African Journal of Traditional, Complementary and Alternative Medicines African Ethnomedicines Network ISSN: 0189-6016 Vol. 2, Num. 2, 2005, pp. 166-176

Research Paper

OUEDRAOGO¹* Maurice, OUEDRAOGO² Sylvin, OUEDRAOGO¹ Lamini, TRAORE² Aristide, BELEMTOUGRI¹ G. Raymond, SAWADOGO¹ L. Laya et, GUISSOU^2,3 I. Pierre.

¹UFR/SVT,Université de Ouagadougou, 03 BP 7021 Burkina Faso. ²Institut de Recherche en Sciences de la Santé (IRSS/CNRST), BP 7192, Ouagadougou, Burkina Faso. ³UFR/SDS Université de Ouagadougou, 03 BP 7021, Burkina Faso.
¹ UFR/SVT Université de Ouagadougou BP 7021 BF.
² IRSS/CNRST BP 7192 Ouagadougou BF
³ UFR/SDS Université de Ouagadougou
BP 7021 BF
E-mail : ouedraogo_maurice@yahoo.fr

Code Number: tc05018

Abstract

The present study was designed to investigate the blocking of calcium by the hydroalcoholic extract of Tapinanthus dodoneifolius (Tapidod), "in vitro", on rat trachea. To evaluate this effect, the contractile activity of tracheal chains from Wistar Kyoto rats was isometrically recorded. On the isolated tracheal rings the extract produced the following effects: (a) a reduction of the contraction obtained by BaCl₂, (b) a bronchorelaxing action, on strips precontracted by KCl, which was not influenced by TEA (3x10^-3 M), (c) a concentration-dependent decrease of the spasm evoked by calcium chloride (CaCl₂) in K⁺-rich Ca²⁺-free physiological salt solution, before and after intracellular calcium depletion (d), an inhibitory effect on contraction induced by acetylcholine in Ca²⁺-free Krebs-Heinseleit solution supplemented with EDTA (5x10^-4M). It is concluded that: 1. The activation of the potassium channels does not play a significant role in the relaxant effect of Tapidod. 2. The antispasmodic property of Tapidod seems to be mediated by the blockade of intracellular Ca²⁺ release. 3. Most likely an inhibition of the intracellular Ca²⁺-regulating proteins is involved.

Keys words: Extract of Tapinanthus dodoneifolius (Tapidod); Trachea; Calcium.

Introduction

Asthma is a worldwide chronic airway inflammatory disease, the etiology of which is not known (Holgate, 1997). The mortality and morbidity associated with this disease have not appreciably declined (Sly and O'Donnell, 1997), and its treatment has not changed over the last two decades (Wenzel, 1998). Therefore, research has not reached its goal, and the treatment of asthma is still seeking for targeted, anti-inflammatory therapy (Vaali, 1999).

Phytotherapy research represents a promising approach to the discovery of biologically active molecules that may be beneficial in the treatment of asthma. Tapinanthus dodoneifolius is extensively used as medicinal plant in traditional medicine in Burkina Faso. According to Boussim (2002) Tapinanthus dodoneifolius is used in the treatment of cholera, asthma and diabetes. The aqueous extract is taken against gynecologic disturbances, digestives disorders and nervous confusions (Nacoulma/Ouédraogo 1996). Cardiovascular properties of T. dodoneifolius have been recently described by Ouédraogo et al. (2005a). Cepeleanu et al. (1994) revealed larvicidal and molluscidal effects of T. dodoneifolius. Preliminary pharmacological investigations on rat trachea indicate that Tapinanthus dodoneifolius total aqueous extract inhibited contractile activity induced by physiological and non-physiological agonists (Ouédraogo et al., 2005b).

Chemical analyses of different extracts from T. dodoneifolius yielded components as triterpenes, sterols, carotenoïdes, saponosides, anthracenosides, anthocyanosides and tannins (Traoré, 2000).

The present study was conducted to analyze comprehensively the relaxant effect and to elucidate the mechanism of action of Tapidod in rat trachea.

Material and Method

Plant and extracts

Tapinanthus dodoneifolius (DC.) Danser (syn: Agelanthus dodoneifolius (DC.) R.M. Polhill& D. Wiens) was collected in Loumbila (Burkina Faso) in April 2002 and taxonomically identified by an expert botanist (Prof Boussim I. J., University of Ouagadougou). A voucher specimen is deposited in the Herbarium of the Department of Vegetal Biology, University of Ouagadougou, Burkina Faso, with the reference no. 002. The drying operation was carried out under room temperature with out exposure to sunlight. Air-dried and powdered plants of T. dodoneifolius (100g) were soaked in chloroform (1000 ml) for 24 hours. After filtration, the remainder was dried at room temperature and extracted in ethanol (1000 ml) for 24 hours. The extract was centrifuged, concentrated by rotary evaporation, frozen and lyophilized.

Tracheal rings were dissected from either male or female Wistar rats (weighing 200-300g) following ethyl carbamate anesthesia and exsanguination by carotid artery transection. Thoracic tracheas were removed rapidly and cleaned of surrounding tissue. The isolated tracheas were cut as spirals according to the method described by Brunelleschi et al. (1987); Hazekamp et al. (2001) and suspended in 20 ml organ bath chambers containing modified Krebs-Henseleit physiological solution at pH of 7.4, continuously oxygenated and maintained at 37° C. The rings were mounted by means of two parallel L-shaped stainless steel holders inserted into the lumen. The upper holder was attached with thread to a transducer while the lower one was tied to a stationary glass rod. The preparations were allowed to equilibrate for at least 90 min while the base-line tension was adjusted progressively to 2g as recommended by Tschirhart et al. (1987). Isometric contraction of the smooth muscle was recorded with chart recorder via a force-displacement transducer (Grass FT 03C).

Acetylcholine and barium chloride are tested by cumulative addition, directly in the tissue bath. The total amount of drugs added did not exceed 5 % of the total volume (1ml) of the bath (Van Rossum, 1963). For the determination of the effects of antagonists on the response to the addition of agonists, tissues were incubated in the presence of antagonists 15 or 30 min prior to the addition of agonists.

To investigate the involvement of voltage-sensitive Kchannels activation in Tapidod-induced relaxation, two K⁺ channel blockers (BaCl₂ and TEA) (Emine et al., 1998; Imaizumi and Watanabe, 1981) and the method of depolarization described by Karaki and Weiss (1988) were used.

In order to study the inhibition of the availability of the calcium by Tapidod three different experimental protocols were applied. These protocols derived from method described by (Lagaud et al. (1996)

In the first experimental condition, concentration-response curves to CaCl₂ were constructed by cumulative application in a K⁺-rich Ca²⁺-free solution. Trachea was previously incubated in a normal physiological salt solution (equilibration period) and depolarized in the K⁺-rich Ca²⁺- free solution. The K⁺-rich Ca²⁺- free solution was obtained by KCl (8x10^-2 M) substitution for an equimolar amount of NaCl, omission of calcium and addition of EDTA (5x10^-4 M) (Guan et al. 1987).

In the second experimental protocol which intended to study the component of the Tapidod-induced relaxation due to the blocking of the Ca²⁺ influx, the first experimental condition was repeated after depletion of intracellular Ca²⁺. Depletion was obtained by repeated exposure of the trachea to acetycholine maximally active concentration (10^-3M), in a Ca²⁺-free solution containing 2mM EDTA , until there was no response to acetylcholine.

To study the component of the Tapidod-induced relaxation due to the inhibition of internal Ca²⁺ release, an intracellular Ca²⁺ mobilizing agent (acetylcholine) was used on rat trachea in Ca²⁺-free solution. Acetylcholine was added cumulatively to the bath. A washout period of 45 min was allowed between each experimental protocol.

The bronchodilatory efficacy of the extract was expressed as E_max=maximal bronchodilatory response, evaluated as % relaxation of the KCl-induced contraction. The potency of the plant extract was expressed as pEC₅₀ = -log EC₅₀ (Jenkinson et al. 1995), calculated as concentrations (g of extract per ml of organ bath) required to produce 50% of E_max (Computer program: GRAPH PAD PRISM^® 2.01).

The Krebs-Heinsleit solution had the following composition (millimolar): NaCl, l18; KCl, 4.75; CaCl₂, 2.5; KH₂PO₄, 1.2; MgSO₄ 7H₂O, 1.2; NaHCO₃, 25 and Glucose, 5.55. Acetylcholine chloride and verapamil were obtained from Sigma. Tetraetylammonium chloride was provided by Fluka. Ethylene Diamine Tetraacetic Acid (EDTA), BaCl₂, CaCl₂ and KCl were purchased from Prolabo. All the drugs were prepared as concentrated stock solution and diluted to the final concentration in Krebs-Heinseleit solution. In preparing the stock solution, all the drugs were dissolved in distilled water. Before each experiment the lyophilized extract was dissolved and diluted in distilled water.

Statistical analysis

Results are expressed as means±SEM of number (n) of experiments. The significance of differences was evaluated by means of ANOVA and student' s t test for unpaired data. P values lower than 0.05 were considered to be significant.

Tapidod effects on the concentration-response curve of BaCl₂

BaCl₂ (10^-4M to 10^-1 M) produced a concentration dependent contraction on rat trachea (pEC₅₀=2.03±0.06). Tapidod reduced significantly the contraction induced by BaCl₂ and caused a rightward shift of the concentration-response curve for BaCl₂. In tracheal rings the maximal contractile response to BaCl₂ in the presence of Tapidod (1x10^-3g/ml) was 59.326±2.54 % of the maximal response observed in its absence (Figure 1).

Effects of Tapidod on the KCl-induced contraction

TEA (3x10^-3 M) potentiated KCl-induced contraction. Tracheal rings, pre-treated by TEA, maximal response to 3x10^-2 M KCl did not differ significantly from 4x10^-2 M KCl-induced contraction without TEA. This equivalence in developed tension has been determined by comparing KCl-induced contraction concentration-response curves in the absence or in the presence of TEA (3x10 M). Tapidod elicited a bronchodilatory effect on rat trachea pre-contracted by KCl (pEC=3.02^-3₅₀±0.13, E=106_max.2±15.22%, in absence of TEA; pEC=2.97₅₀±0.075, E=102.9_max±5.33% in presence of TEA)(Figure 2). In tracheal rings precontracted by KCl 8x10^-2M, the extract produced a concentration-dependant bronchorelaxant response (pEC=2.61₅₀±0.07 and E=107.8_max±7.02%).

Tapidod effects on the concentration-response curve of CaCl₂ in k⁺ rich Ca²⁺-free solution

In trachea bathed by K⁺-rich Ca²⁺-free physiological salt solution, CaCl₂ (10^-5M to 10^-2 M) evoked concentration-dependent spasm (pEC₅₀=3.68±0.06). When the effect of Tapidod (5x10^-4, 2x10^-3, 5x10^-3 g/ml) was evaluated on CaCl₂ cumulative concentration-effect curve a dose-dependant depression of the curve was detected in the high K⁺ Ca²⁺-free solution (pEC₅₀=2.86±0.07 and E_max=103.3±5.09%; pEC₅₀=2.13±0.06 and E_max=38.67±2.11%; pEC₅₀=1.38±0.16 and E_max=33.11±5.14%, respectively) (Fig. 3). Calcium depletion did not influenced Tapidod (2x10^-3g/ml) antispasmodic effect (pEC₅₀=3.01±0.06, E_max=39.96±2.34%) (Figure 4).

Tapidod effects on the concentration-response curve of acetylcholine in Ca²⁺ free solution.

Calcium removal from the bath shifted to the right the concentration-response curve for acetylcholine but did not influenced E_max. Tapidod (1x10^-3g/ml) inhibited the effect of acetylcholine in Ca²⁺-free solution. (pEC₅₀=5.041±0.04 in the absence of Tapidod; pEC₅₀=4.55±0.05 and E_max= 69.40± 2.12% in the presence of Tapidod; Figure 5).

Effect of Verapamil and Tapidod on acetylcholine dose-response curve

The contraction induced by acetylcholine was markedly inhibited by the pre-treatment with Tapidod (pEC₅₀=5.041±0.04 in the absence of Tapidod; pEC₅₀=5.43±0.12 and E_max=71.68±2.76% in presence of Tapidod (1x10^-3 g/ml)) (Figure 6). However, in the presence of verapamil (1x10^-6M), acetylcholine concentration-effect curve was not significantly influenced (pEC₅₀=4.75±0.06 and E_max=106.02±2.33%).

Discussion

The presence of Voltage-dependant K⁺ Channels, Ca²⁺-dependant K⁺ channels and ATP-dependant K⁺ channels in tracheal smooth muscle has been reported by Kotlikoff et al. (1990); Marthan et al. (1989) and Black et al. (1990). Barium, a K⁺ channels blocker (Thirstrup et al., 1997), induced concentration dependant contractile activity inhibited by Tapidod. The blocking of potassium conductance increases the transmembrane Ca²⁺ influx as a result of the cell membrane depolarization (Corrompt et al., 1998), eliciting a contractile response. It has been suggested that the activation of K⁺ channels reduces the voltage-dependant Ca²⁺ influx and [Ca²⁺]i through tonic hyperpolarisation of smooth muscle cells (Kannan and Johnshon, 1995). As Tapidod inhibits the tracheal smooth muscle response to the blocking of K⁺ conductance , we suggest that this extract may act by activating the K⁺ influx or by modulating the Ca²⁺ influx. Moreover, one way to study a possible K⁺ channel opener is to increase the K_e to 40 mM level (Vaali, 1999). Tapidod relaxed 80 mM K⁺-induced contraction indicating that its bronchorelaxing action is not mediated by a K⁺ channels activation.

Tapidod prevented the contraction induced by Ca²⁺on rat tracheal muscle bathed by K⁺-rich Ca²⁺-free physiological salt solution. Intracellular calcium depletion did not influence Tapidod effect. These inhibitory effects indicate a blocking of the Ca²⁺-influx. L-type Ca²⁺ channel blocker (Verapamil, 1x10^-6 M) did not reverse acetylcholine-induced contraction. This result is similar to those mentioned by Foster et al. (1984) and Advenier et al. (1984). However, T. dodoneifolius antagonized acetylcholine indicating that the pharmacodynamic profile of Tapidod differs from that of the L-type Ca²⁺ channels blockers.

In Ca²⁺-free solution Tapidod produced a depression of the contractile response to acetylcholine . Eglen et al. (1994) and Marthan et al. (1987) reported that acetylcholine elicits contractile activity by using calcium from internal stores. One characteristic in smooth muscle is that pharmacomechanical coupling can be seen even when extracellular Ca²⁺ concentration is zero and must therefore depend upon intracellular stores. The depression of the contractile response to acetylcholine in Ca²⁺-free Krebs solution suggests an action on the intracellular mobilization of Ca²⁺ ions.

The two most important factors affecting smooth muscle contraction are [Ca²⁺]_i and the contractile machinery's sensitivity to calcium (Vaali, 1999). As the precise mechanism of action of Tapidod remains to be elucidated, the present results suggest that its inhibitory action may involve its binding to intracellular Ca²⁺-regulating proteins (i.e. calmodulin) thereby preventing Ca²⁺ from binding to these proteins. Similar mechanisms have been reported by Hazekamp et al. (2001) to explain the bronchodilatory effect of Clerodendrum petasites (Verbenaceae) on guinea-pig trachea.

Conclusion

It is beyond the scope of this paper to speculate further on the structure-activity relationship. Tapidod is a crude extract that would contain several constituents able to present additional effects. The purpose of this paper was to demonstrate the Ca²⁺-blocking activity of Tapidod, which relaxes KCl-induced contraction on rat trachea and inhibits agonists-induced contraction. It was concluded that Tapidod effect may involves an intracellular calcium sequestration or/and a calcium-regulating proteins inhibition.

Acknowledgements

This work was supported by grants from IFS.

1. Advenier, C., Cerrina, J., Duroux, P., Floch, A. and Renier, A. (1984). Effects of five different organic calcium antagonists on guinea pig isolated trachea. Br. J. Pharmacol. 82 : 727-733
2. Black, J. .L., Armour, C. L., Johnson, P. R. A., Alouan, L. A. and Barnes P.J. (1990). The action of a potassium channel activator BRL 38227 (Iemakalim) on human airway smooth muscle. Am. Rev. Respiratory Disease. 142: 1384-1389.
3. Boussim, I. .J. (2002). Les phanérogames parasites du Burkina Faso : Inventaire, taxonomie, écologie et quelques aspects de leur biologie. Cas particulier des loranthacées parasites du karité. Thèse de doctorat ès-sciences. Univ. Ouaga. p. 62
4. Brunelleschi, S., Haye-legrand, I., Carlos, L., Norel , X., Benveniste, J. and Brinx, C. (1987). Platelet-Activating Factor-acether-induced relaxation of guinea pig airway muscle: Role of prostaglandin E₂ and the epithelium. J. Pharmacol. Exp. Ther. 243: 356-362
5. Cepleanu, F., Hamburger, M. O., Sordat, B., Msonthi, J. D., Gupta, M. P., Saadou, M. and Hostettmann, K. (1994). Screening of tropical medicinal plants for molluscicidal, larvicidal, fungicidal and cytotoxic activities and brine shrimp toxicity. Int. J. Pharmacol. 323 : 294-307.
6. Corrompt, E., Bessard, G., Lantuejai, S., Naline E., Advenier, C. and Devillier P. (1998). Inhibitory effects of large Ca²⁺-activated K⁺ channel blockers on beta-adrenergic and NO- donor-mediated relaxations of human and guinea pig airway smooth muscles. Naunyn Schmiedebergs Ach. Pharmacol. 357: 77-86.
7. Eglen, R. M., Reddy, H., Watson, N., and Challiss, R. A. J. (1999). Muscarinic acetylcholine receptor subtypes in smooth muscle. Trends Pharmacol. Sci. 15 : 114-119.
8. Emine, S. Z., Sevim, E. and Kazlm, R. T. (1998). Nitric-oxide mediated relaxation induced by bradykinin in the isolated mouse trachea. Eur. J. Pharmacol. 352 (2-3): 263-268.
9. Foster, R. W., Okpalugo, B. I. and Small, R. C. (1984). Antagonism of Ca²⁺ and other actions of verapamil in guinea-pig isolated trachelis. Br. J. Pharmacol. 81: 499-507.
10. Guan, Y. Y., Kwan, C. Y. and Daniel, E. E. (1984). The effects of EGTA on vascular smooth muscle contractility in calcium-free medium. Can. J. Physiol. Pharmacol. 66: 1053-1056.
11. Hazekamp, A., Verpoorte, R. and Panthong A. (2001). Isolation of a bronchodilator flavonoid from the Thai medicinal plant Clerodendrum petasites. J. Ethnopharmacol. 78: 45–49.
12. Holgate, S. T. (1997). The cellular and mediator basis of asthma in relation to natural history. Lancet. 350: (Suppl. 2),S115-S119.
13. Imaizumi, Y. and Watanabe, M. (1981). The effect of tetraethylammonium chloride on potassium permeability in the smooth muscle cell membrane of canine trachea J. Physiol. 316: 33-46.
14. Jenkinson, D. H., Barnard, E. A., Hoyer, D., Humphrey, P. .P. A., Leff, P., and Shankley, N. P. (1995). International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. IX. Recommendations on Terms and Symbols in Quantitative Pharmacology. Pharmacol. Rev. 47 (2): 255-266.
15. Kannan, M. S. and Johnson, D. E. (1995). Modulation of nitric-oxide dependent relaxation of pig tracheal smooth muscle by inhibitors of guanylyl cyclase and calcium activated potassium channels. Life Sci. 56: 2229-2238.
16. Karaki, H. and Weiss, G. B., (1988). Minireview: Calcium release in smooth muscle. Life Sc. 42: 111-122.
17. Kotlikoff, M. I. (1990). Potassium currents in canine airway smooth muscle cells. Am. J. Physiol. 259: L984-L395.
18. Lagaud, G. J. L., Stoclet, J. C. and Andriantsitohaina, R. (1996). Calcium handling and purinoceptor subtypes involved in ATP-induced contraction in rat small mesenteric arteries. J. Physiol. 492.3: 689-703.
19. Marthan, R., Savineau, J. P. and Mironneau, J. (1987). Acetylcholine induced contraction on human isolated bronchial smooth muscle: Role of an intracellular store. Resp. physiol. 67: 127-137.
20. Marthan, R., Martin, C., Amedee, T. and Mironneau J. (1989). Calcium channel currents in isolated smooth muscle cells from human bronchus. J. Appl. Physiol. 66: 1706-17l4.
21. Nacoulma/Ouédraogo, O. G. (1996). Plantes médicinales et pratiques médicinales traditionnelles au Burkina Faso : cas du plateau central. Thèse de doctorat ès-sciences. Université de Ouagadougou, tomes II : 508-515.
22. Ouédraogo, S., Traore, A., Some, N., Lompo, M., Guissou, I. P., Schott, C., Bucher, B. And Andriantsithohaina, R. (2005). Cardiovascular properites of Tapinanthus dodoneifolius (DC Danser). Afr. J. Trad. CAM 2(1): 25-30.
23. Ouédraogo, M., Ouédraogo, S., Traore, A., Lompo, M., Some, N., Sawadogo, L.L. et Guissou, I. P. (2005),. Propriétés bronchodilatatrices de l'extrait aqueux total de Tapinanthus dodoneifolius (DC Danser) sur la trachée isolée de rat. Etnopharmacologia 35: In press.
24. Sly, R. M. and O'Donnell, R. (1997). Stabilization of asthma mortality. Ann. Allergy Asthma Immunol. 78: 347-54.
25. Thirstrup, S., Nielsen-KudsK, J. E., Mellemkjaer, S. (1997). Involvement of K channels in the relaxant effect of vasoactive intestinal peptide and atrial natriuretic peptide in isolated guinea-pig trachea. ⁺Eur. J. Pharmacol. 319: 253-259.
26. Traoré, R. (2000). Etude pharmacologique chez l'animal de l'extrait aqueux deTapinanthus dodoneifolius DC. danser (Loranthaceae) utilisée en tradithérapie anti-asthmatique au Burkina Faso. Thèse de pharmacie. FSS. Université de Ouagadougou, Burkina Faso. 94p.
27. Tschirhart, E., Frossard, N., Bertrand, C. and Landry, Y. (1986). Arachidonic acid metabolites and Airway Epithelium-Dependant Relaxant Factor. J. Pharmacol. Exp. Ther. 243 : 310-316
28. Vaali, K.( 1999). Relaxation mechanisms of Nitric Oxide donors, β₂-adrenoceptor agonist and their synergistic effect in vitro in airway smooth muscle: Role of potassium channels. Academic Dissertation; Institute of Biomedicine. University of Helsinki. 17-19
29. Van Rossum J. M. (1963). Cumulative dose-response curves. II. Technique for the making of dose-response curves in isolated organs and the evaluations of drug parameters. Arch. Int. Pharmacodyn. Ther. 143: 299-330
30. Wenzel, S.E. (1998). Antileukotriene drugs in the management of asthma. J. Am. Med. Ass. 280: 2068-2069.

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