The assessment of the antinociceptive property and redox profile of-cymene and two other monoterpenes, namely (+)-camphene and geranyl acetate, revealed that-cymene possessed the strongest antinociceptive action (50, 100 and 200 mg/kg, i.p.) while (+)-camphene and geranyl acetate (200 mg/kg) displayed a moderate analgesic effect in male Swiss mice tested in the acetic acid-induced writhing and formalin models [ 31 ]. In contrast, (+)-camphene exhibited the most relevant antioxidant effect in vitro detected by two specific assays: the thiobarbituric acid-reactive species (TBARS)—an assay employed to quantify lipid peroxidation [ 32 ]—and the total reactive antioxidant potential (TRAP)/total antioxidante reactivity (TAR)—an assay employed to estimate the nonenzymatic antioxidant capacity of samples [ 33 ]. It also showed the highest scavenging activity against different free radicals, including hydroxyl and superoxide radicals [ 31 ].

Other studies have provided further evidence of the antinociceptive and anti-inflammatory properties of-cymene and the possible role of the opioid system and cytokines in these responses. The antinociceptive effect of-cymene (25–100 mg/kg) on male Swiss mice was demonstrated in the tail flick test, showing increased dose-dependent reaction time, an effect that lasted for five hours and was antagonized by naloxone and by δ, κ and µ-opioid receptor antagonists naltrindole, nor-binaltorphimine (Nor-BNI) and CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide), respectively [ 25 ]. In the assessment of the anti-inflammatory activity, treatment with-cymene (25, 50 or 100 mg/kg, i.p.) decreased mechanical hyperalgesia induced by CG, TNF-α, PGE2, and dopamine. In the CG-induced pleurisy test,-cymene reduced leukocyte (100 mg/kg) and neutrophils (50 and 100 mg/kg) migration to the pleural cavity, and decreased the levels of TNF-α in pleural exudates (25, 50 and 100 mg/kg). Neutrophils, in particular, play an important role at the onset of inflammatory hypernociception by secreting pro-inflammatory cytokines (e.g., TNF-α and IL-1β) and mediators such as prostaglandins [ 26 27 ].-Cymene was also shown to diminish nitric oxide (NO) production in murine macrohages incubated with lipopolysaccharide (LPS) (25, 50 and 100 µg/mL), a component known to stimulate toll-like receptor 4 (TLR-4), leading to the activation of the transcription factor NF-κB [ 25 ]. NF-κB contributes to the production of inflammatory (pro-nociceptive) molecules and enhances inducible nitric oxide synthase (iNOS) activity, thereby increasing NO production [ 28 ], which is believed to act as a mediator of inflammation and to sustain hyperalgesia after CG injection [ 29 ]. Furthermore,-cymene significantly enhanced the c-Fos immunoreactive neurons in the periaqueductal gray [ 25 ], a midbrain region activated by opioid agonists and involved with pain modulation [ 30 ]. Together these findings indicate an anti-inflammatory and antinociceptive action of-cymene and suggest the involvement of descending pain suppression mechanisms since its antinociceptive role through the opioid system was increased by the activation of the periaqueductal gray [ 25 ].

A study developed by Bonjardim and collaborators [ 19 ] revealed that, in the acetic acid-induced writhing and formalin tests, exposure of male Swiss mice to-cymene (50 or 100 mg/kg, i.p.) significantly decreased the number of writhes and the licking time in the first and second phase of the formalin test. In the hot plate test, it increased the latency time of the licking and jumping behavior to thermal stimulus. Additionally,-cymene (25, 50 or 100 mg/kg) produced an anti-inflammatory reaction induced by carrageenan (CG), which led to a marked reduction in leukocyte migration. In mice, intraplantar injection of CG leads to hypernociception and an inflammatory response that involves the release of cytokines by resident or migrating cells initiated by the production of bradykinin [ 20 ]. This is followed by the secretion of prostanoids and sympathomimetic amines, such as dopamine [ 21 ], which stimulate Aδ and C fiber nerve terminals and the release of substance P and neurokinin A, accentuating local blood flow and vascular permeability [ 22 ]. In CG-induced hypernociception, the release of tumor necrosis fator α (TNF-α) and keratinocyte-derived chemokine (KC), for exemple, is accompanied by the secretion of interleukin 1β (IL-1β) [ 23 ] with the subsequent induction of cyclooxigenase-2 (COX-2) expression and the production of protanoids, such as prostagladin E2 (PGE2) [ 24 ].

The aromatic monocyclic monoterepene-cymene [1-methyl-4-(1-methylethyl) benzene] is the biological precursor of carvacrol and is abundantly found in essential oils from many plant species such as(Aubl.) Marchand (Burseraceae) [ 15 ] and(L.) Poit. (Lamiaceae) [ 16 ]. It also occurs naturally in a wide variety of foods, including orange juice, carrots, tangerine, butter and oregano [ 17 ]. The antinociceptive and anti-inflammatory activities of-cymene have been evaluated by different behavioral tests of nociception in rodents, which showed that this monoterpene exerted both peripheral and central antinociceptive action. For instance, the antinociceptive effect of-cymene was demonstrated on an orofacial nociceptive response model through tests involving the subcutaneous administration of formalin, capsaicin, and glutamate into the upper lip of-cymene-pretreated male Swiss mice (25, 50 or 100 mg/kg, i.p.).-Cymene markedly decreased the rubbing behavior induced by all three components; an effect counteracted by nonselective opioid receptor antagonist naloxone, indicating the participation of the opioid system in the antinociceptive response [ 18 ].

The antinociceptive action of carvacrol was further corroborated by a study developed by Luo and colloborators [ 44 ] in the assessment of its activity on glutamatergic spontaneous excitatory transmission in substantia gelatinosa neurons of the spinal dorsal horn, a region believed to modulate nociceptive transmission from the peripheral to the central nervous system [ 44 45 ]. By the use of the patch-clamp method in adult rat spinal cord slices, it was verified that exposure to carvacrol increased the secretion of-glutamate from nerve terminals by activating transient receptor potential cation channels, subfamily A, member 1 (TRPA1), and produced membrane hyperpolarization; an effect that could be contributing to its anti-inflammatory action. Several studies have recognized TRP as important analgesic targets in inflammatory and neurophatic pains [ 46 ]. Another contribution was given by Joca and collaborators [ 47 ] that examined possible mechanisms involved in the effects of carvacrol on the peripheral nervous system. Carvacrol reversibly and dose-dependently suppressed the excitability of the rat sciatic nerve (ICvalue of 0.50 ± 0.04 mM), and prevented the generation of action potentials (IC0.36 ± 0.14 mM) of the intact dorsal root ganglion (DRG) neurons without altering the resting potential and input resistance. Carvacrol also suppressed neuronal excitability by a direct inhibition of the voltage gated sodium current of dissociated DRG neurons (IC0.37 ± 0.05 mM), suggesting a local anesthetic effect of this compound.

In a study by Guimarães and collaborators [ 42 ], the role of carvacrol in the attenuation of mechanical hypernociception and inflammation was investigated in models of hypernociception induced by CG, TNF-α, PGE2 and dopamine, and in models of CG-induced pleurisy, paw edema, and LPS-induced nitrite production in murine macrophages. The administration of carvacrol (50 or 100 mg/kg, i.p.) to male Swiss mice significantly suppressed mechanical hypernociception and paw edema induced by CG and TNF-α (but not PGE2 and dopamine), and markedly reduced TNF-α levels in pleural lavage, blocked leukocytes recruitment, and decreased LPS-induced nitrite production in vitro (carvacrol: 1, 10 or 100 µg/mL). Additionally, Guimarães and collaborators [ 43 ] also demonstrated the antinociceptive effect of carvacrol in the formalin-, capsaicin-, and glutamate-induced orofacial nociception tests in which male Swiss mice exhibited reduced face-rubbing behavior in both phases of the formalin test, and nociception induced by capsaicin and glutamate (carvacrol-25, 50 or 100 mg/kg, i.p.).

Carvacrol (5-isopropyl-2-methylphenol) is a phenolic monoterpene found in essential oils of plants from the generaand(Lamiaceae) [ 34 35 ]. Its phamacological properties include acetylcholinesterase inhibition [ 36 ], and anticonvulsive [ 37 ], anxiolytic [ 38 ], and antinociceptive [ 39 ] action. The antinociceptive activity of carvacrol was demonstrated in male Swiss mice tested in animal models of pain (acetic acid-induced writhing, formalin and hot plate). The data obtained after oral treatment with single doses of carvacrol showed a decrease in the number of constrictions (50, 100 and 200 mg/kg), and the paw-licking time (50 mg/kg, first phase of the formalin test; 100 mg/kg, first and second phases), and an increase in the reaction time at 60 min (50 and 100 mg/kg) in the hot plate test. These effects were not reversed by naloxone and-arginine, suggesting that the antinociceptive action of carvacrol may not be related to the opioid system [ 40 ]. On the other hand, the antinociceptive activity of carvacrol was associated with the inhibition of prostaglandin synthesis [ 39 ] as it possesses an effective ability to suppress COX-2 expression and to activate the peroxisome proliferator-activated receptors (PPAR) α and γ [ 41 ].

2.3. Linalool

50,51,52, Ocimum basilicum L. (Lamiaceae) leaf, on orofacial nociception were addressed in formalin, glutamate and capsaicin tests and in an electrophysiological protocol, which involved the evaluation of the neuronal excitability of the hippocampal dentate gyrus. (−)-Linalool (50, 100 and 200 mg/kg, i.p.) administered to male Swiss mice effectively inhibited the nocifensive face-rubbing behavior in the first and second phase of the formalin test. At high doses, it also reduced nociceptive behavior in neurogenic inflammatory nociception induced by capsaicin and glutamate injection in the perinasal area (right upper lip) [ O. basilicum leaf essential oil, indicating that both the oil and (−)-linalool display modulatory action on neurogenic and inflammatory pain, and that the antinociceptive effect could be related to reduced peripheral and central nerve excitability [ (−)-Linalool is an enantiomer monoterpene present in essential oils of various aromatic plants, such as lavender, rosewood and bergamot [ 48 ], and possesses several pharmacological activities including anti-inflammatory, anxiolytic, anticonvulsant and antinociceptive [ 49 53 ]. The effects of (−)-linalool, extracted from the essential oil ofL. (Lamiaceae) leaf, on orofacial nociception were addressed in formalin, glutamate and capsaicin tests and in an electrophysiological protocol, which involved the evaluation of the neuronal excitability of the hippocampal dentate gyrus. (−)-Linalool (50, 100 and 200 mg/kg, i.p.) administered to male Swiss mice effectively inhibited the nocifensive face-rubbing behavior in the first and second phase of the formalin test. At high doses, it also reduced nociceptive behavior in neurogenic inflammatory nociception induced by capsaicin and glutamate injection in the perinasal area (right upper lip) [ 53 ]. It is believed that these effects are related to possible inhibition of substance P release or blocking effect on its receptor neurokinin-1 (NK-1) [ 54 ]. In addition, the electrophysiological analysis revealed that (−)-linalool inhibited the field potentials activated by the antidromic stimulation of the hylus, suggesting that this compound affects the activation of the voltage-dependent sodium channels present in the granular neurons of the hippocampal dentate gyrus [ 53 55 ]. Similar results were observed with theleaf essential oil, indicating that both the oil and (−)-linalool display modulatory action on neurogenic and inflammatory pain, and that the antinociceptive effect could be related to reduced peripheral and central nerve excitability [ 53 ].

The antinociceptive activity of (±)-linalool was evidenced in the paclitaxel-induced acute pain model in male ddY-strain mice. Intraplantar injection of (±)-linalool (5 and 10 µg/paw) effectively and dose-dependently suppressed behavioral responses of paclitaxel-induced mechanical allodynia and hyperalgesia. (±)-Linalool injected into the ipsilateral paw produced antiallodynia and antihyperalgesia effects whereas no such action was detected in the linalool-injected contralateral paw, suggesting that the effects exerted by this monoterpene may be mediated locally rather than systemically. Moreover, (±)-linalool’s effects were reversed by local (paw plantar surface) administration of naloxone hydrochloride (opioid antagonist) and by naloxone methiodide (peripherally acting opioid receptor antagonist), indicating that (±)-linalool’s peripheral antiallodynia and antihyperalgesia activities could partly involve peripheral opiod mechanisms [ 48 ].

Citrus bergamia (Risso, Rutaceae) is a rich source of linalool. The investigation of their effects on neurophatic hypersensitivity induced by partial sciatic nerve ligation (PSNL) in male ddY-strain mice showed that intraplantar injection of these components into the ipsilateral hindpaw decreased PSNL-induced mechanical allodynia dose-dependently whereas no antinociceptive activity was observed after intraplantar injection into the contralateral hindpaw, further suggesting a local effect of linalool and also of bergamot essential oil [60, Bergamot essential oil extracted from(Risso, Rutaceae) is a rich source of linalool. The investigation of their effects on neurophatic hypersensitivity induced by partial sciatic nerve ligation (PSNL) in male ddY-strain mice showed that intraplantar injection of these components into the ipsilateral hindpaw decreased PSNL-induced mechanical allodynia dose-dependently whereas no antinociceptive activity was observed after intraplantar injection into the contralateral hindpaw, further suggesting a local effect of linalool and also of bergamot essential oil [ 56 ]. The possible involvement of spinal extracellular signal-regulated protein kinase (ERK) in bergamot essential oil and linalool-induced antimechanical nociception indicates that the attenuation of the observed effects entailed inhibition of spinal ERK phosphorylation since intraplantar injection of bergamot essential oil or linalool effectively blocked spinal ERK activation induced by PSNL [ 56 ]. The activation of ERK has been demonstrated in dorsal horn neurons in persistent CG and Freund’s adjuvant-induced inflammatory hyperalgesia [ 57 58 ]. Previous studies have shown that injection of capsaicin into the hindpaw produced ERK activation in the spinal cord, while blockade of spinal ERK1/2 activity via i.t. injection of MEK inhibitor U0126 decreased nocifensive responses induced by formalin, capsaicin, CG or complete Freund’s adjuvant [ 59 61 ].

Corroboration of the local action of bergamot essential oil and linalool was provided by Katsuyama and collaborators [ 62 ]. The nocifensive response to formalin (licking and biting) was considerably decreased in both phases of the formalin test following intraplantar administration of bergamot essential oil or linalool into the ipsilateral, but not the contralateral, hindpaw of male ddY-strain mice. These findings show the peripheral antinociceptive action of both compounds, which was antagonized by intraplantar and i.p. injection of naloxone hydrochloride and naloxone methiodide, and confirm previous reports that suggest the involvement of peripheral opioid receptors in antinociception induced by bergamot essential oil and linalool [ 62 ].

Boswellia carterii is commonly used for topical treatment of pain and inflammation [ In traditional Chinese medicine, frankincense fromis commonly used for topical treatment of pain and inflammation [ 63 ]. A study carried out to investigate the antinociceptive and anti-inflammatory action of frankincense oil and water extracts and three of its main componentes, i.e., linalool, α-pinene and 1-octanol, via xylene-induced ear edema and a formalin-inflamed hindpaw model in male Kunming mice, showed consistent evidence about their anti-inflammatory and analgesic effects. Frankincense oil extract, which contains more linalool, α-pinene and 1-octanol than frankincense water extract, produced a faster and more effective reduction of the swelling and pain than the water extract. In addition, the combination of linalool, α-pinene and 1-octanol exhibited stronger biological effect on hindpaw inflammation and COX-2 overexpression than the three compounds used separately, indicating that they contribute to the topical antinociceptive and anti-inflammatory properties of frankincense by inhibiting COX-2 activation [ 64 ].

A study by Tashiro and collaborators [ 65 ] reported the antinociceptive effect of linalool in a different experimental protocol using vapour exposure mediated by hypothalamic orexin neurons, one of the main mediators in the behavioral responses to pain [ 66 ]. The involvement of these cells was evidenced by a significant increase in the number of c-Fos-expressing orexin neurons, and in linalool odour-exposed and odourless air-exposed orexin neuron-ablated mice that exhibited similar pain behavior in the first and second phase of the formalin test. The confirmation of the contribution of orexinergic transmission was shown in orexin peptide-deficient mice exposed to linalool vapour in which linalool failed to evoke antinociceptive effects after formalin-induced insult, suggesting the participation of orexinergic transmission in linalool odour-induced antinociceptive response. Moreover, linalool odour exposure significantly decreased pain response in both phases of the formalin test in mice (wild type: C57BL16) while, in the hot plate test, it increased the latency of hindpaw withdrawal when compared with the odourless air control following an injurious heat stimulus. In the investigation of the participation of olfactory processing in linalool analgesic effects by chemical nociceptive stimulus (formalin test), pain behavior in olfactory bulbectomized mice under linalool vapour exposure did not differ markedly from the odourless air group in both phases of the test. In the anosmic model using mice with a nonfunctional olfactory epithelium, no effects of linalool vapour were observed, providing further evidence that the olfactory response produced by linalool vapour may play a key role in inducing analgesic effects [ 65 ].

Despite the biological properties of (−)-linalool, its use in the treatment of painful and inflammatory disorders is still limited due to poor oral availability [ 67 68 ]. A comparative study using experimental pain models (i.e., acetic acid-induced writhing, formalin and hot plate) in male Swiss mice examined the antinociceptive effect of (−)-linalool and β-cyclodextrin (β-CD) complexed (−)-linalool (20 or 40 mg/kg, p.o.). Both compounds effectively reduced the nocifensive response in all chemical and heat-induced tests, suggesting the involvement of peripheral and central antinociceptive mechanisms. In the writhing test, the antinociceptive effects were antagonized by naloxone, implying the involvement of the opioidergic neurotransmission pathway. (−)-Linalool and (−)-linalool/β-CD complex also inhibited total leukocyte migration and TNF-α levels in peritoneal fluid in the CG-induced peritonitis protocol. However, (−)-linalool/β-CD complex exhibited stronger antinocicptive effect than (−)-linalool alone, indicating once again that cyclodextrin may become a relevant tool to improve the biological activity of water-insoluble monoterpenes [ 67 ].