The special phytochemical composition of RRR water extract causes the formation of the envelope surrounding gold metal atom and acts as a capping and stabilizing layer that prevents the aggregation of gold nanoparticles and the formation of larger structures. This result is in agreement with Timoszyk & Grochowalska, (2022)30.
Phytochemical compounds have an essential and defensive role against the harmful effects of free radicals and oxidative stress on the constituents of biological systems including lipids, carbohydrates and proteins biomolecules which can oxidize them resulting in cellular and tissue damage and many other diseases including inflammation, degenerative diseases and cancer30.
HPLC analysis of the RRR water extract revealed a complex profile of bioactive polyphenols such as catechins, gallic acid, caffeic acid, and chlorogenic acid, which constitute the primary phytoconstituents. Additional metabolites were detected including syringic acid, rutin, naringenin, vanillin, rosmarinic acid, ferulic acid, hesperetin, quercetin, coumaric acid, cinnamic acid, kaempferol, daidzein, and methyl gallate which assures the extract’s chemical diversity. These hydroxyl-rich polyphenolic compounds are of critical importance in this study, as they serve a dual role: acting as potent reducing agents for the transformation of gold ions into Au0 nanoparticles and subsequently functioning as capping ligands that stabilize the RRR-AuNPs. This diverse antioxidant assembly not only facilitates the green synthesis process but also synergistically enhances the antimicrobial and radical scavenging performance of the resulting biogenic nanomaterials. These findings are in alignment with Goyeneche et al., (2015)31.
The water extract of the roots of R. sativus has been screened for its DPPH free radical scavenging and reducing property, where the phytochemical components’ capacity that neutralise highly reactive free radicals was the main cause of the antioxidant property30. A relatively high antioxidant activity was obtained in this study for both RRR water extract and RRR-AuNPs with the higher value was for the RRR-AuNPs. Data obtained in this study is coincided with Noman et al., (2021)32 which suggested that RRR-AuNPs possess the ability to scavenge free radicals representing a powerful antioxidant, anti-inflammatory, anti-mutagenic and anticancer agent and might explain its anticancer activity towards HeP2G and Caco-2 cell lines that will be discussed later.
Furthermore, the antagonizing effect of polyphenols against radicals results from their prooxidant power which prevents lipid per-oxidation. The oxidation of all o-dihydroxylated phenolic derivatives results in the production of reactive oxygen species (ROS)33.
The AuNPs antioxidant effect was found to be higher than the antioxidant activity of the plant extract individually because the antioxidant phytochemical compounds of RRR extract are adsorbed onto the nanoparticles active surface. The high surface area-to-volume ratio of a nanoparticle and its surface reaction may also affect the interaction and free radicals scavenging activity, revealing a higher antioxidant potency. These results are compatible with32,34,35.
Synthesized nanoparticles have broad-spectrum applications in biomedicine, cosmetics, coating and, packing etc. Each application depends on the even size, composition, shape and stability of the prepared nanoparticles. Medicinal plant is used recently to prepare nanoparticles due to its simplicity, eco-friendless and its valuable content of bioactive compounds which have a strong capacity to bio-reduce gold heavy metal ion (Au+) into a stable nanoparticles metal (Au0)36.
The RRR aqueous extract reduced HAuCl4 leading to the biosynthesis of gold nanoparticles (AuNPs) which was confirmed by the formation of ruby red color after 24 h. The plant extract phytoconstituents are acting as both stabilizing and reducing agents during the nanoparticles’ synthesis. This result is compatible with Madhumithra, et al., (2018)37 which confirmed the formation of gold nanoparticles by the water extract of shells of Pistacia vera plant.
UV/Vis spectroscopy is the primary technique to confirm the nanoparticles’ formation and stability in an aqueous solution. The UV/Vis spectroscopy for greenly synthesized RRR-AuNPs indicated that the AuNPs were formulated successfully by recording peaks in the specific region for gold nanoparticles. It was documented that UV/Vis spectra for the gold nanoparticles exhibited plasmon peak about 525–540 nm and it’s a regular characteristic of spherical shaped AuNPs that have a diameter ranged between 30 and 50 nm38,39.
Predominantly, the RRR-AuNPs spherical shapes as showed by the HRTEM indicate the polydispersity nature of the synthesized nanoparticles. This might be due to the presence of different phytochemical compounds (reducing agent) in RRR extract which coated AuNPs forming particles of uneven size. Bright diffraction rings shown in SAED pattern also confirmed the crystallographic phase of the biosynthesized RRR-AuNPs. These rings appeared from the innermost to the outermost boundary as the (111), (200), (220), and (311) reflections, which perfectly correspond to the face-centered cubic (fcc) crystal lattice of metallic gold31.
Zeta potential is the fundamental factor that manages electrostatic interactions in particle dispersions. The zeta sizer and potential of AuNPs capped with R. sativus root extract were 31 nm and − 36.8mV, respectively. The synthesized AuNPs were very stable when stored in refrigerator and the particles of the colloidal AuNPs solution were not aggregated for several months. The high stability of the colloidal AuNPs suspension can be explained by its very high negative zeta potential value40.
XRD revealed that the peaks for 2θ at 38.191o, 44.391o, 64.583o and 77.679o corresponding to the reflections of the planes (111), (200), (220) and (311) supported the bio-reduction of Au (III) to Au (0) by the effect of the phytochemical compounds present in the root of water extract of R. sativus and also emphasizes the crystalline shape of gold atom41.
The dynamic light scattering (DLS) screening confirmed the size of RRR-AuNPs and was corresponding to those of TEM which indicated their disparate sizes. The uneven size distribution of RRR gold nanoparticles was accounted for the diverse coating of AuNPs with various phytochemicals of R. sativus root extract12.
In FT-IR spectra, the presence of stretching bands of O-H for alcohols or phenols, C = C for alkenes or aromatics and C–O for carboxylic acids suggest that polyphenols and flavonoids were involved in the formulation of AuNPs through the reduction of gold ions to gold atoms. Phenolic compounds are documented to possess an exceptional binding affinity for metallic ions, which directly facilitates the reduction of gold ions to elemental gold while inducing a robust chelation effect. Concurrently, the carboxylate groups serve as natural surfactants during nanoparticles synthesis. The strong affinity of these carboxylate groups for the newly formed AuNPs inhibit nanoparticle aggregation and maintaining long-term colloidal stability31.
Energy-dispersive X-ray (EDX) spectroscopy is a widely utilized analytical technique for identifying the elemental composition and purity of synthesized nanomaterials. EDX characterizes the metallic core alongside any associated organic elements derived from the plant extract. The EDX spectrum of the synthesized RRR-AuNPs displayed a distinct, dominant absorption peak at approximately 2.2 keV, which is uniquely characteristic of metallic gold (AuL-alpha shell energy). The study of Barai et al., (2018) is compatible with our findings41.
AFM analysis is used to estimate the morphology of nanoparticles’ surface. AFM image proved the spherical shape and the rough surface of the gold nanoparticles capped with R. sativus root extract12. AFM has reported versatility in various fields of study representing a dynamic method for conceiving the topographic characteristics of nanoscale. The current findings revealed that the bioactive compounds that capped AuNPs were agglomerated resulting in the synthesis of discrete nanostructures. The dissimilar size and morphology of the AuNPs is attributed to the presence of both single and aggregated nanoparticles. These results are harmonized with Ullah et al., (2021)42.
The surface area measured by the BET method of N2 adsorption–desorption isotherm curves clarified the high surface area of green synthesized RRR-AuNPs. The adsorption isotherm curve of synthesized nanoparticles showed the V-type isotherm at which the effects of intermolecular attraction are great, and the adsorption occurs in pores and capillaries43. This indicates that RRR-AuNPs have a large distance of pores and capillaries.
The current findings reported that the synthesis of AuNPs using water extract of R. sativus root as a reducing agent had a potential antibacterial effect against S. aureus and E. coli. Bacterial colony forming unit counting test showed a high percent of inhibition of the growth of those two bacterial strains after 24 h of treatment by RRR-AuNPs which confirms the powerful antibacterial activity of gold nanoparticles capped with red radish root extract. The zone of inhibition test is unfavourable to be applied in nanoparticle solutions because such solutions are suspension which dispersion in the solidified agar media is imperfect causing inaccurate results; therefore, percent of inhibition test is used instead44.
The antibacterial effect of the gold nanoparticles depends on many factors such as the rate of absorption, metabolites release, metabolic functions and its distribution in the cell. The surface positive charge of gold nanoparticles binds and interacts greatly with the negative charge of surface of bacterial cell wall through active moieties penetrating the cell, where they start to interact with its different components. Hydrophobic AuNPs that have a positive surface charge, can create localized aggregates on the of bacterial cell surface leading to easy penetration of the cell wall. Moreover, the modulation of bacterial cell membrane and the electrostatic interactions were the fundamental mechanisms through which the AuNPs stimulated its antibacterial effect. The bacterial strain can also affect the mode of interaction with bacterial cells30,42.
In addition, RRR water extract showed a week to moderate inhibition activity of growth of S. aureus and E. coli and that was attributed to phytochemical constituents such as phenolics, flavonoids, and glucosinolates present in radish root extract can interact with bacterial cell membranes, increase membrane permeability, disrupt nutrient uptake and many cellular processes, leading to antibacterial effects. These findings are in accordance with Ziemlewska et al., (2024)45.
The minimum inhibitory and bactericidal concentration profiles of the crude RRR water extract and the biogenic RRR-AuNPs revealed distinct susceptibility patterns across the tested strains. The crude RRR water extract exhibited a more pronounced delay in neutralizing the Gram-negative bacteria as compared to the Gram-positive one, requiring substantially higher concentrations to achieve complete bactericidal eradication (MBC) than to merely inhibit vegetative growth (MIC). These findings align with previous literature, which often attributes the relative resistance of Gram-negative bacteria to their complex outer membrane structure. This lipid bilayer effectively acts as a selective permeability barrier, limiting the influx and penetration of various phytogenic antimicrobial agents. Conversely, the phytogenic AuNPs successfully bypassed these structural defences, demonstrating potent bacterial inhibition and eradication at low microgram thresholds. This superior antimicrobial potency can be attributed to the high surface area-to-volume ratio of the nanomaterials, which facilitates more effective interactions with the bacterial cell envelope. Furthermore, their nanoscale size range promotes efficient cellular uptake and penetration, while a synergistic effect between the surface-capped phytoconstituents and the gold core amplifies the overall bactericidal toxicity, as conclusively corroborated by the alignment of the MIC and MBC datasets.
Adegbola et al., (2026) successfully utilized onion peel and bulb to synthesize AuNPs that inhibited the growth of Gram-positive bacteria like S. aureus and Streptococcus pyogenes and Gram-negative bacteria like E. coli and pseudomonas aeruginosa46. Likewise, Nayem et al., (2020) documented that the biogenic synthesis of AuNPs and AgNPs using Amorphophallus paeoniifolius had notable antibacterial effects. This bactericidal performance is driven primarily by their nanoscale morphology and high specific surface area, allowing for enhanced cellular penetration and membrane interactions47.
Time-kill kinetic profiles revealed that green-synthesized RRR-AuNPs possessed significantly accelerated and superior bactericidal potency compared to the crude RRR water extract. Against S. aureus, the nanoparticles achieved complete eradication within 6 h, reducing viable counts to zero, whereas the RRR extract required a full 24 h for clearance. For Gram-negative E. coli, RRR-AuNPs systematically eliminated the population after 24 h. Conversely, the crude RRR water extract exhibited a more protracted and limited antimicrobial effect; while it successfully suppressed the population compared to the untreated control, a flexible bacterial fraction survived, leaving a residual load of 2.99 log10 CFU/mL.
The minimum inhibitory/bactericidal concentrations (MIC/MBC) time-kill profiles conclusively demonstrate the superior antibacterial efficacy of RRR-AuNPs compared to the crude red radish root extract. Driven by their nanoscale dimensions, high surface reactivity, and phytochemical capping layers, these biogenic nanoparticles readily attach to and penetrate the bacterial cell wall, inducing lethal damage to intracellular DNA and proteins. Ultimately, these findings confirm that sustainably synthesized RRR-AuNPs exert enhanced, broad-spectrum bactericidal activity against both Gram-positive and Gram-negative pathogens, representing promising candidates for advanced antimicrobial therapeutics. These results correspond with those of Adegbola et al. (2026), highlighting the potent and rapid antimicrobial action of gold nanoparticles synthesized via eco-friendly onion bulb and peel extracts46.
The MTT test is an in vitro model applied to estimate the cytotoxic impact of substances against various cancer cell lines. The current study suggested the use of RRR-AuNPs in hepatic and colorectal cancer. The treatment of Caco-2 and HepG2 cell lines with the AuNPs capped with R. sativus root extract suppressed the cell viability of cancerous cell highly significantly at 250 µg/mL concentration. These results are harmonized with Abel et al., (2016) and Zhao et al., (2021)12,48.
Moreover, the RRR water extract showed anticancer activity in Caco-2 and HepG2 cell lines and that may be attributed to its high content of glucosinolates, phenolics, flavonoids, isothiocyanates, sulforaphane and others which exhibit anticancer properties. These compounds can trigger apoptosis via mitochondrial pathways, modulate detoxifying enzymes (Phase I/II), arrest the cell cycle (particularly at G2/M phase), and inhibit proliferation and metastasis in various cancer types. Additionally, activation of cellular protective pathways such as Nrf2/AhR contributes to enhanced detoxification and antioxidant response, thereby suppressing tumorigenesis49.
The biocompatibility of the greenly synthesized RRR-AuNPs was critically evaluated against the HEK-293 normal human kidney cell line. In the current study, Although the RRR-AuNPs exhibited a lower IC₅₀ and higher relative cytotoxicity compared to the crude water extract, they maintained a remarkably wide therapeutic window. This was quantified by an antimicrobial selectivity index (SI) which was calculated as the ratio of the IC₅₀ on normal cells to the MIC against pathogenic bacteria. The RRR-AuNPs yielded an SI of approximately 303, indicating that the nanoparticles are over 300 times more toxic to bacteria than to human kidney cells. This high selectivity is likely attributed to the unique surface chemistry provided by the Raphanus sativus capping agents, which may enhance bacterial membrane interaction while remaining relatively inert toward mammalian cell membranes at therapeutic concentrations. Consequently, the high SI value highlights RRR-AuNPs as a highly biocompatible candidate for clinical applications, where potent antibacterial action is required without compromising host cell viability. This methodology is consistent with Belete et al. (2025) which utilize the SI to establish a therapeutic safety margin29. Our observed SI of 303 significantly exceeds the common safety threshold (SI > 2), reinforcing the potential of RRR-AuNPs as a non-toxic antimicrobial agent. Compared to the synthesized nanoparticles, the raw RRR water extract displayed a diminished SI value of 9.73. Although the extract maintains a favorable margin of safety (SI > 1), its therapeutic window is markedly narrower than that of its synthesized nanoparticle counterpart, highlighting the enhanced bioactivity achieved through green nanotechnology. Tiras et al., (2024)50 have previously demonstrated the antiproliferative activity of red radish methanolic extract on Human Embryonic Kidney cells (HEK293) cells with lower IC₅₀ values (160 mg/mL) indicating stronger cytotoxicity.
In the present work, it is suggested that RRR-AuNPs effectively target and infiltrate into tumour cells with higher bioavailability and loading efficiency, confirming its important role in drug delivery targeting into cells and effective release drug applications. Moreover, the significant disparity between the cytotoxic threshold (IC50) of the RRR-AuNPs and the effective therapeutic antibacterial and antioxidant concentrations demonstrates a favorable biocompatibility profile. Consequently, RRR-AuNPs represent a versatile and safe platform for future clinical and biomedical advancements.
The present study possesses several notable strengths, primarily the successful development of a completely green and rapid synthesis of gold nanoparticles using Raphanus sativus root extract, which acts as both a reducing and stabilizing agent without the need for toxic chemicals. A significant strength of this work is the establishment of an exceptionally wide therapeutic window, as evidenced by a Selectivity Index (SI) of 303.04. This demonstrates that the nanoparticles are over 300 times more toxic to bacterial pathogens than to HEK-293 normal cells. Furthermore, the RRR-AuNPs displayed potent, dose-dependent antioxidant and antibacterial activities, particularly against S. aureus and E. coli. However, some limitations must be acknowledged. Although the antimicrobial and cytotoxic profiles were clearly established in vitro, still further in vivo investigations are necessary to evaluate the systemic pharmacokinetics and long-term biocompatibility of these nanoparticles within complex biological systems. Additionally, while XRD and FT-IR provided strong evidence of capping and crystallinity, future studies utilizing XPS could further elucidate the precise chemical states of the surface-bound biomolecules.