CA1063941A - Utero-evacuant extracts from plant substances - Google Patents

Abstract

UTERO-EVACUANT EXTRACTS FROM PLANT SUBSTANCES
ABSTRACT OF THE DISCLOSURE

A method of obtaining utero-evacuant extracts from the zoapatle plant is described. The method involves the extraction of an active principle using an aqueous extraction technique preceded or followed by extraction with water immiscible solvents.
Of the latter, ethyl acetate, chloroform, hexane and benzene are suitable.

Description

This invention relates to a method for extracting a biologically active material from plant products.

The zoapatle plant is a bush of about 2 m. high that grows wild in Mexico. Botanically it is known as Montanoa tomentosa according to Cervantes, Fam. Compositae, Tribe Heliantheae; another variety of the species is Montanoa floribunda.

The plant has been used for centuries in the form of a "tea" or other crude aqueous preparations primarily as a labor lnducer or menses inducer for humans. Its use has been documented in the literature, but definitive chemical and pharmacological studies have not been performed.
What little work of a substantive nature that has been done ; is contradictory in its conclusions.
`;
Natural plant substances are generally kno~7n to be exceedingly complex in their composition. Many compounds of similar chemical and physical properties, as well as those with strikingly dissimilar properties, are normally found in these substances and generally present a difficult separation and identification task. This has no doubt contributed in large measure to the conflicting reports seen in the literature.

A helpful and desirable tool in treating zoapatle for enhancement of the active prlnciple would be a separation process which reduces the quantity of impurities without an attendant significant removal of the active material. Such a procedure should also have the attribute ~F

-2-ORTH ~72 106394~

of slmple, direct and economical operation if lt i8 to be beneficial. The procedure would provide a method for rapidly producing an extract which would be the sub~ect of further reflned purification techniques but which itself has the blological activity above referred to and is itself useful biologically. It would also have the attributes of requiring a lower volume of material to be ingested by the patlent.

In the current folk use of zoapatle, the user lG typically drinks a "tea" brewed from the leaves of the plant by boiling with water in the same fashion one would prepare a hot beverage. She normally does this after having missed a menstrual period and thus is presumably pregnant, although it is Xnown that many frankly pregnant women use the tea to terminate the unwanted pregnancy.
The "tea" obviously contains a mixture of complex materials, some of which may be harmful to the patient. In any case, the tea is bitter tasting and quite ob~ectionable, and since large amounts of the mixture are required, represents an unpleasant form for use. Since such a complex mixture is present, many undesirable materials are ingested which are not necessary to produce the desired effect. 0~ the other hand, the methods of the present invention, contrasted to the aqueous extraction method alone, tend to remove a greater quantity of impurity while retaining biological activity thus reducing the variety of compounds and volume of material that ls required.
' O~ 7 In the method of the present invention, aqueous extraction is used, but lt is always preceded or followed by an organic extraction. For example, suitable methods involve treating a portion of the plant, preferably the leaves, containing the utero-evacuant materials as follows:

(a) aqueous extraction of the leaves of the plant followed by organic solvent extraction of the aqueous layer (b) organic solvent extraction of the leaves of the plant followed by aqueous extraction of the organic layer.

The preferred method of the invention is repre-sented by (a) above. Typically preferred solvents in this process are the water-lmmiscible aliphatic lower chain esters such as methyl acetate, ethyl acetate, butyl acetate and other longer chain esters, aliphatic hydrocarbons such as pentane, hexane and heptane and chlorinated hydrocarbons such as chloroform, carbon tetrachloride, methylene chloride and aromatic hydrocarbons such as benzene, toluene, xylene and the like; higher water immiscible aliphatic alcohols such as butanol and pentanol. Whlle aqueous extraction is an important part of this process, it should be noted that water misclble solvents can be employed with good results in admixture with water during the aqueous extraction phase. Thus, aqueous ethanol or methanol may be suitably employed in the aqueous extraction aspect of this process followed or preceded by organic extraction. In addition, various intermediate steps and techniques may be used to good advantage as will be apparent fr~m a detailed description of the ln~entlon presented below.

~JpTH ~72 In practicing the method of the present invention, one selects a suitable amount, of the order of 3 5 ke. of zoapatle leaves, either dry or fresh, washes them ~rith cold water, optionally subdivides the leaves into small particles and then proceeds to extract them with water preferably hot or organic solvent as indicated generally above. The water extraction step is conveniently done at temperatures of from 25C to 100C and preferably at boiling for ten minutes or more. Prolonged extraction times beyond 2 hours are usually not necessary and are uneconomical.
The aqueous layer thus obtained is then separated from the plant residue giving a dark colored solution.

This solution is then available for the organic solvent extraction step generally described above. In practice it is preferred to use an equal volume of the appropriate organic solvent in extracting the aqueous phase. This aspect of the invention is conveniently per-formed at temperatures ranging from slightly above room - temperature to the boiling point of the particular solvent.
It is preferred to extract at the boiling point of the solvent.
The organic extraction is continued until the desired amount of material is obtained usually of the order of 1 - 2 hours. Organic extracts are combined, evaporated to dryness preferably under reduced pressure, and at temperatures preferably in the range of 30 - 80C.

As stated above, the aqueou~ extraction step may be preceded by the organic solvent extraction step.

In such a case, the conditions recited above ~lith respect to each step, apply equally as well to extraction of leaves or extraction of a leaf extract. In addition, the material resulting from the dual extraction procedure may be further treated with appropriate solvents to improve the quality of the material. This aspect is set forth in the examples, especially Example I.

As a result of the above process, there is obtained a utero-evacuant extract greatly reduced in the amount of impurities when compared to the starting material. The purified form can be orally administered to women to induce labor or abortion or to induce a menstrual period. The material obtained herein has the unique ability to achieve the above results using usually only one oral dose containing about 200 mg. - 500 mg. material. In addition, these results are achieved with minimal side effects and in relatively short periods of time usually of the order of 8 - 12 hours.

The following examples are illustrative of specific embodiments of the present invention.

Example I - Organic Solvent Extraction of Leaves Followed By Aqueous Extraction of Organic Layer 2.0 kg. of dry leaves were refluxed in CHCl3 for one hour. After filtration, the chloroform extract was concentrated to dryness under reduced pressure and temperature f 35 ~ 500C and yielded 196 g. of a dark semisolid mass.
This residue was extracted with hot ethanol-water (1:2).
The aqueous ethanolic phase was re-extracted with CHCl3, the CHC13 layer separated and concentrated to dryness under reduced pressure a~ording a yellow brown syrup. This was 3G dls~olved in ether and the 601ution washed with an aqueous . .

saturated solutlon of sodium bicarbonate, 5~ aqueous æodlum hydroxlde, water, and dried over anhydrous sodlum sulfate.
Evaporation o~ the ether left a yellow oil (11.9 g.).

0.211 Gm. of this material was admlnlstered orally to a woman 11 weeks pregnant diagnosed as carrylng a hidatiform mole. The patient was monitored for changes in arterial blood pressure, maternal heart rate and amniotlc fluid pressure using the procedures described in the American Journal ~f Obstetrics and Gynecology, Vol. 96, No. 6, pages 849 to 856, November 15, 1966, Noreiga Guerra et al. Before admlnl~tration of the medicament, amniotic fluid pressure (uterine contractlon) was a normal 8 to 12 mm.
of mercury without contraction. At about 19 minutes after administration of the medicament, the amnlotlc fluid pressure had increased to 25 mm. of mercury and significant uterine contractlons began. There was no change in heart rate, and no change in arterial blood pressure. This continued for a period of 12 hours at which time the patient aborted.
During this time there was no substantial deviation from the normal blood pressure and heart rate.

Example II - Water Ex~raction of Leaves Followed By Organlc ~olvent Extraction of Water Laver v 634 G. of dry leaves were refluxed with 4.0 1. of water for one hour. The extract was decanted and extracted wlth chloroform. The chloroform phase was dried and filtered, and the solvent was evaporated under reduced presqure, leavlng 4.55 g. of a semisolid mass. The aqueous phaæe was re-extracted with ethyl acetate. The organic pha~e was drled over anhydrouæ sodium sulfate, ~lltered, and concentrated under reduced preææure to yield l.g9 g. Both residues (~rom chloro~orm and ~rom ethyl acetate) were combined and showed strong activity as indicated below.

360 Mg. of the above material were given to a woman 36 weeks pregnant. Observable physiological response began 12 minutes after administration during which time sieniflcant uterine contractlons were observed and documented.
The tonus of the uterus increased to about 50 mm. of mercury.
Uterine activity increased from 170 to 250 Montivideo Units and the frequency increased from 6 to 12 contractions per 10 minute period on average. Thi6 condition continued for 23 hours after administration at which time the woman delivered. This material is considered highly active.

Example III - Water Extraction of Leaves Followed By Ethyl Ao-t-te ~traction of Aqueous Layer ... .
140 G. of dry leaves of ~oapatle and 2.5 1. of water were heated under reflux for one hour. The water extract was concentrated to a total volume of 250 ml., and extracted four times with 750 ml. of ethyl acetate. The extracts were combined and dried over anhydrous sodium - sul~ate. Filtration and removal of the solvent in vacuo furnished 1.3 g. of a dark colored mass. Thiæ was further-extracted with 100 ml. of chloroform, filtered and concen-trated in vacuo. The residue weighed 0.840 g. and was very active as demonstrated below.

- 25 210 Mg. of this material was given orally to a pregnant woman 34 weeks into term. The material was highly - active resulting in delivery 20 hours after administration.
The high activity was evident prior to delivery by a large increa~e in uterlne actlvity from 140 to 270 Montivideo Units O~TH 27~

wlthin a short tlme after admlnistration. Tonus stayed at about 20 mm. of mercury, intensity increased from 50 to 70 mm. of mercury and frequency lncreased from

3 to 5 contractions per 10 minute period on average.

Example IV - Hexane Extraction of Leaves 966 G. o~ dry zoapatle leaves were heated under reflux for three hours with sufflcient hexane to entlrely cover the leaves. After filterlng and concentratlng the resultlng liquld to dryness as prevlously descrlbed, 56.8 gm.
of a dark greenlsh resldue were obtalned. 445 Mg. of this material were administered orally to a pregnant woman 40 weeks lnto term. A slgnificant uterine response was observed to begin after about 10 mlnutes following adminis-tration as follows: The intensity of contractlons increased from 15 mm. of mercury to 50, and the uterine activity from 40 to 220 Montivideo Units, tonus increased from 15 to 30 mm.
of mercury and frequency also increased from 3 to 6 contractions per period of IO minutes.

Example V - Hexane Extraction Followed By Water Extræctlon of the Organic Layer Followed By Ethyl Acetate Extraction of the Water Layer 350 Gm. of dry zoapatle leaves were covered with hexane and the mixture heated until boiling began for 15 to 20 minutes with stirring. The hexane extract was decanted, flltered and treated with an equal volume of water (about 700 ml.). The layers were separated and the hexane layer agaln treated wlth an equal volume of water with stirring. After separation of the layers, the hexane phase uas treated in the same manner one more time. The water layers were all comblned and extracted three times with G~TH 27~

an equal volume of ethyl acetate. The ethyl acetate extracts were combined and evaporated to dryness, yleldlng 6.6 g. of a dark brownish yellow syrup. This material was tested orally as described in Example I and found to be moderately active in producing expulsion of the fetus irom the uterus.

Example VI - Water Extraction of Leaves Followe~ By Benzene Extraction of Active Material -One Kg. of dried zoapatle leaves was heated with 15 1. of water for three hours under reflux (about 92C) at 585 mm. of mercury pressure. The water layer was decanted and filtered through gauze. Thereafter it was treated with about 700 gm. of activat,ed charcoal until flocculation occurred. The mixture was then stirred ior two hours, vacuum filtered rapldly, yielding a clear filtrate. The filtered solid was vacuum dried at room temperature for three days and thereafter suspended in three liters of benzene. Distillation of the benzene caused the elimination of about 200 ml. oi water, yielding a benzene layer which when evaporated to dryness as previously described yielded 3 gm. of an oil. This material was moderately active at a dosage level of 500 mg. when orally administered to a pregnant woman and cauæed expulsion of the fetus within 10 to 20 hours aiter oral administration.

Example VII - Water Extraction of Leaves Followed By Ethyl Acetate Extraction of the Aqueous Layer Two Kg. of fresh zoapatle leaves were covered with 0 (10 to 12 llters of water) and boiled for one hour. The mixture was then filtered through cheese cloth while hot and allowed to cool. The aqueous portion was then extracted ORTH 27~

~063941 twlce with two volumes each of ethyl acetate. The ethyl acetate was then brought to dryness at 50C in vacuo to afford 12 gm. of a dark green mass. The dark mass was treated with 500 ml. benzene and the mixture heated to boiling for about 15 minutes. The benzene layer was decanted and the residue washed with benzene until no further material was extracted. The benzene extracts were combined and reduced to dryness at 50C under vacuum. A
brownish mass resulted which was then successively extracted with separate portions of hexane in the same manner as described for the benzene treatment above until the hexane was colorless. The resulting residue, about 8 gm., has the consistency of thick syrup. This material was then dissolved in acetone (100 ml. or less) and 5 gm. of activated charcoal (Darco) added thereto. The mixture was stirred at room temperature for one hour until the charcoal ~locculated.
The mixture was filtered and the filtrate evaporated to dryness under vacuum and slight heat (30C) to afford 4 to 6 gm. of material which was very active as shown below.

500 Mg. of the above material was administered orally to each of four human females on varying days of their menstrual cycle as indicated:

- Patient AgeWeight Day of CycleBleeding 1 25 40 kg. 11 of 27-28 daywithin 2 hours of cycle administration 2 41 57 kg. 24 of 25 daybegan spotting at cycle 5 hours then regular period 3 25 60 kg. 15 of 28-29 daybegan spotting cycle within 1 hour

4 29 53.5 kg. 22 of 28-30 dayno bleeding until cycle 27th day O~TH 27 In each case signlflcant cramplng wa~ felt and was characterlzed by each patlent as the same feeling a~
encountered at the onset of menstruation. There was a slight el~vation in temperature in each case, but thls subslded after 10 hours.

Uhless otherwise indlcated ln the ~oregolng examples, amblent atmospheric pre~sure was approximately 585 mm. o~ mercury.

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Claims (8)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. The method for extracting utero-evacuant materials from the zoapatle plant, said method being selected from the group consisting of a) treating a portion Or the zoapatle plant containing such utero-evacuant materials with water at an elevated temperature to result thereby in a mixture comprising an aqueous phase containing at least a portion of said utero-evacuant materials and a solid phase, b) separating said aqueous phase from said solid phase, and c) treating said aqueous phase with a water-immiscible organic solvent to separate from said phase at least a portion of said utero-evacuant materials;
or d) treating a portion of the zoapatle plant containing such utero-evacuant materials with a water-immiscible organic solvent to result thereby in a mixture comprising an organic phase containing at least a portion of said utero-evacuant materials and a solid phase, e) separating said organic phase from said solid phase, and f): treating said organic phase with water to separate from said phase at least a portion of said utero-evacuant materials.

2. The method of Claim 1 wherein the zoapatle plant is Montanoa tomentosa or Montanoa floribunda.

3. The method of Claim 2 wherein step (a) is conducted at between 25 - 100°C and step (c) at 30 - 80°C.

4. The method of Claim 2 wherein step (d) is conducted at between 30 - 80°C and step (e) at between 25 - 100°C.

5. The method of Claim 3 or 4 wherein the organic solvent comprises a lower aliphatic ester, an aliphatic hydrocarbon, an aromatic hydrocarbon or an aliphatic alcohol.

6. The method of Claim 5 wherein the organic solvent is ethyl acetate, hexane, chloroform or benzene.

7. A utero-evacuant extract material whenever prepared or produced by the process of claim 3 or by any chemical equivalent thereof.

8. A utero-evacuant extract material whenever prepared or produced by the process of claim 4 or by any chemical equivalent thereof.