In this study, using methodology that combined proteolysis and acetone precipitation, we obtained five polysaccharides from the brown seaweed S. filipendula denominated: SF-0.5v, SF-0.7v, SF-1.0v, SF-1.5v, and SF-2.0v. Chemical analysis of sulfated polysaccharides is summarized in Table 1. The monosaccharide composition of sulfated polysaccharides is also shown in Table 1. Fucose, galactose, glucose, mannose, xylose, and glucuronic acid were found in different amounts in each polysaccharide. Exceptions were mannose and glucuronic acid, which were not detected in SF-1.5 and SF-2.0v, respectively. Data showed that galactose and fucose were the main sugars present in all the polysaccharides, indicating that we obtained five different types of sulfated galactofucans. Since the 1950s [10], several sulfated fucans containing xylose and galactose have been described, but only a few with galactose as the major component have been described in seaweed: Saccharina longicruris [11], Lobophora variegata [12], Spatoglossum schroederi [10], Adenocystis utricularis [13], and Sargassum stenophyllum [14]. Accordingly, we sought to analyze the chemical, anticoagulant, antioxidant, and antiproliferative properties of these heterofucans from S. filipendula.

The amount of total sugar and sulfate increased in polysaccharides obtained with high acetone volumes. The heterofucan SF-2.0v showed the highest total sugar content (66.0%) and sulfate content (17.7%) when compared with other sulfated polysaccharides. On the other hand, SF-0.5v and SF-0.7v exhibited the lowest total sugar content (41.4% and 46.2%, respectively) and sulfate content (10.2% and 10.8%, respectively). Additionally, all the polysaccharides showed low protein contamination, ranging from 0.2 (SF-1.5v) to 0.6% (SF-1.5v). Considering total sugar, sulfate and protein content, total percentage varies from 52.2% to 84.1% in SF-0.5 and SF-2.0, respectively. Most articles that show the proportion of proteins, carbohydrates and heterofucan sulfate exhibit data in the same fashion as we present ours [6,15,16]. The sum of the three components found in the heterofucans of these articles does not approach 100%. This is due to the fact that these polymers are very hygroscopic, absorbing water from the atmosphere very rapidly after lyophilization. Furthermore, because of the negative loads of sulfate clusters and glucoronic acids, metals are not eliminated from fucan structures, even after dialysis. Another important point is the coformation that these polymers exhibit in aqueous solutions, which may capture cations within their structures.

Fucans have a wide variety of biological activities, but their potent anticoagulant action is by far the most widely studied. The anticoagulant activity of heterofucans from S. filipendula was evaluated by PT and aPTT tests to assess the extrinsic and intrinsic pathway of coagulation, respectively. In the present study, none of the sulfated polysaccharides showed anticoagulant activity in any of the tested conditions. A similar observation was reported for a range of sulfated galactans from marine invertebrates and heterofucans from brown seaweeds [3,17]. This result suggests that the anticoagulant effect of fucans is stereospecific and not merely a consequence of their charge density or sulfate content [18]. The position of sulfate groups on sugar residues is also very important for the anticoagulant activity of fucans. Thus, sulfated polysaccharides extracted from S. filipendula likely do not have a favorable structure for interacting with proteins involved in the clotting process and therefore do not exhibit anticoagulant activity.

Several sulfated polysaccharides from marine algae have been described as having antioxidant activity [19–22]. Thus, sargassum heterofucan antioxidant activities were evaluated in different antioxidant assays: total antioxidant capacity (TAC), scavenging hydroxyl and superoxide radicals, power reducing and ferrous chelating.

Antiproliferative activity was performed with HeLa, PC3 and HepG2 cells under the following conditions: 24h of incubation at 0.1, 0.5, 1.0, 1.5 and 2.0 mg/mL (Figure 3). In all tested conditions sulfated polysaccharides from Sargassum filipendula exhibited a dose-dependent effect. The only exception was SF-1.5v, which showed no inhibition to HepG2 cells. PC3 and HepG2 cells were only moderately inhibited by the polysaccharides analyzed. SF-0.7v was the most effective polysaccharide for these cells, inhibiting HepG2 and PC3 by 38.1% and 31.0%, respectively.

The strongest inhibition of sulfated polysaccharides from S. filipendula was observed in HeLa cells. In this experiment, polysaccharides SF-0.5v, SF-0.7v and SF-2.0v can be classified as a moderate activity group when compared with SF-1.0v and SF-1.5v, which are classified as a high activity group. Sulfated polysaccharides from the moderate activity group showed maximum antiproliferative activity, with 37.1% inhibition at 2.0 mg/mL (SF-2.0v). However, it was not possible to determine the IC₅₀ value for these polysaccharides. By contrast, sulfated polysaccharides from the high activity group showed significant activity and IC₅₀ value of 15.69 μM (SF-1.0v) and 13.83 μM (SF-1.5v). However, this IC₅₀ values are not comparable to those of positive controls, doxorubicin (IC₅₀ 6.8 μM) and 5-fluorouracil (IC₅₀ 8.0 μM). On the other hand, the sargassum heterofucans exhibited more potent antiproliferative activity against tumor cells in comparison to heterofucans from other source, like as heterofucan from Cladosiphon okamuranus that inhibits HepG2 proliferation showing IC value of 50 μM [25], and a heterofucan from Ascophyllum nodosum that inhibits HeLa cells proliferation, but this IC₅₀ value was not determinate even using high amounts of polysaccharide [26].

Based on the experiment carried out in the present study, it is clear that the effect of fucans on different cell lines is unequal and therefore there must be a preference for certain cells, that is, the effect of the fucan is cell dependent. A number of in vivo and in vitro studies on the antitumor activity of fucans have been conducted [27,28]. Although the precise mechanisms underlying this activity remain to be determined, a few possibilities have been proposed. It has been speculated that fucans act by inhibiting tumor angiogenesis, modulating host immune systems [27], arresting the cell cycle and/or inducing apoptosis [29]. Thus, further studies are needed to clarify the antiproliferative mechanism of fucans from S. filipendula, especially SF-1.5v, which exhibited the strongest activity.

Iron (II) sulfate, potassium ferricianyde, sulfuric acid and acetonitrile were obtained from Merck (Darmstadt, Germany). Nitro Blue Tetrazolium (NBT), monosaccharides, doxorubicin, 5-fluorouracil, methionine and ammonium molybdate were purchased from Sigma-Aldrich Co. (St. Louis, USA). All other solvents and chemicals were of analytical grade.

The Phaeophyta Sargassum filipendula was collected at Búzios Beach, Nísia Floresta, Brazil. The alga was stored in our laboratory and dried at 50 °C under ventilation in an oven, ground in a blender and incubated with acetone to eliminate lipids and pigments. About 100 g of powdered alga was suspended with five volumes of 0.25 M NaCl and the pH was adjusted to 8.0 with NaOH. Next, 900 mg of Prolav 750 (Prozyn Biosolutions, São Paulo, SP, Brazil), a mixture of alkaline proteases, was added for proteolytic digestion. After incubation for 24 h at 60 °C under agitation and periodical pH adjustments, the mixture was filtered through cheesecloth. The filtrate was fractionated by precipitation with acetone as follows: 0.5 volumes of ice-cold acetone was added to the solution under gentle agitation and maintained at 4 °C for 24 h. The precipitate formed was collected by centrifugation (10000 × g, 20 min), vacuum dried, resuspended in distilled water, and analyzed. The operation was repeated by adding 0.7, 1.0, 1.5 and 2.0 volumes of acetone to the supernatant.

Total sugars were estimated by the phenol-H₂SO₄ reaction [30] using l-fucose as standard. Sulfate content was determined according to the gelatin-barium method [31], using sodium sulfate (1 mg/mL) as standard and after acid hydrolysis of the polysaccharides (4 M HCl, 100 °C, 6 h). Protein content was measured using Spector’s method [32].

The polysaccharides were hydrolyzed with 0.5, 1, 2, and 4 M, respectively, for various lengths of time, (0.5, 1, 2 and 4 h), at 100 °C. Reducing sugars were determined using the Somogyi-Nelson method [33]. After acid hydrolysis, sugar composition was determined by a LaChrom Elite^® HPLC system from VWR-Hitachi with a refractive index detector (RI detector model L-2490). A LichroCART^® 250-4 column (250 mm × 40 mm) packed with Lichrospher^® 100 NH₂ (5 μm) was coupled to the system. The sample mass used was 0.2 mg and analysis time was 25 minutes. The following sugars were analyzed as references: arabinose, fructose, fucose, galactose, glucose, glucosamine, glucuronic acid, mannose, and xylose.

The sulfated polysaccharides were subjected to gel-permeation chromatography on Sephadex G-100 (140 × 1 cm) using 0.2 M acetic acid/0.15M NaCl as eluent. The elution was monitored for total sugar [30] and metachromasia [34]. To estimate the molecular weight of the polysaccharides, dextrans of different molecular weights were used as standards.

Prothrombin time (PT) and activated partial thromboplastin time (aPTT) coagulation assays were performed with a coagulometer as described earlier [35] and measured using normal citrate-treated human plasma. All assays were performed in duplicate and repeated at least three times on different days (n = 6). The results were expressed as aPTT ratio, which was determined as follows: aPTT control time/aPTT sample time.

Five assays were performed to analyze the antioxidant activity of the sulfated polysaccharides obtained: total antioxidant capacity, hydroxyl radical scavenging, superoxide radical scavenging, ferric chelating and reducing power, as previously described [2].

The cells culture (HeLa, HepG2 and PC3) were grown in 75 cm² flasks in DMEM medium. Cells were seeded into 96-well plates at a density of 5 × 10³ cell/well and allowed to attach overnight in 300 μL medium incubated at 37 ºC, 5% CO₂. The sulfated polysaccharides fractions were added at a final concentration of 0.1; 0.5; 1.0; 1.5 and 2.0 mg/mL, for 24 h at 37 ºC and 5% CO₂. After incubation, traces of sulfated polysaccharides fractions were removed by washing the cells twice with 200 μL PBS and applying 100 μL of fresh medium plus and 10 μL of 12 mM MTT dissolved in PBS to determine the effects of the algal sulfated polysaccharides on cell proliferation. Cells were then incubated for 4 h at 37 ºC, 5% CO₂. To solubilize the product of MTT cleavage, 100 μL of isopropanol containing 0.04 N HCl was added to each well and thoroughly mixed using a multichannel pipettor. Within 1 h of HCl-isopropanol addition, the absorbance at 570 nm was read using a Multiskan Ascent Microplate Reader (Thermo Labsystems, Franklin, MA, USA). The percent inhibition of cell proliferation was calculated as follows: %   Inhibition = Abs .   570   nm Control − Abs .   570   nm   sample Abs .   570   nm Control × 100

All data were expressed as mean ± standard deviation. Statistical analysis was done by one-way Anova using the SIGMAStat 2.01 software. Student-Newmans-Keuls post-tests were performed for multiple group comparison. In all cases statistical significance was set at p < 0.05.