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Anti-Inflammatory Effects of an Ethanolic Extract of Guava
(Psidium guajava L.) Leaves In Vitro and In Vivo
Mi Jang,
1,2
Seung-Weon Jeong,
1
Somi K. Cho,
3
Kwang Seok Ahn,
4
Jong Hyun Lee,
4
Deok Chun Yang,
2
and Jong-Chan Kim
1
1
Department of Food Analysis & Standardization, Korea Food Research Institute, Seongnam, Gyeonggi-do, Korea.
2
Department of Oriental Medicinal Material and Processing, College of Life Science, Kyung Hee University, Gyeonggi-do, Korea.
3
Faculty of Biotechnology College of Applied Life Sciences, Jeju National University, Jeju, Korea.
4
Department of Oriental Pathology, College of Oriental Medicine, Kyung Hee University, Seoul, Korea.
ABSTRACT Plant extracts have been used as a source of medicines for a wide variety of human ailments. Among the
numerous traditional medicinal herbs, Psidium guajava L. (Myrtaceae), commonly known as guava, has long been used in
folk medicines as a therapeutic agent for the treatment of numerous diseases in East Asian and other countries. The aim of this
study was to investigate the anti-inflammatory activity of an ethanolic leaf extract of P. guajava (guava) in vitro and in vivo.
Our results demonstrated that guava leaf extract (GLE) significantly inhibited lipopolysaccharide (LPS)-induced production of
nitric oxide and prostaglandin E
2
in a dose-dependent manner. GLE suppressed the expression and activity of both inducible
nitric oxide synthase and cyclooxygenase-2 in part through the downregulation of ERK1/2 activation in RAW264.7 mac-
rophages. Furthermore, GLE exhibited significant anti-inflammatory activity in 2 different animal models—Freund’s complete
adjuvant-induced hyperalgesia in the rat and LPS-induced endotoxic shock in mice.
KEY WORDS: cyclooxygenase-2 health functional food inflammation nitric oxide signaling
INTRODUCTION
The inflammatory response occurs when cells and
body tissues are injured by biological, chemical, or
physical stimuli such as bacteria, trauma, toxins, or heat. It is
one of the most important defense mechanisms, which is
aimed at removal of the injurious stimuli and initiation of
the healing process. Macrophages are key players in vari-
ous inflammatory diseases and in the immune response
where they release proinflammatory mediators and pro-
teins, including interleukin-6 (IL-6), tumor necrosis
factor-a(TNF-a), cyclooxygenase-2 (COX-2), and induc-
ible nitric oxide synthase (iNOS).
1
The RAW264.7 mouse
macrophage cell line, when activated by lipopolysaccha-
ride (LPS), produces proinflammatory cytokines and other
inflammatory mediators, including nitric oxide (NO) and
prostaglandin E
2
(PGE
2
),whicharesynthesizedbyiNOS
and COX-2, respectively.
2
Plant extracts have been used as a source of medicines
for a wide variety of human disorders. Herbal and natural
products have recently received increased attention be-
cause of their biological and pharmacological activities.
3
Among the numerous traditional medicinal herbs, Psidium
guajava L. (Myrtaceae), commonly known as guava, has
long been used in folk medicines as a therapeutic agent
for the treatment of a number of diseases, e.g., as an anti-
inflammatory, for diabetes, rheumatic pain, hypertension,
wounds, ulcers, and reducing fever.
4
Over the last few de-
cades, extracts of guava leaves have been heavily commer-
cialized in Taiwan, Japan, China, and Korea, and these
extracts are commonly taken as dietary supplements showing
various pharmacological effects. The main constituents of
guava leaf extract are a variety of polyphenolics, flavonoids,
and triterpenoids.
5
Although guava leaves have been shown to exert various
physiological effects, little is known about the underlying
pharmacological mechanisms of the ethanolic leaf extract of
P. guajava (guava). In the present study, we evaluated the
inhibitory effect of guava leaf extract (GLE) on inflammatory
biomarkers such as NO and PGE
2
production and iNOS and
COX-2 expression in LPS-stimulated RAW264.7 cells. To
investigate the underlying mechanisms, the involvements of
mitogen activated protein kinases (MAPKs) and nuclear
factor-jB(NF-jB) were examined. Moreover, we investi-
gated the potential therapeutic effects of GLE in 2 different
animal models, Freund’s complete adjuvant (FCA)-induced
hyperalgesia in the rat and LPS-induced endotoxic shock in
mice.
Manuscript received 8 May 2013. Revision accepted 21 February 2014.
Address correspondence to: Jong-Chan Kim, Korea Food Research Institute, 516 Bae-
khyeon-dong Bundang-gu, Gyeonggi-do 463-746, Korea, E-mail: jckim@kfri.re.kr
JOURNAL OF MEDICINAL FOOD
J Med Food 17 (6) 2014, 678–685
#Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2013.2936
678
MATERIALS AND METHODS
Plant materials and extract preparation
Plant material was obtained from the College of Applied
Life Science, Jeju National University, Korea. The plant was
taxonomically identified and authenticated by one author
(S.K.C). The air-dried Jeju guava leaves (20 g) were ex-
tracted with 400 mL of 55% ethanol (v/v) at 47C for 4.9 h
and the extracted solution was then filtered and evaporated.
The extract was then freeze-dried to obtain powder and used
as the GLE.
Cell culture
RAW264.7 macrophages were obtained from the Korean
Cell Line Bank (KCLB, Seoul, Korea) and were maintained
in RPMI 1640 medium (L-glutamine, 25mM HEPES buffer,
and sodium bicarbonate [Gibco-BRL, Grand Island, NY,
USA]) containing antibiotics (100 units/mL penicillin A and
100 lg/mL streptomycin) and 10% heat-inactivated FBS
(Gibco-BRL) at 37C in a humidified incubator (5% CO
2
and 95% air).
Measurement of cell viability
The cytotoxicity of GLE was measured using the colori-
metric MTT assay beforethe biological assay was performed.
6
RAW264.7 cells were plated at a density of 1 ·10
4
cells per
well in a 96-well plate and then treated with GLE (5, 10, 30,
and 50 lg/mL). The cells were incubated for 24 h, and the
medium was replaced with fresh medium containing MTT
solution (Sigma-Aldrich, St. Louis, MO, USA) (2 mg/mL in
PBS) for another 2 h at 37C. The optical density of the cells
was measured using a microplate reader (model 680, Bio-Rad,
Hercules, CA, USA) at 570 nm.
Nitrite assay
The cells were pretreated with the indicated concentra-
tions of GLE for 2 h, and then were induced with a 1 lg/mL
concentration of LPS for an additional 22 h. The inhibitory
effect of GLE on NO production was determined with
Griess reagent, as previously described.
7
Measurement of PGE
2
and IL-6 levels
RAW264.7 macrophage cells were plated at a density of
2·10
5
cells per well in a 24-well plate. The cells were
pretreated with the indicated concentrations of GLE for 2 h
and then induced with 1 lg/mL LPS for an additional 22 h.
The concentrations of PGE
2
and IL-6 were determined using
an enzyme-linked immunosorbent assay (ELISA) kit ac-
cording to the manufacturer’s instructions (R&D Systems,
Minneapolis, MN, USA).
Reverse transcription-polymerase chain reaction
Total RNA was isolated using TRIzol reagent (Gibco). The
concentration and integrity of RNA were determined by
measuring absorbance at 260 and 280 nm and then calculating
the 260/280 nm ratio. The forward and reverse primers for
iNOS were 50-TCT TTG ACG CTC GGA ACT GTA GCA-30
and 50-CGT GAA GCC ATG ACC TTT CGC ATT-30,re-
spectively, and the forward and reverse primers for COX-2 were
50-TTG CTG TAC AAG CAG TGG CAA AGG-30and 50-
AGG ACA AAC ACC GGA GGG AAT CTT-30, respectively.
The forward and reverse primers for GAPDH (used as a control
for the total RNA content of each sample) were 50-AAC TTT
GGC ATT GTG GAA GGG CTC-30and 50-TGG AAG AGT
GGG AGT TGC TGT TGA-30, respectively. Reverse
transcription-polymerase chain reaction (RT-PCR) was
performed using a ONE-STEP RT-PCR PreMix kit (In-
vitrogen, Carlsbad, CA, USA) according to the manufacturer’s
instructions.
Western blot analysis
Equal amounts of lysates resolved on sodium dodecyl-
polyacrylamide gel electrophoresis (SDS-PAGE) were
transferred to a nitrocellulose membrane. The blots were
probed with primary and secondary antibodies, and then the
immunoreactive bands were developed with enhanced
chemiluminescence (ECL, Amersham Biosciences, Little
Chalfont, United Kingdom), as previously described.
7
All of
the primary and secondary antibodies, including iNOS,
COX-2, p-ERK1/2, ERK1/2, p-JNK, JNK, p-p38, p38, IjB-a,
p65, b-actin, and lamin B were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA, USA).
Animals
Male Sprague-Dawley (SD) rats (weighing 200–230 g)
and male BALB/c mice (weighing 20–23 g) were used for
the experiments. Animals were provided standard laboratory
rodent food and water ad libitum and were housed in a
temperature-controlled environment (21 –2C) on a 12-h
light/12-h dark cycle. The experimental protocol was re-
viewed and approved by the Animal Care Committee of
Wonkwang University (WKU13-01) and all experiments
were conducted in accordance with following ethical
guidelines of the International Association for the Study of
Pain for investigations on experimental pain in animals.
8
Thermal hyperalgesia assessment
The experiment was performed using a slight modification
of the procedure described by Hargreaves et al.
9
Rats were
randomly divided into 6 groups (n=7 for each group) ac-
cording to the following treatments: saline, FCA (3 mg/mL),
FCA with diclofenac (30mg/kg), and FCA with GLE (100,
200, and 400 mg/kg). All groups, except for the saline group,
which was similarly injected with saline, were injected
subcutaneously with 25 lL of FCA into the plantar surface of
the right hind paw. Twenty-four hours after starting the ex-
periment, paw withdrawal latency (PWL) from a beam of
infrared radiation was assessed in both experimental and
control groups using the plantar test (Stoelting Stereotaxic
Instrument, Ugo Basile, Chicago, IL, USA). PWL was au-
tomatically measured by the apparatus using an infrared in-
tensity of 70 W. PWL was measured 3 times separated by a
ANTI-INFLAMMATORY EFFECTS OF P. GUAJAVA L. 679
minimum interval of 5 min, and the average PWL was cal-
culated from these 3 measurements. Minimum and maximum
cutoffs were assigned at 1 and 30 sec, respectively.
Survival study
Mice were divided into 3 groups that received saline, LPS
(9 mg/kg), and LPS +GLE (n=15 per group). GLE was
administered orally through an esophageal catheter at a dose
of 400 mg/kg twice (at 24 h and 2 h) before LPS injection,
and then the mice were monitored for 72 h and survival
recorded. Whole blood samples were collected from the
retro-orbital plexus after LPS injection at the 3 h mark and
sera were prepared to measure TNF-aand IL-6 (R&D
Systems, Minneapolis, MN, USA).
Liquid chromatography/tandem mass spectrometry analysis
The content of 11 selected phenolic compounds was in-
vestigated by liquid chromatography (LC)/tandem mass
spectrometry (MS/MS), and standard compounds were
purchased from Sigma-Aldrich (St. Louis, MO, USA). The
ultrahigh performance liquid chromatography (UPLC; Wa-
ters Inc., Milford, MA, USA), coupled with electrospray
ionization (ESI)–MS/MS (Quattro Premier XE), was used
for analysis. The compounds were separated using a Kinetex
C18 (100 mm ·2.1 mm ·2.6 lm) column from Phenomenex
(Phenomenex Inc., Torrance, CA, USA). A mass spectro-
metry system equipped with ESI was operated in negative
ion mode. For each compound, the optimum conditions of
MRM were determined in infusion mode (Table 1).
Statistical analysis
The results are expressed as the mean –SD, and analysis
of variance (ANOVA) with Duncan’s multiple-range test
was used for multiple comparisons. Survival curves were
calculated with Mann-Whitney tests using SPSS software
(SPSS Inc., Chicago, IL, USA). Kaplan-Meier survival
analysis was performed with log-rank tests. Pvalues £.05
were considered statistically significant.
RESULTS
Cytotoxic effects of GLE in RAW264.7 cells
To investigate the effect of GLE on cell viability,
RAW264.7 cells were treated with various concentrations
(5, 10, 30, and 50 lg/mL) of GLE for 24 h, and cytotoxicity
was measured by the MTT assay. We found that GLE had no
effect on cell viability (data not shown), and the doses of
GLE listed above were used in all subsequent experiments.
GLE inhibits NO production by suppressing iNOS
expression in LPS-stimulated RAW264.7 cells
The effects of GLE on LPS-induced NO production in
RAW264.7 cells were investigated by measuring the
amount of nitrite released into the culture medium using the
Griess reaction. As shown in Figure 1A, GLE significantly
inhibited the production of LPS-induced NO in a dose-
dependent manner, with >65% inhibition at a concentration
of 50 lg/mL. As shown in Figure 1B, un-stimulated cells
expressed no detectable levels of iNOS protein; however, in
response to LPS (1 lg/mL), iNOS expression markedly in-
creased. Pretreatment with GLE decreased the levels of iNOS
protein expression in LPS-stimulated cells in a dose-dependent
manner (Fig. 1B). LPS treatment of cells upregulated iNOS
mRNA, whereas pretreatment with GLE inhibited LPS-
mediated iNOS mRNA upregulation in a dose-dependent
manner (Fig. 1C). These results indicate that GLE inhibited
NO production by suppressing iNOS gene expression in
LPS-stimulated RAW264.7 cells.
Table 1. Liquid Chromatography and Mass Spectrometry Characteristics and Contents of Selected Bioactive Compounds
in Guava Leaf Extract
Compound-specific MS/MS parameters
a
Compounds RT (min) MRM (m/z) DP (V) CE (V) Contents
b
(lg/g)
Gallic acid 1.19 169 >125 20 13 213.97 –6.24
(+)-Catechin 2.39 289 >109 35 25 4456.49 –43.27
Chlorogenic acid 2.42 353 >191 20 13 55.86 –2.16
Caffeic acid 2.67 179 >135 40 11 4.54 –0.25
Rutin hydrate 2.88 609 >300 40 40 0.27 –0.01
Luteolin 7-glucoside 2.97 447 >284 40 40 1.91 –0.02
q-Coumaric acid 3.06 163 >119 30 20 1.26 –0.09
Myricetin 3.32 317 >151 40 25 1.70 –0.01
Quercetin 3.67 301 >151 40 25 15.65 –2.98
Luteolin 3.67 285 >133 50 40 0.23 –0.02
Kaempferol 3.99 285 >117 30 40 0.66 –0.02
a
Common parameters for all compounds: Mobile phase: eluent A, formic acid in water (0.1%, v/v); eluent B, formic acid in acetonitrile (0.1%, v/v); A linear
gradient (0–1 min, 10% B; 1–5 min, 50% B; 5–8 min, 100% B; 8–10 min, 10% B); flow rate was 0.25 mL/min. MRM scan mode operated under the following
conditions: capillary voltage 3.20 kV, cone voltage 30 V, source temperature 120C, desolvation temperature 300C and collision gas flow 0.22 mL/min, negative
polarity.
b
Values are mean –SD of triplicate determinations.
RT, retention time; MRM, multiple reaction monitoring; DP, declustering potential; CE, collision energy.
680 JANG ET AL.
GLE inhibits PGE
2
production by suppressing COX-2
expression in LPS-stimulated RAW264.7 cells
Pretreatment of cells with GLE significantly inhibited
LPS-induced PGE
2
production in a concentration-dependent
manner (Fig. 2A). At a GLE concentration of 50 lg/mL,
PGE
2
was reduced to the basal level (Fig. 2A). As shown in
Figure 2B, unstimulated RAW264.7 cells did not express
detectable COX-2 protein; however, COX-2 expression was
markedly increased in response to LPS (1 lg/mL), whereas
pretreatment with GLE suppressed LPS-activated COX-2
expression in a dose-dependent manner (Fig. 2B). We also
attempted to determine whether the expression of COX-2
mRNA paralleled its protein levels. The results showed that
in LPS-treated cells, COX-2 mRNA was upregulated, but
pretreatment with GLE inhibited the LPS-mediated COX-2
mRNA increase in a dose-dependent manner (Fig. 2C).
Inhibition of IL-6 secretion by GLE in LPS-stimulated
RAW264.7 cells
IL-6 is produced in response to infection, burns, or other
tissue damage leading to inflammation. IL-6 levels are in-
dicative of the progression of inflammation. To confirm that
GLE inhibited proinflammatory cytokines, IL-6 concentra-
tion was measured in culture supernatants by ELISA. As
shown in Figure 3, pretreatment with GLE significantly
inhibited IL-6 production in LPS-treated cells in a dose-
dependent manner.
Suppression of LPS-induced EPK1/2 phosphorylation
by GLE
To investigate whether inhibition of NO production
by GLE was mediated by downregulation of MAPKs and
NF-jB activation in RAW264.7 cells, we measured the
phosphorylation of MAPKs (ERK, p38, and JNK), p65,
and IjB-ain LPS-stimulated macrophages. As shown in
Figure 4A, LPS treatment induced phosphorylation of
ERK1/2, p38, and JNK. Pretreatment with GLE suppressed
LPS-induced phosphorylation of ERK1/2 in a dose-
dependent manner, but did not suppress p38 or JNK phos-
phorylation. In addition, GLE did not affect p65 at Ser 276
or IjB-a, which is positively correlated with the extent
of NF-jB activation (Fig. 4B). These results suggest
that GLE inhibits LPS-induced NO production in part
through suppression of the ERK1/2 signaling pathway in
RAW264.7 cells.
Effect of GLE on inflammatory hyperalgesia
FCA injection caused a substantial increase in hyper-
algesia, which was obvious in the first hour, and lasted
for more than 24 h. As shown in Figure 5, injection of
FCA caused a dramatic decrease in PWL at 24 h. Ad-
ministration of GLE significantly alleviated thermal
withdrawal latency in a dose-dependent manner, com-
pared with thermal withdrawal latency in the saline group
(Fig. 5).
FIG. 1. GLE-mediated inhibition of LPS-induced NO and iNOS expression in RAW264.7 macrophages. (A) RAW264.7 cells were pretreated
with different concentrations of GLE for 2 h, and then stimulated with LPS (1 lg/mL) for 22 h. The concentrations of nitrite in the conditioned
media of sample-treated cells were calculated from the values measured compared to those of standard concentrations of sodium nitrite dissolved
in RPMI 1640. Three independent experiments were performed, and the data are presented as the means –SD.
abcde
Values with different letters are
significantly different from cells treated with LPS alone (P<.05). (B) RAW264.7 cells were pretreated with different concentrations of GLE for
2 h, and then stimulated with LPS (1 lg/mL) for another 22 h. Cell lysates were obtained, and iNOS protein levels were analyzed via Western
blotting. Representative results of 4 independent experiments are shown. b-Actin expression was used as an internal control for Western blotting.
(C) RAW264.7 cells were pretreated with the indicated concentrations of GLE for 2 h, and then incubated with LPS (1 lg/mL) for 22 h. Total
RNA was isolated, and levels of iNOS mRNA were measured by RT-PCR. GAPDH levels were measured as a control for the cDNA content of
each sample. The results shown are representative of the three independent experiments. GLE, guava leaf extract; LPS, lipopolysaccharide; NO,
nitric oxide; iNOS, inducible nitric oxide synthase.
ANTI-INFLAMMATORY EFFECTS OF P. GUAJAVA L. 681
Effect of GLE on the survival of mice with endotoxic shock
To determine the protective effect of GLE against LPS-
induced shock in mice, we monitored the effect of GLE on
the mortality of mice with lethal endotoxemia. The survival
rates of the mice after endotoxin injection are shown in
Figure 6A. We found that the LPS-injected mice all died in
the first 35 h after LPS injection, whereas mice administered
GLE (400 mg/kg) had a 67% survival rate after 72 h (Fig.
6A). These results suggest that GLE has a protective effect
against septic shock in mice. We also examined the effect of
GLE on the serum levels of the inflammatory cytokines,
TNF-aand IL-6, in LPS-challenged mice. As shown in Figure
6B and C, serum levels of TNF-aand IL-6 were significantly
elevated 3 h after LPS injection. GLE (400 mg/kg) signifi-
cantly decreased TNF-aand IL-6 levels in mice with LPS-
induced endotoxic shock.
UPLC-ESI-MS/MS identification of bioactive compounds
Guava leaves have been reported to contain numerous
polyphenolic compounds and other chemical compounds
that have been shown to exhibit various pharmacological
effects, including anti-inflammatory and antioxidant activi-
ties.
4
In this study, 11 compounds were detected and iden-
tified, including phenolic acid and flavonoids (Table 1).
GLE shows the highest catechin content, followed by gallic
acid, chlorogenic acid, and quercetin in decreasing order, as
shown in Table 1.
DISCUSSION
The objective of this study was to elucidate the
anti-inflammatory potential of GLE through RAW264.7
macrophage and animal experiments. We conclusively dem-
onstrated that GLE suppressed iNOS mRNA and COX-2
FIG. 2. GLE-mediated inhibition of LPS-induced PGE
2
production and COX-2 expression in RAW264.7 macrophages. (A) RAW264.7 cells were
pretreated with different concentrations of GLE for 2 h, and then stimulated with LPS (1lg/mL) for 22h. Culture media were collected to measure
PGE
2
concentration using the mouse PGE
2
ELISA kit. Three independent experiments were performed, and the data are presented as means –SD.
abcd
Values with different letters are significantly different from cells treated with LPS alone (P<.05). (B) RAW264.7 cells were pretreated with GLE
for 2 h, and then stimulated with LPS for 22h. Cell lysates were obtained, and COX-2 protein levels were analyzed via Western blotting.
Representative results of three independent experiments are shown. b-Actin expression was used as an internal control for Western blotting. (C)
RAW264.7 cells were pretreated with the indicated concentrations of GLE for 2h, and then incubated with LPS (1 lg/mL) for 22 h. Total RNA was
isolated, and COX-2 mRNA levels were measured by RT-PCR. GAPDH mRNA levels were measured as a control for the cDNA content of each
sample. The results shown are representative of the 3 independent experiments. PGE
2
, prostaglandin E
2
; COX-2, cyclooxygenase-2.
FIG. 3. GLE-mediated inhibition of LPS-induced IL-6 production
in RAW264.7 macrophages. Cells were pretreated with GLE (5, 10,
30, or 50 lg/mL) for 2 h, and then stimulated with LPS (1 lg/mL) for
22 h. The amount of IL-6 released was determined with a mouse IL-6
ELISA kit. Three independent experiments were performed, and the
data presented are the means –SD.
abcd
Values with different letters
are significantly different from cells treated with LPS alone (P<.05).
682 JANG ET AL.
mRNA and protein expression in LPS-induced RAW264.7
cells by suppressing ERK1/2 phosphorylation. In addition,
GLE had a protective effect on FCA-induced inflammatory
hyperalgesia in rats and inhibited the LPS-mediated cytokine
release and decreased the mortality rate in LPS-challenged
mice.
NO, which is synthesized by iNOS, is a well-known
proinflammatory mediator that is involved in various
physiological and pathological processes. Recently, sup-
pression of NO production has been emphasized as a new
pharmacological strategy for the treatment of inflammation-
related diseases.
10
Here, we found that GLE inhibited NO
production in LPS-induced RAW264.7 cells by suppressing
iNOS gene expression. These data indicate that GLE is a an
anti-inflammatory agent because the excess NO produced by
iNOS mediates both acute and chronic inflammation.
11
It
has been reported that iNOS inhibitors attenuate osteoar-
thritis,
12
periodontitis,
13
septic shock,
14
and other chronic
inflammatory diseases. Inhibition of iNOS activity is a
significant therapeutic target for the treatment of many
pathological processes.
COX-2 is an enzyme that generates prostaglandins, which
is induced by proinflammatory cytokines and other activa-
tors, such as LPS, resulting in the release of a large amount
of PGE
2
at inflammation sites.
15
Therefore, identification of
COX-2 inhibitors is considered to be a promising approach
to protecting against inflammation and tumorigenesis. We
found that GLE significantly inhibited LPS-stimulated PGE
2
production and COX-2 protein and RNA expression in a
dose-dependent manner, indicating that the actions of GLE
occur at the level of transcription.
Recent studies have found that signaling pathways of
MAPKs play critical roles in the regulation of inflammatory
response and in coordinating the induction of many genes
encoding inflammatory mediators.
16
The 3 major MAPK
pathways consist of a highly conserved family of protein
kinases such as ERK, JNK, and p38. These kinases regulate
immune responses, including proinflammatory cytokine
production, mitosis, differentiation, and cell survival/
apoptosis.
17,18
In this study we found that pretreatment with
GLE suppressed LPS-induced phosphorylation of ERK1/2,
but not of p38 or JNK. The ERK signaling module, which
was the first MAPK cascade to be characterized, is a vital
mediator of cellular fates, including growth, proliferation,
and survival.
19
ERK plays a critical role in COX-2 expres-
sion for LPS-treated RAW264.7 cells and peritoneal mac-
rophages.
20
It has been reported that the ERK1/2 inhibitor,
PD98059, and the p38 inhibitor, SB203580, suppressed
LPS-induced NO production.
21
We also found that GLE did
not affect the phosphorylation of p65 at Ser 276 or IjB-a,
which is positively correlated with the extent of NF-jB
activation. NF-jB regulates the expression of various genes
FIG. 4. Effects of GLE on activation of (A) ERK, JNK, and p38
and (B) IjB-aand translocation of p65 in LPS-stimulated RAW264.7
cells. (A) Cells were preincubated with GLE (5, 10, 30, or 50 lg/mL)
for 2 h, and then stimulated with LPS (1 lg/mL) for 15 min. Whole-
cell extracts were prepared, resolved by SDS-PAGE, and electro-
transferred to a PVDF membrane, and then Western blot analyses
using phosphospecific anti-p38, anti-ERK, and anti-JNK antibodies
were performed. The same membranes were reblotted with anti-p38,
anti-ERK, and anti-JNK antibodies. The results shown are represen-
tative of 3 independent experiments. (B) Cells were preincubated
with GLE (5, 10, 30, or 50 lg/mL) for 2 h, and then stimulated with
LPS (1 lg/mL) for 15 min. Equal amounts of protein were analyzed
using antibodies specific for IjB-aand p65. The results shown are
representative of three independent experiments.
FIG. 5. Effect of different doses of GLE on paw withdrawal latency
(PWL) 24 h after FCA injection. GLE was administered 24 h a fter FCA -
induced hyperalgesia. D ata are expressed a s the mean –SD (n=7).
ab
Values with different letters were significantly different from the saline
group treated with FCA (P<.05). FCA, Freund’s complete adjuvant.
ANTI-INFLAMMATORY EFFECTS OF P. GUAJAVA L. 683
that encode proinflammatory cytokines, adhesion mole-
cules, chemokines, growth factors, and chemoattractants,
such as COX-2, and iNOS.
22
These results suggest that GLE
inhibits LPS-induced NO production, in part, through sup-
pression of the ERK1/2 signaling pathway in RAW264.7
cells. However, we cannot exclude the possible inhibition of
other transcription factors. Thus, these results suggest that
GLE partially modulates biomarkers via blocking ERK1/2
signaling pathway in cells.
In addition to the in vitro study, we also demonstrated the
inhibitory effect of GLE in 2 different experimental models of
inflammation. Freund’s complete adjuvant (Mycobacterium
butycirium) has commonly been used to produce inflammation
in the hind paw of rats
23
and is generally used to induce ar-
thritis in animal models.
24,25
FCA-induced inflammatory
hyperalgesia in the rat is a well-established model for the
evaluation of the analgesic activity of drugs.
26
It has been
shown out that FCA-injected rats have elevated serum IL-6,
severe hyperalgesia, and edema during the first week after
intervention.
27
Our results show that injection of FCA caused a
dramatic decrease in PWL at 24 h. Administration of GLE
significantly alleviated thermal withdrawal latency in a dose-
dependent manner, compared with thermal withdrawal latency
in the saline control group. Intraplantar injection of FCA into
the hind paw of rats induces a persistent localized inflamma-
tory state that is associated with thermal hyperalgesia and in-
creased excitability of spinal cord dorsal horn neurons.
23,28
Septic shock is a systemic response to serious infection
that is generally caused by Gram-negative bacterial endo-
toxins. LPS can induce large amounts of NO and PGE
2
release as well as proinflammatory cytokines in immune-
activated macrophages and at inflammation sites. Excess
production of these immune mediators may result in the
clinical syndrome of sepsis.
29,30
The presence of endotoxins
in experimental animals leads to pathophysiologic changes
that are similar to septic shock syndrome in humans, and
lethal endotoxemia has been extensively used as an exper-
imental model of Gram-negative septic shock.
31
In the present study, we found that GLE exhibited dose-
dependent inhibitory activity in FCA-induced PWL, and
significantly improved the survival rate of mice with lethal
endotoxemia. These results showed that GLE has very
strong anti-inflammatory activity in vitro as well as in vivo,
and could relieve LPS-induced systemic inflammation.
In conclusion, this study provides evidence that GLE
inhibits the secretion of inflammatory mediators, such as NO
and PGE
2
, in LPS-stimulated macrophages. GLE also sup-
pressed LPS-induced iNOS and COX-2 expression through
suppression of the ERK1/2 MAPK signaling pathway. Fur-
thermore, we also demonstrated significant anti-inflammatory
effects of GLE in vivo. Therefore, GLE may have the potential
to prevent inflammatory diseases. Further investigation is
needed to identify the active constituents in Psidium guajava
L. that possess these biological activities.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
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ANTI-INFLAMMATORY EFFECTS OF P. GUAJAVA L. 685
