Seaweeds As A Source Of Bioactive Sulfated Polysaccharides

Sulfated polysaccharides play storage and structural roles in seaweeds and may exhibit many interesting biological properties. As mentioned above, seaweeds are the main source of sulfated polysaccharides in vegetables; thus different amounts of sulfated heteropolysaccharides can be found in green seaweeds (Chlorophyta), while other sulfated polysac-charides such as laminaran, alginate, and fucan are present in brown seaweeds (Phaeophyta) and sulfated galactans such as agar and carra-geenan appear in red seaweeds (Rhodophyta) (Costa et al., 2010).

Several studies have demonstrated that composition—sulfated polysaccharide and other nutrients—and biological properties of seaweed could depend on ripening stage or environmental factors such as geographical localization, seasonal variation, nutritional quality of sea water, and other postharvest factors such as seaweed drying or extraction procedures for phycocolloid preparation (Rioux et al., 2007).

A. Preparation of sulfated polysaccharides from seaweeds

They can be sequentially extracted based on their different solubility. For example, the extraction procedure in the brown seaweed Fucus vesiculosus includes water, acid, and alkali treatments (Ruperez et al., 2002). Thus, laminarans are water soluble, but their solubility depends on branching level: the higher the branching degree, the higher the solubility. Fucans are extracted with diluted hydrochloric acid, while alginates are extracted with alkali. Alginates form insoluble precipitates of alginic acid at low pH, but they are stable in solution between pH 6 and 9. The acid- and alkali-insoluble material from F. vesiculosus contains residual polysaccharides plus cellulose.

For red seaweeds, the solubility of sulfated galactans is dependent on temperature. Thus, highly charged sulfated galactans are soluble in aqueous solution at 20 °C, while those less modified such as agar in Nori (Porphyra spp.) are soluble at 60-80 °C. A neutral galactan from agar, agarose, is soluble at acidic pH. Finally, in most red and brown edible seaweeds, cellulose is the main polysaccharide of the acid- and alkali-insoluble fraction (Rupeerez and Toledano, 2003).

B. Biological activity of sulfated polysaccharides from seaweeds

Bioactivity of sulfated polysaccharides seems to be due to a complex interaction of structural features including sulfation level, distribution of sulfate groups along the polysaccharide backbone, molecular weight, sugar residue composition, and stereochemistry (Jiao et al., 2011). Although research studies dealing with the chemical structure of seaweed polysaccharides have been reported (Deniaud et al., 2003; Lahaye and Robic, 2007; Lahaye et al., 2003; Lechat et al., 2000), relationship between macromolecular structure and biological activity is not clearly established (Jiao et al., 2011).

1. In vitro studies

Relevant pharmacological properties of algal sulfated polysaccharides, such as anticoagulant, antioxidant, antiviral, anticancer, and immunomo-dulating activities, have been reviewed recently (Jiao et al., 2011; Wijesekara et al., 2011). Besides, other less well known biological properties have been described for sulfated polysaccharide, namely, antimicrobial, antiproliferative, anti-inflammatory (Wijesekara et al., 2011), liver protection (Charles and Huang, 2009), effect on glucose (Hoebler et al., 2000; Vaugelade et al., 2000) and lipid metabolism (Amano et al., 2005; Bocanegra et al., 2006; Hoebler et al., 2000; Huang, 2010), and prebiotic effect (Deville et al., 2007).

Anticoagulant. The anticoagulant capacity of sulfated polysaccharides from seaweeds has been the most studied property in an attempt to find an algal substitute for heparin. For example, the anticoagulant activity of fucans was shown to depend on their sugar composition, molecular weight, extent of sulfation, and distribution of sulfate groups in the polysaccharide repeating units (Jiao et al., 2011; Pereira et al., 1999). Marine sulfated polysaccharides other than fucans have also been shown to possess anticoagulant and antithrombotic capacity. Thus, the sulfated galactofucan from a brown seaweed lacks significant anticoagulation activity, making it an ideal candidate as an antithrombotic agent (Rocha et al., 2005). Results suggest that algal sulfated polysaccharides could be an alternative to heparin because they present a promising potential to be used as natural anticoagulant agents in the pharmaceutical industry (Wijesekara et al., 2011). Moreover, the development of antith-rombotic algal polysaccharides would avoid the potential for contamination with prions or viruses ( Jiao et al., 2011) of commercial heparins, currently obtained from pig and bovine intestine.

Antioxidant. Sulfated polysaccharides not only function as dietary fiber, but they also contribute to the antioxidant activity of seaweeds. It has been demonstrated that they exhibit potential antioxidant activity in vitro and several of them derived from brown seaweeds, such as fucoidan, laminaran, and alginic acid, have been shown as potent anti-oxidants (Rocha De Souza et al., 2007; Ruperez et al., 2002; Wang et al., 2008, 2010).

The presence of sulfate groups seems to make feasible the interaction between polysaccharide and target centers of cationic proteins (Mulloy,

2005). Another factor which could specifically modulate the antioxidant activity of sulfated polysaccharide is molecular size (positively at lower size) and the presence of nonsulfated sugar units at polysaccharide terminals (negatively) (Silva et al., 2005). This fact suggests a stereospecificity in anticoagulant activity and not just a quantitative presence of sulfate in the molecule (Costa et al., 2010).

Likewise, the relationship between sulfated polysaccharides and antioxidant capacity in vitro has been shown for red seaweed extracts (Chandini et al., 2008; Rocha De Souza et al., 2007) from Indian seaweeds. Also, the biological activity of sulfated polysaccharides from tropical seaweeds collected in Brazil has been evidenced previously (Costa et al., 2010).

2. In vivo studies in animals and cell model

The detoxifying effect of different seaweeds in a Wistar rat model indicates that the presence of sulfated polysaccharides is crucial in the liver protecting effect of macroalgae (Costa et al., 2010). Other studies have evidenced the protective effect of seaweeds against liver toxicity induced by galactosamine in a rat model, concluding that this protecting effect is partly mediated by fucoidan, a sulfated polysaccharide from the brown seaweed Laminaria (Kawano et al., 2007).

Sulfated polysaccharides from seaweeds are evidenced as protectors of the antioxidant status in a stressed induced rat model (Veena et al., 2007). Besides, the protecting effect of aqueous and organic extracts from brown and green seaweeds against induced oxidation has been studied in cell models (Gunji et al., 2007). Therefore, sulfated polysaccharides from edible seaweeds potentially could be used as natural antioxidants by the food industry (Ruperez et al., 2002).

The influence of seaweed intake on glucose metabolism has been shown in a pig animal model (Amano et al., 2005; Hoebler et al., 2000; Vaugelade et al., 2000). Other studies deal with the effect of edible seaweeds (Kombu (Laminaria spp.) and Nori) and fucoidan from Laminaria japonica on lipid metabolism in a hypercholesterolemic rat model (Amano et al., 2005; Bocanegra et al., 2006; Hoebler et al., 2000) and prebiotic effect (Deville et al., 2007). Prebiotic effect of Laminaria polysaccharide has been shown in the gut metabolism through its effects on mucosal composition, intestinal pH, and short chain fatty acids production (Deville et al., 2007).

III. EDIBLE SEAWEEDS AS POTENTIAL SOURCES OF BIOACTIVE PEPTIDES

Biologically active peptides are food-derived peptides that can exhibit diverse activities, including opiate-like, mineral binding, immunomodu-latory, antimicrobial, antioxidant, antithrombotic, hypocholesterolemic, and blood pressure-lowering actions (Erdmann et al., 2008). Bioactive peptides have been detected in different animal and vegetable protein sources, milk peptides being by far the most commonly known source (Jimeenez-Escrig et al., 2010; Pihlanto et al., 2008).

Heart diseases, such as arteriosclerosis, coronary heart disease, stroke, peripheral arterial disease, and heart failure, may be caused by hypertension or blood pressure greater than 140 mmHg systolic and/or 90 mmHg diastolic pressures (Lo and Li-Chan, 2005). The ACE (dipeptidyl carboxy-peptidase, EC 3.4.15.1) performs an important physiological function in the pathogenesis of cardiovascular and renal diseases through blood pressure regulation. In the renin-angiotensin system, ACE catalyzes the conversion of the inactive decapeptide angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) to the potent vasoconstrictor, the octapeptide angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), by hydrolytic removal of the histidyl leucine group from the C-terminal (Ondetti and Cushman, 1982). Further, ACE is implicated in cell oxidative stress, through the generation of reactive oxygen/nitrogen species (Jung et al., 2006).

Certain biologically active peptides may act as ACE-inhibitory peptides and thus may prevent hypertension and its pathological consequences. ACE-inhibitory peptides from foods are less active than synthetic drugs such as captopril; however, their significance lies in the fact that they meet the need for naturalness and safety (Wu and Ding, 2002).

Edible seaweeds have been considered over the past few decades as promising organisms for providing both novel biologically active substances and essential compounds for human nutrition (MacArtain et al., 2007). However, to date, scarce work of the potential ACE-inhibitory compounds such as biopeptides (Sato et al., 2002a,b; Suetsuna and Nakano, 2000) or phlorotannins (Jung et al., 2006) on seaweeds has been done.

A. In vitro and in vivo evaluation of antihypertensive activities: different approaches

The isolation of protein from seaweeds is a difficult task due to the link between polysaccharides and protein within the seaweed matrix. It is described that the extraction of proteins from the tissues of laminarialean algae (Nagai et al., 2008) or Saccharina japonica (Kim et al., 2011) is difficult due to high levels of nonprotein interfering compounds, mainly viscous polysaccharides. As a consequence, isoelectric point (Ma et al., 1996) or ammonium sulfate saturation (Hernandez-Mireles and Rito-Palomares, 2006) or trichloro acetic acid (Barbarino and Lourenco, 2005) approaches, which are commonly used for protein precipitation, are not completely useful for seaweeds. Thus, to solve this task, different approaches are proposed such as proteolytic treatment of the whole seaweeds followed by filtration and dialysis (Suetsuna and Nakano, 2000) or treatment of seaweed matrix with alginate lyase S to obtain an enriched-protein precipitate which is recovered by centrifugation (Sato et al., 2002a,b).

It is described the identification of ACE-inhibitory peptides derived from Undaria pinnatifida (Wakame), and hypotensive action of orally administered peptides on spontaneously hypertensive rats (SHRs). These studies are based on the previous evidence that dietary ingestion of whole Wakame, one of the most widely eaten brown seaweeds in Japan, has been shown to decrease blood pressure in humans. Specifically, the systolic blood pressure (SBP) of patients decreased significantly after daily oral administration of 3.3 g of dried Wakame after 4 weeks (Nakano et al., 1999). In the work of Suetsuna and Nakano (2000), Wakame powder was digested using pepsin. Then, the filtrate of enzymatic digestion is dialyzed, the outer solution is applied sequentially to a Dowex 50W column H+ form, and peptides were eluted with ammonium solution. After concentration under vacuum, the residue was fractionated on a SP-Sephadex C-25 column and a peptide power was obtained. The fractions having a molecular weight of 300-1000 kDa were collected and concentrated to dryness. The total yield of the peptide powder from 23.6 g of seaweed powder was 3.7 g. The peptides on the most ACE-inhibitory potent fraction were purified further by HPLC with an ODS-5 column. Although approximately 100 peaks were detected by this chromatogra-phy, potent inhibitory peptides were obtained in four peaks. Afterward, using protein sequencing, primary structures of the individual peptides were identified. The amino acid sequences of the peptides were Ala-Ile-Tyr-Lys, Tyr-Lys-Tyr-Tyr, Lys-Phe-Tyr-Gly, and Tyr-Asn-Lys-Leu. All of the active peptides had a tyrosine and lysine residue in the structure. Apart from this research, some peptides with potent ACE-inhibitory activity in vitro or intravenously are inactive in oral administration. Thus, hypotensive activity of each tetrapeptide are evaluated by measuring the SBP on SHR after oral administration of chemically synthesized tetrapeptides [50 mg/kg of body weight (BW)] (Suetsuna and Nakano, 2000). SBP did not change in control rats during the study period (6 h). Captopril (10 mg/kg BW) lowered SBP significantly. A single dose (50 mg/kg BW) of the tetrapeptides significantly reduced SBP in SHR. This work firstly isolated the bioactive peptide and then evaluated the activity of each synthesized peptide in a rat model.

The ACE-inhibitory and antihypertensive activities of Wakame hydro-lysates have been investigated in another study, with a different research design (Sato et al., 2002a,b). To obtain an isolated protein residue, Wakame was treated with alginate lyase S at 45 °C for 18 h and an enriched protein precipitate (46.3% dry matter) was recovered by centrifugation. Then Wakame was hydrolyzed using 17 kinds of proteases at different pH and temperature conditions, and ultrafiltered hydrolysates were tested for the inhibitory activity of the ACE. Among the proteases used in this study,

Wakame hydrolysates of pepsin, protease S and N Amano, and proleather FG-F were able to produce potent ACE inhibitors in vitro. The yield of the different enzymes used ranged from 115 to 239 mg as the weight of the solid contents obtained from 1 g of dried Wakame. In a second step, in order to evaluate the antihypertensive activity in vivo of hydrolysates produced by the four selected proteases, single oral administrations of hydrolysates were given to SHR (n = 6) at dosages of 100 and 1000 mg protein/kg BW. All the Wakame hydrolysates used in this test decreased the SBP in SHR, especially hydrolysates from protease S Amano or proleather FG-F. Digestion stability was evaluated by the change in IC50 values of hydrolysates before and after treatment with gastrointestinal proteases (pepsin, trypsin, and chymotryp-sin) to simulate in vivo resistance to digestion. In addition, a long-term feeding of hydrolysates was assayed on SHR. Seven-week-old SHR were fed a diet containing 0%, 0.01%, 0.1%, and 1.0% of the protease S Amano hydrolysate for 10 weeks. The SBP in the Wakame hydrolysate group tended to be lower than in the control group. Summarizing, there is no correlation between the in vitro and in vivo studies. These results indicated that in vivo experiments— single oral administration test and long-term feeding test—are important for the final evaluation of the antihypertensive effects of peptides. Among 17 proteolytic enzymes tested in vitro, it has been found that hypertension in SHR was suppressed by the Wakame protease S Amano hydrolysates.

Moreover, a study of isolation of potential antihypertensive agents (fucosterol and polyphenols) has been derived from seaweeds: Phaeophyta (Ecklonia stolonifera, E. cava, Pelvetia siliquosa, Hizikiafusiforme, and U. pinna-tifida), Rhodophyta (Gigartina tenella, Gelidium amansii, Chondria crassicaulis, and Porphyra tenera), and Chlorophyta (Capsosiphonfulvescens) (Jung et al., 2006). The study includes the crude extracts of selected edible Korean seaweeds which were screened for ACE-inhibitory activity. Seaweed bio-active constituents are extracted with ethanol followed by partitioning with organic solvents: n-hexane, dichloromethane, ethyl acetate, and n-butanol. Then the fractions extracted were chromatographed over a silica gel column yielding different subfractions and evaluated. In the case of the extract containing phloroglucinol, purification over an RP-18 column is used. Among the tested seaweeds, the ethanol extracts at a concentration of 163.93 mg/mL of E. stolonifera and E. cava appeared to be the most active, with inhibition of 64.86 ± 0.58% and 166.67 ± 4.20%, respectively. With the notable exception of H. fusiforme, the other brown algae P. siliquosa and U. pinnatifida also exhibited favorable ACE-inhibitory activity, between 46% and 53%. Among the red algae tested, only G. amansii exhibited significant ACE-inhibitory effects, with an inhibition of 58.11 ± 1.73%. Column chromatography of the n-hexane and ethyl acetate fractions led to the isolation of fucosterol and six phlorotannins, as phloroglucinol, and its oligomers eckstolonol, eckol, phlorofucofuroeckol A (a pentamer), dieckol (a hexamer), and triphlorethol A (a trimer) from the Ecklonia and

Eisenia species of brown algae. The ACE-inhibitory properties of phlorofu-cofuroeckol A, dieckol, and eckol ranked high, with IC50 values of 12.74 ± 0.15, 34.25 ± 3.56, and 70.82 ± 0.25 mM, respectively. Summarizing, other bioactive compounds, besides peptides, may be responsible for the antihypertensive capacity of seaweeds.

IV. CONCLUSION

The addition to traditional foods of edible seaweeds or seaweed-derived ingredients such as bioactive sulfated polysaccharides or peptides can be considered as a good strategy in order to increase the offer of the functional food market. To date, scarce work of the potential ACE-inhibitory compounds such as biopeptides has been done on seaweeds. The required isolation of protein is a difficult task due to the strong link between polysaccharides and protein in the seaweed matrix, and thus different extraction approaches have been proposed. Regarding antihy-pertensive effects of peptides, there is no correlation between in vitro and in vivo studies. Therefore, further research work on edible seaweeds through the systematic study of their sulfated polysaccharides, biopep-tides, and related biological properties in vitro and especially in vivo will make possible a better knowledge of their potential benefit on human health and will contribute at the same time to their use as natural ingredients for the preparation of novel nutraceuticals.

ACKNOWLEDGMENTS

The Spanish Ministry of Science and Innovation through Project AGL2008-00998 ALI supported this research.

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