Gastric protective activities of sea cucumber ... - ACS Publications

Subscriber access provided by Kaohsiung Medical University

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

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

1 ACS Paragon Plus Environment


1

ABSTRACT

2

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

3

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

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

fucoidans adopted random coil conformations declined with the decreasing of molecular

12

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

13

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

14

the gastric protective activity of sea cucumber fucoidan.

15

KEYWORDS

16

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

17

Sea cucumber; Structure-activity relationship

18


Page 2 of 28

Page 3 of 28


19

INTRODUCTION

20

Fucoidans are polysaccharides containing substantial percentages of L-fucose and

21

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

22

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

23

osteoclastogenesis inhibiting activities4. Recently, their physicochemical properties

24

including viscosity, rheological behavior and thermal stability have also been

25

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

26

a promising functional food ingredient.

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

fucoidans.

39

Gastric ulcer is a pervasive gastric diseases8 which affects approximately 14.5

40

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

41

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



Page 4 of 28

42

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

43

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

44

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

45

Acaudina molpadioides and Hizikia fusiformis are competent to protect gastric tissue

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

glycoside hydrolase for cleaving fucoidan) from marine bacterial strain Wenyingzhuangia

54

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

55

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

56

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

57

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

58

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

59

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

60

MATERIALS AND METHODS

61

Preparation and characterization of low-molecular-weight fucoidans

62

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

The

fucoidanase

(endo-acting

63

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

64

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

Page 5 of 28


65

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

66

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

67

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

68

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

69

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

70

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

71

dialyzed and lyophilized. Monosaccharide composition and sulphate contents18 analysis

72

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

73

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

74

Wenyingzhuangia fucanilytica CZ1127T (formerly named as Flavobacteriaceae

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

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

84

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

85

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

86

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

87

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


88

HPSEC-MALLS-Visc-RI analysis

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

mL g-1, respectively.

97

Molecular morphology observation

98

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

99

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

100

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

101

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

102

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

103

(25 °C, ambient pressure and humidity).

104

Protective effect of fucoidans against ethanol-induced gastric ulcer

105

Animal maintenance

106

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

107

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

108

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

109

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

110

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

Page 6 of 28

Page 7 of 28


111

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

112

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

113

Induction of gastric ulcer and treatment

114

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

115

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

116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

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

124

ether anesthesia.

125

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

126

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

127

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

128

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

129

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

130

stored at -80 °C for biochemical analysis.

131

Gastric ulcer index and histological analysis

132

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

133

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


Page 8 of 28

134

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

135

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

136

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

137

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

138

For histological analysis, stomach samples were routinely processed with

139

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

140

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

141

Japan).

142

Biochemical assays

143

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

144

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

145

dismutase

146

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

147

mRNA expression analysis

(SOD)21,

glutathione

(GSH)22

and

malonaldehyde

(MDA)23

using

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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

156

report24. 8 ACS Paragon Plus Environment

Page 9 of 28

157


Western blotting

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

used as endogenous references.

172 173

Statistical analysis

174

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

175

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

176

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

177

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

178

was considered statistical significance.



179

RESULTS AND DISCUSSION

180

Molecular characteristics of fucoidans

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

characteristics.

198

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

199

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

200

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

201

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

Page 10 of 28

Page 11 of 28


202

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

203

Eq. (2).

204

Rg = kM wα s (nm)

205

   

(1)

α

η = KM wη (mL g −1 ) (2)

206

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

207

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

208

correspond to spheres, random coils, and rigid rods28.

209

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

210

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

211

conformation of linear fucoidan from sea cucumber Acaudina molpadioides and

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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

224

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


225

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

226

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

227

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

228

Protective effects of fucoidans against ethanol-induced gastric ulcer

229

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

230

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

231

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

232

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

Gastric protective activities of sea cucumber ... - ACS Publications

Recommend Documents