Note: Descriptions are shown in the official language in which they were submitted.
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ANTINEOPLASTIC EXTRACT FROM ACHILLEA MILLEFOLIUM
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to isolated and purified
plant extracts, and more particularly to one from
Achillea millefolium to treat and prevent neoplastic
disorders.
(b) Description of Prior Art
Yarrow is an important member of the Asteraceae
branch of the Compositae, the daisy family. Common
names for yarrow include milfoil staunch weed,
nosebleed, soldier's herb, carpenter's wort, thousand
weed, woundwort, bloodwort boomadaran and knight's
milfoil. There are about 100 different species of
yarrow that grow mainly in temperate region of the
world. Yarrow, or Achillea millefolium, is said to have
been used by the Greek hero Achilles to stop the
bleeding of his warrior's wounds.
Yarrow (Achillea millefolium LINNAEUS) is used as a
medicinal plant in different parts of the world, as an
haemostatic, emmenagogue, antipyretic and diaphoretic
in cases of common cold.
An infusion is generally made from Achillea
millefolium, which is also used for lack of appetite,
cramps, flatulence and other stomach-related disorders.
Aboriginal people and pioneers also used yarrow as a
tea to treat digestive disorders and fevers and as a
poultice to treat cuts and burns, and chewed the leaves
to relieve toothache pain. Yarrow has long been
associated with the healing of wounds and the steeming
of blood flow. The existing literature indicates that
yarrow improves colon and liver function, is good
against anemia, liver disease, skin disease, eczema,
liver, psoriasis and rashes, as well as for treating
cold, flu, fever, hypertension, painful menstruation
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and bleeding. The value of yarrow as an anti-spasmodic
and diuretic agent, as well as an anti-inflammatory and
antiseptic compound, has been demonstrated.
The use of yarrow tea against cancer is known.
For example, in Iran, people have been using yarrow tea
for cancer for several hundreds years. Yarrow tea has
been used in different parts of the world for centuries
without manifesting toxicity or side effects, and some
cancer patients in the United States and Canada have
been taking yarrow as an alternative medicine. However,
no proven anticancer activity has been reported.
Antitumor sesquiterpenoids were recently
identified and isolated as methyl esters from Achillea
millefolium, namely achimillic acids A, B, and C,.
These compounds are active against mouse P-388 leukemia
cells in vivo.
Known constituents of yarrow are essential oils,
namely cineol, proazulene and achilleine.
Neoplastic disorders such as cancer are treated
with agents which are generally toxic with severe side
effects.
It would be highly desirable to be provided with
a substantially pure biologically active fraction
isolated from Achillea millefolium that would have an
antineoplastic activity, and that could be used to
treat or prevent diseases such as cancer.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide
purified biologically active fractions isolated from
Achillea millefolium that may be used to treat or
prevent disorders such as cancer.
In accordance with the present invention there
is provided a substantially pure biologically active
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extract isolated from Achillea millefolium, said
extract having an antineoplastic activity.
In accordance with one embodiment of the present
invention, the extract consists of a crude methanol
extract.
In accordance with another embodiment of the
present invention, there is provided the use of such an
extract for the preparation of a medicament for the
treatment and/or prevention of a neoplastic disorder,
such as cancer.
In accordance with another embodiment of the
present invention, there is provided an antineoplastic
composition to treat and/or prevent cancer, said
composition comprising a therapeutically effective
amount of a substantially pure extract isolated from
Achillea millefolium having antineoplastic activity,
and a suitable carrier.
In accordance with another embodiment of the
present invention, there is provided a method for
treating and/or preventing a cancer in a patient, said
method comprising administering to said patient a
therapeutically effective amount of a substantially
pure biologically active extract isolated from Achillea
millefolium with a pharmaceutically acceptable carrier.
The composition may be administered to a patient
susceptible of developing or suspected of having a
cancer, in an amount efficient to treat or prevent the
cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates the tracing obtained with the
analytical HPLCs of the extracts;
Fig. 2 illustrates the fractions obtained with a
large scale;
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Fig. 3 illustrates a dose-response relationship
for a methanol extract; and
Fig. 4 illustrates a dose-response relationship
for fractions of methanol extracts.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, there
are provided purified biologically active fractions
isolated from Achillea millefolium to treat diseases
such as cancer.
Fractions from Achillea millefolium LINNAEUS
have been isolated. The purified fractions were
administered to animals in which cancer was induced. No
toxicity was observed at the doses administered.
Moreover, the isolated organic soluble fractions have
antimetastatic activity in a mouse cancer model. The
isolated active fractions contain biologically active
molecules that may be used to treat diseases including
cancer.
More particularly, the crude methanol fraction
had a good antimetastatic activity in the Lewis lung
carcinoma model.
The animal model published by Tozyo et al. CChem
Pharm Bull, 1994, 42:1096-1100) consists of a mouse
leukemia P388 cell model. Tozyo et al. (supra)
injected both cells and drugs intraperitoneally. This
does not mimic physiological/pharmacological conditions
observed in human cancer. Indeed, the conditions in
Tozyo et al. resemble that of a petri dish where both
the target and the drug are in direct contact.
According to the present invention, the cells are
injected subcutaneously to the Lewis lung carcinoma
model. The cells then invade a distant site, such as
lung, and form metastases. The test article is given
by intraperitoneal route. Accordingly, the active
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components) need to be absorbed, perhaps metabolized,
before acting on primary tumors and/or metastases.
This is closer to human disease in term of the growth
versus multistep mechanisms of invasion.
As may be seen in Fig. 3, a dose-response
relationship was observed.
As may be seen if Fig. 4, the E1, E2 and E4
fractions were the most active in inhibiting lung
metastases.
Molecules) responsible for the biological
activity of the extracts may be identified and
characterized. These) molecules) may then be used to
treat or prevent cancer, leukemias, as well as other
diseases.
The fractions and molecules contained therein
are advantageous over the whole plant or teas made from
the plant.
The present invention will be more readily un-
derstood by referring to the following examples which
are given to illustrate the invention rather than to
limit its scope.
EXAMPLE I
Fractionation
Dried plant was grounded, and then stirred in
methanol at 25°C for 48h. The resulting extract was
filtered and treated with fresh methanol for another
48h. The combined extracts were filtered, evaporated
and analyzed by HPLC. Analytical HPLC (WatersT"" 600,
PhotodiodearrayT"' 996) was performed with two Whatman
PartisilT"" 10 ODS-2 analytical columns in series (4.6 x
250 mm). The gradient used consisted of 25-100%
acetonitrile in water, 50 min gradient at a flow rate
of 1 ml/min. Three fractions were identified according
to retention times, namely the fractions 0-10, 11-22
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and 23-60. The tracing of this analytical HPLC is shown
in Fig. 1.
A large scale was then used. Briefly, 2 grams
from methanol extract were dissolved in glass-distilled
methanol and filtered, and three separations were
performed with one PartisilT"" 10 ODS-2 MAG-20
preparative column (22 x 500 mm) with the following
gradient: 25-1000 acetonitrile in water, 50 min.
gradient at a flow rate of 18 ml/min. Four fractions
were collected for each injection according to the
following retention times: F1: 4.63-15.9; F2: 15.9-
24.4; F3: 24.4-40.2; and F4: 40.2-60. The fractions are
shown in Fig. 2.
The fractions were freshly solubilized in
ethanol (final concentration is less than 20% of
distilled water), and immediately used for in vivo
studies or stored at -80°C.
EXAMPLE II
Lewis lung carcinoma (LLC) cell line and cell culture
The Lewis lung carcinoma (LLC) clone, M47, with
a high metastatic potential to the lung, was
established and characterized (Brodt P, Cancer Res.,
46: 2442, 1986). These cells were confirmed free of
mycoplasma infection. Cells were maintained in RPMI-
1640 medium supplemented with 10% fetal bovine serum
and 1% penicillin-streptomycin, under 5o COz. Cells
were passaged twice a week. Stocks of cells were
generated and stored as early passages (passage no. 8-
10 received as passage no. 1, was considered the
initial stock). Cells were then propagated and stocks
of the same passages were established and stored in
liquid nitrogen for further experiments.
For tumor induction, cells were grown to 700
confluence in complete medium and then collected using
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trypsin-EDTA solution [0.050 trypsin, 0.53 mM EDTA-4Na
in HBSS without Ca'~, Mg'~, and NaHC03; Cellgro no. 25-
052-Li]. Cells were then centrifuged and washed three
times with phosphate buffer solution [D-PBS, Ca+' and
Mg" free; Cellgro no. 21-031-LV], and resuspended at a
dilution of 0.1-1 x 106 cells/0.1 ml. Viability was
examined by Trypan blue staining and only flasks in
which the viability was >95o were used for in vivo
studies.
The C57BL/10 mouse strain from the research
laboratories and incinerators was used, and access to
the animal facility is strictly limited to animal
users. The animal room has two doors, one serving as
the entrance and the other providing direct access to
washing, sterilization and incineration facilities,
which allows an accurate adjustment of environmental
parameters including temperature, humidity, ventilation
and lighting.
EXAMPLE III
Tumor cell inoculation and treatment
Five mice were housed per cage and fed a diet of
animal chow and water ad libitum. After one week of
acclimatization, LLC cells were transplanted
subcutaneously, as a suspension of tumor cells (2-5 x
105 viable cells/0.1 ml) in the axillary region of the
right flank. Animals were subjected daily to general
examination. Tumor growth was monitored every second or
third day using calipers. Tumor were measured along the
longest axis (length) and the perpendicular shortest
axis (width) and the relative tumor volume (in cm3) was
calculated by the formula: [Length (cm) x (width
cm)2]/2. When the tumor reached a size of 0.5-1.0 cm2
(in approximately 2-3 weeks), the mice were randomized
into three groups.
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In the first group, the mice were subjected to
surgery to remove the primary tumor. The mice were
lightly anesthetized with Forane. The skin overlying
the tumor was cleaned with betadine and ethanol in a
laminar flow hood. A small skin incision (0.5-1.0 cm)
was made using a sterile scalpel and the tumor was
carefully separated from the normal tissues (skin and
muscle). LLC (at an early stage of growth; 1-3 weeks)
is a well-localized tumor, and separation was easy to
achieve without any significant damage to normal
tissues. The tumor was removed, weighed and fixed for
histopathology purposes. The wound was closed with
surgical stainless steel clips (AutoclipsT"~; 9 mm; Clay
Adams, Inc., Parsippany, NJ). The site was further
disinfected with BetadineT"~ and the animal was housed as
described earlier.
In the second group, the mice were randomized
after surgery into groups of 5 per cage. The cages were
randomly assigned to specific experimental groups. The
mice were then labeled by numbers using the "ear
punching" method. Mice were checked daily to ensure the
absence of infection. Animals with disconfort were
sacrified immediately. An additional extra-group of
control mice was included to determine the optimal
timing for sacrifice in order to obtain a significant
number of well localized lung metastases. The second
group was subjected to the same experimental procedure
as the first group, with the exception of drug
treatment. Based on the second group, a period of two
weeks after removal of the primary tumor was sufficient
to obtain an average of 20-30 nodules on the lung
surface. Therefore, a two-week period after primary
tumor removal was used to sacrify treated mice.
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EXAMPLE IV
Dosing schedule and treatment
Drugs were given by intraperitoneal (ip) route
(0.5 ml per animal) in daily administration after tumor
cell inoculation. Control animals were given the same
volume of saline solution (0.9% sodium chloride; Abott
Laboratories, lot no. 12 455 WS). The dose of each drug
was normalized to an average of 20 g/body weight/per
animal. The schedules for drug treatment were based
upon conditions described in Figs. 3-4.
EXAMPLE V
Animal sacrifice, tumor/organs preparation
At the end of each experiment, for a total of 5-
8 weeks, animals were sacrified in a CO2 chamber and
autopsied. Tumors, organs or both were removed under
sterile conditions using a laminar flow hood. Tumors
were weighed. Organs (5/group) were examined for gross
pathological changes and then fixed in loo formalin.
Lungs were f fixed in 10 o Bouin' s f fixative diluted in a
formalin solution, and lung surface metastases were
counted using a stereomicroscope at 4x magnification or
a magnifying-glass.
EXAMPLE VI
Statistical analysis
The umpaired Student t-test was used to compare
statistical significance among various groups.
While the invention has been described in con-
nection with specific embodiments thereof, it will be
understood that it is capable of further modifications
and this application is intended to cover any varia-
tions, uses, or adaptations of the invention following,
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in general, the principles of the invention and
including such departures from the present disclosure
as come within known or customary practice within the
art to which the invention pertains and as may be
applied to the essential features hereinbefore set
forth, and as follows in the scope of the appended
claims.
