Biogenic AgNPs from Leptadenia arborea: Integrated antimicrobial, OmpF/Erg11 docking, and membrane-disruptive mechanisms

Abstract

Plant-derived materials offer sustainable platforms for functional nanomaterial development; however, Leptadenia arborea remains poorly explored for silver nanoparticle (AgNP) synthesis, and quantitative approaches linking phytochemistry, antimicrobial efficacy, and cellular damage are limited. This study investigated L. arborea leaf extract as a reducing and stabilizing system for AgNP synthesis. HPLC identified chlorogenic acid (53.8 µg mL⁻¹) and gallic acid (53.3 µg mL⁻¹) as major phytochemicals. Replicated optimization experiments identified 1:50 extract dilution, 5 mM AgNO₃, 70 °C, and 2 mL extract as optimal conditions, producing a surface plasmon resonance peak at 435 nm. TEM revealed predominantly spherical AgNPs (12 to 24 nm), while XRD showed (111), (200), (220), and (311) reflections characteristic of crystalline face-centered cubic silver. AgNPs produced inhibition zones of 15 ± 0.7 mm against Escherichia coli and 30 ± 1.0 mm against Candida albicans, with an MIC of 0.117 µg mL⁻¹ against E. coli. As a key novelty, AEF enables normalized comparison with reference antimicrobials, while NBIP converts SEM-observed cellular damage into a quantitative index. This framework extends conventional inhibition-based assessment by integrating antimicrobial potency with cellular damage and provides a transferable approach for evaluating plant-derived antimicrobial nanomaterials.

Full Article

Biogenic AgNPs from Leptadenia arborea: Integrated Antimicrobial, OmpF/Erg11 Docking, and Membrane-disruptive Mechanisms

Emad Abada ,^a,b,*Aiden M. Hussain ,^c Samira M. Yaseen ,^c

Rasha M. Alzayed ,^d Sondos A. Alhajouj ,^d Tarad Abalkhail ,^e

Shifaa O. Alshammari ,^f Ibrahim Y. Y. Sumaily ^a

Plant-derived materials offer sustainable platforms for functional nanomaterial development; however, Leptadenia arborea remains poorly explored for silver nanoparticle (AgNP) synthesis, and quantitative approaches linking phytochemistry, antimicrobial efficacy, and cellular damage are limited. This study investigated L. arborea leaf extract as a reducing and stabilizing system for AgNP synthesis. HPLC identified chlorogenic acid (53.8 µg mL⁻¹) and gallic acid (53.3 µg mL⁻¹) as major phytochemicals. Replicated optimization experiments identified 1:50 extract dilution, 5 mM AgNO₃, 70 °C, and 2 mL extract as optimal conditions, producing a surface plasmon resonance peak at 435 nm. TEM revealed predominantly spherical AgNPs (12 to 24 nm), while XRD showed (111), (200), (220), and (311) reflections characteristic of crystalline face-centered cubic silver. AgNPs produced inhibition zones of 15 ± 0.7 mm against Escherichia coli and 30 ± 1.0 mm against Candida albicans, with an MIC of 0.117 µg mL⁻¹ against E. coli. As a key novelty, AEF enables normalized comparison with reference antimicrobials, while NBIP converts SEM-observed cellular damage into a quantitative index. This framework extends conventional inhibition-based assessment by integrating antimicrobial potency with cellular damage and provides a transferable approach for evaluating plant-derived antimicrobial nanomaterials.

DOI: 10.15376/biores.21.4.10589-10612

Keywords: Leptadenia arborea; Silver nanoparticles (AgNPs); Green synthesis; Antimicrobial enhancement factor (AEF); Nanoparticle–bacteria interaction potential (NBIP)

Contact information: a: Department of Biology, College of Science, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia; b: Environment and Nature Research Centre, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia; c: Biology Department, College of Education for Pure Sciences/Ibn al-Haitham, University of Baghdad, Baghdad, Iraq; d: Biology Department, College of Science, Jouf University, Sakaka 41412, Saudi Arabia; e: Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Saudi Arabia; f: Department of Biology, College of Science, University of Hafr Al Batin, P.O. Box 1803, Hafr Al Batin, 31991, Saudi Arabia;

* Corresponding author: [email protected]

Graphical Abstract

INTRODUCTION

The search for sustainable and biocompatible approaches to nanomaterial production has intensified in recent years, driving interest toward plant-mediated synthesis of metallic nanoparticles (Jeetkar et al. 2022). In green nanotechnology, plant extracts serve as natural reducing, stabilizing, and capping agents, providing an environmentally safer alternative to conventional chemical synthesis. However, although numerous medicinal plants have been investigated for silver nanoparticle (AgNP) synthesis, differences in their phytochemical composition can markedly influence metal-ion reduction, nanoparticle nucleation, surface stabilization, and ultimately biological activity. Therefore, identifying phytochemically suitable and comparatively underexplored plant resources remains important rather than simply extending AgNP synthesis to additional plant species.

Among the medicinal plants with potential for this purpose, Leptadenia arborea remains largely unexplored despite its recognized ethnomedicinal importance (Kumari et al. 2025). Traditionally used in African herbal medicine, L. arborea is known for its antimicrobial, antioxidant, anti-inflammatory, and wound-healing properties, which have been associated with its diverse phytochemical composition, including alkaloids, flavonoids, phenolic compounds, terpenoids, and glycosides (Vaidyanathan and Lokeswari 2024). This phytochemical richness provides a scientific rationale for selecting L. arborea over more extensively investigated medicinal plants. In particular, phenolic and flavonoid constituents possess electron-donating and metal-binding functional groups that can participate in Ag⁺ reduction and subsequently stabilize newly formed nanoparticles through surface interactions (Kirubakaran et al. 2025). Thus, the combination of redox-active phytochemicals, reported biological properties, and limited investigation of L. arborea as a nanomaterial precursor makes this species particularly relevant for green AgNP fabrication.

Despite extensive research on plant-mediated AgNPs, important methodological gaps remain. Many previous investigations primarily demonstrate nanoparticle formation, physicochemical characterization, and antimicrobial activity without quantitatively connecting nanoparticle performance with microbial cellular damage. Moreover, documented research involving L. arborea remains scarce, and systematic assessment of how its biosynthesized nanoparticles interact with microbial cells or compare with conventional antimicrobial agents is particularly limited (Punz et al. 2025). Conventional antimicrobial studies frequently depend on inhibition-zone measurements, MIC values, and descriptive microscopic observations. Although these approaches establish antimicrobial activity, they provide limited standardized information for quantitatively comparing nanoparticle efficacy and the magnitude of nanoparticle-induced cellular damage (Hoseinzadeh et al. 2017).

The present work addresses these limitations by employing L. arborea leaf extract for AgNP phytofabrication while integrating two quantitative analytical approaches: the Antimicrobial Enhancement Factor (AEF) and Nanoparticle Bacteria Interaction Potential (NBIP) (Chowdhury et al. 2024). The AEF provides a mathematical approach for comparing AgNP antimicrobial activity directly with reference antimicrobial agents, generating a normalized index reflecting relative enhancement or reduction in efficacy (Musoliwa 2022). This approach extends the interpretation of conventional inhibition-zone assays beyond simple diameter comparisons. Similarly, NBIP provides a structured means of evaluating morphological alterations in bacterial cells following AgNP exposure. Rather than relying solely on descriptive scanning electron microscopy (SEM), NBIP expresses the proportion of morphologically damaged cells relative to the total observed population (López-Heras et al. 2015), thereby transforming microscopic observations into a measurable indicator of nanoparticle–cell interactions and facilitating comparison between untreated and treated populations (Alqahtany 2019).

By combining the medicinal and phytochemical potential of L. arborea with these quantitative analytical approaches, the present study establishes an integrated framework for evaluating plant-synthesized nanoparticles (Barathi et al. 2024). The integration of AEF and NBIP strengthens antimicrobial assessment by connecting conventional efficacy measurements with quantitative cellular damage evaluation and contributes additional analytical tools to green nanotechnology (Jangid et al. 2024). Therefore, this study aimed to synthesize and characterize AgNPs using L. arborea leaf extract and quantitatively evaluate their antimicrobial effects. The novelty lies in integrating an underexplored phytochemical-rich botanical resource with normalized antimicrobial comparison and SEM-based quantitative damage assessment, thereby providing a more standardized framework for linking plant-derived nanoparticle fabrication with biological performance.

EXPERIMENTAL

Materials

Fresh leaves of Leptadenia arborea were collected from the Jazan region, Saudi Arabia (16°53’21.69” N) and authenticated at the Plant Herbarium, Jazan University. The leaves were thoroughly washed, air-dried in shade at 27 to 29 °C for one week, and then finely ground into powder for extraction (Abada et al. 2025a) (Fig. 1). The herbarium specimens are prepared with a voucher number (JAZUH 1646) and deposited in Jazan University Herbarium for further reference.

Fig. 1. The fresh leaves of Leptadenia arborea

Methods

Preparation of plant extract

An aqueous extract of Leptadenia arborea was prepared by combining leaf powder with distilled water under heated, stirred conditions to promote phytochemical release. The mixture was clarified through filtration and low-temperature centrifugation. The resulting supernatant, containing the plant’s bioactive compounds, was used as the natural reducing and stabilizing agent for AgNP synthesis (Fig. 2) (Zhang et al. 2024).

Fig. 2. Preparation of leaf extract of Leptadenia arborea

High-performance liquid chromatography analysis

Phytochemical profiling of the aqueous leaf extract of Leptadenia arborea was performed using high-performance liquid chromatography (HPLC; Shimadzu Corporation, Kyoto, Japan). The dried extract was dissolved in distilled water at a concentration of 10 mg/mL, filtered through a 0.22-µm membrane filter, and degassed before analysis. Chromatographic separation was performed using a reverse-phase C18 column (250 × 4.6 mm, 5 µm particle size). The mobile phase consisted of solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile) using the following gradient: 5 to 20% B from 0 to 10 min, 20 to 40% B from 10 to 25 min, and 40 to 60% B from 25 to 35 min. The flow rate was 1.0 mL/min, the injection volume was 20 µL, and the column temperature was maintained at 30 °C. Detection was performed using a UV–Vis detector (SPD-20A, Shimadzu Corporation, Kyoto, Japan) at 280 and 320 nm. Compounds were identified by comparing their retention times with authentic standards and quantified using calibration curves prepared from standard solutions of known concentrations.

Optimization of the synthesis of nanoparticles

The synthesis of AgNPs was optimized by systematically evaluating plant extract dilution, AgNO₃ concentration, temperature, extract volume, and pH. During optimization, one parameter was varied while the remaining experimental conditions were maintained constant. The aqueous L. arborea extract was mixed with AgNO₃ solution under continuous stirring. Development of a brown color was considered the initial visual indication of Ag⁺ reduction and AgNP formation, which was subsequently confirmed by UV–Vis spectroscopy (Abada et al. 2025b). The optimized conditions were subsequently established as a 1:50 plant extract dilution, 5 mM AgNO₃, 2 mL extract, and a reaction temperature of 70 °C. Experimental measurements were performed in triplicate, and results are expressed as mean ± standard deviation (SD).

Effect of different AgNO₃ concentrations

Different AgNO₃ concentrations ranging from 3 to 10 mM were investigated to determine the optimal silver-ion concentration for AgNP formation. The plant extract was combined with each AgNO₃ concentration while the other experimental variables were maintained constant. Following reaction, samples were analyzed by UV–Vis spectroscopy over the wavelength range of 200 to 1000 nm. Formation of AgNPs was evaluated from the characteristic surface plasmon resonance (SPR) response, and 5 mM AgNO₃ was identified as the optimal concentration based on the strongest and most defined SPR signal. Measurements were performed in triplicate.

Effect of different dilutions of the plant extract

Different dilutions of the aqueous L. arborea leaf extract, ranging from 1:1 to 1:100, were investigated to determine the effect of extract concentration on AgNP formation. Each diluted extract was reacted with AgNO₃ while the remaining synthesis parameters were maintained constant. AgNP formation was monitored by UV–Vis spectroscopy, and differences in SPR intensity were compared among the tested dilutions. The 1:50 dilution produced the strongest and most distinct SPR response and was selected as the optimal dilution. Measurements were performed in triplicate.

Effect of temperature

The influence of temperature on AgNP formation was evaluated over the range of 4 to 70 °C. Reaction mixtures containing L. arborea extract and AgNO₃ were maintained at the tested temperatures while the remaining experimental parameters were kept constant. Nanoparticle formation was monitored by UV–Vis spectroscopy. A temperature of 70 °C produced the highest SPR absorbance and was therefore selected as the optimal synthesis temperature. Measurements were performed in triplicate.

Effect of extract volume

Different volumes of L. arborea aqueous extract, ranging from 1 to 5 mL, were evaluated while maintaining the AgNO₃ concentration and other reaction conditions constant. AgNP formation was assessed by UV–Vis spectroscopy through comparison of SPR intensity. An extract volume of 2 mL produced the strongest and most defined SPR signal and was selected for subsequent nanoparticle synthesis. Measurements were performed in triplicate.

Effect of pH

The influence of reaction pH on AgNP formation was investigated over the range of pH 2 to 14. The pH of individual reaction mixtures was adjusted while the remaining synthesis conditions were maintained constant. Following reaction, samples were analyzed by UV–Vis spectroscopy to evaluate changes in SPR intensity and spectral characteristics. The condition producing the strongest SPR response was considered optimal. Measurements were performed in triplicate.

Characterization of the AgNPs

UV–Visible spectroscopy analysis

AgNP formation was confirmed using UV–Vis spectrophotometry. Absorbance spectra were recorded between 200 and 1000 nm, and the appearance of a characteristic SPR band was used as a diagnostic indicator of AgNP formation (Asif et al. 2022). The optimized AgNP preparation exhibited a characteristic SPR maximum near 435 nm. Spectral measurements were performed in triplicate.

Transmission electron microscopy (TEM) analysis

Transmission electron microscopy was used to determine the morphology and particle-size characteristics of the synthesized AgNPs. Imaging was performed using a HITACHI H-800 (Japan) transmission electron microscope operated at an accelerating voltage of 200 kV. Samples were prepared and examined according to the procedure described by Malatesta (2021). TEM micrographs were used to evaluate nanoparticle morphology and size distribution.

Fourier transform infrared spectroscopy (FTIR) analysis

FTIR spectroscopy (IRTracer-100, Shimadzu Corporation, Kyoto, Japan) was used to identify functional groups associated with phytochemicals involved in the reduction and stabilization of AgNPs. Samples were analyzed using the KBr pellet method, and spectra were recorded over the range of 500 to 4000 cm⁻¹. Differences in absorption bands were interpreted to identify functional groups potentially involved in nanoparticle formation and stabilization (Shaheen et al. 2022).

X-ray diffraction (XRD) analysis

X-ray diffraction analysis was performed using a SmartLab diffractometer (Rigaku Corporation, Tokyo, Japan) to determine the crystalline structure of the synthesized AgNPs. The diffraction pattern was analyzed according to established procedures (Ali et al. 2023). Characteristic diffraction reflections were used to identify the crystalline metallic silver phase and its corresponding lattice planes.

Antimicrobial activity test

The antimicrobial activity of the synthesized AgNPs was evaluated against selected pathogenic bacteria and Candida albicans using a diffusion-based assay. Standardized microbial suspensions were inoculated onto appropriate growth media, and AgNPs were applied under identical experimental conditions. Novobiocin was used as the reference antibacterial agent, whereas Amphotericin B was used as the reference antifungal agent for C. albicans. Following incubation, inhibition-zone diameters were measured in millimeters. All antimicrobial assays were performed in triplicate, and results were expressed as mean ± SD (Abada et al. 2025c).

Minimum inhibitory concentration (MIC) test

The minimum inhibitory concentration of AgNPs against Escherichia coli was determined using a broth microdilution approach with reference to CLSI recommendations for antimicrobial susceptibility testing. Serial AgNP concentrations ranging from 1 to 0.007 µg/mL were prepared and inoculated with a standardized bacterial suspension. Appropriate growth and sterility controls were included. Following incubation, the MIC was defined as the lowest AgNP concentration at which no visible bacterial growth was detected. The assay was performed in triplicate (Abada et al. 2024).

Scanning electron microscopy (SEM) of treated cells

Scanning electron microscopy was used to investigate morphological changes in E. coli following exposure to AgNPs. Untreated bacterial cells were used as controls. Samples were prepared according to the procedure described by Lavecchia et al. (2024) and examined using a HITACHI S-4800 scanning electron microscope. Micrographs were evaluated for changes in cell morphology, including surface roughening, shrinkage, deformation, and membrane-associated structural alterations.

Calculation of Antimicrobial Enhancement Factor

The Antimicrobial Enhancement Factor (AEF) was introduced in this study as a dimensionless parameter to quantitatively describe the relative performance of biosynthesized AgNPs compared with conventional antimicrobial agents. Conceptually, AEF is based on the general form of a relative change or relative difference, which is commonly defined as the difference between a new value and a reference value, normalized to the reference:

(1)

In the context of antimicrobial testing, the inhibition zone diameter is widely used as an experimental measure of antimicrobial activity. Therefore, the “new value” was taken as the inhibition zone produced by AgNPs, while the “reference value” was taken as the inhibition zone produced by the standard antibiotic. Denoting the inhibition zone diameter of AgNPs as D_AgNP and that of the corresponding reference antibiotic as D_antibiotic the general relative change expression can be rewritten specifically for antimicrobial activity as:

(2)

This formulation provides a normalized measure of how much the activity of AgNPs deviates from that of the conventional drug. An AEF value of zero indicates that AgNPs and the reference antibiotic exhibit identical inhibition zone diameters. Positive AEF values indicate an enhancement of antimicrobial activity by AgNPs relative to the antibiotic, whereas negative values indicate lower activity of AgNPs compared with the standard drug. This simple but informative index allows direct comparison of AgNP performance across different microorganisms and against different reference agents within a unified mathematical framework (Skwarczynski et al. 2022).

Determination of Nanoparticle–Bacteria Interaction Potential

The interaction between AgNPs and Escherichia coli cells was quantitatively assessed through the Nanoparticle–Bacteria Interaction Potential (NBIP). This parameter was developed to estimate the proportion of bacterial cells exhibiting structural alterations after exposure to AgNPs, based on SEM analysis. For each SEM micrograph, bacterial cells were manually counted and classified into two categories. Damaged cells, which showed membrane deformation, pitting, aggregation, or lysis; and undamaged cells, which retained normal rod-shaped morphology with intact surface structure. Counts were performed for at least five non-overlapping fields per sample to minimize bias.

The NBIP was calculated as the percentage of damaged cells relative to the total number of observed cells using the following relationship,

(3)

where N_damaged is the number of cells exhibiting AgNP-induced structural abnormalities, and N_total is the total number of cells counted in each micrograph. Higher NBIP values indicate a stronger interaction between AgNPs and bacterial cells, reflected by greater morphological disruption (Tiwari et al. 2025).

Molecular Docking of Ag⁺ with Erg11 and OmpF

The crystal structure of Candida albicans lanosterol 14α-demethylase Erg11 and a representative Escherichia coli outer-membrane porin (OmpF) were retrieved from the Protein Data Bank in PDB format. Non-protein molecules (co-crystallized ligands, buffer components, and bulk water) were removed, missing side-chain atoms were rebuilt, and polar hydrogens and Gasteiger charges were added using standard protein-preparation tools.

Silver was modeled as a monovalent Ag⁺ ion. A docking grid was centered on the catalytic cavity of Erg11, encompassing the heme-binding pocket and surrounding residues, and on the inner vestibule/constriction region of OmpF, where permeating molecules interact with charged residues. Docking was performed with AutoDock Vina using an exhaustiveness setting sufficient to sample multiple orientations of the Ag⁺ ion within each binding region. For each target, the lowest-energy pose and the most populated interaction pattern were selected for visualization and qualitative analysis (Pugazhenthi et al. 2024).

Statistical Analysis

Quantitative experiments were performed in triplicate, and results are presented as mean ± standard deviation (SD). Statistical significance was established at p < 0.05. Statistical analyses were performed using the statistical software employed for data analysis.

RESULTS

HPLC Profiling of Leptadenia arborea Aqueous Leaf Extract

The HPLC analysis of the aqueous leaf extract of Leptadenia arborea identified 17 phenolic and flavonoid constituents against external standards (Fig. 3).

Fig. 3. HPLC chromatogram (280 nm) of the aqueous leaf extract of Leptadenia arborea, showing the separation and identification of 17 phenolic and flavonoid compounds based on retention time comparison with authenticated standards

Chlorogenic acid was the most abundant compound at 53.8 µg mL⁻¹, corresponding to 2.7 µg g⁻¹ dry weight (DW), followed closely by gallic acid at 53.3 µg mL⁻¹ (2.7 µg g⁻¹ DW), indicating a phenolic acid–rich chemotype. Catechin was detected at 44.7 µg mL⁻¹ (2.2 µg g⁻¹ DW), while ellagic acid and rutin were present at intermediate concentrations of 35.0 µg mL⁻¹ (1.8 µg g⁻¹ DW) and 28.5 µg mL⁻¹ (1.4 µg g⁻¹ DW), respectively. Additional constituents included ferulic acid (27.0 µg mL⁻¹; 1.3 µg g⁻¹ DW), p-coumaric acid (32.0 µg mL⁻¹; 1.6 µg g⁻¹ DW), rosmarinic acid (40.0 µg mL⁻¹; 2.0 µg g⁻¹ DW), and cinnamic acid (38.0 µg mL⁻¹; 1.9 µg g⁻¹ DW). Minor compounds, such as daidzein, quercetin, kaempferol, hesperetin, naringenin, vanillin, methyl gallate, and syringic acid, were detected at lower levels (< 25 µg mL⁻¹).

Collectively, the aqueous extract is dominated by chlorogenic and gallic acids with substantial contributions from rosmarinic and cinnamic acids, a profile consistent with strong reducing capacity and supportive of its role in AgNP biogenic synthesis.

Effect of Physical Conditions on AgNPs Synthesis

The formation of silver nanoparticles was influenced by several reaction parameters, including extract dilution, extract volume, pH, temperature, and AgNO₃ concentration. When the Leptadenia arborea extract was combined with the silver nitrate solution, the mixture gradually changed from pale yellow to deep brown. This progressive color shift signaled the successful reduction of silver ions and the formation of AgNPs (Fig. 4).

Fig. 4. The synthesis of AgNPs from the leaf extract is illustrated by the color change observed in the tubes: the left tube, which remains yellow, serves as the control, while the right tube, which turns dark brown, indicates the successful synthesis of AgNPs

Effect of Different Plant Extract Dilutions

Different dilutions of Leptadenia arborea leaf extract (1:5, 1:10, 1:20, 1:50, and 1:100) were combined with equal volumes of AgNO₃ solution to assess their influence on nanoparticle formation. A gradual shift in color from pale yellow to brown signaled the successful production of AgNPs, which was later confirmed by a UV–Vis absorption peak at 435 ± 1 nm (n = 3). Among the tested dilutions, the 1:50 ratio generated the strongest and most distinct plasmon resonance peak (Fig. 5).

Fig. 5. Effect of various leaf extract dilutions on the synthesis of AgNPs

Effect of different AgNO₃ concentrations

Varying AgNO₃ concentrations (3 to 10 mM) were tested to determine the most suitable level for AgNP formation, with all samples showing the characteristic SPR peak at 435 ± 2 nm. Among them, 5 mM AgNO₃ produced the strongest and most distinct nanoparticle absorption peak (Fig. 6).

Fig. 6. Influence of AgNO₃ Concentrations on the Formation of AgNPs

Effect of Temperature

The reaction temperature strongly influenced AgNP formation, with all samples showing a characteristic SPR peak at 435 ± 2 nm. The highest absorbance was recorded at 70 °C, indicating the greatest nanoparticle yield under this condition (Fig. 7).

Fig. 7. Impact of temperature on the formation of AgNPs

Effect of Extract Volume

Different extract volumes (1 to 5 mL) produced varying absorbance intensities, with all samples showing a characteristic AgNP peak at 435 ± 2 nm. The 2 mL extract volume generated the highest and sharpest plasmon resonance signal, indicating the most efficient nanoparticle formation. Higher volumes (3 to 5 mL) showed reduced peak intensity, suggesting decreased synthesis efficiency (Fig. 8).

Fig. 8. Effect of different leaf extract volumes on the synthesis of AgNPs

Effect of pH

The pH of the reaction medium had a marked influence on AgNP formation, with all samples showing a characteristic peak near 435 nm. Among the tested conditions, pH 4 ± 0.1 produced the highest absorbance intensity, indicating the most efficient nanoparticle synthesis. Extremely acidic (pH 2 ± 0.1) and strongly alkaline conditions (pH 12 to 14 ± 0.2) resulted in markedly reduced peak intensities (Fig. 9).

Fig. 9. Effect of different pH on the synthesis of AgNPs

Characterization of AgNPs

TEM analysis

The TEM analysis revealed that the synthesized AgNPs were predominantly spherical and well-dispersed, with particle sizes ranging from approximately 12 to 24 ± 0.3 nm. The majority of nanoparticles were in a narrow range between 14 to 17 ± 0.5 nm, indicating a narrow size distribution. These observations confirm the successful formation of uniformly shaped nanoscale silver particles (Fig. 10).

Fig. 10. TEM showed the analysis of green synthesized Leptadenia arborea AgNPs

XRD analysis

The XRD pattern displayed distinct peaks at 2θ values around 38°, 42°, 59°, and 69°, corresponding to the (111), (200), (220), and (311) planes. These reflections confirm the face-centered cubic (fcc) crystalline structure of the synthesized AgNPs. The sharp diffraction peaks indicate high crystallinity and successful nanoparticle formation (Fig. 11).

Fig. 11. XRD pattern of synthesized silver nanoparticles

FTIR analysis

The FTIR spectra of the Leptadenia arborea aqueous extract (control) and the Ag⁺ + extract reaction mixture showed differences in peak position and intensity (Fig. 12). The control extract exhibited a broad absorption band around 3400 cm⁻¹ and additional bands in the regions around 2920, 1590, 1352, and 1114 cm⁻¹. Following reaction with Ag⁺, changes in the intensity and position of these bands were observed, together with distinct low-wavenumber bands at approximately 615 and 460 cm⁻¹. Overall, the FTIR profiles demonstrated measurable spectral differences between the untreated plant extract and the Ag⁺-reacted sample.

Fig. 12. FTIR spectrum of silver nanoparticles synthesized using Leptadenia arborea leaf extract

Antimicrobial Activity of AgNPs

The synthesized AgNPs produced clear inhibition zones against all tested microorganisms. For bacterial strains, the inhibition zones were 15 ± 0.7 mm for Escherichia coli (ATCC93111), 10 ± 0.2 mm for Salmonella typhimurium (ATCC14028), 14 ± 0.7 mm for Listeria monocytogenes (ATCC25923), and 15 ± 0.3 mm for Staphylococcus aureus (ATCC25923). For the fungal strain Candida albicans (ATCC 10231), the AgNPs produced an inhibition zone of 30 ± 1 mm. The reference antibiotic Novobiocin showed inhibition zones of 15 ± 0.2 mm, 20 ± 0.1 mm, 40 ± 0.04 mm, and 30 ± 0.3 mm for E. coli, S. typhimurium, L. monocytogenes, and S. aureus, respectively. For Candida albicans, the reference antifungal Amphotericin B produced an inhibition zone of 20 ± 0.5 mm (Table 1, Figs. 13 and 14).

Table 1. Antimicrobial Activity of AgNPs Against Different Microorganisms

Fig. 13. Antimicrobial activity of AgNPs against different microorganisms

Fig. 14. Antimicrobial activity of AgNPs (15 𝜇g/mL)

MIC Test

The MIC assay was conducted to determine the lowest concentration of AgNPs required to prevent visible bacterial growth. Escherichia coli (ATCC 93111), which was identified as the most sensitive bacterial strain in the antimicrobial screening, was selected for MIC evaluation at concentrations ranging from 1 to 0.007 µg/mL. The results revealed that the MIC for E. coli was 0.117 µg/mL, with visible growth occurring at concentrations lower than this value.

Antimicrobial Enhancement Factor

The AEF was calculated for each tested microorganism based on the inhibition zone diameters of AgNPs and the corresponding reference antibiotics. For Escherichia coli (ATCC 93111), AgNPs and Novobiocin produced identical inhibition zones of 15 mm, resulting in an AEF value of 0.00, indicating no difference in activity. In the case of Salmonella typhimurium (ATCC 14028), AgNPs produced an inhibition zone of 10 mm compared with 20 mm for Novobiocin, giving an AEF of –0.50. For Listeria monocytogenes (ATCC 25923), the inhibition zones of AgNPs and Novobiocin were 14 mm and 40 mm, respectively, corresponding to an AEF of –0.65. Staphylococcus aureus (ATCC 25923) showed inhibition zones of 15 mm for AgNPs and 30 mm for Novobiocin, yielding an AEF value of –0.50. For the fungal strain Candida albicans (ATCC 10231), AgNPs produced an inhibition zone of 30 mm, whereas Amphotericin B produced a 20 mm inhibition zone. This difference resulted in a positive AEF value of 0.50 for C. albicans.

Effect of AgNPs on bacterial cell morphology

The SEM imaging revealed clear morphological damage in E. coli cells following AgNP exposure, including cell shrinkage and surface deformation. In contrast, untreated cells maintained smooth and intact structures. The severity of these alterations increased with higher AgNP concentrations, supporting their antimicrobial activity (Fig. 15a, b).