Journal of Ethnopharmacology 139 (2012) 513518

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Relaxant effects of Artemisia ludoviciana on isolated rat smooth muscle tissues

Samuel Estrada-Soto a, , Amanda Snchez-Recillas a , Gabriel Navarrete-Vzquez a ,
b , Rafael Villalobos-Molina c , Maximiliano Ibarra-Barajas c
Patricia Castillo-Espana
a
b
c

Facultad de Farmacia, Universidad Autnoma del Estado de Morelos, Av. Universidad 1001, Colonia Chamilpa, 62209 Cuernavaca, Morelos, Mexico
Centro de Investigacin en Biotecnologa, Universidad Autnoma del Estado de Morelos, Av. Universidad 1001, Colonia Chamilpa, 62209 Cuernavaca, Morelos, Mexico
Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autnoma de Mxico, Tlalnepantla, Estado de Mxico 54090, Mexico

a r t i c l e

i n f o

Article history:
Received 26 September 2011
Received in revised form
19 November 2011
Accepted 22 November 2011
Available online 30 November 2011
Keywords:
Artemisia ludoviciana
Smooth muscle
Spasmolytic activity
Tracheal relaxation
Vasorelaxant activity

a b s t r a c t
Ethnopharmacological relevance: Artemisia ludoviciana spp. mexicana (Willd. Ex.) Spring D.D. Keck (Asteraceae), known as estafiate is employed for the treatment of diarrhea, dysentery, parasites, abdominal
pain, vomiting, stomach ache, and also as antispasmodic agent. The aim of the present study was to evaluate the relaxant effect of hexanic (HEAl), dichloromethanic (DEAl) and methanolic (MEAl) extracts on
isolated trachea, ileum and aorta rat rings, and to establish the tracheo-relaxant mode of action of DEAl.
Materials and methods: All extracts were investigated based on their capacity of to inhibit the rat ileum
spontaneous contraction, to relax contraction induced by noradrenaline (0.1 !M) on endothelium-intact
and endothelium-denuded thoracic aorta rat rings, and also to inhibit contraction provoked by carbachol
(1 !M) on rat trachea.
Results: Organic extracts had no spasmolytic action on ileum strips compared to positive control
(papaverine, p < 0.05). On the other hand, all extracts induced a significant concentration- and partial
endothelium-dependent vasorelaxant activity. Extracts also showed significant relaxant effect on precontracted tracheal tissue in a concentration-dependent manner. In last two experiments, DEAl was the
most potent and efficient extract; however, it was less potent than papaverine and theophylline, used
as positive controls (p < 0.05). In tracheal preparation, DEAl shifted to the right, in a parallel manner, the
concentrationresponse curves induced by carbachol (p < 0.05). Also, DEAl induced a significant relaxant
effect on the contraction produced by potassium chloride (KCl, 80 mM). Pre-incubation with 1-H-[1,2,4]oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ, 10 !M), indomethacin (10 !M), N" -nitro-l-arginine methyl
ester (l-NAME, 10 !M), glibenclamide (10 !M) and 2-aminopyridine (2-AP, 100 !M) did not modify the
DEAl-relaxant curves.
Conclusions: Functional experiments suggest that the most active extract, DEAl, induced its relaxant effect
by possible muscarinic receptors antagonism and calcium channel blockade in tracheal rings. On the
other hand, significant vasorelaxant activity showed by DEAl is partially endothelium-dependent. Finally,
spasmolytic activity induced by the extracts in the rat ileum was not significant, which suggests that
the antidiarrheic effect of the plant is related to antimicrobial and antiparasitic properties previously
described.
2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Abbreviations:
HEAl, hexanic extract from Artemisia ludoviciana; DEAl,
dichloromethanic extract from Artemisia ludoviciana; MEAl, methanolic extract
from Artemisia ludoviciana; KCl, potassium chloride; ODQ, 1-H-[1,2,4]-oxadiazolo[4,3a]-quinoxalin-1-one; l-NAME, N" -nitro-l-arginine methyl ester; 2-AP, 2aminopyridine; NA, noradrenaline; PDEs, phosphodiesterases; NO, nitric oxide;
cGMP, cyclic guanylyl monophosphatase; Ca2+ , calcium ion; K+ , potassium ion;
cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate;
Ca2+ CaM, ion calcium:calmodulin complex; IP3 , inositol trisphosphate; DAG, diacylglycerol; PKC, protein kinase C.
Corresponding author. Tel.: +52 777 3 29 70 89; fax: +52 777 3 29 70 89.
E-mail address: enoch@uaem.mx (S. Estrada-Soto).
0378-8741/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2011.11.041

Current scientific research has evidenced the pharmacological

importance of molecules derived from natural sources (microorganisms, plants and animals) as therapeutic agents in the treatment
of several pathologies (Butler, 2005). In fact, these agents represent novel candidates for the development of phytomedicines,
which can be used to treat chronic degenerative and infectious diseases with high prevalence in the world population (Newman et al.,
2003).
Diabetes, cardiovascular, respiratory and gastrointestinal diseases are leading causes of morbidity and mortality in Mexico,
according to reports from Health Ministry (INEGI/Health Ministry
of Mexico/CONAPO, 2008). Thus, the Mexican traditional medicine

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has developed therapeutic strategies, through the sustainable use

of medicinal plant preparations, in order to provide alternative
drugs to improve lifestyle in patients (Heinrinch, 2003). In consequence, there are many plant species in traditional medicine
used for a long time in traditional medicine, and the most
abundant come from Anacardiaceae, Apocynaceae, Asteraceae,
Burseraceae, Cactaceae, Euphorbiaceae, Fabaceae, Malphigiaceae
2007).
and Rubiaceae families (Monroy-Ortiz and Castillo-Espana,
Plant species that belong to these plant families have been used
to treat prevalent diseases. Artemisia ludoviciana spp. mexicana
(Willd. Ex.) Spring D.D. Keck (Asteraceae), known as estafiate
is employed for the treatment of diarrhea, dysentery, parasites, abdominal pain, vomiting, and stomach ache. Also, it
is used as antispasmodic agent (Said Fernndez et al., 2005;
2007). There are few systemMonroy-Ortiz and Castillo-Espana,
atic studies that support therapeutic properties of this medicinal
plant, especially for the treatment of gastrointestinal disorders,
such as inhibition of charcoalgum acacia-induced hyperperistalsis (Calzada et al., 2010), anti-Helicobacter pylori activity
(aqueous extract MIC = 125 !g/mL, and methanolic extract MIC=
250 !g/mL) (Castillo-Jurez et al., 2009), and antimicrobial and
antiparasitic activities (Said Fernndez et al., 2005; Calzada et al.,
2006, 2007; Lopez-Lutz et al., 2008). Also, a previous phytochemical study revealed that dichloromethane extract contains
some sesquiterpene lactones and flavonoids (Ruiz-Cancino et al.,
1993).
Current study aimed to evaluate the potential spasmolytic
effects of organic extracts derived from Artemisia ludoviciana as
a source of pharmacological agents that act on diarrhea pathophysiology. Furthermore, because of its antispasmodic properties,
we determined its vasorelaxant effect on endothelium-intact and
endothelium-denuded aorta rat rings, and its relaxant activity and
mode of action on tracheal rings.

2. Materials and methods

2.1. Chemicals and drugs
Carbamylcholine (carbachol), noradrenaline (NA), theophylline,
glibenclamide, 2-aminopyridine, N" -nitro-L-arginine methyl ester
(L-NAME), 1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ),
indomethacine, papaverine, KCl and DMSO were purchased from
SigmaAldrich Co. (St. Louis, MO, USA). All other reagents were analytical grade from local sources. Stock solutions of extracts were
made using distilled water and freshly prepared the same day of
experimentation.

2.2. Plant material and extraction

Artemisia ludoviciana (Astearaceae) was collected in October
2007. Plant material was taken from its native habitat in the
State of Morelos, Mexico, collected and identified by Dr. Patricia
A voucher specimen (27191) was deposited in the
Castillo-Espana.
Centro de Estudios Ambientales e Investigacin Sierra de Huautla
(CEAMISH)-HUMO Herbarium, Cuernavaca, Morelos. The plant
material was manually cleaned of solid residues and dried at room
temperature (25 C) during two weeks. Then, dry plant material
was pulverized (32.4 g), and crude organic extracts were prepared
by successive maceration process using hexane, dichloromethane
and methanol (600 mL each, 3 times for 72 h at room temperature). After filtration, organic extracts were concentrated in vacuo
at 40 C using a Rotavapor (Buchi R-200) and the percentage yields
obtained were 15.4% (MEAl), 12.7% (DEAl) and 5.7% (HEAl), respectively.

2.3. Pharmacological evaluations

2.3.1. Animals
Male Wistar rats (250300 g) were used and maintained under
standard laboratory conditions with free access to food and water.
All animal and procedures were conducted in accordance with our
Federal Regulations for Animal Experimentation and Care (SAGARPA,
NOM-062-ZOO-1999, Mexico), and approved by the Institutional
Animal Care and Use Committee. All experiments were carried out
using six animals per group. All animals of the study were sacrificed
by cervical dislocation.
2.3.2. In vitro rat ileum test
Rat ileum longitudinal preparations were assayed as described
elsewhere (Estrada-Soto et al., 2010). Briefly, after rats were sacrificed tissue segments were dissected out, cleaned and placed in
organ baths containing warmed (37 C) and oxygenated (gas mixture O2 /CO2 , 19:1) RingerKrebs solution (14 mL), whose chemical
composition (mM) was: NaCl, 118; KCl, 4.7; CaCl2 , 2.5; MgSO4 , 1.2;
KH2 PO4 , 1.2; NaHCO3 , 25.0; EDTA, 0.026 and dextrose, 11.1 at pH
7.4. One end of the tissue segment (3 cm) was held to the bottom of organ baths using stainless steel hooks, and the other was
attached to an isometric force transducer (Grass-FT03, Astromed,
West Warwick, RI, USA), connected to a MP100 analyzer (BIOPAC
Instruments, Santa Barbara, CA, USA) under an optimal tension of
1 g, as previously described (Estrada-Soto et al., 2010). After stabilization period (30 min), a 10-min-control time range was recorded.
Test samples were added to organ bath in cumulative quarter-log
concentrations (0.971000 !g/mL). The pharmacological effect on
spontaneous contraction due to organic extracts, as well as positive
control was determined by comparing muscular tone after tissue
contractions, before and after sample addition. Muscular tone was
calculated from tracings using Acknowledge software (BIOPAC ).
2.3.3. Rat aorta ring assay
The vasorelaxant activity was determined using a modified standard protocol of Torres-Piedra et al. (2011). After sacrifice, the
thoracic aorta was removed, cleaned and cut in rings of 45 mm
in length. In addition, for some aortic rings the endothelium layer
was gently removed by manual procedures. Then, each piece of
tissue was suspended in a tissue chamber containing Krebs solution at 37 C, continuously gassed with O2 /CO2 (19:1). Tissues were
placed under a resting tension of 3.0 g and allowed to stabilize for
60 min. The contractions were recorded with an isometric force
transducer (Grass FT03) connected to a MP100 Analyzer. After the
stabilization period, the tissues were stimulated with NA (0.1 !M)
during 10 min, and they were washed with fresh Krebs solution.
This procedure was repeated three times at 30 min intervals before
starting the experiments. The absence of endothelium was confirmed by the lack of the relaxant response induced by carbachol
(1 !M), in the last contraction to assess viability. Finally, all tissues
were contracted with NA and test samples (extracts or positive control) added to the bath in quarter-log cumulative concentrations
(evaluation period). The relaxant effect of the samples was determined by its ability to induce a maximal vascular contraction before
and after their addition.
2.3.4. Rat trachea ring assay
For this purpose a previous protocol was used (Fedan et al.,
2001). After sacrifice, tracheas were dissected and cleaned out
of connective tissue and mucus, and immediately was cut into
45 mm length rings. Then, tissue segments were mounted by
stainless steel hooks, under an optimal tension of 2 g in 10 mL organ
baths containing warmed (37 C) and oxygenated (O2 /CO2 , 19:1)
Krebs solution. Changes in tension were recorded by isometric force
transducers (Grass FT03) connected to a MP100 analyzer. After

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2.3.5. Determination of DEAl mode of action on tracheal in vitro

preparation
In order to establish the DEAl mode of action, following experiments were conducted.

0
20

% Relaxation

stabilization, rings were contracted by carbachol (1 !M) and

washed every 30 min for 2 h. After contraction with carbachol
(1 !M) or KCl (80 mM), the test samples (organics extracts, vehicle
and positive control) were added to the bath in a volume of 100 !L;
then cumulative concentrationresponse curves were obtained for
each ring (3.031000 !g/mL). The relaxant effect of extracts and
positive control (theophylline, 1.67550 !M) were determined by
comparing the muscular tone of the contraction before and after
addition of the test materials. Muscular tone was calculated from
the tracings, using Acknowledge software.

40
60

Papaverine

80

HEAl
DEAl

100

MEAl
0.1

a. For interaction with cholinergic receptors: tissues were incubated with DEAl (50, 100 and 156 !g/mL) during 15 min,
then carbachol (cholinergic agonist) was cumulatively added to
the chambers (0.006 to 540 !M), and concentrationresponse
curves were obtained. Antispasmodic effect of DEAl was determined comparing the contraction induced by carbachol in
absence and presence of DEAl.
b. For interaction with phosphodiesterases (PDEs): tissues were
incubated with DEAl (100 !g/mL) during 15 min, then theophylline (inhibitor of PDEs) was cumulatively added to the
bath (1.67550 !M), and concentrationresponse curves were
obtained. The relaxant effect induced by theophylline was compared in absence and presence of DEAl.
c. For disruption of the NO/cGMP pathway: tissues were incubated with l-NAME (10 !M, NOS inhibitor) or ODQ (10 !M, sGC
inhibitor) during 15 min, and immediately DEAl was added at different concentrations and cumulative concentrationresponse
curves were obtained.
d. For interaction with prostaglandins, tissues were incubated
with indomethacin (10 !M, cyclooxygenases inhibitor), then,
DEAl was added at different concentrations and cumulative
concentrationresponse curves were obtained.
e. In order to know the role of K+ channels on DEAl-induced
relaxation: trachea rings were incubated for 15 min with K+
channel blockers (glibenclamide, 10 !M, and 2-AP, 100 !M),
then DEAl was added at different concentrations and cumulative
concentrationresponse curves were obtained.
2.4. Statistical analysis
All experimental results are expressed as the mean of six
experiments S.E.M. Concentrationresponse curves were plotted
using software MicrocalTM Origin 8.0 (Microcal Software Inc., USA).
Curves were adjusted by non-linear curve fitting sub-program
using the same software. Statistical analysis was performed by OneWay Analysis of Variance and post-hoc Dunnets test (sample vs.
control). p values less than 0.05 were considered to be statistically
significant.
3. Results and discussion
The aerial parts of Artemisia ludoviciana were subjected to
extraction by maceration, in order to analyze relaxing pharmacological properties of these extracts on smooth muscle-containing
tissues (ileum, aorta and trachea), and compare relaxant activity between them. Smooth muscle represents functional cell layer
in organs that modulate relaxation/contraction processes, therefore, scientific search of effective extracts that contain bioactive

10

100

1000

[g/mL]
Fig. 1. Relaxing concentrationresponse curves of extracts obtained from Artemisia
ludoviciana on spontaneous contractions of isolated rat ileum strips. Data are
expressed as the mean S.E.M. of six experiments (p < 0.05).

molecules and modify this action could be an important strategy in

drug discovery.
Extract with major yield was methanolic (MEAl, 15.4%), followed
by dichloromethanic (DEAl, 12.7%), and hexanic (HEAl, 5.7%). HEAl,
DEAl and MEAl were evaluated using rat ileum strips assay, to determine their potential activity as spasmolytic agents for the treatment of diarrhea. Results showed that HEAl (Emax = 41.1 4.9%),
DEAl (Emax = 55.4 3.8%) and MEAl (32.0 3.2%) extracts have
almost the same but slight concentration-dependent inhibition on
spontaneous contraction of ileum strips (Fig. 1), and were less
potent than papaverine (positive control). Results also show that
all organic extracts have effect not greater than 50%, suggesting
that Artemisia ludoviciana could exert its antidiarrheic effect on etiologic agents, such as bacteria and parasites and not in the direct
relaxation of intestinal smooth muscle. Findings are in agreement
with those reported about antimicrobial and antiparasitic activities
(Said Fernndez et al., 2005; Calzada et al., 2006, 2007; Lopez-Lutz
et al., 2008).
Organic extracts were also evaluated to determine the relaxant effect in rat aortic rings, with and without endothelium
contracted with NA (0.1 !M). Results from these evaluations show that DEAl was the most effective and potent
extract (EC50 = 62.6 3.5 !g/mL, Emax = 104.8 4.8%) (Fig. 2b) in
endothelium-intact vascular relaxation compared with HEAl
(EC50 = 37.1 5.2 !g/mL, Emax = 46.1 8.8%) (Fig. 2a) and MEAl
(EC50 = 234.7 4.3 !g/mL, Emax = 77.2 4.9%) (Fig. 2c). All organic
extracts were less potent than positive control used (papaverine, EC50 = 8.3 0.25 !g/mL and Emax = 98 1.02%). Vasorelaxant
effect showed by organic extracts was concentration- and partial
endothelium-dependent. The latter suggests that endotheliumderived factors such as NO or prostacyclin are involved the activity
of the extract (Hernndez-Abreu et al., 2009). In contrast, an
endothelium-independent relaxation is related with a smooth
muscle cells activity, which interfere on contraction process such as
#1 -adrenoceptors antagonism, Ca2+ channel blockade, K+ channel
opening, cAMP or cGMP increment, or Ca2+ CaM complex activity
inhibition (Huang and Ho, 1996; Zhang and Tan, 1998; Maciel et al.,
2004; Zhu et al., 2007; Vergara-Galicia et al., 2010).
All organic extracts induced a significant concentrationdependent relaxation on tracheal rings contracted with carbachol
(1 !M). As it can be seen in Fig. 3, DEAl (EC50 = 91.6 5.2 !g/mL and
Emax = 105.2 2.1%) was the most potent and efficient extract, compared with MEAl (EC50 = 496.6 6.6 !g/mL and Emax = 88.3 6.7%)

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S. Estrada-Soto et al. / Journal of Ethnopharmacology 139 (2012) 513518

20

40

% Relaxation

% Relaxation

20

60
80

Papaverine
HEAl (E+)
HEAl (E-)

100
120

40
60
80
100

10

100

1000

[g/mL]

Papaverine
DEAl (E+)
DEAl (E-)
1

10

100

1000

[g/mL]

% Relaxation

20
40
60

Papaverine
MEAl (E+)
MEAl (E-)

80
100

10

100

1000

[g/mL]
Fig. 2. Relaxing concentrationresponse curves of (a) hexane, (b) dichloromethane and (c) methanol extracts from Artemisia ludoviciana on intact and endothelium-denuded
rat aortic rings contracted with NA (0.1 !M). Data are expressed as the mean S.E.M. of six experiments (p < 0.05).

% Relaxation

20
40
60

Theophylline
HEAl
DEAl
MEAl

80
100
120

0.1

10

100

1000

[g/mL]
Fig. 3. Relaxing concentrationresponse curves of extracts obtained from Artemisia
ludoviciana on rat tracheal rings contracted with carbachol (1.0 !M). Data are
expressed as the mean S.E.M. of six experiments (p < 0.05).

and HEAl (EC50 = 534.0 4.4 !g/mL and Emax = 79.6 5.5%), respectively (p < 0.05). All extracts were less potent than theophylline
(IC50 = 25.5 !g/mL) a positive control used. DEAl was the most
active extract over all in vitro smooth muscle tissues assayed. Since
DEAl showed the best relaxant activity on tracheal rings assay, the
possible functional mode of action of dichloromethanic extract on
trachea was assessed. DEAl caused relaxation of carbachol-induced
contractions, and carbachol + DEAl (50156 !g/mL) significantly
displaced the agonist curve to the right and reaches the maximum
efficacy obtained by carbachol alone, suggesting a competitive
insurmountable antagonist, which is defined as the maximal
response to the agonist depressed. It can result from a temporal
inequilibrium involving a slow offset orthosteric antagonist or
be the result of an allosteric modulation of the receptor (Kenakin
et al., 2006). Also, a non-parallel shift with suppression of the
maximum effect was observed at higher concentration, suggesting
an additional non-competitive inhibition (Brunton et al., 2006) i.e.
a possible Ca2+ antagonism (Fig. 4a). Results indicate that in the
extract exist compounds which are acting as cholinergic receptors
antagonists, mainly on M3 muscarinic subtype, which are coupled
to G#q/11 protein that activates phospholipase C, resulting in the
formation of IP3 and DAG. IP3 causes liberation of Ca2+ from the
internal stores resulting in a rapid, but transient increase in free
cytoplasmic Ca2+ concentration. This leads to myosin light chain
kinase activation via Ca2+ calmodulin, resulting in actinmyosin

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Contraction (g)

0
20

% Relaxation

Carbachol
Carbachol + DEAl [50 g/mL]
Carbachol + DEAl [100 g/mL]
Carbachol + DEAl [156 g/mL]
Carbachol + DEAl [500 g/mL]

80

DEAl
DEAl + Glibenclamide [10 M]
DEAl + 2-Aminopyridine [100 M]

120

1
1E-5

1E-4

1E-3

0.01

0.1

10

100

1000

[g/mL]

[M]

Fig. 5. Relaxing concentrationresponse curves of DEAl obtained from Artemisia

ludoviciana on rat tracheal rings pre-treated with glibenclamide and 2aminopyridine and contracted with carbachol (1.0 !M). Data are expressed as the
mean S.E.M. of six experiments (p < 0.05).

20

20

40

% Relaxation

% Relaxation

60

100

40

60
80

120
1E-6

1E-5

1E-4

1E-3

60
80

Nifedipine
DEAl

100

40

100
0.01

0.1

10

100

1000

[g/mL]
Fig. 4. (a) Concentrationresponse curves of carbachol (CCh) in the absence and
presence of increasing concentrations of DEAl. (b) Relaxing concentrationresponse
curves of DEAl on rat tracheal rings contracted with KCl (80 mM). Data are expressed
as the mean S.E.M. of six experiments (p < 0.05).

interaction and the contractile response. DAG triggers translocation and thereby activation of protein kinase C (PKC), which
is involved in regulation of the contractile machinery in smooth
muscles by promoting the agonist-induced Ca2+ release from
intracellular stores, and additional influx of extracellular Ca2+
enhances the contractile response (Racke and Matthiesen, 2004).
In this context, DEAl was capable to induce relaxation on tracheal
rings contracted with KCl (80 mM), in a concentration-dependent
manner (Fig. 4b), which confirms a second mode of action related
with possible blockade of Ca2+ channel. In this regard it is well
known that high K+ concentration (80 mM) induced a depolarization via voltage-gated Ca2+ channels (Gilani et al., 2005; Soder and
Petkov, 2011). Based on last findings, we explored if K+ channels
are implicated on DEAl relaxant mode of action. Incubation with
glibenclamide and 2-AP did not modify DEAl-induced relaxation
on tracheal rings contracted with carbachol (Fig. 5), which suggest
that no K+ channels are opened due to DEAl (Gilani et al., 2005). On
the other hand, no change occurred to DEAl relaxant curves in the
presence of l-NAME, indomethacin and ODQ (Fig. 6), suggesting
that NO, cGMP or prostaglandin are not involved on DEAl-induced
relaxation. Finally, previous phytochemical study revealed that

DEAl
DEAl + L-NAME [10 M]
DEAl + Indomethacin [10 M]
DEAl + ODQ [10 M]

120
10

100

1000

[g/mL]
Fig. 6. Relaxing concentrationresponse curves of DEAl obtained from Artemisia
ludoviciana on rat tracheal rings pre-treated with l-NAME, indomethacin and ODQ
and pre-contracted with carbachol (1.0 !M). Data are expressed as the mean S.E.M.
of six experiments (p < 0.05).

dichloromethane extract contains some sesquiterpene lactones

and flavonoids (Ruiz-Cancino et al., 1993), which could be related
to cholinergic receptor and/or Ca2+ channel blockade showed by
DEAl. Recently, yomogin, a sesquiterpene lactone and major component of Artemisia vulgaris, were identified to exhibit histamine
H1 receptor antagonism on gut and trachea, which suggest that
this kind of compounds are responsible of the activity (Natividad
et al., 2011). Further phytochemical and pharmacological studies
are necessary for the isolation of bioactive compounds.
4. Conclusions
All the extracts showed smooth muscle relaxant activity, being
DEA1 the most active extract. Functional experiments suggest that
DEAl-relaxant effect is by possible muscarinic receptors antagonism, and also may act by calcium channel blockade in tracheal
rings. Its vasorelaxant activity is in part endothelium-dependent.
The spasmolytic activity induced by the extracts was not significant.

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Acknowledgements
This study was financed by a grant from Apoyo a la Mejora del
Perfil Individual del profesorado de tiempo completo (Fondo para la
Consolidacin de las Universidades Pblicas Estatales y con Apoyo
Solidario Ejercicio 2009), Faculty of Pharmacy Budgets (2010 and
2011), and grants IN224408 from PAPIIT, UNAM, and 47481 from
CONACyT.
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