Fitoterapia 76 (2005) 567 – 573

www.elsevier.com/locate/fitote

Vasorelaxant action of aqueous extract of the leaves

of Persea americana on isolated thoracic rat aorta
Mbang A. Owolabi a,*, Smith I. Jajab, Herbert A.B. Coker a
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Lagos, Lagos, Nigeria
b
Department of Physiology, College of Medicine, University of Lagos, PMB 12003, Lagos, Nigeria
Received 23 June 2004; accepted in revised form 27 April 2005
Available online 28 June 2005

Abstract

The present study investigated the vasorelaxant action of the aqueous leaves extract of Persea
americana on isolated rat aorta. The results showed that the extract produced significant
vasorelaxation and that the effect is dependent on the synthesis or release of endothelium-derived
relaxing factors (EDRFs) as well as the release of prostanoid. The extract also reduced
vasoconstriction probably by inhibiting Ca2+ influx through calcium channels.
D 2005 Elsevier B.V. All rights reserved.

Keywords: Persea americana; Vascular relaxation; Rat aorta; Nitric oxide

1. Introduction

Persea americana Mill (Lauraceae) is a deciduous plant, which is widely distributed

throughout tropical and subtropical Africa. The fruit of the plant is commonly known as
avocado pear. In Nigeria, the leaf is known in common names as Ewé pia (Yoruba), IkvD
eben mbakara (Efik), Akwukwo Ube oyibo (Igbo), and Ganyen piya (Hausa). The root,
bark, fruit, and leaf are used extensively in traditional medicine for the treatment of various
ailments. In Congo Brazzaville, a decoction of the stem bark is taken to relieve cough;

* Corresponding author. Tel.: +234 1 4731816; fax: +234 1 5851432.

E-mail address: mbangsandra@yahoo.com (M.A. Owolabi).

0367-326X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2005.04.020
568 M.A. Owolabi et al. / Fitoterapia 76 (2005) 567–573

while in Mexico, it is used as an aphrodisiac, emmenagogue, to prevent miscarriage, to

speed up postpartum recovery, and in the treatment of haemorrhage between menstrual
periods [1,2]. The leaves are used in Brazil and Jamaica for the treatment of high blood
pressure [3,4]. In Nigeria, several ethnic groups use the leaves of P. americana in the
treatment of hypertension. Adeboye et al. [5] have confirmed that the administration of the
leaf extract of P. americana on anaesthetized normotensive male Sprague–Dawley rats
produced a significant reduction in blood pressure.
However, the possible mechanisms by which P. americana lowers blood pressure have
not been worked out. This study investigates the effects of the aqueous leaf extract of P.
americana on endothelium-intact or -denuded aortic rings. In addition, the effects of l-
NAME or methylene blue or indomethacin on P. americana extract activity were
investigated. Finally, the effects of P. americana on aortic rings precontracted with
noradrenaline or potassium chloride were investigated.

2. Experimental

2.1. Plant

P. americana leaves, collected in the University of Lagos Staff Quarters, Akoka, Lagos
State, Nigeria, in June 1997 in the early hours of the morning, in accordance with the
practice of traditional medicine practitioners, were authenticated by Dr. O. Ugboaja,
Forestry Research Institute of Nigeria (FRIN), Ibadan. A voucher specimen has been
deposited in the FRIN Herbarium (no. FHI 106099).

2.2. Plant extract

P. americana leaves dried at 40 8C for 5 days were ground into fine powder and stored
in an amber bottle. Fine powder material (840 g) was Soxhlet-extracted with distilled
water and filtered. The solution (pH 5.4) was lyophilized, giving 143.7 g of extract
(17.11% wt/wt). A new stock solution was prepared on each day of the experiment.

2.3. Animals

Sprague–Dawley rats of either sex weighing 250–300 g were used for the studies. The
animals were obtained from the Laboratory Animal Center of the College of Medicine,
University of Lagos, Lagos, Nigeria. They were kept in a well-ventilated animal house and
received standard animal chow (Pfizer Feeds Nigeria, PLC) and water ad libitum. Prior to
experimentation, they were fasted overnight with access to water ad libitum.

2.4. Drugs

Noradrenaline, acetylcholine hydrochloride, N G-nitro-l-arginine methylester (l-

NAME), indomethacin were from Sigma Chemical Company (St. Louis MO, USA).
Methylene blue and potassium chloride were from British Drug Houses, UK.
 M.A. Owolabi et al. / Fitoterapia 76 (2005) 567–573 569

2.5. Preparation of aortic rings

The rats were killed and the thoracic aorta were carefully excised and cleaned of
connective tissue and adherent fat. The aortic lumen was carefully flushed with Krebs
solution to free the lumen of its content, cut into 5-mm strips, and transferred into a Krebs
solution [composition (mM): NaCl, 119; KCl, 4.7; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3,
14.9; CaCl2, 1.6; and glucose, 11.5; pH 7.4]. Each segment of the aortic ring was suspended
in a 20-ml organ bath between two stainless-steel wire hooks. One hook was fitted to the ring
at the bottom of the bath, while the second hook was attached to a Grass FT.03 force
transducer connected to a Grass polygraph (Model 7D). Each aortic ring was placed under an
initial tension of 2g, which was kept constant throughout the experiments. The bath
containing the Krebs solution was kept at 37 8C and bubbled with 95% O2 and 5% CO2.
Responses to 1  10 7 M noradrenaline were obtained repeatedly until contractions were
uniform during the initial stabilization period of 90 min. During this period, the bath solution
was renewed every 30 min. Endothelium integrity was assessed by verifying that the
contracted rings relaxed by at least 50% when stimulated with 1  10 6 M acetylcholine.
In another preparation, the vascular endothelium was denuded by gently rubbing the
aortic lumen with an 18-gauge hypodermic needle. Removal of the endothelium was
confirmed by a relaxant response to acetylcholine (1  10 6 M) of less than 10% [6]. The
integrity of the vascular smooth muscle function was assured by contraction response to
6  10 2 M potassium chloride and 10 7 M noradrenaline [7].

2.6. Relaxation experiments

Following the equilibration period, the tissues were precontracted by addition of

noradrenaline 1  10 7 M. Once the tonic responses became stable, at the peak of each
contraction, the cumulative concentration–response curves to the aqueous extract of P.
americana (0.01–12.8 mg/ml) were obtained, in preparations with intact or denuded
endothelium and in the absence or in the presence of N G-nitro-l-arginine methylester (l-
NAME, 10 4 M, a nitric oxide synthetase inhibitor) or methylene blue (10 6 M, soluble
guanylate cyclase inhibitor) or indomethacin (10 5 M, cyclooxygenase inhibitor). All
antagonists were incubated with the tissue 30 min before extract addition. Each
preparation was exposed to only one antagonist.
In another experiment, cumulative concentration–response curves to noradrenaline
(1  10 9 to 1  10 5 M) and potassium chloride (1  10 2 to 8  10 2 M) were obtained
in preparations of rat aortic rings with intact endothelium before or after addition of the aqueous
extract at a final bath concentration of 1 mg/ml or 5 mg/ml. The extract was kept in contact with
the preparation for 30 min and throughout the construction of the second concentration–
response curve. In all cases, responses to each concentration of agonist was expressed as
percent of the maximum contraction obtained in the initial concentration–response curve.

2.7. Statistical analysis

Data are presented as meanF S.E.M. The EC50 values (i.e., the concentration of the
agonist or extract that produced 50% reduction of maximal relaxant responses) were
570 M.A. Owolabi et al. / Fitoterapia 76 (2005) 567–573

100
+E
-E

80

60
% Relaxation

40

20

0
-2 -1 0 1 2
Extract (Log mg/ml)

Fig. 1. Effect of leaves aqueous extract of P. americana on rat aortic rings with intact endothelium (+E) and
denuded endothelium ( E) precontracted with 10 7 M noradrenaline. Values are mean + S.E.M. (n = 6).

determined from the concentration–response curves by linear regression analysis. Statistical

significance of the data for control and treated groups was assessed by Student’s t-test for
paired and unpaired samples. Statistical significance was accepted when P b 0.001.

3. Results and discussion

The cumulative addition of the aqueous extract of P. americana (0.01–12.8 mg/ml)

produced a concentration-related vasorelaxation response in rings of rat aorta with

Table 1
Effect of the inhibitors of endothelium-dependent relaxing factors (EDRFs) on the vasorelaxant action of the
leaves’ aqueous extract of P. americana in rat aortic rings with intact endothelium precontracted with 10 7M
noradrenaline
EDRF inhibitors Concentration (M) EC50 (mg/ml)
Control – 0.88 F 0.03
5
Indomethacin 10 15.9 F 1.2
6
Methylene blue 10 89.6 F 10.9
4
l-NAME 10 127.8 F 6.3
Endothelium-denuded – 2001.1 F 252.2
n = 6 in each group.
Values are mean F S.E.M.
P b 0.001 compared to control.
 M.A. Owolabi et al. / Fitoterapia 76 (2005) 567–573 571

Table 2
Effect of the inhibitors of endothelium-dependent relaxing factors (EDRFs) on the vasorelaxant action induced by
acetylcholine in rat aortic rings
EDRF inhibitors Concentration (M) EC50 (M) E max (%)
Control – 82.7 F 0.11 94.6 F 0.15
5
Indomethacin 10 71.3 F 1.12 87.1 F1.02 (NS)
6
Methylene blue 10 n.d. (18.8%) 18.8 F 0.07***
4
l-NAME 10 n.d. (7.2%) 7.2 F 0.02***
Denuded endothelium – n.d. (9.8%) 9.8 F 0.11***
N = 6 in each group.
Values are mean F S.E.M.
n.d. = not detectable; percentages of maximum response are presented in parentheses.
***
P b 0.001 compared to control.

intact endothelium precontracted with noradrenaline (1  10 7 M), with an EC50 of

0.88 F 0.03 mg/ml. In the endothelium-denuded rings, the vasorelaxant action of the
aqueous extract of P. americana was significantly attenuated (EC50 2001.14 F 252.18
mg/ml; Fig. 1). The vasorelaxant effect of the aqueous extract of P. americana was
also significantly attenuated by l-NAME (10 4 M), methylene blue (10 6 M), or
indomethacin (10 5 M) (Table 1). Cumulative addition of acetylcholine (1.1  10 8 to
1.4  10 5 M) produced relaxation of endothelium-intact rat aortic rings precontracted
with noradrenaline (1  10 7 M). The vasorelaxant effect was significantly reduced by

100 Control
Extract 1 mg/ml
Extract 5 mg/ml

80
% Contraction

60

40

20

0
-10 -9 -8 -7 -6 -5
Log M (Noradrenaline)

Fig. 2. Effect of leaves aqueous extract of P. americana on rat aortic rings with intact endothelium on contraction
induced by noradrenaline. Values are mean + S.E.M. (n = 6).
572 M.A. Owolabi et al. / Fitoterapia 76 (2005) 567–573

120 Control
Extract 1 mg/ml
Extract 5 mg/ml
100

% Contraction 80

60

40

20

0
0 20 40 60 80 100
Potassium chloride (mM)

Fig. 3. Effect of leaves aqueous extract of P. americana on rat aortic rings with intact endothelium on contraction
induced by potassium chloride. Values are mean + S.E.M. (n = 6).

l-NAME (10 4 M) and methylene blue (10 6 M), but not affected by indomethacin
(10 5 M) (Table 2).
The aqueous extract of P. americana (1 mg/ml or 5 mg/ml) produced a rightward shift
of the concentration–response curves to noradrenaline (1  10 9 to 1  10 5 M) and
potassium chloride (10–80 mM) (Figs. 2 and 3).
Taken together, the above results showed that the vasorelaxant effect of the aqueous
leaves extract of P. americana is endothelium-dependent. In fact, this activity was blocked
by l-NAME or methylene blue, suggesting that the vasorelaxation is dependent on the
synthesis and release of endothelium-derived relaxing factors (EDRFs). The blockade by
indomethacin suggests that P. americana may act also by activating PGI2 and PGE2
receptors. The vasorelaxant effect may also be produced by the inhibition of Ca2+
mobilization through voltage-dependent channels and, to a lesser extent, receptor-operated
channels.
These vascular effects provide an explanation of its hypotensive action and a basis for
the use of the extract in the management of high blood pressure in folkloric medicine.

Acknowledgements

The authors are thankful to Prof. O.A. Sofola of the Department of Physiology,
University of Lagos, for allowing the use of his laboratory; Dr. F. Mojuminiyi for his
technical assistance; and Mr. R. Ettarh for reading through the manuscript.
 M.A. Owolabi et al. / Fitoterapia 76 (2005) 567–573 573

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