Bioactive screening and molecular docking of phenolics from Stellaria pallida

Abstract

Stellaria pallida L. (Dumort.) is a species that belongs to the Caryophyllaceae family and has a broad range of secondary metabolites and therapeutic potential. In this study, the aerial parts of Stellaria pallida L. (S. pallida) were extracted by various organic solvents, viz. Chloroform, ethyl acetate, ethanol, and methanol, among others, were analyzed by GC/MS and HPLC-DAD to determine their chemical composition and antimicrobial properties. In addition, an in silico molecular docking analysis by AutoDock Vina was performed to evaluate the potential interactions between the identified phytochemical compounds and selected antimicrobial target proteins. The methanol extract had the maximum total phenolic (58±1.55 mg GAE/g), and the ethanol extract recorded the maximum total values of flavonoid (27.22±0.88 mg QE/g). Phytol, stigmasterol, Neophytadiene, rutin, chlorogenic acid, ferulic acid, apigenin-7-glucoside, and epicatechin were the major chemical compounds detected in the S. pallida plant. Additionally, the methanolic extract of S. pallida possesses more antioxidant activity in both DPPH and H₂O₂ scavenging assays, with IC₅₀ values of 16.24 and 13.54 µg/mL, respectively, compared to other solvent extracts. The antimicrobial evaluation using the agar well diffusion method further confirmed the superior efficacy of the methanolic extract, showing the lowest MIC values against C. albicans (6.25 mg/mL), followed in order by S. aureus and B. subtilis (12.5 mg/mL), while E. coli and K. pneumoniae required higher concentrations (25 mg/mL). Furthermore, molecular docking results revealed strong binding affinities of several identified compounds, particularly rutin and chlorogenic acid, with key antimicrobial target proteins, supporting the observed biological activities. These findings highlight the methanolic extract of S. pallida as a reliable source of natural antioxidants and broad-spectrum antimicrobial activity, in addition to its utility against numerous infectious diseases.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13568-026-02090-w.

Introduction

Medicinal plants are widely distributed worldwide and represent unique natural sources with the potential to yield promising novel antimicrobial drugs, making them a significant field of research (Haikal et al. 2024; Marrelli 2021). The medicinal value of these plants is attributed to their active chemical constituents that are present in their secondary metabolites, (such as phenolics, flavonoids, terpenoids, alkaloids, additionally fatty acids, that have a variety of chemical structural diversities and biological activities, and have demonstrated important potential in the treatment of different diseases in additionally, their low level of side effects (Sun and Shahrajabian 2023). One of the promising plant genera is Stellaria, which is widely distributed in fields, gardens, and urban areas in cold- and temperate-climate regions (Li et al. 2024). Stellaria is a genus in the Caryophyllaceae family and comprises about 120–0 species (Bencheikh et al. 2024). The Stellaria genus is distinguished by a rich phytochemical profile, including flavonoids, saponins, alkaloids, phenolic compounds, tannins, and triterpenoids (Singh et al. 2022). These bioactive natural constituents account for a broad range of pharmacological properties, involving antioxidant, antifungal, antiinflammatory, antidiabetic, antimicrobial, anxiolytic, antiparasitic and anticancer properties, also, it was used for the treatment of respiratory as well as gastrointestinal disorders in traditional and folk medicine (Cusumano et al. 2024; Iboroma et al. 2018; Ibrahim et al. 2023; Lahlou et al. 2024; Miere et al. 2023; Singh et al. 2022). In Egypt, there are two species of Stellaria: Stellaria media L. and Stellaria pallida (Dumort.) Pire. Stellaria pallida is a flowering plant. It is commonly called less chickweed. S. pallida had an abundance of different secondary metabolites and promising therapeutic properties (Abu-Ziada et al. 2015; Wittig 2004).

Natural products, particularly polar solvent extracts that contain numerous phenolic and flavonoid constituents, have long been established for their significant antioxidant and antimicrobial properties, making them promising candidates in the search for new medical treatments. For example, the methanolic extract of Tamarindus indica fruit pulp demonstrated both remarkable antibacterial and antioxidant activities, attributed to its bioactive phytochemical constituents (Almuhayawi et al. 2023; Fagbemi et al. 2022). Similarly, hydro-methanol (80%) extracts of Rosa gallica leaves were found to possess the highest phenolic content alongside potent antioxidant and antimicrobial effects; key phenolic constituents—such as gallic acid, catechin, chlorogenic acid, quercetin, in addition to their glycosides—have been characterized and quantified via HPLC-DAD(Abdelbaky et al. 2021). Furthermore, methanolic extracts from Mediterranean shrubs analyzed by RP-HPLC revealed phenolic acids, such as ferulic and ellagic acids, and flavonoids, including quercetin, which correlated strongly with antioxidant capacity (Luís et al. 2011).

Given this context, the current report aims to evaluate the phytochemical profile and bioactive potential of Stellaria pallida’s aerial parts. Extracts obtained using chloroform, ethyl acetate, ethanol, and methanol were analyzed via GC–MS to determine their chemical constituents. At the same time, HPLC-DAD further examined the most active extract (methanol) for its major phenolic and flavonoid compounds. Additionally, antioxidant activity (DPPH and H₂O₂ assays) and antimicrobial efficacy (agar well diffusion and MIC determination) were evaluated to elucidate the relationship between phytochemical composition and biological activity. This integrated assessment provides insight into the therapeutic potential of S. pallida as a herbal source of pharmacologically valuable antioxidant and antimicrobial constituents. Molecular docking has been used to elucidate potential interactions between the identified phytochemical constituents and a target protein associated with antibacterial and antifungal properties. Stellaria pallida is a poorly studied species with limited information available on its phytochemical profile and biological activities. Although previous studies have reported selected phytochemical constituents and biological activities of Stellaria pallida and related Stellaria species, comprehensive investigations integrating comparative solvent extraction, bioactivity evaluation, quantitative phytochemical profiling, and computational analyses remain limited. In the present study, multiple solvent extracts were systematically assessed for their antioxidant and antimicrobial activities, followed by quantitative HPLC analysis and molecular docking to explore the potential interactions of the identified phenolic compounds with selected microbial targets. This integrated approach provides additional insights into the relationship between phytochemical composition and biological activity and contributes to a broader understanding of the bioactive potential and possible mechanisms associated with S. pallida.

Experimental details

Plant collection

S. pallida aerial parts were gathered from Al-Qasaa, Bahariya Oases, Egypt, at the identified location (282322.4 N 285224 E). The fresh aerial portions of S. pallida have been washed thereafter and dried under room conditions. Following this, the samples were ground into a fine powder, sieved, and stored for further analysis. The present phytochemical and biological evaluations are specific to the plant material collected from the indicated location and during the indicated harvesting period.

Preparation of plant extracts

One hundred grams of powdered air-dried aerial portions of S. pallida plant were exposed to successive extraction via a Soxhlet apparatus with various organic solvents based on their polarity, including chloroform, ethyl acetate, ethanol, and methanol, respectively, at a temperature lower than the boiling point of the solvents. The extracts were collected and dried using a rotary evaporator to produce crude dried extracts, which were stored at 4 °C in dark glasses until further use.

Determination of phenolic contents (TPC)

The phenolic content was determined using the Folin–Ciocalteu method [45]. Briefly, one milliliter (1 mL) of ethanolic extract was mixed with 0.5 mL of Folin–Ciocalteu reagent (2 N) in a ten milliliter (10 mL) volumetric flask. After three minutes, 1 mL of saturated sodium carbonate (Na₂CO₃) solution (20% w/v) was added, and the volume was adjusted with distilled water. The mixture was incubated for 1 h, and absorbance was measured at 725 nm using a Unicam UV–Vis spectrophotometer against deionized water as the blank. A calibration curve was constructed with gallic acid. The phenolic values were represented in milligram gallic acid equivalents (GAE) per gram (g) of dry weight (DW).

Determination of flavonoid contents (TFC)

The Modified AlCl₃ calorimetric technique was used to dissolve 1 mL of extract in 2 mL of methanol. A solution of 5% NaNO3, 5% NaOH, and 7% AlCl3 was prepared in water. A sealed glass vial containing 200 µl of extract and 75 µl of 5% NaNO₃ was left at room temperature for 5 min. After that, we added 1.25 ml of AlCl₃ and 0.5 ml of NaOH to each vial. And then, it was sonicated and incubated. Following incubation, the absorbances of all working and standard solutions were measured at 510 nm against a methanol blank. The quercetin standard calibration curve was used to estimate flavonoid concentration in the extracts, and flavonoid values were reported as milligrams of quercetin equivalent (Qu) per 1 g of dry extract (Chang et al. 2020).

Gas chromatography mass (GC/MS) analysis

GC/MS was performed using a Trace GC1310-ISQ mass spectrometer (Thermo Scientific, USA) with a TG–5MS capillary column (30 m × 0.25 mm × 0.25 μm). The oven program started at 50 °C, increased to 230 °C at 5 °C/min (held 2 min), then to 290 °C at 30 °C/min (held 2 min). Helium was employed as carrier gas (1 mL/min). An injection (1 µL) was carried out in split mode with a 3 min solvent delay. Mass spectra were acquired in full scan mode (m/z 40–1000) at 70 eV. Constituents were identified by comparison with WILEY 09 and NIST 11 databases, and their relative abundances were calculated from peak areas (Mokhtar et al. 2022).

Antioxidant activity

DPPH assay

Antioxidant activity estimation is done by applying the radical scavenging procedure for DPPH. Fundamentally, a 0.1 mM DPPH solution was produced using ethanol. Subsequently, three milliliters (3 mL) of aqueous extract were mixed with 1 mL of this ethanol solution at different concentrations (0.97, 1.95, 3.90, 7.81, 15.62, 31.25, 62.5, 125, 250, and 500 µg/mL). The mixture was combined and incubated with vigorous shaking at 100 rpm for 30 min at 37 °C in the dark. Another group of experiments was carried out under the same conditions and at the same concentrations, using ascorbic acid as a positive control. For the negative control, a test tube containing DPPH and Tris buffer was used in the same setup but without any extracts. After the incubation period, the absorbance of the produced color at 517 nm was measured. The percentages of free radical scavenging were calculated by the following formulas (Munteanu and Apetrei 2021).

1

Hydrogen peroxide (H₂O₂) assay

Hydrogen peroxide is among the most widely used reagents for evaluating free radical scavenging activity—a phosphate-buffered saline solution with 20 mM concentration of H₂O₂ at pH 7.4. Then added to different concentrations of plant extracts (0.97–500 µg/mL) two milliliter (2 mL) of hydrogen peroxide in phosphate buffer and to standard ascorbic acid in 1 mL of ethanol. After that, the solutions were left free for ten minutes. Subsequently, the absorbance was measured at 230 nm.(Jayaprakasha et al. 2004).

2

Where, As is the absorbance when the sample of S. pallida extracts, or the standard has been added, and Ac is the absorbance of the control.

Antimicrobial activity

To investigate the antibacterial activities of S. pallida aerial-section extracts obtained with various solvents, several pathogenic strains were evaluated. These included eukaryotic strains such as unicellular fungi (C. albicans ATCC 10231), Gram-positive bacteria (S. aureus ATCC 6538 and B. subtilis ATCC 6633), and Gram-negative bacteria (K. pneumonia ATCC 2146 and E. coli ATCC 11229). The agar well diffusion method was employed to assess the efficacy.(Humphries et al. 2018; Mahmoud and Selim 2025). Each bacterium used in this study was subcultured on Nutrient Agar (NA) and incubated for 48 h at 37 °C. The cultures were adjusted to the 0.5 McFarland turbidity standard, then inoculated onto 15 cm diameter NA plates. Samples were diluted independently with DMSO to get the required concentrations. A sterile cork borer made four wells on each agar plate, each measuring 0.6 mm in diameter. Then, all extracts were added to the wells at 100 µL each. After an hour of refrigeration, the plates were incubated for 24 h at 35 ± 2 °C, and the diameter for each well’s surrounding clear zone was measured in millimeters. Three duplicate tests were conducted.

Determination of minimum inhibitory concentration (MIC)

Following the preliminary antimicrobial screening, the methanolic extract of S. pallida aerial parts, which showed the highest inhibitory activity, has been selected for further assessment of its minimum inhibitory concentration (MIC) using the agar well diffusion method. Six concentrations of methanol extract (100, 50, 25, 12.5, 6.25, and 3.12 mg mL^− 1) were prepared in dimethyl sulfoxide (DMSO). Fresh cultures of the test organisms [Gram-positive bacteria (Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6633), Gram-negative bacteria (Klebsiella pneumoniae ATCC 2146 and Escherichia coli ATCC 11229), and unicellular fungus (Candida albicans ATCC 10231)] were grown on NA and adjusted to 0.5 McFarland turbidity standards. Sterile Petri dishes (15 cm in diameter) containing solidified agar medium were uniformly inoculated with the microbial suspension. Using a sterile cork borer, wells 6 mm in diameter were drilled into the agar plates, and 100 µL of each extract concentration was added to separate wells. Plates were left at 4 °C for 1 h to allow diffusion of the extract, followed by incubation at 35 ± 2 °C for 24 h. After incubation, the diameters of the inhibition zones surrounding the wells were measured in millimeters.

The MIC was defined as the minimum concentration of the methanolic extract that produced a distinct measurable zone of inhibition against the test organisms. To ensure accuracy and reproducibility, all assays were performed in triplicate. (Hashem et al. 2025; Sulieman et al. 2023).

Quantitative analysis of phenolic compounds by HPLC-DAD analysis

Phenolic and flavonoid compounds in the methanolic extract were quantified using an Agilent 1260 HPLC system equipped with an Eclipse XDB-C18 column. The mobile phase consisted of acetonitrile (A) and 2% acetic acid (B), with a 60-min gradient at 0.8 mL/min. Detection was performed using DAD (Diode Array Detection) at 280 and 320 nm for the benzoic acid and cinnamic acid derivatives, respectively, as well as at 360 nm for flavonoids, with an injection volume of 5 µL, and the column was maintained at 40 °C. Identification was based on retention times and UV spectra compared with standards (Kim et al. 2006).

Computational methods

Molecular docking of synthesized compounds

All protein receptors were obtained from the RCSB Table S1. The protein targets employed in the molecular docking study were selected based on their established biological relevance in microbial survival, pathogenicity, and antimicrobial resistance. OpuAC from Bacillus subtilis is involved in osmoprotectant transport and environmental adaptation. DNA gyrase from Staphylococcus aureus is a validated antibacterial target essential for DNA replication and cell survival. NDM-1 β-lactamase from Klebsiella pneumoniae is a clinically important resistance determinant responsible for β-lactam antibiotic hydrolysis. Dihydrofolate reductase from Escherichia coli plays a central role in folate metabolism and nucleotide biosynthesis. Furthermore, Secreted Aspartyl Proteinase (Sap1-like; PDB ID: 1ZAP) from Candida albicans was selected because it is a major virulence-associated enzyme implicated in host tissue invasion and fungal pathogenicity. These targets were selected to explore the potential interactions of the identified phytochemicals with biologically relevant microbial proteins corresponding to the microorganisms evaluated experimentally. Following this, the structures of the target proteins were preprocessed using PyMOL. The structure of the compound was created using BIOVIA Draw.

Each compound was then converted to the mol2 format using Open Babel [38]. Subsequently, the molecules are converted to the pdbqt format using AutoDock tools. Before docking, AutoDock Vina was used to create ligand-centered maps (Eberhardt et al. 2021). The two-dimensional interactions between the target proteins and the ligands were examined using the Discovery Studio software (“BIOVIA” 2017).

Statistical analyses

All experiments were performed in triplicate, and the results are presented as mean ± standard deviation (SD). Statistical analyses were carried out using the appropriate analysis of variance (ANOVA) models, followed by multiple comparison tests when applicable. Differences were considered statistically significant at p < 0.05. Minitab^® version 18 (2017) was used for all analyses, consistent with our earlier methodology. (Selim et al. 2024, 2026).

Results

The residual yields obtained from the successive solvent extractions are presented in Table 1. Among the tested solvents, methanol produced the highest extractable yield (8.57 g), followed by ethanol, ethyl acetate, and chloroform.

Table 1.

Extraction yield of Stellaria pallida aerial parts using different solvents

Solvents	Sample (g)	Boiling point	Total hours of extraction	Yield (g)
Chloroform	100	60	5 h	2.32
Ethyl acetate	95	67	6 h	2.51
Ethanol	90	76	7 h	7.02
Methanol	83	80	7 h	8.57

The relatively high yield of the methanolic extract suggests the presence of abundant polar bioactive constituents, which aligns with previous findings highlighting the richness of Stellaria species in phenolics, flavonoids, and other polar compounds. Given the recognized therapeutic potential of the genus Stellaria, these results further support its value as a promising source of phytochemicals relevant to the development of new chemotherapeutic agents.

Determination of total phenolic and flavonoid contents

In this study, the total phenolic content (TPC) of S. pallida extracts was quantified via the Folin-Ciocalteu method. Values were calculated from a gallic acid calibration curve (y = 414.08x − 7.3669, R²= 0.9959) and expressed as gallic acid equivalents (GAE) per gram (gm) dry extract weight (Fig. 1A). The value of phenolic content in methanol extracts was higher (581.55 mg GAE/g) than in ethyl acetate, ethanol, and chloroform (39.391.23, 50.181.29, and 54.821.01 mg GAE/g, respectively).

Fig. 1.

A Total phenolic content, B Total flavonoid content of each extract was measured in aerial portions of S. pallida using various solvent fractions in standard equivalents. Data are presented as mean ± SD (n = 3). Different superscript letters indicate statistically significant differences at p < 0.05

The value of phenolic content in methanol extracts was higher and measured as ( 58±1.55 mg GAE/g) content compared to ethyl acetate, ethanol, and chloroform (39.39±1.23, 50.18±1.29 and 54.82±1.01 mg GAE/g) respectively. Comparing the TPCs of the solvents, the order was: metanol > ethyl acetate > ethanol > chloroform. As a foundation for quantitative flavonoid content determination in the chosen plant extracts, flavonoid content was measured using aluminum chloride in a colorimetric method. The results were obtained from the calibration curve (y = 405.08.x + 0.5446, R2 = 0.9934) of quercetin (0–600 µg/mL) and expressed in quercetin equivalents (Qu) per gram dry extract weight ( Fig. 1B) while, the ethanol extract had a higher content of flavoniods (27.22±1.08 mg Qu/g) followed by ethyl acetate extract (24.28±0.88 mg Qu/g). Additionally, the methanol extract had a lower flavonoid content (19.80±0.92 mg Qu/g) compared to the other organic solvents.

GC/Mass analysis

Identification of compounds via GC/MS of the organic extracts revealed 20, 11, 18, and 21 compounds in chloroform, ethyl acetate, ethanol, and methanol, respectively. These constituents belong to diverse chemical classes, including fatty acids, fatty acid esters, hydrocarbons, sterols, terpenoids, and alkaloids. The identified components with their retention time (RT) and peak area (%) are present in Table S2–S5 and Figs. 1S–4S.

The major components present in the methanol extract were prostaglandin A1-biotin (15.74%), 13-docosenamide, (Z) (9.96%), and psi. -carotene, 1,1’,2,2’-tetrahydro-1,1 ‘-dimethoxy (6.54%). The chloroform crude extract was 13-docosenamide, (Z)(9.78%), and 1,2-benzenedicarboxylic acid (6.03%). The ethyl acetate crude extract contained 13-docosenamide (Z) (14.28%), followed by stigmasterol (9.22%) and prostaglandin A1-biotin (5.81%). The ethanol extract contained n-hexadecanoic acid (7.40%), 9-octadecenamide (Z) (6.41%), and chondrillasterol (6.26%).

Antioxidant assay on different solvent extracts of S. pallida

DPPH assay

The antioxidant potential of Stellaria pallida aerial part extracts was evaluated using the DPPH radical scavenging assay across four solvents (chloroform, ethyl acetate, ethanol, and methanol). As illustrated in Fig. 2A–D, all extracts exhibited a clear concentration-dependent increase in scavenging activity; however, their effectiveness varied markedly according to solvent polarity.

Fig. 2.

DPPH assay on different solvent extracts of S. pallida. Each value is the Mean ± SD of triplicate analysis; each set of bars presented on Antioxidant—DPPH on crude extract of A chloroform, B ethyl acetate, C ethanol, D methanol, compared with ascorbic acid as a positive control.

Comparative performance with ascorbic acid (positive control), ascorbic acid consistently possessed the highest scavenging Activity across all concentrations, reaching values above 90% at the maximum tested concentration (500 µg/ml). This confirms its role as a strong antioxidant benchmark. None of the crude extracts matched the potency of ascorbic acid, but their activity patterns suggest the presence of bioactive compounds with significant radical scavenging capacity.

The chloroform extract (Fig. 2A) exhibited the lowest antioxidant activity among the tested solvents, with scavenging Activity ranging from 19.3 ± 0.25% at the lowest concentration to 64.9 ± 0.39% at the highest. This relatively weak Activity suggests that nonpolar or semi-polar phytoconstituents extracted with chloroform may not be the primary contributors to the antioxidant potential of S. pallida. Nonpolar solvents typically extract terpenoids, sterols, and lipophilic compounds, which may have moderate or limited hydrogen-donating ability toward DPPH radicals. On the other hand, the ethyl acetate extract (Fig. 2B) showed a moderate increase in scavenging activity, with values ranging from 22.3 ± 0.1% to 70 ± 0.7%. Although higher than chloroform, it was still significantly lower than the standard. Ethyl acetate is an intermediate-polarity solvent that extracts compounds such as flavonoids and phenolics. The observed Activity suggests that some polyphenolic compounds with antioxidant properties are moderately soluble in this solvent fraction.

Furthermore, the ethanol extract (Fig. 2C) exhibited higher scavenging Activity than both chloroform and ethyl acetate extracts, reaching 71.4 ± 0.2% inhibition at the maximum concentration. Ethanol is a polar solvent capable of extracting phenolics, tannins, and flavonoids—classes of phytochemicals well known for their radical scavenging ability. The comparatively higher performance of ethanol extract indicates that such polar phenolic compounds may constitute the major antioxidant constituents in S. pallida.

The superior antioxidant performance of the methanolic extract may be attributed to the higher recovery of polar phenolic constituents, which are known to act as efficient hydrogen donors (Fig. 2D). This extract approached the Activity of ascorbic acid more closely than the others. Methanol, being more polar than ethanol, effectively extracts a wide range of phenolics, flavonoids, alkaloids, and glycosides. The high Activity observed suggests that S. pallida contains abundant methanol-soluble phenolics and flavonoids, which are highly effective hydrogen or electron donors, capable of neutralizing free radicals.

H₂O₂ assay

The antioxidant effects of crude extracts of S. pallida were evaluated via the H₂O₂ scavenging assay across different solvent systems (chloroform, ethyl acetate, ethanol, and methanol).

Data found in Table 2 indicate that four crude extracts show a clear, monotonic, concentration-dependent increase in H₂O₂ scavenging. The methanol extract gives the highest Activity at every matched dose, followed by ethanol, ethyl acetate, and chloroform, while ascorbic acid remains the most potent positive control.

Table 2.

H₂O₂ assay on different solvent extracts of S. pallida

Conc. (µg/ml)	Scavenging activity %
	Ascorbic acid	Chloroform	Ethyl acetate	Ethanol	Methanol
0.97	47.58 ± 0.9	23.61 ± 0.3	25.17 ± 0.3	28.69 ± 1.6	30.85 ± 0.8
1.95	52.18 ± 0.8	28.61 ± 0.7	30.17 ± 0.1	33.69 ± 0.6	34.11 ± 0.9
3.9	57.68 ± 0.5	33.61 ± 0.3	35.17 ± 0.6	38.69 ± 0.5	39.11 ± 1.2
7.81	62.18 ± 1.5	38.81 ± 0.6	40.37 ± 0.8	43.89 ± 0.4	44.10 ± 0.6
15.62	67.88 ± 0.1	44.01 ± 0.9	45.57 ± 0.7	49.09 ± 1.2	52.14 ± 1.3
31.25	72.08 ± 0.7	48.81 ± 0.7	50.37 ± 0.5	53.89 ± 0.8	55.23 ± 1.2
62.5	77.88 ± 1.3	53.61 ± 0.8	55.17 ± 0.1	58.69 ± 1.3	60.24 ± 1.0
125	82.78 ± 0.6	58.51 ± 0.5	60.07 ± 0.6	63.59 ± 0.8	64.14 ± 0.5
250	87.18 ± 0.5	63.51 ± 1.1	65.07 ± 1.2	68.59 ± 0.7	75.16 ± 0.9
500	93.18 ± 0.1	69.21 ± 0.7	70.77 ± 0.9	74.29 ± 0.6	83.74 ± 0.3
IC50	1.49 µg/ml	38.99 µg/ml	30.05 µg/ml	18.58 µg/ml	13.54 g/ml

Antimicrobial activity

The antimicrobial activity of Stellaria pallida extracts was evaluated using the agar well diffusion assay against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae), and the yeast Candida albicans. All solvent extracts exhibited inhibitory effects; however, the magnitude of activity varied significantly with both the extraction solvent and the tested microorganism (Fig. 3).

Among the tested extracts, the chloroform fraction showed the weakest antimicrobial activity, producing inhibition zones ranging from 8.8 ± 1.0 to 17.67 ± 0.92 mm. The ethyl acetate exhibited moderate activity, with inhibition zones ranging from 12.2 ± 1.05 to 27.3 ± 0.58 mm, depending on the microorganism. Higher inhibitory effects were observed with the ethanol extract, which yielded zones of inhibition ranging from 16.4 ± 1.31 to 27.9 ± 0.78 mm. Notably, the methanolic extract demonstrated the strongest antimicrobial activity, with inhibition zones ranging from 16.4 ± 0.64 mm against K. pneumoniae to 31.6 ± 0.46 mm against C. albicans.

Candida albicans was the most susceptible microorganism, exhibiting the largest inhibition zone (31.6 ± 0.46 mm) in response to the methanolic extract. Strong inhibitory effects were also recorded against S. aureus and E. coli (27.93 ± 0.84 and 23.43 ± 0.51 mm, respectively). In contrast, B. subtilis and K. pneumoniae displayed comparatively lower, yet statistically significant, inhibition zones (16.4 ± 1.31 and 19.30 ± 0.56 mm, respectively).

The two-way analysis of variance (Table S7) revealed highly significant effects of both extract type (F = 359.12, p < 0.001) and the microbial pathogens (F = 440.13, p < 0.001) on the inhibition zones. In addition, the interaction between extracts and microbial pathogens was statistically significant (F = 9.93, p < 0.001), observing that the antimicrobial activity of each extract varied depending on the tested microorganism. The high coefficient of determination (r² = 98.67%) confirms that the model explains nearly all the variability in antimicrobial activity, reflecting the robustness of the experimental design and reliability of the observed differences. These results statistically confirm that the methanolic extract produced significantly greater antimicrobial activity, particularly against C. albicans and S. aureus, compared to other extracts. Although the investigated extracts demonstrated antimicrobial activity against the tested microorganisms, this study represents a preliminary screening. Further microbiological studies, including determination of bactericidal versus bacteriostatic effects, antibiofilm activity, cytotoxicity assessments, selectivity index determination, and mechanistic evaluations, are required to better define the pharmacological and therapeutic relevance of the observed antimicrobial effects.

Minimum inhibitory concentration (MIC) of the methanolic extract

In light of the preliminary antimicrobial screening, which indicated that the methanolic extract of S. pallida aerial parts had the greatest inhibitory effect, this extract was advanced to the next stage of evaluation for determine its MIC using the agar well diffusion method (Fig. 4).

Fig. 3.

Antimicrobial activity of solvent extracts from S. pallida. Mean ± SD (n = 3); different superscripts denote significant differences (p < 0.05). Data are presented as mean ± SD (n = 3). Different superscript letters indicate statistically significant differences at p < 0.05

The finding detected a clear concentration-dependent increase in inhibition zones against all tested microorganisms. At the maximum concentration tested (100 mg/mL), inhibition zones ranged from 18.6 ± 1.15 mm (K. pneumoniae) to 31.8 ± 0.61 mm (C. albicans). The MIC values of the methanolic extract were 12.5 mg/mL (7.26 ± 0.20 mm), 12.5 mg/mL (12.76 ± 0.68 mm), 25 mg/mL (9.26 ± 1.10 mm), 25 mg/mL (7.26 ± 0.20 mm), and 6.25 mg/mL (13.5 ± 0.57 mm) for B. subtilis, S. aureus, E. coli, K. pneumoniae, and C. albicans respectively. Although the present findings demonstrate measurable antimicrobial effects of the investigated extracts, the observed MIC values should be interpreted as indicative of moderate antimicrobial activity. Further studies employing standardized broth microdilution assays, bioassay-guided fractionation, and purified bioactive compounds are required to provide a more comprehensive assessment of antimicrobial potency.

HPLC-DAD analysis

The characterization of the constituents in the methanol extract from aerial parts of S. pallida via high-performance liquid chromatography (HPLC) indicated the presence of twenty phenolic constituents (Table 3 and Figure S5 a–c). The main components were rutin (333.7 µg/g), chlorogenic acid (181.17 µg/g), ferulic acid (168.11 µg/g), apigenin-7-glucoside (143.08 µg/g), epicatechin (129.32 µg/g), and caffeic acid (117.56 µg/g). The minor components were quercetin (1.88 µg/g), followed by cinnamic (3.45 µg/g) and epicatechin gallate (4.89 µg/g).

Table 3.

Chemical composition analysis of phenolic compounds of methanol extract from S. pallida aerial parts by HPLC-DAD

No.	Compound name	MF	MW (g/mol )	RT m	Concentration (µg/g)	Biological activity	References
1	Gallic acid	C7H6O5	170	3.70	6.86	Antimicrobial and antioxidant	Wu et al. (2026)
2	Protocatechuic	C7H6O4	154	6.42	15.72	Antibacterial and antioxidant	Cai et al. (2024); Zhang et al. ( 2021),
3	Gentisic	C7H6O4	154	9.60	14.17	Antioxidant	Cunha et al.  (2024)
4	p-hydroxybenzoic acid	C7H6O3	138	10.0	9.76	Antibacterial and antioxidant	Wang and Jiang (2022)
5	Catechin	ND	ND	11.44	ND	ND	ND
6	Chlorogenic acid	C16H18O9	345	12.69	181.17	Antimicrobial and antibiofilm	Yang et al. ( 2022)
7	Caffeic	C9H8O4	180	13.32	117.56	Antimicrobial	Khan et al. (2021)
8	Syringic	C9H10O5	198	14.71	11.34	Antimicrobial	Minich et al. (2022)
9	Epicatechin	C15H14O6	290.27	14.98	129.32	Antimicrobial	Buchmann et al. (2022)
10	Vanillic	C8H8O3	152	16.15	13.19	Antimicrobial and anticancer	Venkidasamy et al. (2024)
11	Ferulic acid	C10H10O4	194	20.93	168.11	Antimicrobial	Wijayanti et al. (2021)
12	Sinapic	C 11 H 12 O 5	224.2	21.80	108.74	Antimicrobial Antiinflammatory	Mandal et al. (2024)
13	Epicatechin gallate	C22H18O10	442.3	22.48	4.89	ND	ND
14	Rutin	C27H30O16	610.5	23.94	333.70	Antibacterial and antioxidant	Saleemi et al. (2022)
15	p-coumaric	C9H8O3	164	25.54	44.46	antioxidant, antiinflammatory, antimicrobial, and antidiabetic	Venkatesan et al. (2023)
16	Rosmarinic	ND	ND	28.55	ND	ND	ND
17	Apigenin-7-glucoside	C21H20O10	432.38	29.43	143.80	antioxidant,	GÜÇLÜ and ÇINAR AYAN (2023)
18	Cinnamic	C9H8O2	148	35.04	3.45	Antimicrobial	Mingoia et al. (2022)
19	Qurecetin	C7H6O5	302.2	36.34	1.88	Antimicrobial and antioxidant	Dziewońska et al. (2026); Osojnik Črnivec et al. (2021)
20	Apigenin	C7H6O4	270	40.45	12.90	Antimicrobial and anticancer	Imam et al. (2022)
21	Kaempferol	C7H6O4	286	41.16	6.79	Antimicrobial	Periferakis et al. (2022)
22	Chrysin	C15H10O4	254.2	53.24	5.80	Antimicrobial and antioxidant	Adesina et al. (2024)

ND: not detected, MW: Molecular Weight, MF: Molecular Formula

Computational analysis

The molecular docking analysis presented in this study was conducted as a predictive computational approach to explore potential interactions between the identified phytochemicals and selected microbial targets. The obtained docking scores and interaction profiles should not be interpreted as direct evidence of antimicrobial mechanisms, target inhibition, or biological efficacy. Rather, these findings provide preliminary hypotheses that require confirmation through appropriate biochemical, enzymatic, and molecular validation studies.

Docking and molecular interaction of identified compounds

Molecular docking was performed to examine the binding interactions between the selected drugs and protein targets associated with antibacterial activity. This study aimed to explore potential ligand–target interactions and prioritize compounds for future investigation. The docking studies evaluated binding affinities between the drugs and three antimicrobial receptors, as shown in Table S7 and Fig. S6.

Docking and molecular interaction studies with choline-binding protein OpuAC of B. subtilis (PDB ID: 5NXY)

Molecular docking of twenty-three phenolic and flavonoid compounds against the choline-binding protein OpuAC of Bacillus subtilis (PDB ID: 5NXY) revealed binding affinities between − 4.5 and − 9.4 kcal·mol⁻¹ as summarized in Table S7 and illustrated in Fig. 5a–w. The observed variation in docking scores reflects differences in molecular size, aromaticity, and functional group composition, which collectively influence ligand–protein interaction patterns and complex stability.

Fig. 4.

Antimicrobial activity of different concentrations of methanol extract from S. pallida. Data are presented as mean ± SD (n = 3). Different superscript letters indicate statistically significant differences at p < 0.05

Ampicillin, employed as the reference antibiotic, showed a binding affinity of − 6.5 kcal·mol⁻¹ and adopted a stable binding conformation within the OpuAC active site. Its interaction profile was characterized by multiple conventional hydrogen bonds with key residues, including Gln47, Asn107, Asn44, and Thr109, as well as Pi–Pi and Pi- Sigma interactions involving aromatic residues such as Tyr63 and Tyr143. This balanced combination of hydrophilic and hydrophobic interactions served as a benchmark for evaluating the binding performance of the investigated natural compounds.

Several flavonoids demonstrated stronger binding affinities than ampicillin. Quercetin dihydrate showed the highest affinity (− 9.4 kcal·mol⁻¹), followed by kaempferol (− 9.2 kcal·mol⁻¹), chrysin (− 9.1 kcal·mol⁻¹), and apigenin (− 8.9 kcal·mol⁻¹). These constituents formed extensive conventional hydrogen bonds with residues involved in Gln11, Gln47, Asn44, Asn107, and Asp66. Furthermore, their planar aromatic systems enabled pronounced Pi–Pi Stacked and Pi–Pi T-shaped interactions with Tyr109, Tyr189, and Tyr213, as well as Pi-Sulfur interactions with Met13, which collectively contributed to their enhanced binding stability.

Catechin (− 8.3 kcal·mol⁻¹) and epicatechin (− 8.7 kcal·mol⁻¹) also exhibited strong binding profiles supported by dense hydrogen-bonding networks and multiple Pi–Pi Stacked interactions with aromatic residues within the binding cavity. In contrast, epicatechin gallate showed a reduced binding affinity (− 5.8 kcal·mol⁻¹), despite forming several hydrogen bonds, likely due to steric constraints that limited optimal accommodation within the binding site.

On the other hand, glycosylated flavonoids such as apigenin-7-glucoside displayed the weakest binding affinity (− 4.5 kcal·mol⁻¹). Although numerous conventional hydrogen bonds were observed, the bulky sugar moiety hindered effective Pi-based interactions and reduced overall binding stability.

Phenolic acids generally exhibited moderate binding affinities, between 6.0 and − 7.4 kcal·mol⁻¹. Caffeic acid and cinnamic acid showed affinities of − 7.1 kcal·mol⁻¹, while ferulic and chlorogenic acids displayed values around − 7.0 kcal·mol⁻¹. Their binding modes were primarily governed by conventional hydrogen bonds involving carboxyl and hydroxyl groups, with limited Pi-Sigma or Pi-Alkyl interactions involving residues such as Met13 and Tyr63.

Similarly, benzoic acid derivatives, including gallic, gentisic, protocatechuic, syringic, vanillic, and sinapic acids, demonstrated moderate docking scores (− 6.0 to − 7.4 kcal·mol⁻¹). These ingredients created hydrogen bonds with polar residues such as Gln11, Asn44, and Asp66, along with occasional Pi-Anion, Pi–Pi Stacked, or Pi-Alkyl engagement involving tyrosine residues. However, their relatively small aromatic frameworks limited the extent of hydrophobic stabilization within the binding pocket.

Rosmarinic acid exhibited a binding affinity of − 6.5 kcal·mol⁻¹, comparable to that of ampicillin. Although it established multiple conventional hydrogen bonds and Pi-based interactions, the presence of unfavorable contacts likely compromised complex stability.

Overall, the docking results indicate that flavonoids with multiple hydroxyl groups and extended aromatic systems exhibit superior binding affinities for OpuAC compared with simpler phenolic acids and the reference antibiotic. The strong correlation between docking scores and the prevalence of conventional H₂ bonds, Pi–Pi Stacked, Pi–Pi T-shaped, and Pi-Sulfur interactions underscores the importance of both hydrogen-bonding capacity and aromatic-driven stabilization in ligand binding to OpuAC.

Molecular docking analysis against Staphylococcus aureus DNA gyrase (PDB ID: 5CDM)

Molecular docking and computational modeling were carried out to estimate the binding behavior of 22 phenolic and flavonoid compounds toward Staphylococcus aureus DNA gyrase (PDB ID: 5CDM), using ciprofloxacin as a reference antibiotic. The calculated binding affinity values (kcal·mol⁻¹) are summarized in Table S7 and range from − 4.6 to − 6.5 kcal·mol⁻¹ or the tested compounds. In contrast, ciprofloxacin exhibited a comparatively weaker affinity of − 3.7 kcal·mol⁻¹. The interaction patterns observed in the corresponding two-dimensional docking figures, Fig. 6a–w, were fully consistent with these affinity values.

Fig. 5.

a–w 2D views of compounds docked in the binding pocket of the choline-binding protein OpuAC in B. subtilis. Compounds with higher binding affinity are shown in green, those with lower affinity in red, and the reference antibiotic in black

The reference drug ciprofloxacin showed a binding affinity of − 3.7 kcal·mol⁻¹ and interacted with the active site primarily through a limited number of hydrophilic contacts, including conventional hydrogen bonds with residues including ARG47, ARG48, SER158, and ASP37, in addition to Pi–cation interactions involving positively charged residues. Despite its established antibacterial activity, the relatively higher binding energy observed in this docking study suggests weaker stabilization within the gyrase binding pocket than for several tested natural compounds.

Among the investigated ligands, chrysin demonstrated the strongest binding affinity (− 6.5 kcal·mol⁻¹), followed by quercetin dihydrate (− 6.0 kcal·mol⁻¹) and epicatechin gallate (− 5.9 kcal·mol⁻¹). These compounds formed dense networks of hydrophilic interactions, including multiple conventional hydrogen bonds with catalytically relevant residues, which significantly enhanced binding stability. Furthermore, their polyphenolic structures enabled extensive Pi-mediated interactions, such as Pi–cation and Pi–alkyl contacts, reinforcing ligand accommodation within the active site, as clearly illustrated in their respective docking figures.

Additionally, compounds such as apigenin, catechin, chlorogenic acid, kaempferol, and epicatechin, which displayed binding affinities clustered around − 5.6 to − 5.8 kcal·mol⁻¹, exhibited balanced interaction profiles. Their docking poses revealed a combination of hydrogen bonding with polar residues and hydrophobic contacts involving aromatic rings, resulting in moderate yet stable binding conformations. The consistency between docking scores and interaction maps provides computational support for the predicted binding modes.

On the other hand, simpler phenolic acids, including cinnamic acid (− 4.9 kcal·mol⁻¹), p-hydroxybenzoic acid (− 4.6 kcal·mol⁻¹), gallic acid (− 5.0 kcal·mol⁻¹), protocatechuic acid (− 5.0 kcal·mol⁻¹), and gentisic acid (− 4.9 kcal·mol⁻¹), exhibited weaker binding affinities. Van der Waals forces and limited hydrophobic contacts, with fewer hydrogen bonds, dominated their interaction patterns. They reduced Pi interactions, leading to less stable binding within the gyrase pocket.

Furthermore, glycosylated and highly substituted compounds displayed variable behavior. While apigenin-7-glucoside (− 5.2 kcal·mol⁻¹) and rutin acid (− 0.1 kcal·mol⁻¹) formed several hydrophilic contacts due to their multiple hydroxyl groups, excessive molecular size and conformational flexibility appeared to hinder optimal fitting within the active site, resulting in reduced or anomalous binding affinity values, as reflected in both the table and the corresponding figures.

Overall, the docking analysis reveals a clear correlation between binding affinity values (kcal·mol⁻¹) and the nature of ligand–protein interactions observed in Figs. 1–23. Compounds exhibiting stronger affinities consistently formed extensive hydrophilic interaction networks complemented by stabilizing hydrophobic and Pi-mediated contacts. Importantly, several natural compounds exhibited more favorable predicted docking scores than the reference ligand under the applied docking protocol antibiotic ciprofloxacin, highlighting their potential to interact with S. aureus DNA gyrase and warranting further experimental validation.

Molecular docking analysis against K. pneumoniae NDM-1 β-lactamase (4HL2)

Molecular docking analysis was conducted to investigate the binding behavior of twenty-two phenolic compounds against the NDM-1 β-lactamase of Klebsiella pneumoniae (PDB ID: 4HL2), with meropenem included as a reference antibiotic are summarized in Table S7 and illustrated in Fig. 7a–w. The obtained binding affinities (kcal·mol⁻¹) and interaction profiles reveal distinct binding modes, governed by a combination of hydrophilic interactions and hydrophobic contacts within the enzyme’s active site.

Fig. 6.

a–w 2D views of compounds docked in the binding pocket of Staphylococcus aureus DNA gyrase (PDB ID: 5CDM)

Apigenin exhibited a binding affinity of − 5.8 kcal·mol⁻⁻¹ and was stabilized primarily through multiple conventional hydrogen bonds with key residues including ASN220 and LYS211, in addition to carbon hydrogen bonding with GLY219. Furthermore, Pi reinforced hydrophobic stabilization–sulfur interactions involving CYS208 and Pi–Pi stacking with HIS250, indicating effective anchoring of the flavone scaffold within the catalytic pocket.

Similarly, Apigenin-7-glucoside showed a slightly reduced affinity (− 5.2 kcal·mol⁻¹), which can be attributed to its bulkier glycosidic moiety. Despite this, the compound maintained strong hydrophilic engagement through hydrogen bonds with ASN220, LYS211, and ASP124. On the other hand, additional Pi–Pi stacking with HIS250 and Pi–alkyl contacts with CYS208 contributed to its overall stability, as clearly illustrated in the interaction map.

Caffeic acid demonstrated a binding affinity of − 5.6 kcal·mol⁻¹ and interacted dominantly through hydrogen bonding with ASN220 and LYS211. Moreover, Pi–anion interactions with ASP124 and Pi–Pi stacking with HIS250 further enhanced binding, suggesting that the conjugated aromatic system plays a crucial role in maintaining enzyme–ligand complementarity.

Catechin, with an affinity of − 5.7 kcal·mol⁻¹, formed an extensive hydrogen-bonding network involving ASN220, LYS211, and ASP124. Additionally, Pi–Pi T-shaped interactions with HIS122 and Pi–sulfur contacts with CYS208 were observed, providing both directional specificity and hydrophobic stabilization within the active site.

Chlorogenic acid showed an affinity of − 5.8 kcal·mol⁻¹ and was characterized by strong hydrophilic dominance. Multiple hydrogen bonds with ASN220, LYS211, GLY219, and HIS189 were evident. Furthermore, Pi–Pi stacking with HIS250 and Pi–alkyl interactions with VAL73 supported ligand accommodation, highlighting the importance of polyhydroxylated scaffolds in NDM-1 inhibition.

Chrysin exhibited one of the strongest affinities among the tested compounds (− 6.5 kcal·mol⁻¹). This enhanced binding can be attributed to a balanced interaction profile, including hydrogen bonds with LYS211 and ASN220, Pi–Pi stacking with HIS250 and HIS122, and Pi–sulfur interactions with CYS208. Notably, the planar flavone structure enabled optimal Pi-electron overlap within the hydrophobic pocket.

In contrast, Cinnamic acid displayed a weaker affinity (− 4.9 kcal·mol⁻¹), forming limited hydrogen bonding mainly with ASN220. However, Pi–Pi T-shaped interactions with HIS250 and Pi–sulfur contacts with CYS208 partially compensated for the reduced hydrophilic engagement.

Epicatechin and Epicatechin gallate exhibited affinities of − 5.6 and − 5.9 kcal·mol⁻¹, respectively. Both compounds formed dense hydrogen-bond networks involving ASN220, LYS211, and ASP124. Additionally, Epicatechin gallate showed enhanced Pi–Pi stacking with HIS122 and Pi–alkyl interactions with VAL73, explaining its slightly superior affinity compared to Epicatechin.

Ferulic acid demonstrated a binding affinity of − 5.5 kcal·mol⁻¹ and interacted via hydrogen bonds with ASN220 and LYS211, alongside Pi–anion interactions with ASP124 and Pi–Pi stacking with HIS250. These interactions collectively stabilized the ligand within the catalytic groove.

Gallic acid (− 5.0 kcal·mol⁻¹) and Gentisic acid (− 4.9 kcal·mol⁻¹) both showed moderate binding, dominated by hydrogen bonding with ASN220 and LYS211. However, Gallic acid exhibited additional Pi–Pi stacking with HIS250, whereas Gentisic acid relied more on Pi–anion interactions with ASP124.

Kaempferol showed a strong affinity of − 5.7 kcal·mol⁻¹, supported by multiple H₂ bonds with ASN220 and LYS211, in addition to pronounced Pi–Pi stacking with HIS250 and HIS122. Moreover, Pi–sulfur interactions with CYS208 contributed significantly to binding stabilization.

p-Coumaric acid (− 5.1 kcal·mol⁻¹), p-hydroxybenzoic acid (− 4.6 kcal·mol⁻¹), and Protocatechuic acid (− 5.0 kcal·mol⁻¹) displayed variable affinities that were associated with the number and orientation of OH groups. Among them, Protocatechuic acid formed additional Pi–anion interactions with ASP124, resulting in improved binding compared to p-hydroxybenzoic acid.

Quercetin dihydrate exhibited a high affinity of − 6.0 kcal·mol⁻¹ and formed an extensive interaction network. Strong hydrogen bonds with ASN220, LYS211, and GLN123 were observed, alongside Pi–Pi stacking with HIS250 and Pi–sulfur interactions with CYS208. This dense interaction pattern explains its favorable docking score.

Rosmarinic acid (− 5.0 kcal·mol⁻¹) and Rutin acid (− 0.1 kcal·mol⁻¹) showed contrasting behaviors. While Rosmarinic acid maintained hydrogen bonding with ASN220 and Pi–Pi stacking with HIS122, Rutin acid exhibited an extremely poor affinity, likely due to steric hindrance and unfavorable donor–donor interactions, as clearly visible in the interaction diagram.

Sinapic acid, Syringic acid, and Vanillic acid showed affinities of − 5.0, − 5.2, and − 5.2 kcal·mol⁻¹, respectively. These ingredients consistently formed H₂ bonds with ASN220 and LYS211, while Pi–anion interactions with ASP124 and Pi–Pi stacking with HIS250 contributed to moderate stabilization across this subgroup.

Finally, the reference antibiotic meropenem displayed a binding affinity of − 3.7 kcal·mol⁻¹, which was notably weaker than many of the tested phenolic compounds. Although meropenem formed several hydrogen bonds with ASN220, LYS211, and GLY219, its limited hydrophobic engagement within the aromatic pocket reduced its overall binding strength compared to flavonoid-based ligands.

Molecular docking analysis against E. coli dihydrofolate reductase (1RX2)

The docking evaluation of the selected natural compounds against Escherichia coli dihydrofolate reductase (PDB ID: 1RX2) was conducted using trimethoprim as the reference antibiotic, which possessed a binding affinity of − 7.7 kcal·mol⁻¹, as summarized in Table S7 and illustrated in Fig. 8a–w. Accordingly, the binding behavior of all investigated ligands was interpreted relative to this reference value, with particular emphasis on hydrophilic interactions and hydrophobic contacts involving π- π interactions.

Fig. 7.

a–w 2D views of compounds docked in the binding pocket of K. pneumoniae NDM-1 β-lactamase (4HL2)

Apigenin displayed a binding affinity of − 8.5 kcal·mol⁻¹, exceeding that of trimethoprim. Its binding mode was stabilized by conventional hydrogen bonds with residues such as ILE94 and TYR100. Additionally, hydrophobic interactions, including Pi–Pi stacking with PHE31 and Pi–alkyl contacts with ALA7 and ILE5, contributed significantly to its enhanced affinity compared to the reference drug.

Similarly, apigenin-7-glucoside demonstrated a markedly stronger affinity of − 10.2 kcal·mol⁻¹. This improvement can be attributed to the extensive hydrophilic interaction network formed by the glucoside moiety, which enabled multiple hydrogen bonds with SER49, ARG52, and THR46. Furthermore, Pi–Pi stacking and Pi–alkyl interactions with aromatic and aliphatic residues ensured a stable accommodation within the active site, clearly outperforming trimethoprim.

In the case of catechin and epicatechin, binding strengths of − 8.1 and − 8.2 kcal·mol⁻¹ were recorded, sequentially. Both compounds established multiple hydrogen bonds with ASP27, TYR100, and THR46, while hydrophobic stabilization was achieved through Pi – Pi stacking with PHE31 and Pi –alkyl interactions involving ILE5 and ALA7. Notably, epicatechin gallate emerged as one of the most potent ligands, exhibiting an affinity of − 10.2 kcal·mol⁻¹. Its superior performance relative to trimethoprim was primarily driven by the coexistence of strong hydrogen bonding and pronounced hydrophobic interactions, including Pi–Pi stacking and Pi–sulfur interactions with MET20.

On the other hand, kaempferol and quercetin dihydrate showed affinities of − 7.8 and − 8.1 kcal·mol⁻¹, respectively, both comparable to or slightly better than trimethoprim. Their planar aromatic systems facilitated efficient Pi–Pi stacking with PHE31, while additional hydrogen bonds with TYR100 and ILE94 enhanced binding stability. Furthermore, rutin acid demonstrated a high affinity of − 9.4 kcal·mol⁻¹, which significantly surpassed that of the reference antibiotic. This strong binding was attributed to its dense hydrogen-bonding pattern, combined with multiple Pi–alkyl and Pi–Pi interactions.

In contrast, simpler phenolic acids, including caffeic acid, ferulic acid, cinnamic acid, p-coumaric acid, gallic acid, syringic acid, vanillic acid, and p-hydroxybenzoic acid, exhibited lower affinities ranging from − 5.3 to − 6.4 kcal·mol⁻¹. Although these constituents formed H₂ bonds with residues such as ASP27, THR46, and ILE94, their reduced aromatic surface area limited the formation of stabilizing π-hydrophobic interactions, resulting in weaker binding than with trimethoprim.

Overall, the comparative analysis clearly indicates that compounds with extended aromatic frameworks and multiple H2-bond donors and acceptors exhibit binding affinities equal to or greater than that of trimethoprim (− 7.7 kcal·mol⁻¹). Moreover, the consistent correlation between the observed Pi -based hydrophobic interactions in the docking images, in addition to the affinity data listed in the corresponding table, suggests the reliability of the docking results and highlights the potential of selected polyphenolic compounds as potential ligand of E. coli dihydrofolate reductase.

Molecular docking analysis against Candida albicans candidapepsin (Sap1-like) (1ZAP)

The molecular docking analysis against Candida albicans aspartic protease (PDB ID: 1ZAP) revealed distinct binding behaviors among the tested phenolic and flavonoid compounds when compared to the reference antifungal agent Pepstatin A, which possessed a binding affinity of − 7.6 kcal·mol⁻¹. The interaction patterns were dominated by a combination of H₂ bonding, hydrophobic contacts, and aromatic stacking interactions, indicating that the active site of 1ZAP favors ligands capable of establishing both polar and nonpolar contacts Table S7 and Fig. 9a – w.

Fig. 8.

a–w 2D views of compounds docked in the binding pocket of E. coli dihydrofolate reductase (1RX2)

Fig. 9.

a–w 2D views of compounds docked in the binding pocket of Candidapepsin (Sap1-like) (1ZAP) in Candida albicans. Compounds with higher binding affinity are shown in green, those with lower affinity in red, and the reference antibiotic in black

Pepstatin A showed a strong binding mode within the catalytic pocket, characterized by multiple conventional H-bonds with key residues including Asp86, Asp120, Thr221, as well as Ser88, in addition to comprehensive van der Waals contacts with surrounding residues such as Ile30, Ile119, Ile123, and Gly220. These interactions collectively stabilized the ligand within the binding cleft, explaining its high inhibitory potency and justifying its use as a benchmark inhibitor.

Among the tested natural compounds, Apigenin-7-glucoside demonstrated the highest binding affinity (− 9.4 kcal·mol⁻¹), surpassing Pepstatin A. This enhanced affinity was linked to the formation of several H-bonds with catalytic residues and adjacent residues, particularly Asp218, Thr221, and Gly85, alongside stabilizing van der Waals interactions. Furthermore, aromatic stabilization through pi–pi stacking with Tyr84 contributed significantly to the anchoring of the ligand within the active site.

Similarly, Epicatechin gallate exhibited a strong binding affinity of − 8.3 kcal·mol⁻¹, which is also higher than that of Pepstatin A. The docking pose revealed multiple hydrogen bonds involving Asp32, Asp86, and Thr221, as well as hydrophobic contacts with Ile119 and Ile123. The presence of aromatic rings enabled pi–pi stacking interactions with Tyr84, further stabilizing the ligand.

Rutin acid and quercetin dihydrate also displayed remarkable binding affinities of − 8.2 and − 8.0 kcal·mol⁻¹, respectively. In both cases, the interaction networks were enriched with hydrogen bonds to Asp218, Ser88, and Gly220, while pi–pi stacking with Tyr84 and extensive van der Waals interactions within the hydrophobic pocket enhanced binding stability. These features suggest a binding mode comparable to, and in some cases exceeding, that of the reference drug.

On the other hand, kaempferol showed a binding affinity equal to Pepstatin A (− 7.8 kcal·mol⁻¹), forming hydrogen bonds with Asp218 and Thr221 and establishing pi–pi stacking interactions with Tyr84. Although its affinity was slightly higher than that of Pepstatin A, the overall interaction network was less extensive, which may limit its inhibitory efficiency.

Additionally, catechin, epicatechin, chrysin, and chlorogenic acid exhibited binding affinities ranging from − 7.5 to − 7.7 kcal·mol⁻¹, values comparable to those of Pepstatin A. These compounds shared common interaction features, including hydrogen bonding with catalytic aspartate residues and hydrophobic interactions with Ile and Gly residues lining the active site. However, the reduced number of stabilizing aromatic interactions likely accounts for their slightly lower affinities relative to the top-scoring ligands.

In contrast, simpler phenolic acids, including gallic, vanillic acid, syringic acid, p-hydroxybenzoic acid, and cinnamic acid, demonstrated weaker binding affinities (− 5.4 to − 5.8 kcal·mol⁻¹). Their binding modes were primarily governed by limited hydrogen bonding and van der Waals interactions, with minimal contribution from hydrophobic or aromatic stabilization. Consequently, these compounds exhibited reduced binding stability compared to Pepstatin A.

Overall, the docking results indicate that several flavonoid derivatives, particularly Apigenin-7-glucoside, Epicatechin gallate, Rutin acid, and Quercetin dihydrate, bind more strongly to Candida albicans 1ZAP than the reference inhibitor Pepstatin A (− 7.6 kcal·mol⁻¹). The enhanced affinities are attributed to a synergistic combination of conventional H₂-bonds, hydrophobic contacts, and aromatic pi–pi interactions, especially with the key residue Tyr84. These findings suggest that such compounds represent promising candidates for further antifungal investigation targeting Candida albicans. Generally, it should be emphasized that the molecular docking results presented in this study are predictive and should not be considered definitive evidence of biological activity or mechanism of action. Although several identified phytochemicals exhibited favorable binding affinities toward the selected microbial targets, experimental validation through enzyme inhibition assays, target-binding studies, and other mechanistic approaches is required to confirm the biological relevance of these predicted interactions.

Discussion

This gradient in extraction efficiency reflects both the polarity differences among solvents and the diverse phytochemical composition present in the aerial parts of S. pallida, as polar solvents generally extract a broader range of secondary metabolites (Swamy et al. 2017). The relatively high yield of the methanolic extract suggests the presence of abundant polar bioactive constituents, which aligns with previous findings highlighting the richness of Stellaria species in phenolics, flavonoids, and other polar compounds. Given the recognized therapeutic potential of the genus Stellaria, these results further support its value as a promising source of phytochemicals relevant to the development of new chemotherapeutic agents.

Phytochemicals have been used as medicines for countless years because they often play an essential role in a plant’s defense against predators, pathogens, stress, and interspecific threats (Ojha et al. 2023). Phenolic compounds contain hydroxyl groups that may significantly impact antimicrobial Activity (Liu et al., 2020). Comparative literature confirms similar variability across species of the genus Stellaria. (Thakur et al., 2021) estimated lower phenolic and flavonoid contents in the methanolic/water extract of S. monosperma aerial sections as (8.20 ± 0.434 and 1.63 ± 0.110 mg/g), respectively. (Miere Groza et al. 2021) reported that ethanol extract of S media contains moderate phenols and flavonoids levels (17.19 ± 1.32 mg GAE/g and 7.28 ± 1.180 mg QE/g, respectively. (Abu-Ziada et al. 2015) determined total polyphenol and flavonoid contents of S. pallida aerial parts as 19.5 mg/g and 58.5 mg/g, respectively. Our results, reflecting variations in TPC and TFC contents, could be due to differences in environmental conditions, harvesting time, and the type of solvent, and can explain the presence of diverse phytochemical constituents in the aerial sections of S. pallida. It is well established that environmental factors, including climate, soil properties, altitude, seasonal variation, and ecological conditions, may influence the biosynthesis and accumulation of secondary metabolites. Therefore, future investigations should examine the phytochemical variability of S. pallida collected from different geographical regions and harvesting seasons to understand better the influence of environmental factors on its biological properties.

The superior biological activity of the methanolic extract may be attributed to the presence of several bioactive constituents identified by GC-MS analysis. Many of these compounds have previously been associated with antioxidant and antimicrobial properties, suggesting that the observed activities are likely the result of synergistic effects among multiple phytochemicals rather than the action of a single compound. The higher recovery of these constituents by methanol further emphasizes the importance of solvent polarity in determining the functional properties of plant extracts (Mahmoud and Selim 2025).

The effectiveness of DPPH scavenging varied markedly according to solvent polarity. This relatively weak Activity in chloroform suggests that nonpolar or semi-polar phytoconstituents may not be the primary contributors to antioxidant potential in S. pallida. Nonpolar solvents typically extract terpenoids, sterols, and lipophilic compounds, which may have moderate or limited hydrogen-donating ability toward DPPH radicals. Ethanol is a polar solvent capable of extracting phenolics, tannins, and flavonoids—classes well known for their radical scavenging ability. The high Activity observed in methanol suggests that S. pallida contains abundant methanol-soluble phenolics and flavonoids, which are highly effective hydrogen or electron donors. These observations support the well-established concept that polar solvents are generally more effective in extracting phenolic antioxidants from medicinal plants.

Mechanistically, H₂O₂ scavenging by plant extracts is largely attributed to phenolic/flavonoid constituents via single-electron transfer (SET) and hydrogen-atom transfer (HAT) pathways. The solvent effect aligns with phytochemical extraction theory: more polar, protic solvents solubilize polyphenols far better than moderately polar ethyl acetate or nonpolar chloroform; consequently, they deliver higher antioxidant capacity.

The antimicrobial magnitude varied significantly with both the extraction solvent and the tested microorganism. The high coefficient of determination (r² = 98.67%) confirms that the model explains nearly all the variability in antimicrobial activity, reflecting the robustness of the experimental design. Polar solvents generally recover higher amounts of polar bioactives—phenolic acids, flavonoids, tannins—well-known for antimicrobial and membrane-active agents. Plant phenolics/flavonoids disrupt membranes, collapse proton motive force, and impair biofilms—mechanisms documented against Gram-positive and Gram-negative bacteria. More polar constituents typically diffuse farther through the hydrophilic agar matrix, resulting in larger zones of methanol and ethanol (Balouiri et al. 2016). The order of susceptibility highlights C. albicans as the predominant sensitive strain, followed by Gram-positive bacteria. Gram-positive bacteria lack an outer membrane, making them more permeable to phytochemicals.

In contrast, Gram-negative species possess an additional lipopolysaccharide-rich outer membrane that restricts the penetration of phenolic constituents. Although the investigations demonstrated inhibitory activity against representative Gram-positive, Gram-negative, and yeast strains, the present findings should be considered a preliminary assessment of the antimicrobial potential. Further investigations involving a broader collection of bacterial and fungal pathogens, including clinically relevant and multidrug-resistant isolates, are required to evaluate the antimicrobial spectrum of S. pallida extracts comprehensively. Moreover, although the methanolic extract exhibited measurable antimicrobial activity against the tested microorganisms, the obtained MIC values suggest moderate potency when compared with conventional antimicrobial drugs. This observation is not unexpected, as crude plant extracts contain complex mixtures of active and inactive constituents that may dilute the apparent activity of individual bioactive molecules. Further purification and characterization of the active fractions may result in substantially improved antimicrobial efficacy.

HPLC findings confirm that methanolic extract is the most efficient solvent for extracting phenolic and flavonoid substances from S. pallida. The high abundance of bioactive constituents strongly supports the detected antioxidant and antimicrobial agents. The identified phenolic constituents, particularly flavonoids and phenolic acids, have previously been reported to possess antioxidant and antimicrobial properties, which may partly explain the biological activities observed in the present study.

Docking scores reflect differences in molecular size, aromaticity, and functional group composition. Flavonoids possessing multiple hydroxyl groups and extended aromatic systems display superior binding affinities toward OpuAC compared with simpler phenolic acids and ampicillin. Several compounds exhibited favorable predicted binding affinities toward S. aureus DNA gyrase compared with the reference ligand under the applied docking protocol. However, these computational observations should be interpreted cautiously and require experimental validation. It is important to emphasize that the molecular docking results presented herein should be considered predictive rather than confirmatory. Although several compounds exhibited favorable binding affinities and interaction profiles with the selected microbial targets, experimental validation through enzyme inhibition assays, protein-binding studies, and other mechanistic investigations is required to verify the proposed interactions and biological relevance of these computational predictions.

Limitations and future perspectives

Despite the promising antioxidant and antimicrobial activities demonstrated by S. pallida extracts under in vitro conditions, several limitations should be acknowledged when considering their potential therapeutic applications. Further in vivo studies, toxicity assessments, pharmacokinetic investigations, and clinical evaluations are still required to validate their efficacy and safety. One limitation of the present study is that antimicrobial potency was evaluated using agar diffusion and agar dilution approaches, whereas broth microdilution-based MIC determination, which is considered a standardized quantitative method, was not performed. Therefore, the obtained antimicrobial data should be regarded as preliminary indicators of biological activity, and future studies employing broth microdilution assays are recommended to provide more accurate assessments of antimicrobial efficacy.

In addition, cytotoxicity evaluation on normal mammalian cell lines was not conducted. Although the extracts demonstrated promising biological activities in vitro, their safety, selectivity, and potential effects on non-target cells remain to be investigated. Future studies should incorporate cytotoxicity assays and determine selectivity indices to better assess the therapeutic potential of the identified bioactive constituents.

Moreover, although molecular docking provides useful insights into the potential interactions between phytochemical constituents and selected antimicrobial targets, these findings remain predictive in nature. Docking scores and interaction profiles do not constitute direct evidence of enzyme inhibition, target engagement, or antimicrobial mechanisms. Therefore, the present docking results should be considered preliminary computational observations that may guide future investigations. Further experimental studies, including enzyme inhibition assays, protein-binding analyses, and mechanistic validation experiments, are required to confirm the biological relevance of the predicted interactions.

Conclusion

The present study demonstrated that Stellaria pallida (Dumort.) Pire is an abundant source of potential secondary metabolites exhibiting notable antioxidant and antimicrobial properties. Successive extraction using solvents of different polarities revealed that methanol was the most effective solvent, yielding the highest extract recovery and consistently superior biological activities. The presence of active components, including rutin, chlorogenic acid, ferulic acid, and apigenin, was determined in the methanol extract using HPLC, which showed strong radical-scavenging effects in both DPPH and H₂O₂ detection. The methanolic extract of S. pallida demonstrated promising but moderate antimicrobial activity against the tested microorganisms, supporting its potential as a source of bioactive compounds for further phytochemical and pharmacological investigations. The molecular docking analysis provided preliminary computational insights into potential interactions between the identified phytochemicals and selected microbial targets. However, these findings should be regarded as hypothesis-generating observations that require further experimental validation. The results of this study will enhance our understanding of the phytochemistry and biological effects of S. pallida, as well as provide fundamental scientific knowledge to elucidate the bioactive and health-promoting properties for the development of natural products and nutraceutical opportunities. Future studies, however, might focus on developing new medications for pharmaceutical use.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Mohamed T. Selim, Mohamed E. Elnosary, Fatma Sh. Abd El-Gwaid. Idea development, experimental work, method design, detailed analysis, result verification, data management, initial manuscript drafting, and reviewing and revising the written content.

Funding

Not applicable.

Data availability

The data supporting the results of this study can be obtained from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.