employing the EDX detector connected to the SEM instrument.

Measuring Antibacterial Activity
An antibacterial study of Fe 3 O 4 NPs, algae, and plant extracts was performed using the agar well diffusion method against gram-positive (B. pumilus and S. aureus) and gramnegative (E. coli and P. aeruginosa) bacteria. A stock solution of 1 g/10 mL of each sample was used to assess the antibacterial and antioxidant activities. The antimicrobial activity was evaluated in a sterilized laminar flow. The sterilized nutrient agar medium was poured into Petri dishes. The medium was allowed to cool naturally, and the bacterial inoculum was spread gently with the help of cotton buds over the nutrient agar surface. The 6-8 mm wells were made using a sterilized cork borer in the agar medium. The antibiotic rifampicin and sterilized water were used as positive and negative controls, respectively. The algal and plant extracts (20 µL) were used in two wells of their respective plates. Iron oxide NPs were added to the wells at 20 µL, 30 µL, and 40 µL. The Petri plates were sealed cautiously with parafilm. These plates were incubated in an incubator for a day at 37 • C. After a given period, the widths of the growth inhibition zones were scaled in millimeters.

Antioxidant Potential
A 0.12 mg sample of DPPH was measured on a spring balance and mixed with 83 mL of methanol. The reagent bottle containing DPPH was covered with aluminum foil and kept in the dark for some time. A solution containing 1 mg of ascorbic acid in 100 mL of distilled water was used as a standard (control). Five samples of iron oxide NPs in triplets had concentrations of 10 µg/mL, 20 µg/mL, 30 µg/mL, 40 µg/mL, and 50 µg/mL. Methanol with varying concentrations including 690 µg/mL, 680 µg/mL, 670 µg/mL, 660 µg/mL, and 650 µg/mL was added to the 10 µg/mL, 20 µg/mL, 30 µg/mL, 40 µg/mL, and 50 µg/mL of iron oxide NP samples, respectively. Subsequently, 800 µg/mL of DPPH was added to each aliquot of iron oxide NPs and placed in the dark for 30 min. A major change in color from violet/purple to yellow was observed because of the scavenging action of samples. The absorbance was recorded at 517 nm via a spectrophotometer. The antioxidant potential of NPs was compared with that of ascorbic acid. The scavenging activities for iron oxide NPs and ascorbic acid were calculated using the following formula (Equation (1)): Scavenging activity (%) = (A control − A sample)/A control × 100 (1) The same protocol was followed to determine the antioxidant potential of both the algal and plant extracts. After measuring scavenging activity, the IC 50 values for all the desired samples were calculated.

Conclusions
The significance of herbal medicine well recognized in the medical sector because of their multitudinous benefits and limited complications for human health. The current research involves the fabrication of iron oxide NPs via green chemistry by exploiting the extracts of Spirogyra hyaline, a green alga, and Ajuga bracteosa, a medicinal plant. The proposed critically active biomolecules are flavonol glycosides, neo-clerodane, diterpenoids, phytoecdysones, ergosterol, iridoid glycosides, and many other polyphenols. The antioxidant action of the respective NPs, as assessed via the DPPH assay, showed that plant-based Fe 3 O 4 NPs have a stronger ability to restrict oxidative stress than algal-based Fe 3 O 4 NPs. Moreover, multidrug-resistant bacteria enhance the various infections that day-by-day lead to greater mortality around the world. The current biofabricated Fe 3 O 4 NPs may provide an appealing alternative to combat bacteria or act as vehicles for the targeted delivery of drugs. In the current work, it is evinced that both algal-and plant-based Fe 3 O 4 NPs exhibited remarkable antibacterial potency against both gram-positive and gram-negative bacteria. Plant-based Fe 3 O 4 NPs showed many bactericidal effects that were fatal to gram-positive and gram-negative bacterial strains. The efficient antioxidant and remarkable antibacterial actions of plant-based NPs compared to algal-based NPs might be due to a higher num-ber of various bioactive agents in the plant extract. The present study emphasized the importance of green-fabricated Fe 3 O 4 NPs in the biomedical field, particularly as a potent antioxidant and an effective antimicrobial agent.

Data Availability Statement:
We declared that the materials described in the manuscript, including all relevant raw data, will be freely available to any scientist wishing to use them for noncommercial purposes, without breaching participant confidentiality.