U.S. patent application number 13/637966 was filed with the patent office on 2013-01-31 for composition comprising coumestrol or a bean extract containing coumestrol.
This patent application is currently assigned to AMOREPACIFIC CORPORATION. The applicant listed for this patent is Hee Young Jeon, Sang Jun Lee, Dae Bang Seo, Hyun Jung Shin, Hyeon Ju Yeo. Invention is credited to Hee Young Jeon, Sang Jun Lee, Dae Bang Seo, Hyun Jung Shin, Hyeon Ju Yeo.
Application Number | 20130028921 13/637966 |
Document ID | / |
Family ID | 44712773 |
Filed Date | 2013-01-31 |
United States Patent
Application |
20130028921 |
Kind Code |
A1 |
Jeon; Hee Young ; et
al. |
January 31, 2013 |
COMPOSITION COMPRISING COUMESTROL OR A BEAN EXTRACT CONTAINING
COUMESTROL
Abstract
The present invention relates to a composition which comprises
as an active ingredient coumestrol or a bean extract containing
coumestrol, whereby adipocyte differentiation is inhibited, the
immune system of the body is improved, toxic substances are purged,
and neurodegenerative disorders are prevented or improved.
Inventors: |
Jeon; Hee Young;
(Gyeonggi-do, KR) ; Shin; Hyun Jung; (Seoul,
KR) ; Seo; Dae Bang; (Gyeonggi-do, KR) ; Yeo;
Hyeon Ju; (Seoul, KR) ; Lee; Sang Jun;
(Gyeonggi-do, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Jeon; Hee Young
Shin; Hyun Jung
Seo; Dae Bang
Yeo; Hyeon Ju
Lee; Sang Jun |
Gyeonggi-do
Seoul
Gyeonggi-do
Seoul
Gyeonggi-do |
|
KR
KR
KR
KR
KR |
|
|
Assignee: |
AMOREPACIFIC CORPORATION
Seoul
KR
|
Family ID: |
44712773 |
Appl. No.: |
13/637966 |
Filed: |
March 31, 2011 |
PCT Filed: |
March 31, 2011 |
PCT NO: |
PCT/KR2011/002233 |
371 Date: |
September 27, 2012 |
Current U.S.
Class: |
424/184.1 ;
514/453 |
Current CPC
Class: |
A61P 39/02 20180101;
A61P 25/34 20180101; A61P 37/04 20180101; A61P 9/10 20180101; A61P
3/04 20180101; A61P 21/04 20180101; A61K 36/48 20130101; A61P 1/16
20180101; A61P 3/10 20180101; A61P 9/12 20180101; A61P 25/00
20180101; A61P 9/00 20180101; A61P 25/16 20180101; A61K 31/35
20130101; A61P 3/00 20180101; A61P 37/02 20180101; A61P 27/06
20180101; A61K 31/366 20130101; A61P 3/06 20180101; A61P 25/28
20180101 |
Class at
Publication: |
424/184.1 ;
514/453 |
International
Class: |
A61K 31/366 20060101
A61K031/366; A61P 3/04 20060101 A61P003/04; A61P 3/10 20060101
A61P003/10; A61P 1/16 20060101 A61P001/16; A61P 9/00 20060101
A61P009/00; A61P 25/16 20060101 A61P025/16; A61P 9/12 20060101
A61P009/12; A61P 9/10 20060101 A61P009/10; A61P 37/02 20060101
A61P037/02; A61P 25/00 20060101 A61P025/00; A61P 25/28 20060101
A61P025/28; A61K 39/00 20060101 A61K039/00; A61P 3/06 20060101
A61P003/06 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2010 |
KR |
10-2010-0029220 |
Claims
1. A method for inhibiting differentiation of adipocytes comprising
administering an effective amount coumestrol or a bean extract
comprising coumestrol to a subject in such need to activate
mitochondria.
2. The method according to claim 1, wherein the coumestrol or bean
extract comprising coumestrol prevents or improves one or more of
obesity, type 2 diabetes and fatty liver.
3. The method according to claim 1, wherein the coumestrol or bean
extract comprising coumestrol prevents or improves cardiovascular
diseases induced by obesity.
4. The method according to claim 3, wherein the cardiovascular
disease comprises one or more of hyperlipidemia, hypertension,
angina, myocardial infarction and arteriosclerosis.
5. A method for enhancing immunity comprising administering an
effective amount of coumestrol or a bean extract comprising
coumestrol to a subject in such need.
6. The method according to claim 5, wherein the coumestrol or bean
extract comprising coumestrol activates immune cells.
7. A method for detoxifying toxic substances comprising
administering an effective amount of coumestrol or a bean extract
comprising coumestrol to a subject in such need to detoxify toxic
substances.
8. The method according to claim 7, wherein the coumestrol or bean
extract comprising coumestrol detoxifies nicotine.
9. The method according to claim 7, wherein the coumestrol or bean
extract comprising coumestrol prevents or improves vascular
diseases caused by toxic substances.
10. The method according to claim 9, wherein the vascular disease
comprises one or more of blood circulation disorder, hypertension
and arteriosclerosis.
11. A method for preventing or improving degenerative neurological
disorders comprising administering an effective amount of
coumestrol or a bean extract comprising coumestrol to a subject in
such need to prevent or improve degenerative neurological
disorders.
12. The method according to claim 11, wherein the coumestrol or
bean extract comprising coumestrol inhibits the action of
neurotoxins.
13. The method according to claim 11, wherein the degenerative
neurological disorder comprises one or more of dementia,
Alzheimer's disease and Parkinson's disease.
14-15. (canceled)
Description
TECHNICAL FIELD
[0001] The present disclosure relates to a composition comprising
coumestrol or a bean extract comprising coumestrol.
BACKGROUND ART
[0002] Bean is of great nutritional value and contains various
physiologically active and functional substances. Especially, since
the phytoestrogens contained in bean are similar to the estrogen of
mammals including human in structure, they have the effect of
preventing chronic diseases such as hormonal disorders. The
phytoestrogens include in general isoflavone, coumestan and
lignan.
DISCLOSURE
Technical Problem
[0003] The present disclosure is directed to providing a
composition for inhibiting differentiation of adipocytes, enhancing
immunity, detoxifying toxic substances, and preventing or improving
degenerative neurological disorders, comprising coumestrol or a
bean extract comprising coumestrol as an active ingredient.
Technical Solution
[0004] In an aspect, the present disclosure provides a composition
for inhibiting differentiation of adipocytes comprising coumestrol
or a bean extract comprising coumestrol as an active
ingredient.
[0005] In another aspect, the present disclosure provides a
composition for enhancing immunity comprising coumestrol or a bean
extract comprising coumestrol as an active ingredient.
[0006] In another aspect, the present disclosure provides a
composition for detoxifying toxic substances comprising coumestrol
or a bean extract comprising coumestrol as an active
ingredient.
[0007] In another aspect, the present disclosure provides a
composition for preventing or improving degenerative neurological
disorders comprising coumestrol or a bean extract comprising
coumestrol as an active ingredient.
Advantageous Effects
[0008] The composition according to an embodiment of the present
disclosure, which comprises coumestrol or a bean extract comprising
coumestrol as an active ingredient, has the effect of inhibiting
differentiation of adipocytes, enhancing immunity, detoxifying
toxic substances, and preventing or improving degenerative
neurological disorders. The composition is useful in the field of
food or medicine.
DESCRIPTION OF DRAWINGS
[0009] FIG. 1 shows inhibition of adipocyte differentiation by
coumestrol.
[0010] FIG. 2 shows decreased expression of adipocyte marker genes
by coumestrol.
MODE FOR INVENTION
[0011] As used herein, "extract" means a substance extracted from a
natural substance, regardless of extraction method or ingredients.
The term is used in a broad sense including, for example,
ingredients soluble in water or an organic solvent extracted from a
natural substance using the solvent, or specific ingredients of a
natural substance such as oil extracted therefrom.
[0012] Hereinafter, the present disclosure is described in further
detail.
[0013] In an aspect, the present disclosure provides a composition
comprising coumestrol or a bean extract comprising coumestrol as an
active ingredient.
[0014] Coumestrol (CMS;
3,9-dihydroxy-6H-benzofuro(3,2-c)(1)benzopyran-6-one) has a
structure of Chemical Formula 1:
##STR00001##
[0015] Coumestrol is one of phytoestrogens and is usually found in
the seed, root or leaf of plants in the family Leguminosae or
Compositae. It can be classified as a coumestan-like isoflavonoid
and is known to have an estrogenic effect.
[0016] In an exemplary embodiment of the present disclosure, the
composition comprises coumestrol, a natural substance comprising
coumestrol or an extract thereof. In another exemplary embodiment
of the present disclosure, the natural substance comprising
coumestrol may be one or more bean selected from soybean, pea, mung
bean and sprouted beans sprouted therefrom, red clover (alfalfa),
Brussels sprout, or the like. In another exemplary embodiment of
the present disclosure, the natural substance comprising coumestrol
may be bean.
[0017] In an exemplary embodiment of the present disclosure, the
bean may be any plant comprising coumestrol in the family
Leguminosae, without particular limitation. For example, the ban
that can be used in the present disclosure may be one for bean
paste, bean curd, namul, rice cooking or green bean. The bean
varieties for bean paste or bean curd include daepung, hojang,
jangwon, daehwang, sodam, songhak, daewon, jinpum, danbaek, duyu,
shinpaldal, taegwang, manli, jangsu, muhan, baegun, saeal,
hwangkeum and jangyeop. The bean varieties for namul include
shinhwa, sowon, anpyeong, seonam, dachae, sorok, soho, somyeong,
dawon, pungsan-namul, iksan-namul, sobaek-namul, gwangan, danyeop
and eunha The bean varieties for rice cooking include cheongja,
heukcheong, galmi, seonheuk, geomjeong-kong and
ilpumgeomjeong-kong. And, the bean varieties for green bean include
daol, shinrok, saeul, geomjeongeul, seokryang-putkong,
hwaeom-putkong and keuneul. In another exemplary embodiment of the
present disclosure, the bean may be one that can be sprouted and is
resistant to damage from disease and harmful insects. Such bean
varieties include, for example, shinhwa, sowon, anpyeong, seonam,
dachae, sorok, soho, somyeong, dawon, pungsan-namul, iksan-namul,
sobaek-namul, gwangan, danyeop and eunha.
[0018] In an exemplary embodiment of the present disclosure, the
natural substance or the extract thereof may comprise a large
quantity of coumestrol. Specifically, it may comprise 0.01-50 wt %,
more specifically 0.1-30 wt %, of coumestrol based on the total
weight of the natural substance or the extract thereof.
[0019] In an exemplary embodiment of the present disclosure, the
extract of the natural substance comprising coumestrol may be
obtained by extracting the natural substance comprising coumestrol
with water or ethanol at normal or elevated temperature, completely
concentrating the resulting extract and dispersing again in water,
and fractionating with one or more solvent of equal volume selected
from hexane, dichloromethane, chloroform, ethyl acetate, butanol,
ethanol, methanol and water. However, the extraction method is not
limited thereto and any extraction method may be employed.
[0020] In an exemplary embodiment of the present disclosure, the
composition comprising coumestrol or a bean extract comprising
coumestrol as an active ingredient may be a composition for
inhibiting differentiation of adipocytes. Coumestrol inhibits
differentiation of adipocytes and decreases mRNA expression of
molecular markers of adipocyte differentiation and fatty acid
synthesis. Accordingly, the composition comprising coumestrol may
inhibit differentiation of adipocytes and prevent or improve
obesity and various diseases induced by obesity. Examples of such
diseases include type 2 diabetes, fatty liver or cardiovascular
diseases, but are not limited thereto. The cardiovascular disease
includes one or more of hyperlipidemia, hypertension, angina,
myocardial infarction and arteriosclerosis.
[0021] In an exemplary embodiment of the present disclosure, the
composition comprising coumestrol or a bean extract comprising
coumestrol as an active ingredient may be a composition for
enhancing immunity. Coumestrol enhances immunity and activates
immune cells by proliferating the immune cells. Accordingly, the
composition comprising coumestrol may enhance immunity and protect
the body from microbes, bacteria, viruses, etc.
[0022] In an exemplary embodiment of the present disclosure, the
composition comprising coumestrol or a bean extract comprising
coumestrol as an active ingredient may be a composition for
detoxifying toxic substances. The composition may detoxify toxic
substances ingested into the body. Particularly, it has an
excellent ability of detoxifying nicotine. Nicotine is an
addiction-inducing substance contained in cigarettes and may cause
negative cardiovascular effects by stimulating the sympathetic
nervous system. Coumestrol increases viability of vascular
endothelial cells by protecting the vascular endothelial cells from
nicotine or other toxic substances. Accordingly, the composition
comprising coumestrol may detoxify nicotine or other toxic
substances ingested into the body and prevent or improve vascular
diseases caused by the toxic substances. Examples of the vascular
diseases may include blood circulation disorder, hypertension or
arteriosclerosis, but are not limited thereto.
[0023] In an exemplary embodiment of the present disclosure, the
composition comprising coumestrol or a bean extract comprising
coumestrol as an active ingredient may be a composition for
preventing or improving degenerative neurological disorders. The
degenerative neurological disorders refer to the diseases occurring
as a result of degenerative damage and deteriorated function of
nerves or nerve cells. Examples of the degenerative neurological
disorders may include glaucoma, glaucoma, myasthenia gravis,
diabetic neuropathy, cerebrovascular accident, spinal cord injury,
Lou Gehrig's disease (amyotrophic lateral sclerosis; ALS),
Parkinson's disease, Alzheimer's disease or idiopathic dementia,
but are not limited thereto. Coumestrol increases viability of
nerve cells by protecting them from neurotoxins and scavenges
reactive oxygen species produced by neurotoxins. Accordingly, the
composition comprising coumestrol may prevent or improve
degenerative neurological disorders by inhibiting the action of
neurotoxins.
[0024] The composition according to an embodiment of the present
disclosure may comprise 0.001-30 wt %, specifically 0.01-10 wt %,
more specifically 0.1-5 wt %, of coumestrol or a bean extract
comprising coumestrol based on the total weight of the composition.
When the coumestrol or the bean extract comprising coumestrol is
included in the above-described range, the intended effect of the
present disclosure can be adequately achieved while both stability
and safety are satisfied and favorable cost-effectiveness may be
achieved.
[0025] In another aspect, the present disclosure provides a food
composition comprising coumestrol or a bean extract comprising
coumestrol. The food composition may be a health food, functional
food or food additive composition. The food composition may
comprise other ingredients providing synergic effect without
negatively affecting the desired effect. For example, additives
such as fragrance, pigment, sterilizer, antioxidant, antiseptic,
moisturizer, thickener, mineral, emulsifier, synthetic polymer,
etc. may be further included. In addition, auxiliary ingredients
such as water-soluble vitamin, oil-soluble vitamin, polymer
peptide, polymer polysaccharide, and seaweed extract, etc. may be
further included. These ingredients may be selected by those
skilled in the art without difficulty considering the particular
formulation or purpose of use. The addition amount may be
determined within the range not negatively affecting the purpose
and effect of the present disclosure.
[0026] The composition according to the present disclosure may be
prepared into various formulations including solution, emulsion,
viscous mixture, tablet, powder, etc.
[0027] Determination of the dose of the active ingredient is within
the level of those skilled in the art. For example, the dose may be
0.1-5000 mg/kg/day, more specifically 50-500 mg/kg/day, but is not
limited thereto, and may be varied with various factors including
the age, physical condition, complication, etc. of a subject to be
treated.
[0028] In another aspect, the present disclosure provides a
pharmaceutical composition comprising coumestrol or a bean extract
comprising coumestrol. The pharmaceutical composition may further
comprise a pharmaceutical adjuvant such as antiseptic, stabilizer,
hydrating agent, emulsifying accelerator, salt and/or buffer for
controlling osmotic pressure, etc. or other therapeutically useful
substance, and may be prepared into various formulations for oral
or parenteral administration.
[0029] The formulation for oral administration may include, for
example, tablet, pill, hard or soft capsule, liquid, suspension,
emulsion, syrup, powder, dust, granule, pellet, or the like. These
formulations may comprise, in addition to the active ingredient, a
surfactant, a diluent (e.g., lactose, dextrose, sucrose, mannitol,
sorbitol, cellulose or glycine) or a lubricant (e.g., silica, talc,
stearic acid and magnesium or calcium salt thereof or polyethylene
glycol). The tablet may comprise a binder such as magnesium
aluminum silicate, starch paste, gelatin, tragacanth, methyl
cellulose, sodium carboxymethyl cellulose and polyvinylpyrrolidone,
and may occasionally comprise a pharmaceutical additive such as a
disintegrant, e.g. starch, agar, alginic acid or a sodium salt
thereof, an absorbent, a colorant, a flavor, a sweetener, or the
like. The tablet may be prepared according to the commonly employed
mixing, granulation or coating method.
[0030] The formulation for parenteral administration may include,
for example, injection, drop, ointment, lotion, gel, cream, spray,
suspension, emulsion, suppository, patch, etc., but is not limited
thereto.
[0031] A pharmaceutically acceptable amount, i.e. administration
dose, of the dose of the active ingredient will vary depending on
the age, sex and body weight of the subject to be treated, severity
of particular disease or pathological condition to be treated,
administration route and discretion of the prescriber. Those
skilled in the art may determine the administration dose
considering the known bioavailability of coumestrol upon oral
administration (about 11.7%) as well as the above-described
factors. For example, a general administration dose may be
0.01-1000 mg/kg/day, specifically 1-40 mg/kg/day. However, the
described administration dose does not limit the scope of the
present disclosure by any means.
[0032] The features and effects of the present disclosure will be
described in detail through test examples. However, the following
test examples are provided for illustrative purposes only and are
not intended to limit the scope of the present disclosure.
TEST EXAMPLE 1
Evaluation of Effect of Inhibiting Adipocyte Differentiation
[0033] In order to evaluate the coumestrol's effect of inhibiting
adipocyte differentiation, 10 M coumestrol (CMS) was administered
continuously while 3T3-L1 mouse preadipocytes (CL-173, ATCC) were
differentiated into adipocytes. 1 M Rosiglitazone (Rosi), which is
known to promote adipocyte differentiation, was used as positive
control and 10 M berberine (BBR), which is known to inhibit
adipocyte differentiation, was used as negative control. On the day
when adipocyte differentiation was started and on days 2, 4 and 6,
the test substance was administered and the medium was changed. The
adipocyte differentiation was carried out for about 7 days when the
adipocyte differentiation was completed in the positive control
group.
[0034] Then, differentiation of adipocytes was identified by
staining triglycerides in the cells with Oil Red O (Sigma). The
result is shown in FIG. 1. As seen from FIG. 1, the group treated
with coumestrol showed less stained area than those treated with
the positive control rosiglitazone or the negative control
berberine. This means that adipocyte differentiation is remarkably
decreased by coumestrol and the effect is superior to that of
berberine.
[0035] Also, expression of adipocyte-specific genes in the in the
adipocytes was investigated by Q-PCR. The expression level of each
group relative to the untreated group was evaluated and is shown in
FIG. 2. The genes tested were aP2 (fatty acid binding protein 4,
adipocyte specific type), Acrp30 (adiponectin), PPAR (peroxisome
proliferator-activated receptor), SREBP1c (sterol regulatory
element binding protein 1c) and FAS (fatty acid synthase), which
are used as molecular markers of adipocyte differentiation and
fatty acid synthesis. As seen from FIG. 2, coumestrol resulted in
remarkably decreased mRNA expression of the molecular markers of
adipocyte differentiation and fatty acid synthesis, and the effect
was better than that of berberine.
[0036] Thus, coumestrol inhibits adipocyte differentiation and
fatty acid synthesis. Accordingly, the composition comprising
coumestrol may prevent or improve obesity and may prevent or
improve type 2 diabetes, fatty liver, cardiovascular disease, etc.
caused by obesity.
TEST EXAMPLE 2
Evaluation of Immune Cell Activating Effect
[0037] Spleen was aseptically removed from an ICR mouse sacrificed
by cervical dislocation. The spleen was washed with an RPMI 1640
solution and lightly smashed with a sterilized glass rod to isolate
the mouse spleen cells. A suspension of the isolated cells was
passed through a 200-mesh stainless steel sieve (Sigma Chemical
Co., St. Louis, Mo., USA) and centrifuged for 10 minutes at
4.degree. C. and 3,000 rpm in a 50-mL centrifuge tube. The
resulting cell pellets were suspended in a lysing buffer
(Tris-buffered ammonium chloride; 0.87% NH.sub.4Cl, pH 7.2) for 5
minutes to remove red blood cells. The cells were washed twice with
RPMI by centrifugal washing, diluted to 5.0.times.10.sup.6cells/mL
in 10%-FBS RPMI 1640, and seeded on a 96-well plate, with 90 .mu.L
per each well, for measurement of cell proliferating activity.
After preparing a stock solution such that the final concentration
of coumestrol was 0.1 or 1 .mu.M and adding 10 .mu.L to each well,
the cells were cultured for 44 hours and the proliferating activity
of the spleen cells was measured (CCK-8, Dojindo Laboratories,
Japan). 10 .mu.L of 10 .mu.g/mL LPS was used as positive control.
The immune cell proliferating activity (%) of each group relative
to the untreated group was measured and is shown in Table 1.
TABLE-US-00001 TABLE 1 Immune cell proliferating activity (%)
Untreated 100 LPS, 10 .mu.g/mL (positive control) 275 Coumestrol,
0.1 .mu.M 278 Coumestrol, 1 .mu.M 283
[0038] As seen from Table 1, coumestrol promotes proliferation of
immune cells, and the effect is better than that of the positive
control at higher concentration. Accordingly, the composition
comprising coumestrol may provide a superior effect of enhancing
immunity by activating immune cells.
TEST EXAMPLE 3
Evaluation of Nicotine Detoxifying Effect
[0039] Human umbilical vein endothelial cells (HUVECs, CRL-2873,
ATCC) were used to evaluate the coumestrol's effect against
nicotine which is a major toxic substance included in cigarettes.
HUVECs were cultured under the condition of 37.degree. C. and 5%
CO.sub.2 using the microvascular endothelial cell growth medium-2
(EGM-2). The cells were seeded onto a 96-well plate, with
2.times.10.sup.4 cells per well, and cultured for about 24 hours in
a medium containing 10% FBS until .about.80% confluency. After
treating with nicotine to a final concentration of 25 mM, the cells
were cultured for 24 hours with 0.1, 1 or 10 .mu.M coumestrol,
except for the negative control group. The viability (%) of the
vascular endothelial cells was evaluated relative to the untreated
group and is given in Table 2.
TABLE-US-00002 TABLE 2 Viability of vascular endothelial cells (%)
Untreated 100 Negative control 55.1 Coumestrol, 0.1 .mu.M 67.2
Coumestrol, 1 .mu.M 72.2 Coumestrol, 10 .mu.M 88.9
[0040] As seen from Table 2, coumestrol increases the viability of
vascular endothelial cells by detoxifying nicotine. Accordingly,
the composition comprising coumestrol may detoxify toxic substances
ingested into the body and prevent or improve vascular diseases
caused thereby.
TEST EXAMPLE 4
Evaluation of Effect of Inhibiting Neurotoxins
[0041] 1. Evaluation of Effect of Protecting Nerve Cells from
Neurotoxins
[0042] SH-SY5Y cells (CRL-2266, ATCC) were used to evaluate the
coumestrol's effect of protecting nerve cells from neurotoxins. The
cells were cultured in a medium comprising 90% DMEM, 10% fetal calf
serum, 100 IU/mL penicillin and 100 pg/mL streptomycin under the
condition of 37 .degree. C. and 5% CO.sub.2. When the cells grew
confluent, they were harvested with trypsin, diluted on a 96-well
plate to 1.times.10.sup.4 cells per well, and then grafted onto a
culture dish. 24 hours later, after treating the cultured cells
with 0.01, 0.1 or 1 .mu.M coumestrol, the cells were treated with
50 .mu.M 6-OHDA 2 hours later to induce cytotoxicity. A negative
control group was treated only with 50 .mu.M 6-OHDA. The viability
(%) of the nerve cells was measured by MTT assay 24 hours later and
the result is shown in Table 3. Also, mitochondrial potential of
the cells was measured using the JC-1 fluorescent reagent
(Beyotime, China). The effect of inhibiting decline of
mitochondrial potential (%) is shown in Table 4.
TABLE-US-00003 TABLE 3 Viability of nerve cells (%) Untreated 100
Negative control (50 .mu.M 6-OHDA) 78.6 Coumestrol, 0.01 .mu.M 94.4
Coumestrol, 0.1 .mu.M 96.5 Coumestrol, 1 .mu.M 98.2
TABLE-US-00004 TABLE 4 Effect of inhibiting decline of
mitochondrial potential (%) Negative control (50 .mu.M 6-OHDA) 0
Coumestrol, 0.01 .mu.M 13.2 Coumestrol, 0.1 .mu.M 29.1 Coumestrol,
1 .mu.M 43.0
[0043] As seen from Tables 3 and 4, coumestrol increases the
viability of nerve cells in the presence of neurotoxins and has an
excellent effect of inhibiting decline of mitochondrial
potential.
[0044] 2. Evaluation of Effect of Scavenging Reactive Oxygen
Species Produced by Neurotoxins
[0045] The same cells used in Test Example 4-1 were cultured under
the same condition. The cultured cells were treated with 0.01, 0.1
or 1 .mu.M coumestrol and then treated with 50 .mu.M 6-OHDA 2 hours
later to induce increase of reactive oxygen species (ROS) in the
cells. After culturing the cells for 6 hours in an incubator, the
quantity of ROS in the cells was measured using DCFH-DA and the
decrease of the ROS was evaluated relative to the negative control
group. The fluorescence intensity of DCF was measured with the
Wallac VICTOR2 fluorometer (Wallac, Turku, Finland) at excitation
wavelength of 485 nm and emission wavelength of 530 nm. ROS
scavenging effect (%) relative to the negative control group was
calculated from the measured fluorescence intensity. The result is
given in Table 5.
TABLE-US-00005 TABLE 5 ROS scavenging effect (%) Negative control
(50 .mu.M 6-OHDA) 0 Coumestrol, 0.01 .mu.M 73.0 Coumestrol, 0.1
.mu.M 86.7 Coumestrol, 1 .mu.M 93.5
[0046] As seen from Table 5, coumestrol has an excellent effect of
scavenging ROS produced by neurotoxins.
[0047] Accordingly, the composition comprising coumestrol may
prevent or improve degenerative neurological disorders by
inhibiting the action of neurotoxins.
[0048] Formulation examples of the composition according to the
present disclosure are described hereinafter. However, the scope of
the present disclosure is not limited to the following
examples.
FORMULATION EXAMPLE 1
Soft Capsule
[0049] Coumestrol (80 mg), vitamin E (9 mg), vitamin C (9 mg), palm
oil (2 mg), hydrogenated vegetable oil (8 mg), yellow beeswax (4
mg) and lecithin (9 mg) are mixed and prepared into a soft capsule
filling solution according to a commonly employed method. 400 mg of
the solution is filled per capsule. Separately from this, a soft
capsule sheet is prepared using gelatin 66 (wt %), glycerin (24 wt
%) and sorbitol (10 wt %), which is filled with the filling
solution to prepare a soft capsule containing 400 mg of the
composition according to the present disclosure.
FORMULATION EXAMPLE 2
Tablet
[0050] Coumestrol (80 mg), vitamin E (9 mg), vitamin C (9 mg),
galactooligosaccharide (200 mg), lactose (60 mg) and maltose (140
mg) are mixed and granulated using a fluidized bed dryer. After
adding sugar ester (6 mg), the resulting composition (500 mg) is
prepared into a tablet according to a commonly employed method.
FORMULATION EXAMPLE 3
Drink
[0051] Coumestrol (80 mg), vitamin E (9 mg), vitamin C (9 mg),
glucose (10 g), citric acid (0.6 g) and oligosaccharide syrup (25
g) are mixed. After adding purified water (300 mL), 200 mL of the
resulting mixture is filled in a bottle. Then, a drink is prepared
by sterilizing at 130 .degree. C. for 4-5 seconds.
FORMULATION EXAMPLE 4
Granule
[0052] Coumestrol (80 mg), vitamin E (9 mg), vitamin C (9 mg),
anhydrous crystalline glucose (250 mg) and starch (550 mg) are
mixed, granulated using a fluidized bed granulator, and then filled
in a pouch.
FORMULATION EXAMPLE 5
Injection
TABLE-US-00006 [0053] Coumestrol 20 mg Sterilized distilled water
for injection adequate pH adjuster adequate
[0054] An injection is prepared using the above ingredients per
ampule (2 mL) according to a commonly employed method.
[0055] Those skilled in the art will appreciate that the present
disclosure may be changed and modified variously within the scope
of the present disclosure.
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