eISSN: 2455-1716
Amole et al., 2018
DOI:10.21276/ijlssr.2018.4.4.3
Research Article
1
Department of Pharmacology, Therapeutics and Toxicology, Lagos State University College of Medicine PMB 21266,
Lagos Nigeria
2
Department of Pharmacology, Therapeutics and Toxicology College of Medicine of the University of Lagos, Idi Araba,
PMB 12003, Lagos, Nigeria
*Address for Correspondence: Dr. Amole Olufemi Olatokunboh, Associate Professor, Department of Pharmacology,
Therapeutics and Toxicology, Lagos State University College of Medicine PMB 21266, Lagos Nigeria
Received: 28 Feb 2018/ Revised: 26 April 2018/ Accepted: 11 June 2018
ABSTRACT
Background- The mainstay of the treatment of pain and inflammation are opioids, steroids, and non-steroidal anti-inflammatory
drugs. Though, they are effective and readily available with negative and unpleasant effects, more importantly, hepatotoxicity and
nephrotoxicity. Thus, the need for safer and effective therapy in the management of pain and inflammation.
Objective- The work sought to investigate the anti-nociceptive and anti-inflammatory activities of the hydro-ethanolic leaf extract
of Clerodendrum polycephalum (HeCP) in animals.
Methods- HeCP (100, 200 or 400 mg/kg, p.o.) given to mice, 1 h before administer of acetic acid (0.6% v/v, i.p.), formalin (1%v/v,
intraplantar) or capsaicin (1% w/v, intraplantar) for nociceptive behavior in mice while carrageenan (1% w/v in saline, intraplantar)
or cotton pellet (20 mg implanted into both groin) to induce acute or chronic inflammation in rats.
Results- HeCP (100 – 400 mg/kg, p.o.) reduced mean writhes number, duration of paw licking or biting in the acetic acid, formalin
and capsaicin models, respectively, in mice. However, the initial treatment of mice with L-NNA (neuronal nitric oxide synthase
+
inhibitor), naloxone (opioid receptor antagonist), or glibenclamide (ATP-sensitive K channel blocker) prevented HeCP induced
anti-nociception in mice. In contrast, the initial treatment of mice with, sulpiride (dopamine D 2-receptor antagonist) failed to
reverse HeCP-induced antinociception. In the aspect of anti-inflammatory activity, HeCP caused significantly but not
dose-dependent inhibition of edema development in carrageenan-induced inflammation and cotton pellet-induced granuloma
formation in rats.
Conclusion- Findings from this work indicates that the hydroethanolic leaf extract of Clerodendrum polycephalum has
anti-nociceptive and anti-inflammatory possibly due to its polyphenolic constituents.
INTRODUCTION
Medicinal plants have been identified and used are also present in Cameroun, Ghana, Sierra Leone and
throughout human history. Clerodendrum polycephalum Guinea [1,2]. In Nigeria, the Yorubas commonly refer to it
(CP) belongs to the family Lamiacea It is found in tropical as ‘’Aporo’’ which means it kills pain and as an antidotes
and warm temperate regions of the world with most of to venomous stings and bites. It is also used as painkiller
the species occurring in tropical Africa, South Asia, they and medicines for the treatment of paralysis, epilepsy,
convulsion [2]. Painful sensations are the reason for
How to cite this article
Amole OO*, Ishola IO, Akinyede AA, Adewale MT. Anti-Nociceptive physician consultation and it can interfere with a
and Anti-Inflammatory Activities of the Hydroethanolic Extract of person’s quality of life and general well-being [3].
the Leaf of Clerodendrum polycephalum (Lamiaceae) Int. J. Life. Sci.
Therefore, it is almost impossible to imagine a world
Scienti. Res., 2018; 4(4): 1863-1871.
without pain relief; we depend on pain relief drugs to an
unspeakable degree. Similarly, inflammation is a
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protective response that involves immune cells, blood
vessels and molecular mediators; the purpose of
inflammation is to eliminate the initial causes of cell
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Amole et al., 2018
DOI:10.21276/ijlssr.2018.4.4.3
injury, clear out necrotic cells, tissues damaged from the carrageenan, capsaicin, and celecoxib (Sigma Aldrich, St.
original insult and to initiate tissue repair. In spite of the Louis, MO, USA).
ethno-medicinal importance of this plant, very little
information is available on CP in literature. The present Acute toxicity tests- Three groups of 5 mice (n=5) fasted
work sought to determine the effect of CP on acute and/ over-night before the experiment were given doses of
or chronic painful inflammatory conditions as well as its HeCP (1000, 2000, or 5000 mg/kg, p.o.). Animals in the
putative mechanism(s) of action using validated different groups were observed for 2 h post-treatment
pharmacological tools. for behavioral parameters such as convulsion,
piloerection, hyperactivity, food and water intake, and
MATERIALS AND METHODS respiratory pattern. The mice were further observed for
Collection of C. polycephalum- Fresh leaves of up to 24 h and 14 days for any signs of delayed toxicity
Clerodendrum polycephalum were obtained from a farm and mortality.
land at Okeletu, Ijede in Ikorodu, Lagos State, Nigeria in
June, 2016. The botanical identification and Test for analgesia
authentication of the plant was done by Mr. O.O. Mouse writhing test- Mice fasted overnight were divided
Oyebanji, a forestry expert at the Department of Botany into five groups (n=5). The animals were then treated
Herbarium, Faculty of Science, University of Lagos, with distilled water (10ml/kg, p.o.); HeCP (100,200,
Akoka, Lagos, Nigeria, where the herbarium voucher 400mg/kg, p.o.); and diclofenac (50 mg/kg, p.o.; as a
specimen (LUH 7080) was deposited for reference. standard drug). Sixty minutes after treatment was
carried out, mice were administered with acetic acid
Preparation of plant extract- The fresh leaves of (0.6% v/v in saline, 10ml/kg, i.p.). The number of writhes
Clerodendrum polycephalum were dried and milled into (characterized by contraction of the abdominal
the coarse powder and 1.252 kg of the dried leaves was musculature and extension of hind limbs) was then
loaded into a percolator. Extraction was done with 2.8 L counted at 5 min interval for 30 minutes [5].
absolute ethanol in 1.2 L of water for 72h. After
filtration, the residue was discarded and the final filtrate Inhibition % =
was concentrated in a rotary evaporator (40oC, under Number of writhes (control) – Number of writhes
(treatment)
vacuum), yield was 7.65% w/w. The greenish solid
X 100
extract obtained was always reconstituted in distilled Number of writhes (control)
water to appropriate concentrations before
administration to experimental animals. Formalin test- Mice fasted overnight was divided into
five groups of five animals each. The different groups of
Experimental animals- Albino mice (20 – 25g) and Wistar animals were treated with distilled water (10ml/kg, p.o.);
rats (180 – 200g) of either sex used in this study were HeCP (100, 200 or 400 mg/kg, p.o.); and diclofenac
obtained from the Laboratory Animal Centre of the (50mg/kg, p.o.). Sixty minutes after administration,
College of Medicine, University of Lagos, Lagos, Nigeria. formalin (20µL of 1% solution) was injected
The animals were kept in well-ventilated and hygienic subcutaneously into the right hind paw of each mouse.
compartments, maintained under standard The time (in seconds) spent in licking and biting
environmental conditions and fed with standard feed responses of the injected paw, indicative of pain was
(Livestock Feed Plc, Lagos, Nigeria) and water ad libitum. recorded for each animal. The responses of the mice
The experimental procedure adopted in this study was in were observed for 5 min (first phase), 15 – 30 min
accordance with the United States National Institutes of (second phase) and post-formalin injection [6].
Health Guidelines for Care and Use of Laboratory
Animals in Biomedical Research [4]. Capsaicin test- Mice fasted overnight was divided into
five groups of five animals each. The different groups of
Drugs and Chemicals- The chemicals used were acetic animals were treated with distilled water (10 ml/kg,
acid, formaldehyde (May and Baker Ltd, Dagenham, p.o.); HeCP (100, 200 and 400 mg/kg, p.o.); and
England); diclofenac (Total Healthcare, Parwanoo, India), diclofenac (50 mg/kg, p.o.). Sixty minutes after
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Int. J. Life. Sci. Scienti. Res. eISSN: 2455-1716
Amole et al., 2018
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Amole et al., 2018
DOI:10.21276/ijlssr.2018.4.4.3
Vehicle 10 106.0±14.87
HeCP 100 45.20±5.22** 57.40
HeCP 200 77.20±8.26 27.20
Values are expressed as mean ± SEM (n= 5); **P<0.01 versus vehicle treated, control
Formalin- induced nociceptive test- As shown in table 2 licking in vehicle-control treated. However, the
below, injection of formalin into the right hind paw pretreatment of mice with HeCP dose dependently and
produced a marked biphasic response. The first phase significantly reduced the duration of licking by 83.90 and
occurs 5 mins post formalin injection while second phase 86.00% at 400 mg/kg, significantly higher than the effect
seen 15-30 min after formalin administration with of diclofenac 50 mg/kg (37 and 49.30%, respectively).
88.00±20.29 and 146.00±26.41 duration of biting and
0 - 5 min 15 - 30 min
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Amole et al., 2018
DOI:10.21276/ijlssr.2018.4.4.3
Capsaicin-induced nociceptive test- To investigate the HeCP-induced anti-nociception in the mouse writhing
role of vanilloid receptors, capsaicin-induced nociceptive assay (Fig. 1).
test was carried out. Table 3 shows that the intraplantar
injection of capsaicin induced licking or biting reaction
M e a n n u m b e r o f w rith e s in 3 0 m in
150
(22.40±5.93) in vehicle control mice. The pretreatment
of mice with HeCP (100, 200 or 400 mg/kg) produced
##
non-dose related decrease in nociceptive behavior with 100 # #
inhibition).
0
Table 3: Effect of HeCP on capsaicin induced
0
0
0
le
5
0
1
L
7
ic
M
rg
h
nociception in mice
L
e
-N
S
-A
G
V
L
L
H
+ H e C P 1 0 0 m g /k g
0-5 min
Fig 1: Mechanism of HeCP-induced anti-nociception in
Treatment Dose Response Inhibition
mouse writhing assay
(mg/kg) duration (s) (%) ***
Values are expressed as mean±SEM (n= 5). P<0.01 versus vehicle
# ##
Vehicle 10 22.40±5.93 - treated control; P<0.05; P<0.01 versus HeCP (100 mg/kg) treated
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Amole et al., 2018
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Fig. 2: effect of HeCP on granuloma formation in rats. Values are expressed as mean ± SEM (n=5); ***P<0.001;
****
P<0.0001 versus vehicle treated, control
Dose
Treatment 30mins 60mins 90mins 120mins 150mins 180mins
(mg/kg)
of COX are good antinociceptive agents as seen with the and induce peripheral antinociception. The involvement
peripherally acting non-steroidal anti-inflammatory of dopaminergic system was also investigated. Lateral
drugs (NSAIDs), diclofenac. C. polycephalum leaf extract hypothalamus (LH) involves in modulation of tonic pain
made less effective the acetic-acid-induced peripheral regarding the direct and indirect neural connections
nociception indicating the presence of analgesic between the LH and nucleus accumbens (NAc) [6].
principles with ability to attenuate inflammatory- Moreover, blockade of accumbal dopamine receptors
mediated pain. However, the abdominal constriction test attenuated analgesia induced by carbachol injection into
is considered a non-specific test due to its inability to the lateral hypothalamus during both phases of formalin
provide information on the peripheral and/or central test. Effect of blockade of D1- and D2-like dopamine
nociceptive level inhibited by HeCP and to have poor receptors on reduction in anti-nociception was more
specificity as it can give false positive results when used during the late phase. The contribution of D2-like
to test certain non-analgesic drugs such as muscle dopamine receptors to mediation of anti-
relaxants [14]. Thus, the applications of other nociceptive nociception during the late phase was greater than the
models are necessary before the final conclusion on the early phase [17]. In this study, pretreatment of mice with
possible mechanisms of action adopted by HeCP could sulpiride (D2 receptor antagonist) did not affect HeCP-
be drawn. In this work, the formalin-induced paw licking induced anti-nociception.
test was adopted to further determine the Experimental data have indicated a link between the
antinociceptive activity of HeCP. The leaf extract inhibits activation of the NO-cGMP pathway and the opening of
both phases of the formalin-induced nociception, thus, the ATP-sensitive K+ channels [18]. From the results
further suggesting its ability to block the central observed, the ATP-sensitive K+ channel is suggested to
nociceptive center. To further ascertain the mechanism be involved in the mechanism of action of HeCP.
of action of HeCP, it was assessed for pain induced by Pre-treatment with glibenclamide, an ATP-sensitive
capsaicin, a natural product that specifically and directly K+ channel blocker significantly reversed anti-
activates TRPV1 receptor [15]. Here, HeCP showed a nociceptive effect of HeCP. HeCP possibly acts by
significant anti-nociception effect on capsaicin-induced modulating K+ currents through the efflux of K + ions
pain that was maintained for up to 4 h, strengthening the permeating the membrane. Increase in K + ion efflux
hypothesis that the antinociceptive effect of this extract alters the membrane potential to avert from action
is at least partially mediated by the inhibition of TRPV1 potential generation, which results in the decrease of
channel. Overall, findings obtained from the three neurotransmitter release [19]. Other than that, the
nociceptive assays implied that HeCP contains bioactive effect of HeCP through the activation of the
compound(s) with ability to modulate the central and NO-dependent pathway is similar to some
peripheral nociceptive mechanisms. pharmacological studies that have evaluated NO/cGMP
The effectiveness of opioid analgesics (such as activation and the opening K + channels, which relates
morphine) has been overshadowed by many adverse to the opioidergic pathway [20]. The anti-inflammatory
side effects (e.g. respiratory depression, vomiting, activity of C. polycephalum was evaluated in this study
nausea, constipation, tolerance, and dependence). using the carrageenan induced paw oedema and cotton
Hence, the need for the more efficacious drug, wool implantation tests. Inflammation induced by
pretreatment of mice with naloxone prevented the carrageenan is acute, non-immune, and highly
antinociceptive effect of HeCP indicative of opioidergic reproducible and can be quantified by the increase in
system in its mechanism of action, HeCP anti-nociception paw size [21]. It is widely used to access the
was reversed by LNNA (neuronal nitric oxide synthase anti-inflammatory effect of natural products [6].
inhibitor) but not L-Arg. The production of nitric Carrageenan-induced edema is a biphasic event, with the
oxide (NO) from l-arginine is catalyzed by NO synthase involvement of several inflammatory mediators: In the
(NOS), which exists as the following three isoforms: first phase (during the first 1hr after carrageenan
endothelial (eNOS), neuronal (nNOS), and inducible injection), chemical mediators such as histamine and
(iNOS ) [16]. Romero et al. [16] have shown that local serotonin play a role, whereas, in the second phase (3-5)
injection of analgesic drugs activates nNOS to release NO hours after carrageenan injection), kinnins and
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Amole et al., 2018
DOI:10.21276/ijlssr.2018.4.4.3
[21]
prostaglandins are involved . In this study, C. CONTRIBUTION OF AUTHORS
polycephalum showed the significant inhibitory effect on Drs Amole, Akinyede and Ishola designed the work, Data
rat paw edema development in the early and late phase collecction, analysis and interpretation was done by Dr
of carrageenan and inflammation, suggesting possible Amole and Mr Adewale. Drafting and critical revision of
inhibition of serotonin, histamine, kinins and the article for intellectual content and final approval of
prostaglandin release. The cotton pellet-induced the version to be published was done by all the authors.
granuloma formation is an established chronic
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