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methylene chloride stem bark extract of Mammea africana in l -NAME-induced hypertensive rats
Journal of Ethnopharmacology 117 (2008) 446–450
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Anti-hypertensive effects of the methanol/methylene chloride stem bark extract of Mammea africana in l-NAME-induced hypertensive rats P.E. Nguelefack-Mbuyo a , T.B. Nguelefack b,∗ , A.B. Dongmo c , S. Afkir d , A.G.B. Azebaze e , T. Dimo a , A. Legssyer d , A. Kamanyi c , A. Ziyyat d a
Department of Animal Biology and Physiology, Laboratory of Animal Physiology, University of Yaounde I, BP 812 Yaounde, Cameroon Department of Animal Biology, Laboratory of Animal Physiology and Phytopharmacology, University of Dschang, BP 67 Dschang, Cameroon Department of Animal Biology and Physiology, University of Douala, BP 24157 Douala, Cameroon d Department of Biology, Laboratory of Physiology and Ethnopharmacology, University Mohamed 1st, BP 717 Oujda, Morocco e Department of Chemistry, University of Douala, BP 24157 Douala, Cameroon b c
a r t i c l e
i n f o
Article history: Received 2 October 2007 Received in revised form 13 February 2008 Accepted 19 February 2008 Available online 4 March 2008 Keywords: Mammea africana Antihypertensive l-NAME
a b s t r a c t Aim of the study: The methanol/methylene chloride (CH3 OH/CH2 Cl2 ) extract from the stem bark of Mammea africana was showed to possess vasodilating effect in the presence and the absence of N -nitro-l-arginine methyl ester (l-NAME). The present study was designed to evaluate the effects of the methanol/methylene chloride from the stem bark of Mammea africana. Materials and methods: The extract (200 mg/(kg day)) was administered orally in rats treated concurrently with l-NAME (40 mg/(kg day)). l-Arginine (100 mg/(kg day)) and captopril (20 mg/(kg day))were used as positive controls. Bodyweight, systolic arterial blood pressure and heart rate were measured weekly throughout the experiment period (28 days). At the end of treatment, animals were killed and the cardiac mass index evaluated. The aorta was used to evaluate the endothelium-dependant relaxation to carbachol. The aorta contraction induced by noradrenalin was also examined and expressed as a percentage of that induced by KCl. Results: The extract neither affected the body weight nor the heart rate. The extract as captopril completely prevented the development of arterial hypertension. Both the substances failed to restore the endothelium-dependent vascular relaxation and increased the vascular contraction to norepinephrine in relation to KCl contraction. They also significantly reduced the left ventricular hypertrophy induced by l-NAME. Conclusion: These findings are in agreement with the traditional use of Mammea africana in the treatment of arterial hypertension and indicate that it may have a beneficial effect in patients with NO deficiency but will be unable to improve their endothelium-dependent vasorelaxation. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Mammea africana is a plant tree which belongs to Guttiferae family. It is found in the tropical rain forest of Africa, including Cameroon. Leaves and stem bark extracts of the plant are used in traditional medicine for the management of many diseases including stomach pains, rheumatism pains, scarbies, cough and hypertension (Raponda-Walker and Sillans, 1961; Adjanohoun et al., 1996). Phytochemical studies revealed the presence of various constituents, mainly coumarins and flavonoids (Carpenter et al., 1970, 1971; Games, 1972; Crichton and Waterman, 1978; Ouahouo et al.,
∗ Corresponding author. Tel.: +237 77564362/33040218. E-mail address: [email protected] (T.B. Nguelefack). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.02.028
2004). A part from these coumarin derivatives, Mammea africana also contains a range of xanthones typical of the Guttiferae family (Poobrasert et al., 1998; Ouahouo et al., 2004). Earlier pharmacological investigations showed that 4-npropylcoumarins and 4-phenylcoumarins from Mammea africana possess in vitro cancer protective effect, HIV-1-specific reversetranscriptase inhibition, cytotoxic and anti-microbial activities (Patil et al., 1993; Itoigawa et al., 2001; Ouahouo et al., 2004). These two compounds, the CH2 Cl2 fraction as well as the crude methanol/methylene chloride (CH3 OH/CH2 Cl2 ) stem bark extract from where they were isolated possess vasodilating effects. These activities were found to be partially endothelium-dependent and may also results from a calcium antagonist property (Dongmo et al., 2007). In accordance to these findings Lee et al. (2002) demonstrated that a pyracoumarin from Peucedanum japonicum induced vasorelaxation by releasing NO and not prostaglandins
P.E. Nguelefack-Mbuyo et al. / Journal of Ethnopharmacology 117 (2008) 446–450
from endothelium. Thus, it can be thought that Mammea africana extract can ameliorate arterial hypertension in patients with endothelium dysfunction. The present work was therefore, undertaken to evaluate the anti-hypertensive effect of the CH3 OH/CH2 Cl2 stem bark extract of Mammea africana in (N -nitro-l-arginine methyl ester) l-NAME-induced hypertensive rats. The use of the total extract was motivated by the fact that none of the pure compounds obtained exerted a vasorelaxant effect equivalent to that of the crude extract.
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2.4. Vascular reactivity tests
Mammea africana materials were collected on the bank of the river Nyong in Mbalmayo, Cameroon, and identified at the National Herbarium, Cameroon, where a voucher’s specimen was deposited under number 4221/SRF/CAM for future reference. The dried ground stem bark powder (4 kg) was extracted by maceration at room temperature in 10 l of CH2 Cl2 /CH3 OH (1:1) for 24 h. The mixture was filtrated and the filtrate was concentrated to dry to give 900 g of extract. The extract was dissolved in 4% DMSO for the chronic cardiovascular studies.
Two stainless steel wires hooks were inserted into the lumen of aortic ring. The rings were suspended under 2 g tension in individual 11 ml organ bath containing Kreb’s bicarbonate physiological solution of the following composition (mM): NaCl 119, KCl 4.7, CaCl2 1.6, MgSO4 1.2, KH2 PO4 1.2, NaHCO3 25 and glucose 11.1. The physiological solution was continuously aerated with carbogene (95% O2 and 5% CO2 ) and its pH was adjusted to 7.4. The mechanical activity of the tissue recorded isometrically using a force displacement transducer (3302 EMKA Technologies, France) was amplified (Amplifier AC 264 EMKA Technologies, France) and printed on a PL3 JJ Instrument recorder. After 30–45 min equilibration period, the organ was contracted with noradrenalin (10−6 M) and the cumulative concentration–response curve to carbachol (10−9 to 10−5 M) was constructed. The organ was washout several times in intervals of 10 min until it return to its initial tension. The cumulative concentration–response curve to noradrenalin (10−9 to 10−5 M) was then constructed. After further washout, the organ was contracted with KCl (60 mM). The experiment was done in duplicate. The contractile activity of each concentration of norepinephrine was expressed as a percentage of the maximal contraction induced by KCl (60 mM).
2.2. Animals
2.5. Statistical analysis
Male Wistar rats of 12–16 weeks old, weighing 250–300 g were used in these studies. Animals were housed in colony cages, under standard laboratory conditions (12:12 h light/dark cycle) and have free access to standard commercial diet and water.
Values are expressed as means ± S.E.M. Data were analysed using ANOVA repeated measures, with Tukey test as post-hoc test using Graph Pad Bioinstat 3.0. p < 0.05 was considered as statistically significant.
2.3. Blood pressure and heart rate measurements
3. Results
Rats were randomly divided into five groups of six animals each. The first group served as control and received distilled water, the second group received l-NAME at the dose of 40 mg/(kg day), the third group was treated with l-NAME (40 mg/(kg day)) + plant extract at the dose of 200 mg/(kg day), the fourth group was treated with l-NAME (40 mg/(kg day)) + l-arginine (100 mg/(kg day)) and the last one with l-NAME (40 mg/(kg day)) + captopril at the dose of 20 mg/(kg day). All the treatments were given orally at the corresponding volume of 1 ml/100 g body weight. The dose of the extract was obtained from the concentration of pure compounds that exerted the maximal vasorelaxant effect, taking in to consideration the yields of extraction and the fact that about 1–5% of natural molecules such as coumarins are absorbed in the digestive tract (Duthie et al., 1998). Blood pressure and heart rate of rats were recorded indirectly by a sphygmomanometer (Mode 59 Pulse Amplifier, IITC INC Innovators Instrumentation) coupled to a recorder (575 71 TY-Recorder, LH Leybold-heraeus). The sphygmomanometer was equipped with an infrared detector incorporated in a pressure cuff to measure pulse. The lateral counter pressure value corresponding to disappearance of the pressure signal indicates the systolic blood pressure. Pulse rate was expressed as beats per minute. The animals were accustomed for 2 days for blood pressure and heart rate measurement before experiment started. After baseline recordings, the rats were treated as indicated above and systolic blood pressure (SBP), heart rate and body weight were monitored weekly for 4 consecutive weeks. At the end of the treatment, animals were anaesthetized with thiopental (50 mg/kg, i.p.) and exsanguinated. The heart was removed free of fats and connective tissues and weighed. The left and the right ventricles were also dissected and weighed. The thoracic aorta was isolated, free of fats and connective tissues, cut in rings of about 3 mm length and used for vascular reactivity.
3.1. Body weight
2. Materials and methods 2.1. Preparation of plant extract
The body weight increase in all treated animals was lower than in control group meanwhile, only the variation in captopril-treated group was significantly lower compared to that of both control and l-NAME-treated groups all along the treatment period (Fig. 1). 3.2. Blood pressure and heart rate measurements At the beginning of the experiment, the animals have a systolic blood pressure of about 125 mm Hg. The oral administration of l-NAME induced a rapid onset of arterial hypertension which increased progressively from 129.00 ± 1.87 to 190.00 ± 4.18 mm Hg at the end of experimentation. The concomitant administration of l-arginine induced a significant reduction in blood pressure but the value (168.00 ± 2.00) was still significantly higher than
Fig. 1. Effects of chronic treatments on the rat body weight. N = 6, b p < 0.01, c p < 0.001 significantly different compared to control; ˛ p < 0.05, p < 0.001 significantly different compared to l-NAME.
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P.E. Nguelefack-Mbuyo et al. / Journal of Ethnopharmacology 117 (2008) 446–450
Fig. 2. Effects of chronic treatments on the rat systolic blood pressure (SBP). N = 6, a p < 0.05, b p < 0.01, c p < 0.01 significantly different compared to control; ˛ p < 0.05, ˇ p < 0.01, p < 0.001 significantly different compared to l-NAME.
Fig. 4. Vascular reactivity to carbachol of aortas from rats submitted chronically (4 weeks) to different treatments. N = 6, a p < 0.05, c p < 0.01 significantly different compared to control.
Fig. 3. Effects of chronic treatments on the heart, left ventricle + the septum (L. ventricle) and the right ventricle (R. ventricle) weights. N = 6; c p < 0.001 significantly different compared to control; ˇ p < 0.01, p < 0.001 significantly different compared to l-NAME.
Fig. 5. Vascular reactivity to norepinephrine of aortas from rats submitted chronically (4 weeks) to different treatments. Results are expressed as a percentage of maximal contraction induced by KCl (60 mM). N = 6, b p < 0.01 significantly different compared to control.
in control untreated group (130.00 ± 3.54 mm Hg). Excepted the second week in l-NAME + Mammea africana-treated rats, the SBP in animals treated with l-NAME + Mammea africana or with lNAME + captopril was significantly lower than in those receiving only l-NAME and was significantly unchanged compared to control during the all period of treatment (Fig. 2). None of the treatment significantly affected the heart rate during the experiment (Table 1).
of the total heart weight while the right ventricular mass did not significantly change between groups.
3.3. Cardiac mass indexes The total heart, the left ventricle plus ventricular septum and the right ventricle were weighed and expressed in mg/100 g body weight. The total heart weight was found increased in l-NAME and l-NAME + l-arginine-treated groups but this mass was significantly higher only in l-NAME + l-arginine-treated group (373.53 ± 21.80) compared to the control group (290.76 ± 6.75). In animals treated with l-NAME + captopril or l-NAME + Mammea africana, the respective heart mass of 263.16 ± 8.66 and 267.11 ± 0.98 were not significantly different to that of control group but were significantly lower than in l-NAME-treated group (328.86 ± 6.99). As shown in Fig. 3, the changes in the left ventricular mass were similar to that
3.4. Vascular reactivity tests On aortic rings from control animal, precontracted with 1 M of noradrenaline (NA), carbachol induced a concentration dependent relaxant effect with an Emax of 80.54 ± 4.17% (% NA contraction). This carbachol relaxant effect was significantly attenuated in l-NAME-treated animals (Emax = 50.35 ± 4.28%). The addition of larginine on l-NAME treatment slightly ameliorated the relaxation (Emax = 55.99 ± 5.74%) while Mammea africana and captopril instead tend to aggravate the effect of l-NAME Emax to 39.20 ± 3.94% and 35.13 ± 8.84%, respectively (Fig. 4). The contraction response to cumulative concentrations of NA (relative to KCl; 60 mM) was not significantly different between rings from l-NAME-treated rats (Emax = 72.74 ± 20.91%), l-NAME + l-arginine (Emax = 71.96 ± 6.51%) and rings from control (Emax = 58.90 ± 4.63). This contractile effect was significantly greater in rings from l-NAME + captopril (Emax = 101.21 ± 5.97%) or l-NAME + Mammea africana (Emax = 111.16 ± 2.35%)-treated groups compared with rings from the control group (Fig. 5).
Table 1 Effects of chronic treatments on the rat heart rate Treatment
Heart rate (beat/min) W0
Control l-NAME l-NAME + l-arginine l-NAME + captopril l-NAME + Mammea africana W, week.
386 394 360 336 332
W1 ± ± ± ± ±
8.28 12.08 4.47 6.78 10.20
404 378 344 308 290
W2 ± ± ± ± ±
6.00 6.63 9.27 4.90 8.37
396 382 340 314 314
W3 ± ± ± ± ±
10.29 5.83 7.75 8.72 9.80
392 354 350 316 314
W4 ± ± ± ± ±
13.93 11.22 10.49 11.66 6.78
368 382 364 312 312
± ± ± ± ±
8.60 14.28 14.70 7.35 12.00
P.E. Nguelefack-Mbuyo et al. / Journal of Ethnopharmacology 117 (2008) 446–450
4. Discussion It has been obviously demonstrated that chronic administration of l-NAME induced arterial hypertension associated with the deficiency of nitric oxide (NO). The present study analysed the effects of the CH3 OH/CH2 Cl2 stem bark extract of Mammea africana on the arterial hypertension associated with the chronic deficiency of nitric oxide. The results indicate that Mammea africana extract is able to inhibit the arterial hypertension and the cardiac hypertrophy that follow the chronic inhibition of NO production, but could not restore the endothelium vascular relaxation and instead increase the aorta sensitivity to noradrenaline. It was showed that none of the treatment affects the heart rate. This result is in accordance with many previous studies that have shown that, the chronic administration of l-NAME did not alter the heart rate (Fortepiani et al., 2002; Gerova and Kristek, 2001). Since no changes in the heart rate were also found in rats treated concomitantly with l-NAME and Mammea africana extract, it seems that the lowering of blood pressure may be predominantly due to the reduction in peripheral resistance even though Dongmo et al. (2007) showed that this extract possess negative inotropic effects on electrical induced papillary muscle contraction. The results on the isolated aorta rings showed that the carbachol relaxant effect was lower in l-NAME + Mammea africana extract as in l-NAME + carptopril-treated animals compared to rats receiving only l-NAME. This suggests that both the extract and captopril were not able to overcome the NO synthase inhibition induced by l-NAME. Dongmo et al. (2007) have shown that the CH3 OH/CH2 Cl2 extract of the stem bark of Mammea africana possesses two coumarins namely 4-n-propylcoumarin and 4-phenylcoumarin with vasorelaxant effects. They were found to act at least partially, directly on the vascular smooth muscle. The dose of extract used in this study was chosen such that the concentration of the extract inducing the in vitro maximal vasorelaxant effect may be reach in the plasma. If the direct in vitro vasorelaxant effects of these compounds could be conserved in vivo, they may explain the antihypertensive activities of Mammea africana extract. Aortic rings from animals treated with l-NAME + Mammea africana exhibited a significant increase in contraction induced by noradrenaline in relation to KCl contraction. This result suggests an increase in the sensitivity of vascular smooth muscle to receptor-induced contraction in comparison to voltage-induced contraction. The increased sensitivity may be caused by the decreased of the NO availability. l-Arginine which is competitive to l-NAME as substrate of NOS, significantly reduced the SBP at the fourth week of treatment but the SBP values were still significantly higher compared to control. Moreover, the l-arginine supplementation could not significantly ameliorate the relaxation to carbachol. These findings are in accordance with previous works which demonstrated that supply of low-endogenous NO donors, nitrites, nitrates and others do not restore completely the blood pressure to the control values (Gerova and Kristek, 2001; Torok et al., 2002). The elevation of blood pressure in l-NAME model of experimental hypertension is associated with an increase in the rennin-angiotensin system activity (Zanchi et al., 1995; Ortiz et al., 1998; Fortepiani et al., 2002). Indeed, the use of angiotensin-converting enzyme inhibitors and angiotensin-receptor antagonists have been effective in declining high-blood pressure in NO-deficient hypertensive rats (Linz et al., 1995; Soares de Moura and Cardoso, 1997). The present results agree with these data since the administration of captopril, an angiotensin-converting enzyme inhibitor, completely prevent the rise in blood pressure. The Mammea africana extract activity was a likeness to that of captopril: both the drugs reduced blood pressure, did not affect the relaxation to carbachol, increased the contraction
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to NA and reduced the cardiac hypertrophy. These coincidences suggest that Mammea africana extract may possess angiotensinconverting enzyme inhibition activity or may interfere with the rennin-angiotensin system. Indeed, previous work have shown that chronic angiotensin II blockade or converting enzyme inhibition prevents the development of l-NAME-induced hypertension but do not stimulate the reduced synthesis of NO (Takase et al., 1996; Tojo et al., 2001). In the present study, a substantial increase in cardiac mass was observed even if the effect was not statistically significant. The cardiac hypertrophy in l-NAME-treated animals is thought to be compensatory to chronic increase in blood pressure (Devlin et al., 1998). It has been showed that cardiac hypertrophy is always associated with a significant increase in plasma rennin activity (Arnal et al., 1993; Morton et al., 1993; Xu et al., 1995; Devlin et al., 1998). Thus, as suggested by Devlin et al. (1998), activation of the renin-angiotensin system may have a permissive effect for cardiac hypertrophy in l-NAME-induced hypertension. Data from the present work showed that the cardiac hypertrophy was more important in l-NAME + l-arginine-treated group. l-Arginine can act competitively with l-NAME on NO synthase, then reducing the inhibitory effect of l-NAME. It is reported that the plasma rennin activity is higher in animals treated with lower dose of lNAME and that the activity reduced with increasing dose of l-NAME (Fortepiani et al., 2002). This fact can explain the predominant cardiac hypertrophy in l-NAME + l-arginine group if the renninangiotensin system is the principal factor of cardiac hypertrophy. These findings are in agreement with the traditional use of Mammea africana in the treatment of arterial hypertension and indicate that it may have a beneficial effect in patients with NO deficiency but will be unable to improve their endothelium-dependent vasorelaxation. Acknowledgements The authors are very grateful to A.U.F. (Agence Universitaire de la Francophonie) for the fellowship awarded and to the Faculty of Sciences, University Mohamed 1st, Oujda-Morocco for having allowed this work to be taken in his place. References Adjanohoun, J.E., Aboubakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., Enoworock, E.G., Foncho, D., Gbile, Z.O., Kamanyi, A., Kamoukom Jr., Keeta, A., Mbenkum, T., Mbi, C.M., Mbielle, A.L., Mbome, I.L., Mubiru, N.K., Naney, W.L., Nkongmeneck, B., Satabie, B., Sofowa, A., Tanze, V., Wirmum, C.K., 1996. Traditional Medicine and Pharmacopeia-Contribution to Ethnobotanical and Floristic Studies in Cameroon. CNPMS, Porto-Novo, Benin, p. 15. Arnal, J.F., El Amrani, A.I., Chatellier, G., Menard, J., Michel, J.B., 1993. Cardiac weight in hypertension induced by nitric oxide synthase blockade. Hypertension 22, 380–387. Carpenter, L., McGarry, E.J., Scheimann, F., 1970. The neoflavonoids and 4alkylcoumarins from Mammea africana G. Don. Tetrahedron Letters 11, 3936–3986. Carpenter, L., McGarry, E.J., Scheimann, F., 1971. Extractives from Guttiferae. Part XXI. The isolation and structural of nine coumarins from the bark of Mammea africana G. Don. Journal of the Chemical Society, 3783–3790. Crichton, E.G., Waterman, P.G., 1978. Dihydromammea C-OB-new coumarin from the seed of Mammea africana. Phytochemistry 17, 1783–1786. Devlin, A.M., Brosnan, J.M., Graham, D., Morton, J.J., McPhaden, A.R., McIntyre, M., Hamilton, A.C., Reid, J.L., Dominiczak, A.F., 1998. Vascular smooth muscle cell polyploidy and cardiomyocyte hypertrophy due to chronic NOS inhibition in vivo. American Journal of Heart and Circulatory Physiology 247, H52–H59. Dongmo, A.B., Azebaze, A.G.B., Nguelefack, T.B., Ouahou, B.M., Sontia, B., Meyer, M., Nkengfack, A.E., Kamanyi, A., Vierling, W., 2007. Vodilator effect of the extracts and some coumarins from the stem bark of Mammea africana (Guttiferea). Journal of Ethnopharmacology 111, 329–334. Duthie, G.G., Pedersen, M.W., Gardner, P.T., Morrice, P.C., Jenkinson, A.M., McPhail, D.B., Steel, G.M., 1998. The effect of whisky and wine consumption on total phenol content and antioxidant capacity of plasma from healthy volunteers. European Journal of Clinical Nutrition 52, 733–736.
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Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Anti-hypertensive effects of the methanol/methylene chloride stem bark extract of Mammea africana in l-NAME-induced hypertensive rats P.E. Nguelefack-Mbuyo a , T.B. Nguelefack b,∗ , A.B. Dongmo c , S. Afkir d , A.G.B. Azebaze e , T. Dimo a , A. Legssyer d , A. Kamanyi c , A. Ziyyat d a
Department of Animal Biology and Physiology, Laboratory of Animal Physiology, University of Yaounde I, BP 812 Yaounde, Cameroon Department of Animal Biology, Laboratory of Animal Physiology and Phytopharmacology, University of Dschang, BP 67 Dschang, Cameroon Department of Animal Biology and Physiology, University of Douala, BP 24157 Douala, Cameroon d Department of Biology, Laboratory of Physiology and Ethnopharmacology, University Mohamed 1st, BP 717 Oujda, Morocco e Department of Chemistry, University of Douala, BP 24157 Douala, Cameroon b c
a r t i c l e
i n f o
Article history: Received 2 October 2007 Received in revised form 13 February 2008 Accepted 19 February 2008 Available online 4 March 2008 Keywords: Mammea africana Antihypertensive l-NAME
a b s t r a c t Aim of the study: The methanol/methylene chloride (CH3 OH/CH2 Cl2 ) extract from the stem bark of Mammea africana was showed to possess vasodilating effect in the presence and the absence of N -nitro-l-arginine methyl ester (l-NAME). The present study was designed to evaluate the effects of the methanol/methylene chloride from the stem bark of Mammea africana. Materials and methods: The extract (200 mg/(kg day)) was administered orally in rats treated concurrently with l-NAME (40 mg/(kg day)). l-Arginine (100 mg/(kg day)) and captopril (20 mg/(kg day))were used as positive controls. Bodyweight, systolic arterial blood pressure and heart rate were measured weekly throughout the experiment period (28 days). At the end of treatment, animals were killed and the cardiac mass index evaluated. The aorta was used to evaluate the endothelium-dependant relaxation to carbachol. The aorta contraction induced by noradrenalin was also examined and expressed as a percentage of that induced by KCl. Results: The extract neither affected the body weight nor the heart rate. The extract as captopril completely prevented the development of arterial hypertension. Both the substances failed to restore the endothelium-dependent vascular relaxation and increased the vascular contraction to norepinephrine in relation to KCl contraction. They also significantly reduced the left ventricular hypertrophy induced by l-NAME. Conclusion: These findings are in agreement with the traditional use of Mammea africana in the treatment of arterial hypertension and indicate that it may have a beneficial effect in patients with NO deficiency but will be unable to improve their endothelium-dependent vasorelaxation. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Mammea africana is a plant tree which belongs to Guttiferae family. It is found in the tropical rain forest of Africa, including Cameroon. Leaves and stem bark extracts of the plant are used in traditional medicine for the management of many diseases including stomach pains, rheumatism pains, scarbies, cough and hypertension (Raponda-Walker and Sillans, 1961; Adjanohoun et al., 1996). Phytochemical studies revealed the presence of various constituents, mainly coumarins and flavonoids (Carpenter et al., 1970, 1971; Games, 1972; Crichton and Waterman, 1978; Ouahouo et al.,
∗ Corresponding author. Tel.: +237 77564362/33040218. E-mail address: [email protected] (T.B. Nguelefack). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.02.028
2004). A part from these coumarin derivatives, Mammea africana also contains a range of xanthones typical of the Guttiferae family (Poobrasert et al., 1998; Ouahouo et al., 2004). Earlier pharmacological investigations showed that 4-npropylcoumarins and 4-phenylcoumarins from Mammea africana possess in vitro cancer protective effect, HIV-1-specific reversetranscriptase inhibition, cytotoxic and anti-microbial activities (Patil et al., 1993; Itoigawa et al., 2001; Ouahouo et al., 2004). These two compounds, the CH2 Cl2 fraction as well as the crude methanol/methylene chloride (CH3 OH/CH2 Cl2 ) stem bark extract from where they were isolated possess vasodilating effects. These activities were found to be partially endothelium-dependent and may also results from a calcium antagonist property (Dongmo et al., 2007). In accordance to these findings Lee et al. (2002) demonstrated that a pyracoumarin from Peucedanum japonicum induced vasorelaxation by releasing NO and not prostaglandins
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from endothelium. Thus, it can be thought that Mammea africana extract can ameliorate arterial hypertension in patients with endothelium dysfunction. The present work was therefore, undertaken to evaluate the anti-hypertensive effect of the CH3 OH/CH2 Cl2 stem bark extract of Mammea africana in (N -nitro-l-arginine methyl ester) l-NAME-induced hypertensive rats. The use of the total extract was motivated by the fact that none of the pure compounds obtained exerted a vasorelaxant effect equivalent to that of the crude extract.
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2.4. Vascular reactivity tests
Mammea africana materials were collected on the bank of the river Nyong in Mbalmayo, Cameroon, and identified at the National Herbarium, Cameroon, where a voucher’s specimen was deposited under number 4221/SRF/CAM for future reference. The dried ground stem bark powder (4 kg) was extracted by maceration at room temperature in 10 l of CH2 Cl2 /CH3 OH (1:1) for 24 h. The mixture was filtrated and the filtrate was concentrated to dry to give 900 g of extract. The extract was dissolved in 4% DMSO for the chronic cardiovascular studies.
Two stainless steel wires hooks were inserted into the lumen of aortic ring. The rings were suspended under 2 g tension in individual 11 ml organ bath containing Kreb’s bicarbonate physiological solution of the following composition (mM): NaCl 119, KCl 4.7, CaCl2 1.6, MgSO4 1.2, KH2 PO4 1.2, NaHCO3 25 and glucose 11.1. The physiological solution was continuously aerated with carbogene (95% O2 and 5% CO2 ) and its pH was adjusted to 7.4. The mechanical activity of the tissue recorded isometrically using a force displacement transducer (3302 EMKA Technologies, France) was amplified (Amplifier AC 264 EMKA Technologies, France) and printed on a PL3 JJ Instrument recorder. After 30–45 min equilibration period, the organ was contracted with noradrenalin (10−6 M) and the cumulative concentration–response curve to carbachol (10−9 to 10−5 M) was constructed. The organ was washout several times in intervals of 10 min until it return to its initial tension. The cumulative concentration–response curve to noradrenalin (10−9 to 10−5 M) was then constructed. After further washout, the organ was contracted with KCl (60 mM). The experiment was done in duplicate. The contractile activity of each concentration of norepinephrine was expressed as a percentage of the maximal contraction induced by KCl (60 mM).
2.2. Animals
2.5. Statistical analysis
Male Wistar rats of 12–16 weeks old, weighing 250–300 g were used in these studies. Animals were housed in colony cages, under standard laboratory conditions (12:12 h light/dark cycle) and have free access to standard commercial diet and water.
Values are expressed as means ± S.E.M. Data were analysed using ANOVA repeated measures, with Tukey test as post-hoc test using Graph Pad Bioinstat 3.0. p < 0.05 was considered as statistically significant.
2.3. Blood pressure and heart rate measurements
3. Results
Rats were randomly divided into five groups of six animals each. The first group served as control and received distilled water, the second group received l-NAME at the dose of 40 mg/(kg day), the third group was treated with l-NAME (40 mg/(kg day)) + plant extract at the dose of 200 mg/(kg day), the fourth group was treated with l-NAME (40 mg/(kg day)) + l-arginine (100 mg/(kg day)) and the last one with l-NAME (40 mg/(kg day)) + captopril at the dose of 20 mg/(kg day). All the treatments were given orally at the corresponding volume of 1 ml/100 g body weight. The dose of the extract was obtained from the concentration of pure compounds that exerted the maximal vasorelaxant effect, taking in to consideration the yields of extraction and the fact that about 1–5% of natural molecules such as coumarins are absorbed in the digestive tract (Duthie et al., 1998). Blood pressure and heart rate of rats were recorded indirectly by a sphygmomanometer (Mode 59 Pulse Amplifier, IITC INC Innovators Instrumentation) coupled to a recorder (575 71 TY-Recorder, LH Leybold-heraeus). The sphygmomanometer was equipped with an infrared detector incorporated in a pressure cuff to measure pulse. The lateral counter pressure value corresponding to disappearance of the pressure signal indicates the systolic blood pressure. Pulse rate was expressed as beats per minute. The animals were accustomed for 2 days for blood pressure and heart rate measurement before experiment started. After baseline recordings, the rats were treated as indicated above and systolic blood pressure (SBP), heart rate and body weight were monitored weekly for 4 consecutive weeks. At the end of the treatment, animals were anaesthetized with thiopental (50 mg/kg, i.p.) and exsanguinated. The heart was removed free of fats and connective tissues and weighed. The left and the right ventricles were also dissected and weighed. The thoracic aorta was isolated, free of fats and connective tissues, cut in rings of about 3 mm length and used for vascular reactivity.
3.1. Body weight
2. Materials and methods 2.1. Preparation of plant extract
The body weight increase in all treated animals was lower than in control group meanwhile, only the variation in captopril-treated group was significantly lower compared to that of both control and l-NAME-treated groups all along the treatment period (Fig. 1). 3.2. Blood pressure and heart rate measurements At the beginning of the experiment, the animals have a systolic blood pressure of about 125 mm Hg. The oral administration of l-NAME induced a rapid onset of arterial hypertension which increased progressively from 129.00 ± 1.87 to 190.00 ± 4.18 mm Hg at the end of experimentation. The concomitant administration of l-arginine induced a significant reduction in blood pressure but the value (168.00 ± 2.00) was still significantly higher than
Fig. 1. Effects of chronic treatments on the rat body weight. N = 6, b p < 0.01, c p < 0.001 significantly different compared to control; ˛ p < 0.05, p < 0.001 significantly different compared to l-NAME.
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Fig. 2. Effects of chronic treatments on the rat systolic blood pressure (SBP). N = 6, a p < 0.05, b p < 0.01, c p < 0.01 significantly different compared to control; ˛ p < 0.05, ˇ p < 0.01, p < 0.001 significantly different compared to l-NAME.
Fig. 4. Vascular reactivity to carbachol of aortas from rats submitted chronically (4 weeks) to different treatments. N = 6, a p < 0.05, c p < 0.01 significantly different compared to control.
Fig. 3. Effects of chronic treatments on the heart, left ventricle + the septum (L. ventricle) and the right ventricle (R. ventricle) weights. N = 6; c p < 0.001 significantly different compared to control; ˇ p < 0.01, p < 0.001 significantly different compared to l-NAME.
Fig. 5. Vascular reactivity to norepinephrine of aortas from rats submitted chronically (4 weeks) to different treatments. Results are expressed as a percentage of maximal contraction induced by KCl (60 mM). N = 6, b p < 0.01 significantly different compared to control.
in control untreated group (130.00 ± 3.54 mm Hg). Excepted the second week in l-NAME + Mammea africana-treated rats, the SBP in animals treated with l-NAME + Mammea africana or with lNAME + captopril was significantly lower than in those receiving only l-NAME and was significantly unchanged compared to control during the all period of treatment (Fig. 2). None of the treatment significantly affected the heart rate during the experiment (Table 1).
of the total heart weight while the right ventricular mass did not significantly change between groups.
3.3. Cardiac mass indexes The total heart, the left ventricle plus ventricular septum and the right ventricle were weighed and expressed in mg/100 g body weight. The total heart weight was found increased in l-NAME and l-NAME + l-arginine-treated groups but this mass was significantly higher only in l-NAME + l-arginine-treated group (373.53 ± 21.80) compared to the control group (290.76 ± 6.75). In animals treated with l-NAME + captopril or l-NAME + Mammea africana, the respective heart mass of 263.16 ± 8.66 and 267.11 ± 0.98 were not significantly different to that of control group but were significantly lower than in l-NAME-treated group (328.86 ± 6.99). As shown in Fig. 3, the changes in the left ventricular mass were similar to that
3.4. Vascular reactivity tests On aortic rings from control animal, precontracted with 1 M of noradrenaline (NA), carbachol induced a concentration dependent relaxant effect with an Emax of 80.54 ± 4.17% (% NA contraction). This carbachol relaxant effect was significantly attenuated in l-NAME-treated animals (Emax = 50.35 ± 4.28%). The addition of larginine on l-NAME treatment slightly ameliorated the relaxation (Emax = 55.99 ± 5.74%) while Mammea africana and captopril instead tend to aggravate the effect of l-NAME Emax to 39.20 ± 3.94% and 35.13 ± 8.84%, respectively (Fig. 4). The contraction response to cumulative concentrations of NA (relative to KCl; 60 mM) was not significantly different between rings from l-NAME-treated rats (Emax = 72.74 ± 20.91%), l-NAME + l-arginine (Emax = 71.96 ± 6.51%) and rings from control (Emax = 58.90 ± 4.63). This contractile effect was significantly greater in rings from l-NAME + captopril (Emax = 101.21 ± 5.97%) or l-NAME + Mammea africana (Emax = 111.16 ± 2.35%)-treated groups compared with rings from the control group (Fig. 5).
Table 1 Effects of chronic treatments on the rat heart rate Treatment
Heart rate (beat/min) W0
Control l-NAME l-NAME + l-arginine l-NAME + captopril l-NAME + Mammea africana W, week.
386 394 360 336 332
W1 ± ± ± ± ±
8.28 12.08 4.47 6.78 10.20
404 378 344 308 290
W2 ± ± ± ± ±
6.00 6.63 9.27 4.90 8.37
396 382 340 314 314
W3 ± ± ± ± ±
10.29 5.83 7.75 8.72 9.80
392 354 350 316 314
W4 ± ± ± ± ±
13.93 11.22 10.49 11.66 6.78
368 382 364 312 312
± ± ± ± ±
8.60 14.28 14.70 7.35 12.00
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4. Discussion It has been obviously demonstrated that chronic administration of l-NAME induced arterial hypertension associated with the deficiency of nitric oxide (NO). The present study analysed the effects of the CH3 OH/CH2 Cl2 stem bark extract of Mammea africana on the arterial hypertension associated with the chronic deficiency of nitric oxide. The results indicate that Mammea africana extract is able to inhibit the arterial hypertension and the cardiac hypertrophy that follow the chronic inhibition of NO production, but could not restore the endothelium vascular relaxation and instead increase the aorta sensitivity to noradrenaline. It was showed that none of the treatment affects the heart rate. This result is in accordance with many previous studies that have shown that, the chronic administration of l-NAME did not alter the heart rate (Fortepiani et al., 2002; Gerova and Kristek, 2001). Since no changes in the heart rate were also found in rats treated concomitantly with l-NAME and Mammea africana extract, it seems that the lowering of blood pressure may be predominantly due to the reduction in peripheral resistance even though Dongmo et al. (2007) showed that this extract possess negative inotropic effects on electrical induced papillary muscle contraction. The results on the isolated aorta rings showed that the carbachol relaxant effect was lower in l-NAME + Mammea africana extract as in l-NAME + carptopril-treated animals compared to rats receiving only l-NAME. This suggests that both the extract and captopril were not able to overcome the NO synthase inhibition induced by l-NAME. Dongmo et al. (2007) have shown that the CH3 OH/CH2 Cl2 extract of the stem bark of Mammea africana possesses two coumarins namely 4-n-propylcoumarin and 4-phenylcoumarin with vasorelaxant effects. They were found to act at least partially, directly on the vascular smooth muscle. The dose of extract used in this study was chosen such that the concentration of the extract inducing the in vitro maximal vasorelaxant effect may be reach in the plasma. If the direct in vitro vasorelaxant effects of these compounds could be conserved in vivo, they may explain the antihypertensive activities of Mammea africana extract. Aortic rings from animals treated with l-NAME + Mammea africana exhibited a significant increase in contraction induced by noradrenaline in relation to KCl contraction. This result suggests an increase in the sensitivity of vascular smooth muscle to receptor-induced contraction in comparison to voltage-induced contraction. The increased sensitivity may be caused by the decreased of the NO availability. l-Arginine which is competitive to l-NAME as substrate of NOS, significantly reduced the SBP at the fourth week of treatment but the SBP values were still significantly higher compared to control. Moreover, the l-arginine supplementation could not significantly ameliorate the relaxation to carbachol. These findings are in accordance with previous works which demonstrated that supply of low-endogenous NO donors, nitrites, nitrates and others do not restore completely the blood pressure to the control values (Gerova and Kristek, 2001; Torok et al., 2002). The elevation of blood pressure in l-NAME model of experimental hypertension is associated with an increase in the rennin-angiotensin system activity (Zanchi et al., 1995; Ortiz et al., 1998; Fortepiani et al., 2002). Indeed, the use of angiotensin-converting enzyme inhibitors and angiotensin-receptor antagonists have been effective in declining high-blood pressure in NO-deficient hypertensive rats (Linz et al., 1995; Soares de Moura and Cardoso, 1997). The present results agree with these data since the administration of captopril, an angiotensin-converting enzyme inhibitor, completely prevent the rise in blood pressure. The Mammea africana extract activity was a likeness to that of captopril: both the drugs reduced blood pressure, did not affect the relaxation to carbachol, increased the contraction
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to NA and reduced the cardiac hypertrophy. These coincidences suggest that Mammea africana extract may possess angiotensinconverting enzyme inhibition activity or may interfere with the rennin-angiotensin system. Indeed, previous work have shown that chronic angiotensin II blockade or converting enzyme inhibition prevents the development of l-NAME-induced hypertension but do not stimulate the reduced synthesis of NO (Takase et al., 1996; Tojo et al., 2001). In the present study, a substantial increase in cardiac mass was observed even if the effect was not statistically significant. The cardiac hypertrophy in l-NAME-treated animals is thought to be compensatory to chronic increase in blood pressure (Devlin et al., 1998). It has been showed that cardiac hypertrophy is always associated with a significant increase in plasma rennin activity (Arnal et al., 1993; Morton et al., 1993; Xu et al., 1995; Devlin et al., 1998). Thus, as suggested by Devlin et al. (1998), activation of the renin-angiotensin system may have a permissive effect for cardiac hypertrophy in l-NAME-induced hypertension. Data from the present work showed that the cardiac hypertrophy was more important in l-NAME + l-arginine-treated group. l-Arginine can act competitively with l-NAME on NO synthase, then reducing the inhibitory effect of l-NAME. It is reported that the plasma rennin activity is higher in animals treated with lower dose of lNAME and that the activity reduced with increasing dose of l-NAME (Fortepiani et al., 2002). This fact can explain the predominant cardiac hypertrophy in l-NAME + l-arginine group if the renninangiotensin system is the principal factor of cardiac hypertrophy. These findings are in agreement with the traditional use of Mammea africana in the treatment of arterial hypertension and indicate that it may have a beneficial effect in patients with NO deficiency but will be unable to improve their endothelium-dependent vasorelaxation. Acknowledgements The authors are very grateful to A.U.F. (Agence Universitaire de la Francophonie) for the fellowship awarded and to the Faculty of Sciences, University Mohamed 1st, Oujda-Morocco for having allowed this work to be taken in his place. References Adjanohoun, J.E., Aboubakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., Enoworock, E.G., Foncho, D., Gbile, Z.O., Kamanyi, A., Kamoukom Jr., Keeta, A., Mbenkum, T., Mbi, C.M., Mbielle, A.L., Mbome, I.L., Mubiru, N.K., Naney, W.L., Nkongmeneck, B., Satabie, B., Sofowa, A., Tanze, V., Wirmum, C.K., 1996. Traditional Medicine and Pharmacopeia-Contribution to Ethnobotanical and Floristic Studies in Cameroon. CNPMS, Porto-Novo, Benin, p. 15. Arnal, J.F., El Amrani, A.I., Chatellier, G., Menard, J., Michel, J.B., 1993. Cardiac weight in hypertension induced by nitric oxide synthase blockade. Hypertension 22, 380–387. Carpenter, L., McGarry, E.J., Scheimann, F., 1970. The neoflavonoids and 4alkylcoumarins from Mammea africana G. Don. Tetrahedron Letters 11, 3936–3986. Carpenter, L., McGarry, E.J., Scheimann, F., 1971. Extractives from Guttiferae. Part XXI. The isolation and structural of nine coumarins from the bark of Mammea africana G. Don. Journal of the Chemical Society, 3783–3790. Crichton, E.G., Waterman, P.G., 1978. Dihydromammea C-OB-new coumarin from the seed of Mammea africana. Phytochemistry 17, 1783–1786. Devlin, A.M., Brosnan, J.M., Graham, D., Morton, J.J., McPhaden, A.R., McIntyre, M., Hamilton, A.C., Reid, J.L., Dominiczak, A.F., 1998. Vascular smooth muscle cell polyploidy and cardiomyocyte hypertrophy due to chronic NOS inhibition in vivo. American Journal of Heart and Circulatory Physiology 247, H52–H59. Dongmo, A.B., Azebaze, A.G.B., Nguelefack, T.B., Ouahou, B.M., Sontia, B., Meyer, M., Nkengfack, A.E., Kamanyi, A., Vierling, W., 2007. Vodilator effect of the extracts and some coumarins from the stem bark of Mammea africana (Guttiferea). Journal of Ethnopharmacology 111, 329–334. Duthie, G.G., Pedersen, M.W., Gardner, P.T., Morrice, P.C., Jenkinson, A.M., McPhail, D.B., Steel, G.M., 1998. The effect of whisky and wine consumption on total phenol content and antioxidant capacity of plasma from healthy volunteers. European Journal of Clinical Nutrition 52, 733–736.
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