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Afr. J. Biomed. Res. 14 (September 2011); 203 -208

Research Article
Anti-Inflammatory and Analgesic Activities of Ethanolic Leaf
Extract of Calotropis procera
1*
Saba A. B, 2Oguntoke P. C, 1Oridupa O. A
1
Department of Veterinary Physiology, Biochemistry & Pharmacology, University of Ibadan.
2
Department of Vet. Physiology, Biochemistry & Pharmacology, University of Agriculture, Abeokuta, Nigeria

ABSTRACT: Ethanolic extract of the leaf of Calotropis procera was investigated for its anti-inflammatory and analgesic
activities. The extract was evaluated using formalin-induced paw lick, carragenaan-induced paw oedema in Wistar rats, acetic
acid-induced writhing and tail flick tests in mice. Each experiment consisted of thirty animals randomly, but equally divided
into groups of 100mg/kg, 200mg/kg or 400mg/kg body weight (b.w) of extract, Indomethacin (10 mg/kg b.w) or aspirin
(15mg/kg b.w) pre-treated animals and a control group administered with distilled water (10ml/kg b.w). The administration of
the extract was repeated for formalin-induced paw lick and acetic-acid induced writhing models in the presence of an opioid
antagonist, naloxone. The data were analyzed using one way ANOVA and difference of means were considered significant at
p<0.05. The ethanolic extract exhibited potent anti-inflammatory or analgesic effect in this study. Inhibition of formation of
paw oedema by the extract (100mg/kg b.w) was significantly higher than for Indomethacin. Itching was significantly reduced
in rats administered with extract in the early phase of formalin response, and was comparable to Indomethacin (10mg/kg b.w).
100 mg/kg body weight of the extract also inhibited the writhing movement comparably with aspirin (15mg/kg b.w). Same
pattern was also observed with tail flick model in mice. The study showed that the mechanism of action of the analgesic or
anti-inflammatory action of the leaf extract is mediated both centrally and peripherally. The analgesic or anti-inflammatory
effect of the extract was not attenuated by opioid antagonist, naloxone, thus ruling out the involvement of opioid receptors in
the central mechanism of action of the extract. It was therefore concluded that these activities are mediated via interaction with
other nociceptive pathways.

Keywords: Calotropis procera, Extract, Anti-inflammatory, Analgesia

INTRODUCTION Asia and Africa. The fruit of the plant is green with an
ovoid shape. The flesh of the fruit contains a toxic
1
milky sap that is extremely bitter and turns into a gluey
Calotropis procera Ait. is a member of the coating resistant to soap. The milky sap contains a
Asclepiadaceae plant family (Aiton, 2010). It is complex mix of chemicals, some of which are steroidal
commonly referred to as milkweed, giant swallow wort heart poisons known as cardiac aglycones. These
or apple of Sodom. The plants are native to southern belong to the same chemical family found in foxgloves
(Digitalis purpurea). The glycosides found in C.
procera are calotropin, calotoxin, calactin, uscharidin
and voruscharin (Sieber et al., 1982; Daniel, 2006).
The bark and latex are widely used as arrow and spear
poison. The root bark is an emetic and the latex is used
for treatment of skin diseases such as ringworm. The
*Address for correspondence: leaves of the plant were reported to have antioxidant
E-mail; [email protected],
Tel. +2348054138127
and antibacterial activities (Yesmin et al., 2008). The
latex of C. procera were established by Kumar and Roy
(2007) to protect against inflammation and oxidative
stress in monoarthritis induced by Freund’s complete
Received: February 2011; Accepted: May 2011 adjuvant in rats.
 Anti-inflammatory and analgesic effect of Calotropis procera

Previous studies by Smit et al. (1995) and Sehgal et al. above. One hour afterwards, oedema was induced in
(2006) demonstrated the potent cytotoxic activity of the the paw of rats by injection of 0.1ml of 1% carrageenan
flower of C. procera and this was comparable to the into the aponeurosis of the right hind limb of rats as
anticancer effect of cisplastin. Eghianruwa et al. (2006) described by Winter et al. (1962). The linear
also reported that C. procera reduced intestinal transit circumference of the paw was measured at 0 minute, 60
of rats. A closely related plant, C. gigantea has also minutes, 120 minutes and 180 minutes after injection of
been demonstrated to possess potent anti-inflammatory carrageenan. The increase in linear circumference at the
activity which was comparable to that observed for different hours determined the inflammatory reaction
ibuprofen (Awasthi et al., 2009; Bulani et al., 2011). observed as formation of oedema in paw (Calixto et al.,
The current study was designed to evaluate the anti- 2003). Cotton thread was wrapped around the paw and
inflammatory and analgesic potential of the ethanolic the circumference measured with a meter rule as
leaf extract of C. Procera using laboratory rats and described by Hess and Milonig, (1972) and Olajide et
mice. al. (2000). The inhibitory activity was then calculated
as shown below.
Inhibition of oedema = (Ct – Co) control - (Ct – Co)
MATERIALS AND METHODS: test
(Ct – Co) control
Extraction of leaves Where Co = mean paw size in control group
Fresh leaves of Calotropis procera were harvested Ct = mean paw size in treatment group
from University of Abeokuta Research farm, Abeokuta.
They were air dried for four weeks. The dried leaves Formalin paw lick test in rats.
were pulverized and soaked in 96% ethanol for 72 Following an overnight fast, the leaf extract was orally
hours. The suspension was decanted and filtered. The administered to rats in the three treatment groups.
solution obtained was clarified by filtration through Simultaneously, 10mg/kg indomethacin (a non-
celite on water pump and was then concentrated in selective cyclo-oxygenase inhibitor) was administered
vacuo using a rotation evaporator (Rotavapor R-210, to animals in a group, while control animals received
Switzerland) at low temperatures. The remaining distilled water. Thirty minutes after treatment, 50 μl of
moisture was finally removed by placing small volumes 2.5% formalin was injected subcutaneously into the
in porcelain dishes in the oven set at low temperatures sub-plantar surface of the left hind paw of the rats.
of 40 0C. Responses were measured 0-5 minutes (early phase)
and 20-30 minutes (late phase) after formalin injection.
Experimental animals The licking of the injected paw and the duration was
Sixty Wistar rats and sixty Swiss mice of both sexes indicative of pain. The cumulative licking time was
were used for this study. The animals were randomly recorded.
divided into groups of five animals. The animals were
housed at the experimental animal unit of the Faculty of Analgesic study
Veterinary Medicine, University of Agriculture,
Abeokuta. They were fed with pelletized rat and mouse Acetic acid writhing response in mice: Mice were
rations, and allowed water ad libitum. They were kept divided into five groups and were administered with
under 12 hour light: dark conditions. Five groups of extract (100, 200 or 400 mg/kg body weight), aspirin
animals were used for each experiment. Three of the (15mg/kg b.w) or distilled water (10mls/kg b.w)
groups were administered with the leaf extract at respectively as earlier described. One hour after,
100mg/kg, 200mg/kg or 400mg/kg body weight b.w 10ml/kg 0.6% acetic acid was injected intraperitonealy
respectively. Another group was administered with to each mouse. 5 minutes following acetic acid
Indomethacin (10mg/kg b.w) or aspirin (15mg/kg b.w) administration, the number of abdominal constrictions
to serve as positive control while the fifth group was that occurred within subsequent 20 minutes were
administered with distilled water (10mls/kg b.w) to counted and recorded.
serve as negative control.
Tail flick test: This experiment was conducted
Anti-inflammatory study according to the modified method adopted by Sanchez-
Mateo et al. (2006) using hot water bath. Groups of
Carrageenan- induced paw oedema in rats: Animals five mice each were administered with the extract (100,
were fasted overnight and administered with the leaf 200 or 400 mg/kg body weight), aspirin 15mg/kg b.w
extract, Indomethacin or distilled water as mentioned or distilled water (10mls/kg b.w) respectively as earlier
204 Afr. J. Biomed. Res. Vol. 14, No.3, 2011 Saba, Oritoke and Oridupa
 Anti-inflammatory and analgesic effect of Calotropis procera

mentioned. Thereafter, the terminal 2 cm of the mice

tail were immersed in a water bath containing hot water Formalin paw lick test
maintained at 55±1°C by a circutine (Haake-Vison, Rats administered with the leaf extract at 100mg/kg,
Germany). Response to pain was taken as the time 200mg/kg or 400mg/kg b.w demonstrated shorter mean
interval between immersion and withdrawal of the tail itching period at the early phase (48.14±6.38 seconds,
by the mice and these were taken at 30, 60 and 90 68.16±10.46 seconds and 62.48±9.42 seconds) and late
minutes after treatment. phase (14.12±2.63 seconds, 22.06±9.84 seconds and
48.30±7.08 seconds) compared to the control group rats
Naloxone antagonism: Formalin paw lick in rats and at the early (89.46±6.79 seconds) and late phases
acetic acid writhing tests in mice were repeated using (70.50±10.89 seconds) respectively. Rats administered
two groups of animals per experiment. The first group with indomethacin demonstrated non-significantly
of animals were administered with naloxone (1mg/kg (p>0.05) longer mean itching period at the early phase
s/c) 30 minutes before administering the leaf extract at (74.00±8.56 seconds), but a shorter mean itching period
200mg/kg b.w. The second group also received at the late phase (13.58±8.03 seconds) compared to rats
naloxone in similar manner before administration of administered with the leaf extract. Rats administered
distilled water (10mls/kg b.w). with the extract at 200mg/kg but pre-treated with
1mg/kg naloxone demonstrated a non-significantly
Statistical analysis (p>0.05) shorter mean itching period at the early phase
Data were analysed using one way analysis of variance (59.44±3.34 seconds), and a longer mean itching period
(ANOVA) on GraphPad Prism 4.0 version. The result at the late phase (25.68±2.17 seconds) compared to
obtained were expressed as mean values ± standard those administered with the extract only. The mean
error of mean (SEM). The statistical significant itching period of rats administered with naloxone alone
difference between the mean values were determined at was also shorter at both phases (52.89±14.05 seconds
p<0.05. and 6.75±1.13 seconds) compared with rats pre-treated
with extract alone (Table 2).

RESULTS Acetic acid writhing test

The mean number of abdominal writhing movements
Carragenaan-induced paw oedema observed for the rats administered with the leaf extract
Change in paw sizes of those administered with the at 100mg/kg, 200mg/kg or 400mg/kg (16.60±8.81,
extract at 100mg/kg b.w (0.12±0.02 cm, 0.14±0.05 cm, 40.40±4.09 and 23.30±9.88) were significantly lesser
0.22±0.14 cm and 0.09±0.04 cm) were significantly compared to rats in the control group (48.00±4.66), but
lower compared to those that served as control more frequent compared to the rats administered with
(0.28±0.02 cm, 0.66±0.12 cm, 0.62±0.08 cm, and indomethacin (14.00±3.56). Rats administered with the
0.48±0.09 cm) at 0, 60, 120 and 180 minutes extract at 200mg/kg, but pre-treated with naloxone
observation times. Rats administered with extract at exhibited lesser numbers of abdominal writhing
200mg/kg b.w recorded a consistent reduction in the (19.40±0.60) compared to those administered with the
swelling of paw (0.34±10 cm, 0.30±0.09 cm, 0.22±0.10 extract only, except rats administered with the extract at
cm and 0.16±0.08 cm) at the observation times. 100mg/kg. Rats administered with naloxone only
Changes in the paw sizes of rats administered with the exhibited significantly higher number of writhing
extract at 400mg/kg b.w (0.32±0.07 cm, 0.30±0.06 cm, movements (39.40±5.99) than for rats pre-treated with
0.26±0.02 cm and 0.22±0.11 cm) were also lower when the extract (Table 3).
compared to the control rats (Table 1).

Table 1:
Effect of ethanol extract of C. procera or Indomethacin on changes in paw size (cm) in albumen induced paw oedema
test in rats.
Treatment Groups 0 minute 60 minutes 120 minutes 180 minutes
Extract 100mg/kg 0.12±0.0.02a 0.14±0.05ab 0.22±0.14a 0.09±0.04ab
a a
Extract 200mg/kg 0.34±0.10 0.30±0.09 0.22±0.10 0.16±0.08
Extract 400mg/kg 0.32±0.07 0.30±0.06a 0.26±0.02a 0.22±0.11
Indomethacin 10mg/kg 0.28±0.04 0.56±0.08b 0.38±0.07 0.08±0.06b
a a a
Control 0.28±0.02 0.66±0.12 0.62±0.08 0.48±0.09a
Groups with same superscript with control and or Indomethacin in a column are statistically significant at p<0.05
205 Afr. J. Biomed. Res. Vol. 14, No.3, 2011 Saba, Oritoke and Oridupa
 Anti-inflammatory and analgesic effect of Calotropis procera

Table 2: Tail flick

The effect of ethanolic extract of C. procera or Indomethacin Response to thermal pain in the tail flick model was
on cumulative itching period (in seconds) in Formalin paw significantly longer in rats administered with the leaf
lick test in rats extract at 100mg/kg (5.95±1.90 seconds, 3.48±0.32
Treatment Groups Early phase Late phase seconds and 4.20±0.39 seconds), 200mg/kg (3.87±0.45
response response
seconds, 3.65±0.75 seconds and 3.30±0.65 seconds)
Extract (100mg/kg) 48.14±6.38ab 14.12±2.63a and 400mg/kg (2.84±0.28 seconds, 3.38±0.10 seconds
Extract (200mg/kg) 68.16±10.46 22.06±9.84a
and 3.14±0.20 seconds) at 30 minutes, 60 minutes and
Extract (400mg/kg) 62.48±9.42 48.30±7.08
90 minutes post administration compared to rats in the
Indomethacin (10mg/kg) 74.00±8.56b 13.58±8.03
control group (3.32±0.86 seconds, 2.22±0.61 seconds
Control 89.46±6.79a 70.50±10.89a
and 2.04±0.59 seconds). Rats administered with
Naloxone (1mg/kg) 59.44±3.34 25.68±2.17a
+ 15mg/kg b.w of aspirin (4.36±1.14 seconds, 3.76±0.67
Extract (200mg/kg) seconds and 3.78±0.51 seconds) exhibited shorter
Naloxone 1mg/kg 52.89±14.05 6.75±1.13a reaction time compared with rats administered with
Groups with same superscript as control and or 100mg/kg extract, but longer reaction time compared
Indomethacin in a column are statistically significant at with rats administered with extract at 200mg/kg and
p<0.05. 400mg/kg b.w (Table 4).

Table 3: DISCUSSION
The effect of ethanolic extract of C. procera or aspirin on
abdominal writhing in mice injected with acetic acid Findings from this study showed that the ethanolic
intraperitonealy. extract of the leaves of Calotropis procera has potent
Treatment Groups Mean number of writhing anti-inflammatory and analgesic properties. This was
Extract (100mg/kg) 16.60±8.81a confirmed by the observations from all the models of
Extract (200mg/kg) 40.40±4.09 inflammation and analgesia used in this study. The
Extract 400mg/kg 23.30±9.88 extract had a dose-dependent anti-inflammatory and
Aspirin 50mg/kg 14.00±3.56a analgesic effect with the lowest concentration of 100
Control 48.00±4.66a mg/kg body weight having the most potent effects.
Naloxone 1mg/kg + Extract 19.40±0.60 The anti-inflammatory effect of C. procera was
200mg/kg
demonstrated in the carrageenan-induced paw oedema
Naloxone 1mg/kg 39.40±5.99
model where carrageenan was unable to incite the
Groups with same superscript as control and or aspirin in a
expected oedema of the paw in the rats administered
column are statistically significant at p<0.05
with the extract. The formalin paw lick test further
Table 4: demonstrated the probable mechanism of action as
The effect of ethanolic extract of C. procera or aspirin on being mediated via both central and peripheral
response (in seconds) to thermal pain induced by tail flick mechanisms of inflammation. Rats administered with
method in mice the leaf extract licked their paws for a shorter period
Treatment 30minutes 60minutes 90 minutes compared to rats administered with indomethacin at the
Groups early phase, but the rats licked for longer in the late
Extract 5.95±1.90 3.48±0.32 4.20±0.39 phase. This shows that the mechanism of anti-
100mg/kg inflammatory action is mediated more via the central
Extract 3.87±0.45 3.65±0.75 3.30±0.65 than the peripheral mechanism of anti-inflammation.
200mg/kg The two phases of anti-inflammatory response are
Extract 2.84±0.28 3.38±0.10 3.14±0.20 usually due to direct stimulation of nociceptors in the
400mg/kg paw which culminates in centrally mediated pain with
Aspirin 4.36±1.14 3.76±0.67 3.78±0.51
release of substance P in the neurogenic (early) phase.
50mg/kg
The late phase on the other hand is observed as a result
Control 3.32±0.86 2.22±0.61 2.04±0.59
of release of histamine, serotonin, bradykinin and
Groups with same superscript as control and or aspirin in a
column are statistically significant at p<0.05
prostaglandins (Zeashana et al., 2009). Some drugs,
especially opioid analgesic agents, inhibit both phases
equally while peripherally acting drugs such as non-
steroidal anti-inflammatory drugs (NSAIDs) and
steroidal anti-inflammatory only inhibit the late phase
206 Afr. J. Biomed. Res. Vol. 14, No.3, 2011 Saba, Oritoke and Oridupa
 Anti-inflammatory and analgesic effect of Calotropis procera

(García et al., 2004; Zeashana et al., 2009). The elucidate the active principle in leaf of Calotropis
possibility of the extract interacting with opioid procera which hopefully may lead to development of a
receptors was ruled out by the reduced period of itching new anti-inflammatory and analgesic agent.
in rats pre-treated with naloxone; the specific
antagonist for opioid receptors. In this study, naloxone
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