A. squamosa seed extract was procured from Anhui Minmetals Development Ltd. To prepare the A. squamosa seed extract solution, 0.3 g of the powder was dissolved in 100 ml of ddH₂O. For synthesizing AgNPs, 33 ml of A. squamosa seeds extract solution was added with 33 ml of a 0.1 M of silver nitrate (AgNO₃). The pH of the mixture was adjusted to 7.0 and the mixture was heated at 60˚C for 5 min and incubated under the exclusion of light for 24 h. The successful formation of of AgNPs was marked by a color change from light to dark brown.

AuNP synthesis begins with making a gold ion solution by mixing 100 mg of HAuCl₄ (Smart Lab, Indonesia) in 100 ml of ddH₂O. To produce AuNPs, 100 ml of HAuCl₄ solution was combined with 100 ml of A. squamosa seed extract solution. The mixture was then incubated under the exclusion of light for 24 h. The appearance of AuNPs was marked by a color change from light brown to purple.

The synthesis of AgNPs and AuNPs was independently repeated at least three times to assess reproducibility. The consistency and formation of the NPs were confirmed by UV-vis spectrophotometry and particle size analysis (PSA). The presence of AgNPs and AuNPs was initially verified based on their characteristic surface plasmon resonance absorption bands. Functional groups associated with the biomolecules responsible for the reduction and stabilization processes were analyzed using a Spectrum 100 FT-IR spectrometer (PerkinElmer, Inc.) within the wavenumber range of 4,000-400/cm. Furthermore, PSA was employed to determine the hydrodynamic size distribution of the NPs in the liquid phase. This method was commenced with an analysis of refractive index and viscosity in the test samples and was crucial for understanding NP properties such as chemical receptivity, stability and biocompatibility. Subsequently, 2 ml of sample was introduced into a PSA analysis cuvette, where a laser beam was emitted into the sample and the intensity of the reflected light was measured to ascertain particle size and distribution (30). Additionally, NP size distribution was measured using a ViewSizer 3000 NP Size Analyzer (Horiba Scientific). Measurements were performed using blue (B, 445 nm), green (G, 520 nm) and red (R, 635 nm) lasers with pulse durations of 15 msec for each channel (B/G/R: 15/15/15 msec). Transmission electron microscopy (TEM) was used for morphology analysis of the NPs (FEI Tecnai G2 F20 X-TWIN; Thermo Fisher Scientific, Inc.). A small aliquot of NPs was deposited onto a copper grid, allowed to dry at room temperature and subsequently analyzed by TEM. TEM images were obtained from the electron beam transmitted through the thin NP sample. The crystalline properties of the biosynthesized NPs were characterized using a PANalytical Empyrean X-ray Diffractometer (EA Almelo). Before X-ray diffraction (XRD) analysis, the NPs were freeze-dried with a Lyovapor L-200 (Buchi) at temperatures between -55 and -57˚C under a pressure of 2 mbar for 24-48 h. The freeze-dried samples were then subjected to XRD analysis using Cu Kα radiation, with diffraction data collected over a 2θ range of 5˚-80˚.

The Ag content of the NPs was quantified using an Agilent 5100 Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES; Agilent Technologies, Inc.). Prior to analysis, the AgNPs samples were digested in an acidic medium prepared from ultrapure water containing 12% (v/v) hydrochloric acid (37%) and 4% (v/v) nitric acid (65%). The digestion process was carried out at 95±2˚C for 2 h, after which the solution volume was brought to 50 ml using the same acid mixture. The concentration of Ag in the NPs was determined according to the following equation according to the 2018 study by Shahid et al (31):

where Cs represents the Ag concentration measured in the digested solution by ICP-OES (Agilent 5100; Agilent Technologies, Inc.), W is the final volume of the digestion solution (ml) and V denotes the mass of the dried sample used for analysis (0.025 g).

Sterile blank discs injected with 20 µl of AgNPs and AuNPs at a concentration of 100 mg/ml were placed onto sterile Mueller-Hinton Agar (MHA) plates media (Sisco Research Laboratories). Sterile blank discs injected with 20 µl of S. aureus and E. coli culture (10⁸ CFU/ml) were used as a positive control, while sterile nuclease-free water (Promega Corp.) served as a negative control. The plates were then incubated for 24 h at 37˚C, after which bacterial growth was observed.

S. aureus [American Type Culture Collection (ATCC) 25923] and E. coli (ATCC 25922) were obtained from the Applied Microbiology Laboratory at the University-CoE Research Center for Biomolecule Engineering, Universitas Airlangga. To prepare an overnight culture, a single colony of each strain was inoculated into Mueller-Hinton Broth (MHB) (Sisco Research Laboratories) and incubated at 37˚C while shaking at 150 rpm for 24 h. Following this incubation, 60 µl of each overnight culture was transferred into 3 ml of sterile MHB and cultured at 37˚C while shaking at 150 rpm until the optical density (OD) at 600 nm (OD₆₀₀) reached 0.1. The OD was measured using a NanoPhotometer NP80 (Implen GmbH) (32).

A modified method based on the Clinical and Laboratory Standards Institute document M02-ed14(32) was used to assess the antibacterial activity of the A. squamosa seeds extract, AuNPs and AgNPs. Each assay was performed in triplicate. Using a cotton swab, 100 µl of cultured test bacteria were spread onto MHA plates. A total of 20 µl of an antibiotic or NPs at different concentrations were applied to sterile blank discs, which were then placed on the agar media inoculated with the test microbes. Gentamicin (40 mg/ml) was used as a positive control. A caliper was used to measure the clear zone resulting from the inhibition of microbial growth.

The minimum inhibitory concentration (MIC) test was conducted in triplicate by adding different concentrations of the AgNPs from 50 to 0.098 mg/ml to MHB media inoculated with 10⁵ colony-forming units/ml E. coli or S. aureus in a 96-well microplate. The OD at 600 nm was measured using a microplate reader before and after incubation at 37˚C for 24 h. The MIC or the lowest concentration of the AgNPs that prevents bacterial growth is observed when there is no increase in OD₆₀₀ value after 24 h of incubation. Meanwhile, the minimum bactericidal concentration (MBC) was determined by spreading 100 µl of bacterial culture treated by AgNPs at the MIC concentration and two concentrations above it onto MHA plates. The MBC is the lowest concentration that inhibits any bacterial growth on the MHA plates.

To evaluate the anticancer activity and selective cytotoxicity of the NPs, an MTT assay was performed. Human breast adenocarcinoma cells (MCF-7) [American Type Culture Collection (ATCC)] and 293 cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (cat. no. D6429; Sigma-Aldrich; Merck KGaA) supplemented with 10% fetal bovine serum (FBS) (cat. no. F2442; Sigma-Aldrich; Merck KGaA) and 1% penicillin-streptomycin. 293 cells were used as a non-malignant control cell line because they are of human origin, exhibit stable growth characteristics and are widely employed in cytotoxicity studies to evaluate the selectivity and safety of anticancer agents (33). Cells were maintained at 37˚C in a humidified incubator containing 5% CO₂. For the cytotoxicity assay, cells were seeded into 96-well plates at a density of 5x10³ cells per well in 100 µl of complete medium and allowed to attach for 24 h. The culture medium was subsequently replaced with fresh medium containing NFW (control), doxorubicin (10 µg/ml; positive control), AgNO₃, AgNPs or AuNPs at concentrations of 12.5, 25, 50, 100 and 200 µg/ml. Cells were incubated with the treatments for 24, 48 and/or 72 h. Following the treatment period, 10 µl of MTT solution (5 mg/ml in PBS) was added to each well and incubated for 4 h at 37˚C. The resulting formazan crystals were dissolved by adding 100 µl of DMSO (Merck KGaA), followed by incubation in the dark until complete dissolution of the crystals. The absorbance was measured at 570 nm using a microplate reader.

The IC₅₀ values were determined by plotting the percentage of cell viability against the logarithm of the NP concentration and fitting the data using nonlinear regression analysis (four-parameter logistic model) in GraphPad Prism 10 software (Dotmatics). The IC₅₀ value was defined as the concentration required to reduce cell viability by 50% compared with the untreated control. All experiments were performed in triplicate.

Cellular DNA content was assessed by staining with 4',6-diamidino-2-phenylindole (DAPI). In brief, cells were fixed in 4% paraformaldehyde and incubated with 1 µg/ml DAPI C001 (ABP Biosciences) for 20 min at room temperature in the dark. After staining, cells were washed three times with PBS and subjected to cell cycle analysis. Images were acquired using a Cell-Insight CX7 Pro High Content Screening (HCS) System (version 393216; Thermo Fisher Scientific, Inc.) with the assay parameters set to ‘CellCycle.V4’, an autofocus interval of 2, a PRIMEBSI 1.53 camera and a x20 objective lens. Image processing and cell cycle quantification were conducted using Thermo Scientific HCS Studio 5.0 Cell Analysis Software (Thermo Fisher Scientific, Inc.). Cell cycle phases were determined based on DNA content, where cells containing 2N DNA were classified as G1 phase, those with DNA content between 2N and 4N as S phase, and those with 4N DNA as G2/M phase.

MCF-7 cells were cultured in 6-well plates and divided into three groups for a 48-h treatment period: A control group, a doxorubicin-treated group (10 µg/ml) and an AgNPs-treated group (100 µg/ml), with each group prepared in triplicate. The concentrations of AgNPs were determined close to the IC₅₀ value. The RNA was extracted using the Total RNA Mini Kit (cat. no. RTD050; Geneaid). The cDNA was synthesized with ReverTra Ace qPCR RT Master Mix (cat. no. FSQ-201; Toyobo) according to the manufacturer's protocol, with 50 ng RNA contained in each sample. Quantitative PCR (qPCR) analysis was performed using Thunderbird Next SYBR qPCR Mix (cat. no. QPX-201; Toyobo) containing Tris (hydroxymethyl)aminomethane, magnesium sulfate, deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate, SYBR^® Green I and DNA polymerase. Each qPCR reaction mixture consisted of 10 µl of THUNDERBIRD™ Next SYBR^® qPCR Mix, 6 pmol each of forward and reverse primers, 4 ng of cDNA template and nuclease-free water added to a final volume of 20 µl. The qPCR was performed with MyGo Pro Real-Time PCR System (IT-IS Life Science Ltd.) under the following cycling conditions: Predenaturation at 95˚C for 60 sec, followed by 40 cycles of denaturation at 95˚C for 5 sec and annealing/extension at 60˚C for 30 sec. The threshold value was determined automatically using MyGo Pro PCR Software version 3.5 (IT-IS Life Science Ltd.). Forward and reverse primers for c-Myc (MYC), cyclin D1 (CCND1), human epidermal growth factor receptor-2 (HER-2), cyclooxygenase-2 (COX-2), catenin delta-1 (CTNND1), tumor protein P53 (TP53), BCL2 associated X (BAX), BCL2 binding component 3 (BBC3/PUMA), BCL2, actin-b and GAPDH were referenced from previous studies (34-38). The primer sequences for the genes analyzed in this study are provided in Table I. Gene expression analysis was performed using the relative quantification method and the reliability of the qPCR results was verified through melting curve analysis. The fold change in mRNA expression was calculated using the ΔΔCq formula: 2^-ΔΔCq=2^{-[(Cqtarget-Cqreference) sample-(Cqtarget-Cqreference) calibrator]}, where Cq_reference refers to the housekeeping gene and calibrator indicates the control sample (39).

Apoptosis analysis was performed on MCF-7 breast cancer cells using the Annexin V-FITC staining method followed by flow cytometric analysis. In brief, MCF-7 cells treated with AgNPs (100 µg/ml) and untreated control cells were harvested after the designated incubation period. Culture media containing floating cells were first collected into centrifuge tubes to preserve non-adherent apoptotic cells. The remaining adherent cells were washed with PBS and detached using TrypLE™ Express (cat. no. 12604013; Gibco; Thermo Fisher Scientific, Inc.). The enzymatic reaction was neutralized by adding complete culture medium supplemented with FBS. The collected cell suspension was centrifuged at 447 x g for 5 min at 4˚C and the resulting cell pellet was washed twice with cold PBS. Subsequently, the cell pellet was resuspended in 100 µl of 1X Annexin V Binding Buffer to obtain a final cell concentration of 1x10⁵-1x10⁶ cells/ml. A total of 5 µl of Annexin V-FITC reagent was added to each sample, followed by gentle mixing and incubation for 15 min at room temperature (20-25˚C) in the dark. After incubation, 400 µl of 1X Annexin V Binding Buffer was added to each sample and the stained cells were transferred into fluorescence-assisted cell sorting tubes for analysis. Samples were analyzed immediately using a flow cytometer. The distribution of viable, early apoptotic, late apoptotic and necrotic cells was determined based on quadrant analysis of the resulting dot plots, which were generated according to Annexin V fluorescence intensity.

Statistical analyses were performed using GraphPad Prism version 10 (Dotmatics). Data are presented as the mean ± standard deviation (SD) from three independent experiments (n=3), unless otherwise stated. Prior to hypothesis testing, data normality was assessed using the Shapiro-Wilk test. For cell viability assays, DNA content analysis, nuclear morphometric measurements and relative gene expression analyses, statistical differences among groups were determined using one-way ANOVA, followed by Dunnett's multiple-comparisons test. Apoptosis levels between the untreated control and AgNP-treated groups were compared using an unpaired two-tailed Student's t-test. Gene expression data associated with apoptosis-related genes were analyzed using two-way ANOVA followed by Tukey's multiple-comparisons test. Differences were considered statistically significant at P<0.05.

In the present study, a noticeable color transition from light brown to dark brown was observed (Fig. 1A), and a prominent plasmon resonance peak in the 410-420 nm range was detected, confirming the formation of AgNPs (the A. squamosa seeds extract itself did not exhibit any peaks in the 300-600 nm range) (Fig. 1B). This indicates that A. squamosa seeds extract is a highly effective reducing agent for converting silver ions to silver. For AuNPs, the successful synthesis was visually confirmed by a distinct purple color observed after A. squamosa seeds extract solution was mixed with HAuCl₄ solution (Fig. 1D). The plant extract efficiently reduced Au3+ ions to Au0. The UV-Vis spectroscopy analysis of the resulting colloidal solutions (Fig. 1E) revealed absorption peaks in the 520-540 nm range, consistent with the surface plasmon resonance of AuNPs, typically around 520 nm. This resonance is influenced by the size of the NPs, affirming the successful synthesis of AuNPs (30).

To further investigate the size, shape and crystal structure of the NPs, TEM and PSA analyses were employed. TEM images revealed distinct morphological characteristics of the AgNPs and AuNPs. The AgNPs (Fig. 1C) exhibited predominantly spherical morphology with moderate polydispersity and occasional particle aggregation. High-resolution (HR)TEM imaging showed well-defined lattice fringes, confirming the crystalline nature of the NPs. By contrast, the AuNPs (Fig. 1F) appeared more uniform in shape and size, displaying predominantly spherical to quasi-spherical morphologies. The HRTEM image of AuNPs revealed pronounced lattice fringes with a characteristic radial pattern, indicating high crystallinity and ordered atomic arrangements. According to the PSA analysis, the average diameter of AgNPs was 14.05 nm, with sizes ranging from 6 to 25 nm, while AuNPs had an average size of 14.92 nm, ranging from 3.67 to 44.89 nm (Fig. 2A and D). Meanwhile, measurements performed using a ViewSizer 3000 NP Size Analyzer; the mean hydrodynamic diameters of the AgNPs and AuNPs were 40.60±34 and 72.65±46 nm, respectively. For AgNPs, the D10, D50 and D90 values were 22.0, 29.5 and 70.76 nm, respectively, indicating that 10, 50 and 90% of the particles had diameters below these values. However, the D10, D50 and D90 values of AuNPs were 26.7, 61.2 and 132.6 nm, respectively (Fig. 2B and E). X-ray diffraction analysis of the synthesized AgNPs showed broad peaks at 38.29, 45.41, 64.40 and 77.54˚, corresponding to the 111, 200, 220 and 311 planes, indicating a face-centered cubic structure. Similarly, the XRD patterns of the biologically synthesized AuNPs revealed sharp peaks at 38.20, 43.73, 64.77 and 77.72˚, which align with the 111, 200, 220 and 311 planes, confirming their face-centered cubic crystalline structure (Fig. 2C and F).

As shown in Fig. 2G, the synthesized AgNPs and AuNPs had similar peaks to A. squamosa seed extract, indicating that A. squamosa seed extract contributed to the reduction of Ag and Au ions. A broad peak was present at ~3,272/cm that comes from OH stretching, and a smaller peak at 2,923/cm from aliphatic CH. The peaks at 1,010/cm and 1,147/cm confirm the presence of secondary OH groups. These functional groups interact with the silver and gold to form a protective layer around the NPs, which keeps them from clumping together. Previous studies have shown that hydroxyl and phenolic groups tend to bind strongly to noble metals through polar and coordinating interactions (40). The increase in peak intensity and slight shifts in wavelength observed in the NPs spectrum, for example at 3,272, 1,147 and 1,010/cm, probably occur because the seed extract is coordinating with the metals (41). Of note, the AgNPs show a unique strong peak at 1,318/cm that comes from C-O aromatic ester stretching and OH bending, which was not present in A. squamosa seeds extract or AuNPs.

ICP-OES analysis revealed that the synthesized AgNPs contained 6.60 µg/g Ag, confirming the presence of Ag in the NP preparation.

The sterility evaluation of AgNPs demonstrated that AgNPs were sterile, as evidenced by the absence of bacterial growth following inoculation onto MHA medium (Fig. 3A; Table II).

To investigate the antibacterial activity of AgNPs and AuNPs, disk diffusion assays were performed against E. coli and S. aureus. Growth inhibition was observed on plates with 100, 50, 25 and 10% concentrations of 100 mg/ml AgNPs, but not with AuNPs or A. squamosa seeds extract (control) after 24 h of incubation. Table III and Fig. 3B reveal that E. coli had a smaller zone of inhibition compared to S. aureus. The bacterial growth inhibition around the wells was attributed to the diffusion of AgNPs to the MHA plates.

Furthermore, the MIC and MBC values of AgNPs were determined (Table IV). The MIC and MBC are important metrics in antimicrobial evaluation, indicating an agent's capacity to inhibit microbial proliferation and eradicate pathogens, respectively. By definition, the MIC represents the lowest concentration of an antimicrobial agent that prevents visible microbial growth, whereas the MBC is the lowest concentration required to kill 99.9% of the microbial population (42). The correlation between these values reveals the agent's mode of action: An MBC value close to the MIC suggests bactericidal activity, whereas a substantially higher MBC denotes a bacteriostatic effect (43). As presented in Table IV, the AgNPs exhibited antibacterial activity against both tested microorganisms. The MIC values were 0.781 mg/ml for Staphylococcus aureus and 3.125 mg/ml for Escherichia coli. The MBC values for both bacteria were 3.125 mg/ml. The lower MIC value observed for S. aureus indicates that AgNP was more effective against this Gram-positive bacterium than against E. coli, as a lower concentration was required to inhibit bacterial growth. Furthermore, the identical MBC values suggest that a concentration of 3.125 mg/ml was required to achieve bactericidal activity against both bacterial species. The AgNPs exhibited bacteriostatic activity against S. aureus and bactericidal activity against E. coli, as evidenced by the relationship between their MIC and MBC values. In S. aureus, the MBC was 4x higher than the MIC, whereas in E. coli, the MIC and MBC were identical, indicating that the inhibitory concentration was also sufficient to exert a bactericidal effect.

To evaluate the anticancer activity and selective cytotoxicity of the biosynthesized NPs, an MTT assay was performed on the breast cancer cell line MCF-7 and the non-carcinogenic 293 cells. Treatment with A. squamosa extract alone (100 µg/ml) showed limited cytotoxic activity against MCF-7 cells at 24 h, with the cell viability remaining comparable to the control group (>90%). However, prolonged exposure significantly reduced the cell viability to ~55% at both 48 and 72 h (P<0.0001), indicating a time-dependent antiproliferative effect (Fig. 4A). By contrast, AuNPs only reduced the cell viability after 24 h at 100 and 200 µg/ml. By 48 and 72 h, there was no significant difference in cell viability between the AuNP-treated cells and the control (Fig. 4B).

Treatment with AgNO₃ produced strong cytotoxic effects on MCF-7 cells, reducing cell viability to ~40-45% across all tested concentrations after 24 h of treatment (P<0.0001) (Fig. 4C). Among all tested treatments, AgNPs demonstrated the greatest anticancer activity against MCF-7 cells. At 24 h, AgNPs reduced cell viability in a concentration-dependent manner, with higher concentrations decreasing viability to ~55-60% (P<0.0001). The cytotoxic effect became more pronounced after 48 h, where the highest concentration reduced viability to ~25-30% (P<0.0001). Similar effects were maintained at 72 h, indicating sustained growth inhibition and enhanced anticancer activity compared with SQ and AuNPs alone (Fig. 4D).

To assess selectivity, the effects of AgNO₃ and AgNPs were also evaluated in 293 cells. AgNO₃ markedly reduced 293 cell viability, decreasing the proportion of viable cells to ~30-40% at all tested concentrations (Fig. 4E), suggesting considerable toxicity toward normal cells. By contrast, AgNPs displayed substantially lower toxicity toward 293 cells, maintaining cell viability above 70-98% across most concentrations and time-points (Fig. 4F). Although a slight decline in viability was observed at higher concentrations, the reduction was significantly less severe than that observed in MCF-7 cells.

The MTT assay results showed that while the AuNPs weren't toxic to the cells, the AgNPs had a clear effect, and accordingly, the study further focused on measuring their IC₅₀ value. The IC₅₀s of the AgNPs were assessed through a detailed analysis involving five doses and three incubation periods. As shown in Fig. 5, it was found that even high doses of AgNPs did not kill enough cells to determine the IC₅₀ after 24 h-it was >200 µg/ml. However, at 48 and 72 h, the AgNPs were much more effective, as evidenced by the significant reduction of cell viability. The IC₅₀ values were determined to be 106.7 µg/ml at 48 h and 79.5 µg/ml at 72 h. These results show that the AgNPs have the potential to stop cancer cells from growing, suggesting they could be useful in cancer treatments.

To investigate the possible molecular targets of AgNPs in the MCF-7 cell line, cell cycle and RT-qPCR analysis of several identified oncogenes in breast cancer (44-46) was performed, including MYC, HER2, CCND1 and COX-2. The DNA content analysis revealed that AgNPs treatment significantly reduced the percentage of cells below 2N and increased the number of cells with 4N DNA. The RT-qPCR analysis results indicated that AgNPs derived from A. squamosa seed extract effectively reduced the mRNA levels of MYC, CCND1, HER2, COX-2 and CTNND1 (Fig. 6). The Cq values are presented in Table SI. These findings underscore the potential of AgNPs to modulate the expression of critical oncogenes and offer valuable insights into their anti-proliferative mechanisms in cancer cells.

To investigate whether the cytotoxic effect of AgNPs was associated with apoptosis, MCF-7 cells were treated with AgNPs (100 µg/ml) for 48 h and analyzed using Annexin V-FITC flow cytometry. As shown in Fig. 7A and B, AgNP treatment markedly increased the population of Annexin V-positive cells compared with the untreated control. Quantitative analysis revealed that the percentage of apoptotic cells increased significantly from 21.25±0.07% in the control group to 27.3±0.32% following AgNP treatment (P<0.001; Fig. 7B), indicating that AgNPs effectively induced apoptosis in MCF-7 cells.

To further elucidate the molecular mechanisms underlying AgNP-induced apoptosis, the expression of several apoptosis-related genes was evaluated by RT-qPCR (Fig. 7C), and the ΔΔCt values are presented in Table SII. AgNP treatment significantly upregulated the expression of p53 (2.7-fold), BAX (3.8-fold), and the BAX/BCL2 ratio (2.2-fold) relative to the control group (P<0.05-0.001). By contrast, the expression of PUMA was markedly reduced to ~0.05-fold of the control level (P<0.05). These findings suggest that AgNPs induce apoptosis in MCF-7 cells through activation of the p53-mediated apoptotic pathway.