BMC Complementary and Alternative Medicine

Background: Sesamum radiatum Schum. & Thonn. (Pedaliaceae) is an annual herbaceous plant, which belongs to the family Pedaliaceae and genus Sesamum. Sesame is used in traditional medicine in Africa and Asia for many diseases treatment. Sesame plant especially the leaves, seed and oil are consumed locally as a staple food by subsistence farmers. The study analyses the relaxation induced by the aqueous extract of leaves from sesame (ESera), compared with those of acetylcholine (ACh) in the guinea-pig aortic preparations (GPAPs), in order to confirm the use in traditional medicine for cardiovascular diseases.


Background
Sesamum radiatum Schum. & Thonn. (Pedaliaceae), pantotropical plant was used in traditional Medicine to facilitate the delivery in pregnant women [1,2]. Our first investigation with the action of ESera in the cardiovascular system of mammalians showed that ESera as well as acetylcholine, induced arterial hypotension resulting from cardiodepression and vasorelaxation of rabbits and rats [3]. Indeed, Furchgott et al (1980) [4] showed the mechanism of acetylcholine on the vascular smooth muscle. The vasodilatation induced by acetylcholine depended on the functional integrity of the endothelium and implied an endothelial hyperpolarising factor (EDHF) and a release of nitric oxide (NO). For these authors, the releasing effects of acetylcholine were extended to other natural biological substances or from synthesis [5,6].
In view of these findings, the current study pursued three aims. The first goal is to explore whether ESera is able to inhibit GPAPs contractile activity. The second aim was to compare ESera and ACh in their potency and efficacy to relax the contractile force in the presence of nitric oxide. The third aim of the study was to explore whether any effect ESera on aorta smooth muscle is exclusively due to activation of cyclooxygenase pathway in relationship with nitric oxide (NO) or whether there is also cholinoceptorindependent component. Finally, the present study is an approach to elucidate the mechanism underlying ESera's action in the vasoralaxation observed in GPAPs.

Plant
Sesamum radiatum (Schum.) Thonn. (Pedaliaceae) was collected in October 2005 from farms specialized in growing plants for scientific or medical purposes. The leaves of Sesamum radiatum were verified to be identical samples at the specimen herbarium of the Centre National Floristique de Côte d'Ivoire at Cocody University in Abidjan. Voucher specimen were preserved and catalogued in the same herbarium (Centre National Floristique). This pantropical plant was authenticated by a Botanic expert, Prof. Aké-Assi Laurent of Centre National Floristique, UFR-Biosciences, University of Cocody, in Abidjan, Côte d'Ivoire).

Phytochemical screening
The extract and its fractions were tested by Lieberman Bouchard, Ferric chloride, cyanidine stiasny and, Valser-Meyer and Dragendorff Tests to determine the presence of strerols, phenolic compounds, flavonoids, Tannin and alkaloids, respectively.

Preparation of extract of sesame leaves
Sesamum radiatum leaves were stored in cellophane bags. The leaves collected were dried at room temperature (Temperature: 27 ± 3°C). The powdered leaves (100 g) were first macerated for 24 hours in n-hexane to remove chlorophyll and other hexanosolubles substances. The residue was dried and extracted by vigorously shaking in bi-distilled water for 24 hours. After 2-h extraction and filtration, the filtrate was concentrated, by evaporation of the solvent. The drug (ESera) obtained was stored at 4°C until use.

Animals
Guinea-pigs (Cavia porcellus), of both sexes weighing between 350 g and 400 g, were obtained from the Animal House of the Laboratory of Nutrition and Pharmacology of UFR-Biosciences at Cocody university in Abidjan (Ivory Coast). The guinea-pigs were housed in a constant temperature rooms with a light/dark cycle of 14/10 hours. The animals were fed and given water ad libitum until they were used.
Since it has been shown that endothelium can inhibit the vasorelaxator effect of acetylcholine in aorta preparations [7,8], experiments were performed in preparations with intact endothelium as well as in aortae where the endothelium had been removed by gentle rubbing of the intimae with a wooden rod to avoid the inhibitory role of functional endothelium to acetylcholine and Esera. Each strip of aorta was cut into 6-7 mm length. The preparations were suspended between two L-shaped stainless steel hooks in a 10 ml organ bath with Mac Ewen solution at 37°C (pH = 7.4). Each preparation was connected by a silk thread to a force transducer FT30 (Hugo Sachs electronic, Freiburg, Germany) and the isometric force was recorded by a pen-recorder Rikadenki (Freiburg, Germany) on paper enrolled at a speed of 2.5 mm/min. A resting tension of 1 g was maintained, this setting allowed for the optimum observation of maximal contractile response to drugs.

Experimental protocol
After the equilibration period of 60 min, tissues were exposed to ESera, which was added to the bath by means of cumulative methods [9] and to depolarizing potassium solution to test the viability. In most cases, a maximal contractile response ranging from 0.8-1.1 g could be induced. The preparations with a response below 0.6 g were considered insufficiently viable and discarded.
The potassium solution (80 mM K + ) had the same composition as the Mac ewen buffer used, except that NaCl had been completely replaced by an equimolar amount of KCl. Once the contraction had reached a plateau, the preparations were washed with Mac Ewen solution four times and left a further 40 min equilibration period at a re-adjusted tension of 1 g.
To avoid tachyphylaxis, only a single cumulative concentration-response curve (CRC) for drugs was obtained in each preparation. Appropriate controls were run at the same time in different strips obtained from the aorta.
At the end of each experiment, after the drugs had been washed-out with Mac Ewen solution four times, acetylcholine (1 μM) was added when the maximal response to noradrenaline (0. 4 μM) had been obtained, in order to assess the presence or absence of functional endothelium. A rapid and marked reduction of noradrenaline induced tone was taken evidence that a significant amount of functional endothelium was present. The absence of relaxant response was taken as indicative of the disappearance of functional endothelium [8,10].

Influence of the endothelium on the effects of Esera
After the equilibration period of at least 60 min, preparations with or without endothelium were exposed to ESera, which was added to the bath by means of a cumulative method [9], cumulative CRCs for Esera (1 × 10 -7 -0.1 μg/ ml) or ACh (1 × 10 -5 -0.1 μg/ml) were constructed in both endothelium-intact and endothelium-denuded aortic preparations of the same aorta. The drugs were then washed out with Mac Ewen solution four times. Subsequently, noradrenaline (1 μM) -induced contractions were imposed and ACh (1 μM) was added to test whether functional endothelium was present.

Influence of the nitric oxide (NO) synthesis inhibitor on the effects of Esera
After equilibration, GPAP were exposed to a nitric oxide (NO) synthesis inhibitor; N ω -Nitro-L-Arginine (L-NNA) at a concentration of 50 nM for 30 min. Cumulative CRCs of ESera were obtained in the presence of the nitric oxide inhibitor.

Influence of the cyclooxygenase inhibitor on the effects of Esera
After equilibration, the preparations were exposed to a cyclooxygenase inhibitor diclofenac at a concentration of 30 nM for 30 min. Cumulative CRCs of Esera were obtained in the presence of the cyclooxygenase inhibitor.

Influence of the non selective potassic channels blocker on the effects of Esera
After equilibration, the preparations were exposed to non selective potassium channels blocker the Tetra-ethylammonium (TEA) at a concentration of 50 nM for 30 min. Cumulative CRCs of Esera were obtained in the presence of TEA.

Chemicals used
The acetylcholine and the N ω -Nitro-L-Arginine were purchased from Sigma Company (St Louis, Mo, USA). Diclofenac and Tetra-ethyl-ammonium were provided from Sigma-Aldrich (Switzerland). All drugs were dissolved in saline.

Statistics analysis
Responses were expressed as absolute changes in mg of tension. Data are expressed as means ± SEM obtained from n separate experiments. Statistical analysis of the results was determined by using the unpaired Student's ttest. p < 0.05 or less was considered as indicative of significance. The EC 50 (i.e. the concentration of ESera or drug causing half maximum response) values were determined from individual experiments for the complete agonist concentration-response curves by figures carried out respectively using the software GraphPad Prism (San Diego, California, USA). The EC 50 values are reported as geometric means accompanied by their respective 95% confidence limits. All other reported results are means ± s.e.m.

Phytochemical screening
Phytochemical study that the sesame plant is rich in phenolic compounds (phenols, lignans and flavenoids). Sterols were also found in aqueous extract and its fractions.

Extract of Sesamum radiatum Schum. & Thonn. (ESera) induced contractions in endothelium-denuded guinea-pig aortic preparations
In control conditions, regular contractile activity of the isolate aorta was recorded which were constant in all experiments and were not influenced by the addition of vehicle (Mac Ewen's solution (original tracing not shown).
In endothelium-denuded GPAPs, ESera in the same concentrations range induced contractions of isolated aortae. Concentration of ESera of 0.1 μg/ml induced a contractile force of 90.6 ± 6 mg ( Fig. 2A). Contractions were obtained with ACh in the same preparations, which induced con-Mean relaxant concentration-response curve for the effect of the extract of Sesamum radiatum Schum   centration-dependently contractions of endotheliumdenuded GPAPs. Concentration of 7 × 10 -2 nM induced a contractile force of 111.9 ± 9 mg (Fig. 2B).

Inhibitory effects of L-N omega -Nitro-Arginine on ESerainduced vasorelaxation in guinea-pig aorta strips
Our previous experiments showed that ESera and ACh induced vasorelaxation of endothelium-intact guinea-pig aorta smooth muscle. In order to justify the involvement of the possible modulation by the endothelium of the contractile responses to ESera, L-NNA was used.

Inhibitory effects of diclofenac or tetra-ethyl-ammonium on ESera-induced vasorelaxation in guinea-pig aorta strips
Previous studies of the effects of ESera were carried out on the cardiac muscle of rat in the presence of the atropine, antagonist of the muscarinic cholinoceptors. This study showed a partial cholinergic antagonist action of ESera [2].
The relaxations induced by Esera in GPAPs with endothelium were antagonized in a concentration-dependent and reversible manner by both diclofenac (cyclooxygenase inhibitor) and TEA (non-selective K + channel blocker).
In endothelium-intact GPAPs ESera-induced relaxation was significantly affected by the potassium channels blocker TEA (50 nM), but was potentiated by the cyclooxygenase inhibitor, diclofenac (30 nM).
In endothelium-intact GPAPs, TEA at the same concentration inhibited totally the vasodilatory action to ESera. The CRC for ESera was shifted to the right (EC 50 -value = 1.2 × Mean relaxant concentration-response curve for the extract of Sesamum radiatum Schum  10 -2 μg/ml, Fig. 5A). In contrast, TEA caused slightly a rightward shift of the CRCs of ACh-induced relaxation in a concentration-response manner (EC 50 -value = 9.2 nM, Fig. 5B). Interestingly, neither TEA nor diclofenac interfered significantly with the relaxation produced by ACh.

Discussion
Herbal preparations are used since ancient times to maintain health. Also, herbal preparations, if taken in appropriate dose, can lead to a better option for curing various aliment [11,12].
Mean relaxant concentration-response curve for the effect of extract of Sesamum radiatum Schum

B
The results of present study have demonstrated that ESera, besides inducing concentration-dependent relaxation in endothelium-intact GPAPs, at the same concentrations produces concentration-dependent contraction in either endothelium-denuded GPAPs or in the presence of nitric oxide synthesis inhibitor. Esera-induced relaxations in GPAPs with endothelium-intact are counteracted by endothelium.
Leaves of S. radiatum are used traditionally by patients in West Africa and are taken as water decoction [13]. Due to this reason the leaf juice of the plant was evaluated and the data also confirmed the traditional indications. Early investigations [2,14] on the antihypertensive properties of the extracts of S. radiatum leaves by other authors also substantiate the results of our studies in Rabbits [3].
It seems likely that the endothelium-induced inhibition of ESera-effects is mediated by the endogenous vasodilator endothelium derived relaxing factor (EDRF) [10,11]. EDRF is assumed to be identical with NO or a closely related compound and vascular endothelial cells are known to synthesize NO [16,17].
Our findings are the first demonstration of endotheliumintact contractions to ESera in GPAPs.
Sesame had been shown to possess multiple health benefits, both alone and in synergistic combination with others compounds. By influencing pharmacological processes in the body, sesame and its lignans promise to help reduce risk for many of today's most common diseases, including heart disease, obesity, arterial hypertension and inflammatory disorders [23][24][25][26][27][28][29][30][31].
On the other hand, the TEA and the diclofenac differently affect the contractile activity induced either by ACh or by ESera. In separate experiments, after the first CRC of ESera had been obtained in endothelium-intact, 30 nM diclofenac or 50 nM TEA were added and then the second CRC was contracted. It was fund that the maximal responses of second curves were increased in the presence of either antagonist. Diclofenac slightly shifted the second curves of ACh to the right, whereas it had no significant effect on the curves compound. These data, taken together with other results in this study, suggest that the contractile response to ESera is mediated by cyclooxygenase-receptors. Modulating factors which influence these parameters can be released from endothelium or from smooth muscle cells.
The prostaglandins as well as indometacin, a powerful antagonist of the cyclooxygenase did not affect relaxation in response to ACh [32,33].
Destruction of the endothelium or inhibition of NO synthesis significantly enhanced the response to ESera in this preparation. Moreover, similar results were obtained with cyclooxygenase-inhibition or K + channels blocker were obtained with Chen et al. [34] and Niu et al. [35] suggest that the endothelium-denuded affect by NO via K + channels involvement cyclooxygenase pathways.
Our study indicated a significant myorelaxant and supports the traditional use of fresh leaves by Ivorian physicians.
In conclusion, on the basis of beneficial effect of ESera in the literature and our own results of the experiments in the extract of the same species ESera induces a decrease of vascular tone. Once found ESera may be incorporated as antihypertensive agent for the improvement of the patients suffering from cardiovascular diseases.
Chemical and pharmacological studies are now in progress to isolate and to characterize the constituents responsible for such effects, and also to investigate in more detail the mechanisms underlying the relaxant action of the active principle (s) of ESera in GPAPs.