Glucofucogalactan, a heterogeneous low-sulfated

International Journal of Biological Macromolecules 113 (2018) 90–97

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Glucofucogalactan, a heterogeneous low-sulfated polysaccharide from Saccharina japonica and its bioactivity Lihua Geng a,b,c, Quanbin Zhang a,c,⁎, Jing Wang a,c, Weihua Jin d, Tingting Zhao e, Weicheng Hu f a

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China Lab for Marine Biology and Biotechnology, Qingdao National Lab for Marine Sci. & Tech, Qingdao 266071, China University of Chinese Academy of Sciences, Beijing 100049, China d College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China e Beijing Key Lab for Immune-Mediated Inflammatory Diseases, Institute of Clinical Medical Sciences, China–Japan Friendship Hospital, Beijing, China f Jiangsu Collaborative Innovation Center of Regional Modern Agriculture & Environmental protection/Jiangsu Key Laboratory for Eco-Agricultural Biotechnology Around Hongze Lake, Huaiyin Normal University, Huaian 223300, China b c

a r t i c l e

i n f o

Article history: Received 25 November 2017 Received in revised form 13 January 2018 Accepted 1 February 2018 Available online 2 February 2018 Keywords: Saccharina japonica Heteropolysaccharide Aristolochic acid Immunomodulation

a b s t r a c t Crude polysaccharide obtained from Saccharina japonica using acid hydrolysis and precipitation was separated into sulfated fuco-oligosaccharide (HDF1) and heteropolysaccharide (HDF2). To further explore the bioactive fraction, HDF2 was successfully separated using membrane filtration into HDF2A and HDF2B, which differed in chemical composition and molecular weight. The bioactivity of all the fractions was tested in vitro, including immunomodulatory activity in RAW 264.7 cells and the protective activity in aristolochic acid (AA)-induced NRK-52E cell injury. HDF1 and HDF2B (low-molecular weight sulfated fucans/fuco-oligosaccharides) did not increase the nitric oxide production in RAW 264.7 cells, whereas HDF2 and HDF2A exhibited potential immunomodulatory activity. All the tested compounds showed different degrees of protective activity in AA-induced injury; HDF2A exhibited superior protective activity. Through chemical analysis, HPLC analysis, and IR spectroscopy and MS, it was determined that HDF2A was a galactose-enriched heteropolysaccharide- glucofucogalactan with a distinctive 2:1 ratio of galactose to fucose. In addition, HDF2A also contained a high amount of glucose and minor amounts of mannose, rhamnose, and xylose, with a low content of sulfate. Thus, HDF2A, a complex heterogeneous polysaccharide mixture with a unique monosaccharide composition, could be studied for further structural characterization and pharmaceutical applications. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Polysaccharides, widely found in plants, animals, and microorganisms, have been intensively studied because of their bioactivity and medicinal properties. In particular, marine-derived polysaccharides have received unprecedented attention as a potential new class of biomaterial for decades [1,2]. Of these polysaccharides, most are predominantly obtained from algae, both microalgae and macroalgae, including green algae (Chlorophyceae), red algae (Rhodophyceae), and brown algae (Phaeophyceae) [3]. Ulvan, agar, carrageenan, laminaria, fucoidan, and alginate are the major polysaccharides obtained from algae, and a large proportion of these polysaccharides always contain substituent groups such as acetyls and sulfates [4,5]. Notably, the structure of polysaccharides varies with the extraction and purification procedure, degradation method (e.g., enzymatic hydrolysis, acid hydrolysis, or other ⁎ Corresponding author at: Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China. E-mail address: [email protected] (Q. Zhang).

https://doi.org/10.1016/j.ijbiomac.2018.02.008 0141-8130/© 2018 Elsevier B.V. All rights reserved.

physical method, such as microwave or ultrasonication), and separation technique; the structure also varies with changes in chemical composition (i.e., sulfate content, substitutions and position, molecular weight, monosaccharide composition), which is related to the bioactivity to a great extent [6]. Sulfated polysaccharides from Saccharina japonica have complex and diverse structures, which strongly depend on the extraction and purification process and the life stage of the seaweed. Until now, a consistent protocol to determine the structure of the polysaccharide fucoidan from Saccharina japonica has not been established [2,7]. Apart from alginate and fucoidan, certain kinds of heteropolysaccharides, which significantly differed in composition and structure, have been previously reported [8]. Bilan et al. [9] detected three different types of sulfated polysaccharides from the brown alga Saccharina latissima, including fucogalactan, fucoglucuronomannan, and fucoglucuronan, but not fucan. Our laboratory has focused on different polysaccharides from Saccharina japonica for decades, and the structure of different polysaccharides, including fucan, galactofucan, and mannoglucuronan, obtained by anion-exchange chromatography or

L. Geng et al. / International Journal of Biological Macromolecules 113 (2018) 90–97

gel permeation chromatography has been characterized using desulfation, methylation, NMR, and MS [10–13]. However, there are comparatively fewer reports on galactose-enriched polysaccharides from Saccharina japonica. Sulfated polysaccharides possess potential biological activity, such as neuroprotection [14] protective properties in diabetic nephropathy [15], anti-inflammatory, immunomodulatory [16,17], and antioxidant activity [18]. Our previous studies showed that polysaccharides extracted from Saccharina japonica were effective in alleviating the symptoms caused by experimental chronic renal failure in rats [19], and showed protective properties in diabetic nephropathy rats [15,20]. In addition, polysaccharides were reportedly used as immunomodulatory therapeutic agents [21]. It is well known that fucoidan activates mitogen-activated protein kinases (MAPKs), such as p38, (ERK)1/2, and SAPK/JNK to function as immunomodulators, and the sulfate and acetyl groups of fucoidan are involved in nitric oxide-inducing activity in RAW 264.7 cells [22]. A kind of heteropolysaccharide from Laminaria japonica, which was mainly composed of arabinose, mannose, glucose, and galactose in a molar ratio of 1.0:7.8:6.6:0.8, exhibited significant stimulation of macrophages [23]. Sulfated fucans isolated from Ecklonia cava also stimulated RAW 264.7 cells to produce considerable amounts of nitric oxide and cytokines [24]. Aristolochiaceae is extensively studied to be the toxic substance. Although many countries and regions have abandoned such medicines years ago, tiny minority of Chinese patented drugs are still mixed with these toxic substances in the form of aristolochic acids and similar compounds (collectively, AA) [25]. AA can accumulate in kidneys, cause toxic injury, and subsequently induce acute aristolochic acid nephropathy (AAN) [26]. Research has shown that the injury mainly occurs in the renal tubular epithelial cells [27,28]. The present study, differing from previous studies both in methodology and product, optimized the conditions for acid hydrolysis and precipitation of crude polysaccharides from Saccharina japonica and used membrane filtration to obtain galactose-enriched polysaccharides. We obtained a novel sulfated heteropolysaccharide (HDFA) along with different fractions and investigated the protective activity in AAN and immunomodulatory activity of all the fractions obtained, in vitro. 2. Experimental and methods

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to settle overnight. Finally, the solution was filtered and dialyzed, and the dialysate was then concentrated, and crude polysaccharide was obtained after precipitation by 85% ethanol.

2.3. Acid hydrolysis of crude polysaccharide Crude polysaccharide (10 g) was degraded by sulfuric acid (0.2 M, 300 mL) at 80 °C with occasional stirring. At the end of hydrolysis, the solution was neutralized using Ba(OH)2 and centrifuged, and the supernatant was collected and precipitated with different reagents at different final concentrations. The factorial design of the different hydrolysis times and precipitating reagents used is presented in Table 1. After precipitation and centrifugation, the supernatant was evaporated, concentrated, and dried to obtain SPF1, and the precipitate was lyophilized to obtain SPF2. Thus, under the conditions shown in Tables 1, 27 kinds each of SPF1 and SPF2 were obtained.

2.4. Preparation of sulfated fuco-oligosaccharide (HDF1) and low-sulfated heteropolysaccharide (HDF2) Based on the optimization study of degradation and precipitation conditions, the best conditions were determined to be the combination 3–3–2 (Table 1). In brief, crude polysaccharide (10 g) was hydrolyzed by sulfuric acid (0.2 M, 300 mL) at 80 °C with occasional stirring for 7 h. After neutralization and centrifugation, the degradation solution was precipitated with 90% acetonitrile overnight. Next, the supernatant was evaporated, concentrated, and lyophilized to obtain HDF1. The precipitate was dried to obtain HDF2. The yield was calculated according to the dried biomass of crude polysaccharide. 2.5. Fractions separated by membrane filtration A dialysis membrane (MwCO: 1KD, Spectrum, USA) was used to separate HDF2 in the water, and HDF2 was further separated into two fractions. The fractions with low and high molecular weight were concentrated and lyophilized to obtain HDF2A and HDF2B, respectively.

2.1. Materials and chemicals

2.6. General methods

The cultured brown alga S. japonica was collected from Rongcheng (Shandong, China) in June 2015. The fresh algal samples were washed to remove sediment, sun-dried, and separated into pieces for storage. Ethanol, acetonitrile, acetone, sulfuric acid and other reagent for polysaccharide extraction and hydrolysis were purchased from Sinopharm chemical reagent Co., Ltd. (Shanghai, China). All reagents for analysis (GC grade) were purchased from Merck (Darmstadt, Germany). Dulbecco's Modified Eagle Medium for NER-52E cell culture and RPMI 1640 medium for RAW 264.7 cell culture were purchased from Hyclone (Logan, UT, USA). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). AA was purchased from Acros Organics (Thermo Fisher Scientific, USA). Penicillin and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Lipopolysaccharide (LPS), MTT reagent, Griess reagent, dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.6.1. Chemical analysis Sulfated content was determined by the barium chloride–gelatin method of Kawai et al. [30] using K2SO4 as a standard. Uronic acid (UA) was determined by a modified carbazole method using D-glucuronic acid as a standard [31]. Fucose content was analyzed according to the method of Gibbons [32]. The molar ratio of neutral monosaccharides was determined by the HPLC pre-column derivatizing method of Zhang et al. [33]. The molecular weight was analyzed by GPC-HPLC on a Sugar-KS804 column (7 μm, 8.0 × 300 mm) eluted with 0.5 M Na2SO4 at a flow rate of 0.5 mL/min at 30 °C, the standard used was dextral polysaccharide with different molecular weights. Table 1 Condition settings for degradation of crude polysaccharide from seaweed. Condition

2.2. Extraction and purification of crude polysaccharide from S. japonica Crude polysaccharide was extracted from S. japonica with cold water as previously described, with some modifications [29]. In brief, dry brown alga (1 kg) was kept immersed in cold water (3.0 L) twice for 3 h. The combined leaching solution was adjusted to pH = 12 using NaOH, to induce precipitation. Next, the precipitate was collected, HCl was added to adjust the solution pH to 2 and the solution was allowed

Hydrolysis time (h) Precipitating reagent Concentration (%)

Level 1

2

3

3 Ethanol 50

5 Acetonitrile 70

7 Acetone 90

Note: There were 27 kinds of SPF1 and SPF2, which were represented using X-Y-Z. In which, X = 1, 2, 3 and represented the degradation time of 3 h, 5 h, and 7 h, respectively. Y = 1, 2, 3 and represented the concentrations of the precipitating reagent, 50%, 70%, and 90%, respectively. Z = 1, 2, 3 and represented the precipitating reagents ethanol, acetonitrile, and acetone, respectively.

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2.6.2. Conditions of HPLC To evaluate the degree of polymerization and separation, the fractions of HDF were tested by HPLC with an ELSD detector. A Click-maltose column (5 μm, 4.6 × 150 mm) was obtained from the Dalian Institute of Chemical Physics (Acchrom, Dalian, China). Dried test samples were dissolved in 1:1 methanol/water (concentration of the sample was approximately 10 mg/mL) and introduced into the HPLC at flow rate of 1 mL/min in a gradient solution. The oven temperature was 30 °C and the mobile phase buffer was 100 mmol/L formic acid-ammonium formate solution (pH 3.2). The gradient was 0–30 min, water–acetonitrile–buffer: from 10:80:10 to 40:50:10. Each sample was balanced with its initial mobile phase for 15 min before testing. The parameters of the ELSD detector set were as follows: evaporation temperature: 80 °C, nebulizer temperature: 80 °C, gas flow: 1.5 mL/min. 2.6.3. IR spectroscopy All samples were dried to powder and then pressed into KBr pellets. Infrared spectroscopy was carried out using a Nicolet iS 10 FT-IR spectrometer (Thermo Fisher, USA) between the ranges of 400–4000 cm−1. 2.6.4. Mass spectroscopy A small amount of samples was taken into mobile phase (CH3CN/ H2O, 1:1, v/v) and then analyzed by a 6530 Accurate-Mass Q-TOF-MS spectrometer (Agilent, USA). The specific conditions included: flow rate (0.4 mL/min), capillary voltage (4 kV), solvent evaporation temperature (350 °C), ion source temperature (120 °C), gas flow (1 L/min), fragmentor (200 V), and mass range (25–1500 m/z). Data acquisition method for the full scan was in the negative ion mode. All spectra were analyzed with MassHunter workstation software (Agilent technology, USA). 2.7. In vitro immunomodulatory activity assay 2.7.1. Cell culture and treatment A mouse peritoneal macrophage cell line, RAW 264.7, was obtained from the American type culture collection (Manassas, VA, USA). Cells were cultured in RPMI 1640 culture medium (10% heat-inactivated FBS and 1% antibiotic, including 100 U/mL penicillin and 100 μg/mL streptomycin) at 37 °C and 5% CO2 atmosphere. Confluent cells were passaged by scraping them with a sterile scraper [34]. 2.7.2. Cell viability assay The effect of samples on the viability of RAW 264.7 cells was determined using the MTT method. In brief, 100 μL RAW 264.7 cells were seeded into a 96-well plate at a density of 1 × 106 cells/mL. After 24 h incubation, cells were treated with test samples for another 24 h in a final volume of 200 μL. Then, the medium was carefully removed, and 100 μL of MTT (0.5 mg/mL in serum-free medium) was added to each well, followed by an additional 4 h incubation. After incubation, the purple formazan crystals that formed were dissolved in 100 μL of MTT stop solution that contained 10% SDS and 0.01 M hydrochloric acid. The absorption values were measured at 550 nm on a multifunctional microplate reader (Infinite M200 Pro spectrophotometer, Tecan, Switzerland). The optical density of the formazan formed in the control vehicle was taken as 100% viability. The data are expressed as the percentage of the control optical density (OD) values for each experiment by the following formula: Cell viability (%) = ODs / ODv × 100, where ODs and ODv indicate the optical density of cell lines incubated with test samples and control vehicle, respectively. 2.7.3. Determination of nitric oxide (NO) production The production of NO was quantified using the Griess reagent. Cells were seeded at the density of 1 × 105 cells/well in a 96-well culture plate. After 24 h incubation, cells were treated with test samples (100 μg/mL) or lipopolysaccharide (LPS) (1 μg/mL) for 24 h in a final volume

of 200 μL. At the end of incubation, 100 μL of cell supernatant was mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% N-1naphthylenediamine di-hydrochloride in 2.5% phosphoric acid). After incubation for another 10 min, the absorbance was measured. Untreated culture media were used as blanks in all experiments and NO production was calculated with reference to a sodium nitrite standard curve. 2.8. In vitro assay of protective activity in AA-induced injury 2.8.1. Cell culture and treatment The rat kidney tubule epithelial (NRK-52E) cell line was obtained from the China-Japan Relationship Hospital (Beijing, China). NRK-52E cells were cultured in Dulbecco's Modified Eagle Medium containing 4.5 mM glucose supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin and were incubated at 37 °C in a humid atmosphere of 5% CO2. Cells were plated at a density of 2 × 103/mL into 96well cell culture plates for 24 h until the density reached 70% confluence. AA was prepared as a stock solution of 1.0 mg/mL in 100% DMSO. After incubation with serum-free medium for 12 h starvation, NRK52E cells were exposed to different concentrations of AA (0, 10, 20, 30, and 40 μg/mL) for 12 h or 20 μg/mL for different periods (0, 2, 6, 12, 24, 48, and 72 h). To investigate the protective activity in AA-induced injury, all samples were dissolved in sterile water at a concentration of 10 mg/mL. NRK-52E cells were treated with AA (20 μg/mL), alone or in combination with fractions separated from Saccharina japonica at the concentration of 0.5 and 1.0 mg/mL for 12 h, and then the cytotoxic effects were tested. 2.8.2. Cell viability assay The effect of samples on the viability of NRK-52E was determined colorimetrically using the MTT method described in Section 2.6.2. 2.9. Statistical analysis Statistical analyses were performed using Prism 5 (GraphPad software, USA). Data are presented as the mean ± standard error (SE) and all experiments were performed in triplicate. The means of multiple independent variables were compared between the control and treatment groups using a one-way analysis of variance (ANOVA). A P-value of b0.05 was considered to be statistically significant compared to the control group. 3. Results and discussion 3.1. Depolymerization of crude polysaccharide and condition optimization Hydrolysis and precipitation of crude polysaccharide were studied based on the different conditions used. The difficulty of hydrolysis of the glycosidic bond is determined by the type of glycosidic bond, type of monosaccharide, and substituent position and location. Regarding the difficulty of hydrolysis, usually, branched chains of glycosidic bonds are considerably easier to hydrolyze than backbones. In addition, furan glycoside was considerably easier to hydrolyze than pyranoside, ketose was easier than aldose, neutral sugar on the non-reducing end is hydrolyzed easily, and the glycosidic bond connected to uronic acid was relatively difficult to hydrolyze [35]. To determine the optimal acid hydrolysis and precipitation conditions, hydrolysis time (3, 7, or 9 h), precipitating reagent (ethanol, acetonitrile, or acetone) and concentration of the precipitating reagent (50%, 70%, or 90%) were investigated in terms of their effects on crude polysaccharide degradation. With these conditions, 27 kinds of SPF1 and SPF2 each were obtained. The average molecular weight (Mw) of SPF1 was approximately 2 kDa, that of SPF2 was 20 kDa, and both were essentially unaffected by the conditions. The concentration of the precipitating reagent was


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Fig. 1. Chemical analysis results (yield, content of fucose, UA, and sulfate) of all 27 kinds of SPF2.

positively correlated with yield, which was consistent with the principle of precipitation. The chemical analysis result of all SPF2 is shown in Figs. 1 and 2. Different hydrolysis times and precipitating reagents had almost no effect on the chemical composition of SPF2. The glycosidic bond connected to uronic acid was hard to hydrolyze; thus, the SPF2 obtained with the lowest concentration (50%) of precipitating reagents had a higher uronic acid content because most of the uronic acid remained with the backbone, resulting in a slightly higher molecular weight. Fucose mainly linked as a residue at the non-reducing high-sulfated end; thus, the SPF2 obtained with the highest concentration (90%) of precipitating reagent had a higher fucose and sulfate content. Moreover, the content of fucose and sulfate was positively correlated, as anticipated. The monosaccharide composition results showed SPF2 contained a large amount of uronic acid and monosaccharides, mainly fucose (Fuc), mannose (Man), galactose (Gal), and glucose (Glc). The product obtained using the hydrolysis and precipitation conditions in the combination 3–3–2 (Table 1) exhibited substantial differences from all other products. According to our previous study [36], SPF1 obtained using 3–3–2 (HDF1) possessed a higher fucose and sulfate content, and was confirmed to be fuco-oligosaccharide. The 3–3–2 corresponding precipitate was a low-sulfated heteropolysaccharide (HDF2). Table 2 shows the chemical compositions and the weight average molecular weight (Mw) of HDF1 and HDF2, and each step of the production is listed. After degradation and precipitation, the yield of HDF2 obtained from crude polysaccharide was 39.3%. The chemical analysis showed that HDF2 was mainly composed of fucose and galactose (in approximately equal amounts), with several other monosaccharides, such as rhamnose, glucose, and mannose, and a minor amount of sulfated substituents. Thus, the analysis of HDF2 confirmed our speculation of its heterogeneity. Previous research has also revealed, as the present study did, that one type of polysaccharide thus obtained was a heteropolysaccharide with a small amount of fucose and sulfate but a considerable amount of other monosaccharides, especially galactose [37]. 3.2. Fractionated HDF2 derivatives and their molecular characteristics HDF2 was fractionated into HDF2A and HDF2B through membrane filtration, with a yield of 60.0% and 36.2%, respectively. The molecular

weight cut-off (MwCO) of the dialysis membrane was 1 kDa. After filtration, the molecular weight of HDF2A was up to 23.5 kDa, The HPGPC results showed that HDF2A had only one peak, but it was symmetric (data not shown). The chemical analysis and IR spectroscopy (Fig. 3a) showed that the main structure was unchanged, which means HDF2A had the similar chemical composition as HDF2 but had greater homogeneity. On the basis of the chemical analysis results shown in Table 2, the highly sulfated fucose residue fragments had fallen off the backbone or branches during acid hydrolysis and separated to form HDF2B. Thus, the content of fucose and sulfate of HDF2B was considerably higher than that of HDF2A (66.06% and 25.80%, respectively). The results showed that HDF2B was mainly composed of fucose and galactose in a molar ratio of 1.0:0.21, whereas HDF2A was mainly composed of galactose, fucose, and glucose in a molar ratio of 1.0:0.52:0.31. For a more intuitive understanding of the effect of membrane filtration, HDF2A and HDF2B were compared with each other in the MaltoseHPLC spectrum (Fig. 3b). Maltose-bonded silica material, Click-Mal, is one of the S-Glycan series separation materials for oligosaccharide from Acchrom Technology [38]. With the excellent technological advantages in hydrophilic interaction liquid chromatography (HILIC), an earlier peak indicates a smaller degree of polymerization (DP). Analysis of the Click-Mal column showed an evidently lower DP for HDF2B and a centralized peak with homogeneous molecular weight distribution was observed for HDF2A. In general, polysaccharide with relatively high molecular weight could be precipitated by 90% acetonitrile, while sulfated fuco-oligosaccharides with low degree of polymerization mainly remained in the supernatant. However, in the present study, a large amount of fucooligosaccharides was also precipitated by 90% acetonitrile and further separated as HDF2B through dialysis. Since HDF2 was a mixture of sulfated fuco-oligosaccharides (HDF2B, 36.2%) and a low-sulfated heteropolysaccharide with complex sugar constituents (HDF2A, 60.0%), the composition of HDF2 was a combination of HDF2A and HDF2B. Thus the compositions of HDF2A and HDF2B were largely different from HDF2. In conclusion, the method of membrane filtration by dialysis was efficient for fractionation of HDF2. Combined with the results of monosaccharide composition analysis, we speculated that HDF2A was glucofucogalactan, with a distinctive 2:1 ratio of galactose to fucose.

Fig. 2. Monosaccharide composition of all 27 kinds of SPF2 (percentage ratio).

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Table 2 Chemical analysis of HDF derivatives. Total sugar (%)

HDF1 HDF2 HDF2A HDF2B

43.5 53.1 93.2 68.7

Fucose (%)

33.2 21.2 21.5 66.1

Sulfate (%)

13.1 10.2 18.0 25.8

Monosaccharides composition(molar ratio) Man

Rha

Glc A

Glc

Gal

Xyl

Fuc

0 0.27 0.13 0

0.06 0.51 0.19 0.04

0.01 0.20 0.09 0

0.02 0.47 0.31 0.03

0.07 0.82 1 0.21

0.05 0.09 0.10 0.02

1 1 0.52 1

3.3. In vitro immunomodulatory activity of four fractions separated from S. japonica polysaccharide Innate immunity is the first line of defense mediating initial protection to ensure the health of the body, and it is known that macrophages play an important role in innate immunity. Activated macrophages can kill pathogens directly through phagocytosis, which is fatal to the invader. In addition, they also contribute to tissue remodeling, adaptive immunity, and many disease processes [39]. RAW 264.7 mouse murine macrophage cells release a variety of cytokines in response to LPS, and RAW 264.7 cells are a reliable system for the detection of the immune response induced by compounds [40]. Therefore, we employed RAW 264.7 mouse murine macrophage cells to examine the immunomodulatory activity of fractions obtained from crude polysaccharide. MTT assays were employed to determine the toxic effects of various fractions on RAW 264.7. As shown in Fig. 4a, after being pretreated with all the tested HDF derivatives (100 μg/mL) for 24 h, no tested sample showed obvious toxic effects on these cells. To examine the immunomodulatory activity, RAW 264.7 cells were treated with all the fractions, and the production of NO was measured

Mw (kDa)

Yield (%)

2.4 19.6 23.5 0.9

25.5 39.3 60.0 36.2

after 24 h. LPS is a potent activator of macrophages and low doses of LPS are considered to be beneficial for the host as it induces immunostimulation and enhances resistance to invaders [41]. In the present study, cells treated with LPS were used as the positive control, and LPS-stimulated cells increased the NO production as expected. As shown in Fig. 4b, different fractions showed different properties with respect to induction of NO production in RAW 264.7 cells. HDF2, but not HDF1 induced NO production in RAW 264.7 cells. In particular, cells treated with HDF2A showed higher NO production than those treated with HDF2, whereas HDF2B did not exhibit the induction activity. HDF1 and HDF2B possessed lower molecular weight and highly similar chemical composition; thus, we inferred that both were low-molecular weight sulfated fucans/fuco-oligosaccharides, which can explain their lack of NO production-inducing activity in RAW 264.7 cells. Much attention has been given to the effects of sulfated polysaccharides on immune response, but it remains controversial. Currently, the consensus is that sulfated polysaccharides play a dual role, inhibitor and promoter, in immune response [16,42]. Some studies reported that the polysaccharides showed anti-inflammatory effects by inhibiting the transmission of inflammatory signals [17,18]. Some

Fig. 3. a) IR spectrum of HDF2 and two fractions (HDF2A and HDF2B) separated by membrane filtration. b) Click-maltose-HPLC analysis of HDF2A and HDF2B.


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Fig. 4. a) Effects on the growth of RAW 264.7 cells. RAW 264.7 cells were treated with all the fractions obtained from Saccharina japonica (100 μg/mL) crude polysaccharide in a 96-well tissue culture plate for 24 h. Viability of RAW 264.7 cells were measured by the MTT assay. Spectrophotometric analysis was conducted at 490 nm using an Infinite M200 Pro spectrophotometer. The normal values of untreated RAW cells served as control values in the calculation of % inhibition. b) NO production in RAW 264.7 cells induced by all fractions separated from Saccharina japonica crude polysaccharide. RAW 264.7 cells were cultured in the presence of 100 μg/mL test samples or LPS (1 μg/mL) for 24 h. c) Dose-dependent NO production in RAW 264.7 cells induced by HDF2A (0.01, 0.1, 1, 10, and 100 μg/mL) or LPS (1 μg/mL) for 24 h. The production of NO was quantified using the Griess reagent. The absorbance was measured at 550 nm. Untreated culture media were used as blanks in all experiments and NO production was calculated with reference to a sodium nitrite standard curve. Data are presented as the mean ± S.D. from independent experiments.

reports supported that sulfated polysaccharides could be excellent immune regulators by activating monocytic cells and inducing the expression of co-stimulatory molecules [43,44], which is consistent with our results. We found that the HDF2A showed superior NO production-inducing activity. After 24 h incubation, RAW 264.7 cells were exposed to increasing concentrations (0.01, 0.1, 1, 10 and 100 μg/mL) of HDF2A for another 24 h. The NO production in cells treated with HDF2A was significantly increased in a dose-dependent manner (Fig. 4c). All the data showed that HDF2A-treated RAW 264.7 macrophages could generate high amounts of NO, which is considered to be a defense against external pathogens. 3.4. In vitro protective activity of AA-induced injury of four fractions separated from S. japonica polysaccharide In the present study, using previously described methods [45,46] with some modifications, NRK-52E cells (rat renal tubular epithelial cells) were exposed to different concentrations (10, 20, 30, and 40 μg/

mL) of AA for different periods (0, 2, 6, 12, 24, 48, and 72 h). Fig. 5a shows AA-induced cell injury, and AA induced a significant decrease in cell viability in a dose- and time-dependent manner. To investigate the protective effect of all the fractions of crude polysaccharide in AA-induced nephrotoxicity, samples and AA (20 μg/mL) were added to the NRK-52E cell medium followed by another 12 h incubation. A comparison of results of all the separated fractions (Fig. 5b) showed that all four tested samples exhibited protective effects at concentrations of 0.5 and 1 mg/mL (p b 0.01). Remarkably, HDF2A showed a significant protective effect in AA-induced cell injury. In some animal models of induced renal injury, it has been revealed that macrophages induced immunological or non-immunological damage leading to apoptosis [47,48]. The result from Nakagawa et al. [48] suggested the macrophages could release cytokines to suppress the expression of vascular endothelial growth factor (VEGF) in the epithelial cells lining the renal tubule. Based on the results of the present study, we inferred that the protective effect in AA-induced injury may be associated with its immunomodulatory activity.

Fig. 5. Effects on AA-induced renal tubular epithelial cell injury. a) After incubation with serum-free medium for 12 h starvation, NRK-52E cells were exposed to different concentrations of AA (0, 10, 20, 30, and 40 μg/mL) for different periods (0, 2, 6, 12, 24, 48, and 72 h). b) Effects of all four fractions on AA-induced NRK-52E cell injury at low concentration (L: 0.5 mg/mL) and high concentration (H: 1 mg/mL). Viability of NRK-52E cells was measured by MTT assay. Spectrophotometric analysis was carried out at 490 nm. Data are presented as the mean ± S.D. from independent experiments. *P b 0.05 and **P b 0.01 compared with the AA-induced group was considered statistically significant.

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Fig. 6. Negative ion ESI-MS of the heteropolysaccharide HDF2A.

3.5. ESI-MS analysis of HDF2A Mass spectroscopy (MS) with electrospray ionization source (ESIMS) is an important tool for the analysis of heteropolysaccharides [49,50]. In the present study, we found that HDF2A obtained from HDF2 by dialysis could both induce the production of NO in RAW 264.7 cells and protect from AA-induced injury in NRK-52E cells. To gain further understanding, HDF2A was analyzed by ESI-TOF-MS in the negative ion mode. Because of the limitations of the signal response, the larger the molecular weight, the lower the corresponding signal is in the mass spectra. The monosaccharide composition analysis confirmed HDF2A to be a polysaccharide mixture containing a considerable percentage of Gal, Fuc, and Glc, and also minor amounts of Man, Rha, and Xyl. The speculated molecular formula is presented in Fig. 6. Negativeion mode ESI-MS of HDF2A showed that the composition of HDF2A was complex and the main component was [FucSO3]− at m/z 243.0188 and [Fuc2SO3]− at m/z 389.0775. In addition, mass spectrum contained the following two series of singly charged ions: one at m/z 243.188, 389.0775, 535.1331, and 681.1792 corresponding to monosulfated fuco-oligosaccharides [FucnSO3]− (n = 1–4), and another at m/z 259.0156, 421.0657, 583.0093, and 744.9078 corresponding to mono-sulfated galacto-oligosaccharides [GalnSO3]− (n = 1–4). The spectrum showed that HDF2A contained di-sulfated galacto-/fuco-oligosaccharides: fragmentation for the doubly charged ions at m/z 411.9402, 493.0396, 662.9156, and 307.0433 corresponding to [Gal4 (SO3)2]2−, [Gal5(SO3)2]2−, [Gal3(SO3)2]2−, and [Fuc3 (SO3)2]2−, and hetero-oligosaccharides fragmentation for the triply charged ions at m/z 344.9556 and 511.9302, corresponding to tetra-sulfated hetero-oligosaccharides [Gal3Fuc2(SO3)3]3− and [Gal7Fuc(SO3)3]3−. In addition, there were some less intensive ions of hetero-oligosaccharide fragment: [Gal2FucXyl(SO3)2]2− at m/z 390.0758 (−2), [ManXylSO3]− at m/z 405.0712 (−1), and [FucXyl5(SO3)2]2− at m/z 491.0211 (−2). Moreover, here were other less intensive triply and quadruply charged ions corresponding to tetra- or penta-sulfated heterogeneous oligosaccharides (data not shown). The monosaccharide composition results analyzed by PMP-HPLC showed that HDF2A contained not only Fuc and Gal, but high amounts of Glc, and minor amounts of Man, Rha, and Xyl. However, the ion fragment peak observed in mass spectrum was not consistent with the

results, and most of the fragmentation corresponded to sulfated galacto-oligosaccharides and fuco-oligosaccharides. We speculated two reasons for the inconsistency in these results: one reason was that the information regarding the neutral composition is often lost in negative mode ESI-MS, and the sulfate content of HDF2A was low in the present study; thus, the substitution of sulfates was mainly concentrated on the fucose and galactose residues, and the other oligosaccharide signals were not detected. Another reason may be that the structures of heteropolysaccharides with high molecular weight and complicated structure are not suitable to be characterized thoroughly by MS. In addition, sulfated group was considered to exist mainly on fucose or galactose residues but not rhamnose or glucose residues of S. japonica [2,6,7], and we speculated the signal of sulfated homogeneous oligosaccharides in the spectrum were mainly fuco-oligosaccharides and galacto-oligosaccharides. Thus, the structure of HDF2A needs to be further studied using additional methods, including degradation, desulfation, and modification, followed by NMR and electrospray tandem mass spectrometry (ESI-MSn).

4. Conclusion Following degradation and fractionation of the polysaccharides obtained from Saccharina japonica, a novel heteropolysaccharide glucofucogalactan (HDF2A) was obtained, which mainly contained galactose and fucose at a distinctive ratio of 2:1 and a high amount of glucose. Results of the present study showed that glucofucogalactan exhibited superior protective effects in AA-induced NRK-52E cell injury and acted as a promoter of immune response in RAW 264.7 cells, than did other fractions. The low-molecular weight sulfated fucans/fuco-oligosaccharides did not exhibit these activities. Unfortunately, the structure of HDF2A could not be characterized properly because of its high degree of polymerization and heterogeneity and the complexity of the polysaccharide itself. Therefore, the relationship between the protective activity in AA-induced injury and the immunomodulatory activity could not be explained. Thus, we concluded that the activity of HDF2A was a result of synergistic effects. In conclusion, these results warrant further research on HDF2A regarding its structure and potential protective activity in AAN and immunomodulatory activity.


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