A rhamnan-type sulfated polysaccharide with novel structure from Monostroma angicava Kjellm (Chlorophyta) and its bioactivity

Accepted Manuscript Title: A rhamnan-type sulfated polysaccharide with novel structure from Monostroma angicava Kjellm (Chlorophyta) and its bioactivity Authors: Xue Liu, Jiejie Hao, Xiaoxi He, Shuyao Wang, Sujian Cao, Ling Qin, Wenjun Mao PII: DOI: Reference:

S0144-8617(17)30668-9 http://dx.doi.org/doi:10.1016/j.carbpol.2017.06.031 CARP 12419

To appear in: Received date: Revised date: Accepted date:

9-12-2016 17-5-2017 7-6-2017

Please cite this article as: Liu, Xue., Hao, Jiejie., He, Xiaoxi., Wang, Shuyao., Cao, Sujian., Qin, Ling., & Mao, Wenjun., A rhamnan-type sulfated polysaccharide with novel structure from Monostroma angicava Kjellm (Chlorophyta) and its bioactivity.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.06.031 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.

A rhamnan-type sulfated polysaccharide with novel structure from Monostroma angicava Kjellm (Chlorophyta) and its bioactivity

Xue Liu a, Jiejie Hao a*, Xiaoxi He a, Shuyao Wang a, Sujian Cao a, Ling Qina, Wenjun Mao a,b *

a

Key Laboratory of Marine Drugs of Ministry of Education, Shandong Provincial Key Laboratory of

Glycoscience and Glycotechnology, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China b

Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science

and Technology, Qingdao 266237, China

*

Corresponding author. Tel.: +86 532 8203 1560; fax: +86 532 8203 3054. E-mail address:

[email protected] (J.-J. Hao), [email protected] (W.-J. Mao)

Highlights 

Sulfated polysaccharide Ls2-2 was extracted from Monostroma angicava Kjellm (Chlorophyta).



Ls2-2 is a glucuronic acid-containing rhamnan-type sulfated polysaccharide with branches.



Ls2-2 acted as a glucose consumption stimulator with depressing lipid accumulation.



Ls2-2 possessed a high anticoagulant, fibrin(ogen)olytic and thrombolytic activities.



Ls2-2 could be a promising antidiabetic and anticoagulant polysaccharide.

1

Abstract

A homogeneous polysaccharide was obtained from Monostroma angicava Kjellm by water extraction, preparative anion-exchange and size-exclusion chromatography. Results of chemical and spectroscopic analyses showed that the polysaccharide was a glucuronic acid-containing rhamnan-type sulfated polysaccharide. The backbone mainly consisted of →3)-α-L-Rhap-(1→ and →2)-α-L-Rhap-(1→ residues, partially sulfated at C-2 of →3)-α-L-Rhap-(1→ and C-3/C-4 of →2)-α-L-Rhap-(1→. The branching contained unsulfated or monosulfated 3-linked, 2-linked, 4-linked α-L-rhamnose and terminal β-D-glucuronic acid residues. The polysaccharide had strong antidiabetic activity assessed by glucose consumption, total cholesterol and triglyceride levels using human hepatocellular carcinoma (HepG2) and insulin-resistant HepG2 cells. The polysaccharide exhibited high anticoagulant property by activated partial thromboplastin time and thrombin time assays, and possessed high fibrin(ogen)olytic activity evaluated by plasminogen activator inhibitior-1, fibrin(ogen) degradation products and D-dimer levels using rats plasma. The investigation demonstrated that the polysaccharide from Monostroma angicava Kjellm was a novel sulfated rhamnan and could be a potential antidiabetic and anticoagulant polysaccharide.

Keywords: Monostroma angicava Kjellm; Sulfated polysaccharide; Structure; Antidiabetic activity; Anticoagulant property

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1. Introduction

Marine algae are rich in sulfated polysaccharides and show various bioactivities, such as antiviral, antitumor, immunomodulating, anticoagulant, antioxidant, antiinflammatory and antihyperlipidemic properties (Hoang, Kim, Lee, You, & Lee, 2015; Jiao, Yu, Zhang, & Ewart, 2011; Karnjanapratum & You, 2011; Lahaye, & Robic, 2007). The active sulfated polysaccharides from marine algae hold valuable pharmaceutical and biomedical potentials (Wijesekara, Pangestuti, & Kim, 2011). Diabetes is a common endocrine metabolic disease with serious damage to the human health and 95 % of diabetics are type 2 diabetes. Type 2 diabetes is a complex polygenic disease while islet cell function defects and insulin resistance are the two main etiologies, especially insulin resistance (Arvind, Poornima, Sibasis, & Arvind, 2005; Maheshwari & Thuluvath, 2011). High blood sugar, insulin resistance, hyperlipidaemia and other metabolic disorders in type 2 diabetes can cause vascular endothelial injury and dysfunction, and coagulation function obstacle (Carr, 2001). In recent years, antidiabetic activity of marine algal polysaccharides has attracted considerable attention. Some algal polysaccharides, including Laminarin and alginate, have demonstrated better role in lowering blood sugar and treating diabetes complications (Jin et al., 2004; Liu et al., 2014). A brown algal fucoidan from Fucus vesiculosus showed α-glucosidase inhibitor activity (Shan et al., 2016). Zhang et al. (2008) reported that the polysaccharides from algae had better stimulatory activity on insulin secretion in vitro. However, the antidiabetic activity of algal polysaccharide has not yet been fully investigated. The leading causes of death are now diseases that involve heart and blood vessels and as a 3

consequence thrombosis (Melo, Pereira, Fogue, & Mourão, 2004). The sulfated polysaccharides from marine algae are known to be one abundant source of heparinoids (Pomin et al., 2005). Interest in the sulfated polysaccharides from green algae is increasing because of their high anticoagulant activities, especially anticoagulant-active sulfated polysaccharides from Monostromaceae species. Sulfated polysaccharide from Monostroma nitidum is 6-fold more antithrombin-active than heparin (Maeda, Uehara, Harada, Sekiguchi, & Hiraoka., 1991). Two different sulfated polysaccharides from Monostroma species have more potent effect on the inhibition of thrombin than heparin or dermatan sulfate (Hayakawa et al., 2000). The anticoagulant-active sulfated polysaccharides were also isolated from M. nitidum and M. latissimum (Li et al., 2011; Mao et al., 2008). The sulfated polysaccharides from Monostromaceae represent a potential source of anticoagulant to be explored. M. angicava Kjellm (Chlorophyta) is widely distributed in China, and has been used as fundamental source of food and drug in traditional Chinese medicine for thousands of years. In the present study, a novel glucuronic acid-containing rhamnan-type sulfated polysaccharide was isolated from M. angicava Kjellm, a species that is abundant on the coast of Long Island, Yantai, China. The structure, antidiabetic and anticoagulant activities of the polysaccharide were investigated.

2. Materials and methods

2.1. Materials

M. angicava Kjellm was collected from the coast of Jiu Zhang Ya of Long Island (Yantai, China) on May 2012. The raw material was thoroughly washed with tap water, air-dried, milled using a 4

blender, and then stored at room temperature in a dry environment. Q Sepharose Fast Flow and Sephacryl S-400/HR were from GE Health care Life Sciences (Piscataway, NJ, USA). Dialysis membranes (flat width 44 mm, molecular weight cut-off 3500; flat width 31 mm, molecular weight cut-off 100) were from Lvniao (Yantai, China). Pullulan standards (Mw: 21.1, 47.1, 107, 200, 344, and 708 kDa) were from Showa Denko K.K. (Tokyo, Japan). L-rhamnose, L-arabinose, D-xylose, L-fucose, D-mannose, D-galactose, D-glucose, D-glucuronic D-glucosamine

acid, D-galacturonic acid and

were from Sigma (St. Louis, MO, USA). Glucose consumption, total cholesterol (TC)

and triglyceride (TG) levels assay reagents were from Applygen (Beijing, China). Tri-Reagent system and moloneymurineleukeminvirus (M-MLV) reverse transcriptase were from Invitrogen (Carlsbad, CA, USA). SYBR Green polymerase chain reaction (PCR) reagents were from QIAGEN (Hilden, Germany). Activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and fibrinogen (FIB) kits were from MD Pacific (Tianjin, China). Fibrin(ogen) degradation products (FDP) kit was from BIOLINKS CO., LTD. (Tokyo, Japan). Plasminogen activator inhibitior-1 (PAI-1) and D-dimer kits were from Simens Healthcare Diagnostics Products (Marburg, Germany).

2.2. Animals

Male Sprague-Dawley rats (250–280 g) were housed at 23 ± 2 °C under a 12 h light/dark cycle with free access to food and water (Huang, Chou, Wang, & Chen, 2012). The experiments were performed in accordance with the Guidelines of Animal Ethics Committee of Ocean University of China. 5

2.3. Isolation and purification of the sulfated polysaccharide

The milled M. angicava Kjellm (100 g) from Long Island was dipped into 30 volumes of distilled water and extracted at room temperature for 3 h and then centrifuged at 3600 × g for 10 min. The supernatant was collected by centrifugation, concentrated and dialyzed in a cellulose membrane against distilled water at room temperature for three successive days. The retained fraction was recovered, concentrated by rotary evaporation, precipitated by adding four volumes of 95 % ethanol (v/v) and dried at 40 °C to obtain a crude polysaccharide (L), and its weight was 19.20 g. The crude polysaccharide (300 mg/ 4 mL distilled water) was fractionated on a Q Sepharose Fast Flow column (30 cm × 3.5 cm, 20 °C) and eluted with a step-wise gradient of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 M NaCl at a flow rate of 1 mL/min. Eluate was collected by auto-fraction collector (6 mL/tube, Model 2110, Bio-Rad). Total sugar content of the eluate was determined by the phenol–sulfuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). The subfraction (Ls2) eluted with 2.0 M NaCl was pooled, which was the most abundant fraction (5.38 g). The fraction (100 mg/ 2 mL distilled water) was further purified on a Sephacryl S-400/HR column (100 cm × 2.5 cm, 20 °C ) eluted with 0.2 M NH4HCO3 at a flow rate of 0.3 mL/min. Eluate was collected by auto-fraction collector (6 mL/tube, Model 2110, Bio-Rad). The major fractions (Ls2-2) were pooled and freeze–dried (1.91 g).

2.4. Physico-chemical analysis

Total sugar content was determined by the phenol–sulfuric acid method using rhamnose as the standard (Dubois et al., 1956). Protein content was determined according to the method of Bradford 6

(1976). Sulfate ester content was estimated according to the method of Therho and Hartiala (1971). Uronic acid content was determined by the carbazole–sulfuric acid method (Bitter & Muir, 1962). Purity and molecular weight of polysaccharide were assessed by high performance gel permeation chromatography (HPGPC) on a Shodex OHpak SB-804 HQ column (8.0 mm × 300 mm, Tokyo, Japan) (Li et al., 2011). The column calibration was performed with pullulan standards and a refractive index detector (Agilent RID-10A Series), and elution with 0.2 M Na2SO4 at a flow rate of 0.5 mL/min. 20 μL of 1 % sample solutions in 0.2 M Na2SO4 was injected. The molecular weight was estimated by reference to a calibration curve made by pullulan standards (Mw: 21.1, 47.1, 107, 200, 344, and 708 kDa, Showa Denko K.K., Japan).

2.5. Analyses of monosaccharide composition and sugar configuration

Monosaccharide compositions were measured by reversed-phase high performance liquid chromatography (HPLC) after pre-column derivatization (Qi et al., 2012). Briefly, polysaccharide was hydrolyzed with 2 M trifluoroacetic acid at 100 °C for 6 h in a sealed tube. Excess acid was removed by co-distillation with methanol for four times after the hydrolysis was completed. 1 mg of dry hydrolysate was dissolved in 100 μL of 0.3 M NaOH, and then added to 120 μL of 0.5 M methanol solution of 1-phenyl-3-methyl-5-pyrazolone (PMP) at 70 °C for 1 h. Finally, the mixture was added 100 μL of 0.3 M HC1 solution and vigorously shaken and centrifuged for 5 min. The supernatant was filtered through 0.22 μm nylon membranes (Westborough, MA, USA) and 10 μL of the resulting solution was injected into the XDB-C18 column (4.6 mm × 250 mm). The chromatograms were performed on an Agilent 1260 Infinity HPLC instrument fitted with Agilent 7

XDB-UV detector (254 nm). The mobile phase was a mixture of 0.1 M KH2PO4 (pH 6.7)–acetonitrile (83:17). The flow rate was 1.0 mL/min and column temperature was 30 °C. Sugar identification was done by comparison with reference sugars (L-rhamnose, L-arabinose, L-fucose, D-xylose, D-mannose, D-galactose, D-glucose, D-glucuronic D-glucosamine).

acid, D-galacturonic acid and

Calculation of the molar ratio of the monosaccharide was carried out on the basis of

the ratio of peak areas of monosaccharide and correspondent monosaccharide standard. Sugar configuration was determined according to the method of Tanaka, Nakashima, Ueda, Tomii, and Kouno (2007). 5 mg of polysaccharide was hydrolyzed with 2 M trifluoroacetic acid at 105°C for 6 h. Excess acid was removed with methanol in a rotary evaporator. The hydrolysate was heated with L-cysteine methyl ester in pyridine at 60 °C for 60 min. A solution of the o-tolyl isothiocyanate was added to the mixture, and was further heated at 60 °C for 60 min. The reaction mixture was directly analyzed on an Agilent 1260 Infinity HPLC instrument using an Eclipse XDB-C18 column (4.6 mm × 250 mm) and detected by an Agilent XDB-UV detector at 250 nm. Sugar configuration was identified by comparison with reference sugars.

2.6. Reduction of carboxyl groups

The reduction of carboxyl groups in Ls2-2 was carried out according to the method of Taylor and Conrad (1972). Briefly, 20 mg of sample was dissolved in 10 mL water and then incubated with 1 mM of 1-ethyl-3-(3-dimethylaminopropyl) carbodimide (EDC) for 2 h. The pH was kept at 4.75 with 0.1 M HCl using automatictitration during incubation. Reduction was thereafter carried out by slow addition of 2 M NaBH4 solution using a hypodermic syringe. The reduction reaction proceeded 8

at room temperature for 60 min during which 4 M HCl was used to keep the pH at 7.0 with automatic titration. After dialysis overnight against distilled water, the reduction procedure was conducted a second time to afford completely reduced Ls2-2, which was collected, dialyzed, lyophilized and designated as Ls2-2R. The complete reduction was confirmed by negative results in determination of uronic acid for Ls2-2R by the carbazole–sulfuric acid method.

2.7. Desulfation

Desulfation of the sulfated polysaccharide was performed according to the method of Falshaw and Furneaux (1998). Briefly, polysaccharide (30 mg) was dissolved in water and passed through a 732 cation-exchange resin column (H+ form), which was eluted with distilled water. The combined effluent was adjusted to pH 9.0 with pyridine and then lyophilized to give a white powdered pyridinium salt. The product was dissolved in 10 mL of dimethyl sulfoxide containing 10 % (v/v) of anhydrous methanol and 1 % pyridine, and then the solution was shaken at 100 °C for 4 h. After the reaction was completed, the mixture was dialyzed against distilled water for several times and then freeze-dried. Desulfation products of Ls2-2 and Ls2-2R were named as DSLs2-2 and Ls2-2RDs, respectively.

2.8. Methylation analysis

Methylation analysis was performed according to the method of Hakomori (1964) with some modification (Harris, Henry, Blakeney, & Stone, 984). About 100 mg of anhydrous NaH was added 9

to 0.8 mL of DMSO and the mixture was stirred under N2 at about 55 °C using an oil bath. Polysaccharide (2 mg) dissolved in 0.5 mL of DMSO was added into the mixture until it turned to a grey-green solution and stirred at room temperature for 1.5 h. CH3I (1 mL) was then added to the mixture for another 1.5 h. The reaction was terminated by addition of 1 mL of distilled water, and the residue was extracted with CH2Cl2. The extract was washed with distilled water and evaporated to dryness. The completion of methylation was confirmed by IR spectroscopy as the disappearance of OH bands. Then methylated polysaccharide was hydrolyzed with 2 M trifluoroacetic acid at 105 °C for 6 h, and then reduced using NaBH4 followed by acetylation with acetic anhydride. The products were analyzed by gas chromatography–mass spectrometry (GC–MS) on a TRACE 1300 instrument (Thermo Fisher, USA) using a DB 225 fused silica capillary column (0.25 mm × 30 m). Identification of partially methylated alditol acetates was carried out on the basis of retention time and mass fragmentation patterns.

2.9. Preparation of oligosaccharide fractions by mild acid hydrolysis of Ls2-2

The acid hydrolysis of Ls2-2 was performed according to the method of Li et al. (2012) with some modification. The polysaccharide (10 mg/mL) was hydrolyzed with 0.1 M HCl at 60 °C for 9 h. The hydrolysis product was neutralized with 1.0 M NaOH, desalted by dialyzed in a cellulose membrane (100 Da) against distilled water at room temperature for three successive days, concentrated under reduced pressure at 40 °C, and a three-fold volume of 95 % (v/v) ethanol was added. The resulting supernatant and precipitate were recovered by centrifugation (3600 × g, 10 min), designated as Ls2-2-S and Ls2-2-E, respectively. The precipitate Ls2-2-E was washed with ethanol 10

and vacuum-dried. The supernatant Ls2-2-S was concentrated, and fractionated with a Bio-Gel P-4 column (1.6 cm × 100 cm) by elution with 0.2 M NH4HCO3 and detection by phenol–sulfuric acid method. The oligosaccharide fractions were collected, freeze–dried and designated as S1–S4, respectively.

2.10. Spectroscopy analysis

Fourier-transform infrared (FTIR) spectrum was measured on a Nicolet Nexus 470 spectrometer The polysaccharide was mixed with KBr powder, ground and pressed into a 1-mm pellet for FTIR measurements in the frequency range of 4000–500 cm–1 (Shingel, 2002). 1

H and 13C NMR spectra were recorded at 23 °C on an Agilent DD2 500 MHz spectrometer.

Polysaccharide (50 mg) was dissolved in 1 mL of 99 % D2O followed by freeze–dried. The process was repeated twice, and the final sample was dissolved in 0.5 mL of 99.98 % D2O. 2D 1H–1H COSY, 1

H–13C HSQC, 1H–13C HMBC and NOESY experiments were all performed (Petersen et al., 2006).

Chemical shifts are expressed in ppm using acetone as internal standard at 2.225 ppm for 1H and 31.07 ppm for 13C.

2.11. Electrospray mass spectrometry

Negative-ion electrospray mass spectrometry (ESMS) analysis was performed on a Micromass Q-Tof Ultima instrument (Waters, Manchester, UK) (Li et al., 2012; Zhang, Yu, Zhao, Liu, & Guan, 2006). Nitrogen was used as the desolvation and nebulizer gas at a flow rate of 250 L/h and 15 L/h, 11

respectively. The source temperature was 80 °C and the desolvation temperature was 150 °C. Samples were dissolved in acetonitrile/H2O (1:1, v/v), typically at a concentration of 10–20 pmol/μL, of which 10 μL was loop-injected. The mobile phase (acetonitrile/H2O, 1:1, v/v) was delivered by a syringe pump at a flow rate of 10 μL /min. The capillary voltage maintained at 3 kV, and the cone voltage was 30–100 V, depending on the size of the oligosaccharides. For CID MS/MS product-ion scanning, argon was used as the collision gas at a pressure of 1.7 bar and the collision energy was adjusted between 20 and 50 eV for optimal sequence information.

2.12. Effects of Ls2-2 on glucose consumption, TG and TC levels in HepG2 cells and PA-induced insulin resistant HepG2 cells

2.12.1. HepG2 cell culture Human hepatocellular carcinoma (HepG2) cells were obtained from China Center for Type Culture Collection and cultured in minimum essential medium (MEM) supplemented with 10 % (v/v) fetal bovine serum containing 100 U/mL penicillin G and 0.1 mg/mL streptomycin sulfate. Cells were incubated in a humidified atmosphere of 5 % CO2 at 37 °C (Inmaculada et al., 2015). Once the monolayer was approximately 70 % confluent, cells were seeded in different plates of 96-well or 12-well. After 24 h cultivation, the media were replaced with serum-free MEM for 12 h as starvation treatment prior to experiments. Then cells were treated with the polysaccharides in serum-free media. For dose–response experiments, polysaccharides of different concentrations were used. Metformin (1.2 mM) was a positive control and the non-drug control group was treated with distilled water.

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2.12.2. Cytotoxicity assay The cytotoxicity of polysaccharide was measured by the MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) assay (Mothanna et al., 2011). HepG2 cells were seeded in 96-well plates and incubated with different concentrations of Ls2-2 (10, 50, 100, 250, 500, 1000, 2000 μg /mL) for 24 h at 37 °C. Then, culture media were replaced by 200 μL fresh media containing 0.5 mg/mL of MTT. After 4 h incubation at 37 °C, the supernatant was removed and 150 μL DMSO was added to each well to solubilize the formazan crystals. After gentle mixing, the absorbance values were measured at 570 nm using a microplate reader (Bio-Rad, USA).

2.12.3. Measurement of glucose consumption in HepG2 cells HepG2 cells were seeded in 96-well plates and starved for 12 h and then incubated with different concentrations of Ls2-2 ( 25, 50, 100, 200 μg/mL) or metformin. They were divided into control group, positive control group and sample groups. After 4 h cultivation, the glucose content level was determined using commercial kit (Wu et al., 2013).

2.12.4. Measurement of glucose consumption in PA-induced insulin resistant HepG2 cells HepG2 cells were seeded in 96-well plates and starved for 12 h and then incubated in serum-free MEM with different concentrations of Ls2-2 ( 25, 50, 100, 200 μg /mL) or positive drug metformin. They were divided into control group, model group, positive control group and sample groups. Besides the control group, other groups were induced insulin resistance with palmitic acid (PA) at a concentration of 100 μM. After 24 h cultivation, the media were replaced with serum-free MEM for 4 h. Then the glucose content level was determined using commercial kit (Luo et al., 2012; Wu et al., 13

2013).

2.12.5. Measurement of TG and TC levels in PA-induced insulin-resistance HepG2 cells HepG2 cells were seeded in 12-well plates and starved for 12 h and then incubated with different concentrations of polysaccharide Ls2-2 at 25, 50, 100, 200 μg/mL and positive drug metformin for 24 h. They were divided into control group, model group, positive control group and sample groups. Besides the control group, others were induced insulin-resistance with PA at a concentration of 100 μM. TG and TC levels were measured using corresponding commercial kits, and the absorbance was monitored at 550 nm using a microplate reader (Bio-Rad, USA) (Luo et al., 2012).

2.13. Effect of Ls2-2 on the mRNA expressions of key genes related to glucose and lipid metabolism in insulin-resistant HepG2 cells

Effect of Ls2-2 on the mRNA expressions of key genes related to glucose and lipid metabolism in insulin-resistant HepG2 cells was detected by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). For real-time qRT-PCR, total RNA of the HepG2 cells was extracted using the TRIzol reagent and the first-strand cDNA was synthesized using M-MLV reverse transcriptase and oligo (dT) primer. The expression of adenosine 5′-monophosphate-activated protein kinase α2 (AMPKα2), glucose transporter 2 (GLUT2), hepatic lipase (HL), phosphoenolypyruvate carboxykinase (PEPCK) and acetyl-CoA carboxylase 2 (ACC2) mRNA was examined by qPCR using SYBR Green-based assays. Relative expressions were

14

calculated with normalization to β-actine value by using the 2-∆∆Ct method (Hao et al., 2011). The sequences of primers used for quantitative PCR analysis were shown in Supplemental Table 1.

2.14. Assay of anticoagulant activity

Activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT) were assayed by the method of Mourao et al. (1996). For APTT clotting assay, polysaccharide was dissolved in 0.9 % NaCl at different concentrations (10, 20, 50, 100, 150 µg/mL). 90 µL of citrated normal human plasma was mixed with 10 µL of polysaccharide samples and incubated at 37 °C for 1 min. 100 µL of APTT assay reagent (Tianjin MD Pacific, China) pre-warmed at 37 °C for 10 min was added, and incubated at 37 °C for 2 min. Pre-warmed 100 µL of 0.25 M calcium chloride was then added and clotting time was recorded in a SL318 coagulometer (Senlan Medical Science and Trading Co., Ltd, China). For PT clotting assay, 90 µL of human plasma was mixed with 10 µL of polysaccharide samples and incubated at 37 °C for 1 min. Then, 200 µL of pre-warmed PT assay reagent (Tianjin MD Pacific, China) was added and clotting time was recorded. For TT clotting assay, 90 µL of human plasma was mixed with 10 µL of polysaccharide samples and incubated at 37 °C for 1 min. Then, 200 µL of pre-warmed TT assay reagent (Tianjin MD Pacific, China) was added and clotting time was recorded. For fibrinogen (FIB) content assay (Mackie, Lawrie, & Kitchen, 2002), 90 µL of citrated human plasma was mixed with 10 µL of polysaccharide samples, then was diluted with 900 µL of imidazole buffer. 200 µL of the diluted plasma sample was taken and incubated at 37 °C for 3 min, followed by addition of 100 µL of FIB assay reagent and clotting time was recorded. The FIB content was estimated by reference to a calibration curve made by FIB constant value 15

plasma. Heparin (Sigma–Aldrich, USA) was used for the comparison of anticoagulant activity of the polysaccharide. Saline solution (0.9 % NaCl) was used as control.

2.15. Evaluation of fibrin(ogen)olytic activity and thrombolytic activity

The assay of fibrin(ogen)olytic activity was done by PAI-1 (Zucker, Seligsohn, Salomon, & Wolberg, 2014), FDP (Ito et al., 2003) and D-dimer (Pulivarthi & Gurram, 2014). Briefly, male Sprague-Dawley rats were randomly divided into sample groups, positive control group and control group (10 rats/group). The experimental rats were anaesthetized with 15 % urethane, and then injected with Ls2-2 (2.5, 5, 10 mg/kg), urokinase (20000 U/kg) or saline solution. After 30 min to allow for circulation, the rats were secured in the supine position, and the blood was taken from the abdominal aorta. The levels of PAI-1, FDP, and D-dimer in the blood were determined using corresponding commercial kits. Berichrom PAI was used for PAI-1, Latex Test BL-2 P-FDP was used for FDP, and INNOVANCE Dimer was used for D-dimer. PAI-1 assay was performed on a CA-7000 automated blood coagulation analyzer (Sysmex Corporation, Japan). FDP and D-dimer assays were performed on a CS-5100 automated blood coagulation analyzer (Sysmex Corporation, Japan). The assay of thrombolytic activity was done by clot lytic rate (Omura et al., 2005). In briefly, the blood was taken from the abdominal aorta of the male Sprague-Dawley rats, and collected in a silicone tube. The blood was placed at room temperature until the big blood clot was completely formed. The clot was rinsed with saline solution, and the liquid in the surface of the clot was removed by filter paper. Then the clot was cut into appropriate pieces, weighed and put into PE tubes, 16

respectively. The tubes were randomly divided into five experimental groups (10 clots/group): Ls2-2 (5, 10, 20 mg/mL), urokinase (100 U/mL) and saline solution groups. Sample (1 mL) was added to the tube, followed by shaking incubation at 85 rpm for 24 h at 37 °C. The residual clot was drawn from the tube. The liquid in the surface of clot was removed. The wet weight of the residual clot was determined. The clot lytic rate was calculated according to the equation: clot lytic rate (%) = (1–W2/W1) ×100, where W1 is the wet weight of the whole clot, and W2 is the wet weight of the residual clot.

2.16. Statistical analysis

The bioassay results were expressed as means ± standard deviation (SD). Three samples were prepared for assays of every attribute. The experimental data were subjected to an analysis of ANOVA and statistical significance is denoted by asterisks and hashes. * and # represented p< 0.05 while ** and ## were p < 0.01.

3. Results and discussion

3.1. Chemical characteristics of the sulfated polysaccharide Ls2-2

The yield of Ls2-2 from crude polysaccharide (w/w) was about 9.95 %. Ls2-2 appeared as a single peak in the HPGPC chromatogram, and its average molecular weight was about 58.4 kDa based on its retention time in HPGPC chromatogram (Supplemental Fig. 1a). Ls2-2 contained 28.83 % sulfate ester and 5.09 % uronic acid. No protein was detected in Ls2-2. Monosaccharide 17

composition analysis by reversed-phase HPLC demonstrated that Ls2-2 mainly consisted of rhamnose (96.12 %) with minor amount of glucuronic acid (3.88 %) (Supplemental Fig. 1b). HPLC analysis showed that the absolute configuration of rhamnose and glucuronic acid in Ls2-2 was L- and D-configuration,

respectively, due to the retention times were in agreement with retention times of the

derivatives of L-rhamnose and D-glucuronic acid, respectively (Supplemental Fig. 1c). IR spectrum gave some characteristic absorptions from function groups of Ls2-2 (Supplemental Fig. 1d). The signal at 3458 cm–1 was from stretching vibration of O–H, and the signal at 2928 cm–1 was due to stretching vibration of C–H (Mitić et al., 2011). The band at 1051 cm–1 was from the stretching vibration of C–O and change angle vibration of O–H. The bands at 1640 and 1429 cm–1 were contributed by asymmetrical and symmetrical stretching vibration of COO–. Several bands corresponding to sulfate ester groups were also observed. The peak at 850 cm–1 derived from the stretching vibration of C–O–S of sulfate ester in axial position, and 1240 cm–1 was from stretching vibration of S=O of sulfate group. A comparative analysis between the sulfated polysaccharide Ls2-2 and its desulfated product DSLs2-2 provided important information for the linkage position assignments of each monosaccharide and the sulfation position. Total ion gas chromatograms of Ls2-2 and DSLs2-2 on GC-MS were shown in Supplemental Fig. 2A and B. The results showed that Ls2-2 was mainly consisted of (1→3)-linked rhamnose, (1→2)-linked rhamnose and (1→2,3)-linked rhamnose residues, minor amounts of (1→4)-linked rhamnose and (1→2,4)-linked rhamnose residues were also detected (Table 1). Compared with the results for Ls2-2, increased amounts of (1→3)-linked rhamnose and (1→2)-linked rhamnose residues were detected, and decreased amounts of (1→2,3)-linked rhamnose and the disappearance of (1→2,4)-linked rhamnose residues were found in 18

DSLs2-2. Therefore, the sulfate substitutions were at the C-2 of (1→3)-linked rhamnose and C-3 or C-4 of (1→2)-linked rhamnose residues. It could be deduced that 23.52 % of total number of the rhamnose residues in Ls2-2 were substituted by sulfate ester groups, specifically to (1→3)-linked rhamnose residues were about 17.95 %, to (1→2)-linked rhamnose residues were about 5.57 %, respectively. In order to obtain the information for the linkage pattern of glucoronic acid and the sulfation position, mehtylation analysis was also carried out with two polysaccharide fractions, the carboxyl-reduced one (Ls2-2R) and the carboxyl-reduced and desulfated fraction (Ls2-2RDs). Total ion gas chromatograms of Ls2-2R and Ls2-2RDs on GC-MS were shown in Supplemental Fig. 2C–D. Compared with the results for Ls2-2R (Table 1), increased amounts of (1→)-linked glucose residues and the disappearance of (1→2)-linked glucose residues were found in Ls2-2RDs besides the same results obtained from the comparative analysis between Ls2-2 and DSLs2-2. Thus, it was deduced that the glucuronic acid in Ls2-2 was existed in the form of (1→)-linked glucuronic acid residues, with partially sulfate groups at the C-2 .

3.2. NMR spectroscopy analyses of Ls2-2 and DSLs2-2

3.2.1. NMR spectroscopy analysis of DSLs2-2 In the 1H NMR spectrum of DSLs2-2 (Fig. 1a), the lower-field signals at 5.00–5.50 ppm were assigned to be anomeric proton signals of α-L-rhamnopyranose residues. The five anomeric proton signals at 5.00, 5.07, 5.23, 5.29 and 5.38 ppm had relative integrals of 1.00: 1.03: 1.01: 0.21: 0.21, 19

which were labeled A, B, C, D and E, respectively. The signal at 1.35 ppm was assigned to be the proton of CH3 group. Other proton signals at 3.30–4.50 ppm were H2–H5 of the sugar residues. In the anomeric region of the 13C NMR spectrum (Fig. 1b), three main carbon resonances occurred at 103.54, 102.41 and 102.22 ppm. The signals at 70–80 ppm were attributed to C-2–C-5 of the rhamnose residues. The signal at 18.26 ppm was assigned to be the C-6 of the rhamnose residue. The α-anomeric configuration of rhamnopyranose residues was also deduced from H-5 signal at 3.89 ppm and C-5 signal at 70.80 ppm (Cassolato et al., 2008). Due to the relatively low glucuronic acid content and overlapping of signals, it was difficult to identify its chemical shifts. The signal at 105.72 ppm presented in the 13C NMR spectrum could arise from non-reducing terminal β-D-glucuronic acid residues (Cassolato et al., 2008). The signal at 177.33 ppm should be assigned to be the carboxyl group of glucuronic acid (Hu et al., 2016). All the results suggested that the polysaccharide was a rather intricate assemblage despite the simplicity of compositional data. The 1H–1H COSY spectrum (Fig. 1c) allowed the assignment of most of the signals of the proton spin systems. The 1H–13C HSQC spectrum (Fig. 1d) also permitted assignment of the relevant carbon signals. The anomeric proton signals of residues A and B at 5.00 and 5.07 ppm were correlated to the anomeric carbon signal at 103.54 ppm. The substitutions of residues A and B at C-3 were deduced because of the downfield shifts of C-3 (79.43 ppm) as compared with that of parent α-L-rhamnopyranose residue. Thus residues A and B were assigned to be the →3)-α-L-Rhap-(1→ residue (Ropellato et al., 2015). The anomeric proton signals of residues A and B with different chemical shifts probably corresponded to a same type of residue with different environments. The anomeric proton signal of residue C at 5.23 ppm was related to the anomeric carbon signal at 102.41 20

ppm. The correlated signal H-2/C-2 (4.11/79.01 ppm) in residue C further indicated that residue C was the →2)-α-L-Rhap-(1→ residue (Ovod, Zdorovenko, Shashkov, Kocharova, & Knirel, 2004). The anomeric proton signal of residue D at 5.29 ppm was correlated to the anomeric carbon signal at 102.22 ppm. The H-2/C-2 and H-3/C-3 resonances of residue D at 4.11/79.01 ppm and 4.00/79.43 ppm were in agreement with the substitution at C-2 and C-3, so residue D was attributed to the →2,3)-α-L-Rhap-(1→ residue. It is observed that H-1 signal of residue E at 5.38 ppm was correlated to the C-1 resonance at 102.22 ppm, which was attributed to the →2)-α-L-Rhap-(1→ residue with sulfation at C-3 (Li et al., 2011). The signal H-3 (4.61 ppm) of residue E exhibited a down-field shift (0.36 ppm) was due to sulfation of C-3 (Pomin et al., 2005). The result indicated that the desulfation of Ls2-2 was not absolute. But this signal was negligible and most of the sulfation was believed to be removed. More correlation between proton and carbon signals within the sugar residues could be deduced by the 1H–13C HMBC spectrum (Fig. 1e). The anomeric proton signal of residue A at 5.00 ppm was related to the signal at 79.01 ppm which were the C-2 of residues C, D and E, indicating the presence of the sequences →3)-α- L -Rhap-(1→2)-α-L-Rhap→, →3)-α-L-Rhap-(1→2,3)-α-L-Rhap→ and →3)-α-L-Rhap-(1→2)-α-L-Rhap(3SO4)-(1→. The anomeric proton signal of residue B at 5.07 ppm was correlated to the C-3 signals of residues A and D at 79.43 ppm, suggesting the presence of the sequences →3)-α-L-Rhap-(1→3)-α-L-Rhap→ and →3)-α-L-Rhap-(1→2,3)-α-L-Rhap→ . Furthermore, the anomeric proton of residue C at 5.23 ppm was related to the C-3 signal of residues A and B at 79.43 ppm, indicating the presence of the sequence →2)-α-L-Rhap-(1→3)-α-L-Rhap→. In addition, the presences of cross signals H-1(D)/C-2(C), H-1(D)/C-2(E), H-1(E)/C-3(A) and H-1(E)/C-3(B) indicated the possible linkages →2,3)-α-L-Rhap-(1→2)-α-L-Rhap→, 21

→2,3)-α-L-Rhap-(1→2)-α-L-Rhap(3SO4)→ and →2)-α-L-Rhap(3SO4)-(1→3)-α-L-Rhap→.

3.2.2. NMR spectroscopy analysis of Ls2-2 In the 1H spectrum of Ls2-2 (Fig. 2a), six anomeric proton signals occurring at 5.03, 5.07, 5.25, 5.29, 5.33 and 5.50 ppm were assigned to be α-L-rhamnopyranose residues, which were labeled A, B, C, D, E and F, respectively. Their relative integrals were 1.00: 2.40: 2.07: 0.51: 0.92: 1.45. Compared with the anomeric proton signals of DSLs2-2, Ls2-2 had an additional anomeric proton signal at 5.50 ppm, and the anomeric proton signals at 5.33 and 5.50 ppm were in high strength. By combining the information from the 1H NMR, 13C NMR (Fig. 2b), 1H–1H COSY (Fig. 2c), 1H–13C HSQC (Fig. 2d) and 1H–1H NOESY (Fig. 2e), the anomeric proton signal at 5.50 ppm was correlated to the anomeric carbon signal at 100.61 ppm, and residue F was assigned to be the →3)-α-L-Rhap(2SO4)-(1→ residue. The anomeric proton signal at 5.33 ppm was corresponded with the anomeric carbon signal at 100.61 ppm, and residue E was ascribed to the →2)-α-L-Rhap(3SO4)-(1→ residue. According to the assignments of DSLs2-2, the anomeric proton signals at 5.03 and 5.07 ppm were related to the anomeric carbon signals at 103.16 and 102.78 ppm, respectively, thus residues A and B were attributed to the →3)-α-L-Rhap-(1→ residues. The anomeric proton signal at 5.25 ppm was related to the anomeric carbon signal at 101.42 ppm, and residue C was attributed to →2)-α-L-Rhap-(1→ residue. The anomeric proton signal at 5.29 ppm was related to the anomeric carbon signal at 101.42 ppm, and residue D was ascribed to →2,3)-α-L-Rhap-(1→ residue. Even more specifically, the C-3 signals of residues A and B showed the downfield signals at 22

78.30 ppm, which were related to the H-3 signals at 3.92 and 3.96 ppm, respectively. With regard to residue C, its characteristic correlation was the C-2 signal at 78.86 ppm with the H-2 signal at 4.30 ppm. The correlation was also assigned to the residues D, while its another correlation was the H-3 signal at 3.97 ppm to the C-3 signal at 78.30 ppm. By combining the data from the 1H–1H COSY and 1

H–13C HMQC spectra, the assignment of almost all the proton and carbon signals of A–F

monosaccharide units were completed. The sequence of sugar residues in Ls2-2 was determined by 1H–13C HMBC and 1H–1H NOESY spectra. Due to the overlap of H and C signals, accurate assignments were hard from the 1H–13C HMBC spectrum. 1H–1H NOESY spectrum gave abundant information. The anomeric proton signal of residue A was correlated to the H-2 signals of residues C, D and E at 4.30 ppm, indicating the presence of the sequences →3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→, →3)-α-L-Rhap-(1→2,3)-α-L-Rhap-(1→ and →3)-α-L-Rhap-(1→2)-α-L-Rhap(3SO4)-(1→. The anomeric proton signal of residue B was related to the H-3 signals of residues A and F at 3.92 ppm and 4.11 ppm, respectively. Meanwhile the H-3 signal of residue B at 3.96 ppm was correlated to the H-1 signal of residue F at 5.50 ppm, indicating the sequences of →3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→, →3)-α-L-Rhap-(1→3)-α-L-Rhap(2SO4)-(1→ or →3)-α-L-Rhap(2SO4)-(1→3)-α-L-Rhap-(1→. The anomeric proton signals of residues C, D and E at 5.25, 5.29 and 5.33 ppm showed correlations with the H-3 signal of residue B at 3.96 ppm, indicating the presence of the sequences →2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→, →2,3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→ and →2)-α-L-Rhap(3SO4)-(1→3)-α-L-Rhap-(1→. The presence of the sequence →2)-α-L-Rhap-(1→3)-α-L-Rhap(2SO4)-(1→ was also deduced by the cross signal C(H-1)/F(H-3). 23

3.3. Sequence analysis of Ls2-2-derived oligosaccharides by negative-ion ES-CID MS/MS

In order to get more detailed structural information of Ls2-2, the oligosaccharides were prepared by mild acid hydrolysis of Ls2-2. Two types of hydrolysis products, Ls2-2-S and Ls2-2-E, were obtained. HPLC analysis showed that Ls2-2-E was only composed of rhamnose, while the oligosaccharide mixture Ls2-2-S consisted of rhamnose and glucuronic acid. The result suggested that glucuronic acid part was easier to be released from the chain of Ls2-2 by mild acid hydrolysis, and the glucuronic acid part could be located at the side chains of Ls2-2. Ls2-2-S was fractionated by gel filtration chromatography. Finally, four oligosaccharide fractions (S1–S4) were obtained. From the negative-ion ES-MS spectra, S1 was deduced to be a monosulfated rhamnose (Supplemental Fig. 3a), S2 mainly consisted of monosulfated rhamno-disaccharide (Supplemental Fig. 3b), S3 was the mixture of disaccharide and trisaccharide with or without sulfate ester (Supplemental Fig. 3c), S4 was a mixture of multiple oligosaccharides with high polymerization degree (Supplemental Fig. 3d). Simple glucuronic acid units were existed in S2, S3 and S4 in the forms of R+GA, R2+GA, RS+GA, R+GA(S), R2S+GA and R2+GA(S), while RS and R2S were the major components of S1 and S2. R, GA and S represented rhamnopyranose, glucuronic acid and sulfate ester, respectively. The detailed sequences of the oligosaccharides were detected by negative-ion ES-CID-MS/MS because the anionic oligosaccharides readily ionize in negative ES-MS mode. Product-ion spectra of singly charged [M–H]– were mainly chosen for the study as other precursors did not produce fragments that were more structurally informative (Li et al., 2016).

3.3.1. Sequence analysis of RS in the oligosaccharide S1 Structure of monosulfated rhamno-monosaccharide (RS) was deduced by the product-ion 24

spectrum of m/z 243 in the negative-ion mode. In the ES-CID MS/MS spectrum (Fig. 3A), the ions at m/z 183 and 139 were assigned to the fragment ions 0,2A and 0,2X, respectively. The 0,2X at m/z 139 was mainly due to the 2-O-sulfated rhamnose, and the 0,2A at m/z 183 was formed from 4-O-sulfated rhamnose (Tissot, Salpin, Martinez, Gaigeot, & Daniel, 2006). The ion at m/z 97 corresponded to the hydrogenosulfate anion HSO4-, and the ion at m/z 225 was produced by the dehydration. The 3-O-sulfated rhamnose might also be existent.

3.3.2. Sequence analysis of R2S and R+GA in the oligosaccharide S2 The ion at m/z 389 attributed to be monosulfated rhamno-disaccharide was major ion of the oligosaccharide S2. In the ES-CID MS/MS spectrum (Fig. 3B), the ions at m/z 225 and 243 were produced from glycosidic bond cleavage and could be assigned to B1 and C1, respectively. The ions at m/z 285, 315 and 329 were all cross-ring cleavages and assigned to 0,2X0/0,2X1, 0,3X1 and 0,2A2, respectively. The dehydrated form of the precursor ion was also observed at m/z 371. Despite the lack of information fragmentation ions the presence of (1→3) glycosidic bond could not be excluded from the disaccharide fraction (Daniel et al., 2007). The sequences of R2S could be α-L-Rhap(2SO4)-(1→3)-α-L-Rhap, α-L-Rhap(3SO4)-(1→2)-α-L-Rhap and α-L-Rhap(2SO4)-(1→4)-α-L-Rhap. The minor ion at m/z 339 was existed in S2. It was attributed to be a disaccharide which consisted of rhamnose (R) and glucuronic acid (GA), and defined as R+GA. The major ion at m/z 321 was derived from the dehydration of the ion at m/z 339 (Fig. 3C). The 2,5A2 –type ion wasn't observed which only appeared in reducing end GlcA residue. Thus the GlcA was believed to locate at the non-reducing end (Anastyuk, Shevchenko, Nazarenko, Dmitrenok, & Zvyagintseva, 2009). 25

The result is in agreement with NMR spectrum analysis of DSLs2-2. The linkage patterns between Rha and GlcA might be 1→2, 1→3 and 1→4. The structures of R+GA could be β-D-GlcA-(1→2)-α-L-Rhap, β-D-GlcA-(1→3)-α-L-Rhap and/or β-D-GlcA-(1→4)-α-L-Rhap. The former two might be the main structures due to only a minor amount of (1→4)-linked-rhamnopyranose was found in Ls2-2.

3.3.3. Sequence analysis of R2+GA in the oligosaccharide S3 In the ES-CID MS/MS spectrum (Fig. 3D), the ion at m/z 485 showed similar feature with the disaccharide R+GA which gave product ion spectra with fragments B2 (m/z 321), C2 (m/z 339), C1 (m/z 193) ions of the glycosidic bond cleavage, 1,3A2 (m/z 235) from cross-ring cleavage and m/z 467 from dehydration. Hence, the ion at m/z 485 was considered to add another rhamnose to the disaccharides of the ion at m/z 339 as R2+GA. Thus, Rn+GA could be deduced, such as R4+GA and R5+GA.

3.3.4. Sequence analysis of RS+GA, R+GA(S), R2S+GA and R2+GA(S) in the oligosaccharide S4 Structures of RS+GA and R+GA(S) appeared in the form of the ion at m/z 419 (Fig. 3E). In the MS/MS spectrum, the ion at m/z 339 was derived from the loss of SO3, the appearance of the ions at m/z 183, 225 and 243 indicated that the sulfate ester was in the rhamnose residue. The ions at m/z 183 indicated C-4 sulfation as well as 0,2A2 cleavage in which hydrogen of the hydroxyl group at C-3 played an essential role (Saad & Leary, 2004; Tissot, Salpin, Martinez, Gaigeot, & Daniel, 2006), so the linkage pattern between GlcA and Rha was 1→2. Moreover, the sulfation at C-2 might also exist and then the linkage was 1→3. The disappearance of m/z 139 might due to the C-3 substitution. As 26

to the ion at m/z 255, it might arise from the dehydration of sulfated glucuronic acid. In the MS/MS/MS spectrum of the ion at m/z 255 (Supplemental Fig. 4A), the fragment ions at m/z 237, 211, 193 and 175 were assigned to be loss of H2O, CO2, H2O/ CO2 and SO3, respectively, the ion at m/z 113 was formed by losing H2O/ CO2 of the ion at m/z 175. Combined with the methylation analyses of the carboxyl-reduced fraction (Ls2-2R) and the carboxyl-reduced and desulfated fraction (Ls2-2RDs), the sulfate ester could be at C-2 of glucuronic acid. The product-ion spectrum of the ion at m/z 565 (Fig. 3F) gave the detailed information of the structures R2S+GA and R2+GA(S), the ion at m/z 485 was derived from the loss of SO3. The MS/MS/MS spectrum of the ion at m/z 389 (Supplemental Fig. 4B) indicated same fragment ions with the MS/MS spectrum of m/z 389 (Fig. 3) except for the disappearance of the ion at m/z 329. Thus the structure R2S+GA was believed to add GlcA to the structures R2S and R2. In addition, the ion at m/z 255 would arise from the dehydration of sulfated glucuronic acid. The MS/MS/MS spectrum of the ion at m/z 255 showed same fragment ions with the MS/MS/MS spectrum of the ion at m/z 255 in the above mentioned MS study (Supplemental Fig. 4A). The main structures were listed in Fig. 3F.

All above results demonstrated that the backbone of Ls2-2 mainly consisted of →3)-α-L-Rhap-(1→ and →2)-α-L-Rhap-(1→ residues, partially sulfate groups were at C-2 of →3)-α-L-Rhap-(1→, C-3 and C-4 of →2)-α-L-Rhap-(1→ residues. The branching was composed of unsulfated or monosulfated 3-linked, 2-linked, 4-linked α-L-rhamnose and terminal β-D-glucuronic acid residues, and the terminal β-D-glucuronic acid was at non-reducing end. The structure of Ls2-2 from M. angicava Kjellm was different from those of the sulfated polysaccharides previously 27

obtained from Monostroma species though they were mainly composed of →3)-α-L-Rhap-(1→ and/or →2)-α-L-Rhap-(1→ residues (Lee, Koizumi, Hayashi, & Hayashi, 2010; Li et al., 2011). However, the monosaccharide composition, linkage pattern, sugar sequence, branches, sulfate substitution and the substitution degree of sulfate ester groups of sugar residues in Ls2-2 were distinguished from those of the sulfated polysaccharides. Moreover, the structural characteristics of Ls2-2 were different from those of PF2 from M. angicava (Li et al., 2017). Especially, Ls2-2 has the branches consisting of unsulfated or monosulfated 3-linked, 2-linked, 4-linked α-L-rhamnose and terminal β-D-glucuronic acid residues. PF2 was merely consisted of rhamnose, with sulfate substitution only at C-3 of →2)-α-L-Rhap-(1→ residues. The complex structure of sulfated polysaccharides from Monostromaceae depends on various factors such as species, ecophysiological growth conditions, biosynthesis machinery, harvest time and extraction methods (Lahaye & Robic, 2007). The present result suggested that marine green algae Monostroma species could be a potential source of sulfated polysaccharides with novel structures. Further work is required to explore its structural diversity in relation with its functional properties among members of Monostromaceae.

3.4. Effects of Ls2-2 on glucose consumption, TG and TC levels in HepG2 cells and PA-induced insulin resistant HepG2 cells

Antidiabetic activity of the sulfated polysaccharide was evaluated by assays of effects of Ls2-2 on glucose consumption, TG and TC levels in HepG2 cells and PA-induced insulin resistant HepG2 cells using metformin as a reference. Metformin is one of the agents more largely used in the therapy of diabetes. It was known that the insulin resistance is a major pathogenic factor in diabetes mellitus. 28

Therefore prevention of metabolic disorder caused by insulin resistance and improvement of insulin sensitivity are very important for the therapy of type 2 diabetes. In the experiment, insulin resistance model was done using PA. Prior to evaluate the effects of Ls2-2 on glucose consumption, TG and TC levels in HepG2 cells and PA-induced insulin resistant HepG2 cells, the cytotoxicity profile of Ls2-2 was determined by MTT assay. As shown in Fig. 4a, Ls2-2 had no significant cytotoxicity up to 500 μg/mL and could be used in the further experiments.

3.4.1. Effect of Ls2-2 on glucose consumption in HepG2 cells and insulin-resistant HepG2 cells From the Fig. 4b, it was observed that Ls2-2 could improve the glucose consumption level in HepG2 cells. Compared to the control group, the glucose consumption level of Ls2-2 at 200 μg/mL increased 36 % (P<0.01). Furthermore, the glucose consumption levels of Ls2-2 at 50 μg/mL and 100 μg/mL also showed significant increasing (P<0.01). Moreover, the effect of Ls2-2 on glucose consumption level was even better than that of metformin. In order to examine whether or not Ls2-2 could affect glucose consumption level in PA-induced insulin resistant HepG2 cells, 100 μM PA were added to the Ls2-2 and metformin treated groups, and their glucose consumption levels were compared with that of model group. As shown in the Fig. 4c, compared with the control group, the glucose consumption level in the model group significantly decreased (P<0.05), indicating the model establishment was successful. It was noted that Ls2-2 increased glucose consumption level in a dose-dependent manner. At 200 μg/mL, Ls2-2 showed significant effect on improving glucose consumption (P<0.05). Compared to the model group, glucose consumption level of Ls2-2 at 200 μg/mL increased 22 %. The glucose consumption of 29

Ls2-2 was similar to that of the metformin group.

3.4.2. Effect of Ls2-2 on TG and TC levels in PA-induced insulin resistant HepG2 cells In order to investigate whether or not Ls2-2 could improve TG and TC levels in PA-induced insulin resistant HepG2 cells, 100 μM PA were added to the Ls2-2 and metformin treated groups, and their TG and TC levels were compared with that of the model group. As shown in Fig. 4d and e, compared with the control group, TG and TC levels of the model group showed markedly significant increasing (P<0.01), indicating that the model establishment was successful. Compared to the model group, TG levels of Ls2-2 at 25, 50, 100 μg/mL reduced 30 %, 33 % and 32 %, respectively. All Ls2-2 groups showed significant effects on TG level decreasing (P<0.01). Moreover, the abilities of Ls2-2 at 25, 50 and 100 μg/mL on TG level decreasing were better than that of the metformin group. In addition, compared with the model group, Ls2-2 groups also showed significant effects on TC level decreasing (P<0.01). At 25 and 50 μg/mL, the effects of Ls2-2 on TC level decreasing were even better than that of metformin. The results suggested that Ls2-2 significantly increased glucose consumption level in HepG2 cells and PA-induced insulin resistant HepG2 cells. Furthermore, Ls2-2 demonstrated significant effects on TG and TC levels decreasing in PA-induced insulin resistant HepG2 cells. The preventing or delaying the absorption of glucose has been considered a promising approach in the treatment of diabetes and its complications (Levetan & Pierce, 2013). Thus, Ls2-2 could be a potential antidiabetic polysaccharide. To date, little work in the literature is related to the hyperglycemic activity of marine algae polysaccharides on insulin resistance in vitro. The fucoidan FvF from the 30

brown alga Fucus vesiculosus showed a high α-glucosidase inhibitory activity and could decrease the fasting blood glucose level of db/db mice (Shan et al., 2016). The oligomannuronate from the brown alga Laminaria japonica increased insulin stimulated-glucose uptake by 20 % at 50 μM (Hao et al., 2011). Some polysaccharides from other resources were also investigated as antidiabetic drugs. The polysaccharide GFP from the fungus Grifola frondosa, which mainly consisted of glucose and galactose, enhanced the absorption of glucose of HepG2 cells in a dose dependent manner (Ma et al., 2014). Chen et al. (2016) reported that the polysaccharide H-1-2 from Pseudostellaria heterophylla, which was a type of glucan, could significantly increase the glucose consumption of HepG2 cells. Incubation with H-1-2 at the concentrations of 50–600 µg/mL for 24 h showed that glucose consumption of HepG2 cells grew from 6.41 to 15.83 mM. It was noted that the glucose consumption level of Ls2-2 at 200 μg/mL was lower than that of H-1-2. The relationships between the structure and antidiabetes activity of the polysaccharides are complex. An in-depth investigation on the structure-activity relationship of the polysaccharides will aid understanding their antidiabetic activities and may ultimately lead to the development of novel antidiabetic agents.

3.4.3. Effect of Ls2-2 on the mRNA expressions of key genes related to glucose and lipid metabolism in insulin-resistant HepG2 cells

In the present study, the mRNA expressions of key genes involved in glucose and lipid metabolism in insulin-resistant HepG2 cells were detected by real-time RT-PCR. As shown in Fig. 5, Ls2-2 stimulated the expressions of AMPKα2, GLUT2 and HL, as well as inhibited the expressions of PEPCK and ACC2. The AMP-activated protein kinase (AMPK) is an essential regulator of glucose and lipid metabolism, imposing profound influence on lipid oxidation, synthesis and storage 31

(Hao et al., 2015; Lanaspa et al., 2012). The AMPKα2 is an important isoform among the AMPK isoforms in liver (Lanaspa et al., 2012; Muoio, Seefeld, Witters, & Coleman, 1999). In the present results, increased AMPKα2 mRNA was followed by the improved mRNA levels of GLUT2 and HL, which suggested that the up-regulated AMPKα2 by Ls2-2 could lead to the increase of glucose transport and hepatic lipase lipolysis via the GLUT2 and HL pathway, correspondingly. Meanwhile, the increased AMPKα2 mRNA, together with its downstream genes GLUT2 and HL, were closely related to the down-regulated mRNA levels of PEPCK and ACC2, which suggested that Ls2-2 could repress the gluconeogenesis via the decreased hepatic gluconeogenic enzymes PEPCK, as well as increase mitochondrial lipid oxidation via the ACC2 pathway. The results suggested that Ls2-2 ameliorated insulin resistance in HepG2 cells at least partly via the induction of AMPKα2 expression and its downstream GLUT2 and HL, as well as the reduction of PEPCK and ACC2. The representative amplification and melting curves of RT-PCR for corresponding genes were shown in Supplemental Fig. 5.

3.5. Anticoagulant activity of Ls2-2

Anticoagulant activity of Ls2-2 was evaluated by APTT, TT, PT and FIB assays using heparin as a reference. As listed in Table 2, the anticoagulant activity of Ls2-2 was concentration–dependent. APTT was strongly prolonged by Ls2-2, and the signal for clotting time was more than 200 s at 50 µg/mL. Moreover, Ls2-2 also effectively extended the TT, and the signal for clotting time was more than 120 s at 200 µg/mL. However, the effect of Ls2-2 on PT was markedly different from that of heparin. No distinct clotting inhibition was observed in PT assay even at the concentration at which 32

APTT and TT were prolonged. In addition, the decreased FIB level by Ls2-2 was observed with increasing concentration of polysaccharide. The prolongation of APTT indicates the inhibition of the intrinsic and/or common pathway, prolongation of TT suggests inhibition of thrombin activity or fibrin polymerization, whereas no prolongation of PT demonstrates no inhibition of the extrinsic pathway of coagulation. Fibrinogen is the key factor in coagulation. Decreased blood levels of fibrinogen can reduce blood viscosity, prevent red blood cell aggregation and platelet aggregation, and thereby avoid the formation of hypercoagulable states and thrombosis. The present results suggested that Ls2-2 had a high anticoagulant property. The anticoagulant activity of Ls2-2 was different from that of heparin, and high concentrations were required to obtain the same effect as with heparin. Ls2-2 demonstrated a higher APTT activity than the sulfated polysaccharides P from M. latissimum and WF1 from M. nitidum (Mao et al., 2008; Zhang et al., 2008). Moreover, it was observed that the anticoagulant activity of Ls2-2 was similar to that of the sulfated polysaccharide PF2 from M. angicava. However, the anticoagulant activity of Ls2-2 was lower than that of the sulfated polysaccharide PML from M. latissimum (Li et al., 2011), in that both were composed of (1→3)- and (1→2)-linked rhamnose residues, but with different molecular weights. The molecular weight of the sulfated polysaccharide PML from M. latissimum was about 513 kDa. The relationship between structure and anticoagulant activity has been previously investigated in detail for sulfated galactans and sulfated fucans (Pomin, 2014). Each type of polysaccharide may form a particular complex with the plasma inhibitor and the target protease. The paradigm of heparin-antithrombin interaction couldn’t be extended to other sulfated polysaccharides (Melo, Pereira, Fogue, & Mourão, 2004). The structural basis of this interaction is complex because it involves naturally heterogeneous 33

polysaccharides. Further work is required to explore the relationship between the fine structure and anticoagulant activity of the different species of Monostroma sulfated polysaccharide.

3.6. Fibrin(ogen)olytic activity and thrombolytic activity of Ls2-2

The fibrin(ogen)olytic activity and thrombolytic activity of Ls2-2 were evaluated by assays of PAI-1, FDP, D-dimer and clot lytic rate using urokinase as a reference. The PAI-1 is a primary regulator of the fibrinolytic system (Kohler & Grant, 2000). It is becoming a recognized risk factor in the development of cardiovascular disease through it s inhibition of the effects of tissue type plasminogen activator. FDP is different molecular weight degradation fragments of fibrin and fibrinogen, which is released by fibrinolysis, including the terminal degradation products of crosslinked fibrin containing D-dimer and fragment E complex. The D-dimer is a unique marker of fibrin degradation which is released from crosslinked fibrin by the action of plasmin (Adam, Key, & Greenberg, 2009). Compared with the control group, the PAI-1 level was effectively decreased by Ls2-2 (Fig. 6A), and the increased markedly level was observed in FDP assay of Ls2-2 (Fig. 6B), indicating that Ls2-2 had good fibrin(ogen)olytic activity. The treatment with Ls2-2 resulted in significant increasing of the D-dimer level suggesting fibrin(ogen)olytic activity and thrombolytic activity (Fig. 6C). Moreover, It was observed that the decreasing effect of Ls2-2 at 10 mg/kg on PAI-1 was marked higher than that of urokinase, and the increasing effects of Ls2-2 on FDP and D-dimer levels were also higher than that of urokinase in the concentration used in the experiment. The in vitro thrombolytic experiment demonstrated that the clot lytic rates of Ls2-2 at 10, 20 mg/mL were markedly higher than that of urokinase in the concentration used in the experiment (Fig. 6D). These 34

results demonstrated that Ls2-2 could be a potential fibrin(ogen)olytic activity and thrombolytic activity polysaccharide. It was noted that the decreasing effect of Ls2-2 at 10 mg/kg on PAI-1 was stronger than that of the sulfated polysaccharide PF2 from M. angicava, but the increasing effects of Ls2-2 on FDP and D-dimer levels were slightly lower than that of PF2 (Li et al., 2017). The structural characteristics of PF2 were different from those of Ls2-2 though they were mainly composed of →3)-α-L-Rhap-(1→ and/or →2)-α-L-Rhap-(1→ residues. PF2 was a sulfated polysaccharide only consisting of rhamnose, with sulfate substitution merely at C-3 of →2)-α-L-Rhap-(1→ residues. So far, little work in the literature is related to the fibrin(ogen)olytic and thrombolytic activities of sulfated polysaccharides from Monostromaceaea. An in-depth investigation on the fibrin(ogen)olytic and thrombolytic activities of sulfated polysaccharide with different structure will indubitably play an important role in the understanding of fibrin(ogen)olytic and thrombolytic activities.

4. Conclusion

A glucuronic acid-containing rhamnan-type sulfated polysaccharide, Ls2-2, was obtained from marine green alga M. angicava Kjellm. Ls2-2 was mainly composed of →3)-α-L-Rhap-(1→ and →2)-α-L-Rhap-(1→ residues. 23.52 % of total number of the rhamnose residues in Ls2-2 were substituted by sulfate ester groups, specifically to →3)-α-L-Rhap-(1→ residues were about 17.95 %, to →2)-α-L-Rhap-(1→ residues were about 5.57 %, respectively. The branching was composed of unsulfated or monosulfated 3-linked, 2-linked, 4-linked α-L-rhamnose and terminal β-D-glucuronic acid residues, and the terminal β-D-glucuronic acid was at non-reducing end. Ls2-2 was a novel 35

rhamnan-type sulfated polysaccharide distinguishing from other sulfated polysaccharides from Monostromaceae species. Ls2-2 could act as a glucose consumption stimulator with depressing lipid accumulation. Moreover Ls2-2 ameliorated insulin resistance in HepG2 cells at least partly via the induction of AMPKα2 expression and its downstream GLUT2 and HL, as well as the reduction of PEPCK and ACC2. Ls2-2 possessed a high anticoagulant activity, and exhibited fibrin(ogen)olytic activity in vivo and thrombolytic activity. Ls2-2 could be a potential antidiabetic and anticoagulant polysaccharide. An in-depth investigation are underway to characterize the in vivo properties of this active Ls2-2.

Chemical compounds studied in this article

Dimethyl sulfoxide (PubChem CID: 679); D-Glucuronic acid (PubChem CID: 94715); Heparin (PubChem CID: 25244225); Insulin (PubChem CID: 118984380); Metformin (PubChem CID: 4091); Palmitic acid (PubChem CID: 985); Pullunan (PubChem CID: 92024139); Pyridine (PubChem CID: 1049); Rhamnose (PubChem CID: 25310); Urokinase (PubChem CID: 54613018)

Acknowledgements This work was supported by the National Natural Science Foundation of China (41476108), the Science and Technology Development Program of Shandong Province, China (2014GHY115015), NSFC-Shandong Joint Fund for Marine Science Research Centers (U1606403) and the Scientific and Technological Innovation Project of Qingdao National Laboratory for Marine Science and Technology (2015ASKJ02). 36

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45

Figure Captions Fig. 1. NMR spectra of DSLs2-2. Spectra were performed on an Agilent DD2 500M NMR spectrometer. Chemical shifts are referenced to internal acetone at 2.225 ppm for 1H and 31.07 ppm for 13C. (a) 1H NMR spectrum; (b) 13C NMR spectrum; (c) 1H–1H COSY spectrum; (d) 1H–13C HSQC spectrum; (e) 1H–13C HMBC. A–E correspond to →3)-α-L-Rhap-(1→, →3)-α-L-Rhap-(1→, →2)-α-L-Rhap-(1→, →2,3) α-L-Rhap-(1→ and →2)-α-L-Rhap(3SO4)-(1→, respectively. Rhap: rhamnopyranose.

46

Figure 1 47

Fig. 2. NMR spectra of Ls2-2. Spectra were performed on an Agilent DD2 500M NMR spectrometer. Chemical shifts are referenced to internal acetone at 2.225 ppm for 1H and 31.07 ppm for 13C. (a) 1H NMR spectrum; (b) 13C NMR spectrum; (c) 1H–1H COSY spectrum; (d) 1H–13C HSQC spectrum; (e) 1

H–1H NOESY spectrum. A–F correspond to →3)-α-L-Rhap-(1→, →3)-α-L-Rhap-(1→,

→2)-α-L-Rhap-(1→, →2,3)-α-L-Rhap-(1→, →2)-α-L-Rhap(3SO4)-(1→ and →3)-α-L-Rhap(2SO4)-(1→, respectively. Rhap: rhamnopyranose.

48

Figure 2

49

Fig. 3. Negative-ion ES-CID-MS/MS product-ion spectra and assignments of the ions of the major fragment of oligosaccharides. The structure is shown to indicate the proposed fragmentation. (A) RS of S1; (B) R2S of S2; (C) R+GA of S2; (D) R2+GA of S3; (E) RS+GA and R+GA(S) of S4; (F) R2S+GA and R2+GA(S) of S4. R, GA and S represented rhamnopyranose, glucuronic acid and sulfate ester, respectively.

50

Figure 3

51

Fig. 4. Cytotoxicity, hypoglycemic and hypolipidemic activities of Ls2-2. (a) cytotoxicity in HepG2 cells; (b) glucose consumption levels in HepG2 cells; (c) glucose consumption levels in insulin-resistant HepG2 cells; (d) TG levels in insulin-resistant HepG2 cells; (e) TC levels in insulin-resistant HepG2 cells. Significance: *p<0.05, **p<0.01 versus the control group; #p<0.05, ##p<0.01 versus the model group.

52

Figure 4 53

Fig. 5. Effect of Ls2-2 on the mRNA expressions of key genes related to glucose and lipid metabolism in insulin-resistant HepG2 cells. PCR fluorescence products were quantified using SYBR Green. The cycle number at which the various transcripts were detectable was compared with that of β-actin as an internal control. (a) AMPKα2 mRNA expression; (b) GLUT2 mRNA expression; (c) PEPCK mRNA expression; (d) HL mRNA expression; (e) ACC2 mRNA expression. Significance: *p<0.05, **p<0.01 versus the control group; #p<0.05, ##p<0.01 versus the model group.

54

Figure 5 55

Fig. 6. Results of fibrin(ogen)olytic activity and thrombolytic activity assays of Ls2-2. (A) PAI-1, the PAI-1 value of Ls2-2 at 10 mg/kg was up to 0; (B) FDP; (C) D-dimer, the D-dimer levels of control, urokinase, Ls2-2 at 2.5 mg/kg groups were below detection limit in this assay; and (D) clot lytic rate. Significance: *p<0.05, **p <0.01 versus the control group; #p<0.05, ##p <0.01 versus the urokinase group.

56

Figure 6

57

Tables Table 1 Results of methylation analyses of Ls2-2, DSLs2-2, Ls2-2R and Ls2-2RDs. Table 1 Molar percent ratios Methylated alditol acetate

Linkage pattern Ls2-2

DSLs2-2

Ls2-2R

Ls2-2RDs

1,2,5-Tri-O-acetyl-3,4-di-O-methyl-rhamnitol

23.76

29.33

22.80

28.30

→2)-Rhap-(1→

1,4,5-Tri-O-acetyl-2,3-di-O-methyl-rhamnitol

1.20

1.20

1.15

1.15

→4)-Rhap-(1→

1,3,5-Tri-O-acetyl-2,4-di-O-methyl-rhamnitol

40.02

57.97

38.12

56.09

→3)-Rhap-(1→

1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-glucitol

nda

nda

2.86

3.90

Glcp-(1→

1,2,5-Tri-O-acetyl-3,4,6-tri-O-methyl-glucitol

nda

nda

1.07

nda

→2)-Glcp-(1→

1,2,3,5-Tetra-O-acetyl-4-O-methyl-rhamnitol

31.04

11.50

30.08

10.56

→2,3)-Rhap-(1→

1,2,4,5-Tetra-O-acetyl-3-O-methyl-rhamnitol

3.98

nda

3.92

nda

→2,4)-Rhap-(1→

a

Not detected

58

Table 1 Result of anticoagulant activity assay of Ls2-2. Table 2 Sample

Ls2-2

Heparin

Concentration

APTT

TT

PT

FIB

(µg/mL)

(s)

(s)

(s)

(mg/dL)

0

39.9±0.2

11.2±0.8

18.9±1.0

201.5±5.3

5

74.2±1.4

11.5±1.1

19.1±0.2

176.9±5.1

10

92.0±6.8

13.2±2.5

21.7±2.3

161.1±2.1

25

144.1±3.8

14.0±0.1

21.8±2.0

155.1±0.9

50

> 200

21.9±1.67

21.9±0.4

137.5±1.0

100

63.5±0.9

31.6±1.8

103.8±3.7

200

> 120

48.4±2.0

< 80 a

0

39.8±0.3

11.6±0.9

18.2±1.1

204.6±0.1

5

99.8±3.5

16.2±0.6

19.2±0.6

143.8±7.1

10

> 200

103.5±3.5

26.0±1.3

98.7±2.0

> 120

62.9±2.9

< 80

25 50

> 120

100 200 a

Three samples were prepared for assays of every attribute. The results were expressed as means ± standard deviation (SD). a The value went beyond the range of calibration curve made by FIB constant value plasma.

59