U.S. patent application number 12/848431 was filed with the patent office on 2010-11-18 for scavenger of in vivo harmful reactive oxygen species and/or free radicals.
Invention is credited to Wataru Murota, Ikuroh Ohsawa, Shigeo Ohta.
Application Number | 20100291228 12/848431 |
Document ID | / |
Family ID | 37757678 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100291228 |
Kind Code |
A1 |
Ohta; Shigeo ; et
al. |
November 18, 2010 |
SCAVENGER OF IN VIVO HARMFUL REACTIVE OXYGEN SPECIES AND/OR FREE
RADICALS
Abstract
An object of the present invention is to provide a scavenger of
in vivo harmful reactive oxygen species and/or free radicals, which
is capable of effectively reducing the concentrations of in vivo
reactive oxygen species and/or free radicals and exhibiting given
effects such as the suppression of aging process, the prevention of
geriatric or lifestyle-related disease, health promotion, and the
inhibition of oxidative stress by virtue of this reduction in the
concentrations of reactive oxygen species and/or free radicals. The
scavenger of in vivo harmful reactive oxygen species and/or free
radicals of the present invention comprises a liquid or gas
comprising at least a hydrogen molecule. This medium may further
comprise an oxygen molecule. Furthermore, this medium may comprise
water or an aqueous solution or may be a gas. The scavenger of
reactive oxygen species and/or free radicals can be used in the
treatment or prevention of a disorder attributed to reactive oxygen
species and/or free radicals.
Inventors: |
Ohta; Shigeo; (Kanagawa,
JP) ; Murota; Wataru; (Ishikawa, JP) ; Ohsawa;
Ikuroh; (Tokyo, JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
1030 15th Street, N.W.,, Suite 400 East
Washington
DC
20005-1503
US
|
Family ID: |
37757678 |
Appl. No.: |
12/848431 |
Filed: |
August 2, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11990649 |
Apr 9, 2008 |
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PCT/JP2006/316665 |
Aug 18, 2006 |
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12848431 |
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Current U.S.
Class: |
424/600 |
Current CPC
Class: |
A61K 33/00 20130101;
A61P 3/06 20180101; A61P 25/28 20180101; A23L 3/3427 20130101; A61P
25/00 20180101; A61P 9/00 20180101; A23L 33/10 20160801; A61P 35/00
20180101; A61P 25/16 20180101; A61P 29/00 20180101; A61P 9/10
20180101; A23L 2/52 20130101; A61P 3/00 20180101; A61P 27/02
20180101; A61P 21/00 20180101; A61P 39/06 20180101; A61K 33/00
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/600 |
International
Class: |
A61K 33/00 20060101
A61K033/00; A61P 25/00 20060101 A61P025/00; A61P 9/10 20060101
A61P009/10; A61P 27/02 20060101 A61P027/02; A61P 29/00 20060101
A61P029/00; A61P 3/00 20060101 A61P003/00; A61P 25/16 20060101
A61P025/16; A61P 25/28 20060101 A61P025/28; A61P 35/00 20060101
A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 19, 2005 |
JP |
2005-238572 |
Jun 6, 2006 |
JP |
2006-157827 |
Claims
1-33. (canceled)
34. A method of treating or preventing a disorder attributed to
reactive oxygen species and/or free radicals, which comprises
administering a therapeutic or preventive agent comprising a
scavenger of in vivo harmful reactive oxygen and/or free radicals
comprising a liquid comprising at least a hydrogen molecule at a
concentration of 1 to 4% (v/v) to a patient.
35. The method according to claim 34, wherein in vivo harmful
reactive oxygen and/or free radicals is a hydroxyl radical or
peroxynitrite.
36. The method according to claim 34, wherein the disorder
attributed to reactive oxygen species and/or free radicals is
selected from the group consisting of oxidative stress, oxidative
stress-induced cell death, and oxidative stress-induced
mitochondrial dysfunction.
37. The method according to claim 34, wherein the disorder
attributed to reactive oxygen species and/or free radicals is
selected from the group consisting of cerebral infarction,
myocardial infarction, arteriosclerosis, ischemia-reperfusion
injury, disorders caused by artificial dialysis, disorders caused
by organ transplantation, retinal degeneration in premature babies,
acute pneumonopathy, inflammation, and disturbance of lipid
metabolism.
38. The method according to claim 34, wherein the disorder
attributed to reactive oxygen species and/or free radicals is
myopathy after strenuous exercise or oxygen injury caused by oxygen
gas inhalation at a high concentration after exercise.
39. The method according to claim 34, wherein the disorder
attributed to reactive oxygen species and/or free radicals is
selected from the group consisting of neurodegenerative diseases
including Alzheimer's disease, Parkinson's disease, and ALS.
40. The method according to claim 34, wherein the disorder
attributed to reactive oxygen species and/or free radicals is
cancer.
Description
TECHNICAL FIELD
[0001] The present invention relates to a scavenger of reactive
oxygen species and/or free radicals, which reduces the
concentrations of in vivo harmful reactive oxygen species and/or
free radicals. Particularly, the present invention relates to a
scavenger of in vivo harmful reactive oxygen species and/or free
radicals, which is capable of effectively reducing the
concentrations of in vivo reactive oxygen species and/or free
radicals and exhibiting effects such as the suppression of aging
process, the prevention of geriatric or lifestyle-related diseases,
health promotion, and the protection against oxidative stress.
BACKGROUND ART
[0002] It is now accepted widely that the presence of reactive
molecules called reactive oxygen species or free radicals is
generally at least one of causes of many human health abnormalities
including aging, cancers, atherosclerosis, myocardial infarction,
attacks, viral infections, lung abnormalities, bowel diseases, and
neurodegenerative diseases and leads to aging and deterioration in
health. These molecules, which are usual by-products of
physiological reactions, are produced by enormous numbers of enzyme
reactions indispensable to oxygen metabolism, for example, cellular
respiration, or to the functions of the immune system (killing of
foreign substances) and metabolism.
[0003] Particularly, mitochondria, subcellular organelles, transfer
electrons in the electron transport system, while an electron leak
always occurs. 2% to 0.2% of oxygen molecules used in respiration
is reduced into reactive oxygen species. Furthermore, such reactive
oxygen species are universally generated even in general
environments. For example, ambient sources of reactive oxygen
species encompass smoke, ionizing radiation, atmospheric pollution,
chemical agents (carcinogens, many petrochemicals, biocides, dyes,
solvents, cell division inhibitors, etc.), toxic heavy metals, and
oxidized or rancid fats. Examples of the most general reactive
oxygen species include superoxide radicals, hydroxyl radicals,
singlet oxygen, and hydrogen peroxide. The reactive oxygen species
encompass, in the broad sense, nitrogen monoxide, peroxynitrite,
and lipid radicals such as alkoxyl radicals or lipid peroxyl
radicals. For example, superoxide, hydroxyl radicals, nitrogen
monoxide, and lipid radicals such as lipid peroxyl radicals or
alkoxyl radicals are free radical molecules.
[0004] Free radical molecules have oxidative toxicity that causes
structural damage to all biomolecules such as nucleic acids,
proteins, and lipids in living organisms. Such molecular damage
induces cellular abnormalities such as alteration in genetic codes,
abnormalities in enzyme reactions, and lipid membrane degeneration
and causes cytotoxicity. Thus, the free radical molecules have
strong oxidizing power. A disorder caused by this oxidizing power
is generally called oxidative stress. The accumulation of such
oxidative stress may cause neurological disorders, endocrine
disruption, increased allergy, vascular endothelial destruction,
joint destruction, and inflammation at an individual level.
[0005] The oxidative stress is caused by strong oxidizing ability
possessed by excessive reactive oxygen species or free radicals in
cells. Most of superoxide anion radicals (O.sub.2.sup.-.) are
generated by an electron leakage in the process from the Krebs
cycle to the electron transport system in mitochondria. Moreover,
O.sub.2.sup.-. is also generated by oxidase such as NADPH oxidase
or xanthine oxidase. O.sub.2.sup.-. is converted to hydrogen
peroxide by superoxide dismutase. This hydrogen peroxide is further
converted for detoxication to water by glutathione peroxidase or
catalase. Excessive O.sub.2.sup.-. reduces iron or copper, which is
a transition metal. These reduction products react with hydrogen
peroxide through the Fenton reaction to generate hydroxyl radicals
(.OH). .OH, which is the strongest reactive oxygen species,
indiscriminately reacts with nucleic acids, lipids, and proteins. A
mechanism for detoxicating this .OH is unknown. Thus, the removal
of .OH is the most important antioxidation process.
[0006] Protection from the poisonous influence of free radical
molecules is found in molecules called antioxidants from diverse
regions. In vivo free radical molecules and their related
by-products may be converted to less harmful products by
neutralization brought by antioxidants. Such antioxidants can be
enzymes (superoxide dismutase, catalase, glutathione peroxidase,
etc.), essential nutrients (beta-carotene, vitamin C and E,
selenium, etc.), enormous numbers of endogenous substances
(glutathione etc.), or food compounds (bioflavonoid etc.). Thus,
humans have some natural inhibitors against free radical molecules
in their bodies.
[0007] However, individuals suffer a great deal of damage by free
radical molecules, in spite of the presence of such in vivo free
radical inhibitors. Thus, it is obvious that effects including
nutritional supplementation for the prevention of oxidation induced
by free radical molecules delay human aging process and have big
advantages to health promotion and the prevention of disease.
[0008] Meanwhile, the oxidation-reduction potential of hydrogen
molecules is -0.42 V, and the oxidation-reduction potential of
oxygen molecules is +0.82 V. Thus, hydrogen molecules have the
intrinsic ability to reduce oxygen molecules. However, the
oxidation-reduction potentials are indicators for oxidizing or
reducing ability and merely indicate the final stage of
oxidation-reduction reactions in an equilibrium state. Whether or
not oxidation-reduction reactions actually proceed in vivo is
another story. In general, the rapid progress of reactions requires
catalysts or the like or requires promoting reactions at a high
temperature. In cells having complex structures, the progress of
oxidation-reduction reactions often requires their respective
specific enzymes. Thus, it is impossible to predict whether
hydrogen actually exhibits reducing power in vivo.
[0009] For example, according to the oxidation-reduction
potentials, hydrogen and oxygen should be converted to water
through a reaction. However, hydrogen and oxygen molecules
dissolved in water are not converted to water through a reaction.
Likewise, whether hydrogen molecules can reduce reactive oxygen
species or free radicals, as described above, can be confirmed only
by actual experiments. Judging from the oxidation-reduction
potentials, hydrogen molecules are supposed to reduce superoxide,
nitrogen monoxide, and hydrogen peroxide in an equilibrium state.
Meanwhile, superoxide, hydrogen peroxide, and nitrogen monoxide
have been demonstrated to play roles indispensable to living
bodies. These roles are killing effects on invading bacteria,
immune functions, defensive mechanisms against cancers,
vascularization, vasodilation, spermatogenesis, neurotransmission,
and so on. Thus, if hydrogen molecules rapidly eliminate these free
radicals or reactive oxygen species in vivo through reduction, the
hydrogen molecules should sometimes be harmful.
[0010] Water with a low oxidation-reduction potential has
heretofore been prepared by electrolysis (see Patent Documents 1 to
3 below) or by dissolving hydrogen under pressure (see Patent
Document 4 below). Of them, an aqueous drink with a low
oxidation-reduction potential prepared by an electrolysis method
merely exhibits alkaline properties attributed to OH.sup.- ions and
does not contain hydrogen gas at a saturated concentration or
higher. Such an alkaline aqueous drink has reducing power
attributed to the OH.sup.- ions and therefore apparently exhibits
reducing properties. However, this aqueous drink results in a high
oxidation-reduction potential, when rendered neutral. This means
that it exhibits apparent reducing properties. Moreover, the
drinking of alkaline solutions in large amounts presents a health
problem. Particularly, such alkaline solutions put a severe strain
on the kidney and are therefore harmful to persons with renal
damage. On the other hand, the alkaline solutions, if in
appropriate amounts, are observed to have a few effects on persons
with gastric hyperacidity. However, these effects are merely
effects brought by the neutralization of gastric acid by the
alkaline solutions and are not the effects of hydrogen gas or
reducing power.
[0011] Moreover, another known method comprises mixing metal
magnesium into an aqueous drink to thereby obtain reductive water.
In this case, magnesium and OH.sup.- ions are generated
simultaneously with hydrogen gas. Therefore, this reductive water
is alkaline. Magnesium ions, if in appropriate amounts, can be
expected to have health maintenance effects on human bodies,
because they have been applied to laxatives and the like. However,
the ingestion of such alkaline aqueous drinks in large amounts, as
already described, works to inhibit the body function of being
constantly neutral and is therefore dangerous. Rather, a drink
prepared by simply dissolving hydrogen gas does not exhibit
alkaline properties and therefore probably has higher safety.
[0012] Patent Document 1: JP Patent Publication (Kokai) No.
2001-145880A (2001)
[0013] Patent Document 2: JP Patent Publication (Kokai) No.
2001-137852A (2001)
[0014] Patent Document 3: JP Patent Publication (Kokai) No.
2002-254078A (2002)
[0015] Patent Document 4: JP Patent Publication (Kokai) No.
2004-230370A (2004)
DISCLOSURE OF THE INVENTION
[0016] Free radical molecules, as described above, have been known
to have strong oxidizing power and place oxidative stress on living
bodies. It has heretofore been suggested that polyphenols,
vitamins, and the like are effective for the prevention of
oxidative stress. Each of vitamin C and E, which have been accepted
and ingested most widely for the prevention of this oxidative
stress, is highly safe and inexpensive. However, vitamin C is
soluble in water, whereas vitamin E is soluble in fat. Therefore,
both of these vitamins cannot easily reach the internal regions of
cells. Thus, antioxidants that are capable of widely penetrating
into the internal and external regions of cells have been
demanded.
[0017] Meanwhile, not only are free radicals harmful, but also they
have been demonstrated to play roles indispensable to living
bodies. These roles are killing effects on invading bacteria,
immune functions, defensive mechanisms against cancer,
vascularization, vasodilation, spermatogenesis, neurotransmission,
and so on. These roles are brought by superoxide, hydrogen
peroxide, and nitrogen monoxide. Thus, if a substance capable of
selectively removing only highly reactive and highly cytotoxic free
radical species such as hydroxyl radicals, peroxynitrite, and lipid
peroxyl radicals can be found, such a substance can be used safely
in defense against oxidative stress and is therefore desirable.
[0018] Antioxidants including antioxidative vitamin, as described
above, have heretofore been utilized in health promotion as agents
for eliminating free radicals or reactive oxygen species. However,
these agents cannot always easily reach the internal regions of
cells. Alternatively, even if the reductive water as disclosed in
Patent Documents 1 to 4 above is drank, it is obvious that this
reductive water is not capable of widely penetrating into the
internal and external regions of cells, with its reducing
properties maintained. In addition, the structures and components
of living bodies are complicated and are not homogeneous systems.
Therefore, the effects of the reductive water cannot easily be
predicted.
[0019] The present inventors have considered that hydrogen
molecules in a gas state are taken up into living bodies and
rapidly distributed into cells, and such hydrogen molecules can be
expected to eliminate free radicals at a point in time when the
free radicals are generated. Thus, the present inventors have
conducted various studies and have consequently completed the
present invention by confirming that hydrogen molecules can
alleviate damage caused by free radicals in cells; hydrogen
molecules are actually taken up into the bodies of animals
including humans; and hydrogen actually alleviates oxidative stress
in the bodies of animals.
[0020] Specifically, an object of the present invention is to
provide a scavenger of free radicals, which is capable of
effectively reducing the concentrations of in vivo free radicals
and exhibiting given effects such as the suppression of aging
process, the prevention of geriatric or lifestyle-related diseases,
health promotion, and the inhibition of oxidative stress by virtue
of this reduction in the concentrations of free radicals, and to
provide an apparatus for the inhalation of this scavenger of free
radicals.
[0021] O.sub.2.sup.-. or hydrogen peroxide with a low concentration
functions as a signal molecule for controlling a large number of
signal transduction cascades, in spite of its cytotoxicity, and has
physiologically important functions such as the control of
physiological processes, for example, apoptosis, cell growth, and
cell differentiation. Hydrogen peroxide with a high concentration
is converted to hypochlorous acid by myeloperoxidase, and this
hypochlorous acid exhibits antimicrobial effects. Furthermore,
nitrogen monoxide radicals, which are neurotransmitters, play an
important role in vasodilation. By contrast, radicals having
unilateral cytotoxicity, such as .OH, can be neutralized by
hydrogen molecules without inhibiting basic physiological
activities possessed by reactive oxygen species as described above.
The present inventors have completed the present invention by
finding that hydrogen molecules can alleviate .OH-induced
cytotoxicity without influencing other reactive oxygen species, and
hydrogen molecules have the potential of being applicable as an
antioxidant to medical care.
[0022] Specifically, the present invention is as follows:
[0023] [1] A scavenger of in vivo harmful reactive oxygen species
and/or free radicals comprising a liquid comprising at least a
hydrogen molecule.
[0024] [2] The scavenger of in vivo harmful reactive oxygen species
and/or free radicals according to [1], wherein the liquid
comprising at least a hydrogen molecule comprises an aqueous
solution.
[0025] [3] The scavenger of in vivo harmful reactive oxygen species
and/or free radicals according to [2], wherein the hydrogen
molecule comprised therein is supersaturated.
[0026] [4] The scavenger of in vivo harmful reactive oxygen species
and/or free radicals according to [2] or [3], further comprising an
oxygen molecule.
[0027] [5] The scavenger of in vivo harmful reactive oxygen species
and/or free radicals comprising a gas comprising at least a
hydrogen molecule.
[0028] [6] The scavenger of in vivo harmful reactive oxygen species
and/or free radicals according to [5], wherein the gas comprising
at least a hydrogen molecule comprises a mixed gas of hydrogen and
oxygen gases.
[0029] [7] The scavenger of in vivo harmful reactive oxygen species
and/or free radicals according to [5], wherein the gas comprising
at least a hydrogen molecule comprises a mixed gas of hydrogen,
oxygen, and inactive gases.
[0030] [8] The scavenger of in vivo harmful reactive oxygen species
and/or free radicals according to [5], wherein the gas comprising
at least a hydrogen molecule comprises a mixed gas of a hydrogen
gas and air.
[0031] [9] The scavenger of in vivo harmful reactive oxygen species
and/or free radicals according to [5], wherein the gas comprising
at least a hydrogen molecule comprises a mixed gas of hydrogen and
anesthetic gases.
[0032] [10] The scavenger of in vivo harmful reactive oxygen
species and/or free radicals according to any of [5] to [9],
wherein the removing agent comprises the hydrogen gas at a
concentration of 1 to 4% (v/v).
[0033] [11] The scavenger of in vivo harmful reactive oxygen
species and/or free radicals according to any of [5] to [9],
wherein the removing agent comprises the hydrogen gas at a
concentration of 1.5 to 2.5% (v/v).
[0034] [12] The scavenger of in vivo harmful reactive oxygen
species and/or free radicals according to any of [1] to [11],
wherein the active oxygen and/or free radicals are active oxygen
and/or free radicals selected from the group consisting of hydroxyl
radicals, peroxynitrite, alkoxy radicals, and lipid peroxyl
radicals.
[0035] [13] The scavenger of in vivo harmful reactive oxygen
species and/or free radicals according to [12], wherein the active
oxygen and/or free radicals are hydroxyl radicals.
[0036] [14] A pharmaceutical composition comprising the scavenger
of in vivo harmful reactive oxygen species and/or free radicals
according to any of [1] to [13].
[0037] [15] A therapeutic or preventive agent for a disorder
attributed to reactive oxygen species and/or free radicals, the
therapeutic or preventive agent comprising the scavenger of in vivo
harmful reactive oxygen species and/or free radicals according to
any of [1] to [13].
[0038] [16] The therapeutic or preventive agent according to [15],
wherein the disorder attributed to reactive oxygen species and/or
free radicals is selected from the group consisting of oxidative
stress, oxidative stress-induced cell death, and oxidative
stress-induced mitochondrial dysfunction.
[0039] [17] therapeutic or preventive agent according to [15],
wherein the disorder attributed to reactive oxygen species and/or
free radicals is selected from the group consisting of cerebral
infarction, myocardial infarction, arteriosclerosis,
ischemia-reperfusion injury, disorders caused by organ
transplantation, retinal degeneration in premature babies, acute
pneumonopathy, disorders caused by artificial dialysis,
inflammation, and disturbance of lipid metabolism.
[0040] [18] The therapeutic or preventive agent according to [15],
wherein the disorder attributed to reactive oxygen species and/or
free radicals is myopathy after strenuous exercise or oxygen injury
caused by oxygen gas inhalation at a high concentration after
exercise.
[0041] [19] The therapeutic or preventive agent according to [15],
wherein the disorder attributed to reactive oxygen species and/or
free radicals is selected from the group consisting of
neurodegenerative diseases including Alzheimer's disease,
Parkinson's disease, and ALS.
[0042] [20] The therapeutic or preventive agent according to [15],
wherein the disorder attributed to reactive oxygen species and/or
free radicals is cancer.
[0043] [21] A drink suitable for the alleviation or prevention of a
disorder attributed to reactive oxygen species and/or free
radicals, the drink comprising the scavenger of reactive oxygen
species and/or free radicals comprising a liquid comprising at
least a hydrogen molecule according to any of [1] to [4].
[0044] [22] A drink with a label stating that the drink is used for
the scavenging or reduction of in vivo reactive oxygen species
and/or free radicals, the drink comprising the scavenger of
reactive oxygen species and/or free radicals comprising a liquid
comprising at least a hydrogen molecule according to any of [1] to
[4].
[0045] [23] A drink with a label stating that the drink is used for
the alleviation or prevention of a disorder attributed to reactive
oxygen species and/or free radicals, the drink comprising the
scavenger of reactive oxygen species and/or free radicals
comprising a liquid comprising at least a hydrogen molecule
according to any of [1] to [4].
[0046] [24] The drink according to any of [21] to [23], wherein the
drink is a health food, functional food, nutritional supplementary
food, supplement, or food for specified health use.
[0047] [25] A container comprising the scavenger of in vivo harmful
reactive oxygen species and/or free radicals according to any of
[5] to [10].
[0048] [26] The container according to [25], wherein the container
is a hydrogen gas cylinder.
[0049] [27] An apparatus for supplying a scavenger of reactive
oxygen species and/or free radicals to a subject in need of the
treatment or prevention of a disorder attributed to reactive oxygen
species and/or free radicals, the apparatus comprising: a container
comprising the scavenger of in vivo harmful reactive oxygen species
and/or free radicals comprising a gas comprising at least a
hydrogen molecule according to any of [5] to [10]; gas inhalation
means; and a supply duct for supplying a gas in the container to
the inhalation means.
[0050] [28] The apparatus for supplying the scavenger of reactive
oxygen species and/or free radicals to a subject in need of the
treatment or prevention of a disorder attributed to reactive oxygen
species and/or free radicals according to [27], the apparatus
further comprising a container comprising at least one gas selected
from the group consisting of oxygen gas, inactive gases, and air,
wherein the scavenger of in vivo harmful reactive oxygen species
and/or free radicals comprising a gas comprising at least a
hydrogen molecule, and the at least one gas selected from the group
consisting of oxygen gas, inactive gases, and air are supplied
separately or as a mixture to the gas inhalation means.
[0051] [29] The apparatus for supplying the scavenger of reactive
oxygen species and/or free radicals to a subject in need of the
treatment or prevention of a disorder attributed to reactive oxygen
species and/or free radicals according to [27] or [28], the
apparatus further comprising a container comprising an anesthetic
gas, wherein the scavenger of in vivo harmful reactive oxygen
species and/or free radicals comprising a gas comprising at least a
hydrogen molecule, and the anesthetic gas are supplied separately
or as a mixture to the gas inhalation means.
[0052] [30] The apparatus for supplying the scavenger of reactive
oxygen species and/or free radicals to a subject in need of the
treatment or prevention of a disorder attributed to reactive oxygen
species and/or free radicals according to any of [27] to [29],
wherein the container comprising the scavenger of in vivo harmful
reactive oxygen species and/or free radicals comprising a gas
comprising at least a hydrogen molecule comprises 1 to 4% hydrogen
gas.
[0053] [31] The apparatus for supplying the scavenger of reactive
oxygen species and/or free radicals to a subject in need of the
treatment or prevention of a disorder attributed to reactive oxygen
species and/or free radicals according to any of [27] to [30],
wherein the container comprising the scavenger of in vivo harmful
reactive oxygen species and/or free radicals comprising a gas
comprising at least a hydrogen molecule is a hydrogen gas
cylinder.
[0054] [32] The apparatus for supplying the scavenger of reactive
oxygen species and/or free radicals to a subject in need of the
treatment or prevention of a disorder attributed to reactive oxygen
species and/or free radicals according to any of [27] to [31],
wherein the gas inhalation means is a gas inhalation mask.
[0055] [33] The apparatus for supplying the scavenger of reactive
oxygen species and/or free radicals to a subject in need of the
treatment or prevention of a disorder attributed to reactive oxygen
species and/or free radicals according to any of [27] to [31],
wherein the gas inhalation means is a sealed chamber, and the
scavenger of in vivo harmful reactive oxygen species and/or free
radicals comprising a gas comprising at least a hydrogen molecule
is supplied into the sealed chamber to thereby supply the scavenger
of reactive oxygen species and/or free radicals to the subject in
the sealed chamber.
[0056] The present specification encompasses the contents described
in the specifications and/or drawings of Japanese Patent
Application Nos. 2005-2385725 and 2006-157827 that serve as a basis
for the priority of the present application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 is a phase contrast microscopic image of antimycin
A-treated PC 12 cells in a hydrogen-containing medium;
[0058] FIG. 2 is a graph where the number of viable PC12 cells is
compared between a hydrogen-containing medium and a hydrogen-free
medium after antimycin A treatment;
[0059] FIG. 3 is a graph where the number of viable PC12 cells is
compared between a hydrogen-containing medium and a hydrogen-free
medium after menadione treatment;
[0060] FIG. 4 is a graph showing time-dependent changes in the
number of viable PC12 cells in a hydrogen-containing medium and a
hydrogen-free medium after antimycin A or menadione treatment;
[0061] FIG. 5 is a graph where the number of viable PC12 cells is
compared between a hydrogen-containing medium and a vitamin
E-supplemented hydrogen-free medium after antimycin A or menadione
treatment;
[0062] FIG. 6 is a graph where the number of viable PC12 cells is
compared among replacements of a hydrogen-containing medium after
antimycin A treatment;
[0063] FIG. 7 is a graph showing the influence of changes in
dissolved hydrogen concentration on the number of viable PC12 cells
after antimycin A treatment;
[0064] FIG. 8 is an image of mitochondria in antimycin A-treated
PC12 cells in a hydrogen-containing medium;
[0065] FIG. 9 is a graph showing the 4-HNE accumulation inhibitory
effects of hydrogen water in model animals with accelerated
oxidative stress;
[0066] FIG. 10 is a graph showing time-dependent changes in the
amount of 4-HNE accumulated in the blood of hydrogen
water-administered model animals with accelerated oxidative
stress;
[0067] FIG. 11 is an image of hematoxylin-eosin-stained liver
slices showing the ischemia-reperfusion injury alleviating effects
of hydrogen gas inhalation;
[0068] FIG. 12 is a graph showing time-dependent changes in the
breath hydrogen concentrations of 4 persons resulting from the
drinking of hydrogen water;
[0069] FIG. 13 is a diagram showing the removal of hydroxyl
radicals by dissolved hydrogen molecules at room temperature under
neutral conditions;
[0070] FIG. 14 is a diagram showing the removal of hydroxyl
radicals by hydrogen molecules in PC12 cells and showing results of
observing HPF fluorescence with a confocal laser scanning
microscope;
[0071] FIG. 15 is a graph showing the removal of hydroxyl radicals
by hydrogen molecules in PC12 cells and showing results of
quantifying HPF fluorescence intensity;
[0072] FIG. 16 is a diagram showing PC12 cells in which guanine
oxidation has been inhibited by hydrogen molecules;
[0073] FIG. 17 is a graph showing the inhibition of guanine
oxidation in PC12 cells by hydrogen molecules;
[0074] FIG. 18 is a diagram showing PC12 cells in which HNE
production has been inhibited by hydrogen molecules;
[0075] FIG. 19 is a graph showing the inhibition of HNE production
in PC12 cells by hydrogen molecules;
[0076] FIG. 20 is a diagram showing the neuronal cell protective
effects of hydrogen molecules against in vitro ischemia and showing
results of staining cells with HPF after 10 minutes of
reperfusion;
[0077] FIG. 21 is a graph showing the neuronal cell protective
effects of hydrogen molecules against in vitro ischemia and showing
results of measuring HPF fluorescence intensity as to 100 cells by
using NIH Image software;
[0078] FIG. 22 is a diagram showing the increasing effects of
hydrogen molecules on the number of viable neuronal cells;
[0079] FIG. 23 is a graph showing increases in the viability of
neuronal cells by hydrogen molecules in terms of the number of
living cells;
[0080] FIG. 24 is a graph showing increases in the viability of
neuronal cells by hydrogen molecules in terms of mitochondrial
enzyme activity;
[0081] FIG. 25 is a diagram showing the protective effects of the
inhibition of oxidative stress by inhaled hydrogen gas against
ischemia-reperfusion injury and showing results of staining slices
with TTC;
[0082] FIG. 26 is a graph showing the protective effects of the
inhibition of oxidative stress by inhaled hydrogen gas against
ischemia-reperfusion injury and showing a brain infarct volume;
[0083] FIG. 27 is diagram and graph showing protective effects
against ischemia-reperfusion injury in varying hydrogen gas
inhalation periods;
[0084] FIG. 28 is a diagram showing the protective effects of the
inhibition of oxidative stress by inhaled hydrogen gas against
ischemia-reperfusion injury and showing results of staining, with
hematoxylin-eosin, brain coronal sections after one week;
[0085] FIG. 29 is a graph showing the protective effects of the
inhibition of oxidative stress by inhaled hydrogen gas against
ischemia-reperfusion injury and showing results of calculating an
infarct volume after one week, with a visually pale pink region
obtained by hematoxylin-eosin staining as an infarction region;
[0086] FIG. 30 is a graph showing changes in the body temperatures
or body weights of hydrogen-treated or hydrogen-untreated rats;
[0087] FIG. 31 is diagram and graph showing results of
immunostaining the brain with anti-8-OH-G antibody after
ischemia-reperfusion injury in the presence or absence of hydrogen
gas;
[0088] FIG. 32 is diagram and graph showing results of
immunostaining the brain with anti-HNE antibody after
ischemia-reperfusion injury in the presence or absence of hydrogen
gas;
[0089] FIG. 33 is diagram and graph showing results of
immunostaining the brain with anti-GFAP antibody after
ischemia-reperfusion injury in the presence or absence of hydrogen
gas;
[0090] FIG. 34 is a diagram showing results of immunostaining the
brain with microglia-specific anti-Iba-I antibody after
ischemia-reperfusion injury in the presence or absence of hydrogen
gas;
[0091] FIG. 35 is a graph showing the number of immunopositive
cells as a result of staining the brain with microglia-specific
anti-Iba-I antibody after ischemia-reperfusion injury in the
presence or absence of hydrogen gas;
[0092] FIG. 36 is a diagram showing an apparatus of the present
invention;
[0093] FIG. 37 is a diagram showing the genotypes of mice born by
the crossing between SOD (+/-) mice;
[0094] FIG. 38 is a graph showing the effects of hydrogen water on
the genotypes of mice born by the crossing between SOD (+/-)
mice;
[0095] FIG. 39 is a diagram showing an experimental method for
evaluating the carcinogenesis inhibitory effects of hydrogen water;
and
[0096] FIG. 40 is a diagram showing the number of GST-P-positive
cell foci in the livers of hydrogen water-administered or hydrogen
water-unadministered mice (FIG. 40a) and the areas of the positive
foci (FIG. 40b).
BEST MODE FOR CARRYING OUT THE INVENTION
[0097] Hydrogen can remove in vivo reactive oxygen species and/or
free radicals through reduction in a living body.
[0098] Reactive oxygen species refer to molecules that have the
high ability to oxidize other substances by stronger oxidizing
power than that of oxygen and refer to singlet oxygen, hydrogen
peroxide, ozone, superoxide radicals, hydroxyl radicals,
peroxynitrite, and the like. Free radicals encompass nitrogen
monoxide, alkoxyl radicals (lipid radicals; L.), lipid peroxyl
radicals (alkylperoxyl radicals; LOO.), and the like, in addition
to the reactive oxygen species such as superoxide or hydroxyl
radicals. In the present invention, the free radicals also mean, in
the broad sense, free radical molecules including reactive oxygen
species.
[0099] Of them, hydroxyl radicals, peroxynitrite, alkoxy radicals,
and lipid peroxyl radicals are bad reactive oxygen species or free
radicals with cytotoxicity that have harmful effects on living
bodies and can be one of causes of various disorders. In the
present invention, hydrogen exclusively removes these harmful
reactive oxygen species and/or free radicals in a short time
through reduction. On the other hand, reactive oxygen species
and/or free radicals other than the bad reactive oxygen species
and/or free radicals participate in signal transduction and so on
in vivo and have useful functions. Such reactive oxygen species
and/or free radicals are referred to as good reactive oxygen
species and/or free radicals. The good reactive oxygen species
and/or free radicals are not easily removed by even hydrogen. A
scavenger of in vivo harmful reactive oxygen species and/or free
radicals comprising a liquid comprising at least a hydrogen
molecule according to the present invention exclusively removes
only bad reactive oxygen species and/or free radicals harmful to
living bodies. In this context, oxidative stress refers to a
disorder in living bodies brought by the oxidizing power of
reactive oxygen species and/or free radicals.
[0100] Hydrogen molecules are capable of passing through even cell
membranes. Therefore, they enter into cells and remove reactive
oxygen species and/or free radicals in the cells. Moreover, the
hydrogen molecules can also enter into nuclei and mitochondria.
Thus, they can protect genes from reactive oxygen species and/or
free radicals and can inhibit cancer. Moreover, hydrogen is capable
of passing through the blood-brain barrier and can therefore
protect the brain from oxidative stress.
[0101] The liquid comprising at least a hydrogen molecule is
characterized by comprising an aqueous solution. Pure water,
ion-exchanged water, distilled water, a saline, and the like may be
used as a medium forming this aqueous solution. Furthermore, the
scavenger of in vivo harmful free radicals obtained by using pure
water, ion-exchanged water, or distilled water as the medium may be
added for drinking to general aqueous drinks, for example, mineral
water, juice, coffee, and tea. In this context, the drink
encompasses health foods, foods for specified health use, foods
with nutrient function claims, nutritional supplementary foods,
supplements, and the like for drinking. In this context, the foods
for specified health use refer to foods with a label stating that
the food can be ingested for specified health purposes in diets and
expected to satisfy the health purposes by this ingestion. These
drinks may be provided with a label stating that the drink is used
for the removal or reduction of in vivo reactive oxygen species
and/or free radicals or a label stating that the drink is used for
the alleviation or prevention of a disorder attributed to free
radicals. Furthermore, the drinks may be provided with a label
specifically defining disorders attributed to reactive oxygen
species and/or free radicals and stating that the drink is used for
the alleviation or prevention of the defined disorders or may be
provided with a label stating that the drink is useful for
antiaging or antioxidation. Furthermore, the liquid comprising
hydrogen may be used in cosmetics for similar purposes.
[0102] The hydrogen molecule, which may be supersaturated, can be
dissolved in water or an aqueous solution for a certain period. The
water or aqueous solution comprising such a supersaturated hydrogen
molecule can be produced by dissolving hydrogen gas in water or an
aqueous solution under pressure and then removing the pressure. For
example, the aqueous solution may be placed under hydrogen gas
pressure of 0.4 MPa or higher for a few hours, preferably, 1 to 3
hours. The scavenger of in vivo harmful reactive oxygen species
and/or free radicals in an aqueous solution form can also be used
for drinking or can also be used for intravenous injection in the
form of a saline comprising oxygen coexisting therewith. In this
case, administration may be administration using catheters or
administration using injection. After administration, the hydrogen
ingested into living bodies is mostly absorbed into the living
bodies and distributed into the whole bodies via blood. The
distributed hydrogen exhibits its effects therein and is then
excreted together with breath.
[0103] Hydrogen can be dissolved in an amount of approximately 17.5
mL per L of water (approximately 1.6 ppm, approximately 0.8 mM)
under conditions involving a hydrogen pressure of 1 atm and room
temperature. The scavenger of reactive oxygen species and/or free
radicals in an aqueous solution form according to the present
invention comprises 10 mL or higher, preferably 15 mL or higher,
particularly preferably 17.5 mL or higher of hydrogen molecule per
L of aqueous solution. Alternatively, the scavenger of reactive
oxygen species and/or free radicals in an aqueous solution form
according to the present invention comprises 1 ppm or higher,
preferably 1.5 ppm or higher hydrogen molecule. Alternatively, the
scavenger of reactive oxygen species and/or free radicals in an
aqueous solution form according to the present invention comprises
0.1 mM or higher, preferably 0.4 mM or higher, more preferably 0.6
mM, particularly preferably 0.8 mM or higher hydrogen.
[0104] Moreover, the scavenger of reactive oxygen species and/or
free radicals in an aqueous solution form according to the present
invention may comprise an oxygen molecule. The hydrogen and oxygen
molecules coexist in the aqueous solution. However, the hydrogen
and oxygen molecules do not immediately react with each other even
in a mixed state and can coexist stably. However, when gases
contain a large amount of oxygen molecules, it is preferred for
securing safety that a hydrogen content should be set to less than
4.7% (v/v) of the total gas amount. In a use environment with no
safety problem, it is preferred that a hydrogen content should be
as high a concentration as possible. The scavenger of reactive
oxygen species and/or free radicals comprising oxygen may also be
used for drinking or for intravenous injection in the form of a
saline. In administration using injection, such a scavenger
comprising oxygen causes less damage to tissues in living bodies
than that caused by an oxygen molecule-free scavenger, because the
living bodies are not placed under local oxygen-deficient
conditions.
[0105] For a drink of the present invention, it is preferred that
the drink should be stored in a container made of a raw material
impermeable to hydrogen, preferably, aluminum or the like. Examples
of such a container include aluminum pouches. Moreover, it is
preferred that the drink should be stored at a low temperature,
because a larger amount of hydrogen is dissolved at a lower
temperature.
[0106] The scavenger of in vivo harmful reactive oxygen species
and/or free radicals comprising at least a hydrogen molecule
according to the present invention may be in a gas form. In this
case, a hydrogen concentration is 1 to 4.7%, preferably 1 to 4.5%,
more preferably 1 to 4% (v/v), even more preferably 1.5 to 2.5%
(v/v), still even more preferably approximately 2%. It is desired
for securing safety that a hydrogen gas content should be less than
approximately 4.7% (v/v). However, the hydrogen gas content can
also be increased more under safe conditions that make
consideration to generate no static electricity under sealed
conditions. The scavenger of in vivo harmful reactive oxygen
species and/or free radicals comprising at least a hydrogen
molecule according to the present invention may further comprise an
oxygen gas and/or other inactive gases. Such a removing agent
comprising an oxygen gas comprises a mixed gas of hydrogen and
oxygen gases. The oxygen gas is consumed for respiration. A
nitrogen gas, helium gas, argon gas, or the like may be used as the
inactive gas. An inexpensive nitrogen gas is desirable. The content
of this inactive gas can be set arbitrarily by those skilled in the
art so as not to be too large. In consideration of an oxygen gas
concentration for respiration, it is preferred that the inactive
gas content should be 80% (v/v) or lower. Furthermore, the
scavenger of in vivo harmful reactive oxygen species and/or free
radicals comprising at least a hydrogen molecule according to the
present invention may be a mixed gas of a hydrogen gas and air.
Such a mixed gas can be produced easily by appropriately mixing
hydrogen gas with air. Furthermore, the scavenger of in vivo
harmful reactive oxygen species and/or free radicals comprising a
hydrogen molecule according to the present invention may comprise
an anesthetic gas. In this case, the scavenger of in vivo harmful
reactive oxygen species and/or free radicals comprises a mixed gas
of hydrogen and anesthetic gases. Examples of the anesthetic gas
include nitrous oxide.
[0107] The scavenger of in vivo harmful reactive oxygen species
and/or free radicals in a gas form comprising at least a hydrogen
molecule according to the present invention is placed in a
pressure-resistant container, for example, a gas cylinder. The
present invention encompasses even a container comprising the
scavenger of in vivo harmful reactive oxygen species and/or free
radicals in a gas form comprising at least a hydrogen molecule.
[0108] The scavenger of in vivo harmful reactive oxygen species
and/or free radicals in a gas form comprising at least a hydrogen
molecule according to the present invention can be inhaled by a
subject. The inhalation can be performed using inhalation means.
The scavenger of in vivo harmful reactive oxygen species and/or
free radicals may be inhaled via the inhalation means through a
duct from the container comprising the scavenger. The inhalation
means is not limited. Examples thereof include inhalation masks.
The mask, preferably, can cover both the mouth and nose of a
subject. Furthermore, the inhalation means may be a small sealed
chamber that is hermetically sealed. The small chamber has a size
that is large enough to accommodate a subject therein. The
scavenger of in vivo harmful reactive oxygen species and/or free
radicals in a gas form comprising at least a hydrogen molecule
according to the present invention can be supplied to the small
chamber accommodating a subject therein and thereby inhaled by the
subject. One example of such a small chamber includes enclosed
beds. A subject lying in the bed can inhale the scavenger of in
vivo harmful reactive oxygen species and/or free radicals in a gas
form comprising at least a hydrogen molecule according to the
present invention.
[0109] The present invention also encompasses a composition such as
a pharmaceutical composition comprising, as an active ingredient, a
scavenger of in vivo harmful reactive oxygen species and/or free
radicals comprising a liquid comprising at least a hydrogen
molecule. In this case, the scavenger of reactive oxygen species
and/or free radicals may be in a liquid form or in a gas form. Such
a composition can be used for the prevention or treatment of a
disorder attributed to reactive oxygen species and/or free
radicals.
[0110] In this context, the disorder attributed to reactive oxygen
species and/or free radicals refers to a disorder, disease,
dysfunction, or the like, one of causes of which is reactive oxygen
species and/or free radicals. Specific examples thereof include
oxidative stress, oxidative stress-induced cell death, and
oxidative stress-induced mitochondrial dysfunction. Alternative
examples thereof include cerebral infarction, myocardial
infarction, ischemia-reperfusion injury caused by operation or the
like, disorders caused by organ transplantation, retinal
degeneration in premature babies, acute pneumonopathy, disorders
caused by artificial dialysis, and inflammation. Further examples
thereof include myopathy after strenuous exercise and oxygen injury
caused by oxygen gas inhalation at a high concentration after
exercise. Further examples thereof include neurodegenerative
diseases such as Alzheimer's disease, Parkinson's disease, and ALS.
Furthermore, the scavenger of reactive oxygen species and/or free
radicals of the present invention can also be used in the
prevention or treatment of cancer by preventing nuclear DNA
oxidation. Furthermore, reactive oxygen species and/or free
radicals participate in aging process. The scavenger of reactive
oxygen species and/or free radicals of the present invention is
capable of exhibiting antiaging effects by the treatment or
prevention of various disorders associated with aging.
Specifically, the scavenger of reactive oxygen species and/or free
radicals of the present invention can also be used as a composition
useful for antiaging. Of these disorders, cerebral infarction and
myocardial infarction are caused by reactive oxygen species and/or
free radicals occurring due to blood flow under oxygen-deficient
conditions. Alternatively, ischemia-reperfusion injury caused by
operation is caused by reactive oxygen species and/or free radicals
occurring due to blood flow that is stopped during operation and
restarted at the completion of operation. Moreover, in organ
transplantation, an organ to be transplanted is placed under
oxygen-deficient conditions, and disorders are caused by reactive
oxygen species and/or free radicals occurring due to blood flow
after transplantation. Retinal degeneration in premature babies is
a disorder in the retina of premature babies that is caused by
reactive oxygen species easily occurring due to high-concentration
oxygen therapy for the premature babies. Acute pneumonopathy is
caused by reactive oxygen species and/or free radicals occurring
due to high-concentration oxygen therapy. Furthermore, immediately
after strenuous exercise, disorders are caused by reactive oxygen
species and/or free radicals occurring due to oxygen supply by the
termination of the exercise after oxygen-deficient conditions.
[0111] In the present invention, prevention or treatment
encompasses the curing of a disorder by the administration of the
scavenger of reactive oxygen species and/or free radicals to a
subject actually having the disorder, the reduction of risk of a
disorder by the administration of the scavenger of reactive oxygen
species and/or free radicals to a subject at risk of the disorder,
the alleviation of the degree of the disorder, and the suppression
of the disorder.
[0112] The timing of administration or ingestion of these
compositions is not limited. The compositions may be administered
or ingested, when the disorder actually occurs. Alternatively, the
compositions may be administered or ingested immediately before or
after the in vivo occurrence of reactive oxygen species and/or free
radicals. For example, it is preferred that the administration or
ingestion should be performed after strenuous exercise, in oxygen
ingestion after strenuous exercise, after the occurrence of mental
stress or physical stress, and so on. Furthermore, cigarette smoke
contains a large amount of superoxide radicals, which are in turn
converted to hydroxyl radicals in vivo. Gene damage caused by these
hydroxyl radicals is allegedly one of causes of lung cancer. Thus,
the scavenger of reactive oxygen species and/or free radicals of
the present invention can be used in the prevention of lung cancer
in smokers.
[0113] The scavenger of reactive oxygen species and/or free
radicals in a liquid form can be administered by oral
administration or intravenous injection. Alternatively, for local
disease such as cancer, the scavenger of reactive oxygen species
and/or free radicals in a liquid form may be administered directly
to the cancer site. For example, to visceral cancer, the scavenger
of reactive oxygen species and/or free radicals in a liquid form
may be administered by intraperitoneal injection. Alternatively,
the scavenger of reactive oxygen species and/or free radicals in a
gas form may be administered by inhalation.
[0114] The present invention further encompasses an apparatus for
supplying a scavenger of reactive oxygen species and/or free
radicals to a subject in need of the treatment or prevention of a
disorder attributed to reactive oxygen species and/or free
radicals, the apparatus comprising: a container comprising a
scavenger of in vivo harmful reactive oxygen species and/or free
radicals comprising a gas comprising at least a hydrogen molecule;
gas inhalation means; and a supply duct for supplying a gas in the
container to the inhalation means. The container is, for example, a
hydrogen gas cylinder. Moreover, examples of the gas inhalation
means include inhalation masks and sealed small chambers, as
described above. The apparatus may further comprise a container
comprising at least one gas selected from the group consisting of
oxygen gas, inactive gases, air, and anesthetic gases. In this
case, the scavenger of in vivo harmful reactive oxygen species
and/or free radicals comprising a gas comprising at least a
hydrogen molecule, and the at least one gas selected from the group
consisting of oxygen gas, inactive gases, and air may be supplied
separately or as a mixture to the gas inhalation means. FIG. 36
shows a schematic diagram of the apparatus of the present
invention. In the diagram, the apparatus comprises gas inhalation
means 1, a container 2 comprising a scavenger of in vivo harmful
reactive oxygen species and/or free radicals comprising a gas
comprising a hydrogen molecule, a container 3 comprising at least
one gas selected from the group consisting of oxygen gas, inactive
gases, air, and anesthetic gases, and a duct 4. The gas is supplied
to the gas inhalation means through the duct and administered to
the subject.
[0115] Hereinafter, specific embodiments of the present invention
will be described in detail with reference to Examples. However,
the present invention is not intended to be limited to these
Examples below. The present invention can also be applied to
various changes or modifications without departing from technical
principles shown in claims.
[0116] In Examples below, hydrogen and oxygen measurements, the
hydrogen treatment of cultured cells, and the staining of cultured
cells are performed by approaches shown below, unless otherwise
specified. Moreover, detailed conditions are shown in each
Example.
Hydrogen and Oxygen Measurements
[0117] Hydrogen and oxygen concentrations in solutions were
measured with a hydrogen electrode (ABLE & Biott Co., Ltd.) and
an oxygen electrode (Strathkelvin Instruments Ltd.), respectively.
A hydrogen gas concentration was measured by gas chromatography
(TERAMECS CO., LTD., Kyoto, Japan). RF-5300PC (manufactured by
Shimadzu Corp.) was used in fluorescence intensity measurement. In
experiments under solution conditions, solutions were left at a
hydrogen gas pressure of 0.4 MPa for 2 hours to thereby dissolve
hydrogen therein. In measurement experiments on the elimination of
hydroxyl radicals by hydrogen, a phosphate buffer (10 mM, pH 7.4),
ferrous hydroxide (0.1 mM), and HPF (0.4 .mu.M; Daiichi Pure
Chemicals Co., Ltd.) were added to the solution containing
dissolved hydrogen, and a concentration was measured with a
hydrogen electrode. Subsequently, hydrogen peroxide (5 .mu.M) was
added thereto to initiate the Fenton reaction, and the mixture was
gently stirred at 23.degree. C.
Hydrogen Treatment of Cultured Cells
[0118] A DMEM medium was left at a hydrogen gas pressure of 0.4 MPa
for 2 hours to thereby dissolve hydrogen therein. An
oxygen-saturated medium was additionally prepared by aerating a
medium with oxygen gas and mixed with the medium containing
dissolved hydrogen to bring a dissolved oxygen concentration at
25.degree. C. to 8.5 mg/L. Moreover, a hydrogen concentration was
measured with a hydrogen electrode. To generate .OH via
O.sub.2.sup.-., antimycin A was added to cells. PC12 cells were
placed in a sealed container that enclosed gases containing
hydrogen and oxygen at appropriately adjusted concentrations of
dissolved hydrogen and dissolved oxygen. The cells were cultured at
37.degree. C.
Staining of Cultured Cells
[0119] In .OH detection, HPF (0.4 .mu.M) was added to a cell
medium, and the fluorescence image thereof was observed using a
confocal laser scanning microscope (FV300 manufactured by Olympus
Corp.) at an excitation wavelength of 488 nm and an absorption
wavelength of 510 nm. MitoTracker Green (1 .mu.M; Molecular Probes)
and MitoTracker Red (100 nM; Molecular Probes) were used in the
staining of mitochondria. Anti-HNE and anti-8-OH-G antibodies were
purchased from Nikken Seil Co., Ltd. Moreover, anti-TUJ-1 and
anti-GFAP antibodies were purchased from Babco and Immunon,
respectively.
Example 1
[0120] Hydrogen molecules have been known to have reducing power.
However, it is impossible to predict, as described above, whether
hydrogen molecules can reduce, in a short time, molecular species
participating in biologically important oxidation-reduction, and
free radical molecules. Thus, in Example 1, hydrogen water was used
to measure effects on each molecular species.
Preparation of Hydrogen Water
[0121] First, 1 l of water was injected into a 5-l
pressure-resistant bottle (manufactured by UNICONTROLS. CO., LTD.).
Then, hydrogen gas was charged thereinto at a pressure of 0.4 MPa.
After 2 hours, water supplemented with hydrogen was collected from
the bottle under reduced pressure. A hydrogen content was analyzed
using a dissolved hydrogen measuring apparatus (manufactured by
ABLE & Biott Co., Ltd.). Water containing approximately 0.8 mM
saturated hydrogen (hydrogen water) was obtained by this
method.
Measurement of Reactive Oxygen Species Eliminating Effects of
Hydrogen Water
[0122] Subsequently, for nitrogen monoxide NO, 0.1 mM or 1 mM
1-Hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene
(NOC7; manufactured by DOJINDO Co.) serving as an NO donor was
added to the hydrogen water adjusted to pH 7.4 with 0.01 M
phosphate. After reaction at room temperature for 30 minutes, 1
.mu.M 5-(and -6)-chloromethyl-2',7'-dichlorodihydrofluorescein
diacetate (DCFDH; manufactured by Molecular Probe, USA) was
subsequently added thereto. The amount of NO remaining was measured
with a fluorescence analyzer (manufactured by TECAN, Austria) at an
excitation wavelength of 485 nm.
[0123] For peroxynitrite (ONOO.sup.-), a peroxynitrite solution
(manufactured by DOJINDO Co.) was added at a final concentration of
62.3 mM to the hydrogen water adjusted to pH 7.4 with 0.01 M
phosphate. After reaction at room temperature for 10 minutes,
absorbance at 300 nm was subsequently measured using a
spectrophotometer (manufactured by BECKMAN).
[0124] For hydrogen peroxide H.sub.2O.sub.2, hydrogen peroxide
water (manufactured by Wako Pure Chemical Industries, Ltd.) was
added at a final concentration of 100 .mu.M to 1 .mu.M to the
hydrogen water. After reaction at room temperature for 30 minutes,
0.2 M phosphate buffer (pH 7.2) containing an equal amount of 20
.mu.M DCFDA was subsequently added thereto. After reaction for 10
minutes, the amount of H.sub.2O.sub.2 remaining was measured with a
fluorescence analyzer at an excitation wavelength of 485 nm.
[0125] For hydroxyl radicals (.OH), ferrous perchlorate
(manufactured by Aldrich) at a final concentration of 100 .mu.M, 1
mM hydrogen peroxide, and 1 pM
2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF;
manufactured by DOJINDO Co.) were added to the hydrogen water.
After reaction for 10 minutes, the amount of .OH remaining was
measured with a fluorescence analyzer at an excitation wavelength
of 485 nm. HPF is less reactive with hydrogen peroxide and highly
reactive with hydroxyl radicals to emit fluorescence. As a result,
the amount of hydroxyl radicals remaining was measured.
[0126] 0.1 mM 2,2'-azobis(2-amidinopropane) dihydrochloride
(manufactured by Wako Pure Chemical Industries, Ltd.) that
generates lipid peroxyl radicals (alkylperoxyl radicals) (LOO.) as
an indicator for lipid radicals was added to the hydrogen water.
After reaction at room temperature for 1 hour, 0.2 M phosphate
buffer (pH 7.2) containing an equal amount of 20 .mu.M DCFDA was
subsequently added thereto. After reaction for 10 minutes, the
amount of LOO. remaining was measured with a fluorescence analyzer
at an excitation wavelength of 485 nm.
[0127] For superoxide (O.sub.2.sup.-.), 25.times. Reaction Buffer,
Xanthine Solution, and NBT Solution contained in TREVIGEN
Superoxide Dismutase Assay Kit were mixed with the hydrogen water
according to the manufacturer's manual. Then, a Xanthine oxidase
solution was added thereto to thereby generate O.sub.2.sup.-.
NBT-diformazan accumulation was measured over time with a
spectrophotometer at 550 nm.
[0128] For cytochrome c, flavin adenine dinucleotide (FAD),
nicotinamide adenine dinucleotide (NAD.sup.+), 10 .mu.M cytochrome
c (manufactured by Sigma), 1 mM FAD (manufactured by Sigma), or 1
mM NAD.sup.+ (manufactured by Sigma) was dissolved in the hydrogen
water. After reaction at room temperature for 30 minutes,
absorbance at 415, 400, and 340 nm, respectively, were measured
with a spectrophotometer. The results are shown in Table 1. In
Table 1, the eliminated amount is indicated as a value obtained by
subtracting the amount of each molecular species remaining after
the reaction in the hydrogen water from the amount of each
molecular species remaining after the reaction in hydrogen-free
water as 100%.
[0129] The measurement results shown in Table 1 demonstrate things
described below. Specifically, it could be confirmed that the
hydrogen water eliminates the most highly reactive and highly toxic
hydroxyl radicals, peroxynitrite, and lipid peroxyl radicals
through reduction. On the other hand, the elimination of NO,
superoxide, hydrogen peroxide, and the like known to be used in
signal transduction and so on in vivo was not observed. Likewise,
the hydrogen water did not have any influence on cytochrome c, FAD,
and NAD.sup.+ that play a central role in in vivo energy metabolism
through oxidation-reduction reactions.
TABLE-US-00001 TABLE 1 Eliminated amount (%) Active oxygen species
NO <1 ONOO.sup.- 97 H.sub.2O.sub.2 <1 .cndot.OH 69 ROO.cndot.
37.6 O.sub.2.sup.-.cndot. <1 Respiratory chain-related molecular
species Cytochrome c <1 FAD <1 NAD+ <1 * The eliminated
amount is indicated as a value obtained by subtracting the amount
of each molecular species remaining after the reaction in the
hydrogen water from the amount of each molecular species remaining
after the reaction in hydrogen-free water as 100%.
Example 2
[0130] Antimycin A, which is an inhibitor of mitochondrial
respiratory chain complex III, promotes reactive oxygen species
production in cells and induces oxidative stress-induced cell death
as a result. Thus, in Example 2, the defensive effects of hydrogen
against oxidative stress in the presence of antimycin A was
measured.
Preparation of Hydrogen-Containing Medium
[0131] A cell culture medium requires containing oxygen, being
almost neutral, and containing no metal ions with a high
concentration. For preparing a culture medium containing oxygen and
hydrogen molecules coexisting with each other, it can be predicted
to separately dissolve these molecules under pressure according to
the Henry's law. The space in the hydrogen pressure device is
filled with oxygen. Then, hydrogen gas is compressed to 5 atm. The
partial pressure of oxygen gas can be kept at 1 atm. Therefore, an
oxygen concentration necessary for culture can be secured.
Penicillin G (manufactured by Invitrogen) at a final concentration
of 100 units/mL and streptomycin (manufactured by Invitrogen) at a
final concentration of 100 .mu.g/mL were allowed to be contained in
1 l of Dulbecco's Modified Eagle Medium (DMEM; manufactured by
Invitrogen) as a cell culture medium. This mixture was injected
into a 5-l pressure-resistant bottle. Then, hydrogen gas was
charged thereinto at a pressure of 0.4 MPa. After 2 hours, DMEM
supplemented with hydrogen was collected from the bottle under
reduced pressure.
[0132] Alternatively, when hydrogen with a high concentration was
unnecessary, a hydrogen-containing medium having an almost equal
amount of dissolved oxygen to that in DMEM before hydrogen addition
was prepared by the addition of an appropriate amount of
oxygen-saturated DMEM. The dissolved oxygen was kept at a constant
level by measuring a dissolved oxygen concentration in DMEM as a
culture solution by use of Mitocell MT200 (manufactured by CT and C
Co., Ltd.). A hydrogen content was analyzed using the dissolved
hydrogen measuring apparatus. One example of results of measuring
dissolved oxygen and dissolved hydrogen concentrations in the
prepared hydrogen-containing DMEM is shown in Table 2. Horse serum
(manufactured by PAA, Austria) was added at a final concentration
of 1% to this medium to prepare a hydrogen-containing medium
adjusted to a dissolved hydrogen concentration of 0.6 to 0.8 mM and
a dissolved oxygen concentration of 8.6 to 9.3 mg/L. This
hydrogen-containing medium was used in subsequent experiments.
TABLE-US-00002 TABLE 2 Dissolved Dissolved DMEM oxygen (mg/L)
hydrogen (mM) Before hydrogen charging 8.86 0 After hydrogen
charging (1) 5.73 0.7 Oxygen saturation(2) 32.0 0
Hydrogen-containing medium* 8.89 0.6 *(2) was added in an amount of
approximately 1/10 to (1).
Culture of Rat Adrenal Gland-Derived Pheochromocytoma PC12
Strains
[0133] Rat adrenal gland-derived pheochromocytoma PC12 strains were
suspended in DMEM containing 10% fetal bovine serum (manufactured
by EQUITECH-BIO, USA) and 5% horse serum and seeded at a cell
density of 1.times.10.sup.4 cells/cm.sup.2 in a collagen-coated
cell culture dish. The cells were cultured with an incubator at
37.degree. C. under 5% CO.sub.2. To confirm the effects of the
hydrogen-containing medium, the medium was aspirated after
overnight culture, and the cells were washed once with DMEM
containing 1% horse serum and used in subsequent experiments.
[0134] The culture dish in an open system was used in cell culture
using the hydrogen-containing medium. In this case, the cell
culture was designed as follows to prevent hydrogen from being
released from the culture solution: 2 l of hydrogen water with an
appropriate concentration was placed in a 3-l plastic container. A
table was placed on the water surface, and the dish during culture
using the hydrogen-containing medium was left standing thereon. The
cells were cultured at 37.degree. C. in a sealed state.
Hereinafter, the use of hydrogen water was in line with this
method. A dish during culture using hydrogen-free medium was left
standing on a table in a plastic container containing untreated
water instead of the prepared hydrogen water. The cells were
cultured at 37.degree. C.
Examination of Inhibitory Effects of Hydrogen on Cell Death Induced
by Antimycin A
[0135] To measure the defensive effects of hydrogen against
oxidative stress, PC12 cells were cultured on a collagen-coated
24-well cell culture dish (manufactured by IWAKI&Co. Ltd.).
Then, 2 mL of hydrogen-containing medium supplemented or
unsupplemented with antimycin A (manufactured by Sigma) was added
to each well. After 24 hours, the number of viable cells having a
pyramid-shaped cell form was counted under a phase contrast
microscope. In this experiment, hydrogen-free DMEM containing 1%
horse serum (hereinafter, referred to as hydrogen-free medium) was
used as a comparative control. A phase contrast microscopic image
after 24 hours is shown in FIG. 1.
[0136] Among the cells cultured in the hydrogen-free medium
supplemented with 10 .mu.g/mL antimycin A, many dead cells that
were round in shape and small in size were observed, and the number
of pyramid-shaped viable cells was reduced. By contrast, in the
hydrogen-containing medium supplemented with 10 .mu.g/mL antimycin
A, the number of dead cells was smaller, and the proportion of
viable cells was significantly increased as compared with the
hydrogen-free medium. Results of counting the numbers of viable
cells in the hydrogen-containing medium and the hydrogen-free
medium at varying antimycin A concentrations are shown in FIG. 2.
The bar graph shows an average of at least 4 wells, and the error
bars denote standard deviation. The number of viable cells in the
hydrogen-containing medium was significantly increased at both
antimycin A concentrations of 10 .mu.g/mL and 30 .mu.g/mL,
demonstrating the cell death inhibitory effects of hydrogen
addition.
Example 3
Examination of Inhibitory Effects of Hydrogen on Cell Death Induced
by Menadione
[0137] Menadione, which is an inhibitor of mitochondrial
respiratory chain complex I, promotes reactive oxygen species
production in cells and induces oxidative stress-induced cell death
as a result. Thus, to measure the defensive effects of hydrogen
against oxidative stress as in the experiment on antimycin
A-induced cell death shown in Example 2, PC12 cells were cultured
in a collagen-coated 24-well cell culture dish. Then, 2-ml of the
hydrogen-containing medium supplemented or unsupplemented with
menadione (manufactured by Sigma) at varying concentrations was
added to each well. After 24 hours, the number of viable cells
having a pyramid-shaped cell form was counted under a phase
contrast microscope. In this experiment, a hydrogen-free medium was
used as a comparative control. The results are shown in FIG. 3. The
bar graph shows an average of at least 4 wells, and the error bars
denote standard deviation. The number of viable cells in the
hydrogen-containing medium supplemented with 10 .mu.M menadione was
significantly increased, demonstrating the cell death inhibitory
effects of hydrogen addition.
Example 4
Time-Course Analyses of Inhibitory Effects of Hydrogen on Cell
Death Induced by Antimycin A and Menadione
[0138] The defensive effects of hydrogen against oxidative stress
in cell death induced by antimycin A and menadione were measured in
time-course analyses in the same way as in Examples 2 and 3 to
thereby examine the durability of the defensive effects of
hydrogen. PC12 cells were cultured in a collagen-coated 24-well
cell culture dish (manufactured by IWAKI&Co. Ltd.). Then, 2-ml
of the hydrogen-containing medium supplemented or unsupplemented
with 10 .mu.M menadione or 30 .mu.g/mL antimycin A was added to
each well. After 0, 1, 2, and 3 days, the number of viable cells
having a pyramid-shaped cell form was counted under a phase
contrast microscope. The results are shown in FIG. 4. Each mark
shows an average of at least 4 wells. The cell death inhibitory
effects of hydrogen addition could be confirmed even on the 2nd day
or later, demonstrating that the cell death inhibitory effects of
hydrogen addition continues even on the 24th hour or later.
Example 5
Comparative Measurement of Cell Death Inhibitory Effects of
Hydrogen Relative to Vitamin E
[0139] In Example 5, vitamin E, which has been well known as a
substance exhibiting antioxidative effects and has been used
widely, was compared with hydrogen. Cell death was induced by
antimycin A or menadione in the same way as in Examples 2 and 3.
The number of viable cells was compared between the
hydrogen-containing medium and a hydrogen-free medium containing
alpha-tocopherol (vitamin E). First, PC12 cells were cultured in a
collagen-coated 24-well cell culture dish (manufactured by
IWAKI&Co. Ltd.). Then, 2-ml of the hydrogen-containing medium
supplemented or unsupplemented with 3 .mu.M menadione or 10
.mu.g/mL antimycin A or a hydrogen-free medium containing 100 .mu.M
alpha-tocopherol (manufactured by Sigma) was added to each well.
After 24 hours, the number of viable cells having a pyramid-shaped
cell form was counted under a phase contrast microscope. A
hydrogen-free medium was used as a comparative control. The results
are shown in FIG. 5.
[0140] The bar graph shows an average of at least 4 wells, and the
error bars denote standard deviation. The number of viable cells in
the hydrogen-containing medium was significantly increased as
compared with the hydrogen-free medium. By contrast, inhibitory
effects on cell death induced by antimycin A were observed, albeit
weaker than those of hydrogen, in the hydrogen-free medium
containing alpha-tocopherol, while significant effects on menadione
were not observed therein. These results demonstrate that hydrogen
is a more excellent anti-oxidative stress substance than vitamin E
at a cellular level.
Example 6
Measurement of Inhibitory Effects of Hydrogen-Containing Medium
Post-Addition on Cell Death Induced by Antimycin A
[0141] To demonstrate that the inhibitory effects of the
hydrogen-containing medium on cell death induced by antimycin A are
not attributed to the degeneration of antimycin A by hydrogen but
serve as the inhibition of oxidative stress induced by antimycin A,
cells were treated with a hydrogen-free medium containing antimycin
A. After a certain period of time, the hydrogen-free medium was
replaced by the hydrogen-containing medium to examine the presence
or absence of cell death inhibition. PC12 cells were cultured in a
collagen-coated 24-well cell culture dish in the same way as in the
experiment on antimycin A-induced cell death shown in Example 5.
Two mL of hydrogen-free medium supplemented with 30 .mu.g/mL
antimycin A was added to each well. Then, the hydrogen-free medium
was replaced by 2 mL of hydrogen-containing medium or hydrogen-free
medium on the 1st, 3rd, and 6th hours. After 24 hours from
antimycin A addition, the number of viable cells having a
pyramid-shaped cell form was counted under a phase contrast
microscope. The results are shown in FIG. 6.
[0142] The bar graph shows an average of at least 4 wells, and the
error bars denote standard deviation. The number of viable cells in
the replacements to the hydrogen-containing medium after 1 and 3
hours was significantly increased as compared with the
hydrogen-free medium. Even the hydrogen supply after the addition
of antimycin A can inhibit cell death, demonstrating that hydrogen
inhibits oxidative stress secondarily caused by the addition of
antimycin A. Moreover, the effects were not observed after 6 hours.
This is presumably because irreversible cell death was already in
process at this point in time and therefore killed cells at a
certain rate, regardless of the presence or absence of hydrogen
addition.
Example 7
Measurement of Influence of Dissolved Hydrogen Concentration on
Cell Death Inhibitory Effects
[0143] A hydrogen concentration necessary for defense against
oxidative stress in cells was examined PC12 cells were cultured in
a collagen-coated 24-well cell culture dish in the same way as in
the experiment on antimycin A-induced cell death shown in Example
2. Two mL of a medium containing hydrogen at varying concentrations
supplemented with 30 .mu.g/mL antimycin A was added to each well.
After 24 hours, the number of viable cells having a pyramid-shaped
cell form was counted under a phase contrast microscope. The
results are shown in FIG. 7. Cell death inhibition depended on
hydrogen concentrations, demonstrating that the inhibitory effects
are provided by hydrogen. Furthermore, cell death could be
inhibited significantly even at a hydrogen concentration as low as
50 .mu.M corresponding to approximately 1/16 of the saturated
hydrogen concentration.
Example 8
Measurement of Inhibitory Effects of Hydrogen on Mitochondrial
Dysfunction Caused by Oxidative Stress
[0144] Antimycin A or the like accelerates oxidative stress,
damaging mitochondrial functions. Thus, PC12 cells were cultured in
a collagen-coated 35-mm glass bottom dish in the same way as in the
experiment on antimycin A-induced cell death shown in Example 2.
The hydrogen-containing medium containing 10 .mu.g/mL antimycin A
was added thereto. A hydrogen-free medium was used as a comparative
control. After 50 minutes, a mitochondria-specific dye MitoTracker
Green (final concentration: 1 .mu.M; manufactured by Molecular
Probe, USA) and a mitochondrial membrane potential-sensitive dye
MitoTracker Red (final concentration: 100 nM; manufactured by
Molecular Probe, USA) were added thereto, and the cells were
cultured for another 10 minutes. Fluorescence images thereof at
excitation wavelengths of 488 nm and 543 nm were observed with a
confocal laser scanning microscope (manufactured by Olympus Corp.).
The results are shown in FIG. 8.
[0145] In the experiment shown in FIG. 8, antimycin A (10 .mu.g/ml)
was added in the presence or absence of 0.6 mM hydrogen. After 30
minutes, 1 .mu.M MitoTracker Green (MTGreen) and 100 nM MitoTracker
Red (MTRed) were added thereto, and the cells were cultured for
another 10 minutes. Subsequently, cellular fluorescence was
observed with a confocal laser scanning microscope. The scale bar
in the diagram denotes 50 .mu.m. Reductions in MTRed staining
intensity in the absence of hydrogen indicate reductions in
mitochondrial membrane potential. The merged images are rendered
darker green in the absence of hydrogen than in the presence of
hydrogen. This suggests that the hydrogen molecules passed through
the mitochondrial membranes.
[0146] The antimycin A-treated cells in the hydrogen-free medium
assume a mitochondrial form that is round in shape and is torn off,
while the antimycin A-untreated cells assume a mitochondrial form
with a reticular structure. Moreover, MitoTracker Red fluorescence
intensity is also reduced. These results indicate decreases in
mitochondrial functions. By contrast, the antimycin A-treated cells
in the hydrogen-containing medium have both a form and MitoTracker
Red fluorescence intensity close to those of the antimycin
A-untreated cells, demonstrating that decreases in mitochondrial
functions are inhibited. Specifically, the hydrogen molecules
protected the mitochondria.
Example 9
Measurement of Effects of Hydrogen Water on Model Animals with
Accelerated Oxidative Stress
[0147] Transgenic mice expressing the inactive gene of aldehyde
dehydrogenase 2 display accelerated oxidative stress and
aging-related disease (C2 mice; WO2005/020681, A1). These C2 mice
(5 week old, female, four individuals in each group) were permitted
to freely ingest hydrogen water. In an open system, hydrogen is
released and lost from the hydrogen water. Therefore, to keep
hydrogen in a state dissolved in the water over a long time, 2
bearing balls were placed in an outlet for water ingestion to
prevent the water from leaking out and coming into contact with
air. The mice can poke at the bearing balls and drink the water
from the outlet for water ingestion. The amount of water drunk by
the mice was not reduced by this method as compared with in the
open system. In this method, half or more of the hydrogen molecules
remained even after 24 hours. Hydrogen water prepared by a method
equivalent to that in Example 1 was charged into a 100-mL glass
feed-water bottle to completely fill the bottle. The hydrogen water
was replaced once 24 hours. Control water used was water obtained
by eliminating hydrogen gas from the hydrogen water. This control
water used was totally the same as the hydrogen water except for
hydrogen dissolution.
[0148] The C2 mice were permitted to freely ingest the hydrogen or
control water. Changes in the concentration of 4-hydroxy-2-nonenal
(4-HNE), which is harmful aldehyde generated form lipid peroxide
and serves as an indicator for in vivo oxidative stress
accumulation, were measured in the blood and in the femoral muscle.
After 4 weeks, the whole blood was collected from the eyeground.
Moreover, the femoral muscle was immediately frozen with liquid
nitrogen and stored at -80.degree. C. The frozen organ was
disrupted with a hammer. Then, 1 mL of ice-cold buffer (100 mM
sodium chloride, 10 mM Tris-HCl, pH 7.2) was added to a 100 mg
aliquot of the organ. Subsequently, this sample was cut finely with
POLYTRON (manufactured by KINEMATICA AG, Switzerland). The cut
samples were sonicated on ice for 1 minute after the addition of a
1/4 amount of 10% SDS and then centrifuged at 3000 rpm at 4.degree.
C. The supernatant was collected and diluted with a buffer to 10
mg/mL in terms of the amount of proteins extracted to thereby
prepare muscle extracts. 60 .mu.L of whole blood and 200 .mu.L of
muscle extracts were used to measure a 4-HNE concentration with
BIOXYTECH HAE-586 ASSAY KIT (manufactured by OXIS, USA) by a method
described in the manufacturer's manual. The results are shown in
FIG. 9. The amount of 4-HNE in the hydrogen water ingestion group
was significantly reduced in both the blood and the femoral muscle.
These results demonstrate that hydrogen ingested with a drink can
inhibit in vivo oxidative stress in blood and in tissues.
Example 10
Time-Dependent Analyses of Oxidative Stress Inhibitory Effects of
Water with Varying Dissolved Hydrogen Concentrations
[0149] C2 mice (4 week old, male, 4 to 5 individuals in each group)
were permitted to freely ingest water with varying dissolved
hydrogen concentrations according to Example 9. After 1, 2, and 3
weeks, 60 .mu.L of blood was collected from the eyeground. The
amount of 4-HNE was measured according to the method shown in
Example 9. Hydrogen contents in the water provided thereto are high
concentration (H, 1.6 to 1.2 mM), medium concentration (M: 0.7 to
0.9 mM), low concentration (L: 0.3 to 0.5 mM), and a control (C: 0
mM). No difference in the amount of hydrogen water drunk per mouse
was observed among the groups. The results are shown in FIG. 10.
Reductions in the amount of 4-HNE were observed on the 2nd week in
the groups that ingested high-concentration and
medium-concentration hydrogen water. However, such reductions were
not observed for the low-concentration hydrogen water,
demonstrating that the hydrogen water with an almost saturated
concentration or higher is effective for oxidative stress
inhibition. These results also demonstrated that the continuous
drinking of the hydrogen water for approximately 2 weeks is
effective for oxidative stress inhibition.
Example 11
Measurement of Ischemia-Reperfusion Injury Alleviating Effects of
Hydrogen Gas Inhalation
[0150] Mice (C57BL/6 line, 5 week old, male) were put under general
anesthesia by supplying mixed anesthetic gases (oxygen: 0.3 L/min.,
nitrous oxide: 0.7 L/min., Sevofrane (Maruishi Pharmaceutical Co.,
Ltd.): 3%) thereto with a small animal general anesthesia machine
Soft Lander (Neuroscience Inc.). After anesthesia introduction, the
anesthesia was maintained for a long time by supplying thereto a
mixed anesthetic gas containing Sevofrane at a concentration
reduced to 1.5% (oxygen: 0.3 L/min., laughing gas: 0.7 L/min.,
Sevofrane: 1.5%). Surgical skills (reference: Yadav, S. S. et al.,
Transplantation 65, 1433-1436 (1998)) shown below were used to
prepare model animals with local ischemia-reperfusion injury in the
liver. The model animals were examined by hematoxylin-eosin
staining (H&E staining) for tissue degeneration in the ischemic
liver and hepatic parenchymal cell death induced by
ischemia-reperfusion injury.
[0151] The belly was opened in the middle thereof. Three canals,
portal vein, hepatic artery, and bile duct, leading to the left
robe of the liver were together blocked (ischemia start) with a
micro-clamp (FD562; Aesculap, South San Francisco, Calif., USA).
The belly was sutured with a silk thread. After 90 minutes, the
belly was opened again by removing the silk thread used for suture,
and the micro-clamp was removed (reperfusion start). The belly was
sutured again with a silk thread. The left robe of the liver was
reperfused for 3 hours under anesthesia. Hydrogen gas was mixed
with the mixed anesthetic gas and supplied to a hydrogen
gas-supplied group at a designated hydrogen gas flow rate at
designated times. To keep all the mixed gas flow rates at a
constant level, the nitrous oxide flow rate was reduced by the
hydrogen gas flow rate. After reperfusion for 3 hours, the left
robe of the liver (ischemic liver) was excised and cut into fine
strip slices. The slices were fixed by dipping in 10% neutral
buffered formalin solution (Wako Pure Chemical Industries,
Ltd.).
[0152] The fixed ischemic liver slices were subjected to
dehydration with ethyl alcohol, dehydrating agent removal with
xylene, and paraffin infiltration in an automatic embedding
apparatus (SAKURA, Tissue-Tek VIP5). The paraffin-embedded ischemic
liver was cut into slices of 3 .mu.m to 5 .mu.m in thickness with a
sledge microtome. The slices were attached to slide glass. The
paraffin was removed by treatment with xylene and then with ethyl
alcohol, and the slices were washed with running water. The slices
were hematoxylin-stained with a Mayer's hematoxylin solution (Wako
Pure Chemical Industries, Ltd.) and then washed with water.
Subsequently, the slices were eosin-stained with 1% eosin Y
solution (Wako Pure Chemical Industries, Ltd.). After complete
dehydration with ethyl alcohol, the slices received xylene
penetration and were covered with cover glass coated with a
mounting agent Malinol (Muto Pure Chemicals Co., Ltd.) to prepare a
permanent preparation.
[0153] The results are shown in FIG. 11 and Table 3.
TABLE-US-00003 TABLE 3 Hydrogen gas flow rate (L/min.) Degenerated
region Hydrogen gas inhalation period in liver tissues (%) 0 74 0.2
From 5 minutes before reperfusion start to 5 10 minutes after
reperfusion start (10 minutes in total) 0.1 From 5 minutes before
reperfusion start to 5 20 minutes after reperfusion start (10
minutes in total) 0.04 From 10 minutes before reperfusion start 26
through whole reperfusion period (280 minutes in total)
[0154] In FIG. 11, cytoplasmic degeneration (which is not stained
red with eosin and leaves a white patch) in many cells occurs in
the hydrogen gas-untreated liver tissues. Furthermore, many cells
in the hydrogen gas-untreated liver tissues lost their nuclei
(which are stained purple with hematoxylin). Both nuclear and
cytoplasmic degenerations less occurred in the liver tissues of the
mice that inhaled hydrogen gas (0.2 L/min.) for 10 minutes from 5
minutes before reperfusion start to 5 minutes after reperfusion
start. These tissues were maintained favorably. The data of Table 3
shows the proportion of degenerated regions to the whole slice area
as 100% obtained by measuring the area of the degenerated regions
(regions with a white patch) in the liver tissues from the digital
photograph of the whole slice by use of image processing software
NIH Image. Hydrogen gas inhalation alleviated degeneration in the
liver tissues in all the cases. It has been revealed that
ischemia-reperfusion injury is caused by the effects of free
radicals. Therefore, these results demonstrated that the hydrogen
gas inhalation eliminated the free radicals. It can be inferred by
analogy that hydrogen gas inhalation, in general, not only
alleviates ischemia-reperfusion injury but also eliminates free
radicals. Hydrogen generates no fire under 4% hydrogen conditions
and can be utilized safely.
Example 12
Measurement of Eliminating Effects of Hydrogen Ingestion on Free
Radicals after Exercise
[0155] It has been known that strenuous exercise and the sudden
stop of exercise generate free radicals and damage various tissues
including muscles. Hydrogen water ingestion and hydrogen gas
inhalation exhibited eliminating effects on free radicals after
exercise. Rats (Wister Rats, 8 week old, male) were run at a speed
of 40 m/min. for 20 minutes. Immediately afterward, the rats
inhaled air containing 10% hydrogen gas for 30 minutes.
Alternatively, the rats received intraperitoneal injection with a
saline containing supersaturated hydrogen and were laid to rest for
30 minutes. After 30 minutes, the rats were slaughtered. The
skeletal muscle was excised. From H&E staining, the hydrogen
inhalation was confirmed to alleviate damage in the muscular
tissues. Furthermore, deletion mutation in mitochondrial DNA was
detected by a PCR method, indicating that deletion in mitochondrial
DNA was reduced in the rats that ingested hydrogen. These results
demonstrated that hydrogen ingestion maintains muscular tissues
after strenuous exercise. The method was conducted according to
Sakai, Y., Iwamura, Y., Hayashi, J., Yamamoto, N., Ohkoshi, N.,
Nagata, H., Acute exercise causes mitochondrial DNA deletion in rat
skeletal muscle, Muscle and Nerve 22: 256-261, 1999.
Example 13
Demonstration that Hydrogen Molecules are Taken Up into Living
Bodies by Drinking of Hydrogen Water and by Hydrogen Gas
Inhalation
[0156] To examine whether hydrogen molecules are actually taken up
into living bodies by the ingestion of water containing dissolved
hydrogen or by hydrogen gas inhalation, a human breath hydrogen
concentration was examined A breath hydrogen concentration differs
among individuals. Therefore, four subjects with a breath hydrogen
concentration of 10 ppm or lower were targeted by the experiment. A
breath hydrogen concentration before hydrogen water ingestion was
measured with Breath Gas Analyzer (breath hydrogen analyzer)
TGA-2000 (TERAMECS CO., LTD., Kyoto, Japan). Then, the subjects
ingested 10 mL of hydrogen water per kg of body weight by drinking.
Then, the subjects rinsed their mouth with hydrogen-free water to
completely remove the hydrogen water. Time-dependent changes of
breath hydrogen concentration were measured. The breath hydrogen
concentration, though differing among individuals, was increased by
30 ppm or more within 20 minutes after hydrogen water ingestion and
then reduced. Even after 1 hour, the high value was kept as
compared with before hydrogen water ingestion, demonstrating that
hydrogen was taken up into the living bodies, dissolved in blood,
and excreted as breath from the lung. These results demonstrated
that hydrogen was taken up into living bodies within approximately
20 minutes after hydrogen water ingestion and dissolved in blood.
The results are shown in FIG. 12.
[0157] In this experiment, the hydrogen molecules were taken up
into living bodies by the drinking of hydrogen water. By contrast,
when subjects inhale hydrogen gas, for example, a method can be
adopted wherein air and hydrogen gas was supplied separately or as
a mixture prepared in advance into a gas inhalation mask for
covering the mouth and nose of the subjects and inhaled by the
subjects, or wherein air and hydrogen gas was supplied separately
or as a mixture prepared in advance into a sealed container and
inhaled by the subjects lying in a bed within the sealed container.
In this case, hydrogen gas concentrations of approximately 4 vol %
or lower are in no danger of fires or explosion.
Example 14
[0158] Whether hydrogen molecules in a simple solution state reduce
biological substances was examined A neutral solution saturated
with hydrogen molecules does not reduce, at room temperature,
NAD.sup.+, FAD, Fe.sup.3+, Cu.sup.2+, and oxidized cytochrome c
containing trivalent heme iron. Specifically, the hydrogen
molecules are stable in a solution without disrupting
oxidation-reduction reactions. Moreover, the hydrogen molecules do
not reduce hydrogen peroxide, nitrogen monoxide, or O.sub.2.sup.-.
under this condition. These results indicate that the hydrogen
molecules do not neutralize such reactive oxygen species that have
main functions in signal transduction. By contrast, the hydrogen
molecules, as shown in FIG. 13, reduce hydroxyl radicals (.OH)
levels without catalysts under the same condition as above. FIG. 13
shows the removal of .OH by dissolved hydrogen molecules at room
temperature under neutral conditions. The .OH was monitored with a
spectrofluorometer. FIG. 13a shows typical changes over time in
2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF)
fluorescence intensity at each hydrogen molecule concentration. The
base lines 1 and 2 denote changes in hydrogen peroxide-free (1) and
ferrous perchlorate-free (2) HPF fluorescence intensities,
respectively, at a hydrogen concentration of 0.8 mM. FIG. 13b shows
results of determining an average value of initial reaction rates
and standard deviation from 4 independent experiments .OH generated
by the Fenton reaction was monitored with HPF fluorescence. HPF can
specifically detect .OH without detecting hydrogen peroxide or
O.sub.2.sup.-.
Example 15
[0159] Whether hydrogen molecules can neutralize .OH in cultured
cells was examined Spontaneously generated .OH does not reach an
amount that exhibits cytotoxicity and is difficult to detect.
Therefore, .OH was induced by two different methods. In this
context, .OH was detected with a confocal laser scanning microscope
by using HPF as a marker dye. First, hydrogen-saturated and
oxygen-saturated media were prepared and mixed at their respective
appropriate concentrations, with the concentrations monitored in
hydrogen and oxygen electrodes. Subsequently, this mixed medium was
replaced by a PC12 cell medium. Furthermore, the cells were placed
in a container filled with gases adjusted to appropriate hydrogen
and oxygen concentrations. In the PC12 cells, O.sub.2.sup.-. was
generated by the addition of antimycin A, a mitochondrial
respiratory chain inhibitor, and .OH levels were increased through
the Fenton reaction. The treatment thereof with hydrogen molecules
reduced .OH levels, as shown in FIGS. 14 and 15. FIG. 14 shows
results of observing HPF fluorescence with a confocal laser
scanning microscope after 30 minutes of antimycin A addition in the
presence or absence of 0.6 mM hydrogen. The arrows and pikes denote
cells with HPF fluorescence-positive and HPF fluorescence-negative
nuclei, respectively. The scale bar denotes 50 .mu.m. FIG. 15 shows
results of quantifying HPF fluorescence intensity after antimycin A
addition in the presence or absence of 0.6 mM hydrogen. The values
were measured as to 100 cells in each independent experiment using
NIH Image software. The cells were cultured in a sealed container
with an appropriate gas concentration to maintain the initial
hydrogen concentration. Interestingly, the hydrogen molecules
reduced .OH levels in the nuclei (indicated by the pikes in the
right panel of FIG. 14).
[0160] Furthermore, antimycin A was added to cells in the presence
or absence of 0.6 mM hydrogen. After culture for 24 hours, the
cells were stained with anti-8-OH-G and anti-HNE antibodies and
observed with a confocal laser scanning microscope. The results are
shown in FIGS. 16 to 19. The scale bars in FIGS. 16 and 18 denote
100 .mu.m. Immunostaining intensities with anti-8-OH-G (FIG. 17)
and anti-HNE (FIG. 19) antibodies were quantified using NIH Image
software. In the experiments, the results of which are shown in
FIGS. 14 to 19, antimycin A (+), hydrogen (+), and vitamin E (+)
concentrations were 10 .mu.g/ml, 0.6 mM, and 0.1 mM, respectively.
Average values and standard deviations were determined from 4
independent experiments. *P<0.05, **P<0.01,
***P<0.001.
[0161] As seen from reductions in oxidized guanine (8-OH-G) level
in FIGS. 16 and 17, the hydrogen molecules protected nuclear DNA
from oxidation. Moreover, the inhibition of accumulation of
4-hydroxy-2-nonenal (HNE), a final product of lipid peroxide, by
the hydrogen molecules, as shown in FIG. 22, demonstrates that the
hydrogen molecules also inhibit lipid peroxidation.
Example 16
[0162] Rat cerebral cortex-derived primary cultured cells were used
to induce oxidative stress under more physiological conditions. It
has been known that a rapid shift from ischemia to reperfusion
causes an oxidative stress-induced disorder. Thus, the cells were
exposed to an oxygen-glucose-deficient state mimicking ischemia for
60 minutes in a nitrogen or hydrogen atmosphere.
[0163] Rat cerebral cortex-derived primary cultured neuronal cells
were prepared from a 16-day-old fetus. The cerebral cortex was cut
after meninges removal and digested with a protease mixed solution
(SUMILON). The slices were mechanically dissociated with a pipette.
Then, the cells were dispersed in a nerve cell culture medium
(SUMILON) and seeded at a cell density of 5.times.10.sup.4
cells/cm.sup.2 in a plate coated with poly-L-lysine. The medium was
replaced every three days by Neurobasal Medium (Gibco) containing
B-27 (Gibco). The cells cultured for 11 days were used in the
experiment. One day before the creation of an
oxygen-glucose-deficient state, the medium was replaced by
Neurobasal Medium (Gibco) containing B-27 minus AO (Gibco). Quality
control for the neuronal cells was confirmed by staining with
neuron-specific anti-TUJ-1 and astrocyte-specific anti-GFAP
antibodies. 90% or more of the cells were neuronal cells.
[0164] To initiate an oxygen-glucose-deficient state, the cell
medium was replaced by a glucose-free DMEM medium aerated with 95%
nitrogen/5% carbon dioxide or 95% hydrogen/5% carbon dioxide. This
medium was left at 30.degree. C. for 1 hour in 95% nitrogen/5%
carbon dioxide or 95% hydrogen/5% carbon dioxide atmosphere. To
terminate the oxygen-glucose-deficient state, the medium was
replaced again by the medium used before replacement. Culture was
further continued at 37.degree. C. in 95% air/5% carbon dioxide
atmosphere.
[0165] After 10 minutes of the termination of the
oxygen-glucose-deficient state, the amount of .OH was measured
based on HPF fluorescence. As a result, significant increases in
the amount of .OH were observed in the absence of hydrogen but not
observed in the presence of hydrogen, as shown in FIGS. 20 and 21.
FIG. 20 shows results of staining the cells with HPF after 10
minutes of reperfusion (the left panels show fluorescence images,
and the right panels show fluorescence images superimposed on
Nomarski differential interference images). To prepare a control,
cells were treated with a DMEM medium containing glucose and
oxygen, instead of being exposed to an oxygen-glucose-deficient
state. The scale bar in the diagram denotes 100 .mu.m. FIG. 21
shows results of measuring HPF fluorescence intensity as to 100
cells by use of NIH Image software. An average value and standard
deviation was determined from 4 independent experiments.
**P<0.01.
[0166] After 1 day of oxygen-glucose deficiency, the viable
neuronal cells were further detected by staining neuron-specific
anti-TUJ-1 antibody. As a result, the hydrogen molecules increased
the number of viable neuronal cells, as shown in FIG. 22. Moreover,
the hydrogen molecules also increased their viabilities, as shown
in FIGS. 23 and 24. FIG. 23 shows results of counting the number of
viable nerve cells in a certain field under a phase contrast
microscope after 1 day from the oxygen-glucose-deficient state.
FIG. 24 shows results of measuring cell viability by a modified MTT
method. Average values and standard deviations shown in FIGS. 23
and 24 were determined from 4 independent experiments.
.sup.#P<0.0001 in FIG. 23, *P<0.05 in FIG. 24. These results
demonstrate that the hydrogen molecules inhibit oxidative
stress-induced cell death.
Example 17
[0167] To examine whether hydrogen molecules as an antioxidant can
be applied to medical care, examination was conducted using rat
ischemic models.
[0168] In cerebral ischemia, diverse mechanisms generate reactive
oxygen species, and .OH is detected after ischemia-reperfusion.
Rats were rendered locally ischemic for 90 minutes by mild cerebral
artery occlusion and subsequently reperfused for 30 minutes. During
this period, the rats kept inhaling hydrogen, unless otherwise
specified. FK506 (1 mg/kg of body weight) was administered into the
blood once immediately before reperfusion. Edaravone (3 mg/kg of
body weight) was administered into the blood twice immediately
before reperfusion and immediately after reperfusion. After
anesthesia, the rats were maintained at 20.degree. C. under usual
air. The rats inhaled hydrogen molecules for 120 minutes in total,
unless otherwise specified. The rats inhaled a mixed gas of nitrous
oxide (for anesthesia), oxygen, and hydrogen at ratios of 66 to
70%, 30%, and 0 to 4% (v/v), respectively. After 1 day of mild
cerebral artery occlusion, the brain was sliced, and the slices
were stained with 2,3,5-triphenyltetrazolium salt (TTC) capable of
serving as a substrate in mitochondrial respiration process. After
1 week, the animals were put under anesthesia. Then, the brain was
rapidly excised and fixed with 10% formalin. The paraffin-embedded
brain was sliced into a thickness of 6 .mu.m, and the slices were
stained with hematoxylin-eosin. Moreover, VECSTAIN ABC Kit was used
in staining with antibodies. Anti-Iba-I antibodies were purchased
from Wako Pure Chemical Industries, Ltd. Image analysis software
(Mac Scope ver. 2.55; Mitsuya Shoji) was used in slide analysis.
All the animal experiments were conducted according to the
guideline of the animal commission of Nippon Medical School.
[0169] An infarct volume was estimated by measuring visually white
regions in the brain. The results are shown in FIGS. 25 and 26.
FIG. 25 shows results of cutting the brain by coronal section into
6 slices after 1 day of mild cerebral artery occlusion and staining
the slices with TTC. For comparison, two compounds were further
tested. One of the compounds was Edaravone, which has been
recommended in Japan to be used in the treatment of cerebral
infarction. The other compound was FK506, which is under clinical
trial for cerebral infarction in USA. The hydrogen molecules were
more effective for the alleviation of an oxidative disorder than
any of the other compounds tested. FIG. 26 shows a brain infarct
volume. The brain infarct volume was calculated as a total sum of
all the slices according to the equation: infarct
area.times.thickness. The marks E and F in FIG. 26 denote infarct
volumes from Edaravone (6 mg/kg of body weight) and FK506 (1 mg/kg
of body weight), respectively, administered under the optimum
conditions. An average value of infarct volumes and standard
deviation were determined from the values of 6 animals in each
group. *P<0.05, **P<0.01, ***P<0.001 as compared with a
hydrogen gas concentration of 0%. .sup.##P.gtoreq.0.01,
.sup.###P<0.001 as compared with a hydrogen gas concentration of
2%.
[0170] Hydrogen concentration-dependent reductions in infarct
volume were obviously observed, as shown in FIGS. 25 and 26, and
the hydrogen concentration of 2% was most effective (FIG. 26).
[0171] FIG. 27 shows results of performing hydrogen inhalation only
during ischemia but not during reperfusion. Rats were rendered
locally ischemic for 90 minutes by mild cerebral artery occlusion
and subsequently reperfused for 30 minutes. The rats inhaled 2%
hydrogen gas in 3 different periods A, B, and C. FIG. 27a shows a
schematic diagram thereof FIG. 27b shows an infarct volume from
hydrogen gas inhalation in the 3 different periods. The brain was
cut by coronal section into 6 slices after 1 day of mild cerebral
artery occlusion, and the slices were stained with TTC. The brain
infarct volume was calculated as a total sum of all the slices
according to the equation: infarct area.times.thickness.
*P<0.05, **P<0.01, ***P<0.0001 as compared with a hydrogen
gas concentration of 0%. .sup.#P<0.05, .sup.###P<0.0001 as
compared with the period A. The infarct volume is not reduced by
performing hydrogen inhalation only during ischemia but not during
reperfusion, as shown in FIG. 27. These results indicate that the
hydrogen molecules must be present during reperfusion for
exhibiting their protective effects.
[0172] Moreover, the difference in infarct volume between the
hydrogen-treated group and the hydrogen-untreated group was more
significant after 1 week of mild cerebral artery occlusion, as
shown in FIGS. 28 and 29. FIG. 28 shows results of staining, with
hematoxylin-eosin, brain slices obtained by coronal section after 1
week of mild cerebral artery occlusion. The photographs of FIG. 28
are the staining images of 3 different slices obtained by this
method. FIG. 29 shows results of calculating an infarct volume in
the same way as above, with a visually pale pink region obtained by
hematoxylin-eosin staining as an infarction region. An average
value of infarct volumes and standard deviation were determined
from the values of 6 animals in each group. *P<0.05,
**P<0.01, ***P<0.001.
[0173] The hydrogen-treated rats were observed to be improved both
in body weight and in body temperature as compared with the
hydrogen-untreated rats, as shown in FIG. 30. FIGS. 30a and 30b
show changes in body temperature and in body weight, respectively,
by 2% hydrogen gas inhalation (solid line) and non-inhalation
(broken line). An average value and standard deviation were
determined from the values of 6 animals in each group. *P<0.05,
**P<0.01, ***P<0.001. As seen from these results, the
hydrogen molecules not only alleviated an early brain disorder but
also inhibited the progression of the disorder.
[0174] Results of using the brain after 1 week of occlusion to
examine changes produced at a molecular level by the protective
effects of hydrogen molecules, by staining brain slices with
anti-8-OH-G antibody indicating nucleic acid oxidation and anti-HNE
antibody indicating lipid oxidation, and anti-GFAP antibody are
shown in FIGS. 31 to 33. FIG. 31 shows the results obtained by
using anti-8-OH-G antibody. FIG. 32 shows the results obtained by
using anti-HNE antibody. FIG. 33 shows the results obtained by
using astrocyte-specific anti-GFAP antibody. The brain was fixed
after 3 days or 7 days of mild cerebral artery occlusion and
embedded in paraffin. The coronal sections of 6 .mu.m in thickness
were stained with anti-8-OH-G, anti-HNE, or anti-GFAP antibodies.
The left photographs of FIGS. 31 to 33 show the same regions
adjacent to occlusion in the temporal cortical regions. The scale
bar denotes 100 .mu.m. The right graphs of FIGS. 31 to 33 show
results of determining an average value of the number of cells
positive to each antibody in a certain field (0.25 mm.sup.2) and
standard deviation from 6 animals in each group. *P<0.05,
**P<0.01. In the hydrogen-treated rats, all the oxidation marker
stainings were significantly reduced.
[0175] FIG. 34 shows results of staining the same brain regions
with microglia-specific anti-Iba-I antibodies. In the staining
experiment, the brain was fixed after 3 days or 7 days of mild
cerebral artery occlusion and embedded in paraffin. The coronal
sections were stained with each antibody. The photographs of FIG.
34 show the central regions of occlusion in the temporal cortical
regions. The scale bar of FIG. 34 denotes 200 .mu.m. The scale bar
in the inserted photographs of FIG. 34 denotes 100 .mu.m. In FIG.
35, an average value of the number of Iba-1-positive cells in a
certain field and standard deviation were determined from 6 animals
in each group. *P<0.05. The same brain regions were stained with
microglia-specific anti-Iba-I antibodies, as shown in FIGS. 34 and
35. As a result, the staining with anti-Iba-I antibodies was
significantly reduced by hydrogen treatment. Microglia accumulation
serves as an indicator for a brain disorder. These results strongly
suggest that the hydrogen molecules remarkably inhibit oxidative
stress and further a brain disorder.
Example 18
Effects of Hydrogen Water
Increasing in SOD-/SOD-Homozygous Mouse Birth Rate of SOD Knockout
Mice
[0176] MnSOD (manganese superoxide dismutase) present in
mitochondria is an enzyme that converts superoxide (O.sub.2.sup.-.)
generated within mitochondria to hydrogen peroxide. MnSOD gene is
present in the nuclear genome. MnSOD deficiency accumulates
O.sub.2.sup.-., which in turn reacts with NO with increased
frequency and thereby increases harmful ONOO.sup.- (peroxynitrite)
levels, causing cytotoxicity. Alternatively, O.sub.2.sup.-. reduces
transition metals and increases Cu.sup.2+ and Fe.sup.2+ levels.
Therefore, the accelerated Fenton reaction generates harmful
hydroxyl radicals (.OH). Thus, an MnSOD-deficient animal birth rate
is reduced due to stillbirth. Alternatively, MnSOD-deficient
animals, even if born, die within 1 week. The crossing between mice
heterozygously having MnSOD-deficient genes (SOD2 (+/-)) produces
mice with normal MnSOD (SOD2 (+/+)), mice heterozygously having
MnSOD (SOD2 (+/-)), and mice completely deficient in MnSOD (SOD
(-/-)), which are supposed to be born at a 1:2:1 ratio according to
the Mendel's laws. In reality, an MnSOD-deficient mouse birth rate
is reduced due to stillbirth caused by the harmful active oxygen
and free radicals (FIG. 37).
[0177] Sixteen female MnSOD heterozygous mice were divided into 2
groups. In one of the group, the mice drank hydrogen water for 8
days and were crossed. Then, the mice kept drinking hydrogen water
until birth. These two groups resulted in 5 and 6 pregnant mice,
respectively, from which 42 and 45 mice were born. DNA was
extracted from the tails of these neonatal mice, and genotypes were
determined according to a standard method.
[0178] The ratio of the 45 neonatal mice born from the mice that
drank control water were MnSOD (+/+):MnSOD (+/-):MnSOD (-/-) of
14:29:2. On the other hand, the ratio of the 42 neonatal mice born
from the mother mice that drank hydrogen water was 14:20:8. The
number of the MnSOD (-/-) mice born was 8 times larger than that
from the control mice and was statistically significant (FIG.
38).
[0179] These results demonstrate that the hydrogen water alleviates
oxidative stress attributed to O.sub.2.sup.-.
Example 19
Carcinogenesis Inhibitory Effects of Drinking and Intraperitoneal
Administration of Hydrogen Water
[0180] Various medium-term carcinogenicity assays have been
developed in recent years as a detection method for predicting
carcinogenicity. The assays involve: first administering, to
two-stage carcinogenesis models prepared with rodents, known
carcinogens for initiation treatment in an amount that is small
enough not to cause cancer; then administering a test substance
thereto, and detecting the presence or absence of promotion
effects. To detect carcinogenicity, the carcinogenicity of the test
subject is evaluated with attention focused particularly on the
promotion effects.
[0181] A medium-term liver carcinogenicity assay (Ito test) is a
method involving: conducting partial hepatectomy at the early stage
during the promotion stage; and promoting liver cell division at
the regenerative proliferation stage to thereby induce mutant cell
foci in a short time. This assay has allegedly accumulated the
greatest deal of data to this day (Ito N, Tamano S, Shirai T.,
Cancer Sci. 2003 January; 94 (1): 3-8. Review). The assay is
targeted for the rat liver. Among 313 chemicals already detected in
this assay, 60/65 (92%) chemicals regarded as hepatocarcinogens
(including promoters) have been reported to give positive results.
This assay has been said to be a highly reliable and useful
detection method for detecting carcinogens targeted for the liver.
Furthermore, the results of detecting hepatocarcinogens at varying
administration doses by this assay correlates, as to the quantified
value of formation of GST-P-positive cell foci, with the results of
hepatocellular carcinoma incidence shown by long-term
carcinogenicity assay. Moreover, dose correlation has also been
reported (Ogiso, T. et al., Toxicol. Pathol., 13, 257-265,
1985).
[0182] In this Example, a detection method was used wherein MeIQx
that is one of heterocyclic amines and has been reported to have
liver carcinogenicity was administered simultaneously with a test
substance at the promotion stage of the medium-term liver
carcinogenicity assay. This detection method has been developed to
detect the inhibitory effects of a test substance on liver
carcinogenesis. Hirose et al. (Hirose, M. et al., Carcinogenesis,
16, 3049-3055, 1995) have previously found some liver
carcinogenesis inhibitors by use of this model.
[0183] In this Example, the test substances used were a
hydrogen-containing saline (H.sub.2 saline) and hydrogen-containing
water (H.sub.2 water). In this Example, the models were used to
detect the liver carcinogenesis inhibitory effects of the
intraperitoneal administration of the hydrogen-containing saline as
a test substance and the additional administration of the
hydrogen-containing water by drinking.
[0184] For the purpose of examining the presence or absence of the
liver carcinogenesis inhibitory effects of combined administrations
of the hydrogen-containing saline and the hydrogen-containing
water, the medium-term liver carcinogenicity assay using placental
Glutathione S-transferase (GST-P)-positive cell foci as an
indicator was used to conduct quantitative study.
[0185] Six-week-old male F334 rats received: the single
intraperitoneal administration of Diethylnitrosamine (DEN) as an
initiator at a dose of 200 mg/kg for initiation treatment of liver
carcinogenesis; 6-week intraperitoneal administration starting 2
weeks thereafter, wherein the hydrogen-containing saline as a test
substance at a dose of 10 ml/kg was administered as frequently as
twice a day or seven times a week; and the additional
administration of the hydrogen-containing water by drinking. A
control group received the intraperitoneal administration of a
saline and the administration of tap water by drinking. Moreover, a
DEN-untreated control group and a test substance-administered group
were also provided. Each of the DEN-treated and DEN-untreated
groups was further subdivided into a group that received the 6-week
combined administration of the test substance with a
hepatocarcinogen 2-amino-3,8-dimethylimidazo[4,5-J]quinoxaline
(MeIQx) as a promoter at a concentration of 0.02% in feed. After a
lapse of the 3rd week of the experiment (after 1 week of initiation
of test substance administration), partial hepatectomy was
conducted on all the animals. After a lapse of 8 weeks from the
initiation of the experiment (after the completion of the test
substance administration period), the animals were slaughtered and
autopsied. Then, quantitative analysis was conducted on
GST-P-positive cell foci in the liver. FIG. 39 shows the
experimental method.
[0186] Changes in general state attributed to test substance
administration, dead animals, and changes in body weight were not
observed during the administration period. The amount of water
ingested exhibited high value tendency in all the test
substance-administered groups for a period of time in the
administration period. Thus, this tendency was probably brought by
the influence of test substance administration.
[0187] In liver weight and visual pathologic investigations, the
influence of test substance administration was not observed. In
biochemical blood examination as well, changes suggesting the
toxicity of test substance administration were not observed. The
test substance used here presumably inhibits lipid peroxide
generation. Thus, measurement was also performed as to serum lipid
peroxide. However, such inhibition was not observed in the
experiment performed here.
[0188] FIG. 40 shows results of measuring the number of
GST-P-positive cell foci in the liver (FIG. 40a) and the areas of
the positive foci (FIG. 40b). The combined administration of the
hydrogen-containing saline (administered intraperitoneally) and the
hydrogen-containing water (administered by drinking) inhibited,
albeit moderately, the formation of GST-P-positive cell foci in the
DEN-treated group. The inhibition rate thereof was 28.0% for the
number and 25.2% for the area. Moreover, the hydrogen-containing
saline and the hydrogen-containing water also inhibited, albeit
moderately, the formation of GST-P-positive cell foci in the group
that received the administration of MeIQx in addition to DEN. The
inhibition rate thereof was 21.0% for the number and 20.9% for the
area.
[0189] Thus, the hydrogen-containing saline and the
hydrogen-containing water were effective for the inhibition of
carcinogenesis.
INDUSTRIAL APPLICABILITY
[0190] The present invention comprises the constitution described
above. As a result, the present invention, as described in detail
in Examples below, can eliminate in vivo harmful reactive oxygen
species and/or free radicals and can therefore suppress various
adverse effects attributed to the presence of this reactive oxygen
species and/or free radicals. Thus, the present invention exhibits
such excellent effects that it can contribute to the suppression of
human aging process, health promotion, and the prevention of
disease. The results of Examples described later demonstrate that
hydrogen molecules have many advantages as an effective
antioxidant. Specifically, the hydrogen molecules effectively
scavenge .OH at appropriate strength that does not influence
reactive oxygen species in metabolic oxidation-reduction reactions
or cell signaling. Many known antioxidants cannot easily reach
target organelles or tissues. By contrast, the hydrogen molecules
have the property of easily passing through biomembranes and being
effectively distributed in cytoplasm by diffusion. Oxidative stress
caused by inflammation or ischemia-reperfusion is also caused by
other various circumstances. Examples thereof include excessive
exercise, myocardial infarction, operations stopped due to
bleeding, and organ transplantation. Antioxidants that are so
effective yet non-damaging, such as hydrogen molecules, are
applicable in many medical fields by virtue of their convenience.
Hydrogen gas inhalation has already been used for protecting divers
from caisson disease caused by reduced pressure, and their safety
has been confirmed widely. Moreover, hydrogen concentrations used
in treatment according to the present invention are in no danger of
fires or explosion. Furthermore, inhaled hydrogen gas is dissolved
in liquids and easily transferred through blood vessels. Thus,
hydrogen, one of the most well known molecules, is widely
applicable as a safe and effective antioxidant with few side
effects in medical fields.
[0191] All publications, patents, and patent applications cited
herein are incorporated herein by reference in their entirety.
* * * * *