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Year : 2014  |  Volume : 46  |  Issue : 4  |  Page : 404--408

Sildenafil enhances the peripheral antinociceptive effect of ellagic acid in the rat formalin test

Mohammad Taghi Mansouri1, Bahareh Naghizadeh2, Behnam Ghorbanzadeh3,  
1 Department of Pharmacology, School of Medicine, Physiology and Atherosclerosis Research Centers; Pain and Physiology Research Centers, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
2 Department of Pharmacology, School of Medicine, Physiology and Atherosclerosis Research Centers; Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
3 Department of Pharmacology, School of Medicine, Physiology and Atherosclerosis Research Centers, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

Correspondence Address:
Mohammad Taghi Mansouri
Department of Pharmacology, School of Medicine, Physiology and Atherosclerosis Research Centers; Pain and Physiology Research Centers, Ahvaz Jundishapur University of Medical Sciences, Ahvaz


Objective: Ellagic acid (EA), a major polyphenolic compound of pomegranate juice, produces antinociceptive effects, which are mediated through opioidergic and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathways. The present study was conducted to elucidate the peripheral antinociceptive effect of EA alone and in combination with sildenafil in the rat formalin test. Materials and Methods: Pain was produced by intraplantar injection of formalin (2.5%) in rats and nociceptive behavior was measured as the number of flinches every 5 min in 60 min after injection. Results: Local administration of EA and sildenafil dose-dependently increased the nociception threshold in both phases of the test. Moreover, sub-effective doses of sildenafil (25 or 50 mcg/paw, i.p.) significantly and dose-dependently enhanced the antinociception induced by a sub-effective dose of EA (60 mcg/paw, in both phases of the test. The antinociception produced by these drugs alone, or in combination, was due to a peripheral site of action, since the administration in the contralateral paw was ineffective. Conclusion: Our results suggest that EA has local peripheral antinociceptive activity, and enhancement of this effect with sildenafil probably occurs through the inhibition of cGMP metabolism.

How to cite this article:
Mansouri MT, Naghizadeh B, Ghorbanzadeh B. Sildenafil enhances the peripheral antinociceptive effect of ellagic acid in the rat formalin test.Indian J Pharmacol 2014;46:404-408

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Mansouri MT, Naghizadeh B, Ghorbanzadeh B. Sildenafil enhances the peripheral antinociceptive effect of ellagic acid in the rat formalin test. Indian J Pharmacol [serial online] 2014 [cited 2023 Oct 3 ];46:404-408
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There is an important role of cyclic guanosine monophosphate (cGMP) in the antinociceptive activity of drugs such as opioids. [1] Intraplantar injection of dibutyryl-cGMP produced antinociception in inflammatory hyperalgesia in rats has been observed. [2] Therefore, cGMP seems to be critical for the modulation of nociceptive transmission. Guanylyl cyclase catalyzes the formation of cGMP from GTP, whereas cGMP-specific phosphodiesterase (PDE) catalyzes the hydrolysis of cGMP to GMP, thereby ending the signal transduction pathway. The PDE 5 isoenzyme seems to be the most relevant enzyme for cGMP inactivation in cells. [3] Sildenafil (1-[3-(4,7-Dihydro-1-methyl-7-oxo-3-propyl- 1H-pyrazolo[4,3-d] pyrimidin-5-yl)-4-ethoxyphenyl] sulfonyl]- 4- methylpiperazine) is a selective inhibitor of cGMP-specific PDE-5, which is effective in the management of erectile dysfunction in men. [4] It has been reported that this compound has peripheral antinociceptive activity through accumulation of cGMP in cells. Also, it has been shown that peripheral inhibition of PDE-5 produces antinociception and enhances the antinociceptive effect of several analgesics such as morphine, [1] indomethacin, and celecoxib [5] via the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway.

Ellagic acid (EA, 2,3,7,8 tetrahydroxy [1] benzopyranol [5,4,3-cde] [1] benzopyran-5,10-dione) is a polyphenolic compound found in fruits and nuts, including blueberries, blackberries, raspberries, strawberries, pomegranate, and walnuts. [6] It has been reported to show different pharmacological properties including inhibition of tumorigenesis, [7] anti-inflammation and antioxidation, [8] neuroprotection against diabetic neuropathy, [9] and also inhibition of lipopolysaccharide-induced prostaglandin E 2 synthesis in human monocytes. [10]

Nascimento et al., (2008) demonstrated the antinociceptive effect of EA systemic in the formalin model of pain. [11] Moreover, Corbett et al., (2010) showed that EA decreases paw edema induced by 3% carrageenan and may interact with ketorolac as a potent inhibitor of COX enzyme. [12] In a previous study, we demonstrated that EA produces antinociceptive effects in different experimental models of nociception via the opioidergic and NO-cGMP pathways in different animal models of pain. [13]

To characterize the peripheral antinociceptive profile of ellagic acid, we have examined in the present study the effects of intraplantal administration of EA on pain behavior produced by an injection of formalin in rats. The formalin test is widely used as a model of acute inflammatory pain, the closest test to clinical pain. Formalin injection activates peripheral sensory nerves and produces pain responses that involve ongoing peripheral activity and peripheral and central sensitization. [14]

Furthermore, to determine the possible involvement of cGMP pathway, previously shown to be involved in the modulation of nociception, the present study was conducted to elucidate the peripheral antinociceptive effect of EA alone and in combination with sildenafil in the rat formalin test.

 Materials and Methods


Experiments were conducted using adult male Wistar rats (130-160 g) purchased from the central animal house of the Jundishapur University of Medical Sciences, Ahvaz, Iran. They were housed at 22°C ± 2°C under a 12 h light/dark cycles (light from 7:00 to 19:00 h) with free access to food and water ad libitum. All animals were divided randomly into groups of 8 in each, acclimatized and habituated to the laboratory environment for at least one week prior to the experiments and were used only once throughout the experiments. We followed the ethical guidelines for investigation of experimental pain in conscious animals, [15] as well as our Institutional Animal Ethical Committee of Jundishapur University, formed under Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Reg. No. PRC-115) approved the pharmacologic protocols.


Ellagic acid (EA) and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Sildenafil citrate donated by Poursina Pharmaceutical Co., Tehran, Iran) was dissolved in physiological normal saline (0.9%). EA was dissolved in normal saline (0.9%) containing 10% DMSO and buffered to a pH 7.2-7.4. Respective controls received only solvent vehicle. The vehicle alone had no effects per se on the nociceptive responses (data not presented). Drug doses were freshly prepared in such a way that the necessary dose could be injected in a volume 50 μL/paw by intraplantar route.

Formalin Test

Antinociception was assessed using the formalin test. [14] To reduce variability, each rat was acclimatized in an acrylic observation chamber (30 × 30 × 30) for 30 min prior to formalin injection. The rat was injected with 50 μl of diluted formalin 2.5% (in 0.9% saline) beneath the skin of the plantar surface of the right hind paw using a 30-gauge needle. Following formalin injection, the animals were immediately returned to the box and nociceptive behavior was measured as the number of flinches (rapid and brief withdrawal and/or shrinking back of the injected paw) every 5 min for a total of 60 min after injection. The early 5 min post formalin injection is known as the first phase (neurogenic) and the period between 15-60 min as the second phase (inflammation). Finally, the animals were euthanized in a CO 2 chamber.

Experimental Protocols

Sildenafil (25, 50, 100, and 200 mcg/paw) and or EA (30, 60, 100, and 300 mcg/paw) were injected subcutaneously (s.c.) into the plantar surface of the right hind paw 20 min before local injection of formalin. [16] To determine whether a combination of sildenafil with EA enhances the antinociceptive effect, increasing sub-effective doses of sildenafil (25 or 50 mcg/paw, were concurrently administered with a sub-effective dose of EA (60 mcg/paw). The effect of sildenafil and EA, administered s.c. into the plantar surface of the contralateral paw was also studied. To evaluate whether the effect was via a local site of action, formalin was administered s.c. into the plantar surface of right hind paw and the highest dose of the tested drugs in the contralateral (left hind paw). Doses and drug administration schedules were selected based on previous reports [16] and the pilot experience in our laboratory. The vehicles used had no effects per se on the nociceptive threshold in rats. In all experiments, the observer was blind to the treatment.

Statistical Analysis

The cumulative flinching number of each phase of responses was calculated for each rat. All experimental results were represented as the mean ± S.E.M. for 8 animals per group. The area under the number of flinches against time curves (AUC) for the second phase was calculated by the trapezoidal rule (Graphpad Prism 5.0, San Diego, CA, USA). Statistical analyses were performed using analysis of variance (one-way ANOVA) followed by Dunnett's test. Statistical significance was set at P < 0.05.


Peripheral Antinociceptive Effect of Sildenafil and EA Alone

Injection of formalin 2.5% (50 μL/paw) into the hind paw plantar surface of normal rats caused a classical pattern of flinching behavior characterized by a biphasic time-course as previously reported. [14] [Figure 1]a and [Figure 2]a showed the time course of flinching behavior in control and treated rats with increasing doses of sildenafil and EA, respectively. Local ipsilateral, but not contralateral, administration of sildenafil (100 and 200 μg/paw) significantly increased nociceptive threshold in both the first [Figure 1]b and second phases [Figure 1]c of the formalin test. The lower doses (25 and 50 mcg/paw) were ineffective in both phases of the test.{Figure 1}{Figure 2}

In a similar way, ipsilateral, but not contralateral, administration of EA (100 and 300 mcg/paw) reduced flinching behavior during both the first [Figure 2]b and second phases [Figure 2]c of the test. Moreover, lower doses

(30 and 60 mcg/paw) were ineffective.

To ensure that the effects of intraplantar injections of sildenafil and EA were local and not due to systemic diffusion of each compound, the highest doses of sildenafil (200 mcg) and EA (300 mcg) were injected into the hind paw contralateral to the formalin injection. Nociceptive behavior following injection of either sildenafil or EA in the contralateral hind paw was not statistically different when compared with the corresponding vehicle-treated rats [Figure 1] and [Figure 2].

Antinociceptive Effect of the Combination of Sildenafil with EA

In order to examine the effect of sildenafil on EA-induced peripheral antinociception, increasing and inactive doses of sildenafil (25 and 50 mcg/paw, were co-administered with the sub-effective dose of EA (60 mcg/paw, 20 min before formalin injection. The intraplantar co-administration of sildenafil and EA exhibited a marked dose-dependent diminution in the number of flinches during both the first [Figure 3] and second phases [Figure 4] of the formalin test as compared to the effect obtained from the single agents. The antinociceptive effect of the combination was due to a local effect, as administration of the combination to the contralateral paw did not produce any effect [Figure 3] and [Figure 4]. On the other hand, intraplantar co-administration of active doses of EA (100 mcg/paw) and increasing doses of sildenafil (50, 100, and 200 mcg/paw){Figure 3}{Figure 4}

produced a significant antinociception during both the first [Figure 5]a and second phases [Figure 5]b of the formalin test as compared with the active dose of EA alone.{Figure 5}


Formalin-induced nociception consists of two different nociceptive states, and distinct mechanisms underlie the two phases of behavioral response. The first results from direct chemical stimulation of nociceptive primary afferent fibers while the second is represent both inflammation-evoked sensory activity and facilitatory processes in the spinal cord. This pain model has been utilized as a tool for observing the effects of various antinociceptive agents on these two types of pain at once. [17]

In the present work, the effect of ellagic acid was investigated in a rat formalin pain model. Intraplantar injection of EA reduced behavioral signs of formalin-induced nociception in a dose-dependent manner. EA injected into the contralateral paw was not antinociceptive at the greatest dose used in the ipsilateral paw. This finding suggests that EA-induced antinociception may be mediated locally but not systemically. The present study further demonstrated that antinociception induced by intraplantar EA was augmented by combined administration of locally (plantar surface of the paw) injected sildenafil citrate (a potent and selective cGMP-specific PDE-5). These data suggest that intraplantar injection of EA could produce peripheral antinociception that is, at least in part, mediated through cGMP mechanism.

Several evidences have implicated a role of the NO-cGMP signaling in the modulation of peripheral pain perception. Durate et al., (1990) showed that the intraplantar administration of L-arginine produces analgesia in rats with carrageenan-induced hyperalgesia, the effect being antagonized by nitric oxide inhibitors and methylene blue (a soluble guanylyl cyclase inhibitor). [18] Also, it has been reported that the local administration of morphine, nitric oxide (NO) donors, and membrane permeable analogs of cGMP-produced antinociception. [19] Formalin administration may lead to an increase of NO [20] which in turn would lead to formation of cGMP by activation of guanylyl cyclase. Intracellular cGMP concentrations are regulated by the action of guanylyl cyclases and the rate of degradation by cGMP-specific PDEs. PDE-5, -6, and -9 are specific for cGMP, but the PDE-5 isoenzyme seems to be the most relevant enzyme for cGMP degradation in cells. [3] In agreement with previous reports, [1] the present study confirmed that sildenafil produces antinociception when microinjected into peripheral tissues. The fact that sildenafil is a potent, selective, and reversible PDE-5 inhibitor that blocks cGMP hydrolysis (Ki = 3 nM) suggests that sildenafil could produce its antinociceptive effect through the increase in intracellular cGMP levels. Thus, our data confirmed the hypothesis that the local peripheral antinociceptive effect of sildenafil was mediated by inhibiting phosphodiesterase 5 with the consequent increase of cGMP in the subcutaneous tissues.

Our results demonstrated that the local peripheral administration of EA dose-dependently reduced formalin-induced nociceptive behaviors during both phases. EA has been reported to be effective as an antinociceptive compound in several nociceptive assays. [11],[12],[21] Moreover, in our previous work, we demonstrated the participation of both central and peripheral antinociception induced by EA in several pain models, which was mediated through opioidergic and NO-cGMP pathways. [13]

Little information is available concerning the antinociceptive site of action of locally administered EA. The present data, however, do support a peripheral site of action though this needs to be investigated further. In fact, intraplantar injection of EA significantly reduced the formalin-induced nociceptive behavior. EA was injected intraplantarly at a concentration (50 μl of 60-300 mcg, which translates to approximately 0.4-2 mg/kg) much lower than the dose given systemically in other studies. [13],[21] This raises the possibility that local administration of EA at doses that do not produce systemic effects could be used to reduce nociception in localized areas without producing unwanted side effects that are observed following systemic use. In addition, local injection of EA or sildenafil into the contralateral hind paw did not yield antinociceptive effects on formalin-induced nociception, strongly supporting a local effect of EA and sildenafil on cutaneous nociceptors.

In certain cases, co-administering antinociceptive agents results in synergistic effects; therefore, the doses of each drug can be reduced. In the present study, sub-effective doses of sildenafil (25 and 50 mcg/paw) increased the activity of a sub-threshold dose of EA (60 mcg/paw). The effect of the combination was due to a local action, as the contralateral administration of the EA-sildenafil combination did not produce any effect. In addition, the results agree with previous reports on the action of selective (1-[3-chloroanilino]-4- phenylphthalazine, MY5445) and non-selective (caffeine) PDE inhibitors to increase the antinociception produced by some NSAIDs and morphine. [1] However, the present results demonstrate for the first time that an interaction between sildenafil and EA occurs at the local site, and hence involves a peripheral mechanism of action. Sildenafil increases cGMP level. On the other hand, previous observations by our group showed the ability of EA to induce antinociception through opioid receptors. [22] In addition, there is the evidence that EA can directly activate the NO-cGMP pathway in the periphery. [13] Therefore, it is assumed that this increase in cGMP levels produces the observed augmentation between sildenafil and EA.

In conclusion, intraplantar EA and sildenafil reduced the nociceptive response, which was assayed by the formalin test. In addition, the antinociceptive effect of EA was enhanced markedly by the combination of sildenafil. Taken together, the present results indicate that EA produces analgesia, partly at least, through the involvement of cGMP signaling. Nevertheless, further studies are needed to elucidate the detailed mechanisms of the peripheral antinociceptive action of EA. Data suggest that low doses of the EA-sildenafil combination can interact synergistically at local peripheral level and therefore, this drug association may represent a therapeutic advantage for the clinical treatment of inflammatory pain.


This study was supported by grants (PRC-115) from the Physiology Research Center, funded by the Vice Chancellor of Research, Ahvaz Jundishapur University of Medical Sciences (Iran).


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