2.1 Antioxidant Activity, Iron Chelating Properties, and Flavonoid Content of A. serpyllifolia Extract

The IC₅₀ value of the DPPH radical scavenging activity of A. serpyllifolia extract was determined to be 355.54 ± 20.62 μg mL⁻¹, and the IC₅₀ value for iron chelation activity was 5.30 ± 4.44 mg mL⁻¹. These results indicate that the extract exhibits good antioxidant activity and strong iron chelation activity. Additionally, the total flavonoid content of A. serpyllifolia extract was measured to be 75.15 ± 2.73 mg quercetin equivalent (QE) g⁻¹ extract, total phenolic content of extract was measured to be 150.83 ± 11.24 mg gallic acid equivalent (GAE) g⁻¹ extract demonstrating that the extract has a significant flavonoid content (Table 1).

Oxidative stress can result from an imbalance between antioxidants and free radicals in the body,^[16] and plants contain antioxidants that may play a potential role in preventing oxidative damage.^[17] The antioxidant effect of A. serpyllifolia extracts was evaluated using the DPPH and iron chelation assays. The DPPH assay yielded a value of 355.54 ± 20.62 μg mL⁻¹, indicating that the methanol extract exhibits good antioxidant activity. The iron chelation activity was determined to be 2.01 ± 0.36 mg mL⁻¹, demonstrating a strong effect. The iron chelation scavenging effect has the potential to reduce oxidative stress by binding free iron ions.^[18] The DPPH and iron chelation activities may be attributed to the presence of specific chemical compounds such as glycerin,^[19] allyl isothiocyanate,^[20] guanosine,^[21] and levoglucosan,^[22] which exhibit free radical activity. The chemical compounds found in A. serpyllifolia extracts suggest that they may act as free radical scavengers and metal ion chelators, contributing to their antioxidant properties. Natural antioxidants are considered safer compared to synthetic ones.^[23] Variations in antioxidant activity have been observed among Arenaria plant extracts. This difference may be attributed to the chemical compounds present in the plant. These compounds can exhibit synergistic effects, leading to different antioxidant behaviors. Variability in chemical composition may influence antioxidant activity. Antioxidant studies have been conducted on A. serpyllifolia extracts; however, research on the Arenaria genus remains limited. In the study conducted by Kazemi and Firouzeh (2022),^[24] the IC₅₀ value for the antioxidant activity of Arenaria hispanica extract was determined to be 0.69 ± 0.03 mg mL⁻¹, whereas in our study, the antioxidant activity of A. serpyllifolia extract was reported to be IC₅₀ value 355.54 ± 20.62 μg mL⁻¹, indicating a higher antioxidant activity of A. serpyllifolia. The IC₅₀ value of the DPPH radical scavenging activity of Arenaria montana methanol extract was determined to be 0.90 ± 0.01 mg mL⁻¹, as reported by Pereira et al. (2014),^[25] while the antioxidant activity of A. serpyllifolia extract was found to be 355.54 ± 20.62 μg mL⁻¹. This result indicates that A. serpyllifolia exhibits higher antioxidant activity. The differences in antioxidant activity can be attributed to environmental factors, the plant species, climatic conditions, the use of different plant parts, extraction methods, and the applied extraction conditions.^[4]

2.2 Antimicrobial, Antibiofilm, and QS Activity of A. serpyllifolia Extract

The MIC, minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) values of A. serpyllifolia extract against bacterial and fungal isolates are presented in Table 2. The MIC value of the extract against C. violaceum bacterial isolates was found to be <5 mg mL⁻¹. This indicates that the extract exhibits good antimicrobial activity against the C. violaceum bacterial isolate.

A. serpyllifolia extract inhibited biofilm formation by 82.52 ± 2.2% at the MIC dose of 5 mg mL⁻¹. The extract showed the best effect at the sub-MIC dose of 4 mg mL⁻¹, inhibiting biofilm formation by 81.32 ± 2.4%. These results indicate that A. serpyllifolia extract exhibits strong antimicrobial biofilm activity (Table 3).

The potential QS inhibition of plant extracts was evaluated using C. violaceum CV12472 bacteria, which produce purple violacein during growth (Table 4). The MIC, defined as the lowest concentration at which visible bacterial growth was inhibited by the extracts, was determined, and violacein inhibition and QS assays were performed at MIC and sub-MIC levels. The MIC value for the methanol extract against C. violaceum CV12472 was determined to be 5 mg mL⁻¹. At the MIC level, the ethanol extracts inhibited violacein production by 90.76 ± 0.3%. At sub-MIC levels, the violacein production inhibition percentages of the methanol extract were determined as 91.60 ± 0.1%, 91.25 ± 0.0%, 90.83 ± 0.6%, and 80.88 ± 1.9%, respectively (Figure 1).

The effect of A. serpyllifolia extracts on QS-mediated processes, such as the inhibition of violacein production in C. violaceum CV12472, has been investigated. This plant not only kills bacteria but can also reduce their severity by disrupting QS networks. Additionally, it may eliminate bacterial resistance. Antibiotics based solely on bacterial growth inhibition or bactericidal effects can lead to health problems due to the development of resistant strains.^[26-28] Since the methanol extract inhibited violacein formation and QS, this activity can be attributed to some of the identified chemical compounds, such as glycerin,^[29] allyl isothiocyanate,^[30] guanosine,^[31] levoglucosan,^[32] and 2,2-dimethoxybutane;^[33] these compounds are supported by our in silico models. A. serpyllifolia’s antimicrobial activity, antibiofilm activity, and anti-QS effects have been reported for the first time here. The extract of A. serpyllifolia shows strong antimicrobial activity (MIC, 5 mg mL⁻¹) against C. violaceum, compared to other bacterial isolates. Therefore, A. serpyllifolia extracts can be used as a potential solution to reduce microbial resistance and the severity of infections.^[34] The extract inhibited violacein production in C. violaceum (sub-MIC dose; 1 mg mL⁻¹; 80.88 ± 1.9). During bacterial communication, signal molecules can diffuse within colonies, which can regulate microorganism motility. Swarming motility plays a role in QS-mediated biofilm formation.^[26, 35] When microorganisms face challenges, such as antibiotics, they form biofilms to survive. The A. serpyllifolia extract inhibited biofilm activity (sub-MIC dose; 4 mg mL⁻¹), showing good antibiofilm activity against C. violaceum bacterial isolates. Clinopodium nepeta dichloromethane (DCM) extract inhibited violacein production in C. violaceum CV12472 at a MIC concentration of 0.5 mg mL⁻¹ (100.0 ± 0.0%), while the ethyl acetate (AcOEt) extract exhibited inhibition at a MIC concentration of 1 mg mL⁻¹ (18.9 ± 4.1%). At MIC/2, the percentage inhibition of violacein production was 19.2 ± 1.1% and 4.5 ± 0.3% for DCM and AcOEt extracts, respectively.^[4] In our study, the MIC value of A. serpyllifolia extract was determined as 5 mg mL⁻¹, showing an inhibition of 90.76 ± 0.3%. Dose-dependent inhibition rates at sub-MIC concentrations (1–4 mg mL⁻¹) were recorded as 91.60 ± 0.1% and 80.88 ± 1.9%, respectively. Both studies demonstrated the ability to disrupt bacterial QS. In our study, A. serpyllifolia extracts, like the extracts of Searsia leptodictya, Searsia lancea, Searsia batophylla, Searsia pendulina, Bauhinia galpinii, and Bauhinia bowkeri, exhibited antibacterial and antibiofilm activity and were found to modulate the QS mechanism by inhibiting violacein production.^[36]

2.3 Chemical Profiling of A. serpyllifolia via GC-MS Analysis

GC-MS analysis of the methanolic extract of A. serpyllifolia revealed the presence of several semipolar phytochemical constituents (Table 5). A total of seven major compounds were identified, each eluting at distinct retention times. The predominant compound was allyl isothiocyanate, accounting for 23.59% of the total peak area, followed by 2,2-dimethoxybutane at 22.28%. Notable quantities of levoglucosan (18.71%) and methyl butyrate (12.94%) were also detected. Additionally, guanosine (7.58%), glycerin (6.75%), and 2,6,7-trimethyldecane (3.59%) were present in relatively lower proportions. These findings suggest that the methanolic extract of A. serpyllifolia contains a diverse array of semipolar compounds, reflecting its rich phytochemical profile.

Although studies have been conducted on A. serpyllifolia extracts, the chemical composition of this plant has not yet been fully elucidated. The results of the phenolic chemical composition here show that A. serpyllifolia methanol extracts contain various chemical compounds. Using GC-MS analysis, seven chemical compounds were identified, and their quantities were determined.^[37] Careful selection of extraction methods and solvents ensures the maximum yield of target compounds found in plants and helps avoid chemical or biological alterations. In this study, methyl butyrate, glycerin, 2,2-dimethoxybutane, allyl isothiocyanate, 2,6,7-trimethyldecane, guanosine, or levoglucosan were detected in A. serpyllifolia samples collected from Turkey. In one study, 10 compounds were isolated from the extract of Arenaria kansuensis.^[38] The chemical composition of Arenaria montana was found to consist of hydrophilic (sugars, organic acids, and phenolic compounds) and lipophilic (fatty acids and tocopherols) components.^[25] The chemical components of A. kansuensis were identified using high-performance liquid chromatography (HPLC) analysis.^[39] Various chemical compounds of the Arenaria genus have been identified using different analytical methods. The compounds identified by GC-MS in A. serpyllifolia represent an important study in this field.

2.4 Binding Affinity-Based Evaluation of Ligand–target Interactions

Table 6 presents the calculated binding interaction parameters of the selected compounds with the target protein 3QP1. The table includes binding energy values (in kcal mol⁻¹), ligand efficiency (LE), fit quality (FQ), estimated inhibition constants (Ki, in μM), and pIC₅₀ values for each compound .

Among the tested molecules, levoglucosan demonstrated the strongest binding affinity with a binding energy of –6.8 kcal mol⁻¹, an estimated Ki of 10.29 μM, and a pIC₅₀ value of 4.86 (Figure 2). Similarly, guanosine exhibited notable interaction with the target, showing a binding energy of –6.2 kcal mol⁻¹, Ki of 28.35 μM, and a pIC₅₀ of 4.43, indicating favorable inhibitory potential.

In contrast, compounds such as 2,2-dimethoxybutane, glycerin, and methyl butyrate showed significantly weaker binding affinities, with high Ki values of 983.69, 830.83, and 422.80 μM, respectively. These findings suggest that these small and more polar molecules are less effective in interacting with the active site of 3QP1.

In terms of LE, allyl isothiocyanate exhibited the highest LE value (0.850), suggesting that, despite its smaller molecular size, it binds relatively efficiently to the target site. However, its FQ was relatively low (0.229), indicating potential room for structural optimization.

Overall, levoglucosan and guanosine emerged as the most promising candidates due to their strong binding affinities and favorable efficiency metrics, suggesting their potential for further investigation in drug design targeting 3QP1.

2.5 Evaluation of Binding Stability through MD Simulations

To investigate the structural stability and binding characteristics of various small molecules to the ligand-binding domain (LBD) of CviR (PDB ID: 3QP1), MD simulations spanning 100 ns were performed for the protein complexed with seven distinct ligands: 2,2-dimethoxybutane, allyl isothiocyanate, levoglucosan, methyl butyrate, guanosine, glycerin, and 2,6,7-trimethyldecane. The dynamic behavior and interaction energies were analyzed to assess system stability and predict binding affinities.

Evaluation of the protein’s structural integrity commenced with the analysis of the root mean square deviation (RMSD) of the backbone Cα atoms relative to the initial minimized structure (Figure 3A). Across all simulated systems, the protein backbone RMSD values demonstrated convergence after an initial equilibration phase of ≈10–20 ns. Subsequently, the RMSD values remained stable, generally fluctuating within a range of 0.15–0.25 nm, with transient peaks occasionally reaching ≈0.3 nm. This indicates that the overall fold of the CviR LBD was well-maintained throughout the 100 ns simulation period in the presence of all tested ligands, without significant conformational rearrangements. Further supporting global structural stability, the radius of gyration (R_g) of the protein was monitored over time (Figure 3D). The R_g values for all systems fluctuated around a stable average, predominantly within the 1.54–1.64 nm range, confirming that the protein maintained its overall compactness and did not undergo significant unfolding or expansion events upon ligand binding.

The stability of the ligands within the CviR binding pocket was assessed by calculating the RMSD of the ligand heavy atoms after aligning the protein backbone (Figure 3B). This analysis revealed marked differences in ligand behavior. Guanosine (brown), 2,6,7-trimethyldecane (blue), 2,2-dimethoxybutane (black), levoglucosan (yellow), and methyl butyrate (green) exhibited relatively stable binding poses, with RMSD values generally stabilizing below 1.0 nm after initial adjustments. This suggests that these ligands maintained consistent interactions within the binding site. In striking contrast, allyl isothiocyanate (red) displayed exceptionally high and erratic RMSD values, frequently fluctuating between 2 and 6 nm and exhibiting peaks beyond this range. This pronounced instability indicates that allyl isothiocyanate failed to establish a stable binding mode and likely diffused away from, or moved extensively within, the putative binding site during the simulation. Glycerin (pink) also showed considerable mobility, with RMSD values fluctuating significantly, often exceeding 1 nm and reaching peaks near 4 nm, suggesting a less stable or transient interaction compared to the more stably bound ligands.

To probe the local dynamics and flexibility of the protein structure, the root mean square fluctuation (RMSF) was calculated for each residue over the simulation trajectory (Figure 3C). The RMSF profiles were broadly similar across all complexes, indicating that the binding of these diverse ligands did not induce major alterations in the protein’s intrinsic flexibility pattern. As anticipated, regions corresponding to loops (e.g., residues ≈40–50, ≈90–110, ≈120–140, and ≈150–160) and the N- and C-termini exhibited higher RMSF values, signifying greater conformational freedom. Conversely, residues located within secondary structure elements displayed lower RMSF values, reflecting their comparative rigidity. While minor variations in peak heights existed between different complexes, no ligand caused a distinct, widespread change in the flexibility profile, suggesting interactions were largely confined to the binding pocket without inducing major allosteric dynamic shifts captured at this timescale.

Finally, the binding free energies (ΔG_bind) and their constituent energy components were estimated using the molecular mechanics/generalized born surface area (MM/GBSA) method on trajectory snapshots (Table 7 and 8). The calculations predicted significant differences in binding affinity. Guanosine demonstrated the most favorable binding free energy (ΔG_bind = −27.06 ± 2.91 kcal mol⁻¹), followed by 2,6,7-trimethyldecane (ΔG_bind = −22.68 ± 2.41 kcal mol⁻¹) and 2,2-dimethoxybutane (ΔG_bind = −20.03 ± 1.53 kcal mol⁻¹). Methyl butyrate exhibited moderate predicted affinity (ΔG_bind = −10.89 ± 3.93 kcal mol⁻¹). In contrast, levoglucosan (ΔG_bind = +5.2 ± 4.74 kcal mol⁻¹) and glycerin (ΔG_bind = +0.99 ± 0.4 kcal mol⁻¹) showed unfavorable positive or near-zero binding energies, suggesting weak or nonspecific interactions. Strikingly, allyl isothiocyanate yielded a highly unfavorable calculated binding energy (ΔG_bind = +65.89 ± 13.54 kcal mol⁻¹), which is fully consistent with its high RMSD values and observed instability within the binding site.

Decomposition of the binding energy revealed the driving forces for the interactions. The strong affinity of guanosine was attributed to substantial favorable contributions from both van der Waals (ΔE_vdw = −32.6 kcal mol⁻¹) and electrostatic (E_elec = −64.11 kcal mol⁻¹) interactions, which compensated for the significant unfavorable polar solvation energy (E_gb = 62.67 kcal mol⁻¹). For the hydrophobic ligand 2,6,7-trimethyldecane, binding was predominantly driven by favorable van der Waals interactions (ΔE_vdw = −32.72 kcal mol⁻¹) and favorable nonpolar solvation contributions (E_surf = −4.6 kcal mol⁻¹), with negligible electrostatic involvement. 2,2-dimethoxybutane binding involved favorable van der Waals (ΔE_vdw = −21.04 kcal mol⁻¹) and moderate electrostatic (E_elec = −12.5 kcal mol⁻¹) terms. The unfavorable binding of levoglucosan resulted from a large polar desolvation penalty (E_gb = 30.52 kcal mol⁻¹) outweighing its favorable van der Waals and electrostatic terms. The extremely poor calculated affinity for allyl isothiocyanate stemmed from minimal favorable direct interaction terms (ΔE_vdw and E_elec) coupled with potentially unfavorable internal strain or repulsive interactions averaged over its unstable trajectory, as reflected also in its positive E_int value. These energetic analyses, combined with the structural stability data, provide a comprehensive view of the differential interactions between the CviR LBD and the tested ligands.

In our study, an in silico molecular docking analysis was performed to investigate the binding potential of the chemical constituents of A. serpyllifolia extract to the CviR receptor in detail. The calculated binding energy data revealed that the compounds present in the extract could inhibit the CviR receptor with different binding affinities. Levoglucosan exhibited the highest affinity with a binding energy of −6.8 kcal mol⁻¹, while guanosine followed closely with a binding energy of −6.2 kcal mol⁻¹. These findings suggest that both compounds can strongly bind to the CviR receptor and may contribute to possible inhibition mechanisms. Allyl isothiocyanate showed a moderate affinity with a binding energy of −5.1 kcal mol⁻¹, indicating its potential interaction with the receptor. Other compounds, including 2,2-dimethoxybutane, glycerin, methyl butyrate, and 2,6,7-trimethyldecane, exhibited binding energies ranging from −4.1 to −4.7 kcal mol⁻¹, indicating lower affinities for the receptor. This suggests that their impact on CviR inhibition may be more limited compared to other identified compounds. Overall, the results indicate that certain compounds in A. serpyllifolia extract may target QS mechanisms by binding to the CviR receptor and acting as antagonists. In this context, isolating active compounds from the extract could lead to the development of novel antimicrobial agents with QS inhibition potential. By disrupting bacterial communication systems, QS inhibition can help prevent biofilm formation, virulence factor expression, and antibiotic resistance. Therefore, further biological and pharmacological investigations of A. serpyllifolia extract and its chemical constituents could be of great importance for potential therapeutic applications. In a study, it was shown that the components of F. suspense could inhibit CviR, particularly with Pinoresinol’s binding energy of (−26.02 kcal mol⁻¹) and its binding affinity. This result supports the potential of F. suspense components to target QS pathways and disrupt bacterial communication and is consistent with the findings of our study.^[40] Blocking the QS mechanism could be an important approach in treating various infections caused by this organism. Molecular docking analyses confirmed the anti-QS potential of bioactive compounds from Cladosporium spp., with docking scores ranging between –5.2 and –9.5 kcal mol⁻¹, supporting their ability to bind to CviR. The inhibition of the QS mechanism could aid in the treatment of various infections caused by C. violaceum. Molecular docking results have confirmed the anti-QS potential of chemical compounds from Cladosporium spp. by binding to CviR with docking scores ranging from –5.2 to –9.5 kcal mol⁻¹. The binding energies of A. serpyllifolia chemical components to the CviR receptor range from –4.1 to –6.8 kcal mol⁻¹, which is consistent with the results of the study by Murali et al. (2023).^[41]