U.S. patent application number 17/098112 was filed with the patent office on 2021-05-06 for hyaluronic acid production promoting agent.
The applicant listed for this patent is Pharma Foods International Co., Ltd.. Invention is credited to Ji-Yeong An, Masayoshi Aosasa, Seiyu Harada, Hiroaki Iitsuka, Mujo Kim, Isafumi Maru, Toshio Nakamura, Utano Nakamura, Maya Sakashita, Sayuri Tanaka, Kazuya Watabe.
Application Number | 20210128640 17/098112 |
Document ID | / |
Family ID | 1000005340827 |
Filed Date | 2021-05-06 |
![](/patent/app/20210128640/US20210128640A1-20210506\US20210128640A1-2021050)
United States Patent
Application |
20210128640 |
Kind Code |
A1 |
Maru; Isafumi ; et
al. |
May 6, 2021 |
HYALURONIC ACID PRODUCTION PROMOTING AGENT
Abstract
An agent of the present invention comprising poultry feet or a
processed product thereof, preferably a hydrolysate of an extract
of poultry feet, promotes hyaluronic acid production, thereby
exhibiting excellent effects of preventing or treating a joint
disorder, improving skin dryness, wrinkles or skin tension,
moisturizing the skin, etc. The agent is thus useful as a
medicament, a quasi-drug, a cosmetic product, a food product or an
animal feed.
Inventors: |
Maru; Isafumi; (Kyoto,
JP) ; An; Ji-Yeong; (Kyoto, JP) ; Tanaka;
Sayuri; (Kyoto, JP) ; Sakashita; Maya; (Kyoto,
JP) ; Harada; Seiyu; (Kyoto, JP) ; Watabe;
Kazuya; (Kyoto, JP) ; Aosasa; Masayoshi;
(Kyoto, JP) ; Kim; Mujo; (Kyoto, JP) ;
Nakamura; Toshio; (Kyoto, JP) ; Nakamura; Utano;
(Kyoto, JP) ; Iitsuka; Hiroaki; (Kyoto,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pharma Foods International Co., Ltd. |
Kyoto |
|
JP |
|
|
Family ID: |
1000005340827 |
Appl. No.: |
17/098112 |
Filed: |
November 13, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15670918 |
Aug 7, 2017 |
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17098112 |
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PCT/JP2015/084852 |
Dec 11, 2015 |
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15670918 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23C 19/093 20130101;
A61Q 19/08 20130101; A23L 33/18 20160801; A61K 9/2068 20130101;
A61Q 19/007 20130101; A61K 8/60 20130101; A23C 9/123 20130101; A61K
38/014 20130101; A61K 38/012 20130101; A61K 31/7004 20130101; A61K
8/735 20130101; A61K 35/57 20130101; A23L 2/56 20130101; A61K
9/4875 20130101; A23C 19/00 20130101; A61K 8/981 20130101; A23L
2/52 20130101; A23L 33/30 20160801; A61K 8/65 20130101; A23C 9/1322
20130101; A23V 2002/00 20130101; A61K 8/64 20130101; A61K 38/05
20130101; A23L 2/60 20130101 |
International
Class: |
A61K 35/57 20060101
A61K035/57; A23C 19/093 20060101 A23C019/093; A61K 38/01 20060101
A61K038/01; A23C 9/13 20060101 A23C009/13; A23L 2/60 20060101
A23L002/60; A61K 9/20 20060101 A61K009/20; A61K 9/48 20060101
A61K009/48; A61K 8/73 20060101 A61K008/73; A23L 2/56 20060101
A23L002/56; A23L 33/18 20060101 A23L033/18; A61K 8/65 20060101
A61K008/65; A23L 33/00 20060101 A23L033/00; A23C 9/123 20060101
A23C009/123; A23C 19/00 20060101 A23C019/00; A23L 2/52 20060101
A23L002/52; A61K 8/60 20060101 A61K008/60; A61K 8/64 20060101
A61K008/64; A61K 8/98 20060101 A61K008/98; A61K 31/7004 20060101
A61K031/7004; A61K 38/05 20060101 A61K038/05; A61Q 19/00 20060101
A61Q019/00; A61Q 19/08 20060101 A61Q019/08 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 9, 2015 |
JP |
2015-023507 |
Claims
1. (canceled)
2. A method for alleviating bodily pain by promoting hyaluronic
acid production of cartilage or cartilage matrix production in a
mammal, the method comprising: selecting a mammal that has bodily
pain; administering to said selected mammal a peptide of 20 amino
acid residues or less containing the amino acid sequence
phenylalanine-hydroxyproline, or a derivative or a salt of said
peptide, wherein said peptide of 20 amino acid residues or less
containing the amino acid sequence phenylalanine-hydroxyproline, or
the derivative or the salt of said peptide, alleviates bodily pain
by promoting hyaluronic acid production of cartilage or cartilage
matrix production, and wherein said mammal has a joint disorder or
said bodily pain is knee pain.
3. The method of claim 2, wherein a C-terminus of the peptide, or
the derivative or the salt of said peptide, is a carboxy group, a
carboxylate, an amide or an ester.
4. The method of claim 2, wherein an N-terminus of the peptide, or
the derivative or the salt of said peptide, is amino or a protected
amino.
5. The method of claim 2, wherein the peptide, or the derivative or
the salt of said peptide, comprises a protected side chain
substituent or an unprotected side chain substituent.
6. The method of claim 2, wherein the peptide, or the derivative or
the salt of said peptide, is phenylalanine-hydroxyproline
dipeptide.
7. The method of claim 2, wherein the peptide, or the derivative or
the salt of said peptide, is acyclic or cyclic.
8. The method of claim 2, wherein the peptide, or the derivative or
the salt of said peptide, is acyclic.
9. The method of claim 2, wherein the joint disorder is
osteoarthritis, a cartilage defect, a cartilage injury or a
meniscus injury.
10. A method for alleviating bodily pain by promoting hyaluronic
acid production of cartilage or cartilage matrix production in a
mammal, the method comprising: administering
phenylalanine-hydroxyproline dipeptide, or a derivative or a salt
thereof, to a mammal, wherein said phenylalanine-hydroxyproline
dipeptide, or a derivative or a salt thereof, alleviates bodily
pain by promoting hyaluronic acid production of cartilage or
cartilage matrix production, and wherein said mammal has a joint
disorder or said bodily pain is knee pain.
11. The method of claim 10, further comprising selecting a mammal
in need of an agent that promotes hyaluronic acid production of
cartilage.
12. The method of claim 10, wherein a C-terminus of the peptide, or
the derivative or the salt of said peptide, is a carboxy group, a
carboxylate, an amide or an ester.
13. The method of claim 10, wherein an N-terminus of the peptide,
or the derivative or the salt of said peptide, is amino or a
protected amino.
14. The method of claim 10, wherein the peptide, or the derivative
or the salt of said peptide, comprises a protected side chain
substituent or an unprotected side chain substituent.
15. The method of claim 10, wherein the
phenylalanine-hydroxyproline dipeptide, or the derivative or the
salt thereof, is phenylalanine-hydroxyproline dipeptide shown
below. ##STR00001##
16. The method of claim 10, wherein the joint disorder is
osteoarthritis, a cartilage defect, a cartilage injury or a
meniscus injury.
Description
TECHNICAL FIELD
[0001] The present invention relates to a hyaluronic acid
production promoting agent. In particular, the present invention
relates to a hyaluronic acid production promoting agent comprising
poultry feet or a processed product thereof as an active
ingredient.
BACKGROUND ART
[0002] For the prevention or treatment of various chondropathies,
the growth of chondrogenic cells and the expression of their
differentiation function are important. That is, the growth and
maturation of chondrogenic cells are considered to promote normal
growth of bones, facilitate repair of bone fracture, maintain
smooth movement of joints, and restore decreased movement of
joints. Several growth factors for chondrogenic cells have been
reported, including transforming growth factor (TGF)-.beta.1,
insulin-like growth factor (IGF)-1, basic fibroblast growth factor
(bFGF), PTH-related peptide (PTHrP), hepatocyte growth factor
(HGF), and bone morphogenetic protein (BMP). However, clinical
applications of chondrocyte growth-promoting drugs that are
excellent in safety, stability and efficacy have not been
established or practiced yet. Consequently, joint disorders remain
a great burden on humans.
[0003] Knee osteoarthritis patients account for the largest
proportion of joint disorder patients. One of the causes of knee
osteoarthritis is aging, and the incidence of the disease is
expected to increase in this aging society. The incidence rate is
particularly higher in women than men, and the sex ratio of men to
women patients is 1 to 4. Joint disorders in which the main lesion
is degeneration of cartilage (e.g., joint diseases) have
conventionally been prevented or treated mostly with symptomatic
therapy by direct injection of hyaluronic acid into knee joints or
by using antiinflammatories, or with drug therapy using bone
resorption inhibitors such as estrogen and calcitonin, or aspirin,
or nonsteroidal anti-inflammatory drugs (NSAIDs). However, these
therapies are not effective enough, and the drugs are well-known to
cause adverse effects such as digestive tract disorders. Under
these circumstances, there has been a great demand for prophylactic
or alleviating drugs that are highly effectively and safely used
for treatment of joint injuries and joint disorders.
[0004] The inventors have found a beneficial action of an egg yolk
hydrolysate on cartilage (Patent Literature 1). However, there has
been no report on an action of poultry feet or a processed product
thereof on cartilage.
CITATION LIST
Patent Literature
[0005] Patent Literature 1: WO 2014/007318
SUMMARY OF INVENTION
Technical Problem
[0006] An object of the present invention is to provide a novel
hyaluronic acid production promoting agent that can be safely and
effectively used for the prevention, alleviation or treatment of a
joint disorder, or for the improvement of skin conditions, such as
dryness, wrinkles, tension and moisture.
Solution to Problem
[0007] The present invention was made to solve the above problems
and includes the following.
(1) A hyaluronic acid production promoting agent comprising poultry
feet or a processed product thereof. (2) The agent according to the
above (1), which is for the prevention or treatment of a joint
disorder. (3) The agent according to the above (1) or (2), which is
for the alleviation of knee pain or bodily pain or the improvement
of physical functioning. (4) The agent according to the above (1),
which is for the improvement of skin dryness, wrinkles or skin
tension or the moisturization of the skin. (5) The agent according
to any of the above (1) to (4), further comprising
N-acetylglucosamine, glucosamine, or a pharmacologically acceptable
salt thereof. (6) The agent according to any of the above (1) to
(5), wherein the processed product of poultry feet is a hydrolysate
of an extract of poultry feet. (7) The agent according to the above
(6), wherein the poultry feet to be subjected to extraction are
crushed or minced poultry feet. (8) The agent according to the
above (6) or (7), wherein the hydrolysate of the extract, when
subjected to gel filtration chromatography, shows peaks with
molecular weights ranging from 500 to 30000 Da and the area of the
peaks accounts for 60% or more of the total peak area. (9) The
agent according to any of the above (1) to (8), which has a
promoting effect on hyaluronic acid production, on the expression
of a hyaluronic acid synthase gene, or on cartilage formation. (10)
A health food, a food additive, or a dietary supplement, comprising
the agent according to any of the above (1) to (9). (11) The health
food, the food additive or the dietary supplement according to the
above (10), further comprising a pharmaceutical excipient. (12) The
health food, the food additive or the dietary supplement according
to the above (11), wherein the pharmaceutical excipient is in the
form of a liquid or solid. (13) The health food, the food additive
or the dietary supplement according to any of the above (10) to
(12), which is in the form of a tablet or a drink. (14) A
medicament comprising the agent according to any of the above (1)
to (9). (15) The medicament according to the above (14), which is
for the prevention or treatment of a joint disorder. (16) A
cosmetic product or a quasi-drug, comprising the agent according to
any of the above (1) to (9). (17) A method for promoting hyaluronic
acid production, the method comprising administering an effective
amount of the agent according to any of the above (1) to (9) to a
mammal. (18) The agent according to any of the above (1) to (9) for
use in promoting hyaluronic acid production. (19) Use of the agent
according to any of the above (1) to (9) for the production of an
agent for promoting hyaluronic acid production. (20) Use of the
agent according to any of the above (1) to (9) for the promotion of
hyaluronic acid production. (21) A method for promoting hyaluronic
acid production, the method comprising administering an effective
amount of a hyaluronic acid production promoting agent comprising
poultry feet or a processed product thereof to a mammal. (22) The
method according to the above (21), which is for the prevention or
treatment of a joint disorder. (23) The method according to the
above (21), which is for the alleviation of knee pain or bodily
pain or the improvement of physical functioning. (24) The method
according to the above (21), which is for the improvement of skin
dryness, wrinkles or skin tension or the moisturization of the
skin. (25) The method according to the above (21), wherein the
hyaluronic acid production promoting agent further comprises
N-acetylglucosamine, glucosamine, or a pharmacologically acceptable
salt thereof. (26) The method according to the above (21), wherein
the processed product of poultry feet is a hydrolysate of an
extract of poultry feet. (27) The method according to the above
(26), wherein the poultry feet to be subjected to extraction are
crushed or minced poultry feet. (28) The method according to the
above (26), wherein the hydrolysate of the extract, when subjected
to gel filtration chromatography, shows peaks with molecular
weights ranging from 500 to 30000 Da and the area of the peaks
accounts for 60% or more of the total peak area. (29) The method
according to the above (21), which is for the promotion of
expression of a hyaluronic acid synthase gene or the promotion of
cartilage formation. (30) The method for promoting hyaluronic acid
production according to the above (21), wherein the processed
product of poultry feet is a peptide of 100 amino acid residues or
less containing the amino acid sequence
phenylalanine-hydroxyproline and an additional amino acid sequence
and having a promoting effect on hyaluronic acid production, a
derivative thereof, or a salt thereof. (31) The method according to
the above (21), wherein the processed product of poultry feet is a
phenylalanine-hydroxyproline dipeptide, a derivative thereof, or a
salt thereof. (32) A method for promoting hyaluronic acid
production, the method comprising administering, to a mammal, an
effective amount of a peptide of 100 amino acid residues or less
containing the amino acid sequence phenylalanine-hydroxyproline and
an additional amino acid sequence and having a promoting effect on
hyaluronic acid production, a derivative thereof, or a salt
thereof. (33) A method for promoting hyaluronic acid production,
the method comprising administering an effective amount of a
phenylalanine-hydroxyproline dipeptide, a derivative thereof, or a
salt thereof to a mammal.
Advantageous Effects of Invention
[0008] The present invention provides a hyaluronic acid production
promoting agent. Poultry feet or a processed product thereof
containing an active ingredient is a safe, natural material from
poultry that has a long history as food, and can therefore be
widely used in daily consumable products, such as food and drink
products, medicaments, animal feeds, etc. The hyaluronic acid
production promoting agent of the present invention improves skin
dryness, wrinkles or skin tension. The hyaluronic acid production
promoting agent of the present invention can also be used as a skin
moisturizing agent. The hyaluronic acid production promoting agent
of the present invention has effects of alleviating knee pain, knee
conditions and bodily pain and improving physical functioning,
etc., and can therefore be used as a prophylactic or therapeutic
agent for a joint disorder. The hyaluronic acid production
promoting agent of the present invention effectively improves the
quality of life (QOL) of a subject suffering from knee pain. The
hyaluronic acid production promoting agent of the present invention
is particularly effective for women.
BRIEF DESCRIPTION OF DRAWINGS
[0009] FIG. 1 shows the molecular weight distribution of a
hydrolysate of a chicken foot extract (hereinafter simply called a
chicken foot extract hydrolysate).
[0010] FIG. 2 shows the effect of a chicken foot extract
hydrolysate on the expression of a hyaluronic acid synthase gene in
chondrogenic cells.
[0011] FIG. 3 shows a schematic view of circular defects created by
drilling a femoral groove of a rabbit.
[0012] FIG. 4 shows the results of macroscopic observation of
restoration of circular chondral defects after administration of 50
mg/day of a chicken foot extract hydrolysate to a rabbit for three
weeks. To a control group, water was administered.
[0013] FIG. 5 shows Alcian blue-stained cartilage tissue including
circular chondral defects.
[0014] FIG. 6 is a graph showing the comparison of Alcian
blue-stained area of defect sites.
[0015] FIG. 7 shows Safranin 0-stained cartilage tissue including
circular chondral defects.
[0016] FIG. 8 is a graph showing the comparison of Safranin
0-stained area.
[0017] FIG. 9 shows the total score of WOMAC assessment.
[0018] FIG. 10 shows the pain score of WOMAC assessment.
[0019] FIG. 11 shows the daily activity score of WOMAC
assessment.
[0020] FIG. 12 shows the total score of JKOM assessment.
[0021] FIG. 13 shows the pain score of JKOM assessment.
[0022] FIG. 14 shows the knee condition score of JKOM
assessment.
[0023] FIG. 15 shows the physical functioning score of SF-36
assessment.
[0024] FIG. 16 shows the bodily pain score of SF-36 assessment.
[0025] FIG. 17 is a graph showing that a chicken foot extract
hydrolysate promotes the expression of a hyaluronic acid synthase
gene in human dermal fibroblasts.
[0026] FIG. 18 is a graph showing that the application of a chicken
foot extract hydrolysate improves the moisture of human skin.
[0027] FIG. 19 is a graph showing cartilage matrix production by
chondrogenic cells in the presence of a dipeptide consisting of the
amino acid sequence phenylalanine-hydroxyproline (hereinafter also
called a Phe-Hyp dipeptide).
[0028] FIG. 20 is a graph showing SOX9 mRNA expression level in
chondrogenic cells in the presence of a Phe-Hyp dipeptide.
[0029] FIG. 21 is a graph showing Acan mRNA expression level in
chondrogenic cells in the presence of a Phe-Hyp dipeptide.
[0030] FIG. 22 is a graph showing Col X mRNA expression level in
chondrogenic cells in the presence of a Phe-Hyp dipeptide.
[0031] FIG. 23 is a graph showing has 2 mRNA expression level in
chondrogenic cells in the presence of a Phe-Hyp dipeptide.
[0032] FIG. 24 is a graph showing the amount of hyaluronic acid
produced in ex vivo mouse knee joints in the presence of a Phe-Hyp
dipeptide.
DESCRIPTION OF EMBODIMENTS
[0033] The present invention provides a hyaluronic acid production
promoting agent comprising poultry feet or a processed product
thereof as an active ingredient (hereinafter also called an agent
of the present invention). The amount of the poultry feet or a
processed product thereof contained in the agent of the present
invention may be 0.01 to 100% by mass, 0.1 to 50% by mass, or 0.1
to 10% by mass.
Poultry Feet
[0034] Poultry feet are not commonly eaten by people. Poultry feet
are usually disposed as waste, and are therefore usually not
commercially distributed as food. Examples of the poultry include
chickens, ducks, emus, geese, Indian peafowls, mute swans,
ostriches, turkeys, guinea fowls, ring-necked pheasants, golden
pheasants, rheas, quails, etc. Particularly preferred are chickens.
A preferred chicken is Mitsuse chicken. Mitsuse chicken is a breed
derived from French red chickens, and raised mainly in mountainous
regions in the northern Kyushu, such as Saga, Fukuoka, Nagasaki and
Oita prefectures.
[0035] Poultry feet are body parts under the belly and thigh, or a
part thereof. The feet may include the belly and thigh, but
preferably do not include the belly or thigh. The feet may include
bones, spurs, toes, and leg feathers.
[0036] Poultry feet may be in a raw state, or a boiled, roasted or
dried state. Crushing of the feet is performed by a known method.
Crushing may be performed using an instrument, such as a mixer and
a mill.
Processed Product of Poultry Feet
[0037] The poultry feet may be pre-treated before processed into a
processed product. The pre-treatment may be, for example, pH
treatment, removal of impurities, etc. The pH treatment may be
performed by immersing the poultry feet in, for example, an acidic
liquid, an alkaline liquid, a neutral liquid, or the like. The
temperature of the liquid for immersion may be, for example, room
temperature (5 to 35.degree. C.), 0 to 5.degree. C., or 35 to
50.degree. C., but is preferably room temperature (5 to 35.degree.
C.). The duration of the immersion may be, for example, 1 hour to 1
week, or 3 to 18 hours, but is preferably 6 hours to 4 days, more
preferably 1 to 3 days. The pH of the liquid can be adjusted by,
for example, adding a strong acid (e.g., hydrochloric acid), a
strong alkali (sodium hydroxide), or the like to the liquid
containing the poultry feet. The pH of the acidic liquid for
immersion may be 0 to 6, but is preferably 4 to 6. The pH of the
neutral liquid for immersion may be 6 to 8. The pH of the alkaline
liquid for immersion may be 8 to 14, but is preferably 8 to 10. In
cases where the poultry feet are pre-treated in the acidic liquid
or the alkaline liquid, the liquid may later be neutralized with an
alkaline liquid or an acidic liquid. The ratio of the poultry feet
(kg) to the immersion liquid (L) in the pH treatment is preferably
0.1 to 1 kg per liter, and is more preferably 0.4 to 0.6 kg per
liter. The removal of impurities may be performed by filtration
etc. Filtration may be performed by a known method.
[0038] A processed product of the poultry feet may be a processed
product of crushed or minced poultry feet. The crushed or minced
poultry feet are not limited to a particular type, and may be
prepared by a known method. The processed product of the poultry
feet is also not limited to a particular type, but is particularly
preferably, for example, a product obtainable by treating the
poultry feet or the feet in a crushed state by extraction,
decomposition, drying, and/or the like. The processed product of
the poultry feet is preferably a hydrolysate of an extract of the
poultry feet or the feet in a crushed state, and is more preferably
a dried product of a hydrolysate of an extract of the poultry feet
or the feet in a crushed state. The hydrolysate or the dried
product is a peptide-containing mixture. The extraction may be
performed in, for example, water with a neutral pH. The extraction
solvent may be a hydrophilic solvent other than water, and examples
of such an extraction solvent include ethanol, acetone,
tetrahydrofuran, butanol, propanol, and a mixture thereof.
Particularly preferred is a mixture of such a hydrophilic solvent
and water. The extraction temperature may be 5 to 100.degree. C.,
50 to 97.degree. C., 70 to 95.degree. C., or 80 to 90.degree. C.
The extraction time may be 1 hour to 1 week, 1 to 24 hours, 1 to 12
hours, or 5 to 7 hours. Extraction at the above extraction
temperature for the above extraction time will efficiently yield
proteins, peptides, and the like that have a promoting effect on
hyaluronic acid production. Crushed poultry feet are preferably
prepared by crushing the feet with a crushing machine. Examples of
the crushing machine include Waring blenders, mill mixers,
homogenizers, single shaft shredders with a pusher, high-speed
single shaft crushers, medium-speed single shaft crushers,
low-speed single shaft shredders, double shaft shredders, four
shaft shredders, hammer mills, disc mills, press machines, etc. The
size of the crushed poultry feet may be, for example, 0.5 to 3 cm
square, but is preferably 0.5 to 1 cm square. After extraction, the
extract is obtained by, for example, removing the extraction
solvent from the liquid.
[0039] The extract is then usually subjected to hydrolysis.
Hydrolysis may be enzymatic decomposition (using, for example,
endoprotease, carboxypeptidase, collagenase, lipase, etc.) or
chemical decomposition (using, for example, an acid or alkali
etc.), but preferred is enzymatic decomposition. The enzyme may be
a hydrolytic enzyme. Hydrolysis is particularly preferably
performed on the proteins extracted from poultry feet or the feet
in a crushed state. The enzyme used for protein hydrolysis is not
particularly limited, but preferred is an enzyme that has protease
or carboxypeptidase activity and is usable for food production.
Examples of the enzyme include pepsin (EC.3.4.23.1), trypsin
(EC.3.4.21.4), renin (EC.3.4.23.15), rennet, which contains renin
and is used for cheese making, carboxypeptidase A (EC.3.4.17.1),
proteases from Bacillus bacteria (trade name "Alcalase" produced by
Novozymes A/S, trade name "Orientase 22BF" produced by HBI Enzymes
Inc., trade name "Nukureishin" produced by HBI Enzymes Inc., trade
name "Protease S `Amano` G" produced by Amano Enzyme, Inc., trade
name "THERMOASE PC10" produced by Daiwa Fine Chemicals Co., Ltd.,
etc.), proteases from Aspergillus fungi (trade name "Orientase ONS"
produced by HBI Enzymes Inc., trade name "Orientase 20A" produced
by HBI Enzymes Inc., trade name "Protease P `Amano` 3G" produced by
Amano Enzyme, Inc., trade name "Flavourzyme" produced by Novozymes
A/S, etc.), etc. These proteolytic enzymes may be used alone or in
combination of two or more types. Preferred are proteases from
Aspergillus fungi, pepsin, and a combination thereof. The
hydrolysis is preferably performed in an appropriate solvent, such
as water.
[0040] The amount of the enzyme is appropriately adjusted depending
on the conditions of the poultry feet and the enzyme to be used.
For example, when 1 kg of a chicken feet extract is used as the raw
material, the amount of the enzyme used is preferably 100 to 100000
units, and is more preferably 1000 to 30000 units. The enzymatic
reaction temperature and the reaction time also vary depending on
the conditions of the poultry feet and the enzyme to be used.
Preferably, the hydrolysis is performed at about 25 to 75.degree.
C. for about 1 to 24 hours.
[0041] The thus prepared hydrolysate may be desalted if desired and
directly used. Alternatively, the hydrolysate may be used after
purification and/or fractionation by ultrafiltration, gel
filtration, various column chromatographic techniques, membrane
filter filtration, methods utilizing an isoelectric point, etc. A
fraction containing a peptide or protein of interest is preferably
dried to give the agent of the present invention.
[0042] The drying method may be concentration and drying; spray
drying; lyophilization; or the like. The poultry feet or the feet
in a crushed state, an extract thereof, or a hydrolysate thereof
can be dried by a known method. The hydrolysate of extracted
proteins from the poultry feet or the feet in a crushed state is
preferably subjected to drying.
[0043] Preferably, the hydrolysate of the extract of the poultry
feet or the feet in a crushed state, or the dried product of the
hydrolysate, when subjected to gel filtration chromatography, shows
peaks with molecular weights ranging from 500 to 30000 Da and the
area of the peaks accounts for 60% or more of the total peak area.
Preferably, the poultry feet or a processed product thereof, when
subjected to gel filtration chromatography, shows peaks with
molecular weights ranging from 500 to 30000 Da and the area of the
peaks accounts for 70% or more of the total peak area. Preferably,
the poultry feet or a processed product thereof, when subjected to
gel filtration chromatography, shows peaks with molecular weights
ranging from 500 to 30000 Da and the area of the peaks accounts for
75% or more of the total peak area. Preferably, the poultry feet or
a processed product thereof, when subjected to gel filtration
chromatography, shows peaks with molecular weights ranging from 500
to 30000 Da and the area of the peaks accounts for 80% or more of
the total peak area.
[0044] The conditions of the gel filtration chromatography analysis
may be as follows.
[0045] Column: YMC-pack Diol 60 (trade name) (6.times.300 mm) (YMC
Co., Ltd.)
[0046] Eluent: 0.2 M potassium phosphate buffer with 0.2 M NaCl (pH
6.9)/acetonitrile (70:30 v/v)
[0047] Flow rate: 0.7 mL/min
[0048] Detection wavelength: 280 nm
[0049] The processed product of the poultry feet is preferably a
peptide of 100 amino acid residues or less containing the amino
acid sequence phenylalanine-hydroxyproline and an additional amino
acid sequence and having a promoting effect on hyaluronic acid
production, a derivative thereof, or a salt thereof.
[0050] The processed product of the poultry feet is particularly
preferably a peptide consisting of the amino acid sequence
phenylalanine-hydroxyproline (hereinafter also called a Phe-Hyp
dipeptide or a phenylalanine-hydroxyproline dipeptide), a
derivative thereof, or a salt thereof.
[0051] The hydroxyproline herein may be 3-hydroxyproline or
4-hydroxyproline, but is preferably 4-hydroxyproline.
[0052] The amount of the "peptide of 100 amino acid residues or
less containing the amino acid sequence
phenylalanine-hydroxyproline and an additional amino acid sequence
and having a promoting effect on hyaluronic acid production, a
derivative thereof, or a salt thereof", the Phe-Hyp dipeptide, a
derivative thereof, or a salt thereof contained in the hyaluronic
acid production promoting agent is preferably 0.01 to 100% by mass,
1 to 100% by mass, 5 to 100% by mass, 10 to 100% by mass, 20 to
100% by mass, 30 to 100% by mass, 40 to 100% by mass, 50 to 100% by
mass, 60 to 100% by mass, 70 to 100% by mass, 80 to 100% by mass,
or 90 to 100% by mass. The hyaluronic acid production promoting
agent may further comprise a pharmaceutical excipient. Preferred
examples of the excipient include lactose hydrate, starch,
crystalline cellulose, mannitol, anhydrous dibasic calcium
phosphate, sucrose, etc. The pharmaceutical excipient may be in the
form of a liquid or solid. The amount of the pharmaceutical
excipient contained in the hyaluronic acid production promoting
agent may be 0.01 to 50% by mass or 0.01 to 10% by mass.
[0053] The present disclosure also includes a method for promoting
hyaluronic acid production, the method comprising administering, to
a mammal, an effective amount of the peptide of 100 amino acid
residues or less containing the amino acid sequence
phenylalanine-hydroxyproline and an additional amino acid sequence
and having a promoting effect on hyaluronic acid production, a
derivative thereof, or a salt thereof.
[0054] The present disclosure also includes a method for promoting
hyaluronic acid production, the method comprising administering an
effective amount of the Phe-Hyp dipeptide, a derivative thereof, or
a salt thereof to a mammal.
[0055] The present disclosure also includes a method for promoting
hyaluronic acid production, the method comprising administering, to
a mammal, a composition comprising 50 to 99.99% by mass, preferably
90 to 99.99% by mass, of the peptide of 100 amino acid residues or
less containing the amino acid sequence
phenylalanine-hydroxyproline and an additional amino acid sequence
and having a promoting effect on hyaluronic acid production, a
derivative thereof, or a salt thereof. The composition may further
comprise a pharmaceutical excipient. The pharmaceutical excipient
includes those described above, and preferred examples of the
excipient include lactose hydrate, starch, crystalline cellulose,
mannitol, anhydrous dibasic calcium phosphate, sucrose, etc. The
pharmaceutical excipient may be in the form of a liquid or solid.
The amount of the pharmaceutical excipient contained in the
composition may be 0.01 to 50% by mass or 0.01 to 10% by mass.
[0056] The "peptide of 100 amino acid residues or less containing
the amino acid sequence phenylalanine-hydroxyproline and an
additional amino acid sequence and having a promoting effect on
hyaluronic acid production, a derivative thereof, or a salt
thereof", or the "Phe-Hyp dipeptide, a derivative thereof, or a
salt thereof" may be contained in, for example, the poultry feet or
a hydrolysate thereof. Alternatively, the peptide, the Phe-Hyp
dipeptide, a derivative thereof, or a salt thereof may be contained
in a hydrolysate obtainable by hydrolysis, e.g., enzymatic
hydrolysis, of an extract extracted from crushed poultry feet using
an extraction solvent such as water. The peptide of interest, a
derivative thereof, or a salt thereof may be purified by
ultrafiltration or various chromatographic techniques, such as
HPLC.
[0057] In another embodiment, the "peptide of 100 amino acid
residues or less containing the amino acid sequence
phenylalanine-hydroxyproline and an additional amino acid sequence
and having a promoting effect on hyaluronic acid production, a
derivative thereof, or a salt thereof", or the "Phe-Hyp dipeptide,
a derivative thereof, or a salt thereof" can easily be produced by
solid-phase synthesis (the Fmoc or Boc method) or liquid-phase
synthesis following the usual peptide synthesis protocol known in
the art.
[0058] Hereinafter, the "peptide of 100 amino acid residues or less
containing the amino acid sequence phenylalanine-hydroxyproline and
an additional amino acid sequence and having a promoting effect on
hyaluronic acid production" is also referred to as the "peptide
.alpha.".
[0059] Use of the peptide .alpha., a derivative thereof, or a salt
thereof is preferred, and use of the Phe-Hyp dipeptide, a
derivative thereof, or a salt thereof is more preferred, due to the
following advantages: the peptides, a derivative thereof, or a salt
thereof can promote cartilage matrix production by chondrogenic
cells; the peptides, a derivative thereof, or a salt thereof can
enhance the expression of cartilage differentiation-related genes,
such as SOX9 (Sry-type human mobility group box 9), Acan
(aggrecan), Col X (type X collagen) and the has2 gene; the
peptides, a derivative thereof, or a salt thereof can promote
hyaluronic acid production in knee joints; and/or the peptides, a
derivative thereof, or a salt thereof can prevent or treat a joint
disorder; or the effects of the present invention can be better
exhibited by the peptides, a derivative thereof, or a salt
thereof.
[0060] The total number of amino acid residues in the peptide
.alpha., a derivative thereof, or a salt thereof is not
particularly limited, but is preferably 100 or less, more
preferably 50 or less, further preferably 20 or less, further
preferably 10 or less, further preferably 8 or less, further
preferably 5 or less, further preferably 4 or less, or further
preferably 3 or less.
[0061] When the total number of amino acid residues is 3, the
peptide .alpha. is preferably the tripeptide
"glycine-phenylalanine-hydroxyproline", or the tripeptide
"phenylalanine-hydroxyproline-glycine". The effects of the present
invention can be better exhibited by these tripeptides.
[0062] When the total number of amino acid residues is 4 or more,
the peptide .alpha. preferably contains one or more amino acid
sequences selected from the group consisting of the amino acid
sequences Phe-Hyp, Gly-Phe-Hyp, and Phe-Hyp-Gly. In these cases
where the peptide .alpha. contains one or more of these amino acid
sequences, the amino acid sequence(s) may be located at the
C-terminus, N-terminus or in the middle of the peptide a. The
effects of the present invention can be better exhibited by such a
peptide .alpha..
[0063] The C-terminus of a derivative of the peptide .alpha. or of
the Phe-Hyp dipeptide, the peptides each being represented by a
particular amino acid sequence, may be a carboxyl group (--COOH), a
carboxylate (--COO.sup.-), an amide (--CONH.sub.2) or an ester
(--COOR). Examples of R of the ester include C.sub.1-6 alkyl
groups, such as methyl, ethyl, n-propyl, isopropyl and n-butyl;
C.sub.3-8 cycloalkyl groups, such as cyclopentyl and cyclohexyl;
C.sub.6-12 aryl groups, such as phenyl and .alpha.-naphthyl;
C.sub.7-14 aralkyl groups including phenyl-C.sub.1-2 alkyl groups,
such as benzyl and phenethyl, and .alpha.-naphthyl-C.sub.1-2 alkyl
groups, such as .alpha.-naphthylmethyl; and a pivaloyloxymethyl
group, which is commonly used as an ester for oral administration.
Examples of the amide include an amide; an amide substituted with
one or two C.sub.1-6 alkyl groups; an amide substituted with one or
two C.sub.1-6 alkyl groups substituted with a phenyl group; and an
amide that forms a 5- to 7-membered azacycloalkane containing the
nitrogen atom of the amide group.
[0064] When the derivative of the peptide a has a carboxyl group or
a carboxylate at a position other than the C-terminus, the
derivative of the peptide .alpha. also includes derivatives with
amidated or esterified carboxyl or carboxylate.
[0065] The derivative of the peptide a or of the Phe-Hyp dipeptide
also includes derivatives in which the N-terminal amino group is
protected with a protecting group (e.g., a C.sub.1-6 acyl group
including a formyl group and C.sub.2-6 alkanoyl groups such as
acetyl), derivatives in which a N-terminal glutamyl group generated
by in vivo cleavage of the N-terminus is converted to a
pyroglutamate, and derivatives in which a substituent (e.g., --OH,
--SH, an amino group, an imidazole group, an indole group, or a
guanidino group) on an amino acid side chain in the molecule is
protected with a suitable protecting group (e.g., a C.sub.1-6 acyl
group including a formyl group and C.sub.2-6 alkanoyl groups such
as acetyl).
[0066] The side chains of the amino acids constituting the
derivative of the peptide .alpha. or of the Phe-Hyp dipeptide may
be modified with a substituent. Examples of the substituent
include, but are not limited to, a fluorine atom, a chlorine atom,
a bromine atom, a cyano group, a carboxyl group, a hydroxy group, a
nitro group, an alkyl group, a cycloalkyl group, an alkoxy group,
an amino group, and a phosphate group. The side-chain substituent
may be protected with a protecting group. The derivative also
includes glycopeptides, which are peptides having sugar chains.
[0067] The derivative of the peptide a or of the Phe-Hyp dipeptide
may form a salt. The salt is preferably physiologically acceptable.
Examples of the physiologically acceptable salt include salts with
an inorganic or organic acid, such as hydrochloric acid, sulfuric
acid, phosphoric acid, lactic acid, tartaric acid, maleic acid,
fumaric acid, oxalic acid, malic acid, citric acid, oleic acid, and
palmitic acid; salts with a hydroxide or a carbonate of an alkali
metal, such as sodium, potassium and calcium, salts with a
hydroxide or a carbonate of an alkaline earth metal, and salts with
aluminum hydroxide or carbonate; and salts with an organic base,
such as triethylamine, benzylamine, diethanolamine, t-butylamine,
dicyclohexylamine, and arginine.
[0068] The derivative of the peptide .alpha. or of the Phe-Hyp
dipeptide may contain a D-amino acid or a non-naturally occurring
amino acid to the extent that the derivative retains the
characteristics of the original peptide. The peptides of the
present invention or a derivative thereof may contain another
substance linked thereto to the extent that the peptides or the
derivative retains the characteristics of the original peptide.
Examples of the substance linkable to the peptides or a derivative
thereof include other peptides, lipids, sugars, sugar chains, an
acetyl group, and naturally occurring or synthetic polymers. The
peptide a may be subjected to modification such as glycosylation,
side-chain oxidation, and phosphorylation to the extent that the
resulting modified peptide retains the characteristics of the
original peptide.
Hyaluronic Acid Production Promoting Agent
[0069] Poultry feet or a processed product thereof has a promoting
effect on hyaluronic acid production etc., and can serve as a
suitable active ingredient of an agent for improving skin dryness,
wrinkles or skin tension, for moisturizing the skin, for preventing
or treating a joint disorder, or for other purposes. The
prophylactic or therapeutic agent for a joint disorder exhibits
effects of alleviating knee pain, knee conditions and bodily pain
and improving physical functioning, etc. The term "joint disorder"
is interchangeable with the term "cartilage disorder". Examples of
the joint disorder include osteoarthritis, cartilage defects,
cartilage injury, and meniscus injury. The agent of the present
invention has a promoting effect on hyaluronic acid production, on
the expression of hyaluronic acid synthase genes, such as the HAS1
gene, the HAS2 gene and the HAS3 gene, on cartilage formation, etc.
The "agent of the present invention" has a function of "promoting
hyaluronic acid production", a function of "promoting the
expression of a hyaluronic acid synthase gene", a function of
"promoting cartilage formation", etc. Whether a sample has a
promoting effect on hyaluronic acid production can be determined
by, for example, the method described in Test Example 1. For
example, if hyaluronic acid production is greater in the presence
of the sample than in a control in the absence of the sample, the
sample is determined to have a promoting effect on hyaluronic acid
production. Whether a sample has a promoting effect on the
expression of a hyaluronic acid synthase gene can be determined by,
for example, the method described in Test Example 2. For example,
if the expression of a hyaluronic acid synthase gene is greater in
the presence of the sample than in a control in the absence of the
sample, the sample is determined to have a promoting effect on the
expression of the hyaluronic acid synthase gene. Whether a sample
has a promoting effect on cartilage formation can be determined by,
for example, the method described in Test Example 3. For example,
if the Alcian blue- or Safranin O-stained area of cartilage in an
osteochondral defect animal model with administration of the sample
is significantly different from that in the animal model without
administration of the sample, the sample is determined to have a
promoting effect on cartilage formation. Whether a sample has
effects of alleviating knee pain, knee conditions and bodily pain
and improving physical functioning, etc. can be determined by, for
example, the method described in Test Example 4. For example, if an
assessment score measured by WOMAC (Western Ontario and McMaster
Universities Osteoarthritis Index) assessment, SF-36 (MOS 36-Item
Short-Form Health Survey), JKOM (Japanese Knee Osteoarthritis
Measure) assessment, etc. after administration of the sample is
significantly different from that before administration of the
sample, the sample is determined to have effects of alleviating
knee pain, knee conditions and bodily pain and improving physical
functioning, etc. Whether a sample has an effect of improving skin
dryness, wrinkles or skin tension can be determined by, for
example, the method described in Test Example 5(2). For example, if
an assessment score measured by VAS for "skin dryness", "skin
tension", "fine lines at the outer corner of the eyes", etc. after
administration of the sample is significantly different from that
before administration of the sample, the sample is determined to
have an effect of improving the skin conditions.
[0070] The amount of the poultry feet or a processed product
thereof contained in the agent of the present invention is not
particularly limited, but is preferably about 0.05 to about 50% by
mass, more preferably about 0.1 to about 25% by mass. The daily
dose of the poultry feet or a processed product thereof varies
depending on the subject, but in cases where the subject is, for
example, an adult human, the daily dose of the poultry feet or a
processed product thereof, preferably a hydrolysate of an extract
of chicken feet, is typically about 0.05 to about 2000 mg/day, and
is preferably about 0.1 to about 1000 mg/day.
[0071] The daily dose of the "peptide of 100 amino acid residues or
less containing the amino acid sequence
phenylalanine-hydroxyproline and an additional amino acid sequence
and having a promoting effect on hyaluronic acid production, a
derivative thereof, or a salt thereof", or the "Phe-Hyp dipeptide,
a derivative thereof, or a salt thereof" is, for example, when the
subject is an adult human, typically about 0.05 to about 2000
mg/day, preferably about 0.1 to about 1000 mg/day.
Medicament
[0072] The present invention provides a medicament having a
promoting effect on hyaluronic acid production. The medicament of
the present invention may be any type of medicament as long as it
comprises the poultry feet or a processed product thereof. The
medicament of the present invention can be administered to a mammal
via an oral or parenteral route. Examples of oral preparations
include granules, powders, tablets (including sugar-coated
tablets), pills, capsules, syrups, emulsions, suspensions, etc.
Examples of parenteral preparations include injections (e.g.,
subcutaneous, intravenous, intramuscular, and intraperitoneal
injections), intravenous infusions, external preparations (e.g.,
transnasal preparations, transdermal preparations, and ointments),
suppositories (e.g., rectal suppositories, and vaginal
suppositories), etc. These preparations can be produced using a
pharmaceutically acceptable carrier in accordance with the usual
practice in the field. Examples of the pharmaceutically acceptable
carrier include excipients, binders, diluents, additives,
fragrances, buffering agents, thickeners, colorants, stabilizers,
emulsifiers, dispersants, suspending agents, preservatives, etc.
Specific examples of the carrier include magnesium carbonate,
magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch,
gelatin, tragacanth, methylcellulose, sodium carboxymethyl
cellulose, low melting wax, cacao butter, etc.
[0073] The oral solid preparations (tablets, pills, capsules,
powders, granules, etc.) can be produced by mixing the active
ingredient with, for example, an excipient (lactose, mannitol,
glucose, microcrystalline cellulose, starch, etc.), a binder
(hydroxypropyl cellulose, polyvinylpyrrolidone, magnesium
aluminometasilicate, etc.), a disintegrant (calcium carboxymethyl
cellulose etc.), a lubricant (magnesium stearate etc.), a
stabilizer, a solubilizer (glutamic acid, aspartic acid, etc.)
and/or the like, and processing the mixture into the desired dosage
form in the usual manner. If needed, the oral solid preparations
may be covered with a coating material (sucrose, gelatin,
hydroxypropyl cellulose, hydroxypropyl methylcellulose phthalate,
etc.), or the oral solid preparations may be covered with two or
more coating layers.
[0074] The oral liquid preparations (solutions, suspensions,
emulsions, syrups, elixirs, etc.) can be produced by dissolving,
suspending or emulsifying the active ingredient in a commonly used
diluent (purified water, ethanol, a mixture of them, etc.). The
oral liquid preparations may further contain a wetting agent, a
suspending agent, an emulsifier, a sweetener, a flavoring agent, a
fragrance, a preservative, a buffering agent and/or the like.
[0075] The injections include solutions, suspensions, emulsions,
and solid injectable preparations that are intended to be dissolved
or suspended in a solvent at the time of use. The injections can be
produced by dissolving, suspending or emulsifying the active
ingredient in a solvent. Examples of the solvent include distilled
water for injection, physiological saline, vegetable oils, alcohols
such as propylene glycol, polyethylene glycol and ethanol, and a
combination thereof. The injections may further contain a
stabilizer, a solubilizer (glutamic acid, aspartic acid,
polysorbate 80 (registered trademark), etc.), a suspending agent,
an emulsifier, a soothing agent, a buffering agent, a preservative,
and/or the like. The injections are sterilized in the final step of
the production process or produced in an aseptic manner.
Alternatively, sterile solid preparations, for example, lyophilized
preparations may be produced for use as injections. Such sterile
solid preparations are intended to be dissolved in a sterilized or
aseptic distilled water for injection or another solvent at the
time of use.
Dietary Supplement
[0076] The present invention provides a dietary supplement
comprising the agent of the present invention. The dietary
supplement of the present invention may be any type of dietary
supplement as long as it comprises the poultry feet or a processed
product thereof. The dietary supplement of the present invention
may be in the form of an oral solid preparation (a tablet, a pill,
a capsule, a powder, granules, etc.), an oral liquid preparation (a
drink), or the like. These preparations can be produced in the same
manner as in the production of the medicament. The dietary
supplement of the present invention preferably further comprises a
pharmaceutical excipient. The pharmaceutical excipient includes
those described above, and preferred examples of the excipient
include lactose hydrate, starch, crystalline cellulose, mannitol,
anhydrous dibasic calcium phosphate, sucrose, etc. The
pharmaceutical excipient may be in the form of a liquid or
solid.
Health Food
[0077] The present invention provides a health food comprising the
agent of the present invention. The health food of the present
invention may be any type of health food as long as it comprises
the poultry feet or a processed product thereof. The health food of
the present invention may be in the form of an oral solid
preparation (a tablet, a pill, a capsule, a powder, granules,
etc.), an oral liquid preparation (a drink), or the like, and is
preferably in the form of a tablet, a drink, or the like. The
health food of the present invention include functional foods,
foods with functional claims, foods with nutrient function claims,
foods for specified health use, and foods for sick people. The form
of the health food is not particularly limited, and examples
thereof include drinks such as tea drink, soft drink (beverage),
carbonated drink, nutritional drink, fruit juice and lactic drink;
noodles such as buckwheat noodle, wheat noodle, Chinese noodle and
instant noodle; sweets and bakery such as hard candy, candy,
chewing gum, chocolate, snack, biscuit, jelly, jam, cream, baked
sweets and bread; processed fishery and livestock products such as
fish cake, ham and sausage; dairy products such as yogurt,
processed milk and fermented milk (cheese); fats, oils and
processed fat and oil products such as vegetable oil, tempura oil,
margarine, mayonnaise, shortening, whipped cream and dressing;
seasonings such as sauce and dipping sauce; retort pouch food
products such as curry, stew, rice bowl, rice porridge and rice
soup; and frozen desserts such as ice cream, sherbet and shaved
ice. The health food of the present invention preferably further
comprises a pharmaceutical excipient. Examples of the excipient
include lactose hydrate, starch, crystalline cellulose, mannitol,
anhydrous dibasic calcium phosphate, sucrose, etc. The
pharmaceutical excipient may be in the form of a liquid or solid.
The health food of the present invention may be those distributed
from a warehouse.
Food Additive
[0078] The present invention provides a food additive comprising
the agent of the present invention. The food additive of the
present invention may be any type of food additive as long as it
comprises the poultry feet or a processed product thereof. The food
additive of the present invention may be in the form of a solid
preparation (a tablet, a pill, a capsule, a powder, granules,
etc.), a liquid preparation (a drink), or the like. The form of the
food additive of the present invention is not particularly limited,
and may be, for example, a liquid, a paste, a powder, flakes,
granules, etc. The food additive of the present invention also
includes an additive for drinks. The food additive of the present
invention can be produced in accordance with the conventional
production method for food additives. The food additive of the
present invention preferably further comprises a pharmaceutical
excipient. Examples of the excipient include the excipients
described above, for example, lactose hydrate, starch, crystalline
cellulose, mannitol, anhydrous dibasic calcium phosphate, sucrose,
etc. The pharmaceutical excipient may be in the form of a liquid or
solid.
Cosmetic Product or Quasi-Drug
[0079] The present invention provides a cosmetic product or a
quasi-drug comprising the agent of the present invention. The
cosmetic product or the quasi-drug of the present invention may be
any type of cosmetic product or quasi-drug as long as it comprises
the poultry feet or a processed product thereof. The form of the
cosmetic product or the quasi-drug is not particularly limited.
Preferred examples of the form of the cosmetic product or the
quasi-drug include external preparations for skin (facial toners,
milky lotions, foundations, hand creams, essences, etc.), shampoos,
conditioners, hair treatment agents, hair care agents, hair styling
agents, face packs, soaps (including facial washes), body shampoos,
hair growers, bath additives, and a solution. Depending on the
purpose, the cosmetic product or the quasi-drug of the present
invention may further contain an ingredient generally used in
cosmetic products and quasi-drugs, if desired. Examples of such
ingredient include stabilizers, surfactants, lubricants, buffering
agents, sweeteners, flavor improvers, binders, antioxidants,
coating agents, wetting agents, flavoring agents, flavors,
colorants, sugar coating agents, isotonic agents, emulsifiers,
thickeners, pH adjusting agents, excipients, dispersants,
disintegrants, antiseptics, preservatives, solubilizers,
solubilizing agents, oils, moisturizers, ultraviolet absorbers,
fillers, sequestering agents, sunscreen agents, defoaming agents,
softening agents, propellants, acidifying and basifying agents,
silicones, vitamins, dyes, pigments, nanopigments, organic solvents
(such as alcohol), water, etc. Preferred dosage forms include solid
preparations, liquid preparations, lotions, emulsions, gels,
creams, ointments, and aerosols, but the dosage form is not limited
thereto as long as the dosage form is suitable for external
use.
Animal Feed
[0080] The present invention provides an animal feed comprising the
agent of the present invention. The animal feed of the present
invention may be any type of animal feed as long as it comprises
the poultry feet or a processed product thereof. The animal feed of
the present invention may be in the form of a solid preparation (a
tablet, a pill, a capsule, a powder, granules, etc.), a liquid
preparation (a drink), or the like. Specific examples of the animal
feed include animal feeds for domestic animals, such as cattle,
horses, and pigs, animal feeds for poultry, such as chickens, and
animal feeds for companion animals, such as dogs and cats. The
animal feed of the present invention can be produced by adding the
poultry feet or a processed product thereof to an animal feed.
Alternatively, the animal feed of the present invention can be
produced or processed by the conventional method for producing
animal feeds. The animal feed of the present invention preferably
further comprises a pharmaceutical excipient. Examples of the
excipient include lactose hydrate, starch, crystalline cellulose,
mannitol, anhydrous dibasic calcium phosphate, sucrose, etc. The
pharmaceutical excipient may be in the form of a liquid or
solid.
Combination with Other Ingredients
[0081] The poultry feet or a processed product is a multifunctional
component and exhibits various effects. The poultry feet or a
processed product is expected to exhibit a high additive or
synergistic effect in combination with another active ingredient
used for the promotion of hyaluronic acid production. Examples of
another active ingredient for the treatment of chondropathies
include egg yolk hydrolysates, glucosamine, chondroitin, type I
collagen, type II collagen, N-acetylglucosamine, and a
pharmacologically acceptable salt thereof. Particularly preferred
are egg yolk hydrolysates, N-acetylglucosamine, glucosamine, a
pharmacologically acceptable salt thereof, etc. Examples of the
salt include a hydrochloric acid salt, a sulfuric acid salt,
etc.
Therapeutic Method
[0082] The present invention also includes a method for promoting
hyaluronic acid production, the method comprising administering an
effective amount of the poultry feet or a processed product thereof
to a mammal, including a human, in need of the promotion of
chondrocyte formation. The present invention also provides a method
for preventing or treating a joint disorder, a method for improving
skin dryness, wrinkles or skin tension, and a method for
moisturizing the skin, each method comprising administering an
effective amount of the poultry feet or a processed product thereof
to a mammal including a human. The present invention further
includes a non-therapeutic method for preventing or alleviating a
joint disorder, the method comprising orally administering the
poultry feet or a processed product thereof to a human in need of
the promotion of chondrocyte formation. The term "non-therapeutic"
refers to a concept excluding medical practice, i.e., excluding
therapeutic treatment of human or animal bodies.
[0083] The present invention also includes a method for promoting
hyaluronic acid production, the method comprising administering an
effective amount of the agent of the present invention to a mammal.
Examples of the mammal include humans, non-human mammals (for
example, rats, mice, rabbits, sheep, pigs, cattle, cats, dogs,
monkeys, etc.). Preferred are humans, and particularly preferred
are women. The route of administration is not particularly limited,
and may be, for example, oral administration or transdermal
administration (application to the skin).
[0084] Needless to say, the above-described techniques regarding
the pharmaceutical preparations and the above-described embodiments
of the therapeutic methods can also be applied to the "peptide of
100 amino acid residues or less containing the amino acid sequence
phenylalanine-hydroxyproline and an additional amino acid sequence
and having a promoting effect on hyaluronic acid production, a
derivative thereof, or a salt thereof", or the "Phe-Hyp dipeptide,
a derivative thereof, or a salt thereof".
Production Method
[0085] The present invention also includes a method for producing
of the agent of the present invention. The production method of the
present invention preferably comprises the step of crushing the
poultry feet. The production method of the present invention
preferably further comprises the steps of performing extraction
from the obtained crushed poultry feet, decomposing the crushed
poultry feet or the extract thereof, drying the crushed poultry
feet, the extract thereof, or the decomposed product thereof, and
formulating a composition using, as an active ingredient, the
crushed poultry feet, the extract thereof, the decomposed product
thereof, or the dried product thereof.
[0086] The present invention also includes the agent of the present
invention for use in promoting hyaluronic acid production.
[0087] The present invention further includes use of the agent of
the present invention for the production of a hyaluronic acid
production promoting agent.
[0088] The present invention further includes use of the agent of
the present invention for the promotion of hyaluronic acid
production.
EXAMPLES
[0089] The present invention will be described in more detail below
with reference to Examples, but the present invention is not
limited thereto. The sign "%" means % by weight.
Production Example A: Production of Hydrolysate of Chicken Foot
Extract
(1) Preparation of Hydrolysate of Chicken Foot Extract
[0090] Raw Mitsuse chicken feet were crushed into about 1 cm square
pieces with a homogenizer. To 1 kg of the crushed chicken feet, 2 L
of water was added, and hydrochloric acid was then added to adjust
the pH to 4.0. After the mixture was stirred at room temperature
for 2 days, the liquid for immersion was discarded and the chicken
feet were washed with 2 L of water. To the washed chicken feet, 2 L
of water was added, and the mixture was neutralized with sodium
hydroxide. Extraction was performed at 90.degree. C. for 6 hours.
The resulting mixture was filtered to remove the solid matter to
give a chicken foot extract liquid.
(2) Preparation of Hydrolysate of Chicken Foot Extract
[0091] To the chicken foot extract liquid obtained in the above
(1), 10,000 units of an endoprotease was added, and the enzymatic
reaction was allowed to proceed at 50.degree. C. for 3 hours. The
reaction mixture was heated at 90.degree. C., and centrifuged at
3,000.times.g for 20 minutes to remove insoluble matter. After
clarification by filtration, the filtrate was spray-dried by the
conventional method to give about 110 g of a hydrolysate of the
chicken foot extract.
[0092] The molecular weight analysis of the chicken foot extract
hydrolysate was performed by gel filtration chromatography under
the following conditions.
[0093] Column: YMC-pack Diol 60 (trade name) (6.times.300 mm) (YMC
Co., Ltd.)
[0094] Eluent: 0.2 M potassium phosphate buffer with 0.2 M NaCl (pH
6.9)/acetonitrile (70:30)
[0095] Flow rate: 0.7 mL/min
[0096] Detection wavelength: 280 nm
[0097] The results of the molecular weight analysis are shown in
FIG. 1. As shown in FIG. 1, the chicken foot extract hydrolysate of
Example 1 shows peaks with molecular weights ranging from 500 Da to
30,000 Da and the area of the peaks accounts for about 80% or more
of the total peak area, which corresponds to the total amount of
proteins, peptides and amino acids contained in the extract
hydrolysate.
Test Example 1: Examination of Effect of Chicken Foot Extract
Hydrolysate on Hyaluronic Acid Production by Chondrogenic Cells
[0098] This study was performed using the mouse teratoma-derived
chondrogenic culture cell line ATDC5 (RIKEN BANK, RBC0565), which
is capable of differentiating into chondrocyte-like cells. ATDC5
cells were seeded in a 24-well plate at a density of
2.times.10.sup.4 cells/mL per well and cultured in Eagle MEM medium
containing 5% fetal calf serum (FCS) under 5% CO.sub.2 at
37.degree. C. After 3 days of culture, the cells were washed once
with serum-free Eagle MEM medium. After addition of 1 mL of a test
solution, the cells were cultured at 37.degree. C. for 2 days. The
test solution was previously prepared by dissolving a test sample
in serum-free Eagle MEM medium and then sterilizing the solution by
passing it through a 0.45-.mu.m filter. The concentration of the
test sample, i.e., the chicken foot extract hydrolysate etc., in
the test solution was adjusted to a final concentration of 1 mg/mL
in each well. At end of culture, the hyaluronic acid concentration
in the medium was measured with a hyaluronic acid measurement kit
(DuoSet Hyaluronan (R&D Systems)).
[0099] The test samples used were Examples 1 and 2 and Comparative
Examples 1 to 6 as described below.
Example 1: Chicken Foot Extract Hydrolysate
[0100] The chicken foot extract hydrolysate produced in the above
Production Example A was used as Example 1.
Example 2: Chicken Foot Extract Hydrolysate and N-Acetylglucosamine
(GlcNAc)
[0101] A mixture of the chicken foot extract hydrolysate produced
in the above Production Example A and N-acetylglucosamine (GlcNAc)
(at a final concentration of 0.5 mg/mL each) was used.
Comparative Examples 1 to 6: Type I Collagen Peptide Etc.
[0102] Pig skin-derived type I collagen peptide was used as
Comparative Example 1. Fish collagen was used as Comparative
Example 2. Pig cartilage-derived chondroitin sulfate was used as
Comparative Example 3. Salmon-derived proteoglycan was used as
Comparative Example 4. A chicken comb extract was used as
Comparative Example 5. N-acetylglucosamine was used as Comparative
Example 6.
Results
[0103] The results are shown in Table 1. The hyaluronic acid
concentration in the group without addition of the chicken foot
extract hydrolysate was set at 100%, and the hyaluronic acid
concentration in each of the test sample addition groups was
calculated as a percentage relative to that in the group without
the addition of the hydrolysate. As shown in Table 1, the relative
concentration (%) of hyaluronic acid in the group with addition of
the chicken foot extract hydrolysate alone (Example 1) was 350%,
indicating that the hydrolysate has the effect of markedly
promoting hyaluronic acid production by chondrogenic cells. On the
other hand, Comparative Examples 1 to 6 showed lower relative
concentrations (%) than the chicken foot extract hydrolysate group.
The combination of the chicken foot extract hydrolysate and
N-acetylglucosamine (GlcNAc) (Example 2) showed a relative
hyaluronic acid concentration of 760%, which was higher than those
in the chicken foot extract hydrolysate alone (Example 1) and in
N-acetylglucosamine alone (Comparative Example 6). These results
indicated that the combination of the chicken foot extract
hydrolysate and N-acetylglucosamine has a synergistic effect on
hyaluronic acid production.
TABLE-US-00001 TABLE 1 Hyaluronic acid Test sample level (%)
Control No sample added 100 Example 1 Chicken foot extract
hydrolysate (1 mg/mL) 350 Comparative Type I collagen peptide (1
mg/mL) 80 Example 1 Comparative Fish collagen (1 mg/mL) 140 Example
2 Comparative Chondroitin sulfate (1 mg/mL) 60 Example 3
Comparative Proteoglycan (1 mg/mL) 100 Example 4 Comparative
Chicken comb extract (1 mg/mL) 90 Example 5 Comparative
N-acetylglucosamine (GlcNAc) (1 mg/mL) 210 Example 6 Example 2
Chicken foot extract hydrolysate + GlcNAc 760 (at final
concentration of 0.5 mg/mL each)
Test Example 2: Effect of Chicken Foot Extract Hydrolysate on the
Expression of Hyaluronic Acid Synthase Gene in Chondrogenic
Cells
[0104] Chondrogenic cells (ATDC5) at a logarithmic growth phase
were suspended in medium at 2.times.10.sup.4 cells/mL, and 1 mL of
the suspension was added to each well of a 12-well culture plate.
The cells were precultured until confluence. After all medium was
removed, medium containing the chicken foot extract hydrolysate at
the indicated concentrations and insulin at a final concentration
of 10 .mu.g/mL was added. After the cells were incubated for 24
hours, 500 .mu.L/well of ISOGEN II (code No. 311-07361, NIPPON GENE
Co., Ltd.) was added to recover the cells, and RNA was extracted
from the recovered cells in accordance with the manual of ISOGEN
II. cDNA was synthesized from the extracted RNA using Takara
PrimeScript RT reagent Kit (Takara Bio, Inc.). The expression of
the hyaluronic acid synthase gene (has2) in the synthesized cDNA
was measured by real-time PCR (LightCycler (Roche)) using SYBR
Premix Ex Taq (Takara Bio, Inc.). The results are shown in FIG. 2.
The expression of the hyaluronic acid synthase gene increased in a
dose-dependent manner, indicating that the chicken foot extract
hydrolysate enhances the expression of the hyaluronic acid synthase
gene and thereby increases the production of hyaluronic acid.
Test Example 3: Effect of Chicken Foot Extract Hydrolysate in
Rabbit Osteochondral Defect Model
3-1 Test Method
(1) Administration Sample
[0105] The chicken foot extract hydrolysate produced in the above
Production Example A was used for administration. The chicken foot
extract hydrolysate was suspended in purified water at 50 mg/15 mL
(50 mg/day for each animal) at the time of use. The suspension was
used as an administration sample.
(2) Test Animal and Test Conditions
[0106] Rabbits (Slc:JW, male, Japan SLC, Inc.) at 22 weeks old were
purchased. The purchased animals were subjected to a period of
quarantine for five days, followed by a period of acclimation for
seven days. The animals were kept in an animal room maintained at a
temperature of 23.degree. C. and a humidity of 55%. The animals
were individually housed in an aluminum cage. The animals were fed
with 120 g of solid feed (LRC4, Oriental Yeast Co., Ltd.) per day,
and allowed free access to tap water as a drinking water.
(3) Preparation of Rabbit Osteochondral Defect Model
[0107] The animals were anesthetized by intramuscular
administration of a mixed solution of ketamine hydrochloride and
xylazine hydrochloride to the thigh muscle. The femoral regions
were shaved, and the animals were placed supine. The shaved areas
were disinfected with 5% HIBITANE (registered trademark, Sumitomo
Dainippon Pharma Co., Ltd.) diluted to about 10-fold, alcohol for
disinfection, and ISODINE SOLUTION 10%. Lidocaine hydrochloride in
a volume of 2 to 3 mL was subcutaneously administered to the right
and left thighs. The skin and fascia of the both thighs were
incised with a scalpel to expose the knee joints. The incision was
extended along the patellar ligament under the fascia. The joint
capsule was cut to dislocate the patella. Bone defects were created
on the surface of the femoral groove of each femur by drilling two
holes (2 mm in diameter and 4 mm in depth) perpendicular to the
bone surface (FIG. 3). During the procedure, the field of operation
and the circular defects were cleaned with physiological saline
(Otsuka Pharmaceutical Factory) containing enrofloxacin (0.05%).
The incised fascia and skin of the right and left thighs were
sutured with a suture (nylon suture, 4-0, 3-0, Alfresa Pharma
Corporation). ISODINE GEL (Meiji Seika Pharma Co., Ltd.) was
applied to the closed incision. After the operation, enrofloxacin
was subcutaneously administered to the dorsal cervical region for
two days. For prevention of infection of the incision, an
Elizabethan collar was placed around the neck for one week after
the operation.
(4) Group Setting and Test Schedule
[0108] Two groups, i.e., a control group (with administration of
purified water) and a 50 mg/day chicken foot extract hydrolysate
administration group were set up. Two rabbits were used per group
and four circular defects were created per animal, that is, test
was performed at n=8 (two animals per group). Oral gavage (15 mL
per animal) was performed once a day for three weeks, starting from
the next day of the damage development until the day before
dissection. During the administration period, the general
conditions and the incidence of death were observed once a day.
[0109] On the day of dissection of all animals, the animals were
sacrificed by bleeding from the abdominal aorta under anesthesia by
administration of 4% pentobarbital sodium into the auricular vein.
The animals were dissected, and the damaged sites and their
surroundings were examined macroscopically.
(5) Histopathological Analysis and Statistical Analysis
[0110] The femur (including the damaged sites) was harvested, fixed
with 4% paraformaldehyde phosphate buffer, defatted, and
decalcified with K--CX. The specimens were paraffin embedded in the
conventional manner, and sliced into sections containing the center
of the defect site. The sections were stained with HE, Alcian blue
or Safranin O. For the sections stained with Alcian blue or
Safranin O, the stained area was determined with a commercial
software (Microsoft Office Excel 2003).
[0111] The mean and standard deviation were determined for the
Alcian blue-stained area and the Safranin 0-stained area in each
group. The test of significance (t-test) between the control group
and the chicken foot extract hydrolysate administration group was
conducted. The significance level was set at 5%, and if the p-value
was smaller than 5% (p<0.05), it was determined there was a
significant difference.
3-2 Results
[0112] No animals died or went into a near-death state, and no
abnormality was observed in the general conditions during the
administration period. In the macroscopic observation at the time
of dissection, the damaged sites were assessed as follows: score 0:
the circular defect was not healed at all, and score 3: the
circular defect was filled with cartilage matrix and the boundary
of the defect became unclear (healed state), as indicated in FIG.
4. The results revealed that the restoration of the circular
defects was significantly more efficient in the chicken foot
extract hydrolysate administration group than in the control
group.
[0113] For detailed analysis of the circular defects, histological
sections of the bone tissue were prepared and stained with Alcian
blue. As shown in FIG. 5, the circular defects of the chicken foot
extract hydrolysate administration group were intensely stained.
The results of measurement of the stained area of the circular
defects are shown in FIG. 6. As shown in the figure, the stained
area of the circular defects was significantly larger in the
chicken foot extract hydrolysate administration group than in the
control group. The results revealed that the cartilage matrix
components, acid mucopolysaccharides, were increased by oral
administration of the chicken foot extract hydrolysate.
[0114] Similarly, in Safranin O staining, the circular defects of
the chicken foot extract hydrolysate administration group were
intensely stained as shown in FIG. 7. The results of measurement of
the stained area of the circular defects are shown in FIG. 8. As
shown in the figure, the stained area of the circular defects was
significantly larger in the chicken foot extract hydrolysate
administration group than in the control group. The results
revealed that the cartilage matrix components, proteoglycans, were
increased by oral administration of the chicken foot extract
hydrolysate.
Test Example 4: Verification Test of QOL Improving Effect of
Capsules Containing Chicken Foot Extract Hydrolysate when Ingested
by Humans Suffering from Pain in the Knee Joints
4-1 Test Method
[0115] The subjects were healthy Japanese men aged between 45 and
69 and Japanese women aged between 40 and 59 who had not reached
menopause, and all the subjects suffered from pain in the knee
joints. The subjects were divided into groups as below. The chicken
foot extract hydrolysate produced in the above Production Example A
was used for administration.
[0116] Chicken foot extract hydrolysate 50 mg group: a total of 12
subjects, consisting of 5 men and 7 women at an average age of
49.8.+-.5.9 years. One capsule contained 25 mg of the chicken foot
extract hydrolysate, 223 mg of dextrin, and 2 mg of calcium
stearate. The subjects ingested two capsules per day.
[0117] Chicken foot extract hydrolysate 200 mg group: a total of 12
subjects, consisting of 5 men and 7 women at an average age of
49.3.+-.6 0.3 years. One capsule contained 100 mg of the chicken
foot extract hydrolysate, 98 mg of dextrin, and 2 mg of calcium
stearate. The subjects ingested two capsules per day.
[0118] The ingestion period of the capsules was 12 weeks. The
evaluation test was performed before ingestion and 6 and 12 weeks
after ingestion.
[0119] Test method: randomized, double-blind, parallel-group
comparison study.
[0120] Ethical considerations: the test was performed in accordance
with the Declaration of Helsinki.
4-2 Evaluation Methods
(1) WOMAC (Western Ontario and McMaster Universities Osteoarthritis
Index)
[0121] WOMAC was used for the evaluation. WOMAC is a
patient-reported assessment tool, in which a patient answers 24
questions about pain, stiffness (sensation of restriction), and
difficulties in daily activities, and assessment is performed based
on the answers. The subjects rated the assessment items on a
five-point scale, including 5 items for pain, 2 items for
stiffness, and 17 items for difficulties in daily activities. The
rated scores were statistically analyzed. A smaller score indicates
a higher QOL.
(2) JKOM (Japanese Knee Osteoarthritis Measure)
[0122] JKOM is a patient-reported QOL assessment scale specific to
knee osteoarthritis in Japanese. JKOM includes the assessment of a
knee pain by the VAS scale, and questions about pain and stiffness
of the knee joint, difficulties in daily activities, and health
conditions. VAS (Visual Analog Scale) used in this assessment is a
measurement instrument for the amount of sensation that a patient
feels for a particular question. The patient marks, on a 100-mm
straight line, the point that he or she feels represents their
perception of the current state, and the amount of sensation is
determined based on the mark made by the patient. The VAS score is
determined by measuring the distance from the left hand end on the
line to the point that the patient marks. In JKOM, the subjects
answered 25 questions on a five-point scale, including 8 items for
the knee conditions, 10 items for daily activities, 5 items for
going-out activities, and 2 items for health conditions. A smaller
score indicates a higher QOL.
(3) SF-36
[0123] SF-36 (MOS 36-Item Short-Form Health Survey) is a subjective
QOL assessment tool for health conditions. This assessment tool is
widely used in various clinical fields, such as knee osteoarthritis
and psychiatric disorders. SF-36 consists of 36 questions
classified into nine domains: physical functioning, physical role
functioning, bodily pain, general health perceptions, vitality,
social role functioning, emotional role functioning, mental health,
and change in health. A larger score indicates a higher QOL.
Statistical Analysis
[0124] In statistical analysis, in-group comparison was conducted
by Wilcoxon signed-rank test to compare the measured values before
ingestion with those at 6 and 12 weeks after ingestion. Data were
considered significantly different if the critical value was 5% or
less (p<0.05). A significant difference is indicated by a
single, double or triple asterisks: (p<0.05), ** (p<0.01) and
*** (p<0.001). Inter-group comparison was conducted by
Mann-Whitney U test to investigate the changes between before
ingestion and 6 or 12 weeks after ingestion.
4-3 Results (in Men and Women)
[0125] The results of WOMAC assessment are shown in FIGS. 9 to 11.
As shown in FIG. 9, the total score was significantly reduced 6 and
12 weeks after ingestion in the chicken foot extract hydrolysate 50
mg and 200 mg groups as compared with that before ingestion,
indicating the alleviation of knee joint disorders. As apparent
from FIGS. 10 and 11, pain was reduced and daily activities became
easier, confirming that the chicken foot extract hydrolysate is
useful as an agent for improving QOL.
[0126] The results of JKOM assessment are shown in FIGS. 12 to 14.
As evident from FIGS. 12 to 14, JKOM assessment also indicated that
oral ingestion of the chicken foot extract hydrolysate resulted in
the alleviation of knee pain and knee conditions and the reduction
in the JKOM total score, indicating the improvement in QOL.
[0127] The results of SF-36 assessment are shown in FIGS. 15 and
16. As apparent from FIGS. 15 and 16, the bodily pain was reduced
and physical functioning was improved by oral ingestion of the
chicken foot extract hydrolysate.
[0128] The above results indicated that the chicken foot extract
hydrolysate has the effects of alleviating knee pain, knee
conditions and bodily pain and improving physical functioning. Also
indicated is that ingestion of the chicken foot extract hydrolysate
improves daily activities. Therefore, it was evident that the
chicken foot extract hydrolysate is useful as an agent for
improving QOL.
4-4 Results in Women
TABLE-US-00002 [0129] TABLE 2 WOMAC total score In-group
significant difference vs. Inter-group significant before ingestion
difference vs. placebo Women group Yes Yes Men group Yes No 200
mg/day, 12 weeks of ingestion
[0130] The incidence rate of knee osteoarthritis in women is about
4 times higher than that in men, and accordingly high efficacy for
women is desired. Women's data were sampled from the experimental
data, and statistical analysis was performed. In-group comparison
showed significant difference between before and after ingestion in
both men and women groups. On the other hand, inter-group
comparison between the hydrolysate group and the placebo group at
two weeks after ingestion showed that only the women group showed
significant difference in improvement in QOL, in particular, the
WOMAC total score, the difficulty in daily activities and knee
conditions. The analysis revealed that the chicken foot extract
hydrolysate is particularly effective for women, and markedly
reduces knee pain, improves daily activities and alleviates knee
conditions.
Test Example 5: Verification Test of Effect of Chicken Foot Extract
Hydrolysate on the Skin
(1) Cell Assay (In Vitro)
[0131] Human dermal fibroblasts (HS68) were suspended in D-MEM
medium [D-MEM (high glucose) (Wako Pure Chemical Industries, Ltd.)
supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v)
penicillin-streptomycin mixed solution (Nacalai Tesque, Inc.)]. The
suspension was seeded in a 24-well plate at 3.times.10.sup.4
cells/well. The cells were cultured under 5% CO.sub.2 at 37.degree.
C. for 24 hours. The medium was replaced with serum-free D-MEM
medium [D-MEM (high glucose) (Wako Pure Chemical Industries, Ltd.)
supplemented with 1% (v/v) penicillin-streptomycin mixed solution
(Nacalai Tesque, Inc.)], and the chicken foot extract hydrolysate
was added as a sample to the medium at 0.05 mg/mL. To a control
group, no sample was added. The cells were cultured under the same
conditions for 24 hours. The cells were recovered and total RNA was
extracted. cDNA was synthesized from the total RNA by reverse
transcription reaction. Primers for the hyaluronic acid synthase
gene (has2) were added, and real-time PCR was performed using the
cDNA as a template to measure the promotion of the has2 gene
expression by the chicken foot extract hydrolysate.
[0132] The measurement revealed that the addition of the chicken
foot extract hydrolysate at a concentration of 0.05 mg/mL
significantly increased the expression level of has2 (FIG. 17).
[0133] Based on the results, it is expected that the chicken foot
extract hydrolysate will enhance the expression of hyaluronic acid
synthase (HAS2) in dermal cells and promote the production of
hyaluronic acid, thereby increasing moisture and tension of the
skin, leading to improvement in wrinkles and sagging skin.
(2) Oral Ingestion Test on Humans (In Vivo)
[0134] Based on the results of the cell assay, it was expected that
the chicken foot extract hydrolysate would improve moisture and
tension of the skin and wrinkles. To examine these effects, a test
was carried out as described below using subjects consisting of
healthy Japanese men aged between 45 and 69 and Japanese women aged
between 40 and 59 who had not reached menopause, all the subjects
suffering from pain in the knee joints.
[0135] A total of 12 subjects, consisting of 5 men and 7 women at
an average age of 49.8.+-.5.9 years, orally ingested two capsules
per day containing the chicken foot extract hydrolysate (one
capsule contained 25 mg of the chicken foot extract hydrolysate,
223 mg of dextrin, and 2 mg of calcium stearate).
[0136] The ingestion period of the capsule was 12 weeks. The
evaluation test was performed before ingestion and 6 weeks after
ingestion. The VAS questionnaire was used to evaluate the following
items: "skin dryness", "skin tension", and "fine lines at the outer
corner of the eyes". The results are shown in Table 3.
TABLE-US-00003 TABLE 3 Skin conditions (VAS questionnaire, n = 12)
6 weeks after ingestion of Before chicken foot extract Item Unit
ingestion hydrolysate (50 mg/day) Skin dryness mm 57.5 (25.0-66.5)
25.0** (23.0-30.3) Skin tension mm 64.0 (49.0-67.3) 48.5*
(37.5-57.3) Fine lines at outer mm 49.0 (38.5-68.0) 31.5*
(27.5-52.5) corner of eyes Median: the first quartile - the third
quartile *p < 0.05, **p < 0.01 (Wilcoxon signed-rank test vs.
before ingestion)
[0137] As apparent from Table 3, significant improvement was
observed in the "skin dryness" in the chicken foot extract
hydrolysate 50 mg group as compared with before ingestion
(p<0.01). In the same group, significant improvement was also
observed in the "skin tension" and the "fine lines at the outer
corner of the eyes" (p<0.05).
[0138] The above results confirmed that the chicken foot extract
hydrolysate promotes the production of hyaluronic acid in the skin,
increases skin moisture, and improves skin dryness, wrinkles or
skin tension.
(3) Application to Human Skin (In Vivo)
[0139] To evaluate the effect of the chicken foot extract
hydrolysate applied to the skin, an application test of the chicken
foot extract hydrolysate to humans was performed. The subjects were
a total of 10 subjects, consisting of 5 men and 5 women at an
average age of 36.0 years. A 0.1% (w/v) aqueous solution of the
chicken foot extract hydrolysate and a control (water) were applied
to both arms in an amount of 0.05 mL for one week. The skin
moisture content of the applied area was measured with Moisture
Checker MY-808S (Scalar Corporation) before application and after 4
and 7 days of application. The measurement results indicated that
the skin moisture content was significantly increased after 4 and 7
days of application of the solution containing the chicken foot
extract hydrolysate compared with that by the application of water.
The results revealed that the chicken foot extract hydrolysate
increases the moisture content of the skin, which confirmed the
improvement in the moisture of the skin (FIG. 18).
Production Example B: Production of a Sample (a Solution Containing
Phe-Hyp Dipeptide)
[0140] A Phe-Hyp dipeptide, phenylalanyl-4-hydroxyproline, was
produced by liquid-phase synthesis. The obtained Phe-Hyp dipeptide
powder was dissolved in DMEM/F-12 medium to give a sample (a
solution containing the Phe-Hyp dipeptide).
Test Example 6: Promoting Effect of Phe-Hyp Dipeptide on Cartilage
Matrix Production by Chondrogenic Cells
[0141] The mouse chondrogenic cell line ATDC5 (RIKEN BRC No.
RCB0565) was cultured until subconfluence at 37.degree. C. in an
atmosphere of 5 vol % CO.sub.2 and 95 vol % air in DMEM/F-12 medium
supplemented with 5% (w/w) FBS, 1% (w/w) penicillin-streptomycin
solution, 5 .mu.g/mL transferrin, and 3.times.10.sup.-8 M sodium
selenite. The cells were collected by trypsin treatment. The
collected cells were suspended in fresh DMEM/F-12 medium with the
same supplements as above to give a cell suspension
(50.times.10.sup.4 cells/mL). The cell suspension was spot-seeded
at a volume of 20 .mu.L in each well of a 24-well plate, and the
cells were precultured at 37.degree. C. in an atmosphere of 5 vol %
CO.sub.2 and 95 vol % air for 3 hours. The medium in each well was
replaced with fresh DMEM/F-12 medium containing the same
supplements as above and the Phe-Hyp dipeptide (at a final
concentration of 500 .mu.M) and insulin (at a final concentration
of 0.1 .mu.g/mL), and the cells were cultured for 14 days. The
cartilage matrix produced by the chondrogenic cells was stained
with Alcian blue staining solution, the stained cartilage matrix
was quantified with Image J (National Institutes of Health), and
the results were evaluated. The amount of the cartilage matrix
produced by the chondrogenic cells in the sample addition group was
expressed in terms of a relative value, with the amount of the
cartilage matrix in the no sample group taken as 100.
[0142] The results are shown in FIG. 19. The Phe-Hyp dipeptide
promoted cartilage matrix production by chondrogenic cells.
Test Example 7: Examination of Enhancing Effect of Phe-Hyp
Dipeptide on the Expression of Cartilage Differentiation-Related
Genes in Chondrogenic Cells
[0143] The mouse chondrogenic cell line ATDC5 (RIKEN BRC No.
RCB0565) was cultured until subconfluence at 37.degree. C. in an
atmosphere of 5 vol % CO.sub.2 and 95 vol % air in DMEM/F-12 medium
supplemented with 5% (w/w) FBS, 1% (w/w) penicillin-streptomycin
solution, 5 .mu.g/mL transferrin, and 3.times.10.sup.-8 M sodium
selenite. The cells were collected by trypsin treatment. The
collected cells were suspended in fresh DMEM/F-12 medium with the
same supplements as above to give a cell suspension
(10.times.10.sup.4 cells/mL). The cell suspension was seeded at a
volume of 500 .mu.L in each well of a 24-well plate, and the cells
were precultured at 37.degree. C. in an atmosphere of 5 vol %
CO.sub.2 and 95 vol % air for 2 days. The medium in each well was
replaced with fresh DMEM/F-12 medium containing the same
supplements as above and the Phe-Hyp dipeptide (at a final
concentration of 500 .mu.M) and insulin (at a final concentration
of 0.1 .mu.g/mL), and the cells were cultured for 3 days. As a
positive control, 10 .mu.g/mL insulin was used. At end of culture,
total RNA extraction and cDNA synthesis were performed, the
relative mRNA expression levels of cartilage
differentiation-related genes were quantified by real-time PCR and
were normalized to the GAPDH mRNA expression level, and the results
were evaluated. The expression levels in the sample addition groups
were expressed in terms of relative fold change, with the
expression levels in the no sample group taken as 1. Outliers were
rejected by applying the Smirnov-Grubbs test.
[0144] The results are shown in FIGS. 20, 21, 22 and 23. The
Phe-Hyp dipeptide enhanced the expression of the cartilage
differentiation-related genes, SOX9, Acan, Col X and has2. *, * *
and *** indicate the significance level at critical values of 5%,
1%, and 0.1%, respectively.
Test Example 8: Examination of Promoting Effect of Phe-Hyp
Dipeptide on Ex Vivo Hyaluronic Acid Production in Knee Joints of
Mice
[0145] DMEM/F-12 medium supplemented with 1 mg/mL Phe-Hyp
dipeptide, 5% (w/w) FBS, 1% (w/w) penicillin-streptomycin solution,
5 .mu.g/mL transferrin, and 3.times.10.sup.-8 M sodium selenite was
added to each well of a 24-well plate at a volume of 1 mL per well.
The femur and tibia were harvested from the right and left hind
legs of ddY mice (female, retired). The synovial membrane was
incised to expose the cartilage, and the bones were placed in each
well so that only the joint was immersed in the medium. The bones
were incubated at 37.degree. C. in an atmosphere of 5 vol %
CO.sub.2 and 95 vol % air for one day. At end of incubation, the
supernatants were recovered and centrifuged at 1500.times.g at
4.degree. C. for 30 minutes. The supernatants were recovered, the
amount of hyaluronic acid was measured by ELISA and the results
were evaluated. The amount of the hyaluronic acid in the sample
addition group was expressed in terms of a relative value, with the
amount of hyaluronic acid in the no sample group taken as 100.
[0146] The results are shown in FIG. 24. The Phe-Hyp dipeptide
promoted ex vivo hyaluronic acid production. * * indicates the
significance level at a critical value of 1%.
Example 3: Beverage
[0147] A beverage containing the chicken foot extract hydrolysate
produced in the above Production Example A was prepared. Briefly,
ingredients were mixed in accordance with the following formula:
15.0% by mass of saccharide isomerate, 10% by mass of fruit juice,
2.0% by mass of the chicken foot extract hydrolysate, 0.1% by mass
of a flavor, 0.1% by mass of calcium and 72.8% by mass of water.
The mixture was sterilized at 90.degree. C. for 15 seconds with a
plate-type pasteurizer to give a beverage.
Example 4: Yogurt
[0148] A yogurt containing the chicken foot extract hydrolysate
produced in the above Production Example A was prepared. Briefly,
ingredients were mixed in accordance with the following formula:
3.0% by mass of the chicken foot extract hydrolysate, 7% by mass of
sucrose, 0.1% by mass of a flavor, and 89.9% by mass of a yogurt.
The yogurt mixture was filled into a container.
Example 5: Cheese
[0149] A processed cheese containing the chicken foot extract
hydrolysate produced in the above Production Example A was
prepared. Briefly, the following ingredients were mixed: 35% by
mass of Gouda cheese, 35% by mass of Cheddar cheese, 20% by mass of
Parmesan cheese, 2.0% by mass of the chicken foot extract
hydrolysate, 1.0% by mass of calcium phosphate, and 7.0% by mass of
water. The mixture was emulsified at an emulsification temperature
of 85.degree. C. to give a processed cheese.
Example 6: Capsule
[0150] The following ingredients were mixed: 60% by mass of the
chicken foot extract hydrolysate produced in the above Production
Example A, 30% by mass of corn starch and 10% by mass of lactose.
The mixture was filled into empty gelatin capsules (200 mg per
capsule) to give capsules.
Example 7: Tablet
[0151] The following ingredients were mixed: 60% by mass of the
chicken foot extract hydrolysate produced in the above Production
Example A, 18% by mass of hydrogenated maltose, 18% by mass of
crystalline cellulose and 4% by mass of sucrose ester. The mixture
was compressed into tablets (300 mg per tablet).
Examples 8 to 12
[0152] A beverage (Example 8), a yogurt (Example 9), a cheese
(Example 10), a capsule (Example 11), and a tablet (Example 12),
each containing the Phe-Hyp dipeptide, were produced in the same
manner as in Examples 3 to 7 except that the Phe-Hyp dipeptide
produced by liquid-phase synthesis was used in place of the chicken
foot extract hydrolysate.
[0153] The present invention is not limited to each of the
embodiments and Examples described above, and various modifications
are possible within the scope of the claims. Embodiments obtainable
by appropriately combining the technical means disclosed in the
different embodiments of the present invention are also included in
the technical scope of the present invention.
INDUSTRIAL APPLICABILITY
[0154] The present invention is useful as a hyaluronic acid
production promoting agent, in particular, a medicament, a food, a
cosmetic product, a quasi-drug, or an animal feed for promoting
hyaluronic acid production.
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