Note: Descriptions are shown in the official language in which they were submitted.
CA 02477900 2004-08-31
DESCRIPTION
COMPOSITION FOR PREVENTING HYPERTENSION
Technical Field
The present invention relates to a composition for preventing hypertension.
Particularly, the present invention relates to food or a pharmaceutical
composition that
has the effect of inhibiting the elevation of blood pressure via the long-term
oral
ingestion thereof and thus can be used for preventing hypertension.
Background Art
Recently, the need for prevention of lifestyle-related diseases has been
actively
argued. However, the number of patients with arteriosclerosis, that of a
variety of
lifestyle-related diseases recognized as signal symptoms thereof, and that of
patients-to-be thereof have not yet decreased. Thus, analyses have been
undertaken via
a wide variety of approaches in order to treat lifestyle-related diseases, and
the
development of pharmaceutical agents has been extensively advanced.
Hypertension is a symptom of lifestyle-related diseases that has a large
number
of patients or patients-to-be. According to the Japan's national nutrition
survey in 1998
(Kokumin eiyou no genjo (Current Status of National Nutrition), Kenko Eiyo
Joho
Kenkyukai (Society for Health and Nutrition Studies) (ed.), Dai-ichi Shuppan
Publishing,
Co., Ltd., p. S4, 2000), as many as 25.3% of males and 20.6% of females were
evaluated
as being afflicted with hypertension, and 19.8 % of males and 14.5 % of
females were
evaluated as being afflicted with borderline hypertension, based on the blood
pressure
levels of surveyed males and females aged 1S or older. The results of this
survey
indicate that ten million or more people are hypertension patients-to-be in
Japan. This
is a seriously problematic situation. Although the cause for hypertension has
not yet
been clarified, interactions between a predisposing cause (genetic) and the
environment
(lifestyle) are considered to be causative of hypertension.
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CA 02477900 2004-08-31
Development of an agent for ameliorating hypertension, i.e., an
antihypertensive
agent, has been remarkable, and diuretics, sympatholytic agents (al Mockers or
(3
blockers), ACE inhibitors, calcium antagonists, and angiotensin II receptor
antagonists
have been used. These agents, however, generate side effects and thus must be
used by
being prescribed by a specialist with careful and strict control. Real effects
of
antihypertensive agents cannot be attained via ingestion thereof only when a
patient does
not feel well. Antihypertensive agents become effective via the long-term
ingestion in
adequate amounts. Antihypertensive agents are considered to be effective for
prevention. Because they are pharmaceutical agents, however, long-term
ingestion
thereof for a preventive purpose imparts a serious economic burden, such as
increased
medical expenses, on patients. Accordingly, discovery of an inhibitor for the
elevation
of blood pressure, which can be casually used by anybody, is more cost-
effective,
generates no substantial side effects, and can be easily obtained, has been
desired.
Disclosure of the Invention
The objects of the present invention are to discover a substance that inhibits
the
elevation of blood pressure via the long-term oral ingestion thereof,
generates no
substantial side effects, is very cost-effective, and is easily obtainable,
and to provide
food or a pharmaceutical composition comprising such substance.
In order to attain the above objects, the present inventors have conducted
studies
as described below.
(1) Examination of screening system
In order to search for a substance that inhibits the elevation of blood
pressure
from among a variety of substances that can be casually used by anybody, are
inexpensive, generate no substantial side effects, and can be easily obtained,
in vitro
screening that focuses on responses by cultured cells, organs, or enzymes is
usually
carried out. A substance that was found positive via in vitro screening is not
always
found positive via an in vivo experiment, i.e., an animal experiment. In
contrast, a
substance that is effective in vivo may not exhibit its effect via in vitro
screening that is
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CA 02477900 2004-08-31
slightly different from the actual conditions in organisms. Therefore, the
present
inventors were persistent in implementing cost- and time-consuming animal
experiments.
In the animal experiments, they raised test animals for a long time period of
2 months
and conducted screening of a large number of highly safe substances.
Specifically, samples prepared by adding a variety of purified substances to
the
standard feeds to a final concentration of 0.3% and then mixing them were
administered
to SHR rats, i.e., the animal models of essential hypertension, that gradually
develop
hypertension as they grow older week-by-week. The effects of sample
administration
were evaluated by using 1 SHR (spontaneouly hypertensive rat) per sample,
letting it
live for 8 weeks, and comparing its blood pressure level with the mean of the
control
group (6 individuals) to which the purified substance had not been
administered. In
general pharmacological tests, animals, such as rats, are forced to orally
ingest an
aqueous solution of pure reagent every day in predetermined amounts relative
to their
body weights. The present inventors considered that a system in which an
active
substance is administered at substantially the same time as a meal was
suitable in terms
of higher safety. Thus, they adopted a method in which a test substance was
mixed
with feed. As a result of the experiment via this screening system, the
present inventors
found that pure acetic acid added to feed has the effect of alleviating the
elevation of
blood pressure in SHR. They confirmed this finding via another experimentation
utilizing an increased number of rats and simultaneously found that the
increased dose
resulted in improved effects of inhibiting the elevation of blood pressure.
(2) Correlation between hypertension and acetic acid
Concerning the correlation between hypertension and acetic acid, Jinko Zoki
(Artificial Organ), (vol. 21, p. 958, 1992) describes that patients who have
been
receiving dialysis for a long period of time are likely to have recurring
hypertension with
high frequency after acetic acid dialysate containing acetic acid of high
concentration
(35 mM) is changed to bicarbonate dialysate containing acetic acid of medium
concentration (8 mM).
(3) Correlation between acetic acid ingestion and acetic acid concentration in
blood
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Orally ingested acetic acid is known to be rapidly changed into acetyl-CoA in
the body and then metabolized. Also, most thereof is completely degraded to
carbon
dioxide and water. Despite the aforementioned finding, accordingly, it is
impossible to
maintain acetic acid concentration in blood at a constantly high level via
oral ingestion
of an acetic-acid-containing composition alone.
As described in the Reference Example below, the present inventors verified
the
difficulty of maintaining the acetic acid concentration in blood via oral
ingestion of an
aqueous solution of acetic acid in an experiment using pigs. Specifically,
pigs were
forced to orally ingest an aqueous solution of acetic acid. The pigs were
dissected for
analysis over time, and the acetic acid concentrations in the blood of various
sites were
then measured. In this case, pigs were forced to ingest a solution containing
6 g of
acetic acid per 1,000 g of solution (an aqueous solution of acetic acid: about
100 mM) at
one time. The maximal acetic acid concentration in blood was exhibited in the
hepatic
portal vein 10 minutes after ingestion, and the concentration at this site was
0.8 mM.
The concentration was 0.4 mM in the abdominal artery, and it was as low as
0.18 mM in
the postcaval vein. Further, the concentrations were lowered to 0.5 mM, 0.3
mM, and
0.17 mM in the hepatic portal vein, in the abdominal artery, and in the
postcaval vein,
respectively, 30 minutes after ingestion. The following was found based on the
correlation with the dilution of acetic acid with body fluid or the rate of
absorption.
That is, orally ingested acetic acid migrates to the portal vein while being
diluted
125-fold or more, the rate of acetic acid metabolism in the body is rapid, the
acetic acid
concentration in blood is rapidly lowered, and the acetic acid concentration
in the vein
does not substantially change.
Accordingly, it is difficult to maintain highly concentrated acetic acid in
blood
to an extent such that blood pressure is lowered in spite of ingestion of an
acetic acid
solution of ingestibIe concentration.
According to other literature (Masui to sosei (Narcosis and Anabiosis), vol.
26,
p. 63, 1990), drip-feeding of I liter of acetated Ringer's solution at the
time of surgical
operation actually resulted in elevated acetic acid concentration in blood
during the
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operation. However, blood pressure at that time was somewhat elevated instead
of
being lowered. Thus, the fact that blood pressure cannot be lowered with
acetic acid of
a physiological concentration has been verified, and the acetic acid
concentration in
blood was found via experiment to return to the level before the operation
when the
patient was awake after operation. The rate of acetic acid metabolism was,
therefore,
determined to be very rapid. In this literature, 15 males and females were
subjected to
measurement of the acetic acid concentrations in blood immediately before the
initiation
of test drug administration, and the measured values were 0 to 0.5 mg/dl
according to the
graph. When the surgical operation was completed, the acetic acid
concentration was
elevated to 0.5 to 2.5 mg/dI (mean: 1.5 mg/dl) as a result of drip-feeding of
acetic acid,
and the blood pressure level was somewhat elevated in comparison with that
immediately before the initiation of test drug administration, although this
elevation was
not significant. The present inventors independently conducted measurement of
a large
number of acetic acid concentrations in human blood under fasting conditions.
The
maximal concentration was 0.6 mg/d1 (0.1 mM) and the minimal concentration was
0
(undetected), which were consistent with the results attained in the
aforementioned
literature.
The following points were elucidated: (i) acetic acid does not Lower blood
pressure at a concentration in human blood of around 0.1 to 0.4 mM; (ii) the
acetic acid
concentration in human blood under fasting conditions is 0.1 mM or lower; and
(iii) the
acetic acid concentration in blood does not substantially change via oral
ingestion of
vinegar at the time thereof. Specifically, oral ingestion of vinegar cannot
rapidly lower
blood pressure.
(4) Examination of report on the effect of vinegar
An example of a composition comprising highly concentrated acetic acid is
vinegar as a condiment. It is described in health magazines or health-related
books that
ingestion of black vinegar, which is a kind of vinegar, lowers the blood
pressure level of
a hypertensive person. However, no active ingredient thereof is described at
all. In
the academic literature (Kiso to ri~aslzo (Experimental and Clinical
Medicine), vol. 19, p.
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237, 1985), the active ingredient of black vinegar was searched for, although
it has not
yet been identified. Fractions having high activity of inhibiting angiotensin
converting
enzyme (ACE), which plays a role in blood pressure regulation, were subjected
to amino
acid analysis. As a result, a wide variety of amino acids had been detected.
This
suggests that the active ingredient of black vinegar is a peptide or amino
acid.
A substance that inhibits ACE activity in vitro may also be able to lower
blood
pressure in vivo. Accordingly, the degree of the ACE activity inhibited is
also
inspected. Tsuzuki et al. inspected the substance in vinegar that inhibits ACE
activity
(Journal of the Japanese Society for Food Science and Technology, vol. 39, p.
188,
1992), and they concluded that an organic acid is not involved with ACE
inhibition.
Matsui et al. demonstrated the effects of ginseng vinegar to inhibit the
elevation
of blood pressure in a stroke-prone spontaneously hypertensive rat (SHRSP)
(Yakuri to
chiryo (Pharmacology and Treatment), vol. 26, p. 23, 1998), and they stated in
the
"Consideration" section that ginseng vinegar may have an antihypertensive
effect since
the antihypertensive effect of the ginseng extract has already been reported.
Even though vinegar is known to have a antihypertensive effect from the
aforementioned report, vinegar comprises only about 4% to 5% of acetic acid.
Thus, it
is appropriate to consider that such effect was exhibited by "a starting
material or a
functional component generated via processing thereof" instead of "the acetic
acid."
Also, it is difficult to compare the effect attained by a single ingredient
with that attained
by food comprising a wide variety of ingredients such as vinegar. Among the
wide
variety of ingredients, some ingredients can positively act on a certain
symptom while
some other ingredients can adversely act thereon. Therefore, the effect of
food is
integrated effects of all the ingredients thereof.
Thus, the excellent antihypertensive effect attained by the long-term oral
ingestion of a very small amount of pure acetic acid was discovered by the
present
inventors. The present invention has been completed based on such finding.
Acetic acid itself have an acidic taste. Thus, it is practically difficult to
ingest
acetic acid of high concentration. Even if it is well-diluted, it is difficult
to ingest a
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large amount of acetic acid. Accordingly, ingestion of acetic acid within an
adequate
concentration range becomes necessary. In order to ameliorate the acidic
taste, acetic
acid can be neutralized with alkali, or a large amount of other taste
components can be
added to acetic acid. This is not simple, however, since it involves problems
such as
increased harsh unpleasant taste, overconsumption of minerals, or nutritional
imbalances.
In the present invention, therefore, the concentration that was adequate for
ingestion was
also examined, and it was determined that the preferable amount of acetic acid
molecules
to be included was 0.36 g to 30 g per 1,000 g of composition.
This value was converted to human terms. This demonstrates that ingestion of
an average of 0.5 g to 5 g of acetic acid per day by an adult who weighs 60 kg
delays
hypertension. That is, such ingestion is effective for preventing
hypertension.
Further, elevation of blood pressure can be inhibited after acetic acid has
been
continually ingested fox 3 weeks. On the contrary, this effect cannot be
expected to a
significant extent by the short-term ingestion of within 2 weeks. Ingestion of
acetic
acid for a long time period of at least 3 weeks leads to an effect of
inhibiting the
elevation of blood pressure.
Continual ingestion of the composition according to the present invention was
found to prevent a person who may develop hypertension in the future or a
person whose
blood pressure is at a borderline level (a systolic blood pressure of I40 to
180 mmHg)
from developing hypertension, according to the results of the experiment.
The present invention has been completed based on the aforementioned findings.
Specifically, the present invention includes the following inventions.
( 1 ) Food or a pharmaceutical composition for preventing hypertension, which
inhibits the elevation of blood pressure via the long-term oral ingestion
thereof and
comprises at least one member selected from among acetic acid, acetate ion,
and acetate.
(2) The composition according to ( 1 ), which comprises at least one member
selected from among acetic acid, acetate ion, and acetate in amounts of 0.36 g
to 30 g in
total (in terms of acetic acid) per 1,000 g of composition.
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(3) The composition according to (1) or (2), wherein the intake of at least
one
member selected from among acetic acid, acetate ion, and acetate is adjusted
to 0.5 g to 5
g in total (in terms of acetic acid) per day.
(4) The composition according to any of (1) to (3), wherein the period of
ingestion is 3 Weeks or longer.
Hereafter, the present invention is described in detail.
The process for producing acetic acid that is employed in the present
invention
is not particularly limited. It may be produced by synthesis or fermentation.
When it
is used as food, however, use of acetic acid produced by fermentation, i.e.,
vinegar
(fermented vinegar), is preferable from the viewpoint of consumer's
perception.
Especially, brown rice vinegar, the sour taste of which is less likely to
stand out, or cider
vinegar, which has a refreshing flavour, is preferable. The use of a sweetener
or
flavoring agent can provide acetic acid having milder sourness. A variety of
acetates
such as sodium acetate can also be used.
The term "acetic acid concentration" used herein refers to a concentration
that is
represented in terms of acetic acid comprising acetic acid molecules (CH3COOH)
that
are not dissociated, acetate ions (CH3C00-) that are dissociated, and acetate
that is not
dissociated, for the following reasons. Whether the orally ingested acetic
acid is an
aqueous solution of acetic acid with a low pH Level, neutralized acetate (for
example,
sodium acetate), or a dissociated acetate ion, the pH levels in the stomach or
the
intestinal canal after the small intestine where these substances are absorbed
are not
substantially affected by the composition, and are maintained at a
substantially constant
level in each site. Accordingly, the condition of an acetic acid molecule in
the
composition when it is put into the mouth does not affect the absorption of
acetic acid in
the body. Therefore, the composition of the present invention needs to
comprise at
least one member selected from among acetic acid, acetate ion, and acetate
(hereafter it
rnay be occasionally referred to as "acetic acids").
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Acetic acids can be assayed using, for example, a carboxylic acid analyzer
(EYELA S-3000, Tokyo Rikakikai Co., Ltd.). This apparatus separates a variety
of
organic acids using columns and detects organic acids based on the principle
whereby a
reagent specifically reacts with a carboxyl group of organic acid. The use of
this
apparatus enables the quantification of acetic acids contained in the solution
regardless
of the dissociated or non-dissociated state thereof.
The composition of the present invention can be obtained by mixing at least
one
member selected from among acetic acid, acetate ion, and acetate with adequate
amounts
of other starting materials (starting materials for food or pharmaceuticals).
The form of the composition of the present invention is not particularly
limited.
Examples thereof include specified health foods (health foods), vinegared
foods, sushi,
marinades, beverages, and pharmaceuticals (tablets, capsules, powders,
granules, fine
grains, and drinkable preparations). The method for adding acetic acids is not
particularly limited, and it may be carried out via a common technique. When
acetic
acid is used in the form of a solution with a low pH level, such as synthetic
acetic acid or
fermented vinegar, instead of the salt form, attention should be paid to
"sourness" caused
by lowered pH level from the viewpoint of ease of drinking or eating. More
specifically, ingestion of a solution with a low pH level generates a
discomforting
feeling in the throat when the beverage slides down the throat, and the person
gets the
beverage stuck in the throat. When highly concentrated acetic acid is
intended, for
example, utilization of salt of acetic acid and/or encapsulation can be
implemented.
The concentration of acetic acids to be added to the composition must be at
least
6 mM in the case of a liquid. When a solid matter is included, the composition
must
comprise at least 0.36 g of acetic acids per 1,000 g of composition. If the
concentration
is lower than this level, ingestion of very large amounts of foods or
beverages becomes
necessary. Such concentration is calculated and determined based on the
presumption
that the daily intake of the composition is about 1,150 g in the case of foods
and about 1
Iiter in the case of beverages.
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About 0.5 g to 5 g of acetic acid (molecules) must be ingested per day. The
necessary amount of active acetic acid molecules can be ingested by eating one
serving
or more per day in the case of vinegared food or sushi. Also, a necessary
amount of
active acetic acid molecules can be ingested by drinking about 50 ml to 1
liter of a
beverage containing vinegar, such as cider vinegar, per day. Although
ingestion of 5 g
or more acetic acid (molecules) is possible, ingestion thereof in an amount
exceeding 5 g
is not preferable from the viewpoints of a taste of food, ease of eating, or
ease of
drinking.
When highly concentrated and unneutralized acetic acid is used, ingestion must
be made with consideration for a disorder of the alimentary canal, such as
stomach or
intestine. According to the literature (Japan J. Pharmacol., vol. 41, p. 101,
1986; Med.
Sci. Monit., vol. 5, p. 1031, 1999), the acetic acid concentration up to 3%
exhibits the
effect of protecting the gastric mucosa rather than damaging the stomach.
Thus, direct
ingestion of acetic acid at a concentration of approximately 3 % in the form
of a beverage
will not cause any serious problems. There is an academic report concerning
the LDso
of acetic acid which was made using mice (the Journal of the Japanese Society
of
Nutrition and Food Science, vol. 36, p. 283, 1983). Based on those research
results,
ingestion of 5 ml of vinegar per kg of the body weight, i.e., ingestion up to
300 ml of
vinegar at a time for an adult who weighs 60 kg, is not considered to cause a
disorder of
the alimentary canal.
Specifically, acetic acid can exhibit the effect of lowering blood pressure
via the
long-term oral ingestion thereof in amounts of approximately 0.5 g to 5 g per
day. The
term "long-term oral ingestion" refers to continual ingestion for at least 3
weeks. This
is because the blood pressure was verified at a statistically significantly
lower value in
comparison with the control 3 weeks after the meal was changed to the test
meal, based
on the animal experiment.
The composition of the present invention is effective for preventing
hypertension, particularly essential hypertension, the cause of which has not
yet been
CA 02477900 2004-08-31
clarified but which accounts for 90% of hypertension, cerebrovascular
disorder, heart
diseases, and other vascular lesions developed thereby.
Brief Description of the Drawings
Fig. 1 shows the acetic acid concentration in the hepatic portal vein of the
control group and that of the group to which acetic acid had been administered
(10
minutes, 30 minutes, and 60 minutes after ingestion).
Fig. 2 shows the acetic acid concentration in the abdominal artery of the
control
group and that of the group to which acetic acid had been administered (10
minutes, 30
minutes, and 60 minutes after ingestion).
Fig. 3 shows the acetic acid concentration in the large vein of the control
group
and that of the group to which acetic acid had been administered (10 minutes,
30 minutes,
and 60 minutes after ingestion).
Fig. 4 shows change in the body weight of the control group and that of the
group to which acetic acid had been administered.
Fig. 5 shows change in the feed consumption of the control group and that of
the
group to which acetic acid had been administered.
Fig. 6 shows change in the water intake of the control group and that of the
group to which acetic acid had been administered.
Fig. 7 shows change in the blood pressure level of the control group and that
of
the group to which acetic acid had been administered.
Fig. 8 shows change in the heart rate of the control group and that of the
group
to which acetic acid had been administered.
Fig. 9 shows change in the blood pressure level of the control group and that
of
the group to which acetic acid had been administered (addition of 0.36%,
0.72%, 1.5%,
3%, 6%, and 9% acetic acid).
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This description includes part or all of the contents as disclosed in the
descriptions of Japanese Patent Application Nos. 2000-394632 and 2001-298211,
which
are priority documents of the present application.
Preferred Embodiments of the Invention
The present invention is hereafter described with reference to the following
examples and reference example, although the technical scope of the present
invention is
not limited thereto.
[Reference Example] Test for oral in~~estion of acetic acid using-pig
1. Method
(1) Test material
As test materials, 150 ml of distilled water or 150 ml of a 6% (w/w) aqueous
solution prepared by diluting a reagent acetic acid (a special grade) with
distilled water
were prepared and then employed as samples.
(2) Method of administration
As test animals, 5 pigs (with body weights of about 20 kg) were prepared for
each group to which each sample was to be administered. These test animals
were
subjected to fasting from the night before testing. A tranquilizer was
administered on
the day of testing. After the test animals were confirmed to be under
sedation, samples
were administered orally to the stomach.
(3) Sacrifice after administration
The test animals were sacrificed 10 minutes after the administration of water,
and 10 minutes, 30 minutes, and 60 minutes after the administration of an
aqueous
solution of acetic acid.
(4) Items to be assayed
Blood was sampled from the hepatic portal vein, the abdominal artery, and the
postcaval vein, and the acetic acid concentrations in the serums were assayed
by gas
chromatography.
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2. Results
The acetic acid concentrations in serums were as shown below.
(1) Hepatic portal vein: as shown in Fig. 1. The acetic acid concentration of
the group assayed 10 minutes after ingestion was significantly higher than
that of other 3
groups (significant difference was observed via ANOVA: p < 0.05).
(2) Abdominal artery: as shown in Fig. 2. The acetic acid concentration of the
group assayed 10 minutes after ingestion was significantly higher than that of
other 3
groups (significant difference was observed via ANOVA: p < 0.05).
(3) Postcaval vein: as shown in Fig. 3. There was no significant difference
among groups (p > 0.05).
Although the acetic acid concentration became relatively high in the portal
vein
minutes after ingestion of an aqueous solution of about 100 mM acetic acid, it
rapidly
decreased with the elapse of time. As with the case in the portal vein, the
maximal
level was marked in the artery 10 minutes after ingestion, although it
returned to the
ordinary level 30 minutes later. Since acetic acid is rapidly absorbed and
utilized in the
periphery, the acetic acid concentration in the large vein was not
substantially elevated
in spite of acetic acid ingestion. The maximal acetic acid concentration was
marked in
the portal vein 10 minutes after acetic acid ingestion, and the concentration
at that time
was about 0.8 mM. This indicates that the acetic acid concentration is diluted
to 1/125
of that at the time of administration thereof.
Thus, the rate of acetic acid metabolism was found to be rapid, and the
concentration thereof was found to return to a level substantially the same as
that under
fasting conditions 30 minutes after ingestion.
Human clinical study cannot be conducted due to the necessity of sacrificing
subjects. Based on the fact that the body weight of a pig is about 20 kg, the
obtained
results are converted to those in terms of a human adult (60 kg). This would
be
equivalent to a human adult drinking 450 ml of a beverage at one time.
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It was judged that the acetic acid concentration in blood cannot be elevated
to
the concentration thereof in a dialysate used for a dialysis treatment for
humans (8 to 35
mM) via ingestion of acetic acid in the form of beverage.
[Example lj Test for confirming the effect of acetic acid using-SHR
1. Method
(1) Test substance
Acetic acid (reagent, special grade) was diluted with distilled water to
prepare a
% (w/v) aqueous solution, and this aqueous solution was added to powdery feeds
in an
amount of 3% to prepare feeds (a test meal). Also, powdery feed (a control) to
which
no substance had been added was prepared. These feeds were employed as
samples.
(2) Method of administration
As test animals, 6 spontaneous hypertensive rats (SPF, SHR/NCrj, male,
4-week-old, Charles River Japan) were prepared for each group to which each
sample
was to be administered (the group to which the test meal was to be
administered and the
control group). Rats were allowed to freely ingest feeds and tap water.
(3) Period of administration
The day on which the test meal had been first administered was determined to
be
the day 1, and administration was continued for 8 weeks.
(4) Test system
The test animals were kept under the following conditions for 5 days before
the
test.
They were kept at a temperature of 20°C to 26°C and a humidity
of 40% to 70%
and placed in a well-lit place for I Z hours a day and in a dark place for 12
hours a day.
(5) Measurement of blood pressure and heart rate
The blood pressure and the heart rate were noninvasively measured every week.
The blood pressure was measured using an apparatus for noninvasive blood
pressure
measurement by the tail-cuff method. The heart rate was measured by employing
the
pulse of the blood pressure as a trigger. Measurements were carried out 5
times, and
the mean thereof was determined.
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2. Results
(1) Increase in body weight, feed consumption, and water consumption
When the test was initiated, the average body weight of the control group was
102.5 g, and that of the test group was 101.8 g. That is, there was no
statistically
significant difference. As shown in Fig. 4, there was no significant
difference between
the body weight of individuals in the control group and that of the group to
which the
test meal had been administered 8 weeks later {p > 0.05 according to the t-
test).
Also, there was no significant difference in feed consumption (Fig. 5) or
water
consumption (Fig. 6) between two groups (t-test; the p < 0.05 Level was
determined to be
significantly different).
(2) Blood pressure and heart rate
The blood pressure level of the group to which the test meal had been
administered became significantly lower than that of the control group from
the 4th week
to the 8th week. Thus, the elevation of blood pressure was found to be
inhibited (Fig.
7) (p < 0.05).
The heart rate varied in a substantially similar manner as with the case of
the
blood pressure (Fig. 8), although the heart rate did not become significantly
different
from that of the control group at any stage.
[Example 2] Culturing test of SHR using feed with varied doses of acetic acid
The experimental method and conditions were substantially in accordance with
Example 1 except for the lineage of SHR, the kind of feed, and the amount of
acetic acid
added to the feed.
Acetic acid (reagent, special grade) was diluted with distilled water to
prepare a
5% (w/v) aqueous solution. This aqueous solution was added to powdery feeds
(Labo
MR Stock, Nousan Corporation, requiring sterilization before use) in amounts
of 0.36%,
0.72%, 1.5%, 3%, 6%, and 9%, respectively. The resulting feeds having
different
acetic acid concentrations were to be independently administered to the test
groups.
Powdery feeds containing no additives were to be administered to another group
(the
control group). Thus, seven experimental plots Were prepared in total.
CA 02477900 2004-08-31
Animals used were the 4-week-old SPF SHR/Hos rats, and each group consisted
of 6 individuals. The blood pressure was continually measured until the 8th
week (Fig.
9), and the blood pressure level of the group to which the feeds comprising
0.36% acetic
acid had been administered was not significantly different from that of the
control group
at any stage. The blood pressure of the group to which the feeds comprising
0.72%
acetic acid had been administered became significantly lower than that of the
control
group on the 5th week, the blood pressure of the groups to which the feeds
comprising
1.5 % acetic acid had been administered became significantly lower than that
of the
control group on the 4th week, and the blood pressure of the groups to which
the feeds
comprising 3% to 9% acetic acid had been administered became significantly
lower than
that of the control group on the 3rd week (significant difference was observed
via
ANOVA in all these cases: p < 0.05). This demonstrates that the feeds must
comprise
0.72% or more of a 5% acetic acid solution, i.e., 0.3b g or more acetic acid
(molecules)
per 1,000 g of feeds, in order to inhibit the elevation of blood pressure.
There was no
significant difference among groups in terms of the body weights and the feed
consumption. (ANOVA: the body weight of the control group was 71.8 g on the
week 0
and 275.7 g on the 8th week; and the feed consumption of the control group was
15.1 g
on the week 0 and 20.5 g on the 7th week.)
The blood pressure levels of the test groups became significantly lower than
that
of the control group 3 weeks after rats began to eat the acetic acid-
containing feeds.
There was no significant difference until the 2nd week. This indicates that
acetic acid
must be continually ingested for at least 3 weeks and that substantially no
effect is
attained by ingestion thereof for less than 2 weeks.
According to the most recent Japan's national survey (Kokumin eiyou no genjo
(Current Status of National Nutrition), Kenko Eiyo Joho Kenkyukai (Society for
Health
and Nutrition Studies) (ed.), Dai-ichi Shuppan Publishing, Co., Ltd., p. 78,
2000), a
Japanese person ingests 1,116 g of food on average per day except for flavor
enhancers,
nonessential beverages, and beverages such as milk or fruit juice.
Accordingly, if the
content with which the aforementioned significant difference was attained
(0.72% to 9%
16
CA 02477900 2004-08-31
of an aqueous solution of 5% acetic acid contained in the feeds) is converted
in relation
to 1,116 g, the amount of acetic acid (molecules) is about 0.5 g to 5 g.
Specifically,
prevention of hypertension can be expected from the ingestion of acetic acid
(molecules)
in amounts of 0.5 g to 5 g on average per day.
[Example 3) Production example of composition (beverage) and evaluation
thereof
A beverage having the following composition was produced. Specifically, 5 g
of acetic acid (food additive) and 0.2 g of sucralose (food additive) were
added to water,
and these substances were mixed to prepare 1 liter of a solution. Thus, a
beverage was
obtained. The concentration of acetic acid molecules in this beverage was 83
mM.
This beverage has excellent drinkability with moderate sourness and a
refreshing
taste. Daily ingestion thereof in amounts of about 100 ml to 1 liter (0.5 g to
5 g of
acetic acid molecules) for a long period of time (3 weeks or longer) is
considered to be
able to prevent hypertension by the effects attained by acetic acid molecules.
Also, 20 adults participated in a tasting test. The acetic acid content in
1,000 g
of the beverage was set at 5 different levels: 1 g; 5 g; 10 g; 30 g; and 50 g.
The
resulting beverages were then evaluated, and as a result, 19 participants
pointed out a
problem of taste when acetic acid content was 50 g. Only 10 participants
recognized a
problem of taste when acetic acid content was 30 g, and 18 or more
participants
indicated ease of drinking when acetic acid content was 10 g or lower. Thus,
the
preferable acetic acid content in the beverage was determined to be 30 g or
lower per
1,000 g of the beverage.
[Example 4) Production example of food
Acetic-acid-containing food (4 servings) having the following composition was
produced. Specifically, 12 sticks of green asparagus, 2 pieces of bacon, 2 g
of dried
bonito flakes, 30 ml of soy sauce, 2.3 g of acetic acid (food additive), and
45 ml of tap
water were thoroughly mixed with each other to prepare acetic-acid-containing
food.
The concentration of acetic acid molecules in the liquid portion of this food
was about
500 mM (containing about 30 g of acetic acid molecules per liter of liquid).
17
CA 02477900 2004-08-31
This food is a vinegared food having excellent edibility with moderate
sourness
and a refreshing taste. Daily ingestion thereof in amounts of one serving per
day to one
serving per meal, i.e., about three servings per day (0.58 g to 1.73 g of
acetic acid
molecules per day) for a long period of time is considered to reduce the
elevation of
blood pressure by the effects attained by acetic acid molecules, thereby
preventing
hypertension.
[Example 5] Production example of food
Acetic-acid-containing food having the following composition was produced in
the following manner. Specifically, 3 you (translator's note: 1 you = 180 cc)
of rice, 3
you of water, 3 pieces of lightly-salted salmon, 4 pieces of shiitake
mushrooms (long
thin strips), a package of shimeji mushrooms, thinly-sliced fried egg prepared
from 3
eggs, 10 leaves of green perilla (long thin strips), a mixed seasoning A (a
mixture of 2
tablespoonfuls of sake (Japanese rice wine), 2 tablespoonfuls of water, and a
pinch of
salt), and a mixed seasoning B (a mixture of 4 tablespoonfuls of rice vinegar
(acetic acid
concentration of 4.5%), 5 tablespoonfuls of sugar, and 2 teaspoonfuls of salt)
were first
prepared as ingredients.
(1) Roasted lightly-salted salmon was put on rice and rice was cooked in that
state, followed by steaming. (2) The salmon was taken out, bones and skin were
removed, and the fish meat was broken up into large pieces. (3) Shiitake
mushrooms
and shimeji mushrooms were combined and steam-steeped in the seasoning A. (4)
The
cooked rice was dressed with the seasoning B to prepare sushi rice (rice
seasoned with
vinegar). (5) The flaked salmon and the steam-steeped shiitake and shimeji
mushrooms
were added thereto and mixed. The thinly-sliced fried egg and the green
perilla (long
thin strips) were sprinkled thereon.
This food is delicious sushi with moderate sourness and comprises about 2.7 g
of
acetic acid molecules. By eating about 1/4 to 1/3 of the finished food (0.68 g
to 0.9 g
of acetic acid molecules) one to three times a day (0.68 g to 2.7 g of acetic
acid
molecules in total), the elevation of blood pressure is inhibited, and
hypertension can
thereby be prevented.
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(Example 6] Production example of beverage
A beverage having the following composition was produced. Specifically, 5 g
of acetic acid (food additive) and 0.5 g of stevioside (food additive) were
added to water,
and these substances were mixed to prepare 1 liter of a solution. Thus, a
beverage was
produced. The concentration of acetic acid molecules in this beverage was 83
mM.
This beverage has excellent drinkability with moderate sourness and a
refreshing
taste. Daily ingestion thereof in amounts of about 100 ml to 1 liter (0.5 g to
5 g of
acetic acid molecules) for a long period of time is considered to inhibit the
elevation of
blood pressure by the effects attained by acetic acid molecules, thereby
preventing
hypertension.
[Example 7J Production example of beverage
A beverage having the following composition was produced. Specifically, 2
teaspoonfuls of cider vinegar (acetic acid concentration of 5%), 2
teaspoonfuls of honey,
and 150 ml of chilled water were mixed with each other to prepare a solution.
Thus, a
beverage was produced. The concentration of acetic acid molecules in this
beverage
was about 120 mM.
This beverage has excellent drinkability with moderate sourness and a
refreshing
taste. Daily ingestion thereof in amounts of about 100 ml to 700 ml (0.7 g to
5 g of
acetic acid molecules) for a long period of time is considered to inhibit the
elevation of
blood pressure by the effects attained by acetic acid molecules, thereby
preventing
hypertension.
[Example 8] Production example of powder
Acetic acid was allowed to adsorb onto dextrin and then dehydrated. Thus,
powders comprising 15 % (w/w) of acetic acid were prepared. These powders (6 %
(w/w)) were added to powders comprising sugar, skimmed milk powder, and
lactose
(94 % (w/w)), and they were thoroughly mixed with each other to prepare
powders.
Such powders have moderate sourness. Oral ingestion thereof in amounts of
100 g per day (0.9 g of acetic acid molecules) for a long period of time is
considered to
inhibit the elevation of blood pressure, thereby preventing hypertension.
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[Example 9] Production example of tablet
Tablets comprising 1.25 g (25 mg per tablet) of sodium acetate per 100 g
thereof
were produced. Oral ingestion of these tablets in amounts of 70 g per day (35
tablets,
0.63 g of acetic acid molecules) for a long period of time is considered to
inhibit the
elevation of blood pressure, thereby preventing hypertension.
[Example 10] Test for confirming the effect of acetic acid on hypertensive
patient
1. Method
( 1 ) Test beverage
As test beverages, a beverage containing 1.5 g of acetic acid, a beverage
containing 0.75 g of acetic acid and 1 g of lactic acid, and a placebo
beverage containing
2 g of lactic acid instead of acetic acid were used (100 ml/bottle). The
composition of
each test beverage is shown in Table 1.
These 3 beverages had substantially the same appearances, sweet tastes, salty
tastes, and the like, although there were some variations in terms of
sourness. It was
confirmed that they couldn't be distinguished from each other based on their
appearance.
As acetic acid, vinegar having moderate sourness and a preferable flavor
(cider vinegar,
Mizkan, acetic acid concentration of 5% (w/v)) was used. Fermented lactic acid
was
used.
CA 02477900 2004-08-31
Table 1: Test beverage composition
Vinegar-containing beveragePlacebo beverage
High-dose Low-dose
Acetic acid (mg)1500 750 0
Lactic acid (mg)0 1000 2000
Water (g) 94.8 95.5 96.3
0 0 0
Protein (g)
0 0
Fat (g) 0
Carbohydrate 4.2 3.5 2.5
(g)
Ash content (g) 1 1 1
Sodium (mg) 5 5 5
Potassium (mg) 550 550 550
Calcium (mg) 60 60 60
Vitamin C (m 50 50 50
)
Total ( )
102 102 102
Calories (kcal) 15 12 8
(2) Subjects
1) Basis of selection
Patients with mild to moderate hypertension who had continuously had systolic
blood pressure of 140 to 180 mmHg or diastolic blood pressure of 90 to 105
mmHg for 3
months before the initiation of the test were subjected to the test. However,
those
having secondary hypertension, those diagnosed by a doctor as being in
immediate need
of an antihypertensive agent administration, those having alcohol dependence
or severe
anemia, and those afflicted with serious diseases were excluded. Also, those
who had
routinely taken some kind of medicine for internal application and those who
had
routinely taken supplements that might affect blood pressure were excluded.
2) Subjects
Volunteers (57 individuals) who met the aforementioned requirements were
divided into 3 groups independently consisting of 19 individuals so as to have
matching
conditions in terms of systolic blood pressure, diastolic blood pressure, age,
and sex
among groups. Among those 57 individuals, a total of 6 individuals, i.e., 2
individuals
who had failed to drink the test beverage for 8 days or longer during the
ingestion period
21
CA 02477900 2004-08-31
for personal reasons and 4 individuals who could not visit the hospital on the
testing day,
were eliminated as subjects of data analysis.
As a result, the number of available subjects for the analysis was 51. They
were 31 males and 20 females aged 51.2 ~ 8.1. There were no significant
differences
among the 3 groups in terms of age, sex, body weight, BMI, blood pressure, or
pulse.
(3) Outline of test
1) Testing method
The double blind method was adopted. A group to which an
acetic-acid-containing beverage was to be administered, a group to which
another
acetic-acid-containing beverage having a different acetic acid content was to
be
administered, and a group to which a placebo beverage was to be administered
were
subjected to parallel comparison.
2) Testing period
The testing period was a total of 15 weeks consisting of 3 weeks of a
pre-observation period (a non-ingestion period), 8 weeks of an ingestion
period, and 4
weeks of a post-observation period (a non-ingestion period).
3) Method of ingestion
Test beverages were ingested by drinking a bottle of 100 ml of the placebo
beverage, 100 ml of the acetic-acid-containing beverage containing 0.75 g of
acetic acid,
or 100 ml of the acetic-acid-containing beverage containing 1.5 g of acetic
acid once a
day in the morning while maintaining customary eating habits. The subjects
were
instructed to refrain from immoderate eating and drinking and not to change
other
routines during the test period.
(4) Items and method of testing
1) Blood testing and urine testing
Besides the preliminary test conducted before the initiation of the test,
blood
testing and urine testing were carried out two times, i.e., at the end of the
pre-observation period (immediately before the ingestion period) and at the
end of the
ingestion period during the test period.
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Blood sampling was carried out from 8:30 am to 11:00 am under fasting
conditions by refraining from eating and drinking after 9:00 pm on the
previous night.
The blood testing was requested from a company that conducts clinical
examinations.
In order to avoid extrinsic influences on the serum protein level, the serum
lipid level,
and the renin activity, blood sampling was carried out in a sitting position
after the
subject was allowed to rest for 10 minutes or longer.
Urine testing was carried out via a paper test, and glucose, protein,
urobilinogen,
and occult blood were examined.
2) Blood pressure and physical measurement
The blood pressure and the pulse were measured between 8:30 am and 11:30 am,
and every subject was assigned a fixed time for measurement. The subjects were
to
visit the hospital without ingesting anything except for water and the test
beverage on
the testing day, allowed to rest for 10 minutes or longer after the visit, and
subjected to
measurement three times at intervals of 1 minute at the elbow of the left arm
in a sitting
position while being dressed but without wearing shoes. The blood pressure and
the
frequency of pulse marked when the median of the systolic blood pressure had
been
obtained were determined as the data for that day. Blood pressure was measured
8
times in total during the test: before the pre-observation period; immediately
before the
ingestion period; the 2nd, 4th, 6th, and 8th weeks after the initiation of the
ingestion; and
the 2nd and 4th weeks after the completion of the ingestion (the post-
observation
period).
The body weight was measured at the time of diagnosis and interview by a
doctor. When measuring the body weight, it was attempted to avoid the
influence
caused by clothing as great an extent as possible.
3) Diagnosis and interview
Diagnosis and interview were conducted by a doctor, and all adverse events
were recorded. In particular, development of subjective symptoms and that of
side
effects were explored in detail.
(5) Method of analysis
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The comparison of the blood pressure and the heart rate among groups during
the ingestion period was conducted via two-way analysis of variance (ANOVA).
The
primary effects and the interaction between the test beverage ingested and the
period of
ingestion were analyzed. Further, when the primary effects were attained
during the
ingestion period, the measured blood pressure level was compared with the
blood
pressure level immediately before the ingestion via Dunnett's multiple
comparison.
Other measured values and the results of blood testing of the patients were
subjected to a
paired t-test, and interaction with the ingested test beverage was tested by t-
test. The
SPSS Ver. 10 statistical software (SPSS) was used, and the level of
significance was
determined to be 5 % or lower for both tests.
2. Results
(1) Blood pressure and heart rate
Changes in blood pressure levels and heart rates during the test are shown in
Table 2. The systolic blood pressure levels were analyzed with a two-way
analysis of
variance. As a result, changes in the systolic blood pressure levels were
different
among 3 groups during the ingestion period (p < 0.05).
The blood pressure level in the group that had ingested 1.5 g of acetic acid
per
day (hereafter this group is referred to as the "high-dose group") and that in
the group
that had ingested 0.75 g of acetic acid per day (hereafter this group is
referred to as the
"low-dose group") began to become significantly lower from the 6th week after
the
initiation of ingestion to the end of the ingestion period. In contrast, no
significant
change occurred in the blood pressure level of the group that had ingested the
placebo
beverage (hereafter this group is referred to as the "placebo group")
throughout the
ingestion period.
Based on the blood pressure level immediately before ingestion, changes in the
systolic blood pressure level of the high-dose group became significantly
different from
those of the placebo group after continual ingestion for 4 weeks. Also, such
significant
difference was observed after continual ingestion for 6 weeks in the case of
the low-dose
24
CA 02477900 2004-08-31
group (Table 3). There was no significant difference between the high-dose
group and
the low-dose group.
As a result of the two-way analysis of variance for the systolic blood
pressure
level, there was no interaction found between the test beverage and the
ingestion period,
and the primary effect was observed during the ingestion period (p < 0.05).
The blood
pressure level of the high-dose group began to become significantly lower from
4th week
after the initiation of continual ingestion and that of the low-dose group
began to become
significantly lower from 6th week after the initiation of continual ingestion
(p < 0.05).
In contrast, no significant change occurred in the placebo group during the
ingestion
period.
Based on the blood pressure level immediately before ingestion, changes in the
systolic blood pressure level of the high-dose group became significantly
different from
those of the placebo group after continual ingestion for 4 weeks.
CA 02477900 2004-08-31
Table 2: Changes in blood pressure levels and heart rates
3 weeks Baseline (immediately
before
period of
beverage
ingestion
ingestion before ingestion)
2nd week 4th week
SBP High-dose152.1 8.4 151.7 13.2 147.2 14.6141.1 17.3
Low-dose151.7 7.5 15I .8 8.6 151.8 10.9146.1 19.0
(mmHg) Placebo 151.6 7.0 151.1 9.1 150.5 13.6151.8 13.9
DBP High-dose90.8 7.8 89.5 6.1 88.0 7.8 84.5 7.4
**
Low-dose90.9 10.8 89.9 9.6 87.2 1 86.1 11.4
I .3
(mmHg) Placebo 89.8 7.0 90.1 b.0 89.9 t 8.4 90.4 7.7
heart High-dose70.5 7.8 69.1 7.5 70.7 10.3 67.7 10.1
rate
Low-dose72.65.3 71.47.1 74.19.4 71.49.4
(beats/min)Placebo 73.3 5.8 71.4 6.3 71.9 6.5 73.4 8.8
2 weeks after 4 weeks after
the the
Period of
beverage
ingestion
end of ingestionend of ingestion
6th week 8th week
SBP High-dose136.7 15.7 136.9 16.6 148.1 16.5 147.9 16.4
*** **
Low-dose141.7 14.4 141.2 16.9 148.1 11,6 148.6 14.2
** **
(mmHg) Placebo151.8 11.8 152.8 8.5 151.0 10.2 150.2 12.3
DBP High-dose83.1 7.5 83.0 7.1 88.4 6.2 88.4 9.7
*** ***
Low-dose84.8 11.1 84.8 10.1 88.5 11.3 88.9 9.3
* *
(mmHg) Placebo89.6 7.1 89.9 7.5 89.4 7.8 88.4 8.7
heart High-dose68.6 11.7 67.7 10.1 68.3 10.2 68.2 8.5
rate
Low-dose72.8 10.5 70.7 10.0 71.9 7.4 69.6 6.7
(beats/min)Placebo71.4 8.1 72.2 3 7.5 71.1 9.1 71.3 8.5
SBP: Systolic blood pressure
DBP: Diastolic blood pressure
*:p<0.05;**:p<0.01;***:p<0.001
Table 3: Changes in blood pressure levels and heart rates
Base- 2 weeks after 4 weeks after
Period of ingesting beverage
line the end of the end of
ingestion ingestion
2nd week 4th week 6th week 8th week
SBP High- 0.0 -4.4 -10.6 -14.5 -14.8 -3.5 -3.8
11.5 16.3 14.1 17.5 12.1 14.7
dose p<0.05 p<0.001p<0.01
Low- U.U U.U -5.7 -I0.1 -10.6 -3.7 -3.2
9.5 16.7 I 14.8 1 1.3 12.5
1.7
dose p < p < 0,01
0.01
(mmHg) Placebo0.0 -0.6 0.7 0.8 1.7 -0.1 -0.9
10.9 13.5 11.0 10.8 14.9 16_.9
DBP High- 0.0 -1.5 -4.9 -6.4 -6.5 -1.1 -1.1
4.2 6.0 6.1 6.6 5.7 t 6.9
dose p<0.05 p<0.05 p<0.01
Low- 0.0 -2.7 -3.8 -5.1 -5.1 -1.4 -1.0
8.1 11.1 9.6 t 9.3 8.4 t 7.4
dose
(mmHg) Placebo0.0 -0.2 0.3 -0.5 -0.2 -0.7 -1.7
6.8 6,5 7.2 3.7 8.4 6.5
heart High- 0.0 1.6 -1.4 -0.5 -1.4 -0.8 -0.9
rate 5.1 4.6 6.5 5.3 t 4.4 5.2
dose
Low- 0.0 2.7 0.1 1.4 -0.7 0.5 -1.8
3.9 5.4 7.6 t 6.0 5.1 3 5.1
dose
(beats/min)Placebo0.0 0.5 2.0 0.0 0.8 -0.3 -0.1
4.7 5.9 6.9 6.3 4.8 4.3
SBP: Systolic blood pressure
DBP: Diastolic blood pressure
26
CA 02477900 2004-08-31
(2) Body weight and BMI
BMI did not significantly change between the periods before and after
ingestion
in either group.
(3) Blood testing and urine testing
Although there were some significant differences concerning some parameters
between the periods before and after ingestion in all groups, these slight
changes were
within the normal ranges. As a result of investigation regarding each case,
the
measured value did not vary from the normal range to an abnormal range in any
case.
According to the urine testing, examples of occult hematuria or unusual change
were observed in all groups, although this abnormality wasn't problematic.
(4) Diagnosis and interview
No difference was observed in the rate of adverse events occurring among the
groups that had ingested the test beverages. Also, the causal relationship
between the
test beverage and any of the adverse events was judged to be poor. According
to the
diagnosis and interview, no case was observed in any group that should be
especially
noted.
3. Conclusions
Accordingly, continual ingestion of about 0.75 g or 1.5 g of acetic acid per
day
for at least 4 weeks was found to significantly lower the blood pressure level
of a patient
with mild to moderate hypertension. Also, the composition of the present
invention
was found to be effective for such hypertensive patient. Further, an acetic
acid-containing beverage was verified to pose no problems in terms of safety.
All publications, patents, and patent applications cited herein are
incorporated
herein by reference in their entirety.
Industrial Applicability
27
CA 02477900 2004-08-31
The present invention provides a composition that can inhibit the elevation of
blood pressure and can be safely used with a sense of security. The
composition
according to the present invention that can inhibit the elevation of blood
pressure is
particularly effective for inhibiting the elevation of blood pressure of
people who have
borderline blood pressure or who are fated to develop essential hypertension.
28
