U.S. patent application number 13/383987 was filed with the patent office on 2012-06-21 for proteoglycan-containing material.
This patent application is currently assigned to HIROSAKI UNIVERSITY. Invention is credited to Masashi Goto, Seiko Itou, Yohtaro Katagata, Youji Katou, Kazushi Yamamoto.
Application Number | 20120157391 13/383987 |
Document ID | / |
Family ID | 43449495 |
Filed Date | 2012-06-21 |
United States Patent
Application |
20120157391 |
Kind Code |
A1 |
Goto; Masashi ; et
al. |
June 21, 2012 |
PROTEOGLYCAN-CONTAINING MATERIAL
Abstract
The present invention was made in view of an object to produce a
novel proteoglycan-containing material, and find a novel use and/or
a superior effect of the proteoglycan-containing material. The
present invention provides a proteoglycan-containing material
obtained from fish cartilage, wherein the proteoglycan-containing
material comprises an acidic saccharide component having a
molecular weight of not less than 2000 kDa. The
proteoglycan-containing material provides advantageous effects for
skin-moisturizing and skin anti-aging, including a superior skin
fibroblast proliferation effect, an effect of enhancing and
improving the skin barrier function, an effect of enhancing and
improving the skin's capability to produce collagen, a
dermis-thickening inhibition effect, and the like.
Inventors: |
Goto; Masashi;
(Takatsuki-shi, JP) ; Yamamoto; Kazushi;
(Takatsuki-shi, JP) ; Katou; Youji; (Hirosaki-shi,
JP) ; Katagata; Yohtaro; (Hirosaki-shi, JP) ;
Itou; Seiko; (Hirosaki-shi, JP) |
Assignee: |
HIROSAKI UNIVERSITY
SUNSTAR INC.
|
Family ID: |
43449495 |
Appl. No.: |
13/383987 |
Filed: |
July 16, 2010 |
PCT Filed: |
July 16, 2010 |
PCT NO: |
PCT/JP2010/062125 |
371 Date: |
February 29, 2012 |
Current U.S.
Class: |
514/20.9 ;
530/395 |
Current CPC
Class: |
A61Q 19/08 20130101;
A61P 1/02 20180101; A23V 2002/00 20130101; A61K 35/60 20130101;
A61K 8/64 20130101; A23F 3/30 20130101; A61P 17/16 20180101; A61P
17/00 20180101; A61Q 19/00 20130101; A61K 2800/92 20130101; C08B
37/0066 20130101; A23G 4/06 20130101; A23V 2250/543 20130101; A23V
2002/00 20130101; A23L 33/17 20160801; A61K 8/735 20130101; A23V
2200/318 20130101; A61Q 11/00 20130101; A23G 3/36 20130101; A23L
29/275 20160801 |
Class at
Publication: |
514/20.9 ;
530/395 |
International
Class: |
A61K 8/64 20060101
A61K008/64; C07K 14/00 20060101 C07K014/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 16, 2009 |
JP |
2009-168123 |
Claims
1. A proteoglycan-containing material comprising proteoglycan and
acidic saccharide as acidic saccharide components obtained from
fish cartilage, wherein the proteoglycan-containing material
comprises an acidic saccharide component having a molecular weight
of not less than 2000 kDa.
2. The proteoglycan-containing material according to claim 1,
wherein the proteoglycan-containing material comprises an acidic
saccharide component having a molecular weight of not less than
5000 kDa.
3. The proteoglycan-containing material according to claim 1,
wherein 50 mass % or more of acidic saccharide components have a
molecular weight of not less than 2000 kDa.
4. The proteoglycan-containing material according to claim 1,
wherein 20 mass % or more of acidic saccharide components have a
molecular weight of not less than 10000 kDa.
5. The proteoglycan-containing material according to claim 1,
wherein 20 mass % or more of acidic saccharide components is
proteoglycan.
6. A food or beverage composition comprising the
proteoglycan-containing material according to claim 1.
7. An oral composition comprising the proteoglycan-containing
material according to claim 1.
8. A cosmetic composition comprising the proteoglycan-containing
material according to claim 1.
Description
TECHNICAL FIELD
[0001] The present invention relates to a proteoglycan-containing
material. More specifically, the present invention relates to a
proteoglycan-containing material obtained from fish cartilage.
BACKGROUND ART
[0002] Proteoglycan is one of the major biological macromolecules
for forming the substrate of the extracellular matrix of connective
tissue, as with collagen, etc. Proteoglycan was hitherto obtained
by being extracted and isolated from mammal cartilage (in
particular, bovine cartilage). However, since the onset of bovine
spongiform encephalopathy (BSE) was reported, there has been a need
for an alternative source of proteoglycan, and a production method
therefor.
[0003] 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 or sharks. However, due to harvest 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 the solvents used for extraction
have certain levels of toxicity. Therefore, the usage of
proteoglycan products is limited, and they were difficulties in
using them as materials of foodstuff or cosmetics.
[0004] Under such circumstances, there has been active research
into various proteoglycan production methods. For example, Patent
Document 1 discloses a method for purifying proteoglycan. In this
method, salmon nasal cartilage is pulverized and defatted to obtain
defatted dry powder, which is then subjected to extraction using a
solvent. The obtained coarse extract is isolated and purified,
followed by dialysis. However, this method uses an organic solvent
having a certain level of toxicity, such as chloroform or methanol.
Another problem of this method is that a fishy smell remains on the
resulting proteoglycan.
[0005] Patent Document 2 discloses a method for purifying
proteoglycan from salmon nasal cartilage using an acetic acid as an
extraction solvent. Further, Patent Document 3 suggests a
capability to proliferate fibroblasts, and an effect of promoting
hyaluronic acid synthesis of the proteoglycan produced by the
method of Patent Document 2.
[0006] As explained above, there is still ongoing research into the
production, usage, and effects of proteoglycan.
CITATION LIST
Patent Documents
[0007] [Patent Document 1] Japanese Unexamined Patent Publication
No. 2001-172296 [0008] [Patent Document 2] Japanese Unexamined
Patent Publication No. 2002-069097 [0009] [Patent Document 3]
Japanese Unexamined Patent Publication No. 2008-247803
SUMMARY OF INVENTION
Technical Problem
[0010] An object of the present invention is to produce a novel
proteoglycan-containing composition (proteoglycan-containing
material) from fish cartilage using a less-toxic solvent; and to
find a novel usage and a superior effect of the
proteoglycan-containing material.
Solution to Problem
[0011] Surprisingly, the inventors of the present invention found
that an extract containing an acidic saccharide component, which
was obtained by being extracted from fish cartilage using a
particular method, contained proteoglycan having a greater
molecular weight than that of the hitherto-known proteoglycan.
Moreover, the inventors further found that the aforementioned
extract had various superior effects. After several attempts to
further improve the extract, the inventors completed the present
invention.
[0012] In the present specification, acidic saccharide and a
compound containing acidic saccharide as an ingredient are referred
to as an acidic saccharide component. Since proteoglycan has a
structure in which acidic saccharide and protein are bonded,
proteoglycan corresponds to the acidic saccharide component; more
specifically, proteoglycan is a kind of acidic saccharide
component. The proteoglycan-containing material of the present
invention comprises proteoglycan and acidic saccharide as acidic
saccharide components. Thus, the proteoglycan-containing material
of the present invention may also be referred to as an acidic
saccharide-containing composition.
[0013] The present invention includes, for example, the
proteoglycan-containing materials and the compositions containing
proteoglycan-containing materials as set forth in the following
Items.
[0014] [Item 1]
[0015] A proteoglycan-containing material comprising proteoglycan
and acidic saccharide as acidic saccharide components obtained from
fish cartilage, wherein the proteoglycan-containing material
comprises an acidic saccharide component having a molecular weight
of not less than 2000 kDa.
[0016] [Item 2]
[0017] The proteoglycan-containing material according to Item 1,
wherein the proteoglycan-containing material comprises an acidic
saccharide component having a molecular weight of not less than
5000 kDa.
[0018] [Item 3]
[0019] The proteoglycan-containing material according to Item 1 or
2, wherein 50 mass % or more of acidic saccharide components have a
molecular weight of not less than 2000 kDa.
[0020] [Item 4]
[0021] The proteoglycan-containing material according to any one of
Items 1 to 3, wherein 20 mass % or more of acidic saccharide
components have a molecular weight of not less than 10000 kDa.
[0022] [Item 5]
[0023] The proteoglycan-containing material according to any one of
Items 1 to 4, wherein 20 mass % or more of acidic saccharide
components is proteoglycan.
[0024] [Item 6]
[0025] A food or beverage composition comprising the
proteoglycan-containing material according to any one of Items 1 to
5.
[0026] [Item 7]
[0027] An oral composition comprising the proteoglycan-containing
material according to any one of Items 1 to 5.
[0028] [Item 8]
[0029] A cosmetic composition comprising the
proteoglycan-containing material according to any one of Items 1 to
5.
Advantageous Effects of Invention
[0030] The proteoglycan-containing material of the present
invention provides various advantageous effects for
skin-moisturizing and anti-aging, including an excellent skin
fibroblast proliferation effect, an effect of enhancing and
Improving the skin barrier function, an effect of enhancing and
Improving the skin's capability to produce collagen, a
dermis-thickening inhibition effect, and the like.
BRIEF DESCRIPTION OF DRAWINGS
[0031] FIG. 1
[0032] Schematic views showing a procedure of determining a peak
area showing the proteoglycan of the present invention.
[0033] FIG. 2
[0034] Graphs showing results of quantitative analysis with respect
to the amounts of acidic saccharide and protein contained in each
of a plurality of 1 mL eluted fractions obtained from the samples
derived from salmon nasal cartilage powder (the left graph) and a
water extract of salmon nasal cartilage powder (the right graph)
through gel filtration chromatography (Sepharose CL-2B packed
column was used). The quantitative analysis was performed according
to the carbazole-sulfuric acid method and UV absorption method.
[0035] FIG. 3
[0036] Graphs showing results of quantitative analysis with respect
to the amount of acidic saccharide contained in each of a plurality
of eluted fractions obtained from commercially available
proteoglycan (PG-K) and commercially available glycosaminoglycan
(PG-M; commercially available as chondroitin) through gel
filtration chromatography. The left graph shows the results of
PG-M, and the right graph shows the results of PG-K.
[0037] FIG. 4
[0038] Graphs showing results of quantitative analysis with respect
to the amount of saccharide (i.e., amount of dextran) contained in
each of a plurality of eluted fractions obtained from dextran
molecular weight markers through gel filtration chromatography. The
quantitative analysis was performed by a phenol-sulfuric acid
method.
[0039] FIG. 5
[0040] A graph showing an analytical curve based on the liquid
amount at which each molecular weight marker is eluted, according
to the results of FIG. 4.
[0041] FIG. 6
[0042] A graph that summarizes measurement results of acidic
saccharide amounts shown in FIG. 1 and FIG. 2.
[0043] FIG. 7
[0044] A graph showing quantitative analysis with respect to the
amount of acidic saccharide contained in each of a plurality of 1
mL eluted fractions obtained from the samples derived from salmon
nasal cartilage powder through gel filtration chromatography
(Sephacryl S-1000 SF packed column was used). The quantitative
analysis was performed by the carbazole-sulfuric acid method.
[0045] FIG. 8
[0046] A graph showing analysis of capabilities to proliferate
human skin fibroblasts of salmon nasal cartilage powder and a water
extract of salmon nasal cartilage powder.
[0047] FIG. 9
[0048] A graph showing analysis of an influence on the skin barrier
function (TEWL value) by oral administration of salmon nasal
cartilage powder and a water extract of salmon nasal cartilage
powder.
[0049] FIG. 10
[0050] A graph showing analysis of an influence on the skin
elasticity by oral administration of salmon nasal cartilage powder
and a water extract of salmon nasal cartilage powder.
[0051] FIG. 11
[0052] A graph showing analysis of an influence on capability to
produce collagen by oral administration of salmon nasal cartilage
powder, and a water extract of salmon nasal cartilage powder.
[0053] FIG. 12
[0054] A graph showing measurements of back dermis thickness of
mice orally administered with salmon nasal cartilage powder, and a
water extract of salmon nasal cartilage powder, for analysis of an
influence on dermis-thickening by these substances.
[0055] FIG. 13
[0056] A graph showing analysis of an influence on the skin barrier
function (TEWL value) by transdermal administration of salmon nasal
cartilage powder and a water extract of salmon nasal cartilage
powder.
[0057] FIG. 14
[0058] A procedure of fractionation of a water extract of salmon
nasal cartilage powder by way of ion-exchange chromatography and
gel filtration chromatography.
[0059] FIG. 15
[0060] A graph showing analysis of cell proliferation effect of
each fraction of a water extract of salmon nasal cartilage powder;
specifically, a graph showing the cell number of each fraction 7
days after the addition of each sample (50 .mu.g/ml). "PGNP water
extract" is a water extract of salmon nasal cartilage powder.
[0061] FIG. 16
[0062] A graph showing analysis of cell proliferation effect of
each fraction of a water extract of salmon nasal cartilage powder;
specifically, a graph showing the cell number of each fraction 7
days after the addition of each sample (10 .mu.g/ml).
DESCRIPTION OF EMBODIMENTS
[0063] The present invention is more specifically described below.
In the following, the molecular weights and the mean molecular
weights of acidic saccharide and proteoglycan are based on the
measurement values obtained in gel filtration chromatography using
dextran as a molecular weight marker.
[0064] The proteoglycan-containing material of the present
invention is produced from fish cartilage. Examples of fish
include, but are not limited to, trout (humpback salmon, cherry
salmon, satsukimasu salmon, etc.), salmons (chum salmon, sockeye
salmon, silver salmon, chinook salmon, steelhead, etc.), sharks,
and cods. Oncorhynchus (salmonidae), in particular, salmons and
trout, are preferable. The cartilage to be used is also not
limited; however, head cartilage, in particular nasal cartilage, is
preferable. Moreover, since fish heads are usually discarded when
fish is processed into foodstuff, the cost of fish heads is low,
and a large amount of fish head can be stably supplied.
[0065] In the present invention, "acidic saccharide component"
designates acidic saccharide or a compound containing acidic
saccharide as an ingredient. The proteoglycan-containing material
of the present invention contains an acidic saccharide component
(i.e., acidic saccharide or a component containing acidic
saccharide as an ingredient).
[0066] Here, "acidic saccharide" is a polysaccharide containing an
uronic acid. Examples of acidic saccharide contained in the
proteoglycan-containing material of the present invention include
glycosaminoglycans such as hyaluronic acid, chondroitin and the
like. Except for hyaluronic acid, glycosaminoglycan is generally
present by being covalently bonded with protein (i.e., as
proteoglycan).
[0067] A specific example of the compound containing acidic
saccharide as an ingredient is proteoglycan. Proteoglycan has a
structure in which glycosaminoglycan and protein are covalently
bonded. Glycosaminoglycan that forms proteoglycan is acidic
saccharide consisting of a repeating sulfated disaccharide unit.
Specifically, examples thereof include chondroitin sulfate,
dermatan sulfate, and heparan sulfate. That is, proteoglycan is a
compound having a structure in which protein and acidic saccharide
are bonded.
[0068] In the repeating disaccharide structure of the acidic
saccharide component, generally, one of the disaccharides is amino
sugar, and the other is an uronic acid. Therefore, the detection of
acidic saccharide components may be performed using a
carbazole-sulfuric acid method, which is one of the ordinary
methods for detecting uronic acids.
[0069] The carbazole-sulfuric acid method is performed by adding a
carbazole solution, which is a color component of glucuronic acid
(Glc A) and iduronic acid, i.e., uronic acid, to a measurement
specimen, measuring the absorbency using a spectrophotometer, and
plotting an analytical curve using the glucuronic acid standard
solution having a specific concentration, thereby finding a
glucuronic acid content in the specimen. More specifically, the
carbazole-sulfuric acid method can be performed as follows.
[0070] 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
(containing 4 to 40 of uronic acid) is gently layered thereon. The
mixture is well-stirred while being ice-cooled, thereby keeping it
at room temperature or below. After the test tube is covered with a
glass ball lid, 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 an 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.
[0071] The mean molecular weight of the acidic saccharide component
contained in the proteoglycan-containing material of the present
invention is generally about 2000 kDa to 40000 kDa, preferably 2500
kDa to 30000 kDa, more preferably 3000 kDa to 20000 kDa, further
preferably 3000 kDa to 10000 kDa, and further more preferably 4000
kDa to 8000 kDa.
[0072] The proteoglycan-containing material of the present
invention contains an acidic saccharide component having a
molecular weight of not less than 2000 kDa, preferably not less
than 2500 kDa, more preferably not less than 3000 kDa, further
preferably not less than 4000 kDa, and further more preferably not
less than 5000 kDa.
[0073] The proportion of an acidic saccharide component having a
molecular weight of not less than 2000 kDa in the
proteoglycan-containing material of the present invention is
preferably not less than 50 mass %, more preferably not less than
55 mass %, further preferably not less than 60 mass %, and still
further more preferably not less than 65 mass %.
[0074] The proteoglycan contained in the proteoglycan-containing
material of the present invention has a significantly greater
molecular weight (MW) than the hitherto-available proteoglycan.
More specifically, even a small proteoglycan in the
proteoglycan-containing material of the present invention has a
molecular weight of at least about 5000 kDa to 6000 kDa or more.
Thus, the proteoglycan-containing material of the present invention
contains proteoglycan having a molecular weight of about 5000 kDa
or more. For example, the proteoglycan-containing material of the
present invention contains proteoglycan having a molecular amount
of about 5000 kDa to 100000 kDa, preferably 5000 kDa to 90000 kDa,
more preferably 5000 kDa to 80000 kDa, further more preferably 5000
kDa to 70000 kDa. Moreover, although the mean molecular weight of
the proteoglycan contained in the proteoglycan-containing material
of the present invention is not particularly limited, the mean
molecular weight is generally about 6000 kDa to 60000 kDa,
preferably about 7000 kDa to 50000 kDa, more preferably about 9000
kDa to 40000 kDa.
[0075] The proportion of the proteoglycan having a molecular weight
of 6000 kDa or more (further preferably 10000 kDa or more) in the
proteoglycan-containing material of the present invention is
preferably not less than 20 mass % or more, more preferably not
less than 30 mass %.
[0076] The proteoglycan-containing material of the present
invention preferably contains an acidic saccharide component in an
amount of, on a dry mass basis, 15 to 70 mass %, more preferably 30
to 70 mass %. Moreover, the proteoglycan-containing material of the
present invention preferably contains proteoglycan in an amount of,
on a dry mass basis, 4 to 40 mass %, more preferably 10 to 40 mass
%, further preferably 15 to 40 mass %.
[0077] The molecular weight of the acidic saccharide component or
proteoglycan contained in the proteoglycan-containing material of
the present invention can be measured using chromatography or the
like. In particular, the molecular weight is preferably measured by
gel filtration chromatography. More specifically, for example, an
agarose (agarose, cross-linked agarose, etc.) gel matrix may be
used as a carrier of the column, and a phosphate buffer (preferably
containing sodium chloride) may be used as a buffer. Through the
gel filtration of the proteoglycan-containing material, the
molecular weight of the acidic saccharide component or proteoglycan
contained in the composition can be determined according to the
elution volume before elution of the acidic saccharide component or
proteoglycan.
[0078] Because proteoglycan has a structure in which
glycosaminoglycan (mucopolysaccharide) and protein are covalently
bonded, the elution of proteoglycan in the chromatography can be
detected by monitoring the acidic saccharide and protein. More
specifically, a chromatogram obtained by monitoring of acidic
saccharide and a chromatogram obtained by monitoring protein are
overlaid to confirm whether there is an overlapped peak in
substantially the same eluate range in the two chromatograms. If
such a peak is found, the peak is regarded as the detection of
proteoglycan. For example, the carbazole-sulfuric acid method can
be used for monitoring acidic saccharide. The eluted fraction of
chromatography is divided into separate fractions of a
predetermined amount, and the acidic saccharide amount contained in
each fraction is determined using the carbazole-sulfuric acid
method. Further, for example, the monitoring of protein can be
performed according to the UV absorption method (in which protein
quantity is determined by measuring absorbencies of tryptophan,
tyrosine, and phenylalanine having absorptions in the vicinity of
280 nm), ninhydrin reaction, BCA method, Bradford method, Lowry
method, biuret method, and the like. Among these, quantitative
analysis using the UV absorption method is easily done.
[0079] Further, the molecular weight can be determined from the
amount of eluate, as follows. A molecular weight marker is
subjected to gel filtration chromatography in the same manner, and
the liquid measure at which the molecular weight marker is eluted
is measured to plot an analytical curve (elute amount vs. molecular
weight). A suitable molecular weight marker is appropriately
selected and purchased in consideration of the molecular weight of
the proteoglycan-containing material or the type of gel matrix to
be used. For example, a molecular weight marker made of dextran can
be used. Such a molecular weight marker may be purchased from
Sigma-Aldrich Co., etc.
[0080] In addition to the acidic saccharide component, the
proteoglycan-containing material of the present invention also
contains other fish cartilage-derived components. Examples thereof
include proteins such as collagen, and salts.
[0081] In the present invention, the mean molecular weight of a
substance designates a specific molecular weight determined as
follows. A line (bisector) perpendicular to the horizontal axis
(amount of eluate) that divides the peak area in the chromatogram
of the substance obtained by analysis using gel filtration
chromatography is drawn, and a mean molecular weight is determined
from the amount of eluate corresponding to the position of the
bisector, using an analytical curve. More specifically, the mean
molecular weight of a substance designates a molecular weight
determined according to the method disclosed in the "Analysis of
Molecular Weight of Proteoglycan-Containing Materials" section of
the Examples.
[0082] For example, the mean molecular weight of the acidic
saccharide component contained in the proteoglycan-containing
material is found by isolating the proteoglycan-containing material
by gel filtration chromatography, drawing a line that divides the
area of the chromatogram obtained by monitoring the acidic
saccharide component contained therein, and finding the mean
molecular weight by determining a molecular weight from the amount
of eluate corresponding to the position of the bisector using an
analytical curve (in other words, by substituting the amount of
eluate in the equation of the analytical curve). The monitoring of
the acidic saccharide component is more specifically described
below. The proteoglycan-containing material is subjected to gel
filtration chromatography analysis, followed by elution at a
predetermined speed, and the resulting eluate is divided into
separate fractions of a predetermined amount. The fractions are
subjected to quantitative analysis, and the measured amount of
acidic saccharide in each eluate (fraction) is plotted to create a
chromatogram.
[0083] Further, the mean molecular weight of proteoglycan contained
in the proteoglycan-containing material is found by drawing a line
that divides the peak area denoting proteoglycan in a chromatogram
obtained by gel filtration chromatography analysis of the
proteoglycan-containing material, and finding the mean molecular
weight by determining a molecular weight from the amount of eluate
corresponding to the position of the bisector using an analytical
curve.
[0084] As described above, the peak denoting proteoglycan
corresponds to a peak that is substantially overlapped when a
chromatogram obtained by the monitoring of acidic saccharide and a
chromatogram obtained by monitoring protein are overlaid.
[0085] If the rise and the fall of the peak denoting proteoglycan
cannot be specified because they overlap with the peaks of other
acidic saccharide components, the rise and the fall are estimated
based on the shape of the peak. Using the estimated values of the
rise and the fall, the molecular weight and the mean molecular
weight are found. FIG. 1 shows an example of such an estimation.
FIG. 1 is a schematic view showing a method for estimating the peak
shape when the fall of the peak cannot be specified. In FIG. 1, the
line is extended by drawing a downward-sloping curve, thereby
determining the peak shape.
[0086] For both an acidic saccharide component and proteoglycan,
the molecular weight or mean molecular weight is preferably found
by analyzing a chromatogram obtained by monitoring the acidic
saccharide.
[0087] Moreover, by collecting only the fraction corresponding to
the peak denoting proteoglycan during the gel filtration
chromatography, it is possible to purify proteoglycan contained in
the proteoglycan-containing material of the present invention.
Further, by collecting from the initial fraction to the fraction
corresponding to the peak denoting proteoglycan, it is possible to
obtain a proteoglycan-containing material having a further greater
mean molecular weight.
[0088] The proportion of proteoglycan in the acidic saccharide
component contained in proteoglycan-containing material of the
present invention is generally not less than 20 mass %, preferably
20 to 60 mass %, more preferably 25 to 55 mass %, further
preferably 30 to 50 mass %. The proportion of proteoglycan in the
acidic saccharide component can be found from the chromatogram used
to find the molecular weight of the acidic saccharide component or
proteoglycan. More specifically, by finding the proportion of the
peak area showing proteoglycan among the entire area of the
chromatogram obtained by monitoring the acidic saccharide component
of the proteoglycan-containing material, it is possible to find the
proportion of proteoglycan in the acidic saccharide component.
[0089] As described above, the proteoglycan-containing material of
the present invention is produced from fish cartilage. More
specifically, the proteoglycan-containing material of the present
invention can be produced by defatting fish cartilage using
ethanol. Further, the proteoglycan-containing material may be
produced by being extracted from the defatted fish cartilage
through water extraction.
[0090] For example, the proteoglycan-containing material is
produced through the following steps.
Step (1): Water Treatment Step
[0091] Fish tissue containing cartilage (e.g., a fish head) is
Immersed in water for several minutes to several days at room
temperature or a low temperature (about 0 to 40.degree. C.). The
tissue may be allowed to stand still in water, stirred during
immersion, stirred together with water using a line mixer, etc. The
amount of water is not particularly limited; however, it is
preferable to use a sufficient amount of water so that all of the
tissue containing cartilage is immersed therein. Such complete
immersion of the tissue removes the fishy smell from the tissue,
and enables easy removal of the parts other than cartilage because
the tissue is swollen by the infiltration of the water. Before the
immersion in water, it is possible to slice or crack the tissue
containing cartilage beforehand, or to separate removable parts
from the tissue other than cartilage.
[0092] When the tissue is deodorized and desirably swollen by
sufficient immersion in water, lipids, etc., are extracted from
fish tissue containing cartilage (that is, the tissue containing
cartilage is defatted). The extracted lipids are dissolved or
suspended in the water layer, or floats in the water layer as a
lipid layer. By removing the water layer and the lipid layer, the
lipids contained in the tissue containing cartilage are removed. It
is also possible to remove these unwanted substances by
centrifugation.
Step (2): Ethanol Treatment Step
[0093] After removing the lipid layer and the water layer, the
resulting residue (cartilage tissue) is isolated. Ethanol is added
to the residue, and the lipids are further removed by extraction.
By further removing lipids, it is possible to more reliably remove
odor. Hydrous ethanol may also be used. Further, it is preferable
to pulverize the residue (cartilage tissue) before addition of the
organic solvent. The method for pulverizing the tissue is not
limited. For example, the pulverization may be performed using a
device capable of finely pulverizing cartilage materials, such as a
ball mill, swing mill, low-temperature-grinder, freezing grinder,
rotor mill, grind mix, mixer mill, and the like. The particle size
of the pulverized tissue is preferably, but not limited to, about
10 to 500 .mu.m, more preferably about 50 to 250 .mu.m. The
particle size can be measured according to the laser diffraction
scattering method.
[0094] For example, this step may be performed by immersing the
cartilage tissue (preferably, pulverized cartilage tissue) obtained
above in ethanol in a sufficient amount to fully immerse the
tissue; stirring the immersed tissue, or allowing it to stand
still; and then removing the solvent. The immersion is preferably
performed a plurality of times (e.g., 2 to 5 times). It is also
possible to remove lipids by circulating ethanol. This process is
more preferably performed with the Soxhlet extractor or the like.
After such a treatment, solids are separated. The collected solids
may be dried by air-drying or the like to completely remove the
organic solvent.
[0095] The solids thus obtained are used as a
proteoglycan-containing material.
[0096] A more detailed example of Step (2) above may be the
following method comprising the Steps [1] to [9].
[1] Extraneous tissue such as skin or bone is removed from salmon
nasal cartilage, and the resulting cartilage is pulverized with a
meat chopper.
[0097] [2] Tap water or purified water having a pH of 6 to 7.5 in
an amount that is equal or double the amount (volume) of the
cartilage is added to the pulverized salmon nasal cartilage, and
the mixture is sufficiently stirred at 40.degree. C. or below.
[3] After stirring, the mixture is subjected to centrifugation
using a centrifuge to collect solids. [4] Steps [2] and [3] are
performed once or twice. [5] The resulting solids are further
finely pulverized with a wet grinder. [6] Ethanol having a purity
of 95% or more in an amount (volume) that is about ten times the
amount of cartilage is added to the finely pulverized salmon nasal
cartilage about, and the mixture is sufficiently stirred at
40.degree. C. or below. [7] After stirring, the mixture is
subjected to centrifugation using a centrifuge to collect solids.
[8] Steps [6] and [7] are performed 1 to 3 times. [9] The solids
are dried, as necessary. In Step [5], fine pulverization may be
performed after the resulting solids are freeze-dried.
Step (3): Water Extraction Step
[0098] As the proteoglycan-containing material of the present
invention, it is more preferable to use an extract resulting from
additional water extraction. Therefore, the solids obtained in Step
(2) are preferably further subjected to water extraction. For
example, the solids obtained in Step (2) are immersed in a
sufficient amount of water to be completely immersed therein, the
mixture is stirred, and insoluble matter is removed. In this
manner, the obtained solution or a dried product thereof is used as
a proteoglycan-containing material. The pH of the water to be added
is generally 5.5 to 8.0, preferably 6.0 to 7.5, more preferably 6.5
to 7.5. More specifically, for example, the tissue is stirred while
immersed in water for about 30 minutes to 6 hours, or stirred
together with water using a line mixer or the like; and insoluble
matter is removed. After removing the insoluble matter, it is
possible to perform drying by a usual method. Further, it is also
possible to add ethanol in a double to tenfold amount (volume)
after removing the insoluble matter, thereby collecting the
resulting solids. In this case, sodium chloride may be added before
the addition of ethanol.
[0099] A more detailed example of Step (3) above may be the
following method comprising Steps [10] to [12], which are performed
after Steps [1] to [9].
[10] Purified water having a pH value of 6 to 7.5 in an amount
(volume) that is approximately equal or double that of the dried
product obtained in Step [9] is added to the dried product, and the
mixture is sufficiently stirred at 40.degree. C. or lower for about
30 minutes to 48 hours. [11] Centrifugation is performed to remove
solids. [12] The solids are dried, as necessary.
[0100] It is also possible to add ethanol after Step [11], stir the
mixture, and collect the resulting solids.
[0101] As described above, the proteoglycan-containing material of
the present invention may also be produced by Steps (A) and (B), or
by Steps (A) to (C).
(A) A step of purifying fish cartilage (B) A step of removing
lipids from fish cartilage using an organic solvent (C) A step of
further performing water extraction with respect to defatted fish
cartilage, thereby collecting an extract Steps (A), (B), and (C)
correspond respectively to Steps (1), (2), and (3) described
above.
[0102] The proteoglycan-containing material of the present
invention may be suitably used for skin anti-aging. Skin is
constantly exposed to various kinds of damage. For example, in the
dermic layer, the epidermis barrier function decreases due to
external factors (for example, optical radiation such as
ultraviolet) or internal factors (for example, aging). Such damage
causes an increase in transepidermal water loss (TEWL), thereby
causing dry or rough skin. Further, for the same reason, a decrease
in capability to produce collagen, a decrease in skin elasticity,
or dermis-thickening may occur in the dermic layer, thereby
facilitating skin-hardening. This may result in wrinkles or the
like.
[0103] The proteoglycan-containing material of the present
invention exhibits various effects (advantageous effects in terms
of skin moisturizing and anti-aging, such as skin fibroblast
proliferation effect, effects of enhancing and improving the skin
barrier function, effects of enhancing and improving the skin's
capability to produce collagen, dermis-thickening inhibition
effect, and the like) to suppress or treat such skin symptoms. In
particular, the proteoglycan-containing material of the present
invention is suitable to prevent or treat the aforementioned skin
symptoms caused by optical radiation (in particular, ultraviolet
radiation).
[0104] The usage of the proteoglycan-containing material of the
present invention is not limited; however, the
proteoglycan-containing material of the present invention is
particularly useful for products in the oral-care industry,
cosmetic industry, and food and beverage industry. Accordingly, the
present invention includes oral compositions containing the
proteoglycan-containing material of the present invention, cosmetic
compositions containing the proteoglycan-containing material of the
present invention, and food and beverage compositions containing
the proteoglycan-containing material of the present invention.
[0105] The oral compositions oral containing the
proteoglycan-containing material (these oral compositions may be
hereinafter referred to as oral compositions of the present
invention) used for oral-care products can be produced by
appropriately combining the proteoglycan-containing material of the
present invention 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, paste, chewing gums, troches, and tablets,
which may be manufactured by usual methods.
[0106] Such oral compositions of the present invention can be used
by being sprayed into the oral cavity, or as a mouthwash. By such
applications, the oral compositions proliferate, in particular, the
fibroblasts in the oral cavity, thereby enhancing and improving the
skin barrier function and the capability to produce collagen. With
such advantages, the oral compositions of the present invention are
appropriately used for regeneration and anti-aging of oral
tissue.
[0107] The amount of the acidic saccharide component contained in
the proteoglycan-containing material of the oral compositions of
the present invention is not particularly limited; however, the
amount is generally 0.002 to 13 mass %, preferably 0.01 to 5 mass
%, more preferably 0.02 to 3 mass %, based on the entire
composition. Moreover, the amount of proteoglycan contained in the
proteoglycan-containing material of the oral composition is also
not limited; however, the amount is generally 0.001 to 5 mass %,
preferably 0.005 to 2 mass %, more preferably 0.01 to 1 mass %,
based on the entire composition.
[0108] The cosmetic compositions containing the
proteoglycan-containing material of the present invention (these
cosmetic compositions may be hereinafter referred to as cosmetic
compositions of the present invention) used for cosmetic products
can be produced by appropriately combining the
proteoglycan-containing material of the present invention with
cosmetically acceptable media, bases, carriers, or additives; and,
as necessary, other cosmetically acceptable components and
materials by a usual method. More specifically, examples of
cosmetic compositions include emulsions, lotions, creams, serums,
foundations, masks, and sunscreens, which are produced by using the
proteoglycan-containing material of the present invention. Such
cosmetic compositions of the present invention are preferably used
for prevention or treatment of sunburn, moisturizing and anti-aging
of the skin (e.g., prevention or treatment of dry skin, rough skin,
facial wrinkles or sagging skin), and the like.
[0109] The amount of the acidic saccharide component contained in
the proteoglycan-containing material of the cosmetic composition of
the present invention is not particularly limited; however, the
amount is generally 0.002 to 5 mass %, preferably 0.02 to 2 mass %,
more preferably 0.1 to 2 mass %, based on the entire composition.
Moreover, the amount of proteoglycan contained in the
proteoglycan-containing material of the cosmetic compositions is
also not limited; however, the amount is generally 0.001 to 2 mass
%, preferably 0.01 to 1 mass %, more preferably 0.05 to 1 mass %,
based on the entire composition.
[0110] The food and beverage compositions containing the
proteoglycan-containing material (these food and beverage
compositions may be hereinafter referred to as food and beverage
compositions of the present invention) used for food and beverage
products can be produced by appropriately combining the
proteoglycan-containing material of the present invention with
food-hygienically acceptable bases, carriers, or additives; and, as
necessary, other components or materials used for food and
beverages. Examples thereof include fabricated food or beverages
with claimed effects of moisturizing and anti-aging of the skin
(e.g., prevention or treatment of dry skin, rough skin, facial
wrinkles or sagging skin), food with health claims (food with
nutrient function claims, food for specified health uses, etc.),
supplements, weight-reducing food, food for patients, etc., which
contain the proteoglycan-containing material of the present
invention. Moreover, the present invention also includes
moisturizers and skin anti-aging agents formed of the
aforementioned food and beverage compositions of the present
invention. The moisturizers and skin anti-aging agents may be
supplied in the forms of drinks, tablets, capsules, granules,
jelly, troches, or the like for cosmetic or skin anti-aging
purposes (e.g., prevention or treatment of dry skin, rough skin,
facial wrinkles or sagging skin).
[0111] The amount of the acidic saccharide component contained in
the proteoglycan-containing material of the food and beverage
compositions of the present invention is not particularly limited;
however, in the case of a food composition or an agent comprising a
food composition, the amount is generally 0.01 to 50 mass %,
preferably 0.02 to 25 mass %, more preferably 0.1 to 8 mass %,
based on the entire composition or agent. The amount of the acidic
saccharide component contained in the proteoglycan-containing
material of a beverage composition or an agent comprising a
beverage composition is generally 0.002 to 13 mass %, preferably
0.01 to 8 mass %, more preferably 0.1 to 2 mass %, based on the
entire composition or agent. Moreover, the amount of proteoglycan
contained in the proteoglycan-containing material of the food and
beverage compositions is also not limited; however, in the case of
a food composition or an agent comprising a food composition, the
amount is generally 0.005 to 20 mass %, preferably 0.01 to 10 mass
%, more preferably 0.05 to 3 mass %, based on the entire
composition or agent. In the case of a beverage composition or an
agent comprising a beverage composition, the amount is generally
0.001 to 5 mass %, preferably 0.005 to 3 mass %, more preferably
0.01 to 1 mass %, based on the entire composition or agent.
[0112] Furthermore, the proteoglycan-containing material of the
present invention is preferably applied in combination with
hyaluronic acid or collagen. In a combination with these
substances, the effect of the proteoglycan-containing material of
the present invention increases. Therefore, the aforementioned oral
compositions, cosmetics compositions, and food and beverage
compositions of the present invention also preferably comprise a
hyaluronic acid and/or collagen (preferably collagen hydrolysate).
In particular, oral administration of the proteoglycan-containing
material of the present invention together with a hyaluronic acid
is preferable, as it further improves the skin moisturizing and
anti-aging effect. The amount of hyaluronic acid is not particular
limited; however, it is 0.01 to 1 parts by mass, preferably 0.02 to
0.5 part by mass, more preferably 0.05 to 0.2 part by mass, per
part by mass of the proteoglycan-containing material of the present
invention.
[0113] The amount of acidic saccharide components contained in the
oral compositions, cosmetic compositions, and food and beverage
compositions of the present invention can be found, for example,
according to the carbazole-sulfuric acid method. The amount of
acidic saccharide components can also be found by acidic saccharide
detection chromatograms obtained by gel filtration chromatography
of those compositions. Further, the amount of proteoglycan
contained in those compositions can be determined, for example, by
performing gel filtration chromatography of each composition,
overlaying the acidic saccharide detection chromatogram and the
protein detection chromatogram, and detecting and determining the
quantity of proteoglycan represented by an overlapped peak.
[0114] The present invention also includes a method for orally or
transdermally applying the proteoglycan-containing material of the
present invention, thereby obtaining the effects recited in this
specification, i.e., the effect of proliferating fibroblasts, or
the effect of enhancing or improving the skin barrier function. The
method may be performed by directly using the
proteoglycan-containing material of the present invention, or more
preferably using the aforementioned oral compositions or the
cosmetic compositions of the present invention. The subject of the
method is not limited; however, it is more preferable to perform
the method on a person who suffers from a decrease in skin barrier
function due to aging or sunburn. The method may also be applied
for cosmetic purposes. The application amount is also not limited,
and any desired amount may be applied.
[0115] Furthermore, the present invention also includes a method
for orally administering the proteoglycan-containing material of
the present invention, thereby obtaining the effects recited in
this specification, i.e., the effect of proliferating fibroblasts,
the effect of enhancing or improving the skin barrier function, the
effect of enhancing or improving skin elasticity, the effect of
preventing dermis-thickening, or the effect of enhancing or
improving the skin's capability to produce collagen. The method may
be performed by directly using the proteoglycan-containing material
of the present invention, or more preferably using the
aforementioned food and beverage compositions of the present
invention. The subject of the method is not limited; however, it is
more preferable to perform the method on a person who suffers from
a decrease in skin barrier function, or a decrease in skin
elasticity due to aging or sunburn. The method may'also be applied
for cosmetic purposes. The application amount is also not limited,
and any desired amount may be applied.
EXAMPLES
[0116] Hereinafter, the present invention will be described in
detail. However, the present invention is not limited to the
following Examples.
Production of Proteoglycan-Containing Materials
[0117] A head of salmon was immersed in water, and allowed to stand
for one day to swell. Then, tissue other than nasal cartilage was
removed from the head of salmon to obtain salmon nasal cartilage.
The salmon nasal cartilage was crushed into salmon nasal cartilage
powder. After 100 mL of water was added to 100 g of the powder and
gently stirred, the mixture was allowed to stand at room
temperature for 10 minutes, and defatted. Centrifugation (8000 rpm,
30 minutes, room temperature) was then carried out, and the
obtained residue (salmon nasal cartilage defatted powder) was
collected and freeze-dried. Using an ultracentrifugal mill, 9.02 g
of the freeze-dried salmon nasal cartilage defatted powder was
pulverized into fine powder with a particle size of about 100 to
200 .mu.m (measured by the laser diffraction scattering method).
The fine powder was washed with 20 mL of ethanol three times, and
then air-dried to obtain 7.69 g of fine powder containing acidic
saccharide components. This fine powder may be hereinafter referred
to as "salmon nasal cartilage powder." Note that "wash" with
ethanol here means an operation (ethanol precipitation) in which
fine powder is dispersed in ethanol, and then subjected to
centrifugation to collect the precipitate.
[0118] Furthermore, after 1000 mL of purified water at room
temperature (pH 6.5) was added to 20 g of salmon nasal cartilage
powder and stirred for 30 minutes, the mixture was allowed to stand
at room temperature for 10 minutes. This was followed by
centrifugation (8000 rpm, 30 minutes, room temperature). The
supernatant was collected and dried by concentration to obtain
about 7 g of powder containing acidic saccharide components. The
water extract thus obtained may be hereinafter referred to as
"water extract of salmon nasal cartilage powder."
[0119] The salmon nasal cartilage powder contained about 20 mass %
of acidic saccharide components and about 9 mass % of proteoglycan
(that is, about 11 mass % of acidic saccharide such as
glycosaminoglycan was contained therein). Additionally, the water
extract of salmon nasal cartilage powder contained about 35 mass %
of acidic saccharide components and about 15 mass % of proteoglycan
(that is, about 20 mass % of acidic saccharide such as
glycosaminoglycan was contained therein). These percentages were
calculated on the basis that uronic acid (glucuronic acid) was
quantified by the carbazole-sulfuric acid method; further, the
amount (mass) of chondroitin sulfate was determined using the
following formula well known in this quantification method, and the
amount of chondroitin sulfate was defined as the amount of acidic
saccharide components.
The amount of chondroitin sulfate=the amount of glucuronic
acid.times.2.593 [Formula 1]
[0120] Hereinafter, if not otherwise specified, the amount of
acidic saccharide components determined by the carbazole-sulfuric
acid method indicates the amount of chondroitin sulfate determined
in the same manner as above.
[0121] In addition, the proteoglycan content can be calculated from
the area ratio of the peak area showing proteoglycan in a
chromatogram to the entire area of the chromatogram. The
chromatogram is obtained as described below, by conducting gel
filtration chromatography analysis while monitoring the amount of
acidic saccharide components by quantifying uronic acid.
Specifically, the proteoglycan content can be calculated by
multiplying the area ratio by the amount of acidic saccharide
components.
Analysis of Molecular Weight of Proteoglycan-Containing
Materials
[0122] The molecular weight of the obtained proteoglycan-containing
materials was analyzed by gel filtration chromatography. More
specifically, salmon nasal cartilage powder and a water extract of
salmon nasal cartilage powder, used as samples, were subjected to
gel filtration chromatography conducted under the following
conditions; and 1 mL eluted fractions were collected to quantify
the amounts of acidic saccharide and protein contained in each of
the fractions by the carbazole-sulfuric acid method and the UV
absorption method, respectively.
[0123] FIG. 2 shows chromatograms obtained as a result of the gel
filtration chromatography analysis. Note that the chromatograms
that analyze the amount of acidic saccharide show the amount of
glucuronic acid quantified by the carbazole-sulfuric acid method
(not the amount of chondroitin sulfate determined by multiplying
the amount of glucuronic acid by 2.593). With respect to the salmon
nasal cartilage powder, extraction was performed with 4 M guanidine
hydrochloride (4 M GuCl) to increase purity, and the resulting
extract was used as a sample. The extraction was specifically
carried out as follows. 4 M GuCl was added to 1 g of the salmon
nasal cartilage powder and stirred at 4.degree. C. for one day,
followed by centrifugation. A threefold amount of ethanol saturated
with sodium chloride was added to the supernatant, and left to
stand overnight. Then, centrifugation was performed to collect the
precipitate. This precipitate was used as a sample for the gel
filtration chromatography. The water extract of salmon nasal
cartilage powder was used as is, as a sample.
[Gel Filtration Chromatography Conditions]
[0124] Column: Sepharose CL-2B packed column (.phi.1 cm.times.48 cm
column packed with Sepharose CL-2B as a carrier; Sepharose CL-2B
has a dextran fractionation range of 100 to 20,000 kDa, and is
available from, e.g., GE Healthcare; Sepharose CL-2B, CAS registry
No. 65099-79-8, is a 2% crosslinked agarose with a particle size of
60 to 200 .mu.m (measured by the laser diffraction scattering
method)) Buffer: 0.1 M phosphate buffer (pH 7.1, containing 0.2 M
NaCl) Amount of applied sample: 4 mg (dissolved in 1 ml of buffer
for use) Flow rate: 0.15 mL/min Amount of fraction: 1 mL/tube
[0125] In addition, commercially available proteoglycan
(hereinafter sometimes referred to as "PG-K") and commercially
available glycosaminoglycan (chondroitin) (hereinafter sometimes
referred to as "PG-M") were also subjected to gel filtration
chromatography under the same conditions, and the amount of acidic
saccharide contained in each eluted fraction was quantified. FIG. 3
shows the results.
[0126] As shown in FIG. 2, for an amount of eluate in the range of
about 15 to 23 mL, a peak was observed for both saccharide and
protein. Thus, it was found that this peak shows proteoglycan.
[0127] Next, each of the below-described various dextran molecular
weight markers was also subjected to gel filtration chromatography
under the same conditions as described above (except that the
amount of sample was 1 mg), and the amount of saccharide (i.e.,
amount of dextran) contained in each eluted fraction was quantified
by the phenol-sulfuric acid method. More specifically, the amount
of saccharide was quantified as follows, according to the method
described in Hodge, J. E. and Hofreiter, B. T., Methods in
Carbohydrate Chemistry, 1, 338 (1962).
[1] 500 .mu.l of a sample aqueous solution or a standard
monosaccharide (mannose) aqueous solution was placed in a
105.times.15 mm test tube. [2] 500 .mu.l of a phenol reagent (5 v/v
% aqueous phenol solution) was added thereto, and stirred. [3] 2.5
ml of concentrated sulfuric acid was added thereto, and immediately
stirred vigorously for 10 seconds. [4] The mixture was left to
stand for 20 minutes or more at room temperature. [5] The
absorption at 490 nm was measured with a spectrophotometer.
<Dextran Molecular Weight Markers>
[0128] Dextran from Leuconostoc mesenteroides (mol wt
5,000,000-40,000,000) (Sigma-Aldrich Co.) . . . for measuring void
volume, 10000 kDa
Dextran Standard 1,400,000 (Sigma-Aldrich Co.) . . . 1400 kDa
Dextran Standard 670,000 (Sigma-Aldrich Co.) . . . 670 kDa
Dextran Standard 410,000 (Sigma-Aldrich Co.) . . . 410 kDa
Dextran Standard 270,000 (Sigma-Aldrich Co.) . . . 270 kDa
[0129] The dextran marker from Leuconostoc mesenteroides was used
for measuring the void volume of the Sepharose CL-2B packed column
(upper limit of fractionation: 20,000 kDa). To more accurately
measure the void volume, pretreatment was carried out to remove
low-molecular weight dextran contained in the marker. The
pretreatment was performed by eluting the dextran from Leuconostoc
mesenteroides under the conditions described above in "Gel
Filtration Chromatography Conditions," and collecting and
freeze-drying fractions having a molecular weight of 20,000 kDa or
more. More specifically, fractions consisting of the amount of
eluate of from 15 to 19 mL, which corresponded to a first appeared
peak, were collected and freeze-dried (it is believed that dextran
having a molecular weight of 20,000 kDa or more was obtained by
collecting and freeze-drying such fractions. Then, this
freeze-dried product was applied to the column, and measured.
[0130] The resulting chromatograms are shown in FIG. 4A to 4E. FIG.
4A shows measurement of the aforementioned pretreated freeze-dried
product. Since the molecular weight of the pretreated product of
dextran from Leuconostoc mesenteroides in FIG. 4A exceeds the
fractionation range (100 kDa to 20000 kDa) of the Sepharose CL-2B
packed column used, the amount of eluate corresponding to the peak
top position was defined as the amount of eluate at which molecules
of 20000 kDa, which is the exclusion limit of the column, are
eluted. This amount of eluate is interpreted as indicating the void
volume of the column. In each of FIGS. 4B to 4E, the amount of
eluate corresponding to the position of the bisector of the peak
area in the chromatogram designates the amount of eluate at which
molecules of molecular weight of the marker are eluted. FIGS. 4B to
4E respectively show results of measurements for Dextran Standard
1,400,000, 670,000, 410,000, and 270,000. The relationship between
these amounts of eluate and molecular weights were graphed, and a
linear calibration curve was obtained (y=-4.1355 Ln(x)+59.47;
R.sup.2=0.9869) (FIG. 5). From this, it was confirmed that the
molecular weights and the amounts of eluate obtained by the dextran
molecular weight markers are highly correlated.
[0131] Furthermore, it was found from analysis results that there
is a high possibility that an eluate before reaching the void
volume contains proteoglycan (i.e., proteoglycan of greater than
20000 kDa, fractionation limit, exists). Since the fractionation
range of the column used in the gel filtration chromatography was
100 kDa to 20000 kDa, it is highly possible that molecules of 20000
kDa or more were not accurately fractionated. Thus, as in the
above, analysis by gel filtration chromatography under the
following conditions was also performed using salmon nasal
cartilage powder as a sample, and the amount of acidic saccharide
in each fraction was quantified.
[Gel Filtration Chromatography Conditions]
[0132] Column: Sephacryl S-1000 SF packed column (.phi.1
cm.times.48 cm column packed with Sephacryl S-1000 SF as a carrier;
Sephacryl S-1000 SF has a dextran fractionation range of
5.times.10.sup.5 to 1.times.10.sup.8 Da, and is available from,
e.g., GE Healthcare) Buffer: 0.1 M phosphate buffer (pH 7.1,
containing 0.2 M NaCl) Amount of applied sample: 4 mg Flow rate:
0.3 mL/min Amount of fraction: 1 mL/tube
[0133] Furthermore, the below-described molecular weight markers
were subjected to gel filtration chromatography under the same
conditions. The amount of saccharide (i.e., amount of dextran)
contained in each eluted fraction was quantified by the
phenol-sulfuric acid method, and a calibration curve was
prepared.
<Dextran Molecular Weight Markers>
[0134] Dextran from Leuconostoc mesenteroides (mol wt
5,000,000-40,000,000) (Sigma-Aldrich Co.) . . . 10000 kDa
Dextran Standard 1,400,000 (Sigma-Aldrich Co.) . . . 1400 kDa
Dextran Standard 670,000 (Sigma-Aldrich Co.) . . . 670 kDa
[0135] The obtained calibration curve was as follows:
y=-3.8743 Ln(x)+59.887(R.sup.2=0.9961)
[0136] FIG. 6 is a graph that collectively shows the graphs of
measurement results of acid saccharide amounts illustrated in FIGS.
2 and 3, and further shows the relationship between the molecular
weights and the amounts of eluate obtained as described above. As
mentioned above, regarding the salmon nasal cartilage powder and
the water extract of salmon nasal cartilage powder, a peak appears
in an amount of eluate in the range of about 15 to 23 mL in the gel
filtration chromatography analysis. On the other hand, a peak
appears in the range of about 28 to 49 mL for commercially
available proteoglycan PG-M, and in the range of about 18 to 47 mL
for commercially available PG-K. This shows that the salmon nasal
cartilage powder and the water extract of salmon nasal cartilage
powder (i.e., proteoglycan-containing materials of the present
invention) contain very high molecular weight proteoglycan that is
different from hitherto-known proteoglycan.
[0137] In addition, from the calibration curve shown in FIG. 5, it
can be calculated that an amount of eluate of 23 mL corresponds to
a molecular weight of about 6700 kDa. From this, the salmon nasal
cartilage powder and the water extract of salmon nasal cartilage
powder were found to contain proteoglycan of a molecular weight of
about 6000 kDa or more.
[0138] Furthermore, FIG. 7 shows the results of analysis of the
salmon nasal cartilage powder using the Sephacryl S-1000 SF packed
column. In FIG. 7, the rising of the proteoglycan peak from its
first appearance in the chromatogram starts from an amount of
eluate in the range of 15 to 16 mL. The molecular weight
corresponding to this range of amount of eluate was calculated
using the aforementioned calibration curve (y=-3.8743
Ln(x)+59.887), and determined to be about 90000 kDa. Thus, the
salmon nasal cartilage powder was found to contain proteoglycan of
about 90000 kDa.
[0139] From the above, it was confirmed that the salmon nasal
cartilage powder contains proteoglycan of about 6000 to 90000
kDa.
[0140] Furthermore, using the calibration curve shown in FIG. 5,
the mean molecular weight of proteoglycan in each sample was
calculated from the amount of eluate at the peak position in each
corresponding graph in FIG. 6. The mean molecular weight is
commonly calculated from the amount of eluate at the position of
the bisector of a peak area. However, since the proteoglycan peak
in each chromatogram shown in FIG. 6 is nearly symmetrical in
shape, the peak position was defined as a bisector position, and
the mean molecular weight was calculated. More specifically, in
consideration of experimental error and lot difference, values
within .+-.1 mL of the amount of eluate at the peak position were
regarded as y values of the calibration curve, and the range of the
obtained x values was regarded as the mean molecular weight of
proteoglycan in each sample. However, since the upper limit of
fractionation range of the column used (Sepharose CL-2B packed
column) was 20000 kDa, there is a possibility that the upper limit
of the mean molecular weight of proteoglycan obtained from the
analysis was not accurately calculated. Therefore, similarly, the
mean molecular weight of proteoglycan was also determined from the
results obtained when the Sephacryl S-1000 SF packed column was
used. The results are shown in Table 1.
TABLE-US-00001 TABLE 1 Mean molecular weight Mean molecular weight
of proteoglycan of proteoglycan contained (Sepharose contained
(Sephacryl CL-2B packed S-1000 SF column) packed column) Salmon
nasal cartilage 12200 kDa to 19500 kDa 22000 kDa to powder 38000
kDa Water extract of salmon 9700 kDa to 15500 kDa nasal cartilage
powder PG-K 480 kDa to 760 kDa PG-M 90 kDa to 150 kDa
(chondroitin)
[0141] It was found from the above results that the mean molecular
weight of proteoglycan contained in the proteoglycan-containing
materials of the present invention is about 9700 kDa to 38000
kDa.
[0142] Additionally, regarding the salmon nasal cartilage powder
and the water extract of salmon nasal cartilage powder, the amount
of eluate corresponding to the position where the area of the
chromatogram obtained by the analysis with the Sepharose CL-2B
packed column was bisected was determined (dotted arrows in FIG.
6), and the mean molecular weight was obtained from values within
.+-.1 mL of the determined amount of eluate. As a result, the mean
molecular weight of the mixture of acidic saccharide components in
the salmon nasal cartilage powder was about 4800 kDa to 7700 kDa,
and the mean molecular weight of the mixture of acidic saccharide
components in the water extract of salmon nasal cartilage powder
was about 1800 kDa to 4200 kDa.
[0143] Furthermore, regarding the salmon nasal cartilage powder,
the amount of eluate corresponding to the position where the area
of the chromatogram obtained by the analysis with the Sephacryl
S-1000 SF packed column was bisected was determined (dotted arrow
in FIG. 7), and the mean molecular weight was obtained from values
within .+-.1 mL of the determined amount of eluate. As a result,
the mean molecular weight of acidic saccharide components contained
in the salmon nasal cartilage powder was about 3900 kDa to 6600
kDa.
Analysis of Skin Anti-Aging Effect of Proteoglycan-Containing
Materials
Evaluation of Capability to Promote Cell Proliferation
[0144] Salmon nasal cartilage powder, a water extract of salmon
nasal cartilage powder, and PG-K were used as samples, and their
cell proliferation effects were analyzed. More specifically, the
following experiment was carried out. In a culturing dish, human
skin fibroblasts (HDF50: Cell Applications, Inc.) were seeded at
1.0.times.10.sup.4 cells in minimum essential medium (MEM)
containing 10% fetal bovine serum (FBS). Each sample was added
individually to MEM to a concentration of 1 .mu.g/mL or 10
.mu.g/mL. Additionally, a medium to which nothing was added was
used as a control. After the addition, the cells were cultured for
five days. Following the culture, the MEM was removed, and the
cells were detached and suspended with Trypsin-EDTA (Invitrogen).
Afterward, Trypan blue stain (Sigma-Aldrich Co.) was added, and the
number of cells was counted using a Burker-Turk counting
chamber.
[0145] FIG. 8 shows the results. It was found from the results that
the salmon nasal cartilage powder and the water extract of salmon
nasal cartilage powder exhibit significant capability to
proliferate human skin fibroblasts, while PG-K, a commercially
available proteoglycan, does not exhibit the proliferation
capability.
[0146] Moreover, as shown in FIG. 6, there is a great difference in
the molecular weights of the contained components of PG-K, and the
salmon nasal cartilage powder and the water extract of salmon nasal
cartilage powder. In particular, in the chromatogram for PG-K, a
peak for proteoglycan of large molecular weight, which exists in
the chromatograms for the salmon nasal cartilage powder and the
water extract of salmon nasal cartilage powder, is not seen. Thus,
it seemed that the aforementioned capability to proliferate human
skin fibroblasts is attributed to the proteoglycan of large
molecular weight.
Evaluation of Moisturizing and Anti-Skin Aging-Capability Through
Ingestion
<Experimental Animal Used>
[0147] Hairless mice (Hr-/Kud ) (Kyudo Co., Ltd.) were used for an
experiment. Male mice (four weeks old) that were free of the
influence of fluctuations in estrogen on skin conditions were
preliminarily fed, and then used for the experiment.
<Test Method>
[0148] The mice were placed in five feeding cages, as shown in
Table 2 (six mice for one group). In addition, the subjects were
marked on their tail portion to be individually identified. They
continued to be preliminarily fed until they reached seven weeks of
age.
TABLE-US-00002 TABLE 2 UVB Group Abbreviation Evaluation material
irradiation 1 Co-UVB Water (control) Without 2 Co+UVB Water
(control) With 3 HA+UVB Hyaluronic acid With 4 PG+UVB
Proteoglycan-containing material With 5 PG/HA+UVB
Proteoglycan-containing material + With hyaluronic acid
<Preparation of Oral Administration Samples of Evaluation
Materials>
[0149] A 2% dispersion of salmon nasal cartilage powder was
prepared, and centrifugation was performed. The resulting
supernatant was used as an administration sample of
proteoglycan-containing material. This supernatant corresponds to a
water extract of salmon nasal cartilage powder dissolved in water.
The supernatant was evaporated to dryness to give solids. The
supernatant contained about 0.7 mass % of water extract of salmon
nasal cartilage powder. In addition, the amount of acidic
saccharide components contained in the supernatant was quantified
by the carbazole-sulfuric acid method. The amount of acidic
saccharide components was about 0.17 mass % relative to the
supernatant. Moreover, gel filtration chromatography analysis was
conducted to determine the amount of proteoglycan contained in the
supernatant from an area ratio of the obtained chromatogram. The
amount of proteoglycan was about 0.07 mass % relative to the
supernatant.
[0150] A 0.5 mass % aqueous solution of hyaluronic acid was
prepared and used as an administration sample of hyaluronic acid.
Additionally, a 1:1 liquid mixture (mass ratio) of the
administration sample of proteoglycan-containing material and the
administration sample of hyaluronic acid was used as an
administration sample for coadministration of proteoglycan and
hyaluronic acid. To the controls, distilled water was administered.
Note that hyaluronic acid purchased from Nakahara, Co., Ltd. was
used.
<Oral Administration Method>
[0151] When the hairless mice reached seven weeks of age, 0.5 mL of
each administration sample was individually given once a day by
forced oral administration using a sonde. This administration was
continued at a frequency of five times per week (Monday to Friday)
until the end of the experiment.
The amounts of the evaluation materials contained in each of the
administration samples were as follows.
[0152] Hyaluronic acid: 2.5 mg/day
[0153] Proteoglycan-containing material: about 3.5 mg/day (acidic
saccharide components: about 0.83 mg/day, proteoglycan: about 0.33
mg/day)
[0154] Proteoglycan-containing material+hyaluronic acid: about 1.75
mg/day of proteoglycan-containing material (acidic saccharide
components: about 0.42 mg/day, proteoglycan: about 0.17 mg/day),
and 1.25 mg/day of hyaluronic acid
<UVB Irradiation Method>
[0155] UVB irradiation started from four weeks after the start of
the oral administration. The mice were placed in a cage for UVB
irradiation. The cage was placed into an UVB irradiation device to
carry out UVB irradiation five times per week (Monday to Friday),
at an intensity of 1.0 mW/cm.sup.2. The amount of irradiation was
60 mJ/cm.sup.2 during only the first week after the start of the
irradiation, and 120 mJ/cm.sup.2 from the second week onward. The
total amount of UVB irradiation during the 10-week period was 5.7
J/cm.sup.2. Note that UVB was ultraviolet rays with wavelengths
from 280 to 315 nm.
<Evaluation of Skin Barrier Functions>
[0156] Using a Tewameter, a multiprobe-type skin measuring
instrument (MPA580: Courage-Khazaka), transepidermal water loss
(TEWL) was measured at a frequency of once a week to evaluate skin
barrier functions. Three areas on the back of each mouse were
measured, and the average value was calculated. It is indicated
that the larger the TEWL value, the lower the skin barrier
functions (functions that preventingress of foreign matter from
outside the skin into the body, and that prevent the escape of
moisture inside the body to the outside).
[0157] FIG. 9 shows the results of TEWL measurement eight weeks
after the start of the UVB irradiation. In addition, Table 3 shows
relative values of TEWL of mice Groups 3 to 5 four weeks, six
weeks, and eight weeks after the start of the UVB irradiation, with
respect to the TEWL value of an unirradiated control (Group 1:
Co-UVB), which was assumed to be 100; and the TEWL value of an
UVB-irradiated control (Group 2: Co+UVB), which was assumed to be
0. The relative values can be said to indicate a skin barrier
improvement rate (%).
TABLE-US-00003 TABLE 3 <UVB Irradiation> 4 weeks 6 weeks 8
weeks HA+UVB -15 -5 -17 PG+UVB 5.2 3.7 34 PG/HA+UVB 36 35 30
[0158] It was found from these results that the
proteoglycan-containing material from salmon nasal cartilage lowers
the TEWL value, and improves skin barrier functions through oral
administration (FIG. 9 and Table 3). Furthermore, it was found that
ingestion of the proteoglycan-containing material from salmon nasal
cartilage in combination with hyaluronic acid ensures the effect of
improving skin barrier functions at an earlier stage (Table 3).
<Evaluation of Skin Elasticity>
[0159] Skin elasticity was measured with a Cutometer, a
multiprobe-type skin-measuring instrument (MPA580:
Courage-Khazaka). More specifically, four areas on the back of each
mouse were measured, and elasticity (R2 value) was calculated by
the following formula using the obtained Uf value and Ua value.
Note that the Ua value represents return of the skin upon release
from aspiration, and the Uf vale represents extensibility of the
skin upon aspiration.
Elasticity (R2)=Ua/Uf
[0160] FIG. 10 shows the results of analysis conducted eight weeks
after the start of the UVB irradiation (in FIG. 10, UVB is written
as UV). It was suggested from the results that the
proteoglycan-containing material from salmon nasal cartilage has an
effect of improving skin elasticity through oral administration.
Furthermore, it was shown that ingestion of the
proteoglycan-containing material from salmon nasal cartilage in
combination with hyaluronic acid significantly restores skin
elasticity.
<Evaluation of Capability to Produce Collagen>
[0161] Ten weeks after the start of the UVB irradiation, skin
tissue on the back of each mouse was collected. A portion of the
back skin tissue was subjected to formalin fixation (for preparing
skin tissue sections), and the rest was used for quantifying
collagen.
[0162] The thus-obtained skin tissue (for quantifying collagen) was
frozen and pulverized into powder with a cell grinder (auto mill,
TK-AM5) (Tokken), and the powder was dried with a vacuum dryer.
Protease inhibitor (P.I.) cocktail tablets (Complete Mini Easy Pack
(Roche)) were dissolved in 0.5 M acetic acid. This acetic acid
(containing P.I.) was added to the above skin tissue powder, and
stirred at a low temperature. This was followed by centrifugation,
and the supernatant portion (acid soluble collagen extract) was
collected.
[0163] Then, the amount of collagen of the acid-soluble collagen
extract was measured using a kit for quantifying acid-soluble
collagen (Sircol Soluble Collagen Assay (Biocolor)) based on the
manual.
[0164] FIG. 11 shows the results. It was found from the results
that the proteoglycan-containing material from salmon nasal
cartilage significantly improves decrease in capability to produce
collagen in the skin through oral administration.
<Analysis of Dermis-Thickening Inhibition Effect>
[0165] Using the aforementioned formalin-fixed tissue sections,
paraffin-embedded blocks were prepared with an automatic paraffin
fixation device (tissue processor (Tissue-Tek)). Sections were made
with a microtome, stained with Hematoxylin-Eosin (HE), and used as
samples.
[0166] The samples were observed with an optical microscope, and
images were saved on a digital camera. In each of the obtained
images, the thickness of the dermal layer was measured at 10
locations. The average of the measurement values was calculated as
the thickness of the dermal layer. FIG. 12 shows the results (in
FIG. 12, UVB is written as UV). It was found from the results that
the proteoglycan-containing material from salmon nasal cartilage
significantly inhibits dermis-thickening through oral
administration. Furthermore, it was shown that ingestion of the
proteoglycan-containing material from salmon nasal cartilage in
combination with hyaluronic acid enables dermis-thickening to be
significantly inhibited, and that the inhibition capability is
superior to that in cases where only the proteoglycan-containing
material from salmon nasal cartilage was taken.
Evaluation of Moisturizing and Anti-Skin-Aging Capability by
Applying to the Skin
<Experimental Animal Used>
[0167] Hairless mice (Hr-/Kud ) (Kyudo Co., Ltd.) were used for an
experiment. Male mice (four weeks old) that were free of the
influence of fluctuations in estrogen on skin conditions were
preliminarily fed, and then used for the experiment.
<Test Method>
[0168] The mice were placed in four feeding cages, as shown in
Table 4 (five mice for one group). In addition, the subjects were
marked on their tail portion to be individually identified. They
continued to be preliminarily fed until they reached seven weeks of
age.
TABLE-US-00004 TABLE 4 UVB Group Abbreviation Evaluation material
(application sample) irradiation 1 Co-UVB 0.5% xanthan gum aqueous
solution Without (control) 2 Co+UVB 0.5% xanthan gum aqueous
solution With (control) 3 HA+UVB 0.5% hyaluronic acid aqueous
solution With 4 PG+UVB 0.5% salmon nasal cartilage powder With
aqueous solution
<Evaluation of Skin Barrier Functions>
[0169] When the hairless mice reached seven weeks of age, 0.1 mL of
one respective application sample was applied to the back of the
mice once a day, and the hairless mice were subjected to UVB
irradiation in the same manner as described in the section
"Evaluation of Moisturizing and Anti-Skin Aging-Capability Through
Ingestion" above. Five weeks after the start of the UVB
irradiation, transepidermal water loss (TEWL) was measured in the
same manner as described in the aforementioned section "Evaluation
of Moisturizing and Anti-Skin Aging-Capability Through Ingestion."
FIG. 13 shows the results (in FIG. 13, UVB is written as UV). It
was found from the results that the proteoglycan-containing
material from salmon nasal cartilage significantly lowers the TEWL
value, and improves skin barrier functions by application to the
skin.
[0170] As described above, it seemed that the capability to
proliferate human skin fibroblasts is attributed to proteoglycan of
large molecular weight, which is not contained in PG-K. In view of
this, it appeared that various other effects are also attributed to
the proteoglycan.
Fractionation of Water Extract of Salmon Nasal Cartilage Powder and
Effect Verification
[0171] A water extract of salmon nasal cartilage powder was
fractionated using ion-exchange chromatography and gel filtration
chromatography to analyze which fraction has a cell proliferation
effect. FIG. 14 shows a procedure of the fractionation. The
following are the fractionation conditions.
<Ion-Exchange Chromatography>
[0172] A .phi.5.0 cm.times.20 cm column was packed with a carrier
(DEAE Sephacel (GE Healthcare)) to a height of 15 cm. Note that
DEAE is an abbreviation of diethylaminoethyl.
[0173] As a solvent, 7 M urea-50 mM tris-hydrochloric acid buffer
(pH 7.4) was used. Using a solvent in which 0 to 0.75 M sodium
chloride was added to the above solvent, elution was performed by
gradient-elution (linear gradient).
About 100 mg of a water extract of salmon nasal cartilage powder
was dissolved in about 20 ml of the solvent. Afterward, an
operation was carried out according to page 189 of Kiso Seikagaku
Jikkenho (Basic Biochemistry Experimental Method), Vol. 5
(Shishitsu/Toshitsu/Fukugo Toshitsu (Lipids/Carbohydrates/Complex
Carbohydrates)) edited by the Japanese Biochemical Society (Tokyo
Kagaku Dojin); and the water extract of salmon nasal cartilage
powder was fractionated into a protein fraction, a hyaluronic acid
fraction, and a fraction of acidic saccharide with sulfate groups.
Elution of the column was performed at a flow rate of 2.0 ml/min,
and the volume of each of the individual fractions in the following
combined fractions was 16 ml. In this case, the protein fraction
was a combined fraction of Fraction Nos. 16 to 35; the hyaluronic
acid fraction was a combined fraction of Fraction Nos. 37 to 42;
and the fraction of acidic saccharide with sulfate groups was a
combined fraction of Fraction Nos. 52 to 67. Although hyaluronic
acid is also a kind of acidic saccharide, it does not have sulfate
groups. Acidic saccharide (for example, chondroitin sulfate)
contained in proteoglycan has sulfate groups. Additionally, since
molecular polarity increases in the order of protein (in
particular, collagen), hyaluronic acid, and acidic saccharide
having sulfate groups, these three substances can be fractionated
by ion exchange chromatography.
[0174] Protein was quantified by absorbance measurement at 280 nm.
Hyaluronic acid was quantified using a Seikagaku Co. kit for
quantifying hyaluronic acid. Acid saccharide having sulfate groups
was quantified by the carbazole-sulfuric acid method.
[0175] The amounts of protein, hyaluronic acid, and acidic
saccharide having sulfate groups that correspond to the case where
100 mg of the water extract of salmon nasal cartilage powder was
isolated were 0.9 mg, 1.2 mg, and 43.0 mg, respectively.
[0176] Four samples of the thus-obtained protein fraction,
hyaluronic acid'fraction, fraction of acidic saccharide with
sulfate groups, and a water extract of salmon nasal cartilage
powder were used, and the capability to proliferate human skin
fibroblasts of each sample was analyzed in the same manner as in
the section "Evaluation of Capability to Promote Cell
Proliferation" above. FIG. 15 shows the results. Other than the
water extract of salmon nasal cartilage powder, only the fraction
of acidic saccharide with sulfate groups had significantly high
capability to proliferate human skin fibroblasts with respect to
the control. Moreover, the fraction of acidic saccharide with
sulfate groups had high capability to proliferate human skin
fibroblasts, even as compared with the water extract of salmon
nasal cartilage powder. Hence, it seemed that the effect of
proliferating human skin fibroblasts of the water extract of salmon
nasal cartilage powder is attributed to acidic saccharide having
sulfate groups. In addition, it appeared that this effect is
attributable to proteoglycan, due to the fact that the fraction of
acidic saccharide with sulfate groups is rich in proteoglycan.
[0177] In FIGS. 14 to 16, the fraction of acidic saccharide with
sulfate groups is simply written as "acidic saccharide
fraction."
<Gel Filtration Chromatography>
[0178] 43.0 mg of the fraction of acidic saccharide with sulfate
groups obtained as described above was further fractionated by gel
filtration chromatography. More specifically, under the gel
filtration chromatography conditions using a Sepharose CL-2B packed
column as described in the section "Analysis of Molecular Weight of
Proteoglycan-Containing Materials" above, 1 ml of buffer was added
per 5 mg of the fraction of acidic saccharide with sulfate groups,
and dissolved to fractionate the fraction of acidic saccharide with
sulfate groups into a fraction having a molecular weight of 5000
kDa or more, and a fraction having a molecular weight of less than
5000 kDa. The amount of acidic saccharide contained in each
fraction was quantified by the carbazole-sulfuric acid method. The
fraction having a molecular weight of 5000 kDa or more contained
9.7 mg of acidic saccharide, and the fraction having a molecular
weight of less than 5000 kDa contained 16.8 mg of acidic
saccharide.
[0179] FIG. 16 shows results in which the effect of proliferating
human skin fibroblasts of these fractions was analyzed in the same
manner as above. It was shown that the fraction having a molecular
weight of 5000 kDa or more has a high effect of proliferating human
skin fibroblasts, as compared to the fraction having a molecular
weight of less than 5000 kDa. From the results, it appeared that
the effect is largely attributable to proteoglycan. In particular,
it seemed that proteoglycan of large molecular weight (molecular
weight of 5000 kDa or more) contributes to the effect.
[0180] Note that the meanings of the marks "+," "**," "***" in
FIGS. 15 and 16 are the same as those in FIG. 8.
[0181] Formulation examples of oral compositions, cosmetic
compositions, and food or beverage compositions according to the
present invention are described below. Note that % indicates mass
%. Formulation Examples 1 to 7 are for the cosmetic compositions;
Formulation Examples 8 to 16 are for the food or beverage
compositions; and Formulation Examples 17 to 22 are for the oral
compositions.
Methods for producing proteoglycan-containing materials
individually used in each of the following formulation examples are
also described below.
<Proteoglycan-Containing Material A>
[0182] [1] Extraneous tissue such as skin or bone is removed from
salmon nasal cartilage, and the resulting cartilage is crushed with
a meat chopper. [2] Tap water having a pH of 6.0 to 7.5 in an
amount (volume) that is about double or triple the amount of the
cartilage is added to the crushed salmon nasal cartilage, and
sufficiently stirred at a temperature of 40.degree. C. or below
(room temperature). [3] After the stirring, solids are separated
and collected. [4] Steps [2] and [3] are performed twice. [5] The
resulting solids are freeze-dried. [6] The dried solids are finely
pulverized with an atomizer mill. [7] 95% ethanol in an amount
(volume) that is about ten times the amount of the finely
pulverized salmon nasal cartilage is added to the finely pulverized
cartilage, and sufficiently stirred at a temperature of 40.degree.
C. or below. [8] After the stirring, solids are separated and
collected. [9] Steps [7] and [8] are performed twice. [10] The
resulting solids are evaporated to dryness.
<Proteoglycan-Containing Material B>
[0183] [1] Purified water having a pH of 6.0 to 7.0 in an amount
(volume) that is about ten times the amount of
proteoglycan-containing material A is added to
proteoglycan-containing material A, and sufficiently stirred at a
temperature of 40.degree. C. or below (room temperature) for about
30 minutes to 6 hours. [2] After solids are separated and removed,
the resulting solution is dried to obtain solids.
<Proteoglycan-Containing Material C>
[0184] [1] Purified water having a pH of 6.0 to 7.0 in an amount
(volume) that is about ten times the amount of
proteoglycan-containing material A is added to
proteoglycan-containing material A, and sufficiently stirred at a
temperature of 40.degree. C. or below (room temperature) for about
30 minutes to 6 hours. Then, solids are separated and removed, and
an aqueous solution is obtained. [2] Ethanol in an amount that is
about five times the amount of the obtained aqueous solution is
added to the aqueous solution, and sufficiently stirred at a
temperature of 40.degree. C. or below (room temperature). [3] The
resulting solids are collected and dried.
<Proteoglycan-Containing Material D>
[0185] [1] Extraneous tissue such as skin or bone is removed from
salmon nasal cartilage, and the resulting cartilage is crushed with
a meat chopper. [2] Purified water having a pH of 6.5 to 7.5 in an
amount (volume) that is about equal or double the amount of the
cartilage is added to the crushed salmon nasal cartilage, and
sufficiently stirred at a temperature of 40.degree. C. or below
(room temperature). [3] After the stirring, solids are separated
and collected. [4] Steps [2] and [3] are performed three times. [5]
The resulting solids are finely pulverized with a wet grinder. [6]
95% ethanol in an amount (volume) that is about ten times the
amount of the finely pulverized salmon nasal cartilage is added to
the finely pulverized cartilage, and sufficiently stirred at a
temperature of 40.degree. C. or below (room temperature). [7] After
the stirring, solids are separated and collected. [8] Steps [6] and
[7] are performed once. [9] The resulting solids are evaporated to
dryness. [10] Purified water having a pH of 6.5 to 7.5 in an amount
(volume) that is about ten times the amount of the dried product
obtained in the above Step [9] is added to the dried product, and
the dried product is immersed while stirring at a low temperature
for about 12 to 48 hours. [11] After the immersion, solids were
separated and removed, and an aqueous solution is obtained. [12]
Ethyl alcohol in an amount that is about five times the amount of
the aqueous solution was added to the aqueous solution, and
sufficiently stirred at a temperature of 40.degree. C. or below
(room temperature). [13] The resulting solids are collected and
dried.
<Proteoglycan-Containing Material E>
[0186] [1] Extraneous tissue such as skin or bone is removed from
salmon nasal cartilage, and the resulting cartilage is pulverized
with a meat chopper. [2] Tap water having a pH of 6.0 to 7.5 in an
amount (volume) that is about five times the amount of the
cartilage is added to the pulverized salmon nasal cartilage, and
sufficiently stirred at a temperature of 40.degree. C. or below
(room temperature). [3] After the stirring, solids are separated
and collected. [4] Steps [2] and [3] are performed twice. [5] The
resulting solids are finely pulverized with a wet grinder. [6] 95%
ethanol in an amount (volume) that is about five times the amount
of the finely pulverized salmon nasal cartilage is added to the
finely pulverized cartilage, and sufficiently stirred at a
temperature of 40.degree. C. or below (room temperature). [7] After
the stirring, solids are separated and collected. [8] Steps [6] and
[7] are performed twice. [9] Purified water having a pH of 6.0 to
7.0 in an amount (volume) that is about equal or double the amount
of the resulting solids is added to the solids, and sufficiently
stirred at a temperature of 40.degree. C. or below for about 30
minutes to 6 hours. Then, solids are separated and removed.
<Proteoglycan-Containing Material F>
[0187] [1] Extraneous tissue such as skin or bone is removed from
salmon nasal cartilage, and the resulting cartilage is crushed with
a wet grinder. [2] Tap water having a pH of 6.0 to 7.5 in an amount
(volume) that is about five times the amount of the cartilage is
added to the crushed salmon nasal cartilage, and sufficiently
stirred at a temperature of 40.degree. C. or below (room
temperature). [3] After the stirring, solids are separated and
collected. [4] Steps [2] and [3] are performed twice.
[0188] [5] The resulting solids are finely pulverized with a wet
grinder.
[6] Ethanol (product under standards of food additives) in an
amount (volume) that is about five times the amount of the finely
pulverized salmon nasal cartilage is added to the finely pulverized
cartilage, and sufficiently stirred at a temperature of 40.degree.
C. or below (room temperature). [7] After the stirring, solids are
separated and collected. [8] Steps [6] and [7] are performed twice.
[9] Purified water having a pH of 6.0 to 7.0 in an amount (volume)
that is about five times the amount of the resulting solids was
added to the solids, and sufficiently stirred at a temperature of
40.degree. C. or below for about 30 minutes to 6 hours. Then,
solids are separated and removed, and an aqueous solution is
obtained. [10] 95% ethanol in an amount that is about ten times the
amount of the obtained aqueous solution is added to the aqueous
solution, and sufficiently stirred at a temperature of 40.degree.
C. or below (room temperature). The resulting solids are collected
and then evaporated to dryness.
<Proteoglycan-Containing Material G>
[0189] [1] Extraneous tissue such as skin or bone is removed from
salmon nasal cartilage, and the resulting cartilage is crushed with
a wet grinder. [2] Tap water having a pH 6.0 to 7.5 in an amount
(volume) that is about ten times the amount of the cartilage is
added to the crushed salmon nasal cartilage, and sufficiently
stirred at a temperature of 40.degree. C. or below (room
temperature). [3] After the stirring, solids are separated and
collected. [4] Steps [2] and [3] are performed twice. [5] The
resulting solids are finely pulverized with a wet grinder. [6] 95%
ethanol in an amount (volume) that is about three times the amount
of the finely pulverized salmon nasal cartilage was added to the
finely pulverized cartilage, and sufficiently stirred at a
temperature of 40.degree. C. or below (room temperature). [7] After
the stirring, solids are separated and collected. [8] Steps [6] and
[7] are performed three times. [9] Purified water having a pH of
6.0 to 7.0 in an amount (volume) that is about double or triple the
amount of the resulting solids is added to the solids, and
sufficiently stirred at a temperature of 40.degree. C. or below for
about 30 minutes to 6 hours. Then, solids are separated and
removed, and an aqueous solution is obtained. [10] Sodium chloride
is added to the aqueous solution after the separation, and the
solution is saturated with sodium chloride. [11] 95% ethanol in an
amount that is about five times the amount of the aqueous solution
is added to the aqueous solution, and sufficiently stirred at a
temperature of 40.degree. C. or below (room temperature). The
resulting solids are collected and then evaporated to dryness.
Regarding proteoglycan-containing materials A to D, the amount of
acidic saccharide in each material was determined by the
carbazole-sulfuric acid method, and the amount of proteoglycan in
each material was determined from the area ratio of each
corresponding chromatogram. The mass ratio of acidic saccharide and
proteoglycan to each material on a dry mass basis is as follows. A:
Acidic saccharide: about 35% Proteoglycan: about 15% B: Acidic
saccharide: about 45% Proteoglycan: about 18% C: Acidic saccharide:
about 55% Proteoglycan: about 23% D: Acidic saccharide: about 60%
Proteoglycan: about 24%
Formulation Examples
Formulation Example 1
Lotion
TABLE-US-00005 [0190] Components Amount % Proteoglycan-containing
material E 5.0 Ethyl alcohol 20.0 1,3-butylene glycol 5.0
Phenoxyethanol 0.7 Oxyethylene hydrogenated castor oil (60 E.O.)
0.3 Oxyethylene hydrogenated castor oil (40 E.O.) 0.05 Citric acid
0.08 Sodium citrate 0.08 Polyethylene glycol 0.03 (mean molecular
weight: one million) Perfume 0.03 Purified water Balance Total
100.0
Formulation Example 2
Serum
TABLE-US-00006 [0191] Components Amount % Proteoglycan-containing
material G 2.0 Ethyl alcohol 10.0 Concentrated glycerin 10.0
1,3-butylene glycol 6.0 Phenoxyethanol 0.7 Oxyethylene hydrogenated
castor oil (60 E.O.) 0.5 Hydrogenated soybean phospholipid 0.5
Xanthan gum 0.4 Citric acid 0.08 Sodium citrate 0.08 Perfume 0.1
Purified water Balance Total 100.0
Formulation Example 3
Emulsion
TABLE-US-00007 [0192] Components Amount % Proteoglycan-containing
material D 0.2 Ethyl alcohol 10.0 1,3-butylene glycol 5.0
Concentrated glycerin 5.0 Carboxyvinyl polymer 0.5 Phenoxyethanol
0.5 Oxyethylene hydrogenated castor oil (60 E.O.) 0.3 Potassium
hydroxide 0.3 Polyoxyethylene hydrogenated castor oil (40 E.O.) 0.1
Citric acid 0.05 Sodium citrate 0.05 Disodium edetate 0.05 Perfume
0.1 Purified water Balance Total 100.0
Formulation Example 4
Cream
TABLE-US-00008 [0193] Components Amount % Proteoglycan-containing
material A 5.5 Concentrated glycerin 10.0 Olive oil 8.0 Squalane
6.0 Polyglyceryl monostearate 4.0 Lipophilic glyceryl monostearate
4.0 Stearic acid 4.0 Ethyl alcohol 3.0 Cetanol 3.0 Carboxyvinyl
polymer 0.2 Potassium hydroxide 0.6 Perfume 0.1 Purified water
Balance Total 100.0
Formulation Example 5
Hair restorer
TABLE-US-00009 [0194] Components Amount % Proteoglycan-containing
material C 0.05 Polyoxyethylene hydrogenated castor oil (60 E.O.)
0.5 Xanthan gum 0.3 Pyridoxine hydrochloride 0.2 Benzyl nicotinate
0.02 1-menthol 0.1 Perfume 0.03 Purified water Balance Total
100.0
Formulation Example 6
Hair tonic
TABLE-US-00010 [0195] Components Amount % Proteoglycan-containing
material F 0.005 Ethyl alcohol 50.0 Xanthan gum 0.3 Acrylic resin
alkanolamine liquid 0.1 1-menthol 0.2 Perfume 0.01 Purified water
Balance Total 100.0
Formulation Example 7
Lotion for Decreasing the Sizes of Pores of the Skin
TABLE-US-00011 [0196] Components Amount % Proteoglycan-containing
material B 0.1 Ethyl alcohol 15.0 Xanthan gum 0.15 1,3-butylene
glycol 5.0 Purified water Balance Total 100.0
Formulation Example 8
Powdered Brown Rice Beverage
TABLE-US-00012 [0197] Components Amount % Proteoglycan-containing
material C 23.0 Black sesame paste 15.0 Powdered brown sugar 8.0
Soybean powder Balance Total 100.0
Formulation Example 9
Tablet
TABLE-US-00013 [0198] Components Amount % Proteoglycan-containing
material D 40.0 Maltitol 10.0 Lactose 36.0 Sucrose fatty acid ester
5.0 Calcium stearate 4.0 Silicon dioxide 4.0 Powder flavor 1.0
Total 100.0
Formulation Example 10
Powdered Food
TABLE-US-00014 [0199] Components Amount % Proteoglycan-containing
material B 5.5 Fructose 30.0 Dextrin 52.4 Peppermint flavor 3.0
Ascorbic acid 2.5 Sucralose 0.1 Lemon flavor 2.0 Total 100.0
Formulation Example 11
Pill
TABLE-US-00015 [0200] Components Amount % Proteoglycan-containing
material A 23.0 Galactose 30.0 Erythritol 10.0 Sucralose 0.06
Citric acid 5.0 1-menthol 1.0 Sucrose fatty acid ester 5.0
Crystalline cellulose Balance Total 100.0
Formulation Example 12
Candy
TABLE-US-00016 [0201] Components Amount % Proteoglycan-containing
material E 0.5 Reducing maltose starch syrup 52.0 Lactose 10.0
Citric acid 7.0 Peppermint flavor 1.5 Spearmint flavor 1.0 Peach
flavor 2.5 Purified water Balance Total 100.0
Formulation Example 13
Chewable Tablet
TABLE-US-00017 [0202] Components Amount % Proteoglycan-containing
material G 1.0 Glucose 30.0 Erythritol 20.0 Sucrose fatty acid
ester 4.0 Aspartame 0.15 Spearmint flavor 3.0 Crystalline cellulose
Balance Total 100.0
Formulation Example 14
Powdered Tea
TABLE-US-00018 [0203] Components Amount % Proteoglycan-containing
material F 0.01 Oolong tea water extract powder 10.0 Coix
lacryma-jobi var. ma-yuen water extract powder 5.0 Green tea water
extract powder 5.0 Glucose 10.0 Sucralose 0.1 Dextrin Balance Total
100.0
Formulation Example 15
Capsule
TABLE-US-00019 [0204] Components Amount % Proteoglycan-containing
material D 80.0 Crystalline cellulose 20.0 Total 100.0
Formulation Example 16
Chewing Gum
TABLE-US-00020 [0205] Components Amount % Proteoglycan-containing
material B 0.05 Gum base 20.0 Reducing starch syrup 18.0 Flavor 1.0
Powdered sugar Balance Total 100.0
Formulation Example 17
Oral Gel
TABLE-US-00021 [0206] Components Amount % Proteoglycan-containing
material B 0.05 Glycerin 10.0 Propylene glycol 5.0 Hydroxyethyl
cellulose 1.0 Polyoxyethylene (60) hydrogenated castor oil 0.2
Xanthan gum 0.2 Flavor 0.1 Ethyl p-hydroxybenzoate 0.1 Saccharin
sodium 0.01 Purified water Balance Total 100.0
Formulation Example 18
Oral Embrocation
TABLE-US-00022 [0207] Components Amount % Proteoglycan-containing
material D 5.0 Shellac 10.0 Ethyl alcohol 40.0 Hydroxyethyl
cellulose 0.3 Flavor 2.0 Purified water Balance Total 100.0
Formulation Example 19
Mouthwash
TABLE-US-00023 [0208] Components Amount % Proteoglycan-containing
material F 0.01 Glycerin 10.0 Propylene glycol 3.0 Polyoxyethylene
(40) hydrogenated castor oil 0.4 Flavor 0.1 pH regulator Suitable
amount Purified water Balance Total 100.0
Formulation Example 20
Liquid Dentifrice
TABLE-US-00024 [0209] Components Amount % Proteoglycan-containing
material C 0.02 Glycerin 11.0 Propylene glycol 3.0 Polyoxyethylene
(60) hydrogenated castor oil 0.4 Flavor 0.1 Saccharin sodium 0.01
pH regulator Suitable amount Purified water Balance Total 100.0
Total 100.0
Formulation Example 21
Dentifrice
TABLE-US-00025 [0210] Components Amount % Proteoglycan-containing
material A 14.5 Sorbitol 45.0 Abrasive silica 18.0 Thickening
silica 3.0 Polyethylene glycol 400 3.0 Flavor 1.0 Polyoxyethylene
(40) hydrogenated castor oil 0.5 Sodium carboxymethylcellulose 0.4
Saccharin sodium 0.2 pH regulator Suitable amount Purified water
Balance Total 100.0
Formulation Example 22
Mouth Spray
TABLE-US-00026 [0211] Components Amount % Proteoglycan-containing
material G 0.01 Ethanol 30.0 Glycerin 10.0 Polyoxyethylene (60)
hydrogenated castor oil 1.0 Flavor 1.0 1-menthol 0.5 Saccharin
sodium 0.1 pH regulator Suitable amount Purified water Balance
Total 100.0
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