Hot plate test
Treatment with 30, 100, or 300 mg.kg-1 ASE dose-dependently increased the %MPE to 39.1 ± 10.0, 51.9 ± 9.5, or 94.7 ± 4.4 %, respectively (Fig. 1a, n = 10 per group, p < 0.05). The %MPE was also increased by tramadol (2 mg.kg−1) to 78.3 ± 10.3 % (n = 10, p < 0.05). Pre-treatment with i.p. administration of L-NAME (30 mg.kg−1), naloxone (1 mg.kg−1), yohimbine (5 mg.kg−1) or atropine (2 mg.kg−1) reduced the antinociceptive effect of ASE (100 mg.kg−1) from 51.9 ± 9.5 to 8.9 ± 3.3, 8.3 ± 3.3, 13.6 ± 4.1, or 26.2 ± 6.8 %, respectively (Fig. 1b, n = 10 per group, p < 0.05).
Nociception induced by thermal stimulation (hot plate test) is used to evaluate antinociceptive agents that act centrally but not peripherally [19]. This test involves various physiological systems, including cholinergic, adrenergic, opioid, and L-arginine/NO, which may be targets for antinociceptive compounds.
The importance of the sympathetic nervous system in pain modulation has been known since 1904, when Weber [20] demonstrated the antinociceptive effect of epinephrine injected in the spinal cord of a cat. Intrathecal or intraperitoneal administration of α2-adrenoceptor agonists induces significant antinociceptive effects in the hot plate test in rodents [21]. Here, the α2-adrenoceptor antagonist yohimbine inhibited the antinociceptive effect of ASE, supporting involvement of the adrenergic system on pain modulation, consistent with others flavonoids [22]. The antinociceptive effect of ASE is probably dependent on flavonoids content, because flavones [23], and quercetine [24] have similar effect in animals. Specifically, polymeric proanthocyanidins, which are common compounds in our extract, may underlie the antinociceptive effects, as seem with proanthocyanidins obtained from Croton celtidifolius bark [9].
Morphine is considered to be the gold standard drug for systemic pain treatment. However, prolonged use of morphine induces tolerance and hyperalgesia. In the present study naloxone, an opioid antagonist blocked the anti-nociceptive effects of ASE. Opioid mechanisms also modulate the antinociceptive effects of flavones compounds [23] and quercetin [22]. Muscarinic cholinergic receptors are present along the pain pathway from the dorsal root ganglia to somatosensory cortex [25], and muscarinic agonists have antinociceptive effects in rodents [18]. Inhibition of muscarinic receptors by atropine reduced, but did not abolish the antinociceptive effect of ASE. This finding suggests that cholinergic mechanisms may mediate these activities.
The L-arginine–nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway also modulate pain responses [26]. NO activates soluble guanylyl cyclase, leading to the production of cGMPn which activates cGMP-dependent protein kinase to open ATP-sensitive K+ channels, leading to neuronal hyperpolarization and spinal and peripheral antinociception [27]. In this study, the NO synthesis inhibitor L-NAME inhibited the antinociceptive effect of ASE. This inhibition demonstrates the involvement of the L-arginine-NO-pathway to the antinociceptive activities of ASE. Inhibition of NO synthesis antagonizes the activities of several antinociceptive compounds [28].
Taken together, these results indicate that ASE has an antinociceptive effect that is modulated by the cholinergic, adrenergic, opioid, and L-arginine-NO pathways. In addition, reactive oxygen species can enhance nociceptive responses [29], and ASE may block these responses via antioxidant activities and increasing NO-synthase to release NO [30].
Formalin-induced hind paw-licking test
The total amounts of time spent licking, scratching, or biting during the neurogenic and inflammatory phases after intraplantar injection of formalin were 73.1 ± 6.1 s and 207.8 ± 19.0 s, respectively (Fig. 2). Reactivity in the neurogenic phase was not affected by oral administration of the lowest doses of ASE (30 mg.kg−1) or acetylsalicylic acid (150 mg.kg−1), but was reduced by higher doses (100 and 300 mg.kg−1 ASE) to 45.6 ± 5.0 s and 36.4 ± 5.3 s, respectively (p < 0.05). Reactivity in the inflammatory phase was reduced by acetylsalicylic acid to 101.9 ± 14.9 s and by (30, 100, or 300 mg.kg−1 ASE) to 122.5 ± 14.5 s, 90.1 ± 15.2 s and 106.4 ± 11.0 s, respectively ( p < 0.05).
Intraplantar injection of formalin in rodents induces nociceptive-related behavior when assessed over two temporally distinct phases [13]. The first phase is induced by a direct activation of peripheral afferent C-fibers. The second phase is mediated by ongoing stimulation of nociceptors by inflammatory mediators (serotonin, histamine, bradykinin, NO, and prostaglandins) released from injured tissue, leading to activity-dependent sensitization of CNS neurons within the dorsal horn [31]. Local anesthetics and morphine inhibit the first phase whereas NSAIDs inhibit the second inflammatory phase. In this study, we found that ASE inhibited the first phase, probably due to interaction with CNS targets. ASE reduced reactivity in the second phase; this finding suggests that ASE has anti-inflammatory activities, perhaps via inhibition of cyclooxygenase 1 and 2 [4].
Carrageenan-induced pain test
Intraplantar administration of carrageenan reduced paw withdrawal latency to heat stimulation to 55.4 ± 5.7 % of control (Fig. 3). The effect of carrageenan was noted 5 min after administration, sustained for 150 min, and not affected by oral administration of ASE (30 mg.kg−1). However, higher doses of ASE (100 and 300 mg.kg−1) or acetylsalicylic acid (150 mg.kg−1) reduced the effect of carrageenan on paw withdrawal latency.
After carrageenan-induced inflammation, noxious stimuli elicit an enhanced pain response (hyperalgesia) [14]. This enhanced synaptic transmission is essential for central sensitization. ASE prevented the appearance of this sensitization, supporting its antinociceptive effects in inflammatory pain. Some flavonoids in açaí are modulate proinflammatory cytokine production [32]. Carrageenan stimulates the release of tissue necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, with subsequent increases in COX products and IL-8, to stimulate local production of sympathetic amines [33]. Therefore, ASE may block the cascade of cytokine release induced by carrageenan-induced sensitization to produce analgesia in inflammatory pain.
Acetic acid-induced writhing test
ASE at 100 and 300 mg.kg−1 dose-dependently reduced the number of abdominal contractions in response to acetic acid from 61.0 ± 4.8 (saline) to 44.5 ± 4.2 and 26.9 ± 2.5, respectively (p < 0.05). This effect was not significant at the lowest dose of ASE (30 mg.kg−1), which slightly reduced contractions to 50.5 ± 4.4. The reference drug indomethacin (2 mg.kg−1) reduced contractions to 34.4 ± 5.1 (Fig. 4).
The acetic acid-induced writhing test is a screening tool for assessment of antinociceptive and anti-inflammatory agents [34]. Intraperitoneal injection of acetic acid increases pain mediators, such as prostaglandins, lipoxygenase, cyclooxygenase, histamine, serotonin, bradykinin, substance P, IL-1β, IL-8, and TNF-α [34, 35], which increase vascular permeability and reduce the nociceptive threshold, causing stimulation of nociceptive terminals to induce abdominal writhing. The writhing response starts a few minutes after acetic acid injection. Reduction of this behavior is used to test the efficacy of drugs with visceral antinociceptive activity [36]. We measured the writhing response for 20 min starting 10 min after acetic acid injection to avoid counting stress reaction of the animal due to manipulation. We found similar writhing levels to other studies that measured the reaction for 30 min starting 5 min after acetic acid administration [16, 37]. Pre-treatment with ASE reduced the acetic acid-induced writhing response, suggesting reduced synthesis or release of pain modulators.
SNL-induced thermal hyperalgesia and mechanical allodynia
ASE (10, 30, or 100 mg.kg−1) dose-dependently prevented development of thermal hyperalgesia and mechanical allodynia in SNL rats on the ipsilateral side (Fig 5a and b), but no effect was observed on the contralateral side. At 7 days after surgery, the thermal withdrawal duration was reduced from 13.6 ± 0.5 s to 7.4 ± 0.9 s (n = 4). ASE had significant effects from day 1 to 7 of treatment, reaching 13.2 ± 0.4 s. Treatment with 10 or 30 mg.kg−1 ASE was as effective as 10 mg.kg−1 amitriptyline. The mechanical withdrawal threshold was reduced 7 days after surgery from 40.5 ± 0.6 g to 18.8 ± 1.0 g. After 7 days of treatment, ASE (100 mg.kg−1, n = 4) increased this threshold to 32.9 ± 3.2 g, similar to amitriptyline (10 mg.kg−1, n = 4). ASE had no effect on withdrawal duration or withdrawal threshold in the contralateral paw (Fig. 5).
Chronic pain with neuropathic features affects 7-8 % of the general population [38]. Unfortunately, current pharmacotherapies used to treat the main symptoms of this disorder, hyperalgesia and allodynia, are not completely effective. Oral administration of ASE over 7 days prevented the development of thermal hyperalgesia and mechanical allodynia in rats with SNL. Analgesic effects of ASE in this model were observed from 1 to 7 days after treatment with no signs of tolerance, which is a drawback of morphine [39]. Furthermore, side effects such as sedation were not observed after prolonged ASE treatment, providing an advantage over amitriptyline, which is sedative in humans [40]. A combination of the CNS and anti-inflammatory effects of ASE may underlie the antinociceptive effects in rats subjected to SNL.
Flavonoids such as the polyphenolic compounds rutin and quercetin have anti-inflammatory [41], analgesic [42], and antioxidant [43] effects. SNL is a neuropathic pain model used in rats that mimics the pain sensations experienced by human patients [44]. ASE had comparable efficacy to the clinical drug amitriptyline, in treating SNL-induced neuropathic pain. Others flavonoids can impact animal models of neuropathic pain. For example, Azevedo et al. [24] showed that rutin and quercetin prevented thermal and mechanical nociceptive responses in oxaliplatin-induced neuropathic pain in mice by mediating oxidative stress-induced damage.