Planta Med. 57(1991) 519
Activity of Boldine on Rat Ileum H. Speisky', .1. A. Squella2, and Luis J. Náñez-Vergara23 1 2
Biochemical Pharmacology Unit, Nutrition and Food Technology Institute (INTA), University of Chile, Santiago, Chile
Laboratory of Pharmacology & Electrochemistry, Faculty of Chemical & Pharmacological Sciences, University of Chile, P0 Box 233, Santiago, Chile
Address for correspondence Received: October 23, 1990
(2). Official pharmacognostic descriptions have listed boldo
preparations as cholagogue/choleretic, diuretic, sedative and antihelmintic (3—5). Although the pharmacological evi-
Boldine shows a concentration-dependent relaxation effect on the rat ileum preparation with a pD2
of 3.77 0.12. The alkaloid also produces a concentration-dependent parallel shift of the curve-response to acetylcholine, therefore it directly interferes with the cholinergic mechanism associated with the contraction, behaving as a competitive antagonist with a pA2 of 4.78
0.19. On the other hand, boldine produces a nonparallel shift of the contraction response induced by barium, revealing the occurrence of a non-competitive antagonism. This effect could be the result of interfer-
ence with intracellular events associated with the
dence to support these activities is only limited, it seems clear that most of the actions promoted by crude boldo preparations can be reproduced by the administration of pure boldine alone (6—9).
Recently we found that boldine can also act as a very potent antioxidant substance protecting various biological systems from undergoing free-radical-mediated peroxidative damage (10, 11). This latter activity of boldine may possibly be related to some of the liver-protecting effects attributed, in popular medicine, to the administration of boldine-containing crude boldo preparations (9, 12).
barium-induced changes in calcium pools.
Early work conducted by Delourne (13) Key words
suggests that an additional effect of boldine is the inhibition of intestinal muscle activity in the anaesthetized cat prep-
Boldine, Peumus boldus, (S)-2,9-dihy-
aration. In the present study we have employed the rat
droxy-1 ,10-dimethoxyaporphine, muscle-relaxing ef-
fect, contractility of rat ileum.
ileum preparation as an experimental system to examine in vitro the smooth muscle inhibitory effect of boldine, and have addressed the possibility that boldine exerts its effect
through interference with neuromuscular cholinergic Introduction
transmission. Materials and Methods
Boidme [(S)-2,9-dthydroxy-1 ,10-dimethoxy-
aporphine, Figure 1] is the major alkaloid present in the leaves and bark of boldo (Peumus boldus Molina), a widely distributed evergreen tree native of Chile (1).
Pharmaceutical preparations based on boldo have been widely used in South and North America, and Europe for medicinal purposes since the last century
Chemicals Boldine was extracted from the bark of Peumus boldus and crystallized repeatedly from chloroform The alkaloid was chromatographically (TLC) pure and its identity was established by JR and NMR spectroscopy. All other chemicals were of analytical grade.
Biological assay Female Sprague-Dawley rats weighing 250 to
/
CH3
OH
HO
OCH3 OCH3 bol dine
Fig. 1 Chemical structure of boldine [(S)-2,9-dihydroxy-1,10-dimethoxyaporphine]).
350 g, fed ad ithitum, were used. Immediately after sacrifice by cervical dislocation, the intestinal portion corresponding to the ileum was surgically sectioned, placed on ice, and continuously washed throughout with cold saline. After washing, a 2cm length of ileum was ligated at one end, and filled with 0.2 ml of the following buffer (gil): NaC1 6.84; CaCl2 2 H20 0.27; MgC1 ' 6 H20 0.112; KC1 0.44; Na2HPO4'2 H20 0.15; NaHCO3 2.1; glucose 2.0(14). Using an iso-
lated organ system, the preparation was incubated in a glass bath containing 10 ml of the buffer described above through which a (95/5) 02iC0 mixture was bubbled continuously. One of the ends of the ileum segment was attached to a strength transducer (N.K. TB 61 2-T) and the changes in tension were recorded by means of a
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Abstract
520 Planta Med. 57(1991)
H. Speiskyetal.
polygraph (N.K. RM-6000) connected to a carrier amplifier (N.K. AP 620-G). The system used was set up with a scanning speed of
25 mm/mm, a sensitivity of 20, and an initial calibration of 2 g (20mm). Simultaneous monitoring of the biological responses was
carried out using an oscilloscope (N.K. VC-640-6) set up with a scanning speed of 1 division/sec. In order to ensure the stability of the responses before the addition of any drug into the system, each preparation was subjected to a basal tension of I g (10 mm) during a 60 mm period.
Concentration-response curves
basal response condition. Finally, after pre-incubating the preparation with an antagonist for 30 mm, a new concentration-response curve for the agonist was obtained in the presence of a fixed
antagonist concentration. The 30 mm pre-incubation time in the presence of the antagonist was found to produce highly reproducible responses in this system. All drugs used were dissolved in the incubation buffer solution and added directly into the system.
Data processing The effect of each agonist on the preparation is expressed as the fraction of the Ema,, induced by it (E/Em,,,j. EC50
4.2
31
3.4
— LOG(IOLDIt)
Fig. 2 Concentration-response curve for boldine in the rat ileum preparation. Each point represents the mean of ten experiments S.E.M.
values were obtained by plotting the E/Emax ratio versus the logarithm of the agonist concentration (log C). Similarly, from such data, the pD2 values (negative log C for EC50) were estimated.
Sigmoidal concentration-response curves were
linearized by applying logarithms to the data according to Guidicelli (16) and then plotting log EE/Emaxl/[1 E/Emax] versus log C. The antagonist character of boldine was calculated according to the following methods:
(a) Schild's Method (17). After estimating the ratio (X) of EC50 for the agonist in the presence and absence of antagonist, the logarithm of(X—1) was plotted against the concentration of the antagonist (log B). When log (X—1) equals zero, log B represents the pA2 value (18).
(b) Lineweaver-Burk Method (19). The dissociation (and affinity constants) were obtained from plotting 1/[E/Emax]
versus 1/C. All experimental data represent the mean S.E.M.; unless stated otherwise, the significance between mean values was assessed by Student's t test for paired data.
Results Figure 2 shows a smooth muscle relaxing effect of boldine in the isolated rat ileum preparation. This effect of boldine was concentration-dependent within the
range of 1.6• iO M to 4.6. iO M. From the plotted data an EC50 of 1.7. iO- M which corresponds to a PD2 of 3.77 0.12 was estimated.
$4 - LOB (ACH)
Fig. 3 Effect of different boldine concentrations on the concentration-response curves of acetylcholine. (0) Control; () 7.8• 1O M boldine; (D) 2.5- iO- M boldine; (A) 5.0• lO M boldine. Each point represents the mean of five (control) and ten (boldine) experiments S.E.M.
Figure 3 shows the effect of boldine on the
contraction of the ileum preparation induced by acetylcholine. Boldine induced a concentration-dependent shift towards the right of the concentration-response curve of acetyicholine. Within the range of boldine concentrations assayed [7.8• iO M to 5.0. iO M], the maximum original response elicited by acetylcholine was always attained
(control). Following linearization of the sigmoidal concen-
tration-response curves, a comparison of their slopes [con-
trol 1.37; boldine 1.54 (7.8 iO M); 1.57 (2.5 i0 M); 1.65 (5.0. i0 M)1 indicates no significant differences (analysis of variance p> 0.05). After applying the Schild's
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Concentration-response curves were carried out in a cumulative form (15). A maximum effect (Em) of the agonist was assumed when more than two increasing concentrations of the drug under study caused no significant increment in the response of the system. After an Emax was obtained, the preparation was washed with the incubation buffer (three times) and left stabilizing for a period of 30 mm, which allowed the preparation to recover its
Planta Med. 57(1991) 521
Activity of Boldine on Rat Ileum
moidal curves (16) revealed no significant differences. A further indication of this assumption was obtained from a Lineweaver-Burk plot of the data (19) which showed a con-
centration-dependent increase in the slopes of the curves while maintaining the same intercept. A quantitative degree of the competitive antagonist nature of boldine is provided by its pA2 value (4.78 0.19). In parallel studies and employing identical experimental conditions, we have obtained a pA2 of 7.78 0.14 for atropine, a well recognized competitive cholinergic antagonist. Although this latter value is slightly lower than that reported (8.6—9.0) by others (17), it is significantly greater than that estimated for boldine, thus indicating the superior competitive character of atropine.
Our studies also show that boldine can
4.1
Fig. 4 Effect of different boldine concentrations on the concentration-re-
sponse curves of barium. (0) Control; (0)1. 104M boIdine;()2.5• 10M boldine; (•) 5.O• iO M boldine. Each point represents the mean of ten experiments S.E.M.
method to determine the competitive antagonist degree of
boldine a pA2 value of 4.78 0.19 was estimated. We also assessed a possible effect ofboldine
on barium-induced contraction of the ileum preparation.
As shown in Figure 4, the addition of barium (2 iO to 7.0• iO M) to this preparation induced a concentrationdependent contraction which is susceptible to inhibition by
boldine. Increasing boldine concentrations produced a non-parallel shift of the concentration-response curves induced by barium. In addition, boldine clearly prevented barium from eliciting the maximum effect observed under control conditions. Discussion
Results from these studies clearly demonstrate a muscle-relaxing effect of boldine in the isolated rat ileum preparation. This pharmacological effect of boldine was evidenced both as an intrinsic activity capable of relaxing the basal tone of the ileum smooth muscle, and as an activity capable of interfering with the muscle contraction induced by acetylcholine. A comparison of the pD2 and the pA2 values
suggest that a substantially lower dose would be required for boldine to antagonize the acetylcholine-induced contraction. Thus, at therapeutic drug levels one would not expect boldine to exert a significant effect on the basal intestinal tone. Data presented indicate that boldine exerts its inhibitory effect on the acetyicholine-mediated muscle contraction via a competitive antagonist mechanism. In fact, boldine was shown to induce a parallel shift towards the right in the concentration-response curve of acetylcholine while maintaining unaltered the maximum contraction response induced by this agonist. Attesting to the competitive nature of the interaction between acetylcholine and boldine, the analysis of the slopes of the linearized sig-
counteract the barium-induced contraction of the ileum preparation. The effect of barium is thought not to be mediated by an increase in the release of acetylcholine (20),
but rather to result from changes in intracellular pools of calcium relevant to the contraction process. In addition, the barium-induced contraction, which is known to be insensitive to atropine (21), is believed to result from barium directly replacing calcium on its interaction with contractile proteins (20). In contrast to the interaction with acetylcholine, increasing boldine concentrations produced a non-
parallel shift of the contraction response induced by barium, thus revealing the occurrence of a non-competitive antagonism. Consistent with this latter interpretation is the finding that boldine effectively prevents barium from eliciting the maximum effect observed under control conditions.
Therefore, our studies show that, like atropine, boldine blocks the acetylcholine-induced muscle contraction. However, boldine differs from the former by being able to modify
the maximum extent of the barium-induced contraction. Conceivably, this latter effect of boldine is the result of inter-
ference with intracellular events associated with the barium-induced changes in the calcium pool.
Acknowledgments This work was supported by funds provided by FONDECYT (Grant 1047/91).
References 1
2
6 8
10
'
Montes,
M., Wilkomirsky, T. (1985) in: Medicina Tradicional
Chilena, Ed. Universidad de Concepción, Concepción, Chile. Bourgoin, E., Verne, Cl. (1872) J. Pharm. Chim. 16. 192—5. Youngken, H. W. (1943) in: Textbook of Pharmacognosy, 6th Edn., p. 361, Blakinston, Philadelphia. Osol, A., Farrer, G. E. (1955) in: The Dispensatory of United States of America, 25th Edn., p. 1606, Lippincott, Philadelphia. Todd, H. G. (1967) in: Extra Pharmacopoeia, Martindale, 25th Edn., p. 1508, The Pharmaceutical Press, London. Kreitmair, H. (1952) Pharmazie 7,507—11. Jimeno. J. L. D. (1956) Anales Inst. Farmacol. Espan. 5, 395—401. Borkowski, B., Desperak-Naciazek, A., Obojska, K., Szmal, Z. (1966) Dissertations Pharm. 18,455—462. Genest, K., Hughes, D. W. (1968) Can. J. Pharm. Sci. 4,84—92. Speisky. H., Cassels, B. K., Lissi, E. A., Videla, L.A. (1991) Biochem. Pharmacol. 41, 1575—1581. Zanocco, L. A., Jimenez, G., Nuflez-Vergara, L. J. (1990) Thesis
Facultad de Ciencias Quimicas y Farmacéuticas, Universidad de 12 13
Chile. Schindler. H. (1957) Arzneimittel-Forsch. 7, 747—752. Delourne, M. J. (1949) Compt. Rend. 229, 953—8.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
45
522 Planta Med. 57(1991)
16 17
Withmore, D. A., Brookes, L. G., Wheeler, K. P. (1980) J. Pharmac. Pharmacol. 32, 62—3. Van Rossum, J. M. (1963) Arch. mt. Pharmacodyn. 143, 299—330. Guidicelli, J. F. (1971) J. Pharmacol (Paris) 2, 373—380. Arunlakshana, 0., Schild, 0. H. (1958) Br. J. Pharmacol. 14,48—57.
15 19 20 21
Mackay, D. (1978) J. Pharmac. Pharmacol. 30, 312—3.
Marshall, A. G. (1978) in: Biophysical Chemistry, p. 287, John Wiley & Sons, New York. Clement, J. G. (1981) Can. J. Physiol. Pharmacol. 59. 541—547. Clement, J. G. (1980) Can. 1. Physiol. Phannacol. 58, 265—270.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
14
H. Speiskyetal.
Activity of Boldine on Rat Ileum H. Speisky', .1. A. Squella2, and Luis J. Náñez-Vergara23 1 2
Biochemical Pharmacology Unit, Nutrition and Food Technology Institute (INTA), University of Chile, Santiago, Chile
Laboratory of Pharmacology & Electrochemistry, Faculty of Chemical & Pharmacological Sciences, University of Chile, P0 Box 233, Santiago, Chile
Address for correspondence Received: October 23, 1990
(2). Official pharmacognostic descriptions have listed boldo
preparations as cholagogue/choleretic, diuretic, sedative and antihelmintic (3—5). Although the pharmacological evi-
Boldine shows a concentration-dependent relaxation effect on the rat ileum preparation with a pD2
of 3.77 0.12. The alkaloid also produces a concentration-dependent parallel shift of the curve-response to acetylcholine, therefore it directly interferes with the cholinergic mechanism associated with the contraction, behaving as a competitive antagonist with a pA2 of 4.78
0.19. On the other hand, boldine produces a nonparallel shift of the contraction response induced by barium, revealing the occurrence of a non-competitive antagonism. This effect could be the result of interfer-
ence with intracellular events associated with the
dence to support these activities is only limited, it seems clear that most of the actions promoted by crude boldo preparations can be reproduced by the administration of pure boldine alone (6—9).
Recently we found that boldine can also act as a very potent antioxidant substance protecting various biological systems from undergoing free-radical-mediated peroxidative damage (10, 11). This latter activity of boldine may possibly be related to some of the liver-protecting effects attributed, in popular medicine, to the administration of boldine-containing crude boldo preparations (9, 12).
barium-induced changes in calcium pools.
Early work conducted by Delourne (13) Key words
suggests that an additional effect of boldine is the inhibition of intestinal muscle activity in the anaesthetized cat prep-
Boldine, Peumus boldus, (S)-2,9-dihy-
aration. In the present study we have employed the rat
droxy-1 ,10-dimethoxyaporphine, muscle-relaxing ef-
fect, contractility of rat ileum.
ileum preparation as an experimental system to examine in vitro the smooth muscle inhibitory effect of boldine, and have addressed the possibility that boldine exerts its effect
through interference with neuromuscular cholinergic Introduction
transmission. Materials and Methods
Boidme [(S)-2,9-dthydroxy-1 ,10-dimethoxy-
aporphine, Figure 1] is the major alkaloid present in the leaves and bark of boldo (Peumus boldus Molina), a widely distributed evergreen tree native of Chile (1).
Pharmaceutical preparations based on boldo have been widely used in South and North America, and Europe for medicinal purposes since the last century
Chemicals Boldine was extracted from the bark of Peumus boldus and crystallized repeatedly from chloroform The alkaloid was chromatographically (TLC) pure and its identity was established by JR and NMR spectroscopy. All other chemicals were of analytical grade.
Biological assay Female Sprague-Dawley rats weighing 250 to
/
CH3
OH
HO
OCH3 OCH3 bol dine
Fig. 1 Chemical structure of boldine [(S)-2,9-dihydroxy-1,10-dimethoxyaporphine]).
350 g, fed ad ithitum, were used. Immediately after sacrifice by cervical dislocation, the intestinal portion corresponding to the ileum was surgically sectioned, placed on ice, and continuously washed throughout with cold saline. After washing, a 2cm length of ileum was ligated at one end, and filled with 0.2 ml of the following buffer (gil): NaC1 6.84; CaCl2 2 H20 0.27; MgC1 ' 6 H20 0.112; KC1 0.44; Na2HPO4'2 H20 0.15; NaHCO3 2.1; glucose 2.0(14). Using an iso-
lated organ system, the preparation was incubated in a glass bath containing 10 ml of the buffer described above through which a (95/5) 02iC0 mixture was bubbled continuously. One of the ends of the ileum segment was attached to a strength transducer (N.K. TB 61 2-T) and the changes in tension were recorded by means of a
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Abstract
520 Planta Med. 57(1991)
H. Speiskyetal.
polygraph (N.K. RM-6000) connected to a carrier amplifier (N.K. AP 620-G). The system used was set up with a scanning speed of
25 mm/mm, a sensitivity of 20, and an initial calibration of 2 g (20mm). Simultaneous monitoring of the biological responses was
carried out using an oscilloscope (N.K. VC-640-6) set up with a scanning speed of 1 division/sec. In order to ensure the stability of the responses before the addition of any drug into the system, each preparation was subjected to a basal tension of I g (10 mm) during a 60 mm period.
Concentration-response curves
basal response condition. Finally, after pre-incubating the preparation with an antagonist for 30 mm, a new concentration-response curve for the agonist was obtained in the presence of a fixed
antagonist concentration. The 30 mm pre-incubation time in the presence of the antagonist was found to produce highly reproducible responses in this system. All drugs used were dissolved in the incubation buffer solution and added directly into the system.
Data processing The effect of each agonist on the preparation is expressed as the fraction of the Ema,, induced by it (E/Em,,,j. EC50
4.2
31
3.4
— LOG(IOLDIt)
Fig. 2 Concentration-response curve for boldine in the rat ileum preparation. Each point represents the mean of ten experiments S.E.M.
values were obtained by plotting the E/Emax ratio versus the logarithm of the agonist concentration (log C). Similarly, from such data, the pD2 values (negative log C for EC50) were estimated.
Sigmoidal concentration-response curves were
linearized by applying logarithms to the data according to Guidicelli (16) and then plotting log EE/Emaxl/[1 E/Emax] versus log C. The antagonist character of boldine was calculated according to the following methods:
(a) Schild's Method (17). After estimating the ratio (X) of EC50 for the agonist in the presence and absence of antagonist, the logarithm of(X—1) was plotted against the concentration of the antagonist (log B). When log (X—1) equals zero, log B represents the pA2 value (18).
(b) Lineweaver-Burk Method (19). The dissociation (and affinity constants) were obtained from plotting 1/[E/Emax]
versus 1/C. All experimental data represent the mean S.E.M.; unless stated otherwise, the significance between mean values was assessed by Student's t test for paired data.
Results Figure 2 shows a smooth muscle relaxing effect of boldine in the isolated rat ileum preparation. This effect of boldine was concentration-dependent within the
range of 1.6• iO M to 4.6. iO M. From the plotted data an EC50 of 1.7. iO- M which corresponds to a PD2 of 3.77 0.12 was estimated.
$4 - LOB (ACH)
Fig. 3 Effect of different boldine concentrations on the concentration-response curves of acetylcholine. (0) Control; () 7.8• 1O M boldine; (D) 2.5- iO- M boldine; (A) 5.0• lO M boldine. Each point represents the mean of five (control) and ten (boldine) experiments S.E.M.
Figure 3 shows the effect of boldine on the
contraction of the ileum preparation induced by acetylcholine. Boldine induced a concentration-dependent shift towards the right of the concentration-response curve of acetyicholine. Within the range of boldine concentrations assayed [7.8• iO M to 5.0. iO M], the maximum original response elicited by acetylcholine was always attained
(control). Following linearization of the sigmoidal concen-
tration-response curves, a comparison of their slopes [con-
trol 1.37; boldine 1.54 (7.8 iO M); 1.57 (2.5 i0 M); 1.65 (5.0. i0 M)1 indicates no significant differences (analysis of variance p> 0.05). After applying the Schild's
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Concentration-response curves were carried out in a cumulative form (15). A maximum effect (Em) of the agonist was assumed when more than two increasing concentrations of the drug under study caused no significant increment in the response of the system. After an Emax was obtained, the preparation was washed with the incubation buffer (three times) and left stabilizing for a period of 30 mm, which allowed the preparation to recover its
Planta Med. 57(1991) 521
Activity of Boldine on Rat Ileum
moidal curves (16) revealed no significant differences. A further indication of this assumption was obtained from a Lineweaver-Burk plot of the data (19) which showed a con-
centration-dependent increase in the slopes of the curves while maintaining the same intercept. A quantitative degree of the competitive antagonist nature of boldine is provided by its pA2 value (4.78 0.19). In parallel studies and employing identical experimental conditions, we have obtained a pA2 of 7.78 0.14 for atropine, a well recognized competitive cholinergic antagonist. Although this latter value is slightly lower than that reported (8.6—9.0) by others (17), it is significantly greater than that estimated for boldine, thus indicating the superior competitive character of atropine.
Our studies also show that boldine can
4.1
Fig. 4 Effect of different boldine concentrations on the concentration-re-
sponse curves of barium. (0) Control; (0)1. 104M boIdine;()2.5• 10M boldine; (•) 5.O• iO M boldine. Each point represents the mean of ten experiments S.E.M.
method to determine the competitive antagonist degree of
boldine a pA2 value of 4.78 0.19 was estimated. We also assessed a possible effect ofboldine
on barium-induced contraction of the ileum preparation.
As shown in Figure 4, the addition of barium (2 iO to 7.0• iO M) to this preparation induced a concentrationdependent contraction which is susceptible to inhibition by
boldine. Increasing boldine concentrations produced a non-parallel shift of the concentration-response curves induced by barium. In addition, boldine clearly prevented barium from eliciting the maximum effect observed under control conditions. Discussion
Results from these studies clearly demonstrate a muscle-relaxing effect of boldine in the isolated rat ileum preparation. This pharmacological effect of boldine was evidenced both as an intrinsic activity capable of relaxing the basal tone of the ileum smooth muscle, and as an activity capable of interfering with the muscle contraction induced by acetylcholine. A comparison of the pD2 and the pA2 values
suggest that a substantially lower dose would be required for boldine to antagonize the acetylcholine-induced contraction. Thus, at therapeutic drug levels one would not expect boldine to exert a significant effect on the basal intestinal tone. Data presented indicate that boldine exerts its inhibitory effect on the acetyicholine-mediated muscle contraction via a competitive antagonist mechanism. In fact, boldine was shown to induce a parallel shift towards the right in the concentration-response curve of acetylcholine while maintaining unaltered the maximum contraction response induced by this agonist. Attesting to the competitive nature of the interaction between acetylcholine and boldine, the analysis of the slopes of the linearized sig-
counteract the barium-induced contraction of the ileum preparation. The effect of barium is thought not to be mediated by an increase in the release of acetylcholine (20),
but rather to result from changes in intracellular pools of calcium relevant to the contraction process. In addition, the barium-induced contraction, which is known to be insensitive to atropine (21), is believed to result from barium directly replacing calcium on its interaction with contractile proteins (20). In contrast to the interaction with acetylcholine, increasing boldine concentrations produced a non-
parallel shift of the contraction response induced by barium, thus revealing the occurrence of a non-competitive antagonism. Consistent with this latter interpretation is the finding that boldine effectively prevents barium from eliciting the maximum effect observed under control conditions.
Therefore, our studies show that, like atropine, boldine blocks the acetylcholine-induced muscle contraction. However, boldine differs from the former by being able to modify
the maximum extent of the barium-induced contraction. Conceivably, this latter effect of boldine is the result of inter-
ference with intracellular events associated with the barium-induced changes in the calcium pool.
Acknowledgments This work was supported by funds provided by FONDECYT (Grant 1047/91).
References 1
2
6 8
10
'
Montes,
M., Wilkomirsky, T. (1985) in: Medicina Tradicional
Chilena, Ed. Universidad de Concepción, Concepción, Chile. Bourgoin, E., Verne, Cl. (1872) J. Pharm. Chim. 16. 192—5. Youngken, H. W. (1943) in: Textbook of Pharmacognosy, 6th Edn., p. 361, Blakinston, Philadelphia. Osol, A., Farrer, G. E. (1955) in: The Dispensatory of United States of America, 25th Edn., p. 1606, Lippincott, Philadelphia. Todd, H. G. (1967) in: Extra Pharmacopoeia, Martindale, 25th Edn., p. 1508, The Pharmaceutical Press, London. Kreitmair, H. (1952) Pharmazie 7,507—11. Jimeno. J. L. D. (1956) Anales Inst. Farmacol. Espan. 5, 395—401. Borkowski, B., Desperak-Naciazek, A., Obojska, K., Szmal, Z. (1966) Dissertations Pharm. 18,455—462. Genest, K., Hughes, D. W. (1968) Can. J. Pharm. Sci. 4,84—92. Speisky. H., Cassels, B. K., Lissi, E. A., Videla, L.A. (1991) Biochem. Pharmacol. 41, 1575—1581. Zanocco, L. A., Jimenez, G., Nuflez-Vergara, L. J. (1990) Thesis
Facultad de Ciencias Quimicas y Farmacéuticas, Universidad de 12 13
Chile. Schindler. H. (1957) Arzneimittel-Forsch. 7, 747—752. Delourne, M. J. (1949) Compt. Rend. 229, 953—8.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
45
522 Planta Med. 57(1991)
16 17
Withmore, D. A., Brookes, L. G., Wheeler, K. P. (1980) J. Pharmac. Pharmacol. 32, 62—3. Van Rossum, J. M. (1963) Arch. mt. Pharmacodyn. 143, 299—330. Guidicelli, J. F. (1971) J. Pharmacol (Paris) 2, 373—380. Arunlakshana, 0., Schild, 0. H. (1958) Br. J. Pharmacol. 14,48—57.
15 19 20 21
Mackay, D. (1978) J. Pharmac. Pharmacol. 30, 312—3.
Marshall, A. G. (1978) in: Biophysical Chemistry, p. 287, John Wiley & Sons, New York. Clement, J. G. (1981) Can. J. Physiol. Pharmacol. 59. 541—547. Clement, J. G. (1980) Can. 1. Physiol. Phannacol. 58, 265—270.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
14
H. Speiskyetal.
