Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
THERAPEUTIC AGENT FOR POLYCYSTIC OVARY SYNDROME (PCOS)
TECHNICAL FIELD
[0001]
The present invention relates to an effective and safe therapeutic agent for
treating polycystic ovary syndrome (PCOS), which is a cause of female
sterility,
menstrual disorder, acne, excess hair growth, obesity, or the like.
BACKGROUND ART
[0002]
Recent studies have reported that 6% of women of reproductive age suffer from
polycystic ovary syndrome (PCOS). Moreover, it is predicted that the actual
number of
patients with potential polycystic ovary syndrome (PCOS) is much higher than
this.
Polycystic ovary syndrome (PCOS) means the state in which a follicle is not
sufficiently
matured for ovulation to occur and an ovary is filled with numerous small
immature ova.
Thus, ovulation disorder is caused.
[0003]
The reason for this is believed to be because the balance between FSH
(follicle-stimulating hormone) and LH (luteinizing hormone) in gonadotropic
hormones
(gonadotropins), which are secreted from the pituitary gland and required to
induce
ovulation, is not properly maintained. However, there are still many factors
that have
not yet been clarified.
[0004]
Polycystic ovary syndrome (PCOS) results in irregular menstruation, loss of
menstrual periods, menorrhagia, an increase of male hormones (testosterone),
obesity,
sterility, and other problems. As conventional methods for treating polycystic
ovary
syndrome (PCOS), the following medications and surgical procedures are known.
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[0005]
(I) Treatment using an ovulation-inducing agent (clomiphene, Clomid)
Although this is a treatment in which clomiphene acts on the hypothalamic area
to induce ovulation, there are cases in which ovulation does not always occur.
[0006]
(II) Treatment using clomiphene concomitantly with another agent
This is a treatment in which clomiphene is used concomitantly with other
agents such as a steroid hormone and/or HMG (human menopausal gonadotropins),
when
no effects are exhibited by administering clomiphene alone.
[0007]
(III) Treatment using gonadotropin (method in which HMG or FSH is
administered)
This is a treatment method in which a gonadotropic hormone (gonadotropin) is
injected into a patient so as to induce ovulation by stimulating the ovary.
Although this
treatment exhibits relatively high efficacy and a good pregnancy rate, there
are some side
effects such as multiple pregnancy and ovarian hyperstimulation syndrome
(OHSS).
[0008]
(IV) Administration of Shakuyaku-kanzo-to
This is a treatment in which Shakuyaku-kanzo-to, a Chinese herbal medicine, is
administered with few, if any, side effects, but its effect is not
satisfactory.
[0009]
(V) Laparoscopic treatment
This is a treatment in which a laparoscope is used to form a small hole by
radiating laser beams on the surface of the ovary. The problem is that this
treatment is
not a definitive treatment and is invasive to the living body.
[0010]
(VI) Administration of oral contraceptive agents
A treatment using hormonal agents such as oral contraceptive agents or the
like
to cause periodic menstruation is conducted for unmarried or young females
suffering
from polycystic ovary syndrome (PCOS) and who have no desire to bear children
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(pregnancy).
DISCLOSURE OF THE INVENTION
[Problems to be Solved by the Invention]
[0011]
For sterile females suffering from polycystic ovary syndrome (PCOS) and who
desire to bear children, the treatments aiming to induce ovulation as
described above are
mainly employed. Among them, the treatments using the ovulation-inducing
agents as
described in any of (I) to (III) are considered effective, but they still have
a possibility of
causing serious side effects such as multiple pregnancy or ovarian
hyperstimulation
syndrome (OHSS), and thus cause difficult problems.
[0012]
Moreover, there is another problem in the treatment for unmarried females or
young females. Many of these patients are reluctant to consult obstetricians
or
gynecologists and are often left without receiving proper medical attention.
Such
patients will be in danger where symptoms such as hypertrophy of the ovary
membrane
or the like will further progress and, thus, the treatment thereof becomes
even more
difficult.
[0013]
Also, even when unmarried or young female patients consult obstetricians or
gynecologists, both the doctors and the patients tend to avoid, as much as
possible,
treatment using strong agents such as an ovulation-inducing agent or
gonadotropin, as
described in (I) to (III) above. Also, the administration of the oral
contraceptive agents
described in (IV) is not favored by the patients. Accordingly, the patients
are often
diagnosed as having "irregular menstruation" or the like to receive only
temporary relief
against the symptoms.
[0014]
Thus, conventional methods for treating polycystic ovary syndrome (PCOS)
have the problems as described above and cannot satisfy the efficacy and the
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preventability of side effects. Moreover, recent studies have reported that
the patients
affected with polycystic ovary syndrome (PCOS) have a seven-fold risk of
developing
diabetes complications compared with unaffected individuals. As is well known,
diabetes is a serious disease which could result in myocardial infarction,
cerebral stroke,
renal insufficiency, loss of eyesight caused by retinopathy, or the like.
Accordingly, it is
necessary that patients with polycystic ovary syndrome (PCOS) be treated at a
stage
before diabetes complications develop.
[0015]
The present invention has been achieved in consideration of the
above-mentioned situation, and has as an object thereof to provide a
therapeutic agent for
polycystic ovary syndrome (PCOS) which exhibits few, if any, side effects, is
effective at
inducing ovulation, and is safely available to not only females who desire to
bear
children, but also for unmarried or young females.
[Means for Solving the Problems]
[0016]
As a result of extensive investigation on a safe and effective therapeutic
agent
for treating polycystic ovary syndrome (PCOS) to solve the above-mentioned
problems,
the inventors of the present invention have found that an extract of mushrooms
exhibits
activities effective against polycystic ovary syndrome (PCOS), and thereby
completing
the present invention_
[0017]
That is, the present invention relates to a therapeutic agent for treating
polycystic ovary syndrome (PCOS) containing an extract of mushrooms as an
active
ingredient thereof.
[0018]
It is preferable that the mushrooms be at least one selected from the group
consisting of Grifola frondosa, Polyporus umbellatus, Meripilus giganteus,
Grifola
albicans, Lentinus edodes, Agaricus blazei Murill, Agaricus bispirus,
Ganoderma
applanatum, Fomitopsis pinicola, Coriolus versicolor, Ganoderma lucidum,
Pleurotus
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ostreatus, Pleurotus eryngii, Hericium erinaceus, Cordyceps sinensis,
Cordyceps
sobolifera, Auricularia auricula, Tremella fuciformis, and Phellinus linteus.
[0019]
Moreover, it is more preferable that the mushrooms be at least one selected
from the group consisting of Grifola frondosa, Lentinus edodes, Agaricus
blazei Murill,
Ganoderma lucidum, and Pleurotus ostreatus, among which Grifola frondosa is
most
preferable.
[0020]
Also, it is preferable that the above-mentioned extract of mushrooms be
prepared by a process including: (1) a step of treating a raw material of
mushrooms (the
fruiting body and/or mycelium are collectively called "raw material of the
mushrooms")
with ethanol having a concentration of 90% or more, and thereby removing
ethanol-soluble components, (2) a step of extracting the residue with hot-
water thereafter,
adding the ethanol to the obtained hot-water-extracted liquid until a final
ethanol
concentration becomes 50 to 75% by value, removing produced insoluble
components,
and thereby obtaining a supernatant, and (3) a step of collecting a fraction
having a
molecular weight of 14,000 Dalton or more from the obtained supernatant.
[Effects of the Invention]
[0021]
The therapeutic agent for polycystic ovary syndrome (PCOS) obtained under
the present invention contains the extract of mushrooms as the active
ingredient thereof,
which is safe, exhibits few, if any, side effects, demonstrates excellent
effects of inducing
ovulation, and is safely available for not only females who desire to bear
children, but
also for unmarried or young females.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022]
FIG. 1 is a chart drawing indicating a result of an analysis using
high-performance liquid chromatography (HPLC) on an extract of Grifola
frondosa
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obtained in Preparation Example 1. (Analysis Example)
FIG. 2 is a chart drawing indicating a result of an analysis using
high-performance liquid chromatography (HPLC) on an extract of Lentinus edodes
obtained in Preparation Example 3. (Preparation Example 3)
FIG. 3 is a chart drawing indicating a result of an analysis using
high-performance liquid chromatography (HPLC) on an extract of Agaricus blazei
Murill
obtained in Preparation Example 4. (Preparation Example 4)
FIG. 4 is a chart drawing indicating a result of an analysis using
high-performance liquid chromatography (HPLC) on an extract of Ganoderma
lucidum
obtained in Preparation Example 5. (Preparation Example 5)
FIG. 5 is a chart drawing indicating a result of an analysis using
high-performance liquid chromatography (HPLC) on an extract of Pleurotus
ostreatus
obtained in Preparation Example 6. (Preparation Example 6)
BEST MODE FOR CARRYING OUT THE INVENTION
[0023]
The therapeutic agent for polycystic ovary syndrome (PCOS) obtained under
the present invention contains an extract of mushrooms as the active
ingredient thereof.
This extract of mushrooms can be used in a therapeutic treatment for
polycystic ovary
syndrome (PCOS). Also, this extract of mushrooms can be used for preparing
therapeutic agents for treating polycystic ovary syndrome (PCOS).
[0024]
It is preferable that the mushrooms be at least one selected from the group
consisting of Grifola frondosa, Polyporus umbellatus, Meripilus giganteus,
Grifola
albicans, Lentinus edodes, Agaricus blazei Murill, Agaricus bispirus,
Ganoderma
applanatum, Fomitopsis pinicola, Coriolus versicolor, Ganoderma lucidum,
Pleurotus
ostreatus, Pleurotus eryngii, Hericium erinaceus, Cordyceps sinensis,
Cordyceps
sobolifera, Auricularia auricula, Tremella fuciformis, and Phellinus linteus,
in view of
being used as food or medicine and having been highly prized and deemed to
provide
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health benefits since ancient times.
[0025]
Among them, it is more preferable that the mushrooms be at least one selected
from the group consisting of Grifola frondosa, Lentinus edodes, Agaricus
blazei Murill,
Ganoderma lucidum, and Pleurotus ostreatus. Grifola frondosa is most
preferable.
[0026]
These mushrooms contain a glycoprotein, which is a saccharide-bound protein
(conjugated protein), and the extract of the mushrooms according to the
present invention
is deemed to contain this glycoprotein as a main component thereof.
[0027]
The glycoprotein, which is the main component of the extract of mushrooms
under the present invention, is brown and exhibits water-solubility and
thermal stability,
and it is preferable that the mass ratio of a protein portion to a saccharide
portion be in a
range from 75:25 to 90:10, and the distribution of molecular weight be in a
range from
18,000 to 22,000 Dalton, and be positive in a biuret reaction and Fehling's
reaction. The
term "protein portion" used herein means a portion composed of only a
polypeptide chain
in the glycoprotein that is a conjugated protein.
[0028]
It is preferable that the distribution of the molecular weight of glycoprotein
be
in a range from 18,000 to 22,000 Dalton, and it is more preferable that the
average
molecular weight be 20,000. Thermal stability means that bioactivity is not
lost when
left to stand at a temperature from 80 to 130 C for 1 to 5 hours.
[0029]
Also, it is preferable that the extract of mushrooms under the present
invention
be prepared by a process including: (1) a step of treating a raw material of
the mushrooms
(the fruiting body and/or mycelium) with ethanol having a concentration of 90%
or more,
and thereby removing ethanol-soluble components, (2) a step of extracting the
residue
with hot-water thereafter, adding the ethanol to the obtained hot-water-
extracted liquid
until a final ethanol concentration becomes 50 to 75% by value, removing
produced
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insoluble components, and thereby obtaining a supernatant, and (3) a step of
collecting a
fraction having a molecular weight of 14,000 Dalton or more from the obtained
supernatant.
[0030]
The method for producing the extract of mushrooms under the present
invention includes the above-mentioned steps (1) to (3). First of all, the raw
material of
mushrooms used in the present invention is treated with ethanol having a
concentration of
90% or more to remove ethanol-soluble components, as the first step (1).
[0031]
As the raw material of mushrooms used in the present invention, fresh or dried
mushrooms described above can be used without any particular limitations.
Among
them, dried powder is preferably used to conduct effective treatment. The
ethanol
having a concentration of 90% or more may be water containing 90% or more of
ethanol
(an aqueous ethanol). Among them, water containing 95% or more of ethanol is
preferable, and 100% ethanol is most preferable, in view of effectively
removing the
ethanol-soluble components eluted from the raw material of mushrooms.
[0032]
In the treatment of step (1), an ethanol or aqueous ethanol is added to the
raw
material of mushrooms and then stirred. At this time, it is preferable that
the content of
the raw material of mushrooms be 10 to 25% by mass, and more preferably 10 to
12.5%
by mass. Also, although the temperature for the extraction is generally set
from 20 to
70 C, it may be room temperature. Although the treatment time is dependent on
the
state of the raw material of mushrooms used, the treatment temperature, or the
like, it is
preferable to be 1 to 10 hours, and more preferably 2 to 3 hours.
[0033]
By conducting this treatment, components soluble in ethanol are dissolved into
the ethanol or aqueous ethanol from the raw material of mushrooms. Since the
ethanol-soluble components are not required, the components are removed by
filtration or
centrifugation. Among them, centrifugation is preferable in the case that a
large amount
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of the extract is required for experiment or commercialization.
[0034]
Next, in step (2), a residue remaining after the ethanol-extraction in which
the
ethanol-soluble components are removed is subject to a hot-water extraction,
the ethanol
is added to the obtained hot-water-extracted liquid until the final ethanol
concentration
becomes 50 to 75% by value, and produced insoluble components are then removed
to
obtain a supernatant.
[0035]
In the hot-water extraction, water is added to the residue remaining after the
ethanol-extraction in step (1) and then heated. At this time, it is preferable
that the
content of the residue remaining after the ethanol-extraction be 10 to 25% by
mass, and
more preferably 10 to 12.5% by mass. Also, it is preferable that the
temperature for
extraction be 80 to 130 C, and more preferably 100 to 120 C. Also, it is
preferable that
the extraction time be 1 to 5 hour(s), and more preferably 2 to 3 hours.
[0036]
Next, the residue of extraction is filtered off, and the ethanol is added to
the
hot-water-extracted liquid, which is a filtrate obtained by the filtration,
until the final
ethanol concentration becomes 50 to 75% by value. It is preferable that the
hot-water-extracted liquid be concentrated to 1/3 to 1/2 of the original
volume before
adding the ethanol.
[0037]
After adding the ethanol, it is preferable that the production of insoluble
components such as precipitates or floating matter be promoted by allowing it
to stand at
a low temperature, preferably 0 C to room temperature, and more preferably 4
to 10 C,
for 5 to 24 hours, and more preferably 8 to 12 hours. After that, the produced
insoluble
components are removed by centrifugation or filtration to obtain a supernatant
such as a
filtrate or the like. By removing the insoluble components produced by adding
the
ethanol, substances having an immunosuppressive effect or the like can be
substantially
removed from the final product.
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[0038]
Next, in step (3), a fraction having a molecular weight of 14,000 Dalton or
more is collected from the obtained supernatant. It is preferable that the
collection of
the fraction having a molecular weight of 14,000 Dalton or more be carried out
by
conducting dialysis or ultrafiltration. There are no particular limitations
imposed on a
dialysis filter used for this dialysis or ultrafiltration, provided that the
filter has an ability
to collect any fraction with a molecular weight of more than 14,000 Dalton,
and
generally-used filters such as a cellophane filter or collodion filter can be
used.
[0039]
After that, the obtained fraction having a molecular weight of 14,000 or more
may be further purified for the purpose of increasing the purity thereof. As
such a
purifying method, a method usually used for purifying glycoprotein, such as,
for
example, gel filtration chromatography can be used without any particular
limitations.
[0040]
The solvents available for above-mentioned extraction are those approved by
the Japanese Health, Labour and Welfare Ministry to be used for preparing
health food
products or the like, and therefore this extract may also be used as a
material for health
food products.
[0041]
The therapeutic agent for polycystic ovary syndrome (PCOS) under the present
invention may further contain a carrier and/or a diluent which are(is)
pharmaceutically
acceptable in addition to the extract of mushrooms as the active ingredient
thereof. As
the carrier, cellulose, calcium monohydrogen phosphate, sucrose fatty acid
ester, silicon
dioxide, methylcellulose, lactose, or the like can be used. As the diluent,
water,
glycerol, or the like can be used. Also, general additives such as
preservatives,
stabilizers, excipients, binders, disintegrators, sweeteners, or the like may
be further
added.
[0042]
Moreover, the therapeutic agent for polycystic ovary syndrome (PCOS) under
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the present invention may be administered orally, parenterally, or
transdermally. It is
generally desirable that the amount of the active ingredient to be
administered be
properly decided depending on the body weight of the patient, the nature and
the state of
the disease, the administration route, or the like. In the case of oral
administration, the
dose per day is preferably 50 to 800 mg/person, more preferably 100 to 500
mg/person,
and most preferably 200 to 350 mg/person, and this is administered once or in
several
divided doses a day.
[0043]
The extract of mushrooms under the present invention may be used alone or
concomitantly with other pharmacologically active substances. Examples of
forms
suitable for administration include tablets (plain tablets), coated tablets,
capsules,
suppositories, solutions, syrups, emulsions, dispersive powders, and the like.
It is
preferable that the tablets or the like contain the extract of mushrooms under
the present
invention in an amount of 3 to 80% by mass, and more preferably 5 to 50% by
mass.
[0044]
The tablets can be prepared by mixing the extract of mushrooms under the
present invention with at least one active ingredients and known excipients
including
inactive components such as calcium carbonate, calcium phosphate, lactose, or
the like;
disintegrators such as corn starch, alginic acid, or the like; binders such as
starch, gelatin,
or the like; lubricants such as magnesium stearate, talc, or the like; and/or
agents enabling
a delayed release such as carboxymethylcellulose, cellulose acetate phthalate,
polyvinyl
acetate, or the like, or alternatively the tablets can be prepared by mixing
the extract with
another pharmacologically active ingredient. The tablets may be composed of
several
layers.
[0045]
The coated tablet can be prepared by coating a core prepared in the same
manner as that of the tablets with a substance conventionally used for coating
tablets,
such as, for example, gum arabic, talc, titanium dioxide, methylcellulose,
sucrose, or the
like. In order to enable delayed release or avoid incompatibility, the core
may be
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composed of several layers and may contain the above-mentioned excipients for
the
tablets.
[0046]
Also, syrups containing the extract of mushrooms under the present invention
or a mixture of this and other pharmacologically active substances may contain
sweeteners such as saccharin, cyclamate, glycerol, sucrose, or the like, and
flavor-improvers, such as, for example, flavoring agents such as vanillin,
orange extract,
or the like. Suspension auxiliary agents or thickeners such as sodium
carboxymethylcellulose or the like; humectants such as a condensate of
aliphatic alcohol
and ethylene oxide, or the like; or preservatives such as p-hydroxybenzoate or
the like
may also be contained.
[0047]
Also, the capsules containing the extract of mushrooms under the present
invention or a mixture of this and other pharmacologically active ingredients
may be
prepared by, for example, mixing these ingredients with inactive carriers such
as lactose,
sorbitol, or the like, and encapsulating the mixture into a gelatin capsule or
the like.
[0048]
Also, suppositories can be prepared by mixing the extract of mushrooms under
the present invention or a mixture of the extract and other pharmacologically
active
ingredients with conventionally-used carriers, specifically a neutral lipid,
polyethyleneglycol, a derivative thereof, or the like.
[0049]
Also, the extract of mushrooms under the present invention or a mixture of
this
and other pharmacologically active ingredients may be applied to food such as
health
food, functional food, or general food. Since the glycoprotein is contained in
the extract
of mushrooms under the present invention, it is very safe and so the above-
mentioned
food can be taken over an extended period of time. These foods may contain
vitamins,
minerals, herbs, or other nutritional materials, in addition to the above-
mentioned
supplemented components.
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[0050]
The following examples do not limit the scope of the present invention and are
provided merely for illustrative purposes.
[Examples]
[0051]
[Preparation Example 1] Preparation of the extract of mushrooms (Grifola
frondosa)
L of 95% aqueous ethanol was added to 1000 g of dried Grifola frondosa
fruiting body powder, stirred for 2 to 3 hours at room temperature, and then
filtered to
remove the ethanol-soluble components.
[0052]
Then, 5 L of deionized water was added to the obtained residue, and then
extracted for 2 hours while stirring at 100 to 120 C. This hot-water-extracted
liquid was
concentrated to half of the original value, followed by adding ethanol until
the final
ethanol concentration became 50 to 75% by value. This was allowed to stand in
a
low-temperature chamber at 4 to 10 C for 8 to 12 hours, and solid substances
such as
precipitates or floating matter, which were insoluble components, were removed
by
centrifugation to obtain a supernatant.
[0053]
From this supernatant, a fraction having a molecular weight of 14,000 Dalton
or more was collected by dialysis, and thus an extract of Grifola frondosa was
obtained.
[0054]
In order to analyze this extract of Grifola frondosa, it was purified by gel
filtration chromatography, and thus approximately 21 g of a brown substance
was
obtained. This purified extract of Grifola frondosa was tested positive for
the biuret
reaction and Fehling's reaction, and thus was confirmed to contain a protein
portion and
saccharide portion.
[0055]
[Analysis Example] Analysis of purified extract of Grifola frondosa
With respect to the purified extract of Grifola frondosa obtained as described
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above, quantitative analysis of the simple protein portion thereof was carried
out by using
the Bradford method. The analysis on the constituent amino acids of the simple
protein
portion was carried out using an automatic amino-acid analyzer (Hitachi L-
8500A Amino
Acid Analyzer). Also, the quantitative analysis of the saccharide portion was
carried
out by using the phenol-sulfuric acid method, and the analysis with respect to
the
constituent of the saccharide portion was carried out by using high-
performance liquid
chromatography (HPLC). The measurement of the molecular weight was carried out
by
using SDS-PAGE electrophoresis. Also, a 1H-NMR measurement was carried out.
[0056]
Results of the analysis with respect to mass ratios of the simple protein
portion
and the saccharide portion in the purified extract of Grifolafrondosa are
shown in Table
1. The substance was confirmed to be a glycoprotein.
[0057]
Table 1
Sample Protein Saccharide
1 83.8% 16.2%
2 75.8% 24.2%
3 86.7% 13.3%
4 79.8% 20.2%
[0058]
A chart drawing of results of the analysis by high-performance liquid
chromatography (HPLC) on the extract of Grifola frondosa obtained in
Preparation
Example 1 is shown in FIG. 1. In FIG. 1, the peak at 7.189 was identified to
be a
glycoprotein.
[0059]
The glycoprotein, which was the main component of the obtained extract of
Grifolafrondosa, had the following characteristics.
Appearance: Brown and hygroscopic powder
Solubility: Soluble in water and alkaline solution
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Stability: Stable at high temperature and in ethanol
Chemical constituent: Simple protein portion: Saccharide portion = 75:25 to
90:10 (see Table 1)
The constituent amino acids of the protein portion: asparagine, glutamine,
serine, threonine, glycine, alanine, valine, cysteine, methionine, isoleucine,
leucine,
tyrosine, phenylalanine, lysine, histidine, arginine, proline
Components of the saccharide portion: galactose, mannose, glucose,
N-acetyl-glucosamine, fucose
11-1-NMR spectrum: 3.0473, 3.8983, 6.8500, 7.3150
Average molecular weight: 20,000 Dalton
[0060]
[Preparation Example 2] Preparation method of a tablet
A tablet having the following components was prepared using the extract of
Grifolafrondosa which was obtained in Preparation Example 1.
Tablet Per tablet
Active ingredients:
Extract of Grifola frondosa (powdered) 36 mg
Dried Grifola frondosa powder 250 mg
Excipients:
Fine crystalline cellulose 264 mg
Calcium monohydrogen phosphate 20 mg
Sucrose fatty acid ester 20 mg
Fine silica dioxide 10 mg
Coating material:
Methylcellulose 1 to 1.5% by mass of the tablet
[0061]
The dried Grifola frondosa powder means a powder obtained by merely drying
and pulverizing Grifolafrondosa without extracting with ethanol. The
above-mentioned extract of Grifola frondosa (powdered) and the dried Grifola
frondosa
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powder were mixed with the finely pulverized excipients and granulated, dried,
pulverized, and tableted to make tablets having a proper form and size,
followed by
coating the tablets with the coating material.
[0062]
Results of physical analysis and microbial test on the obtained tablet are
shown
in the following. This tablet was confirmed to satisfy the standards defined
in the
Pharmaceutical Affairs Law.
Item of analysis:
Disintegration time Not more than 45 minutes
Weight variation 600 mg 5%
Microbial test:
Total plate count Not more than 3000
Yeast and mold Not more than 300
Salmonella Absent
Escherichia coli Absent
[0063]
[Preparation Examples 3 to 6] Preparation and analysis of extracts from
Lentinus
edodes, Agaricus blazei Murill, Ganoderma lucidum, and Pleurotus ostreatus
Lentinus edodes (Preparation Example 3), Agaricus blazei Murill (Preparation
Example 4), Ganoderma lucidum (Preparation Example 5), and Pleurotus ostreatus
(Preparation Example 6) were used instead of Grifola frondosa, for extraction
in the same
manner as that in Preparation Example 1. The measurements by HPLC were carried
out
in the same manner as in the above-mentioned analysis example. FIG. 2 shows a
chart
drawing of a result of the analysis by HPLC on the extract of Lentinus edodes
extract,
FIG. 3 shows that on Agaricus blazei Murill, FIG. 4 that on Ganoderma lucidum,
and
FIG. 5 shows that on Pleurotus ostreatus. In FIGS. 2 to 5, each peak was
recognized at
approximately the same position of 7.189, which is the peak position of the
extract of
Grifola frondosa in Preparation Example 1, that is, 7.087 (FIG. 2), 7.082
(FIG. 3), 7.096
(FIG. 4), or 7.086 (FIG. 5), and thus a component similar to the extract of
Grifola
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fi-ondosa in Preparation Example 1 was respectively confirmed to be contained.
[0064]
[Test Example 1] Administration to patients suffering from polycystic ovary
syndrome
(PCOS)
Among outpatients diagnosed with polycystic ovary syndrome (PCOS), 18
patients with anovulation over 3 months or more were randomly divided in two
groups.
The tablet prepared in Preparation Example 2 described above (tablet made
under the
present invention) was administered to the test group at a dose of 9 tablets
per day in
three equally divided doses. To the control group, Shakuyaku-kanzo-to was
administered at a dose of 7.5 g per day in three equally divided doses. Each
administration was continued over 12 weeks (three cycles) while the patients
of each
group monitored their basal body temperature to check for the existence of
ovulation.
[0065]
Results of the ovulation rates based on the number of patients showing
ovulation are compared as shown in Table 2. Also, results of the number of
cycles of
ovulation are shown in Table 3.
CA 02590549 2007-06-05
18
[0066]
Table 2
Number of Number of patients
Rate of patients
Treatment
patients showing ovulation
showing ovulation
Tablet according to the
8 6 75%
present invention
Shakuyakukanzoto 10 1 10%
[0067]
Table 3
Number of Number of cycles of
Rate of cycles
Treatment
cycles ovulation of ovulation
Tablet according to the
24 cycles 11 cycles 46%
present invention
Shakuyakukanzoto 30 cycles 3 cycles 10%
[0068]
As shown in Table 2, the comparison of the ovulation rate based on the number
of patients who were administered the tablet prepared in Preparation Example 2
described
above (tablet made under the present invention) with that of those who were
administered
Shakuyaku-kanzo-to revealed that the tablet made under the present invention:
Shakuyaku-kanzo-to = 75% : 10%. Also, as shown in Table 3, the comparison
based on
the number of cycles with ovulation revealed that the tablet made under the
present
invention: Shakuyaku-kanzo-to = 46%: 10%. According to Fisher's exact
probability
test, the p value was less than 0.05, and thus a significant difference was
recognized and
it was confirmed that the therapeutic agent for polycystic ovary syndrome
(PCOS) under
the present invention was superior to Shakuyaku-kanzo-to. Also, side effects
were not
recognized in either group, and thus it was confirmed that the therapeutic
agent for
polycystic ovary syndrome (PCOS) under the present invention is very safe.
[0069]
[Test Example 2] Safety Test of the extract of mushroom
CA 02590549 2007-06-05
19
A safety test was carried out using 10 male and 10 female healthy 4-week-old
ICR mice. These mice were weighed 4 hours after starvation, and a solution in
which
the extract of Grifola frondosa obtained in Preparation Example 1 was
dissolved in
distilled water was forcibly administered to respective male and female mice
at a single
dose of 2,000 mg/kg of body weight using a gastric sonde. To control groups,
0.7 mL
of purified water was administered to male mice and 0.6 mL of purified water
was
administered to female mice in the same manner. The observation period was 14
days
and autopsy was carried out on all mice after the end of the observation
period.
[0070]
The male and female mice did not die during the observation period and no
abnormality was found with respect to their general conditions and body
weight. Under
the autopsy, no abnormality was found in the major organs of any of the tested
mice.
Accordingly, each value of LD50 of the extract of mushrooms according to the
present
invention to be orally administered in a single dose to each male and female
mouse was
considered to be 2,000 mg/kg of body weight or more, and thus it was confirmed
that the
extract of mushrooms according to the present invention was very safe.
INDUSTRIAL APPLICABILITY
[0071]
Since the therapeutic agent for polycystic ovary syndrome (PCOS) under the
present invention contains the extract of mushrooms as the active ingredient
thereof, the
therapeutic agent is safe, exhibits few, if any, side effects, demonstrates
excellent effects
to induce ovulation, and is safely available for not only females who desire
to bear
children, but also for unmarried or young females.
