WO1996010408A1

WO1996010408A1 - Cardioactive compounds isolated from woody perennials

Info

Publication number: WO1996010408A1
Application number: PCT/AU1995/000639
Authority: WO
Inventors: Elizabeth Alexander; Emilio Luciano Ghisalberti; Marcello Pennacchio; Guy Stephen Richmond; Yana Maolana Syah
Original assignee: The Executive Director Of The Western Australian Department Of Conservation And Land Management
Priority date: 1994-09-30
Filing date: 1995-09-28
Publication date: 1996-04-11

Abstract

Glycosides, salts, hydrates, solvates, derivatives, analogues, tautomers, isomers and/or racemates thereof and optionally at least one other compound isolatable from a woody perennial, said glycosides and other compound having cardiogenic activity. Cardioactive agents or pharmaceutical or veterinary compositions containing these compounds are disclosed as well as methods of treatment and/or prophylaxis involving these compounds. Verbascoside and geniposidic acid isolated from Eremophila species (native fuchsia) are preferred.

Description

Cardioactive compounds isolated from woody perennials

The present invention relates generally to cardioactive compounds isolatable from woody perennials, in particular the Eremophila species of the Myoporaceae family, cardioactive agents containing these compounds and the use of these compounds in therapy including the treatment and/or prophylaxis of cardiogenic disorders.

The Myoporaceae family occurs in the south-west Pacific, Hawaii, China and

Japan, on the Indian Ocean islands (Mauritius and Rodrigeuz) and the West Indies, with the greatest centre of diversity being Australia. The family consists of woody shrubs or small trees which typically grow in low rainfall areas and are characterised by viscid to resinous vegetative parts, bracteate flowers and indehiscent woody fruits.

Within the Myoporaceae, the most significant genus is Eremophila (from the Greek: eremos, desert; phileo, to love) founded by Robert Brown in 1810. Eremophila is well represented in the semi-arid and arid regions of Australia. Many of the 212 recognized species have been record as important in the pharmacopoeia of the Australian Aboriginal people¹². Of these species, two featured prominently in their pharmacopoeia.

E. altemifolia R.Br. (narrow leaf fuchsia bush, native honeysuckle) has long been regarded as the "number one medicine"² and was one of the few plants which the Aboriginal people dried, stored and carried with them in case of need. E. altemifolia is a small to medium ornithophilous shrub (2-3m by 2-3m) often found on skeletal soils on hills and in red loams in Central Australia. The leaves are alternate, scattered, linear, terate to narrowly elliptic or oblanceolate and flattened³. 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 periods⁴.

Infusions from the leaves of E. altemifolia 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-being²'^{7,9,12 mά 14}. 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 poultice⁵.

E. longifolia R.Br. was widely used in the "smoke treatment" of newly born babies and their mothers and had sacred and mystical significance to the Aboriginal people¹². Reportedly, decoctions of the leaves were prepared for eye washes¹⁵, as counter-irritants¹² and for skin and body washes¹⁶. A warning that such preparations should not be taken internally has been noted¹⁶.

We have now isolated extracts of Eremophila species and found that they possess cardiogenic activity. The historic use of this species, particularly by Aboriginal communities in central Australia is recognised. Accordingly, the Department of Conservation and Land Management will seek to secure for these communities a fair and reasonable proportion of any royalty received from the present invention.

According to one aspect of the present invention there is provided a glycoside, salts, hydrates, solvates, derivatives, analogues, tautomers, isomers and/or racemates thereof and optionally at least one other compound isolatable from a woody perennial, said glycoside and other compound having cardiogenic activity.

The salts of the glycoside are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present invention. Examples of pharmaceutically acceptable salts include salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric, orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids; or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, trihalomethanesulphonic, toluenesulphonic, - 3 - benzenesulphonic, salicyclic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

The derivatives of the glycoside are preferably pharmaceutically acceptable salts, hydrates or any other compounds which are capable of providing (directly or indirectly) the glycoside.

The term "tautomer" is used herein in its broadest sense to include glycosides which are capable of existing in a state of equilibrium between two isomeric forms. Such compounds may differ in the bond connecting two atoms or groups and the position of these atoms or groups in the compound,

The term "isomer" is used herein in its broadest sense and includes structural, geometric and stereo isomers.

The term "cardiogenic activity" is used herein in its broadest sense and includes any activity which is produced by, has an effect on or has its origin in the heart.

Suitable glycosides include phenylpropanoid glycosides, iridoid glycosides or flavonoid glycosides.

Phenylpropanoid glucosides, salts, hydrates, solvates, derivatives, analogues, tautomers, isomers and/or racemates thereof hereinafter referred to as "PhGs" are natural products having structures derived by the junction of three basic units: (i) a central glucose;

(ii) a C -C2 moiety, usually a dihydroxyphenyl-β-ethanol, bound at the hemiacetalic function of the glucose; and (iii) a Cg-C moiety, usually a hydroxycinnamic acid, acylating one of the remaining hydroxyls of the glucose. The aromatic units can be differently derivatised and other saccharides are usually linked to one or two of the free hydroxyls of the central glucose. The PhG's which may be isolated are conveniently represented by the following general formulae:

wherein R_j is hydrogen, Ac, Rha or Ara;

R₂ is hydrogen, Api, Glu, Rha, Rha(Ac)₃, Xyl (l-3)-Rha, Api (l-3)-Rha, Ara (l-2)-Rha, Ara (l-3)-Rha, Glu (l-2)-Rha, Xyl (l-2)-Rha, Rha (l-2)-Rha or Glu (l-4)-Rha; and R3 is hydrogen, Xyl, Glu, 6-caffeoyl-Glu, Gal, Ara or Api,

wherein Ri is hydrogen or hydroxy;

Ro s hydroxy or methoxy; R₃ s hydroxy;

RA s hydrogen, hydroxy or methoxy; s hydrogen, Rha or Ara (l-2)-Rha; and

*6 s hydrogen,

wherein Ri is Rha or hydrogen; R2 is Rha or hydrogen; R3 is hydrogen or Rha; and R4 is hydrogen or methyl,

wherein Ri is hydroxy, methoxy or hydrogen; R₂ is hydrogen or hydroxy; R3 is hydrogen or methyl; and R4 is hydrogen, Rha, Glu or All,

wherein R_j is hydrogen, hydroxy or methoxy; R2 is hydroxy or methoxy; R3 is hydrogen or Gal; and

R4 is hydrogen, hydroxy or methoxy,

wherein R_j is hydrogen, caffeoyl or Ac; R is hydrogen, caffeoyl, Rha or Rha(Ac)3; R3 is hydrogen or p-coumaroyl; and R4 is hydrogen, Glu, Rha or caffeoyl,

wherein Ri is Xyl or hydrogen; and

R2 is hydrogen, vanilloyl, coumaroyl, feruloyl, cinnamoyl, dimethylcaffeoyl, p- methoxycinnamoyl or cis-p-coumoroyl,

wherein R is hydrogen, Glu or Glu (1-4) Glu and

wherein R is hydrogen or Glu.

In the above general formulae, the following abbreviations have been used: Ac = Acetyl; Rha = Rhamnose; Ara = Arabinose; Api = Apiose; Glu = Glucose; Xyl = Xylose; and Gal = Galactose.

Preferred PhG's which may be isolated are those of the formula (I) defined above including the following: Calceolarioside A Rι=R2=R3=H;

Calceolarioside D and

Calceolarioside E

and R2=Api;

Plantamajoside = Purpureaside A Rι =R3=H and R2"Glu; Conadroside Rι=R₃=H and R =Xyl;

Verbascoside = Acteoside R_j=R3=H and

2'-O-Acetylacteoside R_j=Ac, R2=Rha and R3=H;

Lugrandoside Rι=R2=H and

Forsythoside A = Forsythiaside Rι =R2=H and

Crassifolioside

and

Poliumoside R_j=H and R2=R3=Rha;

2'-O-acetylpoliumoside = Brandioside R_j=Ac and

Ehrenoside Rι =Ara, R2=Rha and

Pedicularioside A

Purpureaside B Rι =H, R₂=Glu and R₃=Rha;

Echinacoside

6-O-caffeoylchinacoside R_j=H,

and R3=6-Caffeoyl-Glu;

Purpureaside C

and R3~Gal;

Angoroside A Rι=H, R2=Rha and R3=Ara; Forsythoside B Rι =H, R₂=Rha and R3=Api;

Arenarioside Rι =H, R2=Rha and R3Xyl;

Pheliposide Rι=Ac, R₂-Rha and

Tubuloside A Rι=Ac, R2=Rha a

Tubuloside C Rι=Ac, R₂=Rha(Ac)₃ and R₃=Glu; Teucrioside Rι=R₃=H and R₂=Xyl(l-3)-Rha;

Myricoside Rι=R₃=H and R₂=Api(l-3)-Rha;

Lavandulfolioside Rι=R3=H and R2=Ara(l-2)-Rha;

Stachyoside A Rι=R₃=H and R₂=Ara(l-3)-Rha;

Phlinoside A Rι=R₃=H and R₂=Glu(l-2)-Rha; Phlinoside B R_!=R₃=H and R₂=Xyl(l-2)-Rha;

Phlinoside C Rι=R₃=H and R₂=Rha(l-2)-Rha;

Rossicaside A R!=R₃=H and R =Glu(l-4)-Rha; and 2-O-acetylrossicaside A Rι=Ac, R₃=H and R₂=Glu(l-4)-Rha.

A particularly preferred PhG which may be isolated is verbascoside represented by the general formula (Ia)

Iridoid glycosides, salts, hydrates, solvates, derivatives, analogues, tautomers, isomers and/or racemates thereof have the following basic carbon skeleton

Iridoid glycosides may be divided into those having eight, nine and ten carbon skeletons. The nine carbon skeleton glycosides may be separated in two subgroups depending on the position of Co which is either on C or Cg. Examples of eight carbon iridoid glycosides include unedoside and stilbericoside. Examples of nine carbon iridoid glycosides having Cg on C4 include strictoside, loasaside, deutziol, mentzeloside (deutzioside), 7-chlorodeutziol, scabroside and decaloside. Examples of nine carbon iridoid glycosides having Co on Cg include 6, 10 bisdeoxyaucubin, antirride, linaride ( 10- deoxyaucubin), gluroside, 6-desoxy-harpagide, mioporoside, reptoside, glucoside VII, ajubol, ajugoside (leonuride), galiridoside, laterioside, iridoid A, harpagide, acetyl harpagide, harpagoside, 8-(O methyl-p-coumaroyl) harpagide, procumbide, antirrinoside, 5-O-β-glucosyl-antirrinoside, linarioside, bartsioside, aucubin (aucuboside, rhinanthin), 10-O-β-glucosyl aucubin, scrophularioside, agnuside, melampyroside, catalpol, globularidin, globularin (scutellaroside-I), kutkoside, picroside-I, O-methyl-catalpol, scutellariosid-II, verproside, verminoside, specioside, veronicoside, minecoside, catalposide, amphicoside (picroside II), 6-α-L-rhamnopyranosyl catalpol, monomelittoside, odontoside, melittoside, macfadienoside, globularimin and globularinin. Examples of ten carbon iridoid glycosides lamioside, lamiol, decapetaloside, villoside, montinioside, valerosidate, syringenone, syringoxide, patrinoside, opulus iridoid I, opulus iridoid III, opulus iridoid II, opulus iridoid IV, penstemide, boschnaloside, ixoroside, tarennoside, tecomoside, gardoside, bisdesoxydihydromonotropein (dexosyloganic acid), deoxyloganin (bisdesoxydihydromonotropein methyl ester), brasoside, dihydrocornin, loganic acid, loganin (loganoside), mussaenoside, ladroside, ketologanin (7-oxologanin), verbenalin (verbenaloside) (cornin), iiastatoside, syringopicroside, ipolamiide, ipolamiidoside, shanzhiside, shanzhiside methyl ester, caryoptoside (5-desoxy-lamiide), gentioside, barlerin, acetyl barlerin, pulchelloside I, pulchelloside-II, lamiide, lamiidoside, durantoside-III, durantoside-II, durantoside-I, lamalbid (lambridoside), phlomiol, adoxoside, geniposidic acid, geniposide, genipin-1-O-β-gentiobioside, plumieride, theveside, theviridoside, deacetyl-asperulosidic acid, scandoside, asperulosidic acid, 6-EPI-paederosidic acid, 10-acetyl scandoside, paederosidic acid, scandoside methyl ester, feretoside, daphylloside, deacetyl-asperuloside, asperuloside, paederoside, monotropein, vaccinioside, monotropein-methyl ester, nyctanthiioside, gardenoside, forsythide, forsythide methyl ester, ixoside, griselinoside and aralidioside.

A particularly preferred iridoid glycoside which may be isolated is geniposidic acid represented by the general formula (Ila)

The woody perennial is preferably capable of growing in semi-arid or arid conditions. Such woody perennials include those of the Myoporacae family, in particular the genus Eremophila. Preferred species of the genus Eremophila include E. altemifolia, E. bignoniiflora, E. cuneifolia, E. dalyana, E. duttonii, E. elderi, E. fraseri, E. freelingii, E. gilesii, E. goodwinii, E. latrobei, E. longifolia, E. maculata, E. mitchellii, E. neglecta, E. oppositifolia, E. paisleyi and E. sturtii. Particularly preferred woody perennials are Eremophila altemifolia R.Br and Eremophila longifolia R.Br. Specimens of these plants are deposited in the Botanic Gardens of Adelaide and State Herbarium under voucher specimen Nos. RJC 8653 and TI2, respectively.

An example of the other compound which may be isolated is mannitol. The presence of mannitol may contribute to the increase of coronary percussion.

Thus, in a preferred embodiment the present invention provides verbascoside, salts, hydrates, solvates, derivatives, analogues, tautomers, isomers and/or racemates thereof and optionally mannitol isolatable from Eremophila altemifolia, said verbascoside and mannitol having cardiogenic activity.

In another preferred embodiment, the present invention provides geniposidic acid salts, hydrates, solvates, derivatives, analogues, tautomers, isomers and/or racemates thereof and optionally at least one other compound isolatable from Eremophilia longifolia, said genisposidic acid and at least one other compound having cardiogenic activity. For ease of reference, the glycoside and the other optional compound will collectively hereinafter be referred to as "compounds of the invention".

The compounds of the invention are preferably isolated from the woody perennial in substantially biologically pure form.

Thus, according to another aspect of the present invention there is provided a substantially biologically pure from of the compounds of the invention.

The present invention also provides a method for the isolation of the compounds of the invention from a woody perennial which comprises extracting the woody perennial with methanol and/or water.

The compounds of the invention are conveniently isolable from the leaves, bark, stems, flowers or roots of the wood perennial. Generally, the plant material is ground to a powder using any suitable apparatus, such as, a grinder. Extracts are then prepared of the ground plant material and the methanol and/or water fraction is subjected to a separation technique, for example, chromatograp y and the fraction recovered with water and/or methanol.

The compounds of the invention as isolated from the woody perennial have been found to possess cardiogenic activity including effects on heart rate, contraction force and coronary perfusion rate (hereinafter referred to as "CPR"). The compound(s) isolated from E. altemifolia increased heart rate, contraction force and CPR. The compounds isolated from E. longifolia decreased heart rate, contraction force and CPR.

Thus, the present invention also provides a cardioactive agent comprising the compounds of the invention suitable for use in therapy including the treatment and/or prophylaxis of cardiogenic disorders.

The term "cardioactive agent" is used herein in its broadest sense and includes any agent capable of acting on the heart. It will be appreciated that this term includes "cardiotonic agents" which are capable of increasing the efficiency of the contractions of the heart muscle.

The present invention further extends to the use of the compounds of the invention in the manufacture of a medicament for the treatment and/or prophylaxis of cardiogenic disorders.

The present invention still further provides a method for the treatment and/or prophylaxis of cardiogenic disorders which comprises administering therapeutically acceptable amounts of the compounds of the invention to a subject in need of such treatment and/or prophylaxis.

The cardiogenic disorders may include any disorder which hinders the heart acting as an efficient pump, such as, for example, angina pectoris, dysrhythmias, tachycardia, myocardial infarction, congestive heart failure and atrial fibrillation.

For ease of reference, the compounds of the invention will hereinafter be referred to as the "active ingredients".

The method of treatment and/or prophylaxis involves the administration of effective amounts of the active ingredients to a subject in need thereof for a time and under conditions sufficient for the cardiogenic disorder to be inhibited, reduced or otherwise ameliorated. The subject may be a human, livestock animal (e.g. sheep, cow or horse), laboratory test animal (e.g. mouse, rabbit or guinea pig) or companion animal (e.g. dog or cat). The present invention, however, is particularly directed to human therapy.

The active ingredients may be administered in a single dose or a series of doses. While it is possible for the active ingredients to be administered alone, it is preferable to present them as a pharmaceutical composition.

Accordingly, the present invention also provides a pharmaceutical composition comprising the active ingredients and one or more pharmaceutically acceptable carriers, adjuvants, diluents and/or excipients and optionally other therapeutic agents.

The carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. Compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredients with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredients may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g inert diluent), preservative, disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredients therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Compositions suitable for topical administration on the skin include creams, ointments, gels, pastes, foam or spray formulations which may include cosmetically acceptable carriers, for example, alcohols, perfumes, surfactants, thickeners, antioxidants and preservatives.

For topical application to the eye, the active ingredient may be in the form of a solution or suspension in a suitable sterile aqueous or non-aqueous vehicle. Additives, for instance buffers, preservatives including bactericidal and fungicidal agents, such as phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorohexidine and thickening agents such as hypromellose may also be included.

Compositions for rectal administration may be presented as a suppository or retention enema with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the active ingredient. Such excipients include cocoa butter or a salicylate.

For intranasal and pulmonary administration, the compounds according to the invention may be formulated as solutions or suspensions for administration via a suitable metered or unit dose device or alternatively as a powder mix with a suitable carrier for administration using a suitable delivery device.

Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredients such carriers as are known in the art to be appropriate.

Compositions suitable for parenteral administration include aqueous and non- aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit- dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage compositions are those containing a daily dose or unit, daily sub-dose, as hereinabove described, or an appropriate fraction thereof, of the active ingredient.

The active ingredients may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary compositions include those adapted for:

(a) oral administration, external application, for example drenches (e.g. aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feedstuffs; pastes for application to the tongue;

(b) parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced into the udder via the teat; (c) topical application, e.g. as a cream, ointment or spray applied to the skin; or (d) intravaginally, e.g. as a pessary, cream or foam. It should be understood that in addition to the active ingredients particularly mentioned above, the compositions of this invention may include other agents conventional in the art having regard to the type of composition in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents.

Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

The invention will now be described with reference to the following Examples. These Examples are not to be construed as limiting the invention in any way.

EXAMPLE 1

In Example 1, reference will be made to the accompanying drawings in which: Fig. 1 is a graph showing the effects of the aqueous extract of Eremophila alte ifolia leaves on force-displacement (mean averages + S.E.M) in which Ea = E. altemifolia water extract; Pr = propranolol; Pa = phentolamine and g = grams;

Fig. 2 is a force-displacement (top), ECG of atria (middle) and ECG of ventricles (bottom) following the injection (arrow) of one ml/min. of the aqueous extract of E. altemifolia;

Fig. 3 is a graph showing the effects of the water extract of Eremophila alte ifolia leaves on heart rate (mean averages + S.E.M) in which Ea = E. altemifolia aqueous extract; Pr = propranolol; Pa = phentolamine and bp = beats per minute; and Fig. 4 is a graph showing the effects of the aqueous extract of Eremophila altemifolia leaves on coronary perfusion (mean averages + S.E.M) in which Ea = E. altemifolia aqueous extract; Pr = propranolol and Pa = phentolamine.

Plant Material and Extract Preparation

Leaf samples of E. altemifolia 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, North Terrace, Adelaide, S.A., 5000 deposited shortly after their collection in November 1993; voucher No. RJC 8653). The leaves were dried at 40°C for 48 hrs and then ground up to a fine powder using a vegetative grinder (Dietz-Motoren KG, Elekromotorenfabrik, West Germany, 220 v, type WRB 80 C12Q SIL). The powder (28. Ig) was successively extracted with hexane (0.48g; 1.7% dry weight), dichloromethane (0.6 lg; 2.2% dry weight), methanol (1.80g; 6.4% dry weight) and water (0.80g; 2.9% dry weight) to generate four fractions.

Isolated Heart Preparation Isolated rat hearts were prepared from 50 male and female albino Wistar rats of body mass between 400-600g. The animals were fed and given water ad libitum until they were sacrificed by spinal dislocation⁸. The hearts were rapidly excised, freed of adhering tissue and mounted intact on the modified Langendorff heart apparatus ^,0 and perfused retrogradely with a modified Krebs-Henseleit solution. A solution of the extract (1ml; 8mg/ml) was administered retrogradely through a polyethylene cannula over a time period of one minute. 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.

Perfusate

The hearts were retrogradely perfused with a saline Krebs-Henseleit solution (pH 7.36) modified according to Alexander et al The solution was kept at a constant temperature of 37°C and a constant pressure of 70cm of water and was aerated with a 95% O2 and 5% CO2 gas mixture (Carbogen) prior to and during the experiment. The Krebs-Henseleit solution consisted of the following (mM): NaCl., 118.0; KC1, 4.7; MgCl₂.6H₂O, 0.5; NaHCO₃, 25.0; NaH₂PO₄, 1.0; Glucose, 10.0 and CaCl₂.2H₂0, 2.2.

Drugs

Phentolamine methane sulphonate (α-adrenergic blocker), propranolol hydrochloride (β-adrenergic blocker), phenylephrine hydrochloride (α-adrenergic agonist) and isoproterenol (β-adrenergic agonist), all purchased from Sigma Chemical Company, were used. Stock solutions of 10^"^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.

Data Recording Electrocardiograms were recorded by micro-electrodes 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-B10N time base unit. Signals from the CRO were directed through to a Narco Biosystems Physiography (Registered Trade Mark), model 7173.

Force of contraction was measured with a Nihon Kohden Kogyo Co., Ltd. force- displacement randucer, model SB-1T-H. The transducer was coupled with the Narco Biosystems Physiography (Registered Trade Mark) (model 7172) and attached perpendicular to the heart by a small hook and thread. 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 the laboratory.

Data Analysis At least six experiments were conducted for each treatment. Paired and unpaired

Student's t-tests were performed on the data. Probabilities of less than 0.05 were considered statistically significant. Results

The administration of a single injection of 1ml of crude aqueous extract (8mg/ml injected at the rate of lml/min) induced a biphasic response in the force of contraction. The response (mean + S.E.M) consisted of an initial increase from 1.03 + 0.04g to 1.30 + 0.07g (26% increase), followed by a decease to 0.88 ± 0.07g (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. During positive inotropism the electrical activity of the atria and ventricles decreased. After one minute, when negative inotropism commenced, the electrical activity of both atria and ventricles increased (Fig. 2). Where arrhythmias were observed (38% of experiments), they occurred during this phase. The ensuing decrease in force persisted for an average of 13 minutes, during which time it gradually returned to the initial force of contraction.

Heart rate was significantly lower (from 195 + 8 bpm to 168 + 11 bpm; 14% decrease) during positive inotropism (Fig. 3). Within a minute tachycardia followed, with heart rate increasing to 230 + 8 bpm (19%). This significantly increased chronotropism (PO.05) coincided with the commencement of the decrease in force- displacement. It progressively returned to 195 + 8 bpm after an average time period of 12 minutes.

A sharp, significant (P < 0.05) increase in coronary perfusion rate, from 9.73 + 0.29ml min to 13.32 + 0.83mlΛnin (37%), occurred concurrently with the decrease in force displacement (Fig. 4). After an average period of 10 minutes, it returned to its initial rate.

Propranolol did not reduce or block the isotropic and chronotropic effects of the aqueous extract. A dose of 1 μM propranolol in the perfusate (enough to completely block the effects of 1 μM 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 μM), like propranolol, did not block the isotropic, 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 (PO.05) to those induced by the extract on its own.

The results show that the crude aqueous extract of E. altemifolia leaves mediates an initial positive isotropic effect followed by an immediate decrease. The decrease occurred simultaneously with increased chronotropism and coronary perfusion rate. All induced effects lasted less than 15 minutes, after which the isolated rat hearts made a full recovery and returned to their initial force and rates of concentration and to their initial coronary perfusion rates. Preliminary tests with the methanol extract of E. altemifolia leaves and methanol extract of the bark resulted in identical responses to those induced by the water extract of the leaves. All these responses are similar to those associated with the catecholamine epinephrine.

Comparisons with data obtained by Pennacchio" suggest that the effects of the crude E. altemifolia leaf extract were similar, although not as pronounced, as those of epinephrine. A single 1ml injection of the hormone (1 μM), when administered to isolated rat hearts, induced a significant increase in force-displacement (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 would suggest 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.

The results of α and β-blockers revealed that the active compound or compounds do not exert their effects through the adrenergic receptors of the isolated rat heart. The α-adrenergic blocker phentolamine, widely used in clinical medicine, did not abolish any of the isotropic or chronotropic effects induced by the crude aqueous extract. Moreover, the increase in coronary perfusion was unaltered by the blocker. Propranolol, a non- specific clinical β-blocker, was also unable to reduce the effects caused by the crude extract. Both blockers did, however, completely antagonise the effects of phenylephrine (α-adrenergic agonist), in the case of phentolamine, and isopropterenol (β-adrenergic agonist) in the case of propranolol.

Relatively little is known abut the secondary metabolites produced by this plant. The essential oil fraction (4%) contains fenchone:

as the major component (44%) and limonene:

(15%) . The furanoid sesquiterpenes 9-hydroxydihydro-myomontanone:

and 4-hydroxydihydromyodesmone:

^ have been detected in the leaf extracts ¹³ and the two flavonoids, galangin-3-methyl ether:

and pinobanksin:

have been isolated from the powdered plant ⁶. It is unlikely that any of these compounds are responsible for the activity observed since these compounds are extracted by hexane and dichloromethane.

EXAMPLE 2

In Example 2, reference will be made to the accompanying drawings in which:

Fig. 5 is a graph showing the changes in heart rate mediated by verbascoside and geniposidic acid in which -O- = verbascoside and -O- = geniposidic acid;

Fig. 6 is a graph showing the changes in contraction force mediated by verbascoside and geniposidic acid in which -O- = verbascoside and -O- = geniposidic acid; and

Fig. 7 is a graph showing the changes in coronary perfusion rate mediated by verbascoside and geniposidic acid in which -D- = verbascoside and -O- = geniposidic acid.

For the sake of clarity, SEM values are given in Tables 1 and 2 below rather than as error bars in Figs. 5 to 7. Table 1

The effects of verbascoside on heart rate, contraction force and coronary perfusion rate

Time (min) Heart Rate ± SEM Force ± SEM CPR ± SEM

-4 192 ± 11 1.15 ±0.10 8.2 ± 0.6

-3 192 ± 11 1.15 ±0.10 8.2 ± 0.6

-2 192 ± 11 1.15 ±0.10 8.2 ± 0.6

-1 192 ± 11 1.15 ±0.10 8.2 ± 0.6

0 192 ± 11 1.15 ±0.10 8.2 ± 0.6

1 237 ± 15 1.32 ±0.14 14.3 ±1.1

2 275 ± 9 1.14 ±0.15 12.8 ± 1.0

3 237 ± 11 1.03 ±0.14 12.5 ± 1.0

4 229 ± 16 1.12 ±0.11 11.5 ±0.8

5 216 ± 12 1.12 ±0.11 10.9 ± 1.0

10 188 ± 8 0.98 ±0.15 9.2 ± 0.8

15 187 ±5 0.94 ±0.15 8.2 ± 0.5

- 25

Table 2

The effects of geniposidic acid on heart rate, contraction force and coronary perfusion rate

Time (min) Heart Rate ± SEM Force ± SEM CPR ± SEM

-4 247 ± 10 1.08 ± 0.05 8.6 ± 1.1

-3 247 ± 10 1.08 ± 0.05 8.6 ± 1.1

-2 247 ± 10 1.08 ± 0.05 8.6 ± 1.1

-1 247 ± 10 1.08 ± 0.05 8.6 ± 1.1

0 247 ± 10 1.08 ± 0.05 8.6 ± 1.1

1 222 ± 11 0.65 ± 0.10 8.1 ± 1.1

2 214 ± 17 0.78 ± 0.11 8.3 ± 1.2

3 222 ± 17 0.85 ± 0.12 9.2 ± 1.3

4 230 ± 14 0.89 ± 0.11 9.1 ± 1.5

5 233 ± 14 0.94 ± 0.12 4.9 ± 0.9

10 229 ± 15 0.86 ± 0.12 5.2 ± 1.2

15 229 ± 15 0.84 ± 0.11 7.6 ± 1.4

Plant Material

Leaf samples of £ altemifolia were collected from a granite outcrop in the Mt. Dimer region, Jaurdi Nature Reserve, approximately 100km NE of Southern Cross, Western Australia in November 1993. 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 E. longifolia samples were collected 24km east of Sandstone, Western Australia in February 1994 and the identification was verified by Dr. Chinnock (voucher No. Tl 2). The leaves were dried at 40°C for 48 hrs and were ground up to a fine powder using a vegetative grinder (Dietz-Mororen KG, Eleckromotoenfabrik, West Germany, 220 v, type WRB 80 C12Q SIL).

Extraction of Plant Material and Fractionation of Extracts a) E. altemifolia. Powdered leaves (85g) of the plant were successively extracted into four fractions with light petroleum (4.7g, 5.5% of dry weight), dichloromethane (4.0g, 4.7%), methanol (20g, 23.5%) and water (9.3g, 10.9%). A portion (15ml) of the total aqueous extract (500ml) was fractionated into three fractions by chromatography on a polyamide column (15g, 120-150 mesh, Koch-Light); fraction 1 (eluted with water); 240mg; fraction 2 (eluted with 50% aqueous methanol); 40mg; and fraction 3 (eluted with methanol): lOmg.

A portion of the methanol extract (5g) was subjected to vacuum liquid chromatography (120g Silicic acid 100 mesh; Mallinckrodt). Discontinuous gradient elution with 10% methanol-dichloromethane to methanol afforded 19 fractions which were grouped on the basis of thin layer chromatography (tic) (Kieselgel 60 F254 aluminium sheets; Merck) into three main fractions; fraction l :500mg; fraction 2:38mg; fraction 3:115mg. Further chromatography of fraction 1 gave 85mg of a mixture of two compounds which exhibited cardioactivity.

b) E. longifolia. Powdered leaves (106g) of the plant were successively extracted with light petroleum (1.8g, 1.7% of dry weight), dichloromethane (0.7g, 0.6%), methanol (40g, 39%) and water (11.6g, 11%). A portion (6g) of the methanol extract was fractionated into methanol-ether soluble (1.9g) and insoluble components (3.7g). The latter fraction was subjected to vacuum liquid chromatography (120g Silicic acid 100 mesh; Mallinckrodt) with gradient elution from 20% dichloromethane-methanol to methanol. Analysis of the fractions by tic showed one fraction (1.6g) to be mainly one component. A portion of this (470mg) was purified by further chromatography to yield a fraction (180mg) which contained the major component of the methanol-ether insoluble fraction.

Analysis of Chromatography Fractions H- and ^C-nuclear magnetic resonance (NMR) spectra were obtained for methanol or D2O solutions using a Bruker AMX 500 or a Bruker AM 300 spectrometer. The identity of the compounds were confirmed by comparison of their spectral parameters with those described in the literature and by comparative tic behaviour with that of standard samples. Isolated Heart Preparation

Isolated rat hearts were prepared from male and female albino Wistar rats of body mass between 400-600g. The animals were fed and given water ad libium until they were sacrificed by spinal dislocation. The hearts were rapidly excised, freed of adhering tissue and mounted intact on a Langendorff heart apparatus and perfused with a modified Krebs-Henseleit solution.

Perfusate

The hearts were retrogradely perfused with a saline Krebs-Henseleit solution (pH 7.36) modified according to Alexander et al The solution was kept at a constant temperature of 37°C and constant pressure of 70 cm of water. It was aerated with a 95% O2 and 5% CO2 gas mixture (Carbogen)prior to and during the experiment. The Krebs-Henseleit solution consisted of the following (mM):NaCl, 118.0; KC1, 4.7; MgCl₂.6H₂O, 0.5; NaHCO₃, 25.0; NaH₂PO₄, 1.0: Glucose, 10.0 and CaCl₂.2H₂0, 2.2.

Test Compounds

Both verbascoside and geniposidic acid were administered through a polyethylene cannula in 1 ml ,in retrograde perfusions of ImM concentration. The freshly prepared solutions were introduced into the system only when the hearts had stabilized after being mounted onto the Langendorff apparatus (usually 30 minutes).

Data Recording

Heart rates were monitored throughout each experiment by electrocardiograms recorded by micro-electrodes attached to the right atrial appendage and to the right and left ventricular walls of the isolated heart. The electrodes were directed through to an analog-to-digital converter (MacLab) and recorded on a Macintosh computer. 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 MacLab and computer set-up and attached perpendicular to the heart by a small hook and thread. Coronary effluent was measured by a drop counter. Data Analysis

At least six experiments were conducted for each treatment. The data was analyzed by paired t test with probabilities of less than 0.05 considered statistically significant. All data is expressed as mean ± SEM.

Results

Following the detection of cardioactive compounds in the methanolic and aqueous extracts of E. altemifolia leaves, a bioassay-guided fractionation was undertaken to individuate the active compound(s). Fractionation of the aqueous extract yielded two compounds which were identified from their *H- and ^C-NMR spectral parameters as verbascoside, fraction 2 and mannitol, fraction 1. Silica gel chromatography of the methanol extract afforded a fraction which exhibited cardioactivity by, from NMR analysis, appeared as a mixture of verbascoside and isoverbascoside in a 3:1 ratio. The identities of verbascoside, isoverbascoside and mannitol were established by comparison of their ^H- and ^C-NMR spectral parameters with those reported in the literature¹⁷-¹⁸*¹⁹ and for verbascoside and mannitol by comparative tic with standard samples.

Preliminary tests with the methanol extract of E. longifolia leaves suggested that there was at least one compound in the extract that mediated in inhibitory effect on the isolated rat heart. The major component of the more polar fraction of this extract was identified as the iridoid glucoside geniposidic acid. The ¹H- and ¹³C-NMR parameters were consistent with those reported in the literature²⁰ ™^{d 2I}. Small amounts of mannitol and verbascoside were also isolated from this extract.

Verbascoside and geniposidic acid were tested separately for their effect on the isolated rat heart. Following the administration of 1ml of 1 mM verbascoside, there were significant increases in chronotropism, inotropism and CPR, all of which occurred within the first two minutes. These effects lasted for several minutes except positive inotropism, which lasted less than a minute. The average increase in chronotropism was as high as 43% (Fig. 5). From an initial rate of 192 ± 11 beats per minute (bpm), it significantly (P = 0.001) increased to 275 ± 9 bpm. The effect lasted for a period of 10 minutes, the heart beat progressively returning to normal within that time and dropping slightly thereafter (Fig. 5). The contraction force significantly (P = 0.016) increased from 1.15 ± 0.1 Og to 1.32 ± 0.14g (18%) in the first minute but returned to the initial value by the second minute (Fig. 6). Following the positive inotropic phase there was a gradual decline in contraction force, which is a common occurrence in the Langendorff heart bioassay. The most pronounced effect mediated by verbascoside was on the CPR (Fig. 7). The initial rate of 8.2 ± 0.6 ml/min significantly (P = 0.002) rose to 14.3 ± 1.1 ml/min (74%). The increase in CPR was the longest lasting effect of the three recorded.

With geniposidic acid, at doses of 1ml of 1 mM solution, the initial contraction force significantly (P = 0.002) decreased from 1.08 ± 0.005 ml/min to 0.65 ± 0.10 ml/min (40% decrease). Although there was a slight recovery from this effect, it did not return to its starting point (Fig. 6). The changes in chronotropism were similar to those of inotropism (Fig. 5). Starting at 247 ± 10 bpm, the heart rate significantly (P = 0.005) decreased to 214 ± 17 bpm (13%) two minutes after geniposidic acid was introduced. During this period CPR also decreased by 6%. After three minutes, the CPR increased slightly (7%) and then decreased again (43%). By five minutes the initial CPR of 8.6 ± 1.1 ml min had dropped significantly (P = 0.004) to 4.9 ± 0.9 ml/min (Fig. 7).

Discussion

From two different species of Eremophila two known compounds have been identified with previously unknown cardioactivity. Both verbascoside and geniposidic acid caused significant, but opposite, changes to occur in the Langendorff rat heart preparation. Significant increases in heart rate, contraction force and CPR all occurred within the first two minutes and generally lasted for about 15 minutes. This suggested that verbascoside was responsible for the activity reported for the original extracts. Mannitol may also have played a small role in the CPR response mediated by these extracts. It was observed that increases in CPR were enhanced when verbascoside and mannitol were co-administered. Similar mannitol-induced increases in CPR has been previously reported. Willerson et al ^r and Fixler et al²³ showed that mannitol improved coronary blood flow in ischaemic myocardium of experimental animals. The results obtained with geniposidic acid suggest that it is the active compound in the methanolic extract of E. longifolia leaves. Geniposidic acid significantly decreased heart rate, contraction force and CPR. Similar responses had been observed using crude methanolic extracts of E. longifolia leaves. However, in those tests, the inhibitory effects were soon followed by stimulatory effects, thus suggesting the presence of either a compound with a biphasic effect or the presence of two or more active compounds. Since geniposidic acid does not exhibit a biphasic effect, this leads us to suggest that the stimulatory effects were mediated by the verbascoside with co-occurs in the leaves of E. longifolia. Geniposidic acid is a member of the large iridoid glucoside group of compounds and is of more limited distribution than verbascoside.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

References

1. Alexander, E., Shavit, G., Oron, Y., Gitter, S. and Korczyn, A.D. (1987) Alpha adrenergic mediation of arrhythmogenic effects in intact perfused rat hearts. Univ. Tel Aviv, Dept. Miscell. Publ 88/1.

2. Barr, A. (1988) Traditional bush medicines: An Aboriginal pharmacopoeia. Aboriginal communities of the Northern Territory of Australia. Greenhouse Publications, Richmond, Victoria, pp 256.

3. Chinnock, R.J. (1986) Family Myoporaceae. In: Jessop, J.P. and Toelken, H.R. (Eds) Flora of South Australia. Part III. Plemoniaceae - Compositae. pp 1325- 21345. South Australian Printing Division Publications, Adelaide.

4. Elliot, W.R., and Jones, D.L. (1984) Encyclopaedia of Australian plants suitable for cultivation. Vol. 3. Lothian Publications, Melbourne, pp 516.

5. 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, pp 166.

6. 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, pp 532-537.

7. Latz, P.K. (1982) Bushfires and bushtucker. MA Thesis, The University of New England, pp 244.

8. Lord, R. (1989). Use of ethanol for euthanasia of mice. Aust. Vet. J. 66:268. 9. Low, T. (1990) Bush medicine: A pharmacopoeia of natural remedies. Collins/ Angus and Robertson Publications, Australia, pp 238.

10. Opie, L.H., Shipp, J.C. and Lebouef, B. (1962). Metabolism of glucose-U-C¹⁴ in perfused rat heart. Am. J. Physiol. 203: 838-843.

11. Pennacchio, M. (1992) A study into alpha adrenergic receptors in the isolated rat heart. B.Sc (Hons) Thesis, Curtin University of Technology pp 92.

12. Richmond, G.S. (1993) A review of the use of Eremophila (Myporaceae) by Australian Aborigines. Journal of Adelaide Botanic Gardens 15, pp 101-107.

13. Sutherland, M.D. and Rodwell J.L. (1989) Terpenoid Chemistry XXVIII. Furanosesquiterpene β-ketols from Myoporum betcheanum, M. deserti, M. montanum and other Myoporaceae. A ustralian Journal of Chemistry 42, pp 1995-

2019.

14. Smith N.M. (1991) Ethnobotanical notes from the Northern Territory, Australia. Journal of the Adelaide Botanic Gardens 14, pp 1-65.

15. O'Connell, J.F., Latz, P.K and Barnett, P. (1983) Traditional and modern plant use among the Alyawara of Central Australia. Economic Botany 31, pp 80-109.

16. Barr, A. (1983) Traditional Aboriginal medicines in the Northern Territory of Australia Conversation Commission of the Northern Territory of Australia,

Darwin, pp 650.

17. Andary, C, Wylde, R., Lafitte, C, Privar, G. and Winternitz, F. (1982) Structures of verbascoside and orobanchoside, caffeic acid sugar esters from Orobanche rapumgenissae. Phytochemistry 21, pp 1123-1127. 18. Numata, A., Pettit, G.R., Nabae, M., Yamamoto, K., Yamamoto, E., Matsumura, E. and Kawano, T. (1987) Assignment of quaternary carbons in aromatic compounds by long-range heteronuclear shift correlated 2D-nmr specrroscopy and its application to acteoside. Agricultural and Biological Chemistry 51, pp 1199- 2201.

19. Usov, A.I. and Chizhov, A.O. (1993) The structure and ¹³C NMR spectra of mannitol oligo-β-D-glucopyranosides isolated from the brown sea weed Chordia filum (L) Lam. Russian Chemical Bulletin 42, pp 1742-1745.

20. Guarnaccia, R., Madyastha, K.M., Tegtmeyer, E. and Coscia, C.J (1972) Geniposidic acid, an iridoid glucoside from Ge ipa americana. Tetrahedron Letters pp 5125-5127.

21. Chaudhuri, R.K., Afifi-Yazar, F.U., Sticher, O. and Winkler, T. (1980) ¹³ C NMR spectroscopy of naturally occurring iridoid glucosides and their acylated derivatives. Tetrahedron 36, pp 2317-2326.

22. Willerson, J.T., Powell, W.T. jr, Guinev, T.E., Sanders, CA. and Leaf, A. (1972). Improvement in myocardial function and coronary blood flow in ischaemic myocardium after mannitol. Journal of Clinical Investigation 51, pp 2988-2999.

23. Fixler, D.E., Watson, J.T., Wheller, J.M. and Willerson, J.T. (1976). Effect of hypertonic mannitol and isoproterenol on regional coronary flow following right ventriculotomy. Circulation 54, pp 26-31.

24. El-Nagger, L.J. and Beal, J. (1980) Iridoids. A review. Journal of Natural Products 43, pp 649-707.

Claims

CLAIMS:

1. A glycoside, salts, hydrates, solvates, derivatives, analogues, tautomers, isomer and/or racemates thereof and optionally at least one other compound isolatable from woody perennial, said glycoside and other compound having cardiogenic activity.

2. A glycoside and optionally at least one other compound according to Claim 1 wherein the glycoside is a phenylpropanoid glycoside, an iridoid glycoside or flavonoid glycoside.

3. A glycoside and optionally at least one other compound according to Claim 2 wherein the phenylpropanoid glycoside is verbascoside represented by the genera formula (Ia)

4. A glycoside and optionally at least one other compound according to Claim 2 wherein the iridoid glycoside is geniposidic acid represented by the general formula (Ila

5. A glycoside and optionally at least one other compound according to any one of the preceding claims, which is isolatable from a woody perennial capable of growing in semi-arid or arid conditions.

6. A glycoside and optionally at least one other compound according to Claim 5, wherein the woody perennial is from the Myoporacae family.

7. A glycoside and optionally at least one other compound according to Claim 6, wherein the woody perennial is from the genus Eremophila of the Myoporacae family.

8. A glycoside and optionally at least one other compound according to any one of Claims 5 to 7, wherein the woody perennial is E. altemifolia, E. bignoniiβora, E. cuneifolia, E. dalyana, E. duttonii, E. elderi, E. fraseri, E. freelingii, E. gilesii, E. goodwinii, E. latrobei, E. longifolia, E. maculata, E. mitchellii, E. neglecta, E. oppositifolia, E. paisleyi or E. sturtii.

9. A glycoside and optionally at least one other compound according to any one of Claims 5 to 8, wherein the woody perennial is Eremophila altemifolia R.Br.

10. A glycoside and optionally at least one other compound according to Claims 5 to 8, wherein the woody perennial is Eremophila longifolia R.Br.

11. A glycoside and optionally at least one other compound according to any one of the preceding claims, wherein the other compound is mannitol.

12. Verbascoside, salts, hydrates, solvates, derivatives, analogues, tautomers, isomers and/or racemates thereof and optionally mannitol isolatable from Eremophila altemifolia, said verbascoside and mannitol having cardiogenic activity.

13. Geniposidic acid, salts, hydrates, solvates, derivatives, analogues, tautomers, isomers and/or racemates thereof and optionally at least one other compound isolatable from Eremophila longifolia, said geniposidic acid and at least one other compound having cardiogenic activity.

14. A substantially biologically pure form of the glycoside and optionally at least one other compound according to any one of Claims 1 to 11.

15. A method for the isolation of the glycoside and optionally at least one other compound according to any one of Claims 1 to 11 from a woody perennial which comprises extracting the woody perennial with methanol and/or water.

16. A cardioactive agent comprising the glycoside and optionally at least one other compound according to any one of Claims 1 to 1 1.

17. A cardioactive agent according to Claim 16 for use in therapy.

18. A cardioactive agent according to Claim 16 for use in the treatment and/or prophylaxis of cardiogenic disorders.

19. A cardioactive agent according to any one of Claims 16 to 18 which is a cardiotonic agent.

20. A pharmaceutical or veterinary composition comprising the glycoside and optionally at least one other compound according to any one of Claims 1 to 1 1 and one or more pharmaceutically or veterinarily acceptable carriers, adjuvants, diluents and/or excipients.

21. A pharmaceutical or veterinary composition according to Claim 20 which further comprises other therapeutic agents.

22. A pharmaceutical or veterinary composition according to Claim 20 or Claim 21 for use in therapy.

23. A pharmaceutical or veterinary composition according to Claim 20 or Claim 21 or use in the treatment and/or prophylaxis of cardiogenic disorders.

24. Use of a glycoside and optionally at least one other compound according to any one of Claims 1 to 8 in the manufacture of a medicament for the treatment and/or prophylaxis of cardiogenic disorders.

25. A method for the treatment and/or prophylaxis of cardiogenic disorders which comprises administering a therapeutically acceptable amount of a glycoside and optionally at least one other compound according to any one of Claims 1 to 8 to a subject in need of such treatment and/or prophylaxis.

26. A method according to Claim 25, wherein the cardiogenic disorder is angina pectoris, dysrhythmias, tachycardia, myocardial infarction, congestive heart failure or atrial fibrillation.