Assessment of biochemical and immunomodulatory activity of sulphated polysaccharides from Ulva intestinalis

Accepted Manuscript Title: Assessment of biochemical and immunomodulatory activity of sulphated polysaccharides from Ulva intestinalis Author: Napassorn Peasura Natta Laohakunjit Orapin Kerdchoechuen Punchira Vongsawasdi Louis Kuoping Chao PII: DOI: Reference:

S0141-8130(16)30473-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.05.062 BIOMAC 6123

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

20-12-2015 12-3-2016 15-5-2016

Please cite this article as: Napassorn Peasura, Natta Laohakunjit, Orapin Kerdchoechuen, Punchira Vongsawasdi, Louis Kuoping Chao, Assessment of biochemical and immunomodulatory activity of sulphated polysaccharides from Ulva intestinalis, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.05.062 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.

1 Assessment of the biochemical and immunomodulatory activity of sulphated polysaccharides from Ulva intestinalis Napassorn Peasura a, Natta Laohakunjit a,*, Orapin Kerdchoechuen a, Punchira Vongsawasdi b

, Louis Kuoping Chao c

a

Division of Biochemical Technology, School of Bioresources and Technology, King

Mongkut’s University of Technology Thonburi, 49 Tientalay 25 Rd., Takham, Bangkhuntien, Bangkok 10150, Thailand b

Department of Microbiology, Faculty of Science, King Mongkut’s University of

Technology Thonburi, 126 Pracha Uthit Rd., Bang Mod, Thung Khru, Bangkok 10140, Thailand c

Department of Cosmeceutics, China Medical University, 91 Hsueh-Shih Road, Taichung,

Taiwan, 40402, R.O.C *Corresponding author: Dr. Natta Laohakunjit, Division of Biochemical Technology, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, 49 Tientalay 25 Rd., Takham, Bangkhuntien, Bangkok 10150, Thailand Email: [email protected], phone: +66 2 470 7752, fax: +66 2 470 7781

2 Highlights  Sulphated polysaccharides from U. intestinalis fractionated on a silica-silica column activated macrophages. 

The molecular weight and sulphate/total sugar ratio affect the immunomodulatory activity of sulphated polysaccharides.



Fractionated sulphated polysaccharides showed higher scavenging activity and immunomodulatory activity than unfractionated sulphated polysaccharides.

Abstract The biochemical characteristics and immunomodulatory activity of sulphated polysaccharides isolated from Ulva intestinalis and fractionated using a silica-silica column were investigated. The unfractionated (USP) and fractionated sulphated polysaccharides (FSP4, FSP30, and FSP32) consisted mostly of carbohydrates (4.84–26.55%) and sulphates (2.85-20.42%). Structural analyses showed that USP, FSP4, FSP30 and FSP32 had molecular weights of 300, 80, 110 and 140 kDa, respectively. FSP30 exhibited the strongest DPPH• radical scavenging activity. Moreover, FSP30 showed stronger immunomodulatory activities than UPS in term of stimulating the production of pro-inflammatory cytokines, including nitric oxide (NO), tumour necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), in macrophage J774A.1 cells. USP and FSP30 were not cytotoxic to mouse macrophage at the tested concentrations (6.25 to 50 µg/mL). The results suggested that U. intestinalis polysaccharides could be explored as potential antioxidant and immunomodulatory agents to be used as complementary medicine or functional foods. Key words: Ulva intestinalis sulphated polysaccharide silica-silica column immunomodulatory activity

3 1. Introduction Sulphated polysaccharides are bioactive macromolecules in which some of the hydroxyl groups of the sugar residues are substituted by sulphate groups. These anionic polysaccharides are ubiquitous in nature and occur in a wide variety of organisms, including mammals, invertebrates and flora [1]. In algae, sulphated polysaccharides are the major component of the cell wall matrix [2]. Sulphated polysaccharides from marine algae have been mainly found in brown algae (Phaeophyta), such as fucoidans, ascophyllan, sargassum and glucuronoxylofucan, and in red algae (Rhodophyta), such as agar and carrageenan [1]. In contrast to sulphated polysaccharides found in brown and red algae, the major sulphated polysaccharide of green algae is more heterogeneous in sugar composition; its three main groups are glucuronoxylorhamnans, glucuronoxylorhamnogalactans and xyloarabinogalactans [3]. These sulphated polysaccharides are of particular interest because they are bioactive, i.e., they exert anticoagulant, antiviral, antitumor and anti-inflammatory activity [4]. Ulva intestinalis is a green seaweed belonging to the family Ulvaceae. They are a rich source of sulphated polysaccharides that are referred to as ulvans. Sulphated polysaccharides mainly contain the sugars rhamnose, xylose, and glucuronic acid, as well as small amounts of other sugars (galactose, arabinose, mannose, and glucose) [5]. Sulphated polysaccharides from U. intestinalis feature a typical sugar backbone, like glucose and rhamnose, and sulphate is attached to rhamnose at C-2 or C-3 [6]. Various monosaccharide components of the sulphated polysaccharide chain, such as L-arabinose, D-glucose, L-mannose and L-rhamnose, promote antioxidant [7] and immunomodulatory activity [8]. Specifically, L-rhamnose and α-Lrhamnose oligosaccharides have been described to strongly interact with specific receptors on several proteins that are involved in immunomodulatory activity [7]. The α-L-rhamnose, a component of glycosides or polysaccharides, is especially distributed in plants and microorganisms and recognized by functional antibodies in humans, which is an

4 important aspect of immunomodulatory activity [9]. Sulphated polysaccharides also exert various biological and pharmacological activities, including immunomodulatory activity [1]. The immunomodulatory activities of sulphated polysaccharides depend on their characteristic parameters, such as the degree of sulphation (DS), carbohydrate content and sulphate content/total sugar ratio [10]. Sulphated polysaccharides from algae have been reported to stimulate the secretory activity of macrophages, induce the production of nitric oxide (NO), and enhance the secretion of cytokines and chemokines, such as tumour necrosis factor (TNF-α) and interleukin IL-1β [11]. Leiro et al. [12] reported that sulphated polysaccharides from Ulva rigida extracted with distilled water and purified by anion exchange were potent immunomodulators, stimulating murine macrophage RAW 264.7 cells and inducing the secretion of nitric oxide (NO). In a previous study, sulphated polysaccharides from Codium fragile extracted with a solvent enhanced the production of pro-inflammatory cytokines from RAW 264.7 cells, including interleukins-1, 6, and 12, tumour necrosis factor-α and anti-inflammatory (IL-10) cytokines [13]. Furthermore, RAW 264.7 cells stimulated with fractionated sulphated polysaccharides from red algae exhibited the increases in TNF-α production than cells stimulated with lipopolysaccharides (LPS), the endotoxin from gram-negative bacteria, which could be elicited a variety of responses in macrophages, including stimulating nitric oxide production [2]. Additionally evidence suggests that sulphated polysaccharides extracted from Capsosiphon fulvescens and polysaccharides fractionated by ion-exchange chromatography might be strong stimulators of RAW264.7 cells, resulting in the production of a considerable amount of NO [14]. According to Paul et al. [15], protein kinase C activation has been identified as an early response in LPS-stimulated macrophages and might be essential for the up-regulation of NO production. Furthermore, the fraction of sulphated polysaccharides

5 separated by DEAE-Sepharose from Prunella vulgaris L. showed stronger immunomodulatory activities than crude sulphated polysaccharides [16]. Although the previous study have explored the antioxidant activity of hot water-extracted sulphated polysaccharides from U. intestinalis [6], the research on the biochemical and immunomodulatory activity of U. intestinalis polysaccharides fractionated with a silica-silica column has not been reported. The present study was designed to explore the immunomodulatory activity of sulphated polysaccharides and fractionated sulphated polysaccharides from U. intestnali. The effects of sulphated polysaccharide fractions on macrophage viability, nitric oxide production, and cytokine production were also monitored in order to explore the immunomodulatory activity and induce transcription of key cytokines for further useful immunopotentiators 2. Materials and methods

2.1. Materials Green seaweed, Ulva intestinalis, was harvested in July 2015 from the Pattani bay in Pattani province, Thailand. The raw material was washed with tap water and air-dried at 60°C. The dried raw material was ground using a universal mill (IKA-M20, Germany), sieved (80 mesh) and stored at −20°C before the extraction of the polysaccharide. 2.2. Extraction of sulphated polysaccharide The fine seaweed powder (20 g) was treated with 95% ethanol (EtOH, 200 mL) at room temperature for 24 h to remove lipophilic pigments (such as chlorophylls and carotenes) and low-molecular-weight proteins [17]. The sample was filtered with 40 mesh of nylon cloth (Industrial Netting, USA) and dried at room temperature. The dried biomass (20 g) was gently mixed with distilled water (400 mL) using a hot plate stirrer bar at 80°C for 24 h. The mixture was then centrifuged at 18,500×g for 10 min, and the supernatant was collected. The

6 supernatant volume was reduced to 100 mL in a rotary evaporator (IKA, RV10, Germany), mixed with 3 volumes of ethanol, and allowed to incubate overnight at 4°C. The precipitated polysaccharide, which was obtained by centrifugation at 6000×g for 15 min, was referred to as unfractionated sulphated polysaccharide (USP). The USP was dried at 60°C for 3 h, stored in plastic bags, and placed in a desiccator for further fractionation (see below). 2.3. Fractionation of the crude sulphated polysaccharides (Refference) The sulphated polysaccharide extract (30 mg) was dissolved in distilled water (10 mL), and the solution was then injected into a silica-silica column (RediSep®, Flash column 12 g, USA). The column was first washed with 1 M NaCl and subsequently with a distilled water gradient; 1 M NaCl/distilled water (80/20, v/v), 1 M NaCl/distilled water (60/40, v/v), 1 M NaCl/distilled water (40/60, v/v) and 1 M NaCl/distilled water (20/80, v/v) at 0.5 mL/min. The absorbance of the eluant at 490 nm was continually monitored. Eluant representing a particular peak in absorbance was collected in 10 tubes, each containing 4 mL of liquid. The total carbohydrate content of an aliquot from each tube was determined using the phenolH2SO4 method [18], and the three tubes with the highest carbohydrate measurements for a particular elution peak were pooled, dialyzed into dialysis bag, and lyophilized as described by Leiro et al. [12]. 2.4 Biochemical characteristics of unfractionated sulphated polysaccharide (USP) and fractionated sulphated polysaccharides (FSP) 2.4.1 Determination of total sugar The total sugar content was determined with the phenol-sulphuric method [18]. In brief, the dried USP (0.03 g) was dissolved in 3 mL of distilled water. The 0.5 mL of the solution was mixed with 0.5 mL of 5% (w/v) phenol and 2.5 mL of 98% concentrated sulphuric acid and immediately vortexed after mixing. After standing for 20 min at room temperature, the absorbance of the sample solution was measured at 490 nm with a spectrophotometer (G-10

7 UV scanning, USA.). Glucose was used as the standard for the calibration curves and expressed as g/100 g dried weight of sample. 2.4.2 Determination of sulphate content The sulphate content was analysed using the barium chloride-gelatine method [19]. Dried sulphated polysaccharide (2 mg) was hydrolysed in 0.2 mL of 1 N HCl at 95˚C in a sealed tube for 5 h. After cooling samples at room temperature, residue was re-dissolved in 1 mL of distilled water. The sulphated polysaccharide solution was mixed with 3.8 mL of 3% (v/v) trichloroacetic acid, and 1 mL of barium chloride-gelatine reagent (2 g of gelatine and 2 g of barium chloride in 400 mL of water at 60-70°C). The mixtures were allowed to stand for 20 min, and their turbidities were measured at 360 nm by spectrophotometry (Genesys 10 UV, Thermo electron cooperation, USA). The results were calculated based on a sodium sulphate standard curve and expressed as gram of sulphate/100 g dried mass of sample. The degree of substitution (DS) was calculated based on the sulphur content according to following equation [20] DS = 1.62× SO42 (%) / (32−1.02× SO42 (%) Degree of sulphation (DS) or degree of substitution of sulphated polysaccharides is defined as the average number of hydroxyl groups (–OH) substituted by ester-linked sulphate groups (–SO3-) per repeating sugar in the polysaccharide backbone. 2.4.3 Determination of molecular weight The average relative molecular weights of the USP and FSP were estimated by High Performance Size Exclusion Chromatography (HPSEC) (Water 600E, USA) using an ultrahydrogel linear column (WAT 011545, Waters, USA). USP and FSP (0.2 g) were dissolved in 10% (w/v) NaCl and filtered through a 0.45-µm nylon cloth [21]. The supernatant was injected, eluted with 0.05 M sodium bicarbonate buffer (pH 11) at a flow

8 rate of 0.6 mL/min and detected with a refractive index detector. Dextrans were used as the relative molecular mass standards (MW: 4.4-401 kDa). 2.4.4 Antioxidant activity based on a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity assay The DPPH radical scavenging assay was conducted using the Shimada method [22]. Aliquots of USP and FSP solutions (2 mL) were separately mixed with 2 mL of 0.1 DPPH in 95% methanol (v/v). After 30 min of incubation, the absorbance of each reaction mixture was measured at 517 nm. The DPPH radical scavenging activities of the USP and FSP fractions were calculated as a percentage according to the following equation [22]: % Scavenging activity = [(ADPPH – Asample+DPPH)/ADPPH] ⨯ 100 2.5 Immunomodulatory activity of unfractionated and fractionated sulphated polysaccharide 2.5.1 Cell lines Murine macrophage J774A.1 cells were obtained from ATCC (Rockville, MD). The cells were placed in RPMI 1640 medium (analytical grade) (Lonza, Walksville, MD, USA.) containing 10% (v/v) fetal bovine serum (Hyclone Co., Logan, UT) and 2 mM L-glutamine (Life Technologies, Inc., MD) and cultured at 37°C in a CO2 incubator where CO2 concentration was held constant at 5%. 2.5.2 Determination of cell viability by alamarBlue® assay The macrophage cell viability was determined based on the percentage of reduction of alamarBlue® as described in Shin et al. [23]. The J774A.1 cells were seeded in a 96-well microplate (1×106 cells/mL) and incubated in RPMI-1640 medium containing 10 µL unfractionated sulphated polysaccharides and fractionated sulphated polysaccharides at concentrations of 6.25, 12.5, 25, 50 and 100 µg/mL. Each concentration was tested in triplicate. The cells were placed for 48 h at 37°C in a 5% CO2 incubator. After this incubation

9 period, 20 µL of alamarBlue® was added to each well, and the mixture was then incubated for 4 h in the dark. The percentage of alamarBlue® reduction was detected with a Synergy HT instrument (BIO-TEK) using KC4 v3.1 software and calculated using the following formula [24]: Cell viability (%) =

(117,216×A570 of sample) – (80,586×A600 of sample ×100 (155,677×A600 of media) – (14,652× A570 of media)

2.5.3 Nitric oxide (NO) production assay NO secretion from macrophages and the nitrite concentration were measured using the Griess reaction as described by Green et al. [25]. The J774A.1 cells were cultured in a 96-well microplate (1×106 cells/mL in a volume of 500 µL) and stimulated with the USP and FSP30 (6.25, 12.5, 25, 50 and 100 µg/mL). Cells treated with 1 µg/mL lipopolysaccharide solution (LPS) (Thermo Fisher Scientific, USA) served as a positive control, and untreated cells (without USP or FSP) served as negative controls. After 24 h of incubation at 37°C, 100 µL of cultured cell supernatant was mixed with 50 µL of Griess reagent (1% (w/v) sulphanilamide, 0.1% (w/v) N-[1-naphthyl]-ethylenediamide dihydrochloride in 5% (w/v) phosphoric acid). After the reaction, products were incubated for 10 min in the dark at room temperature, the absorbance was measured at 550 nm on a microplate reader. The NO secretion from the J774A.1 cells was calculated with reference to a standard curve obtained using NaNO2 (6.25-100 µM). 2.5.4 Tumor Necrosis Factor-alpha (TNF-α) quantification using enzyme linked immunosorbent assay (ELISA) Murine J774A.1 macrophages (1×106 cells/mL) were seeded on a 96-well tissue culture plate in standard medium in the absence (negative control) or presence of varying concentrations of USP or FSP30 (25, 50, 100 and 200 μg/mL). The LPS (1 µg/mL) was used as a positive control. TNF-α secretion was assessed after 6 h incubated at 37 °C under 5% CO2. Specifically, an aliquot of the supernatant from each culture was collected at these time

10 points and maintained at −80 °C for further analysis. The TNF-α concentrations in the supernatant were measured using a mouse TNF-α quantikine ELISA kit (R&D Systems, Inc., Minneapolis, MN), following the manufacturer’s instructions (abcam® , USA) The results are expressed in pg/mL. 2.5.5 Expression of interleukins 1β (IL-1 β) using Western Blot Analysis The expression of IL-1β and β-actin was quantified via western blot analysis as described by Zhang et al. [26]. The cells were cultured (2×106 cells/dish) in 2 mL of RPMI 1640 culture medium containing different concentrations of USP and FSP30 (0, 50, 100 and 200 μg/mL). LPS (1 μg/mL) was used as a positive control. The cells were cultured at 37 °C in a 5% CO2 incubator for 6 h, and the supernatants were lysed with 100 μL cell lysis buffer (0.1 M PMSF, 0.1 M Na3VO3, 0.5 M NaF) containing freshly added 1% (w/v) protease inhibitor (pancreas extract, pronase, thermolysin, chrymotrypsin, and papain), following the manufacturer’s instructions (Roche Diagnostics, Indianapolis, IN). The supernatant was collected after centrifugation at 13,200×g for 20 min and assayed for protein content with a BCA assay kit using bovine serum albumin as a standard (Sigma-Aldrich, USA). After heat denaturation for 7 min, the aliquot containing 40 µg of protein was separated on a 10% (w/v) sodium dodecyl sulphate–polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% (w/v) skim milk in tris buffer saline, 0.1% (v/v), Tween 20 (TBST) (Merck Millipore, Germany) overnight. The blots were probed with primary antibody (rabbit polyclonal antibody) (Thermo Fisher Scientific, USA) prepared in phosphate buffer saline (PBS) at a concentration of 0.1 µg/mL at 4°C for 1.5 h. Subsequently, the membranes were washed with tris buffered saline (TBS) and incubated with an appropriate secondary antibody (horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG) (Thermo Fisher Scientific, USA) in 2% (v/v) PBS for 2 h. After washing the membrane with TBS three times for 10 min, the blots were visualized using an enhanced

11 emission of light between horseradish peroxidase (HRP)-labeled antibodies and substrate (chemiluminescence detection system). The membranes were exposed to X-ray films. The bands were visualized with an LAS-400 image reader (Fujifilm Life Science Co., Japan). 2.6 Statistical analysis All experiments were performed in triplicate in a Randomized Complete Block Design, and the results represented the average of three independent experiments. The statistical analysis was performed using the Statistical Analysis System (SAS, version 6.0) (SAS Institute, Cary, NC, USA). Duncan’s Multiple Range Test was used to determine significant differences between means (p < 0.05). 3. Results and Discussion 3.1 Extraction and fractionation of sulphated polysaccharide Sulphated polysaccharides were extracted from U. intestinalis and designated as unfractionated sulphated polysaccharides (USPs). The USPs were loaded onto a silica-silica column for purification based on the polarity of the sulphated polysaccharide extract, which was successively eluted with progressively lower concentrations of NaCl solution. As shown in Figure 1A, three main fractions, namely FSP4, FSP30 and FSP32, were collected based on the total carbohydrate measured using the phenol–sulphuric acid test. When USPs extract were loaded, and sulphated polysaccharides retained in silica-silica column which contained silanol (≡Si─OH) groups on the surface. The first eluant from fractionation, FSP4 consisted low sulphate content, which it might be slightly polar [27]. It could be explained that Na+ from NaCl directly interacted with sulphate groups by hydrogen bonding in disaccharides back bone structure of sulphated polysaccharides (ONa+SO3-) [28]. These bonds were especially strong intermolecular forces between sulphate group and sodium, resulting in early elute of FSP4. However, FSP32 contained high sulphate which increased expanded sulphated polysaccharides chain by forces among sulphate groups, resulting in the enhancement water

12 solubility and decrease the viscosity [29]. Consequently, distilled water ratio in eluent was increased during the gradient which promoted elution solvent polarity. FSP32 was separated from the column, which it indicated that polar eluent (distilled water) could be more effectively competed with high polar sulphated polysaccharide. It attached the polar group on silica-silica column surface, resulting in polar sulphated polysaccharides (FSP32) separation. The polarity of sulphated polysaccharide decreased in the following order: FSP4 ˂ FSP30 ˂ FPS32, and these results might relate to the high polarity of sulphated polysaccharide (FSP32) which might be attached to the silica more than the low polar fraction (FSP4), resulting in a long retention time [30]. 3.2 Biochemical characterization of unfractionated and fractionated sulphated polysaccharide 3.2.1 Total sugar Total carbohydrate and sulphate contents of the USP and the three fractions (FSP4, FSP30 and FSP32) were determined using the phenol–sulphuric acid test and the barium chloridegelatine method, respectively. Total sugar content values are shown in Table 1. The USP had the highest total sugar content (26.55%) in its readily dissolved the sulphated polysaccharide. Long-chain sulphated polysaccharides should agglomerate and precipitate, resulting in a high total sugar recovery yield [31]. Total sugar of USPs extracted with water at 80°C for 6 h from U. intestinalis was difference from Ulva fasciata (20.63%) [32], and Ulva lactuca (57.4%) [33]. In this study, USPs mainly consisted of rhamnose (12.70%), followed by glucose (11.86%) (data not shown) [6]. The variation of total sugar could be influenced by the growing conditions (water temperature, salinity, light, and nutrients) [34], which might reflect differences in the proportions of cell wall polysaccharides. Moreover, sulphated polysaccharides extracted from U. intestinalis might consist of other compounds, including protein and minerals which was associated tightly with polysaccharides. Due to unremoval of

13 other compound in USP, which could affect the biological activities. Effect of different composition in USPs and its fraction on immunomodulatory activity were studied and compared by cytokine secretion in macrophages. The USP was fractionated on a silica-silica column into three fractions, FSP4, FSP30 and FSP32, with total sugar contents of 4.85%, 8.20% and 13.11%, respectively. According to the analysis of biochemical compositions, the three fractions mainly consisted of carbohydrates, and FSP4 had the lowest total sugar content, whereas FSP32 had the highest total sugar content. 3.2.2 Sulphate content, degree of sulphation (DS) and sulphate/sugar ratio The sulphate contents of USP, FSP4, FSP30 and FSP32 are presented in Table 1. The USP had the highest sulphate content (20.42%), and this difference was significant (p˂0.05) compared with the sulphate contents of FSP4 (2.85%), FSP30 (8.85%), and FSP32 (8.91 %). The sulphate content of sulphated polysaccharides increased in the following order: FPS4 ˂ FSP30 ˂ FSP32. Specifically, the sulphate content directly correlated with the polarity of the eluting solvent. As shown in Table 1, USP showed the highest DS (2.96), which indicated a significantly higher sulphate content compared with DS values for FSP4, FSP30 and FSP32. The DS values of FSP4, FSP30 and FSP32 were 0.16, 0.62, and 0.63, respectively, which their values were related to its polarity. In sulphated polysaccharides extracted from U. intestinalis, the sulphate is linked to the rhamnose residue of the polysaccharide backbone by an ester bond (O–SO3-), which plays an important role in algal biological properties [35,36]. However, FSP30 showed the highest sulphate/sugar ratio (1.08), and FSP4 exhibited the lowest ratio (0.59). Results indicated that fractionation using silica-silica column chromatography yielded sulphated polysaccharides that varied on the relative polarity of their sulphate groups.

14 3.3.3 Molecular weight distribution Molecular weight distribution of the different sulphated polysaccharide fractions was determined by using High Performance Size Exclusion Chromatography (HPSEC). The RI chromatograms for the USP, FSP4, FSP30 and FSP32 are shown in Figure 1B. Based on calibration with dextrans, single peaks corresponding to USP, FSP4, FSP30 and FSFP32 were eluted from the HPSEC column between the elution times of 9 and 17 min, and almost sulphated polysaccharides (FSP4, FSP30 and FSP32) eluted during this interval were high and narrow peaks. This finding suggested that unfractionated and fractionated sulphated polysaccharides might be consist of polymers with homogeneous molecular weight distributions. The molecular weight (Mw) distributions of USP, FSP4, FSP30 and FSP32 were 300, 80, 110 and 140 kDa, respectively. Variations in MW have been found among unfractionated and fractionated sulphated polysaccharides. Differences in biochemical composition, such as differences in the total sugar and sulphate content, have also been found in USP and all fractionated polysaccharides. USP and FSP4 had the highest (300 kDa) and the lowest (80 kDa) molecular weight, respectively. This finding implied that deviations in the molecular weight of sulphated polysaccharides correlated with the biochemical composition. This difference in the elution profiles reflects differences in molecular size and the composition of these extracted sulphated polysaccharides [37].

3.3 In vitro antioxidant activity assay of unfractionated and fractionated sulphated polysaccharide 3.3.1 DPPH∙ radical scavenging activity (1,1-Diphenyl-2-pycrilhydrazyl) assay The free radical scavenging activity of USP, FSP4, FSP30 and FSP32 are presented in Figure 1C. All fractions of the sulphated polysaccharide exhibited DPPH radical-scavenging activity higher than 50%. FSP30 had the highest DPPH radical-scavenging activity (82.23%),

15 followed by FSP32 (72.43%), FSP4 (54.14%) and USP (51.30%). Although all fractions of sulphated polysaccharides with low sulphate contents and molecular weights had significantly higher (p < 0.05) DPPH• scavenging abilities than USP. In this study, FSP30 was found to exhibit the strongest antioxidant activity. The molecular weight of FSP30 was 110 kDa, which was much higher than FSP4 (80 kDa), but lower than USP (330 kDa). These results indicated that the molecular weight of polysaccharides plays an important role in their bioactivities. Sulphated polysaccharides with large molecular weight (330 kDa) might not expose plenty of active moieties, while sulphated polysaccharides with low molecular weight (80 kDa) probably are not able to form a suitable conformation to exhibit antioxidant activity [38]. Thus, a suitable molecular weight is important for sulphated polysaccharide to exhibit antioxidant activity. Moreover, it was believed that low molecular weight polysaccharide molecules would have more reductive -OH terminals available for reacting with radical species [6]. This effect was true for the activities measured by the DPPH radical scavenging method, which might be attributed to their electron donation power to the free radicals, thereby terminating the radical chain reactions [39]. Furthermore, the highest DS value (2.96) and low sulphate/sugar ratio (0.77) in USP showed lower (p<0.05) radical scavenging than FSP30, which its sulphate/sugar ratio was (1.08). Results suggested that the DPPH∙ radical scavenging of sulphated polysaccharides from U. intestinalis depended on the spatial patterns of sulphate groups. Several studies have reported that the antioxidant activity of other algal sulphated polysaccharides also directly correlated with the degree of sulphation [40,41]. The DS may not directly affect the radical-scavenging activity of SPF30, and the molecular weight change induced by sulphation may also contribute to the overall radical-scavenging capacity [42]. Although USP had the highest sulphate content and degree of sulphation, these sulphated polysaccharides had the lowest antioxidant activity. In addition, FSP30 showed significantly

16 higher (p˂ 0.05) radical scavenging than FSP4, FSP32 and USP, but it had a low sulphate content and degree of sulphation. For further study, USP and FSP30 were selected to investigate their immunomodulatory activity.

3.4 Immunomodulatory activity of USP and FSP30 3.4.1 Cell viability The effects of various USP and FSP30 fractions derived from U. intestinalis on the viability of murine macrophages are shown in Figure 2A. USP and FSP30 were not cytotoxic to J774A.1 cells at concentrations ranging from 6.25 to 25 µg/mL. The concentration of sulphated polysaccharides allowed >90% of the cells to remain viable, which was defined as not cytotoxic [43]. Results also showed that USP was not cytotoxic at a concentration of 50 µg/mL, whereas FSP30 at the same concentration was cytotoxic. However, this cytotoxicity was lower than that of LPS (1 µg/mL). At a concentration of 50 µg/mL, USP-treated cells showed greater than 90% viability, whereas the viability of FSP30-treated cells was 85.51%. Furthermore, the viability of cells was inversely correlated with the concentrations of USP and FSP30, indicating a dose-dependent relationship between the viability rates and the concentrations of sulphated polysaccharides. Conversely, a high level of cytotoxicity may indicate that sulphated polysaccharides damaged or interfered with the cell metabolism [44]. In the control group, cell viability was 100% after incubation for 24 h with medium. However, the viability of 1 µg/mL LPS-treated cells was 69.96%, which may be due to differences in the structural components of LPS, including lipid A, core oligosaccharide, and an O-specific polysaccharide chain. Specifically, lipid A is the endotoxic centre of LPS and showed higher activity than the core oligosaccharide and O-specific polysaccharide [45]. This response indicated that macrophages stimulated by LPS undergo cellular apoptosis, a phenomenon called activation-induced cell death [46].

17 3.4.2 Effect of USP and FSP30 on the production of nitric oxide by J774A.1 cells Nitric oxide (NO) is an important inflammatory mediator that can be produced by activated macrophages. It contributes to the killing of tumour cells and pathogenic microorganisms and acts as an intracellular messenger molecule to mediate a variety of biological functions [47]. The NO production induced by USP and FSP30 at different concentrations, expressed as the amount of NO, released from macrophage cells, is shown in Figure 2B. LPS (1 µg/mL) was used as a positive control. The level of NO produced by J774A.1 cells in response to USP and FSP30 was found to be relatively low (2.11 and 2.36 µM, respectively) at the highest concentration (100 µg/mL). In the present study, USP and FSP30 were found to effectively enhance the production of NO, indicating that these agents were able to functionally activate macrophages. USP and FSP30 were able to stimulate the production of NO in J774A.1 cells in a dose-dependent manner at concentrations ranging from 6.25–100 µg/mL, whereas a minimum amount of NO was released when J774A.1 cells were exposed to 6.25 µg/mL FSP30. The increases in the production of NO suggested that USP and FSP30 may activate the bactericidal and tumoricidal activity of macrophages by binding to the receptor of macrophages, activating macrophages by a series of identification steps and transferring bioinformation to exert their immunomodulating activity [48]. LPS activated macrophages, as evidenced by a significant increase in NO production compared with USP- and FSP30-treated cells. LPS is recognized by Toll-like receptor 4 (TLR-4) expressed in macrophages (J774A.1 cells) and activated mitogen activated protein kinases (MAPKs), including extracellular signal-related kinase [49]. These kinases promote the expression of inflammatory genes, such as nitric oxide synthases, which secrete nitric oxide [50]. However, FSP30 significantly reduced cell viability, as observed after 24 h of treatment at a concentration of 100 µg/mL, but 6.25 µg/mL USP treatment maintained high cell viability. This finding may indicate high NO production produced by macrophages can cause damage or interfere with cell metabolism

18 [44]. Although the DS of FSP30 was lower than that of USP, FSP30 induced significantly higher (p˂0.05) NO production (2.36 µM) than USP (2.11 µM) at 100 µg/mL. This finding suggested that other factors, such as the molecular weight and sulphate/total sugar ratio may act synergistically to induce NO release from macrophages [51]. In this study, high sulphate content and molecular weight of USP resulted in less effective than FSP30 [52]. Specifically, USP may be too large to move across the membranes of J774A.1 cells [12]. According to Maeda et al. [53], purified sulphated polysaccharides with an average molecular weight exceeding 100 kDa showed potent immunostimulatory activities in macrophage cells. Specifically, the addition of purified sulphated polysaccharides to murine RAW 264.7 macrophages increased NO production [14]. Conversely, higher molecular weight polysaccharides from Capsosiphon fulvescens were able to induce higher NO production in RAW 264.7 cells, as reported by Karnjanapratum et al. [14]. Although many studies have shown that high- and low-molecular-weight sulphated polysaccharide exert different effects in some diseases models [54], these differences have not yet been fully elucidated, especially in immune cells. Results showed that the production of NO reached a plateau at 100 μg/mL of USP and FSP30. The concentrations lower than 25 μg/mL had very little effect on macrophage activity. Therefore, concentrations of USP and FSP30 ranging from 25–200 μg/mL were used in subsequent experiments. 3.4.3 Effect of USP and FSP30 on tumour necrosis factor alpha Tumour necrosis factor alpha (TNF-α) is a cytokine with tumour necrosis activity that is primarily secreted by macrophages and has been recognized as an important host regulatory molecule. As shown in Figure 2C, USP and FSP30 induced the secretion of TNF-α from J774A.1 cells in a concentration-dependent manner. Specifically, USP and FSP30 significantly stimulated the secretion of TNF-α at concentrations ranging from 25–200

19 µg/mL, and 200 µg/mL FSP30 (551.67 pg/mL) exhibited higher activity than USP (501.67 pg/mL) at the same concentration. Therefore, USP and FSP30 could bind to the receptors on the surfaces of macrophages to stimulate the secretion of TNF-α. The TNF-α could activate macrophages to enhance various functional responses and induce the expression of other inflammatory and immune regulatory mediators [55]. However, FSP30 had a lower DS than USP, but both of these fractions produced similar macrophage activity. Results showed that not only sulphate group but also the proper DS is also necessary for inhibition TNF-α secretion. In addition to the degree of sulphation, the distribution of the sulphated group is an important factor influencing the anticoagulant activity. This result was consistent with those obtained using sulphated polysaccharide extracted from Ganoderma atrum [56]. The present study also showed that FSP30 induced higher TNF-α concentrations than USP, and these two fractions exhibited different molecular weight distributions. This finding indicated that the low molecular weight of FSP30 enhanced the binding affinity of vascular endothelial growth factor to macrophage receptors. This effect may be mediated by the formation of various bridge types, suggesting that lower Mw polysaccharides possess conformations that facilitate their binding capacity [57]. At a concentration of 200 μg/mL, the production of TNF-α in response to USP and FSP30 was higher than that of the LPS-stimulated positive control. Thus, sulphated polysaccharides recognize a range of cell adhesion systems, unlike LPS. Sulphated polysaccharide can bind to cluster of differentiation 2, 3, and 4 (CD2, CD3 and CD4) receptors in T- helper cells, and they can produce cytokines, such as interleukin-1 beta and TNF-α [58, 59]. In addition, the treatment of macrophage cells with LPS (1 µg/mL) caused a significant increase in the production of TNF-α, reaching a value of 476 pg/mL. This finding indicated that LPS-induced TNF-α secretion may be involved in the increases in nitrite production observed in J774A.1 cell cultured treated with LPS. Conversely, TNF-α can upregulate inducible nitric oxide synthase (iNOS), leading to the release of NO [60]. Thus,

20 the secretion of TNF-α in response to USP and FSP30 treatment may explain the increase in the NO production in J774A.1 cells. 3.4.4 Effect of USP and FSP30 on IL-1β expression The IL-1β is an important cytokine for the regulation of immune responses and it is involved in a variety of cellular activities, including the proliferation of T and B lymphocytes [61]. As shown in Figure 3A and B, USP and FSP30 remarkably stimulated J774A.1 cells to release IL-1β, which has been implicated as a key mediator in the response to immunological stimuli. A western blot analysis revealed that the level of IL-1β protein was very low in un-stimulated cells (control), but this level increased upon activation with USP, FSP30 and LPS. The concentrations of IL-1β secreted by RAW 264.7 cells in response to FSP30 were significantly higher (p < 0.05) than those in response to USP at all concentrations. Sulphated polysaccharide enhanced the production of IL-1β in a dose-dependent manner at concentrations ranging from 50−200 μg/mL. These results indicated that FSP30 had better stimulation the immune response via the release of IL-1β than USP. This difference might be related to differences in the molecular weight distribution. Specifically, the molecular weight of FSP30 was lower than that of USP. According to Lake et al. [57], low-Mw sulphated polysaccharides might enhance the binding affinity of vascular endothelial growth factor 165 (VEGF165) to its receptor, possibly via the formation of various bridge types. This relationship suggests that lower Mw polysaccharides possess conformations that facilitate their binding capacity [57]. Sulphated polysaccharides were recognized by specific receptors to activate macrophages. The receptors, such as Toll-like receptor 4, cluster of differentiation 14, complement receptor 3, scavenger receptor, dectin-1 and the mannose receptor, are known as pattern recognition molecules and can recognize foreign ligands during the initial phases of the immune response. The activation of these receptors leads to intracellular signalling cascades, resulting

21 in transcriptional activation and the production of pre-inflammatory cytokines [62]. In the present study, USP and FSP30 were found to enhance the production of TNF-α and IL-1β. The results revealed that both the inducers of IL-l β and TNF-α and the effects of IL-1 and TNF-α on the host were remarkably similar [63, 64]. Moreover, NO production was also induced by USP and FSP30, indicating that these fractions were able to functionally activate macrophages. 4. Conclusions In this study, fractionated sulphated polysaccharides (FSP30) from U. intestinalis extracted with hot water exhibited stronger antioxidant activities than USP and might be potential candidates for antioxidant agents. After purification, FSP30 had a molecular weight of 110 kDa, a sulphate content of 8.85%, a degree of sulphation of 0.62 and the highest sulphate/sugar ratio of 1.08, which significantly affected the DPPH• radical-scavenging activity. These results suggested that U. intestinalis fractionated sulphated polysaccharides could be explored as potential antioxidant, which the suitable molecular weight is important for sulphated polysaccharide to exhibit antioxidant activity. In addition, they could be strong stimulators of J774A.1 cells, resulting in the production of considerable amount of nitric oxide production, TNF-α secretion and IL-1β expression. This could be the potential of sulphated polysaccharides for being as immunomodulatory agents. Acknowledgments This work was supported by the Toward Sustainability Foundation (TSF) Granted Research Project of Japan, the National Research University (NRU) Project of Thailand, and King Mongkut’s University of Technology Thonburi.

22

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30

Figure 1. A) The elution profile of fractionated sulphated polysaccharides from a silica-silica column; B) High Pressure Size Exclusion Chromatographic profile of USP, FSP32, FSP30 and FSP4 and C) DPPH∙ radical-scavenging (%) of unfractionated and fractionated sulphated polysaccharide. Values are presented as the mean ±S.D. (n=3). a, b, c,…Means above the bars of each fraction in DPPH radical scavenging with different letters are significantly different at p < 0.05. Remark: USP = unfractionated sulphated polysaccharides, FSP4 = 4th fractionated sulphated polysaccharides, FSP30 = 30th fractionated sulphated polysaccharides, FSP32 = 32nd fractionated sulphated polysaccharides

31

Figure 2. A) Effect of USP and FSP30 from U. intestinalis on cell viability, B) Effect of the USP and FSP30 on the production of nitric oxide (NO), and C) Effect of the USP and FSP30 on the TNF-α secretion of murine macrophage J774A.1, Values presented as the mean ±S.D. (n=3). a, b, c,…Means above the bars of each parameter with different letters are significantly different at p < 0.05. ND = not detected. Remark: USP = unfractionated sulphated polysaccharides, FSP4 = 4 th fractionated sulphated polysaccharides, FSP30 = 30th fractionated sulphated polysaccharides, FSP32 = 32 nd fractionated sulphated polysaccharides

32 A)

B)

Figure 3. A) Effect of the USP and B) FSP30 on IL-1β secretion into the culture supernatant were measured by Western Blot Analysis.

33 Table 1 Chemical characteristic of unfractionated and fractionated sulphated polysaccharides. Chemical compositions Sulphated Total sugar

Sulphate content

Degree of

Sulphate/sugar

(%)

(%)

sulphation

ratio

polysaccharides

USP

26.55±0.59a 20.42±0.16a

2.96±0.06a

0.77±0.02b

FSP4

4.85±0.14d

2.85±0.05c

0.16±0.1c

0.59±0.03d

FSP30

8.20±0.1c

8.85±0.04b

0.62±0.02b

1.08±0.01a

FSP32

13.11±0.02b 8.91±0.01b

0.63±0.01b

0.68±0.01c

a,b,c…

Means in the same column with different letters are significantly difference at p < 0.05.

Remark: USP = unfractionated sulphated polysaccharides, FSP4 = 4 th fractionated sulphated polysaccharides, FSP30 = 30th fractionated sulphated polysaccharides, FSP32 = 32 nd fractionated sulphated polysaccharides