Antinoceptive effect of triterpenoid [alpha],[beta]-amyrin in rats on orofacial pain induced by formalin and capsaicin.

Abstract

The effects of [alpha],[beta]-amyrin, a pentacyclic triterpene isolated from Protium heptaphylum was investigated on rat model of orofacial pain induced by formalin or capsaicin. Rats were pretreated with [alpha],[beta]-amyrin (10, 30, and l00mg/kg, i.p.), morphine (5 mg/kg, s.c.) or vehicle (3% Tween 80), before formalin (20 [micro]l, 1.5%) or capsaicin (20 [micro]l, 1.5 [micro]g) injection into the right vibrissa. In vehicle-treated controls, formalin induced a biphasic nociceptive face-rubbing behavioral response with an early first phase (0-5 min) and a late second phase (10-20 min) appearance, whereas capsaicin produced an immediate face-rubbing (grooming) behavior that was maximal at 10-20 min. Treatment with [alpha,[beta]-amyrin or morphine significantly inhibited the face-rubbing response in both test models. While morphine produced significant antinociception in both phases of formalin test, [alpha],[beta]-amyrin inhibited only the second phase response, more prominently at 30mg/kg, in a naloxone-sensitive manner. In contrast, [alpha],[beta]-amyrin produced much greater antinociceptive effect at l00mg/kg in the capsaicin test, which was also naloxone-sensitive. These results provide first time evidence to show that [alpha],[beta]-amyrin attenuates orofacial pain atleast, in part, through a peripheral opioid mechanism but warrants further detailed study for its utility in painful orofacial pathologies.

[C] 2007 Published by Elsevier GmbH.

Keywords: Protium heptaphyllum; [alpha],[beta]-Amyrin; Antinociception; Orofacial pain; Formalin; Capsaicin

Introduction

Orofacial pain is a common complaint patients seek medical or dental care. Orofacial pain is frequent with a high prevalence in temporomandibular and dental disorders, malignant diseases, and in chemo-radiotherapy-surgery-associated mucositis and neurotoxicity (Benoliel et al., 2007; Epstein et al., 2007). A wide variety of drugs are indicated for treating orofacial pain, which include NSAIDs, muscle relaxants, opioids and antidepressants but side effects and toxicity are apparent with long-term administration (Dionne, 2001). In recent years, there has been a constant search for alternative drugs that possess greater efficacy and safety in reducing the inflammatory and neuropathic pain and with a strategy to halt the transition from acute to pain chronicity.

Protium heptaphyllum (Aubl.) March., commonly known as almecega, is a widely distributed plant in the Amazon and in Northeast of Brazil. The resin collected from its trunk wood is an effective healing agent of wounds with anti-inflammatory and analgesic properties (Siani et al., 1999). The major component of the resin is the pentacyclic triterpene [alpha],[beta]-amyrin (an isomeric mixture) (Fig. 1), and previous studies described its antimicrobial, anti-inflammatory, gastroprotective and antinociceptive properties (Katerere et al., 2003; Oliveira et al., 2005; Lima-Junior et al., 2007). By virtue of these properties, it is hoped that [alpha],[beta]-amyrin might be an ideal agent to combat orofacial pain and to prove it, the present study investigated its antinociceptive potential in the test models of orofacial pain induced by formalin and capsaicin in rats.

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Materials and methods

Animals

Experiments were performed on male Wistarrats (160-200 g), housed in standard conditions (12-h light/dark cycle and 22[+ or -]2[degrees]C) and had free access to food and water except during the test period. All behavioral tests were carried out between 11:00 and 16:00 h. Animal experiments were conducted in accordance with the guidelines of International Association for the study of pain and the Institutional Animal Care and Use Committee of the Federal University of Ceara approved the experimental protocols.

Plant material and isolation of [alpha],[beta]-Amyrin

The resinous exudate from the trunk wood of Protium heptaphyllum (March.) was collected from the municipal areas of Timon, Maranhao state of Brazil, after its identification by botanist Prof. Roseli Farias de Melo Barros. A voucher sample (#18247) has been deposited at the Herbarium Graziela Barroso of the Federal University of Piaui, Teresina, Brazil. The extraction and isolation of [alpha],[beta]-amyrin (1) from the crude resin was carried out as described earlier (Vieira-Junior et al., 2005) and its structural identity was confirmed by [1.sup.H]- and [13.sup.C]-NMR spectral analysis, based on the method developed by Gallegos and Roque (1990) and in comparison with literature data (Mahato and Sen, 1997).

Formalin test

Orofacial nociception was induced in rats by s.c. injection of 20 [micro]l of 1.5% formalin into the right vibrissa pad, using a 27-gauze needle (Clavelou et al., 1995). This volume and percentage concentration of formalin was selected from our pilot studies that showed a pain-related biphasic behavioral response (face-rubbing) of greater intensity at the periods of 0-5 min (first phase) and 10-15 min (second phase), following its injection into vibrissa pad. Pain was quantified at these time periods by measuring the time (s) that the animals spent face-rubbing in the injected area with its fore- or hindpaws. To assess the effects of test drugs, rats in groups (n = 8) were pretreated systemically with vehicle (3% Tween 80 in distilled water, the solvent for [alpha],[beta]-amyrin), [alpha],[beta]-amyrin (10, 30, and l00mg/kg, i.p.), 2h before the local injection of formalin. Morphine (5 mg/kg, s.c.), administered 30 min before the algogen was included as positive control. In separate experiments, the possible involvement of opioid mechanism was assessed in the antinociception produced by [alpha],[beta]-amyrin or morphine with naloxone (2mg/kg, i.p.), an opioid antagonist, injected simultaneously.

Capsaicin test

The orofacial pain was induced by capsaicin in rats as described earlier (Pellisier et al., 2002). Rats (n = 8) were injected with capsaicin (20 [micro]l, 1.5 [micro]g) subcutaneously into the right vibrissa pad, using a 27 gauze needle. Capsaicin was dissolved in ethanol, dimethyl sulfoxide and distilled water (1:1:8). In pilot studies, rats manifested pain-related face-rubbing/grooming behavior immediately following the injection of capsaicin with a higher intensity at 10-20 min period. Therefore, pain quantification was performed at this time period by measuring the time (s) that the animals spent face-rubbing/grooming the injected area with the fore- or hindpaws. The [alpha],[beta]-amyrin or vehicle were given to animals as described for formalin test, 2h before the local injection of capsaicin. Morphine (5mg/kg, s.c), administered 30 min before the algogen was included as a positive control. To test the possible involvement of opioid mechanism in the antinociception produced by [alpha],[beta]-amyrin or morphine, animals were simultaneously pretreated with the opioid antagonist, naloxone (2mg/kg, i.p.).

Statistical analysis

The results were presented as the mean [+ or -] SEM, and were analyzed using one-way analysis of variance (ANOVA) followed by Student Newman-Keuls post-hoc test for multiple comparisons between individual groups. Differences were considered significant at p < 0.05.

Results and discussion

The results show that pretreatment with [alpha], [beta]-amyrin produces pronounced antinociception as evidenced by decreased face rubbing behavior in capsaicin test and in the second phase of formalin test (Figs. 2 and 3). Algesic effects in both models are said to be due to liberation of mediator substances like sensory neuropeptides and prostaglandins (Henry et al., 1999; Sandor et al., 2007). In formalin-induced orofacial pain, the first phase response (0-5 min) corresponds to acute neurogenic pain sensitive to drugs that interact with opioid system and the second phase response (10-20 min) corresponds to inflammatory pain that can be inhibited by cyclooxygenase inhibitors. Drugs that act primarily as central analgesics could inhibit both phases while peripherally acting drugs inhibit only the second phase algesia (Roseland et al., 1990).

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It was observed that [alpha], [beta]-amyrin was unable to suppress the first phase nociceptive behavior in formalin test (Fig. 2A), while the second phase was inhibited possibly acting via suppression of arachidonate cascade. Consistent with this finding was the observation that systemic administration of ketorolac or diclofenac (10 and 30 mg/kg, i.p.) significantly attenuates formalin-induced nociceptive behaviour in the second phase only (Padi et al., 2006). These results indicate that Prostaglandins (PGs), particularly peripheral PGs are involved in nociceptive behaviour following formalin injection into the vibrissa pad.

The opioids can produce analgesia through peripheral mechanisms in formalin test (Stein et al., 2001), consistent with the observations of Shannon and Lutz (2002) with subcutaneous loperamide, a peripherally acting [mu]-opioid agonist. Peripheral opioid analgesia is undoubtedly of clinical relevance, especially considering that systemic opioid therapy often is hampered by central side effects. [alpha], [beta]-amyrin lacks analgesic action in tests of tail-flick and hot-plate suggesting no central analgesia (Otuki et al., 2005), but interestingly, it displays peripheral analgesia by a naloxone-sensitive mechanism (Fig. 2B) suggesting that it might even have a direct action on the opioid receptor, which needs to be clarified in a future study.

[alpha], [beta]-amyrin inhibits the neurogenic inflammatory pain induced by capsaicin injection into the vibrissa pad, as evidenced by naloxone-reversible suppression of facial grooming (Fig. 3). The inhibitory effect observed with [alpha], [beta]-amyrin on capsaicin- and in the second phase of formalin-induced facial grooming may be a result of its possible inhibition on Substance P release or due to a direct blocking action on its receptor neurokinin-1 (NK-1). In this context, a previous study provided evidence for tonic activation of NK-1 receptors during the second phase of the formalin test in the rat and systemically administered NK-1 receptor antagonist SR14033 blocks only the second phase of the orofacial formalin test (Henry et al., 1999; Luccarini et al., 2003). In a more recent study, we have shown the decreased NK-1 immunoreactivity in animals treated with [alpha], [beta]-amyrin in the rat model of visceral nociception induced by cyclophosphamide (Lima-Junior et al., 2007). At the doses tested, [alpha], [beta]-amyrin lacks overt toxicity and no sedative or motor impairment effects in rodent species (Lima-Junior et al., 2007).

In conclusion, this study demonstrates that [alpha], [beta]-amyrin modulates neuropathic and inflammatory pain in the test models of orofacial pain induced by capsaicin and formalin through a naloxone-senitive mechanism. The results also support the clinical use of peripheral opioids to combat orofacial pain and further suggest that [alpha], [beta]-amyrin has a therapeutic potential for painful facial and dental disorders.

Acknowledgements

This work was supported by grants from CNPq and CAPES, Brazil. We thank Mrs. Dannyella Marques Ferreira for the technical assistance.

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S.A. Holanda Pinto (a), L.M.S. Pinto (a), M.A. Guedes (a), G.M.A. Cunha (a), M.H. Chaves (b), F.A. Santos (a), V.S. Rao (a), (*)

(a) Department of Physiology and Pharmacology, Post-Graduation Programme in Medical Sciences, Faculty of Medicine, Federal. University of Ceara, POB 3157, Rua Cel Nunes de Melo-1127, Porangabussu, 60430-270 Fortaleza, Ceara, Brazil (b) Department of Organic and Inorganic Chemistry, Federal University of Piaui, Teresina, Piaui, Brazil

* Corresponding author. Tel.: [phone]; fax: [phone].

E-mail addresses: [e-mail], viet [e-mail] (V.S. Rao).