The Lebanese Red Algae Jania rubens: Promising Biomolecules against Colon Cancer Cells

Colorectal cancer (CRC) is ranked the second most lethal type of tumor globally. Thus, developing novel anti-cancer therapeutics that are less aggressive and more potent is needed. Recently, natural bioactive molecules are gaining interest as complementary and supportive antineoplastic treatments due to their safety, effectiveness, and low cost. Jania rubens (J. rubens) is a red coral seaweed abundant in the Mediterranean and bears a significant pharmacological essence. Despite its therapeutic potential, the natural biomolecules extracted from this alga are poorly identified. In this study, the proximal analysis revealed high levels of total ash content (66%), 11.3% proteins, 14.5% carbohydrates, and only 4.5% lipids. The elemental identification showed magnesium and calcium were high among its macro minerals, (24 ± 0.5 mg/g) and (33 ± 0.5 mg/g), respectively. The Chlorophyll of J. rubens was dominated by other pigments with (0.82 ± 0.02 mg/g). A 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay identified effective antioxidant activity in various J. rubens extracts. More importantly, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium reduction and wound healing assays indicate that organic extracts from J. rubens significantly counteract the proliferation of colon cancer cell lines (HCT-116 and HT-29) and inhibit their migratory and metastatic properties in a dose and time-dependent manner. Overall, this study provides insight into the physicochemical properties of red seaweed, J. rubens, and identifies its significant antioxidant, cytotoxic, and anti-migratory potential on two colorectal cell lines, HCT-116 and HT-29.


Introduction
Algae are known worldwide as an exciting field of research and an essential source of active constituents with various applications, including in the pharmaceutical, cosmetic, food, and, agricultural industries [1]. Seaweeds are rich in nutrients such as amino acids, fatty acids, lipids, vitamins, and essential minerals [2,3]. In addition, they contain important biogenic metabolites such as polysaccharides, phenols, flavonoids, and carotenoids [4][5][6]. More importantly, accumulating evidence suggests that compounds extracted from seaweeds induce antitumor effects through multiple mechanisms of action, including the inhibition of cancer cell growth, metastasis, and the induction of apoptosis [7].

Physicochemical Analysis of J. rubens
The physicochemical evaluation of J. rubens will allow its standardization as raw material, and it will help quantify many elements in this seaweed, which will identify its nutritional value. The pie chart (Figure 1) reflects the variation in the organic content and inorganic ash present in J. rubens on the Lebanese coast. The proximal analysis of J. rubens powder shows that proteins (11.3%) and carbohydrates (14.5%) were relatively higher than lipids (4.5%).
In addition, the inorganic ash of this algae was found to be very abundant (66%), and it is mainly composed of equal amounts of water-soluble (23.26%) and acid-insoluble ash (24%) ( Table 1) In addition, the inorganic ash of this algae was found to be very abundant (66%), and it is mainly composed of equal amounts of water-soluble (23.26%) and acid-insoluble ash (24%) ( Table 1). Values are reported as mean ± SD; n = 3 refers to three independent experiments. SD: standard deviation.
The elemental analysis, presented in (Table 2), showed the elevated levels of some essential macro-minerals in this seaweed: calcium (33mg/g) and magnesium (24 mg/g). Furthermore, the photosynthetic pigment, chlorophyll (1.04 mg/g), dominated the other pigments in J. rubens (Table 3).

The Antioxidant Effect of J. rubens Extracts
The antioxidant properties of the aqueous and organic extracts of J. rubens were investigated by DPPH free radical scavenging activity. The standard used was vitamin C. The results showed that 750 µg.mL −1 of the DM Soxhlet extract has the highest antioxidant activity (64.62 ± 6.68%) ( Figure 3A). At a concentration of 750 µg.mL −1 , the scavenging activity of the M crude extract and the AQ Soxhlet extract was 61.52 ± 5.44% and 60.44 ± 0.54%, respectively ( Figure 3B and Figure 3C). The extraction methods had no significant influence on the antioxidant activity.

The Antioxidant Effect of J. rubens Extracts
The antioxidant properties of the aqueous and organic extracts of J. rubens were investigated by DPPH free radical scavenging activity. The standard used was vitamin C. The results showed that 750 µg.mL −1 of the DM Soxhlet extract has the highest antioxidant activity (64.62 ± 6.68%) ( Figure 3A). At a concentration of 750 µg.mL −1 , the scavenging activity of the M crude extract and the AQ Soxhlet extract was 61.52 ± 5.44% and 60.44 ± 0.54%, respectively (Figures 3B,C). The extraction methods had no significant influence on the antioxidant activity.

Antibacterial Effect of J. rubens Extracts
The antibacterial activity of the DM, M, and AQ extracts was evaluated against some Gram-positive and Gram-negative bacteria using the agar disc diffusion method (Table  4). Penicillin/streptomycin was used as a positive control. The results showed that J. rubens extracts have no antibacterial activity against any of the bacteria species tested.

Antibacterial Effect of J. rubens Extracts
The antibacterial activity of the DM, M, and AQ extracts was evaluated against some Gram-positive and Gram-negative bacteria using the agar disc diffusion method (Table 4). Penicillin/streptomycin was used as a positive control. The results showed that J. rubens extracts have no antibacterial activity against any of the bacteria species tested.

Zone of Inhibition (mm)
A. Baumannii In the present study, an MTT assay was used to evaluate the impact of J. rubens DM extracts' (0-750 µM at 24 h and 48 h) treatment on the cell viability of two CRC cell lines, the HCT-116 and HT-29 colon cancer cells ( Figure 4). The results showed that DM extracts significantly decreased the cell viability (p < 0.0001) of both treated cell lines at all tested concentrations. Interestingly, the results indicate that DM Soxhlet exhibited a much higher cytotoxic effect compared to the DM crude extract. The decrease in HCT-116 cell viability treated with DM Soxhlet compared with the non-treated control cells at concentrations of 100-250-500-750 µg.mL −1 reached 68%, 65%, 56%, and 32%, respectively, while it was 88%, 87%, 82%, and 78% upon the treatment with DM crude, at the same time point (24 h). Besides, the DM Soxhlet and crude extracts exhibit a time-dependent inhibitory effect on HCT-116 cells. Upon treatment of the cells with 750 µg.mL −1 of DM Soxhlet, the percentage of cell proliferation was 32.11 ± 5.71% at 24 h, to reach only 4.93 ± 1.85% at 48 h. Similarly, DM crude (750 µg.mL −1 ) decreased the cell viability from 78.31 ± 4.45% to 31.99 ± 6.88% at 24 and 48 h, respectively. Further analysis of the results revealed that the DM Soxhlet treatment exhibited a dose-dependent effect on the HCT-116 cells at both time points, 24 and 48 h of treatment (0.05 < p < 0.0001), while DM crude induces a dose-dependent inhibition only after 48 h of treatment (p < 0.0001). Overall, the results indicate that DM Soxhlet is more potent than the crude extract. In addition, the HCT-116 cells (IC 50 equal 499.94 µg.mL −1 ) were more sensible than the HT-29 (531.44 µg.mL −1 ) to treatment ( Table 5). The results were validated with the trypan blue assay (Supplementary Figure S1).

Effect of M extracts on the viability of CRC cell lines
Since M extract is more polar than DM extract and different molecules could be extracted with solvents of a different polarity [23], using the same experimental conditions, our next objective was to investigate the antiproliferative property of the M Soxhlet and crude extracts of J. rubens on the two treated colon cancer cells. The results showed that this extract, both Soxhlet and crude, significantly decreased the cell viability (p < 0.0001 compared to control) at all tested concentrations ( Figure 5). Moreover, M Soxhlet and crude revealed a time-dependent inhibition (0.01 < p < 0.0001). Upon treatment with 750 µg.mL −1 of the M Soxhlet extract, the inhibition of the cell viability was 57.39 ± 5.41% at 24 h and further reduced by 47% to 9.54 ± 4.09% at 48 h. Remarkably, M Soxhlet is more potent on HCT-116 cells than HT-29 with IC 50 equal to 389.73 µg.mL −1 and 666.82 µg.mL −1 , respectively. ous. SD: standard deviation. IC50 refers to the half-maximal inhibitory concentration.

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Effect of M extracts on the viability of CRC cell lines Since M extract is more polar than DM extract and different molecules could be extracted with solvents of a different polarity [23], using the same experimental conditions, our next objective was to investigate the antiproliferative property of the M Soxhlet and crude extracts of J. rubens on the two treated colon cancer cells. The results showed that this extract, both Soxhlet and crude, significantly decreased the cell viability (p < 0.0001 compared to control) at all tested concentrations ( Figure 5). Moreover, M Soxhlet and crude revealed a time-dependent inhibition (0.01 < p < 0.0001). Upon treatment with 750 µg.mL −1 of the M Soxhlet extract, the inhibition of the cell viability was 57.39 ± 5.41% at 24 hours and further reduced by 47% to 9.54 ± 4.09% at 48 hours. Remarkably, M Soxhlet is more potent on HCT-116 cells than HT-29 with IC50 equal to 389.73 µg.mL −1 and 666.82 µg.mL −1 , respectively. Results are presented as the mean ± SD (n ≥ 3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. crude extract. #### p < 0.0001 vs. control group. M: methanol.

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Effect of AQ Extracts on the Viability of CRC Cell Lines We also tested the cytotoxic effect of the aqueous extract. The AQ extracts induced a significant antiproliferative effect, with p < 0.0001 on both CRC cell lines ( Figure 6). However, the results did not reveal any major difference between the two tested cell lines, and the cytotoxic effect of the AQ extracts is neither dose-nor time-dependent. Overall, the cytotoxicity of the AQ extracts is not as pronounced as the one observed with DM and M. ever, the results did not reveal any major difference between the two tested cell lines, and the cytotoxic effect of the AQ extracts is neither dose-nor time-dependent. Overall, the cytotoxicity of the AQ extracts is not as pronounced as the one observed with DM and M.
Subsequently, considering all the results up to this point, we concluded that organic extracts are generally more potent than aqueous extracts on both cell lines, and the Soxhlet extraction method increases the cytotoxic potential of the extracts. Also, the HCT cell line is more sensitive than HT-29 to the organic extract's treatment.  Subsequently, considering all the results up to this point, we concluded that organic extracts are generally more potent than aqueous extracts on both cell lines, and the Soxhlet extraction method increases the cytotoxic potential of the extracts. Also, the HCT cell line is more sensitive than HT-29 to the organic extract's treatment. Since DM and M Soxhlet extracts presented a significant decrease in cell proliferation by MTT assay, we investigated the anti-migratory potential of these extracts on HCT-116 and HT-29 cells by a classic wound-healing assay.
The DM Soxhlet treatment showed remarkable and significant inhibition of cell migration in a dose-dependent manner (0.01 < p < 0.0001). At 24 h, the HCT-116 cells were treated with 100 µg.mL −1 of DM Soxhlet, which migrated at a rate of 22.4% compared to 37.5% for the control; this ratio decreased to 6.11% with 750 µg.mL −1 of the DM Soxhlet treatment (Figure 7). Similarly, a concentration of 750 µg.mL −1 significantly decreased the gap closure rate of the HT-29 cells to 9.59% at 24 h, a percentage four times lower than the control. Thus, DM Soxhlet exerts a potent anti-migratory effect on both cell lines after 24 h.
were treated with 100 µg.mL −1 of DM Soxhlet, which migrated at a rate of 22.4% compared to 37.5% for the control; this ratio decreased to 6.11% with 750 µg.mL −1 of the DM Soxhlet treatment (Figure 7). Similarly, a concentration of 750 µg.mL −1 significantly decreased the gap closure rate of the HT-29 cells to 9.59% at 24 hours, a percentage four times lower than the control. Thus, DM Soxhlet exerts a potent anti-migratory effect on both cell lines after 24 hours.   Regarding the M Soxhlet treatment, the quantitative analysis of the wound area showed that the cells treated with different concentrations (100-750 µg.mL −1 ) exhibited a significant dose-dependent anti-migration effect at 24 h (0.01 < p < 0.0001). The percentage of wound-healing decreased to 18.11% and 7.89% at 100 µg.mL −1 and 750 µg.mL −1, , respectively, compared to 36.28% for the control in the HCT-116 cells (Figure 8). We noted that M Soxhlet showed a significant anti-migration effect at an early point (6 h) at 750 µg.mL −1 (9.27% for HCT-116 and 8.85% for HT-29) compared to 25% for the control.

Discussion
In this study, we first identified the physiochemical characteristics and the antioxidant, cytotoxic, and anti-migratory effects of Lebanese red seaweed, J. rubens. We started by measuring the ash content. Our study revealed that the total ash content of Lebanese J. rubens is 66 %. This amount is considered high compared to other red seaweeds [24] and indicates abundant minerals and nutraceutical products [25,26]. Then, we analyzed the organic content, and we showed that Lebanese J. rubens contain 11.3% proteins and 14.48% carbohydrates. The protein and carbohydrate active biomolecules could have a role in the anticancer and antioxidant effects recorded in this study. Indeed, previous studies have shown that algal proteins possess significant biological potential as anti-in-

Discussion
In this study, we first identified the physiochemical characteristics and the antioxidant, cytotoxic, and anti-migratory effects of Lebanese red seaweed, J. rubens. We started by measuring the ash content. Our study revealed that the total ash content of Lebanese J. rubens is 66 %. This amount is considered high compared to other red seaweeds [24] and indicates abundant minerals and nutraceutical products [25,26]. Then, we analyzed the organic content, and we showed that Lebanese J. rubens contain 11.3% proteins and 14.48% carbohydrates. The protein and carbohydrate active biomolecules could have a role in the anticancer and antioxidant effects recorded in this study. Indeed, previous studies have shown that algal proteins possess significant biological potential as anti-inflammatory, antibacterial, and antioxidant compounds [27,28]. Moreover, the proteins extracted from red algae also have a potential antitumor effect [29,30]. Phycocyanin, a phycobiliprotein present in red algae, possesses an effective anticancer effect against MCF-7 breast cancer cells [31], HT29 colorectal carcinoma cells [32], A549 lung adenocarcinoma cells [33] and others. Moreover, many reports have indicated that red seaweed polysaccharides exert an anticancer effect by activating the immune system and increasing the infiltration of the immune cells into the tumor [34,35]. For example, Porphyra haitanensis polysaccharides exerted inhibitory effects on growth in the HT-29, LoVo, and SW-480 colon cancer cell lines [36].
Moreover, the present study revealed that Lebanese J. rubens is rich in calcium (24 ± 0.5mg/g) and magnesium (33 ± 0.5 mg/g). These elements are essential for numerous human metabolic reactions, such as enzymatic regulation and the metabolism of lipids, carbohydrates, and proteins [37,38]. Also, the high amount of calcium and magnesium in marine seaweeds plays a role in suppressing the growth and differentiation of colon cancer carcinoma [39]. Calcium is a second messenger in many pathways related to cell proliferation and death [40]. Previous studies showed that many natural components induce anticancer properties through Ca-mediated pathways [41][42][43]. Also, magnesium plays a vital role in many physiological activities and promotes an anti-tumor activity by inducing apoptosis, autophagy, and cell cycle arrest [44]. Similarly, clinical studies revealed that magnesium protects against colorectal cancer by inhibiting the expression of c-myc and the ornithine decarboxylase activity in the intestine's mucosal epithelium [45].
We have also shown that J. rubens collected from the Lebanese coast has a four times higher chlorophyll content than the same algae collected from the Egyptian coast (0.25 mg/g) [46]. In general, the chlorophyll content in seaweeds depends on the depth of the algae. The deeper the location, the higher the chlorophyll amount [47]. In our study, J. rubens was collected at 2-3 m deep, and this could explain the high amount of chlorophyll. The role of seaweed pigments is not only to characterize each group of algae but also it has an important role as an antioxidant, anti-inflammatory, and anticancer [47][48][49][50]. Previous studies showed that chlorophyll and carotenoids inhibit cancer proliferation and can induce apoptosis in different cell lines, including colon cancer cells (HT-29, Caco-2), breast cancer cells (MCF-7), T-cell leukemia, and others [51][52][53].
Phytochemicals, such as flavonoids and phenols, are produced by seaweeds [54]. Phenolic compounds are found in large amounts in red seaweeds compared to other groups of macroalgae [55,56]. Phenols are known for their importance as hormones, antioxidants, cofactors, and anti-tumor compounds [3,57]. Our present data show that all organic extracts are rich in flavonoids and phenols, while aqueous extracts contain low levels. The difference in the flavonoid and phenols content between these extracts is due to the polarity of the solvent, the method of extraction, the molecular weight of the phenolic components, the temperature, and the extraction [58]. Some reports demonstrated that flavonoids and phenols work as antioxidants; they can scavenge reactive oxygen species and inhibit lipid peroxidation [59,60]. Furthermore, phenols have been marked as a substantial metabolite for anti-tumor activity [61]. Some studies showed that polyphenols promote anticancer activity via different pathways, including apoptosis, cell cycle arrest, and metastasis [62]. In addition, phenolic compounds possess an anti-migratory effect against H460 lung cancer cells [63]. Our data revealed that phenols content is the highest in M Soxhlet extract (24.26 ± 0.25 mg/g), which could explain its antioxidant, antiproliferative, and antimigratory potentials against colorectal cancer cells.
In addition, the highest antioxidant activity detected, among all extracts, was in the DM Soxhlet one with 64.6% (±6.7). In fact, there is a correlation between its antioxidant and anticancer effects [61,64,65]. Antioxidants inhibit carcinogenic agents, modulate cancer cell signaling, and induce apoptosis and cell cycle arrest [66]. Many studies have shown that red seaweeds exhibit high antioxidant activity, leading to an antiproliferative effect and apoptosis against different cancer cells [67,68]. We believe that the high antioxidant effect of DM Soxhlet plays a role in its effective cytotoxic and anti-migratory properties.
Previous studies showed that seaweeds contain bioactive components with antimicrobial effects. Lebanese J. rubens has no antibacterial effect against bacteria. However, J. rubens, collected from Tunisia and Egypt, showed high potential in inhibiting the growth of different Gram+ and Gram-bacteria [11,69]. This variation may be due to environmental factors since seaweeds are known to change their active components in response to changes related to season, temperature, pollution, and others [1].
Novel CRC treatments are investigated to reduce the side effects and prolong patient survival. The new treatment methodology must include cytotoxicity to limit cancer progression and development, the anti-migratory effect to limit secondary metastasis, and the antioxidant potential to limit cancer-related oxidative stress. The cytotoxicity and antimetastatic effect of Lebanese J. rubens extracts are associated with the presence of anticancer metabolites, including the phenols, flavonoids, and antioxidants detected in this study. Interestingly, DM and M Soxhlet inhibit the cell proliferation of CRC cells more than the crude extracts. Thus, extraction at high temperatures by the Soxhlet apparatus accumulates more bioactive molecules with anti-tumor activity [6]. This concept may be attributed to many reasons, such as the molecules' polarity, size, and lipophilicity [70,71]. In addition, organic extracts (DM and M) are more potent than aqueous extracts. Therefore, we believe that the cytotoxic molecules are more soluble in organic extracts. This result emphasizes the influence of the choice of solvent extraction on the cytotoxic activity of seaweed extracts against different cancer cell lines [23].
For the first time, we studied the effect of Lebanese J. rubens extracts on colon cancer cell migration. Interestingly, DM and M Soxhlet extracts inhibit the migration of cancer cells after 24 h of treatment. Considering all previous results, we believe that DM and M Soxhlet extracts can be used as potential adjuvant treatments in addition to conventional chemotherapy.

Macroalgal Biomass
J. rubens samples were collected from the North Lebanese coast of the Mediterranean region at a depth of 2-3 m. Fresh seaweeds were rinsed thoroughly and air-dried at room temperature, then ground to a fine powder. The herbarium voucher of J. rubens (AZM-1105) was preserved at the Doctoral School of Science and Technology, Lebanese University.

Proximal and Elemental Analysis
Lipids, moisture, total ash, acid-insoluble, and water-soluble ash were quantified according to the methods described by the Association of Official Analytical Chemists [72]. Ashed seaweeds were moistened with distilled water and then dissolved with nitric acid and deionized water for macro and micro elemental analysis. The solution was heated at 100 • C till dry; then, the residue was ashed at 500 • C for one h. Later, the ash was dissolved with HCl and filtered with ashless filter paper. Atomic absorption spectrometry was used, and for each element measured, a standard calibration curve was prepared.

Organic Content Analysis
For total carbohydrates analysis, algal samples were hydrolyzed with concentrated sulfuric acid at 37 • C for one hour, and the acid strength was diluted to 1 M, followed by two hours of boiling. The Dubois phenol-sulfuric acid method was used, and glucose was used as a standard [73]. The absorbances were read at 490 nm.
For lipid analysis, the dried seaweeds were extracted with chloroform/hexane (2:1) and stirred overnight. The mixture was centrifuged, and the collected supernatant containing lipids was evaporated by rotavap, whereas the residue was saved for protein extraction. The lipid yield was determined gravimetrically.
For proteins, ultra-pure water (40 • C) was added to the residue obtained from the previous lipid extraction. The solution was stirred for 24 h at 40 • C, filtered, and then hot ultra-pure water was added. Proteins were precipitated by zinc sulfate and barium hydroxide, followed by centrifugation at 5000× g for 10 min at 4 • C. The resulting protein pellet was lyophilized and quantified using the Bradford method, with bovine serum albumin (BSA) as a standard.

Pigment Analysis
Pigment extracts were isolated using 80% acetone extraction. The extracts were utilized for chlorophyll (Chl) estimation. Using Arnon's equations, the absorbance was read at 645 and 663 nm in an ultraviolet spectrophotometer [74]. Carotenoids were estimated by the method of Kirk and Allen (Kirk and Allen 1965) [75], where the same extract was measured at 480 nm in the spectrophotometer.

Seaweed Solvent Extraction
For crude and Soxhlet extractions, three different solvents were used. These include dichloromethane/methanol (DM, 1:1), methanol (M), and finally, water or aqueous (AQ) solvents. For crude extraction, powdered algae were macerated with each solvent for three days at room temperature in an orbital shaker. Powdered algae were also extracted with the same solvents at an elevated temperature using a Soxhlet extractor for six hours. All extracts were concentrated using a rotary evaporator, and the aqueous extracts were lyophilized.

Total Phenol Content
The extract's total phenolic content (TPC) was determined by the Folin-Ciocalteu method [76]. The extract (100 µL) aliquot was mixed with 750 µL of a ten-fold diluted Folin-Ciocalteu's phenol reagent. After 5 mins, a 7.5% sodium bicarbonate solution was added and then the reaction was allowed to stand for 90 min at room temperatures [77]. The absorbance was measured at 725 nm with a gallic acid standard curve for estimating the TPC concentration in the sample. The phenolic content was calculated as mg gallic acid equivalents GAE per gram of dry algal powder.

Total Flavonoid Content
The aluminum chloride colorimetric assay was implemented to determine the total flavonoid content of algal samples [78]. Each extract's aliquot (0.5 mL) was mixed with 1.5 mL of ethanol and 0.1 mL of 10% aluminum chloride (10%). Then, 0.1 mL of potassium acetate (1M) was added, followed by 2.8 mL of distilled water. The mixture was incubated for 30 mins at room temperature. The absorbance was measured at 415 nm, where quercetin was used as a standard. The concentration of total flavonoid content was expressed as mg quercetin equivalent (QE) per gram of dried algal material.

DPPH Free Radical Scavenging Assay
Seaweed extracts were aliquoted into concentrations (100-750 µg/mL). A methanolic DPPH solution was added to samples and incubated in the dark for 30 min. The absorbance was measured at 517 nm [77]. Vitamin C was used as a standard, and the percentage of free radical scavenging was calculated using the formula: Free radical scavenging (%) = ([control OD − sample OD]/control OD])/100

Antimicrobial Assay
Antibacterial tests were performed using the agar disc diffusion method. Sterile discs were impregnated with J. rubens extracts of a concentration of 1 mg/ml deposited on the surface of the agar medium (Mueller-Hinton Agar, pH 7.4 ± 0.2 at 25 • C) that was previously inoculated with various bacteria strains. The five bacterial strains were obtained from the Health and Environment Microbiology Laboratory of AZM Center, Lebanon: Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumonia, Bacillus cereus, and Acinetobacter baumannii. The results were expressed by measuring the diameters of the bacterial inhibition zone.

Cell Lines and Culture
Colon cancer cells (HT-29 and HCT-116) were purchased from the American Type Culture Collection (ATCC). Cells were cultured in DMEM in a humidified incubator at 37 • C, with 5% CO 2 and 95% air. The media was supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin (100 U·mL −1 ).

Cell Viability Assay
The cell viability assay is an MTT-based method that measures the ability of metabolically active cells to convert tetrazolium salt into formazan blue. HCT-116 and HT-29 cells were seeded overnight in a 96-well plate, to be later treated with different concentrations of J. rubens extracts (100-750 µg.mL −1 ). Cells were treated at two-time points (24 and 48 h), then incubated with MTT for two hours at 37 • C in the dark. The ELISA microplate reader measured absorbances at 570 nm. All determinations were carried out in triplicate.

Trypan Blue Test
Colon cancer cells (HCT-116and HT-29) were seeded at a density of 5 × 104 in a 24-well plate. Based on MTT data, only potent organic extracts were tested to confirm their cytotoxicity using a trypan blue assay: DM and M Soxhlet extracts. After 24 and 48 h, treated and non-treated cells were washed, trypsinized, and stained with trypan blue (0.4%). A hemocytometer, using a light microscope, counted the number of viable versus dead cells. All determinations were carried out in triplicate.

Wound-Healing Migration Assay
HCT-116 and HT-29 cells were seeded in a 24-well plate overnight. First, a scratch wound was applied with a sterile 200 µL tip at confluency. Cell debris was washed twice; then, cells were incubated with the potent organic extracts at different concentrations (0, 100, 250, 500, and 750 µg.mL −1 ). Images of the wounds were taken post 0, 6, and 24 h treatments. Images were captured using a digital camera coupled to a light microscope. The Image J analysis program analyzed the surface area.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27196617/s1. Figure S1: Cytotoxic effect of J. rubens DM and M crude extracts (100-250-500-750 µg.mL −1 ) on colon cancer cells using a trypan blue exclusion assay. Funding: This work was funded by the Lebanese University grant. Data Availability Statement: All data and materials support our published claims and comply with the field standards. The original data can be made available upon request.