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American Journal of Food and Nutrition, 2015, Vol. 3, No. 3, 64-68
Available online at http://pubs.sciepub.com/ajfn/3/3/1
© Science and Education Publishing
DOI:10.12691/ajfn-3-3-1
Study of Antinociceptive Activity of Kaempferia galanga
from Bangladesh in Swiss albino Mice
Pritesh Ranjan Dash1, Mahmuda Nasrin2, Mahmud Tareq Ibn Morshed1, Mohammad Shawkat Ali1,*
1Department of Pharmacy, BRAC University, Dhaka, Bangladesh
2Department of Pharmacy, Jahangirnagar University, Savar, Dhaka, Bangladesh
*Corresponding author: shawkat.ali@bracu.ac.bd
Received March 23, 2015; Revised April 25, 2015; Accepted May 05, 2015
Abstract Kaempferia galanga (K. galanga) (Family: Zingiberaceae) has been used in the folk medicine for the
treatment of inflammation, chest pain, cholera, headache, toothache, hypertension, and abdominal pain. Previous
investigations on this plant suggested that the methanol extract of rhizome showed the analgesic activity. Therefore,
the present study investigated the antinociceptive activity of different extracts of rhizome and leaves of K. galanga.
The antinociceptive activity was evaluated by using acetic acid-induced writhing, hot plate and tail immersion tests
in Swiss albino mice at the doses of 100 and 200 mg/kg body weight p.o. The acetone extract of rhizome (ACR), as
well as petroether fraction (PEF), chloroform fraction (CHF), methanol fraction (MEF) and acetone extract of leaves
(ACL) were examined for antinociceptive activity. In this study, all the extracts displayed significant (p<0.05 and
p<0.001) antinociceptive action in a dose dependent manner. In acetic acid induced writhing method, chloroform
and methanol extract of rhizome (200 mg/kg) showed 81.22% and 70.12% writhing inhibition, respectively whereas
the standard drug Diclofenac-sodium (25 mg/kg) and Aspirin (100 mg/kg) exhibited 80.72% and 61.94% inhibition.
In hot plate and tail immersion tests, the petroether extract of rhizome and acetone extract of leaves (200 mg/kg)
produced maximum 69.41% and 81.69% nociception inhibition of thermal stimulus respectively. In this study
Morphine (5 mg/kg) was used as standard. The present study revealed that the acetone extracts and fractions of
rhizome and leaves of K. galanga possess an antinociceptive property which supports its use in traditional medicine
and suggesting that the plant may be further investigated to discover its pharmacologically active natural products.
Keywords: Kaempferia galanga, Zingiberaceae, writhing test, hot plate test, tail immersion test
Cite This Article: Pritesh Ranjan Dash, Mahmuda Nasrin, Mahmud Tareq Ibn Morshed, and Mohammad
Shawkat Ali, “Study of Antinociceptive Activity of Kaempferia galanga from Bangladesh in Swiss albino Mice.”
American Journal of Food and Nutrition, vol. 3, no. 3 (2015): 64-68. doi: 10.12691/ajfn-3-3-1.
1. Introduction
K. galanga (Chandramulika in Bengali) belonging to
the family of Zingiberaceae is a rhizomatous and small
herb. The rhizomes of this plant are widely used in East
Asia for a wide range of medicinal applications. The most
common indications include rheumatism, asthma,
headaches, cough, toothaches and use as a poultice for the
application on bruises and wound [1]. The aromatic oil is
used as condiment and as a folk medicine. In Bangladesh,
rhizomes juices of K. galanga are used as a remedy for
toothache or a wash for dandruff or scabs on the head. It is
used as stimulant, stomachic and carminative and
externally used to treat abdominal pain, swelling and
rheumatism [2]. Literature survey revealed that extracts of
K. galanga has anti-inflammatory and analgesic,
nematicidal, mosquito repellent and larvicidal,
vasorelaxant, sedative, antineoplastic, antimicrobial , anti-
oxidant properties [3]. It also has antidiarrhoeal and
cytotoxic properties [4,5]. Several compounds has been
isolated from dicloromethane, hexane and methanol
extracts of rhizome K. galanga i.e. ethyl-cinnamate and
ethyl–p—methoxycinnamate [3]. Isolated ethyl-p-
methoxycinnamate from K. galanga extracts responsible
for various pharmacological actions including, nematicidal,
mosquito repellent, anti-neoplastic and anti-microbial
effects whereas ethyl cinnamate, a vital constituent of this
plant, responsible for its vasorelaxant effects [6]. The
major chemical constituents of the volatile oil from dried
rhizome of Kaempferia galanga were ethyl-p-
methoxycinnamate (31.77%), methylcinnamate (23.23%),
carvone (11.13%), eucalyptol (9.59%) and pentadecane
(6.41%), respectively. Other constituents of the rhizome
include cineol, borneol, 3-carene, camphene, kaempferal,
cinnamaldehyde, p-methoxycinnamic acid and ethyl
cinnamate [7]. To date, different extracts of K. galanga
have not been systemically studied. Therefore, as a part of
our continuing studies [4,5,8] on natural products for their
pharmacological properties we investigated acetone
extracts of different parts of the plant of K. galanga for
their antinociceptive activity.
2. Materials and Methods
2.1. Collection of the Plant Material
65 American Journal of Food and Nutrition
The plant of K. galanga was collected from the local
area of Mauoa, Dhaka during December 2011. The
collected plant was then identified by Bushra Khan,
Principal Scientific Officer, Bangladesh National
Herbarium, Mirpur, Dhaka and a voucher specimen has
been deposited (DACB: 36,064) for further reference.
2.2. Extraction and Fractionation of the Plant
Material
The identified plant’s rhizome and leaves were cut into
small pieces separately and then dried in the shade. After
complete drying the rhizome and leaves were reduced to
coarse powder separately with the help of a mechanical
grinder and the powder was stored in a suitable container
for extraction. The dried grinded powder weighed by
rough balance. The plant parts were extracted by a cold
extraction method. The rhizome (900 g) and leaves (200 g)
powder were taken and soaked with 2700 ml and 600 ml
of acetone, respectively, for 3 consecutive days at 25 °C.
The extracts were filtered and evaporated on rotary
evaporator under reduced pressure. Recovered solvent was
again used for percolation for another 3 days. The process
was repeated three times to obtain 58 g rhizome (yield
6.45%) and 4.14 g leaves (yield 2.07%) extracts of K.
galanga. 8g of the rhizome extract was separated for
experimental work and further 50g was used for
fractionation. The rhizome extract was further partitioned
using petroether, chloroform and methanol to obtain
18.72g, 18.42g and 2.86g extracts, respectively.
2.3. Chemicals
Acetic acid was product of Merck, Germany.
Diclofenac sodium and aspirin were purchased from
Square Pharmaceuticals Ltd., Bangladesh; morphine was
purchased from Gonoshasthaya Pharmaceuticals Ltd.,
Bangladesh; 0.9% sodium chloride solution (Normal
saline) was purchased from Orion Infusion Ltd.,
Bangladesh and other reagents were of analytical grade.
2.4. Experimental Animals
For the experiment Swiss albino mice of either sex, 4-5
weeks of age, weighing between 25-30 gm, were collected
from the Animal Research Branch of the International
Center for Diarrhoeal Disease and Research, Bangladesh
(ICDDR, B). Animals were maintained under standard
environmental conditions (temperature: (24.0±1.0º), relative
humidity: 55-65% and 12hrs light/12 hrs dark cycle) and
had free access to fed and water ad libitum. The animals
were acclimatized to laboratory condition for two weeks
prior to experimentation. All the experimental animals were
treated following the Ethical Principles and Guidelines for
Scientific Experiments on Animals (1995) formulated by
the Swiss academy of medical Sciences and the Swiss
academy of Sciences. The University Animal Research
Ethical Committee approved the experimental protocol.
2.5. Drugs and Treatment
After reconstituted into distilled water (1% DMSO was
used to dissolved the extracts) all the extracts were
administered to the mice at 100 and 200 mg/kg per orally
by gavage. The water (5 ml/kg) was administered by
gavage to the control group. All drugs, used as standard,
were dissolved in 0.9% saline and administered
intraperitoneally (i.p.) except aspirin. Diclofenac-sodium
(25 mg/kg i.p.) and morphine (5 mg/kg i.p.) were used as
standard peripheral and central antinociceptive agents,
respectively.
2.6. Acute Toxicity Study
Mice were divided into control and test groups (n=6).
The test groups received the extract per orally at the doses
of 500, 1000, 1500 and 2000 mg/kg. Then the animals
were kept in separate cages and were allowed to food and
ad libitum. The control group received the water. The
animals were observed for possible behavioral changes,
allergic reactions and mortality for the next 72 h [9].
2.7. Antinociceptive Activity Study
2.7.1. Acetic Acid Induced Writhing Test
The method described by Dash et al., (2011) [10] that
was adopted to study the effect of the K. galanga extract
on acetic acid induced writing test. Test samples and
control (n=6) were administered orally 30 min before
intraperitoneal administration of 0.7% acetic acid but
Diclofenac-sodium (i.p) and aspirin (p.o) were
administered 15 min before injection of acetic acid. After
an interval of 5 min, the mice were observed (abdominal
contraction, elongation of the body and extension of the
hind limb were referred as writhing) for the next 10 min.
Percentage inhibition of writhing was calculated using the
following formula:
( ) ( )
( )
( )
%
Mean no. of writhings control
–meanno. of writhing test 100.
Mean no. of writhings control
=
×
Writhing inhibition
2.7.2. Hot Plate Test
The hot plate test was performed according to the
method described by Eddy and Leimbach (1953) [11]. The
animals in the control group received water (5 ml/kg, p.o.)
while the standard groups were treated with morphine
sulphate (5 mg/kg, i.p.). The animals in the test groups
were treated with 100 and 200 mg/kg, per oral of different
extracts of K. galanga. Then the animals were placed on
Eddy’s hot plate kept at a temperature of 52±0.5 0C. A cut
off period of 28s was observed to avoid damage to the
paw. Reaction time was recorded when animals licked
their fore or hind paws, or jumped prior to and 0, 30, 60
and 90 min after the administration of the standard and
test drugs (n=6). Percentage of elongation was calculated
using the following formula:
( )
( ) ( )
( )
Latency test Latency control
% 100.
Latency test
−
= ×Elongation
2.7.3. Tail Immersion Test
The procedure is based on the observation that
morphine like drugs selectively prolongs the reaction time
of the typical tail withdrawal reflex in mice [12].The
animals were treated as discussed above. 1 to 2 cm of the
American Journal of Food and Nutrition 66
tail of mice was immersed in warm water kept constant at
55±1°C. The reaction time was the time taken by the mice
to deflect their tails. The first reading was discarded and
the reaction time was recorded as a mean of the next three
readings. A latency period of 28s was defined as complete
analgesia and the measurement was then stopped to avoid
injury to mice. The latent period of the tail-immersion
response was determined at 0, 30, 60 and 90 min after the
administration of standard and test drugs (n=6).
Percentage of elongation was calculated using the same
formula used in hot plate test.
2.8. Statistical Analysis
The statistical analysis for animal experiment was
carried out using one-way ANOVA followed by Dunnett’s
multiple comparisons. The results obtained were
compared with the control group. P < 0.05 and P < 0.001
were considered to be statistically significant.
3. Results
3.1. Acute Toxicity
Oral administration of different extracts of K. galanga
at the doses of 500–2000 mg/kg did not produce any
mortality or noticeable behavioral changes in mice within
72 hr observation period. Therefore, it can be suggested
that K. galanga have low toxicity profile with LD50 greater
than 2000 mg/kg.
Table 1. Effect of different extracts of K. galanga on acetic acid-induced writhing test in mice.
Group Dose (mg/kg)
No. of writhings
(Mean±SEM)
% of writhing % of writhing inhibition
Control
5ml/kg
50.58±4.18**
100.00
--
Diclofenac-Na
25
9.75±0.77**
19.27
80.72
Aspirin
100
19.25±4.12**
38.06
61.94
ACR 100 17.17±3.19** 33.95 66.05
200
15.08±2.50**
29.81
70.12
PEF
100
25.25±4.22**
43.65
50.07
200
17.58±2.33**
34.76
65.24
CHF
100
22.08±3.53**
37.23
56.35
200
9.5±1.73**
18.78
81.22
MEF
100
18.83±3.30**
37.23
62.77
200 15.08±2.91** 29.81 70.12
ACL
100
22.08±2.55**
43.65
56.35
200
20.08±3.33**
39.70
60.30
Control group received water 5ml/kg body weight (p.o.), standard groups received Diclofenac-Na 25mg/kg (i.p.) and Aspirin 100mg/kg body weight
(p.o.).Standard drugs were administered 15 min before 0.7 % acetic acid administration. Writhing was counted for 15 min, starting after 5 min of acetic
acid administration. Test groups ACR, PEF, CHF, MEF and ACL were treated with 100 and 200 mg/kg body weight of the extracts (p.o.) respectively.
Values are mean ±SEM, (n=6); **p < 0.001, Dunnett t-test as compared to control. ACR=Acetone extract of rhizome, PEF= Petroether fraction of
rhizome, CHF=Chloroform fraction of rhizome, MEF=Methanol fraction of rhizome and ACL=Acetone extract of leaves.
Table 2. Effect of different extracts of K. galanga on hot plate test in mice.
Group Dose (mg/kg) Mean Reaction Time (s)
0 min 30 min 60 min 90 min
Control 5ml/kg 9.67±0.98 8.83±0.60 9.83±1.04 9.33±1.11
Morphine 5 10.83±1.76
17±2.09*
(48.06%)
21.33±2.19*
(53.91%)
16±1.80
(41.68%)
ACR 100 11.5±2.10
13.50±1.94
(34.59%)
16.17±2.33
(39.21%)
21±2.82**
(55.57%)
200 7.83±0.79
13.67±1.89
(35.41%)
21±2.35*
(53.19%)
23.67±1.47**
(60.58%)
PEF 100 8±1.15
11±2.25
(19.73%)
19±2.46*
(48.26%)
22.17±2.02*
(57.92%)
200 8.17±1.30
14.67±2.51
(39.81%)
23.5±2.64**
(58.17%)
30.5±3.54**
(69.41%)
CHF 100 9.83±1.14
13.17±1.42
(32.95%)
19±1.75*
(48.26%)
21.5±1.92**
(56.60%)
200 8.83±0.91
13.5±2.44
(34.59%)
20.83±2.38*
(52.81% )
25±2.76**
(62.68%)
MEF 100 8±0.93
10.5±1.17
(15.90%)
17±2.01
(42.17%)
18.33±1.72*
(49.10%)
200 8.5±1.42
17.5±2.51*
(49.54%)
21.5±3.64*
(54.28%)
21.67±3.21**
(56.94%)
ACL 100 10.17±1.10
12.5±0.43
(29.36%)
22±1.69
(55.32%)
21.5±1.99*
(56.60%)
200 9.33±1.35
14.5±1.05
(39.10%)
25.83±3.27**
(61.94%)
25.33±4.12**
(63.17%)
Control group received water 5ml/kg body weight (p.o.), standard groups received Morphine 5 mg/kg body weight (i.p.), test groups ACR, PEF, CHF,
MEF and ACL were treated with 100 and 200 mg/kg body weight of the extracts (p.o.) respectively. Values are mean ±SEM, (n=6); * p < 0.05, **p <
0.001, Dunnett t-test as compared to control. ACR=Acetone extract of rhizome, PEF= Petroether fraction of rhizome, CHF=Chloroform fraction of
rhizome, MEF=Methanol fraction of rhizome and ACL=Acetone extract of leaves.
67 American Journal of Food and Nutrition
3.2. Antinociceptive Activity
3.2.1. Acetic Acid Induced Writhing Test
The results of the acetic acid induced writhing test
showed that the different extracts of K. galanga at all
doses produced significant (p<0.001) inhibition of writhing
reaction in a dose dependent manner. In this test, ACR, PEF,
CHF, MEF and ACL (200 mg/kg) inhibited maximum
70.12%, 65.24%, 81.22%, 70.12%, and 60.30% writhing
respectively, whereas the writhing inhibition of the
standard drug Diclofenac-Na (25 mg/kg) and Aspirin (100
mg/kg) were 80.72% and 61.94% respectively (Table 1).
3.2.2. Hot Plate Test
Results of hot plate test are presented in Table 2. All
doses of the extracts produced a dose dependent increase
in latency time when compared with the control. The
results were found to be statistically significant (p < 0.05-
0.001). In the hot plate test, ACR, PEF, CHF, MEF and
ACL (200mg/kg) showed maximum 60.58%, 69.41%,
62.68%, 56.94%, and 63.17% nociception inhibition of
thermal stimulus respectively, whereas the standard drug
Morphine (5 mg/kg) displayed maximum 53.91%
nociception inhibition.
3.2.3. Tail Immersion Test
The tail withdrawal reflex time following
administration of the extracts of K. galanga was found to
increase with increasing dose of the sample. In this test
maximum effect was observed after 60 and 90 min of drug
administration. The results were statistically significant (p
< 0.05-0.001). In tail immersion test, maximum 80.67%,
80.69%, 80.90%, 74.53% and 81.69% nociception
inhibition of thermal stimulus were exhibited with the
ACR, PEF, CHF, MEF and ACL (200 mg/kg) respectively.
In this study morphine (69.89% inhibition) was used as
standard (Table 3).
Table 3. Effect of different extracts of K. galanga on tail immersion test
Group Dose (mg/kg) Mean Reaction Time (s)
0 min 30 min 60 min 90 min
Control 5ml/kg 1.77±0.11 1.66±0.15 1.83±0.24 1.77±0.11
Morphine 5 2.44±0.11
4.94±0.63**
(66.39%)
5.11±0.40**
(64.18%)
5.88±0.25**
(69.89%)
ACR 100 1.99±0.15
4.55±0.92*
(63.52%)
5.22±0.44**
(64.94%)
5.94±0.30**
(70.20%)
200 2.28±0.13
4.61±0.20*
(63.99%)
6.61±0.31**
(72.32%)
9.16± 0.47**
(80.67%)
PEF 100 2.22±0.38
2.99±0.34
(44.48%)
4.55±0.57**
(59.78%)
5.44±0.68**
(67.46%)
200 2.05±0.20
4.88±0.37**
(65.98%)
7.22±0.32**
(74.65%)
9.17±0.36**
(80.69%)
CHF 100 1.66±0.17
3.72±0.34*
(55.37%)
4.11±0.33*
(55.47%)
5.77± 0.72**
(69.32%)
200 2.27±0.18
4.94±0.42**
(66.39%)
6.67±0.56**
(72.56%)
9.27±0.53**
(80.90%)
MEF 100 2.33±0.17
2.94±0.10
(43.54%)
3.99±0.35*
(54.14%)
3.99±0.27**
(55.64%)
200 2.05±0.16
4.38±0.51**
(62.10%)
5.77±0.76**
(68.28%)
6.95±0.40**
(74.53%)
ACL 100 2.27±0.55
3.77±0.16**
(55.97%)
5.05±0.44**
(63.76%)
.05±0.76**
(74.89%)
200 2.17±0.18
4.72±0.29**
(64.83%)
7.11±0.25**
(74.26%)
9.67±0.38**
(81.69%)
Control group received water 5ml/kg body weight (p.o.), standard groups received Morphine 5 mg/kg body weight (i.p.), test groups ACR, PEF, CHF,
MEF and ACL were treated with 100 and 200 mg/kg body weight of the extracts (p.o.) respectively. Values are mean ±SEM, (n=6); * p < 0.05, **p <
0.001, Dunnett t-test as compared to control. ACR=Acetone extract of rhizome, PEF= Petroether fraction of rhizome, CHF=Chloroform fraction of
rhizome, MEF=Methanol fraction of rhizome and ACL=Acetone extract of leaves.
4. Discussion
The present study demonstrates that different extracts
of K. galanga possess potent antinociceptive activity in
chemical and heat induced models. No acute toxicity was
observed after oral administration of K. galanga even at
the dose of 2000 mg/kg in mice.
Acetic acid-induced writhing test is a visceral pain
model. The pain is induced at the peritoneal receptors by
the increasing amount of endogenous mediators of pain,
such as prostaglandin E2, prostaglandin E2α, kinins, and
the like [13,14]. The stimulation of the nociceptive
neurons, which is sensitive to NSAIDs and narcotics, by
the endogenous mediators also contribute in the pain
induction [15]. Acetic acid-induced abdominal writhing is
widely used to screen peripherally acting analgesics [16].
The findings of the present study indicate that different
extracts of K. galanga possess a significant peripheral
antinociceptive activity.
The hot plate test is used to evaluate the centrally acting
analgesics [17]. The paw-licking or jumping responses in
hot plate are complex supraspinally organized behavior of
mice [18]. So, a decrease in licking or increase in latency
indicates the centrally acting analgesic properties of the
treatment. The results of the hot plate test showed that K.
galanga extracts produced antinociceptive effect against
heat induced pain. The effect was evident from the
elongation of the latency time till the 4th observation (90
min).
Tail immersion model is an acute pain model. The tail-
withdrawal response is predominantly considered to be
selective for centrally acting analgesics, whereas the
peripherally acting drugs are known to be inactive on
American Journal of Food and Nutrition 68
heat-induced pain [19]. The significant increase (p < 0.05-
0.001) in tail-withdrawal time by the extracts suggests
centrally acting analgesic activity of K. galanga. Both tail
immersion and hot plate test measure the latency time of
mice to thermal stimuli. Therefore, the results of the
present study indicate that the central antinociceptive
effect of K. galanga may be prominent. Influx of calcium
ions at the terminal of the axon of the afferent nerve by
different compounds in the extracts may also decrease the
activity of adenylyl cyclase. This may lead to decreased
cAMP level, potassium ion efflux and subsequent
hyperpolarization of the nerves and give the
antinociceptive effect [20]. The antinociceptive effect of
the extracts in these three models implies that the extracts
contain pharmacologically active phytoconstituents that
may act both centrally and peripherally.
5. Conclusion
Finally it can be concluded that, the different extracts of
K. galanga possesses a significant antinociceptive
activities and the results tend to corroborate the traditional
use of this plant in the treatment of pain. However, further
investigations are required to identify the active
constituents and to verify the therapeutic merits of the
active constituents.
Acknowledgments
The authors are acknowledge to the director of Animal
Research Division of the International Centre for
Diarrhoeal Disease and Research, Bangladesh (ICDDR, B)
for supplying mice and the National Herbarium of
Bangladesh for identifying the plant sample.
Declaration of interest
The authors report no conflicts of interest. The authors
alone are responsible for the content and writing of the
paper.
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