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eISSN: 2231-0541 CAS CODEN: PHARN8 An ELSEVIER Covered Journal
PHARMANEST
An International Journal of Advances in Pharmaceutical Sciences
Volume 4|Issue 4|July-August 2013|Pages 751-763
Original Research Article
ACUTE TOXICITY AND CARDIAC EFFECTS OF A CHROMATOGRAPHIC FRACTION
FROM BIDENS PILOSA L. (ASTERACEAE) LEAVES IN MAMMALS
aLÉANDRE KOUAKOU KOUAKOU, aMATHIEU NAHOUNOU BLÉYÉRÉ, bANDRÉ BROU KONAN, bAUGUSTIN KOUAO AMONKAN, cJEAN CLAUDE KOUAKOU ABO, aPAUL ANGOUÉ YAPO, aETIENNE EHOUAN EHILÉ
aLaboratory of Physiology, Pharmacology and African Pharmacopoeia of UFR-SN, University of Nangui Abrogoua,
02 BP 801 Abidjan 02, Côte d’Ivoire.
bLaboratory of Pharmacology and Nutrition of UFR-Biosciences, University of Felix Houphouet Boigny, Cocody,
22 BP 582 Abidjan 22, Côte d’Ivoire.
cLaboratory of Animal Physiology of UFR-Biosciences, University of Felix Houphouet Boigny, Cocody, 22 BP 582
Abidjan 22, Côte d’Ivoire.
Author for Correspondence: bleyere@yahoo.fr
Received: 01-07-2013 Revised: 07-07-2013
Accepted: 10-07-2013 Available online: 01-09-2013
ABSTRACT
Bidens pilosa L. (Asteraceae) is a plant commomly used in traditional medicine to treat several ailments. The effects of a chromatographic fraction isolated from the aqueous leaf extract (BpF2) were investigated for the acute toxicity in mice, isolated rat heart and rabbit ECG. The graphic method of Miller and Tainter (1944) permitted to determine a LD50 of 429.14 ± 28.11 mg/kg b.w. while the calculation method of Dragsted and Lang (1957) gave 452.50 ± 23.10 mg/kg b.w. as LD50. BpF2 induced negative inotropic and chronotropic effects on isolated rat heart preparations for concentrations ranging from 10-12 to 10-4 mg/ml. Those effects were significantly attenuated by the use of atropine, a muscarinic cholinoceptors antagonist. The dose-response effect (5-25 mg/kg b.w.) on anesthetized rabbit ECG revealed a decrease of P, QRS, T waves and the cardiac frequency. In contrast, PQ interval was increased and QT interval was not significantly affected. These results indicated that BpF2 was relatively toxic but could be used with caution. The cardioinhbition induced suggested the presence of cholinomimetic substances acting via muscarinic M2 receptors and the non significant variation of QT interval was important in so far as an extension of this interval could be source of arrhythmia induction.
Key words: Bidens pilosa, Acute toxicity, ECG, Isolated rat heart, cholinomimetic substances.
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INTRODUCTION
Traditional medicine has been practiced for
years in developing countries, especially in
Africa. According to WHO, this use is
widespread and is becoming increasingly
important in terms of health and economy,
since more than 80% of the population
resort to it for health needs.1 According to
the same source, in Asia and Latin
America, people still use traditional
medicine because of historical
circumstances and cultural beliefs. Bidens
pilosa L. is an herbaceous plant, set up,
with regularly cogged and glabrous leaflets.
Very widespread pantropical species from
Angola to Cameroon, it is also found in Côte
d’Ivoire.2,3 The plant is used in traditional
medicine to treat diverse illnesses such as
diarrhoea and inflammation in Côte d’Ivoire
and is prescribed as anti-poison, against flu
and haemorrhoids in Congo and
Rwanda.2,3,4 Many works carried out on this
plant revealed different properties among
which anti-microbial, anti-inflammatory,
anti bacterial, anti-malarial and anti-gastric
ulcer effects.5,6,7,8,9
The cardiovascular aspect was also subject
to many studies.10,11,12,13,14, 15. Investigations
showed that a chromatographic fraction
from Bidens pilosa L. leaves (BpF2) elicited a
decrease of the blood pressure of rabbit via
cholinomimetic and β-adrenomimetic
agonist substances.14,15 The phytochemical
study of the fraction revealed flavonoids,
polyphenols and catechic tannins.
This study was aimed to evaluate in mice
the acute toxicity of a chromatographic
fraction from the leaves of Bidens pilosa L.
(BpF2) and to investigate its effects on
isolated rat heart and rabbit
electrocardiogram.
MATERIAL AND METHODS
Animals: Rabbits (Oryctolagus cuniculus)
weighing 2 ± 0.2 kg, Mice (Mus musculus)
weighing between 20 and 25 g and rats
(Ratus norvegicus) weighing between 180
and 250 g were used. They were bred in
Animal house of Animal Physiology,
Pharmacology and Phytotherapy of the
University of Nangui Abrogoua (Former
University of Abobo-Adjamé, Abidjan, Côte
d’Ivoire) according to the principles for the
care and use of laboratory animals of the
Ethical Committee of the University (Nangui
Abrogoua, Abidjan, Côte d’Ivoire).
Plant material: The plant material was
described.14,15 Authentication of fresh
leaves of Bidens pilosa L. (Asteracea)
collected from Abidjan, Côte d’Ivoire was
implemented by Prof. Aké-Assi Laurent, an
expert in Botany (Department of Botany,
University of Cocody, Abidjan, Côte
d’Ivoire). In Côte d’Ivoire, Bidens pilosa L.
was first found on November 12, 1966 in
Bondoukou and herbarium specimen
(voucher n° 9266) was made and deposited
at the National Botanic Centre (University
of Cocody, Abidjan, Côte d’Ivoire) and then
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in Kakpin (Côte d’Ivoire) on June 20, 1968.
Herbarium specimen (voucher n° 10286)
was also made and deposited at the
National Botanic Centre (University of
Cocody, Abidjan, Côte d’Ivoire).
Plant extraction: The extraction and the
separation of extracts were as previously
described.14,15 Hundred grams (100g) of
powder from Bidens pilosa L. leaves dried at
room temperature were macerated under
magnetic shaker during 48h in 2 L of
distilled water. The supernatant was filtered
on cotton and filter paper Whatman. Two
litres of distilled water were added to the
base and then mixed during two hours and
also filtered. The filtrates were freeze-dried
using a lyophilisator TELSTAR (Terrassa,
Spain). 0.8 g of the powder obtained were
dissolved in 10 ml of distilled water and
chromatographed on a fine Sephadex G25
column (3 by 20 cm) packed in distilled
water. Elution was carried out with the
same solvent. Fractions (5) of 20 ml each
were collected then freeze-dried. They were
tested on the blood pressure of the rabbit.
The fraction 2 (BpF2) was found to be the
most active.
Chemicals: Atropine was purchased from
Sigma Chemical Company (St Louis, MO,
USA).
Data analysis: Statistical analysis and
graphics were performed with the software
GraphPad Instat and GraphPad Prism 4
(San Diego California, USA) respectively. All
values are expressed as mean ± standard
error on the mean (m ± sem). The
differences observed between the
concentrations were precised by an analysis
of variances (ANOVA) of the multiple test of
comparison of Turkey-Kramer and were
considered statistically significant when
p < 0.05.
Acute toxicity assessment
Mice were divided into 9 groups of 10 mice
(5 males and 5 females). Each mouse in the
first group (control group) was treated with
0.5 ml isotonic solution of NaCl 9 ‰. The
other 8 groups were treated with a single
administration of BpF2 diluted in the
normal saline isotonic solution. The
different doses of BpF2 ranging from 215
mg/kg b.w to 740 mg/kg b.w. and the
normal saline solution of the control group
were administered intraperitoneally (i.p.).
Behavioural changes of the 8 treated
groups were observed every 30 min for a
period of 2 hours after administration of the
extract (Mandal et al., 2001) and mortality
rate were recorded for 24 hours post
treatment.16 The graphic and the
calculation methods were used to
determine the LD50.17,18
Recording of the isolated rat heart
activity
The experimental process used to record
and assess the isolated rat inotropic and
chronotropic activities was as previously
described.13, 19 Rat was anesthetized with
intraperitoneal injection of 20% ethyl
urethane at 1 g/kg body weight. It was then
placed under artificial respiration to avoid
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anoxia of the heart during operation. To do
this, a tracheotomy was performed and a
cannula connected to an air pump was
placed in the trachea. Then, a thoracotomy
was practiced. A hemisection of the aorta
was carried out. A cannula fixed to a
syringe containing heparinized solution
(2500 UI, 0.2 ml/100 g body weight) was
inserted in the aorta. The heart was rapidly
removed and the heparinized solution
injected to dissolve and expel any blood
clots that were probably formed in the heart
to prevent thrombosis in the coronary
circulation. The isolated heart was perfused
with Mac Ewen solution of the following
composition (mM): NaCl 147; KCl 5.6; CaCl2
2.6; NaH2PO4 0.66; CO3NaH 11.9; MgCl2
0.24; C6H12O6 11 with a pH adjusted to 7.4.
The apex of the heart was fixed by a fine
clip and linked to the needle of the
cardiograph for recording. After each
treatment, the heart was washed by
perfusion fluid for 10 min, time within
which the baseline recording was achieved,
and the second dose was then given. The
recording before the direct perfusion of
extract was considered as baseline reading
for each dose (control).
BpF2 was dissolved in Mac Ewen solution.
Registration of the global electrical
activity (ECG) of the rabbit
Methods were as previously
described.19,20,21. The electrocardiogram of
the rabbit was recorded by the technique of
external electrodes used in the human
practices and adapted to the rabbit.20
Briefly, the saphenous vein of the
anesthetized rabbit by an intraperitoneal
injection of 40% ethyl urethane (1 g/kg
body weight) was intubated in order to
administer plant extract.20 The armpits of
the anterior limbs and the groin of the
posterior limbs were shaved and cleaned
with 90% ethanol. After applying
electrolytic dough, four electrodes were put
and bound to the four sockets of the
registration cable connected to the
electrocardiograph (CARDIOFAX ECG-
6851K, Nihon Kohden, Japan). The studied
parameters (P, QRS, T waves, PQ, QT
intervals and cardiac frequency) were
recorded from the DIII derivation of the
standards or bipolar Einthoven derivations
on thermo sensitive paper, at constant
speed (25 mm/s).
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Fig.1.Dose-response effect of BpF2 on isolated rat heart
Increasing concentrations of BpF2 induced a significant (*p < 0.05, n = 4) drop of the amplitude (A) and the frequency (B) of the cardiac activity in a dose-dependent manner. The EC50 determined graphically were 1.98 x 10-10 mg/ml and 1.95 x 10-8 mg/ml respectively for the amplitude and the frequency.
-14 -12 -10 -8 -6 -4 -2
0
10
20
30
40
50
60
70
80
90
100
Log(Concentration of BpF2 in mg/ml)
Log(EC50) = -7,709
*
De
cre
ase
of
fre
qu
en
cy o
f
card
iac
con
trac
tio
ns
(%)
B
-14 -12 -10 -8 -6 -4 -2
0
10
20
30
40
50
60
70
80
90
100
Log(Concentration of BpF2 in mg/ml)Log(EC50) = -9,702
*
*
*
*
De
cre
as
e o
f a
mp
litu
de
of
ca
rdia
c c
on
tra
cti
on
s
(%)
A
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Fig.2.Effect of different concentrations of atropine on the negative inotropic and chronotropic effects induced by BpF2 at 10-4 mg/ml
The negative inotropic and chronotropic effects induced by BpF2 at 10-4 mg/ml were significantly (*p < 0.05, n = 4) inhibited by atropine at 10-10, 10-9 and 10-8 mg/ml.
Table.1.Acute toxicity of BpF2 in mice
Group 1 (Control group) was administered normal saline (NS) intraperitoneally. The 8 other groups received BpF2 (215-740 mg/kg b.w.) intraperitoneally and the mortality rate was evaluated after treatment.
Groups of 10 mice Doseof BpF2 (mg/kg b.w.) Number of mice dead
Mortality %
1 NS 0 0
2 215 0 0
3 290 2 20
4 365 4 40
5 440 5 50
6 515 6 60
7 590 8 80
8 665 10 100
9 740 10 100
0
10
20
30
40
50
Amplitude
Frequency
Concentrations of atropine (mg/ml)
0 10-10 10-910-8
*
** *
*
*
Re
du
cti
on
of
ca
rdia
c a
mp
litu
de
an
d
fre
qu
en
cy
(%)
ind
uc
ed
b
y B
pF
2 a
t 1
0-4
mg
/ml
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Table.2.Dose-response effect of BpF2 on rabbit ECG
BpF2 elicited a decrease of P, QRS, T waves and the heart rate. PQ interval increased. These changes were significant (*p < 0.05, n = 4). QT interval was not significantly affected.
RESULTS
Acute toxicity of BpF2
BpF2, injected at doses below 215 mg/kg of
body weight (b.w.), had no significant
effects on the behaviour of mice. However,
the injection of BpF2 at doses ranging from
215 to 740 mg/kg b.w. caused a decrease
in locomotion and the animals tended to
gather. These effects were observed 25-35
min after injection. Two hours after
injection, the mice that received doses
between 590 and 740 mg/kg b.w. became
completely immobile, refused to feed and to
drink water. The groups which were
administered doses less than 590 mg/kg
b.w. displaced painfully and exhibited
difficulties in feeding and drinking.
Mortality was function to the administered
dose. Indeed, no death was observed for
doses of BPF2 less than 215 mg/kg b.w. in
the treated mice.
For doses of BpF2 above 215 mg/kg b.w.,
the mortality rate of mice increased in a
dose-dependent manner. The first deaths
were noticed between 5 and 6 hours post
treatment. Death occurred after
convulsions and jerky breathing and
animals remained lying down on their back.
At the dose of 740 mg/kg b.w., the
mortality rate was 100%. That dose
corresponded to the lethal dose that caused
mortality of all mice (LD100). Table I shows
the evolution of the mortality of mice for
one experiment. The same experiment was
repeated three times and permitted to
determine the LD50 by the graphic and the
calculation methods. According to the
graphic method the LD50 determined was
429.14 ± 28.11 mg/kg b.w. The calculated
LD50 was 452.50 ± 23.10 mg/kg b.w. There
is no statistical difference between the two
values of LD50 (p > 0.05).
BpF2 (mg/kg b.w.)
P wave (µv)
QRS complex (µv)
T wave (µv)
PQ interval (ms)
QT interval
(ms)
Frequency (cycles/min)
0(Control) 168.75 ±11.96 612.5±65.74 118.75±6.25 85±5 150±5.77 280±4.4
5 125 ±28.86 562.5±65.74 112.5±7.21 90±5.77 150±5.77 269±4.6
15 100 ±14.43 418.75±86.22 81.25±6.25* 100±8.16 155±9.57 264±3.48*
20 75 ±17.67* 312.5±21.65* 68.75±6.25* 120±8.16* 160±8.16 258±3.31*
25 62.5 ±12.5* 193.75±27.71* 0* 125±9.57* -------- 242±2.16*
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Effects of BpF2 on the contractile activity of isolated rat heart
Dose-response effect of BpF2 on isolated
rat heart contractions
The dose-response effect of BpF2 on isolated
rat heart contractions was achieved. This
resulted in a dose-dependent decrease of
the cardiac amplitude and frequency
compared to control recordings, for
concentrations ranging from 10-12 to 10-4
mg/ml. In this range of concentrations, the
decrease of amplitude varied from 11.18 ±
0.93% to 35.30 ± 1.01% and the decrease of
the frequency attained 8.98 ± 0.59% to
42.29 ± 2.77%. These effects were reversible
after returning to Mac Ewen reference
solution.
The dose-response curves (Fig. 1A and B)
for four (n= 4) experiments permitted to
determine the values of EC50 which were
1.98 x 10-10 mg/ml and 1.95 x 10-8 mg/ml
respectively for the amplitude and the
frequency.
Interaction atropine- BpF2 on isolated
rat heart contractions
In this experimentation, a control effect of
BpF2 was implemented at the concentration
of 10-4 mg/ml. At this concentration, BpF2
elicited negative inotropic and chronotropic
effects. Indeed, the amplitude and the
frequency diminished and reached 28 ±
2.15% and 43.55 ± 3.79% respectively. The
treatment of the heart with atropine at 10-
10, 10-9 and 10-8 mg/ml strongly and
significantly (p < 0.05) inhibited the
negative inotropic and chronotropic effects
induced by BpF2 at10-4 mg/ml. Thus, the
amplitude dropped to 14.20 ± 1.53%, 10.73
± 0.72% and 6.02 ± 0.65% respectively for
atropine at 10-10, 10-9 and 10-8 mg/ml. The
same tendency was noticed with the
frequency which values fell to 25.23 ±
0.96%, 17.83 ± 0.94% and 15.79 ± 1.51%
respectively for atropine at 10-10, 10-9 and
10-8 mg/ml (Fig. 2).
Effects of BpF2 on the electrocardiogram
of rabbit
As shown in table 2, the dose-response
effect of BpF2 was carried out on rabbit
ECG. The results indicated that BpF2
triggered some changes in the parameters
of the global electrical activity of the rabbit
heart. The normal ECG values recorded
(control) significantly (p < 0.05) decreased
for P, T, and QRS waves. Indeed, P wave
varied from 125 ± 28.86 to 62.5 ± 12.5 µv
for doses ranging from 5 to 25 mg/kg b.w.
T wave was reduced by increasing doses of
BpF2 from 5 to 25 mg/kg b.w. and dropped
from 112. 5 ± 7.21 to 0 µv. QRS complex
also diminished and reached 562.5 ± 65.74
to 193.75 ± 27.71 µv in the same range of
doses. The cardiac frequency was also
affected by the same doses of BpF2 and
deceased from 269 ± 4.6 to 242 ± 2.16
cycles/min. However, PQ interval was
found to augment significantly (p < 0.05)
from 90 ± 5.77 to 125 ± 9.57 ms for doses
ranging from 5 to 25 mg/kg b.w. whereas
QT interval increased slightly but not
significantly from 150 ± 5.77 to 160 ± 8.16
ms for doses ranging from 5-20 mg/kg b.w.
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DISCUSSION
Toxicological studies showed that doses of
BpF2 ranging from 215 to 740 mg/kg b.w.
induced a decrease in mobility. This fact
can suggest that BpF2 had an effect on the
locomotion system and therefore the
nervous system. In that range of doses, the
mortality rate increased in a dose-
dependent manner to reach 100% at a dose
of 740 mg/kg b.w. The LD50 values of BpF2
determined according to the graphic mode
(429.14 ± 28.11 mg/kg b.w.) and the
calculation mode (452.50 ± 23.10 mg/kg
b.w.) are quite similar. According to the
defined classification, substances that have
a LD50 less than 5 mg/kg b.w. are
considered highly toxic. In contrast, those
which have a LD50 greater than 5000
mg/kg b.w. are called non-toxic
substances. Between these two extremes,
very toxic substances (5 < LD50 < 50 mg/kg
b.w.), toxic substances (50 < LD50 < 500
mg/kg b.w.), or weakly toxic substances
(500 < LD50 < 5000 mg/kg b.w.) can be
distinguished.22 According to this
classification, BpF2, administered
intraperitoneally, is not highly or very toxic
but simply toxic. This toxicity is relatively
low compared to that of other plant
extracts. Indeed, BpF2 is less toxic than the
aqueous extracts of root bark of Swartzia
madagascariensis (Caesalpiniaceae) with a
LD50 equals to 5.99 mg/kg b.w. and the
leaves of Sesamum radiatum (Pedaliaceae)
which LD50 is 184.2 ± 21 mg/kg b.w.21,23
BpF2 is more toxic than the methanol
extract from the stem bark of Erythrina
Senegalensis (Fabaceae) which LD50 is
above 12500 mg/kg b.w. and the seeds
infusion of Securigera securidaca (Fabaceae)
with a LD50 of 1260 mg/kg b.w.19,24
BpF2 caused a dose-dependent decrease in
the amplitude and frequency of the
mechanical activity of the isolated rat heart.
The cardioinhibitory effect induced by this
fraction F2 was strongly reduced in the
presence of increasing doses of atropine.
However, the negative chronotropic and
inotropic effects did not completely vanish.
These results were highly similar to those
obtained with the administration of
acetylcholine, suggesting the presence in
this extract of cardioactive cholinomimetic
substances acting via muscarinic
cholinoceptors. These effects were also
similar to those of aqueous extracts of
plants such as total leaf extract and the
fraction F2 of Mareya micrantha
(Euphorbiaceae) on the isolated rat heart,
bulbs extract of Hypoxis hemerocallidea
Fisch & Mey (Hypoxidaceae) and the leaf
extract from Persea americana Mill
(Lauraceae) on the isolated guinea-pig
atrium.25,26,27,28 The effects of acetylcholine
on the heart activity are well known in
mammals. According to some studies, the
predominating muscarinic receptors are
those of the type M2.29,30 Several authors
indicate that their stimulation leads to a
decrease in contractile force by the
activation of adenyl cyclase and the
activation of a G protein coupled to
potassium channels. Thus, the final
consequences are hyperpolarization and
decreased opportunities for transmission of
action potentials, which limit calcium
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influx.31,32,33,34. However, other authors
observed positive inotropic effects by the
use of muscarinic receptor agonists, and
according to them, these effects were
mediated by M1 and M3 receptors.30,35,36,37
Through the results obtained with BpF2 on
isolated rat heart, it is likely that the
cholinomimetic substances in this fraction
act via M2 receptors. A study showed that
in isolated human myocardium,
acetylcholine had a biphasic effect resulting
in a negative inotropic effect at low doses
(10-9 to 10-6 M) through M2 receptors and
positive inotropic effect at high doses (10-6
to 10-3 M) via M1 receptors.38
On rabbit ECG, BpF2 induced a sharp
reduction of P wave, QRS complex and T
wave. However, this fraction elicited a slight
increase of PQ interval and had no
significant effect on QT interval. These
results revealed that BpF2 could decrease
the depolarization of the atria and
ventricles, ventricular repolarization and
heart rate. The outcome of the whole action
of BpF2 indicated an inhibitory effect of this
substance on the global electrical activity of
the rabbit heart. These results were similar
to those of acetylcholine on the ECG.
Indeed, some authors revealed that on a
heart-lung preparation of dog, acetylcholine
caused a slowing of the pacemaker, a
blockade of atrio-ventricular conduction
associated with prolongation of PR interval
and a decrease in ventricular activity.39,40,41
However, BpF2 did not influence
significantly the duration of QT interval. A
series of studies advocate drug evaluation
against the risk of induction of arrhythmias
due to torsades de pointes in humans. This
requires an assessment of QT interval
which extension is a risk.42,43 The effects of
acetylcholine on the ECG of guinea pig also
were highlighted by an Ivorian reseracher.25
Thus, the presence of cholinomimetic
substances in BpF2 could justify its effects
on rabbit ECG.
CONCLUSION
The active fraction F2 of the aqueous leaf
extract of Bidens pilosa L. is toxic when
administered intraperitoneally. However,
this cannot impede its use in traditional
medicine which should nevertheless be
done with caution. The study of the effects
of BpF2 on the isolated rat heart showed
that this fraction caused cardioinhibition
which was linked to the presence of
cholinomimetic substances. On the
electrical activity of rabbit heart, the
diminution of the activity was due to the
inhibitory effect of BpF2 on ECG
parameters, except QT interval. This
observation seemed particularly interesting
since an extension of QT interval could be a
factor of arrhythmia induction. These
cardioinhibitory effects of BpF2 could
explain, at least in part, the hypotension
induced by this fraction by cholinomimetic
substances in rabbits.
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