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Effect of Azadirachta indica hydroalcoholic leaf extract on the cardiovascular system
ISSN 0306-3623/97 $17.00 + .00 PII S0306-3623(96)00184-X All rights reserved
Gen. Pharmac. Vol. 28, No. 3, pp. 449-451, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. ELSEVIER
Effect of Azadirachta indica Hydroalcoholic Leaf Extract on the Cardiovascular System R. R. Chattopadhyay BIOMETRYRESEARCHUNIT, INDIANSTATISTICAL INSTITUTE, 203, BARRACKPORETRUNKROAD, CALCUTTA700 035, INDIA A B S T R A C T . 1. The effect of Azaclirachta indica hydroalcoholic leaf extract on the cardiovascular system was studied. 2. The leaf extract was found to reduce a dose-dependent hypotensive effect without altering the amplitude or rate of respiration. 3. In isolated frog heart, there was no noticeable change in amplitude of contraction or rate of the heart at lower doses of leaf extract. However, at higher doses, there was temporary cardiac arrest in diastole. 4. The results are discussed. Copyright © 1997 Elsevier Science Inc. tEN PHARMAC28;3:449-451, 1997. KEY WORDS. Azadirachta indica, cardiovascular activity INTRODUCTION
Azadirachta indica, A. Juss. (Meliaceae; Neem) is an indigenous plant widely available in India and Burma. Different parts of this plant have been reported to possess several medicinal properties, such as anticliabetic, antiseptic, healing of wounds, curing of skin disease and antiulcer activity (Chopra et al., 1956; Kirtikar and Basu, 1933). The water-soluble part of an alcoholic extract of leaves of A. indica has been found to possess hypoglycemic, anti-inflammatory, antiserotonin, antifertility, hypotensive and hepatoprotective activity (Chattopadhyay et al., 1987a; 1987b; 1986; Chattopadhyay 1991; 1992; 1993). But the effect ofA. indica leaf extract on the cardiovascular system seems to be dubious; therefore, the present investigation has been designed to study this in experimental animals. MATERIALS AND METHODS
Extraction of plant material Fresh matured leaves of A. indica were collected from the Botanical Garden, Calcutta during September and October 1994 and were botanically identified by a botanist from the University of Kalyani, India. Air-dried powder of leaves of A. indica (1 ks) was extracted by percolation at room temperature with 70% EtOH. The extract was concentrated under reduced pressure (bath temperature 50°C) and, finally, dried in a vacuum dissector. The residue was dissolved in distilled water and filtered. The filtrate was evaporated to dryness. The dried mass (yield=50.2 g) was suitably diluted with normal saline and used in the experiments.
Animals Healthy male cats (2.5-3.0 ks) and frogs (250-300 g) were housed in cages at an ambient temperature of 25±2°C and 45-55% RH, with 12:12L:D cycle. Animals were kept on a standardized diet (Hindustan Lever Ltd., Bombay, India) and tap water ad lib.
Test dose The test doses for crude extract were chosen arbitrarily at 25-400 ms/ks in vivo and ll*g-10 mg in vitro trials. The doses were selected Received 31 October 1995; revised 20 March 1996; accepted 1 April 1996.
on the basis of an acute toxicity study (crude extract given 1 g/ks dose did not have any toxic effect). In the trials on cat blood pressure the drug was given in a volume not exceeding 1 ml.
Cardiovascular study CAT BLOODPRESSURE. Male cats (n= 14) weighing 2.5-3.0 kg were selected for the experiments. Under ether anesthesia, the cat was secured on the experimental table and the femoral vein cannulated with a polythene cannula on one side. This cannula was subsequently used for all drug administration. Chloralose (60 ms/ks IV) was injected for maintenance of anesthesia. A midline incision was made in the front of the neck. The trachea and common carotid arteries were dissected out. A tracheal cannula was introduced into the trachea and an arterial cannula filled with anticoagulant (hepafin) was tied into the common carotid artery. The blood pressure was recorded through a manometer on a moving kymograph. The stability of the preparation was tested with standard doses of known agonists [e.g. acetylcholine (1 ~g/kg) and adrenaline (1 txg/ kg)]. Graded doses of leaf extract (25, 50, 100, 200 and 400 ms/ks) were administered through the femoral vein and the effects on mean arterial blood pressure were recorded. To study the effect of A. indica leaf extract on alpha and beta adrenergic, cholinergic and histaminergic receptors, a dose of 200 ms/ kg of leaf extract was administered 2 min prior to each of the following drugs: noradrenaline acid nitrate (NA) (2 Izg/kg), isoprenaline (2 Izg/kg), acetylcholine (Ach) (1 ~g/kg) and histamine (2 Izg/kg), respectively. In another set of experiments, atropine sulphate (1.5 ms/ks) was administered to block the muscarinic response of Ach, and A. indica leaf extract (200 ms/ks) was administered thereafter. Pheniramine maleate (7.5 ms/ks), a histamine Hi-antagonist, was also administered in separate experiments to block the histamine-induced fall of blood pressure prior to A. indica leaf extract administration. To determine the possibility of beta-adrenergic receptor activation, propanonol (2 ms/ks) was injected prior to administration of A. indica leaf extract (200 ms/ks). CAT RESPIRATION. Respiration was recorded according to the method of Gaddum (194t). A tracheal cannula was tied into the
450
R . R . Chattopadhyay T A B L E 1. Effect of Azadirachta indica leaf extract on pressure response in anesthetised cats Treatment
Dose (mg/kg, IV)
Saline
A. indica F I G U R E 1. Dose-dependent hypotensive activity of A. indica leaf extract in the anesthetized cat. • Normal saline; : 25 mg/kg; "." 50 mg/kg; :: 100 mg/kg; :. : 200 mg/kg; and il 400 mg/kg A. indica leaf extract.
2 25 50 100 200 400
Pressure response (mmHg, mean -+ SEM) 100.5 83.0 79.9 74.1 59.7 35.9
_+ 4.6 _+ 4.35 _+ 4.26 -+ 5.72 _+ 5.65 _+ 4.72
n = 14.
ceptors. A n Hi-receptor antagonist, beta blocker and atropine had no effect on A. indica leaf extract-induced hypotension.
Cat respiration F I G U R E 2. Dose-dependent cardiac depressant activity of A. indica leaf extract on isolated frog heart. - 1 mg; -. 10 mg;
No significant change in amplitude or rate of respiration by A. indica leaf extract (25 to 400 mg/kg) was observed.
"." Atropine (10 lutg).
Isolated frog heart trachea of the anesthetized cat. Through this cannula, the cat inhaled or exhaled v/a one-way valves. The inspiratory air came from an enclosed flask from a very small capillary leak. The pressure change in this flask, brought on by inspiratory withdrawal of air, was recorded through a large tambour writing on smoked paper. A. indica leaf extract was administered in the doses of 25, 50, 100, 200 and 400 mg/kg. A n equal volume of normal saline was used as control. ISOLATEDFROG HEART. The frog's heart was perfused according to the method of Bulbring described by Burn (1952). A pithed frog is laid on its back on a cork board. The heart was dissected out and perfused in Frog-Ringer's at room temperature through a cannula tied into the inferior vena cava, both aorta having been cut. Tracings were recorded through a Starling's heart lever on smoked paper. The effect of A. indica leaf extract in graded doses (1 fxg, 10 Ixg, 50 Ixg, 100 Ixg, 1 mg and 10 mg) were observed and the influence of atropine (10 Ixg) on the effects of A. indica leaf extract was tested. RESULTS
Cat blood pressure A. indica leaf extract produced a sharp and short-lasting dose-dependent fall of blood pressure with a progressive increase in doses (Fig. 1). Generally, the blood pressure returned to normal level within 2-3 min. A. indica leaf extract (200 mg/kg) failed to alter the sensitivity of alpha and beta adrenergic, cholinergic or histaminergic re-
In isolated frog heart with A. indica leaf extract up to a concentration of 100 Ixg, there was no noticeable change in amplitude of contraction or heart rate. With a 1-mg or 10-mg concentration, there was temporary cardiac arrest in diastole with subsequent recovery of contraction by about 30-40 sec; the inhibitory effect was not blocked by atropine (Fig. 2). DISCUSSION In the case of cat blood pressure, A. indica leaf extract produced a dose-dependent fall in blood pressure that was not blocked by anticholinergic, antihistaminergic and adrenergic receptor-blocking agents. The sensitivity of cholinergic, histaminergic and adrenergic receptors toward the respective agonists could not be altered by pretreatment with A. indica leaf extract. In isolated frog heart, A. indica leaf extract induced temporary cardiac arrest in diastole. Systematic chromatographic separation of A. indica leaf extract revealed that the extract contains mainly a mixture of six flavonol-O-glycosides that are known to be responsible for blood sugar-lowering activity against streptozotocin-induced diabetics in rats (Chakraborty et al., 1989). It is presumed that these flavonol-O-glycosides either wholly or partly, or the remaining other constituents of the extract, may be responsible for cardiac-depressant and hypotensive activity. Chemical analysis in this respect is in progress. The possible mechanism of action of the hypotensive and cardiac-depressant activity of A. indica leaf extract cannot be explained at present. Further studies are needed to substantiate these findings.
T A B L E 2. Effect of Azaclirachta inclica leaf extract on perfused frog hearts Dose 1 Ixg to 100 ~g 1 mg 10 mg
n~6.
Heart rate
Contractility
Insignificant Decrease or standstill, followed by increase Standstill
Insignificant Decrease followed by increase Standstill, increase if recovery takes place
Tone No change No change No change or increase
Rhythm Normal Normal, sometimes irregular Sometimes irregular on recovery
Azadirachta Extract and the Cardiovascular System Thus, A. indica leaf extract needs to be further evaluated considering its hypotensive and cardiac-depressant activities. This preliminary report may serve as a footstep in this direction. The author wishes to acknowledge Prof. C. H. Sastry, Head of the Unit, Indian Statistical Institute, Calcutta and Prof. S. K. Maitra, Department of Pharmacology, School of Tropical Medicine, Calcutta, for their whole-hearted cooperation and help to carry out this work.
References Burn J. H. (1952) Practical Pharmacology. Blackwell Scientific Publications, Oxford. Chakraborty T., Verotta L., and Poddar G. (1989) Evaluation of Azadirachta indica leaf extract for hypoglycemic activity in rats. Phytotherapy Res. 3, 30-32. Chattopadhyay R. R., Chattopadhyay R. N., Nandy A. K., Poddar G., and Maitra S. K. (1987a) Preliminary report on antihyperglycemic effect of a fraction of fresh leaves of Azadirachta indica (Beng. Neem). Bull. Calcutta Sch. Trop. Med. 35, 29-33. Chattopadhyay R. R., Chattopadhyay R. N., Nandy A. K., Poddar G. and Maitra S. K. (1987b) Antiinflammatory activity of a fraction of fresh
451 leaves of Azadirachta indica (Beng. Neem). Bull. Calcutta Sch. Trop. Med. 35, 6-8. Chattopadhyay R. R., Chattopadhyay R. N., Nandy A. K., Poddar G. and Maitra S. K. (1986) Antiserotonin activity of a fraction of fresh leaves of Azadirachta indica (Beng. Neem) Bull. Calcutta Sch. Trop. Med. 34, 9-12. Chattopadhyay R. R. (1993) Effect of Azadirachta indica leaf extract on oestrous cycle of rats. Ancient Sci. Life, 12, 435-438. Chattopadhyay R. R. (1991) Studies on alcoholic extract of leaves of Azadirachta indica (Beng. Neem) on carbohydrate metabolism. Ph.D. Thesis, University of Kalyani, India. Chattopadhyay R. R., Sarkar S. K., Ganguly S., Banerjee R.N. and Basu T. K. (1992) Hepatoprotective activity of Azadirachta indica leaf extract on paracetamol induced hepatic damage in rats. Indian J. Exp. Biol. 30, 738-740. Chopra R. N., Nayar S. L. and Chopra I. C (1956) Azadirachta indica. In Glossary of Indian Medicinal Plants (edited by R. N. Chopra, I. C. Chopra, K. L. Handa and L. D. Kapur) 2nd edition, pp. 31, CSIR, New Delhi. Gaddum J. H. (1941) A simple method for measurement of respiration of experimental animals. J. Physiol. (London) 99, 257-261. Kirtikar K. R. and Basu B. D. (1933) Azadirachta indica. In Indian Medicinal Plants (edited by E. Blatter, J. F. Caiux and K. S. Mhaskar) vol. 3, pp. 1965-1967. LM Basu, Allahabad.
Gen. Pharmac. Vol. 28, No. 3, pp. 449-451, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. ELSEVIER
Effect of Azadirachta indica Hydroalcoholic Leaf Extract on the Cardiovascular System R. R. Chattopadhyay BIOMETRYRESEARCHUNIT, INDIANSTATISTICAL INSTITUTE, 203, BARRACKPORETRUNKROAD, CALCUTTA700 035, INDIA A B S T R A C T . 1. The effect of Azaclirachta indica hydroalcoholic leaf extract on the cardiovascular system was studied. 2. The leaf extract was found to reduce a dose-dependent hypotensive effect without altering the amplitude or rate of respiration. 3. In isolated frog heart, there was no noticeable change in amplitude of contraction or rate of the heart at lower doses of leaf extract. However, at higher doses, there was temporary cardiac arrest in diastole. 4. The results are discussed. Copyright © 1997 Elsevier Science Inc. tEN PHARMAC28;3:449-451, 1997. KEY WORDS. Azadirachta indica, cardiovascular activity INTRODUCTION
Azadirachta indica, A. Juss. (Meliaceae; Neem) is an indigenous plant widely available in India and Burma. Different parts of this plant have been reported to possess several medicinal properties, such as anticliabetic, antiseptic, healing of wounds, curing of skin disease and antiulcer activity (Chopra et al., 1956; Kirtikar and Basu, 1933). The water-soluble part of an alcoholic extract of leaves of A. indica has been found to possess hypoglycemic, anti-inflammatory, antiserotonin, antifertility, hypotensive and hepatoprotective activity (Chattopadhyay et al., 1987a; 1987b; 1986; Chattopadhyay 1991; 1992; 1993). But the effect ofA. indica leaf extract on the cardiovascular system seems to be dubious; therefore, the present investigation has been designed to study this in experimental animals. MATERIALS AND METHODS
Extraction of plant material Fresh matured leaves of A. indica were collected from the Botanical Garden, Calcutta during September and October 1994 and were botanically identified by a botanist from the University of Kalyani, India. Air-dried powder of leaves of A. indica (1 ks) was extracted by percolation at room temperature with 70% EtOH. The extract was concentrated under reduced pressure (bath temperature 50°C) and, finally, dried in a vacuum dissector. The residue was dissolved in distilled water and filtered. The filtrate was evaporated to dryness. The dried mass (yield=50.2 g) was suitably diluted with normal saline and used in the experiments.
Animals Healthy male cats (2.5-3.0 ks) and frogs (250-300 g) were housed in cages at an ambient temperature of 25±2°C and 45-55% RH, with 12:12L:D cycle. Animals were kept on a standardized diet (Hindustan Lever Ltd., Bombay, India) and tap water ad lib.
Test dose The test doses for crude extract were chosen arbitrarily at 25-400 ms/ks in vivo and ll*g-10 mg in vitro trials. The doses were selected Received 31 October 1995; revised 20 March 1996; accepted 1 April 1996.
on the basis of an acute toxicity study (crude extract given 1 g/ks dose did not have any toxic effect). In the trials on cat blood pressure the drug was given in a volume not exceeding 1 ml.
Cardiovascular study CAT BLOODPRESSURE. Male cats (n= 14) weighing 2.5-3.0 kg were selected for the experiments. Under ether anesthesia, the cat was secured on the experimental table and the femoral vein cannulated with a polythene cannula on one side. This cannula was subsequently used for all drug administration. Chloralose (60 ms/ks IV) was injected for maintenance of anesthesia. A midline incision was made in the front of the neck. The trachea and common carotid arteries were dissected out. A tracheal cannula was introduced into the trachea and an arterial cannula filled with anticoagulant (hepafin) was tied into the common carotid artery. The blood pressure was recorded through a manometer on a moving kymograph. The stability of the preparation was tested with standard doses of known agonists [e.g. acetylcholine (1 ~g/kg) and adrenaline (1 txg/ kg)]. Graded doses of leaf extract (25, 50, 100, 200 and 400 ms/ks) were administered through the femoral vein and the effects on mean arterial blood pressure were recorded. To study the effect of A. indica leaf extract on alpha and beta adrenergic, cholinergic and histaminergic receptors, a dose of 200 ms/ kg of leaf extract was administered 2 min prior to each of the following drugs: noradrenaline acid nitrate (NA) (2 Izg/kg), isoprenaline (2 Izg/kg), acetylcholine (Ach) (1 ~g/kg) and histamine (2 Izg/kg), respectively. In another set of experiments, atropine sulphate (1.5 ms/ks) was administered to block the muscarinic response of Ach, and A. indica leaf extract (200 ms/ks) was administered thereafter. Pheniramine maleate (7.5 ms/ks), a histamine Hi-antagonist, was also administered in separate experiments to block the histamine-induced fall of blood pressure prior to A. indica leaf extract administration. To determine the possibility of beta-adrenergic receptor activation, propanonol (2 ms/ks) was injected prior to administration of A. indica leaf extract (200 ms/ks). CAT RESPIRATION. Respiration was recorded according to the method of Gaddum (194t). A tracheal cannula was tied into the
450
R . R . Chattopadhyay T A B L E 1. Effect of Azadirachta indica leaf extract on pressure response in anesthetised cats Treatment
Dose (mg/kg, IV)
Saline
A. indica F I G U R E 1. Dose-dependent hypotensive activity of A. indica leaf extract in the anesthetized cat. • Normal saline; : 25 mg/kg; "." 50 mg/kg; :: 100 mg/kg; :. : 200 mg/kg; and il 400 mg/kg A. indica leaf extract.
2 25 50 100 200 400
Pressure response (mmHg, mean -+ SEM) 100.5 83.0 79.9 74.1 59.7 35.9
_+ 4.6 _+ 4.35 _+ 4.26 -+ 5.72 _+ 5.65 _+ 4.72
n = 14.
ceptors. A n Hi-receptor antagonist, beta blocker and atropine had no effect on A. indica leaf extract-induced hypotension.
Cat respiration F I G U R E 2. Dose-dependent cardiac depressant activity of A. indica leaf extract on isolated frog heart. - 1 mg; -. 10 mg;
No significant change in amplitude or rate of respiration by A. indica leaf extract (25 to 400 mg/kg) was observed.
"." Atropine (10 lutg).
Isolated frog heart trachea of the anesthetized cat. Through this cannula, the cat inhaled or exhaled v/a one-way valves. The inspiratory air came from an enclosed flask from a very small capillary leak. The pressure change in this flask, brought on by inspiratory withdrawal of air, was recorded through a large tambour writing on smoked paper. A. indica leaf extract was administered in the doses of 25, 50, 100, 200 and 400 mg/kg. A n equal volume of normal saline was used as control. ISOLATEDFROG HEART. The frog's heart was perfused according to the method of Bulbring described by Burn (1952). A pithed frog is laid on its back on a cork board. The heart was dissected out and perfused in Frog-Ringer's at room temperature through a cannula tied into the inferior vena cava, both aorta having been cut. Tracings were recorded through a Starling's heart lever on smoked paper. The effect of A. indica leaf extract in graded doses (1 fxg, 10 Ixg, 50 Ixg, 100 Ixg, 1 mg and 10 mg) were observed and the influence of atropine (10 Ixg) on the effects of A. indica leaf extract was tested. RESULTS
Cat blood pressure A. indica leaf extract produced a sharp and short-lasting dose-dependent fall of blood pressure with a progressive increase in doses (Fig. 1). Generally, the blood pressure returned to normal level within 2-3 min. A. indica leaf extract (200 mg/kg) failed to alter the sensitivity of alpha and beta adrenergic, cholinergic or histaminergic re-
In isolated frog heart with A. indica leaf extract up to a concentration of 100 Ixg, there was no noticeable change in amplitude of contraction or heart rate. With a 1-mg or 10-mg concentration, there was temporary cardiac arrest in diastole with subsequent recovery of contraction by about 30-40 sec; the inhibitory effect was not blocked by atropine (Fig. 2). DISCUSSION In the case of cat blood pressure, A. indica leaf extract produced a dose-dependent fall in blood pressure that was not blocked by anticholinergic, antihistaminergic and adrenergic receptor-blocking agents. The sensitivity of cholinergic, histaminergic and adrenergic receptors toward the respective agonists could not be altered by pretreatment with A. indica leaf extract. In isolated frog heart, A. indica leaf extract induced temporary cardiac arrest in diastole. Systematic chromatographic separation of A. indica leaf extract revealed that the extract contains mainly a mixture of six flavonol-O-glycosides that are known to be responsible for blood sugar-lowering activity against streptozotocin-induced diabetics in rats (Chakraborty et al., 1989). It is presumed that these flavonol-O-glycosides either wholly or partly, or the remaining other constituents of the extract, may be responsible for cardiac-depressant and hypotensive activity. Chemical analysis in this respect is in progress. The possible mechanism of action of the hypotensive and cardiac-depressant activity of A. indica leaf extract cannot be explained at present. Further studies are needed to substantiate these findings.
T A B L E 2. Effect of Azaclirachta inclica leaf extract on perfused frog hearts Dose 1 Ixg to 100 ~g 1 mg 10 mg
n~6.
Heart rate
Contractility
Insignificant Decrease or standstill, followed by increase Standstill
Insignificant Decrease followed by increase Standstill, increase if recovery takes place
Tone No change No change No change or increase
Rhythm Normal Normal, sometimes irregular Sometimes irregular on recovery
Azadirachta Extract and the Cardiovascular System Thus, A. indica leaf extract needs to be further evaluated considering its hypotensive and cardiac-depressant activities. This preliminary report may serve as a footstep in this direction. The author wishes to acknowledge Prof. C. H. Sastry, Head of the Unit, Indian Statistical Institute, Calcutta and Prof. S. K. Maitra, Department of Pharmacology, School of Tropical Medicine, Calcutta, for their whole-hearted cooperation and help to carry out this work.
References Burn J. H. (1952) Practical Pharmacology. Blackwell Scientific Publications, Oxford. Chakraborty T., Verotta L., and Poddar G. (1989) Evaluation of Azadirachta indica leaf extract for hypoglycemic activity in rats. Phytotherapy Res. 3, 30-32. Chattopadhyay R. R., Chattopadhyay R. N., Nandy A. K., Poddar G., and Maitra S. K. (1987a) Preliminary report on antihyperglycemic effect of a fraction of fresh leaves of Azadirachta indica (Beng. Neem). Bull. Calcutta Sch. Trop. Med. 35, 29-33. Chattopadhyay R. R., Chattopadhyay R. N., Nandy A. K., Poddar G. and Maitra S. K. (1987b) Antiinflammatory activity of a fraction of fresh
451 leaves of Azadirachta indica (Beng. Neem). Bull. Calcutta Sch. Trop. Med. 35, 6-8. Chattopadhyay R. R., Chattopadhyay R. N., Nandy A. K., Poddar G. and Maitra S. K. (1986) Antiserotonin activity of a fraction of fresh leaves of Azadirachta indica (Beng. Neem) Bull. Calcutta Sch. Trop. Med. 34, 9-12. Chattopadhyay R. R. (1993) Effect of Azadirachta indica leaf extract on oestrous cycle of rats. Ancient Sci. Life, 12, 435-438. Chattopadhyay R. R. (1991) Studies on alcoholic extract of leaves of Azadirachta indica (Beng. Neem) on carbohydrate metabolism. Ph.D. Thesis, University of Kalyani, India. Chattopadhyay R. R., Sarkar S. K., Ganguly S., Banerjee R.N. and Basu T. K. (1992) Hepatoprotective activity of Azadirachta indica leaf extract on paracetamol induced hepatic damage in rats. Indian J. Exp. Biol. 30, 738-740. Chopra R. N., Nayar S. L. and Chopra I. C (1956) Azadirachta indica. In Glossary of Indian Medicinal Plants (edited by R. N. Chopra, I. C. Chopra, K. L. Handa and L. D. Kapur) 2nd edition, pp. 31, CSIR, New Delhi. Gaddum J. H. (1941) A simple method for measurement of respiration of experimental animals. J. Physiol. (London) 99, 257-261. Kirtikar K. R. and Basu B. D. (1933) Azadirachta indica. In Indian Medicinal Plants (edited by E. Blatter, J. F. Caiux and K. S. Mhaskar) vol. 3, pp. 1965-1967. LM Basu, Allahabad.