Antinociceptive effects of hydroalcoholic extract from Euterpe oleracea Mart. (Açaí) in a rodent model of acute and neuropathic pain

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 ?2-adrenoceptor agonists
induces significant antinociceptive effects in the hot plate test in rodents 21]. Here, the ?2-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).

Fig. 2. Effect of ASE and acetylsalicylic acid on the formalin test. The bars represent mean?±?SEM
(n?=?10). *P??0.05 versus saline. ANOVA followed by Newman-Keuls test

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.

Fig. 3. Effect anti-hyperalgesic of ASE and acetylsalicylic acid on the carrageenan test.
The points represent the mean?±?SEM (n?=?10). *P??0.05 versus saline. Two-way ANOVA followed by Bonferroni post hoc test

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)-?, interleukin
(IL)-1? 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).

Fig. 4. Effect of ASE and indomethacin on the acetic acid-induced writhing test. The number
of writhing was evaluated during 30 minutes. The bars represent the mean?±?SEM (n?=?10). *P??0.05 versus saline. ANOVA followed by Newman-Keuls test

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-1?,
IL-8, and TNF-? 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).

Fig. 5. Antinociceptive effect of ASE or amitriptyline in SNL rats. The ASE and amitriptyline
were once daily administered by gavage during 7 days. a) Latency in response to thermal stimulation and b) Withdrawal threshold in response to mechanical stimulation applied to the paw of
rats submitted to SNL. The points represent the mean?±?SEM (n?=?4). *P??0.05 versus day 0;
^#P??0.05 versus day 7. Two-way ANOVA followed by Bonferroni post hoc test

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.