Biological and Therapeutic Properties of the Seaweed Polysaccharides Leonel Pereira1*

Home , Chondrus crispus, Eucheuma, Furcellaria, Gelidium amansii, Tokoroten

Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 1 of 50

REVIEW ARTICLE Biological and therapeutic properties of the seaweed polysaccharides Leonel Pereira1*

Author’s affiliations: 1. MARE (Marine and Environmental Sciences Centre), Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal

* Corresponding author: Leonel Pereira, E-mail: [email protected]

Abstract Seaweed polysaccharides isolated from the cell walls of various species of algae, possess immunomodulatory, anti-inflammatory, antiviral, antitumor, antithrombotic, anticoagulant and antioxidant bioactivities. Within the group of polysaccharides extracted from algae there are the phycocolloids. Collo- ids are extracted compounds that form colloidal solutions, an intermediate state between a solution and a suspension; they can be used commercially as thickeners, gelling agents and stabilizers for suspensions and emulsions. Hydrocolloids are carbohydrates that when dissolved in water form viscous solutions. Sulfated galactans (e.g. agars, carrageenans, porphyrans) can be obtained from red algae, (e.g. ulvans) from green algae, alginates and other sulfated polysaccharides (e.g. ascophyllan, laminaran and fucoidan) are obtained from selected brown algae. The historic origin of the main phycocolloids and other seaweed polysaccharides (e.g. agar, carrageenan, alginic acid - alginates), their chemistry, uses and bioactivities are described in this review.

Keywords: Macroalgae; Hydrocolloids; Sulfated galactans; Sulfate content; Bioactivities

1. Introduction a) the green algae are included in the phylum Chlorophyta and their pigmentation is Seaweeds (or macroalgae) are aquatic pho- identical to that of terrestrial plants (chloro- tosynthetic organisms belonging to the do- phyll a, b and carotenoids); main Eukarya and to kingdoms Plantae (green and red algae) and Chromista (brown b) the red algae belong to the phylum Rho- algae). Although classification systems dophyta and their photosynthetic pigments have varied greatly over time and according are chlorophyll a, phycobilins (R-phyco- to the authors, it is generally agreed that: cyanin and R-phycoerythrin) and caroteno-

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 2 of 50 ids, mostly β-carotene, lutein and zeaxan- amount from the algae. All these polysac- thin; charides are water soluble and could be extracted with hot water or alkaline solution c) the brown algae are included in the phy- [11]. lum Ochrophyta (or Heterokontophyta), class Phaeophyceae and their pigments en- The historic origin of the main phycocollo- close chlorophylls a, c and carotenoids, ids (agar, carrageenan, and alginate) and dominated by fucoxanthin [1-3]. other seaweed polysaccharides (e.g. ulvan, porphyran, fucoidan, laminaran, ascophyl- Macroalgae are known to be enriched in lan), their chemistry, uses and bioactivities polysaccharides, with concentrations that are described in this review. can vary in the range of 4% to 76% of dry weight [4]. Globally, these are mainly structural cell wall polysaccharides, although 2. Polysaccharides from green algae considerable amounts of mucopolysaccha- (Chlorophyta) rides and storage polysaccharides can occur in specific species [3,5,6]. 2.1. Ulvan The seaweed hydrocolloids, associated with Ulvan is a sulfated, water-soluble polysac- the cell wall and intercellular spaces, are charide which have physio-chemical and mainly produced by red and brown algae. biological features of great potentials in Several species of red algae (Rhodophyta) food, pharmaceutical, agricultural, and produce galactans (e.g. carrageenans and chemical applications [12,13]. agars) and of brown algae (Phaeophyceae) Ulvan may represent 8 - 29% of algal dry produce uronates (alginates) [7-10]. weight and is produced by some species Sulfated galactans (e.g. agars and carragee- belonging to the Phylum Chlorophyta nans) can be obtained from red algae, and (green algae), mostly belonging to the Class alginates and other sulfated polysaccharides Ulvophyceae [14]. It is mainly made up of (e.g. laminaran and fucoidan) are obtained disaccharide repeating sequences composed from brown algae. Phycocolloids are used of sulfated rhamnose and glucuronic acid, in food industries as natural additives and iduronic acid, or xylose [15,16]. have different European codes: E400 (al- The two major repeating disaccharides are ginic acid), E401 (sodium alginate), E402 aldobiuronic acids designated as: type A, (potassium alginate), E403 (ammonium ulvanobiuronic acid 3-sulfate (A3s) and alginate), E404 (calcium alginate), E405 type B, ulvanobiuronic acid 3-sulfate (B3s) (propylene glycol alginate), E406 (agar), (Figure 1). Partially sulfated xylose residues E407 (carrageenan) and E407a (semi- at O-2 can also occur in place of uronic refined carrageenan or ―processed Eucheu- acids. Low proportions of galactose, glu- ma seaweed‖) [10]. Agar, alginates and cose, mannose and protein were also gener- carrageenans are the ones with the highest ally described as components of ulvan. Ad- economic and commercial significance, ditionally, minor repeating units were being since these polysaccharides exhibit high reported to contain sulfated xylose, replac- molecular weights, high viscosity and ex- ing the iduronic acid or glucuronic acid cellent gelling, stabilizing and emulsifying components [12,13,17]. properties. They are extracted in fairly high

(ulvanobiuronic acid – type A) (ulvanobiuronic acid – type B)

Figure 1 - Idealized structure of the chemical units of Ulvan.

Ulvan have exhibited strong antioxidant stable supramolecular structures via elec- [18,19], anticoagulant, antithrombotic [20], trostatic interactions and could also form antitumor [21], immune-stimulatory [22], stabilized membranes. Ulvan/chitosan poly- anti-inflammatory [22,23], antiviral [24,25], electrolytes were found to mimics the antibacterial [26], antiprotozoal [12], anti- extracellular matrix structure providing hypercholesterolemic [27], and antihyperli- points of attachment for osteoblasts, be- pidemic [28] activities; used also for hyper- lieved to be enhanced by the nanofibrous plasia prevention, gastrointestinal, rege- structure of this construct. It is suggested nerative and nanomedicine applications that this construct could be a suitable scaf- [29,30]. fold for applications in tissue engineering [32,33]. Alves et al. [31] determined the cytotoxicity of ulvan via MTT test (Methyl Thiazolyl Tetrazolium - colorimetric assay for assess- 3. Polysaccharides from red algae (Rho- ing cell metabolic activity), using fibrob- dophyta) last-like cells were incubated with ulvan, in vitro. Quantified protein and total DNA 3.1. Carrageenans stands data were directly correlated with Carrageenan has been used in Europe and hyaluronic acid use as control (non- Asia for centuries as a thickening and stabi- cytotoxic); in the studies conducted by lizing agent in food. In Europe, the use of these researchers [31], ulvan showed en- carrageenan began in Ireland more than 600 couraging results in terms of cytotoxicity. years ago when, in the small village on the Meanwhile, cytotoxicity is one of the most southern coast of Ireland, called "Carrag- important factors to determine the use of a hen", the flans were made by cooking "Irish biomaterial for medical purposes, as in tis- Moss" (Irish Moss - Chondrus crispus) in sue engineering and wound healing [32]. milk. The name carrageenin, the ancient Ulvan can be used in biomedical applica- name of carrageenan, was first used in 1862 tions, especially in tissue engineering. Bio- to designate the extract obtained from C. functionalized ulvan hydrogels with ALP crispus, in reference to the name of the (Alkaline Phosphatase) enzyme served as small Irish town [36]. The procedure for the inducers of mineralization in osteoblastic extraction of carrageenans was described differentiation [32,33] and the cytocompa- for the first time by Schmidt in 1844 [37]. tibility of ulvan as well as it relative mem- The name was later changed to carrageenan brane are promising features for scaffold to comply with the ―-an‖ suffix for the development [34,35]. The inter-molecular names of polysaccharides. charges of ulvan and chitosan (i.e. anionic and cationic charges respectively) produced

The Irish moss has been used in industry sausages, patties and low-fat hamburgers since the 19th Century, in the clarification of [7,9,37,46-48]. beer [39]. The industrial extraction of carrageenan had its start in 1930 in New- The most commonly used commercial car- England, from Chondrus crispus and Mas- rageenans are extracted from Kappaphycus tocarpus stellatus thalli, for the preparation alvarezii and Eucheuma denticulatum [45]. of chocolate milk. The interruption of agar Primarily wild-harvested genera such as imports during World War II, led to its re- Chondrus, Furcellaria, Gigartina, Chon- placement by carrageenan. This situation dracanthus, Sarcothalia, Mazzaella, Iri- was the starting point of a booming industry daea, Mastocarpus, and Tichocarpus are [40]. also mainly cultivated as carrageenan raw materials, and producing countries include Fractionation of crude carrageenan extracts Argentina, Canada, Chile, Denmark, started in the early 1950s [41,42], resulting France, Japan, Mexico, Morocco, Portugal, in the characterization of the different car- North Korea, South Korea, Spain, Russia, rageenan types. A Greek prefix was intro- and the US [8,10,47]. duced to identify the different carrageenans. In the same period, the molecular structure The carrageenan market is the largest of the of carrageenans was determined [43,44]. seaweed-derived, food hydrocolloids mar- The structure of 3,6-anhydro-D-galactose in kets and currently holds the fourth largest kappa (κ) carrageenan, as well as the type share (in $ value) of the wider global hy- of linkages between galactose and anhydro- drocolloids market, behind gelatin, starch galactose rings, was determined. and pectin [49]. In 2014, the global hydrocolloids market was valued at US$ 5.4 Bil- Today, the industrial manufacture of carra- lion but projected to reach US$ 7.6 Billion geenan is no longer limited to extraction by 2020 [50]. Industry estimates for from C. crispus, and numerous red seaweed 2015/2016 put a value of US$ 600-700 Mil- species (Gigartinales, Rhodophyta) are lion on the global carrageenan market with used. For a long period of time, these sea- a projected average growth of ≈ 3% per weeds have been harvested from naturally year (Compound Annual Growth Rate - occurring populations. Seaweed farming CAGR). Most recent market research pre- started almost 200 years ago in Japan. dicted that the global market for carragee- Scientific information about the seaweed nan would approach US$ 1 Billion by 2024 life cycles allowed artificial seeding in the [49,51]. 1950s. Today, lots of seaweed taxa are cultivated, lowering the pressure on naturally Current industry estimates for the global occurring populations [45]. production capacity of carrageenan range from about 70,000-80,000 dry MT per year The modern industry of carrageenans dates [52] to over 110,000 MT per year [53]. Cy- from the 1940‘s where it was found to be berColloids‘ (www.cybercolloids.net) esti- the ideal stabilizer for the suspension of mate for current global usage, as based on cocoa in chocolate milk. In the last decades, end-sales data, is for 68,000 dry MT or ap- due to its physical functional properties, proximately 80% of the estimated produc- such as gelling, thickening, emulsifying and tion capacity (Campbell and Hotchkiss stabilizing abilities, carrageenans have been 2017). Most of this carrageenan (≈ 90%) employed in food industry to improve the comes from species and strains of Kappa- texture of cottage cheese, puddings and phycus and Eucheuma or ―cottonii‖ and dairy desserts, and in the manufacture of ―spinosum‖ respectively as they were re- ferred to historically and still are by the

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 5 of 50 industry. In 2016, CyberColloids estimated included in these estimates. The remainder the global production of Kappaphycus for of the carrageenan produced comes from the carrageenan industry to be in the region cold-water species that are harvested mostly of 1.5 Million wet MT or 200,000 dry MT in South America, approximately 15,000 [51] with most cultivation occurring in In- dry MT and from the dwindling remnants donesia and the Philippines. Eucheuma is of a small, traditional N. Atlantic industry not cultivated on such a large-scale as Kap- which produces only a few hundred dry MT paphycus, current estimates put production annually from Chondrus crispus [54]. for the carrageenan industry in the range of 0.3 Million wet MT or 40,000 dry MT [51]. Main commercial carrageenans (Figure 2) It must be noted that a substantial market are usually classified into κ (kappa), ι (iota), for sea vegetables also exists which is not and λ (lambda) carrageenans [9].

(β) Beta-carrageenan

(κ) Kappa-carrageenan (ι) Iota-carrageenan (λ) Lambda-carrageenan

Figure 2 - Idealized structure of the chemical units of the main types of Carrageenan.

3.2. Biological activities of Carrageenans ―arroz caldo‖ has hypoglycemic effects in normal subjects. A study done on another From a human health perspective, it has seaweed extract showed that when incorpo- been reported that carrageenans have anti- rated into ―puto‖, ―siomai‖ and a meal oxidant, anticancer, antihyperlipidemic, composed of rice and meatballs with sweet- anticoagulant, immunomodulatory, antifun- and-sour sauce, agar had a glucose lowering gal, antiviral properties, gastric discomfort effect in normal people [69]. The study management, and digestive health support conducted by Panlasigui et al. [57] indicates [55-65]. Carrageenans are also classically that regular inclusion of carrageenan in the used as agents for the induction of experi- diet may result in reduced blood cholesterol mental inflammation and inflammatory pain and lipid levels in human beings. [66]. Historically, Irish Moss or Carrageen Previous studies conducted by Panlasigui et (Chondrus crispus and Mastocarpus stella- al. [67], and by Dumelod et al. [68], showed tus) has many medical applications, some that carrageenan incorporated into common of which date from the 1830s. It is still used Philippine foods such as ―fishballs‖ and in Ireland to make traditional medicinal teas

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 6 of 50 and cough medicines to combat colds, and bone fracture [81]. C. ocellatus is an bronchitis, and chronic coughs. It is said to important economic original alga that can be particularly useful for dislodging mucus produce carrageenan. Antitumor, antiviral, and has antiviral properties. Carrageenans anticoagulant, and immunomodulatory ac- are also used as suspension agents and sta- tivities of carrageenan have been found in bilizers in other drugs, lotions and medicin- this species, however, many evidences indi- al creams. Other medical applications are as cated that λ-carrageenan, which contained an anticoagulant in blood products and for 1,4-linked D-galactose 2,6-disulfate units, the treatment of bowel problems such as and highly sulfated group, has more bioac- diarrhea, constipation and dysentery. They tivities than other carrageenan types are also used to make internal poultices to [59,77,79]. control stomach ulcers [70-72]. Carrageenans extracted from several algae, Many reports exist of anticoagulant activity i.e., Chondrus crispus, Mastocarpus stella- and inhibited platelet aggregation of carra- tus, Kappaphycus alvarezii, Eucheuma den- geenan [73-77]. Among the carrageenan ticulatum, Gigartinaceae and Phyllophora- types, λ-carrageenan (primarily from C. ceae, enter cough medicines, toothpastes, crispus) has approximately twice the activi- lotions, sun ray filterers, shaving creams, ty of unfractionated carrageenan and four shampoos, hair conditioners, and deodo- times the activity of κ-carrageenan (Kappa- rants. More than 20% of carrageenan pro- phycus alvarezii – formerly Eucheuma cot- duction is used in pharmacy and cosmetol- toni, and E. denticulatum – formerly E. spi- ogy. They are said to be invaluable for the nosum). The most active carrageenan has manufacture of wash-removable creams and approximately one fifteenth the activity of ointments. Excipients of algal origin are heparin [73], but the sulfated galactan from used in vanishing creams—the rapid evapo- Grateloupia indica collected from Indian ration of the emulsion‘s aqueous phase on waters, exhibited anticoagulant activity as the skin leaves a thin protective medicated potent as heparin [78]. The principal basis oily microfilm. Dried and pulverized Litho- of the anticoagulant activity of carrageenan thamnium and Phymatolithon are used to appeared to be an antithrombotic property make absorbent face and/or beauty masks [73,74]. λ-carrageenan showed greater an- [82,83]. tithrombotic activity than κ-carrageenan, probably due to its higher sulfate content, Carrageenans are selective inhibitors of whereas the activity of the unfractionated several enveloped and nonenveloped virus- material remained between the two. It was es and act predominantly by inhibiting the found that toxicity of carrageenans de- binding or internalization of virus into the pended on the molecular weight and not the host cells [84,85]. Carrageenans are excep- sulfate content. Similar results were ob- tionally potent inhibitor of Human Papillo- tained with λ-carrageenan of Coccotylus mavirus (HPV) in vitro by inhibiting the brodiei (as Phyllophora brodiaei) which initial stage of infection [84]. Notably, they gave the highest blood anticoagulant activi- are also extremely effective against a range ty. Carrageenan of Grateloupia turuturu of sexually transmitted HPV types that lead also showed anticoagulant activity [78-80]. to cervical cancer and genital warts [86,87]. Several in vitro studies suggest that carra- Chondrus ocellatus is distributed sponta- geenans may also have antiviral properties, neously in intertidal zone of the southeast inhibiting the replication of herpes and he- seaside of China [59]. In ancient times the patitis A virus [86,88-90]. More recently, alga was not only edible, but was also used Schiller and coworkers demonstrated that as a medicine to cure chronic constipation carrageenan is an extremely potent infec-

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 7 of 50 tion inhibitor of a broad range of genital centrations about 1000 times higher than Human papillomaviruses (HPVs) and there those required to inhibit Papillomaviruses are indications that carrageenan-based sex- [84]. However, carrageenans may serve as ual lubricant gels may offer protection models for designing novel anti-HIV against HPV transmission [84,91,92]. agents, improving their therapeutic properties through chemical modifications [97]. The antimicrobial activities of numerous algae species have been tested and reported, Carlucci and colleagues [98,99] found that presenting an extended spectrum of action 휆-type carrageenan is active against the against bacteria and fungi [93]. Carragee- replication of HSV upon its firm interaction nans have proved to have effects against that leads to inactivation of HSV virion some bacterial strains such as Salmonella [98]. They also discovered that the 휆- enteritidis, S. typhimurium, Vibrio mimicus, carrageenan and moderately cyclized 휇/휄- Aeromonas hydrophila, Escherichia coli, carrageenan isolated from Gigartina Listeria monocytogenes and Staphylococcus skottsbergii exert promising antiviral activi- aureus. The growth of all the bacterial ties towards diverse strains of Herpes simp- strains except L. monocytogenes was signif- lex virus HSV-1 and HSV-2, during virus icantly inhibited by them, particularly by attachment stage [90,99]. Surprisingly, sim- the ι-carrageenan. A growth inhibition ex- ilar results were reported by different group periment using S. enteritidis showed that of researchers, who analyzed the chemical the inhibitory effect of the carrageenans structure and antiviral activity of carragee- was not bactericidal but bacteriostatic. Re- nan (λ, κ, and ι) against HSV-2 infection moval of the sulfate residues eliminated the [100,101]. The data obtained in the work of bacteriostatic effect of ι-carrageenan, sug- Soares [64] revealed that the viral infection gesting that the sulfate residues in carragee- by Lentivirus was reduced upon exposure to nan play an essential role in this effect [94]. a pre-treatment with extracts from female In 2014, Sebaaly et al. [95] reported that gametophytes (FG) and tetrasporophytes carrageenans isolated from the red alga Co- (T) of Chondracnthus teedei var. lusitani- rallina sp. exhibited antibacterial activity cus. Although the inhibitions were not sta- against Staphylococcus epidermis. Infrared tistically significant, FG (producers of κ/ 휄 spectroscopy (FTIR) showed that the iso- hybrid carrageenan) and T (producers of ξ- lated carrageenan was of λ-type [95]. carrageenan) of C. teedei var. lusitanicus extracts was able to reduce 14% of the virus Researchers from the Population Council infection, and the Tetra extract was able to [96] (http://www.popcouncil.org/), a non- reduce, approximately, 35% of the virus profit research organization in the USA, infection [64]. conducted a clinical trial investigating whether a carrageenan-based sexual lubri- A recent in vitro study conducted by Gras- cant was effective as a topical microbicide sauer and colleagues [85] reported the inhi- by blocking Human Immunodeficiency bitory effects of 휄-carrageenan against Hu- virus (HIV) infection in women. The study man rhinovirus (HRV) proliferation by pre- was conducted from March 2004–2007 in venting the primary phases of virus replica- South Africa and enrolled more than 6000 tion. They have suggested that this effect is volunteers. The results demonstrated that possibly attributed to the suppression of the the experimental microbicide gel was safe allosteric activity of virus particles during for use but does not provide protection their entry [85,102]. Additionally, 휄- against HIV [61,62,96]. These results are carrageenan was proven to be effective consistent with in vitro assays since carra- against dengue virus replication in mosqui- geenan was active against HIV only at con- to and mammalian cells; however, the mode

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 8 of 50 of antiviral action of 휄-carrageenan in both So, the studies of Yuan et al. [112] suggest cell types was interestingly distinctive. In that carrageenan oligosaccharides and their Vero cell line, the inhibitory activity has derivatives show relevant antioxidant ac- been exerted at early stage of virus adhe- tivity both in vitro and in a cell system. sion probably due to some primary recep- tors, whereas in mosquito cell it affected the According Sokolova et al. [113], antioxi- cell proliferation and protein synthesis dant actions of λ-, ξ-, κ-, κ/β- and κ/ι- [103,104]. carrageenans against reactive oxygen/nitrogen species depend on polysaccha- Carrageenan from red marine algae is ride concentration and such structural cha- known to be also a potent inflammatory racteristics as presence of hydrophobic 3,6- agent in rodents, primes mice leucocytes to anhydrogalactose unit, amount and position produce tumor necrosis factor-α (TNF-α) in of sulfate groups, and an oxidant agent, on response to bacterial lipopolysaccharide which sample antioxidant action is directed. [105]. Moreover, some types of carragee- The results of Sokova et al. [113] indicated nans induce potent macrophage activation that carrageenans possess antioxidant ca- [106,107], while some carrageenans and pacity in vitro, and this action notably de- fucoidan appear to inhibit macrophage pends on the structure of the polysaccharide functions [108,109]. However, sulfated itself than the reducing capacity of the po- polysaccharides may have potential bio- lysaccharides. medical applications in stimulating the immune system or in controlling macrophage Affording the works of Sokolova et al. activity to reduce associated negative ef- [114], a carrageenan food supplement fects [22]. brings about the decrease in the level of cholesterol and low-density lipoprotein cho- Exposure of human (and other mammals) lesterol, and the supplementation with car- cells to ultraviolet (UV) light induces vari- rageenans produces moderate modulation of ous deleterious responses. Damage to cells all links of the immunity. by UVA is thought to involve reactive oxygen species, including singlet oxygen, the Until 1935, pneumonia was the leading superoxide and hydroxyl radicals, and hy- recorded cause human death in the USA. drogen peroxide. Some of the major harm- 100 years ago, five of the top ten causes of ful effects are DNA damage, systemic im- death in men were respiratory diseases. mune suppression, and accelerated aging Today, asthma is the leading cause of juve- [110,111]. When thymic lymphocytes were nile school absenteeism and is increasingly exposed to a dose of 90 mJ/cm2 UVA, their an affliction of adults. Red algae (carragee- viability was decreased by about 60%. nophytes) containing carrageenan have However, the viability of lymphocytes was been used for millennia as treatments for significantly increased compared with the respiratory ailments, especially intractable control group when carrageenan oligosac- sinus infections and lingering pneumonias. charides and their derivatives were pre- Asthma was not separated out as such in the administered to cells before UVA radiation. old literature [115]. This phenomenon showed that carrageenan A recent in vivo study in mice has revealed oligosaccharides and their derivatives can that the low molecular weight carrageenans protect the lymphocytes against UVA in- (3, 5, and 10 kDa), as well as acetylated and jury. As like a H2O2 induced oxidative sulfated derivatives, have substantial inhibi- stress model, the chemical modification of tory effects against influenza virus. Fur- carrageenan oligosaccharides also had no thermore, the smallest 휅-carrageenan with significant effect on their protective effect. appropriate sulfation and acetylation degree

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 9 of 50 was the greatest antiviral candidate against fluenza virus illness this combinational influenza virus [116]. therapy meets an urgent medical need [125,126]. Acute viral upper respiratory tract infection, also known as common cold, is the most Marine algae are prolific sources of sulfated frequently observed infectious disease in polysaccharides, which may explain the low human beings. Children get four to eight incidence of certain cancers in countries upper respiratory infections per year and that traditionally consume marine food. adults suffer from two to four episodes per Breast cancer is one of the most common year [117]. In most of cases, common cold types of non-skin cancer in females. In the is caused by respiratory viruses such as study made by Murad et al. [127], extracted rhinovirus, coronavirus, parainfluenza, in- sulfated carrageenan, predominantly con- fluenza, respiratory syncytial virus, adeno- sisting of ι-carrageenan extracted from the virus, enterovirus and metapneumovirus red alga Palisada perforata (as Laurencia [118-121]. According to the work of Koe- papillosa), has demonstrated potential cyto- nighofer et al. [122], the administration of toxic activity against the MDA-MB-231 carrageenan nasal spray in children as well cancer cell line. These findings suggest that as in adults suffering from virus-confirmed sulfated carrageenan may serve as a poten- common cold reduced duration of disease, tial therapeutic agent to target breast cancer increased viral clearance and reduced re- via prompting apoptosis [127]. lapses of symptoms. Carrageenan nasal spray appeared as an effective treatment of λ-Carrageenan is a seaweed polysaccharide common cold in children and adults [122- which has been generally used as pro- 124]. inflammatory agent in the basic research, however, how the immunomodulating ac- A nasal spray containing ι-carrageenan on- tivity of λ-carrageenan affects tumor micro- ly, or together with ―Zanamivir‖, provides environment remains unknown. Luo et al. an easy to apply treatment of upper respira- [128] found that intratumoral injection of λ- tory tract infections in patients under suspi- carrageenan could inhibit tumor growth in cion to be influenza A (H1N1) infected B16-F10 and 4T1 bearing mice and enhance [125,126]. Patients would benefit from the tumor immune response by increasing the fast and efficient treatment of uncompli- number of tumor-infiltrating M1 macro- + + cated influenza in the upper respiratory phages, DCs and more activated CD4 CD8 tract. Due to the faster influenza virus T lymphocytes in spleen. In addition, λ- clearance from the upper respiratory tract carrageenan could enhance the secretion of and the independent antiviral mechanism of IL17A in spleen and significantly increase carrageenan and ―Zanamivir‖ the likelihood the level of TNF-α in tumor, most of which to develop escape mutations against ―Za- was secreted by infiltrating macrophages. namivir‖ will be reduced. Both individual Moreover, λ-carrageenan exhibited an effi- compounds can reduce severity and/or du- cient adjuvant effect in OVA-based preven- ration of the influenza illness and a combi- tative and therapeutic vaccine for cancer nation is expected to work similarly. Addi- treatment, which significantly enhanced the tionally, due to the broad antiviral effec- production of anti-OVA antibody. The tox- tiveness of carrageenan, patients will re- icity analysis suggested that λ-carrageenan ceive in parallel a treatment of concomitant was with a good safety profile. Thus, λ- viral infections. Therefore, patients will carrageenan might be used both as a potent benefit from a decreased probability to de- antitumor agent and an efficient adjuvant in velop complications. In consideration of the cancer immunotherapy [128]. complications known to accompany an in-

Carrageenans extracted from Chondracan- 3.3. Agar thus teedei var. lusitanicus were studied in order to determine their potential antifungal Agar is the Malay name for red algae, also activity [64,65]. FTIR-ATR and FT-Raman called agar-agar and Kanten. It is an extract spectroscopic analysis confirmed the pres- of red algae, sold in granular, powder form, ence of a hybrid κ/ι-carrageenan belonging as flakes or long strips. This colloid is to the gametophyte phase and a hybrid ξ/θ- widely used as a gelling agent and rarely carrageenan in the tetrasporophyte phase eaten on its own. In its pure form, agar is a [7,9,10,129]. κ/ι and ξ/θ-carrageenan in- tasteless and odorless polysaccharide; duced the formation of swollen hyphal which normally contains proteins, vitamins segments in Alternaria infectoria, upon and minerals, as the red algae from which it exposure to 125 μg mL–1 and 60 μg mL−1, is derived [83,131]. respectively. The observed phenotype was Kanten was discovered by accident. For similar to those induced by antifungals tar- more than a thousand years, the Japanese geting the fungal cell wall. When exposed −1 have eaten a dish called ―Tokoroten‖, to 87.5 μg mL of kappa/iota carrageenan, which is prepared for the abolition of red Aspergillus fumigatus hyphae became algae Gelidium amansii (called in Japan as shortened and highly branched, a phenotype ―Tengusa‖), and then letting the mixture commonly observed in response to antifun- stiffen. At some point toward the end of the gals. These morphological alterations were 17th Century, leftover ―Tokoroten‖ away associated with a decrease of the β-glucan outside on a freezing cold day. When it was content in A. infectoria after exposure to −1 found later, it had become a dry, whitish 150 μg mL of kappa/iota and to 100 μg solid [131,132]. mL−1 of ξ/θ-carrageenan. On the other hand, the chitin cell wall content of A. fu- Agar is light in color, semi-transparent, and migatus decreased significantly upon expo- very brittle when dry. When soaked it ab- sure to 150 μg mL−1 of both extracts, which sorbs water and, in contrast to gelatin (pre- triggered an increase in the content of β- pared from animal origin), it must have glucan. Overall, the present work shows allowed to swell up completely before the that carrageenans extracted from C. teedei water is warmed to a temperature above its var. lusitanicus cause alterations on the A. melting point of 85 °C. Agar can be used as fumigatus and A. infectoria cell walls, indi- a gelling agent as it cools [38,131]. cating a marked antifungal activity [64,65]. Agar consists of a mixture of at least two According the works of Azizi et al. [130], polysaccharides, i.e. agarose and agaropec- the slow silver (Ag) release, κ- tin [133]. Typically, agarose is the predo- carrageenan/silver nanoparticles (Ag-NPs) minant fraction of agar (50-90%) [134,135] presented good antibacterial activities and the responsible for its gelling properties against Staphylococcus aureus, Methicillin [135]. It consists of high molecular weight resistant Staphylococcus aureus, Peseudo- polysaccharides composed of repeating monas aeruginosa and Escherichia coli units of (1→3)-β-D-galactopyranosyl- with maximum zones of inhibition 11 +/- 2 (1→4)-3,6-anhydro-α-L galactopyranose mm. Cytotoxicity study showed that the (Figure 3), although some variations can bio-nanocomposite hydrogels with non- occur, depending on factors such as the toxic effect of concentration below 1000 species of seaweed, as well as environmen- μg/mL have great pharmacological poten- tal and seasonal conditions [133]. In turn, tial and a suitable level of safety for use in agaropectin is a less clearly defined, it is a the biological systems [130]. more complex polysaccharide of lower molecular weight than agarose and it has

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 11 of 50 thickening properties [38,133]. Its structure The most important commercial agarophyte is essentially made up of alternating (1→3)- genera are Gelidium, Pterocladiella, Geli- β-D-galactopyranose and of (1→4)-3,6- diella, and Gracilaria. Agar has also been anhydro-α-L-galacto-pyranose residues found in species of Ceramium, Phyllopho- [133,136]. ra, Ahnfeltia, Campylaephora, Acanthopho- lis, and Gracilariopsis [137].

Figure 3 – Idealized structure of the chemical units of Agar

3.4. Biological activities of Agar cate that CDSAM was more useful than standard media in accurately evaluating the Agar has been used as functional dietary efficacy of antifungal drugs. Agar propor- fiber for diabetics in the health food market tion method for drug susceptibility tests has because of its non-nutritive nature. Never- been used since 1957 [142]. More recently, theless, its α-glucosidase inhibitory ability the test has been replaced by more rapid should also be considered for retarding the tests [143]. carbohydrate digestion. Some inhibitors with pseudo-saccharide structure including The possibility to use agar and agarose acarbose and 1-deoxynojirimycin have been beads for sustained release of water soluble used very successfully in controlling dis- drugs has been investigated [144]. Agarose eases such as diabetes, obesity or hyperli- has a significantly lower sulfate content, pemia [138,139]. better optical clarity and increased gel strength with respect to agar, but it is consi- Agar has medicinal or pharmaceutical in- derably more expensive [145]. Agar beads dustrial applications including use as sus- containing phenobarbitone sodium as a wa- pending agent for radiological solutions ter soluble and hypnotic drug were prepared (barium sulfate), as a bulk laxative as it [146]. The encapsulation procedure consists gives a smooth and non-irritating hydrated in dissolving the drug in a hot (around 70 bulk in the digestive tract, and as a forma- °C) agar aqueous solution and then drop- tive ingredient for tablets and capsules to ping the solution in a cold bath containing a carry and release drugs. Pharmaceutical non-solvent for agar (acetone or ethyl ace- grade agar has a viscous consistency. In tate). Agar beads instantaneously form by microbiology, agar is the medium of choice gelation. The results of dissolution and re- for culturing bacteria on solid substrate. lease studies indicated that agar beads could Agar is also used in some molecular micro- be useful for the preparation of sustained biology techniques to obtain DNA informa- release dosage forms, although no many tion [140]. More recently, agar was used in further studies have been developed [147]. a newly-developed medium, i.e., combined deactivators-supplemented agar medium Antitumor activity was found in an agar- (CDSAM), to evaluate the viability of der- type polysaccharide from cold water extrac- matophytes in skin scales [141]. The expe- tion of a Gracilaria species and hydroly- rimental data from this clinical study indi- sates of agar resulted in agaro-oligo-

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 12 of 50 saccharides with activity against α- inflammatory cytokine and an enzyme as- glucosidase and antioxidant ability sociated with the production of nitric oxide [139,148]. It has also been reported that [154]. agar leads to decreases in the concentration of blood glucose and exerts an anti- Among therapeutics for liver diseases, pro- aggregation effect on red blood cells [4]. It tective drugs have been attracted more and has as well employed as an anti-rheumatic more attentions, such as antioxidant preven- agent for prolonged treatments, and in the tion approaches. The in vitro and in vivo stabilization of cholesterol solutions [149]. antioxidative activities of agaro- oligosaccharides with the model related Several antitumor oligosaccharides (GSO) with liver disease was tested by [155]. Aga- include sulfated disaccharide and neoagaro- ro-oligosaccharides derived from red sea- tetraose have been discovered in the Deles- weed polysaccharide have been reported to seriaceae (Ceramiales, Rhodophyta) [150]. possess antioxidant activity. Overall, the These oligosaccharides were prepared by results of the study made by Chen and col- spray dried and proved the physiological leagues [155] indicate that agaro- activity by reducing the number of revertant oligosaccharides can exert their in vitro and colonies of Salmonella typhimurim and in vivo hepatoprotective effect through sca- mildly inhibiting angiogenesis on the venging oxidative damage induced by reac- chicken chorioallantoic membrane (CAM). tive oxygen species (ROS). GSO was assumed to have a desmutagenic activity and no harmful side effects such as Agaro-oligosaccharides and neoagaro- blood coagulation of fibrinolysis. GSO with oligosaccharides have been reported to pos- high safety owed various physiological ac- sess lots of biological activities, such as tivity, which is expected to apply in future antioxidative activities [156], moisturizing food and medical supplies [151]. effect on skin [157], and anti-inflammation effects [158], which suggested that these Agarose can be separated from the agar oligosaccharides merit further investigation with a yield of 42%, and the agar content as functional foods to control inflammation. varied seasonally from 26% - 42% in Geli- Neoagaro-oligosaccharides derived from dium spp. in another experiment [152,153]. agarose have been demonstrated to possess a variety of biological activities, such as In recent years, versatile biological activi- antibacterial and antioxidative activities ties of agaro-oligosaccharides have been [159]. observed, and the structure and bioactivity relationship research also revealed that the functionality is directly correlated to the 3.5. Porphyran degree of polymerization. Agaro- oligosaccharides were hydrolytically ob- Porphyran is a sulfated polysaccharide iso- tained from agar using hydrochloric acid, lated from selected (red) algae of the Order citric acid, and cationic exchange resin (sol- Bangiales, Phylum Rhodophyta, especially id acid) [139]. Agaro-oligosaccharides have from the genus Porphyra/Pyropia been reported to display antioxidant effects, [160,161]. The chemical structure of por- such as inhibiting the production of lipid phyran contains a linear backbone of alter- peroxide radicals and inhibiting the produc- nating 3-linked β-D-galactose and 4-linked tion of nitric oxide (NO) [139]. Agaro- α-L-galactose-6-sulfate or 3,6-anhydro-α-L- oligosaccharides have also been shown to galactose units (Figure 4) [79,162]. suppress the production of a pro-

Figure 4 - Idealized structure of the chemical units of Porphyran (after [327]).

3.6. Biological activities of Porphyran be exploited by ―nutraceutical‖ companies that market them as ―health products‖ Non-starch polysaccharide (NSP) has been [57,68,176,180-187]. consumed for centuries and has been recog- nized as having nutritional benefits. Many Porphyran is effective against contact reports have demonstrated that ingestion of hypersensitivity induced by 2, 4, 6 tri- NSP lowers serum cholesterol and lipo- nitrochlorobenzene. The mechanism is sug- protein levels [170-172], helps to normalize gested to operate by suppressing the serum serum glucose and insulin levels [173] and levels of IgE and IFN-ƴ. Porphyran has promotes normal laxation [174]. Porphyran, excellent inhibitory activities against hyalu- which is considered an NSP, can be ex- ronidase, responsible for the release of his- tracted with hot water from Porphy- tamine from mast cells [188]. ra/Pyropia spp. [161,175], edible species cultivated abundantly in eastern Asia. Por- Some sulfated polysaccharides from red phyran is distributed in the intercellular algae also showed anti-viral activities to- matrix of the algal body and its basic struc- wards viruses responsible for human infec- ture is closely related to that of agar [176]. tious diseases. Porphyran is reported to in- It has also been reported that porphyran hibit HIV reverse transcriptase in vitro. It also has health benefits as other NSPs: e.g., has minimal effects on human DNA and prebiotic activity [177], antitumor activity RNA polymerase activity. Some agaroids, [178,179], and antihyperlipidemic activity such as high molecular weight, galactan [180]. The study in rats [180] suggested that sulfate also have antiviral properties against dietary porphyran prevents hypercholeste- the Herpes simplex virus, human cytomega- rolemia that is induced by feeding diet con- lo virus (HCMV), dengue virus (DENV), taining 1.5% cholesterol 0.5% bile salts. respiratory syncytial virus (RSV) and in- However, little is known about how dietary fluenza virus due to the inhibition of the porphyran affects growth and lipid metabol- initial viral attachment to host cells [189- ism in the feeding of a normal diet [176]. 194]. Porphyran produces hypocholesterolemic Upon the intraperitoneal administration of and hypolipidemic effects due to reduced porphyran to aging mice, the activities of cholesterol absorption in the gut, with in- superoxide dismutase and glutathione pe- creased fecal cholesterol content and a hy- roxidase as well as the total antioxidant poglycemic response. It is also reported to capacity were increased signiﬁcantly and reduce total cholesterol, free cholesterol and the level of lipid peroxidation was the levels of triglycerides and phospholipids signiﬁcantly decreased, suggesting that in the liver. These substances are likely to porphyran has antioxidant activity both in vitro and in vivo [195].

In human gastric cancer cells, porphyran Furthermore, polysaccharides prepared displays antitumor properties, in particular from Pyropia yezoensis (as Porphyra ye- the ester sulfates of the polysaccharides zoensis) considerably inhibited human gas- may be related to the antitumor activities tric and lung cancer cell growth, and degra- reported. The degree of polyanionic proper- dation products of these polysaccharides ties may be intimately related to this activi- resulting from an ultrasonic treatment ty. Porphyran reportedly induced apoptosis significantly increased this antiproliferative by caspase-3 or caspase-9 activation, while eff ect against only human gastric cancer decreasing the Insulin-like, growth Factor-I cells [198]. Porphyra/ Pyropia sp. also re- receptor (IGF-IR) phosphorylation and portedly contains various anticancer com- down regulated a serine/threonine kinase pounds other than porphyrans [199-201]. In (Akt) phosphorylation. The caspase-induce a human case-control study, it was reported apoptosis was activated in a sequential cas- that a high rate of intake of nori (Porphy- cade of cleavages from their inactive forms. ra/Pyropia sp.) may decrease the risk of Activation of caspase-3 leads to the clea- breast cancer [202]. vage of many proteins, one of which is poly (ADP-ribose) polymerase (PARP) [196]. Additionally, porphyrans reportedly im- The cleavage of the poly (ADP-ribose) po- proved lipid and glucose metabolism lymerase is the hallmark of apoptosis. The [203,204] and iron deficiency [205]. In the expression level of antiapoptotic molecules, works of Wang et al. [206] the hypolipi- such as B-cell lymphoma 2 (Bcl-2), gradu- demic and antioxidant effects of porphyran ally decreased, whereas the proapoptotic from the red algae Pyropia haitanensis (as molecule Bcl-2-associated death promoter Porphyra haitanensis) as a dietary supple- (BAD), opposed the action of Bcl-2, which ment were evaluated in mice. The results increased in response to levels of exposure suggested that porphyran could be used as porphyran. Porphyran decreased the expres- functional foods and natural drugs in pre- sion levels in Gastric cancer cell lines venting the hyperlipidemia and this activity (AGS) by negatively regulating the insulin- might be attributed to its antioxidant poten- like, growth Factor-I receptor (IGF-IR) tial [206]. phosphorylation which is a potent mitogen and growth stimulatory factor for several 4. Polysaccharides from brown algae kinds of cells (over-expression and en- (Ochrophyta, Phaeophyceae) hanced activation of which is frequently observed in Human cancers) [196]. Thus, 4.1. Fucoidan Insulin-like, growth Factor-I receptor (IGF- IR) phosphorylation was decreased in por- Fucans are sulfated polysaccharides which phyran-treated AGS cells, which correlated comprise a fucose backbone. One of the with Akt activation. Porphyran down regu- best studied fucans from the brown algae is lates Akt phosphorylation. The PI3- ki- fucoidan, which was first isolated by Kylin nase/Akt pathway is viewed as a key player [207]. The fucoidan from Fucus vesiculosus to cell survival in different systems. The has been available commercially for dec- IGF-I stimulation induced Akt activation ades (Sigma-Aldrich Chemical Company, decreased in cells treated with porphyran, St. Louis, MO, US). Early work on its which suggested that inhibition of Akt structure showed that it contained primarily phosphorylation may be an important me- (1→2) linked 4-O-sulfated fucopyranose chanism for porphyran-induced apoptosis residues (Figure 5). However, 3- linked [196,197]. fucose with 4-sulfated groups was subsequently reported to be present on some of

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 15 of 50 the fucose residues. Additionally, it was nodosum contained a predominant disacc- determined to contain branches every 2-3 haride motif with sulfate at the 2-position of fucose residues. Subsequently, Chevolot the 3-linked fucose and sulfate groups on and colleagues reported that the fucoidan the 2- and 3-positions of the 4-linked fucose from Fucus vesiculosus and Ascophyllum [137,208].

Figure 5 - Idealized structure of the chemical units of Fucoidan

4.2. Biological activities of Fucoidan Several studies reported that the anticoagulant functionalities of fucoidans ex- Fucoidan can be easily ―cooked‖ out of tracted from Fucus vesiculosus and Eklonia most edible brown algae by simmering for cava were due to thrombin-inhibition- 20-40 min. in water. When consumed, it mediated via plasma anti-thrombin-III; their seemed to reduce the intensity of the in- anti-coagulant activity was like that of he- flammatory response and promote more parin [13,213,214]. Moreover, fucoidans rapid tissue healing after wound or surgical from Ascophyllum nodosum, Fucus distich- trauma. This led to recommendations that ous, F. evanescens, F. serratus, Laminaria brown seaweed broth be administered after digitata, Saccharina latissima and S. lon- auto-collision, sports injuries, bruising falls, gissima were also described to possess muscle and joint damage and deep tissue strong anticoagulant activities both in vitro cuts, including surgery [209]. and in vivo models [213,214]. However, the commercial importance of In general, structural-bioactive studies sug- fucoidans is presently much lower than that gested that the anticoagulant /anti-thrombin of seaweed hydrocolloids; these polysac- activities of fucoidans were mainly depen- charides are now attracting considerable dent on the content and/or positioning of attention because of the growing market for sulfate groups, as well as the molecular them as ―bioactive polysaccharides‖ in a weight of the polymers [213]. Likewise, wide arena of diverse applications [210]. their monomeric composition, types of lin- More recently, anticoagulant and antith- kages and branching might also exert mod- rombotic activities are amongst the most erate modulation on the biological proper- studied biological effects of fucoidans. ties of fucoidans [13,210,215]. In this con- Commonly, the anti-coagulant activity of text, it is possible that the greater anticoagu- fucoidans is mediated through the activa- lant/antithrombin activities exhibited by tion of thrombin inhibitors, although direct longer fucoidans are due to the higher con- thrombin inhibition and competitive bind- tent of fucose and sulfate groups ing of fibrinogen to block the actions of [13,209,213,215,216], though this is still thrombin are also possible [210-212]. under debate [3,217].

Fucoidans are also reported to inhibit the Antitumor activities of fucoidans include replication of several enveloped viruses the inhibition of tumor proliferation, the such as Human immune-deficiency and stimulation of tumor cell apoptosis, block- human cytomegalovirus, among others ing of tumor cell metastasis and enhance- [13,213]. The mechanisms for such activity ment of various immune responses [223]. In are thought to occur via inhibition of cell this context, fucoidans from several ma- infection by viral sorption, or hampering of croalgal species (e.g. Saccharina japonica, viral-induced, syncytium formation S. latissima, Laminaria digitata, Fucus ser- [13,215]. Fucoidans from Saccharina japo- ratus, F. distichus and F. vesiculosus) nica, Cladosiphon okamuranus, Adenocys- proved to be useful and were regarded as tis utricularis, Stoechospermum margina- good candidates for future cancer therapy tum, Cystoseira indica, Dictyota mertensii, [213,215]. Besides those, the commercial Lobophora variegata, Fucus vesiculosus, fucoidans branded ―Tokida‖ (from cultured Spatoglossum schroederi and Undaria pin- Cladosiphon okamuranus) and that from natifida showed impressive, positive results the Korean cultured sporophyte (Miyeok- in both in vitro and in vivo models of infec- gui) of U. pinnatifida also revealed promis- tion by poliovirus III, adenovirus III, ing antitumoral activities, as tested in in ECHO6 virus, coxsackie B3 virus, coxsack- vitro models [215]. ie A16, Newcastle disease virus (NDV), Herpes simplex virus (HSV-1, HSV-2), Other important, demonstrated biological HIV and avian reverse transcriptase activities of fucoidans included antioxidant, [13,212,215]. anti-inflammatory and anti-allergic, although others cannot be forgotten, e.g. he- Fucoidan isolated from the brown seaweed pato-protection, cardio-protection, stomach Fucus vesiculosus [218] shows inhibitory protection and anti-obesity [13,213,215]. effect on the replication of DNA viruses: Examples of fucoidans showing promising Herpes virus (HSV-1, HSV-2) and Human antioxidant activities in in vitro models in- cytomegalo virus (HCMV) [218-220]. cluded those obtained from Saccharina These compounds are also active against japonica, Canistrocarpus cervicornis, Fu- RNA viruses: Vesicular stomatitis virus cus vesiculosus, Dictyota cervicornis, Sar- (VSV), Sinbis virus (SINV), and HIV-1 gassum filipendula and Dictyopteris delica- [221]. However, this compound is not ac- tula [13,213,215,224], while the anti- tive against Coxsackievirus, Poliovirus, and inflammatory activity of several fucoidans Parainfluenza virus. A water-soluble, non- (Laminaria digitata, Fucus evanescens, F. carbohydrate component of fucoidan iso- serratus, F. distichus, F. spiralis, Ascophyl- lated from F. vesiculosus can inhibit HIV lum nodosum, Cladosiphon okamuranus, Reverse transcriptase (RT) in vitro at μg Padina gymnospora and Saccharina latis- mL-1 [220]. Pre-incubation of cell-free virus sima) have been described through inhibi- to 200 μg mL-1 causes 100% reduction in tion of leucocyte recruitment, in an inflam- the amount of HIV-1 p24 antigen released. mation model in rats [225]. Moreover, inhi- Tests show that these effects are not due to bition of the expression of inducible nitric the killing of target cells. In fact, fucoidan oxide synthase (iNOS) has also been dem- produced no adverse effects on cell prolife- onstrated for fucoidans, such as that from ration and protein metabolism. The pre- the Sigma-Aldrich Chemical Co. (from F. incubation of target cells with fucoidan pro- vesiculosus). Furthermore, commercial fu- tects them from HIV-1 infection [220]. In coidans (from Mekabu and Sigma-Aldrich addition to its antiviral activity, fucoidan Chemical Co.) together with those isolated possesses low anticoagulation properties from A. nodosum, F. evanescens, C. oka- [212,218-220,222]. muranus and from other several members

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 17 of 50 of the Order Laminariales (Phaeophyceae) Laminarans are basically a class of low- were described to exhibit anti- molecular weight storage β-glucans, which complementary activities, rendering their are composed of (1,3)- β -D-glucan [232]. potential as anti-allergen [13,121,213,215]. They consist of (1,3)- β -D-glucopyranose residues with some 6-O-branching in the The sulfated and acetylated fucoidan frac- main chain and some β-(1,6)-intra-chain tion, containing fucose, galactose, mannose, links are also present. For example, lamina- glucose and uronic acid residues, was iso- ran from Eisenia bicyclis is composed of a lated from the brown alga Padina boryana linear chain of (1,3) and (1,6) linkage in a [226]. The anticancer effect of native and ratio of 2:1 (1,3)- β -D-glucans [233]. The modified fucoidan fractions was studied in molecular weight of laminaran is approx- vitro on the colorectal carcinoma cells imately 5 kDa and is dependent upon the DLD-1 and HCT-116. All fucoidans had no degree of polymerization which is in the cytotoxicity under 400 μg/mL and inhibited range of 20-25 glucose moieties [234,235]. colony formation of cancer cells at concen- The molecular weight of laminaran from tration of 200 μg/mL [226]. Saccharina longicruris is reported to be in the range of 2.89 - 3.32 kDa depending on 4.3. Laminaran the extraction conditions [236], including the solvent type used [228]. Not all lamina- Laminarans are a category of small glucans rans are the same characteristics, different present in either soluble or insoluble form. species of raw material present different The first form is characterized by its com- structures of molecules, therefore different plete solubility in cold water, while the oth- biological properties [228,229]. er is only soluble in hot water [207,298]. This polysaccharide is composed of D- In nature, laminarins exist in either soluble glucose with β-(1,3) linkages, with β-(1,6) or insoluble forms. The soluble form is cha- intra-chain branching (Figure 6) [17,227- racterized by complete solubility in cold 229]. Laminaran, also known as ―lamina- water, and the insoluble form can be dis- rin‖ or ―leucosin‖, was first isolated from solved only in hot-water solutions. The so- members of the Laminariaceae (Phaeophy- lubility of laminarins is determined by the ceae) by Schmiedeberg [230]. Laminaran is branching factor: laminarins with the high- a food reserve of brown algae. It is located est branching are easily dissolved in cold in vacuoles present in cells. Laminaran is water in comparison with laminarins with found in the fronds of Laminaria and Sac- less branching. Moreover, the degree of charina species [231]. polymerization of water-soluble laminarins is approximately 20-25 glucose units [236,237].

Figure 6 - Chemical structure of β-1,3-1,6-glucan (Laminarin, Laminaran) (after [328]).

4.4. Biological activities of Laminaran minarans investigated was reported to be significantly reduced with the formation of The laminaran prepared from kelp by water carbonyl groups. This is suggested to lead extraction were reported to be able to effec- to enhanced antioxidant activities. tively inhibit the adsorption of HIV on lymphocytes and the activity of HIV re- Ermakova et al. [245] demonstrated that verse transcriptase at the concentration of water-soluble laminaran isolated from Eck- 50 μg mL-1, which suggest that laminaran lonia bicyclis (as Eisenia bicyclis) has no polysaccharides possess good inhibitory cytotoxic effect on Human malignant mela- effect on HIV replication [238]. noma (SK-MEL-28) and Human colorectal adenocarcinoma cells (DLD-1) after treat- Laminaran has many reported bioactivities ment for 24, 48, and 72 h (1-200 μg/mL). such as anticancer, anti-inflammatory, anti- However, the authors reported that incuba- coagulant, and antioxidant effects [227]. tion of laminaran with these cancer cells Studies on feeding of laminaran-containing significantly inhibits colony formation (for foods to animals (pigs) indicate that lamina- SK-MEL-28 Human melanoma cells by rans hold promise as a functional ingredient 32% and 42% respectively at 100 and 200 in food applications [239]. μg/mL). Usoltseva and co-workers [246] Algae polysaccharides have numerous bio- also researched the inhibitory effects of logical activities because they enhance ma- laminarans isolated from Alaria marginata crophage immune responses [240]. Similar- and Alaria angusta on colony formation by ly, laminaran can be used to achieve the 3 cancer cell lines: Human colon adenocar- activation of macrophages leading to im- cinoma cells (HT-29), Human breast cancer mune-stimulatory, antitumor and wound- cells (T47D), and Human malignant mela- healing activities [241,242]. Thus, it is also noma (SK-Mel-28). Laminaran concentra- termed as a biological response modifier tions less than 400 μg/mL were not toxic to [243]. The biological activities of lamina- the tested cancer cell lines. However, the rans can be enhanced or modified using authors found that at the concentration of various techniques including irradiation, 100 μg/mL, laminaran has an inhibitory sulfation, reduction and oxidation. During effect on colony formation on HT-29 cells irradiation, the molecular weight of the la- compared to the control group (A. angusta

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 19 of 50 laminaran by 22% and A. marginata lami- adenocarcinoma (DLD-1) cancer cells after naran by 6%). In addition to these signifi- irradiation (4 Gy) showed a synergistic ef- cant results, the authors reported that mod- fect with X-ray irradiation against cancer ifications of laminarans (deacetylation and cells, decreasing the amount and size of desulfation) cause a loss of the anticancer cancer cells colonies [250]. activity under the tested conditions [237]. Ji and Ji [247] studied the anticancer effect 4.5. Alginates of commercial laminaran on human colorectal adenocarcinoma (LoVo) cells. Their ―Alginate‖ is the term usually used for the results revealed that incubation of cancer salts of alginic acid, but it can also refer to cells with laminaran (400-1600 μg/mL) for all the derivatives of alginic acid and algin- 72 h induces apoptosis in LoVo cells. Addi- ic acid itself; in some publications the term tionally, Ji et al. [248] studied anticancer ―algin‖ is used instead of alginate. Chemi- activity of laminaran after modifying the cally, alginates (Figure 7) are linear copo- sulfate content of laminaran by the chloro- lymers of β-D-mannuronic acid (M) and α- sulfonic-acid-pyridine method. The authors L-guluronic acid (G) (1-4)-linked residues, compared the anticancer effects of normal arranged either in heteropolymeric (MG) and modified laminaran on LoVo cells by and/or homopolymeric (M or G) blocks the 3-(4,5-dimethylthiazol-2-yl)-2,5- [251-253]. diphenyltetrazolium bromide (MTT) assay. Alginic acid was discovered in 1883 by At the concentration of 1600 μg/mL, mod- E.C.C. Stanford, a British pharmacist who ified laminaran could reduce the number of called it algin. Alginic acid is extracted as a LoVo cells by 86%. However, at the same mixed salt of sodium and/or potassium, concentration, unmodified laminaran could calcium and magnesium. Since Stanford reduce the cancer cell number by only 38%. discovered algin, the name has been applied Thus, the authors proposed that sulfation of to a number of substances, e.g. alginic acid the structure might increase bioactivities of and all alginates, derived from alginic acid. laminaran [248]. The extraction process is based on the con- In the works of Malyarenko et al. [249], the version of an insoluble mixture of alginic laminarans from brown seaweeds Sacchari- acid salts of the cell wall in a soluble salt na cichorioides, Saccharina japonica, and (alginate) which is appropriate for the water Fucus evanescens were isolated and their extraction [254,255]. Alginic acid is present sulfated derivatives were tested. The latter in the cell walls of brown seaweeds, where inhibited proliferation, colony formation, it is partially responsible for their flexibility and migration of human colorectal adeno- [256]. carcinoma, melanoma, and breast adenocar- While any brown seaweed could be used as cinoma cells in different manners. The sul- a source of alginate, the actual chemical fated laminaran from F. evanescens pos- structure of the alginate varies from one sessed the highest anticancer activity in genus to another, and similar variability is vitro and effectively prevented migration of found in the properties of the alginate that is breast adenocarcinoma cells by inhibiting extracted from the seaweed. Since the main of the Matrix Metalloproteinases 2 and 9 applications of alginate are in thickening activity [249]. aqueous solutions and forming gels, its The effect of laminaran and fucoidan ex- quality is judged on how well it performs in tracted from Dictyota dichotoma (40 these uses [256]. μg/mL) on colony formation of colorectal

25 to 30 years ago almost all extraction of Water-in-oil emulsions such as mayonnaise alginates took place in Europe, USA, and and salad dressings are less likely to sepa- Japan. The major change in the alginates rate into their original oil and water phases industry over the last decade has been the if thickened with alginate. Sodium alginate emergence of producers in China in the is not useful when the emulsion is acidic, 1980s. Initially, production was limited to because insoluble alginic acid forms; for low cost, low quality alginate for the inter- these applications propylene glycol alginate nal, industrial markets produced from the (PGA) is used since this is stable in mild locally cultivated Saccharina japonica. By acid conditions. Alginate improves the tex- the 1990s, Chinese producers were compet- ture, body and sheen of yoghurt, but PGA is ing in western industrial markets to sell also used in the stabilization of milk pro- alginates, primarily based on low [38,257]. teins under acidic conditions, as found in some yoghurts. Some fruit drinks have fruit A high-quality alginate forms strong gels pulp added and it is preferable to keep this and gives thick, aqueous solutions. A good in suspension; addition of sodium alginate, raw material for alginate extraction should or PGA in acidic conditions, can prevent also give a high yield of alginate. Brown sedimentation of the pulp and to create seaweeds that fulfill the above criteria are foams. In chocolate milk, the cocoa can be species of Ascophyllum, Durvillaea, Ecklo- kept in suspension by an alginate/phosphate nia, Fucus, Laminaria, Lessonia, Macro- mixture, although in this application it faces cystis and Sargassum. However, Sargassum strong competition from carrageenan. Small is only used when nothing else is available: amounts of alginate can thicken and stabil- its alginate is usually borderline quality and ize whipped cream [135,259]. the yield usually low [258]. Alginates have several commercial applica- The goal of the extraction process is to ob- tions based on their thickening, gelling, tain dry, powdered, sodium alginate. The emulsifier and stabilizing abilities. They are calcium and magnesium salts do not dis- used in the food industry for improving the solve in water; the sodium salt does. The textural quality of numerous products such rationale behind the extraction of alginate as salad dressing, ice-cream, beer, jelly and from the seaweed is to convert all the algi- lactic drinks, but also in cosmetics, pharma- nate salts to the sodium salt, dissolve this in ceuticals, textiles and painting industries water, and remove the seaweed residue by [5,38,260]. filtration [256].

G M

Figure 7 - Idealized structure of the chemical units of alginic acid

4.5. Biological activities of Alginates low toxicity and immunogenicity [265], together with the ability of preserving the Alginates have anticancer properties biological properties of proteins [266,267], [5,147,261] and a bioactive food additive can act as a coating membrane. A chito- ―Detoxal‖, containing calcium alginate, has san/PEG-alginate microencapsulation anti-toxic effects on hepatitis. This drug process [268], applied to biological macro- decreases the content of lipid peroxidation molecules such as albumin or hirudin [269], products and normalizes the concentrations was reported to be a good candidate for oral of lipids and glycogen in the liver [71,262]. delivery of bioactive peptides. Alginate molecules are playing vital role in Several examples of alginate-encapsulated the fields of pharmacy and medicine. Due drugs, other than proteins, can also be to its ability to retain water, is mainly used found in literature. Qurrat-ul-Ain et al. as gelling and viscosifying agent. In con- [270] reported that alginate microparticles trolled drug delivery systems, alginates can showed better drug bioavailability and re- be used. In this case, the rate of drug release duction of systemic side effects compared depends on the type and molecular weight with free drugs in the treatment of tubercu- of alginates used. Alginates have been losis. Polyelectrolyte coating of alginate mostly used marine derived polymers in the microspheres showed to be a promising tool preparation of drug delivery particles with to achieve release systems characterized by the shape of spheres of different sizes [263]. approximately zero-order release kinetics, Alginate instantaneously forms gel-spheres release up to 100% of entrapped drug (dex- at pH > 6 by ionotropic gelation with diva- amethasone) within 1 month, and improved lent cations such as Ca2+ [264], Ba2+, or 2+ biocompatibility [271]. Composites tech- Zn and for this it is widely used for mi- nology has been applied to alginate for drug croencapsulation of drugs. On the other delivery purposes. As an example, montmo- hand, at low pH, hydration of alginic acid rillonite-alginate nanocomposites have been leads to the formation of a high-viscosity recently proposed as a system for sustained ―acid gel‖. The ability of alginate to form release of B1 and B6 vitamins [272]. The two types of gel dependent on pH, i.e., an vitamins intercalated in the nanocrystals of acid gel and an ionotropic gel, gives the the inorganic phase, and successively the polymer unique properties compared to hybrid B1/B6 montmorillonite (MMT) is neutral macromolecules, and it can be tai- further used for the synthesis of B1/B6- lor-made for many applications [147]. MMT-alginate nanocomposite. The microencapsulation technique has been In their simplest design, oral controlled- developed particularly for the oral delivery release dosage forms made from alginates of proteins, as they are quickly denatured are monolithic tablets in which the drug is and degraded in the hostile environment of homogeneously dispersed. Drug release is the stomach. The protein is encapsulated in controlled by the formation of a hydrated a core material that, in turn, is coated with a viscous layer around the tablet, which acts biocompatible, semi permeable membrane, as a diffusional barrier to drug diffusion and which controls the release rate of the pro- water penetration. Water soluble drugs are tein while protecting it from biodegrada- mainly released by diffusion of dissolved tion. Due to its mild gelation conditions at drug molecules across the gel layer, while neutral pH, alginate gel can act as core ma- poorly soluble drugs are mainly released by terial in this application, while erosion mechanisms. Modulation of drug poly(ethylene glycol) (PEG), which exhi- release rate has been achieved by incorpo- bits properties such as protein resistance, rating pH-independent hydrocolloids gel-

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 22 of 50 ling agents or adding polycationic hydro- Alginate forms mild gelation by addition of colloids such as chitosan [273,274]. Several divalent cation Ca2+ and have been exten- mucoadhesive systems based on alginate sively studied and applied as a biomaterial have been developed [275,276]. The main in wound healing, tissue engineering [282], shortcoming of alginates consists in their orthopedics, and dental implant surgery rapid erosion at neutral pH; furthermore, because of its low toxicity, relatively low the adhesion to mucosal tissues is reduced cost, good biocompatibility, and osteo- when cross-linked with divalent cations. conductivity [32,283]. The thickening, gel- Alginates have been extensively used to forming and stabilizing property of alginate modify the performances of other polysac- adds it amongst the most widely used bio- charides, such as chitosan, through the rea- polymer with broader range of applications lization of alginate coated chitosan micro- including tissue engineering, drug delivery, spheres [277]. In the literature, it is also biosensor, and wound dressing [284]. possible to find acrylic modified polysaccharides developed with the aim to obtain a Composite materials of alginate with ce- finer control over release rate or to improve ramics play a major role in increasing the adhesive properties [278,279]. mechanical strength of extracellular matrix. Injectable calcium phosphate-alginate hy- Due to the large variety of possible chemi- drogel-umbilical cord mesenchymal stem cal compositions and molecular weights of cell (UCMSC) paste reported by Zhao et al. alginate preparations resulted in different [285] showed remarkable osteogenic diffe- effects on biological systems. A biological rentiation with alkaline phosphatase activity effect of alginate initially was hinted at in (ALP), osteocalcin (OC), collagen I, and the transplantation trails of encapsulated mineralization expressions. Bouhadir et al. Langerhans islets for diabetes control. Over [286] introduced hydrogel containing chon- growth of alginate capsule by phagocytes drocytes in to the dorsal region of mice. and fibroblasts resembling foreign After 7 weeks the mice were dissected; hy- body/inflammatory reaction was reported. drogel-containing chondrocytes scaffolds The main hurdles to the widespread use of uncovered and removed. A white opales- islet transplantation for the treatment of cence was observed in which appearance of type 1 diabetes continue to be the insuffi- native cartilage was confirmed by standard cient number of appropriate donors and the trichrome blue staining. Reduction in need for immunosuppression. Microencap- weight of the hydrogel implant within a sulation has been proposed to protect trans- span of 7 weeks was attributed to the de- planted islets from the host‘s immune sys- gradation of the hydrogel and release of tem. The study made by Qi et al. [280] in- oxidized alginate much faster than in vitro vestigated the function of human pancreatic studies on the same construct revealed. islets encapsulated in Ca2+/Ba2+– alginate These results were in contrast with the micro beads intraperitoneally transplanted unmodified alginate and chondrocytes in in diabetic Balb/c mice and concluded that which small cartilage-like tissues were sur- The Ca2+/Ba2+– alginate microbeads can rounded by huge amount of residues from protect human islets from xenogeneic rejec- the alginate. This injectable scaffold could tion in immune competent mice without find way in regeneration of cartilage like immunosuppression. However, grafts ulti- tissues. Moshaverinia et al. [287] developed mately failed likely secondary to a macro- an injectable and biodegradable oxidized phage-mediated foreign body reaction alginate microbeads encapsulation to peri- [281]. odontal ligament and gingival mesenchymal stem cells (GMSCs) and in vitro study

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 23 of 50 showed high level osteo-differentiation and cells, monocytes, macrophages, and den- adipo-differentiation [32,287]. dritic cells) molecules on the surface of lymphocyte (T) cells. Furthermore, there is Alginates, fucoidans and laminaran have huge correlation between the size of the also antibacterial effect. Extracts were sulfated SPMG oligosaccharides and their tested against nine bacteria, including Esce- inhibitory significance that the octa- richia coli, Salmonella, Staphycococcus and saccharide will be the minimal active frag- Listeria. They appeared to be effective ment preventing syncytium formation and against E. coli and Staphylocococcus. So- reducing the p24 core antigen level in HIV- dium alginate seemed to demonstrate a IIIB-infected CEM cells (a cell line derived strong antibacterial element. It not only from human T cells) [295,296]. binds but also kills the bacteria. Studies conducted on seaweed extracts found that Moreover, alginates with molecular weights fucoidan appeared to function as a good greater than or equal to 50 kDa could pre- prebiotic (a substance that encourages the vent obesity, hypocholesterolemia and di- growth of beneficial bacteria in the intes- abetes [281,297-300]. Clinical observations tines). An anti-inflammatory effect from of volunteers who were 25-30% overweight some of the extracts has also been found, showed that alginate, a drug containing and so far, no toxic effects have emerged in alginic acid, significantly decreased body use for human health [281,288,289]. weight [301,302]. Cooking into the bread does not reduce the molecular size of the A prominent marine polysaccharide drug alginate or affect its inhibition properties. named ―911‖ derived from alginate poly- These data demonstrate the robustness of saccharide exhibited promising activity alginates lipase inhibition despite the cook- against Human Immunodeficiency virus ing process and digestion. Therefore, add- (HIV-1) at both chronic infection of Human ing alginate to a bread vehicle may have the Cutaneous T lymphocyte (H9) cells and potential in the treatment for obesity [303]. acute infection of Human Tcell leukemia In type II diabetes treatment, taking 5 g of (MT4) cells in vitro and in vivo [102]. sodium alginate, every morning was found These special effects revealed that ―911‖ to prevent a postprandial increase of glu- drug inhibited the viral replication of HIV cose, insulin, and C-peptide levels and via significantly decrementing the activity slowed down gastric transit [304]. Meal of reverse transcriptase (RTase), disconti- supplemented with 5% Kelp alginates de- nuing the virus adsorption, and improving creased glucose absorption balance over 8 h the defense mechanisms of the host cells in pigs. Similar studies have been done on [290-292]. Alternative inhibitory result was rats and humans [305,306]. also reported later for hepatitis B virus (HBV) that ―911‖ drug could inhibit the Another health effect is that the binding virus replication by suppressing the activity property of alginic acid to divalent metallic of DNA polymerase activity [293]. Wang ions is correlated to the degree of the gela- and colleagues discovered that the sulfated tion or precipitation in the range of polymannuroguluronate (SPMG) [294], the Ba

Canada, the use of alginic acid and its de- 4.6. Ascophyllan rivatives for the treatment of gastritis and gastroduodenal ulcers, as well as the use of Ascophyllan isolated from the brown alga alginates as anti-ulcer remedies, is protected Ascophyllum nodosum (Phaeophyceae) is a by patents [3,281,308-310]. Several prod- fucose-containing sulfated polysaccharide, ucts of alginate containing drugs have been which has similar but distinct characteristic shown to effectively suppress postprandial monosaccharide composition and entire (after eating) and acidic refluxes, binding of chemical structure to fucoidan [322]. Asco- bile acids and duodenal ulcers in humans phyllan (xylofucoglycuronan) has similar [310]. Examples are ―Gaviscon‖ (sodium but obviously distinct composition charac- alginate, sodium bicarbonate, and calcium teristics from those of fucoidans isolated carbonate), ―Algitec‖ (sodium alginate and from A. nodosum and Fucus vesiculosus cimetidine, an H2 antagonist) and ―Gastral- [323,324]. Specifically, ascophyllan has gin‖ (alginic acid, sodium alginate, alumi- fucose and xylose in about equimolecular num hydroxide, magnesium hydroxide and proportion, whereas fucoidans have much calcium carbonate) [262,281,311-313]. higher ratio of fucose than of xylose. The Clinical trials showed that sodium alginate sulfate levels of ascophyllan and fucoidans promotes regeneration of the mucous mem- isolated from A. nodosum and F. vesiculo- brane in the stomach, suppresses inflamma- sus were 9.6, 19.4, and 22.6%, respectively tion, eradicates colonies of Helicobacter [325]. In addition, the apparent molecular pylori in the mucous membrane and norma- mass of ascophyllan estimated by gel filtra- lizes non-specific resistance of the latter in tion chromatography was about 390 kDa, 4 to 15-year-old children. It also promotes which is much higher than that of fucoidan restoration of the intestinal biocenosis [326]. [71,281,314]. Other studies show positive dietary effects of alginates on fecal micro- 4.7. Biological activities of Ascophyllan bial fauna, changes in concentrations of compounds and acids, and prebiotic proper- Mohsin et al. [318] characterize and eva- ties that can promote health [71,315, 316]. luate the antioxidant activity of a sulfated polysaccharide ascophyllan isolated from A study conducted by Zaharudin et al. [317] marine brown algae Padina tetrastromati- investigated the potential of dried edible ca. The results showed that one of the asco- seaweed extracts and alginates for α- phyllan fractions showed stronger free- amylase inhibitory effects. Alginates found radical-scavenging abilities and had good in Laminaria digitata and Undaria pinnati- antioxidant effect. Available data obtained fida (Phaeophyceae) appeared to be potent by in vitro models suggest that there is a inhibitors of α-amylase activity with an IC50 correlation between the sulfate content and of (0.075 +/- 0.010-0.103 +/- 0.017) antioxidant activity. mg/mL, also a mixed-type inhibition. Over- all, the findings provide information that In work carried out by Jiang et al. [319], crude extracts of brown edible seaweeds, ascophyllan and crude extract administered phenolic compounds and alginates are po- via the oral route showed greater antitumor tent α-amylase inhibitors, thereby potential- effects than via IP (Intraperitoneal) route, ly retarding glucose liberation from starches and the tumor sizes in mice treated with and alleviation of postprandial hyperglyce- ascophyllan and crude extract were reduced mia [317]. by a mean of 68.7% and 42.4% by the oral route, and 41.4% and 13.6% by IP route compared to control mice. Splenic natural

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 25 of 50 killer cell activity in the mice treated with Later, the same team [321] evaluated asco- ascophyllan and crude extract by IP route phyllan as an adjuvant for its therapeutic was significantly enhanced, while only a and preventive effect on tumor in a mouse slight increase of this activity was observed melanoma model. Ascophyllan induced in orally-treated mice. Furthermore, in- migration of DCs to spleen and tumor- crease in spleen weight of tumor-bearing draining lymph node (drLN) in a mouse mice was slightly suppressed by oral ad- B16 melanoma model. Moreover, ascophyl- ministration of ascophyllan, whereas IP lan induced activation of dendritic cells administration resulted in further enlarge- (DCs), and promoted IFN-γ- and TNF-α- ment. Analysis of serum cytokines revealed producing murine type 1 helper T cells that oral treatment with ascophyllan re- (Th1) immune responses in tumor-bearing sulted in significant increase of tumor ne- mice. In addition, treatment with a combi- crosis factor-α and interleukin-12 levels. nation of ascophyllan and ovalbumin Since ascophyllan showed no direct cyto- (OVA) in the tumor-bearing mice promoted toxic effect on sarcoma-180 cells, orally- proliferation of OVA-specific CD4 and administered ascophyllan is suggested to CD8 T cells and migration of those cells exhibit its antitumor activity through the into the tumor, consequently inhibiting the activation of the host immune system [319]. tumor growth. Immunization with the combination of ascophyllan and OVA caused Zhang et al. [320] investigated the effect of enhanced OVA-specific antibody produc- ascophyllan, a sulfated polysaccharide puri- tion and memory T cell responses compared fied from Ascophyllum nodosum, on the to OVA immunization alone, and almost maturation of mouse dendritic cells (DCs) completely prevented B16-OVA tumor in vitro and in vivo. Ascophyllan induced growth upon subsequent tumor challenge. up-regulation of co-stimulatory molecules Lastly, the combination of ascophyllan and and production of pro-inflammatory cyto- OVA prevented B16-OVA tumor invasion kines in bone marrow-derived DCs and metastasis into the liver. Therefore, (BMDCs). Interestingly, ascophyllan in- these results demonstrate that ascophyllan duced a higher degree of co-stimulatory can function as an adjuvant to induce DCs molecule up-regulation and pro- activation, antigen specific cytotoxic T inflammatory cytokine production than fu- lymphocyte (CTL) activation, Th1 immune coidan, a marine-derived polysaccharide response and antibody production, and with well-defined effect for promoting DCs hence may be useful as a therapeutic and maturation. Ascophyllan also promoted the preventive tumor vaccine [321]. generation of IFNγ (Interferon gamma)- producing Th1 (murine Type 1 helper T cells) and Tc1 (Type 1 cytotoxic T lympho- 5. Conclusion cytes) cells in the presence of DCs in an IL (cytokines)-12-dependent manner. Finally, Currently, seaweeds are used in many coun- myeloid differentiation primary response 88 tries for very different purposes. Most of (MyD88) signaling pathway was essential macroalgae are used directly for human for DCs maturation induced by ascophyllan. consumption, while the remaining portion is Taken together, these results demonstrate used for industrial exploitation of macroal- that ascophyllan induces DCs maturation, gae derived products, among which poly- and consequently enhances Th1 and Tc1 saccharides have many uses. The polysac- responses in vivo. This knowledge could charides produced by the marine macroal- facilitate the development of novel thera- gae have varied bioactivities, in which they peutic strategies to combat infectious dis- stand out the antioxidant, anticoagulant, eases and cancer [320,329]. antithrombotic, antitumor, anti-inflamma-

Copyright 2018 KEI Journals. All Rights Reserved http://journals.ke-i.org/index.php/IBR Pereira L. International Biology Review, vol. 2, issue 2, June 2018 Page 26 of 50 tory, antiviral, antiprotozoal, and other ac- ninsula. Current Organic Chemistry tivities [329]. Among the various bioactivi- 2014, 18(7), 896-917. ties listed in this review, polysaccharides may be used as potential ingredients in fu- 4. Holdt SL, Kraan S. Bioactive com- ture pharmaceutical products. pounds in seaweed: functional food applications and legislation. Journal of Applied Phycology, 2011, 23(3), Acknowledgements 543-597.

This work had the support of Fundação para 5. Murata M, Nakazoe J. 2001. Pro- a Ciência e Tecnologia (FCT), through the duction and use of marine algae in strategic project UID/MAR/04292/2013 Japan. Japan Agricultural Research granted to MARE. This work had the sup- Quartery, 2001, 35, 281-290. port from the European Structural & In- vestment Funds through the COMPETE 6. Chandini SK, Ganesan P, Bhaskar Programme and from National Funds N. In vitro antioxidant activities of through FCT – Fundação para a Ciência e a three selected brown seaweeds of Tecnologia under the Programme grant India. Food Chem, 2008, 107(2), SAICTPAC/0019/2015. This work is also 707-713. co-financed by the European Regional De- velopment Fund through the Interreg Atlan- 7. Pereira L, Amado AM, Critchley tic Area Programme, under the project AT, van de Velde F, Ribeiro-Claro NASPA. PJA. Identification of selected seaweed polysaccharides (phycocolloids) by vibrational spectroscopy References (FTIR-ATR and FT-Raman). Food Hydrocolloids, 2009, 23, 1903- 1. Pereira L. Guia ilustrado das ma- 1909. croalgas - conhecer e reconhecer al- gumas espécies da flora Portuguesa 8. Bixler HJ, Porse H. A decade of (Illustrated guide of macroalgae - to change in the seaweed hydrocolloid know and recognize some species of industry. Journal of Applied Phy- the Portuguese flora). Imprensa da cology, 2011, 23, 321-335. Universidade de Coimbra, Coimbra, Portugal, 2009, 90 pp. (in Portu- 9. Pereira L, van de Velde F. Portu- guese). guese carrageenophytes: carrageenan composition and geographic 2. Pereira L. Chapter 4 - Cytological distribution of eight species (Gigar- and cytochemical aspects in selected tinales, Rhodophyta). Carbohydrate carrageenophytes (Gigartinales, Polymers, 2011, 84, 614-623. Rhodophyta). In: Heimann, K. and Katsaros, C. (eds.), Advances in Al- 10. Pereira L. Population studies and gal Cell Biology. De Gruyter, Ber- carrageenan properties in eight Gi- lin, 2012, pp. 81-104. gartinales (Rhodophyta) from western coast of Portugal. The Scientific 3. Cardoso SM, Loïc GC, Paulo JS, World Journal, 2013, 11 pp. Rodrigues MS, Pereira OR, Pereira https://www.hindawi.com/journals/t L. Bioproducts from macroalgae: A swj/2013/939830/ review with focus on the Iberian Pe-

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