Tingenone, a pentacyclic triterpene,

induces peripheral antinociception due to

cannabinoid receptors activation in mice

C. C. Veloso, R. C. M. Ferreira,
V. G. Rodrigues, L. P. Duarte, A. Klein,
I. D. Duarte, T. R. L. Romero &
A. C. Perez
Inflammopharmacology
Experimental and Therapeutic Studies

ISSN 0925-4692

Inflammopharmacol
DOI 10.1007/s10787-017-0391-7

1 23
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 Author's personal copy
Inflammopharmacol
DOI 10.1007/s10787-017-0391-7
ORIGINAL ARTICLE
Tingenone, a pentacyclic triterpene, induces peripheral
antinociception due to cannabinoid receptors activation in mice
C. C. Veloso1 • R. C. M. Ferreira2 • V. G. Rodrigues3 • L. P. Duarte3
A. Klein2 • I. D. Duarte2 • T. R. L. Romero2 • A. C. Perez2
Received: 7 June 2017 / Accepted: 23 August 2017
 Springer International Publishing AG 2017
Abstract Several works have shown that triterpenes
induce peripheral antinociception by activation of
cannabinoid receptors and endocannabinoids; besides,
several research groups have reported activation of
cannabinoid receptors in peripheral antinociception. The
aim of this study was to assess the involvement of the
cannabinoid system in the antinociceptive effect induced
by tingenone against hyperalgesia evoked by prostaglandin
E2 (PGE2) at peripheral level. The paw pressure test was
used and the hyperalgesia was induced by intraplantar
injection of PGE2 (2 lg/paw). All drugs were injected
subcutaneously in the hind paws of male Swiss mice.
Tingenone (200 lg/paw) administered into the right hind
paw induced a local antinociceptive effect, that was
antagonized by AM630, a selective antagonist to CB2
cannabinoid receptor. AM251, a selective antagonist to
CB1 cannabinoid receptor, did not alter the peripheral
antinociceptive effect of tingenone. MAFP, a fatty acid
amide hydrolase (FAAH) inhibitor; VDM11, an anan-
damide reuptake inhibitor; and JZL184, monoacylglycerol
lipase (MAGL) inhibitor did not potentiate the peripheral
antinociceptive effect of the lower dose of tingenone
(50 lg/paw). The results suggest that tingenone induced a
& C. C. Veloso
clariceveloso@ufam.edu.br
1
Faculdade de Ciências Farmacêuticas, Universidade Federal
do Amazonas (UFAM), Av. General Rodrigo Octávio Jordão
Ramos, 3000, Coroado I, Manaus, AM 69.077-000, Brazil
2 antagonist for the opioid receptors. Clocinnamox, nal-
Department of Pharmacology, Institute of Biological
Sciences, Federal University of Minas Gerais (UFMG),
Belo Horizonte, MG, Brazil
3
Department of Chemistry, Institute of Exact Sciences,
UFMG, Belo Horizonte, MG, Brazil
Inflammopharmacology
•
peripheral antinociceptive effect via cannabinoid receptor
activation. Therefore, this study suggests a pharmacologi-
cal potential for a new analgesic drug.
Keywords Tingenone
Pentacyclic triterpene

Cannabinoid receptors
Peripheral antinociception
Introduction
The Celastraceae family consists of 98 genera and 1210
species (Spivey et al. 2002; Simmons et al. 2008), that
include Maytenus genus, for the treatment of inflammation
and pain (Martins et al. 2012). Several studies involving
different species of this genus demonstrated anti-inflam-
matory and antinociceptive effects (Jorge et al. 2004; Sosa
et al. 2007; Da Silva et al. 2011; Martins et al. 2012;
Veloso et al. 2014a). Rodrigues et al. (2012) isolated and
characterized secondary metabolites from the roots of the
species M. imbricata, among them tingenone, a pentacyclic
triterpene.
Previous studies demonstrated the antinociceptive effect
of the extracts and tingenone obtained from M. imbricata
roots Veloso et al. (2014a). Besides, it also demonstrated
the peripheral mechanism, involving the activation of the
opioidergic pathway against mechanical hyperalgesia
induced by E2 prostraglandin (PGE2) (Veloso et al. 2014b).
In this study, it was shown that tingenone, when adminis-
trated in the right hind paw, induces a local antinociceptive
effect that was antagonized by naloxone, a nonspecific
trindole, and nor-binaltorphimine, which are specific
antagonists for the l, d, and j receptors, respectively,
reverted the peripheral antinociception induced by tin-
genone. Bestatin, an aminopeptidase inhibitor, an enzyme
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 Author's personal copy
that degrades opioid peptides, intensified the antinocicep-
tive effect of tingenone. Thus, the results suggest the
participation of the opioidergic system in the peripheral
antinociception induced by tingenone. In another study, the
activation of nitric oxide (NO)/cyclic guanosine
monophosphate (cGMP)/channels for sensitive potassium
ATP (KATP) pathways was demonstrated (Veloso et al.
2015).
There are similarities in the mechanism of action of
opioid and CB1/CB2 cannabinoid receptors. They are G
protein-coupled receptors (GPCRs), leading to inhibition of
adenylate cyclase and production of cyclic adenosine
monophosphate (cAMP). Through the same pathway,
opioids (Ferreira et al. 1991; Rodrigues and Duarte 2000;
Pacheco et al. 2005) and cannabinoids (Reis et al. 2011;
Romero et al. 2012; Romero and Duarte 2012) induce
peripheral antinociception by activation of L-arginine/NO/
cGMP/K? channels pathway. Reis et al. (2011) provided
evidence that the peripheral antinociceptive effect of the
cannabinoid receptor agonist, anandamide, is primarily
caused by activation of ATP-sensitive K(?) channels and
does not involve other potassium channels. In other study,
Romero et al. (2012) provided evidence that N-palmitoyl-
ethanolamine may activate neuronal nitric oxide synthase
(nNOS), thus initiating the NO/cGMP pathway and ATP-
sensitive K?-channel activation, inducing peripheral
antinociceptive effects.
Endocannabinoids are not stored in vesicles, but they are
synthesized on demand from polyunsaturated fatty acid pre-
cursors by hydrolases as a consequence of increase in
intracellular Ca2? concentration, from the influx or mobi-
lization of intracellular stocks. The termination of the action of
anandamide is achieved through its reuptake and by inacti-
vation through enzyme fatty acid amide hydrolase (FAAH),
resulting in arachidonic acid and ethanolamine. On the other
hand, 2-AG, under the action of monoacylglycerol lipase
(MAGL), forms arachidonic acid and glycerol (Di Marzo
2009; Kress and Kuner 2009; Stein and Machelska 2011).
Therefore, the aim of this study was to investigate
whether the antinociceptive effect of tingenone also
involves the activation of cannabinoid system.
Materials and methods
Plant material
Roots of Maytenus imbricata (Celastraceae) were carefully
collected to prevent damage to the specimen. The plant
material was identified and a voucher specimen (number
27780) was deposited in the collection of the Herbarium of
the Botanic Department of the Federal University of
Viçosa, Brazil.
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C. C. Veloso et al.
The roots of M. imbricata were dried at room temper-
ature and powdered in a mill. The powder (1.5 kg) was
submitted to extraction in a Soxhlet apparatus with the
organic solvent hexane/ethyl ether (1:1). The filtrate was
removed in a rotator evaporator. The quantity of filtrate
obtained was 16.1 g for hexane/ethyl ether (1:1) extract.
From this extract, 1.5 g of tingenone (Fig. 1) was isolated
and characterized (Rodrigues et al. 2012).
Animals
Male Swiss mice (30–35 g), from the Bioterism Center, were
used in the experiments. They were housed in standard cages
and kept at a constant temperature of 23 C with a 12-h light–
dark cycle and free access to food and tap water. All testing
procedures were in accordance with the ethical guidelines of
the International Association for the Study of Pain (IASP)
(Zimmermann 1983) and approved by Ethics Committee
(115/2012) in Animal Experimentation.
Measurement of hyperalgesia
Hyperalgesia was induced by subcutaneous injection of
prostaglandin E2 (PGE2) (2 lg) into the plantar surface of
the hind paw. Hyperalgesia was measured according to the
rat paw pressure test (Randall and Selitto 1957) adapted to
mice (Kawabata et al. 1992). An analgesimeter was used
(Ugo-Basile, Italy) with a cone-shaped paw-presser with a
rounded tip, which applies a linearly increasing force to the
hind paw. The weight in grams (g) required to elicit the
nociceptive response of paw flexion was determined as the
nociceptive threshold. A cutoff value of 160 g was used to
reduce the possibility of damage to the paws. The noci-
ceptive threshold was measured in the right paw and
determined as the average of the three consecutive trials
Fig. 1 Tingenone structure, a natural pentacyclic triterpene, isolated
from roots of Maytenus imbricata Mart. ex. Reissek
 Author's personal copy
Tingenone, a pentacyclic triterpene, induces peripheral antinociception due to cannabinoid…
recorded before and ten minutes after the third-hour after
PGE2 injection (time in which the maximum antinocicep-
tive effect of tingenone is observed) (Veloso et al. 2014b).
The threshold was calculated as the difference between
these averages (D of nociceptive threshold) and is expres-
sed in grams, concerning four animals per group. D of
nociceptive threshold [0 (zero) means hyperalgesia by
PGE2 injection and decrease of this value means antihy-
peralgesic effect by the tested drug.
Drugs administration
All drugs were injected using a volume of 20 ll/paw. Tin-
genone was dissolved in 20% dimethylsulfoxide (DMSO)
and 1% Tween 20 in isotonic saline. AM630 (6-iodo-2-
methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl(4-ethox-
yphenyl) methanone; Tocris, EUA, Ki = 31.2 nM),
selective antagonist of the CB2 cannabinoid receptor, and
AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3 carboxamide;
Tocris, Ki = 7.49 nM), selective antagonist of the CB1
cannabinoid receptor, were dissolved in 10% DMSO in
isotonic saline. MAFP (5Z,8Z,11Z,14Z-eicosatetraenyl-
phosphonofluoridic acid, methyl ester; Tocris), fatty acid
amide hydrolase (FAAH) inhibitor, was dissolved in 3%
ethanol in isotonic saline. VDM11 (N-(4-hydroxy-2-
methylphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide; Tocris,
Ki[5–10 lM), arachidonoyl ethanolamide (AEA, known as
anandamide) transport inhibitor, and JZL184 (4-nitro-
phenyl-4-(dibenzo[d][1,3]dioxol-5-yl(hy-
droxy)methyl)piperidine-1-carboxylate; Tocris), inhibitor
of monoacylglycerol lipase (MAGL), were dissolved in 10%
tocrisolve and in 10% DMSO in isotonic saline, respectively.
The drug used as a hyperalgesic agent was PGE2 (Sigma,
USA) dissolved in 2% ethanol in isotonic saline.
Experimental protocol
Tingenone (50 and 200 lg/paw) was administered subcu-
taneously in the right hind paw 3 h after local injections of
PGE2. AM251, AM630, MAFP, VDM11, and JZL184
were administered 10 min prior to tingenone. It should be
noted that the protocols concerning dose and time of
administration of each drug used in this study were
obtained from literature data and pilot experiments (Silva
et al. 2012; Romero et al. 2013a; Veloso et al. 2014b).
Statistical analysis
The obtained results were analyzed using GraphPad Prism
6.0 and expressed as mean ± SEM. Statistically differ-
ences between groups were calculated by one-way
ANOVA followed by the Bonferroni test. Statistically
significance was set at p \ 0.05.
Results
To verify the participation of the cannabinoid receptor in
the peripheral antinociceptive effect induced by tingenone,
AM630 and AM251 were used, selective antagonists to
CB2 and CB1 cannabinoid receptors, respectively.
Administration of AM630 (50; 100; and 200 lg/paw), a
selective antagonist to CB2 cannabinoid receptor, pre-
vented in a dose-dependent (p \ 0.05) manner the
peripheral antinociceptive effect of the tingenone
(200 lg/paw; Fig. 2). However, AM251 (80 lg/paw), a
selective antagonist to CB1 cannabinoid receptor, did not
alter the peripheral antinociceptive effect of the triterpene
(200 lg/paw; Fig. 2). It was observed that intraplantar
injection of AM630, when given at the highest dose and
alone, did not induce antinociception or hyperalgesia
(Fig. 2).
To evaluate the involvement of endocannabinoids in the
peripheral antinociceptive effect of tingenone, MAFP
(0.5 lg/paw), FAAH inhibitor, AEA metabolizing enzyme;
Fig. 2 Effect of the intraplantar administration of AM251 and
AM630 on the antinociceptive peripheral effect induced by tingenone.
AM251 and AM630 were administrated 10 min before tingenone.
Each column represents the mean ± SEM of the D, measuring the
nociceptive threshold expressed in grams (g), concerning four
animals. *Statistical significance p \ 0.05 when compared to the
control group PGE2 2 lg ? V1 ? V2 and #p \ 0.05 when compared
to the group PGE2 2 lg ? V1 ? Ting 200 lg. V1 (10% DMSO in
saline); V2 (20% DMSO ? 1% Tween 20 in saline) and Et (ethanol).
D = difference of the nociceptive threshold obtained in the beginning
of the experiment, basal value, before any injection (time zero),
regarding the threshold measured 10 min after the third-hour after the
PGE2 injection (time in which the maximum antinociceptive effect of
tingenone is observed)
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VDM11 (2.5 lg/paw), AEA transport inhibitor; and
JZL184 (3.8 lg/paw) inhibitor of MAGL, 2-arachidonoyl
glycerol (2-AG) metabolizing enzyme, were used. These
drugs did not potentiate the peripheral antinociceptive
effect of the lower dose of tingenone (50 lg/paw) (Figs. 3,
4, and 5, respectively).
Discussion
Several works have shown that triterpenes induce periph-
eral antinociception by activation of the cannabinoid
receptors and endocannabinoids. It was demonstrated that
pentacyclic triterpenes inhibit MAGL, an enzyme that
metabolizes 2-AG endocannabinoid (King et al., 2009;
Stein and Machelska, 2011; Chicca et al. 2012). Dutra et al.
(2011) reported that euphol exhibited pronounced and
long-lasting oral analgesia in several rodent behavior
models of inflammatory and neuropathic persistent pain.
These effects were markedly blocked by CB1 or CB2-se-
lective antagonists. In the present study, the involvement of
CB2 cannabinoid receptor in the peripheral antinociceptive
effect of tingenone was demonstrated, confirmed by
administration of a selective antagonist of CB2 cannabinoid
receptors, AM630, that prevented the peripheral
Fig. 3 Effect of the intraplantar administration of MAFP on the
antinociceptive peripheral effect induced by tingenone. MAFP was
administrated 10 min before tingenone. Each column represents the
mean ± SEM of the D, measuring the nociceptive threshold
expressed in grams (g), concerning four animals. *Statistical signif-
icance p \ 0.05 when compared to the control group PGE2
2 lg ? V1 ? V2. V1 (3% Et in saline); V2 (20% DMSO ? 1%
Tween 20 in saline) and Et (ethanol). D = difference of the
nociceptive threshold obtained in the beginning of the experiment,
basal value, before any injection (time zero), regarding the threshold
measured 10 min after the third-hour after the PGE2 injection (time in
which the maximum antinociceptive effect of tingenone is observed)
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C. C. Veloso et al.
Fig. 4 Effect of the intraplantar administration of VDM11 on the
antinociceptive peripheral effect induced by tingenone. VDM11 was
administrated 10 min before tingenone. Each column represents the
mean ± SEM of the D, measuring the nociceptive threshold
expressed in grams (g), concerning four animals. *Statistical signif-
icance p \ 0.05 when compared to the control group PGE2
2 lg ? V1 ? V2. V1 (10% Tocrisolve in saline); V2 (20%
DMSO ? 1% Tween 20 in saline) and Et (ethanol). D = difference
of the nociceptive threshold obtained in the beginning of the
experiment, basal value, before any injection (time zero), regarding
the threshold measured ten minutes after the third-hour after the PGE2
injection (time in which the maximum antinociceptive effect of
tingenone is observed)
antinociceptive effect of tingenone. On the other hand, the
treatment with the CB1 receptor antagonist (AM251) was
unable to reverse the peripheral antinociceptive effect of
tingenone (200 lg/paw) at the maximum dose, previously
reported to reverse the effect of a CB1 cannabinoid receptor
agonist (Romero et al. 2013a). The selectivity of these
antagonists can be confirmed in the previous studies of
several research groups (Guindon et al. 2011; Reis et al.
2011; Romero et al. 2013a, 2013b; Desroches et al. 2014).
It is known that the end of the process of endocannabinoids
action is via AEA uptake in the synaptic space through
membrane transporters already identified (Kress and Kuner
2009), that may be inhibited by the use of VDM11 (Mur-
illo-Rodrı́guez et al. 2013). After reuptake, AEA is
degraded by FAAH, to arachidonic acid and ethanolamine
(Di Marzo 2009), besides, this enzyme is blocked by
MAFP (Deutsch et al. 2001). Furthermore, other sub-
stances, such as JZL184 (Pihlaja et al. 2015), a selective
MAGL inhibitor, are responsible for the degradation of
another major endocannabinoid, 2-AG.
Thus, a compound that acts through the release of
endocannabinoids will have its antinociceptive effect
potentiated by inhibition of both events (uptake and/or
degradation). In literature, FAAH and MAGL inhibitors,
given systemically, may result in antinociception (Kinsey
 Author's personal copy
Tingenone, a pentacyclic triterpene, induces peripheral antinociception due to cannabinoid…
Fig. 5 Effect of the intraplantar administration of JZL184 on the
antinociceptive peripheral effect induced by tingenone. JZL184 was
administrated 10 min before tingenone. Each column represents the
mean ± SEM of the D, measuring the nociceptive threshold
expressed in grams (g), concerning four animals. *Statistical signif-
icance p \ 0.05 when compared to the control group PGE2
2 lg ? V1 ? V2 and #p \ 0.05 when compared to the group PGE2
2 lg ? V1 ? Ting 200 lg. V1 (20% DMSO in saline); V2 (20%
DMSO ? 1% Tween 20 in saline) and Et (ethanol). D = difference
of the nociceptive threshold obtained in the beginning of the
experiment, basal value, before any injection (time zero), regarding
the threshold measured ten minutes after the third-hour after the PGE2
injection (time in which the maximum antinociceptive effect of
tingenone is observed)
et al. 2009; Clapper et al. 2010; Kinsey et al. 2011a; Kinsey
et al. 2011b). In our study, it was found that neither
VDM11 nor MAFP were able to enhance the antinoci-
ceptive effect of tingenone (Rang et al. 2007). This result
was expected since MAFP is a FAAH inhibitor, the
enzyme responsible for anandamide degradation; VDM11,
anandamide reuptake inhibitor; and JZL184, inhibitor of
MAGL, enzyme that metabolizes 2-AG (Stein and
Machelska 2011), did not potentiate the peripheral
antinociceptive effect of tingenone. Anandamide has more
specificity to CB1 cannabinoid receptor than CB2
cannabinoid receptor (Ki = 89 and 371 nM at CB1 and
CB2, respectively). On the other hand, 2-AG (Ki = 472
and 1400 nM for CB1 and CB2, respectively) acts as a full
and potent agonist at both receptors, unlike anandamide
(Rang et al. 2007); it is interesting to verify whether the
reduction of 2-AG degradation could potentiate the
antinociceptive effect of tingenone. Despite the antihy-
peralgesic effect of 2-AG to be mediated by activation of
CB2 cannabinoid receptor and not by CB1 cannabinoid
receptor (Guindon et al. 2007; Khasabova et al. 2011),
surprisingly JZL184 did not potentiate the peripheral
antinociceptive effect of tingenone. According to our
results, we do not suggest the involvement of AEA or
2-AG, once degradation and transport of these substances
are inhibited, an increase in the antinociceptive effect
induced by tingenone would be expected. We also do not
suggest the involvement of CB1 cannabinoid receptor,
since the pretreatment with the antagonist of the CB1
cannabinoid receptor did not alter the peripheral antinoci-
ceptive effect of tingenone. As degradation and transport
inhibitors of anandamide did not potentiate tingenone-in-
duced antinociception, we hypothesize that tingenone
neither binds to CB1 cannabinoid receptor nor releases
anandamide.
Therefore, according to our results, tingenone presented
specificity to CB2 cannabinoid receptor, in a similar way as
other triterpenes. We observed that 2-AG does not seem to
participate in the antinociception induced by tingenone,
once the inhibition of MAGL did not alter the peripheral
antinociceptive effect, and therefore, our results demon-
strate that the antinociception induced by tingenone is
related to activation of CB2 cannabinoid receptor and not
CB1, perhaps binding directly on CB2 receptor.
Seeking plausible explanations for the action of tin-
genone in the cannabinoid receptors, previous studies
demonstrated that CB2 cannabinoid receptor agonists
induce antinociceptive effects through activation of opioi-
dergic system (Anand et al. 2009). Indeed, Ibrahim et al.
(2005) demonstrated that antinociception induced by
intraplantar injection of specific CB2 cannabinoid receptor
agonist is mediated via stimulation of release of b-endor-
phin by keratinocytes. The participation of cannabinoid in
the peripheral antinociceptive mechanisms of opioids has
also been documented. Pacheco et al. (2008) demonstrated
that antinociception induced by activation of l-opioid
receptors was antagonized by CB1 receptor antagonist and
partially by CB2 cannabinoid receptor antagonist. There-
fore, the activation of cannabinoid receptors releases
endogenous opioid and the activation of cannabinoid
receptors contributes for the peripheral effects of opioids.
In the present study, it was demonstrated that tingenone-
induced peripheral antinociceptive effect with involvement
of CB2 cannabinoid receptor. Veloso et al. (2014b)
demonstrated the involvement of the endogenous opioid
peptides in the tingenone-induced peripheral antinocicep-
tion. Thus, we suggest that tingenone could release
selective endocannabinoids for CB2 cannabinoid receptors,
and contributes, at least in part, for the peripheral
antinociceptive effects of opioids induced by tingenone, or
it might release endogenous opioids which in turn release
endocannabinoids selectively acting on CB2 cannabinoid
receptors. This argument is strengthened, since both opi-
oids (Ferreira et al. 1991; Rodrigues and Duarte 2000;
Pacheco et al. 2005) and cannabinoids (Reis et al. 2011;
Romero et al. 2012; Romero and Duarte 2012) induce
peripheral antinociception by activation of the L-arginine/
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 Author's personal copy
NO/cGMP/K? channels pathway, and by a previous study
that demonstrated the peripheral antinociceptive effect of
tingenone by activation of L-arginine/NO/cGMP/potassium
channels pathway (Veloso et al. 2015). Thus, we suggest
that the activation of cannabinoid receptors, as well as
opioid, could be related to the activation of nitric oxide
pathway and potassium channels.
This is the first study that describes that the peripheral
antinociceptive effect of tingenone is dependent on CB2
cannabinoid receptors activation, but not CB1, discarding
the participation of AEA and 2-AG in this event, with a
possible participation of other endocannabinoids, that may
contribute for the peripheral antinociceptive effects of
opioids induced by tingenone. Therefore, tingenone could
be used in the future as a systemic and local analgesic,
promoting pain relief without some of the psychoactive
effects elicited by activation of CB1 cannabinoid receptors.
Compliance with ethical standards
Conflict of interest The authors declare that there are no conflicts of
interest.
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