CA1083589A - Effective substance from plants belonging to the labiatae family - Google Patents

Abstract

EFFECTIVE SUBSTANCE FROM PLANTS BELONGING TO THE
LABIATAE FAMILY
Abstract of the disclosure:
Extraction processes are described resulting in the isola-tion 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 vaso-dilating and blood pressure lowering effect the compound is useful as a drug for the treatment of hypertension.

Description

` ~83S~9 HOE 75/F 342 The present invention relates to a pharmacologically effective substance isolated from plants belonging to the Labiatae family, to a process for the isolation of that sub-stance from the plant material and to therapeutical prepara-tions containing it as active substance.
The Labiatae family comprises about 3500 plant speciesdistributed 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 plants. There are about nine Coleus species to be found in India bearing the names C. amboinicus, C. forskohlii, C. mala-baricus, C. parviflorus, C. spicatus, C. rotundifolius, C. scu- -tellarioides, C. blumei and CO lacinatus. In the Anisochilus group, the species A~ carnosus and A. verticillatus are the two more commonly growing plants of the thirteen reported to be found in India. The plants of interest in the Lavandula genera are L. bipinatta, L. officinalis, L. gibsoni and L. burmanni.
Leonotis nepetaefolia is the more common plant of the two Leonitis species found to grow in India.
The present invention relates to a process for the isola-tion of a pharmacologically effective substance from the afore-mentioned plants belonging to the Labiatae family. A process for the isolation of a pharmacologically effective substance from Coleus forskohlii belonging to the Labiatae family is ~` preferred.
` Coleus forskohlii is an Indian herb belonging to the 29 Labiatae family and is synonymous with Coleu~ barbatus (Benth.).
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The plants grow in different parts o India and are commonly to be found in the subtropical Himalayan region, the ~ecan Pen-insula, Cujarat, Bihar and South India. The plants are also cultivated in several places in India, namely Bombay, Gujarat and Saurashtra. The morpholigical details and distribution of Coleus rorskohlii are fully described (cf. The Wealth of India, Vol. 11, C.S.I.R., India, 1950, page 308).
A pharmacologically effective substance has been obtained from a plant of the Labiatae family which substance has princi-10 pally a blood pressure lowering property and a positively inotro-pic effect. Preferably the roots of Coleus forskohlii are used for isolation of the pharmacologically effective substance.
The present invention provides a process for the isolation -of a pharmacologically effective substance from a plant of the Labiatae family which comprises extracting the dried and ground plant material with a hydrocarbon and then with solvents like aromatic hydrocarbons, aromatic and aliphatic halogenated hydro-carbons, dialkyl ethers, dialkyl ketones, alkanols, carboxylic acids and their esters or any solvent which will dissolve the - 20 desired pharmacologically effective substance, such solvents being for example dimethyl formamide and dimethyl sulfoxide, evaporating the extract to give a residue, either treating the residue with an alkanol and filtering the suspension to provide an alkanolic solution, or paritioning the residue between a pair of immiscible solvents, only one of which is capable of dissolv-ing the desired pharmacologically effective substance, evaporat-ing the alkanolic solution or the solution containing the de-sired substance to give a residue, subjecting the residue to 29 column chromatography, evaporating the chromatographic frac~ions . . .. . -, .. . - -. , ., . . - .

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containing the desired substance to give a re~idue and recrys-tallizing the residue.
Two operational methods are preferred. In the first one, a plant of the Labiatae family is extracted with a hydrocarbon having 5 to 10 carbon atoms and then with a halogenated hydro-carbon having 1 to 3 carbon atoms and up to 6 halogen atoms, the halogenated hydrocarbon extract is evaporated to give a residue, the residue is extracted with an alkanol having 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 crystal-lized to give the pharmacologically active substance..
More particularly, the invented process comprises extract-ing dried roots of the plant Coleus forskohlii with an ali-phatic halogenated hydrocarbon having 1 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 insolu-ble residue by filtration, evaporating the alkanolic filtrate to give a residue, subjecting the residue to column chromato-graphy, 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 resulting-rssidue.
( In the second operational method, a plant of the Labiatae ; family is extracted with a hydrocarbon having 5 to 10 carbon atoms and then wlth an alkanol havlng 1 to 6 carbon atoms, the ~; 29 alkanolic extract is evaporated to give a residue, the residue . . ~ . ... ; ~ , .. . . . , ,: .
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1~35B9 HOE 75/F 342 is partitioned between a pair of immiscible solvents, one of which must be capable of dissolving the pharmacologically ef-fective substance, the solution is evaporated to give a resi due and the residue so obtained is subjected to column chroma-to~raphy, the eluted fractions containing the product are eva-porated to give a residue and the residue is recrystallized to give the pharmacologically active substance according to the invention.
More particularly, the second invented process 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 pola-rity known to those skilled in the art, evaporating the frac-. 20 tions containing the desired product and recrystallizing the residue.
.; It is advantageous in both operational methods to use the preferably dried.and ground roots of Coleus forskohlii and to . subject these to a previous extraction with a hydrocarbon sol-; . 25 vent 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, pen-tane or hexane in the ratio of 1 : 2 to 1 : 10 parts by weight 29 of plant materlal to solvent.

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For extracting the active substance rom 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.
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 processed different-ly as described in the next two consecutive paragraphs and as depicted in the accompanying flow-sheet diagram of the isola-tLon procedure.

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` ~ ~ 108~-~9 HOÉ 75/F 342 Flow-Sheet Diagram of Isolation Procedure . . .

Defatted, ground Plant Parts ¦ extracted with halogenated alkanol hydrocarbons ~
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Halogenat~ ~d hydrocarbon Alkanol extract extract evapd. to evapd. to . . dryness dryness Residue Residue : treated with Partition . alkanol and between CHC13-filtered H20 I' I I
Alkanolic Residue A~uqous CHC13 layer : solution layer ~ .
evapd. to dryness evapd. to dryness .~ .
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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 re-moval 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 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 ~efera_ly 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 benzene-chloroform and containing the desired compownd as indicated by , . ~ .
thin-layer chromatography are combined and evaporated to dry-.i., .

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ness under reduced pressure, preferably in vacuo, and the re-sidue 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 pharma-cologically active substance of the present invention.
The molecular formula of the active substance is C22H34O7 which has been computed from the molecular weight of 410 mass units determined by mas$ spectrometry and from the following elemental analytical data: C 64 - 65 ~ and H=8 - 8.5 %.
- ~0 C22H34O7 requires C = 64.37 and H - 8.35 %.
The ultraviolet spectrum shows absorption at~max 208 - 210 (~max 900 - 1200) an~ 305 - 310 nm ( ~ max 45 - 55). The infrared and nuclear magnetic resonance spectra are shown in Figs. 1 and 2 of the accompanying drawings. The specific rotation [~]25 is -20 to - -30 (c = 1.7 in CHC13).
The active substance is soluble in organic solvents such as meihanol, ethanol, propanol, acetone, chloroform, methylene chloride, ethyl acetate, benzene and ether.
The active substance exhibits physical and chemical proper-2a ties usually shown by terpenoids. Several crystalline deriva- l ¦
tiyes have been maae some of which are for example (a) a dihydro deriyative~ m.p, 245-8, (~ an acetate, m.p. ~99 - 205, and (c~ a methyl ether, m.p. ~74 - ~76. The spectral and chemical 1 data reveal, in particular, that the active substance is a di- , terpenoid possessing a labd-~4-ene carbon skeleton 'i . I
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1~83S~ HOE 75/F 3~2 , bearing oxygen-containing functional groups and which is substi-tuted in such a way that it also has tihe followiny structural features: (a) two secondary hydroxy groups, - (b) one tertiary hydroxy group, (c) an oxide group, (d) one acetoxy group and (e~ one keto group.
The new active su~stance according to the invention is characterized by a very good hypotensive effect. It has peri-~0 pheral vasodilatation and mild central nervous system depressant activ;ty~
Due to its blood pressure lowering action the active sub-stance of the invention is suitable for the treatment of cardiac and circulatory diseases, for example essential and malignant ~5 hypertonia, cardiac insufficiency, Angina pectoris, and distur-~ ~ances of the peripheral circulation. The active substance can Y be used ~ox therapeutical purposes in combination with other pharmacologically active substances, for example diuretics, anti-arrhythmica, ~-blockers, tranquillisers, cardio-dilatatory agents, hypolipidemics, etc.
:~ The act~ve subsi~ance of the invention can be administered perorally or intravenously. The severity of the disease and the weight of the patient determine the daily dose to be administer-ed, ~hic~ may vary within 25 mg and 1,000 mg.
For the administration per os, tablets and drag~es are con-, sidered ~hich contain the active substance in an amount of 25 to 1,000 mg, and the uisual auxiliaries and carriers, for example talcum, starch, lactose, etc. For intravenous administration 29 solutions or suspensions of the active substance in a pharmaceu-: .

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tically tole~ated vegetahle OIl, ~or ex~ple ~eanut oil or ses~me oil and alcoholic solutions of the acti,~e s~bstance, ~or example et~anol, pro~anediol or glycerol or in m~xtures o~ khe above solvents are considered.
The following Examples ~llustrate the extraction of the new active substance from Coleus forskohl~. The possibilit;es of extraction are, ho~ever, not limited to these examples~
E X ~ M P L E ~:
Dried and ~xound roots of Coleus forskohlii (~2 kg~, ~ere ` ~Q extracted t~ice w;th ~5 l portions of petroleum ether (~.p. 60 -8Q~, The roots ~ere then repeatedly extracted w,i:th ~5 l por~
' tions of meth~lene chloride till they were exhausti,vely extract-ed. 60 l methylene chloride were used. The combined methylene chloride extracts were filtered and evaporated in vacuo. The ~5 residue (ca. 20Q g~ was extracted twice with 3 l portions of `' methanolO The combined methanol extracts were filtered and t~e ` methanolic filtrate ~.~a$ evaporated in vacuo. The residue (ca.
~30 g~ ~as chxomatographed on a silica gel column us~ng as eluants t~e follo~ing solvents; petroleum ether (b~p. 60 - 80l, , 2Q petroleum ether~benzene mixtures, benzene, benzene-ethyl acetate mixture$, ethyl acetate, ethyl acetate-methanol m~xtures and ~, metha,nol~ T~e appropr~ate benzene ethyl acetate eluted ~ractions containin~ the desired compound, as indi:cating by monitorlng the eluted fract~ions by thin-layer chromatography andJor by testi:n~
for pharmacolo~ical activity, were combined and evaporated to dr~ne$s in ~a~uo, The residue ~as recrystallized from ethyl acetate-petroleum ether (b.p. 60 - 8Q~) to give colourless cry-- ~ stals ln a yield of 8.6 g, which cr~stals had the properties 'j~ 2~ indi,cated earlier herein.
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ried and ground roots o~ Coleus forskohlii (12 kg) were extracted twice with 15 1 portions of petroleum ether (b.p. 60 -80~. The roots were then repeatedly extracted with 25 1 of S methanol, till they were exhaus~ve~lextracted. 100 1 of metha-nol were used. The combined methanol extracts were filtered and evaporated ln vacuo. The residue (ca. 650 g) was partitioned between 2 l portions of chloroform and 2 l portions of water each, the c~loroform layer was separated. The aqueous layer was ; ~0 repeatedly extracted with 2 l portions of chloroform. The com-bined chloroform extracts were filtered, dried over sodium sul-fate and evaporated in acuo. The residue (about 300 g) was -chromatograp~ed on a silica gel or alumina column and worked up as in Example 1. Colorless crystals (9.0 g~ were obtained which ~5 had the properties indicated earlier E X A M P L E 3:
~5 kg of dried and ground whole plants of Coleus forskohlli were treated accordîng to the process described in Examples 1 and 2 and yielded 8.5 g of active substance; this active sub-stance had the properties indicated earlier.
X A M P L E 4:
~2 kg o~ 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 - 80l, petroleum ether-benzene mixtures, ~enzene, benzene-chloroform mixtures, chloroform, chloroform-methanol mixtures -~ and methanol. The appropriate b~nzene-chloroform eluted frac-2~ tions after e~aporation to dryness 1 acuo and recrystalliza-.. . . . . ... . . . . . . . .
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~35~ ~F 3 4 2 tion of the residue from ethyl acetate-petroleum sther (b.p, 60 - 80C) gave 8.~ g of act~ve substance which substance had the properties indicated earlier herein.

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Claims (16)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of a pharmacologically active substance isolated from a plant of the Labiatae family, characterized in that it has a melting point between 230 and 232°C
and the molecular formula C22H34O7, in which (a) the plant material of the Labiatae family is extracted with a halogenated hydrocarbon, the residue of the extract is treated with an alkanol, undissolved material is separated and the active substance is isolated from the alcoholic solution, or (b) the plant material of the Labiatae family is extracted with an alkanol, the residue of the extract is partitioned between a pair of immiscible solvents and the active substance is isolated from the phase containing it.

2. A process as claimed in claim 1 in which the preparation is carried out according to reaction (a) and the plant material is first extracted with a hydrocarbon to eliminate accompanying substances.

3. A process as claimed in claim 1 in which the preparation is carried out according to reaction (b) and the plant material is first extracted with a hydrocarbon to eliminate accompanying substances.

4. A pharmacologically active substance isolated from a plant of the Labiatae family, characertized in that it has a melting point between 230 and 232°C and the molecular formula C22H34O7, whenever obtained according to a process as claimed in claim 1, claim 2 or claim 3 or by an obvious chemical equivalent thereof.

5. A process as claimed in claim 1 in which isolation is accomplished by evaporation of the solution and chromatography and crystallization of the residue.

6. A process as claimed in claim 1 in which the active substance is isolated by evaporating the solutions obtained, the residue is subjected to a column chromatography and the resultant eluted fractions containing the product are evapo-rated and the residue produced thereby is recrystallized to give the active substance.

7. A process as claimed in claim 1 in which the halogenated hydrocarbon has from 1 to 3 carbon atoms and from 1 to 6 halogen atoms and the alkanol contains from 1 to 6 carbon atoms.

8. A pharmacologically active substance isolated from a plant of the Labiatae family, characterized in that it has a melting point between 230 and 232°C and the molecular formula C22H34O7, whenever obtained according to a process as claimed in claim 5, claim 6 or claim 7 or by an obvious chemical equivalent thereof.

9. A process as claimed in claim 1 in which the immiscible solvents are chloroform and water or benzene and water.

10. A pharmacologically active substance isolated from a plant of the Labiatae family, characterized in that it has a melting point between 230 and 232°C and the molecular formula C22H34O7, whenever obtained according to a process as claimed in claim 9 or by an obvious chemical equivalent -thereof.

11. A process as claimed in claim 1 in which the plant of the Labiatae family is Coleus forskohlii.

12. A pharmacologically active substance isolated from Coleus forskohlii, characterized in that it has a melting point between 230 and 232°C and the molecular formula C22H34O7, whenever obtained according to a process as claimed in claim 11, or an obvious chemical equivalent thereof.

13. A process as claimed in claim 1 in which the dried roots of Coleus forskohlii are extracted with an aliphatic halo-genated hydrocarbon having 1 to 3 carbon atoms and 1 to 6 halogen atoms, the resultant extract is evaporated to produce a residue, the residue is treated with a lower alkanol having 1 to 6carbon atoms, the resultant insoluble residue is removed by filtration, the alkanoic filtrate produced is evaporated to give a residue, the residue is subjected to column chromatography, the fractions containing the product are evaporated from the resultant eluate to produce a residue and the residue is subsequently recrystal-lized.

14. A pharmacologically active substance isolated from Coleus forskohlii, characterized in that it has a melting point between 230 and 232°C and the molecular formula C22H34O7, whenever obtained according to a process as claimed in claim 13 or by an obvious chemical equivalent thereof.

15. A process as claimed in claim 1 in which the dried roots of Coleus forskholii are extricated with an aliphatic alkanol having 1 to 6 carbon atoms, the resultant extract is evaporated to give a residue, the residue is partitioned between two immiscible solvents, one of which is capable of dissolving the desired active substance, the resultant solvent layer containing the active substances is evaporated to give a residue, the residue is subjected to column chromatography, the fractions containing the product are evaporated from the resultant eluate to produce a residue and the residue is subsequently recrystallized.

16. A pharmacologically active substance isolated from Coleus forskholii, characterized in that it has a melting point between 230 and 232°C and the molecular formula C22H34O7, whenever obtained according to a process as claimed in claim 15 or by an obvious chemical equivalent thereof.