Antioxidant and Antiproliferative Activities of Marine Algae, Gracilaria edulis and Enteromorpha lingulata, from Chennai Coast

Murugan, K.; Iyer, V.V.

INTRODUCTION

Marine algae are a group of aquatic autotrophic organisms that are broadly classified as Chlorophyta (green), Rhodophyta (red seaweeds) or Phaeophyta (brown seaweeds), based on the presence of photosynthetic pigments. Seaweeds are widely included in Japanese and Chinese diet and traditional medicine (Fujiwara-Arasaki et al., 1984; Chengkui et al., 1984) and have developed biological molecules and approaches which help them survive in their harsh and extreme environment. Seaweed sulfated polysaccharides like fucoidan, carrageenan (red seaweed) or algin (brown seaweed) are rich sources of soluble fibers, which have been reported to perform a varied range of functions such as antioxidant, antimutagenic, anticoagulant and antitumor (De Souza et al., 2007; Smit, 2004; Madhusudan et al., 2011). Caulerpenyne from the Caulerpa spp. (green algae) (Fischel et al., 1995) and polysaccharides such as fucoidan and laminarin from brown seaweeds have also been reported to show antitumor activity (Yamasaki-Miyamoto et al., 2009). Therefore, the objective of the present study was to explore marine algae for new compounds possessing antioxidant and antiproliferative activities, so that these compounds could be used as leads for making more potent, selective and less toxic drugs with better therapeutic indices. Gracilaria edulis and Enteromorpha lingulata are edible marine algae. However, in Phillipines, three fatal poisoning cases have been reported during 2002-2003 due to ingestion of G. edulis and Acanthophora spicifera (Yotsu-Yamashita et al., 2004). There are no reports, to date, on the in vitro antiproliferative activity of G. edulis. To the best of our knowledge, this is the first report on investigation into antioxidant and antiproliferative activities of Enteromorpha lingulata and antiproliferative activity of Gracilaria edulis.

MATERIALS AND METHODS

Sample collection: Enteromorpha lingulata and Gracilaria edulis were collected from the Chennai coast of Tamil Nadu, India and identified by Dr. Baluswami, Madras Christian College, Chennai.

Chemicals: Ascorbic acid, beta-carotene, BHA (butylated hydroxyanisole), DPPH (2, 2-diphenyl-1-picrylhydrazyl), gallic acid, linoleic acid, DMEM growth medium and resazurin sodium salt were purchased from Sigma-Aldrich, India. Quercetin was purchased from Fluka, India. All other chemicals were purchased from SRL, India. Bromophenol blue, trichloroacetic acid (TCA) and other cell culture materials were purchased from HiMedia, India. TLC plates (TLC Silica gel 60 F₂₅₄) were purchased from Merck, India. Cell lines were obtained from the National Centre for Cell Sciences in Pune, India.

Algal extract preparation: The samples were washed thoroughly in fresh water and subsequently shade-dried and powdered (Vinayak et al., 2011). Five grams of the dry powder was extracted twice for 16 h each time, with 50 mL of each solvent (methanol, chloroform or ethyl acetate separately) in a rotary shaker at 37°C. The extracts were filtered and the solvent was removed completely by using a rotary evaporator (Buchi Rotavapor R215, Switzerland). The crude extract was stored at -20°C and reconstituted in methanol for assays.

Antioxidant assays
DPPH radical-scavenging activity: The free radical-scavenging potential of the algal extracts was analyzed according to previously reported methods (Zubia et al., 2009; Butkhup and Samappito, 2011; Jain et al., 2011; Amatya and Tuladhar, 2011; Hanachi et al., 2006). The concentrations of M (methanol), C (chloroform) and E (ethyl acetate) extracts taken for the experiment were 10, 50 and 100 μg mL^-1. A methanolic solution of DPPH (200 μL, 20 mg L^-1) was added to 22 μL of each extract in a 96 well plate. Ascorbic acid was used as a positive control while 200 μL of DPPH solution plus 22 μL methanol without any extract was used as a control, to calculate the extent of scavenging. The plate was incubated in the dark for 2 h at room temperature and the absorbance measured at 492 nm spectrophotometrically (Fluostar Optima BMG Labtech Gmbh).

% DPPH radical scavenging activity was determined by the formula:

Where:
A_t	=	Absorbance with extract
A_c	=	Absorbance of control (200 μL of DPPH, 22 μL of methanol)

Beta-carotene bleaching assay: The antioxidant potential of marine algae was measured by modifying the beta-carotene bleaching assay described earlier (Zubia et al., 2009; Duan et al., 2006; Amatya and Tuladhar, 2011; Hanachi et al., 2006). About 210 μL of a solution of beta-carotene (1 mg mL^-1) in chloroform was taken in a round bottom flask containing 5 μL of linoleic acid and 42 μL of Tween-20. The chloroform was removed in a rotary evaporator at 40°C and 10 mL of distilled water was added to form an emulsion with continuous shaking. Approximately 200 μL of the above emulsion was added to 50 μL of extracts (100 μg mL^-1) taken in a 96 well microplate. Emulsion without beta-carotene was used as a blank, BHA was used as a positive control and wells containing the beta-carotene emulsion with methanol instead of extract served to calculate the extent of bleaching. The plate was immediately read at 450 nm (0 h) and after 2 h of incubation at 50°C in the dark. Antioxidant activity (AA) was measured by using the formula:

Where:
A_t0 and A_t2	=	Absorbance with extracts measured at 0 and 2 h, respectively
A_c0 and A_c2	=	Absorbance of control (beta-carotene-containing emulsion and methanol instead of extract) measured at 0 and 2 h, respectively

Total reducing activity: The reducing power of marine algae was determined by using the assay described earlier (Zubia et al., 2009; Ashawat et al., 2007). To 200 μL of the M, C and E extracts, 200 μL of phosphate buffer (200 mM, pH 6.6) and 200 μL of 1% potassium ferricyanide solution were added. The mixture was incubated at 50°C for 30 min and 200 μL of 10% TCA solution added after the mixture had cooled down. After mixing, 125 μL of the above mixture was transferred to a 96 well plate and 20 μL of 0.1% ferric chloride solution was added. The formation of a Prussian blue complex indicated the reducing power of the samples. The absorbance was measured at 620 nm in a plate reader (Fluostar Optima BMG Labtech GmbH). Ascorbic acid was used as positive control and the reaction mixture with methanol instead of the extract was used as (negative) control. The total reducing activity was determined by using the formula:

Where:
A_c	=	Absorbance of control (reaction mixture with methanol instead of extract)
A_t	=	Absorbance with extracts

Total phenolic content: The total phenolic content present in G. edulis and E. lingulata extracts was determined by using the Folin-Ciocalteu method described earlier (Masaldan and Iyer, 2011). Gallic acid (1 mg mL^-1) in methanol was used as a standard in concentrations ranging from 2 to 10 μg mL^-1. To 10 μL of standard/extracts (1 mg mL^-1) in a 2 mL tube were added 25 μL of the Folin-Ciocalteu’s phenol reagent and 50 μL of 25% sodium carbonate solution. The mixture was vortexed and the volume was made up to 1 mL with distilled water. The tubes were then incubated in the dark for 1 h at room temperature and the absorbance was measured at 725 nm in a spectrophotometer (Beckman Coulter Du 730). Quercetin (1 mg mL^-1) in methanol was used as a positive control.

Thin layer chromatography: Thin layer chromatography was used to qualitatively determine the antioxidant activity of selected marine algae by the method described earlier (Masaldan and Iyer, 2011; Jain et al., 2008). Methanolic solutions of M, C and E extracts were spotted on precoated silica gel aluminium sheets. These extracts were resolved separately by using the following three solvent systems listed in the order of increasing polarity: BEA benzene/ethanol/ammonium hydroxide (90:10:1), CEF-chloroform/ethyl acetate/formic acid (5:4:1) and EMW-ethyl acetate/methanol/water (40:5.4:5). The chromatograms obtained were further analyzed with iodine vapor and DPPH solution (0.2% in methanol).

Cell culture: A549 (lung adenocarcinoma) cells were grown in DMEM growth medium and HCT15 (colon adenocarcinoma) cells were grown in RPMI growth medium at 37°C in a 5% CO₂ atmosphere. The medium was supplemented with 10% FBS and penicillin (100 U mL^-1) and streptomycin (100 μg mL^-1). For experiments, A549 (2500) cells and HCT15 (4000) cells in 200 μL media well^-1 were seeded in a 96 well plate.

Growth inhibition assay: The antiproliferative potential of the marine algal extracts was studied in the above cell lines by using a resazurin-based assay (Masaldan and Iyer, 2011; Anoopkumar-Dukie et al., 2005). Resazurin is the active component of the blue redox dye, Alamar Blue^® which is converted into pink resorufin by the oxido-reductases in the cytoplasm, mitochondria and microsomes of viable cells. Cells were seeded in two 96 well plates-T₀ and T_i, with media blank and untreated control and incubated at 37°C in a 5% CO₂atmosphere. After 24 h, the T₀ plate was treated with 10% resazurin (1 mg mL^-1 in 50 mM PBS) for 4 h and the absorbance was measured at 584 nm and 620 nm. At the same time, the T_i plate was treated with 100 μg mL^-1 of M, C and E extracts (in DMSO) and Doxorubicin (positive control). Medium blank with and without 0.5% DMSO (vehicle blank) as well as untreated (negative) control with and without 0.5% DMSO were also included in the T_i plate. After 48 h, the T_i plate was treated with resazurin like the T₀ plate and the absorbance was measured.

% of growth inhibition (GI) was calculated using the formula:

Image for - Antioxidant and Antiproliferative Activities of Marine Algae, Gracilaria edulis and Enteromorpha lingulata, from Chennai Coast

Where:
T₀	=	OD_584-620of untreated cells after 24 h
T_i	=	OD_584-620of cells treated with test compound after 48 h and
C	=	OD_584-620of negative control cells after 48 h (blanks subtracted from all)

Statistical analysis: All experiments were performed in triplicate (n = 3) and expressed as Mean±SEM. Means were compared with SPSS v 16.0 software for Windows (SPSS Inc., Chicago, IL) by using one-way ANOVA followed by the Tukey’s post-hoc test. p<0.05 indicated statistical significance. Pearson correlation coefficient (r) was calculated (p<0.05) to assess the strength of the linear relationship between two variables.

RESULTS

ANOVA analysis indicated that the difference between the group means was statistically significant (p<0.05). However, except for a high positive correlation observed between the means for GI values in HCT15 and TPC values, none of the other Pearson correlation coefficients were significant for any of the other group mean pairs (p>0.05). Tukey’s HSD post-hoc test showed that the differences between the following group mean pairs were statistically significant (p<0.05): DPPH radical scavenging and BCB assay group means; DPPH radical scavenging and A549 GI group means; A549 GI and TRA group means; A549 GI and BCB group means; A549 GI and HCT15 GI group means; HCT15 GI and BCB group means and BCB and TPC group means.

Antioxidant assays
DPPH radical scavenging activity: The antioxidant potential of marine algae was measured in terms of their ability to scavenge the stable DPPH radical (Fig. 1). Enteromorpha lingulata C and E extracts (100 μg mL^-1) showed higher activity of 15.85 and 15.61%, respectively. The M extracts of both E. lingulata and G. edulis (100 μg mL^-1) were almost equal in their DPPH radical-scavenging activity (13.59 and 14.84%, respectively). Lower activity was observed in G. edulis with 100 μg mL^-1 C and E showing 9.01 and 8.47%, respectively. Tukey’s HSD post-hoc test showed that the differences between the DPPH radical scavenging and BCB assay group means and between the DPPH radical scavenging and A549 GI group means were statistically significant (p<0.05).

Thin layer chromatography: The chromatograms showed the presence of organics and DPPH free radical-scavenging molecules when exposed to iodine vapor and DPPH solution, respectively (Fig. 2, 3). All organics appeared as brown spots and the scavenging of the DPPH radical was seen in the form of disappearance of purple color at some spots.


Fig. 1:	DPPH radical scavenging activity of the G. edulis and E. lingulata extracts; M: Methanolic extract, C: Chloroform extract, E: Ethyl acetate extract


Fig. 2(a-b):	TLC analysis of G. edulis: methanol (M), chloroform (C) and ethyl acetate (E) extracts using different eluent systems; A: BEA, B: CEF and C: EMW (from left to right) treated with (a) iodine vapor for all organics and (b) DPPH solution for radical scavenging activity


Fig. 3(a-b):	TLC analysis of E. lingulata: methanol (M), chloroform (C) and ethyl acetate (E) extracts using different eluent systems; A: BEA, B: CEF and C: EMW (from left to right) treated with (a) iodine vapor for all organics and (b) DPPH solution for radical scavenging activity

Our results suggest that DPPH radical-scavenging activity was associated with the more polar components in all extracts because the R_f values of these components increased with increasing polarity of the resolving system. For all three extracts of both algae, E. lingulata extracts showed strong DPPH radical-scavenging activity in TLC and C and E extracts of G. edulis seemed to have stronger DPPH radical-scavenging activity on the TLC plates than the M extract.

Beta-carotene bleaching assay: Antioxidant activity was also examined in terms of inhibition of beta-carotene bleaching by linoleic acid (Fig. 4). The highest inhibitory activity (60%) was found in G. edulis C extract whereas E extract had negligible activity at 100 μg mL^-1.


Fig. 4:	Beta-carotene bleaching inhibition by G. edulis and E. lingulata extracts; M: Methanolic extract, C: Chloroform extract, E: Ethyl acetate extract


Fig. 5:	Total reducing activity of the G. edulis and E. lingulata extracts; M: Methanolic extract, C: Chloroform extract, E: Ethyl acetate extract

G. edulis M and E. lingulata M, C and E extracts had almost similar inhibitory activity of around 40%. Tukey’s HSD post-hoc test showed that the differences between A549 GI and BCB group means; HCT15 GI and BCB group means and BCB and TPC group means were statistically significant (p<0.05).

Total reducing activity: The total antioxidant potential of marine algae was also determined by their reducing power and the results are shown in Fig. 5. The reducing power of the extracts showed concentration dependence with G. edulis C and E extracts having higher values of 38.16 and 37.54% at 200 μg mL^-1, respectively. The C and E extracts of E. lingulata also had similar activity (32.21 and 37.41%, respectively) whereas M extracts of both algae showed lower values: G. edulis M: 17.64%; E. lingulata M: 13.64%. Tukey’s HSD post-hoc test showed that the differences between A549 GI and TRA group means were statistically significant (p<0.05).

Total phenolic content: As phenols from natural sources are considered to be good antioxidants, the antioxidant potential observed in the two marine algae in this study was postulated to be linked to the phenolic constituents.


Fig. 6:	Gallic acid standard curve for total phenolic content

Table 1:	Total phenolic content

M: Methanolic extract, C: Chloroform extract, E: Ethyl acetate extract, Quercetin was used as positive control with GAE 8.25±0.15 at 10 μg mL^-1. All values are represented as Mean±SEM (n = 3)

The phenolic content of the algal extracts was measured in terms of Gallic Acid Equivalents (GAE) from the regression equation y = 0.1249 x+0.0319 of the gallic acid standard curve (Fig. 6). The E extract of E. lingulata had the highest phenolic content of 0.54 μg mL^-1 GAE, whereas G. edulis M extract had the least phenolic content of 0.14 μg mL^-1 GAE for 1 mg mL^-1 of the extracts (Table 1). Tukey’s HSD post-hoc test showed that the differences between TPC and BCB group means were statistically significant (p<0.05). Also, a high positive correlation (Pearson correlation coefficient) was observed between the means for GI values in HCT15 and TPC value, at p<0.05, suggesting a link between the TPC and GI observed in HCT15 cells for all these extracts (in particular, E of E. lingulata).

Growth inhibition assay: The antiproliferative activity of G. edulis and E. lingulata M, C and E extracts showed different trends in HCT 15 and A549 cell lines (Fig. 7). The E. lingulata E extract showed maximal inhibition in the HCT15 cell line of about 19.37% followed by C extract (15.66%); the M extract showed no growth inhibition. G. edulis (E, C and M) extracts showed low growth inhibition of about 7.12, 4.22 and 0.79%, respectively. Neither alga had any growth inhibitory effects on the A549 cell line (Fig. 7). Tukey’s HSD post-hoc test showed that the differences between DPPH radical scavenging and A549 GI group means; A549 GI and TRA group means; A549 GI and BCB group means; A549 GI and HCT15 GI group means and HCT15 GI and BCB group means were statistically significant (p<0.05).


Fig. 7:	Growth inhibition by G. edulis and E. lingulata-M, C, E extracts in HCT15 and A549 cell lines at 100 μg mL^-1

DISCUSSION

Reactive oxygen species (free radicals) are highly reactive chemical molecules that are formed as by-products of normal metabolism and whose levels increase dramatically due to various environmental stresses or damage. They finally cause great damage to the cells by interacting with vital cellular components like DNA or the cell membrane. Antioxidants play an important role in preventing cellular damage-a major feature of cancer, ageing and various other diseases-by neutralizing these free radicals (Chanda et al., 2011; Lompo et al., 2007). Several molecules and antioxidants from natural sources, such as mushrooms and higher plants have been reported and used for therapeutic benefit (Dimitrios, 2006; Samchai et al., 2009; Anokwuru et al., 2011).

Cancer is the most commonly occurring “disease” worldwide of which, lung and colon cancers are widely prevalent (Garcia et al., 2007). Most of the anticancer drugs in clinical use possess deleterious side effects. Hence, the discovery of novel anticancer drug leads with better toxicological and pharmacokinetic profiles is of great importance. Phytochemicals, such as polyphenols, are considered to be good chemo-preventive agents for colorectal cancer (Sale et al., 2005). Several anticancer drugs from plant sources, such as taxol, vinblastine, vincristine and etoposide phosphate, have been in use for the treatment of cancer (Da Rocha et al., 2001). Antioxidant and antiproliferative studies have been performed on various marine algae (seaweeds) all over the world (Vinayak et al., 2011; Zubia et al., 2009; Yuan and Walsh, 2006; Ismail and Hong, 2002). In India too, reports have demonstrated antioxidant activity of marine algae found along the Indian coastline but only few reports describe antiproliferative activity (Delma et al., 2008; Sachindra et al., 2010; Ganesan et al., 2008). Hence, the current study has been conducted to determine the antioxidant and antiproliferative activities of M, C and E extracts of two marine algae from the Chennai coast in Tamil Nadu, India.

Gracilaria edulis and Enteromorpha lingulata are edible marine algae. There are no reports, to date, on the in vitro antiproliferative activity of G. edulis although three fatal poisoning cases have been reported during 2002-2003 in Phillipines, due to ingestion of G. edulis and Acanthophora spicifera (Yotsu-Yamashita et al., 2004). Our TLC results suggest that the relatively polar compounds that have been extracted by all three solvents (methanol, chloroform, ethyl acetate) from both algae are likely to be strong contributors to the observed DPPH radical scavenging activity. While it is difficult to correlate the qualitative results of TLC with the quantitative ones of the DPPH radical-scavenging assay, E. lingulata extracts showed strong DPPH radical-scavenging activity in both TLC and the assay. M extract of G. edulis showed greater DPPH radical-scavenging activity at all concentrations in the assay in contrast to the results of TLC analysis. In contrast, Devi et al. (2008) had reported a value of 25% for the DPPH radical-scavenging activity of the methanolic extract of G. edulis at 100 μg mL^-1. However, they used different concentrations of DPPH and a different incubation period in their assay. No reports were found on the antioxidant activity of E. lingulata.

In contrast to the higher activity seen for E. lingulata in other assays, the BCB assay showed that G. edulis C extract had the highest inhibitory activity of about 60% at 100 μg mL^-1, with the others being almost comparable to the positive control, BHA (100 μg mL^-1, 39%), except for E extract of G. edulis which showed slight pro-oxidant activity.

Total reducing activity of the E extract of E. lingulata was the highest of all, followed by C and E extracts of G. edulis and the C extract of E. lingulata, with the M extracts of both algae showing the least total reducing activity. Total phenolic content was higher for E. lingulata (E>C>M for both algae).

Reports suggest that the widely prevalent cancer of the colon can metastasize into other organs, such as the lungs and vice versa (Dishop and Kuruvilla, 2008). Hence, the antiproliferative effects of the algae were tested on HCT15 (colon carcinoma) and A549 (lung adenocarcinoma) cells. The results show no growth inhibitory effects of any of the extracts on A549 cells; in fact, the E. lingulata extracts (M>C>E) showed varying growth-promoting effects. However, the C and E extracts (E>C) of E. lingulata showed very modest growth inhibition (15.7 and 19.4%) in HCT15 cells. The M, C, E extracts of G. edulis and the M extract of E. lingulata showed very negligible or no inhibitory effects on the growth of HCT15 cells. A higher Pearson correlation coefficient (>0.8) between TPC and HCT15 GI means suggests a role for phenolic compounds in the antiproliferative activity of the E extract of E. lingulata.

CONCLUSION

In summary, the above results suggest that the compounds extracted by methanol, chloroform and ethyl acetate into M, C and E extracts of both algae show different levels of antioxidant activities. Regardless of the differences observed for the various assays, it appears that the relatively more polar components extracted from Enteromorpha lingulata by ethyl acetate and chloroform showed higher antioxidant (DPPH radical-scavenging, TRA) activities than the others. In contrast, the methanolic (M) extracts of both algae, especially of G. edulis, showed the lowest antioxidant activities (DPPH, TRA). The similar trend of total phenolic content (E, C>M and E. lingulata>G. edulis) warrants further investigation into the nature of the compounds in the E and C extracts of E. lingulata that have shown the observed antioxidant and antiproliferative activities (in HCT15 cells).

ACKNOWLEDGMENT

The authors would like to thank the Department of Science and Technology (DST), India, for financial support. We are also grateful to Dr. Baluswami, Madras Christian College, Chennai, for his valuable help in identifying the algae and to Ms. S. Sowmya, VIT University, Vellore, for her valuable inputs into the statistical analysis.