HCT 116 human colorectal carcinoma cells and Vero (African green monkey kidney epithelial) cells were obtained from the National Cancer Institute in Cairo, Egypt. Cis (with the commercial name CISPLATINE MYLAN; Code DCI: 05G111), chitosan, and folic acid (FA) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Tissue culture reagents [DMEM 4.5 g/L Glucose with L-Glutamine, fetal bovine serum (FBS), penicillin/streptomycin, trypsin, dimethyl sulfoxide (DMSO), MTT, Trypsin-EDTA 10x solution] were supplied by Sigma and GIBCO (New York, United States), and Lonza Bioproducts (Verviers, Belgium). Gene JET RNA Purification Kit was obtained from Thermo Scientific (Waltham, MA, United States), while the Quantiscript Reverse Transcription Kit and Quanti-Tect SYBR Green PCR Kit were purchased from Qiagen (Hilden, Germany). Soybean phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL, United States). Primers were synthesized by Integrated DNA Technologies (Coralville, IA, United States). All other chemicals and reagents were of the highest purity available.

Ant was extracted by homogenizing an equal ratio (1/1 w/v) of sample (200 g peeled beetroot fruit) and solvent (200 mL 95% ethanol) for 2 h at 65 ˚C. After the color had changed to dark red, the extract was filtered and evaporated to dryness from ethanol in an oven at 40 °C for 48 h, washed, and dissolved in distilled water, then kept in a dark bottle at 4 °C until used for liposome fabrication (Awad et al., 2024b).

Under acidic conditions, Ant and betacyanin exhibited distinct behaviors that help differentiate them. When treated with hot hydrochloric acid (HCl), Ant remained stable and retained their original color, whereas betacyanin degraded and lost their pigmentation. In contrast, under alkaline conditions (with sodium hydroxide, NaOH), Ant shifted to a blue-green hue that might fade over time, while betacyanin transformed into a stable yellow color. These contrasting responses to pH changes were a useful diagnostic tool for identifying these pigments in plant extracts (Nielson and Harley, 2010).

The total Ant content of beetroot extract was determined using the pH differential method (Jumaa Jandal, 2025). This method relies on the property of Ant to alter their color in response to changes in the pH of the solution. At an acidic pH of approximately 1, the substances appeared red; conversely, at neutral or slightly basic pH levels around 4.5, they exhibited purple or blue. To determine the total Ant content, we measured the absorbance of the extract at two wavelengths (520 nm and 700 nm) and two pH levels (1 and 4.5). Total Ant content (mg/L) was calculated as using this formula: A 520 − A 700 pH 1 − A 520 − A 700 pH 4   × MW × DF × 1000 ϵ × L , where A520 and A700 represent the absorbance values at 520 nm and 700 nm; MW denotes the molecular weight of cyanidin-3-glucoside (which is 449.2 g/mol and serves as a reference compound); DF indicates the dilution factor (which was calculated as 25 divided by 0.5, resulting in a value of 50); ϵ refers to the molar absorptivity of cyanidin-3-glucoside (quantified at 26,900 L/mol/cm); and L signifies the path length of the cuvette, typically 1 cm (Fierri et al., 2023).

The liposomal formulation of Ant, Cis, and their combination (Cis + Ant) was synthesized via a modified thin-film hydration method as previously detailed (Abd El- Salam et al., 2023; Bahrami Parsa et al., 2023). The lipid phase was created in each formulation by dissolving 20 mg of soybean phospholipid in 0.5 mL of chloroform and 3 mL of ethanol while magnetically stirring at 70 °C (600 rpm for 5 min). Subsequently, 20 mg of cholesterol, similarly dissolved in the same solvent combination, was integrated. Hydration was accomplished using 40 mL of distilled water, and surface modification included the addition of 5 mg of folic acid mixed with 20 mL of albumin (50 mg/100 mL). The suspensions were sonicated for 15 min and then dialyzed against distilled water for 24 h before lyophilization and storage at −20 °C. For Ant Ls, 100 mg of Ant was first sonicated in 2 mL (25 mg/100 mL) chitosan before adding the lipid phase. Cis Ls were generated by sonicating 2 mg of Cis in 200 µL of polygalacturonic acid (50 mg/mL) and 1 mL of chitosan (25 mg/100 mL) before their integration into the lipid mixture. The Cis + Ant liposomes were generated by integrating both drug solutions (Ant in chitosan and Cis in polygalacturonic acid/chitosan) into the lipid phase before hydration. This standardized and versatile method facilitated the effective encapsulation of one or two pharmaceuticals into a singular liposomal delivery system.

The prepared liposomes were characterized using transmission electron microscopy (TEM) (Inkson, 2016) and Fourier transform infrared (FTIR) spectroscopy (Mahamuni and Adewuyi, 2009). For TEM analysis, 1 mL of each diluted sample was sonicated for 5 min to ensure homogeneity. The sample was then applied to carbon-coated copper grids, air-dried for 15 min, and negatively stained with phosphotungstic acid. Imaging was performed using a 200 kV JEOL JEM 2100F TEM. For FTIR analysis, surface molecular structures were assessed in the 500–4,000 cm⁻¹ range via the KBr pellet method using a JASCO FTIR spectrometer (Model AUP1200, Japan). Each sample was scanned at least three times across different regions, and representative spectra were analyzed for functional group identification.

The following equation was used: EE (%) = Total   amount   of   Ant   mg − Amount   of   Ant   in   the   supernatant Total   amount   of   Ant   × 100 (Amararathna et al., 2022) to determine the concentration of Ant within the capsule. The total Ant in the extract and supernatant was obtained following the centrifugation of the Ant Ls. The total amount of Ant and the quantity in the supernatant after encapsulation were assessed using the pH differential technique.

The in vitro release of entrapped Ant and Cis from liposomes was checked utilizing the dialysis bag diffusion method (Gupta and Trivedi, 2018). The dialysis tubing was soaked in distilled water overnight to facilitate the complete expansion of the dialysis membrane prior to cutting it into suitable segments. Liposomes containing Ant, Cis, or Cis + Ant (5 mL) were each placed into a sealed membrane dialysis bag. A dialysis bag was immersed in 200 mL of phosphate-buffered saline solution at pH 7.4. Ethanol was incorporated into the medium to ensure the establishment of sink conditions. The system was maintained at 37 ± 0.5 °C with continuous magnetic stirring at 400 rpm. Glass beakers were sealed with Parafilm to prevent the volatilization of alcohol (Yan et al., 2010). To ensure a constant volume of the receptor medium, 3 mL aliquots were removed at designated intervals and replaced with an equivalent volume of fresh media. The collected samples underwent analysis via UV spectroscopy.

Cell viability was assessed with the MTT assay, which evaluates mitochondrial reductase activity in viable cells by converting tetrazolium salts into formazan crystals. Cells were inoculated at a density of 10,000 cells per well in full growth media (DMEM supplemented with 10% FBS and 1% antibiotics) and allowed to adhere for 24 h. Upon reaching 70%–80% confluence, cells were subjected to successive dilutions (0–100 μg/mL) of Cis Ls and/or Ant Ls for a duration of 24 h. After treatment, MTT reagent (5 mg/mL) was introduced, and cells were incubated for 4 h to facilitate formazan crystal formation. The crystals were dissolved in DMSO, and absorbance was recorded at 570 nm using a microplate reader. The half-maximal inhibitory concentration (IC₅₀) was determined using dose-response curves produced with GraphPad Prism software. Cell viability percentages were standardized to untreated controls (deemed 100% viable) using the formula: OD   treated − OD   blank OD   control − OD   blank   × 100 .

All procedures involving animals followed ARRIVE guidelines and were ethically approved by the Animal Ethics Committee at the Faculty of Women for Arts, Science, and Education, Ain Shams University (Approval Code: ASU/W/Sci-5R/23-1-16). Female Swiss albino mice (10–12-week-old and weighing 20–25 g) were housed under a regulated temperature in a 12-12-h light-dark cycle. Before the trial began, each mouse spent 2 weeks acclimating. The animals ate the same food and had full water access. Thirty-five mice were divided into five groups (n = 7 for each group): normal control (Cnt), EAC, Cis Ls, Ant Ls, and Cis + Ant Ls. The Cnt group was given an intraperitoneal (i.p) injection of normal saline (0.9% w/v, 200 μL/mouse). The EAC group was i. p injected with 200 µL containing 1 × 10⁶ EAC cells and received no therapeutic intervention. EAC cells were sourced from three EAC-bearing mice obtained from the National Institute of Oncology at Cairo University, Egypt, after 14 days of intraperitoneal injection. These mice in our lab underwent repeated aseptic intraperitoneal transplanting using the ascitic fluid containing EAC cells. In the Cis Ls group, EAC was induced as in the previous group, but animals were i. p injected with 200 μL (50 mg/kg) Cis Ls for seven consecutive days starting from Day (D) 8 to D14 post-EAC injection (Franco et al., 2021). The Ant Ls group was manipulated as the Cis Ls group, but mice were i. p injected with 200 μL (100 mg/kg) Ant Ls (Carvalho et al., 2017). The Cis + Ant Ls group received an i. p injection of 200 µL containing 1 × 10⁶ EAC cells at D1 and was i. p injected with 50 mg/kg Ant Ls and 50 mg/kg Cis Ls over seven consecutive days from D8 to D14. After the experimental period (2 weeks), mice were euthanized 24 h following the final treatment, and blood samples were subjected to centrifugation in sterile glass tubes for 15 min at 3,000×g to obtain clear, non-hemolyzed sera. Eppendorf tubes labeled accordingly were promptly shipped to −20 °C, and the sera were frozen for subsequent biochemical analysis. Peritoneal fluids containing EAC cells were collected using a sterile syringe for cell counting, viability assessment, RNA extraction, and cellular pathology analysis. Livers and kidneys were promptly excised, and specimens were fixed in 10% formalin for pathological examination.

After euthanasia, the peritoneal cavity was carefully opened, and the ascitic fluid was collected using a sterile syringe. The volume of ascitic fluid was measured directly using a graduated 5 mL syringe and recorded in milliliters for each mouse. EAC cells were counted using a hemocytometer following staining with 0.5% trypan blue. A trypan blue exclusion assay was performed as previously described (Strober, 2015). The viability percentage was calculated as follows: total   number   of   cells − number   of   trypan   blue   dead   cells total   number   of   cells × 100 . (El-Magd et al., 2017).

Real-time PCR was used to assess alterations in the relative expression of the caspase3, Bcl2, IL1β, VEGF, MMP9, Nrf2, and HO-1 genes in EAC cells across all groups. Total RNA was first extracted and then reverse-transcribed into cDNA using kits obtained from Thermo Scientific, United States (#K0731 and #EP0451, respectively). Table 1 lists the primer sequences. The 2^−ΔΔCT approach was used to evaluate the change in gene expression.

Assess hepatic function in female mice by measuring aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Creatinine in serum was measured using an alkaline picrate colorimetric method. The urea levels were estimated calorimetrically by measuring the amount of urea nitrogen in the serum using a commercially available kit (Diamond Diagnostics).

Following ascitic fluid collection, the samples were fixed with ethanol. Cells were dried on a microscope slide before staining with hematoxylin and eosin (H&E). Liver and kidney tissues undergo dehydration using a sequential series of ethanol alcohol concentrations. The specimen was subsequently cleared in xylene, embedded in paraffin wax, sectioned to a thickness of 5 μm, and stained by H&E. All specimens were imaged using a light microscope.

The statistical analysis was conducted using one-way ANOVA with GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, United States), followed by Tukey’s Honestly Significant Difference (Tukey’s HSD) test as a post hoc test. Data were presented as mean ± standard error of the mean (SEM). The significance was defined as P < 0.05.

The colorimetric responses seen under different pH settings validate the existence of Ant in the extract. Following the addition of HCL and a 5-min heating period (Figures 1A–C), the pigments maintained their distinctive red coloration, indicating durability in the presence of hot HCL—a defining property of Ant. Conversely, under alkaline circumstances (Figures 1D–F), the pigments transitioned to a blue-green hue, aligning with the established behavior of Ant in sodium hydroxide, whereby their chromophores experience structural alterations resulting in bathochromic shifts. The lack of pigment degradation in acid or a yellow transition in alkali excludes the existence of betacyanin, which would destabilize acidic environments and maintain a stable yellow hue in basic circumstances. Consequently, the results designate the pigments as Ant.

The chemical structure of Cis was found to be intact inside Ls, according to the FTIR data (Figure 2A). The Cis and Cis Ls exhibited platinum ammonium bonds at 565 cm⁻¹ and 466 cm⁻¹, respectively. Tensional tremors of ammonium groups could be noticed at 3,300 cm⁻¹ (Cis) and 3,400 cm⁻¹ (Cis Ls) (Awad et al., 2024b; Babaei et al., 2019). The Ant and Ant Ls FT-IR spectra were recorded at a resolution of 0.1 cm⁻¹ in the 4,000 cm⁻¹ to 400 cm⁻¹ range (Figure 2B). Ant Ls at 3,277 cm⁻¹ also showed -OH stretching vibrations, which were thought to be responsible for the noticeable broadband at 3,266 cm⁻¹ in the FT-IR spectra of Ant. Ant and Ant Ls showed the C=N stretching band at 1,645 cm⁻¹ and 1,558 cm⁻¹. In contrast to the strong presence in Ant Ls at 1,048 cm⁻¹, the last faint band in Ant at 1,013 cm⁻¹ was thought to be caused by C-H bending (Jeyaram and Geethakrishnan, 2020). It was at 3,302 cm⁻¹ when the stretching vibrations of -OH were detected in the Cis + Ant Ls spectra. Ant and Cis were suggested to be present by the band at 1,405 cm⁻¹. In contrast, Cis was indicated by the band at 1,548 cm⁻¹ (Figure 2B). TEM analysis revealed that Ls were spherical and exhibited a consistent, uniform size distribution (Figure 2C). The diameters of the Ls ranged from 70 to 130 nm, confirming their structural homogeneity and suitability for experimental applications.

The total Ant concentration in the beetroot extract was measured using the pH differential method. This recognized spectrophotometric approach depends on the structural alteration of anthocyanins at varying pH levels. Absorbance studies were conducted at 520 nm and 700 nm in two distinct buffer systems: pH 1.0 (potassium chloride buffer) and pH 4.5 (sodium acetate buffer). The adjusted absorbance values were computed as follows: at pH 1.0, (A520−A700) = 1.226−(−1.045) = 2.271 and at pH 4.5, (A520−A700) = 1.093–0.032 = 1.061 (Figure 3A). Total Ant content = 2.271 − 1.061 × 449.2 × 50 × 1000 26 , 900 × 1 = 1.210 × 834.94 = 1,010.23 mg/L. The beetroot extract contains 1,010.23 mg/L of total Ant, indicating a substantial pigment content that aligns with its deep red hue.

The EE of beetroot Ant was assessed by comparing the crude extract with the post-encapsulation supernatant using the pH differential technique. Preliminary spectrophotometric assessments at pH 1.0 and 4.5 produced corrected absorbance values of 0.086 and 0.002, respectively (Figure 3B), validating accurate anthocyanin characterization with enhanced stability in acidic environments. The examination of the supernatant indicated an unencapsulated Ant concentration of 70.13 mg/L, determined using cyanidin-3-glucoside as a reference (MW = 449.2 g/mol, ε = 26,900 L/mol cm) with a dilution factor of 50. The EE was then determined by comparing the encapsulated Ant content to the total starting content. EE (%) = (Encapsulated Ant/Total Ant) × 100. In contrast to the crude extract’s significant Ant concentration of 1,010.23 mg/L, the lower supernatant value of 940.10 mg/L (93.06%) signifies efficient encapsulation, demonstrating excellent incorporation of Ant into the matrix. These data indicate high EE, implying strong safeguarding of the bioactive chemicals.

The time-dependent release profiles of Cis, Ant, or Cis + Ant from different formulations demonstrated significant variations among treatment groups. The Cis Ls group exhibited a gradual release pattern, reaching 12.17% ± 1.35% at 1 h, 13.75% ± 2.12% at 2 h, and progressively increasing to 34.16% ± 1.74% by 48 h. In contrast, the Ant Ls group showed markedly enhanced release kinetics, with percentages escalating from 8.97% ± 1.65% at 1 h to 59.11% ± 1.87% at 48 h, representing the most substantial cumulative release among all groups. The combination group (Cis + Ant Ls) displayed intermediate release characteristics, maintaining lower but sustained release values throughout the study period (3.82% ± 1.21% at 1 h to 13.68% ± 1.30% at 48 h) (Figures 3C–F). Comparative analysis revealed that all liposomes achieved substantially high release rates. The current data further extends these observations, with the Ant Ls group showing superior release performance, reaching nearly 60% cumulative release by 48 h. Anthocyanin-loaded liposomes showed a mean size of 90 nm and a strong negative zeta potential (−35.35 ± 1.75 mV) as shown in Figure 4.

The MTT assay results demonstrated that Ant Ls exhibited a cytotoxic effect on HCT 116 cells, with IC₅₀ of 67.35 ± 1.86 μg/mL, while Cis Ls showed slightly higher cytotoxicity, with IC₅₀ of 32.65 ± 1.20 μg/mL. The cotreatment (Cis + Ant Ls) further reduced cell viability, yielding the lowest IC₅₀ 27.71 ± 1.01 μg/mL in HCT 116 cells (Figure 5). Conversely, the Vero cells showed no significant (P > 0.05) cytotoxic effects when treated with Cis Ls, Ant Ls, or Cis + Ant Ls resulting in IC₅₀ values of 306.20 ± 2.39, 580.30 ± 4.01, and 423.10 ± 3.25 μg/mL, respectively (Figure 5).

Compared to the untreated EAC group, all three treatment groups (Cis Ls, Ant Ls, and Cis + Ant Ls) showed a notable (P < 0.05) reduction in total tumor cell counts, viable tumor cells, and ascitic fluid volume, along with a significant (P < 0.05) increase in non-viable cells (Figure 6). Among the treated groups, the Cis + Ant Ls combination stood out as the most effective, with the lowest total and viable tumor cell counts and the smallest ascitic fluid volume. The Ant Ls group also showed strong suppression of tumor growth, though slightly less pronounced than the combination treatment, while the Cis Ls group had a more modest effect.

The untreated EAC cells were densely packed and round with intact membranes and homogenous cytoplasm (arrows, Figure 7A), indicating high cell viability and proliferation. EAC treated with Cis Ls displayed notable morphological changes: nuclear shrinkage, vacuoles formed, and membrane irregularities, indicative of apoptosis (arrows, Figure 7B). EAC treated with Ant Ls also showed disintegrated membranes, apoptotic bodies formed, cytoplasmic vacuolization, and other signs of necrosis and apoptosis (arrows, Figure 7C). EAC treated with Cis and Ant Ls demonstrated the most extensive cellular damage, including shrinkage, fragmented cells, and complete loss of structural integrity, reflecting enhanced synergistic cytotoxicity (arrows, Figure 7D).

The EAC-bearing mice exhibited significant hepatic and renal damage, as evidenced by markedly elevated serum ALT, AST, urea, and creatinine levels compared to healthy controls. All treatment regimens significantly (P < 0.05) mitigated hepatic and renal dysfunction. Ant Ls and the combination therapy (Cis + Ant Ls) demonstrated the most potent hepatoprotective effects, reducing ALT, AST, urea, and creatinine to near-normal levels. However, Cis Ls monotherapy provided a more modest improvement (Table 2).

The results indicated that Cis + Ant Ls is the most effective treatment in increasing apoptotic markers Caspase3 (10.41 ± 0.25-fold change) while reducing the anti-apoptotic marker Bcl2 (0.11 ± 0.02-fold change) in EAC cells compared with Cis Ls and Ant Ls (Caspase3, 4.41 ± 0.17 and 3.89 ± 0.14-fold change, and Bcl2, 0.29 ± 0.01 and 0.76 ± 0.03-fold change, respectively) (Figure 8). Moreover, the EAC group exhibited minimal Nrf2 expression (0.70 ± 0.06-fold change) in the EAC cells. Ant Ls substantially enhanced Nrf2 expression (6.36 ± 0.26-fold change). Like the control, Cis Ls exhibited low Nrf2 expression (0.87 ± 0.11-fold change), while Cis + Ant Ls demonstrated the most significant (P < 0.05) Nrf2 expression (7.67 ± 0.15-fold change). Cis Ls reduced HO-1 expression (0.35 ± 0.06-fold change); Ant Ls substantially enhanced HO-1 expression (4.69 ± 0.12-fold change); Cis + Ant Ls significantly (P < 0.05) maximized this expression (6.73 ± 0.14-fold change) (Figure 8).

The EAC group showed the highest VEGF expression (1.00 ± 0.00-fold change). Ant Ls modestly reduced VEGF expression (0.81 ± 0.06-fold change). The Cis Ls group showed a more substantial decrease in VEGF levels (0.34 ± 0.02-fold change) than the EAC group. Additionally, Cis + Ant Ls showed the lowest VEGF expression (0.17 ± 0.04-fold change), indicating a combined, strong suppression of angiogenesis, effectively limiting the tumor’s capacity for growth and spread (Figure 9).

Moreover, the EAC group exhibited the greatest IL1β expression level (1.00 ± 0.00-fold change). Ant Ls reduced IL1β expression (0.49 ± 0.04-fold change) compared to the EAC. Cis Ls diminished IL1β levels (0.88 ± 0.08-fold change). The combination of Cis and Ant Ls resulted in the minimal production of IL1β (0.27 ± 0.05-fold change), suggesting a possible anti-inflammatory action that may hinder tumor development and spread.

The EAC group demonstrated the most significant (P < 0.05) levels of MMP9 expression (1.00 ± 0.00-fold change). Ant Ls exhibited a modest reduction in MMP9 expression (0.62 ± 0.07-fold change). Cis Ls further reduced MMP9 levels (0.44 ± 0.04-fold change). Cis and Ant Ls demonstrated the lowest expression of MMP9 (0.27 ± 0.02-fold change), indicating a significant (P < 0.05) synergistic impact in inhibiting tumor cell migration and metastasis (Figure 9).

In the Cnt group, the liver has a healthy structure with polyhedral-shaped hepatocytes arranged in a cord-like pattern (H) radiating from the intact central vein (CV) and separated by blood sinusoids (S) and an intact portal artery (P) (Figures 10A,B). In the EAC group, notable pathological alterations are evident, including aggregations of polymorphic neoplastic cells (asterisks) around the congested central vein (arrowheads), swelling and vacuolar degeneration of hepatocytes (black arrows), and an area of hepatocellular necrosis (yellow arrows) (Figures 10C,D). Cis Ls treatment decreased neoplastic cell clusters (asterisks) around the congested hepatic blood vessels (arrowheads). However, isolated polymorphic neoplastic cells remained (red arrows), and some hepatocytes exhibited swelling and vacuolar degeneration (black arrows) (Figures 11A,B). Ant Ls therapy has a therapeutic benefit by diminishing neoplastic cell clusters (asterisks) around the mildly congested hepatic blood vessels (arrowheads). Still, some hepatocyte degradation persists (black arrows) in addition to the number of sporadic polymorphic neoplastic cells (red arrow) (Figures 11C,D). Cis + Ant Ls has the most pronounced ameliorative benefits, shown by diminished clusters of degenerative neoplastic cells (red arrows), decreased congestion of hepatic blood vessels (arrowheads), and reduced hepatocellular degeneration symptoms (black arrows) (Figures 11E,F). The findings indicate that Cis and Ant, alone and in conjunction, may provide therapeutic advantages in alleviating liver damage caused by migratory EAC cells, with their combination yielding superior protective effects.

In the Cnt group, the kidney had a robust architecture, featuring intact glomeruli (G) encased by Bowman’s capsules and well-structured proximal and distal convoluted tubules (T) (Figure 12A). In the (EAC) group, pathological alterations are observed, including clusters of polymorphic neoplastic cells (asterisk), degeneration of tubular epithelium (arrowheads), hyaline casts (yellow arrow), atrophied glomeruli, and enlarged capsular space (black arrow) (Figure 12B). Individual treatment with Cis Ls or Ant Ls demonstrated modest enhancement, characterized by reduced clusters of degenerated neoplastic cells (asterisk) around congested renal blood vessels (yellow arrow), accompanied by minimal glomerular shrinkage (black arrow). However, degeneration of the tubular epithelium continues (arrowheads) (Figures 12C,D). The Cis + Ant Ls group had the most pronounced ameliorative impact, characterized by minimal clusters of degenerated neoplastic cells (asterisk), diminished vascular congestion (yellow arrow), intact tubular epithelium (T), and retained glomeruli (G) (Figure 12E). Our data demonstrate that Cis and Ant, either alone or in combination, may help mitigate EAC-induced kidney injury, with the combined treatment offering the most robust protection.