Dietary Cerebroside from Sea Cucumber ... - ACS Publications

Subscriber access provided by Northern Illinois University

Article

Dietary cerebroside from sea cucumber (Stichopus japonicus): absorption and effects on skin barrier and cecal short-chain fatty acids Jingjing Duan, Marina Ishida, Kazuhiko Aida, Tsuyoshi Tsuduki, jin Zhang, Yuki Manabe, Takashi Hirata, and Tatsuya Sugawara J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02564 • Publication Date (Web): 02 Sep 2016 Downloaded from http://pubs.acs.org on September 9, 2016

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 35

Journal of Agricultural and Food Chemistry

Dietary cerebroside from sea cucumber (Stichopus japonicus): absorption and effects on skin barrier and cecal short-chain fatty acids

Jingjing Duan1,2*, Marina Ishida1, Kazuhiko Aida3, Tsuyoshi Tsuduki4, Jin Zhang2, Yuki Manabe1, Takashi Hirata1,5 and Tatsuya Sugawara1*,

1

Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University,

Kyoto 606-8502, Japan 2

Present address of J. Duan: Department of Cardiology, Boston Children's Hospital,

Boston, MA 02115, USA 3

Central Laboratory, Nippon Flour Mill Company Ltd., Kanagawa 243-0041, Japan

4

Laboratory of Food and Biomolecular Science, Graduate School of Life Science and

Agriculture, Tohoku University, Sendai 981-8555, Japan 5

Present address of T. Hirata: Shijonawate Gakuen University, Daito, Osaka 574-0011,

Japan

*Corresponding author: Tatsuya Sugawara (Tel: +81-75-753-6212; Fax: +81-75-753-6212; E-mail: [email protected]) Jingjing Duan (Tel: +1-617-919-4654; Fax: +1-617-731-0787; E-mail: [email protected])

1

ACS Paragon Plus Environment


1

Abstract

2

Sphingolipids from marine source attract more attentions recently because of their

3

distinctiveness on structures and expected functions. In this study, the content and

4

component of cerebroside from sea cucumber Stichopus japonicus was analyzed. The

5

absorption of cerebroside from S. japonicus was investigated with an in vivo lipid

6

absorption assay. Our result revealed that S. japonicus is a rich source of cerebroside

7

that contained considerable amounts of odd carbon chain sphingoid bases. The

8

cumulative recovery of d17:1 and d19:2 consisting cerebrosides were 0.31±0.16% and

9

0.32±0.10%, respectively for 24 h after administration. To the best of our knowledge,

10

this is the first work that shows sphingolipids from marine source could be absorbed

11

in vivo and incorporated into ceramides. In addition, dietary supplementation with sea

12

cucumber cerebroside to hairless mouse improved the skin barrier function and

13

increased short-chain fatty acids in cecal contents, that shown beneficial effects on

14

host.

15 16

Keywords: cerebroside; dietary sphingolipids; sea cucumber Stichopus japonicus;

17

sphingoid base

2


Page 2 of 35

Page 3 of 35


18

Introduction

19

It has been estimated that the consumption of sphingolipids in selected foods

20

has been estimated over 100 g per year by Vesper et al. in 1999.1 Most of the dietary

21

studies to date have been conducted with sphingolipids from milk, soy and other

22

“terrestrial” foods; however, aquatic organisms are also rich sources.2-4 In recent years,

23

researchers found several atypical structures of sphingolipids from squid, octopus, sea

24

cucumbers, and many other marine organisms.3,

25

invertebrates have atypical types of chain length，unsaturation and hydroxylation in

26

sphingoid bases such as 2-amino-4,8,10-octatriene-1,3-diol (d18:3) ， 2-amino-9-

27

methyl-4,8,10- octatriene-1,3-diol (d19:3) and 2-amino-1,3-dihydroxy-4-heptadecene

28

(d17:1).3, 5-9 With the shift of dietary habits of modern humans in recent years, and the

29

development of the sphingolipid analysis platform “sphingolipidomics”,10-11 we

30

believe that humans consume more sphingolipids than currently known and it is thus

31

necessary to re-survey the sphingolipids in diet.

5-7

Sphingolipids of marine

32

Most of the cellular sphingolipids are usually synthesized de novo, however,

33

biosynthesis of sphingolipids can be affected by many dietary factors, including the

34

availability of the precursors and their regulators in metabolic pathways.7,

35

Sphingolipids that are taken up from exogenous sources can be recycled and/or

36

degraded and participate in lipid raft formation.13 Therefore, exogenous (dietary)

37

sphingolipids and modulators of sphingolipid metabolism affect raft related signaling

38

pathways,

39

including glucosylceramide (GlcCer) and galactosylceramide (GalCer), as one of the

which

might

explain

their

biological

3


activities.

12

Cerebroside,


40

most significant components of sphingolipids, has emerged as an important ingredient

41

in our diet. Several bioactivities of dietary cerebrosides, such as their

42

anti-inflammatory,14 improving the barrier function of the skin,15-17 cancer-protective

43

effects on the intestine,18-19 and preventing melanin formation20 have been reported.

44

Cerebrosides from sea cucumbers have unique sphingolipid molecules3, 5 known to

45

have anti-tumor8, 21, anti-adipogenic22, and against oxidative damage23 activities, and

46

can induce apoptosis in human hepatoma HepG2 cells through p-AKT and DR5.24 It

47

has been found that cerebroside from different species of sea cucumbers consists of

48

both GlcCer and GalCer,25-29 whereas there is no report to date that cerebroside from

49

the sea cucumber S. japonicus contains any GalCer. S. japonicus is used in fresh

50

(whole body) or dried (body wall only) as an important food and traditional medicine

51

in Asian countries. 30,31 Its body wall or viscera soft capsules have been consumed as

52

a dietary supplement recently.32 Du et al. found that liposomes prepared from sea

53

cucumber could be transport and uptake in small intestinal epithelial cell models,33

54

that would be beneficial to understanding the fate of dietary sea cucumber

55

sphingolipids. However, in vivo absorption of cerebroside from sea cucumber and its

56

function as a dietary component are still not well understood.

57

In this study, we evaluated the amount of cerebroside from edible sea cucumber

58

S. japonicus and investigated its absorption in the rat intestine by a lipid absorption

59

assay. The effects of dietary cerebroside from sea cucumber on the skin barrier and

60

cecal short-chain fatty acids were investigated with a hairless murine model.

61

Materials and Methods 4


Page 4 of 35

Page 5 of 35


62

Analysis of cerebroside from sea cucumber Stichopus japonicus

63

Dried sea cucumber (Stichopus japonicus) was purchased from a local

64

fisherman in Nagasaki, Japan. Fresh sea cucumber (S. japonicus) was kindly donated

65

by Isonoya Co. Ltd. (Maizuru, Japan). After washing with saline, fresh sea cucumbers

66

were necropsied to acquire different body parts, which were then lyophilized and

67

milled. Total lipids were extracted by Folch’s method and saponified with 0.4 M KOH

68

in methanol at 38 °C for 2 h to remove glycerolipids. The alkali-stable lipid fractions

69

were applied to high-performance liquid chromatography with evaporative light

70

scattering detector (HPLC- ELSD) with a TSKgel CN-80Ts ( 250 × 4.6 i.d. mm, 5 µm)

71

(TOSOH, Tokyo, Japan) and maintained at 40 °C. The mobile phase consisted of

72

hexane-2-propanol (99:1, v/v) and chloroform-methanol (60:40, v/v) with flow rate

73

1.0 mL/min. The amounts of cerebroside were calculated with our previous published

74

quantitative analysis method.4 The cerebroside fractions from body wall and viscera

75

of fresh sea cucumber were collected after HPLC and applied for further structural

76

analysis.

77

A Shimadzu high performance liquid chromatography-ion trap-time of flight

78

mass spectrometer (LCMS-IT-TOF) equipped with an electrospray ionization

79

interface (Shimadzu, Kyoto, Japan) was used for Liquid chromatography-mass

80

spectrometry (LC-MS) analyses. A TSK gel ODS-100Z column (2.0 × 50 mm, 3 µm,

81

Tosoh, Tokyo, Japan) was eluted with methanol/water (95:5, v/v) containing 5 mM

82

ammonium acetate at a flow rate of 0.2 mL/min. The MS was operated under

83

previously reported conditions.3, 34 For the structural analysis of cerebroside from S. 5



84

japonicus, [M+H]+ and [M+H-18]+ (loss of water) in the positive scan mode were

85

used for MS/MS analysis as precursor ions to obtain the product ions. The

86

characteristic signals of sphingoid base moieties were observed as the product ions

87

using the auto MS/MS detection mode. Pairs of the structurally specific product ions

88

of sphingoid bases and their precursor ions were used and calculated for identification

89

of cerebroside molecules (for example, m/z 700.7/264.4, 728.7/264.4 and 810.9/264.4,

90

for d18:1/C16:0, d18:1/C18:0 and d18:1/C24:1 cerebroside, respectively) .34-35 For

91

analysis of sphingolipids in rat lymph, the MS was operated under the same

92

conditions as we reported previously.36

93

Analysis of the sphingoid base fraction prepared from sea cucumber cerebroside

94

The cerebroside powder from dried sea cucumber (S. japonicus) was kindly

95

prepared by Nippon Flour Mills Co. Ltd. (Atsugi, Japan), and its purity was 96% as

96

determined by HPLC.37 The powder was hydrolyzed with 1 M HCl in methanol at

97

70 °C for 18 h to release free sphingoid bases, then o-phthalaldehyde (OPA)

98

derivatives of the free sphingoid bases were quantified with reverse-phase HPLC

99

system consisted of an LC-10AD pump and an RF-10AXL fluorescence detector

100

(Shimadzu). Sphingoid bases were separated with acetonitrile/water (80:20, v/v) on a

101

TSK gel ODS-80Ts column (Tosoh), 4.6 × 250 mm that isocratic analyses were

102

performed at 1.0 mL/min. The OPA derivatives were detected with excitation

103

wavelength and emission wavelength of 334 nm and 440 nm, respectively. 38

104

Evaluation of intestinal absorption of sea cucumber cerebroside

105

Sea cucumber cerebroside absorption was studied via a lipid absorption assay. 6


Page 6 of 35

Page 7 of 35


106

This study was conducted in conformity with the policies and procedures detailed in

107

the Animal Experiment Guidelines of Tohoku University. Surgeries and maintenance

108

of rats and all other procedures were the same as in our previous study

109

male Sprague-Dawley rats (9 weeks old, n=5 for each experimental and control

110

groups) were obtained from Japan SLC (Hamamatsu, Japan) and were housed at 23 ±

111

1 °C with a 12 h light/dark cycle. After acclimatization, the rats were anesthetized and

112

a cannula (SV35, Dural Plastics) was inserted into their left thoracic channel for

113

collecting lymphatic fluid, and a catheter (SP-55, Dural Plastics) was inserted into

114

their stomach. After surgery, each rat was placed in a restraining cage in a warm

115

recovery room. A physiological solution containing 139 mmol/L glucose and 85

116

mmol/L NaCl was infused continuously overnight at a rate of 3 mL/h through the

117

stomach cannula, and the same solution was also provided for drinking. The next

118

morning, after collection of lymph for 2 h as a blank control, the rats were infused

119

with 3 mL emulsions prepared by ultrasonication (containing 200 mg triolein, 50 mg

120

fatty acid-free albumin, 200 mg sodium taurocholate, and with or without 5 mg

121

cerebroside powder from dried sea cucumber as described above) as a single bolus

122

through the stomach catheter. After those emulsions, infusion of the glucose/NaCl

123

solution was continued and subsequently lymph samples were collected in

124

EDTA-containing tubes and stored at -30 °C until analysis. Lipids were extracted and

125

saponified, the alkali-stable fraction was subjected to HPLC analysis for

126

quantification of free sphingoid bases (free sphingoid base fraction-“Free”). A portion

127

of the alkali-stable fraction of lymph extract was degraded with aqueous methanolic 1 7


36

. Briefly,


Page 8 of 35

128

M HCl at 70 °C for 18 h. Free sphingoid bases liberated from the complex

129

sphingolipids were then subjected to HPLC for quantification analysis of total

130

sphingolipids (total sphingoid base fraction-“Total”). The OPA derivatives of the free

131

forms of sphingoid bases were analyzed as described above. The method for

132

quantifying the recovery of sea cucumber sphingoid bases in this study was based on

133

our previously study 36.

134

Effect of dietary cerebroside on the barrier function of skin in a murine model

135

Four-week-old female hairless mice (Hos: HR-1) were purchased from Japan

136

SLC Inc. (Shizuoka, Japan). Animals were kept at 25 °C with a 12 h light/dark cycle

137

and allowed free access to food and water. All experiments were performed according

138

to the guidelines of Kyoto University for the use and care of laboratory animals. After

139

acclimatization, mice were randomly allocated to 2 groups (n=6) with feeding

140

standard AIN-93G (control group) or sea cucumber cerebroside supplemented (SC

141

group) diets. The cerebroside supplemented diet was made with sea cucumber

142

cerebroside powder (0.1% of total weight) that mixed uniformly into 0.1% soybean

143

oil-reduced AIN-93G growth purified diet. After 2 weeks, the dorsal skin of mice was

144

tape-stripped to remove the stratum corneum by using 25 mm × 40 mm adhesive

145

cellophane tape (Nichiban, Tokyo, Japan) until the trans epidermal water

146

loss (TEWL) reached

147

room temperature and 40% relative humidity, every 2 days before skin damage and

148

every 6 h after tape stripping until it recovered to the normal level. Mice were

149

sacrificed by cervical dislocation under isoflurane anesthesia. The cecum of each

60

g/h/m2.15

TEWL

was

measured

8


under 20-22

°C

Page 9 of 35


150

mouse was immediately excised, and cecal content samples were quick frozen in

151

-80 °C until use.

152

Determination of short-chain fatty acids in cecal contents

153

Short-chain fatty acids in cecal contents of hairless mouse were determined by

154

gas chromatography with slight modifications.39 Briefly, 100 µL diethyl ether and 5

155

µL 35% HCl were added to 100 µL 15% cecal content homogenates containing 0.95

156

mM 2-ethylbutyric acid as an internal standard. After centrifugation at 1500×g at 4 °C

157

for 20 min, the diethyl ether layer (upper layer) was collected. The ether extract

158

sample (1 µL) was injected into a gas chromatograph (GC-14B, Shimadzu Co., Kyoto,

159

Japan) equipped with a DB-FFAP capillary column (15 m × 0.530 mm × 0.5 µm,

160

Agilent Technologies, CA, USA). Injector and detector temperatures were 145 °C and

161

175 °C, respectively. The initial oven temperature was 80 °C for 1 min, subsequently

162

increased at a rate of 10 °C/min, and then held at 130 °C for 1 min.

163

Statistical analysis

164

Data are reported as mean ± S.E. Statistical analyses were performed using

165

Student’s t-test to identify levels of significance between the control and the other

166

groups (p

Dietary Cerebroside from Sea Cucumber ... - ACS Publications

Recommend Documents