Vasorelaxant action of aqueous extract of the leaves of Persea americana on isolated thoracic rat aorta Mbang A. Owolabia,*, Smith I. Jajab, Herbert A.B. Cokera aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Lagos, Lagos, Nigeria bDepartment of Physiology, College of Medicine, University of Lagos, PMB 12003, Lagos, Nigeria Received 23 June 2004; accepted in revised form 27 April 2005 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 significantvasorelaxation and that the effect is dependent on the synthesis or release of endothelium-derivedrelaxing factors (EDRFs) as well as the release of prostanoid. The extract also reducedvasoconstriction 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 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 asavocado pear. In Nigeria, the leaf is known in common names as Ewe´ pia (Yoruba), IkvDeben 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 variousailments. 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: (M.A. Owolabi).
0367-326X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2005.04.020 M.A. Owolabi et al. / Fitoterapia 76 (2005) 567–573 while in Mexico, it is used as an aphrodisiac, emmenagogue, to prevent miscarriage, tospeed up postpartum recovery, and in the treatment of haemorrhage between menstrualperiods The leaves are used in Brazil and Jamaica for the treatment of high bloodpressure In Nigeria, several ethnic groups use the leaves of P. americana in thetreatment of hypertension. Adeboye et al. have confirmed that the administration of theleaf extract of P. americana on anaesthetized normotensive male Sprague–Dawley ratsproduced 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 wereinvestigated. Finally, the effects of P. americana on aortic rings precontracted withnoradrenaline or potassium chloride were investigated.
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 thepractice of traditional medicine practitioners, were authenticated by Dr. O. Ugboaja,Forestry Research Institute of Nigeria (FRIN), Ibadan. A voucher specimen has beendeposited in the FRIN Herbarium (no. FHI 106099).
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 distilledwater 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.
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 andreceived standard animal chow (Pfizer Feeds Nigeria, PLC) and water ad libitum. Prior toexperimentation, they were fasted overnight with access to water ad libitum.
Noradrenaline, acetylcholine hydrochloride, NG-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 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 Krebssolution to free the lumen of its content, cut into 5-mm strips, and transferred into a Krebssolution [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 suspendedin a 20-ml organ bath between two stainless-steel wire hooks. One hook was fitted to the ringat the bottom of the bath, while the second hook was attached to a Grass FT.03 forcetransducer connected to a Grass polygraph (Model 7D). Each aortic ring was placed under aninitial tension of 2Âg, which was kept constant throughout the experiments. The bathcontaining the Krebs solution was kept at 37 8C and bubbled with 95% O2 and 5% CO2.
Responses to 1 Â10À 7 M noradrenaline were obtained repeatedly until contractions wereuniform during the initial stabilization period of 90 min. During this period, the bath solutionwas renewed every 30 min. Endothelium integrity was assessed by verifying that thecontracted rings relaxed by at least 50% when stimulated with 1 Â 10À 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 wasconfirmed by a relaxant response to acetylcholine (1 Â 10À 6 M) of less than 10% Theintegrity of the vascular smooth muscle function was assured by contraction response to6 Â 10À 2 M potassium chloride and 10À 7 M noradrenaline Following the equilibration period, the tissues were precontracted by addition of noradrenaline 1 Â10À 7 M. Once the tonic responses became stable, at the peak of eachcontraction, 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 denudedendothelium and in the absence or in the presence of NG-nitro-l-arginine methylester (l-NAME, 10À 4 M, a nitric oxide synthetase inhibitor) or methylene blue (10À 6 M, solubleguanylate cyclase inhibitor) or indomethacin (10À 5 M, cyclooxygenase inhibitor). Allantagonists were incubated with the tissue 30 min before extract addition. Eachpreparation was exposed to only one antagonist.
In another experiment, cumulative concentration–response curves to noradrenaline (1 Â10À 9 to 1 Â10À 5 M) and potassium chloride (1 Â10À 2 to 8 Â 10À 2 M) were obtainedin preparations of rat aortic rings with intact endothelium before or after addition of the aqueousextract at a final bath concentration of 1 mg/ml or 5 mg/ml. The extract was kept in contact withthe 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 aspercent of the maximum contraction obtained in the initial concentration–response curve.
Data are presented as mean F S.E.M. The EC50 values (i.e., the concentration of the agonist or extract that produced 50% reduction of maximal relaxant responses) were M.A. Owolabi et al. / Fitoterapia 76 (2005) 567–573 Fig. 1. Effect of leaves aqueous extract of P. americana on rat aortic rings with intact endothelium (+ E) anddenuded endothelium (À E) precontracted with 10À 7 M noradrenaline. Values are mean + S.E.M. (n = 6).
determined from the concentration–response curves by linear regression analysis. Statisticalsignificance of the data for control and treated groups was assessed by Student’s t-test forpaired and unpaired samples. Statistical significance was accepted when P b 0.001.
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 1Effect of the inhibitors of endothelium-dependent relaxing factors (EDRFs) on the vasorelaxant action of theleaves’ aqueous extract of P. americana in rat aortic rings with intact endothelium precontracted with 10À 7Mnoradrenaline 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 Table 2Effect of the inhibitors of endothelium-dependent relaxing factors (EDRFs) on the vasorelaxant action induced byacetylcholine in rat aortic rings N = 6 in each group.
Values are mean F S.E.M.
n.d. = not detectable; percentages of maximum response are presented in parentheses.
intact endothelium precontracted with noradrenaline (1 Â10À 7 M), with an EC50 of0.88 F 0.03 mg/ml. In the endothelium-denuded rings, the vasorelaxant action of theaqueous extract of P. americana was significantly attenuated (EC50 2001.14 F 252.18mg/ml; The vasorelaxant effect of the aqueous extract of P. americana wasalso significantly attenuated by l-NAME (10À 4 M), methylene blue (10À 6 M), orindomethacin (10À 5 M) (Cumulative addition of acetylcholine (1.1 Â10À 8 to1.4 Â 10À 5 M) produced relaxation of endothelium-intact rat aortic rings precontractedwith noradrenaline (1 Â10À 7 M). The vasorelaxant effect was significantly reduced by Fig. 2. Effect of leaves aqueous extract of P. americana on rat aortic rings with intact endothelium on contractioninduced by noradrenaline. Values are mean + S.E.M. (n = 6).
M.A. Owolabi et al. / Fitoterapia 76 (2005) 567–573 Fig. 3. Effect of leaves aqueous extract of P. americana on rat aortic rings with intact endothelium on contractioninduced by potassium chloride. Values are mean + S.E.M. (n = 6).
l-NAME (10À 4 M) and methylene blue (10À 6 M), but not affected by indomethacin(10À 5 M) ( 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 Â 10À 9 to 1 Â10À 5 M) andpotassium chloride (10–80 mM) ( 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 blockedby l-NAME or methylene blue, suggesting that the vasorelaxation is dependent on thesynthesis and release of endothelium-derived relaxing factors (EDRFs). The blockade byindomethacin suggests that P. americana may act also by activating PGI2 and PGE2receptors. The vasorelaxant effect may also be produced by the inhibition of Ca2+mobilization through voltage-dependent channels and, to a lesser extent, receptor-operatedchannels.
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.
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 histechnical assistance; and Mr. R. Ettarh for reading through the manuscript.
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