HPLC-DAD Characterization and Bioactivity of Hammada scoparia Phenolic Extract from Brezina, Algeria

INTRODUCTION

Reactive Oxygen Species (ROS) are continuously produced during normal cellular metabolism, and when their production exceeds the capacity of endogenous antioxidant defenses, the ensuing oxidative stress leads to lipid peroxidation, protein damage, and DNA mutation, mechanisms involved in aging, cardiovascular disease, and carcinogenesis.¹

Plant-derived polyphenols, a structurally diverse group including phenolic acids, flavonoids, tannins, lignans, and stilbenes, are recognized as active free-radical scavengers owing to their aromatic rings and hydroxyl substituents, and they have consequently attracted considerable interest as natural alternatives to synthetic antioxidants in food and pharmaceutical applications.^1,2 Beyond acting as antioxidants, polyphenolic compounds display notable antimicrobial effect, acting through different mechanisms such as disruption of bacterial membrane integrity, inhibition of protein and nucleic acid synthesis, and induction of intracellular ROS accumulation in microbial cells.³

In the current context of rising antibiotic resistance among both Gram-positive and Gram-negative pathogens, plant extracts rich in phenolic constituents are increasingly explored as complementary or alternative antibacterial agents, since their multi-target mode of action reduces the likelihood of resistance development compared with single-target synthetic antibiotics.^3,4 Parallel to this, several classes of phenolic acids and flavonoids have been shown to interfere with tumor cell proliferation by modulating apoptotic pathways, cell-cycle regulators, and signal transduction cascades, making phenolic-rich plant extracts candidates for complementary anticancer strategies, as illustrated by studies linking polyphenol content to antiproliferative effects against cultured cancer cell lines.⁵

Desert and semi-arid ecosystems host an endemic flora adapted to extreme abiotic stress, including drought, intense UV radiation and salinity, Factors that promote the biosynthesis of secondary metabolites, particularly phenolic compounds, as part of the plant's protective response.² Xerophytic species from these environments therefore represent a comparatively underexplored reservoir of bioactive phytochemicals. Among them, Hammada scoparia (Pomel) Iljin, synonymous with Haloxylon scoparium Pomel and Arthrophytum scoparium, is a shrub of the family Amaranthaceae (formerly Chenopodiaceae) native to the semi-arid and Saharan regions of North Africa and the Middle East, where it is frequently a dominant component of steppe vegetation.⁶

Hammada scoparia occupies an important place in North African traditional medicine, where decoctions of its aerial parts have long been used to treat diabetes, scorpion sting and snakebite, stomachache, oral infections, and inflammatory conditions, and to promote wound healing.⁷ These ethnopharmacological uses have prompted a growing body of phytochemical research. Preliminary screenings have revealed that the plant is rich in tannins, saponins, coumarins, alkaloids, flavonoids, and steroids, alongside appreciable total phenolic and flavonoid contents.^8,9 More detailed chromatographic and spectroscopic investigations have identified individual constituents such as gallic, caffeic, and benzoic acids, rutin, quercetin, myricetin, naringenin, and isorhamnetin glycosides, together with alkaloids unique to the Hammada genus.^9,10 Pharmacological evaluations conducted to date support antidiabetic, antioxidant, antimicrobial, wound-healing, and cytotoxic properties for extracts of this species, reinforcing the relevance of its established applications.^7,11–13

Despite this accumulating evidence, the polyphenolic profile of H. scoparia and its relationship to biological activity remain incompletely characterized, and comparatively few studies have combined precise chromatographic identification of individual phenolic compounds with a parallel assessment of antioxidant, antibacterial, and antiproliferative properties in the same extract. High-performance liquid chromatography coupled with diode-array detection (HPLC-DAD) offers a reliable and widely used approach for the separation, identification, and quantification of individual phenolic acids and flavonoids in complex plant matrices, allowing bioactivity to be more directly related to specific chemical constituents rather than to crude extract content alone.¹¹

Building on this rationale, the present study aimed to determine the total polyphenol content of H. scoparia extract, to establish a detailed phenolic profile using HPLC-DAD, and to evaluate the resulting antioxidant, antibacterial, and antiproliferative activities. This integrated approach is intended to clarify the phytochemical basis of the biological effects reported for this species, especially research on this species remains scarce in western Algeria. To the best of our knowledge, this is the first study providing a detailed HPLC-DAD characterization of phenolic compounds in H. scoparia from the Brézina region (El Bayadh, Algeria), integrated with comprehensive bioactivity evaluation, to provide new insights of the relationship between regional phytochemical variation and biological potential. Furthermore, this study supports its potential valorization in food preservation, antimicrobial therapy, and complementary anticancer approaches.

MATERIAL AND METHODS

Chemicals and reagents

Folin-Ciocalteau’s reagent and 1, 1- diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma Aldrich (Steinheim, Germany). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-Diphenyl-2h-Tetrazolium Bromide (MTT) was obtained from Sigma Aldrich (St. Louis, MO). All other chemicals and reagents used were of analytical grade.

Plant material

The aerial parts of Hammada scoparia (Pomel) Iljin (Figure 1), a member of the Chenopodiaceae family, are locally known in Algeria as ‘Rimth’, were collected from Brezina region, El-Bayadh (Figure 2), Algeria in July 2025. The specimen has been deposited at the Herbarium of the CNDRB (Laguermi Annexe, El-Bayadh, Algeria) for reference, and the identification of the plant was carried out at its seed bank. The plant material was dried for ten days at room temperature under shade, then crushed, ground, and stored in a dry place.

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Ultrasound-assisted extraction

Ten grams of dried plant powder were extracted three times with 100 mL of 70% methanol in an ultrasound bath (30 min, 50ºC, 25 rpm), these selected conditions were chosen based on literature reports, with slight adjustments, demonstrating high efficacy for phenolic retrieval.¹² Extract was filtered, solvent was removed in a rotary evaporator, and crude residue stored at 4ºC in the dark until use in experiments. Primary trials confirmed reproducibility under these conditions The formula below was used to calculate the yield of the extract:¹²

The reproducibility was confirmed with a Relative Standard Deviation (RSD) below 5%. These results support the efficiency of the chosen protocol and provide the basis for subsequent determination of polyphenol content.

Total polyphenols

The total polyphenol content was measured using the Folin-Ciocalteu method with gallic acid as the standard, as highlighted in previous study.¹³ The method is based on the reduction of phosphotungstic acid in a basic medium. In a 5 mL test tube, 20 µL of extract, 1.58 mL of water, 100 µL of Folin-Ciocalteu reagent, and 300 µL of 20% (w/v) Na₂CO₃ solution were mixed. The mixture was incubated in the dark for 2 hr at 25ºC, and absorbance was measured at 765 nm. Results were expressed in milligrams of gallic acid equivalent per gram of dry weight (mg EAG/g of dry weight).

Identification of phenolic compounds by HPLC-DAD

The concentration of phenolic compounds in the H. scoparia methanolic extract were identified using an Agilent 1200 Infinity HPLC system equipped with a Diode-Array Detector (DAD) and a Zorbax C18 column (4.6 mm × 25 cm, 3 µm) at 40 ºC. Gradient elution was performed with two mobile phases: solvent A (water with 0.1% formic acid) and solvent B (acetonitrile) over 50 min, at a flow rate of 0.5 mL/min and injection volume of 5 µL. UV spectra of phenolics were recorded from 190-400 nm, with detection at 254, 280, and 330 nm. Peak identification was established by matching retention time and UV absorption spectra with those of reference standards analyzed under uniform chromatographic conditions; for structurally related polyphenolic compounds, identity was further validated through co-injection (spiking) of the extract with the corresponding reference standard, and no shift or splitting of the peak was observed, reinforcing the accuracy of peak assignment. Calibration curves were established using authentic standards as reported in earlier study,¹⁴ and results were expressed in mg/mL of extract and extrapolated to mg/g of dried plant. Calibration curves were established with standards; phenolics were expressed as mg/mL of extract and calculated as mg/g of dried plant․ Method validation was performed according to ICH guideline with respect to validated HPLC-DAD methods of phenolics analysis․ The standard calibration curves of phenolic standards were linear over 10 to 50 µg/mL with R² values of ≥ 0․99․ The Limit of Detection (LOD) and the Limit of Quantification (LOQ) were determined by a signal to noise ratio of 3 and 10 respectively and were between 0․046 and 0․322 µg/mL․ Intra-day and inter-day variability was determined by %RSD of 6 replicates and 3 replicates respectively․ These were found to be less than 2%․ In recovery studies‚ the addition of all spiked standards into the extract gave a recovery of 95% to 105%․ The system suitability tests and the acceptance criteria have been met‚ and the retention time‚ peak asymmetry‚ theoretical plate count‚ and chromatographic resolution between adjacent peaks exceeded 1.5, confirming adequate separation and reliability of the method. The method specificity was established by comparison of the UV spectra of each identified peak with those of the corresponding standard, and by the lack of co-eluting interfering peaks at the retention times of the target analyte. Consequently, all quantitative data reported herein were determined using this fully validated HPLC-DAD analytical method, confirming reliability of the quantified phenolic concentrations.

Antioxidant Activity

1, 1-Diphenyl-2-Picrylhydrazyl-radical scavenging assay (DPPH)

The free radical scavenging ability was evaluated using the 1, 1-Diphenyl-2-Picrylhydrazyl-radical scavenging assay (DPPH), as described previously,¹⁵ with small modifications. A 60 µM solution of DPPH in 70% methanol (1.950 mL) was mixed with 50 µL of plant extract at concentrations ranging from 2.5 to 200 µg/mL. After incubation for 30 min at room temperature in obscurity, absorbance was measured at 515 nm. Ascorbic acid served as the positive control. The inhibition percentage of DPPH radical was calculated using the formula given as follows:

Where IP is the inhibition percentage, A0 is the absorbance of the DPPH solution without extract, and A is the absorbance of the DPPH solution with the extract.

Ferric Reducing Antioxidant Power assay (FRAP)

The FRAP method was assessed to evaluate the total antioxidant potential of the H. scoparia methanolic extract. Different concentrations of the extract (2.5, 5, 15, 25, 50, 150, and 200 μg/mL) were mixed with 1250 µL of 1% (m/v) potassium ferricyanide and 1250 µL of 0.2 M phosphate buffer (pH 7). The mixture was incubated at 50ºC for 30 min. Subsequently, 1250 µL of 10% (m/v) Trichloroacetic Acid (TCA) was added, and the mixture and centrifuged at 6500 rpm for 10 min. The supernatant was then collected, and 1250 µL of deionized water and 250 µL of ferric chloride reagent were incorporated. The absorbance was assessed at 700 nm.²² Ferric-reducing antioxidant power (FRAP) values were calculated as follows:

Where: A_Control refers to the absorbance measured in the control solution, which contains all reagents except the sample, while A_Sample refers to the absorbance measured in the sample.

Antibacterial activity

Test organisms

Different pathogenic microorganisms, including four Gram‑positive and two Gram‑negative bacterial strains, were tested: Staphylococcus aureus (ATCC 25923), Listeria monocytogenes (CECT148), Clostridium perfringens (CECT486), Bacillus cereus (ATCC11778), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). The test organisms were obtained from American Type Culture Collection (ATCC) and the Spanish Type Culture Collection (CECT).

Agar diffusion method

The antibacterial activity of the extract was evaluated using the agar diffusion method. Bacterial suspensions were standardized to 0.5 McFarland (≈10⁸ CFU/mL) and inoculated onto Mueller-Hinton agar using a cotton swab. Sterile discs (6 mm) impregnated with 10 µL of extract solutions (0.1 g/mL) were placed on the agar surface. In parallel, standard antibiotics (Amoxicillin 30 µg/mL, Erythromycin 15 µg/mL, Amikacin 30 µg/mL, Fusidic acid 10 µg/mL, Ampicillin 10 µg/mL, and Gentamicin 10 µg/mL) were used as positive controls, whereas solvent discs (methanol) were used as negative control. Plates were incubated at 37 ºC for 24 hr, and antibacterial activity was determined by measuring the diameters of inhibition zones. Tests were performed in triplicate, and mean values were used for data analysis.

Liquid micro-dilution method (MIC and MBC)

The Minimum Inhibitory Concentration (MIC) of H. scoparia extract was determined by broth microdilution in sterile 96‑well plates, according to previous research,¹⁶ with slight modifications. The methanolic extract of H. scoparia was serially diluted in Mueller-Hinton broth to final concentrations of 500, 250, 200, 125, 100, 50, 25, 10, 5 and 2.5 mg/mL, and 100 µL of each dilution was distributed per well. Bacterial suspensions standardized to 0.5 McFarland and diluted 1:20 were added (10 µL/well). Negative controls (broth only) and growth controls (broth with inoculum) were included, and Gentamicin (0.0005-0.1 mg/mL) was used as the antibacterial agent. Plates were incubated at 37ºC for 24 hr, and growth was evaluated using iodonitrotetrazolium chloride. MIC was defined as the lowest concentration with no visible growth, while the Minimum Bactericidal Concentration (MBC) was determined by subculturing wells without growth onto agar plates to confirm bactericidal activity.

Antiproliferative activity

Experimental treatments

The H. scoparia extract was dissolved in DMSO to prepare stock solutions at final concentrations of 0.125, 0.160, 0.213, 0.270, and 0.380 mg/mL. The stock solutions were stored at -20ºC and used to prepare the corresponding treatment media concentrations. The final DMSO concentration was kept constant across all treatment groups within a given experiment and did not exceed 0.1%.

Cell line and culture conditions

The human breast cancer MCF-7 cell line was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA; catalog no. HTB-22). ATCC authenticates all cell lines by Short Tandem Repeat (STR) profiling prior to distribution, confirming their identity against the reference profile for MCF-7 (Cellosaurus accession CVCL-0031). Prior to use, cells were tested for mycoplasma contamination using a commercial PCR-based detection kit and confirmed to be mycoplasma-free. The cells were maintained in RPMI-1640 media supplemented with 10% v/v Fetal Bovine Serum (FBS), 100U/mL penicillin, and 0.1 mg/mL streptomycin and maintained at 37ºC in an environment of 95% air and 5% CO₂ in humidified incubator. For subculturing, cells were washed with Ca²⁺- and Mg²⁺-free Phosphate-Buffered Saline (PBS) and were detached by trypsin-EDTA (0.05% trypsin, 0.02% EDTA in 1x PBS) for 2-5 min at 37ºC. Subsequently, trypsin was neutralized with 10% FBS RPMI-1640 media and the detached cells were centrifuged, re-suspended and counted using a hemocytometer. Then cells were seeded at the appropriate number depending on the experiment.

Measurement of viable cell number

Viable cell count was determined using the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-Diphenyl-2h-Tetrazolium Bromide (MTT) colorimetric assay.¹⁷ Cells were seeded at count of 1.5x10⁴ per well in 96-well plates (6 replicates/group) and incubated in 10% growth media (RPMI-1640 media supplemented with 10% FBS) and allowed to attach overnight. The next day, treatment was given in mitogen-free media at the concentrations indicated for plant extract (0, 1, 5, 10, 25, 50, 100, 150 and 200 µg/mL). After 48 hr in culture, the medium was replaced with fresh one and 20 µL of MTT solution was added per well to achieve a final concentration of 0.45 mg/mL. Cells were incubated for 3 hr at 37ºC in a humidified incubator. At the end of incubation period, media was removed and DMSO was added at 100 µL/well to dissolve formazan crystals. Optical density was measured at 490 nm using a microplate reader.

Statistical analysis

All assays were performed in triplicate (technical replicates, n=3) using three aliquots derived from the same extract preparation, with a single measurement taken per aliquot. It should be noted that biological replicates (independently prepared extracts) were not performed in this study; consequently, the reported variability reflects technical replicate variation rather than biological variability. Future studies incorporating multiple independent extractions are recommended to assess biological reproducibility. Data are expressed as mean±Standard Deviation (SD). For the antiproliferative activity, comparisons between treated groups and the control were analyzed using Student’s t-test. Concentrations that induced 50% of cell growth (IC₅₀ values) were determined by non-linear regression curve fitting in GraphPad Prism version 7 (GraphPad Software, San Diego, CA, USA). Two-way ANOVA followed by Šídák's multiple-comparisons test was carried out to assess the antioxidant activity of the extract compared with ascorbic acid. Statistical significance was set at p<0.05, and exact p-values are reported.

RESULTS AND DISCUSSION

Extract yield and total polyphenols

Under the selected extraction conditions, the yield was 11.96±0.10%, with Relative Standard Deviation (RSD) equal to 0.84%, confirming excellent reproducibility of the method. In addition, the extract revealed a high total phenolic content (112.09±0.03 mg GAE/g) (Table 2), closely comparable to those reported in previous Algerian research. A study on methanolic H. scoparia extract from Laghouat, Algeria, found 108.41±4.59 mg GAE/g,¹⁸ while another study reported 228.58±0.69 mg GAE/g in extract from Biskra, Southeastern Algeria.¹⁹ Similarly, a value of 141.61±0.53 mg GAE/g was obtained in the same region.⁷ In contrast, other studies have reported lower phenolic contents. A previous study reported 58.82±0.08 mg GAE/g in methanolic extract from Tunisia,²⁰ while a value of 59.75±1.80 mg GAE/g was reported from methanolic extract in Tunisia.²¹ and 50.08±1.09 mg GAE/g from methanolic extract in Tazarine, southern Morocco.²² This variation in results may be attributed either to environmental conditions affecting the species, or to the differences in the region or the extraction techniques used.²³

Identification of phenolic compounds by HPLC-DAD

The methanolic extract of H. scoparia harvested from the Brezina region (El-Bayadh) in western Algeria reveals a complex phenolic profile, characterized by the predominance of epicatechin and rutin, followed by a moderate quantity of Luteolin, chlorogenic acid, ascorbic acid and apigenin 7-glucoside, with smaller amounts of tyrosol and verbascoside (Table 1; Figures 3 and 4). HPLC-DAD analysis highlights epicatechin (15.045 mg/g) as the most abundant compound, followed by rutin (2.124 mg/g) that contribute significantly to antioxidant activity. Luteolin (0.399 mg/g), chlorogenic acid (0.375 mg/g), ascorbic acid (0.310 mg/g) and apigenin 7-glucoside (0.297 mg/g) are present in moderate concentrations, while tyrosol, verbascoside, hydroxybenzoic acid derivatives and other flavonoids and phenolic acids occur in lower amounts.

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RT: retention time, λ (nm): wavelength in nanometer

These results are consistent with reports from other Algerian regions and other Neighboring countries.^7,23,24 However quantitative variations are evident due to differences in extraction methods, solvent polarity, and ecological factors. A previous study reported the absence of flavonoids in the aqueous extract, but their presence in the methanolic extract of H. scoparia, while alkaloids and saponins occurred in both, demonstrating that the extraction method directly influences phytochemical composition.²⁵

While the present study relied on HPLC-DAD for polyphenol identification and quantification, LC-ESI-MS/MS-based profiling offers a complementary and more structurally resolved analytical perspective. A recent study on Haloxylon scoparium (a synonym of Hammada scoparia) leaf extract used LC-ESI-MS/MS to characterize 27 metabolites, including organic acids, gallic and gentisic acid derivatives, dicaffeoylquinic acid, and several isorhamnetin and quercetin glycosides (e.g., isorhamnetin 3-o-rutinoside, isorhamnetin 3-o-glucoside, and quercetin-o-pentoside-o-deoxyhexoside-hexoside), of which 18 were reported for the first time in the genus Haloxylon.²⁶ These findings are broadly consistent with our HPLC-DAD analysis, which revealed rutin (quercetin-3-o-rutinoside) as one of the predominant polyphenols in H. scoparia, together with apigenin 7-glucoside detected in both studies, supporting the reliability of our compound assignments. However, epicatechin, luteolin, ascorbic acid, tyrosol, and verbascoside detected in the present work were not reported in that LC-MS/MS profile, which may reflect differences in extraction solvent, plant part, or the higher sensitivity of tandem MS in resolving low-abundance compounds compared to UV-based detection. This underscores both the complementary value of the two techniques and an acknowledged limitation of the present method. Together, these results highlight the importance of standardized extraction and analytical procedures to ensure reproducibility and comparability across studies of this species. Finally, it should be noted that compound identification based solely on retention times and UV spectra may not be fully conclusive, and future studies should apply LC-MS/MS for structural confirmation.

Antioxidant activity

The antioxidant activity of plant extract was evaluated by its ability to scavenge DPPH free radicals. The lower IC₅₀ value indicated that the plant extract possessed higher antioxidant activity.²⁷ The methanolic extract of H. scoparia exhibited marked scavenging activity (21.13±0.12 µg/mL), leading to a noticeable reduction in DPPH free radicals. In contrast, ascorbic acid showed a significantly higher scavenging capacity (9.02±0.13 µg/mL) (Figure 5 and Table 2).²⁸ Two-way ANOVA demonstrated significant effects of treatment, concentration, and their interaction on DPPH radical-scavenging activity (p<0.0001 for all). Šídák's multiple-comparisons test showed that the DPPH radical-scavenging activity of ascorbic acid was significantly higher than that of the H. scoparia extract at all tested concentrations (p<0.05).²⁹

These findings are consistent with previous studies on the same species, such as Bouaziz et al. (2016) with 28±0.70 µg/mL (Tunisia) and Benkherara and Bordjiba (2018) with 29.96±0.92 µg/mL (Biskra, Algeria).^19,21 Other studies have reported higher IC₅₀ values, indicating lower antioxidant activity. Bakchiche et al. (2013) found 114±1.5 µg/mL in H. scoparia from Laghouat region, Algeria, while Nounah et al. (2019) obtained 70.60±0.09 µg/mL in Tazarine, Morocco, and Benine et al. (2023) reported 85.75±0.61 µg/mL in methanolic extract from Biskra region, Algeria.^7,18,22

FRAP reducing power

For the FRAP assay, which evaluates the reducing power of antioxidants. The Ferric Reducing Antioxidant Power (FRAP) of the H. scoparia extract and ascorbic acid is shown in Figure 6. H. scoparia methanolic extract showed an EC₅₀ value of 82.10±0.01 μg/mL (p≤0.0001). Although this is higher than that of ascorbic acid (EC₅₀=17.30±0.05 μg/mL), it reflects a moderate reducing capacity compared to standards. The concentration-dependent increase in FRAP response exhibited for the H. scoparia extract shows that its reducing power increased progressively with rising extract concentration, though the response remained weaker than that of ascorbic acid at the equivalent concentrations.

These findings are consistent with previous studies.^7,22, that evaluated extracts of the same species from Algeria and Morocco in FRAP assays, which reported values of 124.04±1.66 mg/g and 67,286±2,380 mg/g of dry weight respectively, indicating that, although its reducing strength is lower than standard antioxidants, it still demonstrates measurable activity (Table 2). The differences observed among studies might be linked variations in geographical origin, extraction conditions, extract composition, assay protocols, and the manner in which FRAP response was expressed, which should be taken into consideration in any direct comparison of these values.

The FRAP results align with the DPPH findings (21.13±0.12 µg/mL for methanolic extract and 9.02±0.13 µg/mL for ascorbic acid), supporting the antioxidant potential of H. scoparia extract. Although the values obtained from DPPH and FRAP assays cannot be directly compared because the two assays rely on different reaction mechanisms and endpoints, the extract’s lower activity relative to ascorbic acid was consistently observed across both assays. The agreement between DPPH and FRAP outcomes (Figures 5 and 6) reinforces the antioxidant activity of H. scoparia extract, as the two assays reflect distinct mechanisms, hydrogen/electron donation to a stable radical versus ferric ion reduction, thereby reducing the likelihood that the observed activity is assay-specific. This complementary response offers stronger evidence that the antioxidant potential of the extract is not restricted to a single reaction mechanism and might reflect the contribution of numerous redox-active constituents present in the H. scoparia methanolic extract.

Furthermore, the statistically significant differences observed between the H. scoparia extract and ascorbic acid across the tested concentrations (two-way ANOVA followed by Šídák's multiple-comparisons test; p<0.05) support the observed variances in reducing capacity. As shown in Figure 6, the differences were not statistically significant at the lowest tested concentrations (2.5 and 5 µg/mL), however significant differences became evident starting at 15 µg/mL and were more marked at higher concentrations, further confirming the concentration-dependent difference in reducing capacity between the extract and the reference standard.

Phenolic compounds have been reported to have various biological properties, including antioxidant activity.^7,19 A prior study revealed that chromatographic separation of Haloxylon scoparium extracts identified fraction, with high antioxidant activity, largely attributed to salsolinol and potentially enhanced by polyphenols or synergistic interactions among constituents.²⁸ In the same way, previous HPLC-DAD profiling of Hammada scoparia correlated its major phenolics (rutin, naringenin, and gallic acid) with potent antioxidant activity.²⁴

The extract of H. scoparia represents an appreciated source of bioactive constituents with notable antioxidant potential, primarily through reduction, chelation, and peroxyl radical scavenging-mechanisms usually attributed to the hydroxyl groups and conjugated ring structures found in phenolic and flavonoid compounds.²⁰ These hydroxyl groups act as hydrogen donors, neutralizing free radicals by forming relatively stable phenoxyl radicals, while the conjugated ring systems allow for electron delocalization, further stabilizing the resulting radical species.²⁰ Additionally, certain phenolic compounds can chelate transition metal ions such as iron and copper, thereby restraining their catalytic role in the generation of reactive oxygen species via Fenton-type reactions.²¹ The activity established in the DPPH assay may reflect a possible synergistic interaction among the extract's phenolic constituents, although this was not directly tested in the present work, and it further supports its potential therapeutic application.^7,18

Antibacterial activity

Evaluation of the in vitro antibacterial activity of methanolic extract of H. scoparia was determined initially by the disc diffusion method against different pathogenic bacterial strains habitually encountered in infectious diseases, and then quantified by measuring the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) using broth microdilution in sterile 96‑well plates. As revealed in Table 3, H. scoparia extract showed modest antibacterial activity against both tested Gram-positive and Gram-negative bacterial species. with mean inhibition zones ranging from 8 to 13 mm compared with their sensitivity to positive control (with inhibition zone up to 36 mm). However, the negative controls used in extraction did not show an antibacterial activity. The MIC and MBC results as the same show a moderate antibacterial effect with values of MIC and MBC ranging from 25 to 100 mg/mL, and 50 to 200 mg/mL respectively (Table 4). Bacillus cereus was the most sensitive pathogenic strain, followed by Pseudomonas aeruginosa and Staphylococcus aureus species, with inhibition zones of 13, 12.5±0.50 and 12 mm respectively, all major agents of nosocomial infections and are well known for their ability to develop antibiotic resistance, highlighting the need for new antimicrobial compounds.

Previous studies were proved the antimicrobial activity of the H. scoparia extract against a range of different pathogenic micro-organisms which support our study,^{11,21,22,28,30,31} though results vary depending on several factors. Differences are often attributed to plant part, phytoconstituents, solvent polarity, bacterial membrane permeability, or genetic factors.³²

Mechanistically, phenolic and flavonoid constituents are thought to exert antibacterial effects through disruption of bacterial cell membrane integrity, inhibition of extracellular microbial enzymes, and interference with bacterial efflux pump systems, among other mechanisms.²¹ Some research highlighted the alkaloid carnegine as principally potent, while other authors linked bacterial sensitivity to flavonoid hydroxyl groups.²¹ Given the phytochemical diversity of H. scoparia extract, its antibacterial activity may also result from synergistic or additive interactions among multiple bioactive compounds rather than a single active constituent, although this was not directly investigated in the present study. Overall, H. scoparia reveals consistent antibacterial activity, but its effectiveness is strongly influenced by extraction methods and phytochemical composition.

These results support the potential relevance of H. scoparia as a candidate source of natural antibacterial agents, particularly in the context of the growing interest in plant-derived alternatives to conventional antibiotics given the growing challenge of antimicrobial resistance. Given that H. scoparia is a characteristic steppe species of the Saharan Atlas in southwestern Algeria, its antibacterial constituents may be synthesized as stress metabolites in response to high temperatures during growth.² Instead, these compounds could be produced following mechanical stress, such as cutting.³³ In reality, the use of natural compounds to control infections has been practiced for centuries. Plant exudates and their constituents are one source of natural substances exhibiting antibacterial action.³⁴

Antiproliferative activity

Effects of the Treatment of the Plant Extract on Viability of Hormone-dependent Breast Cancer Cells

MCF-7 breast cancer cells were seeded at 1.5x10⁴ cells/well in 10% FBS RPMI-1640 media and allowed to attach overnight. Next, cells were treated with vehicle-control or various concentrations of the plant extract for 48 hr period. At the end of the experiment, MTT assay was used to determine the viable cell number. Each experiment was performed in triplicates, and data shown represent mean number of viable cells±SEM. The effect of a range of concentrations of the tested plant extract (H. scoparia) on the viability of MCF-7 breast cancer cells after 48 hr incubation period is shown in Figure 7. The results indicate that methanolic extract of H. scoparia significantly reduced the growth of MCF-7 cells in a dose-dependent fashion in mitogen-free media compared with the untreated control (p<0.05), with increasing statistical significance at higher concentrations. This effect was further confirmed by the estimated IC₅₀ value (50.76 µg/mL) obtained from nonlinear regression analysis (Table 5). These findings indicate the cytotoxic activity of the methanolic extract of H. scoparia, with the extract exhibiting a noticeable antiproliferative effect that reached 67% inhibition at a 1% extract concentration.³⁵ However, the underlying mechanism such as apoptosis, generates ROS, disrupts mitochondrial membrane potential and cell-cycle arrest was not investigated in this study. Further mechanistic assays would be needed to confirm whether similar pathways are involved in the case of H. scoparia. Moreover, the cytotoxic effect of H. scoparia extract on normal human cells is also needed to evaluate its selectivity index and potential toxicity.

In fact, plants have been reported to be rich sources of natural compounds that can exhibit antiproliferative effects thanks to the bioactive phytochemicals they contain.³⁶ They can exhibit anticancer potential through various mechanisms such as arresting the cell cycle by suppressing proliferation in cancer cells; often through interference with cyclin-dependent kinase activity at key cell-cycle checkpoints, including the G1 and G2 phases, inducing apoptosis via activation of intrinsic or extrinsic pathways leading to caspase cascade activation, and preventing metastatic processes by inhibiting cell migration and invasion.³⁶ Given the phenolic and flavonoid content of H. scoparia extract, it is possible that similar mechanisms may contribute to the observed cytotoxic effect; though, this remains speculative, as no mechanistic assays were performed in the present study.

The number of studies reporting the antiproliferative activity of the studied plant on viability of hormone-dependent breast cancer cells is limited. A few studies indicated that polyphenolic extracts of this genus displayed a high activity to inhibit cell proliferation, highlighting the plant's potential in anticancer research. However, relatively there are not many researches that have been published on the phytochemistry of this specie.¹⁹ Further research work on antimelanoma activity of this specie may assure high cytotoxicity. H. scoparia showed more than 80% inhibition in melanoma assays.³⁷ In similar research, H. scoparia flavonoids fraction and its compound rutin were demonstrated to have the capacity to specifically induce apoptosis specially the cytotoxicity of adherent and chemoresistant leukemic progenitors.³⁸ Moreover, previous research has shown that the ethyl acetate fractions of Haloxylon scoparium Pomel exert significant antiproliferative effects against U87 glioblastoma cells, mediated through inhibition of cell migration, suppression of neurosphere formation, and induction of apoptosis.³⁹

This research is to provide an important basis for applying this extract in in vitro anticancer activity against human carcinoma cells. Besides, using desert plant extracts may serve as a promising anticancer drug for cancer therapy. Similarly, supplementary screening of desert plants from western Algeria, using different extraction solvents, diverse plant organs, as well as their harvest in variant seasons, may lead to identification of additional species with therapeutical activities.

The findings of the present study revealed that the phenolic extract of H. scoparia from Brezina, Algeria, possesses a diverse profile of bioactive constituents. These results are in line with previous studies on polyphenol-rich desert plants, further confirming the potential of H. scoparia as a natural source of antioxidant agents. The observed bioactivity confirms the initial hypothesis that phenolic constituents are important in the pharmacological properties of the plant. However, variation in the distribution of some compounds indicates the need for comparative studies with related species to better understand the ecological and biochemical influences. To summarize, the results emphasize the importance of this plant for natural product research and raise new questions regarding its possible uses in nutraceuticals and drug development.

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CONCLUSION

The present study provides new insights into the chemical composition and biological activities of the methanolic extract of Hammada scoparia, a medicinal plant conventionally used in the Brézina region of western Algeria. The findings contribute to the valorization of H. scoparia as a source of bioactive compounds, revealing notable antioxidant, antibacterial, and antiproliferative activities that support its potential for future therapeutic investigations. However, since the cytotoxicity of the extract was not assessed on normal human cell lines, its selectivity toward cancer cells and therapeutic index could not be established, and conclusions regarding its therapeutic potential should therefore be interpreted with caution. As this study was limited to in vitro assessments, further in vivo validation, toxicity evaluation, pharmacokinetic studies, and formulation development are required before considering any pharmaceutical application. More investigations, including selectivity and mechanistic assays, are required to isolate and characterize these bioactive agents, which may represent potential sources for further preclinical drug development research. Moreover, the presence of phenolic constituents and natural antioxidants such as epicatechin and rutin in H. scoparia extract, both widely reported for their antioxidant properties, suggests a possible application as a natural agent for food preservation, deserving further investigation.

ACKNOWLEDGEMENT

The authors owe a depth of gratitude to thank Dr. Arne Saatkamp, Lecturer, Mediterranean Institute of Biodiversity and Ecology (MIBE), Aix Marseille University for his assistance in the plant identification, and also thankful to the Department of Pharmacy and Department of nursing, University of Irbid (JUST), Jordan for facilitating the research.

ABBREVIATIONS

AMC: Amoxicillin-Clavulanic Acid; Asc.Ac.: Ascorbic acid; ATCC: American Type Culture Collection; CECT: Colección Española de Cultivos Tipo (Spanish Type Culture Collection); CFU: Colony‑Forming Units; DMSO: Dimethyl Sulfoxide; DNA: Deoxyribonucleic Acid; DPPH: 2,2-diphenyl-1-picrylhydrazyl; EC₅₀: Effective Concentration 50%; EDTA: Ethylenediaminetetraacetic acid; ER+: Estrogen Receptor Positive; FBS: Fetal Bovine Serum; GAE: Gallic Acid Equivalents; GEN10: Gentamicin; HER2-: Human Epidermal Growth Factor Receptor 2 Negative; HPLC-DAD: High‑Performance Liquid Chromatography-Diode Array Detection; IC₅₀: Inhibitory Concentration 50%; JUST: Jordan University of science and technology; MBC: Minimal Bactericidal Concentration; MCF-7: Michigan Cancer Foundation clone 7; a human breast adenocarcinoma cell line (Breast cancer cell line); Meth. Ext.: methanolic extract; Meth. Ext.: methanolic extract; MIBE: Mediterranean Institute of Biodiversity and Ecology; MIC: Minimal Inhibitory Concentration; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NCDBR: National Center for the Development of Biological Resources; PBS: Phosphate‑Buffered Saline; PR+/-: Progesterone Receptor Positive/Negative; ROS: Reactive oxygen species; RPMI‑1640: Standard cell culture medium (Roswell Park Memorial Institute 1640); SD: standard deviations; SEM: standard error of the mean; TPC: Total phenolic content; UV: Ultraviolet.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

AUTHORS CONTRIBUTION

Mehdi Yamina: Primary and corresponding author; conceived the study, led the analysis, statistical evaluation and interpretation, and wrote and revised the manuscript together with the second co-author. Kebir Nasreddine: Assisted in manuscript evaluation and editing, revised the English language throughout, and supported data evaluation.

ETHICAL APPROVAL / CONSENT STATEMENT

Ethical approval and informed consent were not applicable to this study.

DATA AVAILABILITY STATEMENT

The data supporting the findings of this study are available from the corresponding author upon reasonable request.