Introduction

Owing to their specific chemical structure, gold nanoparticles (AuNPs) have always been regarded as valuable assets, not only economically in ancient times but also biologically in recent eras1. Researchers have employed these NPs in various applications, for example as catalysts2,3, biosensors4,5, photothermal agents6, and antibacterial or antifungal agents1. One important feature of AuNPs is their low toxicity compared to other metal NPs; however, biochemical features are known to vary depending on the shape, size, and concentration of AuNPs7. The morphology of NPs can range from dodecahedron8, octahedron9, triangular10, and spherical11,12 to nanorod13 forms, depending on the synthesis method. Common physical, chemical, or biological production approaches are generally considered expensive and yield environmentally unfriendly products14. On the other hand, the green synthesis method, as a non-toxic, clean, and eco-friendly strategy, uses natural materials such as biomolecules in plant extracts to readily produce nano-scale products with high productivity, cost-effectiveness, and environmental friendliness15,16,17,18,19,20. The main advantages of AuNPs include the relative ease and affordability of production, enhanced effectiveness due to their nanoscale size, low side effects, and superior efficacy in animal and clinical studies21. These features suggest AuNPs as viable alternatives to certain drugs and antibiotics for treating a wide range of infectious diseases of viral, bacterial, fungal, and parasitic origin. However, these applications require addressing challenges, including environmental concerns related to air and water pollution22,23,24. Green synthesis of NPs can be mediated using microorganisms, plants, algae, and bacteria, offering environmentally biocompatible and safe methods25,26,27. Plant extracts have recently received lots of attention for NP production due to the presence of compounds such as flavonoids, ketones, aldehydes, tannins, carboxylic acids, phenolic acids, and proteins that can act as reducing agents28,29.

Bacterial and fungal diseases resistant to common therapeutics pose an emerging threat to the health of populations, requiring the discovery of new therapeutic agents for these pathogens from sources such as plants, animals, and NPs30. Plants are rich sources of structurally complex compounds with antimicrobial properties31. Moreover, AuNPs have good stability and biocompatibility, whose antimicrobial properties can be augmented by modifying their physiochemical characteristics during plant-mediated green synthesis and incorporating them with other antibacterial drug agents (Table 1)32. There have also been efforts to treat drug-resistant candidiasis using nano-scale drugs and medicinal plants33,34. Cystic echinococcosis (CE), also known as hydatid cyst, is another parasitic infection and a major public health problem, especially in developing countries35. The disease is caused by the larval form of Echinococcus granulosus and affects organs such as the liver and lungs36. Given the complications of medications such as albendazole and mebendazole, the role of natural scolicidal agents with fewer side effects and more effectiveness is emerging in preventing and treating these infections19,37,38.

The Salvia genus, with over 900 species across the world, is one of the largest and most important aromatic and medicinal genera of the Lamiaceae plant family39. Due to their biologically active ingredients, species in this genus are used to obtain antibacterial, anti-insect, antifungal40, and antidiabetic41 agents. Recent studies have further reported anticholinesterase, anti-urease, and antioxidant activities for these compounds42,43,44,45. Salvia sclarea L. mediated AuNP synthesis is a novel contribution to green nanotechnology, leveraging a unique plant source to produce biocompatible NPs via an eco-friendly approach. Nonetheless, some limitations in this field, such as the lack of in vivo studies, toxicity concerns, and challenges in scalability and reproducibility, need to be addressed. In this study, we initially used the aqueous flower extract of S. sclarea L., as a novel bio-reducing and stabilizing agent, to synthesize AuNPs and then evaluated their scolicidal, antimicrobial, and antifungal activities. This study aligns with the growing interest in green nanotechnology, which prioritizes environmentally benign, cost-effective, and biologically safe approaches for nanoparticle synthesis. Compared to the current literature, this work stands out for its focus on AuNPs’ scolicidal activity (Fig. 1) and lays a strong foundation for exploring S. sclarea-derived AuNPs as versatile therapeutic agents.

Fig. 1
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An overview of green synthesis of AuNPs and evaluation of their antibacterial, anticandidal, and scolicidal properties.

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Table 1 A summary of studies reporting the green synthesis of AuNPs with focus on their size, shape, and functional properties.
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Materials and methods

Plant material and Preparation of the extract

Salvia sclerae L. plant was prepared from pastures of Kohgiluyeh and Boyer-Ahmad (Moleh Shour mountain pass). The plant was identified with the herbarium number of KBGH1048 at the Herbarium of the Research Center for Agriculture and Natural Resources of Isfahan Province, Iran. The aerial parts of the plant were dried in our laboratory58, and 2 g of the dried plant was dissolved in 50 mL deionized water and heated at 80 °C for 30 min. Next, the aqueous extract of the plant was filtered with Whatman No. 1 filter paper and directly used in next steps59,60. All chemicals and solvents used in this experiment were purchased from Merck, Germany, except HAuCl4, which was prepared from Sigma-Aldrich. Deionized water was used in all assays.

Synthesis of AuNPs

AuNPs were biosynthesized using the aqueous extract of S. sclarea L. aerial parts (10 mL) after adding a diluted HAuCl4 solution (50 mL, 1 mM) at room temperature61. Immediately after addition, the light-yellow solution turned brownish, indicating the synthesis of AuNPs. Stirring continued for 15 min until the reaction was completed. The pH of the mixture was adjusted to 10 using 1 M NaOH solution62. The UV spectrum of the solution was recorded at times 5, 10, and 15 min, and the best time was chosen according to the peak observed in the range of 500–600 nm. The solution was centrifuged at 12,000 rpm for 5 min; NPs were rinsed with deionized water, and adherent parts were separated and dark-dried at room temperature59.

Characterization of AuNPs

The characterization of AuNPs was conducted through spectroscopy and electron microscopy. The UV-visible spectrum (200–700 nm) of diluted AuNPs was recorded using a UV/Vis spectrophotometer (PerkinElmer Lambada 365, USA). A Laser Particle Size Analyzer (DLS Zetasizer, Malvern UK company) was used to analyze AuNPs’ particle size distribution and zeta potential. To determine the molecules involved in the redox reaction, Fourier transform infrared spectroscopy (FT-IR) was performed using a Spectrum Two TM spectrometer (Spectrum Two model, PerkinElmer, USA). AuNPs’ morphology was determined using transmission electron microscopy (TEM: EM10C-100KV- zeiss Germany), field emission scanning electron microscopy (FE-SEM: ZEISS-Germany- model: Sigma VP.), and energy dispersive X-ray energy diffraction spectroscopy (EDX, Mapping). The crystallinity of AuNPs was investigated by X-ray diffraction (XRD: X’ Pert Pro device, Panalytical Company- Netherlands).

Assessment of antibacterial activity

Four bacterial strains, Staphylococcus aureus (ATCC25923), Bacillus cereus (ATCC6633), Escherichia coli (ATCC25922), and Pseudomonas aeruginosa (ATCC27853), were used to study the antibacterial activity of AuNPs. The bacterial strains were obtained from Pasteur Institute, Tehran, Iran, and cultivated in Mueller-Hinton Agar (MHA)63. The minimum inhibitory concentration (MIC) of AuNPs was ascertained using the serial dilution method according to CLSI guidelines. In this study, the antibiotic Gentamicin (64 µg/mL) was used as a positive control, and distilled water was used as a negative control. The initial concentration used was 500 µg/mL for the synthesized AuNPs, and bacterial suspensions were prepared at a concentration of 0.5 McFarland in Mueller-Hinton Broth. After cultivation on MHA, samples were incubated for 24 h at 37 °C. The colonies were counted from the first well without turbidity and evaluated for growth patterns. The last dilution inhibiting the growth of 99.99% of bacteria was regarded as MBC64,65. Finally, the halo diameter and the growth zone of inhibition (ZOI) index were measured. To examine the measured data of antibacterial activity and reach the results (MIC, MBC, and ZOI), all experiments were performed in triplicate, and the average values were reported.

Antifungal assay

Four Candida species, Candida albicans (ATCC10231), C. glabrata (ATCC900300), C. parapsilosis (ATCC22019), and C. krusei (ATCC6258), together with 40 clinical Candida spp. isolated from patients diagnosed with PCR- RFIP were used for antifungal activity assessment. Samples were cultivated on Sabouraud dextrose agar (SDA) and incubated at 35 °C for 24 h, after which the initial suspension was analyzed at the wavelength of 530 nm. The numerical value of 75–78 was suitable for yeast samples. Finally, the suspension was diluted at a ratio of 1:1000 using RPMI66,67,68. To obtain the values (MIC range, MIC50, MIC90, and MICGM), the CLSI M27-A2 guideline was used, in which the data of each candidate strain were measured in 3 replicates.

The initial concentration of the synthesized AuNPs was considered to be 200 µg/mL. After preparing a serial dilution for AuNPs, 100 µL of the solution was poured into the wells of a microplate, to which the fungal suspension was added at the same ratio. The plate was incubated at 35 °C for 24 h. Negative control wells contained no organism, and positive control wells held fungal suspensions without AuNPs. The MIC (i.e., the lowest concentration at which the fungi showed no significant growth after incubation for 24 h was determined optically. The antifungal effects of AuNPs were compared with those of Fluconazole69.

Scolicidal activity

Sheep livers infected with hydatid cysts were obtained from the industrial slaughterhouse of Yasuj City, Iran, to prepare hydatid cysts. The samples were washed several times, poured into test tubes, and prepared for examination. One drop of a solution containing at least 500 protoscolices was tested with 3 replications. The concentrations of AuNPs tested were 20, 40, 80, 160, and 320 µg/mL (as study groups) at times 5, 10, 15, 30, 60, and 120 min, with each exposure repeated three times. Saturated saline was used as the positive control and normal saline as the negative control, and the lethality percentage was determined by counting non-alive protoscolices using eosin vital staining (0.1%) or flame cell activity control70.

Statistical analysis

Data were analyzed by SPSS software using analysis of variance (ANOVA). In addition, Duncan’s test was used to compare the means. Normality was checked using the Shapiro-Wilk test, and Bartlett’s test for homogeneity of variances was used to test if variances were equal across groups.

Results and discussion

Phenolic compounds in plant extract

The Phenolic compounds identified in the S. sclarea extract are shown in Table 2; Fig. 2. A total of 11 polyphenols from the plant extract were determined and quantified by using HPLC. (Table 2). Numerous studies have observed that this group of polyphenol and phenolic compounds are present in the S. sclarea plant, and these compounds have good and valuable antioxidant capacity, which distinguishes this plant among plants of the Salvia genus42,71,72. In general, the large family of lamiaceae and the genus Salvia are used in different fields in different industries, and one of the reasons why this genus is valuable is the presence of phenolic and polyphenolic compounds, which have different quantitative and qualitative values in different species73,74. Numerous studies have shown that compounds such as phenols, flavonoids, and terpenoids found in plant extracts act as reducing and stabilizing agents in the biosynthesis of metal nanoparticles, including gold nanoparticles75,76.

Table 2 Phenolic compounds identified in the aqueous extract of S. sclarea using HPLC.
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Fig. 2
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HPLC Chromatogram of Phenolic compounds detected in the aqueous extract of S. sclarea.

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Green synthesis of AuNPs and their characterization

UV-Visible spectroscopy

To confirm the successful synthesis of AuNPs, UV-Visible spectroscopy was conducted (Fig. 3-a), which showed the specific UV-Visible spectra related to NPs at times 5, 10, and 15 min. As shown in this figure, the absorbance peak at 548 nm revealed an incremental trend as the time increased from 5 min to 15 min. Figure 3-b shows S. sclarea extract without NPs and the extract containing reduced AuNPs 15 min after the reaction. The absorbance at 548 nm and yellow-to-dark discoloration are attributed to the surface plasmon resonance excitation, indicating the reduction of gold ions to AuNPs32,77. Time-dependent kinetics of the production of AuNPs following reduction by the extract at room temperature (Fig. 3c) revealed that the absorbance intensity at 548 nm increased significantly and suddenly during the first 10 min of the reaction, then followed a slower trend. In previous research on the production of AuNPs, these NPs were generated using different methods such as chemical, thermal, or UV reduction78,79,80. In this study, AuNPs were successfully synthesized through reduction by S. sclarea extract at room temperature, offering advantages such as novelty, time-saving, energy-saving, and being environment-friendly, as well as high effectiveness in AuNP synthesis.

Fig. 3
figure 3

The UV-Vis spectra of gold nanoparticles (a). Color change from the original golden to black after synthesis of AuNPs (b). Absorption intensity at 548 nm over time, showing the successful reduction of gold nanoparticles by S. sclarea plant extract (c).

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DLS analysis and zeta potential measurement

Figure 4-a shows the characteristic structure of AuNPs synthesized by the aqueous extract of S. sclarea. During dynamic light scattering (DLS), the light is scattered by NPs existing in a suspension, corresponding to particle diameter and hydrodynamic diameter, which are somewhat different from particle appearance under microscopy visualization81,82. It is crucial for AuNPs to display high stability before they can be widely applied in the field of biomedicine and biotechnology83. The dispersity of the NPs, observed in DLS analysis as overlapped peaks (Fig. 4-a), indicates mean particle sizes of 125.7 and 1029 nm at the first and second peaks, respectively. About 67% of particles were located within the range of the first peak and 33% within the range of the second peak. Overall, the mean diameter of all these NPs was 130.6 nm, and the hydrodynamic diameter (size) widely ranged from 32 nm to up to 3000 nm with a polydispersity index (PDI) of 0.486, indicating a broad particle size distribution. The difference observed in particle sizes between DLS and TEM is due to the presence of a thick layer of the biological compounds of S. sclarea extract on the surface of AuNPs84. Larger-sized aggregates could be formed due to the partial agglomeration of nanoparticles during synthesis or sample preparation. The hydrodynamic diameter calculated in DLS also accounts for the solvation layer, which is highly sensitive to slight aggregation in colloidal systems. Although the majority of AuNPs were within the nanoscale range, the relatively wide PDI suggests that synthesis or dispersion techniques can be further optimized in future studies to achieve narrower size distributions. Nevertheless, the dominant fraction of nanoparticles remains below 200 nm, which is favorable for the intended applications.

In this research, zeta potential was measured as a colloidal stability index and an indicator of the surface charge of AuNPs (Fig. 4-b). Colloidal interactions in a suspension are particularly important to keep particles separate from each other and prevent them from aggregation, requiring the repulsion between particles to reach a maximum value85,86. As shown in Fig. 4-b, the sample’s zeta potential was equal to -16.2 mV, and electrophoretic mobility was obtained as -1.268 µmcm/Vs. The negative value of zeta potential reflects the presence of negatively charged bioactive compounds on the surface of AuNPs, leading to their high stability and efficiency87. This was in agreement with the findings of Balasubramani Sundararajan et al. (2022), B. Sundararajan et al. (2017)88, and Sujitha et al. (2013)49, noting that NPs derived from the aqueous extracts of medicinal, industrial, and toxic plants carried a strong negative electric surface charge, making them stable across a wide range of pH89,90. Additionally, AuNPs produced by green synthesis have shown the presence of a negative surface charge according to zeta potential values91,92. As noted, the tendency of the same charged particles to repel each other is directly related to their zeta potential, drawing a boundary line between the stability or instability of the suspension. In general, NPs with zeta potentials of greater than + 30 mV or less than − 30 mV are relatively stable93. A zeta potential outside the ± 30 mV range can lead to electrostatic repulsive forces between particles in a suspension, resulting in their relative stability. As shown in Fig. 4-b, the AuNPs synthesized showed sub-optimal colloidal stability and tended to attract each other. This instability appeared as the bi-dispersity of particles in DLS analysis. Some of the particles were attracted to each other and formed agglomeration caused by the low zeta potential, generating particles with hydrodynamic diameters of greater than 100 nm.

Altogether, the zeta potential observed (-16.2 mV) for AuNPs suggested their moderate colloidal stability, indicating that electrostatic repulsion between particles was insufficient to fully prevent their aggregation. This moderate stability could affect the uniformity of the nanoparticle suspension, so further surface modifications and optimization of synthesis methods are required to achieve AuNPs with higher absolute zeta potential values and better dispersion stability.

Fig. 4
figure 4

DLS size distribution (a) and zeta potential of gold nanoparticles synthesized by S. sclarea extract (b).

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FTIR analysis

FTIR analysis was used to investigate the chemical bonds and identify the factorial groups and biomolecules existing as possible reducing agents in the extract of S. sclarea. In the FTIR spectrum (Fig. 5), the peaks emerging at 3445 cm− 1 and 3782 cm− 1 were related to the stretching vibrations of O-H and N-H bonds in the structure of phenols, terpenoids, and saponins of the plant extract, as well as the water adsorbed on the surface of AuNPs94,95,96. The peaks located at 2930 cm-1 and 2855 cm− 1 were related to asymmetric and symmetric C-H bond stretching vibrations in the methyl and methylene structures, respectively. Also, the peaks observed at 1740 cm− 1 and 1612 cm− 1 were related to the C = O bond stretching vibration in the carbonyl group, O-H bond bending vibration, or C = C bond stretching vibration in aromatic rings97. The peak located at 2309 cm− 1 was caused by the carbon dioxide remaining in the test chamber98. The peaks at the wavelengths of 1263 cm− 1 and 1362 cm− 1 were related to the C-N bond stretching vibration and C-H bond bending vibration in different compounds, respectively95,99, and 1112 cm− 1 and 998 cm− 1 peaks represented the stretching vibrations of the C-OH and C-O-C bonds, respectively94,99. Finally, the stretching vibration resulting from the binding of AuNPs with the compounds existing in the plant extract was observed at 521 cm− 1100. Kačániová et al.40 reported that compounds with the highest frequency in S. sclarea were monoterpene and sesquiterpene hydrocarbons, oxygenated monoterpenes and sesquiterpenes, aliphatic alcohols, and esters. Therefore, the results of FTIR affirmed the presence of these compounds as the capping agent on the surface of AuNPs.

Fig. 5
figure 5

FTIR spectra of gold nanoparticles synthesized using S. sclarea extract.

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XRD analysis

The crystalline nature of the AuNPs produced by S. sclarea was studied through XRD analysis. As shown in Fig. 6, the matching of visible peaks to reference diffraction patterns using Highscore plus X’Pert software showed that the pattern was associated with the reference code JCPDS No. 00-004-0784 related to the crystal structure of gold96,101. Accordingly, the synthesized AuNPs revealed a cubic crystalline structure with the space group of Fm-3 m. In this structure, diffraction planes111, (200), (220), and (311) have been respectively observed at angles of 38°, 44°, 64° and 77°, with a lattice constant of 4.743 ∘A. Moreover, a broad peak within the angle range of 10° − 25° was observed, which is common during the green synthesis of NPs and can be attributed to the presence of organic groups with amorphous structures on the surface of NPs, strengthening their colloidal stability102,103. According to Scherer’s relation (D = Kλ/(FWHM)×cos(θ))104, the crystal size of the NPs was obtained as 11.2 nm. The XRD pattern indicated the dominance of111 crystalline planes in the structure of AuNPs, further confirming their crystalline nature105. Therefore, it could be concluded that the particles observed in TEM and FE-SEM images are composed of several interconnected crystals, explaining the relatively larger particle sizes detected in these microscopic tests.

Fig. 6
figure 6

XRD spectrum of gold nanoparticles synthesized by S. sclarea extract.

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FESEM and EDX analysis

FESEM imaging is an analytical technique used to determine the mean size and shape of NPs106. Micrographs of AuNPs at different magnifications are shown in Figs. 5-b and 7-a, displaying NPs with semi-spherical morphology and particle sizes of 50–170 nm. The aggregation of some AuNPs could be seen, forming larger particles, which was consistent with the results of DLS. This tendency for agglomeration can be attributed to the presence of plant-derived organic compounds with polar groups on the surface of NPs. Moreover, the zeta potential of AuNPs suggested that they electrostatically tended to reconcile with each other due to the lack of sufficient electrostatic repulsion. To determine the size of the synthesized AuNPs, 100 FE-SEM images were analyzed using the ImageJ software. The histogram of particle size distribution is shown in Fig. 7-c. In this histogram, the horizontal axis shows the particle size visible in the FE-SEM micrograph, and the vertical axis represents the count of NPs detected by software. Accordingly, more than 80% of the particles had a size between 50 and 110 nm, with most of them being at the range of 70–80 nm (Table 3).

Table 3 FE-SEM size distribution data of gold nanoparticles synthesized by S. sclarea extract.
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As shown in Table 3, the mean size of AuNPs was 80.80 nm, and the standard deviation was 22.31 nm. Considering that the standard deviation was less than 30% of the mean, it could be concluded that the particle size distribution was relatively narrow (i.e., a plausible difference between the smallest and largest particles). Moreover, as the mean particle size was almost the same as the median value, particle size distribution seemed to be relatively normal, which could also be seen in the appearance of the histogram (Fig. 7-c).

The EDX and elemental map analysis were also employed to further study AuNPs. According to EDX analysis (Fig. 7-d), the gold element (88.5%) was the main component of AuNPs, evidenced by a sharp absorption peak at 2.15 kv107. Oxygen (1.1%) and carbon (10.3%) found in the 0.5 kv range108 represented the surface modifications of AuNPs by plant-derived compounds. As shown in the elemental analysis map (Figs. 6-f and 7-e), the uniform distribution of gold was obvious throughout the sample, indicating the proper dispersion of AuNPs.

Fig. 7
figure 7

FE-SEM image of AuNPs green-synthesized by S. sclarea (a and b). Size distribution histogram based on FE-SEM image analysis (c). EDX image of AuNPs (d). EDX mapping of the synthesized AuNPs (e and f).

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TEM analysis

The shape, size, and aggregation of the AuNPs biosynthesized by S. sclarea were investigated using TEM (Fig. 8). The active metabolites in the structure of the aqueous extract of S. sclarea were capable of forming dark color NPs with different shapes (triangular, polygonal, and spherical). The NPs showed proper size dispersion within the 20–50 nm range, which agreed with the morphology and particle sizes reported in previous studies109,110,111. Some differences in the size and shape of NPs can be attributed to plant metabolites acting as reducing agents. The biological activity of green-synthesized NPs depends on different factors such as size, shape, surface topology, surface charge, and tendency for aggregation112. A study showed that selenium nanoparticles (SeNPs) possessed higher antimicrobial activity when their diameter was between 21 and 40 nm compared to the 41–50 nm range113. Therefore, optimization of the size, morphology, surface charge, and tendency for aggregation is a vital step toward expanding the biological applications of green-synthesized NPs.

Fig. 8
figure 8

TEM micrographs of gold nanoparticles synthesized by S. Sclarea extract.

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Anti-bacterial activity of green-synthesized AuNPs

Table 4 summarizes the effects of AuNPs green-synthesized by S. sclarea at a concentration of 500 µg/mL on the growth of four standard bacteria compared to gentamicin antibiotic. The AuNPs, at lower concentrations, had the highest inhibitory effects on Gram-positive B. cereus (MIC: 41.66 ± 3.60 µg/mL) and S. aureus (83.33 ± 1.44 µg/mL MIC: ). However, Gram-negative bacteria showed some levels of resistance to AuNPs. Considering the minimum bactericidal concentration (MBC), Gram-positive B. cereus (MBC: 250.00 ± 0.00 µg/mL) and S. aureus (MBC:416.66 ± 28.86 µg/mL) were susceptible to lower concentrations of AuNPs compared to Gram-negative P. aeruginosa and E. coli (MBC > 500 µg/mL). The ZOI values of AuNPs observed against the bacteria were as follows: B. cereus (17.26 ± 0.68 mm), S. aureus (14.83 ± 0.61 mm), P. aeruginosa (11.90 ± 0.52 mm), and E. coli (12.23 ± 0.40 mm, Fig. 9). In comparison with gentamicin, the standard antibiotic showed better antibacterial effects than AuNPs; however, based upon MIC, MBC, and ZOI values at the concentration tested, the AuNPs synthesized by the plant extract had considerable antibacterial effects against both Gram-positive and Gram-negative bacteria (P < 0.01). Studies have proven that when metal ions, such as gold, come into contact with bacteria, they trigger the efflux of intracellular ions, promoting considerable toxicity114. Our results showed that Gram-positive bacteria were more susceptible to AuNPs and gentamicin antibiotic compared to Gram-negative strains. This can be attributed to the absence of an outer membrane in Gram-positive bacteria. Another toxicity mechanism includes the disruption of cellular respiration115. It seems that the antibacterial effects of AuNPs synthesized by plant extracts occur in two steps: (1) AuNPs distort the membrane potential and metabolic processes by reducing the activity of adenosine triphosphate (ATP) synthase and (2) they inhibit protein production by preventing ribosomes from binding to tRNAs116. Recent studies have proven that AuNPs synthesized by green nanotechnology have notable antibacterial and antibiofilm activities117, enabling them as viable tools to fight against a wide range of infectious bacteria118,119,120. Recent research on AuNPs synthesized by Callistemon viminalis121 and Clerodendrum trichotomum122 has demonstrated the high antimicrobial activity of the NPs against both Gram-positive and Gram-negative bacterial strains, including E. coli, S. aureus, Klebsiella pneumoniae, and P. aeruginosa. Likewise, silver nanoparticles synthesized by Lagerstroemia speciosa123 and Celastrus paniculatus Willd.124 exhibited antibacterial and anti-biofilm properties against different strains of K. pneumoniae, S. aureus, Proteus vulgaris, and P. aeruginosa. Recent studies have also highlighted the antibacterial activity and larvicidal effects of nanoemulsions produced from the essential oil of Vitex negundo L. against Aedes aegypti L125. and the antibacterial (Enterococcus faecalis, S. aureus, Salmonella paratyphi, and K. pneumoniae) and larvicidal (Culex quinquefasciatus) effects of nanoemulsions derived from the essential oil of Ocimum basilicum L126. Ultimately, multiple studies have demonstrated that the type of metals used in green synthesis, variations in plant species, and methodological approaches can influence the size and morphology, and thereby the biological effects, of NPs15,127,128,129.

Table 4 Antibacterial activity of AuNPs biosynthesized by S. sclarea extract.
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Fig. 9
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Antimicrobial activity (ZOI) of green-synthesized gold nanoparticles generated by S. sclarea against four Gram-positive and Gram-negative bacteria.

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Antifungal activity of green synthesized AuNPs

Table 5 shows the effects of AuNPs biosynthesized by S. sclarea at a concentration of 80 µg/mL on four Candida species (a total of 40 clinical and standard isolates) compared to fluconazole. According to the MICGM, the biosynthesized AuNPs had more pronounced effects on C. krusei (MICGM: 25 µg/mL) compared to other Candida isolates, but the MICGM was still inferior compared to that of fluconazole (MICGM: 0.125 µg/mL). The highest MIC range of the synthesized AuNPs (50–100 µg/mL) was related to C. albicans and C. parapsilosis, and the lowest range (12.5–50 µg/mL) belonged to C. krusei. The MIC range of fluconazole was still lower for all candida isolates (0.125–0.25 µg/mL). According to the results, the MIC50 of fluconazole was lower for non-albicans Candida species, including C. parapsilosis, C. glabrata, and C. krusei (0.125 µg/mL) than for C. albicans species (0.25 µg/mL). One study evaluating the anticandidal efficiency of AuNPs synthesized by Spirulina maxima reported the MIC and MLC values of 32 and 64 µg/mL for these NPs, respectively130, asserting the better anticandidal effects of AuNPs at lower concentrations. Shirzadi-Ahodashti (2023) evaluated the antifungal effects of AuNPs biosynthesized using Pistacia vera extract, expressing that at the concentrations of 137 and 550 µg/mL, AuNPs could inhibit fungal growth131. In comparison, our results showed superior anti-fungal effects for AuNPs at lower concentrations. The differences observed in the effective fungicidal concentrations of AuNPs could be justified by methodological variations and use of different Candida species (i.e., standard vs. clinical isolates, each of which showing different drug resistance patterns)132. The unique structure of green synthesized AuNPs enable them to be introduced as novel antifungal agents against different candida species133. The toxicity mechanisms can encompass binding to the cell membrane of infectious and pathogenic fungi, destroying the plasma membrane, and inhibiting the growth of these pathogens134. It has been noted that AuNPs synthesized by the aqueous extracts of plants can bind to the cell wall of candida species through electrostatic interactions, leading to an increase in reactive oxygen species (ROS), interruption of cellular growth signals, and induction of apoptosis130. In a study, Shahid Khan et al. (2024) used green synthesis to produce silver and gold nanoparticles using Callistemon viminalis extract and investigated their antimicrobial activities. The results demonstrated that the biosynthesized silver and gold nanoparticles exhibited significant effectiveness against C. albicans, C. krusei, Aspergillus sp., and Trichoderma species121. Another study conducted to evaluate the effectiveness of biosynthesized silver nanoparticles derived from Cardiospermum halicacabum L. extract against a wide range of Gram-positive and Gram-negative bacteria, as well as fungal pathogens responsible for plant infections and diseases, such as Alternaria solani and Fusarium oxysporum, demonstrated that these nanoparticles exhibited significant efficacy against this category of pathogens135. These findings aligned with the results of our study, supporting the fact that green-synthesized metal NPs across different sizes and morphologies can effectively suppress the growth of microorganisms, including Gram-positive and Gram-negative bacteria, as well as various fungal strains136,137,138.

Table 5 The susceptibility indicators (MIC range, MIC50, MIC90, and MICGM) of Candida isolates against green-synthesized AuNPs.
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Scolicidal activity of green synthesized AuNPs

Table 6; Fig. 10 show the results of treatment of hydatid cyst protoscolices with different concentrations (20, 40, 80, 160, and 320 µg/mL) of the synthesized AuNPs at different times (5, 10, 15, 30, 60, and 120 min). The highest percentage of lethality (100%) was observed at the concentration of 160 µg/mL within 30 min and 80 µg/mL within 60 min, and the lowest scolicidal activity (9%) was observed at the concentration of 20 µg/mL within 20 min of exposure (P < 0.05). Therefore, the in-vitro scolicidal effects of AuNPs were dose- and time-dependent (P < 0.05). Today, surgery is considered the most feasible technique for hydatidosis treatment139. During the surgical operation, it is important to prevent patient exposure to protoscolices and prevent their spillage and spread to other organs using scolicidal agents such as formaldehyde, hypertonic salt, silver nitrate, and cetrimide, which are injected into hydatid cysts140. The injection of these chemicals; however, is accompanied by various side effects, such as hepatocyte necrosis141. In this study, the AuNPs prepared by S. sclarea extract showed effective scolicidal effects and could be used before the surgical removal of hydatid cysts. Currently, green synthesized AuNPs are used in the diagnosis, development, and production of several common drugs for parasitic diseases caused by leishmania, giardia, toxoplasma, and Schistosoma142. Çolak et al. (2019) showed that photothermal Au-NCs (0.8 mL) were capable of removing 89.3% of hydatid cyst protoscolices under laboratory conditions within 120 min143. Barabadi et al. (2017) reported that Au-NCs synthesized by Penicillium aculeatum had significant scolicidal effects in vitro, eradicating 90% of protoscolices after 2 h exposure to the dose of 0.3 mg/mL144. In our study, the synthesized AuNPs were capable of killing 100% of hydatid cyst protoscolices at lower concentrations and within a shorter time period compared to previous studies. These differences in scolicidal effects are probably due to factors such as the type of the used plant, synthesis methods of NPs, and duration and concentration of treatment. Previous studies have reported that AuNPs can suppress the activity of metabolic enzymes such as trypanothione reductase and cysteine protease falcipain-2 in parasites such as leishmania and impair protein synthesis and DNA replication145,146,147.

Table 6 Protoscolicidal effects of green-synthesized AuNPs on the protoscolices of hydatid cyst at different times and concentrations.
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Fig. 10
figure 10

Scolicidal effects of biosynthesized AuNPs against protoscolices of hydatid cyst at different concentrations following various exposure times.

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Control + (NaCl 5%); Control – (Normal saline).

Conclusion

In this research, the biosynthesis of AuNPs using the aqueous extract of Salvia sclarea L. was presented as an easy, fast, inexpensive, and environmentally friendly method to produce biologically active nanomaterials. The AuNPs were characterized for physicochemical features, and their antibacterial activity was tested against four Gram-positive and Gram-negative bacterial strains, anti-candida activity against 40 clinical isolates, and scolicidal activity against Echinococcus granulosus of liver hydatid cyst in vitro. Our results showed that the biosynthesized AuNPs had a polygonal and spherical shape and an average size of 20–50 nm and were effective against bacteria (S. aureus, B. cereus, E. coli, and P. aeruginosa), clinical isolates of Candida (C. albicans, C. parapsilosis, C. glabrata, and C. Krusei), and protoscolices of liver hydatid cyst. By introducing a one-step, eco-friendly, cost-effective, and biologically safe method for Salvia sclarea L.-mediated green synthesis of AuNPs, this study offers a compelling contribution to the field of green nanotechnology. The use of Salvia sclarea L. flower extract as a reducing and stabilizing agent highlights the potential of plant-based approaches to produce AuNPs with significant antibacterial, anticandidal, and scolicidal properties. The novelty of this study lies on leveraging Salvia sclarea, a readily available plant, to create stable nanoparticles with promising biomedical applications, aligning with the global push for sustainable nanotechnology. Compared to previous studies, this report excels in using an environmentally benign and simple strategy, avoiding toxic chemicals commonly used in conventional methods. However, limitations such as potential variabilities in plant extracts’ composition, scalability challenges, and lack of mechanistic insights into AuNPs’ bioactivity temper their immediate applicability. Future research should focus on optimizing synthesis parameters, elucidating molecular interactions, and evaluating in vivo efficacy to fully realize the therapeutic potential of AuNPs. Overall, this work underscores the promise of green synthesis in advancing nanotechnology for antimicrobial and antiparasitic applications, paving the way for sustainable and effective biomedical innovations.