Anticancer and antioxidant effects of selected Sri Lankan marine algae

For the investigation of bioactive components from Sri Lankan seaweeds, three species of red algae (Chondrophycus ceylanicus, Gelidiella acerosa, Gracilaria corticata), two species of green algae (Chaetomorpha crassa, Caulerpa racemosa) and one species of brown algae (Sargassum cassifolium) were evaluated. For the bioactivity evaluations, total phenol content (TPC), free radical scavenging activity using electron spin resonance (ESR) spectroscopy and anticancer activity against different cancer cell lines including a human promyelocytic leukemia (HL-60), a human lung carcinoma (A549) and a mouse melanoma (B16F10) were assessed in vitro. Among the extracts, C. racemosa showed significantly higher radical scavenging activity against DPPH (34.34 %), alkyl (85.17 %) and hydroxyl radicals (81.16 %), respectively. The MTT assay confirmed that all the extracts were not cytotoxic at 50 and 100 μg/mL concentrations against vero cells in vitro. In addition, a significant cancer cell growth inhibitory effect (IC 50 value 30.17 μg/mL) was observed by C. racemosa methanol extract against HL-60 cells and it was the highest anticancer effect compared to the other extracts. Apoptotic body formation in HL-60 cells and the accumulation of DNA in sub-G 1 phase were determined by C. racemosa extract in a dose-dependent manner. This study is the first report of Sri Lankan seaweeds with the potential of pharmacological effects including antioxidant and anticancer activity in vitro.


INTRODUCTION
At present, there is a great interest on bioactive natural products obtained from marine organisms.The large and structurally diverse array of marine-derived natural resources (Aneiros & Garateix, 2004), can be described as the largest remaining reservoir of secondary metabolites to be evaluated for future therapeutic needs.Marine seaweeds are considered as rich sources of secondary metabolites, which are considered as key ingredients for pharmaceutical applications.Over many years, marine natural products have been used as the lead compounds for drug discovery all over the world (Samarakoon et al., 2013).
The Sri Lankan coastal waters are rich in marine flora and the most frequent seaweeds have been identified by Boergesen (1936).In fact, the earliest marine algae collection has been reported in early 19 th century by Barton (1903).Baldwin (1991) has reported on 440 taxa of marine algae belonging to 148 genera.More recently, a comprehensive study of marine seaweeds of Sri Lanka has been carried out (Coppejans et al., 2009).There has been a tradition of consuming seaweeds from the Puttalam Lagoon area (Jayasuriya, 1987).Gracilaria species is a popular food item among fishermen and they believe that it has health promoting effects.Among the Sri Lankan seaweeds, Saragassum species (brown algae) are the most abundant type of algae found in the coastal area (Durairatnam, 1961).In addition, Laminaria, Fucus, Ascophyllum and Tubinaria species are also found in the Sri Lankan waters.Alginic acid has been extracted from seaweeds in 1960/1970 and the cultivation of seaweeds has also been reported (Arumugam et al., 1981).
However, bioactivities of the Sri Lankan seaweeds have not been studied extensively and only a few

December 2014
Journal of the National Science Foundation of Sri Lanka 42 (4) reports have been found in the literature.Among them, Premekumara et al. (1995) have reported the postcoital contraceptive mechanism of the crude extract of Sri Lankan marine red algae, Gelidiella acerosa.As a consequence of the tropical climatic conditions in the seas around Sri Lanka such as extreme salinity levels, temperature variations, low light intensities and nutrient deficient habitats, changes would result in the ecological and physiological properties of the marine seaweeds.Therefore, these algae are a rich source of bioactive secondary metabolites and functional ingredients.
In this study, the main objective was to evaluate the antioxidant and anticancer activities of some selected marine algae from Beruwela, Sri Lanka.In addition, the marine algal species with potent anticancer activity of apoptotic body inducing effect against human promyelocytic leukemia cell line (HL 60) was determined.

Seaweed collection and preparation of extracts
Fresh marine seaweeds were collected from the sampling sites at Beruwela coral reef in the Southern Province of Sri Lanka.Collected samples were washed well to remove salt, epiphytes and sand.The seaweeds were identified (Figure 1) by one of the co-authors Dr Kalpa W. Samarakoon based on the descriptions on ABC Taxa (Coppejans et al., 2009) and the herbarium specimens were kept in the research laboratory, Department of Chemistry, University of Colombo.All the samples were stored at -70 °C and air dried at room temperature for 24 h.The frozen and air dried samples were lyophilized and homogenized using a laboratory mechanical blender before extraction.Each homogenized sample was mixed with 80 % methanol and subjected to sonication (ultrasound assisted extraction) at 25 °C for three 90 min periods.The extracts were filtered under vacuum using Whatman No.1 (Whatman Ltd., Maidenstone, England) filter paper and the solvent was evaporated using a rotary evaporator (Fisher Scientific, Loughborough, UK).The collected crude methanolic extracts were used for the in vitro bioassays.

Determination of total phenolic content (TPC)
The total phenolic content of the samples were determined according to Chandler and Dodds (1983).One milliliter of (0.1 mg mL -1 ) sample was mixed in a test tube containing 1 mL of 95 % ethanol, 5 mL of distilled water and 0.5 mL of 50 % Folin-Ciocalteu reagent.The mixture was allowed to react for 5 min, and then 1 mL of 5 % Na 2 CO 3 was added, mixed thoroughly and placed in the dark for 1 h.The absorbance was measured at 725 nm using a UV-vis spectrometer.A gallic acid standard curve was obtained for the calibration of phenolic content.

Cell culture
HL-60 and A549 cells were grown in RPMI-1640 medium.Further more B16F10 and vero cells were cultured in DMEM.Both culture media were supplemented with 1 % antibiotic (100 mL -1 penicillin, 100 μg mL -1 of streptomycin) and 10 % FBS.The cells were incubated and maintained in an atmosphere of 5 % CO 2 at 37 °C.The cells were sub-cultured every 2 days and exponential phase cells were used throughout the experiments.

Determination of cell viability (MTT assay)
The sample cytotoxicity was evaluated on vero cells and cell growth inhibitory activity of the methanol extracts were assessed by colourimetric MTT assay against cancer cells (HL-60, B16F10, and A549) with different concentrations (50, 100 and 200 μg mL -1 ).Suspension cells (HL-60 cells) and attached cells (B16F10 and A549) were seeded in a 96-well plate at a concentration of 2 × 10 4 cells mL −1 and incubated for 24 h and treated with samples with different concentrations and further incubated for 24 h before MTT treatment.MTT stock solution (50 µL; 2 mg mL -1 in PBS) was added to each well to achieve a total reaction volume of 250 µL.After 3 h of incubation, the plates were centrifuged for 10 min at 2000 rpm and the supernatants were aspirated.The formazan crystals in each well were dissolved in DMSO and the amount of purple formazan was assessed by measuring the absorbance at 540 nm.

Nuclear staining with Hoechst 33342
The cell permeable DNA dye, Hoechst 33342 stain was used to examine the nuclear morphology of the cells.Hoechst 33342 is excited by uv light and emits blue fluorescence at 460 − 490 nm.This dye binds preferentially to the adenine-thymine (A-T) regions of the DNA.The stained nuclei were considered as viable.
In addition, the presence of DNA fragmentations and chromatin condensations were visualized as apoptosis (Lizard et al., 1995).HL-60 cells were placed in 24-well plates at a concentration of 2 × 10 4 cells mL −1 .The cells were then treated with different concentrations (50, 100 and 200 µg mL -1 ) of Caulerpa racemosa methanol extract and incubated for an additional 24 h.Next, Hoechst 33342 dye was added to the culture media at a final concentration of 10 µg mL -1 and the plates were incubated for an additional 15 min at 37 ˚C.The stained cells were observed under a fluorescent microscope equipped with a CoolSNAP-Pro colour digital camera in order to determine the degree of nuclear condensation.

Cell cycle analysis
The cell cycle analysis was performed to examine the proportion of apoptotic sub-G 1 hypodiploid cells as described by Nicoletti et al. (1991).The HL-60 cells were seeded on 6-well plates at a concentration of 2 × 10 5 cells mL −1 and treated with different concentrations (50, 100 and 200 µg mL -1 ) of C. racemosa methanol extract.The cells were harvested after 24 h, fixed in 1 mL of 70 % ethanol for 30 min at 4 ˚C, washed twice with PBS followed by incubation in the dark in 1 mL of PBS containing 100 µg of propidium iodide (PI) and 100 µg RNase A for 30 min at 37 ˚C.The flow cytometric analysis was performed with a FACS caliber flow cytometer (Becton-Dickinson, SanJose, CA, USA).The effect on the cell cycle was examined by changes in the percentage of cell distribution at each cell cycle phase, and assessed by histograms generated by the Quest and Mod-Fit computer programmes (Wang et al., 1999).

Statistical analysis
All data are expressed as mean ± standard deviation of three determinations.Statistical comparison was performed via a one-way analysis of variance followed by Duncan's multiple range test (DMRT).Probability values of less than 0.05 (p < 0.05) were considered as significant.

Total phenolic contents (TPC) and free radical scavenging activity (%)
The total phenolic content (TPC) of methanol extracts of selected Sri Lankan marine algae were determined by Folin-Ciocalteu method and the free radical scavenging activity (%) using electron spin resonance (ESR) spectroscopy (Table 1).The highest TPC (%) was observed as 5.13 (%) from Gracillaria corticata and the lowest TPC was recorded from Gelidiella acerosa as 3.64 (%).However, the reported TPC % was not significantly different among the species.Free radical scavenging activities were evaluated by ESR spectroscopic technique using three types of radicals including DPPH, hydroxyl and alkyl radicals.Among all the extracts at 1 mgmL -1 concentration, C. racemosa showed a significant average radical scavenging activity against DPPH (34.34 %), alkyl (85.17 %) and hydroxyl (81.16 %), respectively.In fact, the best alkyl radical scavenging activity was recorded as 91.36 % from Chondrophycus ceylanicus.In general, alkyl and hydroxyl radical scavenging activity (%) of the methanol extracts showed significant differences compared to DPPH radical scavenging activity (Table 1).

Sample cytotoxicity assessment against vero cells
Sample cytotoxicity effect against vero cells was determined by the MTT assay.The methanol extract of marine algae samples were incubated at 50, 100 and 200 μg mL -1 concentrations for 24 h with vero cells and the cell viability (%) was determined in vitro (Figure 2).All samples at 50 and 100 μg mL -1 concentrations showed a high cell viability (%) compared to the controls.Among the samples, C. ceylanicus, Chaetomorpha crassa, Sargassum cassifolium and Gelidiella acerosa indicated the least cytotoxicity on vero cells at 200 μg mL -1 concentration.The methanol extracts of C. racemosa and Gracillaria cortcata showed significant cytotoxic effects at 200 μg mL -1 concentration (Figure 2).

Control
December 2014 Journal of the National Science Foundation of Sri Lanka 42 (4) growth inhibitory activity of the cancer cells are listed in Table 2.According to the results, the lowest cancer cell growth inhibitory activity (IC 50 value 30.17 µg mL -1 ) was reported in C. racemosa.Therefore, the methanol extract of C. racemosa was used to determine the mechanism of anticancer activity against HL-60 cancer cells, and the apoptotic body formation and accumulation of DNA content percentage in the sub-G1 phase of the cell cycle of HL-60 were evaluated.

Apoptosis induced anticancer activity
The cell permeable DNA dye Hoechst 33342 staining was used to examine the nuclear morphology of the cells.The methanol extract of C. racemosa (CRM) at different concentrations (50 ~ 200 µg mL -1 ) was incubated with the HL-60 cells for 24 h, and the apoptosis induced nuclear morphology of HL-60 cells stained with Hoechst 33342

Growth inhibitory activity of Sri Lankan seaweed extracts against HL-60, B16F10 and A549 cancer cell lines
The cell viability (%) of the cancer cells HL-60, B16F10 and A549 were determined by MTT assay with incubation of different concentrations (50, 100, 200 µg mL -1 ) of methanol extract of seaweeds for 24 h.Observed results is shown in Figure 3 (A), (B) and (C), respectively.Among the cancer cells, HL-60 cells were significantly suppressed (p < 0.05) with the C. racemosa extract and C. ceylanicus extract dose-dependently compared to the control.In particular, the highest growth inhibitory activity on HL-60 cells was observed at 200 µg mL -1 of C. racemosa (Figure 3 A), which was approximately 95 %.The same concentration of C. racemosa showed a marked growth inhibitory activity against B16F10 and A459 cancer cells (Figure 3 B and C).The calculated IC 50 values for the

Induction effect of sub-G 1 contents in HL-60 cells
The inhibitory effects on the proliferation of HL-60 cells were evaluated by determining the sub-G 1 content population (%) with respect to the incubated different concentrations of CRM.According to the results, the cell cycle arrest and the accumulation of cells in the sub-G 1 phase increased in a concentration dependent manner [Figures 5 (B), (C) and (D)].Moreover, the apoptotic body formations were reflected in the accumulation of sub-G 1 content and further determined as 3.59, 7.18 and 20.13 % of sub-G 1 population against the CRM concentrations 50, 100 and 200 µg mL -1 , respectively compared to the control (Figure 4 E).These evidences suggest that CRM induce the cell death due to the apoptotic body formations.

DISCUSSION
Reactive oxygen species (ROS) are the major causative agents for oxidative stress in the human body.Some endogenous antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase protect the internal organs and tissues from oxidative damage (Ahn et al., 2004).Hence, antioxidants play an important role in the human body by reducing oxidative reactions.However, imbalances between the endogenous antioxidants and ROS cause serious health issues and disorders such as cancer, cardiovascular disease, hypertension, diabetes mellitus, inflammatory diseases, neurodegenerative diseases and ageing (Valko et al., 2007).Among them, cancer is the leading threat for the world population; the first-leading cause of death in economically developed countries and the secondleading cause of death in developing countries (Ezzati et al., 2002).Therefore, researchers are continually seeking a good source with potent antioxidant ability and cancer suppressing activity as an alternative for dietary supplements.
Among the marine organisms, marine algae are considered as a rich source of natural antioxidants (Ngo et al., 2011).In this study, the methanol extracts of some selected Sri Lankan seaweeds were evaluated to determine their medicinal properties.The most sensitive method for the free radical scavenging activity using electron spin resonance spectroscopy showed that the seaweed extracts have the profound free radical quenching effects.In particular, all the extracts showed the best hydroxyl radical scavenging activity (as higher than 75 % of scavenging effect) at the tested concentrations.The total phenolic content of the extracts did not give the supportive facts that correlate with the determined antioxidant activity.It is believed that a different kind of secondary metabolite could be involved for the antioxidant activity.
Finding cancer cure therapeutics from marine food sources would attract great attention from many scientists.Apoptosis is considered as a programmed cell death and characterized cell changes including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation (Kroemer et al., 1995).Apoptosis provides a conceptual framework to understand cancer genetics and cancer therapy (Lowe & Lin, 2000).In fact, the most cytotoxic anticancer agents induce apoptosis that will compel characteristic cell changes and diminishing cancer cells.In this study, C. racemosa was identified as the best species with apoptotic inducing potency against HL-60 cancer cells.Apoptosis is also an important biological mechanism that effectively plays a role in maintaining cellular integrity and homeostasis (Yan et al., 2008).This process regulates cell division and intrinsic suicide or programmed cell death (Chang & Yang, 2000).This confirms that the observed typical nuclei morphology characteristics induced with the nuclear condensation or DNA fragmentation and sub-G 1 DNA accumulations are dose-dependently related to CRM.
In addition, Ratnasooriya et al. (1991) have investigated the hypotensive activity of a marine red algae, Gracilaria sp.extracts on rats.The same group of researchers has identified the gastro-protective effects of the crude extracts of Jania sp. on ethanol induced gastric lesions in rats (Ratnasooriya et al., 1990).Moreover in another study, the extracts from macro algae collected from different coastal areas of Northern Sri Lanka have been tested for antibacterial activity and some phytochemicals including alkaloids, saponins, flavonoids, tannins and glycosides have been identified (Jayaseelan et al., 2012).A sphingosine derivative isolated from the red algae, G. acerosa hexane fraction has shown to have non-steroidal, anti progesterone contragestative activity as reported by Premakumara et al. (1996).In addition, human sperm mortality stimulation activity has been reported by a sulfono glycolipid isolated from G. acerosa (Premakumara et al., 2001).This is the first study to determine the antioxidant and anticancer

1 Figure 2 :
Figure 2: The effect on cell viability (%) of the methanol extracts of selected Sri Lankan seaweeds against vero cells after incubation at 24 hours and evaluated by MTT assay.Values are expressed as means ± SD in triplicate experiments.

Table 1 :
Free radical scavenging activity (%) and total phenolic content of Sri Lankan seaweed extracts a

Table 2 :
Cancer cell (HL-60) growth inhibition by methanol extracts from selected Sri Lankan seaweeds