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Research article
Xeroderris stuhlmannii (Taub.) Mendonça &E.P.Sousa (Fabaceae):
Evidence of the antihypertensive and antioxidant activities of its
leaf aqueous extract in cadmium chloride hypertensive rats
Augustine Nkojap Kuinze
a
, Edwige Laure Nguemfo
b
, William Nana Yousseu
a
,
Jacquy Joyce Wanche Kojom
c
, Calvin Zangueu Bogning
a
,
Christelle St´
ephanie Sonfack
a
, Willifred Tsopgni Dongmo Tekapi
d
, Timo D. Stark
e
,
Guy Blaise Anatole Azebaze
d
, Alain Bertrand Dongmo
a,*
a
Department of Biology and Physiology of Animal Organisms, Faculty of Science, University of Douala, PO Box 24157, Douala, Cameroon
b
Department of Biological Sciences, Faculty of Medicine and Pharmaceutical Sciences, University of Douala, PO Box 2701, Douala, Cameroon
c
Department of Biology and Physiology of Animal, Faculty of Sciences, University of Yaound´
e I, PO Box 812, Yaound´
e, Cameroon
d
Department of Chemistry, Faculty of Sciences, University of Douala, PO Box 24157, Douala, Cameroon
e
Lehrstuhl für Lebensmittelchemie und Molekulare Sensorik, Technische Universit¨
at München, 85354, Freising, Germany
ARTICLE INFO
Keywords:
X. stuhlmannii
Hypertension
Cadmium chloride
Oxidative stress
Phenols
ABSTRACT
Xeroderris stuhlmannii (Fabaceae) is a medicinal reported in Cameroonian herbal medicine to treat
hypertension. The aim of the study was to assess the antihypertensive and antioxidant activities of
X. stuhlmannii aqueous leaf extract (AEXS) on cadmium chloride-induced hypertensive rats. The in
vitro antioxidant activities of AEXS were investigated for their radical scavenging potency using
2,2-diphenyl-1-picrylhydrazyl (DPPH), Ferric reducing antioxidant power (FRAP), 2,2
′
-azino-bis-
(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS), Nitric oxide (NO) and OH- assays completed
with oxidative stress markers analyses. Antihypertensive activity of AEXS (35, 100, and 300 mg/
kg) was assessed in CdCl
2
induced-hypertensive rats. Antihypertensive activities performed
include systolic (SBP), diastolic blood pressure (DBP) and heart rate (HR) variation, followed by
evaluation of selected biochemical parameters in urine, blood (Alanine aminotransferase (ALT),
aspartate aminotransferase (AST)), creatinine, urea and total protein) and histological examina-
tion of tissue samples (aorta, heart, kidneys and liver). The amount of the phenols of the leaf
extract was estimated in mg gallic acid equivalent and identication of some compounds was
done by UPLC-UV-ESI-TOF-MS. Accordingly, the identied phenols were stuhlmannione A (1),
formononetin (2), stuhlmarotenoid A (3), 9-methoxymaackiain (4), 4-hydroxymaackiain (5) and
7-hydroxy-3
′
,4
′
-methylenedioxy-isoavone (6). The extract exhibited a signicant (P <
0.05–0.001) decrease of SBP, DBP and HR when compare to control. AEXS also reduced (P <
0.05) serum rates of ALT, AST, and urea. The extract showed benecial effects on alterations
observed in the histological structures of the aorta, heart, kidneys and liver. AEXS highlighted
high level of phenols (26.48 ±2.89 mg GAE/g) and a strong antiradical activity on DPPH, ABTS
+
,
OH
−
and NO with IC
50
of 148.8
μ
g/mL, 27.83
μ
g/mL, 22.29
μ
g/mL, 29.84
μ
g/mL respectively. An
optical density of 1.79 nm was obtained with FRAP test. Thus, X. stuhlmannii leaf extract has in
vitro antioxidant and antihypertensive effects that may support its use against hypertension.
* Corresponding author.
E-mail address: alainberd@yahoo.fr (A.B. Dongmo).
Contents lists available at ScienceDirect
Heliyon
journal homepage: www.cell.com/heliyon
https://doi.org/10.1016/j.heliyon.2024.e38075
Received 4 May 2024; Received in revised form 17 September 2024; Accepted 17 September 2024
Heliyon 10 (2024) e38075
Available online 18 September 2024
2405-8440/© 2024 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).
1. Introduction
Hypertension occurs when the blood pressure in vessels becomes too high (140 mmHg SBP and 90 mmHg DBP or higher). It
represents the main cause of premature death worldwide. Reduce the prevalence of hypertension by 33 % within 2010 and 2030 has
become one of the global challenges for non-communicable diseases [1]. An estimation reveals that 1.28 billion of adults aged between
30 and 79 years worldwide are suffering from hypertension, most of them reside in low- and intermediate-income countries [1].
Hypertension has many origins, including exposure to an environmental pollutant such as cadmium (Cd), which is a toxic heavy metal
that could be present in food and tobacco [2]. Its industrial exploitation grew at the beginning of the 20th century. Once absorbed, Cd
could be accumulated in different tissues, especially in the liver and kidneys [2], and therefore cause harmful effects such as kidney
dysfunction [3], pulmonary edema [4], cancer [5] and cardiovascular diseases including hypertension and atherosclerosis [6]. In
addition, numbers of clinical and experimental carried out have correlated a hypertension and Cd presence in organism, although the
biological mechanisms which explain the link between Cd exposure and high blood pressure are unknown. However, it has been
brought evidence that the hypertensive effect of Cd exposure results from complex mechanisms that operate on both vascular smooth
muscle cells and on vascular endothelium. The main factors playing a key role in the cardiovascular complications in living organisms
and which have been the subject of intensive research are reactive nitrogen species (RNS), reactive oxygen species (ROS), and
depletion of antioxidant levels. These factors always lead to a state of oxidant/antioxidant balance [7]. In recent years, numerous
studies have reported the role of oxidative stress as a key mechanism which can explain cadmium chloride toxicity [8]. Oxidative stress
can cause blood pressure increase by several pathways such as causing a decrease in the bioavailability of NO, which promotes
increased peripheral vascular resistance, endothelial dysfunction, and vascular remodeling, platelet and leukocyte adhesion [9].
However, oxidative damage could be suppressed by natural or synthetic antioxidants used for medicinal purposes [10]. Synthetic
antioxidants like butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) are actually not indicated due to their
carcinogenic effects in vivo [11]. They are now being replaced by natural plant-derived antioxidants due to their excellent non-toxic,
free radical detoxifying properties, affordable prices joint to fewer adverse effects [12]. The treatment of hypertension aims to
gradually drop blood pressure and also prevent possible damage of organs. It mainly relies on hygieno-dietetic measures and the use of
antihypertensive drugs. Due to the substantial cost of health services and medicines on the one hand, and unfavourable socio-economic
factors on the other, a large proportion of the population relies on medicinal plants for their health care [13]. Therefore, an interest was
given to the study of many plant properties include the antihypertensive and antioxidant properties of the aqueous extract of the leaves
of X. stuhlmannii (Fabaceae). It is a species found in open forests or Sudano-Guinean wooded savannahs [14]. The plant is particularly
found on well-drained soils, on sandy soils and it is resistant to drought. The species is largely spread in tropical Africa, from eastern
Senegal to Kenya, south of Zimbabwe, Mozambique, Cameroon and northern South Africa. The leaves are used to treat colds, coughs,
wounds, stomach ache, fever, malaria, and hypertension [14]. Phytochemical screening of the ethanolic leaf extract of X. stuhlmannii
indicated the presence of isoavones (avonoids) and rotenoids [15]. Although the virtues of this plant are known empirically to treat
various pathologies, its effect on high blood pressure has not yet been demonstrated. Thus, this study was undertaken to assess the
antioxidant and antihypertensive effects of the aqueous extract from the leaves of X. stuhlmannii against CdCl
2
-induced hypertension in
rats.
2. Materials and methods
2.1. Plant material and extraction procedure
Fresh leaves (Fig. 1A) of X. stuhlmannii, were collected from Bitchoua-Nord, West region of Cameroon in June 2022. The plant was
identied at the National Herbarium of Yaounde-Cameroon, in comparison of the harvested leaves and fruits (Fig. 1B) with the
Voucher’s specimen, deposited under Number 6011/SRFCAM. The leaves were shade-dried and ground into ne powder. The aqueous
extract was obtained by infusing 4.84 g of X. stuhlmannii leaves powder into 200 mL of boiling distilled water (100 ◦C) until complete
cooling. Therefore, extract ltration was performed using Whatman paper n◦3. The ltrate was dried in an oven at 40◦Ϲand 0.973 g of
dried brown powder was obtained, let be a percentage yield of 20.10 %.
Fig. 1. Photographs of the leaves (A) and fruits (B) of Xeroderris stuhlmannii.
A. Nkojap Kuinze et al. Heliyon 10 (2024) e38075
2
2.2. Experimental animals
Wistar Albino rats, aged 10–14 weeks old and weighing between 182 and 225 g were used for antihypertensive effect. They were
housed in a colony inside plexiglass cages, at the animal house of the University of Douala, Cameroon, at room temperature (23–25 ◦C)
with a 12 h dark-light natural cycle. They were fed the standard commercial diet and tap water ad libitum. The institutional ethics
committee of the University of Douala approved the research protocol under the reference N◦3082 CEI-UDo/05/2022/T.
2.3. Drugs and chemicals
Analytical-grade reagents were used in this study. Iron-chloride, sodium chloride, Potassium peroxodisulfate and potassium hy-
droxide were bought from Acros organics (Germany). Amlodipine was purchased from Denk (Germany). Sodium carbonate, Tris,
Folin-Ciolcalteu were from Carl Roth (Germany). 2-deoxy-D-ribose, sodium hydroxide and sodium nitrite were obtained from Alfa
Aesar (Germany). Phosphate buffered saline, Sodium nitrite, and NED were purchased from VWR life science (Belgium). Hydrogen
peroxide HR rapid was purchased from Tintometer group GmbH (Germany). Trichloroacetic acid and gallic acid were from Cayman
chemical company (Germany). Alanine aminotransferase, Aspartate aminotransferase, urea, total protein, and creatinine kits were
purchased from SGM Italia (Roma). Trolox, potassium ferricyanide, 2,2-diphenyl-1-picrylhydrazyl, thiobarbituric acid, cadmium
chloride hydrate, 2,2
′
-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), disodium hydrogen phosphate, ferric chloride, phosphoric
acid and ascorbic acid were obtained from Sigma-Aldrich (Germany).
2.4. Quantication of total phenolic content
The total phenolic content was quantied in extract by using the modied method of Singleton and Rossi [16]. Gallic acid was used
as a standard and the reaction mixture was prepared with 40
μ
L of extract (1 mg/mL), mixed with 3.16 mL of distilled water and 200
μ
L
of 10 % Folin-Ciocalteu reagent was added. The mixture was incubated for 8 min, then 600
μ
L of 20 % of Na
2
CO
3
was added, and
incubated for 30 min at 40◦Ϲ. Experiment was carried out in triplicate and absorbance was measured at 760 nm. The results obtained
for the different concentrations of the standard gallic acid (0–300
μ
g/mL; y =0.0007x +0.0198; R
2
=0.9707) were expressed as the
mg gallic acid equivalent per gram of dry weight [17].
2.5. In vitro assays: assessment of the antioxidant activity of X. stuhlmannii leaf extract
Overall, concentration providing 50 % of radical scavenging activity (IC
50s
) obtained from the tests on DPPH (2,2-diphenyl-1-
picrylhydrazyl radical), ABTS (2,2
′
-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid), OH
−
and NO were determined from the graph
depicting inhibition percentages against extract concentrations. Ascorbic acid or trolox were used as reference compounds.
2.5.1. Scavenging test on 2,2-diphenyl-1-picrylhydrazyl radical
The ability of AEXS to scavenge DPPH radical was determined as described by Brand Williams et al. [18] with modications. For
each test tube containing 1000
μ
L of various concentrations (1-3-10-30-100-300-700 and 1000
μ
g/mL) of the plant extract or ascorbic
acid, 500
μ
L of DPPH solution (0.063 mg/mL) were added and incubated for 20 min at room temperature in the absence of light. The
absorbance was measured against blank (methanol) at 517 nm.
Experiments were run in triplicate and the inhibition percentage was calculated as follows:
%Inhibition of DPPH =Ac −At
Ac×100
Where A
c
and A
t
are the absorbances of the blank and test substances (extract/ascorbic acid), respectively.
2.5.2. Scavenging test on 2,2
′
-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid radical
ABTS radical scavenging assay was determined as described by Re et al. [19] with modications. The solutions of potassium
persulfate (2.45 mM) and ABTS (7 mM) previously dissolved in distilled water, were stirred at equal volume. The mixture was kept to
react in the dark at room temperature within 12–16 h. Distilled water was supplemented to that working solution until an absorbance
of 0.70 ±0.02 (734 nm) was obtained. Thereafter, 1 mL of ABTS solution was stirred with 0.1 mL of extract or trolox at different
concentrations (1–1000
μ
g/mL), and was analyzed after 7 min against the blank (working solution and distilled water). All the
concentrations were tested in triplicate. The results were presented as percentage of inhibition of ABTS:
%Inhibition of ABTS =Ac −At
Ac×100
where A
c
and A
t
are the absorbances of control and test substances (extract/trolox) respectively.
2.5.3. OH radical Scavenging assay
The scavenging of the OH radical produced by Fenton reaction was determined according to the modied method of Kunchandy
and Rao [20]. Five hundred microliter (500
μ
L) of different concentrations (1–1000
μ
g/mL) of extract or trolox were added to 100
μ
L of
A. Nkojap Kuinze et al. Heliyon 10 (2024) e38075
3
2-deoxy-D-ribose (28 mM), 200
μ
L mixture of EDTA (1.04 mM) and FeCl
3
(0.2 mM) [1:1 v/v], 100
μ
L H
2
O
2
(1 mM) and 100
μ
L of
ascorbic acid (1 mM). All the reagents and extract contained in the M1 mixture were diluted in 20 mM of KH
2
PO
4
-KOH buffer, pH 7.4.
This mixture (M1) was incubated at 37◦Ϲfor 1 h. Thiobarbituric acid (1 %, 500
μ
L) and trichloroacetic acid (2.8 %, 500
μ
L) was added
successively to M1 (500
μ
L) and incubated at 100 ◦C for 30 min. After cooling, the absorbance was measured at 532 nm against a
control sample. The assays were performed in triplicate. The results were expressed as a percentage of 2-deoxyribose degradation.
% 2 −deoxyribose degradation =Ac −At
Ac×100
Where A
c
and A
t
are the absorbances of the control and test substances (extract/trolox), respectively.
2.5.4. Ferric reducing antioxidant power
The reducing capacity of the extract was evaluated according to a previously described method with modications [21]. Half
milliliter (0.5 mL) of various concentrations of extract/ascorbic acid (1–300
μ
g/mL), 1.25 mL of phosphate buffer (200 mM, pH =6.6),
and 1.25 mL of potassium ferricyanide (1 %) were introduced in the test tubes. The mixture was incubated at 50◦Ϲfor 20 min, then
1.25 mL trichloroacetic acid (10 %) was added. After centrifugation at 3000 rpm for 10 min, 1.25 mL of supernatant was taken and
1.25 mL of distilled water and 0.25 mL of FeCl
3
(0.1 %) were added. The mixture was incubated for 10 min at 37◦Ϲand the absorbance
was recorded at 700 nm in triplicate against the blank solution. The antioxidant power was estimated by plotting of the variation of the
optical density as a function of the concentration of the assay tubes compared to the standard tubes (ascorbic acid).
2.5.5. Nitric oxide Scavenging test
The nitric oxide (NO) released by sodium nitroprusside was determined using the Griess reaction [22]. A volume of 50
μ
L of sodium
nitroprusside (10 mM in phosphate buffered saline, pH 7.4) was transferred to other tubes containing 950
μ
L of extract or ascorbic acid
at different concentrations (1–1000
μ
g/mL). The mixture reaction was left to stand at room temperature (25◦Ϲ) for 3 h under light.
Thereafter, 500
μ
L of the mixture was removed and then stirred with 500
μ
L of Griess reagent (1 % sulfanilamide prepared in distilled
water and 0.1 % napthylethylenediamine prepared in 2 % phosphoric acid) in dark. The absorbance of the chromatophore was
recorded immediately at 546 nm against the blank (distilled water, sodium nitroprussiate, and Griess reagent) and referred to the
standard solution (ascorbic acid prepared in the same way) absorbance. The experiment was carried out in triplicate [23]. The results
were expressed as percentage of inhibition of nitrite ions by the following formula:
%inhibition of nitrite =Ac −At
Ac×100
Where A
c
and A
t
were the absorbance of the control and test substances (extract/ascorbic acid), respectively.
2.6. Antihypertensive activity of Xeroderris stuhlmannii against cadmium chloride-induced hypertension
2.6.1. Animal preselection, distribution and drug administration
After 5 days acclimatization period, animals were screened to exclude anyone with blood pressure higher than 140/90 mmHg.
Therefore, 48 animals divided into 2 separate groups were fasted 12 h prior to the experiment, which was curative and ongoing during
3 weeks for the preliminary test. Accordingly, the rst group of 06 rats, named normotensive rats (control), daily received NaCl (0.9 %,
i.p) as vehicle. The second group (42 rats) were administrated cadmium chloride (1 mg/kg, i.p daily) to induce hypertension. At the end
of 3 weeks, the pressure of the animals was recorded and those in the second group with blood pressure values higher than or equal to
140/90 mmHg were considered as hypertensive animals and selected for the following experience. Accordingly, 36 rats which were
hypertensive were randomized into 5 groups of 6 rats each, which received different solutions as follows:
- Hypertensive group (control) continuous to receive cadmium chloride (CdCl
2
)
- Standard group which received amlodipine (1 mg/kg body weight) concomitantly with CdCl
2
- Three assay groups which received the extract at different doses of 35, 100 and 300 mg/kg of body weight concomitantly with
CdCl
2
(1 mg/kg, i.p daily).
Throughout the experiment, the rats of group 1 were receiving tap water while the other groups were receiving NaCl (1 %) as
drinking water.
Following animal redistribution, the experiment was extended for more two weeks, blood pressure and heart rate were recorded
twice a week following the non-invasive method with the use of CODA system (Kent Scientic Co, USA) [24].
2.6.2. Sample collection and analysis
At the end of experiment, the urine sample of each animal was collected using metabolic cages and transferred to laboratory tubes.
Animals were anesthetized with dual intraperitoneal injection of diazepam and of ketamine (10 mg/kg; 50 mg/kg) and euthanized by
cervical decapitation. Blood specimen was also collected in heparin-tubes. Blood and urine samples were centrifuged at 3000 rpm for
15 min to obtain plasma and supernatant respectively. Aliquot solutions collected were used to perform various biochemical mea-
surements including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, urea, and total protein. Aorta,
A. Nkojap Kuinze et al. Heliyon 10 (2024) e38075
4
heart, kidneys, and liver tissues were excised quickly, freed of fat and connective tissues, rinsed in physiological saline (0.9 % NaCl)
and weighed. Known weights representative fragments of tissues (aorta and heart) were homogenized in Mac Even or in Tris-HCl 50
mM buffer solution pH =7.4 for liver and kidney (20 %, w/v). After centrifugation at 3000 rpm for 30 min (aorta and heart) and for 15
min (liver and kidney), the supernatant was collected to assess oxidative stress parameters (catalase, malondialdehyde, superoxide
dismutase and nitric oxide).
2.6.3. Histopathological examination
The remaining portions of tissue specimens (aorta, heart, kidneys, and liver) were kept in 10 % buffered formalin (pH =7.4)
solution, dehydrated in graded alcohol and embedded in parafn for further histopathological examination. 5
μ
m sections obtained
were mounted on glass slides and stained with hematoxylin-eosin for observation under an optical microscope ( ×400-magnication).
Parameters such as media diameter in the aorta, aspect of muscle bers in heart, aspect of hepatocytes in liver, and aspect of
glomerulus in the kidney were observed and photographed.
2.7. UPLC-UV-ESI-TOF-MS analysis of X. stuhlmannii leaf extract
Aliquots 2
μ
L of the leaf extract (1 mg/10 mL, 50 % MeCN) were analyzed by means of UPLC-ESI-TOF MS on a Waters Synapt G2-S
HDMS mass spectrometer (Waters, Manchester, UK) coupled to an Acquity UPLC core system (Waters, Milford, MA, USA) equipped
with a 2 ×150 mm, 1.7
μ
m, BEH C18 column (Waters, Manchester) consisting of a binary solvent manager, sample manager and
column oven. Operated with a ow rate of 0.4 mL/min at 50 ◦C, the following gradient was used for chromatography: starting with a
mixture (1/99, v/v) of aqueous HCO
2
H (0.1 % in H
2
O) and MeCN (0.1 % HCO
2
H) for 0.3 min, the MeCN content was increased to 100
% within 8 min, kept constant for 2 min, decreased to 1 % within 1 min and nally kept constant for 1 min at 1 %. Scan time for the MS
e
method (centroid) was set to 0.1 s. Analyses were performed with negative and positive ESI in high resolution mode using the following
ion source parameters: capillary voltage −2.0 or +2.5 kV, sampling cone 50 V, source offset 30 V, source temperature 120 ◦C, des-
olvation temperature 450 ◦C, cone gas ow 2 L/h, nebulizer 6.5 bar and desolvation gas 800 L/h. Data processing was performed by
using Mass Lynx 4.1 SCN 9.16 (Waters, Manchester) and the elemental composition tool for determining the accurate mass. All data
were locked mass corrected on the pentapeptide leucine enkephaline (Tyr-Gly-Gly-Phe-Leu, m/z 554.2615, [M −H]
-
) in a solution (1
Fig. 2. DPPH radical scavenging (a), ferric reducing antioxidant power (b), ABTS radical scavenging (c), NO radical scavenging (d) and OH radical
scavenging (e) capacity of the aqueous leaf extract of X. stuhlmannii and ascorbic acid or Trolox. Data are presented as mean ±SEM, n =3;
b
P <
0.01;
c
P < 0.001 vs, ascorbic acid or Trolox.
A. Nkojap Kuinze et al. Heliyon 10 (2024) e38075
5
ng/
μ
L) of MeCN/0.1 % HCO
2
H (1/1, v/v). Scan time for the lock mass was set to 0.3 s, an interval of 15 s and 3 scans to average with a
mass window of ±0.3 Da. Calibration of the Synapt G2-S in the range from m/z 50 to 1200 was performed using a solution of HCO
2
Na
(5 mmol/L) in 2-propanol/H
2
O (9/1, v/v). The UPLC and Synapt G2-S systems were operated with MassLynx™software (Waters).
Collision energy ramp for MS
e
was set from 20 to 40 eV.
2.8. Statistical analysis
Results in both studies (in vitro and in vivo) were computed as mean ±standard error of the mean. For the in vitro antioxidant assays,
IC
50
values were determined using a nonlinear regression curve followed by normalized and logarithmic transformation. Data from in
vivo experiments were submitted to one-way ANOVA followed by Tukey’s post test for organ weight variation and biochemical pa-
rameters and two-way ANOVA with repeated measures followed by Bonferroni post test for blood pressure and body weight. A result
with P < 0.05 was considered statistically signicant.
3. Results
3.1. Total phenols content
Total phenols content of X. stuhlmannii leaf extract expressed as gallic acid equivalent (GAE) was found to be 26.48 ±2.89 mg GAE/
g.
3.2. Antioxidant activities of X. stuhlmannii leaf extract
3.2.1. Antiradical activity of X. stuhlmannii leaf extract against DPPH
The leaf extract of X. stuhlmannii developed a concentration-dependent scavenging activity on DPPH radical with an inhibitory
concentration 50 (IC
50
) of 148.8
μ
g/mL, while the standard ascorbic acid exhibited an IC
50
of 26.95
μ
g/mL (Fig. 2a).
3.2.2. Antioxidant effect of X. stuhlmannii through ferric reducing power/reducing potential assay
The ferric reducing antioxidant power of the aqueous leaf extract and the standard increased proportionally with the concentration
(Fig. 2b). The optical density of the extract was 1.79 nm, while that of ascorbic acid was 3.33 nm.
3.2.3. Scavenging activity of X. stuhlmannii leaf extract against ABTS radical
The inhibition of the absorbance of ABTS radical by X. stuhlmannii extract and Trolox are represented in Fig. 2c. The extract and
Trolox exhibited signicant scavenging activity on ABTS radical cation at various tested concentrations. The concentration of
X. stuhlmannii extract necessary for 50 % inhibition was found to be 27.83
μ
g/mL while 5.72
μ
g/mL was required for the standard
Trolox.
3.2.4. Scavenging activity of X. stuhlmannii against NO radical
The result of the NO radical scavenging (Fig. 2d) showed that the inhibition of NO release in presence of X. stuhlmannii leaf extract
and ascorbic acid is concentration dependent manner. The IC
50
value obtained for the extract was 29.84
μ
g/mL compared to that of
ascorbic acid of 26.55
μ
g/mL.
Fig. 3. Relative weight of some organs Data are presented as mean ±SEM, n =6;
a
P<0.05,
b
P < 0.01, and
c
P < 0.001: compared to the control;
CdCl
2
=cadmium chloride; X.s =X.s stuhlmannii; Amlo =Amlodipine.
A. Nkojap Kuinze et al. Heliyon 10 (2024) e38075
6
3.2.5. Scavenging activity of X. stuhlmannii against hydroxyl radical
Scavenging ability of hydroxyl radical was concentration dependent for the aqueous extract and Trolox (Fig. 2e). The IC
50
value
obtained with the extract was 22.29
μ
g/mL compared to that of Trolox which was 84.71
μ
g/mL.
The concentration providing 50 % of radical scavenging activity (IC
50
) was obtained using graphpad prism software version 5.03.
3.3. Effect of cadmium chloride and aqueous leaf extract of X. stuhlmannii on organs relative weight, blood pressure and heart rate in rats
Compared to the control the administration of CdCl
2
induced signicant variation of relative weight of the liver (32.36 %; P <
0.001), kidneys (27.40 %; P < 0.001) and lungs (51.45 %; P < 0.001). The concomitant administration of the extract or amlodipine
with CdCl
2
did not induce any signicant variation in the heart, aorta the liver, kidneys, and lungs relative weight compared to the
hypertensive group (CdCl
2
group) (Fig. 3).
Two weeks of treatment with CdCl
2
induced a signicant increase (P < 0.001) of SBP and DBP in rats compared to control.
Concomitant administration of CdCl
2
and aqueous extract of X. stuhlmannii or amlodipine resulted in a progressive decrease (P <
0.001) in SBP and DBP in all groups compared to the hypertensive group. At the end of the rst week of treatment the administration of
the extract (300 mg/kg) induced a signicant fall of the SBP from 158.30 mmHg to 115.10 mmHg (Fig. 4a). At the end of second week
of treatment, the maximum decrease in DBP by 32.19 % as compared to hypertensive group was observed (Fig. 4b).
The heart rate did not change signicantly during experimental induction of hypertension as well as treatment with plant extract.
Only the dose of 100 mg/kg of extract signicantly reduced (P < 0.01) the HR compared to hypertensive group.
3.4. Effect of X. stuhlmannii extract on some enzymes and renal markers
3.4.1. Effect of aqueous extract of X. stuhlmannii on serum transaminases activities
The results revealed that the hypertensive group had signicantly (P < 0.001) increased serum transaminases (ALT and AST)
activity compared to controls. Concomitant administration of CdCl
2
and the aqueous leaf extract of X. stuhlmannii led to a decrease (P
< 0.01) in ALT activity as compared to hypertensive group. This decrease reached 67.22 U/I and 66.81 U/I at doses of 35 and 300 mg/
kg, respectively (Fig. 5a). The administration of the plant extract or amlodipine and cadmium chloride led to a decrease (P < 0.05) in
AST activity compared to the hypertensive group (Fig. 5b).
3.4.2. Effect of extract on some kidneys functions parameters
The repeated administration of cadmium chloride to rats provoked an increase (P<0.05) of the serum creatinin from 0.579 ±
0.064 mg/dL for the control to 1.155 ±0.105 mg/dL in hypertensive rats (Fig. 6a). However, a decrease of urinary creatinin con-
centration was observed in the hypertensive group (32.29 ±3.739 mg/dL) compared to the control (74.41 ±1.568 mg/dL). Compared
to the hypertensive group oral administration of EAXS caused an increase (P>0.05) of urinary creatinin concentration in all the assays
groups (Fig. 6b). The serum urea level in rats was signicantly higher (P < 0.001) in hypertensive group than in control group, with a
percentage of depletion of 40.99 % (Fig. 6c). The treatment of CdCl2 induced-hypertensive rats with EAXS signicantly prevented (P
< 0.05–0.001) the elevation of urea level by 28.32 %, 37.88 %, and 24.20 %, respectively for the doses 35, 100 and 300 mg/kg
compared to the hypertensive group. The reference drug, amlodipine also caused a decrease (P < 0.001) in the urea level.
The glomerular ltration rate (GFR) was low (P < 0.001) in the hypertensive group compared to the control. During the treatment,
the concomitant administration of the extract and CdCl
2
led to an increase (P < 0.05) in this ow rate of 90.07 ±7.22 and 95.05 ±
7.62 mL/min/kg respectively for 100 and 300 mg/kg against 53.23 ±10.85 mL/min/kg for the hypertensive group (Fig. 6d).
Urinary debit was signicantly higher in the hypertensive group (2.903 ±0.474 mL/min/kg) compared to the control (1.449 ±
0.249 mL/min/kg) (Fig. 6e). The plant extract in concomitant administration with CdCl
2
caused a signicant decrease in the urinary
Fig. 4. Effects of different treatments on systolic (a) and diastolic (b) blood pressures Data are presented as mean ±SEM, n =6;
a
P<0.05,
b
P <
0.01 and
c
P < 0.001: compared to the control;
α
P < 0.05;
β
P < 0.01 and
γ
P < 0.001: compared to CdCl
2
; CdCl
2
=Cadmium chloride; X.s =
X. stuhlmannii; Amlo =Amlodipine.
A. Nkojap Kuinze et al. Heliyon 10 (2024) e38075
7
debit in all the assay groups, in the order of 1.483 ±0.262 mL/min/kg for the extract (100 mg/kg) vs. The hypertensive group.
The effects of AEXS on serum proteins are presented in Fig. 6f. No signicant variation between the different batches treated was
observed.
3.5. Effect of the aqueous extract of X. stuhlmannii on oxidative stress markers
The effects of AEXS on catalase and superoxide dismutase (SOD) activities, as well as malondialdehyde (MDA), reduced glutathione
(GSH) and nitrite oxide (NO) concentrations are summarized in Table 1.
Animals only treated with CdCl
2
showed a low catalase activity compared to controls. This decrease (P < 0.001) of activities
Fig. 5. Variation of serum transaminases activities after administration of leaf aqueous extract of X. stuhlmannii in rats Data are presented as mean
±SEM, n =6;
a
P<0.05,
b
P < 0.01 and
c
P < 0.001: compared to the control;
α
P < 0.05 and
β
P < 0.01: compared to CdCl
2
; CdCl
2
=Cadmium
chloride; X.s =X. stuhlmannii; Amlo =Amlodipine.
Fig. 6. Effects of different treatments on serum (a) and urinary (b) creatinine, serum urea (c), glomerular ltration rate (d), urinary debit (e) and
serum protein (f) Data are presented as mean ±SEM, n =6;
a
P<0.05,
b
P < 0.01 and
c
P < 0.001: compared to the control;
α
P < 0.05,
β
P < 0.01
and
γ
P<0.001: compared to CdCl
2
; CdCl
2
=Cadmium chloride; X.s =X. stuhlmannii; Amlo =Amlodipine.
A. Nkojap Kuinze et al. Heliyon 10 (2024) e38075
8
reached 60.75 %, 60.75 %, and 76.23 % for hepatic, renal, and aortic tissue, respectively, compared to control. Administration of the
extract (35 and 100 mg/kg) resulted in a signicant increase in catalase activity in the liver, heart aorta and kidneys tissues compared
to the hypertensive group. Amlodipine (1 mg/kg) used as reference substance also increased the catalase activity with respective
percentages of 70.93 % for the extract in the heart compared to hypertensive group.
It appears that the injection of CdCl
2
led to a reduction (P>0.05) in the activity of SOD in all tissue’s specimen. Treatment with the
extract or amlodipine resulted in an increase (P>0.05) at all doses tested in all organs assayed.
The administration of CdCl
2
dropped GSH levels in the kidneys. This decrease was 49.12 % (P < 0.001) compared to control.
However, administration of the plant extract or amlodipine concomitantly with CdCl
2
resulted in an increase (P < 0.001) in GSH levels
in the liver and kidney.
The treatment with CdCl
2
signicantly increased the activity of MDA in liver. This increase was 0.01196 ±0.001 mmol/g organ for
the control versus 0.00325 ±0.001 mmol/g organ for the hypertensive group. Concomitant administration of the extract with CdCl
2
resulted in a decrease in MDA activity. As for renal MDA, the concomitant administration of the extract and CdCl
2
led to a signicant
decrease (P < 0.001) in the activity of MDA at a dose of 100 mg/kg compared to the control and hypertensive groups, with respective
reduction percentages of 73.21 % and 70.92 %.
The level of NO signicantly (P < 0.01) decreased in the kidneys of rats receiving only CdCl
2
compared to controls. In addition, the
plant extract and amlodipine could not signicantly remediate this decrease in NO levels compared to the hypertensive group.
3.6. Effect of X. stuhlmannii on histological structures of aorta, heart, liver, and kidney
The effects of AEXS on the microarchitecture of the heart, aorta, kidney and liver are represented in Fig. 7. The aorta of control rats
showed a normal size of the media.
CdCl
2
-induced experimental hypertension is linked to the wall hypertrophy. The media size which was 36.50 ±1.00
μ
m for the
control and increased to 68.09 ±3.73
μ
m for hypertensive group (Fig. 7e). After administration of plant extract or amlodipine resulted
in a decrease of the media size of 36.64, 36.77, 35.23 and 36.06 % respectively at the dose of 35, 100, 300 mg/kg and amlodipine
(Fig. 7a).
At the level of heart, the chronic administration of CdCl
2
revealed leukocyte inltration for hypertensive group compared to
control. The different doses of extract and amlodipine presented a similar architecture to control, with normal nuclei and muscle bers
(Fig. 7b).
The liver section of control indicated normal architecture. However, in negative control, we observed a leukocyte inltration
compared to the control. The architecture of rat’s liver that concomitantly received CdCl
2
and extract or amlodipine highlighted a
structural organization similar to those of the control (Fig. 7c).
Table 1
Effect of X. stuhlmannii and amlodipine on oxidative stress markers in cadmium chloride-induced hypertension.
Parameters Organs Control CdCl
2
CdCl
2
+
X.s 35 X.s 100 X.s 300 Amlo
Catalase (
μ
mol of H
2
O
2
/min/g
organ)
Aorta 689.1 ±60.66 163.8 ±
12.69
c
445.7 ±76.26 363.6 ±88.06
a
279.5 ±69
b
287.7 ±
64.94
b
Heart 88.80 ±19.04 43.52 ±8.24 93.62 ±
13.51
α
109.7 ±5.62
β
104.2 ±2.97
β
74.92 ±7.20
Liver 133.1 ±16.24 52.24 ±
10.03
c
106.7 ±7.27
β
53.58 ±11.45
c
35.70 ±7.05
c
56.15 ±4.95
c
Kidneys 91.51 ±5.67 35.92 ±5.05
c
79.30 ±6.44
γ
73.43 ±5.46
β
61.03 ±5.75
a
61.40 ±6.58
a
α
SOD (unity of SOD/g Organ) Heart 327.5 ±29.81 318.2 ±9.66 421.2 ±38.86 449.4 ±56.67 428.4 ±62.15 300.8 ±23.51
Liver 288.9 ±18.27 222.8 ±32.39 178.8 ±11.23 249.8 ±60.16 259.6 ±49.12 207.8 ±39.04
Kidneys 177.3 ±21.18 117.8 ±6.41 122.3 ±7.28 149.2 ±12.13 158.1 ±21.64 146.3 ±9.77
Gluthatione (mmol/g Organ) Aorta 0.47 ±0.06 0.38 ±0.03 0.31 ±0.06 0.58 ±0.08 0.28 ±0.05 0.43 ±0.04
Heart 0.06 ±0.01 0.05 ±0.006 0.04 ±0.005 0.03 ±0.005 0.04 ±0.008 0.04 ±0.004
Liver 0.03 ±0.008 0.03 ±0.003 0.02 ±0.002 0.05 ±0.003 0.02 ±0.002 0.06 ±0.007
bγ
Kidneys 0.022 ±0.002 0.003 ±
0.001
c
0.006 ±
0.001
c
0.009 ±
0.0009
c
0.017 ±0.002
γ
0.016 ±
0.001
aγ
MDA (mmol/g Organ) Liver 0.003 ±0.001 0.011 ±
0.001
b
0.01 ±0.001
a
0.002 ±0.001
β
0.002 ±0.001
β
0.005 ±0.001
Kidneys 0.008 ±
0.0003
0.007 ±
0.0005
0.006 ±
0.0006
0.002 ±0.001 0.005 ±
0.0001
cγ
0.006 ±
0.0005
NO (
μ
mol/g organ) Aorta 2.33 ±0.40 1.44 ±0.25 1.32 ±0.56 1.43 ±0.64 2.23 ±0.19 2.05 ±0.12
Heart 0.16 ±0.004 0.17 ±0.01 0.15 ±0.01 0.18 ±0.01 0.12 ±0.01 0.14 ±0.01
Liver 0.065 ±0.006 0.046 ±0.005 0.062 ±0.004 0.059 ±0.007 0.043 ±0.005 0.044 ±
0.0008
Kidneys 0.084 ±0.005 0.054 ±
0.002
b
0.058 ±
0.005
a
0.052 ±0.006
b
0.056 ±0.004
b
0.054 ±
0.003
b
Data are presented as mean ±SEM, n =6;
a
P<0.05,
b
P < 0.01 and
c
P < 0.001: compared to the control;
α
P < 0.05,
β
P < 0.01 and
γ
P < 0.001:
compared to CdCl
2
; SOD =Super oxide dismutase, MDA =Malondialdehyde, NO =nitric oxide, CdCl
2
=Cadmium chloride; X.s =X. stuhlmannii;
Amlo =Amlodipine.
A. Nkojap Kuinze et al. Heliyon 10 (2024) e38075
9
The kidneys of control animals showed anormal glomeruli and the urinary space as well as convoluted tubules. Hypertensive
animals revealed glomerulosclerosis and leukocyte inltration. The concomitant administration of CdCl
2
and extract or amlodipine
highlight showed structural organization similar to those of the control rats (Fig. 7d).
3.7. UPLC-UV-ESI-TOF-MS analysis of X. stuhlmannii leaf extract
Fig. 8 shows the Base Peak Ion Chromatogram (BPIC) of the aqueous leaf extract of X. stuhlmannii, obtained by UPLC-UV-ESI-TOF-
MS in positive and negative ion modes. This chromatogram allowed us to visualize some of the chemical constituents with broad
polarity ranging from 5 to 100 % of organic solvent. Compounds 1–6were identied via reference compounds [15] as: stuhlmannione
A (1), formononetin (2), stuhlmarotenoid A (3), 9-methoxymaackiain (4), 4-hydroxymaackiain (5) and 7-hydroxy-3
′
,4
′
-methyl-
enedioxy-isoavone (6). (Table 2 and Fig. 7).
Fig. 7. Photomicrographs of Aorta (a), heart (b), liver (c) and kidney (d) (HE, 200×) and the effect of different treatments on media length (e).
Aorta: I =intima, M =media, A =adventice; Heart: No =nucleus, Fm =muscular ber, IL =leukocyte inltration; Liver: Vcl =centrilobular Vein,
He =hepatocyte, IL =leukocyte inltration; kidneys: G =glomerulus, Gse =glomerulosclerosis, EU =urinary space. Data are presented as mean ±
SEM, n =6;
c
P < 0.001: compared to the control;
γ
P < 0.001: compared to CdCl
2
; CdCl
2
=cadmium chloride; X.s =X. stuhlmannii; Amlo
=amlodipine.
A. Nkojap Kuinze et al. Heliyon 10 (2024) e38075
10
4. Discussion
The study purposed to assess the effects of the aqueous leaves extract of X. stuhlmannii against cadmium chloride-induced hy-
pertension in rats and oxidative stress. The aqueous extract was adopted for experiment to follow the traditional use of the plant
material by local population and traditional healer as observed during ethnobotanical investigations. Cd is widely used in various
Fig. 8. Base peak chromatograms of leaf aqueous extract of X. stuhlmannii obtained by UPLC-UV-ESI-TOF-MS. The number on each peak corre-
sponds to the compound numbers presented in Table 2.
A. Nkojap Kuinze et al. Heliyon 10 (2024) e38075
11
industrial applications and is an ubiquitous environmental toxicant, causing oxidative stress and hypertension [25].
CdCl
2
intraperitoneal injection in rats for ve weeks provoked an increase of SBP and DBP compared to controls. By acting as a
partial agonist of calcium channels, cadmium produces a direct contractile response on vascular smooth muscle, or could also act to
alter cation transport, leading to an increase in blood pressure [26–30]. Treatment of hypertensive animals for two weeks with AEXS
lowered blood pressure at all doses tested. This antihypertensive activity could be related to the extract’s interference with calcium
channels or to variable protective effects of its metabolites on the cardiovascular system [31].
When penetrate in the body, cadmium specically accumulates in the heart, aorta, liver, and kidneys where it can induce severe
tissue damage [32]. In this study, a signicant increase in the weight of lungs, liver and kidneys was observed in all assay groups
compared to controls. Thus, once absorbed by the gastrointestinal tract, cadmium is transported in direction to the liver and kidneys.
The high weight of hepatic and renal tissues should be an indication of the presence of this heavy metal bound to metallothionein (MT)
in the cytoplasm of their cells. When the hepatic concentration of MT becomes insufcient to bind to cadmium, it causes hepatocyte
membrane lyses and promotes the release of Cd-MT complexes into the bloodstream in direction to the kidneys [33]. The extract could
not reverse the increase in the relative weight of these organs.
AST and ALT constitute the key functional indicators used to detect liver damage or functional integrity [34]. Previous results
revealed an increase in serum transaminase levels during chronic administration of CdCl
2
in rats [35]. Indeed, the extract counteracted
the liver damaging by activating the thiol group thus preventing the cadmium from binding to the thiol group; or else by inactivating
the Kupffer cells and some inammatory mediators. Alternatively, the extract may provide protection by stabilizing the cell membrane
in liver damage-associated Cd [36,37]. The histological analysis of the rats’liver allowed to conrm this result, since it has been
observed a reduction of leucocytes inltration, a well-differentiated architecture of the centrilobular vein and of hepatocytes in AEXS
treated rats.
Chronic Cd poisoning mainly target kidneys [2]. The exposition of animals to this metal, could led to the development of renal
tubular dysfunction [38]. Our study revealed that the administration of Cd resulted in an increase in serum creatinine, serum urea, and
urinary debit but a decrease in urinary creatinine and GFR. These dysfunctions are caused by the cadmium-thionein complex, which is
very nephrotoxic [39]. Administration of AEXS for a period of two weeks has been able to decline the rate of urea in the blood, as well
as the urinary debit. At the dose of 300 mg/kg, the extract signicantly increased the GFR compared to hypertensive group,
demonstrating the ability of the aqueous extract to remove the creatinine from the blood in the urine. These results were conrmed by
histopathological analysis of the kidneys which showed protective effects in AEXS treated rats compared to the control.
Oxidative stress is another manifestation of CdCl
2
damage playing an important role in the toxicity of many xenobiotics [35].
The ability of Cd to induce oxidative stress has been established in vivo,in vitro, and in some epidemiologic studies [40]. AEXS
exerted a remarkable antioxidant activity in vitro and in vivo.
The in vitro antioxidant effect of AEXS was evaluated using various known assays [41]. From our results, AEXS developed DPPH
radical scavenging activity with an IC
50
lower than that of the ascorbic acid. Indeed, in alcoholic solution, DPPH forms a stable free
radical which can be stabilize by antioxidant chemicals through hydrogen releasing or electrons transferring [42]. The phytochemical
analysis revealed an interesting concentration of phenols in the extract, which are rich in hydroxyl groups [43]. Thus, antioxidant
molecules such as phenols scavenge DPPH radicals by donating hydrogen or electrons [44]. FRAP test was performed and the extract
reveals an activity. Indeed, reducing agents such as OH groups enriched phenols present in extract reduce ferric iron (Fe
3+
) into ferrous
iron (Fe
2+
) by donating electrons [45]. Moreover, the ABTS
+
radical scavenging capacity observed with the AEXS was signicant.
DPPH and ABTS are two free radicals used to bring evidence of the antiradical potential of substances. However, the ABTS assay strictly
relies on hydrogen atom transfer [46]. AEXS has developed lower antiradical activity than the standard (trolox) by reducing ABTS
+
cation radical through donating protons [47]. Excessive generation of NO is related to a number of pathological conditions, including
intracellular oxidative damage and cell death [48,49]. The extract inhibited the nitrite formation by direct competition with oxygen in
its reaction with NO, hence protecting cells and organelles from damages [50,51]. The hydroxyl radical was formed by the
Fe
3+
-ascorbate-EDTA-H
2
O
2
system. The scavenging activity on OH radical was measured through inhibition of deoxyribose degra-
dation [20]. AEXS at all concentrations tested inhibited the degradation of deoxyribose. Thus, when the extract was added to the
reaction mixture, it removed the hydroxyl radicals from the sugar to prevent reaction [50]. Overall, the antioxidant activity of the
Table 2
Identied phenols compounds in X. stuhlmannii leaf extract using UPLC-UV-ESI-TOF-MS.
Compound
Number
Retention time
(min)
MS (m/z) Ion
adducts
Molecular
Formular
Proposed identication References
13.62 387.1077 [M+H]
+
C
20
H
18
O
8
Stuhlmannione A Mekuete et al.
[15]26.11 269.0825 [M+H]
+
C
16
H
12
O
4
Formononetin
36.20 493.1872 [M −H]
-
C
28
H
30
O
8
Stuhlmarotenoid A
47.35 313.2388 [M −H]
-
C
17
H
14
O
6
9-methoxymaackiain Mekuete et al.
[15]
57.54 301.1417 [M+H]
+
C
16
H
12
O
6
4-hydroxymaackiain Mekuete et al.
[15]
68.21 265.1479 [M −H]
-
C
16
H
10
O
4
7-hydroxy-3
′
,4
′
-methylenedioxy-
isoavone
Mekuete et al.
[15]
78.32 341.2670 [M+H]
+
C
19
H
16
O
6
Unknown /
88.46 271.2282 [M −H]
-
C
15
H
14
O
5
Unknown /
98.56 297.2446 [M −H]
-
C
17
H
14
O
5
Unknown /
A. Nkojap Kuinze et al. Heliyon 10 (2024) e38075
12
extract could be partially attributed to its phenols content. Nevertheless, the quantied value (26.48 ±2.89 mg GAE/g) of phenols was
considered not as much, thus explaining the lowest activity of extract compared with the reference compounds (ascorbic acid and
trolox) tested. In addition, the antioxidant activity of phenolic compounds varies remarkably, depending on their chemical structure,
such as the position occupied by the -OH group in the whole molecule [52]. Furthermore, some authors such as Salim et al. [53] and
Lee et al. [54] have shown that the selection of the solvent for extraction is benecial to have a maximum of secondary metabolites
(like phenolic compounds). This could be observed for example by the strong antioxidant power (IC
50
=42,653
μ
g/mL) of the
methanol-chloroform extract of Chenopodium murale as compared to that of ascorbic acid (IC
50
=55,004
μ
g/mL) during DPPH test
[53]. Most of the identied compounds have only have 1 or 2 OH functional groups, which is not too strong for antioxidant. The plant
also contains many other metabolites that have not been identied as contributing to the extract’s biological activity. Phytochemical
studies are underway to provide more information about the plant’s metabolite content.
Antioxidant enzymes contribute as essential part of the cellular defense against reactive oxygen species (ROS) [55]. Cd adminis-
tration was associated with a reduction of aortic, hepatic and renal catalase, renal glutathione, and renal NO compared to control. Cd
has been shown to directly inhibit catalase activity via an interaction between cadmium and the enzyme, which leads to a disruption of
the enzyme’s topography, important for catalytic activity [56]. The treatment with different doses of AEXS had increased the liver and
kidneys catalase. The GSH is a non-enzymatic antioxidant. The administration of Cd to rats reduces renal glutathione compared to
controls. Cd could increase oxidative stress by damaging the antioxidant defense systems of cells by depleting GSH [57]. But, the
administration of the extract at the dose of 300 mg/kg has increased the level of renal GSH. The extract might play an important role in
the metabolism of GSH, thus increasing intracellular antioxidant power [58]. Another study demonstrated that cadmium-induced
hypertension is linked with the decrease of NO [59]. This potent free radical can switch endothelial nitric oxide synthase (eNOS).
The extract was not able to increase the level of renal NO. It may be possible that the extract does not signicantly interfere on
peroxynitrite formation. A direct link exists between the level of tissue damage and the quantity of MDA produced [60]. Hence, the
amount of MDA can be used as an index of peroxidative damage in vivo and the evaluation of the sensibility of tissues to underlying
oxidative stress. The elevated level of MDA in the liver of hypertensive group compared to controls may be due to the increase of
membrane lipid peroxidation. The reduction in MDA levels by AEXS treatment could be due to its ability to attenuate Cd-induced lipid
peroxidation. Histological alterations observed in the aorta section showed the increase of media size of the hypertensive group
compared to controls. For all groups tested, AEXS remedied all alterations, since no signicant difference was observed between the
cardiac architecture of normal control animals and that of all groups tested. The extract could therefore potentially contribute to
restoring Cd-induced cardiac damage [61].
The step of identication of some phenols in AEXS was carried out using UPLC-UV-ESI-TOF-MS data compared to those of the
isolates obtained during previous investigation of X. stuhlmannii leaf extract [15]. These metabolites mainly belong to the isoavonoid
and pterocarpan classes, which are classes of compounds which characterize Fabaceae family. Isoavonoid belongs to the avonoid
family, which is a group of bioactive polyphenolic compounds abundant in dietary plants and herbs. The avonoids proved to have
cardiovascular (hypertension) benets affecting blood pressure [62]. The isoavonoid formononetin attenuates oxidative stress [63]
and hypertension. The antihypertensive effect of this compound may cause vasodilatation, probably due to the inhibition of
voltage-dependent Ca
2+
channels and intracellular Ca
2+
release and by release of NO [64]. Additionally, some studies revealed the
strongest antioxidant potential of compounds belonging to the pterocarpan class [65].
5. Conclusion
We can conclude that the aqueous leaf extract of AEXS exhibited scavenging activities on DPPH, ABTS
+
, OH
−
and NO radicals, and
expressed interesting ferric reducing power. The extract also enhanced signicant decreased of systolic and diastolic blood pressures as
well as heart rate, which is correlated with a reduction in serum transaminases activities, urea concentrations, and urinary debit while
increasing the glomerular ltration rate at all the doses tested. Therefore, AEXS leaf extract has in vitro antioxidant and antihyper-
tensive effects that may support its use against hypertension. Secondary metabolites such as stuhlmannione A (1), formononetin (2),
stuhlmarotenoid A (3), 9-methoxymaackiain (4), 4-hydroxymaackiain (5) and 7-hydroxy-3
′
,4
′
-methylenedioxy-isoavone (6), were
identied in AEXS. They belong to isoavonoid and pterocarpan classes, which are known to possess antioxidant and antihypertensive
effects. We intend in further studied to isolate the identied active compounds and elucidate the mechanism of activities underlining
the observed antioxidant and antihypertensive effects of AEXS which were reported in the current study.
Ethics statement
The Institutional Ethics Committee for Research on Human Health (CEI-UD) of the University of Douala, Cameroon approved (Ref
N
o
3082 CEI-UDo/05/2022/T) all experimental protocols. Procedures and animals handling were conducted according to animal
welfare guidelines of the NIH publications N
o
8083, revised 1978.
Data availability
Data will be made available on request.
A. Nkojap Kuinze et al. Heliyon 10 (2024) e38075
13
CRediT authorship contribution statement
Augustine Nkojap Kuinze: Methodology, Investigation, Conceptualization. Edwige Laure Nguemfo: Conceptualization. William
Nana Yousseu: Writing –review &editing, Writing –original draft, Formal analysis, Data curation. Jacquy Joyce Wanche Kojom:
Methodology, Conceptualization. Calvin Zangueu Bogning: Methodology, Conceptualization. Christelle St´
ephanie Sonfack:
Methodology. Willifred Tsopgni Dongmo Tekapi: Writing –original draft, Formal analysis. Timo D. Stark: Writing –review &
editing, Writing –original draft, Funding acquisition. Guy Blaise Anatole Azebaze: Methodology, Formal analysis. Alain Bertrand
Dongmo: Writing –review &editing, Writing –original draft, Supervision, Conceptualization.
Declaration of competing interest
The authors declare the following nancial interests/personal relationships which may be considered as potential competing in-
terests:DONGMO Alain Bertrand reports equipment, drugs, or supplies was provided by Alexander von Humboldt Foundation. If there
are other authors, they declare that they have no known competing nancial interests or personal relationships that could have
appeared to inuence the work reported in this paper.
Acknowledgements
This work was supported by the Alexander von Humboldt Foundation Germany, through a donation of research equipment
Abbreviations
AEXS Aqueous extract of Xeroderris stuhlmannii
DPPH 2,2-Diphenyl-1-picrylhydrazyl
FRAP Ferric reducing antioxidant power
ABTS 2,2
′
-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid
NO Nitric oxide
IC
50
Median inhibitory concentration
SBP Systolic blood pressure
DBP Diastolic blood pressure
HR Heart rate
ALT Alanine aminotransferase
AST Aspartate aminotransferase
GFR Glomerular ltration rate
SOD Superoxide dismutase
GSH Reduced glutathione
MDA Malondialdehyde
MT Metallothionein
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