Green-synthesized metal oxide nanoparticles enhance the antibacterial activity of Portulaca oleracea through foliar nano-elicitation

Abstract

Acne-associated and other skin-related bacterial infections are increasingly difficult to manage because of antimicrobial resistance and the need for safer therapeutic alternatives. Portulaca oleracea is a medicinal plant with recognized bioactive potential, yet its antibacterial activity can potentially be enhanced through nano-elicitation. Unlike studies that examine nanoparticles as direct antimicrobials, this work investigated green-synthesized metal oxide nanoparticles as foliar elicitors to improve the antibacterial potential of P. oleracea extracts. This approach links sustainable nanoparticle synthesis with medicinal-plant priming to generate extracts with greater activity against acne-associated pathogens. Zinc oxide, magnesium oxide, and alpha-iron oxide nanoparticles were synthesized using aqueous Psidium guajava leaf extract and verified by standard physicochemical characterization. P. oleracea plants received foliar applications of 100 ppm nanoparticle suspensions under greenhouse conditions, after which methanolic aerial-part extracts were evaluated against Staphylococcus aureus, Escherichia coli, and Cutibacterium acnes. Iron oxide treatment produced the strongest response without visible phytotoxicity. By day 25, FeNP-treated plants reached 10.7 ± 0.7 cm in height and 12 ± 1 leaves, compared with 9.1 ± 0.6 cm and 10 ± 1 leaves in untreated controls. Extracts from FeNP-treated plants also showed the highest antibacterial activity, with inhibition zones of 20.0 ± 1.7 mm against S. aureus, 15.7 ± 1.5 mm against E. coli, and 18.3 ± 1.5 mm against C. acnes. These findings show that foliar nano-elicitation, particularly with FeNPs, can enhance plant growth and strengthen the antibacterial potential of P. oleracea. Because phytochemical changes were inferred indirectly from FTIR patterns and antibacterial response rather than direct metabolite quantification, future studies should perform quantitative phytochemical profiling and confirm extract potency through MIC and MBC assays.

Introduction

Acne vulgaris is one of the most common dermatological disorders worldwide and can markedly impair patients’ quality of life through persistent inflammation, post-acne scarring, and psychological distress (Tahir et al., 2025; Wang et al., 2017). Its pathogenesis is multifactorial and involves increased sebum production, follicular hyperkeratinization, microbial colonization, and inflammation (Zhang et al., 2018). Among the microorganisms associated with acne, Cutibacterium acnes (formerly Propionibacterium acnes) is the principal bacterium implicated in disease development because it contributes to inflammation through the production of lipases, proteases, and pro-inflammatory mediators (Platsidaki, 2020). In addition, Staphylococcus aureus may act as a secondary colonizer that can aggravate infection severity and complicate treatment outcomes. Recent nanoparticle-based antimicrobial work has also highlighted the clinical importance of Staphylococcus species in biofilm-associated infections (Mahapatra and Paul 2024; Wu et al. 2025; Lu et al. 2024).

Topical and systemic antibiotics, including clindamycin, erythromycin, and tetracyclines, have long been used in acne management. However, their sustained use has been increasingly challenged by antimicrobial resistance, which reduces treatment efficacy and reinforces the need for alternative or adjunct therapeutic strategies (Nakase et al., 2022; Dreno et al., 2018). In this context, medicinal plants and nanotechnology have emerged as promising complementary approaches. Portulaca oleracea L. is a medicinal plant rich in flavonoids, alkaloids, phenolic acids, and other secondary metabolites with reported antioxidant and antimicrobial potential (Iranshahy et al., 2020). Likewise, metal and metal oxide nanoparticles such as zinc oxide, magnesium oxide, and iron oxide have attracted interest because of their broad-spectrum antimicrobial effects, which may involve membrane disruption, ion release, oxidative stress induction, and interference with essential microbial processes (Vazquez-Muñoz, 2020; Zhao et al. 2025).

Green synthesis has received growing attention as an environmentally friendly and biocompatible route for nanoparticle production. In biological synthesis, natural reducing and stabilizing agents derived from plants or microorganism’s mediate nanoparticle formation, often improving bioactivity while reducing the need for hazardous reagents. A recent study on biologically synthesized selenium nanoparticles described this approach as an eco-friendly strategy that can yield bioactive nanomaterials with low toxicity and enhanced bioavailability, while also emphasizing the role of extracellular enzymes, proteins, and metabolites in nanoparticle formation and capping. Such features make biologically synthesized nanoparticles attractive for biomedical and antimicrobial applications (Wang et al. 2025a, b; Yan et al. 2026a).

Importantly, the biological performance of nanomaterials depends not only on their chemical identity but also on their physicochemical characteristics. Parameters such as shape, size, dissolution behavior, agglomeration state, chemical composition, specific surface area, crystal structure, surface morphology, surface energy, surface coating, and surface charge can strongly influence biological interactions, biodistribution, cellular uptake, oxidative stress generation, and both desired and adverse outcomes (Yan et al., 2026b; Li et al. 2024). Therefore, these properties should be considered when interpreting the antimicrobial activity and biosafety of nanomaterials. This concept is well supported in the nanotoxicology literature, including the review by Samadian et al., which discusses how the unique physicochemical properties of nanomaterials can govern their biological behavior and make them function as “double-edged swords.”

Although plant extracts have frequently been used for the green synthesis of nanoparticles, the use of nanoparticles as foliar nano-elicitors to stimulate secondary metabolism in medicinal plants remains comparatively underexplored. Foliar nanoparticle application may activate plant defense responses and metabolic pathways, thereby increasing the production of endogenous bioactive compounds. This creates an interesting dual opportunity: nanoparticles may act not only as direct antimicrobial agents, but also as indirect enhancers of phytochemical accumulation in medicinal plants.

Despite increasing interest in plant-mediated nanoparticle synthesis and the medicinal potential of Portulaca oleracea L., limited attention has been given to whether foliar application of green-synthesized nanoparticles can enhance the antimicrobial activity of the plant itself. In particular, the effect of nanoparticle foliar elicitation on the subsequent antibacterial activity of P. oleracea extracts against acne-relevant and skin-associated bacteria remains insufficiently studied.

The novelty of the present study lies in integrating two approaches that are usually investigated separately: green synthesis of metal oxide nanoparticles and nanoparticle-mediated elicitation of a medicinal plant. Specifically, zinc oxide (ZnO), magnesium oxide (MgO), and alpha-iron oxide (α-Fe₂O₃) nanoparticles were synthesized using Psidium guajava leaf extract and then applied foliarly to Portulaca oleracea L. to assess whether this treatment enhances the antimicrobial efficacy of the resulting plant extracts.

This work is significant because it advances the interface between phyto-nanotechnology and antimicrobial research. Scientifically, it contributes to understanding how nanoparticle elicitation may modulate plant secondary metabolism and downstream antibacterial performance. Practically, it may support the development of alternative or adjunct antimicrobial strategies relevant to acne-associated and skin-related bacterial infections, especially in the context of increasing concern over antibiotic resistance.

Accordingly, the objectives of this study were to: synthesize ZnO, MgO, and α-Fe₂O₃ nanoparticles using Psidium guajava leaf extract through a green synthesis approach; apply the synthesized nanoparticles foliarly to Portulaca oleracea L. plants as nano-elicitors; evaluate whether foliar nanoparticle treatment enhances the antimicrobial activity of P. oleracea extracts; and compare the antibacterial activity of the treated plant extracts against Cutibacterium acnes (ATCC 6919), Staphylococcus aureus, and Escherichia coli.

Materials and methods

Green synthesis of metallic nanoparticles

The green synthesis of zinc oxide (ZnO), magnesium oxide (MgO), and iron oxide (α-Fe₂O₃, hematite) nanoparticles was carried out using aqueous leaf extract of Psidium guajava as a biological reducing and stabilizing agent, following previously reported plant-mediated synthesis protocols with slight modifications (Salem, 2021; Kumar et al., 2020). Fresh leaves of P. guajava were collected, thoroughly rinsed with tap water followed by distilled water to remove adhering impurities, and shade-dried at ambient temperature until constant weight was achieved. The dried leaves were then pulverized into fine powder using a laboratory grinder.

For extract preparation, 45 g of the powdered leaf material was mixed with 450 mL of distilled water and incubated at 52 °C for 48 h under continuous agitation to facilitate the extraction of bioactive phytochemicals. The resulting mixture was cooled to room temperature and filtered sequentially through muslin cloth and Whatman No. 1 filter paper to obtain a clear aqueous extract. This extract was used immediately for nanoparticle synthesis.

For the synthesis of ZnO nanoparticles, 13.39 g of Zn(NO₃)₂·6 H₂O was added to the plant extract under constant magnetic stirring at 80 °C. Similarly, 11.54 g of Mg(NO₃)₂·6 H₂O and 12.1 g of FeCl₃·6 H₂O were separately added to fresh aliquots of the extract for the synthesis of MgO and α-Fe₂O₃ nanoparticles, respectively. In each case, the reaction mixture was maintained under continuous stirring to ensure efficient interaction between the metal precursor and the phytochemical constituents of the extract. The pH of each reaction system was adjusted to approximately 10 by dropwise addition of 1 M NaOH. The reaction was allowed to proceed for 3 h at 80 °C, during which visible color change and precipitate formation were taken as preliminary indicators of nanoparticle generation.

At the end of the reaction period, the suspensions were cooled to room temperature, and the formed nanoparticles were recovered by centrifugation at 5000 rpm for 10 min. The precipitates were washed repeatedly with distilled water to remove unreacted salts and residual soluble impurities. The washed pellets were dried at 60 °C to constant weight and subsequently calcined at 400 °C for 2 h in order to enhance crystallinity and phase purity. The final nanoparticle powders were stored in sterile airtight containers until further analysis and application.

Characterization of synthesized nanoparticles

The synthesized nanoparticles were subjected to comprehensive physicochemical characterization in order to confirm their formation and determine their optical, structural, and morphological properties. UV-Visible spectroscopy was performed using a Shimadzu UV-1800 spectrophotometer over the wavelength range of 200–800 nm. For analysis, each nanoparticle sample was dispersed in distilled water and homogenized prior to spectral acquisition. The absorbance profiles were recorded to identify characteristic optical signatures associated with nanoparticle formation.

Fourier-transform infrared spectroscopy was carried out using a PerkinElmer Spectrum Two spectrometer in the range of 500–4000 cm⁻¹. FTIR analysis was used to identify the functional groups derived from plant phytochemicals that may have contributed to nanoparticle reduction and stabilization, as well as to detect metal-oxygen stretching vibrations indicative of oxide formation. Structural and crystalline properties were assessed by X-ray diffraction using a Rigaku MiniFlex 600 diffractometer operated with Cu Kα radiation (λ = 1.5406 Å), and diffraction patterns were recorded over a 2θ range of 10°-80°. Crystallite size was estimated from the major diffraction peaks using the Debye-Scherrer equation.

Morphological features of the synthesized nanoparticles were examined by field-emission scanning electron microscopy using a Hitachi SU8000 instrument. This analysis provided information on particle shape, aggregation pattern, and surface texture. Where applicable, elemental composition was additionally assessed during microscopic examination to support identification of the synthesized nanomaterials.

Cultivation of Portulaca oleracea and foliar nanoparticle treatment

Healthy plants of Portulaca oleracea were grown under controlled greenhouse conditions at 28 ± 2 °C under a 12 h light/12 h dark photoperiod. Plants were maintained under uniform irrigation and cultivation conditions throughout the experiment. Prior to treatment, plants of comparable size and growth stage were selected to minimize biological variation among treatment groups.

The plants were randomly distributed into four experimental groups consisting of an untreated control group sprayed with distilled water and three treatment groups sprayed with ZnO, MgO, or α-Fe₂O₃ nanoparticle suspensions, respectively. Each treatment group included three biological replicates. Nanoparticle suspensions were freshly prepared in distilled water at a final concentration of 100 ppm prior to application. Foliar treatment was performed twice weekly for 3 consecutive weeks according to previously described plant treatment strategies with minor modifications (Shang et al., 2019; Zhang et al., 2018). Spraying was applied uniformly to the aerial parts of the plants until near run-off, with care taken to maximize surface coverage. Plant growth parameters, including plant height and number of leaves, were recorded at 5-day intervals over a total experimental period of 25 days. The foliar treatment schedule is presented in Table 1.

Table 1.

Foliar application and monitoring schedule for nanoparticle treatments in Portulaca oleracea

Treatment Group	Nanoparticle Type	Concentration (ppm)	Mode of Application	Initial Treatment	Subsequent Applications	Observation Days	Extract Collection Day	Remarks
Control	—	—	Distilled water spray	Day 0	—	0, 5, 10, 15, 20, 25	Day 25	No nanoparticles applied
ZnNPs	Zinc oxide (ZnO)	100	Foliar spray	Day 0	Days 3, 7, 10, 14, 17, 21, 24	0, 5, 10, 15, 20, 25	Day 25	Normal growth, no toxicity observed
MgNPs	Magnesium oxide (MgO)	100	Foliar spray	Day 0	Days 3, 7, 10, 14, 17, 21, 24	0, 5, 10, 15, 20, 25	Day 25	Slightly improved leaf pigmentation
FeNPs	Iron oxide (α-Fe₂O₃)	100	Foliar spray	Day 0	Days 3, 7, 10, 14, 17, 21, 24	0, 5, 10, 15, 20, 25	Day 25	Good foliar absorption, enhanced vigor

Preparation of methanolic plant extracts

At the completion of the treatment period, the aerial parts of P. oleracea were harvested and dried under shade at room temperature until a constant dry weight was obtained. The dried material was ground into fine powder and subjected to Soxhlet extraction using 80% methanol as the extraction solvent. Extraction was continued for 8 h to ensure exhaustive recovery of methanol-soluble phytoconstituents. The extract was then filtered and concentrated under reduced pressure at 40 °C using a rotary evaporator. The resulting crude extract was collected in sterile glass vials and stored at − 20 °C until use. Prior to antibacterial testing, the extract was reconstituted in dimethyl sulfoxide to the desired concentration (Zhang et al., 2018).

Antibacterial activity assay

The antibacterial activity of the prepared plant extracts was evaluated by the agar well diffusion method as previously described by Balouiri et al. (2016). The test microorganisms used in this study were Staphylococcus aureus ATCC 25,923, Escherichia coli ATCC 25,922, and Cutibacterium acnes ATCC 6919. Fresh bacterial inocula were prepared and adjusted to 0.5 McFarland turbidity standard, corresponding to approximately 1 × 10⁸ CFU/mL, to ensure uniform inoculum density across all tests.

Mueller-Hinton agar plates were prepared under aseptic conditions and inoculated by evenly spreading each bacterial suspension over the agar surface using sterile cotton swabs. Wells of 6 mm diameter were punched into the inoculated agar using a sterile cork borer. Each well was loaded with 100 µL of plant extract solution prepared at a concentration of 100 mg/mL in DMSO. Clindamycin (30 µg) served as the positive control for Gram-positive organisms, whereas ciprofloxacin (5 µg) served as the positive control for E. coli. Sterile DMSO was used as the negative control.

The plates inoculated with S. aureus and E. coli were incubated aerobically at 37 °C for 24 h. Plates inoculated with C. acnes were incubated anaerobically at 37 °C for 48 h using an anaerobic jar equipped with an anaerobic indicator system to confirm oxygen-free conditions. After incubation, the diameters of inhibition zones were measured in millimeters. To obtain the net inhibition value, the diameter of the well was subtracted from the total inhibition zone. All assays were conducted in triplicate under sterile laboratory conditions.

Statistical analysis

All experimental data were generated from three independent biological replicates and are expressed as mean ± standard deviation. Statistical analyses were performed using SPSS version 25.0 (IBM Corp, 2017). Differences among treatment groups were evaluated by one-way analysis of variance followed by Tukey’s honestly significant difference post hoc test. A p-value of less than 0.05 was considered statistically significant. Graphical data presentation was performed using GraphPad Prism version 9.0 (GraphPad Software, 2020).

Results

Synthesis and characterization of nanoparticles

Green synthesis of ZnO, MgO, and α-Fe₂O₃ nanoparticles using Psidium guajava leaf extract was successfully achieved, and the experimental workflow is summarized in Fig. 1. The synthesis sequence included extract preparation, precursor addition, pH adjustment, washing, drying, calcination, and nanoparticle collection.

Fig. 1.

Schematic overview of the green synthesis workflow for ZnO, MgO, and α-Fe₂O₃ nanoparticles using Psidium guajava leaf extract, including extract preparation, precursor addition, pH adjustment, washing, drying, calcination, and final nanoparticle recovery

Preliminary optical characterization by UV-Vis spectroscopy showed peak wavelengths at 198 nm for zinc nanoparticles, 194 nm for magnesium nanoparticles, and 193 nm for iron nanoparticles, as presented in Table 2; Fig. 2. These findings supported the formation of the synthesized nanoparticle preparations and distinguished the three nanoparticle systems at the optical level.

Table 2.

UV–Vis peak wavelengths of synthesized nanoparticles

Nanoparticles Type	UV-vis Peak Wavelength (nm)
Zinc	198
Magnesium	194
Iron	193

Fig. 2.

UV-Vis absorption spectra of the green-synthesized nanoparticles: (a) ZnO nanoparticles (ZnNPs), (b) MgO nanoparticles (MgNPs), and (c) iron oxide nanoparticles (FeNPs)

FTIR analysis further confirmed nanoparticle formation and the involvement of plant-derived biomolecules in reduction and stabilization. As shown in Table 3; Fig. 3, ZnNPs exhibited O–H stretching at 3423–3387 cm⁻¹, C = O stretching at 1541–1529 cm⁻¹, C–N/C–O stretching at 1404–1078 cm⁻¹, and Zn–O vibration at 696–606 cm⁻¹. MgNPs displayed O–H stretching at 3430–3370 cm⁻¹, C = O stretching at 1630–1400 cm⁻¹, C–O–C or C–N stretching at 1110–1020 cm⁻¹, and Mg–O vibration at 670–520 cm⁻¹. FeNPs showed O–H stretching at 3427–3477 cm⁻¹, a band at 2351 cm⁻¹ assigned to C ≡ C or CO₂ asymmetric stretching, C–N or C–O–C vibrations at 1403–1107 cm⁻¹, and Fe–O stretching at 686–560 cm⁻¹. These FTIR features indicated the presence of hydroxyl-containing phytochemicals, proteins, polysaccharides, and metal–oxygen bonds associated with the synthesized oxide nanoparticles.

Table 3.

FTIR peak assignments for synthesized nanoparticles

Nanoparticle Type	Wavenumber (cm⁻¹)	Functional Group Assignment	Possible Compounds	Reference
ZnNPs	3423–3387	O–H stretching	Hydroxyl groups from plant extract	(Rai et al., 2021)
	1541–1529	C = O stretching (amide II)	Protein capping on ZnO NPs	(Gunalan et al., 2012)
	1404–1078	C–N / C–O stretching	Plant metabolites (alcohols, esters)	(Karthik et al., 2020)
	696–606	Zn–O stretching vibration	ZnO nanoparticle core	(Sundrarajan et al., 2015)
MgNPs	3430–3370	O–H stretching	Alcohol/phenol groups in biomolecules	(Karthik et al, 2020)
	1630–1400	C = O stretching (amide I, II)	Proteins / amide linkages	(Dinesh et al., 2021)
	1110–1020	C–O–C or C–N stretching	Polysaccharides / alkaloids	(Salem, 2021)
	670–520	Mg–O stretching vibration	Magnesium oxide lattice vibration	(Alotaibi & Negm 2023)
FeNPs	3427–3477	O–H stretching	Polyphenols / flavonoids in plant extract	(Rai et al, 2021)
	2351	C ≡ C or CO₂ asymmetric stretching	Organic acids or CO₂ adsorption	(Kumar et al., 2020)
	1403–1107	C–N stretching or C–O–C vibrations	Proteins / polysaccharides	(Aisida et al., 2019)
	686–560	Fe–O stretching vibration	Iron oxide (Fe–O bond)	(Gobinath et al. 2015)

Fig. 3.

FTIR spectra of the green-synthesized nanoparticles: (a) ZnNPs, (b) MgNPs, and (c) FeNPs, showing major absorption bands associated with plant-derived biomolecules involved in nanoparticle bioreduction and surface capping/stabilization

FE-SEM analysis demonstrated clear morphological differences among the synthesized nanoparticles, as shown in Fig. 4; Table 4. ZnNPs were irregular in shape, with rough surfaces and moderate clustering, and showed sizes ranging from 206 to 320 nm. MgNPs were spherical, more uniform, and well dispersed, with an average size of 165 nm. FeNPs were also spherical and showed slight aggregation, with sizes of 165 and 176 nm. These observations indicated that particle morphology and aggregation behavior varied according to nanoparticle type.

Fig. 4.

Representative FE-SEM micrographs of the synthesized nanoparticles showing the surface morphology of (a) ZnNPs, (b) MgNPs, and (c) FeNPs

Table 4.

SEM-derived morphology and average particle size (nm) of synthesized nanoparticles

Nanoparticle Type	Shape	Average Size (nm)	Morphology Description
ZnNPs	Irregular	206–320	Rough surface, moderate clusters
MgNPs	Spherical	165	Uniform, well-dispersed
FeNPs	Spherical	165,176	Slightly aggregated

XRD analysis confirmed the crystalline nature of all synthesized nanoparticles, as illustrated in Fig. 5 and summarized in Table 5. The XRD-derived crystallite sizes were 18.58 nm for ZnNPs, 37.68 nm for MgNPs, and 10.44 nm for FeNPs. In Table 5, Zn nanoparticles were described as showing strong plasmon resonance, good crystallinity, and stability; Mg nanoparticles showed prominent O–H stretching linked to phytochemical capping and confirmed MgO formation; and Fe nanoparticles showed broader peaks due to FeO bond vibrations and confirmed iron oxide nanoparticle formation.

Fig. 5.

XRD patterns of the green-synthesized nanoparticles: (a) ZnNPs, (b) MgNPs, and (c) FeNPs, confirming crystalline phase formation and nanoparticle crystallinity

Table 5.

Integrated nanoparticle characterization summary (SEM mean/aggregate size, UV–Vis peak wavelength, XRD crystallite size, and dominant FTIR functional groups)

Nanoparticle Type	Mean Particle Size (nm)	UV-Vis Peak Wavelength (nm)	Crystalline Structure (XRD) size	Major Functional Groups (from FTIR)	Key Observations
Zinc (Zn)	206,320	198	18.58 nm	O–H, C = O, C– N / C–O, Zn–O	Strong plasmon resonance indicating ZnO formation; good crystallinity and Stability.
Magnesium (Mg)	165	194	37.68 nm	O–H, C = O, C– O–C or C–N, Mg–O	Prominent O–H stretching linked to phytochemical capping; confirms MgO nanoparticles formation.
Iron (Fe)	165,176	193	10.44 nm	O–H, C ≡ C or CO₂, C–N or C– O–C, Fe–O	Broader peak due to FeO bond vibrations; confirmed presence of iron oxide nanoparticles.

Growth and physiological response of Portulaca oleracea to foliar nanoparticle treatment

The growth and physiological responses of Portulaca oleracea during foliar nanoparticle treatment are presented in Table 6; Figs. 6 and 7. No visible toxicity was observed in any treatment group throughout the 25-day experimental period. At day 0, all plants showed similar baseline values, with heights ranging from 5.1 ± 0.4 cm to 5.3 ± 0.5 cm and four leaves per plant.

Table 6.

Growth and physiological observations of P. oleracea during 25 days of foliar nanoparticle treatment (100 ppm), reported as mean ± SD (n = 3 plants per treatment), recorded every 5 days according to Table 1

Day	Treatment Group	Plant Height (cm) ± SD	No. of Leaves ± SD	Leaf Color Observation	Overall Growth Response	Visible Toxicity
0	Control	5.2 ± 0.4	4 ± 1	Light green	Normal baseline	None
	FeNPs	5.3 ± 0.5	4 ± 1	Light green	Normal baseline	None
	MgNPs	5.1 ± 0.4	4 ± 1	Light green	Normal baseline	None
	ZnNPs	5.2 ± 0.3	4 ± 1	Light green	Normal baseline	None
5	Control	6.0 ± 0.6	6 ± 1	Slightly green	Normal growth	None
	FeNPs	6.8 ± 0.5	7 ± 1	Deeper green	Improved vigor	None
	MgNPs	6.5 ± 0.4	7 ± 1	Green	Slight enhancement	None
	ZnNPs	6.2 ± 0.5	6 ± 1	Greenish-brown tint	Normal	None
10	Control	7.4 ± 0.5	8 ± 1	Green	Moderate growth	None
	FeNPs	8.6 ± 0.4	9 ± 1	Deep green	Strong growth	None
	MgNPs	8.1 ± 0.6	9 ± 1	Green	Good growth	None
	ZnNPs	7.6 ± 0.5	8 ± 1	Dark green	Normal	None
15	Control	8.3 ± 0.6	9 ± 1	Green	Healthy	None
	FeNPs	9.8 ± 0.5	11 ± 1	Deep green	Excellent vigor	None
	MgNPs	9.2 ± 0.5	10 ± 1	Bright green	Increased vigor	None
	ZnNPs	8.7 ± 0.6	9 ± 1	Dark olive green	Moderate	None
20	Control	8.9 ± 0.7	10 ± 1	Green	Normal	None
	FeNPs	10.4 ± 0.6	12 ± 1	Deep green	Vigorous	None
	MgNPs	9.8 ± 0.6	11 ± 1	Green	Healthy	None
	ZnNPs	9.2 ± 0.7	10 ± 1	Greenish-brown	Normal	None
25	Control	9.1 ± 0.6	10 ± 1	Green	Stable growth	None
	FeNPs	10.7 ± 0.7	12 ± 1	Deep green	Robust	None
	MgNPs	10.1 ± 0.6	11 ± 1	Green	Stable	None
	ZnNPs	9.4 ± 0.7	10 ± 1	Dark green	Moderate	None

Fig. 6.

Representative photographs of Portulaca oleracea plants at day 25 under control treatment (water spray) and foliar nanoparticle treatments (100 ppm), showing overall plant appearance and treatment-related growth differences

Fig. 7.

Growth trajectories of P. oleracea plant height over the 25-day foliar treatment period (100 ppm) in the control and nanoparticle-treated groups (ZnNPs, MgNPs, and FeNPs)

By day 5, FeNP-treated plants reached 6.8 ± 0.5 cm and 7 ± 1 leaves, whereas MgNP-treated plants reached 6.5 ± 0.4 cm and 7 ± 1 leaves. ZnNP-treated plants measured 6.2 ± 0.5 cm with 6 ± 1 leaves, while control plants reached 6.0 ± 0.6 cm with 6 ± 1 leaves. Leaf color observations at this stage indicated deeper green coloration in FeNP-treated plants and slight enhancement in MgNP-treated plants.

At day 10, FeNP-treated plants measured 8.6 ± 0.4 cm with 9 ± 1 leaves, MgNP-treated plants measured 8.1 ± 0.6 cm with 9 ± 1 leaves, ZnNP-treated plants reached 7.6 ± 0.5 cm with 8 ± 1 leaves, and control plants reached 7.4 ± 0.5 cm with 8 ± 1 leaves. FeNP-treated plants were described as showing strong growth, while MgNP-treated plants showed good growth.

At day 15, the same trend persisted. FeNP-treated plants reached 9.8 ± 0.5 cm with 11 ± 1 leaves and were described as showing excellent vigor. MgNP-treated plants reached 9.2 ± 0.5 cm with 10 ± 1 leaves and increased vigor. ZnNP-treated plants reached 8.7 ± 0.6 cm with 9 ± 1 leaves, while controls reached 8.3 ± 0.6 cm with 9 ± 1 leaves.

At day 20, FeNP-treated plants reached 10.4 ± 0.6 cm with 12 ± 1 leaves, MgNP-treated plants reached 9.8 ± 0.6 cm with 11 ± 1 leaves, ZnNP-treated plants reached 9.2 ± 0.7 cm with 10 ± 1 leaves, and control plants reached 8.9 ± 0.7 cm with 10 ± 1 leaves.

At day 25, FeNP-treated plants showed the greatest final growth, reaching 10.7 ± 0.7 cm and 12 ± 1 leaves, followed by MgNP-treated plants at 10.1 ± 0.6 cm and 11 ± 1 leaves. ZnNP-treated plants reached 9.4 ± 0.7 cm and 10 ± 1 leaves, whereas control plants reached 9.1 ± 0.6 cm and 10 ± 1 leaves. FeNP-treated plants were described as robust with deep green leaves, MgNP-treated plants as stable, and ZnNP-treated plants as moderate. These visual and numerical observations are also reflected in Fig. 6 and the height trajectories in Fig. 7.

Antibacterial activity of methanolic extracts from nanoparticle-treated Portulaca oleracea

The antibacterial activity of methanolic extracts from nanoparticle-treated P. oleracea is summarized in Table 7 and illustrated in Figs. 8 and 9. A clear hierarchy of activity was observed across the treatment groups, with FeNP-treated extract showing the highest activity, followed by MgNP-treated extract, ZnNP-treated extract, and control extract.

Table 7.

Antibacterial activity (net zone of inhibition, mm; mean ± SD, n = 3) of methanolic extracts from nanoparticle-treated P. oleracea against S. aureus, E. coli, and C. acnes, including positive antibiotic controls and DMSO negative control

Treatment	Staphylococcus aureus (mm)	Escherichia coli (mm)	Cutibacterium acnes (mm)	Mean ZOI (mm)
FeNP-treated extract	20.0 ± 1.7	15.7 ± 1.5	18.3 ± 1.5	18.0 ± 1.6
MgNP-treated extract	17.3 ± 1.2	13.0 ± 1.0	15.7 ± 1.2	15.3 ± 1.1
ZnNP-treated extract	15.3 ± 0.6	10.7 ± 0.6	14.3 ± 0.6	13.4 ± 0.6
Control extract	1.0 ± 1.0	1.0 ± 1.0	1.7 ± 1.2	---
DMSO (Negative Control)	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0
Clindamycin (Positive Control)	22.3 ± 0.6	---	20.3 ± 0.6	---
ciprofloxacin	---	8.3 ± 0.6	---	---

Fig. 8.

epresentative agar well diffusion images showing inhibition zones produced by methanolic extracts of P. oleracea against (a) Staphylococcus aureus, (b) Escherichia coli, and (c) Cutibacterium acnes in the control and nanoparticle-elicited groups (ZnNP-, MgNP-, and FeNP-treated plants)

Fig. 9.

Antibacterial activity of methanolic extracts from nanoparticle-treated P. oleracea. Bars represent mean zone of inhibition (mm) ± SD against S. aureus, E. coli, and C. acnes (n = 3). Different letters indicate statistically significant differences among treatments according to one-way ANOVA followed by Tukey’s HSD test (p < 0.05)

Against Staphylococcus aureus, the FeNP-treated extract produced a mean inhibition zone of 20.0 ± 1.7 mm, the MgNP-treated extract produced 17.3 ± 1.2 mm, and the ZnNP-treated extract produced 15.3 ± 0.6 mm. The control extract showed only 1.0 ± 1.0 mm inhibition. Clindamycin, used as the positive control, produced an inhibition zone of 22.3 ± 0.6 mm.

Against Escherichia coli, the FeNP-treated extract produced a mean inhibition zone of 15.7 ± 1.5 mm, the MgNP-treated extract produced 13.0 ± 1.0 mm, and the ZnNP-treated extract produced 10.7 ± 0.6 mm. The control extract produced 1.0 ± 1.0 mm, while ciprofloxacin produced 8.3 ± 0.6 mm.

Against Cutibacterium acnes, listed in Table 7; Fig. 8 as Cutibacterium acnes, the FeNP-treated extract produced a mean inhibition zone of 18.3 ± 1.5 mm, the MgNP-treated extract produced 15.7 ± 1.2 mm, and the ZnNP-treated extract produced 14.3 ± 0.6 mm. The control extract produced 1.7 ± 1.2 mm, whereas clindamycin produced 20.3 ± 0.6 mm. DMSO showed no inhibition against any tested bacterium.

The mean zone of inhibition values across bacterial strains were 18.0 ± 1.6 mm for FeNP-treated extract, 15.3 ± 1.1 mm for MgNP-treated extract, and 13.4 ± 0.6 mm for ZnNP-treated extract. These results showed that foliar nanoparticle treatment enhanced the antibacterial activity of P. oleracea, with FeNP treatment producing the strongest effect.

Statistical analysis of antibacterial activity

Pairwise statistical comparison of the mean zone of inhibition values is presented in Table 8. FeNP treatment differed significantly from MgNP treatment by 2.70 ± 0.32 mm with a p-value of 0.002, from ZnNP treatment by 4.60 ± 0.32 mm with p < 0.001, and from the control by 17.03 ± 0.32 mm with p < 0.001. MgNP treatment differed significantly from ZnNP treatment by 1.90 ± 0.32 mm with a p-value of 0.014 and from the control by 14.33 ± 0.32 mm with p < 0.001. ZnNP treatment also differed significantly from the control by 12.43 ± 0.32 mm with p < 0.001. All pairwise comparisons were significant at α = 0.05.

Table 8.

Pairwise comparison of the mean zone of inhibition (mm) of treatments using a Tukey HSD test (data pooled across bacterial strains, n = 9 per group)

Comparison	Mean Difference ± SE	p-value	Significance (α = 0.05)
FeNP vs. MgNP	2.70 ± 0.32	0.002	Significant
FeNP vs. ZnNP	4.60 ± 0.32	< 0.001	Significant
FeNP vs. Control	17.03 ± 0.32	< 0.001	Significant
MgNP vs. ZnNP	1.90 ± 0.32	0.014	Significant
MgNP vs. Control	14.33 ± 0.32	< 0.001	Significant
ZnNP vs. Control	12.43 ± 0.32	< 0.001	Significant

Relationship between crystallite size and antibacterial enhancement

The relationship between nanoparticle crystallite size and mean antibacterial enhancement of P. oleracea extracts is shown in Fig. 10. The figure reports a negative correlation coefficient of r = -0.94267 between nanoparticle crystallite size derived from XRD and mean zone of inhibition across pathogens. Based on Tables 5 and 7, FeNPs showed the smallest XRD crystallite size at 10.44 nm and the highest mean zone of inhibition at 18.0 ± 1.6 mm, ZnNPs showed a crystallite size of 18.58 nm and a mean zone of inhibition of 13.4 ± 0.6 mm, and MgNPs showed a crystallite size of 37.68 nm and a mean zone of inhibition of 15.3 ± 1.1 mm.

Fig. 10.

Association between nanoparticle crystallite size, determined by XRD, and the mean antibacterial enhancement of P. oleracea extracts, expressed as mean zone of inhibition across the tested pathogens

FTIR comparison of control and FeNP-treated Portulaca oleracea extracts

Comparative FTIR analysis of control and FeNP-treated P. oleracea methanolic extracts is presented in Fig. 11; Table 9. The control extract showed a broad band at ~ 3852 cm⁻¹, whereas the FeNP-treated extract showed bands at 3851, 3815, and 3748 cm⁻¹ assigned to O–H stretching of free and hydrogen-bonded hydroxyl groups associated with phenols, alcohols, and flavonoids. Table 9 interprets these shifts and increased intensity as indicating enhanced phenolic content under iron proficiency.

Fig. 11.

FTIR spectra of methanolic extracts of P. oleracea from (a) control plants and (b) FeNP-treated plants, showing spectral shifts and intensity differences associated with foliar nano-elicitation

Table 9.

Comparative FTIR peak analysis of control and FeNP-treated P. oleracea leaf extracts, including wavenumbers (cm⁻¹), functional group assignments, major biomolecule/bond associations, and interpretation of changes under FeNP nano-elicitation

Wavenumber (cm⁻¹) Control (Untreated)	Wavenumber (cm⁻¹) Iron NP- Treated	Functional group assignment	Major biomolecules/bonds	Interpretation (iron proficiency effect)	References
~ 3852	3851, 3815, 3748	O–H stretching (free & hydrogen-bonded hydroxyls)	Phenols, alcohols, flavonoids	Increased intensity and peak shifts indicate enhanced phenolic content under iron proficiency	(Salem, 2021)
~ 2380	2388	C ≡ N stretching / atmospheric CO₂	Organic acids, plant metabolites	Minor shift suggests iron-induced biochemical modulation	(Sutaria et al., 2022)
~ 2064	2002, 1929	C ≡ C / C ≡ N stretching	Alkynes, nitriles	Reduced intensity reflects altered secondary metabolite composition	Socrates 2001
1700–1600	1560	C = O stretching / N–H bending (amide I & II)	Proteins, enzymes	Peak shift indicates iron-mediated changes in protein structure or abundance	(Gunalan et al., 2012)
1450–1400	1398	C–N stretching	Amines, proteins	Higher intensity suggests increased nitrogen-containing biomolecules	(Dinesh et al., 2021)
1200–1000	1204	C–O–C / C–O stretching	Polyphenols, carbohydrates	Confirms enrichment of polyphenolic compounds due to iron treatment	(Sutaria, 2022; Kumar et al., 2020)
< 1000	966, 922	Aromatic C–H bending	Phenolic compounds	Additional bands reflect biochemical enrichment	(Zhou et al., 2015)

Additional spectral differences were observed in several regions. A minor shift from ~ 2380 to 2388 cm⁻¹ was assigned to C ≡ N stretching or atmospheric CO₂ and interpreted as iron-induced biochemical modulation. Changes from ~ 2064 cm⁻¹ in the control to 2002 and 1929 cm⁻¹ in the treated extract were assigned to C ≡ C/C ≡ N stretching and interpreted as altered secondary metabolite composition. The band region 1700–1600 cm⁻¹ in the control shifted to 1560 cm⁻¹ in the treated extract, corresponding to C = O stretching and N–H bending of proteins and enzymes. A shift from 1450 to 1400 cm⁻¹ to 1398 cm⁻¹ was assigned to C–N stretching and associated with increased nitrogen-containing biomolecules. The region 1200–1000 cm⁻¹ shifted to 1204 cm⁻¹ and was assigned to C–O–C/C–O stretching of polyphenols and carbohydrates, while additional bands below 1000 cm⁻¹ at 966 and 922 cm⁻¹ were assigned to aromatic C–H bending of phenolic compounds. Overall, Table 9 interprets these spectral changes as evidence of biochemical enrichment under FeNP nano-elicitation.

Discussion

The present study demonstrated that foliar treatment with green-synthesized metal oxide nanoparticles enhanced the growth response and antibacterial potential of Portulaca oleracea extracts. Among the tested nanoparticle treatments, FeNPs produced the strongest overall response, followed by MgNPs and ZnNPs. This ranking was consistent across plant growth observations, antibacterial activity measurements, and pooled statistical comparisons. FeNP-treated plants showed the greatest final height and leaf number, and FeNP-treated extracts exhibited the largest inhibition zones against S. aureus, E. coli, and Cutibacterium acnes.

The characterization data collectively confirmed successful green synthesis of ZnO, MgO, and α-Fe₂O₃ nanoparticles using Psidium guajava leaf extract. UV-Vis analysis distinguished the three nanoparticle types by their characteristic peak wavelengths, while FTIR spectra indicated the participation of hydroxyl-containing phytochemicals, proteins, polysaccharides, and other plant-derived biomolecules in nanoparticle bioreduction and capping. The presence of Zn–O, Mg–O, and Fe–O vibrational bands confirmed oxide formation. FE-SEM analysis further showed clear morphological variation among the nanoparticles, with ZnNPs appearing more irregular and clustered and MgNPs and FeNPs appearing more spherical. XRD analysis verified nanoscale crystallite formation and showed that FeNPs had the smallest crystallite size among the synthesized materials (Asai et al. 2021; Azwanida 2015; Barbieri et al. 2019). These findings indicate that the plant extract provided both reducing and stabilizing functions during nanoparticle formation and that the resulting materials differed not only in chemical identity but also in size, morphology, and surface-associated biomolecular features. Such physicochemical variation may have contributed to the distinct biological effects observed after foliar application (Ahmed et al. 2025a, b, c; Fadhill and Ahmed 2025; Abas et al. 2025; El-Tayeb et al. 2025).

The growth data showed that foliar nanoparticle application did not induce visible toxicity at 100 ppm and instead promoted plant performance, particularly in the FeNP- and MgNP-treated groups. FeNP-treated plants displayed the highest plant height and leaf number throughout the later stages of the experiment and were described visually as deeper green, vigorous, and robust. MgNP-treated plants also showed improved height, leaf number, and leaf pigmentation relative to the control. ZnNP-treated plants showed a smaller growth-promoting effect. These results suggest that foliar nanoparticle application acted as a biostimulatory treatment under the tested conditions (Ghotekar et al. 2021; Jaiswal et al. 2021; Joseph and Singh 2022). The stronger response observed in the FeNP group may indicate more efficient foliar uptake, stronger elicitation of physiological pathways, or more pronounced modulation of nutrient- or stress-related metabolism. The deep green coloration observed in FeNP-treated plants is consistent with improved physiological status and may reflect enhanced pigment-related or metabolically active growth (Ahmed and Ahmed 2026; Anwer and Ahmed 2024; Samadian et al. 2020; Ahmed et al. 2025d, 2026).

The main biological outcome of the study was the marked increase in antibacterial activity in methanolic extracts from nanoparticle-treated P. oleracea. The strongest activity was consistently observed in the FeNP-treated extract, which produced inhibition zones of 20.0 ± 1.7 mm against S. aureus, 15.7 ± 1.5 mm against E. coli, and 18.3 ± 1.5 mm against Cutibacterium acnes. MgNP-treated and ZnNP-treated extracts also showed enhanced activity compared with the control extract, but the magnitude of the effect was lower than that observed with FeNP treatment.

The pooled mean antibacterial activity values and Tukey HSD results confirmed that all nanoparticle treatments significantly enhanced extract activity compared with the control and that FeNP treatment produced the highest overall antibacterial response. This indicates that foliar nano-elicitation successfully modulated the biological performance of the plant and improved the antibacterial potency of its extracts (Mittal et al. 2020; Mourdikoudis et al. 2018; Phan et al. 2023).

The FTIR comparison between control and FeNP-treated P. oleracea extracts provided further support for biochemical modulation following nano-elicitation. The FeNP-treated extract showed multiple spectral shifts and intensity changes in hydroxyl-, carbonyl-/amide-, amine-, and carbohydrate-associated regions, and Table 9 interpreted these changes as indicating enhanced phenolic content, altered secondary metabolite composition, changes in protein structure or abundance, and enrichment of nitrogen-containing biomolecules and polyphenolic compounds.

Taken together, these spectral changes support the view that FeNP foliar treatment altered the phytochemical profile of P. oleracea, which likely contributed to the stronger antibacterial activity of the resulting extract. The observed enhancement may therefore reflect nanoparticle-mediated stimulation of plant metabolic pathways associated with the biosynthesis or accumulation of antimicrobial constituents (Rastogi et al. 2019; Salem et al. 2022; Singh et al. 2021).

The negative correlation shown in Fig. 10 between crystallite size and mean antibacterial enhancement suggests an association between nanoparticle physicochemical properties and the magnitude of the elicited biological response. FeNPs showed the smallest XRD crystallite size and the greatest antibacterial enhancement, whereas MgNPs showed the largest crystallite size and a lower mean antibacterial effect than FeNPs. ZnNPs showed intermediate crystallite size and lower antibacterial activity than both FeNPs and MgNPs. This pattern suggests that nanoparticle size may be related to the efficiency of foliar nano-elicitation and the extent of downstream phytochemical activation. In the present study, the smallest crystallite size was associated with the strongest antibacterial enhancement, supporting the possibility that more finely crystalline nanoparticles may interact more effectively with plant tissues or induce stronger metabolic responses (Tan et al. 2019; Zu-Man et al. 2024; Zaenglein et al. 2016).

The enhanced activity of nanoparticle-elicited P. oleracea extracts against S. aureus and Cutibacterium acnes is particularly relevant to the dermatological focus of the study. The FeNP-treated extract showed high activity against both organisms and also displayed broad antibacterial activity against E. coli. These results support the potential of nanoparticle-primed medicinal plants as a source of improved plant-derived antibacterial preparations. The findings also reinforce the concept that nanoparticles can be used not only as direct antimicrobial materials but also as foliar elicitors to enhance the medicinal performance of living plants. This expands the potential application of green nanotechnology in pharmaceutical, dermatological, and plant-based antimicrobial research.

A major strength of the study is the integrated design linking nanoparticle synthesis, physicochemical characterization, foliar treatment, plant growth monitoring, antibacterial testing, and comparative FTIR analysis of treated plant extracts. The consistency of the ranking across multiple datasets also strengthens the overall interpretation. The FTIR results indicate biochemical changes following FeNP treatment, and this supports the proposed mechanism of nano-elicitation. However, this mechanistic interpretation remains indirect because the present study did not include quantitative phytochemical profiling of the treated extracts. Therefore, the proposed nano-elicitation effect is currently supported by FTIR-based spectral changes together with the observed biological response, rather than by direct metabolite quantification. Future studies should apply targeted and untargeted phytochemical analyses, such as total phenolic and flavonoid assays, HPLC, LC-MS, or GC-MS, to identify and quantify the specific compounds altered after nanoparticle treatment. Likewise, expanding antibacterial evaluation to MIC and MBC assays would provide additional resolution regarding extract potency and bacteriostatic or bactericidal action.

Conclusion

The study demonstrates that foliar application of green-synthesized metal oxide nanoparticles can enhance both the growth performance and antibacterial potential of Portulaca oleracea. Among the tested treatments, FeNP priming produced the strongest effect, followed by MgNPs and ZnNPs, as reflected in plant height, leaf number, and antibacterial activity against Staphylococcus aureus, Escherichia coli, and Cutibacterium acnes. FeNP-treated plants reached the highest final growth values and yielded extracts with the largest inhibition zones, while all nanoparticle-treated groups performed significantly better than the control. FTIR comparison between control and FeNP-treated extracts further indicated biochemical modulation consistent with enrichment of bioactive plant constituents after nano-elicitation. Together, these findings support the concept that nanoparticles can function not only as antimicrobial materials but also as effective foliar elicitors that improve the medicinal value of plants. This strategy offers promising potential for developing enhanced plant-based therapeutics for skin-associated bacterial infections.

Author contributions

AA, SA, NJ, TA, HQ: methodology, supervision, experimentation, writing, drafting, research design, investigation, data curation; HSEB, IMA, KHA, SD: validation, software; writing, drafting, statistical analysis, validation; NYR, KO, SZ, MSA, MAS: writing, software, resource, research design, validation, data collection, drafting, statistical analysis. All authors reviewed the manuscript.

Funding

This work was supported by Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia (KFU262176). This Publication has been supported by the RUDN University Scientific Projects Grant System, project no. 202787-2-000.

Data availability

The author confirms that all data generated or analyzed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue. So, it is not applicable.

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Not applicable.

Competing interests

The authors declare no competing interests.

Clinical trial number

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