Ethyl rosmarinate relaxes rat aorta by an endothelium-independent pathway

Article abstract of DOI:10.1016/j.ejphar.2015.09.003

Ethyl rosmarinate is an ester derivative of rosmarinic acid, a major constituent of Hyptis suaveolens. The present study investigated the vasorelaxant mechanism of ethyl rosmarinate in isolated rat aortic rings using an organ bath system. Ethyl rosmarinat

Full text of DOI:10.1016/j.ejphar.2015.09.003

European Journal of Pharmacology 766 (2015) 9–15
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Ethyl rosmarinate relaxes rat aorta by an endothelium-independent
pathway
a
b
c
a
Piyawadee Wicha , Jiraporn Tocharus , Archawin Nakaew , Rungusa Pantan ,
c
a,
n
Apichart Suksamrarn , Chainarong Tocharus
Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand
Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 January 2015
Received in revised form
Ethyl rosmarinate is an ester derivative of rosmarinic acid, a major constituent of Hyptis suaveolens. The
present study investigated the vasorelaxant mechanism of ethyl rosmarinate in isolated rat aortic rings
using an organ bath system. Ethyl rosmarinate (0.1 mM–3 mM) produced concentration-dependent re-
25 August 2015
2
laxation in aortic rings pre-contracted with phenylephrine (10 mM), exhibiting a pD value of 4.5670.08 and
Accepted 1 September 2015
Available online 8 September 2015
an Emax value of 93.8275.00% (in endothelium-intact rings), as well as a pD value of 4.4270.05 and an Emax
2
value of 92.1073.78% (in endothelium-denuded rings). In the endothelium-denuded rings, the vasorelaxant
effect of ethyl rosmarinate was reduced by only 4-aminopyridine (1 mM); however, this was not the case
with tetraethylammonium (5 mM), glibenclamide (10 mM), barium chloride (1 mM), and 1H-[1,2,4] ox-
adiazolo [4,3-a]quinoxalin-1-one (ODQ, 1 mM). Ethyl rosmarinate also reduced the contraction induced by
Keywords:
Ethyl rosmarinate
Hypertension
Vasorelaxation
Endothelium-independent
Rat aorta
2
þ
phenylephrine (10 mM) and caffeine (20 mM) in a Ca -free solution, and inhibited the contraction induced
by increasing extracellular Ca2 influx, which was induced by KCl (80 mM). Ethyl rosmarinate (10 mM) in-
hibits concentration-response curves for phenylephrine, while in the same concentration of ethyl rosmar-
inate has no effect on contractions induced by increasing concentrations of calcium in the presence of high
extracellular potassium. Our results suggests that ethyl rosmarinate induces relaxation in aortic rings via an
endothelium-independent pathway, which involves the opening of voltage-gated potassium (Kv) channels
þ
and the blockade of both Ca2 release from intracellular stores and extracellular Ca influx. Moreover, ethyl-
rosmarinate acts on the extracellular Ca2 influx inhibition by interacting with voltage-operated calcium
channels (VOCCs) and receptor-operated calcium channels (ROCCs).
þ
2þ
þ
&
2015 Elsevier B.V. All rights reserved.
1
. Introduction
Rosmarinic acid is a major constituent of several plants, including
Rosmarinus officialis and Hyptis suaveolens, which have long been
used as medicinal plants, herbs, and spices in several countries. The
pharmacological activities of rosmarinic acid have a wide range of
beneficial effects, including anti-oxidative, anti-inflammatory, anti-
bacterial, and antiviral effects (Fadel et al., 2011; Huang and Zheng,
A significant number of health problems worldwide have been
caused by cardiovascular disease, of which hypertension is the
primary cause. Hypertension is estimated to be the cause of ap-
proximately 12.8% of all annual deaths (Chobanian et al., 2003;
Iadecola and Davisson, 2008). Therefore, controlling hypertension
is one means of decreasing the incidences of cardiovascular dis-
ease, as well as promoting health improvements. Relaxation and
contraction of the vasculature are both important factors in the
regulation of blood pressure. Several previous studies have studied
natural products that help to promote vasorelaxant effects with a
minimum of side effects; these products reduce blood pressure in
hypertensive rats (Bankar et al., 2011; Li et al., 2010; Taqvi et al.,
2006; Pandey et al., 1982). Additionally, protection from vascular
dysfunction has been observed in diabetic rats that received ros-
marinic acid, which also reportedly functions as both a cardiopro-
tective agent and a vasodilator (Ferreira et al., 2013; Karthik et al.,
2
011; Sotnikova et al., 2013). Previous studies have described the
vasorelaxant effects of rosmarinic acid in rat aortic rings via en-
dothelium-derived hyperpolarizing factor (EDHF), prostacyclin (PGI
and nitric oxide (NO) (Ersoy et al., 2008).
2
)
2
008; Vergara-Galicia et al., 2008).
Interestingly, rosmarinic acid is a natural compound that has
vasorelaxant effect. However, this compound exhibits low vasor-
elaxant activities. Therefore, we interest to investigate the deri-
vatives of this compound. A number of ester derivatives of
n
Corresponding author. Fax: þ66 53 945304.
E-mail address: chainarongt@hotmail.com (C. Tocharus).
http://dx.doi.org/10.1016/j.ejphar.2015.09.003
014-2999/& 2015 Elsevier B.V. All rights reserved.
0

10
P. Wicha et al. / European Journal of Pharmacology 766 (2015) 9–15
rosmarinic acid were subsequently prepared. Preliminary vasor-
elaxant activity evaluations have indicated that ethyl rosmarinate
exhibits higher activity than rosmarinic acid. It is of special in-
terest to evaluate the role of ethyl rosmarinate in vasorelaxation
and the mechanism responsible for its vasodilatory effects. The
purpose of the present study was to evaluate the relaxant effects
of ethyl rosmarinate and its possible mechanisms of action in rat
thoracic aorta.
1.0 mM MgCl
7.4 at 37 °C, and bubbled with a mixture of 95 O
2
, 11.0 mM glucose, and 18.0 mM CaCl
2
, with a pH of
. The
2
and 5% CO
2
aortic rings were suspended between two parallel stainless-steel
angles. One angle was attached to the organ bath, and another was
connected to a force transducer. The changes in tension were de-
tected using FT-104 isometric force transducers (Iworx System,
Inc., NH, USA) coupled to a Power Lab data acquisition system
(ADInstruments, Sydney, Australia) and connected to a computer
equipped with the Lab Chart 7 Software program (ADInstruments,
Sydney, Australia). A resting tension of 1 g was applied to each ring
and equilibrated for 60–90 min. During equilibration, the Krebs
solution was replaced every 15 min. Following equilibration, en-
dothelial integrity was verified with a sub-maximal pre-contrac-
tion of phenylephrine (10 mM). After the tension stabilized, Ach
(10 mM) was added to the chamber bath to detect and evaluate
either the presence or the absence of the endothelial cells; more
than 90% relaxation was considered characteristic of an en-
dothelium-intact ring, whereas less than 10% relaxation was con-
sidered characteristic of an endothelium-denuded ring. Relaxation
was calculated as the percentage of the maximal contraction in-
duced by phenylephrine. Before each experimental protocol was
performed, the presence of the endothelial cells was verified, and
the aortic rings were exposed with the Krebs solution for at least
2
. Materials and methods
2.1. Chemicals and drugs
The following drugs were used: rosmarinic acid, phenylephr-
ine, acetylcholine (ACh), barium chloride, ethylene glycol-bis( -
β
aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), tetra-
ethylammonium, glibenclamide, 4-aminopyridine, 1H-[1,2,4]ox-
adiazolo[4,3-a]quinoxalin-1-one (ODQ), caffeine, and nifedipine,
and dimethyl sulfoxide (DMSO), all chemicals were provided by
Sigma–Aldrich (St. Louis, MO, USA).
2.2. Preparation of ethyl rosmarinate
30 min.
Rosmarinic acid (1 g) was dissolved in absolute ethanol (25 ml),
and concentrated sulfuric acid (1 ml) was added. The mixture was
kept stirring overnight, and water (20 ml) was then added. The
mixture was extracted with ethyl acetate; the organic layer was
washed with water and dried over anhydrous sodium sulfate, and
the solvent was evaporated. The crude product was purified using
silica column chromatography by eluting with ethyl acetate–me-
thanol (100:1) to yield ethyl rosmarinate (800 mg). The spectro-
scopic (NMR and mass spectra) data were in agreement with the
structure. The structure of ethyl rosmarinate is shown in Fig. 1.
2.5. Experimental protocols
2.5.1. The effects of ethyl rosmarinate-induced relaxation on en-
dothelial cells
To investigate the vasorelaxant effects of ethyl rosmarinate on
endothelium-intact and endothelium-denuded rings, the aortic
rings were pre-contracted with phenylephrine (10 mM) in Krebs
solution. When the aortic rings were stable contracted, ethyl ros-
marinate (0.1 mM–3 mM) was added cumulatively into the organ
bath chamber to induce a concentration-dependent response in
the rings.
2
.3. Experimental animals
2
.5.2. The effects of ethyl rosmarinate on isolated aortic rings pre-
Male Wistar rats (weighing 200–300 g) were obtained from the
contracted with either phenylephrine or KCl
National Laboratory Animal Center, Mahidol University, Salaya, Na-
kornpathom, Thailand. They were housed in a temperature-con-
trolled room (2471 °C) under a timed 12 h light/dark cycle. The
animals were allowed free access to food and water until the day of
the experiment. The experiments were performed in accordance
with the protocol approved by the Faculty of Medicine, Chiang Mai
University Institutional Animal Care and Use Committee.
The endothelium-denuded rings were stable contracted with
either phenylephrine (10 mM) or KCl (80 mM), and then the stable
contracted ethyl rosmarinate (0.1 mM–3 mM) was subsequently
added cumulatively into the organ bath chamber. The results
pertaining to the ability of ethyl rosmarinate to induce vasor-
elaxation in the setting of phenylephrine-induced and KCl-induced
contractions were subsequently compared with the results ob-
tained in the absence of ethyl rosmarinate (control group).
2
.4. Preparation of the rat aortic rings
þ
2
.5.3. The effects of ethyl rosmarinate-induced relaxation on K
channels
The objective of this experiment was to determine whether K
Male Wistar rats were anesthetized by intra-peritoneal injec-
tions with sodium pentobarbital (50 mg/kg). The rats were killed,
and their thoracic aorta were removed. The aorta were carefully
cleaned and cut into 3–5 mm pieces. To obtain endothelium-de-
nuded rings, the endothelium was mechanically removed using a
thin cotton swab. Each ring was immersed in an organ bath
chamber containing Krebs solution composed of 122.0 mM NaCl,
þ
channels were involved in ethyl rosmarinate-induced vasorelaxa-
tion. The endothelium-denuded rings were pre-incubated with K
channel blockers 4-aminopyridine (1 mM), tetraethylammonium
5 mM), barium chloride (1 mM), and glibenclamide (10 mM) for
0 min before contraction was induced with phenylephrine. When
the aortic rings were stable contracted, ethyl rosmarinate (0.1 mM–
þ
(
3
4
2 4 2 4
.9 mM KCl, 10.0 mM HEPES, 0.5 mM KH PO , 0.5 mM NaH PO ,
3
mM) was cumulatively added. The results pertaining to ethyl
þ
rosmarinate-induced vasorelaxation in both the presence of a K
channel blocker and the absence (control) of a K channel blocker
þ
were subsequently compared.
2.5.4. The effects of soluble guanylyl cyclase (sGC) on ethyl rosmar-
inate-induced vasorelaxation
The vasorelaxation effects of ethyl rosmarinate were assessed
using ODQ, sGC inhibitor. The process was conducted by pre-
Fig. 1. The chemical structure of ethyl rosmarinate.

P. Wicha et al. / European Journal of Pharmacology 766 (2015) 9–15
11
incubating the endothelium-denuded aortic rings with ODQ
1 mM) for 30 min before phenylephrine (10 mM) was added for
2.6. Statistical analysis
(
contraction. Ethyl rosmarinate (0.1 mM–3 mM) was added when
the pre-contraction stabilized. The results pertaining to ethyl
rosmarinate-induced vasorelaxation in both the presence of ODQ
and the absence (control) of ODQ were subsequently compared.
The data are presented as means7S.E.M. Statistical sig-
nificance was analyzed using the unpaired Student's t-test. A one-
way-analysis of variance (ANOVA), followed by Dunnett's post hoc
test, was used to compare parameters between two or more
groups. All statistical analyses were performed using SPSS, version
17. A value of Po0.05 was considered statistically significant.
2
þ
2.5.5. The effects of ethyl rosmarinate on intracellular Ca
release
from the sarcoplasmic reticulum (SR)
To determine whether ethyl rosmarinate inhibited the release
of Ca2 from intracellular stores, endothelium-denuded rings
were used in this experiment. Initially, the rings were pre-con-
þ
3
. Results
2
þ
tracted with KCl (80 mM) to elicit Ca
loading into the SR. The
3.1. The effects of the endothelium on ethyl rosmarinate-induced
2
þ
rings were subsequently washed and exposed to a Ca -free Krebs
solution containing EGTA (1 mM). The rings were stimulated via
transient contraction with either phenylephrine (10 mM) or caf-
feine (20 mM) in a Ca2 -free solution. The contractions of both the
agonists were induced before and after pre-incubation with ethyl
rosmarinate (1 mM, 10 mM, and 100 mM). The percentages of con-
traction of both phenylephrine and caffeine in both the presence
and the absence of ethyl rosmarinate were compared.
relaxation in rat aortic rings
To determine whether ethyl rosmarinate-induced vasorelaxa-
tion was endothelium-dependent, the effects of ethyl rosmarinate
on vasorelaxation were analyzed in both endothelium-intact and
endothelium-denuded aortic rings pre-contracted with pheny-
lephrine. The results indicated that ethyl rosmarinate produced
vasorelaxation in a concentration-dependent manner in the set-
ting of phenylephrine-induced contraction in both the en-
dothelium-intact and the endothelium-denuded rings. The con-
centration-response curves for cumulative ethyl rosmarinate were
not different between the endothelium-intact and the en-
dothelium-denuded rings (P40.05) (Fig. 2A), with Emax values of
þ
2
þ
2
.5.6. The effects of ethyl rosmarinate on extracellular Ca influx
To investigate the mechanism of ethyl rosmarinate-induced
2
þ
vasorelaxation relative to Ca
channel activity, the endothelium-
concentration-re-
denuded rings were used to generate a CaCl
sponse curve. When the aortic rings stabilized inthe Ca -free
2
2
þ
9
3.8275.00% and 92.1073.78%, respectively (vs. the control
2
þ
Krebs solution, the rings were placed in Ca -free KCl (80 mM) for
0 min to open the voltage-operated calcium channels (VOCCs) on
the vascular smooth muscle cells. The concentration-response
curves of CaCl (0.01–10 mM) were constructed in both the pre-
sence and the absence of ethyl rosmarinate (1 mM, 10 mM, and
00 mM). The extent of vasoconstriction induced by CaCl in the
group Emax value of 5.7570.93%, Po0.01). The pD values for the
2
2
relaxation of ethyl rosmarinate in both the presence and the ab-
sence of the endothelium were 4.5670.08 and 4.4270.05, re-
spectively. These findings suggest that the vasorelaxant effects of
ethyl rosmarinate are independent of the presence of the
endothelium.
2
1
2
presence and the absence (control) of ethyl rosmarinate was
compared. Moreover, the percentages of contraction induced by
3.2. The vasorelaxant effects of ethyl rosmarinate on isolated aortic
CaCl
2
in nifedipine, a calcium channel blocker, were compared
rings pre-contracted with either phenylephrine or KCl
with those of the treatment group.
To determine the effects of ethyl rosmarinate on voltage-op-
erated calcium channels (VOCCs) and receptor-operated calcium
channels (ROCCs), endothelium-denuded rings were used to assay
the contraction induced by KCl (80 mM) and phenylephrine
2
.5.7. The inhibitory vasoconstriction effect of ethyl rosmarinate
To determine whether ethyl rosmarinate inhibited vasocon-
striction, endothelium-intact rings were used. The aortic rings
were exposed to ethyl rosmarinate (1 mM, 10 mM, and 100 mM) for
2
lephrine (0.1 nM–10 mM). The results are depicted as percentages
of contraction compared with the results obtained in the absence
of ethyl rosmarinate (control).
(10 mM). The results demonstrated that ethyl rosmarinate reduced
0 min before being subjected to vasoconstriction with pheny-
the percentages of phenylephrine- and KCl-induce contraction, as
depicted in Fig. 2B and 2C. Additionally, the Emax values were
92.1073.78% for phenylephrine and 94.1274.50% for KCl, as de-
monstrated in Table 1.
2
.5.8. The effects of rosmarinic acid-induced relaxation on en-
þ
3
.3. The effects of K channel activity on ethyl rosmarinate-induced
dothelial cells
vasorelaxation
To investigate the vasorelaxant effects of rosmarinic acid on
endothelium-intact and endothelium-denuded rings, the aortic
rings were pre-contracted with phenylephrine (10 mM) in Krebs
solution. When the aortic rings were stable contracted, rosmarinic
acid (0.1 mM–3 mM) was added cumulatively into the organ bath
chamber to induce a concentration-dependent response in the
rings.
þ
The objective of this experiment was to determine whether K
channels were involved in ethyl rosmarinate-induced vasorelaxa-
tion. The endothelium-denuded rings were pre-incubated with K
channel blockers, including 4-aminopyridine (1 mM); K
tetraethylammonium (5 mM); Ca blocker, barium chloride
1 mM); KIR blocker and glibenclamide (10 mM); KATP blocker be-
þ
v
blocker,
K
(
2
.5.9. The effects of ethyl rosmarinate and rosmarinic acid on iso-
fore being treated with phenylephrine (10 mM); thereafter, ethyl
rosmarinate (0.1 mM–3 mM) was cumulatively added. The results
demonstrated that inhibition of the vasorelaxant effects of ethyl
rosmarinate was observed in the aortic rings pre-incubated with
4-aminopyridine (Emax¼62.0774.12%), but not with tetra-
ethylammonium, barium chloride, or glibenclamide, which had
lated aortic rings pre-contracted with phenylephrine
The endothelium-intact rings were induce contracted with
phenylephrine (10 mM), and then the stable contracted ethyl ros-
marinate (0.1 mM–3 mM) and rosmarinic acid (0.1 mM–3 mM) was
subsequently added cumulatively into the organ bath chamber.
The results pertaining to the ability of ethyl rosmarinate and ros-
marinic acid to induce vasorelaxation in the setting of pheny-
lephrine-induced contractions were subsequently compared.
Emax values of 95.2876.72%, 90.4374.26%, and 89.4170.24%,
respectively (vs. control group Emax value of 91.7275.21%), as
demonstrated in Fig. 3A.

12
P. Wicha et al. / European Journal of Pharmacology 766 (2015) 9–15
Table 1
Vasorelaxation activity of ethyl rosmarinate against contraction induced by phe-
nylephrine (10 mM) and KCl (80 mM).
Ethyl rosmarinate
against
pD
þEC
2
% Emax
þEC
ꢀEC
ꢀEC
Phenylephrine
KCl
4.5670.08 4.4270.05 93.8275.00 92.1073.78
4.5370.09 4.5670.08 94.5275.37 94.1274.50
This experiment was performed in both endothelium-intact (þEC) and en-
dothelium-denuded (–EC) rings.
The values are expressed as means7S.E.M., n¼6.
Fig. 3. The vasorelaxation induced by ethyl rosmarinate on the aortic rings pre-
incubated with 4-aminopyridine (1 mM), tetraethylammonium (5 mM), barium
chloride (1 mM), and glibenclamide (10 mM) (n¼6) (A). There was no significant
difference between the vasorelaxation effects induced by ethyl rosmarinate in ei-
ther the presence or the absence of ODQ (1 mM) (B). The values are expressed as the
*
**
Fig. 2. The concentration-dependent effects of ethyl rosmarinate on the vascular
tone of endothelium-intact rings and endothelium-denuded rings (A) pre-con-
tracted with phenylephrine (10 mM). Vasorelaxation response induced by ethyl
rosmarinate on the aortic rings pre-contracted with phenylephrine (10 mM) and KCl
means7S.E.M. of six rats. Po0.05 and Po0.01 vs. control group.
3
.5. The effects of ethyl rosmarinate on the SR calcium release in-
duced by either phenylephrine or caffeine
(
80 mM) (B). The data are expressed as the means7S.E.M. of six rats.
An analysis of the inhibitory effects of ethyl rosmarinate on the
2
þ
3
.4. The effects of sGC in ethyl rosmarinate-induced vasorelaxation
transient contraction that occurs secondary to Ca release from the
2
þ
SR was carried out in a Ca -free solution using endothelium-de-
nuded rings treated with phenylephrine (10 mM) and caffeine
(20 mM). The results indicated that pretreatment with ethyl rosmar-
inate (1 mM, 10 mM, and 100 mM) reduced the percentages of pheny-
lephrine-induced contraction to 62.20%, 50.16%, and 27.15%, respec-
tively (vs. control group 91.96%), as demonstrated in Fig. 4. However,
caffeine elicited reduced effects in the aortic rings treated with only
ethyl rosmarinate (100 mM), and had an Emax value of 83.60% com-
pared with 90.39% in the control group as demonstrated in Fig. 4.
To determine the vasorelaxant effects of ethyl rosmarinate on
the sGC pathway, the endothelium-denuded rings were pre-in-
cubated with ODQ (1 mM), sGC inhibitor. The results indicated that
the vasorelaxation effects induced by ethyl rosmarinate were not
significantly different in either the presence or the absence of ODQ
(
control), the Emax values of which were 97.62% and 97.05%, re-
spectively, as demonstrated in Fig. 3B.

P. Wicha et al. / European Journal of Pharmacology 766 (2015) 9–15
13
Table 2
The effect of ethyl rosmarinate on the pD
2
value and Emax of CaCl
2
-induced con-
traction in endothelium-denuded rings in Ca2 -free Krebs solution.
þ
Groups
pD
2
%Emax
Control
3.5970.110
3.6270.066
3.6170.069
10073.78
10072.39
Ethyl rosmarinate1 mM
Ethyl rosmarinate10 mM
Ethyl rosmarinate100 mM
Nifedipine1 mM
97.5572.43
a
a
2.8970.116
2.8270.139
55.8573.76
a
18.15 71.59a
Fig. 4. The inhibition of intracellular Ca2þ store release induced by phenylephrine
10 mM) and caffeine (20 mM) were assessed using endothelium-denuded rings
pre-incubated with ethyl rosmarinate (1 mM, 10 mM, and 100 mM). The results were
The values are expressed as means7S.E.M., n¼6.
Significantly different from the control (Po0.05) (ANOVA followed by Dun-
nett's Multiple Comparison Test).
a
(
compared with the results obtained in the absence of ethyl rosmarinate (control).
*
**
The values are expressed as the means7S.E.M. of six rats. Po0.05 and Po0.01
vs. control group.
2
þ
3
.6. The effects of ethyl rosmarinate on extracellular Ca -induced
contraction
The objective of this experiment was to examine the vasor-
elaxant effects of ethyl rosmarinate on the VOCCs pathway. In the
2
þ
Ca -free KCl solution, the cumulative addition of CaCl
0 mM) induced a progressive increase in aortic rings tension.
Pretreatment with 100 mM ethyl rosmarinate significantly atte-
nuated CaCl -induced contraction compared with the control
group, but 1 mM and 10 mM had no significant effect. Moreover,
2
(0.01–
1
2
2
þ
pretreatment with nifedipine (1 mM), a Ca
inhibited the contraction induced by extracellular Ca (Fig. 5 and
Table 2).
channel blocker, also
2
þ
Fig. 6. The inhibitory contractile effect of ethyl rosmarinate (1 mM, 10 mM, and
1
00 mM) on endothelium-intact induced contraction by cumulative (0.1 nM–10 mM)
3
.7. The inhibitory vasoconstriction effect of ethyl rosmarinate
of phenylephrine. The values are expressed as the means7S.E.M. of six rats.
*
**
Po0.05 and Po0.01 vs. control group.
To determine the inhibitory effects of ethyl rosmarinate on
phenylephrine-induced vasoconstriction, the aortic rings were
pre-incubated with ethyl rosmarinate (1 mM, 10 mM, and 100 mM)
before the addition of phenylephrine (0.1 nM–10 mM) was added
cumulatively. The result indicated that each of the three con-
centrations of ethyl rosmarinate (1 mM, 10 mM, and 100 mM) re-
duced the maximal contraction of phenylephrine, which had Emax
values of 78.0372.97%, 68.072.25%, and 50.7871.46%, respec-
tively (vs. the control group Emax value of 90.4773.21%), as de-
monstrated in Fig. 6.
3
.8. The vasorelaxant effects of rosmarinic acid on isolated aortic
rings pre-contracted with phenylephrine
In Fig. 7A, an investigation was done to find whether ros-
marinic acid which induces vasorelaxation was endothelium-de-
pendent or not. The result showed that rosmarinic acid produced a
concentration-dependent vasorelaxation in endothelium-intact
but was absent in endothelium-denuded vessels. These finding
suggest that the vasorelaxant effect caused by rosmarinic acid is
endothelium-dependent.
3.9. The vasorelaxant effects of ethyl rosmarinate or rosmarinic acid
on isolated aortic rings pre-contracted with phenylephrine
Fig. 7B showed the vasorelaxant effects of rosmarinic acid and
ethyl rosmarinate in aortic rings that were pre-contracted with
phenylephrine. The results showed that ethyl rosmarinate
(
EC50¼30.3870.120 mM) showed more potent vasodilator than
rosmarinic acid (EC50¼346.970.126 mM).
4. Discussion
In this study, we first demonstrated the mechanisms under-
lying the effects of ethyl rosmarinate. Vasorelaxation is regulated
via the endothelium-dependent and endothelium-independent
signaling pathways. The endothelium-dependent pathway re-
quires the regulation of vessel diameter by endothelial cells via the
2
Fig. 5. The effects of ethyl rosmarinate on the CaCl -induced contraction responses
in the endothelium-denuded rings. The concentration-response curves for CaCl
2
were examined in a Ca2 -free KCl solution (80 mM). The curves were determined
þ
in separate control, pre-incubation with ethyl rosmarinate (1 mM, 10 mM, and
2
production and release of several moleculessuch as PGI and NO,
100 mM), and nifedipine (1 mM) (positive control) groups. The results obtained for
both of which trigger vasorelaxation (Sumpio et al., 2002). More-
over, vascular tone is also regulated by smooth muscle cells, the
endothelial-independent pathway. The major function of this
ethyl rosmarinate (100 mM) and nifedipine were significantly different from the
results obtained in the control group. The values are expressed as the means7S.E.
M. of six rats. *Po0.05 and Po0.01 vs. control group.
**

14
P. Wicha et al. / European Journal of Pharmacology 766 (2015) 9–15
demonstrated that pre-incubation with ethyl rosmarinate reduced
the aortic rings tension induced by phenylephrine treatment.
Phenylephrine is an 1-adrenoceptor agonist that induce con-
traction mediated by extracellular Ca
α
2
þ
influx through ROCCs and
release from the specific inositol 1, 4, 5-tripho-
sphate receptor (IP R) channels in the SR membrane (Ferris and
Synder 1992; Hori et al., 1993). Based on the results, it is implying
2
þ
intracellular Ca
3
2
þ
that ethyl rosmarinate may act by inhibiting the Ca
influx
2
þ
through the ROCCs or inhibiting IP3-dependent Ca releases from
SR sensitive to PE. K channels play a vital role in the regulation of
both contractability and vascular tone. The opening of K chan-
nelsin vascular smooth muscle cells increases K efflux, which hy-
þ
þ
þ
perpolarizes the cell membrane and promotes vasorelaxation (Nelson
and Quayle, 1995). Vascular smooth muscle cells express different
þ
types of K channels, including K
v
, KCa,KIR, and KATP, which may
blocked by 4-aminopyridine (1 mM), tetraethylammonium (5 mM),
barium chloride (1 mM), and glibenclamide (10 mM) (Kuriyama et al.,
1995). The vasorelaxant effects of ethyl rosmarinate reduced the ten-
sion in the aortic rings pre-contracted with 4-aminopyridine but not
tetraethylammonium, barium chloride or glibenclamide. These find-
ings suggest that the relaxant effects of ethyl rosmarinate on en-
dothelial-denuded rings are partially mediated by K
opening of K channels in vascular smooth muscle cells results in the
inhibition of Ca
v
channels. The
v
2
þ
2þ
influx via the closureof voltage-dependent Ca
channels, which results in vascular relaxation (Jackson, 2000; Nelson
and Quayle, 1995). Additionally, we studied the role of sGC, which
increases the amount of cyclic guanosine monophosphate (cGMP) the
smooth muscle cells and causes vasorelaxation (Moncada et al., 1989).
There was no significant difference in the vasorelaxation effects in-
duced by ethyl rosmarinate in either the presence orin the absence of
ODQ (control). Therefore, these findings confirm that the effects of
ethyl rosmarinate on sGC activity in aortic rings may be ruled out.
2
þ
The influence of ethyl rosmarinate on Ca
release from in-
tracellular stores sensitive to phenylephrine and caffeine was also
2
þ
studied. Phenylephrine induces the release of Ca from the SR via
Fig. 7. (A) The concentration-dependent effects of rosmarinic acid on the vascular
tone of endothelium-intact rings and endothelium-denuded rings pre-contracted
with phenylephrine (10 mM). The data are expressed as the means7S.E.M. of six
rats. (B) The concentration-dependent effects of ethyl rosmarinate or rosmarinic
acid on endothelium-intact rings. Vasorelaxation response induced by ethyl ros-
marinate or rosmarinic acid on the aortic rings pre-contracted with phenylephrine
2þ
IP
3
, whereas caffeine induces Ca
release from the SR via rya-
nodine receptor (Karaki et al., 1997; McGeown, 2004; Meissner,
994). The results indicated that ethyl rosmarinate (1 mM, 10 mM,
1
and 100 mM) significantly inhibited the transient contraction in-
duced by phenylephrine. These findings were suggestiveof the
possible effects of ethyl rosmarinate on the calcium mobilization
(
10 mM). The data are expressed as the means7S.E.M. of six rats.
3
elicited via IP (inositol 1,4,5-triphosphate) sensitive intracellular
2
þ
pathway is its vasoconstrictor effects, which are caused by both
intracellular and extracellular Ca2 (Clinton, 2003).
Ca
in SR. However, ethyl rosmarinate (100 mM) also attenuated
þ
2þ
the effects exerted by caffeine in Ca -free medium, suggesting
that ethyl rosmarinate may inhibit Ca
sensitive intracellular Ca
together, these results suggest that ethyl rosmarinate may function
as a calcium channel blocker at high concentrations.
Another phenomenon investigated in this study was whether
ethyl rosmarinate-induced vasorelaxation was related to the in-
hibition of extracellular Ca . We observed that ethyl rosmarinate
(100 mM) significantly decreased KCl-induced vascular smooth
muscle contraction in endothelium-denuded rings and sig-
nificantly reduced Ca -induced contraction in aortic rings.
Moreover, nifedipine, an L-type voltage-operated calcium channel
blocker, reduced the contraction of the rings, which confirms the
involvement of L-type voltage-operated calcium channels in the
contractile activityof the rings (Hockerman et al., 1997). The
highest concentrations of ethyl rosmarinate inhibited calcium-in-
flux, which suggests that at these concentrations, the compound
blocks voltage-gated calcium channels.
We determined that ethyl rosmarinate (EC50¼30.387
0.120 mM) exhibited stronger vasorelaxation effects compared
with rosmarinic acid (EC50¼346.970.126 mM) which is a chemical
precursor of ethyl rosmarinate (Fig. 7B). Our results correlated well
with those of previous studies demonstrating that rosmarinic acid
2
þ
In the present study, the effects of ethyl rosmarinate in the
setting of phenylephrine-induced contraction in both en-
dothelium-intact and endothelium-denuded aortic rings were
demonstrated. The findings suggest that the vasorelaxant effects
elicited by ethyl rosmarinate are endothelium-independent.
Therefore, we subsequently determined the mechanisms by which
ethyl rosmarinate acts on rat aortic rings, including whether it
stimulates vascular smooth muscle cells. The vasorelaxation ef-
fects of ethyl rosmarinate were assessed in both phenylephrine
and KCl pre-contracted aortic rings. Vasoconstriction wastriggered
release from caffeine-
2
þ
in SR at a high concentration. Taken
2
þ
2
þ
2
þ
by increasing intracellular Ca concentrations via the influx of
extracellular Ca2 into VOCCs (Van-Breemen and Saida, 1989) for
þ
2
þ
KCl, and ROCCs and the release Ca stored from SR for pheny-
lephrine (Nelson and Quayle, 1995; Ruzycky and Morgan, 1989).
Therefore, to distinguish between the effects of ethyl rosmarinate
on VOCCs and ROCCs, the aortic rings were assessed following
treatment with KCl (80 mM) and phenylephrine (10 mM). The re-
sults indicated that the relaxation elicited in aortic rings pre-
contracted with phenylephrine and KCl was not significantly dif-
ferent following treatment with either agent. These results sug-
gested that ethyl rosmarinate-induced vasorelaxation by inhibit-
ing both VOCCs and ROCCs activity. Moreover, the present study

P. Wicha et al. / European Journal of Pharmacology 766 (2015) 9–15
15
induced vasorelaxation via endothelium-dependent pathways,
mediated by NO, PGI , and EDHF pathways (Ersoy et al., 2008). By
Clinton, W.R., 2003. Smooth muscle contraction and relaxation. Adv. Physiol. Ed. 27,
01–206.
Ersoy, S., Orhan, I., Turan, N.N., Sahan, G., Ark, M., Tosun, F., 2008. Endothelium-
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rat isolated thoracic aorta. Phytomedicine 15, 1087–1092.
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spontaneously penetrates membranes to inhibit lipid peroxidation in situ.
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2
2
contrast, ethyl rosmarinate, the ethyl ester derivative of rosmarinic
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ways (Fig. 7A). Based on these findings, we have provided scien-
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at very low concentrations as a vasodilator that acts via vascular
smooth muscle. However, the relaxant effect of ethyl rosmarinate
on the thoracic aorta, large size arterial, probably does not explain
its potent antihypertensive activity. To achieve its potent blood
pressure lowering effects, ethyl rosmarinate need to further verify
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5
. Conclusion
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Our study demonstrated that ethyl rosmarinate, the ethyl ester
derivative of rosmarinic acid, induces vasorelaxation in rat aortic
rings. The vasorelaxant effects of ethyl rosmarinate were mediated
via endothelium-independent pathways involving the blockageof
extracellular Ca2 influx via the opening of K
þ
channels and in-
v
teractions with both VOCCs and ROCCs. Moreover, the inhibition of
intracellular Ca2 mobilization via the reduction of IP
þ
and caf-
3
514–521.
2
þ
feine-sensitive intracellular Ca release from the SR may also
have contributed to the effects of ethyl rosmarinate.
Kuriyama, H., Kitamura, K., Nabata, H., 1995. Pharmacological and physiological
significance of ion channels are factors that modulate them in vascular tissues.
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Conflict of interest
McGeown, J.G., 2004. Interactions between inositol 1,4,5-trisphosphate receptors
and ryanodine receptors in smooth muscle: one store or two? Cell Calcium 35,
The authors of this manuscript declare that they have no con-
flicts of interest regarding its publication.
6
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þ
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Acknowledgments
This study was supported by The National Research Council of
Thailand, Grant no. 7/2556. Partial support was provided by The
Thailand Research Fund, Thailand (Grant no. DBG5680006) and is
gratefully acknowledged.
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