Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : August 3, 2023
Accepted : September 5, 2023
Published online : September 30, 2023
doi:10.24659/gsr.10.3_94

Original article

Diverse effects of single molecules of rice-derived functional lipids,
glucosylceramides, ceramides, and β-sitosterol glucoside,on epidermal and lung functions
Hiroshi Shimoda 1), Shogo Takeda 1), Kenchi Miyasaka 1), Akari Yoneda 1),
Yoshiaki Manse 2), Toshio Morikawa 2, 3)
1) R&D, Oryza Oil & Fat Chemical Co., Ltd., Aichi, Japan
2) Pharmaceutical Research and Technology Institute, Kindai University, Osaka, Japan3) Antiaging Center, Kindai University, Osaka, Japan

Abstract

Glucosylceramides (GlcCer), ceramides (Cer), and β-sitosterol glucoside (BSG) are produced in the refining process of ricebran oil. GlcCer are used for functional foods that claim to promote skin barrier functions and moisturization. However,
the efficacies of single molecules of GlcCer, Cer, and BSG remain unclear. Therefore, we herein examined the effects ofrice-derived GlcCer, Cer, and BSG on skin. The anti-inflammatory effects of BSG in a mouse pneumonia model were also
investigated. Skin barrier effects were assessed based on transepidermal water loss (TEWL) in a reconstructed humanepidermal keratinization model. The results obtained showed that GlcCer[d18:2 (4E, 8Z )] with C18 to C26 fatty acids,
Cer[t18:0/24:0], and BSG decreased TEWL. The suppressive effect of GlcCer was dependent on the length of fatty acids,
while Cer [t18:0/24:0] and GlcCer[d18:2(4E,8Z )/26:0] exerted similar strong effects. GlcCer[d18:2 (4E, 8Z)] with C18
and C20 fatty acids and Cer[t18:0/24:0] suppressed melanogenesis in B16 melanoma cells. Furthermore, BSG prolonged
the survival of influenza-infected mice and increased blood O 2 saturation in lipopolysaccharide-induced pneumonia
mice. In conclusion, GlcCer promoted barrier function in a fatty acid length-dependent manner and up-regulated theexpression of filaggrin. Cer[t18:0/24:0] strongly promoted barrier functions by increasing the production of Cer[NS/NDS].
GlcCer[d18:2(4E, 8Z )] with C18 and C20 fatty acids and Cer[t18:0/24:0] suppressed melanogenesis. These results suggestthat specific GlcCer and Cer promote epidermal barrier functions and exhibit anti-melanogenic activity, leading to clinicalefficacy. BSG also contributed to epidermal and lung barrier functions. Therefore, these 3 components in rice oil by-products
are beneficial for skin and lung health.

KEY WORDS: glucosylceramide; β-sitosterol glucoside; transepidermal water loss; melanin; pneumonia

Introduction

Glucosylceramides (GlcCer) and β-sitosterol glucoside(BSG) concomitantly exist in a wide variety of plants 1) andexert beneficial effects on health. As functional foods inJapan, GlcCer are added to processed foods to promoteepidermal moisturization and barrier function 2). A clinicalstudy suggested the potential of rice-derived GlcCer and
BSG to suppress transepidermal water loss (TEWL), whichreflects skin barrier functions 3). An increase in the epidermal
production of ceramide (Cer) by GlcCer supplementation has
been suggested as one of the mechanisms responsible for theirpromotion of barrier functions 4). Skin Cer are sphingolipids

that mainly exist in the stratum corneum (SC) and play
pivotal roles in epidermal hydration and barrier functions 5)
with other lipids, including cholesterol, and moisturizing
proteins, such as filaggrin 6). Among Cer, ω-hydroxy Cer,
such as Cer[EOS], which has very long chain fatty acids,
are essential for skin barrier functions 7). In addition, a recent
study demonstrated that Cer[NP] was more important thanCer[NS], the most abundant Cer in SC 8).
Plant-derived GlcCer consist of a sphingoid base andbetween C16 and C26 fatty acids depending on the plant.
Besides GlcCer, Cer without a glucose in their structure,

Correspondence to: Hiroshi Shimoda
Numata 1, Kitagata-cho, Ichinomiya, Aichi 493-8001, Japan,
TEL: +81-586 - 86-5141 FAX: +81- 586 - 86- 6191
e-mail: kaihatsu@mri.biglobe. ne.jp
Co-aouthors; Takeda S, kaihatsu@mri.biglobe.ne.jp;
Miyasaka K, kaihatsu@mri.biglobe.ne.jp; Yoneda A, kaihatsu@mri.biglobe.ne.jp;
Manse Y, manse@phar.kindai.ac.jp; Morikawa T, morikawa@kindai.ac.jp_ 94 _

Glycative Stress Research 2023; 10 (3): 94-109(c) Society for Glycative Stress ResearchGlycative Stress Research

including Cer[t18:0/24:0] (ceramide[AP]), have been identified
as minor constituents in rice 9) and several fermented foods 10).
However, limited information is currently available on theeffects of single molecules of GlcCer and Cer on barrier
functions. Therefore, we isolated 13 GlcCer and 6 Cer fromthe gummy by-products of rice bran oil and examined
their effects on epidermal barrier 11) and melanogenesis 9) toelucidate structure-activity relationships. BSG is a glucosideof β-sitosterol that exerts beneficial effects on health, such as
reductions in cholesterol 12) and lung protective effects against
influenza infection 13). However, few studies have investigatedthe biological effects of BSG even though it is present at
approximately the same amount as GlcCer in rice. Therefore,
we also investigated the epidermal and lung barrier functionsof BSG.

Methods

Preparation of GlcCer and Cer

GlcCer and Cer ( Fig. 1 ) were purified according to ourpreviously described methods 3, 9, 11). The crude GlcCer fraction
manufactured from gummy by-products obtained during therefining process of rice bran oil was used as a starting material.
The fraction (10 g) was applied to flash column chromatography(Yamazen, Osaka, Japan) equipped with a silica gel column(Universal silica gel column, 3L) and evaporative liquid
scattered detector (ELSD, Yamazen). After washing the column
with hexane: ethyl acetate (9 : 1 → 0 : 10) to remove less polar
fractions, the mobile phase was changed to chloroform andmethanol. The composition of the solvent was graduallychanged from chloroform and methanol (10 : 1 → 0 : 10) to
obtain a chloroform: methanol (8 : 2) fraction as the GlcCer
fraction. The fraction was separated and repeatedly purified
by HPLC equipped with an ODS column (Inertsil Prep. ODS
φ20 × 250 mm, GL Science, Tokyo, Japan) and RI detector(Shimazdu RID-20A, Kyoto, Japan). Methanol was used as
the solvent. HPLC fractions were evaporated to obtain GlcCer[d18:2(4E, 8Z )/18: 0] (1, 23.6 mg), GlcCer [t18:1(8Z )/20:0](2, 16 mg), GlcCer[d18:2(4E, 8Z)/20:0] (3, 117.6 mg), GlcCer[d18:2(4E, 8E )/20:0] (4, 56.5 mg), GlcCer [t18:1(8Z)/22:0](5, 32.2 mg), GlcCer[d18:1(4E)/20:0] (6, 6.0 mg), GlcCer[d18:2 (4E, 8Z )/22:0] (7, 29.1 mg), GlcCer [d18: 2 (4E, 8E)/
22:0](8, 4.5 mg), GlcCer[t18:1(8Z )/24:0] (9, 5.8 mg), GlcCer[d18:2(4E, 8Z)/24:0] (10, 28.7 mg), GlcCer [d18:2(4E, 8E )/24:0] (11, 8. 9 mg), GlcCer[t18:1(8Z )/26:0] (12, 8.8 mg),and GlcCer[d18:2(4E, 8Z )/26:0] (13, 3.9 mg).
To obtain Cer, the chloroform: methanol ( 9 : 1) fractionof flash chromatography was purified by HPLC equippedwith a same column described above and MeOH
: tetrahydrofuran (9 : 1) as the mobile phase to obtainCer[t18:0/24:0] (14, 9.9 mg), Cer[t18:0/22:0] (15, 6.2 mg),Cer[t18:0/23:0] (16, 7.4 mg), Cer[t18:0/25:0] (17, 4.6 mg),Cer[t18:1(8Z )/24:0] (18, 5.1 mg), and Cer[t18:0/26:0] (19,5.5 mg). Chemical structures were identified by 1H- and 13CNMR spectra with the referenced values 3, 9, 11).

Evaluation of Skin Barrier Function in
Reconstructed Human Epidermal Keratinization(RHEK) Model by Measuring TEWL

According to the previously described methods 11, 14), A
RHEK model (LabCyte EPI-MODEL) were cultured in a
24 -well culture plate with assay medium under the cup. Afterincubating the plate at 37 ℃ under a 5% CO 2 atmosphere fora day, the RHEK model was treated with solutions of GlcCer(1-13), Cer (14 -19) or BSG (final DMSO concentration:0.1%). Culture times were selected for each experiment. The
RHEK model was cultured for 7 days and then subjected to
TEWL measurements, a lipid analysis, Western blotting, andmicroscopic observations, or for 2 days followed by real-time
RT-PCR.
TEWL measurements were conducted before and 1, 3,
5, and 7 days after the treatment of samples with Tewitro
TW24 (Courage +Khazaka, Cologne, Germany). The RHEK
model was placed on a thermal insulation mat (HIENAI
Mat; Cosmo Bio Co., Ltd., Tokyo, Japan), which maintained
the entire bottom surface at 32 ℃, without a lid for 5 min
before measurements. TEWL was measured for 30 min while
maintaining the bottom surface at 32 ℃, and the mean value
for the last 10 min was used in the analysis. Measurements
were performed under sterile conditions.

Skin Cer Identification and Determination

According to our previously described methods 11, 14). SC
lipids from the RHEK model were extracted using a mixtureof chloroform, methanol, and PBS (1 : 2 : 0.8). Dried SC lipid
samples were subjected to HPTLC to measure Cer contents.

Real Time RT-PCR and Western Blotting Analysis

The mRNA expression of Cer synthesis-related enzymes
in the RHEK model was measured by quantitative real time
RT-PCR according to our previously described method 11, 14).
Extracted RNA from the whole RHEK model was reversetranscribed to obtain cDNA.
The Western blot analysis described in our previous
studies 11, 14) was used to assess protein expression. Proteinsextracted from the RHEK model were electrophoresed on
10 % SDS gels and then separated proteins were transferred
to polyvinylidene difluoride membranes. After blocking
with 5 % skim milk, membranes were treated with a primary
antibody followed by a secondary antibody. Anti-GCS (1 :200), anti-filaggrin (1 : 200), anti-corneodesmosin (1 : 200),
anti- CerS3 (1 : 1,000), anti-GCS (1 : 200), and anti-GAPDH(1 : 1,000) antibodies were used as primary antibodies.
HRP- conjugated goat anti-rabbit IgG (1 : 5,000) and HRPconjugated goat anti-mouse IgG (1 : 5,000) were used as
secondary antibodies. SuperSignal ™ West Pico PLUS
Chemiluminescent Substrate and SuperSignal ™ West Femto
Maximum Sensitivity Substrate were used for detection
solutions.

_ 95 _

Cer, ceramides; GlcCer, glucosylceramides.

Fig. 1. Structures of rice-derived GlcCer (1-13) and Cer (14-19).

Efficacy of Rice Ceramides and Sterol Glucoside_ 96 _Glycative Stress ResearchElectron Microscopic Analysis
Regarding electron microscopic observations of tissues,
the preparation and imaging of pathological sections wereconsigned to Hanaichi UltraStructure Research Institute, Co.,
Ltd. (Aichi, Japan) 11).

Diet-induce Atopic Dermatitis Model in Mice

The experiment was performed in accordance with the
Guidelines for Animal Experimentation (Japan Association
for Laboratory Animal Science, 1987). Male hairless mice
(Hos: HR-1) aged 6 weeks old were fed a special diet (HRAD diet; Nosan Corp., Yokohama, Japan) 15) for 80 days. For
normal group, standard CE -2 non-purified diet was used. At
the same time as the feed was given, GlcCer (10 mg/kg) or
BSG (10 mg/kg) suspended with 5 % gum Arabic in water
was orally given to mice once a day for 80 days. TEWL andepidermal moisture on dorsal area were measured by Vapometer(Keystone scientific Inc., Tokyo, Japan) and corneometer(Courage +Khazaka Electronic GmbH, Köln, Germany),
respectively. For skin filaggrin determination, protein solution
extracted from the skin were electrophoresed and adopted toWestern blotting as described above. Anti-filaggrin (1: 1,000)
and anti-GAPDH (1:1,000) antibodies were used as primary
antibodies.

Melanogenesis and mRNA Expression in B16
Melanoma Cells

B16 melanoma cells (5 × 10 4 cells/mL) were seeded on
a 48-well culture plate (200 μL for melanogenesis) and a 24well culture plate (500 μL for RT-PCR). After an incubationfor 24 h, test samples and theophylline (2 mM) were added
and incubated for 72 h. After a further incubation, medium
was removed from the 48-well plate and distilled water(100 μL/well) was added. Cells were sonicated and 6 N
NaOH (20 μL/well) was added. The absorbance of thesonicated solution was measured with a microplate reader(measurement wavelength: 4 05 nm, reference wavelength:
660 nm).
The mRNA expression was assessed by quantitative
real-time RT-PCR. After extraction, 0.1 μg of total RNA
was reverse transcribed using PrimeScript ™ Reverse
Transcriptase to obtain cDNA. Real-time RT-PCR was
performed using TB Green® Premix Dimer Eraser ™ and
Thermal Cycler Dice® Real-Time System Single (TM 800,
Takara Bio Inc.). The primers were listed in the reference 9).

Tyrosinase Inhibitory Activity andLuciferase Based ATP Assay

Crude tyrosinase solution 9) (50 μL/well, 420 μg protein/mL), a 2 mM L-DOPA (levodopa) solution (4 0 μL/well), anda test sample (10 μL/well) were placed onto a 96-well plate.
The enzyme reaction was performed at 37 °C for 90 min.
After the enzyme reaction, absorbance was measured with amicroplate reader (Measurement wavelength: 475 nm, reference
wavelength: 660 nm).
For luciferase assay, B16 melanoma cells (1.0 × 10 5 cells/mL) were seeded on a 96-well plate (100 μL/well) and incubatedfor 24 h. Medium was then replaced with that containing

theophylline (2 mM) and the test sample. After an incubationfor 24 h, medium was removed and cells were gently washedwith PBS (−). ATP was determined by Glo Lysis Buffer(Promega, Madison, WI, USA) and ATP assay reagent.

Clinical Examination of UV-induced Inflammation
and Pigmentation

This randomized, placebo-controlled, double-blind,parallel-group study was performed according to the
Declaration of Helsinki (2013 revision) and was registeredin the University Hospital Medical Information Network
Clinical Trials Registry (UMIN000042696). Inclusion
criteria were Japanese male and female adults (20 years orolder and 59 years or younger) with a normal skin coloror fair skin who expressed concerns about dry skin. First
selection criteria were individuals with confirmed minimum
erythema dose 1 (MED) among 6 UV irradiated areas. Fortyeight subjects were selected for the present study. Subject
allocation was performed in a manner to prevent significant
differences in the means of 1 MED and forearm TEWL
between groups.
Subjects took one appropriate capsule (Oryza Ceramide®
or placebo) daily after breakfast for 8 weeks. Active capsules
contained 40 mg of Oryza Ceramide®-PCD (comprising 1.2
mg of GlcCer and 0.06 mg of Cer [ t18:0/24:0]) and 160
mg of γ-cyclodextrin. Placebo capsules contained 200 mg
of γ-cyclodextrin. MED and the minimal tanning dose(MTD) were examined at baseline and after 4 and 8 weeksof the intervention as the primary outcome. UV irradiation
was performed on the screening day, baseline day, after 4
weeks, and after 8 weeks. According to references, 6 areas(areas A to F ) were irradiated by UV rays using a UV solar
simulator (WBS-10C×6F-5UV, Ushio Inc., Tokyo, Japan)
from 1074 to 2672.4 mJ/cm2 ·s at intervals of 1.2 times. The
content of melanin and skin redness were measured using amexameter (MX18, Courage+Khazaka Electronic GmbH,Köln, Germany).

Survival Rate of Influenza-infected Mice

The study was performed at Hamri Co. Ltd. (Ibaraki, Japan).
Test samples were orally administered to female BALB/cmice for 7 days and the virus (mutated A/PR/8/34, 7.9×10 6
TCID 50) was then intranasally inoculated. Test samples were
continuously administered once a day and the survival rate
was monitored.

Lipopolysaccharide (LPS)-induced Lung Neutrophil
Accumulation and Pneumonia

According to the method of Rojas et al. 16), BSG wasorally administered to male C57BL/6 mice and LPS (1 mg/kg) was intraperitoneally injected. The lungs were removed
24 h later and specimens were fixed in 4 % paraformaldehyde.
The specimen for microscopic analysis was stained by H.E.
For peneumonia model 17), BSG or dexamethasone was orally
administered to male ICR mice from the day or the day after
the intratracheal administration of LPS (75 μg). Test samples
were administered once a day and oxygen saturation wasmeasured by pulse oximeter (MouseOx, Harvard Apparatus,
Hollison, MA, USA) for 3 min under anesthesia.

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Efficacy of Rice Ceramides and Sterol Glucoside

Results & Discussion

Effects of Rice-derived GlcCer, Cer, and BSG on
Epidermal Barrier Functions

1) Skin Barrier Abilities of GlcCer and Cer
The RHEK model was treated with a sample for 7 days
and TEWL was measured every 2 days. The results obtained
showed that 10 μM of GlcCer with 4E,8Z sphingadienine,such as GlcCer[d18:2(4E,8Z )/18:0] (1), GlcCer[d18:2 (4E/
8Z )/20:0] (3), GlcCer[d18:2(4E/8Z )/22:0] (7), GlcCer[d18:
2 (4E/8Z )/24:0] (10), and GlcCer[d18:2(4E/8Z )/26:0] (13),decreased TEWL after 7 days ( Fig. 2, upper). Structure activity relationships were clearly demonstrated for GlcCer[d18:2(4E, 8Z )] (1, 3, 7, 10, 13), but not GlcCer[t18:1(8Z )] (2,5, 9, 12), GlcCer[d18:2(4E, 8E)](4, 8, 11), or GlcCer[t18:1(4E)](6 ). The effects of active GlcCer (1, 3, 7, 10, 13) on epidermal
barrier functions were dependent on fatty acid lengths, withlonger carbon chains of fatty acids in GlcCer[d18:2(4E,8Z )]more strongly promoting barrier functions. The effects of 10
and 13 with more than C24 fatty acids on barrier functions
were observed in the earlier stages of the culture (Days 1 and
3) and were strong.
Regarding Cer, 10 μg/mL of Cer[t18:0/24:0] (14), Cer[t18:0/23:0] (16), and Cer[t18:1(8Z)/24:0] (18) significantly
reduced TEWL (Fig. 2, lower). However, a clear structureactivity relationship was not confirmed, such as relationships
between activity and fatty acid lengths or the types ofsphingoid bases. Among Cer, Cer (14) at 1, 3, and 10 μg/mL
exerted concentration-dependent effects on barrier functions.
These effects were as strong as those of GlcCer (13) inthe later culture stage (Days 5 and 7 ), but did not decrease
TEWL in the early culture stage. Therefore, the mechanisms
underlying the effects of 13 and 14 appeared to differ.
2) Different Mechanisms for Effects of Cer (14) and
GlcCer (13) on Skin Barrier Functions
We examined Cer contents in the RHEK model treatedwith Cer (14) to elucidate the mechanisms responsible
for enhancements in barrier functions. Cer (14) increased
Cer contents (Fig. 3-a) in the RHEK model, specificallyCer [NS/NDS] 11). Since Cer[NS/NDS] is a major Cer inSC, we examined the mRNA (Fig. 3-b) and protein (Fig.
3-c) expression of enzymes involved in the de novo Cersynthesizing pathway. The results obtained showed that 14
up-regulated the expression of GlcCer synthase (GCS), which
has been shown to facilitate changes from Cer to GlcCerin the stratum granulosum (SG). Therefore, 14 exerted its
effects on barrier functions through Cer production based on
the expression of GCS.
In contrast, since GlcCer (13) which suppressed TEWL
( Fig. 4-a) did not affect Cer contents ( Fig. 4-b), we performed
an electron microscopic analysis to elucidate the underlying
mechanism. The results obtained showed a denser SC imageof the RHEK model treated with 13 (Fig. 4-c, left), suggestingstronger barrier functions than in the control RHEK model.
Moreover, increases in corneodesmosomes (Fig. 4-c, middle)
and the number and area of keratohyalin granules (Fig. 4-c,right) were observed in the RHEK model treated with 13.
Keratohyalin granules primary exist in SG and contribute to
the cross-linking of keratin filaments, which creates atight barrier in the epidermis 18). These granules also produce

filaggrin, a precursor of natural moisturizing factor(NMF) 19). The Western blot analysis showed an increase in
the expression of filaggrin. Therefore, 13 appeared to exert
moisturizing effects by up-regulating the expression of filaggrin(Fig. 4-d, left), thereby providing NMF to SC. In addition, theWestern blot analysis revealed the up-regulated expressionof corneodesmosin (Fig. 4-d, right). Corneodesmosin, a celladhesion molecule, exists in corneodesmosome cores andcontributes to a tight SC structure 20). The mechanism by which
13 exerted moisturizing effects may involve corneodesmosin.
Regarding the mechanisms by which rice-derived Cer
and GlcCer promote barrier functions (Fig. 5 ), the main
mechanism of Cer[t18:0/24:0] (14) involved the SC productionof Cer[NS/NDS] through the expression of GCS. In contrast,
GlcCer with the 4E, 8Z structure exhibited barrier functions,and longer fatty acids enhanced this effect. The mechanismof GlcCer (13) involved enhancing the tight structure of
SC and increasing NMF production without affecting Cercontents in SC.

3) Enhanced Epidermal Barrier Functions by BSG
through the Production of Cer and Filaggrin
BSG (Fig. 6-a) residing with GlcCer in plant resources
is a sterol glucoside and the biological activities despite ofwhich have not been examined as extensively as β-sitosterol.
Therefore, we investigated the effects of BSG on epidermal
barrier functions 14). BSG suppressed TEWL in the cultured
RHEK model at a later stage (Day 7) (Fig. 6-b). The underlying
mechanism involved an increase in the production of Cer[EOS] through ceramide synthase (CerS) -3 and the upregulated expression of GCS (Fig. 6-c, d ). Since Cer[EOS]is an acylated Cer with potent barrier function 21), strong
epidermal protective effects are expected. In an in vivo
evaluation of the effects of BSG on barrier functions using a
diet-induced atopic dermatitis mouse model 22), the consecutive(80 days) oral administration of BSG (10 mg/kg) significantly
increased epidermal moisture and slightly suppressed TEWL
(Fig. 6-e), whereas GlcCer did not improve these parametersin this model. The underlying mechanism involved the up regulated expression of filaggrin ( Fig. 6-f ). Therefore, BSG
strongly enhanced barrier functions in skin.

Anti-melanogenic Effects of GlcCer and Cer

Regarding the other biological activities of GlcCer and
Cer in skin, we investigated their anti-melanogenic effects ontheophylline-induced melanogenesis in B16 melanoma cells 9).
Table 1 shows the results obtained for GlcCer (1-13) and Cer(14 -19). Regarding GlcCer, GlcCer[d18:2(4E,8Z )/18:0] (1)and GlcCer[d18:2(4E,8Z)/20:0] (3) suppressed melanogenesis
with IC 50 values less than 10 μg/mL. GlcCer[d18:2(4E, 8E )/20:0] (4) and GlcCer[d18:1(4E )/20:0] (6) slightly suppressedmelanogenesis at 10 μg/mL. However, GlcCer ( 9 -13) withvery long chain fatty acids did not suppress melanogenesis.
Based on these results, the structure-activity relationshipfor the suppression of melanin synthesis markedly differedfrom that for skin barrier functions. The structure-activity
relationship for anti-melanogenic activity currently remains
unclear; however, the major GlcCer (3) exhibited the strongest
anti-melanogenic activity among rice GlcCer (1-13).

_ 98 _Glycative Stress Research

Gl cC
G lc
e rC( er
( 1-75,) 7)
1- 5,

G l cCeG l cCer ( 9 r- 13( 9 -) 13 )

G lcG
C lc
erC( er
6 , 8( 6) , 8 )Ce rCe( 14
r (- 14
16,- 1816,) 18 )C erC( er17,( 17,19 ) 19 )Ce rCe( 14r )( 14 )Fig. 2. Effects of GlcCer (1-13) and Cer (14-19) on TEWL in the RHEK model.
The RHEK model was treated with each sample ( 1-13 : 10 μM, 14 -19 : 10 μg/mL) for 7 days. Data are expressed as means ± SE ( n =3 - 4 ). Asterisks denote a significant difference from the control group, *p < 0.05, **p < 0.01. Figure is quoted from Ref 11. Cer, ceramides;
GlcCer, glucosylceramides; TEWL, transepidermal water loss; RHEK, reconstructed human epidermal keratinization; SE, standard error.

_ 99 _

Efficacy of Rice Ceramides and Sterol GlucosideaTotal Cer

Cer [NS,NDS]
*

200

100

0

Control

1

3

Cer [NS,NDS] contents(µg/mg protein)

Total Cer contents(µg/mg protein)300

10 (µg/mL)

300

*

200

100

0

Control1

10 (µg/mL)

3

Cer (14 )Cer (14 )

c

b

GAPDH

GCS

4

*

3
2
1
0

0

1

3
Cer (14 )

10 (µg/mL)

Relative protein expression(GCS/GAPDH)

Relative RNA expression(GCS/GAPDH)

GCS

8

1

Control

3

10(µg/mL)Cer (14 )

**

6

**

4
2
0

0

1

3

10 (µg/mL)

Cer (14 )Fig. 3. Effects of Cer (14) on ceramide contents (a) and mRNA (b) and protein (c) expression of GCS in the RHEK
model.
The RHEK model was treated for 2 days (for real-time RT-PCR) or 7 days (for ceramide contents and Western blotting) with 14 (1 –10 μg/mL). Data are expressed as means ± SE (n = 4). Asterisks denote a significant difference from the control group, *p < 0.05, **p< 0.01. Figure is quoted from Ref 11. Cer, ceramides; GlcCer, glucosylceramides; GCS, GlcCer synthase; GAPDH, glyceraldehyde-3phosphate dehydrogenase; RHEK, reconstructed human epidermal keratinization; SE, standard error.

_ 10 0 _Glycative Stress Research

a

b

c

d

Fig. 4. Effects of GlcCer[d18:2(4E, 8Z)/26:0)] (13) on epidermal hydration parameters and factors in the RHEK
model.
a) Changes in TEWL, b) SC Cer contents after a treatment with 13 for 7 days (n = 4), c) electron microscopic images of crosssections of the RHEK model, and d) Protein expression of filaggrin and corneodesmosin (n = 4 -5). All data are expressed asmeans ± SE. Asterisks denote a significant difference from the control group, *p < 0.05, **p < 0.01. Figure is quoted from Ref 11.
TEWL, transepidermal water loss; SC, stratum corneum; Cer, ceramides; GlcCer, glucosylceramides; GAPDH, glyceraldehyde - 3 phosphate dehydrogenase; RHEK, reconstructed human epidermal keratinization; SE, standard error.

_ 101 _

Efficacy of Rice Ceramides and Sterol Glucoside

Enhancement of Cer production
by Cer (14)Serine+Palmitoyl-CoA

S. spinosum

Dihydroceramide
Ceramide
GCS

SMS

Glucosylceramide Sphingomyelin
GBA

GlcCer (13)

S. basale

SPT
CerS

Effects of GlcCer (13) on moisturizing factors

Maturation of
S. corneum

Keratohyalin
granuleCorneodesmosin

Moisture holding

Filaggrin

Moisture retention

S. granulosum
NMF

ASM

Ceramide(Cer [NS/NDS])

TEWL

S. corneum
TEWL

Fig. 5. Potential mechanisms by which Cer[t18:0/24:0] (14) and GlcCer (13) promote barrier functions.

Figure is quoted from Ref 11. Cer, ceramides; GlcCer, glucosylceramides; GCS, GlcCer synthase; SPT, serine palmitoyl-trnsferase;
CerS, ceramide synthase: SMS, sphingomyelin synthase; GBA, β-glucocerebrosidase; ASM, acid sphingomyelinase; TEWL,
transepidermal water loss; NMF, natural moisturizing factor.

_ 10 2 _Glycative Stress Researchab∆TEWL (g/m 2 ∙ h)

4
H
H
H

CH2OH
OO
OH

2
-2
-4

BSG (10μg/mL)

-6

*

-8

- 10

OH

Control

0

OH

0

1

3

5

7

Day

d
*

2.0
1.5
1.0
0.5
0

0

1
3
10
B S G (µ g/m L)

Cer [ EOS ]

GCS
1.6
1.2
0.8
0.4
0

CerS3

CerS3

ß- actinß-actin

0

1
3
10
B S G (µ g/m L)

30
25
20
15
10
5
0

e

*

35

** **
Cer[EOS] contents(µg/mg protein)

CerS3

2.5

Relative protein expression(GCS/ß-actin)

Relative protein expression(CerS3/ß-actin)

c

0

1
3
B S G (µ g/m L)

f

*

25
20
15

**

10
5
0

Normal Control GlcCer BSG

Moisture
40

**
**

30
20

4
3
2
1
0

10
0

Relative protein
expression( Filaggrin/GAPDH)TEWL (g/m2 . h )30

Epidermal moisture ( %)

5

TEWL

10

NormalControl

GlcCerBSG

Filaggrin
Normal Control GlcCer BSG

GAPDH

Fig. 6. Effects of BSG on epidermal moisture and the underlying mechanism.
a) Chemical structure of BSG. b) Effects of BSG on TEWL in the RHEK model ( n = 4). The RHEK model was treated for 7 days
with BSG (10 μg/mL). c, d) Effects on the protein expression of CerS3 and GCS ( n = 5 ) and SC Cer[EOS] contents (n = 6 ) in the
RHEK model. SC Cer[ EOS] was quantified by HPTLC. e, f ) Effects on TEWL, epidermal moisture, and filaggrin expression indiet-induced atopic disease mice (n = 3 -5). BSG (10 mg/kg) or GlcCer (10 mg/kg) was orally administered to mice for 80 days
and TEWL and epidermal moisture were measured. Asterisks denote a significant difference from the control group, *p < 0.05, **:p < 0.01. Figures a ~ d quoted from Ref. 14; e and f are unpublished original data. BSG, β-sitosterol glucoside; CerS, ceramide
synthase; SC, stratum corneum; Cer, ceramides; GlcCer, glucosylceramides; GSC, GlcCer synthase; TEWL, transepidermal water
loss; RHEK, reconstructed human epidermal keratinization; SE, standard error (error bar).

_ 10 3 _Efficacy of Rice Ceramides and Sterol Glucoside

Table 1. Inhibitory activities of GlcCer (1-13) and Cer (14-19) on theophyllinestimulated melanogenesis of B16 melanoma cells.
IC 50 (μM)
GlcCer[d18:2(4E, 8Z )/16:0]

NE

GlcCer[d18:2(4E, 8E)/16:0]

NE

GlcCer[d18:2(4E, 8Z)/18:0] (1)6.6 (69.8 %)GlcCer[d18:2(4E, 8Z )/20:0] (3)GlcCer[d18:2(4E, 8E)/20:0] (4)5.2 (63.4 %)> 10 (45.6 %)GlcCer[d18:1(4E )/20:0] (6 )> 10 (32.7 %)GlcCer[d18:2(4E, 8E)/22:0] (8)NE> 10 (21.0 %)GlcCer[t18:1(8Z )/20:0] (2)NE

GlcCer[t18:1(8Z )/22:0] (5)NE

GlcCer[d18:2(4E, 8Z )/22:0] (7)NE

GlcCer[t18:1(8Z )/24:0] (9)GlcCer[d18:2(4E, 8Z )/24:0] (10)NE

GlcCer[d18:2(4E,8E)/24:0] (11)NE

> 10 (17.8 %)> 10 (22.7 %)GlcCer[t18:1(8Z )/26:0] (12)GlcCer[d18:2(4E, 8Z )/26:0] (13)Cer[t18:0/24:0] (14)3.9 (63.4 %)Cer[t18:0/22:0] (15)NE

Cer[t18:0/23:0] (16)> 10 (11.7 %)Cer[t18:0/25:0] (17)NE

Cer[t18:1(8Z)/24:0] (18)NE

Cer[t18:0/26:0] (19)NE

Each value represents the mean ± SE (n = 4). Values in parentheses are % inhibition of melanogenesis
at 10 μg/mL. Data are quoted from Ref 9. Cer, ceramides; GlcCer, glucosylceramides; IC 50, half
maximal inhibitory concentration; NE, not effective; SE, standard error.

Among Cer, only Cer[t18:0/24:0] (14) strongly suppressedmelanogenesis with an IC 50 value of 3.9 μM. The structureof 14 is the same as ceramide [AP], which is present in skin
SC. Therefore, Cer[t18:0/24:0] was suggested to exert strong
inhibitory effects on innate melanin production in skin.
To elucidate their mechanisms of action, we investigatedwhether 3 and 14 down-regulated the mRNA expression ofmelanogenic molecules. As shown in Fig. 7-a, 14 significantly
down-regulated the mRNA expression of TYRP1, whereas 3
did not. Therefore, the suppression of TYRP1 is a key antimelanogenic mechanism of 14. Fig. 7-b shows the effects of
3 and 14 on tyrosinase activity derived from B16 melanoma
cells. Kojic acid dose-dependently inhibited tyrosinase
activity (IC 50 : 3.6 μM), whereas 3 and 14 did not. Moreover,
14 and emodin suppressed ATP levels, whereas 3 did not(Fig. 7-c). Therefore, the anti-melanogenic effects of 14 were
attributed to reductions in TYRP1 expression and ATP
levels, and not to the inhibition of tyrosinase activity. ATP
is needed for the proliferation of B16 melanoma cells
through P2X7 receptors 23) and melanogenesis 24). However,
the inhibitory mechanism of 3 has not yet been elucidated.
We evaluated the clinical anti-melanogenic effects of
Oryza ceramide ® (OC) standardized with rice lipids in aplacebo-controlled double-blind study on Japanese subjects 9).

The results obtained showed that OC suppressed UV-induced
skin tanning (melanin pigmentation), but not erythema.
Therefore, orally administered rice GlcCer and Cer inhibited
UV- induced melanogenesis (Fig. 8).

Effects of BSG on Influenza Virus- and LPS-induced
Pneumonia in MiceIn addition to the effects of rice lipids on skin barrier
functions, we examined their effects on lung protective andbarrier functions in order to detect other biological activities
of GlcCer and BSG. The effects of GlcCer, BSG, or a crude
mixture of GlcCer and BSG were examined in mice infectedwith influenza virus guided by mortality. The results
obtained showed that the crude mixture of GlcCer and BSG
and purified BSG induced a right shift in the survival curveof mice and the average number of survival days of the micein these groups was significantly higher ( Fig. 9-a, left). In
an additional experiment, BSG treatments induced a rightshift in the survival curve ( Fig. 9-a, right) and significantly
increased the average number of survival days at 30 and 100
mg/kg. Therefore, BSG was effective against fetal pneumonia
in mice induced by influenza infection.
We also investigated the potential of BSG against
bactericidal pneumonia in LPS-induced mice. We
intraperitoneally injected LPS and then orally administered_ 10 4 _Glycative Stress Research

a

b

c

Fig. 7. Effects of GlcCer [d18:2(4E, 8Z)/20:0] (3) and Cer[t18:0/24:0] (14) on the mRNA expression ofmelanogenic factors (a), tyrosinase activity (b), and ATP levels in B16 melanoma cells (c).
a) B16 melanoma cells were treated with 3 or 14 for 72 hr. Each value was corrected by the mRNA expression level of β-actin andshown as a relative value to the control. b) Crude tyrosinase was extracted from B16 melanoma cells treated with theophylline for 72
h. Enzyme solutions were incubated with L-DOPA and test samples for 90 min. c) Cells were treated with 3 or 14 for 24 h. Data areexpressed as the mean ± SE (n = 4). Asterisks denote significant differences from the control at **p < 0.01. Figures are quoted from
Ref 9. Cer, ceramides; GlcCer, glucosylceramides; ATP, adenosine triphosphate; L -DOPA, levodopa; MITF, melanocyte inducing
transcription factor; T Y R, tyrosinase; TYRP1, tyrosinase -related protein 1; SE standard error.

_ 10 5 _Efficacy of Rice Ceramides and Sterol GlucosideabFig. 8. Effects of Oryza ceramide® on skin pigmentation induced by UV irradiation.
a) Changes in melanin frequency in UV-irradiated skin. Each value represents the mean and SD (n = 24). Asterisks denote significant
differences from the control at *p < 0.05 and **p < 0.01. b) The arm skin of 3 typical subjects in each group exposed to UV irradiation
after 8 weeks. Figures are quoted from Ref 9. Cer, ceramide; UV, ultraviolet; SD, standard deviation.

_ 10 6 _Glycative Stress ResearchaSurvival curve
90

80

80

Survival rate ( %)

100

90

Survival rate ( %)

100

70
60
50
40

Control
Crude GlcCer and
BSG (10 mg/kg)
GlcCer ( 5 mg/kg)
BSG (5 mg/kg)

30
20

70
60
50
40

Control
BSG (5 mg/kg)
BSG (10 mg/kg)
BSG (30 mg/kg)
BSG (100 mg/kg)
Favipilavir (30 mg/kg)

30
20

10

10
0

2

4

6

8

10

12

14

0

Days after influenza inoculation

2

4

6

8

10

12

14

Days after influenza inoculation

Average number of survival days
Favipiravir (30 mg/kg)

BSG (5 mg/kg)

BSG (100 mg/kg)

GlcCer (5 mg/kg)

BSG (30 mg/kg)
BSG (10 mg/kg)

Crude GlcCer and BSG (10 mg/kg)

BSG (5 mg/kg)
ControlCont rol
0

2

4

6

8

Days af ter inoculation

10

0

2

4

6

8

10

12

Days af ter inoculation

14

16

b

Given from the day of the LPS inoculation

Given from the day af ter the LPS inoculation
100

95
90
85
80
75
70

2

3

Days after LPS inoculation

4100

O 2 saturation (%)

O 2 saturation (%)

100

O 2 saturation (%)

c

95
90
85
80
75
70

2

3

Days after LPS inoculation

4

95
90
85
80
75
70

2

3

4

Days after LPS inoculation

Fig. 9. Effects of BSG on the survival rate of influenza-infected mice (a), LPS-induced lung neutrophil accumulation(b), and LPS-induced pneumonia (c).
a) Test samples were orally administered to female BALB/c mice for 7 days and the virus (mutated A/PR/8/34, 7. 9 × 10 6 TCID 50)

was then intranasally inoculated. Test samples were continuously administered once a day and the survival rate was monitored.
b) BSG was orally administered to male C57BL/6 mice and LPS (1 mg/kg) was intraperitoneally injected. The lungs were removed
24 hr later and specimens were stained by H. E. c) BSG or dexamethasone was orally administered to male ICR mice from the dayof or the day after the intratracheal administration of LPS (75 μg). Test samples were administered once a day and oxygen saturation
was measured for 3 min under anesthesia. Each value represents the mean or mean and SE (n = 5 -10). Asterisks denote significant
differences from the control at *p < 0.05, **p < 0.01, respectively. These are unpublished original data. BSG, β -sitosterol glucoside;
LPS, lipopolysaccharide; H. E., hematoxylin eosin; SE. standard error.

_ 10 7 _Efficacy of Rice Ceramides and Sterol Glucoside

BSG. A microscopic analysis showed the accumulation ofneutrophils. In contrast, BSG (100 mg/kg) suppressed thealveolar accumulation of neutrophils ( Fig. 9-b). BSG (100mg/kg) was also orally administered to mice and LPS was
intratracheally administered to induce pneumonia. O 2 blood
saturation was then measured. BSG prevented reductionsin the blood O 2 saturation rate in mice with LPS-induced
pneumonia ( Fig. 9-c). These results suggest that BSG nonspecifically ameliorated lung inflammation and improved
respiratory function.

Conclusion
We herein demonstrated that GlcCer[d18:2(4E, 8Z )]exerted fatty acid length-dependent effects on barrier
functions in an epidermis model. The underlying mechanisms
appear to involve increasing the density of SC and upregulating the expression of filaggrin and corneodesmosin.
In contrast, Cer[t18:0/24:0] exerted moisturizing effects bypromoting the production of SC Cer[NS/NDS] through
GCS. Regarding anti-melanogenic effects, GlcCer[d18:2(4E,8Z)] with C18 or C20 fatty acids and Cer[t18:0/24:0]
suppressed melanin production in melanoma cells. Although
the inhibitory mechanism of GlcCer remains unclear, theeffects of Cer[t18:0/24:0] involved down-regulating theexpression of TYRP1 and ATP. Furthermore, BSG not only
enhanced skin barrier functions, but also exerted lungprotective effects.

Acknowledgments
This work was supported by the New Aichi Creative
Research and Development Subsidy (grant numbers: 118-20,
2020) and Oryza Oil & Fat Chemical Co., Ltd.

Conflict of Interest Statement
The authors have indicated no potential conflict ofinterest.

_ 10 8 _Glycative Stress Research

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_ 10 9 _