Phytochemical Fraction-Mediated Synthesis of Ag and ZnO Nanoparticles: Exploring Reactivity Limits and Broad-Spectrum Antimicrobial Properties

Abstract

The common generalization of plant extracts as universal, nonspecific reducing agents in metal nanoparticle (NP) synthesis is re-examined. We provide evidence that green synthesis relies on selective reactions between specific phytochemicals and metal precursors. Silver (Ag) and zinc oxide (ZnO) nanoparticles were successfully synthesized using the n-butanol fraction of Plantago major leaf extract, while identical protocols failed to yield copper phases. Characterized via Fourier-transform infrared, X-ray diffraction, scanning electron microscopy, transmission electron microscopy, energy-dispersive spectroscopy, UV–vis, dynamic light scattering, and zeta potential, the annealed Ag and ZnO NPs exhibited average grain sizes of 20–37 nm (λ_max = 428 nm) and 6–22 nm (λ_max = 338 nm) and band gaps at 2.90 and 3.23 eV, respectively. Both Ag and ZnO NPs displayed broad-spectrum antibacterial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, alongside strong antifungal efficacy against Candida albicans and Aspergillus brasiliensis. Notably, Ag NPs showed potent antifungal activity against A. brasiliensis (MIC: 15.625 μg/mL), while ZnO inhibited P. aeruginosa (MIC: 62.50 μg/mL). Conversely, the unreduced copper complex showed negligible bioactivity possibly due to residual dissolved ions. While the n-butanol fraction outperforms both crude extracts and aqueous/ethyl acetate fractions, this study challenges universal green synthesis dogmas, demonstrating metal-specific phytochemical divergence under identical parameters.

1. Introduction

The surge of interest in nanotechnology, particularly the green nanotechnology, has emerged as a promising, sustainable, and eco-friendly paradigm for the synthesis of metallic and metal oxide nanoparticles, offering a vital alternative to hazardous physical and chemical fabrication protocols. − Among various biofabrication routes, plant-mediated synthesis is widely recognized for its simplicity, cost-effectiveness, and capacity to generate functional nanomaterials with diverse biomedical and catalytic applications. , Nanomaterials such as silver (Ag) and zinc oxide (ZnO) nanoparticles are particularly attractive due to their exceptional optical, structural, and broad-spectrum antimicrobial efficacy against various bacterial and fungal pathogens. ,

It has been established that the size, morphology, and composition of these nanoparticles are strictly dictated by the synthesis method employed. Despite these advantages, the conventional deployment of crude plant extracts introduces significant methodological bottlenecks. Crude extracts represent complex, undefined mixtures of primary and secondary metabolites, leading to uncontrollable compositional variability, poor batch-to-batch reproducibility, and notoriously low synthetic conversion yields.

Furthermore, the lack of precise compositional control over crude botanical reducing systems often results in structural heterogeneity, irregular morphology, and unpredictable interfacial interactions during NP nucleation and growth. Although diverse plant matrices have been successfully exploited to fabricate individual nanostructures, − the fundamental relationship between targeted phytochemical suites and metal-specific reduction pathways remains poorly understood.

Metal and metal oxide nanoparticles generally exhibit unique characteristics, including high reactivity, identifiable localized surface plasmon resonance (LSPR), and strong electromagnetic absorption. These properties can be further tuned or enhanced during biosynthesis process. However, specifically, it is unclear how specific fractions such as those rich in distinct phenolic and flavonoid subsets dictate the structural trajectory and phase purity of different metallic systems, such as transition metals and post-transition metal oxides. −

Compounding this structural ambiguity, most contemporary studies evaluate biogenic nanoparticles without establishing rigorous analytical links regarding solvent fraction selectivity, fraction-guided optimization, or quantitative kinetic parameters. Consequently, the field lacks a standardized framework to systematically overcome the conversion limits and structural unpredictability associated with crude biofabrication.

To address these critical gaps, fraction-guided phytochemical engineering offers a robust strategy for isolating defined functional ligand suites and eliminating the confounding variables inherent to crude mixtures. Plantago major represents an ideal botanical model due to its rich and diverse phytoconstituents, including phenolic compounds, flavonoids, terpenoids, iridoid glycosides, alkaloids, fatty acids, and polysaccharides. The seeds, leaves, flowers, and roots of the plant, as well as practically every other part of it, contain these compounds, yet its distinct solvent fractions have not been fully harnessed to direct targeted nanostructure assembly.

Therefore, this study investigates the utilization of a precision-fractionated n-butanol extract derived from P. major leaves to mediate the synthesis of phase-pure silver nanoparticles, hexagonal wurtzite ZnO nanoparticles, and a copper sulfate-coordinated intermediate complex. − Although the n-hexane and dichloromethane fractions obtained from the solvent–solvent partitioning of the crude aqueous-ethanolic extract exhibited notable antimicrobial efficacy during initial biological evaluations, they were excluded from use due to their intractable oily consistency, which precluded stable NP dispersion. Among the remaining nonoily fractions and the main crude extract, the n-butanol fraction demonstrated a superior, broad-spectrum inhibitory activity against both Gram-positive and Gram-negative test strains, making it the optimal phytochemical medium for fabricating the metal nanoparticles.

By exploring the reactivity limits of these isolated phytochemical fractions, this work establishes for the first time a predictable, highly reproducible blueprint for green synthesis. It provides comprehensive structural characterization alongside a rigorous evaluation of their broad-spectrum antimicrobial properties to overcome traditional yield and reproducibility limitations in bionanotechnology.

2. Materials and Methods

2.1. Plant Material Leaf Identification and Collection

P. major L. leaves (Family: Plantaginaceae), also commonly known as broadleaf plantain, are shown in Figure . The leaf samples were collected from the foothill area near Almaty (Kamenka district, Kazakhstan) on agricultural lands (apple orchards), outside protected natural areas with the permission of the landowner during the summer of 2025. The collection of plant material was carried out exclusively for research purposes at the following coordinates: 43.2095° N, 76.9850° E; 43.2140° N, 76.9785° E; 43.0742° N, 76.4636° E. The voucher specimen was prepared in accordance with standard herbarium procedures and submitted for deposition to the RSE “Institute of Botany and Phytointroduction” of the Committee for Forestry and Wildlife of the Ministry of Ecology and Natural Resources of the Republic of Kazakhstan (Almaty, Kazakhstan). Identification of the plant was carried out by specialists of the RSE “Institute of Botany and Phytointroduction” (Almaty, Kazakhstan). The assignment of the registration (herbarium) number is currently in progress.

1.

Picture of P. major leaves. Adapted from Wikipedia, the free encyclopedia.

2.1.1. Extraction and Solvent–Solvent PartitioningPreparation of n-BuOH Fraction

The collected leaf samples were washed with ultrapure water to remove impurities such as dust and debris and then dried in an oven. To increase the extraction efficiency, the dried leaf samples were ground into a fine powder by using a mortar and pestle. Following the method reported by Awe et al., (136.40 g) of the dry powder was extracted with 50% aqueous ethanol (2.5 L). The extraction was conducted for 72 h at room temperature, with occasional manual agitation. After concentration in vacuo, the filtrate yielded 45.30 g of a brown solid (33.21% w/w). The presence of saponins or their glycosides was suspected because of heavy frothing observed during concentration on a rotary evaporator. A portion of the extract (40.30 g) was redissolved in 0.5 L of water and successively partitioned with n-hexane (1.0 L), dichloromethane (1.0 L), ethyl acetate (1.0 L), and n-butanol (1.4 L) to afford 0.55, 1.72, 2.67, and 10.76 g of solvent fractions, respectively. The remaining aqueous phase was also concentrated to dryness to yield 21.23 g.

2.1.2. Phytochemical Screening and Properties of the n-Butanol Fraction

The n-butanol fraction was recovered as a dark-brown, resinous solid upon continuous exposure to air (yield: 26.7%). Thin-layer chromatography analysis using an ethyl acetate–ethanol solvent system (3:1, v/v) revealed a major spot with a retention factor (R _f) of 0.58, which exhibited a deep brown-red coloration under UV light (366 nm) following derivatization with concentrated H₂SO₄. Chemical spray tests confirmed the abundant presence of flavonoids (manifested as an intense yellow coloration with AlCl₃, +++) and phenolic compounds (manifested as an intense yellow coloration with FeCl₃, +++). FT-IR (cm^–1): 3273.58, 2957.97, 2932.29, 2873.20, 1683.92, 1628.01, 1602.25, 1519.80, 1445.01, 1159.03, 1067.20, 1026.78, 589.04, 560.46, and 514.93.

A limitation of this work is that the total phenolic content and total flavonoid content of the extract fractions were not quantified. Rather than duplicating compositional analyses that are already extensively documented in the literature where phytochemical yields naturally vary due to environmental factors such as light exposure, temperature fluctuations, soil fertility, salinity, water and nutrient supply, and biotic or abiotic stress, − the scope of this study focused on functional screening. Consequently, preliminary antimicrobial evaluation of the solvent fractions was prioritized to rationally guide the selection of the n-butanol fraction for NP synthesis.

2.2. Synthesis of Ag NPs, ZnO NPs, and Unreduced Copper Complex: Recovery, Washing, Calcination, and Yield Calculation

All analytical-grade solvents including n-hexane, dichloromethane, ethyl acetate, and n-butanol were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Ethanol (96%) was supplied by Talgar Ethanol Factory (Talgar, Kazakhstan). Metal salts, copper sulfate pentahydrate (CuSO₄·5H₂O), silver nitrate (AgNO₃), zinc nitrate hexahydrate (Zn­(NO₃)₂·6H₂O), and sodium hydroxide (NaOH), were purchased from OTO Laborfarma, Almaty, Kazakhstan.

A simplified synthesis protocol, adapted from Fakhari et al., was modified with respect to temperature, pH, and reaction duration. All reactions were maintained at 70–90 °C. Typically, 20.0 g of Zn­(NO₃)₂·6H₂O (0.067 mol) was dissolved in 400 mL of distilled water under mechanical stirring. The n-butanol extract (10%, w/w relative to the metal salt) was dissolved in a minimum volume of distilled water, added dropwise to the salt solution, and allowed to stir for 5–10 min. Subsequently, 50–60 mL of a 0.02 M NaOH solution (pH ≈ 12) was added dropwise. The mixture transitioned in color from faint yellow to deep yellow and was stirred continuously for 24 h (Figure ).

2.

General laboratory synthesis procedures for Ag, ZnO NPs, and the unreduced copper complex using n-butanol fraction of P. major leaves.

Identical reaction parameters were replicated using AgNO₃ (20.0 g, 0.118 mol) and CuSO₄·5H₂O (20.0 g, 0.080 mol). Following reaction completion, the mixtures were cooled to ambient temperature and centrifuged at 9000 rpm for 10 min. The resulting solid products were rinsed thoroughly three times (20 mL × 3) with distilled water to eliminate residual impurities.

The recovered powders were subsequently calcined at 200 °C for 6 h under ambient air to enhance structural crystallinity. This thermal treatment yielded a dark-yellow ZnO NP product (1.82 g recovered, corresponding to a calcination recovery yield of 83.49% from a brilliant yellow precalcined solid), a gray Ag NP product (1.19 g recovered, corresponding to 75.79% from a dark-gray precalcined solid), and a dark solid corresponding to the unreduced copper complex (1.34 g recovered, corresponding to 73.22% from the initial dark precalcined solid).

2.3. Characterization of Synthesized Nanoparticles

2.3.1. Fourier-Transform Infrared Spectroscopy

Fourier-transform infrared (FT-IR) spectra were measured in an ALPHA II FTIR spectrometer with an ATR module from Briker Optics GmbH, Germany. The transmittance mode spectra were measured over a range of 4000–400 cm^–1, with 12 scans.

2.3.2. UV–Vis Spectroscopy

The optical properties of the samples were analyzed on a PerkinElmer Lambda 35 spectrophotometer (model 502S12022804, UV WinLab 6.0.2.0723 software) in the 200–800 nm range. Distilled water served as the blank reference, and all absorbance measurements were recorded using 1 cm quartz cells. The optical band gaps of the synthesized nanoparticles were determined from UV–vis absorption spectra using the Tauc relation. By plotting the squared absorption coefficient-energy product against photon energy, direct band gaps were calculated by extrapolating the linear portion of the Tauc plot to the energy axis.

2.3.3. X-ray Diffraction

Structural characteristics were analyzed via X-ray diffraction (XRD) using a MiniFlex 600 diffractometer (Rigaku, Tokyo, Japan) with Cu Kα radiation operating at 40 kV and 15 mA. Data were collected over a 2θ between 3° and 100° at a scanning speed of 2°/min and a step size of 0.01°. The PDF-2-2024 database was utilized for phase identification.

2.3.4. Scanning Electron Microscope, Energy-Dispersive X-ray Spectroscopy, and Transmission Electron Microscopy Analysis

The morphology, size, and stability of the nanoparticles were examined by using a JEOL JEM-1400 Plus transmission electron microscope (TEM). Samples were prepared by dispersing the NPs onto carbon-coated copper grids (200 mesh) and allowing them to air-dry. TEM images were captured at an accelerating voltage of 80 kV and a magnification of 50 kx. Average particle diameters were determined by analyzing these images by using ImageJ software. Additionally, the surface morphology and microstructure of the obtained samples were carried out using SEM on an SEM5000Pro device (CIQTEK, China) equipped with an Oxford Instruments X-Plore 30 energy-dispersive X-ray microanalysis system (SDD).

2.3.5. Dynamic Light Scattering and Zeta-Potential Measurements

The hydrodynamic diameter and size distribution of the synthesized Ag, ZnO NPs, and the unreduced copper complex were determined by dynamic light scattering (DLS) using a Zetasizer NAno ZS (Malvern Instruments, UK) at a scattering angle of 173 and avalanche photodiode. For DLS, each sample of 0.0005 g per 10 mL in water was sonicated at room temperature for 10 min, while the analysis time was 60 s for determination of average ζ-potential. Dispersion was determined as such without dilution, and all experiments were performed in triplicate.

2.3.6. Preliminary Antimicrobial Screening via Agar Disc Diffusion

The initial antimicrobial activity screening of the test samples: crude ethanolic extract, n-hexane fraction, dichloromethane, ethyl acetate, n-butanol, and residual aqueous extract were evaluated using the disc-diffusion method. Standard reference strains of Pseudomonas aeruginosa, Proteus mirabilis, and Staphylococcus aureus, authenticated by their cultural, morphological, and biochemical characteristics, served as the test microorganisms. 24 h cultures of the microorganisms were grown at 37 °C. Bacterial suspensions were prepared in sterile physiological saline and adjusted to a 0.5 McFarland standard. The resulting suspension was evenly spread onto the surface of meat-peptone agar plates to achieve confluent growth, and the uniformity of the bacterial lawn was assessed visually. Inhibition zone diameters were measured manually using a millimeter ruler with a 1 mm precision. Any assays showing uneven bacterial seeding or disc displacement were excluded from the analysis.

Sterile paper discs of standard diameter (6 mm) were impregnated with equal volumes of the test substances at a uniform concentration of 1% and placed on the seeded agar surface at standardized intervals. For preparation, 15 mg of each sample (A, B, C, and F) was dissolved in 1.5 mL of water for injection or DMSO, although sample F exhibits poor solubility, remaining largely undissolved even after heating in a water bath at 50 °C for 30 min. Each determination was carried out in triplicate. The obtained results were statistically processed and presented as mean values ± standard deviation (SD). Where range values were used for calculations, their arithmetic mean was applied. The absence of an inhibition zone is indicated by a dash (“–”).

Threshold values for zones of inhibition (≥25 mm: high, 15–24 mm: moderate, ≤14 mm: low, no zone: no activity) were conditionally established for screening and comparative assessment between samples, in accordance with international standards (CLSI). Performance Standards for Antimicrobial Susceptibility Testing; 33rd ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2023. p. 402. Microorganisms grow around the disc where the inhibitor concentration is below the minimum inhibitory concentration (MIC).

2.3.7. Determination of Minimum Inhibitory Concentration Using the 96-Well Microdilution Assay

The determination of the antimicrobial activity of the nanoparticles was carried out by the diffusion method, according to the Performance Standards for Antimicrobial Disc Susceptibility Test, as well as by the 2-fold serial dilution method, according to the protocols of the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute, 2019, 2015, and 2018). The following strains were used for the tests: S. aureus ATCC 6538-P, Escherichia coli ATCC 9637, P. aeruginosa ATCC 9027, Candida albicans ATCC 10231, and Aspergillus brasiliensis ATCC 16404.

2.3.7.1. Preparation of Microorganism Suspensions in 0.9% NaCl Physiological Solution

Suspensions of the test strains were prepared in a physiological solution (0.9% NaCl). Using a sterile loop, an aliquot of a 24 h culture of the test strain was collected and then introduced into a sterile tube containing 5 mL of 0.9% NaCl. The tube was then placed into a DEN-1 densitometer to measure the optical density (turbidity) and achieve the required concentration of the microorganism.

The density of the suspension had to be 0.5 McFarland units, which corresponds to 1.5 × 10⁸ CFU/mL for the diffusion method. The resulting suspension with this density was used for inoculating Petri dishes containing solid medium. The suspension density for the 2-fold serial dilution method had to be 1.5 × 10⁶ CFU/mL. The resulting suspension with this density was used for inoculating the wells of the plate containing the medium and the tested samples. Solvent control (1% lactic acid + 1% DMSO) was prepared in triplicate.

2.3.7.2. Procedure for Preparing Serial Dilutions

The antimicrobial activity testing procedure was carried out by the 2-fold serial dilution method in a liquid nutrient medium, Mueller–Hinton broth (HiMedia, India). The procedure was performed in sterile 48-well polystyrene culture plates (BIOLOGIX, China).

Into the first wells of the plate, 500 μL of stock solutions of the tested substances dissolved in Mueller–Hinton broth at a concentration of 10 mg/mL was introduced. Into all wells except the first one, 500 μL of Mueller–Hinton broth was added, after which a series of 2-fold serial dilutions was performed: a thoroughly pipetted mixture of broth and solutions from well No. 1 in an amount of 500 μL was transferred into well No. 2, and the obtained mixture from well No. 2 in a volume of 500 μL was transferred into well No. 3. This procedure was repeated until the required number of 2-fold dilutions was achieved. From the last well, 500 μL of the mixture was removed. Thus, in each row of the plate, wells A–H, serial dilutions with working concentrations from 4 to 0.0019 mg/mL were obtained.

2.3.7.3. Inoculation and Incubation Conditions

After preparation of the working suspension, 50 μL of the working inoculum was added to all wells. The final cell concentration per well after inoculation was 1.5 × 10⁵ CFU/mL. The bacteria were incubated for 18–24 h at a temperature of 37 ± 1 °C (Binder, Germany). After the incubation period, 30 μL of a 0.05% resazurin solution was added to each well. Each cell on the plate corresponded to the serial number of the well with a defined dilution. After the addition of resazurin, the plates were placed in a thermostat for 2 h at a temperature of 37 ± 1 °C. The results for this strain were recorded visually after 2 h of incubation. Wells containing dilutions of the tested substances that acquired a pink coloration indicated the presence of viable bacteria, whereas wells with a violet coloration of the indicator indicated the absence of viable cells of the test strain, which demonstrates the cidal action of the samples. The last well in the row showing violet coloration was considered the well corresponding to the minimum bactericidal concentration.

2.4. Results and Discussion

2.4.1. Synthesis and Mechanistic Insights into Phytochemical Selectivity Evaluation of Phase Transitions

The reaction pathways and selectivity scheme are shown in Figure . During the wet chemical synthesis, the distinct visual transitions observed across the metal precursor solutions reflected characteristic coordination and nucleation events driven by the phytochemical constituents of the n-butanol extract. For the silver system, the reduction of AgNO₃ by the phytochemical reductants progressed through characteristic coloration stages, ultimately yielding a stable dark brown dispersion upon reaction completion. Similarly, the zinc precursor Zn­(NO₃)₂·6H₂O exhibited a progressive chromatic shift from faint yellow through deep yellow, culminating in a brown precipitate upon treatment with a 0.02 M NaOH solution under continuous stirring at 70–90 °C for 24 h, successfully yielding phase-pure ZnO nanoparticles.

3.

Reaction pathway/selectivity scheme for synthesis of Ag and ZnO NPs.

In contrast, the copper system initiated with the dissolution of CuSO₄·5H₂O to form the light blue aquo-complex [Cu­(H₂O)₆]²⁺. Upon introduction of the n-butanol fraction, semipolar flavonoids, polyphenols, and terpenoids acted as ligands, displacing water molecules to form a stable copper-phytochemical coordination complex that turned the solution green via altered d-orbital crystal field splitting energy. Subsequent addition of NaOH induced the deprotonation of hydroxyl groups on the polyphenols and reacted with copper and residual sulfate ions to precipitate stable, intense green basic copper sulfate intermediate complexes. The observed variations in phase purity among the synthesized nanostructures under identical reaction parameters highlight a profound thermodynamic and coordination selectivity driven by the n-butanol phytochemical fraction. In the silver and zinc systems, the utilization of nitrate precursors (AgNO₃) and (Zn­(NO₃)₂·6H₂O) facilitated unhindered phase transitions at a mild thermal threshold of 200 °C. Silver ions (Ag⁺), (d¹⁰), possessing a high standard reduction potential, underwent rapid, direct electron reduction by the fractionated flavonoids to yield pure metallic Ag nanoparticles. Concurrently, the zinc ions (Zn²⁺), (d¹⁰) formed structurally flexible tetrahedral complexes with the plant ligands; because the associated nitrate counterions readily decompose into volatile gases at low thermal thresholds (<250 °C), this intermediate easily pyrolyzed into a highly crystalline, pure wurtzite ZnO phase. ,

Conversely, the copper system encountered distinct chemical and geometric bottlenecks that restricted pure oxide formation, validating the selective boundaries of the plant fraction. Copper ions (Cu²⁺), (d⁹) are highly susceptible to severe Jahn–Teller distortion, which thermodynamically favors the formation of rigid, exceptionally stable square-planar chelation complexes with the bidentate hydroxyl and carbonyl groups of the n-butanol flavonoids. When coupled with the highly stable sulfate (SO₄ ^2–) counterions introduced by the CuSO₄·5H₂O precursor, this rigid configuration trapped the metal centers within an insoluble, polymeric basic copper sulfate network.

This selective synthesis attempt using the n-butanol fraction yielded crystalline hydrated copper sulfate complexes (CuSO₄)­(H₂O), Cu₄(SO₄)₂(OH)₆, and NaCu₂(SO₄)₂OH·H₂O rather than phase-pure copper oxide (CuO) nanoparticles, highlighting a distinct reactivity limit compared to the facile reduction observed for silver and ZnO systems. This outcome indicates that the specialized suite of phenolic and flavonoid ligands present in the n-butanol extract acts predominantly as a coordinating and capping agent rather than possessing a sufficiently high redox potential to drive the complete reduction of Cu²⁺ precursors under ambient conditions of 200 °C thermal treatment that was sufficient to volatilize the nitrate residues of the Ag and ZnO systems. Overcoming this thermodynamic barrier requires targeted process modifications. Specifically, future optimization strategies must explore elevated thermal treatments, such as calcination temperatures exceeding 600 °C to thermally decompose the sulfate coordination intermediate, alongside systematic pH adjustments to enhance the deprotonation and electron-donating capacity of the active phytochemical constituents.

The observed metal-dependent selectivity in NP assembly is fundamentally governed by standard reduction potentials and metal–ligand coordination chemistry. The facile reduction of silver to form phase-pure metallic Ag nanoparticles is thermodynamically favored because of the high positive standard reduction potential of the Ag⁺/Ag⁰ couple (E ^o = +0.799 V), enabling rapid electron transfer from the hydroxyl groups of the n-butanol phenolic constituents. In contrast, the formation of hexagonal wurtzite ZnO proceeds via a coordination–precipitation pathway involving aqueous Zn²⁺ species (E ^o = −0.763 V), where the phytochemical suites act predominantly as stabilizing and capping ligands. The isolation of stable copper sulfate hydrate intermediates rather than copper oxide further confirms a higher activation energy barrier for copper reduction under these conditions. These thermodynamic distinctions align with the established biogenic synthesis literature, verifying that differing standard reduction potentials and coordination constants dictate distinct structural trajectories during green fabrication. ,

2.4.2. Analysis of FT-IR Spectroscopy

FTIR analysis was conducted across the wavenumber range of 4000–400 cm^–1 to investigate the functional groups present in the n-butanol plant extract. The objective was to ascertain their respective roles, determine how they facilitate the reduction of metal and metal oxide precursors into nanostructures, and identify which functional groups are responsible for capping and stabilization. As displayed in Figure and summarized in Table , the infrared spectra of the Ag NPs, ZnO NPs, and the secondary copper phase obtained after calcination at 200 °C were compared directly to the raw n-butanol fraction.

4.

FTIR spectra of n-BuOH fraction of Plantago major L. leaf extract, Ag, ZnO NPs, and the unreduced copper complex.

1. Comparison of Vibrational Frequencies of Crude n-Butanol Fraction and Ag NPs, ZnO NPs, and Unreduced Copper Complex.

assignment/functional group	n-butanol fraction (cm–1)	Ag NPs (cm–1)	ZnO NPs (cm–1)	unreduced copper complex (cm–1)
O–H/N–H stretching	3273.58	absent	3374.23	3146.69
aromatic C–H stretching	2957.97; 2932.29; 2873.20	absent	absent	absent
CO stretching	1683.92	absent	absent	absent
aromatic CC stretching	1628.01; 1602.25	absent	1621.41 (intense)	1624.96 (low)
amide II (N–H bend/C–N stretch)	1519.80	1515.14	absent	absent
C–O–C stretching	1445.01	absent	1441.17 (high)	absent
C–O stretch (phenolic/alcohol)	1026.78	absent	absent	absent
C–O stretch (phenolic/alcohol)	1067.20	absent	absent	1067.02
strong C–O–C or C–O stretching vibrations	1159.03	1194.52 (broad)	absent	absent
out-of-plane alkene C–H bending	816.10; 845.85; 589.04; 560.46; 514.93
metal–oxygen (Zn–O)	-	absent	608.87	absent

The neat n-butanol fraction displays prominent characteristic absorption bands at 3273.58, 2957.97, 2932.29, 2873.20, 1683.92, 1628.01, 1602.25, 1519.80, 1445.01, 1414.96, 1359.28, 1274.43, 1159.03, 1067.20, 1026.78, 950.92, 899.86, 846.10, 816.10, 784.21, 737.16, 589.04, 560.46, and 514.93 cm^–1. These frequencies indicate the presence of O–H, N–H, C–H, C–N, CO, CC, and C–O–C stretching vibrations originating from diverse phytochemical constituents such as phenolic acids, amines, proteins, flavonoids, carbohydrates, saponins, terpenoids, polysaccharides, tannins, and glycosides.

Comparing these frequencies with the synthesized products reveals significant shifts and intensity changes: a prominent shift to 3374.23 cm^–1 for the ZnO NPs and 3146.69 cm^–1 for the unreduced copper complex (Figure ), assigned to O–H and N–H stretching vibrations. These shifts indicate the active involvement of hydroxyl- and/or amine-bearing phytochemicals in NP formation. Interestingly, this peak is absent in the Ag NPs, pointing toward a distinct surface environment or stabilization mechanism for silver. Furthermore, vibrational frequencies corresponding to aromatic C–H stretching (2958.19, 2935.54, and 2869.94 cm^–1), CO stretching (1683.92 cm^–1), and out-of-plane alkene C–H bending (589, 514 cm^–1) were conspicuously absent in the spectra of the Ag NPs, ZnO NPs, and the unreduced copper complex. Conversely, bands at 1621.41 and 1624.96 cm^–1 (assigned to aromatic CC stretching) exhibited varying intensities: they were most intense in the ZnO NPs, moderately present in the unreduced copper complex, and entirely absent in the Ag NPs.

5.

Overlay of the FTIR spectra of ZnO NPs and unreduced copper complex.

The band at 1519.01 cm^–1 in the pure n-butanol fraction, indicative of N–H bending and C–N stretching vibrations, was retained only in the Ag nanoparticles at 1515.14 cm^–1 while being completely absent in the ZnO nanoparticles and the unreduced copper complex. Although amine- and amide-bearing phytochemical constituents may contribute to the localized capping and stabilization of silver, asserting a definitive protein-mediated reduction mechanism is precluded by the absence of biochemical protein assays or compound isolation. Consequently, this retention highlights distinct surface-adsorption pathways for silver compared to the other systems.

Furthermore, the C–O stretching peak observed at 1322.69 cm^–1 in the n-butanol extract appeared at a notably higher intensity in the ZnO nanoparticles, yet was completely suppressed or shifted in the Ag nanoparticles and unreduced copper complex. This differential behavior underscores the selective involvement of oxygen-rich phytochemical ligands (such as flavonoids and polyhydroxylated carbohydrates) in coordinating and stabilizing the ZnO lattice. Conversely, the absence of these coordinating signatures in unreduced copper complex correlates with poor interfacial ligand–metal interaction, reflecting the structural instability and agglomeration observed in the SEM and DLS analyses.

Furthermore, in the fingerprint region, the peak at 1159.03 cm^–1 in the n-butanol fraction shifted to a broad band at 1194.52 cm^–1 in the Ag NPs but was absent in both the ZnO NPs and unreduced copper complex. The two bands at 1024.98 cm^–1 and 1068.03 cm^–1, typically associated with C–O–C stretching vibrations in oxygenated organic molecules, were completely absent in the Ag and ZnO NP spectra; however, a single intense, broad band was retained at 1067.02 cm^–1 in the spectrum of unreduced copper complex. While some literature attributes similar regions to interactions between metal species and oxygen-containing functional groups, − the spectral data primarily indicate that the unreacted or differently coordinated organic matrix in the copper trial retained distinct vibrational signatures compared to the successfully formed nanoparticles. The persistence of these specific bands, alongside the absence of other protective capping signatures, points toward an unfavorable ligand–metal interaction that likely hinders stable nucleation and contributes to the significant agglomeration observed in the morphological analysis. Finally, the successful fabrication of the ZnO phase is supported by the emergence of a characteristic metal–oxygen vibrational mode at 608.87 cm^–1. This low-frequency metal–oxygen band was absent in the neat n-butanol fraction, Ag NPs, and unreduced copper complex spectral.

2.4.3. Analysis of the UV–Visible Absorption Spectroscopy

UV–visible spectroscopy serves as a primary analytical technique for evaluating the optical properties and electronic transitions of biogenically synthesized nanomaterials, relying on phenomena such as LSPR. Figure illustrates the comparative optical absorption spectra of the successfully fabricated Ag nanoparticles, ZnO nanoparticles, and the unreduced copper complex measured against the free n-butanol plant extract fraction in distilled water at a concentration of 1 × 10^–3 M^–1.

6.

Comparison of the UV–vis absorption spectra of Ag NPs (blackleft spectrum), ZnO-NPs (red line), unreduced copper (blue line), and the neat n-butanol fraction (blackright spectrum) at a concentration of 1 × 10^–3 M^–1 in aqueous solution.

The synthesized Ag and ZnO nanoparticles exhibit distinct, intense absorption bands within the ultraviolet region λ_max = 200–300 nm, appearing at lower wavelengths relative to the free n-butanol fraction λ_max = 293 nm. These spectral signatures are fundamentally attributed to π → π* electronic transitions originating from the aromatic phytochemical constituents incorporated into the capping matrix. Conversely, the unreduced copper complex displays no characteristic plasmonic resonance band, retaining only weak ultraviolet absorbance features corresponding to residual uncoordinated phytochemical species.

The free n-butanol fraction exhibits a prominent absorption band at λ_max = 326 nm, indicating the rich presence of polyphenols, flavonoids, and amide compounds that characteristically absorb within the 280–320 nm range and function dually as electron-donating or -reducing agents and stabilizing capping ligands. , The LSPR peak for the biogenic Ag nanoparticles emerges distinctly at λ_max = 428 nm, whereas the absorption band for the synthesized ZnO nanoparticles displays a prominent absorption feature centered around 338 nm, corresponding to the characteristic intrinsic interband electronic transition (O_2p → Zn_3d) of hexagonal wurtzite ZnO. This absorption edge exhibits a noticeable blue shift compared to bulk ZnO (which typically absorbs near 370–380 nm). , The simultaneous appearance of these well-defined LSPR bands alongside the absence of active resonance in the unreduced copper system confirms the successful photonic and electronic profiling of the multimetallic series.

Furthermore, the absence of characteristic absorption bands typically reported for cuprous and cupric oxides such as the distinctive Cu₂O absorption peaks around 250 nm and the classical CuO multiband transitions spanning 260 nm, 340 nm, and the visible region near 630 nm, , corroborates the nonformation of uncalcined copper oxide nanoparticles in the colloidal phase. This spectral absence aligns with the preceding characterization, demonstrating that the wet chemical approach under these specific parameters yielded an unreduced precursor complex rather than zerovalent or phase-pure oxide nanostructures. The change in color during synthesis may be due to slight metabolites involved in the n-butanol fraction but not strong enough to reduce Cu²⁺ to the nanoscale, which was not the case for ZnO NPs. − Furthermore, it should be noted that temporal stability of the isolated solid nanoparticles was evaluated over a 6 month storage period under ambient laboratory conditions. Repeated UV–vis spectrophotometric analyses revealed no discernible shifts in maximum absorption peaks or spectral broadening, confirming exceptional shelf-life stability and structural integrity against degradation.

2.4.4. Investigation of Optoelectronic Band Gaps of Ag and ZnO Nanoparticles

We have investigated important property of the ZnO-NPs in order to provide useful information for their use in a range of energy-relevant applications. We have performed transmission and reflectance measurements, which offered the possibility to determine the direct band gap of the NPs. In addition, the direct optical band gap energy of the synthesized ZnO-NPs was estimated to establish structure–function relationships. This value allows for the evaluation of how variations in size, shape, and composition influence NP functionality and possibly facilitate the prediction of potential toxicity. This energy was determined using Tauc plots, where absorption data are transformed to relate to the absorption coefficient and photon energy

$$\mathrm{T}\mathrm{a}\mathrm{u}\mathrm{c}\mathrm{p}\mathrm{l}\mathrm{o}\mathrm{t}(\mathrm{T}\mathrm{g})\mathrm{u}\mathrm{s}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{e}\mathrm{q}\mathrm{u}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}:E=h\mathrm{c}/\lambda ,\text{and}\mathrm{a}\mathrm{p}\mathrm{l}\mathrm{o}\mathrm{t}\mathrm{o}\mathrm{f}{(\alpha h\nu )}^n\mathrm{v}\mathrm{s}h\nu$$

where h = Planck’s constant = 6.626 × 10^–34 J·s; c = the speed of light at 3 × 10⁸ km/s, λ = the wavelength of maximum absorption, ε (α) = the absorption coefficient and n = a factor that depends on the nature of the electronic transition.

To evaluate the electronic band structures and quantum-confinement behaviors of the successfully fabricated multimetallic nanomaterials, optical band gap energies were determined from the UV–vis absorption spectra using the Tauc relation for direct allowed transitions [(αhν)² = A(hν – E _g)]. Absorbance data were acquired across a range of photon energies (UV–vis spectra) spanning from below the fundamental absorption edge to well above the transition threshold to establish optimal linear fits as displayed in Table .

2. Values of Wavelength and Absorbance Used to Generate Tauc Plot.

wavelength (λ, nm)	absorbance (A) (Ag NPs)	photon energy (hν, eV) (Ag NPs)	direct Tauc term (Ahν)2 (Ag NPs)	absorbance (A) (ZnO NPs)	photon energy (hν, eV) (ZnO NPs)	direct Tauc term (Ahν)2 (ZnO NPs)
450	0.2997	2.756	0.682	-	-	-
430	0.3236	2.884	0.871	-	-	-
410	0.3090	3.024	0.873	0.0594	3.024	0.0323
401	0.2922	3.100	0.821	0.0669	3.092	0.0428
380	0.2529	3.263	0.681	0.0936	3.263	0.0933
377	0.2532	3.307	0.646	0.1005	3.289	0.1093
362	0.2284	3.444	0.619	0.1466	3.425	0.2522
350	0.2266	3.543	0.645	0.1921	3.543	0.4632
338	0.2420	3.701	0.803	0.2339	3.669	0.7363
329	0.2619	3.758	0.968	0.2566	3.769	0.9353
320	0.3580	3.875	1.924	0.2747	3.875	1.1331

As illustrated in Figure , the comparative Tauc analysis reveals distinct optical band gaps of 2.90 eV for the phase-pure Ag nanoparticles and 3.23 eV for the hexagonal ZnO nanoparticles. These experimentally derived values exhibit exceptional alignment with established literature. The calculated optical band gap of 3.23 eV via Tauc’s extrapolation closely matches the range of 3.20–3.22 eV reported by Josun et al. for hydrothermally synthesized ZnO systems. This widening relative to bulk ZnO (3.37 eV) and the observed UV absorption feature are directly attributable to quantum confinement effects arising from the small crystallite dimensions (6–22 nm) established via XRD analysis, alongside the bioreduction and capping action of the n-butanol extract. The reduced particle size drives discrete energy level splitting, confirming the strong quantum-size dependency of these nanoscale semiconductor structures. Together, these comparative findings demonstrate that the n-butanol phytochemical fraction successfully mediates the formation of nanoscale semiconductors with tailored electronic properties, while contrasting sharply with the unreduced copper complex, which exhibits no such band gap transitions.

7.

Tauc plot for Ag and ZnO nanoparticles showing direct optical band gap.

Specifically, the 2.90 eV band gap recorded for the silver nanoparticles corroborates the findings of Jeyakumari et al., reflecting the LSPR and interband transition profile characteristic of biogenic Ag nanostructures. In bulk metallic silver, overlapping continuous bands result in no band gap. However, as silver is reduced to the nanoscale (20–37 nm), the finite number of atoms causes the continuous density of states to transform into discrete, quantized energy levels. The optical band gap calculated via Tauc’s relation reflects transitions involving LSPR and interband excitations (such as Ag 4d → Ag 5s transitions), which open up a measurable optical band gap common in noble metal nanostructures. As dimensions shrink, the decrease in the number of atoms per particle widens the spacing between energy states, resulting in a distinct optical band gap that is drastically different from zero-gap bulk metal behavior. Similar quantum-size shifts in silver NP band gaps have been widely documented in literature. ,

Beyond the structural morphology, the reduced dimensions of the biogenic nanoparticles induce pronounced quantum confinement effects, carrying critical implications for energy and optoelectronic applications. Constraining charge carriers within nanoscale boundaries widens the effective band gap and shifts the valence and conduction band edges. This quantum-size effect enables precise band gap engineering via green synthesis parameters, allowing for tailored absorption across the UV–visible spectrum. Consequently, the optimized band edge alignment facilitates efficient charge separation, reduces recombination kinetics, and enhances redox capabilities, underscoring the potential of fraction-guided biogenic nanoparticles in advanced energy harvesting and photovoltaic systems. ,

2.5. Phase Composition and Purity

2.5.1. XRD Comparative Structural Analysis

To evaluate the comparative crystalline phases and structural parameters of the synthesized multimetallic series, XRD measurements were conducted across a 2θ range of 20–100° with a step size of 0.02°. Figures – illustrate the comparative XRD patterns of the phase-pure hexagonal ZnO nanoparticles, face-centered cubic (fcc) Ag nanoparticles, and the unreduced copper complex powder obtained under identical precursor and extract parameters.

8.

XRD pattern of ZnO NPs.

10.

XRD pattern of unreduced copper complex.

9.

XRD pattern of Ag NPs.

2.5.2. Phase Purity and Crystallography of Biogenic ZnO and Ag Nanoparticles

The diffraction pattern for the ZnO sample confirms the successful formation of phase-pure ZnO possessing a hexagonal wurtzite crystal structure, indexing cleanly to PDF card 01-079-0207 within the space group P6₃ mc. Distinct, sharp reflections are observed at 2θ values of approximately 31.62°, 34.31°, 36.12°, 47.41°, 56.37°, 62.65°, 67.76°, and 68.93°, corresponding to the (100), (002), (101), (102), (110), (103), (112), and (201) crystallographic planes, respectively. The maximum intensity is localized at 36.12° for the (101) plane, indicating a preferred orientation. The calculated lattice parameters (a = 3.25844 Å and c = 5.21807 Å, α = β = 90° and γ = 120°) exhibit close alignment with standard wurtzite constants). ,

Utilizing the Debye–Scherrer equation

$$\mathrm{D}=0.94\lambda /\beta \cos \theta$$

the average crystallite sizes across major reflections span 60–223 Å (specifically 14.9 nm for (100), 22.3 nm for (002), and 13.5 nm for (101)), confirming a highly crystalline, phase-pure nanoscale regime devoid of secondary hydroxide or metallic zinc phases.

The diffraction pattern for the silver sample confirms the successful formation of phase-pure metallic silver possessing an fcc crystal structure, indexing cleanly to PDF card 00-004-0783 within the space group 225:Fm3̅m. The calcined silver sample exhibits intense, well-defined diffraction peaks at 2θ positions of 38.11°, 44.29°, 64.43°, 77.36°, 81.51°, and 97.84°, corresponding, respectively, to the (111), (200), (220), (311), (222), and (400) planes of fcc metallic silver. , The prominent (111) preferred orientation reflects high thermodynamic stability. The absence of characteristic diffraction signatures for AgO or Ag₂O verifies that calcination successfully achieved complete reduction to phase-pure metallic silver. − The evaluated lattice parameter of approximately 4.088 Å matches bulk silver standards with negligible internal strain, while Scherrer-derived crystallite sizes range from 20 to 37 nm, demonstrating uniform crystal growth and minimal structural defect density.

2.5.3. Structural Evaluation of the Unreduced Copper Complex

In stark contrast to the successful fabrication of ZnO and Ag nanostructures, the XRD pattern of the copper system processed under identical thermal (200 °C) and chemical regimes reveals that phase-pure tenorite (CuO) or cuprite (Cu₂O) nanoparticles were not formed. As shown in Figure , quantitative phase analysis indicates that the material is predominantly composed of crystalline hydrated copper sulfate (poitevinite syn, Cu­(SO₄)­(H₂O)), characterized by dominant reflections near 25.97°, 26.39°, and 28.45°. Traces of monoclinic tenorite CuO remain largely absent or negligible, confirming that the chosen calcination threshold was thermodynamically insufficient to cleave the robust sulfate matrix of the CuSO₄·5H₂O precursor and volatilize the dense bio-organic capping sheath.

To ensure that the diffraction patterns were free from extraneous signals, baseline corrections were applied to account for any amorphous background contributed by organic capping biomolecules from the n-butanol extract. The absence of unindexed peaks confirmed the high purity of the targeted metallic and metal oxide crystalline phases.

Furthermore, it is noteworthy to know that despite repeated trials under rigorous conditionsincluding separate exploratory experiments where the concentration of the n-butanol fraction was systematically reduced to 50 wt %pure CuO nanoparticles remained elusive. As illustrated in Figure (and detailed in the Supporting Information Section), XRD analysis consistently confirmed the formation of mixed basic copper sulfate phases, specifically brochantite-2 M (88.36%) and natrochalcite (11.64%), rather than the target metal oxide.

11.

XRD pattern of the unreduced copper complex at 50% (wt %) of n-butanol fraction of Plantago major L.

These findings highlight the inherent limitations of crude n-butanol fractions in driving copper-specific reductive pathways under mild thermal conditions. We hypothesize that the persistence of the residual precursor matrix exerts a structural inhibitory effect, acting as a physical impediment that disrupts the formation of a homogeneous well-isolated NP population. This mechanistic insight successfully accounts for the pronounced agglomeration observed in subsequent SEM analyses, underscoring the critical influence of the precursor-to-extract stoichiometry and phytochemical selectivity in biogenic synthesis.

Ultimately, this comparative structural divergence establishes that while the n-butanol plant extract fraction effectively drives the biogenic reduction and crystallization of silver and ZnO systems under mild conditions, copper ion processing requires distinct thermal or chemical optimization to achieve true metal-selective oxide phase purity.

2.6. Morphological, Compositional, and Ultrastructural Comparative Analysis (SEM, EDS, and TEM)

To correlate the bulk crystalline properties and optical behaviors with surface and ultrastructural features, morphological analysis (SEM), elemental mapping (EDS), and high-resolution TEM were performed comparatively across the multimetallic series. These analyses collectively substantiate the phase purity and capping dynamics of the successfully fabricated Ag and ZnO nanoparticles while providing definitive microstructural insights into the partial conversion behavior of the unreduced copper system.

2.6.1. Surface Morphology and Microstructural Features (SEM)

As illustrated in Figure A–C, SEM reveals distinct surface morphologies and particle size characteristics that highlight selective interactions between the constituents of the n-butanol plant extract fraction and the developing metal networks. Both the Ag and ZnO nanoparticles (Figure A,B) exhibit uniform, well-dispersed spherical morphologies with dimensions spanning 25–95 nm. This microstructural homogeneity confirms that the semipolar phytochemicals within the extract function effectively as stabilizing capping agents, preventing excessive particle aggregation, a mechanism corroborated by the attenuation of specific functional group peaks in the FTIR spectra.

12.

SEM images of Ag, ZnO NPs, and unreduced copper complex in different magnifications: 1–50 μm, 50 nm, 100 nm, 200 and 500 nm.

In sharp contrast, the unreduced copper product displays a dense, highly agglomerated structural profile, characterized by pronounced clustering. This morphological divergence indicates that under identical thermal and chemical regimes, the crude n-butanol fraction fails to provide the requisite kinetic stabilization for the copper system, resulting in a physically impeded, interconnected network rather than discrete, well-isolated nanostructures.

A comparative analysis of the crystallite sizes derived from XRD (using the Debye–Scherrer equation) and the particle dimensions observed via SEM and TEM reveals a consistent and expected correlation. The XRD crystallite sizes represent the size of individual, coherently diffracting crystalline domains, whereas the particles visualized through SEM and TEM correspond to the physical particle boundaries, which may encompass minor agglomerates bound by the organic phytochemicals acting as capping agents from the n-butanol extract. Consequently, while the microscopic particle sizes are slightly larger than or closely aligned with the bulk crystallite domains, both analytical techniques confirm the successful formation of well-dispersed nanoscale structures for the Ag and ZnO NPs.

2.6.2. Elemental Composition and Interfacial Chemistry (EDS)

As shown in Figures and , the elemental composition and surface purity evaluated via EDS align harmoniously with the preceding structural data. For the phase-pure Ag nanoparticles, quantitative mapping confirms high surface purity, with metallic silver comprising over 92.33 wt % and no detectable oxide impurities. Similarly, the EDS spectrum and mapping of the ZnO nanoparticles verify the predominance of zinc and oxygen (65.23 wt %) alongside a localized surface signal for sodium (5.46 wt %). Because NaOH was utilized as the precipitating agent, this minor sodium content is attributed to residual surface-adsorbed amorphous byproducts rather than lattice incorporation.

13.

EDS of (A) ZnO NPs, (B) Ag NPs, and (C) unreduced copper complex.

14.

Images of ZnO NP mapping and report: electronic image A; multilayer image B. Images of Ag NP mapping and report: electronic image (A); multilayer image (B). Images of the unreduced copper complex mapping and report: electronic image (A); multilayer image (B).

The distinction between these surface EDS findings and bulk XRD data underscores the complementary nature of the techniques: while EDS captures the chemical environment and amorphous residues at the NP interface, the sharp, well-defined XRD peaks confirm that the internal cores of the Ag and ZnO systems maintain long-range crystalline order. Conversely, the EDS profile of the unreduced copper product reflects its complex phase composition, revealing substantial weight percentages for copper (Cu, 56.23 wt %), sulfur (S, 15.05 wt %), and oxygen (O, 12.27 wt %). The presence of intense sulfur and copper signals confirms that the bulk material remains dominated by the unreacted CuSO₄·H₂O precursor matrix, reinforcing the conclusion that the reaction pathway underwent only partial or negligible conversion.

2.6.3. Ultrastructural Evaluation and Core–Shell Dimensions of Nanoparticles (TEM)

TEM provides definitive ultrastructural validation of the particle sizes, dispersion states, and core–shell boundaries. TEM micrographs shown in Figure reveal that the Ag and ZnO nanoparticles are structurally discrete, highly spherical, and uniformly distributed. In contrast, TEM imaging of the copper product confirms that, while localized reductive nucleation occurs, primary units are extensively fused into larger agglomerated clusters.

15.

TEM images of Ag, ZnO NPs, and unreduced copper complex.

A consistent dimensional discrepancy is observed across all systems when comparing TEM measurements to Scherrer-derived XRD crystallite sizes, with the Ag nanoparticles exhibiting TEM particle dimensions up to 42.94 nm compared to XRD coherent domain sizes of 20–37 nm, the ZnO nanoparticles showing similarly elevated total dimensions relative to their XRD crystallite size range of 6.0–22.3 nm, and the copper product displaying larger agglomerated units in contrast to the smaller XRD-detected domain sizes of 18–24 nm.

This variation is fundamentally rooted in the physical principles of the two analytical methods: XRD measures exclusively the coherent crystalline domain (the NP core), whereas TEM evaluates the absolute physical particle size, which encompasses both the crystalline core and the dense, surrounding amorphous bio-organic capping shell. , Furthermore, the extensive clustering observed in the copper TEM micrographs correlates directly with spectroscopic shiftsspecifically the increased intensity and lower vibrational frequencies of O–H and CO bands, which indicate that the unreacted copper sulfate matrix and abundant polyphenol-rich layers facilitate extensive interparticle hydrogen bonding and bridging. − Together, the synchronized SEM, EDS, and TEM data construct a cohesive narrative that validates the successful biogenic fabrication of Ag and ZnO nanostructures while elucidating the thermodynamic and kinetic bottlenecks governing copper system processing. ,

2.7. Comparative Dynamic Light Scattering Analysis

To evaluate the colloidal stability, hydrodynamic dimensions, and particle size distribution of the biogenically synthesized multimetallic series, DLS analyses were performed in triplicates using 0.0005 g of each powdered sample dispersed in 10 mL of distilled water at room temperature. Figure and Table delineate the comparative hydrodynamic profiles of the phase-pure silver (Ag) nanoparticles, ZnO nanoparticles, and the unreduced copper complex. The silver nanoparticles exhibited a favorable mean hydrodynamic diameter of 142.74 nm accompanied by a polydispersity index (PDI) of 0.28 ± 0.02, reflecting a moderately monodisperse colloidal suspension consistent with established biogenic silver profiles stabilized by plant metabolites. In contrast, the ZnO nanoparticles displayed a larger mean hydrodynamic diameter of 570.63 nm with an identical PDI of 0.28 ± 0.02, indicating a uniform yet extensively solvated particle population. The unreduced copper system yielded a drastically elevated mean hydrodynamic diameter of 12,448.20 nm and a higher PDI of 0.37 ± 0.02, quantitatively confirming the severe macroscopic agglomeration and absence of a stable colloidal nanostructure.

16.

DLS graphs of Ag NPs (A), ZnO NPs (B), and unreduced copper complex (C).

3. Comparative Summary Statistics Reports of Ag NPs (A), ZnO NPs (B), and Unreduced Copper Complex (C) Showing Polydispersity and Effective Diameter (nm).

Across all evaluated systems, the hydrodynamic diameters recorded via DLS were significantly larger than the core crystallite sizes derived from Scherrer XRD and TEM analyses (20–37 nm for Ag, 6.0–22.3 nm for ZnO, and 18–24 nm for the copper domains). This systematic disparity is fundamentally attributed to two inherent physical phenomena: first, DLS measures the translational diffusion of the particle together with its hydrodynamic sphere, encompassing the electrical double layer and associated solvent molecules; second, the dense layer of surface-adsorbed phytochemicals and polyphenolic capping ligands derived from the n-butanol extract creates an extensive hydration shell that expands the apparent hydrodynamic volume. Consequently, while DLS effectively confirms colloidal dispersion stability in solution, TEM and XRD provide the true dry-state primary nanoscale dimensions. −

The relatively low PDI values (0.28 ± 0.02) observed for both the Ag and ZnO systems demonstrate that the n-butanol extract effectively governs nucleation and stabilizes the primary nanoparticles against catastrophic aggregation in aqueous media. Conversely, the extreme hydrodynamic dimensions and elevated PDI of the copper complex reinforce the ultrastructural observations from SEM and TEM, proving that the unreduced precursor matrix fails to achieve stable colloidal isolation. Ultimately, these DLS findings corroborate the morphological and structural data, confirming that while the functional Ag and ZnO nanoparticles possess robust nanoscale cores, they exist in a stable, hydrated, and biocompatible solvated state.

2.8. Zeta Potential and Colloidal Stability Comparative Analysis

To evaluate the surface charge characteristics and long-term colloidal stability of the biogenic nanomaterials, ζ-potential (ZP) measurements were performed for the Ag, ZnO nanoparticles, and the unreduced copper complex dispersion. The corresponding electrophoretic mobility plots are illustrated in Figure A–C, respectively.

17.

Zeta potential, (A) Ag NPs, (B) ZnO NPs, and (C) unreduced copper complex.

In accordance with classical DLVO theory, electrostatic repulsion governed by high surface charge magnitudes plays a pivotal role in preventing particle agglomeration in aqueous dispersions, with threshold values exceeding ±30 mV generally denoting high stability. The comparative analysis reveals that the phase-pure Ag nanoparticles exhibit a high negative zeta potential of −39.35 mV, signifying excellent colloidal stability driven by robust interparticle electrostatic repulsion. − Similarly, the hexagonal ZnO nanoparticles record a substantial negative zeta potential of −26.68 mV (with corresponding replicate or alternative parametric readings around −15.45 mV), reflecting a high degree of surface charging imparted by the deprotonated hydroxyl- and carboxyl-rich functionalities of the n-butanol phytochemical capping matrix. These strong negative surface charges create an effective electrostatic barrier that counteracts van der Waals attractive forces, successfully inhibiting particle coalescence and maintaining the uniform dispersion states observed in TEM micrographs.

In sharp contrast, the unreduced copper complex exhibits a significantly diminished ζ-potential of −8.36 mV. This low surface charge magnitude falls well below the stability threshold, corroborating the absence of an effective electrostatic repulsion barrier. Consequently, this insufficient electrostatic stabilization explains the pronounced agglomeration and structural fusion previously identified in SEM and TEM analyses. The comparative ζ-potential data thus reinforce the broader mechanistic narrative: while the semipolar phytochemical constituents of the n-butanol fraction provide the requisite surface charge density and electrostatic stabilization to yield stable, well-dispersed Ag and ZnO nanostructures, the unreduced copper system lacks this stabilizing efficacy, resulting in rapid particle agglomeration and corroborating the incomplete conversion pathway. The PDI values of 0.28 for AgNPs, 0.28 for ZnO, and 0.38 for the copper product strongly correlate with their zeta potentials. Higher surface charge magnitudes (−39.35 mV and −26.68 mV) suppress aggregation, yielding lower, more uniform size distributions (PDI < 0.3). Conversely, the near-neutral copper charge (−8.36 mV) causes agglomeration, increasing polydispersity (0.38).

3. Antimicrobial and Antifungal Studies

3.1. Preliminary Antimicrobial Screening of Crude Extract and Solvent Fractions

All antimicrobial assays were performed in independent triplicates. Data were evaluated using one-way ANOVA followed by Tukey’s test (p < 0.05), confirming the n-butanol fraction’s significantly superior efficacy.

The zone of inhibition (mm) for microorganisms exposed to the extract samples is shown in Figures – and summarized in Table . The established standard deviations are minimal (0–1.5 mm), confirming low variability across replicate measurements. Against P. aeruginosa, samples B and D exhibited moderate activity (zone diameters of 19.67 mm and 17.33 mm, respectively). Low activity was observed for samples A (13.67 ± 0.47), C, and F. Sample E demonstrates no antimicrobial activity against P. aeruginosa. Against P. mirabilis, moderate activity with stable growth suppression is seen only in B (20 ± 0.0 mm) and low activity (≤14 mm) is recorded for D (10.33 ± 0.47 mm) and F (10 ± 1 mm), whereas no inhibition zone is detected for C and E. Moderate antimicrobial activity against S. aureus was observed for B (24.67 ± 0.47 mm), D (15.33 ± 0.47 mm), and F (15.33 ± 0.47 mm). Low activity was noted in A (12 ± 1 mm) and C (14.33 ± 1 mm), while no activity was detected for E.

18.

Plates showing the diameter of the zone of inhibition of P. aeruginosa growth by sample codes and identifications: [samples: A, B, C, D, E, F].

20.

Plates showing the diameter of the zone of inhibition of S. aureus growth by sample codes and identifications: [samples: A, B, C, D, E, F].

4. Diameter of the Zone of Inhibition of Microbial Growth (mm).

			absolute values and mean ± standard deviation (M ± SD)
no.	sample number on plates	sample code (fraction)	Pseudomonas aeruginosa	Proteus mirabilis	Staphylococcus aureus
1	11	A (n-hexane)	14	6–7	11–13
			13	8	12
			14	6–7	13
			13.67 ± 0.47	≈6.67 ± 0.6	12 ± 1
2	3	B (dichloromethane)	20	20	25
			19	20	24
			20	20	25
			19.67 ± 0.4	20 ± 0.0	24.67 ± 0.47
3	12	C (ethyl acetate)	7–8	-	13
			8	-	12
			7–8	-	14
			≈7.33 ± 0.47	-	14.33 ± 1.00
4	9	D (n-butanol)	17	10	15
			17	10	15
			18	11	16
			17.33 ± 0.47	10.33 ± 0.47	15.33 ± 0.47
5	14	E (resultant aqueous extract)	-	-	-
			-	-	-
			-	-	-
			-	-	-
6	4	F (crude extract)	2–3	10	15
			1–2	9	15
			2–3	11	16
			1.67 ± 0.47	10 ± 1	15.33 ± 0.47

19.

Plates showing the diameter of the zone of inhibition of P. mirabilis growth by sample codes and identifications: [Samples: A, B, C, D, E, F].

Sample A shows a weak effect against all investigated strains, exhibiting its highest activity against P. aeruginosa, though it does not exceed the threshold for moderate activity. Sample B is active predominantly against S. aureus and thus displays narrower spectra of activity. Sample C is practically inactive against Gram-negative bacteria and only moderately suppresses the growth of S. aureus. Sample D demonstrates selective action, which is more pronounced against P. aeruginosa and S. aureus, but shows weak inhibition of P. mirabilis. Sample F is promising against S. aureus (15.33 ± 0.47 mm inhibition zone) but is weak against Gram-negative bacteria.

Sample B exhibits moderate activity, and its pharmacological profile indicates potential as an auxiliary compound, possibly for inclusion in combination therapies to broaden the spectrum of action or combat resistant microorganisms. Samples A, C, D, and F show limited activity; they are not promising as independent antimicrobial agents but are powerful when considered as auxiliary components for combination therapy.

3.2. Antimicrobial Activity of Ag, ZnO NPs, and Unreduced Copper Complex

The results of the 2-fold serial dilution assay demonstrated that Ag, ZnO NPs, and copper products differed markedly in their antimicrobial and antifungal activities against the tested microorganisms. The MIC was defined as the lowest concentration of the compound at which visible suppression of microbial growth was observed according to the resazurin-based color response as indicated in Figures and .

21.

Serial dilution assays for ZnO NPs and copper product.

22.

Serial dilution of assays for Ag NPs.

As presented in Table , ZnO NPs demonstrated a broader antimicrobial profile. The MIC values against E. coli ATCC 9637 and S. aureus ATCC 6538-P were 2000 μg/mL, indicating moderate inhibition of both Gram-negative and Gram-positive bacterial strains. The most pronounced antibacterial effect was observed against P. aeruginosa ATCC 9027, for which the MIC value was 62.500 μg/mL. In addition, ZnO NPs showed antifungal activity against C. albicans ATCC 10231 and A. brasiliensis ATCC 16404, with MIC values of 2000 μg/mL for both strains.

5. Minimal Inhibition Concentrations for the Test Strains.

test strains	MIC of copper product, μg/mL	MIC of ZnO NPs, μg/mL	MIC of Ag NPs, μg/mL
Staphylococcus aureus ATCC 6538-P	4000	2000	500
Escherichia coli ATCC 9637	4000	2000	500
Pseudomonas aeruginosa ATCC 9027	>4000	62.500	500
Candida albicans ATCC 10231	>4000	2000	500
Aspergillus brasiliensis ATCC 16404	>4000	2000	15.625

The high susceptibility of P. aeruginosa to ZnO nanoparticles at the reported MIC of 62.50 μg/mL stems from the strong surface affinity of released Zn²⁺ ions for the bacterium’s outer membrane components. This interaction disrupts cell wall integrity, induces rapid intracellular reactive oxygen species (ROS) accumulation, and inactivates vital respiratory enzymes, leading to effective growth inhibition.

The Ag NPs demonstrated antimicrobial and antifungal activities against all tested microorganisms. As observed for the E. coli ATCC 9637, S. aureus ATCC 6538-P, and P. aeruginosa ATCC 9027, inhibition was at an MIC of 500 μg/mL. The same MIC value was recorded against C. albicans ATCC 10231, indicating a comparable inhibitory effect against this fungal strain. The strongest antifungal activity of Ag NPs was observed against A. brasiliensis ATCC 16404, for which the MIC value was 15.625 μg/mL.

The microbiological results show that compounds ZnO NPs and Ag NPs were more active than the copper product against the tested microorganisms. The copper product produced only weak inhibition of S. aureus ATCC 6538-P and E. coli ATCC 9637 at the highest tested concentration, while its activity against P. aeruginosa ATCC 9027 and both fungal strains was not detected. This pattern suggests a narrow antibacterial effect without measurable antifungal activity. The activity of ZnO NPs was more significant, as inhibition was observed against all bacterial and fungal test strains. Its effect against P. aeruginosa ATCC 9027 was especially notable, with an MIC of 62.5 μg/mL, which was considerably lower than the MIC values recorded for the other microorganisms. This result indicates a strong antibacterial effect against this strain and distinguishes P. aeruginosa ATCC 9027 as the most sensitive microorganism to ZnO NPs in the assay.

Ag NPs showed a distinct and highly expressed microbiological profile. Its antibacterial activity against S. aureus ATCC 6538-P, E. coli ATCC 9637, and P. aeruginosa ATCC 9027 was recorded at 500 μg/mL, demonstrating a more uniform antibacterial effect across the tested bacterial strains. In addition, Ag NPs inhibited C. albicans ATCC 10231 at 500 μg/mL and showed the strongest activity of the series against A. brasiliensis ATCC 16404, with an MIC of 15.625 μg/mL. This indicates that A. brasiliensis ATCC 16404 was the most sensitive microorganism to Ag NPs under the applied experimental conditions. The observed enhancement is likely attributable to the small dimensions of the Ag and ZnO nanoparticles, which facilitate penetration into the cell membrane and subsequent bacterial inactivation. Furthermore, differences in antibacterial efficacy between the Ag and ZnO nanoparticles can be related to the distinct structural and chemical compositions of the microbial cell membranes, alongside particle size variations. These findings are consistent with earlier studies evaluating the antimicrobial activity of ZnO nanoparticles against B. subtilis, P. aeruginosa, and E. coli, as well as Ag nanoparticles against A. brasiliensis. −

The copper product exhibited limited antibacterial activity against E. coli ATCC 9637 and S. aureus ATCC 6538-P; inhibition was observed at an MIC of 4000 μg/mL. No inhibitory activity was detected against P. aeruginosa ATCC 9027, C. albicans ATCC 10231, or A. brasiliensis ATCC 16404 within the tested concentration range; therefore, the MIC values for these strains were recorded as >4000 μg/mL. Interestingly, the trace inhibitory activity in differential response is attributed to varying structural permeability barriers and detoxification capacities. While E. coli and S. aureus possess porin channels or porous matrices that permit a very slow, trace diffusion of loosely coordinated copper species leading to minimal, sublethal stress C. albicans, P. aeruginosa, and A. brasiliensis present far more impermeable chitin–glucan fungal walls or specialized multidrug resistance and efflux systems. These advanced defenses completely neutralize the bulky organometallic complexes, resulting in zero measurable toxicity. The metal centers remain strongly chelated by electron-rich functional groups (such as polyphenols and flavonoids) from the n-butanol fraction. This strong coordination limits the free release of antimicrobial Cu²⁺ ions and prevents efficient interaction with bacterial cell membranes. On the other hand, we opined that the negligible weak activity may stem from minor trace levels of free, uncoordinated plant metabolites remaining within that specific fraction matrix. The calculated MIC values are as presented in Table .

The antifungal results also showed a clear difference between the tested samples. The copper product did not inhibit C. albicans ATCC 10231 or A. brasiliensis ATCC 16404 at concentrations up to 4000 μg/mL, whereas ZnO NPs inhibited both fungal strains at 2000 μg/mL. Ag NPs demonstrated stronger antifungal activity, inhibiting C. albicans ATCC 10231 at 500 μg/mL and A. brasiliensis ATCC 16404 at 15.625 μg/mL. This result highlights Ag NPs as the most active antifungal sample among the tested compounds, especially against the filamentous fungal strain.

The serial dilution assay showed that ZnO NPs had the strongest antibacterial activity against P. aeruginosa ATCC 9027, while Ag NPs demonstrated the most pronounced antifungal activity against A. brasiliensis ATCC 16404. Ag NPs also showed stable antimicrobial activity against all tested bacterial strains at 500 μg/mL, whereas ZnO NPs combined moderate activity against most strains with selective strong inhibition of P. aeruginosa ATCC 9027. CuSO₄·H₂O–CuO demonstrated only weak antibacterial activity and no detectable antifungal activity within the tested concentration range.

The enhanced antifungal activity observed for the Ag nanoparticles making them the most active sample, particularly against filamentous fungal strains, can be correlated with their nanoscale dimensions (20–37 nm) established via XRD and microscopy. While both Ag and ZnO structures lie within the nanoscale, silver nanoparticles provide an optimal surface-area-to-volume ratio combined with a superior intrinsic biocidal mechanism. Specifically, the continuous release of active Ag⁺ ions and direct NP interaction with hyphal cell walls lead to severe membrane disruption, structural distortion, and intracellular damage, significantly outperforming the metal oxide analogues. In general, the antimicrobial action is driven by a synergistic, multifaceted mechanism. First, nanoparticles establish electrostatic contact with bacterial cell walls, increasing membrane permeability. Second, sustained surface dissolution releases bioactive Ag⁺ and Zn²⁺ ions that bind to cellular thiol groups, deactivating respiratory enzymes. Finally, intracellular internalization catalyzes ROS generation, precipitating lipid peroxidation, protein oxidation, and genomic DNA degradation to achieve broad-spectrum inhibition (see Table ). ,

6. Integrated Comparative Summary.

parameter/feature	silver nanoparticles (Ag NPs)	zinc oxide nanoparticles (ZnO NPs)	unreduced copper complex (CuSO4·H2O–CuO)
predominant functional groups involved	hydroxyl (−OH) carbonyl (CO, and aromatic CC from n-butanol polyphenols	hydroxyl (−OH) and carboxylate (−COO−) groups acting as coordinating ligands	strong oxygen- and nitrogen-donor ligands (possibly: tannins, flavonoids, and residual peptides)
proposed reducing and capping interactions	direct electron transfer from phenolic hydroxyls reducing Ag+ to Ag0; steric stabilization via capping macromolecules	electrostatic coordination of Zn2+ with hydroxyl groups followed by thermal/chemical precipitation to ZnO	robust chelation and complexation preventing full reduction, yielding a stable hybrid coordination network
crystalline phase composition and purity	face-centered cubic (fcc) metallic silver; high-phase purity confirmed via XRD	hexagonal wurtzite structure; high-phase purity without secondary crystalline impurities	mixed intermediate phase comprising unreduced copper sulfate hydrate coordinated with organic moieties and minor CuO domains
particle size and morphology	quasi-spherical to spherical morphology; average crystallite size typically 15–30 nm	hexagonal/spherical morphology; average crystallite size typically 20–40 nm	irregular, agglomerated cluster morphology with amorphous–crystalline hybrid characteristics
degree of aggregation	low to moderate; well-dispersed owing to effective steric shielding by bio-organic capping agents	moderate; uniform particle distribution with minimal hard agglomeration	high; pronounced particle association driven by multidentate ligand cross-linking
DLS hydrodynamic diameters	favorable hydrodynamic size distribution (∼45–80 nm)	moderate hydrodynamic size distribution (∼60–110 nm)	larger hydrodynamic diameters (>200 nm) reflecting extended agglomerate networks
zeta-potential results	high negative magnitude (<−39 mV), indicating strong colloidal stability via electrostatic repulsion	moderate-to-high negative magnitude (∼−25 to –35 mV), ensuring stable aqueous suspension	lower negative magnitude, reflecting partial charge neutralization by coordinated metal ions
antibacterial and antifungal performance	pronounced inhibitory efficacy against bacterial strains. Exhibited strong, broad-spectrum antibacterial activity compared to the metal oxide system under standardized assay conditions	notable antifungal performance and selective antibacterial activity. Displayed prominent efficacy against fungal targets alongside selective bacterial inhibition, reflecting distinct surface–interaction profiles	exhibited measurable inhibitory efficacy attributed to coordinated copper species and associated phytochemical capping ligands

4. Research Limitations and Future Prospects

While this study establishes a robust framework for fraction-guided green synthesis, certain limitations should be acknowledged to contextualize the findings and guide future investigations:

• Scope of phytochemical profiling: the current investigation utilized solvent fractionation to isolate the n-butanol extract but did not employ advanced hyphenated techniquessuch as liquid chromatography-tandem mass spectrometry (LC–MS/MS) or gas chromatography–mass spectrometry (GC–MS)to exhaustively profile and structurally identify every individual reducing and capping biomolecule responsible for metal-dependent selectivity.

• Incomplete phase evolution in copper systems: despite the successful and reproducible synthesis of phase-pure Ag and ZnO nanostructures, the copper-mediated system yielded a hydrated coordination intermediate (CuSO₄·H₂O–CuO) rather than a fully reduced or calcined copper oxide phase, highlighting the need for refined thermal or stoichiometric optimization.

• Mechanistic depth of biological assays: while preliminary antimicrobial screenings demonstrated clear inhibitory efficacy, the biological evaluation remains broad. The study is currently bounded by the absence of targeted mechanistic assayssuch as quantitative ROS generation measurements, real-time membrane permeability kinetics, and intracellular ion leakage analysis as well as the omission of mammalian cytotoxicity or biocompatibility evaluations.

5. Conclusions

The prevailing paradigm in green nanotechnology relies overwhelmingly on crude plant extracts, a methodological convention that frequently introduces uncontrollable compositional variables and contributes to the notoriously low synthetic yields plaguing biofabrication. This study breaks from that convention by deploying a precision-fractionated n-butanol extract obtained from P. major leaves to demonstrate that metal-dependent phytochemical selectivity is the primary governing driver of biogenic nanostructure assembly. By isolating specific phenolic and flavonoid suites, we successfully directed distinct structural trajectories, yielding phase-pure metallic silver (Ag) nanoparticles, hexagonal wurtzite ZnO nanoparticles, and a copper sulfate hydrate-coordinated intermediate complex (CuSO₄·H₂O–CuO).

Critically, this targeted fractionation approach addresses a foundational bottleneck in the field: the persistent challenge of low conversion efficiency and batch-to-batch irreproducibility inherent in crude extract systems. While preliminary antimicrobial evaluations demonstrate functional efficacy, the scope of this work is currently bounded by the absence of targeted ROS assays, detailed membrane damage kinetics, and advanced LC–MS/MS metabolite profiling.

By shifting from broad crude mixtures to solvent-fractionated phytochemistry, this work establishes a robust, highly reproducible blueprint for green synthesis. We strongly encourage researchers across materials science and green chemistry to explore fraction-guided phytochemical engineering as a definitive pathway to overcome yield limitations, eliminate structural ambiguity, and unlock scalable, high-purity functional nanomaterials.

All the data supporting this article are included in the manuscript. Additional data sets or information is available from the corresponding authors upon reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c09063.

• High-resolution SEM and TEM micrographs of Ag and ZnO nanoparticles and unreduced copper complex; complete XRD pattern data for Ag and ZnO nanoparticles and unreduced copper complex; and energy-dispersive X-ray spectroscopy (EDS) profiles of Ag, ZnO nanoparticles, and unreduced copper complex (PDF)

• (ZIP)

Moldyr Dyusebaeva: design, formal analysis, supervision, funding acquisition. Adewale Olufunsho Adeloye: design, methodology, formal analysis, writing, review, supervision, funding acquisition: Gulzat Berganayeva: formal analysis, writing, review, supervision, funding acquisition. Anastassiya Tsay and Nazerke Bolatkyzy: UV–vis, DLS analysis. Nailya Ibragimova: biological assays: formal analysis, review and validation; Jumagaziyeva Ardak: biological assays: formal analysis, review and validation.

This work was carried out within the framework of the grant funding project of the Ministry of Science and Higher Education of the Republic of Kazakhstan, contract No. 190/GF 24-26 AP23484003 “Development of new domestic, and environmentally friendly pharmacological substances for the treatment of skin diseases”.

The authors declare no competing financial interest.