Journal of Oleo Science
Copyright ©2020 by Japan Oil Chemists’ Society
doi : 10.5650/jos.ess20050
J. Oleo Sci. 69, (7) 751-757 (2020)

Polar Lipid Fraction from Golden Oyster Mushrooms(Pleurotus citrinopileatus) Suppresses Colon Injuries
from Inflammatory Stresses in vivo and in vitro
Shinji Yamashita1, Takuya Seino1, Miho Inobe1, Mirinthorn Jutanom1,
Satoshi Matsumoto2, and Mikio Kinoshita1＊
1
2

Department of Life and Food Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, JAPANL/S Corporation, 5F, Kyodo Bldg. 13-4 Nihonbashi Kodenma, Chuo-ku, Tokyo 103-0001, JAPAN

Abstract: The rising incidence of inflammatory bowel disease (IBD) in East Asian countries has necessitatedthe implementation of preventive methods in the form of dietary supplementation and changes in dietary
habits. We have previously reported that dietary golden oyster mushroom (Pleurotus citrinopileatus) ethanol
extract (GOMEE) suppresses intestinal inflammation in mouse models of IBD induced by dextran sulfate
sodium salt (DSS). Here, we investigated the components of GOMEE that exert suppressive effects on colon
inflammation in vivo and in vitro. The total lipid fraction was extracted from GOMEE, and the polar andneutral lipid fractions were subsequently separated via solvent fractionation. Mice were assigned to dietarygroups—control, 1% total lipid, 1% polar lipid, or 1% neutral lipid diet—and fed the respective diets for
one week; mice were administered 1.5% DSS in drinking water ad libitum for 20 days. Dietary
supplementation with the total or polar lipid fraction alleviated DSS-induced chorionic crypt injury as
determined by morphological observation, while dietary supplementation with the neutral lipid fraction did
not produce such effects. In the in vitro study, using differentiated Caco-2 cells as the colon model, treatment
with the total or polar lipid fraction suppressed cell decrease by lipopolysaccharide (LPS)-induced apoptosis
whereas treatment with the neutral lipid fraction did not. Moreover, accumulation of glucosylceramide(GlcCer), a fungal sphingolipid, was observed in the intestinal cells after treatment with polar lipid fraction.
These results suggest that the active components of GOMEE that suppress colon inflammation are polar
lipids, especially GlcCer. The structure of mushroom GlcCer differs from that of the plant counterpart andis therefore expected to exert different food functions.
Key words: apoptosis, colitis, inflammation, mushroom, polar lipid, sphingolipid
1 Introduction
The incidence of intestinal impairments, such as coloncancer and inflammatory bowel disease（IBD）, is increasingin East Asian countries, while in the Western countries it
remains high1, 2）. Complete recovery from IBD is difficult,and patients with this disease are at an increased risk ofdeveloping colon cancer3）. We have previously suggested
that the induction of inflammation-related cytokines has aconnection with the formation of aberrant crypt foci
（ACF）,
which are precursors of colon cancer4）. Epidemiologicalstudies indicate that colon cancer development is strongly
associated with diet and thus, diseases related to inflammation in the colon can be prevented through dietary supplementation and changes in dietary habits5）.

Pleurotus citrinopileatus, commonly known as thegolden oyster mushrooms has recently become a popular
delicacy in East Asia due to its flavor and health benefits.
This mushroom and its extract have been shown to exert
various biological effects6−10）. We have previously reported
that dietary golden oyster mushroom ethanol extract（GOMEE）
suppresses intestinal injury in a mouse model of
IBD and that it is rich in sphingolipids, including glucosyl11）
ceramide（GlcCer）.
GlcCer is found on the eukaryotic cell membrane, mostly
in plants and fungi12）. The GlcCer molecule possesses asphingoid base with an amide-linked fatty acid（i.e., a ceramide）and glucose. Dietary GlcCer from plant sources isreported to alleviate colon inflammation in the IBD mouse＊

Correspondence to: Mikio Kinoshita, Department of Life and Food Science, Obihiro University of Agriculture and Veterinary
Medicine, Obihiro, Hokkaido 080-8555, JAPANE-mail: kinosita@obihiro.ac.jp
Accepted March 29, 2020 (received for review February 20, 2020)

Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
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S. Yamashita, T. Seino, M. Inobe et al.

models13）and suppress colon ACF formation and inflammation-related cytokine production in the mouse models ofcolon cancer4）. In vitro experiments indicate that GlcCer
protects the colon surface from the damage caused byvarious drugs14）.
The sphingolipid composition of mushrooms has beenreported to differ from those of plants and mammals 15）.
The major sphingoid base in GlcCer is a 9-methyl-trans4t,8t
4,trans-8-sphingadienine（9-Me d18:2 ）, which is foundonly in fungi such as mushrooms. Moreover, the main sterolin mushrooms is ergosterol, which is not found in plants or
mammals16）.
In this study, we investigated the components in GOMEE
that exert suppressive effects on colon inflammation. The
total lipid, polar lipid, and neutral lipid fractions were prepared from GOMEE. These lipid fractions were supplemented in the diets of mice with colitis induced by dextran
sulfate sodium salt（DSS）（mode of colitis）and used to treat
intestinal cells stimulated with lipopolysaccharide（LPS）（model of intestinal tract inflammation）.

2 Experimental Procedures
2.1 Preparation of lipid fraction from GOMEE
GOMEE was purchased from Three-B Corporation（Sapporo, Japan）. The total lipid fraction was extracted
from GOMEE using the Folch method and separated to thepolar lipid and the neutral lipid fractions by solvent frac17）
tionation with cold acetone（Fig. 1A）.
2.2 Animals and their diet
Male BALB/c mice were obtained from Japan SLC, Inc.

Fig. 1（Shizuoka, Japan）at 4-week old and housed in isolator
cages at 22℃ under a 12 h light/dark cycle. Mice were provided with tap water and CE-diets（ CLEA Japan, Inc.,Tokyo, Japan）ad libitum. After acclimation for one week,
the mice were randomly divided into five groups（n＝5）.
The blank and control groups were fed a control diet basedon AIN-76, which does not contain sphingolipids13）. Groupsof total lipid, polar lipid, or neutral lipid were each fed theAIN-76 diets containing 1％ total lipid, 1％ polar lipid, or
neutral lipid. After following the experimental diet for one
week, mice, excluding those in the blank group, were administered 1.5％ DSS in drinking water. The mice were
killed and dissected after 21 days. All protocols involving
the animals were approved by the Animal Care and Use
Committee and were conducted in accordance with the
Obihiro University Guidelines（Permit Number, 29-196）.
2.3 Histological analysis
The large intestines of the mice were excised under anesthesia using sodium pentobarbital（50 mg/kg BW, i.p.）,and the portion from the cecum to the vent was removed
and rinsed with cold saline. This was followed by an overnight cell fixation with 4％ paraformaldehyde in phosphate-buffered saline. The specimens were subsequently
embedded in paraffin wax, and transverse sections were
cut and stained with hematoxylin and eosin11）.
2.4 Cell culture
Caco-2 cells, obtained from Riken Gene Bank（Tsukuba,
Japan）, were maintained in DMEM supplemented with
10％ FCS, 2 mM glutamine, and 0.1 mM nonessential aminoacids. For routine maintenance, Caco-2 cells on one dishwere trypsinized and passaged to three new dishes, andPreparation（A）and profiles（B）of lipid fractions from golden oyster mushroom ethanol extract.
1. Maize GlcCer fraction; 2. Total lipid fraction from GOMEE; 3. Hydrophilic fraction from GOMEE; 4. Polar lipid
fraction; 5. Neutral lipid fraction. Mobile phase, Chloroform/Methanol（95/12, v/v）
; Detection, 50％ H2SO4. TLC, thinlayer chromatography; GlcCer, glucosylceramides; SG, sterylglycosides; FFA, free fatty acids.

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Mushroom Polar Lipids Suppress Colon Injuries

then maintained in culture to reach confluency. For spontaneous differentiation, the time at which the cells initiallyreached confluency, as determined by light microscopy,
was designated as day 0, and the cells were transfected 21days later.
2.5 Cell countCaco-2 cells were seeded at a density of 2.0×105 cells/
mL in 24-well plates（ Nunc, Rochester, NY）in 1 mL ofculture medium and incubated for 21 days at 37℃ and 5％
CO2. The medium was then replaced with the test medium
containing 0.1％ BSA instead of 10％ FCS. LPS（50 μg/mL）
as an inflammatory stimulator and lipid fractions wereadded for 48 h. After stimulation, cells were rinsed with
PBS
（Nissui, Tokyo, Japan）, trypsinized, and counted using
a counting chamber（improved Neubauer chamber, EMTechcolor; Hirshmann, Eberstadt, Germany）.
2.6 Apoptosis assay
Caco-2 cells were seeded at a density of 1.0×105 cells/
mL on Lab-TekTM Chamber Slides（ Thermo Scientific,
Waltham, MA）in 0.5 mL of culture medium, and subsequently treated as described above. Apoptotic cells weredetected by characteristic fragmented nuclei with 4 ,6-diamidino-2-phenylindole（DAPI）staining, under a fluorescence microscope14）.
2.7 Thin-layer chromatography（TLC）
analysis
To determine the movement of exogenous lipid fractions,
TLC analyses of cells, cell surface, and medium were conducted14）. Briefly, after 48-h cell incubation with 50 μM ofeach lipid fraction in medium or medium only, the total

Fig. 2

lipids in cells, on the cell surface, and in the medium wereextracted. The alkaline-stable lipid fraction was preparedand subjected to TLC with 95:12（v/v）chloroform/methanol
as the solvent.
2.8 Statistical analysis
Differences among all data groups were assessed byusing one-way ANOVA with Fisher s least significant difference test; P values of ＜0.05 were considered to be statistically significant. All data were analyzed using BellCurve for
Excel（Social Survey Research Information Co., Ltd., Tokyo,
Japan）and expressed as means±standard error of themean（SEM）.

3 Results
3.1 Profiles of lipid fractions from GOMEE
The total lipid fraction was obtained at 75 wt％ of
GOMEE and was divided to 25 wt％ polar lipid fraction and
49 wt％ neutral lipid fraction of GOMEE（33 wt％ and 65
wt％ of the total lipid fraction, respectively）. Lipid composition of each GOMEE fraction was determined by TLC（Fig. 1B）. The major components of the polar lipid fraction
were GlcCer and sterylglycoside（ SG）derivatives, while
those of the neutral lipid fraction were free and acyl
sterols, free fatty acids（FFA）, ceramides, and SG derivatives.
3.2 Effect of dietary lipid fractions from GOMEE on
weight gain and other factors by DSS
Figure 2A shows the weight gain of mice during the ex-Effects of dietary lipid fractions on weight gain（A）and large intestinal villi（B）
in DSS-treated mice.
After feeding experimental diets for one week, mice were administered 1.5％ DSS in drinking water for 20 days anddissected on day 21. The colons were stained with hematoxylin and eosin. 1. Blank（non-treated with DSS）; 2.
Control; 3. Total lipid group; 4. Polar lipid group; 5. Neutral lipid group. Mean±SEM（n＝5）. a,bValues with different
superscript letters in the same column differ significantly（p＜0.05）. Body weight of each mouse at 0 day starting todrink DSS＝1.0. B. ×10 objective magnification. Scale bar＝100 μm. DSS, dextran sulfate sodium salt.
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S. Yamashita, T. Seino, M. Inobe et al.

perimental period. Before DSS administration, body weightof animals in all groups increased daily. Although the addition of 1.5％ DSS to the drinking water did not affect bodyweight for 12 days of treatment, body weights of thecontrol group tended to decrease in thereafter. The bodyweights of mice from the groups administered the dietary
lipid fractions of GOMEE were not significantly different
from those of animals in the control group; however,animals in the dietary neutral lipid fraction-supplemented
group showed significantly decreased body weight compared with that of blank animals.
In general, acceleration of colon inflammation inducedcolon constriction and swelling in the spleen. DSS administration significantly reduced the length of the large intestine and increased the spleen weight, and dietary lipid
fractions of GOMEE did not significantly suppress thesefactors by DSS（ data not shown）. Because the dietary
neutral lipid fraction reduced the body weight of DSStreated mice at an early stage, the DSS administration
period in this study was shorter than the 26-day period in
our previous study on GOMEE11）and there was no significant difference in the colon length or the spleen weightFig. 3among groups treated with DSS.
3.3 Effects of dietary lipid fractions of GOMEE on colon
villus damage by DSS
To observe the status of the colon, we examined specimens under a microscope following hematoxylin and eosin
staining（Fig. 2B）. The blank group showed normal crypts
in the mucosa layer, whereas the control group showedmucin depletion and the loss of epithelial cells due to DSS
administration. Supplementation with the dietary total orpolar lipid fraction from GOMEE alleviated injury to thechorionic crypts associated with DSS administration, while
supplementation with dietary neutral lipid fraction did not.
3.4 Effects of lipid fractions of GOMEE on LPS-induced
cell number decrease and apoptosis of intestinal cells
Under the culture conditions used herein, Caco-2 cells
were adherent. Therefore, to determine cell viability andinflammatory stress, the numbers of adherent cells wereassessed. Addition of LPS as an inflammation inducer decreased the number of adherent cells, and treatment withthe total or polar lipid fraction of GOMEE suppressed thisEffects of lipid fractions from GOMEE on the viability（A）and apoptosis（B and C）
of differentiated Caco-2 cells under
LPS-induced inflammation.
After cultivation for 48 h in the presence of LPS and/or 50 μM of each lipid fraction, the number of viable cells werecounted（A）, and apoptotic bodies（characteristic of apoptosis initiation）were observed following DAPI staining（B）
and counted（C）. 1. Blank（non-treated with LPS）
; 2. Control; 3. Total lipid group; 4. Polar lipid group; 5. Neutral lipid
group. A & C. Mean±SEM（n＝4）. a,b,c,dValues with different superscript letters in the same coulmn differ significantly（p＜0.05）. Blank＝1.0. B. ×20 objective magnification. Scale bar＝50 μm. LPS, lipopolysaccharide; DAPI,
4 ,6-diamidino-2-phenylindole.

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Mushroom Polar Lipids Suppress Colon Injuries

decrease（Fig. 3A）. Treatment with the neutral lipid fraction did not significantly suppress the LPS-induced cellnumbers decrease. Without LPS treatment, the cellnumbers in the group treated with the neutral lipid fraction
were also lower when compared to the blank group（data
not shown）.
Induction of the apoptotic pathway leads to the formation of apoptotic bodies characterized by cell fragmentation
around the nucleus. Cells in the blank group had normal
sized nucleus, whereas the control group, treated with
LPS, had normal and small nuclei（Figs. 3B and C）. The
total or polar lipid fraction of GOMEE suppressed the formation of small nuclei, while the neutral lipid fraction did
not. Without the presence of LPS, treatment with theneutral lipid fraction induced the formation of small nuclei
and the numbers of apoptotic cells significantly increased
about fourfold（data not shown）.
3.5 Accumulation of GlcCer in differentiated Caco-2 cells
incubated with lipid fractions determined by TLC
To confirm localization of lipid components in additional
lipid fractions, Caco-2 cells were divided to the medium,surface, and cell parts at 48-h after the addition of eachlipid fraction. In the medium and cell parts, mushroom
GlcCer from GOMEE was detected after the addition ofpolar lipid fraction（Fig. 4）. Mushroom GlcCer was also detected in the medium and cell parts after the addition of
total lipid fractions, but it was not observed in all the parts
after the addition of the neutral lipid fraction（data notshown）.

Fig. 44 Discussion
Dietary golden oyster mushrooms and its hydrophilic
fraction are known to exert several food functions6−11）. We
have previously reported that dietary GOMEE, which ismushroom lipophilic fraction, alleviated intestinal injury inmice with DSS-induced colitis via suppression of intestinal
inflammation. In this study, we investigated the components of GOMEE that exert the suppressive effects oncolon injuries from inflammatory stresses in vivo and invitro. The polar lipid fraction of GOMEE alleviated DSSand LPS-induced colon injuries in vivo and in vitro, respectively. However, the neutral lipid fraction did not exert
suppressive effects in vivo or in vitro, but instead affected
the mice and the cells adversely（Figs. 2, 3, and not showndata）.
According to TLC analysis, the major component of thepolar lipid fraction was GlcCer, whereas those of theneutral lipid fraction were FFA and ceramides（Fig. 1B）.
GlcCer from plants has been reported to suppress intestinal inflammation in vivo and in vitro13, 14）. In this study,
the polar lipid fraction containing GlcCer also suppressed
intestinal inflammation in vivo and in vitro（Figs. 2 and 3）.
4t,8t
However, the main sphingoid base（9-Me d18:2 ）of mushroom GlcCer is different from that of plants, and the specific mushroom GlcCer was reported to exert different
effects from plant GlcCer on the fluidity of phospholipid liposomes18）. GlcCer from golden oyster mushrooms localized in the cell portion（Fig. 4）, while wheat GlcCer, which
8c
as the main sphingoid base,has 8-cis-sphingenine（d18:1 ）localized in the cell surface portion14）. Therefore, GlcCer
from golden oyster mushrooms may have a food function
different from that of plant GlcCer.
In contrast, it is well-known that high level of FFA is cy-

Accumulation of sphingolipids in differentiated Caco-2 cells incubated with polar lipid fraction determined by TLC.
Cells were cultured for 48 h in the presence of 50 μM polar lipid fraction, and then fractionated（3, 4, and 5）. Mobile
phase, Chloroform/Methanol（95/12, v/v）; Detection, 50％ H2SO4. 1. Wheat GlcCer standard; 2. Medium incubatedwith polar lipid fraction of GOMEE; 3. Medium incubated with Caco-2 cells and polar lipid fraction; 4. Surface of thecells incubated with polar lipid fraction; 5. Cells incubated with polar lipid fraction; 6. Medium incubated with cells; 7.
Surface of cells; 8. Cells.
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S. Yamashita, T. Seino, M. Inobe et al.

totoxic19）and ceramide is an apoptotic signal in cells20, 21）. In
addition, sphingosine-1-phosphate（S1P）, a ceramide metabolite, is thought to be a therapeutic target for colitis
because the inhibition of sphingosine kinase 1, which generates S1P from ceramides, reduces immune responses in
DSS-induced colitis 22, 23）and the deficiency of ceramide
synthase 6 protects against the development of colitis
induced by an adoptive transfer method24）. However, when
extrinsic ceramides are added to cells to induce apoptosis,
short-chain ceramides（C2 to 8）are used because ceramides usually have a chain length of C18 to 26 and low cellpermeability25）. Therefore, it is thought that ceramides in
the neutral lipid fraction, made only marginal contributions
to the progression of intestinal inflammation. However, it isreported that a small part of long-chain ceramides canenter the intestinal cells26）; in addition, intestines treatedwith DSS have increased nonspecific cell permeability.
Moreover, a part of ceramides is digested to sphingoidbases by the digestive enzymes and enteric bacteria, which
induce apoptosis in weak cells（ e.g. cancer cells anddamaged cells）27）. Overall, we think that an overdose of
FFA and ceramides in the neutral lipid fraction adversely
affected the intestinal condition.
In conclusion, the total lipid or polar lipid fraction of
GOMEE exerts anti-inflammatory effects in vivo and invitro. These results suggest that the active contents in
GOMEE in the suppression of colon inflammation are polar
lipids, especially GlcCer. The structure of mushroom
GlcCer is different from that of the plant counterpart andis therefore expected to exert different food functions.

Acknowledgement
We would like to thank Dr. Yoshihiko Tokuji, Departmentof Human Science, Obihiro University of Agriculture andVeterinary Medicine, for helping with the preparation of
microscopic slides.

Conflict of Interest
The authors declare no conflict of interest.

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