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(0.24%) as the main constituents of the oil. Initially, mice were subjected to the formalin pain model.
In this test, animals treated with the essential oils eugenol and myrcene showed reduced licking times
of the paw in the first (5–10 min) and second (15–30 min) phases of nociception. On the other hand, in
the hot-plate test, the essential oil increased the latency of paw withdrawal from the hot plate, even
after 4 h of administration. Animals that received 5 or 10 mg/Kg of eugenol or 5 or 10 mg/kg of
myrcene exhibited a significant increased latency to either lick the paw(s), or to jump from the hot
plate. Pre-treatment with naloxone (1 mg/Kg, i.p.) significantly reversed the antinociceptive effects
of the oil, eugenol and myrcene in the hot plate test. These data suggest that the opioid system is
involved in the mediation of the antinociceptive effects of Ocimum gratissimum essential oil and its
isolated active principles, eugenol and myrcene. Opioid receptors (m, κ and d) are located in several
steps of the pain transmission pathway and are responsible for the direct and indirect antinociceptive
activities of opioid agonists [
122
]. Substances that can also activate these receptors should be of great
pharmacological and therapeutic importance [
123
]. Antinociceptive effects of Ocimum micranthum
essential oil (15, 25, 50 and 100 mg/Kg, p.o.) were studied by Pinho et al. [
42
] in mice subjected to the
following pain models: acid-induced writhing, formalin and hot-plate tests. Chemical analyses of the
essential oil revealed the following composition: (E)-methyl cinnamate (33.6%), limonene (12.9%),
carvone (9.6%), β-caryophyllene (8.03%), linalool (7.2%), (Z)-methyl cinnamate (5.92%), β-selinene
(3.95%), α-selinene (2.82%), α-humulene (2.7%) and trans-α-bergamotene (2.68%). Initially, Ocimum
micranthum essential oil significantly reduced the acetic acid-induced writhing responses. Under
formalin-induced nociception, only the second phase was significantly inhibited. No effect was
observed in mice subjected to the hot-plate test. Based on the lack of significant results in the first
phase of the formalin test and in the hot plate model Pinho et al. [
42
] conclude that the essential
oil inhibits nociception of inflammatory origin acting at the peripheral rather than supraspinal
and/or spinal level. However, as β-caryophyllene is found in the composition of the essential
oil, early studies showing that β-caryophyllene has both antinociceptive and anti-inflammatory
actions [
124
–
126
] may be due to its cannabinoid receptor 2 (CB2) activating properties [
127
]. The
formalin model of nociception may not be a reliable model to reveal the actions of substances outside
of certain pharmacological profiles.
3.15. Peperomia serpens (Sw.) Loud Essential Oil
The genus Peperomia, belonging to Piperaceae, comprises an estimated 1500–1700 species [
128
].
Peperomia serpens (Sw.) Loud. In the Amazon rainforest it is known as “carrapatinho” or
“carapitinha” growing wild on differing host trees. The decoction of its leaves is recommended
for anti-inflammatory and analgesic properties, particularly against flu, asthma, cough, earache and
irritation provoked by ant bites [
129
]. Other oils of Peperomia contain mono- and sesquiterpenes,
as in the case of Peperomia serpens, whose main constituents were α-humulene, (E)-caryophyllene,
(E)-nerolidol and (Z)-nerolidol acetate [
130
]. Pinheiro et al. [
43
] evaluated the antinociceptive effect
of the essential oil from the whole plant (31.25, 62.5, 125, 250 and 500 mg/Kg, p.o.) in mice
subjected to the acetic acid-induced writhing, formalin and hot plate tests. GC-MS analysis indicated
the presence of the following major constituents: (E)-Nerolidol (38.0%), ledol (27.1%), α-humulene
(11.5%), (E)-caryophyllene (4.0%) and α-eudesmol (2.7%). Oral pretreatment with Peperomia serpens
(Sw.) Loud essential oil evoked dose-dependent inhibition of acetic acid-induced abdominal writhes
in mice. The writhing test induced by acetic acid in mice is described as a typical model of study
of inflammatory pain, used to screen when evaluating analgesics or anti-inflammatory drugs [
131
].
The local irritation provoked by intraperitoneal injection of acetic acid triggers the liberation a
variety of mediators such bradykinin, substance P and prostaglandins, and especially PGI2, as well
as some cytokines such as IL-1β, TNF-α and IL-8 [
132
]. Such mediators activate chemosensitive
nociceptors that contribute to the development of this type of inflammatory pain, which is known
to be sensitive to non-steroidal anti-inflammatory drugs (NSAIDs). Like indomethacin (10 mg/Kg,
p.o.), the essential oil was able to reduce dose-dependent acetic acid-induced writhing response,
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suggesting a mechanism resulting in peripheral antinociceptive effect. Pretreatment of animals with
the essential oil showed no antinociceptive effect in mice subjected to the hot-plate test. Such results
indicate non-participation in thermal stimulation associated with central neurotransmission where
heat activates nociceptors (Aδ, and C fibers), driving the momentum to the dorsal horn of the
spinal cord subsequently to cortical centers. Finally, in the formalin test, the oil inhibited both first
(neurogenic pain), and second (inflammatory pain) phases of the test. Such effect was not altered
by naloxone pretreatment [
43
]. The formalin test is a very useful method for not only assessing
antinociceptive drugs but also for helping to elucidate their action mechanism. Centrally acting
drugs such as narcotics inhibited both phases equally. Peripheral acting drugs such as NSAIDs and
corticoids inhibited mainly the second phase [
60
]. According to Pinheiro et al. [
43
], Peperomia serpens
(Sw.) Loud essential oil induces its antinociceptive action by direct action on nociceptive afferent
fibers not interacting with the opioid system. In addition, the oil was effective in the second phase of
the formalin test indicating anti-inflammatory activity. These results suggest that the antinociceptive
action of Peperomia serpens (Sw.) Loud essential oil is more related to a peripheral mechanism than a
central one.
3.16. Pimenta pseudocaryophyllus (Gomes) L.R. Landrum Essential Oil
Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae) is a plant popularly known
in Brazil as pau-cravo, louro-cravo, louro, craveiro, craveiro-do-mato, chá-de-bugre and cataia [
133
–
135
].
In folk medicine, the leaves have been used to produce a refreshing drink with calming, diuretic
and aphrodisiac properties, as well as to treat colds with their complications, and digestive and
menstrual problems [
133
–
135
]. De Paula et al. [
44
] studied the antinociceptive effect of the essential
oil obtained from leaves of Pimenta pseudocaryophyllus in mice subjected to the acetic acid-induced
writhing test. GC-MS analysis indicated the presence of oxygenated mono- and sesquiterpenes
(69.65% and 13.7%, respectively), and monoterpene aldehydes neral and geranial were the major
components (27.59% and 36.49%, respectively), which are referred to as citral when their isomers
are mixed [
44
]. The essential oil at doses of 60, 200 and 600 mg/Kg, p.o., showed significant
dose-dependent inhibitory effects on abdominal contortions induced by intraperitoneal acetic acid
in mice. De Paula et al. [
44
] suggest that inhibition by Pimenta pseudocaryophyllus essential oil of the
contortions induced by chemical stimulation in mice in this study may be due to both peripheral and
central mechanisms. However, more investigations are needed to reinforce their case.
3.17. Piper alyreanum C.DC Essential Oil
Piper alyreanum C.DC, a member of the Piperaceae family, is a small tree that is widely
distributed in tropical and subtropical regions, and greatly in North and South America. In Brazil,
it is found in the North, mainly in the Amazon forest and is popularly known as “João brandinho”,
“pimenta longa”, “pimenta longa da mata”, “pimenta de cobra” and “pani-nixpu”. Moreover, this
plant has been used as an immunomodulator, analgesic and antidepressant in folk medicine [
45
].
The antinociceptive effect of the essential oil obtained from the aerial (leaves and stems) of Piper
aleyreanum was evaluated. The essential oil (30, 100, 300 and 1000 mg/Kg, p.o.) was tested in mice
subjected to the formalin test. GC-MS analysis indicated the presence of caryophyllene oxide (11.5%),
β
-pinene (9%), spathulenol (6.7%), camphene (5.2%), β-elemene (4.7%), myrtenal (4.2%), verbenone
(3.3%) and pinocarvone (3.1%) as the major constituents [
45
]. The results indicated that the essential
oil significantly inhibited both the neurogenic and inflammatory phases of formalin-induced licking.
However, its antinociceptive effects were significantly more pronounced in the second phase of this
pain model. Also, it was noted that pre-treatment with the non-selective opioid receptor antagonist
naloxone (1 and 5 mg/Kg, i.p.) did not reverse the antinociception caused by the essential oil [
45
].
Nociception as produced by formalin (first phase) is quite resistant to the majority of NSAIDs,
such as acetylsalicylic acid, indomethacin, paracetamol and diclofenac. However, these drugs can
dose-dependently attenuate the second phase of formalin-induced licking [
25
,
136
,
137
]. Moreover, it
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has also been reported that morphine, some tachykinin receptor antagonists, non-selective excitatory
amino acid antagonists and both B1 and B2 bradykinin receptor antagonists are able to inhibit both
phases of the formalin test [
138
,
139
]. Lima et al. [
45
] suggest that the opioid system is unlikely to be
involved in the antinociceptive action of Piper alyreanum essential oil. This is inferred by the fact that
pre-treatment of animals with naloxone, a nonselective opioid receptor antagonist, did not inhibit the
antinociceptive effect of morphine in the formalin model.
3.18. Satureja hortensis L. Essential Oil
Satureja hortensis L. belongs to the Lamiaceae family and is a well-known medicinal herb in Iran.
Aerial parts of this plant are frequently used as a food additive and also as a traditional remedy to
treat various disorders including cramps, muscle pain, nausea, indigestion, diarrhea and infectious
diseases, based on the antispasmodic, antidiarrheal, antibacterial and antifungal properties of their
constituents [
140
–
142
]. The essential oil (100 and 200 µL/Kg) from the seeds of Satureja hortensis in
mice subjected to the acetic acid-induced writhing and formalin tests was tested [
143
]. A chemical
analysis indicated the presence of γ-terpinene (50.5%) and thymol (32.7%) as the main constituents.
In the acetic acid-induced writhing test, the essential oil significantly inhibited abdominal writhes.
In the formalin test, the essential oil presented antinociceptive activity only in the late phase.
Acetic acid-induced abdominal pain is not a specific model, but because of its similarity to the signs
of human visceral disorders it has been extensively used for the screening of analgesic drugs [
78
,
144
].
In this test, many drugs including opioids, nonsteroidal anti-inflammatory drugs, antispasmodics,
calcium channel blockers and antihistamines show analgesic activity [
57
,
78
]. Pain in the early phase
is predominantly caused by the activation of C-fibers, while in the late phase, a combination of an
inflammatory reaction in peripheral tissue and functional changes in the dorsal horn of the spinal
cord are involved [
65
]. Finally, the authors suggested that the antinociceptive effect of the essential
oil is dependent upon peripheral mechanisms.
3.19. Senecio rufinervis D.C. Essential Oil
Senecio rufinervis D.C. (Asteraceae) is a tall aromatic herb, leaves are shortly stalked, ovate, long
pointed, sharply toothed; the lower surface is white and tomentose except for the nerves. The flowers
are yellow and present as small rounded corymbs [
145
]. The plant grows in Uttarakhand, India
at an altitude of 1800–3000 m and has no traditional or commercial use. Senecio rufinervis is an
aromatic plant containing essential oil which is produced by many plants and confirms analgesic
and antiinflammatory activities [
146
–
151
]. It was studied the antinociceptive effect of the essential
oil from the dried leaves of Senecio rufinervis (25, 50 and 75 mg/Kg, i.p.) in mice subjected to
the acetic acid-induced writhing and hot-plate tests [
46
]. A chemical analisys of the essential oil
showed the presence of germacrene (40.19%) as the major constituent, followed by β-pinene (12.23%),
β
-caryophyllene (6.21%) and β-longipinene (4.15%). In the acetic acid-induced writhing test, the
essential oil significantly and dose-dependently inhibited the acetic acid-induced abdominal twitches.
Similarly, the essential oil significantly increased the latency of reaction time 15 and 30 min after the
administration of drug. Therefore, Mishra et al. [
46
] demonstrated significant analgesic activity in
both pain models. The acetic acid-induced writhing reaction in mice, described as a typical model
for inflammatory pain, has long been used as a screening tool for the assessment of analgesic or
anti-inflammatory properties of new agents [
152
]. The hot-plate method is considered to be selective
for screening of the compound acting through the opioid receptor, but other centrally acting drugs,
including sedatives and muscle relaxants, have also shown activity in this test [
153
]. From the above
results the authors affirm that the oil possesses both peripheral and central analgesic effect. Yet, the
peripheral analgesic effect produced by oil was more pronounced than the central analgesic effect.
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3.20. Tetradenia riparia (Hochst.) Codd Essential Oil
Tetradenia riparia (Hochst.) Codd (Lamiaceae) is an herbaceous shrub that occurs throughout
tropical Africa [
154
,
155
]. T. riparia possesses a variety of medicinal properties. In South Africa,
T. riparia has traditionally been used in the treatment of cough, dropsy, diarrhea, fever, headaches,
malaria and toothache [
154
]. In Brazil, Tetradenia riparia was introduced as an exotic ornamental
plant and is grown in parks, home gardens and orchards in the state of São Paulo. In Brazil, it is
popularly known as incenso, lavândula, limonete, pluma-de-névoa, or falsa mirra and is mainly used
as an ornamental [
155
]. The antinociceptive effect of the essential oil obtained from the leaves of
Tetradenia riparia (10 mg/Kg, p.o.), collected in different seasons, was investigated by Gazim et al. [
47
]
in mice subjected to the acetic acid-induced writhing test. GC-MS analysis indicated that
oxygenated sesquiterpenes were the dominant compounds, such as 14-hydroxy-9-epi-caryophyllene
(18.27%–24.36%), cis-muurolol-5-en-4-α-ol (7.06%–13.78%), α-cadinol (5.36%–8.33%) and ledol
(4.39%–8.74%). The content of the essential oil varied significantly by season. The oxygenated
sesquiterpenes that varied most by season were 14-hydroxy-9-epi-caryophyllene (maximum 24.36%
in spring, and absent in winter) and cis-muurolol-5-en-4-α-ol with a maximum of 13.78% in autumn
and a minimum of 7.06% in winter. In the acetic acid-induced writhing test, the essential oil inhibited
constrictions and this activity was not affected by seasonal variation.
However, no additional
information about the mechanism of action is given by the authors.
3.21. Teucrium stocksianum Essential Oil
The Lamiaceae family is rich in essential oils. The main components of the essential oil
reported from the genus Teucrium are alpha pinene, linalool, carophyllene oxide, germacrene D,
beta-carophyllene and delta-cadinene. Teucrium stocksianum is a species found in North Western
Pakistan. It is a perennial aromatic herb of 10–30 cm height having grayish-white leaves and sessile
flowers. It grows in the shades of the mountains. This plant is used in folk medicine for treating
diarrhea, cough, jaundice and abdominal pain [
156
]. In the work developed by Shah et al. [
48
],
a GC-MS analysis of the essential oil obtained from the aerial parts of Teucrium stocksianum
revealed the presence of δ-cadinene (12.92%), α-pinene (10.3%), myrcene (8.64%), β-caryophyllene
(8.23%), germacrene D (5.18%), limonene (2.36%), elemol (2.13%) and γ-cadinene (1.86%) as the
main compounds. The antinociceptive activity of the essential oil (20, 40, 80 and 160 mg/Kg,
i.p.) was assessed in mice subjected to the acetic acid induced writhing test. In this model of
pain, the oil decreased the number of writhings. The acetic acid induced writhing protocol is
most commonly used for evaluating antinociceptive activity of medicinal plants. In this model,
prostaglandins, initially PGE2 and then PGF2α and free arachidonic acid are released from tissue
phospholipids and, consequently, their levels in the peritoneal fluids increase due to intraperitoneal
administration of the irritant, acetic acid. This results in localized inflammatory response and pain
sensation due to increases in capillary permeability. Substances which counteract this phenomenon
exert antinociceptive effects and reduce pain sensations [
64
]. Despite reporting these effects, more
investigations are needed in order to elucidate the mechanism of action of the material.
3.22. Ugni myricoides (Kunth) O. Berg Essential Oil
The Myrtaceae family comprises a large number of plants, including at least 138 genera and
approximately 3800 species [
157
,
158
]. Several experimental studies have demonstrated the biological
activity of extracts, essential oils, or fractions obtained from different species of this family, such
as Psidium guajava L., Syzygium jambos (L.) Alston, and Plinia glomerata (O. Berg) Amshoff [
3
–
6
].
Ugni myricoides (Kunth) O. Berg (syn. Myrtus myricoides Kunth), one of the four members of this
genus, is found in Brazil, Southern Mexico, Central America, Colombia, Venezuela, Guyana, Ecuador
and Peru [
159
]. The plant can be recognized by its small (1–1.5 cm long), opposite, coriaceous, dark
green, aromatic, obovate to elliptic leaves and by its solitary white or pink flowers. The spherical
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fruits, of about 1 cm in diameter, are edible, fleshy and purple to black in color. Quintão et al. [
49
]
studied the antinociceptive effect of essential oil from the leaves of Ugni myricoides (Kunth) O. Berg
(5, 10, 12.5, 25 and 50 mg/kg, p.o.) in mice subjected to the following pain models: carrageenan- and
CFA (complete Freund’s adjuvant)-induced mechanical hypernociception (evaluated by Von Frey
hairs-induced hindpaw withdrawal response method), and used partial ligation of sciatic nerve
tests. A GC-MS analysis of the essential oil indicated the presence of α-pinene (52.1%), 1,8-cineole
(11.9%), α-humulene (4.6%), caryophyllene oxide + globulol (4.5%), humulene epoxide II (4.2%) and
β
-caryophyllene (2.9%) as the main components. In the first test, oral treatment with the essential oil
was able to significantly inhibit the mechanical hypernociceptive response induced by carrageenan.
This effect was observed for up to 48 h after treatment with the essential oil. Similar results were
obtained with animals injected with CFA, in which Ugni myricoides (Kunth) O. Berg essential oil
postponed the onset of hypernociceptive threshold for up to 24 h after CFA paw injection [
49
].
In addition, the hypernociceptive response evoked by CFA in the mouse paw was strikingly
reduced by the pretreatment of animals with the pure monoterpene compound present as the major
constituent in the oil, α-pinene (5–50 mg/Kg, p.o.), given 24 h before the injection of CFA. In the
partial ligation of sciatic nerve tests, the essential oil was capable of diminishing the hipernociceptive
response induced by this chronic constriction injury. This effect was observed for up to two days
after the end of the treatment, and α-pinene administration was also capable of abolishing the
hypernociceptive response in this model of pain. Quintão et al. [
49
] suggest that oral treatment with
Ugni myricoides essential oil presents important effects in preventing and also reverting mechanical
sensitization caused by inflammatory and neuropathic states. This conclusion is supported by results
showing that the mechanical hypernociception induced by i.pl. injection of carrageenan or CFA in
mice was strikingly reduced by both the essential oil and its major constituent. The injection of
carrageenan into the hindpaws of mice induces a local inflammatory response, characterized by paw
edema, neutrophil migration, and the release of several mediators such as cytokines, which precedes
inflammatory hypernociception [
160
]. Additionally, CFA produces an inflammatory response that
is associated with a striking modification in the activity of superficial (I and II), and deep (V and
VI) laminal dorsal horn neurons receiving noxious inputs [
161
]. Also, chronic constriction nerve
injury (such as partial ligation of the sciatic nerve) produces an inflammatory response that is
associated with modification of the spinal cord neurons, culminating in altered neuronal excitability
and conduction during evoked and spontaneous activity [
162
,
163
]. Quintão et al. [
49
] conclude that
the essential oil obtained from the leaves of Ugni myricoides has relevant oral anti-hypernociceptive
properties for persistent models of inflammatory and neuropathic pain in mice. However, the
mechanism through which Ugni myricoides essential oil exerts its anti-hypernociceptive actions
remains unclear and requires further investigation.
3.23. Valeriana wallichii DC Essential Oil
Valeriana (Valerianaceae) originated from the Latin word “valere” meaning “to be in good
health”, as a source of important phytomedicines, has been used for curing nervous unrest, emotional
troubles, epilepsy, insanity, snake-poisoning, eye-trouble and skin diseases [
164
]. Valeriana wallichii
DC grows wild in the temperate Himalaya at an altitude of 1500–3000 m and is an ingredient of
herbal medicines in Indian systems of medicine. Sah et al. [
50
] evaluated the antinociceptive effect of
the essential oil isolated from the roots and rhizomes of Valeriana wallichii DC chemotype (patchouli
alcohol) (20, 40 and 80 mg/Kg, p.o.) in mice subjected to the acetic acid induced writhing and tail-flick
tests. GC-MS analysis of the essential oil revealed the presence of δ-guaiene (10%), seychellene (8%),
acetoxyl patchouli alcohol (5%) and virdifloral (5%). Initially, the administration of the essential
oil produced a significant inhibition of writhings. The effect was potentiated in the presence of
aspirin (5 mg/Kg, i.p.). The writhing test induced by acetic acid in mice is described as a typical
model of study of inflammatory pain, being used as a screening for evaluation of analgesics or
anti-inflammatory drugs [
131
]. The local irritation provoked by intraperitoneal injection of acetic
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acid triggers liberation of a variety of mediators such bradykinin, substance P and prostaglandins,
(especially PGI2), as well as certain cytokines such as IL-1β, TNF-α and IL-8 [
132
]. Such mediators
activate chemosensitive nociceptors that contribute to the development of this type of inflammatory
pain, which is known to be sensitive to non-steroidal anti-inflammatory drugs (NSAIDs). However,
the essential oil failed to prolong the latency time in the tail-flick model. This pain model is reported
to be a useful test to discriminate analgesic agents acting primarily at the spinal medulla level and
at the higher central nervous system levels, from those acting by peripheral mechanisms, (positive
results indicating central action [
3
]. Based on the results, Sah et al. [
50
] suggest that Valeriana wallichii
DC essential oil possesses peripheral analgesic action and the effect is comparable to aspirin.
3.24. Xylopia laevigata (Mart.) R.E.Fries Essential Oil
The Annonaceae is a large family of tropical and subtropical trees and shrubs, comprising about
135 genera and more than 2500 species [
165
,
166
]. This family is known for its edible fruits and
the medicinal properties of many of its species [
167
]. In the Brazilian Northeast, Xylopia laevigata
(Mart.) R.E. Fries (Annonaceae) is commonly called “meiú” or “pindaíba”, a plant (leaves and
flowers) used popularly to treat painful disorders, heart disease and inflammatory conditions [
51
].
Queiroz et al. [
51
] evaluated the antinociceptive effect of Xylopia laevigata essential oil in mice subjected
to the acetic acid-induced writhings and formalin tests. Chemical analysis indicated the presence
of the following sesquiterpenes: γ-muurolene (17.78%), δ-cadinene (12.23%), bicyclogermacrene
(7.77%), α-copaene (7.17%), germacrene D (6.54%), (E)-caryophyllene (5.87%), γ-cadinene (4.72%),
aromadendrene (4.66%) and γ-amorphene (4.39%).
When tested, the essential oil significantly
inhibited the acetic acid-induced writhings, and the two phases of formalin. It is important to
stress that the antinociceptive effect of the oil was not reversed by naloxone (1.5 mg/Kg, i.p.) in the
formalin test. The acetic acid-induced abdominal constriction test is a standard, simple and sensitive
model for measuring analgesia induced by both opioids and peripherally acting analgesics [
168
].
According to Le Bars et al. [
3
], in the acetic acid test, 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; those behavioral changes are probably in relation to the inhibition in the
peritoneal fluid levels of prostaglandin and cytokines.
This information supports the authors’
conclusion that the essential oil may also participate in the inhibition of prostaglandin synthesis,
since nociceptive mechanisms involve the processing or release of arachidonic acid metabolites via
COX and prostaglandin biosynthesis [
169
]. In addition, the formalin test is sensitive to various classes
of analgesic drugs [
24
]; and is characterized by the first phase (neurogenic), which is evoked by
direct formalin stimulation of the sensorial C-fibers followed by substance P release [
60
], and the
second phase (inflammatory) mainly due to a subsequent inflammatory reaction in the peripheral
tissue mediated by the release of various inflammatory mediators associated with the increased level
of prostaglandin, induction of COX and release of nitric oxide (NO) [
3
]. It is important to note
that the essential oil reduced the production of nitrite, showing it to have a potential role as a NO
scavenging agent, and since NO plays an important role in various types of inflammatory processes,
it is thus possible that the reduction of NO is involved in a potential antinociceptive action of the
Xylopia laevigata essential oil [
51
].
3.25. Vanillosmopsis arborea Baker Essential Oil
Vanillosmopsis arborea Baker is native to the Araripe National Forest, in the Northeast of
Brazil in the state of Ceará. There are few studies concerning the traditional use of this plant.
However, biological and pharmacological studies have shown that its essential oil presents
antimicrobial, antiinflammatory and gastroprotective activities [
170
]. The topical antinociceptive
effect of Vanillosmopsis arborea Baker essential oil (25, 50, 100 and 200 mg/Kg, p.o. or topical)
was studied by Leite et al. [
52
] in mice subjected to formalin and eye wiping (corneal nociception)
tests. The composition (w/w) of Vanillosmopsis arborea Baker essential oil revealed the presence of
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α
-bisabolol (70%). Other identified compounds were α-cadinol (8.4%), elemicin (6.21%), β-bisabolene
(4.46%), δ-guaiene (2.31%), β-cubebene (1.76%) and estragole (1.08%). In the formalin test,
pretreatment with the essential oil (oral and topical) caused significant reductions of both first phase
(neurogenic) and second phase (inflammatory) nociception responses. Such effect may be related, at
least in part, to release of leukotrienes, which decrease the production of inflammatory eicosanoids
and influence the production of arachidonic acid metabolites [
171
]. This antinociceptive effect may
also be related to the high α-bisabolol content in the essential oil, since α-bisabolol possesses visceral
antinociceptive activity [
172
], and is able to reduce neuronal excitability in a concentration-dependent
manner [
107
]. The topically administered essential oil decreased the number of eye wipes induced
through local application of 5 M NaCl solution on the corneal surface. Oral treatment with the oil
also reduced the number of eye wipes. In addition, the antinociceptive effect induced by the essential
oil was significantly inhibited by ondansetron (0.5 mg/Kg, i.p.), PCPA (a tryptophan hydroxylase
inhibitor—100 mg/Kg, i.p.), prazosin (0.15 mg/Kg, i.p.), atropine (0.1 mg/Kg, i.p.) and capsazepine
(5 mg/Kg, i.p.). On the other hand, the administration of glibenclamide (2 mg/Kg, i.p.), naloxone
(2 mg/Kg, i.p.), ruthenium red (5 mg/Kg, s.c.), yohimbine (2 mg/Kg, i.p.), L-NAME (2 mg/Kg,
i.p.) or theophylline (5 mg/Kg, i.p.) did not prevent the essential oil-induced antinociception.
The cornea is used for nociception studies on the trigeminal system [
173
], since corneal nociceptive
receptors have large representation in the trigeminal ganglion through the ophthalmic branch of the
trigeminal nerve [
174
]. Thin myelinated fibres [
175
] as well as unmyelinated fibers in the cornea
respond to chemical, mechanical and thermal noxious stimuli [
176
]. The application of hypertonic
saline to the tongue and cornea transiently activates nociceptive neurons with wide dynamic range
properties in the trigeminal subnucleus caudalis [
177
]. Moreover, infusion of hypertonic saline into
the masseter muscle produces hind paw shaking and activates c-Fos positive neurons in the ipsilateral
trigeminal subnucleus caudalis [
178
]. Taken together, the results indicate that the essential oil of
Vanillosmopsis arborea Baker exerts antinociceptive activity by peripheral and central mechanisms,
possibly mediated by 5-HT, α1, muscarinic and TRPV1 receptors.
3.26. Zingiber oficinalle and Zingiber zerumbet Essential Oils
The Zingiberaceae family is among the most prolific plants in tropical rainforests. Ginger,
the rhizome of Zingiber officinale Roscoe, is one of the most widely used spices and a traditional
remedies in Indian, Chinese and Oriental medicine against pain, inflammation and gastrointestinal
disorders. Ginger oil is produced from fresh rhizomes of Zingiber officinale. It possesses the aroma
and flavor of the spice but lacks the pungency. The essential oil of ginger has been found to possess
antibacterial, antiviral and antifungal properties [
179
,
180
]. Zingiber zerumbet (L.) Smith, locally known
in Malaysia as “lempoyang” is one of the commonly used wild ginger species in Malay traditional
medicine. The concoction of Zingiber zerumbet rhizomes is normally drunk to treat indigestion,
stomach ache, fever, and worm infestation. The young stems, rhizomes and inflorescence are also
used as a poultice for topical applications to treat muscle sprain and as a curative for swelling
sores. The juice extracted from the rhizomes or the cooked rhizomes are usually taken by women
post-partum or post-surgical patients to improve appetite, enhance recovery or healing as well as
to alleviate pain [
181
,
182
]. Jeena et al. [
53
] studied the antinociceptive effect of the essential oil of
ginger (100, 500 and 1000 mg/Kg, i.p.) in mice subjected to the acetic acid-induced writhing test. The
principal constituent of ginger oil was found to be zingiberene (31.08%), a sesquiterpene hydrocarbon,
followed by arcurcumene (15.4%) and α-sesquiphellandrene (14.02%). Other compounds include
bisabolene (13.80%) and sabinene (8.27%). Ginger oil showed marked and significant reduction in
the number of writhings induced by acetic acid. The analgesic activity at all tested doses indicated a
dose dependent relationship. Jeena et al. [
53
] affirm that acetic acid induces pain in the peritoneal
cavity by enhancing levels of endogenous substances like: PGE2 and PGF2 [
63
]. This indicates
that acetic acid acts indirectly in the stimulation of nociceptive neurons by releasing endogenous
mediators and suggests that ginger oil has strong antinociceptive activity.
Its mode of action
Molecules 2016, 21, 20
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might involve inhibition of arachidonic acid synthesis, a metabolite mediated by COX inhibition.
Sulaiman et al. [
54
] investigated the antinociceptive effect of essential oil from the rhizome of Zingiber
zerumbet (30, 100 and 300 mg/Kg, i.p. and p.o.) in mice subjected to the following pain models:
acetic acid-induced abdominal writhing, formalin and hot-plate tests. GC/MS analyses indicated
the presence of zerumbone (36.12%) was the most abundant constituent among the sesquiterpenes,
followed by humulene (10.03%), humulene oxide I (4.08%), humulene oxide II (2.14%), caryophyllene
oxide II (1.66%) and caryophyllene oxide I (1.43%). Among the monoterpenes we found: camphene
(14.29%), borneol (4.78%), camphor (4.18%), eucalyptol (3.85%), α-pinene (3.71%), γ-terpinene
(2.00%), β-phellandrene (1.63%), 1-terpen-4-ol (1.44%), β-myrcene (1.22%) and linalool (1.06%).
Intraperitoneal administration of the essential oil caused dose-dependent inhibition of the writhing
response induced by acetic acid. The oral administration caused a partial but significant inhibition of
the acetic acid-induced pain. This method is very sensitive and able to detect antinociceptive effects
of compounds and dose levels that may appear inactive in other methods like the tail-flick test [
183
].
It has been suggested that acetic acid acts indirectly by releasing endogenous mediators, such as
PGE2 and PGF2α as well as increasing lipoxygenase production in the peritoneum that stimulate the
nociceptive neurons sensitive to nonsteroidal anti-inflammatory drugs [
184
]. Therefore, the results
of the acetic acid-induced abdominal constriction test strongly suggest that the mechanism of action
of the oil may be mediated by lipoxygenases and/or cyclooxygenases’ activity inhibition. In the
formalin test, intraperitoneal pretreatment with different doses of Zingiber zerumbet essential oil had
significant and dose-dependent effects on the duration of licking activity in both early and late phases
of the test. Such effect was reversed significantly by naloxone (5 mg/Kg, i.p.). It is well known that
centrally acting drugs such as narcotics inhibit both nociception phases equally, while peripherally
acting drugs such as acetylsalicylic acid, which block prostaglandin synthesis, only inhibit the second
phase [
60
]. Taken together, Sulaiman et al. [
54
] affirm that the antinociceptive effects of the essential
oil in the writhing test and in both phases of the formalin test strongly suggested that they contained
active analgesic principles acting both centrally and peripherally, it was also implied that the extract
possessed not only antinociceptive but also antiinflammatory activity. This finding is supported, at
least in part, by the results of the hot plate test. In this pain model, the intraperitoneal administration
of the oil increased the latency time to the nociceptive response in the hot plate test significantly.
This effect began early, 30 min after intraperitoneal administration of the essential oil, and persisted
until the fifth hour. The antinociceptive effect was also reversed by naloxone. In a later study,
Khalid et al. [
55
] suggested a mechanism of antinociceptive action for Zingiber zerumbet essential oil
(50, 100, 200 and 300 mg/Kg, i.p. and p.o.). Acetic acid-induced abdominal constriction, capsaicin-,
glutamate- and phorbol 12-myristate 13-acetate-induced paw licking tests in mice were employed in
the study. The essential oil exhibited significant dose-dependent inhibition on abdominal writhing
when administered intraperitoneally.
Similar dose dependent inhibition was also observed in
mice administered orally. Likewise, intraperitoneal administration of Zingiber zerumbet essential
oil at similar doses produced significant dose dependent inhibition of neurogenic pain induced by
intraplantar injection of capsaicine (1.6 µg/paw). It is believed that capsaicin directly activates a
non-selective ionotropic channel in primary sensory neurons, the capsaicin receptor, also known
as the transient receptor potential vanilloid 1 (TRPV1) [
185
]. Therefore, this finding indicates that
the effect of the essential oil may involve, at least in part, TRPV1 receptor inhibition. Similarly,
the essential oil also inhibited pain induced by intraplantar injection of glutamate (10 µM/paw).
It was reported that this nociceptive response caused by glutamate involves peripheral, spinal and
supraspinal sites of action with glutamate receptors (AMPA, kainate and NMDA receptors), which
play an important role in modulating the nociceptive response [
186
]. A similar result was observed
with intraplantar administration of phorbol 12-myristate 13-acetate (a PKC activator at 1.6 µg/paw).
PKC activation is an essential step for the nociceptive effects of numerous stimuli, including those that
are caused by inflammatory mediators. PKC phosphorylates many cellular components, including
membrane bound receptors, ion channels and enzymes, which are known to regulate the excitation
Molecules 2016, 21, 20
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of nociceptors [
187
]. Peripheral introduction of PMA produces nociception, thermal hyperalgesia
and mechanical allodynia in mice and rats. PMA, acting on PKC can directly stimulate TRPV1
channels leading to the propagation of nociceptive impulses [
188
]. This finding can be linked to earlier
findings discussed above. It was also demonstrated that pretreatment with
L
-arginine (100 mg/Kg,
i.p.) significantly reversed the antinociceptive activity induced by the oil. Previous studies have
reported that NOS inhibitors reduced nociception caused by acetic acid [
189
]. NO is a diffusible gas
that permeates cell membranes and is not stored in vesicles. NMDA receptor activation increases
intracellular calcium, which in turn stimulates NOS to catalyze the substrate
L
-arginine to NO
and to
L
-citrulline [
190
]. NO seems to be involved in all three levels of the pain pathway, which
are the peripheral, spinal cord dorsal horn and the cerebral cortex where perception of pain is
processed [
191
]. In addition, pre-treatment with methylene blue (20 mg/Kg, i.p.) significantly
enhanced antinociception produced by the essential oil. It has been suggested that methylene blue
promotes antinociception by sequentially inhibiting peripheral NOS and guanylyl cyclase, resulting
in NO interference [
192
]. The activation or deactivation of noci-responsive neurons is dependent
on the availability of cGMP. Intracellular cGMP concentrations are regulated by the action of
guanylyl cyclase and also by the rate of degradation by cGMP-specific phosphodiesterases [
193
].
Therefore, cGMP is very important for the functioning of nociceptors. Nitric oxide activates guanylyl
cyclase, which in turn catalyses the formation of cGMP from guanosine triphosphate, whereas cyclic
GMP-specific phosphodiesterase catalyzes the hydrolysis of cGMP to GMP, thus consequently ending
the signal transduction [
194
]. Finally, the administration of glibenclamide (10 mg/Kg, i.p.), an
ATP-sensitive K
+
channel antagonist, significantly reversed antinociceptive activity induced by the
essential oil. This finding suggests that the oil exerted its antinociceptive activity through the opening
of ATP sensitive K
+
channel that allows the efflux of K
+
ions, thus leading to membrane repolarization
and/or a hyperpolarization state which reduces the membrane excitability [
195
]. Taken together,
the results indicate that the antinociceptive action of Zingiber zerumbet essential oil involves, at least
in part, the activation of the
L
-arginine/NO/cGMP/ATP-sensitive K
+
channel pathway, apart from
its ability to interact and inactivate the TRPV1 receptor, inactivation of protein kinase C, and also
inhibition of the glutamatergic system.
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