U.S. patent application number 13/576433 was filed with the patent office on 2013-08-01 for age production inhibitor.
This patent application is currently assigned to ORYZA OIL & FAT CHEMICAL CO., LTD.. The applicant listed for this patent is Hiromichi Murai, Hiroshi Shimoda, Takashi Watanabe, Masayuki Yoshikawa. Invention is credited to Hiromichi Murai, Hiroshi Shimoda, Takashi Watanabe, Masayuki Yoshikawa.
Application Number | 20130196937 13/576433 |
Document ID | / |
Family ID | 44367840 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130196937 |
Kind Code |
A1 |
Shimoda; Hiroshi ; et
al. |
August 1, 2013 |
AGE PRODUCTION INHIBITOR
Abstract
Disclosed is an AGE production inhibitor or the like, which
effectively inhibits the production of an advanced glycation end
product (AGE), while having improved biological safety.
Specifically disclosed is an AGE production inhibitor or the like,
which contains en extract of cherry tree (preferably blossoms or
leaves thereof) and/or a processed product of the extract as an
active substance. The AGE production inhibitor or the like
contains, as an active substance, at least one compound that is
selected from among 1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside,
1-O-(E)-Coumaroyl-.beta.-D-glucopyranoside,
1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside, Kaempferol
3-O-.beta.-D-glucopyranoside, Quercetin
3-O-.beta.-D-glucopyranoside, Kaempferol
3-O-(6''-malony)-.beta.-D-glucopyranoside, and Quercetin
3-O-(6''-malony)-.beta.-D-glucopyranoside.
Inventors: |
Shimoda; Hiroshi; (Aichi,
JP) ; Watanabe; Takashi; (Aichi, JP) ; Murai;
Hiromichi; (Aichi, JP) ; Yoshikawa; Masayuki;
(Aichi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Shimoda; Hiroshi
Watanabe; Takashi
Murai; Hiromichi
Yoshikawa; Masayuki |
Aichi
Aichi
Aichi
Aichi |
|
JP
JP
JP
JP |
|
|
Assignee: |
ORYZA OIL & FAT CHEMICAL CO.,
LTD.
Ichinomiya-shi, Aichi
JP
|
Family ID: |
44367840 |
Appl. No.: |
13/576433 |
Filed: |
February 10, 2011 |
PCT Filed: |
February 10, 2011 |
PCT NO: |
PCT/JP2011/052905 |
371 Date: |
October 16, 2012 |
Current U.S.
Class: |
514/27 |
Current CPC
Class: |
A23C 9/13 20130101; C07H
13/04 20130101; A61K 31/7032 20130101; A61K 36/73 20130101; A61P
9/10 20180101; C07H 17/07 20130101; A61P 43/00 20180101; A61P 9/00
20180101; A61K 8/602 20130101; A61K 2236/30 20130101; A61P 25/00
20180101; A23G 3/36 20130101; A61K 36/736 20130101; A61P 17/00
20180101; A61K 31/7048 20130101; A23G 4/06 20130101; A23L 33/105
20160801; A61Q 19/08 20130101; A61K 8/9789 20170801; A61P 3/10
20180101; A23C 2240/15 20130101; A61P 27/02 20180101; A61K 31/7028
20130101; A61P 13/12 20180101; A61K 31/7024 20130101 |
Class at
Publication: |
514/27 |
International
Class: |
A61K 31/7048 20060101
A61K031/7048; A61Q 19/08 20060101 A61Q019/08; A61K 8/60 20060101
A61K008/60 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 10, 2010 |
JP |
2010-027351 |
Feb 25, 2010 |
JP |
2010-039465 |
Claims
1-26. (canceled)
27. The cherry-tree extract of this invention holds all compounds,
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside,
1-O-(E)-Coumaroyl-.beta.-D-glucopyranoside,
1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside, Kaempferol
3-O-.beta.-D-glucopyranoside, Quercetin 3-0,3-D-glucopyranoside,
Kaempferol 3-O-(6''-malony)-.beta.-D-glucopyranoside, Quercetin
3-O-(6''-malony)-.beta.-D-glucopyranoside and said cherry-tree
extract is obtained by a manufacturing method having the following
three steps: (a) extracting one or both cherry blossom extract and
the cherry leaf extract with 20 to 50 wt % hydrous ethanol, (b)
absorbing the extract obtained in step (a) by an absorbent,
comprising porous synthetic resin, elute the extract in water and
remove the eluted extract, and (c) after step (b) further eluting
the eluted extract with a lower alcohol content (carbon numbers 1
to 5), and then concentrate the further eluted extract.
28. An AGE production inhibitor comprising cherry-tree extract
and/or its processed active substance according to claim 27.
29. An apoptosis inhibitor comprising cherry-tree extract and its
processed substance as an active substance, according to claim 27,
which inhibits fibroblast apoptosis caused by the CML-collagen
(Carboxymethyl lysine-collagen) which is an AGE.
30. An human fibroblast-collagen grating-formulation promoter
comprising cherry-tree extract and/or its processed substance as an
active substance according to claim 27.
31. An AGE production inhibitor within the fibroblast comprising
cherry-tree extract and/or its processed substance as an active
substance according to claim 27.
32. An agent, to prevent and cure the diabetic complications,
comprising cherry-tree extract and/or its processed substance as an
active substance according to claim 27.
33. An agent, to prevent the skin from aging and to improve it,
comprising cherry-tree extract and/or its processed substance as an
active substance according to claim 27.
Description
TECHNICAL FIELD
[0001] This invention relates to a new AGE production inhibitor.
Regarding this invention, the AGE production inhibitor has an
inhibitory effect on fibroblast apoptosis which is induced by
CML-collagen (carboxymethyl lysine-collagen), an AGE, and is widely
used as a material for foods, medicines, cosmetics or the like.
TECHNICAL BACKGROUND
[0002] There may still be much unknown about the production process
of Advanced Glycation End-products (hereinafter referred to as
AGE). However, it is thought that the key factor of AGE is a
non-enzymatic glycation. In other words, it is thought that in the
initial-stage reaction of AGE production, an amino group that
exists in protein and an aldehyde group which is a reducing sugar
such as glucose or the like, non-enzymatically react to each other
(i.e. glycation), which produces Amadori-rearrangement products
through Schiff's base. In the late-stage reaction, AGE is produced
after a long period of complex cleavages and condensations.
[0003] Diabetes complication includes retinopathy, nephropathia,
neuropathy, ischemic cardiac disease, cerebrovascular disease and
others. It is known that one of the factors for the cause of these
diseases relates to AGE produced in a hyperglycemic biological
body. (For instance, refer to non-patent reference 1)
[0004] Diabetic nephropathy is renal microvascular damage caused by
diabetes. Its basic pathological change includes the thickening of
the glomerular basement membrane and the expansion of the mesangial
area. Recently a number of diabetic patients stricken with the
end-stage renal disease, caused by diabetic nephropathy increases
and so were treated with dialysis. It is thought that AGE which is
produced by a continuous state of hyperglycemia is involved in the
development of nephropathy which is developed by 1. enhanced
vascular permeability, 2. enhanced deposition of protein and
lipoprotein, 3. inactivation of nitric oxide (NO), and 4.
accelerated production of extracellular matrix. (See Non-patent
Reference 2.)
[0005] For the sake of the prevention of and for the improvement of
diabetic complications, various types of AGE production inhibitors
have been researched and developed.
[0006] For example, it is shown that AGE production that induces
diabetic complications such as nephropathia, retinopathy and others
is inhibited by synthetic drugs (synthetic compounds). (See Patent
References 1 and 2.) However, such synthetic drugs [that inhibit
such] AGE production remains in the developmental stage, and such
synthetic drugs often produce unanticipated side effects.
[0007] Contrarily, research is being done of substances or food
compositions which prevent, control, improve and cure diseases
through a daily dietary way of life, instead of using synthetic
drugs. Patent References 3 to 5, for instance, show compositions of
extracts of plants used for food, which inhibit AGE production.
[0008] However, AGE inhibitory activity of such extracts derived
from plants is generally very low, compared to that of synthetic
drugs (synthetic compounds), and the amount of the extracted active
substance is extremely small.
[0009] To prevent or cure diabetic complications, continuous and
long-term therapy is required. Thus, it is desired to develop an
AGE production inhibitor that both effectively inhibits AGE
production and avoids side effects. However, little is known about
conventional botanical extracts which meet public expectation.
[0010] In recent years, it has been disclosed that AGE relates
closely not only to diabetes but also to the aging of the skin. AGE
increases with aging, and when Maillard reaction occurs in the
collagen part [of the skin] which is the skin protein, the carbonyl
group of glucide, non-enzymatically reacts with either the amino
group of lysine residue or with the guanidyl group or arginine
group in the protein to produce AGE, thus resulting in collagen
cross-linking. Once the link structure is formed, the molecule is
hardened, and inherent elasticity of the skin is lost. Also, such
crosslink substances are treated as foreign substances, and the
volume of secretion of degrading enzyme (collagenase, elastase)
increases, which decreases skin tone and firmness and weakens the
skin, thus resulting in wrinkles, slackness and dullness of the
skin.
[0011] As such, AGE is now recognized as a trigger of diabetes
complications. From the viewpoint of anti-aging care, the
production mechanism of AGE is also focused.
PRIOR ARTS
Patent References
[0012] (Patent Reference 1) JP-A-2000-324629 [0013] (Patent
Reference 2) JP-A-2005-170935 [0014] (Patent Reference 3)
JP-A-2006-28090 [0015] (Patent Reference 4) JP-A-2005-343842 [0016]
(Patent Reference 5) JP-A-2005-320262
Non-Patent References
[0016] [0017] (Non-patent Reference 1) Higasi, T. et. al. (Bio
Clinica, Vol. 12-2, pp. 19-21, 1997) [0018] (Non-patent Reference
2) Baba, T. et. al. (Bio Clinica, Vol. 12-2, pp. 39-48, 1997)
SUMMARY OF THE INVENTION
Problems to be solved by the invention
[0019] In such circumstances, the inventors conducted research on
the components and amount of the components contained in the
extract and of the various plant origins. They focused on "cherry
tree" which contains plenty of polyphenol. After conducting various
experiments, they realized that the extract of cherry blossoms and
cherry leaves, especially, contain phenylpropanoid glycoside
(caffeoyl glucose, coumaroyl glucose, cinnamoyl glucose) and
flavonoid glycoside (kaempferol glucoside, quercetin glucoside,
kaempferol malonyl glucose, quercetin malonyl glucose), which
effectively inhibits AGE production and promotes the formation of
fibroblast-collagen grating.
[0020] Also, the inventors learned of a new physiological activity
of cherry tree extract, which effectively inhibits fibroblast
apoptosis caused by an AGE, CML-collagen (i.e. Carboxymethyl
Lysine-collagen), thus providing this invention.
[0021] This invention is to solve the problems, above, and its
purpose is effectively to inhibit AGE production and to provide a
biologically safe AGE production inhibitor for the human body.
[0022] Also, another purpose of the invention is to provide an
apoptosis inhibitor to inhibit the formation of human fibroblast
apoptosis caused by AGE, such as CML-collagen (Carboxymethyl
lysine-collagen).
[0023] Moreover, another purpose of the invention is to provide a
fibroblast collagen grating formation promoter.
[0024] Still another purpose of the invention is to provide an AGE
production inhibitor effectively to inhibit AGE production within
the fibroblast.
Means to Resolve the Problems
[0025] In light of resolving the above problems, the features of
this invention are as follows.
[0026] 1. The AGE production inhibitor of this invention contains
cherry extract and/or its processed substance as an active
substance.
[0027] 2. The AGE production inhibitor of this invention contains
cherry blossom extract and/or its processed substance as an active
substance.
[0028] 3. The AGE production inhibitor of this invention contains
cherry leaf extract and/or its processed substance as an active
substance.
[0029] 4. The AGE production inhibitor of this invention contains
at least one compound as an active substance selected from among
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside,
1-O-(E)-Coumaroyl-.beta.-D-glucopyranoside,
1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside, Kaempferol
3-.beta.-D-glucopyranoside, Quercetin 3-O-.beta.-D-glucopyranoside,
Kaempferol 3-O-(6''-malony)-.beta.-D-glucopyranoside, and
(Quercetin 3-O-(6''-malony)-.beta.-D-glucopyranoside.
[0030] 5. The AGE production inhibitor of this invention contains
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside, as an active
substance.
[0031] 6. The AGE production inhibitor of this invention contains
Quercetin 3-O-.beta.-D-glucopyranoside as an active substance.
[0032] The AGE production inhibitor of this invention, according to
the above features 1 to 6, allows the edible and safe
cherry-derived extract to inhibit effectively AGE production and to
avoid side effects, thus providing an ingredient for foods,
medicines, cosmetics and other products which are helpful in
effectively preventing or treating diabetic complications.
[0033] 7. The apoptosis inhibitor of this invention contains cherry
extract and/or its processed substance as an active substance,
which inhibits fibroblast apoptosis caused by CML-collagen (i.e.
Carboxymethyl lysine-collagen) which is an AGE.
[0034] 8. The apoptosis inhibitor of this invention contains cherry
blossom extract and/or its processed substance as an active
substance, which inhibits fibroblast apoptosis caused by
CML-collagen (i.e. Carboxymethyl lysine-collagen) which is an
AGE.
[0035] 9. The apoptosis inhibitor of this invention contains cherry
leaf extract and/or its processed substance as an active substance,
which inhibits fibroblast apoptosis caused by CML-collagen (i.e.
Carboxymethyl lysine-collagen) which is an AGE.
[0036] 10. The apoptosis inhibitor of this invention contains at
least one active substance selected from among
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside,
1-O-(E)-Coumaroyl-.beta.-D-glucopyranoside,
1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside, Kaempferol
3-O-.beta.-D-glucopyranoside, Quercetin
3-O-.beta.-D-glucopyranoside, Kaempferol
3-O-(6''-malony)-.beta.-D-glucopyranoside and Quercetin
3-O-(6''-malony)-.beta.-D-glucopyranoside, which inhibits the
fibroblast apoptosis caused by the CML-collagen (i.e. Carboxymethyl
lysine-collagen) which is an AGE.
[0037] 11. The apoptosis inhibitor of this invention contains
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside as an active substance,
which inhibits fibroblast apoptosis caused by CML-collagen (i.e.
Carboxymethyl lysine-collagen) which is an AGE.
[0038] 12. The apoptosis inhibitor of this invention contains
Quercetin 3-O-.beta.-D-glucopyranoside and/or Quercetin
3-O-(6''-malony)-.beta.-D-glucopyranoside as an active substance,
which inhibits fibroblast apoptosis caused by CML-collagen (i.e.
Carboxymethyl lysine-collagen) which is an AGE.
[0039] The AGE production inhibitor of this invention, according to
the above features 7 to 12, allows cherry-derived extract, which is
safe for food, to inhibit effectively the fibroblast apoptosis
caused by CML-collagen (Carboxymethyl lysine-collagen), thus
providing a material for foods, medicines, cosmetics and other
products, which is helpful in effectively preventing or inhibiting
the skin from aging.
[0040] 13. The human fibroblast-collagen grating-formation promoter
of this invention contains cherry-tree extract and/or its processed
substance as an active substance.
[0041] 14. The human fibroblast-collagen grating-formation promoter
of this invention contains cherry-blossom extract and/or its
processed substance as an active substance.
[0042] 15. The human fibroblast-collagen grating-formation promoter
of this invention contains cherry-leaf extract and/or its processed
substance as an active substance.
[0043] 16. The human fibroblast-collagen grating-formation promoter
of this invention contains at least one active substance selected
from among this group of compounds:
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside,
1-O-(E)-Coumaroyl-.beta.-D-glucopyranoside,
1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside, Kaempferol
3-O-.beta.-D-glucopyranoside, Quercetin
3-O-.beta.-D-glucopyranoside, Kaempferol
3-O-(6''-malony)-.beta.-D-glucopyranoside and Quercetin
3-O-(6''-malony)-.beta.-D-glucopyranoside.
[0044] 17. The human fibroblast-collagen grating-formation promoter
of this invention contains
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside as an active
substance.
[0045] 18. The human fibroblast-collagen grating-formulation
promoter of this invention contains Quercetin
3-O-.beta.-D-glucopyranoside as an active substance.
[0046] The human fibroblast-collagen grating-formulation promoter
of this invention, according to the above features 13 to 18, allows
cherry-derived extract, which is safe for food, to promote
effectively human fibroblast collagen grating formulation, thus
providing a material for foods, medicines, cosmetics and other
products which are helpful in effectively preventing or inhibiting
the skin from aging.
[0047] 19. The AGE production inhibitor within the fibroblast of
this invention contains cherry-tree extract and/or its processed
substances as active substances.
[0048] 20. The AGE production inhibitor within the fibroblast of
this invention contains cherry-blossom extract and/or its processed
substance as an active substance.
[0049] 21. The AGE production inhibitor within the fibroblast of
this invention contains cherry-leaf extract and/or its processed
substance as an active substance.
[0050] 22. The AGE production inhibitor within the fibroblast of
this invention contains at least one active substance selected from
among this group of compounds, i.e.
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside,
1-O-(E)-Coumaroyl-.beta.-D-glucopyranoside,
1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside, Kaempferol
3-O-.beta.-D-glucopyranoside, Quercetin
3-O-.beta.-D-glucopyranoside, Kaempferol
3-O-(6''-malony)-.beta.-D-glucopyranoside and Quercetin
3-O-(6''-malony)-.beta.-D-glucopyranoside.
[0051] 23. The AGE production inhibitor within the fibroblast of
this invention contains 1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside
as an active substance.
[0052] 24. The AGE production inhibitor within the fibroblast of
this invention contains Quercetin 3-O-.beta.-D-glucopyranoside as
an active substance.
[0053] The AGE production inhibitor within the fibroblast of this
invention, according to the above features 19 to 24, allows
cherry-derived extract, which is safe for food, to effectively
inhibit AGE production within the fibroblast, thus providing a
material for foods, medicines, cosmetics and other products which
are helpful in effectively preventing or inhibiting the skin from
aging.
[0054] 25. The cherry-tree extract of this invention holds at least
one active substance selected from among this group of compounds,
i.e. 1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside,
1-O-(E)-Coumaroyl-.beta.-D-glucopyranoside,
1-O-(E)-Cinnamoyl-6-D-glucopyranoside, Kaempferol
3-O-.beta.-D-glucopyranoside, Quercetin
3-O-.beta.-D-glucopyranoside, Kaempferol
3-O-(6''-malony)-.beta.-D-glucopyranoside, and Quercetin
3-O-(6''-malony)-.beta.-D-glucopyranoside.
[0055] 26. The cherry-tree extract of this invention holds all of
the substances of this group of compounds, i.e.
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside,
1-O-(E)-Coumaroyl-.beta.-D-glucopyranoside,
1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside, Kaempferol
3-O-.beta.-D-glucopyranoside, Quercetin
3-O-.beta.-D-glucopyranoside, Kaempferol
3-O-(6''-malony)-.beta.-D-glucopyranoside, and Quercetin
3-O-(6''-malony)-.beta.-D-glucopyranoside.
[0056] 27. The cherry-tree extract of this invention holds all
compounds, 1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside,
1-O-(E)-Coumaroyl-.beta.-D-glucopyranoside,
1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside, Kaempferol
3-O-.beta.-D-glucopyranoside, Quercetin
3-O-.beta.-D-glucopyranoside, Kaempferol
3-O-(6''-malony)-.beta.-D-glucopyranoside, Quercetin
3-O-(6''-malony)-.beta.-D-glucopyranoside and said cherry-tree
extract is obtained by a manufacturing method having the following
three steps:
(a) extracting one or both cherry blossom extract and the cherry
leaf extract with 20 to 50 wt % hydrous ethanol, (b) absorbing the
extract obtained in step (a) by an absorbent comprising porous
synthetic resin, elute the extract in water and remove the eluted
extract, and (c) after step (b) further eluting the eluted extract
with a lower alcohol content (carbon numbers 1 to 5), and then
concentrate the further eluted extract.
[0057] 28. The AGE production inhibitor of this invention holds
cherry-tree extract and/or its processed active substance according
to the above feature 27.
[0058] 29. The apoptosis inhibitor of this invention holds
cherry-tree extract and its processed substance as an active
substance, according to the above feature 27, and inhibits
fibroblast apoptosis caused by the CML-collagen (i.e. Carboxymethyl
lysine-collagen) which is an AGE.
[0059] 30. The human fibroblast-collagen grating-formulation
promoter of this invention holds cherry-tree extract and/or its
processed substance as an active substance according to the above
feature 27.
[0060] 31. The AGE production inhibitor within the fibroblast of
this invention holds cherry-tree extract and/or its processed
substance as an active substance according to the above feature
27.
[0061] 32. The agent, to prevent and cure the diabetic
complications, of this invention holds cherry-tree extract and/or
its processed substance as an active substance according to the
above feature 27.
[0062] 33. The agent, to prevent the skin from aging and to improve
it, of this invention holds cherry-tree extract and/or its
processed substance as an active substance according to the above
feature 27.
[0063] The invention, according to the above features 25 to 33,
allows the cherry-derived extract, which is safe for food, to
inhibit AGE production effectively without side effects, thus
providing a material for foods, medicines, cosmetics and other
products which are helpful in effectively preventing or treating
diabetic complications.
[0064] The invention, according to the above features 25 to 33,
also effectively inhibits fibroblast apoptosis caused by
CML-collagen (Carboxymethyl lysine-collagen), thus effectively
promoting the human fibroblast-collagen grating-formulation, thus
effectively inhibiting AGE production within the fibroblast, thus
providing a material for foods, medicines, cosmetics and other
products which are helpful in effectively preventing the aging of
the skin and for improving it.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] FIG. 1 is a chart explaining the search for the substances
contained in the cherry-blossom extract.
[0066] FIG. 2 is a microscopic image of fibroblast caused by
CML-collagen (Carboxymethyl lysine-collagen) regarding the cherry
blossom extract of the example of this invention.
[0067] FIG. 3 is a megascopic image of 24 and 48 hours of a culture
of collagen-grating formulations of fibroblasts glycosylated by
glyoxal.
[0068] FIG. 4 shows microscopic images (.times.100) in 24 hours of
culture according to FIG. 3.
[0069] FIG. 5 shows microscopic images (.times.200) in 48 hours of
culture according to FIG. 3.
[0070] FIG. 6 shows the effect on AGE production (carboxy
methyllsin protein), using Western blotting, within the fibroblast
(human diploid fibroblast) caused by glyoxal.
[0071] FIG. 7 shows the effect on AGE production within the
glyoxal-induced skin, using Western blotting, regarding the cherry
blossom extract as an example of this invention.
[0072] FIG. 8 shows microscopic images (.times.400) of TUNEL
positive cells which identify the effect on dermal cell apoptosis
induced by CML-collagen (Carboxymethyl lysine-collagen), regarding
the cherry blossom extract as an example of this invention.
MODELS FOR CARRYING OUT THE INVENTION
[0073] Hereinafter, examples of this invention are described. Of
this invention, the AGE production inhibitor and fibroblast
apoptosis inhibitor and human fibroblast-collagen
grating-formulation promoter and AGE production inhibitor within
the fibroblast and cherry extract (hereinafter collectively
referred to as the AGE production inhibitor) contain, as a
bioactive substance, at least one substance selected from among
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside,
1-O-(E)-Coumaroyl-.beta.-D-glucopyranoside,
1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside, Kaempferol
3-O-.beta.-D-glucopyranoside, Quercetin
3-O-.beta.-D-glucopyranoside, Kaempferol
3-O-(6''-malony)-.beta.-D-glucopyranoside and Quercetin
3-O-(6''-malony)-.beta.-D-glucopyranoside.
[0074] 1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside is a compound as
described in Formula 1, below.
##STR00001##
[0075] 1-O-(E)-Coumaroyl-3-D-glucopyranoside is a compound as
described in Formula 2, below.
##STR00002##
[0076] 1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside is a compound as
described in Formula 3, below.
##STR00003##
[0077] Kaempferol 3-O-.beta.-D-glucopyranoside is a compound as
described in Formula 4, below.
##STR00004##
[0078] Quercetin 3-O-.beta.-D-glucopyranoside is a compound as
described in Formula 5, below.
##STR00005##
[0079] Kaempferol 3-O-(6''-malony)-.beta.-D-glucopyranoside is a
compound as described in Formula 6, below.
##STR00006##
[0080] Quercetin 3-O-(6''-malony)-.beta.-D-glucopyranoside is a
compound as described in Formula 7, below.
##STR00007##
[0081] It is preferable that AGE production inhibitor of this
invention contain at least the compound
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside (Formula 1) or Quercetin
3-O-.beta.-D-glucopyranoside (Formula 5), which are selected from
among the compounds, as described in Formulae 1 to 7, or especially
the compound, 1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside (Formula
1).
[0082] Any method used in obtaining the compound as described in
Formulae 1 to 7, above, is not specifically limited. Such a
compound is, however, preferably to be extracted from plants, which
makes it simple to obtain. Also, when extracting the compound from
plants, it is preferable to extract it from the cherry tree.
[0083] Cherry blossom (referred to as Sakura in Japanese) is the
generic name of the Genus Cerasus of Rosaceae, excluding apricots
and peaches, and refers to plants belonging to Subgenus
Cerasus.
[0084] The types of cherry blossoms (Sakura) used of this invention
are not specifically limited. Cherry blossoms of this invention
include, for example, prunus yamazakukra (Cerasus jamasakura),
edohigan (Cerasus spachiana Lavalee ex H. Otto var. spachiana forma
ascendens (Makino) H. Ohba), mamezakura (Prunus incisa Thunb. ex
Murray) choujizakura (Prunus apetara Fr. et Sav.), miyamazakura
(Prunus maximowiczii Rupr.), shinamizakura (Prunus pseudo-cerasus
Lindl.) and others, but not limited to these.
[0085] The part of the cherry, which is used for the material of
this invention, is not specifically limited but includes its leaf,
stem, trunk, blossom, fruit and other parts. However, it is
preferable to use the cherry leaf or blossom.
[0086] If extraction is done by using a polar solvent, the type of
such a polar solvent is not limited but includes water, methanol,
ethanol, isopropanol, acetone, 1,3-butylene glycol, ethylene
glycol, propylene glycol, glycerin, acetic acid, ethyl acetate,
ether, hexane and others. Of these, water, methanol, and ethanol
are especially preferable, which efficiently extracts the active
substance. Also, only one or more solvents from among the polar
solvents, above, can be used.
[0087] When using water as an extracting solvent, the extraction
temperature is to be set between 20 and 100 degrees centigrade,
preferably between 40 and 70 degrees centigrade. If the temperature
is too low, the active substance will not efficiently be extracted.
On the other hand, if the temperature is too high, cyanogen
compounds contained in the cherry-tree (sakura) will be eluted and
remain in the water, and the active substance will be degraded in
such a high temperature, which is not preferable, either. The types
of water used for the extraction are not specifically limited but
include tap water, distilled water, mineral water, ionized alkaline
water and others.
[0088] When using aqueous alcohol as an extracting solvent, the
concentration of alcohol is preferably to be set at 25 to 50 wt %
(percent by weight), preferably at 25 to 30 wt %. If the level of
alcohol concentration is below 20 wt %, it will be hard to get a
high level of active substance. Contrarily, if the concentration of
alcohol is over 50 wt %, the yield will be low, due to impurities
or the like.
[0089] Also, in case where the concentration of alcohol is 30 wt %
or more, the extraction temperature is preferably to be set at 0 to
95 degrees centigrade, more preferably at 0 to 60 degrees
centigrade. As for the extraction of aqueous ethanol, it is
preferable to repeat the extractions in various concentrations so
as to improve the content rate of the active substance.
[0090] Also, when conducting an extraction with a polar solvent,
the method of extraction is not specifically limited. The method of
the extraction, for instance, includes a continuous extraction, a
soaking extraction, a countercurrent extraction or any other
extraction which can be used with any optional equipment at room
temperature or by heating under reflux. Also, any one, or any
combination of the above methods can be used for the extraction.
Furthermore, the extraction can be conducted once or more than
once.
[0091] As for the specific extraction method, put the extraction
ingredient into the processing vessel filled with extracting
solvent, and stir it so that the active substance seeps into the
solvent.
[0092] For instance, when using hydrous ethanol as the extracting
solvent, the extraction is conducted with the extracting solvent
approximately 3 to 100 times as much weight as the extraction
ingredient, from one to 150 minutes. After the active substance
seeps into the solvent, filter it and remove the residue to obtain
the extracted liquid. After that, according to the ordinary method,
apply the dilution, concentration, drying, refining method or the
like to the extracted liquid, so as to obtain the extract
containing polyphenol, as described in the above Formulae 1 to 7 or
the like.
[0093] Also, the refining method includes, for example, an
activated carbon treatment, a resin absorption treatment, a silica
gel treatment as well as another method using ion-exchange resin,
and liquid-liquid countercurrent distribution, and membrane
separation, or the like.
[0094] In addition, when the extraction is done by supercritical
extraction, the supercritical fluid is not specifically limited but
includes, for instance, carbon dioxide, nitrogen or the like, of
which one or more fluids can be used at the same time. However,
carbon dioxide is preferable, since it can easily elute the active
substance. Also, the known conventional method of extraction can
also be used. After the extraction, according to the ordinary
method, apply the dilution, concentration, drying, refining method
or the like to the extracted liquid, so as to obtain the extract
containing polyphenol, as described in the above Formulae 1 to 7,
or the like.
[0095] To obtain the polyphenol, as described in the above Formulae
1 to 7, it is preferable to use the following processes:
(a) extract the cherry blossom extract and/or the cherry leaf
extract with 20 to 50 wt % hydrous ethanol, (b) absorb the extract
obtained in Process (a) by absorbent, elute the extract in water
and remove the eluted extract, and (c) elute the extract with an
aqueous solvent, and then concentrate the eluted extract.
[0096] Using the above processes efficiently obtains the extract
containing a high level of polyphenol, as described in the above
Formulae 1 to 7.
[0097] As for the absorbent used in the above referenced process
(b), one or more absorbents can be selected and used from among the
known conventional absorbent such as an ion-exchange resin, a
synthetic absorbent resin, an activated carbon, a chelating resin,
silica gel, an alumina gel series absorbent, a porous glass or the
like. It is preferable to use a column chromatography with porous
synthetic absorbent resin such as DIAION.RTM.HP-20 (Mitsubishi
Chemical Corporation), SEPABEADS.RTM.SP-207 (Mitsubishi Chemical
Corporation) or the like.
[0098] As for the aqueous solvent used in the above referenced
process (c), a lower alcohol content (carbon numbers 1 to 5) can be
used. Methanol and/or ethanol is especially preferable.
[0099] The AGE production inhibitor or the like of this invention
effectively inhibits AGE production and prevents diabetic
complications such as nephropathy, retinopathy or the like. Also,
it especially inhibits AGE production within the fibroblast to
improve the crosslink of the collagen part of the skin, thus
preventing the skin from hardening. Moreover, it inhibits
fibroblast apoptosis occurring after the production of AGE, thus
retaining the elasticity of the skin, thus preventing flecks and
dullness of the skin. Furthermore, it improves collagen
cross-linking after AGE production, thus preventing the skin from
hardening.
[0100] The AGE production inhibitor or the like of this invention
can be used as an ingredient for any food and drink such as, edible
oils (salad oils), confectionary (chewing gums, candies, caramels,
chocolates, cookies, jellies, gummies, tablet shaped sweets or the
like), noodles (Japanese buckwheat noodles called Soba, Japanese
wheat noodles called Udon, Chinese noodles called Ramen or the
like), dairy food (milk, ice cream, yogurt, or the like), seasoning
(fermented bean paste called Miso, Soy sauce called Shoyu, or the
like), soups, drinks (juice, coffee, black tea, green tea,
carbonated drink, sports supplement drinks or the like) including
general foods and healthy food (tablet type, capsule type or the
like), nutritional supplements (nutritious supplement drink or the
like). The AGE production inhibitor or the like of this invention
can be used for the above foods and drinks.
[0101] According to the type of the above foods and drinks, the
following ingredients can be added: Glucose, fructose, sucrose,
maltose, sorbitol, stevioside, corn syrup, lactose, citric acid,
tartaric acid, malic acid, saccinic acid, lactic acid, L-ascorbic
acid, dl-.alpha.-tocopherol, sodium erythorbate, glycerin,
propylene glycol, glycerin fatty acid ester, polyglycerol fatty
acid ester, sucrose fatty acid ester, sorbitan fatty acid ester,
propylene glycol fatty acid ester, Arabian gum, carrageenan,
casein, gelatin, pectine, agar-agar (gelatin made from seaweed),
vitamin B family, nicotinic-acid amide, pantothenate acid calcium,
amino acids, calcium salts, pigment, aroma chemicals,
preservatives, or the like.
[0102] Also, other antioxidants or compounding ingredients of
healthy food include the antioxidant "reduced ascorbic acid" or
vitamin C and also the antioxidants, vitamin E, reduced glutacin,
tocotrienol, vitamin A derivative, lycopene, rutin,
.beta.-cryptoxanthin, astaxanthin, zeaxanthin, fucoxanthin, uric
acid, ubiquinone, coenzyme Q-10, folic acid, garlic extract,
allicin, sesamin, lignans, catechin, isoflavone, chalcone, tannins,
flavonoids, coumarin, isocoumarines, blueberry extract, ingredients
for healthy food (V. (vitamin) A, V.B1, V.B2, V.B6, V.B12, V.C,
V.D, V.E, V.P, choline, niacin, pantothenic acid, calcium folic
acid, EPA, oligosaccharide, dietary fiber, squalene, soybean
lecithin, taurine, dunalliela, protein, octacosanol, DHA, egg-yolk
lecithin, linoleic acid, lactoferrin, magnesium, zinc, chrome,
selenium, kalium, hem iron, oyster extract, chitosan, chitin
oligosaccharides, collagen, chondroitin, elastin, turmeric,
sweetroot, extract of Chinese wolfberry fruit called kukoshi,
cinnamon, may, ginger, bracket fungus, shijimi clam (corbicula
japonica) extract, snapping turtle, sweetroot, lycii fructus,
cinnamomi cortex, hawthorn, plantain, chamomilla, chamomile,
dandelion, hibiscus, honey, pollen, royal jelly, lime, lavender,
rose hip, rosemary, sage, bifidobacteria, streptococcus faecalis,
lactobacillus, wheat germ oil, sesame oil, perilla oil, soybean
oil, medium chain fatty acid, agaricus, ginko biloba extract,
chondroitin, brown rice germ oil, leechee, onion, DHA, EPA, DPA,
rubus suavissimus s.lee, plant worm (cordyceps sineusis saccardo),
garlic, larvae of a bee, papaya, pu-erh-tea, propolis, Acer
nikoense, hericium erinaceum, royal jelly, saw palmetto, hyaluronic
acid, gaba, harp seal oil, shark cartilage, lecithin, phosphatydyl
serine, panax notoginseng, mulberry leaf, soybean extract,
echinacea purpurea, acanthopanax senticosus, barley extract, olive
leaf, olive, gymnema, banaba, salacia reticulata, garcinia,
chitosan, saint john's wort, jujube, carrot, passion flower,
broccoli, placenta, coix lacryma-bobi, grape seed, peanut skin,
bilberry, black cohosh, milk thistle, laurel, sage, rosemary,
apocynum venetum, black vinegar, bitter gourd, maca, carthamus
tinctorius, linseed, oolong tea, flower aculeus, caffeine,
capsaicin, xylo-oligosaccharide, glucosamine, buckwheat, citrus,
dietary fiber, protein, prune, spirulina, young green barley leaf,
nucleic acid, natural yeast, shiitake mushroom (lentinus edodes),
Japanese plum, amino acid, extract of deep sea shark, morinda
citrifolia, oyster, snapping turtle, champinion, common plantain,
acerola, pineapple, banana, peach, apricot, melon, strawberry,
raspberry, orange, fucoidan, acer nikoense, cranberry, zinc, iron,
ceramide, silk peptide, glycine, niacin, chaste tree, ceramide,
L-cysteine, red grape juice, millet, horsetail, bition, centrlla
asiatica, lonicera caerulea, pycnogenol, petasites japonicus,
rhubarb, clove, pu-erh, citric acid, beer yeast, mellilot, black
zinger, ginger, curcuma zedoaria, nattokinase, ang-khak (Chinese
red rice), tocotrienol, lactoferrin, tartary buckwheat, cocoa,
houttuynia cordata, kiwi fruit, piper longum, lotus leaf, pfaffia
and star fruit.
[0103] A more specific use of the extracting method is herein
described. Firstly, spray-dry or freeze-dry the cherry-tree extract
with powdered cellulose, then make it a powder, a granule, a
tablet, or liquid to easily use with different kinds of food and
drinks (ready-to eat meals or the like). Also, it is possible to
dissolve the cherry-tree extract, for instance, oil and fat,
ethanol, glycerin, or a mixture of these substances, and to use
such a liquid for dry food or drinks. Also it is possible to make
it into a powder or granule by mixing it with a binder such as
Arabian gum, dextrin, or the like to add to dry food or drinks.
[0104] The total amount of the active substance of the AGE
production inhibitor or the like of this invention, which is added
to the food and drinks, is preferably 1 to 20 wt % or less, since
the major objective of this invention is health maintenance.
[0105] The AGE production inhibitor or the like of this invention
can be used as the raw material of medicines (including drugs and
quasi-drugs). The AGE production inhibitor or the like of this
invention can be appropriately mixed with raw materials for drug
formulations, for instance, vehicles (glucose, sucrose, white soft
sugar, sodium chloride, starch, calcium carbonate, kaolin,
crystalline cellulose, cacao oil, hydrogenated vegetable oil, talc,
or the like), binders (distilled water, normal saline solution,
ethanol in water, ethanolic solution, simple syrup, dextrose in
water, starch solution, gelatin solution, carboxymethyl cellulose,
potassium phosphate, polyvinyl pyrrolidone, or the like),
disintegrating agents (alginate sodium, agar-agar, sodium hydrogen
carbonate, sodium lauryl sulphate, stearic acid monoglyceride,
starch, lactose, powdered aracia, gelatin, ethanol, or the like),
suppressive agents for disintegration (white soft sugar, stearin,
cacao oil, hydrogenated oil, or the like), absorption promoters
(quaternary ammonium base, sodium lauryl sulphate, or the like),
adsorbents (glycerin, starch, lactose, kaolin, bentonite, silic
acid, or the like), lubricant agents (purified talc, stearate,
polyethyleneglycol, or the like)
[0106] The AGE production inhibitor or the like of this invention
can be orally administered in the form of tablets, pills, soft or
hard capsules, subtle granules, powders, granules, liquids, or the
like. However, it can also be parenterally administered in the
different forms of solution or together with a dispersant, a
suspending agent, a stabilizer, or the like such as a medical skin
patch, a lotion, an ointment, a cream or the like.
[0107] The applied dose can be adjusted according to the method of
administration, the condition of the disease, the age of the
patient, or the like. However, adults can normally take approx. 1
to 1,000 mg of an active substance per day, while children can take
0.5 to 500 mg per day. The compounding ratio of the AGE production
inhibitor or the like of this invention can be adjusted according
to the mode of administration. When the dietetic composition is
orally administered or mucosally administered, the applied dose is
preferably 0.3 to 15.0 wt %. When the dietetic composition is
parenterally administered, the dose is preferably 0.01 to 10 wt %.
The dose varies depending on the conditions. Therefore, a dose
which is less than the above-stated amount may be sufficient, or a
greater amount may sometimes be needed.
[0108] The AGE production inhibitor or the like of this invention
can be mixed with cosmetics such as emulsions, soaps, facial
cleansers, bath agents, creams, skin lotions, colognes, shaving
creams, shaving lotion, beauty oils, tanning lotions, sunscreen
lotions, face powders, foundations, perfumes, facial masks, nail
creams, nail enamels, nail-polish removers, eyebrow pencils,
blushers, eye creams, eye shadows, mascaras, eye liners, sticks,
lip creams, shampoos, hair conditioners, hairdyes, dispersion
liquids, cleansing preparations, or the like. Also, the AGE
production inhibitor or the like of this invention can be mixed
with drugs and quasi-drugs such as ointments, cream
pharmaceuticals, liquids for external use or the like.
[0109] Within the functional range of the AGE production inhibitor
or the like of this invention, the above items for external skin
use can be also mixed with the ingredients of cosmetics,
quasi-drugs, or the like. Those ingredients include, for example,
oil, higher alcohol, fatty acids, ultraviolet absorbers, powders,
pigments, surface active agents, polyhydric alcohol and sugar,
polymers, biologically active ingredients, solvents, antioxidants,
aroma chemicals (perfume material), antiseptics. However, those
ingredients usable in the present invention are not limited to
these examples.
(1) Specific Examples of Oil
Ester-Type Oil Phase Ingredient:
[0110] Triglyceryl 2-ethylhexanoate, cetyl 2-ethylhexanoate,
isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl
stearate, octyl palmitate, isocetyl isostearate, butyl stearate,
butyl myristate, ethyl linoleate, isopropyl linoleate, ethyl
oleate, isocetyl myristate, isostearyl myristate, isostearyl
palmitate, octyldodecyl myristate, isocetyl isostearate, diethyl
sebacate, diisopropyl adipate, isoarachyl neopentanoate,
caprylic-capric acid triglyceride, trimethylolpropane
tri-2-ethylhexanoate, trimethylolpropane triisostearate,
pentaerythritol tetra-2-ethylhexanoate, cetyl caprylate, decyl
laurate, hexyl laurate, decyl myristate, myristyl myristate, cetyl
myristate, stearyl stearate, decyl oleate, cetyl ricinoleate,
isostearyl laurate, isotridecyl myristate, isocetyl myristate,
isostearyl myristate, isocetyl palmitate, isostearyl palmitate,
octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl
oleate, octyldodecyl linoleate, isopropyl isostearate, cetostearyl
2-ethylhexanoate, stearyl 2-ethylhexanoate, hexyl isostearate,
ethylene glycol dioctanoate, ethylene glycol dioleate, propylene
glycol dicaprate, propylene glycol di (caprylate/caprate),
propylene glycol dicaprylate, neopentyl glycol dicaprate, neopentyl
glycol dioctanoate, glyceryl tricaprylate, glyceryl triundecylate,
glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl
neopentanoate, isostearyl octanoate, octyl isononanoate, hexyldecyl
neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate,
isostearyl isostearate, octyldecyl isostearate, polyglycerin
oleate, polyglycerin isostearate, dipropyl carbonate, dialkyl
carbonate (C12-18), triisocetyl citrate, triisoarachyl citrate,
triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl
lactate, octyldecyl lactate, triethyl citrate, acetyltriethyl
citrate, acet-yltributyl citrate, trioctyl citrate, diisostearyl
malate, 2-ethylhexyl hydroxystearate, 2-ethylhexyl succinate,
diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate,
cholesteryl stearate, cholesteryl isostearate, cholesteryl
hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate,
phytosteryl isostearate, phytosteryl oleate, isocetyl
12-stearoylhydroxystearate, stearyl 12-stearoylhydroxystearate and
isostearyl 12-stearoylhydroxystearate. From among the above
ingredients, one only can be used, or two or more can be used
together.
Hydrocarbon-Type Oil Phase Ingredient:
[0111] Squalane, liquid paraffin, .alpha.-olefin oligomer,
isoparaffin, ceresin, paraffin, liquid isoparaffin, polybutene,
microcrystalline wax, Vaseline or the like.
Animal and Plant Oil, Hardened Oil Thereof, and Wax of Natural
Origin:
[0112] Animal oils and hardened oils thereof, such as beef tallow,
hardened beef tallow, lard, hardened lard, horse oil, hardened
horse oil, mink oil, orange roughy oil, fish oil, hardened fish oil
and egg yolk oil; plant oils and hardened oils thereof such as
avocado oil, almond oil, olive oil, cacao oil, apricot kernel oil,
kukui nut oil, sesame oil, wheat germ oil, rice germ oil, rice bran
oil, safflower oil, shea butter, soybean oil, evening primrose oil,
perilla oil, tea seed oil, tsubaki oil (camellia japonica oil),
corn oil, rapeseed oil, hardened rapeseed oil, palm kernel oil,
hardened palm kernel oil, palm oil, hardened palm oil, peanut oil,
hardened peanut oil, castor oil, hydrogenated castor oil, sunflower
oil, grape seed oil, jojoba oil, hardened jojoba oil, macadamia nut
oil, meadowfoam seed oil, cottonseed oil, hardened cottonseed oil,
conoanut oil, hardened cocoanut oil; and waxes such as beeswax,
high acid number beeswax, lanolin, reduced lanolin, hardened
lanolin, liquid lanolin, carnauba wax and montan wax. From among
the above ingredients, one only can be used, or two or more can be
used together.
Silicone-Type Oil Phase Ingredient:
[0113] Dimethylpolysiloxane, methylphenylpolysiloxane,
methylcyclopolysiloxane, octamethylpolysiloxane,
decamethylpolysiloxane, dodecamethylcyclosiloxane,
methylhydrogenpolysiloxane, polyether-modified organopolysiloxane,
dimethylsiloxanemethylcetyloxysiloxane copolymer,
dimethylsiloxane-methylstearoxysiloxane copolymer, alkyl-modified
organopolysiloxane, terminal-modified organopolysiloxane,
amino-modified silicone oil, amino-modified organopolysiloxane,
dimethiconol, silicone gel, acryl silicone, trimethylsiloxysilicic
acid and silicone RTV rubber. From among the above ingredients, one
only can be used, or two or more can be used together.
Fluorine-Type Oil Phase Ingredient:
[0114] Perfluoropolyether, fluorine-modified organopolysiloxane,
fluorinated pitch, fluorocarbon, fluoroalcohol and
fluoroalkyl-polyoxyalkylene-comodified organopolysiloxane. From
among the above ingredients, one only can be used, or two or more
can be used together.
(2) Specific Examples of Higher Alcohol
[0115] Lauryl alcohol, myristyl alcohol, cetyl alcohol, stearyl
alcohol, isostearyl alcohol, oleyl alcohol, behenyl alcohol,
2-ethylhexanol, hexadecyl alcohol and octyl dodecanol. From among
the above ingredients, one only can be used, or two or more can be
used together.
(3) Specific Examples of Fatty Acid
[0116] Caprylic acid, capric acid, undecylenic acid, lauric acid,
myristic acid, palmitic acid, palmitoleic acid, stearic acid,
isostearic acid, oleic acid, linoleic acid, linolenic acid, arachic
acid, arachidonic acid, behenic acid, erucic acid and
2-ethylhexanoic acid. From among the above ingredients, one only
can be used, or two or more can be used together.
(4) Specific Examples of Ultraviolet Absorber
[0117] Para-aminobenzoic acid, amyl para-aminobenzoate,
ethyldihydroxypropyl para-aminobenzoate, glyceryl
para-aminobenzoate, ethyl para-aminobenzoate, octyl
para-aminobenzoate, octyldimethyl para-aminobenzoate, ethylene
glycol salicylate, octyl salicylate, triethanolamine salicylate,
phenyl salicylate, butylphenyl salicylate, benzyl salicylate,
homomethyl salicylate, benzyl cinnamate, octyl
para-methoxycinnamate, 2-ethylhexyl para-methoxycinnamate, glyceryl
mono-2-ethyl hexanoate di-para-methoxycinnamate, isopropyl
para-methoxycinnamate, diethanolamine para-methoxyhydrocinnamate,
diisopropyl diisopropylcinnamic acid ester mixture, urocanic acid,
ethyl urocanate, hydroxymethoxybenzophenone,
hydroxymethoxybenzophenone sulfonic acid and a salt thereof,
dihydroxymethoxybenzophenone, sodium
dihydroxymethoxybenzophenonedisulfonate, dihydroxybenzophenone,
dihydroxydimethoxybenzophenone, hydroxyoctoxybenzophenone,
tetrahydroxybenzophenone, butylmethoxydibenzoylmethane,
2,4,6-trianilino-p-(carbo-2-ethylhexyl-1-oxy)-1,3,5-triazine,
2-(2-hydroxy-5-methylphenyl)benzotriazole, methyl-O-aminobenzoate,
2-ethylhexyl-2-cyano-3,3-diphenylacrylate, phenylbenzimidazole
sulfuric acid, 3-(4-methylbenzylidene)camphor,
isopropyldibenzoylmethane,
4-(3,4-dimethoxyphenylmethylene)-2,5-doxy-1-imidazolidinepropionate,
and polymer derivatives and silane derivatives thereof. From among
the above ingredients, one only can be used, or two or more can be
used together.
(5) Specific Examples of Powder and Pigment
[0118] Pigments such as Food Red 104, Food Red 201, Food Yellow 4,
Food Blue 1 and Food Black 401; lake pigments such as Food Yellow 4
AL lake and Food Yellow 203 BA lake; polymers such as nylon powder,
silk powder, urethane powder, Teflon.RTM. powder, silicone powder,
polymethyl methacrylate powder, cellulose powder, starch, silicone
elastomer spherical powder and polyethylene powder; color pigments
such as yellow iron oxide, red iron oxide, black iron oxide,
chromium oxide, carbon black, ultramarine and iron blue; white
pigments such as zinc oxide, titanium oxide and cerium oxide;
extender pigments such as talc, mica, sericite, kaolin and plate
barium sulfate; pearl pigments such as mica titanium; metal salts
such as barium sulfate, calcium carbonate, magnesium carbonate,
aluminum silicate and magnesium silicate; inorganic powders such as
silica and alumina; metal soaps such as aluminum stearate,
magnesium stearate, zinc palmitate, zinc myristate, magnesium
myristate, zinc laurate and zinc undecylenate; bentonite; smectite;
and boron nitride. From among the above ingredients, one only can
be used, or two or more can be used together.
[0119] The shape (e.g., sphere, bar, needle, plate, amorphous,
scale, spindle) and the particle size of these powders are not
particularly limited. These powders may or may not be previously
surface-treated by a conventionally known surface treatment such as
fluorine compound treatment, silicone treatment, silicone resin
treatment, pendant treatment, saline coupling agent treatment,
titanium coupling agent treatment, lubricant treatment, N-acylated
lysine treatment, polyacrylic acid treatment, metal soap treatment,
amino acid treatment, lecithin treatment, inorganic compound
treatment, plasma treatment and mechanochemical treatment. From
among the above ingredients, one only can be used, or two or more
can be used together.
(6) Specific Examples of Surfactant
Anionic Surfactant:
[0120] Fatty acid soap, a-acyl sulfonate, alkyl sulfonate,
alkylallyl sulfonate, alkylnaphthalene sulfonate, alkyl sulfate,
POE alkyl ether sulfate, alkylamide sulfate, alkyl phosphate, POE
alkyl phosphate, alkylamide phosphate, alkyloylalkyl taurine salt,
N-acylamino acid salt, POE alkyl ether carbonate, alkyl
sulfosuccinate, sodium alkylsulfoacetate, acylated hydrolyzed
collagen peptide salt and perfluoroalkylphosphoric acid ester. From
among the above ingredients, one only can be used, or two or more
can be used together.
Cationic Surfactant:
[0121] Alkyltrimethylammonium chloride, stearyltrimethylammonium
chloride, stearyltrimethylammonium bromide,
cetostearyltrimethylammonium chloride, distearyldimethylammonium
chloride, stearyldimethylbenzylammonium chloride,
behenyltrimethylammonium bromide, benzalkonium chloride, behenic
acid amidopropyldimethyl hydroxypropylammonium chloride,
diethylaminoethylamide stearate, dimethylaminoethylamide stearate,
dimethylaminopropylamide stearate and lanolin derivative quaternary
ammonium salt. From among the above ingredients, one only can be
used, or two or more can be used together.
Amphoteric Surfactant:
[0122] Carboxybetaine type, amidobetaine type, sulfobetaine type,
hydroxysulfobetaine type, amidosulfobetaine type, phosphobetaine
type, aminocarboxylate type, imidazoline derivative type and
amidoamine type. From among the above ingredients, one only can be
used, or two or more can be used together.
Nonionic Surfactant:
[0123] Propylene glycol fatty acid ester, glycerin fatty acid
ester, polyglycerin fatty acid ester, sorbitan fatty acid ester,
POE sorbitan fatty acid ester, POE sorbitol fatty acid ester, POE
glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester,
POE hydrogenated castor oil, POE castor oil, POE-POP copolymer,
POE-POP alkyl ether, polyether-modified silicone lauric acid
alkanolamide, alkylamine oxide and hydrogenated soybean
phospholipid. From among the above ingredients, one only can be
used, or two or more can be used together.
Natural-Type Surfactant:
[0124] Lecithin, saponin and sugar-type surfactant. One ingredient
only can be used, or two or more can be used together.
(7) Specific Examples of Polyhydric Alcohol and Sugar
[0125] Ethylene glycol, diethylene glycol, polyethylene glycol,
propylene glycol, dipropylene glycol, polypropylene glycol,
glycerin, diglycerin, polyglycerin, 3-methyl-1,3-butanediol,
1,3-butylene glycol, sorbitol, mannitol, raffinose, erythritol,
glucose, sucrose, fruit sugar, xylitol, lactose, maltose, maltitol,
trehalose, alkylated trehalose, mixed isomerized sugar, sulfated
trehalose and pullulan. Chemically modified products thereof can
also be used. From among the above ingredients, one only can be
used, or two or more can be used together.
(8) Specific Examples of Polymer Compound
[0126] Anionic polymer compounds such as acrylic acid
ester/methacrylic acid ester copolymer (PLUS-SIZE, produced by
Sogokagaku K. K.), vinyl acetate/crotonic acid copolymer (Resin
28-1310, produced by NSC), vinyl acetate/crotonic acid/vinyl
neodecanate copolymer (28-2930, produced by NSC), methyl vinyl
ether maleic acid half ester (GANTREZ ES, produced by ISP), T-butyl
acrylate/ethyl acrylate/methacrylic acid copolymer (RUBIMER,
produced by BASF), vinylpyrrolidone/vinyl acetate/vinyl propionate
copolymer (RUBISCOL VAP, produced by BASF), vinyl acetate/crotonic
acid copolymer (RUBISET CA, produced by BASF), vinyl
acetate/crotonic acid/vinylpyrrolidone copolymer (RUBISET CAP,
produced by BASF), vinylpyrrolidone/acrylate copolymer (RUBIFLEX,
produced by BASF), acrylate/acrylamide copolymer (ULTRAHOLD,
produced by BASF), vinyl acetate/butyl maleate-isobornyl acrylate
copolymer (ADVANTAGE, produced by ISP), carboxy vinyl polymer
(CARBOPOL, produced by BF Goodrich) and acrylic acid-alkyl
methacrylate copolymer (PAMUREN, produced by BF Goodrich);
amphoteric polymer compounds such as acetic acid amphoteric
compound of dialkylaminoethyl methacrylate polymer (YUKAFORMER,
produced by Mitsubishi Chemical) and octylacrylamide
acrylate/hydroxypropyl acrylate/butylaminoethyl methacrylate
copolymer (AMPHOMER, produced by NSC); cationic polymer compounds
such as quaternized compound of vinylpyrrolidone/dimethylaminoethyl
methacrylate (GAFQUAT, produced by ISP) and methyl vinyl
imidazolium chloride/vinylpyrrolidone copolymer (RUBICOTE, produced
by BASF); and nonionic polymer compounds such as
polyvinylpyrrolidone/vinyl acetate copolymer (RUBISCOL VA, produced
by BASF) and vinylpyrrolidone/dimethylaminoethyl methacrylate
copolymer (COPOLYMER VC713, produced by ISP).
[0127] In addition, polymer compounds of natural origin, such as
cellulose and derivatives thereof, calcium alginate, pullulan,
agar, gelatin, tamarind seed polysaccharides, xanthane gum,
carrageenan, high-methoxyl pectin, low-methoxyl pectin, guar gum,
gum arabi, crystal cellulose, arabino galactan, karaya gum,
tragacanth gum, alginic acid, albumin, casein, cardrun, gellan gum
and dextran, can also be suitably used. From among the above
ingredients, one only can be used, or two or more can be used
together.
(9) Specific Examples of Biologically Active Ingredient
[0128] The biologically active ingredient may include substances
which are capable of imparting some biological activity to skin,
when such a substance is applied to the skin. Specific examples
thereof may include: whitening ingredient, age resistor,
ultraviolet protection, slimming agent, skin tightening agent,
antioxidant, hair restorer, hair growing agent, moisturizer, blood
circulation accelerator, antibacterial agent, bactericide,
desiccant, cooling agent, warming agent, vitamin compound, amino
acid, wound healing accelerator, torpent, analgetic, cell activator
and enzyme ingredient.
[0129] Suitable examples of the ingredient to be blended therefor
may include: angelica extract, avocado extract, hydrangea extract,
althea extract, arnica extract, aloe extract, apricot extract,
apricot core extract, ginkgo extract, fennel extract, turmeric
extract, oolong tea extract, rose fruit extract, echinacea leaf
extract, scutellaria root extract, phellodendron bark extract,
goldthread extract, barley extract, hypericum extract, white nettle
extract, watercress extract, orange extract, sea salt, seaweed
extract, hydrolyzed elastin, hydrolyzed wheat powder, hydrolyzed
silk, chamomile extract, carrot extract, artemisia capillaris
extract, glycyrrhiza extract, sabdariffa extract, pyracantha
fortuneana fruit extract, cinchona extract, cucumber extract,
guanosine, gardenia extract, sasa albo-marginata extract, sophora
root extract, walnut extract, grapefruit extract, clematis extract,
chlorella extract, mulberry bark extract, gentian extract, black
tea extract, yeast extract, burdock extract, fermented rice bran
extract, rice germ oil, comfrey extract, collagen, cowberry
extract, asiasarum root extract, bupleurum falcatum root extract,
umbilical cord extract, salvia extract, saponaria extract, bamboo
grass extract, crataegus extract, zanthoxylum fruit extract,
shiitake mushroom extract, rehmannia root extract, lithospermum
root extract, perilla extract, linden extract, filipendula extract,
peony root extract, calamus rhizome extract, birch extract,
horsetail extract, ivy extract, hawthorn extract, sambucus nigra
extract, yarrow extract, peppermint extract, sage extract, mallow
extract, cnidium rhizome extract, swertia herb extract, soy
extract, jujube extract, wild thyme extract, green tea extract,
clove extract, cogon extract, citrus unshiu peel extract, angelica
root extract, calendula extract, peach seed extract, bitter orange
extract, houttuynia extract, tomato extract, natto extract, ginseng
extract, garlic extract, wild rose extract, hibiscus sabdariffa
flower extract, ophiopogon tuber extract, parsley extract, honey,
witch hazel extract, pellitory extract, isodonis extract,
matricaria extract, loquat extract, coltsfoot extract, butterbur
scape extract, Poria cocos extract, butcher bloom extract, grape
extract, propolis, luffa extract, safflower extract, peppermint
extract, linden extract, peony extract, hop extract, pine extract,
horse chestnut extract, skunk cabbage extract, sapindaceae extract,
balm mint extract, peach extract, cornflower extract, eucalyptus
extract, saxifrage extract, coix seed extract, mugwort extract,
lavender extract, apple extract, lettuce extract, lemon extract,
Chinese milk vetch extract, rose extract, rosemary extract, Roman
chamomile extract and royal jelly extract, strawberry extract,
piper longum extract, lotus leaf extract, pfaffia extract, star
fruit extract or the like.
[0130] Other examples may include biopolymers such as
deoxyribonucleic acid, mucopolysaccharide, sodium hyaluronate,
sodium, elastin, chitin, chitosan and hydrolyzed eggshell membrane;
moisture retentive ingredients such as amino acid, hydrolyzed
peptide, sodium lactate, urea, sodium pyrrolidonecarboxylate,
betaine, whey and trimethylglycine; oily ingredients such as
sphingolipid, ceramide, phytosphingosine, cholesterol, cholesterol
derivatives and phospholipid; anti-inflammatory such as
E-aminocaproic acid, glycyrrhizic acid, -glycyrrhetic acid,
lysozyme chloride, guaiazlene and hydrocortisone; vitamins such as
vitamin A, vitamin B2, vitamin B6, vitamin D, vitamin E, calcium
pantothenate, biotin and nicotinic acid amide; active ingredients
such as allantoin, diisopropylamine dichloroacetate and
4-aminomethylcyclohexanecarboxylic acid; antioxidants such as
tocopherol, carotenoid, flavonoid, tannin, lignin and saponin; cell
activators such as a-hydroxy acid and hydroxy acid; blood
circulation accelerators such as y-orizanol and vitamin E
derivatives; wound healing agents such as retinol and retinol
derivatives; whitening agents such as albumin, kojic acid, placenta
extract, sulfur, ellagic acid, linoleic acid, tranexamic acid and
glutathione; and hair growing agents such as cepharanthine,
glycyrrhiza extract, capsicum tincture, hinokitiol, iodized garlic
extract, pyridoxine hydrochloride, DL-a-tocopherol, DL-a-tocopheryl
acetate, nicotinic acid, nicotinic acid derivatives, calcium
pantothenate, D-pantothenyl alcohol, acetyl pantothenylethyl ether,
biotin, allantoin, isopropylmethylphenol, estradiol, ethynyl
estradiol, capronium chloride, benzalkonium chloride,
diphenhydramine hydrochloride, Takanal, camphor, salicylic acid,
vanillylamide nonylate, vanillylamide nonanoate, pyroctone olamine,
glyceryl pentadecanoate, L-menthol, mononitroguaiacol, resorcinol,
y-aminobutyric acid, benzethonium chloride, mexiletine
hydrochloride, auxin, female hormone, cantharis tincture,
cyclosporine, zinc pyrithione, hydrocortisone, minoxidil,
polyoxyethylene sorbitan monostearate, peppermint oil and
SASANISHIKI extract. From among the above ingredients, one only can
be used, or two or more can be used together.
(10) Specific Examples of Antioxidant
[0131] Sodium hydrogensulfite, sodium sulfite, erythorbic acid,
sodium erythorbate, dilauryl thiodipropionate, tocopherol,
tolylbiguanide, nordihydroguaiaretic acid, parahydroxy anisole,
butylhydroxy anisole, dibutylhydroxy toluene, ascorbyl stearate,
ascorbyl palmitate, octyl gallate, propyl gallate, carotenoid,
flavonoid, tannin, lignin, saponin and plant extracts having
antioxidant effect, such as apple extract and clove extract. From
among the above ingredients, one only can be used, or two or more
can be used together.
(11) Specific Examples of Solvent
[0132] Purified water, ethanol, lower alcohol, ethers, LPG,
fluorocarbon, N-methylpyrrolidone, fluoroalcohol, volatile linear
silicone and next generation freon (such as fluorocarbon,
chlorofluorocarbon, CFC). From among the above ingredients, one
only can be used, or two or more can be used together.
Example
[0133] Examples of this invention are described here, which
identify the AGE inhibitory activity, or the like, of the compound
obtained by this invention and which show that the scope of this
invention is not limited to its products and manufacturing
methods.
(1) Method for Producing Extract of Cherry-Tree (Cherry Blossom,
Cherry Leaf):
[0134] Using cherry blossoms (referred to as "yaezakura" in
Japanese, Prunus lannesiana Wils. cv. Sekiyama), an extraction was
done with 30% (w/w) hydrous ethanol at 60 degree centigrade for one
hour. After that, filtering and concentrating were done to obtain
the cherry-blossom extract of a yield of 5 wt %. In the same way,
cherry-leaf extract was obtained of a yield of 4 wt %. Then, the
cherry-blossom extract and cherry-leaf extract were applied, per
Example 1 and Example 2, respectively. (2) Method for Separating
the Active Substance from Cherry-Blossom Extract:
[0135] The search for substances held in the cherry-blossom extract
is shown in FIG. 1. In other words, Example 1, the cherry-blossom
extract (90.10 grams) is suspended within the chromatography column
(HP-20, Mitsubishi Chemical Corporation) to obtain eluted water
(H2O) [of] (62.16 g, 68.88%), eluted methanol (MeOH) [of] (28.21 g,
31.31%) and eluted acetone (Acetone) [of] (0.60 g, 0.67%).
[0136] Next, repeatedly separate and refine the eluted methanol
(MeOH) of (26.00 g) within the normal-phased silica-gel
chromatography column, within the reverse-phased ODS chromatography
column and within the HPLC (high-performance liquid chromatography
column) to isolate the known three-different phenylpropanoid
glycosides: 1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside (8.73 g,
9.6%), 1-O-(E)-Coumaroyl-.beta.-D-Glucopyranoside (22.1 mg,
0.0245%) and 1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside (21.0 mg,
0.0233%), as well as the known four-different flavonoid glycosides:
Kaempferol 3-O-.beta.-D-glucopyranoside (32.6 mg, 0.0361%),
Quercetin 3-O-.beta.-D-glucopyranoside (86.0 mg, 0.0954%),
Kaempferol 3-O-(6''-malony)-.beta.-D-glucopyranoside (55.3 mg,
0.0613%) and Quercetin 3-O-(6''-malony)-.beta.-D-glucopyranoside
(66.2 mg, 0.0734%). (Examples 3 to 5 and 6 to 9).
[0137] The known compounds, above are identified by comparison of
the literature values of 1H-NMR and .sup.13C-NMR, Mass Spectral
Database and optical rotation. Also, each yield (w/w) of the
isolated substances, as shown in FIG. 1, means an isolated yield of
the cherry-blossom extract, Example 1.
(3) Effect Confirmation Test
(Test 1: Effect of AGE Production Inhibition)
[0138] To confirm the effect of the cherry-blossom extract and the
cherry-leaf extract on the formation of AGE, the following tests
were done.
[0139] Put the sample solution (100 mL) of the concentration of
cherry-blossom extract (Example 1) and cherry-leaf extract (Example
2), as shown in the chart, below, into a phosphate buffer solution
(pH: 7.4, 900 mL) containing D-glucose (10%) and into bovine serum
albumin (fraction 5.1%) and leave it out at a temperature of 60
degrees centigrade for two days. Dilute the reaction solution with
distilled water until the intensity of fluorescent light reaches
about 500, thus determining its intensity (measurement wavelength:
370 nm, excitation wavelength: 440 nm). Also, the extract is to be
diluted with distilled water, and the substance, after being
dissolved in DMSO (dimethyl sulphoxide), is to be diluted with a
phosphate buffer solution until DMSO concentration is of 1%. The
result is shown in Chart 1, below. Each value in Chart 1 indicates
an average value with standard error of the three examples.
Asterisks (*, **) mean a significant difference (i.e. *:
p.sub.<0.05; **: p<0.01) between the processed samples and
the unprocessed samples as determined by Dunnett's Multiple
Comparison Test.
TABLE-US-00001 CHART 1 AGE production inhibitory effect of
cherry-blossom extract and its content substances Inhibition Ratio
(%) IC50 1 (.mu.g/mL) 3 10 30 100 300 (.mu.g/mL) Cherry-blossom
extract -8.6 .+-. 0.5 -14.6 .+-. 0.7 -10.8 .+-. 0.4 -9.9 .+-. 0.6
15.1 .+-. 0.7** 42.6 .+-. 3.2** >300 Cherry-leaf extract -1.4
.+-. 0.1 2.8 .+-. 0.1 7.0 .+-. 0.1 21.6 .+-. 0.4** 37.3 .+-. 1.4**
49.8 .+-. 0.4** >300 1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside
-3.3 .+-. 0.1 0.9 .+-. 0.1 10.7 .+-. 0.1** 19.5 .+-. 0.3** 25.0
.+-. 0.3** 30.0 .+-. 0.4** >300
1-O-(E)-Coumaroyl-.beta.-D-glucopyranoside -3.2 .+-. 0.1 -8.2 .+-.
0.1** -8.6 .+-. 0.1** -8.9 .+-. 0.1** -3.7 .+-. 0.1** 11.6 .+-.
0.1** >300 1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside -8.4 .+-.
0.1 -10.4 .+-. 0.1 -10.9 .+-. 0.4* -7.8 .+-. 0.1 5.7 .+-. 0.1 23.3
.+-. 0.4** >300 Kaempferol 3-O-.beta.-D-glucopyranoside -8.4
.+-. 0.1** -9.1 .+-. 0.2** -2.0 .+-. 0.1 19.4 .+-. 0.1** 45.0 .+-.
0.5** 80.3 .+-. 0.7** 102 Quercetin 3-O-.beta.-D-glucopyranoside
8.5 .+-. 0.1** 6.5 .+-. 0.1* 27.6 .+-. 0.5** 49.8 .+-. 0.7** 74.2
.+-. 1.1** 100.8 .+-. 0.6** 30 Kaempferol
3-O-(6''-malony)-.beta.-D- -5.0 .+-. 0.1 -8.5 .+-. 0.1 0.2 .+-. 0.1
20.5 .+-. 0.3** 50.8 .+-. 0.4** 91.7 .+-. 1.7** 78 glucopyranoside
Quercetin 3-O-(6''-malony)-.beta.-D- -10.4 .+-. 0.9 1.9 .+-. 0.1
20.4 .+-. 0.3** 43.7 .+-. 0.7** 74.6 .+-. 0.7** 103.9 .+-. 3.6** 36
glucopyranoside Aminiguanidine hydrochloride -- -- 1.4 .+-. 0.1
18.1 .+-. 1.1 42.6 .+-. 1.7** 67.7 .+-. 1.6** 138 Significant
difference *p < 0.05, **p < 0.01
[Test 1: Result and Effect of Examples]
[0140] It is identified in Chart 1 that the cherry-blossom extract
(Example 1) and cherry-leaf extract (Example 2), both in
concentration of 100 to 300 .mu.g/mL, inhibit the production of
AGE.
[0141] Also, as shown in Chart 1, the main polyphenol of cherry
blossom, i.e. 1-O-(E)-Caffeoyl-3-D-glucopyranoside (Example 3), in
high concentration of 300 .mu.g/mL, shows a 30% inhibitory
activity. The inhibitory activity of a related compound with less
hydroxyl, i.e. 1-O-(E)-Coumaroyl-.beta.-D-Glucopyranoside (Example
4), and 1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside (Example 5) is
reduced. Contrarily, the inhibitory activity of flavonoid glycoside
is generally much, and the inhibitory activity of Quercetin
3-O-.beta.-D-glucopyranoside (Example 7) and Quercetin
3-O-(6''-malony)-.beta.-D-glucopyranoside (Example 9) is much
(IC50), more than twice as much as the inhibitory activity of other
compounds with Kaempferol as an aglycon. (IC50: half-maximal (50%)
inhibitory concentration)
[Test 2-1: Inhibitory Activity Against Fibroblast Apoptosis Caused
by CML-Collagen]
[0142] It is known that the main AGE factor, CML-collagen
(Carboxymethyl lysine-collagen), found in the skin, damages skin
cells. Here, Test 2-1 verifies the beneficial activity of
cherry-blossom extract (Example 1) upon human fibroblast apoptosis
caused by CML-collagen.
(1) Method of Preparing CML-Collagen
[0143] 50 mg of 1 mM HCL was added to 25 mL of bovine skin-derived
collagen and dissolved for two hours incubation at the temperature
of 37 degrees centigrade, and was shaken as needed. Then, PBS
(pH7.4 25 mL) was added to sodium cyanoborohydride (1.42 g) and to
glyoxylic acid (0.9 g) and mixed so as to react at the temperature
of 37 degrees centigrade for 24 hours. The reaction solution was
then put into a dialysis cell of 10,000 molecular mass for a
dialysis-with-water test to be conducted for one day and for a
dialysis-with-PBS to be conducted for six days. CML-collagen (21
mg) was obtained by freeze-drying the dialysate.
(2) Fibroblast culture
[0144] Prior to the test below, a human diploid fibroblast
(female/age 40/normal skin-derived/PDL20) was cultured in the
medium of fetal calf serum (FCS) with penicillin (100 units/mL) and
streptomycin (100 mg/mL).
[0145] The fibroblast (7.times.104 cells) was suspended in the
D-MEM medium containing FCS (0.5%), penicillin (100 units/mL) and
streptomycin (100 mg/mL) to be seeded within 96-well plates (100
mL/well). After 24 hours of culture, the dissolved CML-collagen
solution (2 mgmL) and the sample solution (13 mL) were added to the
medium and cultured for 24 hours. Collagen, instead of
CML-collagen, was added to Normal group.
(4) Cell Survival Rate and Apoptosis Measurement
[0146] The rate of cell survival was measured by MIT Assay. The
result is shown in Chart 2. At the same time, a microscopic test
was done, of which the result of formazan formation of fibroblast
(.times.100) is shown in FIG. 2. Apoptosis was identified by
measuring active caspase-3/7 by the fluorescence method, of which
the result is shown in Chart 3, below. Each value of Charts 2 and 3
is indicated by the average value with standard error of the six
examples. Asterisks (*, **) mean a significant difference (i.e. *:
p<0.05; **: p<0.01) between the unprocessed samples and
Dunnett's multiple comparison test, and f represents p<0.05 by
Fisher's PLSD Test.
TABLE-US-00002 CHART 2 Effect of cherry-blossom extract against
fibroblast cell survival rate (MTT Assay) Concentration Absorbance
of (.mu.g/mL) formazan formation (570 nm) Normal -- 0.192 .+-.
0.007* Control -- 0.172 .+-. 0.007 Cherry-blossom 10 0.166 .+-.
0.002 extract 30 0.169 .+-. 0.002 100 0.175 .+-. 0.002 300 0.174
.+-. 0.002 1000 0.185 .+-. 0.004 f N = 6, Average value .+-.
standard error *p < 0.05 (Dunnett's Test) f p < 0.05
(Fisger's PLSD Test)
TABLE-US-00003 CHART 3 Effect of cherry-blossom against fibroblast
apoptosis (caspase-3/7 Assay) Concentration Intensity of
fluorescent light (.mu.g/mL) (Excitation 480 nm/Detection 530 nm)
Normal -- 282 .+-. 9* Control -- 331 .+-. 12 Cherry- 10 301 .+-. 13
blossom 30 348 .+-. 11 extract 100 293 .+-. 16 f 300 245 .+-. 12*
1000 139 .+-. 5** N = 6, Average value .+-. standard error, *p <
0.05, **p < 0.01 (Dunnett's Test), f p < 0.05 (Fisher's PLSD
Test)
[Test 2-1: Result and Effect of Examples]
(1) Cell Survival Rate
[0147] According to Chart 2, above, the absorbent rate of the
Control group is higher than that of the Normal group, and the cell
survival rate was lowered due to CML-collagen. As shown in FIG. 2,
the microscopic images identify the inhibition of formazan
accumulation within the cell. By this situation, it is anticipated
that fibroblast apoptosis is induced by CML-collagen. Contrarily,
in the group of which 1000 mg/mL of the cherry-blossom extract
(Example 1) is added, the formazan formation significantly
increased.
(2) Apoptosis Inhibition
[0148] As shown in Chart 3, above, caspase-3/7 activity of the
Control group increases compared to that of the Normal group, and
it is identified that apoptosis is induced by the CML-collagen.
Contrarily, in the Normal group of which the cherry-blossom extract
(more than 100 mL) was added, caspase-3/7 activity decreased. When
the volume of cherry-blossom extract was more than 300 mg/mL,
caspase-3/7 activity was lower in the Control group than that in
the Normal group, which suggests that the cherry-blossom extract
possibly inhibits enzyme activity and enzyme expression of
caspase-3/7.
[Test 2-2: Inhibitory Activity of Cherry-Blossom Extract, and
Cherry-Leaf Extract and their Substances Against Fibroblast
Apoptosis Caused by CML-Collagen]
[0149] Here, in Test 2-2, respecting the cherry-blossom extract
(Example 1), and the cherry-leaf extract (Example 2) and the seven
substances contained in the cherry-blossom extract (Example 1):
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside,
1-O-(E)-Coumaroyl-.beta.-D-Glucopyranosideand,
1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside, Kaempferol
3-O-3-D-glucopyranoside, Quercetin 3-O-.beta.-D-glucopyranoside,
Kaempferol 3-O-(6''-malony)-.beta.-D-glucopyranoside and Quercetin
3-O-(6''-malony)-.beta.-D-glucopyranoside (Examples 3 to 9), the
activity against fibroblast apoptosis, caused by CML-collagen, was
identified in the same way as in Test 2-1.
[0150] Also, in Test 2-2, PDL22-32 cells were used as the normal
diploid fibroblast. As a target for comparison, aminoguanidine
hydrochloride which is a medicine for the glycation inhibitor was
examined in the same way. The result is indicated in Chart 4,
below. Each value of Chart 4 indicates an average value with
standard error of the five examples. Asterisks (*, **) mean a
significant difference (i.e. *: p<0.05; **: p<0.01) between
processed samples and unprocessed samples with CML-collagen,
determined by Dunnett's Multiple Comparison Test.
TABLE-US-00004 CHART 4 Effect of Cherry-blossom/Cherry-leaf extract
against fibroblast apoptosis (Caspase Assay) Inhibition Ratio (%) 1
(.mu.g/mL) 3 10 30 100 Cherry-blossom extract -- 61.8 .+-. 2.6 77.1
.+-. 4.2* Cherry-blossom leaf extract (-13.2 .+-. 0.7) 62.7 .+-.
3.7 85.1 .+-. 3.1 (176.7 .+-. 13.7)** (-24.5 .+-. 0.7) 46.9 .+-.
1.4 58.4 .+-. 1.9* (135.2 .+-. 3.1)**
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside 26.2 .+-. 0.5* 37.6 .+-.
1.2 72.2 .+-. 2.7* 1-O-(E)-Coumaroyl-.beta.-D-glucopyranoside 17.2
.+-. 0.5 7.1 .+-. 0.2 51.1 .+-. 1.9
1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside -11.8 .+-. 0.3 19.7 .+-.
0.9 48.6 .+-. 2.9 Kaempferol 3-O-.beta.-D-glucopyranoside -0.7 .+-.
0.1 27.9 .+-. 1.1 100.7 .+-. 4.2 Quercetin
3-O-.beta.-D-glucopyranoside 44.2 .+-. 1.5* 39.0 .+-. 1.1* 121.5
.+-. 5.4** Kaempferol 3-O-(6''-malony)-.beta.-D-glucopyranoside
-18.9 .+-. 0.6 -17.3 .+-. 0.6 10.5 .+-. 0.5 Quercetin
3-O-(6''-malony)-.beta.-D-glucopyranoside 21.8 .+-. 0.7 36.6 .+-.
1.4 98.4 .+-. 4.4* Aminoguanidine Hydrochloride 104.8 .+-. 34*
Significant difference *p < 0.05, **p < 0.01
[Test 2-2: Result and Effects of Examples]
[0151] As shown in Chart 4, the cherry-blossom extract (Example 1,
10 to 100 mg/mL) lowered caspase-3/7 activity. From this result, it
is suggested that the cherry-blossom extract (Example 1) inhibits
apoptosis caused by CML-collagen. Among the contents, Quercetin
3-O-.beta.-D-glucopyranoside (Example 7), shown to be the strongest
substance in the AGE production inhibitory Test 1, had the
strongest inhibitory activity. Similar compounds like Kaempferol
3-O-.beta.-D-glucopyranoside (Example 6) and Quercetin
3-O-(6''-malony)-.beta.-D-Glucopyranoside (Example 9) showed strong
inhibitory activity. However, Kaempferol
3-O-(6''-malony)-.beta.-D-glucopyranoside (Example 8) showed
relatively weak activity. As for cinnamic acids, Kaempferol
3-O-.beta.-D-glucopyranoside, which contains the highest level of
cinnamic acid, showed especially strong inhibitory activity.
[0152] As such, it is thought that the cherry blossom extract
(Example 1), and the cherry leaf extract (Example 2) and their
active substances have AGE production inhibitory activity which
inhibits fibroblast apoptosis caused by CML-collagen. In light of
the above results, it is seen that these active substances function
as a breaker that degrades CML-collagen, which is an AGE. Thus, the
AGE production inhibitor of this invention can be used to as an AGE
decomposition agent or as an AGE remover.
[Test 3: Promoting Activity of Fibroblast Collagen Grating
Formation]
[0153] When fibroblast is cultured in the presence of collagen
solution, collagen grating formation can be identified. In a
similar test, when adding fibroblast that has been glycosylated by
the intermediate product of glycation (glyoxal), it is known that
the collagen grating formation is inhibited. Here, Test 3 showed
the cherry-blossom extract inhibits formation of the collagen
grating of glycated human fibroblast.
(1) Glycation of Fibroblast
[0154] The infant-derived NB1 RGB (passage number 22) was suspended
in the D-MEM complete media (1.86.times.104 cells/mL). 15 mL each
of it was seeded within each cell-culture dish of 14 cm in
diameter. After 24 hours, each sample and glyoxal were added until
the molecular mass of each dish became 200 mM and incubate the
dishes for five days.
(2) Collagen Grating Formation
[0155] Bovine skin-derived collagen (25 mg) was dissolved in 0.1%
acetic acid (8.3 mL). Then, a 10-times concentration of Hanks
solution (1.66 L) was added. Then, 0.1% acetic acid (3.5 mL) was
added. 1M NaOH was added to neutralize the solution, which was
sterilized by filteration to obtain the collagen solution.
[0156] The mixed solution of collected fibroblast (5.4.times.104
cell) was suspended in FCS (50 .mu.m), and collagen (450 mL) was
seeded within a 24-well cell-culture plate and allowed to culture
for 24 hours and 48 hours. Each culture was observed through a
microscope and by the naked eye. An image observed by the naked eye
is shown in FIG. 3. A microscopic image (.times.100), [taken] after
the 24-hour culture, is shown in FIG. 4. A microscopic image
(.times.200) [taken] after the 48-hour culture, is shown in FIG. 5.
For the purpose of comparison, aminoguanidin hydrochloride which is
an anti-glycation agent was also examined in the same way.
[Test 3: Result and Effects of Examples]
[0157] The result of the fibroblast and collagen solution culture,
as shown in FIG. 3, is that collagen-grating formation was
identified. In FIG. 3, the white hazy part shows the extension and
projection of the fibroblast and collagen grating. The amount of
collagen-grating formation of the Control group which was processed
with glyoxal significantly decreased compared to that of Normal
group. The well of fibroblast, processed with 100 and 1000 mg/mL of
cherry blossom-extract (Example 1) and glyoxal showed an increase
in the amount of grating formation. On the other hand, the well of
fibroblast processed with 100 mg/mL of guanidine hydrochloride,
compared to the control group, showed no increase in the amount of
grating formation. In FIG. 4, showing the microscopic image after
24 hours of culture, it was identified that the fibroblast of the
Normal group and of the Control group and of guanidine
hydrochloride were spherically shaped. However, the well of 100 and
1000 mg/mL of the cherry blossom-extract (Example 1) identified the
extension and projection of the fibroblast. FIG. 5, the 200-times
enlarged microscopic image, after eight hours of culture, showed a
few extensions of the fibroblast of the Control group. However, it
was still weak compared to that of the well of 100 and 1000 mg/mL
of the cherry blossom-extract (Example 1).
[Test 4: AGE Production Inhibitory Activity within the
Fibroblast]
[0158] Test 4 examined the activity of the cherry blossom-extract
(Example 1) and of the 1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside
(Example 3) against the production of AGE (carbon methyl lysine
protein) within the fibroblast (human diploid fibroblast) which was
processed with glyoxal.
[0159] Human diploid fibroblast (passage number 30) was suspended
in a D-MEM complete culture media, and 20 mL each was seeded within
a cell culture dish of 14 cm in diameter. After 48 hours of
culture, samples of various concentrations and of 401M of glyoxal
were added to 80%-confluent cells and cultured for 5 days. After
that, each sample was collected and crushed. Then, protein 10 mg
was examined by the Western Blotting method using an anti-AGE
antibody to detect AGE. The result as shown in FIG. 6 shows a
detection of AGE by the anti-AGE antibody (HRP staining system). In
FIG. 6, CaGlu means 1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside
(Example 3).
[Test 4: Result and Effects of Examples]
[0160] As shown in FIG. 6, the detection of AGE using an anti-AGE
antibody identified the amount of AGE that had been increased by
the stimulation of glyoxal (comparison between Control and
Non-control). Contrarily, the cherry blossom-extract (Example 1) of
10 mg/mL strongly inhibited the production of AGE. The effect of
100 mg/mL of the cherry-blossom extract was reduced. Yet,
inhibitory activity was still found.
1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside (CaClu: Example 3) of 1
and 10 mg/mL inhibited the production of AGE. From these results,
it is now clearly shown at the cellular level that cherry-blossom
extract and its major substances inhibits a production of AGE.
[Test 5: AGE Production Inhibitory Activity by the Intradermal
Administration of Glyoxal]
[0161] After continuously administrating the cherry-blossom extract
(Example 1) in various concentrations, the AGE precursor, glyoxal,
was intradermally administrated to observe its activity in the
production of AGE. Based on this observation, a recommended dose of
the cherry-blossom extract (Example 1) was determined.
[0162] The solution (10, 50 and 100 mg/kg) of the cherry-blossom
extract (Example 1) was administered to a mouse (ICR, male,
5-week-old) once a day for 10 days. After completing the
administration, glyoxal (800 mM 100 mL) was intradermally
administered into the shaved back of the mouse. After 21 hours, the
last administration of cherry-blossom extract (Example 1) was done
to the mouse. Then, after another three hours, the mouse was
immolated with an ether anesthesia, and the glyoxal-administered
part was removed. The removed skin of the mouse was then crushed in
2 mL of lysis buffer (50 mM tris, 150 mM NaCl, 1% Triton X100, pH:
7.2) using Polytron Homogenizer, and the crushed material was
centrifugally separated (1000.times.g, 7 min). Then, the
supernatant was collected and refrigerated at the temperature of 20
degrees Centigrade below zero.
[0163] After measuring the protein content of the frozen
supernatant by the BCA method, the AGE content was measured using
an ELISA kit (Trans Genic. Inc.) for measuring glucose-derived
AGEs. In addition, the AGE was detected by the Western Blotting
method. The result is shown in Chart 5, as well as in FIG. 7.
[Test 5: Result and Effects of Examples]
[0164] As shown in Chart 5 and in FIG. 7, the cherry-blossom
extract (Example 1) indicated no dosage dependency. It was
identified that 10 to 100 mg/kg of the cherry-blossom extract,
orally administration, inhibited the production of AGE. From this
result, the recommended dosage of the cherry blossom-extract
(Example 1) is to be set between 10 and 100 mg/day.
TABLE-US-00005 CHART 5 Effect of cherry-blossom extract against
intradermal AGE production caused by glyoxal (Detected by ELISA)
Dosage amount AGE Inhibition ratio (mg/kg) (pg/mg protein) (%)
Control -- 53 .+-. 12 -- Cherry-blossom 10 50 .+-. 15 5.7 extract
50 18 .+-. 17 66.0 100 30 .+-. 12 43.4 Average value .+-. standard
error (n = 6)
[Test 6: Dermal Cell Apoptosis Inhibitory Activity by Intradermal
Administration of CML-Collagen]
[0165] After the continuous administration of the cherry-blossom
extract in various concentrations, the CML-collagen was
intradermally administrated to observe the activity against the
apoptosis of dermal cells as well as the fibroblast. The test was
conducted in the following method.
[0166] The solution (10, 50 and 100 mg/kg) of cherry-blossom
extract (Example 1) was administered to a mouse (ICR, male,
5-week-old) once a day for 10 days. After completing the
administration, CML-collagen (100 mg/100 mL) was intradermally
administered into the shaved skin area of the mouse where the
biauricular line of the occipital region of the mouse crosses a
midline. Collagen was administered to the Normal group. After 21
hours, the last administration of the cherry-blossom extract
(Example 1) was done to the mouse. Then, after another three hours,
the mouse was immolated after being administrated an ether
anesthesia. The collagen-administered part of the mouse was
removed. The part removed from the mouse was immersed and fixed in
a solution of 4% paraformadehyde, and the cut section was stained
by TUNEL staining. To count the number of dermal cells, which were
confirmed as being positive by the TUNEL staining, the image of the
cross section of the skin was taken by a microscope (.times.400)
and processed with color gradations from purple to orange to
identify the number of cells. To calculate the area of the inner
skin, a printed image of the cross section of the inner skin was
measured by a digital planimeter, and the scale was multiplied. The
result is shown in Chart 6, as well as in FIG. 8. In FIG. 8, the
part marked with a .tangle-solidup. means TUNEL-positive cells.
[Test: Result and Effects of Examples]
[0167] By the administration of CML-collagen, the number of
TUNEL-positive cells in the inner skin increased. To the contrary,
the cherry-blossom extract (Example 1) decreased the number of
TUNEL-positive cells in a dosage-dependent manner. From this
result, it was identified that the cherry-blossom extract (Example
1) inhibited fibroblast apoptosis of the skin caused by the
CML-collagen, in vivo.
TABLE-US-00006 CHART 6 Effect of cherry-blossom extract against
dermal cell apoptosis caused by CML-collagen Dosage Number of TUNEL
amount stained positive Inhibition ratio (mg/kg) cells
(cells/mm.sup.2) (%) Normal -- 27.2 .+-. 1.7 -- Control -- 36.4
.+-. 10.6 -- Cherry-blossom extract 10 22.7 .+-. 5.4 149 50 14.5
.+-. 2.9 238 100 8.8 .+-. 1.7* 300 Average value .+-. standard
error (n = 3), *p < 0.05
[0168] The following charts present examples of the compounds using
cherry-blossom extract (Example 1) as an AGE production inhibitor,
or the like. However the compounds shown below are not limited to
just these.
TABLE-US-00007 CHART 7 Blending example 1: Chewing gums Sugar 53.0
wt % Gum base 20.0 Glucose 10.0 Starch syrup 16.0 Aroma chemical
0.5 AGE production inhibitor, etc 0.5 100.0 wt %
TABLE-US-00008 CHART 8 Blending example 2: Gummies Reduction sugar
40.0 wt % Granulated sugar 20.0 Glucose 20.0 Gelatine 4.7 Water
9.68 Yuzu juice (Citrus junos) 4.0 Yuzu flavor 0.6 Pigment 0.5 AGE
production inhibitor, etc 0.5 100.0 wt %
TABLE-US-00009 CHART 9 Blending example 3: Candies Sugar 50.0 wt %
Starch syrup 33.0 Water 14.4 Organic acid 2.0 Aroma chemical 0.2
AGE production inhibitor, etc 0.4 100.0 wt %
TABLE-US-00010 CHART 10 Blending example 4: Yogurt (Hard type/Soft
type) Milk 41.5 wt % Powdered skim milk 5.8 Sugar 8.0 Agar-agar
0.15 Gelatine 0.1 Lactic acid bacterium 0.005 AGE production
inhibitor, etc 0.4 Aroma chemical Minute amount Water Rest 100.0 wt
%
TABLE-US-00011 CHART 11 Blending example 5: Soft drinks Fructose
glucose solution 30.0 wt % Emulsifying agent 0.5 AGE production
inhibitor, etc 0.3 Aroma chemical Appropriate amount Distilled
water Rest 100.0 wt %
TABLE-US-00012 CHART 12 Blending example 6: Tablet-shaped sweets
Sugar 76.4 wt % Glucose 19.0 Glycerine fatty acid ester 0.2 AGE
production inhibitor, etc 0.5 Distilled water 3.9 100.0 wt %
TABLE-US-00013 CHART 13 Blending example 7: Soft capsules Brown
rice germ oil 47.0 wt % Yuzu (Citrus junos) seed oil 40.0
Emulsifying agent 12.0 AGE production inhibitor, etc 1.0 100.0 wt
%
TABLE-US-00014 CHART 14 Blending example 8: Tablets Lactose 54.0 wt
% Crystaline Cellulose 30.0 Starch splitting product 10.0 Glycerin
fatty acid ester 5.0 AGE production inhibitor, etc 1.0 100.0 wt
%
TABLE-US-00015 CHART 15 Blending example 8: Tablets Lactose 54.0 wt
% Crystaline Cellulose 30.0 Starch splitting product 10.0 Glycerin
fatty acid ester 5.0 AGE production inhibitor, etc 1.0 100.0 wt
%
TABLE-US-00016 CHART 16 Blending example 10: Skin lotions Ethanol
5.0 wt % Glycerin 2.0 1,3-butylene glycol 2.0 Polyethylene oleyl
ether 0.5 Sodium citrate 0.1 Citric acid 0.1 AGE production
inhibitor, etc 0.1 Distilled water Rest 100.0 wt %
TABLE-US-00017 CHART 17 Blending example 11: Body gels Macadamia
nut oil 2.0 wt % Octyl decyl myristate 10.0 Methylphenyl
polysiloxane 5.0 Behenyl alcohol 3.0 Stearic acid 3.0 Bathyl
alcohol 1.0 Glycel monostearate 1.0 Tetra oleic acid
polyoxyethylene 2.0 sorbit Hydrogenated soybean phosphatide 1.0
Ceramide 0.1 Retinol palmitate 0.1 Preservative agent Appropriate
amount Centella asiatica extract 1.0 AGE production inhibitor, etc
1.0 1,3-butylene glycol 5.0 Distilled water Rest 100.0 wt %
TABLE-US-00018 CHART 18 Blending example 12: Cosmetic emulsion
Squalene 4.0 wt % Vaseline 2.5 Cetanol 2.0 Glycerin 2.0 Oleophilic
glycerine monostearate 1.0 Stearic acid 1.0 L-arginine 1.0 AGE
production inhibitor 0.5 Potassium hydroxide 0.1 Aroma chemical
Minute amount Distilled water Rest 100.0 wt %
TABLE-US-00019 CHART 19 Blending example 13: Bath agent (liquid
type) Propylene glycol 50.0 wt % Ethanol 20.0 Sodium sulphate 5.0
AGE production inhibitor 0.5 Lanoline 0.5 Avocado oil agent 0.5
Pigment 1.5 Aroma chemical 22.0 100.0 wt %
[0169] Compounds 1 to 13, above, contain the cherry-blossom extract
(Example 1). Yet, they still may contain cherry-leaf extract
(Example 2) or any one of Examples 3 to
9:1-O-(E)-Caffeoyl-.beta.-D-glucopyranoside,
1-O-(E)-Coumaroyl-.beta.-D-Glucopyrano-side and,
1-O-(E)-Cinnamoyl-.beta.-D-glucopyranoside, Kaempferol
3-O-.beta.-D-glucopyranoside, Quercetin
3-O-.beta.-D-glucopyranoside, Kaempferol
3-O-(6''-malony)-.beta.-D-glucopyranoside and Quercetin
3-O-(6''-malony)-.beta.-D-Glucopyranoside. It is also possible to
use a mixture of the above seven substances (Examples 3 to 9).
INDUSTRIAL APPLICABILITY
[0170] As described above, this invention makes it possible to
inhibit the production of AGE and to prevent cells and
intercellular matrix from suffering damage induced by AGE. From
this result, this invention also makes it possible to effectively
prevent and cure diabetes and diabetic complications, thus
effectively preventing the skin from aging.
* * * * *