Read IJP_June04.pmd text version

Research Paper

Cardiac stimulant activity of Ocimum basilicum Linn. extracts

A. Muralidharan, R. Dhananjayan*

ABSTRACT Department of Pharmacology and Environmental Toxicology, Dr. ALM PG Institute of Basic Medical Sciences, Taramani, Chennai 600113, India. Received: 4.2.2003 Revised: 21.10.2003 Accepted: 2.11.2003 Correspondence to: R. Dhananjayan

E-mail: [email protected]

Objective: To evaluate the cardiac effects of extracts derived from the aerial parts of Ocimum basilicum Linn. Material and Methods: The aerial parts of Ocimum basilicum Linn. were extracted with 95% ethanol and double distilled water. The extracts were screened for their effects on frog-heart in situ preparation. Enzyme studies such as Na+/K+ ATPase, Ca2+ATPase and Mg2+ATPase were done on the heart tissue aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) were estimated in the heart tissue and serum of albino rats after administering the extracts for 7days. Results: The alcoholic extract produced significant positive ionotropic and negative chronotropic actions on frog heart. The positive ionotropic effect was selectively inhibited by nifedipine. A significant decrease in membrane Na+/K+ ATPase, Mg2+ATPase and an increase in Ca2+ATPase pointed the basis for the cardiotonic effect. The aqueous extract produced positive chronotropic and positive ionotropic effects which were antagonized by propranolol indicating that these might have been mediated through ß-adrenergic receptors. Nifedipine also blocks the action of the aqueous extract. Conclusion: The alcoholic extract exhibited a cardiotonic effect and the aqueous extract produced a ß-adrenergic effect. KEY WORDS: Adrenergic activity, cardiotonic, sweet basil

Introduction Ocimum basilicum Linn. (Labiatae), popularly known as "Sweet Basil" is used in both Ayurvedic and Unani systems of Medicine.1 It is a small perennial, tropically growing shrub of Asian origin.2 It has antipyretic, antiemetic, diuretic and cardiotonic3 properties. Despite continuing advances in understanding the basic pharmacology of cardiac glycosides, digitalis intoxication remains a common clinical problem. It necessitates research for new nature based drugs which increase cardiac muscle contractility with a broad therapeutic index. As a part of the screening for a suitable natural drug we have chosen Ocimum basilicum Linn. and evaluated its cardio active potential and its mechanism of action. Material and Methods Preparation of extracts Ocimum basilicum Linn. was collected from the Arignar Anna Hospital for Indian Medicine, Arumbakkam, Chennai,

af ter being duly identified by a Botanist, Prof. R. Rangasamy , University of Madras. The aerial part of the plant was cleaned with water and dried in the shade until a constant weight was obtained. It was extracted with 95% ethanol (Total alcoholic extract = TAE) and then with double distilled water (Total aqueous extract = TAQ). The extracts were concentrated over a water-bath maintained at 550C until a semi solid concentrate masses were obtained. The yields were for TAE, 4.8% and for TAQ, 9.3% of the plant material. F subsequent pharor macological and biochemical studies, 1% of TAE was suspended in 5%gum acacia, as it was not soluble in water and TAQ was being water soluble hence an aqueous solution was used. Drugs Adrenaline, digoxin, propranolol and nifedipine. (Sigma Chemical Co, St.Louis, MI, USA). Animals Frogs of Rana hexadactyla species maintained in the animal house and male Wistar albino rats (150 to 200 g) housed

Indian J Pharmacol

| June 2004 | Vol 36 | Issue 3 | 163-166


Muralidharan A, et al.

in cages at 270 ± 20C on a 12 h light / dark cycle were used for the studies. The animals were fed with food and water ad libitum. The animals were maintained as per the norms of CPCSEA and the experiments were cleared by CPCSEA and the local ethics committee. Frog heart in situ preparation Frogs were pithed and the heart exposed. The inferior vena cava was cannulated for perfusing the heart with the frog's Ringer solution.4 (The composition of the frog Ringer solution in millimoles: NaCl-110; KCl-1.9; CaCl2-1.1; NaHCO 3-2.4; NaH2PO4-0.06; Glucose-11.1). The basal cardiac contraction was recorded on a smoked k ymographic drum after the administration of frog Ringer's solution and gum-acacia (5%). The administration of gum acacia was done to see that it did not contribute to the effects of TAE. The drugs and extracts were administered through the cannula. The average basal heart rate and the contraction amplitude were 70 beats/min and 18 mm respectively. The effects obtained with the drugs and extracts were transposed to the respective percentage of the basal values. Graded dose-response was recorded for each extract (0.5, 1 and 1.5 mg) and the dose which caused the maximum effect was chosen as the experimental dose. The frog heart was washed with the Ringer solution after ever y administration of extracts and drugs till it was brought back to the normal state. The frog heart was perfused with propranolol, a ß -adrenergic blocker at 3 X 10-5 M concentration in frog Ringer solution for 60 seconds followed by the administration of extracts and the recording were noted. Nifedipine, a calcium-channel blocker at 2.88 X 10-5M concentration in frog Ringer solution was administered for 60 seconds followed by extracts and the recordings were noted. Biochemical studies Wistar albino rats were divided into 3 groups of 6 animals each. Group I received with 5% gum acacia suspension which ser ved as control; Group II and Group III were treated with TAE and TAQ at a dose of 100 mg/kg (approximately 1/10 of the LD50) body weight i.p. for 7 days. On the 8th day the animals were sacrificed and the blood and heart-tissue were colTable 1

lected and the serum was separated from the blood. The heart was washed in ice-cold saline and about 100 mg of tissue was weighed and homogenized in chilled 0.1 M Tris-HCl Buffer in Patter-Elvejhem teflon homogenizer. The serum and homogenized samples were assayed for clinical marker enzymes lik e CPK,5 LDH6 and transaminases AST and ALT. 7 Heart homogenate samples were also assayed for Na+ K+ ATPase,8 Ca2+ATPase 9 and Mg2+ATPase.10 Statistical analysis The frog heart in situ experiment and biochemical parameters obtained were subjected to one-way ANOVA followed by Tukey's multiple comparison test. P value < 0.05 was considered significant. Results Total alcoholic extract (TAE) produced significant positive ionotropic and negative chronotropic actions similar to that of digoxin on frog heart. This cardiotonic action was not antago nized by propranolol (Table 1). Nifedipine pretreatment significantly reduced the cardiotonic activity of the extracts. There was a significant decrease in membranous Na+ K+ATPase, and Mg2+ATPase and an increase in Ca2+ ATPase (Figure 1). Total aqueous extract produced a significant increase in the force of contraction as reflected by an increase in the amplitude and heart rate. Both propranolol and nifedipine antagonized the effect of the extract TAQ (Table 1). No significant change was observed in Na+ K+ATPase, Mg2+ATPase and Ca2+ ATPase of the heart (Figure 1). Both the TAE and TAQ did not produce any significant changes in the levels of AST, ALT, LDH and CPK in heart and in serum samples when compared with the control animals (Table 2). Discussion Cardiac glycosides and catecholamines have been used as the main therapeutic drugs in the treatment of congestive cardiac failure.11 However, the dangers of cardiac glycoside intoxication are well documented12 and doubts have been ex-

Effects of the extracts from Ocimum basilicum Linn. on the frog heart in situ preparation

Extracts / drugs

HR% 76.5 ± 2.47 Digoxin (1.28x10 M) Adrenaline (2.5x10-5M) 158.6 ± 5.05b TAE (1 mg/ml) 76.0 ± 2.68a TAQ (1 mg/ml) 121.5 ± 3.67c One-way ANOVA F df P 120.54 3,36 <0.001

-5 a

Frog ringer

FC% 306.0 258.2 261.0 132.8 ± ± ± ± 10.99 8.12b 10.83 b 4.25c


Frog ringer + propranolol (3x10 -5M)

HR% ­ 116.6 ± 3.40a 74.0 ± 2.56b 86.0 ± 5.10b 92.51 5,54 <0.001 FC% ­ 190.8 ± 5.58a 234.0 ± 8.12b 104.0 ± 5.10c 162.01 5,54 <0.001

Frog ringer + nifedipine (2.88x10-5M)

HR% 59.5 ± 2.67 ­ 43.80 ± 2.56b 66.0 ± 5.34a 88.98 5,54 <0.001


FC% 229.4 ± 5.99a ­ 141.8 ± 6.25b 66.0 ± 5.34c 198.99 5,54 <0.001

66.71 3,36 <0.001

Control values: HR: 70.0+1.25 (100%), FC: 18+0.06 (100%). V alues with dif ferent superscripts in a column are significantly different from each other at P<0.05, n=10. V alues are mean± SEM. TAE: Total alcoholic extract. TAQ: Tot al aqueous extract.


Indian J Pharmacol

| June 2004 | Vol 36 | Issue 3 | 163-166

Cardiac activity of Ocimum basilicum

Table 2

Effects of the extracts from Ocimum basilicum Linn. on the marker enzymes in rats

Marker enzymes Group I (Control)

Heart AST ALT LDH CPK 0.193 0.0952 2.92 0.56 ± ± ± ± 0.007 0.006 0.09 0.04 Serum 0.512 0.614 5.220 8.560 ± ± ± ± 0.021 0.034 0.179 0.320 Heart 0.203 0.0978 3.01 0.61 ± ± ± ± 0.007 0.007 0.08 0.04

Group II (TAE)

Serum 0.534 0.598 5.650 8.950 ± ± ± ± 0.025 0.036 0.209 0.310

Group III (TAQ)

Heart 0.205 0.101 2.98 0.57 ± ± ± ± 0.006 0.006 0.07 0.03 Serum 0.528 0.608 5.230 8.980 ± ± ± ± 0.029 0.036 0.181 0.290

N = 6, values are expressed as mean ± SEM, NS: not significant. Enzyme units; Aminotransferases: (AST, ALT) µmolesx10-2 of pyruvate liberated/min/mg protein. LDH: µmoles x 10-1 of pyruvate liberated/min/mg protein. For Heart CPK: µmoles of phosphorus liberated/min/mg protein. For Serum CPK: µmoles x 10-3 of phosphorus liberated/min/mg protein

F df P

16.63 21.61 35.37 2, 15 2, 15 2, 15 <0.001 <0.001 <0.001 One-way ANOVA

Ca2+ current16 as well as in transient Ca2+current15. Ca2+ induced Ca2+ release is a general mechanism that most cells use to amplify Ca2+ signals.16 In heart cells, this mechanism is operated between voltage-gated L-type calcium channels (LCCs) in the plasma membrane and calcium release channel, commonly known as r yanodine receptors in the sarcoplasmic reticulum.17 Nifedipine is a LCC antagonist.16 Since nifedipine, blocks the cardiotonic action of TAE significantly, the extract might have produced its action by opening the voltage sensitive slow Ca2+channel. In connection with the cardiotonic effects obser ved one could see a relationship that exists between the inhibitor y levels of the activities of Mg2+ATPase and Na+ K+ATPase.18 The significant rise in the level of activity of Ca2+ ATPase might be due to the rise of cytosolic Ca2+. 19 The total aqueous extract (TAQ) produced positive chronotropic and ionotropic effects similar to that of adrenaline by stimulating the ß-adrenergic receptors. References

1. 2. 3. Jain ML, Jain SR. Therapeutic utility of Ocimum basilicum Linn. VAR Album. Planta Med 1988;54:66-70. Dhar AK. Sweet Basil: Ocimum basilicum - a review. Journal of Medicinal and Aromatic Plant Sciences 2002;24:738-55. Warrier PK, Nambiar VPK, Ramankutty C. Indian Medicinal Plants. Orient Longman 1995;3:162-3. Burn JH. Practical pharmacology blackwell. Scientific Publications; 1952. p. 1-7. Okinaka S, Kumagi H, Ebashi S, Sugita H, Momoi H, Toyokura Y, et al. Serum creatine phosphokinase. Activity in progressive muscular dystrophy and neuromuscular diseases. Arch Neurol 1961;4:520-5. King J. The dehydrogenases or oxidoreductases. Lactate dehydrogenase In: Practical Clinical Enzymology. London: Van Nostrand, D.Company Ltd; 1965. King J. The Transferases ­alanine and aspartate transaminases In: Practical Clinical Enzymology. London: Van Nostrand, D.Company Ltd; 1965. Bonting SL. Sodium-potassium activated adenosine triphosphatase and cation transport In: Bittar EE, editor. Membrane and ion transport. London: WileyInterscience; 1970. Hjerten S, Pan H. Purification and characterization of two forms of low affinity Ca 2+ATPase from erythrocyte membranes. Biochem Biophys Acta 1983;728: 281-8. Ohinishi T, Suzuki T, Suzuki Y, Ozawa K. Comparative study of plasma membrane Mg2+ ATPase activities in normal, regenerating and malignant cells. Biochem Biophys Acta 1982;684:67-74. Kitada Y, Narimatsu A, Suzuki R, Endoh M, Taira N. Does the positive ionotropic action of a novel cardiotonic agent, MCI-154, involve mechanisms other than cyclic AMP? J Pharmacol Exp Ther 1987;243:639-45. Beller GA, Smith TW, Abelmann WH, Haber E, Hood WB Jr. Digitalis intoxication. A prospective clinical study with serum level correlations. N Engl J Med 1971;284:989-97. Akera T, Brody TM. The role of Na+ K+ATPase in the ionotropic action of digi-

Figure 1: Effects of the extracts of Ocimum basilicum Linn. on heart tissue ATPases of Wistar albino rats. ***P<0.001 when compared to control.

Enzyme units as micro moles of P(i) liberated/ min/mg of protein

pressed about their long-term effectiveness. The use of catecholamines is limited by their insufficient differentiation between positive ionotropic and chronotropic actions, their potential arrhythmogenic properties and tachyphylaxis due to receptor down-regulation.11 Total alcoholic e xtract elicited a powerful cardiotonic effect, which was characterized by positive ionotropic and negative chronotropic actions. This effect was not significantly blocked by propranolol whereas nifedipine, the calcium channel blocker antagonized the effect significantly. The cardiac enzyme profile indicates that TAE exhibited cardiotonic like activity which manifested as a result of general decrease in the activity of Na+ K+ ATPase, and Mg2+ ATPase and an increase in Ca2+ATPase. This inhibition of Na+ K+ ATPase is similar to the action of cardiac glycosides. 13 Cardiac glycosides are specific and unique inhibitors of Na+ K+ ATPase at normal concentrations (10-8 to 10-9M).14 Na+ K+ ATPase inhibition by cardiac glycosides leads ultimately to increase intracellular Ca2+concentrations through Na+/ Ca 2+exchange and an associated increase in slow inward

4. 5.

6. 7. 8.






Indian J Pharmacol

| June 2004 | Vol 36 | Issue 3 | 163-166


Muralidharan A, et al. talis. Pharmacol Rev 1977;29:187-220. 14. Goto A, Yamada K, Yagi N, Yoshioka M, Sugimoto T. Physiology and pharmacology of endogenous digitalis-like factors. Pharmacol Rev 1992;44:377-99. 15. McGarry SJ, Williams AJ. Digoxin activates sarcoplasmic reticulum Ca2+ release channels: a possible role in cardiac ionotropy. Br J Pharmacol 1993;108: 1043-50. 16. Wang SQ, Song LS, Lakatta EG, Cheng H. Ca2+ signaling between single L-type Ca2+ channels and rynodine receptors in heart cells. Nature 2001;410:592-6. 17. Fabiato A. Time and calcium dependence of activation and inactivation of calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinjee cell. J Gen Physiol 1985;85:247-89. 18. Chen CL, Sangiah S, Patterson E, Berlin KD, Garrison GL, Dunn W, et al. Effects of BRB-I-28, a novel antiarrhythmic agent, and its derivatives on cardiac Na+ K+ATPase, Mg 2+ATPase activities and contractile force. Res Commu Chem Pathol Pharmacol 1992;78:3-16. 19. Kelly RA, Smith TW. Pharmacological treatment of Heart failure. In: Goodman, Gillman, editors. The pharmacological basis of therapeutics. 9th ed. McGrawHill; 1996. Chapter 34. p. 811.


The Indian Council of Medical Research (ICMR), the premier national agency for the formulation, promotion and conduct of biomedical research in India invites applications from Indian biomedical scientists for the above fellowship awards for the year 2004-05. Application Form & details are available at ICMR website.

Applications should be submitted to:


International Health Division, Indian Council of Medical Research, V. Ramalingaswami Bhawan, Post Box No. 4911, Ansari Nagar, New Delhi -110029, India.

Last date of receipt of applications : 31st July 2004


Indian J Pharmacol

| June 2004 | Vol 36 | Issue 3 | 163-166



4 pages

Report File (DMCA)

Our content is added by our users. We aim to remove reported files within 1 working day. Please use this link to notify us:

Report this file as copyright or inappropriate


Notice: fwrite(): send of 199 bytes failed with errno=32 Broken pipe in /home/ on line 531