The RAW264.7 and A549 cell lines were independently cultured in DMEM and RPMI-1640 medium, respectively. Each medium was supplemented with 10% FBS and 1% Penicillin–Streptomycin to enhance cell viability and prevent microbial contamination. The cells were maintained under optimal growth conditions: at 37 °C in a humidified incubator with a 5% CO₂ atmosphere.

The in vitro cytotoxicity of the Pk-AuNps was tested by MTT assay (34). Cells were plated at a density of 1 × 10⁴ cells per well in 96-well culture plates (Corning Costar, Lowell, NY, United States) and incubated overnight to allow for attachment. Upon completion of the incubation period, the cells underwent a 24 h treatment regimen with the nanoparticles, with three wells free of Pk-AuNps used as controls. Subsequently, 20 μL of MTT solution (5 mg/mL, in PBS) was introduced to each well for a 3 h interval. The spent media and MTT reagent were then aspirated and superseded with 100 μL of DMSO (10%), allowing an additional 30 min for incubation in dark and shaken. The amount of formazan, an indicator of viable cells, was quantified via a multi-mode microplate reader (Bio-Tek Instruments, Winooski, VT), using test and reference wavelengths of 570 nm and 630 nm, respectively.

The murine macrophage RAW264.7 cells were co-treated with 1 μg/mL LPS and a dose of Pk-AuNps and then subjected to a 24 h incubation period. The supernatant was collected for further analysis. To detect the level of nitric oxide (NO), an equal volume of Griess reagent was combined with 100 μL of the harvested culture supernatant. This mixture was then assessed using a multi-plate reader, set to a wavelength of 540 nm. The levels of Prostaglandin E2 (PGE2) and Tumer Necrosis Factor-α (TNF-α) were determined through Enzyme-Linked Immunosorbent Assay (ELISA) commercially available kits strictly following the manufacturer’s procedures (R&D Systems, Minneapolis, MN, United States) (20).

The capacity of Pk-AuNps to provoke oxidative stress was gaged using the DCFH-DA assay (35). Adherent RAW264.7 cells, following being seeded in a 96-well black/clear bottom plate (Corning Costar, Lowell, NY, United States), were exposed to varied concentrations of Pk-AuNps for 24 h. Subsequently, 100 μL of DCFH-DA reagent, prepared at a concentration of 15 μmol/L, was added and permitted to incubate for 30 min under low-light conditions. Thereupon, the fluorescence, a measure of reactive oxygen species (ROS) generated within the cells, was read using a Synergy™2 microplate reader (Bio-Tek Instruments, Winooski, VT).

RAW264.7 macrophages were seeded at a density of 1 × 10⁶ cells/well in a 6-well culture plate (Corning Costar, Lowell, NY, United States). After an initial overnight incubation, 24 h of treatment with or without diverse doses of Pk-AuNps in the presence or absence of LPS stimulation was added. Total RNA was extracted using the TRIzol reagent. The cDNA synthesis was conducted in accordance with the supplier’s instructions (Thermo Scientific, EU, Lithuania). qPCR was carried out using the primers shown in Table 1 (20). The relative gene expression levels were normalized to the amount of GAPDH. mRNA by the ΔCt method (36).

RAW264.7 cells were cultured overnight on 8-well chamber slides (SPL Life Sciences Co., Ltd., Korea). The cells were pre-conditioned with Pk-AuNps for 2 h to evaluate their potential modulatory effects, followed by a stimulation phase with LPS (1 μg/mL) for an additional 2 h. Subsequently, the cells were meticulously washed with PBS, then fixed with a 3.7% formaldehyde solution to preserve cellular morphology and permeabilized with 0.5% Triton X-100 for 10 min to facilitate antibody penetration. Followed, the slides were incubated overnight at 4 °C with rabbit monoclonal anti-NF-κB p65 antibodies (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, United States). After thorough washing to remove unbound primary antibodies, the slides were incubated in the dark for 1 h with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibodies (1:200; Cell Signaling Technology, Beverly, MA, United States) to detect the primary antibody binding. For nuclear counterstaining, the slides were performed using DAPI, a fluorescent stain, at a concentration of 10 mg/mL for 15 min, allowing for the visualization of cellular nuclei. Finally, the immunofluorescence-stained slides were examined using an inverted research fluorescence microscope, capturing images that reveal the intracellular localization of NF-κB p65 in response to the experimental treatments (37–39).

Employing FE-TEM, we conducted a comprehensive investigation to elucidate the characteristics and dimensionality of the synthesized nanostructures (42), with a particular focus on gold nanoparticles. Our study uniquely demonstrates that the Pk-AuNps predominantly exhibit a quasi-spherical configuration, with sizes precisely ranging from 10 to 20 nm, as clearly portrayed in Figures 1c,d. The controlled synthesis of these nanoparticles achieved by modulating key parameters such as reaction temperature, and incubation time can be readily extended to the tailored production of other gold nanoparticles. This is accomplished by leveraging the same bio-reduction principles mediated by the phytochemical constituents present in the P. koreana root extract, which act as both reducing and stabilizing agents. Such reproducibility and tunability represent an innovative contribution to the field of green nanobiotechnology.

Elemental mapping of the nanoparticles provided a vivid electron imagery, confirming the elemental uniformity across the particles, as depicted in Figures 1d,e. This uniformity is essential for the consistent performance of Pk-AuNps in various applications.

The purity of these metallic nanoparticles was further authenticated through meticulous examination of the EXD spectra, ensuring the high-quality material needed for advanced nanodevices (43). The distinct peaks at 2.15 keV, corresponding to the characteristic absorption edges of metallic gold, were observed in Figure 1f, indicating the exceptional purity of Pk-AuNps. The peak recorded at 8 keV correspond to coper grid used for analysis.

The ability to control the crystallographic facets is pivotal for tailoring the electronic properties of gold nanoparticles, which is an innovative aspect of our methodology, highlighting the potential for their use in nanoscale devices with specific performance criteria.

The XRD pattern of the crystalline Pk-AuNps, with its pronounced and intense diffraction peaks as highlighted in Figure 2a, is indicative of a well-defined crystallographic structure, which is a prerequisite for various high-performance applications (44).

The four characteristic diffraction peaks, indexed to the [111], [200], [220], and [311] lattice, and primarily composed of [111] orientation, align with Bragg’s law and the reference patterns of the Joint Committee on Powder Diffraction Standards (JCPDS No. 04–0784). This precise peak assignment confirms the face-centered cubic (FCC) structure of the gold nanoparticles and reflects a high degree of crystallographic ordering, essential for their robust physical and chemical properties (45, 46). The average diameter of nanoparticles was estimated by Debye–Scherrer equation: Pk-AuNps maintained average crystallite sizes of 6.31 nm. The SAED patterns likewise confirmed the polycrystalline nature of the nanoparticles (47), as shown in Figure 2b.

In addition to the applications in catalysis, sensing, and electronics, the high crystallinity and biocompatibility of these nanoparticles also make them suitable candidates for innovative uses in the food science industry. For instance, the Pk-AuNps could be integrated into active food packaging to enhance the shelf life of perishable goods by preventing microbial contamination. Their inherent antimicrobial properties can be harnessed to create self-sanitizing surfaces that maintain food safety and quality.

Furthermore, the gold nanoparticles’ non-toxic nature and ability to withstand high temperatures suggest their potential suitability for use in food processing environments, where they could contribute to thermal management applications. The innovative synthesis method employed in our study ensures the formation of highly crystalline nanoparticles with a narrow size distribution, which is evident from the sharp and intense peaks in the XRD pattern. This controlled synthesis is essential for achieving the uniformity expected in food-grade nanomaterials—a critical prerequisite for any future functional applications. It should be noted, however, that the actual performance and stability of these nanoparticles under realistic food processing conditions—such as exposure to thermal cycling, shear forces, or variable pH—have not been evaluated in this study and warrant further investigation.

The size distribution profile of the biosynthesized Pk-AuNps was meticulously characterized using DLS, a technique that assesses particle size with respect to volume, number, and intensity, as depicted in Figures 2c–e. The resulting size distribution histogram exposed a moderately polydisperse population, with a Z-average value of 244.8 nm for Pk-AuNps, accompanied by PDI of 0.229. This observation indicates that the nanoparticles produced through the green synthesis process using P. koreana are not monodisperse but exhibit a common distribution type for biological syntheses.

The primary and secondary metabolites of P. koreana, including ranunculin, anemoside A, phenylpropanoids, and flavonoid glycosides, could potentially contribute to the formation of a protective capping layer around the metallic nanoparticles, thereby preventing their agglomeration (6). The discrepancy between the average sizes of the biogenic nanoparticles determined by XRD and FE-TEM versus DLS can be elucidated by the distinct measurement principles and sample states inherent to each technique. XRD anticipates the crystallite size of the nanoparticles in their dried state, providing an insight into the primary particles’ dimensions. FE-TEM further provides two-dimensional projection measurements of individual particles or small agglomerates under high vacuum on dried specimens. In contrast, DLS measures the hydrodynamic diameter in an aqueous suspension. This hydrodynamic diameter encompasses the metallic core, tightly bound hydration layers, and the biomolecular corona derived from the plant metabolites stabilizing the nanoparticles (48). Crucially, DLS exhibits high sensitivity toward any aggregates or agglomerates present in the suspension, with its intensity-weighted distribution heavily influenced by larger species. This phenomenon explains the observed difference where XRD and FE-TEM report the core or near-core dimensions, while DLS reflects the larger hydrodynamic size including the capping layer and potential aggregates in solution. Therefore, this disparity underscores the importance of employing multiple complementary characterization techniques to fully understand the size distribution, surface characteristics, and the interaction of synthesized nanoparticles with their surrounding environment (49, 50).

Moreover, the FE-TEM images corroborated the DLS findings by revealing a range of sizes within the nanoparticle population, confirming the moderate polydispersity. The acknowledgment of this moderate polydispersity is crucial for applications requiring a tailored size distribution, such as nutrient delivery or sensing applications, where nanoparticle interactions with biological systems are influenced by their size and distribution characteristics (51).

Employing FTIR, we characterized the organic molecules on the surface of bio-reduced gold nanoparticles synthesized from P. koreana. The FTIR spectra disclosed a distinctive absorption pattern that corroborated the presence of specific functional groups on the nanoparticle surface, as depicted in Figure 3a. Notably, the bands at 3364–3345 cm⁻¹ corresponded to O–H stretching vibrations (52), indicative of hydroxyl groups, while the regions of 1,626–1,634 cm⁻¹ and 1,016–1,067 cm⁻¹ were attributed to C–H and C═C/C–O groups (53), respectively, reflecting the presence of methylene/aromatic and alkene/ether functionalities. These groups are crucial for the stabilization of Pk-AuNps and are suggested to enhance their biological activity through surface capping. The disappearance of FTIR bands at 1425.55 cm⁻¹ and 767.15 cm⁻¹ in Pk-AuNps, compared to the P. koreana extract, originated from synthesis-driven chemical transformations and surface interactions (54). The 1425.55 cm⁻¹ band, characteristic of aliphatic C-H bending vibrations or −COO⁻ symmetric stretching, vanished due to vibration restriction from hydrophobic adsorption onto Pk-AuNP surfaces or -COOH deprotonation forming coordinating carboxylates. The 767.15 cm⁻¹ band, indicative of aromatic C-H out-of-plane bending in polyphenols, disappeared owing to oxidative aromatic ring degradation or hydrogen-bond-mediated peak broadening/redshift. Concomitant loss of these bands confirms polyphenols mediated metal ion reduction through aromatic electron donation and nanoparticle stabilization via aliphatic chain adsorption or carboxylate coordination. Enhanced spectral changes from purification-induced removal of unbound molecules provide spectroscopic evidence for plant metabolites’ role in Pk-AuNP synthesis (55).

The findings from the FTIR spectra underscore the potential of Pk-AuNps as a nano-carrier platform. The surface composition characterized by FTIR analysis offers a basis for optimizing the biological performance of these nanoparticles (56, 57).

In this current study, RAW264.7 and A549 cells were used as model systems to assess the cytotoxicity of Pk-AuNps. Cell viability was measured in vitro using the MTT assay. Cells were treated with Pk-AuNps at concentrations ranging from 1 to 100 μg/mL for 24 or 48 h. Results showed no significant cytotoxicity in RAW264.7 cells even at the highest concentration tested (50 μg/mL) after 24 h, well, at 48 h in RAW264.7 cells, 100 μg/mL Pk-AuNps showed minimal cytotoxicity (Figure 4a). In contrast, the A549 cell line exhibited significant growth inhibition at 50 μg/mL (Figure 4b). These findings enabled the selection of 50 μg/mL as the maximum concentration for subsequent in vitro experiments.

Macrophages constitute one of the key immune defense mechanisms within the human body endowed with the ability of phagocytosis (63). They are also involved in the inflammatory response by generating both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) through NF-κB activation (2, 64). Previous studies have reported that NO production by iNOS and PGE2 was derived from COX-2 and played an essential role during the inflammatory reaction process. Antigen-presenting cells (APCs), such as macrophages, play a vital role in initiating immune responses (65, 66). The complement system also contributes to the inflammatory process.

We first verified the antioxidant capacity of Pk-AuNps. As shown in Figure 4c, the ROS level decreased in a concentration-dependent manner with increasing concentrations of Pk-AuNPs. At a treatment concentration of 50 μg/mL, the cellular ROS level was significantly reduced. This finding laid the foundation for subsequent research on anti-inflammatory activity. Consequently, to ascertain the anti-inflammatory effect of Pk-AuNps, the levels of NO, TNF-α, and PGE2 were measured. We also undertook the determination of the production of NO, TNF-α, and PGE2 in LPS-induced RAW264.7 cells, with or without Pk-AuNps treatment, using DEX as a positive control.

In Figure 4d, our results indicated an increase in NO levels in the LPS-induced RAW264.7 cells compared with the basal level without LPS (1 μg/mL) treatment. The dose-dependent treatment of Pk-AuNps significantly reduced the NO level. The qPCR analysis in Figure 4g exhibited that the expression of iNOS at the gene level was also suppressed. Figure 4d indicated that the LPS-induced PGE2 production release was decreased by treating with Pk-AuNps in a dose-dependent manner. In addition, the gene expression of COX-2 significantly decreased with the Pk-AuNps treatment of the LPS-stimulated murine macrophages (Figure 4h). Consequently, Pk-AuNps blocked the activities of both iNOS and COX-2 at the mRNA level in the LPS-induced RAW264.7 cells.

In previous research, the extract of P. koreana was found to suppress NO production in LPS-stimulated RAW264.7 cells. Moreover, these oleanane-type triterpenoid saponins showed a considerable degree of activity in inhibiting TNF-α-induced NF-κB activation (64). Notably, Pk-AuNps could significantly inhibit the PGE2 levels in 25 μg/mL (Figure 4e). Activated macrophages and T cells are capable of generating TNF-α and other proinflammatory cytokines as an integral part of the immune response (67, 68). By conducting ELISA to test the production (17) of TNF-α in LPS-induced RAW264.7 cells, we evaluated the efficacy of Pk-AuNps in diminishing proinflammatory mediators. Pk-AuNps reduced the LPS-induced release of TNF-α in a dosage-dependent manner, as depicted in Figure 4e. Then, qPCR was employed to assess the TNF-α gene level. It was observed that TNF-α levels in the stimulated RAW264.7 cells were reduced by Pk-AuNps (Figures 4f,i). The attenuating of TNF-α in our study implied that Pk-AuNps might possess anti-inflammatory attributes (69).

According to the immunofluorescence staining presented in Figure 5, the density of NF-κB (green fluorescence) was conspicuously augmented at 2 h of LPS exposure. Nevertheless, LPS-induced nuclear translocation was markedly inhibited by the co-treatment of Pk-AuNps, the quantities of nuclear NF-κB p65 were escalated following LPS exposure, yet Pk-AuNps significantly impeded this LPS-induced nuclear translocation. These findings align with the concept of immunomodulation, suggesting that Pk-AuNps may have potential in regulating immune responses (64) and inflammatory conditions. Further studies could explore its interaction with immune checkpoints and cytokine networks for a comprehensive understanding of its immunological effects.

Within the framework of the immune system, cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and interferon-γ (IFN-γ) play pivotal roles in regulating inflammatory responses. The major histocompatibility complex (MHC) molecules are indispensable for antigen presentation (70). Regulatory T cells contribute significantly to maintaining immune homeostasis (71). The complement cascade constitutes another crucial element of the immune defense mechanism (72). Future studies could explore the interaction of Pk-AuNps with these immune factors to furnish a more comprehensive comprehension of its immunomodulatory effects.