A novel green synthesis of gold nanoparticles using Syrian bee bread extract: phytochemical analysis, characterization, antimicrobial activity and biocompatibility

Abstract

This study investigates the bioactive composition of Syrian bee bread (SBB) and its application in the green synthesis of gold nanoparticles (AuNPs). For the first time, bee bread was utilized for the synthesis of AuNPs. The SBB extract exhibited high levels of phenolic and flavonoid compounds. High-performance liquid chromatography (HPLC) identified key phenolic flavonoid, and sugar components, while Gas chromatography-mass spectrometry (GC-MS) revealed various bioactive constituents. The synthesis was optimized at pH 9.5, gold salt concentration of 2 mM, SBB extract 1%, and room temperature, yielding small, stable and well-dispersed AuNPs with Z-average of 46.38 ± 6.12 nm, polydispersity index (PDI) of 0.235 ± 0.002, and ζ-potential of − 29.7 mV according to Dynamic Light Scattering (DLS). Ultraviolet-visible spectroscopy (UV-Vis) showed a Localized surface plasmon resonance (LSPR) peak at 524 nm. Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscopy (FESEM) confirmed spherical nanoparticles, with sizes of ~ 33 nm and ~ 28.69 nm, respectively. The SBB-AuNPs displayed antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Klebsiella Spp., and Candida albicans, with inhibition zones ranging from 18 to 27 mm. Importantly, no cytotoxic effects were observed in normal fibroblast cells at concentrations below 100 µg/mL indicating promising biocompatibility. This novel approach yields small, stable, and bioactive nanoparticles with potent antimicrobial properties and high biocompatibility, representing a significant improvement over other bee product-based methods. These findings highlight the potential biomedical applications of SBB-AuNPs, including their incorporation into topical creams or ointments for the prevention and treatment of skin infections, wound-healing formulations, and as antimicrobial agents in medical devices or protective coatings.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12906-026-05383-6.

Introduction

Green synthesis has recently gained considerable attention as a sustainable and eco-friendly alternative to traditional physical and chemical methods for nanoparticle production, utilizing biological sources [1–4]. This eco-friendly approach not only minimizes toxic by-products, environmental pollution, and health hazards, but also provides enhanced control over nanoparticle size, morphology, and stability, while reducing energy and time requirements [5]. Secondary metabolites, such as flavonoids, terpenoids, polyphenols, sugars, alkaloids, and proteins, play a pivotal role in this process by facilitating both the reduction of metal ions and the stabilization of the resulting nanoparticles [6]. Among the various metal nanoparticles synthesized via green synthesis, AuNPs have emerged as particularly promising due to their unique physicochemical and biological properties. Their small size, high surface to volume ratio, excellent biocompatibility, and low biotoxicity make them highly valuable for a broad range of biomedical applications [7]. Their multifunctional biological activities- including antibacterial, antiviral, antifungal, anticancer, antioxidant, and anti-inflammatory effects- have been extensively documented [8–13], highlighting their potential across diverse biomedical and industrial domains [14–19]. Building on the growing interest in biologically mediated nanoparticle synthesis, bee products have gained attention as natural, safe, and sustainable reducing and stabilizing agents. Previous studies have demonstrated successful green synthesis of AuNPs using honey [20], propolis [21] and bee pollen [22] largely attributed to their rich content of phenols, flavonoids, terpenes, proteins, vitamins, and minerals [23]. Bee products differ in their degree of scientific exploration; among them, honey is the most extensively studied, while bee bread remains comparatively underexplored despite its high biological value and unique chemical composition, highlighting a clear novelty gap. Bee bread is produced when honey bees mix collected pollen, which is naturally rich in fats and proteins, with honey and secretions from their salivary glands. This mixture is stored in the hive cells and fermented by lactic acid bacteria (LAB) and yeasts. Serving as the primary food source for larvae and young workers that produce royal jelly, bee bread generally consists of water, proteins, free amino acids, carbohydrates, fatty acids, reducing sugars, vitamin K, antioxidants, and a wide array of minerals, including some rare elements that exceed the mineral content typically found in pollen [24–27]. Its chemical composition and biological activity—including antimicrobial effects against Gram-positive and Gram-negative bacteria and fungi, antioxidant, anticancer, anti-obesity, and antiviral effects are influenced by factors such as pollen type, bee microbiota, fermentation duration, geographic origin, plant source, harvest season, and extraction methodology [28–32].With the rapid advances in green synthesis of nanoparticles, there is a growing need to explore new biological sources that can produce effective and safe nanomaterials. Various bee products such as honey, propolis, and bee pollen have been previously used for the green synthesis of AuNPs due to their rich content of functional compounds. Compared to these bee-derived materials, Syrian bee bread (SBB) obtained from citrus pollen exhibits a uniquely balanced chemical composition, combining phenols, flavonoids, and natural sugars that synergistically enhance nanoparticle reduction and stabilization. However, bee bread remains underexplored compared to other bee products, representing a significant novelty gap and untapped potential. In particular, Syrian bee bread has never been chemically characterized or studied for green synthesis of gold nanoparticles, making this the first report on its use. The bee bread extract was chemically characterized to determine its TPC and TFC, while HPLC was employed to analyze phenolics and reducing sugars. Additionally, GC-MS was used to identify key active constituents. Guided by these chemical analyses, the green synthesis process was optimized by varying key parameters, including pH (7.5, 8.5, 9.5, 10.5), temperature (25, 40, 60, and 80 °C), gold salt concentration (1, 2, 5 mM), and SBB extract concentration (1%, 5%, 10%). The resulting AuNPs were comprehensively characterized using advanced techniques. UV-Vis spectroscopy provided initial confirmation of AuNPs formation, while DLS measured particle-size distribution, and ζ-potential. The surface morphology and structure were examined using AFM and FESEM, providing detailed insights into the physical and structural properties that directly impact their functionality. Furthermore, Energy Dispersive X-ray Spectroscopy (EDS) coupled with FESEM was performed to assess the elemental composition of the nanoparticles, confirming the presence of gold and other constituent elements. To evaluate their biological potential, the antimicrobial activity of these nanoparticles was tested against Gram-negative bacterial strains: P. aeruginosa, E. coli, and Klebsiella spp., as well as the fungal pathogen C. albicans. Furthermore, cytotoxicity was assessed on normal human dermal fibroblast cells using the MTT assay to ensure their biocompatibility. The objective of this study is to demonstrate for the first time, the use of bee bread extract for the green synthesis of stable and bioactive AuNPs, establishing a clear link between the chemical composition of the extract and its role in nanoparticle formation and stabilization. This work not only introduces a novel biological route for AuNPs synthesis but also provides a scientific foundation for future biomedical applications of bee bread–derived nanomaterials.

Materials and methods

All chemical reagents and analytical-grade materials were purchased from Merck and Sigma-Aldrich, and used without any further purification. The chemicals included: hydrogen tetrachloroaurate (III) trihydrate (HAuCl₄·3H₂O), Folin-Ciocalteu reagent, gallic acid, quercetin, sodium carbonate, aluminum chloride, acetonitrile, formic acid, sodium hydroxide, MTT reagent, dimethyl sulfoxide (DMSO), and phosphate-buffered saline (PBS). Deionized water was used for all preparations and dilutions. Normal human dermal fibroblast cells (ATCC^® PCS-201-012™) were obtained from the American Type Culture Collection (ATCC). The cells were cultured and maintained following standard protocols and in accordance with standard ethical guidelines for in vitro research. No new human samples were collected for this study. Statistical analysis was performed using Origin software, applying One-way ANOVA followed by Tukey’s post-hoc test for pairwise comparisons. Results are expressed as mean ± SD, and differences were considered statistically significant at p < 0.05.

Preparation of the aqueous extract of SBB

Fresh SBB was collected in April 2023 from a local apiary located in the countryside of Damascus, Syria. The SBB originated mainly from citrus flower pollen (Citrus spp.), which is expected to influence its phytochemical composition. The sample was immediately stored in sterile, airtight containers at 4 °C to preserve its bioactive constituents until further use. A total of 10 g of SBB were mixed with 100 mL of deionized distilled water (ddH₂O) and stirred continuously using a magnetic stirrer at 350 rpm at room temperature (~ 25 °C) in the dark. The green synthesis of SBB-AuNPs was monitored and completed within 48 h. The mixture was then centrifuged at 4000 rpm for 15 min to remove impurities. The resulting supernatant was collected and stored at 4 °C for subsequent analyses.

Determination of total phenolic content

The TPC of SBB extract was determined using the Folin-Ciocalteu method [33], with slight modifications to adapt the protocol for SBB extract. Specifically, 0.5 mL of the SBB extract was used instead of 5 g of royal jelly, mixed with 1 mL of 10% Folin-Ciocalteu reagent, followed by the addition of 2.5 mL of 7.5% sodium carbonate solution. The reaction mixture was incubated at 45 °C for 30 min (compared to 20 min at room temperature in the original protocol), and the absorbance was measured at 755 nm. Gallic acid was used to generate the calibration curve, and the result was expressed as milligrams of gallic acid equivalent per gram of sample (mg GAE/g). These modifications involved adjustment of sample volume, reagent concentrations, and incubation conditions to suit the SBB extract.

Determination of total flavonoid content

The TFC of the SBB extract was determined using the aluminum chloride colorimetric method [34], with modifications to adapt the protocol for SBB extract. Specifically, 1 mL of 2% methanolic aluminum chloride solution was used instead of the multi-component mixture in the original protocol. The mixture was incubated at room temperature for 45 min, compared to 30 min in the original method. The absorbance was measured at 415 nm using a UV-Vis spectrophotometer. Quercetin was used as reference standard, and the result was expressed as milligrams of quercetin equivalent per gram of sample (mg QE/g). These modifications involved adjusting the reagent composition, concentration, and incubation time to suit the SBB extract.

Determination of sugar content

The sugar content of the SBB extract was determined using high-performance liquid chromatography (HPLC, Agilent 1100). The method was adapted, with slight modifications to suit the SBB extract, from the protocol described by Orascanin et al. (2025) [33] for royal jelly analysis. Specifically, the sample preparation was modified: a smaller sample volume was used, the extraction solvent ratio was adjusted to acetonitrile/deionized water (80:20, v/v) instead of 3:1 water/methanol, and the sample was injected in a 100 µL aliquot. Separation was carried out on an R-NH₂ column maintained at 35 °C (compared to 33 °C in the original protocol) at a flow rate of 1 mL/min. The concentrations of fructose, glucose, sucrose, and raffinose were identified and quantified, and the results were expressed as grams per 100 g of bee bread sample (g/100 g). These modifications involved adjustment of sample volume, solvent composition, injection volume, and column temperature to accommodate the SBB extract characteristics.

Quantitative determination of phenolic profile in SBB extract

The phenolic profile of the SBB extract was analyzed using high-performance liquid chromatography (HPLC, Agilent 1100) equipped with a UV/Visible detector. The method was adapted, with slight modifications to suit the SBB extract, from the previously reported protocol [31]. Specifically, the UV detection wavelength was set at 265 nm instead of 284 nm, the mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (acetonitrile) instead of the water/methanol/acetonitrile mixture, and the flow rate was adjusted to 1 mL/min instead of 0.6 mL/min. Separation was performed on a C18 symmetry column (250 mm × 4.6 mm, 5 μm), and a gradient elution program was applied optimized for SBB extract. The injection volume was 20 µL, and the column temperature was maintained at 30 °C. Compounds were identified by comparing retention times with authentic standards, and calibration curves were used to quantify the compounds.

Gas-chromatography mass spectrometry analysis

The GC-MS analysis of SBB extract was performed using an Agilent GC 6890 N coupled with a 5973 mass selective detector. The method was adapted from previously reported protocols [31] with precise modifications to accommodate the specific properties of SBB extract. A capillary Rtx‑5ms column (30 m × 0.25 mm i.d., 0.25 μm film thickness) was used instead of the DB‑WAX column reported in the reference. Helium was employed as the carrier gas at a constant flow rate of 0.9 mL/min instead of hydrogen at 1.9 mL/min, and split injection at a ratio of 1:20 was applied instead of splitless injection. The oven temperature program was adjusted for the SBB extract, starting at 40 °C for 5 min, then ramping to 200 °C at 5 °C/min, followed by an increase to 250 °C held for 5 min, while the injection volume and the injector and detector temperatures were maintained at 1 µL and 250 °C, respectively. The mass scan range was expanded to m/z 40–550 instead of 60–400 to capture a broader range of compounds present in the extract. Compounds were identified by comparing the mass spectra with those in the NIST and Wiley libraries. These precise adjustments improved reproducibility and optimized separation for the specific chemical profile of the SBB extract.

Optimization and characterization of SBB-AuNPs

AuNPs were synthesized using an aqueous extract of bee bread. In a typical procedure, an aqueous solution of HAuCl₄·3H₂O was added dropwise to the SBB extract in a 1:1 volumetric ratio (20 mL HAuCl₄·3H₂O solution:20 mL extract) under continuous stirring (350 rpm), with a gradual color change from yellow to wine red within approximately 30 min, indicating the formation of AuNPs. The resulting nanoparticles were centrifuged at 9000 rpm for 10 min. To optimize the synthesis conditions for obtaining small, dispersed and stable nanoparticles, several experimental parameters were systematically varied: reaction temperature (25, 40, 60, and 80 °C), solution pH (7.5, 8.5, 9.5, and 10.5), and HAuCl₄·3H₂O solution concentrations (1, 2, and 5 mM), and bee bread extract concentrations (1, 5, and 10%). The optimization process was guided by dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern), which was used to measure the Z-average and PDI. All optimization experiments for pH, temperature, and reactant concentrations were performed in triplicate, and the results are reported as mean± standard deviation (SD) to ensure statistical reliability. These parameters were essential to determine the optimal conditions for nanoparticles size and uniformity. Different techniques were used to characterize the AuNPs, including their optical properties, which were identified by an ultraviolet-visible spectrophotometer (Cary 5000) to measure the absorption peak of AuNPs. Morphological features were analyzed using atomic force microscopy (AFM - Nanosurf Easyscan 2, Tap 190 Al-G NanoSensors™, Switzerland) and field emission scanning electron microscopy (FESEM ZEISS Sigma 300, Baden-Württemberg, Germany) providing detailed insights into the surface topography, shape, and distribution of AuNPs. Furthermore, Energy Dispersive X-ray Spectroscopy (EDS) coupled with FESEM was performed to assess the elemental composition of the nanoparticles, confirming the presence of gold and other constituent elements. These comprehensive analytical approaches ensured accurate characterization of AuNPs, enabling correlation between their physicochemical properties and potential biological activities.

Evaluation of antimicrobial activity of AuNPs synthesized using SBB extract

The pathogenic strains were collected from the central laboratory at Aleppo University Hospital and were subsequently cultured on suitable growth media to ensure the effective development and proliferation of tested bacteria and fungi. Bacterial suspensions were adjusted to 0.5 McFarland standard (~ 1.5 × 10⁸ CFU/mL), while fungal suspensions were prepared at (~ 1 × 10⁶ CFU/mL) before use. The agar well diffusion method was employed to evaluate the antimicrobial effectiveness of AuNPs synthesized using the SBB extract against Gram-negative bacteria (P. aeruginosa, E. coli and Klebsiella spp.), as well as Candida Fungi (C. albicans). Gentamicin was used as standard antibiotic control for the bacterial strains, while Fluconazole was employed as standard antifungal control for C. albicans. Citrus bee bread extract (1%), HAuCl₄·3H₂O solution, and a blank control (culture medium without AuNPs) were used as controls in the experiment to demonstrate that the antimicrobial effects were attributed to the AuNPs themselves rather than the precursors. All tested agents were added in a volume of 100 µL into the wells. The final concentration of gold in AuNPs was ~ 197 µg/mL. Petri dishes were incubated at 37 °C, and inhibition zones were measured after 24 h. All antimicrobial tests were performed in triplicate, and the result to ensure reproducibility of the results.

Evaluation of cytotoxicity of AuNPs using MTT assay

To evaluate the cytotoxic effects of the synthesized AuNPs using SBB extract, normal human dermal fibroblast cells (ATCC^® PCS-201-012™) were seeded at a density of 1 × 10⁴ cells per well in a flat-bottom 96-well microplate and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂ to allow attachment. Cells were then treated with a broader range of AuNPs concentrations (0, 6.25, 12.5, 25, 50, 100, 200, and 400 µg/mL) and incubated for 48 h under the same conditions. Cell viability was assessed using MTT reagent at 0.5 mg/mL, and the resulting formazan crystals were solubilized with DMSO, exactly as described in the reference. The protocol was adapted from Machado et al. (2021) [3] with the key modifications being the use of human dermal fibroblast cells, the application of a wider concentration ranges of nanoparticles, and an extended incubation period of 48 h, while maintaining the MTT concentration and formazan solubilization procedure as in the original protocol. Cell viability was calculated relative to the untreated control group, all experiments were performed in triplicate, and the IC₅₀ values were determined to quantify the cytotoxic thresholds of SBB-AuNPs.

Results and discussion

Total phenolic and flavonoid content

The TPC of the SBB extract was determined using the Folin-Ciocalteu method, with gallic acid serving as a standard. The absorbance values obtained were plotted against known concentrations (0.01, 0.02,0.03,0.04,0.05,0.06,0.07 mg/mL) of gallic acid to generate a calibration curve, yielding the regression equation (y = 8.7071x + 0.0527) with a coefficient of determination (R² = 0.9944) as shown in (Fig. 1, a). Based on this calibration, the TPC of the SBB extract was calculated to be 120.9 ± 2.51 mg gallic acid equivalents (GAE) per gram of extract (mean ± SD, n = 3).

Fig. 1.

Standard calibration curves: (a) Gallic acid for TPC; (b) Quercetin for TFC

Similarly, the TFC was assessed using the aluminum chloride method, employing quercetin as the reference compound. A standard curve was established by plotting absorbance values against quercetin concentrations (0.005,0.01,0.02,0.03,0.04,0.05,0.06 mg/mL), resulting in the regression equation (y = 5.5357x + 0.0804) with linearity (R² = 0.9963) as shown in (Fig. 1, b). The TFC of SBB extract was determined to be 43.89 ± 1.74 mg quercetin equivalents (QE) per gram of extract (mean ± SD, n = 3).

The chemical composition of bee bread extract exhibits significant variability influenced by several factors such as geographical location, floral sources, and extraction technique [25]. This natural variability is largely attributed to differences in floral diversity and environmental conditions. Among the most biologically active constituents are phenolic and flavonoid compounds, which play a key role in the antioxidant potential of bee bread [35]. Several studies have examined the chemical constituents of bee bread from different regions, showing notable variations in TPC and TFC values. For example, Indonesian bee bread exhibited TPC of 4.01–6.5 mg GAE/g and TFC of 0.70–1.93 mg QE/g [36], Greek bee bread 6.49–14.64 mg GAE/g and 2.34–5.49 mg QE/g [37], German samples 386.59–743.73 mg GAE/100 g and 117.05–142.09 mg QE/100 g [38], while Turkish and Anatolian bee bread ranged 18.35 mg GAE/g, 2.82 mg QE/g [39] and 2.041–3.224 mg GAE/g, 0.323–1.903 mg QE/g [40], respectively. These comparisons indicate that the SBB extract analyzed in this study contains relatively high levels of phenolic and flavonoid compounds, which can be attributed to diverse flora and environmental characteristics of the region.

Sugar content

In addition to chemical characterization of the SBB extract, its sugar content was also analyzed using HPLC (Fig. 2). The analysis of free sugars revealed that fructose was the dominant sugar, whereas sucrose appeared in the lowest concentration, likely due to its breakdown by bacteria during the fermentation process [25]. The SBB sample was found to contain fructose, glucose, sucrose and raffinose, with respective amounts of 17.3 ± 1.12 g/100 g, 6.5 ± 0.63 g/100 g, 0.3 ± 0.10 g/100 g, and 0.5 ± 0.14 g/100 g, respectively. The total sugar content was calculated and amounted to 24.6 g/100 g, while the Fructose to Glucose ratio (F/G) was found to be 2.66.

Fig. 2.

Analysis of sugars in SBB extract using HPLC

The levels of fructose, glucose, and sucrose in the SBB extract were notably higher than those reported for Polish bee bread, which contained 11.58 g/100 g of fructose, 5.12 g/100 g of glucose, and 0.11 g/100 g of sucrose, with raffinose undetected, a total sugar content of 16.81 g/100 g, and an F/G ratio of 2.26 [41]. In Portuguese bee bread samples, fructose and glucose were the predominant sugars, ranging from 21.9 to 23.5 g/100 g and 12 to 12.4 g/100 g, respectively, with an average F/G ratio of 1.86. Sucrose content varied between 3.35 and 11.25 g/100 g, while maltulose and maltose were present and raffinose was absent [42]. In Anatolian bee bread, fructose and glucose concentrations ranged from 11.6 to 32.1 g/100 g and 6.21 to 21.5 g/100 g, respectively, with the highest recorded F/G ratio of 2.22, slightly lower than that observed in the SBB extrac[43]. Once again, these compositional differences strongly underline the influence of botanical origin. The floral source of the pollen collected by bees is widely recognized as the principal determinant of sugar content in bee bread samples [44]. These observations collectively highlight the distinctive sugar composition of the SBB sample, reflecting its unique botanical and regional characteristics.

Phenolic profile

A detailed analysis of phenolic compounds in SBB extract was conducted to identify the compounds contributing to its notable antioxidant properties. The results of this analysis are shown in (Fig. 3), where six phenolic compounds were identified, of which kaempferol exhibited the highest concentration at 28.3 mg/g. This was followed by myricetin, p-coumaric acid, quercetin, and rosmarinic acid. Caffeic acid was detected in the lowest concentration among the identified compounds as shown in Table 1. Interestingly, vanillic acid, chrysin, and apigenin were not detected in the current analysis.

Fig. 3.

Analytical HPLC Chromatogram of SBB Extract

Table 1.

HPLC Analysis of Phenolic Compound in SBB Extract

Compounds	Concentrations (mg/g)
Kaempferol	28.3
Myricetin	4.7
p-Coumaric acid	3.7
Quercetin	1.9
Rosmarinic Acid	0.8
Caffeic Acid	0.7
Vanillic Acid	ND
Chrysin	ND
Apigenin	ND
Total	40.1

Comparison with previous studies highlights both similarities and differences in the phenolic composition of bee bread. Some phenolic compounds found in other bee bread samples were also detected in the Syrian SBB extract, although luteolin was absent in SBB [45]. Other studies identified kaempferol, p-coumaric acid, quercetin, and caffeic acid in multiple samples; myricetin appeared in only a few, rosmarinic acid was often absent, while vanillic acid, chrysin, and apigenin were consistently present [46]. In different regional samples, myricetin was detected sporadically, caffeic acid and chrysin in some samples, and p-coumaric acid in all, whereas rosmarinic acid, kaempferol, and apigenin were not detected [47]. Such variations are primarily attributed to differences in pollen type, collection season, and geographical origin [24].

GC-MS analysis

Expanding on phenolic profile, the GC/MS analysis of SBB extract further revealed a rich and diverse composition of volatile and semi-volatile compounds. A total of 18 compounds were identified based on spectral data matched with Wiley and NIST library. The representative gas chromatogram is illustrated in (Fig. 4), while detailed properties and quantitative data are presented in Table 2. The results demonstrated a complex mixture of bioactive constituents, including fatty acids and their esters (palmitic acid, methyl linolenate, ethyl hexadecanoate, and linolenic acid). In addition, a wide range of alkanes was detected (n-nonadecane, n-heneicosane, n-pentacosane, n-hexacosane, n-heptacosane, n-octacosane, n-nonacosane, n-triacontane, n-hentriacontane, and n-tritriacontane). Alkenes such as (9-tricosene, and 9-hentriacontene) were also present, alongside alcohols (tricosen-1-ol), and the notable terpenoids compound (squalene).

Fig. 4.

GC-MS Chromatogram of SBB Extract

Table 2.

GC-MS identified compounds in SBB extract with retention time and area

	Compound	RT(min)	%Area
Fatty Acids	Hexadecanoic acid	6.4	0.06
	Linolenic acid	10.8	9.7
Fatty Acid Esters	Methyl linolenate	7.2	0.02
	Ethyl hexadecanoate	8.3	0.04
Alkanes	n-Nonadecane	5.9	0.07
	n-Heneicosane	9.4	0.8
	n-Pentacosane	13.7	12.2
	n-Hexacosane	14.9	1.5
	n-Heptacosane	16.3	14.1
	n-Octacosane	17.2	1.2
	n-Nonacosane	21.4	14.4
	n-Triacontane	23.8	0.1
	n-Hentriacontane	26.5	10.4
	n-Tritriacontane	30.4	8.9
Alkenes	9-Tricosene	12.3	0.6
	9-Hentriacontene	24.6	5.1
Alcohols	Tricosen-1-ol	27.8	8.7
Terpenoids	Squalene	19.1	0.2

Comparative GC-MS analyses of bee bread from different regions reveal both shared and distinct chemical profiles. Turkish bee bread contained several fatty acids and esters, including palmitic acid, ethyl hexadecanoate, methyl linoleate, and linolenic acid methyl ester, along with terpenoids absent in the current analysis [48]. Other studies reported diverse compounds across bee bread types, encompassing fatty acids, alcohols, alkanes, and alkenes [49], with one analysis identifying 107 compounds including nonane, 1-dodecene, and alcohols [26].

Synthesis of SBB-AuNPs

Based on the chemical analysis of SBB, it was found to contain phenolic compounds, flavonoids, sugars, fatty acids, and other bioactive constituents. These bioactive compounds make SBB a promising candidate for the green synthesis of AuNPs. In this study, AuNPs were synthesized by adding an aqueous solution of HAuCl₄·3H₂O (2mM) into a (1%) aqueous extract of SBB. A gradual color change from yellow to wine red was observed during the incubation period, indicating the successful formation of AuNPs. This change occurred at alkaline conditions (pH 9.5) and at room temperature (~ 25 °C).

It is clear that the bioactive compounds in the SBB extract particularly phenolics and sugars, play a significant role in the reduction of metal ions during nanoparticle synthesis. These constituents interact with metal ions primarily through ionic or electrostatic forces, thereby initiating a redox process in which electrons are transferred from their functional groups to reduce Au^{+ 3} to Au⁰. Beyond acting as reducing agents, these compounds also act as natural capping agents, stabilizing the synthesized nanoparticles and preventing their aggregation. Sugars present in the extract such glucose and fructose, contribute significantly to the reduction process, with glucose exhibiting relatively higher reducing potential attributed to its antioxidant capacity and fewer tautomeric shifts. Phenolics compounds, particularly flavonoids further enhance the reduction capacity by donating hydrogen atoms or electrons, directly facilitating gold ion reduction [50]. For instance, the hydroxyl groups on the B and C rings of kaempferol directly facilitate AuNP formation by participating in gold ion reduction. Similarly, quercetin may enhance the bioreduction process by adsorbing onto the metal surface and chelating gold ions via its carbonyl, hydroxyl, and catechol groups [51]. The oxidized enol forms of flavonoids can further adsorb onto the nanoparticle surface, acting as capping and stabilizing agents that prevent aggregation and improve nanoparticle stability [52]. Consequently, the combined presence of flavonoids, phenols, and sugars in SBB extract likely plays a significant role in both gold ion reduction and nanoparticle stabilization. This aligns with previous reports emphasizing the dual functionality of these compounds in nanoparticle synthesis and may also enhance their biological activity.

Optimization and characterization of SBB-AuNPs

To further evaluate the properties of AuNPs and confirmed their stability, optimization was carried out using DLS. This technique provided precise measurements of the hydrodynamic particle size distribution, PDI, and ζ-potential, which are critical indicators of nanoparticles stability, uniformity, and overall synthesis efficiency.

The green synthesis of AuNPs using the SBB extract was optimized by varying key reaction parameters, including temperature, solution pH, and concentrations of reactants as detailed in Table 3. Among the tested parameters, pH emerged as a particularly influential factor, as it critically affects the green synthesis by modulating the ability and reactivity of functional groups in the reducing agents, thereby impacting the size, morphology, and stability of the nanoparticles [53]. At pH 7.5, the AuNPs exhibited a relatively large size and a high PDI, indicating significant polydispersity and poor stability. As the pH increased to 8.5, a noticeable reduction in particle size was observed, along with improved monodispersity. This trend continued at pH 9.5, where the AuNPs achieved their smallest average size (46.38 nm) and low PDI (0.235 ± 0.002), suggesting optimal nucleation and controlled growth, resulting in more uniform and stable nanoparticles. However, further increasing the pH to 10.5 led to a reversal of this trend, with a marked increase in particle size and PDI, indicating possible nanoparticles aggregation.

Table 3.

Impact of experimental parameters on size and polydispersity index of AuNPs

pH	ZA(nm)	PDI	Temperature	ZA(nm)	PDI
7.5	208.8 ± 23.21	0.519 ± 0.024	~ 25 °C	46.38 ± 6.12	0.235 ± 0.002
8.5	94.56 ± 15.00	0.189 ± 0.032	40 °C	46.39 ± 7.14	0.461 ± 0.014
9.5	46.38 ± 6.12	0.235 ± 0.002	60 °C	64.26 ± 9.00	0.643 ± 0.123
10.5	98.05 ± 14.98	0.570 ± 0.015	80 °C	86.5 ± 12.17	0.658 ± 0.155

Metal salt concentration	ZA (nm)	PDI	SBB concentration	ZA(nm)	PDI
1mM	136.6 ± 19.20	0.254 ± 0.045	1%	46.38 ± 6.12	0.235 ± 0.002
2mM	46.38 ± 6.12	0.235 ± 0.002	5%	330.6 ± 42.58	0.468 ± 0.042
5mM	261.6 ± 33.11	0.372 ± 0.120	10%	1586 ± 56.88	0.907 ± 0.014

Values are expressed as mean ±SD of three independent experiments (n=3)

In parallel with the effect of pH, temperature was also found to exert a significant impact on the green synthesis of nanoparticles. At room temperature, the nanoparticles showed a favorable average size of 46.38 nm and a PDI of 0.235. Increasing the temperature to 40 °C maintained the size (~ 46.39 nm) but increased the PDI to 0.461, indicating a broader size distribution. Further elevation to 60 °C resulted in larger nanoparticles with a markedly higher PDI, reflecting accelerated growth and reduced uniformity. This trend became more pronounced at 80 °C, where nanoparticles size peaked at 86.5 nm, with the highest PDI (0.658), suggesting rapid aggregation. Although moderate temperature elevation may enhance nucleation by increasing kinetic energy, excessive heat can destabilize or degrade the bioactive compounds responsible for reduction and stabilization processes. Maintaining room temperature helps to preserve the stability of these compounds, which in turn contributes to the stability of resulting nanoparticles [54].

Building upon the optimal pH (9.5) and room temperature (~ 25 °C) identified earlier, further optimization focused on evaluating the influence of gold salt and extract concentrations. The concentration of the gold precursor (HAuCl₄·3 H₂O) proved to be critical in determining the efficiency of initial nucleation and subsequent growth. At a lower concentration of 1 mM, relatively large nanoparticles (136.6 nm) were produced with a moderate PDI (0.254), indicating suboptimal nucleation and uncontrolled particle enlargement. Increasing the salt concentration to 2 mM resulted in the production of smaller, more uniform nanoparticles with a lower PDI, suggesting improved nucleation without significant aggregation. Notably, at 5 mM, a faster color change was observed during the reaction, indicating more rapid initiation of the reduction process. However, this concentration also led to significant increase in average size (261.6 nm), suggesting particle aggregation and reduced stability. These findings highlight that while higher concentrations of metal ions may accelerate nucleation and growth, they ultimately affect the properties of the nanoparticles [55].

Controlling extract concentration is a key parameter in the green synthesis of nanoparticles, as it directly influences product quality, size distribution, and colloidal stability. Excessively high extract concentrations may hinder the reduction process and adversely affect nanoparticle formation [50]. This was clearly observed at 10% extract concentration, where the particles exhibited an average size of 1586 nm and an extremely high PDI (0.907), indicating uncontrolled growth and severe aggregation. Reducing the extract concentration to 5% resulted in smaller nanoparticles (330.6 nm) and a lower PDI (0.468) compared to 10%; however, the particles were still relatively large and polydisperse, suggesting that the presence of excess biomolecules continued to disrupt uniform nucleation and stabilization. In contrast, a lower concentration of 1% yielded significantly more favorable outcomes. The resulting AuNPs exhibited a narrow size range (2–7 nm), a Z-average of 46.38 nm, and a low PDI of 0.235. These values reflect a well-regulated synthesis process, characterized by balanced reduction and efficient stabilization. Such findings underscore the importance of optimizing extract concentration to maintain control over nanoparticle formation and to avoid the pitfalls associated with excessive reducing agent content.

Following the optimization process, the ideal sample was further evaluated using DLS measurements to assess particle size distribution, and ζ-potential. As shown in (Fig. 5), the size distribution of AuNPs synthesized using SBB extract showed consistent results across intensity, volume, and number distributions. The AuNPs ranged from 2 nm to 7 nm (according to the number distribution) with Z-average of 46.38 nm, and low PDI of 0.235 ± 0.002, indicating the formation of small, uniformly distributed AuNPs. To further evaluate colloidal stability, ζ-potential measurements were performed immediately after synthesis. A sharp peak at − 29.7 mV was observed (Fig. 6, a), reflecting strong electrostatic repulsion among the negatively charged particles. This repulsion effectively prevented aggregation, suggesting high colloidal stability [56]. Notably, a follow-up measurement conducted two months later showed only a slight decrease in ζ-potential to − 28.8 mV (Fig. 6, b). This minimal shift reflects good stability of the colloidal suspension, suggesting that the bioactive components in SBB extract not only facilitated efficient reduction and capping during synthesis, but also provided surface stabilization over time. These results indicate that the bioactive components in SBB extract, including phenolic compounds, flavonoids, and sugars, not only facilitated efficient reduction and natural capping during synthesis but also contributed to long-term surface stabilization. The composition and high content of these metabolites likely enhanced particle size control and colloidal stability, as supported by the comparative data presented in Table 4 on the use of other bee products in nanoparticle synthesis. This highlights the unique potential of SBB extract in the green synthesis of small and stable AuNPs, emphasizing both its reduction and stabilization roles.

Fig. 5.

Intensity (a), Volume (b), and Number (c) Size Distribution of AuNps Synthesized Using SBB Extract

Fig. 6.

ζ-potential of SBB-AuNPs Immediately after Synthesis (a), and After Two Months (b), Indicating Good Long-Term Colloidal Stability

Table 4.

Nanoparticles synthesized by bee products compared to current study

Extract	Size (nm)	PDI	ζ-potential (mV)	Reference
Bee bread	46.38 ± 6.12	0.235 ± 0.002	–29.7	Current study
Propolis	15.4	0.385	–34.7	[57]
Propolis	108 ± 68	Not detected	–24	[21]
Bee Pollen	52.8 ± 25.9	0.240	Not detected	[22]
Honey	< 67.9	Not detected	Not detected	[58]
Honey	151.4	0.282	− 46.5	[59]
Royal jelly	24.36–68.06	0.4	–26.5	[60]

After successfully optimizing the reaction conditions, it became essential to perform further analyses to confirm the formation of AuNPs and investigate their optical properties. UV–Vis spectroscopy was employed to monitor the LSPR, a distinctive feature of metallic nanoparticles. LSPR is a phenomenon that arises from the collective oscillation of free electrons at the nanoparticle surface under incident electromagnetic light. The resonance frequency is influenced by several factors, including particle size, shape, and interparticle distance. Typically, AuNPs exhibiting a single, well-defined resonance peak around 525 nm are indicative of a spherical shape and small size (ranging from 10 to 70 nm). Conversely, nanoparticles with irregular morphologies or broader size distribution tend to display multiple resonance peaks [61].

In the current study, the green synthesis of AuNPs using SBB extract led to a distinct color change in the solution from pale yellow to wine red which aligns with previous study [61], confirming that such a color change is typical for small, spherical AuNPs. This color shift is a direct indication of LSPR activation, and thus confirms the successful formation of AuNPs. As shown in (Fig. 7), the UV–Vis spectrum displays a single peak at 524 nm, suggesting that the synthesized AuNPs are spherical in shape and well-dispersed. This optical behavior is consistent with the characteristics typically observed in small, uniformly shaped AuNPs, further validating the effectiveness of the green synthesis approach.

Fig. 7.

UV-Vis Absorption Peak of AuNPs Synthesized Using SBB Extract

AFM and FESEM analyses were conducted to gain a detailed understanding of the morphology and surface characteristics of the SBB-AuNPs. The three-dimensional (Fig. 8a) and two-dimensional (Fig. 8b) AFM images clearly revealed uniformly distributed, spherical nanoparticles with smooth surfaces. The average size of most particles was approximately 33 nm, although a fraction of much smaller particles—below 7 nm—was also observed. This narrow size distribution and morphological uniformity highlight the reproducibility and high quality of the biosynthesized nanoparticles. The particle size distribution histogram (Fig. 8c) further supports these findings.

Fig. 8.

3D and 2D AFM Images (a, b), and Particle Size Distribution Histogram (c) of SBB-AuNPs

Complementary FESEM analysis confirmed the nanoparticle morphology. At a magnification of ×60,000, the AuNPs appeared as clusters of spherical, cloud-like structures, with an average size of 28.69 nm (Fig. 9a). However, these apparent aggregates are most likely artifacts resulting from sample preparation—such as solvent evaporation or high sample concentration—rather than actual aggregation in the colloidal state. DLS measurements demonstrated that the nanoparticles remained well-dispersed for up to two months, confirming that the aggregation observed in FESEM images was due to sample processing rather than colloidal instability [62]. It is important to note that DLS measures the hydrodynamic diameter of nanoparticles in solution, reflecting the particle along with any associated solvation layer, whereas AFM and FESEM measurements represent the dry, physical size of the nanoparticles. Consequently, DLS values are typically slightly larger than those obtained from AFM or FESEM, highlighting the distinction between nanoparticles in their dispersed versus solid states.

Fig. 9.

FESEM Image of SBB-AuNPs Showing Size and Surface Morphology

EDS analysis was used to determine the elemental composition and to infer the capping mechanisms of the synthesized nanoparticles. The results confirmed gold as the predominant element, with additional signals for carbon and oxygen derived from bioactive compounds remaining from the SBB extract, acting as natural capping and stabilizing agents. The weight percentages were 58.49% C, 18.82% O, and 22.69% Au (Fig. 9b). The presence of carbon and oxygen indicates that phenolic compounds, flavonoids, and sugars in the SBB matrix effectively adsorbed onto the nanoparticle surfaces, preventing aggregation and enhancing colloidal stability. This composition reflects the intrinsic advantage of green synthesis, yielding naturally capped, stable, and biocompatible nanoparticles without the need for synthetic surfactants.

Antimicrobial activity

Gentamicin was used as a reference antibiotic against bacterial strains, while fluconazole served as an antifungal agent against fungal isolates. These treatments produced inhibition zones that varied according to the tested microorganism. HAuCl₄·3 H₂O was also evaluated and showed limited antimicrobial activity, affecting only E. coli among the tested species. In contrast, the bee bread extract (SBB) was tested at the same concentration used in the biosynthesis process (1%) but did not exhibit any antimicrobial activity against the examined microorganisms. However, higher concentrations of SBB extract might exhibit noticeable inhibitory effects. Testing the extract at this concentration served as a control to confirm that the observed antimicrobial effects are attributable to the formed nanoparticles rather than the original extract components. Additionally, a blank control (solvent only, without active compounds) was tested on separate plates and showed no inhibition, further confirming that the antimicrobial activity arises solely from the tested samples and not from the solvent or assay conditions.

The small spherical morphology and high colloidal stability of the biosynthesized AuNPs play a crucial role in enhancing their biological activity. Due to their reduced size, these nanoparticles can penetrate bacterial cell walls more easily. Furthermore, their higher surface-to-volume ratio promotes stronger interactions with microbial membranes, thereby enhancing their antibacterial potency [63]. AuNPs synthesized using SBB extract demonstrated antimicrobial activity against Gram-negative bacteria and C. albicans (Fig. 10). The inhibition zones for the tested microorganisms are systematically presented in Table 5. Notably, C. albicans exhibited the highest sensitivity, with an inhibition zone of 27 ± 0.50 mm, Meanwhile, Gram-negative strains, such as P. aeruginosa, E. coli and Klebsiella spp. also responded well, although with varying degrees of sensitivity.

Fig. 10.

Antimicrobial Activity of AuNPs Synthesized Using SBB Extract

Table 5.

Inhibition Zones of AuNPs and Controls Against Pathogenic Strains

	Inhibition zones (mm)
strain	SBB-AuNPs	HAuCl4·3H2O2 mM	SBB extract1%	Positive control	P value
P. aeruginosa	18±0.50	ND*	ND*	ND*	3.79×10-11
E. coli	20±0.75	10±0.50	ND*	15±0.0	1.34×10-11
Klebsiella spp.	20±0.00	ND*	ND*	21±0.50	4.34×10-14
C. albicans	27±0.50	ND*	ND*	26±0.25	2.01×10-12

ND*: Not detected (no inhibition zone was observed)

The results of the one-way ANOVA presented in Table 5 revealed highly significant differences among the four treatments for P. aeruginosa, E. coli, Klebsiella spp., and C. albicans. Post-hoc analysis using Tukey’s HSD test (Table 6) indicated that most treatment pairs did not differ significantly, such as AuNPs versus mineral salt, and AuNPs versus extract depending on the organism (p > 0.05). However, significant differences were observed in specific comparisons, including Gentamicin versus AuNPs, extract versus mineral salt, and Fluconazole versus AuNPs (p < 0.05), highlighting the superior antimicrobial activity of AuNPs relative to the other treatments.

Table 6.

Tukey HSD post-hoc analysis for differences in inhibition zones among tested treatments

Strain	Comparison	Mean Diff	P Value	Significant
P. aeruginosa	AuNPs vs. Metal salt	-18	0	Yes
P. aeruginosa	Extract vs. AuNPs	-18	0	Yes
P. aeruginosa	Extract vs. metal salt	0	1	No
P. aeruginosa	Gen vs. AuNPs	-18	0	Yes
P. aeruginosa	Gen vs. Extract	0	1	No
P. aeruginosa	Gen vs. metal salt	0	1	No
E.coli	AuNPs vs. Metal salt	-9.5	0	Yes
E.coli	Extract vs. AuNPs	-20	0	Yes
E.coli	Extract vs. metal salt	-10.5	0	Yes
E.coli	Gen vs. AuNPs	-5	4.53 × 10 − 7	Yes
E.coli	Gen vs. Extract	15	0	Yes
E.coli	Gen vs. metal salt	4.5	1.20 × 10 − 6	Yes
Klebsiella spp.	AuNPs vs. Metal salt	-20	0	Yes
Klebsiella spp.	Extract vs. AuNPs	-20	0	Yes
Klebsiella spp.	Extract vs. metal salt	0	1	No
Klebsiella spp.	Gen vs. AuNPs	0.75	0.00294	Yes
Klebsiella spp.	Gen vs. metal salt	20.75	0	Yes
Klebsiella spp.	Gen vs. Extract	20.75	0	Yes
C. albicans	AuNPs vs. Metal salt	-27	0	Yes
C. albicans	Extract vs. AuNPs	-27	0	Yes
C. albicans	Extract vs. metal salt	0	1	No
C. albicans	Fluc vs. AuNPs	-1	0.02653	Yes
C. albicans	Fluc vs. metal salt	26	0	yes
C. albicans	Fluc vs. Extract	26	0	yes

Although pinpointing the exact mechanism behind the antimicrobial activity of AuNPs remains challenging, several pathways have been proposed (Fig. 11), including oxidative stress, non-oxidative stress, and the release of gold ions. The antibacterial effectiveness of AuNPs is also strongly influenced by their physicochemical properties, such as size, shape, surface charge, and stability. In particular, the small size and high surface-to-volume ratio of these nanoparticles facilitate their penetration into bacterial cell walls and membranes, enhancing their antimicrobial action. Additionally, secondary metabolites present in the extract used during synthesis may also directly influence their antimicrobial effectiveness, which varies depending on the type of microorganism. These bioactive molecules can damage microbial cell walls, increase intracellular calcium ion concentrations, and subsequently trigger apoptosis. They may also disrupt cellular structures and functional proteins, impairing respiration and division. DNA interactions might occur as well, potentially inhibiting transcription and causing mutations. Another critical mechanism involves the generation of reactive oxygen species (ROS), which can damage cellular structures and deactivate antioxidant enzymes, ultimately leading to oxidative imbalance and cell death [63].

Fig. 11.

Schematic representation of the proposed antimicrobial mechanisms of SBB-AuNPs

The greater sensitivity of C. albicans to AuNPs compared to Gram-negative bacteria may be attributed to the more complex structural organization of fungal cells compared to bacteria [64, 65]. This structural complexity of fungi may facilitate enhanced interaction, adhesion, and penetration of nanoparticles, potentially leading to oxidative stress, protein denaturation, enzyme inhibition, and DNA damage. Although these mechanisms were not directly investigated in the present study, they provide a plausible hypothesis for the observed differential sensitivity.

The enhanced antimicrobial performance of the SBB-AuNPs can be attributed to a combination of their physicochemical properties. The nanoparticles exhibited a small and uniform size (PDI = 0.235) and a stable negative surface charge (ζ-potential = − 29.7 mV initially and − 28.8 mV after two months), which promotes better penetration through bacterial cell walls and ensure long-term colloidal stability. In addition, the presence of sugars, phenolics, and flavonoids in the SBB extract, as confirmed by TPC, TFC, and HPLC analyses, likely contributing to the reduction and capping of the nanoparticles, further stabilizing their size and preventing aggregation. These bioactive compounds may also exert a direct antimicrobial effect, enhancing the overall efficacy of the AuNPs against Gram-negative bacteria and C. albicans. Thus, the interplay between the nanoparticles’ physical characteristics and the chemical composition of the SBB extract underlies their observed biological activity. Overall, these results highlight that the antimicrobial activity of SBB-AuNPs is influenced by multiple factors, including nanoparticle size, shape, surface charge, and the presence of bioactive compounds from the SBB extract.

Although several antimicrobial mechanisms have been proposed in the literature (such as oxidative stress, apoptosis, DNA interaction, and disruption of cellular structures), they were not directly investigated in this study and are therefore considered proposed/hypothetical mechanisms. As a limitation of the present work, future studies could perform additional experiments to directly verify these biochemical pathways and their contribution to the observed antimicrobial activity of the nanoparticles.

Several studies have investigated the synthesis of AuNPs and assessed their antimicrobial properties. Mandey et al. (2019) synthesized AuNPs using honey, reporting an average particle size of 219.8 nm with a PDI of 0.249, as measured by a particle size analyzer. These nanoparticles demonstrated antibacterial activity, with inhibition zones of 8.4 mm against E. coli and 7.25 mm against S. aureus [20]. In a related study, Khan et al. (2020) utilized an extract of Clerodendrum inerme leaves for the biosynthesis of spherical AuNPs with an average size of 5.82 nm. The nanoparticles showed antimicrobial activity against a range of Gram-positive, Gram-negative bacteria, and fungi [4]. Similarly, Gouyay et al. (2021) employed the Turkevich approach to synthesize AuNPs with an average size of 13.5 nm and a negative surface charge of − 43 mV, according to DLS measurements. However, these particles exhibited only weak antibacterial activity against both E. coli and S. aureus [8]. In contrast, Aljohani et al. (2022) used propolis extract for the green synthesis of AuNPs, which averaged 15.4 nm in size and carried a ζ-potential of − 34.7 mV. These nanoparticles displayed notable antimicrobial and antifungal activity against various Gram-positive and Gram-negative bacteria as well as C. albicans [57]. In comparison, Kaabour et al. (2025) employed royal jelly extract for the green synthesis of AuNPs, yielding spherical nanoparticles with sizes ranging from 24.36 to 68.06 nm as determined by DLS, while FESEM analysis revealed smaller particle dimensions of approximately 29–34 nm. The synthesized nanoparticles exhibited a ζ-potential of − 26.5 mV, indicating moderate colloidal stability. Notably, these AuNPs demonstrated significant antimicrobial activity against both Gram-positive bacteria, including S. aureus and S. epidermidis, and Gram-negative bacteria such as Klebsiella spp., P. aeruginosa, and E. coli [60]. While many studies have reported that positively charged AuNPs adhere more effectively to bacterial surfaces [66], negatively charged nanoparticles can also display antimicrobial activity depending on the extract used in their synthesis [57, 67]. Moving forward, modifying the surface charge of AuNPs synthesized using SBB extract could be a promising strategy to further enhance their antibacterial performance. Overall, these studies highlight that the antimicrobial effectiveness of AuNPs is not solely determined by the precursor or extract, but is strongly influenced by their physicochemical properties, including size, shape, surface charge, and the presence of bioactive compounds, which collectively govern interactions with microbial cells. As mentioned above, the properties of the extract used in the synthesis process also play a crucial role in determining the effectiveness of the resulting nanoparticles. Often, the antibacterial activity of nanoparticles significantly exceeds that of the extract alone [63]. This was clearly observed when the SBB extract was tested independently against Gram-negative bacteria and C albicans, where no inhibition zones were detected. Similarly, the gold salt solution, when tested at low concentrations, showed either no inhibition zones or only minimal ones. These findings emphasize that the enhanced antimicrobial effects are not due to the extract or precursor alone, but rather to the synergistic properties achieved through nanoparticles synthesis.

Cytotoxicity assessment (MTT Assay)

The initial cytotoxicity evaluation of the SBB-derived AuNPs was performed using the MTT assay on normal human fibroblast cells. As shown in (Fig. 12), the dose–response data revealed that the nanoparticles exhibited no cytotoxic effects at lower concentrations (6.25–50 µg/mL), as cell viability remained consistently high. Starting at 100 µg/mL, a moderate decrease in viability was observed (85.63%), indicating the onset of mild cytotoxic effects. This reduction became more pronounced at the highest tested dose (400 µg/mL), where cell viability dropped to approximately 40.59%, reflecting a clear cytotoxic impact at elevated concentrations. The IC₅₀ value was calculated as 341.9 µg/mL, providing a quantitative measure of the cytotoxic threshold of the synthesized AuNPs. These findings support the preliminary safety of AuNPs within concentrations ≤ 50 µg/mL, suggesting their potential suitability for dermatological applications without compromising the integrity of normal cells. However, the significant decline in viability at higher doses underscores the importance of dose optimization and highlights the potential risks associated with excessive exposure. Statistical analysis of cytotoxicity data was performed using a one-way ANOVA, followed by Tukey’s post hoc test for pairwise comparisons between different doses. The results revealed statistically highly significant differences in cell viability across concentrations (p < 0.0001, R² = 0.9614), confirming that the observed reductions at higher doses were statistically meaningful. Details of the mean, standard deviation, and sample size for each dose are presented in Table 7, while the pairwise comparisons from Tukey’s test are summarized in Table 8. Tukey’s analysis indicated that significant differences in cell viability began to emerge at doses above 50 µg/mL. For instance, comparisons between 0 and 100 µg/mL (p = 0.0374), 0 and 200 µg/mL (p = 0.0007), and 0 and 400 µg/mL (p < 0.0001) showed statistically significant reductions. In contrast, no significant differences were observed among lower doses (0–50 µg/mL), supporting the conclusion that AuNPs are safe within this range. Comparisons between low and intermediate doses, such as 6.25 and 50 µg/mL (p = 0.043), also revealed meaningful differences with increasing concentration, highlighting the dose-dependent impact on cell viability. Overall, this analysis demonstrates a clear correlation between higher AuNP doses and reduced cell viability, while confirming that concentrations ≤ 100 µg/mL maintain primary cellular safety, which is crucial for defining a safe usage range for these nanoparticles.

Fig. 12.

Cell Viability of Fibroblast Cells Treated with AuNPs Synthesized using SBB Extract

Table 7.

The viability of NHDF cells at different SBB-AuNPs concentrations

Dose(µg/ml)	Mean	SD	N
0	100.000000	0.167025	3
6.25	107.328833	4.261597	3
12.5	101.350048	4.494187	3
25	96.046287	8.279125	3
50	93.249759	3.120287	3
100	85.631630	7.927979	3
200	77.531340	1.261012	3
400	40.597878	2.897778	3

Values are expressed as mean ± standard deviation (n = 3)

Table 8.

Tukey’s Post Hoc test for pairwise comparisons of SBB-AuNPs doses

Tukey’s multiple comparisons test	Mean Diff.	Summary	P Value
0 vs. 6.25	-7.329	ns	0.6025
0 vs. 12.5	-1.35	ns	> 0.9999
0 vs. 25	3.954	ns	0.9687
0 vs. 50	6.75	ns	0.6883
0 vs. 100	14.37	*	0.0374
0 vs. 200	22.47	***	0.0007
0 vs. 400	59.4	****	< 0.0001
6.25 vs. 12.5	5.979	ns	0.7947
6.25 vs. 25	11.28	ns	0.1525
6.25 vs. 50	14.08	*	0.043
6.25 vs. 100	21.7	**	0.0011
6.25 vs. 200	29.8	****	< 0.0001
6.25 vs. 400	66.73	****	< 0.0001
12.5 vs. 25	5.304	ns	0.8726
12.5 vs. 50	8.1	ns	0.4887
12.5 vs. 100	15.72	*	0.0195
12.5 vs. 200	23.82	***	0.0004
12.5 vs. 400	60.75	****	< 0.0001
25 vs. 50	2.797	ns	0.9956
25 vs. 100	10.41	ns	0.2178
25 vs. 200	18.51	**	0.005
25 vs. 400	55.45	****	< 0.0001
50 vs. 100	7.618	ns	0.5593
50 vs. 200	15.72	*	0.0195
50 vs. 400	52.65	****	< 0.0001
100 vs. 200	8.1	ns	0.4887
100 vs. 400	45.03	****	< 0.0001
200 vs. 400	36.93	****	< 0.0001

Relative cytotoxicity of the SBB-AuNPs was assessed by comparing different doses

P-values represent the statistical significance between doses, with all treatments performed in triplicate

*p < 0.05, **p < 0.01, ***p < 0.001. Pairwise comparisons were performed using Tukey HSD test

The findings open exciting possibilities for advanced dermatological applications, including therapeutic creams, anti-inflammatory agents, and antimicrobial formulations aimed at protecting skin health. Moving forward, it is essential to transition this promising technology from the laboratory to practical, commercial applications to fully realize the unique therapeutic benefits of these green-synthesized AuNPs.

The cytotoxicity of AuNPs is influenced by several key parameters, including particle size, shape, and administered dose. In the current study, a gradual decrease in cell viability was observed with increasing concentrations, consistent with the findings of Mateo et al.. (2015) [68], who reported that smaller AuNPs (30 nm) exhibited significantly higher cytotoxicity compared to larger counterparts (50–90 nm). These observations underscore the importance of particle size as a critical determinant of biocompatibility. However, size alone does not fully explain the differences in cellular responses. For instance, Huang et al. (2020) [69], pointed out that AuNPs exhibited good biocompatibility with fibroblast cells at an average size (~ 20 nm). This indicates that AuNPs are suitable for dermatological applications requiring high cellular safety. On the other hand, a study by De Faria et al. (2023) [70], showed that PEG-coated AuNPs demonstrated greater biocompatibility with fibroblast cells compared to PEG-coated gold-iron nanoparticles. This suggests that polyethylene glycol alone is insufficient; rather, the specific chemical composition of the coating plays a crucial role in reducing the activation of toxicity mechanisms such as oxidative stress. These findings open new avenues for exploring additional coating agents and their impact on cellular toxicity.

Limitations

This study provides comprehensive insights into the synthesis and characterization of gold nanoparticles using SBB extract, representing the first reports of its kind. Future research could further expand and refine several aspects. Detailed physicochemical analyses, including Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and transmission electron microscopy (TEM), would offer deeper insights of nanoparticle structure, size, and stability. Investigating the chemical profile of bee bread from different regions could identify the specific bioactive compounds responsible for nanoparticle formation, and assessing the antioxidant potential of both the nanoparticles and extracts using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay would further clarify their biological functionality and potential applications. Cytotoxicity evaluations should be extended to wider range of human cell lines and compared with pharmaceutical agents, while high-resolution imaging of nanoparticles–cell interactions could help clarify the associated cellular responses. Antimicrobial activity should also be assessed against a broader spectrum of microbial strains using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays, accompanied by molecular mechanisms, such as gene expression related to oxidative stress or bacterial cell death pathways. Furthermore, in vivo investigations would provide essential data on safety and efficacy, complementing the current in vitro findings and supporting future biomedical applications.

Conclusion

The growing demand for eco-friendly and biocompatible nanomaterials continues to drive the exploration of natural biomaterials for sustainable green synthesis approaches. In this study, SBB was demonstrated for the first time as a promising natural source of bioactive compounds, including phenolics, flavonoids, terpenoids, alongside sugars and fatty acids, which effectively acted as both reducing and stabilizing agents in the green synthesis of AuNPs. Through systematic optimization of critical synthesis parameters (pH, temperature, gold salt concentration, and extract concentration), led to the formation of stable and small nanoparticles with a mean size of 46.38 ± 6.12 nm, low PDI of 0.235 ± 0.002 and a ζ-potential of − 29.7 mV, indicating high colloidal stability according to DLS. UV–Vis analysis indicated spherical AuNPs formation with a characteristic peak at 524 nm. To conclusively confirm this, AFM and FESEM analysis further verified the spherical morphology, revealing average diameters of ~ 33 and ~ 28.69 nm, respectively. The SBB-AuNPs demonstrated antimicrobial activity, with inhibition zones ranging from 18 to 27 mm. Moreover, preliminary in vitro assessments indicated their biocompatibility at doses below 100 µg/mL, as normal fibroblast cells maintained viability without signs of acute toxicity, highlighting a favorable safety profile. Overall, these findings confirm the novelty, stability, and bioactivity of SBB-AuNPs, supporting their potential for biomedical use. Nevertheless, as the present study is limited to in vitro evaluations, future in vivo investigations are essential to validate their safety, efficacy, and mechanism of action. Furthermore, the obtained results indicate potential applications in dermatology, particularly as antimicrobial agents in creams or topical formulations to protect against skin infections. Future work should prioritize scaling up the synthesis process, translating these findings into applicable commercial formulations, and addressing regulatory and dosage optimization challenges to ensure safe and effective therapeutic implementation.

Authors’ contributions

BK wrote the main manuscript text, BK and IA prepared figures, BK and GL contributed to the experimental designs, IA and GL conducted the literature review, BK and IA managed data interpretation; All authors reviewed the manuscript.

Funding

No financial support was received for this research.

Data availability

The data sets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study did not involve the use of human participants, or animals. The cell line used (Normal human dermal fibroblast cells, (ATCC^® PCS-201-012™) is commercially available, and all experiments were performed in accordance with relevant institutional and international guidelines and regulations.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.