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Cardiovascular effects of Tabernaemontana pandacaqui
107
Journal of Ethnopharmacology. 2’7(1989) 107- 119 Elsevier Scientific Publishers Ireland Ltd.
CARDIOVASCULAR PANDACAQUI
EFFECTS OF TARERNAEMONTHA
T. TAESOTIKUL’, A. PANTHONG’, D. KANJANAPOTHP, J.J.C. SCHEFFEW
R. VERPOORTEb and
“Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50002 (ThailandI and bDivision of Pharmacognosy, Center of Bio-Pharmaceutical Sciences, Leiden University, 2.900 RA Leiden (The Netherlandsj (Accepted April 12, 1989)
Summary Intravenous injection of ethanolic extracts of the stem, leaf and flower of Tabernaemontana panohaqui caused hypotension in pentobarbital anesthetized rats. At high doses (100-300 mg/kg), the flower extract showed a transient hypertensive effect preceeding hypotensive activity. The effects of the extracts on the heart rate of anesthetized rats correlated well with the negative chronotropic and inotropic activity observed with isolated atrium. The hypotensive activity was not inhibited by antihistaminic and antimuscarinic agents. The extracts had no effect on the pressor effects induced by norepinephrine or dual carotid occlusion. These results suggest that the hypotensive action of the extracts is not mediated through histaminic and muscarinic receptors stimulation, a-adrenoceptor blockade or interference of sympathetic transmission.
Introduction Plants of the genus Tabernaemontana (syn. Ervatamiu, family Apocynaceael have been used in traditional medicine for the treatment of fever, pain, dysentery (Boonyaratanakornkit and Supawita, 19771, and for healing wounds caused by snake and centipede bites (Jayaweera, 19811.Some of these plants have been claimed to be effective in the treatment of hypertension (Tsiang and Li, 19771.Recent studies of ethanolic extracts of T. divaricata and T. pandacaqui in rats using hippocratic observational screening suggested vasodilator activity (Taesotikul et al, 19891.The present investigation was carried out to determine the effects of the extracts on the cardiovascular system. Correspondence to: A. Panthong. 0378-8741/$04.55 0 1989 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
108
Materials and methods
Plant materials Stems, leaves and flowers of T. pandacaqui Pair. synonomy: Ervatamia pandacaqui (Pair.) 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 the Agricultural University, Wageningen, The Netherlands. Extraction and preparation of samples The ground, dried material of each plant part was macerated in 95% ethanol at room temperature for 24 h. The ethanolic solutions were evaporated under reduced pressure to viscous syrups and then lyophilized. The powders obtained (yields of 9.8%, 13% and 42% for stem, leaf and flower, respectively) were dissolved in distilled water, centrifuged and the supernatant used for all testing. All doses are expressed here in terms of the lyophilized powders. Isolated rat atrium experiment The experiment was carried out according to the method of Robert and Konjovic (1966). Sprague-Dawley rats of either sex, weighing between 200 -300 g, were used. The atria were isolated and mounted in a lo-ml organ bath filled with continuously oxygenated (95% 0, + 50/6 CO,) Feigen’s solution (NaCl = 9.0, KC1 = 0.42, CaCl, = 0.62, NaHCO, = 0.6 and glucose = 1.0 g/l) at a controlled temperature of 33OC. The spontaneous contraction of the atrium was recorded under the resting tension of 750 mg by a polygraph (model 7D, Grass Instrument Co., Quincy, MA) via a force-displacement transducer (FT 03, Grass Instrument Co., Quincy, MA). The preparation was left to equilibrate for at least 30 min. Anesthetized rat experiment Either sex of Sprague-Dawley rats weighing between 200-250 g were anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal injection). The trachea was cannulated with a polyethylene tube to facilitate spontaneous respiration. Drugs and extracts were slowly injected via the cannula inserted into the external jugular vein. The maximum volume of injection was 0.2 ml. The systemic blood pressure was recorded from the femoral artery via the arterial cannula connected to a pressure transducer (model P23 ID, Gould Statham Instruments Inc., Hato Rey, Puerto Rico) and the heart rate was recorded using a tachograph (model 7P 44C, Grass Instrument Co., Quincy, MA) driven by the blood pressure waves. These parameters were displayed on a polygraph. The preparation was allowed to equilibrate for at least 30 min before the experiment was initiated following the methodology of Farris and Griffith (1949).
109
Drug sources
The following reference materials were obtained from the sources specified: acetylcholine chloride (Calbiochem, Los Angeles, CA); atropine sulfate (The Government Pha~aeeutical Organ~ation, Thailand~ chlorpheniramine maleate (Piriton, Glaxo Co., Thailand); histamine dihydrochloride (B grade, Calbiochem, Los Angeles, CA); norepinephrine bitartrate (Levophed, Metro, Manila, Philippines); pentobarbital sodium (Nembutal, Abbott Laboratories, North Chicago, IL). Results Effects
on isolated
rat atl*ia
Stem extract of T. pandacaqui at doses of 2.5, 5.0, 7.5 and 10.0 mglml caused a progressive decrease in the force and rate of atria1 contraction (Fig. 11. The decrease was dose-related k = 0.9961.There was a gradual recovery from the decreased force of contraction whereas the decreased rate persisted until the extract was washed out. At doses lower than 2.5 mglml, the stem extract did not show either a stimulant or depressant effect on the atria1 contraction. The extract of the leaf at doses of 2.5, 5.0 and 7.5 mglml caused a doserelated (r = 0.946, Fig. 21 positive inotropic effect, having a maximal response at the dose of 7.5 mglml, whereas the rate of contraction was not affected Cp > 0.051. With the dose of 10 mglml, a decrease in force and rate were observed after the positive inotropic effect and cardiac arrest ensued with doses higher than 10 mglml. Increases in force as well as rate of atria1 contraction were observed with the flower extract at doses of 0.16,0.31 and 0.63 mglml (Fig. 31. It is interesting to note that at doses of more than 5 mg/ml, the extract caused three phases of response: (il a decrease of force and rate; (ii) recovery of force and rate; and (iii) a decrease of force and the onset of arrhythmia (Fig. 41. Effects
on rat blood pressure
Tables 1 and 2 summarize the effects of stem and leaf extracts of 7! pandacaqui on the blood pressure and the heart rate of pentobarbital anesthetized rats. The extracts exhibited a definite hypotensive effect. At high doses (50 mg/kg of stem extract, 300 mg/kg of leaf extract) the maximal hypotensive effect (58.4% and 58.0%. respectively) was observed with a duration of effect of more than 60 min (Fig. 51. A statistical decrease in heart rate @’ < 0.051 was accompanied by the hypotensive effect of 6.25, 12.5, 25 and 50 mgkg of stem extract and 200 and 300 mg/kg of leaf extract. Transient hypotension (l-5 min duration1 and increased heart rate were observed with low doses (25 and 50 mglkgl of the flower extract. At higher doses (100, 200 and 300 mglkgl a biphasic response of blood pressure was seen: a pressor effect and a subsequent fall (Tables 3 and 41. The fall of blood
110
pressure reached the maximal extent at the dose of 200 mg/kg. It was observed that transient bradycardia or arrhythmia and tachycardia occurred simultaneously with both pressor and depressor phases, respectively, and finally stabilized on a prolonged decrease of heart rate (Fig. 51. The relationship between the doses and hypotensive responses of extracts of T. punducaqui is illustrated in Fig. 6. It was found that the hypotensive
A e $1 i---Tpist)
2.5 mg/ml
8 9 10.0
mg/ml
*.(N=9) . I*8
(N+B)
L
ti. IN=91
Fig. 1. (A) Mean response (+ S.E.M.) of isolated rat atria to stem extract of T. pundaeaqui at doses of 2.5 mg/ml (upper panel) and 10 mg/ml (lower panel). (B) Inotropic (0) and chronotropic (a) effects of various doses of the stem extract. Significant change: *F’ < 0.05; **P < 0.01: ***I’ < 0.001.
2.5 mg/ml r
ip (If1 ,I? ,mg/ml
(N=S)
(N:IO)
g/ml
Fig. 2. (A) Mean response (it S.E.M.) of isolated rat atria to leaf extract of 2’. pamfucaqui at doses of 2.5 mg/ml (upper panel) and 10 mglml (lower panel). (B) Inotropic (0) and chronotropic (k3) effects of various doses of the leaf extract. Significant change: l**P < 0.001.
effect of the extracts from stems, leaves and flowers was clearly dosedependent (7 values of 0.989, 0.992 and 0.971, respectively). When comparing their EC, values, the stem extract was the most potent, and the flower extract was only slightly more potent than the leaf extract. Effects on sympathetic stimulation The three doses (50 mg/kg of stem extract, 300 mg/kg of leaf and flower
112
A z 8I
8I Tp(fl)
0.63
mg/ml
B
(N=8)
me*
T
(N-8)
I 0.16
*‘I
T
3
2.5 mgktl
Fig. 3. (A) Mean response (k S.E.M.) of isolated rat atria to flower extract of T. pandacaqui at doses of 0.16 mg/ml (upper panel) and 0.63 mg/ml (lower panel). (B) Inotropic (0) and chronotropic Cm) effects of various doses of the flower extract. Significant change: **P < 0.01; ***P<
0.001.
extracts) which caused the maximal and most prolonged fall in rat blood pressure were used. As shown in Table 5, during the maximal hypotensive period (about 2.5 min after injection), the pressor responses to the carotid occlusion and norepinephrine (1 rg/kg) were slightly less than those when the
113
tI P
ON BLOOD PRESSURE (BP) AND HEART RATE (HR) OF ANESTHETIZED RATS
37.5 56.5 67.8 84.0 81.2
+ + f f +:
4.7 5.1 4.6 4.8 6.0
Pressure (mmHg) 26.6 42.0 48.6 59.0 58.4
f zt + + f
Change (%)
25 50 100 200 300
1.9 5.4 3.9 5.9 7.4
15.9 30.4 44.6 52.4 58.0
+ + + f k
3.2** 3.9**+ 2.1+** 2.3*++ 1.5***
2-10 10-15 10-20 15->60 >60
f 17.6 zt 15.2 + 9.2 zk 22.6 f 8.7
+ 4.8 + 5.5 +3.2 - 17.4 - 24.5
k + + f f
+ 22.5 +23.7 + 12.5 - 75.0 - 110.0
24.8 42.2 66.0 76.2 85.7
f f f f it
Change (%I
Rate (beats/mini
Duration (min)
Pressure (mmHg)
Change
Change of HR from control
(%I
3.5 3.7* 2.7* 2.1** 3.1+*
3.8 3.7 2.1 5.0’ 2.0*+*
33.5 beatslmin. Values are expressed as means +
Decrease of BP from control
Mean control blood pressure was 147.3 f 14.6 mmHg while mean heart rate was 435.5 f S.E.M.. N = 5. Significant from control: *P < 0.05; **P < 0.01; ***P< 0.001.
Dose kng/kg)
f + f + f
8.2 10.8 12.2 13.4 23.0
14.2 15.9 10.4 8.8 14.2
33.3 43.0 45.0 53.3 78.3
l-2 2-6 5-10 5->60 >60
f f * f f
Change (%I
Rate (beats/mini
Duration (min)
Decrease of HR from control
ON BLOOD PRESSURE (BP) AND HEART RATE (HR) OF ANESTHETIZED RATS
3.1** 3.9*** 3.4+** 3.1*** 1.0***
Decrease of BP from control
EFFECT OF LEAF EXTRACT OF T. PANDACAQUZ
TABLE 2
3.0 6.25 12.5 25.0 50.0
(mg/kg)
Dose
Mean control blood pressure was 139.8 f 14.7 mmHg while mean heart rate was 385.8 + 32.7 beatslmin. Values are expressed as means f S.E.M., N = 5. Significant from control: lP < 0.05; l*P < 0.01; ***P< 0.001.
EFFECT OF STEM EXTRACT OF T. PANDACAQUZ
TABLE 1
E *
Fig. 5. Typical responses of blood pressure (BP) and heart rate (HR) in pentobarbital anesthetized rats to (A) 50 mg/kg of stem extract, (B) 300 mg/kg of leaf extract and (Cl 300 mg/kg of flower extract of T. pandacaqui
carotid occlusion and norepinephrine were performed before the administration of the extracts. However, the results obtained did not show any statistically significant difference P > 0.05). Antagonist studies The effects of two possible antagonists: atropine (an antimuscarinic agent) and chlorpheniramine (an antihistaminic agent) on the hypotensive effect of
OF ANESTHETIZED
RATS FOLLOWING
THE INJECTION
OF THE FLOWER
EXTRACT
RESPONSE
25 50 100 200 300
Dose (mglkg)
+ 50.0 +84.2 +72.5 +84.2 +99.0
f 7.6 f 11.8 2 8.9 + 10.4 f 18.4
+ 12.2 1-22.6 + 16.8 +20.7 + 19.5
2 f f k +
2.0** 4.1** 2.2** 4.1*** 4.1**
0.5- 2 l-2 3-5 4-6 4-6
0 0 - 36.7 f 19.8 -95.8 f 21.3 - 132.0 2 26.4
0 0 - 8.6 * 4.7 -22.7 f 3.1+* -26.6 -t 4.9**
Change (Oh)
Rate (beats/min)
Duration (min)
Rate (beatslmin)
Change (O/o)
Second phase (bradycardia)
First phase (tachycardia)
0 0 5-20 >60 >60
Duration (min)
as means
+
RATS FOLLOWING Values are expressed
OF ANESTHETIZED
Mean control blood pressure was 131.4 + 11.3 mmHg while mean heart rate was 411.3 -C 32.5 beats/mm. S.E.M., N = 5. Significant from control: *P < 0.05; **P < 0.01; ***P < 0.001.
CHANGE OF HEART RATE DURING DEPRESSOR PHASE OF BLOOD PRESSURE THE INJECTION OF THE FLOWER EXTRACT OF T. PANDACAQUI.
TABLE 4
k
OF T.
Mean control blood pressure was 131.4 k 11.3 mmHg while mean heart rate was 411.3 f 32.5 beats/min. Values are expressed as means S.E.M., N = 5. Significant from control: *P < 0.05; **P < 0.01; ***P < 0.001. --_---_-l_ ____-__-_-Pressor phase Dose Depressor phase (mglkg) .~__-.-_-_ Pressure Change Duration Pressure Change Duration ImmHg) (%I kec) (mmHg) (min) (%I ---.-_ -.-- --“.._ ._--- --1- ...~.. _ ._____~ ~..~...__-.-~-----__._. -- . .ll-.._---- -__.-_ 25 0 0 0 - 33.5 2 6.2 - 23.9 -c 4.2** l-3 50 ‘1 0 0 - 43.3 * 6.6 - 35.4 + 4.8** l-5 100 + 16.2 % 3.2 t 7.6 -+ 2.34 15-30 - 53.9 + 2.6 -40.9 f 2.4*** 10-20 200 + 25.3 r 2.7 -t 19.9 f 2.3*** 15--30 - 79.2 f 5.3 - 61.8 + 3.0*** 25->60 300 + 35.5 = 2.9 + 26.4 + 2.8*** 15-30 - 82.6 f 4.8 - 60.5 + 0.9*** >60
PANDA CA Q UI.
CHANGE OF BLOOD PRESSURE
TABLE 3
117
II':"'"
f2.09+33.92r
60c
p
::%~+39.09,
1
2.5
Fig. 6. Relationship and flower (O-01
between doses and hypotensive extracts of 2’. panahcaqui
activity
of stem (e-01,
leaf
(+--+I
stem, leaf and flower extracts of T. pundacaqui are shown in Fig. 7. The extracts were given 5 min after the administration of atropine (2 mg/kg) (1 mg/kg). It was found that atropine and or chlorpheniramine chlorpheniramine, which were demonstrated to block the effects of acetylcholine (5 pg/kg) and histamine (5 pg/kg), respectively, did not affect the hypotensive response to stem (12.5 mg/kg), leaf and flower (100 mg/kg) extracts. TABLE
5
INFLUENCEOF T.PANDACQUIEXTRACTS ONTHERESPONSEOFBLOODPRESSURE (BP) TO SYMPATHETIC AND ADRENOCEPTOR ACTIVATION IN ANESTHETIZED RATS. Carotid arteries were occluded (OCCl bilaterally for 1 min, and norepinephrme [NE, dose of 1 rg/ kg1 was injected intravenously, after the maximal hypotension response to tbe extracts. Values are expressed as means 2 S.E.M., N = 9. Challenge
Part used
Dose (mglkgl
Before extract
After extract
occ
Stem Leaf Flower
50 300 300
25.6 f 1.3 26.8 f 2.7 24.8 + 1.9
22.2 f 24.5 f 22.8 f
NE
Stem Leaf Flower
50 300 300
40.5 f 2.8 43.0 f 2.1 42.6 & 3.7
36.7 + 2.6 39.8 2 2.8 41.0 * 3.0
BP increase from control (e/cl
2.6 1.1 2.0
118
DEPRESSOR EFf ECTS
kontfol
r=-=lr-----l
Jc AC),
St Lf
Fl
Wirt.
St
U
After Chbtph6nimmine
Mt.
F
Fig. 7. Effect of atropine (2 mg/kg) and ehlorpheniramine (1 mg/kg) on hypotensive response to T.
pamhmqui (12.5 mg/kg stem extract, 100 mg/kg leaf and flower extracts). Atropine and chlorpheniramine inhibited the effect of 5 rg/kg acetylcholine (ACU and 5 pg/kg histamine (Hist), respectively, but not those of the extracts. Significant difference from the control effects given before the antagonists: ***P < 0.001.
Discussion
Extracts of T. pandacaqui showed cardiac stimulant as well as depressant effects on spontaneous contraction of rat atria1 strips. The stem extract caused depression of both force and rate. With small to moderate doses of the flower and leaf extracts, an increase in force was observed. High doses of both extracts caused depression of force and rate. Intravenous injection of extracts of T. pandacaqui caused hypotensive responses in anesthetized rats. With the flower extract, doses of 100, 200 and 300 mg/kg produced a transient hypertensive effect before hypotension was seen. This dose-response study suggests that the hypotensive potency of the stem extract is more than that of flower and leaf extracts. It is commonly agreed that the physiological state of the heart, as determined by its force and rate of contraction, influences the maintenance of blood pressure. The results obtained from the isolated rat atrium suggest that the depressant effect of stem extract and high doses of flower and leaf extracts on the heart probably contribute to the hypotensive response. It was found that during the hypotensive response to low doses (25, 50 and 100 mg/kgl of leaf extract, there was no significant increase in heart rate, which also correlated well with the effect of the extract at doses of 2.5, 5, and 10 mglml on the isolated heart. During the hypotensive phase of blood pressure response to flower extract, only tachycardia was seen with low doses (25 and 50 mg/kg).
119
Tachycardia followed by prolonged bradycardia was observed with high doses (100, 200 and 300 mgkg). It is possible that the tachycardia is due to a direct effect on the heart, as seen with the isolated atrium, and/or to a reflex increase in sympathetic activity initiated by the hypotensive effect of the extract. The hypotensive effect of T. pandacaqui was not affected by chlorpheniramine (an antihistaminic) and atropine (an antimuscarinicl. Therefore, it would appear that the extracts exhibit hypotension by actions not mediated through histaminic and muscarinic receptors. In addition, mechanisms such as an a-adrenoceptor blockade and interference with sympathetic transmission can be excluded, since the extracts of T. pandacaqui did not block the pressor effect of norepinephrine and the reflex pressor response to a bilateral occlusion of the common carotid arteries. Acknowledgement
Financial support for the project “Thai Medicinal Plants Medicine” (Account 00960401 by the Dutch Ministry of Foreign the State University of Leiden is gratefully acknowledged.
and Herbal Affairs and
References Boonyaratanakornkit, L. and Supawita, T. (1977) Names of Medicinal Plants and Their Uses. Department of Pharmacognosy, Faculty of Pharmacy, Chulalongkorn University, Bangkok, p. 69. Farris, E.J. and Giffith, J.Q. (1949) The Rat in Laboratory Investigation, 2nd Edn. Hafner Publishing Company, New York, pp. 283-284. Jayaweera, D.M.A.fl981) Medicinal Plants Used in Ceylon, Part 1. National Science Council of Sri Lanka, Colombo, p. 113. Robert, E.M. and Konjovic, J&966) Difference in the chronotropic and inotropic response of the rat atrium to choline esters, cholinesterase inhibitors and certain blocking agents. Journal of Pharmacology and Experimental Therapeutics 169,109- 119. Taesotikul, T., Panthong, A., Kanjanapothi, D., Verpoorte, R., and Scheffer, J.J.C. (1989) Hippocratic screening of ethanolic extracts from two Tabernnemontana species. Journal of Ethnopharmacology 27,99- 106. Tsiang, Y. and Li, P.T. (1977) Flora Republicae Popularis Sinicae, Vol. 63. Kexue Chubanshe, Beijing, pp. 98, 115.
Journal of Ethnopharmacology. 2’7(1989) 107- 119 Elsevier Scientific Publishers Ireland Ltd.
CARDIOVASCULAR PANDACAQUI
EFFECTS OF TARERNAEMONTHA
T. TAESOTIKUL’, A. PANTHONG’, D. KANJANAPOTHP, J.J.C. SCHEFFEW
R. VERPOORTEb and
“Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50002 (ThailandI and bDivision of Pharmacognosy, Center of Bio-Pharmaceutical Sciences, Leiden University, 2.900 RA Leiden (The Netherlandsj (Accepted April 12, 1989)
Summary Intravenous injection of ethanolic extracts of the stem, leaf and flower of Tabernaemontana panohaqui caused hypotension in pentobarbital anesthetized rats. At high doses (100-300 mg/kg), the flower extract showed a transient hypertensive effect preceeding hypotensive activity. The effects of the extracts on the heart rate of anesthetized rats correlated well with the negative chronotropic and inotropic activity observed with isolated atrium. The hypotensive activity was not inhibited by antihistaminic and antimuscarinic agents. The extracts had no effect on the pressor effects induced by norepinephrine or dual carotid occlusion. These results suggest that the hypotensive action of the extracts is not mediated through histaminic and muscarinic receptors stimulation, a-adrenoceptor blockade or interference of sympathetic transmission.
Introduction Plants of the genus Tabernaemontana (syn. Ervatamiu, family Apocynaceael have been used in traditional medicine for the treatment of fever, pain, dysentery (Boonyaratanakornkit and Supawita, 19771, and for healing wounds caused by snake and centipede bites (Jayaweera, 19811.Some of these plants have been claimed to be effective in the treatment of hypertension (Tsiang and Li, 19771.Recent studies of ethanolic extracts of T. divaricata and T. pandacaqui in rats using hippocratic observational screening suggested vasodilator activity (Taesotikul et al, 19891.The present investigation was carried out to determine the effects of the extracts on the cardiovascular system. Correspondence to: A. Panthong. 0378-8741/$04.55 0 1989 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
108
Materials and methods
Plant materials Stems, leaves and flowers of T. pandacaqui Pair. synonomy: Ervatamia pandacaqui (Pair.) 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 the Agricultural University, Wageningen, The Netherlands. Extraction and preparation of samples The ground, dried material of each plant part was macerated in 95% ethanol at room temperature for 24 h. The ethanolic solutions were evaporated under reduced pressure to viscous syrups and then lyophilized. The powders obtained (yields of 9.8%, 13% and 42% for stem, leaf and flower, respectively) were dissolved in distilled water, centrifuged and the supernatant used for all testing. All doses are expressed here in terms of the lyophilized powders. Isolated rat atrium experiment The experiment was carried out according to the method of Robert and Konjovic (1966). Sprague-Dawley rats of either sex, weighing between 200 -300 g, were used. The atria were isolated and mounted in a lo-ml organ bath filled with continuously oxygenated (95% 0, + 50/6 CO,) Feigen’s solution (NaCl = 9.0, KC1 = 0.42, CaCl, = 0.62, NaHCO, = 0.6 and glucose = 1.0 g/l) at a controlled temperature of 33OC. The spontaneous contraction of the atrium was recorded under the resting tension of 750 mg by a polygraph (model 7D, Grass Instrument Co., Quincy, MA) via a force-displacement transducer (FT 03, Grass Instrument Co., Quincy, MA). The preparation was left to equilibrate for at least 30 min. Anesthetized rat experiment Either sex of Sprague-Dawley rats weighing between 200-250 g were anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal injection). The trachea was cannulated with a polyethylene tube to facilitate spontaneous respiration. Drugs and extracts were slowly injected via the cannula inserted into the external jugular vein. The maximum volume of injection was 0.2 ml. The systemic blood pressure was recorded from the femoral artery via the arterial cannula connected to a pressure transducer (model P23 ID, Gould Statham Instruments Inc., Hato Rey, Puerto Rico) and the heart rate was recorded using a tachograph (model 7P 44C, Grass Instrument Co., Quincy, MA) driven by the blood pressure waves. These parameters were displayed on a polygraph. The preparation was allowed to equilibrate for at least 30 min before the experiment was initiated following the methodology of Farris and Griffith (1949).
109
Drug sources
The following reference materials were obtained from the sources specified: acetylcholine chloride (Calbiochem, Los Angeles, CA); atropine sulfate (The Government Pha~aeeutical Organ~ation, Thailand~ chlorpheniramine maleate (Piriton, Glaxo Co., Thailand); histamine dihydrochloride (B grade, Calbiochem, Los Angeles, CA); norepinephrine bitartrate (Levophed, Metro, Manila, Philippines); pentobarbital sodium (Nembutal, Abbott Laboratories, North Chicago, IL). Results Effects
on isolated
rat atl*ia
Stem extract of T. pandacaqui at doses of 2.5, 5.0, 7.5 and 10.0 mglml caused a progressive decrease in the force and rate of atria1 contraction (Fig. 11. The decrease was dose-related k = 0.9961.There was a gradual recovery from the decreased force of contraction whereas the decreased rate persisted until the extract was washed out. At doses lower than 2.5 mglml, the stem extract did not show either a stimulant or depressant effect on the atria1 contraction. The extract of the leaf at doses of 2.5, 5.0 and 7.5 mglml caused a doserelated (r = 0.946, Fig. 21 positive inotropic effect, having a maximal response at the dose of 7.5 mglml, whereas the rate of contraction was not affected Cp > 0.051. With the dose of 10 mglml, a decrease in force and rate were observed after the positive inotropic effect and cardiac arrest ensued with doses higher than 10 mglml. Increases in force as well as rate of atria1 contraction were observed with the flower extract at doses of 0.16,0.31 and 0.63 mglml (Fig. 31. It is interesting to note that at doses of more than 5 mg/ml, the extract caused three phases of response: (il a decrease of force and rate; (ii) recovery of force and rate; and (iii) a decrease of force and the onset of arrhythmia (Fig. 41. Effects
on rat blood pressure
Tables 1 and 2 summarize the effects of stem and leaf extracts of 7! pandacaqui on the blood pressure and the heart rate of pentobarbital anesthetized rats. The extracts exhibited a definite hypotensive effect. At high doses (50 mg/kg of stem extract, 300 mg/kg of leaf extract) the maximal hypotensive effect (58.4% and 58.0%. respectively) was observed with a duration of effect of more than 60 min (Fig. 51. A statistical decrease in heart rate @’ < 0.051 was accompanied by the hypotensive effect of 6.25, 12.5, 25 and 50 mgkg of stem extract and 200 and 300 mg/kg of leaf extract. Transient hypotension (l-5 min duration1 and increased heart rate were observed with low doses (25 and 50 mglkgl of the flower extract. At higher doses (100, 200 and 300 mglkgl a biphasic response of blood pressure was seen: a pressor effect and a subsequent fall (Tables 3 and 41. The fall of blood
110
pressure reached the maximal extent at the dose of 200 mg/kg. It was observed that transient bradycardia or arrhythmia and tachycardia occurred simultaneously with both pressor and depressor phases, respectively, and finally stabilized on a prolonged decrease of heart rate (Fig. 51. The relationship between the doses and hypotensive responses of extracts of T. punducaqui is illustrated in Fig. 6. It was found that the hypotensive
A e $1 i---Tpist)
2.5 mg/ml
8 9 10.0
mg/ml
*.(N=9) . I*8
(N+B)
L
ti. IN=91
Fig. 1. (A) Mean response (+ S.E.M.) of isolated rat atria to stem extract of T. pundaeaqui at doses of 2.5 mg/ml (upper panel) and 10 mg/ml (lower panel). (B) Inotropic (0) and chronotropic (a) effects of various doses of the stem extract. Significant change: *F’ < 0.05; **P < 0.01: ***I’ < 0.001.
2.5 mg/ml r
ip (If1 ,I? ,mg/ml
(N=S)
(N:IO)
g/ml
Fig. 2. (A) Mean response (it S.E.M.) of isolated rat atria to leaf extract of 2’. pamfucaqui at doses of 2.5 mg/ml (upper panel) and 10 mglml (lower panel). (B) Inotropic (0) and chronotropic (k3) effects of various doses of the leaf extract. Significant change: l**P < 0.001.
effect of the extracts from stems, leaves and flowers was clearly dosedependent (7 values of 0.989, 0.992 and 0.971, respectively). When comparing their EC, values, the stem extract was the most potent, and the flower extract was only slightly more potent than the leaf extract. Effects on sympathetic stimulation The three doses (50 mg/kg of stem extract, 300 mg/kg of leaf and flower
112
A z 8I
8I Tp(fl)
0.63
mg/ml
B
(N=8)
me*
T
(N-8)
I 0.16
*‘I
T
3
2.5 mgktl
Fig. 3. (A) Mean response (k S.E.M.) of isolated rat atria to flower extract of T. pandacaqui at doses of 0.16 mg/ml (upper panel) and 0.63 mg/ml (lower panel). (B) Inotropic (0) and chronotropic Cm) effects of various doses of the flower extract. Significant change: **P < 0.01; ***P<
0.001.
extracts) which caused the maximal and most prolonged fall in rat blood pressure were used. As shown in Table 5, during the maximal hypotensive period (about 2.5 min after injection), the pressor responses to the carotid occlusion and norepinephrine (1 rg/kg) were slightly less than those when the
113
tI P
ON BLOOD PRESSURE (BP) AND HEART RATE (HR) OF ANESTHETIZED RATS
37.5 56.5 67.8 84.0 81.2
+ + f f +:
4.7 5.1 4.6 4.8 6.0
Pressure (mmHg) 26.6 42.0 48.6 59.0 58.4
f zt + + f
Change (%)
25 50 100 200 300
1.9 5.4 3.9 5.9 7.4
15.9 30.4 44.6 52.4 58.0
+ + + f k
3.2** 3.9**+ 2.1+** 2.3*++ 1.5***
2-10 10-15 10-20 15->60 >60
f 17.6 zt 15.2 + 9.2 zk 22.6 f 8.7
+ 4.8 + 5.5 +3.2 - 17.4 - 24.5
k + + f f
+ 22.5 +23.7 + 12.5 - 75.0 - 110.0
24.8 42.2 66.0 76.2 85.7
f f f f it
Change (%I
Rate (beats/mini
Duration (min)
Pressure (mmHg)
Change
Change of HR from control
(%I
3.5 3.7* 2.7* 2.1** 3.1+*
3.8 3.7 2.1 5.0’ 2.0*+*
33.5 beatslmin. Values are expressed as means +
Decrease of BP from control
Mean control blood pressure was 147.3 f 14.6 mmHg while mean heart rate was 435.5 f S.E.M.. N = 5. Significant from control: *P < 0.05; **P < 0.01; ***P< 0.001.
Dose kng/kg)
f + f + f
8.2 10.8 12.2 13.4 23.0
14.2 15.9 10.4 8.8 14.2
33.3 43.0 45.0 53.3 78.3
l-2 2-6 5-10 5->60 >60
f f * f f
Change (%I
Rate (beats/mini
Duration (min)
Decrease of HR from control
ON BLOOD PRESSURE (BP) AND HEART RATE (HR) OF ANESTHETIZED RATS
3.1** 3.9*** 3.4+** 3.1*** 1.0***
Decrease of BP from control
EFFECT OF LEAF EXTRACT OF T. PANDACAQUZ
TABLE 2
3.0 6.25 12.5 25.0 50.0
(mg/kg)
Dose
Mean control blood pressure was 139.8 f 14.7 mmHg while mean heart rate was 385.8 + 32.7 beatslmin. Values are expressed as means f S.E.M., N = 5. Significant from control: lP < 0.05; l*P < 0.01; ***P< 0.001.
EFFECT OF STEM EXTRACT OF T. PANDACAQUZ
TABLE 1
E *
Fig. 5. Typical responses of blood pressure (BP) and heart rate (HR) in pentobarbital anesthetized rats to (A) 50 mg/kg of stem extract, (B) 300 mg/kg of leaf extract and (Cl 300 mg/kg of flower extract of T. pandacaqui
carotid occlusion and norepinephrine were performed before the administration of the extracts. However, the results obtained did not show any statistically significant difference P > 0.05). Antagonist studies The effects of two possible antagonists: atropine (an antimuscarinic agent) and chlorpheniramine (an antihistaminic agent) on the hypotensive effect of
OF ANESTHETIZED
RATS FOLLOWING
THE INJECTION
OF THE FLOWER
EXTRACT
RESPONSE
25 50 100 200 300
Dose (mglkg)
+ 50.0 +84.2 +72.5 +84.2 +99.0
f 7.6 f 11.8 2 8.9 + 10.4 f 18.4
+ 12.2 1-22.6 + 16.8 +20.7 + 19.5
2 f f k +
2.0** 4.1** 2.2** 4.1*** 4.1**
0.5- 2 l-2 3-5 4-6 4-6
0 0 - 36.7 f 19.8 -95.8 f 21.3 - 132.0 2 26.4
0 0 - 8.6 * 4.7 -22.7 f 3.1+* -26.6 -t 4.9**
Change (Oh)
Rate (beats/min)
Duration (min)
Rate (beatslmin)
Change (O/o)
Second phase (bradycardia)
First phase (tachycardia)
0 0 5-20 >60 >60
Duration (min)
as means
+
RATS FOLLOWING Values are expressed
OF ANESTHETIZED
Mean control blood pressure was 131.4 + 11.3 mmHg while mean heart rate was 411.3 -C 32.5 beats/mm. S.E.M., N = 5. Significant from control: *P < 0.05; **P < 0.01; ***P < 0.001.
CHANGE OF HEART RATE DURING DEPRESSOR PHASE OF BLOOD PRESSURE THE INJECTION OF THE FLOWER EXTRACT OF T. PANDACAQUI.
TABLE 4
k
OF T.
Mean control blood pressure was 131.4 k 11.3 mmHg while mean heart rate was 411.3 f 32.5 beats/min. Values are expressed as means S.E.M., N = 5. Significant from control: *P < 0.05; **P < 0.01; ***P < 0.001. --_---_-l_ ____-__-_-Pressor phase Dose Depressor phase (mglkg) .~__-.-_-_ Pressure Change Duration Pressure Change Duration ImmHg) (%I kec) (mmHg) (min) (%I ---.-_ -.-- --“.._ ._--- --1- ...~.. _ ._____~ ~..~...__-.-~-----__._. -- . .ll-.._---- -__.-_ 25 0 0 0 - 33.5 2 6.2 - 23.9 -c 4.2** l-3 50 ‘1 0 0 - 43.3 * 6.6 - 35.4 + 4.8** l-5 100 + 16.2 % 3.2 t 7.6 -+ 2.34 15-30 - 53.9 + 2.6 -40.9 f 2.4*** 10-20 200 + 25.3 r 2.7 -t 19.9 f 2.3*** 15--30 - 79.2 f 5.3 - 61.8 + 3.0*** 25->60 300 + 35.5 = 2.9 + 26.4 + 2.8*** 15-30 - 82.6 f 4.8 - 60.5 + 0.9*** >60
PANDA CA Q UI.
CHANGE OF BLOOD PRESSURE
TABLE 3
117
II':"'"
f2.09+33.92r
60c
p
::%~+39.09,
1
2.5
Fig. 6. Relationship and flower (O-01
between doses and hypotensive extracts of 2’. panahcaqui
activity
of stem (e-01,
leaf
(+--+I
stem, leaf and flower extracts of T. pundacaqui are shown in Fig. 7. The extracts were given 5 min after the administration of atropine (2 mg/kg) (1 mg/kg). It was found that atropine and or chlorpheniramine chlorpheniramine, which were demonstrated to block the effects of acetylcholine (5 pg/kg) and histamine (5 pg/kg), respectively, did not affect the hypotensive response to stem (12.5 mg/kg), leaf and flower (100 mg/kg) extracts. TABLE
5
INFLUENCEOF T.PANDACQUIEXTRACTS ONTHERESPONSEOFBLOODPRESSURE (BP) TO SYMPATHETIC AND ADRENOCEPTOR ACTIVATION IN ANESTHETIZED RATS. Carotid arteries were occluded (OCCl bilaterally for 1 min, and norepinephrme [NE, dose of 1 rg/ kg1 was injected intravenously, after the maximal hypotension response to tbe extracts. Values are expressed as means 2 S.E.M., N = 9. Challenge
Part used
Dose (mglkgl
Before extract
After extract
occ
Stem Leaf Flower
50 300 300
25.6 f 1.3 26.8 f 2.7 24.8 + 1.9
22.2 f 24.5 f 22.8 f
NE
Stem Leaf Flower
50 300 300
40.5 f 2.8 43.0 f 2.1 42.6 & 3.7
36.7 + 2.6 39.8 2 2.8 41.0 * 3.0
BP increase from control (e/cl
2.6 1.1 2.0
118
DEPRESSOR EFf ECTS
kontfol
r=-=lr-----l
Jc AC),
St Lf
Fl
Wirt.
St
U
After Chbtph6nimmine
Mt.
F
Fig. 7. Effect of atropine (2 mg/kg) and ehlorpheniramine (1 mg/kg) on hypotensive response to T.
pamhmqui (12.5 mg/kg stem extract, 100 mg/kg leaf and flower extracts). Atropine and chlorpheniramine inhibited the effect of 5 rg/kg acetylcholine (ACU and 5 pg/kg histamine (Hist), respectively, but not those of the extracts. Significant difference from the control effects given before the antagonists: ***P < 0.001.
Discussion
Extracts of T. pandacaqui showed cardiac stimulant as well as depressant effects on spontaneous contraction of rat atria1 strips. The stem extract caused depression of both force and rate. With small to moderate doses of the flower and leaf extracts, an increase in force was observed. High doses of both extracts caused depression of force and rate. Intravenous injection of extracts of T. pandacaqui caused hypotensive responses in anesthetized rats. With the flower extract, doses of 100, 200 and 300 mg/kg produced a transient hypertensive effect before hypotension was seen. This dose-response study suggests that the hypotensive potency of the stem extract is more than that of flower and leaf extracts. It is commonly agreed that the physiological state of the heart, as determined by its force and rate of contraction, influences the maintenance of blood pressure. The results obtained from the isolated rat atrium suggest that the depressant effect of stem extract and high doses of flower and leaf extracts on the heart probably contribute to the hypotensive response. It was found that during the hypotensive response to low doses (25, 50 and 100 mg/kgl of leaf extract, there was no significant increase in heart rate, which also correlated well with the effect of the extract at doses of 2.5, 5, and 10 mglml on the isolated heart. During the hypotensive phase of blood pressure response to flower extract, only tachycardia was seen with low doses (25 and 50 mg/kg).
119
Tachycardia followed by prolonged bradycardia was observed with high doses (100, 200 and 300 mgkg). It is possible that the tachycardia is due to a direct effect on the heart, as seen with the isolated atrium, and/or to a reflex increase in sympathetic activity initiated by the hypotensive effect of the extract. The hypotensive effect of T. pandacaqui was not affected by chlorpheniramine (an antihistaminic) and atropine (an antimuscarinicl. Therefore, it would appear that the extracts exhibit hypotension by actions not mediated through histaminic and muscarinic receptors. In addition, mechanisms such as an a-adrenoceptor blockade and interference with sympathetic transmission can be excluded, since the extracts of T. pandacaqui did not block the pressor effect of norepinephrine and the reflex pressor response to a bilateral occlusion of the common carotid arteries. Acknowledgement
Financial support for the project “Thai Medicinal Plants Medicine” (Account 00960401 by the Dutch Ministry of Foreign the State University of Leiden is gratefully acknowledged.
and Herbal Affairs and
References Boonyaratanakornkit, L. and Supawita, T. (1977) Names of Medicinal Plants and Their Uses. Department of Pharmacognosy, Faculty of Pharmacy, Chulalongkorn University, Bangkok, p. 69. Farris, E.J. and Giffith, J.Q. (1949) The Rat in Laboratory Investigation, 2nd Edn. Hafner Publishing Company, New York, pp. 283-284. Jayaweera, D.M.A.fl981) Medicinal Plants Used in Ceylon, Part 1. National Science Council of Sri Lanka, Colombo, p. 113. Robert, E.M. and Konjovic, J&966) Difference in the chronotropic and inotropic response of the rat atrium to choline esters, cholinesterase inhibitors and certain blocking agents. Journal of Pharmacology and Experimental Therapeutics 169,109- 119. Taesotikul, T., Panthong, A., Kanjanapothi, D., Verpoorte, R., and Scheffer, J.J.C. (1989) Hippocratic screening of ethanolic extracts from two Tabernnemontana species. Journal of Ethnopharmacology 27,99- 106. Tsiang, Y. and Li, P.T. (1977) Flora Republicae Popularis Sinicae, Vol. 63. Kexue Chubanshe, Beijing, pp. 98, 115.