molecules

Article
Antinociceptive Effect of Hinokinin and Kaurenoic
Acid Isolated from Aristolochia odoratissima L.
Rosa Mariana Montiel-Ruiz 1 , Marcos Córdova-de la Cruz 2 , Manasés González-Cortázar 1, *,
Alejandro Zamilpa 1 , Abraham Gómez-Rivera 2 , Ricardo López-Rodríguez 2 ,
Carlos Ernesto Lobato-García 2 and Ever A. Blé-González 2, *
1 Centro de Investigación Biomédica del Sur (CIBIS), Instituto Mexicano del Seguro Social (IMSS),
Argentina No. 1, Col. Centro, Xochitepec 62790, Morelos, Mexico; montielrmariana@gmail.com (R.M.M.-R.);
azamilpa_2000@yahoo.com.mx (A.Z.)
2 Universidad Juárez Autónoma de Tabasco, Carretera Cunduacán-Jalpa Km. 0.5, Cunduacán 86690, Tabasco,
Mexico; marcoscordovaqfb@gmail.com (M.C.-d.l.C.); abgori@gmail.com (A.G.-R.);
richard_lorr@hotmail.com (R.L.-R.); Carlos.lobato@ujat.mx (C.E.L.-G.)
* Correspondence: gmanases@hotmail.com (M.G.-C.); Ever.ble@ujat.mx (E.A.B.-G.);
Tel.: +52-993-358-1500 (ext. 4711) (E.A.B.-G.)

Academic Editor: Natalizia Miceli




Received: 28 February 2020; Accepted: 16 March 2020; Published: 24 March 2020

Abstract: Aristolochia odoratissima L. is employed for the treatment of pain and as an antidote against
the poison of venomous animals in traditional medicine. However, reports have not been found, to
our knowledge, about the evaluation of the antinociceptive activity of extracts nor about the presence
of compounds associated with this activity. Thus, the purpose of this work was to evaluate the
antinociceptive activity of extracts and compounds isolated from the stems of Artistolochia odoratissima
L. The extracts were obtained with solvents of increasing polarity and the compounds were isolated
and characterized by column chromatography, HPLC, and NMR. The antinociceptive activity was
carried out by the formalin test in mice. Ethyl acetate (AoEA) and methanolic (AoM) extracts
decreased the paw licking in both phases of the formalin test. The isolated compounds (kaurenoic
acid and hinokinin) from AoEA showed the highest antinociceptive activity in both phases of the
formalin test. These results confirmed the analgesic effect of this specie described in traditional
medicine and provided a base for a novel analgesic agent. They also allowed an approach for the
development of standardized plant extracts with isolated metabolites.

Keywords: Aristolochia odoratissima L.; antinociception; hinokinin; kaurenoic acid; formalin test

1. Introduction
Pain is a complex experience that involves affective, motivational and cognitive aspects, and act
as an alarm system to minimize the contact with noxious stimuli [1]. However, when pain persists,
it constitutes a public health problem that affects the quality of life of persons who suffer it and therefore,
the presence of pain may become a socioeconomic burden. It has been estimated that in the first two
decades of the XXI century, more than 5.5 billion people (80% of the world population) live in countries
with low or null access to treatment for pain [2]. In addition, studies have shown that approximately
60 million persons suffer from chronic pain. Also, the World Health Organization (WHO) indicates
that, each year, one in 10 adults are diagnosed with chronic pain associated with a particular disease,
such as rheumatoid arthritis, cancer, diabetes, or post-surgical lesions and procedures [3].
The treatment for this problem is mainly constituted by the administration of non-steroidal
anti-inflammatory drugs (NSAIDs), which produce an anti-inflammatory, antipyretic, and analgesic
effect and include a broad variety of formulations and doses [4]. NSAIDs have the capacity to

Molecules 2020, 25, 1454; doi:10.3390/molecules25061454 www.mdpi.com/journal/molecules

Molecules 2020, 25, 1454 2 of 11

block the synthesis of prostaglandins (PGs) by the inhibition of the cyclooxygenase enzymes (COX-1
and COX-2). It is known that COX-1 produces PGs and thromboxane A2, which controls the
mucosal barrier in the gastrointestinal tract, renal homeostasis, platelet aggregation, and other
physiological functions. While, COX-2 produces PGs related with inflammation, pain, and fever [5].
Another important group of analgesics are opioids, which have the capacity to act on central and
peripheral sites, they inhibit pain pathways by binding with opioid receptors [6], which are widely
distributed in the central and peripheral nervous system, and in the gastrointestinal tract [7]. However,
the continuous use of analgesics generates adverse effects, such as gastrointestinal damage, renal
toxicity, tolerance, respiratory depression, and physical dependence [8–10]. Thus, the search for novel
and effective analgesic compounds constitutes a challenge. In this context, pharmacological studies
have been conducted on the extracts of plants employed in traditional medicine. This research have
led to the isolation of compounds which have been identified to exhibit both antinociceptive and
anti-inflammatory effects [11]. For example, phytochemical analysis furofuran lignans isolated from
Pleurothyrium cinereum and Ocotea macrophylla exert a selective inhibitory activity against COX-1 and
COX-2; similarly terpenes such as lemnalol and madecassoside isolated from several plant species
have shown a selective activity against COX-2 [12,13].
Aristolochia odoratissima L. (Aristolochiaceae family) is commonly known as cocoba or guaco in
Tabasco, Mexico. It is a climber plant that is distributed in tropical and subtropical climates in
Mexico. Aristolochia odoratissima is employed for the treatment of pain and as an antidote against
snake venom in traditional medicine [14]. The parts used are the stem and the rhizome as an infusion
in water. Chemical study of the essential oil of the leaf of this plant reported the presence of a high
proportion of sesquiterpenes and, in minor proportion, mono- and diterpenes [15]. To date, a study has
confirmed the effect of Aristolochia odoratissima against snake venom and the isolation of (−)-cubebin
and aristolochic acid from the essential oil [16,17]. To our knowledge, phytochemical analysis and the
antinociceptive activity of Aristolochia odoratissima has not been investigated. Therefore, the present
study was undertaken to demonstrate the antinociceptive effect of extracts and isolated compounds
obtained from the stem of Aristolochia odoratissima using chemical induced nociception in mice.

2. Results

2.1. Antinociceptive Effect of the Extracts of Aristolochia odoratissima

We employed 800 g of dry and ground material for the extraction procedure and we obtained the
following amounts and percentages of each of the extracts: AoEA (17 g, 2.12%); AoM (22 g, 2.75%), and
AoA (20 g, 2.5%). Figure 1 shows the antinociceptive effect of AoEA extract in formalin tests. In the
early phase, formalin administration in the right hind paw of mice produced nociceptive responses of
licking and biting of the injected paw. Administration of AoEA extract (100 mg/kg, i.p.) produced a
significant (p < 0.001) inhibition of the nociceptive response (41%). This effect was higher than the
one produced by the administration of 30 mg/kg of diclofenac (15%) and it was similar to the effect
produced by 5 mg/kg of tramadol (42%). In the late phase, the effect of the AoEA extract in nociceptive
response was significant (p < 0.05 and 0.001) at a dose rate of 30 (55%) and 100 (66%) mg/kg. The effect
of diclofenac (30 mg/kg) and tramadol (5 mg/kg) were 58% and 72%, respectively.
The antinociceptive effect generated by the AoM extract is presented in Figure 2. In phase 1, the 30
and 100 mg/kg doses significantly inhibited pain with percentages values of 31% and 28%, respectively.
However, in phase 2, the 30 mg/kg dose did not have a significant effect. At 30 and 100 mg/kg dose of
AoM, the antinociceptive effect was 48 and 42%, respectively, these values have a significant difference
when compared to the control group (DMSO, 3%) but their effect is lower than the reference drugs.
The effect of the aqueous extract (AoA) is presented in Figure 3. Both the 30 and 100 mg/kg doses
of AoA extract did not show significant difference when compared to the vehicle group in both phases
of the formalin test in mice.
 E a r ly p h a s e L a te p h a s e
V e h (D M S O , 3 % )
D ic (3 0 m g /k g )

T r a (5 m g /k g )

L ic k in g tim e (s )
200
A o E A (3 0 m g /k g )

Molecules 2020, 24, x FOR PEER REVIEW A o E A (1 0 0 m g /k g ) 3 of 11

Molecules 2020, 25, 1454 **
3 of 11
**
**
100
** ** **
300
E a r ly p h a s e L a te p h a s e
V e h (D M S O , 3 % )
D ic (3 0 m g /k g )
0
L ic k in g tim e (s )
T r e a t m e n t (i.p .) T r a (5 m g /k g )
200
A o E A (3 0 m g /k g )

A o E A (1 0 0 m g /k g )
Figure 1. Effect of the ethyl acetate extract of Aristolochia odoratissima
** (AoEA, 30 and 100 mg/kg, i.p.) in the
**
formalin test (2.5%,
100 25 µL/paw) in mice. The data are expressed with
**
the mean ± sem. One-way ANOVA
**
+ Dunnett test, * p < 0.05 and *** p* < 0.001. Veh: Vehicle; Dic:
**
Diclofenac, Tra: Tramadol.

The antinociceptive effect generated by the AoM extract is presented in Figure 2. In phase 1, the 30
0
and 100 mg/kg doses significantly inhibited pain with percentages values of 31% and 28%, respectively.
T r e a t m e n t (i.p .)
However, in phase 2, the 30 mg/kg dose did not have a significant effect. At 30 and 100 mg/kg dose of
AoM, the antinociceptive
Figure 1. Effect of theeffect was 48extract
ethyl acetate and 42%, respectively,
of Aristolochia these (AoEA,
odoratissima values 30
have
anda100
significant difference
mg/kg, i.p.) in
Figure 1. Effect test
of the ethyl25
acetate extract of Aristolochia odoratissima (AoEA, 30 and 100±mg/kg, i.p.) in the
when compared to the control group (DMSO, 3%) but their effect is lower than the reference drugs.
the formalin (2.5%, µL/paw) in mice. The data are expressed with the mean sem. One-way
formalin
ANOVA test
+ (2.5%,
Dunnett 25test,
µL/paw)
* p < in mice.
0.05 and The
** p data are Veh:
< 0.001. expressed with
Vehicle; theDiclofenac,
Dic: mean ± sem.Tra:One-way ANOVA
Tramadol.
+ Dunnett test, * p < 0.05 and ** p < 0.001. Veh: Vehicle; Dic: Diclofenac, Tra: Tramadol.
E a r ly p h a s e L a te p h a s e
300

The antinociceptive effect generated by the AoM extract is presented inV eFigure
h ( D M S2.O In) phase 1, the 30

and 100 mg/kg doses significantly inhibited pain with percentages values of 31% and 28%,
D i c ( 3 0 m g / k g) respectively.
T ra (5 0 m g /k g )
However, in phase 2, the 30 mg/kg dose did not have a significant effect. At 30 and 100 mg/kg dose of
L ic k in g tim e (s )

200 A o M (3 0 m g /k g )
AoM, the antinociceptive effect was 48 and 42%, respectively, these values Ahave a significant difference
o M (1 0 0 m g /k g )
*
when compared to the control group (DMSO, 3%) but their effect
* is lower than the reference drugs.
**
100 *
E a r l y p h a*s e L a t e* p* h a s e
300 **

V e h (D M S O )
D ic (3 0 m g /k g )
0 T ra (5 0 m g /k g )
L ic k in g tim e (s )

200 T r e a t m e n t, i .p . A o M (3 0 m g /k g )
* A o M (1 0 0 m g /k g )
Figure 2. Effect of the methanolic extract of Aristolochia odoratissima
* (AoM, 30 and 100 mg/kg, i.p.) in
Figure 2. Effect of the methanolic extract of Aristolochia
the formalin test (2.5%, 25 µL/paw) in mice. The data
** odoratissima (AoM,
are expressed with30the
and 100 ±
mean mg/kg, i.p.) in the
sem. One-way
Molecules 2020, 24, x 1FOR
0 0 PEERtest,
REVIEW 4 of 11
ANOVA + Dunnett * p < 0.05* and **
* p <
formalin test (2.5%, 25 µL/paw) in mice. The data are expressed
0.001. Veh:
** with the mean ± sem. One-way ANOVA
Vehicle; Dic: Diclofenac, Tra: Tramadol.
**
+ Dunnett test, * p < 0.05 and ** p < 0.001. Veh: Vehicle; Dic: Diclofenac, Tra: Tramadol.
E a r ly p h a s e L a te p h a s e
300
The effect of the
0
aqueous extract (AoA) is presented in Figure 3. Both the V e30
h and 100 mg/kg doses of
D ic (3 0 m g /k g )
AoA extract did not show significant Tdifference
r e a t m e n t , when
i .p . compared to the vehicle group in both phases of
T ra (5 0 m g /k g )
the formalin test in mice. A o A (3 0 m g /k g )
T im e o f lik in g (s )

200 A o A (1 0 0 m g /k g )
Figure 2. Effect of the methanolic extract of Aristolochia odoratissima (AoM, 30 and 100 mg/kg, i.p.) in the
formalin test (2.5%, 25 µL/paw) in mice. The data are expressed with the mean ± sem. One-way ANOVA
+ Dunnett test, * p < 0.05 and ** p < 0.001. Veh: Vehicle; Dic: Diclofenac, Tra: Tramadol.
**
100
The effect of the aqueous extract
** (AoA) is presented* *in Figure 3. Both the 30 and 100 mg/kg doses of
AoA extract did not show significant difference when compared to the vehicle group in both phases of
the formalin test in mice.
0
T r e a t m e n t, i .p .

Figure 3. Effect of the aqueous extract of Aristolochia odoratissima (AoA, 30 and 100 mg/kg, i.p.) in
Figure 3. Effecttest
the formalin of the aqueous
(2.5%, extractinof
25 µL/paw) Aristolochia
mice. The dataodoratissima (AoA,
are expressed 30the
with and 100 ±
mean mg/kg, i.p.) in the
sem. One-way
formalin
ANOVA test
+ (2.5%,
Dunnett 25test,
µL/paw)
* p < in mice.
0.05 and The
** p data are Veh:
< 0.001. expressed with
Vehicle; theDiclofenac,
Dic: mean ± sem.Tra:One-way ANOVA
Tramadol.
+ Dunnett test, * p < 0.05 and ** p < 0.001. Veh: Vehicle; Dic: Diclofenac, Tra: Tramadol.

2.2. Antinociceptive of Kaurenoic Acid (1) and Hinokinin (2) Isolated from Aristolochia odoratissima
The administration of kaurenoic acid and hinokinin at a dose rate of 15 mg/kg (i.p.) produced a
significant (p < 0.001) antinociceptive effect compared to the vehicle group (DMSO, 3%) in both early and
late phases of the experiment. Kaurenoic acid and hinokinin decreased by 38% and 48%, respectively, the
paw licking time in early phase, as well as 69% and 82% respectively, in the late phase of the formalin
 0
T r e a t m e n t, i .p .

Figure 3. Effect of the aqueous extract of Aristolochia odoratissima (AoA, 30 and 100 mg/kg, i.p.) in the
Molecules 2020, 25, 1454 4 of 11
formalin test (2.5%, 25 µL/paw) in mice. The data are expressed with the mean ± sem. One-way ANOVA
+ Dunnett test, * p < 0.05 and ** p < 0.001. Veh: Vehicle; Dic: Diclofenac, Tra: Tramadol.
2.2. Antinociceptive of Kaurenoic Acid (1) and Hinokinin (2) Isolated from Aristolochia odoratissima
2.2. Antinociceptive of Kaurenoic Acid (1) and Hinokinin (2) Isolated from Aristolochia odoratissima
The administration of kaurenoic acid and hinokinin at a dose rate of 15 mg/kg (i.p.) produced
The administration
a significant of kaurenoic
(p < 0.001) acid and effect
antinociceptive hinokinin at a dose
compared rate
to the of 15 group
vehicle mg/kg (DMSO,
(i.p.) produced a
3%) in both
significant
early(pand
< 0.001)
late antinociceptive effect compared
phases of the experiment. to the acid
Kaurenoic vehicle
andgroup (DMSO,
hinokinin 3%) in both
decreased early
by 38% and
and 48%,
late phases
respectively, the paw licking time in early phase, as well as 69% and 82% respectively, in the latethe
of the experiment. Kaurenoic acid and hinokinin decreased by 38% and 48%, respectively, phase
paw licking
of thetime in early
formalin testphase, as well
in mice. Theseasresults
69% and
are82% respectively,
higher in theshown
than the effect late phase of the
by both formalinand
diclofenac
test in mice. These
tramadol results4).are higher than the effect shown by both diclofenac and tramadol (Figure 4).
(Figure

300
E a r ly p h a s e L a te p h a s e V e h (D M S O 3 % )
D ic (3 0 m g /k g )
T ra (5 0 m g /k g )
T im e o f lik in g (s )

K a u r e n o ic a c id (5 m g /k g )
200
K a u r e n o ic a c id (1 5 m g /k g )
H in o k in in (5 m g /k g )
H in o k in in (1 5 m g /k g )
**
100
* **
** *
**

0
T r e a t m e n t, i .p .

Effect4.ofEffect
Figure 4.Figure of the kaurenoic
the kaurenoic acid andacid and hinokinin
hinokinin (5 and
(5 and 15 15 mg/kg,
mg/kg, from Aristolochia
i.p.)Aristolochia
i.p.) from odoratissima
odoratissima in
in the formalin test (2.5%, 25 µL/paw) in mice. The data are expressed with
the formalin test (2.5%, 25 µL/paw) in mice. The data are expressed with the mean ± sem. One-way the mean ± sem. One-way
ANOVAANOVA+ Dunnett + Dunnett
test, * p <test, < 0.05
0.05* pand ** p and
< 0.001. < 0.001.
** p Veh: Veh: Dic:
Vehicle; Vehicle; Dic: Diclofenac,
Diclofenac, Tra: Tramadol.
Tra: Tramadol.
2.3. Characterization by NMR of the Compounds (1–3)
2.3. Characterization by NMR of the Compounds (1–3)
Compound 1 was obtained as dichloromethane-soluble colorless crystals. The 13 C NMR spectrum
Molecules 2020, 24, x FOR PEER REVIEW 5 of 11
Compound
of 1, showed1 was20 obtained as dichloromethane-soluble
characteristic colorlessand
signals for a diterpene structure crystals. The 13to
according C the
NMR spectrum
DEPT of
experiment,
1, showed 20 characteristic signals quaternary
for a diterpene structure and accordingto to the DEPT ten experiment, five as
the spectroscopic
five signals data withtothose
correspond described in the three
carbons, literature [18] lead usmethines,
correspond to indicate that arethis compound
assigned
signals methylenes,
correspond to quaternary carbons, three correspond 1 to methines, ten are assigned as methylenes,
corresponds toand ent-kaur-16-en-19-oic
there are two methyl acid, commonly
groups. The known
H NMRasspectrum
kaurenicexhibited
acid (1) (Figure 5).
a double-linked AB
and there are two methyl groups. The 1H NMR spectrum exhibited a double-linked AB system: [δ 4.81
system: [δ 4.81 (1H, s), 4.75 (1H, s)] (For spectra see Supplementary Figures S1–S6). This evidence and
Compound
(1H, s), (1H,(1):
4.75comparison H-NMR
s)] 1(For (600see
MHz, CDCl3): δ 4.75 (1H, s,S1–S6).
br, H-17a), 4.81 (1H, and
s, br,the
H-17b), 0.96 (3H,
of s, H-
the ofspectra supplementary
the spectroscopic data withFigures
those described This evidence
in the literature [18] comparison
lead us to indicate
20), 1.25 (3H, s, H-18); 13C-NMR (150 MHz, CDCl3): δ 40.7 (C-1), 19.1 (C-2), 37.8 (C-3), 43.7 (C-4), 57.0 (C-
that this compound corresponds to ent-kaur-16-en-19-oic acid, commonly known as kaurenic acid (1)
5), 21.8 (C-6), 41.2 (C-7), 44.2 (C-8), 55.1 (C-9), 39.6 (C-10), 18.4 (C-11), 33.1 (C-12), 43.8 (C-13), 39.7 (C-14),
(Figure 5).
48.9 (C-15), 155.8 (C-16), 102.9 (C-17), 28.9 (C-18), 184.2 (C-19), 15.5 (C-20).

20 11 16 17
14
1
H
3 5

H
OH
18 19
O

Figure 5. Chemical structure of kaurenoic acid (1).

Figure 5. Chemical structure of kaurenoic acid (1).
Compound (1): 1 H-NMR (600 MHz, CDCl3 ): δ 4.75 (1H, s, br, H-17a), 4.81 (1H, s, br, H-17b), 0.96 (3H, s,
Compound 2 was obtained as a dichloromethane-soluble yellow oil. The 13C NMR spectrum of 2
H-20), 1.25 (3H, s, H-18); 13 C-NMR (150 MHz, CDCl3 ): δ 40.7 (C-1), 19.1 (C-2), 37.8 (C-3), 43.7 (C-4),
showed 20 signals: Seven quaternary carbons; eight methines and four methylenes (identified by the
57.0 (C-5), 21.8 (C-6), 41.2 (C-7), 44.2 (C-8), 55.1 (C-9), 39.6 (C-10), 18.4 (C-11), 33.1 (C-12), 43.8 (C-13),
DEPT experiment). The 1H NMR spectrum of 2, displayed two ABX aromatic-ring systems: one in [δ 6.63
39.7 (C-14), 48.9 (C-15), 155.8 (C-16), 102.9 (C-17), 28.9 (C-18), 184.2 (C-19), 15.5 (C-20).
(1H, d, 1.4 Hz, H-2), 6.73 (1H, d, 7.7 Hz, H-5) and 6.60 (1H, dd, 1.4, 7.7 Hz, H-6)], and the other in [δ 6.47
(1H, d, 1.8 Hz, H-2′), 6.7 (1H, d, 8.4 Hz, H-5′) and 6.47 (1H, dd, 2.2, 8 Hz, H-6′)]. According to the HMBC
experiment they are correlated with a methylenedioxy group (-O-CH2-O-) located in δ 5.93 (2H, m) and
with two signals in δ 3.0 (1H, dd, 5.1, 14.3 Hz, H-7a) and 2.84 (1H, dd, 7.3, 14.3 Hz, H-7b), which indicates
the presence of a benzyl fragment that is attached to a tetrahydrofuran ring, resulting in a lignan-type
 Molecules 2020, 25, 1454 5 of 11
Molecules 2020, 24, x FOR PEER REVIEW 5 of 11

the spectroscopic
Compounddata with
2 was those described
obtained in the literature [18]yellow
as a dichloromethane-soluble lead us
oil.toThe
indicate that this
13 C NMR compound
spectrum of 2
corresponds
showed 20to ent-kaur-16-en-19-oic
signals: acid,
Seven quaternary commonly
carbons; eightknown
methinesas and
kaurenic acid (1) (Figure
four methylenes 5).
(identified by the
1 NMR spectrum of 2, displayed two ABX aromatic-ring systems: one in [δ
Compound (1): 1H-NMR
DEPT experiment). (600HMHz,
The CDCl3): δ 4.75 (1H, s, br, H-17a), 4.81 (1H, s, br, H-17b), 0.96 (3H, s, H-
20), 1.25 (3H, s, H-18); C-NMR (150d,MHz,
6.63 (1H, d, 1.4 Hz, H-2),
13 6.73 (1H, 7.7 Hz,CDCl
H-5)3):and
δ 40.7
6.60(C-1), 19.11.4,
(1H, dd, (C-2), 37.8H-6)],
7.7 Hz, (C-3),and
43.7the
(C-4), 57.0
other (C-
in [δ
0 ), 6.7 (1H, d, 8.4 Hz, H-50 ) and 6.47 (1H, dd, 2.2, 8 Hz, H-60 )]. According to the
5), 21.8 (C-6), 41.2 (C-7), 44.2 (C-8), 55.1 (C-9), 39.6 (C-10), 18.4 (C-11), 33.1 (C-12), 43.8 (C-13), 39.7 (C-14),
6.47 (1H, d, 1.8 Hz, H-2
48.9 (C-15),
HMBC 155.8 (C-16),
experiment they102.9 (C-17), 28.9
are correlated (C-18),
with 184.2 (C-19), 15.5
a methylenedioxy group(C-20).
(-O-CH2 -O-) located in δ 5.93 (2H,
m) and with two signals in δ 3.0 (1H, dd, 5.1, 14.3 Hz, H-7a) and 2.84 (1H, dd, 7.3, 14.3 Hz, H-7b), which
indicates the presence of a benzyl fragment that is attached to a tetrahydrofuran ring, resulting in a
20 11 16 17
lignan-type skeleton (For spectra see Supplementary Figures S7–S12). According to these spectroscopic
14
analysis (Table 1) and the comparison with the data described in the literature [19,20] lead us to
1
indicate that this compound corresponds to (3R, 4R)-3,4-bis(1,3-benzodioxol-5-ylmethyl)oxolan-2-one
(2), known as hinokinin (Figure 6). H
3 5
Table 1. NMR spectroscopy data (1 H and 13 C, in CDCl3 ) of compounds 2 and 3.
H
δ 13 C δ 1 H (JOH
in Hz) δ 13 C δ 1 H (J in Hz)
Position
2
18 19 2 3 3
1 131.5 O 134.6
2 109.3 6.63 (1H, d, 1.4) 109.5 6.58 (1H, s, br)
3 147.8 147.9
4 146.4
Figure5. Chemical structure of kaurenoic 146.1
acid (1).
5 108.2 6.73 (1H, d, 7.7) 108.3 6.69 (1H, d, 7.7)
Compound
6 2 was obtained
122.1 asa dichloromethane-soluble
6.60 (1H, dd, 1.4, 7.7) yellow oil. The
122.1 6.5513 C NMR
(1H, d, br, 7.7)spectrum of 2
a 3.0 (1H, dd, 5.1, 14.3) a 2.53 (1H, dd, 6.2, 13.9)
showed 20 signals: Seven quaternaryb carbons;
7 38.3
2.84 (1H, dd,eight
7.3, 14.3)methines and four methylenes
39.3 (identified
b 2.53 (1H, dd, 7.3, 13.5) by the
DEPT experiment).
8 The 1H46.4NMR spectrum 2.53 (1H,of 2, displayed
ddd, 5.1,7.7, 8.0) two ABX52.6 aromatic-ring 2.12systems:
(1H, m) one in [δ 6.63
9 178.3 104.6 5.14 (1H, d, 1.6)
(1H, d, 1.4 Hz,1H-2),
0 6.73 (1H,
131.2 d, 7.7 Hz, H-5) and 6.60 (1H, dd, 1.4, 133.8 7.7 Hz, H-6)], and the other in [δ 6.47
(1H, d, 1.8 Hz,2H-2′), 6.7 (1H,
108.7d, 8.4 Hz, H-5′) and 6.47 (1H, dd, 2.2, 109.28 Hz, H-6′)].6.51 According
(1H, s, br) to the HMBC
0 6.47 (1H, d, 1.8)
30 147.8 147.9
experiment they 40 are correlated
146.2 with a methylenedioxy group (-O-CH 146.1 2-O-) located in δ 5.93 (2H, m) and

with two signals50

in δ 3.0 (1H, dd, 5.1, 14.3 Hz, H-7a) and 2.84 (1H, dd, 7.3, 14.3 Hz,
108.1 6.7 (1H, d, 8.4) 108.4
H-7b), which indicates
6.69 (1H, d, 7.7)
60 121.4 6.47 (1H, dd, 2.2, 8) 121.7 6.50 (1H, d, br, 8.0)
the presence of0 a benzyl fragment that is attached
a 2.60 (1H, dd, 9.9, to a tetrahydrofuran ring,
17.2) resulting
a 2.64 (1H, dd, 7.7,in a lignan-type
13.9)
7 34.7 38.7
skeleton (For 8spectra
0 see supplementary
41.2
Figures S7–S12).
b 2.47 (1H, dd, 8.4, 16.8)
2.46 (1H, m)
According
46.0
to these spectroscopic
b 2.43 (1H, dd, 7.3, 13.5)
2.12 (1H, m)
analysis
(Table 1) and the
90
comparison 71.0
with the adata described
4.13 (1H, dd, 6.9, 9.5)in the literature [19,20]a 3.92
72.3
lead(1H,usdd,
to7.3,
indicate
8.4) that this
compound 1-O-CH
corresponds to (3R, 4R)-3,4-bis(1,3-benzodioxol-5-ylmethyl)oxolan-2-one
b 3.86 (1H, dd, 7.3, 9.1)
(2),
b 3.41 (1H, dd, 8.0, 8.4)
known as
2 -O 100.9 5.93 (2H, m) 101.1 5.93 (2H, m)
hinokinin (Figure
2-O-CH2 -O6). 100.9 5.93 (2H, m) 101.1 5.90 (2H, m)
Compound 2: 1H-NMR (600 MHz, CDCl3) and 13C-NMR (150 MHz, CDCl3) (see Table 1).
H O
O 3 1 7
9
8

8'
O
6 9'
O
H
1'

3'

4' O
O
Figure
Figure 6.
6. Chemical
Chemical structure
structure of
of hinokinin
hinokinin (2).
(2).

Compound
Compound 2: 1 H-NMR
3 was obtained(600 MHz,
as an CDCl3 ) and
oil. Analysis
13
of theC-NMR
1H and (150 MHz,spectra,
13C NMR CDCl3 )exhibited
(see Tablesimilarities
1).
Compound 3 was obtained as an oil. Analysis of the 1 H and 13 C NMR spectra, exhibited
with the spectroscopic signals found for compound 2. However, we found the presence of a ketal group
in similarities with the
δ 104.6, which wasspectroscopic
attributed tosignals found forofcompound
the reduction 2. However,
the carbonyl group ofwethe
found the presence
lactone of
in C-9. This
a ketal group in δ 104.6, which was attributed to the reduction of the carbonyl group of the lactone in
C-9. This structural feature was supported by the observation in the proton spectrum of a signal in δ
Molecules 2020, 24, x FOR PEER REVIEW 6 of 11
Molecules 2020, 25, 1454 6 of 11

structural feature was supported by the observation in the proton spectrum of a signal in δ 5.14 (1H, d,
1.65.14
Hz,(1H,
H-9)d,which possesses
1.6 Hz, a HMBC
H-9) which correlation
possesses a HMBC with a C-O signal
correlation inaδC-O
with 72.3signal
assigned
in δto72.3
C-9′. Analysis
assigned
oftothe
C-9spectra ((For of
0 . Analysis spectra see supplementary
the spectra Figures
((For spectra see S13–S18).) Figures
Supplementary allowedS13–S18).)
us to determine
allowedthat this
us to
compound corresponds to the 8,8′ trans-cubebin (3), known
0 as cubebin (Figure 7).
determine that this compound corresponds to the 8,8 trans-cubebin (3), known as cubebin (Figure 7).
Compound 3: 1H-NMR (600 MHz, CDCl3) and 13C-NMR (150 MHz, CDCl3) (see Table 1) [19,20].
H OH
O 3 1 7 9
8

8'
O
6 9'
O 7'

H
1'

3'

4' O
O
0 trans-cubebin (3).
Figure 7.
Figure 7. Chemical
Chemical structure
structure of 8,8′ trans-cubebin
of 8,8 (3).

Compound 3: 1 H-NMR (600 MHz, CDCl ) and 13 C-NMR (150 MHz, CDCl ) (see Table 1) [19,20].
Table 1. NMR spectroscopy data (31H and 13C, in CDCl3) of compounds 23 and 3.

3. Discussion δ 13C δ 1H (J in Hz) δ 13C δ 1H (J in Hz)

Position
2 2 3 3
Aristolochia odoratissima is widely used in Mexican traditional medicine for the treatment of pain
1 131.5 134.6
and as an2 antidote against
109.3 snake venom. In this
6.63 (1H, work, we evaluated
d, 1.4) 109.5 the antinociceptive
6.58 (1H, s, activity
br) of the
extracts 3and compounds 147.8 isolated from the stem of Aristolochia odoratissima
147.9 using a chemically induced
pain test4 in mice. The 146.4formalin test, a method commonly 146.1 employed to study the antinociceptive
activity 5of drugs, has 108.2
been reported6.73 (1H, d, 7.7)
to produce a characteristic108.3 6.69 (1H,response
biphasic nociceptive d, 7.7) [21,22].
The early6 or neurogenic122.1 phase represents
6.60 (1H,paindd, 1.4, 7.7)
centrally 122.1 evoked by
mediated, 6.55
the(1H, d, br,
direct 7.7)
stimulation of
the formalin in the primary a 3.0 C-fibers,
afferent (1H, dd, 5.1, 14.3)
that results in the release a 2.53
of (1H, dd,P,6.2,
substance 13.9)
serotonin, or
7 38.3 39.3
and/or CGRP (calcitonin gene-related b 2.84 (1H, dd, 7.3,Meanwhile,
peptide). 14.3) b 2.53 (1H, dd, phase,
the late or inflammatory 7.3, 13.5)reflects
8 46.4 2.53 (1H, ddd, 5.1,7.7, 8.0) 52.6 2.12 (1H, m)
pain peripherally mediated, that is primarily due to an inflammatory reaction in the peripheral tissue
9 178.3 104.6 5.14 (1H, d, 1.6)
mediated by the release of various mediators such as PG‘s, COX, and nitric oxide (NO), as well as the
1′ 131.2 133.8
sensitizing
2′ of the neurons
108.7 of the dorsal horn of
6.47 (1H,d, 1.8)the spinal cord
109.2[21] Centrally acting
6.51 (1H, s,drugs,
br) such as
opioids,3′inhibit both147.8phases of pain, while peripherally acting147.9 drugs, such as diclofenac, only inhibits
4′
pain during the late146.2
phase [22,23]. 146.1
The5′ results of 108.1
the present study 6.7revealed
(1H, d, 8.4) 108.4
that the intraperitoneal (i.p.)6.69 (1H, d, 7.7)
administration of the
AoEA and 6′ AoM extracts121.4 of the Aristolochia
6.47 (1H, odoratissima
dd, 2.2, 8) stems 121.7 6.50 (1H, d, br, 8.0)
possesses an antinociceptive effect in the
formalin7′test. AoEA 34.7 showed the agreatest
2.60 (1H, dd, 9.9,
effect 17.2)early and late phases,
in both 38.7
a 2.64 (1H, dd, that
suggesting 7.7, 13.9)
the extract
contains metabolites with the capacity to inhibit nociception caused by the formalin at 7.3,
b 2.47 (1H, dd, 8.4, 16.8) b 2.43 (1H, dd, the 13.5)
peripheral
8′ 41.2 2.46 (1H, m) 46.0 2.12 (1H, m)
and central level. Therefore, the mechanism of action of AoEA could be related to the inhibition of
a 4.13 (1H, dd, 6.9, 9.5) a 3.92 (1H, dd, 7.3, 8.4)
9′
COX-1 and/or COX-2 71.0
and other derived inflammatory mediators 72.3 including histamine and bradykinin.
b 3.86 (1H, dd, 7.3, 9.1) b 3.41 (1H, dd, 8.0, 8.4)
In addition,
1-O-CH2-O the effect of
100.9 AoEA may involve the
5.93 (2H, m) inhibition of the activation of the5.93
101.1 nociceptor
(2H, m) present on
afferent
2-O-CHC-fiber
2-O as well
100.9as the release of 5.93glutamate
(2H, m) and substance 101.1P. 5.90 (2H, m)
The presence of lignans in Aristolochia species has been previously described; and a previous work
3. Discussion reported the presence of (−)-cubebin in the essential oil of Aristolochia odoratissima [24–28].
has already
However, this research presents for the first time (to our knowledge) the presence of kaurenic acid
Aristolochia odoratissima is widely used in Mexican traditional medicine for the treatment of pain and
and hinokinin in this medicinal plant. Both kaurenic acid and hinokinin produced the greatest
as an antidote against snake venom. In this work, we evaluated the antinociceptive activity of the extracts
antinociceptive activity at the highest dose evaluated in the formalin test. Previous studies have
and compounds isolated from the stem of Aristolochia odoratissima using a chemically induced pain test
reported the analgesic activity of kaurenoic acid, a diterpene, in thermal-induced pain (tail-flick
in mice. The formalin test, a method commonly employed to study the antinociceptive activity of drugs,
test), as well as in chemical-induced pain such as the intraplantar administration of capsaicin [29].
The mechanism of action of kaurenoic acid is by inhibiting proinflammatory cytokines and the activation
of the NO-cyclic GMP-ATP-K+ channels signaling pathway [30,31]. Other biological activities that
Molecules 2020, 25, 1454 7 of 11

have been reported for this diterpene include anti-inflammatory, hypotensor, and diuretic effects in
in-vivo models, as well as a smooth-muscle relaxant and cytotoxic activity in in-vitro models [32,33].
Hinokinin showed the greatest antinoceptive activity in both phases of the formalin test. The
results indicate that this compound is able to produce both central and peripheral effects. In
terms of the mechanism of action, it has been demonstrated by in vitro assays that hinokinin and
synthetic derivatives selectively inhibit COX-1 and COX-2 [34–36]. Furthermore, previous studies have
reported that this compound has antitumoral, anti-inflammatory, antimicrobial and anti-trypanosomal
activity [34].
Based on these data, this study supports the antinociceptive effect of Aristolochia odoratissima,
which is employed in traditional medicine. Furthermore, the presence of metabolites such as kaurenic
acid and hinokinin could be related to the biological activity.

4. Materials and Methods

4.1. Plant Material

The stems of Aristolochia odoratissima were collected on May 2016, in Poblado C-9 Francisco I.
Madero, Cárdenas, Tabasco, Mexico (18◦ 90 46.84” N; 93◦ 280 14.88” O; 7.0 m.a.s.l.). A specimen was
deposited at the Herbarium of Academic Division of Biological Sciences of the Juárez Autonomous
University of Tabasco for its taxonomic identification (Voucher No. 036502).

4.2. Extraction and Isolation

The stems of Aristolochia odoratissima were dried at room temperature for 72 h and grounded to a
4 mm particle size (Pulvex brand, Mexico City, Mexico). The dry plant material (800 g) was successively
extracted three times with ethyl acetate (2.0 L, Merck, Mexico City, Mexico) at room temperature, after
each extraction, the solvent was filtered and vacuum-concentrated at 40 ◦ C in a rotary evaporator
(Heidolph G3, Heidolph, Schwabach, Germany) to obtain the ethyl acetate extract (AoEA). To obtain the
methanolic (AoM) and aqueous (AoA) extracts, the plant’s dry residue was macerated with methanol
(2.0 L; Merck, Mexico City, Mexico) and later with water (2.0 L; Merck, Mexico City, Mexico) following
the same procedure previously described. All the extracts were lyophilized (Heto Drywinner DW3,
ThermoFisher SCIENTIFIC, Mexico City, Mexico).

4.3. Isolation and Identification of the Compounds

The AoEA extract (7 g) was adsorbed onto silica gel (7 g, Gel 60, Merck, Mexico City, Mexico) and
placed in a silica gel-packed glass column (600 × 6050 mm) (100 g, 70–230 mesh, Merck, Mexico City,
Mexico) as stationary phase and, as mobile phase, a mixture of n-ethyl hexane-acetate was employed
with an increase in polarity of 5% v/v, collecting 33 fractions of 200 mL each. In the AoEA-3, AoEA-9,
and AoEA-11 fractions, only one TLC point was shown, and it was identified as kaurenoic acid (1;
40 mg), hinokinin (2; 35 mg), and cubebin (3, 12 mg), respectively. Only compounds 1 and 2 were
evaluated in the biological model. The chemical characterization of these structures was carried out by
means of the analysis of the 1D and 2D NMR spectra. The spectra were obtained in a BrukerAdvance III
HD-600 (600 MHz, Bruker, Fällanden, Switserland,) equipment and the solvent employed was CDCl3
for the three compounds. The chemical displacements are reported in ppm, while tetramethylsilane
(TMS) was employed as internal standard.

4.4. Experimentation Animals

Male ICR mice with a weight range of 25–30 g, from Envigo RMS S.A. de C.V. (Mexico City,
Mexico), were used throughout the experiments. These animals were maintained in the Bioterium of
Centro de Investigación Biomédica del Sur (CIBIS-IMSS) under 12 h–12 h light-dark cycle and constant
temperature (23–25 ◦ C) with free access to food and water. The experimental protocol was approved by
the Institutional Commission for Ethics in Research of the Juárez Autonomous University of Tabasco,
Molecules 2020, 25, 1454 8 of 11

and received the registration number DI/524/2020. The animals were treated under the Mexican federal
regulations (Norma Oficial Mexicana for care and use of laboratory animals, NOM-062-ZOO-1999) and
the ethical guidelines for the investigation of pain in conscious animals [37]. The number of animals
and the intensity of the noxious stimuli employed were the minimum necessary to demonstrate the
consistent effects of the pharmacological treatments. Mice were used only once during the experiments
and were euthanized in a CO2 chamber immediately after the experiment.

4.5. Drugs and Reagents

The drugs and reagents employed were acquired from Sigma-Aldrich (Toluca, México):
formaldehyde; dimethylsulfoxide (DMSO), diclofenac (Dic), and tramadol (Tra). For the evaluation
of the antinociceptive activity, diclofenac (30 mg/kg, i.p.) and tramadol (5 mg/kg, i.p.) were used as
positive groups and DMSO (3%, diluted in water) as a negative group.

4.6. The Formalin Test

The method used for this test was similar to that described previously by Dubuisson and
Dennis [38] and modified by Hunskaar and Hole [23]. This experimental test consists of placing
each mouse in an open observation chamber to allow them to accommodate to their surroundings.
Each mouse was removed for subcutaneous (s.c.) administration of 25 µL of diluted 2% formalin into
the right hindpaw with a 30-gauge needle. The time of licking and biting of the injected paw was
defined as a nociceptive response, which was recorded in periods of 5 min during 30 min after the
analgesic injection [23]. Nociceptive response is biphasic: the initial (early phase, 0–5 min) results by the
direct stimulation of nociceptors, it is of rapid onset and great intensity. The second (late phase, 15–30
min) is secondary to inflammatory reactions [22,23]. Groups of five animals were intraperitoneally
(i.p.) administered with: Vehicle (DMSO, 3%), diclofenac (30 mg/kg), tramadol (5 mg/kg), extracts (30
and 100 mg/kg), or compounds (5 and 15 mg/kg), 30 min before to the analgesic stimuli. Doses were
chosen by pilot tests and previous studies [39–41].

4.7. Statistical Analysis

The data are expressed as the mean ± standard error of the mean (SEM), and statistical significance
was determined using an analysis of variance (ANOVA) + Dunnett´s test. Values were considered
significant different at * p < 0.05 and ** p < 0.01. All analysis were performed using by GraphPad Prism
ver. 7.0 statistical program (Graph pad Prism version 7.05 for windows, GraphPad Software, La Jolla,
CA, USA, 2018).

5. Conclusions
The present study demonstrated the antinociceptive potential of the extracts and compounds
isolated from the A. odoratissima stem, mainly from the AoEA extract and hinokinin, which were
active in both phases of the formalin model. Therefore, we conclude that the stems of this plant
can be employed as a therapeutic alternative in the treatment of pain. The results obtained permit
the confirmation of the analgesic use of this species in traditional medicine and provide a base for
new studies on antinociceptive activity, as well as the development of standardized extracts with
isolated metabolites.

Supplementary Materials: The following are available online. Figures S1–S18: 1 H NMR (CDCl3, 600 MHz), 13 C
NMR (CDCl3, 150 MHz), DEPTQ (CDCl3 , 150 MHz), 1 H-1 H COSY NMR (CDCl3 , 600 MHz), HSQC (CDCl3 , 600
MHz) and HMBC (CDCl3 , 600 MHz) of Compounds 1–3.
Author Contributions: Formal analysis: M.G.-C., A.Z., R.L.-R. and C.E.L.-G.; Research: E.A.B.-G.; Methodology,
R.M.M.-R., M.C.-d.l.C., A.G.-R. and E.A.B.-G.; Writing—original draft: R.M.M.-R. and E.A.B.-G.; Writing—review
and editing: M.G.-C. and A.G.-R. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Molecules 2020, 25, 1454 9 of 11

Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds 1–3 are available from the authors.

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