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Purification, structural analysis and mechanism of murine macrophage cell activation by sulfated polysaccharides from Cystoseira indica
Accepted Manuscript Title: Purification, structural analysis and mechanism of murine macrophage cell activation by sulfated polysaccharides from Cystoseira indica Authors: Saman Bahramzadeh, Mehdi Tabarsa, SangGuan You, Changsheng Li, Seraj Bita PII: DOI: Reference:
S0144-8617(18)31198-6 https://doi.org/10.1016/j.carbpol.2018.10.022 CARP 14154
To appear in: Received date: Revised date: Accepted date:
9-8-2018 27-9-2018 8-10-2018
Please cite this article as: Bahramzadeh S, Tabarsa M, You S, Li C, Bita S, Purification, structural analysis and mechanism of murine macrophage cell activation by sulfated polysaccharides from Cystoseira indica, Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.10.022 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.
Purification, structural analysis and mechanism of murine macrophage cell activation by sulfated polysaccharides from Cystoseira indica
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Running title: Immunostimulatory of polysaccharides from Cystoseira indica
Department of Seafood Processing, Faculty of Marine Sciences, Tarbiat Modares University,
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Saman Bahramzadeha, Mehdi Tabarsaa*, SangGuan Youb*, Changsheng Lib, Seraj Bitac
Department of Marine Food Science and Technology, Gangneung-Wonju National University,
Gangneung, Gangwon 25457, Korea
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Chabahar, Iran
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Department of Fisheries, Faculty of Marine Sciences, Chabahar Maritime University,
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c
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b
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P.O. Box 46414-356, Noor, Iran
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*Corresponding
author. Tel.: +981144553101; Fax: +981144550906 (M. Tabarsa); Tel.: +82
33 640 2853; Fax.: +82 33 640 2340 (S.G. You)
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E-mail addresses: [email protected] (M. Tabarsa); [email protected] (S.G. You)
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Highlights
Sulfated polysaccharides were isolated from Cystoseira indica.
Polysaccharides exerted high RAW264.7 macrophage stimulating activity.
RAW264.7 cells were activated through NF-κB and MAPK signaling pathways.
TLR4 was the major cell surface receptor interacting polysaccharides with RAW264.7
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cells.
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The main backbone of the polysaccharides was consisted of (1→3)-fucopyranosyl units.
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Abstract
Sulfated polysaccharides were isolated and purified from the water extract of Cystoseira
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indica using DEAE Sepharose Fast Flow column to evaluate their structure and macrophage
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stimulating capacity. Crude and fractionated polysaccharides, CIF1 and CIF2, were mostly
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composed of neutral sugars (73.1%-78.6%) with relatively lower amounts of acidic sugars (1.3%-9.0%) and sulfate esters (6.9%-9.7%). The polymer chains of polysaccharides were
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mainly built of different levels of glucose (2.1%-30.8%), fucose (17.2%-24.4%), mannose (17.8%-20.6%) and galactose (16.7%-17.3%). The weight average molecular weight (Mw) of
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polysaccharides varied between 573.1 × 103 g/mol to 1146.6 × 103 g/mol. The CIF2 polysaccharide, as the most immunostimulating polysaccharide, remarkably induced the release of nitric oxide and inflammatory cytokines including TNF-α, IL-1β, IL-6 and IL-10 from RAW264.7 murine macrophage cells through NF-κB and PAMKs transduction signaling 2
pathways via cell surface TLR4. The interconnections of sugars in CIF2 polysaccharide were complex with (1→3)-fucopyranose, (1→2,3,4)-glucopyranose, (→1)-galactopyranose, (→1)xylopyranose, (1→2)-rhamnopyranose and (1→2,3)-mannopyranose units being the most
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predominant residues.
Keywords: Cystoseira indica, Polysaccharide, Structure, Immunostimulatory, NF-κB and
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MAPKs signaling pathways
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1. Introduction
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Polysaccharides are a class of naturally available macromolecules which ubiquitously exist
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in plants, microorganisms, algae and animals. Polysaccharides are known to be necessary for
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cell-cell communication, cell adhesion and molecular recognition in the biological systems (Dwek, 1996). In the past decade, a growing number of studies have been paying great amount
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of attention towards polysaccharide isolation from natural resources such as plants, mushrooms,
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bacteria and seaweeds, mostly owed to their versatile pharmacological activities (Yu, Shen, Song, & Xie, 2017). Antitumor, anti-inflammatory, immunostimulatory and anticoagulant
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activities could be listed as the top widely explored physiological properties of polysaccharides (Yu et al., 2017; Borazjani, Tabarsa, You, & Rezaei, 2018). Among these, immunostimulatory polysaccharides are one of the relatively new class of biologically active compounds that have been attracted a tremendous interest for their promising 3
roles in boosting body’s natural resistance against viral and bacterial infections or assisting with the treatment of diseases having suppressed immune system conditions such as cancer and AIDS (Tripathi, Tripathi, Kashyap, & Singh, 2007). By far, various types of glucans (Tabarsa, Shin,
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Lee, Surayot, Park, & You, 2015), mannans (Simões, Nunes, Domingues, & Coimbra, 2012), galactans (Stephanie, Eric, Sophie, Christian, &Yu, 2010) and fucoidans (Borazjani et al., 2018) have been suggested as immunostimulant agents with capacity to interact directly or indirectly with the immune system, triggering a series of cellular and molecular events.
Fucoidans are a group of structurally diverse sulfated polysaccharides from brown seaweeds
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that could be potential immunostimulating polysaccharides for further industrial applications
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(Vo, & Kim, 2013). Regardless of their structural complexities, all fucoidans share a common
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chemical characteristic which is the presence of sulfate-bearing L-fucose units in their polymer
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chains. In fact, fucoidans are heteropolysaccharides in which different levels of mannose,
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galactose, glucose, xylose, rhamnose and arabinose sugars are involved in their structure, in
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addition to fucose units (Borazjani et al., 2018). Although, the interconnections of fucoidan units differ among various brown seaweeds, they are mostly formed of a main backbone consisted of
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either (1→3)-α-L-fucopyranose or alternating (1→3)- and (1→4)-α-L-fucopyranose residues
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(Cumashi, Ushakova, Preobrazhenskaya, D’lncecco, Piccoli, & Totani, 2007). Altogether, there seems to be a consensus that the structure of fucoidans are immensely
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complex and needs an in-depth consideration with respect to extraction, purification and the following chemical analysis prior to any industrial applications (Wu, Sun, Su, Yu, Yu, & Zhang, 2016). The complexity in chemical structure along with the heterogeneity in molecular size greatly contribute to the multifunctionality of fucoidan polysaccharides, specifically to their 4
immunoenhancing activities. Nonetheless, our understanding on the exact activation mechanism and relating structural feature that evokes the target immune cells are incomplete (Zhang, Qi, Guo, Zhou, & Zhang, 2016).
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Therefore, in the present study, we examined whether fudoidan from Cystosira indica has any capacity to activate macrophage cells and if the results are positive what underlying mechanisms play the major roles. To reveal the potential structural feature that contributed to the macrophage stimulation capacity of the fucoidan, a highly purified polysaccharide obtained after DEAE Sepharose Fast Flow chromatography was subjected to extensive molecular and chemical
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characterizations.
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2.1. Samples and reagents
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2. Materials and methods
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Samples of C. indica were collected in December 2016 from the coastline of Chabahar,
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Iran. The raw material was soaked in fresh water, washed and air dried at 60 °C in the oven. The dried sample material was ground using a blender, passed through a 0.5-mm sieve and kept in
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seal sample bags at -20 °C. All chemicals and reagents used in this study were analytical grade.
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2.2. Extraction of polysaccharides
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The extraction of polysaccharides from C. indica was performed using the method
described previously (Borazjani et al., 2018). Briefly, 20 g of dried and milled seaweeds were soaked into 200 mL of 80% ethanol and stirred overnight at room temperature to eliminate lipids, pigments and low molecular weight compounds. The residue was rinsed with acetone and air dried at room temperature in a fume hood. The defatted and depigmented powder (20 g) was 5
suspended in 400 mL of distilled water and the extraction performed at 65 °C under stirring for 2 h. The resultant supernatants were kept after centrifugation at 10 °C and 6080 g for 10 min. Then, ClCa2 (1%; w/w) was added into the concentrate and kept at 4 °C overnight to precipitate
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alginic acid. The precipitation of polysaccharides in the final alginate-free solution was carried out with the addition of ethanol (99%) to reach a final ethanol concentration of 70%. The crude polysaccharide were then washed and dehydrated with ethanol (99%), acetone, and finally dried
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at room temperature.
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2.3. Fractionation of polysaccharides
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Crude polysaccharides were fractionated on a DEAE Sepharose Fast Flow column (17-
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0709-01; GE Healthcare Bio-Science AB, Uppsala, Sweden). The crude polysaccharide (250 mg) was dissolved in 10 mL distilled water at 65 °C for 15 min and immediately passed through
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a 3.0-μm cellulose ester filter membrane. Sample solution was eluted with distilled water and a
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stepwise NaCl gradient (0.5-2 M). Fractions were determined using the phenol-sulfuric acid method after which carbohydrate-positive fractions were pooled, dialyzed and lyophilized
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(Dubois, Gilles, Hamilton, Rebers, & Smith, 1956).
2.4. Chemical characterization of polysaccharides
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The content of neutral sugars in the isolated polysaccharides was quantified using the
phenol–sulfuric acid method and D-fucose as a standard (Dubois et al., 1956). Lowry method was employed to measure the amount of protein using a DC protein assay kit (Bio-Rad, CA, USA) (Lowry, Rosebrough, Farr, & Randall, 1951). The polysaccharides were hydrolyzed with 6
0.5 M HCl and then BaCl2-gelatin method used to measure the amount of sulfate (Dodgson & Price, 1962). The content of uronic acid was determined by a sulfamate/m-hydroxydiphenyl
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assay and glucuronic acid as a standard (Filisetti-Cozzi & Carpita, 1991).
2.5. Monosaccharide composition and methylation analysis
To determine the monosaccharide composition, polysaccharides (2 mg) were hydrolyzed with 4 M TFA at 100 °C for 6 h. The hydrolyzed products were reduced with NaBD 4 and
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acetylated using acetic anhydride. The final derivatives were injected into a gas chromatography
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mass spectrometer (GC–MS) system (6890 N/MSD 5973, Agilent Technologies, Santa Clara,
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CA) equipped with HP-5MS capillary column (30 m 0.25 mm 0.25l m) (Agilent Technologies,
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Santa Clara, CA). The methylation analysis was carried out according to the method of Ciucanu and Kerek (1984) in order to determine the glycosidic linkages. Polysaccharides (3 mg) were
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suspended in 0.5 mL dimethyl sulfoxide (DMSO) which was followed by NaOH (20 mg)
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addition under nitrogen gas. Substitution of hydroxyl groups with methyl groups was performed
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by methyl iodide (CH3I) and incubation at room temperature for 45 min. The methylated products were subjected to hydrolysis, reduction and acetylation to produce partially methylated
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alditol acetates (PMAAs). Finally, the PMAAs were injected into the GC–MS system. Helium
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was used as the carrier gas at a constant flow rate of 1.2 mL/min.
2.6. 1H NMR spectroscopy
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Polysaccharide (20 mg) was solubilized in D2O (0.5 mL) and subjected to the spectrometer at 50 °C. 1H NMR spectrum was recorded on a JEOL ECA-600 spectrometer (JEOL, Akishima,
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Japan) at a base frequency of 600 MHz.
2.7. Determination of molecular properties
The lyophilized polysaccharides (2 mg/ mL) were dissolved in distilled water and heated for 30 sec in a microwave bomb (#4872; Parr Instrument Co., Moline, IL, USA) for complete
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solubilization (Borazjani et al., 2018). Sample solutions (20 μL) were loaded onto a size
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exclusion chromatography (SEC) system operating with 0.15 M NaNO3 and 0.02% NaN3 at a
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flow rate of 0.4 mL/min. The high performance size exclusion chromatography (HPSEC) system
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was consisted of a TSK G5000PW column (7.5 × 600 mm; Toso Biosep, Montgomeryville, PA, USA) connected to multi-angle laser light scattering (HELEOS; Wyatt Technology Corp, Santa
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Barbara, CA, USA) and a refractive index detector (Waters, 2414) (HPSEC-MALLS-RI). The
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weight average molecular weight (Mw) and radius of gyration (Rg) were calculated by ASTRA
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5.3 software (Wyatt Technology Corp.).
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2.8. Polysaccharide effects on RAW264.7 murine macrophage proliferation and nitric oxide
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release
RAW264.7 murine macrophage cells were grown in an RPMI-1640 medium containing
10% fetal bovine serum (FBS) and seeded in 96-well microplate (100-μL volume, 1×104 cells/well). The cells were treated with 100 μL of either polysaccharide samples (10, 25 and 50 8
μg/mL) or culture medium (negative control) for 18 h at 37 °C in a humidified atmosphere containing 5% CO2. The supernatants were mixed with an equal amount of Griess reagent and kept at room temperature for 10 min (Green, Wanger, Glogowski, Skipper, Wishnok, &
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Tannenbaum, 1982). Absorbance was recorded at 540 nm. To evaluate macrophage proliferation, RAW264.7 cells were incubated with different concentrations of polysaccharides for 18 h. Then, medium was discarded and 20 μL of MTT (5 mg/mL) were added into the wells and the plate was kept for 4 h. The medium was discarded and 200 μL of DMSO was added to dissolve the formazan. Absorbance was recorded at 570 nm. Macrophage proliferation was calculated using
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Macrophage proliferation (%) = At/Ac × 100
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the following equation:
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in which At the absorbance of the test group and Ac is the absorbance of the control group.
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2.9.Cytokine gene expressions
RAW264.7 macrophage cells with density of 1 × 105 cells/well were incubated with
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polysaccharides (10, 25 and 50 μg/mL) or LPS (1 μg/mL) at 37 °C for 18 h. Total RNA was
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isolated from RAW264.7 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance to the manufacturer’s protocol. The construction of cDNA was carried out using
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oligo-(dT)20 primer and Superscript III RT (Invitrogen, Carlsbad, CA, USA). The PCR amplification was performed by GoTaq Flexi DNA Polymerase (Promega, Madison, WI, USA) and specific primers. PCR conditions were as follows: 30 cycles of 94 °C for 30 s, 56 °C for 40 s and 72 °C for 1 min which was followed by 72 °C for 10 min. PCR products were separated in 9
1% agarose gel and stained by ethidium bromide. The sequences of primer employed in the
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present study are shown in Table 1.
2.11. Western blot analysis
RAW264.7 macrophage cells were lysed in RIPA buffer containing 50 mM Tris-HCl (pH
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7.4), 150 mM NaCl, 1% Nonidet P-40 and 0.1% sodium dodecyl sulfate. Cell lysates were
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electrophoresed on 10% SDS-PAGE and transferred onto PVDF membranes. Then, the
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membranes were blocked in a 5% fat-free milk dispersed TBST (Tris-buffered saline containing
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Tween 20) at room temperature for 2 h. Subsequently, the blocked membrane was kept with
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primary antibodies including anti-phospho-NF-κB p65, anti-phospho-JNK, anti-phospho-
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ERK1/2, anti-phospho p38. Washed membranes were incubated with HRP-conjugated antirabbit antibody for 1 h at 4 °C. The target proteins were identified by an enhanced
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chemiluminescence (ECL) kit in accordance with the manufacturer’s protocol. Bio-Rad image analysis system (Bio-Rad Laboratories, Hercules, CA) was used to visualize the gel bands and
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software Quantity one (version 4.6, Bio-Rad, USA) was employed to quantify protein
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expression.
2.12. Statistical analyses
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Statistical differences between the test groups were identified by one-way analysis of variance (ANOVA) and Duncan's multiple-range test. All experiments were carried out in triplicate (n=3) the data are presented as mean ± standard deviation (SD). The statistical analysis
value of p < 0.05 was considered to be statistically significant.
3. Results and discussions
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3.1.Isolation, purification and chemical analysis
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was performed using SPSS software (Version 16; SPSS Inc., Chicago, IL, USA). A probability
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A crude polysaccharide was isolated from C. indica using hot water and ethanol
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precipitation. The yield of crude polysaccharide was calculated 5.7% (Table 2). The amount of
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polysaccharides extracted from brown seaweeds greatly varies depending on species. For instance, while the polysaccharide yield achieved in the present study was higher than Ecklonia
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cava (1.8%) and Laminaria japonica (2.3%), it was relatively lower than Agarum cribrosum
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(8.1%) (Wang, Zhang, Zhang, & Li, 2008; Lee, Ko, Ahn, You, Kim, Heu, Kim, Jee, & Jeon,
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2012; Cho, Lee, Kim, & You, 2014). The crude extract from C. indica was chiefly composed of neutral sugars (71.5%) and limited amount of proteins (7.4%). Besides, isolated polysaccharide
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possessed an anionic character mostly originated from the presence of sulfate esters (12.95) and, to a lesser extent, acidic sugars (2.9%). Hence, crude polysaccharides were subjected to a
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purification step using DEAE Sepharose Fast Flow column which successfully produced two fractions designated as CIF1 (34.5%; eluted with 0.5 M NaCl) and CIF2 (55.8%; eluted with 1.0 M NaCl) fractions (Fig. 1). Both fractions contained different amounts of neutral sugars (73.1%78.6%), proteins (5.8%-6.5%), uronic acids (1.3%-9.0%) and sulfate groups (6.9%-9.7%). 11
As presented in Fig. 2A and Table 2, crude polysaccharide was mostly consisted of fucose (35.3%) and galactose (23.2%) monosaccharides followed by lower amounts of mannose, glucose, rhamnose and xylose units. The monosaccharide profiles of fractionated
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polysaccharides were not only different from crude polysaccharides but also were different with one another (Figs. 1B and 1C); while the major sugar of CIF1 polysaccharide was glucose (30.8%) with relatively lower levels of mannose (17.8%), galactose (17.3%) and fucose (17.2%), the CIF2 was mainly consisted of fucose (24.4%), glucose (21.3%) and mannose (20.6%). As noted in the introduction, all polysaccharides from brown seaweeds contain certain amounts of
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sulfate and fucose in their chain structure and these two constituents are believed to significantly
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contribute in the overall biological properties of polysaccharides (Borazjani et al., 2018; Cho et
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al., 2014). In the present study, accordingly, we obtained polysaccharides with different fucose
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and sulfate ratios which might help us better understand their role in cell stimulation.
3.2. Molecular properties of polysaccharides
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The molecular characteristics of crude and fractionated polysaccharides were revealed using
a HPSEC-MALLS-RI system. Fig. 3A shows the refractive index (RI) chromatogram of crude polysaccharide molecules separated on a SEC column as two asymmetrical peaks eluting from 28-31 min and 33-43 min. However, in the RI elution profile of CIF1, the initial small peak with 12
large size mostly reduced and only a single broad peak was observed between the elution times of 31-42 min (Fig. 3B). A narrow symmetrical RI peak, which implies the homogeneity of molecular distribution, was obtained for the CIF2 polysaccharides eluting from 30-49 min (Fig.
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3C). The weight average molecular weight (Mw) of the small peak of crude polysaccharide was 10800.5 × 103 g/mol while most of the polysaccharide molecules involved in the second peak with Mw of 1651.6 × 103 g/mol (Table 2). A considerably smaller average size of molecule was obtained for CIF1 polysaccharides having an Mw of 573.1 × 103 g/mol. As shown in Table 2, the average Mw of CIF2 polysaccharides was 1146.6 × 103 g/mol.
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The radius of gyration (Rg), which was also calculated from the data obtained by MALLS,
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indicates the maximal average distance between the center point of polysaccharide to the outer
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edge of the molecule. The highest Rg values were calculated for peak I (102.4 nm) and peak II
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(105.1 nm) of crude polysaccharides (Table 2). The CIF1 (85.2 nm) and CIF2 (64.7 nm)
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molecules were found to have much smaller Rg. The specific volume of gyration (SVg) is another
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informative molecular feature of polysaccharide molecules which is obtained by the following equations using Mw and Rg inputs:
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SVg = 4/3(Rg × 108)3/(Mw/N) = 2.522 Rg3/Mw
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in which N was the constant value of 6.02 × 1023/mol (Avogadro’s number) and the units for SVg, Mw and Rg were cm3/g, g/mol and nm respectively.
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The SVg value provides an approximate theoretical gyration volume per unit of molar mass
of polysaccharide molecules and inversely indicates the molecular compactness of polysaccharide molecules. As shown in Table 2, the highest SVg value was found for CIF1 (2.7 cm3/g) implying the structural conformation of this polysaccharide as being the most extended 13
polymer measured in this study. On the other hand, the lowest SVg value of CIF2 (0.6 cm3/g) indicated a high degree of molecular compactness of its polysaccharide molecules.
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3.3. RAW264.7 cells proliferation and stimulation by polysaccharides As far as we understand, macrophages are the major players of the cell-mediated immunity and act as key immune effector cells. The hallmark of macrophages is their ability to react to environmental stimuli and rapidly change their physiology. In the present study, all crude and
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fractionated polysaccharides from C. indica were added into the culture medium of RAW264.7
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murine macrophage cells at concentration range of 10-50 µg/mL to evaluate their potential
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proliferation effects. As shown in Fig. 4A, none of the tested polysaccharides had any cytotoxic
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effect on RAW264.7 cells and can be regarded as safe molecules for further cell-based assays.
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cell growth by nearly 25%.
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Besides, CIF2 polysaccharide acted as a proliferation stimuli of RAW264.7 cells which enhanced
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Since one of the most reliable methods to assess classical activation of murine macrophages is examining nitric oxide (NO) production, we determined the amount of NO in the
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culture medium as an indication of polysaccharide stimulation capacity. Accordingly, RAW264.7 cells were incubated with crude polysaccharide and fractions (10, 25 and 50 µg/mL) for 18 h to examine NO production. As shown in Fig. 4B, the crude polysaccharide exerted a weak activation capacity on macrophage cells releasing less than 20 µmol of NO into 14
surrounding medium. The macrophage NO induced by CIF1 polysaccharides was even lower (< 10 µmol) and it seems the inactive portion of the isolated polysaccharides were eluted from the DEAE Sepharose Fast Flow column into CIF1 fraction. Contrarily, a considerable macrophage
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activation was observed when CIF2 polysaccharides were added into the culture medium (p < 0.05) so that an appreciable amount of NO, up to nearly 45 µmol, was produced which was proportionate to that of LPS (Escherichia coli lipopolysaccharides, Sigma, 1.0 μg/mL) used as positive control (50 µmol). The potency of a polysaccharide to stimulate macrophage cell is thought to be closely modulated by one or more of the following structural characteristics:
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sulfate groups, carboxyl groups, molecular weight, conformation, degree of branching and
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monosaccharide composition (Ferreira, Passos, Madureira, Vilanova & Coimbra, 2015). In a
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previous study, we showed that the capability of fucoidan from Sargassum angustifolium to
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activate macrophage cells is governed by the amount of sulfate groups presented in their structure (Borazjani et al., 2018). This was, however, somewhat different for a galactan obtained
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from Opuntia polyacantha where the lower molecular weight polysaccharides more significantly
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induced proinflammatory response in macrophage cells (Schepetkin, Xie, Kirpotina, Klein, Jutila,
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Quinn, 2008). On the contrary to the studies mentioned above, neither sulfate content nor molecular weight were responsible to drive the highest activity of CIF2 polysaccharide; rather,
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their smaller molecular size and higher degree of molecular compactness seemed to be plausible structural determinants. However, this hypothesis needs to be systematically tested with respect
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to molecular conformation of CIF2 polysaccharide. Essentially, NO is a short-lived gaseous radical with robust multifunctional nature acting as both regulator and effector molecules (Chi et al., 2003). While the former role of NO molecules includes immunosuppressive effects on lymphocyte proliferation and modulation of the cytokine 15
response, the latter role is comprised of immunopathologic effects in tissue destruction and immunoprotective activities against pathogens (Tripathi et al., 2007). The up-regulation of cytokines or, in other words, proinflammatory response of immune system, which is the subject
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of the present study, initiates with the activation of macrophages and the synthesis of large amount of NO (Chi et al., 2003). Although, the NO is produced by all three forms of nitric oxide synthase (NOS), inducible NOS (iNOS) is the dominant type of these enzymes during the large production of NO in the event of tissue injury, cancer tumor suppression and antimicrobial activity (Tripathi et al., 2007). In case of the present investigation, this raises the question of
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whether the presence of NO in the culture medium of test groups was due to macrophage
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activation via the up-regulation of iNOS enzyme. Hence, RAW264.7 cells containing 50 μg/mL
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of polysaccharides were incubated with iNOS inhibitor NG-monomethyl-L-arginine. As shown in
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Fig. 4B, the amount of NO released into the medium was considerably blocked by the inhibitor suggesting the activation of RAW264.7 cells through the up-regulation of iNOS genes. In
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addition, CIF2 polysaccharides were selected as the most macrophage stimulating agents for the
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examination of iNOS gene expression in concentrations of 10, 25 and 50 µg/mL. As
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demonstrated in Figs. 5A and 5B, consistent with NO production, the level of iNOS gene expression, amplified with rt-PCR and detected on an agarose gel electrophoresis, substantially
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increased with enhanced polysaccharide concentrations (p < 0.05). This result revealed the production of iNOS-driven NO and thus confirmed the activation of RAW264.7 cells by CIF2
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polysaccharides.
When the gene expressions of proinflammatory cytokines including TNF-α, IL-1β and IL-6 was examined, a concentration-dependent increase was observed in the intensity of detected bands (Figs. 5A and 5B). Nonetheless, it is important to note that although the aforementioned 16
inflammatory cytokines are beneficial at appropriate amounts, their excessive production in a deregulated fashion is toxic and may causes severe inflammatory responses (Chi et al., 2003). Therefore, we examined the potential production of IL-10 and the result of gene expression
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showed the presence of distinct and strong bands on the agarose gel indicating the capability of CIF2 polysaccharide to tightly mediate the inflammatory process (Figs. 5A and 5B). It has been stated that strong inflammatory responses, especially those stimulated via Toll-like receptors (TLRs), simultaneously activate feedback inhibitory process to constrain the magnitude of their impact (Liew, Xu, Brint, & O'Neill, 2005).
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As shown in Figs. 5C and 5D, the Western-blot analysis of the cellular proteins using specific
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antibodies revealed the presence of high amount of phosphorylated nuclear factor-kappa B (p-
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NF-κB) which suggested the leading role of NF-κB signaling pathway in the activation process
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of RAW264.7 cells by CIF2 polysaccharides. Basically, NF-κB is a transcription factor that its
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activation and regulation is hindered in the cytoplasm by a group of inhibitory proteins (iκB).
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Once the macrophage cell is activated, NF-κB /iκB complex undergoes a series of phosphorylation and proteolysis process upon which liberated NF-κB migrates into the nucleus
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and activates the gene expression of proinflammatory cytokines and mediators such as TNF-α and iNOS (Denkers, Butcher, Del Rio, & Kim, 2004). We also searched for phosphorylated-JNK
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(p-JNK), -ERK (p-ERK) and -p38 (p-p38) proteins in the macrophage cells and the positive
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results of constantly increasing band intensities suggested the mitogen activated protein kinase (MAPKs) as being part of the macrophage stimulation mechanism by CIF2 polysaccharides (Figs. 5C and 5D).
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Following the determination of transduction pathways, the interaction mechanism between RAW264.7 macrophage cells and CIF2 polysaccharide molecules was sought using specific antibodies including anti-TLR4, anti-TLR2 and anti-CR3. Interestingly, as shown in Fig. 6, it
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was found that CIF2-induced NO release was notably diminished (51.7%) in the presence of TLR4 antibody (p < 0.05). Nonetheless, the inhibitory effects of anti-TLR2 and anti-CR3 on the production of NO from the CIF2-activated macrophages were not considerable. On the basis of these results, we concluded that TLR4 was the main receptor through which CIF2 polysaccharides interacted with macrophage cells and triggered NF-κB and MAPKs signaling
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pathways leading to initiation of a cascade of gene expressions and the following release of
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proinflammatory mediators. TLRs, with 10 known members, are the most characterized family
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of pattern recognition receptors (PRRs) in mammalian cells among which TLR4 is actively
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engaged in recognition of gram-negative bacteria, activation of dendritic cells and macrophages (Janeway, & Medzhitov, 2002). Previous investigations have also reported that polysaccharides
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can induce TLR4-mediated macrophage activation (Kim, Kim, Lee, Ryu, Kim, Yoon, & Han,
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2012).
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3.4. Linkage analysis of CIF2 polymer chain Partially methylated products of CIF2 polysaccharides were hydrolyzed with acid, reduced
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with NaBD4 and acetylated with acetic anhydride to produce final derivatives generally called partially methylated alditol acetates (PMAAs) which were detected by GC-EIMS. The identification of PAMMs and corresponding deduced glycosidic linkages are presented in Fig. 7A and Table 3. The methylation results were nearly consistent with the overall monosaccharide 18
composition and suggested a very complex structure for the purified CIF2 polysaccharide. The detection of large amount of 2,4-Me2-Fucp in the final derivatives indicated that the backbone of CIF2 polysaccharides was mainly consisted of (1→3)-fucopyranose residues. A portion of fucose
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sugars were involved in the structure of CIF2 as non-reducing terminal units as interpreted from the existence of 2,3,4-Me3-Fucp products. The presence of 2,3,4,6-Me4-Glcp, 4,6-Me2-Glcp and 6-Me-Glcp products suggested the involvement of glucose units in the polysaccharide structure as (→1)-, (1→2,3)- and (1→2,3,4)-glucopyranose residues. Additionally, mannoses were found in the form of 3,4,6-Me3-Manp, 2,3,6-Me3-Manp, 4,6-Me2-Manp, 6-Me-Manp products
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suggesting their connections through (1→2)-, (1→4)-, (1→2,3)- and (1→2,3,4)-mannopyranose.
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The presence of 2,3,4,6-Me4-Galp, 2,4,6-Me3-Glcp, 2,3,4-Me3-Galp, and 2,4-Me2-Galp indicated
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the glycosidic linkages of galactose units as (→1)-, (1→3)-, (1→6)- and (1→3,6)-
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galactopyranose residues. There also were different levels of 2,3,4-Me3-Xylp, 3,4-Me2-Xylp and 3,4-Me2-Rhap indicating the existence of (→1)- and (1→2)-xylopyranose and (1→2)-
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rhamnopyranose residues. In accordance with the glycosidic linkages of CIF2 fucoidan, (1→3)-
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fucopyranose has been found as the major part of the backbone of various fucoidans from
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Sargassum mcclurei, S. japonica and S. latissima (Thinh, Menshova, Ermakova, Anastyuk, Ly, & Zvyagintseva, 2013; Cumashi et al., 2007). In addition, the engagement of galactose units in
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(1→3) and (1→6) glycosidic linkages seems to be a shared characteristic among fucoidans obtained from many types of sources (Thinh et al., 2013; Borazjani et al., 2018). However, we
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think it is noteworthy to point out that, as mentioned above, the methylation analysis of the purified polysaccharide revealed the presence of high amount of 6-Me-Glcp which is relatively high compared to that of other fucoidans. Given the roughly matching ratios of terminal groups and branching points, we assumed that there was a high possibility of existing some sulfate esters 19
on 6-Me-Glcp residues along with other parts being presumably located on fucose and/or galactose units. However, since we were not be able to carry out the desulfation analysis in the present study, further experiments are required to provide assurances.
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Altogether, the present methylation analysis showed that although CIF 2 polysaccharide from C. indica was purified on an anion-exchange column and the RI chromatogram confirmed its molecular homogeneity, its chemical structure was yet very complex. This complexity in the building constituents of CIF2 polysaccharide was also obvious from the overlapping signals of 1
H NMR spectrum (Fig. 7B). However, the presence of different spin systems were observed in
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chemical shifts of 4.7-5.7 ppm corresponding to anomeric protons, 3.0-4.5 ppm corresponding to
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ring protons (H-2-H-5) and 1.3-1.5 ppm corresponding to methyl protons (H-6) of fucose
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residues. It is noteworthy to mention that our attempts to attain 13C NMR and 2D NMR did not
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4. Conclusions
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produce proper results.
A sulfated polysaccharide (CIF2) was successfully purified from C. indica using a DEAE
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Sepharose Fast Flow chromatography column. The CIF2 polysaccharide had a homogenous molecular distribution with the average molecular weight of 1146.6 × 103 g/mol. The building
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blocks of CIF2 polysaccharides were mainly fucose, glucose, mannose and galactose as well as lower amounts of rhamnose and xylose. The greatest amount of nitric oxide was produced from RAW264.7 murine macrophages induced by CIF2 polysaccharides. The interaction of RAW264.7 cells with CIF2 polysaccharides occurred through the cell surface TLR4 initiating the 20
cascade activations of NF-κB and MAPKs signaling pathways and the following proinflammatory response. The intermolecular connections of CIF2 sugars were highly complex and yet sharing the common (1→3)-fucopyranose, (1→3) and (1→6)-galactopyranose residues
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of fucoidans. However, further studies are needed to fully elucidate the sequence and repeating units of CIF2 polysaccharide with aid of tightly controlled hydrolysis using acid or specific enzymes prior to NMR spectroscopy.
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Acknowledgement
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The authors would like to thank Tarbiat Modares University for its partial financial support
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of this work through Graduate Student Research Fund. We are also grateful for the additional
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support provided by Basic Science Research Program through the National Research Foundation
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of Korea (NRF) funded by the Ministry of Education (No. 2018R1A6A1A03023584).
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Wang, J., Zhang, Q., Zhang, Z., & Li, Z. (2008). Antioxidant activity of sulfated polysaccharide
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fractions extracted from Laminaria japonica. International Journal of Biological
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Wu, L., Sun, J., Su, X., Yu, Q., Yu, Q., & Zhang, P. (2016). A review about the development of
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fucoidan in antitumor activity: Progress and challenges. Carbohydrate Polymers, 154, 96111.
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Yu, Y., Shen, M., Song, Q., & Xie, J. (2017). Biological activities and pharmaceutical applications of polysaccharide from natural resources: A review. Carbohydrate Polymers, 183, 91-101.
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Zhang, X., Qi, C., Guo, Y., Zhou, W., & Zhang, Y. (2016). Toll-like receptor 4-related immunostimulatory polysaccharides: Primary structure, activity relationships, and possible
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interaction models. Carbohydrate Polymers, 149, 186-206.
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A
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Figure captions
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Fast Flow column.
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Fig.1. Stepwise elution profile of crude polysaccharide from C. indica on DEAE Sepharose
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Fig. 2. GC chromatograms of monosaccharides of crude, CIF1 and CIF2 polysaccharides.
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28
D
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EP
CC
A
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A
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Fig. 3. Refractive index (RI) profiles of crude, CIF1 and CIF2 polysaccharides on a TSK
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CC
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TE
D
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A
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U
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G5000PW column.
Fig. 4. RAW264.7 murine macrophage proliferation (A) and nitric oxide production (B)
induced by crude, CIF1 and CIF2 polysaccharides. Bars with different letters indicate a significant difference between the test groups (p < 0.05). 29
30
D
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CC
A
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U
N
A
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Fig. 5. The mRNA expression of iNOS, IL-1β, TNF-α, IL-6 and IL10 in the presence of
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CIF2 in RAW264.7 cells (A and B). The activation of NF-κB, JNK, ERK and p38 in RAW264.7
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cells induced by CIF2 (C and D). Bars with different letters indicate a significant difference
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between the test groups (p < 0.05). β-actin was used as a control.
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Fig. 6. The nitric oxide release from RAW264.7 cells in the presence of CIF2 polysaccharide.
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The cells were incubated with antibodies of receptors (TLR4, TLR2 and CR3) for 2 h before
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stimulation with CIF2 polysaccharides. Bars with different letters indicate a significant difference
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CC
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between the test groups (p < 0.05).
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Fig. 7. GC-EIMS chromatogram of PMAAs of CIF2 polysaccharide (A). Helium was used as the carrier gas at a constant flow rate of 1.2 mL/min in GC-EIMS. 1H NMR chromatogram of CIF2 polysaccharide. The polysaccharides were dissolved in D2O and scanned at 50 °C.
33
Table
Table 1
Genes
Primer sequences (5’→3’) Forward
CCCTTCCGAAGTTTCTGGCAGCAGC
Reverse
GGCTGTCAGAGCCTCGTGGCTTTGG
Forward
ATGAGCACAGAAAGCATGATC
Reverse
TACAGG CTTGTCACTCGAATT
Forward
ATGGCAACTATTCCTGAACTCAACT
Reverse
CAGGACAGGTATAGATTCTTTCCTTT
Forward
TTCCTCTCTGCAAGAGACT
Reverse
TGTATCTCTCTGAAGGACT
Forward
TACCTGGTAGAAGTGATGCC
iNOS
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TNF-α
A
N
IL-1β
CATCATGTATGCTTCTATGC
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Reverse
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IL-10
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IL-6
Forward
TGGAATCCTGTGGCATCCATGAAAC
Reverse
TAAAACGCAGCTCAGTAACAGTCCG
A
CC
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β-actin
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Sequence of oligo (dT) primers used for the rt-PCR
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Table 2 Yield, chemical composition and molecular properties of the polysaccharides from C. indica. Crude
CIF1
Neutral sugars (%)
34.5 ± 1.9
71.5 ± 0.27
73.1 ± 0.4
78.6 ± 0.7
6.5 ± 0.4
5.8 ± 0.1
9.0 ± 0.3
1.3 ± 0.4
6.9 ± 0.3
9.7 ± 0.2
11.3 ± 0.2
11.2 ± 0.2
17.2 ± 0.9
24.4 ± 0.3
5.6 ± 0.1
5.8 ± 0.3
14.5 ± 0.3
17.8 ± 0.3
20.6 ± 0.4
14.2 ± 0.5
30.8 ± 1.6
21.3 ± 0.7
23.2 ± 0.6
17.3 ± 0.3
16.7 ± 0.5
Protein (%）
7.4 ± 0.2
Uronic acid (%)
2.9 ± 0.7
Sulfate (%)
12.9 ± 0.5 9.5 ± 0.1
Fucose
35.3 ± 0.3
Xylose
3.2 ± 0.2
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Rhamnose
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Monosaccharide
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composition (%) Glucose Galactose
10800.5 ± 133.6
-
-
Peak II
1651.6 ± 37.5
573.1 ± 6.0
1146.6 ± 29.9
Peak I
102.4 ± 3.7
-
-
Peak II
105.1 ± 5.6
85.2 ± 4.1
64.7 ± 4.9
SVg (cm3/g)
Peak I
0.2 ± 0.0
-
-
Peak II
1.7 ± 0.2
2.7 ± 0.4
0.6 ± 0.1
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Peak I
CC
D
MW × 103 (g/mol)
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Mannose
55.8 ± 1.5
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5.7
Yield (%）
CIF2
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Rg (nm)
A
ND: not detected, Mw: Mean average molecular weight, Rg: Radius of gyration, SVg: Specific volume of gyration
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Table 3
Time PMAAsa (min)
Deduced glycosidic CIF2 linkage
7.88
1,5-di-O-acetyl-2,3,4-tri-O-methyl-Xyl
Xylp-(1→
8.35
1,5-di-O-acetyl-2,3,4-tri-O-methyl-Fuc
9.50
1,2,5-tri-O-acetyl-3,4-di-O-methyl-Xyl
9.88
1,2,5-tri-O-acetyl-3,4-di-O-methyl-Rha
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Glycosidic linkage analysis of the constituent sugars of the fraction F2 from C. indica.
8.09
Fucp-(1→
7.63
→2)-Xylp-(1→
3.27
→2)-Rhap-(1→
7.66
Galp-(1→
8.40
Glcp-(1→
1.91
→2)-Manp-(1→
1.54
→3)-Fucp-(1→
18.21
→4)-Manp-(1→
6.60
→3)-Galp-(1→
4.27
12.31 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-Gal
→6)-Galp-(1→
3.06
13.21 1,2,3,5-tetra-O-acetyl-4,6-di-O-methyl-Man
→2,3)-Manp-(1→
6.72
→2,3)-Glcp-(1→
3.31
14.25 1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl-Gal
→3,6)-Galp-(1→
4.67
14.34 1,2,3,4,5-penta-O-acetyl-6-mono-O-methyl-Man
→2,3,4)-Manp-(1→
2.44
→2,3,4)-Glcp-(1→
12.17
10.17 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-Gal 10.53 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-Glc
A
11.97 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-Man
N
11.78 1,3,5-tri-O-acetyl-2,4-di-O-methyl-Fuc
U
11.71 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl-Man
D
M
12.12 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-Gal
EP
TE
13.41 1,2,3,5-tetra-O-acetyl-4,6-di-O-methyl-Glc
17.59 1,2,3,4,5-penta-O-acetyl-6-mono-O-methyl-Glc Partially O-methylated alditol acetates
A
CC
a
36
S0144-8617(18)31198-6 https://doi.org/10.1016/j.carbpol.2018.10.022 CARP 14154
To appear in: Received date: Revised date: Accepted date:
9-8-2018 27-9-2018 8-10-2018
Please cite this article as: Bahramzadeh S, Tabarsa M, You S, Li C, Bita S, Purification, structural analysis and mechanism of murine macrophage cell activation by sulfated polysaccharides from Cystoseira indica, Carbohydrate Polymers (2018), https://doi.org/10.1016/j.carbpol.2018.10.022 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.
Purification, structural analysis and mechanism of murine macrophage cell activation by sulfated polysaccharides from Cystoseira indica
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Running title: Immunostimulatory of polysaccharides from Cystoseira indica
Department of Seafood Processing, Faculty of Marine Sciences, Tarbiat Modares University,
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a
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Saman Bahramzadeha, Mehdi Tabarsaa*, SangGuan Youb*, Changsheng Lib, Seraj Bitac
Department of Marine Food Science and Technology, Gangneung-Wonju National University,
Gangneung, Gangwon 25457, Korea
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Chabahar, Iran
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Department of Fisheries, Faculty of Marine Sciences, Chabahar Maritime University,
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c
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b
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P.O. Box 46414-356, Noor, Iran
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*Corresponding
author. Tel.: +981144553101; Fax: +981144550906 (M. Tabarsa); Tel.: +82
33 640 2853; Fax.: +82 33 640 2340 (S.G. You)
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E-mail addresses: [email protected] (M. Tabarsa); [email protected] (S.G. You)
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Highlights
Sulfated polysaccharides were isolated from Cystoseira indica.
Polysaccharides exerted high RAW264.7 macrophage stimulating activity.
RAW264.7 cells were activated through NF-κB and MAPK signaling pathways.
TLR4 was the major cell surface receptor interacting polysaccharides with RAW264.7
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cells.
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The main backbone of the polysaccharides was consisted of (1→3)-fucopyranosyl units.
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Abstract
Sulfated polysaccharides were isolated and purified from the water extract of Cystoseira
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indica using DEAE Sepharose Fast Flow column to evaluate their structure and macrophage
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stimulating capacity. Crude and fractionated polysaccharides, CIF1 and CIF2, were mostly
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composed of neutral sugars (73.1%-78.6%) with relatively lower amounts of acidic sugars (1.3%-9.0%) and sulfate esters (6.9%-9.7%). The polymer chains of polysaccharides were
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mainly built of different levels of glucose (2.1%-30.8%), fucose (17.2%-24.4%), mannose (17.8%-20.6%) and galactose (16.7%-17.3%). The weight average molecular weight (Mw) of
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polysaccharides varied between 573.1 × 103 g/mol to 1146.6 × 103 g/mol. The CIF2 polysaccharide, as the most immunostimulating polysaccharide, remarkably induced the release of nitric oxide and inflammatory cytokines including TNF-α, IL-1β, IL-6 and IL-10 from RAW264.7 murine macrophage cells through NF-κB and PAMKs transduction signaling 2
pathways via cell surface TLR4. The interconnections of sugars in CIF2 polysaccharide were complex with (1→3)-fucopyranose, (1→2,3,4)-glucopyranose, (→1)-galactopyranose, (→1)xylopyranose, (1→2)-rhamnopyranose and (1→2,3)-mannopyranose units being the most
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predominant residues.
Keywords: Cystoseira indica, Polysaccharide, Structure, Immunostimulatory, NF-κB and
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MAPKs signaling pathways
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1. Introduction
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Polysaccharides are a class of naturally available macromolecules which ubiquitously exist
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in plants, microorganisms, algae and animals. Polysaccharides are known to be necessary for
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cell-cell communication, cell adhesion and molecular recognition in the biological systems (Dwek, 1996). In the past decade, a growing number of studies have been paying great amount
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of attention towards polysaccharide isolation from natural resources such as plants, mushrooms,
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bacteria and seaweeds, mostly owed to their versatile pharmacological activities (Yu, Shen, Song, & Xie, 2017). Antitumor, anti-inflammatory, immunostimulatory and anticoagulant
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activities could be listed as the top widely explored physiological properties of polysaccharides (Yu et al., 2017; Borazjani, Tabarsa, You, & Rezaei, 2018). Among these, immunostimulatory polysaccharides are one of the relatively new class of biologically active compounds that have been attracted a tremendous interest for their promising 3
roles in boosting body’s natural resistance against viral and bacterial infections or assisting with the treatment of diseases having suppressed immune system conditions such as cancer and AIDS (Tripathi, Tripathi, Kashyap, & Singh, 2007). By far, various types of glucans (Tabarsa, Shin,
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Lee, Surayot, Park, & You, 2015), mannans (Simões, Nunes, Domingues, & Coimbra, 2012), galactans (Stephanie, Eric, Sophie, Christian, &Yu, 2010) and fucoidans (Borazjani et al., 2018) have been suggested as immunostimulant agents with capacity to interact directly or indirectly with the immune system, triggering a series of cellular and molecular events.
Fucoidans are a group of structurally diverse sulfated polysaccharides from brown seaweeds
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that could be potential immunostimulating polysaccharides for further industrial applications
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(Vo, & Kim, 2013). Regardless of their structural complexities, all fucoidans share a common
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chemical characteristic which is the presence of sulfate-bearing L-fucose units in their polymer
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chains. In fact, fucoidans are heteropolysaccharides in which different levels of mannose,
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galactose, glucose, xylose, rhamnose and arabinose sugars are involved in their structure, in
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addition to fucose units (Borazjani et al., 2018). Although, the interconnections of fucoidan units differ among various brown seaweeds, they are mostly formed of a main backbone consisted of
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either (1→3)-α-L-fucopyranose or alternating (1→3)- and (1→4)-α-L-fucopyranose residues
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(Cumashi, Ushakova, Preobrazhenskaya, D’lncecco, Piccoli, & Totani, 2007). Altogether, there seems to be a consensus that the structure of fucoidans are immensely
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complex and needs an in-depth consideration with respect to extraction, purification and the following chemical analysis prior to any industrial applications (Wu, Sun, Su, Yu, Yu, & Zhang, 2016). The complexity in chemical structure along with the heterogeneity in molecular size greatly contribute to the multifunctionality of fucoidan polysaccharides, specifically to their 4
immunoenhancing activities. Nonetheless, our understanding on the exact activation mechanism and relating structural feature that evokes the target immune cells are incomplete (Zhang, Qi, Guo, Zhou, & Zhang, 2016).
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Therefore, in the present study, we examined whether fudoidan from Cystosira indica has any capacity to activate macrophage cells and if the results are positive what underlying mechanisms play the major roles. To reveal the potential structural feature that contributed to the macrophage stimulation capacity of the fucoidan, a highly purified polysaccharide obtained after DEAE Sepharose Fast Flow chromatography was subjected to extensive molecular and chemical
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characterizations.
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2.1. Samples and reagents
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2. Materials and methods
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Samples of C. indica were collected in December 2016 from the coastline of Chabahar,
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Iran. The raw material was soaked in fresh water, washed and air dried at 60 °C in the oven. The dried sample material was ground using a blender, passed through a 0.5-mm sieve and kept in
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seal sample bags at -20 °C. All chemicals and reagents used in this study were analytical grade.
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2.2. Extraction of polysaccharides
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The extraction of polysaccharides from C. indica was performed using the method
described previously (Borazjani et al., 2018). Briefly, 20 g of dried and milled seaweeds were soaked into 200 mL of 80% ethanol and stirred overnight at room temperature to eliminate lipids, pigments and low molecular weight compounds. The residue was rinsed with acetone and air dried at room temperature in a fume hood. The defatted and depigmented powder (20 g) was 5
suspended in 400 mL of distilled water and the extraction performed at 65 °C under stirring for 2 h. The resultant supernatants were kept after centrifugation at 10 °C and 6080 g for 10 min. Then, ClCa2 (1%; w/w) was added into the concentrate and kept at 4 °C overnight to precipitate
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alginic acid. The precipitation of polysaccharides in the final alginate-free solution was carried out with the addition of ethanol (99%) to reach a final ethanol concentration of 70%. The crude polysaccharide were then washed and dehydrated with ethanol (99%), acetone, and finally dried
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at room temperature.
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2.3. Fractionation of polysaccharides
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Crude polysaccharides were fractionated on a DEAE Sepharose Fast Flow column (17-
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0709-01; GE Healthcare Bio-Science AB, Uppsala, Sweden). The crude polysaccharide (250 mg) was dissolved in 10 mL distilled water at 65 °C for 15 min and immediately passed through
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a 3.0-μm cellulose ester filter membrane. Sample solution was eluted with distilled water and a
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stepwise NaCl gradient (0.5-2 M). Fractions were determined using the phenol-sulfuric acid method after which carbohydrate-positive fractions were pooled, dialyzed and lyophilized
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(Dubois, Gilles, Hamilton, Rebers, & Smith, 1956).
2.4. Chemical characterization of polysaccharides
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The content of neutral sugars in the isolated polysaccharides was quantified using the
phenol–sulfuric acid method and D-fucose as a standard (Dubois et al., 1956). Lowry method was employed to measure the amount of protein using a DC protein assay kit (Bio-Rad, CA, USA) (Lowry, Rosebrough, Farr, & Randall, 1951). The polysaccharides were hydrolyzed with 6
0.5 M HCl and then BaCl2-gelatin method used to measure the amount of sulfate (Dodgson & Price, 1962). The content of uronic acid was determined by a sulfamate/m-hydroxydiphenyl
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assay and glucuronic acid as a standard (Filisetti-Cozzi & Carpita, 1991).
2.5. Monosaccharide composition and methylation analysis
To determine the monosaccharide composition, polysaccharides (2 mg) were hydrolyzed with 4 M TFA at 100 °C for 6 h. The hydrolyzed products were reduced with NaBD 4 and
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acetylated using acetic anhydride. The final derivatives were injected into a gas chromatography
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mass spectrometer (GC–MS) system (6890 N/MSD 5973, Agilent Technologies, Santa Clara,
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CA) equipped with HP-5MS capillary column (30 m 0.25 mm 0.25l m) (Agilent Technologies,
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Santa Clara, CA). The methylation analysis was carried out according to the method of Ciucanu and Kerek (1984) in order to determine the glycosidic linkages. Polysaccharides (3 mg) were
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suspended in 0.5 mL dimethyl sulfoxide (DMSO) which was followed by NaOH (20 mg)
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addition under nitrogen gas. Substitution of hydroxyl groups with methyl groups was performed
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by methyl iodide (CH3I) and incubation at room temperature for 45 min. The methylated products were subjected to hydrolysis, reduction and acetylation to produce partially methylated
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alditol acetates (PMAAs). Finally, the PMAAs were injected into the GC–MS system. Helium
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was used as the carrier gas at a constant flow rate of 1.2 mL/min.
2.6. 1H NMR spectroscopy
7
Polysaccharide (20 mg) was solubilized in D2O (0.5 mL) and subjected to the spectrometer at 50 °C. 1H NMR spectrum was recorded on a JEOL ECA-600 spectrometer (JEOL, Akishima,
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Japan) at a base frequency of 600 MHz.
2.7. Determination of molecular properties
The lyophilized polysaccharides (2 mg/ mL) were dissolved in distilled water and heated for 30 sec in a microwave bomb (#4872; Parr Instrument Co., Moline, IL, USA) for complete
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solubilization (Borazjani et al., 2018). Sample solutions (20 μL) were loaded onto a size
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exclusion chromatography (SEC) system operating with 0.15 M NaNO3 and 0.02% NaN3 at a
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flow rate of 0.4 mL/min. The high performance size exclusion chromatography (HPSEC) system
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was consisted of a TSK G5000PW column (7.5 × 600 mm; Toso Biosep, Montgomeryville, PA, USA) connected to multi-angle laser light scattering (HELEOS; Wyatt Technology Corp, Santa
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Barbara, CA, USA) and a refractive index detector (Waters, 2414) (HPSEC-MALLS-RI). The
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weight average molecular weight (Mw) and radius of gyration (Rg) were calculated by ASTRA
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5.3 software (Wyatt Technology Corp.).
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2.8. Polysaccharide effects on RAW264.7 murine macrophage proliferation and nitric oxide
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release
RAW264.7 murine macrophage cells were grown in an RPMI-1640 medium containing
10% fetal bovine serum (FBS) and seeded in 96-well microplate (100-μL volume, 1×104 cells/well). The cells were treated with 100 μL of either polysaccharide samples (10, 25 and 50 8
μg/mL) or culture medium (negative control) for 18 h at 37 °C in a humidified atmosphere containing 5% CO2. The supernatants were mixed with an equal amount of Griess reagent and kept at room temperature for 10 min (Green, Wanger, Glogowski, Skipper, Wishnok, &
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Tannenbaum, 1982). Absorbance was recorded at 540 nm. To evaluate macrophage proliferation, RAW264.7 cells were incubated with different concentrations of polysaccharides for 18 h. Then, medium was discarded and 20 μL of MTT (5 mg/mL) were added into the wells and the plate was kept for 4 h. The medium was discarded and 200 μL of DMSO was added to dissolve the formazan. Absorbance was recorded at 570 nm. Macrophage proliferation was calculated using
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Macrophage proliferation (%) = At/Ac × 100
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the following equation:
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in which At the absorbance of the test group and Ac is the absorbance of the control group.
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2.9.Cytokine gene expressions
RAW264.7 macrophage cells with density of 1 × 105 cells/well were incubated with
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polysaccharides (10, 25 and 50 μg/mL) or LPS (1 μg/mL) at 37 °C for 18 h. Total RNA was
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isolated from RAW264.7 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance to the manufacturer’s protocol. The construction of cDNA was carried out using
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oligo-(dT)20 primer and Superscript III RT (Invitrogen, Carlsbad, CA, USA). The PCR amplification was performed by GoTaq Flexi DNA Polymerase (Promega, Madison, WI, USA) and specific primers. PCR conditions were as follows: 30 cycles of 94 °C for 30 s, 56 °C for 40 s and 72 °C for 1 min which was followed by 72 °C for 10 min. PCR products were separated in 9
1% agarose gel and stained by ethidium bromide. The sequences of primer employed in the
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present study are shown in Table 1.
2.11. Western blot analysis
RAW264.7 macrophage cells were lysed in RIPA buffer containing 50 mM Tris-HCl (pH
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7.4), 150 mM NaCl, 1% Nonidet P-40 and 0.1% sodium dodecyl sulfate. Cell lysates were
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electrophoresed on 10% SDS-PAGE and transferred onto PVDF membranes. Then, the
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membranes were blocked in a 5% fat-free milk dispersed TBST (Tris-buffered saline containing
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Tween 20) at room temperature for 2 h. Subsequently, the blocked membrane was kept with
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primary antibodies including anti-phospho-NF-κB p65, anti-phospho-JNK, anti-phospho-
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ERK1/2, anti-phospho p38. Washed membranes were incubated with HRP-conjugated antirabbit antibody for 1 h at 4 °C. The target proteins were identified by an enhanced
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chemiluminescence (ECL) kit in accordance with the manufacturer’s protocol. Bio-Rad image analysis system (Bio-Rad Laboratories, Hercules, CA) was used to visualize the gel bands and
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software Quantity one (version 4.6, Bio-Rad, USA) was employed to quantify protein
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expression.
2.12. Statistical analyses
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Statistical differences between the test groups were identified by one-way analysis of variance (ANOVA) and Duncan's multiple-range test. All experiments were carried out in triplicate (n=3) the data are presented as mean ± standard deviation (SD). The statistical analysis
value of p < 0.05 was considered to be statistically significant.
3. Results and discussions
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3.1.Isolation, purification and chemical analysis
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was performed using SPSS software (Version 16; SPSS Inc., Chicago, IL, USA). A probability
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A crude polysaccharide was isolated from C. indica using hot water and ethanol
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precipitation. The yield of crude polysaccharide was calculated 5.7% (Table 2). The amount of
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polysaccharides extracted from brown seaweeds greatly varies depending on species. For instance, while the polysaccharide yield achieved in the present study was higher than Ecklonia
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cava (1.8%) and Laminaria japonica (2.3%), it was relatively lower than Agarum cribrosum
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(8.1%) (Wang, Zhang, Zhang, & Li, 2008; Lee, Ko, Ahn, You, Kim, Heu, Kim, Jee, & Jeon,
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2012; Cho, Lee, Kim, & You, 2014). The crude extract from C. indica was chiefly composed of neutral sugars (71.5%) and limited amount of proteins (7.4%). Besides, isolated polysaccharide
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possessed an anionic character mostly originated from the presence of sulfate esters (12.95) and, to a lesser extent, acidic sugars (2.9%). Hence, crude polysaccharides were subjected to a
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purification step using DEAE Sepharose Fast Flow column which successfully produced two fractions designated as CIF1 (34.5%; eluted with 0.5 M NaCl) and CIF2 (55.8%; eluted with 1.0 M NaCl) fractions (Fig. 1). Both fractions contained different amounts of neutral sugars (73.1%78.6%), proteins (5.8%-6.5%), uronic acids (1.3%-9.0%) and sulfate groups (6.9%-9.7%). 11
As presented in Fig. 2A and Table 2, crude polysaccharide was mostly consisted of fucose (35.3%) and galactose (23.2%) monosaccharides followed by lower amounts of mannose, glucose, rhamnose and xylose units. The monosaccharide profiles of fractionated
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polysaccharides were not only different from crude polysaccharides but also were different with one another (Figs. 1B and 1C); while the major sugar of CIF1 polysaccharide was glucose (30.8%) with relatively lower levels of mannose (17.8%), galactose (17.3%) and fucose (17.2%), the CIF2 was mainly consisted of fucose (24.4%), glucose (21.3%) and mannose (20.6%). As noted in the introduction, all polysaccharides from brown seaweeds contain certain amounts of
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sulfate and fucose in their chain structure and these two constituents are believed to significantly
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contribute in the overall biological properties of polysaccharides (Borazjani et al., 2018; Cho et
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al., 2014). In the present study, accordingly, we obtained polysaccharides with different fucose
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and sulfate ratios which might help us better understand their role in cell stimulation.
3.2. Molecular properties of polysaccharides
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The molecular characteristics of crude and fractionated polysaccharides were revealed using
a HPSEC-MALLS-RI system. Fig. 3A shows the refractive index (RI) chromatogram of crude polysaccharide molecules separated on a SEC column as two asymmetrical peaks eluting from 28-31 min and 33-43 min. However, in the RI elution profile of CIF1, the initial small peak with 12
large size mostly reduced and only a single broad peak was observed between the elution times of 31-42 min (Fig. 3B). A narrow symmetrical RI peak, which implies the homogeneity of molecular distribution, was obtained for the CIF2 polysaccharides eluting from 30-49 min (Fig.
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3C). The weight average molecular weight (Mw) of the small peak of crude polysaccharide was 10800.5 × 103 g/mol while most of the polysaccharide molecules involved in the second peak with Mw of 1651.6 × 103 g/mol (Table 2). A considerably smaller average size of molecule was obtained for CIF1 polysaccharides having an Mw of 573.1 × 103 g/mol. As shown in Table 2, the average Mw of CIF2 polysaccharides was 1146.6 × 103 g/mol.
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The radius of gyration (Rg), which was also calculated from the data obtained by MALLS,
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indicates the maximal average distance between the center point of polysaccharide to the outer
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edge of the molecule. The highest Rg values were calculated for peak I (102.4 nm) and peak II
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(105.1 nm) of crude polysaccharides (Table 2). The CIF1 (85.2 nm) and CIF2 (64.7 nm)
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molecules were found to have much smaller Rg. The specific volume of gyration (SVg) is another
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informative molecular feature of polysaccharide molecules which is obtained by the following equations using Mw and Rg inputs:
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SVg = 4/3(Rg × 108)3/(Mw/N) = 2.522 Rg3/Mw
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in which N was the constant value of 6.02 × 1023/mol (Avogadro’s number) and the units for SVg, Mw and Rg were cm3/g, g/mol and nm respectively.
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The SVg value provides an approximate theoretical gyration volume per unit of molar mass
of polysaccharide molecules and inversely indicates the molecular compactness of polysaccharide molecules. As shown in Table 2, the highest SVg value was found for CIF1 (2.7 cm3/g) implying the structural conformation of this polysaccharide as being the most extended 13
polymer measured in this study. On the other hand, the lowest SVg value of CIF2 (0.6 cm3/g) indicated a high degree of molecular compactness of its polysaccharide molecules.
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3.3. RAW264.7 cells proliferation and stimulation by polysaccharides As far as we understand, macrophages are the major players of the cell-mediated immunity and act as key immune effector cells. The hallmark of macrophages is their ability to react to environmental stimuli and rapidly change their physiology. In the present study, all crude and
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fractionated polysaccharides from C. indica were added into the culture medium of RAW264.7
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murine macrophage cells at concentration range of 10-50 µg/mL to evaluate their potential
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proliferation effects. As shown in Fig. 4A, none of the tested polysaccharides had any cytotoxic
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effect on RAW264.7 cells and can be regarded as safe molecules for further cell-based assays.
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cell growth by nearly 25%.
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Besides, CIF2 polysaccharide acted as a proliferation stimuli of RAW264.7 cells which enhanced
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Since one of the most reliable methods to assess classical activation of murine macrophages is examining nitric oxide (NO) production, we determined the amount of NO in the
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culture medium as an indication of polysaccharide stimulation capacity. Accordingly, RAW264.7 cells were incubated with crude polysaccharide and fractions (10, 25 and 50 µg/mL) for 18 h to examine NO production. As shown in Fig. 4B, the crude polysaccharide exerted a weak activation capacity on macrophage cells releasing less than 20 µmol of NO into 14
surrounding medium. The macrophage NO induced by CIF1 polysaccharides was even lower (< 10 µmol) and it seems the inactive portion of the isolated polysaccharides were eluted from the DEAE Sepharose Fast Flow column into CIF1 fraction. Contrarily, a considerable macrophage
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activation was observed when CIF2 polysaccharides were added into the culture medium (p < 0.05) so that an appreciable amount of NO, up to nearly 45 µmol, was produced which was proportionate to that of LPS (Escherichia coli lipopolysaccharides, Sigma, 1.0 μg/mL) used as positive control (50 µmol). The potency of a polysaccharide to stimulate macrophage cell is thought to be closely modulated by one or more of the following structural characteristics:
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sulfate groups, carboxyl groups, molecular weight, conformation, degree of branching and
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monosaccharide composition (Ferreira, Passos, Madureira, Vilanova & Coimbra, 2015). In a
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previous study, we showed that the capability of fucoidan from Sargassum angustifolium to
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activate macrophage cells is governed by the amount of sulfate groups presented in their structure (Borazjani et al., 2018). This was, however, somewhat different for a galactan obtained
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from Opuntia polyacantha where the lower molecular weight polysaccharides more significantly
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induced proinflammatory response in macrophage cells (Schepetkin, Xie, Kirpotina, Klein, Jutila,
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Quinn, 2008). On the contrary to the studies mentioned above, neither sulfate content nor molecular weight were responsible to drive the highest activity of CIF2 polysaccharide; rather,
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their smaller molecular size and higher degree of molecular compactness seemed to be plausible structural determinants. However, this hypothesis needs to be systematically tested with respect
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to molecular conformation of CIF2 polysaccharide. Essentially, NO is a short-lived gaseous radical with robust multifunctional nature acting as both regulator and effector molecules (Chi et al., 2003). While the former role of NO molecules includes immunosuppressive effects on lymphocyte proliferation and modulation of the cytokine 15
response, the latter role is comprised of immunopathologic effects in tissue destruction and immunoprotective activities against pathogens (Tripathi et al., 2007). The up-regulation of cytokines or, in other words, proinflammatory response of immune system, which is the subject
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of the present study, initiates with the activation of macrophages and the synthesis of large amount of NO (Chi et al., 2003). Although, the NO is produced by all three forms of nitric oxide synthase (NOS), inducible NOS (iNOS) is the dominant type of these enzymes during the large production of NO in the event of tissue injury, cancer tumor suppression and antimicrobial activity (Tripathi et al., 2007). In case of the present investigation, this raises the question of
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whether the presence of NO in the culture medium of test groups was due to macrophage
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activation via the up-regulation of iNOS enzyme. Hence, RAW264.7 cells containing 50 μg/mL
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of polysaccharides were incubated with iNOS inhibitor NG-monomethyl-L-arginine. As shown in
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Fig. 4B, the amount of NO released into the medium was considerably blocked by the inhibitor suggesting the activation of RAW264.7 cells through the up-regulation of iNOS genes. In
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addition, CIF2 polysaccharides were selected as the most macrophage stimulating agents for the
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examination of iNOS gene expression in concentrations of 10, 25 and 50 µg/mL. As
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demonstrated in Figs. 5A and 5B, consistent with NO production, the level of iNOS gene expression, amplified with rt-PCR and detected on an agarose gel electrophoresis, substantially
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increased with enhanced polysaccharide concentrations (p < 0.05). This result revealed the production of iNOS-driven NO and thus confirmed the activation of RAW264.7 cells by CIF2
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polysaccharides.
When the gene expressions of proinflammatory cytokines including TNF-α, IL-1β and IL-6 was examined, a concentration-dependent increase was observed in the intensity of detected bands (Figs. 5A and 5B). Nonetheless, it is important to note that although the aforementioned 16
inflammatory cytokines are beneficial at appropriate amounts, their excessive production in a deregulated fashion is toxic and may causes severe inflammatory responses (Chi et al., 2003). Therefore, we examined the potential production of IL-10 and the result of gene expression
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showed the presence of distinct and strong bands on the agarose gel indicating the capability of CIF2 polysaccharide to tightly mediate the inflammatory process (Figs. 5A and 5B). It has been stated that strong inflammatory responses, especially those stimulated via Toll-like receptors (TLRs), simultaneously activate feedback inhibitory process to constrain the magnitude of their impact (Liew, Xu, Brint, & O'Neill, 2005).
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As shown in Figs. 5C and 5D, the Western-blot analysis of the cellular proteins using specific
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antibodies revealed the presence of high amount of phosphorylated nuclear factor-kappa B (p-
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NF-κB) which suggested the leading role of NF-κB signaling pathway in the activation process
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of RAW264.7 cells by CIF2 polysaccharides. Basically, NF-κB is a transcription factor that its
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activation and regulation is hindered in the cytoplasm by a group of inhibitory proteins (iκB).
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Once the macrophage cell is activated, NF-κB /iκB complex undergoes a series of phosphorylation and proteolysis process upon which liberated NF-κB migrates into the nucleus
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and activates the gene expression of proinflammatory cytokines and mediators such as TNF-α and iNOS (Denkers, Butcher, Del Rio, & Kim, 2004). We also searched for phosphorylated-JNK
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(p-JNK), -ERK (p-ERK) and -p38 (p-p38) proteins in the macrophage cells and the positive
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results of constantly increasing band intensities suggested the mitogen activated protein kinase (MAPKs) as being part of the macrophage stimulation mechanism by CIF2 polysaccharides (Figs. 5C and 5D).
17
Following the determination of transduction pathways, the interaction mechanism between RAW264.7 macrophage cells and CIF2 polysaccharide molecules was sought using specific antibodies including anti-TLR4, anti-TLR2 and anti-CR3. Interestingly, as shown in Fig. 6, it
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was found that CIF2-induced NO release was notably diminished (51.7%) in the presence of TLR4 antibody (p < 0.05). Nonetheless, the inhibitory effects of anti-TLR2 and anti-CR3 on the production of NO from the CIF2-activated macrophages were not considerable. On the basis of these results, we concluded that TLR4 was the main receptor through which CIF2 polysaccharides interacted with macrophage cells and triggered NF-κB and MAPKs signaling
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pathways leading to initiation of a cascade of gene expressions and the following release of
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proinflammatory mediators. TLRs, with 10 known members, are the most characterized family
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of pattern recognition receptors (PRRs) in mammalian cells among which TLR4 is actively
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engaged in recognition of gram-negative bacteria, activation of dendritic cells and macrophages (Janeway, & Medzhitov, 2002). Previous investigations have also reported that polysaccharides
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can induce TLR4-mediated macrophage activation (Kim, Kim, Lee, Ryu, Kim, Yoon, & Han,
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2012).
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3.4. Linkage analysis of CIF2 polymer chain Partially methylated products of CIF2 polysaccharides were hydrolyzed with acid, reduced
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with NaBD4 and acetylated with acetic anhydride to produce final derivatives generally called partially methylated alditol acetates (PMAAs) which were detected by GC-EIMS. The identification of PAMMs and corresponding deduced glycosidic linkages are presented in Fig. 7A and Table 3. The methylation results were nearly consistent with the overall monosaccharide 18
composition and suggested a very complex structure for the purified CIF2 polysaccharide. The detection of large amount of 2,4-Me2-Fucp in the final derivatives indicated that the backbone of CIF2 polysaccharides was mainly consisted of (1→3)-fucopyranose residues. A portion of fucose
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sugars were involved in the structure of CIF2 as non-reducing terminal units as interpreted from the existence of 2,3,4-Me3-Fucp products. The presence of 2,3,4,6-Me4-Glcp, 4,6-Me2-Glcp and 6-Me-Glcp products suggested the involvement of glucose units in the polysaccharide structure as (→1)-, (1→2,3)- and (1→2,3,4)-glucopyranose residues. Additionally, mannoses were found in the form of 3,4,6-Me3-Manp, 2,3,6-Me3-Manp, 4,6-Me2-Manp, 6-Me-Manp products
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suggesting their connections through (1→2)-, (1→4)-, (1→2,3)- and (1→2,3,4)-mannopyranose.
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The presence of 2,3,4,6-Me4-Galp, 2,4,6-Me3-Glcp, 2,3,4-Me3-Galp, and 2,4-Me2-Galp indicated
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the glycosidic linkages of galactose units as (→1)-, (1→3)-, (1→6)- and (1→3,6)-
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galactopyranose residues. There also were different levels of 2,3,4-Me3-Xylp, 3,4-Me2-Xylp and 3,4-Me2-Rhap indicating the existence of (→1)- and (1→2)-xylopyranose and (1→2)-
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rhamnopyranose residues. In accordance with the glycosidic linkages of CIF2 fucoidan, (1→3)-
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fucopyranose has been found as the major part of the backbone of various fucoidans from
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Sargassum mcclurei, S. japonica and S. latissima (Thinh, Menshova, Ermakova, Anastyuk, Ly, & Zvyagintseva, 2013; Cumashi et al., 2007). In addition, the engagement of galactose units in
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(1→3) and (1→6) glycosidic linkages seems to be a shared characteristic among fucoidans obtained from many types of sources (Thinh et al., 2013; Borazjani et al., 2018). However, we
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think it is noteworthy to point out that, as mentioned above, the methylation analysis of the purified polysaccharide revealed the presence of high amount of 6-Me-Glcp which is relatively high compared to that of other fucoidans. Given the roughly matching ratios of terminal groups and branching points, we assumed that there was a high possibility of existing some sulfate esters 19
on 6-Me-Glcp residues along with other parts being presumably located on fucose and/or galactose units. However, since we were not be able to carry out the desulfation analysis in the present study, further experiments are required to provide assurances.
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Altogether, the present methylation analysis showed that although CIF 2 polysaccharide from C. indica was purified on an anion-exchange column and the RI chromatogram confirmed its molecular homogeneity, its chemical structure was yet very complex. This complexity in the building constituents of CIF2 polysaccharide was also obvious from the overlapping signals of 1
H NMR spectrum (Fig. 7B). However, the presence of different spin systems were observed in
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chemical shifts of 4.7-5.7 ppm corresponding to anomeric protons, 3.0-4.5 ppm corresponding to
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ring protons (H-2-H-5) and 1.3-1.5 ppm corresponding to methyl protons (H-6) of fucose
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residues. It is noteworthy to mention that our attempts to attain 13C NMR and 2D NMR did not
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4. Conclusions
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produce proper results.
A sulfated polysaccharide (CIF2) was successfully purified from C. indica using a DEAE
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Sepharose Fast Flow chromatography column. The CIF2 polysaccharide had a homogenous molecular distribution with the average molecular weight of 1146.6 × 103 g/mol. The building
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blocks of CIF2 polysaccharides were mainly fucose, glucose, mannose and galactose as well as lower amounts of rhamnose and xylose. The greatest amount of nitric oxide was produced from RAW264.7 murine macrophages induced by CIF2 polysaccharides. The interaction of RAW264.7 cells with CIF2 polysaccharides occurred through the cell surface TLR4 initiating the 20
cascade activations of NF-κB and MAPKs signaling pathways and the following proinflammatory response. The intermolecular connections of CIF2 sugars were highly complex and yet sharing the common (1→3)-fucopyranose, (1→3) and (1→6)-galactopyranose residues
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of fucoidans. However, further studies are needed to fully elucidate the sequence and repeating units of CIF2 polysaccharide with aid of tightly controlled hydrolysis using acid or specific enzymes prior to NMR spectroscopy.
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Acknowledgement
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The authors would like to thank Tarbiat Modares University for its partial financial support
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of this work through Graduate Student Research Fund. We are also grateful for the additional
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support provided by Basic Science Research Program through the National Research Foundation
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of Korea (NRF) funded by the Ministry of Education (No. 2018R1A6A1A03023584).
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Tabarsa, M., Shin, I. S., Lee, J. H., Surayot, U., Park, W., & You, S. (2015). An immuneChlorella
vulgaris
and
structural
characteristics. Food Science and Biotechnology, 24(6), 1933-1941.
Thinh, P. D., Menshova, R. V., Ermakova, S. P., Anastyuk, S. D., Ly, B. M., & Zvyagintseva, T. N. (2013). Structural characteristics and anticancer activity of fucoidan from the brown alga
U
Sargassum mcclurei. Marine Drugs, 11, 1456–1476.
N
Tripathi, P., Tripathi, P., Kashyap, L., & Singh, V. (2007). The role of nitric oxide in
A
inflammatory reactions. FEMS Immunology & Medical Microbiology, 51(3), 443-452.
M
Vo, T. S., & Kim, S. K. (2013). Fucoidans as a natural bioactive ingredient for functional foods. Journal of Functional Foods, 5(1), 16-27.
D
Wang, J., Zhang, Q., Zhang, Z., & Li, Z. (2008). Antioxidant activity of sulfated polysaccharide
TE
fractions extracted from Laminaria japonica. International Journal of Biological
EP
Macromolecules, 42, 127–132.
Wu, L., Sun, J., Su, X., Yu, Q., Yu, Q., & Zhang, P. (2016). A review about the development of
CC
fucoidan in antitumor activity: Progress and challenges. Carbohydrate Polymers, 154, 96111.
A
Yu, Y., Shen, M., Song, Q., & Xie, J. (2017). Biological activities and pharmaceutical applications of polysaccharide from natural resources: A review. Carbohydrate Polymers, 183, 91-101.
24
Zhang, X., Qi, C., Guo, Y., Zhou, W., & Zhang, Y. (2016). Toll-like receptor 4-related immunostimulatory polysaccharides: Primary structure, activity relationships, and possible
A
CC
EP
TE
D
M
A
N
U
SC RI PT
interaction models. Carbohydrate Polymers, 149, 186-206.
25
M
A
N
U
SC RI PT
Figure captions
A
CC
EP
TE
Fast Flow column.
D
Fig.1. Stepwise elution profile of crude polysaccharide from C. indica on DEAE Sepharose
26
SC RI PT U N A M D TE EP CC A
Fig. 2. GC chromatograms of monosaccharides of crude, CIF1 and CIF2 polysaccharides.
27
28
D
TE
EP
CC
A
SC RI PT
U
N
A
M
Fig. 3. Refractive index (RI) profiles of crude, CIF1 and CIF2 polysaccharides on a TSK
A
CC
EP
TE
D
M
A
N
U
SC RI PT
G5000PW column.
Fig. 4. RAW264.7 murine macrophage proliferation (A) and nitric oxide production (B)
induced by crude, CIF1 and CIF2 polysaccharides. Bars with different letters indicate a significant difference between the test groups (p < 0.05). 29
30
D
TE
EP
CC
A
SC RI PT
U
N
A
M
SC RI PT U N A M D TE
Fig. 5. The mRNA expression of iNOS, IL-1β, TNF-α, IL-6 and IL10 in the presence of
EP
CIF2 in RAW264.7 cells (A and B). The activation of NF-κB, JNK, ERK and p38 in RAW264.7
CC
cells induced by CIF2 (C and D). Bars with different letters indicate a significant difference
A
between the test groups (p < 0.05). β-actin was used as a control.
31
SC RI PT
U
Fig. 6. The nitric oxide release from RAW264.7 cells in the presence of CIF2 polysaccharide.
N
The cells were incubated with antibodies of receptors (TLR4, TLR2 and CR3) for 2 h before
A
stimulation with CIF2 polysaccharides. Bars with different letters indicate a significant difference
A
CC
EP
TE
D
M
between the test groups (p < 0.05).
32
SC RI PT U N A M D TE EP CC A
Fig. 7. GC-EIMS chromatogram of PMAAs of CIF2 polysaccharide (A). Helium was used as the carrier gas at a constant flow rate of 1.2 mL/min in GC-EIMS. 1H NMR chromatogram of CIF2 polysaccharide. The polysaccharides were dissolved in D2O and scanned at 50 °C.
33
Table
Table 1
Genes
Primer sequences (5’→3’) Forward
CCCTTCCGAAGTTTCTGGCAGCAGC
Reverse
GGCTGTCAGAGCCTCGTGGCTTTGG
Forward
ATGAGCACAGAAAGCATGATC
Reverse
TACAGG CTTGTCACTCGAATT
Forward
ATGGCAACTATTCCTGAACTCAACT
Reverse
CAGGACAGGTATAGATTCTTTCCTTT
Forward
TTCCTCTCTGCAAGAGACT
Reverse
TGTATCTCTCTGAAGGACT
Forward
TACCTGGTAGAAGTGATGCC
iNOS
U
TNF-α
A
N
IL-1β
CATCATGTATGCTTCTATGC
TE
Reverse
D
IL-10
M
IL-6
Forward
TGGAATCCTGTGGCATCCATGAAAC
Reverse
TAAAACGCAGCTCAGTAACAGTCCG
A
CC
EP
β-actin
SC RI PT
Sequence of oligo (dT) primers used for the rt-PCR
34
Table 2 Yield, chemical composition and molecular properties of the polysaccharides from C. indica. Crude
CIF1
Neutral sugars (%)
34.5 ± 1.9
71.5 ± 0.27
73.1 ± 0.4
78.6 ± 0.7
6.5 ± 0.4
5.8 ± 0.1
9.0 ± 0.3
1.3 ± 0.4
6.9 ± 0.3
9.7 ± 0.2
11.3 ± 0.2
11.2 ± 0.2
17.2 ± 0.9
24.4 ± 0.3
5.6 ± 0.1
5.8 ± 0.3
14.5 ± 0.3
17.8 ± 0.3
20.6 ± 0.4
14.2 ± 0.5
30.8 ± 1.6
21.3 ± 0.7
23.2 ± 0.6
17.3 ± 0.3
16.7 ± 0.5
Protein (%）
7.4 ± 0.2
Uronic acid (%)
2.9 ± 0.7
Sulfate (%)
12.9 ± 0.5 9.5 ± 0.1
Fucose
35.3 ± 0.3
Xylose
3.2 ± 0.2
U
Rhamnose
N
Monosaccharide
A
composition (%) Glucose Galactose
10800.5 ± 133.6
-
-
Peak II
1651.6 ± 37.5
573.1 ± 6.0
1146.6 ± 29.9
Peak I
102.4 ± 3.7
-
-
Peak II
105.1 ± 5.6
85.2 ± 4.1
64.7 ± 4.9
SVg (cm3/g)
Peak I
0.2 ± 0.0
-
-
Peak II
1.7 ± 0.2
2.7 ± 0.4
0.6 ± 0.1
TE
Peak I
CC
D
MW × 103 (g/mol)
M
Mannose
55.8 ± 1.5
SC RI PT
5.7
Yield (%）
CIF2
EP
Rg (nm)
A
ND: not detected, Mw: Mean average molecular weight, Rg: Radius of gyration, SVg: Specific volume of gyration
35
Table 3
Time PMAAsa (min)
Deduced glycosidic CIF2 linkage
7.88
1,5-di-O-acetyl-2,3,4-tri-O-methyl-Xyl
Xylp-(1→
8.35
1,5-di-O-acetyl-2,3,4-tri-O-methyl-Fuc
9.50
1,2,5-tri-O-acetyl-3,4-di-O-methyl-Xyl
9.88
1,2,5-tri-O-acetyl-3,4-di-O-methyl-Rha
SC RI PT
Glycosidic linkage analysis of the constituent sugars of the fraction F2 from C. indica.
8.09
Fucp-(1→
7.63
→2)-Xylp-(1→
3.27
→2)-Rhap-(1→
7.66
Galp-(1→
8.40
Glcp-(1→
1.91
→2)-Manp-(1→
1.54
→3)-Fucp-(1→
18.21
→4)-Manp-(1→
6.60
→3)-Galp-(1→
4.27
12.31 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-Gal
→6)-Galp-(1→
3.06
13.21 1,2,3,5-tetra-O-acetyl-4,6-di-O-methyl-Man
→2,3)-Manp-(1→
6.72
→2,3)-Glcp-(1→
3.31
14.25 1,3,5,6-tetra-O-acetyl-2,4-di-O-methyl-Gal
→3,6)-Galp-(1→
4.67
14.34 1,2,3,4,5-penta-O-acetyl-6-mono-O-methyl-Man
→2,3,4)-Manp-(1→
2.44
→2,3,4)-Glcp-(1→
12.17
10.17 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-Gal 10.53 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-Glc
A
11.97 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-Man
N
11.78 1,3,5-tri-O-acetyl-2,4-di-O-methyl-Fuc
U
11.71 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyl-Man
D
M
12.12 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-Gal
EP
TE
13.41 1,2,3,5-tetra-O-acetyl-4,6-di-O-methyl-Glc
17.59 1,2,3,4,5-penta-O-acetyl-6-mono-O-methyl-Glc Partially O-methylated alditol acetates
A
CC
a
36