Biomedicine & Pharmacotherapy 152 (2022) 113253

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Antinociceptive effect of Mansoa alliacea polar extracts involves opioid

receptors and nitric oxide in experimental nociception in mice
María Guadalupe Valle-Dorado a, Alberto Hernández-León b, Andrés Nani-Vázquez b, Guadalupe
Esther Ángeles-López a, María Eva González-Trujano b, *, Rosa Ventura-Martínez a, *
a
Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de México, Av. Universidad No. 3000, Col. Ciudad Universitaria, Alcaldía
Coyoacán, 04510 Ciudad de México, Mexico
b
Laboratorio de Neurofarmacología de Productos Naturales, Dirección de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatría “Ramón de la Fuente
Muñiz”, Av. México-Xochimilco 101, Col. San Lorenzo Huipulco, 14370 Ciudad de México, Mexico

A R T I C L E I N F O A B S T R A C T

Key words: To evaluate the antinociceptive effect and the possible mechanism of action of two polar extracts of Mansoa
Mansoa alliacea alliacea, a medicinal plant used in Perú, Brazil, and Mexico to treat rheumatic pain, we used the formalin and hot-
Polar extracts plate tests in mice. We found that ethanolic (MA-EtOH) and aqueous (MA-AQ) extracts of M. alliacea induced
Antinociceptive effect
antinociceptive effects in both nociceptive tests. The antinociceptive efficacy of the highest dosage (300 mg/kg)
Nitric oxide pathway
Opioid receptors
of both extracts were also compared by using intraperitoneal and oral administration in the formalin test. Results
Mechanism of action showed that intraperitoneal injection of the two extracts produced better antinociceptive effects than that ob­
tained by their oral administration. The mechanism of action involved in their antinociceptive activity was
determined in the formalin test. Results showed that the presence of A784168 (TRPV1 antagonist) did not alter
the antinociceptive effect induced by any of the M. alliacea extracts, whereas naltrexone (opioid antagonist)
partially prevented the antinociceptive effect only of MA-EtOH in both phases of the formalin test. Furthermore,
the effects of the extracts were diminished by L-NAME (inhibitor of nitric oxide synthase), but not by ODQ
(inhibitor of the soluble guanylyl cyclase) or glibenclamide (blocker of K+ ATP channels) in the neurogenic phase.
However, the effect of MA-AQ was diminished by all the inhibitors in the inflammatory phase. These results
support the use of M. alliacea as a potential natural product with efficacy for pain relief depending on the form of
preparation and the route of administration by involving opioid receptors and the production of nitric oxide.

1. Introduction inflammatory process as well as in the generation of pain [3,4]. How­

Inflammatory pain is a public health problem that affects almost 30%
of the world population [1]. It is known that non-steroidal anti-in­
flammatory drugs (NSAIDs) are the most prescribed medications for the
relief of inflammatory pain [2]. Their analgesic effects are due to the
inhibition of cyclooxygenase (COX), an enzyme involved in the pro­
duction of prostaglandins, a mediator that participates in the
ever, despite their clinical efficacy, most NSAIDs cause adverse effects as
gastric, renal, and/or cardiovascular damage; hence, limiting their
long-term use [5,6]. In addition to NSAIDs, among the therapeutic al­
ternatives for the relief of inflammatory pain, the use of medicinal plants
is also considered [7,8].
Mansoa alliacea (Lam.) AH Gentry (M. alliacea) is a plant native to the
tropical forests of South America such as Perú, Brazil, and Mexico that

Abbreviations: M. alliacea, Mansoa alliacea; MA-EtOH, ethanolic extract of M. alliacea; MA-AQ, aqueous extract of M. alliacea; DIC, diclofenac; COX, cyclo­
oxygenase; CFA, Complete Freund’s adjuvant; NSAIDs, non-steroidal anti-inflammatory drugs; GLIB, glibenclamide; L-NAME, N(G)-L-nitro-arginine methyl ester;
NOS, nitric oxide synthase; ODQ, 1-H-(1,2,4)-oxadiazolo-(4,2-a)-quinoxalin-1-one; NTX, naltrexone; TRPV1, transient receptor potential cation channel subfamily V
member 1; SS, saline solution; i.p., intraperitoneal administration; p.o., oral administration; VEH, vehicle; S.E.M., standard error of the mean; AUC, area under the
curve; ANOVA, analysis of variance; au, area units; TCs, temporal courses; OECD, Organization for Economic Cooperation and Development guideline; LD50, lethal
dose 50.
* Corresponding authors.
E-mail addresses: lvalle-59@hotmail.com (M.G. Valle-Dorado), albertoh-leon@hotmail.com (A. Hernández-León), nani@imp.edu.mx (A. Nani-Vázquez),
geangeles@yahoo.com.mx (G.E. Ángeles-López), evag@imp.edu.mx (M.E. González-Trujano), rventuram7@hotmail.com (R. Ventura-Martínez).

https://doi.org/10.1016/j.biopha.2022.113253
Received 3 May 2022; Received in revised form 1 June 2022; Accepted 2 June 2022
Available online 10 June 2022
0753-3322/© 2022 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
M.G. Valle-Dorado et al.
belongs to the Bignoniaceae family and is popularly known as "ajillo",
“ajo sacha” or false garlic [9]. In the Southern Mexico, M. alliacea grows
wild in the states of Chiapas, Tabasco, and Veracruz [10]. The name of
false garlic is due to the smell like that of bulb garlic (Allium sativum) that
is produced when its leaves are crushed [11]. M. alliacea is a perennial
climbing shrub, with leaves, semi-woody branches, and violet flowers
[12]. In traditional medicine, the infusion of the dried aerial parts of this
plant has been used to treat fever, headache, or rheumatic pain [12,13].
The alcoholic maceration of the bark of the roots and the patch of the
leaves are considered anti-rheumatic and antiarthritic [14]. It has also
been used as insecticidal, antioxidant or as a condiment [14]. Despite its
use for pain relief, in the biomedical literature there is only one report
describing the antiallodynic, antihyperalgesic, and antiinflammatory
effects of an hydroethanolic extract of M. alliacea using a model of
chronic inflammation induced by the intraplantar administration of
Complete Freund’s Adjuvant (CFA) in mice [15]. To date, there are no
studies on the antinociceptive effect of other extracts of this species.
Therefore, the objective of this study was determined the anti­
nociceptive activity of the aqueous and ethanolic extracts of M. alliacea
leaves in the formalin and hot-plate tests, two acute nociceptive pain
models. The antinociceptive efficacy of both extracts was compared
using enteral and parenteral administration, as well as with diclofenac,
an analgesic of clinical use, in the formalin test. The possible mechanism
of action involved in the antinociceptive activity of this medicinal plant
was also analyzed.
2. Material and methods
2.1. Vegetal material and extracts preparation
M. alliacea was collected in March 2019 in Gutiérrez Zamora in the
state of Veracruz in eastern Mexico. It was identified by the taxonomist
Martha Juana Martínez Gordillo and deposited in the Herbarium from
Facultad de Ciencias at UNAM with voucher number 178051.
Ethanolic extract of M. alliacea (MA-EtOH) was prepared with 400 g
of the dried and crushed aerial parts of the plant. The material was
macerated with ethanol (3.14 L) for seven days at room temperature
with occasional agitation. We used gravity filtration to separate the
extract from the plant residues, the preparation was gravity filtered. The
filtrate was concentrated under vacuum, obtaining 18.6 g of a dark
green solid that represents a yield of 4.6% of the crude ethanolic extract.
Aqueous extract of M. alliacea (MA-AQ) was obtained by infusion
with 150 g of the dried and crushed aerial parts in 600 mL of boiling
water for 20 min. The solid residues of the extract were separated by
gravity filtration and the filtrate was lyophilized for 12 h (Heto model,
FD3 Lab). A sample of 5.3 g of the final crude aqueous extract was ob­
tained as a light brown powder, which represented a yield of 3.5%. The
dry extracts were stored at 4 ◦ C until further use.
For their administration in mice, MA-EtOH extract was suspended in
0.5% tween 80 in saline solution (SS, 0.9% NaCl), while MA-AQ extract
was suspended only in SS. The extracts were given intraperitoneally (i.
p.) or by gavage (p.o) in a volume of 10 mL/kg. Control groups received
vehicle by the same route of administration.
2.2. Animals
One hundred seventy-seven male mice (Swiss Webster, Taconic;
25–30 g) obtained from the Bioterium of the Instituto Nacional de Psi­
quiatría (INP) “Ramón de la Fuente Muñiz” and from the Central Bio­
terium of the Facultad de Medicina at Universidad Nacional Autónoma
de México (UNAM) were used in this study. The animals were housed in
a temperature-controlled room (22 ± 1 ◦ C), with access to food and
water ad libitum and an automatic 12-h light/dark cycle. The experi­
mental protocols were carried out under international guidelines for the
use of laboratory animals, in addition to the Official Mexican Standard
for the care and handling of animals [16] and the guidelines of the
2
Biomedicine & Pharmacotherapy 152 (2022) 113253
Institute’s Bioethics Committee (CONBIOETICA-09-CEI-01–20170316).
This protocol was approved by the Ethics and Research Committees as
well as by the Internal Committee for the Care and Use of Laboratory
Animals of the Facultad de Medicina at UNAM (FM/DI/054/2018 and
009-CIC-2018) and by the INP (No. NC123280.0 and NC17073.0).
2.3. Compounds
Diclofenac (DIC) and morphine, used as positive controls, was pur­
chased from Novartis Farmacéutica and Pisa, respectively; A7841683
(6-dihydro-3’-(trifluoromethyl)-N-[4-[(trifluoromethyl) sulfonyl]
phenyl]-[1(2 H), 2’-bipyridine]-4-carboxamide), an antagonist of
TRPV1 receptor, was purchased from TOCRIS Bioscience (Bristol); L-
NAME (N(G)-L-nitro-arginine methyl ester), a non-selective inhibitor of
nitric oxide synthase; ODQ (1-H-(1,2,4)-oxadiazolo-(4,2-a)-quinoxalin-
1-one), a selective inhibitor of soluble guanylate cyclase; naltrexone
(NTX), an antagonist of opioid receptor, and glibenclamide (GLIB), a
blocker of ATP-sensitive potassium channels (K+ ATP) were purchased
from Sigma (Sigma Chemical Co., MO, USA). All compounds were
administered intraperitoneally (i.p.) in a volume of 10 mL/kg using
saline solution (SS, 0.9% NaCl) for their dissolution, except A784168,
which was dissolved with 0.1% DMSO/saline. A 1% formalin solution (J.
T. Baker) was used as the allogeneic agent.
2.4. Nociception tests
2.4.1. Formalin test
In this test, nociception was induced by the intradermal adminis­
tration of 20 µl of 1% formalin into the dorsal surface of the right paw in
mice. Immediately after formalin administration, each mouse was
placed in an acrylic cylinder surrounded by mirrors to quantify the
number of flinches in the administered limb. The number of flinches was
recorded during one minute at 5-minute intervals for a total time of 30
min. The formalin test consists of two phases: an early phase known as
neurogenic (0–10 min), and a late phase known as inflammatory (10–30
min) after administration of the allogeneic agent [17,18]. Animals were
habituated to the acrylic cylinder for at least 30 min before formalin
administration.
2.4.2. Hot-plate test
In this test, animals were placed, one by one, in an open cylinder with
a floor of a metallic plate that was previously heated to 55 ± 1 ◦ C. The
heated plate produces two kinds of behavioral responses: paw licking
and jumping, both are considered nociceptive responses. The latency to
the first nociceptive response was recorded and the animal was imme­
diately removed from the hot plate. The first trial served as the control
reaction time, animals with base line latencies of more than 20 s were
excluded from the study [19].
2.5. Experimental design
To determine the antinociceptive effect of the extracts of M. alliacea
in the formalin test, eight groups of mice were used (n = 6 for group).
Three groups of animals were administered with MA-EtOH (30, 100, and
300 mg/kg), three received MA-AQ (30, 100, and 300 mg/kg), one
group received diclofenac (DIC, 10 mg/kg as a positive control), and
another was the saline group (VEH). All treatments were administered
intraperitoneally (i.p.) 30 min before the administration of formalin. A
decrease in the number of formalin-induced flinches in the presence of
treatments was considered as an antinociceptive effect.
To determine the antinociceptive effects of the extracts of M. alliacea
in the hot plate test, eight groups of mice were used (n = 6 for group).
Three groups of animals were administered with MA-EtOH (100, 300,
and 600 mg/kg), three received MA-AQ (100, 300 and 600 mg/kg), one
group received morphine (10 mg/kg as a positive control), and another
was the saline group (VEH). All treatments were administered
M.G. Valle-Dorado et al.
intraperitoneally (i.p.) 30 min before the hot-plate test. A decrease in the
latency time to licking a paw or jumping was considered as an anti­
nociceptive effect. Measurements were performed 30, 60, 90, and 120
min after treatment, with a cut-off time of 20 s to prevent development
of paw lesions.
To know if the antinociceptive effect of the extracts of M. alliacea was
affected by the route of administration, we evaluated this effect
exploring the highest dosage of MA-EtOH and MA-AQ by intragastric (p.
o.) and intraperitoneal (i.p.) administration used in the formalin test.
For this, two groups of mice with n = 6 for each group were used.
To determine the possible mechanism of action involved in the
antinociceptive effect of MA-ETOH or MA-AQ, we used the formalin test
with dosage of 300 mg/kg, i.p., of each extract. For this, ten groups of
mice with n = 6 for each group were used. Different groups of animals
received intraperitoneally the following treatment: A784168 (antago­
nist of TRPV1 receptor, 1 mg/kg), NTX (antagonist of opioid receptor, 5
mg/kg), L-NAME (inhibitor of nitric oxide synthetase, 20 mg/kg), GLIB
(blocker of K+ATP channel, 10 mg/kg) or ODQ (inhibitor of soluble gua­
nylate cyclase, 5 mg/kg), 15 min before the extracts. The anti­
nociceptive effect of each M. alliacea extract was compared in the
presence or in the absence of antagonists, blockers, or inhibitors. Doses
of all drugs were chosen from previous studies in our laboratory [20,21].
2.6. Acute toxicity study
The oral acute toxicity of the M. alliacea extracts was carried out
according to procedure of Organization for Economic Cooperation and
Development (OECD) guideline No. 425 [22]. Briefly, three groups of
mice were used to determine the acute toxicity of extracts of M. alliacea.
For this, two groups of mice (n = 3 per group) were treated with a single
dose of 2000 mg/kg of each extract (MA-AQ or MA-EtOH) and the other
was administered with vehicle (VEH). The extracts or VEH were
administered orally using gastric feeding tube. All animals were
observed individually during 4 h every 30 min, and daily thereafter, for
a total of 14 days for any clinical signs of toxicity or mortality. The body
weight of all animals was recorded before the administration of treat­
ment (day 0) and every 24 h for 14 days.
After the acute administration of each treatment, locomotor activity
(rotarod test) and exploratory behavioral was recorded every 30 min for
2 h. For rotarod test, animals were placed in the rotarod apparatus (4
bars of 2.5 cm diameter, 25 cm above the floor) which was turned to 10
rpm for a period of 2 min. The duration of permanence in the bar in
seconds (s), and the number of falls of animals was recorded. To assess
the exploratory activity of animal, they were placed in an acrylic cage
with 9 squares of 5 cm and the number of explored squares was
recorded.
2.7. Statistical analysis
Data are expressed as the mean ± standard error of the mean (S.E.
M.) of six mice per group. Considering that 28 groups of animals (n = 6
animals per group) and 3 groups for the acute toxicity (n = 3 animals per
group) were used, the total number of animals used in this study was one
hundred seventy-seven.
Temporal courses (TCs) of the number of flinches in the formalin test
and the latency to licking a paw or jumping in the hot-plate test in the
presence of different treatments were constructed. The areas under the
curve (AUC) of the neurogenic phase (from 0 to 10 min) and of the in­
flammatory phase (from 10 to 30 min) in the formalin test were calcu­
lated from the TCs with the trapezoid addition method [23] and the
corresponding bar graphs were constructed for statistical analysis. Sta­
tistical differences of treatments were obtained through a one-way
analysis of variance (ANOVA) for independent samples followed by
Dunnett’s post-hoc test, which was used only if F achieved P < 0.05 and
there was no significant variance in homogeneity. Differences between
two groups were determined using an unpaired t test of two tailed. Data
3
Biomedicine & Pharmacotherapy 152 (2022) 113253
of the acute toxicity study of both extracts were analyzed with two-way
ANOVA test. Differences between means with a P-value < 0.05 were
considered significant at the 95% confidence level. Statistical analysis
was performed using GraphPad Prism version 9.1.1 (GraphPad Software
INC, La Jolla, CA, USA).
3. Results
3.1. Antinociceptive effect of the ethanolic (MA-EtOH) and aqueous
(MA-AQ) extracts of M. alliacea in the formalin test
Formalin injection produced a rapid flinches response in the
administered limb (28.7 ± 1.3) in the VEH group that decreased at 10
min (8.4 ± 0.8) (neurogenic phase) and increased again until reaching
the maximum number of flinches at 25 min in the inflammatory phase
(17.9 ± 1.1), which was maintained until 30 min (16.6 ± 0.9) (Fig. 1a,
b). The AUC of the nociceptive effect induced by formalin in the animals
with vehicle (VEH) was 160 ± 14.8 au in the neurogenic phase (Fig. 1c,
d) and 280.4 ± 19.0 au in the inflammatory phase (Fig. 1e, f).
The administration of both MA-ETOH and MA-AQ extracts as well as
DIC, used as positive control, decreased the number of flinches induced
by formalin from the first 5 min of evaluation (Fig. 1a, b). This dimi­
nution of the number of flinches was considered as an antinociceptive
effect. To know if this diminution produced by each treatment was
significant, the AUC of each phase in the formalin test was compared.
DIC significantly diminished the AUC of the nociceptive effect
induced by formalin in comparison with VEH in the neurogenic phase
(0–10 min) (92.9 ± 4.3 vs 160 ± 14.8 au, respectively; with P-value =
0.0014, t = 4.351, df = 10; using an unpaired t test of two-tailed)
(Fig. 1c, d) and in the inflammatory phase (10–30 min) (194.2 ± 21.7 vs
280.4 ± 19.0 au, respectively; with P-value = 0.0135, t = 2.99, df = 10;
using an unpaired t test of two-tailed) (Fig. 1e, f).
Regarding the effect of M. alliacea extracts, the analysis of the AUC
(0–10 min) of the neurogenic phase showed that the MA-EtOH extract
significantly diminished the nociceptive behaviors induced by formalin
with all doses administered by i.p. (30, 100, 300 mg/kg) and p.o.
(300 mg/kg) in comparison with that in VEH (99.6 ± 7.8, 81.3 ± 7.6,
61.3 ± 5.7 and, 120.0 ± 3.8 vs 160 ± 14.8 au, respectively; with P-
value <0.0001, F(4,24) = 18.01; using one-way ANOVA followed by
Dunnett’s post-hoc test). Furthermore, the diminution of the nociception
induced by formalin of this extract at 300 mg/kg by p.o. was signifi­
cantly lower than that obtained with the same dosage by i.p. (120 ± 3.8
vs 61.3 ± 5.7 au, respectively; with P-value < 0.0001, t = 8.16, df = 9;
using an unpaired t test of two-tailed) (Fig. 1c).
On the other hand, the analysis of the AUC (0–10 min) of the MA-AQ
extract in the same phase also showed a significant reduction in the
nociceptive behaviors induced by formalin with all doses administered
by i.p. (30, 100, 300 mg/kg) and p.o. (300 mg/kg)) in comparison with
that in VEH (106.7 ± 11.5, 112.5 ± 14.2, 88.3 ± 9.7 and, 78.0 ± 3.5 vs
160 ± 14.8 au, respectively; with P-value = 0.0007, F(4,24) = 7.04;
using one-way ANOVA followed by Dunnett’s post-hoc test). However,
in this case, there was no difference between the effect of the MA-AQ
extract (300 mg/kg) administered p.o. compared to i.p. (78.0 ± 3.5 vs
88.3 ± 9.7 au, respectively; with P-value = 0.3782, t = 0.927, df = 9;
using an unpaired t test of two-tailed) (Fig. 1d).
In relation to the analysis of AUC (10–30 min) of the inflammatory
phase, all doses of the MA-EtOH extract (30, 100, 300 mg/kg, i.p. and,
300 mg/kg, p.o.) significantly reduced the nociception induced by
formalin in comparison with that by VEH (172.9 ± 18.1, 105.8 ± 15.5,
63.8 ± 9.2 and, 169.0 ± 23.8 vs 280.4 ± 19.0 au, respectively; with P-
value < 0.0001, F(4,24) = 23.2; using one-way ANOVA followed by
Dunnett’s post-hoc test). Like in the neurogenic phase, the diminution of
the nociception induced by formalin of 300 mg/kg of this extract by p.o.
was significantly lower than that obtained with the same dosage used by
i.p. way in the inflammatory phase (169.0 ± 23.8 vs 63.8 ± 9.2 au,
respectively; with P-value = 0.0017, t = 4.42, df = 9; using an unpaired
M.G. Valle-Dorado et al. Biomedicine & Pharmacotherapy 152 (2022) 113253

Fig. 1. Temporal courses (TCs) of the number of flinches induced by formalin in mice in the absence (VEH) and in the presence of DIC and different doses of the
ethanolic or aqueous extracts of M. alliacea (MA-EtOH or MA-AQ) (a, b). AUC of the neurogenic phase calculated from TCs of each treatment (0–10 min) (c, d) and,
AUC of the inflammatory phase calculated from TCs of each treatment (10–30 min) (e, f). Each point or bar represents the mean ± S.E.M. of a n = 6 animals per
treatment. *P < 0.05, **P < 0.01 and, ***P < 0.001 using one-way ANOVA followed by Dunnett’s test compared with VEH. ##P < 0.01 and ###P < 0.001 using
an unpaired t test for each comparison.

4
M.G. Valle-Dorado et al.
t test of two-tailed) (Fig. 1e).
In the same phase in the formalin test, the analysis of AUC (10–30 min)
of MA-AQ extract showed that only two doses (100 and 300 mg/kg, i.p.)
significantly reduced the nociception in comparison with that in VEH
(87.5 ± 25.1 and 67.9 ± 17.1 vs 280.4 ± 19.0 au, respectively; with P-
value = 0.0002, F(4,24) = 8.83; using one-way ANOVA followed by
Dunnett’s post-hoc test). Finally, unlike in the neurogenic phase, the
effect of MA-AQ extract (300 mg/kg) by p.o. in the nociception
formalin-induced was significantly lower than that obtained with the
same dosage by i.p. in the inflammatory phase (185.5 ± 16.0 vs 67.9
± 17.1 au, respectively; with P-value = 0.0008, t = 4.93, df = 9; using
an unpaired t test of two-tailed) (Fig. 1 f).
3.2. Antinociceptive effect of the ethanolic (MA-EtOH) and aqueous
(MA-AQ) extracts of M. alliacea in the hot-plate test
On the other hand, in the hot-plate test, the administration of
300 mg/kg of MA-EtOH extract significantly increased the latency at the
first nociceptive response (paw licking) at 60, 90, and 120 min of
evaluation (13.2 ± 1.6, 14.0 ± 0.6 and 13.6 ± 1.4 s, respectively); and
with 600 mg/kg at 90 and 120 min (11.0 ± 2 and 10.7 ± 1.2 s,
respectively); as well as morphine did at 30, 60, 90 and 120 min (14.8
± 1.7, 19.5 ± 0.4, 18.9 ± 0.6 and 19.1 ± 0.8 s, respectively) in com­
parison with that in VEH at the respective time of evaluation: 30, 60, 90
and 120 min (5.8 ± 0.7, 7.7 ± 0.6, 7.4 ± 0.5 and 7.2 ± 0.8 s, respec­
tively) (with P-value < 0.0001, F(3,75) = 16.1; using two-way ANOVA
followed by Dunnett’s multiple comparison test) (Fig. 2a).
Regarding the effect of MA-AQ extract in this test, 100 mg/kg
significantly increased the latency at the first paw licking at 90 min
(12.0 ± 1.0 s), 300 mg/kg at 60 min (13.8 ± 1.6 s) and, 600 mg/kg at
60, 90 and 120 min (14.7 ± 2.4, 14.8 ± 2.0 and 12.2 ± 1.2 s, respec­
tively) in comparison with that in VEH at the respective time of evalu­
ation: 30, 60, 90 and 120 min (5.8 ± 0.7, 7.7 ± 0.6, 7.4 ± 0.5 and 7.2
± 0.8 s, respectively) (with P-value < 0.0001, F(3,100) = 9.94; using
5
Biomedicine & Pharmacotherapy 152 (2022) 113253
two-way ANOVA followed by Dunnett’s multiple comparison test)
(Fig. 2b).
3.3. Participation of TRPV1 channels and opioids receptors in the
antinociceptive effects of M. alliacea extracts
In the temporal courses obtained after the formalin injection,
administration of A784168 (a TRPV1 receptor antagonist) did not alter
the antinociceptive effects induced by MA-EtOH neither MA-AQ ex­
tracts; while naltrexone (NTX, an opioid receptor antagonist) prevented
the antinociceptive effect induced by MA-EtOH but did not alter the
effect of MA-AQ (Fig. 3a, b). To know if some of the pre-treatment with
A78168 or NTX significantly altered the diminution in the number of
flinches produced by MA-EtOH or MA-AQ extracts at 300 mg/kg, i.p.,
their AUCs in each phase of the formalin test were compared.
Results showed that both MA-EtOH and MA-AQ extracts significantly
reduced the AUC (0–10 min) in the neurogenic phase of the number of
flinches produced by formalin in comparison with that in VEH (61.3
± 5.8 vs 160.0 ± 14.8 au with P-value < 0.0001, t = 6.22, df = 10; and,
88.3 ± 9.7 vs 160.0 ± 14.8 au with P-value = 0.0023, t = 4.05, df = 10,
respectively; in both comparisons an unpaired t test of two-tailed was
used) (Fig. 3c, d and 4c, d). Like in the neurogenic phase, both MA-EtOH
and MA-AQ extracts also significantly reduced the AUC (10–30 min) in the
inflammatory phase in the formalin test (63.8 ± 9.2 vs 280.4 ± 19.0 au
with P-value < 0.0001, t = 10.3, df = 10; and, 67.9 ± 17.1 vs 280.4
± 19.0 au with P-value < 0.0001, t = 8.31, df = 10, respectively; in both
comparisons an unpaired t test of two-tailed was used) (Figs. 3e, f and 4
e, f).
Otherwise, in the neurogenic phase in the formalin test the admin­
istration of A784168 did not alter the AUC (0–10 min) produced by MA-
EtOH extract as NTX did in comparison with the extract alone (84.6
± 15.5 and 122.9 ± 13.0 vs 61.3 ± 5.8 au, respectively; P-value =
0.008, F(2,15) = 6.6; using one-way ANOVA followed by Dunnett’s
post-hoc test) (Fig. 3c); while neither A784168 nor NTX altered the AUC
Fig. 2. Latency (s) in the hot-plate test in mice
with VEH or different doses of the ethanolic
extract (MA-EtOH) (a) or of the aqueous extract
(MA-AQ) of M. alliacea (b) evaluated in each
group every 30 min during 2 h. VEH group is
the negative control and morphine group is the
positive control. Each treatment was adminis­
tered 30 min before the test. The cut-off time of
this test in each time of evaluation was 20 s.
Each bar represents the mean ± S.E.M. of a
n = 6 animals per treatment. *P < 0.05,
**P < 0.01 and, ***P < 0.001 using two-way
ANOVA followed by Dunnett’s multiple com­
parison test compared with VEH at the corre­
sponding time.
M.G. Valle-Dorado et al. Biomedicine & Pharmacotherapy 152 (2022) 113253

Fig. 3. Temporal courses (TCs) of the effect of MA-EtOH or MA-AQ on 1% formalin-induced nociception in mice in the presence of NTX (opioids receptor antagonist)
and A784168 (TRPV1 receptor antagonist) (a, b). AUC of the neurogenic phase of the formalin test calculated from TCs of each treatment (0–10 min) (c, d) and, AUC
of the inflammatory phase of the formalin test calculated from TCs of each treatment (10–30 min) (e, f). Each point or bar represents the mean ± S.E.M. of a n = 6
animals per treatment. ###P < 0.001 using an unpaired t test of two-tailed in each comparison; **P < 0.01 using one-way ANOVA followed by Dunnett’s post-hoc
test compared with the extract alone.

6
M.G. Valle-Dorado et al. Biomedicine & Pharmacotherapy 152 (2022) 113253

produced by MA-AQ extract alone (85.8 ± 7.7 and 112.5 ± 17

(0–10 min) did not alter the AUC (10–30 min) of the effect produced by MA-EtOH
vs 88.3 ± 9.7 au, respectively; P-value = 0.261, F(2,15) = 1.47; using extract as NTX did in comparison with the extract alone (56.7 ± 12.9
one-way ANOVA test) (Fig. 3d). and 125.4 ± 17.0 vs 63.8 ± 9.2 au, respectively; P-value = 0.0003, F
While, in the inflammatory phase, the administration of A784168 (2,15) = 14.36; using one-way ANOVA followed by Dunnett’s post-hoc

Fig. 4. Temporal courses (TCs) of the effect of MA-EtOH or MA-AQ on 1% formalin-induced nociception in mice in the presence of L-NAME (SON inhibitor), ODQ
(sGC inhibitor) or GLIB (blocker of K+
ATP channels) (a, b). AUC of the neurogenic phase of the formalin test calculated from TCs of each treatment (0–10 min) (c, d),
and AUC of the inflammatory phase of the formalin test calculated from TCs of each treatment (10–30 min) (e, f). Each point or bar represents the mean ± S.E.M. of a
n = 6 animals. ###P < 0.001 using an unpaired t test of two-tailed in each comparison; **P < 0.01 and ***P < 0.001 using one-way ANOVA followed by Dunnett’s
post-hoc test compared with the extract alone.

7
M.G. Valle-Dorado et al.
test) (Fig. 3e), while neither A784168 nor NTX alter the AUC (10–30 min)
produced by MA-AQ extract alone (81.7 ± 16.6 and 80.4 ± 19.5 vs 67.9
± 17.1 au, respectively; P-value = 0.835, F(2,15) = 0.183; using one-
way ANOVA test) (Fig. 3 f).
3.4. Participation of the NO-cGMP-K+ATP channels pathway in the
antinociceptive effects of M. alliacea extracts
To know the possible participation of the NO-cGMP-K+
ATP pathway in
the antinociceptive effect of MA-EtOH and MA-AQ extracts in the
formalin test, we used several inhibitors of this pathway. Results showed
that the administration of L-NAME (an inhibitor of NO synthase)
partially inhibited the effect of MA-EtOH extract (300 mg/kg, i.p.) on
the nociceptive responses induced by formalin, whereas neither ODQ (a
soluble guanylate cyclase inhibitor) nor GLIB (a K+
ATP channel blocker)
altered it (Fig. 4a). On the other hand, administration of L-NAME, ODQ
and GLIB seemed to alter the effect of MA-AQ extract (300 mg/kg, i.p.)
on the nociceptive responses induced by formalin (Fig. 4b). To know if
the pretreatment with L-NAME, ODQ or GLIB significantly altered the
diminution in the number of flinches produced by MA-EtOH or MA-AQ
extracts in each phase of the formalin test, their AUCs were compared.
In this sense, neither the administration of ODQ nor GLIB alter the
AUC (0–10 min) of the effect produced by MA-EtOH extract in the neuro­
genic phase as L-NAME did in comparison with the extract alone (77.5
Fig. 5. Acute toxicity tests. Exploratory activity of animals administered with an oral dose of 2000 mg/kg of MA-EtOH or MA-AQ on the acrylic cage with 9 squares
of 5 cm each one evaluated in each group every 30 min during 2 h (a). Body weight of animals administered with an oral dose of 2000 mg/kg of MA-EtOH, MA-AQ or
vehicle (VEH). Each point or bar represents the mean ± S.E.M. of a n = 3 animals.
8
Biomedicine & Pharmacotherapy 152 (2022) 113253
± 6.3, 82.5 ± 13.4 and 120.8 ± 12.8 vs 61.3 ± 5.8 au, respectively; P-
value = 0.004, F(3,20) = 6.1; using one-way ANOVA followed by
Dunnett’s post-hoc test) (Fig. 4c). The same situation occurred with the
MA-AQ extract: neither of ODQ nor GLIB alter the AUC (0–10 min) of the
effect produced by the extract as L-NAME did in comparison with the
extract alone (105.8 ± 13.7, 73.8 ± 7.3 and 160.0 ± 12.0 vs 88.3 ± 9.7
au, respectively; P-value = 0.0001, F(3,20) = 11.9; using one-way
ANOVA followed by Dunnett’s post-hoc test) (Fig. 4d).
In regard to the analysis of AUC (10–30 min) in the inflammatory phase,
results showed that neither the administration of L-NAME, ODQ nor
GLIB alter the AUC (10–30 min) of the effect produced by MA-EtOH extract
alone (67.9 ± 19.8, 135.4 ± 29.4 and 150.0 ± 33.6 vs 63.8 ± 9.2 au,
respectively; P-value = 0.073, F(3,20) = 3.25; using one way ANOVA
test) (Fig. 4e); while, on the contrary, the L-NAME, ODQ and GLIB
significantly alter the AUC (10–30 min) of the effect produced by MA-EtOH
extract alone (177.5 ± 8.9, 200.8 ± 34.4 and 180.4 ± 28.3 vs 67.9
± 17.1 au, respectively; P-value = 0.004, F(3,20) = 6.11; using one-way
ANOVA followed by Dunnett’s post-hoc test) (Fig. 4 f).
3.5. Acute toxicity of M. alliacea extracts
The oral dose of 2000 mg/kg of MA-AQ or MA-EtOH extracts did not
cause death in mice during the short and long-term observation period,
and no signs of toxicity were observed in the animals throughout the 14
M.G. Valle-Dorado et al.
days study period. The lethal doses 50 (LD50) of both extracts were
estimated to be greater than 2000 mg/kg. Regarding the exploratory
behavioral, the mice pretreated with MA-AQ or MA-EtOH (2000 mg/kg)
did no show changes in the number of explored squares at any time
evaluated compared with those of the VEH (P-value = 0.671, F(8, 30)
= 0.721; using two-way ANOVA test) (Fig. 5a). Also, in relation to the
locomotor activity, the mice pretreated with MA-AQ or MA-EtOH
(2000 mg/kg) did no show motor performance alterations in the
rotarod test compared with those of VEH since all animals were held in
the rotarod for 2 min at each evaluation time (data not shown). There
were also no differences in the body weight of the animals with any of
the treatments during the 14 days of registration (P-value = 0.9989, F
(28,90) = 0.347; using two-way ANOVA test) (Fig. 5b).
4. Discussion
In the present study, the antinociceptive effects of polar extracts
(ethanol and aqueous) from M. alliacea leaves were demonstrated in the
formalin and in the hot-plate tests in mice: two models that represent an
acute nociceptive pain. M. alliacea has been used in the traditional
medicine [9] for relieving rheumatic-type pain [11,24,25], headache,
muscle fatigue [11], to treat colds, pneumonia [26], fever, and as insect
repellent; as well as a condiment in food preparation due to its smell and
taste of garlic [9,11]. The aerial parts of the plant are used as infusion or
decoction, as well as a preparation using the roots or leaves macerated in
ethanol [14].
Results of this study show that both ethanol and aqueous extracts of
M. alliacea (MA-EtOH and MA-AQ) produced antinociceptive effects in
both phases of the formalin test. The antinociceptive effects of both
extracts were better than those shown by diclofenac, a NSAID. In gen­
eral, these results showed that the antinociceptive effects of both ex­
tracts were better in the inflammatory phase than in the neurogenic
phase of this test. It is well known that formalin test is an assay used as a
model for searching compounds with antinociceptive and anti-
inflammatory effects. In this test, nociception is induced by the intra­
dermal administration of 1% formalin into the dorsal surface of right
paw in mouse. The number of formalin-induced flinches is quantified,
and an increase is related to the degree of nociception [27]. This test is
biphasic where different mechanisms and/or nociceptive mediators
participate and can be distinguished pharmacologically. The first phase
is characterized by central neurogenic pain caused by direct chemical
stimulation of C-fiber nociceptors and occurs within the first (0–10 min)
minutes of formalin administration. The nociceptive behaviors in this
phase can be attenuated with opioid drugs because of their central ef­
fect. The second phase is characterized by an inflammatory process in
which several mediators are involved such as prostaglandins, neuro­
peptides, and nitric oxide. This phase occurs from 10–30 min after the
administration of formalin and the nociceptive behaviors in this phase
can be attenuated by NSAIDs for their peripheral effects [17]. The fact
that both extracts of M. alliacea in this study induced antinociceptive
effects in the two phases of the formalin test suggests that it acts by
central and peripheral mechanisms by blocking inhibitory receptors
and/or by inhibiting the formation and/or release of inflammatory
mediators. To demonstrate that in the antinociceptive effects of
M. alliacea extracts, central mechanisms are involved, the hot-plate test
was used in this study. The hot-plate test is also a quick and relatively
inexpensive way to assess acute, thermal pain, and it has been consid­
ered a sensitive test to repeated measurements. Also, it is well known
that the behavioral responses induced in this thermal nociceptive test
(paw licking and jumping) are considered supraspinally integrated re­
sponses [19]. Results of this study confirmed that in the antinociceptive
effect of both M. alliacea extracts central mechanisms are involved, such
as their effects in the hot-plate test resembling the effect of morphine,
whose antinociceptive effect occur centrally. Morphine is considered the
classic opioid analgesic that produces its analgesic effects by binding to
opioid receptors within the central nervous system (CNS). In this way,
9
Biomedicine & Pharmacotherapy 152 (2022) 113253
morphine activates the descending inhibitory pathway of the CNS and
inhibits the nociceptive afferent neurons of the peripheral nervous sys­
tem, which leads to a reduction of the nociceptive transmission [28].
In this work, we also compared the antinociceptive effects of
M. alliacea prepared in two polar extracts (MA-EtOH and MA-AQ) using
enteral and parenteral routes of administration. Results showed that the
antinociceptive efficacy of both extracts was better by intraperitoneal
than intragastric administration in the formalin test using the same
dosage. As in other extracts of medicinal plants, antinociceptive effects
of M. alliacea extracts might depend on the bioactive constituents pre­
sent in preparations of this species. Phytochemical analysis of an
hydroethanol extract of M. alliacea by UHPLC-MS/MS revelated the
presence of higher concentrations of phenolic compounds such as p-
coumaric acid, luteolin, ferulic acid, chlorogenic acid, and apigenin
[15], a triterpene (betulinic acid) identified in an ethanol extract
through analysis by HPLC-MS [29] and two pyranonaphthoquinonas
(mansonin A and mansonin B) determined by spectroscopic analysis
[30]. In contrast, sulfur compounds have been identified in the essential
oil from M. alliacea fresh leaves, whose characteristic smell to garlic
depends on these kinds of constituents [31] as well as estigmasterol and
γ-sitosterol identified in the no-polar extract of this species [32]. These
kinds of metabolites might be obtained in the aqueous extract that was
prepared by decoction involving boiling water.
One of the bioactive metabolites identified in the polar extracts is
apigenin, whose antinociceptive effect has been reported in the hot-
plate, formalin, and writhing tests in mice [33,34]. Also, the chloro­
genic [35,36] and ferulic acids [37] have produced antinociceptive ef­
fects in a neuropathic pain model induced by ligation of the sciatic
nerve. Whereas luteolin has shown anti-inflammatory effect in the CFA
model in rats [38] and p-coumarinic acid induces analgesic and
anti-inflammatory effects in the gouty arthritis model induced with acid
crystals monosodium uric acid in rats [39].
To know the possible mechanisms involved in the antinociceptive
effects of MA-EtOH and MA-AQ extracts, the participation of TRPV1 and
opioid receptors, as well as the NO-cGMP-K+ ATP pathway were investi­
gated in this work. It is known that opioid receptors are one of the most
important signaling pathways in the descending modulation of noci­
ception [21]. It has also been reported that some natural products such
as flavonoids and terpenoids participated through this signaling
pathway [21,40]. In this study, the administration of naltrexone, a
non-selective antagonist of opioid receptors, produced a partial inhibi­
tion of the antinociceptive effect of MA-ETOH extract but not of the
MA-AQ extract in both phases in the formalin test. These results agreed
with a previous report where the antinociceptive effect of the hydro­
alcoholic extract was mediated by the participation of opioids, mainly δ
receptors [15]. It is important to mention that in binding assays, the
apigenin has shown affinity for δ and μ opioid receptors [41]. In addition
to this, an activation of opioid receptors was involved in the anti­
nociceptive effects showed by luteolin [42], ferulic acid [37], and
chlorogenic acid [43].
On the other hand, the TRPV1 receptor is one of the most intensively
studied channel for being one of the main receptors involved in the
sensitization and the maintenance of nociception in the inflammatory
pain [44]. For this, its participation in the antinociceptive effect of MA
extracts in this study was assessed using a A784168 as specific antago­
nist of this receptor [45]. Results showed that antinociceptive effects of
both extracts of M. alliacea did not change in the presence of this
antagonist, suggesting that the participation of TRPV1 receptors is not
involved in the MA antinociceptive properties.
Evidence in literature indicates that participation of the NO–cGMP
pathway is involved in the antinociceptive activity of several drugs [46,
47]. Nitric oxide (NO) is a small gaseous molecule with a short half-life
that is synthesized by nitric oxide synthetase (NOS) [48]. NO can in­
crease the concentration of cGMP by activation of soluble guanyl cyclase
(sGC) in different types of cells. The increase of cGMP activates the
ATP-sensitive potassium channels (K+ ATP). Finally, the opening of these
M.G. Valle-Dorado et al.
channels induces the membrane hyperpolarization reducing the action
potential transmission abilities of neurons producing analgesia [49,50].
In this study, the inhibition of the NOS using L-NAME significantly
decreased the antinociceptive effect of both MA-EtOH and MA-AQ ex­
tracts in the neurogenic phase of the formalin test, while the adminis­
tration of ODQ (an inhibitor of sGC) or glibenclamide (a blocker of K+ ATP
channels) did not alter the antinociceptive effect of these extracts in this
phase. As previously mentioned, it is known that in the neurogenic
phase of the formalin test, mainly drugs like opioids induce anti­
nociceptive effects. In this sense, the participation of NO-cGMP pathway
in the modulation of antinociceptive activity of morphine has been re­
ported [51]. These results suggest that the antinociceptive effect of ex­
tracts of M. alliacea involve central mechanisms such as the participation
of ON as an analgesic mediator, but it is produced by an independent
mechanism that the classic cGMP-ATP-sensitive K+ channel pathway as
it has been observed with some natural products such as pure com­
pounds or complete extract, for example ursolic acid [20] or Clinacan­
thus nutans methanol extract [52].
On the other hand, the NO-cGMP-K+ ATP pathway did not participate in
the antinociceptive effect of MA-EtOH extract in the inflammatory phase
of the formalin test since L-NAME, ODQ or GLIB did not alter its anti­
nociceptive effect. Whereas this NO-cGMP-K+ ATP pathway participated in
the antinociceptive effect obtained with the MA-AQ extract in the in­
flammatory phase of the formalin test. These results reinforced the po­
tential use of M. alliacea in inflammatory pain and they agree with the
already reported antinociceptive effect of the hydroalcoholic extract of
this species collected in the Amazonian region using the intraplantar
administration of the complete Freund’s adjuvant in mice, a model
representing a condition of chronic inflammatory pain like those pre­
sented by patients with arthritis [15]. Previous studies in cell culture
have shown that apigenin can activate the NO pathway [53,54] and
some other studies indicate that ferulic acid [55] and p-coumarin
decrease NO levels to produce antinociceptive effects [39].
Finally, we also evaluated the possible adverse effects of M. alliacea
extracts and the results of the oral administration of 2000 mg/kg of MA-
AQ and MA-EtOH extracts suggesting that both extracts may be safe at
these doses and the oral LD50 considered greater than 2000 mg/kg in
mice, according to OECD guideline No. 425 [22]. These results are ac­
cording with a study where it was demonstrated that the M. alliacea
hydroethanolic extract did not produce constipation, alterations in the
motor coordination, hypothermia, gastric lesions neither hepatic, nor
renal failure, considering that this species is a safe medicinal plant [15].
5. Conclusions
In conclusion, our study corroborates that M. alliacea prepared in
both ethanolic and aqueous extracts can produce antinociceptive effects
with similar efficacy in acute nociceptive pain, thus contributing to the
scientific evidence that supports the ethnopharmacological knowledge
of this species for pain relief. It seems that concentrations and kinds of
constituents in the polar nature of the extracts can influence the anti­
nociceptive mechanism of action involved. Since for the ethanolic
extract it had influence on the opioid receptors while for the aqueous
extract, the participation of all the NO-cGMP/K+ ATP pathway was
observed.
CRediT authorship contribution statement
Rosa Ventura-Martínez, María E. González-Trujano: Resources,
Writing − review & editing, Visualization; Funding acquisition. María
E. González-Trujano, María G. Valle-Dorado: Conceptualization,
Writing − original draft. María G. Valle-Dorado, Alberto Hernández-
León, Guadalupe E. Ángeles-López: Methodology, Formal analysis,
Investigation. Andrés Nani-Vázquez: Methodology, Collected the me­
dicinal plant and prepared the extracts.
10
Biomedicine & Pharmacotherapy 152 (2022) 113253
Declaration of interest
The authors declare that there is no conflict of interest.
Acknowledgments
The authors would like to thank Mrs. Josefina Bolado, Head of the
Scientific Paper Translation Department, from División de Investigación
at Facultad de Medicina, at UNAM for the diligent proofreading of the
English-language version of this manuscript. We also thanks to Andrea
Bañuelos Sánchez and Miguel A. Zumaya for their technical assistance.
PhD. Maria G. Valle-Dorado thanks the fellowship from Dirección
General de Asuntos del Personal Académico at Universidad Nacional
Autónoma de México (DGAPA-UNAM, 080/2021).
Funding sources
Funding: This work was supported by the Programa de Apoyo a
Proyectos de Investigación e Innovación Tecnológica, UNAM, Mexico
[UNAM-PAPIIT, grant number IN201820]; Consejo Nacional de Ciencia
y Tecnología, Mexico [CONACYT, grant numbers 226454 and 256448];
and Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñiz”,
Mexico [grant numbers NC123280.0 and NC17073.0]. PhD. Maria G.
Valle-Dorado has received the fellowship from Dirección General de
Asuntos del Personal Académico at UNAM, Mexico (DGAPA-UNAM,
080/2021).
Data statement
The datasets generated and/or analyzed during the current study are
available from the corresponding author on reasonable request.
References
[1] A.J. Barragán-Berlanga, S. Mejía-Arango, L.M. Gutiérrez-Robledo, Dolor en adultos
mayores de 50 años: prevalencia y factores asociados [Pain in the elderly:
prevalence and associated factors], Salud Publica Mex. 49 (2007) S488–S494,
https://doi.org/10.1590/s0036-36342007001000008.
[2] V.K. Bournia, G. Kitas, A.D. Protogerou, P.P. Sfikakis, Impact of non-steroidal anti-
inflammatory drugs on cardiovascular risk: Is it the same in osteoarthritis and
rheumatoid arthritis? Mod. Rheumatol. 27 (2017) 559–569, https://doi.org/
10.1080/14397595.2016.1232332.
[3] J.N. Cashman, The mechanisms of action of NSAIDs in analgesia, Drugs 52 (1996)
13–23, https://doi.org/10.2165/00003495-199600525-00004.
[4] N. Leyva-López, E.P. Gutierrez-Grijalva, D.L. Ambriz-Perez, J.B. Heredia,
Flavonoids as cytokine modulators: a possible therapy for inflammation-related
diseases, Int. J. Mol. Sci. 17 (2016) 921, https://doi.org/10.3390/ijms17060921.
[5] M.M. Wolfe, D.R. Lichtenstein, G. Singh, Gastrointestinal toxicity of nonsteroidal
anti-inflammatory drugs, N. Engl. J. Med. 340 (1999) 1888–1899, https://doi.org/
10.1056/NEJM199906173402407.
[6] C.K. O’Neil, J.T. Hanlon, Z.A. Marcum, Adverse effects of analgesics commonly
used by older adults with osteoarthritis: focus on non-opioid and opioid analgesics,
Am. J. Geriatr. Pharmacother. 10 (2012) 331–342, https://doi.org/10.1016/j.
amjopharm.2012.09.004.
[7] J.B. Calixto, Twenty-five years of research on medicinal plants in Latin America: A
personal view, J. Ethnopharmacol. 100 (2005) 131–134, https://doi.org/10.1016/
j.jep.2005.06.004.
[8] B.B. Mishra, V.K. Tiwari, Natural products: An evolving role in future drug
discovery, Eur. J. Med. Chem. 46 (2011) 4769–4807, https://doi.org/10.1016/j.
ejmech.2011.07.057.
[9] L.G. Lohmann, M. Ch. Taylor, A new generic classification of Bignonieae
(Bignoniaceae), Ann. Mo. Bot. Gard. 99 (2014) 348 489, https://doi.org/10.3417/
2003187.
[10] CONABIO. Comisión Nacional para el conocimiento y uso de la biodiversidad.
〈https://enciclovida.mx/especies/202399〉.
[11] M.G. Bichara Zoghbi, J. Oliveira, G.M. Pinheiro Guilhon, The genus Mansoa
(Bignoniaceae): A source of organosulfur compounds, Rev. Bras. Farmacogn. 19
(2009) 795–804, https://doi.org/10.1590/S0102-695X2009000500025.
[12] A. Tasambay-Salazar, L. Scalvenzi, A.S. Piedra-Lascano, M. Radice,
Ethnopharmacology, biological activity and chemical characterization of Mansoa
alliacea, A Rev. A Promis. Plant Amazon. Reg., MOL2NET´17 3 (2017) 15, https://
doi.org/10.3390/mol2net-03-04617.
[13] E. Pagani, J. Santos, E. Rodrigues, Culture-Bound Syndromes of a Brazilian Amazon
Riverine population: Tentative correspondence between traditional and
conventional medicine terms and possible ethnopharmacological implications,
M.G. Valle-Dorado et al.
J. Ethnopharmacol. 203 (2017) 80–89, https://doi.org/10.1016/j.
jep.2017.03.024.
[14] I. Patel, S. Sipai, D. Rathod, G. Shrimali, A. Patel, E. Rami, Phytochemical studies
on Mansoa alliacea (Lam.), Int. J. Adv. Pharm. Res. 4 (2013) 1823–1828.
[15] F.R. Hamann, I. Brusco, G. de Campos Severo, L.M. de Carvalho, H. Faccin,
L. Gobo, S.M. Oliveira, M.A. Rubin, Mansoa alliacea extract presents
antinociceptive effect in a chronic inflammatory pain model in mice through
opioid mechanisms, Neurochem. Int. 122 (2019) 157–169, https://doi.org/
10.1016/j.neuint.2018.11.017.
[16] NOM-062-ZOO,1999, Official Mexican Norm for Animal Care and Management.
Secretaría de salud. Especificaciones técnicas para la producción, cuidado y uso de
los animales de laboratorio. 〈https://www.gob.mx/senasica/documentos/nom-
062-zoo-1999〉.
[17] A. Tjølen, O.G. Berge, S. Hunskaar, J.H. Rosland, K. Hole, The formalin test: an
evaluation of the method, Pain 51 (1992) 5–17, https://doi.org/10.1016/0304-
3959(92)90003-T.
[18] S. Hunskaar, O.G. Berge, K. Hole, Dissociation between antinociceptive and anti-
inflammatory effects of acetylsalicylic acid and indomethacin in the formalin test,
Pain 25 (1986) 125–132, https://doi.org/10.1016/0304-3959(86)90014-X.
[19] N.B. Eddy, D. Leimbach, Synthetic analgesics. II. Dithienylbutenyl- and
dithienylbutylamines, J. Pharm. Exp. Ther. 107 (1953) 385–393.
[20] J. Verano, M.E. González-Trujano, M. Déciga-Campos, R. Ventura-Martínez,
F. Pellicer, Ursolic acid from Agastache mexicana aerial parts produces
antinociceptive activity involving TRPV1 receptors, cGMP and a serotonergic
synergism, Pharmacol. Biochem. Behav. 110 (2013) 255–264, https://doi.org/
10.1016/j.pbb.2013.07.020.
[21] A. Hernandez-Leon, A. Fernández-Guasti, M.E. González-Trujano, Rutin
antinociception involves opioidergic mechanism and descending modulation of
ventrolateral periaqueductal grey matter in rats, Eur. J. Pain. 20 (2016) 274–283,
https://doi.org/10.1002/ejp.720.
[22] OECD. Test No. 425: Acute Oral Toxicity: Up-and-Down Procedure. OECD
Guidelines for the Testing of Chemicals, Section 4, 2008. OECD Publishing, Paris.
〈https://doi.org/10.1787/9789264071049-en〉.
[23] M. Rowland, T.N. Tozer, Clinical Pharmacokinetics: Concepts and Applications,
second ed., Lea & Febiger, Philadelphia, 1989.
[24] H. Itokawa, K. Matsumoto, H. Morita, K. Takeya, Cytotoxic naphtoquinones from
Mansoa alliacea, Phytochem 31 (1992) 1061–1062, https://doi.org/10.1016/0031-
9422(92)80077-R.
[25] J.A. Hasrat, J.P. De Backer, G. Vauquelin, A.J. Vlietinck, Medicinal plants in
Suriname: screening of plant extracts for receptor binding activity, Phytomedicine
4 (1997) 59–65, https://doi.org/10.1016/S0944-7113(97)80029-3.
[26] C. Desmachelier, M. Repetto, J. Coussio, S. Llesuy, G. Ciccia, Total reactive
antioxidant potential (TRAP) and total antioxidant reactivity (TAR) of medicinal
plants used in Southwest Amazonia (Bolivia and Peru), Int. J. Pharm. 35 (1997)
1–9, https://doi.org/10.1076/phbi.35.4.288.13303.
[27] T.J. Coderre, F.V. Abbott, J. Sawynok, Formalin Test, in: G.F. Gebhart, R.F.
Schmidt (Eds), Encyclopedia of Pain, Springer, Berlin, Heidelberg, 2013, pp.
1303–1308. 〈https://doi.org/10.1007/978–3-642–28753-4〉.
[28] P.B. Murphy, S. Bechmann, M.J. Barrett, Morphine. [Updated 2021 May 30]. In:
StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-.
Available from: 〈https://www.ncbi.nlm.nih.gov/books/NBK526115/〉.
[29] L.A. Gobo, C. Viana, A. Lameira, L.M. Carvalho, A liquid chromatography-
atmospheric pressure photoionization tandem mass spectrometric (LC-APPI-MS/
MS) method for the determination of triterpenoids in medicinal plant extracts,
J. Mass. Spectrom. 51 (2016) 558–565, https://doi.org/10.1002/jms.3783.
[30] J. Luo, Z.L. Wu, X.J. Huang, X.Q. Zhang, C.L. Fan, W.C. Ye, Y. Wang, Two new
pyranonaphthoquinones from Mansoa alliacea, Zhongguo Zhong Yao Za Zhi 46
(2021) 3364–3367, https://doi.org/10.19540/j.cnki.cjcmm.20210317.201.
[31] M. Olivera-Condori, J. Flores-Arizaca, T. Vásquez-Zavaleta, E. Ocsa-Borda,
Propiedades Fisicoquímicas y Bioactivas in Vitro del Aceite Esencial de Mansoa
alliacea (LAM.) A. Gentry, Ceprosimad 2 (2013) 96–102. 〈https://journal.cepro
simad.com/index.php/ceprosimad/article/view/12〉.
[32] J.E. Grovas Llamocca, E.A. Condor Cuyubamba, V.M. Reyna Pinedo, I.E. Collantes
Díaz, Esteroles presentes en el extracto apolar de las raíces de ajo sacha Mansoa
alliacea, Rev. Soc. Quím. Perú 84 (2018) 513–521. 〈http://www.scielo.org.pe/sciel
o.php?script=sci_arttext&pid=S1810-634X2018000400011&lng=es&nrm=iso〉.
[33] M.M. Pinheiro, F. Boylan, P.D. Fernandes, Antinociceptive effect of the Orbignya
speciosa Mart. (Babassu) leaves: evidence for the involvement of apigenin, Life Sci.
91 (2012) 293–300, https://doi.org/10.1016/j.lfs.2012.06.013.
[34] G.A. El-Shoubaky, M.M. Abdel-Daim, M.H. Mansour, E.A. Salem, Isolation and
identification of a flavone apigenin from marine red alga Acanthophora spicifera
with antinociceptive and anti-inflammatory activities, J. Exp. Neurosci. 10 (2016)
21–29, https://doi.org/10.4137/JEN.S25096.
[35] K. Hara, Y. Haranishi, K. Kataoka, Y. Takahashi, T. Terada, M. Nakamura, T. Sata,
Chlorogenic acid administered intrathecally alleviates mechanical and cold
hyperalgesia in a rat neuropathic pain model, Eur. J. Pharm. 723 (2014) 459–464,
https://doi.org/10.1016/j.ejphar.2013.10.046.
[36] D. Bagdas, Z. Gul, J.A. Meade, B. Cam, N. Cinkilic, M.S. Gurun, Pharmacologic
overview of chlorogenic acid and its metabolites in chronic pain and inflammation,
Curr. Neuropharmacol. 18 (2020) 216–228, https://doi.org/10.2174/
1570159X17666191021111809.
[37] Y. Xu, D. Lin, X. Yu, X. Xie, L. Wang, L. Lian, N. Fei, J. Chen, N. Zhu, G. Wang,
X. Huang, J. Pan, The antinociceptive effects of ferulic acid on neuropathic pain:
11
Biomedicine & Pharmacotherapy 152 (2022) 113253
involvement of descending monoaminergic system and opioid receptors,
Oncotarget 7 (2016) 20455–20468, https://doi.org/10.18632/oncotarget.7973.
[38] R. Jeyadevi, T. Sivasudha, A. Rameshkumar, L. Dinesh Kumar, Anti-arthritic
activity of the Indian leafy vegetable Cardiospermum halicacabum in Wistar rats and
UPLC–QTOF–MS/MS identification of the putative active phenolic components,
Inflamm. Res. 62 (2013) 115–126, https://doi.org/10.1007/s00011-012-0558-z.
[39] S.J. Pragasan, M. Rasool, Dietary component p-coumaric acid suppresses
monosodium urate crystal-induced inflammation in rats, Inflamm. Res. 62 (2013)
489–498, https://doi.org/10.1007/s00011-013-0602-7.
[40] F. Moreno-Perez, A. Hernandez-Leon, M.G. Valle-Dorado, A. Cano-Martínez,
F. Narváes-González, E. Aguirre-Hernández, H. Salgado-Ceballos, M.E. González-
Trujano, Neo-clerodane diterpenic influence in the antinociceptive and anti-
inflammatory properties of Salvia circinnata Cav, J. Ethnopharmacol. 268 (2021),
113550, https://doi.org/10.1016/j.jep.2020.113550.
[41] D.E. Webster, Y. He, S.N. Chen, G.F. Pauli, N.R. Farnsworth, Z.J. Wang, Opioidergic
mechanisms underlying the actions of Vitex agnus-castus L, Biochem. Pharmacol.
81 (2011) 170–177, https://doi.org/10.1016/j.bcp.2010.09.013.
[42] K. Hara, Y. Haranishi, T. Terada, Y. Takahashi, M. Nakamura, T. Sata, Effects of
intrathecal and intracerebroventricular administration of luteolin in a rat
neuropathic pain model, Pharmacol. Biochem. Behav. 125 (2014) 78–84, https://
doi.org/10.1016/j.pbb.2014.08.011.
[43] O. Guadarrama-Enríquez, M.E. González-Trujano, R. Ventura-Martínez,
R. Rodríguez, G.E. Ángeles-López, R. Reyes-Chilpa, D.A. Moreno, Broccoli sprouts
produce abdominal antinociception but not spasmolytic effects like its bioactive
metabolite sulforaphane, Biomed. Pharmacother. 107 (2018) 1770–1778, https://
doi.org/10.1016/j.biopha.2018.09.010.
[44] M.K. Chung, S.J. Jung, S.B. Oh, Role of TRP channels in pain sensation, Adv. Exp.
Med. Biol. 704 (2011) 615–636, https://doi.org/10.1007/978-94-007-0265-3_33.
[45] M. Cui, P. Honore, C. Zhong, D. Gauvin, J. Mikusa, G. Hernandez, P. Chandran,
A. Gomtsyan, B. Brown, E.K. Bayburt, K. Marsh, B. Bianchi, H. McDonald,
W. Niforatos, T.R. Neelands, R.B. Moreland, M.W. Decker, C.H. Lee, J.P. Sullivan,
C.R. Faltynek, TRPV1 receptors in the CNS play a key role in broad-spectrum
analgesia of TRPV1 antagonists, J. Neurosci. 26 (2006) 9385–9393, https://doi.
org/10.1523/JNEUROSCI.1246-06.2006.
[46] A. Hernández-Pacheco, C.I. Araiza-Saldaña, V. Granados-Soto, T. Mixcoatl-Zecuatl,
Possible participation of the nitric oxide-cyclic GMP-protein kinase G-K+ channels
pathway in the peripheral antinociception of melatonin, Eur. J. Pharmacol. 596
(2008) 70–76, https://doi.org/10.1016/j.ejphar.2008.07.068.
[47] Y. Cury, G. Picolo, V. Pacciari, S. Henrique, Pain and analgesia: The dual effect of
nitric oxide in the nociceptive system, Nitric Oxide 25 (2011) 243–254, https://
doi.org/10.1016/j.niox.2011.06.004.
[48] N. Gamper, L. Ooi, Redox and nitric oxide-mediated regulation of sensory neuron
ion channel function, Antioxid. Redox Signal. 22 (2015) 486–504, https://doi.org/
10.1089/ars.2014.5884.
[49] A.R.A. Rodrigues, I.D.G. Duarte, The peripheral antinociceptive effect induced by
morphine is associated with ATP-sensitive K(+) channels, Br. J. Pharm. 129 (2000)
110–114, https://doi.org/10.1038/sj.bjp.0703038.
[50] F. Gomes, F.Q. Cunha, T.M. Cunha, Peripheral nitric oxide signaling directly blocks
inflammatory pain, Biochem. Pharm. 176 (2020), 113862, https://doi.org/
10.1016/j.bcp.2020.113862.
[51] V. Granados-Soto, M.D.O. Rufino, L.D. Gomes Lopes, S.H. Ferreira, Evidence for the
involvement of the nitric oxide-cGMP pathway in the antinociception of morphine
in the formalin test, Eur. J. Pharmacol. 340 (1997) 177–180, https://doi.org/
10.1016/s0014-2999(97)01399-x.
[52] M.H. Abdul Rahim, Z.A. Zakaria, M.H. Mohd Sani, M.H. Omar, Y. Yakob, M.
S. Cheema, S.M. Ching, Z. Ahmad, A. Abdul Kadir, Methanolic Extract of
Clinacanthus nutans exerts antinociceptive activity via the opioid/nitric oxide-
mediated, but cGMP-Independent, pathways, Evid. Based Complement. Altern.
Med (2016), 1494981, https://doi.org/10.1155/2016/1494981.
[53] J.H. Lee, H.Y. Zhou, S.Y. Cho, Y.S. Kim, Y.S. Lee, C.S. Jeong, Anti-inflammatory
mechanisms of apigenin: inhibition of cyclooxygenase-2 expression, adhesion of
monocytes to human umbilical vein endothelial cells, and expression of cellular
adhesion molecules, Arch. Pharm. Res. 30 (2007) 1318–1327, https://doi.org/
10.1007/BF02980273.
[54] A. Erdogan, A.K. Most, B. Wienecke, A. Fehsecke, C. Leckband, R. Voss, M.T. Grebe,
H. Tillmanns, C.A. Schaefer, C.R. Kuhlmann, Apigenin-induced nitric oxide
production involves calcium-activated potassium channels and is responsible for
antiangiogenic effects, J. Thromb. Haemost. 5 (2007) 1774–1781, https://doi.org/
10.1111/j.1538-7836.2007.02615.x.
[55] M. Aswar, V. Patil, Ferulic acid ameliorates chronic constriction injury induced
painful neuropathy in rats, Inflammopharmacology 24 (2016) 181–188, https://
doi.org/10.1007/s10787-016-0272-5.
Rosa Ventura-Martínez, Departamento de Farmacología, Facultad de Medicina, Uni­
versidad Nacional Autónoma de México, Av. Universidad No. 3000, Col. Ciudad Uni­
versitaria, alcaldía Coyoacán, 04510, Ciudad de México, México. Tel.: + 52 5 55 6232162;
fax: + 52 5 55 6232162. Email: rventuram7 @hotmail.com, rventuram@comunidad.
unam.mx
María Eva González-Trujano, Dirección de Investigaciones en Neurociencias, Instituto
Nacional de Psiquiatría “Ramón de la Fuente Muñiz”, Av. México-Xochimilco 101, Col. San
Lorenzo Huipulco, 14370, Ciudad de México, México. Email: evag@imp.edu.mx