Ecofriendly synthesis of silver nanoparticles using Barleria gibsonii and evaluation of antibacterial antioxidant cytotoxic and catalytic activities.

Introduction
The concept of nanotechnology was first introduced by N. Taniguchi in 1974 at a Tokyo conference, where it was described as the manipulation and processing of matter at the nanoscale levels1. Since then, this area has grown rapidly, with a central focus on the development and use of nanoparticles (NPs) smaller than 100 nm2. The prefix “nano,” originating from Greek and meaning “very small,” reflects the precision with which atoms and molecules can be controlled to synthesize particles within the 1–100 nm range3. Such advancements have influenced a wide range of disciplines, including medicine, pharmaceuticals, biology, chemistry, energy, and materials science3. A particularly notable branch is nanomedicine, which increasingly emphasizes the use of environmentally safe methods for NP synthesis, relying on natural resources such as plants, biopolymers, and microbes as reducing and stabilizing agents4.
The physical and chemical traits of NPs, such as size, shape, and surface composition, are largely determined by the synthesis technique5. Therefore, an ideal synthesis method should be simple, cost-efficient, eco-friendly, and capable of delivering uniform nanoscale particles suitable for targeted applications6. Historically, nanoparticles were unknowingly applied in stained glass, pigments, and construction long before their nanoscale characteristics were scientifically recognized7. With advances in electron microscopy, transition metal NPs became prominent for catalytic and industrial purposes8. Their nanoscale behavior, including quantum effects and a large surface-to-volume ratio, differentiates them from bulk materials and supports their use in medicine, environmental management, and electronics9. Depending on their composition, NPs are typically grouped into four categories: metallic, polymeric, semiconductor, and carbon-based, each offering unique benefits10. However, their capacity to penetrate biological systems raises safety concerns, requiring rigorous environmental and toxicological assessments10,11. Despite these issues, NPs remain central to ongoing technological innovation10.
Nanoparticles are also categorized as inorganic, organic, or carbon-based depending on their structure12. Among various fabrication approaches, biological or “green” synthesis has received significant interest as a sustainable, safer alternative13. This method avoids the use of harmful chemicals, resulting in biocompatible nanoparticles with a range of morphologies and properties14. Biological systems such as bacteria, fungi, algae, and higher plants, which play a vital role in NP synthesis through enzymatic and biomolecular processes involving proteins, alkaloids, and phenolics15–17. Compared to chemical methods, biologically synthesized NPs generally possess reduced toxicity and improved compatibility with biological systems, making them highly relevant in medicine18. For example, bacteria and actinobacteria can tolerate metals and effectively mediate nanoparticle synthesis19, while yeasts, algae, and fungi contribute metabolites and pigments that are useful for nanoparticle stabilization, although pathogenic fungi may limit their safe use20–23.
Biological synthesis of nanoparticles presents several advantages: it is affordable, eco-conscious, scalable, and enables functional versatility. Natural sources, including plants and microorganisms, offer readily available raw materials24–26, and the synthesis approach yields biocompatible nanoparticles suitable for pharmaceutical applications27. Additionally, it allows reproducible large-scale synthesis28 and enhances stability and biological activity through natural biomolecular coatings26.
Among green-synthesized metallic NPs, silver nanoparticles (AgNPs) have gained exceptional attention due to their strong antimicrobial effects, which make them valuable in agriculture, food preservation, and healthcare29,30. Their compatibility further enables use in targeted drug delivery31, while their catalytic activity supports environmental applications such as degradation of pollutants32. Moreover, the tunable optical and electronic properties of these nanoparticles enhance their applicability in sensors and electronic devices33.
The genus Barleria (Acanthaceae), first described by Linnaeus in 1753, ranks as the third-largest genus in the family and is distributed across Asia, Africa, and the Americas34,35. Several Barleria species are pharmacologically recognized for their antimicrobial, anti-inflammatory, anticancer, hepatoprotective, antidiabetic, and antiviral activities36. These biological properties are largely attributed to the abundance of phenolics, flavonoids, iridoids, and other redox-active secondary metabolites reported across the genus, which are known to play a crucial role in metal ion reduction and stabilization during the synthesis of green nanoparticles. Among noble metals, AgNPs stand out due to their distinctive optical behavior, ease of functionalization, and strong antimicrobial potential37. Their nanoscale dimensions and large surface area facilitate effective cellular interactions, leading to enhanced biological performance38. Despite extensive reports on plant-mediated AgNP synthesis from various medicinal plants, many studies remain limited to basic synthesis and antimicrobial screening, with insufficient emphasis on systematic optimization, comprehensive physicochemical characterization, and integrated evaluation of multifunctional biological and catalytic activities. Although zinc oxide nanoparticles (ZnO NPs) have been synthesized from B. gibsonii39, their phytochemical potential has not yet been exploited for the green synthesis of silver nanoparticles, representing a clear and unexplored gap in current nanobiotechnology research.
In this context, Barleria gibsonii was strategically selected as a bioresource due to its underexplored phytochemical profile relative to other well-studied Barleria species, despite its reported medicinal relevance. This study addresses a significant knowledge gap by presenting, for the first time, a comprehensive investigation into the eco-friendly synthesis of AgNPs using the aqueous leaf extract of B. gibsonii. Following phytochemical profiling of the extract and systematic optimization of synthesis parameters, the resulting nanoparticles were subjected to detailed physicochemical characterization (UV–Vis, FT-IR, XRD, TEM, SEM, EDX, DLS, and zeta potential). Their multifunctional performance was rigorously evaluated through antibacterial, antioxidant, and cytotoxic activity against MCF-7 breast cancer cells, along with catalytic degradation of methylene blue. By integrating phytochemical profiling with green nanomaterial synthesis and systematic functional evaluation, this study extends existing plant-mediated AgNP research and highlights B. gibsonii as a promising, yet underexplored, bioresource for sustainable nanotechnological applications.

Methodology

Chemicals and reagents
All chemicals and reagents were of analytical or extra-pure grade. Silver nitrate (AgNO₃, analytical grade) was obtained from Alpha Chemical. Cell-culture components, including MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], nutrient agar, nutrient broth, and Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), along with penicillin–streptomycin, trypsin, and hydrochloric acid (HCl), were supplied by HiMedia Laboratories (India). DPPH (2,2-diphenyl-1-picrylhydrazyl, 95% purity), phosphate-buffered saline (PBS), methylene blue (1% aqueous solution), sodium borohydride (NaBH4), sodium hydroxide (NaOH), and ascorbic acid were procured from SRL Chemicals. Ampicillin and paclitaxel were purchased from a licensed pharmacy. Unless otherwise stated, all solutions were freshly prepared using deionized water.

Plant collection and identification
Fresh samples of Barleria gibsonii Dalzell (Acanthaceae) were collected from the wild in Gujarat, India, with permission from the Gujarat Biodiversity Board, Gandhinagar (Permission Ref. No. GBB/Legal/T-6/1890/2025-26). The plant was taxonomically identified by Prof. Padmanabhi S. Nagar, Department of Botany, The Maharaja Sayajirao University of Baroda, Vadodara, India. Authentication was confirmed at the BARO Herbarium (Department of Botany, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara-390 002, Gujarat, India). A voucher specimen (Herbarium No. SA-11) has been deposited in the BARO Herbarium under accession number AN30787.

Preparation of B. gibsonii extract
Fresh leaves of B. gibsonii were thoroughly rinsed with distilled water to remove surface contaminants, dried in the shade, and pulverized into a fine powder. A total of 100 g of this powdered material was mixed with 200 mL of deionized water and maintained at 60 °C for 30 min under continuous magnetic stirring. The suspension was then passed through a Whatman No. 1 filter paper, and the clear extract was collected and stored at − 4 °C until further use.

Phytochemical analysis

Qualitative phytochemical screening
The qualitative phytochemical screening of the B. gibsonii aqueous extract was performed following the procedures described in our previous study, Ali et al. 202540 and Shaikh et al. 202041.

Quantitative phytochemical analysis
The total phenolic and flavonoid contents of the B. gibsonii aqueous extract were determined using our previously reported method40. The total phenolic content (TPC) was expressed as milligrams of gallic acid equivalents per gram of dry extract (mg GAE/g), while the total flavonoid content (TFC) was expressed as milligrams of quercetin equivalents per gram of dry extract (mg QE/g).

Optimization of B.gb-AgNPs synthesis conditions
The synthesis parameters for the B. gibsonii-mediated silver nanoparticles (B.gb-AgNPs) were systematically optimized to achieve reproducible nanoparticle formation. A range of conditions was investigated to determine their impact on the process. Specifically, the influence of varying silver nitrate (AgNO₃) concentrations (1.25, 2.5, 5, 10, and 20 mM) and extract-to-AgNO₃ ratios (1:1 to 1:5) was evaluated. The optimal pH was assessed by testing values of 3, 6, 7, 8, and 10, with adjustments made using 0.2 M NaOH or 0.2 M HCl. Incubation temperature was also a variable, with trials conducted at 25 °C, 37 °C, 60 °C, and 90 °C, and incubation periods ranged from 10 to 180 min. The success of nanoparticle synthesis was determined by analyzing the absorbance spectra between 300 and 800 nm using a UV–Visible spectrophotometer (Agilent Cary 60)42,43.

Green synthesis of B.gb-AgNPs
B.gb-AgNPs were synthesized by combining 100 mL of B. gibsonii leaf extract with 100 mL of 5 mM AgNO₃ at pH 8. The reaction proceeded at 90 °C for 3 h with magnetic stirring in subdued light. A characteristic color change to dark brown indicated Ag⁺ ion reduction. After a subsequent 24 h incubation in darkness at room temperature, the nanoparticles were pelleted by double centrifugation (10,000 rpm, 10 min), washed with acetone, and dried. The synthesis yield was calculated based on the dry weight of the final product43,44.

Characterization of B.gb-AgNPs

UV–Visible spectrophotometric analysis
The evaluation of optical characteristics of metallic nanoparticles through UV–Visible spectrophotometry serves as a conventional approach, offering insights into their dimensions, configuration, and density. This method records absorption within the 200–800 nm spectrum, where silver nanoparticles commonly display a unique surface plasmon resonance (SPR) band. This SPR peak, resulting from the coordinated vibration of conduction electrons, is particularly responsive to nanoparticle size and structure, serving as an effective marker for AgNP synthesis. In this investigation, the synthesis of B.gb-AgNPs was monitored at room temperature (25 °C) using a UV–Vis spectrophotometer (Agilent Cary 60 model), with spectra recorded over the 300–700 nm range. The formation and optical stability of the AgNPs were confirmed by the appearance of a characteristic surface plasmon resonance (SPR) absorption band in the 400–450 nm region.45.

Fourier transform infrared (FT-IR) spectroscopic analysis
Fourier transform infrared (FT-IR) spectroscopy was employed to identify the functional groups associated with phytochemical biomolecules involved in the reduction and stabilization of B.gb-AgNPs. Finely powdered, dried nanoparticle samples (1–2 mg) were thoroughly mixed with spectroscopic-grade potassium bromide (KBr, 100 mg) and compressed into pellets. FT-IR spectra were recorded using a Bruker Alpha FT-IR spectrometer operating in transmission mode, over the 500–3500 cm⁻1 wavenumber range at a spectral resolution of 4 cm⁻1, with 32 scans accumulated per sample. Data acquisition and processing were carried out using OPUS software. The observed absorption bands were assigned to characteristic functional groups responsible for phytochemical capping and stabilization of the silver nanoparticles.43,45,46

X-ray diffraction (XRD) analysis
The crystalline structure and phase composition of B. gibsonii-mediated silver nanoparticles (B.gb-AgNPs) were analyzed using X-ray diffraction (XRD). Dried nanoparticles were finely powdered and examined with an X’Pert PRO Powder Diffractometer operated at 40 mA and 45 kV, using Cu Kα radiation (λ = 1.54060 Å). Diffraction data were collected over a 2θ range of 5.00–89.99°, with a step size of 0.038° and a step time of 5.715 s. Samples were mounted on a flat holder and analyzed at room temperature (25 °C) under position-sensitive detector (PSD) mode, with a divergence slit of 0.87° and a specimen length of 10 mm.
The average crystallite size of the nanoparticles was calculated using the Debye–Scherrer Eq. (1):where D is the mean crystallite size, K is the Scherrer constant (0.94), λ is the X-ray wavelength (0.1546 nm), β represents the full width at half maximum (FWHM) of the diffraction peak obtained via Gaussian fitting, and θ is the Bragg angle43,44.

Transmission electron microscopy (TEM) analysis
Transmission Electron Microscopy (TEM) was used to examine the morphology, size, and crystallinity of B. gibsonii-mediated silver nanoparticles (B.gb-AgNPs). The analysis was performed using a Tecnai G2 20 S-TWIN (FEI) transmission electron microscope operated at an accelerating voltage of 200 kV, providing high-resolution imaging. For imaging, a sample aliquot was drop-cast onto a carbon-film-coated copper grid, followed by solvent evaporation under ambient atmosphere. TEM imaging provided detailed information on particle shape, size distribution, dispersion, and nanoscale structural characteristics. Furthermore, Selected Area Electron Diffraction (SAED) patterns obtained from TEM analysis confirmed the crystalline nature of the nanoparticles, exhibiting well-defined diffraction rings corresponding to specific lattice planes47.

SEM–EDX analysis
The elemental composition and surface morphology of the synthesized B.gb-AgNPs were examined using a scanning electron microscope equipped with an energy-dispersive X-ray spectroscopy (SEM–EDX) system. SEM and EDX analyses were performed on a Hitachi SU3800 Hi-SEM (Hitachi High-Tech India Pvt. Ltd.) fitted with an Oxford Xplore EDS detector. Before analysis, the nanoparticle samples were sputter-coated with a thin layer of gold to enhance surface conductivity. Elemental spectra were acquired at an accelerating voltage of 30 kV, and qualitative and quantitative elemental compositions were obtained from the recorded spectra48.

Dynamic light scattering (DLS) and Zeta potential analysis
Dynamic Light Scattering (DLS) analysis was conducted on an aqueous suspension of B. gb-AgNPs to measure the hydrodynamic diameter, polydispersity index (PDI), and particle size distribution. The surface charge of the B.gb-AgNPs was measured using zeta potential to assess their colloidal stability44,49.

Minimum inhibitory concentration (MIC) of B.gb-AgNPs
The minimum inhibitory concentration (MIC) was determined using a standardized broth microdilution assay in a 96-well plate. Briefly, 100 µL of Luria–Bertani (LB) broth was dispensed into each well. Test agents—B.gb-AgNPs, ampicillin (positive control), and B. gibsonii extract—were serially diluted two-fold across the plate. Each well was then inoculated with 20 µL of a standardized bacterial suspension (OD600 = 0.5–1.0). Following 24 h of incubation at 37 °C, 20 µL of resazurin solution (0.015% w/v) was added to each well as a metabolic indicator. The MIC was defined as the lowest concentration at which no color change from blue (oxidized, non-metabolized) to pink (reduced, metabolically active) was observed after a further incubation period43.

Antimicrobial activity of B.gb-AgNPs
The antimicrobial potential of B. gibsonii-mediated silver nanoparticles (B.gb-AgNPs) was evaluated using the agar well diffusion method in Staphylococcus aureus (Gram-positive), Pseudomonas stutzeri, and Escherichia coli (Gram-negative)50. Test concentrations of B.gb-AgNPs ranged between 125 and 500 µg/mL, B. gibsonii extract at 5000 µg/mL, with ampicillin employed as the reference standard at 125 µg/mL. Following incubation at 37 °C for 24 h, the diameters of inhibition zones were measured to assess antibacterial activity51.

Antioxidant potential of B.gb-AgNPs
The antioxidant activity of B. gibsonii-mediated silver nanoparticles (B.gb-AgNPs) was evaluated using the DPPH free radical scavenging assay, with ascorbic acid (AA) as the reference standard52. The assay was conducted in a 96-well microplate format. Test solutions of B.gb-AgNPs, B. gibsonii extract, and AA were prepared in methanol at concentrations ranging from 6.25 to 200 μg/mL. Each well received 100 μL of the test sample or standard, while control wells contained methanol alone. Subsequently, 100 μL of freshly prepared 0.1 mM methanolic DPPH solution, protected from light, was added. Methanol served as the blank for the control. The mixtures were incubated in the dark at room temperature for 30 min, after which the absorbance was measured at 517 nm using a Synergy HTX multimode plate reader. The percentage of radical scavenging activity was calculated using Eq. (2):where the control absorbance represented the DPPH–methanol system and the sample absorbance denoted DPPH in the presence of B.gb-AgNPs. All experiments were conducted in triplicate53.

Total antioxidant capacity (TAC)
The total antioxidant capacity (TAC) of the plant extract, nanoparticles, and ascorbic acid (AA) was assessed using the phosphomolybdate method. The reaction mixture contained 0.6 M sulfuric acid, ammonium molybdate, and sodium phosphate. Sample solutions at concentrations ranging from 62.5 to 1000 µg/mL were incubated with the reagent for 90 min at 95 °C. Following cooling, absorbance was measured at 695 nm. Ascorbic acid was used as the reference standard, and TAC values were expressed as ascorbic acid equivalents (µg/mL) after baseline correction with reagent blanks54,55.

Cytotoxicity of B.gb-AgNPs
The cytotoxic potential of B. gibsonii-mediated silver nanoparticles (B.gb-AgNPs) was evaluated on MCF7 breast cancer cells using the MTT assay. MCF-7 cells procured from NCCS, Pune, were grown in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin, and maintained at 37 °C in a humidified incubator with 5% CO2. Cell growth and sterility were regularly monitored under a microscope43.
For the assay, cells were seeded in 96-well plates at a density of 1 × 104 cells per 100 µL per well. After 48 h, the culture medium was replaced, and cells were treated with varying concentrations of B.gb-AgNPs, and B. gibsonii extract (7.8–250 µg/mL), while control wells received PBS. Following 24 h of exposure, 10 µL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated for 4 h. The medium was then removed, and the resulting formazan crystals were dissolved in 100 µL of DMSO. Absorbance was measured at 570 nm using a Synergy HTX multimode plate reader56. The OD was used to determine cell viability via Eq. (3):
OD of the sample represents the treated group, while OD of the control shows the untreated group’s average optical density (OD)57.

Catalytic activity of B.gb-AgNPs
To determine the catalytic potential of the B.gb-AgNPs, we observed their ability to reduce methylene blue (MB) with NaBH4 as a reducing agent. In a 3 mL quartz cuvette, 2 mL of deionized water was mixed with 100 µL of 0.1 mM MB solution, followed by the addition of 500 µL of freshly prepared 0.1 M NaBH₄. Subsequently, 50 µL of B.gb-AgNPs was added, and the progress of MB degradation was monitored using UV–Visible spectroscopy44,58.

Statistical analysis
All experimental data were processed using Excel, GraphPad Prism 9, and OriginPro 2018. Values are presented as mean ± standard deviation (SD) from three independent experiments. Group comparisons were conducted using Student’s t-test and one-way ANOVA, with differences considered statistically significant at p < 0.05.

Results and discussion

Phytochemical analysis

Qualitative phytochemical screening
The qualitative phytochemical profile of the aqueous extract of B. gibsonii (Table 1) showed the presence of a rich profile of phenolic compounds, which are essential in the green synthesis of the silver nanoparticles (B.gb-AgNPs). The extract had a high presence (+ + +) of phenols, moderate presence (+ +) of flavonoids and tannins, and weak presence ( +) of saponins. Alkaloids and terpenoids were not observed.
This phytochemical structure provides a mechanistic explanation for nanoparticle synthesis and stabilization. The hydroxyl groups of phenols, flavonoids, and tannins provide them with the capacity to reduce Ag+ ions to Ag059. This high rate of efficient synthesis is presumably due to their concerted action. Additionally, these polyphenolic compounds, along with saponins, can be utilized as capping agents, releasing onto the surface of nanoparticles to inhibit aggregation and maintain them in a state of colloidal suspension60. Reduction and stabilization are mainly mediated by the polyphenolic constituents, which are ensured by the absence of alkaloids and terpenoids. Comprehensively, the phytochemical profile confirms the traditional application of B. gibsonii and is the reason why it can be successful in the synthesis of stable, bioactive B.gb-AgNPs that have potential biocompatibility improvements in the future61.

Quantitative phytochemical analysis
The quantitative evaluation of the aqueous B. gibsonii leaf extract indicated high levels of phenolic and flavonoid compounds. The calibration curves for quercitrin and gallic acid standards demonstrated excellent linearity for total flavonoid content (TFC) (R2 = 0.9824) and for total phenolic content (TPC) (R2 = 0.9938), respectively (Fig. 1a and b). Based on these standard plots, the extract exhibited a TFC of 38.2 mg quercetin equivalent (QE)/g extract and, a TPC of 70.5 mg gallic acid equivalent (GAE)/g extract and (Fig. 1c). These findings align with the qualitative phytochemical screening and confirm the extract’s strong reducing and stabilizing capacity in the biosynthesis of B. gibsonii-mediated silver nanoparticles (B.gb-AgNPs). The presence of phenolic hydroxyl groups contributed to the reduction of Ag+ to Ag0, while polyphenols and tannins acted as capping and stabilizing agents, preventing nanoparticle aggregation. This phytochemical composition establishes B. gibsonii extract as an effective green reducing agent and suggests that the synthesized nanoparticles may retain the antioxidant characteristics of their phytochemical precursors62.

Optimization of B.gb-AgNPs synthesis parameters
The effects of various parameters on the biosynthesis of silver nanoparticles (B.gb-AgNPs) were investigated under different conditions to achieve a favorable maximum yield of B.gb-AgNPs based on the UV‒Vis results. We started to determine the optimal concentration of AgNO3 for the synthesis of B.gb-AgNPs, and a concentration of 5 mM was determined to be the best concentration with high absorbance at 420 nm (Fig. 2a). Additionally, we obtained a ratio of 1:1 (AgNO3 to the plant extract is a favorable ratio compared with other ratios) (Fig. 2b). Furthermore, the pH of the mixture was adjusted, and pH 10 was the best pH for the synthesis of B.gb-AgNPs, as shown in Fig. 2c. The enhanced nanoparticle synthesis under alkaline conditions can be attributed to increased deprotonation of phenolic and hydroxyl groups in the plant extract, which improves their electron-donating capacity and facilitates more efficient reduction of Ag⁺ ions, while also promoting nanoparticle stabilization. Moreover, 90 °C was the most suitable temperature for synthesizing B.gb-AgNPs (Fig. 2d). Elevated temperature accelerates reaction kinetics, enhances nucleation rates, and promotes uniform nanoparticle growth, resulting in a more intense and stable SPR band. In conclusion, the optimal parameters for the biosynthesis of B.gb-AgNPs were determined to be 5 mM AgNO3, a 1:1 plant extract-to-precursor ratio, pH 10, and a temperature of 90 °C. These conditions collectively produced nanoparticles with a characteristic and intense SPR absorption, confirming a high yield of synthesized nanoparticles43.

Characterization of B.gb-AgNPs

UV–Vis spectrophotometry
The addition of B. gibsonii leaf extract to AgNO₃ solution induced a visible color change from translucent to dark brown, signifying the reduction of silver ions and synthesis of silver nanoparticles (AgNPs). UV–Visible spectral analysis further validated nanoparticle synthesis and stability, revealing a characteristic SPR peak at 420 nm, which is typical for AgNPs (Fig. 3a)63. The characteristic SPR band at 420 nm, which intensified and stabilized as the reaction proceeded, confirms the time-dependent synthesis and final stabilization of AgNPs, indicating the completion of the reduction process64.

FT-IR analysis
FT-IR spectroscopy was performed to identify the functional groups responsible for the bioreduction and stabilization of B. gibsonii-mediated silver nanoparticles (B.gb-AgNPs). The spectrum recorded in the range of 400–4000 cm−1 displayed characteristic absorption bands corresponding to phytochemicals from the plant extract (Fig. 3c). The major peaks were observed at 3431.96, 2925.54, 2851.96, 1723.22, 1629.05, 774.61, and 608.74 cm−165. The broad band at 3431.96 cm−1 corresponds to O–H stretching vibrations of hydroxyl groups, mainly from phenols and alcohols. These hydroxyl groups are strong reducing agents, where their oxidation to carbonyl groups donates electrons for the reduction of Ag⁺ to Ag⁰. Peaks at 2925.54 cm−1 and 2851.96 cm−1 represent C–H stretching of aliphatic hydrocarbons, suggesting the presence of long-chain organic molecules66. These, along with the C=O stretching band at 1723.22 cm−1 (from oxidized groups or inherent esters/carboxylic acids) and the N–H/C=C features at 1629.05 cm⁻1 (from amides/proteins), are instrumental in stabilization67. They create a protective organic layer around the nascent nanoparticles via steric hindrance and potential electrostatic interactions, preventing aggregation and ensuring colloidal stability. The peaks at 774.61 cm−1 and 608.74 cm−1 correspond to C–Cl bending vibrations, suggesting the presence of halogenated secondary metabolites40,68. These spectral features confirm a dual role for the phytoconstituents: hydroxyl-rich phenolics and flavonoids primarily drive the bioreduction, while proteins, aliphatic chains, and carbonyl-containing compounds form a stabilizing capping layer through surface coordination and functionalization69.

XRD analysis
X-ray diffraction (XRD) was used to determine the crystalline structure of the B.gb-AgNPs. The resulting diffractogram (Fig. 3b) displays four prominent diffraction peaks, which are well-matched to the (111), (200), (220), and (311) planes of face-centered cubic (fcc) metallic silver (JCPDS file no. 04–0783). No diffraction peaks corresponding to common silver oxide phases (e.g., Ag₂O, AgO) or other crystalline impurities were detected, confirming the high phase purity of the synthesized AgNPs. The average crystallite size, calculated using the Debye–Scherrer equation, was 41.65 ± 6.48 nm (Table 2). The minor, low-intensity peaks (marked with asterisks) are attributed to the crystalline bio-organic compounds from the B. gibsonii extract acting as capping and stabilizing agents, and do not indicate secondary metallic or oxide phases. The particle size estimated from XRD showed good correlation with TEM observations, confirming the nanoscale dimensions of the synthesized AgNPs43.

TEM analysis
Transmission electron microscopy (TEM) images at different magnifications (Fig. 4a, b) revealed that the B.gb-AgNPs were primarily spherical and well dispersed. The selected area electron diffraction (SAED) pattern (Fig. 4c) exhibited concentric rings with distinct diffraction spots, further confirming the crystalline nature of the nanoparticles, in agreement with XRD results. Analysis of the particle distribution showed an average particle size of 39.6 ± 12.4 nm (Fig. 4d).

SEM–EDX analysis
The morphology and elemental composition of the B.gb-AgNPs were analyzed by scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM–EDX). A representative SEM micrograph (Fig. 5a; 25.0 kV, 10.0 k magnification) shows discrete, quasi-spherical nanoparticles with partial aggregation, a typical outcome of biogenic synthesis due to high surface energy and phytochemical capping. The primary particle size is consistent with TEM data and XRD size. EDX analysis of the same region (Fig. 5b) confirms the predominant presence of silver (Ag: 55.2 wt%, peak at ~ 3.0 keV). Signals from carbon (28.0 wt%) and oxygen (11.7 wt%) are attributed to surface-bound phytochemicals, while a minor gold signal (5.1 wt%) originates from the sputter coating. The absence of other elemental peaks indicates high-purity B.gb-AgNPs, corroborating the XRD results and confirming that silver nanoparticles are stabilized by plant-derived organics 43,48.

DLS and zeta potential
Dynamic Light Scattering (DLS) was used to determine the hydrodynamic diameter of the biosynthesized B.gb-AgNPs. The analysis revealed a Z-average of 175.0 nm and featured a peak at 139.3 nm (Fig. 6a), with a PDI of 0.2, indicating a narrow and nearly monodisperse size distribution. The hydrodynamic diameter obtained by DLS is inherently larger than the metallic core size observed by TEM. This expected discrepancy arises from the different measurement principles, as DLS reports the apparent size of solvated nanoparticles undergoing Brownian motion, which includes the inorganic core, the organic capping layer, and the associated hydration shell, whereas TEM and XRD determine only the dehydrated crystalline silver core. The enlarged hydrodynamic shell is attributed to surface-bound phytoconstituents from the plant extract, such as polyphenols, flavonoids, and proteins, which act as capping and stabilizing agents and are essential for colloidal stability70.
Zeta potential analysis was performed to assess surface charge and stability. The B.gb-AgNPs exhibited a zeta potential of − 25 mV, indicating a moderately high negative surface charge (Fig. 6b). This negative potential generates electrostatic repulsion between particles, thereby preventing aggregation and enhancing long-term dispersion stability. Phytochemicals, particularly flavonoids, phenolics, and carboxylic acid-containing compounds, are likely responsible for both the reduction of Ag⁺ ions to Ag⁰ and the capping of nanoparticles, contributing significantly to their stability71.

Minimum inhibitory concentration (MIC) of B.gb-AgNPs
The minimum inhibitory concentration (MIC) of B. gibsonii–mediated silver nanoparticles (B.gb-AgNPs) was determined against E. coli, S. aureus, and P. stutzeri using a broth microdilution assay with resazurin as a metabolic indicator (Fig. 7a–c). Following 24 h incubation and resazurin addition, the MIC was recorded as the lowest concentration at which no color change from blue to pink was observed.
B.gb-AgNPs inhibited bacterial growth at MIC values of 15.6 µg/mL for E. coli, 31.2 µg/mL for S. aureus, and 15.6 µg/mL for P. stutzeri (Fig. 7b). The Gram-negative strains exhibited lower MIC values than the Gram-positive S. aureus, indicating higher susceptibility. These results demonstrate species-dependent antibacterial activity of B.gb-AgNPs against both Gram-positive and Gram-negative bacteria.

Antibacterial activity of B.gb-AgNPs
The antibacterial activity of B.gb-AgNPs was evaluated against Gram-positive S. aureus and Gram-negative P. stutzeri and E. coli using the agar well diffusion method, with ampicillin as the reference antibiotic. Nanoparticles were tested at concentrations (500, 250, 125) µg/mL (Fig. 8a–c). Figure 8d shows the mean inhibition zone diameters (mm) of B.gb-AgNPs, B. gibsonii extract and ampicillin against different bacterial strains. The diameters of inhibition zones increased with concentration, indicating a dose-dependent antibacterial effect. Among the tested bacterial strains, S. aureus demonstrated the highest sensitivity to both ampicillin and B.gb-AgNPs at 500 µg/mL. In contrast, P. stutzeri showed comparatively lower susceptibility to these treatments but greater sensitivity to the crude plant extract. Notably, E. coli exhibited increased vulnerability at lower B.gb-AgNP concentrations (125–250 µg/mL). These results demonstrate that B.gb-AgNPs possess notable antibacterial potential, primarily mediated through a concentration-dependent mechanism. The biosynthesized B.gb-AgNPs exhibited antibacterial efficacy comparable to the standard antibiotic ampicillin. The precise antibacterial mechanism of B.gb-AgNPs is still not fully established. However, several potential mechanisms have been proposed, including the formation of reactive oxygen species (ROS), the release of silver ions that interact with sulfhydryl groups of proteins leading to structural damage, and the attachment of AgNPs to bacterial cell membranes, resulting in membrane disruption and cell death43. The differential antibacterial efficacy can be rationalized by considering bacterial cell wall architecture. The pronounced activity against S. aureus (Gram-positive) likely stems from the direct interaction of AgNPs with its thick, yet porous, peptidoglycan layer, facilitating membrane disruption. For Gram-negative strains (E. coli, P. stutzeri), the complex outer membrane presents a greater initial barrier to nanoparticle penetration; the significant activity observed, particularly against E. coli, suggests the bio-capped B.gb-AgNPs effectively compromise this barrier. The collective antibacterial mechanism is therefore attributed to a synergy of membrane damage, sustained Ag⁺ ion release, and the induction of oxidative stress, leading to the concentration-dependent bacterial inhibition demonstrated in this study72.
In addition to these mechanisms, the antibacterial efficacy of B.gb-AgNPs is strongly influenced by their nanoscale dimensions and surface characteristics. The relatively small particle size increases the specific surface area, enhancing direct contact with bacterial cell envelopes and facilitating more efficient Ag+ ion release. Moreover, surface-bound phytochemicals acting as capping agents improve nanoparticle stability and may promote stronger electrostatic interactions with negatively charged bacterial membranes, thereby amplifying membrane disruption and intracellular penetration. These size- and surface-dependent effects collectively rationalize the pronounced, concentration-dependent inhibition zones observed in the present study73.

Antioxidant potential of B.gb-AgNPs
The antioxidant capacity of B.gb-AgNPs was assessed using the DPPH free radical scavenging assay, with ascorbic acid employed as the standard reference. The nanoparticles exhibited a concentration-dependent increase in radical scavenging activity across the tested range of 6.25–200 μg/mL. At the highest concentration (200 μg/mL), B.gb-AgNPs achieved a maximum inhibition of 78.68% (Fig. 9a). The IC₅₀ value was determined to be 89.5 μg/mL, compared to 24.4 μg/mL for ascorbic acid and 58.3 μg/mL for plant extract (Fig. 9b)74.

Total antioxidant capacity (TAC)
The total antioxidant capacity of B. gibsonii aqueous extract and its silver nanoparticles (B.gb-AgNPs) was determined using the phosphomolybdate assay, with results expressed as ascorbic acid equivalents (AAE, µg/mL). Both samples displayed a concentration-dependent increase in antioxidant activity across the tested range of 62.5–1000 µg/mL. At the highest concentration (1000 µg/mL), B. gibsonii extract showed the maximum activity of 679.79 ± 5.9 µg/mL AAE, while B.gb-AgNPs exhibited 434.73 ± 35.7 µg/mL AAE. Antioxidant capacity gradually decreased with lower concentrations (Table 3). At all tested levels, the aqueous extract consistently demonstrated higher AAE values than the nanoparticles, indicating stronger antioxidant potential. These results suggest that although both the extract and nanoparticles possess considerable antioxidant activity, the aqueous extract of B. gibsonii remains a more effective natural antioxidant source75.

Cytotoxicity of B.gb-AgNPs
The anticancer potential of B. gibsonii-derived silver nanoparticles (B.gb-AgNPs) was assessed in the MCF-7 breast cancer cell line using the MTT assay. The nanoparticles exhibited a pronounced dose-dependent cytotoxic effect, with cell viability significantly decreasing at concentrations of 7.8–250 µg/mL (Fig. 10a). The IC50 value was determined to be 28.93 µg/mL, reflecting strong cytotoxic activity. For comparison, paclitaxel (IC50 = 18.0 µg/mL) and the plant extract (IC50 = 99.7 µg/mL) were included as controls, highlighting the superior potency of B.gb-AgNPs (Fig. 10b).
Cell morphology analysis, as shown in the accompanying Fig. 11, revealed progressive morphological changes with increasing B.gb-AgNPs concentrations (0–250 µg/mL). The control (PBS) displayed intact, confluent cell layers, while treated cells exhibited dose-dependent alterations, including cell shrinkage, membrane blebbing, and reduced density, indicative of apoptosis and necrosis at higher doses (e.g., 125–250 µg/mL). These morphological shifts corroborate the cytotoxicity data, suggesting that B.gb-AgNPs induce cell death by interfering with critical cellular proteins and causing metabolic disruptions. These in vitro results indicate that B.gb-AgNPs exhibit cytotoxic effects in breast cancer cells. However, further studies are required to elucidate their mechanisms of action and to evaluate in vivo efficacy before any therapeutic relevance can be established.
The B.gb-AgNPs exhibited a markedly lower IC50 value (28.9 µg/mL) than the corresponding crude plant extract (99.7 µg/mL), indicating a substantial enhancement of cytotoxic activity following nanoparticle synthesis. Furthermore, when compared with previously reported plant-mediated AgNPs evaluated against MCF-7 cells (Table 4), the B.gb-AgNPs demonstrated competitive cytotoxic potency, surpassing several reported systems and approaching the activity of other plant-derived silver nanoparticles.
The observed morphological alterations, including cell shrinkage and membrane blebbing at higher concentrations, are consistent with cytotoxic stress and have been widely associated with apoptotic-like cell death in AgNP-treated cancer cells. However, since no molecular or biochemical assays were performed in the present study, these observations should be considered indicative rather than confirmatory. The enhanced cytotoxicity of B.gb-AgNPs may plausibly involve oxidative stress-mediated pathways, mitochondrial dysfunction, or DNA damage, as reported for other plant-mediated AgNP systems, warranting further validation through ROS quantification and apoptosis-specific assays.

Catalytic activity of B.gb-AgNPs
The catalytic performance of B. gibsonii-mediated silver nanoparticles (B.gb-AgNPs) was evaluated through the reduction of methylene blue (MB) using sodium borohydride (NaBH4) as a reducing agent. The reaction progress was monitored using UV–Vis spectroscopy at 30 s intervals, from 30 up to 840 s, to observe the progressive decrease in the characteristic absorption peak of MB.
In the presence of B.gb-AgNPs, the degradation of MB was significantly accelerated, achieving complete decolorization within 840 s (14 min) (Fig. 12a). By contrast, MB reduction in the absence of nanoparticles typically requires a long time under comparable conditions as shown in Fig. 12b. These findings demonstrate the strong catalytic capability of B.gb-AgNPs, highlighting their potential as effective and eco-friendly nanocatalysts for environmental remediation applications.
Based on the experimental results, the catalytic performance of B. gibsonii-mediated silver nanoparticles (B.gb-AgNPs) was demonstrated to be highly effective, facilitating the complete reduction of methylene blue within 14 min. This result aligns favorably with the efficiencies reported for other biogenic silver nanoparticles in previous studies. The performance of B.gb-AgNPs is superior to several other systems, such as those derived from Gmelina arborea or Millettia peguensis, which require 30 min80,81. Furthermore, the degradation time is faster than the 17 min reported for Barleria prattensis AgNPs43 and is comparable to other reported agents from Sargassum coreanum, which achieved decolorization in 20 min82. The 14 min efficiency of B.gb-AgNPs therefore positions them competitively, confirming their strong inherent catalytic potency. These results indicate that B.gb-AgNPs exhibit catalytic activity toward methylene blue degradation, warranting further investigation for potential dye removal applications.

Limitations and future perspectives
This study provides valuable insights into the green synthesis and multifunctional behavior of B.gb-AgNPs; nevertheless, its findings are constrained by several key aspects that define the scope of the present work. The proposed mechanisms of antibacterial and cytotoxic action (e.g., ROS generation, membrane disruption) require direct validation through targeted molecular assays. The anticancer evaluation was conducted exclusively in vitro using a single breast cancer cell line (MCF-7; IC50 = 28.9 µg/mL), with untreated cells serving as the negative control. Evaluation of cytotoxic selectivity against normal human cells was not feasible due to the unavailability of specialized culture media, growth factors, and supplements required for maintaining non-tumorigenic cell lines. Consequently, the therapeutic index and cancer selectivity of B.gb-AgNPs could not be determined in the present study. The catalytic application lacks analysis of long-term nanoparticle stability and environmental impact, including catalyst recyclability and reusability over multiple cycles, as well as evaluation against additional model pollutants such as 4-nitrophenol, and the synthesis process was performed on a laboratory scale. The potential environmental risks and ecotoxicological effects of nanoparticle release following catalytic application were not assessed. Future studies will therefore include cytotoxic selectivity assessment using normal human cell lines (e.g., HEK-293), along with in vivo validation to establish therapeutic relevance and biosafety, as well as systematic investigations of catalyst durability, recyclability, and extended catalytic performance to improve practical applicability.
Addressing these limitations will require interdisciplinary collaboration among chemists, materials scientists, biologists, and environmental engineers to enable mechanistic validation, biosafety assessment, scalability, and real-world translation of green-synthesized nanomaterials.

Conclusion
This study successfully establishes a sustainable and efficient green synthesis protocol for silver nanoparticles (B.gb-AgNPs) using Barleria gibsonii leaf extract. Qualitative phytochemical screening revealed a rich profile of bioactive compounds, including phenols, flavonoids, tannins, and saponins, which are crucial for nanoparticle synthesis. Subsequent quantitative analysis confirmed high concentrations of phenolic (70.5 mg GAE/g) and flavonoid (38.2 mg QE/g) constituents, which are instrumental as both reducing and capping agents. The successful synthesis of B.gb-AgNPs, achieved through systematic optimization, was initially indicated by a visible color change from light to dark brown and confirmed by a characteristic surface plasmon resonance peak at 420 nm in UV–Vis spectroscopy. A multi-technique characterization approach provided a comprehensive analysis of the nanoparticles. FT-IR spectroscopy identified functional groups from phenolics and flavonoids responsible for the reduction and capping processes. XRD analysis, supported by SAED patterns, confirmed a face-centered cubic crystalline structure with an average crystallite size of 41.65 ± 6.48 nm. TEM imaging revealed predominantly spherical morphology and a consistent core size of 39.6 ± 12.4 nm, validating the nanoscale dimensions. Furthermore, SEM–EDX analysis further verified quasi-spherical morphology and high elemental purity of silver, with surface stabilization by plant-derived organic constituents. Additionally, DLS and zeta potential measurements indicated a hydrodynamic diameter of 175 nm and a surface charge of -25 mV, respectively, confirming the strong colloidal stability of the synthesized nanoparticles. The synthesized B.gb-AgNPs exhibited compelling multifunctional performance. They demonstrated potent, dose-dependent antibacterial activity against Gram-positive (S. aureus) and Gram-negative (Ps. stutzeri and E. coli) bacteria, evidenced by large inhibition zones (19–22 mm at 500 μg/mL and 14–16 mm at 125 μg/mL) together with low minimum inhibitory concentration values (15.6–31.2 µg/mL), indicating strong antibacterial efficacy at relatively low nanoparticle doses. Furthermore, the nanoparticles showed significant bioactivity in therapeutic assays, displaying strong antioxidant capacity (IC50 = 89.5 μg/mL) and pronounced cytotoxicity in MCF-7 breast cancer cells (IC50 = 28.93 μg/mL), notably surpassing the efficacy of the crude plant extract (IC50 = 99.7 μg/mL). Beyond biomedical applications, the nanoparticles functioned as highly efficient nanocatalysts, achieving complete degradation of methylene blue within 14 min, underscoring their potential in environmental remediation.
In conclusion, this work establishes B. gibsonii as a novel and effective bio-resource for nanoparticle synthesis and highlights the potential of B.gb-AgNPs as versatile agents for further development in nanomedicine and environmental technology. As outlined in the limitations, future research is essential to elucidate the molecular mechanisms of action, assess in vivo efficacy and biocompatibility, and develop scale-up strategies to realize this potential.