Cardioactive effects of Eremophila alternifolia extracts

~ Journal of ~ ETHNOPHARMACOLOGY ELSEVIER

Journal of Ethnopharmacology47 (1995) 91-95

Cardioactive effects of

Eremophila alternifolia extracts

M a r c e l l o P e n n a c c h i o *a, E l i z a b e t h A l e x a n d e r a, E m i l i o L. G h i s a l b e r t i b, G u y S. R i c h m o n d a aSchool of Environmental Biology, Curtin University of Technology, GPO Box U1987, Perth, Western Australia, 6001, Australia bDepartment of Chemistry, The University of Western Australia. Nedlands, Western Australia, 6009, Australia

Received 18 August 1994; accepted 15 March 1995

Abstract

An aqueous extract obtained from the leaves of the traditional Aboriginal medicinal plant Eremophila alternifolia R.Br. (Myoporaceae) was tested on isolated hearts of normotensive rats using the Langendorff heart preparation. A single injection of the extract into the retrograge perfusion solution induced cardioactivity, consisting of a short initial increase in force of contraction (positive inotropism), followed by a decrease in the force (negative inotropism) with simultaneous increase in heart rate (positive chronotropism) and in coronary perfusion rate. These effects were not mediated by c~- or/~-adrenergic receptors. Keywords: Eremophila alternifolia; Traditional Aboriginal medicines; Cardioactivity; Langendorff heart preparation

1. Introduction

The genus Eremophila R.Br. (Myoporaceae) is well represented in the semi-arid and arid regions of Australia. Many of the 212 recognized species (Chinnock, pers. commun., 1994) have been recorded as important in the pharmacopoeia of the Australian Aboriginal people (Richmond, 1993). In particular, Eremophila alternifolia R.Br. (narrow leaf fuchsia bush, native honeysuckle) has long been regarded as the 'number one medicine' (Barr, 1988) and it was one of the few plants which the Aboriginal people dried, stored and carried with them in case of need. Eremophila alternifolia * Corresponding author.

is a small to medium ornithophilous shrub (2-3 m × 2-3 m) often found on skeletal soils on hills and in red loams in Central Australia. The leaves are alternate, scattered, linear, terete to narrowly elliptic or oblanceolate and flattened (Chinnock, 1986). The tubular flowers, normally appearing in August-September, are carmine or more rarely pink or yellow. It is a hardy species and tolerates light to medium frosts and dry periods (Elliot and Jones, 1984). Infusions from the leaves were used by Aboriginal people both internally and externally as a decongestant, expectorant and analgesic. It was reported that this treatment alleviated colds, influenza, fever and headaches and was used for septic wounds, inducing sleep and general well-

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M. Pennacchio et al./ Journal of Ethnopharmacology 47 (1995) 91-95

being (Latz, 1982; Barr, 1988; Low, 1990; Smith, 1991; Richmond, 1993). In some cases, the leaves were chopped up and mashed to a paste with water and used as 'rubbing medicine' for the head or put into grasses and tied around the head as a poultice (Goddard and Kalotas, 1988). As part of a study aimed at identifying plant extracts with cardiotonic activity, we have examined the cardioactivity of an aqueous extract from the leaves of Eremophila alternifolia on isolated intact hearts of normotensive rats. 2. Materials and methods

2.1. Plant material and extract preparation Leaf samples of Eremophila alternifolia were collected in November 1993 from a granite outcrop in the Mt. Dimer region, Jaurdi Nature Reserve, approximately 100 km NE of Southern Cross, Western. Australia (30°23.77'S,119°53.88'E). Sample identification was confirmed in the field by the Myoporaceae authority, Dr. R.J.Chinnock (Botanic Gardens of Adelaide and State Herbarium; voucher no. RJC 8653). The samples were dried at 40°C for 48 h and the plant material was ground up to a fine powder using a vegetative grinder (Dietz-Motoren KG, Eleckromotorenfabrik, Germany, 220 v, type WRB 80 CI2Q SIL). The powder (28.1 g) was successively extracted with hexane (0.48 g; 1.7% dry weight), dichloromethane (0.61 g; 2.2% dry weight), methanol (1.80 g; 6.4% dry weight) and water (0.80 g; 2.9% dry weight) to generate 4 fractions. 2.2. Isolated heart preparation Isolated rat hearts were prepared from 50 male and female albino Wistar rats of body mass between 400 and 600 g. The animals were fed and given water ad libitum until they were sacrificed by spinal dislocation (Lord, 1989). The hearts were rapidly excised, freed of adhering tissue and mounted intact on the Langendorff heart apparatus (modified by Opie et al., 1962) and perfused retrogradely with a modified Krebs-Henseleit solution. This technique and all experiments were approved by the Animal Ethics and Experimentation Committee (approval no. R 15/93). A solution of the extract (1 ml; 8 mg/ml) was administered

retrogradely through a polyethylene cannula over a time period of 1 min. This minimised the likelihood of errors caused by cold stress since the slow injection allowed the extract enough time to warm to 37°C. Furthermore, this mimicked the cardiovascular conditions encountered in vivo.

2.3. Perfusate The hearts were retrogradely perfused with a saline Krebs-Henseleit solution (pH 7.36) modified according to Alexander et al. (1987). The solution was kept at a constant temperature of 37°C and constant pressure of 70 cm of water and was aerated with a 95% 02 and 5% CO2 gas mixture (Carbogen) prior to and during the experiment. The Krebs-Henseleit solution consisted of the following (mM): NaCI, 118.0; KCI, 4.7; MgCI2.6H20, 0.5; NaHCO3, 25.0; NaH2PO4, 1.0; Glucose, 10.0 and CaCI2.2H20 , 2.2. 2.4. Drugs Phentolamine methane sulphonate (ot-adrenergic blocker), propranolol hydrochloride (/~adrenergic blocker), phenylephrine hydrochloride (ct-adrenergic agonist) and isoproterenol (/~adrenergic agonist), all purchased from Sigma Chemical Company®, were used. Stock solutions of 10-2 M were prepared in deionised water and kept frozen to avoid oxidation. All further dilutions were prepared daily (as they were required) and kept in a dark, cool place (4°C) until used. 2.5. Data recording Electrocardiograms were recorded by microelectrodes attached to the right and left atrial appendage and to the right and left ventricular walls of the isolated heart. The electrodes were coupled with a 5111 Tektronix storage cathode ray oscilloscope (CRO) with a 5-A22N differential two channel amplifier and 5-BION time base unit. Signals from the CRO were directed through to a Narco Biosystems Physiograph®, model 7173. Force of contraction was measured with a Nihon Kohden Kogyo Co. Ltd. force-displacement transducer, model SB-IT-H. The transducer was coupled with the Narco Biosystems Physiograph® (model 7172) and attached perpendicular to the heart by a small hook and thread.

M. Pennacchio et al./ Journal of Ethnopharmacology 47 (1995) 91-95

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The heart was anchored in place with another small hook attached to the apex of the heart and to the stand. Coronary effluent was measured by a drop counter constructed in our laboratory.

2.6. Data analysis At least 6 experiments were conducted for each treatment. Paired and unpaired Student's t-tests were performed on the data. Probabilities of < 0.05 were considered statistically significant.

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3. Results Control

The administration of a single injection of 1 ml of crude aqueous extract (8 mg/ml injected at the rate of 1 ml/min) induced a biphasic response in the force of contraction, The response (mean ± S.E.M.) consisted of an initial increase from 1.03 4- 0.04 g to 1.30 4- 0.07 g tension (26% increase), followed by a decrease to 0.88 4- 0.07 g (14% decrease) (Fig. 1). The increase occurred almost immediately following the infusion of the extract into the perfusate and rarely lasted more than a minute. After 1 min, negative inotropism commenced and persisted for an average of 13 min, during which time it gradually returned to the initial level. Where arrhythmias were observed (38% of experiments), they occurred during this phase.

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Fig. 2. Effects of the aqueous extract of Eremophila alternifolia leaves on heart rate (mean averages ± S.E.M.). Key: Ea, Eremophila alternifolia aqueous extract; Pr, propranolol; Pa, phentolamine.

Heart rate was significantly lower (from 195 ± 8 bpm to 168 ± 11 bpm; 14% decrease) during positive inotropism (Fig. 2). Within a minute tachycardia followed, with the heart rate increasing to 230 4- 8 bpm (19%). This significantly increased chronotropism (P < 0.05) coincided with the commencement of the decrease in forcedisplacement. It progressively returned to 195 4- 8 bpm after an average time period of 12 min.

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Fig. 1. Effects of the aqueous extract of Eremophila alternifolia leaves on force-displacement (mean averages ± S.E.M.). Key: Ea, Eremophila alternifolia aqueous extract; Pr, propranolol; Pa, phentolamine.

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Fig. 3. Effects of the aqueous extract of Eremophila ahernifolia leaves on coronary perfusion (mean averages ± S.E.M.). Key: Ea, Eremophila alternifolia aqueous extract; Pr, propranolol; Pa, phentolamine.

M. Pennacchio et al./ Journal of Ethnopharmacology 47 (1995) 91-95

94

A sharp, significant (P < 0.05) increase in coronary perfusion rate, from 9.73 ± 0.29 ml/min to 13.32 ± 0.83 ml/min (37%), occurred concurrently with the decrease in force-displacement (Fig. 3). After an average period of 10 rain, it returned to its initial rate. Propranolol did not reduce or block the inotropic and chronotropic effects of the aqueous extract. A dose of 1 /~M propranolol in the perfusate (enough to completely block the effects of 1 /~M isoproterenol) did not eliminate the effects mediated by the extract. Arrhythmias generated by the extract (38%) were not abolished in the presence of propranolol. Phentolamine (1/~M), like propranolol, did not block the inotropic, chronotropic or coronary perfusion effects induced by the extract nor did it prevent arrhythmias, t-tests performed on these data suggest that the increases observed were not significantly different (P < 0.05) to those induced by the extract alone.

hearts recovered fully and returned to the original levels of rate and contraction and as well as coronary perfusion rates. Preliminary tests with the methanol extract of Eremophila alternifolia leaves and methanol extract of the bark resulted in identical responses to those induced by the water extract of the leaves. All of these responses are similar to those associated with the catecholamine epinephrine. Comparisons with data obtained by Pennacchio (1992) suggest that the effects of the crude Eremophila alternifolia leaf extract were similar, although not as pronounced, as those of epinephrine. A single 1 ml injection of the neurohormone (1 /~M), when administered to isolated rat hearts, induced a significant increase in forcedisplacement (40%), followed by a reduction in force coinciding with increased chronotropism (50%). No data was available for comparison of coronary perfusion rate. The similarity of these results suggests the involvement of a compound or compounds within the extract that are catecholamine-like in nature. However, the inability of phentolamine and propranolol to block these effects suggests otherwise. Our results with o~- and/3-blockers revealed that the active compound or compounds do not exert their effects through the adrenergic receptors of the isolated rat heart. The ot-adrenergic blocker phentolamine, widely used in clinical medicine, did not abolish any of the inotropic or chronotropic

4. Discussion

Our results show that the crude aqueous extract of Eremophila alternifolia leaves mediates an initial, but transient, positive inotropic effect followed by an immediate decrease. The decrease occurred simultaneously with increased chronotropism and coronary perfusion rate. All induced effects lasted < 15 min, after which the isolated rat

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M. Pennacchio et al./ Journal of Ethnopharmacology 47 (1995) 91-95

effects induced by the crude aqueous extract. Moreover, the increase in coronary perfusion was unaltered by the blocked. Propranolol, a nonspecific clinical /3-blocker, was also unable to reduce the effects caused by the crude extract. However, phentolamine completely antagonized the effects of phenylephrine (ct-adrenergic agonist), and propranolol those of isoproterenol (~adrenergic agonist). Relatively little is known about the secondary metabolites produced by this plant. The essential oil fraction (4%) contains fenchone (1), as the major component (44%), and limonene (2) (15%) (Barr, 1988) (Fig. 4). The furanoid sesquiterpenes, 9-hydroxydihydromyomontanone (3) and 4-hydroxydihydromyodesmone (4) have been detected in the leaf extracts (Sutherland and Rodwell, 1989) and the two flavonoids, galangin-3-methyl ether (5) and pinobanksin (6), have been isolated from the powdered plant (Jefferies et al., 1962). It is unlikely that any of these compounds are responsible for the activity observed since compounds 1-6 are extracted by hexane and dichloromethane. The isolation and identification of the active compound(s) is currently underway. Acknowledgments Dr R.J. Chinnock (Botanic Gardens of Adelaide and State Herbarium) is thanked for assistance in the field and for providing Eremophilataxonomy. The WA Department of Conservation and Land Management (CALM) is thanked for giving permission to carry out field work on Jaurdi Nature Reserve and for allowing sample collection.

References Alexander, E., Shavit, G., Oron, Y., Gitter, S. and Korczyn, A.D. (1987) Alpha adrenergic mediation of arrhythmogenic

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effects in intact perfused rat hearts. University of Tel Aviv, Departmental Miscellaneous Publication No. 88/1. Barr, A. (1988) Traditional bush medicines: an Aboriginal pharmacopoeia. Aboriginal communities of the Northern Territory of Australia. Greenhouse Publications, Richmond, Victoria, 256 pp. Chinnock, R.J. (1986) Family Myoporaceae. In: J.P. Jessop and H.R. Toelken (Eds.), Flora of South Australia. Part IlL Polemoniaceae- Compositae. pp. 1325-1345. South Australian Printing Division Publications, Adelaide. Elliot, W.R. and Jones, D.L. (1984) Encyclopaedia of Australian plants suitable for cultivation. Vol.3. Lothian Publications, Melbourne, 516 pp. Goddard, C. and Kalotas, A. (Eds.), (1988) Punu. Yankunytjatjara Plant Use Traditional methods for preparing food, medicines, utensils and weapons from native plants. Institute for Aboriginal Development, Angus & Robertson Publishers, 166 pp. Jefferies, P.R., Knox, J.R. and Middleton, E.J. (1962) The chemistry of Eremophila species IV. Some flavonoid constituents. Australian Journal of Chemistry 15, 532-537. Latz, P.K. (1982) Bushfires and bushtucker. MA Thesis, The University of New England, 244 pp. Lord, R. (1989) Use of ethanol for euthanasia of mice. Australian Veterinary Journal 66, 268. Low, T. (1990) Bush medicine. a pharmacopoeia of natural remedies. Collins/Angus and Robertson Publications, Australia, 238 pp. Opie, L.H., Shipp, J.C. and Lebouef, B. (1962). Metabolism of glucose-U-C 14 in perfused rat heart. American Journal of Physiology 203, 838-843. Pennacchio, M. (1992) A study into alpha adrenergic receptors in the isolated rat heart. B.Sc. (Hons.) Thesis. Curtin University of Technology. 92 pp. Richmond, G.S. (1993) A review of the uses of Eremophila (Myoporaceae) by Australian Aborigines. Journal of Adelaide Botanic Gardens 15, 101-107. Sutherland, M.D. and Rodwel|, J.L. (1989)Terpenoid Chemistry XXVIII. Furanosesquiterpene/~-ketols from Myoporum betcheanum, M. deserti, M. montanum and other Myoporaceae. Australian Journal of Chemistry 42, 1995-2019. Smith, N.M. (1991) Ethnobotanical notes from the Northern Territory, Australia. Journal of the Adelaide Botanic Gardens 14, 1-65.