Tribulus terrestris (Tt) was used in this study. It is a flowering plant with yellow flowers and is included in numerous traditional medicine formulations. The plant was collected from Pudhur and its surrounding areas in Chennai. It was identified as 387.1753 and authenticated by the Siddha Central Research Institute in Chennai.

The aerial parts of the plant were washed and cleaned with distilled water, then cut into small pieces, shade-dried, and finally powdered. This powder was used in the synthesis of nanoparticles [25].

A beaker containing 100 mL of deionized water and 1g of powdered plant material was heated at 60 °C for 30 minutes. The extract was then stored at ambient temperature, cooled, and filtered through Whatman filter paper. Fe₃O₄-NPs were synthesized from the filtrate. In a conical flask, 50 mL of the extract was mixed with 50 mL of ferric chloride and stirred for 20 minutes using a magnetic stirrer. The reaction mixture was continuously stirred to produce Fe₃O₄-NPs. The appearance of a black color indicated the formation of nanoparticles [26].

Ascorbic acid (vitamin C), Antibiotic-antimycotic solution, Bovine Serum Albumin (BSA), FeCl₃ (ferric chloride), Hydrogen peroxide (H₂O₂), 2,2-Diphenyl-1-picrylhydrazyl (DPPH) were purchased from HiMedia, India. Methyl Thiazole Tetrazolium (MTT) salt, trypsin-EDTA, Dulbecco’s Modified Eagle Medium (DMEM), and 100$\times$Phosphate-buffered saline (PBS) were purchased from Gibco, Thermo Scientific, India [26].

The optical characteristics of the nanoparticles were examined using UV-visible spectroscopy (Shimadzu, Kyoto, Japan). The synthesised Fe₃O₄-NPs were analyzed in this work by identifying their highest absorption peak within a particular wavelength range of 200–700 nm [27]. Fourier-transform infrared spectroscopy (FTIR) (IR Prestige 21, Shimadzu, Kyoto, Japan) was used to analyze the functional groups of the iron oxide nanoparticles with scan rates between 400 and 4000 cm^-1 were used for this analysis [28]. X-ray diffraction (XRD) (PANalytical, Almelo, Netherlands) patterns were utilized to determine the crystal structure of the nanoparticles [29]. Scanning electron microscopy (SEM) (FEI, Quanta 200, Hillsboro, OR, USA) [30] and transmission electron microscopy (TEM) (JEOL Japan, JEM-2100 plus) [31] provided information on the size and morphology of the nanoparticles. The magnetic properties, including magnitude and direction of the magnetic field, were assessed using a vibrating sample magnetometer (VSM) [32].

The protein denaturation assay was conducted using the method outlined by Sultana et al. [35]. The reaction mixture (5 mL) consisted of 0.02 mL of extract, 4.78 mL of phosphate-buffered saline (PBS, pH 6.4), and 0.2 mL of 1% bovine serum albumin. After incubation at 37 °C for 15 minutes, the mixture was heated to 70 °C for 5 minutes and then cooled. The intensity of protein denaturation was measured at 660 nm using a UV-visible spectrometer. The control solution was phosphate buffer. The percentage of inhibition was calculated as follows:

Percentage of Inhibition = [(Ab – As) / Ab] $\times$ 100

where Ab = absorbance of the control sample and As = absorbance of the test samples.

The antibacterial activity of Tribulus-Fe₃O₄-NPs was evaluated using the agar well diffusion technique. Bacterial cultures of Lactobacillus, Vibrio cholerae, Staphylococcus aureus, and Pseudomonas aeruginosa were maintained in nutrient broth. A 24-hour bacterial culture was used to swab Muller Hinton agar plates. Wells (6 mm in diameter) were punched into the agar plates, and 25 µL, 50 µL, and 100 µL of the green-synthesized Fe₃O₄-NPs were added to each well, along with 30 µL of a control antibiotic. The plates were incubated at 37 °C for 24 hours, and the zone of inhibition around the wells was measured [36].

In a 6-well plate, 7.5 $\times$ 10⁴ colon cancer cells were seeded per well and allowed to grow. A scratch was made using micropipette tips (10 µL). Tribulus-Fe₃O₄-NPs were applied to the scratched cells at concentrations of 10, 25, and 50 µg/mL. An untreated well served as a control. The cells were rinsed with 1.5 mL of PBS and incubated in a CO₂ incubator [39].

Molecular docking predicts ligand-protein interactions and is widely used in structural biology and drug development. Protein-ligand interactions are crucial for structure-based drug design and protein function prediction, as noted by Naqvi et al. [40]. PyrX virtual screening tool version 0.8 (https://sourceforge.net/projects/pyrx/) was used for virtual docking, and Discovery Studio was used for interaction analysis.

Statistical analysis was performed using Microsoft Excel 2019 (Redmond, Washington, United States) to calculate standard deviation (SD) and compare test and control sample results. Results were considered statistically significant when the p value was less than 0.05 [42].

The synthesis of iron nanoparticles was indicated by an immediate change in solution color and a decrease in pH after 1 hour of incubation when plant extract was added to the aqueous FeCl₃ solution. The reaction between ferric chloride and Tribulus terrestris aerial extract resulted in a color change from pale orange to dark brown (Fig. 1A,B). This color alter indicates that iron oxide nanoparticles (FeO) are being produced in solution as a result of the reduction of ferric ions (Fe₃+). Ferric chloride solution addition of effects, such as stabilization of the nanoparticles and a reduction in Fe₃+. Fe₃O₄-NPs synthesized from tribulus, were examined for absorption spectrum in the range of 200–800 nm wavelength. Periodically, the biosynthesized Fe₃O₄-NPs were initially, not shown any noticeable peaks. The synthesized T. terrestris-mediated iron nanoparticles (Tt-Fe₃O₄-NPs) showed a clear peak at 290 nm after the incubation of 30 minutes, shown in Fig. 2. Similar results were observed in previous studies, including the synthesis of Fe₃O₄-NPs from Tribulus terrestris leaves [43] and Phyllanthus niruri plant leaf and fruit parts [44].

Fig. 1.

Synthesis of T. terrestris-mediated iron nanoparticles. (A) Before colour change, (B) After colour change.

Fig. 2.

UV-visible spectroscopy analysis of T. terrestris-mediated iron nanoparticles.

FTIR spectroscopy was employed to identify the functional groups involved in the reduction of Fe₃O₄ nanoparticles by Tribulus terrestris and to determine the specific groups responsible for this process. The FTIR spectrum of the T. terrestris extract showed bands at 2970.80 cm⁻¹ and 2885.51 cm⁻¹, corresponding to the C-H stretching of alkanes, indicating the presence of alkane groups. Fig. 3 depicts additional bands at 2121.70 cm⁻¹ (N=N=N stretching, indicating azide groups), 1639.49 cm⁻¹ (C=C stretching of alkenes), 1384.89 cm⁻¹ (C-H bending of alkanes), 1321.24 cm⁻¹ (N-O stretching of nitro compounds), and 1038.99 cm⁻¹ (C-O stretching of primary alcohols). The broad and intense peak at 2970.38 cm⁻¹ suggests the presence of O-H stretching bonds from both alcohol and phenolic groups in the synthesized T. terrestris-Fe₃O₄-NPs.

Fig. 3.

FTIR spectra analysis of T. terrestris-mediated iron nanoparticles. FTIR, Fourier-transform infrared spectroscopy.

Phenolic compounds in plant extracts are often attributed as the primary agents responsible for the reduction of Fe³⁺ to Fe⁰ [45]. Rajendran et al. [46] conducted FTIR analysis on iron oxide nanoparticles synthesized from Sesbania grandiflora (Agati) and observed a significant peak at 1650 cm⁻¹, corresponding to N-H bending, with a visible bandwidth spanning 1500–600 cm⁻¹. Similarly, Pallela et al. [47] identified functional groups in Sida cordifolia through FTIR spectroscopy, noting strong peaks around 2928 cm⁻¹ and 2429 cm⁻¹, which correspond to methyl and amine groups, respectively.

X-ray powder diffraction (XRD) was utilized to evaluate the crystalline structure and phase of the nanoparticles. The XRD pattern of iron oxide nanoparticles synthesized from Tribulus terrestris (T. terrestris)-Fe₃O₄-NPs is shown in Fig. 4. The diffraction peaks observed at 15.96°, 20.67°, 32.31°, and 31.01° correspond to Bragg’s reflections for the (220), (311), (400), (440), and (533), respectively. These peaks are consistent with the Fe₃O₄ nanoparticle data in the JCPDS database. Dhuper et al. [48] performed XRD analysis on Mangifera indica-mediated Fe₃O₄-NPs and found that the X-ray diffraction, using a graphite monochromator, revealed a rhombohedral crystalline structure with a wavelength of 1.54060 Å. Similarly, Patil et al. [49] reported that Triadica procumbens-mediated Fe₃O₄-NPs displayed peaks at 17.24°, 32.5°, and 39.06°, which corresponded to the (311) planes and confirmed the cubic structure of the nanoparticles.

Fig. 4.

XRD analysis of T. terrestris-mediated iron nanoparticles. XRD, X-ray diffraction.

Scanning Electron Microscopy (SEM) provides high-resolution images of nanoparticles, revealing their size, shape, composition, electrical conductivity, topography, and surface features [50]. The SEM analysis of Tribulus terrestris-Fe₃O₄-NPs showed clusters of nanoparticles with an irregular spherical shape (Fig. 5A,B). Transmission Electron Microscopy (TEM) further revealed that the green-synthesized T. terrestris-Fe₃O₄ nanoparticles were predominantly spherical and exhibited accumulation. This morphology is attributed to the hydroxyl groups present in the plant extract. The nanocomposite was composed of iron and oxygen, with a mean diameter of 29 $\pm$ 0.24 nm (Fig. 5C). Fig. 5D shows a histogram of newly synthesized Tt-Fe₃O₄-NPs with a mean diameter. In comparison, Baskar et al. [51] reported that Hypnea valentiae-mediated Fe₃O₄-NPs have a similar value analyzed by TEM analysis and exhibited rectangular shapes as observed by SEM analysis. Similarly, Naseem and Farrukh [52] analyzed nanoparticles from Lawsonia inermis (henna) and found them to clump together, displaying a deformed hexagonal-like morphology. Kheshtzar et al. [53] evaluated nanoparticles produced from Pinus eldarica needles by TEM and observed that they were predominantly spherical with sizes ranging from 8 to 34 nm. Jasminoides leaf extract synthesized Fe-NPs were reported to be globular with a size of 21 nm.

Fig. 5.

SEM and TEM analysis of T. terrestris-mediated iron nanoparticles. (A,B) SEM micrograph images in different magnification. Scale bar: 1 µm. (C) TEM image of T. terrestris-Fe₃O₄-NPs. Scale bar: 200nm. (D) Size distribution histogram.

A prominent method for determining the magnetic properties of materials is vibrating sample magnetometry (VSM). This method involves subjecting samples to a varying electromagnetic field to measure properties such as magnetic retentivity (Mr), coercivity (Hci), and saturation magnetization (Ms). The magnetic properties of Tribulus terrestris-mediated Fe₃O₄-NPs were evaluated using VSM. Fig. 6 illustrates the magnetic behavior of these nanoparticles. The Fe₃O₄-NPs showed a saturation magnetization (Ms) of 14.75 emu/g, a coercivity (Hci) of 285.28 Oe and a magnetic retentivity (Mr) of 1.0244 $\times$ 10⁻⁶ emu/g.

Fig. 6.

Magnetic characterization of T. terrestris-mediated iron nanoparticles by vibrating sample magnetometry(VSM) technique.

Mahdavi et al. [54] studied the magnetic properties of Fe₃O₄-NPs synthesized from Sargassum muticum extract. These nanoparticles exhibited superparamagnetic behavior with a saturation magnetization of 22.1 emu/g, a coercivity of 82.3 Oe, and no remanence, indicating the presence of a single magnetic domain. Similarly, Izadiyan et al. [55] reported high saturation magnetization in iron oxide nanoparticles obtained from Juglans regia. These nanoparticles displayed a high saturation magnetization of 53.32 emu/g and a low coercivity of 32.50 Oe. The magnetization curves confirmed the high saturation magnetization and low coercivity characteristics of the J. regia-derived iron oxide NPs.

Free radicals are molecules that are extremely reactive and possess unpaired electrons. They have the potential to cause cellular injury during the metabolic process. This oxidative stress is mitigated by antioxidants, which are recognized for their ability to neutralize these free radicals. Secondary metabolites in plants often exhibit antioxidant properties, despite being non-nutritive, and can benefit human health when consumed [56].

The DPPH and hydrogen peroxide radical assays were employed to evaluate the antioxidant activity of Tribulus terrestris-mediated Fe₃O₄-NPs. The Fe₃O₄-NPs effectively neutralized free radicals. In the DPPH assay, conducted with doses ranging from 10 to 50 µg/mL, the Fe₃O₄-NPs demonstrated significant radical-scavenging activity, with a peak inhibition of 65.5% at 50 µg/mL. For comparison, standard ascorbic acid (Vitamin-C) showed an inhibition of 70.4% at the same concentration, as illustrated in Fig. 7. Nandini and Subhashini [57] reported a DPPH radical scavenging activity of about 61.4% in Cuminum cyminum, while Deshmukh et al. [58] observed a maximum radical scavenging activity of 60% in fenugreek seed-mediated Fe-NPs.

Fig. 7.

DPPH assay of T. terrestris-mediated iron nanoparticles. DPPH, 2,2-Diphenyl-1 picrylhydrazyl.

In the hydrogen peroxide assay, T. terrestris-Fe₃O₄-NPs exhibited 65.56% inhibition of free radicals at a concentration of 50 µg/mL. Fig. 8 shows that vitamin C suppressed free radicals by 74.23% at the same dose. Essa et al. [59] reported a 42.38% scavenging activity for hydrogen peroxide in Vitis vinifera leaf-mediated Fe-NPs, while Sandhya and Kalaiselvam [60] observed a 44.12% radical scavenging activity in Borassus flabellifer tender seed-synthesized iron nanoparticles. The significant antioxidant activity of Tribulus terrestris is attributed to its numerous phenolic hydroxyl groups [61]. Based on Lushchak [62], antioxidants reduce oxidative damage to biological components and restrict cancer cell development by suppressing free radicals.

Fig. 8.

Hydrogen peroxide assay of T. terrestris-mediated iron nanoparticles.

Tribulus terrestris-synthesized Fe₃O₄-NPs demonstrated the capacity to inhibit the denaturation of bovine serum albumin (BSA). At 50 µg/mL, Fe₃O₄-NPs showed maximum inhibition of 43.28. For comparison, the standard drug diclofenac, at the same concentration of 50 µg/mL, achieved a higher inhibition of 54.57%, as shown in Fig. 9. Khuda et al. [63] investigated the in vitro anti-inflammatory activity of crude extracts from Xanthium strumarium and Achyranthes aspera leaves, reporting a maximum inhibition of 73%. Additionally, Bailey-Shaw et al. [64] studied the anti-inflammatory properties of 99 plant samples. Among these, iron nanoparticles synthesized from Mangifera indica (Julie mango) leaves showed a significant inhibition of 72.60%.

Fig. 9.

Protein denaturation assay of T. terrestris-mediated iron nanoparticles.

The antibacterial efficacy of the synthesized nanoparticles is comparable to that of the original Tribulus terrestris plant extract. This antimicrobial activity arises because the functional groups from the plant extract, which possess inherent herbal qualities, saturate the nanoparticle surfaces. The nanoparticles’ diminutive size enables them to easily pass through the bacterial cell walls, resulting in the demise of the cells [65].

The antibacterial efficacy of the synthesized T. terrestris-Fe₃O₄-NPs was evaluated, as illustrated in Fig. 10. At a concentration of 100 µL, the maximum zones of inhibition were observed against Lactobacillus (35 mm) and Vibrio cholerae (34 mm). Moderate inhibition was noted for Staphylococcus aureus (19 mm) and Pseudomonas aeruginosa (25 mm). The synthesized nanoparticles demonstrated the highest effectiveness against Lactobacillus, as illustrated in Fig. 11.

Fig. 10.

Antibacterial activity of T. terrestris-mediated iron nanoparticles against wound pathogens.

Fig. 11.

Zone of inhibition of T. terrestris-mediated iron nanoparticles.

Kanagasubbulakshmi and Kadirvelu [65] reported similar findings for nanoparticles synthesized from Lagenaria siceraria. Their study showed that these nanoparticles had comparable antibacterial potential to the plant extract, with higher efficacy against Staphylococcus aureus at higher concentrations. At lower concentrations, however, the nanoparticles did not show inhibition, as depicted in Fig. 11. Additionally, Yadav et al. [66] examined the antibacterial efficiency of Aloe vera nanoparticles against Klebsiella pneumoniae and reported a 17-mm maximal inhibitory zone.

The MTT method was used to evaluate the in vitro anticancer effectiveness of T. terrestris-Fe₃O₄-NPs against the HCT-116 colon cancer cell line. The results indicated that higher concentrations of T. terrestris-Fe₃O₄-NPs led to a greater percentage of cell inhibition. The half-inhibitory concentration (IC₅₀) for HCT-116 cells was 25.95 µg/mL, as shown in Fig. 12. In contrast, for the MG-63 (osteosarcoma) cell lines, the IC₅₀ value was 35.36 µg/mL, depicted in Fig. 13. Microscopic images of both cell lines reveal that T. terrestris-Fe₃O₄-NPs are more effective against colon cancer than bone cancer. According to Alkahtane et al. [67] studied the cytotoxicity of the iron oxide nanoparticles on HCT 116 cell lines and it limit proliferation at 55 mg/mL and less toxic for fibroblast cells.

Fig. 12.

Cell Viability (A) Control cells (B) Treated Cells (C) Cytotoxic activity of T. terrestris-mediated iron nanoparticles in HCT-116 cell line. Scale bar: 1 µm.

Fig. 13.

Cell Viability (A) Control cells (B) Treated Cells (C) Cytotoxic activity of T. terrestris-mediated iron nanoparticles in MG-63 cell line. Scale bar: 1 µm.

Yusefi et al. [68] investigated the cytotoxic effects of Fe₃O₄-NPs maintained with Garcinia mangostana fruit peel on HCT-116 and normal CCD112 cell lines. At a dosage of 250 µg/mL, a 68% suppression of colon cancer cells was observed. Chauhan et al. [69] noted that malignant cells, lacking a protective mucus layer, are more susceptible to therapeutic agents like those derived from T. terrestris. Based on these cytotoxicity results, further anticancer screening of T. terrestris-Fe₃O₄-NPs at concentrations of 50, 25, and 10 µg/mL was deemed necessary. According to Ratan et al. [70], plant extracts include biological components that serve as capping and reducing agents in nanoparticles, effectively inhibiting cancer cell development. The dimensions, and morphology of iron nanoparticles are also contributing factors in inducing apoptosis in cancer cells by stimulating the increase of reactive oxygen species (ROS). As per Ansari and Asiri [71], the anticancer mechanism of iron oxide nanoparticles is caused by releasing Fe ions to eliminate cancer cells. It is conceivable that Fe₃O₄-NPs are responsible for the development of reactive oxygen species, which eradicate malignant cells.

The impact of green synthesized T. terrestris-Fe₃O₄-NPs on cancer cell migration was evaluated through a wound healing assay using HCT-116 colon cancer cells. This study showed that T. terrestris-Fe₃O₄-NPs at 10, 25, and 50 µg/mL substantially reduced wound closure compared to normal control cells. Notably, at the highest concentration of 50 µg/mL, the migration of HCT-116 cells was reduced by 90%, as illustrated in Fig. 14. This result indicates that the nanoparticles effectively hindered cancer cell migration, in contrast to the untreated control cells, which showed continued migration and spread.

Fig. 14.

Wound healing assay of T. terrestris-mediated iron nanoparticles. Scale bar: 1 µm.

The results underscore the potential of plant-mediated iron nanoparticles in reducing tumor cell migration. Such nanoparticles may enhance wound healing processes by promoting repair mechanisms and collagen synthesis, thereby improving tensile strength [72]. Adnan et al. [73] conducted a similar wound closure assay with HCT-116, and A549 cells were treated with an ethanolic extract of Selaginella repanda, observing dose-dependent inhibition of cell migration.

The command-line software utilized in this study divided the docking results into an output registry in PDB format and a log file in text format. Given the frequent abnormalities in the Adenomatous Polyposis Coli (APC) pathway in colon cancer, which underscores its significance in colon cancer development [74], the docking analysis was centered on proteins associated with the APC pathway. Binding affinity values derived from molecular docking investigations are outlined in Table 1. The results indicated that coumaric acid effectively targets several proteins involved in colon cancer, including KRT8, TFRC, KLK6, C3AR1, AHSG, GRP, APOC1, and CAV1, with 5-Fluorouracil used as a standard drug for comparison. Figs. 15,16 highlight that coumaric acid demonstrated the highest binding affinities with GRP (6.8), TFRC (6.2), KLK6 (6), and C3AR1 (6) proteins. These results suggest that coumaric acid exhibits substantial binding affinity towards key proteins associated with colon cancer, as evidenced by these bioinformatics analyses. Zhang et al. [75] also investigated the binding affinity of ginger compounds with colon cancer proteins using in silico methods. They found that the targeted proteins TP53, HSP90AA1, and JAK2 exhibited stronger interactions with ginger compounds compared to prototype ligands, supporting the efficacy of targeted therapeutic approaches.

Fig. 15.

Molecular interaction between T. terrestris-mediated iron nanoparticles derived compounds with Adenomatous polyposis coli (APC) pathway proteins (A) TRFC (B) KLK6 (C) C3AR1 and (D) GRP.

Fig. 16.

Molecular interaction between T. terrestris-derived compounds with Adenomatous polyposis coli (APC) proteins (A) AHSG, (B) CAV1, (C) APOC1, (D) KRT8, (E) CAV1.