Phytochemicals and Biofunctional Properties of Portulaca oleracea L.: Integrated in Vitro and in Silico Analyses

Abstract

Portulaca oleracea L. (purslane) is a nutritionally and pharmacologically important medicinal plant traditionally valued for its therapeutic benefits. This study aimed to comprehensively investigate the phytochemical composition, antioxidant, and anti-inflammatory activities of its aqueous extract, supported by in silico analyses. Total phenolic, flavonoid, alkaloid, and tannin contents were quantified as 9.85 mg GAE/g, 4.42 mg naringenin/g, 0.61 mg caffeine/g, and 0.05 mg epicatechin/g, respectively. HPLC profiling identified ferulic acid (268.33 μg/g), allantoin (140.66 μg/g), and oleracein A (321.98 μg/g) as major bioactive constituents. In vitro antioxidant assays (DPPH, ABTS, and FRAP) demonstrated strong radical scavenging and reducing power, while protein denaturation and erythrocyte membrane stabilization tests confirmed significant anti-inflammatory activity. In silico molecular docking analyses revealed high binding affinities of these compounds to inflammation and Helicobacter pylori protein targets, suggesting potential anti-inflammatory and antimicrobial mechanisms at the molecular level. These findings indicate that purslane is a rich source of bioactive molecules capable of exerting potent antioxidant and anti-inflammatory effects. The integration of phytochemical, in vitro, and in silico approaches provides strong scientific evidence supporting its therapeutic potential.

Keywords:
Portulaca oleracea ; phytochemicals; antioxidant; anti-inflammatory; in silico

GRAPHICAL ABSTRACT

Keywords:
Portulaca oleracea ; phytochemicals; antioxidant; anti-inflammatory; in silico

HIGHLIGHTS

• Purslane extract shows strong antioxidant and anti-inflammatory activities.

• Major bioactives identified: oleracein A, ferulic acid, and allantoin.

• In silico results suggest potential anti-inflammatory and antimicrobial targets.

INTRODUCTION

Functional foods are defined not only as nutritional sources that fulfill the body’s daily requirements for essential nutrients but also as dietary components that support physiological functions and contribute to the prevention and management of various diseases [¹-⁴]. In recent years, there has been a growing interest in naturally occurring wild plants rich in phytochemical compounds due to their health-promoting properties. Within this framework, Portulaca oleracea L. (commonly known as purslane), a plant traditionally used in folk medicine, has emerged as one of the high-potential species attracting increasing scientific attention as a functional food [⁵]. Belonging to the Portulacaceae family, purslane is widely consumed worldwide and is recognized for its rich nutritional composition. Historically, it was employed in ancient Greek medicine for the treatment of fever, stomachache, hemorrhoids, and wounds; in ancient Egypt, it served as a vegetable, medicinal, and aromatic plant; and during the Middle Ages, it gained popularity in England [⁶]. Native to the Middle East and India, purslane commonly grows in moist habitats such as stream banks, meadows, and the margins of vegetable gardens in Turkiye, where it is also cultivated on a limited scale in certain regions. Morphologically, purslane is distinguished by its prostrate, smooth, reddish stems and alternate, sessile leaves. Two primary leaf types exist—green and yellow—of which the green type is more vigorous and demonstrates higher flowering potential [⁷]. Nutritionally, purslane is an abundant source of vitamins (A, B1 [thiamine], B2, B6, C, E, niacin, folate, and β-carotene) and essential minerals including calcium (Ca), iron (Fe), magnesium (Mg), potassium (K), phosphorus (P), sodium (Na), copper (Cu), and manganese (Mn) [⁸]. Moreover, it is particularly notable for its content of unsaturated fatty acids, especially alpha-linolenic acid (ALA), a type of omega-3 fatty acid, with approximately 300–400 mg of ALA per 100 g of fresh leaves [⁹]. Due to these bioactive constituents, purslane exhibits considerable therapeutic potential, as evidenced by in vitro and in vivo studies demonstrating its promise in preventing cardiovascular diseases, neurological and allergic disorders, as well as in enhancing immune function [¹⁰]. Phytochemicals present in purslane, while not directly contributing to nutritional requirements, have been associated with pharmacological effects in the prevention of cancer, diabetes, hypertension, and peptic ulcers [¹¹]. Its high fiber content also plays an important role in digestive health [¹²], and its topical applications have traditionally included treatments for burns, acne, eczema, skin infections, and insect bites [¹³].

Antibacterial and antifungal activities of the plant have been verified in various studies. Helicobacter pylori infection represents a significant global health burden, being implicated in the pathogenesis of gastritis, gastroduodenal ulcers, gastric lymphoma, distal gastric cancer, and other gastrointestinal diseases [¹⁴]. The increasing prevalence of antibiotic resistance has markedly reduced the effectiveness of conventional triple or quadruple therapies, with eradication rates often falling below 80%. Additionally, the adverse effects associated with these regimens contribute to low patient compliance [¹⁵]. Consequently, there is an urgent need for alternative therapeutic strategies that are safer, more effective, and less susceptible to resistance development. Recent investigations have underscored the therapeutic promise of natural compounds derived from medicinal plants, emphasizing their low toxicity and high bioactivity, which may be attributed to their evolutionary compatibility with biological systems. In this context, edible and medicinal plants such as purslane are receiving growing attention for their potential prophylactic and therapeutic benefits against H. pylori infection and gastric inflammation [¹⁶]. In the present study, compounds identified by HPLC in the aqueous extracts of purslane cultivated in the Çubuk region of Ankara were subjected to comprehensive in vitro and in silico analyses to assess their potential anti-inflammatory and anti-Helicobacter pylori activities, with a particular focus on their functional and nutraceutical relevance.

MATERIAL AND METHODS

Plant material

The fresh aerial parts of purslane were cultivated under controlled conditions in the greenhouses of the Medicinal and Aromatic Plants Garden at GETAT, Ankara Yıldırım Beyazit University (Ankara, Çubuk), using seeds obtained from the Muratpasa District Directorate of Agriculture, Antalya. The plants were grown by MSc. Agricultural Engineer Zuhal Özkan. When the plant material reached maturity in May 2023, it was harvested, and a dried specimen was deposited in the GETAT Herbarium of Ankara Yıldırım Beyazit University with GETAT-2023-05-01 saving code.

Extraction of the plant

The freshly harvested purslane was washed with distilled water to remove any impurities. After removing excess water, the plants were left to dry in the shade for 48 hours. The dried plant material was then mixed with distilled water at a ratio of 1:100 and subjected to high-speed stirring for 2 hours, followed by maceration at +4°C for 24 hours. The resulting extract was filtered, freeze-dried using a laboratory type freeze dryer and stored in amber-colored bottles at –18°C until further use. The yield of the aqueous extract was calculated as 21.2 % w/w. To ensure batch reproducibility, all extractions were performed in triplicate under identical conditions.

Qualitative phytochemical analysis

Phytochemical screening was performed following standard protocols to identify phenolics, flavonoids, saponins, alkaloids, tannins, glycosides, and terpenoids in the extracts [¹⁷].

Tannins: 1 mL of purslane extract was combined with 2 mL of 5% ferric chloride solution. The development of a dark blue or greenish-black coloration was taken as evidence of tannins.

Saponins: 2 mL of purslane extract were mixed with 2 mL of distilled water and vigorously shaken in a graduated cylinder for 15 min. The formation of a stable foam layer (approximately 1 cm) indicated the presence of saponins.

Flavonoids: 2 mL of purslane extract were treated with 1 mL of 2N sodium hydroxide. The appearance of a yellow coloration confirmed the presence of flavonoids.

Alkaloids: 2 mL of purslane extract were acidified with 2 mL of concentrated hydrochloric acid, followed by the addition of a few drops of Mayer’s reagent. The formation of a green coloration or white precipitate was indicative of alkaloids.

Glycosides: 2 mL of purslane extract were treated with 3 mL of chloroform and 3 mL of 10% ammonium solution. The appearance of pink coloration was considered positive for glycosides.

Terpenoids: A mixture of 0.5 mL of purslane extract and 2 mL of chloroform was layered with 2 mL concentrated sulfuric acid. The development of a red-brown interface confirmed the presence of terpenoids.

Phenolics: 1 mL of purslane extract was mixed with 2 mL of distilled water and a few drops of 10% ferric chloride solution. The appearance of a blue or green coloration indicated phenolic compounds.

Quantitative phytochemical analysis

Total phenolic content (TPC)

The total phenolic content of the purslane extracts was quantified using a modified Folin–Ciocalteu procedure as outlined by Koraqi and coauthors (2024) [¹⁸]. In this assay, 200 μL of extract was mixed with 600 μL of 10% Folin–Ciocalteu reagent and kept in the dark for 5 min. Subsequently, 300 μL of 7.5% (w/v) Na₂CO₃ solution was added, and the mixture was incubated for 1 h in darkness. The absorbance was measured at 756 nm, and gallic acid was used to construct the calibration curve. Results were expressed as milligrams of gallic acid equivalents per gram of plant sample.

Total flavonoid content (TFC)

Flavonoid content was estimated following a modified protocol of Hasnat and coauthors (2024) [¹⁹]. Briefly, 500 μL of extract was combined with 25 μL of 5% sodium nitrite solution and allowed to react for 5 min. Then, 25 μL of 10% aluminum chloride solution was added, followed by 100 μL of 5N sodium hydroxide. The absorbance was determined at 510 nm using naringenin as the standard. Results were expressed as milligrams of naringenin equivalents per gram of plant.

Total alkaloid content

Alkaloid levels were measured using the spectrophotometric approach described by Krizhanovska and coauthors (2021) [²⁰]. 1 mL of extract was dissolved in 3 mL of 2N HCl and filtered. 1 mL of the filtrate was washed three times with 10 mL of chloroform, and the pH was adjusted to neutral using 0.1N NaOH. The neutralized solution was then reacted with 5 mL of bromocresol green reagent and phosphate buffer, followed by vigorous extraction with 10 mL of chloroform. The absorbance of the resulting complex was recorded at 273 nm, with caffeine serving as the calibration standard.

Total condensed tannin content

The condensed tannin concentration was determined according to the vanillin–HCl assay described by Dhull and coauthors (2016), using epicatechin as a standard. In brief, 400 μL of extract was mixed with 3 mL of 4% vanillin (in methanol) and 1.5 mL of concentrated HCl (37%) [²¹]. After incubation for 15 min at room temperature, the absorbance was measured at 500 nm. Results were expressed as mg of epicatechin equivalents per gram of plant.

Determination of bioactive components by HPLC analysis

The analysis of bioactive compounds in the aqueous extract of purslane was performed using a reversed-phase high-performance liquid chromatography (RP-HPLC) method, adapted from Marhri and coauthors (2024) with modifications [²²]. The chromatographic separation was carried out using a Shimadzu HPLC system (Kyoto, Japan) equipped with a GLC Science Inertsil ODS-4 analytical column (250 × 4.6 mm, 5 μm), an auto-sampler, degasser, column oven, and a photodiode array (PDA) detector. The mobile phase consisted of solvent A (0.1% acetic acid in water) and solvent B (acetonitrile), with a gradient elution starting at 70% B and decreasing to 15% B over a 60-minute run. The gradient profile was as follows: 0–15 min, 70% to 65% B; 15–20 min, 65% to 55% B; 20–30 min, 55% to 45% B; 30–35 min, held at 45% B; 35–40 min, 45% to 30% B; 40–45 min, 30% to 15% B; and 45–60 min, maintained at 15% B. The flow rate was set at 0.6 mL/min, and the column temperature was maintained at 40 ± 1°C. Before injection, all extract and standard solutions were filtered through 0.45 μm polytetrafluoroethylene (PTFE) syringe filters. An injection volume of 10 μL was used for all samples. The PDA detector recorded chromatograms in the wavelength range of 190–650 nm. Quantitative analyses were conducted at the maximum absorbance wavelengths specific to each standard compound (254, 280, 320, and 360 nm), and peak identification was based on retention times and UV spectra comparison with standards. Calibration curves and regression parameters for each compound are presented in Table 1. Statistical analyses were performed using Minitab software (Version 17, Minitab Inc., UK). Allantoin was quantified by HPLC in all three replicate batches and used as a standardization marker to confirm reproducibility.

Table 1.
Regression parameters of polyphenolic compounds identified in purslane by HPLC analysis

No.	Compounds	Regression equation y=m(x)+n	Correlation coefficient (r)	Linear calibration rangea (ug mL−1)	LODb (ug mL−1)	LOQb (ug mL−1)
1	Vanillin	y= 10065.74x-3411	0.9991	0.10-200	0.220	0.670
2	Ellagic acid	y= 9876.45x+1865	0.9995	0.50-250	0.930	2.790
3	Gentisic acid	y= 8965.11x+2491	0.9989	0.05-200	0.380	1.150
4	Ferulic acid	y = 533.22x+489.11	0.9992	0.05-250	0.120	0.360
5	4-HBA, 4-hydroxybenzoic acid	y= 1856.30x + 17453.6	0.9998	0.01-100	0.037	0.105
6	2-HBA, 2-hydroxybenzoic acid	y=7934.21x+ 59875	0.9987	0.01-100	0.125	0.752
7	Scopoletin	y= 5812x + 85.73	0.9978	0.05-100	0.085	0.170
8	p-coumaric acid	y=419076x + 23765	0.9969	0.20-250	0.450	1.980
9	Caffeic acid	y=78654.34x - 34568	0.9995	0.05-200	0.257	0.812
10	Allantoin	y=8.98543x + 0.22945	0.9998	0.01-100	0.054	0.160
11	Chlorogenic acid	y=141678x + 7654	0.9997	0.02-200	0.161	0.487
12	Quercetin	y=3487.67x - 245.98	0.9989	0.05-200	0.378	1.445
13	Quercetin-3-O-rhamnoside	y = 0.9715x + 0.0522	0.9991	0.01-100	0.053	0.167
14	Rutin	y=10638.54x+23451	0.9989	0.50-250	1.960	5.980
15	Protocatechuic acid	y=254.98x-56.96	0.9991	0.05-200	0.080	0.245
16	Quercetin-3-O-glucoside	y=1859.45x+1854.98	0.9998	0.20-250	2.875	9.456
17	Apigenin	y=2136.76x+297.56	0.9985	0.20-200	0.085	0.245
18	Epicatechin	y=4278.96x-215.47	0.9972	0.50-200	0.075	0.230
19	Oleracein A	y=29653.87x-674.76	0.9985	0.01-250	0.245	0.725

• ^a
  10 calibration points were studied for linearity range (n=10)
• ^b
  Three replicates were performed (n=3)
• LOD (μg/mL) = 3.3(SD of the response/slope), LOQ (μg/mL) = 10 (SD of the response/slope)

Preparation of standard solutions and construction of calibration curves

Standard compounds used in this study included vanilin, ellagic acid, gentisic acid, ferulic acid, 4-HBA- 4-hydroxybenzoic acid, 2-HBA-2-hydroxybenzoic acid, scopoletin, p-coumaric acid, caffeic acid, allantoin, chlorogenic acid, quercetin, quercetin-3-O-rhamnoside, rutin, protocatechuic acid, quercetin-3-O-glucoside, apigenin, epicatechin and oleracein-A. Prior to chromatographic analysis, all solvents and mobile phases were degassed to remove dissolved gases and filtered through 0.45 μm nylon membrane filters. For the preparation of calibration curves and determination of other validation parameters, all standard solutions were diluted from a 500 μg/mL stock solution using a H₂O–MeOH (20:80, v/v) mixture. Stock solutions and their dilutions were stored at –18°C in the dark. Working solutions were freshly prepared at ten concentration levels ranging from 0.2 to 250 μg/mL by serial dilution of the stock solutions. Calibration curves were constructed by plotting the peak area against the corresponding concentrations for each standard compound and were subsequently modeled (Table 1). All solutions were stored at +4°C throughout their shelf life.

Antioxidant activities

Determination of the DPPH-radical scavenging activity

The ability of the purslane extract to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was evaluated according to the Baliyan and coauthors (2022) [²³]. Briefly, 100 μL of extract was added to 2900 μL of 0.1 mM DPPH solution. The reaction mixtures were incubated in the dark for 40 min, after which the absorbance was measured at 517 nm.

Ferric reducing antioxidant power (FRAP)

The ferric reducing antioxidant power (FRAP) assay was performed with slight modifications according to the procedure described by González-Palma and coauthors (2016) [²⁴]. The FRAP reagent was freshly prepared by mixing 10 mmol/L TPTZ in 40 mmol/L HCl, 20 mmol/L FeCl₃, and phosphate buffer (pH 4) in a 1:1:10 ratio. Then, 100 μL of the extract was added to 2900 μL of the FRAP reagent, and the mixture was incubated at 37 °C in the dark for 15 min and absorbance was measured at 593 nm. The IC₅₀ value, corresponding to an absorbance of 0.5 for each sample, was determined by plotting the absorbance at 593 nm against the extract concentration.

Determination of the ABTS-radical scavenging activity

The radical scavenging activity of the purslane extract against the ABTS⁺ • radical cation was determined with minor modifications to the method described by Mokrani and coauthors (2025) [²⁵]. The ABTS⁺ • solution was generated by reacting 7 mM ABTS with 2.5 mM potassium persulfate (1:1, v/v) in water and incubating the mixture in the dark at room temperature for 24 h. Subsequently, 0.1 mL of the extract was added to the ABTS⁺ • solution and incubated in the dark for 7 min and absorbance was recorded at 734 nm. The percentage inhibition was calculated, and the IC₅₀ value was determined from the dose–response curve plotted between % inhibition and sample concentration. The IC₅₀ denotes the concentration required to quench 50% of ABTS free radicals. All assays were performed in triplicate to ensure accuracy and reproducibility.

Anti-inflammatory activity

Assay of membrane stabilization

To prepare the erythrocyte suspension, 5 mL of freshly collected sheep blood was centrifuged at 5000 rpm for 10 min to isolate red blood cells, which were subsequently washed three times with 10 mM sodium phosphate buffer. From the suspension obtained, 0.25 mL was mixed with 2.5 mL of 50 mM NaCl solution. Plant extracts were then added to the mixture, followed by incubation at room temperature for 10 min and centrifugation. The absorbance of the resulting supernatant was measured at 540 nm, with diclofenac sodium used as the standard [²⁶].

Inhibition of bovine serum albumin denaturation test

Plant extract or diclofenac sodium (standard) at different concentrations was mixed with 0.2% bovine serum albumin (BSA) prepared in Tris-buffered saline (pH 6.8). The samples were incubated at 37 °C for 20 min and subsequently heated for 10 min to induce denaturation above 70 °C. After cooling, the absorbance was measured at 660 nm [²⁷].

In silico analysis

Molecular docking

Chronic inflammation of the gastric mucosa is often associated with H. pylori infection, which predisposes to gastritis, peptic ulcer disease, and, in the long term, the development of gastric malignancies. H. pylori colonize the gastric mucosa, evades the host immune system, neutralizes gastric acid through its urease enzyme, and induces a chronic inflammatory response at the epithelial surface. This process is characterized by cytokine release, oxidative stress, and tissue damage. Based on their documented involvement in inflammatory signaling and H. pylori pathogenicity, RAC-alpha serine/threonine-protein kinase (PDB ID: 4EJN) and N-lysine methyltransferase SMYD2 (PDB ID: 3TG5) were selected as inflammation-related targets, whereas oxygen-insensitive NADPH nitroreductase (PDB ID: 3QDL) and HopQ (PDB ID: 5LP2) were chosen to evaluate anti-H. pylori activity. The three-dimensional structures of these proteins were retrieved from the Protein Data Bank. Protein preparation involved several steps: (i) energy minimization was performed using a steepest descent algorithm with a step size of 0.02 Å and an update interval of 10 steps, for a total of 100 iterations; (ii) removal of water molecules; (iii) deletion of solvent molecules and non-complexed ions; and (iv) addition of polar hydrogens and assignment of AM1-BCC charges as previously described [²⁸]. Ligand structures, corresponding to bioactive compounds identified through HPLC analysis of purslane extracts, were downloaded from the PubChem database in .sdf format. Similar to the proteins, ligands underwent preparation steps including charge assignment based on the Gasteiger method. Semi-flexible molecular docking simulations were carried out using AutoDock Vina to investigate molecular interactions between volatile bioactive compounds and target proteins. Initially, blind docking was performed to explore potential binding sites on the proteins, and the results were cross-referenced with literature data. Subsequently, focused docking was conducted using the identified coordinates. The docked conformations exhibiting the lowest binding energies were visualized using Discovery Studio Visualizer. The inhibition constant (Ki) was calculated using the formula provided below [²⁹],

$\Delta \text{G=-RTlnK}$

ΔG = Binding affinity (kcal/mol) Ki = Inhibition constant R=1.987×10-3 kcalmol-1K-1 (Gas constant) T=298.15 K (absolute temperature)

PASS analysis

The PASS (Prediction of Activity Spectra for Substances) web-based platform is a computational tool that forecasts the probable biological activities of chemical compounds by referencing data from known pharmacologically active molecules [³⁰]. Following the molecular docking evaluations, beta-ionone epoxide—identified as the compound exhibiting the most significant bioactivity potential—was selected for further investigation using PASS. This software generates predictions by assigning two key probabilities: the likelihood of biological activity (Pa) and the likelihood of inactivity (Pi) across a range of pharmacological and biochemical functions.

ADMET profiling

The pharmacokinetic and toxicity-related properties of the selected compounds were evaluated through ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) analysis using the pkCSM online platform (https://biosig.lab.uq.edu.au/pkcsm). To perform the analysis, the SMILES format of the bioactive constituents was obtained from the PubChem database.

Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics v.27. One-way ANOVA followed by Tukey’s post hoc test was applied to determine statistically significant differences among groups (p < 0.05). Data are expressed as mean ± standard deviation (SD).

RESULTS and DISCUSSION

Qualitative analysis of phytochemicals

Preliminary phytochemical screening of purslane extract revealed qualitative distribution of bioactive compounds (Table 2).

Table 2.
Qualitative profiling of bioactive compounds in aqueous extract of purslane

Phytochemical Constituents	Alkaloids	Tannins	Saponins	Phenolics	Flavonoids	Glycosides	Terpenoids
Water Extract of Purslane	++	++	+	++	++	+	+

The qualitative phytochemical screening of the aqueous extract of purslane revealed the presence of diverse classes of secondary metabolites with varying intensities. Alkaloids, tannins, phenolics, and flavonoids were strongly detected (++), indicating that purslane is a rich source of polyphenolic and nitrogen-containing compounds that may contribute to its broad spectrum of biological activities. In addition, saponins, glycosides, and terpenoids were also identified at lower levels (+), suggesting that these constituents, although present in moderate amounts, could synergistically enhance the overall pharmacological potential of the extract. Okafor and coauthors (2014) demonstrated that the aqueous maceration extracts of Nigerian purslane were predominantly characterized by high levels of alkaloids, saponins, proteins, and starch (++), while only modest amounts of flavonoids and tannins (+) were present, highlighting variations in phytochemical composition depending on extraction conditions and plant origin [³¹].

Quantitative Analysis of Phytochemicals

The phytochemical analysis of purslane revealed significant levels of bioactive compounds, with total phenolics at 9.85 ± 0.21 mg GAE/g and flavonoids at 4.42 ± 0.32 mg naringenin/g, highlighting their primary role in the plant’s antioxidant and therapeutic potential. Alkaloids were present at 0.61 ± 0.1 mg caffein/g, and tannins at a low level of 0.05 ± 0.4 mg epicatechin/g, indicating a minor contribution to the overall bioactivity (Figure 1). Methanol extracts of purslane samples from Konya (Turkiye) exhibited total phenolic content ranging from 29.4 to 102.42 mg/100 g [³²]. In Iranian purslane sample methanol extracts, total phenolics and flavonoids were reported as 142.2 mg GAE/g and 81.2 mg QE/g, respectively [³³]. El Kashef and coauthors (2018) observed that Egypt samples showed total phenolic content of 263.52 mg/g in cold water extracts and 228.27 mg/g in hot water extracts, with corresponding flavonoid contents of 60.70 and 57.35 mg/g [³⁴]. In Tunisia, purslane water extracts contained 2.45 mg/g phenolics, 2.83 mg/g flavonoids, and 7.72 mg/g tannins [³⁵]. Additionally, ethanol extracts of purslane have been reported to contain 2.17 mg/g total alkaloids [³⁶].

Figure 1
Quantitative profiling of bioactive compounds in aqueous extract of purslane

HPLC analysis

The HPLC analysis of the aqueous extract of purslane revealed a diverse polyphenolic profile, with a total of 19 compounds identified at varying concentrations (Table 3 and Figure 2). Among the quantified metabolites, oleracein A (321.98 ± 16.87 μg/g DW) and ferulic acid (268.23 ± 9.21 μg/g DW) were the most abundant, clearly indicating that purslane is a rich source of hydroxycinnamic acid derivatives and cyclodopa alkaloids, which are well-known for their potent antioxidants and anti-inflammatory properties [³⁷]. Allantoin (140.66 ± 4.67 μg/g DW) was also present in remarkably high amounts, consistent with its reported role in wound healing and cytoprotective mechanisms. The presence of scopoletin (38.19 ± 2.65 μg/g DW), chlorogenic acid (23.25 ± 1.98 μg/g DW), and vanillin (18.34 ± 1.54 μg/g DW) further supports the pharmacological relevance of the extract, as these compounds have been associated with antimicrobial, hepatoprotective, and neuroprotective effects [⁴]. On the other hand, flavonoids such as rutin, quercetin, and apigenin, detected at relatively low concentrations (<6 μg/g DW), are thought to reflect species-specific variations, as also observed in the qualitative analyses [³⁸].

Figure 2
Chromatogram of the aqueous extract of purslane obtained by HPLC analysis

Table 3.
Quantification of polyphenolic compounds in the purslane extract determined by HPLC

No	Compounds	Polyphenolics amount μg/g (DW)
1	Vanillin	18.34 ± 1.54
2	Ellagic acid	5.23 ± 0.23
3	Gentisic acid	7.39 ± 0.45
4	Ferulic acid	268.23 ± 9.21
5	4-HBA, 4-hydroxybenzoic acid	14.56 ± 0.98
6	2-HBA, 2-hydroxybenzoic acid	8.65 ± 0.39
7	Scopoletin	38.19 ± 2.65
8	p-coumaric acid	2.98 ± 0.13
9	Caffeic acid	5.94 ± 0.19
10	Allantoin	140.66 ± 4.67
11	Chlorogenic acid	23.25 ± 1.98
12	Quercetin	3.67 ± 0.28
13	Quercetin-3-O-rhamnoside	2.68 ± 0.09
14	Rutin	5.01 ± 0.11
15	Protocatechuic acid	11.76 ± 0.87
16	Quercetin-3-O-glucoside	0.12 ± 0.04
17	Apigenin	0.34 ± 0.07
18	Epicatechin	0.48 ± 0.11
19	Oleracein A	321.98 ± 16.87

The predominance of oleracein A and ferulic acid in the purslane extract is consistent with previous phytochemical studies that identified these metabolites as characteristic constituents and emphasized their contribution to the plant’s strong antioxidant capacity. For instance, Ai and coauthors (2015) investigated the polyphenolic content of purslane samples collected from different regions of China and highlighted the significant impact of geographical variation on metabolite levels [³⁹]. While the Beijing and Changde samples were particularly rich in p-coumaric acid (572.9 μg/g) and quercetin-3-O-rhamnoside (385.3 μg/g), our sample was predominantly enriched in ferulic acid (268.73 μg/g) and oleracein A, revealing a distinct phytochemical profile. Other compounds, including caffeic acid (5.94 μg/g vs. 27.2 μg/g), scopoletin (38.19 μg/g vs. 81.89 μg/g), quercetin (3.67 μg/g vs. 32.71 μg/g), and apigenin (0.34 μg/g vs. 164.7 μg/g), were detected at lower concentrations in our extract compared to the Chinese samples. In the methanolic extracts of the Egyptian samples, chlorogenic acid (140.32 ppm) and ellagic acid (54.92 ppm) were reported as the most abundant compounds, whereas other metabolites, including chlorogenic acid (140.32 ppm), caffeic acid (20.13 ppm), and ellagic acid (54.92 ppm), were detected at lower levels in our aqueous extract [⁴⁰]. The ethanolic extracts of the Italian purslane samples were reported to be particularly rich in quercetin (14.14 μg/g), p-coumaric acid (18.77 μg/g), and rutin (6.17 μg/g) [⁴¹]. Other metabolites, including caffeic acid (5.58 μg/g), quercetin (14.14 μg/g), rutin (6.17 μg/g), and p-coumaric acid (18.77 μg/g), were present at comparatively lower concentrations in our aqueous extract. These comparative data indicate that the biochemical composition of purslane may vary depending on geographical and environmental factors, potentially influencing the concentration of specific bioactive compounds [⁴²].

Antioxidant activity

The aqueous extract of purslane exhibited notable antioxidant activity (Figure 3), with IC₅₀ values of 1.17 ± 0.02 mg/ml (DPPH), 1.27 ± 0.01 mg/ml (ABTS), and 3.82 ± 0.1 mg/ml (FRAP). The strongest effect was observed in the DPPH assay, indicating a high hydrogen-donating capacity of the extract, whereas comparatively higher IC₅₀ values in ABTS and FRAP systems. These findings align with the phytochemical composition of purslane, where phenolic and flavonoid constituents are likely the major contributors to its antioxidant potential.

Figure 3
Antioxidant activities (IC50 expressed as mg/ml) of water extracts from purslane

In the study by Mohamed Ahmed and coauthors (2025), methanolic extracts of purslane exhibited 53.23% DPPH inhibition and 147.78 μmol Trolox/100 g in the ABTS assay [³²]. Similarly, Keser and coauthors (2021) reported 47.13% DPPH inhibition in methanolic extracts [⁴³]. Cannavacciuolo and coauthors (2025) investigated Italian purslane and reported IC₅₀ values of 0.23 mg/ml (DPPH) and 0.59 mg/ml (ABTS) in infusion samples, while the hydrolyzed fractions demonstrated IC₅₀ values of 0.17 and 0.87 mg/ml, respectively [⁴⁴]. Compared with these reports, the aqueous extract in the present study indicates a relatively strong antioxidant potential, particularly in the DPPH and ABTS assays. Although slightly less potent than methanolic or hydrolyzed fractions reported in previous studies, the results confirm that purslane is a valuable and readily accessible natural source of phytochemicals and antioxidants, with its activity strongly influenced by the extraction solvent and preparation method.

Anti-inflammatory activity

The inhibition of heat-induced HRBC membrane lysis (stabilization of HRBC membrane) and protein denaturation was considered as measure of anti-inflammatory ability. The extract exhibited 89% inhibition of membrane lysis, indicating a strong ability to protect erythrocyte membranes from hypotonic-induced damage (Figure 4). In the protein denaturation assay, the extract demonstrated 91% inhibition, reflecting its capacity to prevent the denaturation of proteins, a key mechanism in inflammation. For comparison, diclofenac sodium, a standard anti-inflammatory drug, showed 98% inhibition in these assays.

Figure 4
Anti-inflammatory activity of purslane aqueous extract (% inhibition) compared to diclofenac sodium as a standard control

Studies on methanol extracts of purslane have reported protein denaturation inhibition values ranging between 70–80% [⁴⁵,⁴⁶]. Protein denaturation is a key mechanism in inflammatory processes, as the disruption of cellular protein structural integrity facilitates the activation of inflammatory mediators [⁴⁷-⁴⁹]. Therefore, the capacity of plant extracts to inhibit protein denaturation represents a significant indicator of their anti-inflammatory potential. Similarly, membrane stabilization studies evaluate the resistance of erythrocyte membranes to hemolysis, thereby highlighting a mechanism that contributes to inflammation prevention through the maintenance of cellular integrity [⁵⁰]. The effects of purslane extract on both mechanisms support its potential use as a natural anti-inflammatory agent and provide fundamental scientific evidence for pharmacological applications.

Binding energies and inhibition constants

Molecular docking is a well-established in silico approach widely employed in structure-based drug design. This method facilitates the identification of potential therapeutic agents by enabling the prediction of ligand–target interactions at the three-dimensional level. Furthermore, it allows for the elucidation of structure–activity relationships (SAR) without the necessity for prior knowledge of the chemical structures of known target modulators. In recent years, advancements in computational techniques such as molecular modeling, docking algorithms, and molecular dynamics simulations have laid the groundwork for novel strategies aimed at evaluating the pharmacological potential of both synthetic and natural compounds [⁵¹]. As a result of molecular docking analyses, the binding affinities of various phenolic and flavonoid compounds against four different target proteins were assessed. Among these, quercetin-3-O-rhamnoside, oleracein A, and ellagic acid exhibited the highest binding scores overall. Notably, quercetin-3-O-rhamnoside demonstrated the strongest interactions, with binding energies ranging from –11.4 to –10.7 kcal/mol across all target proteins. Similarly, oleracein A and ellagic acid also stood out with significantly high binding affinities, exhibiting ΔG values of –10.7 and –10.0 kcal/mol, respectively. In contrast, certain low molecular weight phenolic compounds such as gentisic acid, ferulic acid, and 2-/4-hydroxybenzoic acid showed weaker binding affinities toward all target proteins, with docking scores ranging between –5.5 and –7.1 kcal/mol (Table 4).

Table 4.
Molecular docking scores and inhibition constants of lead compounds against specific protein targets

Binding Scores (kcal/mol) and Predicted inhibition constant μM
No.	Bioactive Compounds of Purslane	4EJN		3TG5		3QDL		5LP2
1	Vanillin	-8.3	0.872	-6.2	29.25	-.5.9	47.60	-7.1	6.23
2	Ellagic acid	-10.0	0.046	-6.9	9.16	-8.3	0.872	-7.0	6.93
3	Gentisic acid	-6.2	29.25	-6.0	39.81	-5.5	71.31	-6.4	22.13
4	Ferulic acid	-6.4	22.13	-6.2	29.25	-6.0	39.81	-6.2	29.25
5	4-HBA, 4-hydroxybenzoic acid	-7.1	6.23	-6.1	34.32	-5.8	52.96	-6.0	39.81
6	2-HBA, 2-hydroxybenzoic acid	-7.1	6.23	-6.1	34.32	-5.8	52.96	-6.0	39.81
7	Scopoletin	-7.6	2.95	-6.0	39.81	-6.4	22.13	-7.1	6.23
8	p-coumaric acid	-6.6	17.08	-6.1	34.32	-6.5	19.73	-8.2	0.961
9	Caffeic acid	-6.3	25.59	-5.9	47.60	-6.4	22.13	-6.9	9.16
10	Allantoin	-6.3	25.59	-6.1	34.32	-6.5	19.73	-6.7	12.84
11	Chlorogenic acid	-9.4	0.103	-6.9	9.16	-8.3	0.872	-7.1	6.23
12	Quercetin-3-O-rhamnoside	-11.4	0.009	-7.9	2.34	-8.3	0.872	-10.7	0.020
13	Quercetin	-8.2	0.961	-7.0	6.93	-7.9	2.34	-8.9	0.376
14	Rutin	-8.9	0.376	-7.0	6.93	-8.0	1.93	-8.8	0.430
15	Protocatechuic acid	-8.7	0.478	-6.8	10.76	-7.5	3.56	-8.1	1.38
16	Quercetin-3-O-glucoside	-9	0.343	-6.5	19.73	-7.9	2.34	-8.3	0.872
17	Apigenin	-9.4	0.103	-6.4	22.13	-8.0	1.93	-7.8	1.61
18	Epicatechin	-9.1	0.313	-6.3	25.59	-7.3	4.70	-7.9	2.34
19	Oleracein A	-10.7	0.020	-8.6	0.465	-8.4	0.796	-9.1	0.313

According to the analysis of interactions, quercetin-3-O-rhamnoside exhibits numerous strong interactions within the active site of the Akt protein (Figure 5). The ligand binds through conventional hydrogen bonds, π-π stacking, π-alkyl interactions, and van der Waals contacts. Notably, the π-π stacking and hydrogen bonds formed with key residues such as TRP80 and ASP292 contribute significantly to the high affinity of the compound for the protein. Oleracein A demonstrates a stable binding profile in the binding pocket of N-lysine methyltransferase SMYD2 (Fig. 2B) through hydrogen bonds and π-alkyl interactions. Critically, hydrogen bonds with residues ARG19 and ASN206 serve as important anchoring points for its inhibitory activity. Additionally, van der Waals interactions involving residues such as CYS262 further stabilize the position of the ligand. On the other hand, in the interactions of the same ligand with H. pylori RdxA (Fig. 2C), hydrogen bonds and π-alkyl interactions established with residues ALA183, SER46, and SER134 reinforce the binding of the ligand. These interactions are thought to contribute to the potential of the compound to inhibit the enzyme’s activity. Conversely, the interaction of quercetin-3-O-rhamnoside with its target protein (Fig. 2D) revealed unfavorable interactions that indicate thermodynamic instability, suggesting that a long-lasting stable complex may not form [⁵²]. This phenomenon, predominantly observed at residue LYS178, is thought to result from interactions between the positive charge of the lysine side chain and the aromatic rings of the ligand [⁵³].

Figure 5
2D binding interactions of A 4EJN protein with Quercetin-3-O-rhamnoside B 3TG5 protein with Oleracein A C 3QDL protein with Oleracein A and D 5LP2 protein with Quercetin-3-O-rhamnoside.

PASS calculations

PASS prediction analysis revealed that the bioactive compounds of purslane exhibit significant pharmacological activities (Table 5). Among the 19 bioactive compounds evaluated, the highest Pa value was observed for quercetin-3-O-rhamnoside with free radical scavenging activity (Pa = 0.974). This was followed by caffeic acid (Pa = 0.945), p-coumaric acid (Pa = 0.931), and epicatechin (Pa = 0.962). Notably, flavonoids with antioxidant potential (quercetin, rutin, apigenin) and phenolic acids (caffeic acid, ferulic acid, chlorogenic acid) stood out with high Pa values (>0.85). Some compounds, particularly those with superoxide dismutase inhibitory activity (4-HBA and 2-HBA), exhibited remarkably high predicted values (Pa > 0.90).

Table 5.
Results of PASS calculations for inflammation and gastric ulcer properties

No.	Bioactive Compounds of Purslane	Activity	Pa	Pi
1	Vanillin	JAK2 expression inhibitor	0,886	0,004
2	Ellagic acid	Antiinflammatory	0,749	0,010
3	Gentisic acid	Antiinfective	0,847	0,004
4	Ferulic acid	Antiulcerative	0,605	0,011
5	4-HBA, 4-hydroxybenzoic acid	Superoxide dismutase inhibitor	0,906	0,004
6	2-HBA, 2-hydroxybenzoic acid	Superoxide dismutase inhibitor	0,904	0,003
7	Scopoletin	Caspase 3 stimulant	0,866	0,004
8	p-coumaric acid	Mucomembranous protector	0,931	0,004
9	Caffeic acid	Mucomembranous protector	0,945	0,003
10	Allantoin	Omptin inhibitor	0,476	0,088
11	Chlorogenic acid	Lipid peroxidase inhibitor	0,855	0,003
12	Quercetin-3-O-rhamnoside	Free radical scavenger	0,974	0,001
13	Quercetin	Antioxidant	0,872	0,003
14	Rutin	Antiulcerative	0,586	0,012
15	Protocatechuic acid	Antiseptic	0,906	0,003
16	Quercetin-3-O-glucoside	Apoptosis agonist	0,792	0,009
17	Apigenin	TNF expression inhibitor	0,609	0,012
18	Epicatechin	Mucomembranous protector	0,962	0,003
19	Oleracein A	Antiulcerative	0,580	0,013

The obtained PASS prediction results indicate that the phytochemical constituents of purslane may possess diverse biological activities. It is well established that most of the therapeutic properties predicted by the PASS tool have also been demonstrated through in vitro and in vivo studies. In agreement with the prediction results, Kolgazi and coauthors (2021) reported that caffeic acid alleviates gastric mucosal damage in the stomach [⁵⁴]. Similarly, the therapeutic effects of ferulic acid and rutin on gastric ulcers have been documented in both in vitro and in vivo models [⁵⁵,⁵⁶]. Regarding oleraceins, although their high pharmacological potential has been previously recognized [⁵⁷], their potential antibacterial properties [⁴³] and effects on ulcerative colitis have recently emerged as novel research topics [⁵⁸].

ADMET predictions

The ADMET analysis was conducted to evaluate the pharmacokinetic properties of 19 bioactive compounds identified in the extract of purslane (Table 6). The molecular weights of the compounds ranged from 138.12 to 610.52 g/mol, while log P values were between −0.28 and 1.97. Log S values varied from −3.94 to 0.68, indicating that most of the compounds possess a favorable solubility profile under gastrointestinal conditions.

Table 6.
Predicted ADMET properties of the bioactive compounds

No	Compounds	Molecular Weight (g/mol)	Log Po/w (iLOGP)	Log S (Solubility)	GI absorption	BBB permeant	Lipinski	Bioavailability Score	AMES toxicity
1	Vanillin	152.15	1.57	-1.82	High	Yes	Yes; 0 violation	0.55	No
2	Ellagic acid	302.19	0.79	-2.94	High	No	Yes; 0 violation	0.55	No
3	Gentisic acid	154.12	0.49	-2.23	High	No	Yes; 0 violation	0.56	No
4	Ferulic acid	194.18	1.62	-2.11	High	Yes	Yes; 0 violation	0.85	No
5	4-HBA, 4-hydroxybenzoic acid	138.12	0.85	-2.07	High	Yes	Yes; 0 violation	0.85	No
6	2-HBA, 2-hydroxybenzoic acid	138.12	1.13	-2.50	High	Yes	Yes; 0 violation	0.85	No
7	Scopoletin	192.17	1.86	-2.46	High	Yes	Yes; 0 violation	0.55	No
8	p-coumaric acid	164.16	0.95	-2.02	High	Yes	Yes; 0 violation	0.85	No
9	Caffeic acid	180.16	0.97	-1.89	High	No	Yes; 0 violation	0.56	No
10	Allantoin	158.12	-0.28	0.68	Low	No	Yes; 0 violation	0.55	No
11	Chlorogenic acid	354.31	0.87	-1.62	Low	No	Yes; 1 violation	0.11	No
12	Quercetin	302.24	1.63	-3.16	High	No	Yes; 0 violation	0.55	No
13	Quercetin-3-O-rhamnoside	448.38	1.60	-3.33	Low	No	No; 2 violations	0.17	No
14	Rutin	610.52	0.46	-3.30	Low	No	No; 3 violations	0.17	No
15	Protocatechuic acid	154.12	0.66	-1.86	High	No	Yes; 0 violation	0.56	No
16	Quercetin-3-O-glucoside	464.38	0.94	-3.04	Low	No	No; 2 violations	0.17	No
17	Apigenin	270.24	1.89	-3.94	High	No	Yes; 0 violation	0.55	No
18	Epicatechin	290.27	1.47	-2.22	High	No	Yes; 0 violation	0.55	No
19	Oleracein A	503.46	1.97	-2.85	Low	No	No; 3 violations	0.11	No

The ADMET analysis provided important insights into the drug-likeness, pharmacokinetic behavior, and potential safety profiles of the identified bioactive compounds. Most phenolic acids and low-molecular-weight compounds complied fully with Lipinski’s rule of five, showing no violations and favorable molecular weight and lipophilicity values. This indicates good oral drug-likeness and supports their potential for systemic absorption. Compounds such as ferulic acid, p-coumaric acid, 4-hydroxybenzoic acid, 2-hydroxybenzoic acid, and scopoletin exhibited high gastrointestinal absorption and the highest bioavailability scores (0.85), suggesting strong potential for oral administration. In contrast, flavonoid glycosides and high-molecular-weight compounds, including rutin, quercetin-3-O-rhamnoside, and oleracein A, showed lower GI absorption and multiple Lipinski violations, indicating limited oral bioavailability. These compounds may require formulation strategies to enhance their absorption. Regarding safety, none of the evaluated compounds showed AMES toxicity, suggesting a low mutagenic risk. Furthermore, the moderate log P and favorable solubility profiles of most compounds indicate a balanced distribution between aqueous and lipid phases, which may reduce nonspecific tissue accumulation and toxicity risks. Blood–brain barrier permeability was mainly observed for small phenolic acids, whereas larger flavonoids and glycosides were non-permeant. This selective permeability suggests a lower risk of central nervous system side effects for most compounds.

Overall, the ADMET results indicate that purslane extract is particularly rich in phenolic acids with favorable drug-likeness, bioavailability, and safety profiles, supporting its potential application in nutraceutical and functional food development

CONCLUSION

In this study, the aqueous extract of Portulaca oleracea demonstrated substantial antioxidant and anti-inflammatory activities supported by phytochemical, in vitro, and in silico analyses. The extract exhibited notable antioxidant capacity, with IC₅₀ values of 1.17 ± 0.02 mg/mL (DPPH), 1.27 ± 0.01 mg/mL (ABTS), and 3.82 ± 0.10 mg/mL (FRAP), indicating strong radical scavenging and reducing potential. HPLC profiling confirmed the presence of major bioactive constituents, including ferulic acid (268.33 μg/g), oleracein A (321.98 μg/g), and allantoin (140.66 μg/g), which are likely responsible for the observed biological effects. The extract also exhibited pronounced anti-inflammatory activity, achieving 89% inhibition of erythrocyte membrane lysis and 91% inhibition of protein denaturation. Furthermore, molecular docking analyses revealed high binding affinities of these compounds toward inflammation-related and Helicobacter pylori protein targets, suggesting plausible molecular mechanisms underlying the observed activities.

Overall, these findings highlight purslane as a valuable source of functional bioactive compounds with significant antioxidant and anti-inflammatory potential. However, further in vivo studies are required to evaluate toxicity, pharmacokinetics, and therapeutic efficacy. In addition, isolation and detailed characterization of the active constituents are necessary to support future therapeutic and nutraceutical applications.

Funding:

This research received no external funding

Institutional Review Board Statement:

Not applicable

Informed Consent Statement:

Not applicable

Data Availability Statement:

Research data are only available upon request for corresponding author.

Acknowledgments:

This study is derived from the doctoral dissertation of Veliddin Çiftçi, carried out under the supervision of Assoc. Prof. Dr. Nilüfer Vural at Ankara Yıldırım Beyazit University.

Use of Generative Artificial Intelligence

The author declare that did not use the artificial intelligence.

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Editor-in-Chief:

Paulo Vitor Farago

Associate Editor:

Jane Manfron

*

Correspondence: [e-mail] (N.V.)

Conflicts of Interest:

The authors declare no conflict of interest.

Publication Dates

• Publication in this collection
  28 Sept 2026
• Date of issue
  2026

History

• Received
  01 Nov 2025
• Accepted
  31 Mar 2026