Green synthesis of silver nanoparticles using Ginkgo biloba seed extract: Evaluation of antioxidant, anticancer, antifungal, and antibacterial activities

2.4.1 Reaction time

1 mg/mL of GBSE solution and 0.025 M of AgNO₃ diluent were mixed in a 40:1 ratio and placed in a 50 mL beaker with a reaction system of 40 mL. The reaction temperature was 90°C, and the absorbance changes were observed at 30, 60, 120, 180, and 240 min, respectively.

2.4.2 Reaction temperature

1 mg/mL of GBSE solution and 0.025 M of AgNO₃ diluent were mixed in a 40:1 ratio (GBSE:AgNO₃, v/v) and placed in a 50 mL beaker with a reaction system of 40 mL. The reaction time was 180 min, and the absorbance changes were observed at 60, 70, 80, 90, and 95°C, respectively.

2.4.3 Reaction ratio

1 mg/mL of GBSE solution and 0.025 M of AgNO₃ diluent are divided into 10, 20, 40, 80, 160: The volume ratio (GBSE:AgNO₃, v/v) of 1 is mixed (GBSE concentration is 2 mg/mL at 80:1, GBSE concentration is 4 mg/mL at 160:1), and placed in a 50 mL beaker with a reaction system of 40 mL. The reaction temperature was 90°C, and the absorbance change was observed in 180 min.

2.7.1 UV-Vis spectrum

The synthetic GBSE-AgNPs were validated by UV-Vis in the scanning wavelength range of 300–600 nm. The GBSE-AgNPs solution was diluted to different concentrations with deionized water, and the characteristic peaks were observed. Then, the silver ion concentration at different concentrations was measured by inductively coupled plasma emission spectrometry (ICP-OES), and the concentration–absorbance curve was drawn for subsequent detection.

2.7.2 SEM

A small amount of freeze-dried GBSE-AgNPs samples were directly glued to the conductive adhesive, and gold was sprayed for 45 s with 10 mA by Quorum SC7620 sputtering coater. Then, a ZEISS Sigma 300 SEM was used to photograph the morphology of the sample and spot scan of the energy spectrum. The accelerated voltage during the morphology shooting was 3 kV, and the accelerated voltage during the energy spectrum shooting was 15 kV. The detector was a SE2 secondary electron detector.

2.7.3 XRD and X-ray photoelectron spectroscopy (XPS)

The crystal structure of GBSE-AgNPs was verified by XRD (Bruker D8 Advance, Germany) and characterized at a working voltage of 40 kV, a tube current of 40 mA, a scanning range of 10–80°, Cu-kα as a ray source, and a scanning speed of 2°/min. The GBSE-AgNPs solution was freeze-dried to obtain the powder, 100 mg of the powder was placed in the center of a fluted glass substrate, diluted with a slide, and then tested.

After taking an appropriate amount of sample, paste it on the sample tray, and put the sample into the sample chamber of XPS (Thermo Scientific K-Alpha) instrument. When the pressure of the sample chamber is less than 2.0 × 10⁻⁷ mbar, the sample is sent to the analysis chamber, the spot size is 400 μm, and the working voltage is 12 kV. Filament current 6 mA; Full spectrum scanning energy 150 eV, step size 1 eV; Fine spectrum scanning has a throughput of 50 eV and a step size of 0.1 eV.

2.7.4 High-resolution transmission electron microscopy (HRTEM)

The particle size and morphology of GBSE-AgNPs were analyzed by HRTEM (JEOL JEM-F200, Japan), and the elemental composition of GBSE-AgNPs surface was analyzed by energy spectrometer. Part of the sample was dispersed into ethanol solution for ultrasound, and then a few drops of the dispersed liquid were added to the copper net one by one. After drying, the accelerated voltage was 200 kV and the energy spectrum model was JED-2300T. The morphology and energy spectrum line scanning were taken.

2.7.5 FTIR spectroscopy

The organic functional groups of GBSE-AgNPs were analyzed by FTIR spectrometer (Nicolet iS5 FTIR spectrometer, Thermo Fisher scientific Co. Ltd, USA). The absorption bands were studied in the range of 4,000–400 cm⁻¹. The GBSE-AgNPs powder 1 mg obtained from the lyophilized solution was mixed with KBr powder 100–200 mg and pressed into a transparent sheet. The slices were scanned by infrared spectroscopy with a scanning range of 4,000–400 cm⁻¹.

2.7.6 Monosaccharide composition analysis

Set Mannose, Ribose, Rhamnose, Glucuronic acid, Galacturonic acid, N-acetyl-glucosamine, Glucose, and N-acetyl-galactose aminosus, Galactose, Xylose, Arabinose, Fucose are weighed and dissolved in ultra-pure water to prepare a mixed reference solution. The final concentration of the mixed reference solution was 0.4 mg/mL. GBSE-AgNPs (10.0 mg) and trifluoroacetic acid (TFA, 2 M, 5.0 mL) were added into a 10 mL hydrolysis tube, sealed with N2 (10 L/min, 1 min), and then hydrolyzed in a 110°C oven for 2 h. After cooling, open the lid, take out 1 mL and add 1 mL methanol, then dry with N2 in a water bath at 70°C, repeat adding methanol and dry with N2 twice to remove TFA. Adding 1 mL of 0.3 mol/L NaOH solution to fully dissolve the residue, the polysaccharide hydrolysate was determined after some dilution.

400 μL of mixed monosaccharide standard solution or polysaccharide hydrolysate was taken into 5 mL stoppered test tube, and 400 μL of PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution was added, and then mixed in swirl. Reaction was carried out in 70°C water bath for 2 h. Remove and allow it to cool to room temperature. Add 400 μL of 0.3 M of HCl to neutralize (pH 6–7). Add 1,200 μL of water and an equal volume of chloroform, mix thoroughly by swirling and shaking, then allow the mixture to stand. Discard the chloroform phase. Repeat the extraction twice. The aqueous phase was filtered by 0.45μm microporous membrane (water system) for high-performance liquid chromatography (HPLC) sampling analysis.

2.7.7 Analysis of particle size and zeta potential

The particle size distribution and zeta potential of GBSE-AgNPs were measured by dynamic light scattering (DLS) (Malvern Zetasizer Nano ZS90, DLS) analyzer. First, 1 mL of GBSE-AgNPs solution was ultrasounded for 5 min, then placed in a specific sample pool to determine the particle size distribution and zeta potential, then examined at 25°C and repeated three times.

2.7.8 Synchronous thermal analysis (using thermogravimetric [TG] and differential scanning calorimetry [DSC])

The thermal stability of the synthesized GBSE-AgNPs was evaluated by TG and DSC characterization using a TGA (German Netzsch STA 449 F3). GBSE-AgNPs obtained after lyophilization were used for TG and DSC analysis. The 20 mg GBSE-AgNPs was placed in the crucible from 30 to 800°C at a rate of 10°C/min and the nitrogen flow rate was 40 mL/min. Nitrogen levels were checked periodically during the analysis.

2.7.9 Time stability and pH stability

10 mL of GBSE-AgNPs diluent was prepared by deionized water and set aside at 4°C. At a specific time, the spectral morphology of GBSE-AgNPs at 300–600 nm and the absorbance at the characteristic wavelength were detected by UV-Vis to verify the time stability.

10 mL of GBSE-AgNPs concentrate was prepared by high-speed cryogenic centrifuge at 12,000 rpm for 10 min. 10 μL GBSE-AgNPs concentrated liquid was fully mixed with 90 μL deionized water with different pH (1–10), and the spectral morphology and absorbance of GBSE-AgNPs at 300–600 nm and characteristic wavelength were detected to verify pH stability.

2.8.1 Bacterial resistance performance

Sa and E. coli came from the microbiology laboratory of Shaoxing Central Hospital. The above strains were inoculated in LB liquid medium at 37°C overnight in a shaker. Then, the bacterial precipitation was obtained by centrifuge at low temperature and high speed at 4°C and 4,000 rpm. We diluted the bacterial precipitation with sterile normal saline and made the bacterial solution concentration to 1 × 10⁶ CFU/mL, and set aside.

The paper method was used to verify the bacteriostatic zone [27]. The method is as follows: the heated sterile agar medium was poured into the bacterial culture dish and allowed to cool at room temperature, during which it solidified to form an agar plate. Take a sterile cotton swab with a concentration of 1 × 10⁶ CFU/mL and evenly apply it three times on the agar plate, each time in a different direction. Sterile blank drug-sensitive paper with a diameter of 6 mm was immersed in different concentrations of GBSE-AgNPs, and then added to the AGAR dish containing bacteria, cultured at 37°C for 16–18 h. The size of the sterile area treated by GBSE-AgNPs to bacteria was observed at different concentrations.

Minimal inhibitory concentration (MIC) was investigated by a 96-well plate. First, 100 μL of bacterial solution (1 × 10⁶ CFU/mL) was added into the 96-well plate, and then GBSE-AgNPs of different concentrations were added, so that the final concentration was 0, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg/mL at 37°C for 16–18 h. Part of the solution could be seen in the turbid pore chamber. And 5 μL of Azurin indicator liquid (0.016 g dissolved in 100 mL of sterile distilled water) was added and incubated in the dark [28]. Observe the color change of the whole plate, pink is the positive result, and cyan is the running result. A cyan-colored sample located immediately before a sample showing a pink result is identified as the minimum inhibitory concentration (MIC). The minimal bactericidal concentration (MBC) verification was subsequently performed on an agar plate. The cyan result sample before the indicator color was pink was selected, and the sample was coated on an agar plate with a sampling needle dipped in bacterial solution, and cultured at 37°C for 16–18 h. Colony formation was observed [29]. The concentration of the colony-free group was MBC, and all operations were performed three times.

Mix 1 mL of bacterial solution (1 × 10⁶ CFU/mL) with GBSE-AgNPs (128 μg) at 37°C and place overnight in a rocking bed. Then, the bacterial precipitation was obtained by centrifuge at low temperature and high speed at 4°C and 4,000 rpm. The precipitation was collected, washed twice with PBS, the supernatant was discarded, and 2.5% glutaraldehyde fixing solution at room temperature was slowly added along the tube wall and fixed overnight. A few samples were added to the copper net, dried naturally, and observed by TEM.

2.8.2 Antifungal activity

Four common environmental fungi, such as Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, and Penicillium marneffei, were selected for study. A few fungi were selected with sampling needle and inoculated on Sabouraud’s agar plate, cultured at 37°C for 48 h. Next we added 20 μL of GBSE-AgNPs (50, 100, and 200 μg/mL) with different concentrations on the Sabouraud’s agar plate and evenly applied it with a coating stick. Taking the edge of the fungal colony as the target, 1.5 × 1.5 mm² bacteria cake was selected with the inoculum gun and inoculated on the Sabouraud’s agar plate containing GBSE-AgNPs at 37°C for 48 h. The change in fungal size was observed [30].

2.8.3 Anticancer ability

SGC-7901 cells at the logarithmic growth stage were inoculated in 96-well plates at a density of 3 × 10³ cells/well, and cultured in a humid environment composed of 5% CO₂ at 37°C for 24 h. GBSE-AgNPs solutions of different concentrations (0, 1, 2, 3, 4, 5, 6, 7, and 8 μg/mL) prepared with DEME complete medium were added to each well at 100 μL/well. Each group was tested in triplicate. After culture for 24 h, discard the old medium, add 100 μL of 10% CCK-8 solution in the dark, and continue to culture for 1 h. Absorbance at 450 nm was measured with a microplate reader. Cell survival rate (%) was calculated [31,32]. The following formula is used to measure the percentage of rejection:

where A ₀ is the negative control group and A ₁ is the GBSE-AgNPs group.

Apoptosis experiments were performed with Annexin V-FITC/7-AAD fluorescent double-stain apoptosis detection kit [32]. SGC-7901 cells in the logarithmic growth phase were inoculated in 24-well plates at a density of 5 × 10⁴ cells/well, and cultured in a humid environment composed of 5% CO₂ at 37°C for 24 h. The cells were digested with 0.25% pancreatic enzyme without EDTA, centrifuged at 300 × g for 5 min, the supernatant was discarded, the cells were collected, washed once with PBS, and the cells were gently suspended, centrifuged at 300g for 5 min, and the supernatant was discarded. The cells were washed with PBS once, the supernatant was discarded after centrifugation, and the cells were re-suspended by adding 500 μL of diluted 1× Annexin V Binding Buffer. The cell suspension was added with 5 μL Annexin V-FITC stain and 5 μL 7-AAD stain (100 μg/mL). After gentle swirl mixing, incubated at room temperature and away from light for 15–20 min and then immediately studied with the machine.

2.8.4 Free radical scavenging detection

The bleaching rate of 1.1-diphenyl-2-trinitrohydrazine (DPPH) was detected at characteristic wavelengths indicating free radical scavenging tests [33]. The hydrogen atom or electron donor potential of the sample was determined by bleaching the purple DPPH ethyl alcohol solution. The DPPH solution containing 5, 10, 20, 30, 40, 50 μg/mL of GBSE-AgNPs was obtained by adding different concentrations of GBSE-AgNPs to 1 mL of DPPH (0.4 mM) ethanol solution. The combination was vigorously stirred in the dark for 30 min, then the absorbance of the resulting solution was measured using a 517 nm spectrophotometer. Ascorbic acid was the positive control group, deionized water was the negative control group. The following formula is used to measure the percentage inhibition of DPPH free radical scavenging activity:

DPPH free radical scavenging activity percentage = A 0 − A 1 / A 0 × 100 , where A ₀ is the absorbance of the positive control group and A ₁ is the absorbance of the sample group.

2.8.5 Ferric reducing antioxidant power (FRAP) assay

The ferric reducing power was measured as described by Cherian et al. [34]. To 190 µL of FRAP solution (TPTZ [2,4,6-tri(2-pyridyl)-1,3,5-triazine]; 10 mM), acetate buffer (300 mM, pH 3.6), and ferric chloride hexahydrate (FeCl₃·6H₂O; 20 mM) in a 10:1:1 (v/v/v) ratio], 10 µL of GBSE-AgNPs was added and incubated at room temperature for 15 min. The absorbance at 630 nm was recorded, and the reducing antioxidant effect was determined as Trolox equivalent antioxidant capacity (TEAC).

2.8.6 ABTS antioxidant assay

The ABTS assay was conducted according to the protocol outlined by Cherian et al. [34]. The reaction mixture consisted of equal proportions of 7 mM ABTS salt and 2.5 mM potassium persulfate, which was kept in the dark for 14–16 h. Before adding the GBSE-AgNPs, the absorbance was measured at 734 nm and adjusted to 0.7. Different concentrations of GBSE-AgNPs were then added to the reaction mixture and incubated at room temperature for 15 min under dark conditions. The antioxidant effect was assessed using TEAC, and the absorbance was measured at 734 nm (Trolox C equivalent antioxidant capacity, mM).

3.2.1 Reaction time

The effect of reaction time on the synthesis process was studied by measuring the absorbance change at different times (30, 60, 120, 180, and 240 min). The remaining conditions are set as follows: temperature 90°C and volume ratio 40:1 (GBSE:AgNO₃, v/v). As presented in Figure 1f, the formation of GBSE-AgNPs was confirmed by detecting the change in absorbance at ∼415 nm. The results proved that the absorbance changes rapidly with the extension of time (30–240 min). The absorbance changes slowly within 30–60 min. In the period of 60–180 min, the absorbance increased significantly, and the fastest speed was achieved at 120 min, while the value of absorbance increased slowly at 180 min, which seemed to reach a plateau. This may be the reason that in the beginning, there is rapid nucleation, forming many small particles. Over time, these particles grow by consuming more Ag ions, and the number of available ions decreases, slowing the rate at which new particles form and grow. As small particles form, this can lead to an initial increase in particle concentration, followed by a slower growth phase as larger particles grow at the expense of smaller ones [35,36]. At time point of 240 min, the absorbance further improved, and a significant decrease in the volume of the solution was observed compared to 0 min. Considering the decrease in the solution, there was a concentration phenomenon, which affected the absorbance value. It may also be due to the dissolution of smaller NPs after sufficient nucleation, while larger NPs grow faster, which may lead to an observed increase in absorbance [37]. Therefore, considering various factors, the optimal reaction time for RSM is chosen as 180 min.

3.2.2 Reaction temperature

The effect of reaction temperature on synthesis was studied by measuring the absorbance change at different temperatures (60, 70, 80, 90, and 95 min). The other synthesis conditions are fixed. As shown in Figure 1g, when the temperature is 60–80°C, no obvious absorption wavelength can be seen, so the reaction can be considered incomplete. With the increase in reaction temperature, the absorbance value increases gradually, and the absorption peak appears. When the temperature level is 90°C, the absorbance value increases significantly, which shows the dependence of reaction temperature in the preparation process of GBSE-AgNPs. This acceleration is mainly due to the enhancement of the kinetics of the reduction reaction. When heated, the energy provided increases the reaction rate, promoting faster reduction of silver ions (Ag⁰) to silver atoms, and the subsequent nucleation and growth of NPs [38]. However, at 95°C, the maximum absorption peak occurs blue shift, indicating that higher temperature will increase the reaction rate of silver ion reduction to form NPs. This faster reaction rate can lead to smaller particle sizes and result in a blue shift of the surface plasmon resonance (SPR) peak. This indicates that the size of small particles is larger and may still be in the process of nucleation [39,40]. Therefore, the optimal reaction temperature of RSM is 90°C.

3.2.3 Reaction ratio

The effect of reaction ratio on synthesis was studied by detecting the absorbance change at different ratios (10, 20, 40, 80, and 160:1). The other conditions are fixed. As demonstrated in Figure 1h, no obvious SPR was observed when the ratio was 80:1 and 160:1, which may be due to the excessive concentration of GBSE, which covered up the peak of GBSE-AgNPs. Because when the ratio is increased to 80:1 and 160:1, the solubility of GBSE is exceeded, and there will be powder precipitation after standing. At the ratio of 10:1, 20:1, and 40:1, the absorbance values show a proportional dependence, and when the ratio is set at 40:1, the absorbance value is the largest. In addition, the absorbance of 40:1 ratio is larger than that of 80:1 ratio. Therefore, the optimal response ratio for RSM is 40:1.

Therefore, based on the above results, the experimental conditions for RSM are as follows: reaction time was 180 min, reaction temperature was 90°C, and reaction ratio (GBSE:AgNO₃, v/v) was 40:1.

3.3.1 Box-Behnken design

According to the SFE results, three-factor and three-level analysis was carried out through the RSM design, and the design was depicted in Table S1. The reaction ratio is 20:1 in low level, 80:1 in high water, and 50:1 in the middle as system generation. The reaction temperature is 85°C in low level, 95°C in high water, and 90°C in the middle for system generation. The reaction time was 120 min in low level, 240 min in high water, and 180 min in the middle of system generation. With the absorbance value as the response value, 17 tests were analyzed, and the results were exhibited in Table S2.

3.3.2 Model fitting and variance analysis

Regression analysis was performed on the data in Table S2, and the prediction equation was obtained as follows:

where A represents the reaction volume ratio, B represents the reaction temperature, and C represents the reaction time. According to the absolute value of the coefficient before the factor in the equation [41], it is shown that the changes affecting the absorbance value are as follows: reaction temperature > reaction time > reaction proportion.

Through the quadratic model variance analysis on the data in Table S2, the results are demonstrated in Table S3, F-value = 10.41, p < 0.01, and the model is not significant. The Lack of Fit P-value of 0.1167 suggests that the Lack of Fit is not significant relative to the pure error, indicating that the model fits the experimental data well. The R-squared value of 0.9305 indicates that 93.05% of the variability in the response can be explained by the model. The adjusted R-squared value of 0.841 accounts for the number of predictors and suggests a very good fit of the model. Therefore, the model can replace the actual detection to study and predict the synthesis of GBSE-AgNPs process. In addition, according to Table S3, B, C, AC, and B2 were significant factors (p < 0.05). The high F-values of B (40.51) and C (21.41) indicate that these factors have a significant effect on the variability of absorbance values. B has the greatest influence on the absorbance value, F-value is 40.51, p-value is 0.0004, and the significance level is the highest. C is second, F-value is 21.41, p-value is 0.0024, which is also significant. A has a certain effect, but it is not as significant as B and C. F-value is 3.80 and p-value is 0.0923, which is close to the significant level. In addition, the steepness of the 3D graph can be used to predict the influence of two factors on the response value [42]. As can be seen from the 3D response surface and contour map in Figure 1Ia, the slope of the surface is small and the color gradient changes gently. This suggests that the interaction between time and proportion has a relatively small effect on the response value. As can be seen from the 3D response surface and contours in Figure 1Ib, the slope of the surface is large, and the color gradient changes rapidly, especially in the region of high temperature and high proportion. This indicates that the interaction between temperature and proportion has a large influence on the response value. As can be seen from the 3D response surface and contour diagram of Figure 1Ic, the slope of the surface is small, and the color gradient changes gently. This suggests that the interaction between temperature and time has a relatively small effect on the response value. Through comprehensive analysis of the 3D response surface map and contour map, as well as the results of variance analysis, the following conclusions can be drawn: reaction temperature > reaction time > reaction proportion. In the interaction, the interaction between temperature and proportion (AC) has a significant effect on the response value. In the quadratic term, temperature (B²) has a significant effect on the response value. Therefore, in the process of RSM and experimental optimization, we should focus on the control of temperature and time to optimize the response value.

3.3.3 Verification of optimal conditions

According to the prediction equation given by the Design-Expert 10 software, the optimal synthesis conditions can be obtained: reaction temperature, reaction proportion, and reaction time are 94.725°C, 23.165 (v/v), and 235.890 min, respectively. According to the actual situation, the synthesis conditions were adjusted to the reaction temperature, proportion, and time of 94.7°C, 23.2 (v/v), and 236 min, respectively. Three experiments were conducted under these conditions, and the absorbance value was 0.9707. The predicted value is 0.9611, which is basically consistent with the actual value, which indicates the feasibility and accuracy of the model in predicting the optimal conditions for GBSE-AgNPs synthesis.

3.5.1 Antioxidant activities of biosynthesized GBSE-AgNPs

In Figure 4a, DPPH free radical scavenging experiments proved that GBSE-AgNPs possess the dose-dependent scavenging activity on DPPH free radicals. With the increase in GBSE-AgNPs concentration, the inhibitory percentage of DPPH free radical scavenging activity also increased. The scavenging activity of GBSE-AgNPs was lower than that of positive control ascorbate, but higher than that of GBSE, indicating its effective oxygen free radical scavenging ability. Evaluation of oxygen free radical scavenging activity is essential to evaluate the antioxidant potential of GBSE-AgNPs. The ability of GBSE-AgNPs to scavenge DPPH free radicals highlights their potential applications as antioxidants in various industries, including pharmaceuticals, cosmetics, and food preservation.

Figure 4

Bioactivity of GBSE-AgNPs. (a) Antioxidant activity measured using the DPPH assay. (b) IC₅₀ value for gastric cancer cell line 7901. (c) and (d) Analysis of apoptosis. (e) Antibacterial activity assessed using the inhibition zone method. (f) MIC. (g) MBC. (h) Bacterial viability observed under HRTEM.

FRAP assay results (Figure S1) demonstrate the concentration-dependent ferric reducing antioxidant activity (TEAC values) of GBSE, GBSE-AgNPs, and ascorbic acid (positive control). Across all tested concentrations (5–50 µg/mL), ascorbic acid consistently exhibited the highest antioxidant activity, with TEAC values increasing up to approximately 600 µM at the highest concentration (50 µg/mL). In contrast, the TEAC values of GBSE and GBSE-AgNPs were significantly lower. At 50 µg/mL, the TEAC value of GBSE-AgNPs reached approximately 150 µM, while GBSE only achieved 70 µM. This indicates that GBSE-AgNPs demonstrated a modest but statistically significant improvement in antioxidant activity compared to GBSE (**p < 0.01). However, both GBSE and GBSE-AgNPs were markedly less potent than ascorbic acid (^# p < 0.0001). Notably, the enhancement in antioxidant activity observed for GBSE-AgNPs can be attributed to the synergistic interaction between AgNPs and the bioactive phytochemicals in GBSE, which likely enhances their reducing power.

The antioxidant activity of GBSE, GBSE-AgNPs, and ascorbic acid was evaluated using the ABTS radical scavenging assay, and the results are presented in Figure S2. The data demonstrate that GBSE-AgNPs exhibit a dose-dependent antioxidant activity, with increasing concentrations (5–50 μg/mL) resulting in a significant increase in TEAC values (μM). The activity of GBSE-AgNPs was lower than that of the positive control (ascorbic acid), but surpasses that of the GBSE extract alone. And this indicated the enhancement of antioxidant properties. The statistical significance (*p > 0.05, ^# p > 0.0001) clearly illustrates that ascorbic acid remains the most potent antioxidant among the tested groups. GBSE-AgNPs exhibit significantly higher antioxidant activity compared to GBSE, supporting the hypothesis that the bioreduction and capping process involving GBSE contributes to the enhancement of the free radical scavenging potential. The superior antioxidant activity of GBSE-AgNPs can be attributed to the synergistic interaction between AgNPs and the bioactive phytochemicals present in GBSE. These bioactive compounds, including phenolics and flavonoids, are known to donate electrons, stabilizing free radicals, and enhancing antioxidant activity. The dose-dependent increase in activity underscores the potential of GBSE-AgNPs as a promising antioxidant agent [49,50], which could be explored further for applications in oxidative stress-related disorders.

3.5.2 Anti-cancer ability

As exhibited in Figure 4b, CCK-8 detection showed that GBSE-AgNPs had a dose-dependent inhibitory effect on cell viability in SGC-7901 cells. With the increase in GBSE-AgNPs concentration, the percentage of cell viability inhibition also increased, indicating that AgNPs has cytotoxic effect on cancer cells, IC = 4.826 μg/mL. Similarly, apoptosis assay exhibited a significant increase in apoptotic cells after treatment with GBSE-AgNPs compared to the control group, suggesting that induction of apoptosis is the mechanism of action for the anticancer activity of AgNPs (Figure 4c and d). The ability of GBSE-AgNPs to inhibit cell proliferation and induce apoptosis highlights their promise as anticancer agents for further preclinical and clinical studies. Similar anticancer mechanisms have been reported for AgNPs synthesized using other plant extracts, such as A. acuminata  leaf which also demonstrated dose-dependent inhibition and apoptosis induction in gastric cancer cells [51]. However, compared to previously reported IC₅₀ values for AgNPs synthesized using other biological agents (IC₅₀ = 21.05 μg/mL) [52], GBSE-AgNPs exhibit superior potency (IC₅₀ = 4.826 μg/mL), suggesting the unique contribution of the GBSE phytochemicals in enhancing anticancer activity. This underscores the potential advantage of using underutilized resources like GBSE for AgNP synthesis.

3.5.3 Antibacterial activity

A disk assay was used to evaluate the antibacterial activity of GBSE-AgNPs against bacterial strains (Sa and E. coli). As exhibited in Figure 4e, in the bacterial Petri dish containing Sa, obvious bacteria-free zone was observed only around the paper with a concentration of 128 μg/mL GBSE-AgNPs, indicating the antibacterial effect of GBSE-AgNPs on Sa. However, no significant bacteria-free zone was observed around the other concentrations (16, 32, 64 μg/mL). In the bacterial Petri dish containing E. coli, the clear bacteria-free zone was observed around the paper with the concentration of 16, 32, 64,128 μg/mL of GBSE-AgNPs, indicating that GBSE-AgNPs possess a strong antibacterial effect on E. coli.

The MIC and MBC values of GBSE-AgNPs were determined by half dilution method. Specifically, MIC was defined as the lowest concentration of GBSE-AgNPs that completely inhibited visible bacterial growth, while MBC was determined to be the lowest concentration that caused bacterial death. The MIC and MBC values of GBSE-AgNPs for Sa were 32 and 128 μg/mL, respectively, and the MIC and MBC values of GBSE-AgNPs for E. coli were 0.5 and 2 μg/mL, respectively. These results are comparable to or exceed those reported for AgNPs synthesized using Flos Sophorae Immaturus extract and tansy extract, where MIC values for Sa and E. coli ranged between 31.25 and 31.3 μg/mL [53,54]. Interestingly, the significantly lower MIC for GBSE-AgNPs against E. coli suggests that the unique composition of GBSE, particularly the presence of functional groups such as carboxyl or hydroxyl groups [1], may enhance the interaction of NPs with Gram-negative bacteria. This highlights the potential of GBSE as an efficient reducing and stabilizing agent for producing potent antibacterial NPs. The above results demonstrated that GBSE-AgNPs possess strong antibacterial and bactericidal activities against both Gram-positive and Gram-negative bacteria (Figure 4f and g).

Subsequently, we employed HRTEM to examine the morphological changes in bacteria exposed to GBSE-AgNPs. It can be seen that after treatment with GBSE-AgNPs, the bacterial morphology underwent significant changes, including cell membrane damage, cytoplasmic leakage, and cell deformation (Figure 4h). These findings elucidate the antimicrobial mechanism of GBSE-AgNPs, which predominantly involves the disruption of bacterial cell membranes and intracellular components. Such mechanisms are consistent with previous reports on AgNPs synthesized using alternative green methods, further highlighting the enhanced efficacy of GBSE-derived AgNPs [1]. The interaction between GBSE-AgNPs and bacterial cells could be attributed to the presence of functional groups identified in the FTIR analysis, such as hydroxyl and carboxyl groups, which facilitate strong adhesion to bacterial membranes and subsequent cell damage [1].

Furthermore, the morphological changes of bacterial cells exposed to GBSE-AgNPs were observed by SEM. As demonstrated in Figure 5a, the uniform analysis of silver elements on the surface of Staphylococcus aureus proved that atomic percentage (%) was 0.2% and weight percentage (%) was 0.8%. Energy spectrum line scans demonstrated a homogenous distribution of silver across the bacterial surface, supporting the hypothesis that GBSE-AgNPs effectively adhere to the bacterial membrane, facilitating their antibacterial mechanism through membrane disruption and intracellular component leakage. Further analysis by energy spectrum line sweep indicate that the distribution of silver element is uniform. Similarly, EDS data of E. coli are revealed in Figure 5b. Scattered silver elements can be analyzed in E. coli, with atomic percentage (%) of 0.4% and weight percentage (%) of 1.2%. Energy spectrum line scanning further confirmed the uniform distribution of silver elements on E. coli surfaces, emphasizing the ability of GBSE-AgNPs to achieve effective and consistent silver deposition on diverse bacterial species. These findings suggest that the uniform adhesion and deposition of GBSE-AgNPs on bacterial surfaces play a crucial role in their bactericidal activity.

Figure 5

SEM/EDS analysis of Sa (a) and E. coli (b) showing the distribution of silver elements on their surfaces after reaction with GBSE-AgNPs.

3.5.4 Antifungal activity

Four common environmental fungal strains, including Aspergillus niger, Aspergillus fumigatus, Aspergillus flavus, and Penicillium marneffei, were selected to evaluate the antifungal activity of silver nanoparticles. All four fungi are widely present in the environment and are the most common pathogenic fungi in humans [55]. The antifungal activity of GBSE-AgNPs was evaluated by agar diffusion method. As shown in Figure 6, the growth of fungal colonies on Sabouraud’s agar plates containing GBSE-AgNPs was visually observed and compared with control plates without GBSE-AgNPs. Changes in fungal size, morphology, and growth inhibition were qualitatively assessed. GBSE-AgNPs exhibited dose-dependent inhibition of fungal growth. Smaller fungal volumes were observed around the pores containing 50 μg/mL GBSE-AgNPs than in the control group, but were not statistically significant. The fungal volume of 100 and 200 μg/mL GBSE-AgNPs treated pores was significantly reduced compared to the control group, indicating that they had effective antifungal activity against all fungal strains tested. The diameter of the inhibition zone was positively correlated with the concentration of GBSE-AgNPs.

Figure 6

Antifungal activity of GBSE-AgNPs.

Evaluating the antifungal activity of GBSE-AgNPs against common environmental fungal strains is critical for developing effective antifungal strategies applicable in various settings, including indoor environments, healthcare facilities, and agricultural practices. Understanding the antifungal properties of GBSE-AgNPs provides valuable insights into their potential to mitigate fungal contamination and prevent fungus-related diseases.

Silver-containing NPs are widely recognized for their broad-spectrum and potent antimicrobial activities [56]. Even at low concentrations, they exhibit efficacy against diverse pathogenic microorganisms, including bacteria such as E. coli, Klebsiella pneumoniae, and Sa, as well as fungi such as Candida albicans and Aspergillus niger. In this study, the synthesized GBSE-AgNPs demonstrated exceptional antibacterial and antifungal activities, expanding the application scope of AgNPs as antimicrobial agents.

Unlike conventional antibiotics, which are increasingly associated with microbial resistance, AgNPs exhibit multiple antimicrobial mechanisms, leading to limited reports of resistance development [57]. This property highlights their advantage over conventional antimicrobials and underscores their importance in addressing the growing challenge of antimicrobial resistance. Currently, several proposed mechanisms explain the antimicrobial effects of AgNPs. As illustrated in Figure 7, 1. AgNPs continuously release silver ions, which inhibit microbial growth by interfering with key cellular processes [58]. 2. Silver ions denature ribosomes in the cytoplasm, inhibiting protein synthesis [59]. 3. Silver ions adhere to the bacterial cell wall and plasma membrane via electrostatic attraction and their affinity for thiol-containing proteins, increasing membrane permeability and causing cellular envelope destruction [60]. 4. Once internalized, silver ions inactivate respiratory enzymes, produce ROS, and disrupt ATP production, leading to oxidative stress and cellular damage [61]. 5. Silver ions interact with sulfur and phosphorus in DNA, inhibiting DNA replication, causing abnormal cell division, and ultimately leading to cell death. These multifaceted mechanisms explain the observed potent antimicrobial effects of GBSE-AgNPs and their potential to overcome microbial resistance.

Figure 7

Proposed antibacterial mechanism of GBSE-AgNPs.

Previous studies on GBS are limited due to their low polysaccharide content, predominantly composed of mannose, and the low extraction efficiency of these bioactive compounds. In this study, we directly obtained GBSE via a simple water extraction method and synthesized GBSE-AgNPs. Mannose, the primary polysaccharide component, was confirmed through HPLC analysis. By optimizing the preparation process of GBSE-AgNPs using RSM, this study expands the research scope of GBS and highlights their potential as a novel source of bioactive compounds.