Trace Nutrients Research 40 : 46−51（2023）

Original article

Structural analysis of Ceramide Aminoethyl Phosphonate Derived from
Pacific Oyster (Crassostrea gigas) By-productKoki Sugimoto1, 2）, Saki Itonori3）, Ryota Hosomi2）, Tatsuya Ishida4）, Hiroyuki Matsui4）,
Munehiro Yoshida2）, and Kenji Fukunaga2）
1）
Faculty of Food and Nutritional Sciences, Toyo University,
1-1-1, Izumino, Itakura-machi, Ora-gun, Gunma 374-0193, Japan
2）
Faculty of Chemistry, Materials, and Bioengineering, Kansai University,
3-3-35, Yamate-cho, Suita, Osaka 564-8680, Japan
3）
Faculty of Liberal Arts and Education, Shiga University, 2-5-1, Hiratsu, Otsu, Shiga 520-0862, Japan
4）
Central Research Institue, Japan Clinic Co., Ltd.,
Nishi-machi 1, Taishogun, Kita, Kyoto 603-8331, Japan（Received 29 August 2023; accepted 25 September 2023）

Summary
Oysters contain various nutritional and functional components, such as zinc, glycogen, and taurine. Therefore,oyster extract containing extracted and concentrated nutritional and functional components are marketed. However,
the effective use of oyster extract by-products (OEBP) needs to be realized. A previous study revealed that several
types of mollusc bivalves characteristically contain ceramide aminoethyl phosphonate (CAEP), a sphingophosphonolipid, that improves skin barrier function. In this study, we analyzed the chemical structure of CAEP in OEBP todifferentiate it from other marine CAEP. CAEP was purified by alkali and acid treatments, followed by three typesof column chromatography for Pacific oyster by-products. To analyze the chemical structure of CAEP, fatty acidcomposition analysis via methyl esterification and long-chain base composition analysis via trimethylsilyl derivatization
were performed. The results showed that 26% of OEBP-derived phospholipids were CAEP. Chemical structural analysis indicated that the fatty acid composition of the OEBP-derived CAEP was C16:0 (68%), C17:0 (5%), and C18:0(27%). Furthermore, the long-chain base composition of OEBP-derived CAEP was d16:1 (24%), d18:1 (13%), d18:2 (39%),
d20:1 (4%), and d20:2 (12%). These results indicate that most of the long-chain bases of marine CAEP reported todate have carbon numbers of ≤19, whereas Pacific oyster-derived CAEP characteristically contains long-chain baseswith carbon numbers of 20.

Introduction

tion of OEBP is desired from the perspective of environmental issues. Previous studies have found that the tria-

Oysters are known as a “milk of the sea” because theycylglycerol in OEBP contains approximately 10% n-3

contain various nutritional and functional components,

polyunsaturated fatty acids, such as eicosapentaenoic acidsuch as zinc, glycogen, and taurine. Oyster extract con-and docosahexaenoic acid5）. In addition, OEBP is expected

taining various (extracted and concentrated) nutritional

to contain other functional components.

and functional components has been marketed to derive

Scallops (Patinopecten yessoensis) and pearl oysters

optimal benefits of these components. It has been reported(Pinctada martensii), which are molluscan bivalves asthat the oyster-extract has the food functions related

well as oysters, characteristically contain ceramide amino-

platelet aggregation inhibition and hepatoprotective ef-

ethyl phosphonate (CAEP), a sphingophosphonolipid6, 7）.

fect1, 2）. In addition, our research group has reported theMoreover, some bivalve-derived CAEP are composed ofeffects of oyster extract on colorectal precancerous lesions

components such as hydroxy fatty acids and sphinga-

and renal injury in animal studies. However, the oyster

trienine, which are not found in common sphingolipids.

extract by-product (OEBP) is discarded, although it con-Marine-derived CAEP improves skin barrier function6）. In

tains high concentrations of lipids and proteins, which are

addition, the health-promoting functions of sphingolipids

the main components of oyster meat. The effective utiliza-

are dependent on their chemical structure8）. Therefore, we

3, 4）― 46 ―

analyzed the chemical structure of CAEP in OEBP withsphingolipid fraction was fractionated into chloroform/

the aim of making effective use of OEBP in the future.

methanol/water (C/M/W) and ammonium acetate fractions using TOYOPEAL QAE-550 (Tosoh Co., Tokyo,

Materials and Methods

Japan) column chromatography. Elution from the column
was performed using 5-volume of C/M/W (30:60:8) andMaterials

equal-volume of methanol as the neutral solvent and 5-vol-

OEBP derived from the Pacific oyster (C. gigas) wasume of 0.05 M ammonium acetate–methanol solution asprovided by Japan Clinic Co., Ltd. (Kyoto, Japan); it was

the polar solvent. Elution of 0.05 M ammonium acetate

composed of 63.7 wt% moisture, 22.6 wt% crude protein,fraction was concentrated and dialyzed in running water

9.6 wt% crude fat, and 3.3 wt% crude ash. OEBP used infor 2 days, followed by TOYOPEAL DEAE-650 (Tosoh

the experiments were boiled and dehydrated in acetone.

Co.) column chromatography. The fractions that passed

During this process, the acetone layer was extracted tothrough the column in 5-volume of C/M/W (30:60:8) werethe extent that no coloration was observed; thus, ace-collected, concentrated, and subjected to silica gel column

tone-soluble fats and pigments were also removed.

chromatography. Fractions were collected over-time while

The standard CAEP was a scallop-derived substanceC/M (1:1) was flowed through the column, and fractions

prepared independently in our laboratory. Other chemicals

containing with the same Rf values as the standard CAEP

were purchased from Nacalai Tesque, Inc. (Kyoto, Japan);by thin-layer chromatography (TLC) were concentrated to

Merck KGaA (Darmstadt, Germany); and Tokyo Chemical

purify the OEBP-derived CAEP.

Industry Co., Ltd. (Tokyo, Japan).
Phospholipid class analysis
CAEP purification

TLC and infrared absorption (IR) spectral analyses

CAEP was purified according to the method described

were performed to confirm the phospholipid class. The

by Sugita et al. , with some modifications (Fig. 1). In brief,

TLC plate used was silica gel 60 (Merck KGaA), andthe crude complex lipid fraction was extracted from OEBP

C/M/W (60:30:5) was used as the developing solvent.

using a chloroform–methanol (C/M; 2:1) mixture, and the

Spots were detected with 50% (v/v) sulfuric acid and nin-solvent was eluted under reduced pressure (C/M extract

hydrin solutions and identified using standard phospholip-

in Fig. 1). The C/M extract was stirred in acetone, and theids [CAEP, phosphatidylcholine (PC), and phosphatidyleth-insoluble powder was collected as the phospholipid frac-anolamine (PE)]. The phospholipid class composition wastion. To decompose and remove glycerophospholipids, al-calculated based on spot intensity using JustTLC software

kaline saponification and acid treatment (pH = 1) were(version 4.0.3; Lund, Sweden). IR spectra of the OEBP-

performed, and the solution was dialyzed under runningderived CAEP were measured using a Shimadzu Fourier-water for 2 days. The solution in the dialysis membranetransform

was concentrated and acetone was added to prepare an(Shimadzu Co., Kyoto, Japan).

6）

infraredacetone-insoluble powder (sphingolipid fraction). The

321
322

Fig.1.1 Scheme for preparation and fractionation of CAEP from OEBP
Fig.
― 47 ―

spectrophotometer

FTIR-8400 S

MALDI-TOF MS analysis

Fatty acid composition analysis
Two hundred µg of OEBP-derived CAEP was added to
1 M methanol hydrochloride and heated at 100 ℃ for 3 h.

OEBP-derived CAEP was dissolved in 500 µL of metha-

After cooling, fatty acid methyl esters were extracted

nol, and n-acetylation was performed by adding 10 µL ofthree times with 0.2 mL n-hexane and analyzed by gas

pyridine and 50 µL of acetic anhydride for 30 min at room

chromatography (GC) system (GC-2014; Shimadzu Co.) andtemperature. The acetylated CAEP and matrix (α-cya-

GC-mass spectrometry (MS) system (GCMS-QP2010/no-4-hydroxycinnamic acid) were dried on slides and sub-PARVUM2; Shimadzu Co.), equipped with Omegawax®

jected to MALDI-TOF MS (AXIMA® ConfidenceTM;

capillary GC column (cat no. 24152, Merck KGaA). The GC

Shimadzu Co.) analysis. A nitrogen laser (laser wave-

system parameters were set as follows: injector and inter-

length: 377 mm) was used as the ion source, and measure-face temperatures, 250 ℃; column temperature, 120 ℃;

ments were performed in negative-ion mode.

gradual heating to 240 ℃ at a rate of 2 ℃/min, held at

Results and Discussion

240 ℃ for 10 min; and carrier gas, 1 mL/min helium. MS
system parameters were set as follows: ion source temperature, 200 ℃; electronic ionization(EI), 70 eV; full scan

CAEP purification

m/z 45–350 amu. Fatty acid composition was determined

As shown in Fig. 1, 42.3 g of C/M extract and 9.0 g of

by GC analysis, and peak compounds were identified byphospholipid fraction were obtained from 645 g of OEBP.

GC-MS analysis.

TLC results for the phospholipid fraction are shown in
Fig. 2. This fraction comprised 30.7% PC, 26.0% CAEP,Long-chain base composition analysis

and 8.5% PE (Table 1).

Five hundred µg of OEBP-derived CAEP was added to

In addition, 0.9 g of CAEP was purified from 9.0 g of

0.2 mL of 8.6% aqueous methanol hydrochloric acid andphospholipid fraction by this experimental purification

heated at 70℃ for 18 h . After cooling, the fatty acid meth-method. The TLC results for OEBP-derived CAEP areyl esters were removed three times with 0.2 mL n-hexane,

shown in Fig. 3. CAEP is colored by ninhydrin and sulfu-

and methanol was removed from the remaining solution.

ric acid because it contains lipid-containing amino groups.

Then, 0.6 mL of 1 M sodium hydroxide:methanol (3:4, v/v)

The Rf value of the extract was also compared with thatand 0.72 mL of chloroform were added, stirred, and centri-of the standard CAEP, and it was concluded that CAEP

fuged to separate into two layers. The chloroform layer

was purified from OEBP. The reason for the presence of9）

was washed with 0.4 mL of water/methanol (1:1, v/v) andconcentrated under nitrogen to obtain the long-chain base
fraction. Trimethylsilyl-diazomethane was added to theresulting residue and heated at 60 ℃ for 30 min, and thetrimethylsilyl derivative was subjected to GC and GC-MS
analyses to measure long-chain base composition and identify peak compounds, respectively. The analytical column
was nonpolar 5% phenylmethyl silicon chemically bonded(film thickness, 0.25 µm) silica capillary (Shimadzu HiCapCBP 5). The GC system parameters were set as follows:
injector and interface temperatures, 250 ℃; column temperature, 170 ℃; gradual heating to 240 ℃ at a rate of
4 ℃/min, held at 240 ℃ for 20 min; and carrier gas, 1 mL/
min helium. MS system parameters were set as follows:
ion source temperature, 200 ℃; electronic ionization(EI),
70 eV; full scan m/z 45–500 amu.

323
Fig. 2. T hin-layer chromatogram of the phospholipid fraction
324
Fig. 2from the Pacific oyster by-product
Lane 1: phospholipid fraction prepared from OEBP; lane
2: standard CAEP. Plate was developed in chloroform/
methanol/water (60:30:5, v/v/v), and the spots were visualized using 50% (v/v) sulfuric acid solution (A) andninhydrin solution (B).
OEBP, oyster extract by-product.
― 48 ―

Table 2. Ceramide composition of OEBP-derived CAEP
Peak

(wt%)
Fatty acid

A
B
C

C16:0
C17:0
C18:0
Long-chain base
d16:1
d18:1
d18:2
d20:1
d20:2
Unknown

A
C
B
F
E
D

67.7
5.2
27.2
25.3
13.3
41.2
4.2
12.3
3.6

Peaks a–c (fatty acids) and a–f (long-chain bases) in Table 2 correspond to the peaks shown in Fig. 5 A and 5B, respectively.
CAEP, ceramide aminoethyl phosphonate; OEBP, oyster extractby-product.

325
Fig. 3. Thin-layer chromatogram of OEBP-derived CAEP
326
Fig. 3
Lane 1, standard CAEP; lane 2, OEBP-derived CAEP.
Plate was developed in chloroform/methanol/water(60:30:5, v/v/v), and the spots were visualized using 50%(v/v) sulfuric acid solution (A) and ninhydrin solution (B).
CAEP, ceramide aminoethyl phosphonate; OEBP, oyster
extract by-product.

327
328

Fig. 4. Infrared spectra of OEBP-derived CAEP
Fig. 4329

Table 1. Phospholipid class of OEBP-derived phospho19
lipids fraction
Phospholipid class (wt%)
PE
CAEP
PC
Others

330

30.7
26.0
8.5
34.8

CAEP, ceramide aminoethyl phosphonate; OEBP, oyster extractby-product; PC, phosphatidylcholine; PE, phosphatidylethanolamine.

Fig. 5. Gas chromatograms of fatty acid (A) and long-chain baseFig. 5 (B) of OEBP-derived CAEP
A: Fatty acid methyl esters of OEBP-derived CAEP; a,
C16:0; b, C17:0; c, C18:0.
B: trimethylsilylated long-chain bases of OEBP-derived
CAEP; a, d16:1; b, d18:2; c, d18:1; d, unknown; e, d20:2; f,d20:1.
CAEP, ceramide aminoethyl phosphonate; OEBP, oyster
extract by-product.

two spots of CAEP with different Rf values on TLC could
be the difference in the composition ratio of fatty acids andlong-chain bases that compose the ceramides6）.
IR spectra of OEBP-derived CAEP are shown in Fig. 4.
The OEBP-derived CAEP showed absorption at 1180 cm−1
and 1050 cm−1 from the P=O group derived from the phosphate group of the C–P bond and at 1650 cm−1 and1550 cm−1 from amide bonds I and II. These results also

― 49 ―

confirmed that the substance purified from OEBP waschain length.
Moreover, it has been suggested that the chemical

CAEP.

structures of ceramides are similar in the same taxonomic
Ceramide components of CAEP

order7）. Although further information is not available on

The fatty acid and long-chain base comprising the cera-

CAEP in the order Oysteri, to which the Pacific oyster

mide of OEBP-derived CAEP were identified using bothbelongs, it is possible that CAEP in other bivalves belong-

GC and GC-MS analyses. The compositions and chromato-

ing to the order Oysteri also have a chemical structure

grams of fatty acids and long-chain bases obtained by GC

similar to that mentioned in the present results.

analysis are shown in Table 2 and Fig. 5, respectively. The
MS spectrum of fatty acid methyl esters was character-

MALDI-TOF MS analysis

ized by 74 and molecular ion peaks −31 and −43 . Based

There are two measurement modes for MALDI-TOF

on this information, the fatty acid composition of the

MS: positive-ion mode and negative-ion mode, depending

OEBP-derived CAEP was identified as C16:0 (67.7%), C17:0

on the functional group of the analyzed sample. For mea-

(5.2%), and C18:0 (27.2%) (MS spectra not shown). Grand etsurement of lipids with acidic functional groups in the mol-al. reported that the Pacific oyster (C. gigas)-derived

ecule of the sample to be measured, the ionization efficien-

CAEP contained few fatty acids with 20 or 22-carbon

cy is extremely poor in the positive-ion mode7）. In addition,

chains, such as C20:5 and C22:611）. However, no such fatty

it is effective for analyzing phospholipids with free aminoacids were detected in the OEBP-derived CAEP. It is un-groups such as N-acetyl derivatives12）. Therefore, forclear whether this is due to differences in the production

OEBP-derived CAEP, MALDI-TOF MS analysis was per-areas of Pacific oyster or their decomposition during theformed in negative-ion mode after N-acetylation of free

OEBP purification process, so further research is needed

amino groups in the molecule. The MS spectra are shown

to clarify these.

in Fig. 6. Forty-two masses (CH3COO = 42) of more spec-

10）

In addition, the long-chain base composition was identi-

tra based on each molecular species [M-H] were observed

fied from the GC and GC-MS data as a trimethylsilyl de-after N-acetylation, corresponding to the major compo-

rivative. In a previous study, it was found that alkaline

nents of the ceramides shown in Table 1. The main spec-hydrolysis is necessary when the 9-methyl homologue oftra were as follows: a, m/z 656.71 (16:0 fatty acid-d16:1

the 4, 8, 10-trienine base is present in the base composi-

long chain base, calculated value 656.49); b, m/z 670.59

tion . However, OEBP-derived CAEP does not contain a7）

9-methyl homologue of the 4,8,10-trienine base; therefore,
only acid hydrolysis was performed. The MS spectrum of
trimethylsilylated long-chain bases was characterized by
73 and 132 and molecular ion peaks −105 and −132. As
shown in Table 2, the long-chain base composition of
OEBP-derived CAEP was d16:1 (25.3%), d18:1 (13.3%),
d18:2 (41.2%), d20:1 (4.2%), and d20:2 (12.3%). Peak (d) in
Fig. 5 shows the MS spectrum characteristics of a longchain base; however, the chemical structure of this compound could not be determined because the molecular ion
peak was not detectable. The long-chain bases of OEBPderived CAEP were mostly composed of d16:1 and d18:2,
331
which are commonly found in bivalve-derived CAEP.
332
However, the long-chain bases of OEBP-derived CAEP
were also composed of d20:1 and d20:2. Most of the longchain bases of marine-derived CAEP found to date arecomposed of up to 19-carbon chain, and few long-chain
bases with 20-carbon chains have been discovered. The
results of this study indicated that OEBP-derived CAEP
characteristically contains long-chain bases with 20-carbon chains. Further investigation is required to determine
the factors responsible for these differences in the carbon

Fig. 6. N egative-ion mode of matrix-assisted laser desorption/
Fig. 6ionization time-of-flight mass spectrometry spectra ofN-acetylated OEBP-derived CAEPa, m/z 656.71 (16:0 fatty acid-d16:1 long chain base, calculated value 656.49); b, m/z 670.59 (17:0-d16:1, 670.50); c,
m/z 682.49 (16:0-d18:2, 682.52); d, m/z 684.46 (16:0-d18:1
and 18:0-d16:1, 684.52); e, m/z 696.34 (17:0-d18:2, 696.53);
f, m/z 698.33 (17:0-d18:1, 698.53); g, m/z 710.21 (16:0d20:2 and 18:0-d18:2, 710.55); h, m/z 712.22 (16:0-d20:1
and 18:0-d18:1, 712.55); i, m/z 724.09 (17:0-d20:2, 724.57);
j, m/z 726.12 (17:0-d20:1, 726.57); k, m/z 739.94 (18:0d20:2, 738.58); l, m/z 741.90 (18:0-d20:1, 740.58).
CAEP, ceramide aminoethyl phosphonate; OEBP, oyster
extract by-product.

― 50 ―

(17:0-d16:1, 670.50); c, m/z 682.49 (16:0-d18:2, 682.52); d,product Decreases Triacylglycerol Contents by

m/z 684.46 (16:0-d18:1 and 18:0-d16:1, 684.52); e, m/z

Suppressing Acetyl-CoA Carboxylase Activity and696.34 (17:0-d18:2, 696.53); f, m/z 698.33 (17:0-d18:1, 698.53);

Lowering the Stearoyl-CoA Desaturase Index in Rat
Livers. Trace Nutrients Research 32: 20-26.

g, m/z 710.21 (16:0-d20:2 and 18:0-d18:2, 710.55); h, m/z712.22 (16:0-d20:1 and 18:0-d18:1, 712.55); i, m/z 724.09

6)	 Sugita M, Itonori Saki, Makita A, Higashino A,(17:0-d20:2, 724.57); j, m/z 726.12 (17:0-d20:1, 726.57); k,Nozaki H, Kojima H (2008) Structural Analysis ofm/z 739.94 (18:0-d20:2, 738.58); l, m/z 741.90 (18:0-d20:1,

Phosphonolipids of the Giant Ezo Scallop Patinopecten

740.58), confirming the presence of the spectra attributed

yesseoensis. Memoirs of the Faculty of Education,
Shiga University, Natural Science 58: 21-29.

to the product. These results indicated the presence of
CAEP molecular species with all fatty acids and long-7)	 Itonori S, Kitamura T, Tanaka R, Miyagaki N, Saito
H, Sugita M (2006) Aliphatic Components of Phos-chain bases listed in Table 1.
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from the Pearl Oyster, Pinctada martensii. Memoirs

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Intestinal Absorption of Cholesterol and Fat in Rats.
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― 51 ―