Vasorelaxant Activity of Essential Oils from Croton zambesicus and Some of Their Constituents

• Abstract
• Introduction
• Materials and Methods
• Plant material
• Oil isolation
• Gas chromatography
• Compounds identification
• Measurement of aorta contraction
• Data analysis and statistics
• Supporting information
• Results and Discussion
• Acknowledgements
• References

Abstract

In this study, we determined the vasorelaxant activity of essential oils of different samples of Croton zambesicus collected in the same area in Benin at different periods and analysed their compositions by GC‐FID and GC‐MS. 68 compounds were identified among which 20 have not been described previously in this plant's essential oils. We observed quantitative differences among essential oils but all possess significant vasorelaxant activity on intact rat aortae contracted by KCl (IC₅₀ 5.6–11.8 µg/mL). This activity may, at least in part, be explained by the presence of vasorelaxant diterpenes such as ent-18-hydroxy-trachyloban-3-one, isopimara-7,15-dien-3β‐ol, and ent-18-hydroxy-isopimar-7,15-dien-3β‐ol, previously isolated from the dichloromethane extract of the leaves, but also to linalool (IC₅₀ 43.4 µg/mL) and particularly to caryophyllene oxide (IC₅₀ 2.5 µg/mL).

Introduction

Croton zambesicus Muell. Arg. (syn. C. amabilis Muell. Arg., Euphorbiaceae) is a decorative shrub or tree cultivated around villages in West Africa. The leaves, characterised by a pleasant aromatic odour, are used as home deodorant [1].

They are also traditionally used in West and Central Africa as a household remedy to treat urinary infection, fever associated with malaria or convulsions, and dysentery [2]. Furthermore, aqueous decoctions of C. zambesicus leaves are used in Benin to treat hypertension, and baths with these leaves are employed for the same treatment.

The compositions of the essential oils from the flowering tops, stem barks, roots, and leaves are described in the literature. The presence of monoterpenes and sesquiterpenes, as well as some diterpenes was reported in samples from Sudan, Tchad, Cameroon, and Benin [3], [4], [5], [6].

In previous studies, we showed the vasorelaxant activity of some diterpenes contained in the dichloromethane extract of C. zambesicus leaves [7], [8]. In this work we investigated the vasorelaxant activity of essential oils of different samples of leaves on intact rat aortae contracted with KCl and analysed their compositions to search for the presence of vasorelaxant diterpenes. We also analysed the vasorelaxant properties of some of their major constituents.

Materials and Methods

Plant material

Leaves of C. zambesicus were collected in the same area 20 km from Cotonou (Benin) in December 2001, 2003, 2005, as well as in May and December 2006. The samples were identified by Prof. V. Adjakidje from the Abomey-Calavi University in Benin. A voucher specimen was deposited at the herbarium of the Belgian Botanical Garden at Meise (BR S. P. 848.148).

Leaves were dried in the dark at room temperature, then blended to obtain a homogeneous powder and stored at room temperature in a dry and dark place.

Oil isolation

30 g of each leaves sample were hydrodistilled for 4 hours in a Clevenger type apparatus as described in the European Pharmacopoeia (6th edition) [9]. The volume of essential oil was measured by using a Hamilton syringe. The obtained essential oils were stored at + 4 °C until they were tested and analysed by GC‐FID and GC‐MS.

Gas chromatography

GC‐FID analyses were performed on a ThermoFinnigan Focus GC instrument with a FID detector using a DB‐XLB capillary column (column length 15 m × 0.25 mm with 0.25 µm film thickness; J & W Scientific, Agilent Technologies). Samples (1 µL of 10-fold dilutions in n-hexane) were injected in the split mode (split ratio 1/10) under the following conditions: injector temperature, 250 °C; detector temperature, 250 °C; oven temperature: from 60 °C to 240 °C at 3 °C/min. Helium was used as carrier gas with a 1 mL/min flow rate.

GC-EIMS analyses were performed on a TRACE GC 2000 series ThermoQuest instrument equipped with an autosampler AS2000 ThermoQuest and interfaced to a TRACE MS mass spectrometer. Separations were performed on the same column and under the same conditions as above.

Compounds identification

The identification of the constituents was based on the NIST/EPA/NIH mass spectral library (1998, version 1.6) of the GC‐MS instrument integrated with a home-made library of mass spectra of some commercial references and pure compounds isolated from plants analysed in our laboratory. The linear retention indices (LRI) were determined by comparing them to a series of C₅-C₂₈ FAME (fatty acid methyl esters).

Cembrene derivatives were identified in the NIST library on the basis of their molecular weight (typical C20H32 ion at m/z 272) and by comparing fragmentation with literature data [10].

Pure substances (> 99 % – GC‐FID) previously isolated from C. zambesicus [6], [7], [11], [12] dissolved in dichloromethane (10 mg/mL) were used as reference compounds. (±)Linalool (GC, 97 %), β-caryophyllene (GC, ≥ 98.5 %), caryophyllene oxide (GC, 99 %), α-copaene (GC, 95 %), and longifolene (GC, ≥ 99 %) were obtained from Fluka®; marrubenol was isolated from Marrubium vulgare (TLC, > 95 %) as described previously [13].

The percentages of the essential oil constituents were calculated by electronic integration of GC‐FID peak areas using the normalisation method. Values are calculated as means of 3 different injections and the variation coefficients were lower than 2 %.

Measurement of aorta contraction

Endothelium-intact aortic rings (2 mm length) of male Wistar rats weighing 200 to 300 g were suspended under a resting tension of 20 mN in 12.5 mL organ baths filled with a physiological solution [composition (mM): NaCl, 122; KCl, 5.9; NaHCO₃, 15; MgCl₂, 1.25; CaCl₂, 1.25; glucose, 11] maintained at 37 °C and bubbled with a gas mixture of 95 % O₂ and 5 % CO₂. Contractile responses were measured as described previously [7], [8]. When required, endothelium was gently rubbed out, and N-nitro-L-arginine (Sigma-Aldrich) was added to the solutions to inhibit any NO synthase activity. Endothelium integrity was tested by adding acetylcholine (1 µM) to the bath solution when KCl contraction had reached a plateau. In endothelium-intact preparations, acetylcholine relaxed KCl contraction by at least 30 %, while in endothelium-denuded preparations no response to acetylcholine was observed. Contraction was evoked by changing the physiological solution in the bath to a depolarising solution [composition (mM): NaCl, 27; KCl, 100; NaHCO₃, 15; MgCl₂, 1.25; CaCl₂, 1.25; glucose, 11]. Samples were added cumulatively on the plateau of contraction to obtain a final concentration in the bath from 1 to 30 µg/mL for tested oils or from 0.3 to 100 µg/mL for reference compounds. Oils were dissolved in a physiological solution with 2 % Tween 80. Stock solutions at 10 mg/mL of caryophyllene oxide, β-caryophyllene, α-copaene, and linalool were prepared in DMSO while longifolene and marrubenol were dissolved in EtOH. Further dilutions were made in a physiological solution. The amplitude of contraction evoked in the presence of the tested samples was compared to the response obtained before addition of the compound in the bathing solution. Blanks were performed with the solvent alone (the highest concentration was 0.1 %), and inhibition of the contraction measured in the presence of the samples was normalised to time-matched controls receiving the same volume of the solvent. Maximum relaxation measured in the solvent-treated aortic ring was 22 % and was similar with ethanol, Tween 80, or DMSO. Experiments were repeated at least on 3 different aortas.

Data analysis and statistics

Concentration‐response curves were analysed to give the concentration producing 50 % inhibition of the contractile response (IC50) by sigmoidal curve‐fitting analysis. All results are expressed as means ± the standard error of the mean (SEM). IC₅₀ values are expressed as mean values with their 95 % confidence intervals. Differences in the concentration‐response curves were analysed by two‐way analysis of variance, followed by Bonferroni's test with a criterion set for statistical significance at p < 0.05.

Supporting information

Concentration-response curves for essential oils are available as Supporting Information.

Results and Discussion

The essential oils prepared from different samples of the leaves of Croton zambesicus displayed vasorelaxant activity in KCl-contracted rat aorta with similar potency. [Table 1] gives the IC₅₀ values of the vasorelaxant activity of the essential oils prepared from different samples of C. zambesicus leaves collected in the same area in December 2001, 2003, 2005, and 2006 as well as in May 2006. Oil samples produced concentration-dependent relaxations (cfr. Fig. 1S Supporting Information). This effect was significant (p < 0.001) at 1 µg/mL except for the essential oils of December 2001 and 2003 that became significant at 3 µg/mL.

To explain this activity, we analysed the compositions of these essential oils. The results obtained from GC‐FID and GC‐MS allowed us to identify 68 compounds ([Table 2]); all unidentified peaks were small.

The analysed essential oils contained a high amount (from 28 to 57 %) of sesquiterpenes (especially caryophyllene oxide and/or longifolene) and of diterpenes (from 12 to 54 %), especially ent-trachyloban-3-one, ent-18-hydroxy-trachyloban-3-one, and ent-trachyloban-3β-ol, as well as a smaller amount of monoterpenes (from 1 to 9 %). We noted that the diterpene content was more important in the oldest essential oils (except the sample of December 2001 which was prepared in 2002 and conserved at + 6 °C; the other essential oils were prepared at the same time in 2007, so dried leaves were conserved at room temperature from the collected date to 2007). This difference could be due, at least in part, to the evaporation of the most volatile compounds during storage of the leaves.

Among the 68 identified compounds, 20 were found for the first time in C. zambesicus: sabinol, δ-elemene, azulene, isolongifolene, longipinene, τ-gurjunene, longifolene, β-chamigrene, β-eudesmene, ledene oxide (II), bisabolene oxide, aromadendrene oxide, isoaromadendrene epoxide, longipinocarveol, β-eudesmol, globulol, longiverbenone, labdanol, retinol, and labdandiol. The presence of diterpenes previously isolated by our team from the leaves of C. zambesicus was observed in all oil samples especially those from the leaves collected in December 2001 and December 2003. Among them, ent-trachyloban-3-one was the most abundant (28 % and 23 %, respectively), but we observed in most samples a relatively high amount of ent-trachyloban-3β-ol, isopimara-7,15-dien-3β-ol and ent-18-hydroxy-trachyloban-3-one, the last one possessing a marked vasorelaxant activity as described previously [8], [9], [10], [11], [12], [13], [14]. We also detected the presence of ent-18-hydroxy-trachyloban-3β-ol and ent-isopimara-7,15-dien-3β-ol, another vasorelaxant diterpene previously isolated from C. zambesicus [8]. In the essential oils from the leaves collected in December 2006 and in May 2006, which seem to be quite different from the others, we detected smaller amounts of trachylobanes and pimaranes and for the first time significant amounts of cembrene derivatives and of other unknown diterpenes, although in relatively small amounts. The presence of high amounts of labdanol (3.6 %) and labdandiol (10.1 %) was only detected in the essential oil from the December 2006 sample.

All the essential oils except those of the December 2006 sample were particularly rich in caryophyllene oxide. Both samples of 2006 were relatively rich in longifolene (between 13 and 26 %).

Concentration-response curves of the different essential oils (see Supporting Information) did not reveal any significant difference in activity although we observed a marked decrease of the vasorelaxant diterpenes in the oils from the December 2006 and May 2006 samples. As the activities of essential oils were in the same range as those of the pure diterpenes [8], we decided to evaluate the effect of other major constituents of the essential oils that we obtained and those described in the literature [6]: caryophyllene oxide (9), β-caryophyllene (10), α-copaene (11), linalool (12), and longifolene (13) and compared them with those of the diterpenes that we isolated.

Marrubenol (8), a diterpene with known vasorelaxant activity associated with Ca²⁺‐channel antagonist properties, was used as reference compound [13]. The structures of the tested compounds are represented in [Fig. 1].

Caryophyllene oxide, linalool, and α-copaene relaxed K⁺-induced contractions but neither β-caryophyllene nor longifolene showed any effect. The concentration-response curves of compounds 9 and 12 are represented in [Fig. 2] while their IC₅₀ as well as those of the vasorelaxant diterpenes are reported in [Table 3]. Diterpenes 1, 3, 5, and 6 did not display vasorelaxant activities (IC₅₀ > 80 µM). Unfortunately, it was not possible to calculate the IC₅₀ of α-copaene because of solubility problems starting at 10 µg/mL, which inhibited contraction by about 50 %.

KCl-evoked contraction is associated with the activation of voltage-operated calcium channels. The vasorelaxing effect of caryophyllene oxide in KCl-depolarised aorta might be related to the reported inhibitory effect of this compound on calcium channel current in the guinea-pig heart [15]. The relaxing effect of caryophyllene oxide was significantly depressed in the absence of functional endothelium, while the effect of linalool was less affected by the endothelium, but concentration-effect curves with and without endothelium were significantly different (Anova; [Fig. 2]). A similar influence of a functional endothelium is observed with most calcium channel blockers and has been attributed to a synergy between endothelium-released NO and calcium channel blockers [16].

Caryophyllene oxide showed a good inhibitory activity on KCl-induced contraction in a concentration range similar to the most vasorelaxant diterpenes suggesting that it may also be responsible for at least a part of the effect observed with some essential oils. As concentrations vary from 1- to 10-fold, depending on the oil batch, its contribution to the observed effect also varies. Synergy or additivity of activities with other constituents should also be considered, as essential oils, which are complex mixtures, have a vasorelaxant activity at about the same range of concentration and their effect is close to the one of isolated active compounds. Furthermore, the essential oil of the sample of December 2006 does not have a lower activity than the others even though it contains much less identified vasorelaxing compounds. This could perhaps be explained by the presence of pimarane unidentified derivatives or other unidentified compounds.

Relaxant effect of essential oils from several plants has been reported in tracheal and ileal smooth muscles [17]. In this study, we showed for the first time the vasorelaxant properties of different samples of essential oils of C. zambesicus as well as of caryophyllene oxide and identified vasorelaxant diterpenes in oils. Linalool is already known to have moderate antispasmodic activity [18], and caryophyllene oxide has been reported to have a negative inotropic effect in the heart [15], but it is also the first time that it is shown to have a vasorelaxant effect on KCl-induced contraction. Although the concentrations of these compounds do not allow to totally explain the activity of the essential oils, they contribute, at least in part, to the vasorelaxant properties of the leaves of C. zambesicus.

Acknowledgements

We are grateful to the UCL funds for Scientific Research (FSR) and the FNRS for financial support (fund number 9.4553.04).We also thank M.-C. Fayt and R. Colak for their skillful assistance.

References

• 1 Hutchinson J, Dalziel J M. The useful plants of West Tropical Africa. London; The Crown Agents for the Overseas Colonies 1948: 223-224
  Search in Google Scholar
• 2 Adjanohoun E J, Adjakidje V, Ahyi M, Ake Assi L, Akoegninou A, d'Almeida J, Apovo F, Boukef K, Chadare M, Cusse G, Dramane K, Eyme J, Gassit J-N, Gbaguidi N, Goudote E, Guinko P, Houngnon P, Lo I, Keita A, Kiniffo H V, Kone-Bamba D, Musampa Nseyya A, Saadou M, Sodogandji T, de Souza S, Tchabi A, Zinsou Dossa C, Zohoun T. Contribution aux études ethnobotaniques et floristiques en République Populaire du Bénin. Paris; Agence de Coopération Culturelle et Technique 1989: 895
  Search in Google Scholar
• 3 Fekam Boyom F, Keumedjio F, Jazet Dongmo P M, Ngadjui B T, Amvam Zollo P H, Menut C, Bessiere J M. Essential oils from Croton zambesicus Muell. Arg. growing in Cameroon.  Flavour Fragr J. 2002;  17 215
  CrossrefPubMedSearch in Google Scholar
• 4 Mekkawi A G. The essential oil of Croton zambesicus.  Fitoterapia. 1985;  3 181-183
  PubMedSearch in Google Scholar
• 5 Menut C, Lamaty G, Bessière J M, Seuleiman A M, Fendero P, Maidou E, Dénamganaï J. Aromatic plants of tropical Central Africa. XXII. Volatile constituents of Croton aubrevillei and C. zambesicus.  J Essent Oil Res. 1995;  7 419-422
  PubMedSearch in Google Scholar
• 6 Block S, Flamini G, Brkic D, Morelli I, Quetin-Leclerq J. Analysis of the essential oil from leaves of Croton zambesicus Muell. Arg. growing in Benin.  Flavour Fragr J. 2006;  21 222-224
  CrossrefPubMedSearch in Google Scholar
• 7 Baccelli C, Block S, Van Holle B, Schanck A, Chapon D, Tinant B, Meervelt L V, Morel N, Quetin-Leclercq J. Diterpenes isolated from Croton zambesicus inhibit KCl-induced contraction.  Planta Med. 2005;  71 1036-1039
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Baccelli C, Navarro I, Block S, Abad A, Morel N, Quetin-Leclercq J. Vasorelaxant activity of diterpenes from Croton zambesicus and synthetic trachylobanes and their structure-activity relationships.  J Nat Prod. 2007;  70 910-917
  CrossrefPubMedSearch in Google Scholar
• 9 Pharmacopée européenne, 6th edition. Strasbourg; Conseil de l'Europe 2007: 269-270
  Search in Google Scholar
• 10 Takagi Y, Fujimori T, Kaneko H, Kato K. Cembrene from Japanese domestic tobacco, Nicotinan tabacum cv. Suifu.  Agric Biol Chem. 1980;  44 467-468
  PubMedSearch in Google Scholar
• 11 Block S, Stévigny C, De Pauw-Gillet M C, de Hoffmann E, Llabrès G, Adjakidjé V, Quetin-Leclercq J. ent-Trachyloban-3β-ol, a new cytotoxic diterpene from Croton zambesicus.  Planta Med. 2002;  68 647-649
  Thieme ConnectPubMedSearch in Google Scholar
• 12 Block S, Baccelli C, Tinant B, Van Meervelt L, Rozenberg R, Habib Jiwan J L, Llabrès G, De Pauw-Gillet M C, Quetin-Leclercq J. Diterpenes from the leaves of Croton zambesicus.  Phytochemistry. 2004;  65 1165-1171
  CrossrefPubMedSearch in Google Scholar
• 13 El Bardai S, Wibo M, Hamaide M C, Lyoussi B, Quetin-Leclercq J, Morel N. Characterisation of marrubenol, a diterpene extracted from Marrubium vulgare, as an L-type calcium channel blocker.  Br J Pharmacol. 2003;  140 1211-1216
  CrossrefPubMedSearch in Google Scholar
• 14 Martinsen A, Baccelli C, Navarro I, Abad A, Quetin-Leclercq J, Morel N. Vascular activity of a natural diterpene isolated from Croton zambesicus and of a structurally similar synthetic trachylobane.  Vasc Pharmacol. 2010;  52 63-69
  CrossrefPubMedSearch in Google Scholar
• 15 Sensch O, Vierling W, Brandt W, Reiter M. Effects of inhibition of calcium and potassium currents in guinea-pig cardiac contraction: comparison of beta-caryophyllene oxide, eugenol, and nifedipine.  Br J Pharmacol. 2000;  131 1089-1096
  CrossrefPubMedSearch in Google Scholar
• 16 Salomone S, Silva C L, Morel N, Godfraind T. Facilitation of the vasorelaxant action of calcium antagonists by basal nitric oxide in depolarized artery.  Naunyn Schmiedebergs Arch Pharmacol. 1996;  354 505-512
  CrossrefPubMedSearch in Google Scholar
• 17 Reiter M, Brandt W. Relaxant effects on tracheal and ileal smooth muscles of the guinea pig.  Arzneimittelforschung. 1985;  35 408-414
  PubMedSearch in Google Scholar
• 18 Imaseki I, Kitabatake Y. Effect of essential oils and their components on the isolated intestine of mice.  Yakugaku Zasshi. 1962;  82 1326-1328
  PubMedSearch in Google Scholar

Prof. Joelle Quetin-Leclercq

CHAM Unit
Louvain Drug Research Institute
Université catholique de Louvain

Avenue E. Mounier 72–30

1200 Brussels

Belgium

Phone: + 32 27 64 72 30

Fax: + 32 27 64 72 53

Email: joelle.leclercq@uclouvain.be

References

• 1 Hutchinson J, Dalziel J M. The useful plants of West Tropical Africa. London; The Crown Agents for the Overseas Colonies 1948: 223-224
  Search in Google Scholar
• 2 Adjanohoun E J, Adjakidje V, Ahyi M, Ake Assi L, Akoegninou A, d'Almeida J, Apovo F, Boukef K, Chadare M, Cusse G, Dramane K, Eyme J, Gassit J-N, Gbaguidi N, Goudote E, Guinko P, Houngnon P, Lo I, Keita A, Kiniffo H V, Kone-Bamba D, Musampa Nseyya A, Saadou M, Sodogandji T, de Souza S, Tchabi A, Zinsou Dossa C, Zohoun T. Contribution aux études ethnobotaniques et floristiques en République Populaire du Bénin. Paris; Agence de Coopération Culturelle et Technique 1989: 895
  Search in Google Scholar
• 3 Fekam Boyom F, Keumedjio F, Jazet Dongmo P M, Ngadjui B T, Amvam Zollo P H, Menut C, Bessiere J M. Essential oils from Croton zambesicus Muell. Arg. growing in Cameroon.  Flavour Fragr J. 2002;  17 215
  CrossrefPubMedSearch in Google Scholar
• 4 Mekkawi A G. The essential oil of Croton zambesicus.  Fitoterapia. 1985;  3 181-183
  PubMedSearch in Google Scholar
• 5 Menut C, Lamaty G, Bessière J M, Seuleiman A M, Fendero P, Maidou E, Dénamganaï J. Aromatic plants of tropical Central Africa. XXII. Volatile constituents of Croton aubrevillei and C. zambesicus.  J Essent Oil Res. 1995;  7 419-422
  PubMedSearch in Google Scholar
• 6 Block S, Flamini G, Brkic D, Morelli I, Quetin-Leclerq J. Analysis of the essential oil from leaves of Croton zambesicus Muell. Arg. growing in Benin.  Flavour Fragr J. 2006;  21 222-224
  CrossrefPubMedSearch in Google Scholar
• 7 Baccelli C, Block S, Van Holle B, Schanck A, Chapon D, Tinant B, Meervelt L V, Morel N, Quetin-Leclercq J. Diterpenes isolated from Croton zambesicus inhibit KCl-induced contraction.  Planta Med. 2005;  71 1036-1039
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Baccelli C, Navarro I, Block S, Abad A, Morel N, Quetin-Leclercq J. Vasorelaxant activity of diterpenes from Croton zambesicus and synthetic trachylobanes and their structure-activity relationships.  J Nat Prod. 2007;  70 910-917
  CrossrefPubMedSearch in Google Scholar
• 9 Pharmacopée européenne, 6th edition. Strasbourg; Conseil de l'Europe 2007: 269-270
  Search in Google Scholar
• 10 Takagi Y, Fujimori T, Kaneko H, Kato K. Cembrene from Japanese domestic tobacco, Nicotinan tabacum cv. Suifu.  Agric Biol Chem. 1980;  44 467-468
  PubMedSearch in Google Scholar
• 11 Block S, Stévigny C, De Pauw-Gillet M C, de Hoffmann E, Llabrès G, Adjakidjé V, Quetin-Leclercq J. ent-Trachyloban-3β-ol, a new cytotoxic diterpene from Croton zambesicus.  Planta Med. 2002;  68 647-649
  Thieme ConnectPubMedSearch in Google Scholar
• 12 Block S, Baccelli C, Tinant B, Van Meervelt L, Rozenberg R, Habib Jiwan J L, Llabrès G, De Pauw-Gillet M C, Quetin-Leclercq J. Diterpenes from the leaves of Croton zambesicus.  Phytochemistry. 2004;  65 1165-1171
  CrossrefPubMedSearch in Google Scholar
• 13 El Bardai S, Wibo M, Hamaide M C, Lyoussi B, Quetin-Leclercq J, Morel N. Characterisation of marrubenol, a diterpene extracted from Marrubium vulgare, as an L-type calcium channel blocker.  Br J Pharmacol. 2003;  140 1211-1216
  CrossrefPubMedSearch in Google Scholar
• 14 Martinsen A, Baccelli C, Navarro I, Abad A, Quetin-Leclercq J, Morel N. Vascular activity of a natural diterpene isolated from Croton zambesicus and of a structurally similar synthetic trachylobane.  Vasc Pharmacol. 2010;  52 63-69
  CrossrefPubMedSearch in Google Scholar
• 15 Sensch O, Vierling W, Brandt W, Reiter M. Effects of inhibition of calcium and potassium currents in guinea-pig cardiac contraction: comparison of beta-caryophyllene oxide, eugenol, and nifedipine.  Br J Pharmacol. 2000;  131 1089-1096
  CrossrefPubMedSearch in Google Scholar
• 16 Salomone S, Silva C L, Morel N, Godfraind T. Facilitation of the vasorelaxant action of calcium antagonists by basal nitric oxide in depolarized artery.  Naunyn Schmiedebergs Arch Pharmacol. 1996;  354 505-512
  CrossrefPubMedSearch in Google Scholar
• 17 Reiter M, Brandt W. Relaxant effects on tracheal and ileal smooth muscles of the guinea pig.  Arzneimittelforschung. 1985;  35 408-414
  PubMedSearch in Google Scholar
• 18 Imaseki I, Kitabatake Y. Effect of essential oils and their components on the isolated intestine of mice.  Yakugaku Zasshi. 1962;  82 1326-1328
  PubMedSearch in Google Scholar

Prof. Joelle Quetin-Leclercq

CHAM Unit
Louvain Drug Research Institute
Université catholique de Louvain

Avenue E. Mounier 72–30

1200 Brussels

Belgium

Phone: + 32 27 64 72 30

Fax: + 32 27 64 72 53

Email: joelle.leclercq@uclouvain.be

• Supplementary Material