Note: Descriptions are shown in the official language in which they were submitted.
1063941
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
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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.
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1063941
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.
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1063941
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.
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1063941
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
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1063941
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
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1063941
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
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1063941
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
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1063941
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
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~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
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1063941
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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