Structural features and digestive behavior of fucosylated chondroitin

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Functional Structure/Activity Relationships

Structural features and digestive behavior of fucosylated chondroitin sulfate from sea cucumbers Stichopus japonicus Zhenjun Zhu, Xiuping Dong, Chunhong Yang, chunqing ai, Dayong Zhou, Jingfeng Yang, Hui Zhang, Xiaoling Liu, Shuang Song, Hang Xiao, and Bei-Wei Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04996 • Publication Date (Web): 29 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Journal of Agricultural and Food Chemistry

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Structural Features and Digestive Behavior of Fucosylated Chondroitin Sulfate

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from Sea Cucumbers Stichopus Japonicus

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Zhenjun Zhu

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Yang 1,3, Hui Zhang 1, Xiaoling Liu 2, Shuang Song 1,3,4,*, Hang Xiao 4, Beiwei Zhu 1,2,3

1,2,3,4,

Xiuping Dong 1, Chunhong Yan

1,3,

Chunqing Ai

1,3,

Dayong Zhou

1,3,

Jingfeng

5 6

1

7

Polytechnic University, Dalian 116034, China

8

2

College of Light Industry and Food Engineering, Guangxi University, Nanning 530004, China

9

3

National & Local Joint Engineering Laboratory for Marine Bioactive Polysaccharide Development

School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian

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and Application, Dalian 116034, China

11

4

Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

13

*

Corresponding author at: School of Food Science and Technology, Dalian Polytechnic University,

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No.1 Qinggongyuan, Ganjingzi district, Dalian 116034, P. R. China. Tel: +86-411-86323262, Fax:

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+86-411-86323262.

12

E-mail

address:

[email protected]

1

ACS Paragon Plus Environment

(S.

Song).


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ABSTRACT

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Fucosylated chondroitin sulfate from sea cucumber Stichopus japonicus (FCSSJ) has been demonstrated

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with various biological activities, however, its precise structure is still controversial, and digestive

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behavior remains poorly understood. FCSSJ was purified, and its detailed structure was elucidated

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mainly based on the NMR spectroscopic methods. Its main chain was characterized as

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→4)-β-D-GlcA-(1→3)-β-D-GalNAc-(1→ with GalNAc4S6S:GalNAc4S in a ratio of 1.5:1, and three

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types of sulfated fucosyl branches attaching C-3 of GlcA, namely, Fucp2S4S, Fucp3S4S and Fucp4S,

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were found in a ratio of 2:1.5:1. The digestibility of FCSSJ was investigated in vitro, and the unchanged

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molecular weight and reducing sugar content indicated that FCSSJ was not broken down under salivary

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and gastrointestinal digestion. Furthermore, FCSSJ showed a significant inhibitory impact on pancreatic

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lipase dose-dependently but not on α-amylase, indicating that the inhibition of pancreatic lipase by

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FCSSJ might be a pathway for its hypolipidemic effect. These findings propose a fucosylated

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chondroitin sulfate and provide insight into the mechanism of its physiological effects in digestion

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system.

30 31

KEYWORDS: fucosylated chondroitin sulfate, stichopus japonicus, digestion, pancreatic lipase,

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α-amylase

2


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INTRODUCTION

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Fucosylated chondroitin sulfates (FCSs) are structurally unique glycosaminoglycan present in sea

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cucumber species. The structures of FCSs isolated from over 30 sea cucumber species have been

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analyzed so far. Generally, FCSs are composed of a backbone consisting of alternating

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→4)-β-D-GlcA-(1→3)-β-D-GalNAc-(1→ disaccharide units with fucosyl branches and sulfate groups.1

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They from different species of sea cucumber vary in sulfation pattern, presence of branching, and

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molecular weight for both of the backbone and fucosyl branches.2-6 Stichopus japonicus (or

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Apostichopus japonicus) is a species of sea cucumber with highest commercial value and is used as

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dietary delicacy and folk medicine by Asians for several thousands of years. The structure of FCS from

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S. japonicus (named FCSSJ), containing glucuronic acid (GlcA), N-acetylgalactosamine (GalNAc), and

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fucose (Fuc), was discovered for the first time in 1980.7 Four types of chondroitin sulfate (CS)-like

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blocks (CS, CS-A, CS-C, and CS-E) and three types of fucosyl branches (Fuc2S4S, Fuc3S4S, and

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Fuc4S) were then found in the structure of FCSSJ.1, 8 However, detailed structures of FCSSJ which have

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been reported by several research teams differed with each other at some degree. For example,

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Ustyuzhanina et al.9 reported that the backbone of FCSSJ was CS-A- or CS-E-connected at a molar ratio

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of approximately 2:1, and fucosyl branches only connected to O-3 of GlcA, but Guan et al.10 proposed

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that FCSSJ comprised a backbone of CS-E, and another researcher suggested that the fucosyl branches

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could connect to both O-3 of GlcA and O-4/6 of GalNAc in the backbone.11 Given the fact that the

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biological effects of FCSs significantly depend on the structures of the polysaccharides,2,

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important to carry out the structural analysis of FCS from S. japonicus.

12-13

it is

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It has been reported that the bioactive polysaccharides may be degraded by the acidic environment

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and digestive enzymes distributed in the gastrointestinal tract (GIT),14-15 thereby absorbed by the gut

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epithelium.16 The absorbed compounds may directly act on the GIT or be transmitted by systemic

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circulation to target organs, and then exert their health effects on nutrient needs and metabolism to treat

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or prevent diseases.17 Recently, digestion of bioactive polysaccharides and their impacts on

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gastrointestinal associated metabolic diseases have aroused great attention from scholars.15, 18 FCS, as a

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unique sulfated polysaccharide, has been demonstrated to have a wide variety of bioactivities, such as

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anticoagulation, antioxidantion, antiinflammation, antitumor, and hypolipidemic and hypoglycemic

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effects.2, 6, 19-23 However, literatures concerning the salivary, gastric or intestinal digestion of FCS from 3



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S. japonicus were rarely reported, and how they exert their bioactivities in the GIT remains unclear.

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Our recent studies revealed that sulfated polysaccharide from S. japonicus could positively regulate the

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gut microbiota in vivo, thereby exerting an anti-obesity effect, hypoglycemic and hypolipidemic effects

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by the oral routes to experimental animals.24-25 However, studies have shown that the hypolipidemic

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and hypoglycemic effects of carbohydrates may be due to their inhibitory impact on digestive enzymes

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in the GIT.18, 26-27 Therefore, whether these effects of FCS from S. japonicus are also related to their

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inhibition of digestive enzymes is worth exploring.

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In the present study, an FCS from sea cucumber S. japonicus was isolated and characterized mainly

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based on nuclear magnetic resonance (NMR) spectroscopy. In addition, in vitro the digestion process

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of FCSSJ and its impact on the digestion enzymes were investigated to evaluate the physiological

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effects of FCSSJ potentially occurring in the GIT.

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MATERIALS AND METHODS

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Materials. The fresh sea cucumbers (Stichopus japonicus, 4-5 years) were collected in Dalian,

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China. Papain (3500 U/mg), pepsin (400 U/mg), pancreatin (4 U/mg), trypsin (250 U/mg), α-amylase

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(300-1500 U/mg), and pancreatic lipase (100-500 U/mg) were purchased from Sigma-Aldrich Co.

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(Darmstadt, Germany). GlcA, GalNAc, and fucose (Fuc) were obtained from Aladdin Chemical

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Reagent Co., Ltd. (Shanghai, China). Bile salt was bought from Ryon Biological Technology Co., Ltd.

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(Shanghai, China). CS was purchased from ChromaDex, Inc. (California, United States). Acarbose was

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purchased from Bayer Health Care Co., Ltd. (Leverkusen, Germany). Orlistat was purchased from

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Shandong New Times Pharmaceutical Co., Ltd. (Shandong, China). Other chemical reagents were

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obtained from Sangon Biotechnology Co. (Shanghai, China).

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Preparation of FCSSJ. FCSSJ was prepared as previously described with minor modifications.28

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Briefly, sea cucumber was smashed and hydrolyzed with papain. Then, cetylpyridinium chloride was

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added to precipitate the sulfated polysaccharide. It was purified by a DEAE-Sepharose Fast Flow

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column (GE Healthcare, USA), eluted with a linear gradient of 0.7-2.0 mol/L NaCl (in 0.1 mol/L

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sodium acetate, pH 5.0) and detected by the phenol/sulfuric method.29 Fractions containing FCSSJ (the

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second eluting peak, Fig. S1) were collected, dialyzed (cutoff 3 kDa) against distilled water,

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lyophilized, and used in further analysis. 4


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Structural Characterization of FCSSJ. Molecular weight (Mw) distribution: The purity and Mw

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distribution of FCSSJ were determined using the high-performance size- exclusion chromatography

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(HPSEC) system coupled with a refractive index (RI) detector (Waters 2414, USA), a ultra violet (UV)

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detector (Waters 2998, USA), and a multi-angle light scattering (MALS) detector (DAWN HELEOS,

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Wyatt Corp., USA) in series, eluted by 0.1 mol/L NaNO3 (containing 0.05% w/v Na3N as preservative

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against bacteria) at a flow rate of 0.6 mL/min at 35 °C. An Ultrahydrogel Guard column (40 mm ×6

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mm), an Ultrahydrogel 2000 column (300 mm ×7.5 mm), and an Ultrahydrogel 1000 column (300 mm

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×7.5 mm) (Waters, Milford, MA) were adopted. A dn/dc value of 0.138 mL/g referred previous study30

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was used, and the MW was calculated using the ASTRA software (version 6.1, Wyatt Corp.).

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Chemical composition: Monosaccharide composition was assessed by high performance liquid

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chromatography (HPLC) coupled with a photodiode array detector (PAD) and a LXQ linear ion trap

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mass spectrometer (Thermo Fisher Scientific, Basel, Switzerland) after acid hydrolysis and

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derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP).31 The mass spectrometry parameters were

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as follows: spray voltage: 4.5 kV, capillary voltage: 37 V, capillary temperature: 275 °C, sheath gas: 40

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arbitrary units (AU), and auxiliary gas: 10 AU. The electrospray interface was set in positive-ion mode,

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and the scan range was set from m/z 100 to 2000. Uronic acids were measured by the

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3,5-dimethylphenol method with GlcA being the standard.32 Sulfate content was determined according

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to the BaCl2-gelatin method.33

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Fourier transform-infrared (FT-IR) spectroscopy: The sample FCSSJ (2 mg) was mixed with 100 mg

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dried KBr, ground, and punched into 1 mm pellets for FT-IR assay in the frequency range of 400 to

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4000 cm−1. FT-IR spectra were recorded on a Spectrum One-B FTIR Spectrometer (Perkin Elmer,

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USA).

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NMR spectroscopy: FCSSJ (45 mg) was dissolved in 500 μL D2O (99.9%), and exchangeable proton

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was substituted by deuterium after lyophilization for three times. 1H NMR, 13C NMR, 2-dimensional

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heteronuclear single quantum correlation (HSQC), correlation spectroscopy (COSY), and total

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correlation spectroscopy (TOCSY) spectra of FCSSJ were recorded on a 700 MHz Bruker Avance III

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(Bruker, German).

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Simulated Salivary Digestion of FCSSJ. The fresh saliva sample was provided by three healthy nonsmoking volunteers (no previous colonic disease and without antibiotics treatment at least 3 months) 5



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after 2 h of eating and drinking restriction. The spitting protocol for collecting human whole saliva

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samples was carried out as previously reported,34 and the amylase activity of the human saliva was

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carried out by the previous method.35 In vitro saliva digestion was performed by the previous method.14

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Briefly, FCSSJ was dissolved in distilled water at a concentration of 2.0 mg mL-1. The mixtures of 3.0

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mL FCSSJ solution with 3.0 mL saliva and 3.0 mL distilled water with 3.0 mL saliva were added into

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tubes, respectively. Tubes were incubated in an incubator shaker at 37 °C for digestion. Samples (1.5

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mL) were taken from the mixture at 0, 0.5, 1, and 2 h during saliva digestion and then boiled for 10 min

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to inactivate amylase. All the digestions were done in triplicate.

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Simulated Gastrointestinal Digestion of FCSSJ. In vitro gastrointestinal digestion of FCSSJ was

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performed according to the method previously described18 with proper modifications. The simulated

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gastric electrolyte (SGE) solution containing 3.1 g L-1 NaCl, 1.1 g L-1 KCl, 0.15 g L-1 CaCl2·2H2O, and

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0.6 g L-1 NaHCO3 was adjusted to pH 2.0 with 2 mol/L HCl. Then, 4 000 U mL-1 pepsin and 1.5 mg L-1

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CaCl2 were added into the SGE solution, affording the simulated gastric fluid. The simulated small

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intestinal electrolyte (SIE) solution containing 5.4 g L-1 NaCl, 0.65 g L-1 KCl, and 0.33 g L-1

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CaCl2·2H2O was adjusted to pH 7.0 with 1 mol/L NaHCO3. Then, 160 U mL-1 pancreatin based on

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trypsin activity, 2.6 g L-1 bile salt, and 2.5 mg L-1 CaCl2 were added into the SIE solution, affording the

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simulated small intestinal fluid. Simulated gastrointestinal digestion was performed in two consecutive

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processes, first gastric digestion and then small intestinal digestion. For gastric digestion, the mixtures

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of 10 mL FCSSJ aqueous solution (4 mg/mL) with 10 mL simulated gastric fluid and 10 mL distilled

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water with 10 mL simulated gastric fluid were prepared and incubated at 37 °C for 6 h in a shaking

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bath with continuous shaking at 10 g. During the digestion, the pH of the reaction solution was kept at

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2.0. After the gastric digestion, the solution was neutralized with 1.0 mol/L NaHCO3 and added an

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equal volume of the simulated small intestinal fluid. The resulting mixture was incubated at 37 °C for

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another 6 h with continuous shaking at 10 g. The mixture (2 mL) was taken out at 0, 0.5, 1, 2, 4, and 6

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h during gastric and intestinal digestion and then boiled for 10 min to inactivate enzymes. All the

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digestions were done in triplicate.

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Determination of Mw and Reducing Sugar Content of FCSSJ after Digestion. The digestion

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samples were centrifuged (12000 ×g, 10 min), and the supernatant was used for further analysis. For

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the Mw, the supernatant (200 μL) was filtered through a 0.22 μm membrane and analyzed as Section 6


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2.3.1. The content of reducing sugar released in the supernatant was determined by the 3,

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5-dinitrosalicylic acid (DNS) method with glucose as the standard.36

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Impacts of FCSSJ on Pancreatic Lipase and α-Amylase Activities. The impacts of FCSSJ on

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pancreatic lipase and α-amylase activities were performed as previously described

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modifications. Briefly, 1 mL of SIE solution (pH 7.0) was mixed with 1 mL of soybean oil and 1 mL

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aqueous solution of FCSSJ (2, 5 and 8 mg mL-1), CS or orlistat (positive control, 1 mg mL-1). In

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addition, the aqueous solution without polysaccharide was used as a blank control. The solutions were

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firstly incubated at 37 °C for 10 min, and 1 mL of pancreatic lipase (8000 U mL-1) dissolved in SIE

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solution was added. The final mixture was stirred at 37 °C for 60 min and then boiled for 10 min to

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terminate the reaction. The digestion samples were centrifuged (12000 ×g, 10 min), and the free fatty

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acid contents in the supernatant were titrated with 0.05 mol/L NaOH standard solution, using

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phenolphthalein as an indicator.15

18

with some

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For the α-amylase activity, a mixture consisting of 2 mL of starch solution (1%, w/w), 0.8 mL of

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simulated saliva electrolyte (SSE) solution (1.1 g L-1 KCl, 0.5 g L-1 KH2PO4, 1.2 g L-1 NaHCO3, 0.03 g

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L-1 MgCl2·(H2O)6, 5.8 mg L-1 (NH4)2CO3, and 0.22 g L-1 CaCl2·2H2O, pH 7.0), 0.2 mL of α-amylase

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solution (2500 U mL-1) dissolved in SSE solution, and 1 mL of FCSSJ (2, 5 and 8 mg mL-1), CS or

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acarbose (positive control, 5 mg mL-1) dissolved in SSE solution was prepared. In addition, the

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aqueous solution without polysaccharide was used as a blank control. The mixture was performed in a

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magnetic stirrer at 37 °C for 60 min and then terminated by adding 2 mL of 0.2 mol/L NaOH. The

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digestion samples were centrifuged (12000 ×g, 10 min), and the reducing sugar in the supernatant was

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determined by the DNS method as Section 2.6.

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Statistical Analysis. All data were presented as mean ± standard deviation using SPSS v.17.0

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statistical software (SPSS Inc., Chicago, IL, USA). Significant differences among means were

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determined by a Duncan multiple range tests, and differences were considered statistically significant

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at p

Structural features and digestive behavior of fucosylated chondroitin

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