Effect of silymarin on Helicobacter pylori growth and adhesion: in vitro and in silico evaluation

Helicobacter pylori is one of the main risk factors for gastric diseases and gastric cancer due to its ability to colonize the gastric mucosa, induce chronic inflammation, and express virulence adhesins involved in bacterial persistence. Silymarin, a flavonolignan complex extracted from Silybum marianum, exhibits antioxidant, anti-inflammatory, and antimicrobial properties, suggesting potential therapeutic application against H. pylori. This study aimed to evaluate the antibacterial, anti-adhesive, anti-inflammatory, cytotoxic, and in silico interaction profiles of silymarin against H. pylori and its main adhesins BabA, SabA, and HopQ. The antibacterial activity was evaluated by broth microdilution for determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Morphological alterations were analyzed by scanning electron microscopy (SEM). Cytotoxicity was assessed by MTT assay using AGS gastric adenocarcinoma cells and RAW 264.7 macrophages. TNF-α, IL-6, and nitric oxide (NO) production were quantified in stimulated macrophages. Anti-adhesive activity was determined using FITC-labeled H. pylori adhesion assays in AGS cells. Molecular docking and physicochemical predictions were performed for the major flavonolignans present in silymarin. Silymarin exhibited MIC and MBC values of 128 μg mL −1 against H. pylori ATCC 43504, with 90.46% growth inhibition. SEM demonstrated bacterial deformation and coccoid-like morphology after treatment. Silymarin reduced TNF-α, IL-6, and NO production by 80.82%, 76.24%, and 97.50%, respectively. In adhesion assays, silymarin inhibited bacterial attachment by 25.87% ± 3.50%. Molecular docking demonstrated favorable interactions between flavonolignans and H. pylori adhesins. Isosilybinin A showed the highest affinity for BabA (fitness score = 57.75), establishing hydrogen bonds with CYS189 and SER244. Isosilychristin presented the best interaction with SabA (fitness score = 74.79), interacting with LYS152 and GLN159, while silychristin demonstrated the highest affinity for HopQ (fitness score = 66.42), interacting with SER135, ILE130, and ILE242. Silymarin demonstrated significant antibacterial, anti-inflammatory, and anti-adhesive activities against H. pylori ATCC 43504, supporting its potential as a multifunctional natural therapeutic candidate for the management of H. pylori -associated gastric diseases.

Silymarin is a vegetal complex extracted from the fruits, often mistaken as the seeds, of Silybum marianum L. Gaertn, a plant commonly known as milk thistle, which has been used as a natural product for health purposes for centuries (Elateeq et al., 2020). Silymarin can represent up to 70% of the mass of the brute extract from the fruits and seeds of the plant and is composed by a mixture of multiple flavonolignans instead of one single compound. The complex composition mainly includes the enantiomers silybinin A and B, which together can represent up to 70% of the complex composition, the enantiomers isosilybinin A and B, silychristin, isosilychristin, silydianin and taxifolin, which is a direct precursor in the flavonolignans synthesis pathway (Csupor et al., 2016; Lv et al., 2017; Zhang et al., 2024). The complex is known for possessing remarkable antioxidant activity, which is explained by the fact that its main compounds possess a flavonoid moiety in their structure (Vargas-Mendoza, 2014), hence why it has been widely used in formulations aimed at antioxidant, hepatoprotection, anti-inflammatory and antifibrotic purposes, which are all processes that involve oxidant stress in the body (Taleb et al., 2018). In the past decade, studies have demonstrated that silymarin possesses gastroprotective properties by targeting gastric cancer cells in vitro through different mechanisms and by scavenging free radicals that can harm the gastric epithelium (Koltai and Fliegel, 2022), and also that its main compound, silybinin, has demonstrated anti- Helicobacter pylori activity (Bittencourt et al., 2020).

Although several factors are known to impair the protective mechanisms of gastric tissues, including smoking and alcohol consumption, the colonization of the stomach by H. pylori is recognized as the major risk factor for gastric cancer development due to the intense inflammatory response triggered by the bacterium (Elisa et al., 2023). The presence of H. pylori in the stomach is associated with a wide spectrum of gastric disorders, ranging from acute gastritis to chronic gastritis and pangastritis, as well as the development of peptic ulcers, metaplasia, and dysplasia, which may ultimately progress to gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma (Diaconu et al., 2017; Malfertheiner et al., 2023). Owing to its strong association with gastric carcinogenesis, H. pylori has been classified as a Group 1 carcinogen by the World Health Organization since 1994 (Basílio et al., 2018).

Helicobacter pylori is a Gram-negative, spiral-shaped, microaerophilic bacterium characterized by remarkable motility resulting from the presence of up to six polar flagella. This bacterium is particularly recognized for its exceptional ability to survive and persist within the harsh gastric environment, a process largely mediated by its extensive repertoire of virulence factors (Costa et al., 2021; Al-Fakhrany and Elekhnawy, 2023). Approximately 4% of the bacterial genome is estimated to encode outer membrane proteins (OMPs), many of which function as adhesins that mediate bacterial attachment to gastric epithelial cells. This adhesion process is essential for successful colonization, as it enables the bacterium to evade clearance mechanisms such as mucus turnover, gastric acid flow, and peristaltic movements (Ishijima et al., 2011).

The adhesins expressed by H. pylori are capable of recognizing and binding to distinct extracellular structures present on gastric epithelial cells, particularly oligosaccharide antigens and glycoproteins. Among the best-characterized adhesins are the blood group antigen-binding adhesin A (BabA), which preferentially binds to Lewis b (Le b) blood group antigens, and the sialic acid-binding adhesin A (SabA), which primarily recognizes sialyl-Lewis X antigens (Sharndama and Mba, 2022; Šterbenc et al., 2019). In addition, other adhesins interact with host glycoproteins, such as outer membrane protein Q (HopQ), which specifically binds to members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family (Ansari and Yamaoka, 2019). Although the adhesion process itself is not directly responsible for triggering the inflammatory response, the successful interaction between bacterial adhesins and their host receptors is crucial for the activation and delivery of additional virulence factors involved in pathogenesis, including urease and the cytotoxins CagA and VacA (Alipour, 2021).

This infection also triggers an intense inflammatory response in the gastric mucosa, characterized by increased production of several cytokines and inflammatory mediators, including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and nitric oxide (NO). IL-6 and TNF-α are pro-inflammatory cytokines strongly associated with the recruitment of inflammatory cells, tissue damage, and the progression of chronic gastritis, contributing to pathological alterations such as metaplasia, dysplasia, and gastric carcinogenesis (De Bernard and D’Elios, 2010; Yadav et al., 2012). Additionally, the activation of macrophages and gastric epithelial cells by the bacterium stimulates nitric oxide production, a mediator with antimicrobial activity that, when persistently elevated, may contribute to oxidative stress and cellular damage (Butcher et al., 2017). Therefore, the imbalance between pro-inflammatory and anti-inflammatory mediators during H. pylori infection plays a central role in the pathogenesis of the gastric diseases associated with this bacterium.

Over the past 2 decades the treatment against H. pylori infection has been threatened by the emergence of antimicrobial-resistant strains (Chen et al., 2024), the most known case being the resistance against clarithromycin which was present in the classic triple therapy, which consisted of a proton-pump inhibitor and two antibiotics, usually amoxicillin and clarithromycin itself, whose use has since been halted in many places and replaced by quadruple therapy, which includes a bismuth salt in the protocol and replaces amoxicillin and clarithromycin by two other antibiotics, usually tetracycline and metronidazole (Hu et al., 2020; Rokkas et al., 2021). However, metronidazole resistance has already been detected and surpassed that of clarithromycin in multiples territories, which threatens the efficacy of the quadruple therapy regimen (Savoldi et al., 2018; Yu et al., 2024).

In such context, the need for the development of new drugs against H. pylori infection is clear, therefore, the objective of the present study was to evaluate the activity profile of silymarin compounds against H. pylori itself and against its main adhesins, BabA, SabA and HopQ, while also evaluating the anti-inflammatory activity against pro-inflammatory markers present in the inflammatory process of the infection.

Materials and equipment

The following reagents, materials, and equipment were used in this study: silymarin (≥30% silybinin, batch No. BCCH4151, Sigma-Aldrich, St. Louis, MO, United States), amoxicillin (≥95%, batch No. 105M4773V, Sigma-Aldrich, St. Louis, MO, United States), metronidazole (≥98%, batch No. SLBD5470V, Sigma-Aldrich, St. Louis, MO, United States), cisplatin (≥98.2%, batch No. LRAD4567, Sigma-Aldrich, St. Louis, MO, United States), dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, United States), fluorescein isothiocyanate (FITC; Sigma-Aldrich, St. Louis, MO, United States), Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; Vitrocell Embriolife, Campinas, SP, Brazil), fetal bovine serum (FBS; Vitrocell Embriolife, Campinas, SP, Brazil), and Brucella broth base (Himedia, M348-500G, Mumbai, India). Microaerophilic cultivation conditions were maintained using Panasonic incubators (models MCO-50AICL-PA and MCO-5AC-PA). Centrifugation procedures were conducted using an Eppendorf 5810R centrifuge (Hamburg, Germany). Absorbance and fluorescence measurements were obtained using iMark™ Microplate Reader (Bio-Rad, Hercules, CA, United States) and Synergy™ R1 Microplate Reader (BioTek Instruments, Winooski, VT, United States). Sample preparation for scanning electron microscopy was performed using a Desk V™ sputter coater (Denton Vacuum, Moorestown, NJ, United States) and a Tousimis Autosamdri®-815 Series A critical point dryer (Rockville, MD, United States). Ultrastructural analyses were carried out using a JEOL® JSM-6610LV scanning electron microscope (JEOL Ltd., Tokyo, Japan).

Preparation of the compounds’ solutions

Silymarin, amoxicillin, metronidazole and cisplatin were commercially obtained from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO) at the final concentrations of 40 mg mL -1, 0.02 mg mL -1, 10 mg mL -1 and 1 mg mL -1, respectively. The solutions were sterilized by filtration through a 0.22 μm membrane filter to obtain the stock solutions used in the subsequent assays. To minimize DMSO-related toxicity, the final DMSO concentration in all assays did not exceed 2.56% (v/v). Previous experiments demonstrated minimal toxicity of DMSO toward the cell lines and bacterial strain used in this study at concentrations below this threshold, with less than 18% toxicity observed after 72 h of exposure. A vehicle control containing the corresponding concentration of DMSO was included to account for any potential solvent-related effects.

Helicobacter pylori strain and cell line culture

Helicobacter pylori ATCC 43504, characterized as amoxicillin-susceptible and metronidazole-resistant, was kindly provided by the Oswaldo Cruz Foundation (Fiocruz, Rio de Janeiro, Brazil). The bacterial strain was initially cultured on Columbia agar supplemented with 5% sheep blood and subsequently transferred to Brucella broth supplemented with 10% (v/v) fetal bovine serum (FBS). Cultures were incubated at 37 °C under microaerophilic conditions containing 10% CO 2.

Human gastric adenocarcinoma cells (AGS, ATCC CRL-1739) and the murine macrophage cell line RAW 264.7 (RAW, ATCC TIB-71) were obtained from the Cell Bank of Rio de Janeiro, Brazil. Both cell lines were maintained separately in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% (v/v) fetal bovine serum and incubated at 37 °C in a humidified atmosphere containing 5% CO 2.

Minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays

The MIC and MBC were determined using the broth microdilution method established by the Clinical and Laboratory Standards Institute (CLSI, 2024; 2025), with modifications. Briefly, 100 μL of an H. pylori ATCC 43504 suspension (10 6 CFU mL -1) prepared in Brucella broth supplemented with 10% FBS was added to each well of a 96-well microplate, followed by the addition of 100 μL of silymarin at concentrations ranging from 32 μg mL -1 –1024 μg mL -1 prepared in the same medium. For the negative and positive controls, 100 μL of supplemented medium was added in place of the bacterial suspension and the silymarin dilutions, respectively. Supplemented medium containing amoxicillin (0.016–0.5 μg mL -1) and metronidazole (8–256 μg mL -1) was used as antimicrobial standards. The microplate was homogenized, and the absorbance was measured at 620 nm using an iMark™ Bio-Rad microplate reader prior to incubation for 72 h at 37 °C under microaerophilic conditions with 10% CO2. After incubation, the microplate was homogenized again, and absorbance was remeasured under the same conditions to determine the MIC. The MIC was defined as the lowest concentration capable of reducing bacterial growth by at least 90% compared to the positive control, and the results were expressed as percentage of growth inhibition (%).

The MBC was determined by inoculating the contents of the wells corresponding to the MIC concentration onto Columbia agar plates supplemented with 5% sheep blood. The MBC was defined as the lowest concentration capable of completely inhibiting colony formation after incubation for 72 h at 37 °C under microaerophilic conditions with 10% CO 2.

Scanning electron microscopy (SEM) evaluation

The scanning electron microscopy (SEM) analysis was adapted from Chakraborti et al. (2013) and Fischer et al. (2013) and performed to identify morphological alterations in H. pylori ATCC 43504 cultures following treatment with silymarin. The bacteria were cultured in the presence of ½ MIC of silymarin or amoxicillin (antimicrobial standard), while untreated cultures were used as the positive control. The cultures were incubated for 24 h at 37 °C under microaerophilic conditions with 10% CO2. After incubation, the cultures were aliquoted and centrifuged at 800 rpm for 15 min at 18 °C. The supernatant was discarded, and 0.1 M cacodylate buffer containing 2.5% (v/v) glutaraldehyde was added to the cell pellets. The samples were incubated for 2 h at room temperature and subsequently overnight at 4 °C to promote cell fixation and attachment. The pellets were then washed three times with 0.1 M cacodylate buffer.

Subsequently, the samples were post-fixed with 1% (w/v) osmium tetroxide in 0.1 M cacodylate buffer containing 1.25% (w/v) potassium ferrocyanide and incubated for 30 min at room temperature in the dark. After post-fixation, the samples were washed once with 0.1 M cacodylate buffer followed by two washes with ultrapure water. Dehydration was performed through a graded ethanol series (30%, 50%, 70%, 90%, and 100% v/v) for 10 min each at room temperature. The dehydrated samples were mounted onto coverslips and subjected to critical point drying using CO2 (Tousimis Autosamdri® 815 Series A). After drying, the samples were sputter-coated with metal using a Desk V™ sputter coater (Denton Vacuum) and analyzed using a JEOL® JSM-6610LV scanning electron microscope operated at an accelerating voltage of 20 kV with a tungsten filament.

Cytotoxic assay and selectivity index

The MTT-tetrazolium assay was adapted from Mosmann (1983). AGS cells were trypsinized, counted using a Neubauer chamber, and seeded into 96-well tissue culture plates at a density of approximately 10 5 cells mL -1 in 100 μL of medium per well. The plates were incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2 to allow cell attachment and confluence. After incubation, the culture medium was removed, and the cells were treated with DMEM/F-12 medium containing different concentrations of silymarin (6.25–200 μg mL -1). Following 48 h of treatment, the MTT assay was performed by adding 100 μL of 2% MTT solution prepared in culture medium to each well. After 2 h of incubation, the medium was removed, and 100 μL of DMSO was added to solubilize the formed formazan crystals. Absorbance was measured using an iMark™ Bio-Rad microplate reader at 540 nm with a reference wavelength of 620 nm. Cisplatin (1.5625–50 μg mL -1) was used as the cytotoxic standard, while untreated AGS cells were used as the growth control. The same experimental procedure was performed using RAW 264.7 cells as a non-neoplastic cell model. The results were expressed as the half-maximal inhibitory concentration (IC50), defined as the calculated concentration required to reduce MTT reduction by 50%.

To determine the selectivity of silymarin toward neoplastic and non-neoplastic cells, the selectivity index (SI) was calculated according to Badisa et al. (2006). The SI was determined based on the ratio between the IC50 values obtained for non-neoplastic cells (RAW 264.7) and neoplastic cells (AGS), according to the following formula:

Measurement of cytokine (TNF-α and IL-6) and NO productions

Macrophages cells (RAW 264.7) were distributed into 96-well tissue-culture plates, 1 × 10 6 viable cells/mL for interleukin-6 (IL-6), 1 × 10 5 viable cells/mL for tumor necrosis factor-alpha (TNF-α) and nitric oxide (NO) assays. After incubation (37 °C/5%CO2) for cell adhesion, the supernatant was discarded, and the cells were stimulated with a standardized suspension of H. pylori ATCC 43504 and treated with different silymarin concentrations (0.156 μg mL -1 –50 μg mL -1). The plates were incubated overnight under the same conditions. The supernatants were collected for detection and quantification of cytokines TNF-α and IL-6 using the immunoenzymatic method (eBioscience, San Diego, CA, United States of America) and NO by the Griess method (Giustarini et al., 2008). For the cytokine assays, the data were expressed in cytokine concentrations (pg/mL) using a standard curve with purified cytokine. NO results were expressed in concentrations of NO 2 − (μM) using a standard curve with NaNO 2. Macrophages viability was performed to determine the appropriate non-toxic concentrations of compounds and after all immunomodulatory assays through the MTT method.

Adhesion assay

The anti-adhesion assay, based on fluorescence emission by fluorescein isothiocyanate (FITC)-labeled H. pylori, was adapted from Li et al. (2023). A suspension containing 10 6 AGS cells mL -1 was seeded into 96-well tissue culture plates with DMEM/F-12 medium and incubated at 37 °C in a 5% CO 2 atmosphere for 24 h until cell confluence was achieved. The medium was then removed, and the wells were washed with 200 µL of PBS buffer to remove any residual medium. The labeling of H. pylori cultures with FITC was performed by mixing together 0.075 mL of a 20 mg mL -1 FITC solution in DMSO for every mL of an H. pylori ATCC 43504 suspension in Brucella broth supplemented with 10% FBS, resulting in a final FITC concentration of 1.0 μg mL -1. The suspension was incubated for 30 min in the dark at 37 °C in a 10% CO 2 atmosphere. Subsequently, bacterial cells were collected by centrifugation (6000 rpm for 5 min at 18 °C), the supernatant was discarded, and the pellet was washed three times with PBS buffer to remove unbound FITC. The FITC-labeled H. pylori ATCC 43504 cells were then resuspended in Brucella broth supplemented with 10% FBS until an OD 620 of 0.5 was reached.

The FITC-labeled H. pylori ATCC 43504 suspension was aliquoted and incubated with three different concentrations of silymarin, equivalent to the MIC, ½ MIC, and 2× MIC, for 30 min in the dark at 37 °C in a 10% CO 2 atmosphere. Subsequently, 100 µL of the treated suspension was added to the wells containing AGS cells, and the plates were incubated for 90 min under the same conditions. After incubation, the supernatant was removed, and the wells were washed with 200 µL of PBS buffer to eliminate non-adherent bacterial cells. Subsequently, 100 µL of PBS buffer was added to each well, and fluorescence intensity was measured at excitation and emission wavelengths of 485 nm and 530 nm, respectively, using a Synergy™ R1 BioTek microplate reader. The silymarin-free bacterial suspension was used as the negative control. Adhesion inhibition was calculated according to the formula described above.

Prediction of physicochemical properties and drug-likenes

The eight main silymarin compounds, silybinin A and B, isosilybinin A and B, silychristin, isosilychristin, silydianin and taxifolin had their structures drawn in MarvinSketch (Marvin JS 23.16.0v) and then exported in appropriated file formats which were submitted to the platform ADMETlab 3.0 (Dong et al., 2018; Xiong et al., 2021) in order to obtain the prediction of their main physicochemical properties, logP and logS and to the platform SwissADME in order to predict their drug-likeness profile according to Lipinski’s rule of five (Usha et al., 2018).

Molecular docking

Crystallographic structures of the adhesins BabA (PDB code: 4ZH7), SabA (PDB code: 4O5J) and HopQ (PDB code: 6GBG) were obtained from Protein Data Bank (PDB) (Hage et al., 2015; Pang et al., 2014; Moonens et al., 2018). All structures contained their native receptor fragments or natural ligands bound to their respective binding sites, which were removed along with water residues and the proteins protonated to physiological pH of 7.4 in the PDB2PQR software (Jurrus et al., 2018). The eight main components of the silymarin complex were selected based on their report within the literature, which resulted in the selection of silybinin A and B, isosilybinin A and B, silychristin, isosilychristin, silydianin and taxifolin (Csupor et al., 2016; Lv et al., 2017; Zhang et al., 2024), which had their structures drawn in MarvinSketch (Marvin JS 23.16.0v) optimized to 3D configuration on the Avogadro software (1.2.0v) (Hanwell et al., 2012), where the compounds were protonated to physiological pH of 7.4, had their geometry optimized to the most stable conformer and had their force field built according to the MMFF94 model. Molecular docking was performed in GOLD software (version 2025.1.0, Cambridge Crystallographic Data Centre [CCDC], Cambridge, UK) (Liebeschuetz et al., 2012), using 10Å as spherical grid radius around the protein’s binding site, whose coordinates were defined according to the location of the native ligands or receptor fragments present in the original crystallographic structures. Protein structures were considered rigid, whereas ligands were allowed full rotational flexibility. For each ligand, 100 Genetic Algorithm (GA) runs were performed, resulting in a total of 800 generated poses. The ChemPLP scoring function was selected to rank the predicted binding poses, as it estimates pose quality based on empirically derived protein–ligand interaction terms, with higher scores indicating more favorable interactions and better fitting conformations (Sapundzhi et al., 2019). The resulting complexes were subsequently analyzed using Discovery Studio software to evaluate the intermolecular interactions established between the ligands and amino acid residues within the binding sites (Dassault Systèmes, 2023).

Due to the absence of co-crystallized small-molecule inhibitors for BabA, SabA, and HopQ, conventional docking validation through redocking procedures and RMSD calculations was not feasible. Therefore, an additional validation step was performed using a consensus docking strategy employing the independent docking engine AutoDock Vina v1.2.7 (Trott and Olson, 2009). For this procedure, ligands and proteins were prepared using AutoDockTools 1.5.7, where non-polar hydrogen atoms were merged and atomic charges were assigned according to Gasteiger parameters. Molecular docking was subsequently performed using a 20 Å edge cubic grid centered on the binding site, while protein structures were considered rigid and ligands were allowed full rotational flexibility. Binding affinities were expressed as predicted free energies of binding (kcal/mol) calculated using the built-in Vina scoring function, where lower values indicate more favorable protein–ligand interactions and greater thermodynamic stability of the predicted complexes (Eberhardt et al., 2021).

Statistical analysis

Results were expressed as the mean of three independent trials ±standard deviation. All tests were performed in triplicate and repeated at least 3 times. The dose-effect curve (concentration x absorbance) for CI 50 determination, the concentration of the agent that reduces spectrophotometric absorbance by 50%, was obtained by nonlinear regression analysis. The interpretation of the results was performed by one-way or two-way analysis of variance (ANOVA) with Tukey and Bonferroni post hoc test, where the level of significance was set at p ≤ 0.05. For data analysis, the statistical program GraphPad Prism five was used.

Determination of minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays

Silymarin expressed minimum inhibitory concentration of 128 μg mL -1 over H. pylori ATCC 43504, where growth inhibition was verified as being 90.46%, with minimum bactericidal concentration expressed in the same concentration. The silymarin MIC result is shown in Figure 1. Amoxicillin and metronidazole, used as antimicrobial standards, exhibited MIC values of 0.0625 and 256 μg mL -1, respectively, confirming the previously described antimicrobial susceptibility profile of H. pylori ATCC 43504, namely, susceptibility to amoxicillin and resistance to metronidazole.

Morphology cellular by scanning electron microscopy (SEM) evaluation

Figures 2A–C illustrate the morphological alterations observed in Helicobacter pylori ATCC 43504 following treatment with amoxicillin and silymarin, in comparison with the untreated growth control. Figure 2A represents the bacterial growth control and demonstrates the typical morphology of H. pylori, characterized by preserved spiral-shaped bacilli, smooth cell surfaces, and intact cellular organization, indicating normal bacterial viability and structural integrity. Figure 2B, corresponding to treatment with amoxicillin, shows pronounced morphological alterations, including severe deformation of bacterial cells, disruption of membrane integrity, and the presence of coccoid forms. These findings are consistent with the well-established bactericidal mechanism of amoxicillin, which inhibits bacterial cell wall synthesis, ultimately leading to extensive structural damage and bacterial death. Similarly, Figure 2C, representing treatment with silymarin, also demonstrates marked morphological changes in H. pylori cells compared with the untreated control. The treated bacteria exhibited surface irregularities cellular deformation, and the appearance of coccoid-like forms, suggesting impairment of membrane integrity and cellular organization.

When comparing the effects of amoxicillin and silymarin, both treatments were able to induce substantial morphological alterations in H. pylori, particularly regarding loss of the characteristic helical morphology and induction of coccoid forms. However, the alterations induced by amoxicillin appeared to be more pronounced and associated with extensive membrane disruption, whereas silymarin treatment predominantly resulted in surface deformation and structural disorganization.

Cytotoxic assay and selectivity

Silymarin exhibited IC 50 values of 46.27 ± 0.98 μg mL -1 against AGS cells and 98.18 ± 7.41 μg mL -1 against RAW 264.7 macrophages. Consequently, the calculated selectivity index was 2.12 over AGS cells, indicating that silymarin possesses a higher cytotoxic activity toward neoplastic cells than toward non-neoplastic cells. At the same time, the results demonstrated that the cytotoxic activity of silymarin is lower than that of cisplatin, which exhibited IC 50 values of 4.84 ± 0.41 μg mL -1 for AGS cells and 4.29 ± 0.25 μg mL -1 for RAW 264.7 macrophages. Importantly, the concentrations employed in the immunomodulatory assays (up to 50 μg mL -1) maintained cell viability above 90% in RAW 264.7 macrophages, confirming that the reductions observed in TNF-α, IL-6, and NO production were not attributable to nonspecific cytotoxic effects.

Measurement of cytokines TNF-α, IL-6 and NO production

Stimulated RAW 264.7 macrophages produced 80.25 ± 11.67 pg mL -1 of TNF-α in the presence of 50 μg mL -1 silymarin, representing a reduction of 80.82% ± 2.79% compared with the positive control, which produced 444.0 ± 36.06 pg mL -1. Silymarin inhibitory activity on TNF-α production is presented in Figure 3A. Regarding the effects on IL-6 production, stimulated RAW 264.7 macrophages produced 10.70 ± 8.01 pg mL -1 of IL-6 in the presence of 50 μg mL -1 silymarin, corresponding to a reduction of 76.24% ± 17.79% compared with the positive control, which produced 41.2 ± 5.42 pg mL -1 of IL-6 in the absence of silymarin. The inhibitory effect of silymarin on IL-6 production is shown in Figure 3B. Likewise, stimulated RAW 264.7 macrophages produced 0.24 ± 0.28 μM of nitric oxide in the presence of 50 μg mL -1 silymarin, corresponding to a reduction of 97.50% ± 2.19% compared with the positive control, which produced 9.61 ± 4.25 μM of NO in the absence of silymarin. The inhibitory effect of silymarin on nitric oxide production, expressed as nitrite concentration, is shown in Figure 4.

Adhesion assay

Silymarin demonstrated inhibitory activity against H. pylori ATCC 43504 adhesion to AGS gastric epithelial cells in a concentration-dependent manner. Notably, concentrations both below and above the minimum inhibitory concentration (MIC = 128 μg mL -1) were capable of significantly reducing bacterial attachment to host cells. The highest tested concentration, 256 μg mL -1 (corresponding to 2× MIC), exhibited the greatest anti-adhesive effect, inhibiting bacterial adhesion by 25.87% ± 3.50% compared with the untreated control group. The inhibitory effect of silymarin on bacterial adhesion is presented in Figure 5.

Prediction of physicochemical properties and drug-likeness

The predicted physicochemical properties exhibited similar profiles among the evaluated compounds, with logP values ranging from 0.85 for silydianin to 1.85 for silybinin B, and logS values ranging from −4.45 for isosilychristin to −3.33 for taxifolin. Overall, these findings indicate that the compounds display a mildly lipophilic character while maintaining satisfactory aqueous solubility, likely due to the presence of multiple hydroxyl groups within their chemical structures.

Regarding Lipinski’s rule of five, six of the eight evaluated compounds fulfilled all four parameters, whereas silychristin and isosilychristin satisfied three of the four criteria due to the presence of more than five hydroxyl groups. Nevertheless, as Lipinski’s rule allows a single parameter violation, all evaluated compounds were considered compliant with the rule of five, suggesting favorable drug-likeness and potential oral bioavailability. The complete prediction results are presented in Table 1.

Molecular docking

The eight major flavonolignan constituents of the silymarin complex (silybinin A and B, isosilybinin A and B, silychristin, isosilychristin, silydianin and taxifolin) were individually docked into the active binding pockets of the investigated Helicobacter pylori adhesins, demonstrating favorable binding affinities toward these virulence-associated targets as predicted by the GOLD software and successfully validated by the AutoDock Vina software. The results of all the compounds and predicted binding poses and the principal molecular interactions established by the highest-scoring compounds for each target are illustrated in Table 2 and Figures 6A–C, respectively.

For BabA, the calculated fitness scores in the GOLD software ranged from 47.58 for silydianin to 57.75 for isosilybinin A, while the predicted binding affinity on AutoDock Vina for the same compounds was −4.972 kcal/mol and −6.341 kcal/mol, respectively. Among the evaluated compounds, isosilybinin A exhibited the highest predicted affinity, establishing hydrogen bond interactions with the key active-site residues CYS189 and SER244, in addition to hydrophobic interactions with GLY191 and SER234 within the binding cavity (Figure 6A).

Regarding SabA (Figure 6B), the calculated fitness scores in the GOLD software varied from 50.48 for taxifolin to 74.79 for isosilychristin, which displayed the highest binding affinity among all evaluated ligands–target complexes. At the same time, the predicted binding affinity on AutoDock Vina ranged from −6.867 kcal/mol, for taxifolin, and −7.764 kcal/mol, for isosilychristin. Isosilychristin established strong hydrogen bond interactions with the active residues LYS152 and GLN159, indicating favorable stabilization within the SabA binding site.

For HopQ, the obtained fitness scores in the GOLD software ranged from 47.95 for silydianin to 66.42 for silychristin while the predicted binding affinity on AutoDock Vina ranged from −6.800 kcal/mol to −7.753 kcal/mol, for the same compounds, respectively. The highest-scoring compound, silychristin, established weak hydrogen bond interactions with the active residue SER135, while also forming hydrophobic interactions with residues ILE130 and ILE242 located within the binding pocket (Figure 6C). Although fewer polar interactions were observed, the hydrophobic contacts may contribute to the stabilization of the ligand within the active site. Collectively, the molecular docking analyses demonstrated that the evaluated silymarin-derived compounds possess favorable interaction profiles with important H. pylori adhesins, supporting their potential role as anti-adhesive agents capable of impairing bacterial colonization of the gastric epithelium.

The antimicrobial activity of silymarin against Helicobacter pylori ATCC 43504, evidenced by a MIC of 128 μg mL -1 with 90.46% growth inhibition, is consistent with earlier reports on the anti- H. pylori potential of Silybum marianum constituents. Silybinin, the major bioactive component of the silymarin complex, has previously demonstrated anti- H. pylori activity with a MIC of 256 μg mL -1 against the same ATCC 43504 strain, also promoting notable morphological changes in the bacterial cell wall, including blebs and clusters, suggesting interaction with penicillin-binding protein (PBP) subunits (Bittencourt et al., 2020). The lower MIC value obtained in the present study for the crude silymarin mixture may reflect an additive or synergistic contribution of the multiple flavonolignan constituents - including silychristin, silydianin, and isosilybinin - that coexist within the extract. In this regard, studies on the antimicrobial modulatory activity of silymarin and silybinin have highlighted their promising potential when combined with antibacterial drugs, suggesting that the complexity of the mixture may underpin its biological effects (Rakelly de Oliveira et al., 2015).

The morphological analysis by SEM provided important ultrastructural insights into the bactericidal mechanisms of both amoxicillin and silymarin. The untreated control cells displayed the archetypal helical morphology of H. pylori, consistent with standard descriptions of the viable, metabolically active form of the bacterium (Krzyżek and Gościniak, 2018). The morphological alterations induced by silymarin, surface irregularities, cellular deformation, and the emergence of coccoid-like forms, are consistent with a membrane-targeted mechanism of action. Silymarin has been reported to increase membrane permeability by disturbing the membrane structure, thereby allowing free access to molecules across the membrane barrier, as demonstrated through propidium iodide assays and membrane vesicle models (Yun and Lee, 2017). This membrane-disrupting activity likely underlies the surface deformation and structural disorganization observed in silymarin-treated H. pylori cells in the current study. Furthermore, the antibacterial activity of silymarin’s flavonolignan constituents has been attributed to interference with protein and RNA synthesis, as well as alterations in cell membrane permeability that prevent the activity of enzymes required for bacterial energy production (Aldayel, 2023).

The qualitative comparison between the effects of amoxicillin and silymarin on H. pylori ATCC 43504 morphology mirrors observations previously reported for silybinin, the major constituent of silymarin. Silybinin promoted important morphological changes in the bacterial cell wall, including blebs and clusters, suggesting interaction with PBP subunits (Bittencourt et al., 2020). This molecular docking-based hypothesis aligns with the surface deformation pattern observed in the present silymarin-treated cells and warrants further investigation through in silico and molecular target studies to fully characterize the mechanism underpinning the anti- H. pylori activity of silymarin.

The cytotoxicity assay was an essential preliminary step to define the concentration range of silymarin that could be safely employed in the subsequent immunomodulatory assays involving TNF-α, IL-6, and nitric oxide (NO) production. Silymarin exhibited an IC 50 of 46.27 ± 0.98 μg mL -1 in AGS cells and 98.18 ± 7.41 μg mL -1 in RAW 264.7 macrophages, resulting in a selectivity index (SI) of 2.12. This SI indicates that silymarin was approximately two-fold more cytotoxic toward neoplastic AGS cells than toward non-neoplastic RAW 264.7 cells, demonstrating selectivity for the gastric cancer cell line. In contrast, cisplatin exhibited similar IC 50 values in AGS and RAW 264.7 cells, indicating limited selectivity between the 2 cell types. Importantly, the reduced cytotoxicity of silymarin toward RAW 264.7 macrophages support the use of subcytotoxic concentrations in the subsequent immunomodulatory assays. Therefore, these findings were fundamental to establish biologically relevant concentrations capable of evaluating the anti-inflammatory and immunomodulatory effects of silymarin without interference caused by excessive cell death, ensuring greater reliability in the interpretation of TNF-α, IL-6, and NO modulation results.

Unresolved H. pylori infection drives a sustained pro-inflammatory state in the gastric mucosa, characterized by the upregulation of key mediators including TNF-α, IL-6, and nitric oxide (NO), which collectively perpetuate tissue damage and may contribute to carcinogenesis if the infection persists as gastric epithelial cells infected with H. pylori actively secrete these cytokines and chemokines to promote local inflammation and modulate the immune response (Robinson et al., 2015). The present study demonstrates that silymarin, at a concentration of 50 μg mL -1, significantly attenuated the production of all three inflammatory mediators in H. pylori -stimulated RAW 264.7 macrophages, supporting an immunomodulatory role for the silymarin complex that extends beyond its direct antimicrobial activity.

The IL-6 production was reduced from 41.2 ± 5.42 pg mL -1 in the stimulated control to 10.70 ± 8.01 pg mL -1 in the treated group, corresponding to an inhibition of 76.24% ± 17.79%. IL-6 is a pleiotropic cytokine that plays a major role in coordinating both innate and adaptive immune responses: it triggers acute-phase protein synthesis in hepatocytes, promotes the differentiation of naive CD4 + T cells, stimulates immunoglobulin synthesis through B-cell activation, and drives cytotoxic T-cell differentiation (Tanaka et al., 2014). In the context of H. pylori infection, IL-6 assumes particular pathological relevance. Tandem meta-analyses have shown that serum IL-6 levels are significantly elevated upon H. pylori infection, with the magnitude of IL-6 induction correlating with regional gastric cancer incidence (Yu et al., 2023) Furthermore, H. pylori have been shown to establish an IL-6-driven autocrine and paracrine positive feedback loop between macrophages and gastric epithelial cells that promotes carcinogenesis (Reyes, 2023), while IL-6 secreted in the tumor microenvironment activates the JAK2/STAT3 oncogenic signaling pathway, contributing to epithelial-mesenchymal transition and gastric cancer metastasis (Vainer et al., 2018). The capacity of silymarin to suppress IL-6 production in H. pylori -stimulated macrophages therefore suggests a potential role not only in attenuating acute gastric inflammation, but also in disrupting pro-tumorigenic signaling circuits linked to chronic infection.

TNF-α levels were reduced from 444.0 ± 36.06 pg mL -1 in the stimulated control to 80.25 ± 11.67 pg mL -1 in the presence of silymarin, representing an inhibition of 80.82% ± 2.79%. This result holds clinical relevance given that elevated gastric mucosal production of TNF-α has been consistently documented in patients with H. pylori -associated gastritis, with concentrations being significantly higher in cases of active disease accompanied by neutrophilic epithelial infiltration (Brito et al., 2019). TNF-α also plays a pivotal role in activating the NF-κB signaling pathway, which further amplifies the inflammatory cascade and has been implicated in gastric carcinogenesis. The substantial reduction in TNF-α production observed in the present study is consistent with the established mechanism by which silymarin and its constituent silybinin suppress NF-κB/Rel transcription factor activation in macrophages, thereby downregulating the expression of multiple pro-inflammatory genes (Manna et al., 1999; Raina et al., 2011).

Nitric oxide production had an inhibition of 97.50% ± 2.19% in the silymarin-treated group. The inhibitory effect of silymarin on NO production observed here aligns with prior mechanistic evidence demonstrating that silymarin suppresses iNOS mRNA and protein expression in LPS-stimulated RAW 264.7 macrophages through blockade of NF-κB/Rel transcriptional activity (Kim et al., 2015). During H. pylori infection, the inducible nitric oxide synthase (iNOS) enzyme is expressed by neutrophils and epithelial cells recruited to the gastric mucosa, leading to sustained NO production and the formation of reactive nitrogen species such as peroxynitrite, which cause oxidative DNA damage and mucosal injury (Butcher et al., 2017).

Taken together, the immunomodulatory data presented demonstrate that silymarin simultaneously attenuates three major pro-inflammatory mediators involved in the pathogenesis of H. pylori -associated gastric disease. Although the inhibitory values observed for silymarin—particularly for TNF-α (80.82%) and IL-6 (76.24%) — were moderately lower than those previously reported for pure silybinin at the same concentration (100% and 56.83%, respectively; Bittencourt et al., 2020), the silymarin complex demonstrated superior suppression of NO production compared to silybinin alone (97.50% vs. 73.33%), suggesting complementary and potentially additive contributions among its constituent flavonolignans. These findings reinforce the multi-target anti-inflammatory profile of silymarin and support its further investigation as an adjunct candidate in the management of H. pylori -associated gastric pathology.

The silymarin complex could reduce the adhesion of H. pylori ATCC 43504 to AGS gastric cells. Although the anti-adhesive activity observed for silymarin was moderate (25.87% ± 3.50%), similar resulsts against H. pylori have also been reported for other natural compounds, including epigallocatechin gallate from green tea (Lee et al., 2004), cranberry proanthocyanidins (Gotteland et al., 2008), and garlic-derived organosulfur compounds (Takeuchi et al., 2014), which were shown to interfere with bacterial attachment to gastric epithelial cells and reduce colonization capacity. This inhibitory effect may be associated with the ability of silymarin flavonolignans to interact with bacterial adhesins, particularly BabA, SabA, and HopQ, as suggested by the molecular docking results. Since bacterial adhesion is an essential step for gastric colonization and persistence, the reduction in cell attachment observed in this study suggests that silymarin may impair the establishment of infection at its early stages. Furthermore, the anti-adhesive effect of silymarin should be interpreted in conjunction with its antibacterial and anti-inflammatory activities demonstrated in the present study, supporting a multitarget therapeutic profile rather than a selective anti-adhesion mechanism.

The potential inhibitory effect of silymarin on H. pylori adhesins is supported by the molecular docking results obtained for its individual flavonolignan constituents, which demonstrated that all eight tested compounds of the silymarin complex were able to establish favorable interactions with amino acid residues located within the binding sites of the three evaluated H. pylori adhesins. The consensus docking analysis further supported these findings, as compounds with higher fitness scores obtained using GOLD generally exhibited more favorable binding affinities in the AutoDock Vina validation. However, it is important to emphasize that the docking analyses were performed with isolated flavonolignan constituents rather than with the silymarin complex itself and, therefore, do not account for potential synergistic, additive, antagonistic, pharmacokinetic, or matrix-dependent interactions that may occur within the complete phytocomplex. Consequently, these results should be interpreted as mechanistic support for possible ligand-target interactions rather than as direct evidence explaining the anti-adhesive activity experimentally observed for the whole silymarin preparation.

BabA and SabA primarily recognize the human Le b blood antigen and human sialyl-Lewis X antigen, respectively, both of which are oligosaccharides expressed on the surface of gastric epithelial cells (Alipour, 2021). The binding of these adhesins to their natural ligands depends mainly on the establishment of hydrogen bonds with hydroxyl groups present in these host antigens, and since the flavonolignans present in the silymarin complex also contain hydroxyl groups in their chemical structures, they were able to establish similar interactions with key amino acid residues involved in adhesin activity, including SER244 in BabA and GLN159 in SabA. These residues are highly conserved among different H. pylori strains, and their substitution has been associated with a marked reduction in adhesin function (Hage et al., 2015; Pang et al., 2014).

In contrast, HopQ interacts predominantly with glycoproteins belonging to the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. This characteristic explains the broader binding pocket observed for HopQ when compared to BabA and SabA, as well as the involvement of a larger number of amino acid residues in ligand recognition through multiple interaction mechanisms beyond hydrogen bonding alone (Moonens et al., 2018).

The inhibition of H. pylori adhesion to gastric epithelial cells may represent an important therapeutic strategy against infection, since bacterial attachment is a critical early step in colonization and persistence within the gastric mucosa (Amieva and Peek, 2016). Furthermore, several H. pylori adhesins are known to modulate the expression of additional virulence factors, including vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), both of which are strongly associated with morphological alterations and damage to gastric epithelial cells. In particular, successful BabA-mediated adhesion has been reported to enhance the expression and activity of these virulence determinants (Šterbenc et al., 2019). Therefore, inhibition of adhesin binding sites may not only impair bacterial attachment but also indirectly regulate other pathogenic mechanisms associated with H. pylori infection (Peek, 2008).

The in silico prediction of the physicochemical properties of the eight major compounds present in the silymarin complex demonstrated favorable pharmacokinetic characteristics. All compounds exhibited suitable logP and logS values ranging from which are within the range commonly associated with compounds capable of crossing biological membranes without the assistance of transmembrane transporters (Jorgensen and Duffy, 2002). In addition, all evaluated compounds complied with Lipinski’s Rule of Five, which describes important parameters associated with good oral bioavailability. These findings are consistent with the long-standing oral use of silymarin as a phytotherapeutic agent, particularly for the treatment of liver disorders due to its well-established antioxidant properties (Elateeq et al., 2020; Koltai and Fliegel, 2022).

Overall, the commercially standardized silymarin complex demonstrated significant antibacterial and anti-inflammatory activities against H. pylori ATCC 43504. In addition to inhibiting bacterial growth and promoting morphological alterations in the bacterial cell wall, silymarin was also capable of reducing the adhesion of H. pylori ATCC 43504 to AGS gastric cells, suggesting interference with important adhesins involved in bacterial colonization. Molecular docking analyses performed with the major flavonolignan constituents naturally present in the silymarin complex provided complementary mechanistic insights, suggesting potential interactions with the active sites of adhesins such as BabA, SabA, and HopQ. Since bacterial attachment is a critical step for the establishment and persistence of H. pylori infection, the inhibition of cell adhesion observed in this study reinforces the potential of silymarin as a multifunctional therapeutic candidate capable of simultaneously modulating bacterial growth, virulence, and inflammation.