WO2015099201A1 - Nanobubble-containing composition and use thereof - Google Patents
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- WO2015099201A1 WO2015099201A1 PCT/JP2015/050073 JP2015050073W WO2015099201A1 WO 2015099201 A1 WO2015099201 A1 WO 2015099201A1 JP 2015050073 W JP2015050073 W JP 2015050073W WO 2015099201 A1 WO2015099201 A1 WO 2015099201A1
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Definitions
- the present invention relates to a nanobubble-containing composition and its use. More specifically, the present invention relates to a composition containing nanobubbles and activating mitochondria and its use.
- Patent Document 1 discloses a method for promoting the growth of cultured cells by dissolving a gas in a liquid using microbubble or nanobubble technology.
- Patent Document 1 specifically discloses in detail what size and composition of microbubbles and nanobubbles can be used to promote the proliferation of what types of cells. There has been a demand for the development of more practical compositions, methods, and apparatuses. There has also been a demand for a more efficient method for promoting proliferation.
- Hydrogen water, hydrogen oxygen water, etc. are known as products in which gas is mixed with drinking water using microbubbles or nanobubbles (for example, Patent Document 2).
- Patent Document 2 the number of people who feel a satisfactory effect is limited to a part, and the current situation is that they are not so popular. Therefore, microbubbles and nanobubble drinking water that can realize a sufficient effect have been demanded. Also, microbubbles and nanobubble drinking water having new functions have been demanded.
- mitochondria are energy-producing organs, and it is thought that increasing mitochondrial activity can increase energy production and make the body more active.
- drinking water that activates mitochondria has not been developed yet, and development of drinking water that activates mitochondria has been demanded.
- the present invention provides a composition containing nanobubbles containing hydrogen, oxygen, and nitrogen and uses thereof.
- the concentration of each gas is the concentration of both dissolved gas and nanobubbles.
- the mitochondrial activation composition has the same meaning as the mitochondrial activator.
- the cell growth promoter may contain both the compositions (1) and (2).
- the agent means the cell growth promoter of (3).
- the mitochondrial activation composition of (2) is corrected as a mitochondrial activator, the mitochondrial activator is also included in the agent.
- “any one” means “at least any one” and does not mean “only any one”. Accordingly, the technical scope includes two or more or three or more compositions or agents among (1) to (3). The same applies to the following, and “any one” means “(at least) any one”.
- the agent has the same meaning as in (4)
- the solution means the cell preservation solution in (4).
- the solution includes the cryopreservation solution (5) in addition to the cell preservation solution (4).
- (10) A method for preserving cells using the composition, agent or solution of any one of (1) to (5).
- (11) A method for cryopreserving the composition, agent or solution of any one of (1) to (5).
- (12) A method for producing a food or drink, comprising a step of treating the food or drink or its raw material using the composition, agent or liquid of any one of (1) to (5).
- (12) A cell culture medium prepared using the composition of (1).
- (14) A solution for plant hydroponics prepared using the composition of (1).
- (15) A method for growing animal cells using the medium of (13).
- (16) The method for growing animal cells according to (15), wherein the animal cells are immune system cells.
- a prophylactic / therapeutic agent for arteriosclerosis comprising the composition according to (1) or (2) or the cell growth promoter according to (3) as an active ingredient.
- a prophylactic / therapeutic agent for diabetes comprising the composition according to (1) or (2) or the cell growth promoter according to (3) as an active ingredient.
- An injection solution comprising the composition according to (1) or (2).
- composition of the present invention the effects of activating mitochondria, promoting cell proliferation, and reducing damage to cells during cell storage and cryopreservation can be obtained.
- FIG. 1 is a graph showing the growth promoting effect of cell culture of the composition of the present invention.
- FIG. 2 is a diagram showing the antioxidant action of the composition of the present invention.
- FIG. 3 is a diagram showing the influence of the amount of ATP produced on various drugs.
- FIG. 4 is a diagram showing changes in GPT before and after taking the composition of the present invention.
- FIG. 5 is a diagram showing the generation rate of HEL before and after taking the composition of the present invention.
- FIG. 6 is a diagram showing changes in STAS before and after taking the composition of the present invention.
- FIG. 7 is a diagram showing the growth promoting effect of ES cell culture of the composition of the present invention.
- FIG. 8 is a diagram showing the distribution ratio of each cell in lymphocytes by FACS analysis.
- FIG. 9 is a graph showing the growth promoting effects of T cells, B cells, and NK cells when the MCP of the present invention is added, with the control group as 1.
- FIG. 10 is a diagram showing the effect of promoting the proliferation of immune cells of the composition of the present invention.
- FIG. 11 is a diagram showing the effect of promoting the proliferation of immune cells of the composition of the present invention.
- FIG. 12 is a diagram showing the effect of promoting the proliferation of immune cells of the composition of the present invention.
- FIG. 13 is a diagram showing the effect of promoting the proliferation of immune cells of the composition of the present invention.
- FIG. 14 is a diagram showing the effect of the composition of the present invention on arteriosclerosis.
- FIG. 15 is a diagram showing the effect of the composition of the present invention on arteriosclerosis.
- FIG. 10 is a diagram showing the effect of promoting the proliferation of immune cells of the composition of the present invention.
- FIG. 11 is a diagram showing the effect of promoting the proliferation of immune cells of the composition
- FIG. 16 is a diagram showing the effect of the composition of the present invention on type II diabetes.
- FIG. 17 is a diagram showing the results of a blood test after ingestion of MCP.
- FIG. 18 is a graph showing the fibroblast proliferation promoting effect of the composition of the present invention.
- FIG. 19 is a diagram showing the inflammation healing effect of the composition of the present invention.
- the present invention provides a composition containing, as an active ingredient, microbubbles and / or nanobubbles having a particle size of 30 micrometers or less, containing 0.45 to 0.55 ppm of hydrogen, 10 to 12.5 ppm of oxygen, and 7 to 8 ppm of nitrogen.
- This composition can be used for mitochondrial activation, cell growth promotion, cell preservation, cell cryopreservation, food and drink, preparation of injection solution, and the like.
- the preferred concentration of the gas contained in the composition of the present invention is 0.1 to 3.0 ppm for hydrogen, 5 to 20 ppm for oxygen, 3 to 20 ppm for nitrogen, more preferably 0.3 to 1.0 ppm for hydrogen, 7 to 15 ppm for oxygen, 4 to 15 ppm for nitrogen, More preferably, hydrogen is 0.4 to 0.6 ppm, oxygen is 9 to 13 ppm, nitrogen is 5 to 10 ppm, particularly preferably hydrogen is 0.45 to 0.55 ppm, oxygen is 10 to 12.5 ppm, and nitrogen is 7 to 8 ppm.
- concentrations have a moderate effect in each density
- ppm means a part per million and represents the density
- the gas contained in the composition of the present invention has a particle size of greater than 0 and preferably 30 micrometers or less, more preferably 20 micrometers or less, more preferably 10 micrometers or less, even more preferably 1 micrometers or less, Especially preferably, it is 100 micrometers or less, Most preferably, it is 30 nanometers or less.
- the growth efficiency can be increased by using microbubble gas in addition to nanobubbles.
- the size of the microbubble It is preferably 1 to 100 micrometers, more preferably 5 to 80 micrometers, still more preferably 10 to 70 micrometers, particularly preferably 15 to 60 micrometers, and most preferably 20 to 50 micrometers. These do not mean that all the microbubbles or nanobubbles are included in this size, and 60% or more of the entire microbubbles or nanobubbles may be this size.
- composition containing nanobubbles of the present invention is produced by making hydrogen, oxygen, and nitrogen gas into nanobubbles and mixing them in water (for example, ultrapure water).
- water for example, ultrapure water
- the purity of gas is not specifically limited, When using for drinking water etc., it is preferable to use high purity gas.
- Nanobubbles can be generated, for example, by shearing and refining a gas introduced into a liquid, and various nanobubble generators have been developed.
- the nanobubble generator can be used without particular limitation as long as it is a general nanobubble generator.
- the nanobubble generator Bavitas registered trademark, manufactured by Ligaric Co., Ltd.
- the nanobubble generator Nanoaqua registered trademark, (Tec Corporation) is preferably used.
- the form of the composition containing the nanobubble of the present invention is not particularly limited, it is typically water, and can be used for drinking water, medium preparation water, fish ginger water, plant spray water, and the like.
- the configuration of the equipment for producing functional water includes the following. Hydrogen gas cylinder, oxygen gas cylinder, nitrogen gas cylinder or nitrogen gas generator, digital control regulator, ozone water generator. Nano bubble generator, water injection tank, various filter groups. First, sterilize and clean the nanobubble generator, water injection tank, piping, etc. using about 1 ppm of ozone water.
- the configuration of the filter to be passed is a hollow fiber membrane filter, activated carbon filter, and hollow fiber membrane filter arranged in this order as one unit.
- tap water is raw water, it is normal. Use 2 units. The number of units can be appropriately increased or decreased depending on the degree of impurities in the raw water.
- the filtered water that has passed through the filtration unit is passed through a membrane filter (reverse osmosis membrane filter depending on the purpose) of 0.25 ⁇ m to 0.45 ⁇ m, further filtered, and poured into a water injection tank.
- a membrane filter reverse osmosis membrane filter depending on the purpose
- Use various inspection devices that have reached the desired volume of gas in the water and pass them through the 0.25 ⁇ m to 0.45 ⁇ m membrane filter from the water injection tank.
- the present invention provides an activator that activates mitochondria.
- Mitochondrial activation can be measured as an increase in ATP production by measuring ATP production.
- mitochondria activation means that the amount of ATP produced in mitochondria is higher than that in the control group.
- the control group typically refers to normal water (ultra pure water), but is not limited to this, and means an experimental group using a composition having the same composition except that the nanobubbles of the present invention are not included.
- Use of the composition of the present invention has an effect that the amount of ATP produced in mitochondria is higher than that of the control group when oxidative stress is applied.
- composition of the present invention a high cell proliferation promoting effect can be obtained.
- a cell culture medium is prepared using the composition (aqueous solution) of the present invention and the cells are cultured, a higher growth promoting effect is obtained compared to a medium using normal water (ultra pure water).
- iPS cells induced pluripotent stem cells, induced pluripotent stem cells
- the growth promotion effect was approximately doubled when cultured in a medium using normal ultrapure water in 72 hours of culture.
- This cell growth promoter can also be used for proliferating cells such as other cultured cells such as primary cultured cells, undifferentiated cells, hematopoietic cells, fibroblasts, immortalized cells, stem cells and the like.
- the cultured cell is not particularly limited, but refers to a cell that can divide, such as a primary somatic cell, a cell line, an immortalized cell, or a stem cell.
- Stem cells include embryonic stem cells and somatic stem cells, and include, but are not limited to, neural stem cells, liver stem cells, skin stem cells, germ stem cells, iPS cells, and the like.
- a proliferative cell means a cell having proliferative ability, and includes cultured cells of primary somatic cells, undifferentiated cells, progenitor cells, and the like.
- Somatic cells that can be used as starting material for the production of cultured cells can be any cells other than germ cells derived from mammals (eg, mice or humans), such as keratinized epithelial cells (eg, Keratinized epidermal cells), mucosal epithelial cells (eg, epithelial cells of the tongue), exocrine glandular epithelial cells (eg, mammary cells), hormone-secreting cells (eg, adrenal medullary cells), cells for metabolism and storage (eg, Hepatocytes), luminal epithelial cells that make up the interface (eg, type I alveolar cells), luminal epithelial cells of the inner chain (eg, vascular endothelial cells), and cilia cells that have a transport ability (eg, Airway epithelial cells), extracellular matrix secreting cells (eg, fibroblasts), contractile cells (eg, smooth muscle cells), blood and immune system cells (eg, T lymphocytes), sensory cells (eg, ⁇ cells) Auto
- somatic stem cells there is no particular limitation on the degree of cell differentiation, and it can be used as the source of somatic cells, whether it is an undifferentiated progenitor cell (including somatic stem cells) or a terminally differentiated mature cell.
- undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
- an effect of promoting the growth of plant cells can be obtained.
- a growth promoting effect can be obtained by preparing a medium or a hydroponics solution using the composition of the present invention and using it in plants.
- hydroponics by adding air microbubbles (size 10 nm to 30 nm) together with the composition of the present invention, the above-ground dry weight, above-ground fresh weight, below-ground dry weight, and below-ground fresh weight are both 10 to 20%. Increased (see Example 14, Table 7)
- cells can be stored in a state where the activity of the cells is kept high.
- mitochondrial activity can be kept high, and a savory food can be provided.
- umami components such as inosinic acid (IMP), which is a degradation product thereof, also increase. That is, according to the composition of the present invention, a food with increased umami taste can be provided.
- IMP inosinic acid
- the composition of the present invention can be added to foods and drinks as they are, and can be used as a raw material for foods and drinks, or can be used for processing foods and drinks or food materials.
- the food and drink means, though not limited to, drinks and foods that people eat.
- health supplements low-calorie foods, diet foods, bread, dairy products (eg, non-fat milk, yogurt, pudding, soy milk, etc.), noodles (eg, udon, Chinese noodles, wonton, gyoza skin, soba noodles, etc.)
- Confectionery e.g. cookies, Japanese confectionery, steamed bread, potato chips, chocolate, custard cream, etc.
- beverages e.g. soup, vegetable drinks, diet drinks, tea, coffee etc.
- desserts e.g. cakes, mousses etc.
- Examples include frozen confectionery (for example, jelly, ice cream, etc.), sugar beet (for example, tsumire, date roll, fried chicken), cereal, filling, dressing, baked food, meat products (for
- the composition of the present invention can also be used by adding to feed.
- the feed means, but not limited to, animals other than humans, and preferably drinks and foods such as fish, livestock, and pets. Also includes fish food. More preferred are fish ginger, water in aquariums, ponds, and drinks and foods such as cows, pigs, sheep, chickens, horses, dogs and cats.
- the composition of the present invention can also be used as an injectable agent (including an instillation).
- the composition is also used as a prophylactic and / or therapeutic agent for immune cell proliferation promotion, diabetes, arteriosclerosis, inflammation, etc. be able to.
- composition of this invention can also be used for preparation of an injection solution.
- composition of the present invention can provide various diseases, such as immune cell proliferation-promoting action, diabetes / arteriosclerosis / inflammation, and other preventive and therapeutic effects even when a human or animal drinks it as drinking water. .
- Example 1 iPS cell culture medium was prepared as follows. To 1 L of distilled water (DW) or the composition of the present invention (APAS), 12.5 g of Glasgow minimum essential medium (Sigma; G6148) and 2.75 g of sodium bicarbonate (Sigma; S5761) were added and stirred.
- DW distilled water
- APAS composition of the present invention
- G6148 Glasgow minimum essential medium
- S5761 sodium bicarbonate
- Reagents were added to this solution to the following final concentrations: 0.1 mM MEM Non-Essential Amino Acids Solusion (Gibco; 11140), 1 mM Sodium pyruvate solution (Sigma; S8636), 1% Fetal bovine serum (CCB ; 171012), 1% Penicillin Streptomycin (Gibco; 15140), 5.4% Knockout Serum Replacement (Gibco; 10828), 0.1 mM 2-Mercaptoethanol (Wako; 137-06862), 2000 U / ml Leukemia inhibitor factor (Millipore; ESG1107).
- the mixture was stirred until homogeneous and dissolved, and then sterilized by filtration through a 0.45 ⁇ m filter to obtain a cell maintenance medium.
- the culture results are shown in FIG.
- the medium prepared using the composition of the present invention when a medium prepared using normal ultrapure water is used, the medium prepared using the composition of the present invention is more About twice the amount of growth was obtained compared to the control ultrapure water medium.
- Example 2 Examination of antioxidant action using mouse vascular endothelial cell line (MAEC) Water containing the composition of the present invention, nanobubble water (nanobubble water containing air), hydrogen nanobubble water (hydrogen dissolved gas, micro Nanobubble water containing 70% or more of bubbles and nanobubbles) was prepared, respectively, and M199 medium (SIGMA) was prepared, 10% fetal bovine serum, 100 IU / ml penicillin, and 100 ⁇ l / ml streptomycin were added, did. After culturing MAEC for 24 hours in a medium using each water, the medium was replaced with a fresh medium and further cultured for 2 hours.
- SIGMA mouse vascular endothelial cell line
- FIG. 2 shows the ROS detection results.
- Each graph in FIG. 2 shows the number of cells showing a certain fluorescence intensity, and the MFI value shows the average fluorescence intensity.
- NB nanobubble water
- MAEC mouse vascular endothelial cell line
- NAC N-acetylcysteine
- a medium for controlling the antioxidant action was prepared using ordinary water. MAEC cells were cultured in this medium for 24 hours, replaced with a fresh medium using normal water, and further cultured for 2 hours.
- NAC was added to the medium 30 minutes before the treatment with antimycin or hydrogen peroxide so that the final concentration was 5 mmol / L. Thereafter, changes in intracellular ATP amount were measured using luciferase activity as an index (ATPlite, PerkinElmer).
- the measurement result of the amount of ATP in the cell is shown in FIG.
- the amount of ATP was decreased in all of ethanol treatment, antimycin treatment, and hydrogen peroxide treatment as compared with the case of no stimulation. From this result, it can be seen that ethanol, antimycin, and hydrogen peroxide have an inhibitory effect on ATP production activity.
- the antimycin treatment showed a stronger decrease in ATP production activity than the hydrogen peroxide treatment.
- H2NB hydrogen nanobubble water
- O2, H2, N2NB water containing the composition of the present invention
- an increase in ATP production activity was observed when any treatment was performed. That is, it was confirmed that the water containing hydrogen nanobubble water and the composition of the present invention reduces the inhibitory effect on ATP production activity by antimycin or the like to NAC which is an antioxidant.
- the water containing the composition of the present invention was treated with ethanol or hydrogen peroxide, the effect of suppressing ATP production activity was significantly reduced as compared with hydrogen nanobubble water.
- Example 3 Examination of drinking effects in humans A drinking experiment in humans was carried out for the purpose of clarifying the effect of humans on drinking water containing the composition of the present invention.
- the humans who participated in the experiment were 4 males and 1 female aged 40 to 69 years (average age 51.8 years).
- Table 1 shows the age and gender of each participant. All participants were in good health.
- GPT glutanic transaminase
- HEL hexanoyl-lysine
- STAS Serum Total Antioxidant
- GPT GPT is an enzyme mainly present in the liver. When hepatocytes are destroyed, they leak into the blood specifically, and are used as indicators of liver damage caused by hepatitis virus or drugs. In addition, GOT and GPT values are often mildly abnormal in liver fat, and it is also known that fatty liver due to obesity has a slightly higher GPT than GOT.
- GPT uses a silica liquid ALT reagent (manufactured by Kanto Chemical Co., Inc.) with a concentration of serum, Bio Maasesty (JCA-BM8060) (manufactured by JEOL Ltd.), and Olympus AU5431 biochemical automatic analyzer (Olympus Co., Ltd.). ).
- Serum was collected by clotting the collected blood and centrifuging it.
- HEL HEL is a stable initial product derived from lipid peroxide (13-Hydroperoxy-octadecadienoic acid, 13-HPODE) in the process of lipid peroxidation by reactive oxygen species, and conventionally used 4-HNE, MDA, etc. Unlike the aldehyde lipid peroxidation markers, it is a marker that can capture the initial stage of lipid peroxidation.
- STAS STAS is a water-soluble antioxidant in serum and can measure the total antioxidant capacity against oxidative stress.
- Example 4 Two subjects (O, I) were ingested with water containing the composition of the present invention (APAS, O2 / H2 / N2NB water, also referred to as MCP) for 500 months per day for one month, and blood tests were conducted before and after that. It was. Result / Subject O When the values before ingestion of water containing the composition of the present invention were compared, the values were improved with ALP (alkaline phosphatase), ALT, and ⁇ -GTP after ingestion. In addition, the values of total cholesterol, LDL cholesterol and free fatty acids were also improved.
- ALP alkaline phosphatase
- ALT alkaline phosphatase
- ⁇ -GTP after ingestion.
- the values of total cholesterol, LDL cholesterol and free fatty acids were also improved.
- Example 5 In vitro experiment on the proliferation rate of ES cells When the proliferation of mouse ES cells in a medium prepared using DW or APAS (the same composition as the iPS cell medium of Example 1) was examined, the proliferation rate on the third day of culture was 1 In this case, when the medium was prepared using APAS, a growth rate of about 1.5 times was observed (FIG. 7).
- Example 6 Experiment on the type of water added to the in vivo experimental medium for promoting the proliferation of immune cells (T cells, B cells, NK cells) by MCP (1) Raw water (raw water for MCP preparation) (2) Raw water + gas body (no filter filtration) (3) Raw water + gas body + 0.22 ⁇ m filter
- Raw water + gas body + 0.22 ⁇ m filter Rat rearing method SD rat (12w) male was used as an experimental animal. The rats were given water both orally and intraperitoneally. Intraperitoneal administration was performed by intraperitoneal injection. For oral administration, MCP sterilized with a 0.22 ⁇ m filter was used. On the other hand, the provided DW was administered to the control group.
- PBS (RT) was adjusted to 30 ⁇ l / sample and dispensed into an Eppendorf tube at 30 ⁇ l / tube, and the remainder was used for control.
- results The results of FACS analysis are shown in FIG. Further, as shown in FIG. 9, when the MCP administration group was compared with the DW administration group as 1, an increase in all of T cells, B cells, and NK cells was observed. Although there was not much difference in the rate of increase in the number of cells, the rate of increase in the B cell MCP administration group was particularly large. From this example, it was shown that the MCP of the present invention can also be used as an injection.
- Example 7 In this experiment, (1) raw water: MCP preparation raw water, (2) MCP, (3) MCP + 0.22 micron filter: MCP passed through 0.22 micron filter, oral administration and intraperitoneal injection (1 ml X2 / day, morning, night), and the amount of each fraction in rat blood lymphocytes after 7 days of breeding was compared. Observations 7 days after the start of the study showed no signs of bloating or diarrhea due to overdose injections.
- the ratio of each cell number is shown in FIG. 11 and FIG.
- the ratio of the number of T cells, B cells, and NK cells according to FIG. 12 was similar to the ratio known in normal rats.
- the increase rate of B cells was larger than the increase rate of the other two types of cells.
- FIG. 12 shows the ratios of (2) and (3) with respect to (1) of the number of NK cells among the respective cell fractions in the lymphocyte.
- the increase rate of NK cells in (2) was 348% on average for the three individuals measured.
- Experiment B (1) Raw water (raw water for MCP preparation) (2) Raw water + gas body + 0.45 ⁇ m filter (3) Raw water + gas body + 0.22 ⁇ m filter
- the total number of lymphocytes in the raw water + gas body section, which is not filtered is 3.21 times the raw water.
- the amount of proliferation has increased about 3 times or more.
- the growth amount was 1.76 times in the 0.45 ⁇ m filter and 1.66 times in the medium filtered with the 0.22 ⁇ m filter. Filter filtration may result in this because microbubbles are removed.
- the diameter of the gas body is 10 nm to 30 nm.
- Example 9 The effect of gas body administration on T cells, B cells, and NK cells was measured. In the unfiltered system, B cells proliferated best, followed by NK cells and T cells (FIG. 9).
- Example 10 The number of NK cells in raw water + gas administered individuals was compared. The result is shown in FIG. As shown in FIG. 12, when there was no filter, the raw water grew about 3.5 times. In the case with a filter, the 0.45 ⁇ m filter showed 1.93 times the growth, and the 0.22 ⁇ m filter showed 1.64 times the growth.
- Example 11 Effect on obstructive arteriosclerosis of both lower limbs in an 81-year-old woman. He had been hospitalized for osteoarthritis of the knee with osteonecrosis of both knees since the spring of 2011, but seems to cause edema mainly in the right lower limb since the fall of 2013. Became. In February 2014, thrombolytic therapy was performed at the hospital for right limb thromboangiitis. In March 2014, we began drinking mitochondrial water at 500ml / day. As a result, edema was reduced and inflammation was improved.
- Example 12 Subject: 81-year-old female, type II diabetes February 2008, onset of type II diabetes. She was examining the course of treatment with a focus on internal medicine and lifestyle guidance. For oral use, 1.25 mg (3 tablets after 3 tablets) of sulfonyl preparation and 0.2 mg (3 tablets before 3 tablets) postprandial hyperglycemia improving agent are taken. Complications included hyperlipidemia and mild renal function decline. For hyperlipidemia, 10 mg of atorvastatin calcium preparation was taken before sleep.
- Example 13 In order to clarify the effects of MCP on humans when taken by humans, a human MCP drinking test was conducted. The subjects are as follows. 1. 50-year-old male, 2. 45-year-old male, 3. 60-year-old female, 4. 35-year-old female, 5. 63-year-old female, 6. 66-year-old male, 7 25-year-old female Each participant is the same as usual While living a life, MCP was drunk 0,5-1,0 L / day for 4 weeks. Participants were blood collected before the start of drinking and blood was collected again at 4 and 8 weeks after starting drinking. Tests were performed on leukocytes, liver function, lipid-related and diabetes-related items.
- Example 14 The effect on plants was examined using Komatsuna (Brassica rapa var. Perviridis). Cultivation period March 7, 2014 to April 7, 2014 (32 days) Method: 30 L of dechlorinated water was put into a container, and hydroponics fertilizer tank mix F & B (Table 6, manufactured by OAT Agrio Co., Ltd.) was dissolved. The culture solution was adjusted to EC 2.4 during the cultivation period and aerated for 24 hours.
- Treatment group Control group air microbubble (MB) group
- APAS + MB group In the air MB group, the air MB is converted into MB for 20 minutes, and the APAS + MB group is also converted into MB for 20 minutes. Occurred (total of 6 treatments) Survey item Plant height, maximum leaf length, maximum root length, aboveground fresh weight, belowground fresh weight, aboveground dry weight, belowground dry weight
- Example 15 Preparation of experimental medium for promoting fibroblast proliferation in medium supplemented with MCP Control DW 285g DMEM powder 3g NaHCO3 1.11g 300ml total After mixing the above, it was sterilized by filtration with a 0.45 ⁇ m filter. 10% FBS (fetal bovine serum) was prepared in 10 ml or 50 ml, and 1/10 volume was added to the medium. 2.1 x MCP MCP 47.5ml DMEM powder 0.5g NaHCO 3 0.185 g Whole 50ml After mixing the above, it was filtered with a filter. 9 ml of the above solution (DMEM (MCP) and 1 ml of FBS were mixed and used for culture.
- MCP fetal bovine serum
- the mouse fibroblast (MEF) cells that had been cryopreserved were generated and then passaged three or more times before use in this experiment.
- the cells were cultured in DMEM (High Glucose) prepared with a final concentration of 10% FBS.
- the medium was exchanged once every 3 days, and subculture was performed at the time of confluence.
- the MEF cells were subcultured into 12-well plates so that there were 10,000 cells per well. The day after the passage, the MEF cells were washed once with PBS, and DMEM (High Glucose) prepared with 10% FBS and Penicillin / Streptmycin was added using DW and MCP. The analysis was performed on the third day, with the day on which MCP was added as the zeroth day. The medium was changed daily after adding MCP.
- DMEM High Glucose
- Fibroblasts have the function of producing collagen, elastin, hyaluronic acid, etc. in the epithelium.
- collagen has a role of forming a dermal structure by forming a fiber bundle.
- MCP having a fibroblast proliferation function is expected to show an effective inhibitory effect on such skin aging.
- Example 16 A 63-year-old woman who had inflammation in the knee had been inflamed for about 2 years, but drinking 500 ml to 1 L of MCP every day drastically improved inflammation in the first month. Inflammation almost disappeared (FIG. 19).
- the amount of MCP consumed is preferably 150 ml or more, more preferably 300 ml or more, still more preferably 500 ml or more, and still more preferably 1 L or more. All the water taken in a day may be MCP.
- Example 17 When pets were allowed to drink MCP as drinking water every day for 30 days, as shown in Table 9, improvements in disease index values were observed.
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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 μm 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
本発明は、ナノバブル含有組成物およびその用途に関する。より詳しくは、ナノバブルを含有し、ミトコンドリアを活性化する組成物とその用途に関する。
The present invention relates to a nanobubble-containing composition and its use. More specifically, the present invention relates to a composition containing nanobubbles and activating mitochondria and its use.
マイクロバブル、ナノバブルの技術を用いて気体を液体中に溶存させ、培養細胞の増殖を促進する方法としては、例えば、特許文献１のような方法が開示されている。しかしながら、特許文献１には、どのような大きさ、組成のマイクロバブル、ナノバブルを用いれば、どのような種類の細胞の増殖がどの程度促進されるか、などが具体的に詳細に開示されておらず、より実用的な組成物、方法、装置の開発が求められていた。また、さらに効率のよい増殖促進方法も求められていた。
For example, Patent Document 1 discloses a method for promoting the growth of cultured cells by dissolving a gas in a liquid using microbubble or nanobubble technology. However, Patent Document 1 specifically discloses in detail what size and composition of microbubbles and nanobubbles can be used to promote the proliferation of what types of cells. There has been a demand for the development of more practical compositions, methods, and apparatuses. There has also been a demand for a more efficient method for promoting proliferation.
飲料水にマイクロバブル、ナノバブルを用いて気体を混入した製品としては、水素水、水素酸素水等が知られている（例えば、特許文献２）。しかしながら、これらの飲料水では、十分満足できる効果を感じる人は一部に限られ、あまり普及していないのが現状である。したがって、十分な効果を実感できるマイクロバブル、ナノバブル飲料水が求められていた。また、新機能を有するマイクロバブル、ナノバブル飲料水も求められていた。
Hydrogen water, hydrogen oxygen water, etc. are known as products in which gas is mixed with drinking water using microbubbles or nanobubbles (for example, Patent Document 2). However, in these drinking waters, the number of people who feel a satisfactory effect is limited to a part, and the current situation is that they are not so popular. Therefore, microbubbles and nanobubble drinking water that can realize a sufficient effect have been demanded. Also, microbubbles and nanobubble drinking water having new functions have been demanded.
一方、ミトコンドリアはエネルギーを生み出す器官であり、ミトコンドリアの活性を上げることで、エネルギー生産を増やし、より活力のある体にすることができると考えられる。しかしながら、ミトコンドリアを活性化する飲料水についてはまだ開発されておらず、ミトコンドリアを活性化する飲料水の開発が求められていた。
On the other hand, mitochondria are energy-producing organs, and it is thought that increasing mitochondrial activity can increase energy production and make the body more active. However, drinking water that activates mitochondria has not been developed yet, and development of drinking water that activates mitochondria has been demanded.
また、細胞を凍結保存する際に生存率が低下したり、細胞が破壊されて内容物が漏出し、食感が悪化したり、風味が悪くなるという問題があった。そこで、生存率を高くし、風味を良くする細胞保存液の開発が望まれていた。
In addition, when the cells were cryopreserved, there was a problem that the survival rate was reduced, the cells were destroyed and the contents leaked, the texture was deteriorated, and the flavor was deteriorated. Therefore, it has been desired to develop a cell preservation solution that increases the survival rate and improves the flavor.
本発明は、水素、酸素、窒素を含有するナノバブルを含有する組成物とその用途を提供する。
The present invention provides a composition containing nanobubbles containing hydrogen, oxygen, and nitrogen and uses thereof.
本明細書によれば、以下の発明が提供される。
（１）水素0.45～0.55ppm、酸素10～12.5ppm、窒素7～8ppm含有する、粒径３０マイクロメートル以下のマイクロバブルおよび／またはナノバブルを有効成分として含有する組成物。ここで、各気体の濃度は溶存気体とナノバブルの両方を合わせた濃度である。
（２）（１）の組成物を有効成分として含有するミトコンドリア活性化剤組成物。ここで、ミトコンドリア活性化組成物はミトコンドリア活性化剤と同じ意味である。 According to this specification, the following invention is provided.
(1) A composition containing microbubbles and / or nanobubbles having a particle size of 30 micrometers or less, containing 0.45 to 0.55 ppm hydrogen, 10 to 12.5 ppm oxygen, and 7 to 8 ppm nitrogen. Here, the concentration of each gas is the concentration of both dissolved gas and nanobubbles.
(2) A mitochondrial activator composition containing the composition of (1) as an active ingredient. Here, the mitochondrial activation composition has the same meaning as the mitochondrial activator.
（１）水素0.45～0.55ppm、酸素10～12.5ppm、窒素7～8ppm含有する、粒径３０マイクロメートル以下のマイクロバブルおよび／またはナノバブルを有効成分として含有する組成物。ここで、各気体の濃度は溶存気体とナノバブルの両方を合わせた濃度である。
（２）（１）の組成物を有効成分として含有するミトコンドリア活性化剤組成物。ここで、ミトコンドリア活性化組成物はミトコンドリア活性化剤と同じ意味である。 According to this specification, the following invention is provided.
(1) A composition containing microbubbles and / or nanobubbles having a particle size of 30 micrometers or less, containing 0.45 to 0.55 ppm hydrogen, 10 to 12.5 ppm oxygen, and 7 to 8 ppm nitrogen. Here, the concentration of each gas is the concentration of both dissolved gas and nanobubbles.
(2) A mitochondrial activator composition containing the composition of (1) as an active ingredient. Here, the mitochondrial activation composition has the same meaning as the mitochondrial activator.
（３）（１）または（２）の組成物を有効成分として含有する細胞増殖促進剤。この場合、細胞増殖促進剤は（１）および（２）の両方の組成物を含有していてもよい。
（４）（１）～（３）のいずれか１の組成物又は剤を有効成分として含有する細胞保存液。ここで、剤は、（３）の細胞増殖促進剤を意味する。ただし、（２）のミトコンドリア活性化組成物がミトコンドリア活性化剤と補正された場合はミトコンドリア活性化剤も剤に含まれる。また、「いずれか１の」とは、「少なくともいずれか１の」という意味であり、「いずれか１のみの」という意味ではない。したがって、（１）～（３）のうち２以上又は３以上の組成物または剤を含むものも技術的範囲に含まれる。以下も同様であり、「いずれか１の」は「（少なくとも）いずれか１の」の意味である。
（５）（１）～（４）のいずれか１の組成物、剤又は液を有効成分として含有する凍結保存液。ここで、剤は（４）と同じ意味であり、液は、（４）の細胞保存液を意味する。 (3) A cell growth promoter containing the composition of (1) or (2) as an active ingredient. In this case, the cell growth promoter may contain both the compositions (1) and (2).
(4) A cell preservation solution containing the composition or agent of any one of (1) to (3) as an active ingredient. Here, the agent means the cell growth promoter of (3). However, when the mitochondrial activation composition of (2) is corrected as a mitochondrial activator, the mitochondrial activator is also included in the agent. Further, “any one” means “at least any one” and does not mean “only any one”. Accordingly, the technical scope includes two or more or three or more compositions or agents among (1) to (3). The same applies to the following, and “any one” means “(at least) any one”.
(5) A cryopreservation solution containing the composition, agent or solution of any one of (1) to (4) as an active ingredient. Here, the agent has the same meaning as in (4), and the solution means the cell preservation solution in (4).
（４）（１）～（３）のいずれか１の組成物又は剤を有効成分として含有する細胞保存液。ここで、剤は、（３）の細胞増殖促進剤を意味する。ただし、（２）のミトコンドリア活性化組成物がミトコンドリア活性化剤と補正された場合はミトコンドリア活性化剤も剤に含まれる。また、「いずれか１の」とは、「少なくともいずれか１の」という意味であり、「いずれか１のみの」という意味ではない。したがって、（１）～（３）のうち２以上又は３以上の組成物または剤を含むものも技術的範囲に含まれる。以下も同様であり、「いずれか１の」は「（少なくとも）いずれか１の」の意味である。
（５）（１）～（４）のいずれか１の組成物、剤又は液を有効成分として含有する凍結保存液。ここで、剤は（４）と同じ意味であり、液は、（４）の細胞保存液を意味する。 (3) A cell growth promoter containing the composition of (1) or (2) as an active ingredient. In this case, the cell growth promoter may contain both the compositions (1) and (2).
(4) A cell preservation solution containing the composition or agent of any one of (1) to (3) as an active ingredient. Here, the agent means the cell growth promoter of (3). However, when the mitochondrial activation composition of (2) is corrected as a mitochondrial activator, the mitochondrial activator is also included in the agent. Further, “any one” means “at least any one” and does not mean “only any one”. Accordingly, the technical scope includes two or more or three or more compositions or agents among (1) to (3). The same applies to the following, and “any one” means “(at least) any one”.
(5) A cryopreservation solution containing the composition, agent or solution of any one of (1) to (4) as an active ingredient. Here, the agent has the same meaning as in (4), and the solution means the cell preservation solution in (4).
（６）（１）～（５）のいずれか１の組成物、剤又は液を含有する飲食物。ここで、液は（４）の細胞保存液に加えて（５）の凍結保存液も含む。
（７）（１）～（５）のいずれか１の組成物、剤又は液で処理した原材料を使用して製造された飲食物。
（８）（１）～（５）のいずれか１の組成物、剤又は液を用いてミトコンドリアを活性化する方法。
（９）（１）～（５）のいずれか１の組成物、剤又は液を用いて細胞増殖を促進する方法。 (6) A food or drink containing the composition, agent or liquid of any one of (1) to (5). Here, the solution includes the cryopreservation solution (5) in addition to the cell preservation solution (4).
(7) A food or drink produced using a raw material treated with the composition, agent or liquid of any one of (1) to (5).
(8) A method of activating mitochondria using the composition, agent or solution of any one of (1) to (5).
(9) A method of promoting cell proliferation using the composition, agent or solution of any one of (1) to (5).
（７）（１）～（５）のいずれか１の組成物、剤又は液で処理した原材料を使用して製造された飲食物。
（８）（１）～（５）のいずれか１の組成物、剤又は液を用いてミトコンドリアを活性化する方法。
（９）（１）～（５）のいずれか１の組成物、剤又は液を用いて細胞増殖を促進する方法。 (6) A food or drink containing the composition, agent or liquid of any one of (1) to (5). Here, the solution includes the cryopreservation solution (5) in addition to the cell preservation solution (4).
(7) A food or drink produced using a raw material treated with the composition, agent or liquid of any one of (1) to (5).
(8) A method of activating mitochondria using the composition, agent or solution of any one of (1) to (5).
(9) A method of promoting cell proliferation using the composition, agent or solution of any one of (1) to (5).
（１０）（１）～（５）のいずれか１の組成物、剤又は液を用いて細胞を保存する方法。
（１１）（１）～（５）のいずれか１の組成物、剤又は液を用いて凍結保存する方法。
（１２）（１）～（５）のいずれか１の組成物、剤又は液を用いて飲食物またはその原材料を処理する工程を含む、飲食物の製造方法。
（１３）（１）の組成物を用いて調製した細胞培養用培地。
（１４）（１）の組成物を用いて調製した植物水耕栽培用溶液。
（１５）（１３）の培地を用いて動物細胞を増殖させる方法。
（１６）動物細胞が免疫系細胞である、（１５）の動物細胞を増殖させる方法。 (10) A method for preserving cells using the composition, agent or solution of any one of (1) to (5).
(11) A method for cryopreserving the composition, agent or solution of any one of (1) to (5).
(12) A method for producing a food or drink, comprising a step of treating the food or drink or its raw material using the composition, agent or liquid of any one of (1) to (5).
(13) A cell culture medium prepared using the composition of (1).
(14) A solution for plant hydroponics prepared using the composition of (1).
(15) A method for growing animal cells using the medium of (13).
(16) The method for growing animal cells according to (15), wherein the animal cells are immune system cells.
（１１）（１）～（５）のいずれか１の組成物、剤又は液を用いて凍結保存する方法。
（１２）（１）～（５）のいずれか１の組成物、剤又は液を用いて飲食物またはその原材料を処理する工程を含む、飲食物の製造方法。
（１３）（１）の組成物を用いて調製した細胞培養用培地。
（１４）（１）の組成物を用いて調製した植物水耕栽培用溶液。
（１５）（１３）の培地を用いて動物細胞を増殖させる方法。
（１６）動物細胞が免疫系細胞である、（１５）の動物細胞を増殖させる方法。 (10) A method for preserving cells using the composition, agent or solution of any one of (1) to (5).
(11) A method for cryopreserving the composition, agent or solution of any one of (1) to (5).
(12) A method for producing a food or drink, comprising a step of treating the food or drink or its raw material using the composition, agent or liquid of any one of (1) to (5).
(13) A cell culture medium prepared using the composition of (1).
(14) A solution for plant hydroponics prepared using the composition of (1).
(15) A method for growing animal cells using the medium of (13).
(16) The method for growing animal cells according to (15), wherein the animal cells are immune system cells.
（１７）（１）もしくは（２）に記載の組成物、または（３）に記載の細胞増殖促進剤を有効成分とする動脈硬化症の予防・治療剤。
（１８）（１）もしくは（２）に記載の組成物、または（３）に記載の細胞増殖促進剤を有効成分とする糖尿病の予防・治療剤。
（１９）（１）または（２）に記載の組成物を含む注射液。 (17) A prophylactic / therapeutic agent for arteriosclerosis comprising the composition according to (1) or (2) or the cell growth promoter according to (3) as an active ingredient.
(18) A prophylactic / therapeutic agent for diabetes comprising the composition according to (1) or (2) or the cell growth promoter according to (3) as an active ingredient.
(19) An injection solution comprising the composition according to (1) or (2).
（１８）（１）もしくは（２）に記載の組成物、または（３）に記載の細胞増殖促進剤を有効成分とする糖尿病の予防・治療剤。
（１９）（１）または（２）に記載の組成物を含む注射液。 (17) A prophylactic / therapeutic agent for arteriosclerosis comprising the composition according to (1) or (2) or the cell growth promoter according to (3) as an active ingredient.
(18) A prophylactic / therapeutic agent for diabetes comprising the composition according to (1) or (2) or the cell growth promoter according to (3) as an active ingredient.
(19) An injection solution comprising the composition according to (1) or (2).
本発明の組成物によれば、ミトコンドリアを活性化し、細胞増殖を促進し、細胞の保存、凍結保存の際の細胞へのダメージが少なくなるという効果が得られる。
According to the composition of the present invention, the effects of activating mitochondria, promoting cell proliferation, and reducing damage to cells during cell storage and cryopreservation can be obtained.
本発明は、水素0.45～0.55ppm、酸素10～12.5ppm、窒素7～8ppm含有する、粒径３０マイクロメートル以下のマイクロバブルおよび／またはナノバブルを有効成分として含有する組成物を提供する。この組成物は、ミトコンドリア活性化、細胞増殖促進、細胞保存、細胞凍結保存、飲食物、注射液の調製等に利用することができる。
The present invention provides a composition containing, as an active ingredient, microbubbles and / or nanobubbles having a particle size of 30 micrometers or less, containing 0.45 to 0.55 ppm of hydrogen, 10 to 12.5 ppm of oxygen, and 7 to 8 ppm of nitrogen. This composition can be used for mitochondrial activation, cell growth promotion, cell preservation, cell cryopreservation, food and drink, preparation of injection solution, and the like.
本発明の組成物に含まれる気体の好ましい濃度は、水素0.1～3.0ppm、酸素5～20ppm、窒素3～20ppm、より好ましくは、水素0.3～1.0ppm、酸素7～15ppm、窒素4～15ppm、さらに好ましくは、水素0.4～0.6ppm、酸素9～13ppm、窒素5～10ppm、特に好ましくは、水素0.45～0.55ppm、酸素10～12.5ppm、窒素7～8ppmである。なお、これらの濃度は、それぞれの濃度でそれなりの効果を有するため、この範囲内であれば、どこで区切っても本発明の効果を有するものである。なお、ppmとはパート・パー・ミリオンを意味し、溶存気体の濃度を表す。
本発明の組成物に含まれる気体は、粒径が０より大きく、３０マイクロメートル以下が好ましく、より好ましくは２０マイクロメートル以下、さらに好ましくは１０マイクロメートル以下、よりさらに好ましくは１マイクロメートル以下、特に好ましくは１００マイクロメートル以下、最も好ましくは３０ナノメートル以下である。
植物の水耕栽培などにおいては、ナノバブルに加えて、マイクロバブルの気体を用いることで増殖効率を上げることができる。その場合、マイクロバブルの大きさとしては、
１～１００マイクロメートルが好ましく、より好ましくは５～８０マイクロメートル、さらに好ましくは１０～７０マイクロメートル、特に好ましくは１５～６０マイクロメートル、最も好ましくは２０～５０マイクロメートルである。これらは全てのマイクロバブルまたはナノバブルがこの大きさに含まれることを意味するわけではなく、マイクロバブルまたはナノバブル全体の６０％以上がこの大きさであればよい。 The preferred concentration of the gas contained in the composition of the present invention is 0.1 to 3.0 ppm for hydrogen, 5 to 20 ppm for oxygen, 3 to 20 ppm for nitrogen, more preferably 0.3 to 1.0 ppm for hydrogen, 7 to 15 ppm for oxygen, 4 to 15 ppm for nitrogen, More preferably, hydrogen is 0.4 to 0.6 ppm, oxygen is 9 to 13 ppm, nitrogen is 5 to 10 ppm, particularly preferably hydrogen is 0.45 to 0.55 ppm, oxygen is 10 to 12.5 ppm, and nitrogen is 7 to 8 ppm. In addition, since these density | concentrations have a moderate effect in each density | concentration, if it is in this range, it will have the effect of this invention regardless of where it is divided. In addition, ppm means a part per million and represents the density | concentration of dissolved gas.
The gas contained in the composition of the present invention has a particle size of greater than 0 and preferably 30 micrometers or less, more preferably 20 micrometers or less, more preferably 10 micrometers or less, even more preferably 1 micrometers or less, Especially preferably, it is 100 micrometers or less, Most preferably, it is 30 nanometers or less.
In hydroponic cultivation of plants, the growth efficiency can be increased by using microbubble gas in addition to nanobubbles. In that case, as the size of the microbubble,
It is preferably 1 to 100 micrometers, more preferably 5 to 80 micrometers, still more preferably 10 to 70 micrometers, particularly preferably 15 to 60 micrometers, and most preferably 20 to 50 micrometers. These do not mean that all the microbubbles or nanobubbles are included in this size, and 60% or more of the entire microbubbles or nanobubbles may be this size.
本発明の組成物に含まれる気体は、粒径が０より大きく、３０マイクロメートル以下が好ましく、より好ましくは２０マイクロメートル以下、さらに好ましくは１０マイクロメートル以下、よりさらに好ましくは１マイクロメートル以下、特に好ましくは１００マイクロメートル以下、最も好ましくは３０ナノメートル以下である。
植物の水耕栽培などにおいては、ナノバブルに加えて、マイクロバブルの気体を用いることで増殖効率を上げることができる。その場合、マイクロバブルの大きさとしては、
１～１００マイクロメートルが好ましく、より好ましくは５～８０マイクロメートル、さらに好ましくは１０～７０マイクロメートル、特に好ましくは１５～６０マイクロメートル、最も好ましくは２０～５０マイクロメートルである。これらは全てのマイクロバブルまたはナノバブルがこの大きさに含まれることを意味するわけではなく、マイクロバブルまたはナノバブル全体の６０％以上がこの大きさであればよい。 The preferred concentration of the gas contained in the composition of the present invention is 0.1 to 3.0 ppm for hydrogen, 5 to 20 ppm for oxygen, 3 to 20 ppm for nitrogen, more preferably 0.3 to 1.0 ppm for hydrogen, 7 to 15 ppm for oxygen, 4 to 15 ppm for nitrogen, More preferably, hydrogen is 0.4 to 0.6 ppm, oxygen is 9 to 13 ppm, nitrogen is 5 to 10 ppm, particularly preferably hydrogen is 0.45 to 0.55 ppm, oxygen is 10 to 12.5 ppm, and nitrogen is 7 to 8 ppm. In addition, since these density | concentrations have a moderate effect in each density | concentration, if it is in this range, it will have the effect of this invention regardless of where it is divided. In addition, ppm means a part per million and represents the density | concentration of dissolved gas.
The gas contained in the composition of the present invention has a particle size of greater than 0 and preferably 30 micrometers or less, more preferably 20 micrometers or less, more preferably 10 micrometers or less, even more preferably 1 micrometers or less, Especially preferably, it is 100 micrometers or less, Most preferably, it is 30 nanometers or less.
In hydroponic cultivation of plants, the growth efficiency can be increased by using microbubble gas in addition to nanobubbles. In that case, as the size of the microbubble,
It is preferably 1 to 100 micrometers, more preferably 5 to 80 micrometers, still more preferably 10 to 70 micrometers, particularly preferably 15 to 60 micrometers, and most preferably 20 to 50 micrometers. These do not mean that all the microbubbles or nanobubbles are included in this size, and 60% or more of the entire microbubbles or nanobubbles may be this size.
本発明のナノバブルを含有する組成物は、水素、酸素、窒素ガスをナノバブル化して水（例えば、超純水）に混入させることにより製造される。ガスの純度は特に限定されないが、飲料水等に用いる場合は、高純度ガスを用いることが好ましい。
The composition containing nanobubbles of the present invention is produced by making hydrogen, oxygen, and nitrogen gas into nanobubbles and mixing them in water (for example, ultrapure water). Although the purity of gas is not specifically limited, When using for drinking water etc., it is preferable to use high purity gas.
ナノバブルは、例えば、液体中に導入した気体を剪断破壊して微細化すること等により発生させることができ、種々のナノバブル発生装置が開発されている。本発明においてナノバブル発生装置は、一般的なナノバブル発生装置であれば特に制限なく使用できるが、例えば、ナノバブル発生装置バヴィタス（登録商標、株式会社Ｌｉｇａｒｉｃ社製）や、ナノバブル発生装置ナノアクア（登録商標、株式会社テックコーポレーション製）等が好適に用いられる。
本発明のナノバブルを含有する組成物の形態は特に限定されないが、典型的には水であり、飲料水、培地調製用の水、魚類の生簀の水、植物の散布用水等に使用できる。 Nanobubbles can be generated, for example, by shearing and refining a gas introduced into a liquid, and various nanobubble generators have been developed. In the present invention, the nanobubble generator can be used without particular limitation as long as it is a general nanobubble generator. For example, the nanobubble generator Bavitas (registered trademark, manufactured by Ligaric Co., Ltd.), the nanobubble generator Nanoaqua (registered trademark, (Tec Corporation) is preferably used.
Although the form of the composition containing the nanobubble of the present invention is not particularly limited, it is typically water, and can be used for drinking water, medium preparation water, fish ginger water, plant spray water, and the like.
本発明のナノバブルを含有する組成物の形態は特に限定されないが、典型的には水であり、飲料水、培地調製用の水、魚類の生簀の水、植物の散布用水等に使用できる。 Nanobubbles can be generated, for example, by shearing and refining a gas introduced into a liquid, and various nanobubble generators have been developed. In the present invention, the nanobubble generator can be used without particular limitation as long as it is a general nanobubble generator. For example, the nanobubble generator Bavitas (registered trademark, manufactured by Ligaric Co., Ltd.), the nanobubble generator Nanoaqua (registered trademark, (Tec Corporation) is preferably used.
Although the form of the composition containing the nanobubble of the present invention is not particularly limited, it is typically water, and can be used for drinking water, medium preparation water, fish ginger water, plant spray water, and the like.
機能水（ナノバブル水）を製造するための器機の構成は以下のものを含む。水素ガスボンベ、酸素ガスボンベ、窒素ガスボンベ又は窒素ガス発生装置、デジタル制御型レギュレーター、オゾン水発生装置。ナノバブル発生装置、注水タンク、各種フィルター群。
まず、１ppm程度のオゾン水を使用して、ナノバブル発生装置、注水タンク、配管等を殺菌洗浄する。機能水の原水として淡水を使用する場合、通過させるフィルターの構成は中空糸膜フィルター、活性炭フィルター、中空糸膜フィルターをこの順に配列したものを１ユニットとして使用し、水道水が原水の場合は通常２ユニットを使用する。ユニットの数は、原水の不純物の程度により適宜増減させることができる。この濾過ユニットを通過させた濾過水を、0.25μm～0.45μmのメンブレンフィルター（目的によっては逆浸透膜フィルター）を通過させ、さらに濾過し注水タンクに注水する。
デジタル制御型レギュレーターにより調整した濃度のガス体を各ボンベ等からナノバブル発生装置に送り込み、注水タンク内の濾過水に各ガス体とナノバブルを、ナノバブル発生装置と注水タンクとの間で循環させながら、各ガス体が目的の容存量の数値に成るまで容存させる。各種検査器機で、水中のガス体が目的の容存量に達した状態のものを、注水タンクから0.25μm～0.45μmのメンブレンフィルターを通過させて使用する。 The configuration of the equipment for producing functional water (nano bubble water) includes the following. Hydrogen gas cylinder, oxygen gas cylinder, nitrogen gas cylinder or nitrogen gas generator, digital control regulator, ozone water generator. Nano bubble generator, water injection tank, various filter groups.
First, sterilize and clean the nanobubble generator, water injection tank, piping, etc. using about 1 ppm of ozone water. When fresh water is used as raw water for functional water, the configuration of the filter to be passed is a hollow fiber membrane filter, activated carbon filter, and hollow fiber membrane filter arranged in this order as one unit. When tap water is raw water, it is normal. Use 2 units. The number of units can be appropriately increased or decreased depending on the degree of impurities in the raw water. The filtered water that has passed through the filtration unit is passed through a membrane filter (reverse osmosis membrane filter depending on the purpose) of 0.25 μm to 0.45 μm, further filtered, and poured into a water injection tank.
While sending the gas body with the concentration adjusted by the digital control type regulator from each cylinder etc. to the nano bubble generator, circulating each gas body and nano bubble in the filtered water in the water injection tank between the nano bubble generator and water injection tank, Each gas body is allowed to remain until the desired amount is reached. Use various inspection devices that have reached the desired volume of gas in the water and pass them through the 0.25 μm to 0.45 μm membrane filter from the water injection tank.
まず、１ppm程度のオゾン水を使用して、ナノバブル発生装置、注水タンク、配管等を殺菌洗浄する。機能水の原水として淡水を使用する場合、通過させるフィルターの構成は中空糸膜フィルター、活性炭フィルター、中空糸膜フィルターをこの順に配列したものを１ユニットとして使用し、水道水が原水の場合は通常２ユニットを使用する。ユニットの数は、原水の不純物の程度により適宜増減させることができる。この濾過ユニットを通過させた濾過水を、0.25μm～0.45μmのメンブレンフィルター（目的によっては逆浸透膜フィルター）を通過させ、さらに濾過し注水タンクに注水する。
デジタル制御型レギュレーターにより調整した濃度のガス体を各ボンベ等からナノバブル発生装置に送り込み、注水タンク内の濾過水に各ガス体とナノバブルを、ナノバブル発生装置と注水タンクとの間で循環させながら、各ガス体が目的の容存量の数値に成るまで容存させる。各種検査器機で、水中のガス体が目的の容存量に達した状態のものを、注水タンクから0.25μm～0.45μmのメンブレンフィルターを通過させて使用する。 The configuration of the equipment for producing functional water (nano bubble water) includes the following. Hydrogen gas cylinder, oxygen gas cylinder, nitrogen gas cylinder or nitrogen gas generator, digital control regulator, ozone water generator. Nano bubble generator, water injection tank, various filter groups.
First, sterilize and clean the nanobubble generator, water injection tank, piping, etc. using about 1 ppm of ozone water. When fresh water is used as raw water for functional water, the configuration of the filter to be passed is a hollow fiber membrane filter, activated carbon filter, and hollow fiber membrane filter arranged in this order as one unit. When tap water is raw water, it is normal. Use 2 units. The number of units can be appropriately increased or decreased depending on the degree of impurities in the raw water. The filtered water that has passed through the filtration unit is passed through a membrane filter (reverse osmosis membrane filter depending on the purpose) of 0.25 μm to 0.45 μm, further filtered, and poured into a water injection tank.
While sending the gas body with the concentration adjusted by the digital control type regulator from each cylinder etc. to the nano bubble generator, circulating each gas body and nano bubble in the filtered water in the water injection tank between the nano bubble generator and water injection tank, Each gas body is allowed to remain until the desired amount is reached. Use various inspection devices that have reached the desired volume of gas in the water and pass them through the 0.25 μm to 0.45 μm membrane filter from the water injection tank.
本発明は、ミトコンドリアを活性化する活性化剤を提供する。ミトコンドリアの活性化はＡＴＰ産生量を測定することにより、ＡＴＰ産生量の増加として測定できる。
本明細書において、「ミトコンドリア活性化」とは、ミトコンドリアにおけるＡＴＰ産生量が対照区よりも高くなることを言う。対照区とは、典型的には通常の水（超純水）を言うが、これに限られず本発明のナノバブルを含まない以外は同じ組成の組成物を用いる実験区を意味する。本発明の組成物を使用することにより、酸化ストレス負荷時において、ミトコンドリアにおけるＡＴＰ産生量が対照区よりも高くなる効果を有する。 The present invention provides an activator that activates mitochondria. Mitochondrial activation can be measured as an increase in ATP production by measuring ATP production.
In this specification, “mitochondrial activation” means that the amount of ATP produced in mitochondria is higher than that in the control group. The control group typically refers to normal water (ultra pure water), but is not limited to this, and means an experimental group using a composition having the same composition except that the nanobubbles of the present invention are not included. Use of the composition of the present invention has an effect that the amount of ATP produced in mitochondria is higher than that of the control group when oxidative stress is applied.
本明細書において、「ミトコンドリア活性化」とは、ミトコンドリアにおけるＡＴＰ産生量が対照区よりも高くなることを言う。対照区とは、典型的には通常の水（超純水）を言うが、これに限られず本発明のナノバブルを含まない以外は同じ組成の組成物を用いる実験区を意味する。本発明の組成物を使用することにより、酸化ストレス負荷時において、ミトコンドリアにおけるＡＴＰ産生量が対照区よりも高くなる効果を有する。 The present invention provides an activator that activates mitochondria. Mitochondrial activation can be measured as an increase in ATP production by measuring ATP production.
In this specification, “mitochondrial activation” means that the amount of ATP produced in mitochondria is higher than that in the control group. The control group typically refers to normal water (ultra pure water), but is not limited to this, and means an experimental group using a composition having the same composition except that the nanobubbles of the present invention are not included. Use of the composition of the present invention has an effect that the amount of ATP produced in mitochondria is higher than that of the control group when oxidative stress is applied.
本発明の組成物によれば、高い細胞増殖促進効果が得られる。例えば、本発明の組成物（水溶液）を用いて細胞培養用培地を調製し、細胞を培養した場合、通常の水（超純水）を用いた培地に比べ高い増殖促進効果が得られる。
According to the composition of the present invention, a high cell proliferation promoting effect can be obtained. For example, when a cell culture medium is prepared using the composition (aqueous solution) of the present invention and the cells are cultured, a higher growth promoting effect is obtained compared to a medium using normal water (ultra pure water).
例えば、iPS細胞（induced pluripotent stem cells、誘導多能性幹細胞）の培養において、72時間培養で、通常の超純水を用いた培地で培養した場合の約２倍の増殖促進効果が得られた（図１）。この細胞増殖促進剤は、他の培養細胞、例えば、初代培養細胞、未分化細胞、造血細胞、繊維芽細胞、不死化細胞、幹細胞等、増殖可能な細胞にも使用できる。ここで培養細胞とは、特に限定されないが、１次体細胞、株化細胞、不死化細胞、幹細胞等分裂可能な細胞を言う。幹細胞とは、胚性幹細胞および体性幹細胞を含み、例えば、神経幹細胞、肝幹細胞、皮膚幹細胞、生殖幹細胞、iPS細胞等を言うがこれらに限られない。増殖可能な細胞とは、増殖能を有する細胞をいい、１次体細胞の培養細胞、未分化細胞、前駆細胞等が含まれる。
For example, in the cultivation of iPS cells (induced pluripotent stem cells, induced pluripotent stem cells), the growth promotion effect was approximately doubled when cultured in a medium using normal ultrapure water in 72 hours of culture. (FIG. 1). This cell growth promoter can also be used for proliferating cells such as other cultured cells such as primary cultured cells, undifferentiated cells, hematopoietic cells, fibroblasts, immortalized cells, stem cells and the like. Here, the cultured cell is not particularly limited, but refers to a cell that can divide, such as a primary somatic cell, a cell line, an immortalized cell, or a stem cell. Stem cells include embryonic stem cells and somatic stem cells, and include, but are not limited to, neural stem cells, liver stem cells, skin stem cells, germ stem cells, iPS cells, and the like. A proliferative cell means a cell having proliferative ability, and includes cultured cells of primary somatic cells, undifferentiated cells, progenitor cells, and the like.
培養細胞作製のための出発材料として用いることのできる体細胞は、哺乳動物（例えば、マウスまたはヒト）由来の生殖細胞以外のいかなる細胞であってもよく、例えば、角質化する上皮細胞（例、角質化表皮細胞）、粘膜上皮細胞（例、舌表層の上皮細胞）、外分泌腺上皮細胞（例、乳腺細胞）、ホルモン分泌細胞（例、副腎髄質細胞）、代謝・貯蔵用の細胞（例、肝細胞）、境界面を構成する内腔上皮細胞（例、I型肺胞細胞）、内鎖管の内腔上皮細胞（例、血管内皮細胞）、運搬能をもつ繊毛のある細胞（例、気道上皮細胞）、細胞外マトリックス分泌用細胞（例、線維芽細胞）、収縮性細胞（例、平滑筋細胞）、血液と免疫系の細胞（例、Tリンパ球）、感覚に関する細胞（例、桿細胞） 自律神経系ニューロン（例、コリン作動性ニューロン）、感覚器と末梢ニューロンの支持細胞（例、随伴細胞）、中枢神経系の神経細胞とグリア細胞（例、星状グリア細胞）、色素細胞（例、網膜色素上皮細胞）、およびそれらの前駆細胞（組織前駆細胞）等が挙げられる。細胞の分化の程度に特に制限はなく、未分化な前駆細胞（体性幹細胞も含む） であっても、最終分化した成熟細胞であっても、同様に体細胞の起源として使用することができる。ここで未分化な前駆細胞としては、たとえば神経幹細胞、造血幹細胞、間葉系幹細胞、歯髄幹細胞等の組織幹細胞（体性幹細胞）が挙げられる。
Somatic cells that can be used as starting material for the production of cultured cells can be any cells other than germ cells derived from mammals (eg, mice or humans), such as keratinized epithelial cells (eg, Keratinized epidermal cells), mucosal epithelial cells (eg, epithelial cells of the tongue), exocrine glandular epithelial cells (eg, mammary cells), hormone-secreting cells (eg, adrenal medullary cells), cells for metabolism and storage (eg, Hepatocytes), luminal epithelial cells that make up the interface (eg, type I alveolar cells), luminal epithelial cells of the inner chain (eg, vascular endothelial cells), and cilia cells that have a transport ability (eg, Airway epithelial cells), extracellular matrix secreting cells (eg, fibroblasts), contractile cells (eg, smooth muscle cells), blood and immune system cells (eg, T lymphocytes), sensory cells (eg,桿 cells) Autonomic neurons (eg, cholinergic neurons) Urons), sensory organs and peripheral neuron support cells (eg, companion cells), central nervous system neurons and glial cells (eg, astrocytes), pigment cells (eg, retinal pigment epithelial cells), and Examples include progenitor cells (tissue progenitor cells). There is no particular limitation on the degree of cell differentiation, and it can be used as the source of somatic cells, whether it is an undifferentiated progenitor cell (including somatic stem cells) or a terminally differentiated mature cell. . Examples of undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
また、本発明の組成物を用いることで植物細胞の増殖促進効果も得られる。本発明の組成物を用いて培地または水耕栽培溶液を調製し、植物に使用することにより、増殖促進効果が得られる。特に水耕栽培では本発明の組成物とともに空気のマイクロバブル（サイズ１０ｎｍ～３０ｎｍ）を加えることで、地上部乾物重、地上部新鮮重、地下部乾物重、地下部新鮮重とも１０～２０％増加した（実施例１４、表７参照）
In addition, by using the composition of the present invention, an effect of promoting the growth of plant cells can be obtained. A growth promoting effect can be obtained by preparing a medium or a hydroponics solution using the composition of the present invention and using it in plants. Especially in hydroponics, by adding air microbubbles (size 10 nm to 30 nm) together with the composition of the present invention, the above-ground dry weight, above-ground fresh weight, below-ground dry weight, and below-ground fresh weight are both 10 to 20%. Increased (see Example 14, Table 7)
本発明の組成物によれば、細胞の活性を高く保った状態で細胞を保存することができる。例えば、魚類を本発明の組成物を含む水で生育させ、または保存することで、ミトコンドリアの活性を高く保つことができ、風味のよい食品を提供することができる。これは、ミトコンドリアが活性化されるとＡＴＰの産生量が増加し、その分解物であるイノシン酸（ＩＭＰ）等の旨味成分も増加するためと考えられる。すなわち、本発明の組成物によれば、旨味の増加した食品を提供することができる。
According to the composition of the present invention, cells can be stored in a state where the activity of the cells is kept high. For example, by growing or storing fish in water containing the composition of the present invention, mitochondrial activity can be kept high, and a savory food can be provided. This is considered to be because when mitochondria are activated, the amount of ATP produced increases, and umami components such as inosinic acid (IMP), which is a degradation product thereof, also increase. That is, according to the composition of the present invention, a food with increased umami taste can be provided.
本発明の組成物は、飲食物にそのまま添加することもでき、飲食物の原料として使用したり、飲食物又は飲食物原料の加工用に使用することもできる。ここで、飲食物とは、限定されないが、人が食する飲み物および食べ物を意味する。例えば、健康補助食品、低カロリー食品、ダイエット食品、パン、乳製品（例えば、無脂肪乳、ヨーグルト、プリン、豆乳など）、麺（例えば、うどん、中華麺、ワンタン、餃子の皮、蕎麦など）、菓子（例えば、クッキー、和菓子、蒸しパン、ポテトチップス、チョコレート、カスタードクリームなど）、飲料（例えば、スープ、野菜飲料、ダイエットドリンク、紅茶、コーヒーなど）、デザート（例えば、ケーキ、ムースなど）、冷菓子（例えば、ゼリー、アイスクリームなど）、惣菜（例えば、つみれ、伊達巻、から揚げなど）、シリアル、フィリング、ドレッシング、焼き食物、肉製品（例えば、ソーセージなど）が挙げられる。
The composition of the present invention can be added to foods and drinks as they are, and can be used as a raw material for foods and drinks, or can be used for processing foods and drinks or food materials. Here, the food and drink means, though not limited to, drinks and foods that people eat. For example, health supplements, low-calorie foods, diet foods, bread, dairy products (eg, non-fat milk, yogurt, pudding, soy milk, etc.), noodles (eg, udon, Chinese noodles, wonton, gyoza skin, soba noodles, etc.) Confectionery (e.g. cookies, Japanese confectionery, steamed bread, potato chips, chocolate, custard cream, etc.), beverages (e.g. soup, vegetable drinks, diet drinks, tea, coffee etc.), desserts (e.g. cakes, mousses etc.), Examples include frozen confectionery (for example, jelly, ice cream, etc.), sugar beet (for example, tsumire, date roll, fried chicken), cereal, filling, dressing, baked food, meat products (for example, sausage).
本発明の組成物は、飼料に添加して使用することもできる。飼料とは、限定されないが、人を除く動物、好ましくは、魚類、家畜、愛玩動物などの飲み物および食べ物を意味する。また、魚のえさなども含む。より好ましくは、魚類用の生簀、水槽、池等の水、牛、豚、羊、鶏、馬、イヌ、ネコなどの飲み物および食べ物である。
また、本発明の組成物は、注射用剤（点滴剤も含む）としても使用できる。例えば、ペット、家畜、実験動物および／またはヒトなどに本発明の組成物を注射することで、免疫細胞の増殖促進作用、糖尿病・動脈硬化症・炎症などの予防および／または治療薬としても用いることができる。また、また、注射液の調製に本発明の組成物を用いることもできる。
また、本発明の組成物は、人または動物がそのまま飲用水として飲むことによっても種々の疾患、例えば、免疫細胞の増殖促進作用、糖尿病・動脈硬化症・炎症などの予防、治療効果が得られる。 The composition of the present invention can also be used by adding to feed. The feed means, but not limited to, animals other than humans, and preferably drinks and foods such as fish, livestock, and pets. Also includes fish food. More preferred are fish ginger, water in aquariums, ponds, and drinks and foods such as cows, pigs, sheep, chickens, horses, dogs and cats.
The composition of the present invention can also be used as an injectable agent (including an instillation). For example, by injecting the composition of the present invention into pets, domestic animals, laboratory animals, and / or humans, the composition is also used as a prophylactic and / or therapeutic agent for immune cell proliferation promotion, diabetes, arteriosclerosis, inflammation, etc. be able to. Moreover, the composition of this invention can also be used for preparation of an injection solution.
In addition, the composition of the present invention can provide various diseases, such as immune cell proliferation-promoting action, diabetes / arteriosclerosis / inflammation, and other preventive and therapeutic effects even when a human or animal drinks it as drinking water. .
また、本発明の組成物は、注射用剤（点滴剤も含む）としても使用できる。例えば、ペット、家畜、実験動物および／またはヒトなどに本発明の組成物を注射することで、免疫細胞の増殖促進作用、糖尿病・動脈硬化症・炎症などの予防および／または治療薬としても用いることができる。また、また、注射液の調製に本発明の組成物を用いることもできる。
また、本発明の組成物は、人または動物がそのまま飲用水として飲むことによっても種々の疾患、例えば、免疫細胞の増殖促進作用、糖尿病・動脈硬化症・炎症などの予防、治療効果が得られる。 The composition of the present invention can also be used by adding to feed. The feed means, but not limited to, animals other than humans, and preferably drinks and foods such as fish, livestock, and pets. Also includes fish food. More preferred are fish ginger, water in aquariums, ponds, and drinks and foods such as cows, pigs, sheep, chickens, horses, dogs and cats.
The composition of the present invention can also be used as an injectable agent (including an instillation). For example, by injecting the composition of the present invention into pets, domestic animals, laboratory animals, and / or humans, the composition is also used as a prophylactic and / or therapeutic agent for immune cell proliferation promotion, diabetes, arteriosclerosis, inflammation, etc. be able to. Moreover, the composition of this invention can also be used for preparation of an injection solution.
In addition, the composition of the present invention can provide various diseases, such as immune cell proliferation-promoting action, diabetes / arteriosclerosis / inflammation, and other preventive and therapeutic effects even when a human or animal drinks it as drinking water. .
以下に実施例を用いて本発明を説明するが、本発明の範囲は何らこれらの実施例により制限されるものではない。
Hereinafter, the present invention will be described with reference to examples, but the scope of the present invention is not limited by these examples.
（実施例１）
iPS細胞培地は、以下のようにして調製した。1Lの蒸留水（DW）または本発明の組成物（APAS）に12.5gのGlasgow minimum essential medium (Sigma;G6148)と2.75 gのSodium bicarbonate (Sigma;S5761)を加え攪拌した。この溶液に、以下の終濃度になるように各試薬を添加した：0.1mM MEM Non-Essential Amino Acids Solusion (Gibco;11140)と1mM Sodium pyruvate solution (Sigma;S8636)、1% Fetal bovine serum (CCB;171012)、1% PenicillinStreptomycin (Gibco;15140)、5.4% Knockout Serum Replacement (Gibco;10828)、0.1mM 2-Mercaptoethanol (Wako;137-06862)、2000 U/ml Leukemia inhibitor factor (Millipore;ESG1107)。均一になるまで撹拌し、溶解した後、0.45μmフィルターで濾過滅菌し細胞維持培地とした。
培養の結果を図１に示す。その結果、本発明の組成物を用いて培地を作成した場合、通常の超純水を用いて作成した培地を用いた場合とでは、本発明の組成物を用いて作成した培地の方が、対照の超純水培地に比べ約２倍の増殖量が得られた。 Example 1
iPS cell culture medium was prepared as follows. To 1 L of distilled water (DW) or the composition of the present invention (APAS), 12.5 g of Glasgow minimum essential medium (Sigma; G6148) and 2.75 g of sodium bicarbonate (Sigma; S5761) were added and stirred. Reagents were added to this solution to the following final concentrations: 0.1 mM MEM Non-Essential Amino Acids Solusion (Gibco; 11140), 1 mM Sodium pyruvate solution (Sigma; S8636), 1% Fetal bovine serum (CCB ; 171012), 1% Penicillin Streptomycin (Gibco; 15140), 5.4% Knockout Serum Replacement (Gibco; 10828), 0.1 mM 2-Mercaptoethanol (Wako; 137-06862), 2000 U / ml Leukemia inhibitor factor (Millipore; ESG1107). The mixture was stirred until homogeneous and dissolved, and then sterilized by filtration through a 0.45 μm filter to obtain a cell maintenance medium.
The culture results are shown in FIG. As a result, when a medium is prepared using the composition of the present invention, when a medium prepared using normal ultrapure water is used, the medium prepared using the composition of the present invention is more About twice the amount of growth was obtained compared to the control ultrapure water medium.
iPS細胞培地は、以下のようにして調製した。1Lの蒸留水（DW）または本発明の組成物（APAS）に12.5gのGlasgow minimum essential medium (Sigma;G6148)と2.75 gのSodium bicarbonate (Sigma;S5761)を加え攪拌した。この溶液に、以下の終濃度になるように各試薬を添加した：0.1mM MEM Non-Essential Amino Acids Solusion (Gibco;11140)と1mM Sodium pyruvate solution (Sigma;S8636)、1% Fetal bovine serum (CCB;171012)、1% PenicillinStreptomycin (Gibco;15140)、5.4% Knockout Serum Replacement (Gibco;10828)、0.1mM 2-Mercaptoethanol (Wako;137-06862)、2000 U/ml Leukemia inhibitor factor (Millipore;ESG1107)。均一になるまで撹拌し、溶解した後、0.45μmフィルターで濾過滅菌し細胞維持培地とした。
培養の結果を図１に示す。その結果、本発明の組成物を用いて培地を作成した場合、通常の超純水を用いて作成した培地を用いた場合とでは、本発明の組成物を用いて作成した培地の方が、対照の超純水培地に比べ約２倍の増殖量が得られた。 Example 1
iPS cell culture medium was prepared as follows. To 1 L of distilled water (DW) or the composition of the present invention (APAS), 12.5 g of Glasgow minimum essential medium (Sigma; G6148) and 2.75 g of sodium bicarbonate (Sigma; S5761) were added and stirred. Reagents were added to this solution to the following final concentrations: 0.1 mM MEM Non-Essential Amino Acids Solusion (Gibco; 11140), 1 mM Sodium pyruvate solution (Sigma; S8636), 1% Fetal bovine serum (CCB ; 171012), 1% Penicillin Streptomycin (Gibco; 15140), 5.4% Knockout Serum Replacement (Gibco; 10828), 0.1 mM 2-Mercaptoethanol (Wako; 137-06862), 2000 U / ml Leukemia inhibitor factor (Millipore; ESG1107). The mixture was stirred until homogeneous and dissolved, and then sterilized by filtration through a 0.45 μm filter to obtain a cell maintenance medium.
The culture results are shown in FIG. As a result, when a medium is prepared using the composition of the present invention, when a medium prepared using normal ultrapure water is used, the medium prepared using the composition of the present invention is more About twice the amount of growth was obtained compared to the control ultrapure water medium.
（実施例２）マウス血管内皮細胞株（ＭＡＥＣ）を用いた抗酸化作用の検討
本発明の組成物を含む水、ナノバブル水（空気を含むナノバブル水）、水素ナノバブル水（水素を溶存気体、マイクロバブルおよびナノバブルの合計の７０％以上含むナノバブル水）をそれぞれ用いて、Ｍ１９９培地（ＳＩＧＭＡ）を作製し、１０％ウシ胎児血清、１００ＩＵ／ｍｌペニシリン、及び１００μｌ／ｍｌストレプトマイシンを加え、培養用培地とした。それぞれの水を用いた培地でＭＡＥＣを２４時間培養した後、新鮮な培地に交換してさらに２時間培養した。その後３０μｇ／ｍｌアンチマイシンを各々の培地に添加し、３０分間培養した。対照として溶媒として用いたエタノールをアンチマイシン溶液に含まれる用量と同量である０．０６ｖ／ｖ％になるように加えた。アンチマイシンは、ミトコンドリアの電子伝達系を抑制することにより細胞にＲＯＳ（活性酸素種、Reactive Oxygen Species)を産生させる作用があることが知られている。エタノールは一般にアンチマイシンの溶媒として用いられるもので、その溶媒の影響を知るためにアンチマイシンを含まないエタノールのみを加えた場合についてもテストした。産生されたＲＯＳの量を知るために、蛍光強度を指標として、その蛍光強度を有する細胞の数を計測した。具体的には、ＲＯＳを検出するためのプローブとしてＣＭ－Ｈ２ＤＣＦＤＡを用い、各細胞からの蛍光の強度をフローサイトメーター（ＶａｎｔａｇｅＳＥ、Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ）により検出した。 (Example 2) Examination of antioxidant action using mouse vascular endothelial cell line (MAEC) Water containing the composition of the present invention, nanobubble water (nanobubble water containing air), hydrogen nanobubble water (hydrogen dissolved gas, micro Nanobubble water containing 70% or more of bubbles and nanobubbles) was prepared, respectively, and M199 medium (SIGMA) was prepared, 10% fetal bovine serum, 100 IU / ml penicillin, and 100 μl / ml streptomycin were added, did. After culturing MAEC for 24 hours in a medium using each water, the medium was replaced with a fresh medium and further cultured for 2 hours. Thereafter, 30 μg / ml antimycin was added to each medium and incubated for 30 minutes. As a control, ethanol used as a solvent was added so as to be 0.06 v / v% which is the same amount as that contained in the antimycin solution. Antimycin is known to have the effect of causing cells to produce ROS (Reactive Oxygen Species) by suppressing the mitochondrial electron transport system. Ethanol is generally used as a solvent for antimycin, and in order to know the influence of the solvent, the case where only ethanol without antimycin was added was also tested. In order to know the amount of ROS produced, the number of cells having the fluorescence intensity was counted using the fluorescence intensity as an index. Specifically, CM-H2DCFDA was used as a probe for detecting ROS, and the intensity of fluorescence from each cell was detected by a flow cytometer (VantageSE, Becton Dickinson).
本発明の組成物を含む水、ナノバブル水（空気を含むナノバブル水）、水素ナノバブル水（水素を溶存気体、マイクロバブルおよびナノバブルの合計の７０％以上含むナノバブル水）をそれぞれ用いて、Ｍ１９９培地（ＳＩＧＭＡ）を作製し、１０％ウシ胎児血清、１００ＩＵ／ｍｌペニシリン、及び１００μｌ／ｍｌストレプトマイシンを加え、培養用培地とした。それぞれの水を用いた培地でＭＡＥＣを２４時間培養した後、新鮮な培地に交換してさらに２時間培養した。その後３０μｇ／ｍｌアンチマイシンを各々の培地に添加し、３０分間培養した。対照として溶媒として用いたエタノールをアンチマイシン溶液に含まれる用量と同量である０．０６ｖ／ｖ％になるように加えた。アンチマイシンは、ミトコンドリアの電子伝達系を抑制することにより細胞にＲＯＳ（活性酸素種、Reactive Oxygen Species)を産生させる作用があることが知られている。エタノールは一般にアンチマイシンの溶媒として用いられるもので、その溶媒の影響を知るためにアンチマイシンを含まないエタノールのみを加えた場合についてもテストした。産生されたＲＯＳの量を知るために、蛍光強度を指標として、その蛍光強度を有する細胞の数を計測した。具体的には、ＲＯＳを検出するためのプローブとしてＣＭ－Ｈ２ＤＣＦＤＡを用い、各細胞からの蛍光の強度をフローサイトメーター（ＶａｎｔａｇｅＳＥ、Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ）により検出した。 (Example 2) Examination of antioxidant action using mouse vascular endothelial cell line (MAEC) Water containing the composition of the present invention, nanobubble water (nanobubble water containing air), hydrogen nanobubble water (hydrogen dissolved gas, micro Nanobubble water containing 70% or more of bubbles and nanobubbles) was prepared, respectively, and M199 medium (SIGMA) was prepared, 10% fetal bovine serum, 100 IU / ml penicillin, and 100 μl / ml streptomycin were added, did. After culturing MAEC for 24 hours in a medium using each water, the medium was replaced with a fresh medium and further cultured for 2 hours. Thereafter, 30 μg / ml antimycin was added to each medium and incubated for 30 minutes. As a control, ethanol used as a solvent was added so as to be 0.06 v / v% which is the same amount as that contained in the antimycin solution. Antimycin is known to have the effect of causing cells to produce ROS (Reactive Oxygen Species) by suppressing the mitochondrial electron transport system. Ethanol is generally used as a solvent for antimycin, and in order to know the influence of the solvent, the case where only ethanol without antimycin was added was also tested. In order to know the amount of ROS produced, the number of cells having the fluorescence intensity was counted using the fluorescence intensity as an index. Specifically, CM-H2DCFDA was used as a probe for detecting ROS, and the intensity of fluorescence from each cell was detected by a flow cytometer (VantageSE, Becton Dickinson).
ＲＯＳの検出結果を図２に示す。図２の各グラフは、ある蛍光強度を示す細胞数を示すものであり、ＭＦＩの値は平均蛍光強度を示す。ナノバブル水（ＮＢ；上段）を用いた培地でＭＡＥＣ細胞を培養した場合は、無刺激時（左列）と溶媒コントロールのエタノールのみを添加した場合（真中列）とで平均蛍光強度に殆ど変化が認められなかったが、ＲＯＳ産生作用をもつアンチマイシンを添加した場合（右列）には平均蛍光強度が顕著に増強した。これに対して本発明の組成物を含む水（Ｏ２・Ｈ２・Ｎ２ＮＢ；下段）で培養した場合には、ナノバブル水を用いた培地で培養したときよりも、無刺激とアンチマイシン添加のいずれの場合にも低い平均蛍光強度が得られた。また、水素ナノバブル水（Ｈ２ＮＢ；中段）を用いた培地で培養したときと本発明の組成物を含む水を用いた培地で培養したときとを比較すると、本発明の組成物を含む水の方を用いた培地で培養した方が、無刺激の場合、エタノールのみを添加した場合、アンチマイシンを添加した場合の全てにおいて、低い平均蛍光強度を示していた。この結果から本発明の組成物を含む水の強い抗酸化作用が認められた。
Figure 2 shows the ROS detection results. Each graph in FIG. 2 shows the number of cells showing a certain fluorescence intensity, and the MFI value shows the average fluorescence intensity. When MAEC cells were cultured in a medium using nanobubble water (NB; upper row), there was almost no change in average fluorescence intensity between no stimulation (left column) and when only solvent-controlled ethanol was added (middle column). Although not observed, when antimycin having ROS producing activity was added (right column), the average fluorescence intensity was significantly enhanced. On the other hand, when cultured in water containing the composition of the present invention (O 2 · H 2 · N 2 NB; bottom), both non-stimulated and antimycin-added than when cultured in a medium using nanobubble water In some cases, a low average fluorescence intensity was obtained. Further, when culturing in a medium using hydrogen nanobubble water (H2NB; middle stage) and culturing in a medium using water containing the composition of the present invention, the water containing the composition of the present invention is compared. In the case of no stimulation, when ethanol was added alone, and when antimycin was added, the culture medium in the culture medium using the low average fluorescence intensity was shown. From this result, strong antioxidant action of water containing the composition of the present invention was recognized.
（実施例３）マウス血管内皮細胞株（ＭＡＥＣ）における酸化ストレス負荷時のＡＴＰ産生の検討
次に、ＭＡＥＣにおいて酸化ストレス負荷時に低下するミトコンドリアの機能へのＡＴＰ産生活性水の作用を確認するために、細胞内ＡＴＰ産生について検討を行った。ＭＡＥＣ細胞は上記実験と同様に本発明の組成物を含む水、ナノバブル水、水素ナノバブル水を用いて作製した培養用培地にて培養した。それぞれの水を用いた培地でＭＡＥＣを２４時間培養した後、培地をそれぞれの水を用いた新鮮な培地に交換してさらに２時間培養した。次いで、５０μｇ／ｍｌアンチマイシン、０．０６ｖ／ｖ％エタノール、５００μｍｏｌ／Ｌ過酸化水素をそれぞれの培地に添加した。また、抗酸化作用のコントロールとして、広汎な活性酸素種に作用する抗酸化剤のＮ－アセチルシステイン（ＮＡＣ）を用いた。抗酸化作用のコントロール用の培地は、通常の水を用いて作製した。この培地に、ＭＡＥＣ細胞を２４時間培養し、通常の水を用いた新鮮な培地に交換してさらに２時間培養した。次いで、ＮＡＣを、終濃度が５ｍｍｏｌ／Ｌとなるようにアンチマイシンあるいは過酸化水素水処理の３０分前に培地に添加した。その後、細胞内のＡＴＰ量の変化をルシフェラーゼ活性を指標に測定した（ＡＴＰｌｉｔｅ、ＰｅｒｋｉｎＥｌｍｅｒ）。 (Example 3) Examination of ATP production during oxidative stress loading in mouse vascular endothelial cell line (MAEC) Next, in order to confirm the effect of ATP-producing active water on the function of mitochondria that declines during oxidative stress loading in MAEC In addition, intracellular ATP production was examined. MAEC cells were cultured in a culture medium prepared using water, nanobubble water, and hydrogen nanobubble water containing the composition of the present invention, as in the above experiment. After culturing MAEC for 24 hours in a medium using each water, the medium was replaced with a fresh medium using each water and further cultured for 2 hours. Subsequently, 50 μg / ml antimycin, 0.06 v / v% ethanol, 500 μmol / L hydrogen peroxide was added to each medium. As an antioxidant control, N-acetylcysteine (NAC), an antioxidant that acts on a wide range of reactive oxygen species, was used. A medium for controlling the antioxidant action was prepared using ordinary water. MAEC cells were cultured in this medium for 24 hours, replaced with a fresh medium using normal water, and further cultured for 2 hours. Next, NAC was added to the medium 30 minutes before the treatment with antimycin or hydrogen peroxide so that the final concentration was 5 mmol / L. Thereafter, changes in intracellular ATP amount were measured using luciferase activity as an index (ATPlite, PerkinElmer).
次に、ＭＡＥＣにおいて酸化ストレス負荷時に低下するミトコンドリアの機能へのＡＴＰ産生活性水の作用を確認するために、細胞内ＡＴＰ産生について検討を行った。ＭＡＥＣ細胞は上記実験と同様に本発明の組成物を含む水、ナノバブル水、水素ナノバブル水を用いて作製した培養用培地にて培養した。それぞれの水を用いた培地でＭＡＥＣを２４時間培養した後、培地をそれぞれの水を用いた新鮮な培地に交換してさらに２時間培養した。次いで、５０μｇ／ｍｌアンチマイシン、０．０６ｖ／ｖ％エタノール、５００μｍｏｌ／Ｌ過酸化水素をそれぞれの培地に添加した。また、抗酸化作用のコントロールとして、広汎な活性酸素種に作用する抗酸化剤のＮ－アセチルシステイン（ＮＡＣ）を用いた。抗酸化作用のコントロール用の培地は、通常の水を用いて作製した。この培地に、ＭＡＥＣ細胞を２４時間培養し、通常の水を用いた新鮮な培地に交換してさらに２時間培養した。次いで、ＮＡＣを、終濃度が５ｍｍｏｌ／Ｌとなるようにアンチマイシンあるいは過酸化水素水処理の３０分前に培地に添加した。その後、細胞内のＡＴＰ量の変化をルシフェラーゼ活性を指標に測定した（ＡＴＰｌｉｔｅ、ＰｅｒｋｉｎＥｌｍｅｒ）。 (Example 3) Examination of ATP production during oxidative stress loading in mouse vascular endothelial cell line (MAEC) Next, in order to confirm the effect of ATP-producing active water on the function of mitochondria that declines during oxidative stress loading in MAEC In addition, intracellular ATP production was examined. MAEC cells were cultured in a culture medium prepared using water, nanobubble water, and hydrogen nanobubble water containing the composition of the present invention, as in the above experiment. After culturing MAEC for 24 hours in a medium using each water, the medium was replaced with a fresh medium using each water and further cultured for 2 hours. Subsequently, 50 μg / ml antimycin, 0.06 v / v% ethanol, 500 μmol / L hydrogen peroxide was added to each medium. As an antioxidant control, N-acetylcysteine (NAC), an antioxidant that acts on a wide range of reactive oxygen species, was used. A medium for controlling the antioxidant action was prepared using ordinary water. MAEC cells were cultured in this medium for 24 hours, replaced with a fresh medium using normal water, and further cultured for 2 hours. Next, NAC was added to the medium 30 minutes before the treatment with antimycin or hydrogen peroxide so that the final concentration was 5 mmol / L. Thereafter, changes in intracellular ATP amount were measured using luciferase activity as an index (ATPlite, PerkinElmer).
細胞内のＡＴＰ量の測定結果を図３に示す。ナノバブル水（ＮＢ）を用いた培地で培養した細胞では、ＡＴＰ量は、エタノール処理と、アンチマイシン処理、過酸化水素処理の全てで、無刺激の場合と比較して低下が認められた。この結果から、エタノール、アンチマイシン、及び過酸化水素は、ＡＴＰ産生活性の抑制効果を有することがわかる。
The measurement result of the amount of ATP in the cell is shown in FIG. In cells cultured in a medium using nanobubble water (NB), the amount of ATP was decreased in all of ethanol treatment, antimycin treatment, and hydrogen peroxide treatment as compared with the case of no stimulation. From this result, it can be seen that ethanol, antimycin, and hydrogen peroxide have an inhibitory effect on ATP production activity.
アンチマイシン処理では過酸化水素処理よりも強いＡＴＰ産生活性の低下が認められた。水素ナノバブル水（Ｈ２ＮＢ）、本発明の組成物を含む水（Ｏ２・Ｈ２・Ｎ２ＮＢをそれぞれ用いた培地で培養した 細胞では、いずれの処理を行った場合もＡＴＰ産生活性の増加が認められた。すなわち、水素ナノバブル水と本発明の組成物を含む水は、アンチマイシン等によるＡＴＰ産生活性の抑制効果を、抗酸化剤であるＮＡＣと同等もしくはそれ以上に低減させることが認められた。さらに、本発明の組成物を含む水は、エタノール又は過酸化水素で処理した場合に、水素ナノバブル水よりも、有意にＡＴＰ産生活性の抑制効果を低減させた。
The antimycin treatment showed a stronger decrease in ATP production activity than the hydrogen peroxide treatment. In sputum cells cultured in hydrogen nanobubble water (H2NB) and water containing the composition of the present invention (O2, H2, N2NB, respectively), an increase in ATP production activity was observed when any treatment was performed. That is, it was confirmed that the water containing hydrogen nanobubble water and the composition of the present invention reduces the inhibitory effect on ATP production activity by antimycin or the like to NAC which is an antioxidant. Furthermore, when the water containing the composition of the present invention was treated with ethanol or hydrogen peroxide, the effect of suppressing ATP production activity was significantly reduced as compared with hydrogen nanobubble water.
（実施例３）ヒトにおける飲用効果の検討
本発明の組成物を含む水をヒトが飲用した場合の生体に与える効果を明らかにすることを目的とし、ヒトにおける飲用実験を実施した。実験に参加したヒトは、４０～６９歳（平均年齢５１．８歳）の男性４名、女性１名であった。各々の参加者の年齢と性別を表１に示す。全ての参加者は健康な状態であった。 (Example 3) Examination of drinking effects in humans A drinking experiment in humans was carried out for the purpose of clarifying the effect of humans on drinking water containing the composition of the present invention. The humans who participated in the experiment were 4 males and 1 female aged 40 to 69 years (average age 51.8 years). Table 1 shows the age and gender of each participant. All participants were in good health.
本発明の組成物を含む水をヒトが飲用した場合の生体に与える効果を明らかにすることを目的とし、ヒトにおける飲用実験を実施した。実験に参加したヒトは、４０～６９歳（平均年齢５１．８歳）の男性４名、女性１名であった。各々の参加者の年齢と性別を表１に示す。全ての参加者は健康な状態であった。 (Example 3) Examination of drinking effects in humans A drinking experiment in humans was carried out for the purpose of clarifying the effect of humans on drinking water containing the composition of the present invention. The humans who participated in the experiment were 4 males and 1 female aged 40 to 69 years (average age 51.8 years). Table 1 shows the age and gender of each participant. All participants were in good health.
各々の参加者が本発明の組成物を含む水を１Ｌ／日、４週間飲用し、通常と変わらない生活を心がけた。参加者は、飲用開始前に血液を採取され、飲用開始後４週間目に再び血液を採取された。採血の当日は本発明の組成物を含む水を飲用しないこととした。検査は、肝障害の指標であるＧＰＴ（ｇｌｕｔａｍｉｃ ｐｙｒｕｖｉｃ ｔｒａｎｓａｍｉｎａｓｅ）、過酸化脂質マーカーであるＨＥＬ（ｈｅｘａｎｏｙｌ－ｌｙｓｉｎｅ）、及び水溶性 総抗酸化物質として知られているＳＴＡＳ（Ｓｅｒｕｍ Ｔｏｔａｌ Ａｎｔｉｏｘｉｄａｎｔ Ｓｔａｔｕｓ）について行った。
Each participant took 1 L / day of water containing the composition of the present invention for 4 weeks, and tried to live as usual. Participants were blood collected before the start of drinking and blood was collected again 4 weeks after the start of drinking. On the day of blood collection, water containing the composition of the present invention was not drunk. The tests were performed on GPT (glutamic pyranic transaminase), an indicator of liver damage, HEL (hexanoyl-lysine), a lipid peroxide marker, and STAS (Serum Total Antioxidant), which is known as a water-soluble sputum total antioxidant. It was.
（１）ＧＰＴ
ＧＰＴは、主に肝臓に存在する酵素である。肝細胞が破壊されると特異的に血中に漏出することから肝炎ウイルスや薬物などによる肝障害の指標として利用される。この他、肝脂肪ではＧＯＴ、ＧＰＴの値が軽度の異常であることが多く、肥満による脂肪肝は、ＧＯＴよりＧＰＴがやや高くなることも知られている。ＧＰＴは、血清中の濃度をシリカリキッドＡＬＴ試薬（関東化学株式会社製）を使用し、Ｂｉｏ Ｍａｊａｓｅｓｔｙ（ＪＣＡ－ＢＭ８０６０）（日本電子株式会社製）と オリンパスＡＵ５４３１型生化学自動分析装置（オリンパス株式会社製）を用いて測定した。血清は、採取した血液を凝固させて、遠心分離して分離、回収した。測定結果を図４に箱ひげ図で示す。図４を参照すると、本発明の組成物を含む水の摂取後では、ＧＰＴの値が摂取前と比較して明らかに減少していた（Ｐ＝０．０４２）。従って、本発明の組成物を含む水の飲用により、脂肪肝の改善傾向が認められた。 (1) GPT
GPT is an enzyme mainly present in the liver. When hepatocytes are destroyed, they leak into the blood specifically, and are used as indicators of liver damage caused by hepatitis virus or drugs. In addition, GOT and GPT values are often mildly abnormal in liver fat, and it is also known that fatty liver due to obesity has a slightly higher GPT than GOT. GPT uses a silica liquid ALT reagent (manufactured by Kanto Chemical Co., Inc.) with a concentration of serum, Bio Maasesty (JCA-BM8060) (manufactured by JEOL Ltd.), and Olympus AU5431 biochemical automatic analyzer (Olympus Co., Ltd.). ). Serum was collected by clotting the collected blood and centrifuging it. The measurement results are shown in FIG. Referring to FIG. 4, after ingestion of water containing the composition of the present invention, the value of GPT was clearly reduced compared to before ingestion (P = 0.042). Therefore, a tendency to improve fatty liver was observed by drinking water containing the composition of the present invention.
ＧＰＴは、主に肝臓に存在する酵素である。肝細胞が破壊されると特異的に血中に漏出することから肝炎ウイルスや薬物などによる肝障害の指標として利用される。この他、肝脂肪ではＧＯＴ、ＧＰＴの値が軽度の異常であることが多く、肥満による脂肪肝は、ＧＯＴよりＧＰＴがやや高くなることも知られている。ＧＰＴは、血清中の濃度をシリカリキッドＡＬＴ試薬（関東化学株式会社製）を使用し、Ｂｉｏ Ｍａｊａｓｅｓｔｙ（ＪＣＡ－ＢＭ８０６０）（日本電子株式会社製）と オリンパスＡＵ５４３１型生化学自動分析装置（オリンパス株式会社製）を用いて測定した。血清は、採取した血液を凝固させて、遠心分離して分離、回収した。測定結果を図４に箱ひげ図で示す。図４を参照すると、本発明の組成物を含む水の摂取後では、ＧＰＴの値が摂取前と比較して明らかに減少していた（Ｐ＝０．０４２）。従って、本発明の組成物を含む水の飲用により、脂肪肝の改善傾向が認められた。 (1) GPT
GPT is an enzyme mainly present in the liver. When hepatocytes are destroyed, they leak into the blood specifically, and are used as indicators of liver damage caused by hepatitis virus or drugs. In addition, GOT and GPT values are often mildly abnormal in liver fat, and it is also known that fatty liver due to obesity has a slightly higher GPT than GOT. GPT uses a silica liquid ALT reagent (manufactured by Kanto Chemical Co., Inc.) with a concentration of serum, Bio Maasesty (JCA-BM8060) (manufactured by JEOL Ltd.), and Olympus AU5431 biochemical automatic analyzer (Olympus Co., Ltd.). ). Serum was collected by clotting the collected blood and centrifuging it. The measurement results are shown in FIG. Referring to FIG. 4, after ingestion of water containing the composition of the present invention, the value of GPT was clearly reduced compared to before ingestion (P = 0.042). Therefore, a tendency to improve fatty liver was observed by drinking water containing the composition of the present invention.
（２）ＨＥＬ
ＨＥＬは、活性酸素種による脂質の過酸化過程において、脂質ペルオキシド（１３－Ｈｙｄｒｏｐｅｒｏｘｙ－ｏｃｔａｄｅｃａｄｉｅｎｏｉｃ ａｃｉｄ、１３－ＨＰＯＤＥ）に由来する安定な初期生成物であり、従来用いられてきた４－ＨＮＥやＭＤＡ等のアルデヒド系脂質過酸化マーカーとは異なり、脂質過酸化の初期段階を捉えることが可能なマーカーである。ＨＥＬは、血清中の濃度を、ヘキサノイルリジン測定用ＥＬＩＳＡキット（ＫＨＬ－７００、日研ザイル株式会社製）を使用し、全自動マイクロプレートＥＩＡ分析装置ＡＰ９６０（協和メディックス株式会 社製）を用いて測定した。測定結果を図５に箱ひげ図で示す。図５を参照すると、ＨＥＬはＡＴＰ産生活性水の飲用後に有意に減少した（Ｐ＝０．０２８）。従って、本発明の組成物を含む水は、ヒトにおいて脂質過酸化を抑制する効果があることが示された。 (2) HEL
HEL is a stable initial product derived from lipid peroxide (13-Hydroperoxy-octadecadienoic acid, 13-HPODE) in the process of lipid peroxidation by reactive oxygen species, and conventionally used 4-HNE, MDA, etc. Unlike the aldehyde lipid peroxidation markers, it is a marker that can capture the initial stage of lipid peroxidation. HEL uses a fully automated microplate EIA analyzer AP960 (manufactured by Kyowa Medix Co., Ltd.) using an ELISA kit for measuring hexanoyllysine (KHL-700, manufactured by Nikken Zeil Co., Ltd.). Measured. The measurement results are shown in FIG. Referring to FIG. 5, HEL significantly decreased after drinking ATP-producing active water (P = 0.028). Therefore, it was shown that the water containing the composition of the present invention has an effect of suppressing lipid peroxidation in humans.
ＨＥＬは、活性酸素種による脂質の過酸化過程において、脂質ペルオキシド（１３－Ｈｙｄｒｏｐｅｒｏｘｙ－ｏｃｔａｄｅｃａｄｉｅｎｏｉｃ ａｃｉｄ、１３－ＨＰＯＤＥ）に由来する安定な初期生成物であり、従来用いられてきた４－ＨＮＥやＭＤＡ等のアルデヒド系脂質過酸化マーカーとは異なり、脂質過酸化の初期段階を捉えることが可能なマーカーである。ＨＥＬは、血清中の濃度を、ヘキサノイルリジン測定用ＥＬＩＳＡキット（ＫＨＬ－７００、日研ザイル株式会社製）を使用し、全自動マイクロプレートＥＩＡ分析装置ＡＰ９６０（協和メディックス株式会 社製）を用いて測定した。測定結果を図５に箱ひげ図で示す。図５を参照すると、ＨＥＬはＡＴＰ産生活性水の飲用後に有意に減少した（Ｐ＝０．０２８）。従って、本発明の組成物を含む水は、ヒトにおいて脂質過酸化を抑制する効果があることが示された。 (2) HEL
HEL is a stable initial product derived from lipid peroxide (13-Hydroperoxy-octadecadienoic acid, 13-HPODE) in the process of lipid peroxidation by reactive oxygen species, and conventionally used 4-HNE, MDA, etc. Unlike the aldehyde lipid peroxidation markers, it is a marker that can capture the initial stage of lipid peroxidation. HEL uses a fully automated microplate EIA analyzer AP960 (manufactured by Kyowa Medix Co., Ltd.) using an ELISA kit for measuring hexanoyllysine (KHL-700, manufactured by Nikken Zeil Co., Ltd.). Measured. The measurement results are shown in FIG. Referring to FIG. 5, HEL significantly decreased after drinking ATP-producing active water (P = 0.028). Therefore, it was shown that the water containing the composition of the present invention has an effect of suppressing lipid peroxidation in humans.
（３）ＳＴＡＳ
ＳＴＡＳは、血清中の水溶性抗酸化物質であり、酸化ストレスに対する総合的な抗酸化能が測定可能である。ＳＴＡＳは、血清について、ＴＯＴＡＬ ＡＮＴＩＯＸＩＤＡＮＴ ＳＴＡＴＵＳ（ＮＸ２３３２、ＲＡＮＤＯＸ社製）を使用して、７０２０形日立自動分析装置（株式会社 日立ハイテクノロジーズ製）を用いて測定した。測定の結果を図６に箱ひげ図で示す。図６を参照すると、ＳＴＡＳは摂取後に有意に低い値を示していた（Ｐ＝０．０４２）。この結果から、ＳＴＡＳは水溶性の抗酸化物質を総合的に評価していることから、本発明の組成物を含む水により酸化ストレスの消去効果が期待されるヒドロキシラジカルや過酸化亜硝酸以外の活性酸素種の還元に本発明の組成物を含む水が作用していることが推測された。 (3) STAS
STAS is a water-soluble antioxidant in serum and can measure the total antioxidant capacity against oxidative stress. STAS was measured for serum using a TOTAL ANTIOXIDANT STATUS (NX2332, manufactured by RANDOX) using a 7020 Hitachi automatic analyzer (manufactured by Hitachi High-Technologies Corporation). The measurement results are shown in FIG. Referring to FIG. 6, STAS showed a significantly lower value after ingestion (P = 0.042). From these results, since STAS comprehensively evaluates water-soluble antioxidants, water other than hydroxy radicals and peroxynitrite, which are expected to eliminate oxidative stress by water containing the composition of the present invention. It was speculated that water containing the composition of the present invention was acting on the reduction of reactive oxygen species.
ＳＴＡＳは、血清中の水溶性抗酸化物質であり、酸化ストレスに対する総合的な抗酸化能が測定可能である。ＳＴＡＳは、血清について、ＴＯＴＡＬ ＡＮＴＩＯＸＩＤＡＮＴ ＳＴＡＴＵＳ（ＮＸ２３３２、ＲＡＮＤＯＸ社製）を使用して、７０２０形日立自動分析装置（株式会社 日立ハイテクノロジーズ製）を用いて測定した。測定の結果を図６に箱ひげ図で示す。図６を参照すると、ＳＴＡＳは摂取後に有意に低い値を示していた（Ｐ＝０．０４２）。この結果から、ＳＴＡＳは水溶性の抗酸化物質を総合的に評価していることから、本発明の組成物を含む水により酸化ストレスの消去効果が期待されるヒドロキシラジカルや過酸化亜硝酸以外の活性酸素種の還元に本発明の組成物を含む水が作用していることが推測された。 (3) STAS
STAS is a water-soluble antioxidant in serum and can measure the total antioxidant capacity against oxidative stress. STAS was measured for serum using a TOTAL ANTIOXIDANT STATUS (NX2332, manufactured by RANDOX) using a 7020 Hitachi automatic analyzer (manufactured by Hitachi High-Technologies Corporation). The measurement results are shown in FIG. Referring to FIG. 6, STAS showed a significantly lower value after ingestion (P = 0.042). From these results, since STAS comprehensively evaluates water-soluble antioxidants, water other than hydroxy radicals and peroxynitrite, which are expected to eliminate oxidative stress by water containing the composition of the present invention. It was speculated that water containing the composition of the present invention was acting on the reduction of reactive oxygen species.
（実施例４）
２名の被験者（O, I）に本発明の組成物を含む水（ＡＰＡＳ、Ｏ２・Ｈ２・Ｎ２ＮＢ水、ＭＣＰともいう）を１日５００ｍL、１か月間摂取させ、その前後に血液検査を行った。
結果
・被験者 O
本発明の組成物を含む水の摂取前の値を比較すると、摂取後ではALP（アルカリホスファターゼ)、ALT、γ-GTPで値が改善していた。また、総コレステロール、LDLコレステロール、遊離脂肪酸の値においても改善していた。
・被験者 I
本発明の組成物を含む水の摂取前の値を比較すると、摂取後ではALP（アルカリホスファターゼ)、ALT、γ-GTPで値が改善していた。また、総コレステロール、LDLコレステロール、遊離脂肪酸、中性脂肪の値においても改善していた。 Example 4
Two subjects (O, I) were ingested with water containing the composition of the present invention (APAS, O2 / H2 / N2NB water, also referred to as MCP) for 500 months per day for one month, and blood tests were conducted before and after that. It was.
Result / Subject O
When the values before ingestion of water containing the composition of the present invention were compared, the values were improved with ALP (alkaline phosphatase), ALT, and γ-GTP after ingestion. In addition, the values of total cholesterol, LDL cholesterol and free fatty acids were also improved.
・ Subject I
When the values before ingestion of water containing the composition of the present invention were compared, the values were improved with ALP (alkaline phosphatase), ALT, and γ-GTP after ingestion. In addition, the values of total cholesterol, LDL cholesterol, free fatty acid, and neutral fat were also improved.
２名の被験者（O, I）に本発明の組成物を含む水（ＡＰＡＳ、Ｏ２・Ｈ２・Ｎ２ＮＢ水、ＭＣＰともいう）を１日５００ｍL、１か月間摂取させ、その前後に血液検査を行った。
結果
・被験者 O
本発明の組成物を含む水の摂取前の値を比較すると、摂取後ではALP（アルカリホスファターゼ)、ALT、γ-GTPで値が改善していた。また、総コレステロール、LDLコレステロール、遊離脂肪酸の値においても改善していた。
・被験者 I
本発明の組成物を含む水の摂取前の値を比較すると、摂取後ではALP（アルカリホスファターゼ)、ALT、γ-GTPで値が改善していた。また、総コレステロール、LDLコレステロール、遊離脂肪酸、中性脂肪の値においても改善していた。 Example 4
Two subjects (O, I) were ingested with water containing the composition of the present invention (APAS, O2 / H2 / N2NB water, also referred to as MCP) for 500 months per day for one month, and blood tests were conducted before and after that. It was.
Result / Subject O
When the values before ingestion of water containing the composition of the present invention were compared, the values were improved with ALP (alkaline phosphatase), ALT, and γ-GTP after ingestion. In addition, the values of total cholesterol, LDL cholesterol and free fatty acids were also improved.
・ Subject I
When the values before ingestion of water containing the composition of the present invention were compared, the values were improved with ALP (alkaline phosphatase), ALT, and γ-GTP after ingestion. In addition, the values of total cholesterol, LDL cholesterol, free fatty acid, and neutral fat were also improved.
２人の結果から総合的に判断すると、本発明の組成物を含む水を摂取することにより、肝機能の改善が成されたことから、脂質系の臨床検査値が改善したと考えられる。
Judging comprehensively from the results of two people, it is considered that the clinical value of lipid system was improved because the liver function was improved by ingesting water containing the composition of the present invention.
（実施例５）
ＥＳ細胞の増殖率に関するin vitro実験
ＤＷまたはＡＰＡＳを用いて調製した培地（実施例１のiPS細胞培地と同じ組成）におけるマウスＥＳ細胞の増殖を調べたところ、培養３日目の増殖率を１とした場合に、ＡＰＡＳを用いて培地を調製すると、約１．５倍の増殖率が観察された（図７）。 (Example 5)
In vitro experiment on the proliferation rate of ES cells When the proliferation of mouse ES cells in a medium prepared using DW or APAS (the same composition as the iPS cell medium of Example 1) was examined, the proliferation rate on the third day of culture was 1 In this case, when the medium was prepared using APAS, a growth rate of about 1.5 times was observed (FIG. 7).
ＥＳ細胞の増殖率に関するin vitro実験
ＤＷまたはＡＰＡＳを用いて調製した培地（実施例１のiPS細胞培地と同じ組成）におけるマウスＥＳ細胞の増殖を調べたところ、培養３日目の増殖率を１とした場合に、ＡＰＡＳを用いて培地を調製すると、約１．５倍の増殖率が観察された（図７）。 (Example 5)
In vitro experiment on the proliferation rate of ES cells When the proliferation of mouse ES cells in a medium prepared using DW or APAS (the same composition as the iPS cell medium of Example 1) was examined, the proliferation rate on the third day of culture was 1 In this case, when the medium was prepared using APAS, a growth rate of about 1.5 times was observed (FIG. 7).
（実施例６）
ＭＣＰによる免疫細胞（Ｔ細胞、Ｂ細胞、ＮＫ細胞）の増殖促進に関するin vivo実験
培地に添加した水の種類
実験Ａ
（１）原水（ＭＣＰ作成用原水）
（２）原水＋ガス体（フィルタ濾過無し）
（３）原水＋ガス体＋０．２２μｍフィルタ
ラットの飼育方法 実験動物としてはSDラット（12w）オスを用いた。ラットへの水の与え方は経口と腹腔内投与の両方で行った。腹腔内投与は腹腔内注射により行った。経口投与用には0.22μｍフィルターで滅菌したMCPを用いた。その一方で、コントロール群には、備え付けのDWを投与した。腹腔内投与は10×PBSに対してMCPもしくは培養用DWを1×に希釈して用いた（1ml×2/日）。 水交換と注射は朝と夜の1日2回ずつ行った。
実験期間 試験開始から3日目と7日目で評価した。コントロール群（n=2）と試験群（n=2）を1回の評価で使用した。 (Example 6)
Experiment on the type of water added to the in vivo experimental medium for promoting the proliferation of immune cells (T cells, B cells, NK cells) by MCP
(1) Raw water (raw water for MCP preparation)
(2) Raw water + gas body (no filter filtration)
(3) Raw water + gas body + 0.22 μm filter Rat rearing method SD rat (12w) male was used as an experimental animal. The rats were given water both orally and intraperitoneally. Intraperitoneal administration was performed by intraperitoneal injection. For oral administration, MCP sterilized with a 0.22 μm filter was used. On the other hand, the provided DW was administered to the control group. For intraperitoneal administration, MCP or DW for culture was diluted 1 × with 10 × PBS (1 ml × 2 / day). Water exchange and injection were performed twice daily in the morning and at night.
Experimental period Evaluation was performed on the third and seventh days from the start of the test. A control group (n = 2) and a test group (n = 2) were used in one evaluation.
ＭＣＰによる免疫細胞（Ｔ細胞、Ｂ細胞、ＮＫ細胞）の増殖促進に関するin vivo実験
培地に添加した水の種類
実験Ａ
（１）原水（ＭＣＰ作成用原水）
（２）原水＋ガス体（フィルタ濾過無し）
（３）原水＋ガス体＋０．２２μｍフィルタ
ラットの飼育方法 実験動物としてはSDラット（12w）オスを用いた。ラットへの水の与え方は経口と腹腔内投与の両方で行った。腹腔内投与は腹腔内注射により行った。経口投与用には0.22μｍフィルターで滅菌したMCPを用いた。その一方で、コントロール群には、備え付けのDWを投与した。腹腔内投与は10×PBSに対してMCPもしくは培養用DWを1×に希釈して用いた（1ml×2/日）。 水交換と注射は朝と夜の1日2回ずつ行った。
実験期間 試験開始から3日目と7日目で評価した。コントロール群（n=2）と試験群（n=2）を1回の評価で使用した。 (Example 6)
Experiment on the type of water added to the in vivo experimental medium for promoting the proliferation of immune cells (T cells, B cells, NK cells) by MCP
(1) Raw water (raw water for MCP preparation)
(2) Raw water + gas body (no filter filtration)
(3) Raw water + gas body + 0.22 μm filter Rat rearing method SD rat (12w) male was used as an experimental animal. The rats were given water both orally and intraperitoneally. Intraperitoneal administration was performed by intraperitoneal injection. For oral administration, MCP sterilized with a 0.22 μm filter was used. On the other hand, the provided DW was administered to the control group. For intraperitoneal administration, MCP or DW for culture was diluted 1 × with 10 × PBS (1 ml × 2 / day). Water exchange and injection were performed twice daily in the morning and at night.
Experimental period Evaluation was performed on the third and seventh days from the start of the test. A control group (n = 2) and a test group (n = 2) were used in one evaluation.
実験方法
ラットの心採血（4ml）を行い、血液を15mlチューブに加えた。このチューブに1/5倍量のHetaSep（800μl）を加えてよく混合し、インキュベート（37℃, over 2h）した。その後、赤血球層の分離確認を行った。2時間後に、リンパ球成分がチューブ周辺に、赤血球成分がチューブ中央に凝固したため、この時点でピペットで約2mlのリンパ球成分を分離した。次に、リンパ球成分を15mlチューブに 2ml/sampleで回収、遠心（3,000 rpm, 8 min, RT）した。PBS（RT）で 30μl/sampleに調整してエッペンチューブに30μl/tubeで分注、余りはコントロール用に用いた。IOTest（30μl）を各チューブに加え、ピペッティング（Sample:IOTest = 1:1）した。その後、抗体反応を20min/RTで行い、FACS buffer（1×）で洗浄し、遠心（3,000 rpm, 5 min, RT）した。上清を除去後、遮光下でVersaLyse（500μl/tube）を加え、RTで 10min静置した。その後、総細胞数カウントとFACSによる免疫細胞比率の計測を行った。 Experimental Method Rats were bled (4 ml) and blood was added to a 15 ml tube. To this tube, 1/5 volume of HetaSep (800 μl) was added, mixed well, and incubated (37 ° C., over 2 h). Thereafter, separation of the erythrocyte layer was confirmed. Two hours later, the lymphocyte component coagulated around the tube and the erythrocyte component coagulated at the center of the tube. At this point, about 2 ml of the lymphocyte component was separated with a pipette. Next, lymphocyte components were collected in a 15 ml tube at 2 ml / sample and centrifuged (3,000 rpm, 8 min, RT). PBS (RT) was adjusted to 30 μl / sample and dispensed into an Eppendorf tube at 30 μl / tube, and the remainder was used for control. IOTest (30 μl) was added to each tube and pipetted (Sample: IOTest = 1: 1). Thereafter, the antibody reaction was performed at 20 min / RT, washed with FACS buffer (1 ×), and centrifuged (3,000 rpm, 5 min, RT). After removing the supernatant, VersaLyse (500 μl / tube) was added in the dark and left at RT for 10 min. Thereafter, the total cell count and the immune cell ratio by FACS were measured.
ラットの心採血（4ml）を行い、血液を15mlチューブに加えた。このチューブに1/5倍量のHetaSep（800μl）を加えてよく混合し、インキュベート（37℃, over 2h）した。その後、赤血球層の分離確認を行った。2時間後に、リンパ球成分がチューブ周辺に、赤血球成分がチューブ中央に凝固したため、この時点でピペットで約2mlのリンパ球成分を分離した。次に、リンパ球成分を15mlチューブに 2ml/sampleで回収、遠心（3,000 rpm, 8 min, RT）した。PBS（RT）で 30μl/sampleに調整してエッペンチューブに30μl/tubeで分注、余りはコントロール用に用いた。IOTest（30μl）を各チューブに加え、ピペッティング（Sample:IOTest = 1:1）した。その後、抗体反応を20min/RTで行い、FACS buffer（1×）で洗浄し、遠心（3,000 rpm, 5 min, RT）した。上清を除去後、遮光下でVersaLyse（500μl/tube）を加え、RTで 10min静置した。その後、総細胞数カウントとFACSによる免疫細胞比率の計測を行った。 Experimental Method Rats were bled (4 ml) and blood was added to a 15 ml tube. To this tube, 1/5 volume of HetaSep (800 μl) was added, mixed well, and incubated (37 ° C., over 2 h). Thereafter, separation of the erythrocyte layer was confirmed. Two hours later, the lymphocyte component coagulated around the tube and the erythrocyte component coagulated at the center of the tube. At this point, about 2 ml of the lymphocyte component was separated with a pipette. Next, lymphocyte components were collected in a 15 ml tube at 2 ml / sample and centrifuged (3,000 rpm, 8 min, RT). PBS (RT) was adjusted to 30 μl / sample and dispensed into an Eppendorf tube at 30 μl / tube, and the remainder was used for control. IOTest (30 μl) was added to each tube and pipetted (Sample: IOTest = 1: 1). Thereafter, the antibody reaction was performed at 20 min / RT, washed with FACS buffer (1 ×), and centrifuged (3,000 rpm, 5 min, RT). After removing the supernatant, VersaLyse (500 μl / tube) was added in the dark and left at RT for 10 min. Thereafter, the total cell count and the immune cell ratio by FACS were measured.
結果
FACS解析の結果を図８に示す。また、図９に示すように、MCP投与群でDW投与群を１として比較した場合、T細胞、B細胞、NK細胞の全ての増加が認められた。それぞれの細胞数の増加の割合にはあまり大きな差は認められなかったが、その中でもB細胞のMCP投与群での増加率が大きかった。本実施例より、本発明のＭＣＰは注射剤としても使用できることが示された。 Results The results of FACS analysis are shown in FIG. Further, as shown in FIG. 9, when the MCP administration group was compared with the DW administration group as 1, an increase in all of T cells, B cells, and NK cells was observed. Although there was not much difference in the rate of increase in the number of cells, the rate of increase in the B cell MCP administration group was particularly large. From this example, it was shown that the MCP of the present invention can also be used as an injection.
FACS解析の結果を図８に示す。また、図９に示すように、MCP投与群でDW投与群を１として比較した場合、T細胞、B細胞、NK細胞の全ての増加が認められた。それぞれの細胞数の増加の割合にはあまり大きな差は認められなかったが、その中でもB細胞のMCP投与群での増加率が大きかった。本実施例より、本発明のＭＣＰは注射剤としても使用できることが示された。 Results The results of FACS analysis are shown in FIG. Further, as shown in FIG. 9, when the MCP administration group was compared with the DW administration group as 1, an increase in all of T cells, B cells, and NK cells was observed. Although there was not much difference in the rate of increase in the number of cells, the rate of increase in the B cell MCP administration group was particularly large. From this example, it was shown that the MCP of the present invention can also be used as an injection.
（実施例７）
この実験では、（１）原水:MCP作成用原水、（２）MCP、（３）MCP+ 0.22ミクロンフィルター:MCPを0.22ミクロンフィルターに通したものの3種類の水を、経口投与および腹腔内注射（1ml×2/日、朝、夜）でラットに投与し、7日間の飼育後のラット血液リンパ球中の各画分の量の比較を行った。
試験開始7日後の観察では、どの個体も注射の過剰量投与による腹の膨らみや下痢といった症状は認められず元気だった。 (Example 7)
In this experiment, (1) raw water: MCP preparation raw water, (2) MCP, (3) MCP + 0.22 micron filter: MCP passed through 0.22 micron filter, oral administration and intraperitoneal injection (1 ml X2 / day, morning, night), and the amount of each fraction in rat blood lymphocytes after 7 days of breeding was compared.
Observations 7 days after the start of the study showed no signs of bloating or diarrhea due to overdose injections.
この実験では、（１）原水:MCP作成用原水、（２）MCP、（３）MCP+ 0.22ミクロンフィルター:MCPを0.22ミクロンフィルターに通したものの3種類の水を、経口投与および腹腔内注射（1ml×2/日、朝、夜）でラットに投与し、7日間の飼育後のラット血液リンパ球中の各画分の量の比較を行った。
試験開始7日後の観察では、どの個体も注射の過剰量投与による腹の膨らみや下痢といった症状は認められず元気だった。 (Example 7)
In this experiment, (1) raw water: MCP preparation raw water, (2) MCP, (3) MCP + 0.22 micron filter: MCP passed through 0.22 micron filter, oral administration and intraperitoneal injection (1 ml X2 / day, morning, night), and the amount of each fraction in rat blood lymphocytes after 7 days of breeding was compared.
Observations 7 days after the start of the study showed no signs of bloating or diarrhea due to overdose injections.
結果（7日目） 顕微鏡下での細胞数計測時に（２）および（３）を投与した場合にリンパ球数に有意の増加を認めた（図１０）。このことから、MCPは血中リンパ球数を増加させる作用を持つことが明らかになった。具体的な総リンパ球数は、（１）では、50.2x104、（２）では161x104、（３）では83x104であり、（１）を1とした時の比率は、（２）で3.21、（３）で1.66であった。すなわち、MCPを投与した場合のリンパ球数の増加率は、実験に供した3個体の平均で321%であった。3種類の水を投与した場合のリンパ球内のT細胞、B細胞、NK細胞数、および（１）投与群での細胞数を1とした場合の（２）および（３）投与群での各細胞数の比率を図１１および図１２に示す。図１２によるT細胞、B細胞、NK細胞数の比率は、通常のラットで知られている比率に類似したものであった。図１２によると、MCP投与群でのDW投与群に比較したこれら3種類の細胞数の増加率では、B細胞の増加率が他の2種類の細胞の増加率と比較して大きかった。リンパ球内の各細胞画分のうち、NK細胞数の（１）を基準１とした（２）および（３）での比率を図１２に示す。図１２によると、（２）でのNK細胞増加率は計測した3個体の平均で348%であった。実験（１）と（２）の結果の比較検討 いずれの実験においても、MCPを投与した場合に、ガスを加えていない水（DWあるいはMCP作成用原水）を投与した場合と比べて、血液中にT細胞、B細胞、NK細胞の全てが増加していた。これは、MCPの中核成分であるガス体にリンパ球増殖促進作用のあることを示している。また、両実験とも、それぞれの細胞数の増加の割合にはあまり大きな差を示さなかったが、その中でもB細胞の増加率が大きい特徴を示した。
Results (Day 7) When (2) and (3) were administered at the time of counting the number of cells under a microscope, a significant increase was observed in the number of lymphocytes (FIG. 10). This revealed that MCP has the effect of increasing blood lymphocyte count. Specific total lymphocyte count, in (1), 50.2X10 4, (2) in 161X10 4, a 83X10 4 in (3), the ratio of when the 1 (1), (2) It was 1.66 in 3.21 and (3). That is, the increase rate of the number of lymphocytes when MCP was administered was 321% on average for the three individuals subjected to the experiment. The number of T cells, B cells, and NK cells in lymphocytes when three types of water are administered, and (1) (2) and (3) where the number of cells in the administration group is 1. The ratio of each cell number is shown in FIG. 11 and FIG. The ratio of the number of T cells, B cells, and NK cells according to FIG. 12 was similar to the ratio known in normal rats. According to FIG. 12, in the increase rate of these three types of cells in the MCP administration group compared to the DW administration group, the increase rate of B cells was larger than the increase rate of the other two types of cells. FIG. 12 shows the ratios of (2) and (3) with respect to (1) of the number of NK cells among the respective cell fractions in the lymphocyte. According to FIG. 12, the increase rate of NK cells in (2) was 348% on average for the three individuals measured. Comparison of the results of Experiments (1) and (2) In both experiments, when MCP was administered, in the blood, compared to the case where water (DW or raw water for MCP preparation) was not added. T cells, B cells, and NK cells all increased. This indicates that the gas body, which is the core component of MCP, has a lymphocyte proliferation promoting action. In both experiments, there was not much difference in the rate of increase in the number of cells, but among them, the increase rate of B cells was characteristic.
（実施例８）
実験Ｂ
（１）原水（ＭＣＰ作成用原水）
（２）原水＋ガス体＋０．４５μｍフィルタ
（３）原水＋ガス体＋０．２２μｍフィルタ
実験Ｂでは、フィルタを通さない、原水＋ガス体の区で、原水の３．２１倍の総リンパ球数が観察された（図１３）。このことは、増殖量が約３倍以上になったことを意味する。これに対し、原水＋ガス体をフィルタで濾過した培地では、０．４５μｍフィルタでは１．７６倍、０．２２μｍフィルタで濾過した培地では、１．６６倍の増殖量の増加が認められた。フィルタ濾過により、マイクロバブルが除去されるためにこのような結果になった可能性がある。ガス体の直径は、１０ｎｍ～３０ｎｍである。 (Example 8)
Experiment B
(1) Raw water (raw water for MCP preparation)
(2) Raw water + gas body + 0.45 μm filter (3) Raw water + gas body + 0.22 μm filter In Experiment B, the total number of lymphocytes in the raw water + gas body section, which is not filtered, is 3.21 times the raw water. Was observed (FIG. 13). This means that the amount of proliferation has increased about 3 times or more. In contrast, in the medium in which the raw water + gas body was filtered with a filter, the growth amount was 1.76 times in the 0.45 μm filter and 1.66 times in the medium filtered with the 0.22 μm filter. Filter filtration may result in this because microbubbles are removed. The diameter of the gas body is 10 nm to 30 nm.
実験Ｂ
（１）原水（ＭＣＰ作成用原水）
（２）原水＋ガス体＋０．４５μｍフィルタ
（３）原水＋ガス体＋０．２２μｍフィルタ
実験Ｂでは、フィルタを通さない、原水＋ガス体の区で、原水の３．２１倍の総リンパ球数が観察された（図１３）。このことは、増殖量が約３倍以上になったことを意味する。これに対し、原水＋ガス体をフィルタで濾過した培地では、０．４５μｍフィルタでは１．７６倍、０．２２μｍフィルタで濾過した培地では、１．６６倍の増殖量の増加が認められた。フィルタ濾過により、マイクロバブルが除去されるためにこのような結果になった可能性がある。ガス体の直径は、１０ｎｍ～３０ｎｍである。 (Example 8)
Experiment B
(1) Raw water (raw water for MCP preparation)
(2) Raw water + gas body + 0.45 μm filter (3) Raw water + gas body + 0.22 μm filter In Experiment B, the total number of lymphocytes in the raw water + gas body section, which is not filtered, is 3.21 times the raw water. Was observed (FIG. 13). This means that the amount of proliferation has increased about 3 times or more. In contrast, in the medium in which the raw water + gas body was filtered with a filter, the growth amount was 1.76 times in the 0.45 μm filter and 1.66 times in the medium filtered with the 0.22 μm filter. Filter filtration may result in this because microbubbles are removed. The diameter of the gas body is 10 nm to 30 nm.
（実施例９）
Ｔ細胞、Ｂ細胞、ＮＫ細胞へのガス体投与の影響を測定した。フィルタ無しの系では、Ｂ細胞が最もよく増殖し、次いでＮＫ細胞、Ｔ細胞であった（図９）。 Example 9
The effect of gas body administration on T cells, B cells, and NK cells was measured. In the unfiltered system, B cells proliferated best, followed by NK cells and T cells (FIG. 9).
Ｔ細胞、Ｂ細胞、ＮＫ細胞へのガス体投与の影響を測定した。フィルタ無しの系では、Ｂ細胞が最もよく増殖し、次いでＮＫ細胞、Ｔ細胞であった（図９）。 Example 9
The effect of gas body administration on T cells, B cells, and NK cells was measured. In the unfiltered system, B cells proliferated best, followed by NK cells and T cells (FIG. 9).
（実施例１０）
原水＋ガス投与個体でのＮＫ細胞数を比較した。その結果を図１２に示す。図１２に示すように、フィルタなしの場合、原水の約３．５倍増殖した。フィルタありの場合は、０．４５μｍフィルタでは１．９３倍、０．２２μｍフィルタでは、１．６４倍の増殖を示した。 (Example 10)
The number of NK cells in raw water + gas administered individuals was compared. The result is shown in FIG. As shown in FIG. 12, when there was no filter, the raw water grew about 3.5 times. In the case with a filter, the 0.45 μm filter showed 1.93 times the growth, and the 0.22 μm filter showed 1.64 times the growth.
原水＋ガス投与個体でのＮＫ細胞数を比較した。その結果を図１２に示す。図１２に示すように、フィルタなしの場合、原水の約３．５倍増殖した。フィルタありの場合は、０．４５μｍフィルタでは１．９３倍、０．２２μｍフィルタでは、１．６４倍の増殖を示した。 (Example 10)
The number of NK cells in raw water + gas administered individuals was compared. The result is shown in FIG. As shown in FIG. 12, when there was no filter, the raw water grew about 3.5 times. In the case with a filter, the 0.45 μm filter showed 1.93 times the growth, and the 0.22 μm filter showed 1.64 times the growth.
（実施例１１）
81歳女性両下肢閉塞性動脈硬化症に対する効果
平成23年春より両膝の骨壊死を伴った変形性膝関節症で通院していたが、平成25年秋より主に右下肢に浮腫を来すようになった。平成26年2月に右下肢血栓血管炎で入院で血栓融解療法を行った。平成26年3月よりミトコンドリア水を500ml/day飲用を開始した。
その結果、浮腫の軽減、炎症の改善を認めた。動脈硬化の指標であるABI値（足関節/上腕血圧比）の変化においてミトコンドリア水飲用後左側の値の改善が認められた。baPWV（ 動脈コンプライアンスを評価する指標である上腕－足首間脈波伝播速度）の推移では両側に改善が認められた（表３および図１４～１５参照）。
併用薬は、三月のミトコンドリア水飲用以前からの抗凝固製剤は無く、5月より抗血小板凝集剤の投与を始めたので、この度の2014年5月24日のbaPWVの改善についてはミトコンドリア水の影響が関与している可能性が大であると考えられる。すなわち、この症例の動脈硬化改善にはミトコンドリア水の効果が関与していることが示唆された。 (Example 11)
Effect on obstructive arteriosclerosis of both lower limbs in an 81-year-old woman. He had been hospitalized for osteoarthritis of the knee with osteonecrosis of both knees since the spring of 2011, but seems to cause edema mainly in the right lower limb since the fall of 2013. Became. In February 2014, thrombolytic therapy was performed at the hospital for right limb thromboangiitis. In March 2014, we began drinking mitochondrial water at 500ml / day.
As a result, edema was reduced and inflammation was improved. In the change of ABI value (ankle / brachial blood pressure ratio) which is an index of arteriosclerosis, the improvement of the value on the left side after drinking mitochondrial water was observed. Improvements were observed on both sides in the transition of baPWV (upper arm-ankle pulse wave velocity, which is an index for evaluating arterial compliance) (see Table 3 and FIGS. 14 to 15).
There was no anticoagulant preparation before the mitochondrial water drink in March, and the administration of antiplatelet aggregating agent was started in May. Concerning the improvement of baPWV of May 24, 2014, It is likely that the impact is involved. That is, it was suggested that the effect of mitochondrial water is involved in the improvement of arteriosclerosis in this case.
81歳女性両下肢閉塞性動脈硬化症に対する効果
平成23年春より両膝の骨壊死を伴った変形性膝関節症で通院していたが、平成25年秋より主に右下肢に浮腫を来すようになった。平成26年2月に右下肢血栓血管炎で入院で血栓融解療法を行った。平成26年3月よりミトコンドリア水を500ml/day飲用を開始した。
その結果、浮腫の軽減、炎症の改善を認めた。動脈硬化の指標であるABI値（足関節/上腕血圧比）の変化においてミトコンドリア水飲用後左側の値の改善が認められた。baPWV（ 動脈コンプライアンスを評価する指標である上腕－足首間脈波伝播速度）の推移では両側に改善が認められた（表３および図１４～１５参照）。
併用薬は、三月のミトコンドリア水飲用以前からの抗凝固製剤は無く、5月より抗血小板凝集剤の投与を始めたので、この度の2014年5月24日のbaPWVの改善についてはミトコンドリア水の影響が関与している可能性が大であると考えられる。すなわち、この症例の動脈硬化改善にはミトコンドリア水の効果が関与していることが示唆された。 (Example 11)
Effect on obstructive arteriosclerosis of both lower limbs in an 81-year-old woman. He had been hospitalized for osteoarthritis of the knee with osteonecrosis of both knees since the spring of 2011, but seems to cause edema mainly in the right lower limb since the fall of 2013. Became. In February 2014, thrombolytic therapy was performed at the hospital for right limb thromboangiitis. In March 2014, we began drinking mitochondrial water at 500ml / day.
As a result, edema was reduced and inflammation was improved. In the change of ABI value (ankle / brachial blood pressure ratio) which is an index of arteriosclerosis, the improvement of the value on the left side after drinking mitochondrial water was observed. Improvements were observed on both sides in the transition of baPWV (upper arm-ankle pulse wave velocity, which is an index for evaluating arterial compliance) (see Table 3 and FIGS. 14 to 15).
There was no anticoagulant preparation before the mitochondrial water drink in March, and the administration of antiplatelet aggregating agent was started in May. Concerning the improvement of baPWV of May 24, 2014, It is likely that the impact is involved. That is, it was suggested that the effect of mitochondrial water is involved in the improvement of arteriosclerosis in this case.
（実施例１２）
被験者：８１歳女性 II型糖尿病
平成２０年２月II型糖尿病発症。
内服薬、生活指導が中心で治療経過を診ていた。
内服薬はスルフォニル製剤であるダオニール1.25ｍｇ（３錠分３後）および食後過血糖改善剤ベイスン0.2ｍｇ（３錠分３前）を内服。
合併症として高脂血症、軽度腎機能低下を認めていた。
高脂血症にはアトルバスタチンカルシウム製剤１０ｍｇ眠前に内服されていた。 Example 12
Subject: 81-year-old female, type II diabetes February 2008, onset of type II diabetes.
She was examining the course of treatment with a focus on internal medicine and lifestyle guidance.
For oral use, 1.25 mg (3 tablets after 3 tablets) of sulfonyl preparation and 0.2 mg (3 tablets before 3 tablets) postprandial hyperglycemia improving agent are taken.
Complications included hyperlipidemia and mild renal function decline.
For hyperlipidemia, 10 mg of atorvastatin calcium preparation was taken before sleep.
被験者：８１歳女性 II型糖尿病
平成２０年２月II型糖尿病発症。
内服薬、生活指導が中心で治療経過を診ていた。
内服薬はスルフォニル製剤であるダオニール1.25ｍｇ（３錠分３後）および食後過血糖改善剤ベイスン0.2ｍｇ（３錠分３前）を内服。
合併症として高脂血症、軽度腎機能低下を認めていた。
高脂血症にはアトルバスタチンカルシウム製剤１０ｍｇ眠前に内服されていた。 Example 12
Subject: 81-year-old female, type II diabetes February 2008, onset of type II diabetes.
She was examining the course of treatment with a focus on internal medicine and lifestyle guidance.
For oral use, 1.25 mg (3 tablets after 3 tablets) of sulfonyl preparation and 0.2 mg (3 tablets before 3 tablets) postprandial hyperglycemia improving agent are taken.
Complications included hyperlipidemia and mild renal function decline.
For hyperlipidemia, 10 mg of atorvastatin calcium preparation was taken before sleep.
ミトコンドリア水投与の効果
５月８日より５月１６日まで、さらに９月１日より６日まで、入院のうえウロキナーゼ点滴静注による血栓融解療法を行った。上記の間を含め現在に至るまで、ミトコンドリア水500ｍｌ／日の飲用を継続した。ＨｂＡ１ｃ（赤血球の中で体内に酸素を運ぶ役目のヘモグロビンと、血液中のブドウ糖が結合したもの）の値が、飲用前2014/1/8 7.8→2014/9/1 5.8と劇的に改善した（表４および図１６）。内服量は同じで４年間変化がなかった。ミトコンドリア水の影響によりII型糖尿病が改善されたと考えられる。 Effect of mitochondrial water administration From May 8th to May 16th and from September 1st to 6th, thrombolytic therapy by intravenous infusion of urokinase was performed. Until the present time, including the above period, drinking of mitochondrial water 500 ml / day was continued. The value of HbA1c (hemoglobin that carries oxygen into the body in the red blood cells and glucose in the blood) was dramatically improved from 2014/1/8 7.8 to 2014/9/1 5.8 before drinking (Table 4 and FIG. 16). The internal dose was the same and remained unchanged for 4 years. It is thought that type II diabetes was improved by the effect of mitochondrial water.
５月８日より５月１６日まで、さらに９月１日より６日まで、入院のうえウロキナーゼ点滴静注による血栓融解療法を行った。上記の間を含め現在に至るまで、ミトコンドリア水500ｍｌ／日の飲用を継続した。ＨｂＡ１ｃ（赤血球の中で体内に酸素を運ぶ役目のヘモグロビンと、血液中のブドウ糖が結合したもの）の値が、飲用前2014/1/8 7.8→2014/9/1 5.8と劇的に改善した（表４および図１６）。内服量は同じで４年間変化がなかった。ミトコンドリア水の影響によりII型糖尿病が改善されたと考えられる。 Effect of mitochondrial water administration From May 8th to May 16th and from September 1st to 6th, thrombolytic therapy by intravenous infusion of urokinase was performed. Until the present time, including the above period, drinking of mitochondrial water 500 ml / day was continued. The value of HbA1c (hemoglobin that carries oxygen into the body in the red blood cells and glucose in the blood) was dramatically improved from 2014/1/8 7.8 to 2014/9/1 5.8 before drinking (Table 4 and FIG. 16). The internal dose was the same and remained unchanged for 4 years. It is thought that type II diabetes was improved by the effect of mitochondrial water.
（実施例１３)
ヒトが飲用した場合のＭＣＰが生体に与える効果を明らかにすることを目的として、ヒトでのＭＣＰ飲用試験を実施した。被験者は以下のとおりである。
１. ５０歳 男性、 ２. ４５歳 男性、 ３. ６０歳 女性、 ４. ３５歳 女性、 ５. ６３歳 女性、 ６. ６６歳 男性、 ７ ２５歳 女性
各々の参加者は、通常と変わらない生活を送りながら、ＭＣＰを0,5-1,0Ｌ／日、４週間飲用した。参加者は、飲用開始前に血液を採取され、飲用開始後４週間目および８週間目に再び血液を採取された。検査は、白血球、肝機能、脂質関連、糖尿病関連のそれぞれの項目について行った。その結果、肝機能、コレステロール値、中性脂肪値、糖尿病関連の数値などの幅広い項目の数値に改善が認められた。数値の改善は異常値で顕著であるが、正常値についても、より理想的な数値への改善傾向が認められた。とくに数値の改善が顕著に認められる項目はコレステロール値であり、検査したほぼ全ての被験者で改善が認められた。また、ＨＤＬ値について、過去２０年間の健診時に一度も40を上回ったことがない被験者６の数値が４５となったことから、現在、特効薬のないＨＤＬ値の上昇にもＭＣＰが寄与する可能性がある（表５）。 (Example 13)
In order to clarify the effects of MCP on humans when taken by humans, a human MCP drinking test was conducted. The subjects are as follows.
1. 50-year-old male, 2. 45-year-old male, 3. 60-year-old female, 4. 35-year-old female, 5. 63-year-old female, 6. 66-year-old male, 7 25-year-old female Each participant is the same as usual While living a life, MCP was drunk 0,5-1,0 L / day for 4 weeks. Participants were blood collected before the start of drinking and blood was collected again at 4 and 8 weeks after starting drinking. Tests were performed on leukocytes, liver function, lipid-related and diabetes-related items. As a result, improvements were observed in a wide range of values such as liver function, cholesterol levels, triglyceride levels, and diabetes-related values. Although the improvement of the numerical value is remarkable in the abnormal value, the normal value also showed a tendency to improve to a more ideal numerical value. In particular, the item in which the numerical improvement was noticeable was the cholesterol level, and almost all the subjects examined showed improvement. In addition, as for the HDL value, since the numerical value of the subject 6 who has never exceeded 40 at the time of the medical examination for the past 20 years became 45, MCP can also contribute to the increase of the HDL value without a specific medicine at present. (Table 5).
ヒトが飲用した場合のＭＣＰが生体に与える効果を明らかにすることを目的として、ヒトでのＭＣＰ飲用試験を実施した。被験者は以下のとおりである。
１. ５０歳 男性、 ２. ４５歳 男性、 ３. ６０歳 女性、 ４. ３５歳 女性、 ５. ６３歳 女性、 ６. ６６歳 男性、 ７ ２５歳 女性
各々の参加者は、通常と変わらない生活を送りながら、ＭＣＰを0,5-1,0Ｌ／日、４週間飲用した。参加者は、飲用開始前に血液を採取され、飲用開始後４週間目および８週間目に再び血液を採取された。検査は、白血球、肝機能、脂質関連、糖尿病関連のそれぞれの項目について行った。その結果、肝機能、コレステロール値、中性脂肪値、糖尿病関連の数値などの幅広い項目の数値に改善が認められた。数値の改善は異常値で顕著であるが、正常値についても、より理想的な数値への改善傾向が認められた。とくに数値の改善が顕著に認められる項目はコレステロール値であり、検査したほぼ全ての被験者で改善が認められた。また、ＨＤＬ値について、過去２０年間の健診時に一度も40を上回ったことがない被験者６の数値が４５となったことから、現在、特効薬のないＨＤＬ値の上昇にもＭＣＰが寄与する可能性がある（表５）。 (Example 13)
In order to clarify the effects of MCP on humans when taken by humans, a human MCP drinking test was conducted. The subjects are as follows.
1. 50-year-old male, 2. 45-year-old male, 3. 60-year-old female, 4. 35-year-old female, 5. 63-year-old female, 6. 66-year-old male, 7 25-year-old female Each participant is the same as usual While living a life, MCP was drunk 0,5-1,0 L / day for 4 weeks. Participants were blood collected before the start of drinking and blood was collected again at 4 and 8 weeks after starting drinking. Tests were performed on leukocytes, liver function, lipid-related and diabetes-related items. As a result, improvements were observed in a wide range of values such as liver function, cholesterol levels, triglyceride levels, and diabetes-related values. Although the improvement of the numerical value is remarkable in the abnormal value, the normal value also showed a tendency to improve to a more ideal numerical value. In particular, the item in which the numerical improvement was noticeable was the cholesterol level, and almost all the subjects examined showed improvement. In addition, as for the HDL value, since the numerical value of the subject 6 who has never exceeded 40 at the time of the medical examination for the past 20 years became 45, MCP can also contribute to the increase of the HDL value without a specific medicine at present. (Table 5).
（実施例１４）
コマツナ（Brassica rapa var. perviridis、わかみ）を用いて植物への影響を調べた。
栽培期間 ２０１４年３月７日～２０１４年４月７日（３２日間）
方法：コンテナに脱塩素水を３０Ｌ入れ、水耕栽培肥料タンクミックスＦ＆Ｂ（表６、ＯＡＴアグリオ株式会社製）を溶解させた。培養液は、栽培期間中、ＥＣ２．４に調整し、２４時間エアレーションを行った。 (Example 14)
The effect on plants was examined using Komatsuna (Brassica rapa var. Perviridis).
Cultivation period March 7, 2014 to April 7, 2014 (32 days)
Method: 30 L of dechlorinated water was put into a container, and hydroponics fertilizer tank mix F & B (Table 6, manufactured by OAT Agrio Co., Ltd.) was dissolved. The culture solution was adjusted to EC 2.4 during the cultivation period and aerated for 24 hours.
コマツナ（Brassica rapa var. perviridis、わかみ）を用いて植物への影響を調べた。
栽培期間 ２０１４年３月７日～２０１４年４月７日（３２日間）
方法：コンテナに脱塩素水を３０Ｌ入れ、水耕栽培肥料タンクミックスＦ＆Ｂ（表６、ＯＡＴアグリオ株式会社製）を溶解させた。培養液は、栽培期間中、ＥＣ２．４に調整し、２４時間エアレーションを行った。 (Example 14)
The effect on plants was examined using Komatsuna (Brassica rapa var. Perviridis).
Cultivation period March 7, 2014 to April 7, 2014 (32 days)
Method: 30 L of dechlorinated water was put into a container, and hydroponics fertilizer tank mix F & B (Table 6, manufactured by OAT Agrio Co., Ltd.) was dissolved. The culture solution was adjusted to EC 2.4 during the cultivation period and aerated for 24 hours.
ウレタンに種子を播種し、グロスチャンバーにて育苗１０日後、各コンテナに９個体ずつ定植し、３反復（３コンテナ）試験を行った。定植３週間後に収穫した。
処理区 対照区、空気マイクロバブル（ＭＢ）区、ＡＰＡＳ＋ＭＢ区
空気ＭＢ区では、空気ＭＢを２０分間、ＡＰＡＳ＋ＭＢ区もＭＢ化した気体を２０分間、培養中に、定植１週間後から週に３回、発生を行った（計６回の処理）
調査項目 草丈、最大葉長、最大根長、地上部新鮮重、地下部新鮮重、地上部乾物重、地下部乾物重 Seeds were seeded on urethane, and after 10 days of seedling raising in a gross chamber, 9 individuals were planted in each container, and three repetitions (3 containers) were tested. Harvested 3 weeks after planting.
Treatment group Control group, air microbubble (MB) group, APAS + MB group In the air MB group, the air MB is converted into MB for 20 minutes, and the APAS + MB group is also converted into MB for 20 minutes. Occurred (total of 6 treatments)
Survey item Plant height, maximum leaf length, maximum root length, aboveground fresh weight, belowground fresh weight, aboveground dry weight, belowground dry weight
処理区 対照区、空気マイクロバブル（ＭＢ）区、ＡＰＡＳ＋ＭＢ区
空気ＭＢ区では、空気ＭＢを２０分間、ＡＰＡＳ＋ＭＢ区もＭＢ化した気体を２０分間、培養中に、定植１週間後から週に３回、発生を行った（計６回の処理）
調査項目 草丈、最大葉長、最大根長、地上部新鮮重、地下部新鮮重、地上部乾物重、地下部乾物重 Seeds were seeded on urethane, and after 10 days of seedling raising in a gross chamber, 9 individuals were planted in each container, and three repetitions (3 containers) were tested. Harvested 3 weeks after planting.
Treatment group Control group, air microbubble (MB) group, APAS + MB group In the air MB group, the air MB is converted into MB for 20 minutes, and the APAS + MB group is also converted into MB for 20 minutes. Occurred (total of 6 treatments)
Survey item Plant height, maximum leaf length, maximum root length, aboveground fresh weight, belowground fresh weight, aboveground dry weight, belowground dry weight
結果を表７に示す。草丈、最大葉長、最大根長、地上部乾物重および地下部乾物重では、処理区間で有意差は認められなかった。しかしながら、APAS+MB区の地上部新鮮重および地下部新鮮重では、対照区およびMB区と比較して、有意に増加した。APAS+MBで、生育促進効果が認められた。
The results are shown in Table 7. There were no significant differences between the treatment sections for plant height, maximum leaf length, maximum root length, aboveground dry weight, and belowground dry weight. However, the ground fresh weight and the underground fresh weight of APAS + MB increased significantly compared with the control and MB. APAS + MB showed a growth promoting effect.
（実施例１５）
MCP添加培地中での繊維芽細胞増殖促進作用に関する実験
培地の調製
１．コントロール
ＤＷ ２８５ｇ
ＤＭＥＭ粉末 ３ｇ
ＮａＨＣＯ３ １．１１ｇ
全体 ３００ｍｌ
上記を混合後、０．４５μｍフィルタでろ過滅菌した。１０％ＦＢＳ（ウシ胎児血清）は１０ｍｌずつまたは５０ｍｌずつ調製し、培地に１０分の１容量添加した。
２．１×ＭＣＰ
ＭＣＰ ４７．５ｍｌ
ＤＭＥＭ粉末 ０．５ｇ
ＮａＨＣＯ３ ０．１８５ｇ
全体 ５０ｍｌ
上記を混合後、フィルタでろ過した。上記溶液（ＤＭＥＭ（ＭＣＰ）を９ｍｌとＦＢＳ１ｍｌを混合して培養に使用した。
３．２×ＤＭＥＭ＋ＭＣＰ
ＤＷ ９５ｍｌ
ＤＭＥＭ粉末 ２ｇ
ＮａＨＣＯ３ ０．７４ｇ
全体 １００ｍｌ
上記を混合後、フィルタろ過した。その後、ＭＣＰで２倍に希釈した。
ろ過ＭＣＰ ４．５ｍｌ
２×ＤＭＥＭ ４．５ｍｌ
ＦＢＳ １ｍｌ
合計 １０ｍｌ (Example 15)
1. Preparation of experimental medium for promoting fibroblast proliferation in medium supplemented with MCP Control DW 285g
DMEM powder 3g
NaHCO3 1.11g
300ml total
After mixing the above, it was sterilized by filtration with a 0.45 μm filter. 10% FBS (fetal bovine serum) was prepared in 10 ml or 50 ml, and 1/10 volume was added to the medium.
2.1 x MCP
MCP 47.5ml
DMEM powder 0.5g
NaHCO 3 0.185 g
Whole 50ml
After mixing the above, it was filtered with a filter. 9 ml of the above solution (DMEM (MCP) and 1 ml of FBS were mixed and used for culture.
3.2 × DMEM + MCP
DW 95ml
DMEM powder 2g
NaHCO 3 0.74 g
Whole 100ml
The above was mixed and then filtered. Thereafter, it was diluted 2-fold with MCP.
Filtration MCP 4.5ml
2 x DMEM 4.5ml
FBS 1ml
10ml total
MCP添加培地中での繊維芽細胞増殖促進作用に関する実験
培地の調製
１．コントロール
ＤＷ ２８５ｇ
ＤＭＥＭ粉末 ３ｇ
ＮａＨＣＯ３ １．１１ｇ
全体 ３００ｍｌ
上記を混合後、０．４５μｍフィルタでろ過滅菌した。１０％ＦＢＳ（ウシ胎児血清）は１０ｍｌずつまたは５０ｍｌずつ調製し、培地に１０分の１容量添加した。
２．１×ＭＣＰ
ＭＣＰ ４７．５ｍｌ
ＤＭＥＭ粉末 ０．５ｇ
ＮａＨＣＯ３ ０．１８５ｇ
全体 ５０ｍｌ
上記を混合後、フィルタでろ過した。上記溶液（ＤＭＥＭ（ＭＣＰ）を９ｍｌとＦＢＳ１ｍｌを混合して培養に使用した。
３．２×ＤＭＥＭ＋ＭＣＰ
ＤＷ ９５ｍｌ
ＤＭＥＭ粉末 ２ｇ
ＮａＨＣＯ３ ０．７４ｇ
全体 １００ｍｌ
上記を混合後、フィルタろ過した。その後、ＭＣＰで２倍に希釈した。
ろ過ＭＣＰ ４．５ｍｌ
２×ＤＭＥＭ ４．５ｍｌ
ＦＢＳ １ｍｌ
合計 １０ｍｌ (Example 15)
1. Preparation of experimental medium for promoting fibroblast proliferation in medium supplemented with MCP Control DW 285g
DMEM powder 3g
NaHCO3 1.11g
300ml total
After mixing the above, it was sterilized by filtration with a 0.45 μm filter. 10% FBS (fetal bovine serum) was prepared in 10 ml or 50 ml, and 1/10 volume was added to the medium.
2.1 x MCP
MCP 47.5ml
DMEM powder 0.5g
NaHCO 3 0.185 g
Whole 50ml
After mixing the above, it was filtered with a filter. 9 ml of the above solution (DMEM (MCP) and 1 ml of FBS were mixed and used for culture.
3.2 × DMEM + MCP
DW 95ml
DMEM powder 2g
NaHCO 3 0.74 g
Whole 100ml
The above was mixed and then filtered. Thereafter, it was diluted 2-fold with MCP.
Filtration MCP 4.5ml
2 x DMEM 4.5ml
FBS 1ml
10ml total
凍結保存されたマウス繊維芽（MEF)細胞を起こしてから継代を３回以上行ってから本実験に用いた。上述のように終濃度１０％FBSで調製したDMEM（High Glucose）で培養した。培地交換は３日に１回行い、コンフルエントの時点で継代を行った。
The mouse fibroblast (MEF) cells that had been cryopreserved were generated and then passaged three or more times before use in this experiment. As described above, the cells were cultured in DMEM (High Glucose) prepared with a final concentration of 10% FBS. The medium was exchanged once every 3 days, and subculture was performed at the time of confluence.
本実験はまず、１２ウェルプレートにMEF細胞を１ウェルに１万細胞となるように継代を行った。継代翌日、MEF細胞をPBSで一度洗浄し、DWとMCPを用いて終濃度１０％FBSとPenicillin/Streptmycinで調製したDMEM（High Glucose)を加えた。MCPを加えた日を０日目として、３日目に解析を行った。培地はMCPを加えてから毎日交換した。
In this experiment, first, the MEF cells were subcultured into 12-well plates so that there were 10,000 cells per well. The day after the passage, the MEF cells were washed once with PBS, and DMEM (High Glucose) prepared with 10% FBS and Penicillin / Streptmycin was added using DW and MCP. The analysis was performed on the third day, with the day on which MCP was added as the zeroth day. The medium was changed daily after adding MCP.
細胞数測定は０．２５％トリプシンを加え、トリパンブルー染色によって生細胞のみを数えた。
培養実験は、２連で行い、２日間と８日間の培養を行った。これら２回の実験の結果から、MCPには、繊維芽細胞増殖促進作用があることが示唆された（図１８）。その増殖率はDWを培地に加えた場合と比べて１２５～２００％であった（表８）。 For cell count, 0.25% trypsin was added and only live cells were counted by trypan blue staining.
The culture experiment was performed in duplicate, and the culture was performed for 2 days and 8 days. The results of these two experiments suggested that MCP has a fibroblast proliferation promoting action (FIG. 18). The growth rate was 125-200% compared to the case where DW was added to the medium (Table 8).
培養実験は、２連で行い、２日間と８日間の培養を行った。これら２回の実験の結果から、MCPには、繊維芽細胞増殖促進作用があることが示唆された（図１８）。その増殖率はDWを培地に加えた場合と比べて１２５～２００％であった（表８）。 For cell count, 0.25% trypsin was added and only live cells were counted by trypan blue staining.
The culture experiment was performed in duplicate, and the culture was performed for 2 days and 8 days. The results of these two experiments suggested that MCP has a fibroblast proliferation promoting action (FIG. 18). The growth rate was 125-200% compared to the case where DW was added to the medium (Table 8).
実験結果に認められた多少のばらつきは、実験に用いた細胞のロットの違いによるもの、あるいは実験条件の揺らぎなどに起因すると考えられる。
Some variation observed in the experimental results may be due to differences in the lot of cells used in the experiment or fluctuations in the experimental conditions.
繊維芽細胞は上皮でのコラーゲン、エラスチン、ヒアルロン酸などを作り出す働きがある。また、コラーゲンを繊維束にし、真皮構造を形成する役目を持つ。紫外線を大量に浴びたり老化が進むと、規則性をもった繊維が生成できなくなり、シワやたるみなどが生じる。繊維芽細胞増殖機能を持つMCPが、このような皮膚の老化に対する有効な抑制効果を示すことが期待される。
Fibroblasts have the function of producing collagen, elastin, hyaluronic acid, etc. in the epithelium. In addition, collagen has a role of forming a dermal structure by forming a fiber bundle. When a large amount of ultraviolet light is applied or aging progresses, regular fibers cannot be produced, and wrinkles and sagging occur. MCP having a fibroblast proliferation function is expected to show an effective inhibitory effect on such skin aging.
（実施例１６）
膝に炎症を起こした６３歳女性は約２年に渡って炎症が続いていたが、ＭＣＰを毎日５００ｍｌ～１Ｌ飲用することで１カ月目には炎症が大幅に改善され、３か月目には炎症がほぼ消失した（図１９）。ＭＣＰを飲用する量としては、１日１５０ｍｌ以上が好ましく、より好ましくは３００ｍｌ以上、さらに好ましくは５００ｍｌ以上、よりさらに好ましくは１Ｌ以上である。１日に摂取する水分を全てＭＣＰとしてもよい。 (Example 16)
A 63-year-old woman who had inflammation in the knee had been inflamed for about 2 years, but drinking 500 ml to 1 L of MCP every day drastically improved inflammation in the first month. Inflammation almost disappeared (FIG. 19). The amount of MCP consumed is preferably 150 ml or more, more preferably 300 ml or more, still more preferably 500 ml or more, and still more preferably 1 L or more. All the water taken in a day may be MCP.
膝に炎症を起こした６３歳女性は約２年に渡って炎症が続いていたが、ＭＣＰを毎日５００ｍｌ～１Ｌ飲用することで１カ月目には炎症が大幅に改善され、３か月目には炎症がほぼ消失した（図１９）。ＭＣＰを飲用する量としては、１日１５０ｍｌ以上が好ましく、より好ましくは３００ｍｌ以上、さらに好ましくは５００ｍｌ以上、よりさらに好ましくは１Ｌ以上である。１日に摂取する水分を全てＭＣＰとしてもよい。 (Example 16)
A 63-year-old woman who had inflammation in the knee had been inflamed for about 2 years, but drinking 500 ml to 1 L of MCP every day drastically improved inflammation in the first month. Inflammation almost disappeared (FIG. 19). The amount of MCP consumed is preferably 150 ml or more, more preferably 300 ml or more, still more preferably 500 ml or more, and still more preferably 1 L or more. All the water taken in a day may be MCP.
（実施例１７）
ペットにＭＣＰを毎日３０日間飲用水として飲用させたところ、表９のように、疾患の指標数値の改善が認められた。 (Example 17)
When pets were allowed to drink MCP as drinking water every day for 30 days, as shown in Table 9, improvements in disease index values were observed.
ペットにＭＣＰを毎日３０日間飲用水として飲用させたところ、表９のように、疾患の指標数値の改善が認められた。 (Example 17)
When pets were allowed to drink MCP as drinking water every day for 30 days, as shown in Table 9, improvements in disease index values were observed.
Claims (19)
- 水素0.45～0.55ppm、酸素10～12.5ppm、窒素7～8ppm含有する、粒径３０マイクロメートル以下のマイクロバブルおよび／またはナノバブルを有効成分として含有する組成物。 A composition containing microbubbles and / or nanobubbles having a particle size of 30 micrometers or less, containing 0.45 to 0.55 ppm hydrogen, 10 to 12.5 ppm oxygen, and 7 to 8 ppm nitrogen.
- 請求項１の組成物を有効成分として含有するミトコンドリア活性化組成物。 A mitochondrial activation composition comprising the composition of claim 1 as an active ingredient.
- 請求項１又は２の組成物を有効成分として含有する細胞増殖促進剤。 A cell growth promoter comprising the composition of claim 1 or 2 as an active ingredient.
- 請求項１～３のいずれか１項に記載の組成物又は剤を有効成分として含有する細胞保存液。 A cell preservation solution containing the composition or agent according to any one of claims 1 to 3 as an active ingredient.
- 請求項１～４のいずれか１項に記載の組成物、剤又は液を有効成分として含有する凍結保存液。 A cryopreservation solution containing the composition, agent or solution according to any one of claims 1 to 4 as an active ingredient.
- 請求項１～５のいずれか１の組成物、剤又は液を含有する飲食物。 Food or drink containing the composition, agent or liquid according to any one of claims 1 to 5.
- 請求項１～５のいずれか１の組成物、剤又は液で処理した原材料を使用して製造された飲食物。 Food or drink produced using raw materials treated with the composition, agent or liquid according to any one of claims 1 to 5.
- 請求項１～５のいずれか１の組成物、剤又は液を用いてミトコンドリアを活性化する方法。 A method for activating mitochondria using the composition, agent or solution according to any one of claims 1 to 5.
- 請求項１～５のいずれか１の組成物、剤又は液を用いて細胞増殖を促進する方法。 A method for promoting cell proliferation using the composition, agent or solution of any one of claims 1 to 5.
- 請求項１～５のいずれか１の組成物、剤又は液を用いて細胞を保存する方法。 A method for preserving cells using the composition, agent or solution according to any one of claims 1 to 5.
- 請求項１～５のいずれか１の組成物、剤又は液を用いて凍結保存する方法。 A method for cryopreserving the composition, agent or solution according to any one of claims 1 to 5.
- 請求項１～５のいずれか１の組成物、剤又は液を用いて飲食物またはその原材料を処理する工程を含む、飲食物の製造方法。 A method for producing a food or drink, comprising a step of treating the food or drink or its raw materials using the composition, agent or liquid according to any one of claims 1 to 5.
- 請求項１の組成物を用いて調製した細胞培養用培地。 A cell culture medium prepared using the composition of claim 1.
- 請求項１の組成物を用いて調製した植物水耕栽培用溶液。 A solution for plant hydroponics prepared using the composition of claim 1.
- 請求項１３の培地を用いて動物細胞を増殖させる方法。 A method for growing animal cells using the medium of claim 13.
- 動物細胞が免疫系細胞である、請求項１５の動物細胞を増殖させる方法。 The method of proliferating an animal cell according to claim 15, wherein the animal cell is an immune system cell.
- 請求項１もしくは２に記載の組成物、または請求項３に記載の細胞増殖促進剤を有効成分とする動脈硬化症の予防・治療剤。 A prophylactic / therapeutic agent for arteriosclerosis comprising the composition according to claim 1 or 2 or the cell growth promoter according to claim 3 as an active ingredient.
- 請求項１もしくは２に記載の組成物、または請求項３に記載の細胞増殖促進剤を有効成分とする糖尿病の予防・治療剤。 A prophylactic / therapeutic agent for diabetes comprising the composition according to claim 1 or 2 or the cell growth promoter according to claim 3 as an active ingredient.
- 請求項１または２に記載の組成物を含む注射液。 An injection solution comprising the composition according to claim 1 or 2.
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| US15/193,176 US20170056438A1 (en) | 2014-10-17 | 2016-06-27 | Nanobubble-containing composition and use thereof |
| HK16112319.1A HK1223975A1 (en) | 2013-12-27 | 2016-10-26 | Nanobubble-containing composition and use thereof |
| US16/161,192 US20190046563A1 (en) | 2013-12-27 | 2018-10-16 | Nanobubble-containing composition and use thereof |
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| JP2016063804A (en) | 2016-04-28 |
| HK1223975A1 (en) | 2017-08-11 |
| CN105593363A (en) | 2016-05-18 |
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