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H: Health, Nutrition, &
Food
JFS H: Health, Nutrition, and Food
Anti-inflammatory Effects of Limonene
from Yuzu (Citrus junos Tanaka)
Essential Oil on Eosinophils
RYOJI HIROTA,NGATU NLANDU ROGER,HIROYUKI NAKAMURA,HEE-SUN SONG,MASAYOSHI SAWAMU RA,
AND NARUFUMI SUGANUMA
ABSTRACT: Yuzu (Citrus junos Tanaka) has been used as a traditional medicine in Japan. We investigated in vitro
anti-inflammatory effects of limonene from yuzu peel on human eosinophilic leukemia HL-60 clone 15 cells. To ex-
amine anti-inflammatory effects of limonene on the cells, we measured the level of reactive oxygen species (ROS),
monocyte chemoattractant protein-1 (MCP-1), nuclear factor (NF) kappa B, and p38 mitogen-activated protein ki-
nase (MAPK). We found that low concentration of limonene (7.34 mmol/L) inhibited the production of ROS for
eotaxin-stimulated HL-60 clone 15 cells. 14.68 mmol/L concentration of limonene diminished MCP-1 production
via NF-kappa B activation comparable to the addition of the proteasomal inhibitor MG132. In addition, it inhib-
ited cell chemotaxis in a p38 MAPK dependent manner similar to the adding of SB203580. These results suggest
that limonene may have potential anti-inflammatory efficacy for the treatment of bronchial asthma by inhibiting
cytokines, ROS production, and inactivating eosinophil migration.
Keywords: limonene, monocyte chemoattractant protein-1 (MCP-1), nuclear factor (NF) kappa B, p38 mitogen-
activated protein kinase (MAPK), reactive oxygen species (ROS), yuzu (Citrus junos Tanaka) essential oil
Introduction
In bronchial asthma, a marked increase in eosinophils is
observed at the site of inflammation and bone marrow.
Eosinophils, which infiltrate at the site of inflammation, contain
a major basic protein that has cytotoxic properties. By releasing
cytokines (Schmid-Grendelmeier and others 2002), lipid media-
tors (Busse 1998), reactive oxygen species (Albert and others 2003),
and highly charged cytotoxic granular proteins (Marguet and others
2001), activated eosinophils contribute to airway inflammation and
cause damage to the bronchial mucosa (Pegorier and others 2006).
Thus, eosinophils have played a central role in airway inflamma-
tion in bronchial asthma.
The prevalence of asthma appears to have increased continu-
ously since the 1970s in Japan. There are evidences that clearly
implicate household (dust mite, cockroach, and pet) and other en-
vironmental (pollen) allergens in disease development in teenagers
and adults. Air pollution components, such as diesel exhaust par-
ticles (DEPs), formaldehyde, fine particles, and oxidant gases, are
not definitively linked to disease development, although they trig-
ger exacerbations (Pandya and others 2002). Recently, reactive oxy-
gen species (ROS) have been shown to play an important role in
bronchial asthma (Casillas and others 1999; Li and others 2003;
Chan and others 2006; Kim and others 2006; Martinez-Losa and
others 2007). We have reported that the administration of the
MS 20090713 Submitted 7/26/2009, Accepted 1/10/2010.Authors Hirota,
Roger, and Suganuma are with Dept. of Environmental Medicine, Kochi
Medical School, Kohasu, Oko, Nankoku, Kochi 783-8505, Japan. Author
Nakamura is with Dept. of Environmental and Preventive Medicine,
Kanazawa Univ. Graduate School of Medical Science. Author Song is with
Dept. of Food and Nutrition, Kwang Ju Health College. Author Sawamura
is with Major of Food Science, Faculty of Agriculture, Kochi Univ. Direct in-
quiries to author Hirota (E-mail: hirotar@kochi-u.ac.jp).
antioxidant reagent N-acetyl-L-cysteine (NAC) decreased DEPs-
induced eosinophil chemotaxis (Hirota and others 2008).
Natural products in dietary components/foods may aid in the
prevention and the control of diseases. In particular, yuzu (Cit-
rus junos Tanaka) is one of the most interesting natural products
(Sawamura 2005). Yuzu is a small tree that produces yellow-golden
colored citrus fruits resembling small oranges or tangerines. Yuzu
fruit is common a product of Japan and Korea. The peel of the fruit
produces a delightful citrus fragrance with a floral overtone, which
closely resembles to that of the grapefruit. Yuzu has been used in
traditional Chinese medicines, and many Japanese people believe
that a hot yuzu bath improves blood circulation and prevents colds.
Therefore, yuzu has been a favorite fruit in Japan for over a thou-
sand years. In a previous study, limonene, myrcene, terpinorene,
and alpha terpinene extracted from yuzu peel had inhibited the
formation of the carcinogen N-Nitrosodimethylamine in vitro
(Sawamura and others 2005), but other beneficial effects of them
for human health have not been much studied.
In this study, we examined the effects of limonene from yuzu
on human eosinophilic leukemia HL-60 clone 15 cells, since
eosinophilia is an important pathologic feature of asthma.
Materials and Methods
Reagents
The following materials, reagents, and kits were obtained from
commercial sources: HL-60 clone 15 cells (CRL-1964, Ameri-
can Type Culture Collection, Rockville, Md., U.S.A.); RPMI-1640
medium (pH 7.8), fetal calf serum (FCS), Tween 80, n-butyrate,
superoxide dismutase (SOD), phorbol myristate acetate (PMA),
cytochrome c (Sigma, St. Louis, Mo., U.S.A.); MG132, SB203580
(Calbiochem-Novabiochem Corp., San Diego, Calif., U.S.A.);
human recombinant eotaxin and monocyte chemoattractant
C
⃝2010 Institute of Food Technologists R⃝Vol . 75, N r. 3, 20 10—JOURNAL OF FOOD SCIENCE H87
doi: 10.1111/j.1750-3841.2010.01541.x
Further reproduction without permission is prohibited
H: Health, Nutrition, &
Food
Limonene from yuzu essential oil . . .
protein-1 (MCP-1) ELISA kit (R&D SYSTEMS, Minneapolis, Minn.,
U.S.A.); disposable 96-well chemotaxis chambers (KURABO, Os-
aka, Japan); CyQUANT NF Cell Proliferation Assay Kit (Invitrogen,
Eugene, Oreg., U.S.A.); DC Protein Assay kit (Bio-Rad, Richmond,
Calif., U.S.A.); p38 MAPK Assay Kit (Cell Signaling Technology, Inc.,
Danvers, Mass., U.S.A.).
Eosinophil cell culture and differentiation
HL-60 clone 15 cells were maintained in RPMI-1640 medium
(pH 7.8) supplemented with 10% FCS and passaged 2 times per
week in an atmosphere of 1.5% CO2.Intheexperiments,2×
105cells/mL were induced with 0.5 mmol/L n-butyrate for 5 d
to produce differentiated HL-60 clone 15 cells (df-HL-60 clone 15
cells) as previously described (Hirota and others 2008). Df-HL-60
clone 15 cells were assessed by morphology (eosinophil granules
by May-G¨
unwald and eosinophil peroxidase staining) as previously
described (Lopez and others 2003). Peroxidase positive df-HL-60
clone 15 cells induced by n-butyric acid were collected and used
for experiments.
Limonene preparation
Yuzu (Citrus junos Tanaka) fruit was obtained from the Kochi
Fruit Tree Experimental Station in November 2001. Limonene from
yuzu cold-pressed peel were prepared according to the method de-
scribed previously (Sawamura and Kuriyama 1988). Its purity was
78.13% using GC-MS analyses.
Preparation of DEPs suspensions
The DEPs suspension (Natl. Inst. for Environmental Studies,
Tsu kub a, Ib araki ) was p re par ed j ust p rio r to us e. To i ndu ce p ar-
ticle disaggregation, stock solutions of particles were dispersed in
PBS with 0.05% Tween 80 at a concentration of 10 mg/mL, and
then sonicated at output 5 and duty 30 by ultrasonic disruption for
2 min under cooling conditions (Amakawa and others 2003). Differ-
ent concentrations of particles were then diluted using RPMI-1640.
PBS with 0.05% Tween 80 was used as a negative control. For MCP-
1ELISAandnuclearfactor(NF)-kappaBDNAbindingassay,DEPs
(0.1 mg/mL) were added to df-HL-60 clone 15 cells.
Measurement of ROS production
Superoxide release was quantified by superoxide dismutase
(SOD)-inhibitable cytochrome c reduction as described by Mc-
Cord and Fridovich (Fridovich 1974) and by Condino-Neto and
Newburger (Lopez and others 2003). Cytochrome c (0.1 mmol/L)
with and without varying concentrations of limonene were added
into the incubation medium of df-HL-60 clone 15 cells (5 ×
105cells/mL; 0.5 mL) when necessary. Subsequently, cells were
treated immediately with 50 nmol/L PMA with or without 1 nM
Eotaxin. The assays were run in PBS buffer supplemented with 1
mmol/L CaCl2, 1.5 mmol/L MgCl
2and 10 mmol/L glucose at 37 ◦C
in a final volume of 0.6 mL. ROS release was monitored for 30 min.
Measurement of MCP-1 by ELISA
Initially, an effective concentration of limonene for MCP-1 pro-
duction was determined as described subsequently. Df-HL-60
clone 15 cells (1 ×105cells/mL; 1 mL) were pipetted into a 24-well
tissue culture plate (Becton Dickinson Labware, Franklin Lakes,
N.J., U.S.A.) that had been pre-incubated with either medium or
IL-1 (10 ng/mL) for 1 h. These cells were cultured with medium or
varying concentrations of limonene for 48 h at 37 ◦C in 1.5% CO2.
To study the effect of limonene on DEPs-stimulated MCP-1 produc-
tion, df-HL-60 clone 15 cells (1 105cells/mL; 10 mL) were pipetted
into 70 mL Tissue culture flasks (Becton Dickinson Labware) that
had been pre-incubated with either medium or 5 nmol/L MG132
(Calbiochem, La Jolla, Calif., U.S.A.) for 1 h. The cells were cultured
in the following conditions; medium alone as a control, medium
with DEPs only, and medium with 14.68 mM limonene with and
without DEPs for 24 h at 37 ◦C in 1.5% CO2.MCP-1proteinwas
measured in culture supernatants using a commercial ELISA kit ac-
cording to the instructions.
Chemotaxis assay
Df-HL-60 clone 15 cells (5 ×106cells/mL; 5 mL) were pretreated
with either medium or 10 µMSB203580for1h,andthenstimu-
lated with 1 nmol/L human recombinant eotaxin for 1 h at 37 ◦C
in 1.5% CO2. After eotaxin stimulation, the cells were further stimu-
lated with medium or 14.68 mM limonene for 1 h. Chemotactic cells
were evaluated using a microchamber technique (Falk and others
1980). Eotaxin (1 nmol/L) in RPMI-1640 with 0.1% bovine serum
albumin (BSA) was added to the lower compartment of a 96-well
chemotaxis chamber in a total volume of 80 µL. Fifty micro litters
of the cell suspension (2 ×106cells/mL) were added to the upper
compartment of the chamber that had been precoated with BSA
for 2 h at 37 ◦C in 1.5% CO2.Compartmentswereseparatedbya
5-µm pore size polycarbonate, polyvinylpyrrolidone-free filter. The
chamber was incubated for 2 h at 37 ◦C in 1.5% CO2.Afterincu-
bation, the cells in the lower compartment were stained using the
CyQUANT NF Cell Proliferation Assay Kit.
Preparation of nuclear extracts
Df-HL-60 clone 15 cells (5 ×105cells/mL; 20 mL) in 250 mL Tis-
sue culture flasks (Becton Dickinson Labware) were preincubated
with either medium or 5 nmol/L MG132 for 1 h. Thereafter, these
flasks were cultured either with medium or 14.68 mM limonene
for 4 h at 37 ◦C in 1.5% CO2. Each sample was evaluated by cen-
trifugation in cold PBS. Nuclear extracts were prepared according
to the method described by Lee and others (1988) with modifica-
tions (Xu and others 1997). In brief, cells were washed twice with
ice-cold PBS after incubation and suspended in 1 packed cell vol-
ume (PCV) of ice-colded buffer A (10 mmol/L HEPES, 1.5 mmol/L
MgCl2, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol (DTT), 1 µg/mL
leupeptin and aprotinin, pH 7.9) on ice for 10 min and then lysed
by passing the cell suspension through a 27-gauge needle 5 times.
Crude nuclei were washed twice with buffer A to prevent cytosolic
contamination, and the nuclear proteins were extracted with two-
thirds PCV of ice-cold buffer B (20 mmol/L HEPES, 420 mmol/L
KCl, 1.5 mmol/L MgCl2,0.2mmol/Lethylenediaminetetraacetic
acid (EDTA), 0.5 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl
fluoride, 1 µg/mL leupeptin and aprotinin, and 25% glycerol, pH
7.9). Two-thirds PCV of ice-cold buffer C (20 mmol/L HEPES,
0.2 mmol/L EDTA, 0.5 mmol/L DTT, 0.5 mmol/L phenylmethylsul-
fonyl fluoride, 1 µg/mL leupeptin and aprotinin, 20% glycerol, pH
7.9) was added. The mixture was then centrifuged at 14000 rpm at
4◦Cfor15min.Thenuclearproteinsweretransferredtonewtubes
as aliquots and stored at −70 ◦Cuntiluse.Theproteinconcentra-
tion was determined by a DC Protein Assay kit (Bio-Rad).
NF-kappa B DNA binding assay
NF-kappa B is a ubiquitous transcription factor that medi-
ates the inflammatory response. The proteasome inhibitor MG132
inhibits NF-kappa B formation and degradation of its inhibitor
I-kappa B (Ishikawa and others 1999). One microgram of nuclear
extract was used for NF-kappa B DNA binding assay. NF-kappa B
activation in the nuclear extracts was quantified by an EZ-Detect
NF-kappa B p65 assay kit (Pierce, Rockford, Ill., U.S.A.) based on an
ELISA technique. Specifically, an immobilized oligonucleotide con-
taining the NF-kappa B consensus site (5′-GGGACTTTCC-3′)was
H88 JOURNAL OF FOOD SCIENCE—Vol . 75, N r. 3, 20 10
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Limonene from yuzu essential oil . . .
bound to microwell plates. The active form of NF-kappa B in nu-
clear extracts that specifically binds to this oligonucleotide was de-
tected through a primary antibody that recognizes an epitope on
p65 that is accessible only when NF-kappa B is activated and bound
to its target DNA. To confirm specificity of NF-kappa B activity, a
competitive oligonucleotide (wild type) of the consensus site was
added. Following the addition of a HRP-conjugated secondary an-
tibody, the plates were read on an automated plate reader and the
level of nuclear NF-kappa B p65 was expressed as the absorbance
at 450 nm (A450).
Immunoblot analysis of Thr/Tyr-
phosphorylated p38 MAPK
Df-HL-60 clone 15 cells (5 ×106cells/mL; 5 mL) that had been
pre-incubated either with medium or 10 µM SB203580 for 1 h
were stimulated with 1 nmol/L eotaxin for 1 additional h. After eo-
taxin stimulation, the cells were further stimulated with medium or
limonene for 1 h. Phoshorylated-p38 MAPK and p38 MAPK were
analyzed with a commercial kit (Cell Signaling Technology, Inc.).
One microgram of cell lysates was separated by a 5% to 20% gra-
dient sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE) and transferred to membranes. Phosphorylation of
p38 MAPK was measured by western blot using anti-phospho-p38
MAPK antibody, and p38 MAPK was also measured by western
blot using anti-p38 MAPK antibody. The amounts of proteins were
quantified by a chemiluminescence imaging system (AE-6972 Den-
sitograph Lumino-CCD, ATTO, Tokyo, Japan).
Statistical analysis
Three independently replicated analyses were performed for
each experimental condition and time point, unless otherwise
noted. The results were expressed as the mean ±SD. Groups were
compared using Student’s t-test or one-way analysis of variance
(ANOVA) followed by post hoc Dunnett’s test. Statistical signifi-
cance was set at P<0.05.
Results
Inhibitory effect of limonene on ROS
production by df-HL-60 clone 15 cells
PMA-induced ROS production after treatment with limonene
was quantified for n-butyrate-induced df-HL-60 clone 15 cells
(Figure 1). For limonene concentrations greater than 7.34 mM, ROS
production was significantly decreased for df-HL-60 clone 15 cells.
ROS production was significantly increased after stimulation with
eotaxin compared to no eotaxin stimulation. ROS production af-
ter eotaxin stimulation was also significantly decreased after cell
treatment with limonene compared with the condition without
limonene (P<0.05).
Inhibitory effect of limonene on MCP-1
production by df-HL-60 clone 15 cells
MCP-1 production after treatment with limonene was quantified
for df-HL-60 clone 15 cells that were cultured with and without IL-1
(Figure 2). For limonene concentrations greater than 14.68 mmol/L,
MCP-1 production was significantly decreased in both groups with
and without IL-1 compared to the group without limonene treat-
ment (P<0.05).
Limonene inhibits DEPs-induced MCP-1 production
We ne xt e xa mi ne d DE Ps-induc ed M CP- 1 prod uc tion after tre at -
ment with 14.68 mmol/L limonene for df-HL-60 clone 15 cells. As
shown in the absence of limonene in Figure 3, MCP-1 was increased
after stimulation with DEPs compared with that of no DEPs stim-
ulation. In contrast, with limonene treatment, MCP-1 was signifi-
cantly decreased with or without DEPs stimulation compared to the
control group without limonene or DEPs (P<0.01). The addition of
the inhibitor MG132, with DEPs or without DEPs, significantly de-
creased MCP-1 production in both with and without limonene as
compared with the control (P<0.01).
Limonene attenuated NF-kappa B
activity in df-HL-60 clone 15 cells
NF-kappa B activity was measured in the nuclear extracts
from df-HL-60 clone 15 cells that were cultured with limonene
Figure 1 --- Concentration-dependent inhibitory activity
of ROS production by limonene for df-HL-60 clone 15
cells. Cells were treated with various concentrations of
limonene, after which they were stimulated by 50 nmol/L
PMA with 1 nmol/L eotaxin (Eotaxin) or without eotaxin
(Buffer). ROS was detected as described in Materials
and Methods. Results are means ±SEM of 3 separate
experiments done in duplicate. Statistically significant
differences were observed for cells stimulated by eo-
taxin compared to the cells without eotaxin, ∗
P
<0.05;
∗∗
P
<0.01. Statistically significant differences were ob-
served for eotaxin-stimulated cells with limonene treat-
ment compared with cells without limonene, #
P
<0.05;
##
P
<0.01.
Figure 2 --- Concentration-dependent inhibitory activity on
MCP-1 production by limonene for df-HL-60 clone 15
cells. Cells were treated with various concentrations of
limonene with or without IL-1 stimulation, and MCP-1 was
detected as described in Materials and Methods. Results
are means ±SEM of 3 separate experiments done in dupli-
cate. Statistically significant differences were observed
for IL-1-stimulated cells compared with the cells without
IL-1, ∗∗
P
<0.01. Statistically significant differences were
observed for IL-1-stimulated cells with limonene treat-
ment compared with the cells without limonene, #
P
<
0.05; ##
P
<0.01.
Vol . 75, N r. 3, 20 10—JOURNAL OF FOOD SCIENCE H89
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Limonene from yuzu essential oil . . .
(14.68 mmol/L) or without limonene. NF-kappa B activities for 1
µg protein samples are shown in Figure 4. NF-kappa B activity with
the addition of limonene decreased significantly compared with no
addition of limonene (P<0.01). This decrease in NF-kappa B ac-
tivity corresponded to those groups with the addition of Wild and
MG132.
Inhibitory effect of limonene on df-HL-60 clone
15 cell chemotaxis and the p38 MAP kinase
We examined eotaxin-induced chemotaxis after 10 µM
SB203580 treatment for df-HL-60 clone 15 cells with and without
14.68 mmol/L limonene. As shown in Figure 5, the percentage of
chemotaxis in limonene (column 2), SB203580 (column 3), and
limonene with SB203580 (column 4) were relatively low when
compared with medium alone (column 1).
Inhibitory effect of limonene on p38 MAPK
(Thr180/Thr182) activity in df-HL-60 clone 15 cells
We ex am in ed t he e ff ec t of l im on en e (1 4.68 mmol/L) on p38
MAPK (Thr180/Thr182) activity in df-HL-60 clone 15 cells. As
Figure 3 --- Effect of limonene on MCP-1 production for
DEPs-stimulated df-HL-60 clone 15 cells. Cells were
pipetted into culture flasks that had been pre-incubated
with either medium or 5 nmol/L MG132 for 1 h. These cells
were cultured either with medium alone or medium with
14.68 mM limonene, and without or with 0.1 mg/mL DEPs
for 24 h at 37 ◦C in 1.5% CO2.MCP-1levelsinculture
supernatants were determined by ELISA. The results are
means ±SEM of 3 separate experiments done in dupli-
cate. Statistically significant differences were observed
when compared with the medium alone group, ∗∗
P
<0.01.
Figure 4 --- Effect of limonene on NF kappa B activity for
df-HL-60 clone 15 cells. NF-kappa B activity was mea-
sured in the samples in the presence of competitive (Wild)
oligonucleotide or medium as described in Materials and
Methods. Activity results for Medium alone were set to
100%. Results are means ±SEM of 3 separate experi-
ments done in duplicate. Statistically significant differ-
ences were observed when compared with the medium
alone group, ∗∗
P
<0.01.
shown in Figure 6, the levels of the phosphorylated form has
been removed in limonene-treated cells (lane 2) compared with
untreated cells (lane 1) (P<0.01). The phoshorylation levels in
SB203580-pretreated cells (lane 3) and SB203580-limonene-treated
cells (lane 4) were also significantly lower (P<0.01). These results
indicated that the pretreatment with limonene and SB203580 in-
hibited an eotaxin-induced p38 MAPK activity.
Discussion
Inthisstudy,wehaveshownthatlimoneneextractedfromyuzu
essential oil has antioxidant activity, and also reduces eosinophil
chemotaxis and MCP-1 production. Limonene has been used in
Figure 5 ---Effect of limonene on eotaxin-induced df-HL-
60 clone 15 cells chemotaxis. Cells were pretreated for
1hinmediumwithorwithout10µM SB203580, fol-
lowed by stimulation for 1 h with 1 nmol/L eotaxin. Af-
ter eotaxin stimulation, the cells were further stimulated
with medium or limonene (14.68 mmol/L) for 1 h. Eotaxin-
induced chemotaxis was measured as described in Mate-
rials and Methods. Results are means ±SEM of 3 separate
experiments done in duplicate. Statistically significant
differences were observed compared with the SB203580
(−)/Medium group, ∗∗
P
<0.01.
Figure 6 --- Effect of limonene on p38 MAPK activity for df-
HL-60 clone 15 cells. Phosphorylation of protein was mea-
sured by western blot using phospho-p38 MAPK antibody
(upper lanes). Total p38 MAPK protein was also measured
using anti-p38 MAPK antibody (lower lanes). The cells
were cultured with medium alone (lane 1), 14.68 mmol/L
limonene (lane 2), 10 µMSB203580(lane3),and14.68
mmol/L limonene and 10 µMSB203580(lane4).Thefold
increase in amount of phospho-p38 MAPK is indicated
in the lower panel. The amounts of phospho-p38 MAPK
or p38 MAPK which were quantified by a chemilumines-
cence imaging system (AE-6972 Densitograph Lumino-
CCD, ATTO, Tokyo) were shown relative to medium alone.
Three independent experiments gave similar results. Re-
sults are means ±SEM of 3 experiments. Statistically
significant differences were observed compared to the
medium alone group, ∗∗
P
<0.01.
H90 JOURNAL OF FOOD SCIENCE—Vol . 75, N r. 3, 20 10
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Limonene from yuzu essential oil . . .
flavoring agents such as foods, beverages, liqueurs, and confec-
tioneries, and in aromatic agents such as soap, perfumery, and
household products. Furthermore, varieties of products made
from these oils have been used in aromatherapy, and also in
the relaxation and the stabilization of physical and psychological
conditions. Recent studies have shown that essential oil has an
antifungal activity (Behnam and others 2006), antinociception re-
sponse (Peana and others 2004), and stress repression (Nakamura
and others 2009). Thus, limonene has various bioactive properties.
More than 87 flavor compounds (GC peak area; 98.68%) in the
amount of yuzu essential oil were characterized by high-resolution
gas chromatography-mass spectrometry (HRGC-MS) analysis. Of
these, 34 were hydrocarbons including limonene (77.44%), gamma-
terpinene (9.43%), alpha-phellandrene (0.94%), and alpha-pinene
(2.03%), 37 were alcohols including linalool (1.56%), trans-nerolidol
(0.16%), and thymol/L (0.09%) and 16 were aldehydes including de-
canal (0.05%) and octanal (0.01%) (Sawamura 2005).
Inhibitory activity of malonaldehyde formation in various es-
sential oil was evaluated by Malonaldehyde/Gas Chromatography
(MA/GC) Assay (Wei and Shibamoto 2007). Limonene (inhibitory
activity; 74.6%) in celery seed, alpha pinene (33.7%), beta pinene
(1.4%), sabinene (10.8%), myrcene (12.4%), alpha-terpinene (1.3%),
linalool (4.5%), gamma-terpinene (2.4%), terpinolene (1.7%) in ju-
niper berry, alpha pinene (15.5%), beta pinene (11.7%), and beta-
phellandrene (2.9%) in parsley seed were measured.
Many essential oils effectively inhibit pro-inflammatory cy-
tokine production. Alpha-humulene from Cordia verbenacea pre-
vented neutrophil migration into carrageenan-stimulated mouse
air pouches, and TNF-alpha production was significantly de-
creased (Passos and others 2007). 1,8-cineol inhibited TNF-alpha
and IL-1beta in human lymphocytes, and lipopolysaccharide
(LPS)-stimulated monocytes (Juergens and others 2004). Terpinen-
4-ol suppressed the production of TNF-alpha, IL-1beta, IL-8, IL-10,
and PGE2 by LPS-activated monocytes (Hart and others 2000). An
essential oil extracted from Cinnamomum osmophloeum Kaneh
that contains 1,8-cineole, santolina triene, the sesquiterpenes
spathulenol and caryophyllene oxide was effectively inhibitory to
IL-1beta and IL-6 production, but not for TNF-alpha, of LPS-treated
J774A cells (Chao and others 2005) .
MCP-1 is one of the C-C chemokines that is produced sponta-
neously by eosinophilic cells (Wong and others 2005). Eosinophils
may participate in inflammatory/allergic reactions through the
generation of chemokines, such as MCP-1, further augmenting
leukocyte recruitment. (Goldstein and others 1996).
Infections with some bacteria and viruses are thought to exac-
erbate bronchial asthma. For instance, Pseudomonas aeruginosa,
human rhinoviruses (RVs), and double-stranded viral model RNA
are suspicious to exacerbate bronchial asthma (Papadopoulos and
others 2001; Tsuji and others 2005; Yan and others 2008). Some
chemical compounds from essential oils have exhibited antibac-
terial activity and antiviral infection against some organisms such
as Pseudomonas aeruginosa,Streptococcus pneumoniae,Mycobac-
terium smegmatis,andSalmonella typhii, Herpes simplex virus
(HSV), Influenza A virus, Influenza B virus, and Dengue virus type
2 (DENV-2), and Junin virus (JUNV) (Duschatzky and others 2005;
Naser and others 2005; Ooi and others 2006; Hayashi and others
2007; Saddi and others 2007; Koch and others 2008).
In a recent study, 5-O-Methylhirsutanonol (5-MH) which con-
cerned with ROS production in the regulation of NF-kappa B
signaling, suppressed the mRNA expression such as TNF-alpha,
cyclooxygenase (COX)-2 and IL-1 beta. This 5-MH also suppressed
the expression of inflammation-associated genes (Han and others
2008).
DEPs stimulate eotaxin gene expression via NF kappa B-
dependent activation in human airway epithelial cells (Takizawa
and others 2003). Eotaxin primed the production of ROS in a
dose-dependent manner (Honda and Chihara 1999). From our
data, limonene from yuzu essential oil inhibit the DEPs-stimulated
p38 MAPK signaling pathway, and also inhibited eotaxin-induced
chemotaxis by eosinophils. Thus, yuzu essential oil may be a nat-
ural drug for the treatment of bronchial asthma through its role
on ROS production and ameliorating oxidative damage to the lung
(Beck-Speier and others 2005; Risom and others 2005). In fact, ad-
ministration of antioxidant agents decreases eosinophils in lung
(Yamanaka and others 2006) and suppresses lung injury (Kaimul
Ahsan and others 2005; Behndig and others 2006; Nanua and oth-
ers 2006).
Bioflavonoid quercetin dramatically inhibited the IL-1 beta-
induced MCP-1 expression in mesangial cells and isolated
glomeruli. In addition, NF-kappa B inhibitor MG132 diminished
the IL-1 beta-induced expression of MCP-1 in these cells, whereas
c-Jun/AP-1 inhibitor curcumin did not affect this process (Ishikawa
and others 1999).
In this study, we observed that low concentration of limonene
(7.34 mM) decreased the production of ROS in eotaxin-stimulated
HL-60 clone 15 cells. At 14.68 mmol/L concentration, MCP-1 pro-
duction was decreased significantly. By adding the proteasome in-
hibitor MG132, NF-kappa B formation was diminished. By adding
SB203580 which is a specific p38 MAPK inhibitor, chemotaxis
was decreased significantly. Therefore, limonene extracted from
yuzu essential oil has an ability of antioxidant activity to human
eosinophilic cells and it may prevent to damage from DEPs in a
lung. MCP-1 production was also decreased by cell treatment with
limonene, suggesting that limonene might result in the decrease of
monocyte infiltration in lungs. Furthermore, limonene showed the
prevention of eosinophil migration and therefore limonene might
decrease the eosinophil infiltration in asthmatic lungs.
Conclusions
This study suggests that limonene from yuzu essential oil may
have potential efficacy for the treatment of bronchial asthma
through its anti-inflammatory activity by inhibiting cytokines, ROS
production, and inactivating eosinophil migration.
Acknowledgments
This study was supported by a grant of Special Research Project
of Green Science, Kochi Univ. There was no conflict of interest be-
tween the authors.
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