*5.1. Carrageenans*

Carrageenans are sulphated polysaccharides of red algae whose chemical structure is based on a disaccharide repeating unit consisting of two D-galactose residues joined with each other by β-1,4 glycosidic bonds. These units are bound in polysaccharides by α-1,3 bonds [46]. The structural diversity of carrageenans is due to the presence of β-(l,4)-linked residues in the form of 3,6-anhydrogalactose, as well as the number and position of sulphate groups in monosaccharide residues [47]. Regular polysaccharides, the polymer chain of which is built from repeating disaccharide units of the same type, represent a different class of carrageenans. Natural carrageenans are rarely regular, as more often they contain repeating units of several types and have an irregular or hybrid structure (Figure 1), structures of which are explained by the multi-stage biosynthesis of polysaccharides in the algal cell wall.

The variability of the primary structure of carrageenans determines the diversity of their macromolecular organisation and defines a wide range of their biological activity [46]. The uniqueness of these hydrocolloids is in the alternating galactose and 3,6-anhydrogalactose residues, which are linked by α-1,3 and β-1,4 glycosidic bonds. A characteristic feature of carrageenan molecules is the large number of sulphate groups [48].

The quantity and location of sulphuric acid residues determine the type, form, and functions of carrageenans, which are actively used in food industry for the production of meat, dairy, and confectionery to improve the microtexture of nutritive products, i.e., as gelling agents, emulsifiers, and thickeners. Among the polysaccharides of algae, carrageenans are the most studied regarding their toxicity, pyrogenicity, and allergenicity [49]. The safety of their use in food and medical purposes has been confirmed by numerous studies [50]. Among the diverse biological properties of these sulphated polysaccharides, their antiviral, anticoagulant, immunomodulatory, antitumor, and anti-ulcer activity are currently attracting the greatest interest [46,51,52]. Sulphated polysaccharides interact with a variety of eukaryotic cell proteins and have a multidirectional effect on the body's immune

response, both inhibitory and stimulatory, which makes it possible to consider carrageenans as possible immunomodulators. It is assumed that the immunomodulatory effect of carrageenans is initiated by α-Gal-(1,3)-Gal epitopes [53]. Recently, new data have appeared on the antioxidant activity of algae polysaccharides [46,54].

Carrageenans have attracted the attention of researchers investigating the problem of influenza and other acute respiratory viral infections, particularly in terms of the possibility of creating a physical barrier in the nasal cavity against respiratory viruses, including the flu virus [55]. For this purpose, kappa (k), iota (ι), and lambda (λ) carrageenans were used. It is known that carrageenans block the interaction of viruses with cells and also inhibit the formation of a syncytium, induced by influenza A viruses. A. Leibbrand et al. [37] carried out an investigation showing the effectiveness of carrageenan against human influenza A viruses. The authors determined the sensitivity of the H1N1 influenza virus strains, as well as the pandemic H3N2 strain, to carrageenan subtypes ι and k using the plaque formation method in canine kidney epithelial cells (MDCK). The most active in this test was ι-carrageenan (IC50 or 50% inhibitory concentration = 0.04 μg/mL); k-carrageenan was less active (IC50 = 0.3 μg/mL). The purity of the ι- and k-carrageenans used in these studies was above 95%, and the molecular weight of both polymers was more than 100,000 Da. At the concentrations of 40 and 4 μg/mL, ι-carrageenan effectively reduced viral replication by 2–4 log units within 96 h after infection. Thus, it was found that ι-carrageenan contributes to the survival of cells infected with the virus by direct exposure to the virus. In another series of experiments, the same authors investigated the effect of carrageenans on an influenza virus-infected primary cell culture of the human epithelium from the nasal cavity. Under these conditions, ι-carrageenan inhibited the formation of plaques by the pandemic strain H1N1/2009 (IC50 about 0.04 μg/mL). At the same time, an interesting fact was established: to obtain an effect when cells were infected with another virus (A/PR8/34 H1N1), a higher concentration of polysaccharide was required, i.e., the sensitivity of different strains to carrageenan was different.

Carrageenans are high molecular weight compounds and therefore it is unlikely that they can pass through the barriers of the body. However, local administration has a pronounced effect, for example, with influenza infection and other viral diseases of the respiratory system. In this case, carrageenans reduce the spread of the virus in the surface epithelium of the respiratory organs of infected animals and contribute to survival.

Unfortunately, the solubility of carrageenans is limited, especially in aqueous solutions containing potassium and calcium ions [52], since in their presence carrageenans form viscous gels. Another disadvantage of carrageenans is their anticoagulant properties. Despite this, ι-carrageenan has passed clinical trials and a nasal spray based on it has already been successfully sold in Europe for use in viral infections of the respiratory tract in humans. The effectiveness of the spray for ARIs was also reported by Eccles et al. [56]. The authors showed that, compared with persons receiving placebo, patients in the experimental group noted such significantly reduced symptoms of the disease as nasal congestion, runny nose, cough, and sneezing. Moreover, nasal congestion at the end of the observation period was noted by 63.6% of persons in the placebo group and 28.6% of the group receiving carrageenan. Viral capacity in the nasal mucosa in patients treated with the spray was significantly decreased (92%), while placebo treatment did not affect viral replication. The nasal spray was effective when used during the first 48 h after the onset of symptoms. Similar results were obtained by Ludvig et al. [57].

Spray application reduced the expression of pro-inflammatory cytokines and increased the level of IL-1 and IL-12p40 receptor antagonists, which are known to have anti-inflammatory action in the nasal lavage of patients with respiratory viral infections [56]. It is known that IL-12p40 is necessary for inhibiting the hyperactivity of airway and peribronchial fibrosis [58]. The expression of inflammatory mediators during viral infection may complicate underlying diseases in the form of asthma [59,60]. In this regard, a decrease in the intensity of the immune response due to a lower viral load seems to be an attractive property of treatment of ι-carrageenan. To increase their efficiency, oligosaccharides and their sulphated derivatives having a lower molecular weight were obtained from the high molecular weight

ATP carrageenan [61]. So, the oligosaccharide CO-1 with a molecular weight of 1–3 kDa effectively dose-dependently inhibited the replication of influenza A (H1N1) virus in MDCK cells (selectivity index >25.0). CO-1 did not bind to the cell surface, but it was bound to viral particles during the pre-treatment process. Unlike high molecular weight native carrageenan, this oligosaccharide can penetrate into MDCK cells and inhibit the expression of viral proteins and mRNA after its internalization into the cell, but before it leaves the cell, i.e. in one replication cycle. The main factors affecting the antiviral activity of oligosaccharides are the degree of sulphation and Mw. The most active oligosaccharide CO-1 contained 0.8–1.0 mol/mol sulphate, and its molecular weight was 1–3 kDa. The preparation CO-1 and its full sulphated derivative (COS) significantly increased the survival rate of mice infected with a lethal dose of influenza virus and reduced the viral load in the lungs of these animals [62]. Taking into account these findings, the authors proposed using low molecular weight oligosaccharides of carrageenan in the treatment of influenza as an alternative strategy to combat this infection.

Shao et al. [63] investigated the molecular mechanisms of cell protection using k-carrageenan against SW731 influenza virus penetration. The authors showed that the polysaccharide specifically and effectively inhibited the reproduction of the influenza virus. The MDCK cells were infected with various strains of the influenza virus, after which they were treated with carrageenan at different doses. After 24 h, a dose-dependent decrease in the titer of SW731 and CA04 viruses (homologous MDCK) was recorded. The remaining experimental strains of influenza virus (PR8, WSN, ZB07, and H1N1) were insensitive to carrageenan. Thus, k-carrageenan prevented the development of the extraand intracellular stages of influenza virus replication. To determine the stage of reproduction of the virus affected by carrageenan, the authors added polysaccharide to infected cells during the period of adsorption (0 h), internalization (1 h), early replication (2–6 h), and release (8 h). Then, after 24 h, the inhibitory effect was evaluated, in response to treatment at the time points of 0, 1, 2, 4, and 6 h. After 8 h, there was no inhibition. Thus, it was shown that not only extracellular, but also some intracellular stages of influenza virus replication were affected by k-carrageenan. The titer of influenza virus SW731 was decreased in cases where the virus was treated with the polysaccharide before or during infection of the cells. When treating cells with carrageenan, there was no such effect, i.e. the best results were obtained by the action of the polysaccharide at the adsorption stage. The same authors showed that carrageenan does not inactivate the influenza virus, since no differences were found between the pre-treatment group and the adsorption group. Carrageenan specifically inhibited the binding of the HA virus to its receptor, sialic acid. The effect of polysaccharides on the exit stage was insignificant. The authors have proposed the use of carrageenan against the H1N1/2009 influenza virus and other viruses containing HA/H1N1/2009. The carrageenan used in these experiments contained both highand low-molecular components, and therefore it acted on the extracellular and intracellular stages of reproduction.

Other authors [64], investigating the polysaccharides of the red alga *Gyrodinium impudicum*, obtained sulphated galactan conjugated with uronic acid and studied its activity as an anti-influenza agent. As in the studies by Wang W. [61,62] it was shown that the antiviral activity (IC50 against the influenza virus at doses 0.19–0.48 μg/mL) of the galactan is related to its ability to interact with viral particles, which preventing virus adsorption and internalization.

Yu et al. [65] suggested using hybrid carrageenan (ı/κ/ν-carrageenan) as a potential inhibitor of influenza A virus. In this study, the authors obtained three polysaccharides from the red alga *Eucheuma denticulatum* by successive extraction with cold and hot water and an aqueous solution of NaOH: hybrid polysaccharide (EW), preparation EH, containing only ι-carrageenan, and α-1,4α-d-glucan (EA), which consisted of 88% of glucan and 12% carrageenan as an impurity. The molecular weights of the compounds were 480, 580, and 510 kDa, respectively. Antiviral activity against the H1N1 influenza virus was highest when used the hybrid polysaccharide (276.5 μg/mL), and the H1N1 virus suppression index was 52% using a polysaccharide dose of 250 μg/mL. The IC50 for ι-carrageenan EH was 366.4 μg/mL. The polysaccharide EA showed the lowest antiviral activity (IC50 > 430 μg/mL).

The study of Fazekas T. et al. [66] was very important, because it was conducted in a clinical setting with the participation of patients (children and adults aged from 1 to 18 years) with respiratory viral infections, including influenza B. Intranasal spray was used three times a day in for seven days. Symptom dynamics was monitored and viral load was determined. In this study iota-carrageenan did not alleviate symptoms in children with acute symptoms of common cold, but significantly reduced viral load in nasal secretions that may have important implications for future studies. In this study, v-carrageenan as part of the spray did not reduce the severity of symptoms in children with acute cold symptoms, but significantly reduced the viral load in the nasal lavage of patients who received the spray compared with the control group (27% versus 13%, respectively).

A positive evaluation of the effectiveness of nasal sprays in patients with acute respiratory viral infections was provided in two randomized double-blind, placebo-controlled trials by Koenighofer et al. [67]. In patients treated with carrageenan, the duration of the disease decreased by two days, there were fewer relapses, and the body was cleared of viruses more rapidly. The spray was effective in both children and adults. The treatment of patients with influenza by carrageenan gel was shown to significantly facilitate respiratory tract, reduces the duration of illness, and the severity of symptoms of intoxication. Carrageenans were found to provide a more pronounced synergistic effect with anti-influenza drugs with a different mechanism of action.

A number of authors have proposed increasing the effectiveness of the treatment by combining carrageenan with other drugs. Thus, a combined intranasal spray, including carrageenan and zanamivir (an NA inhibitor) was proposed by Morokutti-Kurz et al. [68]. Previously, the authors investigated the efficacy of in vivo and in vitro intranasal administration of zanamivir in different doses for the prevention and treatment of influenza. Their study showed that treatment of animals before infection and 36 h after infection with a virus was not accompanied by adverse events.

Zanamivir and carrageenan separately exhibited different antiviral activity against different strains of the influenza virus. Since the mechanism of action of these agents is quite different, one could expect protection against a wider spectrum of viruses than with their individual use. Both compounds and the complex preparation were non-toxic at the highest concentration (400 μg/mL zanamivir and 533 μg/mL carrageenan). The effectiveness of the suppression of the replication by both substances depended on the virus strain. The IC50 value for zanamivir ranged from 0.18 μg/mL for H5N1 and 22.97 μg/mL for H7N7. The IC50 values for carrageenan ranged from 0.39 μg/mL to 118.40 μg/mL for H1N1 and H7N7, respectively. Thus, zanamivir and carrageenan target different strains of influenza virus to varying degrees and, therefore, they can provide broader anti-influenza activity by acting synergistically. At the same time, the physical interaction of carrageenan with the virus did not violate the inhibition of NA by zanamivir. The effectiveness of the spray increased when ı-and k-carrageenans were used simultaneously. Mice infected with a lethal dose of the influenza virus that received the placebo dies, as did the animals in all groups receiving monotherapy; however, the combined spray statistically significantly increased the survival of animals. The authors believed that if a vaccine does not keep up with a virus that has changed its composition, such a spray will to some extent protect the population from the impending epidemic.

In addition, an interesting combination of two drugs was suggested [69]. For more than 50 years, xylometazoline has been used to relieve vasoconstriction and oedema of the nasal mucosa in acute respiratory viral infections caused by a wide variety of respiratory viruses, including the influenza virus. The authors combined this vasoconstrictor and ı-carrageenan in one preparation, which had an antiviral effect. It was found that the polysaccharide did not reduce the efficacy and safety of xylometazoline, and the antiviral efficacy of ı-carrageenan remained unchanged.

Thus, carrageenan is currently widely used as a therapeutic and prophylactic agent and was also proposed as an integral part of various antiviral drugs. On the other hand, there is evidence in the literature that oral administration of carrageenan by laboratory animals can lead to the development of inflammation of the gastrointestinal tract [52,70]. However, carrageenan compounds are considered safe and approved for use [48,71]. The Joint Expert Committee on Food Additives (JECFA) concluded that the use of carrageenan is acceptable, even in childhood: "The use of carrageenan in a formula for children or for special medical purposes at concentrations up to 1000 mg/mL does not cause concern" [72]. This confusion, it seems, may be due to imperfect terminology. Some authors combine low molecular weight products of carrageenan hydrolysis, such as polyginan and degraded carrageenan, which are clearly toxic, and native non-degraded food carrageenan, which is considered safe, under the general term "carrageenan" [71]. However there have already been reports of the ability of dietary λ-carrageenan to cause enteritis in rats with prolonged oral ingestion [70].

Among the many study associated with influenza infection, we did not find reports of adverse side effects of carrageenans at intranasal use. Moreover, in Europe, as already mentioned above, the use of the spray for intranasal administration as a preventive and therapeutic agent for influenza and ARIs is permitted. Apparently, attention should be paid to reports of negative phenomena associated with the use of carrageenans and to study this issue separately with respect to these infections.

Thus, it was reported that carrageenans created a physical barrier in the nasal cavity against respiratory viruses, including various strains of influenza virus. At an early stage of viral infection carrageenans are directly associated with the influenza virus, preventing its adsorption, penetration, and replication. At the same time, the antiviral effect of carrageenans is specific and is due to the screening of the cellular structures involved in the binding of the virus to its receptors.
