U.S. patent application number 13/294028 was filed with the patent office on 2012-03-29 for prophylactic agent for renal failure.
Invention is credited to Tatsuhiko Hirota, Teppei Nakamura, Kohji Ohki, Toshiaki Takano.
Application Number | 20120077735 13/294028 |
Document ID | / |
Family ID | 39830596 |
Filed Date | 2012-03-29 |
United States Patent
Application |
20120077735 |
Kind Code |
A1 |
Hirota; Tatsuhiko ; et
al. |
March 29, 2012 |
PROPHYLACTIC AGENT FOR RENAL FAILURE
Abstract
The present invention provides an agent having a suppressive
action against kidney glomerular disease and renal
arteriosclerosis, a prophylactic agent for renal failure containing
the above described agent, and functional foods expected to have a
prophylactic effect for renal failure. The agent for preventing
kidney glomerular disease and renal arteriosclerosis comprises Xaa
Pro Pro as an active ingredient.
Inventors: |
Hirota; Tatsuhiko;
(Sagamihara-shi, JP) ; Ohki; Kohji;
(Sagamihara-shi, JP) ; Nakamura; Teppei;
(Sagamihara-shi, JP) ; Takano; Toshiaki;
(Sagamihara-shi, JP) |
Family ID: |
39830596 |
Appl. No.: |
13/294028 |
Filed: |
November 10, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12567628 |
Sep 25, 2009 |
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13294028 |
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PCT/JP2008/055073 |
Mar 19, 2008 |
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12567628 |
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Current U.S.
Class: |
514/1.9 ;
514/15.4 |
Current CPC
Class: |
A23V 2002/00 20130101;
A61P 9/00 20180101; A23V 2002/00 20130101; A61P 9/10 20180101; A23V
2002/00 20130101; A23L 33/18 20160801; A61K 38/06 20130101; A61P
7/00 20180101; A23V 2002/00 20130101; A61P 9/04 20180101; A61P 1/16
20180101; A61P 13/12 20180101; A23V 2002/00 20130101; A23V 2250/064
20130101; A61P 43/00 20180101; A23V 2250/0654 20130101; A23V
2200/32 20130101; A23V 2250/064 20130101; A23V 2250/0626 20130101;
A23V 2200/32 20130101; A23V 2250/0626 20130101; A23V 2200/326
20130101; A23V 2250/0654 20130101; A23V 2250/064 20130101; A23V
2200/326 20130101; A23V 2250/064 20130101 |
Class at
Publication: |
514/1.9 ;
514/15.4 |
International
Class: |
A61K 38/06 20060101
A61K038/06; A61P 13/12 20060101 A61P013/12 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 27, 2007 |
JP |
2007-81638 |
Claims
1. A method for suppressing kidney glomerular disease and renal
arteriosclerosis, comprising administrating Xaa Pro Pro to a
subject.
2. The method for suppressing kidney glomerular disease and renal
arteriosclerosis according to claim 1, wherein said Xaa Pro Pro is
Val Pro Pro and/or Ile Pro Pro.
3. The method for suppressing kidney glomerular disease and renal
arteriosclerosis according to claim 1, wherein said Xaa Pro Pro is
originated from an animal milk casein hydrolysate or a concentrate
thereof.
4. The method for suppressing kidney glomerular disease and renal
arteriosclerosis according to claim 3, wherein said animal milk
casein hydrolysate is a fermented product obtained by fermenting an
animal milk casein with Aspergillus.
5. The method for suppressing kidney glomerular disease and renal
arteriosclerosis according to claim 3, wherein said animal milk
casein hydrolysate is a decomposed product obtained by hydrolyzing
an animal milk casein with an enzyme derived from Aspergillus.
6. The method for suppressing kidney glomerular disease and renal
arteriosclerosis according to claim 5, wherein said enzyme derived
from said Aspergillus is an enzyme derived from Aspergillus
oryzae.
7. The method for suppressing kidney glomerular disease and renal
arteriosclerosis according to claim 1, wherein said Xaa Pro Pro is
a fermented product obtained by fermenting a raw material
containing a milk protein with a bacterium belonging to the species
Lactobacillus helveticus.
8. The method for suppressing kidney glomerular disease and renal
arteriosclerosis according to claim 7, wherein said bacterium
belonging to the species Lactobacillus helveticus is Lactobacillus
helveticus CM4 strain (FERM BP-6060).
9. A method for suppressing renal failure, comprising
administrating Xaa Pro Pro to a subject.
10. The method for suppressing renal failure according to claim 9,
wherein said Xaa Pro Pro is Val Pro Pro and/or Ile Pro Pro.
11. The method for suppressing renal failure according to claim 9,
wherein said Xaa Pro Pro is originated from an animal milk casein
hydrolysate or a concentrate thereof.
12. The method for suppressing renal failure according to claim 11,
wherein said animal milk casein hydrolysate is a fermented product
obtained by fermenting an animal milk casein with Aspergillus.
13. The method for suppressing renal failure according to claim 11,
wherein said animal milk casein hydrolysate is a decomposed product
obtained by hydrolyzing an animal milk casein with an enzyme
derived from Aspergillus.
14. The method for suppressing renal failure according to claim 13,
wherein said enzyme derived from said Aspergillus is an enzyme
derived from Aspergillus oryzae.
15. The method for suppressing renal failure according to claim 9,
wherein said Xaa Pro Pro is a fermented product obtained by
fermenting a raw material containing a milk protein with a
bacterium belonging to the species Lactobacillus helveticus.
16. The method for suppressing renal failure according to claim 15,
wherein said bacterium belonging to the species Lactobacillus
helveticus is Lactobacillus helveticus CM4 strain (FERM BP-6060).
Description
[0001] This application is a Continuation of U.S. application Ser.
No. 12/567,628 filed Sep. 25, 2009, which is a Continuation of
Application No. PCT/JP2008/055073 filed on Mar. 19, 2008, and for
which priority is claimed under 35 U.S.C. .sctn.120. This
application also claims priority of Application No. 2007-081638
filed in Japan on Mar. 27, 2007 under 35 U.S.C. .sctn.119. The
entire content of each of the above-identified applications is
hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to an active ingredient having
an action for preventing kidney glomerular diseases and renal
arteriosclerosis, a prophylactic agent for renal failure containing
the active ingredient, as well as functional foods containing the
active ingredient, which functional food are expected to have a
prophylactic effect on renal failure.
[0003] In addition, the present invention relates to a method for
preventing kidney glomerular diseases and renal arteriosclerosis.
Further, the present invention relates to a method for preventing
renal failure.
BACKGROUND OF THE INVENTION
[0004] Recently, the number of those who have a risk factor
considered to cause heart diseases or renal diseases, such smoking,
hypertension, hyperglycemia, and hyperlipidemia, have increased in
some countries. There is an increasing trend in the number of
patients suffering from heart failure or renal failure. In
addition, these pathologic states are not limited to humans and the
increasing trend in morbidity of such diseases are seen among
animals that closely interact with humans, such as dogs, cats,
other companion animals and pet animals.
[0005] An example of an effective drug for treating heart failure
is an angiotensin converting enzyme inhibitor (ACEI), such as
Enalapril, which inhibits an enzyme converting angiotensin I to
angiotensin II having a hypertensive action (i.e. angiotensin
converting enzyme; ACE), and has a hypotensive action. In addition,
the antihypertensive drug reportedly improves the progression of
renal damage at the same time as lowering blood pressure (J. Clin.
Invest., 77, 1993-2000, 1986). Yet on the other hand, for heart
failure induced by various primary diseases, the ACE inhibitor is
not effective for all cases. Rather there is a risk of developing
acute renal failure accompanying lowering blood pressure. It has
been pointed out that careful administration is required (Saishin
Igaku, 48:1404 to 1409, 1993), That is, the antihypertensive drug
can be insufficient for the prophylaxis of heart failure but also
could rather induce renal failure.
[0006] Renal failure generally refers to conditions where the
kidney cannot filter blood to remove metabolic wastes in the blood
successfully.
[0007] Renal failure is the pathologic state where renal functions
are deteriorated by various renal diseases. Examples of the primary
disease include diabetic nephropathy, chronic glomerulonephritis,
nephrosclerosis, polycystic kidney, chronic pyelonephritis, rapidly
progressive glomerulonephritis, SLE nephritis, glomerulosclerosis,
and renal artery blood flow failure by renal arteriosclerosis. It
has been reported that ACE inhibitors are effective against renal
failure, which has drawn attention. Yet it has pointed out that the
ACE inhibitors could cause acute renal failure and aggravation of
renal damage. And thus it cannot be said that usefulness of these
drugs is satisfactory (Saishin Igaku, 48:1404-1409, 1993). As
described above, because renal failure is caused by various factors
including hyperglycemia and drug damage, improving hypertension
alone is not sufficient to prevent all renal failures originated
from the above described primary disease. On the other hand,
glomerular disease is a common lesion of renal failure and renal
arteriosclerosis is a lesion associated with renal failure. Under
such circumstances, a compound having a suppressive action for
glomerular disease or a compound additionally having a suppressive
action for renal arteriosclerosis can be said to be useful for
prophylaxis and/or treatment of renal failure.
[0008] Meanwhile, it has been reported that a peptide originated
from food materials, such as casein has the ACE inhibitory activity
and such peptides are known to have a hypotensive action. However,
whether or not the peptides have an action for preventing kidney
glomerular diseases and an action for suppressing renal
arteriosclerosis has not been directly demonstrated (Japanese
Patent Publication No. 2782142, J. Dairy Sci. 1995, 78:777-783, J.
Dairy Sci. 1995, 78:1253-1257, Am. J. Clin. Nutr. 1996,
64:767-771). Additionally, as described above, since the
prophylactic and therapeutic effect for renal failure mediated by
ACE inhibition is limited, prophylaxis and treatment of the disease
which are not dependent on the ACE inhibition are desired.
SUMMARY OF THE INVENTION
[0009] The present invention provides a compound or composition
having a suppressive action against kidney glomerular disease and a
suppressive action against renal arteriosclerosis independently of
the ACE inhibitory activity.
[0010] In addition, the present invention provides a prophylactic
agent for renal failure and functional foods expected to have a
prophylactic effect on renal failure containing the above described
compound or composition.
[0011] The present invention provides a method for suppressing
kidney glomerular disease and a method for suppressing renal
arteriosclerosis, which method includes administration of the above
described compound or composition to a subject.
[0012] Accordingly, the present invention provides a method for
preventing renal failure, which method includes administration of
the above described compound or composition to a subject.
[0013] The present invention is also a use of the above described
compound or composition in producing a pharmaceutical for
suppressing kidney glomerular disease and for suppressing renal
arteriosclerosis. In particular, the present invention is a use of
the above described compound or composition in producing a
pharmaceutical for preventing renal failure.
[0014] The present inventors have discovered that a tripeptide
having a specific structure Xaa Pro Pro (wherein Xaa is any
naturally occurring amino acid) has a suppressive action against
kidney glomerular disease and suppressive action against renal
arteriosclerosis, and have reached to invent a pharmaceutical and
functional foods useful for prophylaxis of renal failure. Concrete
contents of the present invention are as follows:
[0015] The present invention provides a suppressive agent against
kidney glomerular disease and renal arteriosclerosis containing Xaa
Pro Pro as an active ingredient.
[0016] the present invention also provides a prophylactic agent for
renal failure having the above described suppressive action against
kidney glomerular disease and renal arteriosclerosis.
[0017] In addition, the present invention provides functional foods
containing the above described suppressive action against kidney
glomerular disease and renal arteriosclerosis.
[0018] The present invention provides a method for suppressing
kidney glomerular disease and renal arteriosclerosis, which method
includes administration of Xaa Pro Pro or a composition containing
Xaa Pro Pro to a subject.
[0019] In addition, the present invention provides a method for
preventing renal failure, which method includes administration of
Xaa Pro Pro or a composition containing Xaa Pro Pro to a
subject.
[0020] The present invention is also a use of Xaa Pro Pro or a
composition containing Xaa Pro Pro in producing a pharmaceutical
for suppressing kidney glomerular disease and renal
arteriosclerosis. In particular, the present invention is also a
use of Xaa Pro Pro or a composition containing Xaa Pro Pro in
producing a pharmaceutical for preventing renal failure
[0021] Preferably Xaa Pro Pro is Val Pro Pro and/or Ile Pro
Pro.
[0022] In other embodiment of the present invention, the active
ingredient Xaa Pro Pro is derived from an animal milk casein
hydrolysate or a concentrate thereof.
[0023] In other further embodiment of the present invention, the
active ingredient Xaa Pro Pro is originated from a fermented
product obtained by fermenting a raw material containing a milk
protein with a bacterium belonging to the species Lactobacillus
helveticus.
[0024] Preferably the bacterium belonging to the species
Lactobacillus helveticus is Lactobacillus helveticus CM4 strain
(FERM BP-6060).
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows the results of Test 1 examining the effect of
Val Pro Pro (VPP) to suppress the thickening of small- and
medium-sized renal arteries wall in rats. The test was conducted
using the group of rats administered with L-NAME (11 rats per
group), the group of rats administered with L-NAME and Enalapril (9
rats per group), and the group of rats administered with L-NAME and
VPP (12 rats per group). The graph represents mean.+-.standard
error. Comparisons were demonstrated by the Mann-Whitney test. The
symbol (*) in the figure indicates statistical significance
(p<0.05).
[0026] FIG. 2 shows the results of Test 1 examining the effect of
Val Pro Pro (VPP) to suppress occurrence of the rat renal
glomerular lesions. Comparisons were demonstrated by the
Mann-Whitney test. The symbol (*) in the figure indicates
statistical significance (p<0.05).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] The agent for preventing kidney glomerular disease and renal
arteriosclerosis according to the present invention comprises a
tripeptide with a specific structure of Xaa Pro Pro as an active
ingredient. The term "agent" herein is not restricted to a
pharmaceutical but refers to a composition such as a pharmaceutical
composition or food composition, or a compound such as a food
additive. The term "agent for preventing kidney glomerular disease
and renal arteriosclerosis" used in the present invention refers to
a compound or composition having an action to suppress progression
of kidney glomerular disease and renal arteriosclerosis lesions, an
action to improve kidney glomerular disease and renal
arteriosclerosis lesions, or a prophylactic effect on the onset of
kidney glomerular disease and renal arteriosclerosis. In addition,
the "glomerular disease" used in the present invention refers to a
disease with lesions occurred in the glomerulus of the kidney, and
histologically, refers to conditions where atrophy of the
glomerulus, glomerulus deterioration, glomerulosclerosis,
alterations in Bowman's capsule, or the like is observed. Concrete
examples of the name of disease include minimal change nephrotic
syndrome, focal glomerular sclerosis, endocapillary proliferative
glomerulonephritis (so-called acute nephritis), IgA
glomerulonephritis (mesangial proliferative glomerulonephritis),
membranous nephropathy, membranoproliferative glomerulonephritis,
and crescentic glomerulonephritis.
[0028] The Xaa in the active ingredient Xaa Pro Pro according to
the present invention may be any naturally occurring amino acid.
Concrete examples include Val Pro Pro (valine proline proline), Ile
Pro Pro (isoleucine proline proline), Ser Pro Pro (serine proline
proline), and Leu Pro Pro (leucine proline proline). Preferred are
Val Pro Pro and Ile Pro Pro with Val Pro Pro being more preferred.
As the active ingredient, the agent may contain a combination of
two or more of the tripeptides Xaa Pro Pro, as exemplified by a
combination of Val Pro Pro and Ile Pro Pro.
[0029] The active ingredient Xaa Pro Pro may be an chemically
synthesized tripeptide or naturally occurring tripeptide.
[0030] As an chemical synthetic method of tripeptide Xaa Pro Pro, a
common method such as solid phase method (t-Boc method and Fmoc
method) and liquid phase method can be employed. For instance, the
peptide may be synthesized using an automatic peptide synthesizer
such as a peptide synthesizer (PSSM-8type) manufactured by Shimadzu
Corporation. With regard to reaction conditions for the peptide
synthesis and the like, those skilled in the art can arbitrarily
set appropriate reaction conditions and the like, based on their
technological common knowledge depending on the synthesis method to
be selected and desired tripeptide Xaa Pro Pro.
[0031] Alternatively, as the naturally occurring peptide, the
tripeptide may be originated from an animal milk casein hydrolysate
or a concentrate thereof, as well as may be originated from a
fermented product obtained by fermenting a food material containing
a protein with fungi or bacteria such as Aspergillus and
Lactobacillus.
[0032] In cases where the animal milk casein hydrolysate or the
concentrate thereof, or the fermented product obtained by
fermenting the raw material containing the milk protein with the
bacterium belonging to the species Lactobacillus helveticus,
besides the tripeptide Xaa Pro Pro which is the active ingredient
according to the present invention, free amino acids may be
contained. Furthermore, in addition to the above described peptide
and free amino acids, for example, lipids, ash, carbohydrates,
dietary fibers, water and the like, all of which are normally
contained in commercially available animal milk caseins or milk
proteins may be contained. Additionally, as required, a part or all
of the appropriate components among these may be taken out.
[0033] The active ingredient Xaa Pro Pro according to the present
invention may be originated from an animal milk casein hydrolysate
or a concentrate thereof obtained by a method of hydrolyzing an
animal milk casein with a group of enzymes yielding Xaa Pro Pro, in
particular Val Pro Pro and Ile Pro Pro and/or a method of
fermenting animal milk with Aspergillus.
[0034] Examples of the animal milk casein include cow milk, horse
milk, goat milk, and ewe milk. In particular, cow milk casein being
preferably used.
[0035] A concentration of casein when the animal milk casein is
hydrolyzed or fermented is not restricted but is preferably 1 to
19% by weight in order to efficiently produce the animal milk
casein degraded product.
[0036] An example of the above mentioned enzyme group is preferably
an enzyme group (X) including a peptidase capable of cleaving
between Pro and Xaa residues at the carboxyl terminus of the Xaa
Pro Pro Xaa sequence.
[0037] The enzyme group (X) is preferably a serine type proteinase
having serine in the active center or a metal proteinase having a
metal in the active center. Examples of the metal proteinase
include neutral protease I, neutral protease II and leucyl
aminopeptidase. It is preferred that at least one type of these
metal proteinases be additionally included in that the desired
hydrolysates can be efficiently obtained in a short time, and even
in a one-step reaction. Additionally, the peptidase capable of
cleaving the above described Pro Xaa sequence is preferably an
enzyme showing the isoelectric point in the acidic region.
[0038] An example of the above described enzyme group or enzyme
group (X) is an enzyme group originated from Aspergillus such as
Aspergillus oryzae. Such an enzyme group includes an enzyme group
which fungus cells were cultured in an appropriate culture medium
and an enzyme produced was extracted with water. Among the enzyme
groups originated from Aspergillus oryzae, an enzyme group showing
the isoelectric point in the acidic region is in particular
preferred.
[0039] As the enzyme group originated from Aspergillus oryzae,
commercially available products can be used. Examples thereof are
Sumizyme FP, LP or MP(all registered trademark, manufactured by
SHINNIHON CHEMICALS Corporation), Umamizyme (registered trademark,
manufactured by Amano Enzyme Inc.), Sternzyme B 11024, PROHIDROXY
AMPL(all trade names, manufactured by Higuchi Shoukai Co., Ltd.),
Orientase ONS (registered trademark, manufactured by Hankyu
Bioindustry CO., LTD), Denatyme AP (registered trademark,
manufactured by Nagase ChemteX Corporation) with Sumizyme FP
(registered trademark, SHINNIHON CHEMICALS Corporation) being
preferably used.
[0040] When these commercially available enzyme groups are used,
optimum conditions are usually pre-determined. Yet, conditions such
as an amount of enzyme to be used and reaction time can be
appropriately altered depending on an enzyme group to be used such
that the above described casein hydrolysate can be obtained.
[0041] An amount of the enzyme group to be added when the above
described animal milk casein is hydrolyzed is, for instance, such
that the weight ratio of enzyme group/animal milk casein in a
aqueous solution in which the animal milk casein is dissolved is
not less than 1/1000, preferably 1/1000 to 1/10, especially
preferably 1/100 to 1/10, further preferably 1/40 to 1/10.
[0042] The reaction conditions can be appropriately selected
depending on an enzyme group to be used such that an intended
casein hydrolysate is obtained. A temperature is usually 25 to
60.degree. C., preferably 45 to 55.degree. C. And pH is 3 to 10,
preferably 5 to 9, particularly preferably 5 to 8. The enzyme
reaction time is usually 2 to 48 hours, preferably 7 to 15
hours.
[0043] Termination of the enzyme reaction can be achieved by
inactivating the enzyme. Usually the enzyme is inactivated at 60 to
110.degree. C. to stop the reaction.
[0044] After the enzyme reaction is terminated, as required, it is
preferred to remove precipitates by centrifugation removal and
various filter treatments.
[0045] Additionally, as required, a peptide having bitterness
and/or smell can be removed from the obtained hydrolysate. The
removal of such a bitterness component and/or smell component can
be done using activated charcoals, hydrophobic resins or the like.
For instance, the removal can be carried out by adding the
activated charcoals to the obtained hydrolysate in 1 to 20% by
weight based on an amount of casein used and by allowing to react
the resulting mixture for 1 to 10 hours. Removal of the activated
charcoals can be carried out by a known method such as
centrifugation and membrane treatment process.
[0046] The thus obtained reaction mixture containing the animal
milk casein hydrolysate or the concentrate thereof can be added as
is to a liquid product such as beverage to be use in the functional
foods. In order to improve versatility of the animal milk casein
hydrolysate, the above described reaction mixture can be, after
concentrated, dried to yield a powdered form.
[0047] The content ratio of Xaa Pro Pro contained in the animal
milk casein hydrolysate or the concentrate thereof is usually not
less than 1% by weight, preferably 1 to 5% by weight based on a
total amount of peptides and free amino acids in the animal milk
casein hydrolysate or the concentrate thereof. By having the
content ratio of not less than 1% by weight, higher actions are
expected. Additionally, either when the content ratio of each of
Ile Pro Pro or Val Pro Pro contained in the animal milk casein
hydrolysate or the concentrate thereof is not less than 0.3% by
weight based on a total amount of peptides and free amino acids in
the animal milk casein hydrolysate or the concentrate thereof, or
when the content ration of a total of Ile Pro Pro and Val Pro Pro
is not less than 0.3% by weight, high effects are expected.
Further, when 0.3% by weight or more of each of Ile Pro Pro and Val
Pro Pro is contained, higher effects are expected.
[0048] The active ingredient Xaa Pro Pro according to the present
invention may be also originated from a fermented product obtained
by fermenting a raw material containing a milk protein with a
bacterium belonging to the species Lactobacillus helveticus.
Although the bacterium belonging to the species Lactobacillus
helveticus is preferably used individually in the fermentation,
other Lactobacillus or the like may be included/contained to the
extent that the desired effects according to the present invention
is not adversely affected.
[0049] As the bacterium belonging to the species Lactobacillus
helveticus, a proteinase-producing bacterium capable of highly
producing Ile Pro Pro and/or Val Pro Pro is preferred. For
instance, a bacterium strain showing a U/OD590 value measured in
accordance with a method by Yamamoto et al. (Yamamoto, N. et al. J.
Biochem.) (1993) 114, 740), which is based on a method by Twining
et al. (Twining, S. Anal. Biochem.) 143 3410 (1984)), of not less
than 400 is preferred.
[0050] An example of such a preferred bacterium strain is
Lactobacillus helveticus CM4 strain (Ministry of International
Trade and Industry, National Institute of Bioscience and
Human-Technology, 1-1-3 Higashi, Tsukuba, Ibaraki, Japan, Zip code
305, (currently, International Patent Organism Depositary, National
Institute of Advanced Industrial Science and Technology, AIST,
Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan, Zip code
305-8566), Accession No.: FERM BP-6060, Date of deposit: Aug. 15,
1997) (hereinafter referred to as CM4 strain). CM4 strain has been
registered under the above described accession number under the
Budapest Treaty concerning an international approval of
microorganism depository on patent procedures, and already
patented.
[0051] The fermented product obtained by fermenting a raw material
containing milk protein with the bacterium belonging to the species
Lactobacillus helveticus can be obtained by adding a fermented milk
starter containing the bacterium strain belonging to the species
Lactobacillus helveticus to a raw material containing the milk
protein and by fermenting the mixture on appropriately selected
conditions such as fermentation temperature.
[0052] A concentrate of the thus obtained fermented product or the
like may be powdered with freeze drying, spray drying, or the like
and may be used as a powder.
[0053] The bacterium belonging to the species Lactobacillus
helveticus is preferably used as a starter with sufficiently high
activities, which is preliminarily pre-cultured. The number of the
bacteria at the beginning is preferably about 10.sup.5 to 10.sup.9
cells/ml.
[0054] The fermented product obtained by fermenting a raw material
containing a milk protein with the bacterium belonging to the
species Lactobacillus helveticus, for example, when used for
functional foods such as Foods for Specified Health Uses, can be
also fermented with yeast in conjunction with the above described
bacterium strain belonging to the species Lactobacillus helveticus
in order to have better flavor and better palatability. An example
of the strain of yeast is preferably the genus Saccharomyces such
as Saccharomyces cerevisiae but not restricted thereto. The content
ratio of the yeast can be appropriately selected depending on a
purpose.
[0055] Examples of the raw material containing the milk protein
include animal milk such as cow milk, horse milk, ewe milk, goat
milk, vegetable milk such as soybean milk, and processed milk of
these milk including skimmed milk, reconstituted milk, powdered
milk, and condensed milk. Cow milk, soybean milk and processed milk
of these milk is preferred and cow milk or processed milk thereof
is in particular preferred.
[0056] Although the solid content concentration of the milk is not
particularly restricted, when the skimmed milk is for instance
used, the solid content concentration of non-fat milk is usually
about 3 to 15% by weight and preferably 6 to 15% by weight for
better productivity.
[0057] The above described fermentation is usually carried out by
standing or stirring fermentation, for example, by a method of
fermenting at a temperature of 25 to 45.degree. C., preferably 30
to 45.degree. C., with a fermentation time of 3 to 72 hours,
preferably 12 to 36 hours, wherein the fermentation is terminated
when lactic acid acidity reaches 1.5% or more.
[0058] The content ratio of Xaa Pro Pro, preferably Ile Pro Pro
and/or Val Pro Pro in the fermented product obtained by fermenting
the raw material containing the milk protein with the bacterium
belonging to the species Lactobacillus helveticus is preferably 10
mg or more, preferably 15 mg or more, in terms of 100 g of a freeze
dried product of the fermented product.
[0059] As for a dose or intake per day of the agent for preventing
kidney glomerular disease and renal arteriosclerosis according to
the present invention, in the case of human, usually a dose of
about 10 .mu.g to 10 g. preferably 1 mg to 5 g, further preferably
3 mg to 1 g of the active ingredient Xaa Pro Pro, preferably Val
Pro Pro and/or Ile Pro Pro may be administrated or taken dividedly
in several times in a day.
[0060] A period of administration or intake of the agent for
preventing kidney glomerular disease and renal arteriosclerosis can
be adjusted in many ways in view of age of humans or animals to be
administrated or to ingest and/or environments for a risk factor
against kidney glomerular disease and renal arteriosclerosis of the
humans or animals. The period can be for example usually not less
than one day, preferably 7 days to 365 days.
[0061] The prophylactic agent for renal failure according to the
present invention comprises the above described tripeptide as an
active ingredient.
[0062] As for a dose or intake per day of the prophylactic agent
for renal failure according to the present invention, in the case
of human, usually a dose of about 10 .mu.g to 10 g. preferably 1 mg
to 5 g, further preferably 3 mg to 1 g of the active ingredient Xaa
Pro Pro, preferably Val Pro Pro and/or Ile Pro Pro may be
administrated or taken dividedly in several times in a day.
[0063] A period of administration or intake of the prophylactic
agent for renal failure can be adjusted in many ways in view of age
of human or animals to be administrated or ingest and/or
environments for a risk factor against renal failure of the human
or animals. The period can be for example usually 1 day or more,
preferably 7 days to 365 days.
[0064] A method for administrating or taking in the agent for
preventing kidney glomerular disease and renal arteriosclerosis, as
well as a prophylactic agent for renal failure according to the
present invention is preferably oral administration.
[0065] A form of the agent for preventing kidney glomerular disease
and renal arteriosclerosis, as well as a prophylactic agent for
renal failure according to the present invention, when used as a
pharmaceutical, can be a form of formulation for oral
administration. Examples thereof include tablet, pill, hard
capsules, soft capsules, microcapsule, powder, pellet, and
solution.
[0066] In the case of preparing as a pharmaceutical, for example,
pharmaceutically acceptable carrier, adjuvant, vehicle, excipient,
antiseptics, stabilizing agent, binder, pH adjusting agent, buffer
agent, thickener, gelatinizer, preservatives, anti-oxidant or the
like can be, as required, used. The pharmaceutical can be produced
in a unit dose form required pharmaceutical formulation
administration generally accepted.
[0067] The foods according to the present invention comprises the
agent for preventing kidney glomerular disease and renal
arteriosclerosis according to the present invention as an active
ingredient, and can be, for example, functional foods, such as
Foods for Specified Health Uses, which has efficacies such as the
suppressive action against kidney glomerular disease and renal
arteriosclerosis and prophylaxis for renal failure.
[0068] An intake to attain such efficacies, for example, in the
light of the fact that the functional foods are daily, continuously
or intermittently taken for a long period of time, in the case of
human, is usually about 10 .mu.g to 10 g per day in terms of an
amount of the active ingredient Xaa Pro Pro or an amount of Val Pro
Pro and/or Ile Pro Pro, preferably 1 mg to 5 g, more preferably 3
mg to 1 g. Depending on the number of intake per day, an intake of
the foods such as the functional foods per once can be reduced
further to a lower amount than the above described amount.
[0069] In cases where the animal milk casein hydrolysate or the
concentrate containing the active ingredient Xaa Pro Pro is used as
is, in the case of human, the hydrolysate or the concentrate
thereof is preferably taken usually in 1 mg to 100 g per day,
particularly in about 100 mg to 10 g.
[0070] In cases where the freeze dried product of the fermented
product containing the active ingredient Xaa Pro Pro is used as is,
in the case of human, usually 1 to 100 g per day in terms of a
dried amount of the freeze dried product of the fermented product,
in particular, about 2 to 50 g is preferably taken.
[0071] A period of the intake of the foods according to the present
invention, for example the functional foods, is not particularly
restricted. The intake for a long period of time is preferred. In
order to attain the above described efficacies, for example, the
period can be usually not less than one day, preferably 7 days to
365 days.
[0072] The foods according to the present invention, for example
the functional foods, comprise the agent for preventing kidney
glomerular disease and renal arteriosclerosis containing the active
ingredient Xaa Pro Pro, preferably Val Pro Pro and/or Ile Pro Pro.
For instance, the animal milk casein hydrolysate or the concentrate
thereof and the fermented product, all of which were obtained in
the above described manner, can be added to a variety of foods as
they are or in the form of powder or granule. If necessary, a
fermented product by Lactobacillus other than Lactobacillus
helveticus, other ingredients used in foods including sugars,
proteins, lipids, vitamins, minerals, or flavors, or additives such
as various carbohydrates, lipids, vitamins, minerals, sweeteners,
flavors, coloring agents, texture improving agents or a mixture
thereof may be added to improve nutritional balance, flavor, and/or
the like.
[0073] The foods according to the present invention, for example
the functional foods, can be any form of solids, gels, or liquids.
Examples thereof include fermented dairy products such as
lactobacillus beverages, a variety of processed foods and
beverages, dried powders, tablets, capsules, and granule. Further,
additional examples can be various beverage, yogurts, fluid diets,
jerrys, candies, retort-packed foods, tablet candies, cookies,
Castella (or Japanese sponge cake), breads, biscuits, and
chocolates.
[0074] The present invention will now be described in detail by way
of examples thereof, but the scope of the present invention is by
no means limited by the examples.
EXAMPLES
[0075] [Example of Peptide Synthesis]
[0076] The active ingredient according to the present invention Ile
Pro Pro and Val Pro Pro were synthesized by the following chemical
synthesizing method (Fmoc method). The synthesis was carried out by
solid phase method using an automatic peptide synthesizer
(PSSM-8type) manufactured by Shimadzu Corporation.
[0077] As a solid phase carrier, 50 mg of a resin (SynProPep
Resin.RTM., manufactured by Shimadzu Corporation) which is a
2-chlorotrityl(2-Chlorotrityl)type polystyrene resin in which
proline protected its amino group with a fluorenylmethyloxy
carbonyl group (hereinafter referred to as Fmoc for short) is
bound, was used. According to the above described amino acid
sequence, Fmoc-Ile, Fmoc-Pro, and Fmoc-Val (100 .mu.mol each) which
are protected their amino group with the Fmoc group were reacted in
the order of the peptide sequence to obtain a peptide-bound resin
in accordance with a conventional method.
[0078] This peptide-bound resin was then suspended in 1 ml of a
reaction mixture A (10 volume % acetic acid, 10 volume %
trifluoroethanol, 80 volume % dichloromethane), allowed to react at
room temperature for 30 to 60 minutes. Thereafter the peptides were
separated from the resin and the reaction mixture A was filtered
with a glass filter. Immediately after the solvent in the filtrate
was removed under reduced pressure, 1 ml of reaction mixture B
(82.5 volume % trifluoroacetic acid, 3 volume % ethyl methyl
sulfide, 5 volume % purified water, 5 volume % thioanisole, 2.5
volume % ethanedithiol, 2 volume % thiophenol) was added and the
mixture was allowed to react at room temperature for six hours to
uncouple the side chain protecting group. To the resultant 10 ml of
anhydrous ether was added to precipitate the peptide, and
centrifuged at 3000 revolutions for five minutes to separate. The
precipitate was washed with anhydrous ether was several times and
then dried by spraying nitrogen gas. The entire volume of the thus
obtained unpurified synthetic peptide was dissolved in 2 ml of 0.1N
hydrochloric acid aqueous solution and then HPLC using a C18
reverse phase column was carried out in accordance with the
conditions below.
[0079] Pump: Type L6200 intelligent pump (Hitachi, Ltd); detector:
Type L4000UV detector (Hitachi, Ltd) for detection of ultra-violet
absorption at 215 nm; column: Micro Bondashere5.mu.C18 (Waters
Corp.); eluting solutions: solution A; 0.1% by weight TFA aqueous
solution, solution B; acetonitrile with 0.1% by weight TFA aqueous
solution; (B/A+B).times.100(%): 0.fwdarw.40% (60 minutes); and flow
rate: 1 ml/minute. The eluted fraction showing the maximum
absorbance was collected and then freeze-dried to obtain the
intended synthetic peptide Ile Pro Pro and Val Pro Pro (5.7 mg and
6.5 mg, respectively). The purified peptide was analyzed using an
automatic protein primary structure analyzer (Type PPSQ-10,
manufactured by Shimadzu Corporation) from the N-terminal of the
peptide. The purified peptide was further analyzed an amino acid
analyzer (Type 800 series, manufactured by JASCO corporation) to
confirm that the peptide was the one as designed.
[0080] [Example of Preparation of Animal Milk Casein
Hydrolysates]
[0081] To Casein originated from cow milk (Nippon NZMP Ltd.) (1 g)
99 g of distilled water adjusted to about 80.degree. C. was added
and the mixture was stirred well. Subsequently, IN sodium hydroxide
(manufactured by Wako Pure Chemical Industries, Ltd.) solution was
added. The mixture was then adjusted to pH 7.0 and a temperature of
20.degree. C. to prepare a substrate solution.
[0082] To the obtained substrate solution a commercially available
enzyme (registered trademark "Sumizyme FP", manufactured by
SHINNIHON CHEMICALS Corporation) was added, which was derived from
Aspergillus oryzae and contains at least metal protease, serine
protease, neutral protease I, neutral protease II and leucyl
aminopeptidase such that the enzyme/casein weight ratio of 1/25 was
attained. The mixture was allowed to react at 50.degree. C. for 14
hours. Subsequently, the enzymes were inactivated by autoclave at
110.degree. C. for ten minutes, thereby obtaining a casein
enzymolytic product solution. Then, the obtained enzymolytic
product solution was dried by spray drying to prepare a powder.
[0083] Components contained in the obtained powder were analyzed.
Proteins were determined by Kjeldahi method and amino acids were
measured by an amino acid analyzer. In addition, a difference after
subtracting an amount of the amino acids from an amount of the
proteins was defined as an amount of peptides. Further, lipids, ash
content and water were determined by acid decomposition method,
direct ashing method, and drying method by heating under
atmospheric pressure, respectively. The remainder after subtracting
the amount of each component from 100% was defined as an amount of
carbohydrates. The results showed that the powder contained 35.8%
by weight amino acids, 45.7% by weight peptides, 6.6% by weight
water contents, 0.2% by weight lipids, 4.1% by weight ash contents
and 7.6% by weight carbohydrates.
[0084] <Measurement of Amino Acids Composing the Peptide>
[0085] The powder prepared above was dissolved in an appropriate
amount of distilled water and analyzed with an automatic peptide
analyzer (trade name PPSQ-10 manufactured by Shimadzu Corporation)
to check in what order amino acid is located from the N-terminal in
the powder. The automatic peptide analyzer does not detect any free
amino acids.
[0086] The total amount of the amino acid located at the 5th
residue position was 120 pmol and the total amount of the amino
acid located at the 6th residue position was 100 pmol. Based on
these results, most peptides in the above described powder were
found to be dipeptides or tripeptides. Additionally, a proportion
of the peptides having Pro as the amino acid at the 2nd residue
position markedly increased to 49.5%. A proportion of the peptides
having Pro as the amino acid at the 3rd residue position was as
high as 29.8%.
[0087] Hence the above described powder contains many tripeptides
of Xaa Pro Pro. Thus these peptides were presumably a peptide
highly resistant to enzymatic degradation actions of proteases in
living bodies.
[0088] <Measurement of Peptides Contained in Enzymolytic
Products>
[0089] For the above described powder of the enzymolytic product,
amounts of the tripeptides shown in Table 1 contained in the powder
was determined, in accordance with a conventional method, using
various chemically synthesized standard peptides. Results are shown
in Table 1.
TABLE-US-00001 TABLE 1 Amount of tripeptide (.mu.g/ml) Peptide
sequence in 10 mg/ml of powder Ser Pro Pro 2.9 Val Pro Pro 29.5 Ile
Pro Pro 28.1 Phe Pro Pro 27.2 Other Xaa Pro Pro 28.8
[0090] The amount of peptides and free amino acids in a solution in
which the above described powder was dissolved and diluted in
distilled water was 8.15 mg/ml, and an amount of the peptides was
4.57 mg/ml, an amount of Xaa Pro in the peptides was 514.5 .mu.g.
The proportion of Xaa Pro based on a total amount of the peptides
and free amino acids in the powder was therefore 6.3% by weight.
Further, an amount of Xaa Pro Pro in the peptide was 116.5 .mu.g
and it was thus confirmed a proportion of Xaa Pro Pro based on a
total amount of the peptides and free amino acids in the powder was
1.4% by weight.
[0091] [Example of Preparation of CM4 Fermented Milk Animal
Feeds]
[0092] Using fermented milk obtained by fermenting a raw material
containing a milk protein with the CM4 strain, animal feeds
containing the active ingredient Xaa Pro Pro according to the
present invention was prepared.
[0093] Commercially available powdered non-fat milk was dissolved
in distilled water to the solid content of 9% (w/w) and then
autoclaved at 105.degree. C. for ten minutes to sterilize under
heating at high temperature. After cooled to room temperature, the
resulting solution was inoculated with a CM4 strain starter
fermentation solution (the number of the bacteria:
5.times.10.sup.8/ml) at 3% (v/w) and left to stand at 37.degree. C.
for 24 hours to yield CM4 fermented milk.
[0094] The obtained CM4 fermented milk was sterilized at a reaching
temperature of 80.degree. C. and then freeze-dried to yield the
powder. The obtained freeze dried powder was mixed with
commercially available powdered feeds (trade name "CE-2",
manufactured by CLEA Japan, Inc.) at the mass ratio of 10:90. The
mixture was formed into solid feeds to obtain CM4 fermented milk
feeds. The feeds contained 34.1 mg/kg of Val Pro Pro and 17.1 mg/kg
of Ile Pro Pro, both of which peptides were originated from the CM4
fermented milk.
[0095] [Test1: Effect of Val Pro Pro (VPP) to Suppress Kidney
Glomerular Disease and Renal Arteriosclerosis]
[0096] In order to evaluate the effect of the tripeptide Val Pro
Pro (VPP) to suppress kidney glomerular disease and renal
arteriosclerosis, a test on the frequency of occurrence of renal
glomerular lesions and the degree of thickening of the small and
middle renal artrey wall was carried out.
[0097] The test was carried out using three groups of male Wistar
rats of seven weeks old (Japan SLC, Inc.), each of which group
consist of 9 to 12 rats. After acclimation for a week, the animals
received: water with L-NAME (manufactured by Sigma) being dissolved
at a concentration of 1 g/L; water with L-NAME and VPP being
dissolved at a concentration of 1 g/L and 0.3 g/L respectively; or
water with L-NAME and angiotensin converting enzyme (ACE)
inhibitor, Enalapril at a concentration of 1 g/L and 0.5 mg/L
respectively, ad libitum for eight weeks. The dose of Enalapril was
determined such that its activity to inhibit ACE was equivalent to
VPP's.
[0098] The rats were sacrificed by exsanguination under
diethylether anesthesia. The kidney was enucleated and then fixed
with 10% neutral buffered formalin solution. Tissue samples were
prepared from the fixed kidney by excising in parallel with the
minor axis such that the tip of renal papilla was included. The
specimen were embedded in paraffin and cut into thin sections with
a thickness of 2.0 to 2.5 .mu.m using a microtome. Two to five
sections per rat were prepared. The thin sliced sections were
subjected to hematoxylin eosin staining or periodic acid
methenamine silver staining (PAM staining). In accordance with a
method by Foglieni et al. (Chiara Foglieni, et al., "Protective
effect of EDTA preadministration on renal ischemia" BMC Nephrology
2006, 7:5, [online], Mar. 15, 2006, BioMed Central Ltd,
http://www.biomedcentral.com/bmcnephrol/), a histopathological
examination was, with keeping the name of each sample concealed,
carried out by a pathologist who was not involved in the test.
[0099] Under a microscopic observation, the glomerulus that
atrophic lesions, deterioration of the glomerulus,
glomerulosclerosis, alteration of Bowman's capsule or the like was
observed was defined as the glomerulus with lesions, in accordance
with the definition of the glomerular lesions described in a book
by Enomoto et al. (edited by Makoto Enomoto, Yuzo Hayashi, and
Hisako Tanaka, "Pathological tissues of experimental animals",
Softscience, pages 380-396). The atrophic lesion refers to
conditions where the size ratio between the glomerulus and Bowman's
capsule is altered due to glomerular atrophy. The deterioration of
the glomerulus refers to conditions where the blood capillary
cavity of the glomerulus is disappeared and glomerular functions
are lost. The glomerulosclerosis refers to conditions where
increased mesangial matrix was observed without accompanying
hyperplasia of collagen fiber. The alteration of Bowman's capsule
refers to conditions where hypertrophy of epithelial cells and
uptake of lipid droplets and/or protein particles are observed. In
one whole section, the frequency of occurrence of these glomerular
lesions was scored from 0 to 3, and a score was determined for each
individual rat. The thus determined score for each individual rat
was subjected to statistical analysis and a score for each group
was determined.
[0100] Score 0: A case where one or two glomerular lesions owing to
physiologic alterations were observed.
[0101] Score 1: A case where one or two focal regions in the
glomerulus were observed, which regions were a limited and small
range.
[0102] Score 2: A case where two to four focal regions in the
glomerulus were observed, which regions were a middle sized
range.
[0103] Score 3: A case where five or more focal regions in the
glomerulus were observed in a wide range.
[0104] The focal regions herein indicate that a plural of lesions
are, on the basis of pathological findings, observed in a certain
area in one whole section.
[0105] The thickening of the small-, and mid -sized artery wall
existing in the renal cortex was also evaluated by a tissue
pathological test in the same manner as the observation of the
glomerular lesions. Under a microscopic observation, all arteries
observed in one whole section were compared with arteries observed
at the virtually same location in the specimen from a normal rat of
the same age in weeks. The thickness of the small- and mid-sized
artery wall was scored from 0 to 3 as described below and a score
for each individual rat was determined. The determined score for
each individual rat was subjected to statistical analysis and a
score for each group was determined.
[0106] Score 0: A case where only the thickening equal to the
normal rat in one whole section was observed.
[0107] Score 1: A case where the thickening was observed in a mild
degree or at low frequency, compared with the normal rat.
[0108] Score 2: A case where the thickening was observed in a
moderate degree or at moderate frequency, compared with the normal
rat.
[0109] Score 3: A case where the thickening was observed a high
degree or at high frequency, compared with the normal rat.
[0110] The obtained results are shown in FIG. 1 and FIG. 2. As
shown in FIG. 1, compared with the group of rats fed with L-NAME
alone, the group of rats fed with VPP showed a decrease in the
degree of thickening of the small- and mid-sized artery wall, which
indicates that VPP has the effect to suppress renal
arteriosclerosis. Further, as shown in FIG. 2, the intake of VPP
resulted in suppression of the frequency of occurrence of the
glomerular lesions, indicating the effect of VPP to suppress kidney
glomerular diseases. Accordingly, since VPP suppresses the
thickening of the small- and mid-sized artery wall as well as
occurrence of the glomerular lesions VPP is proven to be effective
on the prophylaxis of renal failure. Judging from the fact that
Enalapril did not show the suppressive effect against either renal
arteriosclerosis or glomerular diseases, it was confirmed that the
effect of VPP was not dependent on the ACE inhibition
[0111] [Test 2: Effect of Ile Pro Pro (IPP) to Suppress Kidney
Glomerular Disease and Renal Arteriosclerosis]
[0112] The tripeptide Ile Pro Pro (IPP) was also tested for renal
pathologic state, in order to evaluate its effect to suppress
kidney glomerular disease and renal arteriosclerosis.
[0113] The test was carried out using three groups of male Wistar
rats of seven weeks old (Japan SLC, Inc.), each of which group
consist of 9 to 12 rats. After habituation breeding for a week,
drinking water with L-NAME (manufactured by Sigma) being dissolved
at a concentration of 1 g/L; drinking water with L-NAME and IPP
being dissolved at a concentration of 1 g/L and 0.3 g/L
respectively; drinking water with L-NAME and angiotensin converting
enzyme (ACE) inhibitor, Enalapril at a concentration of 1 g/L and
0.5 mg/L respectively, were supplied ad libitum for eight weeks.
The dose of Enalapril was determined such that its activity to
inhibit ACE was equivalent to the activity of IPP to inhibit
ACE.
[0114] The rats were sacrificed by exsanguination under
diethylether anesthesia. The kidney was enucleated and then fixed
with 10% neutral buffered formalin solution. Tissue samples were
prepared from the fixed kidney by excising in parallel with the
minor axis such that the tip of renal papilla was included. The
specimen were embedded in paraffin and cut into thin sections with
a thickness of 2.0 to 2.5 .mu.m using a microtome. Two to five
sections per rat were prepared. The thin sliced sections were
subjected to hematoxylin eosin staining or periodic acid
methenamine silver staining (PAM staining). In accordance with a
method by Foglieni et al. (Chiara Foglieni, et al., "Protective
effect of EDTA preadministration on renal ischemia" BMC Nephrology
2006, 7:5, [online], Mar. 15, 2006, BioMed Central Ltd,
http://www.biomedcentral.com/bmcnephrol/), a histopathological
examination was, with keeping the name of each sample concealed,
carried out by a pathologist who was not involved in the test.
[0115] Under a microscopic observation, the glomerulus that
atrophic lesions, deterioration of the glomerulus,
glomerulosclerosis, alteration of Bowman's capsule or the like was
observed was defined as the glomerulus with lesions, in accordance
with the definition of the glomerular lesions described in a book
by Enomoto et al. (edited by Makoto Enomoto, Yuzo Hayashi, and
Hisako Tanaka, "Pathological tissues of experimental animals",
Softscience, pages 380-396). The atrophic lesion refers to
conditions where the size ratio between the glomerulus and Bowman's
capsule is altered due to glomerular atrophy. The deterioration of
the glomerulus refers to conditions where the blood capillary
cavity of the glomerulus is disappeared and glomerular functions
are lost. The glomerulosclerosis refers to conditions where
increased mesangial matrix was observed without accompanying
hyperplasia of collagen fiber. The alteration of Bowman's capsule
refers to conditions where hypertrophy of epithelial cells and
uptake of lipid droplets and/or protein particles are observed. In
one whole section, the frequency of occurrence of these glomerular
lesions was scored from 0 to 3, and a score was determined for each
individual rat.
[0116] Score 0: A case where one or two glomerular lesions owing to
physiologic alterations were observed.
[0117] Score 1: A case where one or two focal regions in the
glomerulus were observed, which regions were a limited and small
range.
[0118] Score 2: A case where two to four focal regions in the
glomerulus were observed, which regions were a middle sized
range.
[0119] Score 3: A case where not less than five focal regions in
the glomerulus were observed in a wide range.
[0120] The focal regions herein indicate that a plural of lesions
are, on the basis of pathological findings, observed in a certain
area in one whole section.
[0121] The thickening of the renal artery wall at the hilum
existing in the renal medulla and the thickening of the small and
middle artery wall existing in the renal cortex were also evaluated
by a tissue pathological test in the same manner as the observation
of the glomerular lesions. Under a microscopic observation, all
arteries observed in one whole section were compared with arteries
observed at the virtually same location in the specimen from a
normal rat of the same age in weeks. The thickening of the renal
artery wall at the hilum and the thickening of the small- and
mid-sized artery wall were scored from 0 to 3 as described below
and a score for each individual rat was determined. The determined
score for each individual rat was subjected to statistical analysis
and a score for each group was determined.
[0122] Score 0: A case where only the thickening equal to the
normal rat in one whole section was observed.
[0123] Score 1: A case where the thickening was observed in a mild
degree or at low frequency, compared with the normal rat.
[0124] Score 2: A case where the thickening was observed in a
moderate degree or at moderate frequency, compared with the normal
rat.
[0125] Score 3: A case where the thickening was observed a high
degree or at high frequency, compared with the normal rat.
[0126] These lesions of the glomerulus, the thickening of the renal
artery wall at the hilum and the thickening of the small- and
mid-sized artrey wall were individually scored. A rat with the
score of not less than 1 in all three items was defined as one
having the pathologic state. Using the number of the rats with and
without the pathologic state, comparisons by likelihood-ratio test
were made.
TABLE-US-00002 TABLE 2 Evaluation results on rat renal pathologic
state Administrated group With pathologic state Without pathologic
state L-NAME 4 rats 7 rats L-NAME and 0 rats 9 rats IPP (*) L-NAME
and 2 rats 7 rats Enalapril (*) p < 0.05
[0127] These results revealed that IPP also suppressed the onset of
the pathologic state of the glomerulus and the renal artery, and
thus IPP is effective on the prophylaxis of renal failure. In
addition, like in Test 1, Enalapril herein did not exhibit such an
effect. It was hence confirmed that the effect was not dependent on
the ACE inhibition.
[0128] Since the agent for preventing kidney glomerular diseases
and renal arteriosclerosis according to the present invention shows
effectiveness, even when angiotensin converting enzyme (ACE)
inhibitors do not exhibit the suppressive action, the agent is of
great use as a pharmaceutical for the prophylaxis and/or treatment
of renal failure, in particular as a prophylactic agent, which
agent is not dependent on the ACE inhibitory activity. In addition,
the active ingredient is a naturally occurring tripeptide
originated from foods and the like, it is expected that the
pharmaceutical with high efficacy and with little concern for side
effects is provided.
[0129] Furthermore, by using or adding the agent for preventing
kidney glomerular diseases and renal arteriosclerosis according to
the present invention in foods or beverages, the functional foods
expected to have the prophylactic effect for renal failure are
provided.
REFERENCES
[0130] 1. Japanese Patent No 2782142 [0131] 2. J. Clin. Invest. 77,
1993-2000, 1986 [0132] 3. Saishin Igaku, 48:1404 1409, 1993 [0133]
4. J. Dairy Sci. 1995, 78:777-783 [0134] 5. J. Dairy Sci. 1995,
78:1253-1257 [0135] 6. Am. J. Clin. Nutr. 1996, 64:767-771
Sequence CWU 1
1
114PRTArtificialrecognition sequence for peptidase 1Xaa Pro Pro
Xaa1
* * * * *
References