Abstract

Seaweeds have a growing number of successful applications in the food industry, medicine and in the cosmetic industry, which increases the importance of evaluating their chemical composition. In the present study, three common Egyptian seaweeds (Dictyota dichotoma, Turbinaria decurrens and Laurencia obtusa) were collected from the Red Sea coast, Suez, Egypt. The chemical profile of the three seaweeds was studied beside the antioxidant activity of their extracts. The results indicated that the amount of carbohydrate was greater than the protein and lipid contents in the three seaweeds, with a natural richness in minerals and antioxidants besides considerable amounts of monounsaturated and polyunsaturated fatty acids, including Omega-3 and Omega-6 fatty acids. All essential amino acids for human were found in the three seaweeds, with significant amounts of aspartic and glutamic acids. Furthermore, the results of the antioxidant activity assays were consistent with the antioxidant contents (phenols, flavonoids, alkaloids, vitamin C, carotenoids) of each seaweed. D. dichotoma was the most valuable seaweed of the three species studied, due to its relatively high protein content of 7.28 ± 0.25%, moderate carbohydrate content of 25.35 ± 0.32%, and highest pigment and antioxidant contents. In conclusion, these three seaweeds, especially Dictyota dichotoma, have an interesting chemical composition with a prospective nutritional and pharmaceutical value.

Keywords:
Marine macroalgae; Chemical constituents; Amino acid profile; Fatty acid profile; Antioxidant activity

Resumo

Algas marinhas têm um número crescente de aplicações bem-sucedidas nas indústrias de alimentos, de produtos farmacêuticos e de cosméticos, o que aumenta a importância de se avaliar sua composição química. No presente estudo, três algas marinhas comuns no Egito (Dictyota dichotoma, Turbinaria decurrens e Laurencia obtusa) foram coletadas do Mar Vermelho, Suez, Egito. O perfil químico de cada alga marinha foi estudado como também a atividade antioxidante dos seus extratos. Os resultados indicaram que as quantidades de carboidratos foram maiores que os conteúdos de proteína e lipídeo nas três algas marinhas, com uma riqueza natural em minerais e antioxidantes, além de quantias razoáveis de ácidos graxos monoinsaturados e poli-insaturados, incluindo os ácidos graxos Omega-3 e Omega-6. Todos os aminoácidos essenciais para humanos foram encontrados nas três algas marinhas, com quantias significantes dos ácidos aspártico e glutâmico. Ademais, os resultados obtidos nos ensaios de atividade antioxidante foram de acordo com os conteúdos antioxidantes (fenóis, flavonoides, alcaloides, vitamina C e carotenoides) de cada alga marinha. Das três espécies de algas marinhas estudadas, D. dichotoma apresentou o maior valor devido ao seu conteúdo relativamente alto de proteína, de 7,28 ± 0,25%, ao conteúdo moderado de carboidrato, de 25,35 ± 0,32%, e conteúdos mais altos de pigmentos e antioxidantes. Em conclusão, as três algas marinhas, e especialmente Dictyota dichotoma, têm composições químicas interessantes, cujos valores apresentam perspectivas nutricionais e farmacêuticas.

Palavras-chave:
Macroalgas marinhas; Constituintes químicos; Perfil de aminoácidos; Perfil de ácidos graxos; Atividade antioxidante

1 Introduction

Marine macroalgae, known as seaweeds, are aquatic organisms very similar to plants, but they live in coastal regions. This group includes at least 8000 species (Lüning et al., 1990Lüning, K., Yarish, C., Kirkman, H., (1990). Seaweeds: Their environment, biogeography and ecophysiology. Chicester: Wiley.), which are divided into three categories; green, brown and red. Their classification is based mainly on the colour of the thallus, pigment composition, ultrastructure and biochemical features (Dawczynski et al., 2007Dawczynski, C., Schubert, R., & Jahreis, G. (2007). Amino acids, fatty acids, and dietary fibre in edible seaweed products. Food Chemistry, 103(3), 891-899. http://dx.doi.org/10.1016/j.foodchem.2006.09.041
http://dx.doi.org/10.1016/j.foodchem.200... ; Guiry, 2017Guiry, M. D. (2017). The seaweed site: Information on marine algae. Retrieved 2017, October 10, from http://www.seaweed.ie-/index.php.
http://www.seaweed.ie-/index.php... ). Several articles reported on the chemical composition of different seaweed species collected from different parts of the world, to evaluate their commercial and nutritional value (Renaud & Luong-Van, 2006Renaud, S. M., & Luong-Van, J. T. (2006). Seasonal variation in the chemical composition of Tropical Australian Marine Macroalgae. Journal of Applied Phycology, 18(3-5), 381-387. http://dx.doi.org/10.1007/s10811-006-9034-x
http://dx.doi.org/10.1007/s10811-006-903... ; Banerjee et al., 2009Banerjee, K., Ghosh, R., Homechaudhuri, S., & Mitra, A. (2009). Seasonal variation in the biochemical composition of red seaweed (Catenella repens) from Gangetic delta, northeast coast of India. Journal of Earth System Science, 118(5), 497-505. http://dx.doi.org/10.1007/s12040-009-0045-2
http://dx.doi.org/10.1007/s12040-009-004... ; Gressler et al., 2011Gressler, V., Fujii, M. T., Martins, A. P., Colepicolo, P., Mancini-Filho, J., & Pinto, E. (2011). Biochemical composition of two red seaweed species grown on the Brazilian coast. Journal of the Science of Food and Agriculture, 91(9), 1687-1692. PMid:21495035. http://dx.doi.org/10.1002/jsfa.4370
http://dx.doi.org/10.1002/jsfa.4370... ; Ahmad et al., 2012Ahmad, F., Sulaiman, M. R., Saimon, W., Yee, C. F., & Matanjun, P. (2012). Proximate compositions and total phenolic contents of selected edible seaweed from Semporna, Sabah, Malaysia. Borneo Science., 31, 74-83.; Rohani-Ghadikolaei et al., 2011Rohani-Ghadikolaei, K., Abdulalian, E., Ng, W., 2011. Evaluation of the proximate, fatty acid and mineral composition of representative green, brown and red seaweeds from the Persian Gulf of Iran as potential food and feed resources. Journal of Food Science and Technology, 49(6), 774-780.; Khairy & El-Shafay, 2013Khairy, H. M., & El-Shafay, S. M. (2013). Seasonal variations in the biochemical composition of some common seaweed species from the coast of Abu Qir Bay, Alexandria, Egypt. Oceanologia, 55(2), 435-452. http://dx.doi.org/10.5697/oc.55-2.435
http://dx.doi.org/10.5697/oc.55-2.435... ). The seaweeds investigated showed variable chemical compositions which depended on the species, collection season, geographic habitat and other physicochemical parameters (Marinho-Soriano et al., 2006Marinho-Soriano, E., Fonseca, P., Carneiro, M., Moreira, W., 2006. Seasonal variation in the chemical composition of two tropical seaweeds. Bioresource Technology, 97(18), 2402-2406.; Marsham et al., 2007Marsham, S., Scott, G. W., & Tobin, M. L. (2007). Comparison of nutritive chemistry of a range of temperate seaweeds. Food Chemistry, 100(4), 1331-1336. http://dx.doi.org/10.1016/j.foodchem.2005.11.029
http://dx.doi.org/10.1016/j.foodchem.200... ; Dawczynski et al., 2007Dawczynski, C., Schubert, R., & Jahreis, G. (2007). Amino acids, fatty acids, and dietary fibre in edible seaweed products. Food Chemistry, 103(3), 891-899. http://dx.doi.org/10.1016/j.foodchem.2006.09.041
http://dx.doi.org/10.1016/j.foodchem.200... ). Seaweeds are being used in the food and cosmetic industries, in agriculture (Kelman et al., 2012Kelman, D., Posner, E. K., Mcdermid, K. J., Tabandera, N. K., Wright, P. R., Wright, A. D. (2012). Antioxidant activity of Hawaiian marine algae. Marine Drugs, 10(12), 403-416.; Guiry, 2017Guiry, M. D. (2017). The seaweed site: Information on marine algae. Retrieved 2017, October 10, from http://www.seaweed.ie-/index.php.
http://www.seaweed.ie-/index.php... ) and as a promising source for biofuel production (Gosch et al., 2012Gosch, B. J., Magnusson, M., Paul, N. A., & Nys, R. D. (2012). Total lipid and fatty acid composition of seaweeds for the selection of species for oil-based biofuel and bioproducts. Global Change Biology, Bioenergy, 4(6), 919-930. http://dx.doi.org/10.1111/j.1757-1707.2012.01175.x
http://dx.doi.org/10.1111/j.1757-1707.20... ).

In general, seaweeds contain 90% water; and therefore the chemical composition is usually evaluated based on its dry weight. This includes the contents of carbohydrate, protein, lipids, minerals, vitamins and other metabolites with health benefits (Zvyagintseva et al., 2005Zvyagintseva, T. N., Shevchenko, N. M., Nazarenko, E. L., Gorbach, V. I., Urvantseva, A. M., Kiseleva, M. I., & Isakov, V. V. (2005). Water-soluble polysaccharides of some brown algae of the Russian Far-East. Structure and biological action of low-molecular mass polyuronans. Journal of Experimental Marine Biology and Ecology, 320(2), 123-131. http://dx.doi.org/10.1016/j.jembe.2004.12.027
http://dx.doi.org/10.1016/j.jembe.2004.1... ; Artan et al., 2008Artan, M., Li, Y., Karadeniz, F., Lee, S., Kim, M., & Kim, S. (2008). Anti-HIV-1 activity of phloroglucinol derivative, 6,6′-bieckol, from Ecklonia cava. Bioorganic & Medicinal Chemistry, 16(17), 7921-7926. PMid:18693022. http://dx.doi.org/10.1016/j.bmc.2008.07.078
http://dx.doi.org/10.1016/j.bmc.2008.07.... ; Choi et al., 2009Choi, E., Hwang, H., Kim, I., Nam, T., 2009. Protective effects of a polysaccharide from Hizikia fusiformis against ethanol toxicity in rats. Food and Chemical Toxicology, 47(1), 134-139.). Carbohydrates are considered the major fraction of the dry weight of seaweeds and can reach up to 50%. Most of the seaweed carbohydrates are categorized as dietary fibres since they are not digested by the human gastrointestinal tract (Rupérez & Saura-Calixto, 2001Rupérez, P., & Saura-Calixto, F. (2001). Dietary fibre and physicochemical properties of edible Spanish seaweeds. European Food Research and Technology, 212(3), 349-354. http://dx.doi.org/10.1007/s002170000264
http://dx.doi.org/10.1007/s002170000264... ). The protein content can vary between 3% to 15% (Fleurence, 1999Fleurence, J. (1999). Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends Food Science & Technology, 10(1), 25-28.) and may reach up to 47% in some cases, with large amounts of essential amino acids (García-Casal et al., 2007García-Casal, M. N., Pereira, A. C., Leets, I., Ramìrez, J., & Quiroga, M. F. (2007). High iron content and bioavailability in humans from four species of marine algae. The Journal of Nutrition, 137(12), 2691-2695. PMid:18029485. http://dx.doi.org/10.1093/jn/137.12.2691
http://dx.doi.org/10.1093/jn/137.12.2691... ). The lipid contents are generally low (Kumari et al., 2010Kumari, P., Kumar, M., Gupta, V., Reddy, C., Jha, B., 2010. Tropical marine macroalgae as potential sources of nutritionally important PUFAs. Food Chemistry. 120(3), 749-757.) and range from 1% to 3% of the dry weight. Also, seaweeds usually have large amounts of trace elements and essential minerals important for human needs, including large amounts of potassium and calcium (Ruperez, 2002Ruperez, P. (2002). Mineral content of edible marine seaweeds. Food Chemistry, 79(1), 23-26. http://dx.doi.org/10.1016/S0308-8146(02)00171-1
http://dx.doi.org/10.1016/S0308-8146(02)... ).

Furthermore, marine macroalgae have become a really promising alternative source of bioactive compounds, since they are able to produce a great variety of secondary metabolites characterized by a broad spectrum of biological behaviours such as antibacterial, antiviral and antifungal properties (Marinho-Soriano et al., 2006Marinho-Soriano, E., Fonseca, P., Carneiro, M., Moreira, W., 2006. Seasonal variation in the chemical composition of two tropical seaweeds. Bioresource Technology, 97(18), 2402-2406.; Yaich et al., 2013Yaich, H., Garna, H., Besbes, S., Paquot, M., Blecker, C., & Attia, H. (2013). Effect of extraction conditions on the yield and purity of ulvan extracted from Ulva lactuca. Food Hydrocolloids, 31(2), 375-382. http://dx.doi.org/10.1016/j.foodhyd.2012.11.013
http://dx.doi.org/10.1016/j.foodhyd.2012... ). However, there is no fixed value even for the same genus or species; and only the range or ratio of a certain chemical component in a given taxon can be known. Antioxidants are probably among the most interesting metabolites extracted from seaweeds, and they are valuable in the treatment of various serious diseases (Kohen & Nyska, 2002Kohen, R., & Nyska, A. (2002). Invited review: Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicologic Pathology, 30(6), 620-650. PMid:12512863. http://dx.doi.org/10.1080/01926230290166724
http://dx.doi.org/10.1080/01926230290166... ). The algae produce antioxidants in large amounts to protect the functional macromolecules inside their cells from oxidation via reactive oxygen species (ROS), which causes structural damage inside the algal cells. Thus seaweeds are considered to be rich in natural antioxidants (Lim et al., 2002Lim, S. N., Cheung, P. C., Ooi, V. E., & Ang, P. O. (2002). Evaluation of antioxidative activity of extracts from a Brown Seaweed, Sargassum siliquastrum. Journal of Agricultural and Food Chemistry, 50(13), 3862-3866. PMid:12059172. http://dx.doi.org/10.1021/jf020096b
http://dx.doi.org/10.1021/jf020096b... ; Kuda et al., 2005Kuda, T., Tsunekawa, M., Goto, H., Araki, Y., 2005. Antioxidant properties of four edible algae harvested in the Noto Peninsula, Japan. Journal of Food Composition and Analysis, 18(7), 625-633.; Yuan et al., 2005Yuan, Y. V., Bone, D. E., & Carrington, M. F. (2005). Antioxidant activity of dulse (Palmaria palmata) extract evaluated in vitro. Food Chemistry, 91(3), 485-494. http://dx.doi.org/10.1016/j.foodchem.2004.04.039
http://dx.doi.org/10.1016/j.foodchem.200... ; Duan et al., 2006Duan, X., Zhang, W., Li, X., Wang, B. (2006). Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food Chemistry, 95(1), 37-43.) such as ascorbic acid, phenols, flavonoids (Wu et al., 2010Wu, S.C., Wang, F.J., Pan, C.L. (2010). The composition of antioxidative properties of seaweed oligosaccharides fermented by two lactic acid bacteria. Journal of Marine Science and Technology, 18(4), 537-545.) and carotenoids (Plaza et al., 2008Plaza, M., Cifuentes, A., & Ibanez, E. (2008). In the search of new functional food ingredients from algae. Trends in Food Science & Technology, 19(1), 31-39. http://dx.doi.org/10.1016/j.tifs.2007.07.012
http://dx.doi.org/10.1016/j.tifs.2007.07... ), which have been found in brown, red and green algae (Cox et al., 2012Cox, S., Gupta, S., & Abu-Ghannam, N. (2012). Effect of different rehydration temperatures on the moisture, content of phenolic compounds, antioxidant capacity and textural properties of edible Irish brown seaweed. Lebensmittel-Wissenschaft + Technologie, 47(2), 300-307. http://dx.doi.org/10.1016/j.lwt.2012.01.023
http://dx.doi.org/10.1016/j.lwt.2012.01.... ).

Certain seaweed species, not included in this study, may contain toxic metabolites, which may prevent their use as food or animal feedstuffs. For example, Caulerpa taxifolia contains sesquiterpenic toxins such as caulerpenyne (Guerriero et al., 1992Guerriero, A., Meinesz, A., Dambrosio, M., & Pietra, F. (1992). Isolation of toxic and potentially toxic Sesqui - and Monoterpenes from the Tropical Green Seaweed Caulerpa taxifolia which has invaded the region of Cap Martin and Monaco. Helvetica Chimica Acta, 75(3), 689-695. http://dx.doi.org/10.1002/hlca.19920750303
http://dx.doi.org/10.1002/hlca.199207503... ). However, such toxic compounds may be successfully used in other applications such as drug formulations for certain diseases, for instance due to their anticancer activity (Barbier et al., 2001Barbier, P., Guise, S., Huitorel, P., Amade, P., Pesando, D., Briand, C., & Peyrot, V. (2001). Caulerpenyne from Caulerpa taxifolia has an antiproliferative activity on tumor cell line SK-N-SH and modifies the microtubule network. Life Sciences, 70(4), 415-429. PMid:11798011. http://dx.doi.org/10.1016/S0024-3205(01)01396-0
http://dx.doi.org/10.1016/S0024-3205(01)... ), or even used to synthesize metallic nanoparticles (Aboelfetoh et al., 2017Aboelfetoh, E. F., El-Shenody, R. A., & Ghobara, M. M. (2017). Eco-friendly synthesis of silver nanoparticles using green algae (Caulerpa serrulata): reaction optimization, catalytic and antibacterial activities. Environmental Monitoring and Assessment, 189(7), 349. PMid:28646435. http://dx.doi.org/10.1007/s10661-017-6033-0
http://dx.doi.org/10.1007/s10661-017-603... ). Therefore, more attention should be paid to the choice of suitable seaweed species for use as food.

In the present study, the chemical compositions of three Egyptian seaweeds; Dictyota dichotoma, Turbinaria decurrens and Laurencia obtusa (Figure 1) were estimated, and their use proposed as food additives and as promising sources of antioxidants with a view to the nutrition and health of both humans and animals.

2 Materials and methods

2.1 Collection of the seaweed samples

Sufficient amounts of the three marine macroalgae were collected from the Red Sea coast of Suez, Suez Bay, Egypt (29º 55' N, 32º 28' E) during June 2015. The samples were washed thoroughly with distilled water directly after collection to remove epiphytes and excess salts, placed in sterilized polyethylene bags and transferred to the laboratory in an icebox for the experimental work. In the laboratory, the samples were washed with sterile distilled water, air dried, oven dried at 45 °C to 50 °C, cut into small pieces, and then ground to a fine powder using a tissue grinder. The samples were identified according to Aleem (1978)Aleem, A. A. (1978). Contributions to the study of marine algae of the Red Sea, III-marine algae from Obhor, in the vicinity of Jeddah, Saudi Arabia. King Abdul Aziz University (Jeddah, Saudi Arabia). Faculty of Science Bulletin, 2, 99-118. and Guiry & Guiry (2011)Guiry, M., & Guiry, G. (2011). AlgaeBase. National University of Ireland. Retrieved in 2015, June 25, from http://www.algaebase.org.
http://www.algaebase.org... as belonging to two algal divisions (See Figure 1): Phaeophyta (Dictyota dichotoma, Turbinaria decurrens) and Rhodophyta (Laurencia obtusa).

2.2 Analysis of the chemical composition

2.2.1 Extraction and estimation of the carbohydrate content

The total carbohydrate content was quantitatively determined by the phenol-sulphuric acid method as described by Kochert (1978)Kochert, G. (1978). Carbohydrate determination by the phenol–sulfuric acid method. In J.A. Hellebust & J.S. Craigie (Eds.), Handbook of phycology methods: physiological and biochemical methods (pp. 95-97). London, UK: Cambridge University Press., after extraction by 1 N NaOH in a boiling water bath for 2 hours as described by Payne & Stewart (1988)Payne, J. K., & Stewart, J. R. (1988). The chemical composition of the thallus wall of Characiosiphon rivularis (Characiosiphonaceae, Chlorophyta). Phycologia, 27(1), 43-49. http://dx.doi.org/10.2216/i0031-8884-27-1-43.1
http://dx.doi.org/10.2216/i0031-8884-27-... . The content was calculated in percentage using a standard glucose curve.

2.2.2 Extraction and estimation of the protein and amino acid contents

The protein content was extracted from about 0.1 g powder. The extraction was carried out by adding 1 N NaOH to the seaweed powder and leaving the mixture in a boiling water bath for 2 hours as described by Payne & Stewart (1988)Payne, J. K., & Stewart, J. R. (1988). The chemical composition of the thallus wall of Characiosiphon rivularis (Characiosiphonaceae, Chlorophyta). Phycologia, 27(1), 43-49. http://dx.doi.org/10.2216/i0031-8884-27-1-43.1
http://dx.doi.org/10.2216/i0031-8884-27-... . After centrifugation, the total soluble protein content was estimated using the Bradford method (Bradford, 1976Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72(1-2), 248-254.) and expressed as a percentage of the algal dry weight. Furthermore, the amino acid profile was determined according to the methods described by Khairy & El-Shafay (2013)Khairy, H. M., & El-Shafay, S. M. (2013). Seasonal variations in the biochemical composition of some common seaweed species from the coast of Abu Qir Bay, Alexandria, Egypt. Oceanologia, 55(2), 435-452. http://dx.doi.org/10.5697/oc.55-2.435
http://dx.doi.org/10.5697/oc.55-2.435... . The algal samples (3 g) were prepared for hydrolysis according to Blackburn (1978)Blackburn, S. (1978). Sample preparation and hydrolytic methods. In S. Blackburn (Ed.), Amino acid determination methods and techniques (pp. 7-37). New York: Marcel Dekker Inc. and the amino acids determined using an amino acid analyser (AAA).

2.2.3 Extraction and estimation of the lipid and fatty acid contents

The total lipid content was extracted using chloroform-methanol according to the modified Folch method (Folch et al., 1957Folch, J., Lees, M., & Stanley, G. H. S. (1957). A simple method for the isolation and purification of total lipids from animal tissues. The Journal of Biological Chemistry, 226(1), 497-509. PMid:13428781.), and expressed as a percentage. After methylation according to Francavilla et al. (2013)Francavilla, M., Franchi, M., Monteleone, M., Caroppo, C., 2013. The red seaweed Gracilaria gracilis as a multi products source. Marine Drugs, 11(10), 3754-3776., the fatty acid contents of the lipids were analysed using a GC-MS spectrophotometer (Clarus 580, 560 S Perkin Elmer). The injection volume was 0.1 ul at 220 ºC, the carrier gas was helium and the flow rate 0.2 mL/min.

2.2.4 Extraction and estimation of the pigments

The total carotene and chlorophyll (Chlorophyll a, Chlorophyll b) contents were extracted according to Khuantrairong & Traichaiyaporn (2012)Khuantrairong, T., & Traichaiyaporn, S. (2012). Enhancement of carotenoid and chlorophyll content of an edible freshwater alga (Kai: Cladophora sp.) by supplementary inorganic phosphate and investigation of its biomass production. Maejo International Journal of Science and Technology, 6(01), 1-11., as a modified method of Yoshii et al. (2004)Yoshii, Y., Hanyuda, T., Wakana, I., Miyaji, K., Arai, S., Ueda, K., & Inouye, I. (2004). Carotenoid compositions of Cladophora balls (Aegagropila linnaei) and some members of the Cladophorales (Ulvophyceae, Chlorophyta): their taxonomic and evolutionary implication. Journal of Phycology, 40(6), 1170-1177. http://dx.doi.org/10.1111/j.1529-8817.2004.03210.x
http://dx.doi.org/10.1111/j.1529-8817.20... and of Dere et al. (1998)Dere, S., Günes, T., & Sivaci, R. (1998). Spectrophotometric determination of chlorophyll-a, b and total carotenoid contents of some algae species using different solvents. Turkish Journal of Botany, 22, 13-17.. The total carotene content and chlorophylls a and b were determined spectrophotometrically using a UV-visible spectrophotometer at 470, 645 and 662 nm, respectively, according to the equations 1-3 proposed by Wellburn & Lichtenthaler (1984)Wellburn, A. R., & Lichtenthaler, H. 1984. Formulae and program to determine total carotenoids and Chlorophylls A and B of leaf extracts in different solvents. In C. Sybesma (Eds.), Advances in Photosynthesis Research (Advances in Agricultural Biotechnology, Vol. 2). Dordrecht: Springer. http://dx.doi.org/10.1007/978-94-017-6368-4_3.
http://dx.doi.org/10.1007/978-94-017-636... :

Where (A₆₆₂) is the absorbance at 662 nm, (A₆₄₅) is the absorbance at 645nm, (A₄₇₀) is the absorbance at 470 nm, (C_a) is chlorophyll a and (C_b) is chlorophyll b. The results were expressed as (μg g^-1) of seaweed.

2.2.5 Mineral analysis

About 10 ml of a diacid mixture (2:5 of nitric and perchloric acids) were added to 0.2 g powder and left for a few hours. The mixture was then placed on a hot plate and the contents digested by increasing the temperature. The digestion was continued until the contents became colourless. The digested material was filtered through Whatman Nº. 40 paper and the filtrate diluted to a suitable volume and fed into an inductively coupled plasma spectrometer (ICP) (Perkin Elmer emission spectrophotometer-6000 series), according to Allen et al. (1997)Allen, L. B., Siitonen, P. H., & Thompson, H. C. (1997). Methods for the determination of arsenic, cadmium, copper, lead, and tin in sucrose, corn syrups, and high-fructose corn syrups by inductively coupled plasma atomic emission spectrometry. Journal of Agricultural and Food Chemistry, 45(1), 162-165. http://dx.doi.org/10.1021/jf960376p
http://dx.doi.org/10.1021/jf960376p... .

2.3 Secondary metabolites analysis

2.3.1 Estimation of the total phenolic compound content

The total phenolic compound content was determined using the Folin–Ciocalteau phenol reagent according to the method described by Kumar & Balamuruga (2015)Kumar, N. S., & Balamuruga, V. (2015). In-vitro antioxidant activity, total phenolic and total flavonoid contents of flower extract of Calotropis gigantea. Research Journal of Phytochemistry, 9(3), 137-143. http://dx.doi.org/10.3923/rjphyto.2015.137.143
http://dx.doi.org/10.3923/rjphyto.2015.1... . The total phenol content of the samples was expressed as mg gallic acid equivalents per gram of dry weight (mg GAE g^-1 dry weight).

2.3.2 Total flavonoid content

The total flavonoid content was determined according to the method of Chang et al. (2002)Chang, C. C., Yang, M. H., Wen, H. M., & Chern, J. C. (2002). Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Yao Wu Shi Pin Fen Xi, 10, 178-182., using quercetin as the standard. The flavonoid content was expressed as mg quercetin equivalents per gram of extract (mg QE g^-1 extract).

2.3.3 Estimation of the total alkaloid content

The total alkaloid content was measured quantitatively according to the method described by Harborne (2008)Harborne, J. B. (2008). Phytochemical methods: A guide to modern techniques of plant analysis. New Delhi, India: Springer. and expressed as milligram per gram of dry weight (mg g^-1 dry weight).

2.3.4 Ascorbic acid (Vitamin C) content

The Aascorbic acid content was estimated according to Oser (1979)Oser, B. L. 1979. Hawk’s Physiological Chemistry. New York, USA: Mc Graw Hill. and calculated as milligrams per gram of dry weight (mg g^-1 dry weight) using an ascorbic acid calibration curve.

2.4 Antioxidant activity assays

2.4.1 Determination of DPPH free radical scavenging activity

The radical scavenging activity of the methanolic seaweed extract against DPPH radicals was determined using the method of Blois (1958)Blois, M.S. (1958). Antioxidant determinations by the use of a stable free radical. Nature, 181(4617), 1199-1200. and Yamasaki et al. (1994)Yamasaki, K., Hashimoto, A., Kokusenya, Y., Miyamoto, T., & Sato, T. (1994). Electrochemical method for estimating the antioxidative effects of methanol extracts of crude drugs. Chemical & Pharmaceutical Bulletin, 42(8), 1663-1665. PMid:7954918. http://dx.doi.org/10.1248/cpb.42.1663
http://dx.doi.org/10.1248/cpb.42.1663... and calculated using the following equation 4:

Where (A control) is the absorbance of the control reaction and (A test) is the absorbance in the presence of the extracts.

2.4.2 Total Antioxidant Capacity (TAC)

The total antioxidant activity of the seaweed extracts was determined using the phosphomolybdate assay method (PMA) (Ahmad et al., 2012Ahmad, F., Sulaiman, M. R., Saimon, W., Yee, C. F., & Matanjun, P. (2012). Proximate compositions and total phenolic contents of selected edible seaweed from Semporna, Sabah, Malaysia. Borneo Science., 31, 74-83.) and expressed in mg ascorbic acid equivalents (AAE).

2.5 Statistical analysis

All the experiments were carried out in triplicate and the results expressed as the means ± standard deviation (SD). Statistically significant differences between the seaweed parameters studied were identified by the analysis of variance (one-way ANOVA) using the SPSS software for windows. When the F values showed significance, the individual means were compared using the Duncan multiple range test (DMRT) method for data with significant differences. Significant differences were considered when p < 0.05.

3 Results and discussion

3.1 Chemical composition of the seaweeds studied

Protein, carbohydrate and lipids are the major vital chemical constituents of seaweeds. Figure 2 shows the carbohydrate, protein and lipid contents of the three seaweeds, showing highly significant differences (p≤ 0.05), the fractions of each component varying between the three species depending on the taxon (Yaich et al., 2011Yaich, H., Garna, H., Besbes, S., Paquot, M., Blecker, C., & Attia, H. (2011). Chemical composition and functional properties of Ulva lactuca seaweed collected in Tunisia. Food Chemistry, 128(4), 895-901. http://dx.doi.org/10.1016/j.foodchem.2011.03.114
http://dx.doi.org/10.1016/j.foodchem.201... ). The results indicated that the carbohydrate contents were greater than the protein and lipid contents.

The average carbohydrate contents of the seaweeds studied ranged from 20.6% to 33%. The maximum value was recorded for T. decurrens and the minimum for L. obtusa. This large amount of carbohydrate is important for the metabolism of the organism, since it supplies the energy needed for respiration and other metabolic processes and usually includes considerable amounts of polysaccharides, for example alginate and fucoidan in the brown seaweeds and carrageenan in the red seaweeds. These polysaccharides are edible and show promising biomedical applications including antiviral, antibacterial, anticoagulant and anticancer properties, the use as drug coatings in drug delivery systems and antioxidant activities (Venkatesan et al., 2017Venkatesan, J., Anil, S., & Kim, S. (2017). Seaweed polysaccharides: Isolation, biological and biomedical applications. Amsterdam, Germany: Elsevier.).

In general, the protein content of seaweeds is relatively low, between 3% to 15% of the dry weight (Fleurence, 1999Fleurence, J. (1999). Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends Food Science & Technology, 10(1), 25-28.), similar to the results obtained in the present study (Figure 2). According to the literature, the red seaweeds usually contain a larger protein fraction than both green and brown seaweeds (Cian et al., 2015Cian, R. E., Drago, S. R., de Medina, F. S., & Martínez-Augustin, O. (2015). Proteins and carbohydrates from red seaweeds: evidence for beneficial effects on gut function and microbiota. Marine Drugs, 13(8), 5358-5383. PMid:26308006. http://dx.doi.org/10.3390/md13085358
http://dx.doi.org/10.3390/md13085358... ; Pangestuti & Kim, 2015Pangestuti, R., Kim, S., 2015. Seaweed proteins, peptides, and amino acids. In B. K. Tiwari & D. J. Troy, Seaweed Sustainability (pp. 125-140). San Diego: Academic Press. http://dx.doi.org/10.1016/B978-0-12-418697-2.00006-4.
http://dx.doi.org/10.1016/B978-0-12-4186... ), but the results obtained here showed the largest protein fraction in the brown seaweed (D. dichotoma), and the lowest amount in the red seaweed (L. obtusa), results consistent with those of Parthiban et al. (2013)Parthiban, C., Saranya, C., Girija, K., Hemalatha, A., Suresh, M., & Anantharaman, P. (2013). Biochemical composition of some selected seaweeds from Tuticorin coast. Adv. Applied Scientific Research, 4(3), 362-366.. The reason for these differences could be that the protein content of seaweeds varies between species and even within the same species due to different habitats, time of the year and levels of maturity (Stirk et al., 2007Stirk, W. A., Reinecke, D. L., & Staden, J. V. (2007). Seasonal variation in antifungal, antibacterial and acetylcholinesterase activity in seven South African seaweeds. Journal of Applied Phycology, 19(3), 271-276. http://dx.doi.org/10.1007/s10811-006-9134-7
http://dx.doi.org/10.1007/s10811-006-913... ; Gressler et al., 2010Gressler, V., Yokoya, N. S., Fujii, M. T., Colepicolo, P., Filho, J. M., Torres, R. P., & Pinto, E. (2010). Lipid, fatty acid, protein, amino acid and ash contents in four Brazilian red algae species. Food Chemistry, 120(2), 585-590. http://dx.doi.org/10.1016/j.foodchem.2009.10.028
http://dx.doi.org/10.1016/j.foodchem.200... ).

The amino acid profile analysis was carried out to determine the nutritional value of the three seaweed protein contents, and Table 1 shows the amino acid contents of the seaweeds studied. These amino acids may occur in the combined form or in the free state (Munda & Gubenšek, 1976Munda, I. M., & Gubenšek, F. (1976). The amino acid composition of some common Marine Algae from Iceland. Botanica Marina, 19(2), http://dx.doi.org/10.1515/botm.1976.19.2.85
http://dx.doi.org/10.1515/botm.1976.19.2... ; Dave & Parekh, 1978Dave, M. J., & Parekh, R. G. (1978). Amino acids of green alga Ulva. Botanica Marina, 21(5), 323-326. http://dx.doi.org/10.1515/botm.1978.21.5.323
http://dx.doi.org/10.1515/botm.1978.21.5... ). Ten essential and six non-essential amino acids were detected in the protein hydrolysates of the seaweeds, including all the essential amino acids, and they were especially rich in aspartic and glutamic acids, also showing significant amounts of proline and alanine (Table 1), similar to results from previous studies (Fleurence et al., 2012Fleurence, J., Morançais, M., Dumay, J., Decottignies, P., Turpin, V., Munier, M., Garcia-Bueno, N., & Jaouen, P. (2012). What are the prospects for using seaweed in human nutrition and for marine animals raised through aquaculture? Trends in Food Science & Technology, 27(1), 57-61. http://dx.doi.org/10.1016/j.tifs.2012.03.004
http://dx.doi.org/10.1016/j.tifs.2012.03... ; Cian et al., 2013Cian, R.E., Fajardo, M.A., Alaiz, M., Vioque, J., González, R.J., Drago, S.R., 2013. Chemical composition, nutritional and antioxidant properties of the red edible seaweed Porphyra columbina. International Journal of Food Sciences and Nutrition, 65(3), 299-305. doi:10.3109-/09637486.2013.854746). The amino acid profiles of the seaweeds studied were comparable with the FAO reference pattern (Food and Agriculture Organization of the United Nations, 1981Food and Agriculture Organization of the United Nations – FAO. (1981). Amino acid scoring patterns (20 p.). Rome: FAO/WHO/UNU.) and the amino acid profiles of other food proteins (Orr & Watt, 1968Orr, M. L., & Watt, B. K. 1968. Amino acid content of foods (82 p.). Washington, DC: USDA, Home Economics Research Report.) indicating the high nutritional value of seaweed protein, which can provide the total amount of essential amino acids required as food.

The average total lipid content of the three seaweeds studied varied from 1.7% to 7.5% (Figure 2). The maximum lipid content was recorded in D. dichotoma followed by the red algae L. obtusa, while the minimum lipid content was found in T. decurrens. The total lipid content of seaweeds is generally lower than 5% of the dry weight in most species (Kumari et al., 2010Kumari, P., Kumar, M., Gupta, V., Reddy, C., Jha, B., 2010. Tropical marine macroalgae as potential sources of nutritionally important PUFAs. Food Chemistry. 120(3), 749-757.; Gosch et al., 2012Gosch, B. J., Magnusson, M., Paul, N. A., & Nys, R. D. (2012). Total lipid and fatty acid composition of seaweeds for the selection of species for oil-based biofuel and bioproducts. Global Change Biology, Bioenergy, 4(6), 919-930. http://dx.doi.org/10.1111/j.1757-1707.2012.01175.x
http://dx.doi.org/10.1111/j.1757-1707.20... ). However, some species are different, such as the members of the order Dictyotales, which can have total lipid contents of up to 20% of the dry weight (Gosch et al., 2012Gosch, B. J., Magnusson, M., Paul, N. A., & Nys, R. D. (2012). Total lipid and fatty acid composition of seaweeds for the selection of species for oil-based biofuel and bioproducts. Global Change Biology, Bioenergy, 4(6), 919-930. http://dx.doi.org/10.1111/j.1757-1707.2012.01175.x
http://dx.doi.org/10.1111/j.1757-1707.20... ), which explains the relatively high total lipid content (7.5% ± 0.24%) recorded in D. dichotoma. Furthermore, the results obtained for L. Obtusa were consistent with the total lipid fraction found in the two Laurencia species previously studied, which varied between 1.1% and 6.4% of the dry weight (Gressler et al., 2010Gressler, V., Yokoya, N. S., Fujii, M. T., Colepicolo, P., Filho, J. M., Torres, R. P., & Pinto, E. (2010). Lipid, fatty acid, protein, amino acid and ash contents in four Brazilian red algae species. Food Chemistry, 120(2), 585-590. http://dx.doi.org/10.1016/j.foodchem.2009.10.028
http://dx.doi.org/10.1016/j.foodchem.200... ).

The results obtained in the fatty acid analyses indicated that saturated fatty acids (SFAs) were predominant in the three seaweeds studied (Table 2). L.obtusa recorded the highest percent followed by T. decurrens and D. dichotoma. These results are in agreement with previous studies (Khotimchenko, 1995Khotimchenko, S. V. (1995). Fatty acid composition of green algae of the genus Caulerpa. Botanica Marina, 38(1-6), 509-512. http://dx.doi.org/10.1515/botm.1995.38.1-6.509
http://dx.doi.org/10.1515/botm.1995.38.1... ; Vaskovsky et al., 1996Vaskovsky, V. E., Khotimchenko, S. V., Xia, B., & Hefang, L. (1996). Polar lipids and fatty acids of some marine macrophytes from the Yellow Sea. Phytochemistry, 42(5), 1347-1356. http://dx.doi.org/10.1016/0031-9422(96)00117-3
http://dx.doi.org/10.1016/0031-9422(96)0... ), where SFAs were found to be the dominate fraction amongst the total fatty acids in seaweeds. Palmitic acid (C16:0) was the largest fraction amongst the SFAs, showing 50.86%, 42.89% and 26.51% for T. decurrens, L.obtusa and D. dichotoma, respectively, followed by myristic acid (C14:0) and stearic acid (C18:0). Palmitic acid was also the major SFA fraction in Gellidum micropterum (Venkatesalu et al., 2004Venkatesalu, V., Sundaramoorthy, P., Anantharaj, M., Gopalakrishnan, M., & Chandrasekaran, M. (2004). Studies on the fatty acid composition of marine Algae of Rameswaram coast. Seaweed Res.Util., 26(1-2), 83-86.) and in Porphyra spp. (Sanchez-Machado et al., 2004Sanchez-Machado, D. I., López-Cervantes, J., López-Hern’andez, J., & Paseiro-Losada, P. (2004). Fatty acids, total lipid, protein and ash contents of processed edible seaweeds. Food Chemistry, 85(3), 439-444. http://dx.doi.org/10.1016/j.foodchem.2003.08.001
http://dx.doi.org/10.1016/j.foodchem.200... ). In addition, some brown seaweeds were found to be rich in myristic acid (Gosch et al., 2012Gosch, B. J., Magnusson, M., Paul, N. A., & Nys, R. D. (2012). Total lipid and fatty acid composition of seaweeds for the selection of species for oil-based biofuel and bioproducts. Global Change Biology, Bioenergy, 4(6), 919-930. http://dx.doi.org/10.1111/j.1757-1707.2012.01175.x
http://dx.doi.org/10.1111/j.1757-1707.20... ).

The mononunsaturated fatty acids (MUSFA) were in second place in the fatty acid content of the three seaweeds, and similar results were obtained by Khairy & El-Shafay (2013)Khairy, H. M., & El-Shafay, S. M. (2013). Seasonal variations in the biochemical composition of some common seaweed species from the coast of Abu Qir Bay, Alexandria, Egypt. Oceanologia, 55(2), 435-452. http://dx.doi.org/10.5697/oc.55-2.435
http://dx.doi.org/10.5697/oc.55-2.435... . The brown seaweeds recorded the highest percentage, while the red seaweeds recorded the lowest one. Oleic acid, an omega-9 fatty acid, presented the highest percentage amongst the MUSFA in all the seaweeds studied, in agreement with the results of Dawczynski et al. (2007)Dawczynski, C., Schubert, R., & Jahreis, G. (2007). Amino acids, fatty acids, and dietary fibre in edible seaweed products. Food Chemistry, 103(3), 891-899. http://dx.doi.org/10.1016/j.foodchem.2006.09.041
http://dx.doi.org/10.1016/j.foodchem.200... , who claimed that oleic acid represented the highest fraction of the MUSFA in Porphyra spp. It is important to mention that the fatty acid contents of the three seaweeds may indicate their suitability for biofuel production, since the three species show very high percentages of SFAs as compared to the MUSFA and PUSFA (Hu et al., 2008Hu, Q., Sommerfeld, M., Jarvis, E., Ghirardi, M., Posewitz, M., Seibert, M., & Darzins, A. (2008). Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. The Plant Journal, 54(4), 621-639. PMid:18476868. http://dx.doi.org/10.1111/j.1365-313X.2008.03492.x
http://dx.doi.org/10.1111/j.1365-313X.20... ; Knothe, 2008Knothe, G. (2008). “Designer” Biodiesel: Optimizing fatty ester composition to improve fuel properties. Energy & Fuels, 22(2), 1358-1364. http://dx.doi.org/10.1021/ef700639e
http://dx.doi.org/10.1021/ef700639e... ), with a relatively high content of MUSFA which would enhance the quality of the biofuel (Knothe, 2008Knothe, G. (2008). “Designer” Biodiesel: Optimizing fatty ester composition to improve fuel properties. Energy & Fuels, 22(2), 1358-1364. http://dx.doi.org/10.1021/ef700639e
http://dx.doi.org/10.1021/ef700639e... ).

Concerning the polyunsaturated fatty acids (PUSFA), the brown algae D. dichotoma attained the highest percent followed by the red algae L. obtusa, and T. decurrens recorded the lowest percent. The Omega-3 fatty acids (α-Linolenic acid and Eicosatrienoic acid) were only found in the brown D. dichotoma and T. decurrens. Meanwhile decosahexaenoic acid (DHA), an omega-6 fatty acid, was only found in the red algae L.obtusa. These results are consistent with those of Khairy & El-Shafay (2013)Khairy, H. M., & El-Shafay, S. M. (2013). Seasonal variations in the biochemical composition of some common seaweed species from the coast of Abu Qir Bay, Alexandria, Egypt. Oceanologia, 55(2), 435-452. http://dx.doi.org/10.5697/oc.55-2.435
http://dx.doi.org/10.5697/oc.55-2.435... who reported that DHA was the predominant component of PUFA in the red algae Jania rubens. Furthermore, linoleic acid, an omega-6 fatty acid, was present in all the seaweeds studied. Recently, the importance of the ω-6/ω-3 ratio has been widely discussed in scientific reports. The original value of 1 of the ω-6/ω-3 ratio involved the balance of intake of both PUSFA ω-6 and ω-3 fatty acids (Francavilla et al., 2013Francavilla, M., Franchi, M., Monteleone, M., Caroppo, C., 2013. The red seaweed Gracilaria gracilis as a multi products source. Marine Drugs, 11(10), 3754-3776.). The ω6/ω3 ratios found here were 0.161, 0.114 and 0.987 for D. dichotoma, T. decurren and L.obtusa respectively. According to WHO, this ratio should not be higher than 10 in the diets (Sánchez-Machado et al., 2004), which endorses the need for further investigations concerning the use of the seaweeds studied for nutritive purposes. The PUFAs were reported to play key roles in cellular and tissue metabolism, including the regulation of cell membrane fluidity, electron and oxygen transport and thermal adaptation, and could reduce the risk of coronary heart disease (Funk, 2001Funk, C. D. (2001). Prostaglandins and leukotrienes: advances in eicosanoids biology. Science, 294(5548), 1871-1875. PMid:11729303. http://dx.doi.org/10.1126/science.294.5548.1871
http://dx.doi.org/10.1126/science.294.55... ; Mozaffarian et al., 2005Mozaffarian, D., Ascherio, A., Hu, F. B., Stampfer, M. J., Willett, W. C., Siscovick, D. S., & Rimm, E. B. (2005). Interplay between different polyunsaturated fatty acids and risk of coronary heart disease in men. Circulation, 111(2), 157-164. PMid:15630029. http://dx.doi.org/10.1161/01.CIR.0000152099.87287.83
http://dx.doi.org/10.1161/01.CIR.0000152... ).

3.2 Pigment contents

Table 3 shows the estimated contents for the photosynthetic pigments chlorophyll “a” and chlorophyll “b”, and the total carotenoid contents. D. dichotoma showed the highest chlorophyll “a”, chlorophyll “b” and carotenoid contents, followed by the other two species. T. decurrens and L. obtusa which showed quite similar results; although T. decurrens showed slightly, but still significantly lower contents than the red seaweed. Similarly, Utami et al. (2017)Utami, S., Anggoro, S., Soeprobowati, T.R., 2017. Diversity of invasive plants in the Panjang Island Reserve Jepara Central Java, Indonesia. Advanced Science Letters, 23(7): 6493-6494. found that the pigment concentrations in Dictyota were higher than those in Turbinaria. In general, the variation in pigment content is associated with the species type, light intensity, water depth and temperature (Sampath-Wiley et al., 2008Sampath-Wiley, P., Neefus, C. D., & Jahnke, L. S. (2008). Seasonal effects of sun exposure and emersion on intertidal seaweed physiology: fluctuations in antioxidant contents, photosynthetic pigments and photosynthetic efficiency in the red alga Porphyra umbilicalis Kützing (Rhodophyta, Bangiales). Journal of Experimental Marine Biology and Ecology, 361(2), 83-91. http://dx.doi.org/10.1016/j.jembe.2008.05.001
http://dx.doi.org/10.1016/j.jembe.2008.0... ; Schmid et al., 2017Schmid, M., Guihéneuf, F., & Stengel, D. B. (2017). Ecological and commercial implications of temporal and spatial variability in the composition of pigments and fatty acids in five Irish macroalgae. Marine Biology, 164(8), 158. http://dx.doi.org/10.1007/s00227-017-3188-8
http://dx.doi.org/10.1007/s00227-017-318... ).

Photosynthetic pigments could be a promising component for applications in the food industry and pharmaceutical fields, since the pigments could help in cell communication, human health maintenance, and also probably have antimicrobial activities (Plaza et al., 2010Plaza, M., Santoyo, S., Jaime, L., García-Blairsy Reinac, G., Herrero, M., Señoráns, F. J., & Ibánez, E. (2010). Screening for bioactive compounds from algae. Journal of Pharmaceutical and Biomedical Analysis, 51(2), 450-455. PMid:19375880. http://dx.doi.org/10.1016/j.jpba.2009.03.016
http://dx.doi.org/10.1016/j.jpba.2009.03... ). Furthermore, carotenoids have major functions as antioxidants, that could play a role in preventing human diseases linked to oxidative stress (Mitra et al., 2006Mitra, A., Basu, S., Banerjee, K., & Banerjee, A. (2006). Impact of tidal submergence on astaxanthin content of mangroves. Ultra Scientist of Physical Sciences, 18, 117-122.).

3.3 Mineral analysis

Table 4 presents the results obtained in the analysis of minerals. T. decurrens showed the highest contents of potassium, sodium and iron, while L.obtusa recoded the highest nitrogen and zinc contents. The nitrogen, phosphorus, magnesium and copper contents showed slight variations between the three species. In general, seaweeds are rich in minerals (Cian et al., 2015Cian, R. E., Drago, S. R., de Medina, F. S., & Martínez-Augustin, O. (2015). Proteins and carbohydrates from red seaweeds: evidence for beneficial effects on gut function and microbiota. Marine Drugs, 13(8), 5358-5383. PMid:26308006. http://dx.doi.org/10.3390/md13085358
http://dx.doi.org/10.3390/md13085358... ), especially in sodium and iodine, due to their high polysaccharide content (Lahaye, 1991Lahaye, M., 1991. Marine algae as sources of fibres: Determination of soluble and insoluble dietary fibre contents in some ‘sea vegetables’. Journal of the Science of Food and Agriculture. 54(4), 587-594.). The heavy metal content in seaweeds reflects its concentration in the medium, and the capacity of the seaweed to chelate them.

The Na/K ratio ranged from 1.1 to 4.1 in the species studied, representing desirable values for considering these species as food to provide a balanced Na/K ratio (Insel et al., 2007Insel, P., Ross, D., McMahon, K., & Bernstein, M. (2007). Nutrition (3rd ed.). Sudbury, Canada: Jones and Bartlett Publishers.). Potassium, sodium and chlorides are responsible for maintaining the body fluid balance by decreasing sodium absorption and increasing potassium absorption in the gastrointestinal tract, whereas potassium together with calcium and magnesium are implicated in lowering the blood pressure and lessening the risk of strokes (Vaskonen, 2003Vaskonen, T. (2003). Dietary minerals and modification of cardiovascular risk factors. The Journal of Nutritional Biochemistry, 14(9), 492-506. PMid:14505811. http://dx.doi.org/10.1016/S0955-2863(03)00074-3
http://dx.doi.org/10.1016/S0955-2863(03)... ; Smith et al., 2010Smith, J., Summers, G., & Wong, R. (2010). Nutrient and heavy metal content of edible seaweeds in New Zealand. New Zealand Journal of Crop and Horticultural Science, 38(1), 19-28. http://dx.doi.org/10.1080/01140671003619290
http://dx.doi.org/10.1080/01140671003619... ).

The high iron levels present in seaweeds could compete with other sources, such as meat and spinach, by comparing with the data reported by Tee et al. (1988)Tee, E. S., Mohd Ismail, N., Mohd Nasir, A., & Khatijah, I. 1988. Nutrient composition of Malaysian foods. Kuala Lumpur: ASEAN Sub-Committee on Protein, Food Habits Research and Development.. The iron content of edible seaweeds could take part as a vital constituent in hemoglobin biosynthesis, and also interfere in many other human and metabolic processes.

Furthermore, the high zinc concentration present in the red seaweed, L.obtusa, is important for enzyme function, protein stability and in the regulation of gene expression (Smith et al., 2010Smith, J., Summers, G., & Wong, R. (2010). Nutrient and heavy metal content of edible seaweeds in New Zealand. New Zealand Journal of Crop and Horticultural Science, 38(1), 19-28. http://dx.doi.org/10.1080/01140671003619290
http://dx.doi.org/10.1080/01140671003619... ).

3.4 Antioxidant contents

Natural antioxidants are not limited to terrestrial sources and reports have shown that seaweeds are also rich in natural antioxidant compounds (Lim et al. 2002Lim, S. N., Cheung, P. C., Ooi, V. E., & Ang, P. O. (2002). Evaluation of antioxidative activity of extracts from a Brown Seaweed, Sargassum siliquastrum. Journal of Agricultural and Food Chemistry, 50(13), 3862-3866. PMid:12059172. http://dx.doi.org/10.1021/jf020096b
http://dx.doi.org/10.1021/jf020096b... ; Duan et al., 2006Duan, X., Zhang, W., Li, X., Wang, B. (2006). Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food Chemistry, 95(1), 37-43.; Kuda et al., 2007Kuda, T., Kunii, T., Goto, H., Suzuki, T., & Yano, T. (2007). Varieties of antioxidant and antibacterial properties of Ecklonia stolonifera and Ecklonia kurome products harvested and processed in the Noto peninsula, Japan. Food Chemistry, 103(3), 900-905.http://dx.doi.org/10.1016/j.foodchem.2006.09.042
http://dx.doi.org/10.1016/j.foodchem.200... ) including phenolic compounds, flavonoids, alkaloids and vitamin C. These antioxidants aid in the treatment of various serious diseases such as cancer and the cardiovascular and aging diseases, by scavenging ROS and free radicals (Kohen & Nyska, 2002Kohen, R., & Nyska, A. (2002). Invited review: Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification. Toxicologic Pathology, 30(6), 620-650. PMid:12512863. http://dx.doi.org/10.1080/01926230290166724
http://dx.doi.org/10.1080/01926230290166... ).

Phenolic compounds are commonly found in plants as well as in seaweeds and have been reported to have a wide range of biological activities including antioxidant properties (Duan et al., 2006Duan, X., Zhang, W., Li, X., Wang, B. (2006). Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food Chemistry, 95(1), 37-43.; Kuda et al., 2007Kuda, T., Kunii, T., Goto, H., Suzuki, T., & Yano, T. (2007). Varieties of antioxidant and antibacterial properties of Ecklonia stolonifera and Ecklonia kurome products harvested and processed in the Noto peninsula, Japan. Food Chemistry, 103(3), 900-905.http://dx.doi.org/10.1016/j.foodchem.2006.09.042
http://dx.doi.org/10.1016/j.foodchem.200... ; Wang et al., 2009Wang, B. G., Zhang, W. W., Duan, X. J., & Li, X. M. (2009). In vitro antioxidative activities of extract and semi-purified fractions of the marine red alga, Rhodomelaconfervoides (Rhodomelaceae). Food Chemistry, 113(4), 1101-1105. http://dx.doi.org/10.1016/j.foodchem.2008.08.078
http://dx.doi.org/10.1016/j.foodchem.200... ). The total phenolic compound content (TPC) of the seaweeds studied ranged from 16.87 ± 3.2 to 474.46 ± 29.3 mg GAE g^-1 (Table 5), and that of the D. dichotoma extract was significantly higher. This higher TPC of the D. dichotoma extract resulted in its greater antioxidant capacity.

Table 5 shows the total flavonoid and total alkaloid contents of the seaweed extracts . The D. dichotoma extract showed higher total flavonoid and total alkaloid contents. Flavonoids have been reported to be antioxidants, scavengers of a wide range of ROS and inhibitors of lipid peroxidation, and also to be potential therapeutic agents against a wide variety of diseases (Ross & Kasum, 2002Ross, J. A., & Kasum, C. M. (2002). Dietary flavonoids: bioavailability, metabolic effects, and safety. Annual Review of Nutrition, 22(1), 19-34. PMid:12055336. http://dx.doi.org/10.1146/annurev.nutr.22.111401.144957
http://dx.doi.org/10.1146/annurev.nutr.2... ; Williams et al., 2004Williams, R. J., Spencer, J. P., & Rice-Evans, C. (2004). Flavonoids: antioxidants or signalling molecules? Free Radical Biology & Medicine, 36(7), 838-849. PMid:15019969. http://dx.doi.org/10.1016/j.freeradbiomed.2004.01.001
http://dx.doi.org/10.1016/j.freeradbiome... ).

The vitamin C (ascorbic acid) contents presented in table 5 show similar results for both D. dichotoma and L. obtusa, while T. decurrens showed a much smaller amount. Vitamin C is also one of the important antioxidants that are helpful in the growth and repairing of different body tissues, including cartilage, bones, tendons, ligaments and teeth.

3.5 Antioxidant activity assays

Two simple methods were used to evaluate the antioxidant capacity of the algal extracts including the DPPH free radical scavenging activity and the total antioxidant activity of seaweeds and the results are presented in Table 5. Higher values were observed in the D. dichotoma extract, followed by those of L. obtusa and T. decurrens. These results are consistent with those of Parthiban et al. (2013)Parthiban, C., Saranya, C., Girija, K., Hemalatha, A., Suresh, M., & Anantharaman, P. (2013). Biochemical composition of some selected seaweeds from Tuticorin coast. Adv. Applied Scientific Research, 4(3), 362-366. who found that the acetone extract from D. dichotoma showed strong DPPH radical scavenging activity.

The antioxidant activity of the three seaweeds arose from their chemical compositions including their pigment contents (such as carotenoids), vitamin and vitamin precursor contents (such as ascorbic acid), total phenolic compound contents (such as polyphenolics, hydroquinones and total flavonoids), total alkaloid content andthose of other antioxidative substances, which directly or indirectly contribute to the scavenging of both ROS and free radicals (Shahidi, 2008Shahidi, F. (2008). Antioxidants: extraction, identification, application and efficacy measurement. Elec. J. Env. Agricult. Food Chem., 7(8), 3325-3330.). Several reports have shown a close relationship between the total phenolic compound content and high antioxidant activity, and many researchers have demonstrated that phenolic compounds are one of the most effective antioxidants in marine algae (Luo et al., 2010Luo, H. Y., Wang, B., Yu, C. G., Qu, Y. L., & Su, C. L. (2010). Evaluation of antioxidant activities of five selected brown seaweeds from China. Journal of Medicinal Plants Research, 4(18), 2557-2565., Zakaria et al., 2011Zakaria, N. A., Ibrahim, D., Sulaiman, S. F., & Supardy, A. (2011). Assessment of antioxidant activity, total phenolic content and in vitro toxicity of Malaysian red seaweed, Acanthophora spicifera. Journal of Chemical and Pharmaceutical Research, 3(3), 182-191.).

In agreement with these facts, D. dichotoma showed the highest contents of total phenolic compounds, total flavonoids, ascorbic acid, total alkaloids, pigments, (including carotenoids) and total lipids, followed by L. obtusa, which could be the reason for the higher DPPH scavenging and antioxidant activities of its extract. The brown seaweed, T. decurrens, showed the lowest contents of all the antioxidant compounds, and hence its extract showed the lowest antioxidant activity amongst the three seaweeds. The results indicated that the brown alga D. dichotoma can be used as a source of natural antioxidant compounds since its extracts exhibited significant antioxidant activity.

4 Conclusion

In conclusion, the present study provided evidence that the seaweeds under investigation (Dictyota dichotoma, Turbinaria decurrens and Laurencia obtusa) showed promising potential for use as food, animal feed and in medical applications due to their chemical contents. Carbohydrate represented the largest fraction of the dry weight of the seaweeds, followed by proteins and then lipids, except for D. dichotoma, where the lipid content was very close to the protein content. All the essential amino acids were present in the protein fractions, which were especially rich in aspartic and glutamic acids. The saturated fatty acids predominated in all the three seaweeds with an abundance of palmitic acid, followed by the monounsaturated fatty acids with an abundance of oleic acid, and finally the polyunsaturated fatty acids with an abundance of the omega 3 and omega 6 fatty acids. Also, they are a remarkable natural source of minerals and of some antioxidant compounds (phenols, flavonoids, alkaloids, carotenes, Vitamin C) that could be used as functional ingredients and provide dietary alternatives.

D. dichotoma was found to be the most nutritionally rich species, with appreciable protein contents (with large amounts of essential amino acids), a moderate carbohydrate content (with a useful polysaccharide content), large amount of polyunsaturated fatty acids (especially omega-3), with a suitable ω6/ω3 ratio, and also relatively high levels of minerals (with the required Na/K balance) and high antioxidant activity, which makes this species very valuable for human health as a low-calorie food.

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