Phytochemical, in silico, and in vitro studies of wheatgrass (Triticum aestivum L.) juice powder.

Introduction
Cancer, one of modern medicine’s most complex and challenging diseases, significantly affects global health systems. According to the World Health Organization (WHO) GLOBOCAN 2020 report, 19.3 million new cancer cases were detected worldwide in that year, resulting in approximately 10 million deaths1,2. These statistics clearly reveal the devastating effect of cancer on human health on a global scale. This alarming incidence and mortality highlight the persistent unmet medical need for novel, safe, and mechanistically effective therapeutic approaches.
Gaziantep, located in the Southeastern Anatolia Region of Turkey (37°04′N 37°23′E), is an important center for grain production given its semiarid climate and rich agricultural heritage. The unique microclimate of the region is an important contributing factor to the development of the genetic and phytochemical characteristics of the Karakılçık wheat variety. In this study, we used dried wheatgrass juice powder obtained from the Karakılçık wheat variety grown in this region2,3. This genotype is genetically conserved and phytochemically distinct, potentially contributing to a unique bioactive profile with therapeutic relevance.
Breast cancer is the most common and deadly type of cancer in women. It represents a serious public health problem, with 2.3 million new cases and 685,000 deaths being recorded worldwide in 20221. The complex molecular nature of the disease creates significant challenges in treatment approaches, the most prominent example being associated with triple-negative breast cancer (TNBC), an extremely aggressive subtype of this malignancy. TNBC accounts for approximately 10%–15% of all breast cancer cases and is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression3,4. Triple-negative breast cancer (TNBC), accounting for approximately 10–15% of cases, is the most aggressive subtype and lacks expression of ER, PR, and HER2 receptors.³˒⁴ Current clinical approaches for TNBC treatment include surgical intervention, radiotherapy, chemotherapy, and targeted molecular strategies. However, the molecular profile of TNBC severely limits the efficacy of therapeutic options5. Existing chemotherapeutic protocols are associated with significant systemic toxicity, including cardiotoxicity, myelosuppression, alopecia, and gastrointestinal complications, substantially impairing patient quality of life.⁶ Thus, the identification of novel, biocompatible, and multifunctional therapeutic agents remains a critical clinical priority. Chemotherapeutic protocols can seriously harm healthy cell populations, thereby significantly affecting the quality of life of patients. Side effects such as nausea, vomiting, hair loss, myelosuppression, and cardiotoxicity constitute the most important disadvantages of current treatment approaches6.
Oxidative stress, defined as an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense mechanisms, is a complex biological process that plays a critical role in cancer pathogenesis7. Epidemiological and experimental studies have shown that chronic oxidative stress can trigger carcinogenesis through DNA damage, protein oxidation, and lipid peroxidation8,9. This process plays an important role in the initiation and progression of critical oncogenic processes such as cellular transformation, proliferation, and metastasis. It further contributes to oncogenic signaling, cellular transformation, and metastatic progression. Phenolic compounds are plant-derived secondary metabolites with the ability to neutralize free radicals, reduce oxidative stress, prevent cellular damage, and inhibit cell proliferation. These antioxidants and antiproliferative properties make them a promising area of ​​research in cancer treatment10,11. Emerging evidence suggests that their therapeutic potential may be mediated through multi-target molecular modulation, including interference with redox-sensitive pathways, inflammatory mediators, and cell cycle regulators. Recent studies have revealed that these compounds exert their therapeutic effects through multiple mechanisms, including the prevention of oxidative damage, modulation of signaling pathways, suppression of inflammatory processes, and regulation of the cell cycle12,13.
Wheatgrass (Triticum aestivum L.) is a functional food notable for its high nutritional content and range of bioactive components14. Its antioxidant, antimicrobial, and potential anticancer properties make it a promising nutraceutical agent15,16. However, current literature is limited regarding the mechanistic evaluation of genotype-specific wheatgrass extracts using comprehensive analytical and computational approaches. The positive health effects of this plant are primarily attributable to its phenolic compounds, vitamins, minerals, and enzymes.
In this study, we sought to comprehensively characterize the phytochemical composition, antioxidant potential, and possible anticancer mechanisms of wheatgrass juice powder obtained from the genetically intact Karakılçık wheat variety. We investigated in depth the molecular-level properties and biological activities of its phenolic compounds, integrating modern analytical techniques and advanced computational methods. This study presents several highly distinctive contributions to the existing body of literature. Unlike previous reports investigating the general bioactivity of wheatgrass extracts, the present research provides the first comprehensive mechanistic analysis of dried wheatgrass juice powder specifically derived from the genetically conserved Karakılçık wheat variety. By combining high-resolution phenolic profiling through high performance liquid chromatography-diode array detector (HPLC-DAD) with advanced DFT-based quantum chemical modeling and multi-target molecular docking simulations against cancer- and oxidative stress–associated proteins, the study integrates experimental and computational evidence to elucidate molecular-level biological effects. Furthermore, the direct correlation of these theoretical predictions with in vitro cytotoxicity outcomes in MCF-7 cells represents a novel, multi-disciplinary approach that has not previously been applied to this wheat genotype. Accordingly, the work offers original mechanistic insight and supports the potential development of Karakılçık wheatgrass juice powder as a promising nutraceutical candidate for future preventive or adjuvant cancer therapy. To the best of our knowledge, this is the first study to apply an integrated framework combining high-resolution phenolic profiling, DFT-based quantum chemical modeling, multi-target molecular docking against cancer- and oxidative stress-related proteins, and in vitro cytotoxicity assessment on MCF-7 cells.
The main objective of this study is to elucidate the molecular-level antioxidant capacity and anticancer mechanisms of Karakılçık wheatgrass juice powder and to provide mechanistic evidence supporting its potential development as a novel nutraceutical candidate for future cancer prevention and adjuvant therapeutic strategies.
The main objective of this study was to reveal the potential therapeutic properties of wheatgrass juice powder at the molecular level and provide new information that will potentially contribute to future cancer treatment strategies.

Materials and methods

Phenolic compound extraction
The extraction of wheatgrass juice powder was conducted using the ultrasonic-assisted extraction (UAE) method17. Wheatgrass juice powder (2 g) was mixed with 50 mL of distilled water, which served as the solvent18. Extraction was performed at 50 °C for 3 h. Subsequently, the solution was centrifuged at 9,500 rpm for 30 min, the supernatant was removed using a centrifugal vacuum concentrator, and a stock solution of wheatgrass juice powder was prepared by dissolving 0.5 g of the extract in 10 mL of distilled water. To sterilize the extract and prevent potential damage to the liquid chromatography device during injection, the solution was filtered through a 0.45-µm membrane filter19. All prepared extracts were stored at 4 °C. The extraction yield was determined according to the following formula20:

HPLC analysis of the phenolic compounds
Quantitative analysis of catechin, esculetin, caffeic acid, coumarin, ellagic acid, quercetin, and apigenin in the wheatgrass juice powder extract was performed using HPLC, with a total analysis time of 23 min. The phenolic compounds were analyzed using a Shimadzu High-Performance Liquid Chromatography (HPLC) system equipped with an LC-10AD VP pump, a CTO-10AS column oven, a DGU-20 A degasser, and an SPD-M20A diode array detector (DAD). Chromatographic separation was performed with an X-Terra C18 column (250 × 4.6 mm, 5 μm) supplied by Waters. The HPLC method was applied as previously described21, with minor modifications. The mobile phase consisted of deionized water containing 0.1% orthophosphoric acid (phase A) and ethanol (phase B). The gradient elution program was as follows: 0–7 min, 20%–30% B; 7–15 min, 30%–45% B; 15–21 min, 45%–60% B; and 21–25 min, 60%–20% B. The flow rate was set at 0.85 mL/min, the column temperature was maintained at 20 °C, and the injection volume was 20 µL. Analyses were conducted at 230 nm. To determine the linearity range and obtain standard curve equations for each phenolic compound, HPLC analyses were performed in triplicate using at least five different concentrations22. Quantification of each phenolic compound in the extract was carried out using regression equations derived from the standard curves, correlating peak areas with corresponding concentrations.

Antioxidant activity

DPPH radical scavenging activity
2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a stable free radical with a deep purple color commonly used to assess the antioxidant capacity of wheatgrass juice powder extracts. When antioxidants donate hydrogen atoms or electrons, DPPH undergoes a color change, which can be quantified using a UV-vis spectrophotometer at wavelengths between 515 and 520 nm22. Wheatgrass juice powder extracts at concentrations ranging from 50 to 2500 µg/mL were prepared by diluting the 50 mg/mL wheatgrass juice powder stock solution. L-Ascorbic acid was used as the positive control, and distilled water served as the blank. A volume of 100 µL of each sample was added to each well of a 96-well microplate, followed by the addition of 100 µL of 0.2 mM DPPH (in ethanol). The microplates were incubated for 30 min in a shaking incubator shielded from light. After incubation, the absorbance of each well at 517 nm was measured using a spectrophotometer. The DPPH radical scavenging activity was calculated as a percentage according to the following equation:

where Ablank is the absorbance of the blank (distilled water + DPPH solution), Asample is the absorbance of the sample (wheatgrass extract + DPPH solution).

Total phenolic content
The total phenolic content (TPC) of the samples was determined using a slightly modified Folin-Ciocalteu colorimetric method, as described by Kaur et al.23, with gallic acid as the standard phenolic compound. A calibration curve was prepared using five concentrations (0.10–250 µg/mL) of gallic acid. For the assay, 100 µL of the sample solution was mixed with 200 µL of 10% (v/v) Folin-Ciocalteu reagent, and thoroughly vortexed. The mixture was incubated at room temperature for 5 min to allow the initial reaction to occur. Subsequently, 800 µL of 2% (w/v) sodium carbonate (Na2CO3) solution was added, and the mixture was incubated on a shaker for 90 min at ambient temperature to ensure reaction completion. Finally, the absorbance of the resulting solution was measured at 765 nm using a spectrophotometer. Each sample was analyzed in triplicate, and the measurements were repeated on two separate days to ensure accuracy and reproducibility. The TPC was calculated using the gallic acid calibration curve (y = 0.0485x + 0.0096) and expressed as milligrams of gallic acid equivalents (GAE) per gram of the dry weight of the sample (mg GAE/g).

In Silico studies

Geometric optimization and molecular docking analysis
The major compounds obtained from HPLC analysis—caffeic acid (CID: 689043), catechin (CID: 73160), coumarin (CID: 323), apigenin (CID: 5280443), esculetin (CID: 5281416), quercetin (CID: 5280343), and ellagic acid (CID: 5281855)—were retrieved from the PubChem database. Density functional theory (DFT), one of the most frequently used quantum chemical methods, is the preferred method for geometry optimization of organic molecules. The hybrid functional Becke-3-Lee–Yang–Parr (B3LYP), preferred in optimization studies, is also widely used in the literature due to its accuracy and reliable description of energy surfaces in organic molecules. The 6–311 + + G(d, p) basis set is preferred because it includes polarization and diffusion functions. All the compounds were optimized with the Gaussian09 program24 using the DFT/B3LYP method and the 6–311 + + G(d, p) basis set.
All the compounds were prepared as ligands for molecular docking using AutoDockTools 1.5.7. For theoretical investigation of antioxidant and anticancer properties, target receptors were carefully selected and downloaded from the Protein Data Bank (PDB). Superoxide dismutase (SOD; PDB ID: 1CB4), catalase (CAT; PDB ID: 1QQW), and Kelch-like ECH-associated protein 1 (KEAP1; PDB ID: 5CGJ) were selected for antioxidant studies. Estrogen receptor alpha (ERα; PDB ID: 1A52), epidermal growth factor receptor (EGFR; PDB ID: 1M17), inhibitor of nuclear factor kappa-B kinase subunit beta (IKK-β; PDB ID: 4KIK), and C-X-C chemokine receptor type 4 (CXCR-4; PDB ID: 3ODU) were selected as the receptors for anticancer studies, based on relevant cell experiments. All selected receptors were prepared with AutoDockTools 1.5.7 by adding polar hydrogens and removing water molecules and other ligands. After preparation, molecular docking studies were carried out using AutoDock Vina25.

Absorption, distribution, metabolism, elimination, and toxicity (ADMET) analysis
ADMET analysis was performed using two primary computational tools, namely, the Schrödinger QikProp module (Schrödinger Release 2025-2: QikProp, Schrödinger, LLC, New York, NY, 2025) and the OSIRIS Property Explorer (https://www.organic-chemistry.org/prog/peo/). This analysis provided a thorough evaluation of the pharmacokinetic and toxicological properties of the phenolic compounds.
The OSIRIS Property Explorer was specifically used for toxicity risk prediction, evaluating potential mutagenic, tumorigenic, irritant, and reproductive toxicity risks for each phenolic compound. Pharmaceutical suitability was rigorously evaluated using multiple established criteria, including Lipinski’s rule of five and the Veber, Egan, and Muegge rules. These computational strategies offered comprehensive insights into the potential therapeutic applications and safety profiles of the phenolic compounds identified in Karakılçık wheatgrass juice powder.

In vitro studies
The human breast cancer cell line MCF-7 (originally from ATCC HTB-22) was used for in vitro cytotoxicity studies. The cells were maintained in high glucose DMEM (Gibco) containing 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) and incubated at 37 °C with 5% CO₂. For the MTT assay, cells were seeded in a 96-well culture plate at a density of 1 × 104 cells per well and incubated for 24 h. Following incubation, the medium was replaced with fresh medium containing different concentrations (0.025, 0.05, 0.1, 0.25, 0.5, and 1 mg/mL) of wheatgrass juice powder, prepared in 0.05% alcohol. At the end of the experimental period, 40 µL of MTT (Sigma) was applied to each well to generate formazan crystals, after which the cells were incubated for 4 h at 37 °C. Subsequently, 160 µL of DMSO (Merck) was applied to each well, and the cells were incubated overnight. Absorbance was measured at 570 nm using an ELISA reader (BioTek, ELx800). The viability of the control group at each time point was accepted as 100% and the viability of the experimental groups was determined accordingly. The experiment was independently repeated in triplicate with ten technical replicates.

Statistical analysis
The significance of in vitro cytotoxicity tests was determined using one-way ANOVA, followed by Dunnett’s multiple comparison post-hoc test. P-values of < 0.05 were considered statistically significant. All calculations were performed using GraphPad Prism software (version 6.0, GraphPad Software, Inc., USA).

Results

HPLC analysis
The concentrations of catechin, esculetin, caffeic acid, coumarin, ellagic acid, quercetin, and apigenin in the wheatgrass juice powder extract were quantified using an HPLC-DAD method. Quantification was performed based on the regression equations obtained from the standard calibration curves. All calibration curves exhibited high linearity (r² ≥ 0.9995), confirming the reliability of the analytical method. The retention times, correlation coefficients, LOD and LOQ values of the standards were given in Table 1.

The extraction yield was calculated based on the established formula. According to this calculation, the extraction yield was found to be 6.56%. This value reflects the efficiency of the extraction process under the applied conditions. The concentrations of the phenolic compounds are summarized in Table 2, and all analyses were conducted in triplicate to ensure methodological reliability. Among the detected phenolics, caffeic acid was found to be the dominant compound, with a concentration of 2.323 ± 0.008 µg/mg, which is substantially higher than the levels of the other quantified compounds. Additionally, catechin was identified as the second most abundant compound (0.962 ± 0.012 µg/mg) (Fig. 1).

Antioxidant activity
The in vitro antioxidant activity of wheatgrass juice powder extract was assessed using DPPH and TPC assays. The extracts were tested for their ability to inhibit DPPH at various concentrations. Furthermore, the IC50 value, which represents the concentration of the sample needed to reduce 50% of DPPH free radicals, was calculated for both the extract and the positive control. The IC50 values of wheatgrass juice powder extract and the positive control for DPPH were found to be 202.711 and 3.515 µg/mL, respectively (Table 3).

The TPC of the samples was assessed using Folin-Ciocalteu’s reagent, with the results expressed as GAEs. The TPC calibration curve exhibited high linearity, with an r² value of 0.999. The TPC of the wheatgrass juice powder extract was determined to be 69.787 mg GAE/g.

In Silico studies

Geometric Optimization and Molecular Docking analyses
The 3D structures of the seven phenolic compounds (apigenin, caffeic acid, catechin, coumarin, ellagic acid, esculetin, and quercetin) identified in Karakılçık wheatgrass juice powder were optimized using the Gaussian 09 software package. Optimization was performed using DFT at the B3LYP/6–311 + + G(d, p) level (Table 2 and Fig. S1-S7). This process yielded stable conformations with minimized energy, providing a reliable foundation for subsequent analyses. The optimized three-dimensional structures of the compounds showed no geometric distortions and provided a consistent starting geometry for evaluating the interactions of the compounds with target proteins.
After the optimization study, molecular docking analyses were performed to investigate potential interactions between the identified phenolic compounds and key receptor targets associated with cancer and oxidative stress pathways.
The estrogen receptor is highly expressed in most breast cancer cases and is crucial for cell survival and proliferation26,27. EGFR is another important target macromolecule in anticancer investigations. Its expression is elevated in many tumors and its activation is associated with tumor growth, invasion, and metastasis27–30. IKK-β, a key regulator of the canonical NF-κB pathway, is a macromolecule reported to be a drug target in pathologies such as chronic inflammatory diseases and cancer31. The CXCR-4 receptor is an important therapeutic target that is highly expressed in cancer cells and plays a critical role in cancer cell metastasis, migration, and proliferation32,33. Molecular docking analyses revealed that all major compounds exhibited very good binding affinities in their interactions with EGFR and IKK-β, based on the calculated RMSD values (Table 5). The compounds displaying the highest binding affinity for both EGFR and IKK-β were apigenin, catechin, ellagic acid, and quercetin.

The binding affinities of the seven phenolic compounds for four anticancer protein targets (ER, EGFR, human IKK-β, and CXCR-4) were systematically evaluated (Table 5). Apigenin consistently showed strong binding affinities with all targets, ranging from − 8.4 to − 9.6 kcal/mol, and demonstrated the most compatible binding profiles with human IKK-β and EGFR (Figs. 2 and 3). Notably, apigenin formed hydrogen bonds with the Thr-830, Lys-721, Ala-719, and Thr-766 residues of EGFR (Table 5). For IKK-β, we observed that apigenin interacted with Cys-99, and pi-sigma interactions were predominant in its overall binding profile with IKK-β. Furthermore, the calculated binding affinity of −9.6 kcal/mol for apigenin with IKK-β suggests that apigenin can effectively inhibit IKK-β activity. Quercetin showed varying binding affinities with all targets, ranging from − 8.5 kcal/mol to −9.3 kcal/mol, with the strongest binding profiles with human IKK-β and EGFR (Figures S2–S5). Quercetin was observed to form a hydrogen bond with Cys-99 in the ATP binding site of IKK-β. Pi-alkyl, pi-sigma, and van der Waals interactions were predominant in the molecular docking investigation of quercetin with IKK-β. For the quercetin-EGFR interaction, molecular docking analysis showed that quercetin formed hydrogen bonds with the Thr-830, Met-769, Lys-721, Ala-719, and Thr-766 residues of EGFR. Catechin showed binding energies with anticancer targets in the range of − 9.1 to − 7.9 kcal/mol (Figs. S2–S5), with the highest affinities observed for EGFR (− 8.5 kcal/mol) and human IKK-β (− 9.1 kcal/mol). Catechin was noted to form hydrogen bonds with Lys-721, Ala-719, Thr-766, Asp-831, and Met-769 of EGFR. Catechin, which displayed very strong binding energy with human IKK-β, hydrogen bonded with Asp-103, Cys-99, Met-96, and Lys-44 of IKK-β, and also formed pi-sulfur and pi-alkyl interactions with Met-96 and Lys-44 of the kinase, respectively. Ellagic acid demonstrated high binding affinity with all anticancer targets, with the highest binding affinity of −9.4 kcal/mol being obtained in a molecular docking study with human IKK-β (Figure S4). Ellagic acid also exhibited high binding affinities with CXCR-4 (−9.2 kcal/mol), ER (−9.1 kcal/mol), and EGFR (−8.8 kcal/mol). Additionally, among the major compounds, esculetin, coumarin, and caffeic acid had lower binding affinity values ​​than other compounds. (Figures S2-S5 and Table 5).

SOD is an antioxidant enzyme that plays a role in the defense against oxidative stress, protecting against oxidative damage, particularly that induced by reactive oxygen species (ROS)35–38. Another antioxidant enzyme, catalase, plays an essential protective role against cellular oxidative stress and helps reduce the effects of ROS39,40. Catalase works together with other antioxidant enzymes, such as glutathione peroxidase (GPx) and SOD41. KEAP1, meanwhile, is a well-characterized regulator of the cellular response to oxidative stress, functioning alongside these three enzymes42. Based on the binding energies observed in molecular docking studies involving these macromolecules, functioning as receptors, and the major compounds of wheatgrass, acting as ligands, catalase was considered to be the most prominent receptor (Table 4). KEAP1 also displayed significantly high binding energies in its interactions with the wheatgrass compound ligands. The binding affinities of the major compounds in Karakılçık wheatgrass with the three antioxidant receptors (SOD, catalase, and KEAP 1) are presented in Table 4.

Among the seven parent compounds identified, ellagic acid showed the best binding affinities with catalase and KEAP1 (Figs. 4 and 5). Ellagic acid showed the strongest binding energy (− 11.5 kcal/mol) with catalase compared to the other compounds. For ellagic acid, the interactions with catalase consisted of hydrogen bonds, π-π stacking, π-alkyl interactions, and van der Waals forces. In particular, residues Arg-72, Arg-365, Ala-133, Arg-112, and Tyr-358 provided stability through hydrogen bond interactions, while π interactions and van der Waals forces contributed to complex stabilization. Arg-72, Ala-133, and Tyr-358, which were involved in hydrogen bond formation, also participated in π-alkyl interactions and π-π stacking, contributing to the strong binding energy. The receptor with the best binding affinity value after catalase was KEAP1, with ellagic acid again associated with the best binding energy (− 9.8 kcal/mol), while catechin, quercetin, and apigenin achieved the strongest binding profiles, with binding energies of − 9.6, − 9.1, and − 9.0 kcal/mol, respectively. Examination of the binding profile of KEAP1 and ellagic acid showed that hydrogen bonding interactions were predominant, with residues Gly-367, Val-465, Val-604, and Val-606 in KEAP1 being involved in these interactions. Additionally, weak carbon-hydrogen bond interactions, involving Gly-464, Gly-605, and Gly-603, contributed to the overall score in this docking analysis. Quercetin exhibited strong binding affinity values with all three antioxidant receptors (SOD, catalase, and KEAP1) (Table 6 and Figs. S6–S8). Quercetin displayed a binding energy of − 10.4 kcal/mol with catalase and formed hydrogen bonds with residues Asn-148, Gly-147, Tyr-358, and His-362. It also formed an unfavorable donor-donor interaction with Ala-133, with a distance of 2.22 Å, as well as π-alkyl interactions. Quercetin had a binding affinity value of − 9.1 kcal/mol with KEAP1, interacting via hydrogen bonds (Ala-510, Val-465, and Leu-365) and π-sulfur (Cys-513), carbon-H-bond (Gly-464), and van der Waals interactions. Like ellagic acid and quercetin, catechin also showed strong binding affinity values with catalase and KEAP1 (Figs. S7 and S8). Its binding affinity value with SOD was − 8.1 kcal/mol. Like catechin, apigenin exhibited a strong binding profile with catalase and KEAP1. It also presented a binding energy of − 7.9 kcal/mol with SOD. Additionally, molecular docking studies were performed with caffeic acid, esculetin, and coumarin with the antioxidant receptor, and among them, esculetin was found to have a better binding affinity value than the others (Table 6 and Figures S6–S8).

ADMET analysis
To evaluate the pharmaceutical potential of the seven phenolic constituents, comprehensive ADMET profiling was conducted using Schrödinger’s QikProp module and OSIRIS Property Explorer (Tables 7 and 8). For drug-likeness assessment, we employed established screening criteria, including Lipinski’s rule of five, which stipulates molecular weight ≤ 500 Da, LogP ≤ 5, hydrogen bond donors ≤ 5, and acceptors ≤ 10 for optimal oral bioavailability. Except for quercetin, which exhibited a single violation (6 hydrogen bond donors), all compounds demonstrated compliance with Lipinski’s parameters. Physicochemical properties, including molecular mass (146.145–318.366 Da) and polar surface area, fell within acceptable pharmaceutical ranges.

Pharmacokinetic analysis revealed distinct compound-specific profiles. Lipophilicity (LogP) ranged from − 1.620 (quercetin) to 1.624 (apigenin), while aqueous solubility (LogS) values were generally favorable (− 3.323 to − 0.946). Membrane permeability studies using Caco-2 and MDCK cell models identified pronounced variation among the compounds, with coumarin exhibiting excellent permeating ability (2080 nm/s for Caco-2 cells and 1091 nm/s for MDCK cells), whereas ellagic acid demonstrated poor penetration (8 nm/s for Caco-2 cells and 2 nm/s for MDCK cells). Predicted human oral absorption ranged from 34% (quercetin) to 94% (coumarin). Cardiotoxicity assessment indicated minimal risk of hERG K+ channel blockage (LogIC50: −1.276 to − 5.142).
In the toxicological evaluation, caffeic acid and ellagic acid exhibited optimal safety profiles, with no mutagenic, tumorigenic, irritant, or reproductive toxicity risks. Conversely, coumarin presented a high-risk potential across several toxicity categories, while apigenin raised concerns regarding mutagenic and tumorigenic effects. These favorable ADMET characteristics position caffeic acid, the predominant phenolic constituent, as the most promising therapeutic candidate among the phenolic compounds.
The ADMET analysis results revealed that the phenolic components of Karakılçık wheatgrass juice powder possess varying pharmacokinetic and toxicological properties (Tables 7 and 8). Caffeic acid, found in the highest concentration in wheatgrass juice powder, stands out for its low toxicity risk and good drug-likeness properties, supporting its potential therapeutic application. In contrast, compounds that showed strong protein binding affinity, such as ellagic acid and quercetin, exhibited relatively low membrane-permeating ability and oral bioavailability, which may limit their in vivo efficacy. This suggests that the therapeutic effect of wheatgrass juice powder may arise from synergistic interactions between components with different pharmacokinetic profiles rather than from a single compound.

Cell culture results
In our study, the effects of different concentrations of wheatgrass juice powder on the viability of the human breast cancer cell line, MCF-7, were evaluated by MTT assay. The percent viability of cells treated with wheatgrass juice powder was determined at the 24-, 48-, and 72-h time points. At low concentrations (0.025, 0.05, 0.1, and 0.25 mg/mL), no significant difference in cell numbers was observed between the treatment groups and the control group at any of the sampling time points. However, at the 1 mg/mL concentration, a significant decrease (p < 0.01) in cell viability was recorded at both the 24- and 48-hour time points compared to that in control cells, thereby showing a concentration-dependent effect. At the 72-h time point, there was a significant decline in cell viability with the 0.25 and 0.5 mg/mL concentrations. These findings suggested that the tested compound used in this study exerts cytotoxic effects on MCF-7 cells at a certain threshold concentration, with the effect becoming more pronounced in a time-dependent manner.
The dose and time-dependent changes in viability observed in MCF-7 breast cancer cells (Fig. 6) indicate that Karakılçık wheatgrass juice powder may possess a complex mechanism of action. The significant decline in cell viability (p < 0.01) at the high concentration of 1 mg/mL after 24 and 48 h of incubation parallels the strong interactions with cancer-related proteins, such as topoisomerase I and IKK-β, identified in molecular docking studies. The increase in cell viability observed at lower concentrations (0.25 and 0.5 mg/mL) after 72 h of treatment may be related to a hormetic effect of low-dose phenolic compounds or the activation of adaptive cellular stress response mechanisms.

The findings of this study provide compelling evidence for the potential therapeutic applications of Karakılçık wheatgrass juice powder in breast cancer treatment through both direct antiproliferative mechanisms and antioxidant activity. The integration of in silico molecular docking analyses with experimental biochemical and cell culture studies offers valuable insights into its underlying mechanism of action.

Discussion

HPLC analysis
The HPLC analysis revealed a well-defined and analytically robust phenolic profile in the wheatgrass juice powder extract, with calibration curves showing excellent linearity (r² ≥ 0.9995). Among the identified compounds, caffeic acid was the dominant phenolic (2.323 ± 0.008 µg/mg). The second most abundant compound, catechin (0.962 ± 0.012 µg/mg). The low LOD values observed in this study (0.363–8.639 ng/mL) confirm the high sensitivity of the method and are comparable or superior to those reported in prior analytical investigations of wheatgrass phenolics43,44. Additionally, the retention time distribution was consistent with polarity-based elution patterns described in other HPLC studies of phenolic acids and flavonoids, supporting the adequacy of the chromatographic separation.

Antioxidant activity
The antioxidant capacity and phenolic composition of wheatgrass are known to vary considerably depending on cultivar characteristics, agronomic conditions, and extraction methods. Therefore, comparing the present findings with previously published data is essential to contextualize the potency and biochemical richness of the wheatgrass juice powder analyzed in this study.
Yoon et al. (2024) evaluated the antioxidant activity of two wheat cultivars using the DPPH assay and reported IC₅₀ values of 14.14 mg/mL and 16.12 mg/mL. Such high IC₅₀ values indicate relatively weak antioxidant capacity, as greater sample concentrations are required to achieve 50% radical neutralization45. In contrast, the IC₅₀ value obtained in the present study (0.202 mg/mL) demonstrates significantly stronger radical-scavenging activity, highlighting the superior antioxidant potential of wheatgrass juice powder under the tested conditions. Similarly, Jabeen et al. (2020) reported a total phenolic content (TPC) of 15.20 mg/g and an IC₅₀ of 1.48 mg/g for lyophilized wheatgrass juice powder, reflecting only moderate antioxidant activity46. The extract analyzed in our study exhibited markedly higher phenolic content (69.787 mg GAE/g) alongside a much lower IC₅₀ value, further confirming its enhanced antioxidant performance.
Previous research has generally reported lower TPC values for wheatgrass compared to those observed here. Ove et al. (2021) found TPC values ranging between 9.15 ± 0.18 and 42.09 ± 2.73 mg GAE/g depending on the solvent system and sample form, while Kaur et al. (2021) reported a lower range of 3.08 ± 0.04 to 14.62 ± 0.14 mg GAE/g DW across different wheat varieties23,47. The substantially higher TPC recorded in our study suggests that the juicing–dehydration process may concentrate phenolic constituents more effectively. Additionally, the use of aqueous ultrasound-assisted extraction likely enhanced the release of water-soluble phenolics such as caffeic acid, identified as the dominant compound in our HPLC analysis. The consistent relationship between elevated TPC values and reduced IC₅₀ results aligns with the well-established link between phenolic density and antioxidant functionality.
Plant-derived antioxidants play a critical role in mitigating oxidative stress induced by reactive oxygen species (ROS). As with other phenolic-rich botanical extracts, the antioxidant activity of wheatgrass juice powder can largely be attributed to its phenolic constituents, which participate in electron-transfer and hydrogen-atom donation mechanisms. In this study, the strong coherence between DPPH radical-scavenging activity and TPC supports this relationship. Phenolic acids and flavonoids detected in the extract—including caffeic acid, catechin, and quercetin—are widely recognized for their potent radical-neutralizing capacity, and their presence is consistent with the observed antioxidant behavior. Because the reducing power of phenolic compounds is strongly influenced by their structural features, compounds such as caffeic acid, which contain multiple hydroxyl groups, likely contributed more significantly to the antioxidant activity than phenolics present at lower concentrations. Overall, the positive correlation between phenolic composition and DPPH scavenging efficiency confirms that phenolic constituents are the primary determinants of the antioxidant potential of wheatgrass juice powder.

Geometric Optimization and Molecular Docking analyses
Geometry optimization is an important preparatory step for molecular docking, as the reliability of predicted binding modes is correlated with the accuracy of the starting ligand structure. In this study, the geometry optimization studies performed ensured that major compounds achieved a physically meaningful configuration before docking into the active sites of target proteins. The chosen method and basis set (DFT/B3LYP-6311 + + G(d, p)) are frequently used in the literature for modeling organic compounds48,49. Therefore, the compounds optimized with DFT provided a reliable and consistent basis for molecular docking studies and played a role in predicting the binding affinities and pose stability results obtained.
The strong binding affinities obtained from molecular docking studies theoretically demonstrated apigenin’s potential as a versatile inhibitory molecule for selected anticancer targets. Apigenin’s interaction with key residues in IKK-β and EGFR supports its ability to form stable and functionally relevant complexes. Notably, apigenin’s binding affinity for EGFR was determined to be significantly higher than the binding affinity of the EGFR-targeting drug erlotinib reported in the literature34,35, suggesting that apigenin may exhibit similar or improved inhibitory potential. The calculated binding affinity of −9.6 kcal/mol for IKK-β also suggests that apigenin may be a promising inhibitor of this kinase, given the critical role of Cys-99 in ligand recognition. These findings highlight the theoretical anticancer potential of apigenin through multiple target interactions. The binding affinities obtained in docking studies for quercetin indicate that it may have strong interaction potential with selected anticancer targets. Quercetin’s interaction with Cys-99 in IKK-β is noteworthy, as this residue is critical for inhibitor binding in the ATP-binding pocket. Similarly, quercetin’s interaction with key residues in EGFR, similar to erlotinib34,35, supports its potential as an EGFR inhibitor. Overall, these findings suggest that quercetin could theoretically act as an effective inhibitor of multiple anticancer targets. The binding energy distribution obtained for catechin demonstrates a strong and stable interaction profile between multiple anticancer targets, particularly EGFR and human IKK-β. Interaction with key residues in EGFR, such as erlotinib34,35, supports catechin’s potential as an EGFR-interacting molecule. The observation that catechin exhibits a more favorable binding affinity than erlotinib at the same active site further strengthens this notion34,35. Similarly, catechin’s interaction with key residues such as Cys-99 and Asp-103 in the ATP-binding pocket of IKK-β highlights catechin’s capacity to form energetically favorable and functionally meaningful interactions. The presence of pi-sulfur and pi-alkyl interactions contributes to the overall stability of the catechin-IKK-β complex. Taken together, these findings suggest that catechin has theoretical potential as a multitarget anticancer agent. The consistently high binding affinities observed for ellagic acid across all four anticancer targets demonstrate its strong theoretical potential as a multitarget anticancer agent. This suggests that ellagic acid’s inhibitory potential may extend to multiple signaling pathways involved in cancer progression. The binding affinities of esculetin, coumarin, and caffeic acid were weaker than those of other major compounds. However, this does not imply that the compounds are ineffective; their binding affinities with multiple targets suggest that these compounds may have potential as multitarget modulators. However, their potential is lower compared to the more potent interacting phenolic compounds identified in this study.
Ellagic acid exhibited strong binding affinities, particularly in molecular docking studies with catalase and KEAP1, suggesting that this compound may function in antioxidant defense pathways. The diverse interactions ellagic acid forms with catalase (hydrogen bonding, π interactions, and van der Waals interactions) suggest that the compound tends to form a tightly stabilized complex. The involvement of key residues such as Arg-72, Ala-133, and Tyr-358 in multiple interaction types further supports the compound’s potent inhibitory potential. Overall, these findings suggest that ellagic acid may have antioxidant potential. The strong binding affinities observed for ellagic acid and other phenolic compounds (e.g., catechin, quercetin, apigenin) suggest that these molecules can form stable complexes with KEAP1. The presence of hydrogen bonds, particularly with residues Gly-367, Val-465, Val-604, and Val-606, suggests that ellagic acid can form strong interactions within the KEAP1 binding pocket. The contribution of additional weak carbon-hydrogen bonds further stabilizes the complex. The similarity of these interactions to those of the reference compound (51M)50 supports the possibility that compounds such as ellagic acid, catechin, and quercetin may play a role in KEAP1 activity and suggests possible antioxidant effects due to their interaction with this receptor.
Molecular docking results indicate that phenolic compounds such as ellagic acid, quercetin, catechin, and apigenin exhibit strong binding affinities with both anticancer targets and important antioxidant enzymes. Hydrogen bonding, π-interactions, and vdw interactions contribute to the stabilization of the complexes, suggesting that these compounds may have the potential to modulate multiple targets.
Literature studies have also reported that quercetin is an antioxidant and may have an anticancer effect in breast cancer treatment51,52; ellagic acid, due to its antioxidant and anticarcinogenic properties, can significantly affect tumor growth53,54; catechin is a powerful antioxidant and effectively kills MCF-7 cells55; and apigenin also exhibits antioxidant and anticancer effects56,57. This theoretical step, supported by literature studies, is also supported by the antioxidant and anticancer studies of experimental wheatgrass juice powder in this study.

ADMET analysis
The ADMET profiling of the seven phenolic constituents provides critical insights into their pharmacokinetic behavior and potential therapeutic applicability. Drug-likeness assessment revealed that all compounds except quercetin adhered to Lipinski’s rule of five, a widely accepted criterion for predicting oral bioavailability58. Quercetin’s violation stems from its six hydrogen bond donors, which exceeds the threshold of five. However, this single violation does not necessarily preclude its biological activity, as several clinically approved drugs also exhibit minor rule violations while maintaining therapeutic efficacy59,60.
The lipophilicity values (LogP) observed in this study ranged from − 1.620 (quercetin) to 1.624 (apigenin), indicating diverse membrane permeability characteristics. Compounds with LogP values between − 0.4 and + 5.6 are generally considered to have favorable absorption profiles61. Apigenin’s relatively higher lipophilicity suggests superior membrane penetration capacity, which is consistent with previous reports demonstrating its enhanced cellular uptake in cancer models62. Conversely, quercetin’s negative LogP reflects its hydrophilic nature, potentially limiting passive diffusion across lipid bilayers but favoring active transport mechanisms63.
Membrane permeability predictions using Caco-2 and MDCK cell models revealed substantial variation among the compounds. Coumarin exhibited exceptional permeability (2080 nm/s for Caco-2; 1091 nm/s for MDCK), whereas ellagic acid demonstrated poor penetration capacity (8 nm/s for Caco-2; 2 nm/s for MDCK). According to established permeability classifications, compounds with Caco-2 values > 100 nm/s are considered highly permeable, while those < 10 nm/s exhibit poor absorption64,65. These findings suggest that while coumarin may achieve rapid systemic circulation following oral administration, ellagic acid’s bioavailability may be significantly limited, potentially requiring formulation strategies such as nanoencapsulation to enhance absorption66,67.
Cardiotoxicity assessment via hERG K+ channel blockage prediction is crucial, as hERG inhibition can lead to potentially fatal arrhythmias68. All compounds in this study exhibited LogIC values between − 1.276 and − 5.142, indicating minimal risk of cardiotoxic effects. Compounds with LogIC values > − 5 are generally considered safe from a cardiac perspective69. This favorable safety profile further supports the therapeutic critical role of Cys-99 in ligand recognition potential of wheatgrass phenolics.
The toxicological evaluation revealed compound-specific risk profiles. Caffeic acid and ellagic acid demonstrated optimal safety characteristics with no predicted mutagenic, tumorigenic, irritant, or reproductive toxicity risks. These findings align with extensive literature documenting the safety and chemopreventive properties of caffeic acid in various preclinical and clinical studies70,71. In contrast, coumarin exhibited high-risk potential across multiple toxicity categories. Although coumarin occurs naturally in many plants, chronic exposure has been associated with hepatotoxicity in rodent models, leading to regulatory restrictions in several countries72,73. Similarly, apigenin raised concerns regarding mutagenic and tumorigenic potential in the OSIRIS prediction model74. However, it is important to note that computational toxicity predictions do not always correlate with experimental outcomes, and numerous studies have demonstrated apigenin’s safety and anticancer efficacy both in vitro and in vivo75,76.
The divergence between high binding affinity and poor bioavailability observed for compounds such as ellagic acid and quercetin highlights an important consideration in natural product drug discovery. While these compounds exhibit strong theoretical interactions with cancer-related targets, their limited membrane permeability and oral absorption may restrict their systemic bioavailability11,77. This pharmacokinetic limitation suggests that the overall therapeutic effect of wheatgrass juice powder likely results from synergistic interactions among multiple bioactive constituents rather than the action of a single compound. For instance, caffeic acid’s high abundance, favorable ADMET profile, and demonstrated bioactivity position it as a key contributor to the observed cytotoxic effects, while compounds like quercetin and ellagic acid may exert localized effects in the gastrointestinal tract or function as prodrugs that undergo metabolic activation78,79.
Future research should focus on experimental validation of these in silico predictions through pharmacokinetic studies, including plasma concentration measurements, tissue distribution analysis, and metabolic profiling. Additionally, formulation strategies such as liposomal encapsulation, complexation with cyclodextrins, or development of structural analogs with improved bioavailability may enhance the therapeutic potential of compounds with unfavorable pharmacokinetic profiles80,81.

Cell culture results
To provide a deeper insight into the observed cytotoxicity, it is essential to consider its correlation with the specific phenolic constituents identified in the wheatgrass juice powder. Our HPLC analysis identified caffeic acid as the predominant compound (2.323 ± 0.008 µg/mg), suggesting that the significant reduction in MCF-7 cell viability, particularly at 1 mg/mL concentration, is strongly associated with the high abundance of this phenolic acid. Caffeic acid is well-documented to induce apoptosis via mitochondrial pathways and cell cycle arrest in breast cancer cells through ROS modulation70,82. Interestingly, while quercetin was present in lower quantities (0.044 ± 0.001 µg/mg) compared to caffeic acid and catechin, quercetin is a potent inhibitor of the PI3K/Akt/mTOR signaling pathway, while ellagic acid exerts anti-proliferative effects by inducing DNA damage and inhibiting angiogenesis. Our in silico findings revealed that it exhibits superior binding affinity to key cancer-related targets, such as IKK-β (− 9.3 kcal/mol) and EGFR (− 8.9 kcal/mol). This suggests that while caffeic acid may drive the quantitative cytotoxic effect due to its high concentration, quercetin likely contributes to the overall bioactivity through high-affinity interactions with specific molecular targets. Therefore, the observed cytotoxicity is likely driven by the combined contribution of the dominant caffeic acid and the highly potent quercetin, reflecting the bioactive potential of the extract as a whole rather than the isolated effect of a single compound.
When evaluating the biological activity of the wheatgrass juice powder, it is crucial to benchmark these findings against existing literature on Triticum aestivum. Regarding cytotoxicity, our results on MCF-7 cells are consistent with the pioneering findings of Kulkarni et al. (2006),83 who demonstrated the antiproliferative potential of wheatgrass extracts on breast cancer cells. However, a notable distinction in our study is the observation of significant cytotoxicity at 1 mg/mL concentration. This potency appears comparable to, and in some cases higher than, standard aqueous extracts reported in similar studies, likely due to the concentration of bioactive phenolics in the lyophilized juice powder form compared to fresh juice extracts84. Furthermore, the dominance of caffeic acid in our HPLC profile aligns with the phenolic patterns reported in high-quality wheatgrass cultivars, confirming that the production process effectively preserved the heat-sensitive antioxidant compounds responsible for the observed bioactivity.
Despite the promising findings, this study has certain limitations. First, the biological activity was assessed using a crude extract rather than isolated pure compounds; thus, while we hypothesize a combined action of phenolics, the exact contribution of each individual molecule requires further validation. Second, the in vitro cytotoxicity was limited to the MCF-7 cell line; testing on normal cell lines (to assess selectivity) and other cancer types would provide a broader safety and efficacy profile. Third, while molecular docking provides valuable mechanistic insights, these theoretical interactions need to be confirmed through wet-lab experiments such as Western Blotting or enzymatic assays. Future studies should focus on addressing these gaps by incorporating bioavailability studies to assess the absorption of these phenolics and in vivo models to validate the chemopreventive potential of wheatgrass juice powder in a systemic physiological environment.

Conclusion
Using a multifaceted approach combining in silico, biochemical, and cell culture methodologies, we demonstrated that wheatgrass juice powder contains bioactive phenolic compounds that can interact with key proteins involved in cancer progression and oxidative stress modulation. In silico studies, we hypothesized that natural phenolics could exhibit both anticancer and antioxidant activity, and we evaluated their potential to serve as multi-targeted agents. This integrative study also provides convincing evidence for the potential therapeutic applications of Karakılçık wheatgrass juice powder in breast cancer treatment. In molecular docking analyses, seven major constituents of wheatgrass were docked with anticancer and antioxidant receptors, and specific interaction profiles were identified for each receptor-ligand pair. The observed cytotoxic effect on MCF-7 breast cancer cells at high concentrations of wheatgrass juice powder provided preliminary validation of its anticancer potential, while its robust antioxidant capacity supported its role in mitigating oxidative damage. In conclusion, this study not only advances the understanding of the phytochemical properties and biological activities of Karakılçık wheatgrass juice powder but also highlights the broader value of integrating traditional agricultural resources with modern scientific methodologies in the discovery and development of novel therapeutic agents for cancer treatment. Future research directions should include in vivo studies to validate the observed anticancer effects and assess potential synergistic interactions with established chemotherapeutic agents.

Supplementary Information
Below is the link to the electronic supplementary material.