Eco-friendly one-pot synthesis of silver nanoparticles via microwave irradiation using Artocarpus camansi extract: Characterization, antioxidant, and antibacterial applications

2.1 Tools

The tools utilized in this study include glassware (borosil and pyrex), blender (Philips), Whatman filter paper, Laminar Air Flow Cabinet (Astec HLF 1200L), microplate (Iwaki), Vortex (Vortex V-1plus), centrifuge (Eppendorf centrifuge 5804), micropipette (DragonLAB), analytical balance (Sartorius), magnetic stirrer (IKA ®C-MAG HS-7), microwave (Sharp R-21-DOS), oven (MemmertUN110), liquid chromatography-high resolution mass spectrometry (LC-HRMS) (ThermoScientific), UV-visible spectrophotometer (Shimadzu UV-1800 and Orion Aquamate 8000 UV-Vis), Fourier transform-infrared (FT-IR) spectrophotometer (Shimatzu IRPrestige-21), Particle Size Analyzer (PSA) (Fritsch), Zeta Potential (Nano Particles Analyzer Horibo SZ-100), Scanner Electron Microscope-Dispersive X-ray spectroscopy (SEM-EDX) Spectrum (SEM SU3500), Transmission Electron Microscopy (TEM) (HR TEM H9500) and X-Ray Diffraction (XRD) (Bruker D8 Advance).

2.2 Materials

Kluwih leaves (A. camansi Blanco) were obtained from Sibiru-biru village, Sumatera Utara, Indonesia, and identified at Medanense Herbarium, Universitas Sumatera Utara, on June 12, 2023, with the number 1142/MEDA/2023. Other materials used were deionized water, silver nitrate (AgNO₃) (Smart-Lab), clindamycin (PT Promedraharjo), crystal violet, DPPH (TCL), Folin-Ciocalteu (Sigma), NaCl 0.9 % (Otsuka), quercetin (Sigma), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Propionibacterium acnes (ATCC 11827), and Salmonella typhi (ATCC 6539), nutrient broth, and nutrient agar (Himedia).

2.3 Preparation of the extract

The leaves of A. camansi Blanco were meticulously cleaned using tap water, wet sorted, dried to a brittle consistency in a drying cabinet, and subsequently ground into powder. To prepare the aqueous extract, 10 g of the powder was combined with 1000 mL of deionized water. This mixture was heated for 30 min at 90°C on a hotplate. After cooling, the solution was filtered. Aqueous extract was ready for the synthesis process (Amar et al., 2025).

2.4 LC-HRMS analysis

The phytochemical examination was conducted utilizing the TSQ Exactive technique (Thermo) and an Accucore C-18 column, measuring 100 × 2.1 mm with a particle size of 1.5 µm (ThermoScientific) at AR Laboratory, IPB University. The mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) were employed in a gradient technique. The flow rate was established at 0.2 mL/min, with an injection volume of 2 µL and a duration of analysis of 33 min. The mass spectrometry ionization source utilized was electrospray ionization (ESI) in negative ionization mode, with a mass-to-charge ratio (m/z) range of 100-1500 Da. Additional parameters were established: capillary temperature at 320°C; spray voltage at 3.8 kV; sheath and auxiliary gas flow rates at 15 and 3 mL/min, respectively; separation power at 70,000 FWHM; scan type designated as complete MS/dd MS2. The outcomes were processed and analyzed with CompoundDiscoverer 2.2 software (Waruwu et al., 2025).

2.5 TPC (Total Phenolic Content) analysis

The TPC in ACE samples was measured using the Folin-Ciocalteu method. Briefly, 0.1 mL of ACE was added to the test tube, followed by 7.9 mL of distilled water and 0.5 mL of Folin-Ciocalteu reagent, and stirred for 1 min, and then 1.5 mL of Na₂CO₃ (20%). Then, it was incubated in the dark for 90 min. Further analysis was carried out using a UV-visible spectrophotometer at an absorbance of 775 nm. Gallic acid was used with a series of concentrations (50, 25, 12.5, 6.25, and 3.125 µg/mL) to determine the TPC, carried out in milligram equivalents per gram (mg GAE/g). The data were analyzed in triplicate and presented as the mean ± SD. Total phenolic concentration was evaluated using the equation below:

$\text{TPC}=\frac{\text{C }\times \text{V}\times \text{F}}{\text{M}}$

C = phenolic concentration (µg/mL), V = sample (mL), F = dilution factor, and M = sample weight (g) (Amalia et al., 2024).

2.6 TFC (Total Flavonoids Content) analysis

For this, 2 mL of ACE was combined with 0.1 mL of AlCl₃ (10%), 0.1 mL of CH₃COONa, and 2.8 mL of distilled water. It was then incubated for 40 min at room temperature in the dark and then analyzed using a UV-visible spectrophotometer at an absorbance of 432 nm. Quercetin was used to compare with a series of concentrations (250, 125, 62.5, 31.25, and 15.625 µg/mL) to determine TFC in milligram equivalents per gram of sample (mg QE/g). Data were analyzed in triplicate and presented as the mean ± SD. TFC was evaluated using the following equation:

$\text{TFC}=\frac{\text{C }\times \text{V}\times \text{F}}{\text{M}}$

C = flavonoids concentration (µg/mL), V = sample (mL), F = dilution factor, and M = sample weight (g) (Amalia et al., 2024).

2.7 Synthesis of AgNPs

The synthesis began with the preparation of an AgNO₃ solution (2 mM) by dissolving 0.017 g of AgNO₃ in 500 mL of deionized water. A 50 mL portion of this solution was mixed with 1 mL of ACE and stirred for 15 min. The mixture was then irradiated using a microwave oven at 450 W for three 1-min cycles, with 30-s intervals between each cycle. The intermittent irradiation was intentionally applied instead of continuous exposure to prevent overheating and uncontrolled evaporation of the reaction mixture. Preliminary trials indicated that continuous irradiation at the same power level led to rapid boiling and partial degradation of plant metabolites, which affected nanoparticle stability and size uniformity. In contrast, cyclic irradiation ensured more homogeneous heat distribution and controlled reduction of Ag⁺ to Ag⁰, resulting in well-dispersed nanoparticles. The selected irradiation condition (3 × 1 min, 450 W) was based on preliminary optimization and previous reports on microwave-assisted green synthesis of AgNPs using plant extracts. After irradiation, the solution was centrifuged for 15 min at 8000 rpm, and the obtained precipitates were washed three times with deionized water and dried at 70°C to yield ACE-AgNPs powder (Maryani & Septama, 2022; Amar et al., 2025).

2.8 Characterization of AgNPs

AgNPs from ACE (ACE-AgNPs) were characterized using several instrumental analyses, including UV-Vis spectroscopy, PSA, FT-IR, zeta potential, SEM-EDX, TEM, and XRD. The Shimadzu UV-1800 and Orion Aquamate 8000 UV-Visible were employed to measure the sample's wavelength, with the AgNPs sample exhibiting wavelengths between 400-500 nm. Absorption bands within the 200-800 nm range are typically ideal for particle characterization. Shimadzu IRPrestige-21 was used to identify functional groups in ACE extracts and ACE-AgNPs. AgNPs were monitored over a range from 4000 cm^-1 and 400 cm^-1. Fritsch for analyzing colloidal suspensions of AgNPs in determining the size of the resulting particles. Nano Particle Analyzer Horibo SZ-100 is used for colloidal stability or zeta potential value of nanoparticle aggregation. The SEM-EDX Spectrum (SEM SU3500) is used to analyze shapes, morphology, and validate the presence of silver and other elements in particles. HR TEM Talos F200C for determining the morphology, structure, and size of AgNPs. he SEM and TEM results were analyzed using ImageJ software. XRD Bruker D8 Advance (2θ range of 2–80°) was used to identify the crystal phase and analyze the particle size, and then it was calculated using the Scherrer equation:

$D=\frac{K\lambda }{\beta \cos \theta }$

D = average diameter of ACE-AgNPs, K= constant depends on the crystallite shape (0.9), λ = X-ray wavelength, β = FWHM (full width at half maximum) of XRD diffraction, and θ = diffraction angle, which is also called Bragg angle (Waruwu et al., 2025).

2.9 Antioxidant activity assay

The antioxidant activity of ACE and ACE-AgNP (at concentrations 25, 50, 100, 200, and 400 µg/mL, respectively) was assessed by DPPH assay. Quercetin (0, 0.625, 1.25, 2.5, and 5 µg/mL) was used to compare the results. Next, the DPPH solution was mixed at each concentration and incubated at room temperature in the dark. Then, the absorbance was measured with a spectrophotometer at 516 nm. The determination was performed in triplicate and reported as the average ±SD. These values ​​were then applied to generate the IC₅₀ value, indicating the sample concentration required to scavenge 50% DPPH free radicals (Waruwu et al., 2025).

2.10 Antibacterial activity

The microdilution method determined the minimum inhibitory concentration (MIC) value. First, a bacterial inoculum was prepared and adjusted with sterile NaCl solution to the McFarland 0.5 standard, equivalent to 1×10⁸ CFU/mL. The suspension was then diluted with NaCl to achieve a bacterial concentration of 1×10⁶ CFU/mL. A mixture of media and samples was placed in a 96-well plate, employing a two-fold dilution technique to generate concentrations ranging from 500 µg/mL to 0.9 µg/mL. The bacterial suspension was added to the wells and incubated at 37°C for 24 h. The MIC value was determined by the absence of visible bacterial growth. The Minimum Bacterial Concentration (MBC) value was determined using the streak method. After identifying the wells corresponding to 1 MIC and 2 MIC, the liquid from these wells was transferred onto agar plates using a loop needle and incubated for 24 h at 37°C. The MBC value was indicated by the lack of bacterial growth on the agar medium (Maryani & Septama, 2022; Waruwu et al., 2025). The data were analyzed in triplicate.

2.11 DNA and protein leakage test

The test involved preparing a bacterial suspension, which was incubated for 24 h. Afterward, the pellet and supernatant were separated by centrifugation at 3500 rpm for 20 min. The pellet was then washed with phosphate buffer (pH 7), followed by the addition of ACE-AgNPs and clindamycin samples at various concentrations (½ MIC, 1 MIC, 2 MIC, and 4 MIC). The mixture was incubated at 37°C for 24 h. After incubation, the solution was centrifuged again at 3500 rpm for 20 min. The supernatant was analyzed for absorbance at 260 nm (DNA) and 280 nm (protein) using a UV-visible spectrophotometer. The data were analyzed in triplicate and presented as the mean ± standard deviation (SD) (Waruwu et al., 2025).

2.12 Biofilm formation assay

A biofilm assay was performed to evaluate the impact of ACE-AgNPs on the biofilm formation of test bacteria using a 24-well plate. A bacterial suspension (1 × 10⁶ CFU/mL) was prepared in 0.9% NaCl solution. A mixture of nutrient broth (NB) and samples with varying concentrations of ACE-AgNPs and clindamycin (¼ MIC, ½ MIC, 1 MIC, and 2 MIC) was prepared in the wells. Then, 1 mL of the bacterial suspension was added to each well and incubated for 24 h at 37°C. After incubation, the liquid was discarded, and the wells were washed twice with deionized water. Next, 1 mL of 0.3% crystal violet solution was added to the wells, followed by incubation for 30 min at room temperature. The plate was rinsed with deionized water, and 2 mL of ethanol was added to each well to dissolve the stained biofilm. Biofilm formation was measured at a wavelength of 600 nm using a UV-visible spectrophotometer (Maryani & Septama, 2022). The percentage of biofilm was calculated using the equation below. The data were analyzed in triplicate and expressed as the mean ± standard deviation (SD).

$\text{\% biofilm =}\frac{\text{bacterial absorbance - sample absorbance}}{\text{bacterial absorbance }}\text{×100}$

3.1 LC-HRMS analysis

Phytochemical constituent analysis showed the identified compounds in ACE based on LC-HRMS analysis (Fig. 1). Some compounds were identified as polyphenols, like chlorogenic acid, neochlorogenic acid, and caffeic acid, which belong to the phenolic group, and rutin, quercetin, and 6.8-diprenylnaringenin, which belong to the flavonoid group (Table 1). These compounds can facilitate the green synthesis of AgNPs by enhancing their reducing capacity, leading to the rapid and effective production of AgNPs. Polyphenols act as effective reducing and binding agents, promoting the environmentally sustainable synthesis of nanoparticles. Polyphenolic compounds enhance the stability of nanoparticles, preserving their size and structure throughout time. The antioxidant characteristics of polyphenols augment the recovery capabilities of produced nanoparticles, rendering them effective in diverse applications (Swilam & Nematallah, 2020).

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Fig. 1.

LC-HRMS ACE analysis results.

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Previous studies have reported that chlorogenic acid can function as a reducing agent in the synthesis of AgNPs, with its C=O and hydroxyl groups involved in complex interactions with the nanoparticle surface. In addition, modification of chlorogenic acid with thiol groups (thiolated chlorogenic acid) showed that this compound can also act as a capping agent, increasing the stability of nanoparticles (Nayak et al., 2022; Ceylan et al., 2021). For caffeic acid, recent studies have shown that this compound can synthesize AgNPs directly and participate in functional coatings on the nanoparticle surface (Kurt et al., 2023; Pushpanathan et al., 2025). Quercetin was reported to donate electrons to Ag⁺, so the formation of AgNPs was rapid (Sivakumar, Park, & Lee, 2023). Meanwhile, rutin was reported to act not only as a reducing agent but also as a capping agent, evidenced by a relatively high zeta-potential value indicating particle stability (Kubavat et al., 2022). Quercetin-loaded AgNPs showed consistent size and high stability, which can be attributed to the interaction of hydroxyl and carbonyl groups of quercetin with the AgNPs surface, slowing down aggregation (Sharma et al., 2023). The combination of redox activity and binding ability of these specific compounds supports uniform and stable ACE-AgNPs.

3.2 Results of TPC and TFC

In the TPC test, the Folin-Ciocalteu reagent measures total phenol compound content and phenolic compounds in the reagent sample, changing it from yellow to blue. The addition of sodium carbonate solution increases the color concentration, where the more phenolic compounds, the more concentrated the blue color produced. Meanwhile, in the TFC test, the addition of AlCl₃ is used to measure the total flavonoid compound content, where AlCl₃ interacts with the hydroxyl group (-OH) on the aromatic ring of flavonoids, especially at the C-3 or C-5 position, producing a yellowish complex. ACE showed high phenolic (94.84 ± 0.52 mg GAE/g) and flavonoid (8.30 ± 0.21 mg QE/g). Both compounds are phytochemicals commonly found in plant extracts and contribute significantly to reducing silver ions (Ag⁺) in forming AgNPs. Polyphenol compounds contribute by interacting with silver metal ions to form colloidal suspensions (Gecer et al., 2023; Waruwu et al., 2025). The proposed mechanism of ACE-AgNP formation has been provided in Fig. 2.

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Fig. 2.

Proposed mechanism of ACE-AgNPs formation.

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Many studies have shown that plants rich in phenols and flavonoids can be selected and used as bioreductors to synthesize AgNO₃. Bioactive compounds such as phenols can act as silver ion reducers, capping and stabilizing agents. Flavonoids are compounds often reported to contribute to green synthesis by reducing and stabilizing silver ions. The functional hydroxyl groups in flavonoids are responsible for color changes during synthesis. The -OH and C-O groups contribute to reducing Ag⁺ and stabilizing the formed nanoparticles. Total phenol and flavonoid tests were conducted to identify the presence of these two compounds in plants intended for synthesis, as they play a role in the biosynthesis of AgNPs (Baran et al., 2023; Waruwu et al., 2025).

3.3 Characterization of ACE-AgNPs results

3.3.1 Analysis using UV-visible spectroscopy

AgNPs formation was identified in solution using a UV-visible spectrophotometer. The mixture of ACE with AgNO₃ solution after microwave irradiation causes a color change from pale yellow to brownish, which can be observed directly. This color change, associated with surface plasmon resonance (SPR), occurs during the reaction process, indicating the formation of AgNPs. Fig. 3 displays the UV absorbance spectra of AgNO₃, ACE, and ACE-AgNPs, with peak absorbance at 235 nm (ACE) and 441 nm (ACE-AgNPs). The color alteration and shift of the absorbance peak indicate that AgNPs have been formed, where Ag⁺ has been reduced to Ag⁰ (Krishnamoorthy et al., 2023; Waruwu et al., 2025).

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Fig. 3.

UV-visible spectrophotometry of AgNO₃, ACE, and ACE-AgNPs.

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3.3.2 FT-IR analysis

Identification of the functional groups in ACE that contribute to the reduction and capping processes in AgNPs biosynthesis was conducted using FT-IR (Fig. 4). The spectra showed O–H stretching vibrations of alcohols and phenolics at 3392 and 3423 cm⁻¹, C–H stretching of methyl groups at 2922 and 2314 cm⁻¹, C=O stretching of proteins at 1637 and 1606 cm⁻¹, and C–O stretching of alcohols and phenols at 1037 and 1056 cm⁻¹. Shifts and changes in intensity of these peaks after nanoparticle formation indicate the involvement of hydroxyl (–OH) and carbonyl (C=O) groups of phenolic and flavonoid compounds, such as quercetin, rutin, and chlorogenic acid, in the reduction of Ag⁺ to Ag⁰ and the subsequent stabilization of the nanoparticles. The –OH groups donate electrons to reduce Ag⁺ ions, while the C=O and remaining –OH groups coordinate with the nanoparticle surface, forming a capping layer that prevents aggregation (Maryani & Septama, 2022). This mechanism, illustrated in Fig. 2, is consistent with previous findings on the role of polyphenolic moieties in the green synthesis of AgNPs (Swilam & Nematallah, 2020; Gecer, & Erenler, 2023).

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Fig. 4.

FT-IR of ACE and ACE-AgNPs.

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The microwave-assisted synthesis method was used in this study because it offers a faster, more efficient, and environmentally friendly process compared to conventional methods. Mechanistically, microwave irradiation produces uniform volumetric heating through the direct interaction between electromagnetic waves and polar molecules in the reaction solution. This differs from conventional heating (such as on a hot plate or water bath), where conduction transfers heat from the surface to the interior, often creating an uneven temperature gradient. This uniform heating accelerates the simultaneous nucleation of silver throughout the medium, resulting in smaller, narrowly distributed particles. In addition to accelerating nucleation, microwaves also increase the reduction rate of Ag⁺ ions to Ag⁰ by increasing the kinetic energy of natural reducing molecules in plant extracts. Microwave energy facilitates the activation of hydroxyl and carbonyl groups in compounds such as quercetin, rutin, and chlorogenic acid, accelerating electron transfer to silver ions (Amar et al., 2025; Magdy, Aboelkassim, & Belal, 2025). This rapid and homogeneous heating reduces the possibility of aggregation during particle growth, resulting in more stable and well-dispersed nanoparticles, as confirmed by the morphology and UV–Vis results showing narrow plasmon peaks.

Unlike other environmentally friendly methods, such as stirring, heating, or ultrasonic-assisted synthesis, microwave irradiation produces a significantly shorter reaction time (±3 min) with minimal energy consumption. Previous studies have reported that microwave heating can control particle size between 10 and 40 nm with a uniform distribution (Hassanisaadi et al., 2022; Maryani & Septama, 2022). This study chose the optimum conditions (3 × 1 min at 450 W) based on preliminary test results indicating that continuous irradiation tends to cause rapid evaporation and degradation of active compounds. Cyclic (intermittent) irradiation allows for a short cooling process that maintains the phytochemical stability of the extract while maintaining the efficiency of Ag⁺ ion reduction. Thus, the microwave-assisted method accelerates the formation of AgNPs and improves particle size homogeneity and colloidal stability, potentially enhancing biological activity due to a higher specific surface area (Oe et al., 2023; Zhang et al., 2024). This approach places ACE-mediated AgNPs synthesis in the rapid and sustainable nanofabrication category, which provides advantages over other conventional heating-based green approaches.

3.3.3 PSA and zeta potential analysis

The detected particle size was 42.13 nm (Fig. 5) and is still within the nanoparticle size requirements of 1-100 nm. The particle size confirms that the resulting particles are nanometer-sized. Particle size analysis is conducted to determine the size of the produced particles by measuring the colloidal solution (Amar et al., 2025; Waruwu et al., 2025).

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Fig. 5.

PSA of ACE-AgNPs.

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The surface of the ACE-AgNPs obtained has a strong negative charge, with a zeta potential value of -43.8 mV (Fig. 6). The zeta potential value can affect nanoparticles' stability; a decrease in this value can cause particles to attract each other and cause flocculation. A high zeta potential charge (positive or negative) will remain stable, while a low value tends to experience flocculation. The ideal zeta potential value of particles must be higher than the charge of the dispersing medium to prevent aggregation. Particles with a negative charge will produce a strong repulsive force between one particle and another, thus preventing aggregation and ensuring high stability. Nanoparticles with a zeta potential below -30 mV or above +30 mV possess enough charge to repel each other, providing high stability in suspension and reducing the likelihood of precipitation (Badmus et al., 2020).

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Fig. 6.

Zeta potential of ACE-AgNPs.

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3.3.4 SEM-EDX analysis

Morphological analysis and elemental composition of ACE-AgNPs were performed using SEM-EDX. The particle size, measured with ImageJ, showed an average size of 34.21 ± 14.21 nm (Fig. 7). Additionally, EDX analysis identified the presence of silver, chlorine, and oxygen in the sample, with corresponding elemental compositions of 82.22%, 12.26%, and 5.52%, respectively (Fig. 8). An Ag peak also supports this result at 3 keV, where the peak confirms the presence of the synthesized AgNPs element. In the synthesis process, elements such as oxygen and chloride may function as capping agents (Maryani & Septama, 2022; Amar et al., 2025).

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Fig. 7.

SEM of ACE-AgNPs.

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Fig. 8.

EDX of ACE-AgNPs.

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3.3.5 TEM analysis

Determination of the structure, morphology, and size of AgNPs was more clearly carried out using TEM (Fig. 9). The average particle size at a magnification of 100-50 nm showed that most synthetic nanoparticles were spherical. Nanoparticles with other shapes, such as triangles, rods, and quasi-spherical, were also observed. Nanoparticles synthesized using biological systems can produce various shapes and sizes (depending on the phytochemical compounds that function as reducing and binding agents) (Gecer & Erenler, 2023). Analysis with TEM tends to produce smaller particle sizes than PSA measurement results; this can occur because PSA analysis is carried out in solution. In contrast, electron microscopy analysis is carried out in a vacuum and on dry samples, which may cause changes in the structure or size of the particles compared to their original conditions.

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Fig. 9.

TEM of ACE-AgNPs (50 nm).

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3.3.6 XRD analysis

Fig. 10 shows five distinct diffraction peaks of 2θ value of ACE-AgNPs (38.13°, 44.18°, 64.48°, 77.33°, and 81.29) based on XRD analysis. These points correspond to (111), (200), (202), (311), and (222). Furthermore, the results were compared with JCPDS No. 89-101-3772, showing that the produced ACE-AgNPs had a surface-centered cubic (FCC) shape, resulting in crystals with a size of 9 nm.

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Fig. 10.

XRD of ACE-AgNPs.

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Differences in particle size obtained from PSA (42.13 nm), SEM (34.21 ± 14.21 nm), and XRD (9 nm) are common because each characterization method measures different parameters. PSA determines particle size based on the hydrodynamic diameter of the particles in the colloidal medium. This value not only reflects the Ag⁰ core but also includes the solvent layer and stabilizing molecules (capping agents) from ACE attached to the surface of the nanoparticles, so that the size value obtained from PSA tends to be larger. In contrast, SEM measurements are carried out on dry particles, so they only show the actual physical size and morphology without the influence of the solvent layer (Jia et al., 2023). The SEM value, slightly smaller than PSA, indicates that the particles remain relatively uniform and do not experience significant aggregation (Jagadeesh, Rangappa, & Siengchin, 2024). Meanwhile, the size value from XRD (9 nm) represents the crystallite size, namely the dimensions of the single crystal domain within a particle (Gesing & Robben, 2024). A single silver particle can consist of several aggregated nanocrystals, so the crystallite size calculated using the Scherrer equation is usually smaller than the particle size observed using SEM or PSA (Waruwu et al., 2025). Thus, the size variations between PSA, SEM, and XRD reflect differences in measurement principles, not inconsistencies in the results. These three methods complement each other in providing a comprehensive picture of the physical and structural properties of the resulting AgNPs.

3.4 Antioxidant activity results

A low IC₅₀ value (Table 2) indicates that the sample has high antioxidant activity, an IC₅₀ value <50 µg/mL indicates potent activity, 50-100 µg/mL indicates vigorous activity, 101-150 µg/mL indicates moderate activity, and >150 µg/mL indicates weak activity (Satria et al., 2024). ACE-AgNPs have a more substantial effect than the extract but a weaker effect than quercetin. These findings suggest that the presence of AgNPs may enhance antioxidant activity. Several studies have indicated that the increase in antioxidant activity results from the combined action of polyphenols, which act as antioxidant agents, and AgNPs, which function as catalysts (Swilam & Nematallah, 2020). Both phytochemicals and silver ions can exhibit antioxidant properties through single electron transfer and hydrogen atom donation (Gecer & Erenler, 2023). Furthermore, the greater antioxidant activity of ACE-AgNPs compared to ACE could be attributed the adsorption of bioactive compounds from the leaf extracts onto the nanoparticles, as opposed to the spherical nanoparticles.

Claims of the antioxidant activity of plant extract-based AgNPs have been supported by several studies showing that the presence of phenolic metabolites directly contributes to their free radical scavenging ability (Mortazavi-Derazkola et al., 2020; Maryani & Septama, 2022; Baran et al., 2023). Compounds such as chlorogenic acid and quercetin enhance the antioxidant activity of nanoparticles through electron regeneration and interaction with ROS. These results are consistent with the scavenging activity observed in this study.

3.5 Antibacterial activity

The MIC value of the sample was determined using the microdilution method against gram-negative bacteria (E. coli and S. typhi) and gram-positive bacteria (S. aureus and P. acnes) (Table 3). The MIC values ​​of ACE-AgNPs were obtained at 1.9 µg/mL (E. coli and S. aureus), 7.8 µg/mL (P. acne), and 15.6 µg/mL (S. typhi). As a comparison, clindamycin showed MIC values ​​of 0.9, 31.25, 250, and 250 µg/mL, respectively. Meanwhile, ACE did not demonstrate antibacterial activity against the four bacterial strains, which was indicated by the formation of turbidity and sediment in all wells. After determining the MIC value of the sample, MBC testing was conducted at 1 and 2 MIC. The results revealed bacterial growth at 1 MIC, while no growth was observed at 2 MIC, indicating that the MBC value for ACE-AgNPs and clindamycin was 2 MIC. Repetition of the antibacterial assays yielded consistent results across all replicates, confirming the reproducibility of the experimental procedures. These findings suggest that ACE-AgNPs exhibit effective antibacterial activity against both gram-negative and gram-positive bacteria. Similar antibacterial effects have been reported for AgNPs synthesized from Zanthoxylum acanthopodium against S. aureus (MIC 1.9 µg/mL, MBC 3.9 µg/mL), Methicillin-Resistant Staphylococcus aureus (MIC 3.9 µg/mL, MBC 7.8 µg/mL), K. pneumoniae (MIC 7.8 µg/mL, MBC 15.6 µg/mL), P. aeruginosa (MIC 1.9 µg/mL, MBC 3.9 µg/mL) (Amar et al., 2025). Desmodium triquetrum DC against B. subtilis (MIC 3.9 µg/mL, MBC 7.8 µg/mL), P. aeruginosa (MIC 7.8 µg/mL, MBC 15.1 µg/mL), E. coli (MIC 15.1 µg/mL, MBC 15.1 µg/mL), K. pneumoniae (MIC 31.2 µg/mL, MBC 31.2 µg/mL) (Maryani & Septama, 2022). Brassica oleracea against S. epidermidis (MIC 6.25 μg/ml), B. fragilis (MIC 50 μg/mL), S. pneumoniae (MIC 25 μg/mL), S. aureus (MIC 25 μg/mL), K. pneumoniae (MIC 25 μg/mL), E. coli (MIC 25 μg/mL), E. faecalis (MIC 23.6 μg/mL), P. mirabilis (MIC 12.5 μg/mL), P. aeruginosa (12.5 μg/mL) (Ansar et al., 2020). Crataegus microphylla against S. aureus (MIC 14 μg/mL, MBC 112.5 μg/mL), Enterococcus (MIC 7 μg/mL, MBC 225 μg/mL), Pseudomonas (MIC 3.5 μg/mL, MBC 28 μg/mL), E. coli (MIC 3.5 μg/mL, MBC 56 μg/mL), Klebsiella (MIC 56 μg/mL, MBC 450 μg/mL), A. baumannii (MIC 3.5 μg/mL, MBC 112.5 μg/mL), P. mirabilis (MIC 14 μg/mL, MBC 28 μg/mL (Mortazavi-Derazkola et al., 2020). Pisum sativum L against P. aeruginosa (MIC 50 μg/mL, MBC 100 μg/mL), E. coli (MIC 50 μg/mL, MBC 150 μg/mL), E. faecalis (75 μg/mL, MBC 125 μg/mL), S. gordonii (MIC 12.5 μg/mL, MBC 75 μg/mL), S. aureus (MRSA) (MIC 12.5 μg/mL, MBC 75 μg/mL), and S. aureus (MIC 6.25 μg/mL, MBC 50 μg/mL) (Alarjani et al., 2022). The antibacterial action of AgNPs is associated with the release of silver ions, which attach to the cell wall, causing membrane disruption, interfering with DNA replication, inhibiting cell reproduction, and increasing the production of ROS, which causes oxidative stress and potentially leads to the death of microorganisms. The size of the nanoparticles also affects the antibacterial activity. In this study, we obtained the size of ACE-AgNPs with PSA, which is 42.13 nm. The most effective antibacterial AgNPs usually have a size between 10-50 nm. This size provides a very high surface area to volume ratio, thus increasing the interaction with bacterial cells (Ali et al., 2024; Waruwu et al., 2025).

The antibacterial and antibiofilm activities of AgNPs synthesized by ACE also align with recent reports on plant-mediated AgNPs. Nanoparticles with polyphenol coatings have been shown to suppress biofilm formation through disruption of quorum sensing and increased cell membrane permeability (Ansar et al., 2020; Gecer & Erenler, 2023; Amar et al., 2025). The combination of small particle size and phenolic groups on the surface of AgNPs is believed to enhance the antimicrobial effects observed in this study.

3.6 Assessment of DNA, protein leakage, and biofilm formation assay

The cell leakage test was analyzed using UV-visible spectroscopy with 260 (nucleic acid) and 280 (protein) wavelengths. Deionized water, serving as a control (untreated), showed no absorbance. The results of ACE-AgNPs (Table 4) showed an increase in absorbance along with the increasing concentration of ACE-AgNPs, which was the same thing that happened to clindamycin. These results show that AgNPs can trigger the release of metabolite compounds from bacterial cells, which is indicated by an increase in absorbance value. Elevated absorbance indicates cell leakage, revealing the escape of genetic material such as nucleic acids and proteins. The presence of these biomolecules confirms cell damage or altered membrane permeability, ultimately resulting in bacterial cell death. AgNPs can attach to microbial cell walls, disrupting membrane permeability and respiration. Additionally, they interfere with ribosomes and the cytoplasm, inhibiting protein synthesis, which impairs metabolic activity and leads to the destruction and death of microorganisms (Amar et al., 2025).

Table 4 shows the percentage of biofilm formation against bacteria after being treated with ACE-AgNPs, significantly reducing biofilm formation, especially at higher concentrations. The effectiveness of biofilm inhibition differs based on the strain of bacteria, with S. aureus showing the highest sensitivity to treatment with ACE-AgNPs. In contrast, S. typhi showed higher resistance to the effects of these nanoparticles. Clindamycin also effectively inhibited biofilm formation, but ACE-AgNPs appeared superior, especially at high concentrations, especially against E. coli and P. acne; this suggests that ACE-AgNPs have great potential as a more potent antibacterial and biofilm inhibitor agent than Clindamycin, especially in the case of bacteria that are more sensitive to these nanoparticles. The exact mechanism by which AgNPs inhibit biofilm formation remains unclear. However, the antibiofilm activity of AgNPs is likely related to the release of high amounts of silver ions, which can kill planktonic bacterial cells in the biological film system (Maryani & Septama, 2022). This study demonstrates that the effectiveness of biofilm inhibition by AgNPs is strongly influenced by the sample concentration. Higher concentrations of AgNPs or extracts tested, the stronger the inhibitory effect on biofilm formation. These findings reveal a positive correlation between AgNPs concentration and anti-biofilm activity, where increasing the concentration strengthens the effectiveness of AgNPs in disrupting the formation of biofilm structures. This potential illustrates that AgNPs can be developed as an alternative or additional component in antibiofilm therapy, especially to overcome biofilm resistance to conventional treatments. The antibiofilm mechanism proposed in this study is inferred from the literature, which attributes the inhibition to the gradual release of Ag⁺ ions from AgNPs (Dhaka et al., 2023). However, since no direct quantification of released silver ions was performed in this work, the proposed mechanism remains hypothetical and warrants further confirmation.