- Email: [email protected]
Hypotensive effect of the hydroalcoholic extract from Jacaranda mimosaefolia leaves in rats
Journal of Ethnopharmacology 97 (2005) 301–304
Hypotensive effect of the hydroalcoholic extract from Jacaranda mimosaefolia leaves in rats Pilar Nicasioa , Mariana Meckesb,∗ a
Centro de Investigaci´on Biom´edica del Sur (IMSS), Argentina No. 1, Centro, 62790 Xochitepec, Morelos, M´exico b Unidad de Investigaci´ on M´edica en Farmacolog´ıa de Productos Naturales, Hospital de Pediatr´ıa, Centro M´edico Nacional Siglo XXI (IMSS), Avenida Cuauht´emoc 330, Colonia Doctores, 06725 Distrito Federal, M´exico Received 30 November 2003; received in revised form 1 November 2004; accepted 15 November 2004 Available online 13 January 2005
Abstract Hypothermic and cardiovascular activities of the methanol extract of Jacaranda mimosaefolia leaves were tested. To evaluate the hypotensive properties, anesthetized rats were used and temperature, blood pressure, and cardiac frequency were recorded. In addition, the in vitro effect produced by the extract on induced contraction with norepinephrine (NE) in rat aorta rings was evaluated. The extract produced a significant hypothermic effect with a maximum at 2 h, an effect which was accompanied by hypotension and low cardiac frequency, physiological conditions that were again re-established to the following 2 h. In isolated aorta preparations norepinephrine antagonistic effect was not correlated with the presence of Ca2+ , pD2 for NE was modified by the extract, an effect that could explain a blockade of the adrenergic receptors. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Hypotension; Cardiovascular activity; Isolated aorta; Medicinal plant; Jacaranda mimosaefolia
1. Introduction The genus Jacaranda (Bignoniaceae) is widely distributed in the tropical and subtropical areas of the world. Some species are used in traditional medicine of different countries to cure wounds, ulcers, and as an astringent in diarrhoea and dysentery (Gambaro et al., 1988; Mahran et al., 1991). Jacaranda mimosaefolia D. Don is an ornamental tree with attractive foliage and flowers, known in Mexico as “jacaranda”; the leaves and flowers, and the seeds are used in the state of Morelos to treat hypertension and amoebic infections (Osuna and Lozoya, 1989; Monroy-Ort´ıs and CastilloEspa˜na, 2000). Pharmacological evaluation on Jacaranda caucana revealed antiprotozoal (Weniger et al., 2001), cytotoxic (CA9KB cells), and antitumor (leuk-P388 in mouse) properties (Ogura et al., 1977). Jacaranone was demonstrated to be the
∗ Corresponding author. Tel.: +52 55 56 27 69 00x3234/3235; fax: +52 55 57 61 09 52. E-mail address: [email protected] (M. Meckes).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.11.017
compound responsible for the cytotoxic and antitumor activities of the plant (Ogura et al., 1977). The preliminary screening for potential antimicrobial activity of Jacaranda mimosaefolia grown in Egypt, showed that aqueous extract from the twigs and the seeds were active against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Bacillus cereus, Klebsiella pneumoniae, Pseudomonas sp., Mycobacterium phlei and Neisseria sp., although it had no effect on the growth of Candida sp. (Mahran et al., 1991). Jacaranone and scutellarein-7-glucuronide (Sankara et al., 1972), a phenyl-propanoid with hypotensive and analgesic properties, have been isolated from the leaves; 1-hydroxy4-oxo-2,5-cyclohexadien-1-acetate ethyl ester has also been detected in the fruits. Moreover, verbascoside, phenylacetic-glucoside and a new glucose ester named jacaranose have been isolated from the leaves (Gambaro et al., 1988). It has been assumed that jacaranose interacts with jacaranone to produce the jacaranoside which is metabolized in the human digestive tract and induces the formation of a lactonic compound that has antiulcer properties (Sankara et al., 1973). In an attempt to establish validation of the attributed hypotensive properties of the plant, a biological evaluation of
302
P. Nicasio, M. Meckes / Journal of Ethnopharmacology 97 (2005) 301–304
the methanol crude extract of Jacaranda mimosaefolia was carried out using different experimental models.
2. Materials and methods 2.1. Plant material Samples of Jacaranda mimosaefolia D. Don were collected in the State of Morelos in June 2002; the plant was taxonomically certified by Professor Abigail Aguilar, IMSSM Herbarium Director, based on specimen vouchers that were deposited for reference under the code numbers 11051, 11052, 11053. 2.2. Extract preparation The leaves of flowering Jacaranda mimosaefolia (1.7 kg) were dried at 27 ◦ C protected from direct light. The powdered material was extracted at room temperature by percolation with methanol–water (80:20) and concentrated under low pressure. The solid residue (510 g) was maintained at −20 ◦ C for further pharmacological tests. 2.3. Animal conditions Male Wistar rats (300–350 g) were maintained in controlled environmental conditions with a 12-h light/dark cycle with free access to standard rodent pellet food (Harlan Teklad) and water ad libitum, and the animals were fasted for 12 h before experimentation. In all in vivo studies, methanol extract was administered to the animals at lower doses to the previously determined LD50 = 655 mg/kg (601–714 mg/kg) in rats. The Institutional Ethical Committee approved the study. 2.4. Hypothermic effect Groups of ten male adult Wistar rats were used. Different doses (100–400 mg/kg) of the extract dissolved in water, were i.p.-administered in a volume not exceeding 2 ml. Rectal temperature was measured by means of a Cole Parmer thermistor, before and after the administration of the extract, each hour during the first 4 h, and finally at 24 h.
brated at 20 mmHg/cm (0–160 mmHg). Arterial pressure was recorded every 15 min during the 4 h of observation. Simultaneously, cardiac frequency was determined with the aid of pin electrodes placed in the V4 thoracic derivation (Guerrero et al., 2001; Jim´enez et al., 2001). Once the animals were stabilized, different doses of the extract dissolved in water (100–400 mg/kg) were i.p.-administered, whereby five animals were used for each dose. 2.6. Rat isolated aorta Following the described methodology in the literature (Dimo et al., 1998; Tsai et al., 1999; Guerrero et al., 2001), the rat thoracic aorta was excised; adhering tissues, as well as endothelium, were removed and then cut into rings of 5–6 mm. The rings were supported with steal hooks, one of which was attached to the bottom of an organ bath and the other connected to a Grass FT03C transducer under a resting tension of 1.5 g in 10 ml of modified Krebs Henseleit buffer solution (in mM): 122.0 NaCl; 4.7 KCl; 2.0 CaCl2 ; 1.2 MgCl2 ; 1.2 KH2 PO4 ; 15.0 NaHCO3 ; 11.5 glucose and 0.026 EDTA; at pH 7.4. The organ bath was maintained at 37 ◦ C and bubbled continuously with 95% O2 and 5% CO2 . After a period of stabilization (60 min), maximal contraction was induced by three consecutive depolarization with 70 mM KCl solution (Emax = 100%). Once the preparation had been washed and the tissue had been recovered, a contractile response was induced with 1 × 10−7 M norepinephrine (NE). The stepwise increase of the extract concentration (0.04–0.2 mg/ml) produced relaxation of the tissue. A second concentration–response curve was obtained using a Ca2+ -free medium (Vinet et al., 1991) and the results were expressed as a percentage relative to the maximum response with K+ . Cumulative concentration–response curves were also obtained by stepwise increase concentration of NE (1 × 10−9 M to 1 × 10−6 M) in the absence and presence of a single dose of the extract (previously determined as the EC50 in the relaxation experiments). According to Vinet et al. (1991) the effective removal of endothelium is demonstrated by adding acetylcholine, therefore, the functional integrity of the endothelium was assessed at the end of each experiment by recording the relaxation induced by acetylcholine (1 × 10−5 M to 1 × 10−4 M).
2.5. Cardiovascular activity
3. Results
Groups of five male Wistar rats each were used. Fasted animals were anesthetized with sodium pentobarbital solution (26 mg/kg i.p.) and a femoral vein catheter was introduced. Anesthesia was maintained throughout the experiment, administering every 2 h a pentobarbital solution (0.26/0.2 mg/ml). The trachea was exposed and the carotid artery was cannulated with a polyethylene catheter to be connected to a Statham P23 DC transducer, and the blood pressure was recorded using a Grass Model 7D polygraph cali-
3.1. Body temperature The intraperitoneal administration of the extract in normothermic rats produced a decrease of the corporal temperature (Fig. 1), reaching a maximum state of hypothermia in the following 1–2 h after treatment with all the extract concentrations. After 3 h, there was a tendency of recovery in this parameter; nevertheless, the effect was long-lasting. Even at the lowest concentration (100 mg/ml) the registered corporal
P. Nicasio, M. Meckes / Journal of Ethnopharmacology 97 (2005) 301–304
Fig. 1. Effects of Jacaranda mimosaefolia leaves extract on rat corporal temperature. Each value represents the mean ± S.E.M. of the rectal temperature of 10 rats of i.p. extract administration. Control (C) or untreated animals (N = 10).
temperature after 4 h (36.3 ± 0.31 ◦ C) was lower than the ini-
tial value (37.1 ± 0.34 ◦ C) before the administration of the extract. The hypothermic effect exhibited a dose-dependent relationship and, at 1 h registration, with the highest concentration employed here (400 mg/kg) the temperature decreased to 34 ± 0.45 ◦ C (Fig. 1); at 24 h the animals recovered more or less the initial value (36.8 ± 0.37 ◦ C). No mortality was detected during the following 2 weeks, the period in which the treated animals were maintained under observation. 3.2. Cardiovascular parameters With respect to the cardiovascular parameters, the crude extract induced bradycardia, which is dose-dependent, during the first 2 h, with a maximum intensity at 2 h after the administration of the extract (Fig. 2). The effect was abrupt and long-lasting and a complete recovery of cardiac frequency to basal levels was not observed during the following 3 h of the study. On the other hand, hypotensive effect was also dosedependent, and arterial pressure reached a maximum decrease of 25% with respect to the initial basal level, 1 h after the ad-
Fig. 2. Effects of Jacaranda mimosaefolia leaves extract on rat cardiac frequency. Each value represents the mean ± S.E.M. of the cardiac frequency of five rats after i.p. extract administration. C is the control group (N = 10).
303
Fig. 3. Effects of Jacaranda mimosaefolia leaves extract on rat mean arterial pressure (MAP). Each value represents the mean ± S.E.M. of the MAP of five rats of i.p. extract administration. C is the control group (N = 10).
ministration of the extract. The preparation remained stable at 90 mmHg in the following 2 and 3 h (Fig. 3). 3.3. Rat isolated aorta Rat aortic rings incubated with NE at 1 × 10−7 M in a medium with and without Ca2+ , developed a contractile tension of 1332 ± 104 and 516 ± 95 (mg of developed tension/mg of wet tissue weight), respectively. The extract relaxed NE-contractile responses in a concentration-dependent manner. This effect was also observed when the tissue was incubated in a Ca2+ -free medium. The difference between the calculated EC50 ’s, namely 0.13 mg/ml with Ca2+ and 0.11 mg/ml without Ca2+ , was not significant at p < 0.05 (Fig. 4). Moreover, the addition of the crude extract (EC50 = 0.13 mg/ml with Ca2+ ) induced a decrease in the contractile response to NE (1 × 10−6 M) and the contractile–response curves showed a displacement to the right (Fig. 5). The extract
Fig. 4. Relaxation induced by Jacaranda mimosaefolia leaves extract on rat aortic rings pre-contracted with norepinephrine 1 × 10−7 M in a medium with (䊉) and without () Ca2+ . Relaxation is expressed as percentage respect the maximal contraction achieved with NE. Each point represents the mean ± S.E.M. (N = 12).
304
P. Nicasio, M. Meckes / Journal of Ethnopharmacology 97 (2005) 301–304
The herein demonstrated pharmacological properties serve to explain the wide use of Jacaranda mimosaefolia leaves by Mexican populations to treat hypertension. However, further research is needed to determine which compound or compounds are responsible for the pharmacological effect and to ascertain with accuracy its precise mechanism of action, i.e., whether it is a ␣-1 or ␣-2 adrenergic blocker. Finally, based on the obtained results it was not clear whether the hypothermic effect is due to a direct action of the extract on the central nervous system or due to a consequence of the long-lasting hypotension experimented by the animals.
References Fig. 5. Concentration–response curves of norepinephrine (NE) on ratisolated aortic rings in the absence () and presence of (0.13 mg/ml) Jacaranda mimosaefolia leaves extract ( ). The developed tension of each accumulative concentration is expressed as percentage of the maximal contractile response achieved by a 70 mM KCl depolarizing solution in each aortic ring. Each point represents the mean ± S.E.M. (N = 12).
significantly reduced NE pD2 value from 8.2 to 6.7 (−log of the mean molar concentration of the agonist producing 50% of the maximal response to 1 × 10−6 M NE). For the control, pD2 value was similar to that already reported in the literature (Vinet et al., 1991). In order to get the same response with the extract, it was necessary to increase the NE concentration about 100 times.
4. Discussion and conclusions According to the registered cardiovascular parameters, hypotension, already described for some of the isolated compounds of Jacaranda mimosaefolia, was corroborated in the pharmacological evaluation of the crude aqueous methanol extract. Moreover, the recorded bradycardia and the induced relaxation of the NE-contracted aorta in vitro, could be attributed to the notable hypotension registered in the animals treated with this extract. Contractions induced by NE are due partly to calcium release from intracellular storage sites and, partly, to the influx of extracellular calcium into the cell via receptor channels following ␣1 -receptors activation. In the present experiment, the contracted rings with NE were relaxed with and without calcium. Furthermore, the rightward displacement of the concentration–response curves to NE in the presence of the extract seems to indicate a mechanism of blockade of ␣adrenergic receptors. This assumption is supported by the fact that calculated EC50 for the extract was unchanged by calcium removal.
Dimo, T., Rakootorina, S., Kamgang, R., Tan, P.V., Kamany, A., Bopelet, M., 1998. Effect of leaf aqueous extract of Bidens pilosa (Asteraceae) on KCl and norepinephrine-induced contractions of rat aorta. Journal of Ethnopharmacology 60, 179–182. Gambaro, V., Garbarino, J.A., Galeffi, C., Nicoletti, M., Messana, I., Marini-Bettolo, G.B., 1988. Constituents of Jacaranda mimosaefolia. Revista Latinoamericana de Qu´ımica (M´exico) 19, 17–19. Guerrero, M.F., Carr´on, R., Mart´ın, M.L., San Rom´an, L., Reguero, M.T., 2001. Antihypertensive and vasorelaxant effects of aqueous extracts from Croton schiedeanus Shlecht in rats. Journal of Ethnopharmacology 75, 33–36. Jim´enez, G., Hasegawa, M., Rodr´ıguez, M., Estrada, O., M´endez, J., Castillo, A., G´onzalez-M´ujica, F., Motta, N., V´asquez, J., RomeroVecchione, E., 2001. Biological screening of plants of the Venezuelan Amazons. Journal of Ethnopharmacology 77, 77–83. Mahran, G.H., El-Fishawy, A.M., Hosny, A.M.S., Hilal, A.M., 1991. Phytochemical and antimicrobial study of Jacaranda mimosaefolia D. Don grown in Egypt. Herba Hungarica 30, 98–104. Monroy-Ort´ıs, C., Castillo-Espa˜na, P., 2000. Plantas medicinales utilizadas en el Estado de Morelos. Centro de Investigaciones Biol´ogicas, Universidad Aut´onoma del Estado de Morelos, p. 87. Ogura, M., Cordell, G.A., Farnsworth, N.R., 1977. Potential anticancer agents IV. Constituents of Jacaranda caucana (Bignoniaceae). Lloydia 40, 157–168. Osuna, L., Lozoya, X., 1989. Plantas medicinales usadas por la medicina tradicional para el tratamiento de padecimientos gastrointestinales infecciosos. Revista Medica del IMSS (M´exico) 27, 305– 311. Sankara Subramian, S., Nagarajan, S., Sulochana, N., 1972. Flavonoids of eight Bignoniaceous Plants. Phytochemistry 11, 1499. Sankara Subramian, S., Nagarajan, S., Sulochana, N., 1973. Hydroquinone from the leaves of Jacaranda mimosaefolia (Bignoniaceae). Phytochemistry 12, 220–221. Tsai, H.Y., Lin, Y.T., Chen, C.F., Tsai, C.H., Chen, Y.F., 1999. Effects of veratrine and paeoniflorin on the isolated rat aorta. Journal of Ethnopharmacology 66, 249–255. Vinet, R., Brieva, C., Pinardi, G., Penna, M., 1991. Influence of extracellular Ca2+ on the modulation of alpha-adrenergic-induced contractions in rat aorta. General Pharmacology 22, 365. Weniger, B., Robledo, S., Arango, G.J., Deharo, E., Arag´on, R., Mu˜noz, V., Callapa, J., Lobstein, A., Anton, R., 2001. Antiprotozoal activities of Colombian plants. Journal of Ethnopharmacology 78, 193– 200.
Hypotensive effect of the hydroalcoholic extract from Jacaranda mimosaefolia leaves in rats Pilar Nicasioa , Mariana Meckesb,∗ a
Centro de Investigaci´on Biom´edica del Sur (IMSS), Argentina No. 1, Centro, 62790 Xochitepec, Morelos, M´exico b Unidad de Investigaci´ on M´edica en Farmacolog´ıa de Productos Naturales, Hospital de Pediatr´ıa, Centro M´edico Nacional Siglo XXI (IMSS), Avenida Cuauht´emoc 330, Colonia Doctores, 06725 Distrito Federal, M´exico Received 30 November 2003; received in revised form 1 November 2004; accepted 15 November 2004 Available online 13 January 2005
Abstract Hypothermic and cardiovascular activities of the methanol extract of Jacaranda mimosaefolia leaves were tested. To evaluate the hypotensive properties, anesthetized rats were used and temperature, blood pressure, and cardiac frequency were recorded. In addition, the in vitro effect produced by the extract on induced contraction with norepinephrine (NE) in rat aorta rings was evaluated. The extract produced a significant hypothermic effect with a maximum at 2 h, an effect which was accompanied by hypotension and low cardiac frequency, physiological conditions that were again re-established to the following 2 h. In isolated aorta preparations norepinephrine antagonistic effect was not correlated with the presence of Ca2+ , pD2 for NE was modified by the extract, an effect that could explain a blockade of the adrenergic receptors. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Hypotension; Cardiovascular activity; Isolated aorta; Medicinal plant; Jacaranda mimosaefolia
1. Introduction The genus Jacaranda (Bignoniaceae) is widely distributed in the tropical and subtropical areas of the world. Some species are used in traditional medicine of different countries to cure wounds, ulcers, and as an astringent in diarrhoea and dysentery (Gambaro et al., 1988; Mahran et al., 1991). Jacaranda mimosaefolia D. Don is an ornamental tree with attractive foliage and flowers, known in Mexico as “jacaranda”; the leaves and flowers, and the seeds are used in the state of Morelos to treat hypertension and amoebic infections (Osuna and Lozoya, 1989; Monroy-Ort´ıs and CastilloEspa˜na, 2000). Pharmacological evaluation on Jacaranda caucana revealed antiprotozoal (Weniger et al., 2001), cytotoxic (CA9KB cells), and antitumor (leuk-P388 in mouse) properties (Ogura et al., 1977). Jacaranone was demonstrated to be the
∗ Corresponding author. Tel.: +52 55 56 27 69 00x3234/3235; fax: +52 55 57 61 09 52. E-mail address: [email protected] (M. Meckes).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.11.017
compound responsible for the cytotoxic and antitumor activities of the plant (Ogura et al., 1977). The preliminary screening for potential antimicrobial activity of Jacaranda mimosaefolia grown in Egypt, showed that aqueous extract from the twigs and the seeds were active against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Bacillus cereus, Klebsiella pneumoniae, Pseudomonas sp., Mycobacterium phlei and Neisseria sp., although it had no effect on the growth of Candida sp. (Mahran et al., 1991). Jacaranone and scutellarein-7-glucuronide (Sankara et al., 1972), a phenyl-propanoid with hypotensive and analgesic properties, have been isolated from the leaves; 1-hydroxy4-oxo-2,5-cyclohexadien-1-acetate ethyl ester has also been detected in the fruits. Moreover, verbascoside, phenylacetic-glucoside and a new glucose ester named jacaranose have been isolated from the leaves (Gambaro et al., 1988). It has been assumed that jacaranose interacts with jacaranone to produce the jacaranoside which is metabolized in the human digestive tract and induces the formation of a lactonic compound that has antiulcer properties (Sankara et al., 1973). In an attempt to establish validation of the attributed hypotensive properties of the plant, a biological evaluation of
302
P. Nicasio, M. Meckes / Journal of Ethnopharmacology 97 (2005) 301–304
the methanol crude extract of Jacaranda mimosaefolia was carried out using different experimental models.
2. Materials and methods 2.1. Plant material Samples of Jacaranda mimosaefolia D. Don were collected in the State of Morelos in June 2002; the plant was taxonomically certified by Professor Abigail Aguilar, IMSSM Herbarium Director, based on specimen vouchers that were deposited for reference under the code numbers 11051, 11052, 11053. 2.2. Extract preparation The leaves of flowering Jacaranda mimosaefolia (1.7 kg) were dried at 27 ◦ C protected from direct light. The powdered material was extracted at room temperature by percolation with methanol–water (80:20) and concentrated under low pressure. The solid residue (510 g) was maintained at −20 ◦ C for further pharmacological tests. 2.3. Animal conditions Male Wistar rats (300–350 g) were maintained in controlled environmental conditions with a 12-h light/dark cycle with free access to standard rodent pellet food (Harlan Teklad) and water ad libitum, and the animals were fasted for 12 h before experimentation. In all in vivo studies, methanol extract was administered to the animals at lower doses to the previously determined LD50 = 655 mg/kg (601–714 mg/kg) in rats. The Institutional Ethical Committee approved the study. 2.4. Hypothermic effect Groups of ten male adult Wistar rats were used. Different doses (100–400 mg/kg) of the extract dissolved in water, were i.p.-administered in a volume not exceeding 2 ml. Rectal temperature was measured by means of a Cole Parmer thermistor, before and after the administration of the extract, each hour during the first 4 h, and finally at 24 h.
brated at 20 mmHg/cm (0–160 mmHg). Arterial pressure was recorded every 15 min during the 4 h of observation. Simultaneously, cardiac frequency was determined with the aid of pin electrodes placed in the V4 thoracic derivation (Guerrero et al., 2001; Jim´enez et al., 2001). Once the animals were stabilized, different doses of the extract dissolved in water (100–400 mg/kg) were i.p.-administered, whereby five animals were used for each dose. 2.6. Rat isolated aorta Following the described methodology in the literature (Dimo et al., 1998; Tsai et al., 1999; Guerrero et al., 2001), the rat thoracic aorta was excised; adhering tissues, as well as endothelium, were removed and then cut into rings of 5–6 mm. The rings were supported with steal hooks, one of which was attached to the bottom of an organ bath and the other connected to a Grass FT03C transducer under a resting tension of 1.5 g in 10 ml of modified Krebs Henseleit buffer solution (in mM): 122.0 NaCl; 4.7 KCl; 2.0 CaCl2 ; 1.2 MgCl2 ; 1.2 KH2 PO4 ; 15.0 NaHCO3 ; 11.5 glucose and 0.026 EDTA; at pH 7.4. The organ bath was maintained at 37 ◦ C and bubbled continuously with 95% O2 and 5% CO2 . After a period of stabilization (60 min), maximal contraction was induced by three consecutive depolarization with 70 mM KCl solution (Emax = 100%). Once the preparation had been washed and the tissue had been recovered, a contractile response was induced with 1 × 10−7 M norepinephrine (NE). The stepwise increase of the extract concentration (0.04–0.2 mg/ml) produced relaxation of the tissue. A second concentration–response curve was obtained using a Ca2+ -free medium (Vinet et al., 1991) and the results were expressed as a percentage relative to the maximum response with K+ . Cumulative concentration–response curves were also obtained by stepwise increase concentration of NE (1 × 10−9 M to 1 × 10−6 M) in the absence and presence of a single dose of the extract (previously determined as the EC50 in the relaxation experiments). According to Vinet et al. (1991) the effective removal of endothelium is demonstrated by adding acetylcholine, therefore, the functional integrity of the endothelium was assessed at the end of each experiment by recording the relaxation induced by acetylcholine (1 × 10−5 M to 1 × 10−4 M).
2.5. Cardiovascular activity
3. Results
Groups of five male Wistar rats each were used. Fasted animals were anesthetized with sodium pentobarbital solution (26 mg/kg i.p.) and a femoral vein catheter was introduced. Anesthesia was maintained throughout the experiment, administering every 2 h a pentobarbital solution (0.26/0.2 mg/ml). The trachea was exposed and the carotid artery was cannulated with a polyethylene catheter to be connected to a Statham P23 DC transducer, and the blood pressure was recorded using a Grass Model 7D polygraph cali-
3.1. Body temperature The intraperitoneal administration of the extract in normothermic rats produced a decrease of the corporal temperature (Fig. 1), reaching a maximum state of hypothermia in the following 1–2 h after treatment with all the extract concentrations. After 3 h, there was a tendency of recovery in this parameter; nevertheless, the effect was long-lasting. Even at the lowest concentration (100 mg/ml) the registered corporal
P. Nicasio, M. Meckes / Journal of Ethnopharmacology 97 (2005) 301–304
Fig. 1. Effects of Jacaranda mimosaefolia leaves extract on rat corporal temperature. Each value represents the mean ± S.E.M. of the rectal temperature of 10 rats of i.p. extract administration. Control (C) or untreated animals (N = 10).
temperature after 4 h (36.3 ± 0.31 ◦ C) was lower than the ini-
tial value (37.1 ± 0.34 ◦ C) before the administration of the extract. The hypothermic effect exhibited a dose-dependent relationship and, at 1 h registration, with the highest concentration employed here (400 mg/kg) the temperature decreased to 34 ± 0.45 ◦ C (Fig. 1); at 24 h the animals recovered more or less the initial value (36.8 ± 0.37 ◦ C). No mortality was detected during the following 2 weeks, the period in which the treated animals were maintained under observation. 3.2. Cardiovascular parameters With respect to the cardiovascular parameters, the crude extract induced bradycardia, which is dose-dependent, during the first 2 h, with a maximum intensity at 2 h after the administration of the extract (Fig. 2). The effect was abrupt and long-lasting and a complete recovery of cardiac frequency to basal levels was not observed during the following 3 h of the study. On the other hand, hypotensive effect was also dosedependent, and arterial pressure reached a maximum decrease of 25% with respect to the initial basal level, 1 h after the ad-
Fig. 2. Effects of Jacaranda mimosaefolia leaves extract on rat cardiac frequency. Each value represents the mean ± S.E.M. of the cardiac frequency of five rats after i.p. extract administration. C is the control group (N = 10).
303
Fig. 3. Effects of Jacaranda mimosaefolia leaves extract on rat mean arterial pressure (MAP). Each value represents the mean ± S.E.M. of the MAP of five rats of i.p. extract administration. C is the control group (N = 10).
ministration of the extract. The preparation remained stable at 90 mmHg in the following 2 and 3 h (Fig. 3). 3.3. Rat isolated aorta Rat aortic rings incubated with NE at 1 × 10−7 M in a medium with and without Ca2+ , developed a contractile tension of 1332 ± 104 and 516 ± 95 (mg of developed tension/mg of wet tissue weight), respectively. The extract relaxed NE-contractile responses in a concentration-dependent manner. This effect was also observed when the tissue was incubated in a Ca2+ -free medium. The difference between the calculated EC50 ’s, namely 0.13 mg/ml with Ca2+ and 0.11 mg/ml without Ca2+ , was not significant at p < 0.05 (Fig. 4). Moreover, the addition of the crude extract (EC50 = 0.13 mg/ml with Ca2+ ) induced a decrease in the contractile response to NE (1 × 10−6 M) and the contractile–response curves showed a displacement to the right (Fig. 5). The extract
Fig. 4. Relaxation induced by Jacaranda mimosaefolia leaves extract on rat aortic rings pre-contracted with norepinephrine 1 × 10−7 M in a medium with (䊉) and without () Ca2+ . Relaxation is expressed as percentage respect the maximal contraction achieved with NE. Each point represents the mean ± S.E.M. (N = 12).
304
P. Nicasio, M. Meckes / Journal of Ethnopharmacology 97 (2005) 301–304
The herein demonstrated pharmacological properties serve to explain the wide use of Jacaranda mimosaefolia leaves by Mexican populations to treat hypertension. However, further research is needed to determine which compound or compounds are responsible for the pharmacological effect and to ascertain with accuracy its precise mechanism of action, i.e., whether it is a ␣-1 or ␣-2 adrenergic blocker. Finally, based on the obtained results it was not clear whether the hypothermic effect is due to a direct action of the extract on the central nervous system or due to a consequence of the long-lasting hypotension experimented by the animals.
References Fig. 5. Concentration–response curves of norepinephrine (NE) on ratisolated aortic rings in the absence () and presence of (0.13 mg/ml) Jacaranda mimosaefolia leaves extract ( ). The developed tension of each accumulative concentration is expressed as percentage of the maximal contractile response achieved by a 70 mM KCl depolarizing solution in each aortic ring. Each point represents the mean ± S.E.M. (N = 12).
significantly reduced NE pD2 value from 8.2 to 6.7 (−log of the mean molar concentration of the agonist producing 50% of the maximal response to 1 × 10−6 M NE). For the control, pD2 value was similar to that already reported in the literature (Vinet et al., 1991). In order to get the same response with the extract, it was necessary to increase the NE concentration about 100 times.
4. Discussion and conclusions According to the registered cardiovascular parameters, hypotension, already described for some of the isolated compounds of Jacaranda mimosaefolia, was corroborated in the pharmacological evaluation of the crude aqueous methanol extract. Moreover, the recorded bradycardia and the induced relaxation of the NE-contracted aorta in vitro, could be attributed to the notable hypotension registered in the animals treated with this extract. Contractions induced by NE are due partly to calcium release from intracellular storage sites and, partly, to the influx of extracellular calcium into the cell via receptor channels following ␣1 -receptors activation. In the present experiment, the contracted rings with NE were relaxed with and without calcium. Furthermore, the rightward displacement of the concentration–response curves to NE in the presence of the extract seems to indicate a mechanism of blockade of ␣adrenergic receptors. This assumption is supported by the fact that calculated EC50 for the extract was unchanged by calcium removal.
Dimo, T., Rakootorina, S., Kamgang, R., Tan, P.V., Kamany, A., Bopelet, M., 1998. Effect of leaf aqueous extract of Bidens pilosa (Asteraceae) on KCl and norepinephrine-induced contractions of rat aorta. Journal of Ethnopharmacology 60, 179–182. Gambaro, V., Garbarino, J.A., Galeffi, C., Nicoletti, M., Messana, I., Marini-Bettolo, G.B., 1988. Constituents of Jacaranda mimosaefolia. Revista Latinoamericana de Qu´ımica (M´exico) 19, 17–19. Guerrero, M.F., Carr´on, R., Mart´ın, M.L., San Rom´an, L., Reguero, M.T., 2001. Antihypertensive and vasorelaxant effects of aqueous extracts from Croton schiedeanus Shlecht in rats. Journal of Ethnopharmacology 75, 33–36. Jim´enez, G., Hasegawa, M., Rodr´ıguez, M., Estrada, O., M´endez, J., Castillo, A., G´onzalez-M´ujica, F., Motta, N., V´asquez, J., RomeroVecchione, E., 2001. Biological screening of plants of the Venezuelan Amazons. Journal of Ethnopharmacology 77, 77–83. Mahran, G.H., El-Fishawy, A.M., Hosny, A.M.S., Hilal, A.M., 1991. Phytochemical and antimicrobial study of Jacaranda mimosaefolia D. Don grown in Egypt. Herba Hungarica 30, 98–104. Monroy-Ort´ıs, C., Castillo-Espa˜na, P., 2000. Plantas medicinales utilizadas en el Estado de Morelos. Centro de Investigaciones Biol´ogicas, Universidad Aut´onoma del Estado de Morelos, p. 87. Ogura, M., Cordell, G.A., Farnsworth, N.R., 1977. Potential anticancer agents IV. Constituents of Jacaranda caucana (Bignoniaceae). Lloydia 40, 157–168. Osuna, L., Lozoya, X., 1989. Plantas medicinales usadas por la medicina tradicional para el tratamiento de padecimientos gastrointestinales infecciosos. Revista Medica del IMSS (M´exico) 27, 305– 311. Sankara Subramian, S., Nagarajan, S., Sulochana, N., 1972. Flavonoids of eight Bignoniaceous Plants. Phytochemistry 11, 1499. Sankara Subramian, S., Nagarajan, S., Sulochana, N., 1973. Hydroquinone from the leaves of Jacaranda mimosaefolia (Bignoniaceae). Phytochemistry 12, 220–221. Tsai, H.Y., Lin, Y.T., Chen, C.F., Tsai, C.H., Chen, Y.F., 1999. Effects of veratrine and paeoniflorin on the isolated rat aorta. Journal of Ethnopharmacology 66, 249–255. Vinet, R., Brieva, C., Pinardi, G., Penna, M., 1991. Influence of extracellular Ca2+ on the modulation of alpha-adrenergic-induced contractions in rat aorta. General Pharmacology 22, 365. Weniger, B., Robledo, S., Arango, G.J., Deharo, E., Arag´on, R., Mu˜noz, V., Callapa, J., Lobstein, A., Anton, R., 2001. Antiprotozoal activities of Colombian plants. Journal of Ethnopharmacology 78, 193– 200.