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P a g e | 751 eISSN: 2231-0541

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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: [email protected] 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 M 2 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.

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences

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Volume 4 Issue 4 July-August 2013 Available online: www.pharmanest.net

P a g e | 752 INTRODUCTION Traditional medicine has been practiced for

This study was aimed to evaluate in mice

years in developing countries, especially in

the acute toxicity of a chromatographic

Africa. According to WHO, this use is

fraction from the leaves of Bidens pilosa L.

widespread and is becoming increasingly

(BpF2) and to investigate its effects on

important in terms of health and economy,

isolated

since more than 80% of the population

electrocardiogram.

resort to it for health needs.1 According to the

same

and

rabbit

MATERIAL AND METHODS

Asia

still

use

traditional

Animals: Rabbits (Oryctolagus cuniculus)

of

historical

weighing 2 ± 0.2 kg, Mice (Mus musculus)

circumstances and cultural beliefs. Bidens

weighing between 20 and 25 g and rats

pilosa L. is an herbaceous plant, set up,

(Ratus norvegicus) weighing between 180

with regularly cogged and glabrous leaflets.

and 250 g were used. They were bred in

Very widespread pantropical species from

Animal

Angola to Cameroon, it is also found in Côte

Pharmacology and Phytotherapy of the

d’Ivoire.2,3 The plant is used in traditional

University of Nangui Abrogoua (Former

medicine to treat diverse illnesses such as

University of Abobo-Adjamé, Abidjan, Côte

diarrhoea and inflammation in Côte d’Ivoire

d’Ivoire) according to the principles for the

and is prescribed as anti-poison, against flu

care and use of laboratory animals of the

and

Ethical Committee of the University (Nangui

medicine

people

because

haemorrhoids

in

and

heart

in

America,

source,

rat

Latin

Congo

and

Rwanda.2,3,4 Many works carried out on this

house

of

Animal

Physiology,

Abrogoua, Abidjan, Côte d’Ivoire).

plant revealed different properties among which

anti-microbial,

Plant material: The plant material was

anti-inflammatory,

described.14,15

anti bacterial, anti-malarial and anti-gastric

leaves

ulcer effects.5,6,7,8,9

of

Authentication

Bidens

pilosa

L.

of

fresh

(Asteracea)

collected from Abidjan, Côte d’Ivoire was The cardiovascular aspect was also subject

implemented by Prof. Aké-Assi Laurent, an

to many studies.10,11,12,13,14, 15. Investigations

expert in Botany (Department of Botany,

showed that a chromatographic fraction

University

from Bidens pilosa L. leaves (BpF2) elicited a

d’Ivoire). In Côte d’Ivoire, Bidens pilosa L.

decrease of the blood pressure of rabbit via

was first found on November 12, 1966 in

cholinomimetic

Bondoukou

and

β-adrenomimetic

of

Cocody,

and

Abidjan,

herbarium

Côte

specimen

agonist substances.14,15 The phytochemical

(voucher n° 9266) was made and deposited

study of the fraction revealed flavonoids,

at the National Botanic Centre (University

polyphenols and catechic tannins.

of Cocody, Abidjan, Côte d’Ivoire) and then


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P a g e | 753 in Kakpin (Côte d’Ivoire) on June 20, 1968.

differences

Herbarium specimen (voucher n° 10286)

concentrations were precised by an analysis

was

the

of variances (ANOVA) of the multiple test of

of

comparison of Turkey-Kramer and were

also

National

made Botanic

and deposited at Centre

(University

Cocody, Abidjan, Côte d’Ivoire).

between

considered statistically

the

significant when

p < 0.05.

Plant extraction: The extraction and the separation of extracts were as previously

Acute toxicity assessment

described.14,15 Hundred grams (100g) of

Mice were divided into 9 groups of 10 mice

powder from Bidens pilosa L. leaves dried at

(5 males and 5 females). Each mouse in the

room temperature were macerated under

first group (control group) was treated with

magnetic shaker during 48h in 2 L of

0.5 ml isotonic solution of NaCl 9 ‰ . The

distilled water. The supernatant was filtered

other 8 groups were treated with a single

on cotton and filter paper Whatman. Two

administration

litres of distilled water were added to the

normal

base and then mixed during two hours and

of

saline

BpF2 diluted

isotonic

in

solution.

the The

different doses of BpF2 ranging from 215

also filtered. The filtrates were freeze-dried

mg/kg b.w to 740 mg/kg b.w. and the

using a lyophilisator TELSTAR (Terrassa,

normal saline solution of the control group

Spain). 0.8 g of the powder obtained were

were administered intraperitoneally (i.p.).

dissolved in 10 ml of distilled water and

Behavioural

chromatographed on a fine Sephadex G25

changes

of

the

8

treated

groups were observed every 30 min for a

column (3 by 20 cm) packed in distilled

period of 2 hours after administration of the

water. Elution was carried out with the

extract (Mandal et al., 2001) and mortality

same solvent. Fractions (5) of 20 ml each

rate were recorded for 24 hours post

were collected then freeze-dried. They were

treatment.16

tested on the blood pressure of the rabbit.

calculation

The fraction 2 (BpF2) was found to be the

The

graphic

methods

were

and

the

used

to

determine the LD50.17,18

most active.

Recording

Chemicals: Atropine was purchased from

of

the

isolated

rat

heart

activity

Sigma Chemical Company (St Louis, MO, USA).

The experimental process used to record and assess the isolated rat inotropic and

Data analysis: Statistical analysis and

chronotropic activities was as previously

graphics were performed with the software

described.13,

GraphPad Instat and GraphPad Prism 4

the

mean

(m

±

sem).

Rat was anesthetized with injection

of

20%

placed under artificial respiration to avoid

The


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ethyl

urethane at 1 g/kg body weight. It was then

values are expressed as mean ± standard on

19

intraperitoneal

(San Diego California, USA) respectively. All error

observed

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P a g e | 754 anoxia of the heart during operation. To do

BpF2 was dissolved in Mac Ewen solution.

this, a tracheotomy was performed and a

Registration

cannula connected to an air pump was

of

the

global

electrical

activity (ECG) of the rabbit

placed in the trachea. Then, a thoracotomy was practiced. A hemisection of the aorta

Methods

was carried out. A cannula fixed to a

described.19,20,21.

syringe

solution

the rabbit was recorded by the technique of

(2500 UI, 0.2 ml/100 g body weight) was

external electrodes used in the human

inserted in the aorta. The heart was rapidly

practices and adapted to the rabbit.20

removed

Briefly,

containing

and

the

heparinized

heparinized

solution

were

the

as

previously

The electrocardiogram of

saphenous

vein

of

the

injected to dissolve and expel any blood

anesthetized rabbit by an intraperitoneal

clots that were probably formed in the heart

injection of 40% ethyl urethane (1 g/kg

to prevent thrombosis in the coronary

body weight) was intubated in order to

circulation. The isolated heart was perfused

administer plant extract.20 The armpits of

with Mac Ewen solution of the following

the anterior limbs and the groin of the

composition (mM): NaCl 147; KCl 5.6; CaCl2

posterior limbs were shaved and cleaned

2.6; NaH2PO4 0.66; CO3NaH 11.9; MgCl2

with

0.24; C6H12O6 11 with a pH adjusted to 7.4.

electrolytic dough, four electrodes were put

The apex of the heart was fixed by a fine

and bound to the four sockets of the

clip and linked to the needle of the

registration

cardiograph

electrocardiograph

treatment,

for the

recording. heart

was

After

each

washed

90%

ethanol.

cable

After

connected

applying

to

(CARDIOFAX

the ECG-

by

6851K, Nihon Kohden, Japan). The studied

perfusion fluid for 10 min, time within

parameters (P, QRS, T waves, PQ, QT

which the baseline recording was achieved,

intervals

and the second dose was then given. The

recorded from the DIII derivation of the

recording before the direct perfusion of

standards or bipolar Einthoven derivations

extract was considered as baseline reading

on thermo sensitive paper, at constant

for each dose (control).

speed (25 mm/s).

and

cardiac

frequency)


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were

P a g e | 755

Decrease of amplitude of cardiac contractions (%)

A

*

100 90 80 70 60 50 40 30 20 10 0

*

* * -14

-12

-10

-8

-6

-4

-2

Log(Concentration of BpF 2 in mg/ml)

Log(EC50 ) = -9,702

B

* Decrease of frequency of cardiac contractions (%)

100 90 80 70 60 50 40 30 20 10 0 -14

-12

-10

-8

-6

-4

-2

Log(Conce ntration of BpF 2 in mg/ml) Log(EC50 ) = -7,709

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.


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Reduction of cardiac amplitude and frequency (%) induced by BpF 2 at 10 -4 mg/ml

P a g e | 756

50 40 Amplitude Frequency

30

* 20

*

*

*

*

10

*

0 0

10-9

10-10

10-8

Concentrations of atropine (mg/ml)

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 BpF 2 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

Groups of 10 mice

Doseof BpF2 (mg/kg b.w.)

Number of mice dead

Mortality %

1 2 3 4 5 6 7 8 9

NS 215 290 365 440 515 590 665 740

0 0 2 4 5 6 8 10 10

0 0 20 40 50 60 80 100 100

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.


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P a g e | 757

Table.2.Dose-response effect of BpF2 on rabbit ECG BpF2 (mg/kg b.w.) 0(Control)

P wave (µv)

QRS complex (µv)

T wave (µv)

PQ interval (ms)

168.75 ±11.96

612.5±65.74

118.75±6.25

85±5

QT interval (ms) 150±5.77

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*

Frequency (cycles/min) 280±4.4

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

For doses of BpF2 above 215 mg/kg b.w., the mortality rate of mice increased in a

BpF2, injected at doses below 215 mg/kg of

dose-dependent manner. The first deaths

body weight (b.w.), had no significant

were noticed between 5 and 6 hours post

effects on the behaviour of mice. However,

treatment.

the injection of BpF2 at doses ranging from

convulsions

215 to 740 mg/kg b.w. caused a decrease

At the

gather. These effects were observed 25-35 after

injection.

Two

hours

groups

which

displaced

difficulties

in

painfully feeding

and and

rate

was

100%.

That

one experiment. The same experiment was

were

repeated three times and permitted to determine the LD50 by the graphic and the

exhibited

calculation

drinking.

methods.

According

to

the

graphic method the LD50 determined was

Mortality was function to the administered

429.14 ± 28.11 mg/kg b.w. The calculated

dose. Indeed, no death was observed for

LD50 was 452.50 ± 23.10 mg/kg b.w. There

doses of BPF2 less than 215 mg/kg b.w. in

is no statistical difference between the two

the treated mice.

values of LD50 (p > 0.05).


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dose

the evolution of the mortality of mice for

administered doses less than 590 mg/kg b.w.

and

mortality of all mice (LD100). Table I shows

completely immobile, refused to feed and to The

breathing

corresponded to the lethal dose that caused

between 590 and 740 mg/kg b.w. became water.

jerky

after

dose of 740 mg/kg b.w., the

mortality

after

injection, the mice that received doses

drink

and

occurred

animals remained lying down on their back.

in locomotion and the animals tended to min

Death

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P a g e | 758 Effects of BpF2 on the activity of isolated rat heart

contractile

negative inotropic and chronotropic effects induced by BpF2 at10-4 mg/ml. Thus, the

Dose-response effect of BpF2 on isolated

amplitude dropped to 14.20 ± 1.53%, 10.73

rat heart contractions

± 0.72% and 6.02 ± 0.65% respectively for atropine at 10-10, 10-9 and 10-8 mg/ml. The

The dose-response effect of BpF2 on isolated

same

rat heart contractions was achieved. This cardiac

compared

amplitude

to

control

and

noticed

with

the

0.96%, 17.83 ± 0.94% and 15.79 ± 1.51%

frequency

recordings,

was

frequency which values fell to 25.23 ±

resulted in a dose-dependent decrease of the

tendency

respectively for atropine at 10-10, 10-9 and

for

10-8 mg/ml (Fig. 2).

concentrations ranging from 10-12 to 10-4 mg/ml. In this range of concentrations, the

Effects of BpF2 on the electrocardiogram

decrease of amplitude varied from 11.18 ±

of rabbit

0.93% to 35.30 ± 1.01% and the decrease of

As shown in table 2, the dose-response

the frequency attained 8.98 ± 0.59% to

effect of BpF2 was carried out on rabbit

42.29 ± 2.77%. These effects were reversible

ECG. The

after returning to Mac Ewen reference

results indicated that BpF2

triggered some changes in the parameters

solution.

of the global electrical activity of the rabbit

The dose-response curves (Fig. 1A and B)

heart. The normal ECG values recorded

for four (n= 4) experiments permitted to

(control) significantly (p < 0.05) decreased

determine the values of EC50 which were

for P, T, and QRS waves. Indeed, P wave

1.98 x

10-10

mg/ml and 1.95 x

10-8

mg/ml

varied from 125 ± 28.86 to 62.5 ± 12.5 µv

respectively for the amplitude and the

for doses ranging from 5 to 25 mg/kg b.w.

frequency.

T wave was reduced by increasing doses of BpF2 from 5 to 25 mg/kg b.w. and dropped

Interaction atropine- BpF2 on isolated

from 112. 5 ± 7.21 to 0 µv. QRS complex

rat heart contractions

also diminished and reached 562.5 ± 65.74

In this experimentation, a control effect of

to 193.75 ± 27.71 µv in the same range of

BpF2 was implemented at the concentration

doses. The cardiac frequency was also

of 10-4 mg/ml. At this concentration, BpF2

affected by the same doses of BpF2 and

elicited negative inotropic and chronotropic

deceased from 269 ± 4.6 to 242 ± 2.16

effects. Indeed, the amplitude and the

cycles/min.

frequency diminished and reached 28 ±

found to augment significantly (p < 0.05)

2.15% and 43.55 ± 3.79% respectively. The

from 90 ± 5.77 to 125 ± 9.57 ms for doses

treatment of the heart with atropine at 10-

ranging from 5 to 25 mg/kg b.w. whereas

10,

QT interval increased slightly but not

10-9 and 10-8 mg/ml strongly and

However,

PQ

interval

was

significantly from 150 ± 5.77 to 160 ± 8.16

significantly (p < 0.05) inhibited the

ms for doses ranging from 5-20 mg/kg b.w. PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences

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P a g e | 759 DISCUSSION

above 12500 mg/kg b.w. and the seeds infusion of Securigera securidaca (Fabaceae)

Toxicological studies showed that doses of

with a LD50 of 1260 mg/kg b.w.19,24

BpF2 ranging from 215 to 740 mg/kg b.w. induced a decrease in mobility. This fact

BpF2 caused a dose-dependent decrease in

can suggest that BpF2 had an effect on the

the

locomotion

the

mechanical activity of the isolated rat heart.

nervous system. In that range of doses, the

The cardioinhibitory effect induced by this

mortality

dose-

fraction F2 was strongly reduced in the

dependent manner to reach 100% at a dose

presence of increasing doses of atropine.

of 740 mg/kg b.w. The LD50 values of BpF2

However, the negative chronotropic and

determined according to the graphic mode

inotropic effects did not completely vanish.

(429.14 ± 28.11 mg/kg b.w.) and the

These results were highly similar to those

calculation mode (452.50 ± 23.10 mg/kg

obtained

b.w.) are quite similar. According to the

acetylcholine, suggesting the presence in

defined classification, substances that have

this extract of cardioactive cholinomimetic

a

substances

LD50

system rate

less

and

therefore

increased

than

5

in

mg/kg

a

b.w.

are

amplitude

and

with

frequency

the

of

the

administration

acting

via

of

muscarinic

considered highly toxic. In contrast, those

cholinoceptors. These

which have a LD50 greater than 5000

similar to those of aqueous extracts of

mg/kg

plants such as total leaf extract and the

b.w.

are

called

non-toxic

fraction

very toxic substances (5 < LD50 < 50 mg/kg

(Euphorbiaceae) on the isolated rat heart,

b.w.), toxic substances (50 < LD50 < 500

bulbs extract of Hypoxis hemerocallidea

mg/kg b.w.), or weakly toxic substances

Fisch & Mey (Hypoxidaceae) and the leaf

(500 < LD50 < 5000 mg/kg b.w.) can be

extract

distinguished.22

According

(Lauraceae)

classification,

BpF2,

this

administered

of

from

Persea

on

atrium.25,26,27,28

Mareya

also

substances. Between these two extremes,

to

F2

effects were

the

micrantha

americana

isolated

Mill

guinea-pig

The effects of acetylcholine

intraperitoneally, is not highly or very toxic

on the heart activity are well known in

but simply toxic. This toxicity is relatively

mammals. According to some studies, the

low

plant

predominating muscarinic receptors are

extracts. Indeed, BpF2 is less toxic than the

those of the type M2.29,30 Several authors

aqueous extracts of root bark of Swartzia

indicate that their stimulation leads to a

madagascariensis (Caesalpiniaceae) with a

decrease

LD50 equals to 5.99 mg/kg b.w. and the

activation

of

adenyl

leaves of Sesamum radiatum (Pedaliaceae)

activation

of

a

which LD50 is 184.2 ± 21 mg/kg b.w.21,23

potassium

BpF2 is more toxic than the methanol

consequences are hyperpolarization and

extract from the stem bark of Erythrina

decreased opportunities for transmission of

Senegalensis

action

compared

to

that

(Fabaceae)

of

other

which

LD50

is

in

contractile G

cyclase protein

channels.

potentials,

force

Thus,

which

by

the

and

the

coupled the

limit

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final

calcium

PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences Volume 4 Issue 4 July-August 2013 Available online: www.pharmanest.net

to

P a g e | 760 influx.31,32,33,34.

However,

other

authors

However,

BpF2

did

not

influence

observed positive inotropic effects by the

significantly the duration of QT interval. A

use of muscarinic receptor agonists, and

series of studies advocate drug evaluation

according

to

them,

these

were

against the risk of induction of arrhythmias

receptors.30,35,36,37

due to torsades de pointes in humans. This

Through the results obtained with BpF2 on

requires an assessment of QT interval

isolated rat heart, it is likely that the

which extension is a risk.42,43 The effects of

cholinomimetic substances in this fraction

acetylcholine on the ECG of guinea pig also

act via M2 receptors. A study showed that

were highlighted by an Ivorian reseracher.25

in

Thus,

mediated by M1 and M3

isolated

effects

human

myocardium,

the

presence

of

cholinomimetic

acetylcholine had a biphasic effect resulting

substances in BpF2 could justify its effects

in a negative inotropic effect at low doses

on rabbit ECG.

(10-9 to 10-6 M) through M2 receptors and

CONCLUSION

positive inotropic effect at high doses (10-6 to 10-3 M) via M1 receptors.38

The active fraction F2 of the aqueous leaf extract of Bidens pilosa L. is toxic when

On rabbit ECG, BpF2 induced a sharp

administered

reduction of P wave, QRS complex and T of

PQ

interval

and

had

medicine which should nevertheless be

no

done with caution. The study of the effects

significant effect on QT interval. These

of BpF2 on the isolated rat heart showed

results revealed that BpF2 could decrease the

depolarization

of

the

atria

However,

this cannot impede its use in traditional

wave. However, this fraction elicited a slight increase

intraperitoneally.

that this fraction caused cardioinhibition

and

which was

ventricles, ventricular repolarization and

linked to

cholinomimetic

heart rate. The outcome of the whole action

electrical

of BpF2 indicated an inhibitory effect of this

the

substances.

activity

of

rabbit

presence On

the

heart,

the

diminution of the activity was due to the

substance on the global electrical activity of

inhibitory

the rabbit heart. These results were similar

parameters,

to those of acetylcholine on the ECG.

effect

of

except

BpF2 QT

on

interval.

ECG This

observation seemed particularly interesting

Indeed, some authors revealed that on a

since an extension of QT interval could be a

heart-lung preparation of dog, acetylcholine

factor

caused a slowing of the pacemaker, a

of

arrhythmia

cardioinhibitory

blockade of atrio-ventricular conduction

effects

induction. of

BpF2

These could

explain, at least in part, the hypotension

associated with prolongation of PR interval

induced by this fraction by cholinomimetic

and a decrease in ventricular activity.39,40,41

substances in rabbits.


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