Effects of Hydrocotyle bonariensis Comm ex Lamm (Araliaceae) leaves

extract on the prevention of glucose-induced hypertension in wistar rats.

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Komla Kabouaa, Tcha Pakoussia, Aklesso Mouzoua, Balakiyém Kadissolia, Mindede Assiha,
Aboudoulatif Diallob, G'massampou Datagnia, Patric Boisc, Kwashie Eklu-Gadegbekua.

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o a : Laboratoire de Physiologie-Pharmacologie, FDS, Université de Lomé, BP
1515, Lomé, Togo
o b : Laboratoire de Pharmacologie-toxicologie, FSS, Université de Lomé, BP
1515, Lomé, Togo
o c : Laboratoire de Signalisation et Transport Ionique Membranaire, Pôle
Biosanté, Université de Poitiers, BP 86000, Poitiers, France

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Corresponding author : Komla Kaboua. Email : innocentfokomlan@gmail.com; tel : 00228
90 59 50 93

Highlights:

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Hydrocotyle bonariensis prevents the development of high blood pressure ;
 Hydrocotyle bonariensis has beneficial effects on biological markers of cardiovascular
diseases ;
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 The crude hydroethanol extract of hydrocotyle bonariensis contains 27 bioactive
compounds.

ABSTRACT
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Ethnopharmacological relevance
Pharmacological research, mainly focused on natural substances in Africa, contributes to the
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optimisation of traditional know-how by applying scientific biomedical methods. Thus, the

pharmacological study of medicinal plants with anti-hypertensive properties remains a major
priority in order to find convincing solutions against hypertension, which is not only the main
risk factor for cardiovascular diseases but also one of the main causes of death in the world.
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The aim of the study

This study evaluates the preventive potential of Hydrocotyle bonariensis against hypertension
induced in wistar rats by chronic dietary glucose overload. This plant is traditionally used in
the prevention and treatment of this pathology. Next, the phytochemical content of the plant
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was explored.
Material and methods
The impact of the hydroethanol extract of H. bonariensis on the prevention of induced
hypertension in wistar rats was evaluated by oral administration of different doses of this
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extract, compared to positive and negative controls. Blood pressure levels and cardiovascular
biomarkers were compared. The phytochemical study of the extract was done by HPLC
chromatography and then HPLC coupled with mass spectrometry.

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 Results
We confirmed the capacity of the extract to inhibit the development of arterial hypertension by

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intensive chronic glucose consumption in wistar rats. In addition to lowering blood pressure,
the extract was found to have mainly beneficial antioxidant, anti-inflammatory, lipid and
nephroprotective biochemical effects. HPLC analysis alone revealed five major peaks while
LCMS revealed 33 compounds in the crude extract.

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Conclusion
The present study demonstrated that Hydrocotyle bonariensis, a medicinal plant traditionally
used to prevent and treat hypertension, is endowed with hypertension preventive capacity and
reduces cardiovascular risk factors. These effects are thought to be due to its richness in
phytochemicals as revealed by chromatographic analysis. Our observations justify the

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traditional use of this plant for the prevention and treatment of hypertension.

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GRAPHICAL ABSTRACT

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 1. INTRODUCTION

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Highly prevalent and a major risk factor for cardiovascular disease (CVD), hypertension (HTA)
is the leading cause of death worldwide (WHO, 2021). Despite technological developments in
favour of conventional medicine in recent years, this disease, like all non-communicable
chronic diseases, is still poorly controlled. In Africa and Asia, 80% of the population still use

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traditional rather than modern medicines for primary health care (WHO, 2020). These
populations use medicinal plants as first-line treatment for various conditions including
hypertension. These treatments are poorly evaluated, both in terms of frequency of use and
benefit/risk ratio for the populations (Fyhrquist et al., 2006). Natural products play a major role
in the discovery of new drugs. They are therefore a significant source of potential remedies to
combat the development of various forms of resistance to current treatments (Koehn et al.,

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2005). Hydrocotyle bonariensis Comm. ex Lam. is one of the medicinal plants of the Araliaceae
family highly valued in the traditional management of several diseases in Africa. It is a glabrous

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herbaceous perennial plant with numerous horizontal creeping stems rooted at the nodes. It
grows preferentially in large sandy and gravelly riparian areas (Hackbart & Cordazzo, 2003).
According to Burkill (1985) and Monyn et al.(2016), Hydrocotyle bonariensis is used by
traditional medicine practitioners to treat high blood pressure. It is also used as a sedative,
against kidney and liver problems, tuberculosis, rheumatoid arthritis and many other conditions
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(Evan, 1992; Masoumian et al., 2011). Our previous work has demonstrated its anti-arrhythmic
cardiac effects (Kaboua et al, 2021a) as well as its safety by oral administration (Kaboua et al,
2021b). Other pharmacological studies of this plant have revealed its antioxidant and anti-
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inflammatory properties (Masoumian et al., 2011). But the evaluation of its antihypertensive
properties by experimental induction of hypertension in laboratory animals has not yet been
scientifically studied. Considering the intensity of the impact of hypertension on the health of
populations, we set ourselves the objective of evaluating the preventive anti-hypertensive
properties of the hydro-ethanolic extract of the leaves of Hydrocotyle bonariensis, by
experimental induction of hypertension based on a chronic oral administration of D-glucose
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10%. More specifically, the preventive effects of this extract will be evaluated on biological
markers and cardiovascular risk factors at the end of the experimental period. A preliminary
phytochemical study of this plant will also be carried out through a chromatographic analysis
of it’s crude extract used for the pharmacological studies.
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Keywords: Hydrocotyle bonariensis, hypertension, D-glucose, risordan, HPLC analysis

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2. MATERIAL AND METHODS

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2.1. Preparation of hydro-ethanolic extract

The leaves of Hydrocotyle bonariensis were collected in the botanical garden of the Faculty of
Sciences of the University of Lomé. These leaves were identified at the Laboratory of Botany
and Plant Ecology of the Faculty of Sciences of the University of Lomé and deposited in the
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herbarium under the identification number TG 15183. After harvesting, they are washed
abundantly with tap water and then dried under air-conditioning at 20°C in the dark.

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 The leaves of Hydrocotyle bonariensis (Araliacea) after 3 weeks of drying were pulverised and
then 320 grams were macerated for 72 hours with intermittent stirring in a hydro-ethanol solvent

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in an equal volume ratio (1:1). After maceration, the solution was filtered through filter paper
and cotton. The filtrate obtained was evaporated under vacuum using a rotavapor at 45ºC. 68.28
grams of extract were obtained, giving a yield of 21.34%. The extract obtained was kept cool
for the preparation of the different concentrations to be used for the tests.

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2.2. Experimental induction of hypertension
2.2.1 Preparation of the animals
Wistar Albino rats, weighing between 180 and 250 g, were used. The animals were reared in
the animal house of the Physiology-Pharmacology Laboratory of the Faculty of Sciences

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(University of Lomé), under standard light (natural day/night cycle of 12 h) and temperature
(25°C). The rats were fed with a standard diet. Principles of laboratory animal care as described

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in institutional guidelines and ethics of Laboratory of Physiology/Pharmacology of University
of Lome-Togo (ref: 001/2012/ CB-FDS-UL) were followed.

2.2.2 Experimental design er

The effect of H. bonariensis extract on D-glucose induced hypertension was evaluated.
Normotensive rats were randomly divided into five groups of six animals each, which received
different gavage treatments. Group I received tap water and served as a negative control (NC),
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group II received a 10% solution of D-glucose in drinking water as a positive control (PC).
Groups III and IV were the treatment groups which each received 10% D-glucose in drinking
water and the plant extract by gavage at 200 mg/kg/day and 400 mg/kg/day, respectively. Group
V received D-glucose in drinking water and risordan at 10 mg/Kg by gavage. The experiments
lasted 3 weeks. At the end of the experiment, mean arterial pressure (MAP), systolic blood
pressure (SBP) and diastolic blood pressure ( DBP) were measured as described by Kadissoli
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et al. (2019).
After blood pressure measurement, blood was collected from the retroorbital sinus. The serum
was separated and different types of cardiovascular biomarkers were measured. The antioxidant
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potential of the extract was determined in the serum using the "MAK334 Antioxidant Assay
Kit, Sigma-Aldrich". This kit measures the total antioxidant capacity in which Cu2+ is reduced
to Cu+ by an antioxidant. The resulting Cu+ specifically forms a coloured complex with a dye
reagent. The intensity of the colour at 570 nm is proportional to the TAC in the sample. Lipid
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markers such as total cholesterol, high density lipoprotein (HDL), low density lipoprotein
(LDL) and triglyceride levels were measured. C-reactive protein (CRP), leukocyte
sedimentation rate (VS) and white blood cell levels were measured as markers of inflammation.
As biochemical factors, creatinine, urea and creatine kinase were measured. Blood glucose and
ionogram were also measured as indicators of cardiovascular disorders.
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2.3 phytochemical study of the extract

2.3.1 HPLC analysis
In order to confirm the chemical composition of the extract, we performed an HPLC analysis
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using the method of Millot et al, (2017). The analysis was performed on a Dionex UHPLC
U3000RS system equipped with a LPG-3400RS quaternary pump, a WPS-300T RSLC
automatic injector, a TCC-300SD column oven and a UHPLC+ DAD-3000 diode array detector

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 (ThermoFisher SA, Voisins le Bretonneux, France). The system was equipped with an
Accucore aQ C18 column (15 cm × 3 mm i.d., particle size 2.6 µm), itself protected by a

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Securiguard (Thermofisher). The mobile phases were solvent A 0.1% trifluoroacetic acid (TFA)
in water and solvent B acetonitrile. The gradient was set as follows: The initial acetonitrile
content was 0%, increased to 10% in 5.24 min, 15% in 0.32 min, 20% in 0.31 min, and 100%
in 0.32 min and held for 4 min. The flow rate was set at 0.8 mL/min, the column temperature
was set at 40 °C and the injection volume was 8 µL. An HPLC chromatogram of H. bonariensis

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was recorded at 220 nm and is shown in Figure X

2.3.2 Liquid chromatographic coupled with mass spectrometry (LCMS) analysis

Chromatographic separation was performed using a Waters Acquity UPLC system on an

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Acquity BEH C18 column (1.7 μm, 2.1 x 100 mm, Waters, Milford, MA) eluted at a constant
flow rate of 0.4 mL/min and placed at 40°C. Separation was carried out by a mobile phase

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gradient with solvent A consisting of water containing 0.1% (v/v) formic acid and solvent B
consisting of acetonitrile containing 0.1% formic acid according to the following schedule: 10%
B for 10 min, followed by a 30 min linear gradient passing through 100% B, held at 100% B
for 5 min before returning to 10% B in 15 min. Mass spectrometric detection of the compounds
in the crude H. bonariensis extract (prepared as described in Herbiniere et al., 2005) was
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performed by electrospray ionisation (ESI-MS) with a Xevo Q-TOF mass spectrometer
(Waters, Milford, MA, USA). The samples were suspended in a methanol/acetonitrile/water
(75%/20%/5%)v/v mixture. LC-MS mass spectra were performed in positive mode with a cone
voltage of 40 V. The electrospray voltage was set at 3.0 kV, the source temperature at 120 °C
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and the desolvation temperature at 450 °C. Data acquisition and control of the apparatus was
performed using MassLynx software version 4.1 (Waters, Milford, MA, USA).

2.4 Statistical analysis

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The results are expressed as mean ± standard error of the mean. They are processed with
GraphPad Prism 8.0 software, which was also used to construct the histograms. Analysis of
variance (ANOVA) is used. The results are significant if the p-value < 0.05.
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3. RESULTS
3.1 Effect of the extract on blood pressure
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Chronic administration of 10% glucose solution to normotensive rats for 3 weeks caused a
significant mean increase in MAP, DBP, and SBP of 60.95 mmHg; 60.71 mmHg; and 61.43
mmHg, respectively, compared with the control (Figure1). In NTRs treated concomitantly with
D-glucose solution and plant extract (200 and 400 mg/kg) or risordan (10 mg/kg) for the same
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experimental duration, it was observed that both plant extract and risordan are able to prevent
the elevation of MBP, DBP, and SBP caused by chronic Dglucose consumption. Compared
with rats treated with glucose alone, MBP decreased by 49.78 mmHg and 60.65 mmHg at 200
and 400 mg/kg, respectively. A proportional decrease in DBP and SBP was observed under the
same conditions. Risordan caused a mean reduction in MBP of 37.27 mmHg compared with
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controls. Dglucose administration induced hypertension in wistar rats. This induction was
significantly reduced in the presence of the extract at treated doses but the reduction was not
significant in the presence of risordan 10 mg/kg.

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 MBP

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SBP
DPB

200 **

**
**

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Blood pressure (mmHg)

# #
150 #

##
##

##
100

v
50

re
0
G ord (G r

iso % ater

iso % ater
+H 0 m g

+H 0 m g

+H 0 m g
00 kg

kg

00 kg

kg

00 kg

kg
+H 0 m )

+H 0 m )

+H 0 m )
% an 1 0%

% n 1 0%

% n 1 0%
is % ate

% B20 g/k

% B20 g/k

% B20 g/k
B4 g/

g/

B4 g/

g/

B4 g/

g/
w

w
m

m
1

1
(G

(G
+R 10

+R 10

+R 10
10 da

10 da
r

r
% se

er
os

os
o
G luc

G luc

uc
10

10

10

10
l
G

G
%

%
G

G
10

10

10
G

Figure1: Effects of hydroethanol extract of H. bonariensis on blood pressure of glucose-treated rats. Each
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bar represents the mean ± S.E.M (2ways ANOVA + Turkey test) n=5. **P<0.01 compared with the normotensive
rat group. #P<0.5; ##P<0.01 compared with the glucose-treated rat group. MBP: mean blood pressure; SBP:
systolic blood pressure; DBP: diastolic blood pressure.

3.2 Effects of H. bonariensis extract on total antioxidant capacity in rats treated with 10% D-
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glucose
Figure 2 shows a significant decrease in the TAC of rats after 3 weeks of treatment with 10%
D-glucose compared to control. This decrease is significantly corrected in rats concomitantly
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treated with D-glucose and H. bonariensis extract at doses of 200gm/kg and 400mg/kg,
respectively by 71.77% and 75.71% compared to rats treated with D-glucose alone.
Simultaneous treatment of rats with D-glucose 10% and risordan 10mg/kg induced an
improvement in TAC but which was not significant compared to rats treated with D-glucose
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alone.
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 ####
####
150

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water
Glucose 10% (G10%)
% of change of TAC

G10%+Risordan 10mg/kg
100
G10%+HB 200 mg/kg

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G10%+HB 400 mg/kg
****

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Figure 2: Effects of H. bonariensis hydroethanol extract on total antioxidant capacity of glucose-treated

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rats. Each bar represents the mean ± S.E.M (ANOVA 2ways + Turkey test) n=5. ****P<0.0001 compared with
the normotensive rat group. ####P<0.0001; ####P<0.0001 compared with the glucose-treated rat group. TAC:
total antioxidant capacity; HB: Hydrocotyle bonariensis

3.3 Effects of H. bonariensis extract on some markers of inflammation in D-glucose treated

rats 10%.
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Chronic administration of a 10% glucose solution to normotensive rats for 3 weeks caused a
slight increase in C-reactive protein (CRP) and leukocyte count compared with the control. The
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increase in CRP was significantly corrected by 88.52% in the group of rats concomitantly
treated with 10% glucose and H. bonariensis extract at the dose of 400 mg/kg (Figure3).
Compared to the negative control group, the sedimentation rate of the glucose-treated group of
rats was very significantly elevated (250% compared to the distilled water-treated rats).
Risordan 10 mg/k, H.bonariensis extract at doses of 200 mg/kg and 400 mg/kg prevented the
increase of SV of rats treated simultaneously with glucose and these products by 150%, 250%
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and 275% respectively compared to rats treated with glucose only. Thus, the variatioin in withe
blood cell count was small and not significant when comparing treated rats to controls. In
contrast, the increase in SV and CRP of glucose-only treated rats was significantly inhibited in
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rats treated with the extract and in rats treated with risordan, a reference vasodilator.
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 % of change of inflammation markers CRP
500

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SR

****
RBC
400

300

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##

####
####
200
#
100

v
G sord (G er

G sord (G er

G sord (G er
g

g
g
kg

00 /kg

kg

00 /kg

kg
G +H 10 m )

G + H 10 m )

G +H 10 m )
% an 10%

% an 10%

% an 10%
% 20 g/k

% 20 g/k

% 20 g/k
k
% at

% at

% at
/
g/

g/

g/
B4 mg

B4 mg

B4 mg
w

w
m

m
+H 0
00

+H 0

+H 0

re
10 B

10 B

10 B
+R 10

+R 10

+R 10
% se

% se

% se
10 co

10 co

10 co
i

i
10

10

10
G lu

G lu

G lu
G

Figure: Effects of H. bonariensis hydroethanol extract on some markers of inflammation in glucose-treated

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rats (RHG). Each bar represents the mean ± S.E.M (2ways ANOVA + Turkey test) n=5. ****P<0.0001 compared
to normotensive rat group (RNT). #<P; ##P<0.01; ####P<0.0001 compared to the glucose-treated group (RHG).
CRP: C-reactive protein; SR: sedimentation rate; RBC: red blood cell count; HB: Hydrocotyle bonariensis.
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3.4 Effects of H. bonariensis extract on some lipid markers in D-glucose-treated rats
Figure 4 shows that the changes in lipid markers of rats treated with glucose alone and rats treated with
glucose and extract (or glucose and risordan) simultaneously, including triglyceride, total cholesterol
and HDL levels were not significant. However, HDL levels decreased by 45.73% in rats treated with
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risordan 10 mg/kg (positive control) plus 10% glucose, compared to negative controls. Analysis of LDL
levels revealed that LDL levels were significantly elevated in rats treated with 10% glucose alone
compared to rats treated with water (negative controls). Compared to the group of rats treated only with
10% glucose, the LDL level of rats treated simultaneously with glucose and 200mg/kg and then
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400mg/kg of extract was significantly decreased by 130.7% and 118.6% respectively. In rats treated
with risordan 10mg/kg, the LDL level was decreased by 83.46%. The variation in lipid markers therefore
results in a significant decrease in LDL levels in rats treated with H. bonariensis extract.
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 TRG

ed
TC

HDL
250 LDL
% of change of lipid markers

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200

150

###
#

###
100

v
50

re
0
10 da (G er

10 d a (G e r

10 da (G er

10 da (G er
+H 00 /kg

+H 00 /kg

+H 00 /kg

+H 00 /kg
m g
kg

m g
kg

m g
kg

m g
kg
)

)
G +H 10 %

G +H 10 0%

G +H 10 %
00 /k

00 /k

00 /k

00 /k
t

t
g/

g/

g/

g/
i 0% wa

i 0% wa

i 0% wa

i 0% wa
% B2 mg

% B2 mg

% B2 mg

% B2 mg
B4 mg

B4 mg

B4 mg

B4 mg
% n 10

% n 10

% n 10
% n 1

G +H 10
+R 1

+R 1

+R 1

+R 1
G sor

G sor

G sor

G sor
% se

% os e

% ose

% ose
10 co

10 c

10 c

10 c
10

10

10

10
G Glu

G lu

G Glu

G Glu
er
G

Figure 4: Effects of H. bonariensis hydroethanol extract on lipid markers in glucose treated rats. Each bar
represents the mean ± S.E.M. (2ways ANOVA + Turkey test) n=5. *P<0.5 compared to normotensive group
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(RNT). #P<0.5; ###P<0.001; compared to the glucose-treated group (RHG). TRG: total triglycerides; CT: total
cholesterol; HDL: high density lipoprotein; LDL: low density lipoprotein, HB: Hydrocotyle bonariensis

3.5 Effects of H. bonariensis extract on some biochemical markers in rats treated with 10%
D-glucose
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Figure 5 shows the effect of H. bonariensis extract on some nephrological (urea and creatinine)
and cardiac (Creatine Phosphokinase) biochemical markers. Chronic administration of glucose
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resulted in a slight increase in urea and creatinine in rats treated with 10% glucose only. This
increase was not significantly reduced in rats treated simultaneously with glucose and plant
extract at 200 mg/kg and 400 mg/kg. In the analysis of CPK blood levels, the group of rats that
received only 10% glucose for 3 weeks had a significantly elevated CPK level of 69.95%
compared to the negative controls. Rats treated simultaneously with 10% glucose and risordan
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10 mg/kg, H. bonariensis extract 200 mg/kg and 400 mg/kg had a decrease in CPK levels of
32.70%, 67.10% and 88.34% respectively. This decrease was significant in rats.
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 urea

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Creatinine
% of change of biochemical markers

250
CPK

200
*

iew
150

##
100

50

v
0
G o rd (G e r

G o rd (G e r

G o rd (G e r
G +H 0 m )

G +H 0 m )

G +H 0 m )
0 0 /kg

kg

0 0 kg

kg

0 0 kg

kg
+H 0 0 kg

re
0%

0%

0%
% 2 0 g /k

% 2 0 g /k
is 0 % a t

is 0 % a t

is 0 % a t
g/

/
g/

/
g/
g/

B4 m g

B4 m g
w

w
10 a n 1

10 a n 1

10 a n 1
m

m
+H 0

+H 0
1

1
B2

B4

B
+R e 1

+R 1

+R e 1
% se
os

os
%

%
10 co
10

10

10
c

c
lu

lu

lu
G

G
%

%
10

10
G

er
Figure 5: Effects of H. bonariensis hydroethanol extract on some biochemical markers in glucose treated
rats. Each bar represents the mean ± S.E.M. (2ways ANOVA + Turkey test) n=5. *P<0.5 compared to
normotensive group (RNT). #P<0.5; ##P<0.01; compared to glucose-treated group (RHG). CPK: creatine
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phosphokinase; HB: Hydrocotyle bonariensis.

3.6 Effects of H. bonariensis extract on blood glucose levels in D-glucose treated rats
Chronic administration of 10% D-glucose to rats for 3 consecutive weeks resulted in a non-
significant increase in blood glucose levels (Figure 6). This non-significant increase observed
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in rats treated with glucose alone was non-significantly reduced when the glucose treatment
was simultaneously combined with treatment with risordan 10mg/kg, a conventional
vasodilator, or combined with treatment with hydroethanol extract of H. bonariensis at doses
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of 200 mg/kg and 400 mg/kg.

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10

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 water

ed
Glucose 10% (G10%)

G10%+Risordan 10mg/kg
Blood glucose concentration (g/l)

2.5
G10%+HB 200 mg/kg

2.0 G10%+HB 400 mg/kg

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1.5

1.0

0.5

v
0.0

re
5
Figure: Effects of H. bonariensis hydroethanol extract on blood glucose levels in glucose-treated
rats. Each bar represents the mean ± S.E.M. (2ways ANOVA + Turkey test. HB: Hydrocotyle
bonariensis er
3.7 Effects of H. bonariensis extract on the ionogram in D-glucose treated rats
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Figure 7 shows the effects of H. bonariensis extract on the variation of Ca2+, K+, Na+ and Cl-
ion concentration. The analysis of this figure reveals that the treatment of the rats with 10%
glucose alone or with glucose combined with risordan as well as with H. bonariensis extract did
not influence the plasma concentration of the rats after 3 weeks of treatment. No variation was
therefore noted in the ionogram.
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Ca2+
K+
Na+
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200 Cl-
Ionogram (mmol/l)

150
rin

100

50
ep

0
r

r
% B2 m )

% B2 m )

% 2 m )

% B2 m )
m g
kg

m g
kg

m g
kg

m g
kg
B4 m g

B4 m g

B4 m g

B4 m g
10 H 0 %

10 H 0 %

10 H 0 %

10 H 0 %
1 0 d (G te

1 0 d (G te

1 0 d a (G te

1 0 d a (G a te
0 0 g /k

0 0 g /k

0 0 g /k

0 0 g /k
+H 0 0 g /k

+H 0 0 g /k

+H 0 0 g /k

+H 0 0 g /k
g/

g/

g/

g/
G is o r 0 % wa

G s o r % wa

G s o r 0 % wa
G %+ n 1 10

G %+ n 1 10

G %+ n 1 10

G %+ n 1 10
is 0 % w
Pr

B
10
a

a
1

1
G or
+R e

+R e

+R e

+R e
% os

% os

% os

% os
i

i
10 c

10 c

1 0 lu c

10 c
G G lu

G G lu
l
G

G
G

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 Figure 7: Effects of H. bonariensis hydroethanol extract on the ionogram in glucose treated rats.
Each bar represents the mean ± S.E.M. (2ways ANOVA + Turkey test. HB: Hydrocotyle bonariensis.

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3.8 HPLC analysis
HPLC analysis of the freeze-dried hydroethanol extract showed the presence of five major

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polyphenol peaks at 220 nm and 265 nm. At this spectrum, the extract also showed small peaks
which indicate the presence of a small amount of other phenolic/polyphenolic compounds.
(Figure 8).

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Figure 8: HPLC chromatogram of the hydroethanol extract of H.bonariensis, recorded at 220 nm (black)
and 265 nm (pink).
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3.9 LCMSanalysis
The separation of the phytoactive compounds present in the hydroethanolic extract of H.
bonariensis was performed by LC-MS. The identification of the compounds is done on the basis
of their retention time, mass and spectrum. The peaks separated on the 0-100% acetonitrile
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gradient (see Figure 9). Mass spectrometry used positive ionisation (m/z)-1. The MS data
obtained with the individual compounds are summarised in Table 1, 27 compounds are
identified but not known.
ep
Pr

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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=3977444
 XEVO-QTOFMS#YAA141HB
20210519_Innocent_C18_pos_9 TOF MS ES+
BPI
100
39.72 3.43e3
28.53
39.64

ed
40.20

32.75
32.89

0.96 36.78
37.88
13.34 34.58
%

12.40 14.64

iew
22.81 29.03 36.49
15.56 22.16
5.59 17.82 27.01 29.44
8.82 21.94
24.82 26.26 30.24
3.57 19.97
16.15
20.47 23.82

0 Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

Figure 9: Recording of the LCMS analysis of the hydroethanol extract of H. bonariensis leaves.

v
Table1: Identification of compounds in the hydroethanol extract of H. bonariensis leaves. Eight major peaks
were identified, each corresponding to a different compound in the H. bonariensis extract. The retention time, the
spectrum and mass (m/z value) are specific to a particular compound.

re
COMPONENT
COMPONENT CORRESPONDING
RETENTION
MASSE (m/z) SPECTRUM (peak CORRESPONDING
TIME (min)
intensity in nm) er COMPONENTS
3.57 350.2515 553 Component 1
8.82 438.2906 683 Component 2
12.4 482.2979 1436 Component 3
pe
13.34 526.3063 1765 Component 4
13.87 570.3175 1622 Component 5
14.64 484.097 1467 Component 6
15.14 382.086 525 Component 7
15.79 657.271 775 Component 8
17.82 427.2553 819 Component 9
ot

18.18 304.1074 687 Component 10

18.88 472.3943 176 Component 11
19.97 370.262 498 Component 12
tn

20.47 419.2592 231 Component 13

23.82 440.241 286 Component 14
24.82 359.2148 586 Component 15
26.26 365.3825 614 Component 16
rin

28.53 457.287 3289 Component 17

32.75 508.2777 2574 Component 18
33.31 318.2371 1651 Component 19
33.86 486.1569 767 Component 20
ep

34.58 351.3064 1793 Component 21

39.72 571.406 3433 Component 22
40.2 414.2969 2884 Component 23
41.62 708.1108 3446 Component 24
Pr

42.37 685.3888 3983 Component 25

44.04 781.115 2904 Component 26
47.238 508.506 3841 Component 27

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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=3977444
 4. DISCUSSION
The antihypertensive effects of the hydro-ethanolic extract of the leaves of H. bonariensis were

ed
evaluated in wistar rats made experimentally hypertensive by glucose. In our study, the effects
of this extract were evaluated on D-glucose-induced hypertension as a preventive treatment,
during oral administration for 3 weeks. The results of this study showed that the administration
of H. bonariensis extract (200 and 400 mg/kg) prevented the rise in blood pressure in rats on a

iew
high glucose diet. This beneficial effect may involve a number of complex mechanisms. Indeed,
abnormal glucose metabolism is often accompanied by hypertension, and this association is as
well documented as the insulin resistance syndrome (Ferrannini et al., 1987; Hwang et al.,
1987). Experimental studies have shown that glucose and fructose contribute to the increase in
blood pressure (El Midaoui and De Champlain, 2002) through several mechanisms including
dyslipidaemia (Reaven and Ho, 1991), sodium retention, increased blood volume (Baum,

v
1987), stimulation of the sympathetic nervous system (Bunag et al., 1983), endothelial
dysfunction (Tomiyama et al., 2000), and increased oxidative stress (Cai and Harisson, 2000).

re
However, we evaluated the impact of the extract on some biological markers of cardiovascular
disease in rats treated with the extract, compared to controls, in order to better understand these
mechanisms.
For this reason, we evaluated the antioxidant capacity of the extract in this study of experimental
er
induction of hypertension by D-glucose by determining the total antioxidant capacity (TAC) in
the serum of treated rats compared to controls. Indeed, increased oxidative stress and excessive
free radical formation is implicated in the development of atherosclerotic lesions (Kattoo et al.,
2019). It is also well established that excessive sugar consumption is associated with increased
pe
production of reactive forms of oxygen in tissues (El Midaoui and De Champlain, 2002).
Furthermore, in hypertensive patients, the decrease in antioxidant defences is well documented
(Rodrigo et al., 2007). Our results showed that in this study model of hypertension, the
significantly reduced TAC of rats treated with glucose alone is very significantly corrected in
rats treated with the extract. These results suggest that the antioxidant properties of H.
bonariensis are a contributing factor to its beneficial effects on hypertension. They suggest that
ot

the extract induces an antioxidant effect probably due to the richness of this plant in bioactive
antioxidant compounds, which inhibit the excessive production of free radicals, as shown in the
studies of Massoumian et al (2011).
tn

In recent years, there has been considerable interest in the association between inflammation
and cardiovascular disease (Willerson et al., 2004). We therefore wished to study the
preventive potential of inflammation by analysing some biological data indicative of the
inflammatory state of the rats at the end of the 3 weeks of the experiment. Several biological
rin

markers, including C-reactive protein, erythrocyte sedimentation rate (ESR) and leukocyte
count, have been shown to be predictors of cardiovascular disease (Gillum & Makuc, 1995;
Andresdottir et al., 2003; Danesh et al., 2004). Our results indicate a non-significant elevation
of CRP and NGB, but a very significant elevation of SV in rats treated only with glucose. In
contrast, the levels of these markers were low in rats treated with 200 and 400 mg/kg of H.
ep

bonariensis extract after 3 weeks of treatment. These results indicate an anti-inflammatory

effect of the extract in rats. These effects are thought to be due to the presence of bioactive
phytochemicals with anti-inflammatory effects in the extract, which prevented or inhibited the
development of systemic inflammation in these animals (Massoumian et al., 2011).
Pr

Dyslipidemias result from abnormalities in the distribution of lipids (a collection of various

molecules such as free fatty acids, cholesterol, triglycerides and phospholipids) in the blood.
High blood levels of total cholesterol, triglycerides and/or LDL, as well as low levels of HDL,

14

This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=3977444
 are thought to be associated with a significant increase in the risk of high blood pressure (Dalal
et al., 2012). Thus, we wished to evaluate the impact of H. bonariensis extract on the lipid

ed
profile of rats at the end of the experiment. To do this, we measured the blood levels of
triglycerides, total cholesterol, HDL and LDL at the end of the 3 weeks from the blood sera of
the rats used in the experiment. The results we obtained show a significant increase in LDL
levels in the rats treated with glucose alone. This increase was also observed in triglyceride and
total cholesterol levels, but was not significant compared to the control group. Rats treated with

iew
H. bonariensis extract in addition to glucose administration had significantly lower LDL levels
compared to rats treated with glucose alone. HDL levels were slightly reduced in rats treated
with glucose alone, but this reduction was corrected in rats treated with H. bonariensis extract.
We conclude that the lipid metabolic disorder created by chronic administration of 10% glucose
to rats would be slowed in rats given glucose but treated with 200 mg/kg and 400 mg/kg of H.
bonariensis extract. Similar studies with other plants have shown beneficial effects on these

v
same lipid factors in the context of glucose-based experimental induction of hypertension (Tom
et al, 2011; kadissoli et al., 2019).

re
A relationship between the kidney and blood pressure (BP) has been established: on the one
hand, renal dysfunction can cause an increase in BP, while on the other hand, high BP
accelerates the loss of function of the diseased kidney (Hall, 2003). In order to determine the
potential impact of glucose administration as well as that of H. bonariensis extract on renal
er
function, we analysed some markers of renal function impairment, namely urea and creatinine.
Our results show a slight increase in urea and creatinine levels in the rats that took only 10%
glucose. For the rats treated with the extract plus glucose administration, the urea and creatinine
levels are relatively low, compared to the rats that took glucose only. Based on these results,
pe
we believe that H. bonariensis extract has an inhibitory effect on the mechanisms by which
altered renal function leads to the development of hypertension. This preventive effect would
be due to the presence in the extract of phytoactives with protective actions on renal and
cardiovascular functions such as polyphenols ( Kaboua et al., 2021b). In addition to renal
function, it is hypothesised that high activity of creatine kinase, the central enzyme regulating
energy metabolism, facilitates the development of hypertension (Brewster et al., 2006).
ot

Relatively high activity of the enzyme, especially in resistance arteries, would enhance pressor
responses, increasing blood pressure (Teixeira & Borges, 2012). For this purpose, we included
the analysis of the plasma concentration of creatine phospokinase (CPK) among the
tn

biochemical markers investigated in this study. In our analysis, the significant elevation of CPK
levels in rats that received only 10% glucose for 3 weeks was significantly reduced in rats
treated with H. bonariensis extract in addition to chronic glucose administration. This relatively
low level of CPK in rats treated with the extract demonstrates the protective effect of the extract
against cardiovascular damage that could be caused by chronic intensive glucose consumption.
rin

This action of the extract would be justified by its richness in compounds which would have a
biochemical impact on the metabolic energy regulation beneficial to the cardiovascular
function. Experimental pharmacological studies conducted on cardiovascular pathologies
involving renal function or energy metabolism have shown reducing effects on blood levels of
creatinine, urea, uric acid as well as CPK, by medicinal plant extracts (Kadissoli et al., 2019;
ep

(Yanardağ et al., 2002; Nahar & Akhter, 2018).

Although the direct link between blood glucose and the etiology of hypertension is not yet very
clearly established (Feldstein et al., 2008), hyperglycaemia is considered a factor in metabolic
and lipid disorders, increasing cardiovascular risk in both diabetic and non-diabetic subjects
Pr

(Maqbool et al., 2018. To investigate the potential impact of H. bonariensis extract on blood
glucose levels in rats that were used in the experiment to induce hypertension by chronic
administration of 10% gulucose, we performed a comparative analysis of blood glucose levels

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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=3977444
 in rats treated with the extract in addition to glucose, compared to rats that received glucose
alone. This study revealed a non-significant increase in blood glucose levels in the rats treated

ed
with chronic glucose alone, an increase not observed in the rats treated with the extract plus
glucose. This study confirms other studies previously conducted, which have shown a
modulation of blood glucose levels by medicinal and condiment plants (Kendler, 1987; Díaz-
de-Cerio et al., 2017).

iew
Electrolity disorders also remain a significant factor in terms of hypertension risk (Gobin et al.,
2014): Calcium in addition to its role on bone mass, promotes the relaxation of the artery walls
in case of vasoconstriction which is responsible for arterial hypertension. As for magnesium,
often associated with potassium, whose action it facilitates, it favours the dilation of the vessels
and therefore a drop in blood pressure. Chlorine is correlated with sodium in order to detect a
possible acid-base imbalance involved in hypertension and whose effects are felt in the kidney,
leading to renal insufficiency. Consequently, the study of the blood ionogram provides

v
information on the balance between the different ions in the plasma, the pertirbation of which
is an indicator of cardiovascular alteration (Semondji, 2018). We therefore evaluated the effect

re
of H. bonariensis extract on rats subjected to chronic administration of 10% glucose for 3
weeks. The analysis of our results reveals no variation in the blood levels of the ions measured
at the end of the experiment, namely Ca2+, K+, Na+ and Cl-. Neither in the rats treated with
glucose alone, nor in the rats treated simultaneously with glucose and the plant extract. This
er
would mean that the experimental conditions did not significantly affect the electrolyte balance
of the rats.
Risordan, containing mainly isosorbide dinitrate, is a vasodilator of the nitrate family (Thadani
et al., 1982). We used this drug as a reference product in our experimental study of induction
pe
of hypertension by chronic glucose overload in oral administration. The effects of the
hydroethanol extract of the leaves of Hydrocotyle bonariensis were therefore compared with
the effects of risordan, the latter serving as a positive control. All the parameters analysed were
compared between the group of rats treated simultaneously with 10% glucose and risordan and
those treated simultaneously with glucose and the two doses of extract studied (200 mg/kg and
ot

400 mg/kg). The results of this study show a non-significant decrease in MAP, DBP and SAP,
whereas it was significant for both doses of the extract. The results of the antioxidant capacity
analysis show a non-significant improvement in TAC of risordan treated rats. This
improvement was highly significant in the extract-treated rats. The results of the inflammation
tn

markers show a non-significant decrease in CRP in risordan-treated rats but a significant

decrease in rats treated with 400 mg/kg of H. bonariensis; a significant decrease in SV in
risordan-treated rats and a very significant decrease in rats treated with both doses of the extract;
a slight decrease in the number of leukocytes in risordan-treated rats, but a slightly larger
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decrease in rats treated with both doses of the extract. Regarding lipid markers, risordan caused
a non-significant decrease in triglyceride levels which was relatively greater than that of H.
bonariensis extract. Risordan rather decreased the HDL level while the extract had a very small
impact. LDL levels were significantly reduced by risordan and very significantly by both doses
of extract. The data of the few biochemical markers analysed indicate a weak impact on the
ep

blood urea level and a non-significant decrease of the creatinine and CPK levels. However, the
extract caused a significant decrease in CPK levels at both doses of the extract, and a non-
significant but larger decrease in blood urea and creatinine levels. The extract also resulted in a
greater decrease in blood glucose than risordan, although no significant decrease was observed.
The ionogram analysis indicates that both risordan and the extract did not influence the
Pr

extracellular concentration of Ca2+, K+, Na+ and Cl-.

An overall observation and comparative analysis of these results states that the hydroethanol
extract of H. bonariensis leaves at 200 mg/kg and 400 mg/kg would have a greater anti-
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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=3977444
 hypertensive preventive effect than risordan (nitrate isosorbite) at 10 mg/mg. The data for all
parameters analysed indicate that risordan has a milder effect on cardiovascular biomarkers

ed
than the extract at the doses used. These effects are thought to be a consequence of the multi-
targeting of the extract, due to its diverse bioactive phytochemical composition. As risordan has
a targeted endothiolar action, it is reasonable to note a low level of its actions on the various
cardiovascular risk factors. Another reason for the relatively low level of action of risordan
compared to the extract would be the dose administered. Not only is the indicated dosage for

iew
risordan 2 to 3 times daily, but the doses used of the extract seem to be quite high compared to
the administered dose of risordan. A decrease in the dose of the extract could have a similar
effect on some factors as the extract doses.
The literature indicates that more than 200 vasodilatory secondary metabolites have been
identified in plants over the last three decades, noting their structural diversity and multiple
mechanisms of action. Nevertheless, their chemical natures have allowed them to be classified.

v
From HPLC and LCMS analyses of H. bonariensis extract, peaks corresponding to the major
bioactive phytochemicals were identified. However, this analysis does not allow a complete

re
identification with precise molar masses nor the nomenclature of the components of this extract.
This will require fractionation of the extract followed by further LCMS analysis. Nevertheless,
previous phytochemical studies of H. bonariensis leaves and rhyzomes have revealed the
presence of alkaloids, flavonoids, tannins, phenolic compounds, terpenoids and saponins (Ajani
er
et al., 2009; Massoumian et al., 2011; Tabopda et al., 2012; kaboua et al., 2021b). It is evident
that most compounds with cardiovascular or antuhypertensive activity are alkaloids, flavonoids
or terpenoids (Luna-Vázquez et al., 2018). Although the mechanisms of action of these
molecules are very diverse, most of their antihypertensive effects involve both arterial smooth
pe
muscle and vascular endothelium (Luna-Vázquez et al., 2013; Victório et al., 2009). These
bioactive metabolites induce vasodilation by several pathways at once, either by activating at
least the nitric oxide/cGMP pathway or by blocking voltage-gated calcium channels (Mouzou
et al., 2009; Bartáková et al., 2021 ). The presence in this plant of vasodilator compounds such
as flavonoids, alkaloids and terpenoids would justify the effects we observed.
ot

5. CONCLUSION
Hydrocotyle bonariensis leaf hydroethanol extract has comparatively better antihypertensive
preventive effects than risordan which has a targeted action. This extract is reported to have
tn

positive cardiovascular effects including anti-oxidant, anti-inflammatory, lipid-lowering and

biochemical properties. These effects observed in this study would be due to the bioactive
phytochemicals revealed by the analysis of this extract, which could justify its use in traditional
medicine in the treatment of hypertension. However, there is a need to complement these studies
with a curative study protocol and also using other methods of induction of hypertension.
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Furthermore, it is imperative to conduct a phytochemical analytical study of the extract, which

may lead to the isolation of the pharmacological molecules.

Author contributions
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K. Kaboua, A. Mouzou, P. Bois, T. Pakoussi and K. Eklu-Gadegbeku conceived the project

and defined research methodology; K. Kaboua, A. Diallo, B. Kadissoli, G. Datagni and M.
Assih performed the experiments; K. Kaboua, P. Bois, T. Pakoussi and K. Eklu-Gadegbeku and
A. Mouzou wrote the paper.
Pr

Acknowledgement

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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=3977444
 We would like to thank M. Mamatchi, K. Atsu, M. K. Dossou-Yovo and C-A. Chapotte-
Baldacci for their expertise. The work is supported by Campus France and La Fondation Pierre

ed
Fabre.

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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=3977444