Antioxidant activity of Rhodophyceae extracts from Atlantic and Mediterranean Coasts of Morocco

The evaluation of the antioxidant activity of ten aqueous and methanol extracts of the red seaweeds, Pterosiphonia complanata, Boergeseniella thuyoides, Sphaerococcus coronopifolius, Asparagopsis armata, Halopitys incurvus, Hypnea musciformis, Gelidium spinulosum, Plocamium cartilagineum, Gelidium pulchellum and Ceramium rubrum was realized through three different tests. Using the DPPH (2,2-diphényl-1-picrylhydrazyl) test, four methanol extracts allowed the transformation of DPPH radical in reduced form with an EC50 between 96 and 862 μg.mL -1 . With respect to the β-carotene test, 7 methanol extracts showed activity against peroxide radicals with an EC50 between 9 and 176 μg.mL -1 . In the deoxyribose test, the inhibition percentage of hydroxyl radicals varies between 25 and 68% for five aqueous extracts; the most important being the extract of A. armata.


INTRODUCTION
Seaweeds as natural sources with a high degree of bioavailability of trace elements are strongly advised for fast grown children and pregnant women.In contrast to their use as a source of food, marine algae are widely used in the life science as the source of compounds with diverse structural forms and biological activities (Athukorala et al., 2006;Bouhlal et al., 2010a;b;2011).Seaweeds are considered to be a rich source of antioxidants (Cahyana et al., 1992;Devi et al., 2011;Kelman et al., 2012;Murugan and Iyer, 2012;Kim et al., 2012).Consequently, antioxidant activity is intensively focused due to the currently growing demand from the pharmaceutical industry where there is interest in antiaging and anticarcinogenic natural bioactive compounds, which possess health benefits (Heo et al., 2005;Murugan and Iyer, 2012).
Those compounds are widely distributed in plants or seaweeds and are known to exhibit higher antioxidative activities.The activities have been reported through various methods of reactive oxygen species scavenging activity and the inhibition of lipid peroxidation (Yan et al., 1998;Athukorala et al., 2003a;Athukorala et al., 2003b;Heo et al., 2003;Siriwardhana et al., 2003;Siriwardhana et al., 2004;Heo et al., 2005).
The objective of the present study was to investigate the antioxidant properties of ten different Moroccan red seaweeds using three different reactive oxygen species scavenging assays such as DPPH free radical, Deoxyribose and assay with β-carotene-linoleic acid system.

Seaweed material
Seaweeds were collected by hand using Scuba diving or snorkeling (1 to 4 m depth) and preserved on ice until further processing.Ten species were sampled between 2006 and 2007 at various sites, along the Strait of Gibraltar (Ksar-sghir, Belyounech) and on the Atlantic coast (Sidi Bouzid) (Table 1).The taxonomic identification of species was done by experts in these fields, using standard literature and taxonomic keys.Voucher specimens of all species tested are deposited in the herbarium of our Laboratory of Applied Algology-Mycology, Department of Biology, Faculty of Sciences, Abdelmalek Essâadi University, 93002 Tetouan, Morocco (Table 1).Two species belong to the Gelidiales order, three to Gigartinales order, four to Ceramiales order and one to Bonnemaisoniales.

Preparation of extracts
The samples were collection, rinsed with sterile seawater to remove associated debris and necrotic parts.Epiphytes were removed from the algae and the surface microflora was removed by soaking the algal samples for ten minutes with 30% ethanol.The samples were shade dried, cut into small pieces and powdered in a mixer grinder.The powder obtained was preserved cold in -12°C.Samples (5 g) were extracted with solvent methanol (100%) or with water for 8 h using a soxhlet apparatus.The resulting organic extracts were concentrated to dryness under reduced pressure at 30 to 35C° with a rotary evaporator and aqueous extracts were lyophilized.Each residue was weighed and stored in sealed vials in a freezer until being tested.All extracts were stored at (-4°C) (Ozdemir et al., 2004).
To extract polysaccharides from Sphaerococcus coronopifolius and Boergeseniella thuyoides (collected at Belyounech) and after the cleaning and the drying, the seaweed was depigmented with absolute ethanol and acetone.The polysaccharides from seaweed (20 g) were extracted in hot distilled water (1.5 L) at 80 °C for 4 h with magnetic stirring.Insoluble residues were eliminated by filtration and centrifugation (20 min, 30,000 g).The supernatant was poured into 2 volumes of absolute ethanol during one night at 4°C.The precipitate were recovered and washed by absolute acetone, dried overnight at 50°C, weighed and ground to a powder.The polysaccharide was redissolved in distilled water (Bourgougnon, 1994;Haslin et al., 2000).

Antioxidant assay for DPPH radical-scavenging activity
The scavenging effects of samples for DPPH radical were monitored according to the method of the previous report of Duan et al. (2006).Briefly, a 1 ml aliquot in 99% of methanol of test sample was added to 1.0 ml of 0.25 mM DPPH solution (in methanol).The mixture was agitated using a vortex for 1 min and then left to stand at room temperature for 30 min in the darkness, and its absorbance was read at 517 nm.The ability to scavenge the DPPH radical was calculated using the following equation: Where the A control is the absorbance of the control (DPPH solution without sample), the Asample is the absorbance of the test sample (DPPH solution plus test sample), and the synthetic antioxidants, BHT and BHA were used as positive controls.

Antioxidant assay with β-carotene-linoleic acid system
The antioxidant activities of marine algae using the β-carotenelinoleic acid system was measured as in the method as reported by Jayaprakasha et al. (2001) and Chew et al., (2008) with some modification.Briefly, 200 µl of a solution of β-carotene in chloroform (1 mg/ml) in a flask containing 20 mg of linoleic acid and 200 mg of Tween-40.The chloroform was removed by rotary evaporator under vacuum at 50°C for 5 min, and 50 ml distilled water were added slowly to the semi-solid residue with vigorous agitation to form an emulsion.The tubes of spectrophotometer were charged with 200 µl of test sample and 3.8 ml of the emulsion, and the absorbance was measured at 470 nm, immediately, against a blank consisting of the emulsion without β-carotene.The tubes with sample were conserved at room temperature (50°C), and the absorbance measurements were conducted again at 60 min intervals up to 470 min.All determinations were carried out in triplicate.The antioxidant activity (AA) of the extracts was evaluated in terms of bleaching of β-carotene using the following formula:
After that, the tubes were removed and cooled; the absorbance was measured at 532 nm by a spectrophotometer.Mannitol was used as a positive control.

Free radical scavenging activity calculation:
The free radical scavenging activity I(%) was calculated as follows: For the deoxyribose assay, Ai = the absorbance of water extract in deoxyribose assay; Aj = the absorbance of the same water extract mixed with 1 ml distilled water, 1 ml TBA solution and 1 ml TCA solution; Ac = the absorbance of control solution (distilled water) in the assay system.

Antioxidant assay for DPPH radical-scavenging activity
DPPH is a useful reagent for investigating the free radical-scavenging activities of compounds.In DPPH test, the extracts were able to reduce the stable radical DPPH .to the yellow-coloured diphenylpicrylhydrazine.The method is based on the reduction of alcoholic DPPH solution in the presence of a hydrogen-donating antioxidant due to the formation of the non-radical form DPPH-H by the reaction (Shon et al., 2003;Duan et al., 2006).
According to the results (Table 2), we can consider that the methanol extracts of Pterosiphonia complanata and B. thuyoides have significant scavenging activity with EC 50 of 96 and 132 µg.mL -1 , respectively.

Antioxidant assay with β-carotene-linoleic acid system
The antioxidant activity of carotenoids is based on the radical adducts of carotenoids with free radical from linoleic acid.The linoleic acid free radical attacks the highly unsaturated β-carotene models.The presence of different antioxidants can hinder the extent of β-carotenebleaching by neutralizing the linoleate-and other free radicals formed in the system (Shon et al., 2003;Duan et al., 2006;Chew et al., 2008).The antioxidant activities of methanol extracts from ten species, positive control (BHT and BHA) are presented in In conclusion, in this test of ß-carotene, seven extracts showed an activity towards the radicals peroxides with a EC 50 between 9 and 176 µg.mL -1 and three extracts presented no efficiency in the range of concentrations used.

Deoxyribose test
The evaluation of antioxidant activity in the deoxyribose test involves the hydroxyl radical the most active of reactive oxygen derivatives (Yan et al., 1999;Chandini et al., 2008).The effect of ten aqueous extracts (1 mg.mL -1 ) and two polysaccharides on the radical scavenging of hydroxyl (OH) to prevent oxidative degradation of the substrate deoxyribose was determined (Figure 1).The highest percentage of inhibition was obtained by the aqueous extract of A. armata (68.76%), followed by that of B. thuyoides (PS) (35.05%),P. cartilagineum (28.84%), S. coronopifolius (PS) (25.88%),P. complanata (25.37%), S. coronopifolius (24.30%) and B. thuyoides (19.13%).It may be noted that the percentages Rhimou et al. 113 of inhibition of the extracts described above have values superior to the mannitol used as positive control that presents an inhibition of 18.37%.In this test of deoxyribose, there is liberation of the radicals of the hydroxyl group.The aqueous extracts of A. armata, B. thuyoides, P. cartilagineum, P. complanata, S. coronopifolius were involved in the inhibition of the formation of OH groups.Extracts of H. musciformis, G. pulchellum, H. incurvus showed an average percentage of inhibition and the two extracts of C. rubrum and G. spinulosum showed a lower percentage of inhibition towards this type of free radicals.
The antioxidants are effective in protecting the body against damage by reactive oxygen derivatives.There is an increasing interest in natural antioxidants because of health problems and toxicity of synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), generally used in the food domain associated with lipids (Howell, 1986;Ito et al., 1986;Amarowicz et al., 2000).Many natural antioxidants have already been isolated from plants such as oleaginous seeds, cereals, vegetables, leaves, roots, spices and herbs (Wettasinghe and Shahidi, 1999;Shon et al., 2003).Among the most important natural antioxidants are the phenolic compounds, they are widely distributed in plants.
The plants contain various phenolic compounds, including simple phenolics, phenolic acids, anthocyanins, hydroxycinnamic acid derivatives, and flavonoids.All the phenolic classes present structural characteristics of free radical scavengers and have potential antioxidant in the food sector (Bandoniene and Murkovic, 2002;Devi et al., 2011;Lopez et al., 2011).However, natural antioxidants are not limited to the ground sources.Some algae are also considered a rich source of antioxidants (Lim et al., 2002).For example, chlorophylls, carotenoids, tocopherol derivatives such as vitamin E, and isoprenoids extracted from marine algae have shown their efficiency (Shahidi et al., 1992;Farombi et al., 2000;Kaur and Kapoor, 2001;Takamatsu et al., 2003;Canadanovic-Brunet et al., 2006).The antioxidant effect of natural phenolic compounds has previously been studied in relation to the prevention of coronary diseases and cancer, as well as for degenerative disorders relative to the age of brain (Gilani et al., 2000;Kahkonen et al., 2001;Stoclet et al., 2004;Cole et al., 2005;Fraga, 2007;Fusco et al., 2007;  Miyamoto et al., 2007;Stevenson and Hurst, 2007).As part of the screening of antioxidant activity of seaweed collected on the coast of Morocco, we studied ten methanol extracts of algae using the activity of DPPH radical trapping, β-carotene-linoleate assay and the deoxyribose test.
Species belonging to the family of Rhodomelaceae are known for their richness in phenolic compounds, especially bromophenols (De Carvalho and Roque, 2000;Zhao et al., 2004).In our study, the methanol extract of P. complanata (Rhodomelaceae) showed better results than the other species in the tests of DPPH (96.07 µg.mL - 1 ) and β-carotene (9 µg.mL -1 ).In other studies, the crude extract of methanol: chloroform (2:1) and ethyl acetatesoluble fraction of Polysiphonia urceolata showed higher antioxidant activity than the control BHT in the DPPH analysis model and higher than standard antioxidants, gallic acid (GA) and ascorbic acid (ACAA) in the trial of βcarotene (Duan et al., 2006).Bromophenols isolated from P. urceolata were evaluated for their power and DPPH radical-scavenging and showed significant activity with EC 50 values in the range of 9.67 to 21.90 µM, compared to positive control (BHT with EC 50 of 83.84 µM) (Li et al., 2007).The urceolatin of P. urceolata showed a DPPH radical-scavenging activity with an EC 50 of 7.9 µM, ten times higher than the positive control BHT (Li et al., 2008).The fractions of ethyl acetate obtained after separation of the methanol-chloroform (2:1) extract from Rhodomela confervoides gave a strong antioxidant activity in both antioxidants models tested, free radical DPPH and the method of bleaching β-carotene (Wang et al., 2009).The aqueous, methanol and hexane extracts of Digenea simplex (Rhodomelaceae, Ceramiales) gave a percentage inhibition of DPPH radical of 42.3, 41.0, 21.5%, respectively (Al-Amoudi et al., 2009).With respect to Acanthophora spicefera, ethyl acetate fraction obtained from the methanol extract showed a strong antioxidant activity (equivalent to 32.01 mg ascorbic acid / g extract) while the fraction of petroleum ether showed an inhibition of 12% against the DPPH radical (Ganesan et al., 2008).
Moreover, the porphyran, a sulfated polysaccharide extracted from Porphyra haitanesis (Bangiaceae, Bangiales) delayed the aging process in Rhimou et al. 115 mice by increasing the amount of antioxidant enzymes and thereby reducing the risk of peroxidation lipid (Zhang et al., 2003;Zhang et al., 2010).Polysaccharides enhance the antioxidant activity because they exhibited the easy abstraction of anomeric hydrogen from the internal units of monosaccharides.The sulfated polysaccharides of Gracilaria caudata (Gracilariaceae, Gracilariales) gave inhibitions of the hydroxyl radical of 1.4, 2.4, 3.8, 8% for concentrations of 0.05, 0.1, 0.25, 0.5 mg.mL -1 , respectively (Costa et al., 2010).Therefore, algal polysaccharides of S. coronopifolius and B. thuyoides also can be possible active principles by their antioxidant activity moderate.

Conclusion
In our study, the best antioxidant activities were shown by aqueous extracts of A. armata and B. thuyoides with 68 and 35% of inhibition of the hydroxyl groups (OH.) and methanolic extract of P. complanata against the peroxide radicals with an EC 50 of 9 µg.mL -1 and against the DPPH radical with an EC 50 of 96 µg.mL -1 .
AA(%) = 100 [(DRc -DRs)/DRc] with DRc = Degradation rate of the control and DRs = degradation rate of the test.Degradation rate: DR = [ln (a/b)/T] with a = absorbance at t= 0 and b = absorbance at t= T.

Figure 1 .
Figure 1.Scavenging activity of hydroxyl radical (%) of ten aqueous extracts and two polysaccharides of red algae (PS: Polysaccharide).

Table 1 .
List of marine algae screened for antioxidant activity.

Table 2 .
Evaluation of the antioxidant activity in the DPPH (EC 50 ) of ten methanolic extracts.

Table 3 .
Evaluation of antioxidant activity by β-carotene (EC 50 ) with ten methanol extracts of red algae.

Table 3
. The extract of P. complanata presents a relatively important activity (EC 50 of 9.0 µg.mL -1 ) in comparison with the values of BHT and BHA, followed by extracts from G. pulchellum, H. incurvus, B. thuyoides, C. rubrum, G. spinulosum and H. musciformis.While the extracts of P. cartilagineum, A. armata and S. coronopifolius present an EC 50 superior to 1 mg.mL -1 .