U.S. patent application number 13/322381 was filed with the patent office on 2012-03-22 for composition containing a bean extract for improving blood circulation and increasing vascular health.
Invention is credited to Yeon Su Jeong, Kyung Mi Joo, Yung Hyup Joo, Chae Wook Kim, Jin Kwan Kim, Sang Jun Lee, Kyung Min Lim, Young-Ho Park, Dae-Bang Seo, Hyun Jung Shin.
Application Number | 20120070520 13/322381 |
Document ID | / |
Family ID | 43223244 |
Filed Date | 2012-03-22 |
United States Patent
Application |
20120070520 |
Kind Code |
A1 |
Shin; Hyun Jung ; et
al. |
March 22, 2012 |
COMPOSITION CONTAINING A BEAN EXTRACT FOR IMPROVING BLOOD
CIRCULATION AND INCREASING VASCULAR HEALTH
Abstract
The present invention relates to a composition containing a bean
extract extracted by low-concentration, low-grade alcohol or
fractions thereof. The composition exhibits excellent effects in
improving blood circulation, improving obesity, and preventing
diabetes, hyperlipidemia and the like, and exhibits the effects of
alleviating or treating the symptoms of diabetes, hyperlipidemia,
and the like. The present invention also relates to a method for
improving blood circulation and vascular health.
Inventors: |
Shin; Hyun Jung; (Seoul,
KR) ; Kim; Jin Kwan; (Gyeonggi-do, KR) ; Kim;
Chae Wook; (Gyeonggi-do, KR) ; Joo; Kyung Mi;
(Gyeonggi-do, KR) ; Jeong; Yeon Su; (Gyeonggi-do,
KR) ; Lim; Kyung Min; (Gyeonggi-do, KR) ; Seo;
Dae-Bang; (Gyeonggi-do, KR) ; Joo; Yung Hyup;
(Gyeonggi-do, KR) ; Lee; Sang Jun; (Gyeonggi-do,
KR) ; Park; Young-Ho; (Seoul, KR) |
Family ID: |
43223244 |
Appl. No.: |
13/322381 |
Filed: |
May 26, 2010 |
PCT Filed: |
May 26, 2010 |
PCT NO: |
PCT/KR2010/003335 |
371 Date: |
November 23, 2011 |
Current U.S.
Class: |
424/757 ;
424/725; 514/46 |
Current CPC
Class: |
A61K 36/48 20130101;
A61P 25/06 20180101; A61P 9/08 20180101; A23L 33/105 20160801; A61P
9/10 20180101; A23L 2/52 20130101; A61P 9/12 20180101; A61P 3/04
20180101; A61P 3/06 20180101; A61P 3/10 20180101; A61P 7/02
20180101; A61P 9/14 20180101; A61P 7/06 20180101; A61P 7/00
20180101; A61P 9/00 20180101; A61K 2236/333 20130101 |
Class at
Publication: |
424/757 ;
424/725; 514/46 |
International
Class: |
A61K 36/48 20060101
A61K036/48; A61K 31/7076 20060101 A61K031/7076; A61P 7/00 20060101
A61P007/00; A61P 7/02 20060101 A61P007/02; A61P 25/06 20060101
A61P025/06; A61P 3/10 20060101 A61P003/10; A61P 9/10 20060101
A61P009/10; A61P 9/12 20060101 A61P009/12; A61P 7/06 20060101
A61P007/06; A61P 3/06 20060101 A61P003/06; A61K 36/00 20060101
A61K036/00; A61P 3/04 20060101 A61P003/04 |
Foreign Application Data
Date |
Code |
Application Number |
May 26, 2009 |
KR |
1020090046107 |
Claims
1. A method for improving blood circulation of a subject,
comprising administering to the subject an effective amount of a
bean extract extracted with a C.sub.1-C.sub.5 alcohol with a
concentration of 1-70% (v/v) or a fraction thereof, wherein the
bean extract or the fraction thereof improves blood circulation in
the subject.
2. The method according to claim 1, wherein the C.sub.1-C.sub.5
alcohol is at least one selected from a group consisting of
methanol, ethanol, isopropyl alcohol, n-propyl alcohol, n-butanol
and isobutanol.
3. The method according to claim 2, wherein the C.sub.1-C.sub.5
alcohol is ethanol.
4. The method according to claim 1, wherein the bean extract or the
fraction thereof comprises adenosine.
5. The method according to claim 4, wherein the adenosine is
included in an amount of 0.01-1.0 wt % based on the weight of the
bean extract or the fraction thereof.
6. The method according to claim 1, wherein the bean is at least
one selected from a group consisting of Seoritae, Seomoktae, Black
soybean, blue bean, yellow bean, field bean, kidney bean, pinto
bean, small red bean, small black bean, sprouting bean and
soybean.
7. The method according to claim 1, wherein the bean is Seoritae or
field bean.
8. The method according to claim 1, wherein the fraction is an
ethyl acetate or butanol fraction of a C.sub.1-C.sub.5 alcohol
extract.
9. The method according to claim 1, wherein the bean extract or the
fraction thereof improves blood circulation through suppression of
blood clotting, suppression of vasoconstriction or suppression of
cholesterol generation.
10. The method according to claim 1, wherein the bean extract or
the fraction thereof improves vascular health.
11. (canceled)
12. The method according to claim 1, wherein the method is for
preventing, alleviating or treating obesity, diabetes, stroke,
cerebral hemorrhage, arteriosclerosis, angina, myocardial
infarction, hypertension, anemia, migraine or hyperlipidemia.
13. A health food composition comprising a bean extract extracted
with a C.sub.1-C.sub.5 alcohol with a concentration of 1-70% (v/v)
or a fraction thereof.
14. (canceled)
15. The health food composition according to claim 13, wherein the
bean is at least one selected from a group consisting of Seoritae,
Seomoktae, Black soybean, blue bean, yellow bean, field bean,
kidney bean, pinto bean, small red bean, small black bean,
sprouting bean and soybean.
16. The health food composition according to claim 15, wherein the
bean is Seoritae or field bean.
17. The health food composition according to claim 13, wherein the
fraction is an ethyl acetate or butanol fraction of a
C.sub.1-C.sub.5 alcohol extract.
18. A pharmaceutical composition comprising a bean extract
extracted with a C.sub.1-C.sub.5 alcohol with a concentration of
1-70% (v/v) or a fraction thereof.
19. The pharmaceutical composition according to claim 18, wherein
the bean is at least one selected from a group consisting of
Seoritae, Seomoktae, Black soybean, blue bean, yellow bean, field
bean, kidney bean, pinto bean, small red bean, small black bean,
sprouting bean and soybean.
20. The pharmaceutical composition according to claim 19, wherein
the bean is Seoritae or field bean.
21. The pharmaceutical composition according to claim 18, wherein
the fraction is an ethyl acetate or butanol fraction of a
C.sub.1-C.sub.5 alcohol extract.
Description
TECHNICAL FIELD
[0001] The present disclosure relates to a composition containing a
bean extract for improving blood circulation and vascular
health.
BACKGROUND ART
[0002] Modern people take in more fats with the change in eating
habits, but they tend to exercise less and suffer from various
stresses. With the change in the dietary lives, various diseases
including hypertension, arteriosclerosis and blood circulation
disorder are increasing. In particular, the blood circulation
disorder is known to cause such symptoms as decline of memory,
lethargy, lack of concentration, chronic fatigue, and the like.
[0003] Blood circulation refers to the flow of blood in the body in
specific directions, and the blood circulation disorder refers to a
condition in which the blood vessels become inelastic and
cholesterol, etc. is deposited in the inner wall of the blood
vessels, leading to narrowing of the blood vessels and interrupting
blood circulation.
[0004] The diseases caused by the blood circulation disorder
include cardiovascular diseases such as hyperlipidemia,
arteriosclerosis, myocardial infarction, cerebral thrombosis, etc.
Among the cardiovascular diseases, hypertension, arteriosclerosis,
heart disease and stroke are one of the most important causes of
death in the elderly people.
[0005] As such, if the blood circulation disorder is left
untreated, it will lead to difficulties in maintaining normal lives
and, in severe cases, various types of diseases and even death.
Accordingly, it is thought that prevention is more important than
treatment of the diseases caused by the blood circulation disorder.
Although drugs for cardiovascular diseases are used clinically at
present, they are expensive and may cause various side effects.
DISCLOSURE
Technical Problem
[0006] The present disclosure is directed to providing a
composition for improving blood circulation.
[0007] The present disclosure is also directed to providing a
composition for improving vascular health.
[0008] The present disclosure is also directed to providing a
pharmaceutical composition for improving blood circulation.
[0009] The present disclosure is also directed to providing a
pharmaceutical composition for improving vascular health.
[0010] The present disclosure is also directed to providing a
health food composition for improving blood circulation.
[0011] The present disclosure is also directed to providing a
health food composition for improving vascular health.
Technical Solution
[0012] In one general aspect, the present disclosure provides a
composition containing a bean extract extracted with a
low-concentration, lower alcohol or a fraction thereof as an active
ingredient.
Advantageous Effects
[0013] The composition according to the present disclosure has
superior effect of improving blood circulation and is effective for
prevention, amelioration or treatment of cardiovascular diseases,
including obesity, diabetes, hyperlipidemia, etc.
Best Mode
[0014] The previous studies on beans are concentrated on isolation
and purification of pharmacologically active ingredients from bean,
and studies about the medical use of the bean itself are
insufficient. In addition, the commonly employed extraction method
was one using a high-concentration organic solvent.
[0015] A bean extract contains a lot of ingredients not known as
yet, and some of them exhibit useful pharmacological effect for the
human body. The inventors of the present disclosure have acquired a
bean extract using a low-concentration, lower alcohol as extraction
solvent, unlike in the extraction method commonly employed for
extraction of natural products or herbs, and have shown that the
extract exhibits stronger antithrombotic effect than one extracted
using a high-concentration organic solvent.
[0016] A composition according to an embodiment of the present
disclosure comprises a bean extract extracted using a
low-concentration organic solvent or a fraction of the extracted
bean extract. In an exemplary embodiment, the organic solvent may
be a C.sub.1-C.sub.5 alcohol, although not being limited thereto.
The C.sub.1-C.sub.5 alcohol may be, for example, at least one
selected from a group consisting of methanol, ethanol, isopropyl
alcohol, n-propyl alcohol, n-butanol and isobutanol, specifically
ethanol. In another exemplary embodiment, the concentration of the
C.sub.1-C.sub.5 alcohol may be 1-70% (v/v), specifically 1-40%
(v/v), more specifically 5-25% (v/v), more specifically 7-20%
(v/v). For example, the solvent may be 10% or 20% (v/v)
ethanol.
[0017] The composition according to the present disclosure is
obtained by extracting bean using the low-concentration, lower
alcohol. Through various researches and repeated experiments, the
inventors of the present disclosure have identified that a bean
extract extracted using a lower alcohol among various organic
solvents, for example, ethanol, particularly low-concentration
ethanol, has the effect of improving blood circulation and vascular
health and thus being useful in alleviation or treatment of
obesity, diabetes or hyperlipidemia.
[0018] The fraction of the bean extract refers to a component
isolated from further fractionation of the bean extract. In an
exemplary embodiment, the fraction of the bean extract may be an
ethyl acetate or butanol fraction, specifically an ethyl acetate
fraction, of the C.sub.1-C.sub.5 alcohol extract. The inventors of
the present disclosure have obtained various fractions from the
bean extract extracted using the low-concentration ethanol. As a
result, an ethyl acetate or butanol fraction showed better effect
of improving blood circulation than a water fraction. Especially,
the ethyl acetate fraction showed a very superior effect.
[0019] The composition comprising the bean extract or the fraction
thereof according to the present disclosure has an effect of
inhibiting blood clotting by suppressing platelet aggregation as
well as an effect of inducing vasodilation by suppressing
constriction of blood vessels. Further, the composition has an
effect of reducing lipids in blood and the liver by suppressing
increase of cholesterol. Accordingly, the composition is effective
in improving blood circulation and vascular health and may be
effectively used to treat or prevent obesity, diabetes,
hyperlipidemia, or the like.
[0020] In an exemplary embodiment, the bean extract or the fraction
thereof may comprise adenosine as a marker or functional component.
The inventors of the present disclosure have separated and purified
the bean extract or the fraction thereof. After measuring
activities of the separated and purified products, it was confirmed
that adenosine is included therein. In an exemplary embodiment,
adenosine may be included in an amount of 0.01-1.0 wt %, more
specifically 0.1-0.6 wt %, based on the weight of the bean extract
or the fraction thereof. That is to say, the bean extract extracted
using the low-concentration, lower alcohol according to the present
disclosure contains relatively large amount of active ingredients
such as adenosine.
[0021] The bean is not particularly limited as long as the bean
extract or the fraction thereof has an effect of improving blood
circulation. In an exemplary embodiment, the bean may be black bean
or colored bean. In another exemplary embodiment, the bean may be
at least one selected from a group consisting of Seoritae (Glycin
max MERR), Seomoktae (Rhynchosia Nolubilis), Black soybean (Glycine
max(L.) Merr.), blue bean (Glycime max MERR), yellow bean (Glycime
max MERR), field bean (Vicia faba), kidney bean (Phaseolus
vulgaris), pinto bean (Phaseolus vulgaris L.), small red bean
(Vigna angularis), small black bean (Phaseolus angularis
.F.WIGHT.), sprouting bean (Glycine max (L.) Merr.) and soybean
(Glycine max). More specifically, the bean may be seoritae or field
bean.
[0022] As used herein the term "black bean" collectively refers to
a bean whose grain exhibits black color. The black bean is not
particularly limited. For example, it may be black bean (Glycine
max), Seoritae (Glycin max MERR), Seomoktae (Rhynchosia Nolubilis),
Black soybean (Glycine max(L.) Merr.), or the like. The black bean
may be called differently in different regions. And, the term
"black bean extract" collectively refers to a substance extracted
from black bean. For example, it includes a substance extracted
using an organic solvent and also covers various fractions of the
extract.
[0023] Also, as used herein the term "colored bean" collectively
refers to a bean whose grain exhibits deep color, not only black
color but also red, yellow or blue color. Examples of the colored
bean include Seoritae (Glycin max MERR), Seomoktae (Rhynchosia
Nolubilis), Black soybean (Glycine max(L.) Merr.), blue bean
(Glycime max MERR), yellow bean (Glycime max MERR), field bean
(Vicia faba), kidney bean (Phaseolus vulgaris), pinto bean
(Phaseolus vulgaris L.), small red bean (Vigna angularis), small
black bean (Phaseolus angularis .F.WIGHT), sprouting bean (Glycine
max (L) Merr.) and soybean (Glycine max), although not being
limited thereto. The colored bean may be called differently in
different regions. And, the term "colored bean extract"
collectively refers to a substance extracted from colored bean. For
example, it includes a substance extracted using an organic solvent
and also covers various fractions of the extract.
[0024] The present disclosure provides a pharmaceutical composition
comprising the afore-described composition. In an exemplary
embodiment, the pharmaceutical composition may be a pharmaceutical
composition for improving blood circulation, preventing
cardiovascular disease, or alleviating or treating related
symptoms. The pharmaceutical composition according to the present
disclosure has the effect of preventing blood clotting, suppressing
vasoconstriction and/or reducing cholesterol. Specifically, the
pharmaceutical composition may be a pharmaceutical composition for
improving blood circulation via antithrombotic effect, or
alleviating or treating cardiovascular diseases including obesity,
diabetes, hyperlipidemia, etc. The cardiovascular diseases may
include, for example, obesity, diabetes, stroke, cerebral
hemorrhage, arteriosclerosis, angina, myocardial infarction,
hypertension, anemia, migraine, hyperlipidemia, or the like.
[0025] When the composition according to the present disclosure is
used as a medicine, it may be prepared in the form of solid,
semisolid or liquid by adding a commonly used organic or inorganic
carrier for oral or parenteral administration.
[0026] A formulation for oral administration may be in the form of
tablet, pill, granule, soft/hard capsule, powder, fine granule,
dust, emulsion, syrup, pellet, or the like. A formulation for
parenteral administration may be in the form of injection, drip,
ointment, lotion, spray, suspension, emulsion, suppository, or the
like. The active ingredient of the present disclosure may be easily
prepared into such formulation according to a commonly employed
method, and commonly used adjuvants such as surfactant, excipient,
colorant, fragrance, preservative, stabilizer, buffering agent,
suspending agent, etc. may be adequately used.
[0027] The pharmaceutical composition may be administered orally or
parenterally, e.g., rectally, topically, transdermally,
intravenously, intramuscularly, intraabdominally or
subcutaneously.
[0028] An administration dose of the active ingredient will vary
depending on the age, sex and body weight of the subject,
particular disease or pathological condition to be treated,
severity of the disease or pathological condition, administration
route, or decision by a physician. The determination of the
administration dose based on such factors is within the knowledge
of those skilled in the art. A general administration dose is
0.001-2000 mg/kg/day, more specifically 0.5-1500 mg/kg/day.
[0029] In an exemplary embodiment, the present disclosure provides
a food additive, a functional food or a health food comprising the
composition according to the present disclosure. Specifically, the
composition may be a health food composition for improving blood
circulation, or alleviating or treating cardiovascular diseases
including obesity, diabetes, hyperlipidemia, etc. The
cardiovascular diseases may include, for example, obesity,
diabetes, stroke, cerebral hemorrhage, arteriosclerosis, angina,
myocardial infarction, hypertension, anemia, migraine,
hyperlipidemia, or the like.
[0030] The present disclosure provides various types of food
additive or functional food comprising the afore-described
composition according to the present disclosure. The composition
may be processed into fermented milk, cheese, yogurt, juice,
probiotic, dietary supplement or other food additives.
[0031] In an exemplary embodiment, the composition may further
comprise other ingredients providing synergic effect within the
range not negatively affecting the main effect desired by the
present disclosure. For example, it may further comprise such
additives as fragrance, pigment, sterilizer, antioxidant,
antiseptic, humectant, thickener, mineral, emulsifier, synthetic
polymer, etc. for improvement of physical properties. In addition,
it may further comprise such auxiliary ingredients as water-soluble
vitamin, oil-soluble vitamin, polypeptide, polysaccharide, seaweed
extract, or the like. Those skilled in the art will select and mix
these ingredients without difficulty considering the formulation
type or purpose of use, and their content may be determined within
the range not negatively affecting the purpose and effect of the
present disclosure. For example, those ingredients may be added in
an amount of 0.01-5 wt %, more specifically 0.01-3 wt %, based on
the total weight of the composition.
[0032] The composition according to the present disclosure may be
in various forms, including solution, emulsion, viscous mixture,
tablet, powder, etc., and may be administered in various manners,
including drinking, injection, spraying, squeezing, etc.
MODE FOR INVENTION
[0033] The examples and experiments will now be described. The
following examples and experiments are for illustrative purposes
only and not intended to limit the scope of this disclosure.
EXAMPLE 1
Preparation of 10% Ethanol Seoritae (Glycin max MERR) Extract
[0034] Dried seoritae (1 kg) was immersed in 10% ethanol solution
(10 L) at 50.degree. C. After extracting 3 times for 5 hours under
reflux and allowing to stand at room temperature for 12 hours, the
extract was filtered, concentrated under reduced pressure and
lyophilized to prepare a powder sample. The yield was 7-20% and the
prepared powder was kept at low temperature until use.
EXAMPLE 2
Preparation of 20% Ethanol Seoritae (Glycin max MERR) Extract
[0035] Dried seoritae (1 kg) was immersed in 20% ethanol solution
(10 L) at 50.degree. C. After extracting 3 times for 5 hours under
reflux and allowing to stand at room temperature for 12 hours, the
extract was filtered, concentrated under reduced pressure and
lyophilized to prepare a powder sample. The yield was 7-20% and the
prepared powder was kept at low temperature until use.
EXAMPLE 3
Preparation of 50% Ethanol Seoritae (Glycin max MERR) Extract
[0036] Dried seoritae (1 kg) was immersed in 50% ethanol solution
(10 L) at 50.degree. C. After extracting 3 times for 5 hours under
reflux and allowing to stand at room temperature for 12 hours, the
extract was filtered, concentrated under reduced pressure and
lyophilized to prepare a powder sample. The yield was 7-20% and the
prepared powder was kept at low temperature until use.
EXAMPLE 4
Preparation of 70% Ethanol Seoritae (Glycin max MERR) Extract
[0037] Dried seoritae (1 kg) was immersed in 70% ethanol solution
(10 L) at 50.degree. C. After extracting 3 times for 5 hours under
reflux and allowing to stand at room temperature for 12 hours, the
extract was filtered, concentrated under reduced pressure and
lyophilized to prepare a powder sample. The yield was 7-20% and the
prepared powder was kept at low temperature until use.
EXAMPLE 5
Preparation of 20% Ethanol Seomoktae (Rhynchosia Nolubilis)
Extract
[0038] Dried seomoktae (1 kg) was immersed in 20% ethanol solution
(5 L) at 60.degree. C. After extracting for 3 hours under reflux
and allowing to stand at room temperature for a predetermined time,
the extract was filtered, concentrated under reduced pressure and
lyophilized to prepare a powder sample. The yield was 3-15% and the
prepared powder was kept at low temperature until use.
EXAMPLE 6
Preparation of 20% Ethanol Kidney Bean (Phaseolus vulgaris)
extract
[0039] Dried kidney bean (300 g) was immersed in 20% ethanol
solution (1.5 L) at 60.degree. C. After extracting for 3 hours
under reflux and allowing to stand at room temperature for a
predetermined time, the extract was filtered, concentrated under
reduced pressure and lyophilized to prepare a powder sample. The
yield was 3-15% and the prepared powder was kept at low temperature
until use.
EXAMPLE 7
Preparation of 20% Ethanol Pinto Bean (Phaseolus vulgaris L.)
Extract
[0040] Dried pinto bean (300 g) was immersed in 20% ethanol
solution (1.5 L) at 60.degree. C. After extracting for 3 hours
under reflux and allowing to stand at room temperature for a
predetermined time, the extract was filtered, concentrated under
reduced pressure and lyophilized to prepare a powder sample. The
yield was 3-15% and the prepared powder was kept at low temperature
until use.
EXAMPLE 8
Preparation of 20% Ethanol Sprouting Bean (Glycine max (L.) Merr.)
Extract
[0041] Dried sprouting bean (300 g) was immersed in 20% ethanol
solution (1.5 L) at 60.degree. C. After extracting for 3 hours
under reflux and allowing to stand at room temperature for a
predetermined time, the extract was filtered, concentrated under
reduced pressure and lyophilized to prepare a powder sample. The
yield was 3-15% and the prepared powder was kept at low temperature
until use.
EXAMPLE 9
Preparation of 20% Ethanol Yellow Bean (Glycime max MERR)
Extract
[0042] Dried yellow bean (1 kg) was immersed in 20% ethanol
solution (5 L) at 60.degree. C. After extracting for 2 times for 3
hours under reflux and allowing to stand at room temperature for a
predetermined time, the extract was filtered, concentrated under
reduced pressure and lyophilized to prepare a powder sample. The
yield was 3-15% and the prepared powder was kept in a refrigerator
until use.
EXAMPLE 10
Preparation of 20% Ethanol Blue Bean (Glycime max MERR) Extract
[0043] Dried blue bean (300 g) was immersed in 20% ethanol solution
(1.5 L) at 60.degree. C. After extracting for 3 hours under reflux
and allowing to stand at room temperature for a predetermined time,
the extract was filtered, concentrated under reduced pressure and
lyophilized to prepare a powder sample. The yield was 3-15% and the
prepared powder was kept in a refrigerator until use.
EXAMPLE 11
Preparation of 20% Ethanol Field Bean (Vicia faba) Extract
[0044] Dried field bean (1 kg) was immersed in 20% ethanol solution
(5 L) at 60.degree. C. After extracting for 3 hours under reflux
and allowing to stand at room temperature for a predetermined time,
the extract was filtered, concentrated under reduced pressure and
lyophilized to prepare a powder sample. The yield was 3-15% and the
prepared powder was kept at low temperature until use.
EXAMPLE 12
Preparation of 20% Ethanol Soybean (Glycine max) Extract
[0045] Dried soybean (1 kg) was immersed in 20% ethanol solution (5
L) at 60.degree. C. After extracting for 3 hours under reflux and
allowing to stand at room temperature for a predetermined time, the
extract was filtered, concentrated under reduced pressure and
lyophilized to prepare a powder sample. The yield was 3-15% and the
prepared powder was kept at low temperature until use.
EXAMPLE 13
Preparation of 20% Ethanol Small Black Bean (Phaseolus angularis
.F.WIGHT.) Extract
[0046] Dried geodu (300 g) was immersed in 20% ethanol solution
(1.5 L) at 60.degree. C. After extracting for 3 hours under reflux
and allowing to stand at room temperature for a predetermined time,
the extract was filtered, concentrated under reduced pressure and
lyophilized to prepare a powder sample. The yield was 3-15% and the
prepared powder was kept in a refrigerator until use.
EXAMPLE 14
Preparation of 20% Ethanol Small Red Bean (Vigna angularis)
Extract
[0047] Dried red bean (300 g) was immersed in 20% ethanol solution
(1.5 L) at 60.degree. C. After extracting for 3 hours under reflux
and allowing to stand at room temperature for a predetermined time,
the extract was filtered, concentrated under reduced pressure and
lyophilized to prepare a powder sample. The yield was 3-15% and the
prepared powder was kept at low temperature until use.
EXAMPLE 15
Preparation of 20% Ethanol Black Soybean (Glycine max(L.) Merr.)
Extract
[0048] Dried heuktae (300 g) was immersed in 20% ethanol solution
(1.5 L) at 60.degree. C. After extracting for 3 hours under reflux
and allowing to stand at room temperature for a predetermined time,
the extract was filtered, concentrated under reduced pressure and
lyophilized to prepare a powder sample. The yield was 3-15% and the
prepared powder was kept at low temperature until use.
EXAMPLE 16
Preparation of Seoritae (Glycin max MERR) Butanol Extract
[0049] The 20% ethanol seoritae extract (25 g) obtained in Example
2 was dissolved in distilled water (250 mL). After extracting 2
times with n-butanol (250 mL) using a separatory funnel, the
resulting butanol layer was concentrated under reduced pressure and
lyophilized to prepare a sample. The yield was 5-15% and the
prepared sample was kept in a refrigerator until use.
EXAMPLE 17
Preparation of Seomoktae (Rhynchosia Nolubilis) Butanol Extract
[0050] The 20% ethanol seomoktae extract (1 g) obtained in Example
5 was dissolved in distilled water (10 mL). After extracting 2
times with n-butanol (10 mL) using a separatory funnel, the
resulting butanol layer was concentrated under reduced pressure and
lyophilized to prepare a sample. The yield was 5-15% and the
prepared sample was kept in a refrigerator until use.
EXAMPLE 18
Preparation of Field Bean (Vicia faba) Butanol Extract
[0051] The 20% ethanol field bean extract (25 g) obtained in
Example 11 was dissolved in distilled water (250 mL). After
extracting 2 times with n-butanol (250 mL) using a separatory
funnel, the resulting butanol layer was concentrated under reduced
pressure and lyophilized to prepare a sample. The yield was 5-15%
and the prepared sample was kept in a refrigerator until use.
EXAMPLE 19
Preparation of Blue Bean (Glycime max MERR) Butanol Extract
[0052] The 20% ethanol blue bean extract (1 g) obtained in Example
10 was dissolved in distilled water (10 mL). After extracting 2
times with n-butanol (10 mL) using a separatory funnel, the
resulting butanol layer was concentrated under reduced pressure and
lyophilized to prepare a sample. The yield was 5-15% and the
prepared sample was kept at low temperature until use.
EXAMPLE 20
Preparation of Yellow Bean (Glycime max MERR) Butanol Extract
[0053] The 20% ethanol yellow bean extract (1 g) obtained in
Example 9 was dissolved in distilled water (10 mL). After
extracting 2 times with n-butanol (10 mL) using a separatory
funnel, the resulting butanol layer was concentrated under reduced
pressure and lyophilized to prepare a sample. The yield was 5-15%
and the prepared sample was kept at low temperature until use.
EXAMPLE 21
Preparation of Sprouting Bean (Glycine max (L.) Merr.) Butanol
Extract
[0054] The 20% ethanol sprouting bean extract (1 g) obtained in
Example 8 was dissolved in distilled water (10 mL). After
extracting 2 times with n-butanol (10 mL) using a separatory
funnel, the resulting butanol layer was concentrated under reduced
pressure and lyophilized to prepare a sample. The yield was 5-15%
and the prepared sample was kept at low temperature until use.
EXAMPLE 22
Preparation of Soybean (Glycine max) Butanol Extract
[0055] The 20% ethanol soybean extract (25 g) obtained in Example
12 was dissolved in distilled water (250 mL). After extracting 2
times with n-butanol (250 mL) using a separatory funnel, the
resulting butanol layer was concentrated under reduced pressure and
lyophilized to prepare a sample. The yield was 5-15% and the
prepared sample was kept at low temperature until use.
EXAMPLE 23
Preparation of Kidney Bean (Phaseolus vulgaris) Butanol Extract
[0056] The 20% ethanol kidney bean extract (1 g) obtained in
Example 6 was dissolved in distilled water (10 mL). After
extracting 2 times with n-butanol (10 mL) using a separatory
funnel, the resulting butanol layer was concentrated under reduced
pressure and lyophilized to prepare a sample. The yield was 5-15%
and the prepared sample was kept at low temperature until use.
EXAMPLE 24
Preparation of Fractions of Seoritae (Glycin max MERR) Extract
[0057] Dried seoritae (1 kg) was immersed in 20% ethanol solution
(10 L) at 50.degree. C. After extracting 3 times for 5 hours under
reflux and allowing to stand at room temperature for 12 hours, the
extract was filtered and concentrated under reduced pressure.
[0058] After adding 5 times the volume of ethyl acetate solution to
the concentrated filtrate and allowing to stand at room so that
separation occurs between an ethyl acetate and a water layer, only
the ethyl acetate layer was taken and lyophilized to prepare
fractions. The prepared powder was kept at low temperature until
use.
COMPARATIVE EXAMPLE
Preparation of Seoritae (Glycin max MERR) Extract Using Water
[0059] Dried seoritae (1 kg) was immersed in 20% water (10 L) at
100.degree. C. After extracting 3 times for 5 hours under reflux
and allowing to stand at room temperature for 12 hours, the extract
was filtered, concentrated under reduced pressure and lyophilized
to prepare a powder sample. The yield was 7-20% and the prepared
powder was kept at low temperature until use.
TEST EXAMPLE 1
Inhibition of Human Platelet Aggregation Induced by Collagen of
Seoritae (Glycin max MERR) Extract
[0060] Experiment was performed as follows in order to compare the
activity of the extracts depending on ethanol concentration.
[0061] In order to isolate human platelet-rich plasma (hereinafter,
PRP), blood was taken from the vein of a healthy man who did not
take drugs over 2 weeks using 3.2% sodium citrate as anticoagulant.
The blood (150 g) was centrifuged for 15 minutes. After separating
the supernatant (PRP), the residue was centrifuged again to
separate platelet-poor plasma (hereinafter, PPP). The number of
platelets in the separated PRP was counted under an optical
microscope, and the PRP was diluted with the PPP such that
3.times.10.sup.8 platelets were included per 1 mL.
[0062] The platelet aggregation activity was evaluated by measuring
the change in absorbance using the lumi-aggregometer (Chrono-Log
Co., USA). After adding 100 pg/mL of the seoritae extract to the
PRP, followed by incubation in a thermomixer for 10 minutes, the
incubated PRP (495 .mu.L) was put in a silicone-coated cuvette for
measurement of platelet aggregation and further incubated for 1
minute until the temperature reached 37.degree. C. Then, after
adding collagen which induces platelet aggregation with a
concentration of 5 .mu.g/mL at which the maximum aggregation is
achieved, the reaction was observed for 5 minutes. The result is
given in Table 1. In Table 1, the inhibition of platelet
aggregation (%) is a value relative to that of the control group
treated only with collagen as 0%.
TABLE-US-00001 TABLE 1 Inhibition of platelet aggregation (%) Water
extract 8.3 .+-. 2.5 10% ethanol extract 74.6 .+-. 6.4 20% ethanol
extract 40.1 .+-. 12.2 50% ethanol extract 18.8 .+-. 10.9 70%
ethanol extract 12.5 .+-. 8.4
[0063] As seen from Table 1, the 10% and 20% ethanol extracts
showed better platelet aggregation inhibition effect than other
extracts. Especially, the 10% ethanol extract showed the best
effect.
[0064] Also, an experiment was performed to identify the
concentration dependence of the platelet aggregation inhibition
effect for the 20% ethanol extract. First, the same experiment was
performed as described above after adding the extract at
concentrations of 10, 25, 50 and 100 .mu.g/mL. The inhibition of
platelet aggregation was evaluated relative to that of the control
group treated only with collagen as 0%. As seen from FIG. 1, the
20% ethanol extract showed a concentration-dependent platelet
aggregation inhibition effect, demonstrating superior effect
against blood clotting.
TEST EXAMPLE 2
Inhibition of Human Platelet Aggregation Induced by Collagen of
Field Bean (Vida faba) Extract
[0065] Experiment was performed as follows in order to compare the
activity of the extracts depending on ethanol concentration.
[0066] In order to isolate human PRP, blood was taken from the vein
of a healthy man who did not take drugs over 2 weeks using 3.2%
sodium citrate as anticoagulant. The blood (150 g) was centrifuged
for 15 minutes. After separating the supernatant (PRP), the residue
was centrifuged again to separate PPP. The number of platelets in
the separated PRP was counted, and the PRP was diluted with the PPP
such that 3.times.10.sup.8 platelets were included per 1 mL.
[0067] The platelet aggregation activity was evaluated by measuring
the change in absorbance using the lumi-aggregometer (Chrono-Log
Co., USA). After adding 200 mg/mL of the field bean extract to the
PRP, which had been incubated in a thermomixer at 37.degree. C. for
2 minutes, followed by incubation for 7 minutes, the incubated PRP
(500 .mu.L) was put in a silicone-coated cuvette for measurement of
platelet aggregation and further incubated for 3 minutes. Then,
after adding collagen which induces platelet aggregation with a
concentration of 1-3 .mu.g/mL, i.e. the lowest concentration at
which the maximum aggregation is achieved, the reaction was
observed for 6 minutes. The result is given in Table 2. In Table 2,
the inhibition of platelet aggregation (%) is a value relative to
that of the control group treated only with collagen as 0%.
TABLE-US-00002 TABLE 2 Inhibition of platelet aggregation (%) Water
extract 69.0 10% ethanol extract 57.3 20% ethanol extract 76.3 30%
ethanol extract 38.7 40% ethanol extract 34.7 50% ethanol extract
39.7 70% ethanol extract 10.7
[0068] As seen from Table 2, the 10% and 20% ethanol extracts
showed better platelet aggregation inhibition effect than other
extracts. Especially, the 20% ethanol extract showed the best
platelet aggregation inhibition effect.
TEST EXAMPLE 3
Human Platelet Aggregation Inhibition Effect of Different Beans
[0069] Under the 20% ethanol extraction condition showing the best
activity, experiment was performed as follows in order to
investigate the platelet aggregation inhibition of black bean. All
the beans used in the experiment were produced in Korea.
[0070] Experiment was performed for seoritae, seomoktae and Black
soybean. The beans were extracted in the same manner as in Example
2. In order to investigate the platelet aggregation inhibition
effect, experiment was performed using 100 .mu.g/mL bean extract
under the same condition as in Test Example 1. The result is given
in Table 3.
TABLE-US-00003 TABLE 3 Beans Inhibition of platelet aggregation (%)
Seoritae 45.31 .+-. 9.67 Seomoktae 42.86 .+-. 11.02 Black soybean
41.78 .+-. 8.49
[0071] As seen from Table 3, the black beans seoritae, seomoktae
and heuktae showed high platelet aggregation inhibition effect.
Accordingly, it was confirmed that the low-concentration ethanol
extracts of seoritae, seomoktae and heuktae have superior platelet
aggregation inhibition effect.
TEST EXAMPLE 4
Human Platelet Aggregation Inhibition Effect of Different Beans
[0072] Under the 20% ethanol extraction condition showing the best
activity, experiment was performed as follows in order to
investigate the platelet aggregation inhibition of black bean. All
the beans used in the experiment were produced in Korea.
[0073] Experiment was performed for small black bean, soybean,
seoritae, seomoktae, field bean, kidney bean, small red bean, blue
bean, sprouting bean and yellow bean. The beans were extracted in
the same manner as described above. In order to investigate the
platelet aggregation inhibition effect, experiment was performed
using 200 .mu.g/mL bean extract under the same condition as in Test
Example 2. The result is given in Table 4.
TABLE-US-00004 TABLE 4 Beans Inhibition of platelet aggregation (%)
Small black bean 31.0 Soybean 19.3 Seoritae 65.3 Field bean 76.3
Kidney bean 31.0 Small red bean 32.0 Blue bean 16.5 Sprouting bean
37.5 Yellow bean 2.5
[0074] As seen from Table 4, seoritae, seomoktae and field bean
showed superior platelet aggregation inhibition effect.
TEST EXAMPLE 5
Inhibition Specificity for Different Causes of Platelet
Aggregation
[0075] Under the 20% ethanol extraction condition, it was
investigated whether the seoritae extract show specific inhibition
effect for different causes of platelet aggregation.
[0076] It is known that platelet aggregation is induced by
collagens which are exposed to the bloodstream when a blood vessel
is damaged, ADPs secreted by the platelets, stress by the
bloodstream (shear stress; hereinafter, SS), and thrombins.
Accordingly, it was investigated whether the 20% low-concentration
ethanol seoritae extract has inhibition specificity for collagen,
ADP, thrombin and shear stress (SS). The evaluation was performed
in the same manner as in Test Example 1. ADP, thrombin or shear
stress was applied instead of collagen. The result is given in
Table 5.
TABLE-US-00005 TABLE 5 Collagen ADP Thrombin Shear stress
Inhibition 61.38 .+-. 10.44 8.25 .+-. 3.10 19.61 .+-. 4.52 12.36
.+-. 3.68 of platelet aggregation (%)
[0077] As seen from Table 5, the 20% ethanol seoritae extract
specifically inhibited platelet aggregation induced by collagen,
with little effect of inhibiting platelet aggregation induced by
other stimulations, i.e. ADP, thrombin or shear stress.
TEST EXAMPLE 6
Active Substance Expression and Secretion Inhibition Effect After
Platelet Aggregation
[0078] When platelets are coagulated by stimulation such as
collagen, they express specific proteins on their surface or
secrete specific substances out of cells. Accordingly, it was
investigated whether the 20% low-concentration ethanol seoritae
extract can reduce the expression of P-selectin and secretion of
serotonin into the bloodstream by suppressing platelet
aggregation.
[0079] After adding the sample to the same PRP as that of Test
Example 1, followed by incubation for 10 minutes in an incubator
and reaction for 6 minutes after adding 10 .mu.g/mL of collagen,
the extract was added to the test tube together with anti-CD42b-PE
and anti-CD62P-FITC and allowed to react for 20 minutes in the
shade. Then, Tyrode's buffer (500 .mu.L) was added to terminate the
reaction.
[0080] Measurement was made using a fluorescence-activated cell
sorter (FACS; BD Bioscience, USA). The degree of P-selectin
expression was determined by measuring the reduction of
fluorescence due to the expression of P-selectin. The result is
given in Table 6. The inhibition of P-selectin expression is given
relative to the fluorescence of the extract-untreated group as
100.
TABLE-US-00006 TABLE 6 Treatment concentration (.mu.g/mL) 10 25 50
100 Inhibition of P-selectin 13.8 .+-. 2.3 19.8 .+-. 1.6 24.1. .+-.
2.2 36.9 .+-. 1.9 expression (%)
[0081] As seen from Table 6, the 20% ethanol seoritae extract
decreased the expression of P-selectin on the surface of platelets
in a concentration-dependent manner.
[0082] The secretion of serotonin was measured using the
radioisotope method. After adding 0.5 .mu.Ci/mL of
.sup.14C-serotonin (Amersham Bioscience, CFA170) to the same PRP as
that of Test Example 1, followed by treatment at 37.degree. C. for
45 minutes and further treatment at 37.degree. C. for 10 minutes
after adding the extract, 2 .mu.g/mL of collagen was added and
allowed to react for 6 minutes. After terminating the reaction by
adding EDTA, centrifugation was carried out at 12000.times.g for 2
minutes. The quantity of secreted of [.sup.14C]-serotonin in the
supernatant was measured using a liquid scintillation counter
(Wallac 1409, Perkin Elmer, USA). The inhibition of serotonin
secretion was calculated relative to that of the extract-untreated
group as 100. The result is given in Table 7.
TABLE-US-00007 TABLE 7 Treatment concentration (.mu.g/mL) 10 25 50
100 Inhibition of serotonin 9.8 .+-. 2.5 17.7 .+-. 3.2 38.0. .+-.
2.2 59.4 .+-. 1.3 secretion (%)
[0083] As seen from Table 7, the 20% ethanol seoritae extract
decreased the secretion of serotonin into the bloodstream in a
concentration-dependent manner.
TEST EXAMPLE 7
Inhibition of Blood Clotting in Vein in SD Rat
[0084] The blood clotting inhibition effect of the 20% ethanol
seoritae extract in vivo was investigated as follows. Male Sprague
Dawley (hereinafter, SD) rats weighing 220-250 g were used.
[0085] 0, 50 or 100 .mu.g/mL of the seoritae extract dissolved in
saline (300 .mu.L) was orally administered to the SD rat. 1 hour
later, sodium pentobarbital (50 mg/kg) was abdominally administered
and the rat was generally anesthetized. After cutting the abdomen
open, the adipose tissue was removed so that the caudal vena cava
could be seen well. Care was taken to avoid damage to the nearby
blood vessels when removing the adipose tissue. A filter paper
soaked with 5% FeCl.sub.3 solution was put on the caudal vena cava
for 5 minutes and then removed. 30 minutes later, the caudal vena
cava containing blood clots was ligated at 12 mm length and then
incised. After transferring the blood clots to saline, water was
removed and the weight was measured. The result is given in FIG.
2.
[0086] Referring to FIG. 2, it can be seen that the oral
administration of the 100 mg/kg seoritae extract significantly
inhibits blood clotting.
[0087] Also, the 50 or 100 mg/kg extract was orally administered
for 14 days, and the result is shown in FIG. 3. Differently from
the result 1 hour after the administration, both the 50 and 100
mg/kg extract significantly inhibited blood clotting. Accordingly,
it can be seen that the seoritae extract extracted using the
low-concentration ethanol is a superior anticoagulant.
TEST EXAMPLE 8
Human Platelet Aggregation Inhibition Effect of Seoritae
Extract
[0088] It was investigated which fraction exhibits superior
activity under the 20% ethanol extraction condition.
[0089] First, ethyl acetate, butanol and water fractions were
prepared in the same manner as in Example 25. The platelet
aggregation inhibition effect of each fraction (100 .mu.g/mL) was
evaluated in the same manner as in Test Example 1. The result is
given in Table 8.
TABLE-US-00008 TABLE 8 Inhibition of platelet aggregation (%) Ethyl
acetate fraction 95.2 .+-. 0.9 Butanol fraction 76.8 .+-. 6.9 Water
fraction 4.7 .+-. 5.2
[0090] As seen from Table 8, the ethyl acetate fraction showed the
best platelet aggregation inhibition effect, followed by butanol
and water fractions. Accordingly, it though that the platelet
aggregation inhibition effect of the low-concentration ethanol
extract is derived from the active ingredients included in the
ethyl acetate fraction.
[0091] In the ethyl acetate fraction that exhibited the best
effect, the activity was concentration-dependent, as shown in FIG.
4. That is to say, the platelet aggregation inhibition effect
increased in the order of 10, 25, 50 and 100 .mu.g/mL of the ethyl
acetate fraction, as seen from FIG. 4.
TEST EXAMPLE 9
Platelet Aggregation Inhibition Effect of Butanol Fractions of
Different Beans
[0092] Experiment was performed as follows in order to identify the
platelet aggregation inhibition effect of the butanol fraction of
different beans.
[0093] The platelet aggregation inhibition effect of the butanol
fraction (200 .mu.g/mL) of soybean, seomoktae, seoritae, field
bean, kidney bean, blue bean, sprouting bean and yellow bean was
evaluated in the same manner as in Test Example 2. The result is
given in Table 9.
TABLE-US-00009 TABLE 9 Inhibition of platelet aggregation (%)
Soybean 94.0 Seomoktae 90.5 Seoritae 72.7 Field bean 88.5 Kidney
bean 77.5 Blue bean 91.5 Sprouting bean 90.5 Yellow bean 88.5
[0094] As seen from Table 9, the butanol fractions of all of the
beans showed superior platelet aggregation inhibition effect.
TEST EXAMPLE 10
Specificity of Platelet Aggregation Inhibition of Ethyl Acetate
Fraction for Different Causes
[0095] Experiment was performed in the same manner as in Test
Example 5 to investigate the specificity of platelet aggregation
inhibition of ethyl acetate fractions (50 and 100 .mu.g/mL) for
different causes. The result is given in Table 10.
TABLE-US-00010 TABLE 10 Shear Conc. Collagen ADP Thrombin stress
Inhibition 50 .mu.g/mL 80.6 .+-. 0.8 25.1 .+-. 9.2 0.6 .+-. 3.3
16.0 .+-. 2.3 of platelet 100 .mu.g/mL 94.0 .+-. 1.9 53.7 .+-. 6.3
9.1 .+-. 7.7 36.9 .+-. 3.0 aggre- gation (%)
[0096] As seen from Table 10, the ethyl acetate fraction
specifically inhibited the activity of collagen in a
concentration-dependent manner.
TEST EXAMPLE 11
Inhibition Effect of Ethyl Acetate Fraction Against Expression and
Secretion of Active Substances After Platelet Aggregation
[0097] Experiment was performed in order to investigate the ethyl
acetate fraction of seoritae of reducing expression of P-selectin,
secretion of serotonin and generation of thromboxane after platelet
aggregation. Experiment for P-selectin and serotonin was performed
in the same manner as in Test Example 6. The inhibition of
thromboxane generation was evaluated by adding the ethyl acetate
fraction to the PRP used in Test Example 1, incubating at
37.degree. C. for 10 minutes, adding collagen (10 .mu.g/mL) and
then further incubating for 6 minutes. Some of the sample was added
to a test tube containing EDTA and indomethacin (final conc.=50
.mu.M) to terminate the reaction. After centrifuging at
12000.times.g for 2 minutes, the supernatant was subjected to
enzyme immunoassay for quantification of the produced
thromboxane.
[0098] The inhibition of P-selectin expression by the ethyl acetate
fraction is shown Table 11, the inhibition of serotonin secretion
in Table 12, and the inhibition of thromboxane generation in Table
13.
TABLE-US-00011 TABLE 11 Concentration (.mu.g/mL) 10 25 50 100
Inhibition of P-selectin 16.2 .+-. 2.3 39.0 .+-. 1.5 58.8. .+-. 0.3
73.7 .+-. 0.2 expression (%)
TABLE-US-00012 TABLE 12 Concentration (.mu.g/mL) 10 25 50 100
Inhibition of serotonin 12.3 .+-. 2.3 40.3 .+-. 2.0 66.8 .+-. 3.1
89.6 .+-. 0.6 secretion (%)
TABLE-US-00013 TABLE 13 Concentration (.mu.g/mL) 10 25 50 100
Inhibition of 2.6 .+-. 0.5 5.4 .+-. 3.7 11.4. .+-. 3.2 38.3 .+-.
3.6 thromboxane generation (%)
[0099] As seen from Tables 11, 12 and 13, the ethyl acetate
fraction of seoritae reduced the expression of P-selectin,
inhibited the secretion of serotonin and inhibited the generation
of thromboxane after platelet aggregation, in a
concentration-dependent manner.
TEST EXAMPLE 12
Inhibition of Ethyl Acetate Fraction Against Blood Clotting in Vein
of SD Rat
[0100] Experiment was performed in the same manner as in Test
Example 7 in order to investigate whether the administration of the
ethyl acetate fraction actually provides blood clotting inhibition
effect in vivo. The oral administration dose was 10, 25 and 50
mg/kg, and the amount of produced blood clots was determined 1 hour
after the administration. The result is shown in FIG. 5.
[0101] As seen from FIG. 5, the oral administration of the ethyl
acetate fraction inhibited blood clotting in a
concentration-dependent manner.
TEST EXAMPLE 13
Inhibition Effect Against Aggregation of Platelets Derived from SD
Rat
[0102] Experiment was performed in the same manner as in Test
Example 1 in order to investigate whether the low-concentration
ethanol extract and the ethyl acetate fraction of seoritae is
effective in inhibiting the aggregation of platelets derived from
SD rat. The treatment concentration was 10, 25, 50 and 100
.mu.g/mL, and the result is given in Table 14.
TABLE-US-00014 TABLE 14 Concentration (.mu.g/mL) 10 25 50 100
Inhibition of Low- 3.7 .+-. 1.2 17.1 .+-. 2.3 36.3 .+-. 5.7 46.5
.+-. 5.4 platelet concen- aggregation tration extract (%) Ethyl
17.1 .+-. 6.9 48.6 .+-. 3.8 66.1 .+-. 2.9 80.0 .+-. 4.4 acetate
fraction
[0103] As seen from Table 14, not only the ethanol
low-concentration extract but also the ethyl acetate fraction
showed very superior effect of inhibiting the aggregation of
platelets derived from SD rat, in a concentration-dependent
manner.
TEST EXAMPLE 14
Observation of Vasoconstriction Inhibition Effect Using Blood
Vessel Ring
[0104] Male SD rats for experiment weighing 250-300 g were acquired
from Daehan Biolink (Seoul, Korea) and kept under the condition of
22.+-.2.degree. C. and 45-55% humidity, with 12/12-hr light/dark
cycles (light from 7 a.m. to 7 p.m.). Feed (Purina Korea, Seoul,
Korea) and water were given freely during an accommodation period
of 1 week.
[0105] After sacrificing the rat by bleeding, the chest was cut
open and the thoracic aorta was taken out quickly and transferred
to KR buffer [composition (mM): NaCl 115.5, KCl 4.6,
KH.sub.2PO.sub.4 1.2, MgSO.sub.4 1.2, CaCl.sub.2 2.5, NaHCO.sub.3
25.0, Ca.sup.2+ EDTA 0.026 glucose 11.1; pH 7.4] saturated with 95%
O.sub.2/5% CO.sub.2 mixture gas. A 3-4 mm long blood vessel ring
was prepared by removing the blood and neighboring adipose and
connective tissues in the blood vessel. After applying tension
gradually to the blood vessel ring for initial 30 minutes until the
equilibrium was reached, it was constricted with 10.sup.-6 M
phenylephrine and then dilated with 10.sup.-6 M acetylcholine. The
one with vasodilation of 80% or more was used. Maximum
vasoconstriction was induced by replacing the buffer in a water
bath with KR buffer containing 90 mM KCl saturated with 95%
O.sub.2/5% CO.sub.2 mixture gas. After pretreating the blood vessel
for 30 minutes with the seoritae extract at different
concentrations, vasoconstriction was induced in the water bath with
phenylephrine of gradually increasing concentrations. The
vasoconstriction induced by phenylephrine and 90 mM KCl is shown in
Table 15 and Table 16.
TABLE-US-00015 TABLE 15 Phenylephrine Seoritae extract
concentration (.mu.M) Control (constriction %) (constriction %) 0.1
15.26 .+-. 7.04 0.39 .+-. 0.27 1 60.35 .+-. 4.83 16.30 .+-. 3.44 10
92.13 .+-. 8.46 40.02 .+-. 7.57 100 97.69 .+-. 10.54 53.68 .+-.
10.08
[0106] As seen from Table 15, the vasoconstriction increased with
the concentration of the phenylephrine. The treatment with the
seoritae extract at 100 .mu.g/mL resulted in a
concentration-dependent vasoconstriction inhibition effect.
TABLE-US-00016 TABLE 16 Seoritae extract (.mu.g/mL) Control 25 50
Constriction (%) 87.55 .+-. 16.01 70.45 .+-. 9.56 59.6 .+-.
6.69
[0107] As seen from Table 16, the treatment with the seoritae
extract at 25 and 50 .mu.g/mL resulted in a concentration-dependent
vasoconstriction inhibition effect. Accordingly, it can be seen
that the 20% ethanol seoritae extract provides very superior
vasoconstriction inhibition effect.
TEST EXAMPLE 15
Lowering of Serum and Liver Lipid Levels in Animal Model
[0108] 8-week-old female rats weighing 250-300 g were kept in
polycarbonate cages, 8 heads per cage, maintained at
22.+-.2.degree. C. and 55.+-.15% relative humidity with 12/12-hr
light/dark cycles. Normal diet or high-cholesterol diet was
provided and water was given freely.
[0109] Fenofibric acid (200 mg/kg) used for treatment of
hyperlipidemia was suspended in 1% methyl cellulose (MC) and orally
administered as a positive control. The seoritae extract (200
mg/kg) was orally administered once a day for 4 weeks. After
fasting for 12 hours, blood sample was taken from the retro-orbital
plexus and centrifuged for 10 minutes at 10000 rcf (relative
centrifugal force). After the centrifugation, total cholesterol,
LDL-cholesterol, HDL-cholesterol and triglyceride levels in the
obtained serum were evaluated.
[0110] The analysis was performed using an automated hematology
analyzer and the Roche diagnostic kit. The result is shown in Table
17 (serum lipids) and Table 18 (liver lipids).
TABLE-US-00017 TABLE 17 (mg/ Seoritae dL) Normal Control Positive
control extract Total 114.25 .+-. 2.41 218.57 .+-. 20.44 112.42
.+-. 13.33 156.42 .+-. 10.21 cho- les- terol HDL- 84.85 .+-. 0.35
55.72 .+-. 3.56 87.89 .+-. 6.84 50.35 .+-. 6.88 cho- les- terol
LDL- 30.5 .+-. 6.42 134.75 .+-. 9.41 75.34 .+-. 2.80 97.21 .+-.
10.44 cho- les- terol
[0111] As seen from Table 17, the control group rats fed with the
high-cholesterol diet for 4 weeks showed about 2 times higher total
cholesterol level and about 4.4 times higher LDL-cholesterol level
in serum as compared to the normal group. This shows that
hyperlipidemia was induced well by the high-cholesterol diet. The
group treated with the seoritae extract as well as the
high-cholesterol diet showed about 28% decreased total cholesterol
level (156.42 mg/dL) and about 28% decreased LDL-cholesterol level
as compared to the control group.
TABLE-US-00018 TABLE 18 Positive Seoritae (.mu.g/mg) Normal Control
control extract Total 5.81 .+-. 0.85 27.96 .+-. 2.46 15.43 .+-.
0.52 13.50 .+-. 7.62 cholesterol Triglyceride 199.91 .+-. 24.33
230.35 .+-. 10.53 185.39 .+-. 23.84 172.32 .+-. 11.16
[0112] Total cholesterol and triglyceride levels in the liver are
shown in Table 18. It was confirmed that fatty liver was induced by
the high-cholesterol diet in the control group. The seoritae
extract-treated group showed 52% decreased total cholesterol level
and 25% decreased triglyceride level. Accordingly, it was confirmed
that the seoritae extract has superior effect of improving serum
and liver lipid levels in vivo.
TEST EXAMPLE 16
Analysis of Active Ingredient in Seoritae Extract Fractions
[0113] In order to analyze the active ingredient of the seoritae
extract, assay-guided fractionation was carried out for the ethyl
acetate fractions. The overall scheme of isolating and purifying
the active ingredient of the ethyl acetate fraction is shown in
FIG. 6.
[0114] Referring to FIG. 6, solid-phase extraction was carried out
for the ethyl acetate fraction A-2 of the 20% ethanol seoritae
extract obtained in Example 25. During the solid-phase extraction,
50-100% (v/v) methanol (MeOH) solution was used as the extraction
solvent. The solid-phase extracted extract was fractionated to A-3
through A-9. Platelet aggregation was evaluated for the extract,
and the result is shown in FIG. 7. As seen from FIG. 7, the
extracts A-3 and A-4 showed relatively low platelet
aggregation.
[0115] A mixture of the extracts A-3 and A-4 that showed relatively
low platelet aggregation was subjected to column chromatography
(Sephadex LH 20 column chromatography) for separation of the
components. The separated components were numbered from A-3-1 to
A-3-10. Platelet aggregation was evaluated again for A-3-1 through
A-3-10, and the result is shown in FIG. 8. As seen from FIG. 8, the
sample A-3-1 showed the lowest platelet aggregation among A-3-1
through A-3-10.
[0116] The sample A-3-1 that showed the lowest platelet aggregation
was subjected to high-performance liquid chromatography
(preparative ODS HPLC) for separation of the active ingredients.
The separated components were numbered from A-3-1-1through A-3-1-7.
Platelet aggregation was evaluated for the components, and the
result is shown in FIG. 9. As seen from FIG. 9, A-3-1-3 showed the
lowest platelet aggregation. To conclude, A-3-1-3 showed the best
platelet aggregation inhibition activity.
[0117] The platelet aggregation inhibition activity of A-3-1-3 was
compared with that of standard adenosine. The result is shown in
FIG. 10. As seen from FIG. 10, A-3-1-3 showed almost the same
effect as that of adenosine, suggesting that A-3-1-3 comprises
adenosine. The chemical structure of adenosine is as follows:
##STR00001##
TEST EXAMPLE 17
Measurement of Adenosine Content in 20% Ethanol Seoritae
Extract
[0118] The activity of the 20% ethanol seoritae extract was
compared with that of standard adenosine, and the result is shown
in FIG. 11 and FIG. 12. Referring to FIG. 11 and FIG. 12, the
content of adenosine in the 20% ethanol seoritae extract is
calculated as 0.35-0.5% from the comparison with the standard
adenosine. That is to say, the seoritae extract extracted using the
low-concentration, lower alcohol has a very high adenosine content.
In addition, the blood clotting inhibition of the seoritae extract
is better than that of adenosine alone. Accordingly, a more stable
inhibition of blood clotting can be expected from the seoritae
extract as compared to when adenosine is used alone.
TEST EXAMPLE 18
Measurement of Adenosine Content in 20% Ethanol Bean Extracts
[0119] Adenosine content in the 20% ethanol extracts of various
beans including seoritae was measured. The result is given in Table
19.
TABLE-US-00019 TABLE 19 Beans Adenosine content (%) Seoritae 0.36
Seomoktae 0.20 Blue bean 0.19 Yellow bean 0.16 Kidney bean 0.29
Sprouting bean 0.15 Field bean 0.34
[0120] As seen from Table 19, the adenosine content was high with
0.15% or above in various beans. In particular the seoritae and
field bean extracts showed relatively high adenosine content.
TEST EXAMPLE 19
Blood Clotting Inhibition Activity of Seoritae (Glycin max MERR)
Extract in Red Blood Cells
[0121] It was investigated whether the bean extract affects the
exposure of red blood cells to phosphatidylserine (hereinafter, PS)
and the generation of microvesicles (hereinafter, MV) by
lysophosphatidic acid (hereinafter, LPA) and phosphatidic acid
(hereinafter, PA) which are produced during inflammation.
[0122] In general, it is known that blood clotting is facilitated
when red blood cells are exposed to PS or MV is generated. After
treating with the seoritae extract in inflammatory state,
inhibition of PS exposure and MV generation induced by LPA and PA
were measured. Specifically, 1 hour after adding the seoritae
extract (100 .mu.g/mL), 50 .mu.M LPA or 25 .mu.M PA was added for
15 minutes to induce PS exposure and MV generation. Then, the
degree of PS exposure and MV generation was compared between the
case where the seoritae extract was treated (+) and untreated (-).
The degree of PS exposure is shown in FIG. 13, and the degree of MV
generation is shown in FIG. 14.
[0123] As seen from FIGS. 13 and 14, the seoritae extract inhibited
the PS exposure and MV generation induced by LPA and PA.
[0124] Further, the degree of thrombin generation was measured by
prothrombinase assay from the change in procoagulant activity
mediated by PS exposure. The result is shown in FIG. 15. In the
figure, (+) is the case where the seoritae extract was treated, and
(-) is the case where it was untreated. As seen from FIG. 15, the
treatment with the seoritae extract inhibited the thrombin
generation by LPA and PA.
TEST EXAMPLE 20
Inhibition of Blood Clotting by Seoritae Extract
[0125] Coagulation is a procedure whereby blood clots are formed as
coagulation factors which are zymogens are sequentially activated
to turn fibrinogen to fibrin. An intrinsic pathway also called the
contact system and an extrinsic pathway initiated by the activation
of tissue factors lead to the formation of blood clots.
[0126] The effect of the 20% ethanol seoritae extract on the
extrinsic pathway or the intrinsic pathway of the coagulation
cascade was evaluated by the prothrombin time (PT) and the
activated partial thromboplastin time (hereinafter, aPTT).
[0127] PT was measured as follows. Blood was taken from the rat and
centrifuged using 3.8% sodium citrate as anticoagulant. After
adding RecombiPlasTin to the supernatant, the time to fibrin
formation was measured using a fibrinometer. A longer time means
better inhibition of blood clotting.
[0128] aPTT was measured as follows. Blood was taken from the rat
and centrifuged using 3.8% sodium citrate as anticoagulant. After
adding 20 mM calcium chloride (CaCl.sub.2) solution to the
supernatant, 100 .mu.L of the serum was taken and the time to
fibrin formation was measured using a fibrinometer.
[0129] The PT measurement result is shown in FIG. 16, and the aPTT
measurement result is shown in FIG. 17.
[0130] As seen from FIG. 16 and FIG. 17, the 20% ethanol seoritae
extract had no effect on PT or aPTT. Accordingly, since the
seoritae extract does not affect the normal coagulation of blood,
it will not result in the side effects of the known anticoagulants
(e.g., aspirin) such as interruption of hemostasis or excessive
bleeding.
TEST EXAMPLE 21
Effect of Seoritae (Glycin max MERR) Extract on Bleeding Time
[0131] Bleeding time was compared for the cases where 20% ethanol
seoritae extract, aspirin or clopidogrel was treated. The result is
shown in FIG. 18.
[0132] As seen from FIG. 18, aspirin or clopidogrel resulted in
significant increase of bleeding time, whereas the seoritae extract
resulted in little change in bleeding time. The bleeding time was
almost unchanged even when the treatment amount was increased
5-fold from 100 mg/kg to 500 mg/kg. Accordingly, it can be seen
that side effects such as increased bleeding time do not occur even
when the administration dose or treatment amount is increased.
[0133] Hereinafter, the formulation examples of compositions
comprising the bean extract according to the present disclosure
will be described in detail. The following examples are for
illustrative purposes only and not intended to limit the scope of
the present disclosure.
FORMULATION EXAMPLE 1
Soft Capsule
[0134] Seoritae extract (100 mg) was mixed with soybean extract (50
mg), soybean oil (180 mg), red ginseng extract (50 mg), palm oil (2
mg), hydrogenated palm oil (8 mg), beeswax (4 mg) and lecithin (6
mg), and filled in a soft capsule according to a commonly employed
method.
FORMULATION EXAMPLE 2
Tablet
[0135] Field bean extract (100 mg) was mixed with soybean extract
(50 mg), glucose (100 mg), red ginseng extract (50 mg), starch (96
mg) and magnesium stearate (4 mg). After forming granules by adding
30% ethanol (40 mg), followed by drying at 60.degree. C., a tablet
was prepared using a tablet making machine.
FORMULATION EXAMPLE 3
Granule
[0136] Seoritae extract (100 mg) was mixed with soybean extract (50
mg), glucose (100 mg), red ginseng extract (50 mg) and starch (600
mg). After forming granules by adding 30% ethanol (100 mg),
followed by drying at 60.degree. C., the granules were filled in a
pouch. The final weight was 1 g.
FORMULATION EXAMPLE 4
Drink
[0137] Seoritae extract (100 mg) was mixed with soybean extract (50
mg), glucose (10 g), red ginseng extract (50 mg), citric acid (2 g)
and purified water (187.8 g), and filled in a bottle. The final
volume was 200 mL.
FORMULATION EXAMPLE 5
Health Food
TABLE-US-00020 [0138] Seoritae extract 1000 mg Vitamins Vitamin A
acetate 70 .mu.g Vitamin E 1.0 mg Vitamin B.sub.1 0.13 mg Vitamin
B.sub.2 0.15 mg Vitamin B.sub.6 0.5 mg Vitamin B.sub.12 0.2 .mu.g
Vitamin C 10 mg Biotin 10 .mu.g Nicotinamide 1.7 mg Folic acid 50
.mu.g Calcium pantothenate 0.5 mg Minerals Ferrous sulfate 1.75 mg
Zinc oxide 0.82 mg Magnesium carbonate 25.3 mg Potassium phosphate
monobasic 15 mg Calcium phosphate dibasic 55 mg Potassium citrate
90 mg Calcium carbonate 100 mg Magnesium chloride 24.8 mg
[0139] The exemplary contents of the vitamins and minerals in the
health food may be changed as desired. The above ingredients were
mixed and prepared into granules according to the commonly employed
health food preparation method for use in the preparation of the
health food composition.
FORMULATION EXAMPLE 6
Health Drink
TABLE-US-00021 [0140] Field bean extract 1000 mg Citric acid 1000
mg Oligosaccharide 100 g Concentrated plum extract 2 g Taurine 1 g
Purified water to make 900 mL
[0141] The above ingredients were mixed and heated at 85.degree. C.
for about 1 hour with stirring according to the commonly employed
health drink preparation method. The resulting solution was put in
a sterilized 2-L container, sealed and sterilized, and then kept in
a refrigerator for use in the preparation of the health drink
composition.
[0142] The composition of the health drink may be changed
considering regional or ethnic preferences, such as particular
customers, country, purpose of use, or the like.
[0143] The composition according to the present disclosure may be
widely applicable in the field of medicine, food and others.
* * * * *