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Antinociceptive, anti-inflammatory and antipyretic effects of Muntingia

calabura aqueous extract in animal models

Article  in  Journal of Natural Medicines · August 2007

DOI: 10.1007/s11418-007-0167-2

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J Nat Med (2007) 61:443–448
DOI 10.1007/s11418-007-0167-2
NOTE
Antinociceptive, anti-inflammatory and antipyretic effects
of Muntingia calabura aqueous extract in animal models
Z. A. Zakaria Æ N. A. Mohd Nor Hazalin Æ
S. N. H. Mohd Zaid Æ M. Abdul Ghani Æ
M. H. Hassan Æ H. K. Gopalan Æ M. R. Sulaiman
Received: 5 February 2007 / Accepted: 1 May 2007 / Published online: 10 July 2007

The Japanese Society of Pharmacognosy and Springer 2007
Abstract The present study was carried out to elucidate the
potential of aqueous extract of Muntingia calabura leaves
aqueous extract (MCAE) as antinociceptive, anti-inflam-
matory and antipyretic agents using the formalin-, carra-
geenan-induced paw edema- and brewer’s yeast-induced
pyrexia tests in rats. The extract was prepared by soaking the
dried powdered leaves of M. calabura in distilled water
(dH2O) overnight. The supernatant obtained, considered as a
stock solution (100% concentration/strength), was then
diluted to concentrations of 10% and 50% and used together
in all experimental models. The MCAE, at concentrations of
10%, 50% and 100%, were found to show significant
antinociceptive, anti-inflammatory and antipyretic activities
in all tests. However, all of the activities occurred in a
concentration-independent manner. The 50% and 100%
concentrations of MCAE produced insignificant antino-
ciceptive and antipyretic activities, respectively. Although
the 100% concentration of MCAE produced significant
(P<0.05) anti-inflammatory activity, the activity was lower
than that of the 10% and 50% concentrations of MCAE.
Z. A. Zakaria (&)
Faculty of Pharmacy, Universiti Teknologi MARA,
40450 Shah Alam, Selangor, Malaysia
e-mail: shaza8174@yahoo.com
N. A. M. Nor Hazalin
S. N. H. M. Zaid

M. A. Ghani
M. H. Hassan
H. K. Gopalan
Faculty of Biotechnology and Life Sciences,
Universiti Industri Selangor, Jalan Zirkon A7/A,
Seksyen 7, 40000 Shah Alam, Selangor, Malaysia
M. R. Sulaiman
Department of Biomedical Sciences,
Faculty of Medicine and Health Science,
Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia
Based on the results, we conclude that the M. calabura leaves
possessed antinociceptive, anti-inflammatory and antipy-
retic activities and, thus, justifies the Peruvian folklore
claims of its medicinal values.
Keywords Muntingia calabura
Aqueous extract

Antinociceptive
Anti-inflammatory
Antipyretic
Introduction
Muntingia calabura L., locally known as Kerukup siam and
belonging to the Elaeocarpaceae family, has been claimed
in Peruvian folklore medicine to possess medicinal values
[1, 2]. Its leaves, in particular, are drunk as a tea-like
beverage [1] and has been claimed to provide relief from
gastric ulcers or to reduce swelling of the prostate gland if
either boiled or steeped in water, respectively [1].
Furthermore, the leaves are also taken to alleviate head-
aches and colds [2, 3]. Other than that, its flowers are
claimed to possess antiseptic properties, while its infusion
is said to have antispasmodic activity and its boiled barks
are used to reduce swelling in the lower extremities [1].
Scientifically, the leaves and roots of M. calabura were
reported to possess anti-tumour properties [4, 5]. Recently,
we have reported on the antinociceptive activity of M. cala-
bura leaves assessed using the abdominal constriction test
and that the activity is mediated through L-arginine/nitric
oxide/cyclic guanosine monophosphate pathway [6]. Ear-
lier, we have also reported on the M. calabura leaves’ anti-
bacterial activity [7, 8]. Based on the facts that M. calabura
are wildly cultivated in Malaysia and that no extensive
studies have been carried out to explore its pharmacological
benefits, we take the opportunity to evaluate and establish
some of the basic pharmacological properties of the plant.
123
444
The aim of the present study is to determine the antinoci-
ceptive, anti-inflammatory and antipyretic activities of
M. calabura leaves aqueous extract (MCAE) in rats using
the respective formalin-, carrageenan-induced paw edema-
and brewer’s yeast-induced pyrexia tests.
Methods
Experimental animals
Male Sprague Dawley rats (180–200 g; 8–10 weeks old),
obtained from the Veterinary Animal Unit, Faculty of
Veterinary Medicine, Universiti Putra Malaysia (UPM),
Malaysia, and kept at room temperature (27±2C; 70–80%
humidity; 12-h light/darkness cycle) in the animal holding
unit (UPM) were supplied with food and water ad libitum
up to the beginning of the experiments. The rats were, at all
times, handled in accordance with current UPM guidelines
for the care of laboratory animals and the ethical guidelines
for investigations of experimental pain in conscious ani-
mals. All experiments were conducted between 09.30 h and
18.30 h to minimise the effects of environmental changes.
Preparation of drugs
Acetylsalicylic acid (ASA) (100 mg/kg; Bayer, Singapore),
morphine sulphate (5 mg/kg; Sigma, Germany), used for
the purposed of comparison, were prepared by dissolving
them in distilled water (dH2O).
Plant material
The leaves of M. calabura were collected in the months of
July and August 2005 from its natural habitat in Shah
Alam, Selangor, Malaysia, and were identified by
Mr. Shamsul Khamis, a botanist from the Institute of
Bioscience, UPM, Malaysia. A voucher specimen (SK 964/
04) was deposited at the Herbarium of the Laboratory of
Natural Products, Institute of Bioscience, UPM, Serdang,
Selangor, Malaysia.
Phytochemical screening of the M. calabura leaves
The phytochemical screening of the M. calabura leaves
was carried out according to the standard screening tests
and conventional protocols as described by Ikhiri et al. [9].
Preparation of Muntingia calabura aqueous
extract (MCAE)
Fresh leaves of M. calabura were washed and rinsed with
water to remove all dirt and unwanted particles and were
123
J Nat Med (2007) 61:443–448
then oven-dried for 72 h at a temperature of 50C. The
dried leaves were then ground into small particles and the
MCAE was prepared by soaking the air-dried powdered
leaves of M. calabura (20 g) with dH2O at a ratio of 1:20
(w/v) for 72 h. The supernatant was collected and filtered
using Whatman No. 1 filter paper, while the remaining
plant residue was kept in an oven. The filtered supernatant
obtained, considered as stock solution with 100% concen-
tration/strength, was diluted with dH2O to concentrations
of 10% and 50% for the antinociceptive, anti-inflammatory
and antipyretic studies. Thirty millilitres of the obtained
supernatant was also subjected to a freeze-drying process
and yielded 0.81 g of crude dried MCAE (percentage of
yield is 4.1%). Based on the amount of crude dried
MCAE obtained, it was estimated that the 10%, 50% and
100% concentrations of MCAE were approximately
equivalent to the dosages of 27 mg/kg, 135 mg/kg and
270 mg/kg, respectively. The extract was administered
subcutaneously in all assays to standardise the route of
administration between the extract and the standard drugs,
particularly morphine, which is well known to produce best
antinociceptive effect via a systemic (i.e. subcutaneous,
s.c.) route. This will allow appropriate comparison to be
made on their activity at the end of the experiment. Other
than that, 5 mg of the crude dried MCAE was also
dissolved in 0.5 ml of filtered dH2O and filtered using a
0.45-lm membrane filter for high performance liquid
chromatography (HPLC) profiling.
Instrumentation for HPLC profiling of the MCAE
HPLC profiling of the MCAE was carried out at the Labo-
ratory of Phytomedicine, Forest Research Institute of
Malaysia, Kepong, Malaysia. A Waters Delta 600 with 600
Controller and Waters 2996 Photodiode Array (Milford,
MA, USA) equipped with an autosampler, online degasser
and column heater was used for the HPLC analysis. The
data were analysed and processed using the installed Mil-
lennium 32 Software (Waters Product). The sample was
separated on a minibore Phenomenex Luna 5-lm C18
column with dimension 250·4.60 mm using a one-step
linear gradient. The solvents were: (A) 0.1% ortho-phos-
phoric acid and (B) acetonitrile. The elution system was as
follows: 0–12 min, 0–15% of B; 12–22 min, 15–25% of B;
22–35 min, 25–15% of B. The flow rate was1.0 ml/min,
the injection volume was 10 ll and the column oven
temperature was set at 27C. The eluant was monitored at
wavelengths of 200 nm, 254 nm and 330 nm.
Formalin test
The formalin test was carried out as described by Hun-
skaar and Hole [10] but with some slight modifications.
J Nat Med (2007) 61:443–448
Pain was induced by injecting 50 ll of 5% formalin
(Sigma, USA) in the subplantar region of the left hind
paw. Rats (n=5) were given (s.c.) extract/drugs 30 min
prior to formalin injection. The rats were individually
placed in a transparent Plexiglass cage observation
chamber. The amount of time that the animal spent
licking the injected paw, considered as an indicator of
pain, was recorded for a duration of 30 min following the
formalin injection. The early phase and late phase of
nociception was measured between 0–5 min and 15–
30 min after the formalin injection, respectively. ASA
(100 mg/kg; s.c.) and morphine (5 mg/kg; s.c.) were used
as reference drugs. All of the tested solutions were
administered at a volume of 10 ml/kg.
Anti-inflammatory assay
The carrageenan-induced paw edema test was carried out
as described by Chakraborty et al. [11] but with some
slight modifications. Acute inflammation was produced by
the subplantar injection of 0.1 ml of 1% suspension of
carrageenan (Sigma, USA) in dH2O on the right hind paw
of the rats (n=5), 30 min after the administration (s.c.) of
extract/drugs. The thickness of the paw was measured
using a caliper before (BF) and after carrageenan (i.p.)
treatment at 0, 30, 60, 120, 180, 240, 300, 360, 420 and
480 min. ASA (100 mg/kg; s.c.) was used as a reference
drug. All of the tested solutions were administered at a
volume of 10 ml/kg.
Antipyretic assay
The brewer’s yeast-induced pyrexia test was carried out
according to the method described by Reanmongkol
et al. [12] but with some slight modifications. Pyrexia
was induced by injecting (i.p.) 10% (w/v) Brewer’s yeast
(BY) (Sigma, USA) suspension (10 ml/kg) 30 min after
the s.c. administration of the extract/drug. The rectal
temperature of each rat (n=5) was measured before (BF)
and after BY treatment at 0, 30, 60, 120, 180, 240, 300,
360, 420 and 480 min using a digital thermometer (SK-
1250MC, Sato Keiryoki Mfg. Co., Ltd., Japan). ASA
(100 mg/kg; s.c.) was used as a reference drug. All of
the tested solutions were administered at a volume of
10 ml/kg.
Statistical analysis
The results are presented as mean±standard error of the
mean (SEM). The one-way analysis of variance (ANOVA)
test with Dunnett’s post-hoc test was used to analyse and
compare the data, with P<0.05 being used as the limit of
significance.
445
Results
Phytochemical screening of the M. calabura leaves
The phytochemical screening of the leaves of M. calabura
has demonstrated the present of flavonoids, saponins, tan-
nins, steroids and triterpenes but no alkaloids.
HPLC profiling of the MCAE
The HPLC profiles of MCAE were measured at three
different wavelengths, namely, 200 nm, 254 nm and
330 nm (Fig. 1). Comparison with three types of standard
flavonoids, namely, catechin, rutin and fisetin, were carried
out at 200 nm, as those standards were best detected at this
wavelength. Based on the wavelength comparison between
the major peaks in the MCAE and the standards, the types
of flavonoids present in the extract are suggested to be
different form that of the selected standards (data not
shown).
Pharmacological studies on the MCAE
Figure 2 shows the antinociceptive activity of MCAE as-
sessed using the formalin test in rats. The extract exhibited
concentration-independent antinociceptive activity in both
phases of nociception. All concentrations of MCAE
showed significant (P<0.05) activity in the early phase and,
except for the 135 mg/kg measure of MCAE, the rest
concentrations of the extract produced significant (P<0.05)
antinociceptive activity in the late phase. In addition, the
extract activity, at all concentrations used, was lower than
that of 5 mg/kg morphine.
Figure 3 shows the anti-inflammatory activity of MCAE
assessed using the carrageenan-induced paw edema test in
rats. The extract was found to exhibit a concentration-
Fig. 1 The high performance liquid chromatography (HPLC) profiles
of Muntingia calabura leaves aqueous extract (MCAE) at wave-
lengths of 200 nm, 254 nm and 330 nm
123
446 J Nat Med (2007) 61:443–448

90 41

80
40

Rectal Temperature (°C)

Latency of Discomfort (s)

#
70 #
#
60 * 39 # *
* # * *
* * #
50 * #
*
* 38 * *
40 * *
* *
* * *
30 37 *
*
*#
*
20 *
36
10 *

0 35
Early Late 0 60 120 180 240 300 360 420 480
Phase of Nociception Time Interval (min)
dH2O 5 mg/kg Morphine 100 mg/kg ASA dH2O 100 mg/kg ASA 27 mg/kg MCAE 135 mg/kg MCAE 270 mg/kg MCAE
27 mg/kg MCAE 135 mg/kg MCAE 270 mg/kg MCAE # Data with this superscript differed significantly (p<0.05) when compared to the data at the interval time of 0 min.
* Data with this superscript differed significantly (p<0.05) when compared to the control group (dH2O-treated) of the respective interval time.
* Significant (P<0.05) when compared to the respective control group

Fig. 4 The antipyretic profile of MCAE assessed by the Brewer’s

Fig. 2 The antinociceptive profile of MCAE assessed by the formalin yeast-induced pyrexia test in rats. # Data differed significantly
test in rats. The asterisk (*) indicates significance (P<0.05) when (P<0.05) when compared to the data at the time interval of 0 min.*
compared to the respective control group Data differed significantly (P<0.05) when compared to the control
group (dH2O-treated) of the respective time interval

0.7

no activity at all. The 27 mg/kg and 135 mg/kg measures

Thickness of Paw Edema (mm)

0.65
#
# # #
of MCAE were found to produce significant (P<0.05)
#
0.6
* #
# antipyretic activity after 240 min of their respective
#
* *
*
*
* *
* * #
administration, which lasted until the end of the experi-
0.55 * * * *
*
* * *
* * * ment. Their activity was, however, less effective than that
* * *
0.5 of the 100 mg/kg of ASA.
0.45

0.4
BF 0 60
dH2O 100 mg/kg ASA
# Data with this superscript differed significantly ( p<0.05) when compared to the data obtained before (BF) carrageenan administration.
* Data with this superscript differed significantly ( p<0.05) when compared to the control group (dH2O-treated) of the respective interval time.
Fig. 3 The anti-inflammatory profile of MCAE assessed by the
carrageenan-induced paw edema test in rats. # Data differed
significantly (P<0.05) when compared to the data obtained before
(BF) carrageenan administration.* Data differed significantly
(P<0.05) when compared to the control group (dH2O-treated) of the
respective time interval
independent anti-inflammatory activity, with the highest
concentration of MCAE producing activity that is lower
than that of the other concentrations of MCAE. Except for
the 270 mg/kg measure of MCAE, whose activity dimin-
ished after 360 min of its administration, the 27 mg/kg and
135 mg/kg measures of MCAE activity were completely
lost after 420 min of their administration. Interestingly, the
27 mg/kg and 135 mg/kg measures of MCAE produced
activity that was significantly (P<0.05) greater than that of
100 mg/kg of ASA between the time intervals of 180 min
and 240 min.
Figure 4 shows the antipyretic activity of MCAE
assessed using the BY-induced pyrexia test in rats. The
extract exhibited concentration-independent antipyretic
activity, with the 270 mg/kg measure of MCAE producing
Discussion
120 180 240 300 360 420 480
Time Interval (minutes)
27 mg/kg MCAE 135 mg/kg MCAE 270 mg/kg MCAE The present study has demonstrated that the aqueous
extract of M. calabura possessed antinociceptive, anti-
inflammatory and antipyretic properties. This finding was
in line with claims made earlier on the plant’s potential to
relieve gastric ulcers, headaches and colds [1–3]. The
observed pharmacological activities, particularly the
antinociceptive activity, of the extract were concomitant
with our recent report on M. calabura antinociceptive
activity [6]. The ability of the extracts/drugs to show
antinociceptive activity when assessed using the abdominal
constriction and hot plate tests has been associated with
their ability to affect the peripheral and central nociceptive
mechanism [13–15], a mechanism shown by opioid drugs
like morphine.
According to Hunskaar et al. [16], the formalin test
produces a distinct biphasic nociceptive response and is
widely used to elucidate the non-anti-inflammatory,
antinociceptive properties of extracts/drugs. The adminis-
tration of formalin will lead to an immediate and intense
increase in the C-fibre afferent spontaneous activity and
evokes a distinct quantifiable behaviour indicative of pain
[17, 18]. The early and late phases have different properties
and are very useful for assessing the potency and mecha-
nisms of analgesic agents. The early phase is caused by a

123
J Nat Med (2007) 61:443–448
direct effect of formalin on nociceptors, whereas the late
phase is a tonic response involving the inflammatory pro-
cesses and activation of the neurons in the dorsal horns of
the spinal cord [19]. Centrally acting drugs (i.e. morphine)
have been proven to inhibit both phases, while peripherally
acting drugs (i.e. ASA) inhibit only the late phase of the
said test [20, 21]. Based on the results obtained, the MCAE
inhibits both phases of the formalin-induced nociception,
indicating the involvement of the central mechanism that is
in line with our study using the hot plate test [22].
The carrageenan-induced rat paw edema test has been
widely used to evaluate the anti-inflammatory potency of
extracts/drugs [20, 23]. The edema development depends
on the participation of kinins and polymorphonuclear leu-
cocytes with their pro-inflammatory factors, such as pro-
staglandins [24]. The significant reduction of the thickness
of paw edema when compared to the control group indi-
cated the anti-inflammatory activity of MCAE [25, 26].
This finding has scientifically confirmed the folklore use of
M. calabura leaves in the treatment of pain associated with
gastric ulcers [1] via the central mechanism.
The MCAE also exhibited an antipyretic activity, which
confirmed the traditional uses of the plant in the treatment
of headaches and colds [1]. The fact that pyretic activity
involved inhibition of the activity of prostaglandins syn-
thesised within the central nervous system (CNS) [27] and
that the blood–brain barrier (BBB) prevents drugs mole-
cules or other chemicals from entering the CNS [28], our
finding indicated the extract’s ability to enter the CNS and,
thus, could also be used to explain the extracts’ morphine-
like ability seen with the formalin test, as well as the hot
plate test [22].
The concentration-independent activity seen with the
MCAE could be associated with the therapeutic window
phenomenon [29]. Certain extracts/drugs exhibited a
desired therapeutic effect only over a narrow range of drug
doses or plasma drug concentrations and if the doses were
below or above this narrow therapeutic range, suboptimal
beneficial effects or a decline in effects will be produced.
On the other hand, certain drugs have been reported to
cause deactivation of the receptor when present at high
concentrations within the biological system [30] and this
phenomenon could be related to the anti-inflammatory and
antipyretic activities of MCAE.
Earlier studies on MCAE antinociceptive activity has
demonstrated the involvement of, at least in part, opioid-
receptor [22] and the L-arginine/NO/cGMP pathway [6].
Several bioactive compounds have been isolated and
identified from the leaves of M. calabura. Kaneda et al.
[4] and Su et al. [5] have described the presence of
various types of anti-tumour-bearing flavanones and
flavones in the leaves of M. calabura. Interestingly,
flavonoids [31, 32], triterpenes [33, 34] and tannins [35,
447
36], in part, have been reported to show anti-inflammatory
activity, while, according to Attaway and Zaborsky [37],
compounds that possess an anti-inflammatory activity also
possessed antinociceptive activity, supporting our previ-
ous and recent findings. Although the actual mechanisms
of activities of the bioactive compounds were yet to be
proven, the report made by Dawson and Snyder [38] that
the in vivo anti-inflammatory activity of flavonoids and
flavones derivatives occur through the modulation of pro-
inflammatory gene expression, such as inducible NO
synthase and cyclooxygenase-2, could be used to support
our findings. In conclusion, this study demonstrated that
the MCAE possesses antinociceptive, anti-inflammatory
and antipyretic activities, which could be due to the
synergistic effect of flavonoids, saponins, tannins and
steroids, and, thus, can justify the traditional uses of the
plant to treat various ailments related to pain and
inflammation.
Acknowledgements This study was supported by a research grant
of the Universiti Industri Selangor, Malaysia (project code number:
03013; project vote number: 3090103013). The authors would like to
thank the Universiti Putra Malaysia for the use of their facilities.
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