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Structural characterization and effect on anti-angiogenic activity of a fucoidan from Sargassum fusiforme
Accepted Manuscript Title: Structural characterization and effect on anti-angiogenic activity of a fucoidan from Sargassum fusiforme Author: Qifei Cong Huanjun Chen Wenfeng Liao Fei Xiao Peipei Wang Yi Qin Qun Dong Kan Ding PII: DOI: Reference:
S0144-8617(15)00952-2 http://dx.doi.org/doi:10.1016/j.carbpol.2015.09.087 CARP 10389
To appear in: Received date: Revised date: Accepted date:
5-5-2015 22-9-2015 24-9-2015
Please cite this article as: Cong, Q., Chen, H., Liao, W., Xiao, F., Wang, P., Qin, Y., Dong, Q., and Ding, K.,Structural characterization and effect on anti-angiogenic activity of a fucoidan from Sargassum fusiforme, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.09.087 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Structural characterization and effect on anti-angiogenic activity of a
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fucoidan from Sargassum fusiforme
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Qifei Conga,1, Huanjun Chena,1, Wenfeng Liaoa, Fei Xiaoa, Peipei Wanga, Yi Qina, Qun Dong*a, Kan
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Ding*a a
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Glycochemistry and Glycobiology Lab, Shanghai Institute of Materia Medica, Chinese Academy of
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Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
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Corresponding Author
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Qun Dong: e-mail: [email protected]; Tel: +86-21-50806600 ext. 3203; Fax: +86-21-50806928
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Qifei Cong and Huanjun Chen contributed equally to this work.
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Kan Ding: e-mail: [email protected]; Tel: +86-21-50806928; Fax: +86-21-50806928
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Abstract
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A fucoidan FP08S2 was isolated from the boiling-water extract of Sargassum fusiforme, purified by
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CaCl2 precipitation and chromatography on DEAE-cellulose and Sephacryl S-300. FP08S2 contained
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fucose, xylose, galactose, mannose, glucuronic acid, and 20.8% sulfate. The sulfate groups were
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attached to diverse positions of fucose, xylose, mannose, and galactose residues. The backbone of
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FP08S2 consisted of alternate 1, 2-linked α-D-Manp and 1, 4-linked β-D-GlcpA. Sugar composition
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analysis and ESI-MS revealed that the oligosaccharides from branches contained fucose, xylose,
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galactose, glucuronic acid and sulfate. FP08S2 could significantly inhibit tube formation and
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migration of human microvascular endothelial cells (HMEC-1) dose-dependently. These results
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suggested that the fucoidan FP08S2 from brown seaweeds Sargassum fusiforme could be a potent
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anti-angiogenic agent.
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Key words: Sargassum fusiforme; fucoidan; structure; anti-angiogenic activity
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Highlights
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A fucoidan from Sargassum fusiforme was isolated and characterized;
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Backbone was alternate 1, 2-linked α-D-Manp and 1, 4-linked β-D-GlcpA;
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Branches contain fucose, xylose, galactose, glucuronic acid and sulfate group;
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Fucoidan could significantly inhibit the tube formation;
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Oligosaccharide fractions exhibited the anti-angiogenesis activities.
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1. Introduction
Fucoidan refers to a family of natural polysaccharides that are characteristic of rich content of
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fucose and sulfate ester substituent. In recent years, more complex fucoidans have been identified
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(Shevchenko et al., 2015). Four typical structures of fucoidans from some brown seaweed species
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were proposed by previous researches (Ale, Mikkelsen & Meyer, 2011; Li, Lu, Wei & Zhao, 2008;
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Ustyuzhanina et al., 2014). (1) A fucan sulfate consisting of a 1, 3-linked α-L-Fucp backbone or a
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linear backbone of alternating 1, 3- and 1, 4-linked α-L-fucopyranose, with sulfate ester substituted at
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C-4 and C-2 (Bilan et al., 2002; Shevchenko et al., 2015); (2) A fucogalactan containing a
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(1→6)-β-D-Galp and/or (1→2)-β-D-Manp backbone, with branches consisting of terminal galactose
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and fucose attached at C-4 or C-2 (Duarte, Cardoso, Noseda & Cerezo, 2001); (3) A
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fucoglucuronomannan composed of a backbone of alternating 1, 2-linked α-D-Manp and 1, 4-linked
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β-D-GlcpA, with α-L-Fucp substituted at C-3 of 1, 2-linked α-D-Manp as branches (Li, Wei, Sun & Xu,
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2006; Sakai et al., 2003); (4) A fucoglucuronan having a 1, 3- or 1, 4-linked β-D-GlcpA backbone with
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branches of α-L-Fucp or β-D-Xyl attached to C-2 or C-4 (Bilan et al., 2010; Silva et al., 2005).
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Fucoidan from brown alga was found to have various biological activities, including antitumor
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(Senthilkumar, Manivasagan, Venkatesan & Kim, 2013), immunomodulatory (Kim & Joo, 2008),
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antioxidant (Lim et al., 2014), antivirus (Synytsya et al., 2014), anticoagulant (Chandia & Matsuhiro,
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2008), and anti-inflammatory (Lee et al., 2012). Due to these various biological activities, structures
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and properties of these polysaccharides have been intensively investigated.
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Sargassum fusiforme is a brown alga distributed mainly along the coastline of China, Korea, and
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Japan (Cong, Xiao, Liao, Dong & Ding., 2014). It was used as a traditional Chinese medicine to treat
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tumor, scrofula, edema, beriberi, and chronic bronchitis. Previous researches reported an alginate from 4
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Sargassum fusiforme (Mao, Li, Gu, Fang & Xing, 2004), which could decrease the level of total
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cholesterol, triglyceride, and low density lipoprotein-cholesterol. Li et al isolated a fucoidan from
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Sargassum fusiforme and found that the fucoidan could prolong the blood coagulation time (Li, Zhao
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& Wei, 2008). Other researchers suggested that the polysaccharides have anti-tumor properties in vivo
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and in vitro (Chen et al., 2012a), anti-oxidant (Wang et al., 2013), immuno-stimulating (Chen et al.,
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2012b; Hu et al., 2014), as well as anti-HIV activities (Paskaleva et al., 2006).
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Unfortunately, in most cases, the chemical structures of fucoidan from Sargassum fusiforme are
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not fully elucidated. In order to understand the relationship between the structure of polysaccharide
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and its biological effect, this study was dedicated to characterization of the structural features, and to
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evaluation of the anti-angiogenic activity of a fucoidan extracted from Sargassum fusiforme.
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2. Materials and methods
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2.1. Materials
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The raw material of Sargassum fusiforme (3.0 kg) was purchased from Hu Qing Yu Tang
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Drugstore. DEAE-cellulose 32 was purchased from Whatman Co., and Sephacryl S-300 HR was from
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GE Healthcare Life Sciences. Bio Gel P-2 was purchased from Bio-Rad. Polysaccharide calibration kit
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including pullulans of different molecular weights (Mw 180, 667, 6000, 11300, 21700, 48800, 113000,
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210000, 393000, and 805000) was from Varian Medical Systems Inc. (s)-(+)-1-amino-2-propanol was
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purchased from Sigma-Aldrich. Other reagents were of analytical grade from Sinopharm Chemical
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Reagent Co. Ltd.
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2.2. Isolation and purification of fucoidan from Sargassum fusiforme.
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The extraction of crude polysaccharide SFbWP was referenced to our previous report (Cong et al., 5
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2014). To remove most alginate, SFbWP was dissolved in water (2%, w/v), then 3 volumes of a 2%
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aqueous CaCl2 (w/v) were added with stirring. After centrifugation, the supernatant was dialyzed
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against tap water. The retentate was precipitated with 4 volumes of 95% ethanol, and the precipitate
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was collected and dried in vacuum to obtain SFbWP-FP.
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The schematic procedure for the extraction and purification of the polysaccharide was shown in
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Fig. 1. Briefly, SFbWP-FP (12.1 g) was fractionated on a DEAE-cellulose column (50 cm × 5 cm, Cl-
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form), eluted stepwise with water, 0.2, 0.4, 0.8, and 1.6 M aqueous NaCl, and finally with 0.3 M
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NaOH. The eluate was monitored by phenol-sulfuric acid method for carbohydrate content and
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appropriately pooled, giving five fractions (Fig. S.1.A), FPW (91.3 mg, 0.8% of SFbWP-FP), FP02
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(1.5 g, 12.3%), FP04 (1.0 g, 8.4%), FP08 (3.0 g, 24.9%), FP16 (405.0 mg, 3.3%), and the NaOH
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eluate FPB (223.0 mg, 1.8%). The major fraction, FP08 (1.9 g), was further fractionated by gel
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permeation chromatography on a Sephacryl S-300 column (100 cm × 2.6 cm), equilibrated and eluted
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with 0.2 M NaCl, and detected by a differential refraction detector, into three fractions (Fig. S.1.B).
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The major fraction was collected, dialyzed, and designated as FP08S2 (996.5 mg).
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Fig. 1.
2.3. Physicochemical properties
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The neutral carbohydrate was determined using phenol-sulfuric acid method with D-glucose as the
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standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), and uronic acid content measured by the
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m-hydroxybiphenyl method using D-glucuronic acid as the standard (Blumenkrantz & Asboe-Hansen,
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1973). Sulfate content was determined by elemental analysis, which was conducted on an Elementar
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Vario EL CUBE instrument (Elementar, Germany). Specific rotation was estimated with an Autopol
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VI instrument (Rudoph Research Analytical) at 25 oC at a wavelength of 589 nm. Homogeneity and 6
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molecular weight were determined by high performance gel permeation chromatography (HPGPC) on
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an Agilent 1260 HPLC system, with 0.1 M NaNO3 used as the mobile phase. Other manipulation was
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referenced to previous report (Xu, Dong, Qiu, Cong & Ding, 2010).
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2.4. Monosaccharide composition analyses and determination of absolute configuration
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Monosaccharide composition of polysaccharides was analyzed by gas chromatography (GC).
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Briefly, 2 mg of polysaccharide was hydrolyzed with 2 M TFA at 110 oC for 4 h. The hydrolyzates
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were reduced with sodium borohydride and subsequently converted into alditol acetates as described
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(Xu et al., 2010). For quantification of uronic acid, FP08S2 was subjected to carboxyl-reduction as
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described by Taylor & Conrad (Taylor & Conrad, 1972). The carboxyl reduced product was analyzed
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as described above.
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Absolute configuration of monosaccharide constituents was determined as the method described
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(Li et al., 2014). Briefly, three reagents of solution A [ (s)-(+)-1-amino-2-propanol: absolute methanol,
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1:8], solution B (3%, NaBH3CN, m/v), and solution C (glacial acetic acid: absolute methanol, 1:4)
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were prepared. 2.0 mg of the polysaccharide was dissolved in TFA (2 M, 4 mL) and hydrolyzed at 110
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re-dissolved in water and lyophilized. Then 25 μL of solution A, B, and C were added into the
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lyophilized sample and dried monosaccharide standards, respectively. The mixed solutions were
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transferred into ampoule bottle and sealed to react in a water bath at 65 oC for 1.5 h. Then the reaction
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solution was evaporated to dryness with methanol. 0.2 mL of anhydrous pyridine and acetic anhydride
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were added into the mixture respectively and were allowed to react at 100 oC for 1 h. Then, 5 mL of
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distilled water was added into the reaction solution and cooled for 30 min. The sample solution was
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followed by extraction with 5 mL of chloroform. Finally, the chloroform layer was dried with
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C for 4 h, the hydrolysate was evaporated to dryness with methanol. Subsequently, the residue was
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anhydrous sodium sulfate and analyzed by GC-MS.
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2.5. Methylation analysis The polysaccharide was methylated by the modified Ciucanu and Kerek method (Ciucanu &
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Kerek, 1984). Briefly, the polysaccharide (10 mg), was dissolved in DMSO (2 mL) at room
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temperature, and stirred vigorously with powered sodium hydroxide for 10 min. Iodomethane (0.3 mL)
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was added dropwise under ice bath, and stirred for 30 min at room temperature. The mixture was
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dialyzed and the retentate was freeze-dried to yield the methylated polysaccharide. The partially
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methylated alditol acetates (PMAAs) were prepared and analyzed by GC-MS (Wang, He & Huang,
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2007).
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2.6. Infrared spectroscopy (IR) and electrospray ionization mass spectrometry (ESI-MS)
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The IR spectra were measured as the previous report (Cong et al., 2014). Methylated
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polysaccharide was measured by Nujol method. The ESI-MS experiment was performed by a Finnigan
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LCQ/DECA mass spectrometer in the negative ion mode, with nitrogen used as the nebulizer gas, and
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the capillary temperature was kept at 280 oC.
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2.7. NMR analysis
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The polysaccharides (30 mg) were deuterium-exchanged and dissolved in 0.5 ml of D2O (99.8%
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D). 1H NMR, 13C NMR were recorded at 25 oC on a Bruker AVANCE III NMR spectrometer at 500
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MHz with a cryogenic NMR probe. The HOD signal (4.85 ppm in 1H NMR) and acetone (31.50 ppm
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in
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were also performed to provide 1H-1H COSY, hetero-nuclear single quantum coherence (HSQC), and
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hetero-nuclear multiple bond correlation (HMBC) spectra. Data were analyzed on MestReNova
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V7.0.0-8331 software.
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C NMR) signals were employed as the internal standards, respectively. 2D NMR experiments
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2.8. Desulfation of polysaccharide Briefly, the polysaccharide (100 mg) was dissolved in 10 mL of water and passed through a
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column of 732# sulphonic exchange resin (H+) (1.6 cm × 50 cm). The eluate was neutralized with
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pyridine and lyophilized. Other manipulation was referenced to previous report (Li et al., 2006). The
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desulfated product was dialyzed against water (MWCO 500). The retentate was lyophilized to give the
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desulfated polysaccharide, designated as FP08S2-DS.
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2.9. Partial acid hydrolysis
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Three aliquots of FP08S2 (100 mg) were each dissolved in 0.05 M, 0.1 M and 2.0 M TFA and
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heated at 100 oC for 1 h. After removing TFA, the residue was re-dissolved in water (5 mL), and
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dialyzed against water (MWCO 3500 Da). The dialyzate and retentate from 0.05 M, 0.1 M, and 2.0 M
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TFA treatment were designated as F005MDO and F005MDI, F01MDO and F01MDI, F2MDO and
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F2MDI, respectively. By using a Bio-Gel P-2 column, F005MDO was resolved into two fractions
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detected by phenol-sulfuric acid method, F005MDO1 and F005MDO2. In addition, F01MDO was
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fractionated with an online RI detector into four fractions, F01MDO1, F01MDO2, F01MDO3, and
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F01MDO4. Similarly, F2MDO was fractionated into two fractions, F2MDO1, and F2MDO2,
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respectively.
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2.10. Cell lines and culture conditions
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Human microvascular endothelial cells (HMEC-1) were cultured in MCDB 131 medium (Gibco
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BRL, U.S.A.) as described in previous reports (Qiu, Yang, Pei, Zhang & Ding, 2010). The cells were
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incubated at 37 oC with 5% CO2 under a humidified atmosphere.
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2.11. Tube formation assay
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The effect of fucoidan FP08S2 on angiogenesis in vitro was examined by the HMEC-1 cells 9
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capillary-like tube formation assay. Briefly, A 96-well plate pre-coated with 50 μL Matrigel per well
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was prepared and solidified at 37 oC for 30 min. HMEC-1 cells (5 × 104 cells/well) and samples
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(FP08S2 and FP08S2-DS of 25, 50, 100, and 200 μg/mL, partial acid hydrolysis products of 500
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μg/mL) were added into each well correspondingly and incubated for another 12 h. The enclosed
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capillary networks of tube formation were recorded by an Olympus IX51 digital camera (Tokyo
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Japan).
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2.12. Wound healing assay of HMEC-1 cells
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HMEC-1 cells (5 × 105 cells/well) were cultured into a 6-well culture plate. After 24 h, the
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confluent monolayer was scraped using a yellow pipette tip to create a wound gap and rinsed with
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PBS thrice carefully. Then 0, 0.4 and 0.8 mg/mL of FP08S2 prepared in new MCDB 131 medium
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were added into each well. After incubation for 12 h, three randomly selected regions around the cross
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were photographed. The migration of cells through the wound area was then estimated quantitatively
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using Image-Pro Plus software.
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2.13. Cell proliferation assay of HMEC-1 cells
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A MTT assay was determined to evaluate the cell proliferation of HMEC-1 cells. Briefly,
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HMEC-1 cells (5 × 103 cells/well) were cultured in 96-well plates with MCDB 131 medium for 24 h.
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The medium was discarded and exchanged by 0, 25, 50, 100, 200, 400, 800 μg/mL of FP08S2
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dissolved in MCDB131 medium. After 72 h, 10 μL of 5 mg/mL MTT was added into each well and
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kept for 4 h at 37 oC. The medium was discarded and the formazan crystal was dissolved in 150 μL of
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DMSO. The solution was vibrated for 20 min and the absorption at 490 nm was measured to calculate
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the cell viability ratio of HMEC-1 cells.
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3. Results and Discussion
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3.1. Structure elucidation of FP08S2 The crude polysaccharide (SFbWP) was extracted from Sargassum fusiforme with boiling water,
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and then it was treated by 2% CaCl2 precipitation to remove alginate fraction. The fraction obtained
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from the supernatant (SFbWP-FP) was fractionated by anion-exchange chromatography on a
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DEAE-cellulose 32 column (Fig. S.1.A). FP08 was obtained as the major fraction from 0.8 M NaCl
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eluate, which (1.9 g) was further purified by gel filtration chromatography on a Sephacryl S-300 HR
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column to afford a homogeneous polysaccharide FP08S2 (996.5 mg) (Fig. S. 1. B).
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The neutral sugar and uronic acid content of FP08S2 were determined to be 16.8% and 34.6%,
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respectively, and the sulfate content (as SO42-) was calculated to be 20.8%. The specific rotation [α]D
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was -56.2o (c 0.5, H2O). HPGPC analysis (Fig. S.2.A) indicated the polysaccharide was homogeneous
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for only one symmetrical peak appeared except for the salt peak. The apparent molecular weight was
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estimated to be 47.5 kDa by reference to the pullulan standards. The monosaccharide composition
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analysis showed that FP08S2 is composed of fucose, xylose, mannose, galactose and glucuronic acid,
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in a ratio of 36.6: 18.3: 7.0: 19.1: 19.1 (Table 1). The content of fucose was similar to that reported in
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literature (Li et al., 2006), but the contents of xylose, mannose, and glucuronic acid were intensively
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different from Li’s report. In addition, rhamnose and arabinose were not detected. In addition, the
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absolute configurations of monosaccharide constituents were determined to be L-fucose, D-xylose,
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D-mannose,
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D-configuration
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and
D-galactose.
Absolute configuration of glucuronic acid was demonstrated to be
from the reduction product of FP08S2 (FP08S2-R). Data were shown in Fig. S.3. Table 1.
IR spectrum of FP08S2 was shown in Fig. S.2.B. The strong absorption at 1259.3 cm-1 could be 11
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attributed to asymmetric O=S=O stretching vibration of sulfate esters. In addition, an intense
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absorption at 1737.6 cm-1 indicated the presence of uronic acid. In the fingerprint region (750-960
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cm-1), the distinct absorption at 842.7 cm-1 but no trace at 824 cm-1 revealed that the sulfate esters were
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mainly substituted at the axial C-4 of fucopyranose residues (Bilan et al., 2002). After desulfated, the
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absorption at 1259.3 cm-1 disappeared in the IR spectrum of FP08S2-DS (Fig. S.2.B). Elemental
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analysis showed that the sulfur content of FP08S2-DS was reduced to 0.9%, indicating 87% of the
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sulfate groups had been removed. As shown in Table 1, FP08S2-DS contained fucose, xylose,
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mannose, galactose and glucuronic acid, in a ratio of 31.8: 17.2: 8.2: 21.8: 21.1.
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The glycosyl linkages of FP08S2 were determined by methylation analyses, along with
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FP08S2-DS, as shown in Table 2. For the native polysaccharide, the fucose residues were found to be
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present in complicated glycosyl linkages, including terminal, 1, 3-, 1, 4-, 1, 2, 3-, and 1, 3, 4-linked
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Fucp, largely due to sulfate substitution at different positions. According to previous reports (Bilan et
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al., 2004; Chandia et al., 2008; Ale et al., 2011; Chen et al., 2012; Yu et al., 2013), most fucoidans
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contain 1, 3-linked Fucp residues with sulfate attached mainly to C-4 or C-2. Our results indicated that
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the 1, 3-linked 2-sulfated fucopyranosyl residue accounted for the major linkage type for fucose. After
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desulfation, the proportion of 1, 3-linked fucose residues increased while the 1, 3, 4-linked fucose
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decreased distinctly, indicating that the sulfate groups were also attached to C-4 of 1, 3-linked Fucp. In
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addition, some of the sulfate groups were probably substituted at C-4 of non-reducing terminal fucose
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residues. After desulfation, the content of terminal xylose increased in the expense of 1, 4-linked
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xylose, indicating that the terminal xylose residues were partially sulfated at C-4. 1, 2, 4, 6-linked
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mannose units disappeared after desulfation, and the content of 1, 2, 3-linked mannose decreased, but
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the content of 1, 2-linked and 1, 2, 6-linked mannose increased slightly. These indicated that some
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sulfate groups were linked to C-3/4/6 of 1, 2-linked mannose. After desulfation, 1, 3, 6-linked and 1, 2,
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4, 6-linked galactose disappeared, while the content of 1, 6- and 1, 2, 4-linked galactose increased
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slightly, indicating that the sulfate were probably attached to C-6 of 1, 2, 4-linked galactose, C-3 of 1,
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6-linked galactose and C-2 and/or C-4 of 1, 6-linked galactose. In addition, 1, 3, 4-linked galactose
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disappeared, replaced by 1, 3-linked galactose, indicating that sulfate groups were attached to C-4 of 1,
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3-linked Gal. Linkage analysis of carboxyl-reduced, desulfated product showed terminal and 1,
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4-linked glucose, indicating that glucuronic acid in FP08S2 was present as terminal and 1, 4-linked. Table 2.
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3.2. Structural analysis of the partial acid hydrolysis-derived polysaccharides
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Like other fucoidans from brown seaweeds, FP08S2 displayed heavily overlapped resonances in
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its 1H and 13C NMR spectra (Fig. S.4.A and D), mainly due to the complex glycosyl composition and
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diverse sulfation substitution.
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To further elucidate the structure, FP08S2 was subjected to partial acid hydrolysis to afford
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oligosaccharides from branches, partially hydrolyzed with 0.05 M, 0.1 M and 2 M TFA, respectively.
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Sugar composition analysis of partial acid hydrolysis products of dialyzate and retentate was
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summarized in Table 1, and ESI-MS analysis of dialyzate fractions was shown in Fig. 2.
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Fig. 2.
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F005MDO1 had a higher content of α-L-Fucp, but other residues like xylose, galactose and
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glucuronic acid were also in substantial amounts. ESI-MS analyses (Fig. 2) revealed the most
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intensive ion at m/z 535.2 was assigned to monosulfated fucotrioligosaccharide [Fuc3SO3]-. The
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fucoxylooligosaccharide was identified by [Fuc3XylSO3]- ion at m/z 667.2 and [Fuc4XylSO3]- ion at
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m/z 813.1. The ion at m/z 843.0 was assigned to a hexose-containing oligosaccharide [Fuc4GalSO3]-. In 13
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addition, the sulfated disaccharide [FucGlcASO3]- ion was also observed at m/z 419.2. In comparison,
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F005MDO2 was only composed of fucose and xylose. ESI-MS analysis revealed that the signals of
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monosulfated fucose [FucSO3]- at m/z 243.1 and monosulfated xylofucooligosaccharide [Fuc2XylSO3]-
259
ion at m/z 520.7 were observed. In addition, Both F01MDO1 and F01MDO2 contained fucose and
260
glucuronic acid, in a molar ratio of 1: 1. ESI-MS revealed intensive [FucGlcASO3]- ion at m/z 419.4
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for F01DMO1 and at m/z 419.5 for F01MDO2, respectively. In addition, [Fuc2GlcASO3]- ion at m/z
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565.4 and [Fuc2GlcA2SO3]- ion at m/z 741.4 were also observed from F01MDO1. However, F01MDO3
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contained fucose and galactose, in a molar ratio of 3:2. F01MDO4 had a higher percentage of fucose,
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as well as substantial content of xylose and galactose. For F01MDO3, the three negative ions at m/z
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243.3, 389.4 and 405.3 corresponded to [FucSO3]-, [Fuc2SO3]- and [FucGalSO3]-. For F01MDO4, the
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[Fuc2SO3]- ion at m/z 388.9 confirmed the presence of monosulfated fuco-disaccharide. Meanwhile,
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monosulfated galactose was also observed from [GalSO3]- ion at m/z 259.3. The ion at m/z 442.5
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corresponded to trisaccharide [GalXyl2]-. The two oligosaccharide fractions of FP08S2 after treated
269
with 2M TFA partial hydrolysis both contain fucose, xylose and galactose. For F2MDO1, the main
270
component was a monosulfated galacto-tetrasaccharide [Gal4SO3]- at m/z 744.9. The ion at m/z 586.5
271
was attributed to disulfated xylofucotrisacchride [FucXyl2(SO3)2]-. For F2MDO2, the signal at m/z
272
243.1 of highest intensity corresponded to monosulfated fucose. Other ions at m/z 405.1, 421.2, and
273
828.7 were attributed to the [FucGalSO3]-, [Gal2SO3]-, and [Fuc3XylGalSO3]-, respectively.
Ac ce pt e
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274
With the increasing TFA concentration, fucose, galactose, and xylose were identified in the
275
released oligosaccharides, indicating that they were present mainly in branches. However, mannose
276
and glucuronic acid remained in the degraded polysaccharide (Table 1), indicating that they were
277
present in the core structure. 14
Page 14 of 35
Compared to FP08S2 and the retentate F01MDI, F2MDI presented a well-resolved 1H NMR
279
spectrum (Fig. S.4.B and C), in which the anomeric resonances corresponding to Fuc, Xyl, and Gal
280
largely disappeared, indicating that they had been mostly removed by acid treatment. It was reasonable
281
to suggest that F2MDI represented the core structure of FP08S2. The apparent molecular weight of
282
F2MDI was estimated to be 12.4 kDa by HPGPC. Sulfate content of F2MDI is calculated to be 7.5%
283
by elemental analysis. The carboxyl-reduced F2MDI-R was found to contain Fuc, Man, Gal and Glc,
284
in a mole ratio of 5.6: 32.2: 9.3: 53.0, as shown in Table 1. Methylation analysis (Table 2) showed that
285
F2MDI-R consisted mainly of 1, 2- and 1, 2, 6-linked Manp, and 1, 4-linked GlcpA. Other minor
286
residues involved 1, 3-linked Fucp, terminal Galp, and terminal GlcpA. According to methylation
287
analysis, it could be inferred that F2MDI possessed a backbone composed of 1, 2-linked Manp and 1,
288
4-linked GlcpA. 1, 3-linked Fucp, terminal Galp, and terminal GlcpA residues were probably present
289
as the component residues in branches.
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In the 1H NMR (Fig. 3.A), the two intense anomeric signals at δ 5.40 and 4.56 ppm could be
291
assigned to α-Manp and β-GlcpA, respectively. The minor signals at δ 1.28-1.31 ppm were ascribed to
292
H6 of Fuc. Other signals were assigned according to the 2D homonuclear 1H/1H COSY and shown in
293
Table 3. The 13C NMR spectrum (Fig. 3.B) of F2MDI showed four intensive signals in the anomeric
294
region at 99.95-102.99 ppm. The resonances at 102.78 and 102.99 ppm were ascribed to 1, 4-linked
295
and terminal β-D-GlcpA, while those at 99.95 and 100.39 ppm were assigned to 1, 2- and 1, 2, 6-linked
296
α-D-Manp. The two resonances at 173.58 and 173.92 ppm were assigned to terminal and 1, 4-linked
297
GlcpA, respectively. The two methylene signals at δ 67.65 and 61.08 ppm in DEPT spectrum could be
298
assigned to C6 of 1, 2, 6-, and 1, 2-linked Manp residues, respectively. The weak resonance at 93.3
299
ppm was assigned to C1 of 1, 3-linked α-L-Fucp, and the resonance at 17.73 ppm was apparently from
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Page 15 of 35
300
C6 of Fuc. In addition, the weak resonance at 103.5 ppm was ascribed to anomeric carbon of terminal
301
β-D-Galp. Table 3.
303
Further spectral assignment was aided by COSY, HSQC, and HMBC spectra (Fig. 3), and the
304
detailed assignments for NMR data of F2MDI were concluded in Table 3 (Bilan et al., 2010; Li et al.,
305
2006). HMBC spectrum (Fig. 3.E) revealed the sequence of the glycosyl residues in F2MDI. The
306
correlation at 3.85/99.95, 100.39 ppm indicated that the H4 of 1, 4-linked β-GlcpA was correlated with
307
C1 of 1, 2-linked and 1, 2, 6-linked α-Manp residues. On the other hand, C1 of 1, 4-linked β-GlcpA
308
was correlated with H2 of 1, 2- and 1, 2, 6-linked α-Manp. These transglycosidic correlations between
309
1, 2-linked α-Manp and 1, 4-linked β-GlcpA suggested that they were probably connected alternately
310
in F2MDI as the backbone, similar as that in previous report (Li et al., 2006). Li et al (Li et al., 2006)
311
reported a core structure of fucoidan isolated from Sargassum fusiforme composed of alternating 1,
312
2-linked α-Manp and 1, 4-linked β-GlcpA units with minor 1, 4-linked β-Galp, branched at C-3 of 1,
313
2-linked Manp. Wang et al (Wang et al., 2012) confirmed the alternate disaccharide repeat of
314
→4)-β-GlcA(1→2)-α-Man(1→. However, our study indicated some different features in backbone of
315
FP08S2, as indicated by NMR spectra, especially for the signals of C6 of 1, 2, 6-linked α-Manp as
316
well as galactose residues. According to the previous NMR analyses of a sulfated trisaccharide
317
obtained from fucoglucuronomannan(Sakai et al., 2003), the resonance at 4.19/4.35ppm was ascribed
318
to H-6 of 6-O-sulfated D-Manp. And the chemical shift of C-6 of the 6-O-sulfated D-Manp was at
319
67.65ppm from the HSQC spectrum (Figure 3.D). In addition, the sulfate content of F2MDI was
320
determined up to 7.5% by elemental analysis. The sulfation group was substituted at C-6 of 1, 2,
321
6-Man from NMR analyses. The ratio of 1, 2, 6-Man was up to 15.7% from methylation results.
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Page 16 of 35
Therefore, the sulfated content was calculated to be 5.2% from methylation analyses, consistent with
323
the result from elemental analysis. Taken together, the substitution at C-6 of 1, 2, 6-Man was not
324
derived from the substitution of the other neighbouring glycosyl residues, but from the substitution of
325
sulfate group. In addition, C1 of terminal β-Galp in the branches correlated with H3 of 1, 3-linked
326
α-Fucp.
cr
Fig. 3.
327
3.3. Anti-angiogenic activity of FP08S2
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As shown in Fig. 4.A and B, FP08S2 at 25 μg/mL to 200 μg/mL inhibited the tube formation in a
330
dose-dependent manner. A complete disruption on network formation of HMEC-1 cells could be
331
observed at 200 μg/mL. FP08S2 exhibited a measurable decline in tube formation with increasing
332
concentration of FP08S2 (Fig. 4.B). In contrast, for the desulfated derivative, FP08S2-DS, no
333
significant inhibition was observed even at 200 μg/mL (Fig. 4.A and B). The results indicated that
334
sulfate group played an important role in the anti-angiogenic activity. In order to understand to what
335
degree of the oligosaccharide branches and the backbone of FP08S2 contribute to the inhibitory effect
336
on tube formation, the partial acid hydrolysis products of 0.05 M, 0.1 M, and 2 M TFA treatment were
337
subjected to tube formation assay at 0.5 mg/mL, respectively. The results were shown in Fig. 4.C and
338
D. Compared with the control, the three retentate fractions, F005MDI, F01MDI and F2MDI exhibited
339
a significant inhibition on tube formation. Intriguingly, the disruption of enclosed capillary network
340
was decreased for the degraded polysaccharide derived from high TFA concentration. It indicated that
341
the branches might also have a significant effect on the anti-angiogenic activity of FP08S2. In addition,
342
F005MDO1 and F01MDO1 exhibited a significant inhibition on tube formation. Given the
343
compositions of these two fractions, it suggested that not only the sulfate group but also glucuronic
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Page 17 of 35
344
acid influenced the tube formation significantly. Fig. 4.
345
In wound healing assay, as shown in Fig. 5.A and B, the migration of HMEC-1 cells was
347
substantially impaired with FP08S2 treatment compared with the control. In untreated primary
348
HMEC-1 cells, 64.7% (± 1.9) of the wound was closed after 12 h. In contrast, treated with fucoidan
349
FP08S2 (0.4 mg/mL), only 38.1% (± 2.7) of the wound was enclosed. With treatment of FP08S2 (0.8
350
mg/mL), only 16.6% (± 2.7) of the wound was closed. Thus FP08S2 significantly inhibited the
351
migration of HMEC-1 cells in a dose-dependent manner (Fig. 5.B).
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To investigate whether the inhibitory effects on capillary network and migration were the result
353
of inhibition of HMEC-1 cells proliferation, we analyzed the viability of HMEC-1 cells treated with
354
FP08S2 (25 - 800 μg/mL) in vitro. Fig. 5.C showed that native fucoidan FP08S2 showed no significant
355
cytotoxicity on HMEC-1 cells.
357 358
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an
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Fig. 5.
4. Discussion
359
Fucoidans are sulfated homo- and hetero-polysaccharides, mainly composed of α-L-fucopyranose
360
residues, which may be partially sulfated and/or acetylated (Shevchenko et al., 2015). In the present
361
study, a novel fucoidan FP08S2 was purified and characterized from Sargassum fusiforme. FP08S2
362
was shown to have a glucuronomannan backbone, composed of alternating 1, 2-linked α-D-Manp and
363
1, 4-linked β-D-GlcpA. The outer branches are composed mainly of 1, 3-linked α-L-Fucp residues
364
highly sulfated at O-2 and O-4, or as fucogalactan and fucoglucuronan, and 4-O partially sulfated
365
terminal Xylp. 18
Page 18 of 35
Angiogenesis involves the progression of proliferation, differentiation, and migration of mature
367
endothelial cells and is regulated by various endothelium stimulating angiogenic factors such as VEGF
368
and FGF (Kim, Park, Kang, Kim & Lee, 2014). Heparin and heparan sulfate are highly sulfated
369
polysaccharides. It has been reported that N-sulfated glucosamine and 2-O-sulfated iduronate residues
370
are necessary for the angiogenic factors binding to HSPG. This suggested sulfation of polysaccharides
371
was required for the anti-angiogenesis effect. Multiple structural factors may influence the biological
372
activities of fucoidans, including monosaccharide composition, sulfation, glycosyl linkages, branches,
373
and molecular weight. Matsubara et al. (Matsubara et al., 2005) reported the mild acid hydrolysis
374
product of fucoidan, containing 33.2% of sulfate content with a molecular weight of 30 kDa, exhibited
375
inhibition of angiogenesis in vitro. In contrast, the other product with a molecular weight of 15-20 kDa
376
and a low degree of sulfation (8.2%) did not inhibit HUVEC tube formation. In our study, the native
377
fucoidan of 47.5 kDa, with a degree of sulfation (20.8%), could significantly disrupt the capillary
378
network. The partial acid hydrolysis products demonstrated a weak inhibitory effect on tube formation
379
compared to FP08S2. In addition, the derivative F2MDI, with a molecular weight of 12.4 kDa and
380
7.5% of sulfate content, exhibited the lowest inhibition among the three degraded polysaccharides,
381
suggesting that molecular weight and sulfation played a key role in the anti-angiogenic activity of
382
fucoidan. In addition, we observed that some oligosaccharide fractions derived from branches also
383
demonstrated a significant inhibitory effect on tube formation. Based on the structural analysis of these
384
oligosaccharides, it could be suggested that the high degree of sulfation and/or uronic acid residues in
385
branches contributed to the anti-angiogenic activity of FP08S2. Moreover, previous studies have
386
reported that increased negative charge resulting from sulfation or oversulfation might enhance the
387
formation of fucoidan-protein complexes (Ale et al., 2011; Koyanaqi, Taniqawa, Nakagawa, Soeda, &
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Page 19 of 35
Shimeno, 2003). It had been studied that the spatial orientation of the negative charges on the fucoidan
389
exhibited a significant effect on the binding potency of fucoidan to VEGF165. The interaction of
390
fucoidan with higher sulfated content with VEGF165 resulted in the formation of highly stable
391
complexes. Therefore, the complexes disrupted the binding of VEGF165 to VEGF receptor-2
392
(VEGFR-2) (Koyanaqi et al., 2003). It indicated that the potent biological effect was dependent on
393
sulfation property, which was consistent with our result.
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In conclusion, a fucoidan FP08S2 isolated from Sargassum fusiforme was shown to have a
395
glucuronomannan backbone, composed of alternating 1, 2-linked α-D-Manp and 1, 4-linked β-D-GlcpA.
396
The outer branches were composed mainly of 1, 3-linked α-L-Fucp residues highly sulfated at C-2 and
397
C-4, or as fucogalactan and fucoglucuronan, and C-4 partially sulfated terminal Xylp. FP08S2
398
demonstrated a significant inhibition on tube formation and migration of HMEC-1 cells
399
dose-dependently.
401
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394
Acknowledgements
402
This research was supported by National Natural Science Foundation of China (NSFC)
403
(31230022), New Drug Creation and Manufacturing Program (2012ZX09301001-003), National
404
Science Fund for Distinguished Young Scholars (81125025) in China.
405 406
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Supporting information
521
(1) Figure S.1: The elution profile of SFbWP-FP on DEAE-cellulose 32 column, as determined by
522
phenol sulfuric acid method (A). The elution profile of FP08 on Sephacryl S-300 HR column with RI
523
detection (B).
524
(2) Figure S.2: HPGPC profile (A) of FP08S2 and IR spectra (B) for FP08S2 and FP08S2-DS.
525
(3) Figure S.3: Absolute configuration of FP08S2 and its reduction product FP08S2-R comparing to D-
526
and L- configurations of all monosaccharide standards.
527
(4) Figure S.4: 1H NMR spectra of FP08S2 (A), F01MDI (B) and F2MDI (C). 13C NMR spectrum of
528
FP08S2 (D).
an
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M
529
Figure captions
531
Figure 1. The scheme of extraction, isolation and purification of fucoidan, FP08S2, from Sargassum
532
fusiforme.
533
Figure 2. The ESI-MS fragment pattern of acid degraded oligosaccharide fractions in negative ion
534
mode.
535
Figure 3. 1D and 2D NMR spectra of F2MDI. 1H-NMR (A); 13C-NMR (B); 1H-1H COSY (C); HSQC
536
(D); HMBC(E); Legend for the residues in F2MDI. a: 1, 2-linked α-D-Manp; b: 1, 2, 6-linked
537
α-D-Manp; c: 1, 4-linked β-D-GlcpA; d: terminal β-D-GlcpA; e: 1, 3-linked α-L-Fucp; f: terminal
538
β-D-Galp.
539
Figure 4. Effect of FP08S2 and its derivatives on tube formation of HMEC-1 cells. (A) HMEC-1 cells
540
treated with FP08S2 and its desulfated derivative FP08S2-DS at different concentrations (25, 50, 100,
541
and 200 μg/mL). (B) Quantitative measurement of tube numbers. (C) Effect on anti-angiogenesis
Ac ce pt e
d
530
26
Page 26 of 35
activity of oligosaccharide fractions and retentate fractions after treatment of 0.05 M, 0.1 M, and 2.0
543
M TFA. Concentration of all samples is 500 μg/mL. (D) Quantitative measurement of tube numbers.
544
The values represent mean±S.D. * P ＜ 0.05; ** P ＜ 0.01, as determined by unpaired t-test.
545
Figure 5. (A) The effect of FP08S2 (0.4 mg/mL and 0.8 mg/mL) on migration of HMEC-1 cells after
546
incubating for 12 h in a wound healing assay. (B) Quantification of FP08S2 on HMEC-1 cells
547
migration in the wound healing assay. (C) The cell viability of HMEC-1 cells after treatment with 25,
548
50, 100, 200, 400, 800 μg/mL of FP08S2 for 72 h. The values represent mean±S.D. * P ＜ 0.05; **
549
P ＜ 0.01, as determined by unpaired t-test.
us
cr
ip t
542
Ac ce pt e
d
M
an
550
27
Page 27 of 35
550 551
Tables
552
Table 1. Molecular weight and sugar composition of FP08S2 and partially acid hydrolysis products Sugar compositions (mol % of all sugars) 4
Molecular weight (/10 Da) Man 7.0
4.75
36.6
18.3
FP08S2-DS
3.07
31.8
17.2
F005MDI
3.58
38.1
15.3
F01MDI
1.95
18.3
F2MDI
1.24
an 9.4
88.3
11.7
19.1
19.1
21.8
21.1
19.1
27.5
ND
18.9
48.2
ND
32.2
9.3
53.0
6.1
3.2
53.0
47.0
F01MDO2
48.6
51.4
F01MDO3
59.6
F01MDO4
57.8
28.0
14.2
F2MDO1
43.7
23.1
33.2
F2MDO2
60.4
24.7
14.9
Ac ce pt e
F01MDO1
81.3
M
F005MDO2
5.6
d
F005MDO1
14.6
GlcA
8.2
us
FP08S2
Gal
ip t
Xyl
cr
Fuc
40.4
553 554 555
28
Page 28 of 35
Table 2. The results for linkage analyses for FP08S2, FP08S2-DS and F2MDI-R Molar ratios (mol %) FP08S2
FP08S2-DS
T-Xyl
5.8
11.8
1, 4-Xylp
16.5
6.5
T-Fucp
5.6
9.5
1, 3-Fucp
8.0
11.1
1, 4-Fucp
4.9
3.0
1, 3, 4-Fucp
10.1
2.4
1, 2, 3-Fucp
17.2
5.0
1, 2-Manp
0.8
3.7
1, 3-Manp
0.8
0.3
1, 2, 6-Manp
0.5
0.9
1, 2, 3-Manp
5.1
3.9
1, 2, 4, 6-Manp
2.9
T-Galp
3.4
cr us
an
15.7
6.1
0.7
1, 4-Galp
0.8
1, 6-Galp
3.8
1, 2, 4-Galp
1.0
1, 3, 4-Galp
1.0
1, 3, 6-Galp
1.7
1, 2, 4, 6-Galp
10.1
1.3
Ac ce pt e
557
17.7
0.7
1, 3-Galp
556
6.2
M
1, 2-Galp
6.5
F2MDI-R
ip t
Linkages
d
555
7.6 1.8
T-Glcp
2.5
8.6
1, 4-Glcp
20.9
43.3
29
Page 29 of 35
557 558
Table 3. 1H and 13C NMR spectral assignments for F2MDI Chemical shifts (ppm)
D
β- D-GlcpA-(1→
E
→3)-α-L-Fucp-(1→
F
β- D-Galp-(1→
H
5.41
4.19
3.82
3.79
3.73
3.79/3.85
C
99.95
78.77 70.56
67.1
74.32
61.08
H
5.39
4.19
3.82
3.77
4.19/4.35
C H C H C
3.82
ip t
→4)-β- D-GlcpA-(1→
6
100.39 78.86 70.64 67.33 70.62 4.53
3.45
3.73
3.85
4.06
102.78 73.67 76.79 78.26 75.23 4.59
3.45
3.73
3.61
173.92
3.99
102.99 73.61 76.72 72.57 75.94 4.06
67.65
cr
C
5
us
→2, 6)-α- D-Manp-(1→
4
H
5.3
4.05
C
93.3
70.03 79.46
H
4.89
3.63
C
103.5
73.48 74.28 70.63 76.67
173.58
4.09
4.36
1.28
69.5
68.15
17.73
4.04
3.71
3.77/3.87
M
B
3
3.77
61.69
d
→2)-α-D-Manp-(1→
2
Ac ce pt e
559
A
1
an
Residues
30
Page 30 of 35
Ac
ce
pt
ed
M
an
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i
Figure(1)
Page 31 of 35
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ce
pt
ed
M
an
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i
Figure(2)
Page 32 of 35
Ac
ce
pt
ed
M
an
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Figure(3)
Page 33 of 35
Ac
ce
pt
ed
M
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Figure(4)
Page 34 of 35
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ed
M
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Figure(5)
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S0144-8617(15)00952-2 http://dx.doi.org/doi:10.1016/j.carbpol.2015.09.087 CARP 10389
To appear in: Received date: Revised date: Accepted date:
5-5-2015 22-9-2015 24-9-2015
Please cite this article as: Cong, Q., Chen, H., Liao, W., Xiao, F., Wang, P., Qin, Y., Dong, Q., and Ding, K.,Structural characterization and effect on anti-angiogenic activity of a fucoidan from Sargassum fusiforme, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.09.087 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Structural characterization and effect on anti-angiogenic activity of a
2
fucoidan from Sargassum fusiforme
3
Qifei Conga,1, Huanjun Chena,1, Wenfeng Liaoa, Fei Xiaoa, Peipei Wanga, Yi Qina, Qun Dong*a, Kan
4
Ding*a a
5 6
ip t
1
Glycochemistry and Glycobiology Lab, Shanghai Institute of Materia Medica, Chinese Academy of
cr
Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
us
7
Corresponding Author
9
Qun Dong: e-mail: [email protected]; Tel: +86-21-50806600 ext. 3203; Fax: +86-21-50806928
12 13
1
Qifei Cong and Huanjun Chen contributed equally to this work.
d
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M
Kan Ding: e-mail: [email protected]; Tel: +86-21-50806928; Fax: +86-21-50806928
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an
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1
Page 1 of 35
Abstract
14
A fucoidan FP08S2 was isolated from the boiling-water extract of Sargassum fusiforme, purified by
15
CaCl2 precipitation and chromatography on DEAE-cellulose and Sephacryl S-300. FP08S2 contained
16
fucose, xylose, galactose, mannose, glucuronic acid, and 20.8% sulfate. The sulfate groups were
17
attached to diverse positions of fucose, xylose, mannose, and galactose residues. The backbone of
18
FP08S2 consisted of alternate 1, 2-linked α-D-Manp and 1, 4-linked β-D-GlcpA. Sugar composition
19
analysis and ESI-MS revealed that the oligosaccharides from branches contained fucose, xylose,
20
galactose, glucuronic acid and sulfate. FP08S2 could significantly inhibit tube formation and
21
migration of human microvascular endothelial cells (HMEC-1) dose-dependently. These results
22
suggested that the fucoidan FP08S2 from brown seaweeds Sargassum fusiforme could be a potent
23
anti-angiogenic agent.
26 27
Key words: Sargassum fusiforme; fucoidan; structure; anti-angiogenic activity
Ac ce pt e
25
d
24
M
an
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13
Highlights
28
A fucoidan from Sargassum fusiforme was isolated and characterized;
29
Backbone was alternate 1, 2-linked α-D-Manp and 1, 4-linked β-D-GlcpA;
30
Branches contain fucose, xylose, galactose, glucuronic acid and sulfate group;
31
Fucoidan could significantly inhibit the tube formation;
32
Oligosaccharide fractions exhibited the anti-angiogenesis activities.
33 34 2
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35
36
3
Page 3 of 35
36
1. Introduction
Fucoidan refers to a family of natural polysaccharides that are characteristic of rich content of
38
fucose and sulfate ester substituent. In recent years, more complex fucoidans have been identified
39
(Shevchenko et al., 2015). Four typical structures of fucoidans from some brown seaweed species
40
were proposed by previous researches (Ale, Mikkelsen & Meyer, 2011; Li, Lu, Wei & Zhao, 2008;
41
Ustyuzhanina et al., 2014). (1) A fucan sulfate consisting of a 1, 3-linked α-L-Fucp backbone or a
42
linear backbone of alternating 1, 3- and 1, 4-linked α-L-fucopyranose, with sulfate ester substituted at
43
C-4 and C-2 (Bilan et al., 2002; Shevchenko et al., 2015); (2) A fucogalactan containing a
44
(1→6)-β-D-Galp and/or (1→2)-β-D-Manp backbone, with branches consisting of terminal galactose
45
and fucose attached at C-4 or C-2 (Duarte, Cardoso, Noseda & Cerezo, 2001); (3) A
46
fucoglucuronomannan composed of a backbone of alternating 1, 2-linked α-D-Manp and 1, 4-linked
47
β-D-GlcpA, with α-L-Fucp substituted at C-3 of 1, 2-linked α-D-Manp as branches (Li, Wei, Sun & Xu,
48
2006; Sakai et al., 2003); (4) A fucoglucuronan having a 1, 3- or 1, 4-linked β-D-GlcpA backbone with
49
branches of α-L-Fucp or β-D-Xyl attached to C-2 or C-4 (Bilan et al., 2010; Silva et al., 2005).
Ac ce pt e
d
M
an
us
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37
50
Fucoidan from brown alga was found to have various biological activities, including antitumor
51
(Senthilkumar, Manivasagan, Venkatesan & Kim, 2013), immunomodulatory (Kim & Joo, 2008),
52
antioxidant (Lim et al., 2014), antivirus (Synytsya et al., 2014), anticoagulant (Chandia & Matsuhiro,
53
2008), and anti-inflammatory (Lee et al., 2012). Due to these various biological activities, structures
54
and properties of these polysaccharides have been intensively investigated.
55
Sargassum fusiforme is a brown alga distributed mainly along the coastline of China, Korea, and
56
Japan (Cong, Xiao, Liao, Dong & Ding., 2014). It was used as a traditional Chinese medicine to treat
57
tumor, scrofula, edema, beriberi, and chronic bronchitis. Previous researches reported an alginate from 4
Page 4 of 35
Sargassum fusiforme (Mao, Li, Gu, Fang & Xing, 2004), which could decrease the level of total
59
cholesterol, triglyceride, and low density lipoprotein-cholesterol. Li et al isolated a fucoidan from
60
Sargassum fusiforme and found that the fucoidan could prolong the blood coagulation time (Li, Zhao
61
& Wei, 2008). Other researchers suggested that the polysaccharides have anti-tumor properties in vivo
62
and in vitro (Chen et al., 2012a), anti-oxidant (Wang et al., 2013), immuno-stimulating (Chen et al.,
63
2012b; Hu et al., 2014), as well as anti-HIV activities (Paskaleva et al., 2006).
cr
ip t
58
Unfortunately, in most cases, the chemical structures of fucoidan from Sargassum fusiforme are
65
not fully elucidated. In order to understand the relationship between the structure of polysaccharide
66
and its biological effect, this study was dedicated to characterization of the structural features, and to
67
evaluation of the anti-angiogenic activity of a fucoidan extracted from Sargassum fusiforme.
68
2. Materials and methods
70
2.1. Materials
Ac ce pt e
69
d
M
an
us
64
71
The raw material of Sargassum fusiforme (3.0 kg) was purchased from Hu Qing Yu Tang
72
Drugstore. DEAE-cellulose 32 was purchased from Whatman Co., and Sephacryl S-300 HR was from
73
GE Healthcare Life Sciences. Bio Gel P-2 was purchased from Bio-Rad. Polysaccharide calibration kit
74
including pullulans of different molecular weights (Mw 180, 667, 6000, 11300, 21700, 48800, 113000,
75
210000, 393000, and 805000) was from Varian Medical Systems Inc. (s)-(+)-1-amino-2-propanol was
76
purchased from Sigma-Aldrich. Other reagents were of analytical grade from Sinopharm Chemical
77
Reagent Co. Ltd.
78
2.2. Isolation and purification of fucoidan from Sargassum fusiforme.
79
The extraction of crude polysaccharide SFbWP was referenced to our previous report (Cong et al., 5
Page 5 of 35
2014). To remove most alginate, SFbWP was dissolved in water (2%, w/v), then 3 volumes of a 2%
81
aqueous CaCl2 (w/v) were added with stirring. After centrifugation, the supernatant was dialyzed
82
against tap water. The retentate was precipitated with 4 volumes of 95% ethanol, and the precipitate
83
was collected and dried in vacuum to obtain SFbWP-FP.
ip t
80
The schematic procedure for the extraction and purification of the polysaccharide was shown in
85
Fig. 1. Briefly, SFbWP-FP (12.1 g) was fractionated on a DEAE-cellulose column (50 cm × 5 cm, Cl-
86
form), eluted stepwise with water, 0.2, 0.4, 0.8, and 1.6 M aqueous NaCl, and finally with 0.3 M
87
NaOH. The eluate was monitored by phenol-sulfuric acid method for carbohydrate content and
88
appropriately pooled, giving five fractions (Fig. S.1.A), FPW (91.3 mg, 0.8% of SFbWP-FP), FP02
89
(1.5 g, 12.3%), FP04 (1.0 g, 8.4%), FP08 (3.0 g, 24.9%), FP16 (405.0 mg, 3.3%), and the NaOH
90
eluate FPB (223.0 mg, 1.8%). The major fraction, FP08 (1.9 g), was further fractionated by gel
91
permeation chromatography on a Sephacryl S-300 column (100 cm × 2.6 cm), equilibrated and eluted
92
with 0.2 M NaCl, and detected by a differential refraction detector, into three fractions (Fig. S.1.B).
93
The major fraction was collected, dialyzed, and designated as FP08S2 (996.5 mg).
95
us
an
M
d
Ac ce pt e
94
cr
84
Fig. 1.
2.3. Physicochemical properties
96
The neutral carbohydrate was determined using phenol-sulfuric acid method with D-glucose as the
97
standard (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956), and uronic acid content measured by the
98
m-hydroxybiphenyl method using D-glucuronic acid as the standard (Blumenkrantz & Asboe-Hansen,
99
1973). Sulfate content was determined by elemental analysis, which was conducted on an Elementar
100
Vario EL CUBE instrument (Elementar, Germany). Specific rotation was estimated with an Autopol
101
VI instrument (Rudoph Research Analytical) at 25 oC at a wavelength of 589 nm. Homogeneity and 6
Page 6 of 35
molecular weight were determined by high performance gel permeation chromatography (HPGPC) on
103
an Agilent 1260 HPLC system, with 0.1 M NaNO3 used as the mobile phase. Other manipulation was
104
referenced to previous report (Xu, Dong, Qiu, Cong & Ding, 2010).
105
2.4. Monosaccharide composition analyses and determination of absolute configuration
ip t
102
Monosaccharide composition of polysaccharides was analyzed by gas chromatography (GC).
107
Briefly, 2 mg of polysaccharide was hydrolyzed with 2 M TFA at 110 oC for 4 h. The hydrolyzates
108
were reduced with sodium borohydride and subsequently converted into alditol acetates as described
109
(Xu et al., 2010). For quantification of uronic acid, FP08S2 was subjected to carboxyl-reduction as
110
described by Taylor & Conrad (Taylor & Conrad, 1972). The carboxyl reduced product was analyzed
111
as described above.
M
an
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cr
106
Absolute configuration of monosaccharide constituents was determined as the method described
113
(Li et al., 2014). Briefly, three reagents of solution A [ (s)-(+)-1-amino-2-propanol: absolute methanol,
114
1:8], solution B (3%, NaBH3CN, m/v), and solution C (glacial acetic acid: absolute methanol, 1:4)
115
were prepared. 2.0 mg of the polysaccharide was dissolved in TFA (2 M, 4 mL) and hydrolyzed at 110
116
o
117
re-dissolved in water and lyophilized. Then 25 μL of solution A, B, and C were added into the
118
lyophilized sample and dried monosaccharide standards, respectively. The mixed solutions were
119
transferred into ampoule bottle and sealed to react in a water bath at 65 oC for 1.5 h. Then the reaction
120
solution was evaporated to dryness with methanol. 0.2 mL of anhydrous pyridine and acetic anhydride
121
were added into the mixture respectively and were allowed to react at 100 oC for 1 h. Then, 5 mL of
122
distilled water was added into the reaction solution and cooled for 30 min. The sample solution was
123
followed by extraction with 5 mL of chloroform. Finally, the chloroform layer was dried with
Ac ce pt e
d
112
C for 4 h, the hydrolysate was evaporated to dryness with methanol. Subsequently, the residue was
7
Page 7 of 35
124
anhydrous sodium sulfate and analyzed by GC-MS.
125
2.5. Methylation analysis The polysaccharide was methylated by the modified Ciucanu and Kerek method (Ciucanu &
127
Kerek, 1984). Briefly, the polysaccharide (10 mg), was dissolved in DMSO (2 mL) at room
128
temperature, and stirred vigorously with powered sodium hydroxide for 10 min. Iodomethane (0.3 mL)
129
was added dropwise under ice bath, and stirred for 30 min at room temperature. The mixture was
130
dialyzed and the retentate was freeze-dried to yield the methylated polysaccharide. The partially
131
methylated alditol acetates (PMAAs) were prepared and analyzed by GC-MS (Wang, He & Huang,
132
2007).
133
2.6. Infrared spectroscopy (IR) and electrospray ionization mass spectrometry (ESI-MS)
M
an
us
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126
The IR spectra were measured as the previous report (Cong et al., 2014). Methylated
135
polysaccharide was measured by Nujol method. The ESI-MS experiment was performed by a Finnigan
136
LCQ/DECA mass spectrometer in the negative ion mode, with nitrogen used as the nebulizer gas, and
137
the capillary temperature was kept at 280 oC.
138
2.7. NMR analysis
Ac ce pt e
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134
139
The polysaccharides (30 mg) were deuterium-exchanged and dissolved in 0.5 ml of D2O (99.8%
140
D). 1H NMR, 13C NMR were recorded at 25 oC on a Bruker AVANCE III NMR spectrometer at 500
141
MHz with a cryogenic NMR probe. The HOD signal (4.85 ppm in 1H NMR) and acetone (31.50 ppm
142
in
143
were also performed to provide 1H-1H COSY, hetero-nuclear single quantum coherence (HSQC), and
144
hetero-nuclear multiple bond correlation (HMBC) spectra. Data were analyzed on MestReNova
145
V7.0.0-8331 software.
13
C NMR) signals were employed as the internal standards, respectively. 2D NMR experiments
8
Page 8 of 35
146
2.8. Desulfation of polysaccharide Briefly, the polysaccharide (100 mg) was dissolved in 10 mL of water and passed through a
148
column of 732# sulphonic exchange resin (H+) (1.6 cm × 50 cm). The eluate was neutralized with
149
pyridine and lyophilized. Other manipulation was referenced to previous report (Li et al., 2006). The
150
desulfated product was dialyzed against water (MWCO 500). The retentate was lyophilized to give the
151
desulfated polysaccharide, designated as FP08S2-DS.
152
2.9. Partial acid hydrolysis
us
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147
Three aliquots of FP08S2 (100 mg) were each dissolved in 0.05 M, 0.1 M and 2.0 M TFA and
154
heated at 100 oC for 1 h. After removing TFA, the residue was re-dissolved in water (5 mL), and
155
dialyzed against water (MWCO 3500 Da). The dialyzate and retentate from 0.05 M, 0.1 M, and 2.0 M
156
TFA treatment were designated as F005MDO and F005MDI, F01MDO and F01MDI, F2MDO and
157
F2MDI, respectively. By using a Bio-Gel P-2 column, F005MDO was resolved into two fractions
158
detected by phenol-sulfuric acid method, F005MDO1 and F005MDO2. In addition, F01MDO was
159
fractionated with an online RI detector into four fractions, F01MDO1, F01MDO2, F01MDO3, and
160
F01MDO4. Similarly, F2MDO was fractionated into two fractions, F2MDO1, and F2MDO2,
161
respectively.
162
2.10. Cell lines and culture conditions
Ac ce pt e
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M
an
153
163
Human microvascular endothelial cells (HMEC-1) were cultured in MCDB 131 medium (Gibco
164
BRL, U.S.A.) as described in previous reports (Qiu, Yang, Pei, Zhang & Ding, 2010). The cells were
165
incubated at 37 oC with 5% CO2 under a humidified atmosphere.
166
2.11. Tube formation assay
167
The effect of fucoidan FP08S2 on angiogenesis in vitro was examined by the HMEC-1 cells 9
Page 9 of 35
capillary-like tube formation assay. Briefly, A 96-well plate pre-coated with 50 μL Matrigel per well
169
was prepared and solidified at 37 oC for 30 min. HMEC-1 cells (5 × 104 cells/well) and samples
170
(FP08S2 and FP08S2-DS of 25, 50, 100, and 200 μg/mL, partial acid hydrolysis products of 500
171
μg/mL) were added into each well correspondingly and incubated for another 12 h. The enclosed
172
capillary networks of tube formation were recorded by an Olympus IX51 digital camera (Tokyo
173
Japan).
174
2.12. Wound healing assay of HMEC-1 cells
us
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168
HMEC-1 cells (5 × 105 cells/well) were cultured into a 6-well culture plate. After 24 h, the
176
confluent monolayer was scraped using a yellow pipette tip to create a wound gap and rinsed with
177
PBS thrice carefully. Then 0, 0.4 and 0.8 mg/mL of FP08S2 prepared in new MCDB 131 medium
178
were added into each well. After incubation for 12 h, three randomly selected regions around the cross
179
were photographed. The migration of cells through the wound area was then estimated quantitatively
180
using Image-Pro Plus software.
181
2.13. Cell proliferation assay of HMEC-1 cells
Ac ce pt e
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M
an
175
182
A MTT assay was determined to evaluate the cell proliferation of HMEC-1 cells. Briefly,
183
HMEC-1 cells (5 × 103 cells/well) were cultured in 96-well plates with MCDB 131 medium for 24 h.
184
The medium was discarded and exchanged by 0, 25, 50, 100, 200, 400, 800 μg/mL of FP08S2
185
dissolved in MCDB131 medium. After 72 h, 10 μL of 5 mg/mL MTT was added into each well and
186
kept for 4 h at 37 oC. The medium was discarded and the formazan crystal was dissolved in 150 μL of
187
DMSO. The solution was vibrated for 20 min and the absorption at 490 nm was measured to calculate
188
the cell viability ratio of HMEC-1 cells.
189 10
Page 10 of 35
190
3. Results and Discussion
191
3.1. Structure elucidation of FP08S2 The crude polysaccharide (SFbWP) was extracted from Sargassum fusiforme with boiling water,
193
and then it was treated by 2% CaCl2 precipitation to remove alginate fraction. The fraction obtained
194
from the supernatant (SFbWP-FP) was fractionated by anion-exchange chromatography on a
195
DEAE-cellulose 32 column (Fig. S.1.A). FP08 was obtained as the major fraction from 0.8 M NaCl
196
eluate, which (1.9 g) was further purified by gel filtration chromatography on a Sephacryl S-300 HR
197
column to afford a homogeneous polysaccharide FP08S2 (996.5 mg) (Fig. S. 1. B).
us
cr
ip t
192
The neutral sugar and uronic acid content of FP08S2 were determined to be 16.8% and 34.6%,
199
respectively, and the sulfate content (as SO42-) was calculated to be 20.8%. The specific rotation [α]D
200
was -56.2o (c 0.5, H2O). HPGPC analysis (Fig. S.2.A) indicated the polysaccharide was homogeneous
201
for only one symmetrical peak appeared except for the salt peak. The apparent molecular weight was
202
estimated to be 47.5 kDa by reference to the pullulan standards. The monosaccharide composition
203
analysis showed that FP08S2 is composed of fucose, xylose, mannose, galactose and glucuronic acid,
204
in a ratio of 36.6: 18.3: 7.0: 19.1: 19.1 (Table 1). The content of fucose was similar to that reported in
205
literature (Li et al., 2006), but the contents of xylose, mannose, and glucuronic acid were intensively
206
different from Li’s report. In addition, rhamnose and arabinose were not detected. In addition, the
207
absolute configurations of monosaccharide constituents were determined to be L-fucose, D-xylose,
208
D-mannose,
209
D-configuration
210 211
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an
198
and
D-galactose.
Absolute configuration of glucuronic acid was demonstrated to be
from the reduction product of FP08S2 (FP08S2-R). Data were shown in Fig. S.3. Table 1.
IR spectrum of FP08S2 was shown in Fig. S.2.B. The strong absorption at 1259.3 cm-1 could be 11
Page 11 of 35
attributed to asymmetric O=S=O stretching vibration of sulfate esters. In addition, an intense
213
absorption at 1737.6 cm-1 indicated the presence of uronic acid. In the fingerprint region (750-960
214
cm-1), the distinct absorption at 842.7 cm-1 but no trace at 824 cm-1 revealed that the sulfate esters were
215
mainly substituted at the axial C-4 of fucopyranose residues (Bilan et al., 2002). After desulfated, the
216
absorption at 1259.3 cm-1 disappeared in the IR spectrum of FP08S2-DS (Fig. S.2.B). Elemental
217
analysis showed that the sulfur content of FP08S2-DS was reduced to 0.9%, indicating 87% of the
218
sulfate groups had been removed. As shown in Table 1, FP08S2-DS contained fucose, xylose,
219
mannose, galactose and glucuronic acid, in a ratio of 31.8: 17.2: 8.2: 21.8: 21.1.
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212
The glycosyl linkages of FP08S2 were determined by methylation analyses, along with
221
FP08S2-DS, as shown in Table 2. For the native polysaccharide, the fucose residues were found to be
222
present in complicated glycosyl linkages, including terminal, 1, 3-, 1, 4-, 1, 2, 3-, and 1, 3, 4-linked
223
Fucp, largely due to sulfate substitution at different positions. According to previous reports (Bilan et
224
al., 2004; Chandia et al., 2008; Ale et al., 2011; Chen et al., 2012; Yu et al., 2013), most fucoidans
225
contain 1, 3-linked Fucp residues with sulfate attached mainly to C-4 or C-2. Our results indicated that
226
the 1, 3-linked 2-sulfated fucopyranosyl residue accounted for the major linkage type for fucose. After
227
desulfation, the proportion of 1, 3-linked fucose residues increased while the 1, 3, 4-linked fucose
228
decreased distinctly, indicating that the sulfate groups were also attached to C-4 of 1, 3-linked Fucp. In
229
addition, some of the sulfate groups were probably substituted at C-4 of non-reducing terminal fucose
230
residues. After desulfation, the content of terminal xylose increased in the expense of 1, 4-linked
231
xylose, indicating that the terminal xylose residues were partially sulfated at C-4. 1, 2, 4, 6-linked
232
mannose units disappeared after desulfation, and the content of 1, 2, 3-linked mannose decreased, but
233
the content of 1, 2-linked and 1, 2, 6-linked mannose increased slightly. These indicated that some
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Page 12 of 35
sulfate groups were linked to C-3/4/6 of 1, 2-linked mannose. After desulfation, 1, 3, 6-linked and 1, 2,
235
4, 6-linked galactose disappeared, while the content of 1, 6- and 1, 2, 4-linked galactose increased
236
slightly, indicating that the sulfate were probably attached to C-6 of 1, 2, 4-linked galactose, C-3 of 1,
237
6-linked galactose and C-2 and/or C-4 of 1, 6-linked galactose. In addition, 1, 3, 4-linked galactose
238
disappeared, replaced by 1, 3-linked galactose, indicating that sulfate groups were attached to C-4 of 1,
239
3-linked Gal. Linkage analysis of carboxyl-reduced, desulfated product showed terminal and 1,
240
4-linked glucose, indicating that glucuronic acid in FP08S2 was present as terminal and 1, 4-linked. Table 2.
241
3.2. Structural analysis of the partial acid hydrolysis-derived polysaccharides
an
242
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234
Like other fucoidans from brown seaweeds, FP08S2 displayed heavily overlapped resonances in
244
its 1H and 13C NMR spectra (Fig. S.4.A and D), mainly due to the complex glycosyl composition and
245
diverse sulfation substitution.
d
M
243
To further elucidate the structure, FP08S2 was subjected to partial acid hydrolysis to afford
247
oligosaccharides from branches, partially hydrolyzed with 0.05 M, 0.1 M and 2 M TFA, respectively.
248
Sugar composition analysis of partial acid hydrolysis products of dialyzate and retentate was
249
summarized in Table 1, and ESI-MS analysis of dialyzate fractions was shown in Fig. 2.
250
Ac ce pt e
246
Fig. 2.
251
F005MDO1 had a higher content of α-L-Fucp, but other residues like xylose, galactose and
252
glucuronic acid were also in substantial amounts. ESI-MS analyses (Fig. 2) revealed the most
253
intensive ion at m/z 535.2 was assigned to monosulfated fucotrioligosaccharide [Fuc3SO3]-. The
254
fucoxylooligosaccharide was identified by [Fuc3XylSO3]- ion at m/z 667.2 and [Fuc4XylSO3]- ion at
255
m/z 813.1. The ion at m/z 843.0 was assigned to a hexose-containing oligosaccharide [Fuc4GalSO3]-. In 13
Page 13 of 35
addition, the sulfated disaccharide [FucGlcASO3]- ion was also observed at m/z 419.2. In comparison,
257
F005MDO2 was only composed of fucose and xylose. ESI-MS analysis revealed that the signals of
258
monosulfated fucose [FucSO3]- at m/z 243.1 and monosulfated xylofucooligosaccharide [Fuc2XylSO3]-
259
ion at m/z 520.7 were observed. In addition, Both F01MDO1 and F01MDO2 contained fucose and
260
glucuronic acid, in a molar ratio of 1: 1. ESI-MS revealed intensive [FucGlcASO3]- ion at m/z 419.4
261
for F01DMO1 and at m/z 419.5 for F01MDO2, respectively. In addition, [Fuc2GlcASO3]- ion at m/z
262
565.4 and [Fuc2GlcA2SO3]- ion at m/z 741.4 were also observed from F01MDO1. However, F01MDO3
263
contained fucose and galactose, in a molar ratio of 3:2. F01MDO4 had a higher percentage of fucose,
264
as well as substantial content of xylose and galactose. For F01MDO3, the three negative ions at m/z
265
243.3, 389.4 and 405.3 corresponded to [FucSO3]-, [Fuc2SO3]- and [FucGalSO3]-. For F01MDO4, the
266
[Fuc2SO3]- ion at m/z 388.9 confirmed the presence of monosulfated fuco-disaccharide. Meanwhile,
267
monosulfated galactose was also observed from [GalSO3]- ion at m/z 259.3. The ion at m/z 442.5
268
corresponded to trisaccharide [GalXyl2]-. The two oligosaccharide fractions of FP08S2 after treated
269
with 2M TFA partial hydrolysis both contain fucose, xylose and galactose. For F2MDO1, the main
270
component was a monosulfated galacto-tetrasaccharide [Gal4SO3]- at m/z 744.9. The ion at m/z 586.5
271
was attributed to disulfated xylofucotrisacchride [FucXyl2(SO3)2]-. For F2MDO2, the signal at m/z
272
243.1 of highest intensity corresponded to monosulfated fucose. Other ions at m/z 405.1, 421.2, and
273
828.7 were attributed to the [FucGalSO3]-, [Gal2SO3]-, and [Fuc3XylGalSO3]-, respectively.
Ac ce pt e
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256
274
With the increasing TFA concentration, fucose, galactose, and xylose were identified in the
275
released oligosaccharides, indicating that they were present mainly in branches. However, mannose
276
and glucuronic acid remained in the degraded polysaccharide (Table 1), indicating that they were
277
present in the core structure. 14
Page 14 of 35
Compared to FP08S2 and the retentate F01MDI, F2MDI presented a well-resolved 1H NMR
279
spectrum (Fig. S.4.B and C), in which the anomeric resonances corresponding to Fuc, Xyl, and Gal
280
largely disappeared, indicating that they had been mostly removed by acid treatment. It was reasonable
281
to suggest that F2MDI represented the core structure of FP08S2. The apparent molecular weight of
282
F2MDI was estimated to be 12.4 kDa by HPGPC. Sulfate content of F2MDI is calculated to be 7.5%
283
by elemental analysis. The carboxyl-reduced F2MDI-R was found to contain Fuc, Man, Gal and Glc,
284
in a mole ratio of 5.6: 32.2: 9.3: 53.0, as shown in Table 1. Methylation analysis (Table 2) showed that
285
F2MDI-R consisted mainly of 1, 2- and 1, 2, 6-linked Manp, and 1, 4-linked GlcpA. Other minor
286
residues involved 1, 3-linked Fucp, terminal Galp, and terminal GlcpA. According to methylation
287
analysis, it could be inferred that F2MDI possessed a backbone composed of 1, 2-linked Manp and 1,
288
4-linked GlcpA. 1, 3-linked Fucp, terminal Galp, and terminal GlcpA residues were probably present
289
as the component residues in branches.
d
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278
In the 1H NMR (Fig. 3.A), the two intense anomeric signals at δ 5.40 and 4.56 ppm could be
291
assigned to α-Manp and β-GlcpA, respectively. The minor signals at δ 1.28-1.31 ppm were ascribed to
292
H6 of Fuc. Other signals were assigned according to the 2D homonuclear 1H/1H COSY and shown in
293
Table 3. The 13C NMR spectrum (Fig. 3.B) of F2MDI showed four intensive signals in the anomeric
294
region at 99.95-102.99 ppm. The resonances at 102.78 and 102.99 ppm were ascribed to 1, 4-linked
295
and terminal β-D-GlcpA, while those at 99.95 and 100.39 ppm were assigned to 1, 2- and 1, 2, 6-linked
296
α-D-Manp. The two resonances at 173.58 and 173.92 ppm were assigned to terminal and 1, 4-linked
297
GlcpA, respectively. The two methylene signals at δ 67.65 and 61.08 ppm in DEPT spectrum could be
298
assigned to C6 of 1, 2, 6-, and 1, 2-linked Manp residues, respectively. The weak resonance at 93.3
299
ppm was assigned to C1 of 1, 3-linked α-L-Fucp, and the resonance at 17.73 ppm was apparently from
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290
15
Page 15 of 35
300
C6 of Fuc. In addition, the weak resonance at 103.5 ppm was ascribed to anomeric carbon of terminal
301
β-D-Galp. Table 3.
303
Further spectral assignment was aided by COSY, HSQC, and HMBC spectra (Fig. 3), and the
304
detailed assignments for NMR data of F2MDI were concluded in Table 3 (Bilan et al., 2010; Li et al.,
305
2006). HMBC spectrum (Fig. 3.E) revealed the sequence of the glycosyl residues in F2MDI. The
306
correlation at 3.85/99.95, 100.39 ppm indicated that the H4 of 1, 4-linked β-GlcpA was correlated with
307
C1 of 1, 2-linked and 1, 2, 6-linked α-Manp residues. On the other hand, C1 of 1, 4-linked β-GlcpA
308
was correlated with H2 of 1, 2- and 1, 2, 6-linked α-Manp. These transglycosidic correlations between
309
1, 2-linked α-Manp and 1, 4-linked β-GlcpA suggested that they were probably connected alternately
310
in F2MDI as the backbone, similar as that in previous report (Li et al., 2006). Li et al (Li et al., 2006)
311
reported a core structure of fucoidan isolated from Sargassum fusiforme composed of alternating 1,
312
2-linked α-Manp and 1, 4-linked β-GlcpA units with minor 1, 4-linked β-Galp, branched at C-3 of 1,
313
2-linked Manp. Wang et al (Wang et al., 2012) confirmed the alternate disaccharide repeat of
314
→4)-β-GlcA(1→2)-α-Man(1→. However, our study indicated some different features in backbone of
315
FP08S2, as indicated by NMR spectra, especially for the signals of C6 of 1, 2, 6-linked α-Manp as
316
well as galactose residues. According to the previous NMR analyses of a sulfated trisaccharide
317
obtained from fucoglucuronomannan(Sakai et al., 2003), the resonance at 4.19/4.35ppm was ascribed
318
to H-6 of 6-O-sulfated D-Manp. And the chemical shift of C-6 of the 6-O-sulfated D-Manp was at
319
67.65ppm from the HSQC spectrum (Figure 3.D). In addition, the sulfate content of F2MDI was
320
determined up to 7.5% by elemental analysis. The sulfation group was substituted at C-6 of 1, 2,
321
6-Man from NMR analyses. The ratio of 1, 2, 6-Man was up to 15.7% from methylation results.
Ac ce pt e
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302
16
Page 16 of 35
Therefore, the sulfated content was calculated to be 5.2% from methylation analyses, consistent with
323
the result from elemental analysis. Taken together, the substitution at C-6 of 1, 2, 6-Man was not
324
derived from the substitution of the other neighbouring glycosyl residues, but from the substitution of
325
sulfate group. In addition, C1 of terminal β-Galp in the branches correlated with H3 of 1, 3-linked
326
α-Fucp.
cr
Fig. 3.
327
3.3. Anti-angiogenic activity of FP08S2
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322
As shown in Fig. 4.A and B, FP08S2 at 25 μg/mL to 200 μg/mL inhibited the tube formation in a
330
dose-dependent manner. A complete disruption on network formation of HMEC-1 cells could be
331
observed at 200 μg/mL. FP08S2 exhibited a measurable decline in tube formation with increasing
332
concentration of FP08S2 (Fig. 4.B). In contrast, for the desulfated derivative, FP08S2-DS, no
333
significant inhibition was observed even at 200 μg/mL (Fig. 4.A and B). The results indicated that
334
sulfate group played an important role in the anti-angiogenic activity. In order to understand to what
335
degree of the oligosaccharide branches and the backbone of FP08S2 contribute to the inhibitory effect
336
on tube formation, the partial acid hydrolysis products of 0.05 M, 0.1 M, and 2 M TFA treatment were
337
subjected to tube formation assay at 0.5 mg/mL, respectively. The results were shown in Fig. 4.C and
338
D. Compared with the control, the three retentate fractions, F005MDI, F01MDI and F2MDI exhibited
339
a significant inhibition on tube formation. Intriguingly, the disruption of enclosed capillary network
340
was decreased for the degraded polysaccharide derived from high TFA concentration. It indicated that
341
the branches might also have a significant effect on the anti-angiogenic activity of FP08S2. In addition,
342
F005MDO1 and F01MDO1 exhibited a significant inhibition on tube formation. Given the
343
compositions of these two fractions, it suggested that not only the sulfate group but also glucuronic
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17
Page 17 of 35
344
acid influenced the tube formation significantly. Fig. 4.
345
In wound healing assay, as shown in Fig. 5.A and B, the migration of HMEC-1 cells was
347
substantially impaired with FP08S2 treatment compared with the control. In untreated primary
348
HMEC-1 cells, 64.7% (± 1.9) of the wound was closed after 12 h. In contrast, treated with fucoidan
349
FP08S2 (0.4 mg/mL), only 38.1% (± 2.7) of the wound was enclosed. With treatment of FP08S2 (0.8
350
mg/mL), only 16.6% (± 2.7) of the wound was closed. Thus FP08S2 significantly inhibited the
351
migration of HMEC-1 cells in a dose-dependent manner (Fig. 5.B).
us
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346
To investigate whether the inhibitory effects on capillary network and migration were the result
353
of inhibition of HMEC-1 cells proliferation, we analyzed the viability of HMEC-1 cells treated with
354
FP08S2 (25 - 800 μg/mL) in vitro. Fig. 5.C showed that native fucoidan FP08S2 showed no significant
355
cytotoxicity on HMEC-1 cells.
357 358
M
d Ac ce pt e
356
an
352
Fig. 5.
4. Discussion
359
Fucoidans are sulfated homo- and hetero-polysaccharides, mainly composed of α-L-fucopyranose
360
residues, which may be partially sulfated and/or acetylated (Shevchenko et al., 2015). In the present
361
study, a novel fucoidan FP08S2 was purified and characterized from Sargassum fusiforme. FP08S2
362
was shown to have a glucuronomannan backbone, composed of alternating 1, 2-linked α-D-Manp and
363
1, 4-linked β-D-GlcpA. The outer branches are composed mainly of 1, 3-linked α-L-Fucp residues
364
highly sulfated at O-2 and O-4, or as fucogalactan and fucoglucuronan, and 4-O partially sulfated
365
terminal Xylp. 18
Page 18 of 35
Angiogenesis involves the progression of proliferation, differentiation, and migration of mature
367
endothelial cells and is regulated by various endothelium stimulating angiogenic factors such as VEGF
368
and FGF (Kim, Park, Kang, Kim & Lee, 2014). Heparin and heparan sulfate are highly sulfated
369
polysaccharides. It has been reported that N-sulfated glucosamine and 2-O-sulfated iduronate residues
370
are necessary for the angiogenic factors binding to HSPG. This suggested sulfation of polysaccharides
371
was required for the anti-angiogenesis effect. Multiple structural factors may influence the biological
372
activities of fucoidans, including monosaccharide composition, sulfation, glycosyl linkages, branches,
373
and molecular weight. Matsubara et al. (Matsubara et al., 2005) reported the mild acid hydrolysis
374
product of fucoidan, containing 33.2% of sulfate content with a molecular weight of 30 kDa, exhibited
375
inhibition of angiogenesis in vitro. In contrast, the other product with a molecular weight of 15-20 kDa
376
and a low degree of sulfation (8.2%) did not inhibit HUVEC tube formation. In our study, the native
377
fucoidan of 47.5 kDa, with a degree of sulfation (20.8%), could significantly disrupt the capillary
378
network. The partial acid hydrolysis products demonstrated a weak inhibitory effect on tube formation
379
compared to FP08S2. In addition, the derivative F2MDI, with a molecular weight of 12.4 kDa and
380
7.5% of sulfate content, exhibited the lowest inhibition among the three degraded polysaccharides,
381
suggesting that molecular weight and sulfation played a key role in the anti-angiogenic activity of
382
fucoidan. In addition, we observed that some oligosaccharide fractions derived from branches also
383
demonstrated a significant inhibitory effect on tube formation. Based on the structural analysis of these
384
oligosaccharides, it could be suggested that the high degree of sulfation and/or uronic acid residues in
385
branches contributed to the anti-angiogenic activity of FP08S2. Moreover, previous studies have
386
reported that increased negative charge resulting from sulfation or oversulfation might enhance the
387
formation of fucoidan-protein complexes (Ale et al., 2011; Koyanaqi, Taniqawa, Nakagawa, Soeda, &
Ac ce pt e
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19
Page 19 of 35
Shimeno, 2003). It had been studied that the spatial orientation of the negative charges on the fucoidan
389
exhibited a significant effect on the binding potency of fucoidan to VEGF165. The interaction of
390
fucoidan with higher sulfated content with VEGF165 resulted in the formation of highly stable
391
complexes. Therefore, the complexes disrupted the binding of VEGF165 to VEGF receptor-2
392
(VEGFR-2) (Koyanaqi et al., 2003). It indicated that the potent biological effect was dependent on
393
sulfation property, which was consistent with our result.
cr
ip t
388
In conclusion, a fucoidan FP08S2 isolated from Sargassum fusiforme was shown to have a
395
glucuronomannan backbone, composed of alternating 1, 2-linked α-D-Manp and 1, 4-linked β-D-GlcpA.
396
The outer branches were composed mainly of 1, 3-linked α-L-Fucp residues highly sulfated at C-2 and
397
C-4, or as fucogalactan and fucoglucuronan, and C-4 partially sulfated terminal Xylp. FP08S2
398
demonstrated a significant inhibition on tube formation and migration of HMEC-1 cells
399
dose-dependently.
401
an
M
d Ac ce pt e
400
us
394
Acknowledgements
402
This research was supported by National Natural Science Foundation of China (NSFC)
403
(31230022), New Drug Creation and Manufacturing Program (2012ZX09301001-003), National
404
Science Fund for Distinguished Young Scholars (81125025) in China.
405 406
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and antioxidant capacity of fucoidan from selected Malaysian seaweeds. Food Hydrocolloids, 42,
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Mao, W., Li, B., Gu, Q., Fang, Y., & Xing, H. (2004). Preliminary studies on the chemical
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an
M
purified from
Aconitum coreanum alleviates
d
type polysaccharide
Ac ce pt e
A RG-II
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cr
ip t
454
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Paskaleva, E. E., Lin, X., Li, W., Cotter, R., Klein, M. T., Roberge, E., Yu, E. K., Clark, B., Veille, J. C.,
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Liu, Y., Lee, D. Y. W., & Canki, M. (2006). Inhibition of highly productive HIV-1 infection in T cells,
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primary human macrophages, microglia, and astrocytes by Sargassum fusiforme. AIDS Research and
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fucoglucuronomannan (SFGM) lyase digests brown algal SFGM into trisaccharides. Marine
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Senthilkumar, K., Manivasagan, P., Venkatesan, J., & Kim, S. K. (2013). Brown seaweed fucoidan:
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Supporting information
521
(1) Figure S.1: The elution profile of SFbWP-FP on DEAE-cellulose 32 column, as determined by
522
phenol sulfuric acid method (A). The elution profile of FP08 on Sephacryl S-300 HR column with RI
523
detection (B).
524
(2) Figure S.2: HPGPC profile (A) of FP08S2 and IR spectra (B) for FP08S2 and FP08S2-DS.
525
(3) Figure S.3: Absolute configuration of FP08S2 and its reduction product FP08S2-R comparing to D-
526
and L- configurations of all monosaccharide standards.
527
(4) Figure S.4: 1H NMR spectra of FP08S2 (A), F01MDI (B) and F2MDI (C). 13C NMR spectrum of
528
FP08S2 (D).
an
us
cr
ip t
520
M
529
Figure captions
531
Figure 1. The scheme of extraction, isolation and purification of fucoidan, FP08S2, from Sargassum
532
fusiforme.
533
Figure 2. The ESI-MS fragment pattern of acid degraded oligosaccharide fractions in negative ion
534
mode.
535
Figure 3. 1D and 2D NMR spectra of F2MDI. 1H-NMR (A); 13C-NMR (B); 1H-1H COSY (C); HSQC
536
(D); HMBC(E); Legend for the residues in F2MDI. a: 1, 2-linked α-D-Manp; b: 1, 2, 6-linked
537
α-D-Manp; c: 1, 4-linked β-D-GlcpA; d: terminal β-D-GlcpA; e: 1, 3-linked α-L-Fucp; f: terminal
538
β-D-Galp.
539
Figure 4. Effect of FP08S2 and its derivatives on tube formation of HMEC-1 cells. (A) HMEC-1 cells
540
treated with FP08S2 and its desulfated derivative FP08S2-DS at different concentrations (25, 50, 100,
541
and 200 μg/mL). (B) Quantitative measurement of tube numbers. (C) Effect on anti-angiogenesis
Ac ce pt e
d
530
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activity of oligosaccharide fractions and retentate fractions after treatment of 0.05 M, 0.1 M, and 2.0
543
M TFA. Concentration of all samples is 500 μg/mL. (D) Quantitative measurement of tube numbers.
544
The values represent mean±S.D. * P ＜ 0.05; ** P ＜ 0.01, as determined by unpaired t-test.
545
Figure 5. (A) The effect of FP08S2 (0.4 mg/mL and 0.8 mg/mL) on migration of HMEC-1 cells after
546
incubating for 12 h in a wound healing assay. (B) Quantification of FP08S2 on HMEC-1 cells
547
migration in the wound healing assay. (C) The cell viability of HMEC-1 cells after treatment with 25,
548
50, 100, 200, 400, 800 μg/mL of FP08S2 for 72 h. The values represent mean±S.D. * P ＜ 0.05; **
549
P ＜ 0.01, as determined by unpaired t-test.
us
cr
ip t
542
Ac ce pt e
d
M
an
550
27
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550 551
Tables
552
Table 1. Molecular weight and sugar composition of FP08S2 and partially acid hydrolysis products Sugar compositions (mol % of all sugars) 4
Molecular weight (/10 Da) Man 7.0
4.75
36.6
18.3
FP08S2-DS
3.07
31.8
17.2
F005MDI
3.58
38.1
15.3
F01MDI
1.95
18.3
F2MDI
1.24
an 9.4
88.3
11.7
19.1
19.1
21.8
21.1
19.1
27.5
ND
18.9
48.2
ND
32.2
9.3
53.0
6.1
3.2
53.0
47.0
F01MDO2
48.6
51.4
F01MDO3
59.6
F01MDO4
57.8
28.0
14.2
F2MDO1
43.7
23.1
33.2
F2MDO2
60.4
24.7
14.9
Ac ce pt e
F01MDO1
81.3
M
F005MDO2
5.6
d
F005MDO1
14.6
GlcA
8.2
us
FP08S2
Gal
ip t
Xyl
cr
Fuc
40.4
553 554 555
28
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Table 2. The results for linkage analyses for FP08S2, FP08S2-DS and F2MDI-R Molar ratios (mol %) FP08S2
FP08S2-DS
T-Xyl
5.8
11.8
1, 4-Xylp
16.5
6.5
T-Fucp
5.6
9.5
1, 3-Fucp
8.0
11.1
1, 4-Fucp
4.9
3.0
1, 3, 4-Fucp
10.1
2.4
1, 2, 3-Fucp
17.2
5.0
1, 2-Manp
0.8
3.7
1, 3-Manp
0.8
0.3
1, 2, 6-Manp
0.5
0.9
1, 2, 3-Manp
5.1
3.9
1, 2, 4, 6-Manp
2.9
T-Galp
3.4
cr us
an
15.7
6.1
0.7
1, 4-Galp
0.8
1, 6-Galp
3.8
1, 2, 4-Galp
1.0
1, 3, 4-Galp
1.0
1, 3, 6-Galp
1.7
1, 2, 4, 6-Galp
10.1
1.3
Ac ce pt e
557
17.7
0.7
1, 3-Galp
556
6.2
M
1, 2-Galp
6.5
F2MDI-R
ip t
Linkages
d
555
7.6 1.8
T-Glcp
2.5
8.6
1, 4-Glcp
20.9
43.3
29
Page 29 of 35
557 558
Table 3. 1H and 13C NMR spectral assignments for F2MDI Chemical shifts (ppm)
D
β- D-GlcpA-(1→
E
→3)-α-L-Fucp-(1→
F
β- D-Galp-(1→
H
5.41
4.19
3.82
3.79
3.73
3.79/3.85
C
99.95
78.77 70.56
67.1
74.32
61.08
H
5.39
4.19
3.82
3.77
4.19/4.35
C H C H C
3.82
ip t
→4)-β- D-GlcpA-(1→
6
100.39 78.86 70.64 67.33 70.62 4.53
3.45
3.73
3.85
4.06
102.78 73.67 76.79 78.26 75.23 4.59
3.45
3.73
3.61
173.92
3.99
102.99 73.61 76.72 72.57 75.94 4.06
67.65
cr
C
5
us
→2, 6)-α- D-Manp-(1→
4
H
5.3
4.05
C
93.3
70.03 79.46
H
4.89
3.63
C
103.5
73.48 74.28 70.63 76.67
173.58
4.09
4.36
1.28
69.5
68.15
17.73
4.04
3.71
3.77/3.87
M
B
3
3.77
61.69
d
→2)-α-D-Manp-(1→
2
Ac ce pt e
559
A
1
an
Residues
30
Page 30 of 35
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Figure(1)
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Figure(2)
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ce
pt
ed
M
an
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Figure(3)
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pt
ed
M
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Figure(4)
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Figure(5)
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