Research Paper

Cardiac activity of Ocimum basilicum Linn. extracts A. Muralidharan, R. Dhananjayan*

ABSTRACT

Objective: To evaluate the cardiac effects of extracts derived from the aerial parts of Ocimum Department of basilicum Linn. and Material and Methods: The aerial parts of Ocimum basilicum Linn. were extracted with 95% Environmental ethanol and double distilled water. The extracts were screened for their effects on frog- in situ Toxicology, Dr. ALM PG preparation. Enzyme studies such as Na+/K+ ATPase, Ca2+ATPase and Mg2+ATPase were done on Institute of Basic Medical Sciences, the heart tissue aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase Taramani, Chennai - (LDH) and creatine phosphokinase (CPK) were estimated in the heart tissue and serum of albino 600113, India. rats after administering the extracts for 7days. Results: The alcoholic extract produced significant positive ionotropic and negative Received: 4.2.2003 actions on frog heart. The positive ionotropic effect was selectively inhibited by nifedipine. A Revised: 21.10.2003 significant decrease in membrane Na+/K+ ATPase, Mg2+ATPase and an increase in Ca2+ATPase Accepted: 2.11.2003 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 Correspondence to: have been mediated through ß-adrenergic receptors. Nifedipine also blocks the action of the R. Dhananjayan aqueous extract. E-mail: Conclusion: The alcoholic extract exhibited a cardiotonic effect and the aqueous extract pro- [email protected] duced a ß-adrenergic effect.

KEY WORDS: Adrenergic activity, cardiotonic, sweet basil

Introduction after being duly identified by a Botanist, Prof. R. Rangasamy, University of Madras. The aerial part of the plant was cleaned Ocimum basilicum Linn. (Labiatae), popularly known as with water and dried in the shade until a constant weight was “Sweet Basil” is used in both Ayurvedic and Unani systems of obtained. It was extracted with 95% ethanol (Total alcoholic Medicine.1 It is a small perennial, tropically growing shrub of extract = TAE) and then with double distilled water (Total Asian origin.2 It has antipyretic, , and car- aqueous extract = TAQ). The extracts were concentrated over diotonic3 properties. a water-bath maintained at 550C until a semi solid concen- Despite continuing advances in understanding the basic trate masses were obtained. The yields were for TAE, 4.8% pharmacology of cardiac glycosides, digitalis intoxication re- and for TAQ, 9.3% of the plant material. For subsequent phar- mains a common clinical problem. It necessitates research macological and biochemical studies, 1% of TAE was sus- for new nature based which increase cardiac muscle pended in 5%gum acacia, as it was not soluble in water and contractility with a broad therapeutic index. As a part of the TAQ was being water soluble hence an aqueous solution was screening for a suitable natural we have chosen Ocimum used. basilicum Linn. and evaluated its cardio active potential and its mechanism of action. Drugs , digoxin, propranolol and nifedipine. (Sigma Material and Methods Chemical Co, St.Louis, MI, USA).

Preparation of extracts Animals Ocimum basilicum Linn. was collected from the Arignar Frogs of Rana hexadactyla species maintained in the ani- Anna Hospital for Indian Medicine, Arumbakkam, Chennai, mal house and male Wistar albino rats (150 to 200 g) housed

Indian J Pharmacol | June 2004 | Vol 36 | Issue 3 | 163-166 163 Muralidharan A, et al. in cages at 270 ± 20C on a 12 h light / dark cycle were used for lected and the serum was separated from the . The heart the studies. The animals were fed with food and water ad libi- was washed in ice-cold saline and about 100 mg of tissue was tum. The animals were maintained as per the norms of CPCSEA weighed and homogenized in chilled 0.1 M Tris-HCl Buffer in and the experiments were cleared by CPCSEA and the local Patter-Elvejhem teflon homogenizer. The serum and homog- ethics committee. enized samples were assayed for clinical marker enzymes like CPK,5 LDH6 and transaminases AST and ALT.7 Heart homoge- Frog heart in situ preparation nate samples were also assayed for Na+ K+ATPase,8 Frogs were pithed and the heart exposed. The inferior vena Ca2+ATPase 9 and Mg2+ATPase.10 cava was cannulated for perfusing the heart with the frog’s Ringer solution.4 (The composition of the frog Ringer solution Statistical analysis in millimoles: NaCl-110; KCl-1.9; CaCl2-1.1; NaHCO 3-2.4; The frog heart in situ experiment and biochemical param-

NaH2PO4-0.06; Glucose-11.1). The basal cardiac contraction eters obtained were subjected to one-way ANOVA followed by was recorded on a smoked kymographic drum after the ad- Tukey’s multiple comparison test. P value < 0.05 was consid- ministration of frog Ringer’s solution and gum-acacia (5%). ered significant. The administration of gum acacia was done to see that it did not contribute to the effects of TAE. The drugs and extracts Results were administered through the cannula. The average basal heart rate and the contraction amplitude were 70 beats/min Total alcoholic extract (TAE) produced significant positive and 18 mm respectively. The effects obtained with the drugs ionotropic and negative chronotropic actions similar to that of and extracts were transposed to the respective percentage of digoxin on frog heart. This cardiotonic action was not antago- the basal values. Graded dose-response was recorded for each nized by propranolol (Table 1). Nifedipine pretreatment sig- extract (0.5, 1 and 1.5 mg) and the dose which caused the nificantly reduced the cardiotonic activity of the extracts. There maximum effect was chosen as the experimental dose. The was a significant decrease in membranous Na+ K+ATPase, and frog heart was washed with the Ringer solution after every Mg2+ATPase and an increase in Ca2+ ATPase (Figure 1). Total administration of extracts and drugs till it was brought back aqueous extract produced a significant increase in the force to the normal state. of contraction as reflected by an increase in the amplitude The frog heart was perfused with propranolol, a ß-adren- and heart rate. Both propranolol and nifedipine antagonized ergic blocker at 3 X 10-5 M concentration in frog Ringer solu- the effect of the extract TAQ (Table 1). No significant change tion for 60 seconds followed by the administration of extracts was observed in Na+ K+ATPase, Mg2+ATPase and Ca2+ ATPase and the recording were noted. Nifedipine, a calcium-channel of the heart (Figure 1). blocker at 2.88 X 10-5M concentration in frog Ringer solution Both the TAE and TAQ did not produce any significant was administered for 60 seconds followed by extracts and the changes in the levels of AST, ALT, LDH and CPK in heart and in recordings were noted. serum samples when compared with the control animals (Table 2). Biochemical studies Wistar albino rats were divided into 3 groups of 6 animals Discussion each. Group I received with 5% gum acacia suspension which served as control; Group II and Group III were treated with Cardiac glycosides and catecholamines have been used as TAE and TAQ at a dose of 100 mg/kg (approximately 1/10 of the main therapeutic drugs in the treatment of congestive car- th 11 the LD50) body weight i.p. for 7 days. On the 8 day the ani- diac failure. However, the dangers of in- mals were sacrificed and the blood and heart-tissue were col- toxication are well documented12 and doubts have been ex-

Table 1

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

Extracts / drugs Frog ringer Frog ringer + propranolol (3x10 -5M) Frog ringer + nifedipine (2.88x10-5M) HR% FC% HR% FC% HR% FC% Digoxin (1.28x10-5M) 76.5 ± 2.47a 306.0 ± 10.99 a – – 59.5 ± 2.67a 229.4 ± 5.99a Adrenaline (2.5x10-5M) 158.6 ± 5.05b 258.2 ± 8.12b 116.6 ± 3.40a 190.8 ± 5.58a –– TAE (1 mg/ml) 76.0 ± 2.68a 261.0 ± 10.83 b 74.0 ± 2.56b 234.0 ± 8.12b 43.80 ± 2.56b 141.8 ± 6.25b TAQ (1 mg/ml) 121.5 ± 3.67c 132.8 ± 4.25c 86.0 ± 5.10b 104.0 ± 5.10c 66.0 ± 5.34a 66.0 ± 5.34c

One-way ANOVA F 120.54 66.71 92.51 162.01 88.98 198.99 df 3,36 3,36 5,54 5,54 5,54 5,54 P <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

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

164 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) Group II (TAE) Group III (TAQ) Heart Serum Heart Serum Heart Serum AST 0.193 ± 0.007 0.512 ± 0.021 0.203 ± 0.007 0.534 ± 0.025 0.205 ± 0.006 0.528 ± 0.029 ALT 0.0952 ± 0.006 0.614 ± 0.034 0.0978 ± 0.007 0.598 ± 0.036 0.101 ± 0.006 0.608 ± 0.036 LDH 2.92 ± 0.09 5.220 ± 0.179 3.01 ± 0.08 5.650 ± 0.209 2.98 ± 0.07 5.230 ± 0.181 CPK 0.56 ± 0.04 8.560 ± 0.320 0.61 ± 0.04 8.950 ± 0.310 0.57 ± 0.03 8.980 ± 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

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 oper- ated between voltage-gated L-type calcium channels (LCCs) in the plasma membrane and calcium release channel, com- monly known as ryanodine receptors in the sarcoplasmic re- ticulum.17 Nifedipine is a LCC antagonist.16 Since nifedipine, blocks the cardiotonic action of TAE significantly, the extract

min/mg of protein might have produced its action by opening the voltage sensi- tive slow Ca2+channel. In connection with the cardiotonic ef- fects observed one could see a relationship that exists be- 2+ Enzyme units as micro moles of P(i) liberated/ units Enzyme tween the inhibitory levels of the activities of Mg 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 chrono- F 16.63 21.61 35.37 tropic and ionotropic effects similar to that of adrenaline by df 2, 15 2, 15 2, 15 stimulating the ß-adrenergic receptors. P <0.001 <0.001 <0.001 One-way ANOVA References

Figure 1: Effects of the extracts of Ocimum basilicum Linn. on 1. Jain ML, Jain SR. Therapeutic utility of Ocimum basilicum Linn. VAR Album. Planta Med 1988;54:66-70. heart tissue ATPases of Wistar albino rats. ***P<0.001 when compared to control. 2. Dhar AK. Sweet Basil: Ocimum basilicum- a review. Journal of Medicinal and Aromatic Plant Sciences 2002;24:738-55. 3. Warrier PK, Nambiar VPK, Ramankutty C. Indian Medicinal Plants. Orient Longman 1995;3:162-3. pressed about their long-term effectiveness. The use of 4. Burn JH. Practical pharmacology blackwell. Scientific Publications; 1952. p. 1-7. 5. Okinaka S, Kumagi H, Ebashi S, Sugita H, Momoi H, Toyokura Y, et al. Serum catecholamines is limited by their insufficient differentiation creatine phosphokinase. Activity in progressive muscular dystrophy and neu- between positive ionotropic and chronotropic actions, their romuscular diseases. Arch Neurol 1961;4:520-5. potential arrhythmogenic properties and tachyphylaxis due to 6. King J. The dehydrogenases or oxidoreductases. Lactate dehydrogenase In: receptor down-regulation.11 Practical Clinical Enzymology. London: Van Nostrand, D.Company Ltd; 1965. Total alcoholic extract elicited a powerful cardiotonic ef- 7. King J. The Transferases –alanine and aspartate transaminases In: Practical Clinical Enzymology. London: Van Nostrand, D.Company Ltd; 1965. fect, which was characterized by positive ionotropic and nega- 8. Bonting SL. Sodium-potassium activated adenosine triphosphatase and cation tive chronotropic actions. This effect was not significantly transport In: Bittar EE, editor. Membrane and ion transport. London: Wiley- blocked by propranolol whereas nifedipine, the calcium chan- Interscience; 1970. nel blocker antagonized the effect significantly. 9. Hjerten S, Pan H. Purification and characterization of two forms of low affinity The cardiac enzyme profile indicates that TAE exhibited Ca 2+ATPase from erythrocyte membranes. Biochem Biophys Acta 1983;728: 281-8. cardiotonic like activity which manifested as a result of gen- 10. Ohinishi T, Suzuki T, Suzuki Y, Ozawa K. Comparative study of plasma mem- + + 2+ eral decrease in the activity of Na K ATPase,and Mg ATPase brane Mg2+ ATPase activities in normal, regenerating and malignant cells. and an increase in Ca2+ATPase. This inhibition of Na+ K+ATPase Biochem Biophys Acta 1982;684:67-74. is similar to the action of cardiac glycosides.13 Cardiac 11. Kitada Y, Narimatsu A, Suzuki R, Endoh M, Taira N. Does the positive ionotropic glycosides are specific and unique inhibitors of Na+ K+ATPase action of a novel cardiotonic agent, MCI-154, involve mechanisms other than at normal concentrations (10-8 to 10-9M).14 cyclic AMP? J Pharmacol Exp Ther 1987;243:639-45. 12. Beller GA, Smith TW, Abelmann WH, Haber E, Hood WB Jr. Digitalis intoxica- + + Na K ATPase inhibition by cardiac glycosides leads ulti- tion. A prospective clinical study with serum level correlations. N Engl J Med mately to increase intracellular Ca2+concentrations through 1971;284:989-97.

Na+/ Ca 2+exchange and an associated increase in slow inward 13. Akera T, Brody TM. The role of Na+ K+ATPase in the ionotropic action of digi-

Indian J Pharmacol | June 2004 | Vol 36 | Issue 3 | 163-166 165 Muralidharan A, et al.

talis. Pharmacol Rev 1977;29:187-220. cium-induced release of calcium from the sarcoplasmic reticulum of a skinned 14. Goto A, Yamada K, Yagi N, Yoshioka M, Sugimoto T. Physiology and pharma- canine cardiac Purkinjee cell. J Gen Physiol 1985;85:247-89. cology of endogenous digitalis-like factors. Pharmacol Rev 1992;44:377-99. 18. Chen CL, Sangiah S, Patterson E, Berlin KD, Garrison GL, Dunn W, et al. 15. McGarry SJ, Williams AJ. Digoxin activates sarcoplasmic reticulum Ca2+ re- Effects of BRB-I-28, a novel , and its derivatives on car-

lease channels: a possible role in cardiac ionotropy. Br J Pharmacol 1993;108: diac Na+ K+ATPase, Mg 2+ATPase activities and contractile force. Res Commu 1043-50. Chem Pathol Pharmacol 1992;78:3-16.

16. Wang SQ, Song LS, Lakatta EG, Cheng H. Ca2+ signaling between single L-type 19. Kelly RA, Smith TW. Pharmacological treatment of Heart failure. In: Goodman, Ca2+ channels and rynodine receptors in heart cells. Nature 2001;410:592-6. Gillman, editors. The pharmacological basis of therapeutics. 9th ed. McGraw- 17. Fabiato A. Time and calcium dependence of activation and inactivation of cal- Hill; 1996. Chapter 34. p. 811.

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