Gastric protective activities of sea cucumber fucoidans with different

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Functional Structure/Activity Relationships

Gastric protective activities of sea cucumber fucoidans with different molecular weight and chain conformations: a structure-activity relationship investigation Xiaoqi Xu, Yaoguang Chang, Changhu Xue, Jingfeng Wang, and Jingjing Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01497 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 4, 2018

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Journal of Agricultural and Food Chemistry

Gastric protective activities of sea cucumber fucoidans with different molecular weight and chain conformations: a structure-activity relationship investigation Xiaoqi Xuab, Yaoguang Changa*, Changhu Xuea*, Jingfeng Wanga, Jingjing Shena

a. College of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China b. College of Food Science and Light Industry, Nanjing Tech University, 30 South Puzhu Road, Nanjing, 211816, China

*

Corresponding author. E-mail address: [email protected]; [email protected].

Tel.: +86 532 82032597

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ABSTRACT

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A variety of bioactivities have been established for fucoidan extracted from sea

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cucumber, whereas its structure-activity relationships were seldom investigated. In this

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study, sea cucumber (Thelenota ananas) fucoidans with different molecular weight were

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prepared with enzymatic degradation. The chain stiffness and molecular size decreased

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with the decreasing of molecular weight. Fucoidans with molecular weight of 1380.0 kDa,

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828.7 kDa and 483.0 kDa exhibited random coil conformations, while fucoidan

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molecular of 215.0 kDa existed as sphere in solution. All examined fucoidans could

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effectively prevent the ethanol-induced gastric ulcer, of which mechanism involved anti-

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oxidation and anti-inflammation. Within the range of the study, the performance of

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fucoidans adopted random coil conformations declined with the decreasing of molecular

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weight, and the performance recovered when the chain conformation transited from coil

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to sphere, indicating the subtle influences of molecular weight and chain conformation on

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the gastric protective activity of sea cucumber fucoidan.

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KEYWORDS

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Fucoidan; Ethanol-induced gastric ulcer; Molecular weight; Chain conformation;

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Sea cucumber; Structure-activity relationship

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INTRODUCTION

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Fucoidans are polysaccharides containing substantial percentages of L-fucose and

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sulphate groups, widely existed in sea cucumbers and algae1. Various bioactivities of sea

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cucumber fucoidans have been verified, such as anticoagulant2, gastric protective3 and

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osteoclastogenesis inhibiting activities4. Recently, their physicochemical properties

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including viscosity, rheological behavior and thermal stability have also been

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investigated5. These properties manifested that sea cucumber fucoidans could be used as

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a promising functional food ingredient.

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Biological activities of polysaccharides greatly depend on their structural features. It

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has been widely verified that molecular weight and chain conformation are important

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factors significantly influencing functions of polysaccharides. For instance, for a fucoidan

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isolated from marine brown algae L. japonica, molecular weight (Mw) of 20-30 kDa is the

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critical point for its proangiogenic effect, and higher or lower Mw will lead to decrease in

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the bioactivity6. After the chain conformation of lentinan ((1→3)-β-D-glucan) transiting

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from triple helical to single chains, the anti-tumor activities significantly decline7. Such

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knowledge on structure-activity relationships is beneficial to the modifications of

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polysaccharides for reaching their maximum effects, which is particularly useful in the

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development of functional foods. Although several bioactivities have been established,

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there are only few studies involving structure-activity relationships for sea cucumber

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fucoidans.

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Gastric ulcer is a pervasive gastric diseases8 which affects approximately 14.5

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million people worldwide9. It could be induced by various aggressive factors including

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ethanol10. Since alcohol abuse is considered as a serious public health problem



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throughout the world11, ethanol-induced gastric ulcer model are widely applied to

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evaluate the gastric protective functions of food and pharmaceutical ingredients12, 13. It

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has been verified that fucoidans extracted from some sea cucumber and algae such as

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Acaudina molpadioides and Hizikia fusiformis are competent to protect gastric tissue

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from the damage of ethanol-induced ulcer3, 14. Nevertheless, up to date, few reports reveal

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the relevance of the gastroprotective activities of fucoidans to their structural features.

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This study was aimed to investigate the structure-activity relationships existed in the

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gastric protective effects of sea cucumber fucoidans. Fucoidan obtained from the body

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wall of sea cucumber Thelenota ananas (Ta-FUC) was utilized as material, of which

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primary structure has been clarified as [→3-α-L-Fucp-1→3-α-L-Fucp-1→3-αL-

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Fucp2,4(OSO3-)-1→3-α-L-Fucp2(OSO3-)-1→]n15.

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glycoside hydrolase for cleaving fucoidan) from marine bacterial strain Wenyingzhuangia

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fucanilytica CZ1127T 15-17 was employed to prepare low molecular weight fucoidans (Ta-

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LMFs) with different Mw. The structural features of Ta-FUC and Ta-LMFs including Mw

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and chain conformation were characterized, and their performance and mechanism for

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protecting gastric tissue were investigated by using an ethanol-induced gastric ulcer

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animal model. The results would provide a better understanding of structure-activity

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relationships for sea cucumber fucoidans, which would facilitate their further utilization.

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MATERIALS AND METHODS

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Preparation and characterization of low-molecular-weight fucoidans

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Preparation of fucoidan from T. ananas and low-molecular-weight fucoidans

The

fucoidanase

(endo-acting

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Sea cucumber T. ananas was harvested from the South China Sea, in April 2012.

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Ta-FUC was prepared according to previously described method15. Briefly, the body wall 4 ACS Paragon Plus Environment

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of the T. ananas was dried, milled and hydrolyzed with papain; Then cetylpyridinium

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chloride was utilized to precipitate the crude sulphated polysaccharides (CSP). The CSP

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was applied to an Express-Ion D (Whatman, USA) column (2.6 × 30 cm), and fractions

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(NaCl concentration 1.2–1.5 M) containing fucoidan were collected, lyophilized. The

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fractions were subsequently purified by a Sepharcral S-500 (GE Healthcare, USA)

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column eluting with 0.2 M NH4HCO3, and finally purified fucoidan was collected,

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dialyzed and lyophilized. Monosaccharide composition and sulphate contents18 analysis

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showed that L-fucose was the only constituting monosaccharide of Ta-FUC, and the

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sulphate content was 28.2 ± 3.5%. Intracellular enzyme of marine bacterial stain

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Wenyingzhuangia fucanilytica CZ1127T (formerly named as Flavobacteriaceae

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CZ1127)18 was obtained according to Yu’s method15.

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50 mL intracellular enzyme was added to 50 mL 0.4% Ta-FUC solution (pH 7.2)

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containing 0.3 M NaCl and 20 mM Tris–HCl. The enzymatic hydrolysis reaction was

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incubated at 35 °C for different time (4 h, 6 h and 10 h) to obtain low-molecular-weight

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fucoidans (LMFs) with different Mw. At the end of reaction, the mixture solution was

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kept at 100 °C for 10 min to stop the enzymatic reaction. Products were subsequently

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purified with HiPrep 26/60 Sephacryl S-300/400/500 HR column (GE, USA) based on

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their approximate molecular weight, with 0.2 M NH4HCO3 as eluate at a flow rate of 1.3

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mL min-1. Fractions around the major elution peak were pooled, dialyzed (cut-off 3.5

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kDa), lyophilized and utilized in the following experiments, which were nominated as

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Ta-LMF1, Ta-LMF2 and Ta-LMF3 in the increasing of reaction time. The sulphate

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content of Ta-LMFs and Ta-FUC was estimated by the BaCl2-gelatin method18, to

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determine whether desulphation occurred during the reaction. 5 ACS Paragon Plus Environment


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HPSEC-MALLS-Visc-RI analysis

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The Mw, z-average radius of gyration (Rg), hydrodynamic radius (Rh) and intrinsic

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viscosity ([η]) were estimated by the HPSEC-MALLS-Visc-RI system as described in

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our previous report5. Briefly, a 10 mM phosphate buffer saline (pH 7.4) containing 150

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mM NaCl (PBS) was employed as the eluent. The flow rate was set at 0.4 mL min-1. And

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the column temperature was 25 °C. The Zimm method was adopted to calculate Mw and

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Rg. The dn/dc value of Ta-FUC, Ta-LMF1, Ta-LMF2, and Ta-LMF3 was 0.1240 ±

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0.0005 mL g-1, 0.1106 ± 0.0007 mL g-1, 0.1247 ± 0.0003 mL g-1, and 0.1147 ± 0.0002

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mL g-1, respectively.

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Molecular morphology observation

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Molecular morphology of Ta-FUC and Ta-LMFs was investigated by using an

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atomic force microscope (AFM) (5400, Agilent Technologies, USA). Ta-FUC and Ta-

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LMFs were dissolved in deioned water at the concentration of 10 µg mL-1. Ten

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microliters of sample solution were added onto muscovite mica substrate and were dried

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for more than 1.5 h. The images of samples were recorded under tapping mode in air

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(25 °C, ambient pressure and humidity).

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Protective effect of fucoidans against ethanol-induced gastric ulcer

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Animal maintenance

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All the animal study procedures were permitted by the ethical committee of

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experimental animal care at Ocean University of China (Qingdao, China). Sprague–

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Dawley rats (Male, weight of 130–140 g, 5 weeks old) were supplied by Vital River

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Laboratory Animal Technology Co. Ltd (permit No.: SCXK2012-0001, Beijing, China).

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All of the rats were acclimatized under a 12/12 h light/dark cycle at constant temperature 6 ACS Paragon Plus Environment

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of 24 °C with relative humidity of 65 ± 15%, and provided with standard laboratory pellet

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chow (Kangda, Jinan, China) and fresh water.

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Induction of gastric ulcer and treatment

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After an acclimation period of 7 days, the rats were divided into 6 groups (12

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animals per group) with similar mean body weight (185 ± 2 g) randomly. The groups

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were designated as follows: normal, model, Ta-FUC, Ta-LMF1, Ta-LMF2, and Ta-

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LMF3 groups. After deprived of food and water for 18 h, rats of the normal and model

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groups were orally administrated with 6 mL kg-1 body weight normal saline (e.g., 9 g L-1

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NaCl solution) through a gavage needle; meanwhile, animals in the other groups were

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accordingly given oral administration of saline solution containing 50 mg mL-1 Ta-FUC

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or Ta-LMFs (6 mL kg-1 body weight). One hour later, rats of model and sample groups

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received 4 mL kg-1 body weight 80% ethanol to induce gastric ulcer19. After another one

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hour, all the animals were sacrificed by bleeding from the abdominal aorta under diethyl

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ether anesthesia.

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The stomachs of rats were dissected out. Half of them in each group were

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immediately filled with 10% phosphate-buffered formalin (pH 7.0) and submerged in the

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same solution. After 30 min, opened along the greater curvature of stomachs and washed

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with cold saline for ulcer index evaluation and histological analysis. Meanwhile the other

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stomachs in each group were opened, washed, frozen immediately in liquid nitrogen and

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stored at -80 °C for biochemical analysis.

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Gastric ulcer index and histological analysis

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Areas of gastric ulcer lesions and glandular stomach were estimated by a system

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consisted of stereomicroscope (Olympus SZ61: Olympus Optical Co. Ltd., Tokyo, Japan) 7 ACS Paragon Plus Environment


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a digital microscope camera (Olympus DP70: Olympus Optical Co. Ltd., Tokyo, Japan)

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and a digital image analysis program (Image pro plus software: Olympus Optical Co. Ltd.,

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Tokyo, Japan). Gastric ulcer of rats was evaluated according to ulcer index (UI) which

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was calculated as follows: UI = (total ulcerated area / total mucosa area) × 100%.

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For histological analysis, stomach samples were routinely processed with

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hematoxylin and eosin stain20. The histological changes of the stomach tissue were

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examined under a light microscopy (Olympus BX-41: Olympus Optical Co. Ltd., Tokyo,

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Japan).

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Biochemical assays

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A stomach segment was dissolved by homogenization in cold saline and centrifuged

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at 10,000 g for 15 min at 4 °C. The supernatant was obtained to determinate superoxide

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dismutase

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corresponding assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

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mRNA expression analysis

(SOD)21,

glutathione

(GSH)22

and

malonaldehyde

(MDA)23

using

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Total RNA of the stomach tissue was extracted by using the Trizol reagent (Thermo

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Fisher Scientific Inc., MA, USA) according to the manufacturer’s methods. The mRNA

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expressions of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were

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subsequently investigated by using quantitative real time-PCR (qRT-PCR), operated with

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the Fast Start Universal SYBR Green Master Mix (F. Hoffmann-La Roche Ltd,

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Genentech, CA, USA) and the Bio-Rad iCycler iQ5 system. The final data for IL-6 and

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TNF-α were normalized using the β-actin as endogenous reference. The sequences of

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primers for β-actin, IL-6 and TNF-α are identical with those described in a previous

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report24. 8 ACS Paragon Plus Environment

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Western blotting

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The expression level of NF-κB p65 protein was investigated by western blotting3.

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Briefly, each frozen stomach tissue was homogenized in RIPA buffer (pH 7.4), which

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composed of 50 mM Tris, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1% Triton X-100,

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1% sodium deoxycholate and 0.1% SDS. The mixture was then centrifuged at 12,000 g

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for 15 min at 4 °C. After denaturation, proteins were separated by SDS-PAGE and

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thereafter transferred to polyvinyl difluoride membrane (EMD Millipore, MA, USA). The

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membrane was blocked with 5% BSA (BD, Franklin Lakes, NJ, USA) for 2 h and then

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incubated with primary antibodies against NF-κB p65 or β-actin (Abcam, Cambridge,

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USA) for 12 h at 4 °C, and the secondary antibody (Jackson Immuno Research

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Laboratories, West Grove, PA, USA), successively. Thereafter, the membrane was

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visualized by an enhanced chemiluminescence kit (Applygen, Beijing, China).

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Autoradiograms were recorded by a gel-imaging system (Tanon T4100; Tanon Inc.,

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Shanghai, China) and analyzed with the Image J 1.44 software. Bands of β-actin were

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used as endogenous references.

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Statistical analysis

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In this study, experimental data were performed in sextuplicate (n = 6). The results

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in tables and graphs were represented as mean values with standard deviation. Statistical

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comparison was tested using variance (ANOVA) analysis followed by Duncan’s multiple

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range tests with SPSS software (Statistics 19.0, SPSS Inc., Chicago, IL, USA). p < 0.05

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was considered statistical significance.



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RESULTS AND DISCUSSION

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Molecular characteristics of fucoidans

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Ta-FUC, Ta-LMF1, Ta-LMF2 and Ta-LMF3 showed narrowly distributed peaks

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with relatively low polydispersity index values (Table 1) on their HPSEC chromatograms

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(Fig. 1A-D), which indicated that all the fucoidan samples were well purified. The values

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of Mw, Rg and Rh for the fucoidan samples were calculated based on the HPSEC-MALLS-

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Vis-RI analysis and listed in Table 1. The Mw of fucoidan gradually decreased with the

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increasing of reaction time, which was attributed to the cleavage of glycoside linkage in

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fucoidan during the enzymatic degradation. Fucoidan with the higher Mw exhibited the

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higher Rg and Rh. A larger dimension would cause an earlier elution on the HPSEC

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column, which was also observed in the chromatograms (Fig. 1A-D).

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The four fucoidan samples exhibited significantly difference in Mw, Rg and Rh

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compared with each other, while sulphate contents of all the fucoidans were similar

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(Table 1). It confirmed that the sulphate groups were not hydrolyzed during the

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degradation. Sulfate groups profoundly impact bioactivities of fucoidans, such as their

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anticoagulant, antiviral, and anti-parasitological activities25. The maintenance of sulfate

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groups would be beneficial to the following study on the relationship between Mw and

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functions, without concerning the influences brought by changes in primary structural

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characteristics.

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The chain conformation of Ta-FUC and Ta-LMFs were deduced from the

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dependence of Rg on Mw and the dependence of [η] on Mw which were estimated from

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each slice of HPSEC-MALLS-Vis-RI chromatograms. The double logarithmic plots (Fig.

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1E) of Rg versus Mw could be described as Eq. (1). And the double logarithmic plots (Fig. 10 ACS Paragon Plus Environment

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1F) of [η] versus Mw, i.e., the Mark-Houwink-Sakurada equations26 could be expressed as

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Eq. (2).

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Rg = kM wα s (nm)

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   

(1)

α

η = KM wη (mL g −1 ) (2)

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Generally the αs value of 0.33, 0.5–0.6, and 1.0 indicates a sphere, a flexible chain, and a

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rigid rod27, respectively. The αη values of ∼0, 0.5–0.8, and up to 1.8 separately

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correspond to spheres, random coils, and rigid rods28.

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The αs and αη of Ta-FUC were 0.60 and 0.92 respectively. Both of them indicated

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that Ta-FUC adopted a random coil conformation in PBS, which was similar to the chain

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conformation of linear fucoidan from sea cucumber Acaudina molpadioides and

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Isostichopus badionotus5. The αs values of Ta-LMF1 and Ta-LMF2 were 0.58 and 0.35,

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and their αη values were 0.77 and 0.63 respectively. Those results revealed that Ta-LMF1

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and Ta-LMF2 also adopted random coil conformations in PBS as Ta-FUC. Nevertheless,

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Ta-LMF1 and Ta-LMF2 were more flexible than Ta-FUC, which was proven by the

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declining tendency in αs and αη with the decreasing of Mw and molecular size.

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The αs (0.17) and αη (0.41) of Ta-LMF3 showed that it exhibited a sphere

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conformation, which was distinct from the random coil conformations of the other three

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fucoidans. This transition of chain conformation from random coil to sphere was also

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supported by the observations of AFM which is a suitable tool for examining

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macromolecule surface topological features. Ta-FUC appeared as elongated chains in

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AFM images (Fig. 2A), Ta-LMF1 (Fig. 2B) and Ta-LMF2 (Fig. 2C) existed as chains

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with more coiling or bending compared with Ta-FUC, and molecules of Ta-LMF3

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showed as spheres (Fig. 2D). The transition could be again attributed to the gradually 11 ACS Paragon Plus Environment


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increasing in degradation degree and the corresponding decreasing in chain stiffness.

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However, it should be noted that the observed size of fucoidan in AFM might be higher

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than Rg and Rh values due to the broaden effect of AFM method.

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Protective effects of fucoidans against ethanol-induced gastric ulcer

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The ethanol-induced hemorrhagic ulcer of stomach surface were observed in rats of

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model group with the UI reached 14.2 ± 2.2% (presentative images of the

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stereomicroscopic observation was shown in supplementary data Fig. S1). The UI values

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of pretreated groups were all significantly lower than that of model group (p

Gastric protective activities of sea cucumber fucoidans with different

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