Journal of Ethnopharmacology 153 (2014) 400407

Contents lists available at ScienceDirect

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

Antihypertensive activities of the aqueous extract of Kalanchoe pinnata

(Crassulaceae) in high salt-loaded rats
Orelien Sylvain Mtopi Bopda a,n, Frida Longo b, Thierry Ndzana Bella d,
Protais Marcellin Ohandja Edzah b, Germain Sotoing Tawe a,c, Danielle Claude Bilanda d,
Esther Ngo Lemba Tom b, Pierre Kamtchouing d, Theophile Dimo d
a

Department of Zoology and Animal Physiology, Faculty of Science, University of Buea, P.O. Box 63 Buea, Cameroon
Department of Biological Sciences, ENS, University of Yaounde, P.O. Box 3805 Yaound, Cameroon
c
Institut National de la Sant et de la Recherche Mdicale, Unit 836, LabEx Ion Channels, Science and Therapeutics, Grenoble Institut de Neurosciences,
Universit Joseph Fourier, Chemin Fortun Ferrini, Site sant de la Tronche, P.O. Box 170, 38042 Cedex 9, Grenoble, France
d
Department of Animal Biology and Physiology, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon
b

art ic l e i nf o

a b s t r a c t

Article history:
Received 23 November 2013
Received in revised form
30 January 2014
Accepted 18 February 2014
Available online 26 February 2014

Ethnopharmacological relevance: The leaves of Kalanchoe pinnata (Crassulaceae) are used in Cameroon
folk medicine to manage many diseases such as cardiovascular dysfunctions. In this work, we aimed to
evaluate the activities of aqueous leaf extract of Kalanchoe pinnata on the blood pressure of normotensive
rat (NTR) and salt hypertensive rats (SHR), as well as its antioxidant properties.
Materials and methods: Hypertension was induced in rats by oral administration of 18% NaCl for 4 weeks.
For the preventive study, three groups of rats received 18% NaCl solution and the plant extract at 25 mg/
kg/day, 50 mg/kg/day or 100 mg/kg/day by gavage. Two positive control groups received 18% NaCl
solution and either spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) by gavage for 4 weeks.
At the end of this experimental period, systolic arterial pressure (SAP), diastolic arterial pressure (DAP)
and heart rate (HR) were measured by the invasive method. Some oxidative stress biomarkers (reduced
glutathione (GSH), superoxide dismutase (SOD), nitric monoxide (NO) were evaluated in heart, aorta,
liver and kidney. NO level was indirectly evaluated by measuring nitrite concentration.
Results: Kalanchoe pinnata extract prevented signicantly the increase of systolic and diastolic arterial
pressures in high salt-loaded rats (SHR). In SHR, concomitant administration of Kalanchoe pinnata at 25,
50 and 100 mg/kg/day signicantly prevented the increase in blood pressure by 32%, 24% and 47% (for
SAP); 35%, 33% and 56% (for DAP), respectively. No signicant change was recorded in heart rate of those
rats. The plant extract improved antioxidant status in various organs, but more potently in aorta. Thus,
antioxidant and modulatory effects of Kalanchoe pinnata at the vasculature might be of preponderant
contribution to its overall antihypertensive activity.
Conclusion: The work demonstrated that the concomitant administration of high-salt and the aqueous
extract of Kalanchoe pinnata elicits prevention of salt-induced hypertension in rat. This antihypertensive
activity is associated with an improvement of antioxidant status. Overall, results justify and support the
use of Kalanchoe pinnata as antihypertensive medicine.
& 2014 Elsevier Ireland Ltd. All rights reserved.

Chemical compounds studied in this article:

Sodium chloride (PubChem CID: 5234)
Spironolactone (PubChem CID: 5833)
Eupressyl (PubChem CID: 5639)
Urethane (PubChem CID: 5641)
Heparin (PubChem CID: 25244225)
Sulfanilamide (PubChem CID: 5333)
N-(1-Naphthyl) ethylenediamine
dihydrochloride (CID: 15106)
Orthophosphoric acid (CID: 1004)
Keywords:
Kalanchoe pinnata
Salt hypertensive rat
Antihypertensive
Antioxidant

1. Introduction

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase;

DAP, diastolic arterial pressure; GSH, reduced glutathione; HR, heart rate; NO, nitric
monoxide; NO2
, nitrite; NTR, normotensive rats; SAP, systolic arterial pressure;
SHR, salt hypertensive rats; SOD, superoxide dismutase
n
Corresponding author. Tel.: 237 75 07 21 74.
E-mail addresses: boresyl@yahoo.fr (O.S.M. Bopda), longofri@yahoo.fr (F. Longo),
Thierrybel2001@yahoo.fr (T.N. Bella), edzahprotais@yahoo.fr (P.M.O. Edzah),
taiwe_sotoing@yahoo.fr (G.S. Tawe), danielleclaudeb@yahoo.fr (D.C. Bilanda),
Esther_ngotom@yahoo.com (E.N.L. Tom), pikam55@yahoo.fr (P. Kamtchouing),
dimo59@yahoo.com (T. Dimo).
http://dx.doi.org/10.1016/j.jep.2014.02.041
0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved.

Hypertension is currently increasing in world population.

In 2000, hypertension already affected 26.4% of the overall adult
population worldwide, and this ratio was predicted to be increased
by about 60% in 2025 (Kearney, 2005). Strongly concerned are
economically developing countries (Kearney, 2005; Balde et al.,
2006; Fezeu et al., 2010) like Cameroon. In 2003, a prevalence of
25.6% and 23.1% was reported respectively in male and female
subjects from Cameroonian urban area (Kamadjeu et al., 2006).
Parts of the reasons of increase in the hypertension prevalence are

O.S.M. Bopda et al. / Journal of Ethnopharmacology 153 (2014) 400407

the changes in population lifestyle, which include a diet rich in

salt, sugar and high fat processed foods as well as sedentary
behaviour. Since the proportion of hypertensive people worldwide
is predicted to rise up dangerously, the detection, prevention,
treatment and control of this burden status must be a top priority
(Kearney, 2005). However, because the cost of modern drug
therapy is prohibitive, many patients turn to traditional herbal
medicine for the management of Hypertension (Adjanohoun et al.,
1996; Dimo et al., 2007; Bopda et al., 2007). Thus scientic
validation and rationalisation of medicinal plants need to be
intensied.
Kalanchoe pinnata, also named Bryophyllum pinnatum, belongs
to the family Crassulaceae whose species possess an array of
medicinal effects. They are used in folk medicine as stomach pain
relief, against gastritis, diarrhoea, bilharzias, dysmenorrhoea, liver
disorders, fever, female infertility, genitourinary infections, snake
and scorpio bites, leprosy, cough, asthma, kidney stones, arthritis,
cardiovascular diseases and general tiredness (Hutchings et al.,
1996; Van Wyk et al., 1997; Quazi Majaz et al., 2011). Furthermore,
pharmacological activities (antidiabetic, antioxidant, antineoplastic, immunomodulatory, antilipidaemic, antiallergic, antiviral, antitumoral, hepatoprotective and antithrombotic) of Kalanchoe
pinnata have been reported (Supratman et al., 2001; Yadav and
Dixit, 2003; Quazi Majaz et al., 2011). In some parts of the south,
west and centre Provinces of Cameroon, the folk medical practice
considers the leaves of Kalanchoe pinnata as a useful remedy
against diabetes and hypertension. Phytochemical analysis of
Kalanchoe pinnata leaf extracts showed the presence of alkaloids,
saponins, tanins, sterols, cardiac glycosides, avonoids, vitamins,
and mineral salts (Supratman et al., 2001; Muzitano et al., 2006;
Quazi Majaz et al., 2011).
It has been reported for long now that excess load of salt (NaCl)
leads to hypertension with increase of peripheral resistance
(Guyton, 1989; Dimo et al., 1999; Badyal et al., 2003; Bopda
et al., 2007), as well as oxidative stress (Kitiyakara et al., 2003;
Banday et al., 2007). In this work, we aimed to evaluate the
antihypertensive activities of aqueous leaf extract of Kalanchoe
pinnata in normotensive Wistar rats (NTR) and in salt hypertensive
rats (SHR), as well as its antioxidant properties.

2. Materials and methods

2.1. Animals
Three months old male albinos Wistar rats, weighting between
180 and 250 g were used. They were carefully handled according
to guidelines for the care and use of laboratory animals approved
by the Japanese Pharmacological Society (1987) and the International Guiding Principles for Biomedical Research Involving Animals developed by the Council for International Organizations of
Medical Sciences (CIOMS, 1985). Animals were raised in the
Animal House of ENS, University of Yaound I in plastic cages,
under standard light (12-hour day/night natural cycle) and temperature (25 1C). Rats were fed with standard diet and water
ad-libitum.

401

Normotensive rats were randomly divided into seven groups of

ve animals each. One group, neutral control, received tap water
and served as normotensive rats (NTR), one group received 18%
NaCl. Salt-induced hypertension was obtained by administration
of 18% NaCl (1 mL/100 gbw) to normotensive Wistar rats using
a gastric pipe. Gavage was done daily, between 08:00 and
09:30 a.m., for 4 weeks. Rats with systolic blood pressure level
above 140 mmHg and/or diastolic blood pressure level above
90 mmHg were considered hypertensive. Three groups received
18% NaCl solution and the plant extract at 25 mg/kg/day, 50 mg/
kg/day or 100 mg/kg/day by gavage. Two positive control groups
received 18% NaCl solution and either spironolactone (0.71 mg/kg)
or eupressyl (0.86 mg/kg) by gavage. At the end of this experimental period, systolic arterial pressure (SAP), diastolic arterial
pressure (DAP) and heart rate (HR) were measured. Biochemical
parameters were also evaluated in heart, aorta, liver and kidney.
Table 1 shows the sharing of rats in the various groups.
2.2. Plant material and extraction
Fresh leaves of Kalanchoe pinnata were harvested in Yaound
(Cameroon), in July 2010, and authenticated at the South western
Cameroon Herbarium, Limbe, where a specimen has been deposited (Voucher Number SCA2770). The material (2 kg) was pounded
by means of porcelain laboratory pounding cup, and then mixed
with water (3 L). This step was followed by ltration and lyophilisation. A 59.2 g (yield of 2.96%) of dried powder was obtained.
The aqueous extract was dissolved in a given volume of distilled
water, giving rise to the stock solution (1 g/mL) for subsequent use.
2.3. Effects of Kalanchoe pinnata on blood pressure and heart rate
During the experiment, weight and food intake (weekly) and
water consumption (daily) were assessed. At the end of the
4-weeks treatment, we evaluated the effect of aqueous extract of
Kalanchoe pinnata on salt-induced hypertension. Rats were anaesthetised using an intraperitoneal injection of 15% urethane
(1.5 g/kg). The trachea was exposed and cannulated to facilitate
spontaneous respiration. A polyethylene catheter (PE 50) was
inserted into the right femoral vein and a bolus injection of 10%
heparin (0.1 mL/100 gbw) was immediately administered. The
invasive method was used for the recording of cardiovascular
parameters. To that effect, a catheter connected to a pressure
transducer was inserted into the left carotid artery. The transducer
was coupled with a Biopac Student Lab MP35 hemodynamic
recorder and a computer. The Biopac Student Lab MP35 is a
multipurpose device that helps to record simultaneously the
systolic blood pressure (SAP), diastolic blood pressure (DAP) and
heart rate (HR). Values were always recorded after 3060 min
stabilisation (Dimo et al., 2003).
2.4. Effects of Kalanchoe pinnata on biochemical parameters
At the end of the treatment with Kalanchoe pinnata extract,
heart, aorta, liver and kidney were dissected out and homogenised
in Mc Even solution for heart and aorta or in TrisHCl 50 mM

Table 1
Allocation of rats for various treatments.
Groups

NTR

Treatment Water
(10 mL/kg/
day)

SHR

Kalanchoe pinnata
(25 mg/kg)

NaCl 18%
NaCl 18% Kalanchoe
(10 mL/kg/day) pinnata (25 mg/kg/day)

Kalanchoe pinnata
(50 mg/kg)

Kalanchoe pinnata
(100 mg/kg)

Spironolactone
(0.71 mg/kg)

Eupressyl
(0.86 mg/kg)

NaCl 18% Kalanchoe

pinnata (50 mg/kg/day)

NaCl 18% Kalanchoe

pinnata (100 mg/kg/day)

NaCl 18%
Spironolactone
(0.71 mg/kg/day)

NaCl 18% Eupressyl

(0.86 mg/kg/day)

402

O.S.M. Bopda et al. / Journal of Ethnopharmacology 153 (2014) 400407

buffer solution for liver and kidney (20%, w/v). Blood levels of
creatinine, bilirubin (Bartels et al., 1972), alanine aminotransferase
(ALT) and aspartate aminotransferase (AST) (Reitman and Frankel,
1957) were assayed. Tissue levels of reduced glutathione (GSH)
and superoxide dismutase activity (SOD) were assayed using
colorimetric method as described by Jollow et al. (1974) and
Misra and Fridovich (1972), respectively. Nitric oxide (NO) level
was indirectly assayed by measuring the concentration of nitrite
(NO2
), its stable metabolite, using Griess reagent method (Green
et al., 1982; Ikeda et al., 2003; Patil et al., 2009). Nitrite contents of
tissues were determined by mixing 0.5 mL of homogenate with an
equal volume of Griess reagent (1% sulfanilamide and 0.1%
naphthyl ethylenediamine dihydrochloride in 2.5% orthophosphoric acid). The reading of the absorbance was done at 546 nm.
Nitrite levels were expressed in mmoles per milligram of protein.

change of heart rate, compared to NTR. In SHR, heart rate

increased nonsignicantly (P 40.05) by 9% (from 310 720 to
3397 24 BPM), as compared to NTR. Also, from the value of
310 720 BPM in NTR, heart rate still remained at 314 715 BPM,
after a 4-week treatment with NaCl (18%) plus Kalanchoe pinnata
(100 mg/kg/day). Similar results were recorded in animal group
administered with NaCl (18%) plus spironolactone (0.71 mg/kg/
day) but not in that which received NaCl (18%) plus eupressyl
(0.86 mg/kg/day) (Table 2)
Data are given as mean 7 SEM of the heart rate in various
groups of rat. n 5.nP o0.05 vs. Normotensive Rats (NTR). During
four weeks, NTR received only tap water, SHR received only NaCl
(18%), while other groups received Kalanchoe pinnata extract
(25100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl
(0.86 mg/kg/day) in addition to NaCl (18%).

2.5. Statistical analysis

3.3. Effects of Kalanchoe pinnata on some biochemical parameters

Results are expressed as mean 7SEM. One-way analysis of

variance (ANOVA) followed by Dunnett's test was used for statistical evaluation. P values less than 0.05 were considered
signicant.

3. Results
3.1. Effects of Kalanchoe pinnata on body weight and water
consumption
After four weeks of treatment with NaCl (18%) and NaCl (18%)
plus Kalanchoe pinnata extract (25100 mg/kg), no signicant
difference was observed between body weights in normotensive
(NTR), hypertensive untreated (SHR) and hypertensive treated rats.
Likewise, no signicant variation was observed between estimated
food consumption in various groups (data not shown). Conversely,
Salt-loaded Hypertensive Rats (SHR) showed increase of water
consumption, compared to Normotensive Rats (NTR). From a
volume of 11 71 mL/day/rat in NTR, water consumption rose up
to 31 72 mL/day/rat in SHR, corresponding to a 182% increase
(P o0.001). Kalanchoe pinnata (25100 mg/kg/day) prevented this
increase of water consumption. In rats administered with the
doses 25, 50 and 100 mg/kg/day, water consumption dropped
respectively to 13 72, 14 72 and 12 71 mL/day/rat, corresponding
to a signicant (P o0.001) reduction of 58%, 55% and 61% respectively, compared to SHR. Similar results were obtained in rats
treated with NaCl (18%) plus reference drugs (spironolactone,
0.71 mg/kg/day; eupressyl, 0.86 mg/kg/day) (Fig. 1).
3.2. Effects of Kalanchoe pinnata on arterial pressure and heart rate
Subchronic administration of NaCl (18%) to rats for four weeks
increases systolic arterial pressure (SAP), diastolic arterial pressure
(DAP) and heart rate (HR). SAP and DAP signicantly varied
from 12475 mmHg (NTR) to 156 78 mmHg (SHR) and from
897 4 mmHg (NTR) to 13575 mmHg (SHR), corresponding to
26% (Po 0.01) and 52% (P o0.001) increases, respectively. In SHR,
concomitant administration of Kalanchoe pinnata at 25, 50 and
100 mg/kg/day signicantly prevented the increase in blood pressure by 32%, 24% and 47% (for SAP); 35%, 33% and 56% (for DAP),
respectively. It is worth noting that at 25 and 50 mg/kg/day, SAP
value was reduced, signicantly (Po 0.05), below NTR level.
Similar results were observed in reference animal groups which
received simultaneously NaCl (18%) and either spironolactone
(0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) (Fig. 2).
Treatment with NaCl (18%) and NaCl (18%) plus Kalanchoe
pinnata extract (25100 mg/kg) did not display any signicant

3.3.1. Effects on some oxidative stress biomarkers

Fig. 3 shows that reduced glutathione (GSH) level was signicantly (Po0.01) decreased in aorta of SHR by 76% (from 5075 to
1277 mmol/L), as compared to NTR. Kalanchoe pinnata extract (25
100 mg/kg/day) signicantly reduced that drop (i.e. increased GSH
level) in the aorta of SHR. At the higher dose of 100 mg/kg/day
the extract even permitted a total recovery of glutathione level. The
level of glutathione was enhanced from 1277 to 5374 mmol/L,
corresponding to an increase of 341%, as compared to SHR
(Po0.001). Similar results were observed with spironolactone
(0.71 mg/kg/day). No signicant decrease of the level of glutathione
was noticed in other organs of SHR (heart, liver and kidney)
However, the plant extract, at the dose of 25 mg/kg/day, increased
the level of that biomarker, especially in heart and kidney. In heart,
the increase was by 107% (from 1572 to 3176 mmol/L, Po0.05),
meanwhile in kidney it was 93% (from 1574 to 2971 mmol/L,
Po0.05), as compared to SHR. Similar results were observed with
spironolactone (0.71 mg/kg/day), but not with eupressyl (0.86 mg/
kg/day).
Salt-loaded animals exhibited a signicant decrease in aorta,
heart, liver and kidney SOD activities, as compared to NTR.
Kalanchoe pinnata extract (25100 mg/kg/day) reduced that fall
(i.e. increase SOD activity) in all organs of SHR. At the higher dose
of 100 mg/kg/day the rise of SOD activity in aorta, heart, liver and
kidney corresponded to 103% (from 29 73 to 59 73 U/mg proteins, Po 0.001), 71% (from 21 72 to 36 75 U/mg proteins,
P4 0.05), 86% (from 227 3 to 41 73 U/mg proteins, P o0.001)
and 76% (from 21 71 to 37 73 U/mg proteins, P o0.01), respectively. Similar results were observed with spironolactone
(0.71 mg/kg/day) and eupressyl (0.86 mg/kg/day) in all organs
(Fig. 4).
Fig. 5 shows that in salt-loaded hypertensive rats, nitrite (NO2
)
levels were signicantly (P o0.001) decreased in aorta, liver and
kidney but not in heart, as compared to NTR. In aorta and kidney,
Kalanchoe pinnata extract at the moderate dose of 50 mg/kg/day
displayed a more potent effect. In fact, Kalanchoe pinnata (50 mg/
kg/day) signicantly (Po 0.01) increased the concentration of
NO2
, in both organs. The level of the biomarker rose by 395%
in aorta and 23% in kidney, compared to SHR. Similar results
were observed with spironolactone (0.71 mg/kg/day) in both
organ types.
3.3.2. Effects on creatinine, bilirubin, AST and ALT levels
Salt-loaded rats displayed a signicant (P o0.001) increase in
blood levels of creatinine and bilirubin, as compared to NTR.
Kalanchoe pinnata extract (25, 50 and 100 mg/kg/day) signicantly
reduced the rise of concentration of creatinine, meanwhile only

Water consump on
(mL/day/rat)

O.S.M. Bopda et al. / Journal of Ethnopharmacology 153 (2014) 400407

35
30
25
20
15
10
5
0

403

Animal
groups

Fig. 1. Effects of Kalanchoe pinnata extract on water consumption in different groups of rat. Each bar represents the mean7 SEM, n 5. nPo 0.001 vs. Normotensive Rats
(NTR), P o 0.001 vs. Salt-loaded Hypertensive Rats (SHR). During four weeks, NTR received only tap water, SHR received only NaCl (18%), while other groups received
Kalanchoe pinnata extract (25100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

Arterial pressure (mmHg)

200
DAP (mmHg)

175
150

**

SAP (mmHg)

***

##
##

125

###

##

100

###

###

##

##

##

*
###

75

***

50
25

Animal
group

Fig. 2. Effects of Kalanchoe pinnata extract on arterial pressure in different groups of rat. Each bar represents the mean 7 SEM, n5. nP o0.05, nnPo 0.01 and nnnP o0.001 vs.
Normotensive Rats (NTR). Po 0.01 and Po 0.001 vs. Salt Hypertensive Rats (SHR). During four weeks, NTR received only tap water, SHR received only NaCl (18%), while
other groups received Kalanchoe pinnata extract (25100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

Table 2
Effect of Kalanchoe pinnata extract on the heart rate in different groups of rat.
SHR

Kalanchoe pinnata
(25mg/kg)

Animal
group

NTR

HR (BPM)

3107 20 339 7 24 3357 8

Kalanchoe pinnata
(50mg/kg)

Kalanchoe pinnata
(100mg/kg)

Spironolactone
(0.71mg/kg)

Eupresssyl
(0.86mg/kg)

329 724

3147 15

366 718

4017 23n

Data are given as mean7 SEM of the heart rate in various groups of rat. n5. nPo 0.05 vs. Normotensive Rats (NTR). During four weeks, NTR received only tap water, SHR
received only NaCl (18%), while other groups received Kalanchoe pinnata extract (25100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in
addition to NaCl (18%).

the dose of 25 mg/kg/day had such effect on bilirubin level. At that

lower dose of 25 mg/kg/day the reduction corresponded to 56%
(P o0.001) and 44% (Po 0.01) for creatinine and bilirubin, respectively. Similar results were observed in animals treated with NaCl
(18%) and spironolactone (0.71 mg/kg/day) (Fig. 6).
Fig. 7 shows that in SHR, AST level increased signicantly
(Po0.01) while ALT level increased nonsignicantly, compared to
NTR. Kalanchoe pinnata extract at the higher dose of 100 mg/kg/day
reduced AST level from 0.05370.003 to 0.02270.003 IU/L,
corresponding to a 58% reduction (Po0.01). Similar results were
observed in both positive control groups.

4. Discussion
This study investigated for the rst time the effects of aqueous
leaf extract of Kalanchoe pinnata on salt-loaded hypertension. The
outcome is that the aqueous extract of Kalanchoe pinnata induces
an antihypertensive activity in salt-loaded hypertensive rats (SHR).
Moreover, the antihypertensive effect of the extract is associated
with antioxidant properties in hypertensive rats.
In our study, hypertension was induced by administering 18%
NaCl to normotensive rats. At the rst look, this dose may seem too
high, but it is worth noting that between administration of salt

O.S.M. Bopda et al. / Journal of Ethnopharmacology 153 (2014) 400407

GSH Concentration (mmol/L)

404

NTR
SHR
K. pinnata (25mg/kg)
K. pinnata (50mg/kg)
K. pinnata (100mg/kg)
Spironolactone (0.71mg/kg)
Eupresssyl (0.86mg/kg)

100
90
80
70
60
50
40
30
20
10
0

Organ type
Aorta

Heart

Liver

Kidney

SOD activity (Units/mg proteins)

Fig. 3. Effects of Kalanchoe pinnata extract on reduced glutathione concentration in different groups of rat. Each bar represents the mean7 SEM, n 5. nP o 0.05,nnPo 0.01
and nnnPo 0.001 vs. Normotensive Rats (NTR). Po 0.05, Po 0.01 and Po 0.001 vs. Salt Hypertensive Rats (SHR). During four weeks, NTR received only tap water, SHR
received only NaCl (18%), while other groups received Kalanchoe pinnata extract (25100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in
addition to NaCl (18%).

NTR
SHR
K. pinnata (25mg/kg)
K. pinnata (50mg/kg)
K. pinnata (100mg/kg)
Spironolactone (0.71mg/kg)
Eupresssyl (0.86mg/kg)

100
90
80
70
60
50
40
30
20
10
0

Organ type
Aorta

Heart

Liver

Kidney

Fig. 4. Effects of Kalanchoe pinnata extract on superoxide dismutase activity in different groups of rat. Each bar represents the mean 7SEM, n5. nnPo 0.01 and nnnPo 0.001
vs. Normotensive Rats (NTR). Po 0.01 and Po 0.001 vs. Salt Hypertensive Rats (SHR). During four weeks, NTR received only tap water, SHR received only NaCl (18%), while
other groups received Kalanchoe pinnata extract (25100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

NO2-concentration (mmol/mg proteins)

140
120

##

**

100

## ##

** **

* * *
***
*** **

***

***

NTR
SHR
K. pinnata (25mg/kg)
K. pinnata (50mg/kg)
K. pinnata (100mg/kg)
Spironolactone (0.71mg/kg)
Eupresssyl (0.86mg/kg)

80
##

60
40

***

*
20
0
Aorta

Heart

Liver

Kidney

Fig. 5. Effects of Kalanchoe pinnata extract on nitrite (NO2
) concentration in different groups of rat. Each bar represents the mean 7SEM, n 5. nP o 0.05, nnPo 0.01 and
nnn
P o0.001 vs. Normotensive Rats (NTR). P o0.05 and Po 0.001 vs. Hypertensive Rats (SHR). During four weeks, NTR received only tap water, SHR received only NaCl
(18%), while other groups received Kalanchoe pinnata extract (25100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

solutions, rats had access to normal water ad-libitum. This model

presents as advantages the accuracy of dosage, the monitoring of
precise time for salt ingestion by animals and the fast (4 weeks)
occurrence of hypertension in animals. Neither in NaCl-loaded
hypertensive rats nor in NaCl plus extract-treated rats was observed
any signicant change of body weight and food consumption.
Conversely, salt treatment caused increase of water consumption

in rats. It is worth noticing that they also urinated consequently.

This diuretic behaviour accounts for an obvious homoeostatic need
of their organism. High blood pressure is a major risk for stroke,
coronary diseases and renal dysfunction. Excess salt accumulation
in internal medium is well known to cause hypertension (Bayorh
et al., 2004; Banday et al., 2007; Bopda et al., 2007) frequently
associated with increase in oxidative stress (Bayorh et al., 2004;

Blood levels (mg/mL) of creatinine and bilirubin

O.S.M. Bopda et al. / Journal of Ethnopharmacology 153 (2014) 400407

16.00
15.50
15.00
14.50
14.00
13.50
13.00
12.50
12.00
11.50
11.00
10.50
10.00
9.50
9.00
8.50
8.00
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00

405

Creatinine (mg/mL)
Bilirubin (mg/mL)

###

###

Animal group

Blood levels (IU/L) of AST and ALT

Fig. 6. Effects of Kalanchoe pinnata extract on creatinine and bilirubin levels in different groups of rat. Each bar represents the mean 7SEM, n 5. nnPo 0.01and nnnPo 0.001
vs. Normotensive Rats (NTR). P o 0.05, Po 0.01 and Po 0.001 vs. Salt Hypertensive Rats (SHR). During four weeks, NTR received only tap water, HSR received only NaCl
(18%), while other groups received Kalanchoe pinnata extract (25100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

0.070
0.060
0.050
#

**

**

**

**

AST

ALT

0.040
0.030

##

##

##

0.020
0.010
0.000

Animal
group

Fig. 7. Effects of Kalanchoe pinnata extract on AST and ALT levels in different groups of rat. Each bar represents the mean7 SEM, n5. nPo 0.05 and nnPo 0.01 vs.
Normotensive Rats (NTR). Po 0.05 and Po 0.01 vs. Salt Hypertensive Rats (SHR). During four weeks, NTR received only tap water, SHR received only NaCl (18%), while
other groups received Kalanchoe pinnata extract (25100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

Wilcox, 2005; Banday et al., 2007) and hardening of blood vessels

(Guyton, 1989; Dimo et al., 1999; Badyal et al., 2003; Bopda et al.,
2007). In the present study, NaCl (18%) induced a signicant
increase in systolic and diastolic blood pressure. Our results
demonstrated that the concomitant subchronic (four weeks)
administration of aqueous extract of Kalanchoe pinnata led to
reduction of the increase in both systolic and diastolic arterial blood
pressure. The higher dose of 100 mg/kg/day appeared to be the
most potent and the effect of extract on blood pressure was

comparable to that induced by the 1-adrenoceptor antagonist,

eupressyl, and aldosterone antagonist, spironolactone (Lechat et al.,
1990; Givertz, 2001). From the above, one might promptly hypothesise that the mechanism involved in this antihypertensive effect
could be either vasodilation or increase of natriuresis and diuresis.
Antihypertensive plant extracts act, in general, either by cardiodepression, increase of diuresis or vasorelaxation (Corallo et al., 1997;
Dimo et al., 2007; Bopda et al., 2007, 2011; Tom et al., 2011;
Nguelefack-Mbuyo et al., 2012). Because Kalanchoe pinnata extract

406

O.S.M. Bopda et al. / Journal of Ethnopharmacology 153 (2014) 400407

did not modify heart rate, we suggest that organs targeted by

Kalanchoe pinnata extract should be primarily kidney and blood
vessels.
From our results, more can be known with regard to mechanism.
An important aspect to consider here is the reduction of oxidative
stress by the plant extract. Indeed, it is well-established that high
salt consumption is associated to increased tissue production of
reactive oxygen species (ROS) (Bayorh et al., 2004; Wilcox, 2005;
Banday et al., 2007). Lower activity of antioxidants has been
reported in various animal models of hypertension (Zhou et al.,
2002; Bayorh et al., 2004; Sei et al., 2010). Accordingly, our results
showed that several biomarkers (reduced glutathione, superoxide
dismutase and nitrite) of oxidative stress are impaired in rats on
high salt diet. Reduced glutathione (GSH) is an endogenous and
ubiquitous antioxidant produced in eukaryote's cells, which contributes in maintaining the integrity of cell against oxidation (Stein
et al., 1989; Zhou et al., 2002). Our data reported a decrease in GSH
levels in aorta of high salt-hypertensive rats. This drop was well
corrected by plant extract. The plant extract might also be able to
enhance the basal level of GSH in normotensive and hypertensive
rats, as exemplied by heart and kidneys recorded values. The
activity of superoxide dismutase (SOD) was measured. SOD is the
enzyme that catalyzes the dismutation of the O2
free radical in
water and hydrogen peroxide (Faraci and Didion, 2004; Wang et al.,
2006). Our data revealed decreased SOD activity in all organs of
hypertensive rats. Long lasting stress due to subchronic administration of salt to rats induces a massive and toxic amount of oxygen
active species, leading to destruction of SOD; which therefore
undergoes a drop of its concentration. Indeed, salt intake enhances
generation of O2
accompanied by enhanced expression and activity
of NADPH oxidase with diminished expression of SOD in the
vasculature and kidneys; this contributes in the pathogenesis of
several models of hypertension (Kitiyakara et al., 2003). The fact
that subchronic treatment with the aqueous extract of Kalanchoe
pinnata signicantly prevented the fall in SOD level, as the case was
for GSH too, conrmed antioxidant properties of the plant extract.
In addition to the above, the level of NO2
, a stable metabolite of NO
(Ikeda et al., 2003) was investigated. We recorded a drop of that
biomarker in all organs of high salt hypertensive rats, except heart.
NO is not as stable as NO2
, but is the active biomarker in the
vessels. NO is a major down-regulator of vascular tone in humans
(Calver et al., 1993; Corvol, 1993; Nguelefack-Mbuyo et al., 2012). In
animal models of salt-sensitive hypertension, the increase in blood
pressure after salt loading is characterized by reduced NO production (Bayorh et al., 2004; Banday et al., 2007). Interaction of ROS,
particularly superoxide with NO, leads to the production of peroxynitrite, which is a highly cytotoxic reactive compound (Zhou et al.,
2002). Interest is focused on overexpression of NADPH oxidase
components, or underexpression of SOD, as potential prohypertensive mechanisms in models of salt or mineralocorticoid-induced
hypertension. Since SOD catalyzes the conversion of O2
to H2O2,
the dominant effect of extracellular and intracellular SOD activity
normally is to metabolize O2
and thereby prevent hypertension
(Wilcox, 2005). There is no consensus on whether oxidative stress
in animal models such as excess salt hypertensive rats is a cause or
a consequence of hypertension, but at least it is now an agreement
that prevention of oxidative stress contributes to a drop of blood
pressure in such hypertensive rats (Wilcox, 2005). Thus antioxidant
properties of Kalanchoe pinnata might represent other strong
mechanisms involved in its antihypertensive activity. However,
because NO activity contributes to antihypertensive effects by
vasodilation (Corvol, 1993; Nguelefack-Mbuyo et al., 2012) it will
be very interesting to carry out in vitro experiments using vascular
rings, to assess the endothelium-dependent vasoreactivity of high
salt hypertensive rats treated with Kalanchoe pinnata aqueous
extract.

In addition to prooxydants/antioxydants disturbances due to NaCl

18% consumption, disturbance of the production or bioavailability of
AST, a hepatic enzyme, was also recorded. High salt intake induced
increase of plasma AST, as a footprint of an increase of cytolysis.
Concomitant administration of NaCl 18% and extract (25 and 100 mg/
kg/day) permitted to reduce signicantly the increase of AST, in
hypertensive rats. This result suggests that plant extract might
partially protect various tissues, including hepatic tissues, from high
salt-induced injuries.
Prolonged administration of NaCl 18% induced a signicant
increase of plasma creatinine. This is likely to be caused by an
alteration of glomerular ltration (Hadj-Assa et al., 1994).
A decrease of glomerular ltration by at least 50% can induce a
hypercreatinemia (Kingdom et al., 2003). As Kalanchoe pinnata
extract prevented that hypercreatinemia, it might prevent any
eventual disruption of glomerular ltration. Plasma level of
bilirubin was also signicantly increased in NaCl 18% loaded rats.
This variation was inhibited by Kalanchoe pinnata extract (25 mg/
kg/day), suggesting that Kalanchoe pinnata up-regulates red blood
cells production and/or bioavailability. These protective activities
are consistent with antioxidant properties of the plant extract.
Moreover, these results while arguing in favour of a very low
toxicity of Kalanchoe pinnata aqueous extract, when used at
therapeutic doses, also conrm hepatoprotective properties of
the plant leaves as reported by Yadav and Dixit (2003), Quazi
Majaz et al. (2011) and Biswas et al. (2011). The presence of
compounds such as avonoids and polyphenols in Kalanchoe
pinnata (Harlalka et al., 2007; Biswas et al., 2011) argues in favour
of its displayed hepatoprotective, antioxidant and antihypertensive properties.

5. Conclusion
This study demonstrates the antihypertensive effects of aqueous extract of the leaves of Kalanchoe pinnata in salt hypertensive
rats. Our results provide justication and support for the use of
this extract as traditional medicine against hypertension in
Cameroon. The effect on blood pressure might be partly due to
the improvement of the antioxidant status in hypertensive organisms. As the antioxidant mechanism seems to be emphasised at
the level of the aorta, in vitro investigations of the endotheliumdependent vasodilation properties of Kalanchoe pinnata should be
strongly envisaged.

Acknowledgements
The authors are very grateful to Prof. Georges Chuyong (H.O.D.,
Botany and Plant Physiology, University of Buea) and Miss MarieClaire Veranso (Ph.D. student in the same Department) for their
assistance in the preliminary identication of the plant.
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