Hindawi
BioMed Research International
Volume 2021, Article ID 4730341, 14 pages
https://doi.org/10.1155/2021/4730341
Research Article
Vasorelaxant-Mediated Antihypertensive Effect of the Leaf
Aqueous Extract from Stephania abyssinica (Dillon & A. Rich)
Walp (Menispermaceae) in Rat
Chamberlin Fodem , Elvine Pami Nguelefack-Mbuyo , Magloire Kanyou Ndjenda II ,
Albert Kamanyi , and Télesphore Benoit Nguelefack
Research Unit of Neuro-Inflammation and Cardiovascular Pharmacology, Faculty of Science, University of Dschang,
P.O. Box 67 Dschang, Cameroon
Correspondence should be addressed to Télesphore Benoit Nguelefack; nguelefack@yahoo.fr
Received 13 July 2021; Accepted 17 September 2021; Published 8 October 2021
Academic Editor: Valeria Pasciu
Copyright © 2021 Chamberlin Fodem et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Stephania abyssinica is a medicinal plant used in Cameroon alternative medicine to treat arterial hypertension (AHT). Previous
in vitro studies demonstrated the endothelium nitric oxide-independent vasorelaxant property of the aqueous extract from
Stephania abyssinica (AESA). But its effect on AHT is unknown. The present study was undertaken to explore other
vasorelaxant mechanisms and to determine the antihypertensive effects of AESA in male Wistar rats. Phytochemical analysis of
AESA was carried out using the liquid chromatography-mass spectrometry (LC-MS) method. The vasorelaxant effects of AESA
(1-1000 μg/mL) were studied on rat isolated thoracic aorta rings, in the absence or presence of indomethacin (10 μM) or
methylene blue (10 μM). The inhibitory effect of AESA on phenylephrine (PE, 10 μM) or KCl- (60 mM) induced contraction as
well as the intracellular calcium release was also evaluated. The in vivo antihypertensive activity of AESA (43, 86, or
172 mg/kg/day) or captopril (20 mg/kg/day) administered orally was assessed in L-NAME- (40 mg/kg/day) treated rats. Blood
pressure and heart rate (HR) were measured at the end of each week while serum or urinary nitric oxide (NO), creatinine, and
glomerular filtration rate (GFR) were determined at the end of the 6 weeks of treatment, as well as histological analysis of the
heart and the kidney. The LC-MS profiling of AESA identified 9 compounds including 7 alkaloids. AESA produced a
concentration-dependent relaxation on contraction induced either by PE and KCl, which was significantly reduced in
endothelium-denuded vessels, as well as in vessels pretreated with indomethacin and methylene blue. Moreover, AESA
inhibited the intracellular Ca2+ release-induced contraction. In vivo, AESA reduced the AHT, heart rate (HR), and ventricular
hypertrophy and increased serum NO, urine creatinine, and GFR. AESA also ameliorated heart and kidney lesions as
compared to the L-NAME group. These findings supported the use of AESA as a potential antihypertensive drug.
1. Introduction
Cardiovascular diseases (CVDs) represent the first cause of
death globally and account for approximately 18.6 million
deaths each year making nearly 32% of all global deaths
worldwide [1, 2]. Of these deaths, 10.8 million (19.2% of all
deaths) are due to complications of arterial hypertension
[3]. Arterial hypertension (AHT) affects 31.1% of adults
(1.39 billion) worldwide [4]. It is the leading cause of CVDs
and then responsible for 13% of premature deaths in devel-
oped and developing countries [5]. Recently, the definition
of AHT has shifted from 140/90 mmHg to 130/80 mmHg
for systolic/diastolic blood pressure, making almost half of
the adult population hypertensive [6]. Although efforts have
been made to reduce the burden of AHT, more and more
people are diagnosed with AHT with a drastic increase in
low- and middle-income countries including sub-Saharan
African countries. AHT is, therefore, a real public health
challenge. In sub-Saharan Africa, the prevalence of AHT
has reached 25.4% and it is projected that by the year 2030,
2
a 66% increase in AHT prevalence will be recorded if adequate measures are not taken [7]. AHT is particularly
severe in African descent in whom a rapid onset, poor
control, and early end-stage organ damage are observed
[8, 9]. Poor AHT control leads to drastic outcomes including kidney failure, coronary heart disease, atherosclerosis,
myocardial infarction, stroke, blindness and premature
mortality, and disability [10, 11].
The main feature of essential hypertension which makes
up to 90 to 95% of all AHT cases [12, 13] is increased vascular resistance due to imbalance between vasoconstricting
and vasodilating substances produced by the endothelium
known as endothelial dysfunction [14–16]. Thus, targeting
endothelial dysfunction appeared as a good therapeutic
option. Endothelial dysfunction can be induced experimentally by blocking the production of nitric oxide (NO), the
main endothelial vasodilating factor using Nω-Nitro-L-Arginine Methyl Ester (L-NAME). This model is well accepted by
the scientific community as it mimics hypertension in
humans [17, 18]. Chronic administration of L-NAME has
been associated with structural, functional, and biochemical
alterations at the level of the heart, aorta, and kidney [18–20].
Although many antihypertensive drugs have been manufactured, the effective control of AHT is poorly achieved
in patients from low- and middle-income countries due to
several limitations such as resistance to therapy, inaccessibility, toxicity, high cost, and low compliance [21–23].
The search for new drugs, especially biological active compounds from natural sources, is of great interest for the development of low cost, more efficient, lesser side effect, and
better-tolerated medication. Complementary and alternative
therapies or pharmacological validation of ethnomedical
medicine could greatly benefit patients in poor economic
situations [24, 25].
Natural substances with vasorelaxant activities have
been the focus of studies in the last decades and are recognized for their efficacy in preventing and treating hypertension [24, 26]. Stephania abyssinica (Dillon & A. Rich) Walp
(Menispermaceae) is a twining liana rich in bioactive alkaloids, flavonoids, lignans, steroids, terpenoids, and coumarins [27–29].
The plant is widely used in African folk medicine for the
treatment of various ailments including asthma, hyperglycemia, sleep disturbances, inflammation, men impotence, miscarriage, and liver diseases [28, 30–34]. S. abyssinica is also
used to treat heart complaints [35], and in the West region
of Cameroon, the aqueous extract from the fresh leaves of
S. abyssinica is administered orally for the management of
cardiovascular disorders including arterial hypertension.
Previous in vitro studies demonstrated that the aqueous
extract from S. abyssinica possesses endothelium nitric
oxide-independent vasorelaxant effects [14]. However, other
vasorelaxant mechanisms were still to be determined as well
as the effect of S. abyssinica extract on the cardiovascular
system. Therefore, the purpose of this study was to explore
the antihypertensive properties of the aqueous extract from
S. abyssinica in the L-NAME-induced hypertensive rat
model and determine other vasorelaxant mechanisms and
the protecting effect of the extract on selected target organs,
BioMed Research International
assuming that this might contribute to the antihypertensive
effect of the plant extract.
2. Materials and Methods
2.1. Drugs and Chemicals. Calcium chloride (CaCl2), N-(1naphthyl)-ethylenediamine dihydrochloride (NED), magnesium sulfate (MgSO4), potassium chloride (KCl), sodium
hydrogenocarbonate (NaHCO3), magnesium chloride (MgCl2),
and methylene blue were purchased from MERCK (Germany).
Sodium chloride (NaCl), dihydrogen phosphate (H2PO4), and
D(+)-glucose were purchased from ROTH (Germany). Ethylenediaminetetraacetic acid (EDTA) was purchased from Fluka
Chemika (Switzerland). Disodium hydrogen phosphate
(Na2HPO4) was provided by Riedel de Haën AG. L-NAME,
indomethacin, carbachol, creatinine, captopril, and phenylephrine were purchased from Sigma-Aldrich (Taufkirchen,
Germany).
2.2. Plant Material and Extraction. The plant material collected in the West of Cameroon (Foréké-Dschang) in April
2018 was identified in Cameroon National Herbarium under
the voucher specimen number 542/HNC. The aqueous
extract was prepared using the protocol previously described
by [14]. Fresh leaves of S. abyssinica (1 kg) were crushed
twice in 2.5 L distilled water giving 5 L of solution. The solution obtained was filtered with Whatman No. 3 filter paper
and lyophilized. This process yielded 47.4 g (4.74%) of dry
powder which was stored at 4°C until use. For the in vitro
experimentations, the powder (700 mg) was dissolved in
10 mL of distilled water to give a stock solution (70 mg/mL).
For the in vivo experimentations, the powder (0.172 g) was
dissolved in 10 mL distilled water to give a stock solution
(172 mg/kg pc). Further dilution was made from stock solutions as needed.
2.3. Liquid Chromatography-Mass Spectrometry Profiling
of the Stephania abyssinica Leaf Aqueous Extract. The
phytochemical profiling of the aqueous extract from the
leaves of Stephania abyssinica was carried out using liquid
chromatography-mass spectrometry (LC-MS) technique
[36]. The high-resolution mass spectrum was obtained using
a QTOF spectrometer (Bruker, Germany) equipped with a
hot electrospray ionization source. It was set in positive mode
(mass range: 100-1500, with a scan speed of 1.00 Hz) with
automatic gain control to provide high-accuracy mass measurements with a deviation of 0.40 ppm using sodium
formate as a calibrant. A spray voltage of 4.5 kV and a capillary temperature of 200°C were used, with nitrogen as a gas
sheath (10 L/min).
The spectrometer was connected to a UHPLC Ultimate
3000 system (Thermo Fisher, USA) consisting of an LC
pump, an iodine detector array (DAD) (λ: 190-600 nm), an
automatic injector sample (10 μL), and a heating column
(40°C). Separations were performed using Synergi MAXRP 100A (50 × 2 mm; particle size 2.5 μm) with a gradient
of H2O (+0.1% HCOOH) (A)/acetonitrile (+0.1% HCOOH)
(B) (circulation speed 500 μL/min, injection volume 20 μL).
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The sample was analyzed using a gradient programmed
as follows: 95% A isocratic (1.5 minutes) and linear gradient
up to 100% B (6 minutes). After 2 minutes of 100% B
isocratic, the system was returned to its initial condition
(90% A) (1 minute) and was equilibrated for 1 minute.
The raw formulas, obtained from LC-MS, were first
introduced into the PubMed, Google Scholar, and Scopus
search engines to seek publications on the phytochemistry
of Stephania abyssinica and to a certain extent on Menispermaceae. Secondly, the molecular formulas were introduced
into the PubChem and ChemSpider search engines to obtain
different nomenclature corresponding to each of the formulas. The names obtained were entered once again in the
PubMed, Google Scholar, and Scopus engines in combination with the name of the plant or the family. Compounds
that had a correspondence in these publications were identified as such.
2.4. Animal Housing. Wistar rats of both sexes aged 8-10
weeks and weighing 150 to 200 g were randomly selected
from our colony. They were raised in the animal house of
the Faculty of Sciences, University of Dschang, Cameroon,
in plastic cages. Rats were housed 4 per cage and exposed
to a natural light-dark cycle (~12 h/12 h). They were maintained at a room temperature of 22 ± 3° C with free access
to food and water ad libitum. Experimental protocols used
in this study were approved by the Laboratory Committee
(Laboratory of Animal Physiology and Phytopharmacology,
Department of Animal Biology, University of Dschang,
Cameroon) according to the standard ethical guidelines for
laboratory animal use and care as described by the law
2010/63/EU of the European Parliament and of the Council
of 22 September 2010 on the protection of animals used for
scientific purposes.
2.5. Screening of the Vasorelaxant Mechanism of the Aqueous
Extract from S. abyssinica
2.5.1. Aorta Isolation and Mounting. The aorta rings were
prepared as previously described by Nguelefack et al. [14]
and suspended at a resting force of 2 g in an oxygenated
tissue bath (95% O2, 5% CO2) containing 10 mL Krebs
solution, maintained at 37°C and pH 7.4. Changes in force
were recorded isometrically using a force transducer connected to a kymograph with a data acquisition software
(SPELL Advanced Kymograph Data Acquisition software,
MDE Heidelberg). Aortic rings were equilibrated for 60 min
during which the solution was renewed every 15 min.
2.5.2. Elucidation of the Vasorelaxant Mechanisms of the
Aqueous Extract from S. abyssinica. After the equilibration
period, the functional integrity of the endothelium was
assessed as follows: the aortic rings were precontracted with
10 μM phenylephrine (PE), and when the contraction
reached a plateau, carbachol (10-5 M) was added. The presence of endothelium was evidenced by a relaxation of at least
60% and considered destroyed when carbachol induced less
than 10% relaxation. After this, the effect of cumulative
concentrations of the aqueous extract of S. abyssinica (101000 μg/mL) was tested on endothelium-intact aortic rings
3
precontracted with PE or KCl (60 mM) in the presence or
absence of indomethacin (10 μM), a cyclooxygenase inhibitor, and methylene blue (10 μM), a guanylate cyclase inhibitor. Rings were preincubated with each inhibitor for 20
minutes prior to precontraction with PE [14]. To evaluate
the nontoxic effect of AESA, aortic rings were subjected to
KCl or PE contraction after being exposed to the plant
extract.
The effect of AESA on intracellular calcium was investigated as described by Chen et al. [24] with slight modifications. Briefly, after 1-hour stabilization period in normal
Krebs solution, endothelium-denuded aortic rings were
contracted with KCl. When the contraction reaches a plateau, the rings were washed in normal Krebs for 15 minutes
and then transferred into Ca2+-free Krebs (+1 mM EDTA)
for another 15 minutes. PE (10 μM) was then added to
induce the first transient contraction (T1). Following
another washing in normal Krebs for 15 minutes and into
Ca2+-free Krebs for another 15 minutes, rings were then
incubated in the absence or presence of AESA (100 and
300 μg/mL) for 20 minutes before the second transient contraction (T2) induced by PE (10 μM). T1 and T2 were
expressed as a percentage of the contraction induced by
60 mM KCl in normal Krebs, and the ratio of the second
transient contraction to the first (T2/T1) was calculated.
2.6. Evaluation of the Antihypertensive Effect of the Aqueous
Extract of S. abyssinica
2.6.1. Animal Grouping and Dosing. Before the beginning of
the experiment, male rats were acclimatized to the indirect
blood pressure and heart rate recording using the tail-cuff
plethysmography method (IITC Life Science, Woodland
Hills, CA, USA). After measuring the baseline value for blood
pressure and heart rate, rats were randomly assigned to two
lots: lot A (normal control) (n = 8) and lot B (L-NAME
hypertensive rats). Lot A received distilled water (10 mL/kg)
while lot B was chronically administered with L-NAME
(40 mg/kg pc/day) once daily for 3 consecutive weeks. At
the end of the third week, rats of lot A became the normal
control (group 1) and continue receiving distilled water; animals of lot B were distributed into 5 groups (groups 2 to 6)
and treated for 3 other consecutive weeks as follows:
(i) Group 1: normal control rats administered only DW
(ii) Group 2: L-NAME (40 mg/kg/day) + distilled water
(iii) Group 3: L-NAME
(20 mg/kg/day)
(40 mg/kg/day) + captopril
(iv) Group 4: L-NAME
(43 mg/kg/day)
(40 mg/kg/day) + AESA
(v) Group 5: L-NAME
(86 mg/kg/day)
(40 mg/kg/day) + AESA
(vi) Group 6: L-NAME
(172 mg/kg/day)
(40 mg/kg/day) + AESA
The dose applied by the traditional healers was calculated to be 86 mg/kg/day. This dose was then divided and
4
multiplied by two to obtain the other doses. All drugs were
administered by gavage once a day. Body weight, blood pressure, and heart rate were weekly monitored, and at the end
of the experiment, 24 h urine samples were collected, centrifuged at 3000 rpm for 15 min at 4°C (TGL-16M centrifuge,
Loncare), and stored at -20°C for subsequent determination
of nitric oxide (NO), creatinine, and proteins. After urine
collection, animals were anesthetized by intraperitoneal
injection of sodium thiopental (50 mg/kg). Blood samples
were collected from the abdominal artery in heparinized
tubes and centrifuged as described before, and the serum
was collected and kept at -20°C for subsequent NO, creatinine, and protein quantification. The heart, kidney, and
thoracic aorta were isolated, washed in saline, and weighed.
The heart and the kidney were fixed in 10% buffered formalin for histological assessment.
2.6.2. Determination of NO, Creatinine, and Proteins. Serum
and urine NO content was quantified according to a previously described method (Fofié et al. [36]). Serum and urine
creatinine was measured spectrophotometrically using Jaffe’s
reaction method [37]. Serum protein was quantified according to the Biuret method [38] while urinary protein excretion was assessed according to the Bradford method [39].
2.7. Histological Analysis. The heart and kidney were fixed in
10% formalin, embedded in paraffin, cut transversally into
4 μm sections, and stained with hematoxylin and eosin
(H&E). Structural abnormalities were visualized using a light
microscope (DN-107T).
2.8. Statistical Analysis. Results were expressed as mean ±
standard error of the mean (SEM), and the analysis was
performed through GraphPad Prism 8.4.2 (GraphPad,
USA). The differences of continuous variables among various groups were tested using one-way analysis of variance
(ANOVA), followed by post hoc Tukey’s multiple comparison test. Two-way ANOVA repeated measures followed by
the Bonferroni post hoc test were used to analyze data with
two variables. Data on NO release were analyzed with the
Mann-Whitney test. Statistical significance was assigned
for p values of less than 0.05.
3. Results
3.1. Phytochemical Composition of the Aqueous Extract from
the Leaves of S. abyssinica. High-performance liquid chromatography gave a chromatogram showing many peaks
with a retention time ranging 0 and 7 minutes.
The positive mode mass spectrum was used to determine
the crude formula of nine compounds on the basis of ions
and fragment ions corresponding to the peak observed at
each retention time (TR) (Figure 1). Thus, the following
molecular formulas were determined: (1) C4H9NO2 (TR:
0.4 min, m/z: 104.07), (2) C19H15N3O3 (TR: 0.8 min, m/z:
334.11), (3) C21H23N3O2 (TR: 1.2 min, m/z: 334.19), (4)
C24H27N3O3 (TR: 3.1, m/z: 406.21), (5) C24H25N3O3 (TR:
3.2, m/z: 404.19), (6) C28H37NO5 (TR: 3.9 min, m/z:
468.27), (7) C30H39NO5 (TR: 4.3 min, m/z: 494.29), (8)
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C41H36O4 (TR: 5.3 min, m/z: 593.26), and (9) C54H66NO8
(TR: 6.3 min, m/z: 891.49) (Figure 1(a)).
Exception for compounds (1) and (8) which appeared,
respectively, at 0.4- and 5.3-minute retention time, all other
compounds were alkaloids. The positive mode mass spectrum shows an ion at m/z 104.07 ½M + H+ at the retention
time of 0.4 minutes, with fragment ions at m/z 87.00,
138.56, 176.73, and 219.58 corresponding to the compound
of the molecular formula C4H9NO2. After analysis and comparison of the spectra of the crude formulas and of the compounds already isolated from the genus Stephania and the
Menispermaceae family, the compound (1) was identified
as γ-aminobutyric acid (GABA) (Figure 1(b)), given that
derivatives of GABA have been isolated from Stephania
rotunda [40]. Research has not matched any other chemical
formula with already isolated compounds from the genus
Stephania or the Menispermaceae family.
3.2. Vasorelaxant Mechanisms of the Aqueous Extract of S.
abyssinica. The vasorelaxant effects of AESA were already
demonstrated but the endothelial mediators involved in
these effects as well as the participation of the intracellular
calcium pathways were unknown. These sets of experiments
were then undertaken to evaluate these aspects. Aortic rings
with intact endothelium precontracted with KCl or phenylephrine (PE) were significantly and concentration-dependently
relaxed by AESA (10-1000 μg/mL) (Figure 2(a)) with respective
EC50 of 134.70 and 126.00 μg/mL and Emax of 99:72 ± 5:96
and 99:40 ± 1:85%. No significant difference was observed
between the effects of AESA on KCl- and PE-induced contraction (Figure 2(a)). Aortic rings reacted normally to both KCl
and PE after being exposed to extracts and washed.
The endothelium destruction significantly (p < 0:01)
inhibited the AESA-induced relaxation in aortic rings precontracted with PE. EC50 was increased from 126.00 to
285.60 μg/mL while Emax was reduced from 99:40 ± 1:85%
to 65:07 ± 5:11% (Figure 2(b)).
The effects of AESA on PGI2/Cox and cGMP pathways
were investigated by using, respectively, indomethacin and
methylene blue as inhibitors. Results are presented in
Figure 2(c). Pretreatment with indomethacin, a nonselective
cyclooxygenase inhibitor (10 μM), or with methylene blue, a
guanylate cyclase inhibitor (10 μM), significantly (p < 0:001)
reduced the vasorelaxant response to AESA. EC50 increased
from 126 to 1726 μg/mL and 1850 μg/mL while Emax was
reduced from 99:40 ± 1:85% to 42:08 ± 3:68% and 36:94 ±
3:82%, respectively. None of the two substances was able to
completely inhibit the effect of AESA. Surprisingly, the combination of indomethacin and methylene blue rather
enhanced the vasorelaxant activity produced by AESA.
EC50 was reduced from 126.00 to 49.27 μg/mL while Emax
was increased from 99:40 ± 1:85% to 112:25 ± 5:03%.
To determine whether AESA affects intracellular calcium
release, its effect was also investigated on intracellular
calcium release-induced contractions. The addition of PE in
Ca2+-free Krebs evoked a contraction of about 40.14% of
the maximal contraction induced with KCl in normal Krebs.
Preincubation of aorta rings with AESA significantly
(p < 0:001) and dose-dependently reduced the maximal
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Intens.
×107
1.0
5
0.8
0.6
4
1
0.4
0.2
O
9
2
8
7
6
3
NH2
HO
0.0
1
2
3
Blank
Aqueous extract S. abyssinica
4
5
Time (min)
(a)
6
7
8
9
(b)
Figure 1: LC-MS chromatogram of aqueous extract from the leaves of Stephania abyssinica (a) and chemical structure of compound (1) γaminobutyric acid (GABA) (b).
contraction induced by PE. The effect of the AESA at the
concentration of 300 μg/mL was also significantly (p < 0:05)
high as compared to the dose of 100 μg/mL (Figure 2(d)).
(86 mg/kg) in lowering heart rate was significant (p < 0:001)
compared to the LN group. There was no significant difference between the effects of the three doses of AESA used.
3.3. Antihypertensive Activity of the Aqueous Extract from
S. abyssinica
3.3.3. Effect of the Aqueous Extract from S. abyssinica on the
Body Weight and Organ Mass. As shown in Table 1, there
was no significant difference in body mass gain. However,
the gain in the L-NAME group was reduced by 32.78% as
compared to the normal control. Only AESA at 43 and
86 mg/kg protected against this weight loss by up to
51.66% as compared to the L-NAME-treated group.
Chronic administration of L-NAME alone for 6 consecutive weeks induced a 24.56% increase in cardiac mass and a
27.26% augmentation of the left ventricular mass compared
to the normal control group (Table 1). Only captopril was
able to significantly (p < 0:05) reverse cardiac hypertrophy
elicited by L-NAME.
Following L-NAME administration, the aorta mass
significantly increased (p < 0:01) compared to the normal
control. Captopril as well as AESA at doses of 43 and
86 mg/kg completely reversed L-NAME-evoked aorta
hypertrophy. No significant change (p > 0:05) in kidney
mass was observed throughout the experiment period
(Table 1).
3.3.1. Effect of AESA on Systolic and Diastolic Blood Pressure.
As shown in Figure 3, there was no significant difference in
systolic (SBP) and diastolic blood pressure (DBP) among the
different treatment groups at baseline. The chronic administration of L-NAME for 6 consecutive weeks induced a progressive and significant (p < 0:001) increase in SBP and
DBP. SBP rose from 120:75 ± 0:84 mmHg in normal control
rats to 174:25 ± 1:88 mmHg in L-NAME-treated rats. DBP
reached 120:50 ± 4:96 mmHg in the L-NAME group as compared to 81:62 ± 0:92 mmHg in the normal control group.
The administration of AESA induced a dose-dependent
decrease in both SBP and DBP. SBP returned to nearly baseline value following AESA treatment while the DBP returned
to the baseline value at doses of 86 and 172 mg/kg. Although
the blood pressure of animals treated with captopril was
significantly low (p < 0:001) compared to that of L-NAMEtreated rats, it is important to notice that at the end of the
experiment, a rebound effect was observed.
3.3.2. Effect of the Aqueous Extract from S. abyssinica on
Heart Rate. As shown in Figure 4, L-NAME administration
induced an increase (p < 0:05) in heart rate compared to control (412:25 ± 20:46 vs. 354:87 ± 7:25 beats/min). All the
treatments coadministered with L-NAME during the last
three weeks evoked a decrease in heart rate. Captopril as well
as AESA (43, 86, and 172 mg/kg) caused a reduction in heart
rate (respectively, 371:12 ± 16:90, 373:12 ± 12:14, 340:25 ±
9:62, and 369:75 ± 7:20 beats/min). The effect of AESA
3.3.4. Effect of the Aqueous Extract from S. abyssinica on
Serum and Urine NO Levels. As depicted in Figure 5, serum
and urinary NO levels were significantly reduced (p < 0:001)
when L-NAME was administered alone to animals. The
coadministration of L-NAME with the plant extract significantly increased serum NO compared to both L-NAME
and normal control groups (p < 0:001). On the same line,
AESA at the doses of 43 and 86 mg/kg significantly
(p < 0:05 and p < 0:01) corrected the effect of L-NAME on
urinary NO.
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Relaxation
(% of maximal contraction)
100
80
60
40
20
80
60
⁎⁎⁎
40
20
0
0
1.0
1.5
2.0
2.5
log [AESA] (𝜇g/mL)
1.0
3.0
1.5
2.0
2.5
log [AESA] (𝜇g/mL)
KCl+ AESA
PE+AESA
AESA
-Endo+AESA
(a)
(b)
120
3.0
50
% of KCl maximal contraction
100
⁎
60
⁎⁎⁎
40
⁎⁎⁎
⁎⁎⁎
20
⁎⁎⁎
0
⁎⁎⁎
1.0
1.5
2.0
2.5
MB+AESA
30
$
20
⁎⁎⁎
⁎⁎⁎
10
0
3.0
log [AESA] (𝜇g/mL)
AESA
40
Indo+AESA
MB+Indo+AESA
(c)
AESA300+PE
80
PE
Relaxation
(% of maximal contraction)
100
AESA100+PE
Relaxation
(% of maximal contraction)
120
(d)
Relaxation
(% of maximal contraction)
120
100
80
60
40
20
0
–9
–8
–7
–6
–5
log [AESA] (𝜇g/mL)
–4
Carbachol
Nifedipine
(e)
Figure 2: Effects of AESA on intact aortic rings precontracted with KCl or with phenylephrine (a), endothelium-denuded aortic rings
precontracted with phenylephrine (b), intact aortic ring preincubated with indomethacin, methylene blue, and indomethacin + methylene
blue (c), and on the intracellular Ca2+-release component of PE-induced contraction (d). Panel (e) presents the effects of reference
substances (carbachol and nifedipine) on aortic rings precontracted with phenylephrine. Each point represents the mean ± SEM of six
different experiments from six rats. Data were analyzed using ANOVA two-way with Bonferroni (a–c) or ANOVA one-way with
Tukey’s multiple comparison test (d). ∗ p < 0:05 and ∗∗∗ p < 0:001, significantly different compared to the effect of AESA without
antagonists. $p < 0:05 significant difference between the two concentrations.
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130
⁎⁎⁎
Start of the treatment
⁎⁎⁎
160
⁎⁎⁎
𝛾
𝛾
⁎⁎⁎
𝛾
⁎⁎⁎⁎
140
⁎⁎⁎
⁎⁎⁎
⁎
𝛾
Diastolic blood pressure (mmHg)
Systolic blood pressure (mmHg)
180
⁎⁎⁎
120
110
Start of the treatment
100
90
⁎⁎⁎
⁎⁎⁎
𝛾
⁎⁎⁎
𝛾
⁎⁎⁎
𝛾
⁎⁎⁎
⁎⁎
⁎
𝛾
80
120
0
1
2
3
4
Time (weeks)
5
6
LN+AESA 43
LN+AESA 86
LN+AESA 172
NT
LN
LN+CAPTO
0
1
2
3
4
Time (weeks)
6
LN+EASA 43
LN+EASA 86
LN+AESA 172
NT
LN
LN+CAPTO
(a)
5
(b)
Figure 3: Effect of the leaf aqueous extract of Stephania abyssinica (AESA) on blood pressure of rats rendered hypertensive by chronic LNAME administration. Values are expressed as mean ± SEM. n = 8; data were analyzed using ANOVA two-way with Bonferroni. ∗∗∗ p <
0:001 significant difference compared to the normotensive control group; γ p < 0:001 significant difference compared to the L-NAME
control group. NT = normotensive control; LN = L-NAME control; CAPTO = captopril; AESA 43, AESA 86, and AESA 172 = aqueous
extract of Stephania abyssinica at the doses of 43, 86, and 172 mg/kg, respectively.
Heart rate (beats/min)
450
has a high content of nitric oxide. AESA released NO in a
concentration-dependent manner and released more NO than
sodium nitroprusside at equivalent concentrations.
⁎
400
350
𝛾
300
S0
S3
S6
Time (weeks)
NT
LN
LN+CAPTO
LN+EASA 43
LN+AESA 86
LN+AESA 172
Figure 4: Effect of chronic administration of L-NAME alone and in
combination with captopril (CAPTO) or the leaf aqueous extract of
Stephania abyssinica (AESA) on heart rate. Values are expressed as
mean ± SEM. n = 8; data were analyzed using ANOVA two-way
with Bonferroni. ∗ p < 0:05 compared to the normotensive control
group. γ p < 0:001 significant difference compared to the LNAME control group. NT = normotensive control; LN = L-NAME
control; CAPTO = captopril; AESA 43, AESA 86, and AESA
172 = aqueous extract of Stephania abyssinica at 43, 86, and
172 mg/kg, respectively.
3.3.5. NO Content of the Aqueous Extract from S. abyssinica.
To better understand the pharmacological effects of the plant
extract, we assessed whether it could contain or release nitric
oxide. As shown in Figure 6, titration revealed that AESA
3.3.6. Effect of the Aqueous Extract of S. abyssinica on Kidney
Function. Table 2 shows the effects of S. abyssinica aqueous
extract on some parameters of kidney function. It can be
observed that L-NAME administration did not affect rats’
serum proteins compared to the normotensive control
group. In the same line, captopril and AESA treatments
did not influence serum protein level except the dose
172 mg/kg which induced a significant (p < 0:05) serum protein increase. On the other hand, rats that receive L-NAME
alone have an increased protein excretion, high serum creatinine levels, and reduced creatinine excretion and GFR while
the urine output was unchanged. Captopril reduced the
effect of L-NAME on proteinuria and increased creatinine
excretion in urine as well as GFR. The plant extract exacerbated the effect of L-NAME on proteinuria and GFR, especially at the dose of 172 mg/kg.
3.4. Effects of the Aqueous Extract of S. abyssinica on
Histology of the Heart and Kidney. The inspection of the
heart histopathological slices showed normal structure of
the myocardium in rat of the normotensive group
(Figure 7(a)) while leukocyte infiltration was observed in
the L-NAME hypertensive group (Figure 7(b)). Captopril
and AESA- (43, 86, and 172 mg/kg) treated groups showed
a significant reduction in inflammatory cell infiltration
(Figures 7(c)–7(f)).
Figure 8 depicts representative photomicrographs of the
kidneys from the normotensive control and various
8
BioMed Research International
Table 1: Effect of L-NAME alone and in combination with captopril (CAPTO) or the leaf aqueous extract of Stephania abyssinica (AESA)
on relative organs’ mass.
NT
LN
LN + CAPTO
LN + AESA 43
LN + AESA 86
LN + AESA 172
Body weight gain (g)
42:00 ± 4:62
28:25 ± 2:03
26:38 ± 4:32
39:75 ± 5:73
45:13 ± 8:11
23:63 ± 3:81
Heart (mg/cm)
222:00 ± 8:53
242:00 ± 3:53
206:00 ± 7:09 α
251:00 ± 7:73
239:00 ± 9:15
228:00 ± 9:40
Left vent. (mg/cm)
158:00 ± 4:03
178:00 ± 3:37
154:00 ± 6:77
186:00 ± 6:13
171:00 ± 8:61
176:00 ± 6:32
∗∗
α
Aortae (mg/cm)
20:00 ± 1:21
Kidneys (mg/cm)
458:00 ± 20:00
27:60 ± 1:96
17:60 ± 1:10
473:00 ± 39:5
493 ± 52:10
21:60 ± 1:65
19:10 ± 1:52
22:6 ± 1:35
556:00 ± 70:80
473:00 ± 19:2
488:00 ± 32:7
Values are expressed as mean ± SEM. The relative mass was calculated by dividing the absolute mass by the tibia length. n = 8; data were analyzed using
ANOVA one-way with Tukey’s multiple comparison test. ∗ p < 0:05, ∗∗ p < 0:01, and ∗∗∗ p < 0:001 compared to the normotensive control group; α p < 0:05
compared to the L-NAME (LN) control group. NT = normotensive control; LN = L-NAME control; LN + CAPTO = captopril; LN + AESA 43, LN + AESA
86, and LN + AESA 172 = aqueous extract of Stephania abyssinica at 43, 86, and 172 mg/kg, respectively.
𝛾
⁎⁎⁎
12
𝛾
⁎⁎⁎
𝛾
⁎⁎⁎
10
8
𝛽
⁎⁎⁎
𝛾
4
⁎⁎⁎
2
𝛾
𝛾
0.10
⁎⁎⁎
⁎
𝛼
0.05
AESA 172
AESA 86
AESA 43
CAPTO
AESA 172
AESA 86
AESA 43
CAPTO
H2O
NT
L-NAME (40 mg/kg/day, p.o)
H2O
0.00
0
NT
6
0.15
Urine NO level (𝜇M/ml)
Serum NO level (𝜇M/ml)
14
L-NAME (40 mg/kg/day, p.o)
(a)
(b)
Figure 5: Effect of AESA on serum (a) and urine (b) nitric oxide concentration in L-NAME-induced hypertensive rats. Values are expressed
as mean ± SEM. n = 8; data were analyzed using ANOVA one-way with Tukey’s multiple comparison test. ∗ p < 0:05 and ∗∗∗ p < 0:001
compared to the normotensive control group. α p < 0:05, β p < 0:01, and γ p < 0:001 compared to the L-NAME (LN) control group.
NT = normotensive control; LN = L-NAME control; LN + CAPTO = captopril; LN + AESA 43, LN + AESA 86, and LN + AESA
172 = aqueous extract of Stephania abyssinica at 43, 86, and 172 mg/kg, respectively.
1.0
⁎⁎
Nitric oxide (𝜇M)
0.8
0.6
0.4
treatment groups. No histopathological change was observed
in the kidneys of the normotensive group (Figure 8(a)). However, the kidneys from L-NAME-induced hypertensive rats
showed histopathological lesions including leukocyte infiltration, arterial wall thickening, and tubular disorganization
(Figure 8(b)). These renal histopathological lesions induced
by the L-NAME chronic administration were attenuated in
the captopril and AESA-treated group (Figures 8(c)–8(f)).
0.2
4. Discussion
0.0
30
100
300
Concentration (𝜇g/mL)
Nitroprussiate
AESA
Figure 6: NO content of the aqueous extract of S. abyssinica.
Values are expressed as mean ± SEM. N = 4. Data were analyzed
by paired concentration using the Mann-Whitney test. ∗∗ p < 0:01
significant difference between the two substances.
In a previous study, we showed that AESA possesses endothelium nitric oxide-independent vasorelaxant effects on
the isolated rat thoracic aorta but neither propranolol,
tetraethylammonium, nor glibenclamide could completely
block the vasorelaxant activity of the extract. More, AESA
was unable to completely suppress extracellular calciuminduced vascular contraction [14]. We then hypothesized
that AESA may possess additional mechanisms that might
trigger its vasorelaxant activity. Besides, AESA is used by
local populations to treat arterial hypertension. It was subsequently thought that its potential antihypertensive effect may
BioMed Research International
9
Table 2: Effect of AESA on kidney function in L-NAME-induced hypertensive rats.
NT
LN
LN + CAPTO
LN + AESA 43
LN + AESA 86
LN + AESA 172
Serum protein (nmol/μL)
13:44 ± 0:17
13:84 ± 0:57
13:18 ± 0:25
13:62 ± 0:61
32 ± 0:33
15:13 ± 0:45∗
Urine protein (nmol/μL)
7:42 ± 2:74
19:27 ± 4:16
13:09 ± 2:84
24:34 ± 6:63
31:68 ± 3:94∗∗
∗∗∗
γ
Serum creatinine (μg/mL)
3:99 ± 0:14
7:094 ± 0:12
Urine creatinine (μg/mL)
15:13 ± 1:11
8:25 ± 0:56∗∗∗
Urine volume (mL/24 h)
22:38 ± 3:58
19:18 ± 2:22
18:86 ± 2:38
GFR (μL/min)
41:60 ± 4:09
20:30 ± 1:96∗
85:8 ± 1:96∗∗∗
4:18 ± 0:33
27:97 ± 0:79∗∗∗
γ
γ
34:940 ± 3:91∗∗∗
β
γ
5:73 ± 0:35
4:96 ± 0:52
13:71 ± 0:95
26:55 ± 1:07∗∗∗
20:79 ± 2:93
12:49 ± 0:68
19:29 ± 2:36
29:3 ± 2:90
22:1 ± 4:00
17:7 ± 2:52∗
3:74 ± 0:42
δ
6:89 ± 0:47∗∗∗
γ
Values are expressed as mean ± SEM. n = 8; data were analyzed using ANOVA one-way with Tukey’s multiple comparison test. ∗ p < 0:05, ∗∗ p < 0:01, and
p < 0:001, compared to the normotensive control group; α p < 0:05, β p < 0:01, and γ p < 0:001, compared to the L-NAME (LN) control group.
NT = normotensive control; LN = L-NAME control; LN + CAPTO = captopril; LN + AESA 43, LN + AESA 86, and LN + AESA 172 = aqueous extract of
Stephania abyssinica at 43, 86, and 172 mg/kg, respectively.
∗∗∗
be supported by the upmentioned vasodilating properties.
The present work was undertaken to evaluate this hypothesis.
AESA relaxant effect was significantly affected by the removal
of the vascular endothelium and pretreatment with indomethacin, methylene blue, or the combination of both antagonists. In addition, AESA significantly inhibited the
intracellular calcium-induced vascular contraction and
reduced L-NAME-induced arterial hypertension.
It was observed in the present study that AESA similarly
relaxed aortic rings precontracted with KCl or PE. This
result is in accordance with previous observation [14] and
confirmed that AESA is capable to relax both voltageoperated calcium channels and receptor-operated calcium
channels. Vascular relaxation depends on two main pathways, the release of endothelial relaxing factors or the direct
effect on the vascular smooth muscles. To evaluate whether
the endothelium mediators contribute to the vasorelaxant
effect of AESA, the plant extract was tested on
endothelium-denuded aortic ring precontracted with PE.
The removal of the endothelium partially inhibited the
vasorelaxant effect of AESA suggesting the implication of
endothelium relaxing factors in the vasorelaxation induced
by AESA.
Mechanisms by which plant extracts or natural products
can induce endothelium-dependent vasorelaxation involve
nitric oxide (NO), prostacyclin (PGI2), or endothelialderived hyperpolarizing factor [41, 42]. Among these
endothelium-derived relaxing factors, only NO and PGI2
are well characterized [43]. In a previous study, it was shown
that endothelial NO did not mediate AESA-evoked vasorelaxation [14]. So we hypothesized that PGI2 might be
responsible for the endothelium-mediated AESA relaxation.
PGI2 is primarily synthesized from arachidonic acid catalyzed by cyclooxygenase (COX) [42]. The incubation of
intact aortic rings with indomethacin, a COX inhibition,
before the AESA challenge significantly reduced the vasorelaxant activity of AESA suggesting that PGI2 mediates
AESA-induced vasorelaxation of aortic rings.
Even with the strong inhibitory effect of indomethacin,
AESA was still able to elicit about 40% relaxation, suggesting
other mechanistic pathways. AESA was then tested in the
presence of methylene blue, an inhibitor of soluble guanylate
cyclase. The activation of soluble guanylate cyclase results in
the generation of cyclic guanosine monophosphate (cGMP).
The increase in intracellular cGMP concentration activates
cGMP-dependent protein kinase (PKG), which causes vasorelaxation via the modulation of Ca2+ channels as well as by
decreasing the Ca2+ sensitivity of the vascular smooth muscle contractile proteins [44]. Unexpectedly, the AESA effect
was strongly reduced by the methylene blue, suggesting the
involvement of the sGC/cGMP pathway in the vasorelaxation activity of AESA. Moreover, when the aortic rings were
preincubated with both indomethacin and methylene blue,
the combination of these two inhibitors highly potentiated
the vasorelaxant activity of AESA. This suggests that when
PGI2 synthesis and soluble guanylate cyclase are both inhibited, AESA induced vasorelaxation by other mechanisms. In
a previous study, we demonstrated that glibenclamide (an
ATP-sensitive K+ channel blocker) greatly reduced AESAinduced aortic muscle relaxation [14]. Thus, the activation
of KATP channels resulting in membrane hyperpolarization
might explain the COX and cGMP-independent vasorelaxation observed in this study. Some studies have shown that
when basal NO synthesis is blocked, relaxation is due to a
combination of both potassium channel and guanylyl
cyclase activation [45]. A charybdotoxin-sensitive K+ channel has found to be also implicated in the vasorelaxation
with the NO donors [46]. NO has been shown to stimulate
BKCa channels independently of cyclic GMP in vascular
smooth muscle cells [47–49]. It was somehow contradictory
that the sGC/cGMP pathway contributes to the vasorelaxant
effect of AESA which has been considered endothelial NOindependent. To better understand this, we quantified the
NO content/release by AESA and realized that it is highly
rich in NO. NO donors have been found to evoke smooth
muscle cell hyperpolarization through activation ATPsensitive potassium channels (KATP) [50, 51], voltageactivated potassium channels (KV) [52, 53], inwardly rectifying potassium channels (Kir) [54, 55], two-pore-domain
potassium channels (K2P) [56], and BKCa [57, 58]. Therefore, AESA might be a direct NO donor and could, therefore,
exert a direct hyperpolarizing mechanism on smooth muscle
cells.
Vascular smooth muscle contraction is triggered by an
increase in intracellular Ca2+ contraction, resulting from an
increase in calcium influx and/or intracellular stores’
10
BioMed Research International
(a)
(b)
(c)
(d)
(e)
(f)
Figure 7: Photomicrographs of histopathological changes in the heart: (a) control, no observable changes; (b) L-NAME alone, showing
important inflammatory cell infiltration (blue arrows); (c) L-NAME + captopril; (d–f) L-NAME + AESA at respective doses 43, 86, and
172 mg/kg, showing reduced inflammatory cell infiltration. H&E, mag 400x.
calcium release. Furthermore, this is part of the vasorelaxant
mechanism of NO. In the present study, the effect of AESA
on intracellular calcium was investigated. Preincubation of
aortic rings with AESA significantly reduced PE-induced
contraction. This result suggests that AESA may cause the
vasorelaxation of aortic smooth muscle by also inhibiting
the release of Ca2+ from intracellular stores.
It is well-known that arterial hypertension (AHT) is
associated with vascular changes characterized by endothelial dysfunction, increased vascular contraction, and arterial
remodeling [59, 60]. Taking into account the vasorelaxant
effect of AESA as demonstrated in this study, we hypothe-
sized that this extract may improve hemodynamic, functional, and structural abnormalities in a rat model of
hypertension. Thus, the antihypertensive effect of AESA
was examined in vivo, using the L-NAME-induced hypertension model in rats, which provides a reliable model of
hypertension with pronounced target organ damages that
mimic AHT seen in humans.
In this study, the chronic oral administration of LNAME was associated with a significant rise in BP and pulse
rate compared with the normotensive control rats, validating
the induction of hypertension. Administration of AESA and
captopril (a control hypotensive drug) for three weeks
BioMed Research International
11
(a)
(b)
(c)
(d)
(e)
(f)
Figure 8: Representative photomicrographs of kidneys from the experimental rats. (a) Kidneys of control rats showing normal kidney
histological architecture. (b) Kidneys from L-NAME-treated rat leukocyte infiltration (blue arrows), arterial wall thickening (red arrow),
and tubular disorganization (yellow arrow). (c) Kidneys from L-NAME + captopril and (d, e) L-NAME + AESA- (43 and 86 mg/kg)
treated groups showing attenuated infiltrations and lesions. Although rats receiving L-NAME + AESA at 172 mg/kg showed almost no
infiltration, the histological architecture was strongly affected. H&E, mag 400x.
caused a significant decline in BP and pulse rate in the hypertensive rats. Various studies have suggested that chronic
NOS inhibition with L-NAME increases sympathetic activity
release and heart rate [42, 61]. Therefore, sympathoexcitation suppression and vasodilation could be a possible mechanism of the significant decrease in arterial pressure and
heart rate induced by AESA in L-NAME-treated rats.
L-NAME treatment significantly reduced serum NO,
urinary creatinine and NO, and glomerular filtration rate
(GFR) but increased serum creatinine and urinary proteins. AESA significantly increased the bioavailability of
NO; this may be related to its high content in nitric as
observed in in vitro testing. These results further indicate
that the antihypertensive effect of AESA is at least partially
related to the increase in NO bioavailability and, thus, to
its vasorelaxant activity. Although captopril and the lower
doses of AESA tended to reverse the renal effects of LNAME, the plant extract at the dose of 172 mg/kg worsens
the parameters. These findings indicate that the higher dose
of AESA although very efficient on AHT might be harmful
for kidney function. Indeed, the histological analysis of the
kidney revealed that AESA at 43 and 86 mg/kg corrected
the renal alterations induced by L-NAME. Though there
was a significant reduction of inflammatory cell infiltration
in the kidney of the rats receiving AESA at 172 mg/kg, the
microarchitecture of the organ was disorganized.
L-NAME-induced pressure overload is associated with
cardiac and arterial remodeling and stiffening which may
initiate pathological changes in the cardiovascular and renal
systems. L-NAME administration increased the heart and
12
aorta masses. This is a well-known phenomenon in LNAME hypertensive rats [62]. AESA tended to reduce the
aorta mass but not that of the heart. This result is a bit hard
to understand given that the same mediators are involved in
the remodeling of both tissues. Besides, it has been demonstrated that increased NO bioavailability reduces the LNAME’s proliferative effect in the heart and aorta [63, 64].
Surprisingly, AESA increased the NO availability but failed
to reduce L-NAME-induced cardiac hypertrophy.
The histopathological examination of the heart of the
L-NAME hypertensive group revealed pronounce leukocyte infiltration. Various studies demonstrated that NOdeficient hypertension by L-NAME resulted in marked cardiac inflammation due to a significant increase in the cardiac
density of macrophages and T-cells that produce several
cytokines [65]. AESA administration reduced leukocyte infiltration. These findings suggest its cardioprotective effect.
Nevertheless, the plant extract was unable to reduce the
cardiac mass.
5. Conclusion
These findings showed that AESA exerted effective vasorelaxant effects in isolated rat thoracic aorta rings. This vasorelaxation could be mediated partly by an endotheliumdependent mechanism involving the prostacyclin pathway
and partly by an endothelium-independent mechanism
through direct activation of sGC/cGMP and K+ channels
in vascular smooth muscle, leading to inhibition of Ca2+
influx from the extracellular milieu and IP3-sensitive intracellular Ca2+ release. The vasorelaxant properties of AESA
in vitro may be translated to a potent antihypertensive effect
in vivo. Therefore, AESA may be a valuable drug candidate
for arterial hypertension. However, caution should be taken
with higher doses as they may induce kidney damage.
Data Availability
The data used and analyzed in this study are available from
the corresponding author on reasonable request.
Ethical Approval
Experimental protocols used herein were approved by the
laboratory committee, Faculty of Science, University of
Dschang, and conformed to the internationally accepted
standard ethical guidelines for laboratory animal use and
care as described in the European Community guidelines
2010/63/EU.
Conflicts of Interest
The authors declare that there is no conflict of interest.
Authors’ Contributions
TBN, CF, EPN-M, and AK conceived the work. CF and
MKN collected the data. TBN and CF analyzed the results.
CF and EPN-M drafted the manuscript. TBN refined the
BioMed Research International
manuscript. All the authors revised the manuscript for its
intellectual content and approved the final version.
Acknowledgments
This work was supported by a research grant from the International Foundation for Science (IFS) awarded (F/5495-2).
We thank M. Takala Jean Pierre (CHU-Yaoundé) for his
technical assistance in preparing histology slices.
References
[1] A. Dzudie, J. M. Fourie, W. Scholtz, O. Scarlatescu, G. Nel, and
S. Kingue, “Cameroon country report PASCAR and WHF,”
Cardiovascular Diseases Scorecard Project, vol. 31, no. 2,
pp. 103–110, 2020.
[2] A. E. Schutte, N. Srinivasapura Venkateshmurthy, S. Mohan,
and D. Prabhakaran, “Hypertension in low- and middleincome countries,” Circulation Research, vol. 128, no. 7,
pp. 808–826, 2021.
[3] Institute for Health Metrics and Evaluation, High systolic blood
pressure-level 2 risk, IHME, University of Washington, 2019,
June 2021, http://www.healthdata.org/results/gbd_
summaries/2019/high-systolic-blood-pressure-level-2-risk.
[4] K. T. Mills, J. D. Bundy, T. N. Kelly et al., “Global disparities of
hypertension prevalence and Control,” Circulation, vol. 134,
no. 6, pp. 441–450, 2016.
[5] M. Kiber, M. Wube, H. Temesgen, W. Woyraw, and Y. A.
Belay, “Prevalence of hypertension and its associated factors
among adults in Debre Markos Town, Northwest Ethiopia:
community based cross-sectional study,” BMC Research Notes,
vol. 12, no. 1, pp. 10–15, 2019.
[6] P. K. Whelton, R. M. Carey, W. S. Aronow et al., “2017
ACC/AHA/AAPA/ABC/ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA guideline for the prevention, detection, evaluation,
and management of high blood pressure in adults: a report of
the American College of Cardiology/American Heart Association task force on clinical practice guidelines,” Circulation,
vol. 138, pp. e484–e594, 2018.
[7] S. Okello, A. Muhihi, S. F. Mohamed et al., “Hypertension
prevalence, awareness, treatment, and control and predicted
10- year CVD risk: a cross-sectional study of seven communities in East and West Africa (SevenCEWA),” BMC Public
Health, vol. 20, no. 1, p. 1706, 2020.
[8] L. M. Brewster, G. A. van Montfrans, G. P. Oehlers, and Y. K.
Seedat, “Systematic review: antihypertensive drug therapy in
patients of African and South Asian ethnicity,” Internal and
Emergency Medicine, vol. 11, no. 3, pp. 355–374, 2016.
[9] F. Wyss, A. Coca, P. Lopez-Jaramillo, and C. Ponte-Negretti,
“Position statement of the Interamerican Society of Cardiology
(IASC) on the current guidelines for the prevention, diagnosis
and treatment of arterial hypertension 2017-2020,” International Journal of Cardiology, Hypertension, vol. 6, article
100041, 2020.
[10] M. J. Sorrentino, “The evolution from hypertension to heart
failure,” Heart Failure Clinics, vol. 15, no. 4, pp. 447–453, 2019.
[11] F. D. Fuchs and P. K. Whelton, “High blood pressure and cardiovascular disease,” Hypertension, vol. 75, no. 2, pp. 285–292,
2020.
[12] N. Aydoğdu, Y. Ö. Yalçınkaya, E. Taştekin, P. Tayfur, O. Kaya,
and N. Kandemir, “The effects of irisin on Nω-nitro-L-
BioMed Research International
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
arginine methyl ester hydrochloride-induced hypertension in
rats,” Balkan Medical Journal, vol. 36, no. 6, pp. 337–346, 2019.
F. Princewel, S. N. Cumber, J. A. Kimbi et al., “Prevalence and
risk factors associated with hypertension among adults in a
rural setting: the case of Ombe, Cameroon,” The Pan African
Medical Journal, vol. 34, p. 147, 2019.
T. Nguelefack, C. Fodem, E. Nguelefack-Mbuyo et al., “Endothelium nitric oxide-independent vasorelaxant effects of the
aqueous extract from Stephania abyssinica on the isolated rat
thoracic aorta,” Journal of Complementary and Integrative
Medicine, vol. 12, no. 1, pp. 15–21, 2015.
S. B. A. Cau, P. R. B. Evora, and R. C. Tostes, “Vasoconstrictor
substances produced by the endothelium,” in Endothelium and
Cardiovascular Diseases, pp. 115–125, Elsevier Inc., 2018.
I. K. Kwaifa, H. Bahari, Y. K. Yong, and S. M. Noor, “Endothelial dysfunction in obesity-induced inflammation: molecular
mechanisms and clinical implications,” Biomolecules, vol. 10,
no. 2, p. 291, 2020.
V. Ramanathan and M. Thekkumalai, “Role of chrysin on
hepatic and renal activities of N ω - nitro-l-arginine-methylester induced hypertensive rats,” International Journal of Nutrition, Pharmacology, Neurological Diseases, vol. 4, no. 1, p. 58,
2014.
L. O. Lerman, T. W. Kurtz, R. M. Touyz et al., “Animal models
of hypertension: a scientific statement from the American
Heart Association,” Hypertension, vol. 73, no. 6, pp. e87–
e120, 2019.
M. Majzunova, M. Kvandova, A. Berenyiova, P. Balis,
I. Dovinova, and S. Cacanyiova, “Chronic NOS inhibition
affects oxidative state and antioxidant response differently in
the kidneys of young normotensive and hypertensive rats,”
Oxidative Medicine and Cellular Longevity, vol. 2019, Article
ID 5349398, 10 pages, 2019.
R. Veerappan and T. Malarvili, “Chrysin pretreatment
improves angiotensin system, cGMP concentration in LNAME induced hypertensive rats,” Indian Journal of Clinical
Biochemistry, vol. 34, no. 3, pp. 288–295, 2019.
D. Macquart de Terline, A. Kane, K. E. Kramoh et al., “Factors
associated with poor adherence to medication among hypertensive patients in twelve low and middle income subSaharan countries,” PLoS One, vol. 14, no. 7, article
e0219266, 2019.
D. Kostova, G. Spencer, A. E. Moran et al., “The costeffectiveness of hypertension management in low-income
and middle-income countries: a review,” BMJ Global Health,
vol. 5, no. 9, article e002213, 2020.
E. D. Brouwer, D. Watkins, Z. Olson, J. Goett, R. Nugent, and
C. Levin, “Provider costs for prevention and treatment of cardiovascular and related conditions in low- and middle-income
countries: a systematic review,” BMC Public Health, vol. 15,
p. 1183, 2015.
C. Chen, C. Guo, J. Gao et al., “Vasorelaxant and antihypertensive effects of Tianshu Capsule on rats: An in vitro and in vivo
approach,” Biomedicine & Pharmacotherapy, vol. 111,
pp. 188–197, 2019.
J. Tomé-Carneiro and F. Visioli, “Polyphenol-based nutraceuticals for the prevention and treatment of cardiovascular disease: review of human evidence,” Phytomedicine, vol. 23,
no. 11, pp. 1145–1174, 2016.
F. Tang, H. Yan, L. Wang et al., “Review of natural resources
with vasodilation: traditional medicinal plants, natural prod-
13
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
ucts, and their mechanism and clinical efficacy,” Frontiers in
Pharmacology, vol. 12, article 627458, 2021.
S. M. Kupchan, A. J. Liepa, and T. Fujita, “New phenolic hasubanan alkaloids from Stephania abyssinica,” Journal of
Organic Chemistry, vol. 38, no. 1, pp. 151–153, 1973.
A. P. Washe and D. Fanta, “Hepatoprotective activities and
bioactive constituents of Stephania abyssinica,” Journal of
Pharmaceutical Research International, vol. 10, no. 6, pp. 1–
9, 2016.
E. Dagne, A. Gunatilaka, D. Kingston, and M. Alemu, “4 ′ OMethylstephavanine from Stephania abbysinica,” Journal of
Natural Products, vol. 56, no. 11, pp. 2022–2025, 1993.
B. Tamene, A floristic analysis and ethnobotanical study of the
semi-wetland of Cheffa area, South Welo, Ethiopia, [M.S. thesis], Addis Ababa University, Addis Ababa, 2000.
S. W. Province, L. Zapfack, J. S. O. Ayeni, S. Besong, and
M. Mdaihli, Ethnobotanical survey of the takamanda forest
reserve, Consultancy Report Submitted to PROFA (MINEFGTZ) Mamfe, S. W., Province Cameroon, 2001.
K. Asres, F. Bucar, T. Kartnig, M. Witvrouw, C. Pannecouque,
and E. De Clercq, “Antiviral activity against human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) of ethnobotanically selected Ethiopian medicinal plants,” Phytotherapy
Research, vol. 15, no. 1, pp. 62–69, 2001.
M. Saravanakumar and B. Raja, “Veratric acid, a phenolic acid
attenuates blood pressure and oxidative stress in L-NAME
induced hypertensive rats,” European Journal of Pharmacology, vol. 671, no. 1–3, pp. 87–94, 2011.
F. W. Muregi, S. C. Chhabra, E. N. Njagi et al., “Anti-plasmodial activity of some Kenyan medicinal plant extracts singly
and in combination with chloroquine,” Phytotherapy Research,
vol. 18, no. 5, pp. 379–384, 2004.
G. H. Schmelzer, A. Gurib-Fakim, R. Arroo et al., Plant
Resources of Tropical Africa 11(1): Medicinal Plants 1, PROTA
Foundation, 2008.
C. K. Fofié, E. P. Nguelefack-Mbuyo, N. Tsabang, A. Kamanyi,
and T. B. Nguelefack, “Hypoglycemic properties of the aqueous extract from the stem bark of Ceiba pentandra in
dexamethasone-induced insulin resistant rats,” Evidence-based
Complementary and Alternative Medicine: Ecam, vol. 2018,
article 4234981, pp. 1–11, 2018.
B. D. Toora and G. Rajagopal, “Measurement of creatinine
by Jaffe’s reaction–determination of concentration of sodium
hydroxide required for maximum color development in
standard, urine and protein free filtrate of serum,” Indian
Journal of Experimental Biology, vol. 40, no. 3, pp. 352–
354, 2002.
A. G. Gornall, C. J. Bardawill, and M. M. David, “Determination of serum proteins by means of the biuret reaction,” The
Journal of Biological Chemistry, vol. 177, no. 2, pp. 751–766,
1949.
M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding,” Analytical Biochemistry, vol. 72,
no. 1-2, pp. 248–254, 1976.
C. Desgrouas, N. Taudon, S. S. Bun et al., “Ethnobotany, phytochemistry and pharmacology of Stephania rotunda Lour,”
Journal of Ethnopharmacology, vol. 154, no. 3, pp. 537–563,
2014.
Y. Zhao, J. Ge, X. Li et al., “Vasodilatory effect of formaldehyde
via the NO/cGMP pathway and the regulation of expression of
14
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
BioMed Research International
KATP, BKCa and L-type Ca2+ channels,” Toxicology Letters,
vol. 312, pp. 55–64, 2019.
D. Aekthammarat, P. Tangsucharit, P. Pannangpetch,
T. Sriwantana, and N. Sibmooh, “Moringa oleifera leaf extract
enhances endothelial nitric oxide production leading to relaxation of resistance artery and lowering of arterial blood pressure,” Biomedicine & Pharmacotherapy, vol. 130, article
110605, 2020.
M. F. Yam, C. S. Tan, and R. Shibao, “Vasorelaxant effect of
sinensetin via the NO/sGC/cGMP pathway and potassium
and calcium channels,” Hypertension Research, vol. 41,
no. 10, pp. 787–797, 2018.
S. Wisutthathum, C. Demougeot, P. Totoson et al., “Eulophia
macrobulbon extract relaxes rat isolated pulmonary artery
and protects against monocrotaline-induced pulmonary arterial hypertension,” Phytomedicine, vol. 50, pp. 157–165, 2018.
F. Plane, A. Hurrell, J. Y. Jeremy, and C. J. Garland, “Evidence
that potassium channels make a major contribution to SIN-1evoked relaxation of rat isolated mesenteric artery,” British
Journal of Pharmacology, vol. 119, no. 8, pp. 1557–1562, 1996.
R. A. Bialecki and C. Stinson-Fisher, “KCa channel antagonists
reduce NO donor-mediated relaxation of vascular and tracheal
smooth muscle,” American Journal of Physiology-Lung Cellular and Molecular Physiology, vol. 268, no. 1, pp. L152–L159,
1995.
V. M. Bolotina, S. Najibi, J. J. Palacino, P. J. Pagano, and R. A.
Cohen, “Nitric oxide directly activates calcium-dependent
potassium channels in vascular smooth muscle,” Nature,
vol. 368, no. 6474, pp. 850–853, 1994.
A. Abderrahmane, D. Salvail, M. Dumoulin, J. Garon,
A. Cadieux, and E. Rousseau, “Direct activation of KCaChannel in airway smooth muscle by nitric oxide: involvement of a
nitrothiosylation mechanism?,” American Journal of Respiratory Cell and Molecular Biology, vol. 19, no. 3, pp. 485–497,
1998.
D. K. Mistry and C. J. Garland, “Nitric oxide (NO)-induced
activation of large conductance Ca2+-dependent K+ channels
(BKCa) in smooth muscle cells isolated from the rat mesenteric
artery,” British Journal of Pharmacology, vol. 124, no. 6,
pp. 1131–1140, 1998.
H. C. Parkington, H. A. Coleman, and M. Tare, “Prostacyclin
and endothelium-dependent hyperpolarization,” Pharmacological Research, vol. 49, no. 6, pp. 509–514, 2004.
C. Corriu, M. Félétou, G. Edwards, A. H. Weston, and P. M.
Vanhoutte, “Differential effects of prostacyclin and iloprost
in the isolated carotid artery of the guinea-pig,” European
Journal of Pharmacology, vol. 426, no. 1-2, pp. 89–94, 2001.
X. J. Yuan, M. L. Tod, L. J. Rubin, and M. P. Blaustein, “NO
hyperpolarizes pulmonary artery smooth muscle cells and
decreases the intracellular Ca2+ concentration by activating
voltage-gated K+ channels,” Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 19,
pp. 10489–10494, 1996.
P. L. Li, A. P. Zou, and W. B. Campbell, “Regulation of potassium channels in coronary arterial smooth muscle by
endothelium-derived vasodilators,” Hypertension, vol. 29,
no. 1, pp. 262–267, 1997.
R. Schubert, U. Krien, I. Wulfsen et al., “Nitric oxide donor
sodium nitroprusside dilates rat small arteries by activation
of inward rectifier potassium channels,” Hypertension,
vol. 43, no. 4, pp. 891–896, 2004.
[55] N. Orie, C. Fry, and L. Clapp, “Evidence that inward rectifier
K+ channels mediate relaxation by the PGI2 receptor agonist
cicaprost via a cyclic AMP-independent mechanism,” Cardiovascular Research, vol. 69, no. 1, pp. 107–115, 2006.
[56] A. Olschewski, Y. Li, B. Tang et al., “Impact of TASK-1 in
human pulmonary artery smooth muscle cells,” Circulation
Research, vol. 98, no. 8, pp. 1072–1080, 2006.
[57] L. H. Clapp, S. Turcato, S. Hall, and M. Baloch, “Evidence that
Ca2+-activated K+ channels play a major role in mediating the
vascular effects of iloprost and cicaprost,” European Journal of
Pharmacology, vol. 356, no. 2-3, pp. 215–224, 1998.
[58] J. Quignard, M. Félétou, C. Corriu et al., “3-Morpholinosydnonimine (SIN-1) and K+ channels in smooth muscle cells of the
rabbit and guinea pig carotid arteries,” European Journal of
Pharmacology, vol. 399, no. 1, pp. 9–16, 2000.
[59] R. M. Touyz, R. Alves-Lopes, F. J. Rios et al., “Vascular smooth
muscle contraction in hypertension,” Cardiovascular Research,
vol. 114, no. 4, pp. 529–539, 2018.
[60] P. M. Vanhoutte, “Nitric oxide: from good to bad,” Annals of
Vascular Diseases, vol. 11, no. 1, pp. 41–51, 2018.
[61] S. K. Kanthlal, J. Joseph, B. Paul, V. M, and P. Uma Devi,
“Antioxidant and vasorelaxant effects of aqueous extract of
large cardamom in L-NAME induced hypertensive rats,”
Clinical and Experimental Hypertension, vol. 42, no. 7,
pp. 581–589, 2020.
[62] P. Sventek, A. Turgeon, and E. L. Schiffrin, “Vascular
endothelin-1 gene expression and effect on blood pressure of
chronic ETAEndothelin receptor antagonism after nitric oxide
synthase inhibition WithL-NAME in normal rats,” Circulation, vol. 95, no. 1, pp. 240–244, 1997.
[63] O.' Pecháňová, I. Bernátová, V. Pelouch, and F. Šimko,
“Protein remodelling of the heart in NO-deficient hypertension: the effect of captopril,” Journal of Molecular and Cellular
Cardiology, vol. 29, no. 12, pp. 3365–3374, 1997.
[64] I. Bernátová, O. Pechánová, and F. Simko, “Effect of captopril
in L-NAME-induced hypertension on the rat myocardium,
aorta, brain and kidney,” Experimental Physiology, vol. 84,
no. 6, pp. 1095–1105, 1999.
[65] L. A. Biwer, K. M. D'souza, A. Abidali et al., “Time course of
cardiac inflammation during nitric oxide synthase inhibition
in SHR: impact of prior transient ACE inhibition,” Hypertension Research, vol. 39, no. 1, pp. 8–18, 2016.