Structural characterization and anticoagulant activity of two polysaccharides from Patinopecten yessoensis viscera

International Journal of Biological Macromolecules 136 (2019) 579–585

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Structural characterization and anticoagulant activity of two polysaccharides from Patinopecten yessoensis viscera Shuang Song a,b,c,⁎, Linlin Wang a,b,c, Lilong Wang a,b,c, Qi Yu a,b,c, Chunqing Ai a,b,c, Yinghuan Fu a,b,c, Chunhong Yan a,b,c, Chengrong Wen a,b,c, Zhenjun Zhu a,b,d a

School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, PR China National Engineering Research Center of Seafood, Dalian 116034, PR China National & Local Joint Engineering Laboratory for Marine Bioactive Polysaccharide Development and Application, Dalian Polytechnic University, Dalian 116034, PR China d College of Light Industry and Food Engineering, Guangxi University, Nanning 530004, PR China b c

a r t i c l e

i n f o

Article history: Received 5 May 2019 Received in revised form 7 June 2019 Accepted 16 June 2019 Available online 17 June 2019 Keywords: Polysaccharide Scallop viscera Chemical structure Antithrombotic activity Anticoagulant activity

a b s t r a c t In the present study, two polysaccharides, SVP2–1 and SVP2–2, were isolated from Patinopecten yessoensis viscera and purified by using DEAE-52 cellulose and Sepharose CL-6B. Both SVP2–1 and SVP2–2 could extend activated partial thromboplastin time (APTT) and thrombin time (TT) and inhibit the transformation of fibrinogen into fibrin (FIB) concentration-dependently, indicating they inhibited clotting and thrombin through intrinsic and common pathways. Of note, SVP2–2 had stronger anticoagulant activity than SVP2–1, and its backbone was determined as →6)-α-Manp (1 → 2)-α-Galp(1 → with Xyl or Glc substituted at C4 of Gal. Based on monosaccharide composition analysis, methylation analysis, and NMR analysis. Further comparison of their monosaccharide analysis and NMR spectra indicates SVP2–1 and SVP2–2 possess the same core structure features, so the higher sulfate content and lower molecular weight may be the possible reasons for the stronger anticoagulant capability of SVP2–2. The present study suggests acidic polysaccharides from scallop viscera as promising anticoagulant candidates. © 2019 Published by Elsevier B.V.

1. Introduction Cardiovascular diseases including heart diseases and stroke related to thrombosis, are the leading causes of death and have caused millions of deaths in recent years. What's the worse, about 52 million may die of cardiovascular diseases in 2030 as expected by the World Health Organization [1]. The imbalance between coagulation with anticoagulation and abnormal coagulation-fibrinolysis can lead to the formation of thrombus [2]. In most of these diseases, anticoagulation and fibrinolytic medicine are applied to make the blood flowing in the normal state [3]. Heparin, a sulfated glycan, is the most widely used anticoagulant and antithrombotic agent at present, but it has several side effects such as bleeding and thrombocytopenia [4–6]. Moreover, the source of raw materials for commercial heparin is porcine or bovine intestine or bovine lung tissue, and the appearance of some infectious diseases, such as bovine spongiform encephalopathy and foot-and-mouth diseases, has limited the production of heparin from porcine and bovine. In addition, porcine heparin also has problems which associated with religious

⁎ Corresponding author at: School of Food Science and Technology, Dalian Polytechnic University, No.1 Qinggongyuan, Ganjingzi district, Dalian 116034, PR China. E-mail address: [email protected] (S. Song).

https://doi.org/10.1016/j.ijbiomac.2019.06.116 0141-8130/© 2019 Published by Elsevier B.V.

limitations on its uses. Hence, more efforts are needed to explore effective alternatives for traditional anticoagulant drugs. Marine organisms are favorable sources of structurally diverse bioactive compounds, which may be beneficial to human nutrition and health. Acidic polysaccharides in animal-origin seafood have been extensively exploited recently because of their significant bioactivities including anticoagulation [7], antithrombosis [8], anti-inflammation [9], and antioxidation [10]. Thus, animal-origin seafoods or their processing by-products could be promising resources for bioactive acidic polysaccharides. Scallop (Patinopecten yessoensis) is a kind of most economically important shellfish, widely cultured, and distributed along the coasts of China, Russia, Japan, and the Korean peninsula. The total output of scallop has reached about 1.88 million tons in China (FAO 2016). Adductor muscle is the main edible and processed part of scallop with high nutritional value, and is considered as a delicacy in the tradition. During industrial processing of scallop, a large number of viscera mainly composed of midgut glands and gonads, accounting for N30% of the scallop body, are usually discarded as wastes. The scallop viscera contain many bioactive substances, such as polysaccharides [11], protein/peptide [12], lipid [13], enzymes [14], and carotenoids [15]. Therefore, these by-products are potential resources for the production of valuable bio-products, and utilization instead of discard of these wastes could reduce the negative impact on environment.

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In the present work, two novel anticoagulant-active acidic polysaccharides were isolated from the viscera of Patinopecten yessoensis. Their structures were investigated by a combination of chemical and spectroscopic methods such as mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. In addition, the anticoagulant activities of the obtained acid polysaccharides were evaluated through activated partial thromboplastin time (APTT), the prothrombin time (PT), the thrombin time (TT), and transformation of fibrinogen into fibrin (FIB) activities. 2. Materials and methods 2.1. Materials Viscera of scallop (Patinopecten yessoensis) collected from Dalian, China, were provided by Yiwangyu Marine Science and Technology Corporation. The standard monosaccharides including galacturonic acid (GalA), glucuronic acid (GlcA), glucose (Glc), galactose (Gal), arabinose (Ara), rhamnose (Rha), mannose (Man), fucose (Fuc), and xylose (Xyl) were purchased from Sigma Chemical Co. (Shanghai, China). A polysaccharide from abalone (Haliotis discus hannai Ino) gonad with backbone of →4)-GlcA(1 → 2)-Man(1 → was isolated and elucidated in our laboratory [16]. Heparin and hyaluronic acid were purchased from Sangon Biotechnology Co. (Shanghai, China). Chondroitin sulfate from shark cartilage was purchased from Sigma-Aldrich (St. Louis, MO, USA). Trypsin and papain were obtained from Sangon Biotechnology Co. (Shanghai, China). APTT, PT, and TT assay reagents were supplied by Shanghai Sun Biotechnology Co. (Shanghai, China). 2.2. Preparation of polysaccharides The viscera of scallop were dried and ground. The powder (1500 g) was defatted with alcohol and n-hexane (1:2), then air-dried. The obtained powder was digested at 37 °C for 4 h in a mixture of 1200 mL Cys-EDTA-2Na solution (0.05 mol/L) and 4800 mL phosphate buffer (pH 8.0, 0.05 mol/L) after adding 0.5% trypsin (w/w). Then, 0.5% papain

was added to the solution for incubation at 65 °C for 3 h. The supernatant collected after centrifugation (10,000 rpm × 15 min) was mixed with 2400 mL of 10% cetylpyridinium chloride (CPC) solution. The resulted precipitation was dissolved in 2250 mL of 3 mol/L NaCl-ethanol (100:15 v/v) solution. After adding 4500 mL of 95% ethanol solution, the mixture was kept at 4 °C for 24 h. Finally, the precipitate was collected by centrifugation (8000 rpm × 15 min), dialyzed, concentrated, and lyophilized to obtain crude polysaccharide sample. The crude polysaccharide (0.2 g) was subjected to a DEAE-cellulose anion exchange column and eluted by 0.25 mol/L, 0.5 mol/L, 0.75 mol/L, 1.0 mol/L, and 2.0 mol/L NaCl solutions sequentially to afford SVP1, SVP2, SVP3, SVP4, and SVP5, respectively (Fig. 1A). The fraction SVP-2 was loaded onto CL-6B column and washed with 0.15 mol/L NaCl (Fig. 1B). Fractions were combined according to the result of phenol sulfuric acid determination. Eluting fractions (Nos. 22–24) for SVP2–1 and eluting fractions (Nos. 34–44) for SVP2–2 were collected, concentrated and lyophilized to obtain purified polysaccharides. SVP2–2 was hydrolyzed with 0.5 mol/L TFA at l00 °C for 1 h, and dialyzed (cut-off Mw 3500 Da) against water to obtain its backbone [16].

2.3. Analysis of chemical composition The molecular weight of polysaccharides was determined by a high-performance size exclusion chromatograph by using Waters Alliance 2695 HPLC (Milford, MA, USA) system connected with a Waters Alliance 2414 refractive index detector and TSK-gel G4000 column (7.8 mm × 300 mm). The molecular weight was calculated by the standard curve prepared with dextran standards with different molecular weights (5, 12, 25, 50, 150, 410, and 670 kDa). The sulfate content was measured by the barium chloride-gelatine method by using K2SO4 as standard [17]. The uronic acid content was determined through the carbazole sulfuric acid method [18] by using glucuronic acid as standard. Protein content was determined according to the Bradford method [19] by using bovine serum albumin as standard.

Fig. 1. Purification and molecular weight distribution of polysaccharides from viscera of P. yessoensis (SVP). (A) Stepwise elution curve of crude SVP on a DEAE-cellulose column; (B) elution profile of SVP2 on a CL-6B filtration column, and high performance gel-permeation chromatography (HPGPC) profiles of SVP2–1 (C) and SVP2–2 (D).

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Table 2 Chemical composition, molecular weight, and monosaccharide composition of SVP2–1 and SVP2–2.

Fig. 2. Inhibitory effects of SVP2–1 and SVP2–2 on coagulation of fibrinogen.

2.4. Monosaccharide composition analysis The component monosaccharides of SVP2–1 and SVP2–2 were identified and quantified by GC analysis as described by previously [20] with some modifications. Each sample (5 mg) was hydrolyzed with 1 mL of 2 mol/L TFA at 120 °C for 2 h. The dried hydrolysate was dissolved with 1 mL of 0.5 mol/L Na2CO3. After being reduced with NaBH4 at 65 °C for 1.5 h, monosaccharides were converted into the alditol acetates by treatment with pyridine and acetic anhydride at 85 °C for 2 h. After being extracted by chloroform for three times, the organic phase was detected by GC (Shimadzu, Kyoto, Japan), which was equipped with Rtx-5 column (30 m × 0.25 mm × 0.25 μm). 2.5. FT-IR analysis The samples each (2 mg) were mixed with 100 mg of dried potassium bromide (KBr) and compressed to prepare a salt disc for reading the spectrum further. The spectra were recorded by a FTIR Spectrometer (Perkin Elmer, Waltham, MA, USA) in the range of 4000–400 cm−1.

Chemical composition

SVP2–1

SVP2–2

Protein content [% wt] Uronic acid content [% wt] Sulfate content [% wt] Monosaccharide composition [% wt]

3.70 ± 0.10 13.33 ± 0.79 8.10 ± 0.18 5.85 2.44 8.54 12.20 7.80 17.07 12.93 10.24 22.92

5.50 ± 0.05 11.10 ± 0.52 13.80 ± 0.56 3.76 3.76 8.27 11.28 6.39 9.40 16.92 11.65 28.57

GlcN GalN Rha Fuc Ara Xyl Man Glc Gal

water to end the reaction. The methylation procedure was repeated three times, and the completion of methylation was detected by FT-IR. The methylated polysaccharide was hydrolyzed in 3 mL of formic acid at 100 °C for 3 h and further hydrolyzed in 2 mL of 2 mol/L trichloroacetic acid at 121 °C for 3 h. After being reduced with 60 mg NaBH4 at 60 °C for 2 h, monosaccharides were converted into the alditol acetates by reacting with pyridine and acetic anhydride at 100 °C for 2 h. After being mixed, the organic phase was analysed by GC–MS (Agilent Technologies Co., USA) equipped with a HP-5MS column (30 m × 0.25 mm × 0.25 μm). The temperature was 160 °C initially for 1 min, increased to 210 °C at 1 °C /min, held at 210 °C for 50 min, increased to 213 °C at 0.4 °C /min and held for 7 min. 2.7. Nuclear magnetic resonance (NMR) spectroscopy SVP2–1 (50 mg) and SVP2–2 (50 mg) were dissolved in 1 mL of D2O (99.9%) individually and lyophilized, and this procedure was repeated three times. Then the polysaccharide was dissolved in 0.5 mL of D2O for analysis. The 1D and 2D NMR spectra were recorded on 400 MHz NMR spectrometer (Bruker, Rheinstetten, Germany). 2.8. Assay of the ability to inhibit the thrombin-catalyzed coagulation of fibrinogen

2.6. Methylation and GC–MS analysis Methylation analysis of the polysaccharides was performed according to the method of Needs and Selvendran [21] with minor modifications. The polysaccharides (2 mg) were dissolved with 2 mL of anhydrous DMSO, and then 0.1 g dried NaOH was added. After ultrasound for 1 h, 1 mL of CH3I was added and the mixture was ultrasound for another hour. Finally, the solution was mixed with 3 mL of distilled

The ability of polysaccharides to inhibit the coagulation of fibrinogen was measured according to Zhang et al. [22] with minor modifications. A microplate reader was set at 37 °C, at a wavelength of 405 nm. The fibrinogen, thrombin, and samples were all dissolved in 0.05 M Tris–HCl buffer (pH 7.2) containing 0.12 mM NaCl. A 0.1% fibrinogen solution (140 μL) and 40 μL of samples with various concentrations (0.1, 0.2, 0.5, 1.0, 1.5, 2.0, and 2.5 mg/mL) were added into the plate wells,

Table 1 Analysis of the anticoagulant activity by APTT, TT and PT on the polysaccharides SVP2–1 and SVP2–2. Each clotting time were expressed as mean ± SD (n = 3). Samples were all compared with blank control (0 μg/mL). Samples

0 μg/mL

10 μg/mL

100 μg/mL

200 μg/mL

1000 μg/mL

2000 μg/mL

106.5 ± 4.6 24.0 ± 2.6 23.4 ± 2.8 8.0 ± 0.3 9.5 ± 0.7 8.8 ± 0.3 117.3 ± 9.0 13.4 ± 0.7 15.4 ± 1.1

N200⁎⁎ 36.5 ± 2.0 33.4 ± 1.1 15.2 ± 1.8 10.3 ± 0.1 9.9 ± 0.6 N200⁎⁎ 17.0 ± 1.1 16.0 ± 1.0

– 51.5 ± 1.1 67.1 ± 1.0 51.4 ± 2.7⁎ 10.7 ± 0.6⁎

– 108.2 ± 3.6⁎⁎ 130.1 ± 1.7⁎ – 10.7 ± 0.1⁎⁎

10.5 ± 0.3 – 22.0 ± 1.7 24.7 ± 0.9⁎

10.4 ± 0.4 – 38.9 ± 1.3⁎ 40.4 ± 0.8⁎⁎

– N200⁎⁎ N200⁎⁎ – 10.7 ± 0.6⁎ 10.8 ± 0.1⁎⁎

Clotting time (s) APTT

PT

TT

Heparin SVP2–1 SVP2–2 Heparin SVP2–1 SVP2–2 Heparin SVP2–1 SVP2–2

20.8 ± 1.9

8.4 ± 1.4

12.7 ± 1.5

Note: – Indicates the value was not evaluated. ⁎ p b 0.05. ⁎⁎ p b 0.01.

– 44.7 ± 1.0⁎⁎ 46.4 ± 1.2⁎⁎

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2.10. Statistical analysis Statistical evaluation was carried out with SPSS 20 (IBM, Chicago, USA). The results are expressed as the mean ± standard deviation (S.D.). The statistical significance of difference was determined by the non-parametric Kruskal-Wallis test with the significance level of *p b 0.05 and **p b 0.01, and samples were all compared with blank control (0 μg/mL).

3. Results and discussion 3.1. Isolation, purification, and molecular weights of SVP2–1and SVP2–2

Fig. 3. The FT-IR spectra of SVP2–1 and SVP2–2.

mixed, and then the absorbance of the sample blank was measured. After that, 10 μL of thrombin solution (12 IU/mL) was added to the wells to start the reaction of thrombin-catalyzed coagulation of fibrinogen. After incubation for 10 min, the absorbance of the sample was recorded again. 40 μL of Tris–HCl buffer (pH 7.2, 0.05 mol/L) was in the place of sample solutions for the measurement of the absorbance of the blank control and the control. Heparin was used as a positive control. The inhibitory effects were calculated by the following equation:

Inhibitory effect; % ¼

½C−CB−½S−SB  100 C−CB

S, SB, C, and CB were short for the sample, sample blank, control, and control blank absorbance, respectively. 2.9. Anticoagulant activity assay Activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) were performed by the method reported [23]. The polysaccharides were formulated into physiological solutions of 10, 100, 200, 1000, and 2000 μg/mL with 0.9% NaCl for assay. For the APTT assay, 100 μL of APTT reagent (Tianjin MD Pacific, China) was added to the mixture of 80 μL of normal rabbit plasma and 20 μL of sample solution for 3 min incubation at 37 °C, and then 100 μL of 0.025 mol/L calcium chloride (37 °C) was added and recorded by the coagulometer (Infinite M200, Shanghai, China). For the PT assay, 80 μL of citrated normal rabbit plasma was mixed with 20 μL of samples and incubated at 37 °C for 1 min, and then, 200 μL of pre-warmed PT assay reagent was added and clotting time was recorded. For the TT assay, a mixture of 160 μL of citrated normal rabbit plasma and 40 μL of samples was incubated at 37 °C for 60 s, and then 200 μL of pre-warmed TT assay reagent was added and clotting time was recorded. Heparin was used as positive control and the normal saline was used as control.

The crude polysaccharides were obtained from the dried viscera of P. yessoensis by enzymatic extraction and ethanol precipitation with a yield of approximate 1% (w/w). After protein removal, the crude SVP was subjected to a DEAE–cellulose column (Fig. 1A). SVP2 was obtained as the major fraction which was further purified by Sepharose CL-6B to provide fractions SVP2–1 and SVP2–2 (Fig. 1B). Both of the two polysaccharide fractions showed a single symmetrical peak in HPGPC analysis (Fig. 1C and D) indicating their homogeneity. Furthermore, the average molecular weights of SVP2–1 and SVP2–2 were determined as 630 kDa and 63 kDa, respectively.

3.2. Inhibitory effects on coagulation of fibrinogen Active thrombin can turn soluble fibrinogen into insoluble fibrin, which contributes to the subsequent clotting, so the inhibition of this process could prolong the clotting time. In the present study, inhibitory effects of SVP2–1 and SVP2–2 on coagulation of fibrinogen were evaluated with heparin as reference. As shown in Fig. 2, the inhibitory effects of SVP2–1 and SVP2–2 were evident at all tested concentrations. The inhibitory effect of SVP2–1 reached about 85% at a concentration of 2.5 mg/mL, while SVP2–2 could inhibit the formation of fibrin almost completely at concentrations of ≥1.0 mg/mL. The results showed that both SVP2–1 and SVP2–2 had antithrombotic activity, and SVP2–2 was more active than SVP2–1.

3.3. Anticoagulant activity Anticoagulant activities of SVP2–1 and SVP2–2 based on APTT, TT, and PT assays were shown in Table 1. No clotting inhibition was observed in PT test of SVP2–1 and SVP2–2 in the range of concentrations used in the experiment, suggesting that SVP2–1 and SVP2–2 cannot inhibit extrinsic pathway of coagulation. SVP2–1 and SVP2–2 with concentrations ≥10 μg/mL could prolong APTT and TT in a concentration dependent manner. Moreover, they are more potential in prolonging APTT of plasma, and the APTT was extended over 200 s with 2 mg/mL of polysaccharides. The prolongations of APTT and TT suggested that SVP2–1 and SVP2–2 could inhibit the intrinsic coagulation pathway and thrombin-mediated fibrin formation. Of note, SVP2–2 was more effective in prolonging APTT than SVP2–1.

Table 3 Monosaccharide and methylation analysis of SVP2–2 backbone. Monosaccharide compositions

Methylation analysis

Fractions

Molar ratio (%)

Retention time (min)

Methylated derivative

Deduced linkage

Molar ratio

Xyl Glc Man Gal

11.49 37.93 13.79 36.78

13.411 16.575 21.186 20.944 23.176

2,3,4-Me3-Xyl 2,3,4,6-Me4-Glc 2,3,4-Me3-Man 3,4,6-Me3-Gal 2,6-Me3-Gal

Xlyf(1→ Glcp(1→ →6)Manp(1→ →2)Galp(1→ →3,4)Galp(1→

1.0 1.2 3.3 2.1 1.1

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Fig. 4. 1H NMR (A), 13C NMR (B), HSQC (C), and H1-H1 COSY (D) spectra of SVP2–2 and proposed structure of SVP2–2 backbone (E). Signals designated with G and M refer to those produced by Gal and Man.

3.4. Chemical composition The chemical compositions of SVP2–1 and SVP2–2 are shown in Table 2. Their protein contents were both lower than 6%. Their sulfate contents were around 10%, and SVP2–2 was more highly sulfated than Table 4 Assignments of 1H and 13C NMR signals of SVP2–2. Structural units

→2)-α-Galp-(1 → (G) →6)-α-Manp-(1 → (M)

Chemical shift H/C

1

2

3

4

5

6

H C H C

5.35 98.1 5.13 100.1

3.22 81.7 4.10 69.5

3.67 72.9 3.92 70.2

3.51 69.0 3.53 72.2

4.11 70.2 3.24 73.1

3.87 60.0 4.19 66.0

SVP2–1, which may be led to more antithrombotic active of SVP2–2 than SVP2–1 [24,25]. A small fraction of uronic acid was observed in SVP2–1 and SVP2–2, and total of 9 species of the other monosaccharides were detected for both polysaccharides but with varying molar ratios. The FT-IR spectra of SVP2–1 and SVP2–2 were also analysed and they exhibited great similarity as shown in Fig. 3. Both of them displayed a broad stretching intense peak at 3428.4 cm−1 for the hydroxyl group and a weak C\\H stretching vibration band at 2940.7 cm−1 [26]. The peaks between 1100.0 and 1000.0 cm−1 belonged to C_O and C\\C stretching vibration in pyranoid ring and C\\O\\C stretching vibration of glycosidic bonds [27]. The intense peaks around 1640.0 cm−1 were attributed to the C_O of uronic acids [20]. In addition, the peaks around 1247.6 cm−1 were caused by the stretching vibration of S_O of sulfate group, which is typical for sulfated polysaccharides [28]. The peak for S_O of SVP2–2 was more intensive

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Fig. 5. 1H NMR (A) and HSQC (B) spectra of SVP2–1.

than that of SVP2–1, which is consistent with their sulfate contents mentioned above. 3.5. Monosaccharide composition analysis and methylation analysis of SVP2–2 backbone To further characterize the structure of SVP2–2, which showed more satisfactory anticoagulant activity, the backbone of SVP2–2 was prepared by mild acid hydrolysis and elucidated by monosaccharide composition analysis and methylation analysis. As shown in Table 3, four monosaccharide compositions, Xly, Man, Glc, and Gal, were observed, where Man and Gal are the major component monosaccharides in the backbone of SVP2–2. Further methylation analysis revealed the linkage pattern of these monosaccharides as shown in Table 3. Thus, the backbone structure of SVP2–2 was determined as →2)Galp(1 → 6)Manp(1 → with Xly or Glc attached to the C4 position of →2)Galp(1 → (Fig. 4E). 3.6. NMR spectroscopy analysis of SVP2–2 1

H NMR, 13C NMR, 1H\\1H COSY, and HSQC were applied to elucidate the structure of SVP2–2. In 1H NMR spectrum of SVP2–2 (Fig. 4A), methyl protons (CH3) of GalNAc and Fuc could be readily identified from the signals at 1.97 ppm and 1.26 ppm, respectively [29]. The chemical shift at 5.35 ppm and 5.13 ppm could be assigned to the anomeric protons [30]. As shown in Fig. 4B, several anomeric carbon signals presented in the range of 95.0–105.0 ppm, and non-anomeric carbon signals appeared from 59.0 ppm to 82.0 ppm. The chemical shift at 175.0 ppm was recognized as carbonyl carbon (C_O) of β-GalNAc or uronic acids [31]. Signals at 17.0 ppm and 20.3 ppm could be attributed to the methyl group of Fuc residue and O-acetyl groups of GalNAc residue, respectively. The HSQC spectrum showed correlations between carbon and its attached protons, and the cross peaks of COSY implied the adjacent protons. In the HSQC spectrum of SVP2–2 (Fig. 4C), a peak at 5.35/ 98.1 ppm was marked in the anomeric region, and then the signals of other protons and carbons (from H-2/C-2 to H-6/C-6) in the same sugar residue were assigned (Table 4) based on the cross peaks in 1 H\\1H COSY (Fig. 4D) as well as HSQC. Notably, the higher chemical shift of C-2 at 81.1 ppm indicated the glycosidic linkage at C-2. By comparing 1H and 13C chemical shifts of this residue with those in literature report [32], the residue was then determined as →2)-α-Galp-(1→. Similarly, another set of NMR signals for a sugar residue was also recognized and assigned (Table 4). The lower field shift of C-6 at 60.0 ppm compared with free hexose suggested that C-6 is involved in glycosidic bonds. Then, this residue could be determined as →6)-α-Manp-(1 → by comparing its NMR data with those reported previously [33,34], and it is also a major component monosaccharide residue in SVP2–2

backbone. Thus, combining the analysis result of Section 3.5, the structure of the backbone of SVP2–2 is proposed in Fig. 4E. Of note, besides the monosaccharides in the backbone, other monosaccharides (e.g. GalNAc and Fuc) as well as sulfate groups also exist in the structure of SVP2–2, but their linkages have not been determined yet. 3.7. Structural comparison of SVP2–1 with SVP2–2 More structural information of SVP2–1 was also necessary for a better understanding of structure-activity relationship of the polysaccharides. As shown in Fig. 5, 1H NMR and HSQC spectra of SVP2–1 all showed great similarity with those of SVP2–2. Some identical cross peaks could be observed in the HSQC spectra of SVP2–1 and SVP2–2, such as G-H1/C1 at 5.35/98.1 ppm, G-H2/C2 at 3.22/81.1 ppm, M-H2/ C2 at 4.10/69.5 ppm, CH3 of GalNAc and Fuc at 1.97 and 1.26 ppm, C_O at 175.0 ppm. These observations suggest SVP2–1 and SVP2–2 possess common monosaccharide residues, which is consistent with results of component monosaccharide analysis. Thus, it could be inferred that SVP2–1 has a similar structure with that of SVP2–2, but varies in the molecular weight and sulfate content, which may result in the differences in the anticoagulant activities of SVP2–1 and SVP2–2. Of note, the sulfate group has been reported to take an important role in anticoagulant activities due to its negative charge [35], and the plasma inhibitor and the target protease may form a particular complex with the sulfated polysaccharides [29,36]. Therefore, the stronger anticoagulant activity of SVP2–2 is possibly due to its higher sulfation degree compared with SVP2–1. In addition, molecular weight may also influence the anticoagulant activity [37]. However, because of the complexity of structures of SVP2–1 and SVP2–2, more efforts are still needed to reveal their activity and structure relationship. 4. Conclusion In this study, the scallop visceral polysaccharides SVP2–1 and SVP2–2 were isolated from Patinopecten yessoensis viscera. The backbone of SVP2–2 was elucidated as →6)-α-Manp-(1 → 2)-α-Galp-(1 → with Xyl or Glc substituted at C-4 of Gal. The structures of SVP2–1 and SVP2–2 had great similarity, and SVP2–2 with a lower molecular weight and higher sulfate content showed stronger anticoagulant capability. The present study may shed a light on the utilization of the scallop viscera which is the by-product of scallop processing. Acknowledgement This work was funded by the National Key Research and Development Program of China (Nos. 2018YFD0901000 and 2017YFD0400203) and National Natural Science Foundation of China (No. 31701601).

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