CN105593363A - Nanobubble-containing composition and use thereof - Google Patents

Nanobubble-containing composition and use thereof Download PDF

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Publication number
CN105593363A
CN105593363A CN201580001311.2A CN201580001311A CN105593363A CN 105593363 A CN105593363 A CN 105593363A CN 201580001311 A CN201580001311 A CN 201580001311A CN 105593363 A CN105593363 A CN 105593363A
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China
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cell
composition
water
agent
liquid
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Chinese (zh)
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亀井一郎
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AQUA ZEST CORP
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AQUA ZEST CORP
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A23L2/54—Mixing with gases
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/16—Inorganic salts, minerals or trace elements
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P30/00—Shaping or working of foodstuffs characterised by the process or apparatus
    • A23P30/40—Foaming or whipping
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Abstract

[Problem] The present invention provides a composition containing nanobubbles containing hydrogen, oxygen, and nitrogen, and the use thereof. [Solution] A composition containing, as an active ingredient, nanobubbles having a diameter of 30 Mum or less and containing 0.45-0.55 ppm of hydrogen, 10-12.5 ppm of oxygen, and 7-8 ppm of nitrogen. A mitochondria-activating composition, cell growth promoting agent, cell preservation liquid or cryopreservation liquid containing this composition, and a method for using this composition, agent, or liquid. A food or beverage, or a food or beverage raw material, produced using this composition.

Description

Containing the composition and use thereof of nano bubble
Technical field
The present invention relates to a kind of composition and use thereof containing nano bubble. In particular toA kind of contain nano bubble, activate mitochondrial composition and use thereof.
Background technology
As adopting microbubble, nano bubble technology, gas dissolution is cultivated with promotion in liquidThe method of the propagation of cell, for example, disclose the such method of patent documentation 1. But, speciallyIn profit document 1, use the microbubble of which kind of size, composition, nano bubble can promote which kind of kindThe cell proliferation of class is to which kind of degree etc., all with no specific disclosure of, thereby it is more practical to seek exploitationComposition, method, device. In addition, further seek effectively to breed promotion method.
As the product that adopts microbubble, nano bubble to entrained gas in drinking water, known hydrogenAir water, hydrogen-oxygen air water etc. (for example, patent documentation 2). But, the showing of these drinking watersShape is, only limits to some people and can feel fully to meet effect, and not very general. Therefore schemeAsk microbubble, the nano bubble drinking water that can really experience abundant effect. In addition also scheme,Ask microbubble, the nano bubble drinking water with new function.
On the other hand, mitochondria is energy-producing organ, it is believed that by improving mitochondrialActivity, can increase the generation of energy, makes health more have vigor. But, activate mitochondrialDrinking water is not developed so far, thereby seeks the mitochondrial drinking water of exploitation activation.
In addition, cell, in the time of freezing preservation, exists survival rate reduction, destroyed the making of cell to includeThe problem of thing leakage, mouthfeel variation, local flavor variation. Therefore, wish exploitation a kind of survival rate high,Sapid cell-preservation liquid.
Prior art document
Patent documentation
Patent documentation 1: Patent 2009-234683 communique
Patent documentation 2: Patent 2008-56741 communique
Summary of the invention
The technical problem to be solved in the present invention
The invention provides a kind of composition that comprises the nano bubble that contains hydrogen, oxygen, nitrogenAnd uses thereof.
The technological means of technical solution problem
According to this description, provide following invention.
(1) composition, its comprise as active ingredient, contain 0.45～0.55ppmHydrogen, 10～12.5ppm oxygen, 7～8ppm nitrogen and particle diameter are the microbubble below 30 μ mAnd/or nano bubble. Wherein, the concentration of each gas is dissolved gas and nano bubbleTotal concentration.
(2) a mitochondria activated compositions, it contains composition in (1) as effectivelyComposition. Wherein, mitochondria activated compositions and mitochondria activator are equivalent in meaning.
(3) cell growth promoter, its composition containing in (1) or (2) is doneFor active ingredient. In the case, cell growth promoter can contain (1) and (2) twoPerson's composition.
(4) cell-preservation liquid, its contain any composition in (1)～(3) orAgent is as active ingredient. Wherein, agent means the cell growth promoter in (3). But,When mitochondria activated compositions in (2) is renamed as mitochondria activator, mitochondria is livedAgent is also contained in agent. In addition, " any " refers to the meaning of " at least any one ",It not the meaning of " only any one ". Therefore, contain (1)～two or more or 3 in (3)Composition more than kind or the cell-preservation liquid of agent are also contained in technical scope. Below also phaseWith, " any " refers to the meaning of " (at least) any one ".
(5) a freezing preservation liquid, its contain any composition in (1)～(4),Agent or liquid are as active ingredient. Wherein, agent is equivalent in meaning with (4), and liquid means the thin of (4)Born of the same parents preserve liquid.
(6) a kind of diet (Japanese :), its contain any composition in (1)～(5),Agent or liquid. Wherein, except the cell-preservation liquid in (4), liquid also comprises freezing in (5)Preserve liquid.
(7) a kind of diet (Japanese: Drink food), it is by using in (1)～(5)The raw material of any composition, agent or liquid processing are prepared from.
(8) a kind of mitochondrial method of activation, it uses any group in (1)～(5)Compound, agent or liquid.
(9) promote the method for cell proliferation, it uses any in (1)～(5)Composition, agent or liquid.
(10) preserve the method for cell, it uses any group in (1)～(5)Compound, agent or liquid.
(11) method for freezing preservation, it uses any group in (1)～(5)Compound, agent or liquid.
(12) manufacture method for diet, it comprises following operation: use (1)～(5)In any composition, agent or liquid diet or its raw material are processed.
(13) a cell cultivation culture medium, it is by using the composition preparation of (1)Form.
(14) a vegetation water cultivation solution, it is joined by the composition that uses (1)System forms.
(15) method that zooblast is bred, it uses the culture medium in (13).
(16) method that makes zooblast propagation in (15), wherein, zooblast is for exempting fromEpidemic disease cell.
(17) prevention of arteriosclerosis and a therapeutic agent, it is with described in (1) or (2)Composition or (3) described cell growth promoter as active ingredient.
(18) prevention of diabetes and a therapeutic agent, it is with the group (1) or (2) Suo ShuCell growth promoter described in compound or (3) is as active ingredient.
(19) parenteral solution, it contains (1) or (2) described composition.
Invention effect
According to composition of the present invention, can obtain following effect: activation of wire plastochondria, promotes thinBorn of the same parents' propagation, reduces the infringement to cell in the time of cell preservation, freezing preservation.
Brief description of the drawings
Fig. 1 is the figure that represents the propagation facilitation effect of the cell cultivation of the present composition;
Fig. 2 is the figure that represents the antioxidation of the present composition;
Fig. 3 is the figure of expression for the impact of the ATP generation of various medicaments;
Fig. 4 is the figure that represents the GPT variation of picked-up present composition front and back;
Fig. 5 is the figure that represents the HEL formation speed of picked-up present composition front and back;
Fig. 6 is the figure that represents the STAS variation of picked-up present composition front and back;
Fig. 7 is the propagation facilitation effect that represents the cell cultivation of the ES cell of the present compositionFigure;
Fig. 8 is the figure of each cell distribution ratio in the lymphocyte that represents to resolve through FACS;
Fig. 9 is for to be made as 1 by check plot, the T while having represented to add MCP of the present invention with ratioThe figure of the propagation facilitation effect of cell, B cell, NK cell;
Figure 10 is the figure that represents the propagation facilitation effect of the immunocyte of the present composition;
Figure 11 is the figure that represents the propagation facilitation effect of the immunocyte of the present composition;
Figure 12 is the figure that represents the propagation facilitation effect of the immunocyte of the present composition;
Figure 13 is the figure that represents the propagation facilitation effect of the immunocyte of the present composition;
Figure 14 represents the figure of the present composition for the effect of arteriosclerosis;
Figure 15 represents the figure of the present composition for the effect of arteriosclerosis;
Figure 16 represents the figure of the present composition for the effect of type ii diabetes;
Figure 17 is the figure that represents the hematological results after MCP picked-up;
Figure 18 is the figure that represents fibroblastic propagation facilitation effect of the present composition;
Figure 19 is the figure that represents the inflammation curative effect of the present composition;
Detailed description of the invention
The invention provides a kind of composition, its comprise as active ingredient, contain0.45～0.55ppm hydrogen, 10～12.5ppm oxygen, 7～8ppm nitrogen and particle diameter are 30 μ mFollowing microbubble and/or nano bubble. Said composition is for mitochondria activation, promotion cellThe preparation of propagation, cell preservation, cell freezing preservation, diet, parenteral solution etc.
In composition of the present invention the preferred concentration of contained gas be 0.1～3.0ppm hydrogen,5～20ppm oxygen, 3～20ppm nitrogen, more preferably 0.3～1.0ppm hydrogen, 7～15ppmOxygen, 4～15ppm nitrogen, more preferably 0.4～0.6ppm hydrogen, 9～13ppm oxygenGas, 5～10ppm nitrogen, be particularly preferably 0.45～0.55ppm hydrogen, 10～12.5ppm oxygenGas, 7～8ppm nitrogen. In addition, these concentration have corresponding effect under concentration separately,Therefore as long as within the scope of this, no matter divide wherefrom, all there is effect of the present invention. In addition,Ppm means partpermillion (1,000,000/), represents the concentration of dissolved gas.
In composition of the present invention, the particle diameter of contained gas is preferably more than below 0,30 μ m,More preferably, below 20 μ m, more preferably, below 10 μ m, be further preferably 1 μ mBelow, be particularly preferably below 100 μ m, most preferably be below 30nm.
In the hydroponic culture of plant etc., except nano bubble, can be by using micro-gasThe gas of bubble improves proliferate efficiency. In the case, as the size of microbubble, be preferably1～100 μ m, more preferably 5～80 μ m, more preferably 10～70 μ m, are particularly preferably15～60 μ m, most preferably are 20～50 μ m. They not refer to all microbubbles or nano bubble equalBe contained in this size, if microbubble or nano bubble all more than 60% for this reason size beCan.
The composition that contains nano bubble of the present invention is by receiving hydrogen, oxygen, nitrogen gasRice bubble is also sneaked into (for example, ultra-pure water) in water and is prepared. The purity of gas is not specialRestriction, for drinking water etc. in the situation that, is preferably used high-pure gas.
Nano bubble for example can be by carrying out failure by shear and trickle to the gas importing in liquidChange waits and produces, and is developing various nano-bubble generating apparatus. Nano bubble in the present inventionGenerating means uses as long as common nano-bubble generating apparatus can not add special restriction,For example, can use nano-bubble generating apparatus BUVITAS (registration mark, Co., Ltd.Ligaric society system) or nano-bubble generating apparatus NANOACQUA (registration mark, strain formulaThe TechCorporation of commercial firm system) etc.
The form of the composition that contains nano bubble of the present invention has no particular limits, typicalFor water, can be used in the existence container of drinking water, culture medium preparation water, fish water, plantThe sprinkle water of thing etc.
Formation for the preparation of the machine of function water (nano bubble water) comprises with lower component: hydrogenGas cylinder, oxygen cylinder, nitrogen cylinder or nitrogen gas generating means, digital-control type adjuster, Ozone WaterGenerating means. Nano-bubble generating apparatus, water injection tank, various filter set.
First, use about 1ppm Ozone Water, to nano-bubble generating apparatus, water fillingTank, pipe arrangement etc. carry out sterilization cleaning. In the case of by fresh water as the former water of function water, instituteThe formation of the filter passing through is with hollow fiber membrane filter, charcoal filter, hollow fibreTactic group of dimension film filter uses as a unit, the feelings that are former water at running waterUnder condition, conventionally use two unit. The quantity of unit can be carried out according to the degree of the impurity of former waterSuitably increase and decrease. The filtered water that has made to pass through above-mentioned filter element is passed through the thin of 0.25 μ m～0.45 μ mFilm filter (can be the anti-film filter that soaks into according to object), further filters and injects noteIn water pot.
The gas of from each bottle, concentration being adjusted by digital-control type adjuster is sent into nano bubble and is sent outGenerating apparatus circulates each gas and nano bubble between nano-bubble generating apparatus and water injection tankTime, in the filtered water in water injection tank, hold each gas to target saturation numerical value. RespectivelyPlant in checkout equipment, the gas in water passes through from water injection tank with the state that reaches target saturationThe membrane filter of 0.25 μ m～0.45 μ m and using.
The invention provides the mitochondrial activator of a kind of activation. Can pass through to measure ATP generation,Measure mitochondrial activation with the increase of ATP generation.
In this manual, " mitochondria activation " refer to ATP generation in mitochondria and rightCompare and increase according to district. Check plot typical case refers to common water (ultra-pure water), but is not limited to this,Also refer to except not containing the experiment that uses the composition of same composition nano bubble of the present inventionDistrict. The composition of the application of the invention, in the time that oxidative stress is loaded, has in mitochondriaThe effect that ATP generation increases compared with check plot.
According to composition of the present invention, can obtain high cell proliferation facilitation effect. For example, existUse composition of the present invention (aqueous solution) preparation cell to cultivate and carry out cultured cell with culture mediumIn situation, compared with using the culture medium of common water (ultra-pure water), can obtain high increasingGrow facilitation effect.
For example, at iPS cell, (inducedpluripotentstemcells, inductivity is multi-functionalStem cell) cultivation in, cultivate by 72 hours, obtained and used common ultra-pure waterThe propagation facilitation effect (Fig. 1) of approximately 2 times when culture medium is cultivated. This cell proliferation is shortEnter agent and also can be used for other cultured cell, for example just subtituted culturing cell, neoblast, hematopoiesisIn the fertile cells such as cell, fibroblast, immortalized cells, stem cell. Herein,Cultured cell has no particular limits, but refer to primary body cell (Japanese: 1 body Fine born of the same parents),Fissionable cells such as established cell line (Japanese: strain Fine born of the same parents), immortalized cells, stem cell.Stem cell contains embryonic stem cell and adult stem cell, for example NSC, liver stem cells,Skin progenitor cell, germline stem cell, iPS cell etc., but be not limited to this. Fertile thinBorn of the same parents refer to have the cell of multiplication capacity, comprise primary somatic cultured cell, neoblast,CFU-GM etc.
Can be to come from lactation as the body cell of the original material of making cultured cellFor example, any cell beyond the reproduction cell of animal (, mouse or people), for example, can listCornified epithelial cell (for example, keratinization epidermal cell), mucomembranous epithelial cell are (for example,The epithelial cell on tongue top layer), exocrine gland epithelial cell (for example, mammary glandular cell), hormone divide(for example, liver is thin to secrete cell (for example, adrenal medullary cell), metabolism and storage cellBorn of the same parents), form inner chamber epithelial cell (for example, I type alveole cell), the locking circulation of boundary faceSystem cavity epithelial cell (for example, vascular endothelial cell), possess the fibre with transporting capacityCell (for example, tracheal epithelial cell), the Extracellular Matrix Secretion cell of hair (for example, becomeFibrocyte), shrinkage cell (for example, smooth muscle cell), hemic and immune systems thinBorn of the same parents' (for example, T lymphocyte), relate to cell (for example, rhabdocyte), the plant of sensationThe supportint cell of property neuron (for example, cholinergic neuron), perceptron and nerve ending unitThe nerve cell of (for example, satellite cell), central nervous system and Deiter's cells (exampleAs, astrocyte), chromatophore (for example, retinal pigment epithelium) and itCFU-GM (tissue CFU-GM) etc. The degree of Cell Differentiation has no particular limits, notThe mature cell that the CFU-GM (comprising adult stem cell) of differentiation and final differentiation obtain all canSimilarly use as somatic source. Herein, as undifferentiated CFU-GM, for example canList the tissues such as NSC, candidate stem cell, mescenchymal stem cell, dental pulp stem cellStem cell (adult stem cell).
In addition, the composition of the application of the invention, can obtain and promote what plant cell was bredEffect. Use composition of the present invention preparation culture medium or hydroponic culture solution, by for plantingThing, can obtain propagation facilitation effect. Particularly in hydroponic culture, by add this simultaneouslyThe composition of invention and the microbubble (size is 10nm～30nm) of air, aerial part dry weight,Aerial part fresh weight, under ground portion dry weight, under ground portion fresh weight have all increased by 10～20% (ginsengsSee embodiment 14, table 7).
According to composition of the present invention, can preserve carefully with the state that highly keeps cytoactiveBorn of the same parents. For example, by fish being raised in the water that contains the present composition or being preserved, canHighly keep mitochondrial activity, sapid food is provided. This is considered to due to because of workChange mitochondria and increased the generation of ATP, as the inosinicacid (IMP) of its cataboliteAlso the reason increasing Deng delicate flavour composition. ,, according to composition of the present invention, can provide delicate flavourThe food increasing.
Composition of the present invention can directly make an addition in diet, also can be used as the raw material of dietUse, or for the processing of diet or dietary materials. Herein, diet is not limited, and meansThe drink that people are edible and food. For example, can list healthy supplement, low-calorie diet,Diet food, bread, dairy products (such as defatted milk, Yoghourt, pudding, soya-bean milk etc.), face(for example crow winter, Chinese face, won ton, dumpling wrapper, buckwheat etc.), dessert (for example cookies,Japanese dessert, steamed bun, potato chips, chocolate, Ka Shida sauce sandwich biscuits (custardcream)Deng), beverage (such as soup, vegetable beverage, weight-reducing drinks, black tea, coffee etc.), dessert (exampleAs cake, mousse etc.), frozen dessert (such as jelly, ice cream etc.), garnishes (for example fishGruel, egg roll, chicken nugget etc.), oatmeal, filling, baste, baking food, meat products (exampleAs sausage etc.).
Composition of the present invention can also be added in feed and use. Feed is also unrestricted, meaningFor the animal except people, be preferably beverage and the food of fish, domestic animal, pet animal etc.In addition also comprise bait. The more preferably water in the existence container of fish, tank, pond etc., ox,Beverage and the food of pig, sheep, chicken, horse, dog, cat etc.
In addition, composition of the present invention can be used as injection (comprising drops) use. For example,By to injection compositions of the present invention such as pet, domestic animal, animal used as test and/or people, also canEnough Effect of promoting growths as immunocyte, diabetes, arteriosclerosis, inflammation etc. pre-Anti-and/or curative uses. In addition, composition of the present invention can be used for the preparation of parenteral solution.
In addition, composition of the present invention is by directly drinking as drinking water to human or animal, alsoCan obtain Effect of promoting growth, diabetes, the artery sclerosis of for example immunocyte of various diseasesThe prevention of disease, inflammation etc., result for the treatment of.
The present invention will be described by the following examples, but that scope of the present invention is not subject to is anyDescribed embodiment limits.
Embodiment
(embodiment 1)
IPS cell culture medium is prepared in the following manner. Stay water (DW) or basis to the steaming of 1LInvention composition (APAS) in add 12.5g Glasgow Minimum Essential Medium (Sigma,G6148) and 2.75g sodium acid carbonate (Sigma, S5761), stir. In this solution,Add the nonessential amino of MEM of each reagent: 0.1mM to form the mode of following ultimate densityThe Sodium Pyruvate solution (Sigma, S8636) of acid solution (Gibco, 11140) and 1mM,1% hyclone (CCB, 171012), 1% mycillin (Gibco, 15140),5.4% Knockout serum substitute (Gibco, 10828), the 2-sulfydryl second of 0.1mMThe LIF ELISA of alcohol (Wako, 137-06862), 2000U/ml (Millipore,ESG1107). Be stirred to after even and dissolving, with 0.45 μ m filter filtration sterilization, asCell maintain base.
The result of cultivating as shown in Figure 1. Its result is to use composition of the present invention to makeIn the situation of culture medium, make in the situation of culture medium with using common ultra-pure water, use thisThe culture medium that bright composition is made is compared with the ultra-pure water culture medium contrasting, and has obtained approximately 2 timesProliferative amount.
(embodiment 2) used the antioxygen of Mouse Endothelial strain (MAEC) to turn intoWith research
Use respectively the water, nano bubble water (the aeriferous nanometer that contain composition of the present inventionAir-bubble), hydrogen nano bubble water (contains closing of dissolved gas, microbubble and nano bubbleThe nano bubble water of more than 70% hydrogen of meter) making M199 culture medium (SIGMA),Add 10% N of tire serum, 100IU/ml penicillin and 100 μ l/ml streptomysins, as cultivationUse culture medium. After MAEC being carried out cultivating for 24 hours in the culture medium that has used various water,Be replaced by fresh culture medium, further carry out cultivating for 2 hours. Then in each culture medium, addAdd 30 μ g/mL antimycins, cultivate 30 minutes. In contrast, by as the ethanol of solvent withAdd with the mode of the 0.06v/v% of contained consumption equivalent in antimycin solution. Known anti-mildewElement has by suppressing mitochondrial electron transport chain, make to produce in cell ROS (active oxygenBunch, ReactiveOxygenSpecies) effect. Ethanol is usually used as the solvent of antimycinUse, therefore in order to understand the impact of this solvent, tested and only added the not ethanol containing antimycinSituation. In order to learn the amount of produced ROS, using fluorescence intensity as index, measuring and calculatingThere is the cell quantity of this fluorescence intensity. Particularly, use CM-H2DCFDA as useIn the probe that detects ROS, by flow cytometer (VantageSE, BectonDickinson)Detect the fluorescence intensity from each cell.
The testing result of ROS as shown in Figure 2. It is strong that each chart of Fig. 2 shows certain fluorescence of embodimentThe cell number of degree, the value representation average fluorescent strength of MFI. Use nano bubble water (NB,Epimere) culture medium in situation that MAEC cell is cultivated, (a left side when non-stimulatedRow) and only added in the situation (center row) of solvent control ethanol, almost unconfirmed to flatAll fluorescence intensity changes, on the situation (right side of having added the antimycin with ROS generation effectRow) under, average fluorescent strength significantly strengthens. In contrast, contain the present invention in useThe water (O2H2N2NB, hypomere) of the composition situation of cultivating under, with useWhen cultivating, the culture medium of nano bubble water compares, non-stimulated and add antimycin arbitraryIn situation, all obtain low average fluorescent strength. In addition, using hydrogen nano bubble waterWhen cultivating, the culture medium in (H2NB, stage casing) contains composition of the present invention with usingWhen the culture medium of water is cultivated, compare, non-stimulated situation, only add ethanol situation,Add in the whole circumstances of situation of antimycin, the water that use contains composition of the present inventionCulture medium is cultivated and is demonstrated low average fluorescent strength. Thus results verification contain thisThe strong antioxidant action of the water of bright composition.
Oxidative stress load in (embodiment 3) Mouse Endothelial strain (MAEC)Time ATP produce research
Then, in order to confirm that ATP produces active water when oxidative stress in MAEC is loadedThe effect of the mitochondrial function declining, produces and is studied ATP in cell. With above-mentionedTest identical, by the water, nano bubble water, the hydrogen nanometer gas that contain composition of the present inventionThe cultivation culture medium of soaked making, cultivates MAEC cell. Each by having usedThe culture medium of planting water carries out, after cultivation in 24 hours, culture medium being replaced by and having been used to MAECThe fresh culture of various water, further carries out cultivating for 2 hours. Then, in each culture mediumAdd 50 μ g/ml antimycins, 0.06v/v% ethanol, 500 μ mol/L hydrogen peroxide. In addition,As the contrast of antioxidation, use the antioxidant that acts on conventional reactive oxygen speciesN-acetylcystein (NAC). The contrast of antioxidation is used common water with culture mediumMake. In this culture medium, MAEC cell is carried out cultivating for 24 hours, be replaced by and usedThe fresh culture of common water further carries out cultivating for 2 hours. Then, in antimycin or mistakeBefore 30 minutes of hydrogen oxide water treatment, so that the mode that ultimate density is 5mmol/L is to cultivationIn base, add NAC. Then, taking uciferase activity as the intracellular ATP amount of index determiningVariation (ATPlite, PerkinElmer).
The measurement result of intracellular ATP amount as shown in Figure 3. Using nano bubble water(NB), in the cell of cultivating in culture medium, confirm compared with non-stimulated situation,ATP amount reduces under all situations of Ethanol Treatment, antimycin processing, hydrogen peroxide treatment.Result is known thus, and ethanol, antimycin and hydrogen peroxide have ATP and produce active inhibitionEffect.
In antimycin is processed, confirm the ATP stronger than hydrogen peroxide treatment and produce activeReduction. Use respectively hydrogen nano bubble water (H2NB), containing composition of the present inventionThe cell cultivated of the culture medium of water (O2H2N2NB) in, carry out any placeThe situation of reason has all confirmed ATP and has produced active increase. That is, can confirm, hydrogen is receivedRice air-bubble and the water that contains composition of the present invention produce ATP that antimycin etc. causes to liveProperty inhibition had and antioxidant NAC same degree or greatly reduce. MoreFurther, in the situation that processing by ethanol or hydrogen peroxide, containing the present compositionWater and hydrogen nano bubble water ratio, ATP produce active inhibition also significantly reduce.
(embodiment 3) people's the research of drinking effect
Taking clear and definite people in the case of drink containing the water of the present composition to the effect of human body asObject, has implemented people's the experiment of drinking. The people who participates in the experiment is 40～69 years old (mean age51.8 years old) 4 male sex and 1 women. Each participant's age and sex are as shown in table 1.All participants are health status.
[table 1]
Sex Age (year)
1 Man 40
2 Man 69
3 Man 50
4 Female 47
5 Man 53
Each participant drinks the water 1L containing the present composition every day, drinks 4 weeks, make great efforts intoCapable and identical at ordinary times life. Participant, drinking blood sampling before beginning, is drinking beginning the rear the 4thZhou Zaidu takes a blood sample. Do not drink the water containing the present composition same day in blood sampling. To liver diseaseSick index GPT (glutamic-pyruvic transaminase), lipid peroxide mark HEL (acetyl-l-lysine),And STAS (the serum total antioxidation shape being widely known by the people as water-soluble anti-oxidant ingredientsState (TotalAntioxidantStatus)) check.
(1)GPT
GPT is a kind of enzyme being mainly present in liver. Due to meeting in the time that liver cell is destroyedIn blood, leak specifically, be therefore used as the finger of hepatitis viruse or drug induced liver diseasesMark. In addition, people also know in liver fat, the value mile abnormality of GOT, GPTSituation in the majority, in the fatty liver that obesity causes, GPT is slightly higher than GOT. Under GPT passes throughThe mode of stating is measured: use SilicaLiquidALT reagent (Kanto Kagaku K. K.'s system),By BioMajasesty (JCA-BM8060) (Jeol Ltd.'s system) and OlympusAU5421 type automatic clinical chemistry analyzer (Olympus Co., Ltd. system) is measured in serumConcentration. Make the blood clotting of taking, separate and reclaim serum with centrifugation. Measurement result is as figureShown in 4 casees line charts. With reference to Fig. 4, after the water that contains the present composition in picked-up, GPTValue with picked-up before compared with have obvious minimizing (P=0.042). Therefore, contain this by drinkingThe water of invention composition, the improvement that can confirm fatty liver is inclined to.
(2)HEL
HEL is in the peroxidating process of the lipid causing because of reactive oxygen species, derives from lipidThe stable initial stage of peroxide (13-hydrogen peroxide-octadecadienoic acid, 13-HPODE)Product, different from the aldehydes such as 4-HNE or the MDA lipid peroxidation mark that used in the past,It is for catching the mark of lipid peroxidation initial stage. HEL uses hexanoyl lysine to surveySurely use ELISA kit (KHL-700, Nikken Seil Co., Ltd. system), pass through automaticallyMicrowell plate EIA analytical equipment AP960 (consonance Medics Co., Ltd. system) measures in serumConcentration. Measurement result is as shown in Fig. 5 case line chart. With reference to Fig. 5, HEL is drinking ATPAfter producing active water, there is remarkable minimizing (P=0.028). Therefore, contain the present invention's combinationThe water meter of thing reveals the effect in human body with inhibition lipid peroxidation.
(3)STAS
STAS is the water-soluble polyphenoils in serum, can measure combining for oxidative stressClosing property oxidation resistance. STAS uses total antioxidant status (NX2332, RANDOXSociety's system), by 7020 automatic analysing apparatus (Co., Ltd. Hitachis of type HitachiHigh-Technologies system) serum is measured. Measurement result is as shown in Fig. 6 case line chart.With reference to Fig. 6, STAS shows remarkable low value (P=0.042) after picked-up. According to this resultCan infer, STAS is by water miscible polyphenoils is carried out to comprehensive evaluation,Can expect the water that contains the present composition oxidative stress eradicating efficacy, hydroxyl freeIn the reduction of the reactive oxygen species beyond base or peroxinitrites, contain the present compositionWater in action.
(embodiment 4)
Allow 2 experimental subjects (O, I) absorb 500mL every day and contain the present compositionWater (APAS, O2H2N2NB water, also referred to as MCP), absorbs one month, to taking the photographBefore and after getting, carry out blood test.
Result
Experimental subjects O
Compare with the value before the water that contains the present composition in picked-up, after picked-up,In the value of ALP (alkaline phosphatase), ALT, γ-GTP, be improved. In addition, T-CHOL,In the value of LDL-C, free fatty, be also improved.
Experimental subjects I
Compare with the value before the water that contains the present composition in picked-up, after picked-up,In the value of ALP (alkaline phosphatase), ALT, γ-GTP, be improved. In addition, T-CHOL,In the value of LDL-C, free fatty, triglycerides, be also improved.
Carrying out comprehensive judgement by 2 people's result can think, contains of the present invention group by picked-upThe water of compound, has formed the improvement to liver function, has improved the clinical examination value of lipid.
[table 2]
(embodiment 5)
About the experiment in vitro of ES cell proliferation rate
(cultivate with the iPS cell of embodiment 1 at the culture medium that uses DW or APAS preparationThe composition that base is identical) in when the propagation of investigation ES cells, will cultivate the propagation of the 3rd dayRate is set as in 1 situation, if use APAS preparation culture medium, observes approximately 1.5 timesThe rate of increase (Fig. 7).
(embodiment 6)
Immunocyte (T cell, B cell, the NK cell) propagation causing about MCP is shortExperiment in the body entering
Be added into the kind of the water of culture medium
Experiment A
(1) former water (the former water of MCP preparation)
(2) former water+gas (filtering without filter)
(3) former water+gas+0.22 μ m filter
The method for breeding of rat: use male SD rat (12 weeks) as animal used as test. ToRat gives the method for water and carries out in two kinds of modes of administration in per os and abdominal cavity. In abdominal cavity, administration is logicalCrossing intraperitoneal injection carries out. Oral administration with in, use go out by 0.22 μ m filterThe MCP of bacterium. On the other hand, in control group, the DW that administration is purchased. Administration in abdominal cavityBe by MCP or cultivate with DW dilution as with respect to 10 × PBS being 1 × use (1ml × 2/Day). The replacing of water and injection are according to sooner or later carrying out for twice every day.
Experimental session: to starting the 3rd day and evaluate for the 7th day from experiment. Control group (n=2)With test group (n=2) for once evaluate.
Experimental technique
Carry out the heart blood sampling (4ml) of rat, blood is added in 15ml pipe, in this pipeAdd the HetaSep of 1/5 times of amount (800 μ l), fully to mix, cultivate that (37 DEG C, 2 is littleTime more than). Then the separation of, carrying out red blood cell layer is confirmed. After 2 hours, because lymph is thinBorn of the same parents' composition is set in pipe periphery, and red blood cell component is set in pipe central authorities, therefore now by moving liquidDevice separates the lymphocyte composition of about 2ml. Then, in 15ml pipe, reclaim with 2ml/ sampleLymphocyte composition, carries out centrifugal (3,000rpm, 8 minutes, RT). By PBS (RT)Be adjusted to 30 μ l/ samples, in centrifuge tube (Eppendorftube), carry out dispensing with 30 μ l/ pipes,All the other are with comparing. To adding IOTest in each pipe, (30 μ l), move liquid (examinationSample: IOTest=1:1). Then, carry out antibody response with 20 minutes/RT, cushion with FACSLiquid (1 ×) cleans, and carries out centrifugal (3,000rpm, 5 minutes, RT). Remove after supernatant,Under shading, add VersaLyse (500 μ l/ pipe), under RT, leave standstill 10 minutes. Then, enterThe immunocyte ratio measuring and calculating of the total cell number counting of row and FACS.
Result
The result that FACS resolves as shown in Figure 8. In addition as shown in Figure 9, throw at MCP,With in group, DW administration group is made as to 1 while comparing, confirm T cell, B cell,NK cell all increases. There is too big difference although do not confirm the increase ratio of each cell number,But among this, the increment rate in the MCP administration group of B cell is large. By the present embodiment,Show that MCP of the present invention also can be used as injection and uses.
(embodiment 7)
In this experiment, (1) former water: for the preparation of former water, (2) MCP, (3) of MCPMCP+0.22 μ m filter: three kinds of water that MCP has been passed through to 0.22 μ m filter are with warpMouthful administration and intraperitoneal injection (1ml × 2/ day, early, evening), to rat administration, were raised 7 daysThe amount of the each component in rear rat blood lymphocyte compares.
In observation after 7 days on-tests, all individualities are all healthy, unconfirmed arriving because of injectionThe abdominal distension that causes of excessive administration or the symptom of diarrhoea.
Result (the 7th day): while calculating cell number under the microscope, observed in administration (2)And the remarkable increase (Figure 10) of lymphocyte number in the situation of (3). Thus, clear and definiteMCP has the effect that increases blood lymphocyte number. Concrete total lymphocyte number is:(1) in, be 50.2 × 104Individual, in (2), be 161 × 104Individual, in (3), be 83 × 104Individual, the ratio that (1) is made as at 1 o'clock is: in (2), being 3.21, is 1.66 in (3). ,The in the situation that of administration MCP, the increment rate of lymphocyte number is individual for 3 that testMean value 321%. In administration T cell, the B in the lymphocyte three kinds of water in the situation thatThe quantity of cell, NK cell and the cell number in (1) administration group is made as to (2) of at 1 o'clockThe ratio of the each cell number in administration group and (3) administration group is illustrated in Figure 11 and Figure 12. FigureKnown in 12 in the ratio of the quantity of T cell, B cell, NK cell and common ratRatio is similar. According to Figure 12, comparing with DW administration group in MCP administration groupIn the increment rate of these three kinds of cell number, the increment rate of the increment rate of B cell and other two kinds of cellsCompare large. In each cell grade (Japanese: each Fine born of the same parents draw and divide) in lymphocyte, with(1) of NK cell number as the ratio in (2) and (3) of benchmark 1 as shown in figure 12.According to Figure 12, the NK cell increment rate in (2) is individual average of measuring and calculating obtain 3Value 348%. The comparative studies of the result of experiment (1) and (2): in all tests,In the situation of administration MCP, the water that does not add gas with administration is (for the preparation of DW or MCPFormer water) situation compare, the T cell in blood, B cell, NK cell all increase.This represents to have lymphopoiesis facilitation as the gas of the core of MCP. ThisOutward, in two experiments, the increase ratio of each cell number does not demonstrate large difference, but at itIn embody the large feature of increment rate of B cell.
(embodiment 8)
Experiment B
(1) former water (for the preparation of the former water of MCP)
(2) former water+gas+0.45 μ m filter
(3) former water+gas+0.22 μ m filter
In experiment B, in the district of the former water+gas by filter not, observe formerThe total lymphocyte number (Figure 13) of 3.21 times of water. This means that proliferative amount has become approximately 3 timesAbove. In contrast, with filter, former water+gas is being carried out to the culture medium filteringIn, in the culture medium filtering with 0.45 μ m filter, confirm the increasing of the proliferative amount of 1.76 timesAdd, in the culture medium filtering with 0.22 μ m filter, confirm the increasing of the proliferative amount of 1.66 timesAdd. Exist due to filtered, removed by filter thereby microbubble caused this result canCan property. The diameter of gas is 10nm～30nm.
(embodiment 9)
Measure and carry out the impact of gas administration to T cell, B cell, NK cell. Do not havingIn the system of filter, B cell proliferation is maximum, is then NK cell, T cell (Fig. 9).
(embodiment 10)
The relatively NK cell number in Yuan Shui+gas administration individuality. Its result is as Figure 12 instituteShow. As shown in figure 12, in the situation that there is no filter, bred former water approximately 3.5 times.In the situation that having filter, in 0.45 μ m filter, show the propagation of 1.93 times, 0.22 μ mIn filter, show the propagation of 1.64 times.
(embodiment 11)
To the effect of 81 years old two lower limb Arteriosclerosis obliterans of women
It is put down into 23 year spring in Japan and starts the morphotropism knee joint disease because following two kneecap necrosisGo to hospital, put down into 25 year autumn in Japan and start mainly to occur right lower extremity edema. Put down in JapanBe in hospital in February, 26 because of right lower extremity thromboangiitis, carries out thromboembolism treatment. Put down in JapanStart to drink every day 500ml mitochondria water in March, 26.
Its result is to confirm edema and alleviate, inflammation improvement. As artery sclerosis indexIn the variation of ABI value (ankle angiosthenia/BA ratio), confirm mitochondria water and drinkThe improvement of the value in rear left side. At baPWV (as evaluating between the upper arm ankle of arterial compliance indexPulse wave conduction speed) development in, confirmed in both sides and improved (with reference to table 3 and figure14～15)。
Before drinking mitochondria water since March, in drug combination, there is no anticoagulation preparation,Carry out the administration of anti-platelet aggregation agent since May, therefore, for specifically 2014The improvement of the baPWV on May 24,, thinks that the possibility of the impact that is subject to mitochondria water is large.The artery sclerosis that, has implied this case is improved relevant with the effect of mitochondria water.
[table 3]
UT=time msPWV=pulse wave conduction speed ABI=ankle SAP/arteria brachialis systolic pressure of upwards beating
Right ankle UT Left ankle UT Right baPWV Left baPWV Right ABI Left ABI
2013/5/24 192 185 1546 2425 0.91 1.08
2013/10/4 230 209 1719 2929 0.87 1.17
2014/5/1 181 131 1657 2780 0.92 0.98
2014/9/5 179 149 1325 2004 0.93 1.26
(embodiment 12)
Experimental subjects: 81 years old women's type ii diabetes
Put down in February, 20 type ii diabetes morbidity in Japan.
The diagnosis and treatment of the therapeutic process centered by medicine for oral administration, Life Guidance are carried out.
Medicine for oral administration be 1.25mg sulphonyl preparation Daonil (3 on the one, points 3 times, after meal)And 0.2mg postprandial hyperglycemia-improving agent Basen (3 on the one, points 3 times, ante cibum), inClothes.
Confirm as the hyperlipidemia of complication, slight poor kidney.
To hyperlipidemia, before sleeping, take 10mg Atorvastatin calcium preparation orally.
The effect of mitochondria water administration
May, 8～May 16 and September 1～6 were passed through urokinase after being admitted to hospitalThromboembolism treatment is carried out in drip-feed. Comprise the above-mentioned time until now, drink lasting every day500mL mitochondria water. HbA1c is (by the blood that is object to delivering oxygen in body in red blood cellGlucose in Lactoferrin and blood is combined into) value occur from drinking 2014/1/87.8 to 2014/9/1 5.8 huge improvement (table 4 and Figure 16). Oral dosage is identical, 4Unchanged in year. This is considered to be changed because the impact of mitochondria water makes type ii diabetesKind.
[table 4]
Mitochondria water is drunk the course of the HbA1c of front and back
2014/1/8 7.8
2014/3/7 7.6
2014/5/8 6.9
2014/6/4 6.5
2014/9/1 5.8
(embodiment 13)
Drink effect that in situation, MCP brings health as object using clear and definite people, implement people'sMCP drinks test. Experimental subjects is as described below.
1.50 years old male sex, 2.45 years old male sex, 3.60 years old women, 4.35 years old women, 5.63Year women, 6.66 years old male sex, 7.25 years old women
When each participant carries out with in the past constant life, in 4 weeks, drink MCP every day0.5～1.0L. Participant drink start before blood sampling, and start the 4th week and the 8th afterwards drinkingZhou Zaici blood sampling. Projects that leucocyte, liver function, lipid are relevant, diabetes are relevant are enteredRow checks. Its result is to confirm liver function, cholesterol value, triglycerides value, diabetesThe improvement of numerical value in the project of the broad range such as relevant numerical value. The improvement of numerical value is in exceptional valueSignificantly, for normal value, confirm the improvement tendency to better numerical value, particularly confirmThe project that significant numerical value improves is cholesterol value, in checked nearly all experimental subjectsConfirm improvement. In addition, for HDL value, never arrived when health check-up in 20 years in the past,The numerical value of 40 experimental subjects 6 has arrived 45, and therefore, MCP may contribute to do not having at presentThere is the rising (table 5) of the HDL value of specific drug.
[table 5]
(embodiment 14)
Use little Song dish (Brassicarapavar.perviridis, わ か body) investigation to plantImpact.
The cultivation phase: April 77 days～2014 March (32 days) in 2014
Method: add 30L dechlorination water in container, make hydroponic culture fertilizer TankmixF&B(table 6, OATAgrio Co., Ltd. system) dissolves. Nutrient solution is interim being adjusted into of cultivationEC2.4, carries out within 24 hours, ventilate (aeration).
At polyurethane upper seeding wheel, after 10 days growing seedlings carried out in growth in case, in each containerEach field planting 9 strain individualities, carry out 3 times (3 containers) and repeat test. In field planting after 3 weeksGather in the crops.
Treatment region: check plot, air microbubble (MB) district, APAS+MB district
In cultivation, start 1 week from field planting 3 times weekly, in air MB district to airMB carries out the generation of 20 minutes, in APAS+MB district, the gas of MBization is also carried out to 20Minute the generation processing of 6 times (amount to)
Survey item: plant height, maximum blade length, maximum root length, aerial part fresh weight, undergroundPart fresh weight, aerial part dry weight, under ground portion dry weight
Result is as shown in table 7, for plant height, maximum blade length, maximum root length, aerial partFresh weight and under ground portion dry weight, unconfirmed to significant difference between treatment region. ButThe aerial part fresh weight in APAS+MB district and under ground portion fresh weight are compared with JiMB district, check plot, there is remarkable increase. In APAS+MB, confirm growth-promoting effect.
[table 6]
TankmixF&B concentrated stock solution becomes component (g/100L)
(according to the record of the HP of OATAgrio Co., Ltd.)
[table 7]
* different English alphabets is illustrated in the conspicuousness (Fisher least significant difference method) of 5% level
* ± after numeric representation standard error (Fisher least significant difference method)
(embodiment 15)
About the reality of the fibroblast proliferation facilitation in the culture medium that has added MCPTest
The preparation of culture medium
1. contrast
Carrying out after above-mentioned mixing, carrying out filtration sterilization with 0.45 μ m filter. With every 10mlOr every 50ml prepares 10%FBS (hyclone), hold to adding 1/10th in culture mediumAmount.
2.1×MCP
After mentioned component is mixed, filter with filter. By above-mentioned 9ml solution (DMEM(MCP) mix with 1mlFBS, for cultivating.
3.2×DMEM+MCP
After mentioned component is mixed, filter with filter. Afterwards with MCP dilution 2Doubly.
Recover mouse embryo fibroblast (MEF) cell of freezing preservation, carries out three times to uploadIn generation, is rear for this experiment. Use the FBS that is 10% by final concentration as above to prepareDMEM (high sugar (HighGlucose)) cultivates. Within every 3 days, carry out a subculture moreChange, in the time merging (confluent), go down to posterity.
First this experiment on 12 orifice plates, enters in the mode that comprises 10,000 MEF cells in 1 holeRow goes down to posterity. Going down to posterity second day, cleaning MEF cell once with PBS, with DW and MCP,The FBS that to add by final concentration be 10% and the DMEM (height of the dual anti-solution preparation of mycillinSugar). The date that adds MCP is made as to the 0th day, resolved at the 3rd day. From addingMCP starts to change culture medium every day.
Cell number is determined as, and adds 0.25% trypsase (trypsin), passes through trypan blue(trypanblue) dyeing, only counts living cells.
Culture experiment is carried out with 2 groups, carries out the cultivation of 2 days and 8 days. From these 2 times experimentAs a result, implied that MCP has fibroblast proliferation facilitation (Figure 18). Its propagationRate, with add the situation of DW to culture medium compared with, is 125～200% (tables 8).
[table 8]
In experimental result, confirm a little deviation, be considered to owing to criticizing for the cell of testingInferior difference, or because the fluctuation of experiment condition is caused.
Fibroblast has collagen, elastin laminin, hyaluronic acid of being created on epithelium etc.Function. In addition there is using collagen as fibre bundle,, form the effect of dermis.If it is aging that Large Amount of Irradiated ultraviolet ray promotes, cannot generate the fiber with systematicness, produce wrinkleLine or lax etc. The MCP with function of fibroblasts is expected to show to above-mentioned skinEffective inhibition that skin is aging.
(embodiment 16)
63 years old women that knee is violated inflammation continued the inflammation of approximately 2 years, by drinking every dayThe MCP of 500ml～1L, is greatly improved 1st month inflammation. 3rd month inflammationBasic disappear (Figure 19). As the amount of drinking MCP, preferably more than 150ml every day, morePreferably more than 300ml, more preferably more than 500ml, be further preferably 1L withOn. The moisture absorbing every day can be MCP entirely.
(embodiment 17)
MCP is drunk by pet as the drinking water of 30 days every day, as shown in table 9, confirmTo the improvement of illness index value.
[table 9]

Claims (19)

1. a composition, its comprise as active ingredient, contain 0.45～0.55ppm hydrogenGas, 10～12.5ppm oxygen, 7～8ppm nitrogen and particle diameter be microbubble below 30 μ m and/ or nano bubble.
2. a mitochondria activated compositions, it contains composition claimed in claim 1 and doesFor active ingredient.
3. a cell growth promoter, it contains the composition described in claim 1 or 2As active ingredient.
4. a cell-preservation liquid, it contains the combination described in any one in claim 1～3Thing or agent are as active ingredient.
5. a freezing preservation liquid, it contains the combination described in any one in claim 1～4Thing, agent or liquid are as active ingredient.
6. a diet, it contains the composition described in any one, agent in claim 1～5Or liquid.
7. a diet, it is by using the combination described in any one in claim 1～5The raw material of thing, agent or liquid processing and manufacturing.
8. the mitochondrial method of activation, its right to use requires in 1～5 described in any oneComposition, agent or liquid.
9. promote a method for cell proliferation, its right to use requires any one institute in 1～5Composition, agent or the liquid stated.
10. preserve a method for cell, in its right to use requirement 1～5 described in any oneComposition, agent or liquid.
The method of 11. 1 kinds of freezing preservations, in its right to use requirement 1～5 described in any oneComposition, agent or liquid.
The manufacture method of 12. 1 kinds of diet, it comprises following operation: right to use requires 1～5Composition, agent or liquid described in middle any one, process diet or its raw material.
13. 1 kinds of cell cultivation culture mediums, it requires the combination described in 1 by right to useThing and preparing.
14. 1 kinds of vegetation water cultivation solution, it requires the group described in 1 by right to useCompound and preparing.
15. 1 kinds make the method for zooblast propagation, and its right to use requires the training described in 13Support base.
16. methods that make zooblast propagation according to claim 15, wherein, instituteStating zooblast is immunocyte.
The prevention of 17. 1 kinds of arteriosclerosis and therapeutic agent, it is with described in claim 1 or 2Composition or cell growth promoter claimed in claim 3 as active ingredient.
The prevention of 18. 1 kinds of diabetes and therapeutic agent, it is with the group described in claim 1 or 2Compound or cell growth promoter claimed in claim 3 are as active ingredient.
19. 1 kinds of parenteral solutions, it contains the composition described in claim 1 or 2.
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