Dietary Cerebroside from Sea Cucumber - ACS Publications

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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

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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

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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

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

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

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

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

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

12

cucumber cerebroside to hairless mouse improved the skin barrier function and

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

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host.

15 16

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

17

sphingoid base

2


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Introduction

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

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

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

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

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

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cucumbers, and many other marine organisms.3,

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

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

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

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

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

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

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necessary to re-survey the sphingolipids in diet.

5-7

Sphingolipids of marine

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

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

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

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

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

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sphingolipids and modulators of sphingolipid metabolism affect raft related signaling

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pathways,

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

which

might

explain

their

biological

3


activities.

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Cerebroside,


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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function as a dietary component are still not well understood.

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

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

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

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

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Materials and Methods 4


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Analysis of cerebroside from sea cucumber Stichopus japonicus

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

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

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

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

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

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

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were applied to high-performance liquid chromatography with evaporative light

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

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

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

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

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

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

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analysis.

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

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

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

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

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

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

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



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

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

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

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

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

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

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

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

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conditions as we reported previously.36

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Analysis of the sphingoid base fraction prepared from sea cucumber cerebroside

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

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

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

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

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

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

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

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

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

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

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Evaluation of intestinal absorption of sea cucumber cerebroside

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


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

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the Animal Experiment Guidelines of Tohoku University. Surgeries and maintenance

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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solution was continued and subsequently lymph samples were collected in

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

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

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

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


36

. Briefly,


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

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sphingolipids were then subjected to HPLC for quantification analysis of total

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

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

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

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our previously study 36.

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

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

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

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

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

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

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

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

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

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

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

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

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loss (TEWL) reached

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

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

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

60

g/h/m2.15

TEWL

was

measured

8


under 20-22

°C

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

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-80 °C until use.

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Determination of short-chain fatty acids in cecal contents

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

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

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

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

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

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

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

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

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

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

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Statistical analysis

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

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

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groups (p

Dietary Cerebroside from Sea Cucumber - ACS Publications

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