NZ614126B2 - Method for mass preparation of proteoglycan - Google Patents
Method for mass preparation of proteoglycan Download PDFInfo
- Publication number
- NZ614126B2 NZ614126B2 NZ614126A NZ61412612A NZ614126B2 NZ 614126 B2 NZ614126 B2 NZ 614126B2 NZ 614126 A NZ614126 A NZ 614126A NZ 61412612 A NZ61412612 A NZ 61412612A NZ 614126 B2 NZ614126 B2 NZ 614126B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- proteoglycan
- cartilage
- frozen
- small pieces
- heating
- Prior art date
Links
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- 210000000845 Cartilage Anatomy 0.000 claims abstract description 95
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- 238000010438 heat treatment Methods 0.000 claims abstract description 48
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4725—Proteoglycans, e.g. aggreccan
Abstract
Disclosed is a method for extracting proteoglycan from fish cartilage, comprising the step of heating small pieces of frozen fish cartilage in water, wherein each small piece of frozen fish cartilage is 0.001 to 0.5 g.
Description
DESCRIPTION
Title of Invention: METHOD FOR MASS PREPARATION OF PROTEOGLYCAN
Technical Field
[0001]
The present invention relates generally to a method for
preparing proteoglycan. More specifically, the present invention
relates to, in particular, a proteoglycan extraction method from
fish cartilage enabling mass preparation of high-molecular-weight
proteoglycan with high efficiency.
Background Art
Proteoglycan is one of the major biological
macromolecules for forming the substrate of the extracellular
matrix of connective tissue, as are collagen and others.
Proteoglycan was hitherto obtained by being extracted and
isolated from mammal cartilage (in particular, bovine cartilage).
However, ever since the occurrence of bovine spongiform
encephalopathy (BSE) was reported, the use of bovine cartilage
has been avoided. Thus, there has been a need for an alternative
source of proteoglycan, and a production method for the
alternative source. In addition, since the production cost for
proteoglycan is very high due to the complexity of the existing
proteoglycan extraction step and the low yield, industrial
application of proteoglycan has not been fully accomplished. Thus,
there has been a need for a simpler method ensuring a greater
yield.
As an alternative source for proteoglycan, aquatic
animal tissue is attracting attention. Therefore, there have been
attempts to extract proteoglycan from cartilage of aquatic
animals, such as whales and sharks. However, due to catch
restrictions placed on these aquatic animals, it has been
difficult to produce a large amount of proteoglycan. Moreover,
the extraction and isolation of proteoglycan is complicated, and
some solvents, etc., used for extraction have relatively high
toxicity.
Under such circumstances, there have been attempts to
extract proteoglycan from the large amount of discarded aquatic
animal tissues (e.g., salmon nasal cartilage). Patent Document 1
discloses a method for producing a composition (nasal cartilage
powder) containing proteoglycan from salmon nasal cartilage. In
particular, since proteoglycan derived from salmon nasal
cartilage is assumed to have an effect for treating or preventing
inflammatory enteric diseases, demand for proteoglycan is
increasing. However, the method of Patent Document 1 cannot
ensure sufficiently high production efficiency; thus, the method
has yet to be improved in terms of efficiency.
Citation List
▪ Patent Documents
Patent Document 1: JP2009-173702A
Summary of Invention
▪ Technical Problem
An object of the present invention is to efficiently
extract proteoglycan from aquatic animal tissues and/or to at
least provide the public with a useful choice.
▪ Solution to Problem
[0007]
Surprisingly, the inventors of the present invention
found that a method of freezing fish cartilage, pulverizing the
cartilage into small pieces, and heating the pieces in water
enables extraction of proteoglycan with high efficiency. After
several attempts to further improve the method, the inventors
completed the present invention.
Specifically, the present invention relates to a method
for extracting proteoglycan from fish cartilage, comprising the
step of (A) heating small pieces of frozen fish cartilage in
water, wherein each small piece of frozen fish cartilage is 0.001
to 0.5 g.
[0008a]
In another embodiment the present invention relates to a
method for increasing efficiency in proteoglycan extraction from
fish cartilage, comprising the step of heating small pieces of
frozen fish cartilage in water, wherein each small piece of
frozen fish cartilage is 0.001 to 0.5 g.
[0008b]
Certain statements that appear below are broader than what
appears in the statements of the invention above. These
statements are provided in the interests of providing the reader
with a better understanding of the invention and its practice.
The reader is directed to the accompanying claim set which
defines the scope of the invention.
[0008c]
Also described herein are proteoglycan extraction methods
(i.e., proteoglycan production methods) and methods for improving
efficiency in proteoglycan extraction set forth in the following
Items 1 to 11.
It is noted here that the claims which define the scope of
this invention are set out on pages 27-28. Further embodiments
described below as a set of Items are provided in the interests
of providing the reader with a better understanding of the
invention and its practice, and are illustrative only.
Item 1
A method for extracting proteoglycan from fish
cartilage, comprising the step of (A) heating small pieces of
frozen fish cartilage in water.
Item 2
The method according to Item 1, wherein each small
piece of frozen fish cartilage is 0.001 to 0.5 g.
Item 3
The method according to Item 1 or 2, wherein the small
pieces of frozen fish cartilage are obtained by freezing fish
cartilage and then pulverizing the frozen fish cartilage.
Item 4
The method according to Item 3, wherein the fish
cartilage is defatted fish nasal cartilage.
Item 5
The method according to any one of Items 1 to 4,
wherein the extracted proteoglycan contains proteoglycan having a
molecular weight of not less than 900,000 (90 × 10).
Item 6
The method according to any one of Items 1 to 5,
wherein the heating in Step (A) is performed for more than 3
hours.
Item 7
The method according to any one of Items 1 to 6,
wherein the temperature of water used in the heating in Step (A)
is not less than 80˚C.
Item 8
The method according to any one of Items 1 to 7,
wherein the mass ratio of all the small pieces of frozen fish
cartilage to water is 1:1 to 1:10.
Item 9
The method according to any one of Items 1 to 8,
wherein the heating in Step (A) is continued until the mass of a
residue collected after the heating is equal to or less than the
mass of the heated small pieces of frozen fish cartilage.
Item 10
The method according to Item 9, wherein the residue
collected after the heating is a precipitate obtained by
centrifugation at 5000 rpm, 4˚C, for 20 minutes.
Item 11
A method for increasing efficiency in proteoglycan
extraction from fish cartilage, comprising the step of heating
small pieces of frozen fish cartilage in water.
▪ Advantageous Effects of Invention
The proteoglycan extraction method of the present
invention enables easy extraction of proteoglycan from fish
cartilage with very high efficiency. In particular, the
proteoglycan extraction method of the present invention enables
extraction of high-molecular-weight proteoglycan. Further, since
in the extraction method of the present invention, extraction is
performed by using only water, the method ensures safety in the
extraction and safety of the resulting proteoglycan product,
compared with hitherto known extraction methods using organic
solvents or acids/alkali. Furthermore, the step of removing the
organic solvents is usually cumbersome, but this step is not
necessary in the extraction method of the present invention. In
addition to, or in alternative of the advantageous effects
described above, an advantage of the invention is to at least
provide the public with a useful choice.
Brief Description of Drawings
Fig. 1a is a photo of a frozen salmon nasal cartilage
block placed in a tray.
Fig. 1b is a photo of small pieces of frozen salmon
nasal cartilage placed in a plastic bag.
Fig. 1c is a photo of defatted salmon nasal cartilage
powder placed in a plastic bag.
Fig. 2 is a graph showing change in yield of
proteoglycan over time when water was added to small pieces of
frozen salmon nasal cartilage, and extraction was performed at
100°C.
Fig. 3 is a graph showing a comparison of the yields of
uronic acid (indicated as “GlcA”, an abbreviation for glucuronic
acid) and the yields of protein (indicated as “Protein”) when
proteoglycan is extracted from defatted salmon nasal cartilage
powder (“Powder”), frozen salmon nasal cartilage blocks
(“Blocks”), or small pieces of frozen salmon nasal cartilage
(“Small pieces”). The yields of GlcA and protein are represented
in terms of amounts extracted per 100 g of frozen salmon nasal
cartilage blocks. The amount of uronic acid reflects the amount
of proteoglycan.
Fig. 4 is a chromatogram drawn based on measurement
values obtained by separating collected proteoglycan-containing
supernatant extracted from small pieces of frozen salmon nasal
cartilage by gel filtration chromatography and measuring the
amount of uronic acid (GlcA) in each fraction and absorbency at
280 nm of each fraction.
Fig. 5 is a chromatogram drawn based on measurement
values obtained by separating collected proteoglycan-containing
supernatant extracted from small pieces of frozen salmon nasal
cartilage by anion exchange chromatography and measuring the
amount of uronic acid (GlcA) in each fraction and absorbency at
280 nm of each fraction.
Description of Embodiments
The present invention is described in more detail below.
The term “mass” in this specification is equivalent to “weight”.
The proteoglycan extraction method of the present
invention is a method for extracting proteoglycan from fish
cartilage. The method comprises the step of (A) heating small
pieces of frozen fish cartilage in water.
The fish cartilage is cartilage obtained from fish,
preferably from Oncorhynchus (Salmonidae). Examples of the fish
include trout (humpback salmon, cherry salmon, satsukimasu salmon,
etc.), salmon (chum salmon, sockeye salmon, silver salmon,
chinook salmon, steelhead, etc.), shark, and cod. Salmon and
trout are particularly preferable. The cartilage to be used is
not particularly limited; however, head cartilage, in particular,
nasal cartilage, is preferable. Moreover, since fish heads are
usually discarded when fish is processed into food products, the
cost of fish heads is low, and a large amount of fish heads can
be stably supplied.
The small frozen pieces of fish cartilage can be
obtained either by (i) freezing fish cartilage and then
pulverizing it into small pieces or (ii) pulverizing fish
cartilage into small pieces and then freezing it. It is also
possible to use (iii) frozen fish cartilage itself. The small
frozen pieces of fish cartilage obtained by method (i) are
particularly preferable in the present invention. The present
invention also encompasses a proteoglycan extraction method
further comprising, before Step (A), the steps of (α) freezing
fish cartilage and/or (β) pulverizing fish cartilage into small
pieces.
The freezing method is not particularly limited, and
any known freezing method can be used. For example, a method of
storing fish cartilage in a freezer at about -20 to -80°C for
about 24 to 72 hours can be used.
The pulverization may be performed using a known method.
For example, the pulverization of fish cartilage (preferably
frozen fish cartilage) into powder may be performed using known
devices such as a blender or a mill. The pulverization is
preferably performed at a low temperature (e.g., not more than
4°C).
Each small piece of frozen fish cartilage is preferably
about 0.001 to 0.5 g, more preferably about 0.005 to 0.3 g,
further preferably about 0.001 to 0.1 g. The pulverization of
fish cartilage into small pieces is preferably performed in a
manner enabling production of such small pieces of frozen fish
cartilage. Further, if the small pieces of frozen fish cartilage
having a weight in the above range are obtained after freezing
fish cartilage, it is not necessary to perform the pulverization.
Although it is not particularly limited, the small pieces of
frozen fish cartilage having a weight in the above range are
preferably not less than 50 mass%, more preferably not less than
70 mass%, further preferably not less than 90 mass% among all the
small pieces of frozen fish cartilage subjected to heating. This
ratio is found by randomly selecting 20 pieces from all the small
pieces of frozen fish cartilage subjected to heating, measuring
the mass of each of the 20 small pieces, and calculating the
proportion (%) of the pieces that have a weight in the above
range in the 20 small pieces. “All the small pieces” herein
refers to a group of small pieces, i.e., an collection consisting
of multiple small pieces.
Although it is not particularly limited thereto,
defatted fish cartilage is preferably used. By using defatted
fish cartilage, a highly purified proteoglycan-containing fish
cartilage extract that incorporates less lipid can be obtained.
The defatting may be performed by using a known method. For
example, a method of running fish cartilage under water (e.g.,
tap water) for about 1 to 24 hours can be performed. Preparation
of fish cartilage can be performed using a known method,
including a method of immersing fish tissues (preferably a fish
head) in water for about 1 to 24 hours to make the tissues swell,
and removing tissues other than cartilage (preferably nasal
cartilage), and a method of thawing a frozen salmon head, then
immediately separating the nasal cartilage and running the nasal
cartilage under water for about 1 to 24 hours, thereby washing
and defatting the cartilage. If the cartilage has residual flesh,
it is preferable to remove the flesh with tweezers or the like.
The proteoglycan extracted by the proteoglycan
extraction method of the present invention contains a high-
molecular-weight proteoglycan. The inventors of the present
invention suggest that the effect of treating and preventing
inflammatory enteric diseases of the proteoglycan increases as
the molecular weight of proteoglycan increases. Thus, the present
invention, which enables production of high-molecular-weight
proteoglycan, is advantageous also in this regard. The term
“high-molecular-weight proteoglycan” used herein specifically
refers to a proteoglycan having a molecular weight of not less
than 900,000 (90 × 10 ), preferably not less than 1,000,000 (100 ×
), more preferably not less than 1,200,000 (120 × 10 ). The
present invention is assumed to be capable of producing a
proteoglycan having a molecular weight of not less than 2,500,000
(250 × 10 ), or even 5,000,000 (500 × 10). The preferable high-
molecular-weight proteoglycan is determined by subjecting the
proteoglycan-containing extract obtained by the proteoglycan
extraction method of the present invention to gel filtration
chromatography under the following conditions, determining the
uronic acid amount (reflecting the proteoglycan amount) in each
fraction by using a carbazole-sulfuric acid method, creating a
chromatogram based on the determined uronic acid amounts, and
confirming that the peak of the chromatogram is equal to or more
than the above range of molecular weight (not less than 900,000
(90 × 10 ), preferably not less than 1,000,000 (100 × 10 ), more
preferably not less than 1,200,000 (120 × 10)). Such a
chromatogram based on the uronic acid amount may be hereinafter
referred to as “proteoglycan uronic acid amount chromatogram.
Further, it is also possible to make a chromatogram (reflecting
the protein amount) based on the absorbencies by measuring the
absorbencies of the fractions at 280 nm, and then finding the
relative values of the protein amounts based on the measurement
results (i.e., the measurement values are assumed to be values
that reflect the protein amounts). Hereinafter, such a
chromatogram may be referred to as “proteoglycan protein amount
chromatogram”.
Gel Filtration Chromatography
Column: Sepharose CL-4B packed column (1-cm dia. x 38.5 cm column
packed with Sepharose CL-4B as a carrier. Sepharose CL-4B is
available from, for example, GE Healthcare and other companies.
Sepharose CL-4B, CAS registry No. 619709, is a 4% crosslinked
agarose with a particle size of 40 to 165 μm (measured by the
laser diffraction scattering method).)
Buffer: 0.1 M phosphate buffer (pH of 7.0, containing 0.2 M NaCl)
Amount of fraction: 1 mL/tube
Molecular weight analytical curve: An analytical curve
for use is prepared by subjecting the various dextran molecular
weight markers described below to gel filtration chromatography
under the same conditions as described above and measuring the
absorbency (which reflects the amount of dextran) of each
fraction by the phenol-sulfuric acid method, which is a well-
known method for detecting sugar chains.
Dextran Molecular Weight Markers
Dextran Standard 1,400,000 (Sigma) 1400 kDa
Dextran Standard 670,000 (Sigma) 670 kDa
Dextran Standard 410,000 (Sigma) 410 kDa
Dextran Standard 270,000 (Sigma) 270 kDa
Quantification of dextran (absorbency measurement) is performed
as follows, according to the method described in Hodge, J. E.,
and Hofreiter, B. T., Method in Carbohydrate Chemistry, 1, 338
(1962).
500 μl of a sample aqueous solution or a standard
monosaccharide (mannose) aqueous solution is placed in a 105 × 15
mm test tube.
500 μl of a phenol reagent (5 v/v% aqueous phenol solution)
is added thereto, and the mixture is stirred.
2.5 mL of concentrated sulfuric acid is added thereto, and
immediately the mixture is stirred vigorously for 10 seconds.
The mixture is left to stand for 20 minutes or more at room
temperature.
[5] The absorbency at 490 nm is measured with a spectrophotometer.
The carbazole-sulfuric acid method refers to a well-
known method performed by adding a carbazole solution, which is a
color component of uronic acid (glucuronic acid (Glc A), iduronic
acid, etc.), to a measurement specimen, and measuring the
absorbency by using a spectrophotometer. An analytical curve is
plotted using the glucuronic acid standard solution having a
specific concentration, thereby finding the glucuronic acid
content in the specimen. More specifically, the carbazole-
sulfuric acid method is performed as follows. 2.5 ml of a reagent
obtained by dissolving 0.95 g of sodium borate decahydrate in 100
ml of a concentrated sulfuric acid is placed in a test tube and
ice-cooled. 0.5 ml of a test object (preferably containing 2 to
μg of uronic acid) is gently layered thereon. The mixture is
stirred well while being ice-cooled, thereby keeping it at room
temperature or below. After the test tube is covered with a glass
ball, the test tube is heated in a boiling water bath for 10
minutes, followed by water cooling to decrease the temperature to
room temperature. Then, 0.1 ml of a reagent obtained by
dissolving 125 mg of carbazole in 100 ml of anhydrous methyl
alcohol is added and mixed therewith, and the mixture is heated
in a boiling water bath for 15 minutes. Thereafter, the mixture
is water-cooled to room temperature, and the absorbency at 530 nm
is measured. In the blank test, 0.5 ml of distilled water is used.
Simultaneously, an analytical curve is plotted using a glucuronic
acid.
The heating in Step (A) is performed to the extent in
which the effect of the present invention is ensured. Although
the heating conditions are not limited insofar as the effect of
the present invention is obtained, an example of the heating
conditions is as follows. Although it depends on the heating
temperature, the heating time is preferably more than 3 hours,
more preferably not less than 3.5 hours, further preferably not
less than 4 hours. Although it depends on the heating time, the
temperature of water used for the heating is preferably not less
than 80°C, more preferably not less than 90°C, further preferably
a boiling temperature (100°C or more under 1 atmospheric
pressure).
[0025]
Further, although the amount of small pieces of frozen
fish cartilage to be subjected to heating and the water amount
may be suitably determined, it is preferable to immerse all the
small pieces in water. Specifically, the mass ratio of all the
small pieces to water (all the small pieces:water) is preferably
about 1:1 to 1:10.
When the small pieces of frozen fish cartilage are
immersed in water, the small fish cartilage pieces swell as the
water permeates into the pieces. Further, during the heating, the
small swollen fish cartilage pieces are gradually softened and
deformed, and finally become a thick fluid partially dissolved in
water. Therefore, the appropriate heating level may be determined
according to the mass of the small swollen fish cartilage pieces
after the heating (i.e., the residue remaining after the heating).
More specifically, in the present invention, it is preferable to
perform the heating until the mass of all the small swollen fish
cartilage pieces (i.e., residue) resulting from the heating falls
below the total mass of the small pieces of frozen fish cartilage
subjected to the heating; more preferably, the heating is
performed until the mass of all the small swollen fish cartilage
pieces (i.e., residue) resulting from the heating falls to 70% or
less, more preferably 50 mass% or less, of the total mass of the
small pieces of frozen fish cartilage used for the heating.
[0027]
The expression “all the small swollen fish cartilage
pieces” (i.e., residue) herein refers to a precipitate obtained
by 20-minute centrifugation at 5000 rpm and 4°C (the precipitate
obtained by removing the solution after the centrifugation).
[0028]
The liquid portion (water) of the product obtained
after Step (A) contains a large amount of proteoglycan. Therefore,
by collecting the liquid portion, it is possible to obtain a
proteoglycan-containing extract. The collection of the liquid
portion is performed by removing the supernatant through, for
example, a centrifugation treatment (preferably the
centrifugation under the above conditions). The liquid
(supernatant) may be used as it is, or it may further be purified
by a known method. It is also possible to concentrate the liquid
by distillation, freeze-drying, or the like. It is also possible
to powderize the liquid according to the freeze-drying method or
the spray drying method. The other processes may also be
performed insofar as the effects of the present invention are not
impaired. The proteoglycan thus obtained may be used as, for
example, materials for food, cosmetics, medicinal products, and
the like.
The usage of the proteoglycan obtained by the method of
the present invention is not limited; however, since the
proteoglycan provides the aforementioned effects, the
proteoglycan obtained by the present invention is suitable for
compositions for external use or oral compositions. More
specifically, a preferable usage is a composition for external
use or an oral composition containing the proteoglycan obtained
by the method of the present invention. The proteoglycan-
containing extract may be used directly as a composition for
external use or as an oral composition. The composition for
external use or an oral composition may be used, for example, as
a medicinal composition, a quasi-drug composition, a cosmetic
composition, or a food composition. These may be produced by a
standard method using the proteoglycan obtained by the method of
the present invention. They are particularly useful for products
in the oral-care industry, cosmetics industry, and food and drink
industry.
[0030]
The oral compositions containing the proteoglycan-
containing extract obtained by the method of the present
invention (which may be hereinafter referred to as oral
compositions as described herein) used in the oral-care industry
may be the proteoglycan-containing extract itself, or a
composition produced by appropriately combining the proteoglycan-
containing extract with other components (e.g., abrasives,
foaming agents, cleaners, surfactants, wetting agents, pH
adjusters, thickeners, flavoring agents, and the like) generally
used for oral compositions. Examples of the oral composition
products include paste agents, ointments, gels, embrocations,
sprays, supplements, liquids, mouthwashes, pasta, chewing gum,
troches, and tablets, which may be manufactured by standard
methods.
[0031]
Such oral compositions can be preferably used for
alleviation of inflammation in oral tissues, or anti-aging in
oral tissues. More specifically, the oral compositions described
herein encompass an inflammation alleviation oral composition and
an anti-aging oral composition.
The cosmetic composition (hereinafter may be referred
to as “cosmetic composition”) containing the proteoglycan-
containing extract obtained by the method of the present
invention may be the proteoglycan-containing extract described
herein itself, or a composition produced by appropriately
combining the proteoglycan-containing extract with cosmetically
acceptable media, bases, carriers, additives, or other
cosmetically acceptable components or materials using a standard
method. More specifically, the cosmetic compositions produced by
incorporating the proteoglycan-containing extract described
herein include emulsions, lotions, creams, serums, foundation,
face masks, and sunscreens. Such a cosmetic composition as
described herein may be preferably used for alleviation of
inflammation or for anti-aging. Examples of preferable usages
include compositions for sun protection, sunburn care,
moisturizing and anti-aging of the skin (e.g., prevention or
alleviation of dry skin, rough skin, facial wrinkles, or sagging
skin).
[0033]
The food and beverage compositions (food and beverages)
containing the proteoglycan-containing extract obtained by the
method of the present invention (which may be hereinafter
referred to as food and beverage compositions) used in the food
industry may be the proteoglycan-containing extract itself, or a
composition produced by appropriately combining the proteoglycan-
containing extract with bases, carriers or additives that are
acceptable in terms of food hygiene, or other components or
materials that are used for food and beverages. Examples of these
include processed food and beverages containing the proteoglycan-
containing extract with claimed effects of moisturizing and anti-
aging of the skin (e.g., prevention or alleviation of dry skin,
rough skin, facial wrinkles, or sagging skin), health food (food
with nutrient function claims, food for specific health uses,
etc.), dietary supplements, beauty food, and food for patients.
Moreover, also described herein are moisturizers and skin anti-
aging agents formed of the aforementioned food and beverage
compositions. The moisturizers and skin anti-aging agents may be
supplied in the forms of drinks, pills, tablets, capsules,
granules, jelly, troches, or the like for cosmetic or skin anti-
aging purposes (e.g., prevention or alleviation of dry skin,
rough skin, facial wrinkles, or sagging skin).
The amount of the proteoglycan-containing extract
contained in the oral compositions, cosmetic compositions, or
food or beverage compositions described herein is, for example,
but not limited to, generally 0.001 to 100 mass%, preferably 0.01
to 95 mass%, based on the entire composition.
The proteoglycan-containing extract obtained by the
proteoglycan extraction method of the present invention contains
a proteoglycan extracted with high efficiency. More specifically,
based on the uronic acid amount (i.e., based on the uronic acid
amount found by the carbazole-sulfuric acid method), not less
than 60 mass%, preferably not less than 70 mass%, more preferably
not less than 80 mass%, further preferably not less than 90 mass%
of the proteoglycan contained in the small pieces of frozen fish
cartilage used for the extraction can be extracted by the method
of the present invention. Further, as described above, the
proteoglycan obtained by the proteoglycan extraction method of
the present invention is a high-molecular-weight proteoglycan.
More specifically, the proteoglycan-containing extract obtained
by the proteoglycan extraction method of the present invention
preferably contains a proteoglycan having a molecular weight of
not less than 900,000 (90×10) (more preferably a molecular weight
of not less than 1,000,000 (100×10 ), further preferably a
molecular weight of not less than 1,200,000 (120×10)). The
proteoglycan having the molecular weight of not less than the
above range is preferably not less than 60 mass%, more preferably
not less than 70 mass%, further preferably not less than 80 mass%,
further more preferably not less than 90 mass% of all the
proteoglycan extracted. The proportion can be found from a peak
area in the aforementioned proteoglycan uronic acid amount
chromatogram by calculating a proportion of the area of a
proteoglycan having a molecular weight of not less than the above
range. More specifically, the proportion can be found by
calculating a proportion of the area of a proteoglycan having a
molecular weight of not less than the above range based on the
entire peak area of the proteoglycan uronic acid amount
chromatogram.
Further, described herein is a method for increasing
efficiency in proteoglycan extraction from fish cartilage,
comprising the step of heating small pieces of frozen fish
cartilage in water. In this method, the preparation of small
pieces of frozen fish cartilage, the heating, the measurement of
proteoglycan extraction efficiency, and the like may be performed
using the aforementioned methods and conditions.
According to the methods described above, the present
invention enables easy extraction of proteoglycan from fish
cartilage with very high efficiency, as well as increasing
efficiency in proteoglycan extraction from fish cartilage. In
particular, the method of the present invention enables
extraction of high-molecular-weight proteoglycan. Although a
restrictive interpretation is not desired, the present invention
succeeded in such a highly efficient extraction of proteoglycan
(in particular, a high-molecular-weight proteoglycan) not only by
using small pieces of fish cartilage, but also by pulverizing
frozen fish cartilage into small pieces. More specifically, in
this method, the function of the enzyme (in particular, the
enzyme for decomposing proteoglycan) contained in the bone tissue
is assumed to be suppressed, and the heating process is assumed
to further deactivate the enzyme. In this view, it is further
assumed that the fish cartilage is preferably handled at a low
temperature, and that the water used for extraction is preferably
heated to a high temperature at the time of addition of the small
pieces of frozen fish cartilage.
Examples
The present invention is described below in more detail.
However, the scope of the invention is not limited to these
Examples.
Analysis of Extraction of Proteoglycan from Salmon Nasal
Cartilage
Samples Used for Extraction
The following three types ((1) to (3)) of samples
derived from salmon nasal cartilage were used for analyzing
extraction of proteoglycan. Salmon nasal cartilage used for the
analysis was obtained by separating nasal cartilage immediately
after thawing a frozen salmon head, washing and defatting the
nasal cartilage by running it under water for 6 hours, removing
pieces of flesh and the like with tweezers, and washing the nasal
cartilage with water by hand.
(1) Frozen Salmon Nasal Cartilage Blocks
Salmon nasal cartilage was stored and frozen in a
freezer, and the frozen nasal cartilage was used as a frozen
salmon nasal cartilage block. The frozen salmon nasal cartilage
block had a size of about 2.5 × 1.5 cm to about 4.5 × 2 cm and a
weight of about 1.71 g to about 6.91 g. (The average weight of 7
blocks was 3.701 g.), although the size and weight depend on the
size of the salmon head used. Fig. 1a shows a photo of a frozen
salmon nasal cartilage block.
(2) Small Pieces of Frozen Salmon Nasal Cartilage
Frozen salmon nasal cartilage blocks from item (1)
above were placed in a blender and crushed for 10 seconds to
prepare small pieces of frozen salmon nasal cartilage. Fig. 1b
shows a photo of small pieces of frozen salmon nasal cartilage.
Twenty pieces were randomly collected, and the size and the
weight of each piece were analyzed. Each piece had a size of
about 0.2 cm to about 0.7 cm and a weight of about 0.0116 g to
about 0.0890 g. (The average weight of 20 pieces was 0.033 g.)
From 100 g of the frozen salmon nasal cartilage blocks, 95.6 g of
small pieces of frozen salmon nasal cartilage was obtained.
(3) Defatted Salmon Nasal Cartilage Powder
Proteoglycan composition powder was prepared using
frozen salmon nasal cartilage from item (1) above by the method
disclosed in Example 1 of Patent Document 1 (JP2009-173702A).
This powder was used as defatted salmon nasal cartilage powder.
From 100 g of the frozen salmon nasal cartilage blocks, 5.83 g of
defatted salmon nasal cartilage powder was obtained. Fig. 1c
shows a photo of defatted salmon nasal cartilage powder.
The following is an excerpt from the disclosure of
Example 1 of Patent Document 1.
“Frozen salmon nasal cartilage (100 g) was crushed, and
an equal volume of tap water at 15°C was added to the
crushed salmon nasal cartilage. The mixture was gently
stirred to mix it thoroughly, and the mixture, which was
maintained at about 5°C, was immediately centrifuged
with a centrifugal separator at 9,000 rpm at 4°C for 30
minutes to separate lipid and other components including
proteoglycan. Three layers were obtained after the
centrifugation. The lipid layer in the upper layer and
the aqueous layer in the middle layer were removed, and
the precipitate was collected. The precipitate was
freeze-dried and then pulverized with a centrifugal mill
to prepare a water-defatted fine powder. At this stage,
some of the fine powder was subjected to ether
extraction for measurement of lipid, and it was found
that 8.8% lipid remained, that the removal rate was
75.0% when lipid before defatting was defined as 100%,
and that the lipid had a faint foul odor. Subsequently,
a 10-fold volume of ethanol was added to the water-
defatted fine powder to dissolve and extract the lipid
with the foul odor. This operation was repeated twice.
The ethanol solution was filtered off and the solvent
was evaporated to obtain a pale yellow-brown, odorless
proteoglycan composition powder. The yield relative to
the salmon nasal cartilage was 58.7% (dry basis), and
the proteoglycan content was 77.7%. The foul odor of the
proteoglycan composition powder completely disappeared.”
The “frozen salmon nasal cartilage” of Example 1 of Patent
Document 1 corresponds to the “frozen salmon nasal cartilage
blocks” described above. The term “dry basis” means a dry mass
basis.
Analysis of Proteoglycan Extraction Using Small Pieces of Frozen
Salmon Nasal Cartilage
Extraction of proteoglycan was attempted by adding
water to small pieces of frozen salmon nasal cartilage prepared
as described above in item (2), and heating the mixture at 100°C.
More specifically, the analysis was performed as follows. Four
samples were prepared. Each sample was prepared by adding 60 mL
of distilled water to about 12 g of the small pieces of frozen
salmon nasal cartilage. These four samples were heated at 100°C
for 1 hour, 2 hours, 3 hours, and 4 hours, respectively, and each
sample was centrifuged with a centrifugal separator at 5,000 rpm
at 4°C for 20 minutes to remove insoluble matter (residue) and
collect the supernatant. After the amount of the collected
supernatant was measured, the amount of proteoglycan in the
supernatant was measured as a uronic acid equivalent by the
carbazole-sulfuric acid method. (In tables and figures, uronic
acid is represented by the term “GlcA,” an abbreviation of
glucuronic acid.) In addition, the amount of protein in the
collected supernatant was measured by the Bradford method. The
results are shown in Table 1. Fig. 2 is a graphical
representation of Table 1.
[0041]
Table 1
Heating Amount Per gram of GlcA/protein Residue
time of small pieces of amount (g)
(amount of liquid frozen salmon and state
small (mL) nasal cartilage
pieces
GlcA Protein
(g)) (mg) (mg)
1 hour 33.83
31.0 4.507 0.441 10.22
Slightly soft
(12.261 g)
28.49
Jelly-like
state; shape
2 hours
40.0 8.069 0.883 9.14 easily
(12.322 g)
collapses
when pressed
with a finger
21.79
3 hours
45.5 8.636 1.039 8.31
About to lose
(12.599 g)
its shape
.56
4 hours
No shape,
61.5 15.592 3.401 4.58
mushy; small
(12.405 g)
amount
From Table 1 and Fig. 2, it was found that the
extraction amount of proteoglycan increases as the heating time
is increased. It was also found that heating for more than 3
hours (e.g., 4 hours), in particular, increases the extraction
amount of proteoglycan significantly.
Comparison of Small Pieces of Frozen Salmon Nasal Cartilage,
Frozen Salmon Nasal Cartilage Blocks, and Defatted Salmon Nasal
Cartilage Powder
Proteoglycan was also extracted from the frozen salmon
nasal cartilage blocks from item (1) and from the defatted salmon
nasal cartilage powder from item (3) under the same conditions as
for the small pieces of frozen salmon nasal cartilage from item
(2) above, and the extraction amounts thereof were compared. The
results are shown in Table 2. In Table 2, the amount of extracted
proteoglycan and the amount of extracted protein are represented
in terms of amounts per 100 g of the frozen salmon nasal
cartilage blocks. Fig. 3 is a graphical representation of Table 2.
The descriptions of the samples (1 to 9) shown in Fig. 3
correspond to the descriptions in the Graph No. column in Table 2.
Table 2
Extraction amounts per 100 g of the Frozen Salmon Nasal Cartilage
Blocks (defatted salmon nasal cartilage powder: 5.83 g, small
pieces of frozen salmon nasal cartilage: 95.6 g)
Graph Heating Heating GlcA/protein
GlcA Protein
No. temperature time
(mg) (mg)
Defatted
1 4°C 2 hours 186.68 45.88 4.07
salmon
nasal
2 100°C 2 hours 581.43 136.25 4.27
cartilage
powder
3 100°C 4 hours 733.15 215.30 3.41
Frozen
4 4°C 2 hours 96.90 - -
salmon
nasal
100°C 2 hours 499.90 96.90 5.16
cartilage
blocks
6 100°C 4 hours 832.10 234.85 3.54
Small
7 4°C 2 hours 150.67 18.55 8.12
pieces of
frozen
8 100°C 2 hours 771.40 84.41 9.14
salmon
nasal
9 100°C 4 hours 1490.60 325.14 4.58
cartilage
From Table 2 and Fig. 3, it was found that the amount
of extracted glucuronic acid (reflecting the amount of
proteoglycan) significantly increased when the small pieces of
frozen salmon nasal cartilage were heated at 100°C in water for 4
hours. In particular, considering the fact that the amount of
uronic acid (reflecting the amount of proteoglycan) contained per
100 g of the salmon nasal cartilage was about 1.6 g (value
determined using the carbazole-sulfuric acid method by crushing
the nasal cartilage after freeze-drying and by performing
extraction with 4 M guanidine hydrochloride/50 mM acetate buffer),
the amount of uronic acid extracted at 100°C for 4 hours from the
frozen salmon nasal cartilage blocks was 832 mg (extraction
percentage: 52%), whereas the amount of uronic acid extracted at
100°C for 4 hours from the small pieces of frozen salmon nasal
cartilage was 1490.6 mg, and the extraction percentage thereof
was 93.2%. Proteoglycan extraction methods with such high
extraction efficiency have not been known. It can be said that
the present invention has made such high extraction efficacy
possible for the first time. Note that since 5.83 g of the
defatted salmon nasal cartilage powder was obtained from 100 g of
the frozen salmon nasal cartilage blocks, the amount of uronic
acid obtained when the defatted salmon nasal cartilage powder was
subjected to extraction at 100°C for 4 hours was 733 mg in terms
of the amount per 100 g of the nasal cartilage, and the
extraction percentage thereof was 45.8%.
Analysis of Molecular Weight of Proteoglycan
Proteoglycan extracted as described above was analyzed
for molecular weight. More specifically, the analysis was
performed as follows. The collected supernatant obtained in
“Analysis of Proteoglycan Extraction Using Small Pieces of Frozen
Salmon Nasal Cartilage” described above, which contains
proteoglycan extracted from the small pieces of frozen salmon
nasal cartilage of item (2), was separated into fractions by gel
filtration chromatography under the following conditions. The
amount of uronic acid (reflecting the amount of proteoglycan)
contained in each fraction was quantified by the carbazole-
sulfuric acid method. In addition, absorbency at 280 nm of each
fraction was measured, and the absorbency was defined as a value
reflecting the amount of protein contained therein. Based on
these results, a proteoglycan uronic acid amount chromatogram and
a proteoglycan protein amount chromatogram were drawn. The
results are shown in Fig. 4.
Gel Filtration Chromatography
Column: Sepharose CL-4B packed column (1-cm dia. × 38.5 cm column
packed with Sepharose CL-4B as a carrier. Sepharose CL-4B is
available from, for example, GE Healthcare and other companies.
Sepharose CL-4B, CAS registry No. 619709, is a 4% crosslinked
agarose with a particle size of 40 to 165 μm (measured by the
laser diffraction scattering method).)
Buffer: 0.1 M phosphate buffer (pH of 7.0, containing 0.2 M NaCl)
Amount of applied sample: about 0.5 mL of the collected
supernatant (uronic acid amount: about 1 mg)
Flow rate: about 0.15 mL/min
Amount of fraction: 1 mL/tube
Molecular weight analytical curve: An analytical curve
was prepared by subjecting the various dextran molecular weight
markers described below to gel filtration chromatography under
the same conditions as described above (except that the amount of
the applied sample was 1 mg) and quantifying the amount of
saccharide (i.e., amount of dextran) contained in each eluted
fraction by the phenol-sulfuric acid method.
Dextran Molecular Weight Markers
Dextran Standard 1,400,000 (Sigma) 1400 kDa
Dextran Standard 670,000 (Sigma) 670 kDa
Dextran Standard 410,000 (Sigma) 410 kDa
Dextran Standard 270,000 (Sigma) 270 kDa
Dextran was quantified, specifically, as follows, according to
the method described in Hodge, J. E., and Hofreiter, B. T.,
Method in Carbohydrate Chemistry, 1, 338 (1962).
500 μl of a sample aqueous solution or a standard
monosaccharide (mannose) aqueous solution was placed in a 105 ×
mm test tube.
[2] 500 μl of a phenol reagent (5 v/v% aqueous phenol solution)
was added thereto, and the mixture was stirred.
2.5 mL of concentrated sulfuric acid was added thereto, and
immediately the mixture was stirred vigorously for 10 seconds.
The mixture was left to stand for 20 minutes or more at room
temperature.
The absorbency at 490 nm was measured with a
spectrophotometer.
As shown in Fig. 4, the peaks for uronic acid and
protein are observed around fraction Nos. 10 to 25 (i.e., peaks
for uronic acid and protein overlap). Also from these results, it
was confirmed that these fractions contained proteoglycan. This
is because it is believed that proteoglycan is contained in a
fraction in which both uronic acid and protein are detected,
since proteoglycan has a structure in which a number of sugar
chains (containing a large amount of uronic acid as constituent
sugar) are bonded to a protein (core protein) that serves as a
core. It was also believed that from the molecular weight
analytical curve, the molecular weight of the component contained
in fraction No. 20 was about 900,000 (90 × 10 ), and the molecular
weight of the component contained in fraction No. 30 was about
150,000 (15 × 10 ). Accordingly, it was found that the collected
supernatant obtained from the small pieces of frozen salmon nasal
cartilage contained high-molecular-weight proteoglycan having a
molecular weight of not less than about 900,000 (90 × 10 ). In the
figures, the fraction No. is indicated as “Tube No.”, and these
are synonymous.
In addition, the collected proteoglycan-containing
supernatant extracted from the small pieces of frozen salmon
nasal cartilage was separated into fractions by anion exchange
chromatography under the following conditions, and a proteoglycan
uronic acid amount chromatogram and a proteoglycan protein amount
chromatogram were drawn in the same manner as described above. It
was found that the peak for uronic acid and the peak for protein
overlapped (Fig. 5, left-right arrow). This also provides
evidence that proteoglycan was contained in the collected
supernatant.
Anion Exchange Chromatography
Column: DEAE Sephacel packed column (2.5-cm dia. × 10 cm column
packed with DEAE (diethylaminoethyl) Sephacel as a carrier. DEAE
Sephacel is available from, for example, GE Healthcare and other
companies.)
Buffer: 7 M Urea/50 mM Tris-HCl buffer (pH of 7.4) (Linear
gradient elution is performed with 0.1 M NaCl.)
Amount of fraction: 10 mL/tube
The term “comprising” as used in this specification and
claims means “consisting at least in part of”. When interpreting
statements in this specification, and claims which include the
term “comprising”, it is to be understood that other features
that are additional to the features prefaced by this term in each
statement or claim may also be present. Related terms such as
“comprise” and “comprised” are to be interpreted in similar
manner.
In this specification where reference has been made to
patent specifications, other external documents, or other sources
of information, this is generally for the purpose of providing a
context for discussing the features of the invention. Unless
specifically stated otherwise, reference to such external
documents is not to be construed as an admission that such
documents, or such sources of information, in any jurisdiction,
are prior art, or form part of the common general knowledge in
the art.
Claims (11)
- Claim 1 A method for extracting proteoglycan from fish 5 cartilage, comprising the step of (A) heating small pieces of frozen fish cartilage in water, wherein each small piece of frozen fish cartilage is 0.001 to 0.5 g.
- Claim 2 10 The method according to claim 1, wherein the small pieces of frozen fish cartilage is obtained by freezing fish cartilage and then pulverizing the frozen fish cartilage.
- Claim 3 15 The method according to claim 2, wherein the fish cartilage is defatted fish nasal cartilage.
- Claim 4 The method according to any one of claims 1 and 2, 20 wherein the extracted proteoglycan contains having a molecular weight of not less than 900,000 (90 × 10 ).
- Claim 5 The method according to any one of claims 1 to 4, 25 wherein the heating in Step (A) is performed for more than 3 hours.
- Claim 6 The method according to any one of claims 1 to 5, 30 wherein the temperature of water used in the heating in Step (A) is not less than 80˚C.
- Claim 7 The method according to any one of claims 1 to 6, 35 wherein a mass ratio of all the small pieces of frozen fish cartilage to water is 1:1 to 1:10.
- Claim 8 The method according to any one of claims 1 to 7, 5 wherein the heating in Step (A) is continued until the mass of a residue collected after the heating is equal to or less than the mass of the heated small pieces of frozen fish cartilage.
- Claim 9 10 The method according to claim 8, wherein the residue collected after the heating is a precipitate obtained by centrifugation at 5000 rpm, 4˚C, for 20 minutes.
- Claim 10 15 A method for increasing efficiency in proteoglycan extraction from fish cartilage, comprising the step of heating small pieces of frozen fish cartilage in water, wherein each small piece of frozen fish cartilage is 0.001 to 0.5 g. 20
- Claim 11 A method as defined in any one of claims 1 to 10 substantially as herein described with reference to any example thereof.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2011-009272 | 2011-01-19 | ||
JP2011009272 | 2011-01-19 | ||
PCT/JP2012/051120 WO2012099216A1 (en) | 2011-01-19 | 2012-01-19 | Method for mass preparation of proteoglycan |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ614126A NZ614126A (en) | 2014-06-27 |
NZ614126B2 true NZ614126B2 (en) | 2014-09-30 |
Family
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