U.S. patent application number 12/567322 was filed with the patent office on 2010-01-28 for prophylactic agent for heart failure.
Invention is credited to Tatsuhiko HIROTA, Teppei Nakamura, Kohji Ohki, Toshiaki Takano.
Application Number | 20100022747 12/567322 |
Document ID | / |
Family ID | 39830596 |
Filed Date | 2010-01-28 |
United States Patent
Application |
20100022747 |
Kind Code |
A1 |
HIROTA; Tatsuhiko ; et
al. |
January 28, 2010 |
PROPHYLACTIC AGENT FOR HEART FAILURE
Abstract
The present invention provides an agent having a suppressive
action against the thickening of the heart wall, a prophylactic
agent for heart failure containing the above described agent, and
functional foods expected to have a prophylactic effect for heart
failure. The agent for suppressing heart wall thickening 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) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
39830596 |
Appl. No.: |
12/567322 |
Filed: |
September 25, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/JP2008/055072 |
Mar 19, 2008 |
|
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12567322 |
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Current U.S.
Class: |
530/331 |
Current CPC
Class: |
A23V 2002/00 20130101;
A23V 2002/00 20130101; A61K 38/06 20130101; A61P 1/16 20180101;
A23V 2002/00 20130101; A23V 2200/32 20130101; A23V 2200/32
20130101; A23V 2250/0626 20130101; A23V 2250/0654 20130101; A23V
2250/064 20130101; A23V 2200/326 20130101; A23V 2250/064 20130101;
A23V 2250/064 20130101; A23V 2200/326 20130101; A23V 2250/0654
20130101; A23V 2250/0626 20130101; A23V 2250/064 20130101; A23V
2002/00 20130101; A23V 2002/00 20130101; A61P 13/12 20180101; A23L
33/18 20160801 |
Class at
Publication: |
530/331 |
International
Class: |
C07K 5/08 20060101
C07K005/08; A61P 9/00 20060101 A61P009/00; A61P 9/04 20060101
A61P009/04 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 27, 2007 |
JP |
2007-081638 |
Claims
1. An agent for suppressing heart wall thickening, comprising Xaa
Pro Pro as an active ingredient.
2. The agent for suppressing heart wall thickening according to
claim 1, wherein said Xaa Pro Pro is Val Pro Pro and/or Ile Pro
Pro.
3. The agent for suppressing heart wall thickening according to
claim 1, wherein said Xaa Pro Pro is derived from an animal milk
casein hydrolysate or a concentrate thereof.
4. The agent for suppressing heart wall thickening according to
claim 3, wherein said animal milk casein hydrolysate is a fermented
product obtained by fermenting said animal milk casein with
Aspergillus.
5. The agent for suppressing heart wall thickening according to
claim 3 wherein said animal milk casein hydrolysate is a hydrolyzed
product obtained by degrading said animal milk casein with an
enzyme derived from Aspergillus.
6. The agent for suppressing heart wall thickening according to
claim 5, wherein said enzyme derived from said Aspergillus is an
enzyme derived from Aspergillus oryzae.
7. The agent for suppressing heart wall thickening 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 agent for suppressing heart wall thickening according to
claim 7, wherein said bacterium belonging to the species
Lactobacillus helveticus is Lactobacillus helveticus CM4 strain
(FERM BP-6060).
9. A prophylactic agent for cardiac hypertrophy comprising said
agent for suppressing heart wall thickening according to claim 1 as
an active ingredient.
10. A prophylactic agent for heart failure comprising said agent
for suppressing heart wall thickening according to claim 1 as an
active ingredient.
11. A functional food comprising said agent for suppressing heart
wall thickening according to claim 1.
12. The functional food according to claim 11, which indicates
efficacy for prophylaxis of heart failure.
13. The agent for suppressing heart wall thickening according to
claim 2, wherein said Xaa Pro Pro is derived from an animal milk
casein hydrolysate or a concentrate thereof.
14. The agent for suppressing heart wall thickening according to
claim 2, 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.
15. A prophylactic agent for cardiac hypertrophy comprising said
agent for suppressing heart wall thickening according to claim 2 as
an active ingredient.
16. A prophylactic agent for cardiac hypertrophy comprising said
agent for suppressing heart wall thickening according to claim 3 as
an active ingredient.
17. A prophylactic agent for cardiac hypertrophy comprising said
agent for suppressing heart wall thickening according to claim 4 as
an active ingredient.
18. A prophylactic agent for cardiac hypertrophy comprising said
agent for suppressing heart wall thickening according to claim 5 as
an active ingredient.
19. A prophylactic agent for cardiac hypertrophy comprising said
agent for suppressing heart wall thickening according to claim 6 as
an active ingredient.
20. A prophylactic agent for cardiac hypertrophy comprising said
agent for suppressing heart wall thickening according to claim 7 as
an active ingredient.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to an active ingredient having
a suppressive action on cardiac wall thickening, a prophylactic
agent for cardiac hypertrophy and a prophylactic agent for heart
failure containing the active ingredient, as well as functional
foods containing the active ingredient, the functional foods being
expected to have a prophylactic effect against cardiac hypertrophy
and heart failure.
[0002] Also, the present invention relates to a method for
suppressing cardiac wall thickening. Further, the present invention
relates to a method for preventing cardiac hypertrophy and heart
failure.
BACKGROUND OF THE INVENTION
[0003] Recently, the number of those who have a risk factor
considered to cause heart diseases, such as smoking, hypertension,
hyperglycemia, and hyperlipidemia, have increased in some
countries. There is an increasing trend in the number of patients
suffering from heart failure. In addition, these pathologic states
are not limited to human and the increasing trend in morbidity of
such diseases are seen among animals that closely interact with
human, such as dogs, cats, other companion animals and pet
animals.
[0004] Since the pathologic state, classification, and progression
of heart failure varies, the disease cannot be readily defined. Yet
it can be roughly divided into two types: systolic heart failure
and diastolic heart failure. In some cases, these two types
simultaneously occur. Systolic heart failure occurs when the heart
fails to contract normally. The heart can take in blood but cannot
fully pump out adequate blood due to weakened cardiac muscles. As a
result, the volume of the blood pumping out to the whole body and
lungs decreases and the heart, in particular the left ventricle,
can become hypertrophic. On the other hand, diastolic heart failure
occurs when the heart wall becomes too stiff to fill up the heart
with blood. As a result, blood dams up in the left atrium and lung
blood vessels, which could cause congestion. Thus, heart failure
generally refers to a condition that heart's pump functions become
impaired and hence the heart become incapable of pumping a
sufficient amount of blood. Heart failure could cause other changes
which further deteriorate functions of the heart, including a
decrease in blood volume and congestion of blood in the veins and
lungs.
[0005] Examples of primary diseases that may cause heart failure
include increased pressure overload by hypertension, aortostenosis
and the like, cardiac hypertrophy with heart wall thickening by
volume overload and the like by valvular disease. Meanwhile, after
the onset of myocardial infarction, heart wall thickening may be
developed in injury tissues as a spontaneous reaction for
functional recovery. Also, agnogenic myocardiopathy of unexplained
origins, such as myocardosis, may also cause heart wall
hypertrophy. Continuous mechanical load to the heart triggers a
gradual decrease in the contractile power of the cardiac muscles,
followed by reduction in cardiac performance, and eventually
progresses to ventricular arrhythmia, heart ischemia, coronary
artery disease, and congestive heart failure.
[0006] Considering the situation, a compound having the suppressive
action against heart wall thickening is useful for prophylaxis and
treatment of cardiac hypertrophy and heart failure.
[0007] As a method for improving heart wall thickening, in the case
of cardiac hypertrophy caused by hypertension and the like, for
instance, treating stem-cause hypertension can improve heart wall
thickening in some cases. However, the action is not satisfactory
and also some antihypertensive drugs have no effects (Am J.
Hypertens. 1997 August; 10(8):913.about.20). Also, even when
hypertension is improved, heart wall thickening remains unimproved
in many cases. In such cases there is still a risk for the onset of
heart failure.
[0008] Effective drugs for treating heart failure include
angiotensin converting enzyme inhibitors (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 inhibitors are
not effective for all cases. Rather there is a risk of developing
acute renal failure accompanying lowering blood pressure and
therefore 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.
[0009] 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 they have a suppressive action against heart wall
thickening or not has not been directly demonstrated (Japanese
Patent Publication No. 2782142, J. Dairy Sci. 1995, 78:777-78, 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 heart 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
[0010] The present invention provides a compound or composition
having a suppressive action against heart wall thickening
independently of the ACE inhibitory activity.
[0011] Further the present invention provides a prophylactic agent
for heart failure containing the above described compound or
composition.
[0012] In addition, the present invention provides functional foods
expected to have a prophylactic effect for heart failure containing
the above described compound or composition.
[0013] Further the present invention provides a method for
suppressing heart wall thickening, which method includes
administration of the above described compound or composition to a
subject.
[0014] In addition, the present invention provides a method for
preventing heart failure, which method includes administration of
the above described compound or composition to a subject.
[0015] The present invention is also a use of the above described
compound or composition in production of a pharmaceutical for
suppressing heart wall thickening. In particular, the present
invention is a use of the above described compound or composition
in production of a pharmaceutical for preventing cardiac
hypertrophy. Yet, the present invention is a use of the above
described compound or composition in producing a pharmaceutical for
preventing heart failure.
[0016] 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
heart wall thickening, and have reached to invent a pharmaceutical
and functional foods useful for prophylaxis of heart failure.
Concrete Contents of the Present Invention are as Follows:
[0017] The present invention provides an agent for suppressing
heart wall thickening containing Xaa Pro Pro as an active
ingredient.
[0018] The present invention also provides a prophylactic agent for
cardiac hypertrophy having the above described suppressive action
against heart wall thickening.
[0019] In addition, the present invention provides a prophylactic
agent for heart failure having the above described suppressive
action against heart wall thickening.
[0020] In addition, the present invention provides functional foods
containing a prophylactic agent for heart wall thickening.
[0021] The present invention provides a method for suppressing
heart wall thickening, which method includes administration of Xaa
Pro Pro or a composition containing Xaa Pro Pro to a subject.
[0022] In addition, the present invention also provides a method
for preventing cardiac hypertrophy, which method includes
administration of Xaa Pro Pro or a composition containing Xaa Pro
Pro to a subject.
[0023] In addition, the present invention provides a method for
preventing heart failure, which method includes administration of
Xaa Pro Pro or a composition containing Xaa Pro Pro to a
subject.
[0024] The present invention is also a use of Xaa Pro Pro or a
composition containing Xaa Pro Pro in producing a pharmaceutical
for suppressing heart wall thickening. 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 cardiac
hypertrophy. The present invention is also a use of Xaa Pro Pro or
a composition containing Xaa Pro Pro in producing a pharmaceutical
for preventing heart failure.
[0025] Preferably Xaa Pro Pro is Val Pro Pro and/or Ile Pro
Pro.
[0026] 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.
[0027] 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.
[0028] Preferably the bacterium belonging to the species
Lactobacillus helveticus is Lactobacillus helveticus CM4 strain
(FERM BP-6060).
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows the results of Test 1 examining the effect of
Val Pro Pro (VPP) to suppress rat left ventricle wall thickening.
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 one-sided t-test. The symbol (*) indicates statistical
significance (p<0.05).
[0030] FIG. 2 shows the results of Test 2 examining the effect of
Val Pro Pro (VPP) to suppress rat right ventricle wall thickening.
The test was conducted using the group of rats administered with
L-NAME (11 rats per group), the group of rats administrated 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 one-sided t-test. The symbol (*) indicates statistical
significance (p<0.05).
[0031] FIG. 3 shows the results of Test 3 examining the effect of
Ile Pro Pro (IPP) to suppress rat left ventricle wall thickening.
The test was conducted using the group of rats administered with
L-NAME (9 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 IPP (11 rats per group). The graph
represents mean.+-.standard error. Comparisons were demonstrated by
the one-sided t-test. The symbol (#) indicates statistical
significance (p<0.1).
[0032] FIG. 4 shows the results of Test 5 examining the effect of
Ile Pro Pro (IPP) to suppress rat heart septal wall thickening. The
test was conducted using the group of rats administrated with
L-NAME (9 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 IPP (11 rats per group). The graph
represents mean.+-.standard error. Comparisons were demonstrated by
the one-sided t-test. The symbol (*) indicates significant
difference (p<0.05).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0033] The agent for suppressing heart wall thickening 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 suppressing heart wall thickening" used in the present
invention refers to a compound or composition having an action to
suppress progress of lesions of heart wall thickening, an action to
improve lesions of heart wall thickening, or a prophylactic effect
for the onset of heart wall thickening.
[0034] 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.
[0035] The active ingredient Xaa Pro Pro may be an chemically
synthesized tripeptide or naturally occurring tripeptide.
[0036] As an chemical synthetic method of tripeptide Xaa Pro Pro, a
common method such as solid phase method (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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] Examples of the animal milk casein include cow milk, horse
milk, goat milk, and ewe milk. In particular, cow milk casein being
preferably used.
[0041] 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.
[0042] An example of 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.
[0043] 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.
[0044] 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 fungi 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.
[0045] 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 B11024, 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] After the enzyme reaction is terminated, as required, it is
preferred to remove precipitates by centrifugation removal and
various filter treatments.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] As for a dose or intake per day of the agent for suppressing
heart wall thickening 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.
[0066] A period of administration or intake of the agent for
suppressing heart wall thickening 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 for heart wall thickening of
the humans or animals. The period can be for example usually one
day or more, preferably 7 days to 365 days.
[0067] The prophylactic agent for heart failure according to the
present invention comprises the above described tripeptide as an
active ingredient.
[0068] As for a dose or intake per day of the prophylactic agent
for heart 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.
[0069] A period of administration or intake of the prophylactic
agent for heart 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 heart failure of the human
or animals. The period can be for example usually 1 day or more,
preferably 7 days to 365 days.
[0070] The prophylactic agent for cardiac hypertrophy according to
the present invention contains the above described tripeptide as an
active ingredient.
[0071] A dose or intake and period of administration or intake of
the prophylactic agent for cardiac hypertrophy according to the
present invention are same as those of the prophylactic agent for
heart failure according to the present invention.
[0072] A method of administration or intake of the agent for
suppressing heart wall thickening, prophylactic agent for cardiac
hypertrophy and prophylactic agent for heart failure according to
the present invention is preferably oral administration.
[0073] A form of the agent for suppressing heart wall thickening,
prophylactic agent for cardiac hypertrophy, prophylactic agent for
heart failure according to the present invention, when used as a
pharmaceutical, is a form of formulation for oral administration.
Examples thereof include tablet, pill, hard capsules, soft
capsules, microcapsule, powder, pellet, and solution.
[0074] 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.
[0075] The foods according to the present invention comprises the
agent for suppressing heart wall thickening 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 the action for suppressing heart wall
thickening, prophylaxis for cardiac hypertrophy, and prophylaxis
for heart failure.
[0076] 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 amount described
above.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] The foods according to the present invention, for example
functional foods, comprise the agent for suppressing heart wall
thickening 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.
[0081] 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,
jellys, candies, retort-packed foods, tablet candies, cookies,
Castella (or Japanese sponge cake), breads, biscuits, and
chocolates.
[0082] 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
Example of Peptide Synthesis
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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.
[Example of Preparation of Animal Milk Casein Hydrolysates]
[0087] 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, 1N 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.
[0088] 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.
[0089] 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.
<Measurement of Amino Acids Composing the Peptide>
[0090] 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.
[0091] 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%.
[0092] 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.
<Measurement of Peptides Contained in Enzymolytic
Products>
[0093] 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
[0094] 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.
[Example of Preparation of CM4 Fermented Milk Animal Feeds]
[0095] 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.
[0096] 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.
[0097] 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.
[Test 1: Effect of Val Pro Pro (VPP) to Suppress Left Ventricle
Wall Thickening]
[0098] The tripeptide Val Pro Pro (VPP) was tested for the effect
of suppressing the thickening of the left ventricle. The test was
carried out using three groups of male Wistar rats of seven weeks
old (Japan SLC, Inc.), each of which group consisted of 9 to 12
rats. After acclimation for a week, the animals were received:
water with a nitrogen monoxide synthesis inhibitor,
NG-nitro-L-arginine methylester hydrochloride (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 (AGE) inhibitor, Enalapril at a concentration of 1 g/L and
0.5 mg/L respectively, was supplied ad libitum for eight weeks. The
dose of Enalapril was determined such that its activity to inhibit
ACE was equivalent to VPP's.
[0099] The rats were sacrificed by exsanguination under
diethylether anesthesia. The heart was enucleated and then fixed
with 10% neutral buffered formalin solution. Annularly-shaped
tissue samples were prepared from the fixed heart by horizontally
excising the lower part of the atria such that the right ventricle,
septum, left ventricle, and coronary artery were included. The
specimen were embedded in paraffin and cut into thin sections with
a thickness of 3.0 to 3.5 .mu.m using a microtome. Two to five
sections per rat were prepared. The sliced thin sections were
stained with hematoxylin and eosin and the thickness of the left
ventricle wall was measured. As for the measurement of the
thickness of the ventricle wall, the thickness of a plural of the
points per one section, each of which point was considered to have
an average thickness, was actually measured with an ocular
micrometer under a microscope. The average thickness was defined as
the measured value of the section and subjected to statistical
analysis. The measurement and evaluation of the thickness was
carried out by a pathologist who was not informed of the name of
each sample.
[0100] The obtained results are shown in FIG. 1. Compared with the
group of rats fed with L-NAME alone, the group of rats fed with
L-NAME and VPP showed a decrease in the thickness of the left
ventricle wall, indicating that VPP suppressed the thickening of
the left ventricle wall and is thus effective in the prophylaxis of
heart failure. In addition, since Enalapril of which dose was set
so as to have an equivalent ACE inhibitory activity to VPP did not
exhibit the effect, it was confirmed that the effect did not depend
on the ACE inhibition.
[Test 2: Effect of Val Pro Pro (VPP) to Suppress Right Ventricle
Wall Thickening]
[0101] In order to check the effect of the tripeptide Val Pro Pro
(VPP) to suppress the thickening of the right ventricle wall as
well, a test was carried out in the same manner as Test 1 and
measurement and evaluation were made. The obtained results are
shown in FIG. 2. Compared with the group fed with L-NAME alone, the
group fed with L-NAME and VPP showed a decrease in the thickness of
the right ventricle wall. Hence, VPP also suppressed the thickening
of the right ventricle wall and was proven to be effective in the
prophylaxis of heart failure. Like in Test 1, Enalapril herein did
not show such an effect, it was confirmed that the effect did not
depend on the ACE inhibition.
[Test3: Effect of Ile Pro Pro (IPP) to Suppress Left Ventricle Wall
Thickening]
[0102] The tripeptide Ile Pro Pro (IPP) was also tested for the
suppressive effect against the thickening of the ventricle wall.
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 11 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 IPP 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 the activity of IPP to
inhibit ACE.
[0103] The rats were sacrificed by exsanguination under
diethylether anesthesia. The heart was enucleated and then fixed
with 10% neutral buffered formalin solution. Annularly-shaped
tissue samples were prepared from the fixed heart by horizontally
excising the lower part of the atria such that the right ventricle,
septum, left ventricle, and coronary artery were included. The
specimen were embedded in paraffin and cut into thin sections with
a thickness of 3.0 to 3.5 .mu.m using a microtome. Two to five
sections per rat were prepared. The sliced thin sections were
stained with hematoxylin and eosin and the thickness of the left
ventricle wall was measured. As for the measurement of the
thickness of the ventricle wall, the thickness of a plural of the
points for one section, each of which point was considered to have
an average thickness, was actually measured with an ocular
micrometer under a microscope. The average thickness was defined as
the measured value of the section and subjected to statistical
analysis. The measurement and evaluation of the thickness was
carried out by a pathologist who was not informed of the name of
each sample.
[0104] The obtained results are shown in FIG. 3. Compared with the
group of rats fed with L-NAME alone, the group of rats fed with
L-NAME and IPP showed a decrease in the thickness of the left
ventricle wall, indicating that IPP suppressed the thickening of
the left ventricle wall and is thus effective in the prophylaxis of
heart failure. In addition, since Enalapril, of which dose was set
so as to have an equivalent activity to IPP in terms of ACE
inhibition, did not exhibit such an effect, it was confirmed that
the effect did not depend on the ACE inhibition.
[Test 4: Effect of Ile Pro Pro (IPP) to Suppress Right Ventricle
Wall Thickening]
[0105] In order also to check the effect of the tripeptide Ile Pro
Pro (IPP) to suppress the thickening of the right ventricle wall, a
test was carried out in the same manner as in Test 3, and
measurement and evaluation were made.
[0106] 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 animal
received: water with L-NAME (manufactured by Sigma) being dissolved
at a concentration of 1 g/L; water with L-NAME and IPP 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
the activity of IPP to inhibit ACE.
[0107] The rats were sacrificed by exsanguination under
diethylether anesthesia. The heart was enucleated and then fixed
with 10% neutral buffered formalin solution. Annularly-shaped
tissue samples were prepared from the fixed heart by horizontally
excising the lower part of the atria such that the right ventricle,
septum, left ventricle, and coronary artery were included. The
specimen were embedded in paraffin and cut into thin sections with
a thickness of 3.0 to 3.5 .mu.m using a microtome. Two to five
sections per rat were prepared. Slices of the thin sections were
stained with hematoxylin and eosin and the thickness of the right
ventricle wall was measured. As for the measurement of the
thickness of the ventricle wall, the thickness of a plural of the
points for one section, each of which point was considered to have
an average thickness, was actually measured with an ocular
micrometer under a microscope. The average thickness was defined as
the measured value of the section and the thickness of the
ventricle wall of each individual was determined. The measurement
and evaluation of the thickness was carried out by a pathologist
who was not informed of the name of each sample.
[0108] The evaluation of the thickening of the ventricle wall
involved the measurement of the thickness of the ventricle wall of
six rats of the same age in weeks, which rats were fed with general
tap water with no L-NAME being added. The average of those was
defined as a reference value. In cases where the thickness of its
ventricle wall was larger than the reference value, a rat was
defined as one with the thickening, whereas in cases where the
thickness of its ventricle wall was equal to or smaller than the
reference value, a rat was considered to be normal. Using the
number of the rats with the thickening and the normal rats,
comparisons by likelihood-ratio test were made. The results were
shown in Table 2 below.
TABLE-US-00002 TABLE 2 Evaluation results of the thickening of rat
right ventricle wall Administrated group Thickening Positive Normal
L-NAME 5 rats 6 rats L-NAME and 0 rats 9 rats IPP (**) L-NAME and 1
rat.sup. 8 rats Enalapril (**) p < 0.01
[0109] The obtained results revealed that IPP also suppressed the
thickening of the right ventricle wall and thus IPP is also
effective on the prophylaxis of heart failure. In addition, since
Enalapril, of which dose was set so as to have an equivalent
activity to IPP in terms of ACE inhibition, did not exhibit such an
effect, it was confirmed that the effect did not depend on the ACE
inhibition.
[Test 5: Effect of Ile Pro Pro (IPP) to Suppress Heart Septal Wall
Thickening]
[0110] In order to check the effect of the tripeptide Ile Pro Pro
(IPP) to suppress the thickening of the heart septal wall as well,
a test was carried out in the same manner as Test 3 and measurement
and evaluation were made.
[0111] The obtained results are shown in FIG. 4. Compared with the
group fed with L-NAME alone, the group fed with L-NAME and IPP
showed a decrease in the thickness of the heart septal wall, thus
indicating that IPP suppressed the thickening of the heart septal
wall and was proven to be effective in the prophylaxis of heart
failure. In addition, since Enalapril, of which dose was set so as
to have an equivalent ACE inhibitory activity to IPP, did not
exhibit the effect, it was confirmed that the effect did not depend
on the ACE inhibition.
[0112] Since the agent for suppressing heart wall thickening
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 cardiac hypertrophy and
heart 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. Furthermore, by using or adding the agent for suppressing
heart wall thickening according to the present invention in foods
or beverages, the functional foods expected to have the
prophylactic effect for cardiac hypertrophy and heart failure are
provided.
REFERENCE
[0113] 1. Japanese Patent No. 2782142 [0114] 2. Am J. Hypertens.
1997 August; 10(8):913.about.20 [0115] 3. J. Clin. Invest. 77,
1993-2000, 1986 [0116] 4. Saishin Igaku 48:1404.about.1409, 1993
[0117] 5. J. Dairy Sci. 1995, 78:777-783 [0118] 6. J. Dairy Sci.
1995, 78:1253-1257 [0119] 7. Am. J. Clin. Nutr. 1996, 64:767-771
Sequence CWU 1
1
114PRTArtificialrecognition sequence for peptidase 1Xaa Pro Pro
Xaa1
* * * * *