U.S. patent application number 15/580565 was filed with the patent office on 2018-06-21 for composition for inhibiting and preventing myopathy, containing bean leaf extract as active ingredient.
This patent application is currently assigned to AMOREPACIFIC CORPORATION. The applicant listed for this patent is AMOREPACIFIC CORPORATION. Invention is credited to Hee Young JEON, Young-Gyu KANG, Byung Gyu KIM, Soo Hyun KIM, Chan Woong PARK.
Application Number | 20180169166 15/580565 |
Document ID | / |
Family ID | 57607808 |
Filed Date | 2018-06-21 |
United States Patent
Application |
20180169166 |
Kind Code |
A1 |
KIM; Byung Gyu ; et
al. |
June 21, 2018 |
COMPOSITION FOR INHIBITING AND PREVENTING MYOPATHY, CONTAINING BEAN
LEAF EXTRACT AS ACTIVE INGREDIENT
Abstract
The present invention relates to a composition for inhibiting
and preventing myopathy having a bean leaf extract as an active
ingredient. The composition has an effect of inhibiting the
overexpression of Atrogin1 and Murf1 specifically expressed in
myocyte and restoring cell activity of myocyte to inhibit and
prevent muscle loss by including the extract of bean leaf at stage
R6 to R8 during the growth stage of bean.
Inventors: |
KIM; Byung Gyu; (Yongin-si,
Gyeonggi-do, KR) ; KANG; Young-Gyu; (Yongin-si,
Gyeonggi-do, KR) ; KIM; Soo Hyun; (Yongin-si,
Gyeonggi-do, KR) ; PARK; Chan Woong; (Yongin-si,
Gyeonggi-do, KR) ; JEON; Hee Young; (Yongin-si,
Gyeonggi-do, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AMOREPACIFIC CORPORATION |
Seoul |
|
KR |
|
|
Assignee: |
AMOREPACIFIC CORPORATION
Seoul
KR
|
Family ID: |
57607808 |
Appl. No.: |
15/580565 |
Filed: |
May 9, 2016 |
PCT Filed: |
May 9, 2016 |
PCT NO: |
PCT/KR2016/004828 |
371 Date: |
December 7, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61Q 19/00 20130101;
A61K 8/9789 20170801; A61K 36/48 20130101; A61K 2236/39 20130101;
A61K 8/96 20130101; A61P 21/00 20180101; A61K 2236/51 20130101 |
International
Class: |
A61K 36/48 20060101
A61K036/48; A61K 8/96 20060101 A61K008/96; A61Q 19/00 20060101
A61Q019/00; A61P 21/00 20060101 A61P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 30, 2015 |
KR |
10-2015-0092701 |
Claims
1. A composition for inhibiting and preventing myopathy comprising
a bean leaf extract as an active ingredient.
2. The composition for inhibiting and preventing myopathy according
to claim 1, wherein the myopathy is at least one selected from the
group consisting of sarcopenia, muscle loss and amyotrophic
diseases.
3. The composition for inhibiting and preventing myopathy according
to claim 1, wherein the myopathy is senile myopathy.
4. The composition for inhibiting and preventing myopathy according
to claim 1, wherein the composition increases cell activity of
myocyte.
5. The composition for inhibiting and preventing myopathy according
to claim 1, wherein the composition inhibits expression or activity
of ubiquitin ligase of skeletal muscle.
6. The composition for inhibiting and preventing myopathy according
to claim 1, wherein the composition inhibits expression of Atrogin1
or Murf1 gene.
7. The composition for inhibiting and preventing myopathy according
to claim 1, wherein the bean leaf extract is an extract of bean
leaf corresponding to any one of stage R6 to R8 during the growth
stage of bean.
8. The composition for inhibiting and preventing myopathy according
to claim 7, wherein the extract of bean leaf is an extract of bean
leaf at stage R7 during the growth stage of bean.
9. The composition for inhibiting and preventing myopathy according
to claim 7, wherein the bean is at least one selected from the
group consisting of Rhynchosia Nolubilis, Glycine max(L.) Merr.,
Vicia faba, Phaseolus vulgaris, Phaseolus vulgaris L., Vigna
angularis, Phaseolus angularis W.F. WIGHT., Pisum sativum L. and
Glycine max MERR.
10. The composition for inhibiting and preventing myopathy
according to claim 1, wherein the bean leaf extract is extracted by
a solvent selected from the group consisting of water, an organic
solvent or a mixture thereof.
11. The composition for inhibiting and preventing myopathy
according to claim 1, wherein the bean leaf extract is extracted
sequentially by ethanol and ethyl acetate.
12. The composition for inhibiting and preventing myopathy
according to claim 1, wherein the bean leaf extract is obtained by
a method which comprises the steps of: extracting bean leaf with
water, an organic solvent or a mixture thereof to obtain the first
extract; adding an organic solvent to the first extract to obtain
the second extract; and concentrating under reduced pressure and
drying the final extract containing the first and second
extracts.
13. The composition for inhibiting and preventing myopathy
according to claim 1, wherein the bean leaf extract is at least one
selected from the group consisting of extract of raw bean leaf, and
fractions, dried products, dried fractions, fermented products and
concentrates thereof.
14. The composition for inhibiting and preventing myopathy
according to claim 1, wherein the composition is any one of a food
composition, a pharmaceutical composition and a cosmetic
composition.
15. The composition for inhibiting and preventing myopathy
according to claim 14, wherein the pharmaceutical composition is a
composition of an oral formulation.
Description
TECHNICAL FIELD
[0001] This application claims the benefit of priority based on
Korean Patent Application No. 10-2015-0092701, filed on Jun. 30,
2015, the entire contents of which are incorporated herein by
reference.
[0002] The present invention relates to a composition for
inhibiting and preventing myopathy comprising bean leaf extract as
an active ingredient, which can be usefully utilized in the field
of preparing food, pharmaceutical or cosmetic composition for
myopathy.
BACKGROUND ART
[0003] Human muscles grow and become strong until the age of 30
after the birth of human, but after 30 years of age, muscle density
and function gradually begin to weaken. In particular, if there is
no consistent physical exercise, after 30 years of age, the amount
of muscle is reduced by 3 to 5% every 10 years. And even if the
exercise continues, a certain degree of muscle loss occurs. Most of
these muscle losses are a phenomenon mainly caused by aging, but
the detailed mechanism of such a phenomenon has various
aspects.
[0004] Unlike muscle loss due to normal simple aging, sarcopenia by
the pathological aspect directly induces muscle weakness and
increases the risk of falls or fractures, also causes an outbreak
of myopathy and decrease and impairment in various body functions,
and increases risk of diabetes and cardiovascular diseases by 8.2
times or more, thereby generally increasing risk of death.
[0005] Therefore, it has been considered that the inhibition and
prevention of muscle loss is one of the important challenges
directly linked to improvement and prevention of various other
organically related muscular diseases as well as to general health
problems. Also, at present, the population of Korea is rapidly
aging due to the decrease in fertility rate and the extension of
average life expectancy, and in particular, according to the data
of the Korean National Statistical Office in 2010, the elderly
population aged 65 or older is expected to reach 15.7% in 2020 and
24.1% in 2030 compared to 9.1% in 2005. This increase corresponds
to the fastest growth rate among OECD countries and is also
classified as an urgent challenge in terms of social welfare.
[0006] According to this trend, many studies are under way to
inhibit and prevent muscle loss. Among them, the representative
study is the study related to branched-chain amino acid (BCAA)
containing leucine, one of the essential amino acids that make up
muscles. However, according to this study, leucine was reported to
have little effect on skeletal muscle mass changes in the elderly.
Thus, it is expected that this study will be difficult to become a
fundamental solution of the sarcopenia.
[0007] Meanwhile, recently, specific functions of Atrogin1 and
Murf1 have been elucidated with regard to the sarcopenia, and they
are attracting attention. The Atrogin1 and Murf1 are ubiquitin
ligases specifically expressed in myocyte, and this increase in
expression is known to cause muscle loss due to ubiquitination and
proteasome-dependent degradation of muscle proteins.
[0008] In the process of finding ways to solve muscle loss from a
new perspective, the present inventors have found that using a
composition comprising a bean leaf extract as an active ingredient
in relation to the above mechanism has a considerable effect on the
inhibition of muscle loss, and thus completed the present
invention.
PRIOR ART LITERATURE
[0009] Formulations for prevention or treatment of obesity,
hyperlipidemia, arteriosclerosis, fatty liver, diabetes mellitus or
metabolic syndrome comprising extracts of Glycine max leaves as an
active ingredient (Korean Patent Laid-Open No.
10-2012-0022085).
Disclosure
Technical Problem
[0010] In order to solve the above problems, it is an object of the
present invention to provide a composition for inhibiting and
preventing myopathy which comprises an bean leaf extract as an
active ingredient.
[0011] More particularly, it is an object of the present invention
to provide a substance capable of inhibiting and preventing muscle
loss.
[0012] In addition, it is another object of the present invention
to provide a food composition, a pharmaceutical composition or a
cosmetic composition for inhibiting and preventing myopathy
comprising naturally derived extract of bean leaf, which is easy to
obtain and has minimal side effects, as an active ingredient.
Technical Solution
[0013] In order to achieve the above object, the present invention
provides a composition for inhibiting and preventing myopathy
comprising a bean leaf extract as an active ingredient.
[0014] The bean leaf extract may be an extract of bean leaf at
stage R6 to R8 during the growth stages of bean.
[0015] The bean leaf extract may be at least one selected from the
group consisting of a raw extract of bean leaf, and a fraction, a
dried product, a dried fraction, a fermented product and a
concentrate thereof.
[0016] The composition can be utilized in the form of food,
pharmaceutical or cosmetic composition.
Advantageous Effects
[0017] The present invention provides a composition capable of
inhibiting and preventing myopathy through the solution of the
above problem.
[0018] More specifically, the present invention provides a
substance capable of directly or indirectly inhibiting and
preventing muscle loss by providing a substance capable of
improving or preventing various myopathy which comprises an extract
of bean leaf as an active ingredient.
[0019] The bean leaf extract is a natural component, has few side
effects on the human body and is easy to obtain. In addition it is
economical to prepare because it is made by fully utilizing bean
leaf that has not been used industrially in the prior art.
DESCRIPTION OF DRAWINGS
[0020] FIG. 1 shows the results of comparative analysis of
components of (a) bean leaf extract and (b) bean extract by using
HPLC.
[0021] FIG. 2 is a graph showing relative cell activity (%) of the
experimental group, the control group and the comparison group in
comparison with the normal myocyte (control).
[0022] FIG. 3 is a graph showing relative expression amounts (%) of
Atrogin1 gene (mRNA) of the experimental group, the control group
and the comparison group in comparison with normal myocyte
(control).
[0023] FIG. 4 is a graph showing relative expression amounts (%) of
Murf1 gene (mRNA) of the experimental group, the control group and
the comparison group in comparison with normal myocyte
(control).
BEST MODE
[0024] The present invention provides a composition for inhibiting
and preventing myopathy comprising a bean leaf extract as an active
ingredient.
[0025] The term "myopathy" used herein should be understood to
include at least all of the myopathies directly or indirectly
related to the expression of the Atrogin1 or Murf1 protein as
described below. The term "myopathy" refers to, but is not limited
to, all myopathies, which are accompanied by, or directly or
indirectly related mainly to sarcopenia, muscle loss and
amyotrophic diseases.
[0026] Hereinafter, the contents of the present invention will be
described in more detail. It is to be understood, however, that the
following describes only the most representative embodiments in
order to facilitate understanding of the present invention, and the
scope of the present invention is not limited thereto, and covers
the entire scope equivalent to the following.
[0027] In the present invention, the term "bean" is not limited in
its kind and may be, for example, but is not limited to, at least
one selected from the group consisting of Rhynchosia Nolubilis,
Glycine max(L.) Merr., Vicia faba, Phaseolus vulgaris, Phaseolus
vulgaris L., Vigna angularis, Phaseolus angularis W.F. WIGHT.,
Pisum sativum L. and Glycine max MERR. In addition, shape of the
bean can be any form suitable for extracting the active
ingredient.
[0028] However, an object of extraction in the present invention is
limited to "bean leaf." The reason for this is that, as shown in
Example 1 and FIG. 1 to be described later, the constituents of
bean and bean leaf are different to each other. And the effect of
inhibition and prevention of the myopathy is significant in the
bean leaf extract compared with the bean extract. In addition, the
present invention is economical and easy to prepare because it can
utilize the bean leaf which are conventionally less industrially
utilized and discarded.
[0029] In order to enhance the inhibition and prevention of
myopathy, the bean leaf extract is an extract of bean leaf
corresponding to any one of stage R6 to R8 during the whole growth
stage of bean as follows.
[0030] Stage VE: 1 to 2 Weeks after seeding. Cotyledons emerge from
the soil
[0031] Stage VC: The cotyledons spread out, one node of stem grows
on it, and outer leaves are come into.
[0032] Stage V1: One node is generated from the first outer leaves
and three leaves are created.
[0033] Stage V2: One node is further created at the stage V1 and
three leaves are further created.
[0034] Stage V3: One node is further created at the stage V2 and
three leaves are further created.
[0035] Stage V4: One node is further created at the stage V3 and
three leaves are further created.
[0036] Stage R2: A state where the flowers of bean are in full
bloom.
[0037] Stage R4: A state where the creation of bean pods is
completed.
[0038] Stage R5: A state where beans are formed in the bean
pods.
[0039] Stage R6: A state where green beans have been produced in
the bean pods.
[0040] Stage R7: A state where the bean pods and beans turn
yellow.
[0041] Stage R8: A state where the bean pods and beans become
completely yellow as the leaves fall.
[0042] The bean leaf extract at stage R7 to R8 is preferable, and
the bean leaf extract at stage R7 is more preferable. The bean leaf
at stage R7 to R8 are sometimes referred to as "autumn bean leaf"
because they are bean leaf at a stage in which the color of the
leaf changes to yellow.
[0043] In this specification, the term "extract" means a component
substance extracted from the natural material, regardless of the
extraction method, the extraction solvent, the extracted components
or the form of the extract. For example, the extract is a broad
concept, including all the extract, for example, extracts of
components dissolved in a solvent from natural materials using
water or an organic solvent, and those obtained by extracting only
a specific component of the natural material, i.e., a specific
component such as oil and also includes all of the substances
obtained by, for example, processing after extraction.
Specifically, the extract of the present invention may be at least
a form selected from the group consisting of extract of raw bean
leaf, additional processed or fermented or enzymatically treated
fractions, dried products, dried fractions, fermented products and
concentrates.
[0044] The method of obtaining the bean leaf extract of the present
invention is not limited to any particular method as long as it is
a commonly used method in the art. For example, the bean leaf
extract can be normally obtained by the method comprising placing
the powder of washed, dried and pulverized bean leaves in water or
organic solvent, separating the residue and filtrate after
extraction and precipitation by filtration and centrifugation, and
concentrating the separated filtrate under reduced pressure.
[0045] A solvent for the extraction may be at least one selected
from water, ethanol, methanol, butanol, ether, ethyl acetate,
chloroform or a mixture of these organic solvents and water. And it
is preferable to use water or ethanol having a concentration of 30
to 70% in consideration of the safety of raw materials. It is
effective to use a mixture of each filtrate obtained through
repeated extraction. For example, after obtaining the first extract
from ethanol as described above, ethyl acetate was added to the
residue to obtain the second extract, and further extraction was
repeated once or twice for the residue from the previous step, and
then the filtrates of the extracts thus obtained are mixed.
[0046] After obtaining the extract, a liquid substance therefrom
can be obtained by chilling at room temperature, heating and
filtration which are conventional methods known in the art, or the
process of evaporating, spray drying or lyophilizing the solvent
may be further performed.
[0047] There is no particular limitation on the concentration of
extract. However, referring to Example 2 or Experimental Examples 1
and 2 to be described later, the effect is usually sufficient at
around 100 ppm. And specifically, various concentrations are
available depending on the symptom and degree of the disease and
the mode of providing the composition.
[0048] The bean leaf extract of the present invention inhibits
expression or activity of ubiquitin ligase of skeletal muscle to
inhibit and prevent myopathy. Generally, the ubiquitin ligase in
the human body is an enzyme that induces degradation of the protein
by attaching ubiquitin to the surface of the protein to be
destroyed as a kind of death signal. In particular, there are
ubiquitin ligases, called Atrogin1 and Murf1, which are
specifically expressed when the skeletal muscle is reduced.
However, unlike conventional ubiquitin ligases that induce the
death of unnecessary proteins, the Atrogin1 and Murf1 tend to be
overexpressed in patients with myopathy accompanied mainly by
pathological muscle loss or atrophy and are now known to be one of
the direct molecular biologic causes that cause the sarcopenia.
When the Atrogin1 and Murf1 are overexpressed, the degradation
pathway is activated too much as compared with the synthesis of
myocyte, and thus normal myocyte loss occurs.
[0049] In connection with the above mechanism, the bean leaf
extract of the present invention may prevent muscle loss and may
further improve and prevent other myopathy organically associated
therewith by inhibiting the overexpression of the Atrogin1 and
Murf1 genes.
[0050] Considering that the majority of muscle loss diseases are
classified as a type of degenerative disease in accordance to
aging, the composition of the present invention can be positively
utilized for a senior group.
[0051] The composition of the present invention may be provided in
any one formulation of food, pharmaceutical and cosmetic
compositions.
[0052] The food composition of the present invention, which is one
form of utilization, is mainly a composition of a health functional
food, may contain the bean leaf extract as an active ingredient and
may be blended into a conventional food composition as it is or
with other food or a component of such food. There is no particular
limitation on the type of the above-mentioned food and the food may
include beverages, tea, drinks, alcoholic beverages, vitamin
complexes and the like and they may be used in any form that is
commonly recognized.
[0053] The pharmaceutical composition of the present invention,
which is another form of utilization, includes the bean leaf
extract as an active ingredient and may further include at least
one other pharmaceutically acceptable carrier. For example, saline,
sterile water, Ringer's solution, buffered saline, dextrose
solution, maltodextrin solution, glycerol, ethanol or a mixture of
two or more thereof can be used, and other conventional additives
such as an antioxidant, a buffer, a bacteriostatic agent and the
like may be added as needed.
[0054] The pharmaceutical composition can be formulated in
injectable formulation such as aqueous solutions, suspensions,
emulsions and the like or oral formulation by additionally adding
diluents, dispersants, surfactants, binders and lubricants.
[0055] The dosage formulation for oral administration may be at
least one selected from the group consisting of tablets, pills,
granules, fine granules, pulvis, powders, soft capsules, hard
capsules, emulsions, syrups, and drinks.
[0056] The cosmetic composition of the present invention, which is
still another form of utilization, includes the bean leaf extract
as an active ingredient and may further comprise the other
functional additives and all components which may be included in
the general cosmetic composition.
[0057] The functional additive may be any one or more of, for
example, water-soluble vitamins, oil-soluble vitamins, polymer
peptides, polymeric polysaccharides, sphingolipids and seaweed
extracts. Also, if necessary, it is possible to additionally add
oil and fat components, moisturizers, emollient agents,
surfactants, organic and inorganic pigments, organic powder,
ultraviolet absorbers, antiseptics, bactericides, antioxidants,
plant extracts, pH adjusting agents, alcohol, coloring matters,
flavoring agents, blood circulation promoting agents, cool-feeling
agents, antiperspirant agents, purified water and the like.
[0058] The formulation of the cosmetic composition can be
appropriately selected according to application characteristics,
and may be, for example, in the formulation of skin lotion, skin
softener, skin toner, astringent, lotion, milk lotion, moisturizing
lotion, nutrition lotion, massage cream, nutrition cream,
moisturizing cream, hand cream, foundation, powder, essence,
nutritional essence, pack, soap, cleansing foam, cleansing lotion,
cleansing cream, body lotion and body cleanser, spray and the
like.
[0059] Hereinafter, Examples 1 to 2 and Experimental Examples 1 to
2 according to the present invention are described. The following
Examples and Experimental Examples are only examples related to the
practice and effect of the present invention, and the scope and
effect of the present invention are not limited thereto.
EXAMPLE 1
Comparison of Components of Bean Leaf Extract and Bean Extract
[0060] To confirm that constituents of bean leaf extract and bean
extract are not the same, component analysis using HPLC (high
performance liquid chromatography) was performed.
[0061] HPLC Analysis Condition
[0062] The bean leaf extract and the bean extract were dissolved in
70% ethanol to make 10,000 ppm solution. Thereafter, HPLC (Waters
2695 model) was used and component analysis was performed using a
detector (Waters 2996 PDA detector). The stationary phase was a
Mightysil RP-18 GP 250-4.6 (5 .mu.m) column from Kanto Chemical
Co., and the mobile phase was a mixture of water and 0.1% acetic
acid solution to acetonitrile.
[0063] HPLC Analysis Result
[0064] The results of the HPLC component analysis are shown in Fig.
In FIG. 1, (a) is the bean leaf extract and (b) is the bean
extract.
[0065] According to FIG. 1, the bean leaf extract and the bean
extract exhibit different separation patterns. It can be clearly
seen that the peaks appear to be totally different at each time
point, and thus the constituents of the bean extract and the bean
leaf extract are not the same with each other.
EXAMPLE 2
Preparation of Bean Leaf Extract
[0066] Each of bean leaf collected at the growing stage (stage
VC/V2/V4/R2/R4/R6/R7, 7 stages in total) was washed with purified
water, dried and then pulverized. 100 g of the powder of bean leaf
was added to 1 liter of a 70% by weight aqueous ethanol solution,
extracted at room temperature (25.degree. C.) for 12 hours, and
then filtered through a 300-mesh filter cloth.
[0067] The extract was put into a 3 liters separatory funnel, and 1
liter of ethyl acetate was added, and thereafter stirred to mix
while shaking, and then take the upper layer (ethyl acetate layer)
when completely separated into two layers. The lower layer is
extracted twice again with a separatory funnel. Each of the
separated upper layers was combined and concentrated under reduced
pressure to 50 by using a distillation apparatus equipped with a
cooling condenser and dried. Thus, extracts of bean leaves of each
step were combined to give 10.3 g.
EXPERIMENTAL EXAMPLE 1
Evaluation for Ability to Recover Cell Activity of Myocyte
[0068] In the present Experimental Example 1, the experiments were
conducted to evaluate the recovery efficacy of the bean leaf
extract on the decrease in cell activity of myocyte in a model of
muscle loss induced by Dexamethasone.
[0069] Dexamethasone is a kind of glucocorticoid, and increases
expression of the Atrogin1 and Murf1 in vitro and in vivo to
accelerate proteolytic degradation, thereby resulting in decrease
in skeletal muscle, induction of myocyte death, and reduction of
cell activity. Accordingly, an experimental model of dexamethasone
to myocyte is a useful model for evaluating the function of
sarcopenia and is widely used.
[0070] The C2C12 (mouse myoblast) used in Experimental Example 1
was purchased from American Type Culture Collection (Manassas, Va.,
USA) and the cells were cultured in DMEM containing 10% FBS, 100
units/mL Penicillin, and 100 mg/mL streptomycin. Specific
experimental methods are as follows.
[0071] First, to prepare the experimental group, C2C12 was plated
on a 12-well plate and cultured until the cells were confluent at
80 to 90% of the plate. Subsequently, the differentiation of the
cells was induced by exchanging with 2% horse serum and DMEM
medium. After the medium was changed and then cultivation was
further performed for 5 to 7 days to complete the differentiation
of the cells, the final concentration of dexamethasone was adjusted
to 1 .mu.M. At the same time, the extracts of bean leaves at stage
VC/V2/V4/R2/R4/R6/R7 obtained in Example 2 were adjusted to a final
concentration of 100 ppm.
[0072] Meanwhile, as a control group, the cells without any
treatment other than dexamethasone were used, and as a comparison
group, the cells treated with dexibuprofen at a final concentration
of 100 .mu.M instead of the extracts of bean leaf, which is known
to be effective for suppressing inflammation and effective for
muscle pain, were used.
[0073] After 24 hours of treatment with the material, the medium
was replaced with medium containing 10% solution of CCK8
(cholecystokinin 8), and allowed to react for 10 minutes, and the
cell activity was measured by measuring the absorbance at 450 nm.
To increase the reliability, each sample was repeatedly measured
three times to derive the average value.
[0074] The results of the experiment are shown in FIG. 2 and Table
1 below.
TABLE-US-00001 TABLE 1 Comparison Experimental group group
Treatment VC V2 V4 R2 R4 R6 R7 Dexibuprofen material Change 0.4
-1.3 -2.0 2.5 6.2 9..1 18.1 -4.1 in cell activity relative to
control group (%)
[0075] FIG. 2 is a graph showing relative cell activities of
experimental group, control group (Dex) and comparison group
(dexibuprofen) relative normal myocyte (control). Table 1 shows
change in cell activity of the experimental group and the
comparison group relative to the control group.
[0076] Referring to FIG. 2, it is confirmed that the control group
treated with the dexamethasone has decreased myocyte activity in
comparison with the normal myocyte. Compared with this control
group, the experimental group treated with the bean leaf extracts
shows that the activity of myocyte is restored as a whole.
[0077] Specifically, as shown in Table 1, the experimental groups
treated with the extract of bean leaf at stage R4 to R7 show a
significant increase in the activity of myocyte by 6.2 to 18.1%
compared to the control group. Especially, the extract of bean leaf
at stage R7 shows very effective cell activity recovery.
[0078] Thus, it is confirmed that the bean leaf extract of the
present invention has inhibition and prevention efficacy of muscle
loss.
[0079] Meanwhile, in the group treated with the dexibuprofen as a
comparison group, no recovery of myocyte activity is observed, and
thus it can be seen that common muscle soreness inhibitors are not
suitable for inhibition or prevention of muscle loss, and their
mechanisms of action are also different with each other.
EXPERIMENTAL EXAMPLE 2
Evaluation of Inhibition of Expression of Atrogin1 and Murf1 Gene
(mRNA)
[0080] In the present Experimental Example 2, experiments were
conducted to evaluate the inhibitory efficacy of the bean leaf
extract against the overexpression of Atrogin1 and Murf1 mRNA in
the muscle loss induced model by dexamethasone.
[0081] The experimental group, the control group and the comparison
group of Experimental Example 2 were prepared in the same manner as
Experimental Example 1.
[0082] Provided that, when 6 hours had elapsed since the treatment
with each final material, RNA was extracted with trizol reagent
(TRIzol agent, Invitrogen) after washing twice with cold
saline,
[0083] Subsequently, cDNA was synthesized using 1 .mu.g/.mu.l of
the extracted and quantified RNA and a reverse transcription system
(Promega). Expression patterns of each gene were measured using
primers and probes (Applied biosystems) previously designed for
genes of the synthesized cDNA and Atrogin1, Murf1, and GAPD. At
this time, polymerase chain reaction (PCR) and analysis were
performed using a Rotor-Gene 3000 system (Corbett Research, Sydney,
Australia). To increase the reliability, each sample was repeatedly
measured three times to derive the average value.
[0084] The experimental results are shown in FIGS. 3 and 4 and
Table 2 and Table 3 below.
TABLE-US-00002 TABLE 2 Comparison Experimental group group
Treatment material VC V2 V4 R2 R4 R6 R7 Dexibupropene Change in
Atrogin1 -2.0 -4.7 -0.6 -8.2 -14.9 -16.2 -31.6 -2.6 gene expression
relative to control group (%)
TABLE-US-00003 TABLE 3 Comparison Experimental group group
Treatment material VC V2 V4 R2 R4 R6 R7 Dexibupropene Change in
-4.2 -2.4 -8.5 -9.9 -13.7 -17.5 -24.1 -3.3 Murf1 gene expression
relative to control group (%)
[0085] FIGS. 3 and 4 are graphs showing relative amounts of the
gene (mRNA) expression of Atrogin1 and Murf1 of experimental group,
control group (Dex) and comparison group (dexibuprofen) relative to
normal myocyte (control). Tables 2 and 3 show change in the
Atrogin1 and Murf1 gene (mRNA) expression of the experimental group
and the comparison group relative to the control group.
[0086] Referring to FIGS. 3 and 4, it is confirmed that the
Atrogin1 and Murf1 genes were overexpressed in the control group
treated with the dexamethasone compared to the normal myocyte.
Compared with the control group, it is exhibited that the
expression levels of the Atrogin1 and Murf1 genes are reduced
overall in the experimental group further treated with the bean
leaf extract.
[0087] Specifically, as shown in Table 2 above, the expression
level of the gene of Atrogin1 in the experimental group treated
with the extract of bean leaf at stage R2 to R7 is significantly
reduced by 8.2 to 31.6% in comparison with the control group, and
especially, such reduction is very effective in experimental group
treated with extract of bean leaf at stage R7.
[0088] In addition, as shown in Table 3 above, the expression level
of the gene of Murf1 in the experimental group treated with the
extract of bean leaf at stage V4 to R7 is significantly reduced by
8.5 to 24.1% in comparison with the control group, and especially,
such reduction is very effective in experimental group treated with
extract of bean leaf at stage R7.
[0089] Meanwhile, it can be seen that in the comparison group
treated with the dexibuprofen, the expression inhibition effects of
the Atrogin1 and Murf1 genes are insignificant, and thus, general
muscle pain suppressing substances are not suitable for inhibition
or prevention of muscle loss, and their mechanisms of action are
also different.
[0090] From the above results, it is expected that since the
composition containing the bean leaf extract of the present
invention as an active ingredient has excellent efficacy in the
inhibition and prevention of myopathy, it is highly likely to be
industrially applicable in various forms such as food,
pharmaceutical or cosmetic composition.
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