For millennia, medicinal plants have served as a crucial source for treatment and illness prevention in various traditional healthcare systems. They encompass several bioactive substances that may enhance their therapeutic benefits (Manisha et al., 2025). Due to their accessibility, cultural acceptance and varied biological characteristics, these plants remain integral to primary healthcare in numerous nations, especially in areas with restricted access to contemporary pharmaceuticals (Karunamoorthi et al., 2013). Moreover, increasing concerns about the limitations, safety and efficacy of conventional synthetic drugs have led to a renewed interest in the therapeutic potential of medicinal plants and their bioactive components (Dubey et al., 2026). The integration of traditional knowledge with contemporary scientific validation of these plants offers a distinctive opportunity for the future of healthcare (Manisha et al., 2025).
       
The biological efficacy of medicinal plants is predominantly associated with secondary metabolites, encompassing phenolic chemicals, flavonoids, alkaloids, tannins, terpenoids, saponins and glycosides (Alamgir, 2018). These chemicals are significant for plant defense and survival and may demonstrate pharmacological effects in humans and animals (Aljawdah et al., 2025). Phenolic chemicals and flavonoids are recognized for their protective, antioxidant, anti-inflammatory and antibacterial characteristics (Chagas et al., 2022).
       
Scientific research on medicinal plants is critical since it helps to validate their traditional applications and discover the active chemicals responsible for their biological effects (Dewangan et al., 2026). As a result, phytochemical analysis, including the identification of active chemicals, notably total phenolic and flavonoid content, is widely employed to evaluate the chemical potential of plant extracts (Sembiring et al., 2018). These assays provide important information on the potential medicinal and antioxidant effects of these plants.
       
The antiparasitic and anthelmintic capabilities of these medicinal herbs are also being investigated. Plant secondary metabolites, such as tannins, flavonoids, phenols and saponins, can harm parasites by harming their outer surface, interfering with their metabolic processes, limiting their motility, or causing paralysis and death (Zaman et al., 2020). As a result, these medicinal herbs may be a great resource for developing new natural antiparasitic medicines.
       
As a result, medicinal plants continue to be a significant topic of scientific inquiry due to the natural substances they contain that may have therapeutic properties. However, the use of these plants should be accompanied by adequate phytochemical, pharmacological and toxicological research to ensure their safety, efficacy and possible medical applications.
       
Z. propinquum is a medicinal plant from the Zygophyllaceae family that is adapted to desert environments. Zygophyllum species are found in arid and semi-arid regions (Manzoor et al., 2017) and have traditionally been utilized for a variety of therapeutic purposes, including antioxidant, anti-inflammatory, antibacterial and anthelmintic properties (Albarakaty et al., 2025). This genus’ therapeutic value stems mostly from its abundance of secondary metabolites, including phenolic chemicals, flavonoids, tannins, alkaloids, terpenoids and saponins (Alamholo, 2024).
       
This plant contains several significant phytochemical categories, including phenolic chemicals and flavonoids. These chemicals are well-known for their antioxidant properties and capacity to protect cells from oxidative damage (Shawky et al., 2019). Thus, quantifying the total phenolic and flavonoid content is critical for assessing the phytochemical quality and possible biological activity of plant extracts. Previous research has found phenolics and flavonoids in Zygophyllum species, indicating that these compounds may play a role in the pharmacological actions of these plants.
       
Plant extracts’ anthelmintic action is frequently associated with biologically active secondary metabolites such as tannins, flavonoids, phenols and saponins (Ikbal et al., 2020). These substances may modify the surface structure of parasitic worms, interfere with energy metabolism, reduce motility, or cause paralysis and death. As a result, calculating the time necessary for paralysis and death is an effective way to assess the efficacy of plant extracts as anthelmintics (Ishnava and Konar, 2020).
       
Although the phytochemical diversity and biological activities of several Zygophyllum species have been investigated, the chemical composition and biological properties of Z. propinquum remain insufficiently characterized, particularly with respect to its effects on mammalian cancer-cell viability and its activity in preliminary worm-based screening models. Therefore, the present study aimed to characterize the methanolic leaf extract of Z. propinquum using complementary chemical and in vitro approaches. The specific objectives were to: (i) qualitatively profile GC-MS-detectable constituents; (ii) quantify total phenolic content (TPC) and total flavonoid content (TFC); (iii) assess the concentration-dependent effect of the extract on MCF-7 breast adenocarcinoma cell viability using the MTT assay; (iv) determine paralysis and mortality times in the free-living annelid E. fetida as a preliminary surrogate worm model and (v) qualitatively evaluate histological alterations in extract-treated worms.

Plant collection and preparation of the methanolic leaf extract
 
Fresh leaves of Z. propinquum were collected in March 2025 during the spring season from Al-Quway’iyah, Riyadh Province, Saudi Arabia (24°02′47"N, 45°15′56"E). The botanical identity of the specimen was authenticated by Dr. Rajakrishnan Rajagopal, Department of Botany and Microbiology, King Saud University and a voucher specimen was deposited in the herbarium under voucher No. 24708.
       
The leaves were washed thoroughly under running tap water, rinsed several times with distilled water and air-dried at room temperature. The dried leaves were then ground into a fine powder using an electric grinder.
       
A total of 50 g of powdered leaf material was extracted with 500 mL of 70% methanol at a plant material-to-solvent ratio of 1:10 (w/v) by cold maceration at 4°C for 24 h. The extraction procedure was performed twice and the combined filtrates were concentrated under reduced pressure using a rotary vacuum evaporator at 50°C to remove methanol. The resulting crude extract was lyophilized and stored at -20°C until further analysis (Bennour et al., 2020). All phytochemical and biological assays were performed using aliquots from the same extraction batch.
 
Gas chromatography-mass spectrometry (GC-MS) analysis
 
GC-MS analysis was performed using an Agilent 7890B gas chromatograph coupled to an Agilent 5977A mass-selective detector (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was achieved using a TR-5MS capillary column (Thermo scientific; 30 m × 0.25 mm i.d., 0.25 µm film thickness) containing a 5% phenyl polysilphenylene-siloxane stationary phase.
       
The methanolic extract was prepared at a concentration of 10 ppm and a 1 µL aliquot was injected in splitless mode. The injector temperature was maintained at 250°C and helium was used as the carrier gas at a constant flow rate of 1.0 mL/min. The oven temperature was initially maintained at 60°C for 5 min, increased to 280°C at a rate of 5°C/min and then held at 280°C for an additional 6 min, giving a total chromatographic run time of approximately 55 min.
       
The mass spectrometer was operated in electron ionization (EI) mode at 70 eV. The transfer-line, ion-source and quadrupole temperatures were maintained at 280, 230 and 150°C, respectively. Mass spectra were acquired in full-scan mode over an m/z range of 50-1000 at a scan rate of 2 scans/s. Compound identification was based on comparison of the acquired mass spectra with reference spectra in the NIST and Wiley mass spectral libraries. Because authentic reference standards were not analyzed, all reported compound assignments were considered tentative. The GC-MS analysis was performed as a single analytical determination (n = 1) for qualitative phytochemical profiling.
 
Determination of total flavonoid and total phenolic Content
 
Total flavonoid content (TFC) and total phenolic content (TPC) of the methanolic extract of Z. propinquum were determined spectrophotometrically with minor modifications of the method described by Aziz et al., (2024). The dry extract was prepared at a concentration of 2 mg/mL and measurements were performed in triplicate (n = 3). Results were expressed as mean±standard deviation (SD).
       
For TFC determination, 1 mL of the extract solution (2 mg/mL) or quercetin standard solution (3.125-50 µg/mL) was mixed with 1 mL of 2% aluminum chloride solution. A reagent blank was prepared by replacing the 1 mL sample aliquot with 1 mL methanol while maintaining all other assay components and experimental conditions. After incubation for 15 min at room temperature, absorbance was measured at 430 nm. The quercetin calibration curve was described by the linear regression equation:
 

 y = 0.04008x -0.07886 (R² = 0.9920)

 
Where,
y= Blank-corrected absorbance.
x= Quercetin concentration (µg/mL).
       
TFC was expressed as mg quercetin equivalents per g dry extract (mg QE/g dry extract). For TPC determination, 100 µL of the extract solution or gallic acid standard solution (15.625-500 µg/mL) was mixed with 0.1 mL of folin-ciocalteu reagent and allowed to react for 8 min. A reagent blank was prepared by replacing the sample aliquot with 100 µL methanol. Subsequently, 0.3 mL of 20% sodium carbonate solution was added and the mixture was incubated at room temperature in the dark for 30 min. Absorbance was measured at 720 nm. The gallic acid calibration curve was described by:
 

y = 0.004711x + 0.04869 (R² = 0.9987)

 
Where,
y= The blank-corrected absorbance.
x= The gallic acid concentration (µg/mL).
       
TPC was expressed as mg gallic acid equivalents per g dry extract (mg GAE/g dry extract).
       
The equivalent concentration obtained from the respective calibration curve was normalized to the dry extract according to:

 
Where,
C= The equivalent concentration obtained from the calibration curve (mg/mL).
V= The volume of extract used in the assay (mL).
m= The mass of dry extract represented in the analyzed aliquot (g).
 
Cytotoxicity evaluation
 
MCF-7 human breast adenocarcinoma cells (ATCC HTB-22) were obtained from the American type culture collection (ATCC). The cell line was authenticated and confirmed to be free from mycoplasma contamination before use. The cells were cultivated in RPMI-1640 medium supplemented with 10% fetal bovine serum and 50 μg/mL gentamicin. In a humidified incubator with 5% CO₂, cell cultures were subcultured two or three times each week at 37°C. Cells between passages 20 and 22 were used for the experiments.
 
MTT assay
 
The effect of the methanolic extract of Z. propinquum on MCF-7 cell viability was evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay according to Siddiqui et al., (2008), with minor modifications. The dried plant extract was dissolved in DMSO to prepare a stock solution. Actively proliferating cells were harvested using 0.25% trypsin-EDTA and seeded into 96-well plates at a density of 10,000 cells per well.
       
After 24 h of incubation, cells were exposed to the Z. propinquum extract at concentrations ranging from 15.6 to 1000 µg/mL for an additional 24 h. The final DMSO concentration in the culture medium did not exceed 0.1% at any extract concentration. Untreated control cells were maintained under the same culture conditions without plant extract. Following treatment, 10 µL of MTT solution (5 mg/mL) was added to each well and the plates were incubated for 4 h. The culture supernatant was then removed and the resulting formazan crystals were dissolved in 200 µL DMSO. Absorbance was measured at 550 nm using a microplate reader. Measurements were performed in triplicate (n = 3). Cell viability was calculated relative to the untreated control according to:

 
Where,
 A_treated and A_control= The absorbance values of extract-treated and untreated control cells, respectively.
       
Because cell viability did not decrease to 50% at any experimentally tested concentration, a numerical IC₅₀ value was not estimated by extrapolation. The IC₅₀ was therefore reported conservatively as >1000 µg/mL under the present experimental conditions.
 
Anthelmintic activity
 
Adult E. fetida earthworms were collected from Riyadh, Saudi Arabia. Species identity was confirmed by an expert from the College of Food and Agriculture Sciences, King Saud University. Adult worms approximately 7 cm in body length were used in the experiment. Worm body weight was not recorded in the original experimental records. The worms were rinsed with distilled water to remove adhering substrate and acclimated at ambient laboratory temperature for 30 min before the experiment.
       
The biological activity of ZPPE was evaluated in vitro using adult E. fetida as a preliminary free-living annelid surrogate worm model according to the method described by George and Kousalya (2018), with minor modifications. Aqueous solutions of ZPPE were prepared in distilled water at concentrations of 100 and 200 mg/mL. Mebendazole at 10 mg/mL was used as the positive control, whereas distilled water served as the negative control.
       
Each treatment condition was conducted in a single exposure vessel containing 20 mL of the respective test solution and five worms (five worm-level observations per condition; total n = 20 worms). Because all five worms within a treatment condition shared the same exposure vessel, they were not considered independent treatment replicates.
       
Time to paralysis was recorded when no spontaneous movement was observed and no movement occurred following vigorous shaking. Time to death was recorded when no movement occurred following vigorous shaking and subsequent immersion in warm water at 50°C. The longest recorded event time in the experimental dataset was 33.40 min. No paralysis or mortality occurred in any of the distilled-water control worms throughout the same observation period. therefore, these observations were treated as right-censored at 33.40 min for descriptive time-to-event visualization..
       
To prepare paraffin sections, 3 worms per group from the distilled-water control, ZPPE 200 mg/mL and mebendazole 10 mg/mL groups were examined. The 200 mg/mL extract concentration was selected for histological examination because it produced the more pronounced biological response in the preliminary worm assay, with shorter observed paralysis and mortality times than the 100 mg/mL concentration.
       
Tissue was sampled from the mid-body region and fixed in 10% buffered neutral formalin. Paraffin blocks were sectioned at 4 µm using a rotary microtome and sections were stained with hematoxylin and eosin (H and E) (Drury and Wallington, 1980). Three sections per worm and three microscopic fields per section were examined at 400× magnification. Histological assessment was blinded. No quantitative histological scoring, morphometry, or image-analysis procedure was performed; therefore, the observations were interpreted descriptively. Sections were examined using light microscopy (Olympus BX61, Tokyo, Japan) and captured using a digital camera (DP 73) attached to the microscope.
 
Data analysis
 
Quantitative data are presented as mean±standard deviation (SD), with the number of replicate measurements indicated for each assay. For the MTT assay, cell viability was expressed as a percentage relative to the untreated control. Because 50% cell viability was not reached within the experimentally tested concentration range of 15.6-1000 µg/mL, a numerical IC₅₀  value was not estimated by extrapolation; the IC₅₀  was therefore reported as >1000 µg/mL. Paralysis and mortality times in E. fetida were summarized descriptively at the worm level using individual event times, mean±SD and median values. Kaplan-meier curves were used only as descriptive visualizations of the observed time-to-event patterns. Histological observations were qualitative and were not subjected to inferential statistical analysis.

GC-MS profiling of the methanolic extract of Z. propinquum
 
GC-MS analysis revealed three non-silylated compounds that were retained as tentative assignments after conservative re-evaluation of the chromatographic profile and exclusion of siloxane-related artifacts, silylated assignments and other analytically implausible signals. These compounds were n-undecane (RT 11.395 min), methyl hexadecanoate (RT 32.157 min) and methyl stearate (RT 35.949 min). Their relative peak areas were 0.41%, 0.83% and 0.70%, respectively, based on the total integrated chromatographic peak area before artifact exclusion. The hydrocarbon n-undecane was distinguished from methyl hexadecanoate and methyl stearate, which were classified as fatty acid methyl esters. The retention times, peak areas, molecular characteristics and identification status of the retained compounds are summarized in Table 1 and the total ion chromatogram is presented in Fig 1.




       
Compound assignments are considered tentative because authentic reference standards were not analyzed. Siloxane-related peaks and silylated assignments were excluded during re-evaluation of the chromatogram. Assignments reported by the spectral library as TMS/TBDMS derivatives were excluded because no derivatization procedure was performed on the sample. Library match-score values and experimental retention indices were not available from the original analysis and therefore were not retrospectively assigned or reported.
 
Total flavonoid and total phenolic contents
 
The methanolic extract of Z. propinquum was analyzed spectrophotometrically for total flavonoid and total phenolic contents (Fig 2). Total flavonoid content was 1.91±0.04 mg QE/g dry extract, whereas total phenolic content was 1.98±0.07 mg GAE/g dry extract. Values represent mean±SD of triplicate measurements (n = 3).


 
Cytotoxicity against MCF-7 (Breast cancer)
 
The effect of the methanolic extract of Z. propinquum on MCF-7 cell viability was evaluated using the MTT assay. As shown in Fig 3, cell viability showed a modest concentration-dependent decrease after 24 h of exposure. The cell viability was 100.00±3.33% in the untreated control and 100.63± 2.84, 90.34±4.46, 85.49±2.07, 83.43±1.87, 82.26±2.43, 80.27±1.29 and 71.40±3.00% at 15.6, 31.2, 62.5, 125, 250, 500 and 1000 µg/mL, respectively (n = 3). Cell viability was calculated relative to the untreated control using the following formula:

Where,
A treated and A control= The absorbance of treated extract and untreated control cells, respectively.


       
The cell viability decreased with an increase in the extract concentration, but at the highest tested concentration of 1000 µg/mL, the cell viability was 71.40± 3.00%. Thus, 50% viability was not reached in the experimentally tested range and the IC₅₀ is reported as >1000 µg/mL.
 
Surrogate anthelmintic screening and histopathological evaluation
 
Paralysis and mortality of E. fetida were evaluated as exploratory time-to-event outcomes, with five worm-level observations per treatment condition in Fig 4 and 5. The higher ZPPE concentration was associated with markedly shorter event times. At 100 mg/mL, the mean time to paralysis was 26.50±2.75 min (median, 26.20 min) and the mean time to mortality was 29.06±3.36 min (median, 28.50 min). At 200 mg/mL, the corresponding times were 7.70±1.80 min (median, 8.32 min) for paralysis and 9.19±1.66 min (median, 9.34 min) for mortality. Mebendazole (10 mg/mL) produced the shortest event times, with paralysis occurring at 4.00±2.10 min (median, 4.52 min) and mortality at 5.31±2.12 min (median, 6.28 min). No paralysis or mortality occurred in the distilled-water control group during the 33.40-min observation period. therefore, these observations were right-censored at 33.40 min.




       
Descriptively, the higher extract concentration showed shorter observed paralysis and mortality times than the lower concentration, whereas mebendazole showed the shortest event times. The median paralysis and mortality times at 200 mg/mL were 17.88 and 19.16 min shorter, respectively, than at 100 mg/mL. Compared with ZPPE at 200 mg/mL, mebendazole showed median paralysis and mortality times that were 3.80 and 3.06 min shorter, respectively.  Because the five worms within each condition were exposed together in a single vessel, these between-condition differences are presented descriptively and were not subjected to inferential statistical testing.These findings show an observed concentration-related pattern of ZPPE in the E. fetida surrogate model, while mebendazole showed the more rapidly occurring responses under the present experimental conditions.
       
Qualitative histological examination of the mid-body region showed distinct morphological differences among the examined groups (Fig 6). Sections from distilled-water control worms showed an intact cuticle and relatively well-organized underlying muscular layers. In worms exposed to ZPPE at 200 mg/mL, visible disruption and deformation of the cuticular and muscular architecture were observed. Mebendazole-treated worms showed more marked disruption of cuticular and muscular organization. These observations were interpreted descriptively because no quantitative histological scoring, morphometric measurement, or image-analysis procedure was performed. Accordingly, the histological findings provide qualitative evidence of treatment-associated structural alterations in the E. fetida body wall but should not be interpreted as quantitative evidence of tissue-damage severity.


       
The present study provides an exploratory chemical and biological characterization of the methanolic leaf extract of Z. propinquum. Particular emphasis was placed on conservative interpretation of the GC-MS profile, quantitative determination of total phenolic and flavonoid contents, assessment of MCF-7 cell viability and evaluation of biological effects in the free-living annelid E. fetida as a surrogate worm model. The findings should therefore be interpreted as preliminary rather than as evidence of therapeutic anticancer or anthelmintic efficacy.
       
Following conservative re-evaluation of the GC-MS chromatogram, only three non-silylated compounds were retained as tentative library-based assignments: n-undecane, methyl hexadecanoate and methyl stearate. Their relative peak areas were 0.41%, 0.83% and 0.70%, respectively, corresponding collectively to approximately 1.94% of the total integrated chromatographic peak area before artifact exclusion. Methyl hexadecanoate and methyl stearate are fatty acid methyl esters, whereas n-undecane is a hydrocarbon. Fatty-acid derivatives have previously been reported in phytochemical investigations of Zygophyllum species, supporting the general plausibility of this chemical class in the genus. However, because authentic reference standards were not analyzed and experimental retention indices and library match scores were unavailable, the present GC-MS assignments should be regarded as tentative rather than definitive identifications. Accordingly, no biological activity is attributed specifically to any of these individual compounds.
       
The methanolic extract contained measurable total phenolic and flavonoid constituents, with values of 1.98± 0.07 mg GAE/g dry extract and 1.91±0.04 mg QE/g dry extract, respectively in Fig 2. Phenolic and flavonoid compounds are widely reported in Zygophyllum species and have been associated with diverse biological activities (Shawky et al., 2019). Nevertheless, the concentrations observed in the present study were substantially lower than those reported for some other Zygophyllum species. For example, Albarakaty et al., (2025) reported 156.15 mg GAE/g dry extract and 18.5 mg QE/g dry extract for Zygophyllum coccineum, whereas Touaibia and Abdellali (2025) reported 183.22 mg GAE/g and 101.13 mg QE/g for methanolic extracts of Zygophyllum cornutum leaves. Such differences may reflect species-specific composition as well as differences in plant material, extraction procedures, analytical conditions and calibration methods. Therefore, the present TPC and TFC values are best interpreted as quantitative measures of the tested Z. propinquum extract rather than as evidence of a particularly high phenolic or flavonoid content.
       
In the MTT assay, Z. propinquum extract produced a modest concentration-dependent reduction in MCF-7 cell viability over the tested range. Cell viability remained 71.40 ±3.00% at the highest concentration of 1000 µg/mL and 50% viability was not reached. Consequently, a numerical IC₅₀ value could not be determined within the experimental range and was conservatively reported as >1000 µg/mL. These findings indicate a measurable effect on MCF-7 cell viability but do not establish potent cytotoxic or anticancer activity. Because the MTT assay provides an indirect measure of cellular metabolic activity, the observed reduction in viability cannot by itself establish apoptosis, necrosis, cell-cycle arrest, or any specific mechanism of cell death. Studies of other medicinal-plant extracts have reported associations between phytochemical composition and effects on cancer-cell viability (Aziz et al., 2024; Almarfadi et al., 2022); however, the present study does not establish which constituents, if any, are responsible for the observed MCF-7 response. In addition, selectivity toward malignant versus non-malignant cells was not established. Further studies using fractionated extracts, isolated compounds, additional cancer-cell models and appropriate non-malignant cell controls would therefore be required before any anticancer relevance could be inferred.
       
The use of E. fetida in the present study was intended to provide a preliminary free-living annelid model with readily observable paralysis and mortality endpoints. Descriptive time-to-event patterns showed shorter observed paralysis and mortality times at 200 mg/mL than at 100 mg/mL, whereas mebendazole showed the shortest event times. Because each treatment condition was represented by a single shared exposure vessel containing five worms, these between-condition differences are interpreted descriptively rather than inferentially. The distilled- water control worms remained free of paralysis and mortality throughout the 33.40-min observation period and were therefore treated as right-censored observations rather than being assigned an artificial event time of zero.
       
The concentration-related response observed in E. fetida is broadly consistent with previous studies in which plant extracts produced dose-dependent effects on worm motility and survival. For example, Bazán et al. (2025) reported paralysis and mortality in E. fetida following exposure to methanolic extracts of Campomanesia species. Direct potency comparisons among plant extracts should nevertheless be made cautiously because the species examined, extraction procedures, concentrations, formulations and experimental conditions differ substantially among studies. Importantly, E. fetida is a free-living annelid and not a parasitic helminth. The present findings therefore demonstrate biological activity only within this surrogate model and cannot establish efficacy against parasitic worms or predict therapeutic effectiveness in infected hosts. Although tannins and saponins have been reported in other Zygophyllum species, neither class was quantified or identified in the present extract. Accordingly, potential roles of these or other constituents in enzyme inhibition, altered membrane permeability, oxidative damage, tubulin interactions, or other mechanisms remain untested hypotheses that require targeted chemical and mechanistic investigation.
       
Qualitative histological examination provided complementary morphological observations. Distilled-water control worms showed an intact cuticle and organized underlying muscle layers, whereas worms exposed to ZPPE at 200 mg/mL showed visible disruption and deformation of cuticular and muscular architecture. Mebendazole-treated worms showed more marked disruption of the same structures. Because the histological assessment was qualitative and no morphometric measurements, quantitative image analysis, or numerical scoring system were applied, these observations should not be interpreted as quantitative evidence of tissue-damage severity or as proof of a specific mechanism of action. Instead, they provide descriptive evidence of treatment-associated structural alterations in the E. fetida body wall.
       
Several limitations should be considered when interpreting the present findings. GC-MS profiling was based on a single analytical determination and compound assignments were tentative because authentic standards, experimental retention indices and library match scores were unavailable. The MCF-7 experiment did not establish a numerical IC₅₀ within the tested concentration range or demonstrate selectivity relative to non-malignant cells. The absence of documented vehicle-only and positive cytotoxicity controls, together with the lack of a non-malignant cell comparator, further limits interpretation of the MCF-7 findings. The E. fetida assay used a single shared exposure vessel per treatment condition containing five worms; therefore, independent treatment-level replication was not available and the worm findings are descriptive and exploratory. The assay also represents a surrogate rather than a parasitic helminth model and worm body weight was not recorded in the original experimental records.
       
Histological observations were qualitative and were performed on selected treatment groups rather than constituting a quantitative dose-response assessment. These limitations emphasize the exploratory nature of the study and the need for additional chemical confirmation, bioactivity-guided fractionation, validated parasitic-helminth models and more comprehensive cellular and mechanistic studies.

The present study provides an exploratory chemical and biological characterization of the methanolic leaf extract of Z. propinquum. Conservative re-evaluation of the GC–MS profile retained three non-silylated compounds as tentative library-based assignments: n-undecane, methyl hexadecanoate and methyl stearate. The extract contained measurable total phenolic and flavonoid constituents, with values of 1.98±0.07 mg GAE/g dry extract and 1.91±0.04 mg QE/g dry extract, respectively.
       
In MCF-7 cells, the extract produced a modest concentration-dependent reduction in cell viability; however, 50% viability was not reached within the tested concentration range and the IC₅₀ was therefore conservatively reported as >1000 µg/mL. In the Eisenia fetida surrogate worm model, the higher extract concentration produced shorter paralysis and mortality times than the lower concentration, whereas mebendazole remained the more rapidly acting treatment. Qualitative histological examination also revealed treatment-associated alterations in the cuticular and muscular architecture of extract-exposed worms.
       
These findings should be interpreted as preliminary. The E. fetida assay represents a free-living annelid surrogate model and does not establish efficacy against parasitic helminths, while the MCF-7 findings do not demonstrate potent or selective anticancer activity. Further studies should include confirmatory phytochemical identification using authentic standards, bioactivity-guided fractionation, evaluation in validated parasitic helminth models, appropriate non-malignant cell controls, mechanistic investigations and, where justified, controlled in vivo studies.

The authors extend their appreciation to King Saud University for funding this work through the Ongoing Research Funding program (ORF-2026-1078), King Saud University, Riyadh, Saudi Arabia.
 
Declaration of AI involvement in writing
 
The authors acknowledge the use of AI to assist in language refinement.
 
Ethical approval
 
Not applicable.

The authors declare that they have no conflict of interest.