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Anti-inflammatory Effects of Limonene from Yuzu (Citrus junos Tanaka) Essential Oil on Eosinophils



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 examine 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 kinase (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 inhibited 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.
H: Health, Nutrition, &
JFS H: Health, Nutrition, and Food
Anti-inflammatory Effects of Limonene
from Yuzu (Citrus junos Tanaka)
Essential Oil on Eosinophils
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
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:
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
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
2010 Institute of Food Technologists RVol . 75, N r. 3, 20 10JOURNAL OF FOOD SCIENCE H87
doi: 10.1111/j.1750-3841.2010.01541.x
Further reproduction without permission is prohibited
H: Health, Nutrition, &
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-
(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
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
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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.
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,
<0.01. Statistically significant differences were ob-
served for eotaxin-stimulated cells with limonene treat-
ment compared with cells without limonene, #
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, ∗∗
<0.01. Statistically significant differences were
observed for IL-1-stimulated cells with limonene treat-
ment compared with the cells without limonene, #
0.05; ##
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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
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, ∗∗
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, ∗∗
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.
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, ∗∗
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, ∗∗
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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
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.
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.
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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... It has also been found that LM has antimicrobial properties that can work against foodborne pathogens [4]. Besides, LM contains anti-inflammatory properties and has been counted in bronchial asthma treatment [5]. In addition, LM is safe to consume because it is listed as generally considered safe (GRAS) [6]. ...
... We found that the addition of SB in disperse phase alongside Tween 80 helped to produce stable LM-loaded nanoemulsions, whereas CP had no effect. Emulsions prepared with LM blended SB were stable across the whole range of pH (3)(4)(5)(6)(7)(8)(9), and NaCl (0-500 mM) levels tested. In addition, the emulsions stored at 5 °C and 25 °C were remarkably physically stable in terms of the droplet size range (122-130 nm) over the entire period of 30 days of storage but highly unstable at 50 °C. ...
... We found that the addition of SB in disperse phase alongside Tween 80 helped to produce stable LM-loaded nanoemulsions, whereas CP had no effect. Emulsions prepared with LM blended SB were stable across the whole range of pH (3)(4)(5)(6)(7)(8)(9), and NaCl (0-500 mM) levels tested. In addition, the emulsions stored at 5 • C and 25 • C were remarkably physically stable in terms of the droplet size range (122-130 nm) over the entire period of 30 days of storage but highly unstable at 50 • C. ...
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The present study aimed to investigate the physical stability in terms of (droplet size, pH, and ionic strength) and chemical stability in terms of (retention) of D-limonene (LM) in the nanoemulsions after emulsification as well as after storing them for 30 days under different temperatures (5 °C, 25 °C, and 50 °C). LM is a cyclic monoterpene and a major component extracted from citrus fruits. The modification of disperse phase with soybean oil (SB) and a nonionic emulsifier (Tween 80) was adequate to prepare stable LM-loaded nanoemulsions. LM blended with SB-loaded nanoemulsions were stable against droplet growth over pH (3–9) and ionic strength (0–500 mM NaCl). Regarding long-term storage, the prepared nanoemulsions demonstrated excellent physical stability with droplet size ranging from 120–130 nm during 30 days of storage at both 5 °C and 25 °C; however, oiling off started in the emulsions, which were stored at 50 °C from day 10. On the other hand, the retention of LM in the emulsions was significantly impacted by storage temperature. Nanoemulsions stored at 5 °C had the highest retention of 91%, while nanoemulsions stored at 25 °C had the lowest retention of 82%.
... [103] In another development, essential oil from Yuzu was reported to possess anti-inflammatory properties by reducing eosinophil migration and inhibiting cytokine production, hence a potential agent for treating bronchial asthma. [105] Most of the anti-inflammatory and anti-nociceptive activities mediated by the various CEOs listed above worked much more effectively on acute pain and inflammation but few works have assessed their efficacy in chronic pain and chronic inflammatory models. ...
... Reduction in eosinophil migration Inhibits cytokine production [105] showed a significant antioxidant activity which was comparable to that of ascorbic acid. [109] In another study, the antioxidant effect of citrus fruit peels essential oil was also found to be superior to Trolox in terms of activity. ...
... [102] In another development, evaluation via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test also demonstrated that Kumquat essential oil possesses considerable radical scavenging activity. [73,112] Yuzu citrus fruit peels essential oil is also known to inhibit the production of radical oxygen species due to the presence of limonene [105] (Table 4). ...
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Citrus essential oil (CEO) is obtained from the fruit of Genus Citrus, a flowering plant shrub in the family of the Rutaceae (Eremocitrus or Microcitrus) and extensively used in food, chemical industry, and traditional medicinal treatment owing to its pleasant aroma, antioxidant, and antiseptic properties. This review presents a botanical description, distribution, traditional uses, chemical composition, bioactive components, and the therapeutic uses as well as toxicological effects of the CEO. The objective was achieved via a comprehensive literature search of electronic databases such as Science Direct, PubMed, Web of Science, Wiley, ACS, Springer, Taylor and Francis, Google Scholar, SCOPUS, conference proceedings, thesis, and books until 2022 for publications. Citrus essential oils and their constituents are extracted and isolated either from the fruit peels, seeds, leaves, or flowers of the citrus plants. A comparative study of the sources of CEO confirmed its origin, ethnopharmacological and therapeutic uses. Over 2000 secondary metabolites have been isolated, with the main active constituents: being terpenes, monoterpenes, sesquiterpenes, and diterpenes. A comprehensive literature review revealed vast therapeutic benefits of CEO. Incomplete data report on in vitro and in vivo trials especially, on dosage, positive and negative control groups, intervention time, toxicity studies, phytochemical profiling, and clinical trials seem to be a knowledge gap.
... Recently, studies have shown that D-limonene (LIM), a natural monocyclic monoterpene widely found in citrus fruits, has powerful antioxidant and anti-inflammatory properties (Roberto et al., 2010;Santana et al., 2020;Vieira et al., 2018). In addition, Hirota et al. (2010) demonstrated that it anti-inflammatory effects involves cytokines and reactive oxygen species (ROS) inhibition and inactivation of eosinophil migration (Hirota et al., 2010). LIM also presented a protective effect on acute lung injury induced by lipopolysaccharides (LPS) (Chi et al., 2013). ...
... Recently, studies have shown that D-limonene (LIM), a natural monocyclic monoterpene widely found in citrus fruits, has powerful antioxidant and anti-inflammatory properties (Roberto et al., 2010;Santana et al., 2020;Vieira et al., 2018). In addition, Hirota et al. (2010) demonstrated that it anti-inflammatory effects involves cytokines and reactive oxygen species (ROS) inhibition and inactivation of eosinophil migration (Hirota et al., 2010). LIM also presented a protective effect on acute lung injury induced by lipopolysaccharides (LPS) (Chi et al., 2013). ...
... Sá et al. (2013) suggested that LIM could be used as a potential anti-inflammatory agent for the treatment of bronchial asthma by suppressing cytokines, ROS production, and inactivating eosinophil migration, and Chi et al. (2013) demonstrated that LIM reduced lipopolysaccharide-induced inflammatory responses by suppressing MAPK and NF-κB pathways in acute lung injury. These findings are also confirmed by other studies in animals and humans, such as those by Hirota et al. (2010); Santana et al. (2020) and Yoon et al., 2010). ...
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Background Despite some advances in the discovery of novel therapies, smoke inhalation injury remains a difficult to treat critical health issue due to its physiopathological complexity. Natural products, such as D-limonene (LIM), are becoming an important potential source of new treatments in many health problems. LIM has been shown to have antioxidant, anti-inflammatory and protective effects in respect of several diseases, including respiratory conditions Objective The aim of this study was to evaluate the effects of inhaled LIM on acute smoke-induced lung injury in rats Methods Thirty minutes after smoke inhalation, adult male Wistar rats were treated with vehicle or LIM (0.01 mg/kg) for 30 min. Blood samples, and the liver, lungs, and trachea were collected for analysis Results The results showed that LIM minimized the injuries, reducing oxidative and inflammatory damage by improving catalase (CAT) (p < 0.05), and superoxide dismutase (SOD) activities (trachea: p < 0.01; lung: p < 0.05) and reducing interleukin-1 beta (IL-1β) level (p < 0.01) caused by smoke inhalation. LIM was also able to ameliorate damage in both trachea and lung tissues Conclusions The results indicate that LIM has a beneficial effect on lung injury, mainly by reducing oxidative stress, anti-inflammatory response and histological damage.
... Another study investigating limonene's anti-inflammatory response on human eosinophilic leukemia HL-60 clone 15 cells revealed interesting results. Hirota et al. [127] reported that a low concentration of limonene (7.34 mmol/L) can inhibit ROS production for eotaxin-stimulated HL-60 clone 15 cells. ...
... Furthermore, NF-κB formation was also diminished upon the addition of proteasome inhibitor MG132. The limonene can inhibit DEP induced p38 MAPK signaling pathway and inhibit eotaxin-induced chemotaxis by eosinophils [127]. Citrus EO components exhibit antioxidative activities against the oxidation of linoleic acid. ...
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Citrus is one of the main fruit crops cultivated in tropical and subtropical regions worldwide. Approximately half (40–47%) of the fruit mass is inedible and discarded as waste after processing, which causes pollution to the environment. Essential oils (EOs) are aromatic compounds found in significant quantities in oil sacs or oil glands present in the leaves, flowers, and fruit peels (mainly the flavedo part). Citrus EO is a complex mixture of ~400 compounds and has been found to be useful in aromatic infusions for personal health care, perfumes, pharmaceuticals, color enhancers in foods and beverages, and aromatherapy. The citrus EOs possess a pleasant scent, and impart relaxing, calming, mood-uplifting, and cheer-enhancing effects. In aromatherapy, it is applied either in message oils or in diffusion sprays for homes and vehicle sittings. The diffusion creates a fresh feeling and enhances relaxation from stress and anxiety and helps uplifting mood and boosting emotional and physical energy. This review presents a comprehensive outlook on the composition, properties, characterization, and mechanism of action of the citrus EOs in various health-related issues, with a focus on its antioxidant properties.
... Citrus junos Tanaka, also known as yuzu, is a citrus fruit mainly cultivated in Japan and Korea and widely used during the preparation of tea and sauce. Traditionally, it is used to treat bronchial and respiratory illnesses [5]. Previous investigations have shown that yuzu ...
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In the wake of the COVID-19 pandemic, lung disorders have become a major health concern for humans. Allergic asthma is the most prevalent form of asthma, and its treatments target the inflammation process. Despite significant developments in the diagnosis and management of allergic asthma, side effects are a major concern. Additionally, its extreme heterogeneity impedes the efficacy of the majority of treatments. Thus, newer, safer therapeutic substances, such as natural products, are desired. Citrus junos Tanaka has traditionally been utilized as an anti-inflammatory, sedative, antipyretic, and antitoxic substance. In this study, the protective effects of Citrus junos Tanaka peel extract (B215) against lung inflammation were examined, and efforts were made to understand the underlying protective mechanism using an HDM-induced lung inflammation murine model. The administration of B215 reduced immune cell infiltration in the lungs, plasma IgE levels, airway resistance, mucus hypersecretions, and cytokine production. These favorable effects alleviated HDM-induced lung inflammation by modulating the NF-κB signaling pathway. Hence, B215 might be a promising functional food to treat lung inflammation without adverse effects.
... These cells are capable of producing ROS, pro-inflammatory cytokines, and granular proteins with high cytotoxic potential. Similarly, fractalkine is a chemoattractant of monocytes, T cells, and natural killers under the induction of proinflammatory cytokines (49,50). These anti-inflammatory activities are greater in the groups treated with GRP I and GRP II, suggesting that the polysaccharide fractions of G. resinaceum would exert their anti-inflammatory activities in vivo by inhibiting the synthesis and production of prostaglandins and lysosomal enzymes and by regulating the levels of cytokines and chemokines, thus preventing massive invasion of immune cells at the site of inflammation. ...
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Introduction: Ganoderma resinaceum is used to treat oxidative and inflammatory-related diseases such as cardiovascular and liver diseases. Thus, this study aimed to evaluate the antioxidant and anti-inflammatory activities of different extracts from G. resinaceum fruiting bodies. Methods: Aqueous crude (GRT), mycelial (MYC), exopolysaccharide (EPS I, EPS II) and water-soluble polysaccharide-rich (GRP I and GRP II) extracts of G. resinaceum were assessed for their free radical scavenging and metal chelating ions assays. The in vitro anti-inflammatory activity was evaluated by stabilization of erythrocytes' membranes and protein denaturation assays. For the in vivo study, paw oedema was induced by administration of κ-carrageenan (0.1 mL; 1%) to male Wistar rats aged 4 to 6 weeks. Animals were pre-treated with G. resinaceum extracts (125 mg/kg) and diclofenac sodium (20 mg/kg). Inflammatory cytokine and chemokine levels were determined, and histological analysis of paw tissue was performed. Results: G. resinaceum polysaccharide-rich extracts (GRP I and GRP II) showed the best bioactivities. They scavenged DPPH (1,1-diphenyl-2-picrylhydrazyl, ABTS (2,2-azino-bis-3-ethylbenzylthiazoline-6-sulfonic acid, and NO (nitric oxide) radicals, and chelated ferrous ions, stabilized murine erythrocyte membranes, and inhibited protein denaturation. At 125 mg/kg, GRP I and GRP II restored the microarchitecture with a weak infiltration of immune cells in the subcutaneous tissues. Moreover, they decreased the overproduction of proinflammatory cytokines growth colony-stimulating factor (G-CSF), interferon gamma (IFNγ), tumour necrosis factor alpha (TNFα), chemokines (eotaxin, fractalkine) and increased the levels of anti-inflammatory cytokines (IL-10, IL-12p70).
... By observing the effect of nutmeg EO on human fibroblasts, Matulyte et al. (24) reported that nutmeg EO had anti-inflammatory effect, protected cell viability and reduced the release of IL-6. Limonene in citrus EO inhibited TNF-α, IL-1β, IL-8, and IL-10 to inhibit p38 mitogen activated protein kinase signaling pathway and regulate chemokine induced chemotaxis (25). In addition, limonene might reduce the infiltration of monocytes and eosinophils. ...
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Inflammatory diseases are a major threat to public health. Natural plant essential oils (EOs) possess anti-inflammatory and anti-oxidative activities. The objective of this study was to investigate the anti-inflammatory effect and mode of action of lemon essential oil (LEO), and its main active component, d-limonene, with different doses on intestinal inflammation of mice. Sixty-four 5-week-old male balb/c mice weighing 22.0 ± 1.5 g were randomly assigned into one of 8 treatments (n = 8/treatment), including normal saline group (NS), Escherichia coli (E. coli) group, and either LEO and d-limonene essential oil (DEO) group supplemented at 300, 600, and 1,200 mg/kg of BW, respectively. After the pre-feeding period, the mice were fasted for 12 h, the mice in the NS group and the E. coli group were gavaged with normal saline, and the mice in the LEO group and DEO group were gavaged with respective dose of EOs for 1 week. One hour after the end of gavage on the 7th day, except that the mice in the normal saline group were intraperitoneally injected with normal saline, the mice in the other groups were intraperitoneally injected with the same concentration of E. coli (108 cfu/ml, 0.15 ml per mouse). The antioxidant indexes were measured including superoxide dismutase (SOD), malondialdehyde (MDA), and myeloperoxidase (MPO) in plasma obtained by taking blood from mouse eyeballs. The inflammatory indexes were measured including interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor alpha (TNF-α) in plasma. The tight junction protein indicators were tested include zona occludens 1 protein (ZO-1), occludin and claudin in mouse duodenum. We found that all of the above indexes for E. coli group were different (P< 0.05) with the NS group. The interaction of EO and dose (E × D) were significant (P < 0.01) for all of the indexes. In addition, LEO at 300 mg/kg BW and DEO at 600 mg/kg BW had better antioxidant and anti-inflammation activity on the infected mice, which reduced (P < 0.05) the plasma concentrations of MDA, MPO, TNF-α, IL-1β, and IL-6, but increased (P < 0.01) the concentrations of SOD. Hematoxylin-eosin (H&E) staining of duodenum observation showed that LEO and DEO reduced inflammatory cell infiltration and maintain the orderly arrangement of epithelial cells. Moreover, supplementation of LEO at 600 mg/kg and DEO at 300 mg/kg BW alleviated (P < 0.05) intestinal barrier injury for increasing the relative expression of ZO-1, occludin and claudin mRNA in mice duodenum. These results showed that the pre-treatment with LEO and DEO had protection of intestinal tissue and inflammation in E. coli infected mice. Both LEO and DEO exhibited activity of antioxidant, anti-inflammatory and alleviating intestinal injury, whereas, compared with DEO, LEO can be active at a lower dosage. Furthermore, as the main active component of LEO, the d-limonene appeared to play not only the major role, but also the joint action with other active components of LEO.
In this study, tarragon (Artemisia dracunculus L.) the amount of antioxidants and phenolic substances of water-based extracts in various concentrations (25g/L, 50g/L, 100g/L) with the aroma components of the plant was determined. Various food pathogens (Escherichia coli ATCC 25922, Salmonella enterica ATCC 13076, Listeria monocytogenes ATCC 43251) and bacterial strains ((Gram-negative (Vibrio harveyi (KF443058), Vibrio vulnificus (KF443056), Aeromonas veronii (KF443053), Vibrio anguillarum (NR 029254.1) and Vibrio campbellii (MH231447.1), Vibrio rotiferianus (NR 042081.1), Vibrio ponticus (NR 029032.1), Psychrobacter marincola (NR 025458.1), Pseudoalteromonas prydzensis (NR 044803.1), Pseudoalteromonas mariniglutinosa (NR 028992.1) and Gram-positive (Bacillus thuringiensis (NR 043403.1)) obtained from naturally infected Dicentrarchus labrax fish were determined by the disk diffusion method on their antimicrobial properties. As a result of the study, antioxidant values were found to be 88.5% at maximum concentrations of 10%, while the total phenolic substance content was determined between 3.75-5.06 mg GAE/g values. The main component of the tarragon plant was terpinyl acetate (23.16%), followed by α-terpineol (20.08%), anethole-(Z) (8.93%), limonene (5.20%), spathulenol (4.47%), ısoeugenol (3.73%), valeric acid (3.40%), eucalyptol (3.26%). No antimicrobial activity was determined on the test microorganisms used in the study.
Background Cymbopogon khasianus is a widely used industrial and pharmacologically important aromatic grass species. Objective The present investigation was designed to study and compare the elemicin rich Cymbopogon khasianus essential oil (EREO) and its pharmacological effects, genotoxicity with pure compound elemicin Materials and Methods Chemical composition, identification was performed using GC/MS and NMR techniques. 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), reducing power assay for antioxidant; albumin denaturation, protease inhibitor for anti-inflammatory; acetylcholinesterase (AChE) for anti-cholinesterase; amylase inhibitory for anti-diabetic; tyrosine inhibitory for skin whitening; disc diffusion and minimum inhibitory concentration assay for antimicrobial, and Allium cepa assay for genotoxicity were used. Results GC/MS analysis identified 38 compounds; among them elemicin 72.34%, D-limonene 3.81%; methyl eugenol 3.34% were the major compounds. A significant amount of antioxidant activity IC50 31.38 µg/mL; anti-inflammatory activity (protein denaturation assay) IC50 16.77 µg/mL; protease inihibitor assay IC50 51.08 µg/mL; anticholinesterase IC50 12.095 µg/mL; antidiabetic activity IC50 17.36 µg/mL; and anti-tyrosinase activity IC50 17.69 µg/mL were reported. Antimicrobial activity analysis against 13 microbial strains revealed negative effect. Genotoxicity study using Allium cepa assay revealed negative toxicity of EREO with aberration percentage of 04.30% and pure elemicin 05.30% which was very low in comparison to ethyl-methanesulfonate (EMS) 13.90%. Conclusion To the best of our knowledge this is the first scientific evaluation of novel elemicin rich EREO pharmacological properties and to compare with pure compound elemicin. Together, it can be stated that EREO possesses antioxidant, anti-inflammatory, anti-cholinesterase activities way better than pure compound elemicin as well as standard drugs used.
Ethnopharmacological relevance Inflammation is the body's normal protective response to injury and is stimulated by pathogens, toxic compounds, damaged cells or radiation, promoting healing and restoring homeostasis to the injured tissue. Leaves of Piper gaudichaudianum Kunth, known as “pariparoba” are widely used in folk medicine for the relief of toothache, while the fresh roots are used as anti-inflammatory and to treat liver disorders. For P. mikanianum (Kunth) Steud is known as “aguaxima”, is widely used in the treatment of inflammation, rheumatism and ulcer, with its roots being used for stomach disorders and as a diaphoretic in intermittent fevers. Aim of the study Therefore, this work aims to chemically characterize the essential oil of Piper gaudichaudianum and Piper mikanianum, as well as the evaluation of neutrophil antichemotactic activity of both essential oils in order to complement the information of its traditional use, taking the leaves as plant material and, with that, corroborating its use in folk medicine for the treatment of inflammatory diseases. Materials and methods The essential oil from leaves of both Piper species were obtained from crushed fresh samples, by hydrodistillation using a Clevenger type-apparatus for 4 h. The yield determination was performed as volume/weight (v/w) and in triplicate. The amount of essential oil obtained was quantified in mL. The identification and quantification of the compounds was performed using gas chromatography–mass spectrometry (GC–MS) and gas chromatography–flame ionization detection (GC-FID). The in vitro anti-inflammatory activity was evaluated using the model of modified Boyden chamber. In this test the essential oils were tested for their ability to inhibit leukocyte chemotaxis stimulated by Escherichia coli lipopolysaccharide. Results The chemical composition of the essential oils revealed the identification of 26 constituents for P. gaudichaudianum being the sesquiterpenes β-selinene (14.0%) and viridiflorene (10.5%) the main compounds, followed by caryophyllene oxide (9.3%) and (E)-nerolidol (9.0%). For P. mikanianum essential oil, β-myrcene (17.2%) and bicyclogermacrene (26.3%) were the major components in the monoterpenes and sesquiterpene fractions, respectively. The essential oils were also tested for their ability to inhibit neutrophil chemotaxis in vitro when stimulated by Escherichia coli lipopolysaccharide. Both essential oils showed antichemotactic effect with reduction in migration of 0–72.2% for P. gaudichaudianum and 8.6–100% for P. mikanianum to same concentrations, suggesting a response to acute inflammatory processes. Conclusions Since up to date there is no report of this biological activities by this mechanism (antichemotactic assay) for essential oils this species. These results showed that the essential oils of P. gaudichaudianum and P. mikanianum have a great capacity to inhibit neutrophil chemotaxis in an inflammatory process, in a dose-dependent way, suggesting anti-inflammatory potential, by preventing its accumulation at the injury site with the possibility of tissue damage. Findings of these studies support the traditional use of these species in the treatment of inflammatory processes.
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Objective and Design: To evaluate potential anti-inflammatory properties of tea tree oil, the essential oil steam distilled from the Australian native plant, Melaleuca alternifolia.¶Material and Methods: The ability of tea tree oil to reduce the production in vitro of tumour necrosis factor-α (TNFα), interleukin (IL)-1β, IL-8, IL-10 and prostaglandin E2 (PGE2) by lipopolysaccharide (LPS)-activated human peripheral blood monocytes was examined.¶Results: Tea tree oil emulsified by sonication in a glass tube into culture medium containing 10% fetal calf serum (FCS) was toxic for monocytes at a concentration of 0.016% v/v. However, the water soluble components of tea tree oil at concentrations equivalent to 0.125% significantly suppressed LPS-induced production of TNFα, IL-1β and IL-10 (by approximately 50%) and PGE2 (by approximately 30%) after 40 h. Gas chromatography/ mass spectrometry identified terpinen-4-ol (42%), α-terpineol (3%) and 1,8-cineole (2%, respectively, of tea tree oil) as the water soluble components of tea tree oil. When these components were examined individually, only terpinen-4-ol suppressed the production after 40 h of TNFα, IL-1β, IL-8, IL-10 and PGE2 by LPS-activated monocytes. Conclusion: The water-soluble components of tea tree oil can suppress pro-inflammatory mediator production by activated human monocytes.
The aim of this study was to investigate the effect of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in HL-60 clone 15 cells as they differentiate along the eosinophilic lineage. The results were compared to the eosoniphilic inducers interleukin-5 (IL-5) and butyric acid. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) or IL-5 (200 pM) caused a significant increase in the expression of the eosinophil peroxidase (EPO) and the major basic protein (MBP) genes. Similar results were observed when the cells were cultured with 0.5 mM butyric acid for 5 days. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) also caused a significant increase in superoxide release by HL-60 clone 15 cells after 2 days compared with control or with butyric acid-induced cells. After 5 days, these cytokines and butyric acid induced an even stronger release of superoxide. HL-60 clone 15 cells cultured with IFN-gamma and TNF-alpha for 2 days showed a significant increase in gp91-phox gene expression. We conclude that IFN-gamma and TNF-alpha are sufficient to induce the differentiation of HL-60 clone 15 cells to the eosinophilic lineage and to upregulate gp91-phox gene expression and activity of the NADPH oxidase system.
The volatile samples of the pummelo (Citrus grandis Osbeck forma Tosa-buntan) were obtained by cold-pressed procedure and steam distillation under reduced pressure with subsequent solvent extraction. Extracts were analyzed by glass capillary gas chromatography and mass spectrometric techniques. A total of 28 compounds were identified and quantitated on the basis of two kinds of internal standards. Nootkatone is one of the characteristic components of the pummelo's aroma, in common with the grapefruit. 1,8-Cineole overlapping with limonene on the gas chromatogram was quantitatively determined by mass fragmentography.
Background: Diesel emission particulates (DEP) exert effects on the immune system and act as an adjuvant which enhances allergic inflammation. Animal and human models have delineated the effects of DEP chemicals in enhancing IgE production and promoting T-helper cell-2 (Th2) differentiation. An important primary effect that can explain the DEP-associated humoral and cellular immune responses is the induction of macrophage responses by DEP chemicals. This includes effects on macrophage production of cytokines and chemokines, which may play a role in enhancing allergic inflammation. A potent mechanism in macrophages exposed to DEP chemicals involves the generation of reactive oxygen species (ROS), leading to cellular activation or apoptosis which can be abrogated by antioxidants. Conclusion: These findings may establish a role for antioxidant therapy in diminishing the effects of particulate pollutants in asthma.
The general antifungal activity of essential oils is well documented. The advantage of essential oils is their bioactivity in the vapor phase, a characteristic that makes them attractive as possible fumigants for stored product protection. Essential oils of aerial parts of Mentha piperita and Lavendula angustifolia were obtained with hydrodistillation and oils composition identified with GC-MS. Menthanol (36.24%) and menthone (32.42%) were the major compounds of the M. piperata essential oil. The essential oil of L. angustifolia was rich in linalool (49.2%) , linalyl acetate (12.3%), Lavendul acetate (6.5%), 4-terpineol (5.9%). Fungal toxicity of the essential oils were evaluated against three pathogenic fungi (Rhizopus stolonifer, Botrytis cinerea and Aspergillus niger) in vitro. Plate assayes showed that the different concentrations of essential oils have antifungal activity against these fungi, and the essential oil of L. angustifolia showed stronger fungistatic activity. Lavendula oil exhibited complete growth inhibition of all pathogens at 1000 ppm and minimum EC50 (311.24 ppm) resulted on B. cinerea.
As an attempt to quantitatively analyze the physiopsychological effects elicited by odorants, white blood cells and gene expression were profiled in the whole blood of the rats exposed to (R)-(-)-linalool during restraint stress for 2 h. In neutrophils and lymphocytes, significant changes caused by the restraint were repressed by their exposure to the odorant. This indicates that inhalation attenuates stress-induced changes. Significant changes on the stress-induced variations were induced by inhalation in 115 gene expression levels. Of those, 109 genes were down-regulated, whereas the remaining 6 were up-regulated. These findings show that (R)-(-)-linalool inhalation represses stress-induced effects on the profiles of both blood cells and gene expression. Furthermore, these results suggest the possibility that the odorant-induced effects can be quantitatively evaluated by analyzing the profiles of blood cells and gene expression.
A convenient and rapid method for preparing soluble extracts from the nuclei of as few as 3 x 10(7) mammalian cells (miniextract procedure) is described. By several criteria, miniextracts are comparable to nuclear extracts prepared from large numbers of cells by the conventional procedure. Miniextracts are able to support efficient transcription of a variety of class II promoters. In addition, DNase I footprinting and gel retardation assays can be performed directly in miniextracts, enabling the detection of sequence-specific DNA-binding proteins. Besides transcription, miniextracts efficiently carry out pre-mRNA splicing and allow formation and fractionation of previously characterized splicing complexes. The small-scale procedure enables simultaneous preparation of multiple extracts from a variety of cell types under different experimental conditions. Moreover, the use of small amounts of cells allows minimal expenditure of valuable or expensive materials such as radioactive compounds. Consequently, the procedure is highly advantageous for biochemical analysis of transcription and RNA processing in mammalian cells.
There are numerous characteristics in which mitochondria resemble bacteria and differ from the enveloping eukaryotic cell. These similarities and differences have been offered in support of the symbiotic origin of mitochondria. Such evidence, no matter how striking, can be faulted as representing retained primitive genome if the characteristics being compared evolved prior to the divergence of protoeukaryotes from prokaryotes. In contrast, if a characteristic evolved after this evolutionary divergence, in response to a relatively recent environmental change, it could serve as a clear marker of the symbiotic event. The enzyme superoxide dismutase, which serves as a defense against oxygen toxicity, need not have existed prior to the accumulation of photosynthetic oxygen. It probably evolved after the appearance of blue-green algae and it was apparently evolved independently by prokaryotes and by protoeukaryotes. The superoxide dismutases found in prokaryotes and in mitochondria are remarkably similar in gross properties and in amino acid sequence; whereas the corresponding enzyme of the eukaryotic cytoplasm is entirely different. This represents support for the symbiotic origin of mitochondria which is not easily argued away.
We designed a 48-well chemotaxis chamber to minimize manipulation time and amount of material required by the larger blindwell or Boyden chemotaxis chamber. Cell and chemoattractant dose-response curves showed that results were comparable to our better than those obtained with blindwell chambers. The volume of chemoattractant per well is 25 microliter; the number of cells can be as low as 10,000. The time needed for setting up this multiwell unit and for staining the membrane filter sheet is negligible. Combined with the use of an image analyzer to count the number of migrated cells, the method is suitable for clinical research on the functional state of monocytes in large groups of patients.
Inducible nitric oxide synthase (iNOS) is expressed in response to cytokines by a number of cell types participating in CNS inflammation, including brain cerebral endothelial cells. NF-kappaB, a transcription factor, mediates effector actions of pro-inflammatory cytokines. A combination of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) enhanced the expression of iNOS in murine cerebral endothelial cells (MCECs). In an attempt to modulate TNF-alpha+IFN-gamma induced expression of iNOS in MCECs, we designed a double-strand hairpin (hp) oligonucleotide carrying the NF-kappaB motif. This hp oligonucleotide inhibited NF-kappaB binding activity and decreased both iNOS mRNA and protein expression induced by TNF-alpha+IFN-gamma. As a control, a mutant hp oligonucleotide was without effect. The present study confirms the role of transcription factor NF-kappaB in iNOS expression induced by TNF-alpha+IFN-gamma in MCECs. More importantly, it demonstrates that an appropriately designed hp oligonucleotide is an effective tool to modulate iNOS expression and may be of potential pharmacological use.