Antioxidant, antiproliferative, genotoxic and cytoprotective effects of the methanolic extract of Padina tetrastromatica on human breast adenocarcinoma and embryonic fibroblast cell lines

Abstract

The in vitro antioxidant, antiproliferative, genotoxic and cytoprotective effects of the methanolic extract of the seaweed Padina tetrastromatica were assayed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide, nitric oxide and hydroxyl radical scavenging assays, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and comet assays. The methanolic extract of P. tetrastromatica showed considerable antioxidant activity through inhibition of DPPH and hydroxyl radicals, with median inhibitory concentration (IC₅₀) values of 45.57 ± 1.63 and 36.58 ± 2.13 µg/ml, respectively. Treatment of the human breast adenocarcinoma cell line MCF-7 with the methanolic extract, at a concentration range of 0–500 µg/ml, showed considerable antiproliferative effects, with an IC₅₀ value of 125 ± 2.03 µg/ml. The genotoxic and cytoprotective effects of the extract on the human breast adenocarcinoma cell line MCF-7 and the embryonic fibroblast cell line 3T3-L1 were evaluated by the in vitro comet assay, and the extract demonstrated a genotoxic effect on MCF-7 and antigenotoxic and cytoprotective effects on 3T3-L1 cells. The methanolic extract of P. tetrastromatica was more effective in inducing a genotoxic effect in MCF-7 cells and a cytoprotective effect in 3T3-L1 cells, compared with the standard cancer chemotherapy drug doxorubicin, which induced greater DNA damage in the normal 3T3 cells.

Keywords:

• antioxidant
• antiproliferative
• Padina tetrastromatica
• genotoxic
• cytoprotective
• comet assay

Introduction

Cancer is by far one of the most worrying health issues and continues to be the leading cause of mortality worldwide, accounting for 7.9 million deaths in 2007. This figure is predicted to continue rising, with an estimated 12 million deaths in 2030 (Pandey & Madhuri 2009). Over 100 types of cancer have been documented. Breast cancer tops the chart as the most prevalent cancer today, followed by prostate cancer, colon or rectal cancer, lung cancer and ovarian cancer (Kintzios & Barberaki 2004). Breast cancer is the second leading cause of cancer-related deaths among women globally, with the oestrogen receptor-positive type accounting for over 50% of all breast carcinomas (Gresham et al. 2008).

The marine environment is often thought of as a tranquil habitat. However, this is not so for marine organisms. For these organisms, the marine environment is a highly perilous place where predators are abundant and competition is intense. Marine organisms, such as seaweeds and sponges, nourish themselves through the process of osmosis, despite not having roots, leaves or vascular systems like terrestrial plants (Gupta & Abu-Ghannam 2011). Owing to the complications and diversity of the environment, marine organisms have developed a range of complex mechanisms for survival and defence purposes. Marine phytochemicals may be far more powerful than phytochemicals from terrestrial organisms owing to their ability to kill prey rapidly. Therefore, they may serve as potent anticancer agents (Wu et al. 2000). In recent years, much attention has been devoted to natural antioxidants. An impressive number of antioxidants has been isolated from marine algae. Like all photosynthesizing plants, marine algae are exposed to high levels of oxidative stress, including a combination of light, high oxygen concentration and temperature fluctuations, all of which lead to the formation of free radicals and other strong oxidizing agents (Dykens et al. 1992).

Padina tetrastromatica (Class, Phaeophyceae; Family, Dictyotaceae) is also regarded as a taxonomic synonym of Padina antillarum (Guiry & Guiry 2011). Padina tetrastromatica is a striped, yellowish-brown, fan-shaped alga which turns olive green upon drying (Shaikhi et al. 1991). It is irregularly cleft into narrow lobes with involuted apical margins (Mica 1966). It can be found growing in shallow and sand-covered rocky areas (Shameel 1990). The bioactivities of P. tetrastromatica have been extensively studied. The many health-promoting properties of this seaweed include antimicrobial, antioxidant and free radical scavenging properties, anti-hepatitis B virus activity, hepatoprotective properties and anti-pancreatic cancer activity (Subramaniam et al. 2011; Aravindan et al. 2013; Clara et al. 2014; Mohsin et al. 2014; Ponnanikajamideen et al. 2014). Several studies have been conducted on the antioxidant activities and probably the anticancer activity of P. tetrastromatica. However, to the authors' knowledge, there is virtually no scientific literature pertaining to the antioxidant, antiproliferative and antigenotoxic activities of the methanolic extract of P. tetrastromatica obtained by the single extraction method. A single extraction method was used in this study instead of sequential extraction as some of the compounds may act synergistically to exert their bioactivities. Methanol was chosen for the extraction of the entire constituent of polar compounds in P. tetrastromatica in this study.

Numerous methods have been developed for the evaluation of antioxidant activities of compounds and complex mixtures from natural resources. There is no single universal method for identifying all possible mechanisms characterizing antioxidant activity, despite the emergence of various antioxidant activity determination methods (Frankel & Meyer 2000). Therefore, this study aims to evaluate the antioxidant activity of the methanolic extract of P. tetrastromatica using several different methods.

The MTT assay is a colorimetric assay for measuring enzyme activity in the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In this assay, reduction of the soluble yellow tetrazolium salt (MTT) occurs in the mitochondria of metabolically active cells to form insoluble purple formazan crystals, which can be solubilized into a coloured solution by the addition of solubilisation solutions such as detergents or dimethyl sulfoxide (DMSO) (Berridge et al. 2005). The coloured solution can be quantified by measuring the absorbance at a wavelength between 500 and 600 nm with a spectrophotometer. The colour intensity is directly proportional to the number of viable cells. The comet assay is a method used for determining and measuring DNA damage at the cellular level. The alkaline comet assay enables the detection of a broad range of DNA lesions, which include single strand breaks (SSBs) with alkali-labile sites (ALSs), DNA–DNA and DNA–protein cross-links, and particular types of base disorder (Hashim et al. 2013).

Materials and methods

Sample preparation

The seaweed P. tetrastromatica was collected from the coastal regions of the West Coast of Malaysia. It was identified and a voucher specimen deposited at the Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman, Kampar, Malaysia. The seaweed was washed, cleaned and cut into smaller pieces before being freeze-dried and subsequently ground into powder. The powder was extracted with methanol. Ground material (50 g) was extracted with 500 ml of the solvent for 24 h at room temperature. The filtrate was filtered through Whatman No. 1 filter paper and then concentrated with a rotary evaporator. The concentrated crude extract was dissolved in 10% DMSO.

Determination of total phenolic content

The total phenolic content (TPC) was determined by the method described by Siddhuraju and Becker (2007) with slight modifications in the concentration of Na₂CO₃. The assay involves Folin–Ciocalteu (FC) reagent and gallic acid as the standard. Quercetin and rutin were used as positive controls. One millilitre of 10% FC reagent was added to 20 µl of extract or standard, mixed well and incubated for 5 min before adding 700 µl of 10% Na₂CO₃. The solutions were further incubated for 2 h before reading the absorbance at 765 nm. Gallic acid in the range of 20–200 mg/l was used to construct a calibration curve. TPC values are expressed as mg gallic acid equivalents (GAE)/g dried weight. The experiments were performed in triplicate.

Determination of total flavonoid content

The aluminium chloride colorimetric method of Chang et al. (2002) was used for the determination of total flavonoid content (TFC). Quercetin was used as the standard and catechin was used as the positive control. First, 60 µl of methanol was mixed with 20 µl of extract or standard, 4 µl of 10% aluminium chloride, 4 µl of 1.0 M potassium acetate and 122 µl of MilliQ water. The mixtures were incubated at room temperature for 30 min before reading the absorbance at 415 nm. A calibration curve was prepared using catechin at concentrations of 12.5–100 µg/ml in methanol. TFC was expressed as mg/g dried weight. All experiments were performed in triplicate.

1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity

1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical was used in the evaluation of radical scavenging activity of the extracts, as described by Costa et al. (2010) with minor changes in the DPPH concentration. The reaction mixtures, containing 120 µl of 0.04 mg/ml DPPH solution in methanol, were mixed with 20 µl of different concentrations (25–400 µg/ml) of the extracts and shaken vigorously before being incubated in the dark for 20 min. Quercetin and rutin were the positive controls. Reduction in the absorbance of DPPH was measured against a reagent blank at 517 nm. The radical scavenging activity was calculated using Equation (1): (1) where A_blank and A_{sample/positive controls} denote the absorbance of the blank and the absorbances of samples or positive controls, respectively.

Superoxide anion radical scavenging activity

The superoxide anion scavenging activity was assayed according to the method of Shaikhi et al. (1991) with slight modifications in the nitroblue tetrazolium (NBT) and phenazine methosulfate (PMS) concentrations. The reaction mixture containing 25 µl of NBT solution (150 µM NBT in 100 mM phosphate buffer, pH 7.4), 2 µl of PMS solution (60 µM PMS in 100 mM phosphate buffer, pH 7.4) and 20 µl of NADH solution (468 µM in 100 mM phosphate buffer, pH 7.4) was added to different concentrations (25–400 µg/ml) of the extracts. The mixture was incubated in the dark for 10 min at 25°C and the absorbance read at 560 nm. Quercetin and rutin were the positive controls. All experiments were carried out in triplicate and results were expressed as percentage inhibition of superoxide anion radical using Equation (2): (2)

Nitric oxide scavenging activity

The nitric oxide scavenging activity was determined using the method of Rai et al. (2006). First, 10 µl of 10 mM sodium nitroprusside (SNP) in phosphate buffer was mixed with 10 µl of different concentrations (25–400 µg/ml) of extracts. The mixture was incubated in the dark at room temperature for 2.5 h. Quercetin and rutin were used as positive controls. After the incubation period, 40 µl of sulfanilic acid reagent (0.33% sulfanilic acid in 20% glacial acetic acid) was added to the mixture and further incubated for 5 min, after which 40 µl of 0.1% naphthyl ethylene diamine dihydrochloride was added, mixed and incubated for another 30 min at 25°C. The absorbance of the chromophore formed was read at 540 nm. All determinations were performed in triplicate and results were expressed as percentage of nitric oxide scavenging using Equation (3)(3) : (3)

Hydroxyl radical scavenging activity

The site-specific hydroxyl radical scavenging assay was determined as described by Halliwell et al. (1987). The reaction mixture containing 23.8 µl of 100 mM FeCl₃ solution, 23.8 µl of 1.25 mM H₂O₂ solution, 23.8 µl of 2.25 mM deoxyribose and 23.8 µl of 100 mM ascorbic acid was added to 5 µl of different concentrations (25–400 µg/ml) of extracts. The mixture was incubated at 37°C for 1 h, after which 100 µl of 0.5% of thiobarbituric acid (TBA) in 25 mM NaOH and 100 µl of 2.8% trichloroacetic acid (TCA) were added. The resulting mixture was then boiled at 100°C for 15 min and subsequently cooled on ice before taking the absorbance readings at 550 nm. Quercetin and rutin were used as positive controls. All determinations were performed in triplicate and results were expressed as percentage of hydroxyl radical scavenging activity, calculated using Equation (4): (4)

Cell culture

The normal human breast cell line 184B5, human breast adenocarcinoma cell line MCF-7 and embryonic fibroblast cell line 3T3 were purchased from American Type Culture Collection (ATCC, VA, USA). 184B5 and MCF-7 were maintained in RPMI-1640 (Roswell Park Memorial Institute) while 3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% foetal bovine serum (FBS), glutamine, penicillin and streptomycin at 37°C in a humidified atmosphere with 5% carbon dioxide (CO₂).

Antiproliferative activity

The methanolic extract was dissolved in DMSO and added to medium to make the final concentration of DMSO less than 1%/well. Cell proliferation was determined by the MTT assay. MCF-7 cells were plated at a density of 1 × 10⁴ cells/well in a 96-well plate. The cells were grown for 24 h and subsequently treated with the extracts at doses of 100, 200, 300, 400 and 500 µg/ml for 48 h. Doxorubicin was used as the positive control while the vehicle, DMSO, was used as the negative control. After incubation, 10 µl/well of MTT was added and further incubated for 4 h at 37°C. Spent medium was aspirated and the insoluble formazan dye was dissolved with DMSO. The absorbance of the coloured product was read at 595 nm.

Alkaline comet assay

DNA damage was determined by the alkaline comet assay according to the method of Singh et al. (1988) with slight modifications. The methanolic extract of P. tetrastromatica (100 µg/ml) was used to treat MCF-7 and 3T3 cells for comet assay analysis. Doxorubicin (0.2 µg/ml) was used as the positive control. Treated and non-treated MCF-7 and 3T3 cells were trypsinized and harvested. The pellets were resuspended in 1 ml of 100 mM of hydrogen peroxide (H₂O₂) and kept on ice for 4 h. A total of 60 mg of low-melting agarose and 80 mg of normal-melting agarose were dissolved in 10 ml of distilled water each and heated to 60°C. Low-melting agarose was spread across the microscope slides. Cell suspensions were mixed with 100 µl of normal-melting agarose and spread on the slides precoated with low-melting agarose. Low-melting agarose was spread above the suspension, forming a sandwiched layer. The slides were then incubated in lysis buffer containing 1% sodium dodecyl sulfate (SDS), 2.5 M of sodium chloride (NaCl), 100 mM of disodium salt of ethylenediaminetetraacetic acid (Na₂EDTA), 1% Triton X-100 and 10% DMSO at 4°C for 1 h. The slides were arranged in an electrophoretic tank filled with prechilled electrophoretic buffer (1 mM of Na₂EDTA and 300 mM NaOH) and incubated for 20 min. Electrophoresis was carried out at 25 V (300 mA) for 20 min using a power supply (CBS Scientific Company, San Diego, CA, USA). The slides were washed with 0.4 M of Tris (pH 7.5) and stained with 20 µg/ml of ethidium bromide after electrophoresis. The slides were viewed using an Olympus BX50 fluorescence microscope. The comet tail length was measured using the eyepiece micrometer and the DNA damage was calculated according to Equation (5): (5)

Statistical analysis

All data are reported as mean ± standard deviation (SD) (n = 3). Statistical significance and differences between control and treated samples were tested by the Student's t test. A p-value of less than 0.05 or 0.01 was considered significant.

Results and discussion

Antioxidant activities

The total phenolic and flavonoid contents (TPC and TFC) and the median inhibitory concentration (IC₅₀) values of the free radical scavenging assays for the methanolic extract of P. tetrastromatica are summarized in Table 1.

Table 1. Median inhibitory concentration (IC₅₀) values of the methanolic extract of Padina tetrastromatica in free radical scavenging assays.

	P. tetrastromatica	Quercetin	Rutin	Catechin
TPC (mg GAE/g)	97.5 ± 1.51	980.8 ± 1.98	437.6 ± 2.05	–
TFC (mg/g dried weight)	69.2 ± 1.30	–	–	56.7 ± 1.36
IC50 of DPPH radical scavenging activity (µg/ml)	45.57 ± 1.63	21.67 ± 2.89	23.33 ± 2.89	–
IC50 of superoxide anion scavenging activity (µg/ml)	101.33 ± 1.24	260.00 ± 0	223.33 ± 2.89	–
IC50 of nitric oxide scavenging activity (µg/ml)	116.04 ± 1.59	20.00 ± 0	20.00 ± 0	–
IC50 of hydroxyl radical scavenging activity (µg/ml)	36.58 ± 2.13	20.00 ± 0	101.67 ± 2.89	–

Note: Each value is expressed as mean ± SD (n = 3).

Quercetin and rutin were used as positive controls for all scavenging assays and for the determination of total phenolic content (TPC), whereas catechin was used as a positive control for the determination of total flavonoid content (TFC). GAE = gallic acid equivalents; DPPH = 1,1-diphenyl-2-picrylhydrazyl.

Conventional cancer therapies have not changed dramatically for the past 25 years, despite significant advances in the understanding of the molecular aspects and pathogenesis of cancer. The toxicity and adverse side-effects brought about by conventional therapies have resulted in a remarkable increase in the clinical testing of natural products as alternative or complementary therapies in combination with existing therapies (Shaker & Melake 2012).

The percentage yield of P. tetrastromatica extracted with methanol was 8.71%. Methanol is considered a suitable solvent for the extraction of polyphenolic compounds because of its ability to inhibit the action of polyphenol oxidase, which leads to the oxidation of polyphenols. Methanolic extracts have a very high TPC as phenolic compounds are typically more polar compounds (Yermilo et al. 1995). The high TPC value in P. tetrastromatica is attributable to the presence of phlorotannins, bipolar polyphenols commonly found in brown seaweeds. Phlorotannins show antioxidative properties due to the presence of multiple phenolic groups that assist the algae to overcome oxidative stress arising from their environment (Airanthi et al. 2010). The TPC and TFC of the methanolic extract of P. tetrastromatica are closely linked to the antioxidant properties of the extract (Ashraf et al. 2011).

The results in Table 1 suggest that the extract may elicit its antioxidant activity through the inhibition of DPPH and hydroxyl radicals. The free radical scavenging assay using the DPPH radical is a preliminary test for the analysis of the antioxidant potential of extracts. The assay has been used extensively as it allows high-throughput screening and has high sensitivity for the detection of active ingredients even at low concentrations. Antioxidant activity is related to the presence of bioactive compounds such as phenolics, flavonols and flavonoids. Polyphenols and anthocyanins scavenge DPPH by the donation of hydrogen, thus reducing DPPH (DPPH-H) (Luo et al. 2010). Hydroxyl radicals are generated site-specifically, whereby unchelated iron ions are weakly associated with deoxyribose. These iron ions then react with H₂O₂ through the Fenton reaction, forming hydroxyl radicals that launch an immediate attack on the deoxyribose. When the methanolic extract of P. tetrastromatica was added to the reaction mixture, it removed the hydroxyl radicals from deoxyribose, thus directing the damage towards itself and preventing the reaction (Halliwell et al. 1987).

Antiproliferative activity

Antiproliferative activity was observed in MCF-7 cells treated with the methanolic extract of P. tetrastromatica (IC₅₀ value of 125 ± 2.03 µg/ml) compared with the vehicle control (Figure 1). No cytotoxicity was observed in extract-treated 184B5 cells. MCF-7 and 184B5 cells were treated with doxorubicin as a positive control. The drug showed IC₅₀ values at 0.21 ± 1.25 µg/ml and 0.02 ± 1.97 µg/ml in MCF-7 and 184B5 cells, respectively.

Figure 1. Percentage of inhibition of MCF-7 cells versus different concentrations of the methanolic extract of Padina tetrastromatica. MCF-7 cells were treated with various concentrations of the extract (100, 200, 300, 400 and 500 µg/ml) or 0.1% dimethyl sulfoxide (DMSO; vehicle control) for 48 h. Cell viability of MCF-7 cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

From the results obtained, the methanolic extract of P. tetrastromatica is more appropriate for use as a chemotherapeutic agent than the standard drug, doxorubicin. This is because treatment with doxorubicin in normal breast cells (184B5) results in high toxicity in the cells. The presence of active phytochemicals such as quinine and alkaloids contributes to the antiproliferative activity of the methanolic extract of P. tetrastromatica. Interference in DNA and RNA replication and mitochondrial oxidative pathways, as well as the formation of peroxide, superoxide and hydroxyl radicals in the cell, are responsible for the antiproliferative activity conferred by quinine derivatives. In contrast, antiproliferative activity of alkaloids is due in part to the inhibition of mitotic spindle fibre formation, which is required for cell division (Solanki et al. 2008).

Alkaline comet assay

The methanolic extract of P. tetrastromatica exerted significant dose-dependent genotoxic activity on H₂O₂-challenged MCF-7 cells, and antigenotoxic and cytoprotective effects (p < 0.05) on H₂O₂-challenged 3T3 cells with no evidence of genotoxicity on the negative control when evaluated by the comet assay. Incubation of both cell lines with the extract induced greater DNA damage in the MCF-7 cell line than the normal 3T3 cells, at all concentrations tested, compared with the negative control. Doxorubicin induced greater DNA damage in 3T3 cells than in MCF-7 cells. In treated 3T3 cells, the length of the comet tail and the percentage of DNA damage (genotoxicity) were inversely proportional to increasing concentration of the methanolic extract of P. tetrastromatica, while the opposite was true for treated MCF-7 cells. In contrast, the percentage of DNA protection (cytoprotective effect) in treated 3T3 cells increased dose-dependently with the extract concentration, while the reverse was true for treated MCF-7 cells (Figures 2–4).

Figure 2. Comet tail length of the extract-treated 3T3-L1 and MCF-7 cells relative to hydrogen peroxide (H₂O₂)-treated and untreated negative controls. 3T3-L1 and MCF-7 cells were treated with various concentrations of the extract (5, 10, 15, 20 and 25 µg/ml) or 0.2 µg/ml of doxorubicin, as the positive control. Untreated cells and cells treated with 100 mM of H₂O₂ served as negative controls. Data are shown as mean ± SD. *p < 0.05, **p < 0.01.

Figure 3. Percentage of DNA damage in extract-treated 3T3-L1 and MCF-7 cells relative to hydrogen peroxide (H₂O₂)-treated and untreated negative controls. 3T3-L1 and MCF-7 cells were treated with various concentrations of the extract (5, 10, 15, 20 and 25 µg/ml) or 0.2 µg/ml of doxorubicin, as the positive control. Untreated cells and cells treated with 100 mM of H₂O₂ served as negative controls. Data are shown as mean ± SD. *p < 0.05, **p < 0.01.

Figure 4. Percentage of DNA protection in extract-treated 3T3-L1 and MCF-7 cells relative to hydrogen peroxide (H₂O₂)-treated and untreated negative controls. 3T3-L1 and MCF-7 cells were treated with various concentrations of the extract (5, 10, 15, 20 and 25 µg/ml) or 0.2 µg/ml of doxorubicin, as the positive control. Untreated cells and cells treated with 100 mM of H₂O₂ served as negative controls. Data are shown as mean ± SD. *p < 0.05, **p < 0.01.

In this study, the alkaline comet assay was used to evaluate the genotoxic and cytoprotective effects of the methanolic extract of P. tetrastromatica by measuring single and double strand breaks in DNA. Migration of the broken ends of the negatively charged DNA molecule in the electric field towards the anode leads to the formation of comets (Collins 2004). Evaluation of the potential genotoxicity of natural products is vital because DNA damage may result in critical mutations and, hence, in an increased risk of cancer development. Direct interaction of DNA with a DNA-reactive agent is one of several pathways that cause primary DNA damage, and can be measured as an endpoint in the in the comet assay. These include DNA strand breaks which reflect repair incisions, and alkali-labile sites may also occur owing to other less direct events such as cytotoxicity (Celik 2012). The present findings showed that treatment of 3T3-L1 cells with the methanolic extract of P. tetrastromatica significantly decreased the comet tail length and percentage of DNA damage, implying the antigenotoxicity of the extract on the cells. Treatment of the MCF-7 cell line with the extract induced DNA damage in a dose-dependent manner (Figures 2 and 3). The cancer chemotherapy drug doxorubicin induced greater DNA damage in the normal 3T3 cells than in the MCF-7 cell line, compared with treatment with the methanolic extract of P. tetrastromatica. In addition, the methanolic extract also demonstrated cytoprotective effects on 3T3-L1 cells with increasing extract concentrations. In contrast, treatment of MCF-7 cells with the extract demonstrated minimal cytoprotectivity (Figure 4). Therefore, this extract is more effective in inducing DNA damage in MCF-7 cells at concentrations above 10 µg/ml, compared with doxorubicin. On the other hand, treatment of the extract in normal 3T3 cells at all tested concentrations induced higher cytoprotective effects than doxorubicin. Thus, it can be postulated that the methanolic extract of P. tetrastromatica is more efficient in inducing genotoxicity and cytoprotectivity in MCF-7 and 3T3 cells, respectively.

Flavonoids are phytochemicals, which are considered natural antioxidants. The conferment of cytoprotective effects on 3T3-L1 cells against H₂O₂-induced oxidative stress upon treatment with the extract may be due to the presence of flavonoids (Zhao & Zhang 2009). In accordance with the findings reported by Aherne et al. (2007), the antigenotoxic and cytoprotective effects of the P. tetrastromatica methanol extract could be due in part to their reducing power and free radical scavenging efficiency as a consequence of their phenolic and/or non-phenolic constituents.

Conclusions

To the best of the authors' knowledge, this is the first report of the antiproliferative and antigenotoxic effects of the methanolic extract of P. tetrastromatica in MCF-7 cells, as well as the cytoprotective properties of the extract on 3T3 cells. Overall, this study provides important findings pertaining to the potential of P. tetrastromatica as a beneficial chemotherapeutic agent for breast cancer treatment. Further studies involving the molecular mechanism of action of the isolated compounds, and in vivo pharmacological and toxicological investigations are essential for thorough comprehension of the medicinal application of P. tetrastromatica.

Disclosure statement

No potential conflict of interest was reported by the authors.

ORCID

M.S. Kanthimathi http://orcid.org/0000-0002-3404-3178

Additional information

Funding

This study was supported by University of Malaya Research University grants [RG004/09AFR, RG341/11HTM, PV019/2012A] and an FRGS Fundamental Research Grant, FRGS, from the Ministry of Higher Education, Malaysia [FP065/2007C].