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Article
Marjoram Relaxes Rat Thoracic Aorta Via a
PI3-K/eNOS/cGMP Pathway
Adnan Badran 1, Elias Baydoun 2, Ali Samaha 3,4, Gianfranco Pintus 5,6 , Joelle Mesmar 2,
Rabah Iratni 7, Khodr Issa 8, * and Ali H. Eid 8, *
1Department of Nutrition, University of Petra, Amman, P.O. Box 961343, Amman 11196, Jordan;
abadran@uop.edu.jo
2Department of Biology, American University of Beirut, Beirut P.O. Box 11-0236, Lebanon;
eliasbay@aub.edu.lb (E.B.); joellemesmar@gmail.com (J.M.)
3Department of Biomedical Sciences, Lebanese International University, Beirut, P.O. Box 146404 Mazraa,
Lebanon; ali.samaha@liu.edu.lb
4Faculty of Public Health IV, Lebanese University, Beirut, P.O. Box 6573/14 Badaro, Lebanon
5Department of Biomedical Sciences, Qatar University, Doha P.O. Box 2713, Qatar; gpintus@qu.edu.qa
6Biomedical Research Center, Qatar University, Doha P.O. Box 2713, Qatar
7Department of Biology, United Arab Emirates University, Al Ain P.O. Box 15551, UAE; r_iratni@uaeu.ac.ae
8Department of Pharmacology and Toxicology, American University of Beirut,
Beirut P.O. Box 11-0236, Lebanon
*Correspondence: ki12@aub.edu.lb (K.I.); ae81@aub.edu.lb (A.H.E.);
Tel.: +961-1-35000 (ext. 4891) (K.I. & A.H.E.)
Received: 15 April 2019; Accepted: 29 May 2019; Published: 11 June 2019
Abstract:
Despite pharmacotherapeutic advances, cardiovascular disease (CVD) remains the primary
cause of global mortality. Alternative approaches, such as herbal medicine, continue to be sought to
reduce this burden. Origanum majorana is recognized for many medicinal values, yet its vasculoprotective
effects remain poorly investigated. Here, we subjected rat thoracic aortae to increasing doses of
an ethanolic extract of Origanum majorana (OME). OME induced relaxation in a dose-dependent
manner in endothelium-intact rings. This relaxation was significantly blunted in denuded rings.
N(
ω
)-nitro-l-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ)
significantly reduced the OME-induced vasorelaxation. Cyclic guanosine monophosphate (cGMP) levels
were also increased by OME. Moreover, wortmannin or LY294002 significantly reduced OME-induced
vasorelaxation. Blockers of ATP-sensitive or Ca
2+
-activated potassium channels such as glibenclamide or
tetraethylamonium (TEA), respectively, did not significantly affect OME-induced relaxation. Similarly,
verapamil, a Ca
2+
channel blocker, indomethacin, a non-selective cyclooxygenase inhibitor, and
pyrilamine, a H1 histamine receptor blocker, did not significantly modulate the observed relaxation.
Taken together, our results show that OME induces vasorelaxation via an endothelium-dependent
mechanism involving the phosphoinositide 3-kinase (PI3-K)/endothelial nitric oxide (NO) synthase
(eNOS)/cGMP pathway. Our findings further support the medicinal value of marjoram and provide a
basis for its beneficial intake. Although consuming marjoram may have an antihypertensive effect,
further studies are needed to better determine its effects in different vascular beds.
Keywords: marjoram; hypertension; vasorelaxation; PI3-K; nitric oxide; cGMP
1. Introduction
Cardiovascular disease (CVD) continues to cause more mortalities than any other disease [
1
].
Despite major pharmacotherapeutic advances, chronically elevated blood pressure persists as a major
contributor to CVD-associated mortality [
2
]. Although many antihypertensive drugs continue to be
Biomolecules 2019,9, 227; doi:10.3390/biom9060227 www.mdpi.com/journal/biomolecules
Biomolecules 2019,9, 227 2 of 16
developed, these synthetic drugs are not without many deleterious adverse effects. In addition to
these synthetic drugs, the use of herbal medicine and plant-based therapies is increasing in many
countries [
3
]. In recent years, more interest has been given to these therapies, largely due to their ease
of availability, presumed low toxicity, and documented benefits [
4
–
8
]. Indeed, many of these plants
have been employed for the particular treatment or management of cardiovascular disease [5–9].
Origanum majorana L. is a perennial subshrub with a distinguished fragrant foliage. It is native to
southern Europe and the Mediterranean. It has been traditionally used as a folk medicine for many
ailments, such as indigestion, headache and asthma [
10
,
11
]. Interestingly, the genus Origanum possesses
bountiful biological properties, such as antioxidant, anti-inflammatory and anticholinesterase effects,
as well as protection against aging and neurodegenerative disease [
12
]. In addition, marjoram elicits
an
in vitro
microbicidal activity [
13
,
14
]. We have also shown that marjoram inhibits the malignant
phenotype of breast cancer cells [
15
,
16
]. Relevantly, it has been reported that marjoram is used in the
management/treatment of hypertension, although the mechanism of action was not elucidated [17].
Bioactives of marjoram, such as the monoterpene carvacrol, exhibit a potent inhibitory effect
against bacterial and fungal infections, as well as antispasmodic effects [
13
]. Moreover, carvacrol can
quench free radicals and ameliorate hypercontraction of aortic vasculature [
18
]. More importantly,
carvacrol is known to play an important role in endothelium-dependent vasorelaxation of rat aortae
via a mechanism that apparently involves potassium and calcium channels [
19
,
20
]. Similarly, thymol,
another monoterpe found in marjoram, appears to induce relaxation of isolated rat aortae [
20
].
Hesperetin, another bioactive of marjoram [
21
], reduces systolic blood pressure in spontaneously
hypertensive rats via an endothelium-dependent mechanism [22].
Despite these beneficial values of O. majorana or its bioactives, and to the best of our knowledge,
no studies have so far investigated the vascular activity caused by O. majorana. Accordingly, the present
study was undertaken to investigate and evaluate the potential vasorelaxatory effects of O. majorana on
isolated rat aortae.
2. Materials and Methods
2.1. Preparation of the Extract
Origanum majorana, locally grown and commonly available, was collected in August 2014. Dried
leaves were ground with a mortar and pestle, and the powder extracted with 70% EtOH for 3 h in a
reflux apparatus at 70
±
5
◦
C. Following filtration, the extract was collected and evaporated at 60
◦
C,
and then it was lyophilized in a freeze-dryer to obtain a powder of the crude extract. This powder was
weighed and kept at −20 ◦C until further use.
2.2. Animals
The study protocol was approved by the scientific committee on animal care and use in the
Faculty of Public Health at the Lebanese University (Permit number for Samaha Marjoram Project:
UL/FSPIV/07/2011). Experiments were performed in conformity with the National institutes of health
legislation on the use of laboratory animals. Every effort was taken to minimize animal suffering.
Male Sprague-Dawley (SD) rats (230–290 g body weight; 6–8 weeks old) were housed in an animal
laboratory at an ambient temperature of 23
±
2
◦
C, under a 12 h:12 h dark: light cycle. Animals had
access to a standard animal chow and tap water ad libitum.
2.3. Drugs and Chemicals
Acetylcholine, norepinephrine, tetraethylammonium (TEA), 1H-[1,2,4]oxadiazolo[4,3-alpha]
quinoxalin-1-one (ODQ), atropine, 3-isobutyl-1-methylxanthine (IBMX), indomethacin, pyrilamine,
verapamil, and N
ω
-nitro-l-arginine methyl ester (L-NAME) were purchased from Sigma-Aldrich Co.
(St. Louis, MO, USA). LY294002 and Wortmannin was obtained from Tocris (Abingdon, UK) and Alexis
Biochemicals (Lausen, Switzerland), respectively.
Biomolecules 2019,9, 227 3 of 16
2.4. Arterial Ring Preparation and Vascular Reactivity
Male SD rats were euthanized by an overdose of pentobarbital (50 mg/kg body weight). Thoracic
aortae were quickly dissected into a cold modified Krebs-Henseleit (KHB) buffer, as in our recent
papers [
7
,
8
]. Aortae were cleaned of fat and other adhering connective tissues and cut into ring
segments, which were suspended for the measurement of isometric force in organ chambers filled with
KHB (pH 7.4), maintained at 37
◦
C, and bubbled with a gas mixture of 95% O
2
—5% CO
2
. The KH
solution contained (in 10
−3
M): NaCl (130), KCl (4.7), CaCl
2·
2H
2
O (1.6), Mg
2
SO
4
7H
2
O (1.17), KH
2
PO
4
(1.18), NaHCO3(14.9), and glucose (5.5).
During the equilibration period (at least 30 min), the rings underwent isometric resting tension
in a stepwise manner, as we recently described [
7
,
8
]. Cumulative concentrations (0.01–1 mg/mL,)
of O. majorana (OME) were added, where at each dose of OME, the response curve would reach a
plateau before the subsequent dose was added. To determine the endothelium-dependent relaxation,
aortic rings with denuded endothelium were pre-contracted with norepinephrine (NE; 3
µ
M) and then
exposed to incrementally increased concentrations of OME. As we recently published [
8
], denudation
was done by gently rubbing, in a to and from motion, the lumen of the ring with a roughened piece of
stainless wire. Generation of less than 15% relaxation with acetylcholine is considered indicative of a
successful denudation.
When assessing the potential role of nitric oxide (NO), endothelium-intact rings were pre-incubated
with L-NAME (100
µ
M), a NO synthase inhibitor, for 30 min. Rings were then contracted with NE,
after which OME was added.
When determining the role of prostaglandins, histamine H1-receptors, muscarinic receptors,
guanylate cyclase, or potassium channels in OME-induced relaxation, the following inhibitors were
used: indomethacin (10
µ
M), a non-selective cyclooxygenase inhibitor; pyrilamine (10
µ
M), a histamine
H1-receptor antagonist or atropine (10
µ
M), a muscarinic receptor antagonist; oxadiazole quinoxaline
(ODQ, 10
µ
M), a soluble guanylate cyclase inhibitor; glibenclamide (10
µ
M), an ATP-sensitive potassium
channels blocker; or tetraethylammonium (100
µ
M), a non-selective inhibitor of Ca
2+
-activated
potassium channels. When the role of calcium channels was to be assessed, and as previously
described [
7
,
8
,
23
], rings were pre-incubated with verapamil hydrochloride (1
µ
M), a blocker of L-type
calcium channels before the addition of OME.
To assess the potential role of phosphoinositide 3-kinase (PI3K)/Akt signaling, rings were
pre-incubated with the PI3K inhibitors, LY294002 (10
µ
M) or wortmannin (100 nM), for 30 min.
A cumulative dose response curve with increasing concentrations of OME followed.
To ensure that OME did not impart a significant irreversible effect on vascular responsiveness,
aortic rings that had been exposed to OME were washed and exposed to norepinephrine (NE, 3
µ
M) or
acetylcholine (ACh, 10 µM) anew. Finally, KCl (80 mM) was used to confirm viability of the rings.
2.5. cGMP Assay
Rings that had been pre-equilibrated were incubated in IBMX (a phosphodiesterase inhibitor;
0.1 mM) for 30 min before subsequent addition of NE. Then, these rings were re-equilibrated for
an additional 30 min before adding OME. Tissues were then frozen in liquid nitrogen to stop the
reaction, homogenized in trichloroacetic acid, and centrifuged at 10,000
×
gfor 10 min. The supernatant
was then extracted with water-saturated diethylether. Quantitation of protein or cyclic guanosine
monophosphate (cGMP) in the extract was done by Bradford’s method or a specific immunoassay
(GE Healthcare Life Sciences), Pittsburgh, PA, USA), respectively. Results are plotted as picomoles of
cGMP per milligram of protein.
2.6. Graphing and Statistical Analysis
Graphing and determination of 50% effective dose (pED50) and E
MAX
, along with the confidence
intervals, were performed by GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA).
Biomolecules 2019,9, 227 4 of 16
For statistical analysis, the same software package was used. Data are presented as means
±
standard
error (SE). Student’s t-test was used. If comparison between more than two means was needed,
Analysis of variance (ANOVA) was used—either a one-way ANOVA with Tukey’s post hoc test or a
two-way ANOVA with Sidak’s multiple comparison post hoc test. A p-value of less than 0.05 was
considered statistically significant.
3. Results
3.1. Effect of Origanum majorana Extract on Norepinephrine-Induced Contraction
We first determined the concentration-dependent vasorelaxatory effects of OME (0.001–1 mg/mL)
on endothelium-intact aortic rings. OME caused a dose-dependent relaxation with the maximal
relaxant effect being 90
±
5%, and a pED
50
of
−
3.98, with a 95% confidence interval of
−
4.0 to
−
3.9
(Figure 1). The vehicle (ethanol) did not impart any significant effect on the rings.
Biomolecules 2019, 9, 227 4 of 16
Analysis of variance (ANOVA) was used—either a one-way ANOVA with Tukey’s post hoc test or a
two-way ANOVA with Sidak’s multiple comparison post hoc test. A p-value of less than 0.05 was
considered statistically significant.
3. Results
3.1. Effect of Origanum majorana Extract on Norepinephrine-Induced Contraction
We first determined the concentration-dependent vasorelaxatory effects of OME (0.001–1 mg/mL)
on endothelium-intact aortic rings. OME caused a dose-dependent relaxation with the maximal
relaxant effect being 90 ± 5%, and a pED50 of −3.98, with a 95% confidence interval of −4.0 to −3.9
(Figure 1). The vehicle (ethanol) did not impart any significant effect on the rings.
Figure 1. Effect of Origanum majorana (OME) extract on the vasorelaxation of aortic rings. Cumulative
dose response curve for OME-induced relaxation of rat aortic rings was determined. Data expressed
are mean ± standard error of the mean (SEM, n = 7).
3.2. Effect of Origanum majorana Extract on Endothelium-Intact or Endothelium-Denuded Aortic Rings
Pre-Contracted with Norepinephrine
We next wished to determine the potential role of endothelium in OME-induced relaxation. In
both endothelium-intact and endothelium-denuded aortic rings, OME caused a dose-dependent
relaxation (Figure 2). The maximal relaxant effect was 95 ± 3% or 46 ± 4.0% for endothelium-intact or
endothelium-denuded aortic rings, respectively (Figure 2). A pED50 of −3.96 with a 95% confidence
interval of −4.0 to −3.9 was obtained for the endothelium intact rings, while a pED50 of −3.79 with a
95% confidence interval of −3.9 to −3.6 was obtained for the endothelium-denuded ones. This
significant difference between the two conditions clearly demonstrates that endothelium is, at least
in part, involved in OME-induced relaxation.
Figure 1.
Effect of Origanum majorana (OME) extract on the vasorelaxation of aortic rings. Cumulative
dose response curve for OME-induced relaxation of rat aortic rings was determined. Data expressed
are mean ±standard error of the mean (SEM, n=7).
3.2. Effect of Origanum majorana Extract on Endothelium-Intact or Endothelium-Denuded Aortic Rings
Pre-Contracted with Norepinephrine
We next wished to determine the potential role of endothelium in OME-induced relaxation.
In both endothelium-intact and endothelium-denuded aortic rings, OME caused a dose-dependent
relaxation (Figure 2). The maximal relaxant effect was 95
±
3% or 46
±
4.0% for endothelium-intact or
endothelium-denuded aortic rings, respectively (Figure 2). A pED
50
of
−
3.96 with a 95% confidence
interval of
−
4.0 to
−
3.9 was obtained for the endothelium intact rings, while a pED
50
of
−
3.79 with a 95%
confidence interval of
−
3.9 to
−
3.6 was obtained for the endothelium-denuded ones. This significant
difference between the two conditions clearly demonstrates that endothelium is, at least in part,
involved in OME-induced relaxation.
Biomolecules 2019,9, 227 5 of 16
Biomolecules 2019, 9, 227 5 of 16
Figure 2. Role of endothelium in OME-induced relaxation. Cumulative dose-response curves for
OME in isolated norepinephrine (NE)-pre-contracted rat aortic rings either with intact (+E; black) or
denuded endothelium (-E; red). Data are expressed as mean ± SEM (n = 7; p < 0.01 for +E vs. –E).
3.3 Role of Nitric Oxide and cGMP in Origanum majorana Extract-Induced Aortic Relaxation
NO and cGMP are two important signalling molecules that play a vital role in vasorelaxation.
We next investigated the role of these molecules in the vasorelaxant effect of OME. OME-induced
relaxation of endothelium-intact aortic rings was significantly diminished by the presence of L-
NAME or ODQ. In the presence or absence of L-NAME, the maximal relaxant effect was 54 ± 5% or
94 ± 3%, respectively (Figure 3A), and pED50 was −3.7 or −3.9 with confidence intervals of −3.9 to −3.5
or −4 to −3.9, respectively. Likewise, the maximal relaxant effect in the presence or absence of ODQ
was 52 ± 7% or 96 ± 4%, respectively (Figure 3B), and pED50 was −3.5 or −3.9 with confidence intervals
of −3.7 to −3.3 or −4 to −3.8, respectively. Taken together, these data support the notion that both NO
and cGMP play a significant role in OME-induced vasorelaxation.
A B
Figure 2.
Role of endothelium in OME-induced relaxation. Cumulative dose-response curves for OME
in isolated norepinephrine (NE)-pre-contracted rat aortic rings either with intact (+E; black) or denuded
endothelium (-E; red). Data are expressed as mean ±SEM (n=7; p<0.01 for +E vs. -E).
3.3. Role of Nitric Oxide and cGMP in Origanum majorana Extract-Induced Aortic Relaxation
NO and cGMP are two important signalling molecules that play a vital role in vasorelaxation.
We next investigated the role of these molecules in the vasorelaxant effect of OME. OME-induced
relaxation of endothelium-intact aortic rings was significantly diminished by the presence of L-NAME
or ODQ. In the presence or absence of L-NAME, the maximal relaxant effect was 54
±
5% or 94
±
3%,
respectively (Figure 3A), and pED
50
was
−
3.7 or
−
3.9 with confidence intervals of
−
3.9 to
−
3.5 or
−
4 to
−
3.9, respectively. Likewise, the maximal relaxant effect in the presence or absence of ODQ was 52
±
7% or 96
±
4%, respectively (Figure 3B), and pED
50
was
−
3.5 or
−
3.9 with confidence intervals of
−
3.7
to
−
3.3 or
−
4 to
−
3.8, respectively. Taken together, these data support the notion that both NO and
cGMP play a significant role in OME-induced vasorelaxation.
Biomolecules 2019, 9, 227 5 of 16
Figure 2. Role of endothelium in OME-induced relaxation. Cumulative dose-response curves for
OME in isolated norepinephrine (NE)-pre-contracted rat aortic rings either with intact (+E; black) or
denuded endothelium (-E; red). Data are expressed as mean ± SEM (n = 7; p < 0.01 for +E vs. –E).
3.3 Role of Nitric Oxide and cGMP in Origanum majorana Extract-Induced Aortic Relaxation
NO and cGMP are two important signalling molecules that play a vital role in vasorelaxation.
We next investigated the role of these molecules in the vasorelaxant effect of OME. OME-induced
relaxation of endothelium-intact aortic rings was significantly diminished by the presence of L-
NAME or ODQ. In the presence or absence of L-NAME, the maximal relaxant effect was 54 ± 5% or
94 ± 3%, respectively (Figure 3A), and pED50 was −3.7 or −3.9 with confidence intervals of −3.9 to −3.5
or −4 to −3.9, respectively. Likewise, the maximal relaxant effect in the presence or absence of ODQ
was 52 ± 7% or 96 ± 4%, respectively (Figure 3B), and pED50 was −3.5 or −3.9 with confidence intervals
of −3.7 to −3.3 or −4 to −3.8, respectively. Taken together, these data support the notion that both NO
and cGMP play a significant role in OME-induced vasorelaxation.
A B
Figure 3.
Role of nitrous oxide (NO) or cGMP in OME-induced relaxation. (
A
) Endothelium-intact
rings were treated with cumulative doses of OME in the presence (red) or the absence (black) of
N
ω
-nitro-l-arginine methyl ester (L-NAME) (inhibitor of eNOS, 100
µ
M). Data represent mean
±
SEM
(p<0.01 for OME alone vs. L-NAME plus OME; n=7). (
B
) Endothelium-intact rings were treated
with cumulative doses of OME in the presence (red) or absence (black) of ODQ (inhibitor of soluble
guanylate cyclase, 1 µM). Data represent mean ±SEM (p<0.01 for OME vs. ODQ plus OME; n=6).
Biomolecules 2019,9, 227 6 of 16
3.4. Effect of Origanum majorana Extract on the Production of cGMP in Aortic Rings
Because OME-induced vasorelaxation was significantly reduced by L-NAME and ODQ, we next
wished to determine if OME modulates the level of cGMP. Treatment with OME caused a significant and
dose-dependent increase in the levels of cGMP (Figure 4). In control rings (vehicle-treated), the cGMP
level was 2.8
±
0.8 (mean
±
standard error of the mean (SEM)) picomole/mg protein. However, in
rings treated with 0.3 mg/mL OME, the cGMP level rose to a dramatic level of 28
±
5 (mean
±
SEM)
picomole/mg protein (p<0.001). Importantly, L-NAME and ODQ significantly inhibited OME-induced
production of cGMP (data not shown).
Biomolecules 2019, 9, 227 6 of 16
Figure 3. Role of nitrous oxide (NO) or cGMP in OME-induced relaxation. (A) Endothelium-intact
rings were treated with cumulative doses of OME in the presence (red) or the absence (black) of Nω-
nitro-L-arginine methyl ester (L-NAME) (inhibitor of eNOS, 100 µM). Data represent mean ± SEM (p
< 0.01 for OME alone vs. L-NAME plus OME; n = 7). (B) Endothelium-intact rings were treated with
cumulative doses of OME in the presence (red) or absence (black) of ODQ (inhibitor of soluble
guanylate cyclase, 1 µM). Data represent mean ± SEM (p < 0.01 for OME vs. ODQ plus OME; n = 6).
3.4. Effect of Origanum majorana Extract on the Production of cGMP in Aortic Rings
Because OME-induced vasorelaxation was significantly reduced by L-NAME and ODQ, we next
wished to determine if OME modulates the level of cGMP. Treatment with OME caused a significant
and dose-dependent increase in the levels of cGMP (Figure 4). In control rings (vehicle-treated), the
cGMP level was 2.8 ± 0.8 (mean ± standard error of the mean (SEM)) picomole/mg protein. However,
in rings treated with 0.3 mg/mL OME, the cGMP level rose to a dramatic level of 28 ± 5 (mean ± SEM)
picomole/mg protein (p < 0.001). Importantly, L-NAME and ODQ significantly inhibited OME-
induced production of cGMP (data not shown).
Figure 4. Effect of OME on cGMP levels. Rings were treated without (control) or with increasing
concentrations of OME. cGMP levels were quantitated by an immunoassay. Data are shown as mean
± SEM, n = 5. (* p < 0.01; ** p < 0.001).
3.5. Involvement of the PI3K Signaling Pathway in the Origanum majorana Extract-Induced Relaxation of
Aortic Rings
Overwhelming evidence documents the contribution of the PI3K-Akt signaling to endothelium-
dependent aortic relaxation. To determine if this signaling pathway is involved in OME-induced
relaxation, we employed two inhibitors, wortmannin and LY294002. Indeed, pre-incubation with
either inhibitor instigated a significant reduction in OME-induced vasorelaxation. In rings incubated
in the absence of either inhibitor, the maximal relaxant effect was 91 ± 3% and pED50 was −4.1 with
confidence interval of −4.1 to −3.9. In wortmannin pre-incubated rings, the maximal relaxant effect
Figure 4.
Effect of OME on cGMP levels. Rings were treated without (control) or with increasing
concentrations of OME. cGMP levels were quantitated by an immunoassay. Data are shown as mean
±
SEM, n=5. (* p<0.01; ** p<0.001).
3.5. Involvement of the PI3K Signaling Pathway in the Origanum majorana Extract-Induced Relaxation of
Aortic Rings
Overwhelming evidence documents the contribution of the PI3K-Akt signaling to
endothelium-dependent aortic relaxation. To determine if this signaling pathway is involved
in OME-induced relaxation, we employed two inhibitors, wortmannin and LY294002. Indeed,
pre-incubation with either inhibitor instigated a significant reduction in OME-induced vasorelaxation.
In rings incubated in the absence of either inhibitor, the maximal relaxant effect was 91
±
3% and pED
50
was
−
4.1 with confidence interval of
−
4.1 to
−
3.9. In wortmannin pre-incubated rings, the maximal
relaxant effect was 53
±
6% (Figure 5) and pED
50
was
−
3.5 with confidence interval of 3.7–3.4. Similar
results were obtained when rings were pre-incubated with LY294002 (10
µ
M), where the maximal
effect was 58%, pED
50
was
−
3.7 and a 95% confidence interval of
−
3.8 to
−
6 (Figure 5). Together, our
data clearly allude to the role of the PI3K signaling in OME-induced aortic relaxation.
Biomolecules 2019,9, 227 7 of 16
Biomolecules 2019, 9, 227 7 of 16
was 53 ± 6% (Figure 5) and pED50 was −3.5 with confidence interval of 3.7–3.4. Similar results were
obtained when rings were pre-incubated with LY294002 (10 µM), where the maximal effect was 58%,
pED50 was −3.7 and a 95% confidence interval of −3.8 to −6 (Figure 5). Together, our data clearly allude
to the role of the PI3K signaling in OME-induced aortic relaxation.
Figure 5. Effect of the PI3K pathway on OME-induced vasorelaxation. Rings with intact endothelium
were treated with OME in the absence (black) or presence of phosphoinositide 3-kinase (PI3-K)
inhibitors: Wortmannin (0.1 µM; red) or LY29400 (10 µM; blue). Data presented as mean ± SEM (n =
5; p < 0.01 for OME vs. Wortmannin + OME or LY29400 + OME).
3.6. Effect of K+ Channel Blockers on Origanum majorana Extract-Induced Relaxation
Potassium channels are known to play an important role in vasodilation [24–26]. Thus, we
wished to determine the of role of these channels in OME-induced relaxation. The vasorelaxant
effects of OME were not altered by incubation with K+ channel blockers tetraethylammonium (TEA,
5 mM) or glibenclamide (Glib, 10 µM). Indeed, in the presence or absence of glibenclamide, the OME-
induced maximal relaxant effect was 90 ± 4% or 92 ± 4% (Figure 6A), and pED50 was −3.7 or −4.1 with
confidence intervals of −3.9 to −3.7 or −4 to −3.8, respectively. Similarly, incubation with TEA did not
affect OME-induced relaxation of endothelium-intact aortic rings (Figure 6B). In the presence of TEA,
the maximal effect was 88%, pED50 was −4, and the 95% confidence interval was −4.1 to −4. Together,
these data argue for the absence of a role for potassium channels in OME-induced relaxation.
Figure 5.
Effect of the PI3K pathway on OME-induced vasorelaxation. Rings with intact endothelium
were treated with OME in the absence (black) or presence of phosphoinositide 3-kinase (PI3-K) inhibitors:
Wortmannin (0.1
µ
M; red) or LY29400 (10
µ
M; blue). Data presented as mean
±
SEM (n=5; p<0.01 for
OME vs. Wortmannin +OME or LY29400 +OME).
3.6. Effect of K+Channel Blockers on Origanum majorana Extract-Induced Relaxation
Potassium channels are known to play an important role in vasodilation [
24
–
26
]. Thus, we wished
to determine the of role of these channels in OME-induced relaxation. The vasorelaxant effects of
OME were not altered by incubation with K
+
channel blockers tetraethylammonium (TEA, 5 mM) or
glibenclamide (Glib, 10
µ
M). Indeed, in the presence or absence of glibenclamide, the OME-induced
maximal relaxant effect was 90
±
4% or 92
±
4% (Figure 6A), and pED
50
was
−
3.7 or
−
4.1 with
confidence intervals of
−
3.9 to
−
3.7 or
−
4 to
−
3.8, respectively. Similarly, incubation with TEA did not
affect OME-induced relaxation of endothelium-intact aortic rings (Figure 6B). In the presence of TEA,
the maximal effect was 88%, pED
50
was
−
4, and the 95% confidence interval was
−
4.1 to
−
4. Together,
these data argue for the absence of a role for potassium channels in OME-induced relaxation.
Biomolecules 2019, 9, 227 8 of 16
Figure 6. Effect of potassium channel inhibitors on OME-induced vasorelaxation. Rings with intact
endothelium were treated with OME in the absence (OME; black) or presence of (A) 10 µM of
glibenclamide (Glib + OME; red), or (B) 100 µM of tetraethylammonium (TEA + OME; red). Data
represent mean ± SEM (n = 6). p > 0.05 for OME alone vs. either Glib + OME or OME + TEA.
3.7. Effects of Ca
2+
Channel Blockers on Origanum majorana Extract-Induced Relaxation
To determine if calcium channels contribute to OME-induced relaxation, we pre-incubated
aortic rings with verapamil (1 µM) before adding OME. There was no significant difference between
the control and treated vessels (p > 0.05) (Figure 7). Indeed, the maximal relaxant effect in vehicle-
treated or verapamil-incubated rings was not affected (91 ± 4% vs. 92 ± 3%) (Figure 7). pED
50
was −4.0
or −3.9 with confidence intervals of −4.1 to −3.9 or −3.9 to −3.8 in the absence or presence of verapamil,
respectively.
Figure 7. Role of calcium channels in OME-induced relaxation of aortic rings. Endothelium-intact
aortic rings were pre-incubated with OME in the absence (OME; black) or presence of verapamil
(verap) (1 µM; verap + OME; red). Data are presented as mean ± SEM (n = 5; p > 0.05).
A B
Figure 6.
Effect of potassium channel inhibitors on OME-induced vasorelaxation. Rings with intact
endothelium were treated with OME in the absence (OME; black) or presence of (
A
) 10
µ
M of
glibenclamide (Glib +OME; red), or (
B
) 100
µ
M of tetraethylammonium (TEA +OME; red). Data
represent mean ±SEM (n=6). p>0.05 for OME alone vs. either Glib +OME or OME +TEA.
Biomolecules 2019,9, 227 8 of 16
3.7. Effects of Ca2+Channel Blockers on Origanum majorana Extract-Induced Relaxation
To determine if calcium channels contribute to OME-induced relaxation, we pre-incubated aortic
rings with verapamil (1
µ
M) before adding OME. There was no significant difference between the
control and treated vessels (p>0.05) (Figure 7). Indeed, the maximal relaxant effect in vehicle-treated or
verapamil-incubated rings was not affected (91
±
4% vs. 92
±
3%) (Figure 7). pED
50
was
−
4.0 or
−
3.9 with
confidence intervals of
−
4.1 to
−
3.9 or
−
3.9 to
−
3.8 in the absence or presence of verapamil, respectively.
Biomolecules 2019, 9, 227 8 of 16
Figure 6. Effect of potassium channel inhibitors on OME-induced vasorelaxation. Rings with intact
endothelium were treated with OME in the absence (OME; black) or presence of (A) 10 µM of
glibenclamide (Glib + OME; red), or (B) 100 µM of tetraethylammonium (TEA + OME; red). Data
represent mean ± SEM (n = 6). p > 0.05 for OME alone vs. either Glib + OME or OME + TEA.
3.7. Effects of Ca
2+
Channel Blockers on Origanum majorana Extract-Induced Relaxation
To determine if calcium channels contribute to OME-induced relaxation, we pre-incubated
aortic rings with verapamil (1 µM) before adding OME. There was no significant difference between
the control and treated vessels (p > 0.05) (Figure 7). Indeed, the maximal relaxant effect in vehicle-
treated or verapamil-incubated rings was not affected (91 ± 4% vs. 92 ± 3%) (Figure 7). pED
50
was −4.0
or −3.9 with confidence intervals of −4.1 to −3.9 or −3.9 to −3.8 in the absence or presence of verapamil,
respectively.
Figure 7. Role of calcium channels in OME-induced relaxation of aortic rings. Endothelium-intact
aortic rings were pre-incubated with OME in the absence (OME; black) or presence of verapamil
(verap) (1 µM; verap + OME; red). Data are presented as mean ± SEM (n = 5; p > 0.05).
A B
Figure 7.
Role of calcium channels in OME-induced relaxation of aortic rings. Endothelium-intact
aortic rings were pre-incubated with OME in the absence (OME; black) or presence of verapamil (verap)
(1 µM; verap +OME; red). Data are presented as mean ±SEM (n=5; p>0.05).
3.8. Effect of Atropine and Pyrilamine on Origanum majorana Extract-Induced Relaxation
Endothelial muscarinic M3 receptors have been shown to promote vasorelaxation by virtue of
their ability to increase NO release [
27
]. We thus wished to examine the effect of muscarinic receptors
on OME-induced relaxation. Aortic rings with intact endothelium were pre-treated with atropine
(1
µ
M) for 30 min before NE (3
µ
M) pre-contraction. In the absence of atropine, the maximal relaxant
effect was 96
±
7% and a pED
50
of
−
3.98 with confidence intervals of
−
4.1 to
−
3.8 (Figure 8A). However,
in the presence of atropine, the maximal relaxant effect was dramatically reduced to 43
±
9% and a
pED
50
of
−
3.8 with confidence intervals of
−
4.3 to
−
3.4. This clearly shows that muscarinic receptors
are involved in OME-induced relaxation.
Endothelial histaminergic receptors, particularly H1 G protein-coupled receptors, are known to
initiate vasorelaxation [
28
]. We thus investigated the influence of pyrilamine (also called mepyramine),
a blocker of histamine H1-receptors on OME-induced relaxation. Compared to the control, incubation
with pyrilamine did not significantly modulate OME-induced relaxation of endothelium-intact rings.
In the absence of pyrilamine, the maximal relaxant effect was 88.8
±
6.9% and a pED
50
of
−
3.88 with
confidence intervals of
−
4.0 to
−
3.7 (Figure 8B). In the presence of pyrilamine, there was only a marginal
and insignificant reduction in the maximal relaxation value, which was noted to be 75
±
6%. pED
50
of
pyrilamine-treated rings was found to be
−
3.82 with confidence intervals of
−
3.9 to
−
3.6, which is very
similar to that obtained in the vehicle-treated rings (Figure 8B). Thus, histaminergic receptors do not
seem to have a significant effect on OME-induced relaxation (p>0.05).
Biomolecules 2019,9, 227 9 of 16
Biomolecules 2019, 9, 227 9 of 16
3.8. Effect of Atropine and Pyrilamine on Origanum majorana Extract-Induced Relaxation
Endothelial muscarinic M3 receptors have been shown to promote vasorelaxation by virtue of their
ability to increase NO release [27]. We thus wished to examine the effect of muscarinic receptors on
OME-induced relaxation. Aortic rings with intact endothelium were pre-treated with atropine (1 µM)
for 30 min before NE (3 µM) pre-contraction. In the absence of atropine, the maximal relaxant effect
was 96 ± 7% and a pED50 of −3.98 with confidence intervals of −4.1 to −3.8 (Figure 8A). However, in
the presence of atropine, the maximal relaxant effect was dramatically reduced to 43 ± 9% and a pED50
of −3.8 with confidence intervals of −4.3 to −3.4. This clearly shows that muscarinic receptors are
involved in OME-induced relaxation.
Endothelial histaminergic receptors, particularly H1 G protein-coupled receptors, are known to
initiate vasorelaxation [28]. We thus investigated the influence of pyrilamine (also called
mepyramine), a blocker of histamine H1-receptors on OME-induced relaxation. Compared to the
control, incubation with pyrilamine did not significantly modulate OME-induced relaxation of
endothelium-intact rings. In the absence of pyrilamine, the maximal relaxant effect was 88.8 ± 6.9%
and a pED50 of −3.88 with confidence intervals of −4.0 to −3.7 (Figure 8B). In the presence of
pyrilamine, there was only a marginal and insignificant reduction in the maximal relaxation value,
which was noted to be 75 ± 6%. pED50 of pyrilamine-treated rings was found to be −3.82 with
confidence intervals of −3.9 to −3.6, which is very similar to that obtained in the vehicle-treated rings
(Figure 8B). Thus, histaminergic receptors do not seem to have a significant effect on OME-induced
relaxation (p > 0.05).
Figure 8. Involvement of histaminic or muscarinic receptors in OME-induced relaxation. Rings with
intact endothelium were incubated with cumulative doses of OME in the absence (OME; black) or
presence of (A) 10 µM of atropine (natural alkaloid with antagonistic properties at muscarinic
acetylcholine receptors; atropine + OME; red) or (B) 10 µM of pyrilamine (blocker of H1 histamine
receptors; pyrilamine + OME; red). Data showed represent mean ± SEM (n = 6 or 5 for atropine or
pyrilamine-treated rings, respectively). p < 0.05 for OME alone vs. either atropine + OME; p > 0.05 for
OME alone vs. either pyrilamine + OME.
3.9. Effect of Cyclooxygenase Pathway on Origanum majorana Extract-Induced Relaxation
Vascular tone is greatly affected by prostanoids generated via the activity of cyclocoxygenases
[29]. We thus sought to determine the potential role of the cyclooxygenases in OME-induced relaxation.
In the absence of indomethacin, the maximal relaxant effect was 90.9 ± 2.8% and a pED50 of −3.95 with
confidence intervals of −4.0 to −3.9 (Figure 9). In the presence of indomethacin, the maximal relaxant
effect was 89.6 ± 1.9% and a pED50 of −3.9 with confidence intervals of −3.9 to −3.8 (Figure 9). Therefore,
cyclooxygenases do not play an important role in OME-induced relaxation (p > 0.05) (Figure 9).
Figure 8.
Involvement of histaminic or muscarinic receptors in OME-induced relaxation. Rings
with intact endothelium were incubated with cumulative doses of OME in the absence (OME; black)
or presence of (
A
) 10
µ
M of atropine (natural alkaloid with antagonistic properties at muscarinic
acetylcholine receptors; atropine +OME; red) or (
B
) 10
µ
M of pyrilamine (blocker of H
1
histamine
receptors; pyrilamine +OME; red). Data showed represent mean
±
SEM (n=6 or 5 for atropine or
pyrilamine-treated rings, respectively). p<0.05 for OME alone vs. either atropine +OME; p>0.05 for
OME alone vs. either pyrilamine +OME.
3.9. Effect of Cyclooxygenase Pathway on Origanum majorana Extract-Induced Relaxation
Vascular tone is greatly affected by prostanoids generated via the activity of cyclocoxygenases [
29
].
We thus sought to determine the potential role of the cyclooxygenases in OME-induced relaxation.
In the absence of indomethacin, the maximal relaxant effect was 90.9
±
2.8% and a pED
50
of
−
3.95 with
confidence intervals of
−
4.0 to
−
3.9 (Figure 9). In the presence of indomethacin, the maximal relaxant
effect was 89.6
±
1.9% and a pED
50
of
−
3.9 with confidence intervals of
−
3.9 to
−
3.8 (Figure 9). Therefore,
cyclooxygenases do not play an important role in OME-induced relaxation (p>0.05) (Figure 9).
Biomolecules 2019, 9, 227 10 of 16
Figure 9. Effect of cyclooxygenase inhibition by indomethacin on OME-induced aortic relaxation.
Rings with intact endothelium were pre-treated without (OME; black) or with indomethacin (a
cyclooxygenase (COX)1/2 inhibitor, 10 µM; INDO + OME; red) followed by the addition of cumulative
doses of OME. Data showed represent mean ± SEM (n = 5; p > 0.05).
4. Discussion
In this report, we elucidated the underlying signaling mechanisms implicated in rat aortic
relaxation induced by the leaf extract of O. majorana, commonly known as marjoram. This plant
contains a bounty of bioactives, such as thymol, carvacrol, p-cymene, sabinene hydrate, rosmarinic
acid, γ-terpinene, ursolic acid, and many others [30,31]. These constituents possess antibacterial,
antifungal, and antispasmodic activities, in addition to their ability to scavenge free radicals, inhibit
acetylcholinesterase, and depress cardiac activity [32,33]. Some of these bioactives, like carvacrol,
thymol, ursolic acid, and hesperetin indeed exert vasorelaxing activities [19,20,22,34]. In addition to
these effects, we have recently shown that this plant can significantly inhibit the malignant phenotype
of cancer cells [15,16]. To the best of our knowledge, this is the first report that dissects the mechanism
for the vasodilator effects of marjoram. It is important to note that using the whole plant, part of it,
or a crude extract may provide benefits over the use of individual bioactives. This is partly due to the
notion that these herbs are commonly grown and consumed by people in rural areas where access to
modern medicine is limited. In addition, these extracts can easily be prepared at home and are thus
significantly less costly, making them relatively more appealing to be used. Importantly, oftentimes,
the plant as a whole, or as a crude extract, may prove more effective than individual bioactives, likely
due to a potential synergy between these molecules.
There is an intricate coordination between vasoconstrictors and vasodilators in regulating
vasotone. The maestro of this well-orchestrated balance is the endothelial cell layer. Indeed, this layer
secretes many vasoconstrictors, the most potent of which is endothelin, as well as vasodilators, the
most prominent of which is NO. In endothelial cells, NO is generated from L-arginine by endothelial
NO synthase (eNOS) activation, which is stimulated by the calcium-calmodulin complex or activated
via the PI3K/Akt pathway [35]. The role of NO in vasorelaxation is overwhelmingly established and
documented. Many stimuli, including the vascular endothelial growth factor, β-agonists, and shear-
stress signals regulate NO production via a pathway involving phosphoinositide 3-kinase (PI3-
K)/Akt signaling. This pathway is known to play an important role in endothelium-mediated
Figure 9.
Effect of cyclooxygenase inhibition by indomethacin on OME-induced aortic relaxation. Rings
with intact endothelium were pre-treated without (OME; black) or with indomethacin (a cyclooxygenase
(COX)1/2 inhibitor, 10
µ
M; INDO +OME; red) followed by the addition of cumulative doses of OME.
Data showed represent mean ±SEM (n=5; p>0.05).
Biomolecules 2019,9, 227 10 of 16
4. Discussion
In this report, we elucidated the underlying signaling mechanisms implicated in rat aortic
relaxation induced by the leaf extract of O. majorana, commonly known as marjoram. This plant
contains a bounty of bioactives, such as thymol, carvacrol, p-cymene, sabinene hydrate, rosmarinic
acid,
γ
-terpinene, ursolic acid, and many others [
30
,
31
]. These constituents possess antibacterial,
antifungal, and antispasmodic activities, in addition to their ability to scavenge free radicals, inhibit
acetylcholinesterase, and depress cardiac activity [
32
,
33
]. Some of these bioactives, like carvacrol,
thymol, ursolic acid, and hesperetin indeed exert vasorelaxing activities [
19
,
20
,
22
,
34
]. In addition to
these effects, we have recently shown that this plant can significantly inhibit the malignant phenotype
of cancer cells [
15
,
16
]. To the best of our knowledge, this is the first report that dissects the mechanism
for the vasodilator effects of marjoram. It is important to note that using the whole plant, part of it,
or a crude extract may provide benefits over the use of individual bioactives. This is partly due to the
notion that these herbs are commonly grown and consumed by people in rural areas where access to
modern medicine is limited. In addition, these extracts can easily be prepared at home and are thus
significantly less costly, making them relatively more appealing to be used. Importantly, oftentimes, the
plant as a whole, or as a crude extract, may prove more effective than individual bioactives, likely due
to a potential synergy between these molecules.
There is an intricate coordination between vasoconstrictors and vasodilators in regulating vasotone.
The maestro of this well-orchestrated balance is the endothelial cell layer. Indeed, this layer secretes
many vasoconstrictors, the most potent of which is endothelin, as well as vasodilators, the most
prominent of which is NO. In endothelial cells, NO is generated from l-arginine by endothelial NO
synthase (eNOS) activation, which is stimulated by the calcium-calmodulin complex or activated
via the PI3K/Akt pathway [
35
]. The role of NO in vasorelaxation is overwhelmingly established
and documented. Many stimuli, including the vascular endothelial growth factor,
β
-agonists, and
shear-stress signals regulate NO production via a pathway involving phosphoinositide 3-kinase
(PI3-K)/Akt signaling. This pathway is known to play an important role in endothelium-mediated
vasorelaxation [
36
]. Indeed, the PI3-K/Akt pathway activates eNOS via phosphorylating serine residue
at position 1177 [
37
–
39
]. Here, when we incubated rings with PI3-K/Akt inhibitors, there was a
significant reduction in the OME-induced relaxation. In an ischemia/reperfusion injury model, it was
recently reported that ginsenosides of panax ginseng promote coronary arterial flow by activating the
PI3K/Akt/eNOS cascade. This establishes the cardioprotective effects conferred by isolated extracts of
ginseng [
40
]. Similarly, in another ischemia-reperfusion injury model, stimulation of PI3K/Akt/NO
cascade by a ginsenoside metabolite bestowed cardioprotection [41].
Nitric oxide is known to activate soluble guanylate cyclase (sGC) in vascular smooth muscle cells
(VSMCs) and to promote the synthesis of a secondary intracellular messenger, cGMP. This cGMP elicits
many biological effects including relaxation of vascular smooth musculature [
42
–
44
] and consequent
cGMP-mediated vasodilation [
45
–
47
]. In this context, our data reveals that pretreatment with L-NAME,
a NOS inhibitor, or ODQ, a sGC inhibitor, affect the vasorelaxant effects of OME. These results are a clear
indication that the vasorelaxant effect of OME are directly related to the NO-cGMP pathway. Our results
are similar to other reports showing several plant species causing aortic relaxation through NO/cGMP.
Some of these plant species include Schizophyllum commune [
48
], Salvia fruticosa L. [
7
], Euphorbia humifusa
Willd [
49
], Alpiniae zerumbet (Pers.) B. L. Burtt & R. M. Sm. (Zingiberaceae) [
50
], Rhus coriaria L. [
8
],
and Mansoa hirsuta D.C. [
51
]. Furthermore, here in our study, we report that OME increased production
of cGMP, clearly supporting the involvement of sGC-cGMP in OME-induced relaxation.
In VSMCs, the membrane potential is dynamically regulated via changes in K
+
channel activity,
which greatly influences the function of voltage-dependent calcium channels [
52
,
53
]. Interestingly,
NO is known to have a direct effect on activation of the K
+
channels [
54
]. Here, we employed two
K
+
channel blockers, namely glibenclamide, a highly selective blocker of ATP-sensitive K+channels,
and TEA, a blocker of voltage-sensitive K
+
channels [
55
]. Our results show neither glibenclamide
nor TEA appreciably modulated OME-induced effects. Thus, K
+
channels (K
+ATP
or K
V
channels)
Biomolecules 2019,9, 227 11 of 16
are not involved in OME-induced aortic relaxation. This was very surprising to us due to the greatly
significant role of these channels in vasorelaxation. Indeed, these channels play a critical role in
regulating plasma membrane potential, and hence determining vascular tone [
56
–
58
]. It is already
established that NO directly activates calcium-dependent potassium channels in VSMCs [
59
], leading
to vasorelaxation by a hyperpolarization mechanism [
60
]. Our results argue for the absence of a role for
Ca
2+
-dependent or ATP-driven potassium channels in OME-induced aortic relaxation. Our findings
differ from other studies that show that certain plant extracts produce an endothelium-dependent
vasodilatory effect via activation of the potassium channels. However, our results are in accordance
with previous studies [
61
], which report that stimulation of potassium channels is not necessarily
implicated in vasorelaxation. It is not unreasonable that activation of the NO/cGMP pathway produces
vasodilation through alternative mechanisms.
The role of Ca
2+
in VSMC contraction/relaxation is well-known. The two main sources for
this Ca
2+
are the extracellular space and intracellular stores like the sarcoplasmic reticulum. Other
mechanisms, such as those involving protein kinase C (PKC) or Ca
2+
sensitization, are also implicated
in VSMC contractility [
62
]. By blocking calcium channels, danshen has been shown to potentiate
endothelium-independent relaxation of rat coronary arteries [
63
]. However, our results show that
inhibiting calcium channels did not alter OME-induced relaxation, suggesting that mechanisms
independent of these channels are involved. This is similar to recently reported findings showing that
L-type calcium channels may not affect extract-induced relaxation [
7
,
8
]. It is worth mentioning here
that one of the limitations of this study is that we did not assess the effect of depleting extracellular
calcium on the vasorelaxation involved. However, due to the fact that L-type calcium channels were not
involved, one may argue that the absence or presence of extracellular calcium is unlikely to be involved.
Vasorelaxant responses initiated by histamine are known to be mediated by histamine H1
receptors [
28
], which are G protein-coupled receptors. In this report, we did not find any contributory
role for these receptors in OME-induced relaxation. This is similar to other studies that have used
other plants species such as S. fruticosa,Salvia miltiorrhiza, or R. coriaria [7,8,64].
Cyclooxygenases (COX-1 and COX-2) convert arachidonic acid to active prostanoids. These
prostanoids are well-known to play important roles in vascular physiology and pathology, including
vasorelaxatory mechanisms [
65
]. Prostacyclin (PGI
2
) is a potent vasorelaxant prostaglandin that is
released by the endothelium. It increases the intracellular accumulation of 3
0
-5
0
-cyclic adenosine
monophosphate (cAMP), which is a powerful vasodilator as well as a modulator of many mechanisms
in VSMCs [
66
–
73
]. In our study, pre-treatment with indomethacin, a non-selective cyclooxygenase
inhibitor, did not affect the vasorelaxant effects of OME. This result suggested that the vasorelaxant
effect of OME did not have a relationship with the vascular prostacyclin pathway. This is similar to
other reports showing no role for prostaglandins in relaxation of rat femoral arteries or aortic rings
in response to danshen or S. fruticosa extract, respectively [
7
,
74
]. Similarly, we recently reported no
role for these prostanoids in R. coriaria-induced aortic relaxation [8]. Contextually, our results are not
surprising since danshen, S. fruticosa,R. coriaria and marjoram, all contain rosmarinic acid, which is
known to inhibit both COX-1 and COX-2 [75].
Finally, an endothelium-independent effect may be involved, though this needs further
experimentation. It is possible that the residual vasorelaxation is due to increased activity of adenylate
cyclase. This pathway has indeed been reported to underlie the vasodilatory mechanism of others herbs,
such as R. coriaria [8]. Importantly, two bioactives found in marjoram, namely kaempferol and luteolin,
have been reported to contribute to vasodilatation of rat aortae by inhibiting phosphodiesterases [
76
].
As is well known, inhibiting phosphodiesterases leads to increased accumulation of cAMP, which itself
is a potent vasodilator. However, more experiments are warranted before such an assertion is validated.
5. Conclusions
In conclusion, our results show that the vasorelaxant effect of OME was due to activation of the
PI-3K pathway, as well as an endothelium-dependent increase in the accumulation of cGMP. Together,
Biomolecules 2019,9, 227 12 of 16
our findings support the use of marjoram in the management/treatment of hypertension, [
17
]. More
research, particularly in animal models of hypertension, is needed to better support the antihypertensive
potential of this herb. It is important to note that studies directed at determining which bioactives
reach the bloodstream after oral consumption of marjoram or its extract are warranted.
Author Contributions:
Conceptualization, A.H.E.; Data curation, A.B., E.B. and A.S.; Formal analysis, G.P., J.M.,
R.I. and A.H.E.; Funding acquisition, A.B., E.B. and A.H.E.; Investigation, A.S. and A.H.E.; Methodology, R.I.
and K.I.; Project administration, A.B.; Resources, A.B. and A.H.E.; Writing—Original draft, K.I. and A.H.E.;
Writing—Review & editing, A.B., E.B., A.S., G.P., J.M., R.I. and A.H.E.
Funding:
This research was funded by a grant (Fund #: 5/4/2019) from the University of Petra (Aman, Jordan) to
A.B., E.B. and A.H.E.
Conflicts of Interest: The authors declare no conflict of interest.
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