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Journal of Ethnopharmacology 134 (2011) 305–312
Contents lists available at ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
Anti-inflammatory and antinociceptive properties of the leaves of Eriobotrya
japonica
Dong Seok Cha, Jae Soon Eun, Hoon Jeon ∗
College of Pharmacy, Woosuk University, Chonbuk 565-701, Republic of Korea
a r t i c l e
i n f o
Article history:
Received 22 June 2010
Received in revised form 9 December 2010
Accepted 10 December 2010
Available online 21 December 2010
Keywords:
Eriobotrya japonica
Anti-inflammatory
Antinociceptive
a b s t r a c t
Aim of the study: The leaves of Eriobotrya japonica Lindl. have been widely used as a traditional medicine
for the treatment of many diseases including coughs and asthma. The present study was designed to
validate the anti-inflammatory and antinociceptive properties of the n-BuOH fraction of E. japonica (LEJ)
leaves.
Materials and methods: The anti-inflammatory properties of LEJ were studied using IFN-␥/LPS activated
murine peritoneal macrophage model. The antinociceptive effects of LEJ were assessed using experimental models of pain, including thermal nociception methods, such as the tail immersion test and the
hotplate test, and chemical nociception induced by intraperitoneal acetic acid and subplantar formalin
in mice. To examine the possible connection of the opioid receptor to the antinociceptive activity of LEJ,
we performed a combination test with naloxone, a nonselective opioid receptor antagonist.
Results: In the IFN-␥ and LPS-activated murine peritoneal macrophage model, LEJ suppressed NO production and iNOS expression via down-regulation of NF-␬B activation. It also attenuated the expression of
COX-2 and the secretion of pro-inflammatory cytokines like TNF-␣ and IL-6. Moreover, LEJ also demonstrated strong and dose-dependent antinociceptive activity compared to tramadol and indomethacin in
various experimental pain models. In a combination test using naloxone, diminished analgesic activities of LEJ were observed, indicating that the antinociceptive activity of LEJ is connected with the opioid
receptor.
Conclusions: The results indicate that LEJ had potent inhibitory effects on the inflammatory mediators
including nitric oxide, iNOS, COX-2, TNF-␣ and IL-6 via the attenuation of NF-␬B translocation to the
nucleus. LEJ also showed excellent antinociceptive activity in both central and peripheral mechanism as
a weak opioid agonist. Based on these results, LEJ may possibly be used as an anti-inflammatory and an
analgesic agent for the treatment of pains and inflammatory diseases.
© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Eriobotrya japonica Lindl., also known as ‘loquat’, belongs to the
Rosaceae family. The plant is an evergreen shrub or small tree with
narrow leaves that are dark green on the upper surface and have
a lighter color under surface. The plant originated in south-eastern
China and later became naturalized in Korea, Japan, India and many
other countries. In Korea, the leaves of E. japonica have been widely
used as a traditional medicine with beneficial effects for pain and
chronic inflammatory diseases including headache, low back pain,
dysmenorrhoea, asthma, phlegm and chronic bronchitis (Ito et al.,
2000).
Various triterpenes, sesquiterpenes, flavonoids, tannins and
megastigmane glycosides were analyzed from LEJ, and some of
∗ Corresponding author. Tel.: +82 06 290 1576; fax: +82 06 290 1577.
E-mail address: hoonj6343@hanmail.net (H. Jeon).
0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2010.12.017
them have been found to possess anti-tumor, antiviral, hypoglycemic and anti-inflammatory properties (Shimizu et al., 1986;
De Tommasi et al., 1991; Ito et al., 2000; Taniguchi et al., 2002; Kim
and Shin, 2009).
During the inflammatory process, macrophages produce
nitric oxide, pro-inflammatory enzymes and cytokines. Because
overproduction of these inflammatory mediators might cause
inflammatory damage, many studies have attempted to find materials from traditional plant-derived medicines which selectively
modulate these mediators (Lee et al., 2005). Previously, triterpene acids from LEJ have been reported to have an inhibitory
effect on inflammatory cytokine secretion and the MAPK signal
transduction pathway (Huang et al., 2009). An anti-inflammatory
effect in rat model of chronic bronchitis has also been observed
(Ge et al., 2009). In addition, Banno et al. (2005) demonstrated
that LEJ has anti-inflammatory activity in a TPA-induced inflammation model. These reports strongly suggest that LEJ is an
anti-inflammatory agent. However, there have been no investiga-
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tions of LEJ’s anti-inflammatory activity in the IFN-␥/LPS activated
murine macrophage model.
Despite recent advances in developing new therapies, there
is still a need for effective painkillers. In this regard, new drugs
originated from natural products have received a lot of attention
and many plant-derived compounds have effective antinociceptive activities (Calixto et al., 2000). The plant used in this study,
LEJ, was traditionally used as a painkiller. Nevertheless, there is no
scientific report available in the literature on the antinociceptive
activity of LEJ. Therefore, in the present study, we investigated the
anti-inflammatory and antinociceptive effects of LEJ.
2. Materials and methods
cal density (OD) at 540 nm using an automated microplate reader
(GENios, Tecan, Austria).
2.4.3. Measurement of nitrite concentration
Peritoneal macrophages (3 × 105 cells/well) were cultured with
various concentrations of LEJ. The cells were then stimulated with
rIFN-␥ (20 U/ml). After 6 h, the cells were finally treated with LPS
(10 ␮g/ml). NO synthesis in cell cultures was measured with a
microplate assay method. To measure nitrite, 100 ␮l aliquots were
removed from conditioned medium and incubated with an equal
volume of Griess reagent at room temperature for 10 min. The
absorbance at 540 nm was determined by a microplate reader. The
quantity of NO2 − was calculated by using sodium nitrite as a standard.
2.1. Plant material
The plant materials were purchased from Hainyakupsa (Chonbuk, South Korea) in October 2009. The plant was identified by Dr.
Dae Keun Kim, College of Pharmacy, Woosuk University, Republic of
Korea. A voucher specimen (WOPE057) has been deposited at the
Department of Oriental Pharmacy, College of Pharmacy, Woosuk
University.
2.2. Extraction and fractionation of plant material
We extracted the dried sample (2000 g) using 12,000 ml of
MeOH with 2 h sonication. The extract was concentrated into 60.7 g
(yield: 3.035%) using a rotary evaporator. Then, the sample was subjected to successive solvent partitioning to yield n-hexane (0.83 g),
CH2 Cl2 (16.4 g), EtOAc (21 g) and n-BuOH (15.4 g) soluble fractions.
Because the preliminary experiments for anti-inflammatory and
antinociceptive activity of the fractions showed that the n-BuOH
fraction was the most potent, further studies were conducted using
the n-BuOH fraction.
2.3. Animals
ICR mice (six-week-old males and females) weighing 20–25 g
and C57BL/6 mice (five-week-old) weighing 18–22 g were supplied by Damul Science (Dajeon, Korea). All animals were housed
at 22 ± 1 ◦ C with a 12 h light/dark cycle and fed a standard pellet
diet with tap water ad libitum. For the purpose of isolating peritoneal macrophages, the C57BL/6 mice were given intraperitoneal
(i.p.) injections three days earlier with 2.5 ml of thioglycollate (TG)
solution. The experimental protocols complied with the recommendations of the International Association for the Study of Pain
(Zimmermann, 1983).
2.4. Anti-inflammatory study
2.4.1. Isolation and culture of mouse macrophages
TG-elicited macrophages were harvested three days after i.p.
injection of TG and isolated. Using 8 ml of PBS containing 10 U/ml
heparin, peritoneal lavage was performed. Then, the cells were
distributed in FBS-free DMEM and maintained at 37 ◦ C in a humidified atmosphere of 5% CO2 . After 3 h, the cells were washed three
times with PBS to remove non-adherent cells and equilibrated with
DMEM that contained 10% heat-inactivated FBS before treatment.
2.4.2. Determination of cell viability
Cell respiration, an indicator of cell viability, was measured by
the mitochondrial dependent reduction of 3-(3,4-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan, as
described by Mosmann (1983). The extent of the reduction of MTT
to formazan within cells was quantified by measuring the opti-
2.4.4. Preparation of nuclear extracts
Nuclear extracts were prepared as described previously (Baek
et al., 2002). Briefly, the cells were allowed to swell by adding
lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM
dithiothreitol and 0.5 mM phenylmethylsulfonylfluoride). Pellets
containing crude nuclei were resuspended in extraction buffer
(20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride) and incubated
for 30 min on ice. The samples were centrifuged at 12,000 rpm for
10 min to obtain the supernatant containing nuclear extracts.
2.4.5. Western blot analysis
Whole cell lysates were made by boiling peritoneal
macrophages in sample buffer (62.5 mM Tris–HCl pH 6.8,
2% sodium dodecyl sulfate (SDS), 20% glycerol and 10% 2mercaptoethanol). Proteins in the cell lysates were then separated
by 10% SDS-polyacrylamide gel electrophoresis and transferred
to nitrocellulose paper. The membrane was then blocked with
5% skim milk for 2 h at room temperature and then incubated
with anti-iNOS, anti-NF-␬B (Santa Cruz Biotechnology, CA, USA)
and anti-COX-2 (Pierce Biotechnology, IL, USA). After washing the
membrane in phosphate buffered saline (PBS) containing 0.05%
tween 20 three times, the blot was incubated with HRP-conjugated
anti-rabbit and anti-mouse (Amersham Biosciences, Little Chalfont, UK) and the target proteins were visualized by an enhanced
chemiluminesence detection system (Millipore Corporation, MI,
USA).
2.4.6. Assay of cytokine release
Peritoneal macrophages (3 × 105 cells/well) were treated with
various with concentrations of LEJ. The cells were then stimulated with rIFN-␥ (20 U/ml) plus LPS (10 ␮g/ml) and incubated for
24 h. The amount of TNF-␣ and IL-6 in the supernatants (from
3 × 105 cells/ml, culture medium DMEM with 10% FBS) were measured by a sandwich enzyme-linked immunosorbent assay (ELISA)
according to the manufacturer’s protocol (BD biosciences, CA, USA).
Absorption of the avidin–horseradish peroxidase color reaction
was measured at 405 nm and compared to the serial dilutions of
recombinant mouse TNF-␣ and IL-6, which were used as standards.
2.5. Antinociceptive study
2.5.1. Grouping and drug administration
Animals were randomly assigned into several groups, each consisting of eight or ten mice for analgesic tests. Negative controls
were treated with the same volume of distilled water which was
used for reconstituting the drug. Positive controls were treated with
standard drugs: tramadol (i.p.) or indomethacin (p.o.). Treatment
groups in each test were treated orally with different doses of LEJ.
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2.5.2. Acute toxicity test
To evaluate possible toxicity, the acute toxicity test was carried
out. Mice (n = 6) were tested by administering different doses of
LEJ and the doses were increased or decreased according to the
response of the animal (Bruce, 1985). The control group received
only the equal volume of distilled water. All of the groups were
observed for any gross effect or mortality for a 24 h period.
2.5.3. Tail immersion test
In the present study, the tail immersion test was performed
according to the procedures used by Wang et al. (2000) with minor
modification. Briefly, the lower two-thirds of mouse’s tail was
immersed on a water bath set at temperature of 50 ± 0.2 ◦ C. The
reaction time, i.e. the amount of time it takes the animal to withdraw its tail, was measured 0, 30, 60, 90 and 120 min after the
administration of LEJ (250, 500 mg/kg; p.o.), tramadol (10 mg/kg;
i.p.) and vehicle (D.W). To avoid tissue injury, the cut-off time was
20 s.
2.5.4. Hot plate test
The hot plate test (Franzotti et al., 2000) was carried out on
groups of male and female mice using a hot plate apparatus,
maintained at 55 ± 1 ◦ C. Only mice that showed initial nociceptive
responses (licking of the forepaws or jumping) between 7 and 15 s
were used for additional experiments. The chosen mice were pretreated with LEJ (250, 500 mg/kg; p.o.) or vehicle (D.W), and 30 min
later the measurements were taken. A tramadol (10 mg/kg; i.p.)
treated animal group was included as a positive control. To examine
the possible connection of endogenous opioids to antinociceptive
activity, LEJ and tramadol were investigated in groups of mice pretreated with naloxone (5 mg/kg; i.p.). The cut-off time was set at
30 s to minimize skin damages. The reaction time was calculated as
described for the tail immersion test.
2.5.5. Acetic acid-induced writhing test
Antinociceptive activity of LEJ was detected as previously
described (Olajide et al., 2000). The response to an intraperitoneal
injection of acetic acid solution (1% in 0.9% saline), which consisted of abdominal constrictions and hind limb stretching, was
measured for each mouse starting 5 min after the acetic acid injection and was measured for an additional 20 min. Each experimental
group was treated orally with vehicle (D.W), LEJ (250, 500 mg/kg)
or indomethacin (10 mg/kg) 1 h prior to the acetic acid injection.
To examine the possible connection of endogenous opioids to the
antinociceptive activity, the LEJ group was pretreated with naloxone (5 mg/kg; i.p.).
2.5.6. Formalin test
In the formalin test (Santos and Calixto, 1997), groups of mice
were treated orally with vehicle (D.W) or LEJ (250, 500 mg/kg). After
30 min, each mouse was treated with 20 ␮l of 5% formalin (in 0.9%
saline, subplantar) into the right hind-paw. The duration of paw
licking (s) was used as an index to measure the pain response during the 0–5 min period (first phase, neurogenic) and the 20–35 min
period (second phase, inflammatory) after formalin injection. Tramadol and indomethacin were used as positive control drugs and
were administrated 30 min before the test at a dose of 10 mg/kg,
i.p. and p.o., respectively.
2.6. Statistical analysis
The results are expressed as the mean ± S.D. or mean ± S.E.M.
depending on the experiments. Data between groups were analyzed by a Student’s unpaired two-tailed t-test and p-values less
than 0.01 were considered significant. The intensity of the bands
Fig. 1. Effects of LEJ on NO production in rIFN-␥/LPS-activated mouse peritoneal
macrophages. Peritoneal macrophages (3 × 105 cells/well) were cultured with various concentration of LEJ or 0.05% DMSO, a vehicle. After 30 min, macrophages were
stimulated with rIFN-␥ (20 U/ml) for 6 h and then stimulated with LPS (10 ␮g/ml).
After 48 h of culture, NO release was measured by the Griess method (nitrite). The
amount of NO (nitrite) released into the medium is presented as the mean ± S.D.
of three independent experiments with duplicates in each run. The nitrite volume
was determined by using a sodium nitrite (NaNO2 ) standard curve; *p < 0.01 and
**p < 0.001 compared to the rIFN-␥/LPS-treated control group.
obtained from Western blotting studies was estimated with ImageQuantTL (GE Healthcare, Sweden) and the values were expressed
as mean ± standard error.
3. Results
3.1. Effects of LEJ on NO production
To determine the effect of LEJ on the production of NO in
rIFN-␥/LPS-activated mouse peritoneal macrophages, nitrite accumulation was measured by the Griess reaction. We pre-treated the
cells in the presence or absence of various concentrations of LEJ,
then stimulated the cells with rIFN-␥ (20 U/ml) and LPS (10 ␮g/ml).
The amount of NO in unstimulated cells was 3.3 ± 0.5 ␮M. When
mouse peritoneal macrophages were primed for 6 h with murine
rIFN-␥ and then treated with LPS for 48 h, NO production was
increased about thirty folds (90 ± 0.5 ␮M). As shown in Fig. 1, LEJ
suppressed NO production in a dose-dependent manner and about
87.7% (p < 0.001) inhibition of NO production occurred at a concentration of 500 ␮g/ml.
3.2. Effects of LEJ on cell viability
To determine the effects of LEJ on the viability of mouse
peritoneal macrophages, we carried out a MTT assay. When the
cells were treated with various concentrations of LEJ (125, 250,
500 ␮g/ml), there was no effect on cell viability (data not shown).
Therefore, the inhibitory effect of LEJ on NO production was not due
to the cytotoxicity of LEJ.
3.3. Effects of LEJ on iNOS and COX-2 expression
To investigate the mechanism of the LEJ on the inhibition of NO
production, the following experiment was performed. We investigated the effect of LEJ at a translational level with Western blotting.
As shown in Fig. 2, the expression of iNOS protein was markedly
increased after the 24 h rIFN-␥ (20 U/ml) and LPS (10 ␮g/ml) challenge. This enhanced expression of iNOS protein was significantly
reduced by LEJ in a dose-dependent manner. ␤-Actin, a cytosolic
protein, was used as a control to confirm that there were no differences in protein level between each group. Because an increase in
COX-2 expression is associated with inflammatory responses, we
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Fig. 3. Effects of LEJ on rIFN-␥/LPS-activated TNF-␣ and IL-6 secretion in mouse peritoneal macrophages. Peritoneal macrophages (5 × 106 cells/well) were pretreated
with LEJ or 0.05% DMSO, 30 min prior to rIFN-␥ (20 U/ml) stimulation. After 6 h,
macrophages were then stimulated with LPS (10 ␮g/ml). The amount of TNF-␣ (A)
and IL-6 (B) secretion in LPS stimulated mouse peritoneal macrophages was measured by ELISA after a 24 h incubation. The data are represented as mean ± S.D. of
three independent experiments with aduplicate in each run. The cytokine secretion
level was determined by using recombinant murine TNF-␣ and IL-6 standard curves,
respectively; *p < 0.01 compared to rIFN-␥/LPS-treated control group.
Fig. 2. Effects of LEJ on the expression of iNOS and COX-2 in rIFN-␥/LPS activated peritoneal macrophages. Peritoneal macrophages (5 × 106 cells/well) were
pretreated with LEJ or 0.05% DMSO, 30 min prior to rIFN-␥ (20 U/ml) stimulation.
After 6 h, macrophages were then stimulated with LPS (10 ␮g/ml) for 24 h. The
protein extracts were prepared and samples were analyzed for iNOS and COX-2
expression by Western blotting as described in Section 2. The expression of iNOS
and COX-2 was quantified by densitometric analysis. The expression levels of the
rIFN-␥/LPS treated control cells were considered to be 100% for the percentage
calculations.
also investigated the inhibitory effect of LEJ on COX-2 expression. As
seen in Fig. 2, the increased amount COX-2 protein expression from
rIFN-␥ (20 U/ml) plus LPS (10 ␮g/ml) activation was suppressed by
LEJ dose-dependently.
3.4. Effects of LEJ on cytokine secretion
We examined the inhibitory effect of LEJ on rIFN-␥/LPS-induced
TNF-␣ and IL-6 secretion. Mouse peritoneal macrophages secreted
low levels of TNF-␣ and IL-6 after a 24 h incubation with medium
alone. The basal levels of TNF-␣ and IL-6 did not changed when the
cells were incubated with LEJ alone. When the cells were treated
with rIFN-␥ (20 U/ml) plus LPS (10 ␮g/ml) for 24 h, TNF-␣ and IL-6
levels drastically increased in the cells and pre-treatment of cells
with various concentration of LEJ (125, 250, 500 ␮g/ml) significantly inhibited TNF-␣ (Fig. 3A) and IL-6 (Fig. 3B) induction in
activated mouse peritoneal macrophages.
3.5. Effects of LEJ on NF-ÄB translocation
Many previous studies demonstrated that NF-␬B is an important
transcriptional factor for the expression of various inflammatory
mediators (Henkel et al., 1993; Ghosh et al., 1998). Thus, we evaluated the influence of LEJ on the distribution of the NF-␬B subunit
(p65). Nuclear and cytosolic extracts were prepared and subjected
to immunoblot analysis. As shown in Fig. 4, co-incubation with
LPS plus LEJ the NF-␬B protein level decreased in the nucleus and
increased in the cytosol, respectively. These findings strongly suggest that LEJ inhibited the LPS-stimulated transcriptional activity
of NF-␬B by suppressing phosphorylation-dependent proteolysis
of IkB-␣ in the cells.
3.6. Acute toxicity
To test possible toxicity of LEJ in animals, 2000 mg/kg of LEJ was
administered to the mice. The treated mice did not present any
behavioral alterations, convulsions or death during the assessment
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309
(47.72%, p < 0.01). Tramadol (10 mg/kg), the reference drug, also
exhibited powerful activity which was recorded 30 min after drug
treatment (56.60%, p < 0.01). However, no differences in time latencies were observed in the vehicle-treated control group.
3.8. Analgesic activity of LEJ in the hot plate test
In the hot plate test, pre-treatments of LEJ generated significantly increased analgesia in a dose-dependent manner. The
analgesic effects of LEJ (250, 500 mg/kg) occurred between 30 and
90 min and maximum analgesia was reached at 60 min (71.62% and
77.92%, respectively) (Table 2). Tramadol also caused significant
antinociception (98.20%). In combination studies using naloxone,
an opioid receptor antagonist, the analgesic activity of tramadol
was diminished by naloxone to 19.37%. Interestingly, naloxone
antagonized LEJ (500 mg/kg) antinociception (−55.50%) only at the
30 min time point but not at 60, 90 or 120 min.
3.9. Analgesic activity of LEJ in the acetic acid test
An intraperitoneal injection of 1% acetic acid into mice causes
44.75 ± 2.85 writhing in an interval of 20 min and mice treated with
LEJ showed a significant decrease (when compared to the vehicletreated group) in the mean number of writhing (Table 3). The
data showed that the protective effect of LEJ was dose-dependent.
A 31.56% (p < 0.001) reduction was observed for the 250 mg/kg
dose and a 71.50% (p < 0.001) reduction occurred for the 500 mg/kg
dose. The reference drug, indomethacin (10 mg/kg), had 57.82%
(p < 0.001) inhibition which is lower than the inhibition observed
for the high concentration of LEJ. In combination studies, similar to
the hot plate test, naloxone significantly antagonized LEJ antinociception.
3.10. Analgesic activity of LEJ in the formalin test
In the formalin test, the vehicle-treated mice had mean licking times of 111.22 ± 6.62 s in the first phase (0–5 min) and
113.44 ± 9.67 s in the second phase (15–30 min). As shown in
Table 4, mice pre-treated with LEJ showed a reduced response
to formalin in both the first phase (neurogenic-induced) (29.36%,
42.87%) and the second phase (inflammatory) (49.44%, 74.84%), for
the 250 and 500 mg/kg doses, respectively. The reference drug,
tramadol, also significantly blocked the pain formalin-response
in both phases (first-phase, 53.66 ± 7.17 s and second-phase,
13.57 ± 4.93 s). However, indomethacine was significantly active
(59.72%, p < 0.001) only in the second phase.
Fig. 4. Effects of LEJ on NF-␬B translocation by LPS-stimulated peritoneal
macrophages. Peritoneal macrophages (5 × 106 cells/well) were pretreated with LEJ
or 0.05% DMSO for 30 min and then stimulated with rIFN-␥ (20 U/ml) for 2 h. After
1 h stimulation with LPS (10 ␮g/ml), the nuclear extracts were prepared and samples were analyzed by Western blotting as described in the Method section and
quantified by densitometry.
period (24 h). These results suggest that oral doses of 2000 mg/kg
of body weight or less are safe to use in mice.
3.7. Analgesic activity of LEJ in the tail immersion test
4. Discussion
In the tail immersion test, LEJ was antinociceptive in a dosedependent manner (Table 1). The most prominent analgesic activity
for the lower concentration of LEJ (250 mg/kg) was observed 90 min
after oral administration (23.06%). In contrast, pre-treatment with a
high concentration of LEJ (500 mg/kg) significantly delayed reaction
times to a nociceptive stimulus 60 min after oral administration
The dried leaves of E. japonica Lindl. (Rosaceae) are a well-known
oriental medicine, containing various beneficial compounds, with
diverse pharmacological effects and has been used to treat many
different diseases. Nevertheless, the previous reports on LEJ are still
not enough to understand how LEJ treatment works on inflam-
Table 1
Effect of LEJ on nociceptive responses in the tail immersion test.
Treatment
Dose (mg/kg)
Latency period (min)
0
Vehicle
Tramadol
LEJ
LEJ
–
10
250
500
4.14
4.88
4.57
4.31
30
±
±
±
±
0.28
0.36
0.23
0.31
4.29
7.65
5.22
5.05
60
±
±
±
±
0.34
0.85*
0.37
0.47
4.25
6.50
5.22
6.36
90
±
±
±
±
0.40
0.82
0.22
0.50*
4.46
5.61
5.62
6.22
120
±
±
±
±
0.35
0.45
0.38
0.76
Values expressed as mean ± S.E.M. and units are in seconds (n = 10–12). Differences between groups were statistically analyzed by the Student’s t-test.
*
p < 0.01 compared to vehicle-treated group.
4.68
4.90
5.17
5.15
±
±
±
±
0.47
0.28
0.47
0.44
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Table 2
Effect of LEJ on nociceptive responses in the hotplate test.
Treatment
Dose (mg/kg)
Latency period (min)
Vehicle
Tramadol
LEJ
LEJ
Naloxone
+Tramadol
Naloxone
+LEJ
–
10
250
500
11.15
11.87
11.34
12.01
5 + 10
11.96 ± 0.64
19.71 ± 1.81**
18.23 ± 0.96**
14.30 ± 1.13
13.89 ± 1.09
5 + 500
11.04 ± 0.65
12.41 ± 0.92
20.86 ± 1.37**
15.39 ± 1.31*
11.91 ± 0.96
0
30
±
±
±
±
0.63
0.69
0.50
0.62
11.10
23.53
16.69
19.30
60
±
±
±
±
0.76
1.25**
1.44**
1.97**
11.31
18.81
19.46
21.37
90
±
±
±
±
0.81
1.13**
1.74**
1.78**
120
12.16
16.77
17.09
15.61
±
±
±
±
0.60
1.27**
1.74*
1.55*
12.06
13.78
15.23
12.82
±
±
±
±
1.22
0.89
1.45*
1.15
Values expressed as mean ± S.E.M. and units are in seconds (n = 11–15). Differences between groups were statistically analyzed by the Student’s t-test.
*
p < 0.01 compared to vehicle-treated group.
**
p < 0.001 compared to vehicle-treated group.
Table 3
Effect of LEJ on nociceptive responses in the acetic acid-induced writhing test.
Treatment
Dose (mg/kg)
Number of writhings
(5–25 min)
Vehicle
Indomethacin
LEJ
LEJ
Naloxone + LEJ
–
10
250
500
5 + 500
44.75
18.87
30.62
12.75
26.50
±
±
±
±
±
2.85
2.84
2.05
1.96
2.29
Inhibition (%)
–
57.82**
31.56**
71.50**
40.78**
Values expressed as mean ± S.E.M. (n = 8). Differences between groups were statistically analyzed by the Student’s t-test.
**
p < 0.001 compared to vehicle-treated group.
matory diseases and pain. Thus, the present study was designed
to validate the n-BuOH fraction of E. japonica (LEJ) leaves as an
anti-inflammatory and antinociceptive agent.
We evaluated LEJ’s anti-inflammatory properties using the
rIFN-␥ and LPS-induced murine peritoneal macrophage model. In
macrophages, nitric oxide (NO) is produced from l-arginine and
contributes to the immune defense against viruses, bacteria and
other parasites (Seo et al., 2001). However, large amounts of NO
act as toxic radicals and can cause tissue and cell damage. Thus,
over-production of NO plays a central role in the pathogenesis
of inflammation and results in septic shock, neurologic disorders,
rheumatoid arthritis and autoimmune diseases (Thiemermann and
Vane, 1990). Therefore, to avoid NO production, the use of exogenous modulators is necessary. In this study, IFN-␥ and LPS-induced
NO production was inhibited dose-dependently by LEJ without
notable cytotoxicity.
NO is produced by one of three kinds of NO synthase (NOS):
neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS
(iNOS). Unlike nNOS or eNOS, the level of iNOS plays a crucial
role in the excess production of NO in activated macrophages.
Therefore, suppression of NO production via inhibition of iNOS
expression might be an attractive therapeutic target for the treatment of inflammation. Thus the possibility that LEJ could inhibit
iNOS expression was examined and LEJ suppressed the expression
of iNOS significantly.
Cyclooxygenase-2 (COX-2), another key enzyme in inflammation, is the rate-limiting enzyme that catalyzes the formation
of prostaglandins (PGs) from arachidonic acid. Levels of PGs
increase early in the course of the inflammation (Wallace, 1999).
Since, COX-2 is induced by stimulation in inflammatory cells,
inhibitors of COX-2 induction might candidate for a nonsteroidal
anti-inflammatory drug (NSAID). We documented the increased
production of COX-2 protein by macrophages exposed to IFN-␥
and LPS. IFN-␥ and LPS in combination with LEJ led to a reduction
in COX-2 expression. Thus, it seems quite reasonable to speculate
that LEJ may inhibit PGE2 production. However, further studies are
required to determine whether LEJ is a selective inhibitor of COX-2.
Cytokines are the physiological messengers of the inflammatory
response. The macrophage-derived mediators, TNF-␣ and IL-6 are
thought to play an important role in the inflammatory response
based on the appearance of these cytokines at inflammatory sites
and their ability to induce certain mechanisms in the inflammatory response (Park et al., 2002). TNF-␣ has been documented as
a pathogenic factor in several inflammatory diseases, including
arthritic diseases, inflammatory bowel diseases, Type 1 diabetes
mellitus, multiple sclerosis and Guillain–Barre syndrome (O’Shea
et al., 2002). The secretion of IL-6 has been found to play a central role in the regulation of defense mechanisms, haematopoiesis
and most importantly, the production of acute phase proteins (Park
et al., 1999). Mouse peritoneal macrophages secreted low levels of
TNF-␣ and IL-6 after a 24 h incubation with medium alone. When
the cells were treated with rIFN-␥ and LPS for 24 h, TNF-␣ and IL-6
levels drastically increased in these cells. However, pre-treatment
with LEJ significantly inhibited the increase in TNF-␣ and IL-6 levels.
The induction of pro-inflammatory mediators is largely regulated by transcriptional activation (Park et al., 2006). Nuclear factor
kappa B (NF-␬B) is essential for the transcription of genes that
encode a number of pro-inflammatory molecules which participate in the acute inflammatory response, including iNOS, COX-2,
TNF-␣ and IL-6 (Muller et al., 1993). In unstimulated cells, NF␬B is present in the cytoplasm and interacts with the inhibitory
Table 4
Effect of LEJ on nociceptive response in the formalin test.
Treatment
Dose (mg/kg)
Vehicle
Tramadol
Indomethacin
LEJ
LEJ
–
10
10
250
500
Early phase (0–5 min)
Licking time (s)
111.22
53.66
103.5
78.55
63.53
±
±
±
±
±
6.62
7.17
4.71
5.79
8.00
Late phase (15–30 min)
Inhibition (%)
Licking time (s)
–
51.75**
7.34
29.36*
42.87**
113.44
13.57
45.68
57.34
28.53
Values expressed as mean ± S.E.M. (n = 8–9). Differences between groups were statistically analyzed by the Student’s t-test.
*
p < 0.01 compared to vehicle-treated group.
**
p < 0.001 compared to vehicle-treated group.
±
±
±
±
±
9.67
4.93
8.70
9.50
9.31
Inhibition (%)
–
88.03**
59.72**
49.44**
74.84**
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D.S. Cha et al. / Journal of Ethnopharmacology 134 (2011) 305–312
protein, I␬B. However, in an active state, following the induction
of NF-␬B by appropriate extracellular stimulation, NF-␬B translocates to the nucleus through the phosphorylation, ubiquitation
and degradation of I␬B␣. NF-␬B also acts on the pro-inflammatory
gene promoter to activate transcription (Park et al., 2006). The
translocation of NF-␬B to nucleus was markedly increased after the
rIFN-␥ and LPS challenge. This increase was significantly reduced
by LEJ. On the contrary, LEJ generated a potent increase in NF-␬B
protein levels in the cytosol. These results strongly suggest that
LEJ suppressed various pro-inflammatory mediators via decreased
NF-␬B translocation and DNA binding activities. Similar to the
results described above, Kim and Shin (2009) revealed that the antiinflammatory activity of the loquat leaf was correlated with down
regulation of p38 MAPK, ERK and NF-␬B activation in mast cells.
Huang et al. (2009) also demonstrated that the triterpene acids of
the loquat leaf attenuate the MAPK signal transduction pathway in
alveolar macrophages from rats with chronic bronchitis. Moreover,
Lee et al. (2008) reported that the triterpenes, especially ursolic
acid, inhibited lipopolysaccharide-induced cytokines and inducible
enzyme production via the nuclear factor-kappaB signaling pathway in lung epithelial cells. In the present study, we also isolated
triterpenes from the EtOAc fraction (data not shown). Therefore, it
could be assumed that not only triterpenes in the EtOAc fraction
but also other compounds in LEJ may possess anti-inflammatory
potential.
Next, we investigated the antinociceptive properties of LEJ.
Thermal nociception models such as tail immersion and the hotplate tests were used to determine central antinociceptive activity.
LEJ showed analgesic effect in both the tail immersion and hot plate
tests, implicating both spinal and supraspinal analgesic pathways.
In both tests, tramadol exhibited a rapid effect with a maximum
peak in a short amount of time, which is similar to the action of
opioid agonists (e.g. morphine). In contrast, LEJ reached the maximum analgesic level 60 min after administration. This difference
in the maximum analgesic point could be explained by the methods of drug administration (i.p. or p.o.) or the metabolic rate of
each drug. Moreover, in the hotplate test, the antinociceptive action
of tramadol was reduced, but was still present, when the nonselective opioid receptor antagonist naloxone was applied. These
results correlate with previous studies that showed tramadol is a
partial opioid agonist with both opioid and non-opioid properties
(Yalcin and Aksu, 2005). Interestingly, the analgesic effect of LEJ was
also blocked by naloxone at the 30 min time point, suggesting that
at least some of the antinociceptive effects of LEJ are mediated via
activation of opioid receptors. However, at the other time points,
naloxone was unable to antagonize the analgesic effect of LEJ, which
indicates that LEJ-induced antinociception is largely affected by a
non-opioid mechanism of action. Thus, these results showed that
LEJ acted as a weak opioid receptor agonist.
The effects of LEJ on peripheral nociception were determined
using the acetic acid-induced writhing model which is frequently
used to estimate both the central and peripheral analgesic effects
of drugs (Fukawa et al., 1980). The acetic acid-induced writhing
test has been associated with an increased level of prostaglandins
(PGs), especially PGE2 , in peritoneal fluids (Derardt et al., 1980).
PGs induce abdominal constrictions by activating and sensitizing
peripheral chemosensitive nociceptors (Dirig et al., 1998) which
are largely associated with the development of inflammatory pain
(Bley et al., 1998). Therefore, one of the possible analgesic mechanisms related to this test is the inhibition of the COX enzyme.
Non-steroidal anti-inflammatory drugs exert their peripheral analgesic potential through inhibition of PG synthesis, and in the
present study, indomethacin produced a significant decrease in the
writhing response. LEJ also showed potent inhibition of acetic acidinduced abdominal constrictions in a dose-dependent manner. The
antinociceptive potential of LEJ could be explained by its inhibitory
311
action on COX-2 expression, which was described above. Moreover,
in a combination study, naloxone exhibited moderate inhibition of
the analgesic effect of LEJ. Therefore, it is likely that the opioid system is also involved in the peripheral antinociceptive actions of LEJ,
at least in part.
Because the acetic acid-induced writhing test was not a distinct
test to indicate if the analgesic effects of LEJ resulted from central or peripheral mechanisms, the formalin test was conducted. A
subcutaneous injection of formalin, used as a peripheral noxious
stimulus, causes biphasic nociceptive responses which involve two
different mechanisms (Hunskaar and Hole, 1987). The first phase
(neurogenic pain) is caused by the direct chemical stimulation of
nociceptive afferent fibers, predominantly C fibers, which can be
suppressed by opiates like morphine (Amaral et al., 2007). On the
other hand, the second phase (inflammatory pain) results from
the action of inflammatory mediators such as prostaglandins, serotonin, histamine and bradykinin in the peripheral tissues (Hunskaar
and Hole, 1987) and from functional changes in the spinal dorsal
horn (Dalal et al., 1999). The results of the present study have shown
that tramadol, a central analgesic drug, is effective in preventing
both the early and late phases of formalin-induced nociception,
whereas indomethacin, a NSAID, suppressed nociceptive activity mainly in the later phase. These results are quite reasonable
because many reports suggest that drugs which primarily act on
the central nervous system are inhibited equally in both phases,
whereas drugs which act peripherally, such as steroids and NSAIDs,
can slightly inhibit the early phase of the formalin test (Hunskaar
et al., 1985; Trongsakul et al., 2003; Vontagu et al., 2004). In this test,
it was observed that LEJ could reduce the duration of the paw licking
time in both the first phase and the second phase, demonstrating
that LEJ can suppress neurogenic and inflammatory nociception.
These data provided further confirmation that LEJ has a central
mechanism of action, which was shown in the tail-immersion and
hot plate tests. Furthermore, in agreement with the results from
the acetic acid test, LEJ also displayed better peripheral analgesic
effect than indomethacin. Based on these findings, it may be concluded that LEJ has excellent antinociceptive properties involving
central and peripheral mechanisms.
In summary, in the IFN-␥ and LPS-induced mouse peritoneal
macrophage model, LEJ showed significant inhibitory effects on
proinflammatory mediators including NO, IL-6, TNF-␣, iNOS and
COX-2 via the down regulation of NF-␬B translocation to the
nucleus. In several thermal and chemical nociception tests, LEJ
also exhibited potent antinociceptive activities on both central and
peripheral mechanism. In addition, a combination test with naloxone revealed that LEJ acts as a weak opioid receptor agonist. Based
on these various properties, LEJ may hold great promise for treating
inflammatory diseases and pain as an effective immunomodulatory
and analgesic agent.
Acknowledgements
This work was supported by the grant of the Korean Ministry
of Education, Science and Technology (The Regional Core Research
Program/Healthcare Technology Development) and was also supported by the research grant from Woosuk University.
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