Structure Analysis and Anti-Tumor and Anti-Angiogenic Activities of Sulfated Galactofucan Extracted from Sargassum thunbergii

Sulfated galactofucan (ST-2) was obtained from Sargassum thunbergii. It was then desulfated to obtain ST-2-DS, and autohydrolyzed and precipitated by ethanol to obtain the supernatant (ST-2-S) and precipitate (ST-2-C). ST-2-C was further fractionated by gel chromatography into two fractions, ST-2-H (high molecular weight) and ST-2-L (low molecular weight). Mass spectrometry (MS) of ST-2-DS was performed to elucidate the backbone of ST-2. It was shown that ST-2-DS contained a backbone of alternating galactopyranose residues (Gal)n (n ≤ 3) and fucopyranose residues (Fuc)n. In addition, ST-2-S was also determined by MS to elucidate the branches of ST-2. It was suggested that sulfated fuco-oligomers might be the branches of ST-2. Compared to the NMR spectra of ST-2-H, the spectra of ST-2-L was more recognizable. It was shown that ST-2-L contain a backbone of (Gal)n and (Fuc)n, sulfated mainly at C4 of Fuc, and interspersed with galactose (the linkages were likely to be 1→2 and 1→6). Therefore, ST-2 might contain a backbone of (Gal)n (n ≤ 3) and (Fuc)n. The sulfation pattern was mainly at C4 of fucopyranose and partially at C4 of galactopyranose, and the branches were mainly sulfated fuco-oligomers. Finally, the anti-tumor and anti-angiogenic activities of ST-2 and its derivates were determined. It was shown that the low molecular-weight sulfated galactofucan, with higher fucose content, had better anti-angiogenic and anti-tumor activities.


Introduction
Fucoidans are one type of water-soluble polysaccharide and are synthesized by brown algae. They are heteropolysaccharides containing not only fucose but also residues of galactose, mannose, rhamnose, glucuronica acid, glucose, xylose, and others. Based on the monosaccharides, fucoidans are divided into two types: sulfated heteropolysaccharides and sulfated galactofucans/sulfated fucans, which are the most well-known of the fucoidans. Mass spectroscopy (MS) was performed to determine the chemical composition. MS is widely used for the analysis of heteropolysaccharides due to its speed, sensitivity, and accuracy. However, polysaccharides with high molecular weights or a high degree of sulfate substitutions are not suitable. Thus, ST-2 was partly desulfated. Figure 2 shows the negative-ion mode electrospray ionization mass spectrometry (ESI-MS) spectrum of ST-2-DS. ST-2-DS contained a series of monosulfated fuco-oligosaccharides, methyl glycosides of mono-sulfated fuco-oligosaccharides. Moreover, the mixture also contained methyl glycosides of mono-sulfated and di-sulfated galacto-fucooligosaccharides (hexose was found to be galactose-based on the monosaccharide analysis). The proposed compositions of the ions are summarized in Table 1.  Mass spectroscopy (MS) was performed to determine the chemical composition. MS is widely used for the analysis of heteropolysaccharides due to its speed, sensitivity, and accuracy. However, polysaccharides with high molecular weights or a high degree of sulfate substitutions are not suitable. Thus, ST-2 was partly desulfated. Figure 2 shows the negative-ion mode electrospray ionization mass spectrometry (ESI-MS) spectrum of ST-2-DS. ST-2-DS contained a series of mono-sulfated fuco-oligosaccharides, methyl glycosides of mono-sulfated fuco-oligosaccharides. Moreover, the mixture also contained methyl glycosides of mono-sulfated and di-sulfated galacto-fuco-oligosaccharides (hexose was found to be galactose-based on the monosaccharide analysis). The proposed compositions of the ions are summarized in Table 1.  To elucidate the structural features of the proposed compositions, we performed mass spectrometry in tandem with collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS). Figure 3A shows the fragmentation pattern for the ion at m/z 403.089 (−1), which corresponded to [Me Fuc 2 (SO 3 H)-H] − . The identification of B-type ions at m/z 225.005 (−1) and 371.059 (−1) indicated that the sulfate was located at the non-reducing end, while the Y-type ion at m/z 257.030 (−1) suggested that the sulfate was located at the reducing end. Therefore, we concluded that Me (Fuc) 2 (SO 3 H) represented a mixture of the isomers Fuc(SO 3 H) → Fuc-OMe and Fuc → Fuc(SO 3 H)-OMe.
In addition to the sulfated fuco-oligosaccharides and methyl glycosides of sulfated fuco-oligosaccharides, we also detected methyl glycosides of sulfated galacto-fuco-oligosaccharides.  -H] − , respectively, indicating that the sulfate was substituted at the reducing end or non-reducing end. Thus, we speculated that the ion at m/z 330.048 (-2) was also a mixture of isomers: The ESI-CID-MS/MS spectrum of the ion at m/z 581.136 (−1) ([Me Gal2Fuc (SO3H)-H] − ) is shown in Figure 3D. The ions at m/z 549.129 (−2) (indicating a loss of MeOH (32 Da)), 403.067 (−1) (indicating a loss of methyl fucose (178 Da)), and 241.011 (−1) (a dehydrated sulfated galactose) represent a B3type ion, B2-type ion, and B1-type ion, respectively; indicating that the ion at m/z 581.136 (−1) was Gal(SO3H) → Gal → Fuc-OMe. The fragment ions at m/z 435.095 (−1) and 419.099 (−1) could be explained through Y2 fragmentation, where they would be derived from the loss of the dehydrated fucose (146 Da) and dehydrated galactose (162 Da), suggesting that both galactose and fucose were at the non-reducing end. The presence of the ion at m/z 273.044(−1), corresponding to [Me GalSO3H-H] − , confirmed that the ion at m/z 581.136 (−1) was a mixture of Gal → Fuc → Gal(SO3H)-OMe and Fuc → Gal → Gal(SO3H)-OMe. Other isomers could not be excluded. Thus, it was concluded that the ion respectively, indicating that the sulfate was substituted at the reducing end or non-reducing end. Thus, we speculated that the ion at m/z 330.048 (-2) was also a mixture of isomers: The  Figure 3E. The B-type ions indicated that the ion at m/z 889.244 was a mixture of isomers: Gal(SO 3 H) → Gal → Fuc → Gal → Fuc-OMe and Gal(SO 3 H) → Gal → Gal → Fuc → Fuc-OMe. The presences of the Y' 4 -type ion and the Y" 4 -type ion indicated that the non-reducing end was Gal residue and Fuc residue. In addition, the Y1-type ion corresponded to [Me GalSO 3 H-H] − . However, other ions could not confirm the sequence of residues. Thus, it was concluded that the ion at m/z 889.244 was a mixture of 20 isomers (it was emphasized that three continuous Gal residues were present in the backbone of sulfated galactofucan).
From the above discussion, it was concluded that the sulfated galactofucan from S. thunbergii Autohydrolysis reactions have been used for the structural determination of several polysaccharides [32][33][34][35]. To obtain the precise structure of sulfated galactofucan, an autohydrolysis reaction was performed. Two fractionations (ST-2-S and ST-2-C) were obtained. MS was performed to analyze ST-2-S in Figure 4. It was shown that ST-2-S contained mainly sulfated fuco-oligomers, accompanied by slightly sulfated fuco-xylo-oligomers. The proposed compositions of the ions are also summarized in Table 1. It was proposed that the major components of ST-2-S were the branches of ST-2, while the structure of ST-2-C was the backbone of ST-2 after autohydrolysis. Thus, it was concluded that ST-2 might have branches with sulfated fuco-oligomers.  Figure 3E. The B-type ions indicated that the ion at m/z 889.244 was a mixture of isomers: Gal(SO3H) → Gal → Fuc → Gal → Fuc-OMe and Gal(SO3 H) → Gal → Gal → Fuc → Fuc-OMe. The presences of the Y'4-type ion and the Y''4-type ion indicated that the non-reducing end was Gal residue and Fuc residue. In addition, the Y1-type ion corresponded to [Me GalSO3H-H] − . However, other ions could not confirm the sequence of residues. Thus, it was concluded that the ion at m/z 889.244 was a mixture of 20 isomers (it was emphasized that three continuous Gal residues were present in the backbone of sulfated galactofucan).
From the above discussion, it was concluded that the sulfated galactofucan from S. thunbergii Autohydrolysis reactions have been used for the structural determination of several polysaccharides [32][33][34][35]. To obtain the precise structure of sulfated galactofucan, an autohydrolysis reaction was performed. Two fractionations (ST-2-S and ST-2-C) were obtained. MS was performed to analyze ST-2-S in Figure 4. It was shown that ST-2-S contained mainly sulfated fuco-oligomers, accompanied by slightly sulfated fuco-xylo-oligomers. The proposed compositions of the ions are also summarized in Table 1. It was proposed that the major components of ST-2-S were the branches of ST-2, while the structure of ST-2-C was the backbone of ST-2 after autohydrolysis. Thus, it was concluded that ST-2 might have branches with sulfated fuco-oligomers.  Figure 1D) was similar to that of ST-2, suggesting that ST-2-L had similar functional groups to those of ST-2 (the sulfation of ST-2-C was mainly at the axial C-4 positions). Thus, it was concluded that both ST-2-H and ST-2-L were sulfated galactofucans. Based on the above speculation, ST-2-C was considered to be the backbone of ST-2. Therefore, Gal residues must be interspersed in the backbone, which was in accordance with the previous study [8]. The molar ratio    Figure 1D) was similar to that of ST-2, suggesting that ST-2-L had similar functional groups to those of ST-2 (the sulfation of ST-2-C was mainly at the axial C-4 positions). Thus, it was concluded that both ST-2-H and ST-2-L were sulfated galactofucans. Based on the above speculation, ST-2-C was considered to be the backbone of ST-2. Therefore, Gal residues must be interspersed in the backbone, which was in accordance with the previous study [8]. The molar ratio of sulfate to Hex (Fuc and Gal) residues of ST-2-H was 1.32, suggesting that every Hex (Fuc and Gal) residue has at least one sulfate group. The molar ratio of sulfate to Hex (Fuc and Gal) residues of ST-2-L was 0.89, suggesting that every Hex (Fuc and Gal) residue did not have one sulfate group. However, when the Gal residues did not have sulfate groups (as was confirmed by the NMR results in Figure 5), the molar ratio of sulfate to Fuc residues of ST-2-L was 1.04, suggesting that every Fuc residue has one sulfate group.
Mar. Drugs 2019, 17, x FOR PEER REVIEW 10 of 18 of sulfate to Hex (Fuc and Gal) residues of ST-2-H was 1.32, suggesting that every Hex (Fuc and Gal) residue has at least one sulfate group. The molar ratio of sulfate to Hex (Fuc and Gal) residues of ST-2-L was 0.89, suggesting that every Hex (Fuc and Gal) residue did not have one sulfate group. However, when the Gal residues did not have sulfate groups (as was confirmed by the NMR results in Figure 5), the molar ratio of sulfate to Fuc residues of ST-2-L was 1.04, suggesting that every Fuc residue has one sulfate group. ppm that were characteristic of 3-linked α-L-fucopyranose sulfated at C4, and weaker resonances with chemical shifts of 61.55 and 65.57 (C-6) that corresponded to 2-linked and 6-linked α-Dgalactopyranose, respectively. The proposed structure scheme of ST-2-L was shown in Figure 6. Therefore, it was concluded that ST-2 contained various sulfated galacto-fucans. However, they had the same backbone of alternating (Gal)n (n ≤ 3) and (Fuc)n. In addition, they also had branches, including sulfated fuco-oligomers, sulfated galacto-oligomers and xylo-oligomers. In a previous study [36], it was found that ST-1 might also contain sulfated galactofucan, which consists of a backbone of alternating (Gal)n and (Fuc)n, sulfated randomly on Gal and mainly on C2 in Fuc. Thus, it was hypothesized that Sargassum thunbergii might synthesize different kinds of sulfated galactofucan, which consists of a backbone of alternating (Gal)n and (Fuc)n with different molecular weights. In addition, the sulfation pattern was mainly at C4 of fucopyranose and randomly of galactopyranose. Moreover, the branches were mainly sulfated fuco-oligomers.  Figure 7A that the activities of ST-2-L were similar to those of ST-2, which were higher than ST-2-H. The major differences between ST-2-H and ST-2-L were the molecular weights and the fucose content. In a previous study [22], it was shown that there was no change in the anti-tumor activity at high concentrations (higher than 1 mg/mL). Thus, it was suggested that higher fucose content in sulfated galactofucan leads to better activity. For anti-angiogenic activities, it can be seen in Figure 7B that ST-2-L has the best activities, followed by ST-2 and ST-2-H, suggesting that low molecular-weight sulfated galactofucan, with higher fucose content, has better anti-angiogenic activities. Therefore, it was concluded that ST-2 contained various sulfated galacto-fucans. However, they had the same backbone of alternating (Gal) n (n ≤ 3) and (Fuc) n . In addition, they also had branches, including sulfated fuco-oligomers, sulfated galacto-oligomers and xylo-oligomers. In a previous study [36], it was found that ST-1 might also contain sulfated galactofucan, which consists of a backbone of alternating (Gal) n and (Fuc) n , sulfated randomly on Gal and mainly on C2 in Fuc. Thus, it was hypothesized that Sargassum thunbergii might synthesize different kinds of sulfated galactofucan, which consists of a backbone of alternating (Gal) n and (Fuc) n with different molecular weights. In addition, the sulfation pattern was mainly at C4 of fucopyranose and randomly of galactopyranose. Moreover, the branches were mainly sulfated fuco-oligomers.  Figure 7A that the activities of ST-2-L were similar to those of ST-2, which were higher than ST-2-H. The major differences between ST-2-H and ST-2-L were the molecular weights and the fucose content. In a previous study [22], it was shown that there was no change in the anti-tumor activity at high concentrations (higher than 1 mg/mL). Thus, it was suggested that higher fucose content in sulfated galactofucan leads to better activity. For anti-angiogenic activities, it can be seen in Figure 7B that ST-2-L has the best activities, followed by ST-2 and ST-2-H, suggesting that low molecular-weight sulfated galactofucan, with higher fucose content, has better anti-angiogenic activities. Mar. Drugs 2019, 17, x FOR PEER REVIEW 13 of 18

Preparation and Purification of Polysaccharides
Polysaccharide (ST) was prepared according to a previous study [36]. Briefly, algae (100 g) were cut into pieces and treated with 85% ethanol three times to remove the pigment. Crude polysaccharide was extracted from the residual material with hot water (3 L) for 4 h. The extract solution was filtered with Celite and concentrated. Further elimination of alginate was achieved using 20% ethanol with MgCl 2 (0.05 mol/L). After removing the alginate, the supernatant fluid was ultra-filtered. Finally, the dialysate was concentrated, and crude polysaccharide was obtained by ethanol precipitation, namely ST (The yield was 1.30%).

Preparation of Desulfated Polysaccharides
The desulfated polysaccharide was prepared according to a previously described method [37]. Briefly, ST-2 (1 g) was dissolved in distilled water (100 mL) and mixed with cationic resin (H + ) for 3 h. After filtration, the solution was neutralized with pyridinium and lyophilized. The solution was dissolved in dimethyl sulfoxide (DMSO) (St. Louis, Missouri, MO, USA): methanol (9:1; v/v, 20 mL) and heated at 80 • C for 5 h, and the desulfated product (ST-2-DS) (the yield was 21.69%) was dialyzed and lyophilized.

Depolymerization of ST-2 by Autohydrolysis
The autohydrolysis was performed according to the modified method [38]. Briefly, ST-2 (0.8 g) was changed to the H + form using a column of cation exchange and left for 72 h at room temperature. The mixture was neutralized with 5% NH 4

Compositional Analysis
The sulfated contents were preformed by ion chromatography on a Shodex IC SI-52 4E column (4.0 × 250 mm, Showa Denko K.K., Tokyo, Japan) and eluted with 3.6 mM Na 2 CO 3 at a flow rate of 0.8 mL/min at 45 • C. The molar ratio of monosaccharides and fucose content was determined as described by Zhang et al. [39]. Briefly, polysaccharides (10 mg/mL) were hydrolyzed by trifluoroacetic acid (2 M) under a nitrogen atmosphere for 4 h at 110 • C. Then, the hydrolyzed mixture was neutralized to pH 7 with sodium hydroxide. Later, the mixture was converted into its PMP derivatives and separated by high performance liquid chromatography (HPLC) chromatography on an YMC Pack ODS AQ column (4.6 × 250 mm, YMC, Kyoto, Japan). Uronic acid (UA) concentration was determined by a modified carbazole method [40]. The molecular weights of the polysaccharides were evaluated by gel permeation chromatography (GPC)-HPLC on a TSK G3000 PWxl column (7 µm 7.8 × 300 mm, TOSOH, Tokyo, Japan), with elution in 0.05 M Na 2 SO 4 at a flow rate of 0.5 mL/min at 40 • C with refractive index detection. Ten different molecular weight dextrans, purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), were used as weight standards.

IR and MS Analysis of Oligosaccharides
Infrared spectra (IR) were recorded from powder in KBr pellets on a Nicolet-360 fourier transform infrared spectrometer (Nicolet, Pleasanton, CA, USA) between 400 and 4000 cm −1 (36 scans, at a resolution of 6 cm −1 ).
ESI-MS and ESI-CID-MS/MS were performed on an LTQ ORBITRAP XL (Thermo Scientific, Waltham, MA, USA) (after installation parameters were modified). The samples were dissolved in CH 3 CN-H 2 O (1:1, v/v). The solution was centrifuged for 10 min at 10,000 rpm, and the supernatant was analyzed. Mass spectra were registered in the negative ion mode at a flow rate of 5 µL min −1 . The capillary voltage was set to −3000 V, and the cone voltage was set at −50 V. The source temperature was 80 • C, and the desolvation temperature was 150 • C. The collision energy was optimized between 10 and 50 eV. All spectra were analyzed by Xcalibur.
NMR and two-dimensional spectra were recorded with a Bruker AVANCE III (Bruker BioSpin, Billerica, MA, USA) at 600 MHz and 25 • C. The chemical shifts were adjusted to the internal standard (deuterated acetone, 2.05 and 29.92 ppm, respectively).

Anti-Tumor and Anti-Angiogenic Activities
Anti-tumor activities of polysaccharides against human lung cancer A549 cells and anti-angiogenic activities against human umbilical vein endothelial (HUVEC) cells were determined. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay was used to measure cell viability. Briefly, cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and penicillin-streptomycin (100 units/mL) in an atmosphere of 5% CO 2 at 37 • C. The cells (100 µL) were then seeded in a 96-well plate at a density of 1 × 10 4 cells/well for 24 h. Subsequently, the cells were divided into the following three groups: (1) blank group, which only contained medium for 24 h; (2) control group in which cells were added for 24 h; and (3) experimental groups in which cells and polysaccharides at different concentrations (0.95, 1.82, and 2.61 mg/mL) were cultivated in medium for 24 h and 48 h. After removal of the media, 10 µL of MTT (5 mg/mL) was added to each well. After 4 h of incubation, the supernatants were removed, and dimethyl sulfoxide (DMSO) (100 µL) was added. Next, the absorbance was measured at 490 nm, and the inhibition rate was determined using the following equation: Cell Inhibition rate (%) = (Ac − A1)/(Ac − A0) × 100, where A0 was the absorbance of the blank, A1 was the absorbance in the presence of samples, and Ac was the absorbance of the control.

Statistical Analysis
All data are shown as the mean ± standard deviation (SD). Significant differences between experimental groups were determined by one-way ANOVA, and differences were considered to be statistically significant if p < 0.05. All calculations were performed using SPSS 16.0 statistical software.

Conclusions
In this study, polysaccharide (ST-2) was extracted and fractionated from S. thunbergii with hot water. Then, ST-2 was desulfated to obtain ST-2-DS. ST-2-S and ST-2-C were obtained after autohydrolysis. Finally, ST-2-C was fractionated into two fractions (ST-2-H and ST-2-L) by gel chromatography. ST-2-DS and ST-2-S were determined by MS. It was shown that ST-2 contained a backbone of alternating (Gal) n (n ≤ 3) and (Fuc) n , branched with sulfated fuco-oligomers. ST-2-L was determined by NMR. It was found that ST-2-L contained 3-linked α-L-fucopyranose sulfated at C4 and interspersed with galactose (the linkages might be 2-linked and 6-linked). Combined with the IR results, it was concluded that ST-2 contained a backbone of (Gal) n (n ≤ 3) and (Fuc) n . In addition, the sulfation pattern was mainly at C4 of fucopyranose and randomly at C4 of galactopyranose. Moreover, the branches were mainly sulfated fuco-oligomers. Finally, the anti-tumor and anti-angiogenic activities of ST-2 and its derivates were determined. It was shown that the low molecular-weight sulfated galactofucan, with higher fucose content, had better anti-angiogenic and anti-tumor activities.