Next Article in Journal
A Review of the Phytochemistry and Pharmacology of the Fruit of Siraitia grosvenorii (Swingle): A Traditional Chinese Medicinal Food
Next Article in Special Issue
The Use of Photodynamic Therapy in the Treatment of Brain Tumors—A Review of the Literature
Previous Article in Journal
Pomegranate Flower Extract—The Health-Promoting Properties Optimized by Application of the Box–Behnken Design
Previous Article in Special Issue
Multinuclear MRI in Drug Discovery
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 27
Issue 19
10.3390/molecules27196617
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Mariam Rifi
1,
Zeina Radwan
2,
Reem AlMonla
1,
Ziad Fajloun
1,3,
Jean Marc Sabatier
4,
Achraf Kouzayha
1,5,
Marwan El-Sabban
2,
Hiba Mawlawi
1,6,* and
Zeina Dassouki
1,3,*
1
Laboratory of Applied Biotechnology (LBA3B), AZM Center for Research in Biotechnology and its Applications, Doctoral School for Sciences and Technology, Lebanese University, Tripoli 1300, Lebanon
2
Department of Anatomy, Cell Biology, and Physiological Sciences, Faculty of Medicine, American University of Beirut, Beirut 1107 2020, Lebanon
3
Department of Biology, Faculty of Sciences 3, Lebanese University, Campus Michel Slayman Ras Maska, Tripoli 1352, Lebanon
4
CNRS UMR 7051, INP, Inst. Neurophysiopathol., Aix-Marseille Université, 13385 Marseille, France
5
Department of Biochemistry, Faculty of Sciences 3, Lebanese University, Campus Michel Slayman Ras Maska, Tripoli 1352, Lebanon
6
Faculty of Public Health III, Lebanese University, Tripoli 1310, Lebanon
*
Authors to whom correspondence should be addressed.
Molecules 2022, 27(19), 6617; https://doi.org/10.3390/molecules27196617
Submission received: 4 September 2022 / Revised: 28 September 2022 / Accepted: 29 September 2022 / Published: 5 October 2022
(This article belongs to the Special Issue Anticancer Drug Discovery and Development II)
Browse Figures
Review Reports Versions Notes

Abstract

:
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.

1. 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].
The Lebanese coast is affluent in seaweed, with more than ninety-four macroalgae belonging to the different algal groups (green, brown, and red algae) [8]. The biodiversity of the Lebanese coast is related to its location at the warmest corner of the Mediterranean Sea and its proximity to the northern gate of the Suez Canal, which allows the interaction between the Red and the Mediterranean Sea [6,9]. Rhodophyta or red algae are the sources of more than 1658 natural products [10]; J. rubens is the most common red seaweed in the eastern part of the Mediterranean. Different studies have been performed around the Mediterranean coast to identify the bioactive components of J. rubens and their biological effects. A survey completed in Egypt showed that J. rubens of the Alexandrian coast has a molluscicidal impact [2]. Extracts of J. rubens collected from the Izmir coast in Turkey and from the Tunisian coast near Bizerte have antimicrobial effects [11,12]. More importantly, studies on J. rubens of the Mediterranean Sea in Alexandria, Egypt, and the Red Sea in Hurghada, Egypt have identified different bioactive components with cytotoxic effects [13,14,15]. However, J. rubens of the Lebanese coast has not been investigated yet.
According to the National Comprehensive Cancer Network’s (NCCN) latest review, CRC is the second leading cause of cancer mortality and the fourth most identified cancer in the United States of America (USA). In the MENA region (the Middle East and North Africa region), Lebanon has the highest cancer incidence [16]. The standard CRC treatments are surgery, chemotherapy, and radiation or a combination of more than one mode depending on the stage, location, metastasis, and the patient’s health condition [17]. Traditional chemotherapeutic agents aim to inhibit tumor progression and metastasis; however, they also affect the tumor cell environment and lead to serious side effects [18]. Targeted therapy has increased the survival rate in CRC patients; however, it may have limited efficacy in some patients and may develop drug resistance [19]. To reduce the complications of conventional cancer treatments, researchers have headed towards more natural compounds with lower costs and fewer side effects that can increase the potential for complete recovery [18,20]. Specifically, interest in seaweeds has increased as reliable alternative compounds that can assist cancer treatment [21]. However, the research on seaweed-derived bioactive agents and their antitumor potentials is still in its infancy [22]. Although J. rubens is highly abundant on the Lebanese coast, this alga has been poorly studied. In this study, we will identify the physicochemical properties of J. rubens extracted from the northern Lebanese coast. We will also research the antioxidative, bactericidal, cytotoxic, and anti-migratory potential of different J. rubens extracts against colon cancer.

2. Results

2.1. 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).
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).

2.2. The Phenol and Flavonoid Content of J. rubens Extracts

The concentration of the phenols and flavonoids in J. rubens varied from one extract to another. The highest phenolic content was recorded in methanol (M) Soxhlet, with (24.3 ± 0.25 mg/g). However, the flavonoid content was highly abundant in the dichloromethane/methanol (DM, 1:1) crude organic extract (42.29 ± 0.6 mg/g), followed by the M Soxhlet extract (28.8 ± 2.5 mg/g) (Figure 2).

2.3. 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.

2.4. 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.

2.5. Anti-Proliferative Activity

2.5.1. J. rubens Extracts Inhibit the Proliferation of HCT-116 and HT-29 Cells

  • Effect of DM extracts on the viability of CRC cell lines
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 (IC50 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 IC50 equal to 389.73 µg.mL−1 and 666.82 µg.mL−1, respectively.
  • 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.
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.

2.5.2. DM Soxhlet and M Soxhlet Extracts of J. rubens Reduce the Migration Ability of HT-29 and HCT-116 Cells

  • The Effect of DM Soxhlet extracts on the migration ability of HT-29 and HCT-116 cells
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.
  • Anti-migratory effect of M Soxhlet extracts against HCT-116 and HT-29
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.

3. 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 anti-migratory 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.

4. Materials and Methods

4.1. 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.

4.2. Physicochemical Analysis

4.2.1. 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.

4.2.2. 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.

4.2.3. 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.

4.3. 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.

4.4. 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.

4.5. 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.

4.6. 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

4.7. 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.

4.8. 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% CO2 and 95% air. The media was supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin (100 U·mL−1).

4.8.1. 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.

4.8.2. 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.

4.8.3. 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.

4.9. Reagents

We used, in this study, the following reagents: Sulfuric acid (Sigma-Aldrich, St. Louis, MO, USA), nitric acid (Sigma Aldrich), hydrochloric acid, glucose, zinc sulfate, barium hydroxide, potassium acetate (Laboratory chemicals-Lebanon), bovine serum albumin (Sigma-Aldrich CAS 9048-46-8), acetone (Supelco), dichloromethane (Sigma-Aldrich), methanol (Sigma-Aldrich), Folin–Ciocalteu phenol (Sigma-Aldrich CAS 47641), ethanol (Sigma -Aldrich), aluminum chloride (Sigma-Aldrich), 2,2-diphenyl-1-picrylhydrazyl (Sigma-Aldrich), quercetin (Sigma-Aldrich), Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich CAS D5796), fetal bovine serum (Sigma-Aldrich CAS F9665), phosphate-buffered saline (Sigma-Aldrich, CAS 806552), penicillin/streptomycin (Sigma Aldrich, P4333), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich CAS M56655), and trypsin (Sigma Aldrich CAS T3924).

4.10. Statistical Analysis

All statistical analyses (t-test, one-way ANOVA, and two-way ANOVA) were performed using GraphPad Prism 7 (version 7.0, USA). Probability values below 0.05 (* p < 0.05) were considered as significant, and values below 0.01 (** p < 0.01), 0.001 (*** p < 0.001), and 0.0001 (**** p < 0.0001) were considered as highly significant. The quantitative data are expressed as means ± SD from the indicated set of experiments.

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.

Author Contributions

M.R., Z.R. and R.A. performed the experiments and wrote the manuscript; Z.D., H.M. and M.E.-S. designed the study and wrote the manuscript; Z.F., A.K. and J.M.S. participated in the study design and approved the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

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.

Acknowledgments

The authors would like to express gratitude to the Doctoral School for Sciences and Technology (EDST) for providing many reagents and their complete support (Lebanese University, Azm center, Tripoli, Lebanon).

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds’ J. rubens extracts are available from the authors.

References

  1. Stengel, D.B.; Connan, S. Marine Algae: A Source of Biomass for Biotechnological Applications. Methods Mol. Biol. 2015, 1308, 1–37. [Google Scholar] [CrossRef] [PubMed]
  2. Rashad, S.; El-Chaghaby, G.A. Marine Algae in Egypt: Distribution, phytochemical composition and biological uses as bioactive resources (a review). Egypt. J. Aquat. Biol. Fish. 2020, 24, 147–160. [Google Scholar] [CrossRef]
  3. Cotas, J.; Leandro, A.; Pacheco, D.; Gonçalves, A.M.M.; Pereira, L. A Comprehensive Review of the Nutraceutical and Therapeutic Applications of Red Seaweeds (Rhodophyta). Life 2020, 10, 19. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Ganesan, A.R.; Tiwari, U.; Rajauria, G. Seaweed nutraceuticals and their therapeutic role in disease prevention. Food Sci. Hum. Wellness 2019, 8, 252–263. [Google Scholar] [CrossRef]
  5. El-Chaghaby, G.A.; Rashad, S.; Abdel-Kader, S.F.; Rawash, E.-S.A.; Moneem, M.A. Assessment of phytochemical components, proximate composition and antioxidant properties of Scenedesmus obliquus, Chlorella vulgaris and Spirulina platensis algae extracts. Egypt. J. Aquat. Biol. Fish. 2019, 23, 521–526. [Google Scholar] [CrossRef] [Green Version]
  6. Al Monla, R.; Dassouki, Z.; Kouzayha, A.; Salma, Y.; Gali-Muhtasib, H.; Mawlawi, H. The Cytotoxic and Apoptotic Effects of the Brown Algae Colpomenia sinuosa are Mediated by the Generation of Reactive Oxygen Species. Molecules 2020, 25, 1993. [Google Scholar] [CrossRef]
  7. Fu, Y.; Xie, D.; Zhu, Y.; Zhang, X.; Yue, H.; Zhu, K.; Pi, Z.; Dai, Y. Anti-colorectal cancer effects of seaweed-derived bioactive compounds. Front. Med. 2022, 9, 988507. [Google Scholar] [CrossRef]
  8. Kanaan, H.; Belous, O.; Chokr, A. Diversity Investigation of the Seaweeds Growing on the Lebanese Coast. J. Mar. Sci. Res. Dev. 2015, 5, 156. [Google Scholar] [CrossRef] [Green Version]
  9. Harmelin, J.G.; Bitar, G.; Zibrowius, H. High xenodiversity versus low native diversity in the south-eastern Mediterranean: Bryozoans from the coastal zone of Lebanon. Mediterr. Mar. Sci. 2016, 17, 417–439. [Google Scholar] [CrossRef] [Green Version]
  10. Leal, M.C.; Munro, M.H.; Blunt, J.W.; Puga, J.; Jesus, B.; Calado, R.; Rosa, R.; Madeira, C. Biogeography and biodiscovery hotspots of macroalgal marine natural products. Nat. Prod. Rep. 2013, 30, 1380–1390. [Google Scholar] [CrossRef]
  11. Ali, A.I.-B.; El Bour, M.; Ktari, L.; Bolhuis, H.; Ahmed, M.; Boudabbous, A.; Stal, L.J. Jania rubens-associated bacteria: Molecular identification and antimicrobial activity. J. Appl. Phycol. 2012, 24, 525–534. [Google Scholar] [CrossRef]
  12. Karabay-Yavasoglu, N.U.; Sukatar, A.; Ozdemir, G.; Horzum, Z. Antimicrobial activity of volatile components and various extracts of the red alga Jania rubens. Phytother. Res. PTR 2007, 21, 153–156. [Google Scholar] [CrossRef] [PubMed]
  13. Ktari, L.; Blond, A.; Guyot, M. 16Beta-hydroxy-5alpha-cholestane-3,6-dione, a novel cytotoxic oxysterol from the red alga Jania rubens. Bioorganic Med. Chem. Lett. 2000, 10, 2563–2565. [Google Scholar] [CrossRef]
  14. Awad, N.E. Bioactive brominated diterpenes from the marine red alga Jania rubens (L.) Lamx. Phytother. Res. PTR 2004, 18, 275–279. [Google Scholar] [CrossRef] [PubMed]
  15. Gheda, S.; El-Sheekh, M.; Abou-Zeid, A. In vitro anticancer activity of polysaccharide extracted from red alga Jania rubens against breast and colon cancer cell lines. Asian Pac. J. Trop. Med. 2018, 11, 583–589. [Google Scholar] [CrossRef]
  16. Khachfe, H.H.; Rahal, Z.; Sammouri, J.; Kheil, M.; Baydoun, H.; Chatila, D.; Dirawi, H.; Fouad, F.M. Cancer in Lebanon: A Review of Incidence Rates from 2008 to 2015 and Projections till 2025. South Asian J. Cancer 2020, 9, 147–152. [Google Scholar] [CrossRef] [PubMed]
  17. Alzahrani, S.M.; Al Doghaither, H.A.; Al-Ghafari, A.B. General insight into cancer: An overview of colorectal cancer (Review). Mol. Clin. Oncol. 2021, 15, 271. [Google Scholar] [CrossRef] [PubMed]
  18. Rejhová, A.; Opattová, A.; Čumová, A.; Slíva, D.; Vodička, P. Natural compounds and combination therapy in colorectal cancer treatment. Eur. J. Med. Chem. 2018, 144, 582–594. [Google Scholar] [CrossRef] [PubMed]
  19. Köhne, C.H. Successes and limitations of targeted cancer therapy in colon cancer. Prog. Tumor Res. 2014, 41, 36–50. [Google Scholar] [CrossRef] [PubMed]
  20. Aiello, P.; Sharghi, M.; Mansourkhani, S.M.; Ardekan, A.P.; Jouybari, L.; Daraei, N.; Peiro, K.; Mohamadian, S.; Rezaei, M.; Heidari, M.; et al. Medicinal Plants in the Prevention and Treatment of Colon Cancer. Oxid. Med. Cell. Longev. 2019, 2019, 2075614. [Google Scholar] [CrossRef] [PubMed]
  21. Mehra, R.; Bhushan, S.; Bast, F.; Singh, S. Marine macroalga Caulerpa: Role of its metabolites in modulating cancer signaling. Mol. Biol. Rep. 2019, 46, 3545–3555. [Google Scholar] [CrossRef]
  22. Lomartire, S.; Gonçalves, A.M.M. An Overview of Potential Seaweed-Derived Bioactive Compounds for Pharmaceutical Applications. Mar. Drugs 2022, 20, 141. [Google Scholar] [CrossRef]
  23. Tannourya, M.Y.; Eliaa, J.M.; Saabc, A.M.; Makhloufb, H.Y.; Abboudd, J.S.; DaouChaboa, R.J.; Diab-Assafb, M. Evaluation of Cytotoxic Activity of Sargassum vulgare from the Lebanese Coast against Jurkat Cancer Cell Line. J. Appl. Pharm. Sci. 2016, 6, 108–112. [Google Scholar] [CrossRef] [Green Version]
  24. Rawiwan, P.; Peng, Y.; Paramayuda, I.G.P.B.; Quek, S.Y. Red seaweed: A promising alternative protein source for global food sustainability. Trends Food Sci. Technol. 2022, 123, 37–56. [Google Scholar] [CrossRef]
  25. Vilcanqui, Y.; Mamani-Apaza, L.O.; Flores, M.; Ortiz-Viedma, J.; Romero, N.; Mariotti-Celis, M.S.; Huamán-Castilla, N.L. Chemical Characterization of Brown and Red Seaweed from Southern Peru, a Sustainable Source of Bioactive and Nutraceutical Compounds. Agronomy 2021, 11, 1669. [Google Scholar] [CrossRef]
  26. Khoza, M.; Kayitesi, E.; Dlamini, B.C. Physicochemical Characteristics, Microstructure and Health Promoting Properties of Green Banana Flour. Foods 2021, 10, 2894. [Google Scholar] [CrossRef] [PubMed]
  27. Carpena, M.; Caleja, C.; Pereira, E.; Pereira, C.; Ćirić, A.; Soković, M.; Soria-Lopez, A.; Fraga-Corral, M.; Simal-Gandara, J.; Ferreira, I.C.F.R.; et al. Red Seaweeds as a Source of Nutrients and Bioactive Compounds: Optimization of the Extraction. Chemosensors 2021, 9, 132. [Google Scholar] [CrossRef]
  28. Trigueros, E.; Sanz, M.T.; Alonso-Riaño, P.; Beltrán, S.; Ramos, C.; Melgosa, R. Recovery of the protein fraction with high antioxidant activity from red seaweed industrial solid residue after agar extraction by subcritical water treatment. J. Appl. Phycol. 2021, 33, 1181–1194. [Google Scholar] [CrossRef]
  29. Sugrani, A.; Natsir, H.; Djide, M.N.; Ahmad, A. Biofunctional protein fraction from red algae (Rhodophyta) Eucheuma spinosum as an antibacterial and anticancer drug agent. Int. Res. J. Pharm. 2019, 10, 64–69. [Google Scholar] [CrossRef]
  30. Ismail, M.M.; Alotaibi, B.S.; El-Sheekh, M.M. Therapeutic Uses of Red Macroalgae. Molecules 2020, 25, 4411. [Google Scholar] [CrossRef]
  31. Li, B.; Chu, X.; Gao, M.; Li, W. Apoptotic mechanism of MCF-7 breast cells in vivo and in vitro induced by photodynamic therapy with C-phycocyanin. Acta Biochim. Biophys. Sin. 2010, 42, 80–89. [Google Scholar] [CrossRef] [Green Version]
  32. Wang, H.; Liu, Y.; Gao, X.; Carter, C.L.; Liu, Z.R. The recombinant beta subunit of C-phycocyanin inhibits cell proliferation and induces apoptosis. Cancer Lett. 2007, 247, 150–158. [Google Scholar] [CrossRef]
  33. Jiang, L.; Wang, Y.; Yin, Q.; Liu, G.; Liu, H.; Huang, Y.; Li, B. Phycocyanin: A Potential Drug for Cancer Treatment. J. Cancer 2017, 8, 3416–3429. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Qiu, S.M.; Aweya, J.J.; Liu, X.; Liu, Y.; Tang, S.; Zhang, W.; Cheong, K.L. Bioactive polysaccharides from red seaweed as potent food supplements: A systematic review of their extraction, purification, and biological activities. Carbohydr. Polym. 2022, 275, 118696. [Google Scholar] [CrossRef]
  35. Luo, M.; Shao, B.; Nie, W.; Wei, X.W.; Li, Y.L.; Wang, B.L.; He, Z.Y.; Liang, X.; Ye, T.H.; Wei, Y.Q. Antitumor and Adjuvant Activity of λ-carrageenan by Stimulating Immune Response in Cancer Immunotherapy. Sci. Rep. 2015, 5, 11062. [Google Scholar] [CrossRef] [PubMed]
  36. Yao, W.Z.; Veeraperumal, S.; Qiu, H.M.; Chen, X.Q.; Cheong, K.L. Anti-cancer effects of Porphyra haitanensis polysaccharides on human colon cancer cells via cell cycle arrest and apoptosis without causing adverse effects in vitro. 3 Biotech 2020, 10, 386. [Google Scholar] [CrossRef] [PubMed]
  37. Rossi, A.; Pizzo, P.; Filadi, R. Calcium, mitochondria and cell metabolism: A functional triangle in bioenergetics. Biochim. Biophys. Acta Mol. Cell Res. 2019, 1866, 1068–1078. [Google Scholar] [CrossRef]
  38. Barbagallo, M.; Veronese, N.; Dominguez, L.J. Magnesium in Aging, Health and Diseases. Nutrients 2021, 13, 463. [Google Scholar] [CrossRef]
  39. Ahmed, H.H.; Hegazi, M.M.; Abd-Alla, H.I.; Eskander, E.F.; Ellithey, M.S. Antitumour and antioxidant activity of some Red Sea seaweeds in Ehrlich ascites carcinoma in vivo. Z. Nat. C J. Biosci. 2011, 66, 367–376. [Google Scholar] [CrossRef]
  40. Capiod, T.; Shuba, Y.; Skryma, R.; Prevarskaya, N. Calcium signalling and cancer cell growth. Sub-Cell. Biochem. 2007, 45, 405–427. [Google Scholar] [CrossRef]
  41. Danese, A.; Leo, S.; Rimessi, A.; Wieckowski, M.R.; Fiorica, F.; Giorgi, C.; Pinton, P. Cell death as a result of calcium signaling modulation: A cancer-centric prospective. Biochim. Biophys. Acta. Mol. Cell Res. 2021, 1868, 119061. [Google Scholar] [CrossRef]
  42. Varghese, E.; Samuel, S.M.; Sadiq, Z.; Kubatka, P.; Liskova, A.; Benacka, J.; Pazinka, P.; Kruzliak, P.; Büsselberg, D. Anti-Cancer Agents in Proliferation and Cell Death: The Calcium Connection. Int. J. Mol. Sci. 2019, 20, 3017. [Google Scholar] [CrossRef] [Green Version]
  43. Bae, H.; Lee, W.; Song, J.; Hong, T.; Kim, M.H.; Ham, J.; Song, G.; Lim, W. Polydatin Counteracts 5-Fluorouracil Resistance by Enhancing Apoptosis via Calcium Influx in Colon Cancer. Antioxidants 2021, 10, 1477. [Google Scholar] [CrossRef] [PubMed]
  44. Li, T.; Yu, Y.; Shi, H.; Cao, Y.; Liu, X.; Hao, Z.; Ren, Y.; Qin, G.; Huang, Y.; Wang, B. Magnesium in Combinatorial With Valproic Acid Suppressed the Proliferation and Migration of Human Bladder Cancer Cells. Front. Oncol. 2020, 10, 589112. [Google Scholar] [CrossRef]
  45. Lin, J.; Cook, N.R.; Lee, I.M.; Manson, J.E.; Buring, J.E.; Zhang, S.M. Total magnesium intake and colorectal cancer incidence in women. Cancer Epidemiol. Biomark. Prev. Publ. Am. Assoc. Cancer Res. Cosponsored Am. Soc. Prev. Oncol. 2006, 15, 2006–2009. [Google Scholar] [CrossRef] [Green Version]
  46. El-Din, S.M.M.; El-Ahwany, A.M.D. Bioactivity and phytochemical constituents of marine red seaweeds (Jania rubens, Corallina mediterranea and Pterocladia capillacea). J. Taibah Univ. Sci. 2016, 10, 471–484. [Google Scholar] [CrossRef] [Green Version]
  47. Ganesan, A.R.; Kannan, M.; Rajan, D.K.; Pillay, A.A.; Shanmugam, M.; Sathishkumar, P.; Johansen, J.; Tiwari, B.K. Phycoerythrin: A pink pigment from red sources (rhodophyta) for a greener biorefining approach to food applications. Crit. Rev. Food Sci. Nutr. 2022, 1–19. [Google Scholar] [CrossRef]
  48. Li, W.; Su, H.N.; Pu, Y.; Chen, J.; Liu, L.N.; Liu, Q.; Qin, S. Phycobiliproteins: Molecular structure, production, applications, and prospects. Biotechnol. Adv. 2019, 37, 340–353. [Google Scholar] [CrossRef]
  49. Pagels, F.; Guedes, A.C.; Amaro, H.M.; Kijjoa, A.; Vasconcelos, V. Phycobiliproteins from cyanobacteria: Chemistry and biotechnological applications. Biotechnol. Adv. 2019, 37, 422–443. [Google Scholar] [CrossRef]
  50. Freitas, M.V.; Pacheco, D.; Cotas, J.; Mouga, T.; Afonso, C.; Pereira, L. Red Seaweed Pigments from a Biotechnological Perspective. Phycology 2022, 2, 1–29. [Google Scholar] [CrossRef]
  51. Vijay, K.; Sowmya, P.R.; Arathi, B.P.; Shilpa, S.; Shwetha, H.J.; Raju, M.; Baskaran, V.; Lakshminarayana, R. Low-dose doxorubicin with carotenoids selectively alters redox status and upregulates oxidative stress-mediated apoptosis in breast cancer cells. Food Chem. Toxicol. Int. J. Publ. Br. Ind. Biol. Res. Assoc. 2018, 118, 675–690. [Google Scholar] [CrossRef] [PubMed]
  52. Castro-Puyana, M.; Pérez-Sánchez, A.; Valdés, A.; Ibrahim, O.H.M.; Suarez-Álvarez, S.; Ferragut, J.A.; Micol, V.; Cifuentes, A.; Ibáñez, E.; García-Cañas, V. Pressurized liquid extraction of Neochloris oleoabundans for the recovery of bioactive carotenoids with anti-proliferative activity against human colon cancer cells. Food Res. Int. 2017, 99, 1048–1055. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Pemmaraju, D.; Appidi, T.; Minhas, G.; Singh, S.P.; Khan, N.; Pal, M.; Srivastava, R.; Rengan, A.K. Chlorophyll rich biomolecular fraction of A. cadamba loaded into polymeric nanosystem coupled with Photothermal Therapy: A synergistic approach for cancer theranostics. Int. J. Biol. Macromol. 2018, 110, 383–391. [Google Scholar] [CrossRef]
  54. Chakraborty, K.; Joseph, D.; Praveen, N.K. Antioxidant activities and phenolic contents of three red seaweeds (Division: Rhodophyta) harvested from the Gulf of Mannar of Peninsular India. J. Food Sci. Technol. 2015, 52, 1924–1935. [Google Scholar] [CrossRef] [Green Version]
  55. Khairy, H.M.; El-Sheikh, M.A. Antioxidant activity and mineral composition of three Mediterranean common seaweeds from Abu-Qir Bay, Egypt. Saudi J. Biol. Sci. 2015, 22, 623–630. [Google Scholar] [CrossRef] [Green Version]
  56. Cotas, J.; Leandro, A.; Monteiro, P.; Pacheco, D.; Figueirinha, A.; Gonçalves, A.M.M.; da Silva, G.J.; Pereira, L. Seaweed Phenolics: From Extraction to Applications. Mar. Drugs 2020, 18, 384. [Google Scholar] [CrossRef]
  57. Liu, M.; Hansen, P.E.; Lin, X. Bromophenols in marine algae and their bioactivities. Mar. Drugs 2011, 9, 1273–1292. [Google Scholar] [CrossRef] [Green Version]
  58. Skarpalezos, D.; Detsi, A. Deep Eutectic Solvents as Extraction Media for Valuable Flavonoids from Natural Sources. Appl. Sci. 2019, 9, 4169. [Google Scholar] [CrossRef] [Green Version]
  59. Kosanić, M.; Ranković, B.; Stanojković, T. Biological activities of two macroalgae from Adriatic coast of Montenegro. Saudi J. Biol. Sci. 2015, 22, 390–397. [Google Scholar] [CrossRef] [Green Version]
  60. Ismail, M.M.; Gheda, S.F.; Pereira, L. Variation in bioactive compounds in some seaweeds from Abo Qir bay, Alexandria, Egypt. Rend. Lincei 2016, 27, 269–279. [Google Scholar] [CrossRef]
  61. Grigalius, I.; Petrikaite, V. Relationship between Antioxidant and Anticancer Activity of Trihydroxyflavones. Molecules 2017, 22, 2169. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Alghazeer, R.; Enaeli, M.; Howell, N.K. Anticancer and Antioxidant Activities of Some Algae from Western Libyan Coast. Nat. Sci. 2016, 10, 85881. [Google Scholar] [CrossRef] [Green Version]
  63. Klongkumnuankarn, P.; Busaranon, K.; Chanvorachote, P.; Sritularak, B.; Jongbunprasert, V.; Likhitwitayawuid, K. Cytotoxic and Antimigratory Activities of Phenolic Compounds from Dendrobium brymerianum. Evid. Based Complement. Altern. Med. eCAM 2015, 2015, 350410. [Google Scholar] [CrossRef] [Green Version]
  64. Singh, K.; Bhori, M.; Kasu, Y.A.; Bhat, G.; Marar, T. Antioxidants as precision weapons in war against cancer chemotherapy induced toxicity—Exploring the armoury of obscurity. Saudi Pharm. J. SPJ Off. Publ. Saudi Pharm. Soc. 2018, 26, 177–190. [Google Scholar] [CrossRef]
  65. Haq, S.H.; Al-Ruwaished, G.; Al-Mutlaq, M.A.; Naji, S.A.; Al-Mogren, M.; Al-Rashed, S.; Ain, Q.T.; Al-Amro, A.A.; Al-Mussallam, A. Antioxidant, Anticancer Activity and Phytochemical Analysis of Green Algae, Chaetomorpha Collected from the Arabian Gulf. Sci. Rep. 2019, 9, 18906. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  66. Valadez-Vega, C.; Delgado-Olivares, L.; González, J.A.M.; García, E.A.; Ibarra, J.R.V.; Moreno, E.R.; Gutiérrez, M.S.; Martínez, M.T.S.; Clara, Z.P.; Ramos, Z.C. The role of natural antioxidants in cancer disease. The Role of Natural Antioxidants in Cancer Disease. In Oxidative Stress and Chronic Degenerative Diseases—A Role for Antioxidants; IntechOpen: London, UK, 2013. [Google Scholar] [CrossRef] [Green Version]
  67. Neethu, P.V.; Suthindhiran, K.; Jayasri, M.A. Antioxidant and Antiproliferative Activity of Asparagopsis taxiformis. Pharmacogn. Res. 2017, 9, 238–246. [Google Scholar] [CrossRef] [Green Version]
  68. Tanna, B.; Choudhary, B.; Mishra, A.; Yadav, S.; Chauhan, O.P.; Elansary, H.O.; Shokralla, S.; El-Abedin, T.K.Z.; Mahmoud, E.A. Biochemical and Anti-proliferative activities of seven abundant tropical red seaweeds confirm nutraceutical potential of Grateloupia indica. Arab. J. Chem. 2022, 15, 103868. [Google Scholar] [CrossRef]
  69. Saber, H.; Alwaleed, E.A.; Ebnalwaled, K.A.; Sayed, A.; Salem, W. Efficacy of silver nanoparticles mediated by Jania rubens and Sargassum dentifolium macroalgae; Characterization and biomedical applications. Egypt. J. Basic Appl. Sci. 2017, 4, 249–255. [Google Scholar] [CrossRef] [Green Version]
  70. Yang, N.J.; Hinner, M.J. Getting across the cell membrane: An overview for small molecules, peptides, and proteins. Methods Mol. Biol. 2015, 1266, 29–53. [Google Scholar] [CrossRef]
  71. Bowe, C.L.; Mokhtarzadeh, L.; Venkatesan, P.; Babu, S.; Axelrod, H.R.; Sofia, M.J.; Kakarla, R.; Chan, T.Y.; Kim, J.S.; Lee, H.J.; et al. Design of compounds that increase the absorption of polar molecules. Proc. Natl. Acad. Sci. USA 1997, 94, 12218–12223. [Google Scholar] [CrossRef] [Green Version]
  72. Thiex, N.; Novotny, L.; Crawford, A. Determination of Ash in Animal Feed: AOAC Official Method 942.05 Revisited. J. AOAC Int. 2019, 95, 1392–1397. [Google Scholar] [CrossRef]
  73. Zavřel, T.; Očenášová, P.; Sinetova, M.A.; Červený, J. Determination of Storage (Starch/Glycogen) and Total Saccharides Content in Algae and Cyanobacteria by a Phenol-Sulfuric Acid Method. Bio Protoc. 2018, 8, e2966. [Google Scholar] [CrossRef]
  74. Arnon, D.I. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 1949, 24, 1–15. [Google Scholar] [CrossRef] [Green Version]
  75. Kirk, J.T.; Allen, R.L. Dependence of chloroplast pigment synthesis on protein synthesis: Effect of actidione. Biochem. Biophys. Res. Commun. 1965, 21, 523–530. [Google Scholar] [CrossRef]
  76. Dahmoune, F.; Nayak, B.; Moussi, K.; Remini, H.; Madani, K. Optimization of microwave-assisted extraction of polyphenols from Myrtus communis L. leaves. Food Chem. 2015, 166, 585–595. [Google Scholar] [CrossRef]
  77. Al Monla, R.M.; Dassouki, Z.T.; Gali-Muhtasib, H.; Mawlawi, H.R. Chemical analysis and biological potentials of extracts from Colpomenia sinuosa. Pharmacogn. Res. 2020, 12, 272–277. [Google Scholar] [CrossRef]
  78. Chandra, S.; Khan, S.; Avula, B.; Lata, H.; Yang, M.H.; Elsohly, M.A.; Khan, I.A. Assessment of total phenolic and flavonoid content, antioxidant properties, and yield of aeroponically and conventionally grown leafy vegetables and fruit crops: A comparative study. Evid. Based Complement. Altern. Med. eCAM 2014, 2014, 253875. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Molecules 27 06617 g001 550
Figure 1. Pie chart showing the organic (proteins, lipids, and carbohydrates) and ash (water-soluble and acid-insoluble) content of the Lebanese red seaweed, J. rubens.
Figure 1. Pie chart showing the organic (proteins, lipids, and carbohydrates) and ash (water-soluble and acid-insoluble) content of the Lebanese red seaweed, J. rubens.
Molecules 27 06617 g001
Molecules 27 06617 g002 550
Figure 2. Total phenolic and flavonoid content of J. rubens various extracts. (A) The extract’s total phenolic content was determined by the Folin–Ciocalteu method and calculated as mg gallic acid equivalents GAE per gram of dry algal. (B) The extract’s total flavonoid content was measured by an aluminum chloride colorimetric assay and expressed as mg quercetin equivalent (QE) per gram of dried algal material. Values are reported as the mean ± SD (n = 3). AQ: aqueous; DM; dichloromethane/methanol; M: methanol.
Figure 2. Total phenolic and flavonoid content of J. rubens various extracts. (A) The extract’s total phenolic content was determined by the Folin–Ciocalteu method and calculated as mg gallic acid equivalents GAE per gram of dry algal. (B) The extract’s total flavonoid content was measured by an aluminum chloride colorimetric assay and expressed as mg quercetin equivalent (QE) per gram of dried algal material. Values are reported as the mean ± SD (n = 3). AQ: aqueous; DM; dichloromethane/methanol; M: methanol.
Molecules 27 06617 g002
Molecules 27 06617 g003 550
Figure 3. The antioxidant activity of J. rubens extracts: Determination of DPPH scavenging activity of different concentrations of J. rubens extracts (100, 250, 500, and 750 µg.mL−1). DPPH percentage inhibition of (A) DM extracts: DM Soxhlet and DM crude; (B) M extracts: M Soxhlet and M crude; (C) AQ extracts: AQ Soxhlet and AQ crude. Vitamin C was used as the standard. The absorbance was measured at 517 nm. Values are reported as the mean ± SD (n = 3). DM: dichloromethane/methanol (1:1); M: methanol; AQ: aqueous.
Figure 3. The antioxidant activity of J. rubens extracts: Determination of DPPH scavenging activity of different concentrations of J. rubens extracts (100, 250, 500, and 750 µg.mL−1). DPPH percentage inhibition of (A) DM extracts: DM Soxhlet and DM crude; (B) M extracts: M Soxhlet and M crude; (C) AQ extracts: AQ Soxhlet and AQ crude. Vitamin C was used as the standard. The absorbance was measured at 517 nm. Values are reported as the mean ± SD (n = 3). DM: dichloromethane/methanol (1:1); M: methanol; AQ: aqueous.
Molecules 27 06617 g003
Molecules 27 06617 g004 550
Figure 4. Effect of DM extracts on the viability of CRC cell lines. HCT-116 and HT-29 cells were seeded in 96-well plates and treated subsequently with different concentrations of DM extracts (0–100–250–500–750 µg.mL−1). MTT tetrazolium reduction assay was used to measure HCT-116 and HT-29 cell viability after 24 h and 48 h treatment. (A,B) Effect of DM extract (Soxhlet and crude) on HCT-116 cells at 24 h and 48 h. (C,D) Effect of DM extract (Soxhlet and crude) on HT-29 at 24 h and 48 h. The percentage of cell viability was calculated considering the value of the control as 100%. Results are presented as the mean ± SD (n ≥ 3). ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. crude extract. #### p < 0.0001 vs. control group. DM: dichloromethane/methanol.
Figure 4. Effect of DM extracts on the viability of CRC cell lines. HCT-116 and HT-29 cells were seeded in 96-well plates and treated subsequently with different concentrations of DM extracts (0–100–250–500–750 µg.mL−1). MTT tetrazolium reduction assay was used to measure HCT-116 and HT-29 cell viability after 24 h and 48 h treatment. (A,B) Effect of DM extract (Soxhlet and crude) on HCT-116 cells at 24 h and 48 h. (C,D) Effect of DM extract (Soxhlet and crude) on HT-29 at 24 h and 48 h. The percentage of cell viability was calculated considering the value of the control as 100%. Results are presented as the mean ± SD (n ≥ 3). ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. crude extract. #### p < 0.0001 vs. control group. DM: dichloromethane/methanol.
Molecules 27 06617 g004
Molecules 27 06617 g005 550
Figure 5. Effect of M extracts on the viability of CRC cell lines. HCT-116 and HT-29 cells were seeded in 96-well plates and treated subsequently with different concentrations of M extracts (0-100-250-500-750 µg.mL−1). MTT tetrazolium reduction assay was used to measure HCT-116 and HT-29 cell viability after 24 h and 48 h treatment. (A,B) Effect of M extract (Soxhlet and crude) on HCT-116 cells at 24 h and 48 h. (C,D) Effect of M extract (Soxhlet and crude) on HT-29 at 24 h and 48 h. The percentage of cell viability was calculated considering the value of the control as 100%. 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.
Figure 5. Effect of M extracts on the viability of CRC cell lines. HCT-116 and HT-29 cells were seeded in 96-well plates and treated subsequently with different concentrations of M extracts (0-100-250-500-750 µg.mL−1). MTT tetrazolium reduction assay was used to measure HCT-116 and HT-29 cell viability after 24 h and 48 h treatment. (A,B) Effect of M extract (Soxhlet and crude) on HCT-116 cells at 24 h and 48 h. (C,D) Effect of M extract (Soxhlet and crude) on HT-29 at 24 h and 48 h. The percentage of cell viability was calculated considering the value of the control as 100%. 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.
Molecules 27 06617 g005
Molecules 27 06617 g006 550
Figure 6. Effect of AQ extracts on the viability of CRC cell lines. HCT-116 and HT-29 cells were seeded in 96-well plates and treated subsequently with different concentrations of AQ extracts (0-100-250-500-750 µg.mL−1). MTT tetrazolium reduction assay was used to measure HCT-116 and HT-29 cell viability after 24 h and 48 h treatment. (A,B) Effect of AQ extract (Soxhlet and crude) on HCT-116 cells at 24 h and 48 h. (C,D) Effect of AQ extract (Soxhlet and crude) on HT-29 at 24 h and 48 h. The percentage of cell viability was calculated considering the value of the control as 100%. 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. AQ: aqueous.
Figure 6. Effect of AQ extracts on the viability of CRC cell lines. HCT-116 and HT-29 cells were seeded in 96-well plates and treated subsequently with different concentrations of AQ extracts (0-100-250-500-750 µg.mL−1). MTT tetrazolium reduction assay was used to measure HCT-116 and HT-29 cell viability after 24 h and 48 h treatment. (A,B) Effect of AQ extract (Soxhlet and crude) on HCT-116 cells at 24 h and 48 h. (C,D) Effect of AQ extract (Soxhlet and crude) on HT-29 at 24 h and 48 h. The percentage of cell viability was calculated considering the value of the control as 100%. 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. AQ: aqueous.
Molecules 27 06617 g006
Molecules 27 06617 g007 550
Figure 7. Wound healing assay showing that DM Soxhlet (100–250–500–750 µg.mL−1) inhibits the migration of HCT-116 and HT-29 cells after treatment for 24 h. (A,B) DM Soxhlet treatment of HCT-116: (A) Percentage of wound-healing at 6 h and 24 h. (B) Images of scratched HCT-116 cells taken at 0 h, 6 h, and 24 h. (C,D) DM Soxhlet treatment of HT-29. (C) Percentage of wound-healing at 6 h and 24 h. (D) Images of scratched HT-29 cells taken at 0 h, 6 h, and 24 h. Representative images were captured by a Leica microscope. Data are reported as mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. control group. DM: dichloromethane/methanol.
Figure 7. Wound healing assay showing that DM Soxhlet (100–250–500–750 µg.mL−1) inhibits the migration of HCT-116 and HT-29 cells after treatment for 24 h. (A,B) DM Soxhlet treatment of HCT-116: (A) Percentage of wound-healing at 6 h and 24 h. (B) Images of scratched HCT-116 cells taken at 0 h, 6 h, and 24 h. (C,D) DM Soxhlet treatment of HT-29. (C) Percentage of wound-healing at 6 h and 24 h. (D) Images of scratched HT-29 cells taken at 0 h, 6 h, and 24 h. Representative images were captured by a Leica microscope. Data are reported as mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. control group. DM: dichloromethane/methanol.
Molecules 27 06617 g007
Molecules 27 06617 g008 550
Figure 8. Wound-healing assay showing that M Soxhlet (100–250–500–750 µg.mL−1) inhibits the migration of HCT-116 and HT-29 cells after treatment for 24 h. (A,B) M Soxhlet treatment of HCT-116 cells. (A) Percentage of wound-healing at 6 h and 24 h. (B) Images of scratched HCT-116 cells taken at 0 h, 6 h, and 24 h. (C,D) M Soxhlet treatment of HT-29 cells. (C) Percentage of wound-healing at 6 h and 24 h. (D) Images of scratched HT-29 cells taken at 0 h, 6 h, and 24 h. Representative images were captured by a Leica microscope. Data are reported as mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. control group. M: methanol.
Figure 8. Wound-healing assay showing that M Soxhlet (100–250–500–750 µg.mL−1) inhibits the migration of HCT-116 and HT-29 cells after treatment for 24 h. (A,B) M Soxhlet treatment of HCT-116 cells. (A) Percentage of wound-healing at 6 h and 24 h. (B) Images of scratched HCT-116 cells taken at 0 h, 6 h, and 24 h. (C,D) M Soxhlet treatment of HT-29 cells. (C) Percentage of wound-healing at 6 h and 24 h. (D) Images of scratched HT-29 cells taken at 0 h, 6 h, and 24 h. Representative images were captured by a Leica microscope. Data are reported as mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. control group. M: methanol.
Molecules 27 06617 g008
Table
Table 1. Proximal analysis of J. rubens.
Table 1. Proximal analysis of J. rubens.
Proximal AnalysisResults % (w/w)SD
Ash Content66.081.59
Humidity2.310.46
Water-Soluble Ash23.261.67
Acid-Insoluble Ash24.042.21
Total Carbohydrates14.486.63
Total Lipids4.52.13
Total Proteins11.31.74
Values are reported as mean ± SD; n = 3 refers to three independent experiments. SD: standard deviation.
Table
Table 2. Elemental composition of J. rubens by atomic absorption spectroscopy.
Table 2. Elemental composition of J. rubens by atomic absorption spectroscopy.
Trace MineralsConcentration (mg/g)
Fe0.4 ± 0.04
Cu0.03 ± 0.01
Zn0.036 ± 0.13
Ca24 ± 0.5
Mg33 ± 0.5
Values are reported as mean ± SD (n = 3). Fe: iron; Cu: copper; Zn: zinc; Ca: calcium; Mg: magnesium.
Table
Table 3. Quantitative analysis of the photosynthetic pigments present in J. rubens.
Table 3. Quantitative analysis of the photosynthetic pigments present in J. rubens.
Type of PigmentConcentration
Chlorophyll a (mg/g)0.82 ± 0.02
Total Chlorophyll (mg/g)1.04 ± 2.27
Chlorophyll c1 + c2 (µg/g)4.68 ± 0.01
Carotenoids (mg/g)0.14 ± 0.01
Values are reported as mean ± SD (n = 3).
Table
Table 4. Antibacterial effect of J. rubens extracts.
Table 4. Antibacterial effect of J. rubens extracts.
Bacterial StrainsAQ ExtractDM ExtractM ExtractPositive Control
Zone of Inhibition (mm)
A. Baumannii (G-)---15
P. Aeruginosa (G-)---21
E. Coli (G-)---28
B. Cereus (G+)---28
S. Pneumonia (G+)---28
G+: Gram-positive; G-: Gram-negative. Positive control: penicillin/streptomycin. Data were reported as mean ± SD (n = 3). DM: dichloromethane/methanol; M: methanol; AQ: aqueous; SD: standard deviation.
Table
Table 5. Cytotoxic activity of organic extracts on CRC cell lines.
Table 5. Cytotoxic activity of organic extracts on CRC cell lines.
In Vitro Cytotoxicity, IC50 (µg/mL)
ExtractsHCT-116HT-29
DM Soxhlet499.94531.44
DM Crude2603.541560.38
M Soxhlet389.73666.82
M Crude1578.131662.74
AQ Soxhlet1490.371657.26
AQ Crude1629.451240.42
Data were reported as mean ± SD (n = 3). DM: dichloromethane/methanol; M: methanol; AQ: aqueous. SD: standard deviation. IC50 refers to the half-maximal inhibitory concentration.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Rifi, M.; Radwan, Z.; AlMonla, R.; Fajloun, Z.; Sabatier, J.M.; Kouzayha, A.; El-Sabban, M.; Mawlawi, H.; Dassouki, Z. The Lebanese Red Algae Jania rubens: Promising Biomolecules against Colon Cancer Cells. Molecules 2022, 27, 6617. https://doi.org/10.3390/molecules27196617

AMA Style

Rifi M, Radwan Z, AlMonla R, Fajloun Z, Sabatier JM, Kouzayha A, El-Sabban M, Mawlawi H, Dassouki Z. The Lebanese Red Algae Jania rubens: Promising Biomolecules against Colon Cancer Cells. Molecules. 2022; 27(19):6617. https://doi.org/10.3390/molecules27196617

Chicago/Turabian Style

Rifi, Mariam, Zeina Radwan, Reem AlMonla, Ziad Fajloun, Jean Marc Sabatier, Achraf Kouzayha, Marwan El-Sabban, Hiba Mawlawi, and Zeina Dassouki. 2022. "The Lebanese Red Algae Jania rubens: Promising Biomolecules against Colon Cancer Cells" Molecules 27, no. 19: 6617. https://doi.org/10.3390/molecules27196617

Article Metrics

Back to TopTop