Journal of Ethnopharmacology 142 (2012) 804–810

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Antinociceptive activity of methanol extract of flowers of Impatiens balsamina

Mohammad Zafar Imam a,n, Nazmun Nahar a, Saleha Akter b, Md. Sohel Rana c
a
Department of Pharmacy, Stamford University Bangladesh, 51, Siddeswari Road, Dhaka-1217, Bangladesh
b
Department of Pharmacy, Primeasia University, HBR Tower, 9, Banani C/A, Dhaka-1213, Bangladesh
c
Department of Pharmacy, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Impatiens balsamina Linn. (Balsaminaceae), an annual herb locally
Received 10 February 2012 called ‘‘Dopati’’, is cultivated as an ornamental garden plant in Bangladesh. Flowers of the plant are
Received in revised form used in folk medicine to treat lumbago, neuralgia, burns and scalds.
23 April 2012
Aim of the study: This study evaluated the antinociceptive effect of the methanol extract of I. balsamina
Accepted 3 June 2012
flowers (MIB).
Available online 12 June 2012
Materials and methods: The extract was evaluated for antinociceptive activity using chemical- and heat-
Keywords: induced pain models such as acetic acid-induced writhing, hot plate, tail immersion and formalin test.
Impatiens balsamina Linn. To verify the possible involvement of opioid receptor in the central antinociceptive effect of MIB,
Balsaminaceae
naloxone was used to antagonize the effect. The effect of MIB on central nervous system (CNS) was also
Medicinal plant
studied using hole cross and open field tests.
Pain
Opioid system Results: MIB demonstrated strong and dose-dependent antinociceptive activity in all the chemical- and
heat-induced mice models (p o0.05). These findings imply the involvement of both peripheral and
central antinociceptive mechanisms. The use of naloxone confirmed the association of opioid receptors
in the central antinociceptive effect. MIB also showed significant central nervous system depressant
effect (po 0.05).
Conclusion: This study reported the peripheral and central antinociceptive activity of the flowers of
I. balsamina and rationalized the traditional use of the flower in the treatment of different painful
conditions.
& 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Lawsone, lawsone methyl ether and methylene-3,30 -bilawsone

Impatiens balsamina Linn. (Balsaminaceae) is an annual herb
grown as ornamental garden plant. It is commonly known as
Garden balsam and is locally called ‘‘Dopati’’ in Bangladesh. The
plant is traditionally used in thorn or glass-puncture wounds,
abscesses (Sakunphueak and Panichayupakaranant, 2011), scro-
fulosis, carbuncles, dysentery (Kang and Moon, 1992), rheuma-
tism, isthmus and crural aches, fractures, superficial infections,
fingernail inflammation (Jiang Su New Medicinal College, 2003),
tumor, difficult labor and puerperal pain (Yang et al., 2001) and as
emetic, cathartic, diuretic, and for pain in the joints (Ghani, 2003).
The aerial parts of I. balsamina are used for the treatment of
articular rheumatism, bruises and beriberi (Ishiguro et al., 1992).
Flowers are used to treat lumbago, neuralgia, burns and scalds
(Ghani, 2003). Juice of petals is used for topical application on the
skin to alleviate lesions of several types of dermatitis including
urticaria (Ishiguro et al., 1992).
n
Corresponding author. Tel.: þ880 1816684184; fax: þ880 29355967176.
E-mail address: zafarimam@gmail.com (M.Z. Imam).
have been isolated from the leaf (Sakunphueak and
Panichayupakaranant, 2011). The aerial parts of the plant contain
2-methoxy-1, 4-naphthoquinone (Mori et al., 2011) and bisnaphtho-
quinone derivative called impatienol (Ishiguro et al., 2000). The
flowers have been reported to contain flavonols (Clevenger, 1958),
flavonoid pigments (Hargen, 1966), phenolic compounds (Bohm and
Towers, 1962), lawsone methyl ether, a naphthoquinone (Little
et al., 1948), kaempferol 3-rutinoside, 2-hydroxy 1,4-naphthoqui-
none (Ishiguro et al., 1994; Fukumoto et al., 1995), impatienolate,
balsaminolate (Oku and Ishiguro, 2002), a flavonoid-3-b-D-glucosi-
dase (Boylen et al., 1969), p-coumaric and ferulic acids (Mansell and
Kemerer, 1970), kaempferol 3-[200 -rhamnosyl-300 -glucosyl] glucoside
(Fukumoto et al., 1994). Antimicrobial polypeptides (Thevissen et al.,
2005), baccharane glycosides called hosenkosides, a viscous oil,
alpha-amyrin, beta-sitosterol, alpha-spinasterol, balsaminasterol
and an anthraquinone glycoside (Ghani, 2003) have been isolated
from seeds (Shoji et al., 1994). Dinaphthofuran-7,12-dione deriva-
tives named balsaminones A and B have been isolated from the
pericarp of I. balsamina (Ishiguro et al., 1998).
Pharmacological studies have proven the antianaphylactic
(Ishiguro et al., 1992), antipruritic, and antidermatitic effect of

0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2012.06.004
 M.Z. Imam et al. / Journal of Ethnopharmacology 142 (2012) 804–810
petal extract, kaempferol 3-rutinoside and 2-hydroxy-1,4-
naphthoquinone (lawsone) (Oku and Ishiguro, 2001) and anti-
microbial activity (Sakunphueak and Panichayupakaranant, 2011)
cyclooxygenase-2 inhibitory effect of isolated naphthoquinones
(Oku and Ishiguro, 2002). Polypeptides isolated from seeds have
been reported to possess antimicrobial activity (Thevissen et al.,
2005). Anti-platelet-activating factor has also been reported (Oku
and Ishiguro, 1999). Impatienol, a bisnaphthoquinone derivative,
has been found to inhibit testosterone 5a-reductase (Ishiguro
et al., 2000).
The use of I. balsamina flowers in different painful conditions
in folk medicine (Ghani, 2003), lack of scientific study reporting
its antinociceptive activity and cyclooxygenase-2 inhibitory effect
of isolated compounds convinced to design the present study to
evaluate the effect of methanol extract of I. balsamina (MIB)
flowers using different peripheral and central pain models
in mice.
2. Materials and methods
2.1. Plant material and extraction
The flowers of I. balsamina were collected from DC Hill nursery,
Nandon Kanon, Chittagong, Bangladesh in March, 2011. The
collected samples were then identified by Sarder Nasir Uddin,
Senior Scientific Officer, Bangladesh National Herbarium, Mirpur,
Dhaka, Bangladesh. A voucher specimen (DACB: 35448) has been
deposited in the Herbarium for further reference. Powdered dried
flowers (55 g) were macerated with 260 ml of methanol with
occasional stirring at 25 72 1C for 3 days. The extract was then
filtered using a Buchner funnel and a sterilized cotton filter. The
solvent was completely removed by rotary evaporator and 9.8 g
extract (Yield 17.82%) was obtained. This crude extract was used
for the acute toxicity, antinociceptive and CNS depressant activity
studies.
2.2. Chemicals and drugs
The following drugs and chemicals were used in the current
study: morphine sulphate, diclofenac sodium, naloxone (Sigma,
USA), acetic acid (Merck, Germany), methanol (Merck, Germany),
formalin (Merck, Germany), Folin–Ciocalteu reagent (Merck,
Mumbai, India), sodium carbonate (E. Merck (India) limited),
aluminum chloride (Fine Chemicals, India), potassium acetate
(E. Merck (India) limited), and quercetin (Sigma Chemicals, USA).
2.3. Animals
Swiss albino mice (20–25 g) were collected from Animal
Resources Branch of the International Center for Diarrhoeal
Disease Research, Bangladesh (ICDDR, B). The animals were kept
in standard laboratory conditions (relative humidity 55–60%;
room temperature 2572 1C; 12 h light/dark cycle) and were
provided with standard diet (ICDDR, B formulated) and clean
water ad libitum during acclimatization period. The animals were
acclimatized to the laboratory environment for a period of 14 days
prior to performing the experiments. The animals were fasted
overnight before the experiments. 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.
All experimental protocols were approved by the Institutional
Ethics Committee (SUB/IAEC/11.01).
805
2.4. Drugs and treatments
The standard drug morphine sulphate (5 mg/kg) used in hot
plate and tail immersion tests and diclofenac sodium (10 mg/kg)
in writhing test were administered intraperitoneally 15 min
before the experiments while the animals in control group
received vehicle orally (0.9% saline water) at the dose of 10 ml/
kg body weight 30 min before the experiments. MIB was orally
administered to the test animals 30 min before the experiments
at the doses of 50, 100, 200, and 400 mg/kg body weight in the
chemical-induced pain models and at the doses of 100, 200, and
400 mg/kg in heat-induced pain and CNS depressant activity
tests. Naloxone, a non-selective opioid receptor antagonist, was
injected intraperitoneally at 2 mg/kg dose 15 min before the
administration of morphine sulphate or MIB (100, 200, and
400 mg/kg) to investigate the involvement of opioid receptor
system.
2.5. Acute toxicity test
Mice were divided into control and six test groups (n¼ 5). The
test groups received MIB orally at 500, 1000, 2000, 3000, 4000,
and 5000 mg/kg body weight. After gavage the animals were kept
in separate cages and were allowed to food and water ad libitum.
The animals were then observed for possible behavioral changes,
allergic reactions (skin rash, itching) and mortality for the next
72 h (Walker et al., 2008).
2.6. Phytochemical analysis
2.6.1. Phytochemical screening
The crude methanol extract of I. balsamina flowers (MIB) were
qualitatively tested for the detection of carbohydrates, saponins,
flavonoids, tannins, alkaloids, glycosides, glucosides, reducing
sugars, proteins, and steroids following standard procedures
(Ghani, 2003).
2.6.2. Determination of phenolic content
The content of total phenolic compounds of the extract was
determined by Folin–Ciocalteu reagent (Singelton et al., 1999).
1 ml of extract (200 mg/ml) was mixed with 500 ml of Folin–
Ciocalteu reagent and 4 ml of 7.5% sodium carbonate solution.
The mixture was then incubated for 1 h at 20 1C. The absorbance
of the solution was measured at 765 nm against blank. The total
content of phenolic compounds was calculated in gallic acid
equivalents (GAE) using the formula: A¼(C  V)/m; where A is
the total content of phenolic compounds, mg/g plant extract in
GAE; C is the concentration of gallic acid established from the
calibration curve, mg/ml; V is the volume of extract in ml and m is
the weight of plant extract in g.
2.6.3. Determination of flavonoid content
One ml of extract solution (100 mg/ml) was mixed with 3 ml
methanol, 200 ml (10%) aluminum chloride solutions, and 200 ml
(1 M) potassium acetate solutions. Then 5.6 ml of distilled water
was added to the mixture and incubated for 30 min at room
temperature. The absorbance of the solution was measured at
415 nm against blank. The total flavonoid content was calculated
in quercetin equivalents (Chang et al., 2002).
2.7. Pharmacological tests
2.7.1. Acetic acid-induced writhing test
Acetic acid-induced writhing test was performed to evaluate
the peripheral and central antinociceptive activity of MIB in
806 M.Z. Imam et al. / Journal of Ethnopharmacology 142 (2012) 804–810

chemical induced pain. The mice were treated with drug or middle of the open field. Then the number of squares visited by
extract and then the writhing was induced by injecting 0.7% the mice was counted for 3 min at 0, 30, 60, 90, and 120 min after
acetic acid after 15 and 30 min, respectively, at the dose 10 ml/kg the treatments (Gupta et al., 1971).
body weight. Five minutes after the injection of acetic acid, the
mice were observed and the number of writhing was counted for 2.8. Statistical analysis
10 min (Vogel, 2007). The contraction of the abdomen, elongation
of the body, twisting of the trunk and/or pelvis ending with the The results are presented as mean7SEM. The statistical
extension of the limbs were considered as complete writhing. analysis of the results was performed using one way analysis of
variance (ANOVA) followed by Dunnett’s post hoc test or Bonfer-
2.7.2. Hot plate test roni’s test as appropriate using SPSS 11.5 software. Differences
The mice that showed forepaw licking, withdrawal of the between groups were considered significant at a level of p o0.05.
paw(s) or jumping response within 15 s on hot plate kept at a
temperature of 55 71 1C were selected for this study 24 h prior to
3. Results
the experiment. Mice were fasted overnight with water given ad
libitum. The animals were treated with morphine or extract and
3.1. Phytochemical analysis
were placed on Eddy’s hot plate kept at a temperature of
5571 1C. A cut off period of 20 s was maintained to avoid paw
Preliminary phytochemical screening of the crude extract
tissue damage (Eddy and Leimbach, 1953). The response in the
of I. balsamina revealed the presence of alkaloids, glycosides,
form of forepaw licking, withdrawal of the paw(s) or jumping was
steroids, carbohydrates, saponins, and tannins. Total phenols
recorded at 30, 60, 90, and 120 min following treatment.
and flavonoids were calculated as 103.2671.63 mgGAE/gextract
Then percentage of the maximal possible effect (%MPE) was
and 64.6972.04 mgQE/gextract, respectively.
calculated using the following formula (Coelho et al., 2005):
%MPE¼[(Postdrug latency predrug latency)/(Cutoff period-
predrug latency)]  100. 3.2. Acute toxicity

Administration of MIB at the doses of 500–5000 mg/kg orally

2.7.3. Tail immersion test
did not show any mortality, behavioral changes or allergic
To evaluate the central analgesic property the tail immersion
manifestations during the 72 h observation period after adminis-
test was performed. This procedure is based on the observation
tration. Therefore, it can be assumed that MIB possess low toxicity
that morphine like drugs prolongs the tail withdrawal time from
profile and the LD50 is more than 5000 mg/kg.
hot water in mice (Toma et al., 2003). One to two cm of tail of the
mice pretreated with morphine or MIB were immersed in warm
3.3. Acetic acid-induced writhing
water kept constant at 5271 1C. The latency between tail sub-
mersion and deflection of tail was recorded. Mice that showed a
Oral administration of MIB (100, 200, and 400 mg/kg) resulted
latency period between 1.5 and 3.5 s were selected for this
in a significant reduction (p o0.001) of acetic acid-induced wri-
experiment and the pre-treatment latency was recorded.
thing compared to the control group in mice (Table 1). From the
A latency period of 20 s was maintained to avoid tail tissue damage
percentage inhibition of abdominal constriction it can be
in mice. The latency period of the tail-withdrawal response was
observed that the antinociceptive activity of MIB was dose
taken as the index of antinociception and was determined at 30, 60,
dependent. Diclofenac sodium (10 mg/kg), the standard drug,
90, and 120 min after the administration of the drug and extract.
inhibited 81.55% writhing in comparison to the control group.
The %MPE was calculated from the latency periods.
MIB showed very strong writhing inhibitory effect as the 100 mg/
kg dose showed a similar effect like Diclofenac (79.17%) and the
2.7.4. Formalin test 200, and 400 mg/kg doses showed better antinociceptive activity
Mice were injected with 20 ml of 1.35% formalin (0.5% for- (91.07 and 94.94%).
maldehyde) into the sub-plantar region of the right hind paw
30 min after MIB treatment and 15 min after injection of mor-
3.4. Hot plate test
phine. Licking of the injected paw was recorded as nociceptive
response at 0–5 min (early phase) and 15–25 min (late phase)
The results of the antinociceptive activity of MIB and standard
after formalin injection (Coelho et al., 2005).
drug (Morphine) in hot plate test are given in Table 2. MIB at 200
and 400 mg/kg doses significantly increased the reaction time to
2.7.5. Hole cross test
The method was carried out as described by Takagi et al.
(1971). A cage (30  20  14 cm3) with a fixed partition in the
middle, having a hole of 3 cm diameter was used. Mice were Table 1
Effects of MIB and diclofenac sodium on the acetic acid-induced writhing response
treated with vehicle, extract or diazepam and were placed in one
in mice.
side of the cage. Then the number of passage of a mouse through
the hole from one chamber to other was counted for a period of Treatment Dose (mg/kg) Number of writhing Inhibition (%)
3 min at 0, 30, 60, 90, and 120 min after the treatments.
Vehicle – 33.60 70.83 –
Diclofenac sodium 10 6.20 71.71n 81.55
2.7.6. Open field test MIB 50 20.10 72.44n 40.18
Open field behavioral test is routinely used to evaluate both MIB 100 7.00 72.46n 79.17
locomotor activity and emotionality in rodents. The open field MIB 200 3.00 70.32n 91.07
MIB 400 1.70 70.12n 94.94
apparatus consisted of a wooden field of half square meter, with a
series of squares alternatively painted in black and white. It had a Each value is presented as the mean 7SEM (n¼ 5). MIB ¼Methanol extract of I.
50 cm high wall and was placed in a dimly lit room. Mice were balsamina flowers.
treated with vehicle, extract or diazepam and were placed in the n
p o0.001 compared with the control group (Dunnett’s test).
 M.Z. Imam et al. / Journal of Ethnopharmacology 142 (2012) 804–810 807

Table 2
Antinociceptive effect of I. balsamina flower extract, morphine, and reversal effect of naloxone in hot plate test.

Treatment Dose (mg/kg) Latency period (s) (%MPE)

Pretreatment 30 min 45 min 60 min 90 min 120 min

Vehicle – 2.22 70.17 2.38 70.22 2.397 0.13 2.44 7 0.35 2.27 7 0.21 2.63 7 0.27
Morphine 5 (i.p.) 2.36 70.20 9.57 70.38** (40.91) 9.227 0.45** (38.81) 9.87 7 0.63** (42.55) 8.42 7 0.48** (34.31) 4.28 7 0.34 (10.87)
MIB 100 2.23 70.24 2.54 70.12 (1.66) 3.427 0.26 (6.68) 3.98 7 0.41 (9.71) 4.17 7 0.57 (10.86) 3.99 7 0.47 (9.84)
MIB 200 2.23 70.25 2.97 70.26 (4.11) 3.687 0.31* (8.05) 4.48 7 0.49* (12.55) 5.28 7 0.85** (17.03) 3.94 7 0.76 (9.33)
MIB 400 2.35 70.09 3.84 70.42** (8.43) 5.477 .43** (17.70) 5.54 7 0.31** (18.06) 5.71 7 0.50** (18.96) 5.027 0.28** (15.12)
NLX 2 (i.p.) 2.67 70.44 2.52 70.67 2.627 0.35 2.33 7 0.30 2.45 7 0.30 1.85 7 0.36
NLX þMorphine 2þ 5 2.85 70.31 2.54 70.50a ( 1.78) 2.987 0.25a (0.76) 2.78 7 0.31a ( 0.40) 2.26 7 0.15a (  3.46) 2.73 7 0.35a (  0.68)
NLX þMIB 2þ 100 2.93 70.25 2.34 70.37 (  3.42) 2.227 0.22b (  4.16) 2.74 7 0.48 (  1.07) 2.73 7 0.42 ( 1.18) 2.93 7 0.81 (0.04)
NLX þMIB 2þ 200 2.32 70.31 2.13 70.11c (  1.07) 2.277 0.34c ( 0.28) 2.44 7 0.39c (0.68) 2.18 7 0.27c ( 0.77) 2.27 7 0.30 (  0.30)
NLX þMIB 2þ 400 2.76 70.32 3.07 70.45 (1.79) 3.257 0.42d (2.84) 3.11 7 0.32d (2.06) 2.97 7 0.34d (1.24) 2.047 0.21d ( 4.15)

Each value is presented as the mean 7SEM (n¼5). MIB ¼Methanol extract of I. balsamina flowers; NLX¼ Naloxone.
n
p o 0.05 compared with the control group (Dunnett’s test).
nn
p o0.01 compared with the control group (Dunnett’s test).
a
p o 0.001 compared with the morphine group (Bonferroni’s test).
b
p o0.05 compared with the MIB 100 group (Bonferroni’s test).
c
po 0.05 compared with the MIB 200 group (Bonferroni’s test).
d
po 0.01 compared with the MIB 400 group (Bonferroni’s test).

Table 3
Antinociceptive effect of I. balsamina flower extract, morphine, and reversal effect of naloxone in tail immersion test.

Treatment Dose (mg/kg) Latency period (s) (%MPE)

Pretreatment 30 min 45 min 60 min 90 min 120 min

Vehicle  2.13 7 0.20 2.14 7 0.12 2.49 70.17 2.11 7 0.26 2.23 7 0.16 2.49 70.23
Morphine 5 (i.p.) 2.80 7 0.25 9.63 7 0.49* (39.61) 11.17 70.35* (48.66) 11.13 7 0.33* (48.41) 7.22 7 0.32* (25.62) 2.81 70.23 ( 0.08)
MIB 100 2.87 7 0.27 4.72 7 0.26* (10.76) 5.54 70.27* (15.44) 5.84 7 0.37* (17.21) 5.35 7 0.47* (14.47) 4.70 70.44* (10.67)
MIB 200 2.71 7 0.21 4.86 7 0.47* (12.50) 5.71 70.49* (17.42) 5.90 7 0.46* (18.53) 6.067 0.49* (19.31) 5.50 70.43* (16.03)
MIB 400 2.71 7 0.19 6.64 7 0.22* (22.71) 6.81 70.45* (23.74) 6.03 7 0.29* (19.17) 6.19 7 0.41* (20.10) 5.56 70.26* (16.39)
NLX 2 (i.p.) 2.98 7 0.38 2.92 7 0.34 2.82 70.15 2.81 7 0.17 2.88 7 0.18 2.64 70.33
NLX þMorphine 2 þ5 2.71 7 0.29 2.93 7 0.39a (1.27) 2.88 70.36a (0.97) 2.98 7 0.26a (1.55) 2.93 7 0.20a (1.23) 2.89 70.42 (1.04)
NLX þMIB 2 þ100 3.11 7 0.11 3.507 0.41b (2.36) 3.20 70.33b (0.58) 3.47 7 0.19b (2.15) 2.93 7 0.25b ( 1.05) 2.25 70.30b ( 5.07)
NLX þMIB 2 þ200 3.09 7 0.14 2.66 7 0.31c ( 2.54) 2.47 70.24c (  3.67) 2.76 7 0.40c (  1.92) 3.25 7 0.24c (0.98) 2.95 70.49c (  0.83)
NLX þMIB 2 þ400 2.85 7 0.43 3.107 0.40d (1.48) 2.79 70.39d (  0.34) 3.08 7 0.37d (1.38) 3.207 0.61d (2.04) 2.99 70.41d (0.84)

Each value is presented as the mean 7SEM (n¼5). MIB ¼Methanol extract of I. balsamina flowers; NLX¼ Naloxone.
n
p o 0.001 compared with the control group (Dunnett’s test).
a
p o 0.001 compared with the morphine group (Bonferroni’s test).
b
p o0.05 compared with the MIB 100 group (Bonferroni’s test).
c
po 0.01 compared with the MIB 200 group (Bonferroni’s test).
d
po 0.01 compared with the MIB 400 group (Bonferroni’s test).

the thermal stimulus (p o0.05). The antinociceptive effect was
dose-dependent as we observed stronger effect at 400 mg/kg dose
than 200 mg/kg dose. The standard drug (Morphine) showed
highest %MPE values at all the observation periods. The extract
also showed significant %MPE at 100, 200, and 400 mg/kg doses
(po0.05) (Table 2). Naloxone exerted significant (p o0.05) antag-
onistic effect on the antinociceptive activity of MIB at all three
doses and of morphine (Table 2).
3.6. Formalin test
Oral administration of MIB at the doses of 100, 200, and
400 mg/kg significantly (p o0.001) reduced the formalin-induced
paw licking in both early and late phases of the test (Table 4).
Morphine demonstrated complete inhibition of licking in late
phase. MIB has shown a dose dependent increase in the licking
inhibition in both phases.

3.7. Hole cross test

3.5. Tail immersion test
The antinociceptive activity of MIB and morphine demon-
strated in tail immersion test are given in Table 3. MIB at all
three doses (100, 200, and 400 mg/kg) significantly increased the
latency period to hot-water induced thermal stimuli (p o0.001)
in a dose-dependent manner. Morphine showed highest %MPE
values while the extract also showed significant %MPE at 100,
200, and 400 mg/kg doses (p o0.001) (Table 3) at different
observation time. Naloxone exerted significant (p o0.05) antag-
onistic effect on the antinociceptive activity of MIB at all three
doses and of morphine throughout the observation periods
(Table 3).
MIB at the test doses (100, 200, and 400 mg/kg) did not
produce significant decrease of movement in comparison to the
control group in the first 60 min (Table 5). However, significant
decrease in movement was observed at 90 and 120 min after
treatment of with MIB (p o0.05). The standard drug, diazepam,
decreased the number of hole crossed throughout the observation
period.
3.8. Open field test
In open field test, MIB at 100, 200, and 400 mg/kg doses
produced significant inhibition of locomotion (po0.05).
808 M.Z. Imam et al. / Journal of Ethnopharmacology 142 (2012) 804–810

Table 4
Antinociceptive effect of I. balsamina flower extract and morphine in formalin-induced paw licking test in mice.

Treatment Dose (mg/kg) Licking of the hind paw

Early phase (0–5 min) % inhibition Late phase (15–30 min) % inhibition

Vehicle 10 157.80 79.74 – 166.60 7 9.10 –

Morphine 5 47.80 73.48n 69.71 0.00 7 0.00n 100.00
Diclofenac sodium 10 122.60 711.27nn 22.31 3.407 1.44n 97.96
MIB 50 107.00 75.77n 32.19 106.80 7 7.59n 35.89
MIB 100 92.20 78.35n 41.57 93.007 4.28n 44.18
MIB 200 85.00 75.50n 46.13 76.20 7 2.22n 54.26
MIB 400 69.60 73.94n 55.89 47.40 7 5.66n 71.55

Each value is presented as the mean 7SEM (n¼ 5). MIB ¼Methanol extract of I. balsamina flowers.
n
po 0.001 compared with the control group (Dunnett’s test).
nn
p o 0.05 compared with the control group (Dunnett’s test).

Table 5
Effect of I. balsamina flower extract and diazepam in hole cross test.

Treatment Dose (mg/kg) Number of hole crossed (% inhibition)

0 min 30 min 60 min 90 min 120 min

Vehicle – 11.80 7 1.07 10.40 7 1.21 7.407 0.75 7.20 70.86 7.007 0.45
Diazepam 1 12.60 7 1.75 5.80 7 0.86n (44.23) 3.007 0.71n (59.46) 2.20 71.20n (69.44) 1.007 0.32n (85.71)
MIB 100 100 13.20 7 0.58 7.40 7 0.51 (28.85) 6.007 0.71 (18.92) 4.00 70.71 (44.44) 3.60 7 0.93n (48.57)
MIB 200 200 14.40 7 0.81 6.60 7 1.44 (36.54) 4.607 1.03 (37.84) 3.20 71.28n (55.56) 3.40 7 0.81n (51.43)
MIB 400 400 12.60 7 1.89 5.80 7 1.46n (44.23) 4.007 1.05 (45.95) 2.60 71.03n (63.89) 3.007 0.36n (57.14)

Each value is presented as mean 7SEM (n¼ 5). Each value is presented as the mean 7 SEM. MIB ¼Methanol extract of I. balsamina flowers.
n
p o 0.05 compared with the control group (Dunnett’s test).

Table 6
Effect of I. balsamina flower extract and diazepam in open field test.

Treatment Dose (mg/kg) Number of square crossed (% inhibition)

0 min 30 min 60 min 90 min 120 min

Vehicle – 81.407 3.59 69.20 7 5.81 52.00 7 2.17 39.60 7 2.27 36.20 71.74
Diazepam 1 81.807 5.39 36.60 7 6.67n (47.11) 27.60 7 6.92n (46.92) 14.60 7 5.20n (63.13) 15.40 76.94n(57.46)
MIB 100 100 86.407 5.53 43.007 2.98n (37.86) 25.60 7 1.33n (50.77) 21.60 7 3.37n (45.45) 21.20 73.34 (41.44)
MIB 200 200 84.407 6.85 28.20 7 3.54n (59.25) 22.60 7 3.40n (56.54) 21.80 7 2.29n (44.95) 20.00 72.98 (44.75)
MIB 400 400 86.007 8.02 24.80 7 3.99n (64.16) 21.40 7 6.27n (58.85) 15.40 7 6.95n (61.11) 16.80 71.33n (53.59)

Each value is presented as mean 7SEM (n¼ 5). Each value is presented as the mean 7 SEM. MIB ¼Methanol extract of I. balsamina flowers.
n
p o 0.05 compared with the control group (Dunnett’s test).

Significant depressant effect was demonstrated from the 30 min
observation to 90 min observation period (Table 6).
4. Discussion
The present study demonstrates that oral administration of
MIB elicits a potent and dose-dependent antinociceptive effect in
both chemical- and thermal-induced nociception. The findings of
this study indicate the peripheral and central antinociceptive
effect of MIB at different doses in mice models. The major findings
of the present work are that (1) the LD50 of MIB is not less than 5 g
in mice; (2) oral administration of MIB significantly inhibited the
acetic acid-induced abdominal constriction; (3) oral administra-
tion of MIB significantly increased the latency period to the
thermal stimuli suggesting the central activity of MIB; (4) MIB
caused significant inhibition of pain response (paw licking) in
both early and late phases after the intraplantar injection of
formalin; (5) the central antinociceptive effect of MIB can be
effectively antagonized by the use of naloxone suggesting invol-
vement of opioid receptor; (6) oral administration of MIB also
caused the depression of the central nervous system.
Oral administration of MIB produced significant peripheral and
central antinociceptive effect in acetic acid-induced writhing test.
This inflammatory pain model has been used to assess the central
and peripheral antinociceptive or anti-inflammatory activity of
new agents (De Souza et al., 2009). Intraperitoneal administration
of acetic acid causes an increase in cyclooxygenase (COX),
lipoxygenase (LOX), prostaglandins (PGs), histamine, serotonin,
bradykinin, substance P, IL-1b, IL-8, TNF-a in the peripheral tissue
fluid. Increased level of these mediators causes the excitation of
primary afferent nociceptors entering dorsal horn of the central
nervous system (Ikeda et al., 2001). These mechanisms are
associated with the development of inflammatory pain and
abdominal constriction (Bley et al., 1998). The potent antinoci-
ceptive effect of MIB in the acetic acid-induced model, therefore,
suggests that MIB may be involved in the inhibition of COX, LOX
and other inflammatory mediators resulting interrupted signal
 M.Z. Imam et al. / Journal of Ethnopharmacology 142 (2012) 804–810
transduction in primary afferent nociceptors. The COX-2 inhibi-
tory effect of 1,4-naphthoquinones isolated from I. balsamina has
already been reported by Oku and Ishiguro (2002), that justifies
the use of the corolla in articular rheumatism, pain, and swelling.
Our findings also provide evidences supporting the use of
I. balsamina in such painful and inflammatory conditions.
The significant increase in latency time in hot plate by MIB
(po0.05) at different doses suggests the profound central anti-
nociceptive activity of MIB (Table 2). The effect is further
supported by the results observed in the tail immersion test
(Table 3) as the tail withdrawal response in hot water-induced
pain is selective only for centrally acting analgesics, while the
peripherally acting agents are inactive (Srinivasan et al., 2003).
The hot plate demonstrates supraspinal reflex mediated by m1-
and m2-opioid receptors while the tail immersion monitors a
spinal reflex involving m2- and d-opioid receptors (Jinsmaa et al.,
2005, 2004; Arslan and Bektas, 2010). The findings of the present
study indicate that the central antinociceptive effect of MIB may
be due to its effect on m-opioid receptors of spinal as well as
supraspinal system. Influx of calcium ions at the terminal of the
axon of the afferent nerve by compounds in MIB may also
decrease the activity of adenylyl cyclase. This may lead to
decreased cAMP level, potassium ion efflux, and subsequent
hyperpolarization of the nerves which give the antinociceptive
effect (Yaksh, 1999). The antinociceptive activity of the extract
was antagonized by naloxone in the thermal pain models.
Naloxone significantly antagonized the antinociceptive activity
of morphine and extract at all doses in both hot plate and tail
immersion tests. This clearly suggests the direct involvement of
the activation of opioid receptors in the antinociceptive action of
the extract.
In the formalin test, MIB significantly reduced the licking of
the injected paw. The formalin-induced paw licking test evaluates
antinociceptive property in two distinct phases. In the early
phase, neurogenic pain is induced by direct chemical stimulation
of the sensory afferent fibers, particularly C-fibers. The involve-
ment of substance P and bradykinin has also been reported. In the
late phase, inflammatory pain is induced due to production and
action of different inflammatory mediators like prostaglandins
(PGs), histamine, bradykinin, and serotonin in peripheral tissues
(Le Bars et al., 2001; Parada et al., 2001) and also due to functional
changes in the dorsal horn of the spinal cord (Dalal et al., 1999).
The results of the present study have demonstrated that the
number of paw licking was significantly reduced by MIB in both
neurogenic and inflammatory pain phases (p o0.001) in a dose
dependent manner. However, the effect was more pronounce in
the late phase. Centrally acting analgesic drugs inhibit both the
phases of formalin-induced paw-licking, while peripherally acting
analgesics inhibit only the late phase responses (Tjolsen et al.,
1992). Morphine, a centrally acting opioid analgesic, effectively
prevented both the early and late phase paw-licking responses.
On the other hand, diclofenac sodium significantly inhibited the
late phase pain response. The early phase finding of formalin test
further confirms the central antinociceptive effect of MIB that we
have observed in hot plate and tail immersion tests. The late
phase response as well as the antinociceptive effect observed in
the acetic acid-induced writhing test is due to the inhibition of
the inflammatory mediators (Oku and Ishiguro, 2002).
Preliminary phytochemical screening of MIB revealed the
presence of alkaloids, glycosides, steroids, carbohydrates, sapo-
nins, and tannins. A considerable amount of phenols and flavo-
noids were also quantified in the extract in this study. Plant
materials containing phenols, flavonoids, and tannins have been
reported to possess analgesic property (Starec et al., 1988; Mills
and Bone, 2000; Morteza-Semnani et al., 2006). The phytochem-
icals, particularly the phenolics, flavonoids, and tannins, could be
809
attributed to the antinociceptive activity of the extract. 1,4-
naphthoquinones namely, impatienolate and balsaminolate, is
also reported to be involved in cyclooxygenase-2 inhibition that
reduces inflammation and pain (Oku and Ishiguro, 2002).
MIB has also been found to depress the central nervous system
in hole cross and open field tests. The depressant effect was clear
from the decrease in locomotion activity of mice throughout the
observation period. MIB may induce the depressant effect by
potentiating gamma aminobutyric acid (GABA) mediated post-
synaptic inhibition through allosteric modification of GABA
receptors. Phytochemical analysis showed a good amount of
phenols and flavonoids in MIB. Flavonoids have been reported
to act as ligands for the GABAA receptor in brain like benzodia-
zepine-like molecules (Trofimiuk et al., 2005). The depressant
effect may be due to action of alkaloids on the cerebral mechan-
ism involved in sleep regulation (N’Gouemo et al., 1994). Non-
specific CNS depression can also be attributed to the tannin
(Takahashi et al., 1986).
In conclusion, the results of the present study indicated that I.
balsamina flowers possess strong antinociceptive and CNS depres-
sant activities. The effect is rapid, long-lasting and statistically
significant at all the experimental doses. The effect is mediated by
the inhibition of opioid receptor as well as the peripheral
inflammatory mediators such as cyclooxygenase-2. These findings
justify the use of I. balsamina in folk medicine to treat lumbago,
neuralgia, burns and scalds. However, further chemical studies
are required to isolate the bioactive compound(s) and elucidate
the precise molecular mechanisms responsible for the pharma-
cological activities of the plant. Though the involvement of opioid
receptor has been determined using naloxone, further studies are
needed using different antagonists (such as adrenergic, seroto-
nergic, etc.) to completely understand the exact mechanisms of its
antinociceptive activity. It seems quite possible that I. balsamina
contains chemical constituent(s) with analgesic property which
may be used as lead compound for new drug development.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.
Acknowledgments
The authors are grateful to Professor Dr. Abdul Ghani, Chairman,
Department of Pharmacy, Stamford University Bangladesh for his
permission to use the facilities of the Pharmacology Laboratory.
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