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COMPARISON OF ANALGESIC AND ANTI-INFLAMMATORY EFFECTS OF
RESVERATROL AND CYCLO-OXYGENASE (COX) INHIBITORS (DICLOFENAC
AND ETERICOXIB) IN WISTAR RATS
Elizabeth Ogboli-Nwasor*, Ahmed-Sherif Isa**, John Francis E Dingwoke***, Abdullahi Umar
Hussein**
Department of Anaesthesia*, Human Physiology** and Biochemistry, Ahmadu Bello University,
Zaria, Nigeria
ABSTRACT
BACKGROUND: Resveratrol is a biomolecule that has been demonstrated in numerous studies
to possess therapeutic potentials with multiple targets. Despite the reported low bioavailability,
its cardioprotective, antioxidant, anticancer, neuroprotective anti-inflammatory and analgesic
properties have been described.
OBJECTIVE: To investigate the systemic analgesic and anti- inflammatory effects of
Resveratrol in comparison to Diclofenac and Etoricoxib.
METHODOLOGY: Following institutional ethics approval, the study was conducted in the
human physiology research laboratory. All the animal care and treatment procedures were
respected following the guidelines established by the Ahmadu Bello University Animal Research
committee. Every effort to minimize the number of animals used and their suffering was
employed. Forty –two (42) Wistar rats were assigned into 7 groups (n = 6) as follows; CMC,
Diclofenac, Etoricoxib, RSV 25 mg/kg, RSV 50 mg/kg, RSV 100 mg/kg.
Formalin test was used to evaluate pain response to chemical pain. The rats were administered
distilled water, CMC, Diclofenac, Etoricoxib, or resveratrol (25, 50, or 100mg/kg) orally. After 1
1
hour, 50µl of freshly prepared 2.5% Formalin solution was injected subcutaneously into the
dorsal surface of the right hind paw of the animal using a syringe with a 26-gauge needle.
The severity of pain response was recorded for each rat based. The diameter of the right hand
paw was recorded every thirty minutes for four hours and the findings recorded. Statistical
analysis was done using ANOVA and Kruskal wallis tests.
RESULTS: Showed a significant change (p < 0.05) in nociceptive score in the rats in the early
phase, but no significant change in the late phase in the formalin tests. Anti-inflammatory studies
showed that RSV 50 mg/kg has a significantly potent (P <0.05) anti-inflammatory effect when
compared to control but similar in activity to etoricoxib. TNF α and IL-2 concentration did not
significantly differ between the groups (p > 0.05).
CONCLUSION: Findings suggest that resveratrol may elicit its analgesic actions by inhibiting
direct activation of nociceptors, and not through its anti-inflammatory action as the late phase
results from inflammatory mediators which correspond to results on IL-2 and TNF-α. It may be
suggested that systemic resveratrol at a dose of 50 mg/kg significantly reduces inflammation
comparable to etoricoxib, but possibly less potent than diclofenac. Thus systemic resveratrol may
be useful for the management of acute post-operative pain. Additional studies are needed to
further delineate the degrees of potency of each agent.
Keywords: Analgesia, anti-inflammatory, Diclofenac, Etoricoxib, Resveratrol, Post-operative
pain management.
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INTRODUCTION
Resveratrol is a naturally occurring phytoalexin present in grapes and wine1. It is a natural
polyphenol that protects from cancer and cardiovascular diseases2. Resveratrol is able to induce
apoptotic cell death and it inhibits the cyclooxygenase (COX) cascade. It has been reported that
resveratrol inhibits the activity of COX-2 and suppresses the activation of COX-2 gene
expression3. Etoricoxib is a selective cyclooxygenase (COX) inhibitor. Similar to diclofenac,
Etoricoxib is a non-steroidal anti-inflammatory drug that eases pain and swelling (inflammation)
in conditions like osteoarthritis, rheumatoid, and ankylosing spondylitis. Etoricoxib works by
blocking the effect of a natural chemical called cyclooxygenase-2 (COX-2) enzyme. This
enzyme catalyzes the synthesis of chemical called prostaglandins in the body, which causes pain
and inflammation. Adaptive pain contributes to survival by protecting the individual from injury
or promoting healing when injury has occurred (symptomatic pain), maladaptive pain is an
expression of the pathologic operation of the nervous system (pain as a disease).
Natural products are believed to be important sources of new chemical substances with potential
therapeutic efficacy. Taking into account that the most important analgesic prototypes (e.g.
salicylic acid and morphine) were originally derived from plant sources, the study of natural
products with analgesic and anti-inflammatory properties is a fruitful research strategy in the
search of new analgesic and anti-inflammatory drugs4. This study was designed to investigate the
systemic analgesic and anti-inflammatory properties of Resveratrol in comparison to Diclofenac
and Etoricoxib.
MATERIALS AND METHOD
Following institutional ethics approval, the study was conducted in the Human physiology
research laboratory of the Ahmadu Bello University Zaria. All the animal care and treatment
procedure were respected following the guidelines established by the University’s Animal
Research Committee. Efforts were employed to minimize number of animals used and their
sufferings. Forty two (42) adult male Wistar rats weighing 100 to 120g were used for the
experiments. The animals were maintained under normal laboratory conditions of humidity,
temperature and light for 7 days prior to commencement of the experiments, and allowed free
access to food and water ad Libitum. The animals were divided into seven groups (n = 6) in
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separate cages and treated orally as follows: Distiled water (control); Carbixymethylcellulose
(CMC); Diclofenac; Etoricoxib; Resveratrol (25mg/Kg); Resveratrol (50mg/Kg); and
Resveratrol (100mg/Kg). Thirty minutes after treatment, 50 μl of a freshly prepared 2.5%
solution of formalin was injected subcutaneously on the plantar surface of the left hind paw of
each rat. The rats were placed individually in an observation chamber and monitored after 5
minutes, and then at the end of 45 minutes. The severity of pain response was recorded for each
rat based on the following scale: (0) rat walk or stand firmly on the injected paw; (1) the injected
paw is favored or partially elevated; (2) the injected paw is clearly lifted off the floor; (3) the rat
lick, chew or shake the injected paw5. Paw diameter was measured with a vernier caliper at 30
minutes interval. Increase in diameter of the injected paw was taken as indicator of paw edema6.
Data obtained from the experiments was pooled and expressed as mean ± standard error of mean
(SEM). The results were analyzed statistically using one-way analysis of variance (ANOVA;
95% confidence interval). Values of p<0.05 were considered to imply statistical significance.
RESULTS:
In the formalin induced nociception test, there was a significant change in the early phase of the
test (p<0.05), but no significant change in the late phase (Fig. 1). Result of the formalin induced
paw oedema shows significant difference among the groups (Table1). Concentrations of TNF-α
and IL-2 did not significantly differ between the groups.
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Early Phase
RSV 100 mg/kg
RSV 50 mg/kg
RSV 25 mg/kg
Etoricoxib
Diclofenac
CMC
Control
RSV 100 mg/kg
RSV 50 mg/kg
RSV 25 mg/kg
Etoricoxib
Diclofenac
CMC
Control
Nociceptive scores
35
30
25
20
15
10
5
0
Late Phase
Treatment Groups
Fig. 1: Early and late phase formalin induced nociception in rats
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Control
CMC
Diclofenac
Etoricoxib
RSV 25 mg/kg
RSV 50 mg/kg
RSV 100 mg/kg
P value
Early Phase
Late Phase
21.08
26.75
21.83
31.67
26.75
10.50
11.92
0.008
19.25
25.75
22.50
14.58
29.92
16.00
22.50
0.180
Early and late phase formalin induced nociception in rats in Table format
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Table 1: Formalin induced paw oedema in rats
0 min
30 min
60 min
90 min
120 min
150 min
180 min
210 min
240 min
Control
3.10 ± 0.15
5.07 ± 0.20
5.92 ± 0.08
7.17 ± 0.17
6.50 ± 0.13
5.88 ± 0.05
6.12 ± 0.08
5.93 ± 0.09
5.45 ±0.19
CMC
3.75 ± 0.11
5.00 ± 0.00
5.00 ± 0.15
4.72 ± 0.09*
4.85 ± 0.08*
4.35 ± 0.08*
4.75 ±0.19*
4.40 ± 0.17*
4.03 ±0.02*
Diclofenac
4.00 ± 0.00
4.40 ± 0.28*
4.42 ± 0.13*b
4.53 ± 0.17*b
5.05 ± 0.17*
3.75 ± 0.15*b
3.60 ± 0.20*b
3.37 ± 0.20*b
2.95 ± 0.02*b
Etoricoxib
4.07 ± 0.15
3.15 ±0.08*a
3.45 ± 0.18*a
3.50 ± 0.18*a
5.53 ±0.21*
5.43 ± 0.23a
4.90 ± 0.24*a
5.32 ± 0.18a
5.50 ± 0.18a
RSV 25 mg/kg
4.32 ± 0.18
6.13 ± 0.18*b
6.33 ± 0.25ab
6.37 ± 0.17ab
6.97 ± 0.37ab
6.20 ± 0.39a
6.22 ± 0.33ab
6.58 ± 0.26ab
6.50 ± 0.32*ab
RSV 50 mg/kg
4.07 ± 0.03
2.98 ± 0.03*a
3.43 ± 0.19*a
3.40 ± 0.19*a
5.35 ± 0.16*a
4.98 ± 0.07*a
4.62 ± 0.20*
4.97 ± 0.06*a
5.18 ± 0.17a
RSV 100 mg/kg
4.03 ± 0.25
6.12 ± 0.22*ab
5.67 ± 0.18ab
6.05 ± 0.28*ab
6.00 ± 0.20a
5.87 ± 0.32a
5.97 ± 0.28ab
6.17 ± 0.14ab
6.27 ± 0.20*a
*= indicate significance when compared to control
a,b
=Different supescripts indicate significant difference
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Table 2: ELISA results
Control
CMC
Diclofenac
Etoricoxib
RSV 25 mg/kg
RSV 50 mg/kg
RSV 100 mg/kg
P value
TNF
IL-2
314.98 ± 29.61
293.23 ± 14.88
303.50 ± 16.87
288.27 ± 20.65
309.88 ± 9.32
315.62 ± 13.09
376.18 ± 28.69
0.084
479.62 ± 42.38
477.60 ± 23.65
513.88 ± 29.37
501.43 ± 14.91
456.73 ± 29.17
554.83 ± 45.04
481.65 ± 31.86
0.454
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Paw diameter (cm)
7
6
5
4
3
2
1
0
0 min
30 min
60 min
90 min 120 min 150 min 180 min 210 min 240 min
Time (min)
Control
CMC
Diclofenac
RSV 25 mg/kg
RSV 50 mg/kg
RSV 100 mg/kg
Etoricoxib
Formalin induced paw oedema in rats (Table 1), Presented in graph format
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Discussion
An understanding of the analgesic and anti-inflammatory potential of resveratrol is an important
future paradigm for pain relief. The analgesic and anti-inflammatory effects of resveratrol and
cyclo-oxygenase (COX) inhibitors (diclofenac and etericoxib) were studied and compared in an
animal model of male wistar rats. Flinching behavior was the factor that determined nociception.
It has been validated that this assay is an adequate factor for the characterization of
antinociceptive agents7.
Result of the formalin test (presented in fig. 1), when analyzed using Kruskal wallis, revealed
that the first phase was significant, based on the mean rank. As can be observed, resveratrol had
lower pain scores when compared to other groups indicating that resveratrol exhibited analgesic
effect by reducing the behavior associated with pain (0). This was substantiated as the rats stood
firmly on the injected paw and walked; (1) the injected paw was favored or partially elevated; (2)
the injected paw was clearly lifted off the floor; (3) the rat licked, chewed or shook the injected
paw.). This clearly indicate that resveratrol may have acted peripherally7, 1 and not centrally
since there was no significant difference in the late phase, as shown on Fig. 1, even though the
lowest pain scores were in resveratrol at 50mg/kg and etoricoxib. In a similar study8, the mean
firing frequency of spinal trigeminal nucleus caudalis (SpVc) wide dynamic range neuron in
response to both non-noxious and noxious mechanical stimuli was inhibited by resveratrol at 0.52mg/kg, intravenously, suggesting that resveratrol may be used as a complementary and
alternative agent for treatment of trigeminal nociceptive pain. Taken together it may be
suggested that resveratrol (50 mg/kg) had the most potent analgesic effect, compared to the COX
inhibitors, this corresponds to a finding that resveratrol induced a prolonged antinociceptive
effect, which was correlated to the inhibition of COX-2 mRNA increase in dorsal root ganglion9
The observed analgesic and anti-inflammatory effect of resveratrol indicates that there was
reduction in the generation of prostaglandins from arachidonic acid, due to possible inhibition of
isoform 2 of cyclooxygenase (COX-2). Although the selectivity of resveratrol towards the
cyclooxygenase isoforms is still not clearly established10,11. This can be substantiated by the fact
that resveratrol was able to induce apoptotic cell death and inhibits the cyclooxygenase (COX)
cascade12.
At 0 minute before the administration of formalin there was no difference in the paw diameter,
after 30 minutes, all the groups with “asterisk” were significantly lower when compared to the
control group (that did not receive any drug, hence, the paw diameter was not expected to
reduce), except for RSV at 25 mg/kg and 100 mg/kg which were significantly higher. The paw
diameter at 50mg/kg of RSV was similar to that of etoricoxib (as there was no significant
difference when compared to RSV and this is indicated by the similar superscript a), shown on
Table 1. This observed similarity could have occurred possibly because both resveratrol and
etoricixib exhibits their same analgesic and anti-inflammatory via the same or similar mechanism
of action. However, our result reveals that resveratrol exhibited more potent analgesia that the
COX inhibitors.
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Putting all together after the four hours on induction of formalin, diclofenac and etoricoxib
reduced paw diameter as expected since they are known drugs (standard), they act most probably
by selectively inhibiting the isoform 2 of cyclooxygenase (COX-2). RSV 50mg/kg also exhibited
anti-inflammatory effect by reducing the paw diameter especially after 90 minutes of formalin
injection, meaning it was most effective for the duration of 90 minutes probably because of the
longer half-life of resveratrol. Our results indicate that resveratrol and etoricoxib possess
perhaps, similar mode of action. As there were no significant difference between resveratrol and
etoricoxib suggests that resveratrol may have acted most probably through the inhibition of
cyclooxygenase-2 (COX 2), an enzyme responsible for inflammation and pain.
The ELISA test result (Table 2) obtained in studying the profile of tumor necrosis factor alpha
(TNF), a cell signaling protein (cytokin) involved in systematic inflammation and one of the
cytokins that make up the acute phase reaction shows no significant difference in TNF alpha and
interleukin 2 (IL-2, which induces the stimulation of inflammatory and immune reactions) even
though etoricoxib, diclofenac and resveratrol had low TNF alpha concentration. This could be
attributed to the mechanism of action of cyclooxygenase, TNF and IL-2.
Summarily, our result suggest that resveratrol may elicit its analgesic actions by inhibiting direct
activation of nociceptors, and not through its anti-inflammatory action as the late phase results
from inflammatory mediators which correspond to results on IL-2 and TNF-α. It may be
suggested that systemic resveratrol at a dose of 50 mg/kg significantly reduces inflammation
comparable to etoricoxib, but possibly less potent than diclofenac. Thus systemic resveratrol may
be useful for the management of acute post-operative pain. Additional studies are needed to
further delineate the degrees of potency of each agent.
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REFERENCES
Granados-Soto V, Argu¨elles CF, Ortiz MI. The peripheral antinociceptive of
resveratrol is associated with the activation of potassium channel.
Neuropharmacology. 2002; (43): 917-923.
Yaici Djouer, Gerard Benoit, Dan Benhamou, Jean-Xavier Mazoit. Antinociceptive of
resveratrol carrageenan-evolked hyperalgesia in rat: prolonged effects related to COX-2
expression impairment. Pain (2008); (140): 273-284.
Subbaramaiah K, Chung WJ, Michaluart P, Telang N, Tanabe P, Inoue H. Resveratrol inhibits
cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary
epithelial cells. Journal of Biological Chemistry, 1998; (273):21875-82.
Shanmugasundaram P, Venkataraman S. Anti-noceceptive Activity of Hygrophila auriculata
(Shum) Heine. African Journal of Traditional, Complementary and Alternative
Medicines. 2005; 2(1): 62-69.
Yerima M, Magaji MG, Yaro AH, Tanko Y, Mohammed MM. Analgesic and Anti-Inflammatory
activities of the Methanolic Leaves extract of Securinega virosa (Euphorbiaceae).
Nigerian Journal of Pharmaceutical Sciences. 2009; 8(1): 47-53.
Ojewola GS, Ukachukwu SN, Okulonye EI. Cottonseed Meal as Substitute for Soyabean Meal in
Broiler Ration. International Journal of Poultry Science. 2006; (5)4: 360-364.
Torres-Lopez JE, Ortiz MI, Castaneda-Hernandez G, Alonso-Lopez R, Asomoza-Espinosa R,
Granados-Soto V. Comparison of the antinociceptive effect of celecoxib, diclofenac and
resveratrol in the formalin test. Life Sciences, 2002 (70): 1669-1676.
Takehana S, Sekiguchi K, Inoue M, Kubota Y, Ito Y, Yui K., Shimazu Y, Takeda M. Systemic
administration of resveratrol suppress the nociceptive neuronal activity of spinal
trigeminal nucleus caudalis in rats. Brain Research Bulletin, 2016 (120):117-122.
Pharm-Macou TA, Beloeil H, Sun X, Gentili M, Yaici D, Benoit G, Benhamou D, Mazoit J.
Antinociceptive effect of resveratrol in carrageenan-evoked hyperalgesia in rats:
Prolonged effect related to COX-2 expression impairment. Pain, 2008 (140): 274-283.
Szewczuk LM, Forti, Stivala LA, Penning TM. Resveratrol is a peroxidase-mediated inactivator
of COX-1 but not COX-2: a mechanistic approach to the design of COX-1 selective
agents. Journal of Biological Chemistry. 2004 (279): 22727-37.
12
Montiel-Ruiz RM, Granados-Soto V, Garcia-Jimenez S, Reyes-Garcia G, Flores-Murrieta FJ,
Deciga-Campos M. Synergistic interaction of diclofenac, benfotiamine, and resveratrol
in experimental acute pain. Drug Development Research, 2011 (72): 397-404.
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