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Cardiovascular activity of the crude alkaloidal fraction from Tabernaemontana pandacaqui in the rat
Journal of Ethnopharmacology 59 (1998) 131 – 137
Cardiovascular activity of the crude alkaloidal fraction from Tabernaemontana pandacaqui in the rat T. Taesotikul a, A. Panthong a,*, D. Kanjanapothi a, R. Verpoorte b, J.J.C. Scheffer b b
a Department of Pharmacology, Faculty of Medicine, Chiang Mai Uni6ersity, Chiang Mai 50200, Thailand Leiden/Amsterdam Center for Drug Research, Di6ision of Pharmacognosy, Leiden Uni6ersity, P.O. Box 9502, 2300 RA Leiden, The Netherlands
Received 4 April 1997; received in revised form 2 September 1997; accepted 3 September 1997
Abstract The effects of a crude alkaloidal (CA) fraction from the stem of Tabernaemontana pandacaqui Poir. on the blood pressure and heart rate were investigated in conscious as well as anesthetized rats. The CA fraction exerted a hypotensive activity in both experimental models. In pentobarbital anesthetized rats, an intravenous administration of the CA fraction caused two consecutive hypotensive and bradycardiac responses. In order to investigate the mechanism of the responses, the effect of the CA fraction on the blood pressure and the heart rate was tested in various experimental animals such as pithed rats, reserpinized rats under pentobarbital anesthesia and atropine- or chlorpheniramine-treated rats under pentobarbital anesthesia. The results obtained suggest that the hypotensive and bradycardiac responses of the first phase might involve cholinergic and central mechanisms, whereas those of the second phase involve mechanisms which are mediated by central, biogenic amines, acetylcholine and histamine. © 1998 Elsevier Science Ireland Ltd. Keywords: Tabernaemontana pandacaqui; Crude alkaloidal fraction; Hypotension; Bradycardia activity
1. Introduction Many of alkaloids from plants have been shown to act on the cardiovascular system. Plants of the genus Tabernaemontana, which contain a variety of alkaloids (van Beek et al., 1984) have been reported as antihypertensive (Tsiang and Li, * Corresponding author
1977). In our previous studies, the alcoholic extracts from various parts of T. pandacaqui showed a hypotensive activity in pentobarbital anesthetized rats, and the most potent activity was found in the stem part (Taesotikul et al., 1989). This study was carried out to test the crude alkaloidal (CA) fraction from the stem part of T. pandacaqui for hypotensive activity and to assess the possible mechanism(s) of the activity.
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2. Materials and methods
2.1. Plant material Stems of T. pandacaqui Poir. (synonym: Er6atamia pandacaqui (Poir.) Pichon), were collected in Chiang Mai, Thailand. Voucher specimens (PHCO-CM-012) were deposited at the Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Thailand, and at the Herbarium of Agricultural University, Wageningen, The Netherlands.
2.2. Preparation of CA fraction The dried stem of T. pandacaqui was extracted with 95% ethanol and the alkaloids were separated by an acid–base extraction in the usual manner, and the yield of CA fraction obtained was 0.41% of dry plant material. For the pharmacological testing the CA fraction was dissolved in 0.1 N HCl/distilled water (1:10).
2.3. Animals Sprague–Dawley rats purchased from the National Laboratory Animal Center, Salaya, Mahidol University, Nakorn Pathom, Thailand, were used. All animals were acclimatized for at least 1 week before starting of experiments.
2.4. Systolic blood pressure (SBP) of conscious rats Rats of either sex weighing 200 – 250 g were used. SBP and pulse rate were measured by means of the tail cuff method using Bloodless Pressure Recorder 8006 (Ugo Basile, Milano). The SBP was recorded 5 min before and 5, 10, 20 and 30 min after an intraperitoneal administration of the CA fraction or control vehicle.
2.5. Blood pressure and heart rate of pentobarbital anesthetized rats and pithed rats Rats of either sex weighing between 200 and 250 g were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). The trachea was cannulated with a
polyethylene tube to facilitate spontaneous respiration. Drugs and CA fraction were slowly injected via a cannula inserted into the external jugular vein. The maximum volume of injection was 0.5 ml. The systemic blood pressure was recorded from the femoral artery via an arterial cannula connected to a pressure transducer (model P23 ID, Gould Statham Instruments, Hato Rey, Puerto Rico) and the heart rate was recorded using a tachograph (model 7P 44C Grass Instrument, Quincy, MA) driven by the blood pressure waves. The preparation was allowed to equilibrate for at least 30 min before the experiment was started. In some experiments in which reserpinized rats were used, the rats were pretreated with reserpine at a dose of 5 mg/kg, i.p. once a day, starting 2 days before the experiment. Experiments using pithed rats were carried out according to the method of Gillespie and Muir (1967) with slight modification. A pithed rod of 2 mm diameter and about 25 cm long was inserted obliquely into the eye socket and passed through the whole length of the spinal canal under the ether anesthesia.
2.6. Drugs The following drugs were used: atropine sulfate (The Government Pharmaceutical Organization, Thailand), chlorpheniramine maleate (Piriton, Glaxo, Thailand), norepinephrine (Levophed, Metro, Manila, Philippines), pentobarbital sodium (Nembutal, Abbott Laboratories, North Chicago, IL) and reserpine (Sigma, St. Louis, MO).
2.7. Statistical analysis Student’s t test was used to determine a significant difference between a control group and drugor CA fraction-treated group.
3. Results
3.1. Effect of CA fraction on systolic blood pressure (SBP) and heart rate in conscious rats The effect of an intraperitoneal injection of
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Fig. 1. Time course of the hypotensive action induced by the CA fraction in conscious rats. Numbers in the parentheses represents the heart rate (beats/min) and expressed as mean 9 S.E.M. (N= 5). Significant difference from the initial level: *** P B0.001, ** P B 0.01, * P B0.05. —— = vehicle-control group, + ——+ =CA 500 mg/kg and *——* =CA 750 mg/kg.
the CA fraction on the SBP and heart rate of conscious rats is shown in Fig. 1. In the vehicle-control group, an administration of vehicle did not cause any significant change of SBP. Injection of the CA fraction resulted in a fall in SBP reaching its maximal level at 5 mins after the injection. At the doses of 500 and 750
mg/kg the fall of SBP of 32.0 9 3.2 and 44.89 8.2 mmHg from the initial level of 145.09 8.2 and 141.29 6.7 mmHg, respectively, were observed. The SBP gradually returned to the initial level within 20 min after the injection of the CA fraction. A significant increase (P B 0.05) in heart rate
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Table 1 Dose (mg/kg)
Initial level (mmHg)
Decrease from initial level (mmHg)
(%)
Duration (min)
40.8 92.6 45.3 93.2 55.1 91.8 70.3 92.4
4.6 9 0.4 13.0 9 4.3 25.2 9 7.1 \60
1st Hypotensive phase 2nd Hypotensive phase (a) Effect of CA fraction on mean arterial blood pressure in pentobarbital anesthetized rats 6.25 (6) 148.09 606 81.99 8.5 61.0 95.8 12.5 (10) 142.79 4.1 67.49 4.5 65.0 95.8 25 (11) 146.09 2.8 75.39 3.9 80.8 92.6 50.0 (8) 145.39 5.2 72.29 5.0 102.5 95.9 Dose (mg/kg)
Initial level (beats/min)
Decrease from initial level (beats/min)
(%)
(b) Change of heart rate during the prolonged hypotensive phase (2nd phase) of anesthetized rats following the injection of CA fraction 6.25 (6) 400.0917.7 85.89 18.7 20.9 9 4.2 12.5 (10) 397.5911.1 75.59 16.5 18.99 4.0 25.0 (11) 409.1910.7 103.69 14.7 25.0 9 3.3 50.0 (8) 408.1915.1 154.49 13.6 38.6 9 3.7 Values are expressed as means 9 S.E.M. The number of rats in each group is shown in parenthesis.
18.7% occurred concomitantly during the hypotensive effect of 500 mg/kg of the CA fraction. At 30 min after the injection of CA injection at the dose of 750 mg/kg, a significant decrease (PB 0.01) in heart rate of 8.7% was observed, even though at this time a non-significant change of SBP from the initial level was noted.
3.2. Effect on mean arterial blood pressure and heart rate in pentobarbital anesthetized rats An intravenous injection of the CA fraction produced two phases of hypotensive and bradycardiac responses. The duration of the hypotension and bradycardia of the first phase was brief ( 15 s), whereas that of the second phase was prolonged and dose related (r =0.997). At a dose of 25 mg/kg (Table 1), the CA fraction caused a decrease of blood pressure and heart rate of 55 and 25%, respectively, and the former effect lasted about 25 min.
3.3. Effect on mean arterial blood pressure and heart rate in pithed rats The mean arterial blood pressure and heart rate of pithed rats was 55.3 mmHg and 176 beats/min, respectively (Table 2). An injection of the CA fraction produced an increase in blood pressure (with a duration of 1–5 min) and a slight decrease in heart rate. The response was then followed by a prolonged decrease in blood pressure that was accompanied by a marked increase in heart rate.
3.4. Effect on mean arterial blood pressure and heart rate in reserpinized rats under pentobarbital anesthesia The mean arterial blood pressure and heart rate of reserpinized rats under pentobarbital anesthesia were 88 mmHg and 240 beats/min, respectively. An administration of the CA fraction caused a fall followed by a prolonged increase in blood pressure and heart rate (Table 2). The pattern of the first fall in blood pressure and heart
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Table 2 Effect of CA fraction at a dose of 25 mg/kg on mean arterial blood pressure (MABP) and heart rate (HR) in normotensive anesthetized, reserpinized and pithed rats, respectively Experiment
Normotensive rats Reserpinized rats Pithed rats
N
11 7 5
Initial level
146.092.8 (400.09 17.7) 88.19 2.6* (240.79 6.8*) 55.39 5.5* (176.09 13.2*)
Change of MABP and HR from the initial level (%) 1st phase
2nd phase
−51.59 2.8 (\−50) −45.296.4 (\−50) +67.8910.0* (−16.29 2.3*)
−55.191.8 (−25.093.3) +31.894.6* (+10.393.3*) −31.396.9** (+61.59 19.9*)
Values are expressed as means9 S.E.M. The numbers in parenthesis are HR of the rat. The initial level of MABP and HR are expressed as mmHg and beats/min, respectively. −, decrease in MABP and HR; +, increase in MABP and HR. Significant difference from normotensive rat: * PB0.001 and ** PB0.01.
rate was similar to that of the first hypotensive and bradycardial responses when it was tested in the normotensive rats.
3.5. Effect on mean arterial blood pressure and heart rate in atropine- and chlorpheniraminetreated rats under pentobarbital anesthesia Intravenous administration of atropine (2 mg/ kg) and chlorpheniramine (2 mg/kg) was used to test the effect on the response of blood pressure and heart rate to the CA fraction. CA fraction at the dose of 50 mg/kg was given intravenously 3 min after the antagonist. As shown in Table 3, only atropine could antagonize the first hypotensive and bradycardiac responses to the CA fraction (P B0.001). The prolonged hypotensive response in phase two was partially reduced by atropine (PB 0.05) and by chlorpheniramine (PB 0.01), but the two antagonists did not affect the bradycardiac response in this phase.
3.6. Influence of CA fraction on the response to bilateral carotid occlusion and norepinephrine in normotensi6e rats under pentobarbital anesthesia Pressor and tachycardiac responses to a bilateral carotid occlusion (OCC) and to norepinephrine administration (NE, 1 mg/kg) were tested during the maximum hypotensive period (after about 5 min) caused by the CA fraction at a dose of 50 mg/kg. It was found that the responses to OCC and NE were markedly potentiated by the CA fraction (Table 4).
4. Discussion The CA fraction from the stem of T. pandacaqui exhibited a hypotensive activity when tested in conscious as well as in pentobarbital-anesthetized rats. An intraperitoneal administration of a low dose (500 mg/kg) of the CA fraction in conscious rats caused a hypotensive response which was accompanied with tachycardia, whereas a higher dose (750 mg/kg) did not cause a significant change of heart rate even though a greater decrease in blood pressure occurred. The tachycardia may be due to a reflex occurring as a response to the fall in blood pressure; the higher dose of the CA fraction might cause a myocardiac depression which could overcome the reflex. In anesthetized rats, an intravenous injection of the CA fraction elicited two phases of hypotensive and bradycardiac responses. In the first phase, the responses were brisk, whereas those of the second phase were more prolonged. Only the hypotensive response of the second phase was dose-dependent. The patterns of the responses of blood pressure and heart rate to the CA fraction were similar to those to the alcoholic extract (Taesotikul et al., 1989). It is therefore suggested that the alkaloid components of T. pandacaqui play a role in the hypotensive and bradycardiac activities. The first phase of hypotensive and bradycardiac responses to the CA fraction was almost completely blocked by atropine but not by chlorpheniramine administration. In addition, in the experiments using pithed rats, a hypertensive response was
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Table 3 Effect of CA fraction at a dose of 50 mg/kg on mean arterial blood pressure (MABP) and heart rate (HR) in normotensive, atropine and chlorpheniramine treated anesthetized rats Experiment
N
Initial level
Decrease from initial level (%) 1st Hypotensive phase
Normotensive rats Atropine treated rats Chlorpheniramine treated rats
8 8 8
145.3 9 5.2 (408.09 15.1) 50.3 94.1 (\50) 151.09 6.0 (428.79 11.8) 16.5 93.9* (14.99 1.6*) 168.99 2.8** (446.9 9 6.7***) 49.0 9 5.1 (\50)
2nd Hypotensive phase 70.3 9 2.4 (38.69 3.7) 55.8 9 4.8*** (31.0 94.8) 55.9 9 3.9** (36.2 9 3.1)
Atropine (2 mg/kg) and chlorpheniramine (2 mg/kg) were intravenously administered 3 min before the CA fraction. Values are expressed as means 9 S.E.M. The numbers in parenthesis are HR of the rat. The initial level of MABP and HR are expressed as mmHg and beats/min, respectively. Significant difference from normotensive rat: * PB0.001, ** PB0.01 and *** PB0.05.
observed instead. The results suggest that hypotensive response could be due to cholinergic and central mechanisms, and not to a histaminic mechanism. However, the central mechanism for this first hypotensive response is not mediated through the biogenic amines since the response is still present in reserpinized rats. With respect to the cholinergic mechanisms, it is possible that they might involve a central vagal activation, and/or peripheral stimulation of muscarinic receptors of the heart and vascular smooth muscle. During the second phase of hypotensive response, the pressor effect of NE and reflex pressor response to a bilateral occlusion of the common carotid arteries were still persisted. These findings therefore exclude mechanisms
such as an alpha-adrenergic receptor blockade and interference with sympathetic transmission. In addition, the pressor response to NE was found to be enhanced by the CA fraction. The response was similar to the response to NE in the presence of an uptake-1 inhibitor (Macmillan, 1959). Biogenic amines such as serotonin was found to exhibit hypotensive effect by the mechanisms mediated by 5-HT1 receptors (Burkhalter et al., 1995). The prolonged hypotensive and bradycardiac responses of the second phase were found to change into hypertension and tachycardia in reserpinized rats. These results suggest that the prolonged hypotension and bradycardia elicited by the CA fraction is mediated through biogenic amines which possibly be serotonin. In addition,
Table 4 Influence of the CA fraction on the response of blood pressure (BP) and heart rate (HR) to carotid artery occlusion (OCC) and norepinephrine (NE) in pentobarbital anesthetized rats Challenge
Increase from the level just before the challenge BP (%)
OCC NE
HR (%)
Before CA
After CA
Before CA
After CA
18.9 9 3.0 35.4 93.7
32.2 9 5.2** 86.69 12.0**
3.7 90.7 3.9 90.9
10.7 92.1** 11.8 91.7*
Carotid arteries were occluded bilaterally for 1 min and NE at the dose of 1 mg/kg was intravenously administered, after the maximum hypotension response was induced by the CA fraction at the dose of 50 mg/kg. Values are expressed as means 9S.E.M., N= 9. Significant difference from before CA: * PB0.001 and ** PB0.01.
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the central, cholinergic and histaminic mechanisms might also contribute to the hypotensive and bradycardiac effects of the CA fraction since the effects were partially reduced in pithed rats as well as in atropine- and chlorpheniramine-treated rats. In anesthetized rats with an intact CNS, the CA fraction caused hypotension and bradycardia. In contrast, the CA fraction caused hypertension and tachycardia when tested in pithed rats. It is suggested that the CA fraction exerted hypotension and bradycardiac effects through mechanism(s) involving the CNS, and that the CA fraction contained alkaloids with hypertensive as well as hypotensive activities, and also with positive and negative chronotropic activities. It is also possible that the hypertension and tachycardia result from peripheral vasoconstriction and positive chronotropic activities. In conclusion, the hypotensive and bradycardiac activities of T. pandacaqui are most likely due to its alkaloid components; central and/or peripheral mechanisms being involved in the activities. However, among the alkaloids present, some of them might possess a cholinergic-like, and might show vasoconstrictor and cardiac stimulant actions. Identification of the active alkaloid(s) awaits further pharmacological evaluation of purified fraction(s) of the CA fraction tested.
.
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Acknowledgements Financial support for the project ‘Thai Medicinal Plants and Herbal Medicine’ (Account 0096040) by the Dutch Ministry of Foreign Affairs and the State University of Leiden is greatefully acknowledged.
References Burkhalter, A., Julius, D., Frick, O.L., 1995. Histamine, serotonin, and the ergot alkaloids. In: Katzung, B.G. (Ed.), Basic and Clinical Pharmacology, 6th ed. Appleton and Lange, East Norwalk, USA, p. 263. Gillespie, J.S., Muir, T.C., 1967. A method of stimulating the complete sympathetic outflow from the spinal cord to blood vessels in the pithed rats. British Journal of Pharmacology 30, 78 – 87. Taesotikul, T., Panthong, A., Kanjanpothi, D., Verpoorte, R., Scheffer, J.J.C., 1989. Cardiovascular effects of Tabernaemontana pandacaqui Poir. Journal of Ethnopharmacology 27 (1-2), 107 – 119. Tsiang, Y., Li, P.T., 1977. Flora Republicae Popularis Sinicae, vol. 63. Kexue Chubanshe, Beijng, pp. 98, 119. Macmillan, W.H., 1959. A hypothesis concerning the effects of cocaine on the action of sympathomimetic amines. British Journal of Pharmacology 14, 385 – 391. van Beek, T.A., Verpoorte, R., Baerheim-Svendsen, A., 1984. Tabernaemontana (Apocynaceae): a review of its taxonomy, phytochemistry, ethnobotany and pharmacology. Journal of Ethnopharmacology 10, 1 – 156.
Cardiovascular activity of the crude alkaloidal fraction from Tabernaemontana pandacaqui in the rat T. Taesotikul a, A. Panthong a,*, D. Kanjanapothi a, R. Verpoorte b, J.J.C. Scheffer b b
a Department of Pharmacology, Faculty of Medicine, Chiang Mai Uni6ersity, Chiang Mai 50200, Thailand Leiden/Amsterdam Center for Drug Research, Di6ision of Pharmacognosy, Leiden Uni6ersity, P.O. Box 9502, 2300 RA Leiden, The Netherlands
Received 4 April 1997; received in revised form 2 September 1997; accepted 3 September 1997
Abstract The effects of a crude alkaloidal (CA) fraction from the stem of Tabernaemontana pandacaqui Poir. on the blood pressure and heart rate were investigated in conscious as well as anesthetized rats. The CA fraction exerted a hypotensive activity in both experimental models. In pentobarbital anesthetized rats, an intravenous administration of the CA fraction caused two consecutive hypotensive and bradycardiac responses. In order to investigate the mechanism of the responses, the effect of the CA fraction on the blood pressure and the heart rate was tested in various experimental animals such as pithed rats, reserpinized rats under pentobarbital anesthesia and atropine- or chlorpheniramine-treated rats under pentobarbital anesthesia. The results obtained suggest that the hypotensive and bradycardiac responses of the first phase might involve cholinergic and central mechanisms, whereas those of the second phase involve mechanisms which are mediated by central, biogenic amines, acetylcholine and histamine. © 1998 Elsevier Science Ireland Ltd. Keywords: Tabernaemontana pandacaqui; Crude alkaloidal fraction; Hypotension; Bradycardia activity
1. Introduction Many of alkaloids from plants have been shown to act on the cardiovascular system. Plants of the genus Tabernaemontana, which contain a variety of alkaloids (van Beek et al., 1984) have been reported as antihypertensive (Tsiang and Li, * Corresponding author
1977). In our previous studies, the alcoholic extracts from various parts of T. pandacaqui showed a hypotensive activity in pentobarbital anesthetized rats, and the most potent activity was found in the stem part (Taesotikul et al., 1989). This study was carried out to test the crude alkaloidal (CA) fraction from the stem part of T. pandacaqui for hypotensive activity and to assess the possible mechanism(s) of the activity.
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2. Materials and methods
2.1. Plant material Stems of T. pandacaqui Poir. (synonym: Er6atamia pandacaqui (Poir.) Pichon), were collected in Chiang Mai, Thailand. Voucher specimens (PHCO-CM-012) were deposited at the Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Thailand, and at the Herbarium of Agricultural University, Wageningen, The Netherlands.
2.2. Preparation of CA fraction The dried stem of T. pandacaqui was extracted with 95% ethanol and the alkaloids were separated by an acid–base extraction in the usual manner, and the yield of CA fraction obtained was 0.41% of dry plant material. For the pharmacological testing the CA fraction was dissolved in 0.1 N HCl/distilled water (1:10).
2.3. Animals Sprague–Dawley rats purchased from the National Laboratory Animal Center, Salaya, Mahidol University, Nakorn Pathom, Thailand, were used. All animals were acclimatized for at least 1 week before starting of experiments.
2.4. Systolic blood pressure (SBP) of conscious rats Rats of either sex weighing 200 – 250 g were used. SBP and pulse rate were measured by means of the tail cuff method using Bloodless Pressure Recorder 8006 (Ugo Basile, Milano). The SBP was recorded 5 min before and 5, 10, 20 and 30 min after an intraperitoneal administration of the CA fraction or control vehicle.
2.5. Blood pressure and heart rate of pentobarbital anesthetized rats and pithed rats Rats of either sex weighing between 200 and 250 g were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). The trachea was cannulated with a
polyethylene tube to facilitate spontaneous respiration. Drugs and CA fraction were slowly injected via a cannula inserted into the external jugular vein. The maximum volume of injection was 0.5 ml. The systemic blood pressure was recorded from the femoral artery via an arterial cannula connected to a pressure transducer (model P23 ID, Gould Statham Instruments, Hato Rey, Puerto Rico) and the heart rate was recorded using a tachograph (model 7P 44C Grass Instrument, Quincy, MA) driven by the blood pressure waves. The preparation was allowed to equilibrate for at least 30 min before the experiment was started. In some experiments in which reserpinized rats were used, the rats were pretreated with reserpine at a dose of 5 mg/kg, i.p. once a day, starting 2 days before the experiment. Experiments using pithed rats were carried out according to the method of Gillespie and Muir (1967) with slight modification. A pithed rod of 2 mm diameter and about 25 cm long was inserted obliquely into the eye socket and passed through the whole length of the spinal canal under the ether anesthesia.
2.6. Drugs The following drugs were used: atropine sulfate (The Government Pharmaceutical Organization, Thailand), chlorpheniramine maleate (Piriton, Glaxo, Thailand), norepinephrine (Levophed, Metro, Manila, Philippines), pentobarbital sodium (Nembutal, Abbott Laboratories, North Chicago, IL) and reserpine (Sigma, St. Louis, MO).
2.7. Statistical analysis Student’s t test was used to determine a significant difference between a control group and drugor CA fraction-treated group.
3. Results
3.1. Effect of CA fraction on systolic blood pressure (SBP) and heart rate in conscious rats The effect of an intraperitoneal injection of
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Fig. 1. Time course of the hypotensive action induced by the CA fraction in conscious rats. Numbers in the parentheses represents the heart rate (beats/min) and expressed as mean 9 S.E.M. (N= 5). Significant difference from the initial level: *** P B0.001, ** P B 0.01, * P B0.05. —— = vehicle-control group, + ——+ =CA 500 mg/kg and *——* =CA 750 mg/kg.
the CA fraction on the SBP and heart rate of conscious rats is shown in Fig. 1. In the vehicle-control group, an administration of vehicle did not cause any significant change of SBP. Injection of the CA fraction resulted in a fall in SBP reaching its maximal level at 5 mins after the injection. At the doses of 500 and 750
mg/kg the fall of SBP of 32.0 9 3.2 and 44.89 8.2 mmHg from the initial level of 145.09 8.2 and 141.29 6.7 mmHg, respectively, were observed. The SBP gradually returned to the initial level within 20 min after the injection of the CA fraction. A significant increase (P B 0.05) in heart rate
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Table 1 Dose (mg/kg)
Initial level (mmHg)
Decrease from initial level (mmHg)
(%)
Duration (min)
40.8 92.6 45.3 93.2 55.1 91.8 70.3 92.4
4.6 9 0.4 13.0 9 4.3 25.2 9 7.1 \60
1st Hypotensive phase 2nd Hypotensive phase (a) Effect of CA fraction on mean arterial blood pressure in pentobarbital anesthetized rats 6.25 (6) 148.09 606 81.99 8.5 61.0 95.8 12.5 (10) 142.79 4.1 67.49 4.5 65.0 95.8 25 (11) 146.09 2.8 75.39 3.9 80.8 92.6 50.0 (8) 145.39 5.2 72.29 5.0 102.5 95.9 Dose (mg/kg)
Initial level (beats/min)
Decrease from initial level (beats/min)
(%)
(b) Change of heart rate during the prolonged hypotensive phase (2nd phase) of anesthetized rats following the injection of CA fraction 6.25 (6) 400.0917.7 85.89 18.7 20.9 9 4.2 12.5 (10) 397.5911.1 75.59 16.5 18.99 4.0 25.0 (11) 409.1910.7 103.69 14.7 25.0 9 3.3 50.0 (8) 408.1915.1 154.49 13.6 38.6 9 3.7 Values are expressed as means 9 S.E.M. The number of rats in each group is shown in parenthesis.
18.7% occurred concomitantly during the hypotensive effect of 500 mg/kg of the CA fraction. At 30 min after the injection of CA injection at the dose of 750 mg/kg, a significant decrease (PB 0.01) in heart rate of 8.7% was observed, even though at this time a non-significant change of SBP from the initial level was noted.
3.2. Effect on mean arterial blood pressure and heart rate in pentobarbital anesthetized rats An intravenous injection of the CA fraction produced two phases of hypotensive and bradycardiac responses. The duration of the hypotension and bradycardia of the first phase was brief ( 15 s), whereas that of the second phase was prolonged and dose related (r =0.997). At a dose of 25 mg/kg (Table 1), the CA fraction caused a decrease of blood pressure and heart rate of 55 and 25%, respectively, and the former effect lasted about 25 min.
3.3. Effect on mean arterial blood pressure and heart rate in pithed rats The mean arterial blood pressure and heart rate of pithed rats was 55.3 mmHg and 176 beats/min, respectively (Table 2). An injection of the CA fraction produced an increase in blood pressure (with a duration of 1–5 min) and a slight decrease in heart rate. The response was then followed by a prolonged decrease in blood pressure that was accompanied by a marked increase in heart rate.
3.4. Effect on mean arterial blood pressure and heart rate in reserpinized rats under pentobarbital anesthesia The mean arterial blood pressure and heart rate of reserpinized rats under pentobarbital anesthesia were 88 mmHg and 240 beats/min, respectively. An administration of the CA fraction caused a fall followed by a prolonged increase in blood pressure and heart rate (Table 2). The pattern of the first fall in blood pressure and heart
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Table 2 Effect of CA fraction at a dose of 25 mg/kg on mean arterial blood pressure (MABP) and heart rate (HR) in normotensive anesthetized, reserpinized and pithed rats, respectively Experiment
Normotensive rats Reserpinized rats Pithed rats
N
11 7 5
Initial level
146.092.8 (400.09 17.7) 88.19 2.6* (240.79 6.8*) 55.39 5.5* (176.09 13.2*)
Change of MABP and HR from the initial level (%) 1st phase
2nd phase
−51.59 2.8 (\−50) −45.296.4 (\−50) +67.8910.0* (−16.29 2.3*)
−55.191.8 (−25.093.3) +31.894.6* (+10.393.3*) −31.396.9** (+61.59 19.9*)
Values are expressed as means9 S.E.M. The numbers in parenthesis are HR of the rat. The initial level of MABP and HR are expressed as mmHg and beats/min, respectively. −, decrease in MABP and HR; +, increase in MABP and HR. Significant difference from normotensive rat: * PB0.001 and ** PB0.01.
rate was similar to that of the first hypotensive and bradycardial responses when it was tested in the normotensive rats.
3.5. Effect on mean arterial blood pressure and heart rate in atropine- and chlorpheniraminetreated rats under pentobarbital anesthesia Intravenous administration of atropine (2 mg/ kg) and chlorpheniramine (2 mg/kg) was used to test the effect on the response of blood pressure and heart rate to the CA fraction. CA fraction at the dose of 50 mg/kg was given intravenously 3 min after the antagonist. As shown in Table 3, only atropine could antagonize the first hypotensive and bradycardiac responses to the CA fraction (P B0.001). The prolonged hypotensive response in phase two was partially reduced by atropine (PB 0.05) and by chlorpheniramine (PB 0.01), but the two antagonists did not affect the bradycardiac response in this phase.
3.6. Influence of CA fraction on the response to bilateral carotid occlusion and norepinephrine in normotensi6e rats under pentobarbital anesthesia Pressor and tachycardiac responses to a bilateral carotid occlusion (OCC) and to norepinephrine administration (NE, 1 mg/kg) were tested during the maximum hypotensive period (after about 5 min) caused by the CA fraction at a dose of 50 mg/kg. It was found that the responses to OCC and NE were markedly potentiated by the CA fraction (Table 4).
4. Discussion The CA fraction from the stem of T. pandacaqui exhibited a hypotensive activity when tested in conscious as well as in pentobarbital-anesthetized rats. An intraperitoneal administration of a low dose (500 mg/kg) of the CA fraction in conscious rats caused a hypotensive response which was accompanied with tachycardia, whereas a higher dose (750 mg/kg) did not cause a significant change of heart rate even though a greater decrease in blood pressure occurred. The tachycardia may be due to a reflex occurring as a response to the fall in blood pressure; the higher dose of the CA fraction might cause a myocardiac depression which could overcome the reflex. In anesthetized rats, an intravenous injection of the CA fraction elicited two phases of hypotensive and bradycardiac responses. In the first phase, the responses were brisk, whereas those of the second phase were more prolonged. Only the hypotensive response of the second phase was dose-dependent. The patterns of the responses of blood pressure and heart rate to the CA fraction were similar to those to the alcoholic extract (Taesotikul et al., 1989). It is therefore suggested that the alkaloid components of T. pandacaqui play a role in the hypotensive and bradycardiac activities. The first phase of hypotensive and bradycardiac responses to the CA fraction was almost completely blocked by atropine but not by chlorpheniramine administration. In addition, in the experiments using pithed rats, a hypertensive response was
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Table 3 Effect of CA fraction at a dose of 50 mg/kg on mean arterial blood pressure (MABP) and heart rate (HR) in normotensive, atropine and chlorpheniramine treated anesthetized rats Experiment
N
Initial level
Decrease from initial level (%) 1st Hypotensive phase
Normotensive rats Atropine treated rats Chlorpheniramine treated rats
8 8 8
145.3 9 5.2 (408.09 15.1) 50.3 94.1 (\50) 151.09 6.0 (428.79 11.8) 16.5 93.9* (14.99 1.6*) 168.99 2.8** (446.9 9 6.7***) 49.0 9 5.1 (\50)
2nd Hypotensive phase 70.3 9 2.4 (38.69 3.7) 55.8 9 4.8*** (31.0 94.8) 55.9 9 3.9** (36.2 9 3.1)
Atropine (2 mg/kg) and chlorpheniramine (2 mg/kg) were intravenously administered 3 min before the CA fraction. Values are expressed as means 9 S.E.M. The numbers in parenthesis are HR of the rat. The initial level of MABP and HR are expressed as mmHg and beats/min, respectively. Significant difference from normotensive rat: * PB0.001, ** PB0.01 and *** PB0.05.
observed instead. The results suggest that hypotensive response could be due to cholinergic and central mechanisms, and not to a histaminic mechanism. However, the central mechanism for this first hypotensive response is not mediated through the biogenic amines since the response is still present in reserpinized rats. With respect to the cholinergic mechanisms, it is possible that they might involve a central vagal activation, and/or peripheral stimulation of muscarinic receptors of the heart and vascular smooth muscle. During the second phase of hypotensive response, the pressor effect of NE and reflex pressor response to a bilateral occlusion of the common carotid arteries were still persisted. These findings therefore exclude mechanisms
such as an alpha-adrenergic receptor blockade and interference with sympathetic transmission. In addition, the pressor response to NE was found to be enhanced by the CA fraction. The response was similar to the response to NE in the presence of an uptake-1 inhibitor (Macmillan, 1959). Biogenic amines such as serotonin was found to exhibit hypotensive effect by the mechanisms mediated by 5-HT1 receptors (Burkhalter et al., 1995). The prolonged hypotensive and bradycardiac responses of the second phase were found to change into hypertension and tachycardia in reserpinized rats. These results suggest that the prolonged hypotension and bradycardia elicited by the CA fraction is mediated through biogenic amines which possibly be serotonin. In addition,
Table 4 Influence of the CA fraction on the response of blood pressure (BP) and heart rate (HR) to carotid artery occlusion (OCC) and norepinephrine (NE) in pentobarbital anesthetized rats Challenge
Increase from the level just before the challenge BP (%)
OCC NE
HR (%)
Before CA
After CA
Before CA
After CA
18.9 9 3.0 35.4 93.7
32.2 9 5.2** 86.69 12.0**
3.7 90.7 3.9 90.9
10.7 92.1** 11.8 91.7*
Carotid arteries were occluded bilaterally for 1 min and NE at the dose of 1 mg/kg was intravenously administered, after the maximum hypotension response was induced by the CA fraction at the dose of 50 mg/kg. Values are expressed as means 9S.E.M., N= 9. Significant difference from before CA: * PB0.001 and ** PB0.01.
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the central, cholinergic and histaminic mechanisms might also contribute to the hypotensive and bradycardiac effects of the CA fraction since the effects were partially reduced in pithed rats as well as in atropine- and chlorpheniramine-treated rats. In anesthetized rats with an intact CNS, the CA fraction caused hypotension and bradycardia. In contrast, the CA fraction caused hypertension and tachycardia when tested in pithed rats. It is suggested that the CA fraction exerted hypotension and bradycardiac effects through mechanism(s) involving the CNS, and that the CA fraction contained alkaloids with hypertensive as well as hypotensive activities, and also with positive and negative chronotropic activities. It is also possible that the hypertension and tachycardia result from peripheral vasoconstriction and positive chronotropic activities. In conclusion, the hypotensive and bradycardiac activities of T. pandacaqui are most likely due to its alkaloid components; central and/or peripheral mechanisms being involved in the activities. However, among the alkaloids present, some of them might possess a cholinergic-like, and might show vasoconstrictor and cardiac stimulant actions. Identification of the active alkaloid(s) awaits further pharmacological evaluation of purified fraction(s) of the CA fraction tested.
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Acknowledgements Financial support for the project ‘Thai Medicinal Plants and Herbal Medicine’ (Account 0096040) by the Dutch Ministry of Foreign Affairs and the State University of Leiden is greatefully acknowledged.
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