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Print version ISSN 0102-695X
Rev. bras. farmacogn. vol.21 no.3 Curitiba May/June 2011 Epub Apr 20, 2011
Lucindo J. Quintans-JúniorI; Adriana G. GuimarãesI; Marilia T. de SantanaI; Bruno E.S. AraújoI; Flávia V. MoreiraI; Leonardo R. BonjardimI; Adriano A. S. AraújoI; Jullyana S. SiqueiraI; Ângelo R. AntoniolliI; Marco A. BotelhoII; Jackson R. G. S. AlmeidaIII; Márcio R. V. SantosI
IDepartamento de Fisiologia, Universidade Federal de Sergipe, Brazil
IILaboratório de Biotecnologia, Instituto Federal de Educação Ciência e Tecnologia do Ceará, Brazil
IIINúcleo de Estudos e Pesquisas de Plantas Medicinais, Universidade Federal do Vale do São Francisco, Brazil
Citral (CIT), which contains the chiral enantiomers, neral (cis) and geranial (trans), is the majority monoterpene from Lippia alba and Cymbopogon citratus. The present study aimed to evaluate CIT for antinociceptive and anti-inflammatory activities in rodents. Antinociceptive and anti-inflammatory effects were studied by measuring nociception through acetic acid and formalin tests, while inflammation was verified by inducing peritonitis and paw edema with carrageenan. All tested doses of CIT had significant protection (p<0.001) against acetic acid (0.8%) induced nociceptive behavior and the effects were also similar to morphine while formalin induced nociception was significantly protected (p<0.05) only at higher dose (200 mg/kg) of CIT in the first phase of the test. CIT significantly reduce (p<0.001) nociceptive behavior emanating from inflammation in second phase at all the doses.The pretreatment with CIT (100 and 200 mg/kg) significantly reduced the paw edema induced by carrageenan. Moreover, systemic treatment with CIT (100 and 200 mg/kg) significantly reduced (p<0.001) the leukocyte migration in the carrageenan-induced migration to the peritoneal cavity. Our investigation shows that CIT possess significant central and peripheral antinociceptive effects. It was also verified an anti-inflammatory activity. All together these results suggest that CIT might represent important tool for treatment of painful conditions.
Keywords: monoterpene citral antinociceptive anti-inflammatory
Treatments of some painful conditions, such as inflammatory and neurogenic pain, are often unsatisfactory because its etiology is heterogeneous and the underlying pathophysiology mechanisms are complex (Batista et al., 2010). Moreover, currently available drugs that provide relief from these painful conditions are effective only in a fraction of patients. In general, these drugs present low efficacy and numerous side effects (Basbaum, 1999; Mendell & Sahenk, 2003). In this context, natural products that present fewer side effects emerge as interesting therapeutic resources for the development of new drugs for the management of certain painful states (McCurdy & Scully, 2005; Batista et al., 2010).
An increasing number of studies have demonstrated that essential oils derived plant exhibit a variety of biological properties, such as analgesic (McCurdy & Scully, 2005), hypotensive (Menezes et al., 2010) and anticonvulsant activities (Quintans-Júnior et al., 2008a; Silva et al., 2010). Monoterpenes are the primary components of these essential oils and the effects of many medicinal herbs have been attributed to them (Lima-Accioly et al., 2006; Quintans-Júnior et al., 2008b), including the studies realized by our group (De Sousa et al., 2006; 2007; Melo et al., 2010; Quintans-Júnior et al., 2010; Guimarães et al., 2010).
In this regard, preliminary pharmacological screening realized with the essential oil of the Cymbopogon winterianus (rich in citronellal, citronellol and citral) demonstrates CNS depressant, anticonvulsant, hypotensive, vasorelaxat and antinociceptive activities in rodents (Quintans-Júnior et al., 2008a; Menezes et al., 2010; Leite et al., 2010). Gurgel do Vale et al. (2002) demonstrates that Lippia alba essential oil (rich in citral and myrcene) possess anxiolytic and depressant effects in experimental animals. In same study, authors showed that isolated compound (citral) possess CNS depressant activity in rodents. However, little is known about the possible antinociceptive and anti-inflammatory activities of citral (CIT).
Thus, the purpose of the present study was to evaluate both antinociceptive and anti-inflammatory activities of CIT in rodents.
Material and Methods
Acetic acid, carrageenan, diazepam, citral (minimum 95%, mixture of cis (1a) and trans (1b) isomers, 3,7-dimethyl-2,6-octadienal (1)), and polyoxyethylene-sorbitan monolate (Tween 80) were purchased from Sigma (USA). Morphine (MOR) and Indomethacin (INDO), were purchased from União Química Farmacêutica Nacional (Brazil). The citral (CIT) was administered intraperitoneally (i.p.) in volumes of 0.1 mL/10 g and the doses (50, 100 or 200 mg/kg) were adjusted for those respective volumes.
Male Swiss mice (31-36 g) and male Wistar rats (150-170 g), 2-3 months of age, were used throughout this study. The animals were randomly housed in appropriate cages at 21±2 ºC on a 12 h light/dark cycle (lights on 6:00-18:00 h) with free access to food and water. Experiments were carried out between 9:00 am and 14:00 pm in a quiet room. Nociceptive and inflammatory tests were carried out by the same visual observer and all efforts were made to minimize the number of animals used as well as any discomfort. Experimental protocols were approved by the Animal Care and Use Committee (CEPA/UFS # 26/09) at the Federal University of Sergipe and handling procedures were in accordance with International Association for the Study of Pain guidelines for the use of animals in pain research (Zimmermann, 1983).
Acetic acid-induced writhing
The acetic acid test was done using the method described by Koster et al. (1959) and modified by Gaertner et al. (1999). Initially, the mice were divided into five groups (n=8, per group). Subsequently, CIT (50, 100 or 200 mg/kg), vehicle (saline+Tween 80 0.2%), and MOR (5 mg/kg) were administered i.p. 30 min before an injection of 0.25 mL per animal of the acetic acid (0.8%). Each animal was isolated in an individual observation chamber and 5 min after acetic acid injection the cumulative number of writhing responses was recorded for 15 min.
Formalin induced nociception
The observation chamber was a glass box of 30 cm diameter on an acrylic transparent plate floor. Beneath the floor, a mirror was mounted at a 45º angle to allow clear observation of the paws of the mice. The animals were treated with the vehicle (saline+Tween 80 0.2%), CIT (50, 100, and 200 mg/kg, i.p.), or the reference drug (MOR, 5 mg/kg, i.p.) 30 min before the formalin injection. Each mouse was placed in the chamber more than 5 min before treatment in order to allow acclimatization to the new environment. The formalin test was carried out as described by Hunskaar & Hole (1987). Twenty microliters of a 1.0% formalin solution (0.92% formaldehyde) in a phosphate-buffer (pH 7.2) were injected into the dorsal surface of the left hind paw using a microsyringe with a 26-gauge needle. Each animal was then returned to the chamber and the amount of time that the animal spent licking the injected paw was considered to be indicative of pain. Two distinct phases of intensive licking activity were identified: an early acute phase and a late or tonic phase (0-5 and 15-30 min after formalin injection, respectively) (Le Bars et al., 2001).
Leukocyte migration to the peritoneal cavity
The leukocyte migration was induced by injection of carrageenan (1% 0.25 mL/cavity) into the peritoneal cavity of mice 30 min after administration of CIT (50, 100, and 200 mg/kg, i.p.) or dexamethasone (2 mg/kg, s.c., n=6 per group) by modification of the technique previously described by Bastos et al. (2007). The mice were euthanized by cervical dislocation 4 h after carrageenan injection. Shortly after, phosphate buffered saline (PBS) containing EDTA (1 mM, i.p., 10 mL) was injected. Immediately a brief massage was done for further fluid collection, which was centrifuged (2000 rpm, 5 min) at room temperature. The supernatant was disposed and 1 mL of PBS was introduced to the precipitate. An aliquot of 10 µL from this suspension was dissolved in 200 µL of Turk solution and the total cells were counted in a Neubauer chamber, under optic microscopy. The results were expressed as the number of leukocytes/mL. The percentage of the leukocyte inhibition =(1-T/C) x 100, where T represents the treated groups leukocyte counts and C represents the control group leukocyte counts.
Carrageenan-induced paw edema test
Acute hind paw edema was produced by injecting 0.1 mL of carrageenan (1%, prepared as a suspension in distillate water plus Tween-80 at 0.2%) locally into the subplantar aponeurosis of the right hind paw of rats (Winter et al., 1962). Animals were divided into five groups (n=6, per group). CIT (50, 100 or 200 mg/kg, i.p.), vehicle (saline+Tween 80 0.2%) or the standard drug indomethacin (INDO, 10 mg/kg, p.o.) was administered before the carrageenan-induced paw edema. CIT and INDO were administered 1 h prior to injection of carrageenan (Amresh et al., 2007). Right paw volume was measured by the dislocation of the water column of a plethysmograph (EFF-304, Insight®, Brazil) immediately after carrageenin application (time zero) and at 1, 2, 3, and 4 h after its administration.
The data obtained were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's test for antinociceptive and anti-inflammatory activities. In all cases differences were considered significant if p<0.05. The percentile of inhibition of edema volume between treated and control group (vehicle) was calculated using the following formula: Inhibition % = 100 x (Vc - Vt)/Vc. Where Vc and Vt represent mean increase in paw volume in control and treated groups, respectively.
Results and Discussion
Initially, antinociceptive activity of CIT was evaluated by using the writhing test. Intraperitoneal (i.p.) administration of CIT, in all doses, 30 min. before the acid injection, produced a significant (p<0.001) inhibition of acetic acid-induced abdominal constrictions in mice (Figure 1). MOR (5 mg/kg), a standard drug, used as positive control, also produced significant inhibition of acetic acid-induced writhing response.
Acetic acid-induced abdominal constriction is a standard, simple, and sensitive test for measuring analgesia induced by both opioids and peripherally acting analgesics (Hunskaar & Hole, 1987). In acetic acid-induced abdominal writhing, pain is elicited by the injection of an irritant such as acetic acid into the peritoneal cavity which produces episodes of characteristic stretching (writhing) movements, and inhibition of the number of episodes by analgesics is easily quantifiable. Moreover, it is already well documented that the quantification of prostaglandins by radioimmunoassay in the peritoneal exudates of rodents obtained after the i.p. administration of acetic acid demonstrated high levels of prostaglandins PGE2 and PGF2α during 0.5 h after stimulus (Deraedt et al., 1980). These results support the hypothesis of CIT participation in the inhibition of prostaglandin synthesis; the nociceptive mechanism involves the process or release of arachidonic acid metabolites via cyclooxygenase (COX) and prostaglandin biosynthesis (Duarte et al., 1988) during abdominal writhing induced by acetic acid. Although, this method presents a good sensitivity, it shows poor specificity (Le Bars et al., 2001).
Thus, to avoid misinterpretation of the results, in the present study we confirmed the antinociceptive effect of CIT in a model of inflammatory nociception, the formalin test, which has two distinctive phases that can possibly indicate different types of nociception (Hunskaar & Hole, 1987). CIT produces antinociceptive effects on both first (0-5 min) and second phases (15-30 min) of formalin induced nociception. The early phase, named neurogenic nociception, is a result of direct stimulation of nociceptors and reflects centrally mediated nociception; the late phase, named inflammatory nociception, is caused by local inflammation with a release of inflammatory and hyperalgesic mediators (Hunskaar & Hole, 1987). First phase was blocked only at 200 mg/kg (p<0.001) whereas the higher doses of CIT (100 and 200 mg/kg) significantly (p<0.001) blocked the inflammatory nociception. CIT was found to inhibit the inflammation induced nociception better than the neurogenic nociception. MOR (5 mg/kg) was significantly active (p<0.001) in the both phases of in formalin test (Figure 2).
In this regards, Waning et al. (2007) demonstrated that the capsaicin-sensitive transient receptor potential vaniloid 1 (TRPV1), which plays an important role in pain transduction, is one of the Ca+2 influx channels involved in cell migration. It was also shown that TRPV1 activation increases the expression of the TNF receptor 1 (TNFR1) by a ROS-dependent mechanism (Ma et al., 2009). According to Stotz et al. (2008) partial TRPV1 agonists, such as CIT, might be useful in anti-inflammatory and analgesic compounds.
Since in previous nociception tests, CIT showed better action in inflammatory pain, we verified the effects of CIT in tests of acute inflammation. Initially, we performed the carrageenan-induced leukocytes migration to the peritoneal cavity. Figure 3 shows the inhibitory effect of CIT (higher doses) on leukocytes migration after 4 h of the stimulus (p<0.001). Standard drug, dexamethasone (Dexa, 2 mg/kg, s.c.) significantly inhibited (p<0.001) the carrageenan-induced leukocytes migration to the peritoneal cavity.
Cell recruitment during inflammation depends on the orchestrated release of local mediators that are responsible for local vascular and tissue changes as well as for the recruitment of host defense cells (Luster et al., 2005). The inflammation induced by carrageenan involves cell migration, plasma exsudation and production of mediators, such as nitric oxide, prostaglandin E2, interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α (Salvemini et al., 1996; Loram et al., 2007). Those mediators are able to recruit leukocytes, such as neutrophils, in several experimental models. CIT inhibited leukocyte migration induced by i.p. injection of carrageenan (in peritonitis model) in higher doses. A putative mechanism associated with this activity may be inhibition of the synthesis of many inflammatory mediators whose involvement in the cell migration is well established. In this regards, Katsukawa et al. (2010) identified CIT as a suppressor of COX-2 and an activator of peroxisome proliferator-activated receptors (PPAR) α and γ;. Moreover, Lee et al. (2008) showed that CIT inhibits NO generation through suppression of iNOS expression via inhibition of NF-κB activation.
Besides, an additionally test was performed to verify the effects of CIT in the inflammation, by using carrageenan-induced paw edema test. The initial phase of carrageenan paw edema is mediated by histamine and serotonin, while the mediators in the later phase were suggested to be arachidonate metabolites (prostaglandins and leukotrienes) producing an edema dependent on the mobilization of neutrophils (Vinegar et al., 1987; Hwang et al.; 1996). Moreover, it should be taken into consideration that the mechanism involved in the genesis of the carrageenan-induced edema can cause the release of prostaglandins and kinins, among other substances (Garcia-Leme et al., 1973). In our experiments the edematous response was significantly suppressed in rats pre-treated with the CIT in the first phase of the edema, suggesting an inhibitory effect on the release of histamine and/or serotonin. In addition, CIT showed a significant inhibition of the edema in the second and third phase of edema, suggesting an inhibition of 5-lipoxygenase and/or cyclooxygenase, both enzymes involved in the formation of prostaglandins and leukotrienes. This edematous response was also significantly reduced in rats pre-treated with indomethacin, a compound known to be a cyclooxygenase inhibitor.
It can be concluded that CIT is endowed with peripheral antinociceptive property as well as anti-inflammatory activity. The precise mechanisms through which CIT exerts its action are currently under investigation, but possibly it could be related to the arachidonic acid cascade and/or modulation of pro-inflammatory molecules production. Therefore, this compound will be of potential benefit in the management of inflammatory pain conditions.
We thank Mr Osvaldo A. Santos for the technical support. This work was supported by grants from the Research Supporting Foundation of the State of Sergipe (Fundação de Apoio à Pesquisa e à Inovação Tecnológica do Estado de Sergipe) [grant number 019.203.00860/2009-6].
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Received 21 Aug 2010
Accepted 25 Oct 2010
Lucindo J. Quintans-Júnior
Departamento de Fisiologia, Universidade Federal de Sergipe
Campus Universitário "Prof. Aloísio de Campos"
Av. Marechal Rondon, s/n, Jardim Rosa Elze
49.000-100 São Cristóvão-SE, Brazil
Tel/Fax: +55 79 2105 6645; +55 79 2105 6474