The monoterpene alkaloid cantleyine from Strychnos trinervis root and its spasmolytic properties

Phytomedicine, Vol. 6(3), pp. 169-176

Phytomedicine

© Urban & Fischer Verlag 1999

http://www.urbanfischer.de/journals/phytomed

The monoterpene alkaloid cantleyine from Strychnos trinervis root and its spasmolytic properties T. M. S. cia Silva+, B. A. cia Silva, R. Mukherjee Laborat6rio de Tecnologia Farmaceutica, Universidade Federal da Paraiba, J0<10 Pessoa, PB, Brazil

Summary Cantleyine, a monoterpene alkaloid isolated from the root bark of Strychnos trinervis, was submitted to a broad spectrum pharmacological screening, in which the principal effect observed was a nonspecific relaxation of isolated smooth muscles. Cantleyine relaxed (IC 50 2.1 X 10-4 M) the guinea-pig trachea, pre-contracted by carbachol and antagonized in a nonspecific manner; carbachol (IC 50 2.1 X 10-4 M) and histamine (IC 50 1.4 X 10-4 M) induced contractions in the guinea-pig ileum; and phenylephrine (IC 50 3.8 X 10-4 M) responses in the rat aorta. Cantleyine antagonized (pD' 2' 3.82) cumulative concentration response curves to histamine in the ileum in a noncompetitive, reversible (slope, 4.84) and concentration dependent manner. The tonic contractions induced by histamine and KCl were also inhibited in a concentration-dependent and reversible manner (IC 50 7.2 X 10-5 and 1.8 X 10-4 M, respectively), suggesting that cantleyine should be acting on voltage-dependent Ca 2 + channels. This hypothesis was confirmed by the observation that cantleyine inhibited (pD' 2' 3.35), in a concentration dependent manner, the CaCl 2 induced contraction in depolarizing medium. These results suggest that cantleyine produces nonspecific spasmolytic effects in smooth muscle and that in guinea-pig ileum this effect is, in part, due to the inhibition of Ca+ 2 influx through voltage-dependent Ca 2 +channels. Key words: Strychnos trinervis; cantleyine; spasmolytic actions; Ca+ 2 channels.

Introduction The search for new pharmacologically active compounds from Brazilian Strychnos, particularly from species found in the Northeast of the country, has generated a number of publications, dealing mainly with isolation, structure elucidation, and general pharmacological studies (Mukherjee, 1993; Mukherjee, 1997a), mechanistic mass spectrometry (Laprevote et aI., 1992), and confirmation of the identity and structures of indole alkaloids using high-field two-dimensional nuclear magnetic resonance spectroscopy (Mukherjee et aI., 1997b). A recent attempt to modify the isolation procedure led to the detection of cantleyine, the monoterpene pyridine alkaloid (1), in Strychnos trinervis roots collected in September 1995, from two different places +This work was submitted to the Universidade Federal da Paraiba, Joao Pessoa, PB, Brazil, in partial fulfilment of an M.Sc. in Chemistry and Pharmacology of Natural Products.

near Recife, capital of the northeastern state of Pernambuco. A computer aided search [November 10, 1997, NAPRALERT, College of Pharmacy, The University of Illinois at Chicago, Chicago, Illinois 60612-7231, U.S.A.], showed that cantleyine has been isolated from a number of Loganiaceae species, as well as Apocynaceae, Dipsacaceae, Goodeniaceae, Icacinaceae, Oleaceae, Rubiaceae, and Scrophulariaceae plants. How.' '

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ever, of the Loganiacrae species, cantleyine was isolated from African and Asian plants only and this represents the first report of its occurrence in a Strychnos species of the Americas. The NAPRALERT search also failed to show any work concerning the pharmacology of cantleyine. Therefore, we became interested in a pharmacological study of cantleyine and this paper reports the isolation and identification of the alkaloid and attempts to clarify the mechanism of smooth-muscle relaxation produced by cantleyine (1), in relation to Ca2+.

Materials and methods Plant materials

Fresh roots of S. trinervis were collected from CM de Cruz, sttio do Sr. Jose Correa (1.0 kg), and from Ava<;oiaba (1.1 kg) near Recife, Pernambuco, in September 1995. Extraction

The pulverized, dried roots (590 and 605 g, respectively) were macerated with NH 40H (10%, 50 ml in each case) and percolated at room temperature with EtOAc until the extracts became light maroon and yielded a faint Dragendorff test. The total extracts were then concentrated in a rotary evaporator in vacuo at 40°C to dark syrups (37 and 29 g, respectively) which were then agitated vigorously with H 2S0 4 (2%, 1 1 in each case) until the aqueous extracts responded negative to the Dragendorff test. After agitating the aqueous extracts with active charcoal (2 g), the extracts were filtered over Celite and the gummy masses over the filter papers were discarded. Isolation and identification of cantleyine

The aqueous extracts were then basified at 0-5 °C by dropwise addition of concentrated NH40H to pH 9.5-10, and then extracted exhaustively with CHC1 3 (41). The CHC1 3 extracts were then washed with distilled water, dried over anhydrous Na2S04, and concentrated in a rotary evaporator in vacuo at 40°C to dark syrups (8.5 and 14 g, respectively). The total alkaloids were then fractionated over LH-20 with CHC1 3 to polar and nonpolar fractions. The polar fractions were then chromatographed over neutral Brockmann alumina, grade II-III (Merck) using solvents of increasing polarity followed by subsequent purification and crystallization from EtOAc-benzene mixtures, yielding cantleyine as colorless needles (59 and 136 mg, 0.01 and 0.022%, respectively), C 11 H 13N0 3 , m.p. 130-132 °C, m/z (M+) 207.0897 (found), 207.0895 (calculated), and [alba -42.9 (c = 0.415; MeOH). Comparison of UV, IR, lH and 13C NMR, and mass spectral fragmentation with published data (Hart et al., 1969; Sevenet et al., 1970) established the identity of cantleyine.

Animals

Home-bred and home grown male Wistar rats (Rattus norvegicus, 150-200 g) and guinea-pig (Cavia porceIus, 300-400 g) of both sexes were use for all the experiments. The animals were housed under a 12 hour (06:00-18:00 h) light-dark cycle and had free access to normal food and tap water. General

The neutral solution of cantleyine used was prepared by dissolving pure cantleyine in 0.1 N HCl having 0.1 % cremophor and diluting further with distilled water. The composition (mM) of the physiological salt solutions used for the isolated tissues was as follows: Kreb's solution: NaCl 118.00, NaHC0 3 25.00, KH 2P04.H20 1.10, CaC12.2H20 2.52, KCl 4.55, MgS0 4.7H20 5.70, and glucose 11.00. Tyrode solution: NaCl 137.00, NaHC0 3 11.90, NaH2P04.H20 0.36, CaC12.2H20 1.36, KC12.70, MgC1 2.6H20 0.49, and glucose 5.50. Modified Tyrode solution (High-K+ Ca2+-free solution): NaCl 70.00, NaHC0 3 11.90, NaH 2P0 4.H20 0.41, KCl 70.00, MgC1 2.6H20 0.49, and glucose 5.50. Kreb's-Henseleit solution: NaCl 2.33, 110.88, NaHC0 3 25.00, KH 2P0 4.H20 CaC12.H20 2.49, KCl 5.90, MgS0 4.7H20 1.07, and glucose 11.51. The solutions were bubbled with a 95% O 2 and 5% CO 2 gas mixture and maintained at 37°C for all the experiments unless otherwise specified. The strips were equilibrated for 1 h under a resting tension of 1 g, and during this time the bath medium was changed every 15 min. to protect against interfering metabolites (Altura and Altura, 1970). Force generation was monitored using an isometric transducer coupled to a physiograph (Ugo Basile). Drugs

CaC12.2H210, KCl, NaCl, NaHC0 3, NaH2P04.H20, KH2P0 4.H20, MgCl 2.6H20, MgS0 4.7H20, glucose, and acetylcholine hydrochloride (Merck), phenylephrine L (-) hydrochloride (Pfizer), carbachol, isoprenaline hydrochloride, cremophor, and histamine dihydrochloride (Sigma). Data analysis

Unless otherwise specified, values are expressed as mean ± s.e. mean. Statistical analysis were performed using a paired Student's t-test when appropriate, and was considered significant when probability (p) was <0.05. Schild plots were analyzed by linear regression. Antagonism was judged to be non-competitive when the slope of the Schild's plot was significantly different from unity and depression of the maximum response was observed.

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Cantleyine (-log [M]) Fig. 2. Effect of several concentrations of candeyine on the phenylephrine (1 11M) induced tonic contraction of the rat aorta. Values are mean ± S. e. of mean for 4 experiments.

Effect of cantleyine on guinea-pig trachea precontracted with 1 IJM carbachol

Effect of cantleyine on carbachol and histamineinduced contractions in the guinea-pig ileum

The method used in the present study was similar to that described previously (Emmerson and Mackay, 1979). Guinea-pigs were killed by stunning and bleeding, and the tracheas were excised from the animals. The tracheal ring was opened by cutting longitudinally through the cartilaginous region, the epithelium was dissected away and a muscle strip with two segments of cartilage was removed and placed vertically in a organ bath containing Kreb's-Henseleit solution, in order to measure the isometric tension. Two simple concentration-response curves were obtained for carbachol (1 11M), and during stabilization of the tonic phase of the second carbachol response, cantleyine was added cumulatively. Relaxation was expressed as a percentage reversal of the initial contraction elicited by 1 11M carbachol. IC 50 values were obtained graphically from concentration-inhibition curves.

Guinea-pigs werde killed as described for the trachea. The distal ileum was existed rapidly, and washed thoroughly in Tyrode solution at room temperature. Segments of ileum (2-3 cm) were suspended in a 5 ml organ bath which contained Tyrode solution maintained at 37°C, and allowed to equilibrate for 30 min. Two simple concentration-response curves were obtained for both carbachol and histamine. Cantleyine was then added and after an incubation period of 15 min. (time required to produce maximum effects), a third concentration-response curve was constructed in the presence of cantleyine. The tissue was washed when the agonist responses had returned to resting level. The procedure was repeated in the absence and presence of various concentrations of cantleyine. Inhibition was measured by comparing the response before and after addition of the alkaloid in the organ bath, and IC 50 val-

Table 1. Effect of candeyine on contractions induced by carbachol and histamine in the guinea-pig ileum (n = 4). Candeyine (M)

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ues were obtained graphically from simple concentration-response curves. Effect of cantleyine on isolated rat aortic rings

The rats were killed by stunning and bleeding; aortic rings (2-3 cm) were obtained free from connective tissue and fat. The tissues were suspended by platinum hooks in a 5 ml organ bath containing Kreb's-Henseleit solution for isometric tension recording. Two submaximal (65-75% maximal) tonic responses to phenylephrine (1 pM) which stabilized in 15 min. were obtained initially. A third response was then obtained and cantleyine was added cumulatively in an attempt to obtain a concentration-inhibition curve. Relaxation was expressed as a percentage reversal of the initial contraction elicited by 1 pM phenylephrine. IC 50 values were obtained graphically from concentration-inhibition curves. Mechanism of action of cantleyine on guinea pig ileum:

• Characteristic of the blockade on histamine induced contractions: The strips were prepared as described before. Two cumulative concentration-response curves of approximately equal magnitude were obtained with histamine. After washing, the ileum was incubated with cantleyine for 15 min. and a third concentrationresponse curve for histamine was constructed in the presence of cantleyine. The tissue was washed and, when the response to histamine was recovered after re-

Fig. 4. Effect of cantleyine on histamine (1 JIM) - induced tonic contractions in guinea-pig ileum. Values are mean ± s.e. of mean for 4 experiments. peated washings, the procedure was repeated using different concentrations of the alkaloid. Antagonism was measured by calculating pD' 2 values (Van Rossum, 1963) defined as the -log of the antagonist molar concentration which produces 50% of the maximum effect produced by the agonist.

• Effect of cantleyine on tonic contractions induced by histamine and KCI: After stabilization of the preparations, an isometric contraction was elicited to a concentration of KCI (40 mM) or histamine (1 pM) which gave a response at 60-75% of the maximum (determined from preliminary experiments). KCl or histamine remained in contact with the preparation until a plateau of contraction was reached (approximately 8 min.), after which time the tissue was washed. The plateau characterizes the tonic component of the contraction. After a further 30 min., the process was repeated and at the plateau of contraction, cantleyine was added cumulatively. Subsequent concentrations were added only after the response to the previous concentration became stable. Relaxation was expressed as described above.

• Effect of cantleyine on voltage-dependent Ca 2+ channels: The strips were prepared as described before. To assess the effect of cantleyine on the influx of Ca 2+ through voltage-dependent channels (Goodfraind et al., 1986; Weiss, 1981), the strips were bathed for 30 min. in the Tyrode solution and then exposed for 45-60 min. to high-K+ Ca2 +-free depolarizing solution (Modified Tyrode solution). In general, two cumulative concentration-response curves to CaCl2 were obtained at 60 min. intervals in each preparation (Van Rossum, 1963). After obtaining the first curve, washing and after complete relaxation, different concentrations of

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cantleyine were added to the bath and left in contact with the tissue for 15 min. Then a second cumulative concentration-response curve to CaCl 2 was obtained in the presence of the alkaloid. The maximal contraction obtained with the first concentration-response curve to CaCl 2 was taken as 100%, and all contractions were calculated as a function of this value. Each preparation was exposed to only one concentration of cantleyine. Antagonism was measured as indicated before.

Results Effect of cantleyine on guinea-pig trachea precontracted with 1 ~M carbachol

Cantleyine (2.4 X 10-5_ 4.9 X 10-4 M) relaxed in a concentration-dependent manner, the guinea-pig trachea that had been precontracted with carbachol (1 pM, Figure 1). This effect was reversible, and the IC 50 value was 2.1 X 10-4 M. Effect of cantleyine on carbachol and histamineinduced contractions in the guinea-pig ileum

Cantleyine (4.9 X 10-5 - 4,9 X 10-4 M) inhibited the phasic contractions induced by both carbachol and histamine Cfable 1). The corresponding values of IC 50 obtained graphically were 2.1 X 10-4 and 1.4 X 10-4 M for carbachol and histamine, respectively. Effect cantleyine on isolated rat aortic rings

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tion-dependent relaxation of the tissue and the IC50 value was 3.8 x 10-4 M (Figure 2). This effect was endothelium-independent. Mechanism of action of cantleyine on guinea-pig ileum

• Characteristic of the blockade on histamine induced contractions: Cantleyine (6.0 x 1O-S, 1.2 X 10-4, and 2.4 X 10-4 M) antagonized the cumulative concentration-response curves to histamine (Figure 3). The antagonism was non-competitive in nature as there was a non-parallel shift to the right and suppression of the maximal response. The antagonism became reversible 30 min. after the removal of cantleyine from the bath; this was evident in the recovery of the tissue in response to stimuli. Analysis of the data showed a linear regression and the value of correlation coefficient (r) was 0.96. The pD'2 and Schild slope values were 3.82 and 4.84, respectively.

• Effect of cantleyine on tonic cntractions induced by histamine and KCI: Cumulative addition of cantleyine during the development of the tonic component of the contractions resulted in a concentration-dependent relaxation in the guinea-pig ileum that had been precontracted with both 1 pM histamine (Figure 4) and 40 mM KCI (Figure 5). The threshold concentration of cantleyine was 2.4 X 10-5 M for both histamine and KCI induced contractions, but maximal relaxation occurred at 2.4 X 10-4 M for histamine and 4.9 X 10-4 M

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for KCI (Figures 4 and 5). The IC 50 values were 7.2 X 10-5 and 1.8 X 10-4 M for histamine and KCI, respectively.

• Effect of cantleyine on voltage-dependent Ca 2+ channels: Figure 6 shows the mean cumulative concentration-response curves for CaClz alone and in the presence of different concentrations of cantleyine (1.2 X 10-4,2.4 X 10-4,3.6 X 10-4 and 4.9 X 10-4 M). Cantleyine produced a non-parallel and concentrationdependent rightward displacement of the concentration-response curve to CaClz significantly reducing the maximal response. Analysis of the data showed a linear regression and the value of correlation coefficient (r) was 0.95. The pD'z and Schild slope values were 3.35 and 3.00, respectively, indicating a non-competitive blockade.

Discussion In the present study, we have investigated the effects of cantleyine on vascular and visceral smooth muscles, and the most important finding in this work is the demonstration that cantleyine exerts a nonselective spasmolytic action, and that this effect is due in part to the inhibition of Ca+z influx through voltage-dependent Ca+z channels. As cantleyine inhibited the contractions induced by different agonists in the several smooth muscles assayed (Figures 1 and 2, and Table 1), it would be reasonable to affirm that cantleyine has selectivity neither to agonist nor to tissue. Moreover, as the IC 50 values were not significantly different, it can be suggested that cantleyine may be acting by a similar mechanism of action in the tissues studied. Schild analysis of the data showed that inhibition of guinea-pig contractile response to histamine produced by cantleyine, at least within the range of concentrations employed, involves a simple noncompetitive blockade (Figure 3), suggesting that the spasmolytic effect induced by cantleyine does not require receptor occupancy. In addition, this result could explain the nonselective action of cantleyine in relation to agonist. Thus, it is probable that cantleyine can be acting at another step of the events cascade that leads to smoothmuscle contraction. Smooth muscle contractions are initiated when the concentration of Caz+ in the cell cytoplasm attains a threshold level which is reached by transmembrane influx of Ca z+through voltage dependent or receptor operated channels or due to release of Caz+from intracellular stores, mainly of the sarcoplasmic reticulum and mitochondria (Garret and Grisham, 1995). Thus, a decrease in intracellular Caz+concentration is an essential factor for producing smooth muscle relaxation. Stimu-

Ii that lead to contraction and relaxation in the smooth muscle involve two kinds of couplings: electromechanical, when the contraction involves change in the membrane potential, and pharmacomechanical, when the contraction evoked by agonist is larger than would be expected from the change in membrane potential (Rembold, 1996). The contraction of smooth muscles in response to various agents is frequently composed of two phases: a fast "phasic" component followed by a slower, more sustained "tonic" component. This biphasic response has been attributed to a dual source of Caz+in smooth muscles (for a review see van Breemen et al., 1979; Bolton, 1979). According to this hypothesis, the phasic component would result from Ca z+ release from intracellular stores, and the tonic component would be a consequence of Ca2+influx across the cell membrane. There is good evidence that the tonic response may be maintained by Ca 2+influx through receptor-operated or voltage-dependent channels. In the guinea-pig ileum, the phasic component is reached at 15 sec. after the contact of the stimulus with the tissue, while the tonic component is reached after 8 min. under the same conditions. There is much evidence that a number of compounds derived from medicinal plants modify the Caz+metabolism in cells (Drummond and Hughes, 1987; Freitas et al., 1996; Medeiros et al., 1991; Oliveira et al., 1996; Silva et al., 1993). Structurally, cantleyine has a pyridine nucleus. It is expected that cantleyine should present some similarity with compounds that act as blockers of voltage-dependent Caz+channels (Spedding and Paoletti, 1992) or openers of potassium channels (Edwards and Weston, 1990). In order to verify whether cantleyine acts on Caz+influx across the membrane, we evaluated its effect on the tonic component of the contractile response induced by both histamine (pharmacomechanical and electromechanical coupling) and KCI (electromechanical coupling) in the guinea-pig ileum. As shown in Figure 4 and 5, cantleyine relaxed in a concentration-dependent manner the ileum precontracted with both histamine (IC50 7.2 X 10-5 M) and KCI (1.2 X 10-4 M). Independently of the contraction being evoked by either pharmacomechanical or electromechanical coupling, the maintenance of the tonic component involves activation of the voltage-dependent Ca 2 + channels (Rembold, 1996). Therefore we can postulate that cantleyine may be acting to block these channels to produce nonselective spasmolytic effects. The confirmation of this hypothesis came from the observation that cantleyine inhibited in a concentration-dependent manner the CaClz induced contractions in the guinea-pig ileum in depolarizing medium (Figure 6). Whether the alkaloid inhibits the Caz+influx across the membrane acting directly on voltage-dependent

The monoterpene alkaloid cantleyine from Strychnos trinervis root and its spasmolytic properties Ca2+ channels or indirectly blocking protein kinase C, or opening potassium channels, are some questions that need to be answered. The most plausible hypothesis seems to be the third one, since cantleyine has a pyridine nucleus, structural feature common to most of the potassium channel openers (Edwards and Weston, 1990). Calcium channel blockers like verapamil (Silva et aI., 1993; Calixto and Loch, 1985), and nifedipine (Calixto and Loch, 1985) exhibit non competitive antagonism in smooth muscles, similar to that exhibited by cantleyine in the present case. In both cases, reversibility is referred to the observation of the removal of blocker restoring the full activity of the system. This feature is very desirable when investigating the mechanism of action of drugs which could be of potential therapeutic use. In conclusion, we have shown that cantleyine produces nonselective spasmolytic effects, and that in guinea-pig ileum this effect is due in part to the inhibition of Ca 2+ influx through voltage-dependent Ca2+ channels. However, we do not discard other possible mechanisms that have not been studied yet. Acknowledgements The authors wish to express their sincere thanks to Professor Norman R. Farnsworth, College of Pharmacy, The University of Illinois at Chicago, Chicago, Illinois 60612-7231, U.S.A., and Dr. Bhupesh C. Das, Institut de Chimie des Substances Naturelles, C.N.R.S., 91198 Gif-sur-Yvette, France, for all the spectral data and the optical rotation. Thanks are also accorded to Dr. AIda de A. Chiappeta, Departamento de Antibi6ticos, Universidade Federal de Pernambuco, 50037 Recife, PE, Brazil, for the identification and collection of the plant materials. Financial assistance from CNPq, Brazil, is gratefully acknowledged.

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Freitas, M. R.; Cortes, S. E; Thomas, G.; Barbosa-Filho, ]. M.: Modification of Ca 2 + metabolism in the rat rabbit aorta as a mechanism of spasmolytic action of warifteine, a bisbenzylisoquinoline alkaloid isolated from the leaves of Cissampelos sympodialis Eichl. (Minispermaceae) J. Pharm. Pharmacal. 48: 335-339, 1996. Garret, R. H.; Grisham, C. M.: "Muscle contraction", in: Molecular aspects of cell biology. Philadelphia: Saunders 1156-1179,1995. Goodfraind, T.; Miller, R.; Wibo, M.: Calcium antagonism and calcium entry blockade. Pharmacol. Rev. 38: 321-345, 1986. Hart, N. K.; Johns, S. R.; Lamberton,]. A.: Alkaloids of ]asminum species (Family Oleaceae). Aust. J. Chem. 22: 1283-1290,1969. Laprevote, 0.; Girard, c.; Das, B. c.; Mukherjee, R.: Usefulness of Fast-atom Bombardment and Collision-activated Dissociation Linked Scan (B/E) Mass Spectrometry for Structural Investigation of Indole Alkaloids. Rapid Communications in Mass Spectrometry 6: 318-321,1992. Medeiros, C. L. c.; Thomas, G.; Mukherjee, R.: The source of Ca 2+ for the spasmolytic actions of longicaudatine, a bisindole alkaloid isolated from Strychnos trinervis (Veil.) Mart. (Loganiaceae). Phytother. Res. 5: 24-28, 1991. Mukherjee, R.: Indole alkaloids of Strychnos from the northeast of Brazil, in Traditional Medicine. Ed. Mukherjee, B. Oxford & IBH Publishing Co. Pvt. Ltd., New Delhi, 229-234, 1993. Mukherjee, R.: Chemistry and pharmacology of some Strychnos (Loganiaceae) alkaloids from the northeast of Brazil. 10CD CIJS: Chemistry, Biological and Pharmacological Properties of Medicinal Plants from the Americas. Panama City, February 23-26, 1997a. Mukherjee, R.; da Silva, T. M. S.; Guimaraes,]. B. L.; Oliveira E. de ].; Keifer, P. A.; Shoolery, ]. N.: Tertiary Alkaloid Fraction of Strychnos atlantica: Confirmation of the Identity and Structures of Indole Alkaloids by High Field Nuclear Magnetic Resonance Spectroscopy. Phytochemical Anal. 8: 115-119, 1997b. Oliveira, E. ].; Medeiros, I. A.; Mukherjee, R.: Hypotensive and spasmolytic effects of normacusina B from Strychnos atlantica root. Phytomedicine 3: 45-49, 1996. Rembold, C. M.: "Electromechanical and pharmacomechanical coupling", in Baniny, M., Biochemistry of Smooth Muscle Contraction. San Diego, Academic Press, 227-39, 1996. Sevenet, T.; Das, B. c.; Parello,].; Potier, P.: Cantleyine, nouvel alcaloide monoterpenique de Cantleya carniculata (Becc.) Howard, Icacinacees. Bull. Soc. Chim. Fr. 8-9: 3120-3122,1970. Silva, B. A.; Araujo-Filho, A., P.; Mukherjee, R.; Chiappeta, A., A.: Bisnordihydrotoxiferine and vellosimine from Strychnos divaricans root: Spasmolytic properties of Bisnordihydrotoxiferine. Phytother. Res. 7: 419-424, 1993. Spedding, M.; Paoletti, R.: Classification of calcium channels and the sites of action of drugs modifying function. Pharmacol. Rev. 44: 363-376, 1992. Van Breemen, c.; Aaronson, P.; Loutzenhiser, R.: Sodium-calcium interaction in mammalian smooth muscle. Pharmacal. Rev. 30: 167-208, 1979.

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Address R. Mukherjee, Laborat6rio de Tecnologia Farmaceutica, Universidade Federal da Paraiba, Caixa Postal 5009,58051-970 Joao Pessoa, PB, Brazil.