Enhanced Hypotensive Effects of the Essential Oil of Ocimum gratissimum Leaves and its Main Constituent, Eugenol, in DOCA-Salt Hypertensive Conscious Rats

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

The cardiovascular effects of intravenous (i. v.) treatment with the essential oil of Ocimum gratissimum (EOOG) and its main constituent, eugenol (Eug) were investigated in the experimental model of deoxycorticosterone acetate (DOCA-salt)-hypertensive rats. In both conscious DOCA-salt hypertensive rats and their uninephrectomized controls, i. v. bolus injections of EOOG (1 - 20 mg/kg) or Eug (1 - 10 mg/kg) induced dose-dependent hypotension and bradycardia. Treatment with DOCA-salt significantly enhanced the maximal decreases in mean aortic pressure (MAP) elicited by hexamethonium (30 mg/kg, i. v.) as well as the hypotensive responses to both EOOG and Eug without affecting the bradycardia. However, the enhancement of EOOG-induced hypotension in hypertensive rats remained unaffected by i. v. pretreatment with either hexamethonium (30 mg/kg) or methylatropine (1 mg/kg). These results show that i. v. treatment with EOOG or Eug dose-dependently decreased blood pressure in conscious DOCA-salt hypertensive rats, and this action is enhanced when compared with uninephrectomized controls. This enhancement appears related mainly to an increase in EOOG-induced vascular smooth relaxation rather than to enhanced sympathetic nervous system activity in this hypertensive model.

Ocimum gratissimum L. (Labiatae) is an aromatic plant abundant in north-eastern Brazil, where it is commonly known as ”alfavaca”. The plant is commonly used in cooking for flavouring. In folk medicine, infusions or decoctions of leaves from O. gratissimum are commonly used for the treatment of digestive problems and as a mouth antiseptic [1]. Leaves of O. gratissimum have an essential oil content of 0.2 % of the plant dry weight, comprised principally of mono- and sesquiterpenes [2]. Recently, we showed that intravenous (i. v.) treatment of normotensive rats with EOOG and its main constituent, eugenol (Eug; 43.70 %) dose-dependently decreased mean aortic pressure (MAP) [3], [4]. Such an effect remained unaffected by bilateral vagotomy or i. v. hexamethonium pretreatment, suggesting that it may result from a vasodilatory action of EOOG directly upon vascular smooth muscle rather than withdrawal of sympathetic tone [4]. Therefore, the present investigation was undertaken to gain further support for this hypothesis by assessing the influence of deoxycorticosterone-acetate (DOCA)-salt treatment, which increases basal sympathetic activity [5], on EOOG-induced hypotension.

Baseline MAP in DOCA-salt conscious rats was 166 ± 3 mmHg (n = 31 rats), and was significantly (p < 0.001) higher than that measured in their uninephrectomized controls (122 ± 2 mmHg, n = 20 rats). However, baseline heart rate (HR) values in hypertensive rats (375 ± 7 beats/min) were not statistically different from those in control rats (360 ± 7 beats/min). In both groups studied, i. v. injections of EOOG (1 - 20 mg/kg) or Eug (1 - 10 mg/kg) induced immediate and dose-dependent (p < 0.001) hypotension (Figs. [1] A and [2] A, respectively) and bradycardia (Figs. [1] B and [2] B, respectively), which peaked at the first 20 - 30 s after administration. Only the maximal percent decreases in MAP evoked by EOOG and Eug (Figs. [1] and [2], respectively) were significantly (p < 0.001) enhanced by DOCA-salt hypertension. Furthermore, unlike normotensive rats, hypotensive responses to EOOG in DOCA-salt rats remained significantly (p < 0.05) reduced during a period of 1 - 5 min after the administration of the highest dose of EOOG (20 mg/kg). Maximal percent and absolute decreases in MAP elicited by hexamethonium (30 mg/kg, i. v.) in DOCA-salt hypertensive (-52 ± 6 % and -92 ± 12 mmHg, respectively) were also significantly (p < 0.001) greater than those recorded in uninephrectomized controls (-32 ± 3 % and -38 ± 4 mmHg, respectively). Pretreatment of DOCA-salt hypertensive rats with either methylatropine (1 mg/kg, i. v.) or hexamethonium (30 mg/kg, i. v.) significantly and similarly reduced the EOOG-induced bradycardia (Fig. [3] B, p < 0.001). However, neither pretreatment affected the EOOG-induced hypotension (Fig. [3] A), the magnitude of which remained statistically (p < 0.001) greater than in controls.

The current study shows that i. v. treatment of conscious DOCA-salt hypertensive rats and their uninephrectomized controls with EOOG or Eug dose-dependently decreased MAP and HR, as was reported in intact, normotensive rats [3], [4]. Treatment with DOCA-salt significantly enhanced the hypotensive responses to EOOG and Eug, an effect that could be related to the increased sympathetic activity in this hypertensive model, as evidenced by the significant increases in the hypotensive responses to hexamethonium in DOCA-salt hypertensive rats. However, this enhancement appears to be independent of the degree of vascular tone because it was unaffected after blockade of ganglionic transmission with hexamethonium.

Cardiovascular effects of EOOG in DOCA-salt rats appear to be partly attributable to the actions of its main constituent Eug, as previously discussed for normotensive rats [4]. This constituent has been reported to induce vasorelaxant effects on rat [11] and rabbit [12] thoracic aorta as well as on rat mesenteric vascular bed [3], [13]. In the current study, EOOG-induced hypotension was more potent on diastolic arterial blood pressure (data not shown). These in vitro data, together with the in vivo present findings, give further support to the hypothesis that the hypotensive response to EOOG is due to a decrease in peripheral vascular resistances. Vascular muscarinic receptors that normally mediate hypotension are probably not involved, since pretreatment of hypertensive rats with methylatropine did not affect the EOOG-induced hypotension. Thus, enhanced EOOG-induced hypotension in DOCA-salt hypertensive rats appear most likely related to an increased vascular smooth muscle relaxation induced by EOOG. Further investigations using isolated aortae from DOCA-salt hypertensive rats are needed to corroborate this hypothesis.

As was reported for intact normotensive rats [4], bradycardic effects of EOOG in DOCA-salt hypertensive rats appear to be dependent on an intact and operational parasympathetic nerve drive to the heart since they were significantly and similarly reduced by i. v. hexamethonium and methylatropine. The findings that DOCA-salt treatment enhanced EOOG-induced hypotension without affecting bradycardia, and that i. v. hexamethonium or methylaptropine reduced the EOOG-induced bradycardia without affecting hypotension, give further support to the concept of independent mechanisms for EOOG-induced hypotension and bradycardia [4].

The current study shows that DOCA-salt hypertension enhances the EOOG-induced hypotension. This enhancement remained unaffected by hexamethonium, suggesting that it is mainly related to increased vascular responsiveness to EOOG rather than to enhanced sympathetic nervous system activity in this hypertensive model. The present findings may add antihypertensive activity to the list of therapeutic uses of O. gratissimum in folk medicine.

Materials and Methods

EOOG was obtained as previously described [4], [6]. Analytical conditions, composition of EOOG and retention indices of its constituents have been previously described [4]. Eugenol (Sigma) and EOOG were dissolved in Tween (2 %) and saline. All other drugs (Sigma) were dissolved in saline and administered in a volume of 1 mL/kg body weight.

Male Wistar rats (220 - 250 g) were cared for in compliance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institute of Health (NIH Publication 85 - 23, revised 1996). DOCA-salt and their uninephrectomized controls were obtained after 4-week period treatment with, respectively, DOCA and vehicle (olive oil), as previously described [7]. On the last day of treatment, rats of both groups were anesthetised with sodium pentobarbital (50 mg/kg, i. p.) and two catheters were implanted in the abdominal aorta (for the recording of MAP) and in the inferior vena cava (for drug administration), as previously described [3], [4], [7]. Twenty-fours hours later, baseline MAP and HR values were recorded on a Gilson model 5/6H (Medical Electronics Inc., USA), as previously described [3], [4], [7]. Their maximal changes elicited by i. v. hexamethonium (30 mg/kg) or increasing bolus (100 μL) doses (1 - 20 mg/kg) of EOOG and Eug (1 - 10 mg/kg) were determined in both groups. Similar changes were also studied in DOCA-salt hypertensive rats pretreated 10 min before EOOG (1 - 20 mg/kg, i. v.) with vehicle (1 mL/kg), hexamethonium (30 mg/kg, i. v.) [8] or methylatropine (1 mg/kg, i. v.) [9]. In DOCA-salt hypertensive rats (n = 5), sodium nitroprusside (20 μg/kg) was used as a positive control [10] and induced a decrease in MAP of 35.0 ± 1.9 % (mean ± SEM, n = 5).

Data are expressed as means ± the standard error of the mean. Significance (p < 0.05) of the results was assessed by means of unpaired Student’s t-test, Mann-Whitney U-test and one-way or two-way analysis of variance (ANOVA), followed by Dunnett’s test where appropriate.

Acknowledgements

The authors thank Dr. Selene Maia de Morais (Department of Physics and Chemistry, State University of Ceará, Brazil) for kindly providing us with EOOG and Rodrigo José Bezerra de Siqueira for his technical assistance. This work was supported by the Conselho Nacional de Pesquisa (CNPq) (Edital Universal, Pronex) and the Federal University of Pernambuco (UFPE). LFL Interaminense and RJB de Siqueira are MSc fellows.

References

• 1 Matos F JA. Farmácias vivas. In: Edicões UFC Fortaleza, CE, Brazil; 2001
  Search in Google Scholar
• 2 Pessoa L M, Morais S M, Bevilaqua C M, Luciano J H. Anthelmintic activity of essential oil of Ocimum gratissimum Linn. and eugenol against Haemonchus contortus .  Veterinary Parasitology. 2002;  109 59-63
  CrossrefPubMedSearch in Google Scholar
• 3 Lahlou S, Interaminense L FL, Magalhães P JC, Leal-Cardoso J H, Duarte G P. Cardiovascular effects of eugenol, a phenolic compound present in many plant essential oils, in normotensive rats.  Journal of Cardiovascular Pharmacology. 2004;  43 250-7
  PubMedSearch in Google Scholar
• 4 Lahlou S, Interaminense L FL, Leal-Cardoso J H, Morais S M, Duarte G P. Cardiovascular effects of the essential oil of Ocimum gratissimum leaves in rats: role of the autonomic nervous system.  Clinical and Experimental Pharmacology and Physiology. 2004;  31 219-25
  CrossrefPubMedSearch in Google Scholar
• 5 Nagahama S, Chen Y F, Oparil S. Enhanced depressor effect of bromocriptine in the DOCA-salt hypertensive rat.  American Journal of Physiology. 1985;  249 H64-70
  PubMedSearch in Google Scholar
• 6 Craveiro A A, Matos F JA, Alencar J W. Simple and inexpensive steam-generator for essential oils extraction.  Journal of Chemical Education. 1976;  53 562
  PubMedSearch in Google Scholar
• 7 Lahlou S, Leal-Cardoso J H, Magalhães P JC. Essential oil of Croton nepetaefolius decreases blood pressure through an action upon vascular smooth muscle: studies in DOCA-salt hypertensive rats.  Planta Medica. 2000;  66 138-43
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Sapru H N, Gonzalez E R, Krieger A J. Greater splanchnic nerve activity in the rat.  Brain Research Bulletin. 1982;  8 267-72
  CrossrefPubMedSearch in Google Scholar
• 9 Vasquez E C, Krieger E M. Decreased chronotropic responses to adrenergic stimulation following sinoaortic denervation in rat.  Brazilian Journal of Medical and Biological Research. 1997;  15 377-87
  PubMedSearch in Google Scholar
• 10 de Barros B F, Toledo JC J r, Franco D W, Tfouni E, Krieger M H. A new inorganic vasodilator, trans-{Ru(NO)(NH₃)₄[P(OEt)₃]}[PF₆]₃: hypotensive effect of endothelium-dependent and -independent vasodilators in different hypertensive animals models.  Nitric Oxide. 2002;  7 50-6
  CrossrefPubMedSearch in Google Scholar
• 11 Damiani C E, Rossoni L V, Vassallo D V. Vasorelaxant effects of eugenol on rat thoracic aorta.  Vascular Pharmacology. 2003;  40 59-66
  CrossrefPubMedSearch in Google Scholar
• 12 Nishijima H, Uchida R, Kameyama K, Kawakami N, Ohkubo T, Kitamura K. Mechanisms mediating the vasorelaxing action of eugenol, a pungent oil, on rabbit arterial tissue.  Japanese Journal of Pharmacology. 1999;  79 327-34
  PubMedSearch in Google Scholar
• 13 Criddle D N, Madeira S V, Soares de Moura R. Endothelium-dependent and -independent vasodilator effects of eugenol in the rat mesenteric vascular bed.  Journal of Pharmacy and Pharmacology. 2003;  55 359-65
  CrossrefPubMedSearch in Google Scholar

Prof. Saad Lahlou

Department of Physiology and Pharmacology

Center of Biological Sciences

Federal University of Pernambuco

50670-901 Recife

Pernambuco-PE

Brazil

Fax: +55 81-2126-8976

Email: lahlou@ufpe.br

References

• 1 Matos F JA. Farmácias vivas. In: Edicões UFC Fortaleza, CE, Brazil; 2001
  Search in Google Scholar
• 2 Pessoa L M, Morais S M, Bevilaqua C M, Luciano J H. Anthelmintic activity of essential oil of Ocimum gratissimum Linn. and eugenol against Haemonchus contortus .  Veterinary Parasitology. 2002;  109 59-63
  CrossrefPubMedSearch in Google Scholar
• 3 Lahlou S, Interaminense L FL, Magalhães P JC, Leal-Cardoso J H, Duarte G P. Cardiovascular effects of eugenol, a phenolic compound present in many plant essential oils, in normotensive rats.  Journal of Cardiovascular Pharmacology. 2004;  43 250-7
  PubMedSearch in Google Scholar
• 4 Lahlou S, Interaminense L FL, Leal-Cardoso J H, Morais S M, Duarte G P. Cardiovascular effects of the essential oil of Ocimum gratissimum leaves in rats: role of the autonomic nervous system.  Clinical and Experimental Pharmacology and Physiology. 2004;  31 219-25
  CrossrefPubMedSearch in Google Scholar
• 5 Nagahama S, Chen Y F, Oparil S. Enhanced depressor effect of bromocriptine in the DOCA-salt hypertensive rat.  American Journal of Physiology. 1985;  249 H64-70
  PubMedSearch in Google Scholar
• 6 Craveiro A A, Matos F JA, Alencar J W. Simple and inexpensive steam-generator for essential oils extraction.  Journal of Chemical Education. 1976;  53 562
  PubMedSearch in Google Scholar
• 7 Lahlou S, Leal-Cardoso J H, Magalhães P JC. Essential oil of Croton nepetaefolius decreases blood pressure through an action upon vascular smooth muscle: studies in DOCA-salt hypertensive rats.  Planta Medica. 2000;  66 138-43
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Sapru H N, Gonzalez E R, Krieger A J. Greater splanchnic nerve activity in the rat.  Brain Research Bulletin. 1982;  8 267-72
  CrossrefPubMedSearch in Google Scholar
• 9 Vasquez E C, Krieger E M. Decreased chronotropic responses to adrenergic stimulation following sinoaortic denervation in rat.  Brazilian Journal of Medical and Biological Research. 1997;  15 377-87
  PubMedSearch in Google Scholar
• 10 de Barros B F, Toledo JC J r, Franco D W, Tfouni E, Krieger M H. A new inorganic vasodilator, trans-{Ru(NO)(NH₃)₄[P(OEt)₃]}[PF₆]₃: hypotensive effect of endothelium-dependent and -independent vasodilators in different hypertensive animals models.  Nitric Oxide. 2002;  7 50-6
  CrossrefPubMedSearch in Google Scholar
• 11 Damiani C E, Rossoni L V, Vassallo D V. Vasorelaxant effects of eugenol on rat thoracic aorta.  Vascular Pharmacology. 2003;  40 59-66
  CrossrefPubMedSearch in Google Scholar
• 12 Nishijima H, Uchida R, Kameyama K, Kawakami N, Ohkubo T, Kitamura K. Mechanisms mediating the vasorelaxing action of eugenol, a pungent oil, on rabbit arterial tissue.  Japanese Journal of Pharmacology. 1999;  79 327-34
  PubMedSearch in Google Scholar
• 13 Criddle D N, Madeira S V, Soares de Moura R. Endothelium-dependent and -independent vasodilator effects of eugenol in the rat mesenteric vascular bed.  Journal of Pharmacy and Pharmacology. 2003;  55 359-65
  CrossrefPubMedSearch in Google Scholar

Prof. Saad Lahlou

Department of Physiology and Pharmacology

Center of Biological Sciences

Federal University of Pernambuco

50670-901 Recife

Pernambuco-PE

Brazil

Fax: +55 81-2126-8976

Email: lahlou@ufpe.br