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In vivo Evaluation of the Pharmacological Activities of
Syzygium samarangense (Blume) Merr. & L.M. Perry
Rajib Majumder, Nur-E-Hasnat, Md. Ashraf-Uz-Zaman and Md. Badrul Alam
Graduate School of Biotechnology, Yeungnam University, Geyoungsan, South Korea
Department of Pharmacy, Atish Dipankar University of Science and Technology, Dhaka, Bangladesh
Department of Clinical Pharmacy and Pharmacology, University of Dhaka, Dhaka, Bangladesh
Graduate school of Food Science and Biotechnology, Kyungpook National University, Daegu, South Korea
present studies were carried out to investigate the analgesic, anti-inflammatory, CNS depression as well as the
anti-diarrheal activity of the methanolic extract of Syzygium samarangense leaves (MSSL) and barks (MSSB).
MSSL and MSSB were used to investigate the analgesic effect by acetic acid induced writhing and formalin
induced licking method whereas carrageenan induced inflammation was used for anti-inflammatory activity.
CNS depression activities were evaluated in hole-cross and open field test methods. Furthermore castor
oil-induced diarrhea and charcoal-induced gastrointestinal motility were used to investigate the anti-diarrheal
activity of MSSL and MSSB. MSSBat doses of 100, 200 and 300 mg/kg body weight p.o., significantly (p<0.05)
reduced the writhing caused by acetic acid and the number of licks induced by formalin in a dose dependent
manner. At 400 mg/kg doses of MSSB showed highest anti-inflammatory activity (% inhibition 72.82%) after
4 hrs. A statically significant CNS depression activity was also observed in both hole cross and open field tests
in a dose dependent manner. MSSL and MSSB significantly reduced the frequency and severity of diarrhea in
test animals throughout the study period in a dose dependent manner and also showed a significant (p<0.05)
reduction in the gastrointestinal motility in charcoal meal test. Altogether, these results suggest that the
use of this species in traditional medicine.
methoxychalcone; six quercetin glycosides: reynoutrin;
one flavanone: (S)-pinocembrin and two phenolic acids:
is a plant species in the Myrtaceae, native to an area
gallic acid and ellagic acid were isolated from the pulp and
that includes the Malay Peninsula and the Andaman
seeds of the fruits of S. samarangense . Furthermore,
and Nicobar Islands, but introduced in prehistoric times
to a wider area  and now widely cultivated in the
lrhamnopyranoside; desmethoxymatteucinol; 4,6-
tropical countries. Leaves of Syzygium samarangense
have been reported to have antibacterial , antidiabetic
betulinate; oleanolic acid; jacoumaric acid; ursolic acid;
 anti-diarrheal , antioxidant , analgesic and
arjunolicacid; samarangenin A and samarangenin
anti-inflammation  as well as immunostimulant 
B were also isolated. In addition from the hexane
activities. Three C-methylated chalcones; 2',4'-dihydroxy-
extract of the leaves a mixture of a-carotene and ß-
carotene;lupeol; botulin;epi-betulinic acid; 2,4-dihydroxy-
hyperin; myricitrin; quercitrin; quercetin and guaijaverin,
Institutional Animal Ethics Committee, Atish Dipankar
University of Science & Technology, Dhaka, Bangladesh.
Animal treatment and maintenance for acute toxicity and
glucosidewere also isolated .
Principle of Laboratory Animal Care (NIH publication
Literature reviews indicated that no combined studies
No. 85-23, revised 1985) and the Animal Care and Use
in analgesic, anti-inflammatory, anti-diarrheal and CNS
Guidelines of Atish Dipankar University of Science and
depression effects of the bark of Syzygium samarangense
Technology, Dhaka, Bangladesh.
have so far been undertaken. Taking this in view and as
a part of our ongoing research  on Bangladeshi
Acute Toxicity Study: Acute oral toxicity assay was
medicinal plants, the present study was aimed to
performed in healthy adult male and non-pregnant adult
evaluate the comparison of analgesic, anti-inflammatory,
female albino Swiss mice (28-32g) divided into different
anti-diarrheal and CNS depression activities of the
groups. The test was performed using increasing oral
methanolic extract of the leaves and bark of Syzygium
dose of the MSSL and MSSB (50, 100, 200, 500, 1000
samarangense in different experimental models.
mg/kg body weight p.o.), in 20 ml/kg volume to different
allowed to feed ad libitum, kept under regular observation
of Syzygium samarangense were collected from
Ramna, Dhaka, Bangladesh in July, 2012 and identified by
DR. M.A. Razzaque Shah PhD, Tissue Culture Specialist,
activity of the samples was studied using acetic
BRAC Plant Biotechnology Laboratory, Bangladesh and
acid-induced writhing model in mice. The animals were
the voucher specimen no. maintained in our laboratory
divided into eight groups with five mice in each group.
for future reference. The both plant materials were
Group I animals received vehicle (1% Tween 80 in water,
shade-dried with occasional shifting and then powdered
p.o.), animals of Group II received Diclofenac-Na at 10
with a mechanical grinder, passing through sieve#40 and
mg/kg body weight while animals of groups III, IV, V, VI,
stored in an air-tight container. The dried powder material
VII and VIII were treated with 100, 200 and 300 mg/kg
of leaves (1.0 kg) and barks (1.0 kg) was refluxed with
body weight (p.o.) of the MSSL and MSSB, respectively.
MeOH for three hours. The total filtrate was concentrated
Test samples and vehicle were administered orally 30 min
to dryness; in vacuo at 40°C to render the MeOH extract
before intra-peritoneal administration of 0.7% v/v acetic
160 g and 180 g for leaves and barks, respectively.
acid but Diclofenac-Na was administered intra-peritonially
Chemicals: Acetic acid, formalin and castor oil as well as
5 min, the mice were observed for specific contraction of
carrageenan were purchased from E. Merck (Germany).
body referred to as ‘writhing’ for the next 10 min .
Loperamide, Diazepam, Indomethacin,
Diclofenac-Na were collected from Square
Formalin Test: The anti-nociceptive activity of the drugs
Pharmaceuticals Ltd., Bangladesh. All other chemicals and
was determined using the formalin test described by
reagents were of analytical grade.
Dubuission and Dennis. Control group received 2.5%
Experimental Animals: Young Long-Evans rats of
dorsal surface of the right hind paw 30 min after
either sex weighing about 140-160 g and Swiss Albino
administration of MSSL and MSSB (100, 200 and 300
mice (25-30g) were used for assessing biological activity.
mg/kg, body weight p.o. respectively) and 15 min after
The animals were maintained under standard laboratory
administration of Diclofenac Na (10 mg/kg, body weight
conditions and had free access to food and water ad
p.o.). The mice were observed for 30 min after the injection
of formalin and the amount of time spent licking the
environment for 7 days prior to experimental session.
injected hind paw was recorded. The first 5 min post
The animals were divided into different groups, each
formalin injection is referred to as the early phase and the
consisting of five animals which were fasted overnight
period between 15 and 30 min as the late phase. The total
prior to the experiments. Experiments on animals were
time spent licking or biting the injured paw (pain behavior)
performed in accordance with guidelines of the
test groups. Normal group received water. The mice were
for 48 hrs, for any mortality or behavioral changes .
15 min before injection of acetic acid. After an interval of
formalin, 20 µl of 2.5% formalin was injected into the
performed according to the method described by
Shoba and Thomas . Briefly, mice fasted for 24 hrs
(140-160 g) of both sexes were divided into six groups of
were randomly allocated to four groups of five animals
five animals each. The test groups received 100, 200 and
each. The animals were all screened initially by giving
300 mg/kg body weight p.o. of the extract MSSL and
0.5 ml of castor oil. Only those showing diarrhea were
MSSB. The reference group received Indomethacin
selected for the final experiment. Group I received 1%
(10 mg/kg body weight, p.o.) while the control group
CMC (10 ml/kg, p.o.),Groups III, IV, V,VI, VII and VIII were
received 3 ml/kg body weight normal saline. After 30 min,
treated with 100, 200 and 300 mg/kg body weight (p.o.)
0.1 ml 1% carrageenan suspension in normal saline was
of the MSSL and MSSB, respectively. Group II was given
injected into the sub-planatar tissue of the right hind paw.
Loperamide (3 mg/ kg, p.o.) in suspension. After 60 min,
The paw volume was measured at 1, 2, 3 and 4 hr after
each animal was given 0.5 ml of castor oil, each animal was
carrageenan injection using a micrometer screw gauge.
placed in an individual cage, the floor of which was lined
The percentage inhibition of the inflammation was
with blotting paper which was changed every hour,
calculated from the formula:
observed for 4 hrs and the characteristic diarrheal
% inhibition = (1-D D ) x 100
into four groups of five mice each and each animal was
of the control group of mice at a given time, D was the
average inflammation of the drug treated (i.e., extract or
reference Indomethacin) mice at the same time .
or vehicle. Group I was administered 1% CMC (10 ml/kg
CNS Depression Activity
and VIII were treated with 100, 200 and 300 mg/kg body
weight (p.o.) of the MSSL and MSSB, respectively. Group
described by Takagi et al. . The animals were divided
II received atropine sulfate (0.1 mg/kg body weight p.o.)
into different groups and each group contains 6 animals.
as the standard drug. After 30 min, animals were killed by
The control group received vehicle (1% Tween 80 in water
light ether anaesthesia and the intestine was removed
at the dose of 10 ml/kg p.o.) whereas the test group
without stretching and placed lengthwise on moist filter
received MSSL and MSSB extract (at the doses of 100, 200
paper. The intestinal transit was calculated as a
and 300 mg/kg p.o.) and standard group received
percentage of the distance travelled by the charcoal meal
Diazepam at the dose of 1mg/kg body weight p.o. orally.
compared to the length of the small intestine .
Each animal was then placed on one side of the chamber
and the number of passages of each animal through the
hole from one chamber to the other was recorded for 3 min
mean ± SEM (Standard Error Mean) of three replicate
on 0, 30, 60, 90 and 120 min during the study period.
experiments. The analysis was performed by using SPSS
Open Field Test: This experiment was carried out as
Inc, Chicago). Results related to the reducing power
described by Gupta et al. . The animals were divided
activities were statistically analyzed by applying the
into control standard and test groups (n = 6 per group).
Student t-test and p<0.05 were considered to be
statistically significant. All in vivo data are subjected to
at the dose of 10 ml/kg p.o.). The test group received the
ANOVA followed by Dunnett’s test and p<0.05 were
crude extract (at the doses of 100, 200 and 300 mg/kg p.o)
considered to be statistically significant.
and standard group received Diazepam at the dose of
1mg/kg body weight orally. The animals were placed on
the floor of an open field (100 cm×100 cm×40 cm hr)
divided into a series of squares. The number of squares
Acute Toxicity Studies: The acute toxicity studies mainly
visited by each animal was counted for 3 min on 0, 30, 60,
aim at establishing the therapeutic index, i.e., the ratio
90, 120, 180 and 240 min during the study period.
between the pharmacologically effective dose and the
MSSL and MSSB were safe up to a dose of 1000 mg/kg
(p.o.) body weight. Behavior of the animals was closely
droppings were recorded.
Effect on Gastrointestinal Motility: Animals were divided
body weight p. o.) and animals in groups III, IV, V,VI, VII
statistical package for WINDOWS (version 16.0; SPSS
lethal dose on the same strain and species. Both extract of
No. of writhing
32.77 ± 1.10
8.70 ± 1.30
27.09 ± 1.16
16.19 ± 2.16
11.37 ± 1.96
25.02 ± 1.06
14.10 ± 1.24
9.10 ± 1.04
Values are mean ± SEM, (n = 5), p<0.05, as compared to vehicle control (One way ANOVA followed by Dunnet test). Group I animals received vehicle
(1% Tween 80 in water), group II received Diclofenac Na as standard 10 mg/kg body weight (p.o.), groups III, IV, V, VI, VII and VIII were treated with 100,
200 and 300 mg/kg body weight (p.o.) of MSSL and MSSB, respectively.
Table 2: Effects of methanolic extract of MSSL and MSSB in the hind paw licking in the formalin test in mice
Late phase (Sec)
40.16 ± 1.82
11.03 ± 0.36*
31.16 ± 1.82
19.23 ± 1.12*
15.83 ± 1.22*
26.20 ± 1.39
27.16 ± 1.42
12.16 ± 1.99*
11.13 ± 1.36*
Values are mean ± SEM, (n = 5); * p <0.05 as compared to vehicle control (One way ANOVA followed by Dunnet test). Group I animals received vehicle
(1% Tween 80 in water), group II received Diclofenac Na as standard 10 mg/kg body weight p.o., groups III, IV, V, VI, VII and VIII were treated with 100,
200 and 300 mg/kg body weight (p.o.) of MSSL and MSSB,respectively.
Table 3: Effect of methanolic extract of the MSSL and MSSB on hole cross test in mice
Number of Movements
17.00 ± 1.71
17.20 ± 0.96
18.60 ± 1.12
15.00 ± 1.15
14.80 ± 1.50
13.10 ± 1.11
10.20 ± 0.96
9.60 ± 1.12*
15.10 ± 1.50
13.20 ± 0.65
13.10 ± 1.50
Values are mean ± SEM, (n = 6); * p <0.05, as compared to vehicle control (One way ANOVA followed by Dunnet test). Group I animals received vehicle
(1% Tween 80 in water), group II received Diazepam 1 mg/kg body weight, groups III, IV, V, VI, VII and VIII were treated with 100, 200 and 300 mg/kg
body weight p.o. of the MSSL and MSSB, respectively.
Table 4: Effects of methanolic extract of the MSSL and MSSB on open field test in mice
118.4 ± 1.20
117.2 ± 1.15
110.4 ± 0.81
117.8 ± 1.43
116.2 ± 1.15
121.2 ± 1.15
111.4 ± 1.81
117.8 ± 1.13
body weight p.o.of the MSSL and MSSB, respectively.
Onset of diarrhea (min)
No. of faeces in 4 hrs
% inhibition of defaecation
25.45 ± 1.19
21.16 ± 0.68
30.67 ± 2.73
20.02 ± 1.05
51.23 ± 3.03
65.13 ± 3.03
35.83 ± 3.03
Values are presented as mean ± SEM, (n=5); p<0.05, respectively, compared to control by student’s t-test. Group I animals received vehicle (1% CMC in
water), group II received Loperamide 10 mg/kg body weight p.o., groups III, IV, V, VI, VII and VIII were treated with 100, 200 and 300 mg/kg body weight
Fig. 1: Effects of the extracts of MSSL and MSSB on carrageenan induced paw edema test. Values are mean ±SEM, (n
= 5); *P<0.05 **P<0.005 as compared to vehicle control (One way ANOVA followed by Dunnet test). Group
I animals received vehicle (1% CMC in water), group II received Indomethacin 10 mg/kg body weight p.o., groups
III, IV, V, VI, VII and VIII were treated with 100, 200 and 300 mg/kg body weight (p.o.) of MSSL and MSSB,
Fig. 2: Effect on gastrointestinal motility of MSSL and MSSB values are presented as mean ± SEM, (n=5); p<0.05,
respectively, compared to control by student’s t-test. Group I animals received vehicle (1% CMC in water), group
200 and 300 mg/kg body weight (p.o.) of MSSL and MSSB, respectively
dependent manner when compared with the untreated
during the next 48 hrs. The extract did not cause mortality
controls. At 300 mg/kg doses, MSSL showed 59.31% and
in mice during 48 hrs observation but little behavioral
MSSB 80.57% reduction in the number of fecal episodes,
changes, locomotor ataxia, diarrhea and weight loss were
whereas Loperamide offered 81.52% protection (Table 5).
observed. Food and water intake had no significant
difference among the group studied.
showed significant difference compared with control
(p< 0.05). The intestinal transit of charcoal meal was 75.
the effect of both extracts of MSSL and MSSB on acetic
37% in control group, but at 300 mg/kg b.wt. the dose of
acid induced writhing in mice. The analgesic activity of
MSSB was 27.68% (Fig. 2).
MSSL and MSSB was significantly (p<0.05) inhibited
writhing response induced by acetic acid in a dose
procedure to evaluate peripherally acting analgesics and
MSSB significantly (P<0.05) suppressed the licking
represents pain sensation by triggering localized
activity in either phase of the formalin-induced pain in
inflammatory response mediated by peritoneal mast cells,
mice in a dose dependant manner (Table 2). MSSB at the
acid sensing ion channels and the prostaglandin
dose of 300 mg/kg body weight (p.o.) showed the almost
pathways . It is well known that non-steroidal
similar analgesic activity against both phases of formalin-
anti-inflammatory and analgesic drugs mitigate the
induced pain than that of the standard drug diclofenac
inflammatory pain by inhibiting the formation of pain
mediators at the peripheral target sites where
significant role in the pain process . MSSL and MSSB
anti-inflammatory activity of MSSL and MSSB. Both
might have exerted its peripheral antinociceptive action by
extracts showed dose dependent anti-inflammatory
interfering with the local reaction caused by the irritant or
activity and statistically significant (P<0.05). At 300 mg/kg
by inhibiting the synthesis, release and/or antagonizing
dose, MSSB showed remarkable anti-inflammatory effects
the action of pain mediators at the target sites. However,
(% inhibition 72.82%) compared with the Indomethacin
the biphasic formalin model is represented by neurogenic
(% of inhibition 75.02%).
(0-5 min) and inflammatory pain (15-30 min), respectively
pains by the extract might imply that it contains active
analgesic principle that may be acting both centrally and
MSSB extracts exhibited statistically significant (P<0.05)
peripherally. This is an indication that the extract can be
of decrease in the movements of the test animals at all
used to manage acute as well as chronic pain. Since
dose levels tested and followed a dose-dependent
flavonoids inhibiting the writhing will have analgesic
response. The depressing effect was most intense during
effect preferably by inhibition of prostaglandin synthesis,
the second (60 min) and third (90 min) observation periods
a peripheral mechanism of pain inhibition  and
in both extract (Table 3).
previous studies [6, 9] have evidenced the presence of
augmenting the analgesic activity.
a similar trend to the ones observed in the open-field test.
Carrageenan induced edema has been commonly
They were statistically significant for all dose levels and
used as an experimental animal model for acute
followed a dose-dependent response. The depressing
inflammation and is believed to be biphasic. The early
effect was most intense during the second (60 min) and
phase (1-2hrs) of the carrageenan model is mainly
third (90 min) observation periods (Table 4).
mediated by histamine, serotonin and increased synthesis
The late phase is sustained by prostaglandin release and
mediated by bradykinin, leukotrienes, polymorphonuclear
reduced the number of diarrheal episodes in a dose
cells and prostaglandins produced by tissue macrophages
gastrointestinal transit experiment, the treated groups
Acetic acid induced writhing response is a sensitive
prostaglandins and bradykinin are proposed to play a
. The suppression of neurogenic and inflammatory
flavonoid in S. samarangensemay play the vital role for
of prostaglandins in the damaged tissue surroundings.
which was used as a diarrhea inducing agent in the
induced by carrageenan in the second phase and this
experimental protocol. Several mechanisms have been
finding suggests a possible inhibition of cyclooxygenase
previously proposed to explain the diarrheal effect of
synthesis by the extracts and this effect is similar to that
castor oil including inhibition of intestinal Na , K -
produced by non-steroidal anti-inflammatory drugs
ATPase activity to reduce normal fluid absorption,
such as Indomethacin, whose mechanism of action is
activation of adenylate cyclase or mucosal cAMP
inhibition of the cyclooxygenase enzyme. Flavonoids,
mediated active secretion, stimulation of prostaglandin
Cycloartenyl stearate, lupenyl stearate, sitosteryl stearate
formation, platelet activating factor and recently nitric
and 24-methylenecycloartanyl stearate from the
oxide has been claimed to contribute to the diarrhoeal
effect of castor oil . However, it is well evident that
anti-inflammatory activities at different doses form 
castor oil produces diarrhea due to its most active
and also well known for their ability to inhibit pain
component recinoleic acid which causes irritation and
perception as well as anti-inflammatory properties due to
inflammation of the intestinal mucosa, leading to release
their inhibitory effects on enzymes involved in the
of prostaglandins, which results in stimulation of
production of the chemical mediator of inflammation.
secretion . Previously flavonoids isolated from
This hypothesis is strongly supported by the previous
Syzygium samarangense were tested for a possible
study, which has shown that MSSL and MSSB possess
spasmolytic activity . These indicate that the presence
anti-inflammatory activity due to the presence of high
of compounds with spasmolytic and calcium antagonist
flavonoids content .
activity may be responsible for the anti-diarrheal effect. In
Locomotor activity considered as an increase in
the small intestinal transit test, both extracts of MSSL and
alertness and decrease in locomotor activity indicated
MSSB suppressed the propulsion of charcoal marker in a
sedative effect . Extracts of MSSL and MSSB
dose dependent manner. This finding suggests that the
decreased locomotor activity indicates its CNS depressant
extracts act on all parts of the intestine. A decrease in the
activity. Gamma-aminobutyric acid (GABA) is the major
motility of gut muscles increases the stay of substances
inhibitory neurotransmitter in the central nervous system.
in the intestineallows better water absorption . It is
Different anxiolytic, muscle relaxant, sedative-hypnotic
therefore presumed that the reduction in the intestinal
drugs are elucidation their action through GABA,
propulsive movement in the charcoal meal model may be
therefore it is possible that extracts of MSSL and MSSB
due to antispasmodic properties of the extracts.
may act by potentiating GABAergic inhibition in the CNS
via membrane hyper-polarization which lead to a decrease
in the firing rate of critical neurons in the brain or may be
due to direct activation of GABA receptor by the extracts.
In summary, the methanolic extract of
Many research showed that plant containing flavonoids,
Syzygium samarangense showed significant analgesic,
saponins and tannins are useful in many CNS disorders
anti-inflammatory, CNS depression and anti-diarrheal
. Earlier investigation on phyto-constituents and
properties. Further investigations are required to find
plants suggests that many flavonoids and neuroactive
the active component of the extract in order to confirm
steroids were found to be ligands for the GABA
the mechanism of action in the development of a
receptors in the central nervous system; which led to
assume that they can act as Benzodiazepine like molecules
large dose (300 mg/kg, b. wt.) exhibited significant
anti-diarrheal effects in experimental models. With respect
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