molecules

Article
Antispasmodic Effects and Action Mechanism of
Essential Oil of Chrysactinia mexicana A. Gray on
Rabbit Ileum
Daniel Zavala-Mendoza 1,2,† , Laura Grasa 1,3,4, *, Miguel Ángel Zavala-Sánchez 5 ,
Salud Pérez-Gutiérrez 5 and María Divina Murillo 1,3
1 Departamento de Farmacología y Fisiología (Fisiología), Facultad de Veterinaria, Universidad de Zaragoza,
Miguel Servet 177, 50013 Zaragoza, Spain; [email protected] (D.Z.-M.);
[email protected] (M.D.M.)
2 Dirección General de Servicios Educativos Iztapalapa, Av. Rojo Gómez No. 1149, Col. Barrio San Pedro,
Del Iztapalapa 09300 México, DF, Mexico
3 Instituto de Investigación Sanitaria de Aragón (IIS-Aragón), 50013 Zaragoza, Spain
4 Instituto Agroalimentario de Aragón (IA2), 50013 Zaragoza, Spain
5 Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana-Xochimilco, Calzada del
Hueso 1100, Colonia Villa Quietud, Coyoacán, 09340 Mexico, DF, Mexico;
[email protected] (M.Á.Z.-S.); [email protected] (S.P.-G.)
* Correspondence: [email protected]; Tel.: +34-976-762832
† Present Address: División de Ciencias Biológicas y de la Salud, Universidad de la Costa,
Carretera al Libramiento Paraje de las Pulgas s/n, Colonia Cuauhtémoc, Santiago Pinotepa Nacional,
Oaxaca 71600, Mexico.

Academic Editor: Luca Forti

Received: 22 March 2016; Accepted: 12 June 2016; Published: 16 June 2016

Abstract: The Chrysactinia mexicana A. Gray (C. mexicana) plant is used in folk medicine to treat
fever and rheumatism; it is used as a diuretic, antispasmodic; and it is used for its aphrodisiac
properties. This study investigates the effects of the essential oil of C. mexicana (EOCM) on the
contractility of rabbit ileum and the mechanisms of action involved. Muscle contractility studies
in vitro in an organ bath to evaluate the response to EOCM were performed in the rabbit ileum. EOCM
(1–100 µg¨ mL´1 ) reduced the amplitude and area under the curve of spontaneous contractions of
the ileum. The contractions induced by carbachol 1 µM, potassium chloride (KCl) 60 mM or Bay
K8644 1 µM were reduced by EOCM (30 µg¨ mL´1 ). Apamin 1 µM and charybdotoxin 0.01 µM
decreased the inhibition induced by EOCM. The d-cAMP 1 µM decreased the inhibition induced by
EOCM. L-NNA 10 µM, Rp-8-Br-PET-cGMPS 1 µM, D,L-propargylglycine 2 mM, or aminooxyacetic
acid hemihydrochloride 2 mM did not modify the EOCM effect. In conclusion, EOCM induces an
antispasmodic effect and could be used in the treatment of intestinal spasms or diarrhea processes.
This effect would be mediated by Ca2+ , Ca2+ -activated K+ channels and cAMP.

Keywords: Chrysactinia mexicana; essential oil; antispasmodic effect; ileum; rabbit; intestinal motility

1. Introduction
Chrysactinia mexicana A. Gray (Asteraceae) is a plant distributed throughout central and northern
Mexico [1]. This plant is used to treat respiratory diseases, skin infections, fever and rheumatism [2].
Furthermore, it has diuretic, antispasmodic and aphrodisiac properties, and it has been used as a
general tonic, an energetic or as a stimulating agent [3]. Several antifungal functions or activities of
C. mexicana essential oil, isolated by Cardenas and coworkers, have also been reported [4]. Although the
toxicity of the essential oil of C. mexicana has not been reported, an aqueous extract of C. mexicana has
shown no toxicity in mice. Mice treated with the aqueous extract of C. mexicana by gavage (dose range

Molecules 2016, 21, 783; doi:10.3390/molecules21060783 www.mdpi.com/journal/molecules

Molecules 2016, 21, 783 2 of 12

1–200 mg¨ mL´1 ) showed no signs of neurotoxicity, such as convulsion, stereotyped behavior or ataxia.
The LD50 of the extract was estimated to be greater than 5000 mg¨ Kg´1 . In addition, this extract did not
produce damage in the hepatic functions or in the morphology of the liver and the kidney [3]. Previous
studies have shown that among the chemical constituents of C. mexicana are flavonoid glycosides [5],
thiophenes [6] and terpenes [7]. More recently, eucalyptol, piperitone, and linalyl acetate were found
to be major components of essential oil of C. mexicana [4,8]. Guerra-Boone and coworkers showed
that sylvestrene and limonene are main components of the essential oil of C. mexicana [9]. The LD50
of eucalyptol, one of the major components of the EOCM, is estimated as 3849 mg¨ kg´1 . Subacute
toxicity studies showed that the dose of 21.38 mg¨ kg´1 per day of eucalyptol causes no damage on
mouse liver and kidneys [10].
The rabbit small intestine represents a good model to study the intestinal motility. First, the intestinal
motility of rabbit exhibits cyclic, phasic and rhythmic spontaneous contractions that are highly
reproducible. Second, this model is widely used to study different substances or new drugs. Finally,
our group has characterized these spontaneous contractions and we have studied the mechanisms of
action involved in them. The spontaneous motility and contractility of rabbit intestine is mediated by
the influx of extracellular Ca2+ , intracellular Ca2+ release [11] and K+ channels [12]. Different agents
induce relaxing responses in the rabbit intestine as sodium nitroprusside (nitric oxide donor), trolox
(analogue of vitamin E) or quercetin and genistein (flavonoids) [13–15].
Folk medicine has been using C. mexicana for the treatment of different diseases and symptoms,
but many of these effects have not been scientifically investigated. In this present work, we studied the
effect of EOCM on the longitudinal smooth muscle of rabbit ileum and its possible mechanisms of action.

2. Results

2.1. Effect of EOCM on Ileum Motility

We studied the effect of EOCM on the spontaneous motility of rabbit ileum. EOCM (1–100 µg¨ mL´1 )
reduced the area under the curve (AUC) (Figure 1a, b) and the amplitude but not the frequency of
spontaneous contractions of longitudinal smooth muscle of the rabbit ileum (Table 1 and Figure 1a).
This effect was concentration-dependent and reversible after washing. As EOCM was dissolved in
DMSO, we studied the effect per se of DMSO on the spontaneous contractions of the ileum. The AUC,
amplitude and the frequency of spontaneous contractions on longitudinal smooth muscle were not
modified in the presence of DMSO (Table 2).

Table 1. Effect of EOCM on the amplitude (%) and frequency (%) of spontaneous contractions of
longitudinal smooth muscle of ileum. Results are mean percentage values, with respect to spontaneous
contractions in Krebs (control) ˘ SEM. The number of segments taken from four rabbits is in parentheses.

Conditions Amplitude % Frequency %

Control 100 ˘ 7.5 (14) 100 ˘ 10.9 (14)
EOCM 1 µg¨ mL´1 84.9 ˘ 3.4 (14) * 96.7 ˘ 1.9 (14)
EOCM 3 µg¨ mL´1 72.2 ˘ 4.2 (14) * 97.3 ˘ 3.7 (14)
EOCM 10 µg¨ mL´1 62.9 ˘ 6.1 (14) ** 95.5 ˘ 5.4 (14)
EOCM 30 µg¨ mL´1 40.1 ˘ 5.4 (14) ** 112.3 ˘ 31 (14)
EOCM 100 µg¨ mL´1 13.1 ˘ 2.7 (14) ** 94.8 ˘ 41 (14)
* p < 0.05; ** p < 0.01 vs. control.
Molecules 2016, 21, 783 3 of 12
Molecules 2016, 21, 783 3 of 12

FigureFigure 1 Effect
1. Effect of EOCM,
of EOCM, Ringer
Ringer Krebs
Krebs free-Ca 2+ 2+and
free-Ca and1 1mM
mMEGTA,
EGTA,andand papaverine
papaverine on onlongitudinal
longitudinalsmooth
smooth muscle
muscle of rabbit ileum.
of rabbit (a) Recordings
ileum. of cumulative
(a) Recordings of cumulative
concentration-response
concentration-response curvecurve of EOCM
of EOCM (1–100 (1–100
µM);µM); (b) Histograms
(b) Histograms showing
showing the values
the values of cumulative
of cumulative concentration-response
concentration-response curve
curve of EOCM
of EOCM (1–100
(1–100 µM).µM).
TheThecolumns
columns show the mean values, and vertical bars indicate SEM of 14 segments; (c) Recording of EOCM ´1 30 µg·mL −1; (d) Recording of Ringer Krebs2+
show the mean values, and vertical bars indicate SEM of 14 segments; (c) Recording of EOCM 30 µg¨ mL ; (d) Recording of Ringer Krebs free-Ca and 1 mM EGTA free-Ca 2+ and

1 );
(0 Ca2+ mM (e)EGTA (0 Ca2+of); papaverine
Recording (e) Recording0.6
ofmM.
papaverine 0.6 mM.indicate
Arrowheads Arrowheads indicate of
the addition theagents.
addition
* pof<agents.
0.05; *** pp <
< 0.05;
0.01 **
vs.pcontrol.
< 0.01 vs. control.
Molecules 2016, 21, 783 4 of 12

Table 2. Effect of DMSO at several volumes on the area under the curve (AUC, %), amplitude (%) and
frequency (%) of spontaneous contractions of longitudinal smooth muscle of the ileum. Results are
mean percentage values with respect to spontaneous contractions in Krebs (control) ˘ SEM. The number
of segments taken from four rabbits is in parentheses.

Conditions AUC (%) Amplitude (%) Frequency (%)

Control 100 (15) 100 (15) 100 (15)
DMSO 10 µL 100.8 ˘ 1.3 (15) 104 ˘ 2.7 (15) 99.5 ˘ 1.0 (15)
DMSO 20 µL 100.7 ˘ 2.7 (15) 104.2 ˘ 6.3 (15) 98.1 ˘ 1.9 (15)
DMSO 30 µL 99.0 ˘ 2.0 (15) 104.1 ˘ 6.3 (15) 97.7 ˘ 2.0 (15)
DMSO 40 µL 99.0 ˘ 2.1 (15) 96.8 ˘ 4.0 (15) 100 ˘ 1.3 (15)
DMSO 50 µL 95.2 ˘ 2.4 (15) 95.4 ˘ 5.8 (15) 99.2 ˘ 2.7 (15)

2.2. Comparison between the Effects of Free-Calcium Krebs, Papaverine and EOCM on Smooth Muscle of Ileum
We compared the effect of EOCM with the effects of free-Ca2+ Krebs and papaverine on
spontaneous motility. In free-Ca2+ Krebs with EGTA 1 mM, the amplitude, frequency and the
AUC of spontaneous contractions were reduced, with the inhibition of the AUC approximately
70% (29.2% ˘ 5.9%, n = 23) (Figure 1d). Papaverine 0.6 mM (a smooth muscle relaxant) reduced the
amplitude, frequency, tone and AUC (38.2% ˘ 4.1%, n = 25) of spontaneous contractions of the ileum
(Figure 1e). EOCM 30 µg¨ mL´1 , as mentioned above, decreased the spontaneous contractions, thus
evoking a relaxing response (Figure 1c). The shape of the response on the smooth muscle of the ileum
to the three agents was different, with the inhibition evoked by EOCM, which is lower than free-Ca2+
Krebs with EGTA or papaverine.

2.3. Effect of EOCM on Carbachol- or KCl-Induced Contractions

To examine the effect of EOCM on contractile agonists, segments of ileum were pre-incubated
with EOCM before the addition of carbachol or KCl. The contractile responses induced by carbachol
1 µM or KCl 60 mM were reduced after the addition of EOCM (30 µg¨ mL´1 ) on the longitudinal muscle
of the ileum (Figure 2a–d). Furthermore, the previous incubation of EOCM for 15 min decreased the
contractions induced by carbachol 1 µM (75.7% ˘ 7.3%, n = 16, p < 0.05 vs. control) or KCl 60 mM
(21.9% ˘ 5.1%, n = 9, p < 0.001 vs. control).

2.4. Role of Ca2+ on the Effect of EOCM

The role of Ca2+ on the EOCM relaxant effect was tested on segments of ileum, which were
incubated with different agonists and antagonists of calcium. The incubation of ileal segments with
Bay K8644 1 µM (an activator of Ca2+ ) for 15 min increased the AUC of spontaneous contractions of the
ileum. The addition of EOCM (30 µg¨ mL´1 ) after Bay K8644 restored the control values (Figure 2e,f).
The addition of verapamil 0.1 µM (an antagonist of voltage-dependent Ca2+ channels) after Bay
K8644 also restored the control values (101.7% ˘ 12.3%, n = 6). Trifluoperazine 1 µM (a calmodulin
antagonist) did not modify the EOCM effect on the AUC of the spontaneous contractions of the ileum
(trifluoperazine 87.4% ˘ 5.6%, EOCM 56.0% ˘ 7.5% vs. trifluoperazine + EOCM 52.9% ˘ 7.2%, n = 6).

2.5. Role of K+ Channels on the Effect of EOCM

In order to study the role of K+ channels on the EOCM relaxant effect, segments of ileum
were incubated with different inhibitors of K+ channels. The effect of K+ channel inhibitors on
spontaneous contractions and EOCM (30 µg¨ mL´1 ) response in the ileum is shown in Figure 3A.
Apamin 1 µM (Ap, a potent and selective inhibitor of small-conductance Ca2+ -activated K+ channels)
increased the AUC of the spontaneous contractions of ileum, indicating a per se effect of this K+
channel inhibitor on spontaneous contractions (Figure 3A). However, Tram-34 1 µM (Tr, a selective
and potent inhibitor of intermediate-conductance Ca2+ -activated K+ channels), charybdotoxin
Molecules 2016, 21, 783 5 of 12

0.01 µM (ChTx, a specific inhibitor of intermediate- and large-conductance Ca2+ -activated

Molecules 2016, 21, 783
K+
5 of 12
+
channels), glibenclamide 0.1 µM (Gb, an inhibitor of ATP-dependent K channels) and quinine
10 µM (Qn, an inhibitor of voltage-sensitive + channels) did not modify the AUC on spontaneous
voltage-sensitive K+ channels) did not modifyKthe AUC on spontaneous contractions of ileum. Only
contractions of ileum. Only apamin and charybdotoxin reduced itreduced
apamin and charybdotoxin reduced it slightly, but they significantly slightly,the
but they significantly
EOCM effect on the
reduced the EOCM effect on the AUC of the ileum
AUC of the ileum spontaneous contractions (Figure 3A). spontaneous contractions (Figure 3A).

Figure 2. Effect
Effect ofof carbachol
carbachol 11 µM,
µM, KCl
KCl 6060 mM,
mM, Bay
Bay K8644
K8644 11 µM before the addition of EOCM
µg¨ mL−1´1ononlongitudinal
30 µg·mL longitudinalsmooth
smoothmuscle
muscleofofrabbit
rabbitileum.
ileum.(a,c,e)
(a,c,e)Recordings
Recordings ofof carbachol,
carbachol, KCl
KCl and
Bay K8644 before the addition of EOCM; (b,d,f) (b,d,f) Histograms
Histograms showing
showing the
the values
values of
of control
control (c),
(c),carbachol,
carbachol,
and carbachol
carbachol ++EOCM;
EOCM;KCl, KCl,and
and KClKCl+ EOCM;
+ EOCM; andand
BayBay
K8644, andand
K8644, Bay Bay
+ EOCM,
+ EOCM,respectively. The
respectively.
columns showshow
The columns the mean values,
the mean and vertical
values, bars indicate
and vertical SEM ofSEM
bars indicate 14 segments. Arrowheads
of 14 segments. indicate
Arrowheads
the addition
indicate of agents.of* agents.
the addition p < 0.05;* **
p <p 0.05;
< 0.01;
** ***
p <p0.01;
< 0.001
*** vs.
p <control.
0.001 vs. control.
Molecules 2016, 21, 783 6 of 12
Molecules 2016, 21, 783 6 of 12

Figure
Figure 3. (A)
3. (A) Histogramsshowing
Histograms showing the
the effect
effect of
of KK+ channel
+
channelinhibitors
inhibitorsapamin
apamin(Ap),(Ap),
Tram-34,
Tram-34,
charybdotoxin
charybdotoxin (ChTx),
(ChTx), glibenclamide(Gb)
glibenclamide (Gb) and
and quinine
quinine (Qn)(Qn)ononthetheinhibition induced
inhibition inducedby EOCM
by EOCM
30 µg·mL−1 on the longitudinal smooth muscle of rabbit ileum; (B) Histograms of the effect of
30 µg¨ mL´1 on the longitudinal smooth muscle of rabbit ileum; (B) Histograms of the effect of
cAMP-related agents d-cAMP, H-89 and forskolin (Fk) on the inhibition induced by EOCM 30 µg·mL−1
cAMP-related agents d-cAMP, H-89 and forskolin (Fk) on the inhibition induced by EOCM 30 µg¨ mL´1
on the longitudinal smooth muscle of rabbit ileum. The columns show the mean values, and vertical
on the longitudinal smooth muscle of rabbit ileum. The columns show the mean values, and vertical
bars indicate SEM of 14 segments. * p < 0.05; ** p < 0.01, *** p < 0.001 vs. control.
bars indicate SEM of 14 segments. * p < 0.05; ** p < 0.01, *** p < 0.001 vs. control.
2.6. Role of cAMP on the Effect of EOCM
2.6. Role ofWe
cAMP on the Effect of EOCM
investigated the involvement of cAMP on the EOCM relaxant response in the rabbit ileum.
The investigated
We addition of d-cAMP 1 µM (a cAMP
the involvement ofanalog)
cAMP or onH-89 1 µM (a potent
the EOCM inhibitor
relaxant of cAMP-dependent
response in the rabbit ileum.
protein kinase) did not modify the AUC of the ileal spontaneous
The addition of d-cAMP 1 µM (a cAMP analog) or H-89 1 µM (a potent inhibitorcontractions. The previous incubation
of cAMP-dependent
with d-cAMP for 15 min decreased the EOCM (30 µg·mL−1) and induced inhibition on the AUC of
protein kinase) did not modify the AUC of the ileal spontaneous contractions. The previous incubation
spontaneous contractions of the ileum (Figure 3B).
with d-cAMP for 15 min decreased the EOCM (30 µg¨ mL´1 ) and induced inhibition on the AUC of
Forskolin 10 µM (an activator of adenil cyclase) reduced the AUC of spontaneous contractions
spontaneous contractions
of the ileum. The previousof the ileum (Figure
incubation 3B). with forskolin did not modify the effect of EOCM
of the ileum
Forskolin
(30 µg·mL−110 µM
) on the(an
AUCactivator of adenilcontractions
of spontaneous cyclase) reduced the AUC
of the ileum of 3B).
(Figure spontaneous contractions of
the ileum. The previous incubation of the ileum with forskolin did not modify the effect of EOCM
2.7.mL
(30 µg¨ ´1of
Role Nitric
) on theOxide
AUCand cGMP on the Effect
of spontaneous of EOCM of the ileum (Figure 3B).
contractions
We investigated the involvement of nitric oxide and cGMP on the EOCM relaxant response in
2.7. Role of Nitric Oxide and cGMP on the Effect of EOCM
the rabbit ileum. The AUC of spontaneous contractions on the longitudinal muscle of the ileum was not
modified
We by L-NNA
investigated 10involvement
the µM (a NOS inhibitor),
of nitricODQ 1 µM
oxide and(acGMP
potent and selective
on the EOCM inhibitor of guanylyl
relaxant response in
the rabbit ileum. The AUC of spontaneous contractions on the longitudinal muscle of thebyileum
cyclase) and chelerythrine 1 µM (a protein kinase C inhibitor) (Figure 4A), but was diminished
Rp-8-Br-PET-cGMPS 1 µM (a cGMP analog) and KT-5823 1 µM (a selective inhibitor of protein kinase G)
was not modified by L-NNA 10 µM (a NOS inhibitor), ODQ 1 µM (a potent and selective inhibitor
(Figure 4B). The incubation of ileum segments for 15 min with L-NNA, ODQ, chelerythrine, Rp-8-Br-
of guanylyl cyclase) and chelerythrine 1 µM (a protein kinase C inhibitor) (Figure 4A), but was
PET-cGMP or KT-5823 did not modify the EOCM effect on the AUC of spontaneous contractions
diminished by Rp-8-Br-PET-cGMPS 1 µM (a cGMP analog) and KT-5823 1 µM (a selective inhibitor of
(Figure 4A,B).
protein kinase G) (Figure 4B). The incubation of ileum segments for 15 min with L-NNA, ODQ,
chelerythrine, Rp-8-Br-PET-cGMP or KT-5823 did not modify the EOCM effect on the AUC of
spontaneous contractions (Figure 4A,B).
Molecules 2016, 21, 783 7 of 12
Molecules 2016, 21, 783 7 of 12

Figure 4.
Figure 4. (A)
(A) Histograms
Histograms showing
showing thethe effects
effects of
of LL-NNA,
-NNA, ODQ,ODQ, and
and chelerythrine
chelerythrine on
on the
the inhibition
inhibition
induced by
induced by EOCM
EOCM30 30µg¨ mL on
µg·mL −1
´1 onthethelongitudinal
longitudinal smooth
smooth muscle
muscle of rabbit ileum; (B) Histograms
Histograms of of
the effects
the effects of
ofRp-8-Br-PET-cGMPS
Rp-8-Br-PET-cGMPSand andKT-5823
KT-5823on onthe
theinhibition
inhibitioninduced
inducedbybyEOCM
EOCM3030µg¨ µg·mL on
mL´1−1 on
the longitudinal
the longitudinal smooth
smooth muscle
muscle of
of rabbit
rabbit ileum.
ileum. The
The columns
columns show
show the
the mean
mean values,
values, and
and vertical
vertical bars
bars
indicate SEM of eight segments. *
indicate SEM of eight segments. * p <p < 0.05; ** p < 0.01, *** p < 0.001 vs. control.
< 0.001 vs. control.

2.8. Role
2.8. Role of
of other
other Agents
Agents on
on the
the Effect
Effect of
of EOCM
EOCM
Finally,we
Finally, weexamined
examined whether
whether myosin
myosin light
light chain
chain phosphatase,
phosphatase, Rho-kinase
Rho-kinase or or hydrogen
hydrogen sulphide
sulphide
participated in the EOCM relaxant effect in the rabbit ileum. Okadaic acid 0.1 µM (Ok, aa potent
participated in the EOCM relaxant effect in the rabbit ileum. Okadaic acid 0.1 µM (Ok, potent
inhibitor of myosin light chain phosphatase) did not alter the AUC of
inhibitor of myosin light chain phosphatase) did not alter the AUC of spontaneous contractions of spontaneous contractions of
the ileum
the ileum in in the
the absence
absence (control
(control100
100vs.
vs.okadaic
okadaicacidacid100.9%
100.9%˘± 2.8%,2.8%, nn = = 7)
7) or the presence
or the presence of of EOAM
EOAM
(EOCM 59.2% ± 8.6% vs. okadaic acid + EOCM 56.9% ± 9.2%, n =
(EOCM 59.2% ˘ 8.6% vs. okadaic acid + EOCM 56.9% ˘ 9.2%, n = 7). Y-27632 1 µM (an inhibitor 7). Y-27632 1 µM (an inhibitor of
Rho-kinase) reduced the AUC of spontaneous contractions of the ileum
of Rho-kinase) reduced the AUC of spontaneous contractions of the ileum (control 100%, Y-27632 (control 100%, Y-27632 80.2%
± 4.6%˘with
80.2% 4.6%p with
< 0.05pvs. control,
< 0.05 n = 7). Y-27632
vs. control, 1 µM did
n = 7). Y-27632 not did
1 µM modify the inhibition
not modify inducedinduced
the inhibition by EOCM by
(30 µg·mL −1) (EOCM ´ 1 56.7% ± 5.2%, Y-27632+EOCM 47.1% ± 6.1% p <
EOCM (30 µg¨ mL ) (EOCM 56.7% ˘ 5.2%, Y-27632+EOCM 47.1% ˘ 6.1% p < 0.001 vs. control, n = 7).0.001 vs. control, n = 7).
DD,,L -propargylglycine 22 mM
L-propargylglycine mM (PAG,
(PAG, an an irreversible
irreversible inhibitor
inhibitor of of the
the enzyme
enzyme cystathionine
cystathionine γ-lyase),
γ-lyase),
aminooxyacetic acid hemihydrochloride 2 mM (AOAA, an inhibitor
aminooxyacetic acid hemihydrochloride 2 mM (AOAA, an inhibitor of cystathionine β-synthase),of cystathionine β-synthase), PGA
2 mM+ AOAA 2 mM, tetrodotoxin (TTX) 1 µM + L-NNA 10 µM+PGA
PGA 2 mM+ AOAA 2 mM, tetrodotoxin (TTX) 1 µM + L-NNA 10 µM+PGA 2 mM, TTX 1 µM + L-NNA 2 mM, TTX 1 µM + L-NNA 10 µM
+ AOAA
10 µM + AOAA 2 mM or TTXor
2 mM 1 µM
TTX+1LµM
-NNA + L10 µM +10PGA
-NNA µM 2+ mM
PGA+2AOAA mM + 2AOAA mM did not alter
2 mM did not thealter
AUCthe of
the spontaneous contractions of the ileum (control 100% vs. PAG 106.0%
AUC of the spontaneous contractions of the ileum (control 100% vs. PAG 106.0% ˘ 5.4%, AOAA 94.5% ± 5.4%, AOAA 94.5% ± 3.8%,
PAG
˘ + AOAA
3.8%, PAG +89.6% AOAA ± 3.3%,
89.6%TTX + L-NNA
˘ 3.3%, TTX ++PGA L -NNA97.5% ± 4.1%,
+ PGA TTX˘+ 4.1%,
97.5% L-NNA + AOAA
TTX + L-NNA 94.8% ± 7.0%,
+ AOAA
TTX + ˘
94.8% L-NNA
7.0%, +TTX PGA+ +LAOAA
-NNA +87.7%
PGA±+10.6%;AOAA n =87.7%
8). Furthermore,
˘ 10.6%; n ileal = 8).segments
Furthermore,incubated
ileal for 15 min
segments
with PAG, AOAA, PAG + AOAA, TTX + L-NNA + PGA, TTX + L-NNA + AOAA or TTX + L-NNA +
PGA + AOAA did not modify the EOCM (30 µg·mL−1)-evoked inhibition on the AUC of the ileal
Molecules 2016, 21, 783 8 of 12

incubated for 15 min with PAG, AOAA, PAG + AOAA, TTX + L-NNA + PGA, TTX + L-NNA + AOAA
or TTX + L-NNA + PGA + AOAA did not modify the EOCM (30 µg¨ mL´1 )-evoked inhibition on the
AUC of the ileal spontaneous contractions (EOCM 51.1% ˘ 4.9% vs. PAG + EOCM 50.1% ˘ 7.3%,
AOAA + EOCM 43.7% ˘ 4.6%, PGA + AOAA + EOCM 42.6% ˘ 3.3%, TTX + L-NNA + PGA + EOCM
43.1% ˘ 4.3%, TTX + L-NNA + AOAA + EOCM 45.1% ˘ 4.8%, TTX + L-NNA + PGA + AOAA +
EOCM 31.0% ˘ 4.4% p < 0.001, n ě 7).

3. Discussion
Chrysactinia mexicana is a plant used in folk medicine with antispasmodic actions. EOCM induced
relaxation on the longitudinal smooth muscle of the rabbit ileum. The relaxations of intestinal muscle
are caused by inhibition of the influx or release of Ca2+ to cytosol [11]. Furthermore, cAMP participates
in the relaxations of the smooth muscle evoked by different agents. The relaxing response of the rabbit’s
ileal smooth muscle agrees with the antispasmodic function attributed to C. mexicana as a medicinal
plant, although this effect has not been previously tested. The EOCM-induced relaxation on the smooth
muscle of the ileum was lower than the relaxation evoked in free-Ca2+ Krebs with EGTA. The EOCM
relaxation was similar in shape to the papaverine response on the ileal smooth muscle, although
papaverine evoked a decrease in muscle tone (Figure 1). Papaverine is a relaxant agent of smooth
muscle and it is used as a positive control for comparing relaxing responses. The relaxing response
obtained in this study agrees with the relaxing effect observed with the extract of Artemisia ludoviciana,
Artemisia maritima and Ferula heuffelii in the intestine [16–18].
Contractile agents such as carbachol or KCl induced contractions or spasms in intestinal smooth
muscle. In this study, the carbachol- or KCl-induced contractions were decreased by EOCM.
Furthermore, the increase of spontaneous contractions in the rabbit ileum induced by Bay K8644
was restored to control values by EOCM or verapamil but the contractions were not modified by
trifluoperazine. This finding concurs with the essential oil of Pistacia integerrima, which induces the
inhibition of the transient contraction of acetylcholine and the relaxation of the Bay K8644-precontracted
isolated guinea pig ileum, and these authors suggest that this effect is related to its ability to inhibit the
L-subtype Ca2+ channels [19]. Furthermore, the extracts of Artemisia maritima and Artemisia ludoviciana
induce relaxing effects on the contraction produced by potassium chloride in rabbit jejunum and rat
ileum [16,17]. Our results suggest that the effect induced by EOCM on spontaneous contractions and
on carbachol- or K+ -induced contractions are antispasmodic or relaxing responses in the longitudinal
smooth muscle of the rabbit ileum. Moreover, EOCM and verapamil reduced the increase of the AUC
on the smooth muscle of the ileum evoked by the analog of Ca2+ , Bay K8644, and in these effects the
Ca2+ enter is involved.
In this study, apamin and charybdotoxin partly decreased but tram-34, glibenclamide or quinine
did not alter the effect of EOCM on the smooth muscle of the ileum. Similar results were obtained
with glibenclamide or TEA (a non-selective inhibitor of K+ channels), agents that did not inhibit the
spasmolytic activity of Artemisia copa [20] or Mentha pulegium [17]. Also, dichloromethanic extract of
Artemisia ludoviciana spp. mexicana inhibits the rat ileum spontaneous contraction and this inhibition
was not modified by ODQ, indometacine, L-NAME, glibenclamide and 2-aminopyridine [17].
On the other hand, dibutyryl-cAMP and H-89 reduced but forskolin increased the inhibition
induced by EOCM. Furthermore, Rp-8-Br-PET-cGMPS, ODQ, chelerythrine, KT-5823, or L-NNA did
not modify the EOCM-induced effect on spontaneous contractions in the ileum. Similar results
were obtained with L-NAME or ODQ which did not modify the inhibitory effect evoked by
Achillea millefolium and Artemisia ludoviciana in the rat ileum [17,21]. We propose that the EOCM
effect is mediated in part by cAMP, but not cGMP or NO.
Contractile stimuli can sensitize myosin to Ca2+ by activating RhoA/Rho-kinase which inhibits
myosin light chain phosphatase (MLCP) activity. Conversely, smooth muscle relaxation results
from a decrease in the concentration of cytosolic Ca2+ and/or a reduced Ca2+ sensitivity of the
contractile apparatus.
Molecules 2016, 21, 783 9 of 12

In this way, we examined other possible mechanisms in the EOCM effect. Okadaic acid (inhibitor
of MLCP) did not alter the spontaneous contractions of the rabbit ileum and the EOCM effect. Y-27632
(inhibitor of Rho-kinase) inhibited the spontaneous contractions of the rabbit ileum, but it did not
modify the EOCM effect. Similar results were obtained in inhibiting Rho-kinase or MCLP and it had
no effect on the nitric oxide–induced relaxation in the circular smooth muscle of the rat colon [22].
PAG (inhibitor of the enzyme cystathionine γ-lyase), AOAA (inhibitor of cystathionine β-synthase)
or PAG + AOAA, did not alter the spontaneous contractions of the rabbit ileum and the EOCM effect.
These agents inhibit the synthesis of hydrogen sulfide. Hydrogen sulfide has been suggested as a
gaseous neuromodulator in mammals and evokes the relaxations of smooth muscle [23].
Previously, it has been described that the extract of the aerial part of C. mexicana has different
components such as flavonoid glycosides [5], thiophenes [6], terpenes [7]. Furthermore, the chemical
composition of the essential oil of C. mexicana isolated in this work was analyzed by gas
chromatography–mass spectrometry; the components were eucalyptol (28.7%), α-terpeniol (9.9%) and
piperitone (52.1%) [8]. Some components of C. mexicana have been described in other plants. The effects
of the essential oil of Eucalyptus tereticornis, with the main constituent 1,8-cineole or eucalyptol,
enhanced and reduced acetylcholine-induced contractions dependent on the concentration, at lower
and higher concentrations respectively, on guinea pig tracheal smooth muscle [24]. The addition of
1,8-cineole relaxes the tracheal rings’ carbachol-precontracted or K+ contractions [25]. Pure commercial
piperitone, eucalyptol, and alpha-terpineol, which are the major constituents of Casimiroa pringlei
essential oil, cause an antispasmodic effect on the rat uterus [26]. Flavonoids are phenolic compounds
with effects on intestinal peristalsis [27]. Flavonoids such as genistein and quercetin reduced the
amplitude of spontaneous contractions in the smooth muscle of rabbit duodenum, but they did not
modify the frequency, these effects being similar to those found for EOCM. The effect of genistein is
mediated by Ca2+ and K+ channels, while the effect of quercetin is mediated by cAMP and protein
kinase A [15]. Trolox decreases the amplitude and frequency of spontaneous contractions and the
acetylcholine-induced contractions [14]. However, sodium nitroprusside reduces the amplitude,
frequency and tone of spontaneous contractions [13]. The EOCM-induced relaxation may be due to
components such as piperitone, eucalyptol and alpha-terpineol, which are described as relaxing agents.
In conclusion, the antispasmodic effect induced by EOCM involves Ca2+ , Ca2+ -activated K+
channels and cAMP and might be used in the treatment of intestinal spasms or diarrhea processes.

4. Materials and Methods

4.1. Plant Material

C. mexicana leaves were collected in July 2012, in Guadalcázar, state of San Luis Potosí (Mexico).
The material was authenticated by taxonomist José García-Pérez and a voucher specimen (SLPM37571)
was deposited at the Isidro Palacios Herbarium of the Universidad Autónoma de San Luis Potosí.
Essential oil of Chrysactinia mexicana Gray (EOCM) was isolated and labeled in Laboratory N008
(Organic Chemistry Investigation and Natural Products), Department of Biological Systems of
Universidad Autónoma Metropolitana Unidad Xochimilco (Mexico) [8].

4.2. Preparation of EOCM for the Test

Dimethyl sulfoxide (DMSO) was used to solve the EOCM. Cumulative concentration-response
curves to DMSO were also performed to test the per se effect of DMSO.

4.3. Animals
All procedures were carried out under Project License PI03/14, approved by the in-house Ethics
Committee for Animal Experiments, from the University of Zaragoza, Zaragoza, Spain. The care and
use of animals were performed accordingly, with the Spanish Policy for Animal Protection RD 53/2013,
which meets the European Union Directive 2010/63 on the protection of animals used for experimental
Molecules 2016, 21, 783 10 of 12

and other scientific purposes. Twenty male New Zealand rabbits weighing 2–2.5 kg were kept in a
standard rabbit fodder and had free access to water.

4.4. Preparation of Ileum Segments and Experimental Protocols

Whole thickness segments (10 mm long) of ileum were removed and vertically suspended in
a thermostatically controlled organ bath, containing Krebs solution (in mM: NaCl 120, KCl 4.70,
CaCl2 2.40, MgSO4 1.20, NaHCO3 24.50, KH2 PO4 1.00 and glucose 5.60) at 37 ˝ C to achieve pH 7.4,
and continuously gassed with 95% O2 and 5% CO2 . Each segment was connected to an isometric
force transducer (Pioden UF1, Graham Bell House, Canterbury, UK). The segments were stretched
passively to an initial tension of 2 g. The mechanical activity was amplified (The MacLab Bridge Amp,
AD Instruments Inc., Milford, MA, USA), with a range of 2 mV and recorded for further analysis using
The MacLab System 8e software (AD Instruments Inc.). The segments were allowed to equilibrate in
Krebs solution for 45 min before use.
After the adaptation period, the spontaneous contractions of longitudinal smooth muscle from
ileum were recorded and several experimental protocols were followed:
To study the effect of EOCM, we added EOCM (1–100 µg¨ mL´1 ) every 5 min and the cumulative
concentration-response curves were performed.
To compare the effect of EOCM with free Ca2+ conditions, papaverine 0.6 mM or free Ca2+ Krebs
Ringer with EGTA 1 mM was added at the end of some experiments.
To examine the effect of EOCM on contractile agonists, segments of ileum were pre-incubated with
EOCM (30 µg¨ mL´1 ) for 15 min before the addition of carbachol (1 µM) or KCl (60 mM). Some ileum
segments were pre-contracted with carbachol (1 µM) or KCl (60 mM) and after 1 min of incubation,
EOCM (30 µg¨ mL´1 ) was added.
In order to study the role of Ca2+ or K+ channels on EOCM effect in the spontaneous contractions
of ileum, agents such as Bay K8644, trifuoperazine, apamin, tram-34, charybdotoxin, glibenclamide
or quinine were added to the bath, 15 min before EOCM (30 µg¨ mL´1 ). Some segments were also
incubated with Bay K8644 for 15 min before the addition of verapamil.
To investigate the involvement of cAMP, cGMP or NO on EOCM responses in the ileum, several
agents were added to the bath such as d-cAMP, H-89, forskolin, ODQ(1H-[1,2,4]Oxadiazolo[4,3-α]
quinoxalin-1-one), cherelythrine, Rp-8-Br-PET-cGMPS, KT-5823, or L-NNA for 15 min before EOCM
(30 µg¨ mL´1 ).
Finally, to examine whether myosin light chain phosphatase, Rho-kinase or hydrogen sulphide
participated in EOCM effect on the spontaneous contractions of ileum, some segments were incubated
for 15 min with okadaic acid, Y-27632, D,L-propargylglycine or aminooxyacetic acid hemihydrochloride
before the addition of EOCM (30 µg¨ mL´1 ).

4.5. Analysis of Data

All intestinal segments included in the analysis showed spontaneous contractions. Thus, each
segment served as its own control. The amplitude (in g) and the frequency (contractions per minute,
cpm) of spontaneous contractions were measured as previously described [12]. EOCM, ACh, carbachol,
KCl or Bay K8644 induced motor responses were measured as the Area Under the Curve (AUC) or as
integrated mechanical activity per second, expressed as g s´1 and normalized per gram of wet tissue as
follows: AUC = AUC1 ´ AUC0 , where AUC1 is the area under the curve per second per gram, either
during the first 3 min of response to EOCM, acetylcholine (ACh), carbachol or KCl administration,
and AUC0 is the area under curve per second per gram of the spontaneous motility, 3 min before
adding EOCM or other substances. The AUC values were calculated using a baseline of 0 gram [28].
Data are given as mean percentage with respect to control ˘ SEM, with n denoting the number of
preparations. Comparisons between means were made, using one-way Analysis of Variance (ANOVA)
tests, with p-values determined using Bonferroni’s post hoc test. The data was analyzed using GraphPad
Prism 5.02, and the differences between p-values < 0.05 were considered statistically significant.
Molecules 2016, 21, 783 11 of 12

4.6. Chemicals
Acetylcholine (ACh), carbamoylcholine chloride (carbachol), Bay K8644, trifluoperazine
dihydrochloride, apamin, charybdotoxin (ChTX), glibenclamide, quinine, verapamil, papaverine
dihydrochloride, H-89 dihydrochloride hydrate, KT-5823, chelerythrine chloride, (+)-(R)-trans-4-
(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632),
dibutyryl-cAMP (d-cAMP), D , L-propargylglycine (PAG), aminooxyacetic acid hemihydrochloride
(AOAA), tetrodotoxin (TTX) and NG -nitro-L arginine (L-NNA) were obtained from Sigma (Madrid,
Spain). Tram-34, 1H-[1,2,4]oxadiazolo [4,3-α]quinoxalin-1-one (ODQ), Rp-8-Br-PET-cGMPS, okadaic
acid and forskolin were purchased from Tocris (Madrid, Spain). All chemicals were of analytical grade.
Bay K8644 was dissolved in ethanol. Apamin was diluted in acetic acid. Glibenclamide, tram-34,
forskolin (Fk), okadaic acid and ODQ were prepared in dimethyl sulfoxide. All other chemicals were
dissolved in distilled water.

Acknowledgments: This work was funded by a grant from Gobierno de Aragón, España (B61/2014) and
Fondo Social Europeo. An individual grant to stay with Daniel Zavala Mendoza was provided by Experimental
Biology Ph.D Program, Universidad Autónoma Metropolitana-Iztapalapa, Mexico.
Author Contributions: D.Z.M. performed the experiments and analyzed the data. M.A.Z.S. and S.P.G. gave
advice on obtaining the essential oil of Chrysactinia mexicana. M.D.M. designed the research study and wrote the
paper. L.G. revised the manuscript for important intellectual contents.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the EOCM are available from the authors.

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