2.1 Preparation of plant extract

The S. officinalis dried leaves were procured at a local market located in Riyadh, Saudi Arabia. The verification of plant samples' identity was conducted by the herbarium affiliated with the department of Botany and Microbiology. The dried leaves of S. officinalis underwent a triple cleaning method, which including the use of distilled water after an initial wash with tap water. Subsequently, the desiccated leaves were subjected to natural air drying in the surrounding environment. The leaves underwent a process of pulverization, resulting in the production of a finely powdered substance with a consistent texture, achieved by the use of a mechanical blender. A flask with a capacity of 500 ml was used to accommodate a quantity of 50 g of plant powder together with 200 ml of distilled water. The flask was exposed to a temperature of 60 °C for a period of 30 min using a hot plate. The flask was thereafter subjected to continuous agitation for a period of 24 h at a temperature of 25 °C using a magnetic stirrer. Subsequently, the combination was subjected to filtration using Whatman filter paper (1) to acquire a pure filtrate and eliminate any residual contaminants. Following that, the extract was subjected to sterilization through filtering with a 0.45 µm Millipore membrane filter. Following this, the produced extracts were stored at a temperature of 4 °C for future research.

2.2 Biosynthesis of Fe₂O₃ nanoparticles

Ferric nitrate (Fe(NO₃)₃·9H₂O) of purity ≥ 98 % was purchased from Sigma Aldrich, U.K. For synthesis of IONPs, 0.01 M Ferric nitrate (Fe(NO₃)₃·9H₂O) solution will be added to the prepared aqueous extract of S. officinalis in a 1:1 proportion. The concentration of the plant extract was 7.8 g/L and the reaction was done under room temperature (25 °C ± 2). Formation of black color indicated the formation of IONPs. The reduced solution was centrifuged at 10,000 rpm for 10 min and supernatant was discarded. The pellets were washed thrice with distilled water for removal of impurities. Finally, the biogenic IONPs were dried in an oven at 32 °C, yielding a black powder and the reaction yield was detected to be 9.33 mg/ml. The pH of the reaction was monitored and seen to rise from an initial value of 1.98, corresponding to the pH of the ferric nitrate solution, to a final value of 5.56, representing the pH of the reaction mixture at the end of the reaction and after addition of plant extract. According to a recent study by Akintelu et al. (2021), it was found that the synthesis of IONPs using plant extract is not favored under extreme acidic and basic conditions. In this context, it was observed that the addition of plant extract to the reaction mixture resulted in an increase in pH value, which potentially stimulated the formation of biogenic IONPs.

2.3 Physicochemical characterization of the biogenic IONPs

Different methods were used to analyze the biogenic IONPs, including UV–Vis spectroscopy, which was used to assess the optical characteristics of the IONPs. The morphology and size distribution of the biologically synthesized iron oxide nanoparticles (IONPs) were analyzed using a Transmission Electron Microscope (TEM) (model JEM1011, JEOL, Tokyo, Japan). In addition, the elemental composition of IONPs was evaluated using Energy-Dispersive X-ray (EDX) analysis. In addition, a Fourier transform infrared spectroscopy (FTIR) study was used to identify the main functional groups found in the biofabricated IONPs. The biogenic iron oxide nanoparticles (IONPs) were subjected to X-ray powder diffraction (XRD) analysis to confirm their crystalline structure and determine their crystal size. The Zeta sizer equipment (Malvern Instruments Ltd; zs90, Worcestershire, UK) was used to evaluate the zeta potential value and hydrodynamic diameter of IONPs.

2.4 Evaluation of antibacterial efficiency of the biosynthesized IONPs

The susceptibility of three nosocomial microbial strains, namely methicillin-resistant Staphylococcus aureus (ATCC 43300), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 9027) to the biogenic IONPs was investigated. P. aeruginosa (ATCC 9027) and MRSA (ATCC 43300) strains were reported as MDR pathogens in a previous study (Yassin et al., 2022). The bacterial suspension was prepared by utilizing a sterile saline solution with a concentration of 0.85 %. This was achieved by selecting fresh colonies from 24-hour cultures and immersing them into the saline solution. To achieve viable bacterial cell count of 1.0 × 10⁸, the turbidity of the microbial suspension was adjusted using a 0.5 McFarland standard. The bacterial suspension (0.5 mL) was transferred and evenly distributed onto freshly prepared Mueller-Hinton agar (MHA) medium. Tigecycline, which was used as standard antibacterial agent, was acquired from Sigma–Aldrich (St. Louis, MO, USA). Subsequently, sterile filter paper discs (8 mm in diameter) were impregnated with tigecycline to attain final concentration of 15 μg/disk, serving as positive controls. Furthermore, 8 mm diameter sterile filter paper discs were impregnated with 100 and 200 μg of the biosynthesized IONPs following their dispersion in methanol solvent. Filter paper discs saturated with methanol alone were employed as negative controls. Subsequently, the loaded discs were positioned on top of the seeded layer of MHA plates. Following this, the plates were refrigerated for 4 h to facilitate the diffusion of the biogenic IONPs through the medium. The measurement of inhibition zones was conducted using a Vernier caliper following a 24-hour incubation period at 35 °C. The broth microdilution assay was employed to determine the minimum inhibitory concentration of the biogenic IONPs produced from the leaf extract of S. officinalis. Furthermore, the determination of the minimum bactericidal concentration (MBC) was conducted by culturing the inoculums obtained from wells with minimum inhibitory concentration (MIC) over freshly prepared Mueller-Hinton agar (MHA) plates. These plates were then incubated at a temperature of 35 °C for a period of overnight incubation. Finally, the plates were examined to assess the presence or absence of bacterial growth. The minimum concentration of biogenic IONPs at which no bacterial growth was observed were recorded as MBC.

2.5 Determination of synergistic patterns of the biogenic IONPs with tigecycline

The standard disc diffusion method was employed to assess the combined antibacterial efficacy of biogenic IONPs (200 µg/disk) and tigecycline antibiotic (15 µg/disk) in a synergistic manner. The sterile filter paper discs, measuring 8 mm in diameter, were loaded with 200 µg of IONPs. In another group, the discs were impregnated with both the tigecycline antibiotic and IONPs. Furthermore, the experimental setup involved the loading of control discs with tigecycline antibiotic and methanol solvent, representing the positive and negative controls, respectively. Subsequently, the loaded discs were positioned onto the seeded layer of MHA plates, following the previously outlined procedure. Subsequently, the percentage of synergism (%) was calculated using the equation provided below: $$\mathit{Synergism}(\%)=(B-A)/A\times 100$$ Whereas, A: referred to the inhibition zone diameter of tigecycline antibiotic and B: referred to the inhibition zone diameter of the combined tigecycline and the biogenic IONPs.

The increase in fold of inhibition area (IFA) value was calculated according to the following formula: (IFA) = (B² − A²)/A², whereas A: referred to the inhibition zone diameter of tigecycline antibiotic and B: referred to the inhibition zone diameter of the combined tigecycline and the biogenic IONPs.

2.6 Statistical analysis

The study data were subjected to statistical analysis using GraphPad Prism version 8.0 (GraphPad Software, Inc., La Jolla, CA, USA) through the application of the Tukey test in a One-way ANOVA with a significance level of 0.05. The experiments were conducted in triplicate, and the resulting data were reported as the mean of triplicate measurements ± the standard error. The particle size distribution histogram, FTIR and XRD pattern were generated using OriginPro 2018 software.

3.1 Green synthesis of IONPs

The water extract of S. officinalis leaves was utilized for the green synthesis of IONPs as seen in Fig. 1a. The plant extract of S. officinalis act as a reducing agent of ferric nitrate solution resulting in formation of IONPs.

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Fig. 1

a. Green biofabrication of IONPs using S. officinalis extract. (A): Water extract of S. officinalis, (B): Ferric nitrate solution, (C): IONPs; Fig. 1b. UV–Vis spectrum of the biofabricated IONPs (black line) and S. officinalis extract (red line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.2 UV spectral analysis

UV– Vis spectral analysis of the biosynthesized IONPs revealed the presence of two absorption peaks at 244 and 349 nm (Fig. 1b). However, UV-analysis of the plant extract affirmed the formation of UV absorption band at 242 nm. The band noticed at λ_max around 200 could be assigned to adsorption of phytochemical constituents as flavonoids, polyphenolic compounds, and heteroatoms as N, S, O and unsaturated groups. Taken together, the absorption peak found at 349 nm could be assigned to the surface plasmon resonance of biogenic IONPs.

3.3 TEM analysis of the biofabricated IONPs

TEM analysis was conducted to estimate the shape and size of the phyto-synthesized IONPs. In this context, the average particle size was detected to be 42.327 nm (Fig. 2a). Moreover, TEM micrographs revealed that the nanoparticles exhibit a spherical morphology, with certain particles exhibiting agglomeration. Particle size distribution histogram indicated that the size of the phyto-synthesized IONPs ranged from 10 to 80 nm in diameter with average particle size of 42.327 nm (Fig. 2b).

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Fig. 2

(A) TEM micrograph of the biosynthesized IONPs, (B) Particle size distribution of the biogenic IONPs.

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3.4 EDX analysis of biogenic IONPs

The elemental analysis of the phyto-synthesized IONPs was distinguished using EDX analysis. In this context, the characteristic signals of Fe were actually detected at 0.8, 6.4 and 7.0 keV for Fe L_a, Fe K_a and Fe K_b whereas the signals of C and O were detected at 0.3 and 0.5 keV, respectively (Fig. 3). Moreover, the prominent peak of silicon was detected at 1.7 keV, which could be allotted to the capping molecules of S. officinalis utilized in the synthesis procedure. The carbon peak was accredited to the carbon tape that was used for positioning the biogenic IONPs on the sample holder. Accordingly, the mass percentage (%) of Fe in IONPs was recalculated and the detected mass % was detected to be 56.46 % after excluding the mass % of C element.

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Fig. 3

Elemental composition of the biofabricated IONPs.

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3.5 FTIR analysis of the phyto-synthesized IONPs

Fourier-transform infrared spectroscopy analysis was conducted to determine the main functional groups of the phyto-synthesized IONPs. FTIR spectra of the biologically synthesized IONPs exhibited the existence of six absorption peaks at the following wavenumbers: 3434.75, 1627.30, 1384.17, 1261.97, 1048.24, and 586.21 cm⁻¹, correspondingly. (Fig. 4). The broad band detected at 3434.75 cm⁻¹ could be assigned to O–H stretching of phenolic compounds whereas the peak noticed at 1627.30 cm⁻¹ might be attributed to C = C stretching vibrations of aromatic compounds as flavonoids and polyphenolic compounds. Additionally, it is worth noting that the band detected at a wavenumber of 1384.17 cm⁻¹ may be attributed to the stretching vibrations of C–H bonds in aldehydes. Similarly, the peaks observed at 1261.97 cm⁻¹ can be attributed to the stretching vibrations of C–N bonds in aromatic amines. Likewise, the band seen at a wavenumber of 1048.24 cm⁻¹ may be attributed to the stretching vibration of the carbon–nitrogen bond in amines. The broadband seen at a wavenumber of 586.21 cm⁻¹ may be attributed to the Fe-O stretching vibration in hematite (Fe₂O₃).

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Fig. 4

FTIR spectrum of the biosynthesized IONPs.

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3.6 XRD analysis of the biosynthesized IONPs

XRD analysis affirmed the formation of nine distinct diffraction peaks at 2 theta degrees of 24.80°, 33.41°, 35.03°, 41.28°, 49.15°, 53.41°, 57.37°, 62.40° and 64.31°, assigned to the lattice planes of (0 1 2), (1 0 4), (1 1 0), (2 0 2), (0 2 4), (1 1 6), (2 1 1), (2 1 4) and (3 0 0), respectively (Fig. 5).

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Fig. 5

XRD pattern of the biogenic IONPs.

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3.7 Zeta potential and zetasizer analysis of the phyto-synthesized IONPs

The hydrodynamic diameter of the IONPs synthesized using S. officinalis leaves was detected to be 681.4 nm (Fig. 6A), which was notably higher than the diameter measured by transmission electron microscopy (TEM). The biogenic IONPs surface charge was found to be –5.48 mV (Fig. 6B).

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Fig. 6

(A)The hydrodynamic diameter of the biogenic IONPs, (B) Zeta potential of the biofabricated IONPs.

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3.8 Antibacterial effectiveness of the phyto-synthesized IONPs

The antibacterial effectiveness of the greenly synthesized IONPs was evaluated against E. coli, P. aeruginosa and MRSA strains using the disk diffusion method (Fig. 7). In this setting, the biogenic IONPs (200 µg/disk) synthesized using S. officinalis extract revealed antibacterial efficiency against E. coli and P. aeruginosa strains demonstrating relative inhibitory zones of 21.14 ± 0.16 and 17.26 ± 0.26 mm, respectively (Table 1). Moreover, the biogenic IONPs revealed antibacterial efficiency against the tested MRSA stain recording relative inhibitory zone of 9.17 ± 0.48, 20.56 ± 0.62 mm, respectively. However, faint inhibitory zones were detected at 100 µg/disk against bacterial pathogens. The minimum inhibitory concentration of the phyto-synthesized IONPs was detected to be 80 µg/ml against E. coli strain whereas the minimum bactericidal concentration was detected to be 160 µg/ml.

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Fig. 7

Antimicrobial activity of the phyto-synthesized IONPs (IONPs) against E. coli and P. aeruginosa strains.

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3.9 Synergistic antibacterial effectiveness of biogenic IONPs with tigecycline

The synergistic antibacterial efficiency of the biogenic IONPs (200 µg/disk) with tigecycline antibiotic was estimated using disk diffusion method. In this context, the highest synergistic percentage of tigecycline + IONPs combination was detected against 67.15 % against E. coli strain whereas the lowest synergistic percentage was detected against P. aeruginosa strain, with relative percentage of 38.85 %. Moreover, the tigecycline + IONPs combination exposed a synergistic antibacterial efficiency against MRSA strain with relative synergism percentage of 51.6 % (Table 2).