African Journal of Biotechnology Vol. 10(1), pp. 54-64, 3 January, 2011 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2011 Academic Journals Full Length Research Paper

Antispasmodic and bronchodilator activities of Taxodium mucronatum Ten leaf extract

Alma Rosa Cortés-Arroyo1*, Adriana M. Domínguez-Ramírez1, Martín Gómez-Hernández1, José R. Medina López1, Marcela Hurtado y de la Peña1 and Francisco J. López-Muñoz2.

1Departamento Sistemas Biológicos, Universidad Autónoma Metropolitana-Xochimilco, Calzada del Hueso 1100,

Colonia Villa Quietud. C.P. 04960, México, D.F., México. 2Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados-Unidad Sur, Calzada Tenorios

235, Colonia Granjas Coapa. C.P. 14330, México, D.F., México.

Accepted 2 November, 2010

The relaxant effects of the Taxodium mucronatum Ten leaf hexane extract on intestinal and tracheal smooth muscle were evaluated in vitro by testing spontaneous contractions of rabbit jejunum and agonist-induced contractions of guinea pig ileum and rat trachea. The extract produced a concentration-dependent relaxation of the spontaneous contractions of rabbit jejunum (EC50: 4.9 ±±±± 0.5 µg/ml) that was equipotent to papaverine. Following incubation of guinea pig ileum with the extract, the concentration-response curves to acetylcholine, histamine, 5-hydroxytryptamine and Ca2+ were displaced to the right and the maximum response was significantly reduced. K+-induced contraction of the ileal tissue was completely abolished with 31.6 µg/ml of the extract. In rat tracheal rings, the extract inhibited both 1 µM carbachol (EC50: 33.9 ±±±± 2.5 µg/ml) and 60 mM K+ (EC50: 20.6 ±±±± 1.1 µg/ml)-induced contractions. It also caused the concentration-response to Ca2+ curves to shift to the right in a non-competitive manner. These results demonstrate a non-specific relaxant effect that could be mediated through inhibition of calcium influx via both voltage- and receptor-gated calcium channels. The relaxant activity induced by this extract provides a rational basis for the traditional use of T. mucronatum to treat disorders of the gastrointestinal and respiratory tracts. Key words: Taxodium mucronatum, Cupresaceae, muscular relaxant, antispasmodic, bronchodilator.

INTRODUCTION Antispasmodics are muscular relaxants that are used to relieve cramps or spasms of the stomach, intestines and bladder. They are commonly used for the treatment of different gastrointestinal disorders, including diarrhea and irritable bowel syndrome, which affect millions of people. For example, the Mexican National Health Survey 2000 documented the occurrence of 131 million episodes of diarrhea per year (corresponding to 1.35 events per person per year) (Flores, 2006). Bronchial antispasmodics are also muscular relaxants that are used to treat asthma, bronchitis and many lung diseases. They improve breathing by relaxing or dilating tissues in the bronchial *Corresponding author: E-mail: cortesar@correo.xoc.uam.mx. Tel: (5255)54837213. Fax: (5255)54837237.

tubes. Both the prevalence of and the mortality asso-ciated with asthma appear to have increased in many parts of the world; this chronic respiratory disease affects 300 million people globally (Dougherty and Fahy, 2009).

The Mexican cypress Taxodium mucronatum ten, from the family Cupresaceae, inhabits riparian wetlands from northern Mexico to Guatemala. This species is the national tree of Mexico, and in addition to its historical and cultural importance, it also has great ecological value (Suzán-Azpiri et al., 2007). These trees reach a conside-rable size and age and they are the largest and oldest species of tree in Mexico (Contreras-Medina and Luna-Vega, 2007).

T. mucronatum, popularly known as Montezuma bald cypress, Mexican cypress, sabino or “ahuehuete" (in the Nahuatl language), is widely used in folk medicine.

Several parts of the tree were used by the Aztecs; in

particular, the gummy resin that can be obtained from a cut tree or its burned wood was used as a medicine. Pieces of the burned bark were placed directly on sores, burns and ulcerations of the skin. In addition, chest afflictions could be cured by inhaling the smoke from burning wood and branches. Currently, the bark, branches, leaves and fruits of this tree are used to create infusions or ointments for the treatment of wounds, gout, cardiac diseases, hemorrhoids, ulcers and varices, to relieve rheumatic pain, or as antispasmodics for the treatment of diarrhea and bronchial problems (Márquez et al., 1999, González et al., 2004). Martínez (2005) reported that resinous parts of the leaves and fruits can cure herpes and leg tumors and reduce inflammation and rapidly resolve articular diseases. Infusions of the leaves have also been reported as hypoglycemic (Andrade-Cetto and Heinrich, 2005).

The only published pharmacological study on this species detailed a vasorelaxant effect of the aqueous extract from the aerial part of the tree (Perusquía et al., 1995). Although the effect was studied on isolated rat thoracic aorta, this previous study did not attempt to elucidate the mechanism of action. Phytochemical studies of T. mucronatum have revealed the presence of multiple constituents, including the following biflavones: amen-toflavone (AMF1), 4���-methylamentoflavone (podocar-pusflavone A, AMF2) and 7��,4���-dimethylamentoflavone (podocarpusflavone B, AMF3). These were isolated from acetone extracts of leaves and branches and subsequently identified (Ishratullah et al., 1978; Zhang et al., 2005). In addition, the diterpenoid 8�-hydroxypimar-15-en-19-oic acid and some quercetin glycosides have been isolated from its leaves and fruits (Ramos et al., 1984; Khabir et al., 1986).

Specifically, the aim of this study was to test the anti-spasmodic and bronchodilator actions of the hexane extract of Taxodium mucronatum leaves on isolated preparations (rabbit jejunum, guinea pig ileum and rat trachea) and to determine the mechanisms of action. MATERIALS AND METHODS Plant material and extraction T. mucronatum was collected from the Autonomous Metropolitan University Campus Xochimilco gardens, Mexico City, in September 2007. It was identified by Angélica Ramírez Roa MSc from the National Herbarium, Institute of Biology, National Autonomous University of Mexico, where a voucher specimen was deposited (1214077). The plant was washed with water and air-dried. Leaves were separated and milled to a coarse powder. To extract the resinous components, the powdered leaves (500 g) were then extracted twice for 4 h under reflux with hexane. The extract was filtered and dried on a rotary evaporator. A dark, yellow residue (34.6 g; yield: 6.9%) was obtained. This residue was washed with methanol (3 × 600 ml). The insoluble material was removed by brief centrifugation and subsequently discarded due to its ineffectiveness as a muscular relaxant. The methanolic solution was again evaporated, and the resulting residue was used as a crude extract

Cortés-Arroyo et al. 55 (25 g; yield: 6%). Dried extract was stored at −20°C until needed. T. mucronatum extract and polyvinylpyrrolidone-30K, used as a co-solvent (1:4 W/W), were weighed and dissolved separately in the minimum volume of methanol necessary. They were then quantita-tively mixed and dried under vacuum conditions. The residue was dissolved in distilled water prior to being used in the in vitro experiments. Polyvinylpyrrolidone did not affect the contractile responses of any of the isolated preparations at their final bath concentrations. Chemicals and drugs All chemicals used were reagent grade from Merck, Mexico. Acetylcholine, histamine, 5-hydroxytryptamine, carbachol, papaverine, verapamil, phentolamine, propranolol, N(G)-nitro-L-arginine methyl ester (L-NAME), bradykinin, 4-aminopyridine, glibenclamide and tetraethylammonium chloride were obtained from Sigma Chemical Company, USA. Acetylcholine, histamine and carbachol were dissolved in physiological solution, adjusted to pH 4, with ascorbic acid and kept at 4°C until use. All other drugs were prepared in suitable physiological saline solutions, except glibenclamide and papaverine, which were dissolved in dimethyl sulfoxide. All solutions were prepared on the day of the experiment. Dimethyl sulfoxide, at the concentrations used in this study, did not produce any detectable effect on the contractile responses. Animals Male New Zealand white rabbits (2 to 2.5 kg), Dunkin Hartley guinea pigs of both sexes (400 to 600 g) and male Wistar rats (200 to 250 g) were obtained from Laboratory Animal Production Unit of Autonomous Metropolitan University-Xochimilco. They were housed under controlled environmental conditions at 23 to 25°C with 12/12 h light/dark cycles. The animals were given a standard diet and had free access to water. Food was withdrawn 12 h prior to each experiment with intestine preparations. Rabbits were sacrificed by a blow to the nape of the neck followed by exsanguination, and guinea pigs and rats were euthanized with CO2. All animals were handled in accordance with Mexican federal regulations for the care and use of laboratory animals (NOM-062-ZOO-1999). The Institutional Animal Care and Use Committee approved all of the experimental protocols. The number of experimental animals was kept to a minimum. Isolated tissue preparations Rabbit jejunum and guinea pig ileum Several segments (approximately 1 cm in length) of rabbit jejunum and guinea pig ileum (avoiding the 10 cm closest to the caecum) were rapidly removed. The tissue was cleaned and tied with fine thread at both ends, and then mounted longitudinally in a 10 ml organ chamber. It was allowed to equilibrate for 30 to 60 min in Tyrode solution (with regular washed every 15 min) at 37 ± 0.5°C with continuous aeration provided by a gas mixture of 95% O2 to 5% CO2. Initial tensions of 0.5 g and 1 g were applied to the jejunum and ileum, respectively. The composition of the Tyrode solution was (mM): NaCl (136.9), KCl (2.68), CaCl2 (1.8), MgSO4 (1.05), NaHCO3 (11.9), NaH2PO4 (0.42) and glucose (5.5); pH 7.4 (Aoki et al., 2008). The experiments were performed on three or four segments of tissues taken from each of six animals. The force of contraction was recorded simultaneously on an eight-channel Narco-Biosystem physiograph, equipped with isometric force-displacement transducers (Narco F-60).

56 Afr. J. Biotechnol. Rat trachea The trachea was dissected out and the adherent tissue was removed. Each trachea was then cut transversely into four approximately equal segments, each containing two or three rings of cartilage. Special care was taken to preserve the endothelium. The tracheal rings were suspended between stainless steel hooks, in 10 ml organ baths that contained Krebs solution of the following composition (mM): NaCl (118), KCl (4.7), CaCl2 (2.5), MgSO4 (1.2), NaHCO3 (25.0), KH2PO4 (1.2) and glucose (11.1); pH 7.4. The rings were maintained at 37 ± 0.5°C with 95% O2 to 5% CO2 (Gilani et al., 2005). A tension of 1 g was applied to the tracheal preparations, which were then allowed to equilibrate for 60 min before initiation of the study. The bath solution was changed each 15 min. The experiments in calcium-free conditions were performed using Krebs solution in which CaCl2 was omitted and 0.05 mM EGTA and K+ (60 mM) were added. KCl was replaced by an equimolar concentration of NaCl. In some experiments the epithelial cells were removed mechanically by rubbing the internal surface of the trachea with a cotton-stem. The absence of functional epithelium was confirmed by the inability of bradykinin (10 µM) to induce relaxation (Bolton et al., 1984). Only one experimental protocol was performed on each tracheal ring. The force of contraction was measured with isometric force-displacement transducers (Narco F60) and registered on a physiograph. Experimental protocols Effect on spontaneous contractility in rabbit jejunum After the equilibration period, spontaneous contractions of rabbit jejunum were recorded for 10 min. A concentration-response curve for the relaxant effect of the extract on spontaneous activity was generated by adding the extract (0.6 to 31.6 µg/ml) cumulatively to the organ bath. Each concentration was left in contact with the tissue for 15 min before the next concentration was added. Papaverine (0.04 to 37.6 mg/ml) and verapamil (1.6 to 491.1 ng/ml) were used as standard relaxant controls. The effects of phentolamine (2 µM) and propranolol (10 µM) on the relaxant action of the extract were determined (Cortés et al., 2006); each concentration of the antagonist was added to the bath 30 min before the concentration-response curves of the extract were determined. The response was evaluated as a percentage of the basal spontaneous contractions. Effect on guinea pig ileum Due to its behavior as a quiescent smooth muscle preparation under these experimental conditions, guinea pig ileum was used in another set of experiments. This model is considered to be more useful than rabbit jejunum for the evaluation of spasmogenic activity in the presence of an agonist (Gilani et al., 2005). The antispas-modic effect of the T. mucronatum extract (3.2, 10.0 and 31.6 �g/ml) was tested on the contractions produced by different exogenous spasmogens. After an initial equilibration period of about 30 min, non-cumulative concentration response curves were determined for acetylcholine (1 × 10− 9 to 3.2 × 10− 5 M), histamine (1 × 10− 9 to 3.2 × 10− 5 M) or 5-hydroxytryptamine (2.2 × 10− 8 to 1 × 10− 5 M). The drug contact time with the tissue was approximately 30 to 60 s and the addition of each drug occurred 4 min after the two wash-out periods (Cortés et al., 2004). After 60 min of equilibration, the concentration-response curve was repeated in the presence of either 3.2 or 10 µg/ml of extract. The maximum response obtained from the first curve was taken as the 100% response value. Each preparation was exposed to only one agonist and one extract concentration.

Spasmolytic effect on K+-depolarized guinea pig ileum The spasmolytic effect was studied using K+-depolarized guinea pig ileum. A concentration-response curve was obtained by cumulative addition of T. mucronatum extract (0.6 to 31.6 µg/ml) at 15 to 20 min intervals after the normal Tyrode solution had been replaced with high K+ Tyrode solution (60 mM). The high K+ solution was prepared by equimolar replacement of NaCl with KCl in the physiological solution. Papaverine at a range of 0.07 to 3.8 µg/ml (0.2 to 10 µM) was used as a control. The magnitude of the tissue relaxation was expressed as a percentage of the high K+-induced contraction (Cortés et al., 2006). Effect on extracellular calcium in guinea pig ileum To assess whether the spasmolytic activity of T. mucronatum extract was caused by Ca2+ channel blockade, the guinea pig ileum was allowed to stabilize in normal Tyrode solution before being exposed to Ca2+ free Tyrode solution for 30 min, which removed calcium from the tissues. This solution was replaced with high K+ (60 mM) and Ca2+ free Tyrode solution to induce depolarization. Following the incubation period of 20 min and after the absence of spontaneous tissue contractions had been confirmed, control concentration-response curves to Ca2+ were obtained by adding Ca2+ in a cumulative fashion (3.2 × 10− 5 to 3.2 × 10− 2 M) every 3 min. Following 60 min of stabilization in normal Tyrode solution, the concentration-response curve to Ca2+ was repeated, using the same procedure, after 15 min incubations with 3.2, 10 or 31.6 µg/ml of the extract (Cortés et al., 2006) or papaverine (0.7, 1.2, 2.1 µg/ml). Only one concentration of the extract or papaverine was tested on each ileal segment. Bronchodilator effect on rat tracheal rings The relaxant effect of T. mucronatum extract on carbachol and K+ induced contraction of rat tracheal rings was determined. The tissues were stabilized for 60 min in Krebs solution, and then responses to carbachol (1 µM) were obtained every 30 min until the contraction amplitude became uniform (3 to 4 times). Once the contraction amplitude had plateaued (20 to 30 min), a concen-tration-response curve was constructed by adding cumulative concentrations of the extract (3.2 to 100.0 �g/ml) every 15 min (Ramanitrahasimbola et al., 2005). Because the contractile response was not sustained long enough to construct a full concentration-response curve (2 h approximately), a different protocol using K+ (60 mM) as the spasmogen was carried out. Non-cumulative concentration-response curves were obtained. The trachea rings were preincubated for 15 min with different concen-trations of the extract (3.2 to 100 �g/ml) and stimulated with K+. To test for recovery of the contractile response, a reproducible K+ contraction response was obtained before subsequent concen-trations were applied. The same protocols described above were used to determine the effects of papaverine (0.4 to 21.1 µg/ml) on carbachol and high K+ induced contraction. Some experiments were carried out using endothelium-free tracheal rings in the presence of L-NAME (10 µM). To evaluate the involvement of K+ channels in the relaxant effect on carbachol-induced contraction, some rings were preincubated for 20 min with tetraethylammonium (10 mM), 4-aminopyridine (10 mM) or glibenclamide (10 µM) before addition of carbachol and performance of the extract concentration-response curve. Effect of extracellular calcium on rat trachea Tracheal rings were allowed to stabilize in normal Krebs solution

Cortés-Arroyo et al. 57

Figure 1. Concentration-response curves of T. mucronatum leaf extract (T muc.), papaverine and verapamil on spontaneous contraction of isolated rabbit jejunum. EC50 values for extract, papaverine and verapamil, were 4.9 ± 0.5 µg/ml, 5.3 ± 0.2 µg/ml and 35.9 ± 4.7 ng/ml, respectively. Data are mean ± SEM, n = 6.

and then contracted with carbachol (1 µM), 3 to 4 times each, for a period of approximately 30 min until the maximum contractile response was obtained. The tissues were washed and bathed in Ca2+ free Krebs solution (60 mM KCl plus 0.05 mM EGTA) for 20 min. To obtain control concentration-response curves for Ca2+, incrementally increasing concentrations of CaCl2 were added (3.2 × 10− 5 to 1 × 10− 2 M) every 3 min (Ramanitrahasimbola et al., 2005). The preparations were washed and after an additional 60 min interval, the procedure was repeated using rings preincubated for 20 min with T. mucronatum extract (3.2, 5.6, 10.0 or 17.8 µg/ml). Only one concentration of the extract was tested on each tracheal ring. Statistical analysis The results of experiments obtained from at least four different animals are presented as mean ± SEM. The EC50 values for two experimental conditions were compared using a two-tail non-paired Student’s t-test. A Welch-adjusted one-way ANOVA followed by the Dunnett T3 test for multiple comparisons was used to compare the EC50 values of the extract alone and more than two different agonists or possible antagonists. Probability values < 0.05 were considered statistically significant. Analysis was performed using SPSS for Windows, version 13.0 (SPSS Inc., Chicago, IL, USA). The concentration required to produce 50% of the maximum effect (EC50) was calculated by applying a nonlinear curve-fitting procedure to individual curves using Sigma Plot version 9.0 (Systat Software, Inc., Richmond, CA, USA). Data were fitted to a three-parameter logistic function: E = Emax / (1+ (EC50 / [A])n). E is the response observed, Emax is the maximal response, [A] is the concentration of the agonist, EC50 is the [A] eliciting one-half of

the maximal response and n is the slope of the curve. RESULTS Effect on spontaneous contractility of rabbit jejunum The extract of T. mucronatum demonstrated antispas-modic activity by inhibiting the spontaneous contractions of isolated rabbit jejunum; the resting tension of the tissue, but not the contraction frequency, was slightly altered. The maximum effect of each concentration was reached after 12 to 15 min. The relaxant effect on sponta-neous contraction was concentration-dependent across a range of 0.58 to 31.6 µg/ml (EC50: 4.9 ± 0.5 µg/ml). On the basis of half maximal effective concen-tration (EC50) values calculated as micrograms per milliliter, the extract was equipotent to papaverine (EC50: 5.3 ± 0.2 µg/ml; P > 0.05, Dunnett T3 test), and it was less potent than verapamil (EC50: 35.9 ± 4.7 ng/ml; P < 0.05), which were both used as standard relaxant drugs (Figure 1). A concentration of 31.6 µg/ml of the extract completely abolished spontaneous activity, and full functional reco-very was obtained after 60 min of rest and washing with fresh Tyrode solution at 15 min intervals. The relaxant effect produced by the extract was not altered in the presence of 2 µM phentolamine (EC50: 3.4 ± 0.4 µg/ml) or 10 µM propranolol (EC50: 6.1 ± 1.2 µg/ml). There were no significant differences among EC50 values of the extract in the absence and presence of phenols-amine or 10 µM

58 Afr. J. Biotechnol. propranolol (Welch-ANOVA, P > 0.05). Effect on guinea pig ileum Acetylcholine, histamine, and 5-hydroxytryptamine caused rapid contraction of isolated guinea pig ileum that reached a peak within 30 sec of contact. The T. mucro-natum extract reduced the tissue response to these agonist compounds in a concentration-dependent manner (Figure 2A, B and C). The antispasmodic effect in preparations that were preincubated with the extract was more pronounced in those that were subsequently contracted by 5-hydroxytryptamine than in those that were stimulated with either acetylcholine or histamine. The maximum contraction observed in the presence of 10 µg/ml of the extract was 34.2 ± 8.1% of the maximum produced with acetylcholine alone and 34.4 ± 5.4% of the maximum effect produced with histamine alone. However, the contractile response to 5-hydroxytryptamine was almost abolished (4.2 ± 2.2% of baseline response) in the presence of 10 µg/ml of extract. Spasmolytic effect on K+ depolarized guinea pig ileum A high K+ concentration is known to cause smooth muscle contractions by opening voltage-dependent Ca2+ channels, with the ensuing influx of extracellular Ca2+ causing a contractile effect (Bolton, 1979). To study the effect of the extract on depolarized tissues, high K+ was used as a spasmogen on guinea pig ileum. T. mucro-natum extract diminished the sustained contraction of guinea pig ileum that was precontracted with high K+ (60 mM). This spasmolytic effect was concentration-depen-dent in the range of 0.6 to 31.6 µg/ml (EC50: 4.8 ± 0.5 µg/ml; Figure 3). The extract was significantly less potent than papaverine (P < 0.001, unpaired t test) at reducing high K+-induced muscle contractions (papaverine EC50: 0.9 ± 0.1 µg/ml). During the course of the experiments, there were no significant changes in the K+ response of preparations treated with an equivalent volume of the vehicle. Effect on extracellular calcium in guinea pig ileum A contractile response was obtained by the addition of Ca2+ (1 × 10 −4 to 3 × 10 −2 M) to a previously decalcified guinea pig ileum. Under the same conditions, T. mucronatum extract induced a shift to the right and a depression of the maximal responses of the concentration-response curves elicited by Ca2+ (Figure 4A). This inhibition profile was similar to that produced by papaverine (Figure 4B).

Bronchodilator effect on rat trachea The effects of the T. mucronatum leaf extract (3.2 to 100 �g/ml) on contractions induced in isolated rat trachea by carbachol (1 �M) and high K+ (60 mM) were tested. T. mucronatum extract produced a concentration-dependent bronchodilator effect (Figure 5A); it inhibited the contrac-tions evoked by both carbachol and K+ with comparable efficacy. Its inhibitory action on muscle contractions induced by high K+ (EC50: 20.6 ± 1.1 µg/ml) was slightly but significantly higher (1.6 fold) than on contractions induced by carbachol (EC50: 33.9 ± 2.5 µg/ml; P < 0.001). Full functional recovery was obtained after a 90 to 120 min rest period that included washing with fresh Krebs solution every 15 min. Papaverine (Figure 5B) inhibited the K+- (EC50: 2.8 ± 0.2 µg/ml) and carbachol-evoked (EC50: 3.5 ± 0.3 µg/ml) contractions with comparable potency and efficacy (P > 0.05). When EC50 values were considered in micrograms per milliliter, T. mucronatum extract was less potent (6 to 7 fold) than papaverine at reducing contractions of both spasmogens under these experimental conditions.

Neither mechanical removal of epithelium (EC50: 37.9 ± 2.5 µg/ml) nor preincubation with the classical NO-synthase inhibitor L-NAME (10 µM) (EC50: 33.07 ± 2.9 µg/ml) significantly altered the relaxant effect of the extract on airway smooth muscle that was precontracted with carbachol (P > 0.05). Moreover, the tracheal relaxa-tion induced by T. mucronatum could not be inhibited by addition of any of the following K+ channel blockers: an ATP-sensitive K+ channel blocker (glibenclamide, 10 µM), a selective voltage-sensitive K+ channel blocker (4-aminopyridine, 10 mM) or a non-selective K+ channel blocker (tetraethylammonium, 10 mM) (data not shown). Effect on extracellular calcium in rat trachea To corroborate the effect of the extract on the extra-cellular mobilization of Ca2+ in the isolated rat trachea, the tissue was maintained in Ca2+ free solution and stimulated with Ca2+ in the presence of T. mucronatum extract (3.2, 5.6, 10.0 or 17.8 µg/ml). Pretreatment with the extract inhibited Ca2+ evoked muscle contraction in a concentration-dependent and non-competitive manner (Figure 6). DISCUSSION Taxodium mucronatum has traditionally been used to treat digestive and respiratory disorders such as diarrhea, colic, bronchitis and asthma. Several parts of the tree are used for these and other conditions such as cardiac and articular diseases. However, the underlying pharmaco-logical mechanisms of this plant remain unclear. In the present study, we demonstrate that the hexane extract of

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Figure 2. The effect of preincubation with T. mucronatum leaf extract (T muc.) at 3.2 and 10 µg/ml on the concentration-response curves of isolated guinea pig ileum to: A) acetylcholine, B) histamine and C) 5-hydroxytryptamine. Data are represented as the percentage of the maximum response of each agonist in the presence of the vehicle (Control). Mean ± SEM, n = 6.

60 Afr. J. Biotechnol.

Figure 3. Concentration-response curves of T. mucronatum leaf extract (T muc.) and papaverine on isolated guinea pig ileum depolarized with K+ (60 mM). EC50 values were 4.8 ± 0.5 µg/ml and 0.9 ± 0.1 µg/ml for extract and papaverine, respectively. Mean ± SEM, n = 6.

resinous leaves of T. mucronatum contains a compound or compounds that have a relaxant effect on intestinal and airway smooth muscle. These results provide a pharmacological basis for the medicinal use of T. mucronatum as an antispasmodic and a bronchodilator.

The extract of T. mucronatum leaves had a relaxant action on the spontaneous contractions of rabbit jejunum. This effect was not due to postsynaptic stimulation of adrenoreceptors since preincubation of the tissues with phentolamine (2 µM) and 10 µM propranolol (1 µM) had no effect on the response to the extract. The spasmolytic effect was similar to that of papaverine, a non-specific smooth muscle relaxant and significantly greater than that produced by verapamil (a calcium channel antagonist). This suggests the possibility of a mechanism of action related to receptors or Ca2+ channels.

The mechanism of action was further assessed using guinea pig ileum. The marked inhibition of acetylcholine-, histamine and 5-hydroxytryptamine-induced contractions by T. mucronatum extract suggests a non-specific anta-gonism of these spasmogens that may involve activation of receptor-gated cation channels and voltage-gated Ca2+ channels (Foster et al., 1983). Although the dependence of smooth muscle contraction on increased cytosolic Ca2+ is widely accepted, evidence exists for significant variations in the degree of participation of extracellular and intracellular Ca2+. The increased intracellular Ca2+ may result from either influx via voltage-dependent Ca2+ channels (VDCCs) or release from intracellular stores in the sarcoplasmic reticulum. Periodic depolarization and repolarization regulate the spontaneous movements of

the intestine, and at the height of depolarization, the action potentials appear as a rapid influx of Ca2+ via VDCCs (Carl et al., 1996). Although the best-characterized calcium entry pathway utilizes VDCCs, other types of non-voltage-gated Ca2+ permeable channels are present. These include the receptor-operated Ca2+ channels (ROCCs) that are activated by agonists of a variety of G-protein-coupled receptors, and store-operated calcium channels (SOCCs) that are activated by depletion of calcium stores within the sarcoplasmic reticulum (McFadzean and Gibson, 2002).

Increased extracellular K+ concentration in smooth muscle results in depolarization that accompanies the contraction, which depends on extracellular Ca2+ entering via VDCCs. Accordingly, a high K+ concentration is com-monly used as a non-selective stimulant, a depolarizer and a pharmacological tool to open voltage-dependent Ca2+ channels in smooth muscle preparations (Kamizaki et al., 1988). Therefore, substances that can inhibit high K+-induced contractions are considered to be Ca2+ channel blockers (Karaki and Weiss, 1988). Thus, the effect of T. mucronatum extract was tested on depola-rized guinea pig ileum; the extract completely abolished the contractile response to K+. However, the extract was less potent than papaverine, whose mechanism of relaxation is thought to involve either intracellular accumulation of cAMP and/or cGMP due to the inhibition of phosphodiesterase (PDE) and its effect on Ca2+ movement (Shimizu et al., 2000, Kaneda et al., 2005). To verify the action of plant extract on VDCCs, guinea pig ileum segments were treated with different concentration

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Figure 4. Cumulative concentration-response curves of Ca2+ in the absence (control) and the presence of A) extract of T. mucronatum leaves (T muc., n = 4) at 3.2, 10.0 and 31.6 µg/ml and B) papaverine (PAP, n = 6) at 0.7, 1.2 and 2.1 µg/ml, in isolated guinea pig ileum. Data are expressed as a percentage of the maximum control contraction in each experiment. Mean ± SEM.

of Ca2+ in calcium-free and high K+ medium. Under such conditions, T. mucronatum suppressed Ca2+ responses in a non-competitive manner that possibly reflected restricted Ca2+ influx through VDCCs. A similar pattern was observed using papaverine as a control. It is known that papaverine shifts the concentration-response curve for Ca2+ in this and other depolarized tissues (Takayanagi et al., 1977; Huddart and Saad, 1980). This relaxant activity provides a rational basis for the traditional use of T. mucronatum as an antispasmodic in gastrointestinal disorders.

Based on the traditional use of T. mucronatum as a muscle relaxant in respiratory diseases, the relaxant effect induced by the extract was tested on carbachol- or K+ induced contractions in rat trachea. Carbachol and KCl are considered to induce airway smooth muscle contractions via distinct mechanisms. In most smooth muscles, carbachol-induced contraction is generally resistant to agents that block voltage-dependent Ca2+ channels. This is thought to occur because contractions are produced by Ca2+ influx through a special receptor- operated pathway and/or by Ca2+ released from the

62 Afr. J. Biotechnol.

Figure 5. Relaxant effect of A) T. mucronatum leaf extract and B) papaverine on epithelium-intact rat tracheal rings precontracted with 1 µM carbachol (CCh) or 60 mM K+. Results are expressed as a percentage of the response induced by CCh or K+ before addition of the relaxant agents. Mean ± SEM, n = 6.

sarcoplasmic reticulum (Takemoto, et al., 1998; McFadzean and Gibson, 2002). In contrast, other agents such as high concentrations of K+ induce contraction by depolarizing the cell membrane in a graded manner that results in activation of VDCCs (Nielsen-Kudsk, et al., 1986; Karaki et al, 1997).

T. mucronatum extract caused a complete relaxation of both carbachol- and K+ induced airway smooth muscle contraction. Although it was slightly but significantly more potent at relaxing trachea rings that were precontracted with K+ than those that were pretreated with carbachol (1.6 fold), this finding cannot be used to argue in favor of

preferential involvement of specific mechanisms. Further support for the extract’s inhibitory action on the influx of Ca2+ (similar to ileum guinea pig) was confirmed. Prein-cubation with the extract shifted the Ca2+ concentration-response curves to the right in a non-competitive manner, similar to that seen with papaverine. Kaneda et al. (2005) demonstrated that the relaxant mechanism of papaverine on carbachol-induced contraction in the bovine trachea is mainly due to increases of cAMP content caused by inhibition of phosphodiesterase. This mechanism is partially related to the activation of large conductance Ca2+ activated K+ (BK) channels (Kaneda et al., 2005).

Cortés-Arroyo et al. 63

Figure 6. Effect of increasing concentrations of T. mucronatum leaf extract (T muc., at 3.2-17.8 µg/ml) on the cumulative concentration-response curve for Ca2+ in rat tracheal rings incubated in Ca2+ free solution. Data are expressed as a percentage of the maximum contraction obtained in the absence of the extract. Mean ± SEM, n = 8.

The mechanism of action of the extract was not related to nitric oxide synthase because neither the mechanical removal of epithelium nor L-NAME preincubation signifi-cantly altered the relaxant effect of the extract on airway smooth muscle precontracted with carbachol (Hatziefthimiou et al., 2002). The relaxant effect observed here is in agreement with the vasodilator effect produced by the aqueous extract of the aerial part of T. mucro-natum on isolated rat aorta stimulated with noradrenaline (Perusquía et al., 1995). However, the relaxant effect on carbachol-induced contraction in rat trachea preparations (EC50: 33.9 ± 2.5 µg/ml) observed in this study was significantly greater than that reported by Perusquía et al. (1995) (EC50: 3400 µg/ml). This could be explained by the higher sensitivity of the tissue or because the active compounds are primarily extracted with non-polar solvents such as hexane. The addition of an ATP-sensitive K+ channel blocker (glibenclamide, 10 µM), a selective voltage-sensitive K+ channel blocker (4-aminopyridine, 10 mM) or a non-selective K+ channel blocker (tetraethylammonium, 10 mM) did not affect the tracheal relaxation induced by T. mucronatum. The long-term functional recovery of these tissues suggests that caution should be exercised when using this species for traditional medicine.

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