*5.2. Fucoidans*

Fucoidans are highly sulphated, usually branched polysaccharides, often containing, in addition to fucose residues, glucose, galactose, xylose, mannose, and uronic acids, as well as acetyl groups [45]. The structures of polysaccharides from brown algae are very diverse and depends on the type of alga, its reproductive status, and other abiotic factors. In fact, each new polysaccharide isolated from algae is a new substance, and its molecule contain unique structural elements. This is why the determination of the structure of fucoidans, as well as the clarification of the structure/function relationship of these polysaccharides, is extremely difficult.

It should be noted that the effectiveness of fucoidans of brown algae as potential anti-influenza agents has been studied quite actively in recent years due to the polyvalence of their effects (antiviral, antibacterial, anti-inflammatory, immunomodulatory effects, etc.), as well as the fact that fucoidans penetrate biological membranes. Efficiency of oral administration of fucoidan is confirmed by data of its transformation in macroorganism, which are presented in articles [73,74]. The possibility of the appearance of fucoidan derivatives in the peripheral blood was confirmed by Irhimeh M.R. et al. [73]. Authors using monoclonal antibodies to highly sulfated fucoidan found its derivatives in plasma of healthy participants who took orally for 12 days at 3 g/d Undaria algae powder, containing 10% of fucoidan derivatives and purified galactofucane sulfate. The average concentration of fucoidan detected in plasma was 4.002 mg/L and 12.989 mg/L, respectively. Tokita Y. et al. [74] also found fucoidan from Cladosiphon okamuranus in the serum and urine of healthy participants 6 and 9 h after ingestion of the polysaccharide orally. These facts indicate the possibility of degradation of fucoidan molecules in the human body and the participation of its derived structures in the implementation of antiviral properties.

After administration, these polysaccharides can be detected in the urine and serum [74,75]. Histological studies using monoclonal antibodies against fucoidan made it possible to detect it in the small intestine, the epithelial cells of the jejunum, in mononuclear cells, and in sinusoidal non-parenchymal cells of the liver [76]. The same authors established the active transport of fucoidan through a monolayer of Caco2 cells in vitro and the excretion of fucoidan in the urine of a patient after oral administration. The level of fucoidan increased from 3 to 9 h after administration [77]. To prevent the destruction of fucoidan in the stomach, it is suggested to enclose it in chitosan nanocapsules.

A study on the anti-influenza activity of polysaccharides from the sporophylls of the brown alga *Undaria pinnatifida* allowed Synytsya et al. [78] to establish that in mice, infected in vivo with avian influenza A viruses (subtypes H5N3 and H7N2), the level of virus replication decreased and the production of specific antibodies increased. Oral administration of the polysaccharide blocked the release of the virus from cells and significantly increased the titer of virus-neutralizing antibodies and IgA. This polysaccharide presents as a low molecular weight (Mw 9 kDa) fucogalactan, consisting of partially sulphated and acetylated fucose and galactose residues in approximately equal amounts and having a complex structure. Previously, Hayashi et al. [79] investigated the effectiveness of this *O*-acetylated sulphated fucogalactan in immunocompetent and immunocompromised mice infected with a lethal dose of influenza virus. The use of this polysaccharides reduced virus replication, weight loss, and mortality in animals of both groups and increased their lifespan. Oral administration of fucoidan caused an increase in the titre of neutralizing antibodies in the blood and mucous membranes. In immunocompromised mice, drug-resistant viruses often multiply after treatment with oseltamivir. No resistant viruses were isolated from mice treated with fucoidan. The authors proposed the combined treatment with oseltamivir and fucoidan, because in this case there was no recurrence of influenza virus reproduction, as is sometimes the case when treating only with oseltamivir. Combined treatment with fucoidan and oseltamivir was thus recommended by the authors as a new treatment strategy for influenza infection.

The fucoidan from the brown alga *Kjelmaniella crassifolia* (Mw about 536 kDa, sulphate content 30.1%, purity more than 98%) is a glucuronomanan with branches in the form of oligosaccharides at position 3 of the fucose residues. Oligosaccharides (degree of polymerisation from 0 to 6) consist of 3-linked glucose residues, sulphated at positions 2 and 4 [62]. Intranasal (for four days) application of fucoidan increased the survival of mice (80% versus 30%) and their lifespan and reduced the viral load of the lungs in influenza-infected animals compared to the control group (*p* < 0.05). When treated with oseltamivir alone, 90% of the mice survived. All influenza viruses used in the experiment were sensitive to treatment with fucoidan, but the most susceptible virus was H1N1 (Ca109) (IC50 < 6.5 μg/mL). Treatment with fucoidan reduced the severity of flu symptoms and pathological changes in the lungs. One valuable quality of fucoidan was the lack of formation of resistant strains of the virus under the action of this polysaccharide. In the supernatants of spleen cells, the levels of interferon-gamma (IFN-γ) and interleukin-2 (IL-2) increased following treatment with fucoidan compared with the control animals. In addition, a direct effect of fucoidan on viral particles was found. It was shown that pre-incubation of the virus with fucoidan at concentrations of 31.25–250 μg/mL significantly reduced the number of plaques in MCDK cell culture, i.e. this polysaccharide can inactivate viral particles by direct contact. This polysaccharide inhibited the activation of the epidermal growth factor receptor (EGFR-epidermal growth factor receptor) and was able to bind to viral NA and inhibit its activity. In this regard, the authors believed that such inhibitors of the EGFR pathway and NA can be used alone or with other drugs to block the processes of penetration and the release of influenza A virus from cells. The investigated fucoidan is a potential candidate for creating a medicine in the form of a spray or drops.

Using SPEV cell culture sensitive to the reproduction of influenza A (H5N1), Makarenkova et al. [80] investigated the in vitro antiviral effect of a fucoidan from the brown seaweed *Laminaria japonica* against the H5N1 influenza virus. The results showed that the fucoidan did not possess cytotoxic properties in concentrations from 500 μg/50 μL to 125 μg/50 μL and did not change the morphological properties of the SPEV cell culture. Fucoidan had a virucidal effect and suppressed the infectious properties of the H5N1 flu virus (a decrease in virus titer of 3.0–3.3 log units relative to the control), but did not protect the cell culture against cytopathogenic effects of the influenza A virus at 48 and 72 h after infection. At the same time, the fucoidan showed antiviral activity at an early stage of infection, i.e. during the first 24 h. The application of fucoidan to the cell culture in various concentrations an hour before the virus was introduced resulted in a decrease in the titer of the influenza virus by 2.3–3.3 log units. With simultaneous introduction of influenza A virus and fucoidan into the cell culture, the virus titer was decreased by 2.3–2.8 log units relative to the control. These results open up prospects in terms of developing new approaches to interrupting virus adsorption by sensitive cells.

A number of reports have been devoted to comparative studies of the anti-influenza effectiveness of polysaccharides from several families of algae [81]. Song et al. [82] assessed *Grateloupia filicina* (family Rhodophita), *Ulva pertusa* (family Chlorophyta), and *Sargassum qingdaoense* (Ochrophyta) in their studies. The yield of polysaccharide was 19.7% (*G. filicina*, GFP), 12.1% (*U. pertusa*, UPP), and 7.2% (*S. qingdaoense*, SQP). The content of sulphate groups in the polysaccharide was also different: 13.54% in UPP, 19.89% in GFP, and 5.64% in SQP. The structure of all three polysaccharides was established, and their biological activities were investigated in vivo and in vitro. The safe concentration for SQP and UPP was 5 mg/mL, and for GFP this was 2.5 μg/mL. The in vitro antiviral effects were evaluated against the H9N2 influenza virus. In the hemagglutination test, the most active were UPP and SQP. Under the action of these polysaccharides, the titer of influenza B virus decreased significantly. GFP was the most active in reducing virus replication and SQP was the least active. The most effective dose of the polysaccharide was 20 μg/mL. Using real-time polymerase chain reaction (PCR), it was shown that the expression of the H9N2 gene was significantly reduced under the influence of the studied polysaccharides. The best inhibitory effect was observed with a GFP dose of 20 μg/mL. In the same study, the authors showed that all three polysaccharides had immunomodulatory potential as the studied polysaccharides were active in the spleen lymphocyte proliferation test. The greatest activity in this test was shown by SQP, the effect of which was dose-dependent. Maximum values were obtained when using the sulphated polysacchrides at a dose of 500 μg/mL. In the experimental group of mice treated with the polysaccharide, the levels of IFN-γ and IL-4 were significantly increased (*p* < 0.05). All polysaccharides increased numbers of CD3+ and CD4+ lymphocytes in the blood compared to controls, but only SQP increased the level of CD8+ cells. Thus, the best effect was obtained with the polysaccharide of the brown alga *S. quingdaoense,* especially at a dose of 50 mg/kg. The authors attributed this phenomenon to the presence of fucose residues in its structure, which play a significant role in immunomodulation [83]. The content of fucose in the PCA was 0.02, 0.05, and 1% for UPP, GFP, and SQP, respectively. The authors also observed immunological phenomena as such the proliferation of spleen cells and the humoral immune response, connected with the presence of fucose in these preparations. The more pronounced suppression of the replication of the influenza virus by the GFP polysaccharide was explained by the higher content of sulphate groups in the structure of this polysaccharide [84,85]. The authors suggest the use of all three polysaccharides as a potential alternative to vaccination, as well as to suppress the replication of the influenza virus.

In another study [81], as a result of a comparative study of sulphated polysaccharides activity from algae of different families (red algae: *Polysiphonia lanosa*, *Furcellaria lumbricalis*, and *Palmaria palmate*; brown algae: *Ascophyllum nodosum* and *Fucus vesiculosis*; green alga: *Ulva latuca*), it was found that fucoidans from brown algae, i.e., *F. vesiculosis* and *A. nodosum*, had the highest anti-influenza activity. The total sugar content in the polysaccharides studied varied from 15.4% (*U. latuca*) to 91.4% (*F. lumbricalis*). Galactans (agars or carrageenans) were mainly isolated from *P. lanosa*, xylans from *P. palmate*, and fucoidans from brown algae. Heteropolysaccharides were isolated from green algae.

The interaction between the H5N1 influenza virus and fucoidan were investigated by Bobrovnitsky [86]. For the first time, the authors visualized this process by scanning probe microscopy, which provides information about the surface microrelief and measures the length and height of observed objects. In this case, measurements were made of the height of viral particles before and after treatment with fucoidans from two types of algae. The concentration of fucoidans was 1 and 100 ng/mL. It was found that the average size of the virus particles after treatment with fucoidans changed significantly. In the case of a lower concentration, the average height of the particles increased from 40 to 45 nm. With increasing concentrations, the height of the virus particles reached 50 nm. These results indicate an interaction between the positively charged groups of lipoproteins of the viral envelope and the negatively charged sulphate groups of fucoidans; this interaction may be the cause of the antiviral effect of polysaccharides. The antiviral effect of these compounds is probably due to the encapsulation of viral particles and their deactivation as a result of this. Adhesion of fucoidan on the surface of viral particles is an irreversible process.

Based on this evidence, fucoidans have not only a direct effect on influenza viruses but also affect the processes of viral attachment and replication, interact with neuraminidase, and inhibit the release of viruses from cells [62,80]. In addition, they promote antiviral immunity, enhance antioxidant protection, and reduce the appearance of inflammation. Numerous studies [75,85,87,88] have demonstrated the influence of these compounds on factors important in innate and adaptive immunity, such as the antioxidant system. Another positive quality of fucoidans is their antibacterial action, which in some cases will allow them to be used to prevent bacterial complications, which often aggravates the course of influenza infection.

#### **6. Lectins**

Lectins are widespread carbohydrate-binding proteins and glycoproteins that can specifically and reversibly non-covalently bind mono- and oligosaccharides, both in solution and localized on the cell surface [89,90]. In this way, lectins contribute the so-called first line of defence against bacteria and viruses. These compounds exhibit high specificity in relation of glycoconjugates of bacteria and viruses. Most studied and characterized lectins have been isolated from higher plants; lectins from algae have been studied less thoroughly. However, the observed antiviral and antitumor effects of these compounds have led scientists to look at them from a new perspective. It was previously known that lectins have two or more carbohydrate-binding sites [91]. Connecting to the surface of microorganisms, the lectin can agglutinate and prevent the spread of pathogens throughout the body. In recent years, new families of lectins have been found in the cyanobacterium *Oscillatoria agardhii* (OAA) [92–95], as well as in the red algae *Eucheuma serra* and *Kappaphycus alvaresii* (KAA-2) [96]. They usually have two or four tandem repeats consisting of highly conserved sequences, but do not have homology with other protein families. The uniqueness of these lectins is that they bind carbohydrates with exceptionally high specificity for high-mannose (HM) glucans in the trisaccharide core, including Manα(1-3) Manα(1-6)Man. At low nanomolar levels, these lectins have potential antiviral activity against the influenza virus due to the recognition of HM-glucans in the composition of the glycoproteins of the spikes of the influenza virus.

Mu et al. [97] isolated lectin HRL40 from the green alga *Halimeda renschii*, which was highly specific to HM-N-glycans with (1,3)-bound monosaccharide residues. Lectin HRL40, by binding to the hemagglutinin of the virus, effectively inhibited (with an ED50 2.45 nM) the infectious process in NCI-H292 cells caused by the influenza A/H3N2/Udom/72 virus. Additionally, a lectin with anti-influenza activity was obtained by Sato et al. [98] from the red alga *Eucheuma serra*. This compound, called by the authors a "high mannose-specific lectin" and designated as KAA-2, effectively inhibited the entry of the influenza virus into cells. The carbohydrate-binding profile of this lectin was determined by centrifugation and ultrafiltration. KAA-2 was associated exclusively with high mannose N-glycans, but not with other glycans. The authors tested this lectin against various strains of influenza virus, including the pandemic variant H1N1-2009. With the immunofluorescent method, it was shown that lectin prevented the virus from entering the host cells. Using ELISA, it was found that the lectin KAA-2 was directly associated with the HA of the influenza virus. It was proposed the use of this lectin as a future means of preventing influenza infection.

In the study by Sato et al. [99], the anti-influenza activity of lectins with various carbohydrate specificities was investigated on MDCK cells using different strains (clinical isolates) of the influenza virus (H1N1-2009, A/Oita/ou1P3-3/09). The best results in terms of inhibiting influenza infection were obtained with the HM-binding lectin ESA-2. The EC50 in this case was 12.4 nM. This lectin recognized the branched structure of HM-glycans, including the trisaccharide containing Manα(1-3)Manα(1-6)Man in the D2 branch as a primary target. The direct interaction between the lectin ESA-2 with the viral envelope glycoprotein HA was demonstrated by ELISA. This interaction was effectively suppressed by glycoproteins carrying HM-glycans, suggesting that ESA-2 binds to the HA of the influenza virus through HM-glycans. The lectin inhibited the penetration of the virus into cells most effectively when simultaneously introducing the virus and the lectin into the cell culture. When processing ESA-2 cells, viral antigens were not detected in the cells, which indicated that this lectin inhibited the initial stages of virus penetration into the cells. At the same time, no cytopathic effect was observed in infected cells. The antiviral profile of ESA-2 was similar to the lectin KAA-2 from *Kappaphycus alvarezii*, which belongs to the same anti-HIV lectin family [98]. The lectin was non-toxic up to 1000 nM (the highest dose used in this experiment). Sensitivity to lectin depended on the strain of influenza virus. The most susceptible were strains A/Philippines/2/82 (EC50 17.2 ± 3.9) and WSN/33 (EC50 34.6 ± 2.7 nM). In this case it was also proposed to use the lectin ESA-2 in the future as a disinfectant or prophylactic agent.
