Publication 15 LAFMC 2014

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PHARMANEST - An International Journal of Advances in Pharmaceutical Sciences

Volume 4 |Issue 4| July-August 2013

Available online: www.pharmanest.net

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: [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 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

P a g e | 760




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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