Radical Scavenging and Anticancer Activities of Lebanese Sarcopoterium spinosum Fruit Extracts

Abstract

Objectives

Sarcopoterium spinosum represents a medicinal Mediterranean species of significant scientific interest, owing to its rich phytochemical composition and well-documented health-promoting properties. Most studies have focused on plant material from Palestine, Jordan, and Turkey, mainly on its aerial parts and roots, while the fruit material from Lebanon remains largely underexplored. Given that oxidative stress is a major contributor to the pathogenesis of various diseases, including cancer, we assessed the radical scavenging and anticancer activities of extracts from Lebanese Sarcopoterium spinosum fruits.

Methods

Phytochemicals were extracted from these fruits using three solvents: ethanol, water, and a mixture of both, and the chemical composition of the extracts was evaluated by qualitative phytochemical screening. The total phenolic and flavonoid contents were quantified using the Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. The radical scavenging activity of the three fruit extracts was assessed by the ability to neutralize DPPH free radicals. Moreover, their inhibitory effect on the viability of non-cancerous intestinal cells as well as on colorectal and breast cancer cells was evaluated using the MTT assay.

Results

Our findings showed that the ethanol/water solvent was the most effective at extracting phenolic and flavonoid compounds from the fruits. Moreover, the three fruit extracts demonstrated effective DPPH free radical scavenging potential, with the ethanol/water and ethanol extracts exhibiting the greatest effects. Furthermore, the three fruit extracts inhibited the viability of colorectal and breast cancer cells with the ethanol/water extract displaying the most potent effect. Although the three fruit extracts did not have hemolytic activity, they reduced the viability of non-cancerous intestinal cells, with the ethanol extract exerting the highest effect.

Conclusion

Our findings suggest that despite the promising radical scavenging and anticancer potential of Sarcopoterium spinosum fruit extracts, their effects on non-cancerous cells warrant careful evaluation and further safety studies.

Keywords

anticancer activity breast cancer colorectal cancer flavonoids phenolics radical scavenging activity

I. Introduction

Oxidative stress has emerged as a central pathological mechanism that induces molecular and cellular dysfunction, thereby contributing to the initiation and progression of a wide spectrum of human diseases, ranging from neurodegenerative disorders to cancer. It is defined as the imbalance between the generation of reactive oxygen species (ROS) and their elimination by the endogenous antioxidant mechanisms leading to the pathological accumulation of ROS. At the DNA level, ROS reacts with nitrogenous bases and deoxyribose inducing significant oxidative reactions and thereby leading to mutations, carcinogenesis, and hereditary diseases.¹ Beyond nucleic acid damage, ROS attack lipids, initiating peroxidation, thereby altering membrane structure, impacting its fluidity and impairing its integrity.¹ Likewise, ROS damage the structural integrity of proteins, resulting in the loss of catalytic activity of numerous enzymes and alterations in the regulation of metabolic pathways.¹ The contributions of ROS to the development of diverse pathologies across different organs have been comprehensively reviewed elsewhere.² Therefore, the effective elimination of excess ROS is essential for protecting macromolecules from oxidative damage and contributes to the prevention and treatment of ROS-associated diseases.

Over the past decades, plant extracts have been extensively demonstrated to be rich sources of antioxidants and free radical scavengers, exhibiting potent antioxidant activity capable of mitigating oxidative stress and treating its related diseases. For example, in an alloxan-induced diabetic rat model, plant extracts showed hypoglycemic and anti-diabetic effects and attenuated oxidative stress by enhancing the activities of antioxidant enzymes and lowering oxidative damage markers in the liver, kidney, and spleen.³ Similarly, in a paraquat-induced Parkinson’s disease rat model, plant extracts improved behavioral performance and alleviated oxidative stress through increased activities of antioxidant enzymes and a decreased oxidative damage marker in the brain.⁴ In addition, plant extracts demonstrated effective antioxidant and anti-inflammatory activities in carrageenan-induced mice paw edema.⁵ Interestingly, these health-beneficial effects were primarily attributed to the presence of phenolic and flavonoid compounds within the plant extracts.^3-5

Among the large varieties of plants found in the Mediterranean region, Sarcopoterium spinosum is recognized as a species of particular pharmacological relevance due to its wide range of health-promoting properties. In particular, the antidiabetic effect of its roots and aerial parts has been extensively documented.^6-10 Importantly, numerous studies have demonstrated that the aerial parts of this plant are rich in phenolic and flavonoid compounds and exhibit antioxidant activity.^11-16 This activity may contribute to its other reported biological effects, including anti-obesity potential,¹¹ antibacterial activity,¹² hepatoprotective effects in carbon tetrachloride- induced hepatic damage in rats,¹³ and inhibitory activity on cancer cell viability,^15,16 although this relationship has not yet been well-established. Besides exploring the health-benefits of roots and aerial parts of S. spinosum, recent research also evaluated extract from its fruits which were also found to have antioxidant and antisteatotic effects in addition to anti-inflammatory activities that ameliorated the dysfunction of endothelial cells.^17,18

Despite the wide range of reported biological activities, it is important to note that most investigations on S. spinosum have focused on specimens collected from Palestine, Jordan, Turkey, Italy, and Libya. In contrast, research on S. spinosum grown in Lebanon remains limited. To date, only three studies have addressed its antioxidant potential and associated biological activities: one examining the anticancer activity of the aerial part extracts¹⁵ and two others assessing the anti-inflammatory¹⁷ and antisteatotic¹⁸ effects of fruit extracts in vitro. However, the inhibitory effect of Lebanese S. spinosum fruit extracts on cancer, an oxidative stress-related disease, remains unexplored highlighting a critical gap in the current body of evidence.

Notably, phytochemical investigations have already shown that Lebanese S. spinosum fruits are rich in polyphenols.^17,18 Given the well-documented role of polyphenols in modulating cancer-related molecular pathways,¹⁹ the phytochemical profile of Lebanese S. spinosum fruits suggests strong anticancer potential. The ethanolic extract of this fruit revealed a complex composition comprising 24 distinct compounds, of which 17 were successfully identified. In particular, ellagitannins constituted the predominant group of phenolic compounds and included casuarictin isomers, corilagin, pedunculagin, and castalagin/vescalagin. Flavonoids represented the second most abundant group, with quercetin glucuronide and quercetin diglucoside identified as the principal constituents. The ethanolic extract also contained triterpenes including tormentic acid and several of its derivatives.¹⁸

To address this research gap, and in light of its rich phytochemical composition, the aim of this study was to investigate the anticancer activity of Lebanese S. spinosum fruit extracts prepared using three different solvents: ethanol, water, and a 50% (v/v) ethanol/water mixture. Specifically, we aimed to quantify their total phenolic and flavonoid contents, and evaluate their radical scavenging and hemolytic activities. We also aimed to assess their inhibitory effects on the viability of colorectal and breast cancer cells, as well as non-cancerous intestinal cells, in order to determine their selectivity toward cancer cells.

II. Material and Methods

1. Fruit Collection and Extraction

The S. spinosum fruits were collected from the wild in the south region of Lebanon in summer 2020. Plant identification was performed by Professor George Tohme, taxonomist and president of the National Council for Scientific Research (CNRS), Lebanon. Classification was based on the criteria described in his 2014 work Illustrated Flora of Lebanon, using morphological characteristics and binocular examination. A voucher specimen (R5.36) representative of the studied taxon was deposited in the Lebanon National Herbarium at the Faculty of Sciences, Lebanese University. The fruits were cleaned, and shade dried at room temperature. Subsequently, they were ground into a fine powder with particle sizes in the sub-millimeter range. The powdered material was preserved in clean plastic bags, and kept away from light, heat, and moisture until extraction. Extraction of the bioactive compounds from the fruits was done via the sonication method and using different solvents: distilled water, ethanol (99.7%) and a 50% (v/v) ethanol/water mixture. The latter was selected as a representative intermediate polarity system to enable broad-spectrum extraction of phytochemicals. A total of 196 g of the fruits dry weight of S. spinosum was dissolved in 1.96 L of each solvent. The three extracts were then placed on the sonicator (40 KHz) for 3 hours at 60°C, 35°C and 45°C for the water, ethanol, and ethanol/water extract, respectively. The ultrasound-assisted extraction was performed at 40 KHz to enhance cavitation and extraction efficiency. In addition, extraction temperatures were selected given the reported enhanced extraction of flavonoids and phenolic acids at moderate temperatures (40-60 °C).²⁰

The resulting suspensions were filtered through sterile filter paper into sterile beakers, then concentrated using a rotary evaporator followed by lyophilization.

2. Phytochemical Screening

Phytochemical screening of the fruit extract concentrates was carried out to explore the presence of different groups of secondary metabolites.

a. Detection of phenols

5 mL of each of the three extracts were mixed with 1 mL of ferric chloride (1%) and 1 mL of potassium ferricyanide (1%). The formation of a greenish blue color indicated a positive result.²¹

b. Detection of tannins

1 mL of each of the three extracts was mixed with 1 mL of ferric chloride (1%). The formation of a blue color indicated a positive result.²¹

c. Detection of phlobatannins

1 mL of each of the three extracts was mixed with 1 mL of hydrogen chloride ( 1%). The mixture was boiled for 5 minutes and then allowed to cool. The formation of a red precipitate indicated a positive result.²¹

d. Detection of flavonoids

1 mL of each of the three extracts was mixed with 5 mL of potassium hydroxide (50%). The formation of a yellow color indicated a positive result.²¹

e. Detection of flavonones

1 mL of each of the three extracts was mixed with 1 mL of concentrated sulfuric acid. The formation of a purple red color indicated a positive result.²¹

f. Detection of anthocyanins

1 mL of each of the three extracts was mixed with 1 mL of sodium hydroxide (10%). The formation of a blue color indicated a positive result.²¹

g. Detection of reducing sugars

0.5 mL of each of the three extracts were mixed with 1 mL of distilled water and 5-8 drops of Fehling’s solution (A and B), followed by boiling for 5 minutes. The formation of a brick-red precipitate indicated a positive result.²¹

h. Detection of proteins and amino acids

1 mL of each of the three extracts was mixed with 1 mL of 0.25% ninhydrin solution. The mixture was then boiled for 5 minutes. The formation of a blue color indicated a positive result.²¹

i. Detection of resins

1 mL of each of the three extracts was mixed with 0.5 mL of acetone. A small amount of water was then added to the mixture, followed by agitation. The appearance of turbidity confirmed the presence of resins.²¹

j. Detection of terpenoids

1 mL of each of the three extracts was mixed with 2 mL of chloroform, followed by the careful addition of 3 mL of concentrated sulfuric acid. The formation of a reddish-brown color at the surface indicated a positive result.²¹

k. Detection of diterpenes

1 mL of each of the three extracts was dissolved in distilled water, then mixed with 1 mL of copper acetate solution. The formation of a green color indicated a positive result.²¹

l. Detection of quinones

1 mL of each of the three extracts was mixed with 1 mL of concentrated hydrogen chloride. The formation of a yellow color or precipitate indicated a positive result.²¹

m. Detection of anthraquinones

1 mL of each of the three extracts was mixed with 1 mL of 10% hydrogen chloride . The mixture was then boiled for 5 minutes. The formation of a precipitate indicated a positive result.²¹

n. Detection of sterols and steroids

1 mL of each of the three extracts was mixed with 2 mL of chloroform and 1 mL of concentrated sulfuric acid. The formation of an upper red layer and a greenish-yellow fluorescent acid layer indicated a positive result.²¹

o. Detection of cardiac glycosides

2 mL of each of the three extracts were mixed with 1 mL of acetic acid and one drop of 5% ferric chloride, followed by the addition of 1 mL of concentrated sulfuric acid. The appearance of purple, brown, and green rings indicated a positive result.²¹

p. Detection of carbohydrates

1 mL of each of the three extracts was mixed with few drops of α-naphthol, followed by concentrated sulfuric acid. The formation of a purple ring indicated a positive result.²²

q. Detection of saponins

2 mL of each of the three extracts were vigorously shaken for 5 minutes using a vortex mixer; the formation of a stable layer of foam indicated a positive result.²¹

r. Detection of fixed oils and fatty acids

A few drops of each of the three extracts were placed on a filter paper. The appearance of a persistent oil spot after drying indicated a positive result.²¹

3. Total Phenolic Content

The level of polyphenols in fruit extracts was evaluated using the Folin-Ciocalteu method, as previously described,^23,24 with some modifications. In brief, for each solvent (water, ethanol, and ethanol/water), 100 µL of S. spinosum fruit extract (1 mg/mL) was mixed with 0.5 mL of 1:10 diluted Folin–Ciocalteu reagent and 1.5 mL of 3% (w/v) sodium carbonate. The mixture was then incubated in the dark at room temperature for 15 minutes. The absorbance of the blue-colored solution of all samples was measured at 760 nm using a spectrophotometer. Gallic acid was employed as a reference standard. The total phenolic content was determined in triplicate and expressed as milligrams of gallic acid equivalent per gram of dry fruit extract (mg GAE/g of extract). The blank was prepared by adding the respective solvent for each extract to 0.5 mL Folin-Ciocalteu reagent and 1.5 mL of 3% (w/v) sodium carbonate.

4. Total Flavonoid Content

The total flavonoid content of each extract was determined using the aluminum chloride colorimetric method, as previously described,^24,25 with some modifications. Briefly, extracts were prepared at 10 mg/mL. For the assay, 0.5 mL of each extract or 2.0 mL of quercetin standard solution was mixed with 0.2 mL of 10% (w/v) aluminum chloride and 0.2 mL of 1 M potassium acetate. After 5 minutes, 1 mL of 1 M sodium hydroxide and 2.1 mL of distilled water were added. The mixtures were incubated at room temperature in the dark for 15 minutes, resulting in the development of an orange-yellow color. The absorbance was measured at 510 nm using a spectrophotometer, with distilled water used as the blank. The quercetin calibration curve (100–1000 μg/mL in methanol) was used to quantify total flavonoid content. All samples were analyzed in triplicate, and results were expressed as milligrams of quercetin equivalent per gram of dry fruit extract (mg QE/g of extract) based on the standard curve.

5. Radical Scavenging Activity

The free radical scavenging activity of the fruit extracts was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, as previously described,^26,27 with some modifications. Briefly, 1 mL of DPPH solution (0.052 mg/mL in ethanol) was mixed with 100 μL of fruit extracts at concentrations of 0.0625, 0.125, 0.25, 0.5, and 1 mg/mL. The mixtures were shaken thoroughly and incubated in the dark at room temperature for 10 minutes. Absorbance was then measured at 515 nm using a spectrophotometer. Negative controls were prepared by mixing 1 mL of DPPH solution with 100 μL of the corresponding extract solvent, and the respective solvents were used as blanks. Each sample was analyzed in triplicate. Ascorbic acid was employed as a standard reference compound.

The DPPH scavenging ability of fruit extracts was calculated using the following equation:

% of DPPH Inhibition = \frac{(A_{0} - A_{1})}{(A_{0})} \times 100

Where: A₀ = Absorbance of the control and A₁ = Absorbance of the extract

6. Hemolytic Activity

The hemolytic activity of fruit extracts was determined according to a previously described method,²⁸ with some modifications. Fresh human blood samples were centrifuged at 2,500 rpm for 10 minutes at 4°C. Erythrocytes were separated from plasma and washed three times with 1X phosphate-buffered saline (PBS, pH 7.4). After each wash, cells were pelleted by centrifugation at 2,500 rpm for 12 minutes at 4°C, and the supernatant was discarded. The resulting erythrocyte pellet was resuspended in PBS to prepare a 5% (v/v) cell suspension. Subsequently, 100 μL of fruit extracts at various concentrations (10–200 μg/mL, diluted in 1X PBS) were added to 1 mL of the erythrocyte suspension. The mixtures were incubated at 37°C for 90 minutes with gentle shaking every 30 min. Following incubation, the tubes were centrifuged at 2,500 rpm for 10 min at 4°C, and the absorbance of the supernatant was measured at 540 nm using a spectrophotometer. Erythrocytes maintained solely in PBS served as the negative control (0% hemolysis), while 1% SDS was used as a positive control representing 100% hemolysis. All experiments were performed in triplicate, and hemolysis was expressed as a percentage according to the following equation where OD denotes optical density:

% Hemolysis = \frac{OD of extract - OD of negati ve control}{OD of positi ve control - OD of negati ve control} \times 100

7. Cell Culture

Human colorectal cancer cells (HCT116), human breast cancer cells (MDA-MB-231), and human non-cancerous small intestine epithelial cells (FHs74Int) were obtained from the American University of Beirut in Lebanon. Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and incubated at 37 °C in a humidified atmosphere containing 5 % CO₂. Non-cancerous cells were also supplemented with insulin.

8. Cytotoxic Activity

Cytotoxicity of S. spinosum fruit extracts was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method, as previously described,²⁹ with some modifications. A density of 10⁴ cells/well was seeded in 96-well microtiter plates and incubated overnight. The cells were then treated with increasing concentrations 0, 25, 50, 75, 100, 150 µg/mL of each extract for 24, 48 and 72 hours. After treatment, cells were incubated with 20 µL of MTT reagent ( 5 mg/mL) at 37 °C for 3 hours then with 100 µL of isopropanol for 1 hour. The MTT optical density (OD) was then measured by a spectrophotometer at 515 nm. The results are expressed as percentage of viable cells with respect to the untreated control using this formula:

% viability = [mean OD treatment / mean OD control] \times 100

IC₅₀ values were estimated using linear regression analysis in Microsoft Excel. A linear trendline was fitted to the percentage cell viability versus extract concentration, and the IC₅₀ was calculated as the concentration corresponding to 50% cell viability.

9. Statistical Analysis

All assays were performed in triplicate. Values were then presented as average values along with their standard derivations (Mean ± SD). IC₅₀ values, means, standard deviations as well as graph plots were analyzed using Microsoft Excel 2013. The correlation between DPPH scavenging activity, total phenolic and total flavonoid contents was determined using Pearson’s correlation coefficient (r).

III. Results

1. Phytochemical Screening

Phytochemical screening of the three extracts of S. spinosum fruits revealed that they are enriched with various classes of phytochemical compounds including phenols, tannins, phlobatannins, flavonoids, flavonones, anthocyanins in addition to reducing sugars, proteins, resins, terpenoids, steroids, glycosides, and oils as depicted in Table 1. However, saponins and diterpenes were not detected in the three extracts.

Table 1.

Phytochemical Screening of the Different Sarcopoterium spinosum Fruit Extracts

Compounds	Samples
Compounds	Water extract	Ethanol extract	Ethanol/water extract
Phenols	+	+	+
Tannins	+	+	+
Phlobatannins	+	+	+
Flavonoids	+	+	+
Flavanones	+	+	+
Anthocyanins	+	+	+
Reducing Sugar	+	+	+
Proteins and amino acids	+	+	+
Resins	+	+	+
Terpenoids	+	+	+
Diterpenes	−	−	−
Quinones	+	+	+
Anthraquinones	+	+	+
Sterols and steroids	+	+	+
Cardiac glycosides	+	+	+
Carbohydrates	+	+	+
Saponins	−	−	−
Fixed oils and fatty acids	+	+	+

Note. Negative sign (−) indicates absence; positive sign (+) indicates presence.

2. Total Phenolic Content

Total phenolic content was reported as mg gallic acid equivalent/g of dry weight by reference to a standard curve of gallic acid (y= 0.1973x; R² = 0.9722). The concentration of this phytochemical in the tested extracts varied based on the solvent used in the extraction process. The phenolic content reported in the ethanol/water extract was higher than that found in the ethanol and water extracts by 17 and 44%, respectively (Table 2).

Table 2.

Total Phenolic and Flavonoid Contents of the Different Sarcopoterium spinosum Fruit Extracts

	Phenolic content (mg gallic acid equivalent/g dry extract)	Flavonoid content (mg quercetin equivalent/g dry extract)
Water extract	5.6 ± 0.19	1.91 ± 0.05
Ethanol extract	6.85 ± 0.09	2.58 ± 0.1
Ethanol/water extract	8.04 ± 0.08	2.75 ± 0.04

3. Total Flavonoid Content

The total flavonoid content was expressed as mg quercetin equivalent/g of dry weight and derived from a standard curve of quercetin (y = 0.0005x + 0.0281; R² = 0.9971). The flavonoid content found in the three tested extracts also differed based on the solvent used in the extraction process. The flavonoid content reported in the ethanol/water extract was greater than that found in the ethanol and water extracts by 7 and 44%, respectively (Table 2).

Our results suggested that ethanol/water solvent was the most effective at extracting phenolic and flavonoid compounds from S. spinosum fruits.

4. Radical Scavenging Activity

We assessed the free radical scavenging ability of S. spinosum fruit extracts using DPPH radical scavenging assay. The three S. spinosum fruit extracts induced a dose and solvent -dependent DPPH quenching effect indicating antioxidant potential. The ethanol/water and ethanol extracts exhibited a greater radical scavenging effect than the water extract as evidenced by the higher percentages of antiradical activity reported at all the concentrations ranging from 62.5-500 µg/mL. The ethanol/water and ethanol extracts inhibited DPPH free radicals to almost similar levels at 125-500 µg/mL. In addition, the ethanol/water and ethanol extracts displayed a maximum of DPPH inhibition of 71.5 and 75.7% at 500 µg/mL, respectively which was close to that of the reference compound ascorbic acid (74.11%). While the water extract displayed a maximum of DPPH inhibition of 65.9% at 500 µg/mL (Figure 1). Consistent with these findings, the ethanol/water extract exhibited the highest activity, with an IC₅₀ of 82 µg/mL, followed by the ethanol extract (108 µg/mL) and the water extract (209 µg/mL).

Figure 1.

DPPH free radical scavenging activity of the different Sarcopoterium spinosum fruit extracts. Each of the water, ethanol and ethanol/water fruit extracts of Sarcopoterium spinosum (62.5-500 µg/mL) was mixed with DPPH and the absorbance of the mixture was measured after 10 min. The values are expressed as percentage of DPPH inhibition relative to the control and ascorbic acid was used as reference. Each value represents the mean ± SD of n=1 experiment performed in triplicate.

Interestingly, DPPH scavenging efficiency of S. spinosum fruit extracts showed strong positive correlation with the total phenolic content (r = 0.93) and a moderately strong positive correlation with total flavonoid content (r = 0.77), suggesting that these phytochemicals may be responsible for the radical scavenging activity of the extracts.

5. Hemolytic Activity

To examine if S. spinosum fruit extracts are toxic to human red blood cells, we assessed their hemolytic effect at concentrations ranging from 100- 200 µg/mL. The ethanol/water, ethanol and water fruit extracts induced less than 5% hemolysis at the tested concentrations, indicating that these extracts are not toxic to red blood cells at these doses (Figure 2).

Figure 2.

In vitro hemolytic activity of each of the Sarcopoterium spinosum fruit extracts. Washed fresh human blood was incubated with each of water, ethanol and ethanol/water extracts of Sarcopoterium spinosum fruits (100-200 µg/mL) for 90 min. The samples were then centrifuged, and the absorbance of the supernatant was measured. The values are expressed as percentages of red blood cells (RBC) hemolysis with respect to the positive control (SDS 1%). Each value is obtained from n=1 experiment performed in monoplicate.

6. Anticancer Activity

We investigated the anticancer potential of the ethanol/water, ethanol and water extracts of S. spinosum fruit in vitro. HCT-116 human colorectal cancer cells and MDA-MB-231 human breast cancer cells were treated with different concentrations of the natural agents ranging from 0 to 150 µg/mL for 24, 48 and 72 hours after which cell viability was evaluated by MTT assay.

Treatment of colorectal cancer cells with the three extracts resulted in a dose and solvent -dependent reduction of cell viability. The ethanol/water extract displayed the most potent effect at 48- and 72-hours post-treatment. Treating HCT-116 cells with 150 µg/mL of S. spinosum ethanol/water, ethanol and water extracts for 72 hours reduced cell viability by 78%, 64% and 58%, respectively. The IC₅₀ value of each of S. spinosum ethanol/water, ethanol and water extracts at 72 hours was 93.11, 123, and 129.21 μg/mL, respectively (Figure 3A).

Figure 3.

Anticancer activity of the different Sarcopoterium spinosum fruit extracts against HCT-116 and MDA-MB-231 cancer cells. (A) HCT-116 colorectal cancer cells were treated with each of the water, ethanol and ethanol/water extracts of Sarcopoterium spinosum fruits (0-150 µg/mL) for 24, 48 and 72 hours; (B) MDA-MB-231 breast cancer cells were treated with each of these extracts of Sarcopoterium spinosum fruits (0-150 µg/mL) for the same time points; Cell viability was then determined using MTT assay. The values are expressed as percentage of viable cells relative to untreated control. Each value represents the mean ± SD of n = 1 experiment performed in triplicate.

On the other hand, treatment of breast cancer cells with the three extracts resulted in a dose and solvent -dependent reduction of cell viability. The ethanol/water and water extracts displayed the strongest effect at 72 hours post-treatment. Treating MDA-MB-231 cells with 150 µg/mL of S. spinosum ethanol/water, ethanol and water extracts for 72 hours reduced cell viability by 43%, 22% and 42%, respectively. The IC₅₀ value of each of S. spinosum extracts at 72 hours was higher than 150 µg/mL. Besides, it is important to note that colorectal cancer cells were more sensitive to these extracts compared to breast cancer cells.

We also assessed the anticancer effect of 0-150 μg/mL of each of the three fruit extracts on FHs74Int non-cancerous intestinal cells. Surprisingly, results showed a dose and solvent dependent decrease in the viability of these non-cancerous cells. Treating FHs74Int cells with 150 µg/mL of S. spinosum ethanol/water, ethanol and water extracts for 24 hours decreased cell viability by 34.4%, 65% and 26.5%, respectively. The IC₅₀ value of S. spinosum ethanol extracts at 24 hours was 113.8 μg/mL, while that of each of ethanol/water and water extracts was higher than 150 µg/mL (Figure 4).

Figure 4.

Anticancer activity of the different Sarcopoterium spinosum fruit extracts against FHs74Int non-cancerous small intestine epithelial cells. FHs74Int cells were treated with each of the water, ethanol and ethanol/water extracts of Sarcopoterium spinosum fruit (0-150 µg/mL) for 24 hours. Cell viability was then determined using MTT assay. The values are expressed as percentage of viable cells relative to untreated control. Each value represents the mean ± SD of n = 1 experiment performed in triplicate.

IV. Discussion

Extensive research has highlighted that plants and fruits are abundant sources of bioactive compounds with antioxidant and therapeutic properties,³⁰ making them promising candidates to counteract ROS and alleviate oxidative stress-associated disorders including cancer. Here, our study provides additional insight by demonstrating, for the first time, the radical scavenging and anticancer potential of fruit extracts obtained from the Lebanese S. spinosum plant. However, although the fruit extracts did not exhibit hemolytic activity, they reduced the viability of non-cancerous intestinal cells, suggesting that the therapeutic use of this plant’s fruits should be done with careful consideration.

First, the selection of extraction conditions in the present study was guided by solvent polarity, ultrasound parameters, and temperature, all of which are known to significantly influence extraction efficiency. We extracted bioactive compounds from S. spinosum fruits using three different solvents ethanol, water, and a 50% (v/v) ethanol/water mixture to compare their efficiency in extracting bioactive compounds from the fruit material. The selection of these solvents was determined by two principal factors: (1) the non-toxicity of water and ethanol and their suitability for food-related applications; (2) greater effectiveness of binary solvent systems in extracting bioactive compounds from plant materials compared to their pure counterparts.³¹ Gadjalova et al. (2019) reported the extraction of phenolic compounds from medicinal plants using water at 60 °C under ultrasound at 35 KHz.³² In addition, ethanol has recently been shown to be effective in extracting bioactive compounds from Lebanese S. spinosum fruits.¹⁸ However, to the best of our knowledge, the use of a binary ethanol/water mixture for the extraction of bioactive compounds from the fruits of this species has not yet been investigated. Although the optimal ethanol-water ratio may vary depending on the plant matrix, a 50:50 (v/v) mixture was selected as a representative intermediate polarity system to extract both polar and moderately polar phytochemicals. A previous study has also reported improved extraction efficiency at this composition compared to a higher ethanol concentration.³³ While such findings were obtained from different plant parts, they support the use of intermediate solvent systems for efficient phytochemical recovery.

Moreover, ultrasound-assisted extraction typically operates within a frequency range of 20-120 KHz for the recovery of bioactive compounds from fruit and vegetable by-products.³⁴ Literature reports the use of low ultrasound frequencies, typically in the range of 20-40 KHz, for ultrasound-assisted extraction of bioactive compounds (reviewed in³⁴). This could possibly be due to their stronger cavitation effects³⁴ and enhanced cell disruption efficiency. Within this range, the present study selected 40 KHz.

Furthermore, the extraction efficiency of flavonoids and phenolic acids increases at moderate temperatures (40-60 °C), while temperatures exceeding 80 °C may result in their degradation. (reviewed in²⁰). Although the plant material was ground, the fruit pericarp has a rigid structure, and residual cell wall components may still limit complete solvent penetration. Therefore, a higher temperature (60 °C) was applied for aqueous extraction to enhance tissue softening and facilitate the release of intracellular compounds. Lower temperatures were used for ethanol-containing solvents due to their superior penetration capacity and to minimize solvent evaporation.

The enhanced performance of the ethanol/water extract observed in this study may be attributed to the synergistic effects of water and ethanol as extraction solvents. Water contributes by acting as a swelling agent for plant tissues, increasing the contact surface³⁵ and facilitating solvent penetration. It also efficiently solubilizes highly polar and ionic phenolic compounds.³⁶ However, its extraction efficiency is limited by its inability to effectively disrupt hydrophobic interactions within the plant matrix, which may result in limited solubilization of phenolics and reduced recovery of moderately polar flavonoids.^36,37 In contrast, ethanol, due to its moderate polarity and amphiphilic nature, can effectively disrupt ester and hydrophobic interactions between phytochemicals, thereby promoting their release from the plant matrix.³⁶ It has also been reported to break lipid-protein-phenolic complexes, enhancing the desorption of bound phenolics.³⁶ Consequently, the combination of water and ethanol in a binary solvent system allows for the efficient extraction of both free and bound phenolic compounds. This synergistic effect likely explains the higher phenolic and flavonoid contents observed in the ethanol/water extract in the present study.

Second, we performed qualitative phytochemical screening to explore the constituents of water, ethanol and ethanol/water extracts of S. spinosum fruits. Our findings demonstrated the presence of phenolic and flavonoid compounds in addition to other secondary metabolites in the three extracts, suggesting their potential antioxidant activity. Therefore, the total phenolic and flavonoid contents of the three extracts were subsequently quantified. As anticipated, the binary mixture of water and ethanol resulted in the extraction of greater phenolic and flavonoid compounds compared to their pure counterparts which is in line with previous reports.^38,39 Besides, although we started with twice the weight of fruits compared to that used by Zbeeb et al. (2024), the total phenolic and flavonoid contents in our ethanolic extract were markedly lower than those reported in their study on ethanolic extracts of Lebanese S. spinosum fruits.¹⁸ This discrepancy may be attributed to differences in extraction techniques and extraction temperatures between both studies. Moreover, other factors related to the plant growing conditions could affect their phenolic content including soil nutrients, temperature, water, and fruit maturity stage.⁴⁰ On the other hand, the extracts obtained from the aerial parts of S. spinosum cultivated in different countries exhibited higher phenolic contents. For example, total phenolic content reached 635.2 ± 12.9 mg GAE/g extract in leaves of S. spinosum from Turkey extracted with 80% aqueous ethanol,¹² 364.6 ± 13.5 mg GAE/g extract in methanolic extracts of aerial parts from Greece,¹⁴ and 61.71 ± 2.7 mg GAE/g extract in methanolic extracts of aerial parts from Libya.¹⁶

Third, we assessed the radical scavenging potential of ethanol/water, ethanol and water extracts of S. spinosum fruits using DPPH assay. The ethanol and ethanol/water extracts demonstrated similar levels of radical scavenging activity, both of which were superior to that of the water extract. This could be possibly due to the greater phenolic and flavonoid contents detected in the ethanol and ethanol/water extracts compared to those observed in the water extract. Interestingly, DPPH scavenging activity correlated positively with total phenolic and flavonoid contents, suggesting that these phytochemicals may contribute to the radical scavenging activity of the extracts. Our findings are in line with the study conducted by Zbeeb et al. (2024) who demonstrated the DPPH scavenging activity of the ethanol extract of the Lebanese S. spinosum fruits.¹⁸ Mechanistically, phenols can scavenge DPPH free radicals via two pathways including direct hydrogen atom transfer or sequential proton loss and subsequent electron transfer.⁴¹

In addition to their radical scavenging potential, phenolic-rich extracts have also been reported to have anticancer activity in various types of neoplasms.¹⁹ Increasing evidence suggests that phenolics and flavonoids can interfere with multiple hallmarks of cancer, including the inhibition of cell proliferation, induction of apoptosis, arrest of cell cycle, and modulation of key signaling pathways.⁴² Consistent with these findings, our results showed that S. spinosum fruits extracts inhibited the viability of colon and breast cancer cells with the binary ethanol/water solvent mixture yielding the strongest effect. This could be possibly due to the extraction of higher amounts of phenolic and flavonoid compounds by this binary solvent mixture.

Although the phytochemical screening performed in this study was qualitative and did not allow the identification of specific compounds, the detected classes of compounds possibly account for the observed anticancer activity of the fruit extracts. Compounds belonging to these classes, namely tannins, flavanones, terpenoids, and quinones, have been extensively reported to exert antiproliferative and pro-apoptotic effects in breast and colon cancer cells. For example, tannic acid and tannin-rich extracts have been shown to suppress the proliferation of MDA-MB-231 cells and HCT-116 cancer cells, respectively.^43,44 Flavanones such as hesperetin inhibited the proliferation of MDA-MB-231 and HT-29 cells and induced mitochondrial apoptosis.^45,46 Similarly, terpenoids such as ascaridole exerted a cytotoxic effect on HCT-8 colon cancer cells,⁴⁷ while carvacrol inhibited the proliferation of MDA-MB-231 cells and induced apoptosis through the intrinsic mitochondrial pathway.⁴⁸ Quinones such as antroquinonol D, emodin, and physcion have also demonstrated inhibitory effects on MDA-MB-231 and HCT-116 cell proliferation.^49-53 Taken together, these findings provide a plausible mechanistic basis for the observed activity in this study, while highlighting that the precise bioactive constituents of S. spinosum fruits remain to be identified. Therefore, the observed anticancer activity could be possibly driven by the combined and potentially synergistic actions of multiple constituents within the detected phytochemical classes in the extracts.

On the other hand, Loizzo et al. (2013) demonstrated the antiproliferative activity of the methanolic extract obtained from the aerial parts of S. spinosum collected in Kfar Aaqab (Mount Lebanon) against several cancer cell lines, including human renal cell adenocarcinoma, amelanotic melanoma, malignant melanoma, breast adenocarcinoma (MCF-7), prostate carcinoma, and human epithelial carcinoma. Notably, the extract exhibited its strongest inhibitory effect against malignant melanoma and prostate carcinoma cells. Interestingly, tormentic acid was identified as major component of the aerial parts of S. spinosum. It exhibited cytotoxic activity against all tested cell lines, and was speculated to play a key role in the cytotoxic properties of S. spinosum aerial parts.¹⁵ In another report, Hudec at al. (2021) demonstrated that the ethyl acetate fraction of the aerial parts of S. spinosum collected in Palestine and the three major compounds identified in it (stachydrine, benzalkonium chloride, and rutin) significantly inhibited the growth of cancer cells especially cervical adenocarcinoma (HeLa) and mammary gland adenocarcinoma cell lines (MCF-7). Moreover, the mixture of stachydrine and benzalkonium chloride exerted a significant synergistic inhibitory effect on the growth of acute T-lymphoblastic leukemia (Jurkat), HeLa, and mammary gland adenocarcinoma cell lines (MCF-7 and MDA-MB-231).⁵⁴

Next, we tested the hemolytic activity of S. spinosum fruits extracts as well as their effect on non-cancerous cell viability to evaluate their safety and selectivity toward cancer cells, respectively. The three extracts did not induce hemolysis of red blood cells but reduced the viability of non-cancerous intestinal cells (FHs74Int cells), indicating a lack of selectivity toward cancer cells. This lack of selectivity toward non-cancerous cells represents a key limitation, suggesting that the crude extracts may not be suitable as direct anticancer agents without further purification or fractionation. It should be noted that these observations are based on preliminary in vitro data and warrant further confirmation in in vivo models. In the same line, Hudec at al. (2021) reported that benzalkonium chloride as well as its combination with stachydrine inhibited the proliferation of normal murine fibroblasts (3T3 cells).⁵⁴ In contrast, Loizzo et al. (2013) showed that extracts obtained from the aerial parts of S. spinosum as well as their fractions and isolated compound (tormentic acid) did not affect the proliferation of normal skin fibroblasts (142BR cells).¹⁵ These findings emphasize that the biological activity of a plant of the same species is variable and depends on factors such as the plant part, collection location, extraction solvent, and whether the material is evaluated as a crude extract, fraction, or isolated compounds.

V. Conclusion

The water, ethanol, ethanol/water extracts of Lebanese S. spinosum fruits exhibited promising radical scavenging and anticancer potential. The ethanol/water extract displayed the most prominent effects which could be possibly due to its higher potency in extracting bioactive compounds compared to single solvents. Although these extracts did not have hemolytic activity, they reduced the viability of non-cancerous cells which must be carefully considered. Additionally, although our results are preliminary and need to be confirmed in animal models, they highlight the significant importance of assessing plant toxicity profile. To leverage the anticancer potential of the fruit extracts, further studies should be conducted to isolate and characterize its bioactive compounds, in order to identify the molecules that have anticancer efficacy while exhibiting minimal toxicity toward normal cells.

Footnotes

ORCID iDs

Zaynab Fatfat

Maamoun Fatfat

Hala Khalife

Hala Gali-Muhtasib

Funding

This project was funded by the Lebanese University.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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