A Review with Special Focus on the Iberian Peninsula

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Current Organic Chemistry, 2014, 18(7), 896-917 Bioproducts from Seaweeds: A Review With Special Focus On The Iberian Peninsula

Susana M. Cardosoa,*, Loïc G. Carvalhob, Paulo J. Silvab, Mara S. Rodriguesc, Olívia R. Pereiraa,c and Leonel Pereirab aCERNAS, School of Agriculture, Polytechnic Institute of Coimbra, Bencanta, 3045-601 Coimbra, Portugal; bIMAR (Institute of Marine Research), Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, Apartado 3046, 3001- 401 Coimbra, Portugal; cDTDT, School of Health Sciences, Polytechnic Institute of Bragança, Av. D. Afonso V, 5300-121 Bragança, Portugal

Abstract: Seaweeds, i.e. macroalgae that occupy the littoral zone, are a great source of compounds with diverse applications; their types and content greatly determine the potential applications and commercial values. Algal polysaccharides, namely the hydrocolloids: agar, alginate and carrageenan, as well as other non-jellifying polysaccharides and oligosaccharides, are valuable bioproducts. Likewise, pigments, proteins, amino acids and phenolic compounds are also important, exploitable compounds. For the longest time the dominant market for macroalgae has been the food industry. More recently, several other industries have increased their interest in algal-derived products, e.g. cosmetics, pharmaceuticals and more recently, as a s ource of feedstock for biorefinery applications. This manuscript reviews the chemical composition of dominant macroalgae, as well as their potential added-value products and applications. Particular attention is devoted to the macroalgal species from the Iberian Peninsula. This is located in the Southwest of Europe and is influenced by the distinct climates of the Mediterranean Sea and the Atlantic Ocean, representing a rich spot of marine floral biodiversity.

Keywords: Seaweeds, macroalgae, hydrocolloids, phenolic compounds, proteins, bioactives, fucoidans, ulvans, laminarans.

1. INTRODUCTION drugs of marine origin and the concomitant, exponential investiga- tion fo cusing that issue is p erhaps d elivering its first fruits. This is Seaweeds ( or m acroalgae) ar e aquatic, phot osynthetic or gan- the case for the beneficial properties o f various kelps (Laminaria isms belonging to the Eukaryota Domain and the Kingdoms Plantae spp. a nd Saccharina sp p.) for th e t reatment o f g oiter, w hich ar e (the gree n a nd r ed al gae) and t he Chr omista ( the br own a lgae). now known to be due to the relatively high iodine levels in these Although classification systems have changed over time, it is gen- macroalgae [4]. erally accepted that: a) the green algae are included in the phylum Chlorophyta and their pigmentation is identical to that of terrestrial Today, seaweeds are used in many countries for very different plants (i.e. chlorophylls a, b a nd carotenoids); b) the red algae be- purposes, i ncluding t heir di rect c onsumption a s f ood or s upple- long to the phylum Rhodophyta and their photosynthetic pigments ments (by animals and humans), as feedstock for the extraction of are chlorophyll a and the phycobilins ( i.e. R -phycocyanin and R- phycocolloids, or for their bioactive components and as biostimu- phycoerythrin) and carotenoids, mostly -carotene, lutein and zeax- lants and biofertilizers. Notably, direct use as food has strong roots anthin and c) the brown algae are included in the Phylum Ochro- in the East Asia, whereas the West seems to be more committed to phyta ( or t he H eterokontophyta), C lass Pha eophyceae and t heir the extraction of the h ydrocolloids, namely carrageenan, agar and pigments include the chlorophylls a and c, as well as carotenoids, alginate ( European re gistration nu mbers - E407, E406 a nd E40 0, dominated by fucoxanthin [1, 2]. respectively) [3, 5]. In addition, m any seaweeds are receiving increasing a ttention as a potential, r enewable sources f or t he f ood Seaweeds are fundamental to the food chain of all aquatic eco- industry, as a feed for livestock and as food directly [6]. Industrial- systems. A s pri mary pr oducers t hey pr oduce oxyge n a nd or ganic ized countries are currently increasing efforts regarding the manu- compounds which serve as the basic trophic level or food for many facturing of hi gh-value p roducts der ived fr om algae, since t hese other living beings. They have also found a role of great importance contain chemical components (e.g. polysaccharides, proteins, lipids to mankind. Indeed, coastal communities have been using macroal- and pol yphenols) w ith a w ide ra nge of bi ological ac tivities. Thi s gae in th e p reparation o f h ome medicines fo r th e treatment o f d is- range of activities l eads t o pr omising a pplications i n nut raceuti- tinct ailments for centuries. Green algae are useful as anthelmintics, cal/functional food, cosmetic, and pharmaceutical industries [7-9]. astringents and to treat gout, while brown algae are commonly used (Table 1) reviews t he seaweed or ders reported from t he I berian in th e tr eatment o f rh eumatic p rocesses, arteriosclerosis, m enstrual Peninsula with some known bioactivities. disorders, hype rtension, gas tric ul cers, goi ter, s kin di seases a nd syphilis. In tu rn, r ed alg ae can b e u sed a s an ticoagulants, anti- Moreover, as world energy demands continue to rise and fossil helmintics and for treating gastritis and diarrhea [3]. These applica- fuel resources are increasingly reduced, macroalgae have attracted tions are ba sed on t he em pirical know ledge of m any ge nerations attention, as a possible renewable feedstock to biorefinery applica- and, in m ost cases, the bioactive compounds and their respective tions, for the production of multiple streams of commercial interest mechanisms of action remain unknown. Still, the recent interest for including biofuels such as bioethanol and biogas [10-12], p articu- larly because they have considerable contents of carbohydrates. In this field, macroalgae have several advantages over terrestrial bio- *Address c orrespondence t o t his a uthor a t t he CERNAS - E scola S uperior A grária, mass, primarily because of their potentially high yields, no competi- Instituto Politécnico de Coimbra, Bencanta, 3045-601 Coimbra, Portugal; Tel: +351 239 802940; Fax: +351 273 239 802979; E-mail: [email protected] tion with food crops for the use of arable land and fresh water re-

Table 1. Sytematics of ord ers of I berian P eninsula s eaweeds w ith [6]. The farming of seaweeds has expanded rapidly as demand has some documented bioactivity (after [2]). outstripped the supply available from natural resources. Com mercial har vesting oc curs i n a bout 35 c ountries, s pread be tween t he Phylum/Class Order Genera Species Northern and Southern Hemispheres, in waters ranging from cold, through temperate, to tropical [17]. The consumption and utilization Chlorophyta Bryopsidales 4 13 of seaweed worldwide are associated with a myriad of products that Dasycladale 1 1 generate n early U S$ 8 billion p er y ear [6]. A lmost 9 0 p ercent are food pr oducts for hum an consumption; t he re mainder i s for t he Siphonocladales 1 1 hydrocolloid industry focused on a gar, carrageenan and alginates. Cladophorales 2 9 Macroalgae for direct or indirect consumption (i.e. coloring, flavor- Ulvales 2 8 ing agents and biologically active compounds sold as dietary supplements) have been gaining market share, mainly due to the recog- Ulotrichales 1 1 nition of Man´s seaweed traditional uses in their daily lives. Rhodophyta 1.1. The Macroalgal Biodiversity of the Iberian Peninsula Bangiales 2 4

Ahnfeltiales 1 1 The Ib erian P eninsula is lo cated in the warm temperate, Medi- terranean-Atlantic r egion a nd t he I berian c oasts ar e unde r uni que Bonnemaisoniales 2 3 circumstances, receiving climatic influences from the North Atlan- Ceramiales 22 29 tic O cean ( western, nor th c oasts, a nd a djacent i slands) a nd t he Mediterranean Sea (southern, eastern coasts, and adjacent islands), Corallinales 7 14 thus generating a sharp latitudinal gradient in the macroalgal flora. Gelidiales 3 7 Along the coastline, rocky shores are separated by e xtended areas Gigartinales 18 22 of sandy beaches. Most of the beaches from the western coasts are very exposed and the algae which do occur in the intertidal zone are Gracilariales 2 5 mainly found closest to the low tide level. The intertidal algal flora Halimeniales 1 2 of the northern zone is similar to that of the coast of Central Europe i.e. Nemaliales 5 6 ( B rittany, Fr ance and t he southern pa rts of the B ritish I sles), while the intertidal algal flora of southwestern and eastern coasts is Neamatomales 1 1 very different, responding to a marked influence from the Mediter- Palmariales 2 2 ranean and th e North West African C oast species. T emperate species gra dually dec line in num ber s outhwards along t he W estern Plocamiales 1 1 Iberian coast, where some taxa have their southern limit [18, 19]. Rhodymeniales 5 5 The intertidal flora of the northwestern zones are dominated by Heterokontophyta/ Ascophyllum nodosum, Bifurcaria bifurcata, Himanthalia elongata,

Phaeophyceae Saccorhiza polyschides ( Phaeophyceae, Fuc ales), Gelidium corneum, Gelidium pulchellum ( Rhodophyta, G elidiales), Chondrus Cutleriales 2 2 crispus, Mastocarpus stellatus, Calliblepharis jubata, Gigartina Desmarestiales 1 2 pistillata, Chondracanthus acicularis ( Rhodophyta, Gigartinales), Dictyotales 7 15 Osmundea pinnatifida, Pterosiphonia complanata ( Rhodophyta, Ceramiales) an d Corallina elongata ( Rhodophyta, Cor allinales). Ectocarpales 9 10 The s outhwestern zones ar e dom inated by Corallina elongata Fucales 8 30 (Rhodophyta, Cor allinales), Caulacanthus ustulatus, Chondracan- thus acicularis ( Rhodophyta, Gigartinales), Gelidium pusillum, Laminariales 6 9 Osmundea pinnatifida and Chondria coerulescens ( Rhodophyta, Ralfsiales 1 1 Ceramiales). Codium adherens ( Chlorophyta, Bryopsidales) m ay Sphacelariales 3 3 have a significant presence on the rocky shores in this location [18, 19]. In the southward direction of the Iberian coasts, the number of sources, and utilization of carbon dioxide as the only carbon input species of red algae increase due to the presence of warmer waters [13]. Despite their merits, most macroalgae-based biofuels are rela- and i n a reas subject t o l ess a nthropogenic pr essure, w here t hey tively une xplored re sources. The m ain rea son i s that m acroalgae naturally dominate in numerically over the brown algae and green have several carbohydrates which are distinct from those of terres- algae. With increasing disturbances, the num ber o f red algal taxa trial bi omass sources. H ence, improvement of terrestrial-based decline, ultimately affecting diversity and species richness [20, 21]. technologies in macroalgae or the development of new more effec- Seaweeds a re r esponsible f or a s ignificant proportion of the tive technologies are needed and these are still under evaluation [2, primary production of the Iberian Peninsula. Its traditional co llec- 13-15]. One technical solution that would speed the economic vi- tion and uses were described in the fourteenth century and, in par- ability of this process would be the co-production of biofuels with ticular, th e harvesting of kelp, w hich is st ill done in the north of other higher value products, e.g. the extraction of high-value com- Portugal, was regulated in 1308 by K ing D. Dinis. This usage was ponents or the production of animal feed biomass [16]. constant until the twentieth century [18, 22]. By that time, the lack The industry uses 7.5-8 million tonnes of wet seaweed annually of Japanese Agar during World War II allowed for the emergence [17]. This is harvested either from naturally grown (wild) seaweed, of an Portuguese agar industry, due to the abundance and quality of or from, open-water, cultivated (marine agronomy, farmed) crops local red seaweeds (mainly Gelidium corneum a nd Pterocladiella

Bioproducts from Iberian Peninsula Seaweeds Current Organic Chemistry, 2014, Vol. 18, No. 7 898 capillacea) [23]. However, the unfavourable international economic refined c arrageenan or processed eu cheuma s eaweed). N ote that conditions led to the marked reduction of this industry; today only presently hydr ocolloids are ga ining e ven m ore va lue i n t he food one company persists (i.e. Iberagar - Luso-Spanish Society of Ma- industry (and others) as a result of their potencial as robust func- rine Col loids, SA ) [3, 22] . I beragar i s actually Po rtugal's l eading tional food ingredients. company engaged in the manufacture and distribution of hydrocolloids d erived f rom se aweeds. Ib eragar was created t hrough t he 2.1.1. Sulfated Galactans merger of two companies, B iomar and AGC, and w as established Sulfated galactans are abundant in red algae (but also found in shortly after World War II. In 1970, Iberagar acquired a Japanese brown and green algae), being the most common the agarans (agar) company, Unialgas, and transferred its operations to a plant in Bar- and the carrageenans. Typically, these polysaccharides have a linear reiro, 30 km from Lisbon. backbone with repeating disaccharide units which are m ade of al- The carrageenan production in Iberian Peninsula began in the ternating 3 -linked -D-galactopyranose a nd 4-linked - 1960's, w hen a factory (CEAMSA, M arine A lgae Com pany) was galactopyranose or 3, 6-anhydro--galactopyranose r esidues [36]. established in Galicia, Spain. At the beginning, supply was domi- This “masked repeating” unit of disaccharides was first reported for nated by the local resources of C. crispus and M. stellatus combined the agar-like porphyran stereochemistry [37, 38]. with carrageenophytes imported from Canada and USA. Maximum level of exploitation was attained the 1970's [18, 24]. This activity 2.1.2. Agar declined in the 1980's, mainly due to the competition with develop- This hydrocolloid was the first to be discovered for applications ing t ropical c ountries t hat pr oduced Euchema and Kappaphycus and rec eived it s n ame f rom M alaysia, w here it m eans “r ed alg a”. [24, 25]. The s tructure w as in itially b elieved to b e a s imple, su lfated p oly- Recently some young companies (i.e. Algaplus, Wedotech, Al- galactose pol ymer, but l ater s tudies d emonstrated t hat agar con- gaFuel, among others) are initiating activities in order to harness the sisted of a mixture of at least two polysaccharides, i.e. ag arose an d biotechnological potential of the Iberian m arine flora. In this con- agaropectin [39]. Typically, agarose is the predominant fraction of text, the present manuscript describes the major groups of seaweed- agar (50-90% [40, 41]) and also the responsible for its gelling prop- derived c ompounds t hat ha ve bee n s uccessfully c ommercialized erties [ 41]. It consists of hi gh molecular w eight pol ysaccharides and/or appear to be good ca ndidates f or f uture exploitation a nd composed o f re peating uni ts of (13)--D-galactopyranosyl- commercialization. (14)-3,6-anhydro--L-galactopyranose ( Fig. 1), a lthough some variations c an occur, depending on factors s uch a s t he s pecies of seaweed, as well as environmental and seasonal conditions [39]. In 2. POLYSACCHARIDES turn, agaropectin is a less clearly defined polysaccharide o f lower Macroalgae are known to be rich in polysaccharides, with con- molecular weight than agarose and it has thickening properties [5, centrations that can vary in the range of 4 t o 76% of dry w eight 39]. Its structure is essentially m ade up of alternating (13)--D- [26]. Globally, these are mainly structural cell wall polysaccharides, galactopyranose a nd of ( 14)-3,6-anhydro--L-galacto-pyranose although considerable amounts of mucopolysaccharides and storage residues, where the former can be modified by a cidic side-groups polysaccharides can occur in specific species [27, 28]. which are p rimarily sulfate (up to 32%), plus uronate o r pyruvate groups, as well by non-ionic methoxyl groups [39, 42]. 2.1. Macroalgal Hydrocolloids (Phycocolloids) OH Of all the polysaccharides, macroalgal hydrocolloids, or phyco- CH2OH O O colloids, a re by far t he m ost re levant i n terms of t heir i ndustrial O O O commercialization, with an estimated global value of approximately O OH n $US 1 billion in 2009 and representing more than half of the non- OH food macroalgal market products [29, 30]. Fig. (1). Idealized structure of the chemical units of agarose. Macroalgal hydrocolloids are high molecular weight, structural The gelation mechanism o f ag ar is based on th e ab ility of aga- polysaccharides, found i n t he cell w all o f fr eshwater and m arine rose to form double-helix networks, in w hich each chain forms a algae that t ypically form c olloidal s olutions, i.e. a n intermediate left-handed, three-fold helix [43]. The l atter becomes stable in the state between a solution and a suspension. This property provides presence o f w ater m olecules, bound i nside o f t he doubl e hel ical polysaccharides w ith th e ab ility to be used as thickeners, gelling cavity [44], together w ith exterior, hydroxyl groups w hich permit agents a nd stabilizers f or s uspensions a nd emulsions in diverse the aggregation of up to 10000 helices, with concomitant formation industries, including t he f ood, bi otechnological, pai nt, t extile a nd of microdomains of spherical microgels [45]. biomedics (Table 2). To t he present d ay, hydrocolloids of significant commercial value include the sulfated galactans, agar and car- Remarkably, th e r eversible g els o f agar are formed b y s imply rageenans (obtained fr om re d al gae) a nd t he al ginates (obtained cooling hot aqueous extraction solutions. In general, the agar gels are strong, but their rheological strength is greatly affected by s ul- from brown algae). These are extracted in fairly high amounts from fate su bstitution l evels ( i.e. st ronger gel s are obt ained for t hose various al gal ra w m aterials, w ith m aximum extraction yi elds o b- agars with lower sulfate levels) [39]. Other physicochemical factors tained with hot water or alkaline solutions [31]. affecting the gelling properties of agar include the molecular weight Specific European codes for different phycocolloids, as used in and substitution [46]. Indeed, agar may be modified by substitution varied food industries as natural additives, are E400 (alginic acid), of s ulfate, pyr uvate, ur onate or m ethoxyl gr oups. M odern al kali E401 (sodium alginate), E402 ( potassium alginate), E403 ( ammo- treatment methods tend to increase the level of anhydrous bridging nium a lginate), E404 ( calcium al ginate), E405 ( propylene gl ycol in the m olecule, resulting in an enhancement of ge l strength [39, alginate), E406 (a gar), E407 ( carrageenan) a nd E407A (se mi- 47].

899 Current Organic Chemistry, 2014, Vol. 18, No. 7 Cardoso et al.

Table 2. Applications of Macroalgal Phycocolloids (after [32-35]).

Use Phycocolloid Function

Food additives agar Baked food Improving quality and controlling moisture kappa, iota, lambda carrageenan alginate Promotes flocculation and sedimentation of suspended Beer and wine kappa solids alginate Canned and processed meat Hold the liquid inside the meat and texturing kappa Cheese ka ppa Texturing Chocolate milk kappa, lambda Keep the cocoa in suspension Cold preparation puddings kappa, iota, lambda Thicken and gelling Condensed milk iota, lambda Emulsify Dairy Creams kappa, iota Stabilize the emulsion Fillings for pies and cakes kappa G ive body and texture Frozen fish alginate Adhesion and moisture retention kappa + iota Gelled water-based desserts Gelling kappa + iota + CF agar Gums and sweets Gelling, texturing iota Hot preparation flans kappa, kappa + iota Gelling and improve the mouth-feel Jelly tarts kappa Gelling agar Juices Viscosity, emulsifier kappa, lambda Low calorie gelatins kappa + iota Gelling Milk ice-cream kappa + GG, CF, X Stabilize the emulsion and prevent ice crystals formation Milkshakes lambda Stabilize the emulsion Salad dressings iota Stabilize the suspension agar Sauces and condiments Thicken kappa Soymilk kappa + iota Stabilize the emulsion and improve the mouth-feel Cosmetics Shampoos alginate Vitalization interface Toothpaste carrageenan Increase viscosity Lotions alginate Emulsification, elasticity and skin firmness Lipsticks alginate Elasticity, viscosity Medicinal and Pharmaceutical uses Dental mould alginate Form retention alginate Laxatives Indigestibility and lubrication carrageenan alginate Tablets Encapsulation carrageenan Metal poisoning carrageenan Binds metal HSV alginate Inhibit virus Industrial and Lab Uses Paints alginate Viscosity and suspension, glazing Textiles agar, carrageenan Sizing and glazing

Paper making alginate, agar, carrageenan Viscosity and thickening

Analytical separation alginate, carrageenan Gelling

Bacteriological media agar Gelling

Electrophoresis gel agar, carrageenan Gelling Non-seaweed colloids: CF - Carob flour; GG - Guar gum; X - Xanthan

Bioproducts from Iberian Peninsula Seaweeds Current Organic Chemistry, 2014, Vol. 18, No. 7 900

Agar was, and still is prepared and sold as an extract in solution from a co ld water ex traction of Gracilaria dominguensis, wh ich (hot) or i n g el form (cold), to be use d promptly in areas n ear the inhibited the t ransplantation of Ehrlich ascites carcinoma in m ice factories [6]. The product was known as “tokoroten”. Its industriali- [54]. Agar o-oligosaccharides ( AGO), obt ained by hydrolysis of zation as a dry and stable product began in the early 18th century agar, h ave b een s hown t o suppress t he pr oduction of a p ro- and since then, has been called "Kanten". Presently, "agar-agar" and inflammatory cy tokine an d an en zyme a ssociated with th e p roduc- “agar” ar e th e most ac cepted worldwide te rms. However, it is al so tion of ni tric o xide ( patented by Enoki et al. [5 5]). This an ti- called “ gelosa” i n French- a nd Portuguese-speaking c ountries [6, inflammatory activity o f A GOs w as recently r eported in r ats w ith 31, 48]. chemically-induced c olitis ( by 2, 4,6-trinitrobenzene s ulfonic ac id Agar pr oduction by m odern t echniques of industrial fr eezing (TNBS)) and the results suggested that the o ral administration of (the gel is slowly frozen in order to eliminate water), started in Cali- AGOs could be a possible therapeutic strategy for the treatment of fornia by Matsuoka, who registered his patents in the United States the in flammatory b owel d isease [56]. A GOs’ activity ag ainst - in 1921 and 1922 [39, 48, 49]. During the Second World War, the glucosidase as well as its antioxidant ability has also been demon- lack of a vailable agar w as a s timulus f or c ountries w ith c oastal strated [57]. resources of Gelidum corneum (fo rmerly Gelidium sesquipedale), Currently, a gar i s extracted f rom s pecies of Gelidium and which i s v ery similar to th e Gelidium pacificum used by t he Japa- Gracilaria. Species of Pterocladiella, ar e clo sely related to Gelid- nese industry. In Portugal, the agar industry was started in Oporto, ium and sm all q uantities o f th ese a re co llected, m ainly in th e by Lour eiro, w hile in pa rallel, J. Mejias and F. Ca brero ( Spain) Azores (Portugal) and New Zealand. Gelidiella acerosa is the main started t he es tablishment of the I berian agar i ndustry [48]. Other source of agar in India. Ahnfeltia species have been used in both European countries which did not have access to agarophytes tried Russia ( in par ticular t he i sland of Sa khalin) a nd J apan. Gelidium to prepare agar substitutes from other seaweeds [31, 48]. spp. and Gracilaria spp. are collected in Portugal, Morocco, Tuni- Currently, t he f reeze-thawing technology remains i n use, a l- sia and Chile for agar production [58, 59]. Along the Iberian Penin- though m ost processors have adopted the press/syneresis t echnol- sula, the main agarophytes present are Gelidium corneum, G. spino- ogy (i.e. a method through which the absorbed water can be elimi- sum, G. pulchellum, Pterocladiella capillacea, Gracilaria gracilis, nated by m eans of an applied force), or alternatively, a mixture of G. multipartita, G. vermiculophylla, Gelidiella acerosa and the t wo technologies [5, 48] . While the basi c p rocesses m ay not Ahnfeltia plicata (see Table 3) [3, 4, 19]. have c hanged, i mprovements i n pre sses and f reezing e quipment 2.1.3. Carrageenans must be noted. H igh-pressure m embrane presses have greatly improved the dewatering of agar and thereby reduced energy require- The first formal recognition of the gelling properties of boiled Fucus crispus ments for final drying, prior to milling to powder [29]. w ere discovered by Tur ner in 1809, and this muci- laginous matter w as n amed “carrageenin” by P ereira (1840). The About 90% of the agar produced globally is for food applica- gelatinous, hot w ater-soluble m ucilage o f Chondrus crispus wa s tions [ 16]. The or igin of a gar as a f ood i ngredient w as i n A sia, first isolated by Sc hmidt (1844). Coincidentally, in the same year, where i t ha s bee n c onsumed f or s everal ce nturies [ 5]. A gar has Forchhammer reported on the high sulphur content of the ash from excellent qua lities as a t hickening, stabilizing a nd ge lling a gent, C. crispus [60]. The term “carrageenin” was later ch anged to “car- making it a crucial ingredient in the preparation of processed foods rageenan” so as to comply with the “-an” suffix of terminology as including fruit jellies, dairy products, fruit pastilles, chewing gum, applied to polysaccharides [1, 5]. canned meats, soups, confectionery and baked goods, icings, frozen and salted fish (Table 2) [39, 41]. Moreover, agar has satiating and Chemically, carrageenans ar e h igh m olecular w eight, sulfated gut-regulating properties c ontributing to its c haracteristics a s an D-galactans composed of repeating disaccharide units with alternat- ideal fiber ingredient in the preparation of low-calorie food prod- ing ( 13)- -D-galactose an d (1 4)- -D-galactose o r (14)-3,6- ucts. Furthermore, agar is tasteless and hence it does not interfere anhydro- -D-galactose residues. There are at least 15 different car- with the flavors of foodstuffs, in contrast to some of its competitive rageenan structures that are normally classified on the basis of dis- gums, where the addition of calcium or potassium salts is required tinct features, including the number and position of sulfate groups, to fo rm g els. I t i s also im portant th at ag ar h as b een c lassified a s the pres ence of 3, 6-anhydro-D-galactose a nd the c onformation of GRAS ( Generally R ecognized as S afe) by t he U nited St ates of the pyranose ring [61, 62]. America, Food a nd Drug Administration (FAD), which has estab- The three most relevant commercial carrageenans are kappa (), lished maximum usage levels, depending on particular applications iota ( ) a nd l ambda ( ) carrageenans. The i dealized, d isaccharide [6]. repeating uni ts of t hese car rageenans are gi ven i n ( Fig. 2). - In addition to food applications, about 10% of all agar is cur- carrageenans ha ve al ternating ( 13)- -D-galactose-4-sulfate a nd rently being used for biotechnological applications (e.g. preparation (14)-3,6-anhydro- -D-galactose units [37], w hile the -carragee- of i nert, s olidified c ulture m edia f or bact eria, m icroalgae, f ungi, nans have an additional su lfate group on C-2(O) of the (14)-3,6- tissue culture as well as for separation of macromolecules by elec- anhydro- -D-galactose s ugars, res ulting i n t wo s ulfates per di sac- trophoresis) [ 5, 16] . A lthough, agar applications ar e expected t o charide repeating unit [37]. Moreover, the -carrageenans h ave an increase in the near future, mainly because of the health-associated additional sulfate group linked to the C-6 position of the 4-linked properties c laimed f or t he gel. I ndeed, a gars are not digested by residue, but in turn this is a (14)- - D-galactopyranose [63]. humans and t herefore can be re garded as di etary fibers [50-52]. It should noted that in general seaweeds do not produce these These are water-soluble and were found to be effective in the reduc- idealized and pure carrageenans, but more likely a range of hybrid tion of obesity, hypercholesterolemia, di abetes [53] and intestinal structures and/or precursors (Fig. 2). When exposed to alkali condi- cancer [28]. I t h as b een r eported that ag ar co nsumption l eads to a tions, the precursors (particularly m u and nu f orms) are m odified decrease in the concentration of blood glucose and causes an anti- into ka ppa and i ota, res pectively, t hrough f ormation of the 3 ,6- aggregation effect on r ed blood cells [28]. Antitumor activity was anhydrogalactose b ridge [64-66]. O ther e xisting ca rrageenans in- associated with a highly sulfated, agar-type polysaccharides derived clude the xi (), theta () and beta () (Fig. 2).

901 Current Organic Chemistry, 2014, Vol. 18, No. 7 Cardoso et al.

- O3SO OH

O - O OSO3 - O O3SO OH O O OH OH- O O HO O O - O3SO Enzyme OH - OSO3

Nu-carrageenan Iota-carrageenan

OH OH OH OH O OSO - O 3 O O O - O OH O - O O OSO3 OSO - - HO Enzyme 3 OSO3 - O3SO

Lambda-carrageenan Theta-carrageenan

- OH O3SO O OSO - 3 - OH O O O3SO O - O OH OH O O O HO O OH Enzyme OH - OSO3

Mu-carrageenan Kappa-carrageenan Fig. (2). Idealized structure of the chemical units of nu (), iota (), lambda (), theta (), mu (μ), kappa (), xi () and beta () carrageenans.

The main structural characteristics affecting the chemical and were found to be the ideal stabilizer for the suspension of cocoa in functional properties of carrageenans are the number and position milk chocolate [1, 72]. These polysaccharides are “generally re- of the ester sulfate groups on the repeating galactose units [67]. In garded as safe” (GRAS) and are presently the third most utilized general, the higher the sulfate levels, the lower the temperature of stabilizer/emulsifier agents in the food industry (after gelatin of solubility and the gel strength of the carrageenan [47]. animal origin and starch of plant origin) [32]. The most common Indeed, although all carrageenans are highly flexible molecules, food applications of carrageenans include dairy creams, dessert able to wind around each other to form double-helical zones, the mousses, salad dressings, bakery fillings, ice cream and instant and -carrageenans form gels, whereas -carrageenan does not. Gel desserts (Table 2). Carrageenan gels/emulsifiers also have several formation in -carrageenan requires a gel-inducing agent and in- applications in other diverse industries, including the cosmetic, volves a coil-to-double helix conformational change, followed by pharmaceutical, textile and paints [16, 26]. the formation of an infinite network through aggregation of ordered In addition, the biological properties of carrageens also provide molecules [68, 69]. The strongest gels of -carrageenan are formed them several (potential) applications. Carrageenans are traditionally with K+ rather than Na+, Li+, Mg2+, Ca2+, or Sr2+ [70, 71]. In con- used in the treatment of bowel problems such as diarrhea, dysentery trast, -carrageenan gels are formed in the presence of Ca2+. In this and to make internal poultices to control stomach ulcers [73]. Also, particular case, the 2-sulfate group on the outside of the - carrageenan-bearing seaweeds, namely Irish Moss (Chondrus cris- carrageenan molecule does not allow the helices to aggregate as pus and Mastocarpus stellatus) are traditionally consumed in the well as those of -carrageenan, although additional bonds occur form of teas and other kind of medicines to combat colds, bronchi- through calcium interactions [47]. As a result, -carrageenan gels tis and cronic coughs [26]. Moreover, the anticoagulant activity of are more elastic than those of -carrageenan. At the industrial level, carrageenans and inhibition of human blood platelet aggregation carrageenan gels can be recovered by alcohol precipitation, drum has been reported [74]. Among the carrageenans, -carrageenan drying or freezing. from C. crispus has approximately twice the activity of unfraction- The modern carrageenan industry dates from the 1940´s, receiv- ated carrageenan and four times the activity of -carrageenan (Kap- ing its impetus from numerous dairy applications. Carrageenans paphycus alvarezii – formerly Eucheuma cottonii). The most active

Bioproducts from Iberian Peninsula Seaweeds Current Organic Chemistry, 2014, Vol. 18, No. 7 902

Table 3. Documented and potential Iberian sources of seaweeds products with current or future commercial significance.

Compound Uses Documented Sources Potential Sources

Gelidium corneum (formerly G. sesquiped- Gracilaria spp., Gelidium spinosum, G. pulchel- Agar Thickener, emulsifier, and gelling agent. ale), Gelidium microdon, Pterocladiella lum, Gelidiella acerosa, and Ahnfeltia plicata capillacea (Rhodophyta) (Rhodophyta)

Thickener and emulsifier. Ascophyllum nodosum, Bifurcaria bifurcata, Drug delivery systems. Alginate Laminaria spp. (Phaeophyceae) Laminaria spp., and Saccorhiza polyschides Relevant to meat processing and pet food (Phaeophyceae) production.

Thickener, emulsifier, and gelling agent. Chondrus crispus, Mastocarpus stellatus Chondracanthus spp., Calliblepharis spp., Carrageenan Pet food production. (Rhodophyta) Gigartina pistillata (Rhodophyta) Antiviral.

Anti-aging, antimicrobial, antitumor, antico- Fucoidan Fucales and Laminariales (Phaeophyceae) agulant, anti-inflammatory, contraceptive.

Anti-obesity, antidiabetic, anti-inflammatory, Ascophyllum nodosum, Himanthalia elongata, Fucoxanthin antimalaria, anti-aging, antitumor and neuro- Fucus spp., Laminaria spp., and Undaria pin- protective. natifida (Phaeophyceae)

Used in the cosmetic industry for production Corallina elongata, Gracilaria gracilis, Grate- Phycoerythrin of lipsticks, eyeliners and other cosmetics. loupia turuturu, and Palmaria palmata (Rhodo- Potential use on nutritional supplements. phyta) Phlorotannins Antioxidant, anti-inflammatory, algicidal and Brown alga bactericidal.

Dictyotaceae (Phaeophycaeae) and Lauren- Antitumor, antiviral, and antifouling agent. Terpenoids cia/Osmundea spp. (Rhodophyta)

Antiviral, antitumor, anticoagulant, anti- Ulvan Ulva spp. and other Ulvales (Chlorophyta) hyperlipidemic, and immuno-stimulating.

Chondrus crispus, Mastocarpus stellatus, Chondracanthus spp., Gracilaria spp., Grate- Dietary fibers, proteins, Palmaria palmata, Porphyra spp. (Rhodo- loupia turuturu, Osmundea pinnatifida (Rhodo- Gastronomic use. and vitamins phyta), Laminaria spp., Saccharina latis- phyta), Codium tomentosum, Ulva spp. (Chloro- sima (Phaeophyceae) phyta), Undaria pinnatifida (Phaeophyceae) carrageenan has approximately 1/15 of the activity of heparin [74], gartina skottsbergii, Sarcothalia crispata and Mazzaella laminaroi- an animal-derived highly sulfated, glycosaminoglycan, widely used des are being harvested from natural populations in Chile and Peru, as an in jectable anticoagulant. I t s eems th at th is b iological a ctivity though the recent earthquake in Chile (February 27th, 2010) caused is based on the antithrombotic properties of carrageean [26]. Addi- the elevation of intertidal areas and the consequent large losses of tionally, a pplications of ca rrageenan ge ls f rom C. crispus ma y harvestable biomass [5]. Small quantities of Gigartina canaliculata block the transmission of the human papillomavirus types (that can are h arvested in M exico while Hypnea musciformis has been used cause cervical cancer), of HIV, as well as other sexually transmitted in Brazil [1, 16]. diseases (STD), viruses such as gonorrhoea, genital warts and the Large c arrageenan p rocessors h ave fuelled increased f arming herpes simplex virus (HSV) [26]. activities o f Kappaphycus alvarezii (commercial n ame “cottonii”) The first source of carrageenans was the red seaweed Chondrus and Eucheuma denticulatum (commercial na me “spinosum”) i n crispus, w hich continues to be used in restricted quantities. Beta- several countries including Indonesia, M alaysia, Ph ilippines, T an- phycus gelatinum is used for the extraction of -carrageenan. Pres- zania, K iribati, F iji, K enya and M adagascar [16]. I ndonesia has ently, wild-harvested genera such as Chondrus, Furcellaria, Gigar- recently overtaken the Philippines as the world‘s largest producer of tina Chondracanthus Sarcothalia Mazzaella Iridaea Mastocar- , , , , , dried carrageenophyte biomass. pus, a nd Tichocarpus are, so me of th em, al so cultivated as car- Shortages of carrageenan-producing sea weeds suddenly a p- rageenan ra w materials [64]. P roducing countries i nclude A rgen- tina, C anada, Chi le, D enmark, Fra nce, Ja pan, M exico, M orocco, peared i n m id-2007, c onsequently doubl ing t he pr ice of ca r- Portugal, N orth K orea, South K orea, Spain, Russia, and the U SA rageenans [61]. Probably, this reduced access to carrageenophytes [1, 29]. biomass re sulted f rom a c ombination of e nvironmental fa ctors. Monocultures of s ome ca rrageenophytes, s uch a s Kappaphycus Some South American red algae, which w ere only used tradi- alvarezii tionally in minor quantities, have more recently attracted attention , ha ve encountered s everal p roblems w hen s ubmitted t o from carrageenan producers, as they seek to increase diversification environmental ch anges a s w ell a s an in creased su sceptibility t o of raw material supplies of carrageenophytes with different physical diseases. The problems with ice-ice and epiphytes have resulted in functionalities o f th eir ex tracted g els [16, 6 1]. In th is co ntext, Gi- large scale crop losses [75-77].

903 Current Organic Chemistry, 2014, Vol. 18, No. 7 Cardoso et al.

-OOC -OOC OH OH O OH O OH O O O O O O OH O OH

OH - OH OOC -OOC G GGG -OOC -OOC OH OH HO HO O O O O O O O O HO O HO OH OH -OOC -OOC M M M M -OOC HO -OOC O O OH O O HO - OH O OH OOC O O O OH -OOC OH O OH M G M G Fig. (3). Idealized structure of the chemical units of poly L-guluronic acid (G blocks), poly D-mannuronic acid (M blocks) and alternate D-mannuronic acid and L-guluronic (GM blocks) in alginates.

The m ain carrageenophytes fr om t he Iberian Pe ninsula are Ionic cross-linking with divalent ions (e.g. calcium) is the most Chondrus crispus, Gigartina pistillata, Calliblepharis jubata, C. common method of obtaining hydrogels from an aqueous alginate ciliata, Chondracanthus teedei var. lusitanicus, C. acicularis, Mas- solution, in a m odel t hat i s t ermed “e gg-box” [ 86, 87] . In t his tocarpus stellatus, Gymnogongrus crenulatus, Ahnfeltiopsis de- model, the divalent cations are trapped in a stable, continuous and voniensis a nd Caulacanthus ustulatus (T able 3) [ 1, 64, 23] . It thermo-irreversible, t hree di mensional ne twork, a llowing i nterac- should be noted that, in addition to th e traditionally h arvested c ar- tion with COO- groups of gul uronate residues, of two adjacent G- rageenophytes in the northwest of the Iberian Peninsula (i.e. north- block pol ymers chains (junctions). Thi s results in a ge l st ructure ern coast of Portugal and Galicia) [18, 78, 79], C. teedei var. lusi- [87]. tanicus i s c learly a potential s ource of i ndustrial c o-polymers of Algins/alginates are commercially av ailable in b oth a cid an d carrageenan from the Iberian Peninsula. This algae has a high con- salt fo rms. T hese ar e ty pically ex tracted b y tre ating th e se aweeds tent of kappa/iota and xi/theta carrageenans and it is widespread on with aqueous a lkali s olutions (NaOH) [88] that c onverts a ll the the north coast of Iberian Peninsula [80, 81]. alginate to the sodium salt. Later the salt is dissolved in water and 2.1.4. Uronates (Alginates) separated from the seaweed residue by f iltration [16, 89, 90]. The alginate salt can be transformed into alginic acid by treatment with “Alginate” is the term usually used for the salts of alginic acid, dilute HCl [84]. although this is also commonly used to refer to all the derivatives of About 30 ye ars a go, almost al l extraction of al ginates t ook alginic a cid and t o alginic acid i tself. Some a uthors use t he t erm place i n Eur ope, U SA a nd J apan. Thi s pi cture i s now c hanging “algin” ( i.e. the name given by E. C.C. Stanford to alginic acid by since the emergence of producers in China in the 1980´s [29]. Ini- the time of its discovery, in the 1880´s) [82]. Alginic acid is present tially, this production was limited to low cost (low quality) alginate in the cell walls of brown seaweeds, where it is partially responsible for the internal, industrial markets produced from locally cultivated for th eir flexibility. In th is co ntext, b rown se aweeds th at g row i n Saccharina japonica. In the 1990´s, Chinese producers were com- more turbulent conditions usually have higher alginate content than peting i n w estern, i ndustrial m arkets t o se ll al ginates, p rimarily those in calmer waters [16]. based on low cost [5]. Chemically, alginates are linear copolymers of -D-mannuronic Alginates have several commercial applications based on t heir acid (M ) and -L-guluronic ac id ( G) (14)-linked r esidues, a r- thickening, gelling, e mulsifier and s tabilizing a bilities. They a re ranged either in heteropolymeric (MG) and/or homopolymeric (M used in the food industry for improving the textural quality of nu- or G ) bl ocks (see Fi g. 3) [ 61, 79, 83] . A lginates e xtracted fr om merous products such as salad dressing, ice-cream, beer, jelly and different sources differ in their M and G ratios, as well as on the lactic d rinks, b ut a lso in co smetics, p harmaceuticals, textiles an d length of eac h block. It is noted that more than 200 di stinct algi- painting industries (Table 2) [28, 91]. nates are pr esently pr oduced [84]. I mportantly, m annuronic ac id residues establish -(14) linkages, while guluronic acid forms - Moreover, due to its outstanding properties in terms of biocom- (14) linkages. As a consequence, M-block segments have a linear patibility, biodegradability, non-antigenicity and ch elating abilities, and flexible conformation w hereas the G-block segments cause a the use of a lginates i n a var iety of bi omedical applications (e.g. folded and rigid structural conformation, which is responsible for a tissue engineering, drug delivery and in some formulations of pre- pronounced stiffness of the polymer [47]. It is accepted that only G- venting gastric reflux) is growing [84]. The use of alginates and/or blocks participate in the gel formation and hence, their length is a alginate derivatives as remedies for the t reatment of ga stritis and main factor affecting the functional properties of the gels [85]. gastroduodenal ul cers i s pr otected by pa tents i n se veral c ountries

Bioproducts from Iberian Peninsula Seaweeds Current Organic Chemistry, 2014, Vol. 18, No. 7 904

-OOC O O O O O O O O O HO HO O - OH OSO3 OH - OH OSO3 OH COO- n n A3s B3s

O O O O O O O O O HO O HO OH - OH - OSO - OH OSO3 OSO3 3

n n U3s U2's3s Fig. (4). Structure of the main repeating disaccharides found in ulvan, ulvanobiuronic acids A3s and B3s, and ulvanobioses U3S and U2s 3s.

[72]. A lso, num erous pr oducts of al ginate-containing dr ugs, l ike ents of the polymer [96, 98]. The pr esence of i duronic acid in the “Gaviscon”, have been shown to effectively suppress postprandial ulvan polysaccharide chain is a striking characteristic of these poly- (after ea ting) acidic refluxes, bi nding of bi le acids a nd duode nal saccharides a nd i s uni que a mongst algae [99]. Thi s fe ature a lso ulcers in humans [92]. renders u lvans a close similarity to m ammalian gl ycosaminogly- The bi nding c apacity of a lginates a lso i ncludes c holes- cans. terol/lipids that are th en el iminated fro m th e d igestive sy stem an d In general, the structure of ulvans is influenced by t axonomy result in hypocholesterolemic and hypolipidemic responses, as well (i.e. species used) and ecophysiology (i.e. geographical distribution as an antihypertension effects [72]. This i s often coupled w ith an of s pecies, age/maturity, e nvironmental c onditions, sea sonality, increase in the faecal cholesterol content and a hypogl ycaemic re- etc.) factors [97]. In addition, methods of extraction also have im- sponse. Furthermore, and since alginates bind to divalent m etallic pacts. Thi s obvi ously ha s an e normous i mpact on va riability a nd ions, heavy m etals taken into the human body ar e g elated or ren- hampers th e e stablishment o f an a ccurate structure for th e u lvans. dered in soluble in th e in testines and can not b e ab sorbed in to th e Nevertheless, it i s now ge nerally ac cepted t hat t he bac kbone of body tissue [93]. ulvans is mainly composed of repeating sequences of aldobiuronic Additional biological properties of alginates that might potenti- acid d isaccharides, in p articular o f -D-glucuronosyluronic a cid- ate their applications in the future include their antibacterial activity (14)--L-rhamnosyl-3-sulfate [( -D-GlcpA-(14)--L-Rha-3- - [26], anticancer [28], antitoxic effects on hepatitis [93] and preven- SO3 ), na med as ul vanobiuronic ac id A or A 3s] or of -L- tion of obesity and diabetes [94]. iduronosyluronic acid-(14)--L-rhamnosyl-3-sulfate [(-L-IdopA- p - A good r aw material for alginate extraction should also give a (14)- -L-Rha -3-SO3 ), named as ulvanobiuronic acid B or B3s] high pol ysaccharide y ield. Br own se aweeds t hat fulfill t he above (see Fig. 4). Additionally, other repeating units in ulvans have been criterion i nclude s pecies of Ascophyllum, Durvillaea, Ecklonia, reported, nam ely t he ul vanobiose 3 -sulfate [ U3s, 4)- -D-Xyl- - Fucus, Laminaria, Saccharina, Lessonia, Macrocystis and Sargas- (14)- -L-Rha-3-SO3 -(1]] a nd ul vanobiose 2’ ,3-disulfate - - sum. However, Sargassum is only used as a “l ast resource” because [U2´s3s, 4)- -D-Xyl-2-SO3 -(14)- -L-Rha-3-SO3 -(1] [9 7- its alginate is usually of borderline quality and the yield is also low 100]. [16, 95]. Ulvans form thermoreversible gels, without thermal hysteresis, Ascophyllum nodosum, Fucus spp., Laminaria hyperborea, L. by a uni que a nd c omplex m echanism w hich i s bel ieved t o occ ur ochroleuca, Sargassum vulgare, S. flavifolium, S. muticum, Sac- through t he formation of bor ate es ters w ith ul van 1, 2-diols, f ol- via 2+ corhiza polyschides, Saccharina latissima, Bifurcaria bifurcata and lowed by cross-linking Ca ions [101]. Optimal gelling condi- Padina pavonica r epresent t he m ain al ginophytes w hich occ ur tions thus require the presence of boric acid and calcium ions, at along the Iberian Peninsula (Table 3) [3, 4, 19]. slightly basic conditions (pH 7.5). Since the gel is thermoreversible, the “junction-zones” that crosslink the polymer are thought to in- 2.1.5. Ulvans clude w eak linkages, probably based on l abile borate ester groups The name “ulvan”, as first proposed by L ahaye and Axelos in and io nic in teractions th at are e asily d isrupted b y th ermal t reat- 1993, refers to acidic w ater-soluble single sulfated heteropolysac- ments [97]. charides w hich ar e p resent in th e cell w alls o f g reen se aweeds Overall, the gelling properties of ul vans offer them a potential (Ulva, previously known as Enteromorpha), where it contributes to application where texture needs to be precisely controlled (by pH or the m aintenance o f the osmolar stability and the protection of the temperature), such as th ose d esigned fo r th e release o f entrapped cell [96]. The yield of ulvans ranges from 8 to 29% of the algal dry molecules or par ticles unde r s pecific physi cochemical c onditions weight [97]. [26, 102]. Com mercial applications of these gels are undoubtedly The s ugar composition of ul vans i s extremely var iable but fewer than those o f other hydrocolloids, although other properties uronic acids (i.e. glucuronic acid and iduronic acid), sulfated rham- of t hese pol ysaccharides pr ovide them w ith pot ential industrial nose, xylose and glucose have been identified as the main constitu- applications in several areas, including the chemical, pharmaceuti-

905 Current Organic Chemistry, 2014, Vol. 18, No. 7 Cardoso et al. cal, biomedical and agricultural, amongst others [103-105]. Indeed, the ulvan chemical bonds within the cell wall and hence facilitate fine c hemicals m ay b e pr oduced from rare, sugar prec ursors o b- their solubilization [99]. A n increased yield of u lvans i s l ikewise tained from ulvan biomass. Ulvan is enriched in rhamnose (a rare obtained for low pH extraction solutions, due to the de-stabilisation sugar) which is used in the synthesis of aromas [97]. The produc- of ulvan aggregates [98]. In general, the most common method for tion of this sugar from Monostroma has been patented. L-rhamnose extracting u lvans employs the use of hi gh temperature water (80- is also an essential component of the surface antigens of many mi- 90°C), containing ammonium oxalate as a divalent cation chelator, croorganisms, and is specifically recognized by a number of mam- followed by the recovery of the polysaccharides by ethanol precipi- malians l ectins. H ence, u lvan applications i n ph armaceutical d o- tation [102]. A dditional purification processes are fundamental t o mains ar e expected to in crease [97]. A part from th e rh amnosyl clean-up the contaminating matter, such as lipids and pigments (as units, ulvan is also a source of iduronic acid, which is another rare removed by Soxhlet extraction, or by organic solvents - acetone or sugar re quired i n t he s ynthesis of he parin a nalogues w ith a nti- ethanol), proteins (proteinase k) and starch (-amylase) [99]. thrombotic activities [100]. Regarding t he m acroalgae of t he I berian Pe ninsula, ul vans Besides monomers, it has been demonstrated that ulvan (poly- could be produced from Ulva clathrata, U. compressa, U. intesti- mer) and its oligosaccharides have applications related to their bio- nalis, U. lactuca, U. linza, U. prolifera, U. rigida ( Chlorophyta, logical pr operties, i ncluding a ntitumor a nd i mmune m odulation, Ulvales) and Monostroma latissimum (Chlorophyta, U lotrichales). anticoagulant, antioxidant, strain-specific anti-influenza, hepatopro- These sp ecies a re d istributed o n an almost worldwide b asis, g row- tection, protection of t he colonic mucosa, modulation of lipid me- ing in the intertidal and subtidal zones, attached to hard substrata or tabolism and a decrease of the atherogenic index [96, 97, 106]. as free-living forms. In addition, they are also considered opportun- Notably, t he s trategy of c hemical cr oss-linking ul vans ove r- istic seaweeds and proliferate in eutrophic coastal w aters. D espite comes previous limitations for their application in tissue engineer- this, a nd t aking i nto account i ts bi otechnological pot ential, gr een ing (due to mechanical instability and uncontrolled di ssolution i n algae remain l argely u nexploited in the commercial a rena, p rovid- physiological c onditions) [107]. Alves a nd c o-workers c ombined ing an opening window of opportunity for future research [99]. ulvan w ith pol y-D,L-lactic ac id ( PDLLA) i n or der t o pr oduce a novel scaffold for bone-tissue engineering applications. This matrix 2.2. Fucoidans was then characterized (by micro-computed tomography, mechani- Fucoidans a re a co mplex se ries o f su lfated p olysaccharides cal compression testing, w ater uptake and degradation testing and found widely in the cell walls of brown seaweeds, where they are cytotoxicity assays); the results demonstrated appropriate physico- thought to play a protective role against the effects of d esiccation chemical and cy tocompatible f eatures fo r th e en visaged ap plica- [26]. These pol ysaccharides were f irst isolated by K ylin in 1913 tions [104]. In addition, ulvan particles loaded with dexamethasone and named as “fucoidin”. Presently, they are mainly named accord- dispersed w ithin th e P DLLA m atrix sh owed an ad equate r elease ing t o t he I UPAC t erminology ( fucoidans), although ot her t erms profile of the steroid drug, suggesting that this system can be poten- such as f ucans, fucosans, f ucose containing pol ymers or sulfated tially suitable for localized drug delivery [104]. Cross-linked ulvan fucans have also been adopted [110]. membranes also confirmed their potential as drug delivery devices and suggest a great potential of these natural sulfated polysaccha- For th e majority o f a lgal so urces, the chemical composition o f rides in wound dressings [105]. These results further contribute to fucoidans i s mainly co mposed o f fucose an d su lfate, to gether w ith the establishment of ulvan as a potential novel biomaterial. minor amounts of distinct molecules (Table 4), that can vary from monosaccharides ( i.e. m annose, gl ucose, g alactose, xyl ose, etc.), Applications of ul vans in animal feed detoxification were also acidic monosaccharides, acetyl groups to proteins [110]. Fucoidans patented, bas ed on t he ca pacity of u lvan to i ntercalate into clay. with a low fucose content are also found in nature. This property opens the way for the synthesis of new nanocompo- sites o f in terest w ith u se in d ifferent a reas. Moreover, b esides th e Notably, the structure of f ucoidans has been described to vary traditional u se as a fertilizer, the elicitation of pl ant d efenses has significantly b etween m acroalgal species and ev en w ithin species, been added to ul van bi oactivities, i ncluding ni trogen uptake i m- even though the latter can b e m ainly attributed to the distinct ex- provement and disease resistance [97]. traction conditions applied [9, 110]. The m echanisms b y w hich u lvans in terfere w ith th e d ifferent Regardless of the enormous v ariability of fucoidans, two gen- biological systems are yet to be identified. They may do it in difer- eral groups are assumed for their backbones: The type I polymers, ent ways, su ch a s ta rgeting specific ce ll receptors w here ulvan which c ontain a l arge pr oportion of re peating ( 13)-linked -L- competes amongst other molecules and/or physicochemical proper- fucopyranose re sidues and t he t ype I I polymers which typically ties related to particular ion-exchange. The latter mentioned interac- have alternating (13)- and (14)-linked -L-fucopyranose resi- tions are at the basis of choice of these seaweeds as bioindicators dues. In such polymers, sulfation may occur at positions 2, 3 and 4 for monitoring coastal water heavy metal pollutions [73, 106, 108] and the monosaccharides are associated via -12, -13, or - and could be further exploited to develop ion exchangers from ul- 14 glycosidic bonds [113]. In general, Type I fucoidans are found valean cell walls, with particular ion selectivity for industrial efflu- in Saccharina latissima, Analipus japonicas, Chorda filum, Cla- ents de-pollution or the enrichment of food, feed or soils with spe- dosiphon okamuranus and Laminaria digitata, w hile T ype II fu- cific trace mineral elements [109]. coidans include those isolated from the order of Fucales (i.e. Asco- phyllum nodosum Fucus In ge neral, ul vans ca n be s olubilized by hot w ater [99], w ith a nd s pecies) [37, 114] . I ndividual higher m olecular w eight pol ysaccharides b eing obt ained i n t he representative structures of Type I and II fucoidans are depicted in range 80- 90°C, w hen c ompared t o t hose extracted w ith t empera- (Fig. 5). tures above 100°C [97]. Ulvans can also be extracted with sodium Fucoidans with a high content of uronic acid (UA) and hexose carbonate solution, with calcium chelating agents (e.g. ammonium may ha ve a bac kbone bu ilt o f a lternating U A-hexose, d ue to th e oxalate or ethylenediaminetetraacetic) or with acidic solutions [98, high stability of this structure. Other monosaccharide residues are 100]. Calcium chelating agents sequester calcium ions and disrupt normally observed in the fucoidans branches [110].

Bioproducts from Iberian Peninsula Seaweeds Current Organic Chemistry, 2014, Vol. 18, No. 7 906

Table 4. Composition of some fucoidans and/or water extracts from brown seaweed (based on [110-112]).

Seaweed Specie Order Chemical Composition*

Adenocystis utricularis Ectocarpales fucose, galactose, mannose, xylose, GlcA, sulfate

Ascophyllum nodosum** Fucales fucose (49%), xylose (10%), GlcA (11%), sulfate

Bifurcaria bifurcata** Fucales fucose, xylose, mannose, glucose, galactose, sulfate

Chorda filum** Laminariales fucose, xylose, mannose, glucose, galactose, uronic acid, sulfate

Cladosiphon okamuranus Ectocarpales fucose, glucose, uronic acid, sulfate

Dictyota menstrualis** Dictyotales fucose/xylose/galactose/sulfate (1/0.5/2/2)

Ecklonia kurome Laminariales fucose, galactose, mannose, GlcA, glucose, xylose, sulfate

Fucus distichus Fucales fucose/sulfate/acetate (1/1.21/0.08)

Fucus evanescens Fucales fucose/sulfate/acetate (1/1.23/0.36)

Fucus serratus** Fucales fucose/sulfate/acetate (1/1/0.1)

Fucus spiralis** Fucales fucose, xylose, mannose, glucose, galactose, uronic acid, sulfate

Fucus vesiculosus** Fucales fucose,sulfate

Himanthalia elongata** Fucales fucose, xylose, GlcA, sulfate

Laminaria hyperborea** (formerly Laminaria cloustonii) Laminariales fucose, galactose, xylose, uronic acid, sulfate

Laminaria digitata** Laminariales fucose, xylose, mannose, glucose, galactose, uronic acid, sulfate

Lessonia flavicans (formerly Lessonia vadosa) Laminariales fucose/sulfate (1/1.12)

Macrocystis pyrifera Laminariales fucose/galactose (18/1), sulfate

Padina pavonica** Dictyotales fucose/galactose, sulfate (9/1/9)

Saccharina angustata (formerly Laminaria angustata) Laminariales fucose, galactose, mannose, xylose, GlcA, sulfate

Saccharina religiosa (formerly Laminaria religiosa) Laminariales fucose, xylose, mannose, glucose, rahmnose, uronic acid, sulfate

Sargassum acinarium (formerly Sargassum linifolium) Fucales fucose, mannose, galactose, xylose, uronic acid

Sargassum fusiforme (formerly Hizikia fusiformis) Fucales fucose/xylose/uronic acid/galactose/sulfate (1/0.8/0.7/0.8/0.4) and (1/0.3/0.4/1.5/1.3)

Sargassum stenophyllum Fucales fucose, galactose, mannose, sulfate

Silvetia babingtonii (formerly Pelvetia wrightii) Fucales fucose/galactose (10/1), sulfate

Undaria pinnatifida** Laminariales fucose, mannose, xylose, rhamnose, galactose, glucose, sulfate

Undaria pinnatifida (Mekabu) Laminariales fucose/galactose (1/1.1), sulfate

*Fucoidans were obtained by acidified or alkali/water solutions, followed by precipitation, mostly with ethanol. **Present in the Iberian flora.

Fucus vesiculosus is the seaweed m ost en riched in fu coidans being randomly sulfated or ace tylated in the position 4 (see struc- (up to 20% on a dry w eight basis). Thi s polysaccharide w as first ture in Fig. 5B) [ 110, 120] . I nstead, F. distichus i s m ainly co m- - believed to comprise a linear structure mainly composed of (12)- posed of di saccharide repeating units 3)--L-Fucp-(2,4-di-SO3 )- - linked 4-O-sulfated fucopyranose residues [115], but later Patankar (14)--L-Fucp-(2-SO3 )-(1, where only slight variations m ight et al. [116] rebuilt its structure model and established that the back- exist by r andom ac etylation a nd s ulfation of se veral di saccharide bone of t his f ucoidan w as a f ucose pol ymer, bonde d t hrough - repeating units (see structure in Fig. 5B) [37, 121]. (13) w ith a s ulfate gr oup, s ubstituted a t C-4 , in sev eral fu cose The fu coidan from F. serratus L . h as a b ranched structure. Its residues and with branched fucose (linked to fucose) moieties ap- core ba ckbone, as de picted i n ( Fig. 5B), i s m ainly c omposed of pearing in every 2-3 residues. More recently, Chevolot et al. [117] 3)--L-Fucp-(14)--L-Fucp-(1 repeating units, in which half reported that the fucoidan from F. vesiculosus (and of Ascophyllum of t he 3-l inked residues ar e s ubstituted by -L-Fucp-(14)--L- nodosum) ha ve a core di saccharide m otif of Type II c ontaining Fucp-(13)--L-Fucp-(1 t rifucoside units at C -4 [119]. Sulfate sulfate at the 2-position of the 3-linked fucose and sulfate groups on groups oc cupy m ainly C -2 and s ometimes C -4, a lthough 3, 4- the 2- a nd 3-positions of the 4-linked fucose (see structure in Fig. diglycosylated and some terminal fucose residues m ay be nonsul- 5B) [37, 118]. fated. A cetate gr oups oc cupy C -4 of 3- linked Fuc a nd C -3 of 4- Structural features of fucoidans from other Fucus sp ecies have linked Fuc in a ratio of about 7:3 [119]. also been described. As reported by Bi lan et al. [119-121], the fu- Atypical fucoidans w ere de scribed f or se veral sea weeds, e.g. coidans fro m F. evanescens C. Ag, F. distichus and F. serratus L. Himanthalia elongata and Bifurcaria bifurcata, fo r w hich the fu- are a lso co mposed o f fu cose, su lfate an d a cetate, w ith st ructural coidan structure h as a m ain bac kbone of ( 12)- a nd ( 13)- variations. In p articular, the fu coidan from F. evanescens has a alternating fucose residues with sulfation at C-4 and (14)-GlcA - linear backbone of alternating 3)--L-Fucp-(2-SO3 )-(14)--L- and (14)-Xyl linked non-sulfated residues appear at the periphery - Fucp(2-SO3 )-(1 disaccharides, with the 3-linked fucose residues of highly branched molecules [110, 122].

907 Current Organic Chemistry, 2014, Vol. 18, No. 7 Cardoso et al.

A O

O - OSO3 O O - O3SO O O O OH O OH HO O O HO - O O3SO -O SO O OH 3 O O - O - OSO3 O OSO3 OH OH - - O3SO O O3SO O - O3SO O O O O O O - O OSO3 O HO - - HO COO O3SO OH O -O SO O HO 3 O OH OH OH O OH O Saccharina latissima Chladosiphon okamuranus - O3SO Chorda filum O O O B O OSO - O O 3 O OSO - - 3 OH OSO3 O OH O - O - OSO3 OSO3 O O OSO - O OH 3 O OSO - O - 3 O OSO3 R - O O3SO O OSO - HO O 3 O - O - OSO3 - O3SO O OSO O - 3 OSO3 O OH OH - O O OSO3 - O OSO3 O OSO - O OH 3 O OSO - O - 3 O OSO3 R - O O3SO O HO O OSO - O 3 OSO - O 3 O OSO - O - 3 HO HO OSO3 O OH O - O OSO3 R = -L-Fucp-(1 4)--L- -Fucp-(1 4)--L-Fucp-(1 Ascophyllum nodosum Fucus evanescens Fucus distichus Fucus serratus Fucus vesiculosus Fig. (5). Representative chemical structures of Type I (A) and Type II (B) fucose-containing sulfate polysaccharides described in several seaweed species.

The extraction method for fucoidans can be quite simple. A na- In general, structural-bioactive studies suggested that the anti- tive extraction with hot water can result in a good m ethod, but an coagulant/antithrombin activities of fucoidans are mainly dependent acid e xtraction or a c ombined hot a cidic extraction w ith e thanol on the content and/or positioning of sulfate groups, as w ell as the precipitation is the most commonly applied method [110, 113]. The molecular w eight of t he pol ymer [110]. M oreover, m onomeric variation of extraction methods is known to result in the extraction composition, types of linkages and branching might exert moderate of structurally distinct fucoidans [110, 113]. modulation on bi ological properties of fucoidans [30, 37, 114]. In The co mmercial im portance o f fu coidans is p resently m uch this co ntext, it i s p ossible th at th e g reater an ticoagu- lower than that of seaweed hydrocolloids however, these polysac- lant/antithrombin activities exhibited by longer fucoidans are due to charides are attracting considerable attention because of the grow- the hi gher content of f ucose a nd sulfate groups [37, 110, 114] , ing market fo r them a s b ioactive p olysaccharides in wide areas o f though this is still under debate. applications [30]. More recently, anticoagulant and antithrombotic Fucoidans are also reported to inhibit the replication of several activities are the most studied effects of fucoidans. Commonly, th e enveloped vi ruses s uch a s hum an i mmunodeficiency and hum an anticoagulant activity of fucoidans is mediated through the activa- cytomegalovirus, among others [37, 110]. The mechanisms for such tion of thrombin inhibitors, although direct thrombin inhibition and activity are thought to occur via inhibition of cell infection by viral competitive b inding of fibrinogen to block thrombin’s actions are sorption or due to hampering of viral-induced syncytium formation also possible [30, 123]. [110, 114] . Fuc oidans from Saccharina japonica, Cladosiphon Previous studies reported that the anticoagulant functionalities okamuranus, Adenocystis utricularis, Stoechospermum margi- of fucoidans ex tracted fro m F. vesiculosus and Eklonia cava were natum, Cystoseira indica, Dictyota mertensii, Lobophora variegata, due t o t hrombin-inhibition-mediated via p lasma an tithrombin-III, Fucus vesiculosus, Spatoglossum schroederi and Undaria pinnati- and their anticoagulant activity was similar to that of heparin [37, fida (cultivated plus wild types) showed impressive positive results 110, 114] . M oreover, fucoidans fr om Saccharina longissima, S. in vitro and in vivo models of infection by poliovirus III, adenovirus latissima, L. digitata, F. serratus, F. distichus, F. evanescens and A. III, ECH O6 virus, coxsackie B3 vi rus, coxsackie A 16, N ewcastle nodosum were also described to reveal strong anticoagulant activity Disease Virus (NDV), HSV-1, HSV-2, HIV and avian reverse tran- in vitro and in vivo models [110, 113]. scriptase [110, 114].

Bioproducts from Iberian Peninsula Seaweeds Current Organic Chemistry, 2014, Vol. 18, No. 7 908

Antitumor activities o f fucoidans include th e in hibition o f t u- Laminarans can occur in soluble or insoluble forms, being the mor proliferation, the stimulation of tumor cells apoptosis, blocking first totally soluble in cold water while the second can be s olubi- of tumor cell m etastasis and enhancement of va rious immune re- lized with hot water [127]. The solubility is influenced by br anch- sponses [124]. I n this context, f ucoidans f rom s everal macroalgal ing, w ith b etter s olubility be ing obse rved f or h igher b ranched species (e.g. Saccharina japonica, S. latissima, Laminaria digitata, polymers [127]. Laminarans contain polymeric chains of two types, Fucus serratus, F. distichus and F. vesiculosus) proved to be useful i.e, th e G-chains which ar e b uilt o nly o f Glcp residues and the M- and are regarded as good candidates for future cancer therapy [110, chains, w ith 1 -O-substituted, D-mannitol re sidue at the terminal 114]. Be sides those, t he commercial f ucoidans bra nded Toki da reducing e nd ( Fig. 6) [ 128]. La minarans f rom di fferent s eaweeds (from cultured Cladosiphon okamuranus) and that from the Korean may v ary with r egard to their structural features, such as th e M :G cultured s porophyll (Miyeokgui) of Undaria pinnatifida al so re- ratio, degree of branching and molecular weight [103, 127]. vealed promising antitumoral activities, as te sted in in vitro models [114]. A OH OH OH OH Other important biological activities of fucoidans include anti- HO O OH O oxidant, anti-inflammatory and anti-allergic, although others cannot HO O O HO be ove rlooked ( e.g. he patoprotection, cardioprotection, st omach OH OH OH OH n protection and anti-obesity) [37, 110, 114]. Examples of fucoidans M-Chains showing pr omising a ntioxidant i n in vitro m odels i nclude t hose obtained from S. japonica, Canistrocarpus cervicornis, F. vesiculo- Mannitol sus, Dictyota cervicornis, Sargassum filipendula, Dictyopteris deli- catula and S. japonica [37, 110, 114], while the anti-inflammatory B OH OH activity o f sev eral fu coidans ( Saccharina latissima, L. digitata, HO O OH O Fucus evanescens, F. serratus, F. distichus, F. spiralis, Ascophyl- HO O OH HO O O lum nodosum, Cladosiphon okamuranus and Padina gymnospora) OH HO OH has b een d escribed t o o ccur t hrough t he i nhibition of leucocyte OH n recruitment in an inflammation model in rats [108]. Moreover, in- G-Chains Glucose hibition of the expression of inducible nitric oxide synthase (iNOS) has a lso b een d emonstrated fo r fu coidans, such a s th at fro m th e Fig. (6). Chemical structures of laminarans of two types of chains: mannitol Sigma-Aldrich Chem ical Co. (from F. vesiculosus). F urthermore, (A) o r ( B) glucose is attached to the reducing end of the M-chains or G- commercial fucoidans (from Mekabu and Sigma-Aldrich Chemical chains, respectively. Co) to gether w ith th ose iso lated fro m A. nodosum, F. evanescens, Laminarans are presently attracting commercial interest because C. okamuranus and from other several brown algae of Laminariales of some of their (or their derivatives) potential biological activities. order w ere described to exhibit anticomplementary activities, ren- These include antioxidant, antitumor, antimicrobial, immune modu- dering them potential as anti-allergic agents [37, 110, 114]. lation, drug delivery and anticoagulant properties [26, 129, 130]. In Overall, fucoidans h ave a num ber of pot ential applications, particular, in vitro studies revealed the interaction of laminarins in mainly associated to their claimed h ealth benefits. A lthough there tumor cell metabolism, which suggest active functions in metabolic are o nly a few s tudies fo cusing o n fu coidans i solated fro m Ib erian pathways l eading to apoptotic cell death of the tumor cells [130, brown seaweeds (mainly Ascophyllum nodosum, see T able 3) th e 131]. Lee et al. [132] also reported that laminaran stimulated and previously r eported da ta obt ained f rom m acroalgae of t he sa me strengthed t he i mmune s ystem t hrough m etabolic e xpression a nd species (with an origin outside Iberian Peninsula), genera or or der pathway interactions. Moreover, as for other seaweed polysaccha- might indicate promising perspectives for Iberian specimens. Stud- rides, laminarans are not digested by t he human digestive system, ies with Iberian fucoidan-bearing macroalgae should be undertaken, i.e. i t is a na tural fiber [125] a nd he nce i mproves the gas tro- in order to prove their pot ential for commercial exploitation (e.g. intestinal health by de creasing the production of putrefactive com- nutraceuticals, s upplements o r e ven i ncorporated i nto pr ocessed pounds, know n to induce cancer. Furthermore, they stimulate the foods). Moreover, t he cl aims f or h ealth b enefits of seaweeds are growth of favorable intestinal m icrobiota such as Bifidobacterium probably t he bes t w ay to stimulate t heir c onsumption as f ood i n strains [125, 133- 135]. N ote t hat antioxidant fi bers are not onl y occidental countries, including the Iberian Peninsula. Candidates of important for human digestive health, but also for animals, includ- edible brown macroalgae, occurring on the Iberian Peninsula com- ing l ivestock a nd fish. The fact t hat laminarans ha ve antioxidant prise: Fucus vesiculosus, Undaria pinnatifida, Saccharina latis- properties might even attract the meat industry to feed their animals sima, Laminaria s p. and Himanthalia elongata. N ote th at th ese with supplements containing laminarans since, besides the healthy such ut ilization w ould not onl y contribute t o a healthier di et but improvement of the animals’ intestines, it acts on t he conservation also to positive development for the local economy. of t he m eat after t he animal i s sl aughtered [133-135]. I n t his in- stance, application of the polysaccharides could be an alternative to 2.3. Laminarans other synthetic antioxidants and antibiotics with known toxicity in Laminarans are the main storage polysaccharide of brown algae the meat industry. e.g. Laminaria Saccharina ( spp., spp.) [26, 125]. Their content can There are even feasible ap plications o f l aminarans in th e agri- represent up t o 32 -35% of dr y w eight, w ith va riations o ccurring cultural field. Th ese polysaccharides h ave been shown to provide between growth seasons of the seaweed [26, 103]. Laminarans are protection a gainst p athogens due to t he stimulation of specific small glucans, with a degree of polymerization varying between 20 metabolic pathways of plants which result in the expression of spe- to 50 uni ts. Th e st ructure of these pol ysaccharides includes - cific compounds known to trigger the defense responses of plants to (13)-linked gl ucose, containing r andomly -(16) in tra-chain branching [126]. pathogens [103].

909 Current Organic Chemistry, 2014, Vol. 18, No. 7 Cardoso et al.

HO OCOCH3

Fig. (7). Chemical structure of fucoxanthin [147].

The m ain Iberian m embers o f th e L aminariales o rder in clude: backbone. D egradation m ight be t riggered by diverse external Saccharina latissima, Laminaria hyperborea, L. ochroleuca and L. agents such as high temperature, high pressure, light and the pres- digitata; and th e m ajor member o f th e Tilopteridales o rder is Sac- ence of acid or oxygen. In this sense, storage and processing condi- corhiza polyschides. The i ndustrial application of t hese seaweeds tions can compromise the stability of fucoxanthin resulting in oxi- locally are m ostly focused on bi oethanol a nd bi ogas p roduction dative de gradation a nd i somerization [ 26]. I ndeed, t he l evels of [136]. There are also applications for the cultivation of these brown fucoxanthin w ere reported t o significantly dec rease after dr ying seaweeds with salmon, where the macrolagae can enrich the waters [149]. Moreover, light and pH decreases were reported to degrade of the aquaculture site with oxygen while also providing bioreme- the pi gment, poss ibly related t o trans-cis i somerization reactions diation services and reducing local pollution (eutrophication) from [150]. These modifications currently limit the use of pure fucoxan- the salmon. T his system h as b een c alled th e in tegrated, m ulti- thin as a n ingredient o f f unctional f ood pre parations [151]. It i s trophic aquaculture ( IMTA) s ystem. The seaweed bi omass pr o- noteworthy that the stability of fucoxanthin might be improved in duced in this system can be sold as a direct food resource [137]. the presence of other organic ingredients such as polyphenols [151]. Moreover, h arvested L. hyperborea is commonly used for supple- The content of fucoxanthin in seaweeds greatly differs between mentation of pig food [133, 138] and in cosmetics [61]. species, with reported contents between 0.022 and 3.7 mg g-1 of dry weight [26, 152-154]. Also, the fucoxanthin content can be highly 3. PIGMENTS variable during the season and life cycle of the macroalga. In gen- As previously mentioned, seaweeds are generally classified ac- eral, the levels of fucoxanthin increase from winter to spring (ma- cording to their pigment composition, which include chlorophylls, ture phase of the sporophyte) and decrease during summer (during carotenoids and phycobilins. The majority of these compounds have the senescence phase) [154, 155]. been c ommercialized f or m any ye ars f or c oloring pur poses, but Several in vitro and in vivo experiments suggested that fucoxan- importantly, th e in terest in th eir co mmercial ap plications has sig- thin ex erts important h ealth-promoting ac tivities, mainly d ue to it s nificantly increased in recent decades as promising applications in antioxidant properties [156]. The pigment has been shown to pos- human health are being established. In this context, fucoxanthin is sess a st rong ability to scavenge or quench DPPH• radicals, nitro- probably the main macroalgal pigment under the spotlight of sev- benzene with linoleic a cids r adical a dduct ( NB-L) and 12-doxyl- eral industries. steric acid (1 2-DS). It is generally a ccepted that it s an tioxidant ca- pacities are closely associated to the presence of the unique double 3.1. Fucoxanthin allenic carbon i n i ts structure t hat combined w ith t wo hydr oxyl groups, confer additional stability and resonance stabilization [157]. Fucoxanthin is an orange-colored accessory pigment, belonging to t he xa nthophylls (c arotenoids) and one of t he m ost abundant, Besides being a good a ntioxidant, bioactivities reported for fu- representing about 10% of their total natural production [139-143]. coxanthin also include anti-obesity, antidiabetic, anti-inflammatory, It is the major carotenoid of edible brown seaweeds, where it binds antimalarial, anti-aging, antitumural and protective effects on liver, to several proteins, together with chlorophyll a i n the thylakoid of brain, bones, skin and eyes [26, 30, 151, 157, 158]. A s a result of its chloroplasts. Fucoxanthin was first isolated from the marine brown claimed health-associated properties, the pigment is being evaluated seaweeds: Fucus, Dictyota, and Laminaria by Willstätter and Page for further use as a f ood supplement, as a therapeutic agent in the in 1914 [144] and its complete structure was elucidated by Englert treatment of obe sity, m etabolic s yndrome, di abetes a nd w rinkle et al. [145]. Fr om t he structural poi nt of vi ew, t his compound is formation [30]. unique, with an unusual allenic bond and some oxygenic functional Fucoxanthin, as other algal carotenoids, is commonly extracted groups such as epoxide, hydroxyl, carbonyl and carboxyl moieties with hexane and other non-polar solvents, by liquid solvent extrac- (Fig. 7) [146]. tion. T he so lvent d isrupts th e cell m embranes an d d issolves lipids, Due to the presence of double bonds in the polyene chain of the lipoproteins and the membranes of chloroplasts [159, 160]. Special carotenoid, fucoxanthin m ight exist in trans and/or cis configura- care must be taken in the extraction procedure (e.g. low temperature tions. A s fo r carotenes in g eneral, t he trans forms of f ucoxanthin and being kept in the dark) due to the high instability of f ucoxan- are thermodynamically more stable than the cis counterparts due to thin. Alternative methods such as the enzyme-assisted and micro- reduced steric hindrance [148]. Accordingly, all-trans fucoxanthin wave-assisted extractions a nd pre ssurized l iquid extraction t ech- has been isolated from several seaweed sources and in particular, it niques have been used in an attempt to minimize the degradation of has been shown to account for 88% of the total fucoxanthin in fresh the fucoxanthin [140, 156, 161, 162]. U. pinnatifida [26]. As re ported [ 139-143, 147] , t he s pecies Undaria pinnatifida, Fucoxanthin i s extremely vul nerable to de gradation w hich Fucus vesiculosus, Sargassum siliquastrum, S. fulvellum, S. fusi- mainly occ urs by oxi dative cleavage a nd/or e poxidation o f t he forme, Himanthalia elongata, Eisenia bicyclis, Laminaria digitata,

Bioproducts from Iberian Peninsula Seaweeds Current Organic Chemistry, 2014, Vol. 18, No. 7 910

Saccharina japonica and Ascophyllum nodosum can be ca ndidates enization, o r th e u se o f enzymes (e.g. l yzozymes) [168]. Phyc o- for f ucoxanthin e xtraction. Fr om t he re ported s pecies, t he ones biliproteins ar e th en p urified, u sually b y ch romatographic m ethods present in Ib eria P eninsula a re th e A. nodosum, H. elongata, F. [170], or by the use of novel techniques such as immuno-absorption vesiculosus, L. digitata and U. pinnatifida [71]. and genetic recombination. From the reported data, Corallina elongata, Gracilaria gracilis, 3.2. Phycobiliproteins Grateloupia turuturu and Palmaria palmata are present on the Ibe- Red algae are rich in phycobiliproteins, i.e., water soluble pig- rian Peninsula and hence, can be considered potential candidates for ments found in the cytoplasm or in the stroma of the chloroplasts, the extraction and applications of phycobiliproteins [4]. which ar e formed by c omplexes of phyc obilins w ith c ovalently bound proteins. Che mically, phyc obilins a re open-chain t etrapyr- 4. PROTEINS role chromophores bearing A, B, C and D rings. These chromopho- Besides the commercial applications of phycobiliproteins, mac- res l ink to the polypeptide chain at conserved positions either b y roalgae ha ve ot her pr oteic compounds w hich ha ve a pr omising one cysteinyl thioester linkage through the vinyl substituent on the potential for exploitation. Indeed, macroalgae (in particular certain pyrrole ring A or occasionally, by t wo cysteinyl thioester linkages of the green and reds) have relatively high protein contents, ranging through the vinyl substituent on both A and D pyrrole rings [163]. from 9-26% (w⁄w) dry weight (green) or reaching up to 47% (w⁄w) The phycobilins a re th e m ain co mponent d etermining th e co lor o f dry weight (red) [174]. Some of these contents are even higher than phycobiliproteins. Based on their ab sorption properties they can be those found in high-protein conventional foods, e.g. eggs, beans or blue (phycocyanobilin), red (phycoerythrobilin), yellow (phycouro- fish [174, 175]. A ccording to the literature, the levels o f seaweed bilin) or purple (phycobiliviolin). Molecular pigments are organized proteins are determined by seasonality, with the highest contents in in su pra-molecular co mplexes (i.e. phycobilisomes) and they exert general being observed for the winter period [174, 176]. a fundamental role in the photosynthetic process of the red algae. R-phycoerythrin (Fig. 8) is the most common phycobiliprotein Additionally, the majority of seaweeds contain all the essential in many red algae, w ith levels, on a dry weight basis, of approxi- amino acids. In particular, isoleucine and threonine can be found at e.g. Palmaria palmata mately 0.2% for Polysiphonia stricta and Pyropia (Porphyra) ye- similar levels ( in ) to those found in legumes, e.g. Ulva pertusa zoensis, 12% for Palmaria palmata and Gracilaria gracilis and of while histidine levels ( in ) can be as high as those 0.5% for G. tikvahiae [164-168]. found in egg proteins [174]. Some seaweeds (mainly browns) may also have high levels of ac idic amino acids, i.e. a spartic a cid an d O glutamic acid [174]. The latter have much interest in flavor devel- HO O OH opment processes and in particular, glutamic acid (mainly responsible for the taste sensation of ‘umami’) is presently being used as a food additive i n the f orm of i ts s odium s alt ( E-621) [ 177]. The combined gl utamic ac id and a spartic acid l evels account f or 22 - 44% wet weight of the total amino acid fraction in Fucus sp . an d NH HN 39-41% in Sargassum spp., whilst both amino acids together repre- O N H sent 18% of the total amino acid content in L. digitata proteins [26]. N O Likewise, certain m acroalgae produce several u seful enzymes, from w hich ha loperoxidases ar e pr obably of m ajor re levance. Haloperoxidases are rare enzymes that catalyse the oxidation of a

halide ( i.e., chloride, bromide, or iodide) by hydr ogen peroxide, a Fig. (8). Chemical structure of R-phycoerythrin [169]. process that results in the concomitant halogenation of organic sub- strates [178]. These enzymes are crucial in the synthesis of com- R-phycoerythrin, t ogether w ith ot her phyc obiliproteins, h ave pounds of biological importance that are difficult to be synthesized been used for decades as n atural colorants in foods (e.g chewing by the conventional methods of or ganic chemistry [178]. Haloper- gum, ice creams, soft drinks, fermented milk products, milk shakes, oxidases also have powerful applications in qualitative and quanti- desserts, j ellies and coated sweet cakes [170, 171]), cosmetic and tative diagnostic assays (e.g. glucose, uric acid and cholesterol), as pharmaceutical products. In general, the colors are very stable and it generates intense colored products with appropriate subtrates. tolerate high temperatures, pH changes and light [171]. Moreover, R-phycoerythrin h as s pecialized a pplications in a nalytical t ech- In addition, some macroalgal protein hydrolysates and their as- niques such as flow cytometry, cell sorting and histochemistry [30]. sociated pe ptides m ight e xert i mportant bi oactive pr operties, pro- C-phycocyanin, R- and B -phycoerythrin are currently used in the viding t hem w ith c ommercial pr ospects as f unctional foods. The cosmetic i ndustry for production of l ipsticks, e yeliners a nd other main b eneficial pr operties of pr otein of macroalgal ori gin w ere high value cosmetics [168]. recently reviewed by H arnedy a nd Fi tzGerald [8]. Th ese include Biological properties of phycoerythrin and/or phycobiliproteins ACE-inhibitory, a ntihypertensive, a ntioxidant, antitumor, antity- include a ntioxidant, a nti-inflammatory, neuroprotective, immuno- rosinase, anticoagulant, c alcium-precipitation-inhibitory, a n- modulator, antiviral, antitumor, cardiovascular and liver protection timutagenic, plasma- and hepatic-cholesterol reducing, blood-sugar- [168, 171- 173]. D ue t o their bi ological pr operties, m any pa tents lowering, and superoxide dismutase (SOD)-like activities. have been established, towards applications of these pigments for The main seaweeds with high protein content from the Iberian nutritional supplements and therapeutic agents [170]. Peninsula are: Porphyra (Pyropia) umbilicalis (i.e. A tlantic n ori), Typically, extraction of phycobiliproteins comprises the disrup- Palmaria palmata (Rhodophyta), and Himanthalia elongata, Sac- tion of cells and a primary isolation from the algae by chemical and charina latissima, Undaria pinnatifida ( Phaeophyceae), and Ulva physical t echniques. The extraction yield can be improved by t he compressa (Chlorophyta) [5, 30]. "Atlantic nori" from the Iberian addition of other processes such as freezing, sonication and homog- Peninsula i s, fo r n ow, a w ild seaweed (as compared to J apanese

911 Current Organic Chemistry, 2014, Vol. 18, No. 7 Cardoso et al.

OH HO HO OH

HO OH OH HO OH OH O OH HO O OH HO OH

HO OH OH OH OH HO OH Phloroglucinol Tetrafucol A Eckol OH OH OH OH HO OH

HO O OH O OH OH OH OH HO OH O OH

Fucophloroethol A HO OH HO OH OH

Fucodiphloroethol G OH O HO OH O OH

OH O O O O OH OH OH OH

O O HO O OH OH OH 7-Phloroeckol OH HO OH OH

Phlorofucofuroeckol A OH O HO OH O OH

Triphloroethol-A O O OH HO OH O OH O OH

OH O OH OH O OH Dieckol OH

HO O OH Fig. (9). Chemical structure of phlorotannins (adapted from [185]). equivalent species w hich have been cultivated since the X V cen- Phlorotannins are only found in the brown algae and have been tury). Note t hat nor i i s one of t he m ost a ppreciated a nd hi ghly identified in d istinct f amilies, in cluding: th e A lariaceae, F ucaceae commercially valued algae, due to its high content in minerals and and Sargassaceae. In particular, the species Eisenia bicyclis, Ishige proteins, together with intense flavor, aroma and smooth texture. okamurae, Sargassum thunbergii, S. fusiforme, Undaria pinnatifida and Saccharina japonica, as well as algae belonging to the genera 5. PHLOROTANNINS Cystophora a nd Ecklonia, ar e recognized as good s ources of phlorotannins [183]. Relevant phlorotannins encountered in Ecklo- Chemically, phenolic compounds are organic compounds char- nia cava [184], E. stolonifera [185], Eisenia bicyclis a nd Fucus acterized by a n a romatic r ing w ith one or m ore hydr oxyl gr oups vesiculosus [ 186] are re presented i n ( Fig. 10) an d comprise: that can oc cur i n di fferent chemical structures [179-181] va rying phloroglucinol, t etrafucol A , f ucophlorethol A , f ucodiphloroethol between si mple phe nolic m olecules t o complex hi gh-molecular G, phl orofucofuroeckol A , 7 -phloroeckol, ec kol, di eckol a nd weight pol ymers (126-650 kD a) [182]. Phl orotannins consist of triphlorethol-A. oligomers or polymers of phloroglucinol (Fig. 9), which are known The l evels of phl orotannins var ies g reatly be tween di fferent to b e lo cated in sp ecial v esicles ( i.e. phys odes) of t he cells [26, taxonomic groups and geographical areas. Variations also occur in 110]. the same species and depending on f actors such as plant size, age,

Bioproducts from Iberian Peninsula Seaweeds Current Organic Chemistry, 2014, Vol. 18, No. 7 912

Table 5. Mineral composition of some Iberian Peninsula, edible seaweeds (g.100 g1 DWa or mg. 100 g1 DWb). Adapted from [5].

Species Na a Ka Pa Ca a Mg a Fe b Zn b Mn b Cu b Ib

Green seaweed

Caulerpa racemosa 2.6 0.32 0.03 1.9 0.38 – 1.6 30 – 81 1 – 7 4.91 0.6 – 0.8 -

Ulva lactuca - 0.14 0.84 - 66 - - - -

U. rigida 1.6 1. 6 0.21 0. 52 2.1 283 0.6 1.6 0.5 -

Brown seaweed

Fucus vesiculosus 2.5 – 5.5 2.5 – 4.3 0.32 0.72 – 0.94 0.67 – 1.0 4 – 11 3.71 5.50 <0.5 14.5

Himanthalia elongata 4.1 8. 3 0.24 0. 72 0.44 59 - - - 14.7

Laminaria digitata 3.8 11. 6 - 1.0 0.66 3.29 1.77 <0.5 <0.5 -

Undaria pinnatifida 1.6 – 7.0 5.5 – 6.8 0.24 – 0.45 0.68 – 1.4 0.41 – 0.69 1.54 – 30 0.94 0.33 0.19 22 – 30

Red seaweed

Chondrus crispus 1.2 – 4.3 1.4 – 3.2 0.14 0.42 – 1.1 0.60-0.73 4 – 17 7.14 1.32 <0.5 24.5

Gracilaria spp. 5.5 3. 4 - 0.40 0.57 3.65 4.35 - - -

Palmaria palmata 1.6 – 2.5 7.0 – 9.0 0.24 0.56 – 1.2 0.17-0.61 50 2.86 1.14 0.376 10 – 100

Porphyra umbilicalis 0.94 2.0 0. 24 0.33 0.37 23 - - - 17.3 Na – sodium; K – potassium; P – phosphorus; Ca – calcium; Mg – magnesium; Fe – iron; Zn – zinc; Mn – manganese; Cu – copper; I – iodine. tissue type and environmental factors such as nutrient, light, salin- ver, it is also a result of seaweed’s oceanic residence time and type ity, water depth and season [187]. of processing [202]. The interest in phlorotannins h as increased in the last decade The va lue of edible se aweeds in hum an nut rition i s base d, due to their potential biological activities, as d escribed by se veral among others (e.g., dietary fiber, vitamins, etc.), on their high con- authors. In more detail, the compounds eckol, doxinodehydroeckol tent in several (essential) minerals, namely: Na, Mg, P, K, I, Fe, and and dieckol from E. bicyclis have b een shown to have interesting Zn [202]. Seaweeds also contain l arge am ounts of t race elements deodorizing ef fects [168]. M oreover, phl orotannin compounds [198] that are scarce in vegetables and hence, algal-based supple- identified in d ifferent Ecklonia s pecies w ere re ported t o pos sess ments can provide to humans the daily requirements of these min- important antimicrobial activities against several pathogens [188], erals [203]. as well as an antidiabetic effect in in vitro and in vivo models [189], Seaweeds h ave h igher m ineral co ntent th an ed ible t errestrial antioxidant [183], hypnot ic [190], h epatoprotective [191], anti- plants and animals. Indeed, mean ash (and thereby mineral) content inflammatory [192] and for lowering blood pressure [193] capaci- of most traditional vegetables is frequently much lower than that of ties. O verall, these claimed effects for phlorotannins render these seaweeds (e.g. 10.4% in potatoes, 2.6% in sweet corn, 7.1% in car- metabolites w ith m any potential future applications in several in- rots and i n t omatoes) [ 202]. O nly t he hi gher val ues obser ved i n dustries. spinach (20%) are comparable to those of seaweeds [202]. Rupérez Traditionally, phlorotannins have been extracted using ethanol, [198] also showed that ash content of many algae was elevated and methanol or aqueous acetone as a s olvent [139, 186], while addi- higher in b rown (i.e. 30.1-39.3%) rather than in red (20.6-21.1%) tional purification is commonly achieved by chromatographic tech- seaweeds. niques [194, 195] . D ue t o t he l ow st ability of phl orotannins, t he Most algae display higher Na and K values than those reported extracts are frequently obtained with nitrogen or by adding K2S2O5 in ve getables. H owever, their N a/K ra tios are u sually l ow [204]. or ascorbic acid in order to prevent oxidation [196, 197]. Additionally, most edible algae have higher levels of Mg (0.5-100g-1 DW) than terrestrial plants and animals [205]. Ca is one of the ma- 6. MINERALS jor elements in algae and is present at concentrations of about 0.4-2 -1 Marine algae are known to contain a wide variety and high lev- g.100g DW (Table 5). The Ca and P contents of certain seaweeds els of ce rtain m inerals (t his m ay va ry f orm 8 -40% of a lgal dry are higher than those of apples, oranges, carrots, and potatoes. High weight (DW) and have therefore been employed as m ineral addi- Ca/P ratio in algae (3:5) could compensate the deficit of Ca in sev- tives to feed and food supplements [198]. Their high mineral con- eral f oods, s uch as ce reals a nd m eats [201]. I odine (I ) can re ach tent is related to their capacity to retain inorganic marine substances high levels in certain brown algae [206], whereas green algae pre- [199] due to the ionic and exchange capacity features of their cell sent l ow or none xistent val ues ( Table 5). M embers of the g enus Laminaria surface polysaccharides [200]. are the strongest iodine accumulators among all living systems (the accumulation can be up t o 30,000 times greater than Nevertheless, the mineral composition of macroalgae varies ac- the surrounding sea water, and brown algal tissue can represent a cording to phylum and even amongst species [201] as well as many major source of this element [207]. The uptake of dietary iodide by other factors s uch as e nvironmental a nd phys iological variations, the human and animal thyroid leading to thyroid hormone forma- geographic harvesting site, seasonality and wave exposure. Moreo- tion, is a well-established phenomenon. L. digitata is widely used as

913 Current Organic Chemistry, 2014, Vol. 18, No. 7 Cardoso et al. a health supplement for myxoedema and for the treatment of goiter [4] P ereira, L. Guia Ilustrado das Macroalgas - Conhecer e reconhecer algumas espécies da flora portuguesa; Imprensa da Universidade de Coimbra: Coim- [208]. It has bee n reported that U. pinnatifida (or i ts e quivalent bra, 2009b. iodine content) inhibited t umorogenesis i n rats w ith car cinogen- [5] Pereira, L. A review of the nutrient composition of selected edible seaweeds. induced mammary tumors, although the mode of action is not un- In: Seaweed; Pomin V . H ., E d.; N ova Science Publishers Inc: N ew Y ork, 2011; pp. 15-47. derstood [209]. [6] FAO The state of world fisheries and aquaculture 2010, FAO: Rome, 2010; One negative aspect of seaweed consumption could involve the pp. 197. [7] Laurienzo, P. 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Received: October 08, 2013 R evised: February 13, 2014 Accepted: February 14, 2014