J Appl Phycol DOI 10.1007/s10811-017-1218-z

Immunomodulatory effects and antimicrobial activity of heterofucans from Sargassum filipendula Cinthia Beatrice Silva Telles 1,2 & Carolina Mendes-Aguiar 3 & Gabriel Pereira Fidelis 1 & Amanda Piccoli Frasson 4 & Wogelsanger Oliveira Pereira 5 & Katia Castanho Scortecci 1 & Rafael Barros Gomes Camara 1 & Leonardo Thiago Duarte Barreto Nobre 1 & Leandro Silva Costa 1,6 & Tiana Tasca 4 & Hugo Alexandre Oliveira Rocha 1,2

Received: 23 March 2017 / Revised and accepted: 26 June 2017 # Springer Science+Business Media B.V. 2017

Abstract Parasitic diseases are a human health problem mainly in low-income areas. The drugs available for the treatment of these diseases are far from satisfactory due to high costs, toxicity, and drug resistance. Sulfated polysaccharides are a complex group of bioactive polymers and can be obtained from seaweeds. The heterofucans from Sargassum filipendula (SF) present strong antiproliferative and antioxidant activities. However, their immunomodulatory and antimicrobial capacity have not been evaluated until now. In this study, five sulfated fucose-rich fractions were isolated (named SF0.5V, SF0.7V, SF1.0V, SF1.5V, and SF2.0V). The chemical composition showed slight differences among polysaccharides and,

consequently, biological activity of these polymers. Three fractions (SF0.5V, SF0.7V, and SF1.0V) showed a strong immunomodulatory activity enhancing the release of nitric oxide (NO) by murine macrophages (RAW 264.7), though only SF0.5V was able to induce interleukin-6 (IL-6) and TNF-α release from RAW cells. The sugar to sulfate ratio was not correlated with these activities. Meanwhile, the contents of xylose (P = 0.98 for NO; P = 0.98 for IL-6; P = 0.96 for TNF-α) and glucuronic acid (P = 0.91 for NO; P = 0.9190 for IL-6; P = 0.79 for TNF-α) were strongly positively correlated. SF0.7V and SF1.0V inhibited biofilm formation by Klebsiella pneumoniae (4.2 and 6.8%, respectively), whereas SF0.5V

* Hugo Alexandre Oliveira Rocha [email protected] Cinthia Beatrice Silva Telles [email protected]

Tiana Tasca [email protected] 1

Departamento de Bioquímica, Laboratório de Biotecnologia de Polímeros Naturais (BIOPOL), Centro de Biociências, Universidade Federal do Rio Grande do Norte (UFRN), Natal, Rio Grande do Norte-RN 59078-970, Brazil

2

Programa de Pós-graduação em Ciências da Saúde, Universidade Federal do Rio Grande do Norte (UFRN), Natal, Rio Grande do Norte-RN 59078-970, Brazil

3

Instituto de Medicina Tropical do Rio Grande do Norte, Universidade Federal do Rio Grande do Norte (UFRN), Natal, Rio Grande do Norte-RN 59078-970, Brazil

4

Faculdade de Farmácia da Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, Porto Alegre, RS 90610-000, Brazil

5

Departamento de Ciências Biomédicas, Faculdade de Ciência da Saude, Universidade de Estado do Rio Grande do Norte (UERN), Mossoró, Rio Grande do Norte-RN 59610-210, Brazil

6

Instituto Federal de Educação, Ciência e Tecnologia do Rio Grande do Norte (IFRN), Ceará-Mirim, Rio Grande do Norte-RN 59580-000, Brazil

Carolina Mendes-Aguiar [email protected] Gabriel Pereira Fidelis [email protected] Amanda Piccoli Frasson [email protected] Wogelsanger Oliveira Pereira [email protected] Katia Castanho Scortecci [email protected] Rafael Barros Gomes Camara [email protected] Leonardo Thiago Duarte Barreto Nobre [email protected] Leandro Silva Costa [email protected]

J Appl Phycol

showed inhibitory effect (~50%) on biofilm formation by Staphylococcus epidermidis. Furthermore, SF0.7V and SF1.0V showed high inhibition capacity on the survival of the protozoan Trichomonas vaginalis. The sugar to sulfate ratio was positively correlated (P = 0.60) with this activity. The results demonstrate the spectrum of action of these sulfated polysaccharides obtained from SF and show their potential as immunomodulatory and microbicidal agents. Keywords Fucoidan . Trichomonas vaginalis . Antibiofilm . Nitric oxide

Introduction The immune response performs a fundamental role in the defense against infectious agents and it constitutes the most important prevention against the occurrence of infection spreads that are normally associated with a high mortality rate (Machado et al. 2004). The establishment of an infection, in a vulnerable host, covers several mechanisms; one of the most relevant is the way that the microorganism interacts with the immune system and its response against the invader (Ayres and Schneider 2012). Parasites are highly diversified organisms that have developed different strategies to infect their hosts. These infectious agents vary from single-celled organisms such as bacteria and protozoa, to multicelled organisms such as nematodes and helminths (worms). Bacteria are microorganisms capable of replicating inside the host cells as much as in the extracellular environments, blood circulation, intestinal lumen, and airways, among others (Ayres and Schneider 2012). On the other hand, protozoa are infectious intracellular agents that normally infect the host for long periods of time (Machado et al. 2004). The immune mechanisms involved in fighting bacterial infections and those caused by protozoa vary; generally, intracellular parasites are eliminated through mechanisms mediated by the cell and the extracellular ones via mechanisms that mainly involve the complement system and antibodies (Janeway et al. 2001). The drugs available for the treatment of parasite-related diseases are far from satisfactory due to their high production costs and toxicity, as well as the appearance of resistance (Helms et al. 2008; Limban and Chifiriuc 2011; Chouhan et al. 2014; Innocente et al. 2014). The increase in knowledge about clinical immunology is revealing that the physiopathology of illnesses can be caused by exacerbation, as well as by immunodeficiencies of the immune response. Modern immunologic therapy is divided into two basic groups of immunomodulators: the immunostimulants, which lead to the increase of innate and adaptive immunity, and the immunosuppressives that diminish the activity on the immune system (Uthaisangsook et al. 2002). Natural products are important sources of innovative therapeutic agents for infectious

diseases, cancer, lipid disorders, and immunomodulation (Altmann 2001). Sulfated polysaccharides are a complex group of bioactive polymers in which some of the hydroxyl groups from sugar waste are replaced by sulfate groups. Seaweeds are the main non-animal sources to obtain these anionic polysaccharides; in this group, we find the Fucans, which are a family of polysaccharides that contain L-fucose in its constitution (Albuquerque et al. 2004). Over the last few years, a variety of groups reported that sulfated polysaccharides obtained from Phaeophyta species, especially from the genus Sargassum, show various biological activities: Sargassum horneri (Hoshino et al. 1998); S. tenerrimum (Sinha et al. 2010); S. patens (Zhu et al. 2003); S. fusiforme (Bhadja et al. 2016), S. wightii (Josephine et al. 2007), S. vulgare (Dore et al. 2013), S. siliquosum (Diamond 1957). Our group assessed the heterofucans from Sargassum filipendula (Costa et al. 2011), a common seaweed along the northeastern coast of Brazil, and demonstrated that these polymers are bioactive molecules with strong antiproliferative and antioxidant activities. However, the immunomodulator and antimicrobial activities of the sulfated polysaccharides from S. filipendula have not yet been examined. In this context, the objective of this study was to obtain these sulfated polysaccharides from S. filipendula and evaluate their immunomudulating and antimicrobial activities facing Trichomonas vaginalis, Staphylococcus epidermidis, and Klebsiella pneumonia (KPC).

Materials and methods Materials Bromide of 3-(4,5-dimetiltiazol-2-il)-2-5-diphenyltetrazoliumbromide (MTT), Griess reagent, methanol PA, and medium of bacterial cultivation Luria Bertani (LB), Serum AB (SIGMA), and metronidazole were from Sigma Chemical Company, USA. Medium of cellular culture (RPMI 1640) (developed by Roswell Park Memorial Institute) and DMEM (Dulbecco’s modified Eagle’s medium), trypsin, and bovine fetal serum (BFS) were obtained from CULTILAB (Campinas-SP, Brazil). L-glutamine, gentamicin, penicillin, streptomycin, sodium bicarbonate, HEPES, sodium pyruvate, and saline solution tamponed with phosphate-buffered saline (PBS) were from Invitrogen Corporation (USA). ELISA kits (for TNF-α and IL-6) were from BD Biosciences. FicollHypaque was from GE Healthcare. Panoptic dye was from NewProv. Half Schneider’s insect was obtained from Gibco. All of the other solvents and chemical products were of analytical grade.

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Biological material The cell line of murine macrophages RAW 264.7 (ATCC number TIB-71) was donated by Dr. Carmen Ferreira (Biochemistry Department, UNICAMP, Brazil). The protozoan Trichomonas vaginalis (ATCC number 30236) was given by Dr. Tiana Tasca (Clinical Analysis and Toxicology Laboratory, Faculdade de Farmácia, UFRGS, Brazil). The bacterial strains Staphylococcus epidermidis (ATCC 35984) and the clinical isolate of Klebsiella pneumoniae were given by Dr. Alexandre Jose Macedo (Biotechnology Center and Faculdade de Farmácia, UFRGS, Brazil). Maintenance of cell lines The cell lines of murine macrophages (RAW 264.7) were cultivated in supplemented DMEM with 10% of BFS and antibiotics (100 U mL−1 of penicillin and 100 μg mL−1 of streptomycin). The cells were maintained as cultures in monolayers at a humidified atmosphere of 5% of CO2 at 37 °C. Trichomonas vaginalis were cultivated in vitro in the TYM (trypticase-yeast extract-maltose) medium, pH 6.0, supplemented with 10% (v/v) of serum inactivated by heat, and incubated at 37 °C (Diamond 1957). The organisms in logarithmic growing phase, displaying more than 95% of viability and normal morphology, collected retracted, centrifuged, and suspended again in medium TYM for utilization in tests. Staphylococcus epidermidis and the clinic isolate of K. pneumoniae (KPC) were utilized as biofilm former bacterial models. Staphylococcus epidermidis and K. pneumoniae were cultivated in LB medium at 37 °C under agitation of 150 rpm (Shaker Série Excella E25; New Brunswick Scientific) and adjusted to an OD 600 equivalent to 108 CFU mL−1 for utilization in antibacterial and antibiofilm trials.

positive control those in the presence of LPS (2 μg mL−1). The analysis was carried out in the microplate reader Multiskan Ascent (Thermo Labsystems, USA) with absorbance at 540 nm. NaNO2 was used to generate the standard curve.

Cytokine analysis RAW 264.7 (3 × 105 cells mL−1) cells treated with different heterofucans in the concentrations of 0.125, 0.25, and 0.5 mg mL−1 were cultivated in 24-well plates. After 24 h, the supernatants were collected. They then were centrifuged at 1500×g for 5 min. The levels of TNF-α and IL-6 were determined by utilizing specific ELISA kits (immunoabsorbent enzymatic test); the negative control consists of untreated cells with the heterofucans and positive control those in the presence of LPS (2 μg mL−1). The plate was read at 450 nm, with remediation at 570 and 590 nm.

Cytotoxicity tests in macrophages The cytotoxicity in RAW 264.7 cells was measured by the MTT test as described previously by Telles and collaborators (Telles et al. 2011). The cells were cultivated in 96-well plates to a density of 5 × 103 cells well−1 with the heterofucans in different concentrations (0.125, 0.25, 0.5, and 1.0 mg mL−1) for 24 h at 37 °C and 5% of CO2. After incubation, 100 μL of MTT was added to each well, incubated for 4 h at 37 °C and 5% of CO2, in the dark. The product MTT-formazan, dissolved in 100 mL of ethanol, was estimated by the measurement of the absorbance to 570 nm in a Multiskan Ascent (Thermo Labsystems, USA) microplate reader.

Extraction of sulfated polysaccharides (heterofucans)

Anti-Trichomonas vaginalis assay

Sargassum filipendula was collected at Búzios beach, Nísia Floresta, Rio Grande do Norte, Brazil. The heterofucans SF0.5V, SF0.7V, SF1.0V, SF1.5V, and SF2.0V of S. filipendula were obtained utilizing the methodology described by Costa and collaborators (Costa et al. 2011).

Heterofucans from S. filipendula were analyzed against T. vaginalis trophozoites (ATCC 30236). In 96-well microplates, 50 μL well−1 of solutions containing the different heterofucans and 150 μL well−1 of the suspension of trophozoites were added, resulting in a final volume of 200 μL containing 2.5 × 105 trophozoites mL−1 and 2.0 mg mL−1 of the heterofucans to be tested. In control cultures, heterofucan samples were substituted by distilled water. The plates were incubated for 24 h at 37 °C. Then 20 μL of a solution of resarzurin at 0.1 mg mL−1 in phosphate-buffered saline was added in each well. After 1 h incubation at 37 °C, the fluorescence of each well was measured in a fluorescence spectrophotometer (Spectramax Gemini XS–Molecular Devices Cooperation, USA), the quantification of viable trophozoites was carried out as described by Duarte et al. (2009).

Production of nitric oxide (NO) The production of NO was analyzed through quantification of nitrite production by the Griess reaction (Green et al. 1982). To measure the production of nitrite, aliquots of 100 μL— obtained from supernatants of the cultures to be dosed—were incubated with equal volumes of Griess reagent and were incubated at room temperature for 10 min. The negative control consists of untreated cells with the heterofucans and

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Antibiofilm assay

Statistical analysis

Antibiofilm activity was measured as described by MeloSilveira and collaborators (Jiang et al. 2013). Eighty microliters of bacterial suspension (S. epidermidis ATCC-35984 or a clinic isolate of K. pneumoniae type 174), 80 μL of the heterofucans (0.5, 1.0, 1.5, and 2.0 mg mL−1), and 40 μL of tryptone soy broth (TSB) (Oxoid Ltd., England) were added to the 96-well plate and incubated (37 °C for 24 h). Then, the content of the wells was removed and the plate was washed three times with sterile saline. The remaining adhered bacteria were fixed at 60 °C for 1 h. The biofilm formed was dyed with 0.4% crystal violet for 15 min at room temperature. The crystal violet bonded to the cells/biofilm was solubilized with 99.5% of DMSO and read at 570 nm (Spectramax M2e multimode Microplate Reader, Molecular Devices, USA). The controls (samples containing no fucans) were considered as 100% of the formation of biofilm and the values obtained for the extract were the average of three experiments.

All the data are expressed in average ± standard deviation. The analysis was carried out by analysis of variance. StudentNewman-Keuls posttests were done for multiple comparison by group. In all cases, statistical significance was established at P < 0.05.

Bacterial inhibition assay The experiment was carried out as described by Melo-Silveira et al. (2012). The bacterial growth of S. epidermidis and a clinical isolate of K. pneumoniae was evaluated by the difference of absorbance measured at 600 nm at the end and at the beginning of the incubation time, in 96-well polystyrene microtitration plates. Different concentrations of the heterofucans (0.5, 1.0, 1.5, and 2.0 mg mL−1) were incubated in the presence of each bacterial strain. The control with distilled water was considered as 100% of bacterial growth. All of the experiments were carried out in triplicate.

Chemical characterization of fucans Chemical characteristics of heterofucans SF0.5V, SF0.7V, SF1.0V, SF1.5V, and SF2.0V obtained from S. filipendula are presented in Table 1. The heterofucans SF0.5V and SF2.0V were the ones that had the lowest sugar to sulfate ratio, indicating that the amount of sulfate by sugar residue is bigger when compared to that of the other heterofucans. The monosaccharides fucose, galactose, glucose, and xylose were found in different amounts in all polysaccharides. Mannose and glucuronic acid are also present in almost all of the heterofucans, except for SF1.5V, that does not have mannose residues, and SF2.0V that does not have glucuronic acid in its structure. Comparing the data described in Table 1 with the data published by Costa et al. (2011), it is possible to verify that both are very similar. The small differences between our data and the data presented by Costa and colleagues may be due to the fact that seaweeds were collected in different years. These fucans present antioxidant and antitumor activities; however, other activities have not been assessed yet; therefore, we verified whether these fucans had immunomodulator and antimicrobial effects. Imunomodulator effect of the sulphated heterofucans from S. filipendula in murine macrophages (RAW 264.7)

Pearson correlation coefficient The Pearson correlation coefficient was calculated for the sulfate to sugar ratio, xylose, fucose, mannose, or glucuronic acid and the results of the biological assays obtained with fucans from S. filipendula.

Table 1 Chemical characteristics of the heterofucans from S. filipendula

Results and discussion

Macrophages are essential for the maintenance of homeostasis and play a main role in the defense of the host against pathogens. In this process, activated macrophages release some

Heterofucans

Total sugar/sulfate (%/%)

Fuc:Gal:Glc:Man:Xyl:Gluc acid (molar ratio)

SF 0.5V SF0.7V SF1.0V SF1.5V SF2.0V

4.06 4.27 4.69 5.27 3.72

1.0:1.4:0.5:0.3:1.0:1.0 1.0:1.2:0.8:0.2:0.6:0.7 1.0:1.2:0.4:0.2:0.3:0.6 1.0:1.0:0.3:0.0:0.1:0.4 1.0:2.0:0.4:0.5:0.2:0.0

Fuc fucose, Gal galactose, Man mannose, Xyl xylose, Gluc acid glucuronic acid

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Among the properties featured as important for a fucan to interfere in the amount of NO releases, it is important to mention the degree of sulfation. Nevertheless, the results obtained with the heterofucans (SF0.5V and SF2.0V) were intriguing; despite the two having similar sugar to sulfate ratios, they had different effects in the immunostimulation of macrophages and release of NO, which means that one of them is a stimulator (SF0.5V), whereas the other one (SF2.0V) does not affect the amount of NO in the environment. In addition, the Pearson correlation coefficient was negative (P = −0.33), suggesting that Sargassum fucan sulfation is not an important factor for these polymers to stimulate NO release. According to Leiro et al. (2007), sulfate groups are important points so that a sulfated polysaccharide is able to stimulate RAW 264.7 cells to release NO to the extracellular environment. These authors showed that sulfated polysaccharides of the Ulva rigida were stimulating agents of RAW cells; however, when they were desulfated, their activity was lost. Jiang et al. (2013) also demonstrated that when disulfating ascophylan, a homofucan obtained from A. nodosum, its RAW macrophages’ stimulating activity was significantly decreased when compared to that of the native ascophylan. Despite many authors finding that the amount of sulfate groups present in the polysaccharide is important to its action, the position in which these groups are distributed in the molecule is a much more determining factor so that a sulfated polysaccharide can show higher or lower activity (HarounBouhedja et al. 2000; Barahona et al. 2012; Liang et al. 2014). Therefore, we believe that this would be the reason that makes SF0.5V and SF2.0V, which have a similar sugar to sulfate ratio, show distinct activities.

immunomodulator factors, such as NO, IL-6, and TNF-α, among others (Gamal-Eldeen et al. 2007). Therefore, initially, we evaluated the effect of heterofucans in murine macrophages (RAW 264.7) in culture conditions. Production of NO The production of NO by RAW 264.7 cells, stimulated by the different heterofucans (0.125, 0.25, and 0.5 mg mL−1) is demonstrated in Fig. 1. After a 24-h treatment, the level of NO in the cultures stimulated with the heterofucans SF1.5V and SF2.0V was similar to that observed for the non-stimulated cells (control group). On the other hand, the amount of NO present in the supernatant of the macrophages increased considerably when the cells were incubated with SF0.5V, SF0.7V, and SF1.0V, and this effect had a tendency to be dose-dependent. It is important to stress that the RAW cells, in the presence of SF0.5V (0.5 mg mL−1), made the amount of NO in extracellular environment similar to the one found in the extracellular environment of the cells that was treated with the positive control, which suggests a strong immunostimulating action of the macrophages by this heterofucan. Other groups also have shown that different fucans have distinct potentials as immunostimulating agents, e.g., fucans obtained from Ascophyllum nodosum and Fucus vesiculosus increased the amount of NO released to the extracellular environment when in contact with RAW cells. However, the Ascophyllum fucan was six times more potent than that of Fucus (Jiang et al. 2011). The presented information clearly indicates that the potency of stimulation of the RAW cells carried out by the fucans in order to release NO is not the same and depends on the properties of each fucan, as was observed with the heterofucans from S. filipendula.

pM NaNo2 . cells -1

6

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5

4

a *#

a *#

3

a *# a *#

a *#

2

a *#

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1

0 0.125 0.25 NC

PC

SF0.5v

0.5 0.125 0.25 SF0.7v

0.5 0.125 0.25 SF1.0v

0.5 0.125 0.25 SF1.5v

0.5 0.125 0.25

0.5

SF2.0v

Concentraon (mg.mL -1) Fig. 1 Effect of the heterofucans from S. filipendula over the release of NO by RAW264.7 cells. The data are presented as average ± standard deviation (n = 3). The letters a and b indicate a significant difference (P < 0.05) between the concentration of the heterofucans. Number signs indicate the significant difference (P < 0.05) between the concentration of

the heterofucan and positive control. Asterisks indicate significant difference (P < 0.05) between the concentration of the heterofucan and negative control. NC negative control; PC positive control (LPS 2 μg mL−1)

J Appl Phycol

The Sargassum fucans with a higher xylose and glucuronic acid content exhibited greater NO stimulatory action, as confirmed by the high positive Pearson correlation coefficient between the xylose (P = 0.98) or glucuronic acid (P = 0.92) and NO levels. Unfortunately, we did find other articles showing correlation between these monosaccharides and NO stimulatory action of fucans. However, polysaccharides containing xylose and glucuronic acid, such as xylan from corn cobs, have shown great NO stimulatory effect (Albuquerque et al. 2013). Production of the cytokines TNF-α and IL-6 In Fig. 2, we show that the cells RAW 264.7, when exposed to the heterofucans SF1.5V and SF2.0V, did not promote alteration on the level of TNF-α and IL-6 in the extracellular environment, proving, together with the previous data, that these polymers possibly do not act as immunomodulator agents. The heterofucan SF1.0Valso did not induce the cells RAW 264.7 to produce and release a significant amount of TNF-α and IL-6. This characteristic is not entirely of these fucans; another heterofucan, extracted from Dictyota menstrualis, was also not able to interfere on the production of these two cytokines (Nakayasu et al. 2009). The presented data lead to the observation that the immunomodulator mechanism of SF1.0V would be centered in its capacity to interfere on the production and release of NO. The fucans that altered the highest amount of TNF-α and IL-6 were SF0.5V and SF0.7V. In both the cases, the TNF-α was the cytokine that was most affected by the presence of the fucans. In addition, we found a Pearson correlation coefficient between the xylose (P = 0.956) or glucuronic acid

Fig. 2 Effect of the heterofucans from S. filipendula over the release of TNF-α and IL-6 by RAW 264.7 cells. The data are presented as average ± standard deviation (n = 3). The letters a and b indicate a significant difference (P < 0.05) between the concentration of the heterofucans. Number signs indicate significant difference (P < 0.05)

(P = 0.796) and TNF-α levels as well as the xylose (P = 0.98) or glucuronic acid (P = 0.91) and IL-6 levels. For SF0.5V, it was observed that the presence of 0.25 mg mL−1 of this fucan increased in about a thousand times the amount of TNF-α in the extracellular environment in comparison with the amount of TNF-α found in the control group. This result was very expressive, since other fucans from F. vesiculosus (Jiang et al. 2013) and A. nodosum (Nakayasu et al. 2009), in the concentration of 0.2 mg mL−1, were only capable of increasing the amount of TNF-α in the extracellular environment by 20 times. Do et al. (2010), evaluating the effect of the homofucan extracted from F. vesiculosus in the induction of the production of NO by macrophages (RAW 264.7 and primary peritoneal cells), observed that this fucan made the cells that were being studied increase the release of NO to the extracellular environment, as well as TNF-α. These authors suggest that TNF-α has a synergic effect and increases the release of NO to the extracellular environment even more, which would explain the effect of SF0.5V and SF0.7V. In other words, the way these fucans stimulate the release of TNF-α induces a higher release of NO. This also explains why SF1.0V stimulates the release of a smaller amount of NO, because that fucan does not affect the release of TNF-α. Different groups that study fucans state that they are immunomodulators because they induce the activation in vitro of murine macrophages (RAW 264.7) leading to the increase of the production of NO and cytokines such as TNF-α and IL-6 (Nakamura et al. 2006; Teruya et al. 2010; Jiang et al. 2013; Cho et al. 2014). This leads us to propose that S. filipendula synthesizes imunomodulator fucans.

between the effect of the heterofucan and positive control. Asterisks indicate significant difference (P < 0.05) between the effect of the heterofucan and negative control. NC negative control; PC positive control (LPS 2 μg mL−1)

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Cytotoxicity of the heterofucans related to RAW 264.7 cells In order to evaluate whether the increase in the production of these chemical mediators, NO and the cytokines (TNF-α and IL-6), was not a response arising from the toxicity of the heterofucans, we evaluated the cytotoxicity of these heterofucans facing the same lineage used in previous trials. The data are in Fig. 3. By analyzing Fig. 4, it was possible to observe that under all the conditions tested, the heterofucans did not compromise the cellular viability of the phagocytic cells, suggesting that these polymers do not have a cytotoxic effect over RAW 264.7 cells. Considering the cited aspects, we suggest that the increase in the release of NO, TNF-α, and IL-6 results from the immunomodulating capacity of the heterofucans facing the line of macrophages. Antimicrobial activity Considering that heterofucans from S. filipendula showed to be immunomodulator agents, we also evaluated the effectiveness of these heterofucans against various parasites, for instance, T. vaginalis, K. pneumoniae, and S. epidermidis. Anti-Trichomonas vaginalis activity Heterofucans from S. filipendula were analyzed against trophozoites of T. vaginalis (Fig. 4). The screening revealed that the heterofucans SF0.5V and SF2.0V did not present inhibitory activity against the trophozoites. Whereas, SF0.7V, SF1.0V, and SF1.5V presented anti-T. vaginalis activity after the 24-h treatment. The Pearson correlation coefficient for the sugar to sulfate ratio and the anti-T. vaginalis activity showed a positive correlation (P = 0.60), suggesting that fucan sulfation is an important factor in the anti-T. vaginalis activity of these Sargassum fucans.

Fig. 3 Effect of the heterofucans from S. filipendula over the proliferation of RAW 264.7 cells. The data are presented as average ± standard deviation (n = 3). There were no meaningful differences in all of the tested concentrations

The action of the heterofucans as strong inhibitory agents of the flagellate protozoan T. vaginalis is of key importance since the treatment of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in the world (Sharafi et al. 2013), is essentially based in the use of the drug 5-nitroimidazole (Helms et al. 2008). However, some reports have demonstrated the appearance of resistant T. vaginalis isolates (Blaha et al. 2006). Therefore, it is necessary to search for a new therapeutic arsenal. Besides, there is no study that has demonstrated the activity of polysaccharides against T. vaginalis, so this is the first study that shows that polysaccharides obtained from seaweed (SF0.7 V, SF1.0 V, and SF1.5 V) have cytotoxic action against this pathogenic protozoan.

Antibacterial and antibiofilm activities All of the heterofucans from S. filipendula were evaluated as to their antibacterial and inhibitory capacity concerning the formation of bacterial biofilms (Table 2). In this study, no heterofucan of S. filipendula was shown to be effective in the fighting of bacterial growth; also, they did not considerably inhibit the formation of biofilms, promoted by the association of bacteria of the species K. pneumoniae. The heterofucans also did not have antibacterial activity against S. epidermidis. However, SF0.5V presented antibiofilm activity, inhibiting about 50% of biofilm formation by these bacteria. The difference in the activity of SF0.5V in the formation of biofilms between the two species of bacteria studied may result from the structural difference presented by these two bacterial groups, since S. epidermidis is gram positive, whereas K. pneumoniae is a Gram-negative bacterium, and therefore has a membrane external to the bacterial cellular wall, thus having binders distinct from the ones presented by S. epidermidis.

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Fig. 4 Effect of different heterofucans from S. filipendula (2 mg mL−1) against isolates of T. vaginalis sensitive to metronidazol. Control: Trophozoites in untreated culture (no presence of heterofucans). The data represent averages ± standard deviation of at least three experiments. Number signs indicate significant difference (P < 0.05) between the concentration of the heterofucan and control

Considering that the heterofucan SF0.5V does not have bactericidal activity (Table 2), its antibiofilm activity of S. epidermidis should be mediated by other mechanism unrelated to the inhibition of bacterial growth. Some studies show that the charge of the surface is an important parameter to the formation of biofilms. Positively charged surfaces promote stronger bacterial adhesion, probably due to attractive electrostatic forces (Gottenbos et al. 1999). This way, sulfated polysaccharides or with carboxylic groups, when bound to surfaces, would provide negative charges to the surface and would affect bacterial adhesion by electrostatic repulsion. As an example, we have the effect of two ulvans (sulfated polysaccharides) extracted from the green seaweeds Ulva rotundata and Ulva compressa. They were efficient in the reduction of the colonization of titanium substrate

Table 2 Antibacterial and antibiofilm activities of the heterofucans from S. filipendula

by S. epidermidis, reducing 96% of the initial adhesion (Gadenne et al. 2013). However, we do not completely agree with this hypothesis, since only one of the five tested heterofucans presents antibiofilm activity. Probably, as in other sulfated polysaccharides activities, the distribution of negative charges by the molecule is a factor that is more important than the simple fact of having a negative charge for a polysaccharide to present antibiofilm activity. Another possibility that cannot be excluded would be that of the ability of the fucan to bond to the bacterial surface. Some studies indicate that the acting mechanism of some polysaccharides happens through a competitive inhibition of carbohydrate-protein interactions. As an example, Zinger-Yosovich and Gilboa-Garber (2009) observed that the adhesion that depends on the lectin of Pseudomonas aeruginosa to human cells is effectively inhibited by galactomannans. That way, antibiofilm polysaccharides would block the sugar-binding proteins present on the surface of bacteria, or adhesins present in fimbria and pili (Zinger-Yosovich and Gilboa-Garber 2009). It is intended, in the future, to produce antibodies antiSF0.5V and use them as a tool to discover its molecular antibiofilm target. This antibiofilm activity presented by the heterofucan SF05V is fundamentally important since biofilms hamper the arrival of antimicrobial drugs and even phagocytic cells to the infection site. This can be harmful to health, as in the case of bacterial pellicles that develop on teeth— the origin of cavities—and other problems related to the mouth, lungs, urinary catheters, and contact lenses, which can cause serious infections on tissues (osteomyelitis and endocarditis) and rejection of prosthetic materials (Bjarnsholt 2013; Mohammadi et al. 2015).

Microorganisma, b Samples

KPC producing Klebsiella pneumoniae (clinical isolate)

Staphylococcus epidermidis (ATCC 35984)

S. filipendula

Antibacterial (%)

Antibiofilm (%)

Antibacterial (%)

Antibiofilm (%)

SF0.5V SF0.7V

0 0

0 4.17 ± 0.03

0 0

46.2 ± 0.04 0

SF1.0V SF1.5V SF2.0V

0 0 0

6.75 ± 0.02 0 0

0 0 0

0 0 0

Rifampicin (Sigma-Aldrich Co., USA) was used as control antibiotic a

The data are the average values of three determinations ± SD

The results were obtained in the concentration of 2 mg mL−1 ; other tested concentrations did not have significant activity b

J Appl Phycol

Conclusions The heterofucans from S. filipendula presented distinct activities as stimulators of the immune system and antimicrobial agents. The heterofucans SF0.5V, SF0.7V, and SF1.0V were able to act in the activation of murine macrophages promoting increase in the release of the chemical mediators that are important for fighting intracellular parasites. In addition, SF0.5V presented antibiofilm activity facing the strain of S. epidermidis whereas SF0.7V and SF1.0V reduced almost completely the viability of the protozoan T. vaginalis. Therefore, these fucans act as double-acting drugs; they stimulate the immune system to fight the pathogens, and at the same time, they directly combat these pathogens. All five heterofucans obtained from S. filipendula showed different/specific levels of activities in the trials carried out, again making it evident that their biological activities depend on their structural characteristics. Results such as these reflect the great spectrum of action of these sulfated polysaccharides, but also intensify the need of further studies to elucidate the complete structure of these polysaccharides, configuration of glycosidic bonds, their position as well as the position of sulfate groups and ramification points. Acknowledgments Research was supported by Ministério de Ciência, Tecnologia e Informação (MCTI), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil. Hugo A O Rocha, L. S Costa, and Tiana Tasca are CNPq fellowship-honored researchers. Cinthia Telles had a Ph.D. scholarship from CAPES and Gabriel Fidelis has a Ph.D. scholarship from CAPES. Leonardo Nobre has a postdoctoral fellowship from CAPES. This research was submitted to the Graduate Program in Health Science at the Federal University of Rio Grande do Norte as part of the D. Sc. thesis of C.B.S.T. Author contributions Conceived and designed the experiments: C.B.S.T, T.T., L.S.C., and H.A.O.R. Performed the experiments: C.B.S.T., G.P.F., C.M.A., and A.P.F. Analyzed the data: C.B.S.T. and H.A.O.R. Contributed reagents/materials/analysis tools: L.S.C., T.T., W.O.P, L.S.C., and H.A.O.R. Wrote the paper: C.B.S.T., T.T., L.S.C., L.T.D.B.N., and H.A.O.R. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

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