Two sulfated polysaccharides (SPs), F2 and F3, isolated from Codium isthmocladum were found to contain galactose, sulfate, and pyruvate. The apparent molecular weights of F2 and F3 were determined to be 62 and 61 kDa, respectively.
Carbohydrate Polymers 222 (2019) 115010 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Pharmacological prospection and structural characterization of two purified sulfated and pyruvylated homogalactans from green algae Codium isthmocladum T Diego Araujo Sabrya, Sara Lima Cordeirob, Cynthia Haynara Ferreira Silvaa, Eduardo Henrique Cunha Fariasc, Guilherme Lanzi Sassakid, Helena Bonciani Nadere, ⁎ Hugo Alexandre Oliveira Rochaa, a Department of Biochemistry, Universidade Federal Rio Grande Norte, Natal, Rio Grande Norte, 59.078-970, Brazil Instituto Federal de Educaỗóo, Ciência e Tecnologia Rio Grande Norte, Macau, Rio Grande Norte, Brazil Centro Universitário Rio Grande Norte, Natal, Rio Grande Norte, Brazil d Department of Biochemistry and Molecular Biology, Universidade Federal Paraná, Curitiba, Paraná, 81.531-980, Brazil e Discipline of Molecular Biology, Universidade Federal de São Paulo, São Paulo, São Paulo, 04.044-020, Brazil b c ARTICLE INFO ABSTRACT Keywords: NMR HSQCed Anticoagulant activity Clexane® aPTT Two sulfated polysaccharides (SPs), F2 and F3, isolated from Codium isthmocladum were found to contain galactose, sulfate, and pyruvate The apparent molecular weights of F2 and F3 were determined to be 62 and 61 kDa, respectively NMR spectroscopy combined with chemical analysis showed that F2 and F3 have the same structural features However, F3 showed higher sulfate/sugar ratio (1/2.6) than F2 (1/4) F2 and F3 are essentially (1 → 3)-β-D-galactans with some branching at C6 Pyruvylation occurs at O3 and O4, forming 3,4-O-(1carboxyethylidene)-β-D-Galp residues; some of these pyruvylated residues contain sulfate groups at C6 Some non-branching residues contain sulfate at C4 None of the SPs exhibited antioxidant activity MTT results indicated that mg/mL of both SPs about 40% of PANC-1 cell viability At 10 μg/mL, F2 and F3 had 1.7-fold longer clotting times compared to that of Clexane® at the same concentration The higher sulfate content of F3 is not a determining factor for pharmacological activities of galactans, considering that both F2 and F3 exerted the effects Introduction Green seaweed from the genus Codium are widely distributed worldwide, except for the polar regions Codium comprises 125 species that are found in marine environments (Verbruggen et al., 2007) The Brazilian coast is home to a diverse range of Codium species, including Codium decorticatum, C intertextum, C profundum, C repens, C spongiosum, C taylori, C tomentosum, and C isthmocladum (De OliveiraCarvalho, de, Pereira, & Pedroche, 2010; De Oliveira-Carvalho, Oliveira, Pereira, & Verbruggen, 2012; Farias et al., 2008) Several studies investigated the genus Codium and evaluated the pharmacological/biological potential of the sulfated polysaccharides (SPs) that are present in its cell wall (Ciancia et al., 2007; Estevez, Fernández, Kasulin, Dupree, & Estevez, 2009; Fernández, Raffo, Alberghina, & Ciancia, 2015) SPs obtained from different Codium species showed diverse pharmacological applications as ⁎ immunostimulants (Lee, Ohta, Hayashi, & Hayashi, 2010), anticoagulants (Li et al., 2015), anti-obesity agents, and protective agents for liver-kidney functions (Kolsi et al., 2017) The diversity of pharmacological applications of Codium species could be attributed to the heterogeneity and complexity of their SPs Codium species have been described to produce different types of SPs, such as sulfated mannans (Fernández, Estevez, Cerezo, & Ciancia, 2012), arabinans (Hayakawa et al., 2000), and galactans (Farias et al., 2008) Of these, many structural studies have been conducted on sulfated galactans (SGs) in Codium species Li et al (2015) reported a SG from C divarticum whose main chain is formed by →3-β-D-galactopyranose→1 residues and substituted at the C4 position by branches of 1→-β-D-galactopyranose units or sulfate esters Meanwhile, SGs from C decorticatum are composed of 3-, 6-, and 3,6-O-linked β-D-galactopyranose residues sulfated at C6 and predominantly at C4 In addition, these SGs contain pyruvate ketals both linked to O-3 and O-4 as linked Corresponding author E-mail address: hugo@cb.ufrn.br (H.A Oliveira Rocha) https://doi.org/10.1016/j.carbpol.2019.115010 Received 15 March 2019; Received in revised form 19 June 2019; Accepted 19 June 2019 Available online 20 June 2019 0144-8617/ © 2019 Elsevier Ltd All rights reserved Carbohydrate Polymers 222 (2019) 115010 D.A Sabry, et al to O-4 and O-6 (Fernández et al., 2015) Other studies reported highly sulfated galactans from C fragile and C vermilara, composed of →3-β-Dgalactopyranose→1 residue, with sulfate esters at the C4 and C6 positions These SGs are also pyruvylated at O-3 and O-4 (Ciancia et al., 2007) The structures of sulfated polysaccharides directly affect their biological activities because of the main structure, molecular weight, degree of sulfation, monosaccharide composition, and glycosidic linkages (Costa et al., 2010) However, few studies investigated the relationship between structure and biological activity Meanwhile, the purification process plays an important role in structural elucidation (Fernández et al., 2015) Farias et al (2008) described a method to obtain five SP-rich fractions named F0.3v, F0.5v, F0.7v, F0.9v, and F1.2v, respectively, from the green seaweed C isthmocladum Gel electrophoresis and monosaccharide composition analyses revealed that each fraction contained different SPs In addition, the authors purified and investigated the chemical structure of two preponderantly 4-O-sulfated, 3-linked SGs from F0.9v Among the other fractions, F0.5v showed a potential pharmacological use However, electrophoretic analysis revealed that F0.5v has heterogeneous profile and does require further purification steps Therefore, we purified SPs from the F0.5v fraction from C isthmocladum, proposed their chemical features, and additionally evaluated their pharmacological potential as antioxidant, antiproliferative, and anticoagulant compounds F0.3v, F0.5v, F0.7v, F0.9v and F1.2v respectively After the addition of each acetone volume, the mixture was maintained at °C for 24 h and then collected by centrifugation (for 20 at 10,000 x g), followed by drying under vacuum atmosphere 2.3 Anion exchange chromatography (AEC) 10 g of F0.5v was dissolved to a final concentration of 10 mg/mL and added to 300 mL of MP500 Lewatit resin The mixture was complexed for 18 h under gentle agitation at room temperature The mixture was then applied to a glass column (25 x cm i.d.) Firstly, the mixture was washed with 300 mL of NaCl 0.3 M to remove unbound compounds Chromatography was then carried out by a stepwise gradient elution with seven different concentrations of NaCl (0.3 M, 0.5 M, 0.7 M, 1.0 M, 1.5 M, 2.0 M and 3.0 M) The column was washed three times with each NaCl molar concentration, and the eluate was precipitated separately with volumes of methanol at °C and collected by centrifugation (15 at 10,000 x g) after 12 h, followed by drying under vacuum atmosphere 2.4 Agarose gel electrophoresis All precipitate fractions obtained from AEC were analyzed by agarose gel electrophoresis as described previously (Dietrich & Dietrich, 1976) Samples (50 μg) were applied to a 0.6% (w/v) agarose gel and ran for h at 110 V in 0.05 M 1,3 diaminopropane/acetate (PDA) buffer at pH 9.0 After the run, the sulfated polysaccharides were fixed in the gel with 0.1% CTV solution for 12 h Following the fixation, the gel was dried under airflow and stained with 0.1% (w/v) toluidine blue in a solution of acetic acid:ethanol:water (0.1:5:4.9, v/v) Material and methods 2.1 Chemicals and reagents Toluidine blue, deuterium oxide (D2O), 1,3-diaminopropane, sodium pyruvate, cresol red, Sephadex G-100 medium, acetic anhydride (Ac2O), Coomassie brilliant blue R-250, D-Galactose, D-Glucose, LArabinose, D-Mannose, L-Fucose, D-Xylose, and molecular weight dextran standards were purchased from Sigma (St Louis, MO, USA) Methanol, ethanol, acetone, acetic acid, sulfuric acid, pyridine, N-cetylN,N,N-trimethylammonium bromide (CTV) were purchased from CRQ (São Paulo, SP, Brazil) Maxatase, an alkaline protease from Esporobacillus sp, was purchased from BioBrás (Montes Claros, MG, Brazil) MP500 Lewatit resin was purchased from Bayer Chemicals (São Paulo, SP, Brazil) Panc-1 cells were purchased from Rio de Janeiro Cell Bank (Rio de Janeiro, RJ, Brazil) All other solvents and chemicals were of analytical grade 2.5 Physicochemical analysis of F2 and F3 Total sugar content measurement was performed by phenol-sulphuric acid method using galactose as a reference sugar (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956) Sulfate content was determined by BaCl2-gelatin method (Dodgson & Price, 1962) Protein content was measured as described by Bradford (1976) Pyruvate content was determined by high performance liquid chromatography (HPLC) measurement described by Ohta, Lee, Hayashi, and Hayashi (2009) Molecular weight and homogeneity of F2 and F3 were determined by gel permeation chromatography (GPC) Each fraction was dissolved to a final concentration of 10 mg/mL and applied to a column containing Sephadex G-100 (130 x cm i.d.) GPC was performed using an isocratic elution mode The molecular weight was estimated by reference to a calibration curve made by dextran sulfate standards (10, 40, 70, 147 and 500 kDa) And homogeneity of F2 and F3 was evaluated by chromatographic profile Monosaccharide composition was determined by gas chromatography (GC) after derivatization F2 and F3 were submitted to hydrolysis with M HCl for h at 100 °C The hydrolyzed material was reduced with mg NaBH4 and acetylated with Ac2O 50% (v/v) in pyridine for 30 at 100 °C (Sassaki et al., 2008), yielding alditol acetates The product was resuspended with chloroform and analyzed by GC–MS in Varian Saturn 2000R in a DB225MS column (30 m x0.25 mm i.d.), using a temperature programming at 40 °C/min rate from 50 to 220 °C maintained for 40 min, with He as gas carrier at mL/min flow rate (Sassaki, Gorin, Souza, Czelusniak, & Iacomini, 2005) Identification and quantitation of monosaccharides were performed by comparison with typical retention time of reference sugars: D-Galactose, D-Glucose, L-Arabinose, D-Mannose, L-Fucose and D-Xylose 2.2 Seaweed samples Specimens of Codium isthmocladum (Vickers) were collected from Pirambúzios beach, Nísia Floresta, Rio Grande Norte, Brazil The seaweed was identified according to its morphology (Wynne, 1986) by Dr Valquíria Pereira Medeiros (Federal University of Juiz de Fora, MG, Brazil) and a voucher specimen was deposited in the UFRN Herbarium of the Institute of Biosciences, Federal University of Rio Grande Norte, under the registration code UFRN25933 The material collection occurred under authorization of Brazilian National Management System Genetic Heritage and Associated Traditional Knowledge (loose translation) SISGEN n° A0D4240 The seaweed was washed with tap water, air-dried in an aerated stove at 50 °C and crushed in a blender The dried powder was incubated with acetone to remove pigments and lipids (Leite et al., 1998) Powdered alga was then submitted to proteolytic digestion by alkaline protease and maxatase (pH 8.0) to solubilize sulfated polysaccharides The digestion was conducted by adding three volumes of 250 mM NaCl to powdered alga, adjusting pH to 8.0 and finally adding maxatase After incubating with protease for 24 h at 60 °C, the mixture was filtered and fractionated by precipitation with different volumes of cold acetone: 1:0.3, 1:0.5, 1:0.7, 1:0.9 and 1:0.1.2 (v/v) as established by Farias et al (2008), who named the fractions as 2.6 Desulfation Desulfation of sulfated F2 and F3 was performed as described by Carbohydrate Polymers 222 (2019) 115010 D.A Sabry, et al Nagasawa, Inoue, and Tokuyasu (1979) Briefly, each sulfated polysaccharide (10 mg) was dissolved in distilled water and was added to cation-exchange resin (H+ form) The mixture was filtered, the pH of the supernatant was adjusted to 7.0 with pyridine and then freeze-dried to give the pyridinium salt The product was dissolved in mL of dimethyl sulfoxide (DMSO) containing 10% (v/v) of methanol and then the solution was heated for h at 100 °C After the reaction was completed, the desulfated polysaccharides were dialyzed (MWCO 6–8 kDa) against distilled water and freeze-dried The products were named DSF2 and DSF3, referring to desulfated F2 and F3, respectively and incubated at 95 °C for 90 Once the tubes were at room temperature 200 μL of each tube was collected and measured at an absorbance of 695 nm The results were expressed as ascorbic acid equivalent as of a standard curve 2.11 Anticoagulant test F2 and F3 anticoagulant test was performed in vitro by aPTT (activated partial thromboplastin time) test using a Labtest kit (Lagoa Santa, MG, Brazil), in a Drake coagulometer (São Paulo, SP, Brazil) and a pool of citrated human plasma For the aPTT test, plasma (100 μL) was incubated at 37 °C with saline or SP (100 μL) for and then rabbit cephalin (100 μL) was added After 2.5 min, 25 mM CaCl2 (100 μL) was added and the clotting time measured Clexane was used as a positive control on aPTT test For PT test, plasma (100 μL) was incubated at 37 °C with saline or SP (100 μL) for Thromboplastin (100 μL) was then added and the clotting time was measured 2.7 Methylation analysis F2, F3 and their respective desulfated derivatives were per-O-methylated by the NaOH-DMSO-CH3I method described by Ciucanu and Kerek (1984) Briefly, each sample was dissolved in DMSO to a final concentration of 15 mg/mL and then mL of methyl iodide was added followed by powdered NaOH (30 mg) The mixture was submitted to vigorous mechanical agitation for 30 The product was then dissolved in distilled water in an ice bath and the pH was adjusted to 7.0 with acetic acid The material was dialyzed (MWCO 6–8 kDa), freezedried and the whole process was then repeated twice Per-O-methylated derivatives were then hydrolyzed by formic acid 45% (v/v) for 15 h at 100 °C The product was reduced and acetylated as previously described by Sassaki et al (2008), resulting to partially O-methylated alditol acetates and analyzed by GC–MS as previously described The compounds were identified by their typical retention times and electron impact fragmentation spectra (Sassaki et al., 2005) 2.12 Statistical analysis The experiments performed were expressed as mean ± standard deviation To test differences between the tested concentrations as well as treatments with different polysaccharides, one-way analysis of variance (ANOVA) and Newman-Keuls post-test was performed using GraphPad Prism 5.01 2007 (GraphPad Software, Inc.), and results were considered statistically significant when p ≤ 0.05 Results and discussion 2.8 NMR spectroscopy 3.1 Isolation and physicochemical characterization of sulfated polysaccharides isolated from C isthmocladum NMR 1D and 2D spectra were recorded at 30 °C using an Avance III Bruker 400 MHz spectrometer equipped with a mm inverse probe Polysaccharide (30 mg) was submitted to deuterium-exchange and lyophilized three times with D2O 99% (v/v) The chemical shift of 1H and 13C are expressed in δ (ppm) relative to TMSP (trimethylilsilylpropionate) as an internal standard (δ =0 ppm) Only 2D-NMR HMBC was carried out at 65 °C, all the other experiments were performed at 30 °C 1D-NMR 1H and 2D-NMR spectra (1H/1H COSY, 1H/1H TOCSY, H/13C HSQC and 1H/13C HMBC) were analyzed using TopSpin Bruker software version 3.2pl6 Firstly, 1H chemical shifts assignments were determined by 2D COSY and TOCSY spectra analysis 1H/13C chemical shift correlations were later determined by HSQC analysis and multiple bonding connections were obtained by HMBC experiment 2D NMR experiments were acquired using 24 scans per series of 4096 × 360 data points Five sulfated polysaccharide-rich fractions, named F0.3v, F0.5v, F0.7v, F0.9v, and F1.2v, were obtained from the green seaweed C isthmocladum (Vickers), following the same starting material and precipitation method reported by Farias et al (2008) In addition, these five fractions yielded 17, 30, 27, 21, and 5% (w/w), respectively To confirm the presence of sulfated polysaccharides (SP), the fractions were subjected to agarose electrophoresis in PDA buffer, and their migration profiles were analyzed (Fig 1) After toluidine blue staining, we observed bands that correspond to the predominant SP population in each of the fractions The F0.3v and F1.2v bands were weakly stained, indicating low SP concentrations in these fractions, whereas F0.5v, F0.7v, and F0.9v produced more intense bands, indicating higher SP content Due the PDA buffer, the SPs with the same structure have similar interaction with the diamine and consequently show the same electrophoretic mobility (Presa et al., 2018) The electrophoretic band from F0.7v is composed of a mixture of F0.9v and the band from F0.5v with more mobility In addition, the SPs present in F0.9v and F1.2v showed similar electrophoretic mobilities, indicating similarities in SP composition, consistent with the results reported in previous characterization studies (Farias et al., 2008) The SPs present in F0.7v, F0.9v, and F1.2v showed similar electrophoretic mobilities, indicating similarities in SP composition, consistent with the results reported in previous characterization studies (Farias et al., 2008) F0.5v produced two distinct electrophoretic bands, indicating the presence of two distinct SP populations In addition, the electrophoretic mobilities of the bands were different from those observed in the F0.7v, F0.9v, and F1.2v fractions, indicating the presence of unknown SPs in F0.5v Therefore, F0.5v was further subjected to anion-exchange chromatography (AEC) to isolate these two SP families by stepwise gradient elution AEC of F0.5v produced seven subfractions named F0.3, F0.5, F0.7, F1, F1.5, F2, and F3 The fractions were analyzed by agarose electrophoresis, and migration profile analysis showed that F2 and F3 migrate 2.9 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test assay For the tests, × 103 cells/well were grown in 96-well plates with DMEM medium containing the samples in concentrations of 0.05, 0.1, 0.25, 0.5 and mg/mL for 24 and 48 h (each concentration in triplicate) After treatment, MTT (5 mg/mL) was added and cells were incubated for h at 37 °C The cell capacity to reduce MTT was determined by the colorimetric test of MTT as described earlier by Mosmann (1983) 2.10 Determination of total antioxidant capacity Total antioxidant capacity (TAC) was evaluated by colorimetric assay based on the reduction of molybdenum (Mo) VI to V by sulfated polysaccharides and subsequent formation of a green phosphate/Mo(V) complex as used by Costa et al (2010) The reagent solution containing sulfuric acid (0.6 M), sodium phosphate (28 mM) and ammonium molybdate (4 mM) was added in tubes containing F2 and F3 (10 mg/mL), Carbohydrate Polymers 222 (2019) 115010 D.A Sabry, et al 3.2 Structural characterization of F2 and DSF2 from C isthmocladum by GC–MS and NMR spectroscopy One-dimensional 1H-NMR analysis showed that F2 and F3 have the same chemical shift fingerprint (Fig 4), indicating that the two SGs have the same structural features with slightly different sulfate contents, as shown in Table Because of the high similarity in structure and yield, all subsequent structural characterizations were conducted using only F2 To obtain information on linkage position and sulfation pattern of F2, we performed a comparative GC–MS analysis between F2 and its desulfated derivative (DSF2) per-O-methylated alditol acetates The identified alditol acetates and their respective quantities are listed in Table F2 showed higher content of non-methylated residues even after three rounds of methylation, which could be attributed to the highly branched structure or conformation of F2 that blocks efficient methylation In addition, the high sulfate/sugar ratio (1:4) and presence of pyruvate groups in F2 can prevent methylation process Nevertheless, certain indications about the structure of F2 cannot be determined Only few residues were found to contain the 3-O-methylation, which indicated that F2 predominantly consists of →3)-Galp-(1→ units (Table 2) Moreover, DSF2 showed higher contents of 2,4-di-O- and 2,6-di-Omethyl-galactitol, indicating the presence of sulfate groups at the C4 and C6 positions In addition, DSF2 showed higher content of 2,4,6-triO-methyl-galactitol residues, which also indicated presence of sulfate esters at C6 in F2 Desulfation can also eliminate pyruvylation, as evidenced by the reduction in 2-O-methyl-galactitol at the same proportion as the increase in 2,3,4-di-O-methyl-galactitol, indicating the presence of substituents at positions and Desulfation facilitated the determination of sulfate groups because of variations in the number of methylated residues between F2 and DSF2 units (Table 2) Fig Electrophoretic migration of acetone fractions from Codium isthmocladum The crude polysaccharides before acetone precipitation (CP), and fractions precipitated with 0.3, 0.5, 0.7, 0.9, and 1.2 volumes of acetone (50 μg each) were applied at agarose gel electrophoresis from the origin (Or) to the positive pole (+) 3.3 Structural characterization of F2 and DSF2 from C isthmocladum by NMR spectroscopy The 1H-NMR spectra (Fig 4) of F2 and F3 showed no distinguishable anomeric signals, which is typical of SP spectra However, an anomeric region from δ 4.40 to 4.90 ppm, which represents the characteristic region of β-anomers, can be determined However, the 1HNMR spectra show the presence of a strong peak at δ 1.63 ppm, indicating pyruvylation We performed a more detailed structural analysis of F2 by two-dimensional NMR spectroscopy (2D-NMR) All 2DNMR spectra are shown in Fig Two major spin systems are evident from the HSQC spectrum of F2, named unit A and unit B, which have anomeric hydrogen signals at δ 4.84 and 4.54 ppm, respectively We observed another spin system with lower intensity, unit C, in which an anomeric signal at δ 4.71 ppm was observed All units consist of β-Dgalactose residues, and their respective chemical shifts were determined by combined analysis of the COSY, TOCSY, HSQC, and HMBC spectra (Table 3) The signal detected at δ 1.63/23.4 ppm was attributed to the CH3 groups of pyruvate, which are common in SPs obtained from green seaweeds (Arata et al., 2015; Ciancia et al., 2007, 2012; Farias et al., 2008; Li et al., 2015) This cross-peak also indicate S configuration at C2 from pyruvate (Garegg, Lindberg, & Kvarnström, 1979) Unit A shows the H1/C1 correlation at δ 4.84/102.7 ppm, indicating the presence of strong electronegative substituents in this unit The signals at positions and are shifted to high-frequency field Given that H3/C3 (δ 4.26/78.8 ppm) and H4/C4 (δ 4.18/75.2 ppm) represent the chemical shift of 3,4-O-(1-carboxyethylidene)-β-D-Galp residues, also known as 3,4-pyruvylated-β-D-Galp residues, the results are consistent with those reported by Fernández et al (2015) In addition, HMBC spectra showed a cross-peak correlation between H3 and carbonyl carbon (107.8 ppm) from pyruvate groups, thereby confirming Fig Electrophoretic migration of AEC fractions F0.5v, and AEC subfractions (F0.3, F0.5, F0.7, F1, F1.5, F2 and F3 M) (50 μg each) were applied at agarose gel electrophoresis from the origin (Or) to the positive pole (+) as a single concentric band, indicating that F2 and F3 were obtained with high purity (Fig 2) Furthermore, these two fractions yielded 43% and 39% (w/w) of the total SPs present in F0.5v Both F2 and F3 were further analyzed by gel permeation chromatography (GPC) in a Sephadex G-100 column to determine their molecular weights (Fig 3) The chromatogram showed a single and symmetrical peak, indicating that the SPs were successfully purified from F0.5v by AEC Furthermore, the chromatogram obtained from GPC was used to calculate the apparent molecular weight using a regression equation determined using different molecular weight standards (Fig 3) Thus, the molecular weights of F2 and F3 were estimated to be approximately 62 and 61 kDa, respectively Results of the chemical analyses are plotted in Table F2 and F3 are both composed only of galactose, sulfate, and pyruvate However, the SPs showed differences in the proportions of sulfate and pyruvate; F3 showed higher sulfate/sugar and pyruvate/sugar ratios than F2 F2 showed a sulfate/sugar ratio of 1:4 and pyruvate/sugar ratio of 1:12 On the other hand, F3 had a sulfate/sugar ratio of approximately 1:2.6 and pyruvate/sugar ratio of 1:8 No protein traces were detected for both SPs (Table 1) Carbohydrate Polymers 222 (2019) 115010 D.A Sabry, et al Fig Homogeneity and molecular weight determination of SP, F2, and F3, from C isthmocladum The GPC chromatogram of F2 and F3 on a Sephadex G-100 column and the standard curve of molecular weight (kDa) glycosidic linkage bonding different units A weak cross-peak H3 (B)/ C1 (A) allowed the determination of the bonding between unit A and B, as a 3,4-Pyr-β-D-Galp-(1→3)-β-D-Galp4S-(1→ fragment, with some of these pyruvylated units being sulfated at C6 and absence of sulfation at C4 Meanwhile, a strong cross-peak H2 (C)/C1 (B) allowed the determination of unit B and C bond as the fragment →3)-β-D-Galp4S-(1→ 3,6)-β-D-Galp-(1→ Fig shows the proposed structure of F2 and F3 based on GCeMS and NMR data analysis Similar to the current findings, Farias et al (2008) successfully purified two similar pyruvylated SGs, named SG1 and SG2, from C isthmocladum using the same procedure Therefore, combined ice-cold acetone precipitation, anion exchange chromatography, and gel electrophoresis in PDA buffer is an effective method for the purification of SPs from seaweed SG1 (14 kDa) and SG2 (20 kDa) belong to the same SG family, which are known to be predominantly composed of 4-sulfated, 3-linked β-D-Galp units In addition, SG1/SG2 contain pyruvate groups that form five-membered cyclic ketals as 3,4-O-(1-carboxyethylidene)-β-D-galactose residues These structural features were also identified in F2 and F3, indicating that belong to the same SG family However, we observed differences between F1/F2 and SG1/SG2 polysaccharides F1/F2 and SG1/SG2 were obtained from two different fractions, namely, F0.5 and F0.9, respectively Moreover, the molecular weight of SG1/SG2 is lower than that determined for F1/F2, indicating differences in structure although they belong to the same SG family In fact, SG1/SG2 showed some 4,6-sulfated β-D-Galp units, whereas F1/F2 did not On the other hand, F1/F2 was found to contain pyruvylated 6sulfated β-D-Galp units, which were not detected in SG1/SG2 Other representatives of this SG family were also described from several species of the genus Codium (Ciancia et al., 2007; Estevez et al., 2009; Fernández et al., 2015; Li et al., 2015) Farias et al (2008) showed that acetone fractions from C isthmocladum have different monosaccharide compositions However, some Table Chemical composition of F2 and F3 from Codium isthmocladum SP F2 F3 Sulfate/sugar ratio 1:4 1:2.6 Pyruvate/sugar ratio 1:12 1:8 Protein (μg) nd nd Molar ratio Gal SO4 Pyr 1 0.6 0.8 0.15 0.17 nd = not detected; Gal = galactose; Pyr = pyruvate; SO4 = sulfate the presence of a pyruvate group linked to this unit Moreover, some H6/C6 were shifted to high-frequency (δ 4.33/67.4 ppm), which is typically observed in the presence of sulfate group as confirmed by HSQC spectrum of DSF2 (data not shown), in which the signal was not detected, indicating that some units A also contain sulfation at C6, named A6S The above findings were confirmed by HMBC spectra analysis, which shows a cross-peak between H6 (δ 4.33 ppm) from sulfated residues and C4 (δ 75.2 ppm) from pyruvylated residues (Fig 4) Unit B showed an anomeric correlation at δ 4.54/103.4 ppm The cross-peak of H4/C4 (δ 4.86/77.8 ppm) was observed at the high frequency region, indicating the presence of sulfation at this position Furthermore, H3/C3 (δ 4.09/77.4 ppm) and H5/C5 (δ 3.85/74.8 ppm) signals were also shifted to higher ppm values because of the presence of a nearby sulfate group Determination of the remaining chemical shifts resulted in defining unit B as →3)-β-D-Galp4S-(1→ residue based on the results obtained by Fernández et al (2015) Unit C showed an anomeric cross-peak at δ 4.71/103.8 ppm Chemical shifts of H4/C4 (δ 4.30/70.9) and H6/C6 (δ 4.23/67.4) indicated the absence of sulfation in this residue because these signals are located in regions of lower frequency compared to sulfated residues (Bilan, Vinogradova, Shashkov, & Usov, 2007) In addition, the HMBC spectrum (1H/13C) shows the sequence of Carbohydrate Polymers 222 (2019) 115010 D.A Sabry, et al Fig NMR spectra of SGs from C isthmocladum First panel shows the comparison of the 1H-NMR spectra of F2 and F3 All other panels show the results of 2D-NMR of F2 Letters refer to the spin system, and numbers refer to the position at spin system A: 3,4-Pyr-β-D-Galp; A6S: 3,4-Pyr-β-D-Galp6S; B: →3)-β-D-Galp4S-(1→; C: → 3,6)-β-D-Galp4S-(1→ Galp: galactopyranose Table Methylation analysis of sulfated galactan (F2) obtained from Codium isthmocladum and their desulfated derivative (mol %) O-methylated alditol acetate F2 DSF2 Linkage pattern 0-Me-Gal 2-Me -Gal 2,4-Me2-Gal 2,6-Me2-Gal 2,3,4-Me3-Gal 2,3,6-Me3-Gal 2,4,6-Me3-Gal 2,3,4,6-Me4-Gal 40 28 6 0 27 16 27 15 →2,3,4,6)-Galp-(1→ →3,4,6)-Galp-(1→ →3,6)-Galp-(1→ →3,4)-Galp-(1→ →6)-Galp-(1→ →4)-Galp-(1→ →3)-Galp-(1→ Non-reducing end Table Chemical shift assignments of the NMR spectra of SP (F2 and F3) from C isthmocladum based on 2D-NMR experiments Unit A fractions contained arabinose and mannose residues, in addition to galactose residues Furthermore, results from agarose gel electrophoresis demonstrated the presence of bands with different mobilities, indicating that Codium can synthesize other SGs or other types of SPs Considering that sulfated mannans (Fernández et al., 2012), and sulfated arabinans (Hayakawa et al., 2000) were described in Codium, the current findings are consistent with those reported for other seaweeds, wherein the same species were found to produce different SPs (Skriptsova, 2015) We aim to evaluate the other fractions from C isthmocladum and verify the above findings in future studies Chemical shifts, δ (ppm)a Structural unit 3,4-Pyr-β-D-Galpc c A6S 3,4-Pyr-β-D-Galp6S B →3)-β-D-Galp4S-(1→ C →3,6)-β-D-Galp-(1→ Ref.b H1 C1 H2 C2 H3 C3 H4 C4 H5 C5 H6 C6 4.84 102.7 4.84 102.7 4.54 103.4 4.71 103.8 3.55 73.6 3.55 73.6 3.76 70.8 3.76 70.8 4.26 78.8 4.26 78.8 4.09 77.4 3.86 82.4 4.18 75.2 4.18 75.2 4.86 77.8 4.30 70.9 4.05 73.3 4.05 73.3 3.85 74.8 3.85 74.8 3.89 61.1 4.33 67.4 3.80 61.4 4.23 67.4 1 2 a Chemical shifts are relative to the internal standard, trimethylsilyl propionic acid (δ =0 ppm) b References used to elucidate structural units based on similarity in chemical shifts: (1) Fernández et al (2015) and (2) Bilan et al (2007) c Signals at δ 107.8 ppm and 1.63/23.4 correspond to C2 and C3/H3 of pyruvic acid ketal linked to O-3 and O-4 of terminal galactose units ductal adenocarcinoma cell line (PANC-1) were evaluated by MTT assay after 24 and 48 h of treatment The viabilities of PANC-1 cells were reduced in the presence of F2 in a dose-dependent manner (Fig 6) However, we observed no time-dependent effects on PANC-1 viability in the presence of F2 We observed approximately 40% inhibition of PANC-1 viability using mg/mL F2 at both treatment 3.4 Cell viability in the presence of F2 and F3 The antiproliferative activities of F2 and F3 against the pancreas Carbohydrate Polymers 222 (2019) 115010 D.A Sabry, et al Fig Proposed structure of galactans F2 and F3 from Codium isthmocladum Unit A: 3,4-Pyr-β-D-Galp; Unit B: →3)-β-D-Galp4S-(1→; Unit C: →3,6)-β-D-Galp-(1→ Radicals position (R) can represent a hydrogen nucleus (H) or SO3 group In cases where unit A contains an OSO3 group, this unit is called A6S fine structure such as the type of glycosidic linkage, molecular weight, and the sulfate pattern, are important characteristics for the bioactivity of sulfated polysaccharides 3.5 Antioxidant activities of F2 and F3 We evaluated the antioxidant activities of F2 and F3 by the TAC assay F2 and F3 did not effectively prevent molybdenum reduction, exhibiting less than ascorbic acid equivalents (mg/g) of F2 or F3 (data not shown) Costa et al (2010), observed antioxidant activity below 10 ascorbic acid equivalents (mg/g) of crude SPs from C isthmocladum using the same assay, which is not a strong result However, comparing the results of F2 and F3 with crude SPs, it is likely that components present in the crude SPs (other than both F2 and F3) exhibit slightly stronger capacity to inhibit molybdenum reduction The amount and distribution of sulfate groups in the polysaccharide molecule are very important for it to exhibit strong antioxidant activity (Patel, 2012) However, the structural requirements, such as the presence of disulfated monosaccharides or specific sulfation in a specific position, have not been defined yet In this context, the contribution of this study is that the presence of C4 and C6 sulfation, besides the presence of 3,4-O-linked pyruvate are not important requirements for the antioxidant activity of sulfated galactans 3.6 Anticoagulant activities of F2 and F3 The anticoagulant activities of F2 and F3 were evaluated by aPTT and PT assays The clotting times of both SPs did not change based on the PT test, indicating that these SPs did not act on the extrinsic pathway of coagulation Meanwhile, F2 and F3 were effectively in prolonging the clotting time on intrinsic pathway of coagulation in a dose-dependent manner based on the aPTT test At the concentration of 10 μg/mL, both SPs showed 1.7-fold longer clotting times compared to Clexane®, a commercial heparin (Fig 7A) Furthermore, converting F2, F3, and Clexane® to molar concentrations at the dose of 10 μg/mL, and analyzing the [Clexane®]/[SP] molar ratio, we observed that Clexane® reached about 50% of F2 or F3 activity, using almost 14-fold higher molar concentration than F2 or F3 (Fig 7B) Matsubara et al (2001) evaluating the anticoagulant properties of SGs extracted from C cylindricum, using 15 μg/mL prolonged clotting time over 300 s F2 and F3 from C isthmocladum reached the same prolongation using 10 μg/mL However, Matsubara samples showed high protein content and the authors did not rule out whether proteins influenced this activity On the other hand, F2 and F3 were not Fig Cell viability of PANC-1 cells in the presence of F2 and F3 after 24 h (Panel A) and 48 h of treatment (Panel B) * symbol indicates significant difference between F2 and F3 treatment from negative control (p ≤ 0.05) periods On the other hand, F3 reduced the cell viability of PANC-1 in a dose- and time-dependent manner However, F3 exerted weaker effects on cell viability compared to F2 at the same concentrations Sulfated galactans from green seaweed Udotea flabellum (from 0.1 to mg/mL) did not affect B16-F10 melanoma cells viability (Marques et al., 2019) This indicates that only the type of monosaccharide and the presence of sulfate are not determinant requirements for the antiproliferative activity of SPs, which strengthen that other factors into polysaccharide Carbohydrate Polymers 222 (2019) 115010 D.A Sabry, et al Higher Level Personal Development Coordination, in loose translation), Programa Ciờncias Mar (AUXPE-CIMAR-1956/2014), Programa Nacional de Cooperaỗóo Acadờmica (CAPES/PROCAD n 2965/2014), and Ministộrio de Ciờncia, Tecnologia, Inovaỗừes e Comunicaỗừes (MCTIC) (Science and Technology Ministry in Brazil, in loose translation) for financial support H A O Rocha, H B Nader, and G L Sassaki are CNPq fellowship honored researchers D Sabry had a Ms.C and pH.D scholarship from CNPq and currently has a post-doctoral fellowship from PNPD-CAPES C Silva has a Ms.C scholarship from CAPES References Arata, P X., Quintana, I., Canelón, D J., Vera, B E., 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each SP were analyzed by anticoagulant evaluation Panel B: [Clexane®]/[SP] molar ratio based on F2, F3, and Clexane® at the dose of 10 μg/mL [Clexane®]/[Clexane®] ratio correspond to (data not shown) contaminated by proteins Arata et al (2015) evaluating the anticoagulant potential of F1, a sulfated and pyruvylated galactan from green seaweed Penicillus capitatus, observed a weak anticoagulation activity F1 from P capitatus is composed of galactose residues 3-, 6-, and 3,6-linked, showing 4,6-Olinked pyruvate groups in part sulfated at C2 Furthermore, F1 prolonged weakly the aPTT anticoagulant test time, did not reach 100 s using 100 μg/mL Meanwhile, F2 and F3 from Codium isthmocladum prolonged clotting time over 100 s, using only 2.5 μg/mL This stronger anticoagulant response might be related to the presence of sulfation at C4 and 3,4-O-linked pyruvate groups in part sulfated at C6 Conclusions Two sulfated galactans, namely, F2 and F3, were purified from the green seaweed Codium isthmocladum using combination of ice-cold acetone precipitation and anion exchange chromatography F2 and F3 were found to be pyruvylated and formed five-membered cyclic ketals (S configuration) as 3,4-O-(1′carboxy)-ethylidene-β-D-galactose residues The only significant difference between F2 and F3 is the sulfate/ sugar ratio, in which F3 showed 1/2.6 while F2 showed 1/4 Furthermore, NMR analysis showed that both SGs are linked-(1→3)-βD-galactans, with branching at C6 and sulfation at O4 None of the SGs presented significant activity on MTT or TAC essay On the other hand, both F2 and F3 showed stronger anticoagulant activity than Clexane®, a commercial heparin Taken together, the current findings highlight the potential application these of SGs as anticoagulants agents Acknowledgements The authors wish to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (Edital Universal, n 408369/2016-7), Coordenaỗóo de Aperfeiỗoamento Pessoal de Nớvel Superior (CAPES 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benthic marine algae of the tropical and subtropical western Atlantic Canadian Journal of Botany, 64(10), 2239–2281 https://doi.org/10 1139/b86-298 ... presence of sulfation at C4 and 3,4-O-linked pyruvate groups in part sulfated at C6 Conclusions Two sulfated galactans, namely, F2 and F3, were purified from the green seaweed Codium isthmocladum. .. Fig Homogeneity and molecular weight determination of SP, F2, and F3, from C isthmocladum The GPC chromatogram of F2 and F3 on a Sephadex G-100 column and the standard curve of molecular weight... potential of F1, a sulfated and pyruvylated galactan from green seaweed Penicillus capitatus, observed a weak anticoagulation activity F1 from P capitatus is composed of galactose residues 3-, 6-, and