IE43545B1 - Pharmacologically active substance obtainable from plants - Google Patents

Abstract

EFFECTIVE SUBSTANCE FROM PLANTS BELONGING TO THE LABIATAE FAMILY of the disclosure: Extraction processes are described resulting in the isolation of a pharmacologically active substance from a plant of the Labiatae family. Chemical and physical properties of this compound are given in the specification. Because of its vasodilating and blood pressure lowering effect the compound is useful as a drug for the treatment of hypertension.

Description

The present invention relates to a pharmacologically active effective substance obtainable from plants belonging to the Labiatae family, to a process for the isolation of that substance from the plant material and to pharmaceutical - preparations containing it as active substance.

The present invention provides a pharmacologically active substance obtainable from a plant of the Labiatae family and which has the following properties: (i) a melting point within the range of from 230 to \ 232°C, (ii) a molecular formula of C22H34°7* (iii) a specific rotation [a]25 of -20 to -30° (0=1.7 in CHC13), (iv) an ultraviolet spectrum showing absorption at λ max . 208—210 (ξ max 900—1200) and 305—310 nm (ξ max 45—55). (v) an infra red spectrum as shown in Figure 1 of the accompanying drawings, and (vi) a nuclear magnetic resonance spectrum as shown in Figure 2 of the accompanying drawings.

· The Labiatae family comprises about 3500 plant species distributed over about 180 genera. Among these 180 genera are the plants classified as Plectranthus, Coleus, Anisochilus, Lavandula and Leonitis. About thirty Plectranthus species are found to grow in India, of which P. macranthus, P. mollis, P. stocksii, P. coetsa, and P. incanus are the more common — f.' cj plants. There are about nine coleus species to be found in India bearing the names c. antboinicus, C. forskohlii, c. ma iabaricus, C. parviflorus, C. spicatus, C. rotundifolius, C. scutellarioides, C. blumel and C. lacinatus. In the Anlaochilus group, the species A. carnosus and A. verticlllatus are the two more commonly growing plants of the thirteen reported to be found in India. The plants of interest in the Lavandtla genera are L. biplnatta, L. officinalis, L. giasoni and L. bvraanni, Leonftis nepetaefplia is the more ccamt» plant of the two Leonltis species found to grow in India. coleus forskohlii is a particularly good source of the pharmacologically active substance of the invention. This plant is an Indian herb belonging to the Labiatae family and is synonymous with Coleus barbatus (Benth.). The plants grow in various parts of India and are commonly to be found in the subtropical Himalayan region, the Decean Peninsula, Gujarat, Sihar and South India. The plants are also cultivated in several places in India, namely Bombay, Gujarat and Saurashtra. The morphological details and distribution of Coleus forskohlii are fully described (cf. The Wealth of India, Vol. 11, C.S.I.R., India, 1950, page 503).

A pharmacologically active substance of the invention has principally a blood pressure lowering property and a positive inotropic effect. Preferably the roots of Coleus forskohlii are used for isolation of the pharmacologically active substance.

The present invention provides a process for the isolation of the pharmacologically active substance wherein plant material from a plant of the Labiatae family is extracted with an organic solvent and the active substance is isolated from the resulting extracts. - 4 Suitable solvents are, for example, aromatic hydrocarbons, aromatic and aliphatic halogenated hydrocarbons, dialkyl ethers, dialkyl ketones, alkanols, carboxylic acids and their esters and any other solvent which will dissolve the desired pharmacologically active substance, such solvents being for example dimethyl formamide and dimethyl sulfoxide.

To isolate the active substance it is advantageous to concentrate the extract to give a residue and then to treat the residue with an alkanol. Any undissolved material is removed, to give an alkanolic solution which may then be subjected to chromatography, especially column chromatography. Generally the alkanolic solution is first concentrated to give a residue which is then subjected to chromatography.

Alternatively, the extract containing the active substance is concentrated to give a residue which is then partitioned between a pair of immiscible solvents only one of which is capable of dissolving the active substance. The solution containing the active substance may then be subjected to chromatography as described above. Of course, the residue of the above alkanolic solution may be subjected to partitioning, if desired.

The active substance, can, if desired, be purified by crystallisation. This is preferably carried out after chromatography for example, by evaporating the chromatographic fractions.

Two operational methods are preferred, in the first one, a plant of the Labiatae family is extracted with a hydrocarbon having 5 to lo carbon atoms and then with a halogenated hydrocarbon having 1 to 3 carbon atoms and up to 6 halogen atoms, the halogenated hydrocarbon extract is evaporated to give a residue, ths residue is extracted with an alkanol naving 1 to 6 carbon atoms, the alkanol extract is evaporated to give a residue, and the resulting residue is subjected to column chromatography, the eluted fractions containing the product are evaporated to give a residue and the residue is crystallized to give the pharmacologically active substance.

The first method according to tha invention preferably comprises extracting dried roots of tha plant Coleus fors.koh.lii with an aliphatic halogenated hydrocarbon having I to 3 carbon atoms and up to 6 halogen atoms, preferably chlorine atoms, evaporating the extract to give a residue, treating the residue with a lower alkanol having 1 to 6 carbon atoms, removing the insoluble residue by filtration, evaporating the alkanolic filtrate to give a residue, subjecting the residue to column chromatography, preferably on silica gel or alumina using aa eluants tho usual organic solvents of increasing polarity known to those skilled in the art, evaporating the fractions containing the desired product and recrystallising the .resulting residue, Xn the second operational method, a plant of the Labiatae family is extracted with a hydrocarbon having 5 to 10 carbon atoms and then with an alkanol having 1 to 6 carbon atoms, the alkanolic extract is evaporated to give a residue, tha residue is partitioned between a pair of immiscible solvents, one of 'which must be capable of dissolving the pharmacologically effective substance, the solution is evaporated to give a residue and the residue so obtained is subjected to column chromatography, the eluted fractions containing the product are evaporated to give a residue and the residue is recrystaliised to give the pharmacologically 4354S - 6 active substance according to the invention.

The second operational method preferably comprises extracting the'dried roots of the plant Coleus forskohlii with an aliphatic alkanol having 1 to 6 carbon atoms, preferably methanol or ethanol, evaporating the extract to give a residue, partitioning the residue between two immiscible solvents, one of which being capable of dissolving the desired substance, preferably chloroform/water or benzene/water, evaporating the organic solvent layer to give a residue, subjecting the residue to column chromatography, preferably on silica gel or alumina, using as eluants the usual organic solvents of increasing polarity known to those skilled in the art, evaporating the fractions containing the desired product and recrystallizing the residue.

It is advantageous in all methods to use the preferably dried and ground roots of Coleus forskohlii and to subject these to a previous extraction with a hydrocarbon solvent in order to eliminate the main part of the plant fats and waxes present. There are advantageously used hydrocarbons having 5 to 10 carbon atoms, preferably petroleum ether, pentane or hexane in the ratio of 1:2 to 1:10 parts by weight of plant material to solvent.

For extracting the active substance from Coleus forskohlii, aliphatic halogenated hydrocarbons having 1 to 3 carbon atoms and up to 6 halogen atoms, preferably up to 6 chlorine atoms or alkanols having 1 to 6 carbon atoms are used. From these solvents chlorinated alkanes, especially methylene chloride and chloroform or alkanols, especially methanol and ethanol, are preferred. The extracting agents are preferably used in ) the ratio of 1:2 to 1:10 parts by Weight of plant material to extracting agent. 3 S g The halogenated hydrocarbon extract or the alkanol extract is concentrated under reduced pressure, for example, in vacuo to give a residue. Depending on the extract from which the residues are obtained, the residues are now proces5 sed differently as described in the next two consecutive paragraphs and as depicted in the following flow diagram of the isolation procedure.

FLOW DIAGRAM OF ISOLATION PROCEDURE Halogenated hydrocarbon extract evapd. to dryness Residue treated with alkanol and filtered Alkanol extract evapd. to dryness Residue Partition between CHC1 -H 0 or C6H6 fy Alkanolic Residue solution evapd. to dryness Residue I Chromatography crystallization Product Aqueous Chloroform or layer benzene layer evapd. to dryness Residue Chromatography crystallization Product 4354S - 8 The residue from the halogenated hydrocarbon extract is extracted with a lower alkanol having from 1—6 carbon atoms, such as for example methanol which is preferred, until the removal of alkanol soluble material is complete. The volume of alkanol used is preferably in the ratio of 1:10 to 1:40 parts by weight of residue to alkanol. The alkanolic extracts are combined and filtered to give an alkanolic solution.

The second process for treating the residue from the 0 alkanol extract is partitioning the residue between a pair of immiscible solvents, one of which is capable of dissolving the desired pharmacologically effective substance, and the other is a solvent in which the desired substance is insoluble, such a pair of solvents being preferably chloroform and water. An approximately 1:1 (V:V) mixture of chloroform—water is prefer ably used. The chloroform layer is separated.

The resulting alkanolic solution of the first process or the organic solvent layer of the second process are evaporated to dryness under reduced pressure, preferably in vacuo and the residue is subjected to chromatography, preferably on a column of silica gel or alumina using as eluants organic solvents or mixtures of these solvents of increasing polarity known to those skilled in the art. The preferred organic solvents used as eluants for column chromatography are: petroleum ether (B.p. 60—80°C), petroleum ether-benzene mixtures, benzene, benzene-ethyl acetate or benzene-chloroform mixtures, ethyl acetate-methanol or chloroform-methanol mixtures and methanol. The fractions eluted by benzene-ethyl acetate or benzenechloroform and containing the desired compound as indicated by thin-layer chromatography are combined and evaporated to dryness under reduced pressure, preferably in vacuo, and the residue is recrystallized from organic solvents, preferably ethyl acetate/petroleum ether (b.p. 60—80°C) to give a colorless crystalline product melting at 230—232°c which is the pharmacologically active substance of the present invention.

The molecular formula of the active substance is C22H34°7 which has been computed from the molecular weight of 410 mass units determined by mass spectrometry and from the following elemental analytical data: C 64—65% and 11=8—8.5%. The ultraviolet spectrum shows absorption at λ max 208—210 (ξ max 900~ 1200) and 305—310 nm (ξ max 45—55). The adsorption is often observed at λ max 210 (ξ max 1000) and 305 nm (ξ max 50). The infrared and nuclear magnetic resonance spectra are shown in Figs. 1 and 2 of the accompanying drawings. The specific rota25 o tion [a] is -20 to -30 (C=1.7 in CHCl^), and may be observed to be -25 to -27° (0=1.68 in CHClg).

The active substance is soluble in organic solvents such as methanol, ethanol, propanol, acetone, chloroform, methylene chloride, ethyl acetate, benzene and ether.

The spectral and chemical data reveal that the active 20 substance has the following structure: - 10 It is to be understood that the formula includes all possible stereoisomers of the compound and mixtures thereof.

The active substance according to the invention is characterised by a very good hypotensive effect. It has peripheral vasodilation and mild central nervous system depressant activity.

Due to its blood pressure lowering action the active substance of the invention is suitable for the treatment of cardiac and circulatory diseases, for example essential and ) malignant hypertonia, cardiac insufficiency, Angina pectoris, and disturbances of the peripheral circulation. The severity of the disease and the weight of the patient determine the daily dose to be administered, which may vary within 25 mg and 1,000 mg. > As will be appreciated by those skilled in the art, the positive inotropic effect of the substance will be useful as an adjunct therapy to cardioactive glycosides in congestive heart failure, in collapse due to haemorrhage and in surgical shock.

The invention accordingly provides a pharmaceutical preparation which comprises the active substance of the invention in admixture or conjunction with a pharmaceutically suitable carrier. The preparation may be in a form suitable for enteral, preferably oral, administration, or for administration by injection or infusion, generally intravenously. The enteral preparations are preferably in Unit dosage form, for example, tablets, capsules and dragees, which advantageously comprise from 25 to 1,000 mg of the active substance per unit dose. For intravenous administration the preparations are, for example, solutions or suspensions of the active substance in a pharmaceutically suitable vegetable oil, for example peanut oil or sesame oil and alcoholic solutions of the active substance, for example, solutions in ethanol, propanediol or glycerol or in mixtures of two or more of the above solvents.

The pharmaceutical preparations of the invention may also comprise one or more further pharmacologically active substances. For example, diuretics, antiarrhythmic agents, β-blockers, tranquillisers, cardio-dilatatory agents and hypolipidaemic agents. The following Examples illustrate the invention.

EXAMPLE 1 Dried and ground roots of Coleus forskohlii (12 kg) were extracted twice with 15 1 portions of petroleum ether (b.p. 60— 80°C). The roots were then repeatedly extracted with 15 1 portions of methylene chloride till they were exhaustively extracted. 60 1 methylene chloride were used. The combined methylene chloride extracts were filtered and evaporated in vacuo. The residue (ca. 200 g) was extracted twice with 3 1 portions of methanol. The combined methanol extracts were filtered and the methanolic filtrate was evaporated in vacuo.

The residue (ca. 130g) was chromatographed on a silica gel column using as eluants the following solvents: petroleum (b.p. 60—80°C), petroleum ether-benzene mixtures, benzene, benzene-ethyl acetate mixtures, ethyl acetate, ethyl acetatemethanol mixtures and methanol. The appropriate benzene-ethyl acetate eluted fractions containing the desired compound, as indicating by monitoring the eluted fractions by thin-layer chromatography and/or by testing for pharmacological activity, were combined and evaporated to dryness in vacuo. The residue was recrystallized from ethyl acetate-petroleum ether (b.p. 60— 80°C) to give colourless crystals in a yield of 8.6 g, which crystals had the properties indicated earlier herein. - 12 EXAMPLE 2 Dried and ground roots of Coleus forskohlii (12 kg) were extracted twice with 15 1 portions of petroleum ether (b.p. 60— 80°C). The roots were then repeatedly extracted with 25 1 of methanol, till they were exhaustively extracted. 100 1 of methanol were used. The combined methanol extracts were filtered and evaporated in vacuo. The residue (ca. 650 g) was partitioned between 2 1 portions of chloroform and 2 1 portions of water each, the chloroform layer was separated. The aqueous layer was repeatedly extracted with 2 1 portions of chloroform. The combined chloroform extracts were filtered, dried over sodium sulfate and evaporated in vacuo. The residue (about 300 g) was chromatographed on a silica gel or alumina column and worked up as in Example 1. Colorless crystals (9.0 g) were obtained which had the properties indicated earlier.

EXAMPLE 3 kg of dried and ground whole plants of Coleus forskohlii were treated according to the process described in Examples 1 and 2 and yielded 8.5 g of active substance; this active sub3 stance had the properties indicated earlier.

EXAMPLE 4 kg of dried and ground roots of Coleus forskohlii were extracted as described in the first paragraph of Example 1.

The residue (ca. 130 g) was chromatographed on an alumina column using as eluants the following solvents: petroleum ether (b.p. 60—8o°C), petroleum ether-benzene mixtures, benzene, benzene-chloroform mixtures, chloroform, chloroformmethanol mixtures and methanol. The appropriate benzenechloroform eluted fractions after evaporation to dryness in vacuo and recrystallization of the residue from ethyl acetatepetroleum ether (b.p. 60—80°C) gave 8.1 g of active substance which substance had the properties indicated earlier herein.

Claims (3)

1. CLAIMS:1. A pharmacologically active substance obtainable from a plant of the Labiatae family and which has the following properties: (i) a melting point within the range of from 230 to 232°C, (ii) a molecular formula of

2. A process for the isolation of the active substance claimed in claim 1, wherein plant material from a plant of the Labiatae family is extracted with an organic solvent and the active substance is isolated from the resulting extract. 3. A process as claimed in claim 2, wherein the organic solvent is an aromatic hydrocarbon, an aromatic or aliphatic halogenated hydrocarbon, a dialkyl ether, a dialkyl ketone, an alkanol, a carboxylic acid or an ester thereof, dimethyl formamide or dimethyl sulphoxide. 4. A process as claimed in claim 3, wherein the solvent is a halogenated hydrocarbon. 5. A process as claimed in claim 4, wherein the solvent is an aliphatic halogenated hydrocarbon having up to 3 carbon atoms and up to 6 halogen atoms. 6. A process as claimed in claim 5, wherein the halogen - 14 atoms are chlorine atoms. 7. A process as claimed in claim 5 or claim 6, wherein the solvent is methylene chloride or chloroform. 8. A process as claimed in claim 3, wherein the solvent is an alkanol having up to 6 cartoon atoms. 9. A process as claimed in claim 2 or claim 3, wherein the extract is concentrated to give a residue which is treated with an alkanol, undissolved material is removed and the active substance is isolated from the resulting alkanolic solution. 0 10. A process as claimed in claim 9, wherein the alkanol has up to 6 carbon atoms. 11. A process as claimed in claim 9 or claim 10, wherein the solvent used to produce the extract is as defined in any one of claims 4 to 7. 5 12. A process as claimed in claim 2 or claim 3, wherein the extract is concentrated to give a residue, the residue is partitioned between two immiscible solvents, and the active substance is isolated from the phase containing it. 13. A process as claimed in claim 12, wherein the two 0 immiscible solvents are chloroform and water, or benzene and water. 14. A process as claimed in claim 12 or claim 13, wherein the solvent used to produce the extract is an alkanol having up to 6 carbon atoms. 3 15. A process as claimed in any one of claims 2 to 14, wherein the active substance is isolated from the solution containing it by chromatography and, if desired, crystallisation. 16. A process as claimed in claim 15, wherein the resulting alkanolic solution of any one of claims 9 to 11 or the 4 3 5 4 5 - 15 resulting organic solution of any one of claims 12 to 14 is concentrated to give a residue which is subjected to column chromatography, the eluted fractions containing the active substance are evaporated and, if desired, the resulting residue is recrystallised. 17. A process as claimed in claim 16, wherein the absorbent for the column chromatography is silica gel or alumina. 18. A process as claimed in any one of claims 2 to 17, wherein the plant material is subjected to a preliminary extraction with a hydrocarbon. 19. A process as claimed in claim 18, wherein the hydrocarbon has 5 to 10 carbon atoms. 20. A process as claimed in any one of claims 2 to 19, wherein the organic solvent and the optional hydrocarbon is each used in a ratio of 1:2 to 1:10 parts by weight plant material:solvent or hydrocarbon. 21. A process as claimed in any one of claims 2 to 20, wherein the plant material is the roots of the plant. 22. A process as claimed in any one of claims 2 to 21, wherein the plant of the Labiatae family is Coleus forskohlii. 23. A process as claimed in claim 2, carried out substantially aa described in any one of the Examples herein. 24. The substance claimed in claim 1, whenever produced by a process as claimed in any one of claims 2 to 23. 25. A pharmaceutical preparation which comprises the active substance claimed in claim 1 or claim 24 in admixture or conjunction with a pharmaceutically suitable carrier. - 16 26. A pharmaceutical preparation as claimed in claim 25, in a form suitable for enteral administration. 27. A pharmaceutical preparation as claimed in claim 26, in unit dosage form. 28. A pharmaceutical preparation as claimed in claim 27, which comprises from 25 to 1000 mg of the active substance per unit dose. 29. A pharmaceutical preparation as claimed in claim 25, in a form suitable for administration by injection or infusion. 02 / (iii) a specific rotation [a] 25 of -20 to -30° (C=1.7 D in CHC1 3 ), (iv) an ultraviolet spectrum showing absorption at λ max 208—210 (ζ max 900—1200) and 305—310 nm (ξ max 45—55), (v) an infra red spectrum as shown in Figure 1 of the accompanying drawings, and (vi) a nuclear magnetic resonance spectrum as shown in Figure 2 of the accompanying drawings,

3. 30. A pharmaceutical preparation as claimed in any one of claims 25 to 29, which also comprises one or more other pharmaceutically active substances.