Anti-cancer potential of crude extracts from Dictyota bartayresiana in triple-negative breast cancer
Abstract
This study aims to isolate and characterize secondary metabolites from the brown algae Dictyota bartayresiana and evaluate its cytotoxicity under in vitro conditions. Phytochemical analyses were extracted using dichloromethane and ethyl acetate as solvents. The results showed the presence of various compounds such as terpenoids, flavonoids, phenols, and alkaloids. Gas chromatography-mass spectrometry was utilized to characterize the extracts. The cytotoxicity evaluation of the crude extracts by MTT assay showed the inhibitory concentration (IC50) for both extracts in the MDA-MB-231 cell line to be 1 µg/mL. The studies in cell biology indicated a change in cell morphology including detached cells with a round morphology, indicating the induction of apoptosis. Wound healing assay showed that both extracts inhibited the invasion of MDA-MB-231 cells (0.75 µg/mL).
Introduction
Breast cancer has topped lung cancer as the most spotted cancer, with 2.3 million new cases (11.7%) (Sung et al., 2021). The variations between and within tumors, as well as between patients, breast cancer is recognized as a heterogeneous illness (intratumor heterogeneity). This variation is seen in cancer cells as they express hormone receptors, the human epithelial growth factor 2 oncoprotein, and other biomarkers. When taken together, these elements form the foundation for personalized therapies for breast cancer (Turashvili and Brogi, 2017). Hormone receptor-positive/ERBB2 negative (70% of patients), ERBB2 positive (15–25% of patients), and triple-negative (15%) are the three main subtypes of breast cancer based on the above characteristics (Waks and Winer, 2019).
Among the newly reported cases of breast cancer, 15–20% are diagnosed as triple-negative. As a result, the prognosis for these patients is dismal, with a low 5-year survival rate and a significant recurrence rate even after adjuvant therapy (de Ruijter et al., 2011). Standard treatments for triple-negative breast cancer include chemotherapy or radiotherapy following surgery. However, these treatments often have severe side effects, and leave the body prone to relapse and a higher risk of infections as they tend to weaken the body’s immune system. Moreover, hormonal therapy or trastuzumab-based therapy cannot be used in triple-negative breast cancer treatment due to the lack of the three receptors above.
In recent decades, there has been a rise in the field of marine pharmacology. These organisms have a rich chemical diversity of secondary metabolites with a wide range of bioactive properties and processes, including the ability to halt the cell cycle, induce apoptosis, and inhibit proliferation and metastasis. Their extensive concentrations in the marine ecosystem, aquatic species are currently being explored as a viable source of bioactive compounds. Abectedin, tisotumab vedotin, plitidepsin, and cytarabine are only a few of the sea-based medicines that are approved by the FDA and the European Medicines Agency (Ruiz-Torres et al., 2017).
The marine brown algae are known as an excellent source of anti-cancerous biopotentials. For instance, many members of the family Dictyotaceae produce several secondary metabolites, mainly diterpenes, making Dictyota the richest genus of brown algae in this family (Rushdi et al., 2022). The structural diversity of the cyclic diterpenes produced by this family is exceptional. Antimicrobial, anti-inflammatory, and anti-cancer properties are just some of the many pharmacological properties attributed to diterpenes (Nyirimigabo et al., 2015). Although approximately 225 direct species can be traced back to Dictyota J. V. Lamouroux, only a fraction of them have been analyzed so far.
This paper primarily aims to investigate the brown algae Dictyota bartayresiana, popularly termed "forded sea tumbleweed," which is ubiquitous in tropical and warm water (Bogaert et al., 2020). The current study involved the extraction of D. bartayresiana using solvents and subsequent experimentation to detect the phytoconstituents present and the algae's anti-cancer efficacy against the MDA-MB-231 cell line.
Materials and Methods
Collection of algae
The marine seaweed D. bartayresiana was obtained from the Mandapam coast of Tamil Nadu, India. A voucher specimen (SLS2022001) of the algae was deposited at the School of Life Sciences, BSACIST, Vandalur, India.
Extraction of phytochemicals
The algae D. bartayresiana was shade-dried for 2 weeks and grinded to form a coarse powder. The dried algal powder (20 g) was extracted sequentially with dichloromethane (100 mL) and ethyl acetate (175 mL) for 6 days in an incubator shaker at 37ºC at 100 rpm. The mixture was filtered using Whatman filter paper (Sigma Aldrich) and evaporated in a rotary evaporator. The crude extracts were further dried until complete solvent evaporation and subsequently stored at a temperature of 4˚C for further analyses. The yield extract was calculated as a percentage using the following formula:
Dry weight of extract
%Yield = -------------------------------- x 100
Dry weight of material
The extracts were used after being reconstituted in 1 mg/mL of methanol for all analyses.
Phytochemical screening
The obtained dichloromethane and ethyl acetate extracts of D. bartayresiana were subjected to phytochemical screening according to the following standard methods (Yadav et al., 2011).
Test for tannins and phenols (Ferric chloride test)
About 2-3 drops of 2% ferric chloride solution were added to 2 mL of the crude extracts. The appearance of blue-green or black color indicated the presence of tannins and phenols.
Test for flavonoids (Shinoda test)
Fragments of magnesium ribbon were added to 2 mL of the crude extracts, followed by a drop-wise addition of concentrated HCl. The development of pink, red or orange color confirmed the presence of flavonoids.
Test for terpenoids (Salkowski test)
Chloroform (2 mL) was mixed with 2 mL of the extracts and about 3 mL of concentrated H2SO4 was added carefully to form a layer. The formation of a reddish-brown color indicated the presence of terpenoids.
Test for saponins (Froth test)
Distilled water (1 mL) was added to 2 mL of the extracts and shaken vigorously. The formation of stable froth or foam indicated the presence of saponins.
Test for alkaloids
About 2 mL of 1% HCl was added to 2 mL of the extracts. The mixture was heated gently before adding 2 mL of Mayer's and Wagner's reagents. Turbidity and resulting precipitate indicated the presence of alkaloids.
Test for cardiac glycosides (Keller-Kilani test)
A mixture of 2 mL of glacial acetic acid containing 1-2 drops of a 2% FeCl₃ solution was added to 2 mL of the crude extracts. The mixture was then transferred to a test tube containing 2 mL of concentrated H2SO4. The appearance of a brown ring at the interphase confirms the presence of cardiac glycosides.
Test for amino acids (Ninhydrin test)
The crude extracts (2 mL) were boiled with 2 mL of 0.2%ninhydrin. The formation of a violet color indica-ted the presence of amino acids.
Test for carbohydrates (Molisch's test)
Molisch's reagent (2-3 drops) was mixed with 2 mL of the crude extracts, followed by the careful addition of 2 mL of concentrated H₂SO₄ along the side of the test tube. The appearance of a violet or purple ring at the interface of the two liquid layers confirmed the presence of carbohydrates.
Box I: Wound healing assay
Requirements
Benchtop centrifuge; Breast cancer cell line (MDA-MB-231); Centrifuge tube (15 mL); DEDB extract (1 µg/mL); EEDB extract (1 µg/mL); High-glucose DMEM medium; Incubator; Micropipettes (100-1000 µL); PBS medium; T flask (25 mL); 6-Well culture plate
Procedure
Step 1: MDA-MB-231 cells were seeded in a six well plate at a density of 3 x 105 cells/well.
Step 2: The cells were allowed to adhere for 24 hours in a CO2 incubator.
Step 3: Cell confluency was checked under a microscope. When the cells reached 70% confluency the media was removed.
Step 4: A thin straight scratch was made in each well with the help of a 200 µL micropipette tip.
Step 5: The detached cells were removed by consequent washing with PBS medium.
Step 6: The cells were treated with 0.75 µg/mL of DEDB and EEDB extracts.
Step 7: The cell migration rate was monitored by taking images at hour 0 and hour 24 after incubation.
Step 8: The cells that have not been treated with DEDB and EEDB extracts served as control, and the images that taken at specific intervals were compared to the control.
Reference
Cory, 2011
GC-MS analysis
For the identification of the algal constituents and chemical composition of D. bartayresiana, the extracts were characterized by employing the use of GC-MS (Shimadzu, QP2010) plus equipped with a thermal desorption analyser (TD-20) and a liquid autosampler. The pressure was maintained at 51.5 kPa, with 54.0 mL/min flow volume. The ion source of the mass spectrometer was held at 250ºC with an interface temperature of 260ºC. The threshold was set at 0 with solvent cut time being 2 min. The unknown peaks in the mass spectra of the compounds were collected and then by compared their retention time, area under the peak, peak height, and mass fragmentation spectra with that of established compounds found in the National Institute of Standards and Technology (NIST) collection, the phytoconstituents were determined.
Cell culture
The study relied on MDA-MB-231 cell lines which were offered for the study by Dr. P. Ashok Kumar, School of Life Sciences, BSACIST, Chennai. The cells were subcultured in a high-glucose DMEM (HiMedia, USA)enriched with 10%FBS (HiMedia, USA) and 1%antibiotic cocktail (HiMedia, USA) and subsequently incubated at 37ºC with 5%carbon dioxide incubator (Eppendorf, USA).
In vitro analysis of cell viability
For the cell viability assay, 4 x 103 cells/well were sown in a 96-well plate, and the plate was left for the cells to adhere. The cells were treated with different concentrations of dichloromethane or ethyl acetate extract (0.1-5 µg/mL). The cells were incubated for 24 hours, and then their vitality was evaluated using the MTT assay. To carry out the essay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent was added to the cells and left for incubation at 37ºC for 2 hours. During this period, MTT undergoes reduction to form formazon crystals. Following this, isopropanol was added as a solubilizing agent to facilitate the extraction of the formazon product from cells for further quantitative analysis. Doxorubicin was used as the standard reference, while the cells that were not treated were considered the control (Begum et al., 2017).
Cell biology study
The cells were analysed for morphological changes under phase contrast microscope after treatment with algal extracts. The cells were subcultured (4 x 105 cells/ well) in a 6-well plate, and treated with the inhibitory concentration (IC50) of dichloromethane or ethyl acetate extract. The cells were then left for incubation for 24 hours. After incubation, the cells were again observed, and images were captured.
Statistical analysis
The experiments were carried out thrice in triplicates and the values were expressed as mean ± SD. IBM SPSS 23 Student t-test was used to observe the level of significance, with a p-value of <0.05 indicating statistical significance.
Results
Phytochemicals identified
The dry weights of the dichloromethane and ethyl acetate extracts obtained from the extraction process were 0.47 g (yield, 1.5%) and 0.11 g (yield, 0.4%), respectively. The decrease in yield percentage could be attributed to the difference in polarity between the solvents – dichloromethane < ethyl acetate. The phytochemical analysis verified the presence of secondary metabolites in both extracts. Terpenoids, steroids, flavonoids, phenols, and cardiac glycosides were identified in the crude extracts. Both dichloromethane and ethyl acetate were positive for terpenoids and saponins. However, ethyl acetate extract showed the presence of flavonoids and phenols whereas dichloromethane was positive for cardiac glycosides and alkaloids (Table I).
Table I: Phytochemical analysis of the crude extracts of Dictyota bartayresiana
| Phytoconstituent | Dichloromethane | Ethyl acetate |
|---|---|---|
| Tannins | - | - |
| Flavonoids | - | + |
| Terpenoids | + | + |
| Saponins | + | + |
| Cardiac glycosides | + | + |
| Carbohydrates | - | + |
| Amino acids | - | - |
| Phenols | + | + |
| Alkaloids | - | - |
| + Presence, - Absence | ||
GC-MS analysis to identify the phytoconstituents
Fifty-four peaks that designate various phytoconstituents were spotted in the GC-MS chromatogram of dichloromethane extract (Table II). Out of 54 compounds, pentadecanoic acid exhibited a higher prevalence with a peak area of 40.5% while the remaining compounds’ peak area fell shorter than 5.0%. Such other compounds include 5,9-methanobenzo-cycloocten-5(1H)-ol,2,3,4,6,7,8,9,10-octahydro-2,2,8,8,9-pentamethyl- with a peak area of 4.2%, L-ascorbic acid with 3.5% peak area, decaethylene glycol with 3.0% peak area and hexadecenoic acid with 2.6% peak area. In a correspondent manner, 56 compounds were found in the chromatographic profile of the ethyl acetate extract (Table II). Among these, the compound regis-tered the highest peak area of 9.2%, demonstrating its abundance, was 1-methyl-3-(2,6,6-trimethyl-1-cyclo-hexen-1-yl) propyl acetate. This was followed by n-hexadecanoic acid with an 8.4% peak area, 3,5-di-tert-butyl-4-hydroxy-phenyl propionic acid with a 6.8% peak area, thunbergol with a 6.3% peak area. The compounds that had peak area as low as 0.05% and below include heptadecane, 2-isopropyl-5-methyl-1-heptanol, and 3-isopropoxy-1,1,1, 7,7,7-hexamethyl-3,5,-5-tris(trimethyl-siloxy)tetrasiloxane. Further, compounds such as neophytadiene, testolactone and octa-decanoic acid had peak areas ranging between 2 and 5%.
Table II: GC-MS profile of compounds in two different extracts of Dictyota bartayresiana
| Retention time (min) | Name of the compound | Molecular formula | Molecular weight | %Present |
|---|---|---|---|---|
| Dichloromethane extract | ||||
| 5.851 | 2-Bromo-3,3,3-trifluoropropyl fluorosulfate | C3H3BrF4O3S | 274 | 0.17 |
| 6.085 | Phosphorothioic acid, o,o-dimethyl s-[2-(methylamino)-2-oxoethyl] ester | C5H12NO4PS | 213 | 0.47 |
| 10.141 | [1,3,5]-Triazine-2-carbonitrile, 4-methoxy-6-(pyrrolidin-1-yl)- | C9H11N5O | 205 | 0.48 |
| 13.257 | 5-Chloro-3-(4-chlorophenyl)-benzo[c]isoxazole | C13H7Cl2NO | 263 | 1.81 |
| 16.384 | Heptasiloxane, 1,1,3,3,5,5,7,7,9, 9,11,11,13,13-tetradecamethyl- | C14H44O6Si7 | 504 | 1.03 |
| 17.271 | 1-Dodecanol, 3,7,11-trimethyl- | C15H32O | 228 | 0.66 |
| 19.366 | Proscillaridin | C30H42O8 | 530 | 0.19 |
| 20.02 | Pentadecanoic acid | C15H30O2 | 242 | 40.48 |
| 20.38 | 3,6,9,12-Tetraoxatetradecan-1-ol,14-[4-(1,1,3,3-tetramethylbutyl) phenoxy]- | C24H42O6 | 426 | 2.34 |
| 21.315 | 7-Hexadecenal, (Z) | C16H30O | 238 | 1.37 |
| 23.68 | Valeric acid, 2,3-epoxy-3,4-dimethyl-, ethyl ester, cis- | C9H16O3 | 172 | 1.23 |
| Ethyl acetate extract | ||||
| 18.491 | Neophytadiene | C20H38 | 278 | 2.76 |
| 20.313 | 3,5-Di-tert-Butyl-4-hydroxyphenylpropionic acid | C17H26O3 | 278 | 6.82 |
| 20.675 | Linalol oxide,trifluoroacetate | C12H17F3O3 | 266 | 0.3 |
| 22.225 | α- Santalol | C15H24O | 220 | 0.65 |
| 22.526 | 1-Methyl-3-(2,6,6-trimethyl-1-cyclohexan-1-yl) propyl acetate | C15H26O2 | 238 | 9.23 |
| 22.646 | 1H-Purin-6-amine, [(2-fluorophenyl) methyl]- | C12H10FN5 | 243 | 0.6 |
| 23.626 | 4,8,13-Cyclotetradecatriene-1,3-diol, 1,5,9-trimethyl-12-(1-methylethyl)- | C20H34O2 | 306 | 3.14 |
| 24.245 | Testolactone | C19H24O3 | 300 | 5.91 |
| 26.007 | Thunbergol | C20H34O | 290 | 6.34 |
Effects on cell viability
MDA-MB-231 cells were treated with increasing concentrations of dichloromethane extract and EEDB, ranging from 0.1 to 5 µg/mL for 24 hours and cell death was measured with the help of MTT assay. Cell viability assay results displayed that both the extracts exhibited a dose-dependent response and induced 50% cytotoxicity in the MDA-MB-231 cell line. The IC50 that caused 50% cell death was determined as 1 µg/mL for dichloromethane and ethyl acetate extracts. The IC50 for doxorubicin was also identified as 1 µg/mL. Cell viability seemed to decrease with concentrations >1 µg/mL of both extracts (Table III). Under a phase contrast microscope, cells treated with both extracts shrunk in size, indicating the loss of cell morphology with alterations in the cell shape and size as compared to the untreated control cells (Figure 1).
Figure 1: Phase contrast microscopic views after exposure to dichloromethane and ethyl acetate extracts Dictyota bartayresiana. Doxorubicin was used as standard. Magnification at 10x
Effects on the migration of cells
The wound healing assay showed extract-treated cells had fewer cells migrating into the wound than the control cells, suggesting that both extracts have anti-migratory properties. The percent migration was calculated as 65% for dichloromethane extract and 51.3% for ethyl acetate extract (Figure 2).
Figure 2: Wound healing assay. The assay was carried out to test the anti-migratory effect of dichloromethane and ethyl acetate extracts of Dictyota bartayresiana. The migration of cells into the wound region was observed in control and dichloromethane and ethyl acetate-treated cells at 0 and 24 hours. Magnification at 10x
Table III: Cytotoxic activity of extracts of Dictyota bartayresiana against MDA-MB-231 cell line
| %Cell viability | ||
|---|---|---|
| Concentration (μg/mL) | Dichloromethane | Ethyl acetate |
| 0.5 | 79.7 ± 2.2a | 59.8 ± 3.4 |
| 0.75 | 51.0 ± 4.0a | 58.4 ± 2.3 |
| 1 | 50.1 ± 0.5 | 50.8 ± 2.1 |
| 1.5 | 46.4 ± 1.3 | 33.5 ± 0.5a |
| 2 | 43.2 ± 1.5 | 33.4 ± 0.9 |
| 2.5 | 42.5 ± 1.7 | 32.1 ± 0.7 |
| 3 | 41.4 ± 1.1 | 28.3 ± 0.8 |
| Data are mean ± SD. For standard doxorubicin (1 μg/mL) cell viability (%) = 50.8 ± 0.6; Superscript "a" means p<0.05 compared to control group | ||
Discussion
The findings of this study show the potent cytotoxic and anti-migratory effects of the crude extracts from the brown algae D. bartayresiana on the triple negative breast cancer cell line MDA-MB-231. With an IC50 of 1 µg/mL, the dichloromethane and ethyl acetate extracts demonstrated a significant ability to induce apoptosis and inhibit migration of the cancer cells. Both extracts exhibited dose-dependent cytotoxicity, altering the morphology of MDA-MB-231 cells and reducing their migratory abilities in the wound healing assay. Moreover, the phytochemical screening revealed the presence of diverse bioactive compounds such as terpenoids, flavonoids, phenols, and alkaloids, with the ethyl acetate extract being specifically rich in terpenoids. These findings suggest that D. bartayresiana holds promise as a potential candidate for the development of novel therapeutic agents targeting triple-negative breast cancer cell lines.
The Dictyota genus has been explored generously for a variety of its properties. For instance, D. dichotoma, being one of the highly researched species in this genus, has been tested on various grounds for its anti-cancerous property among many (El-Shaibany et al., 2022, Usolteva et al., 2018, Malyarenko et al., 2019). Moreover, the genus is known for its rich reserve of terpenes (Rushdi et al., 2022). Terpenoids, in particular, have shown immense potential in inducing apoptosis and inhibiting proliferation across various cancer models, and they have been attributed to the genus’s therapeutic potential. However, the species D. bartay-resiana has not been analyzed for its cytotoxic activity, specifically against triple-negative breast cancer cell lines, and further, remains to be deeply unexplored. Previously, only two studies were found to have examined its anti-cancer effects against HT29 (Bharathi et al., 2022) and DLA (Antonysamy et al., 2015) cell lines using nanoparticles synthesized from the algae. In another study (Dubey et al., 2022), the group explicitly isolated and extracted fucoidans from Dictyota bartay-resiana. Fucoidans, being polar compounds, are generally extracted using aqueous solvents under slightly acidic conditions. By employing such techniques, the research group isolated the sulfated polysaccharide effectively, and further investigated its antioxidant and cytotoxic potential in the SK-Hep-1 cell line, producing significant results indicating the same. Moreover, the study further showed that fucoidan treatment induced not only apoptosis but also activated autophagy in the cancer cell. In contrast, our analysis did not detect the presence of sulfated polysaccharides or fucoidans in the extracts since we employed ethyl acetate and dichloro-methane for extraction. These solvents are non-polar and therefore not ideal for extracting polar compounds like fucoidans. Despite the lack of relevant data on D. bartayresiana to draw conclusive findings, the results of this study align with the more generalized characteristics found in the genus.
The MDA-MB-231 cell line was found to be sensitive to both the extracts’ cytotoxicity, and the extracts further displayed anti-migratory ability. This could be attributed to the identified bioactive compounds. A major component in dichloromethane extract, pentadecanoic acid, is known to modulate lipid metabolism and induce apoptosis. The cytotoxicity of pentadecanoic acid has further been linked to the inhibition of the Jak2/Stat3 signaling pathway in a prior study (To et al., 2020). However, several compounds found in dichloro-methane extract have not been thoroughly investigated in prior research, which indicates the likelihood that any of these compounds could be responsible independently or synergistically for the biological activity noted in this study. The high incidence of terpenoids in the ethyl acetate extract raises the possibility of the biological activity exhibited by ethyl acetate in this study being accredited to its rich terpenoid content. These included thunbergol, α-santalol, neophytadiene, linalool oxide, isoaromadendrene epoxide, heptatriacotanol. Thunbergol, also referred to as isocembrol, is a monocyclic diterpene alcohol that has been exploited in the treatment of cancer and was previously claimed as a TNF inhibitor (Li et al, 2020). Another intriguing terpenoid found here is α-santalol, which is a naturally occurring sesquiterpene that is generally derived from sandalwood oils. In a prior investigation, alpha-santalol derived from sandalwood oil was tested against human prostate cancer cells, and reports of apoptosis induction in the cell line through caspase-3 activation (Bomma-reddy et al., 2012) were made. The compound, neophytadiene is also a diterpene, which has also been reported in a previous study to possess anti-inflammatory, antioxidant and cardioprotective properties (Bhardwaj et al., 2020). Linalol oxide, a monoterpene compound, is known for its aromatic power and antimicrobial properties (Griffin et al., 1999). Findings from the above prior investigations into these com-pounds only reinforce their role in promoting cell death.
However, ethyl acetate extract was more effective in inhibiting cell migration by around 13% than dichloromethane extract. This difference could be attributed to their distinct chemical compositions. According to the GCMS results, ethyl acetate extract contained a more diverse array of compounds. In contrast, the majority of dichloromethane extract’s composition is taken up by pentadecanoic acid which accounted for approximately 40% of its content. This high concentration of pentadecanoic acid likely contributes to its cytotoxic effects but may also be accredited to the difference observed in its anti-migratory effects. The polarity of the solvents could be linked to the difference in these chemical com-positions between the extracts since by utilizing solvents with varying polarity, it is possible to extract diverse classes of phytocompounds (Wakeel et al., 2019). Being a nonpolar solvent, dichloromethane extracted compounds like pentadecanoic acid, which are less polar. With its comparatively higher polarity, ethyl acetate extracted a wider range of bioactive molecules, including diverse terpenoids. A few compounds such as 3-Isopropoxy-1,1,1,7,7,7-hexamethyl-3,5,5-tris(trimethylsiloxy)tetrasiloxane and hexadecenoic acid overlapped in both the extracts. The selective solubility of compounds explains the minimal overlap in their GCMS profile and further points to a polaritydriven extraction process which hints at the importance of sound solvent selection for isolating compounds with specific bioactivities.
However, this study has a few limitations. The lack of comparative studies conducted on the same species, (D. bartayresiana) against a broad range of breast cancer cell lines, severely limits the generalizability of the findings. Secondly, while GC-MS analysis revealed rich reserves of phytocompounds, the specific bioactive compounds responsible for the observed effects remain unidentified, which warrants further purification and charac-terization studies. Thirdly, the underlying mechanisms through which the compounds exert their effects were unexplored in this study; however, investigating these mechanisms in future research could provide profound insights and enhance our understanding. Finally, the study was not conducted on animal models due to limited research conditions.
Conclusion
Both dichloromethane and ethyl acetate extracts of D. bartayresiana contain active phytoconstituents, with the ethyl acetate extract being particularly rich in terpenes. The MDA-MB-231 cell line was sensitive to the extract’s cytotoxicity, and the extracts displayed anti-migratory ability.
Ethical Issue
Not applicable
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