Journal of Ethnopharmacology 57 (1997) 189 – 196
Cardiovascular pharmacology of aqueous extract of the leaves of Bridelia atro6iridis Muell. Arg. (Euphorbiaceae) in the rat A. Corallo a,*, S. Foungbe´ a, M. Davy b, Y. Cohen b a
Laboratory of Physiology-Pharmacology, Faculty of Pharmacy, Uni6ersity of Abidijan, 01 BPV 34, Abidjin, Coˆte d’I6oire b Laboratory of Pharmacolgy, Faculty of Pharmacy, Uni6ersity of Paris-Sud, 92296, Chatenay-Malabry, France Received 10 December 1996; received in revised form 23 May 1997; accepted 23 June 1997
Abstract Lyophilised decoction (10%) of the leaves of Bridelia atro6iridis Muell. Arg. (Euphorbiaceae) was studied in the rat cardiovascular system. In vivo, the extract (15 and 30 mg/kg) caused a decrease of arterial pressure and a decrease of heart rate in an anaesthetized rat (ethylcarbamate 1.2 g/kg). If administrations of Bridelia were repeated (three times) a tachyphylaxie phenomena was observed. After administrations of adrenaline, noradrenaline, acethylcholine and isoprenaline the extract had no effect on the action of these neuromediators on blood pressure. In vitro the extract induced dose-dependent negative inotropic and chronotropic effects in isolated rat heart. It was ineffective in rat aorta preparations. Bridelia seemed to have a direct effect on rat heart. Hypotension is not due to an action on the vessels. The extract did not appear to interact with adrenergic nor cholinergic receptors. However, the extract was able to potentiate barium chloride induced contractions of rat aorta preparations. The extract might act through potential dependent calcium channels © 1997 Elsevier Science Ireland Ltd. Keywords: Bridelia atro6iridis; Blood pressure; Cardiovascular activity; Neuromediators
1. Introduction An aqueous extract of the leaves of Bridelia atro6iridis Muell. Arg. (Euphorbiaceae) had been reported in a previous paper to exhibit important
* Corresponding author.
uterotonic activity justifying its traditional use in north Coˆte d’Ivoire to facilitate difficult labour or to produce abortion (Corallo et al., 1985). Uterotonic contractions induced by Bridelia were not due to adrenergic nor cholinergic receptors stimulation (Corallo et al., 1988; Corallo, 1990). The cellular basis of this activity had been attributed to calcium entry through potential calcium channels (Corallo et al., 1991). Biphasic effect in isolated intestine had been related to two chemical
0378-8741/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 3 7 8 - 8 7 4 1 ( 9 7 ) 0 0 0 7 1 - 8
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A. Corallo et al. / Journal of Ethnopharmacology 57 (1997) 189–196
Fig. 1. Effect of aqueous extract of Bridelia (15 mg/kg) on arterial pressure (AP) and heart rate (HR) in the rat. a1 – a2, Control arterial pressure; b1 – b2, control heart rate. (A) AP, hypotension 10 s after intravenous injection of extract (initial AP, 145 mmHg; minimal AP, 65 mmHg; duration, 8 min). (B) HR, bradycardia 30 s after intravenous injection of extract (initial rate, 400 beats/min; minimal heart rate, 230 beats/min; duration, more than 10 min. (C) Time (min).
compounds (not yet identified) with contracting and relaxing activity respectively (Corallo et al., 1989). The present study was designed to examine in vivo and in vitro cardiovascular effects of lyophilised extract of the leaves of Bridelia atro6iridis. We needed more information and confirmation about the action mechanism already found with uterotonic activity. For that reason, firstly, it was necessary to verify the action of Bridelia on adrenergic and cholinergic receptors of rat aortic vessels or rat heart. Secondly, it was also necessary to examine action of Bridelia on potential dependent calcium channels of the cardiovascular system. Lastly it was interesting to establish, if the extract presented dangerous secondary effects on the cardiovascular system in vivo.
2. Materials and methods
2.1. Plant material Leaves of Bridelia atro6iridis Muell. Arg. (Euphorbiaceae) were collected throughout the year from Zuenoula (north Cote d’Ivoire), then dried (at 30°C) and grounded to powder (this plant is repertoried under no. 1002 at the Floristic National Centre of Abidjan by L. Ake Assi).
2.2. Leaf extract preparation A 10% decoction was prepared, then frozen, lyophilised and stored. The extract was diluted daily in the adequate concentration of physiological solution.
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Table 1 Effect of aqueous extract of Bridelia on arterial pressure and heart rate in the rat at 15 and 30 mg/kg (n= 7) Injection of Bridelia
First injection Second injection Third injection
Inhibition of arterial pressure
Inhibition of heart rate
15 mg/kg
30 mg/kg
15 mg/kg
30 mg/kg
33 911 16 911 7 910
49 98 19 9 8 696
12 99 9 96 5 95
13 96 2 94 1 92
Values are expressed as mean 9 S.E.M. (%).
2.3. Reference drugs The drugs used in the present work were: Acetylcholine chloride (Sigma), adrenaline tartrate (Aguettant), noradrenaline bitartrate (Levophed: Winthrop), isoprenaline chlorhydrate (Isuprel: Winthrop), heparine (Roche), barium chloride (Prolabo), atropine sulfate (Prolabo) and ethylcarbamate (Prolabo).
2.4. Statistical analysis Results are expressed as mean9S.E.M (n = seven experiments) and analysed statistically using Student’s t-test or Wilcoxon Mann Whitney test and P B0.05 was regarded as significant.
2.5. Anaesthetized rat experiments (in 6i6o) Sprague-Dawley male rats weighing between 300 – 500 g were anaesthetized with ethylcarbamate (1.2 g/kg, intraperitoneal injection). The trachea was cannulated with polyethylene tube (3 mm diameter) to facilitate spontaneous respiration. Drugs and aqueous extract were injected via a cannula inserted into the pudendal penis vein. The maximum volume of injection was 0.1 ml for 100 g body weight. Arterial pressure was monitored from the left carotid artery via the arterial cannula connected to a pressure transducer (AITT Cannon). Heart rate (from electrocardiogram obtained in D2) was recorded on a physiograph (Desk model DMP 4B). All drugs were dissolved in saline solution (NaCl 0.9%).
2.6. Cardio6ascular acti6ity tests (in 6i6o) Three series of tests were carried out:
2.6.1. Blood pressure and heart rate responses to Iyophilised aqueous extract of Bridelia (EaB) The extract of Bridelia (15 mg/kg) was administered 10 min after saline administration. This experiment was repeated three times in the same animal (n= 7). The same experiment was realised in another animal at the dose of 30 mg/kg (n= 7). 2.6.2. Neuromediators and Bridelia using the following sequence A first series of neuromediators: Noradrenaline (3 mg/kg), adrenaline (3 mg/kg), isoprenaline (3 mg/kg) and acetylcholine (l mg/kg), were administered at 10 min intervals. They were followed by one Bridilia injection at the dose of 15 mg/kg. A second series (30 min later) then a third series (1 h later) of the same neuromediators were administered after the Bridelia injection. 2.6.3. Neuromediatiors and Bridelia using a different sequence A first series of the same neuromediators, in the same order, at the same doses (separated by 10 min intervals) were administered and followed by a first Bridelia injection (15 mg/kg). At 10 min following the first injection of Bridelia, a second series of neuromediators were administered, followed by a second Bridelia administration. At the end, a third series of neuromediators were administered just 10 min after the second Bridelia injection.
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Table 2 Interaction between neuromediators and aqueous extract of Bridelia (15 mg/kg; one injection) on arterial pressure and heart rate in the rat (n = 7) Neuromediators
Noradrenaline (3 mg/kg) Adrenaline (3 mg/ kg) Isoprenaline (3 mg/ kg) Acetylcholine (1 mg/ kg)
First series of neuromediators
Second series of neuromediators after one injection of Bridelia
Third series of neuromediators
MAP−IAP
MHR−IAP
MAP−IAP
MHR−IHR
MAP−IHR
MAP−IHR
309 10
99 3
31 923
7 94
31 9 14
11914
509 7
793
539 18
10 97
58 9 17
19 910
46 9 9
159 5
419 13
23 917
46 9 10
20910
489 5
49 4
439 10
396
46 9 6
5 93
Values expressed as mean 9S.E.M. (%). MAP, maximal or minimal pressure; MHR, maximal or minimal heart rate; AP, initial arterial pressure; IHR, initial heart rate; −, decrease of arterial pressure.
2.7. Rat heart (in 6itro) From anaesthetized Sprague-Dawley rat (ethylcarbamate) weighing 300 – 400 g the aortic artery is cannulated with heparinized polyethylene tubing connected immediately after extraction with physiological MacEwen’s solution with the following composition (mM/l): NaCl, 129.91; KCl, 5.63; H2NaPO4, 1.16; MgCl2, 0.52; HNaCO3, 11.0; CaCl2, 2.16; D-glucose, 5.55. The isolated heart is continuously perfused and gazed with air at 37°C (Langendorff’s technique). Mechanical activity was recorded on a kymograph using a frontal writing point connected to a lever of a cardiograph. For 10 min normal mechanical activity is observed before Bridelia increasing concentrations injections. Then a return to normal saline solution is recorded.
pended (Colot, 1972) in a 10 ml organ bath containing Krebs Heinseleit’s solution at 37°C of following composition (mM/l): NaCl, 35.86; KCl, 91.27; K2HPO4, 23.89; MgSO4 · 7H2O, 49.87; CaCl2, 68.46; NaHCO3, 23.80; D-glucose, 7.22 and gazed with a mixture of 95% O2 and 5% CO2. Aortic segments were connected to a Grass transducer and responses recorded on a polygraph Racia TAO PJ 70.
2.9.1. Direct effect of Bridelia Two series of tests were conducted: A first series with increasing concentrations of extract (1.87× 10 − 5 3.75× 10 − 5, 7.5× 10 − 5, 1.5× 10 − 4, 3 × 10 − 4, 6 ×10 − 4 g/ml) and a second series with cumulative concentrations added each min in the organ bath (final concentration 1.87× 10 − 3 g/ml).
2.8. Interaction of atropine sulfate with Bridelia Atropine sulfate (10 − 2 g/ml) was added 1 min before Bridelia administration at the concentration (9 ×10 − 3 g/ml) which elicited a maximum effect on the perfused rat heart.
2.9. Aortic artery Rat aortic artery segments (3 cm) were sus-
2.9.2. Interaction between agonists and aqueous extract Aortic artery contractions (20% of maximum contraction) were elicited by agonists (noradrenaline, 10 − 5 g/ml; adrenaline, 10 − 5 g/ml; and barium chloride 400 mg/ml) before Bridelia extract addition. It was added every min in cumulative concentrations (final concentration 1.87× 10 − 3 g/ml).
19 9 9 21 9 14 45 911 41910 209 11 31 9 17 43 9 8 39 915
698 11 9 9 19 9 11 7 97
37 9 18 319 15 35 9 18 29920
MAP−IAPR
75911 669 11 589 15 409 20 299 16 29 4
10.1 19.1 28.4* 48.7* 62.1* 96.9*
% Inhibition of maximal hear rate
239 7
MeanA 269 7°
Noradrenaline +Bridelia
Adrenaline
17 95
Increase of contraction (%) 10 9 7
° No significance. * Significant with P =0.05 (Student’s test) and Wilcoxon Mann Witney’s test. A 9 S.E.M. of contraction amplitude.
Noradrenaline
Agonists
19 95°
Adrenaline +Bridelia
12 914
Increase of contraction (%)
11 9 4
Barium chloride
239 7*
Barium chloride+Bridelia
52 9 10
Increase of contraction (%)
Table 5 Interaction between agonists noradraline (10−5 g/ml), adrenaline (10−5 g/ml), barium chloride (400 mg/ml) and increasing cumulative concentration of aqueous extract of Bridelia in rat aorta (in vitro)
17.0 39.0 50* 57* 76* 98*
10 9 4 7 91 6 91 4 91 3 91 0.29 1
83 918 82 9 17 81 917 79 9 18 77 916 76 9 16
139 4 129 3 1294 119 3 1294 119 4
After Bridelia injection
Before Bridelia injection
% Inhibition of maximal contraction
Before Bridelia injection
After Bridelia injection
Heart rate (beat/min)
Contraction amplitude (mm)
Values are expressed as mean 9 S.E.M. (%). * Significant with P= 0.05 (Student’s test).
2.70×10−4 5.60×10−4 1.12×10−3 2.25×10−3 4.50×10−3 9.00×10−3
Bridelia concentrations (g ml−1)
597 897 23916 7 94
MHR−IHR
Third series of neuromediators after a second injection of Bridelia
Table 4 Effect of aqueous extract of Bridelia (increasing concentration) on heart contraction amplitude and heart rate (beat/min) in vitro
5 93 493 13 9 7 20 9 6
MHR−IHR
MAP−IAP
MAP−IAP
MHR−IHR
Second series of neuromediators after a first injection of Bridelia
First series of neuromediators
Values are expressed as mean 9S.E.M. (%).
Noradrenaline (3 mg/kg) Adrenaline (3 mg/kg) Isoprenaline (3 mg/kg) Acetylcholine (1 mg/kg)
Neuromediators
Table 3 Interaction between neuromediators and aqueous extract of Bridelia (15 mg/kg; one injection) on arterial pressure and heart rate in the rat (n= 7)
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Fig. 2. Negative inotropic and chronotropic effect of aqueous extract of Bridelia (9 ×10 − 3 g/ml) in isolated rat heart. (1) Start and; (2) end of extract administration.
3. Results
3.1. Blood pressure and heart rate responses to Bridelia In anaesthetized rat the extract of Bridelia at the dose of 15 and 30 mg/kg induced a significant decrease of blood pressure and heart rate (Fig. 1) with a tachyphylaxie phenomena when the same dose were repeated several times (Table 1). Cardiovascular effects started at 3.75 mg/kg. Arterial pressure effects were observed immediately after injection and during 109 2 min but 30 s later and during 14 9 2 min for heart rate. Maximum effects were reached about 3 – 5 min. Full recovery appeared 8 – 12 min later. Hypotension obtained with aqueous extract at 15 and 30 m/kg was more important than the decrease of heart rate (33911, 49.98 and 129 9%, respectively (Table 1)).
3.2. Interactions between neuromediators and Bridelia (one injection or two injections) Effect of neuromediators (noradrenaline, adrenaline, acetylcholine, isoprenaline) on blood pressure and heart rate, used before or after Bridelia administration, (one injection after 10 min; or two injections: The first one after 30 min and the second one after 1 h, was not significant in both experiments (Tables 2 and 3).
3.3. Rat heart Extract of Bridelia produced a negative inotropic and chronotropic effect in rat heart (Table
4 and Fig. 2). The heart activity stopped at the dose of 9×10 − 3 g/ml and did not recover initial activity after a return to a saline solution. An example of this response is shown in Fig. 2. The effect of Bridelia was not inhibited by atropine sulfate (10 − 2 g/ml).
3.4. Aortic artery Bridelia had no direct action on aortic artery as well with increasing concentrations as with increasing cumulative concentrations (final concentration in the bath 1.87 × 10 − 3 g/ml) and did not increase the 20% contraction induced by agonists (noradrenaline, 10 − 5 g/ml and adrenaline, 10 − 5 g/ml), but potentiated significally 20% barium chloride contraction (Table 5 and Fig. 3). 4. Discussion and conclusions The present results indicated that aqueous extract of Bridelia induced a dose dependent decrease of blood pressure and heart rate (in vivo and in vitro). It had no direct effect on rat aorta preparations rich in alpha adrenergic receptors (Decker et al., 1984; Descombes and Stocklet, 1985; Gerald et al., 1985; Malta et al., 1986; Robert et al., 1981). It did not potentiate rat aorta contractions induced by adrenaline and noradrenaline. The extract did not interact with neuromediators in vivo and in vitro. Bradycardia was not inhibited by atropine sulfate. All these results indicated that Bridelia activity was not mediated through sympathetic nor cholinergic receptors. They strengthen and confirm those obtained with rat uterus preparations (Corallo, 1990).
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Fig. 3. Effect of aqueous extract of Bridelia in increasing cumulative concentrations in the presence of barium chloride (400 mg/ml) in isolated rat aorta. (A) Administration of barium chloride. (AB) 20% aorta contraction induced by barium chloride. (BC) Administration of extract (increasing cumulative concentrations one to six, each min final concentration, 1.87 ×10 − 3 g/ml) during constant contraction level of barium chloride. (CD) Potentiation of barium chloride contraction 1 min after the last addition of Bridelia.
However, at high dose (1.83× 10 − 3 g/ml), extract of Bridelia increased the 20% contractions induced by barium chloride in rat aorta preparations. It has been demonstrated that barium opens calcium channels and acts on contractile proteins (Hansen et al., 1984; Kreye et al., 1984, 1986; Satoh et al., 1987). Bridelia might stimulate calcium entry through potential calcium channels in rat aorta but at higher concentration (1.83 × 10 − 3 g/ml) than that used with uterine preparations. This result confirms also that observed in rat uterus (Corallo et al., 1991). Hypotension might be elicited not by a direct action on vascular smooth muscles, but by the potent direct activity of Bridelia on the heart. Cardiovascular effects (hypotension and a sharp decrease in heart rate) indicate that traditional use of Bridelia by African women is not without potential danger.
Acknowledgements We are grateful to our colleagues of Chatenay Malabry in particular: Dr. M. Davy and Prof. Y. Cohen for their help in this study.
References Colot, M., 1972. Notions Techniques de Pharmacologic Generale. Ed. Masson, Paris, pp. 30 – 50. Corallo, A., 1990. Contribution a` l’e´valuation de l’activite´ ute´rotonique, chez le rat, de Bridelia atroviridis Muell. Arg. (Euphorbiace´es) utilise´e traditionnellement en Coˆte d’Ivoire. Doctorat de l’Universite´ de Paris Sud, Faculte´ de Chatenay Malabry (Sciences Pharmaceutiques), no. 137. Corallo, A., Founge, S., Declume, C., 1985. Etude expe´rimentale de l’activite´ ute´rotonique de plantes utilise´es traditionnellement en Coˆte d’Ivoire. Revue Medicale de Coˆte d’Ivoire 73, 90. Corallo, A., et al., 1988. Etude expe´rimentale de l’activite´ uterotonique de Bridelia atro6iridis et Phyllanthus discoideus (Euphorbiace´es). Mise en e´vidence d’un effet au niveau des re´cepteurs alpha-adre´nergiques. Annales Pharmaceutiques Franc¸aises 46 (3), 171 – 177. Corallo, A. et al., 1989. Proprie´te´s pharmacologiques de deux principes ute´rotoniques extraits des feuilles de Bridelia atro6iridis Muell. Arg. (Euphorbiace´es): Effet sur le duode´num isole´ de rat. Communication 7e` Journe´es Me´dicales de Coˆte d’Ivoire 16 – 19 Janvier. Corallo, A., Savineau, J.P., Tricoche, R., Foungbe, S., 1991. The uterotonic action of the aqueous extract of Bridelia atro6iridis in the rat. Fundamental Clinical Pharmacology 5, 319 – 329. Decker, N., Ehrhardt, J.D., Leclec, G., Schwartz, J., 1984. Postjunctional alpha-adrenoreceptors alpha 1 and alpha 2 subtypes in rat vasculature in vitro and in vivo. NaunynSchmiedeberg’s Archives of Pharmacology 326, 1 – 6.
A. Corallo et al. / Journal of Ethnopharmacology 57 (1997) 189–196
196
Descombes, J.J., Stocklet, J.C.I., 1985. Characterisation of two distincts alpha-adrenoreceptor binding sites in smooth muscle cells membranes from rat and bovine aorta. Naunyn-Schmiedeberg’s Archives of Pharmacology 329, 282 – 288. Gerald, O., et al., 1985. Enhancement of alpha 1 and alpha 2 adrenergic agonist. Induced vasoconstriction by removal of endothelium in rat aorta. Journal of Pharmacology and Experimental Therapeutics 232 (3), 682–687. Hansen, T.R., Dineen, D.X., Petrak, R., 1984. Mechanism of action of barium ion on rat aortic smooth muscle. American Journal of Physiology 246, 235–241. Kreye, V.A.W., Roegg, J.C., Hofmann, F., 1984. Effect of calcium antagonist and barium ions on calmodulin-dependent contractions of chemically skinned renal arteries of the rabbit. In: Calcium Regulations in Smooth Muscles. Inserm, Paris, 124 pp. 455–462.
.
Kreye, V.A.W., et al., 1986. Banum can replace calcium in calmodulin-dependent contractions of skinned renal arteries. Pflu¨gers Archive European Journal of Physiology 406, 308 – 311. Malta, E., Schini, V., Miller, C., 1986. Role of efficiency in the assessment of the actions of alphaadrenoreceptor agonists in rat aorta with endothelium. Journal of Pharmacy and Pharmacology 38, 909 – 1213. Robert, R., et al., 1981. Postsynaptic alpha-adrenergic receptor subtypes differentiated by yohimbine in tissues from the rat. Existence of alpha 2 adrenergic receptors in rat aorta. Journal of Pharmacology and Experimental Therapeutics 217 (2), 235 – 240. Satoh, S., Kubota, Y., Itoh, T., Kuriyama, H., 1987. Mechanism of the barium induced contraction in smooth muscle cells of the rabbit mesenteric artery. Journal of General Physiology 88, 369 – 392.