For the extraction process, 400 g of the powdered plant material (leaves and stem) were soaked in 1,500 mL of petroleum ether (boiling point 30°C–60°C) to remove non-polar materials. The mixture was shaken frequently over 3 days. After filtering, the petroleum ether extract was set aside for further analysis, and the remaining plant material was spread on paper to evaporate any remaining solvent. The dried plant powder was then subjected to Soxhlet extraction using 80% hydroethanolic Solvent (a solution prepared with 80% ethanol and 20% water) for 12 h. The resulting extract was filtered, and the filtrates were combined. The solvent was removed under vacuum using a rotary evaporator, yielding a dark greenish residue. This residue was suspended in 500 mL of water and successively partitioned with chloroform, ethyl acetate, and n-butanol (3 × 500 mL for each fraction) using a separating funnel. The chloroform and ethyl acetate fractions were dried over anhydrous sodium sulfate, filtered, and evaporated to dryness using a rotary evaporator. The scheme of extraction is shown in Supplementary Figure S1.

In order to extract the roots, 150 g of powdered Capparis spinosa root were soaked in 500 mL of petroleum ether for 3 days to remove non-polar compounds. During this process, the mixture was shaken at intervals. After 3 days, the petroleum ether was separated by filtration, and the filtrate was removed from the filter paper and taken away for some hours to allow evaporation. After the hydroethanolic extract was concentrated, the solid plant residue was further liquidated by 80% ethanol for 12 h in a soxhlet thimble. The hydroethanolic extract was filtered, and the solvent was evaporated under vacuum using a rotary evaporator. This residue was dissolved in water 250 mL and further separated into several portions using chloroform, ethyl acetate, and n-butyl alcohol fractions as in the previous work for leaves and stem fractions. The fractions of petroleum ether, chloroform, ethyl acetate, and n-butanol were collected and preserved for further evaluation. Schematic diagram for fractionation of roots of Caparis spinosa crude extracts is shown in Supplementary Figure S2.

The GC-MS analysis of petroleum ether and chloroform fractions from both the aerial parts and roots of Capparis spinosa was conducted using an Agilent GC-MS system, equipped with an Agilent 190915-433UI HP-5ms Ultra Inert column.

Instrument Setup and Operating Conditions:

Column: Agilent 190915-433UI (HP-5ms Ultra Inert).

Injection Mode: Front Split/Splitless (SSZ).

Carrier Gas: High-purity helium (99.995%).

Flow Rate: 0.9 mL/min.

Carrier Gas Pressure: 7.037 psi.

Average Linear Velocity: 34,772 cm/s.

Hold-Up Time: 1.4379 min.

Temperature Program:

Initial Oven Temperature: 60°C.

Temperature Ramp 1: Increase from 60°C to 150°C at 3°C/min, hold for 10 min.

Temperature Ramp 2: Increase from 150°C to 300°C at 10°C/min.

Detection and Data Analysis:

Detector: Mass Selective Detector (MSD).

Ionization Mode: Electron Ionization (EI) at 70 eV.

Sample Injection: 1 µL of 1% extract solution (diluted in respective solvents) was injected in splitless mode.

Compound Identification: Based on mass spectral fragmentation patterns using NIST and Wiley spectral libraries.

Quantification: Relative abundance of detected compounds was expressed as a percentage based on peak area in the chromatogram (Seetharaman et al., 2025).

Antioxidant properties of the aerial parts, roots, resveratrol, astragalin, and rutin fractions were assessed using the DPPH radical scavenging assay. DPPH assay was performed using the following procedure (Brand-Williams et al., 1995; Baliyan et al., 2022): An amount of 0.5 mg DPPH was dissolved in 10 mL of ethanol to give a 1 mg/mL DPPH stock concentration. This solution was stored in a dark glass bottle to avoid proper temperature conditions and/or sunlight to keep it stable throughout the experiment. Four sample solutions were prepared with the following concentrations: 1 mg/mL, 0.1 mg/mL, 0.01 mg/mL, and 0.001 mg/mL in ethanol. These concentrations of the samples made it possible to use the scavenging activity of each of the samples over a range of dilutions. The DPPH assay was performed in a 96-well microplate, which included the addition of 200 µL of DPPH solution in each well as a source of free radicals. Later, 100 µL of different sample solutions at different concentrations was added in the respective wells after the DPPH solution was added. As a control, 100 µL of ethanol was placed instead of the sample in separate wells to assess the DPPH absorbance without the addition of any antioxidant. Further blanks were carried out using 200 µL of ethanol instead of DPPH solution along with 100 µL of each of the samples, to take care of the probable absorbance of the samples themselves ethanol. Mixtures were then incubated in the dark at room temperature for 30 min to allow the reaction of the DPPH radicals with possible antioxidants in the samples in solution. Following this period, the absorbance of each of the mixtures was measured at 517 nm using a UV-Vis microplate reader. The percentage of DPPH radical scavenging activity was calculated using the following formula: DPPH Scavenging Activity  % = A  control  – A  sample  /   A  control × 100

In this formula, A control refers to the absorbance of DPPH solution with ethanol whereas A sample refers to the absorbance of DPPH solution absorbed in the sample. In this manner, a calculation of the percentage of DPPH radical scavenging activity for each sample was done along with IC₅₀. The lower the IC₅₀ value of the samples, the greater the antioxidant activity since it conveyed that less of the sample was needed to reduce the DPPH radicals by 50%. The DPPH scavenging effects of the aerial part, roots extract, resveratrol, astragalin, and rutin were analyzed based on their IC₅₀ or extent of activity at different concentrations. They were also compared to assess their DPPH free radical scavenging ability in depth and anti-oxidative capacity as well (Moukette et al., 2015).

The antioxidant activities of the aerial part of the plant, root, resveratrol, astragalin, and rutin were evaluated using the ABTS radical cation (ABTS⋅⁺) assay. The ABTS⋅⁺ solution preparation method was done according to (Ilyasov et al., 2020) which includes:39.2 mg ABTS dissolved in distilled water and subsequently completed with a 6.7 mM potassium persulfate solution in equal volume (We Prepared a 6.7 mM potassium persulfate solution by dissolving 17.6 mg of potassium persulfate in 5 mL of distilled water.). The solution was allowed to stand in the darkroom at a temperature for 12–16 h to form the ABTS radical cation. This resulting solution was then further diluted with ethanol to measure the maximum probable absorbance density of about 0.70 (±0.02) at 734 nm. Trolox standards were prepared in ethanol from 0.01 to 0.1 mM and sample solutions were prepared in ethanol at required concentrations. In the assay, 200 µL sample solution was taken in each cuvette, and 2 mL ABTS⋅⁺ solution was added to each tube or cuvette for mixing. Controls were prepared by mixing 2 mL of ABTS⋅⁺ solution with 200 µL of ethanol, while blanks were prepared by mixing 2 mL of ethanol with 200 µL of each sample solution (without ABTS⋅⁺). The mixtures were incubated in the dark at room temperature for 30 min before the absorbance was measured at 734 nm using a UV-Vis spectrophotometer. The percentage of ABTS radical scavenging activity was calculated using the following formula: ABTS Scavenging Activity  % = A  control − A  sample  A  control  × 100 Where A control is the absorbance of the ABTS⋅⁺ solution with ethanol, and A sample is the absorbance of the ABTS⋅⁺ solution with the sample. IC₅₀ was determined by plotting the percentage of scavenging activity against the concentrations of the samples; IC₅₀ is the concentration at which it shows 50% scavenging of the ABTS radicals. Based on IC₅₀ values, and for some samples at definite concentrations, the antioxidant activities of the five tested samples were compared (Rumpf et al., 2023).

The ferric reducing antioxidant power (FRAP) assay was selected to evaluate the antioxidant activity of the aerial part, root, and fractions of resveratrol, astragalin, and rutin. For this assay, the reagents were prepared according to Skroza et al. (2015) with some modifications: The acetate buffer was prepared by adding approximately 16 mL of glacial acetic acid to 3.1 g of sodium acetate trihydrate and volumetrically making up to 1 L with distilled water. A TPTZ solution was prepared by dissolving 0.031 g in 10 mL of 40 mM HCl and a ferric chloride solution was prepared by dissolving 0.054 g of FeCl₃·6H₂O in 10 mL of distilled water. The FRAP working solution was made up immediately before use by mixing acetate buffer, TPTZ solution, and ferric chloride solution in a ratio of 10:1:1. Sample solutions from root extract, leaf extract, resveratrol, astragalin, and rutin were prepared in ethanol at a concentration of choice. The assay was carried out by first dispensing 180 µL of the FRAP working solution into each well of a 96-well microplate before adding 20 µL of each sample solution or trolox standard to the wells. The plate was then incubated for half an hour at 37°C before the absorbance reading was undertaken at 593 nm using a microplate reader. This absorbance was employed to estimate the reducing power of samples according to their capability to convert Fe³⁺ to Fe²⁺ in the presence of TPTZ (Benzie and Devaki, 2018).

Cupric reducing antioxidant capacity (CUPRAC) assay was utilized for screening additional radical scavenging activity of the aerial part, root, resveratrol, astragalin, and rutin. To perform the CUPRAC assay the following reagents were prepared according to Apak et al. (2010) 0.4262 g of copper (II) chloride dihydrate (CuCl₂·2H₂O) was dissolved in 200 mL of distilled water to make a 0.01 M copper (II) chloride solution. For preparing 1 M ammonium acetate buffer (pH 7.0), 7.708 g of ammonium acetate was dissolved in 100 mL of distilled water, and about 60 mL of this buffer was supplemented with 0.0075 M neocuproine solution prepared by dissolving 0.039 g of neocuproine in 20 mL of ethanol. Trolox standard solutions were prepared in ethanol with concentrations ranging from 0.01 to 0.1 mM. Sample solutions from root extract, leaf extract, resveratrol, astragalin, and rutin were also prepared in ethanol at desired concentrations. For the assay, 150 µL of the pre-mixed reagent solution [copper (II) chloride, ammonium acetate, and neocuproine] was pipetted into each well of a 96-well microplate, followed by the addition of 50 µL of each sample solution or standard Trolox solution. The microplate was incubated at room temperature for 30 min in the dark to allow the reaction to occur. The absorbance was measured at 450 nm using a UV-Vis microplate reader. The antioxidant activity was calculated using the formula: CUPRAC Activity  % =   A  sample  –   A  blank  /   A  control  × 100 Where A sample is the absorbance of the sample solution, A blank is the absorbance of the blank (without CUPRAC), and A control is the absorbance of the Trolox standard. The IC₅₀ value was determined by plotting the CUPRAC activity percentage against the sample concentrations where this value represents the concentration necessary to achieve 50% of the maximum CUPRAC activity observed from the standard. The antioxidant activities of the five samples were also compared based on IC₅₀ values or CUPRAC activities at a given concentration (Suktham et al., 2019).

Cell lines were purchased from the Holding Company for Biological Products and Vaccines (VACSERA, Giza, Egypt). HCT-116 and WI-38 cells were cultured in RPMI-1640 medium. Enhanced with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture (100 IU/mL penicillin and 0.1 mg/mL streptomycin) (Calmeiro et al., 2021).

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma) was dissolved in PBS at a concentration of 5 mg/mL and filtered to sterilize and remove a small amount of insoluble residue present in some batches of MTT. At the times indicated below, the original MTT solution (10/∼1 per 100/∼1 medium) was added to all test wells and the plates were incubated at 37°C for 4 h. Isopropanol acid (100/∼1 of 0.04 N HCI in isopropanol) was added to all wells and mixed well to dissolve the dark blue crystals. After a few minutes at room temperature to ensure all crystals were dissolved, the plates were read on a Dynatech MR580 Microelisa Reader, using a test wavelength of 570 nm, a reference wavelength of 630 nm, and a calibration setting of 1.99 (or 1.00 if the samples were strongly colored). Plates were typically read within 1 h of adding isopropanol.

The MTT assay was used to evaluate the proliferation of control and treated cells according to Ghasemi et al. (2023). 2.6∼3 × 10⁴ cells were added to each well of a 96-well tissue culture plate containing the appropriate media and grown for 24 h. Drug stock solutions were prepared in DMSO. Eight concentrations (300, 100, 30, 10, 3, 1, 0.3, and 0.1 μg/mL) were prepared for each compound in the growth media. The Nutlin was used as a reference compound. Cells were then treated for 72 h. Freshly prepared MTT salt (3-(4,5-dimethylthiazol-2yl)-2,5- diphenyltetrazolium bromide) (5 mg/mL; Sigma) was then added to each well to give a final concentration of 0.5 μg/μL. The plates were incubated for 4 h and the formation of formazan crystals was checked using an inverted microscope. An Equal volume of 1:1 (200 μL) DMSO and isopropanol mixture was added to each well and incubated for 30–45 min. Cell proliferation was detected by measuring the absorbance of each well at 590 nm using Multiskan^® EX (Thermo Scientific, United States) Microplate Reader. The Experiment was performed three times in triplicates (Ghasemi et al., 2023).

The DPPH method, an established approach for evaluating the free radical scavenging activity, was carried out on the samples in different concentrations (1.0 mg/mL, 0.1 mg/mL, 0.01 mg/mL, and 0.001 mg/mL) in the present study. Supplementary Table S7 also shows the percent inhibition at these concentrations for each of the samples representing all the likely ranges of their antioxidant potentials.

As shown in Supplementary Table S7, the root extract exhibited the highest level of antioxidant activity at the highest concentration (97% inhibition at 1 mg/mL). Rutin also showed strong antioxidant activity, achieving 93% inhibition at 0.1 mg/mL and 84% at 1 mg/mL. Resveratrol followed closely, with 77% inhibition at 1 mg/mL and 84% at 0.1 mg/mL. The aerial parts demonstrated moderate activity, with 75% inhibition at the highest concentration. Astragalin, however, showed the weakest activity, with only 11% inhibition at 1 mg/mL and progressively lower values at lower concentrations. The IC₅₀ value represents the concentration required to inhibit 50% of the DPPH radicals. Lower IC₅₀ values indicate higher antioxidant potency, as less compound is required to achieve significant inhibition (Djeghim et al., 2024). The IC₅₀ values for each sample were determined from the data and are depicted in Figure 3. The DPPH radical scavenging assay was conducted to evaluate the antioxidant activity of the tested samples. The IC₅₀ values indicate the concentration required to scavenge 50% of DPPH radicals, with lower values representing higher antioxidant potential. Among the tested compounds, Rutin (IC₅₀ = 0.013 mg/mL) and Roots (IC₅₀ = 0.017 mg/mL) exhibited the strongest antioxidant activity, followed by Resveratrol (IC₅₀ = 0.032 mg/mL) and Leaves (IC₅₀ = 0.12 mg/mL), while Astragalin displayed the weakest activity (IC₅₀ = 1.2 mg/mL). Statistical analysis using one-way ANOVA and Tukey’s multiple comparison tests confirmed that the differences in antioxidant potential were statistically significant (p < 0.0001) between Astragalin and all other compounds, indicating its significantly lower free radical scavenging capacity. However, no significant differences were observed between Roots and Rutin (p > 0.9999), Leaves and Roots (p = 0.639), or Leaves and Resveratrol (p = 0.791), suggesting comparable antioxidant activity among these compounds. These results highlight the strong radical scavenging properties of Rutin and Roots, while Astragalin exhibited significantly lower antioxidant potential compared to the other samples.

The further evaluation of the antioxidant properties of the aerial parts, roots, and Resveratrol, Astragalin, and Rutin fractions was carried out using the ABTS radical cation method, which is often used in the estimation of the free radical scavenging potential of the plant. Supplementary Table S8 depicts the percentage of ABTS radicals that were effectively inhibited by the four different concentrations of the samples (1.0 mg/mL, 0.1 mg/mL, 0.01 mg/mL, and 0.001 mg/mL). According to the data in Supplementary Table S8, Resveratrol exhibited the highest antioxidant activity across all concentrations, particularly at 1 mg/mL, where it achieved 92% inhibition of ABTS radicals. The root extract followed closely, demonstrating 88% inhibition at the same concentration. Rutin also displayed strong antioxidant potential, with 78% inhibition at 1 mg/mL. However, its activity diminished more rapidly at lower concentrations compared to resveratrol and roots. In comparison, the extracts obtained from the aerial parts were only moderately active, achieving 37% inhibition at 1 mg/mL concentration and this activity decreased at lower concentrations. Astragalin demonstrated the weakest antioxidant activity, achieving only 25% inhibition at 1 mg/mL, and showing minimal effectiveness at lower concentrations.

The results from the ABTS assay, as visualized in Figure 4, provide further insights into the antioxidant capacity of the samples. The ABTS radical scavenging assay was performed to evaluate the antioxidant potential of the tested samples. The IC₅₀ values revealed that Resveratrol (IC₅₀ = 0.1 mg/mL) exhibited the highest antioxidant activity, followed closely by Roots (IC₅₀ = 0.11 mg/mL), Leaves (IC₅₀ = 0.12 mg/mL), and Rutin (IC₅₀ = 0.125 mg/mL), where Astragalin displayed the weakest antioxidant activity (IC₅₀ = 1.1 mg/mL). Statistical analysis using one-way ANOVA and Tukey’s multiple comparison test showed no significant differences between Leaves, Roots, Rutin, and Resveratrol (p > 0.9999), indicating similar antioxidant potential among these samples. However, Astragalin exhibited significantly lower antioxidant activity compared to all other tested compounds (p < 0.0001), confirming its weaker radical scavenging capacity. These findings reinforce the strong antioxidant potential of Leaves, Roots, Rutin, and Resveratrol, with Astragalin demonstrating a markedly lower ability to neutralize ABTS radicals.

The Ferric Reducing Antioxidant Power (FRAP) assay quantified the antioxidant potential of the aerial parts, roots, resveratrol, astragalin, and rutin. This assay evaluates the activity of samples in the reduction of ferric ions (Fe³⁺) to ferrous ions (Fe²⁺) as a defined measure of antioxidant activity. Supplementary Table S9 shows the percentage of ferric ion reduction at various concentrations for each of the samples. At the highest concentration of 1 mg/mL, the root extract was found to have the highest level of antioxidant activity with 82% ferric ion reduction. This was followed by resveratrol at 61% and Rutin at 47% respectively. The aerial parts exhibited moderate antioxidant activity with 34% ferric ion reduction, while Astragalin had the least activity reducing 12% of the ferric ions. At lower concentrations such as (0.01 mg/mL and 0.001 mg/mL), the samples in general showed a significant reduction in antioxidant activities, with some being negative values, showing a lack of or very little activity. This highlights the importance of concentration in the investigation of antioxidant screening by FRAP where high amounts of the materials are essential to produce enhanced antioxidant activities. The results presented in Figure 5 show IC₅₀ of aerial parts, roots, Resveratrol, Astragalin, and Rutin as a free radical scavenging effect. The Ferric Reducing Antioxidant Power (FRAP) assay was performed to assess the reducing capacity of the tested samples. The IC₅₀ values indicated that Roots (IC₅₀ = 0.36 mg/mL) exhibited the highest reducing power, followed by Resveratrol (IC₅₀ = 0.7 mg/mL), Rutin (IC₅₀ = 1.06 mg/mL), and Leaves (IC₅₀ = 1.4 mg/mL), Astragalin showed the weakest reducing capacity (IC₅₀ = 4.08 mg/mL). Statistical analysis using one-way ANOVA and Tukey’s multiple comparison tests confirmed significant differences (p < 0.0001) between Leaves and Roots, as well as between Astragalin and all other tested samples, indicating substantial variations in antioxidant potential. Additionally, significant differences were observed between Rutin and Resveratrol (p = 0.0164) and between Roots and Resveratrol (p = 0.0035), highlighting their distinct reducing capacities. These results suggest that Roots possess the strongest ferric reducing power, followed by Resveratrol, while Astragalin exhibited significantly lower antioxidant activity compared to all other samples.

The evaluation of the antioxidant activities of different fractions of the aerial part, root, resveratrol, astragalin, and rutin was carried out using the Cupric Reducing Antioxidant Capacity (CUPRAC) assay. This assay evaluates the ability of antioxidant compounds to reduce the cupric ions (Cu²⁺) to the cuprous ions (Cu⁺) which gives a good measure of its antioxidant activities. Supplementary Table S10 presents the percentage of reduction at various concentrations for each sample. At the maximum concentration (1 mg/mL), the antioxidant activity was the most substantial as 95% of the root extract was reduced with the decreasing order of activity following 94% of rutin and 93% of resveratrol. Astragalin could also display potent antioxidant effects with only an 85% reduction, whereas the aerial parts exhibited moderate activity with a 68% reduction. Decreased values of antioxidant activities were observed in samples at lower concentrations with the negative reduction values occurring at 0.01 mg/mL, and 0.001 mg/mL suggesting no activity or negligible at those concentrations.

The IC₅₀ values, which represent the concentration required to achieve 50% inhibition, provide a more detailed comparison of the antioxidant efficacy of the samples. Figure 6 illustrates the IC₅₀ values for the aerial parts, roots, resveratrol, astragalin, and rutin. The CUPRAC assay results indicate the antioxidant capacities of Leaves, Roots, Ruth, Astragalin, and Resveratrol, evaluated through % inhibition at concentrations of 0.001, 0.01, 0.01 and 1.0 mg/mL. All samples showed no statistically significant differences (marked as “ns”) in % inhibition across the tested concentrations, suggesting comparable short-range antioxidant effects at these specific doses. The CUPRAC assay was conducted to evaluate the antioxidant capacity of the tested samples. The IC₅₀ values revealed that Roots (IC₅₀ = 0.06 mg/mL) exhibited the strongest antioxidant activity, followed by Rutin (IC₅₀ = 0.09 mg/mL) and Resveratrol (IC₅₀ = 0.12 mg/mL). Astragalin (IC₅₀ = 0.126 mg/mL) demonstrated slightly weaker activity, while Leaves (IC₅₀ = 0.7 mg/mL) showed the lowest antioxidant potential among the tested samples. These findings highlight the superior antioxidant activity of Roots, likely due to the presence of highly effective reducing agents, as measured by the CUPRAC method.

Statistical analysis using one-way ANOVA revealed no significant differences in antioxidant activity between Roots, Rutin, Resveratrol, and Astragalin (p > 0.05).

Interpretation of Pearson Correlation Coefficients in the Context of Antioxidant Assays, A comparative analysis of the antioxidant potential among the samples is illustrated in Figure 7.

The Pearson correlation matrix in the provided figure represents the relationships between four used antioxidant assays DPPH, FRAP, ABTS and CUPRAC.

Each of these assays measures antioxidant potential but through different mechanisms. Correlations among them indicate similarities or differences in how antioxidants behave in different experimental conditions.

Correlations among them indicate similarities or differences in how antioxidants behave in different experimental conditions.

1. Strong Positive Correlation: DPPH vs. CUPRAC (r = 0.67)

The IC50 value for DPPH and CUPRAC shows strong linear correlation, suggesting that samples have high activity in DPPH also will have high activity in CUPRAC.

2. Moderate Negative Correlation: DPPH vs. FRAP (r = −0.50)

A moderate inverse relationship indicates that samples with lower IC₅₀ values in DPPH (higher antioxidant activity) tend to have higher IC₅₀ values in FRAP meaning lower antioxidant activity 3. Weak Negative Correlation: DPPH vs ABTS (r = −0.10)

The weak negative correlation suggests minimal relationship between these assays in terms of IC₅₀ values.

4. Weak Positive Correlation: FRAP vs CUPRAC (r = 0.27)

A weak but positive association exists between these two assays, indicating that some trends in antioxidant activity are shared.

5. Moderate Negative Correlation: ABTS vs. CUPRAC (r = −0.41)

A moderate inverse correlation implies that high antioxidant activity in ABTS (low IC₅₀) is moderately associated with lower antioxidant activity in CUPRAC.

6. Weak Negative Correlation: FRAP vs. ABTS (r = −0.30)

A weak inverse correlation suggests that there is little overlap in the antioxidant mechanisms measured by FRAP and ABTS.