Study on the antiinflammatory activity of Artocarpus altilis leaves extract in mice

Abstract

In addition to the body's response to injury, inflammation is a physiological process underlying the progression various diseases. Investigation for the discovery and development of antiinflammatory agents from natural sources is a promising strategy for curing inflammatory diseases. Breadfruit or Artocarpus altilis, is an Indonesian native plant traditionally used for the treatment of various disease including inflammatory-related diseases such as arthritis, tumors, pain, gastritis and atherosclerosis. Scientific evidence regarding the antiinflammatory effect of A.altilis leaf is limited. This study aimed to investigate the antiinflammatory activities of A.altilis leaf extract (AAE). The expression and the activity of COX, the enzyme responsible for prostaglandins formation, were also evaluated. The A.altilis leaf was extracted in ethyl acetate and the dried extract was used for the experiments in mice models. We employed a carrageenan-induced paw edema in mice to evaluate the antiinflammatory activities. The level of COX-2 expression in the paw tissues were determined using immunohistochemistry, whereas COX-2 inhibitory activity was tested on in vitro enzymatic assays. We found that AAE at the doses of 250, 500 and 1000 mg/kgBW significantly reduced the volume of paw edema until 6 hours of observation. In vitro enzymatic assays revealed that AAE has a lower IC50 against COX-2 compared to COX-1, suggesting the higher selectivity for COX-2. In addition, the level of COX-2 expression in the hind paws was also significantly reduced upon AAE treatment in dose-dependent manner. These indicated that AAE has a potency to be further developed as antiinflammatory agent or as a source of lead compounds acting as antiinflammation. © 2015, International Journal of Pharmacognosy and Phytochemical Research. All rights reserved.

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International Journal of Pharmacognosy and Phytochemical Research 2015; 7(6); 1080-1085
ISSN: 0975-4873
Research Article
*Author for Correspondence
Study on the Antiinflammatory Activity of Artocarpus altilis Leaves
Extract in Mice
Fakhrudin, N*1,2, Hastuti, S1, Andriani, A1, Widyarini, S3, Nurrochmad, A1
1Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta, 55281, Indonesia; 2Center for Natural
Antiinfective Research, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta, 55281, Indonesia;
3Department of Veterinary Pathology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Sekip Utara,
Yogyakarta, 55281, Indonesia.
Available Online:11th October, 2015
ABSTRACT
In addition to the body's response to injury, inflammation is a physiological process underlying the progression various
diseases. Investigation for the discovery and development of antiinflammatory agents from natural sources is a promising
strategy for curing inflammatory diseases. Breadfruit or Artocarpus altilis, is an Indonesian native plant traditionally
used for the treatment of various disease including inflammatory-related diseases such as arthritis, tumors, pain, gastritis
and atherosclerosis. Scientific evidence regarding the antiinflammatory effect of A.altilis leaf is limited. This study aimed
to investigate the antiinflammatory activities of A.altilis leaf extract (AAE). The expression and the activity of COX, the
enzyme responsible for prostaglandins formation, were also evaluated. The A.altilis leaf was extracted in ethyl acetate
and the dried extract was used for the experiments in mice models. We employed a carrageenan-induced paw edema in
mice to evaluate the antiinflammatory activities. The level of COX-2 expression in the paw tissues were determined
using immunohistochemistry, whereas COX-2 inhibitory activity was tested on in vitro enzymatic assays. We found that
AAE at the doses of 250, 500 and 1000 mg/kgBW significantly reduced the volume of paw edema until 6 hours of
observation. In vitro enzymatic assays revealed that AAE has a lower IC50 against COX-2 compared to COX-1,
suggesting the higher selectivity for COX-2. In addition, the level of COX-2 expression in the hind paws was also
significantly reduced upon AAE treatment in dose-dependent manner. These indicated that AAE has a potency to be
further developed as antiinflammatory agent or as a source of lead compounds acting as antiinflammation.
Keyword: Artocarpis altilis, antiinflammation, cyclooxygenase
INTRODUCTION
Inflammation is a body response to injuries induced by a
variousstimuli, such as infectious agents from
microorganisms, noxious substances, physical damages,
and changes induced by malignant cells. Various
pathological conditions, including atherosclerosis, sepsis,
cancer, arthritis and metabolic syndromes are related with
inflammatory condition1. Currently, no satisfying drug is
available for the treatment of these inflammatory-related
diseases. There are two antiinflammatory drugs available
in the clinic: corticosteroids and non-steroidal
antiinflammatory drugs (NSAID). Despite the fact that
corticosteroids and NSAID (non-steroidal
antiinflammatory drugs) remain the common choice for
the treatment of inflammatory diseases, the usage of these
drugs are restricted by their undesirable side effects and
the limited potency to reduce the symptoms of
inflammation. Moreover, chronic use of corticosteroids
antiinflammatory drugs has been limited as they exhibited
a weight gain, osteoporosis and immunosuppressive
effects. Whereas high dose NSAID medication leads to
gastrointestinal tract-related toxicities2. Consequently, the
development of novel potential antiinflammatory agents
with a desirable side effect is a great of interest.
Many therapeutic targets have been identified to interfere
the inflammatory processes. One of the most important
therapeutic target is cyclooxygenase (COX)3. COX-1 is
constitutively expressed, whereas COX-2 is an inducible
enzyme expressed during the inflammatory processes4.
COX-2 is responsible for the formation of prostaglandins
and has a key role for therapeutic intervention in pain and
inflammatory-related diseases5,6. Inhibition of COX-2
activity has become an important target for combating
inflammatory disorders3,7,8. COX-2 was the established
therapeutic target in inflammation and aspirin is the fist
NSAID inhibitor of COX-2. Inhibition of COX-2 activity
interferes the production of prostaglandins, the
inflammatory mediators that play a crucial role in the
development of inflammatory responses and varios
pathophysiological conditions5,9. Unfortunately, the use
of aspirin is associated with the undisired side effects
including renal toxicity and gastrointestinal bleeding10. In
addition, the newer selective COX-2 inhibitor, the
“coxib“ derivatives, still exhibits severe cardiovascular
side effects11-13. Consequently, the exploration for finding

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IJPPR, Volume 7, Issue 6 : December 2015 Page 1081
the alternative therapeutic agent targeting COX-2 is a
promising research.
Medicinal plants have significant contribution to the
drugs development and discovery and still provide
abundant and promising source for lead structures as drug
candidates. Many plants have been used by folk for
decades to treat various human diseases including
inflammatory diseases. Artocarpus altilis (Moraceae),
commonly known in Indonesia as Sukun, is a flowering
tree native to Indonesia and New Guinea, and spreading
throughout Southeast Asia and Africa. Indonesian people
consume the starchy fruit part after boiling and frying it at
all stage of growth as it provides high amount of fibers
and carbohydrates as well as contains protein, vitamins,
calcium, magnesium, potassium, copper, iron, niacin,
thiamin, riboflavin, lutein, and phenolics14,15. Although
the fruit of A. altilis has been acknowledges as a potential
food source for food security for the growing global
population15, the leaf part is underutilized and known to
be a non-toxic suggesting the safety in therapeutic uses16.
The leaves of A. altilis have been traditionally used by
folk for treating various disorders such as hypertension,
liver cirrhosis, diabetes, hypercholesterolemia, and also
used in inflammatory conditions such as arthritis, pain,
gastritis and stroke17,18. Previous studies showed that A.
altilis contains various non-polar a non-glycoside
prenylated flavonoids19-22 responsible for several
pharmacological activities including anticancer23,24,
antiausteric19, antioxidant25, antiinflammation26,
antiplatelet22 and antiatherosclerotic27. The leaves were
also known to contain lutein, sitosterol, squalene,
unsaturated triglycerides, polyprenol, unsaturated fatty
acids28. Previous study demonstrated that AAE showed a
promising antihypertension activity via inhibition of
angiotensin converting enzyme activity29 and exhibited
antiatherosclerosis activity30. However, only little
scientific data is available regarding the antiinflammatory
activity of AAE both in vitro and in vivo studies. In this
study, we investigated the antiinflammatory activity of
AAE in mice and the effects on the expression and the
activity of cyclooxygenase-2 (COX-2).
MATERIALS AND METHODS
Plant material
The main material used in this study is Artocarpus altilis
leaves. The leaves were harvested from Melati district,
Sleman, Yogyakarta, Indonesia. Plant identification was
done by the botanist at the Department of Pharmaceutical
Biology, Faculty of Pharmacy, Universitas Gadjah Mada,
Indonesia. Freshly harvested leaves were dried at 50°C in
the oven for 48 h and further grinded before the
extraction.
Reagents and Chemicals
Indomethacin and carrageenan were purchased from
Sigma Aldrich (Saint Louis, MO, USA), ethyl acetate and
aquadest were obtained from local supplier (Brataco
Chemika, Yogyakarta, Indonesia), whereas dimethyl
sulfoxide and the PBS components were obtained from
EMerck (Darmstadt, Germany). Colorimetric-based COX
Inhibitor Screening Assay was purchased from Caymann
Chemical (Michigan, USA, catalog number 760111) and
COX-2 antibody was obtained from Santa Cruz
Biotechnology (Texas, USA, catalog number sc-1747-R).
Extraction
The extraction of plant material was done using
maceration method. The dried A. altilis leaves (500 gram)
were macerated three times in ethyl acetate (2.5 L). After
filtration, the solvent was evaporated using a vacuum
rotary evaporator instrument under a reduced pressure to
dryness.
Animals
Male BALB/c mice (20-30 g) were used in this study.
They were housed in a controlled environment and fed
with standard pellet diet and water ad libitum. After
acclimatization for at least one week before the
experimental session, the mice were randomized and
divided into 6 groups. All the experimental protocols
were performed according to the guidelines approved by
Institutional Animal Ethic Committee (number 192/KEC-
LPPT/IX/2014), Universitas Gadjah Mada, Indonesia.
Carrageenan-induced paw edema
The acute inflammatory activity was evaluated using
Carrageenan-induced paw edema assay as previously
described31. Paw edema was induced by subplantar
injection of freshly prepared 200 μL carrageenan 1%
(solution in distilled water) in to the right hind paws. The
animals were randomly divided into 5 groups of 5
animals each. All groups, except the untreated group,
were given single dose of extracts (250, 500 or 1000
mg/kgBW), solvent or indomethacin (5 mg/kgBW), 30
minutes prior to paw edema induction using carrageenan.
The volume of the paw edema was measured using
plethysmometer every 30 minutes for 6 hours after
carrageenan administration. The antiinflammatory
activity (percentage activity) was determined based on
the paw edema volume differences between the extracts-
treated and the solvent-treated groups after 6 hours.
Immunohistochemistry of COX-2
After the measurement of paws edema, the mice were
sacrified and the soft plantar region sections of the hind
paw were cut and fixed in 10% buffered formalin for 24
hours and further embedded with parafin32. The parafin-
embedded blocks were cut with a thickness of 4 mm,
flattened and then attached to the glass slides coated with
poly-lysine. Antigen recovery was done by addition of
xylol and ethanol to deparafinize and dehydrate the
sections, respectively, and further washed in a phosphate
buffered saline (PBS) solution. The sections were
incubated for 10 minutes in a peroxidase blocking
solution containing 3% H2O2 to abolish endogenous
peroxidase activity and further incubated 10 minutes in a
bloking buffer containing 5% bovine serum albumin
(BSA). The sections were incubated overnight at 4°C
with diluted mice primary antibody (anti COX-2, 1:250
dilution in PBS-BSA) and then washed in PBS for 5
minutes. The secondary biotinylated universal antibody
(antiIgG) at 1:200 dilution was added and the sections
were incubated for 5 minutes at room temperature.
Following washingwith PBS for 5 minutes, the sections
were incubated for 10 minutes in the conjugated-

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IJPPR, Volume 7, Issue 6 : December 2015 Page 1082
streptavidin peroxidase complex. After another 5 minutes
washing with PBS, the sections were stained by
incubation (10 minutes) in peroxidase substrate solution
containing 3,3'diaminobenzidine-peroxide (DAB) at 1:9
dilution, and counter-stained with Mayer hematoxylin
(100 µl for 2 minutes). The stained sections were then
dehydrated using ethanol and xylol, added mounting
media and embedded in microscope slides for
immunohistochemistry analisis. When the cytoplasm was
stained brown, COX-2 immunostaiting was considered
positive. COX-2 expression was evaluated based on the
percentage of positive cells according to the previous
method32,33. The percentage of COX-2 expression was
determined by calculating the COX-2 expressing cells
(positive cells) in the extracts-treated groups compared to
the solvent-treated group. The cell counting was done in a
light microscope observed under 1000X magnification at
5 different fields.
COX-1 and COX-2 enzymatic assays
The COX-1 and COX-2 inhibitory activities of the
extracts were evaluated using a colorimetric-based COX
Inhibitor Screening Assay (Caymann, catalog number
760111) that employs peroxidase component of
cyclooxygenase. The activity of peroxidase is measured
colorimetrically at 590 nm based on the generation of
oxidized N,N,N',N'-tetramethyl-p-phenylenediamine
Figure 1. The AUC for time-response curves of paw edema on carrageenan-induced paw edema in mice (5 animals
per group). The A. altilis extract (AAE) were tested at 250, 500 and 1000 mg/kgBW and indomethacin (5 mg/kgBW)
was used as a positive control.
Figure 2. Antiinflammatory activity of A. altilis extract
on carrageenan-induced paw edema in mice (5 animals
per group). The extracts were tested at 250, 500 and
1000 mg/kgBW and indomethacin (5 mg/kgBW) was
used as a positive control. The values are mean ±
standard errors. * p< 0.05; ** p< 0.01
(ANOVA/Dunnett, compared to solvent-treated group).
Figure 3. Effect of A. altilis extract on the inhibition of
COX-2 expression on carrageenan-induced paw edema in
mice (5 animals per group). Mice paws were cut and fixed
in buffered formalin and COX-2 expression was quantified.
The extracts were tested at 250, 500 and 1000 mg/kgBW
and indomethacin (5 mg/kgBW) was used as a positive
control. The values are mean ± standard errors. * p< 0.05;
** p< 0.01 (ANOVA/Dunnett, compared to solvent-treated
group).

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(TMPD)34. The assay was performed according to the
manufacturer's spesified protocol.
Statistical Analysis
Data from all experiments were presented as mean values
with ± standard error mean (SEM). Statistical analysis
was performed by one-way analysis of variance
(ANOVA) with Dunnet post hoc test, p < 0.05 was
considered statistically significant.
RESULTS AND DISCUSSION
The leaf of A. altilis is used in Indonesia as an herbal
medicine preparation to treat various diseases including
inflammatory diseases. Although the leaves were
traditionally prepared for the medication as a decoct
dosage form, our preliminary study revealed that the A.
altilis leaves ethyl acetate extract (AAE) demonstrated a
higher antiinflammatory activity compared to the aquoeus
extract35. In this present study, we examined the
antiinflammatory effects of AAE in mice using acute
experimental model of inflammation. We employed
carrageenan-induced paw edema which represents a
common simple method for antiinflammatory evaluation
and then we studied the more specific antiinflammatory
effect on the COX-2 expression and activity. We found
that AAE exhibited antiinflammatory activity by reducing
carrageenan-induced paw edema in dose-dependent
manner (Figure 1 and 2). Paw edema is a common feature
for an acute inflammatory process36. Thus, the reduction
of carrageenan-induced edema volume upon AAE
treatment indicated that the extract has an acute anti-
inflammatory activity. However, the activity is lower
compared to the positive control, indomethacin.
One of the key factor responsible for the progression of
inflammatory process is cyclooxygenase (COX) enzymes,
especially COX-2. This inducible enzyme is over
expressed in inflammation and is known to be responsible
for the formation of prostaglandins from arachidonic acid.
Thus, we investigated the effect of AAE on the COX-2
expression and activity. COX-2 expression in the soft
plantar region sections of the hind paw was investigated
using immunohistochemistry, whereas the COX-2
enzymatic activity was evaluated using a colorimetric-
based COX inhibitor assay. Our study indicated that
COX-2 expression was reduced (Figure 3 and 4) and the
COX-2 activity was also inhibited (Figure 5) upon AAE
treatment in dose-dependent manner. Although
indomethacin (5 mg/kgBW) demonstrated stronger
activity, the higher concentration of indomethacin or the
extract might be required to completely inhibit COX-2
expression to the lower level. These results indicated that
Figure 4. Representative pictures showing COX-2 expression in the soft plantar region sections of the hind paw. The
pictures were taken in a light microscope observed under 1000X magnification at 5 different fields. The cox-2
expressing cells were colored in dark brown. A: solvent-treated goup, B: indomethacin-treated group, C: AAE 250
mg/kgBW, D: AAE 250 mg/kgBW.

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Figure 5. Artocarpus altillis extract inhibited COX-1
and COX-2 activity in vitro. The extract was was
dissolved in DMSO and prepared in 5 different
concentrations, each in a triplicate (n=3). The values are
means ± standard errors.
AAE, at least in part, exerts antiinflammatory activity via
targeting both the expression and activity of COX-2.
To determine the selectivity of AAE on COXs inhibition,
we also investigated the inhibitory effect of the extract on
COX-1 (Figure 5). Interestingly, AAE demonstrated a
potent COX-2 inhibition activity (IC50: 3,17µg/ml) in
comparison to that of COX-1 (IC50: 23,45), indicating
that this extract contains a promising compound with a
high potency to be developed as a selective COX-2
inhibitor. These indicate that AAE exerted
antiinflammatory activity, at least partly by inhibiting the
activity and the expression of COX-2. As COX-2 has a
crucial role in inflammation and it still represents a
potential therapeutic target for inflammation37,38, the
inhibition of COX-2 activity and expression upon AAE
treatment makes A. altilis as a potential source of natural
compounds with a promising antiinflammatory activity.
To determine the selectivity of AAE against COX-1 and
COX-2, we tested the potency of AAE in inhibiting
COX-1 and COX-2 in in vitro enzymatic assays. Figure 5
demonstrates that AAE inhibited both COX-1 (IC50:
23.45 μg/ml) and COX-2 (IC50: 3.17 μg/ml).
Interestingly, AAE shows more potent inhibition against
COX-2 compared to COX-1, indicating that this extract
contains a promising compound with a high potency to be
developed as a selective COX-2 inhibitor.
Our study clearly demonstrates that the AAE exhibited a
promising antiinflammatory activity in an experimental
model of acute inflammation in mice. These results are in
accordance with the previous study showing that A. altilis
contains several antiinflammatory flavonoids with a
distinct mechanism of action39. Artocarpin, a prenylated
flavonoid isolated from A. altilis was also claimed to be a
compound responsible for the antiinflammatory activities
as it decreased the level of TNFα and IL-1β, a major pro-
inflammatory transcription factor and a pro-inflammatory
cytokine, respectively26. These findings provide the
scientific evidence for the traditional use of A. altilis
leaves for the treatment of inflammatory diseases17.
In summary, we demonstrate that AAE exhibited
antiinflammatory activity in mice experimental models. It
inhibited carrageenan-induced paw edema and reduced
the expression and activity of COX-2. Additionally, it
showed a higher selectivity against COX-2 in comparison
to COX-1. These findings suggested that A. altilis leaves
could be a potential source for the discovery of novel
antiinflammatory compounds for drugs or dietary
supplements.
ACKNOWLEDMENT
This research was supported by Hibah Penelitian
Unggulan Perguruan Tinggi (PUPT), the Directorate
General of Higher Education, Ministry of Education and
Culture, Republic of Indonesia, (Grant number: LPPM-
UGM/344/LIT/2014). We also thank Setiono for an
excellent technical support.
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