Journal of Ethnopharmacology 242 (2019) 112039

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

Antihypertensive activity of Petroselinum crispum through inhibition of T

vascular calcium channels in rats
Mohammed Ajebli, Mohamed Eddouks∗
Faculty of Sciences and Techniques Errachidia, Moulay Ismail University, BP 509, Boutalamine, 52000, Errachidia, Morocco

ARTICLE INFO ABSTRACT

Keywords: Ethnopharmacological relevance: Parsley (Petroselinum crispum; P. crispum) is among the popular aromatic vege-
Hypertension tables and a part of the daily diet in the Mediterranean area. This plant is widely used in alternative medicine as
Vasorelaxant a remedy against hypertension.
L-NAME The aim of the study: The aim of the study was to evaluate the antihypertensive activity of the aqueous extract of
Calcium
this plant.
Material and methods: In the current study, the aqueous extract of the aerial parts of parsley (AEPC) was pre-
pared and its antihypertensive activity was evaluated using in vivo and in vitro studies. In the in vivo investigation,
anesthetized L-NAME-hypertensive and normotensive rats received orally AEPC (160 mg/kg) during 6 h for the
acute experiment and during seven days for the sub-chronic treatment. Thereafter, systolic, diastolic, mean
arterial blood pressure and heart rate were recorded using a tail cuff and a computer-assisted monitoring device.
Concerning the in vitro investigation, isolated thoracic aortic rings were suspended in a tissue bath and the
tension changes were recorded to a data acquisition system.
Results: The results indicated that AEPC extract decreased the systolic, diastolic, mean arterial blood pressure in
normotensive and hypertensive rats. The data revealed that parsley extract exerts its hypotensive effects through
vasodilatory properties via an endothelium-independent pathway. More interestingly, the study demonstrated
here that the vasorelaxing ability of AEPC is exerted through both Voltage Operated and Receptor Operated
Calcium Channels (VOCC and ROCC).
Conclusion: The study illustrates the beneficial action of P. crispum as an antihypertensive agent.

1. Introduction agonists, peripheral adrenergic neuronal blocking agents, central/per-

According to World Health Organization (WHO), hypertension, also
known as high or raised blood pressure, is a condition in which the
blood vessels have persistently raised pressure. It contributes to the
burden of heart disease, stroke and kidney failure and premature
mortality and disability. It disproportionately affects populations in low
and middle-income countries where health systems are fragile (WHO,
2013). The treatment of hypertension is becoming more difficult be-
cause of the lower blood pressure (BP) targets set by evidence-based
guideline recommendations (Krum and Martin, 2007).
Medicinal plants are used by different populations throughout the
globe for treating blood pressure problems based either on folkloric
practices or on phytotherapeutic usages. There are currently adequate
therapeutic options for patients with hypertension and many conven-
tional antihypertensive agents are used for treating hypertension like
thiazide, loop, and potassium-sparing diuretics, central α2-adrenergic
ipheral adrenergic neuronal-blocking agents, direct-acting vasodilators,
etc. However these drugs have some side effects like muscle cramps,
dizziness, extreme tiredness, dehydration, blurred vision, abnormal
heart rate, skin rash, etc (Singh et al., 2015). While some herbs and
their derivatives are often safe and do not lead to such side effects.
Petroselinum crispum (Mill.) Fuss. is a medicinal and aromatic plant
commonly known as parsley and belonging to the Apiaceae family.
Parsley is a popular aromatic vegetable and part of the daily diet in
Morocco and around the world. This herb is widely consumed as spice
and flavoring agent used for improving the taste of a multitude of
modern and traditional Moroccan foods. Parsley is also commonly used
in Morocco for managing a range of illnesses such as gastrointestinal
disorders, diabetes mellitus, skin diseases, fever, ulcer, rheumatism and
hypertension (Eddouks et al., 2017). Furthermore, it has been used in
different regions across the world as carminative, gastrotonic, diuretic,
antiseptic of urinary tract, anti-urolithiasis, anti-dote, and anti-

∗
Corresponding author.
E-mail addresses: mohammedajebli@gmail.com (M. Ajebli), mohamed.eddouks@laposte.net (M. Eddouks).

https://doi.org/10.1016/j.jep.2019.112039
Received 3 January 2019; Received in revised form 23 June 2019; Accepted 23 June 2019
Available online 26 June 2019
0378-8741/ © 2019 Elsevier B.V. All rights reserved.
M. Ajebli and M. Eddouks
Abbreviations
AEPC aerial parts of parsley
BP blood pressure
CCBs calcium channel blockers
cGMP cyclic guanosine monophosphate
DBP diastolic blood pressure
EP epinephrine
HVA high voltage-activated
HR heart rate
inflammatory, and for the treatment of amenorrhea, dysmenorrheal,
gastrointestinal disorder, hypertension, cardiac disease, urinary dis-
ease, otitis, sniffle, diabetes, and also various dermal disease in tradi-
tional and folklore medicines (Farzaei et al., 2013). Phenolic com-
pounds and flavonoids particularly apigenin apiin and 6"-Acetylapiin
essential oil mainly myristicin and apiol and coumarins are the active
compounds identified in Petroselinum crispum.
Parsley is well known to be used traditionally to manage hy-
pertension in Morocco. The hypotensive effect of a similar species
(Petroselium sativa) has been demonstrated in normal rats (Campos
et al., 2009). However, until now no pharmacological study was con-
ducted for testing the antihypertensive ability of Petroselinum crispum.
Thus, this study was designed to evaluate the hypotensive effect of the
aqueous extract of Petroselinum crispum in L-NAME-induced hyperten-
sive and normotensive rats and to determine the pharmacological ef-
fects of this extract on rat isolated thoracic aorta.
2. Material and methods
2.1. Plant material and preparation of the aqueous extract
Petroselinum crispum (Mill.) Fuss. was purchased fresh from a local
market in Tafilalet region (semi-arid area) of Morocco in March 2017
and air-dried at 40 °C. The plant was taxonomically identified and au-
thenticated and voucher specimen was deposited at the herbarium of
the Faculty of Sciences and Techniques Errachidia (ME1703). Plant
material was prepared according to the traditional method used in
Morocco (decoction): 1 g of powdered leaves mixed with 100 ml dis-
tilled water was boiled for 10 min and then cooled for 15 min.
Thereafter the aqueous extract was filtered using a Millipore filter
(Millipore 0.2 mm, St Quentin en Yvelines, France) to remove particu-
late matter. Finally, the filtration of the extract was lyophilized in a
Freeze dryer (Labcono, Boyer, Casablanca, Morocco). The yield of the
plant extract was 30.6% after lyophilization. The dose administered
was 160 mg of lyophilized aqueous extract per kg of body weight
(Ajebli and Eddouks, 2017). This dose was selected after preliminary
experiments as it showed optimal responses.
2.2. Experimental animals
Healthy albino adult male rats (Wistar strain) weight about
180–280 g were obtained from experimental center of Missour Morocco
and kept in individual polyethylene cages and maintained in standard
condition, and the animals were fed ad libitum with normal laboratory
chow standard pellet diet. Rats were allowed at least 3 weeks for a
better acclimation from shipping stress.
2.3. Chemical reagents and drugs
Acetylcholine chloride, norepinephrine, N-nitro- l-arginine methyl
ester (L-NAME) and indomethacin were purchased from Sigma
Chemical Co. (St. Louis, USA). All other reagents were of analytical
grade from local sources. All cited drugs were dissolved in distilled
2
Journal of Ethnopharmacology 242 (2019) 112039
L-Name N-nitro- l-arginine methyl ester
LVA low voltage-activated
MBP mean blood pressure
NSAID nonsteroidal anti-inflammatory drug
NO nitric oxide
P. crispum Petroselinum crispum
SBP systolic blood pressure
VSM vascular smooth muscle
WHO World Health Organization
water.
2.4. Experimental protocol
2.4.1. Hypotensive activity
Hypertension was induced in male albino Wistar rats by repeated
(once a day during a week) oral administration of L-NAME (80 mg/kg
body weight) dissolved in distillated water; normal control rats re-
ceived distilled water as vehicle. The animals which were confirmed to
be hypertensive by the raised systolic blood pressure ≥150 mmHg were
used for the study.
Normotensive and hypertensive rats were randomly assigned to
three different groups consisting of six rats each. One control group
received distilled water a second treated group received the aqueous
extract of aerial parts of P. crispum (AEPC) at a dose of 160 mg/kg body
weight and the third group received a reference drug (lasilix at a dose of
20 mg/kg). For single oral administration distilled water (control), la-
silix or the aqueous extract were administered and systolic blood
pressure (SBP) mean blood pressure (MBP) and heart rate (HR) of an-
imals were measured. For repeated oral administration rats were
treated once daily for 7 days and the three last parameters were fol-
lowed during this period. The rats (n = 6 in each group) were treated
orally. All experiments were performed after anesthetizing animals
using ether in order to avoid immobilization stress induction. For es-
timation of blood pressure rats were placed in the NIBP restrainers and
appropriate cuff with sensor was then attached on the tail and warmed
to about 35 °C for 15–20 min to allow reproducible blood pressure
measurements (three measurements per animal per session) using a tail
cuff and a computer-assisted monitoring device (Harvard, Boyer,
Casablanca, Morocco). The tail cuff was inflated to a pressure above the
expected systolic blood pressure (200 mmHg) and slowly released
during which the pulses were recorded by using PowerLab data ac-
quisition system and LabChart 5.0 software (Harvard, Boyer,
Casablanca, Morocco). Systolic blood pressure (SBP), mean blood
pressure (MBP) and heart rate (HR) were measured directly using pulse
tracing while diastolic blood pressure (DBP) was calculated from SBP
and MBP using formula: DBP= (3MBP-SBP)/2. After inflating the cuff
to 200 mmHg pressure, when all pulses disappear, the cuff was deflated
slowly but linearly. The pressure at which the pulses started to appear is
considered as SBP. Then the amplitude of the pulses continues to in-
crease gradually. When this amplitude reaches its maximum, it corre-
sponds to the mean blood pressure (MBP). All blood pressure mea-
surements were carried out at the same time of day for the sub-chronic
test.
2.4.2. Vasorelaxant activity
Male Wistar rats (260–300 g) were raised and housed (n = 6 per
cage) in rooms with a controlled light/dark cycle at 25 °C with food and
water ad libitum. Animals were sacrificed by stunning and ex-
sanguination after anesthesia by pentobarbital sodium. Aortas were
immediately excised and placed in cold buffer, then, quickly removed
and carefully cleaned of adhering fat and connective tissue. The isolated
arteries were transversally cut into rings 2–3 mm length and suspended
M. Ajebli and M. Eddouks Journal of Ethnopharmacology 242 (2019) 112039

at a resting force of 2 g in an tissue bath oxygenated (95% O2, 5% CO2) of the EP and KCl contraction. Each cumulative dose-effect curve for
and containing 40 ml Krebs-Henseleit solution (KH), maintained at AEPC-induced relaxation was plotted contingent on application of sig-
37 °C and pH 7.4 and containing (mM): NaCl (118), KCl (4.50), NaHCO3 moidal curve fitting and non-linear regression using Prism version 7
(25), MgSO4 (1.2), CaCl2 (1.8), NaHPO4 (1.2) and glucose (11). (GraphPad Softare Inc., San Diego, CA., U.S.A.) to generate the para-
Changes in force were recorded isometrically using force transducer meters Rmax (maximal relaxant response) and IC50 (the concentration
(LCM Systems Ltd, Erma, Tokyo). Aortic rings were mounted on two of AEPC dose required to give 50% of the maximum aortic-relaxant
stainless steel hooks, one fixed to the bottom of the chamber and the response). Differences were considered to be statistically significant
other to a UF1 force transducer connected to a PowerLab/400 when p < 0.05.
ADInstrument data acquisition system (Harvard, Boyer, Casablanca,
Morocco) in order to record signal corresponding to the isometric 3. Results
tension. The study was performed in endothelium intact aorta. In ex-
periment with endothelium intact aorta the integrity of endothelium 3.1. Effect of AEPC on body weight
was confirmed if acetylcholine (10 μM) could cause relaxation (40 or
60%) of aorta precontracted with Epinephrine (EP; 10–5 M) (Désiré Oral administration of AEPC (160 mg/kg) during 7 days of the sub-
et al., 2013). On the other hand, experiments with endothelium de- chronic test did not cause any significant change in body weight, either
nuded aorta, the endothelium was removed mechanically by gently for normal or hypertensive groups. The same result was observed in
rubbing the luminal surface with a wooden object before mounting it in control group treated by lasilix as compared to control untreated (data
the tissue bath chamber. The endothelium removal was checked by not shown).
acetylcholine with failed to produced relaxation of EP induced con-
tractions.
3.2. Hypotensive activity
Optimal tension selected from preliminary experiments was the
tension which gave the greatest response to 10−5 M EP. The rings were
3.2.1. Single oral administration
given 2 g (100%) of initial tension and allowed to equilibrate for 1 h.
Daily administration of L-NAME increased systolic blood pressure
Thirty minutes after setting up the tissue bath aortas rings were con-
levels in rats when compared to the control group. Table 1 represents
tracted with 10−5 M EP or 80 mM potassium chloride (KCl) solution to
the effect of the aqueous extract of aerial part of P. crispum on blood
test their contractile responses. After exposure to 10−5 M EP tissues
pressure levels on both normal and L-NAME-hypertensive rats after a
were washed three times with Krebs solution to restore basal tension
single oral administration (6 h of treatment) of the P. crispum extract at
and this solution was renewed every 40 min.
a dose of 160 mg/kg. In normal groups no change was observed con-
In order to determine the cumulative dose-dependent curves for
cerning arterial blood pressure levels (systolic, mean and diastolic
AEPC-induced relaxation and after equilibration rings were contracted
by EP (10 μM) and KCl (80 mM). After the plateau was reached AEPC
Table 1
was added cumulatively (0.02–2.5 μg/ml) at each dose of AEPC the
Effect of single oral administration of AEPC (160 mg/kg) on systolic, mean,
curve was allowed to reach a plateau before the addition of subsequent
diastolic arterial blood pressure (mm Hg) and heart rate (bmp) in anesthetized
dose. To determine the mechanistic route involved in the relaxant effect normal and L-NAME-induced hypertensive rats. Data are expressed as
induced by AEPC on EP pre-contracted rat aortic rings, the following means ± SEM, n = 6. *p < 0.05, **p < 0.01, ***p < 0.001,
experiment was carried out: 20 min before adding EP aortic rings were ****p < 0.0001.
pre-incubated for 20 min with one of various standard drugs. Drugs
Systolic blood pressure (mm Hg)
used for pre-incubation were: 1) 10−4 M L-NAME, a direct inhibitor of
nitric oxide (NO) synthase. 2) 10 μM glibenclamide an ATP-sensitive Control AEPC Lasilix
K+ channel blocker. 3) 10 μM indomethacin a prostaglandin synthesis
inhibitor. 4) 10 μM propranolol a beta-blocker. 5) 10 μM methylene Normal t0 132.00 ± 6.00 136.00 ± 6.00 120.00 ± 7.00
t6 138.00 ± 8.00 120.00 ± 13.00 111.00 ± 7.00
blue (MB), a NO-cyclic guanosine monophosphate (cGMP) blocker. 6) L-NAME t0 180.00 ± 5.00 166.00 ± 7.00 169.00 ± 7.00
10 μM nifedipine a calcium channel blocker. After the addition of EP t6 175.00 ± 7.00 138.00 ± 8.00** 142.00 ± 6.00**
(10−5 M) aortic relaxation was carried out by cumulative addition of
AEPC (0.02–2.5 μg/ml). The vasorelaxant effect on the aortic rings was Mean blood pressure (mm Hg)
calculated as a percentage of contraction in response to EP. Before
Control AEPC Lasilix
changing any of the compounds used in this experiment, the aorta ring
was washed three times (aorta ring should be incubated 5 min intervals Normal t0 115.00 ± 7.00 117.00 ± 7.00 108.00 ± 6.00
in each bath). The control was performed by cumulative addition of t6 113.00 ± 6.00 101.00 ± 12.00 97.00 ± 2.00
vehicle (distillated water) (0.02–2.5 μg/ml) to aortic rings pre-con- L-NAME t0 156.00 ± 6.00 137.00 ± 2.00 150.00 ± 3.00
t6 157.00 ± 6.00 108.00 ± 6.00** 125.00 ± 6.00*
tracted by EP (10−5 M) and KCl (80 mM) (Anwar et al., 2017). In order
to determine whether calcium channels are involved in the vasodilator Diastolic blood pressure (mm Hg)
activity of AEPC or not the effect of AEPC (IC50 = 0.380 μg/ml) was
tested after EP or KCl pre-treatment in a buffer containing CaCl2 or Control AEPC Lasilix
Ca2+ free. The contraction response induced by CaCl2 (0.3–10 mM) in
Normal t0 106.00 ± 5.00 105.00 ± 8.00 103.00 ± 5.00
the aortic rings pre-treated by EP (10 μM) or KCl (80 mM) was eval- t6 101.00 ± 6.00 91.00 ± 12.00 91.00 ± 4.00
uated as well as in Ca2+-free KH buffer. The contraction responses in- L-NAME t0 144.00 ± 7.00 122.00 ± 2.00 152.00 ± 5.00
duced by CaCl2 were expressed as percentages in the presence and t6 148.00 ± 10.00 93.00 ± 6.00** 129.00 ± 4.00*
absence of AEPC pre-treatment (Kim et al., 2017).
Heart rate (bpm)

2.5. Statistical analysis Control AEPC Lasilix

All the values ware expressed as mean ± SEM. Statistical sig- Normal t0 320.00 ± 10.00 332.00 ± 15.00 349.00 ± 19.00
t6 306.00 ± 8.00 328.00 ± 17.00 321.00 ± 9.00
nificance was tested between more than two groups using two-way
L-NAME t0 312.00 ± 15.00 325.00 ± 15.00 348.00 ± 22.00
ANOVA followed by the Bonferroni multiple comparisons test. t6 318.00 ± 12.00 318.00 ± 18.00 311.00 ± 17.00
Relaxations induced by AEPC were expressed as a percentage decrease

3
M. Ajebli and M. Eddouks Journal of Ethnopharmacology 242 (2019) 112039

blood pressure) at the end of the acute experiment (6 h after the single
oral administration of AEPC), the same effect was observed for the
standard drug lasilix for normotensive animals. Whereas a significant
reduction of systolic, mean and diastolic blood pressure (p < 0.01) was
observed at the end of the experiment (after 6 h of treatment) in hy-
pertensive rats treated by AEPC in comparison with baseline values
(T0). Lasilix also provoked a significant reduction of systolic blood
pressure (p < 0.01) mean and diastolic blood pressure after 6 h of oral
administration of the standard drug (p < 0.05).
Table 1 illustrates also the evaluation of heart rate 6 h after a single
oral administration of both AEPC and lasilix. Results showed non-sig-
nificant effect in normal and hypertensive rats concerning this para-
meter.
3.2.2. Repeated oral administration
3.2.2.1. Effect of AEPC on arterial blood pressure and heart rate. In this
sub-chronic assay AEPC was administered orally at a dose of 160 mg/kg
body weight to anesthetized normal and L-NAME-induced hypertension
during one week. Table 2 shows the systolic arterial blood pressure
evolution on normotensive rats after the oral administration of AEPC.
The results show that this aqueous extract provoked a significant
decrease of systolic arterial blood pressure after 2 and 4 days of
treatment (p < 0.01 and p < 0.05 respectively); while for
hypertensive group a significant decrease was observed in the 4th
and 7th days (p < 0.001 and p < 0.01) when compared to the control
group. However standard drug used in this study (lasilix) leads to a fall
on systolic blood pressure in hypertensive rats at the 4th and 7th days
(p < 0.0001) but no change was observed for normotensive rats during
this period. Concerning the mean arterial blood pressure, a significant
reduction was produced at the 2nd and 4th days after repeated oral
administration of AEPC (p < 0.01) in normotensive rats. In
hypertensive rats this effect was interestingly manifested 4 and 7
days after the oral administration (p < 0.001 and 0.01 respectively).
Lasilix caused a severe reduction of MBP at the 4th and 7th day
(p < 0.0001) in hypertensive animals, while no change was noted for
normotensive rats. Similarly a significant diminution of diastolic
arterial blood pressure was observed in normotensive group treated
by AEPC during the sub-chronic period this significant reduction was

Table 2
Effect of repeated oral administration of AEPC (160 mg/kg) on systolic, mean, diastolic arterial blood pressure (mm Hg) and heart rate (bmp) in anesthetized normal
and L-NAME-induced hypertensive rats. Data are expressed as means ± SEM, n = 6. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Systolic blood pressure (mm Hg)

Control AEPC Lasilix

Normal D0 134.00 ± 7.00 138.00 ± 6.00 120.00 ± 7.00

D2 136.00 ± 5.00 112.00 ± 6.00 115.00 ± 5.00
D4 140.00 ± 3.00 111.00 ± 7.00** 108.00 ± 3.00
D7 135.00 ± 6.00 120.00 ± 4.00** 99.00 ± 4.00*
L-NAME D0 180.00 ± 5.00 166.00 ± 3.00 169.00 ± 6.00
D2 176.00 ± 5.00 152.00 ± 3.00 122.00 ± 2.00****
D4 173.00 ± 9.00 132.00 ± 4.00**** 120.00 ± 4.00****
D7 172.00 ± 6.00 118.00 ± 7.00**** 109.00 ± 3.00****

Mean blood pressure (mm Hg)

Control AEPC Lasilix

Normal D0 111.00 ± 4.00 115.00 ± 7.00 109.00 ± 7.00

D2 112.00 ± 5.00 88.00 ± 6.00** 107.00 ± 5.00
D4 117.00 ± 6.00 92.00 ± 7.00** 101.00 ± 3.00
D7 112.00 ± 4.00 99.00 ± 5.00 96.00 ± 2.00
L-NAME D0 156.00 ± 6.00 137.00 ± 3.00 150.00 ± 3.00
D2 149.00 ± 6.00 129.00 ± 3.00 148.00 ± 5.00
D4 148.00 ± 9.00 106.00 ± 4.00**** 103.00 ± 5.00****
D7 147.00 ± 6.00 102.00 ± 3.00**** 95.00 ± 4.00****

Diastolic blood pressure (mm Hg)

Control AEPC Lasilix

Normal D0 100.00 ± 3.00 103.00 ± 8.00 103.00 ± 6.00

D2 101.00 ± 5.00 76.00 ± 7.00 98.00 ± 5.00
D4 106.00 ± 3.00 82.00 ± 6.00** 97.00 ± 6.00
D7 102.00 ± 8.00 89.00 ± 6.00* 95.00 ± 4.00
L-NAME D0 144.00 ± 7.00 122.00 ± 2.00 152.00 ± 5.00
D2 135.00 ± 7.00 118.00 ± 3.00 103.00 ± 4.00
D4 135.00 ± 6.00 93.00 ± 4.00*** 100.00 ± 4.00****
D7 136.00 ± 6.00 94.00 ± 7.00** 98.00 ± 3.00****

Heart rate (bpm)

Control AEPC Lasilix

Normal D0 312.00 ± 15.00 325.00 ± 15.00 348.00 ± 22.00

D2 320.00 ± 14.00 305.00 ± 14.00 341.00 ± 16.00
D4 312.00 ± 16.00 310.00 ± 16.00 341.00 ± 17.00
D7 319.00 ± 8.00 320.00 ± 6.00 305.00 ± 8.00*
L-NAME D0 308.00 ± 12.00 310.00 ± 15.00 348.00 ± 15.00
D2 310.00 ± 15.00 314.00 ± 10.00 343.00 ± 13.00
D4 307.00 ± 13.00 312.00 ± 12.00 315.00 ± 11.00
D7 318.00 ± 18.00 309.00 ± 16.00 311.00 ± 10.00

4
M. Ajebli and M. Eddouks
Fig. 1. Effect of AEPC extract on EP or KCl-induced contraction in aortic rings. Cumulative dose-dependent curves for AEPC-induced relaxation and of the vehicle
(distillated water) in rat aortic rings. n = 6. A: pre-contraction with epinephrine (10 μM); B: pre-contraction KCl (80 mM). *p < 0.05, ****p < 0.0001.
marked at the 2nd and 4th days (p < 0.01 and p < 0.05 respectively).
In addition, AEPC revealed a significant reduction of DBP at the 4th and
the 7th of sub-chronic study (p < 0.0001 and 0.001 respectively) in
hypertensive rats. Lasilix has led to a fall on DBP for the last group at
the 4th and 7th day while no change was revealed for normotensive
treated with this reference drug.
HR did not change after repeated oral administration of AEPC in
both normal and hypertensive rats during the sub-chronic period. The
same result was noticed for lasilix except for normotensive rats at the
7th day in which a decrease on HR was observed (p < 0.05).
3.3. Vasorelaxant activity
3.3.1. Effect of AEPC on relaxation of rat isolated aortic rings
In this series of experiments the vasorelaxant effect of AEPC on
vascular smooth muscle was tested using rat aortic rings. As illustrated
in Figs. 1 and 2 AEPC-induced relaxation in a dose-dependent manner
for vessels. In aortic rings pre-contracted by epinephrine a significant
decrease was observed (p < 0.0001) in the vasorelaxation produced by
AEPC from a dose of 0.02–2.5 μg/ml (Fig. 1A). The relevant parameter
values are IC50 = 0.38 ± 0.07 μg/ml with 95% confidence interval:
0.194–2.122 μg/ml. Similarly, Rmax was 133.60 ± 8.67%
5
Journal of Ethnopharmacology 242 (2019) 112039
(p < 0.0001). Whereas in aortic rings pre-contracted by KCl (Fig. 1B) a
significant decrease from p < 0.05 to p < 0.0001 was noticed. The
appropriate parameter values are IC50 = 0.56 ± 0.09 μg/ml with 95%
confidence interval: 0.38–0.53 μg/ml. Similarly, Rmax was
125.80 ± 6.13% (p < 0.0001). In both endothelium-intact and en-
dothelium denuded aortic rings, AEPC promoted the vasorelaxant effect
with similar Rmax values (144.10 ± 10.49 and 135.20 ± 17.20%
respectively); In addition, no significant difference was found between
the IC50 values (0.39 ± 0.06 and 0.36 ± 0.09 μg/ml, respectively)
(Figs. 3 and 4).
3.3.2. Aortic responses to AEPC in the absence and presence of L-NAME or
indomethacin
To establish if nitric oxide (NO) participates to the AEPC-induced
relaxation the aortic rings were exposed to L-NAME. In L-NAME-treated
aortic rings the vasorelaxant effect of AEPC was potentiated (p < 0.01)
(Fig. 5). In vehicle-treated rings the values were:
IC50 = 0.38 ± 0.07 μg/ml with 95% confidence interval:
0.194–2.122 μg/ml, while in L-NAME-treated aortas, the data were:
IC50 = 0.024 ± 0.06 μg/ml with 95% confidence interval:
0.019–0.053 μg/ml. On the other hand, the vasorelaxant effect of AEPC
on the prostacyclin pathway were examined in aortic rings pre-
Fig. 2. Representative tracings illustrating the re-
laxation response produced by the application of
AEPC on epinephrine and KCl-precontracted rat
aortic rings. AEPC produced a dose-dependent va-
sorelaxant response in epinephrine-precontracted rat
aortic rings (A); 1: addition of epinephrine, 2 to 9:
addition to different doses of AEPC (0.02–2.5 μg/ml)
and KCl-precontracted rat aortic rings (B); 1: addi-
tion of KCl, 2 to 9: addition to different doses of
AEPC (0.02–2.5 μg/ml).
M. Ajebli and M. Eddouks Journal of Ethnopharmacology 242 (2019) 112039

activated potassium channels caused no effect on relaxant responses to

AEPC. Indeed, in vehicle-treated rings the values were:
IC50 = 0.38 ± 0.07 μg/ml with 95% confidence interval:
0.194–2.122 μg/ml. The following results were obtained with the same
rings pre-treated with glibenclamide an ATP-sensitive potassium
channels blocker where IC50 was 0.27 ± 0.12 with 95% confidence
interval of 0.15–0.37 μg/ml. Whereas Rmax was 133.60 ± 8.67% for
inhibitor-independent relaxation and 167.60 ± 6.87% relaxation in
the presence of glibenclamide (p > 0.05) (Fig. 6A). The results showed
also that the effect of propranolol did not affect the AEPC activity
(Fig. 6B). In vehicle-treated rings the values were: IC50 = 0.38 ±
0.07 μg/ml with 95% confidence interval: 0.194–2.122 μg/ml whereas
AEPC-pretreatment of the rings produced an IC50 value of
0.087 ± 0.10 with 95% confidence interval of 0.01–0.23 μg/ml. Si-
milarly, Rmax was 132.40 ± 10.77% for inhibitor-independent relaxa-
tion and 136.60 ± 20.13% of relaxation in the presence of propra-
nolol. These results show clearly that the vasorelaxing ability of AEPC
has not been affected by the presence of glibenclamide or propranolol.

3.3.4. Involvement of NO-cyclic guanosine monophosphate (cGMP) and

calcium channel blocker pathway
In this experiment the results showed that methylene blue (MB), a
NO-cyclic guanosine monophosphate (cGMP) blocker, did not alter
Fig. 3. Effect of AEPC on tension in EP (10 μM) pre-contracted aortic rings with
relaxant responses of rat aortic rings in response to AEPC (p > 0.05).
endothelium (E+) or without endothelium (E−). Tension (%) indicates the
In AEPC-treated rings, the values were: IC50 = 0.38 ± 0.07 μg/ml with
percentage of EP-induced contraction. Values are expressed as the
mean ± SEM. 95% confidence interval: 0.194–2.122 μg/ml. While in Nifedipine-
treated rings the values were: IC50 = 0.403 ± 4.478 μg/ml with 95%
confidence interval: 0.329–0.496 μg/ml. Similarly Rmax was
incubated with indomethacin (1 μM) for 15 min before EP (10 μM) pre-
132.40 ± 10.77% for untreated relaxation and 133.60 ± 7.21% for
contraction. IC50 was 0.145 ± 0.28 with 95% confidence interval of
MB-exposed relaxation (Fig. 7A). In addition, the results showed that
0.012–0.242 μg/ml (Fig. 5A). Likewise, Rmax was 132.40 ± 10.77% or
nifedipine, a calcium channel blocker, did not significantly alter re-
89.5 ± 11.5% in the presence of indomethacin (Fig. 5B); this result
laxant responses of rat aortic rings in response to AEPC (p > 0.05). In
suggested that the vasorelaxant effect of AEPC is independent of vas-
AEPC-treated rings the values were: IC50 = 0.38 ± 0.07 μg/ml with
cular prostacyclin pathway. These results indicate that L-NAME a NO
95% confidence interval: 0.194–2.122 μg/ml. While in nifedipine-
inhibitor potentiated the vasorelaxant effect of AEPC while in-
treated rings the values were IC50 = 0.36 ± 0.20 μg/ml with 95%
domethacin, a prostaglandin synthesis inhibitor, did not significantly
confidence interval: 0.21–2.71 μg/ml. Similarly, Rmax was
alter relaxant responses of rat aortic rings to AEPC.
132.40 ± 10.77% for untreated relaxation and 146.80 ± 19.18% for
nifedipine-exposed relaxation (Fig. 7B). These results show that MB and
3.3.3. Role of potassium channels and β-adrenergic receptors nifedipine did not alter vasodilator activity of AEPC.
Interestingly, prior inhibition of ATP-dependent or calcium-

Fig. 4. Representative tracings illustrating

the vasorelaxant effect of acetylcholine
(ACh) (10 μM) on endothelium-intact (A)
and endothelium-denuded (B) preparations
of rat aortic rings, precontracted with EP
(10 μM). And concentration–response
curves showing the vasorelaxant effect of
AEPC in endothelium-intact (C) and en-
dothelium-denuded (D) preparations of rat
aortic rings, precontracted with EP (10 μM).
1 to 10: addition to different doses of AEPC
(0.02–2.5 μg/ml).

6
M. Ajebli and M. Eddouks Journal of Ethnopharmacology 242 (2019) 112039

Fig. 5. Effect of different drugs incubation on AEPC- relaxation of EP-induced contraction in aortic rings. Aortic rings were incubated with cumulative doses of AEPC in the
absence (circles) or presence (squares) of the following drugs: (A) L-NAME, (B) Indomethacin, Data represent mean ± SEM (n = 6) for AEPC versus drug plus AEPC.

3.3.5. Effect of AEPC on extracellular Ca2+-induced contraction (Figs. 8 and 9). These results suggest that AEPC significantly inhibited
The vasorelaxant effect of AEPC (IC50 = 0.380 μg/ml) on extra- the entry of extracellular Ca2+.
cellular Ca2+-induced contractions by EP or KCl pre-treatment was
tested. We examined the contraction response induced by calcium
4. Discussion
chloride (CaCl2, 0.3–10 mM) in aortic rings by EP (10 μM) or KCl
(80 mM) pre-contraction in Ca2+-free KH buffer, with and without
Petroselinum crispum is an aromatic and medicinal plant which has
(control) AEPC preincubation for 15 min. Compared to the control, the
been used from centuries both for nutritional as well as medicinal
contraction responses induced by CaCl2 were calculated as a percentage
purposes. This herb is used for managing a wide range of illnesses in-
in the presence and absence (control) of AEPC pre-treatment. In Ca2+-
cluding hypertension. High blood pressure is a risk factor for coronary
free KH buffer the cumulative addition of CaCl2 (0.3–10 mM) induced
heart disease, stroke and renal vascular disease (Patel et al., 2012). This
progressively increased tension in the rat thoracic aorta rings (Fig. 6).
study was carried out to assess the blood pressure lowering effect of the
As shown in Fig. 6, AEPC (IC50 = 0.380 μg/ml) pre-incubation sig-
aqueous extract of Petroselinum crispum on normotensive and L-NAME-
nificantly inhibited the contractions induced by extracellular CaCl2
induced hypertensive rats. Oral administration of Nω-nitro-L-arginine
(0.3–10 mM) in both aortic rings pre-contracted by EP (Fig. 8A) and
methyl ester (L-NAME) during one week led to induce hypertension in
those pre-contracted by KCl (Fig. 8B). However, it is well noted that the
male Wistar rats. This compound is used as an inhibitor of nitric oxide
inhibition provoked by AEPC in EP-precontracted aortas was more
synthase (NOS) activity both in vitro and in vivo. Acute and chronic L-
pronounced than the inhibition caused in KCl-pre-contracted aortas
NAME treatment caused a change in blood pressure and vascular

Fig. 6. Effect of different drugs incubation on AEPC-

relaxation of EP-induced contraction in aortic rings.
Aortic rings were incubated with cumulative doses of
AEPC in the absence (circles) or presence (squares)
of the following drugs: (A) Glibenclamide, (B)
Propranolol. Data represent mean ± SEM (n = 6)
for AEPC versus drug plus AEPC.

7
M. Ajebli and M. Eddouks Journal of Ethnopharmacology 242 (2019) 112039

Fig. 7. Effect of methylene blue (MB) drug incubation on AEPC- relaxation of EP-induced contraction in aortic rings. Aortic rings were incubated with cumulative
doses of AEPC in the absence (circles) or presence (squares) of the drug. Data represent mean ± SEM (n = 6; for AEPC versus drug plus AEPC).

Fig. 8. Inhibitory effect of AEPC (IC50 μg/mL) on the

contraction induced by extracellular Ca2+ in rat
thoracic aorta rings that were pre-contracted with EP
(10 μM) (A) or KCl (80 mM) (B) in the presence
(squares) or absence (circles) of AEPC. Values are
expressed as mean ± SEM (n = 6). ***p < 0.001,
****p < 0.0001 vs. control.

Fig. 9. Representative tracings showing in-

hibitory effect of AEPC (IC50 0.3 μg/m L)
on the contraction induced by extracellular
Ca2+ addition (0.3–10 mM) in rat aortic
rings pre-contracted with epinephrine (NP,
10 μm) (A); 1: addition of AEPC, 2: addition
of epinephrine, 3 to 7: addition of Ca2+
concentrations and KCl (80 mM) (B); 1: ad-
dition of AEPC, 2: addition of KCl, 3 to 7:
addition of different concentrations of Ca2+
in Ca2+-free Krebs-Henseleit solution.
Illustrations C and D represent controls
(addition of extracellular Ca2+ concentra-
tions in aortic rings after pre-contraction by
epinephrine and KCl respectively without
pre-incubation in AEPC).

8
M. Ajebli and M. Eddouks
reactivity due to decreased nitric oxide (NO) bioavailability (Kopincová
et al., 2012). Lasilix was used in this study as a standard drug. This
synthetic drug is usually used to treat high blood pressure. Interest-
ingly, lowering high blood pressure helps to prevent strokes, heart at-
tacks and kidney problems. Lasilix, like other loop diuretics, is known
to act by inhibiting the luminal Na-K-Cl cotransporter in the thick as-
cending limb of the loop of Henle, by binding to the chloride transport
channel, thus causing sodium, chloride, and potassium loss in urine.
P. crispum is one of the important spices and flavoring embellishing
Moroccan and Mediterranean cuisine. Although P. crispum is commonly
used in traditional Moroccan medicine as antihypertensive therapy, the
pharmacological evidences of its antihypertensive activity are lacking.
The present study demonstrates that AEPC produced a significant re-
duction in blood pressure parameters (SBP, MBP and DBP) in normal
and L-NAME-hypertensive rats following a repeated oral administration
of this extract. In contrast, a single oral administration of AEPC led to a
lowering effect in SBP, MBP and DBP in hypertensive rats without af-
fecting arterial blood pressure parameters for normotensive rats. It
seems that the spectacular effect of AEPC was recorded in mean arterial
blood pressure of L-NAME hypertensive rats 4 and 7 days after the oral
administration of this extract with a maximum of reduction, which
reached 35 and 31 mm Hg respectively (p < 0.0001). The aqueous
extract was found more efficacious in lowering arterial blood pressure
in hypertensive than in normotensive rats. Several previous studies
reported that secondary metabolites of medicinal herbs are capable to
exert antihypertensive effects by different pathways (Rawat et al.,
2016).
In order to seek one of the possible mechanistic actions by which
this medicinal herb elicits its antihypertensive effect, an in vitro study
was designed to evaluate the vasorelaxant activity on aortic rings iso-
lated from Wistar rats. The second part of the present study reports the
vasorelaxant action of AEPC on the aortic rings at the accumulative
concentrations ranging between 0.020 μg/ml and 2.5 μg/ml. This in-
vestigation demonstrated that AEPC was able to act in aortic rings
producing a concentration-dependent depressant effect on KCl- and EP-
induced contractions. In addition, it provides evidence that AEPC de-
creases tension in both endothelium-intact and endothelium-denuded
aortic rings. In our test, the functional removal of endothelium did not
significantly attenuate the AEPC-induced relaxation in aortic rings
precontracted with EP, thus suggesting that the vasorelaxant effect of
AEPC was endothelium-independent, suggesting also that its me-
chanism did not involve the presence of endothelium-derived relaxing
factor (EDRF) in vascular endothelium. In fact, endothelial function is
associated with a multitude of physiological processes that maintain
healthy homeostasis of the vascular wall. Furthermore, exposure of the
endothelium to cardiac risk factors results in endothelial dysfunction
and it is associated with an alteration in the balance of vasoactive
substances produced by endothelial cells (Muhiddin and Arshed, 2011).
Six reference drugs were used in this experiment in order to explore
the pathway involved in the vasorelaxing action of AEPC (L-Name,
Indomethacin, Methylene blue, Indomethacin, Glibenclamide and
Nifedipine). The present study showed that the six reference drugs used
had no effect on the vasorelaxant effect of AEPC except the potentiating
effect of L-NAME on the vasorelaxant effect of AEPC. This finding in-
dicates that the vasorelaxant effect of AEPC was not mediated through
the direct NO pathway, NO-cGMP pathway, vascular cyclooxygenase
pathway, β-adrenergic receptors pathway, K+ channels pathway or L-
type calcium channels pathway.
In order to determine if calcium channels incorporated into the
vascular smooth muscle cells are involved in this vasodilatation ac-
tivity, the vasorelaxant effect of AEPC (IC50 = 0.380 μg/mL) on ex-
tracellular Ca2+-induced contractions was examined after EP or KCl
9
Journal of Ethnopharmacology 242 (2019) 112039
pre-treatment respectively. The contraction response induced by CaCl2
was evaluated in the aortic rings after EP or KCl pre-contraction in
Ca2+-free KH buffer with and without AEPC preincubation. Smooth
muscle is known to contract in response to the activation of voltage-
dependent and receptor-operated Ca2+ channels (Rüegg et al., 1989;
Horowitz et al., 1996; Taggart et al., 1997; Karaki et al., 1997). Find-
ings of the last assay demonstrated that AEPC relaxed the aortic rings
by blocking the entry of extracellular Ca2+. AEPC inhibited the EP
(10−5 M) and less intensely the KCl (80 mM)-induced contractions.
These results are consistent with a previous study which has demon-
strated that hydroalcoholic extract of parsley seeds is responsible of the
relaxation in isolated adult male Wistar rat's ileum by blocking of vol-
tage-gated calcium channels (Moazedi et al., 2007). In the presence of
EP the CaCl2-induced contractions were significantly altered at the
CaCl2 concentration of 10 mM; while, in the same condition, the con-
traction was decreased in the presence of KCl, when compared to the
control groups. EP induced the influx of extracellular Ca2+ (Yu and
Bose, 1991). Thus, the present study demonstrates that AEPC lowers
blood pressure by a direct effect on the vascular smooth muscle leading
to vasodilation via blocking Ca2+ channels. Many studies have reported
the vasorelaxing ability via inhibition of Ca2+ channels of a number of
medicinal plants and their derivatives. Accordingly, an essential oil
(citronellol) constituent from the medicinal plants Cymbopogon citratus,
Cymbopogon winterianus and Lippia alba was demonstrated to possess
antihypertensive properties by its effect on Ca2+ channels leading to a
smooth muscle vasodilation (Zhang et al., 2010). As it has been de-
monstrated previously, a similar species (Petroselium sativum) exhibited
hypotensive and diuretic effects in normal rats (Campos et al., 2009).
Similarly, P. crispum extract could lower blood pressure via the stimu-
lation of diuresis. The possible pathway implicated by AEPC for its
hypotensive activity is schematically represented in Fig. 10. In the
current study, it was revealed that the vasorelaxant effect of AEPC in
presence of L-NAME (Fig. 5A) has been significantly and unusually
potentialized. In fact, L-NAME is known as an inhibitor of nitric oxide
synthase (NOS). Accordingly, the presence of this drug in contact with
the aorta leads to a vasoconstriction. However, when L-NAME was
added before the application of different doses of AEPC has led to po-
tentiating the vasodilator effect of our extract. In the same context, it
was demonstrated that low doses of L-NAME enhanced endothelium-
dependent vasorelaxation and reduced vasoconstriction of the femoral
artery (Bernatova et al., 2007). Additional mechanistic and analytic
studies are necessary to explain this potentiating effect of L-NAME at
low doses in the presence of P. crispum extract.
Finally, the role of parsley (P. crispum) in managing hypertension
can still be defined more clearly with other appropriate studies.
5. Conclusion
In the current study, the effect of the aqueous P. crispum extract on
blood pressure was investigated in L-NAME and normotensive as well as
its effect on vasorelaxant activity in vascular aortic rings. The study
showed that the aqueous P. crispum extract induced a decrease of blood
pressure parameters in both L-NAME and normotensive rats. In addi-
tion, the extract induced a pronounced vasorelaxation response in
vascular aortic segments of rats and this hypotensive ability is at least
related to relaxation of the aortic rings through blockage of Ca2+ entry.
Furthermore, AEPC induces vasorelaxation via an endothelium-in-
dependent pathway. The study demonstrates also that the vasorelaxant
effect of AEPC was not mediated through NO-cGMP pathway, vascular
cyclooxygenase pathway, β-adrenergic receptors pathway, K+ chan-
nels pathway or L-type calcium channels pathway. The anti-
hypertensive activity of the AEPC may be through multiple pathways,
M. Ajebli and M. Eddouks
Fig. 10. Schematic illustration of the suggested mechanism of action involved by AEPC bioactive component (s) in the vasorelaxant and thereafter the hypotensive
activity. VOCC: Voltage Operated Calcium Channels; ROCC: Voltage Operated calcium Channels.
including increase synthesis of Nitric Oxide, inhibition of Ca2+ entry
channels such as voltage-operated Ca2+ channels (VOCC) and receptor-
operated Ca2+ channels (ROCC). Since the AEPC causes dose-depen-
dent relaxation of both EP and KCl-induced contraction, the most
probable mode of action is through ROCC and VOCC (Fig. 10). The
results confirm and support the potential for AEPC as antihypertensive
agent. Other phytochemical and pre-clinical studies are required to
determine the active phytocompounds involved in this antihypertensive
activity.
Funding
This study was funded by National Center of Scientific Research and
Technology. Morocco (grant number PPR/2015/35).
Conflicts of interest
Authors have no conflict of interest.
Ethical approval
All applicable guidelines for the care and use of animals were fol-
lowed (FSTE/2015).
Author's contributions
Mohamed Eddouks, designed and supervised the study. He con-
tributed to the writing of the manuscript. Mohammed Ajebli realized
the experiments.
Author's information
ME, Pharmacology, Ph.D., Professor.
10
Journal of Ethnopharmacology 242 (2019) 112039
MA, Pharmacology, Phytochemistry, Ph.D.
ME is supervisor of the PhD thesis of MA among the Team of
Physiology and Endocrine Pharmacology at Faculty of Sciences and
Techniques Errachidia. The objective of the team is to identify plants of
interest by ethnobotanical surveys, evaluating their pharmacological
activity and active molecules. The main aimed pathologies are diabetes,
hypertension and obesity.
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