Ultrafine bubble-containing solution, beverages containing the same, and pharmaceuticals

Abstract

[Problem] Because ultrafine bubbles can persist for a long period of time, it is also possible to maintain the antioxidant capacity thereof for a long period of time. However, the effect that ultrafine bubbles of various gases have on active oxygen scavenging is unknown, so this problem was studied. [Solution] By means of this ultrafine bubble-containing solution, this beverage containing said solution, and this drug, it is possible to provide a ultrafine bubble-containing solution, a beverage containing this, and a drug which have active oxygen scavenging capacity, which have a cytotoxic effect on cancer cells and an antitumor effect on cancer-bearing mice, and which contain carbon dioxide and / or hydrogen gas inside of the ultrafine bubbles.

Description

[Technical Field]

[0001] The present invention relates to an ultrafine bubble-containing solution, and a beverage and pharmaceutical product containing the ultrafine bubble-containing solution, and more particularly to an ultrafine bubble-containing solution containing at least one of carbon dioxide and hydrogen, a beverage containing the same, and a pharmaceutical product. [Background technology]

[0002] Reactive oxygen species (hereinafter also referred to as "ROS") are causative substances of many diseases, including cancer, and the elimination of reactive oxygen species is increasingly considered one approach to disease prevention and treatment.

[0003] To efficiently eliminate reactive oxygen species, efficient intake of reactive oxygen species-scavenging components is necessary. However, the intake of reactive oxygen species-scavenging components from food or beverages is limited and in trace amounts, making it impossible to expect sustained effects. Among reactive oxygen species, hydroxyl radicals (OH·) and superoxide anion radicals (·O2) are particularly problematic. - It is said that the elimination of ) is important for the prevention and treatment of disease.

[0004] On the other hand, ultrafine bubbles (hereinafter also referred to as "UFB (Ultrafine Bubble)") have a high negative zeta potential in neutral solutions and possess antioxidant properties. UFBs decrease in size over time and are said to be able to survive even after one month.

[0005] The sustained antioxidant capacity of ultrafine bubbles has not yet been sufficiently investigated. For example, while hydrogen has high reducing power, the antioxidant capacity of nanofine bubble hydrogen water (an aqueous solution of ultrafine bubbles containing hydrogen) has not yet been adequately studied.

[0006] Uemura et al. claim to have developed a technology to eject large quantities of ultrafine bubbles through resonant foaming and vacuum cavitation, and further state that "hydrogen ultrafine bubble water has antioxidant properties and can be used to prevent so-called lifestyle-related diseases such as hypertension, hyperlipidemia, diabetes, heart disease, and cerebral infarction in today's aging society, as well as to prevent cancer" (see Patent Document 1 below). However, this claim lacks scientific basis and has not been proven.

[0007] Regarding the creation of ultrafine bubbles, Ahmed et al. investigated aqueous solutions containing ultrafine bubbles of air, nitrogen, and oxygen using cylindrical ceramic nanofiltration membranes, rather than conventional generation methods using fluid dynamics, acoustics, particles, or optical cavitation, and reported that the size of air bubbles can be reduced to the nanoscale (see Non-Patent Document 1 below). [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2016-104474 [Non-patent literature]

[0009] [Non-Patent Document 1] Ahmed AKB, Shi X., Likun Hua L., Leidy Manzueta L., Weihua Qing W., Marhaba T., Wen Zhang, W., J. of Agricultural and Food Chemistry, 2018, 66(20), 5117-5124. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] As mentioned above, since ultrafine bubbles can exist for long periods, their antioxidant capacity may also be long-lasting. However, the effect of ultrafine bubbles of various gases on reactive oxygen species scavenging is unknown, and investigating this has been a challenge. [Means for solving the problem]

[0011] In view of the above issues, the inventors investigated the antioxidant capacity and effects on various diseases of nanofine bubbles of various gases, and found that a solution containing ultrafine bubbles that include at least one of carbon dioxide and hydrogen gas inside the ultrafine bubbles has sustained ROS scavenging ability, cytotoxic effects on cancer cells, and antitumor effects on tumor-bearing mice.

[0012] Therefore, the object of the present invention is to provide an ultrafine bubble-containing solution that has reactive oxygen species scavenging ability, a cytotoxic effect on cancer cells, and an antitumor effect on tumor-bearing mice, in which at least one of carbon dioxide and hydrogen gas is contained within the ultrafine bubble, as well as a beverage and a pharmaceutical product containing the same.

[0013] In other words, the present invention is as follows: [1] An ultrafine bubble-containing solution in which at least one of the gases, carbon dioxide and hydrogen, is contained within the ultrafine bubbles. [2] The ultrafine bubble-containing solution according to [1], which contains both carbon dioxide and hydrogen gases inside the ultrafine bubbles. [3] An ultrafine bubble-containing solution according to [1] or [2], wherein the ultrafine bubbles contain carbon dioxide inside and the 50% average particle size of the ultrafine bubbles is in the range of 50 nm to 300 nm. [4] The ultrafine bubble-containing solution according to any one of [1] to [3], wherein the 50% average particle diameter of the ultrafine bubbles containing hydrogen inside the ultrafine bubbles is in the range of 10 nm to 500 nm. [5] The ultrafine bubble-containing solution according to any one of [1] to [4], wherein the content of the ultrafine bubbles containing carbon dioxide inside the ultrafine bubbles is in the range of 100 million bubbles / mL to 10 billion bubbles / mL. [6] The ultrafine bubble-containing solution according to any one of [1] to [5], wherein the content of the ultrafine bubbles containing hydrogen inside the ultrafine bubbles is in the range of 10 million bubbles / mL to 100 billion bubbles / mL. [7] The ultrafine bubble-containing solution according to any one of [1] to [6], wherein the gas inside the ultrafine bubbles is carbon dioxide or hydrogen generated by at least one selected from the group consisting of passage through porous ceramics, passage through porous plastic membranes, double-bottle hydrogen generators, pressurized dissolution systems, and spiral flow systems. [8] The ultrafine bubble-containing solution according to any one of [1] to [6], wherein the carbon dioxide inside the ultrafine bubbles is carbon dioxide generated by passing carbon dioxide through porous ceramics. [9] The ultrafine bubble-containing solution according to any one of [1] to [6], wherein the hydrogen inside the ultrafine bubbles is hydrogen generated by electrolysis using a double-bottle hydrogen generator.

[10] The ultrafine bubble-containing solution according to any one of [1] to [6], wherein the gas inside the ultrafine bubbles is both carbon dioxide and hydrogen generated by at least one selected from the group consisting of pressurized dissolution systems and spiral flow systems.

[11] An ultrafine bubble-containing beverage comprising the ultrafine bubble-containing solution according to any one of [1] to

[10] .

[12] An ultrafine bubble-containing pharmaceutical comprising an ultrafine bubble-containing solution described in any one of items [1] to

[10] .

[13] The aforementioned pharmaceutical product is used for the prevention and treatment of diseases caused by reactive oxygen species, as described in

[12] , and contains ultrafine bubbles.

[14] The ultrafine bubble-containing pharmaceutical according to

[13] , wherein the disease caused by the reactive oxygen species is selected from the group consisting of geriatric diseases and cancer. [Effects of the Invention]

[0014] The present invention provides an ultrafine bubble-containing solution, a beverage and a pharmaceutical product containing the same, which has reactive oxygen species scavenging ability, a cytotoxic effect on cancer cells, and an antitumor effect on tumor-bearing mice, and which contains at least one of carbon dioxide and hydrogen gases within the ultrafine bubbles. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows an apparatus for producing ultrafine bubbles of carbon dioxide according to the present invention. [Figure 2] This figure shows an apparatus for producing ultrafine bubble hydrogen according to the present invention. [Figure 3] This figure shows the main components of the IFA measuring device for ultrafine bubbles according to the present invention. [Figure 4] This figure shows the particle size distribution of ultrafine bubbles according to the present invention. [Figure 5] This figure shows the stability of ultrafine bubble hydrogen according to the present invention. [Figure 6] This figure shows the antioxidant capacity of the ultrafine bubbles according to the present invention. [Figure 7] This figure shows the antioxidant capacity of the ultrafine bubbles according to the present invention. [Figure 8] This figure shows G-CYPMPO and adducts of reactive oxygen species in the method for detecting reactive oxygen species (spin trap method / ESR method) according to the present invention. [Figure 9] This figure shows the cytotoxic effect of the ultrafine bubble hydrogen-containing solution according to the present invention on cancer cells. [Figure 10] This figure shows the antitumor effect of the ultrafine bubbles according to the present invention in tumor-bearing mice. [Modes for carrying out the invention]

[0016] Embodiments of the present invention will be described in detail below.

[0017] (Solution containing ultrafine bubbles) The ultrafine bubble-containing solution according to the present invention is characterized by being a solution containing ultrafine bubbles that contain at least one of the gases, carbon dioxide and hydrogen, inside the ultrafine bubbles.

[0018] The ultrafine bubble-containing solution according to the present invention may also be an ultrafine bubble-containing solution that contains both carbon dioxide and hydrogen gases inside the ultrafine bubbles (hereinafter also referred to as "ultrafine bubble carbon dioxide and hydrogen mixed solution").

[0019] In the aforementioned ultrafine bubble-containing solution, at least one of carbon dioxide and hydrogen gas may be contained within each ultrafine bubble contained in the solution.

[0020] A bubble is a closed space consisting of a gas surrounded by something other than a gas; a bubble completely surrounded by a liquid is called a suspended gas. Of these suspended gases, bubbles with a diameter of 1 micrometer or less are called ultrafine bubbles (Fine Bubble Society Federation).

[0021] Because ultrafine bubbles are extremely small, they are known to carry a negative potential called the zeta potential, and can remain in a liquid for an extremely long time.

[0022] Hereinafter, ultrafine bubble-containing solutions containing carbon dioxide, hydrogen, and both carbon dioxide and hydrogen within the ultrafine bubbles will be referred to as ultrafine bubble carbon dioxide, ultrafine bubble hydrogen, and ultrafine bubble carbon dioxide and hydrogen mixed solution (as described above), respectively.

[0023] Examples of liquids for the solution containing ultrafine bubble carbon dioxide and ultrafine bubble hydrogen include water, salt solutions containing various ions, organic acid solutions, amino acid solutions, mixed solutions of water and water-soluble organic solvents, and organic solvents. Among these, various aqueous solutions are preferred for the ultrafine bubble-containing solution.

[0024] In the present invention, it is preferable that the ultrafine bubbles also have a specific 50% average particle size. By having gas contained inside the ultrafine bubbles with a particle size smaller than the specific 50% average particle size, the stability of the ultrafine bubbles is improved, and they can exist as ultrafine bubbles for a long time. In the present invention, the 50% average particle size usually refers to the 50% average particle size with respect to the number of ultrafine bubbles.

[0025] The 50% average particle size of the ultrafine bubbles containing carbon dioxide is preferably in the range of 50 nm to 300 nm, and more preferably in the range of 50 nm to 150 nm. This 50% average particle size range allows the bubbles to exist as ultrafine bubbles for several days. It is difficult to create ultrafine carbon dioxide bubbles with a 50% average particle size of less than 50 nm.

[0026] The 50% average particle size of the ultrafine bubbles containing hydrogen is preferably in the range of 10 nm to 500 nm, and more preferably in the range of 10 nm to 150 nm. This 50% average particle size range allows the bubbles to exist as ultrafine bubbles for several days. It is difficult to create ultrafine bubbles with a 50% average particle size of less than 10 nm.

[0027] In the present invention, the ultrafine bubble-containing solution preferably has a specific ultrafine bubble content. For the antioxidant capacity of the ultrafine bubble-containing solution to be exhibited, a certain amount of gas must be contained in the ultrafine bubbles.

[0028] In the ultrafine bubble-containing solution according to the present invention, the content of the ultrafine bubbles containing carbon dioxide inside is preferably in the range of 100 million bubbles / mL to 10 billion bubbles / mL, and more preferably in the range of 1 billion bubbles / mL to 10 billion bubbles / mL.

[0029] In the ultrafine bubble-containing solution according to the present invention, the content of ultrafine bubbles containing hydrogen inside is preferably in the range of 0.1 billion / mL to 100 billion / mL, and more preferably in the range of 100 million / mL to 50 billion / mL.

[0030] (Preparation of an ultrafine bubble-containing solution) Various methods are known for preparing the aforementioned ultrafine bubble-containing solution, including the spiral flow system method, the pressurized dissolution system method, the ultrasonic wave method, passing through porous ceramics, passing through porous plastic membranes, and double-bottle hydrogen generators.

[0031] The ultrafine bubble carbon dioxide can be produced by various methods. In particular, it is preferable to produce a solution containing the ultrafine bubble carbon dioxide according to the present invention by passing carbon dioxide from a pressurized tank through porous ceramics (the porous ceramic passage method) or through a porous plastic membrane, and then blowing it into a liquid (see Figure 1). As the porous plastic membrane, a membrane that removes ordinary fine particles can also be used for nanobubble generation.

[0032] The aforementioned ultrafine bubble hydrogen can be produced by various methods. Among these, the ultrafine bubble hydrogen produced by electrolysis (the aforementioned electrolytic method for fine bubble production) using a double bottle hydrogen generator (manufactured by Woo, Gas & Water Double Hydrogen Bottle®) is preferred. The double bottle hydrogen generator can be used to create a solution containing ultrafine bubble hydrogen (see Figure 2).

[0033] The ultrafine bubble carbon dioxide and hydrogen mixed solution can be produced by passing carbon dioxide through porous ceramics (the porous ceramic passage method) or through a porous plastic membrane, and then blowing it into the ultrafine bubble hydrogen-containing solution, or by generating ultrafine bubble hydrogen from the ultrafine bubble carbon dioxide using a double-bottle hydrogen generator.

[0034] Furthermore, the ultrafine bubble carbon dioxide and hydrogen mixed solution can be produced by generating it using at least one method selected from the group consisting of the pressurized dissolution system and the spiral flow system. These methods allow for the production of large quantities of the ultrafine bubble carbon dioxide and hydrogen mixed solution.

[0035] (Method for measuring the properties of ultrafine bubble-containing solutions) Known methods for characterizing ultrafine bubbles include the Resonant Mass Method, Particle Trajectory Method, Laser Diffraction Method, Dynamic Light Scattering Method (DLS Method), and Interactive Force Apparatus Method (IFA Method).

[0036] The properties of the ultrafine bubbles in this invention can be measured by various methods. In particular, for measuring the ultrafine bubbles of this invention, it is preferable to use the following interaction force apparatus (IFA) measurement method, as it is possible to measure ultrafine bubbles with particle sizes as small as those measured by the dynamic light scattering (DLS) method and has high accuracy.

[0037] In the present invention, the particle size distribution of ultrafine bubbles was measured in water using the IFA measuring device shown in Figure 3 (see Figures 4 and 5). According to this measuring device, "the particle size distribution of fine bubbles ranging from a few nanometers to several hundred micrometers can be measured with high accuracy, and measurements can be performed without being affected by particle concentration, light transmittance of the liquid, or refractive index" (Japanese Patent No. 6502657, page 3, paragraph

[0009] ). An example of this measuring device is shown in Figure 3. The measured values ​​were in good agreement with those obtained by the dynamic light scattering (DLS) method.

[0038] The aforementioned interaction force measurement method is disclosed in Japanese Patent Application Publication No. 2016-109453 (Title of Invention: "Method and Apparatus for Measuring Particle Size Distribution of Fine Bubbles," Patent No. 6502657).

[0039] (Characteristics of ultrafine bubble-containing solutions) Ultrafine bubble particle size The particle size of the ultrafine bubbles was measured using the interaction force apparatus method described above. The particle size of the ultrafine carbon dioxide bubbles (50% average diameter of the bubble size) was measured to be 115 nm, and the particle size of the ultrafine hydrogen bubbles (50% average diameter of the bubble size) was measured to be 130 nm (see Figure 4).

[0040] Stability of ultrafine bubbles The change in particle size of the ultrafine bubbles over time was measured using the interaction force apparatus method described above, and their stability (persistence) was evaluated. The ultrafine carbon dioxide bubbles existed in distilled water for several days and disappeared after 4 days. The stability of the ultrafine carbon dioxide bubbles in distilled water was several days, indicating stability due to the ultrafine bubbles. The ultrafine hydrogen bubbles had a particle size of 25 nm after 40 days, and it was found that the particle size decreased over time. It was found that the ultrafine hydrogen bubbles remained stable even after 40 days (see Figure 5). A comparison of these stabilities with the stability of other bubbles is shown in Table 1.

[0041] The content of ultrafine bubble carbon dioxide and ultrafine bubble hydrogen in the aforementioned ultrafine bubble-containing solution was in the range of 100 million particles / mL to 10 billion particles / mL and 10 million particles / mL to 100 billion particles / mL, respectively, based on particle size and density measurements.

[0042] Table 1 summarizes the content, particle size, and stability of the ultrafine bubble-containing solution. As is clear from Table 1, the ultrafine bubbles according to the present invention have a sufficiently small particle size and content within it, and can exist stably for a long period of time.

[0043] [Table 1]

[0044] (Performance of ultrafine bubble-containing solution) The reactive oxygen species scavenging ability of the ultrafine bubble-containing solution according to the present invention. The ultrafine bubble-containing solution according to the present invention has the ability to scavenge reactive oxygen species (ROS). In particular, the ultrafine bubble carbon dioxide-containing solution has the ability to scavenge hydroxyl radicals (OH·), and the ultrafine bubble hydrogen-containing solution has the ability to scavenge superoxide anion radicals (·O2). - The elimination ability of ) was remarkable.

[0045] In other words, ultrafine bubble carbon dioxide produced by passing carbon dioxide from a pressurized tank through porous ceramics (the porous ceramic passage method) (hereinafter referred to as "porous ceramic carbon dioxide UFB") had a high ability to scavenge hydroxyl radicals (OH·) generated using hydrogen peroxide and an ultraviolet lamp. On the other hand, ultrafine bubble hydrogen produced by electrolysis using a double-bottle hydrogen generator (Gas & Water Double Hydrogen Bottle®) (the fine bubble production electrolysis method) (hereinafter referred to as "electrolyzed hydrogen UFB") did not show the ability to scavenge hydroxyl radicals (OH·) (see Figure 6 above).

[0046] Furthermore, the electrolytic hydrogen UFB contains superoxide anion radicals (·O2 - High elimination ability was observed in the porous ceramic carbon dioxide UFB. Ultrafine bubble hydrogen showed the highest elimination ability 24 hours after creation. On the other hand, the porous ceramic carbon dioxide UFB showed high elimination ability in superoxide anion radicals (·O2 - The ability to eliminate ) was not observed (see Figure 7 above).

[0047] The above are hydroxyl radicals (OH·) and superoxide anion radicals (·O2) -The measurement was performed by using G-CYPMPO(registered trademark):2-(5,5-dimethyl-2-oxo-2-l5-[1,3,2]dioxaphosphinan-2-yl)-2-methyl-3,4-dihydro-2H-pyrro-line N-oxide (Chemical Formula 1 below) as a spin trapping agent, and recording the ESR spectrum of the spin-trapping adduct using an ESR spectrometer (JEOL JES-TE25X).

[0048] [ka]

[0049] In a hydrogen peroxide solution (0.1 w / v%) under UV irradiation, i.e., the adduct between hydroxyl radical (OH·) and G-CYPMPO®, and in the hypoxanthine / xanthine oxidase system, i.e., the superoxide anion radical (·O2 - Figure 8 shows the ESR spectrum of the adduct between ) and G-CYPMPO(registered trademark). In the figure, the diamond symbol (◆) represents the superoxide anion radical (·O2 - The peaks of the adducts (indicated by the ) and the peaks of the hydroxyl radical (OH·) adducts (indicated by the inverted triangle (▼)) are shown. The peak-to-peak intensities of both peaks are used to measure the amount of reactive oxygen species.

[0050] Kohri's ESR spin trap method, a representative technique, was used for data analysis. The peak-to-peak intensity of selected ESR lines of the free radical adducts was tracked in the presence and absence of antioxidants.

[0051] In the presence of a spin trapping agent (SP) and an antioxidant (AO), the following free radicals (R) may undergo a wrapping reaction. R+SP→R Velocity constant of the added material: tksp (1) Rate constant for R+AO→product: k AO (2) When I0 and I are the ESR peak heights in the presence of ST alone and ST+AO respectively, the amount of product in formula (2) is I0-I. Therefore, I0 / I-1 is calculated to quantify the free radical scavenging capacity.

[0052] To quantify the free radical scavenging capacity, the oxidant species is mixed with the free radical generation system several hours before ESR measurement. When I0 and I are the ESR peak heights in the presence of ST alone and ST+oxidant respectively, the amount of the free radical generation system oxidized by the oxidant species is I0-I. Therefore, I0 / I-1 is calculated to quantify the free radical scavenging capacity.

[0053] Cytotoxic effects on cancer cells (in vitro) Among the ultrafine bubble-containing solutions according to the present invention, when cancer cells were cultured in the medium containing the electrolytic hydrogen UFB, the survival rate of the cancer cells was significantly reduced to 80%. This decrease in survival rate was not observed with UFB that does not contain electrolytic hydrogen. From this, the cytotoxic effect on cancer cells was confirmed for the ultrafine bubble hydrogen-containing solution (Figure 9).

[0054] Antitumor effect in tumor-bearing mice (in vivo) Among the ultrafine bubble-containing solutions according to the present invention, the antitumor effect of the drinking water containing the electrolytic hydrogen UFB was confirmed in tumor-bearing mice (Lewis lung cancer-bearing 57BL / 6J mice). In the group of the ultrafine bubble hydrogen-containing solution, a tumor growth inhibitory effect was observed up to day 19 after transplantation, as compared with the control group (see Figure 10).

[0055] As described above, the ultrafine bubble-containing solution according to the present invention has a particle size of less than 200 nm, and therefore has high stability, allowing its performance to be maintained for a long time. For this reason, the ultrafine bubble-containing solution according to the present invention is effective against reactive oxygen species (ROS), particularly hydroxyl radicals (OH·) and superoxide anion radicals (·O2). - It possesses antioxidant activity against ). Furthermore, the ultrafine bubble-containing solution according to the present invention has been found to have cancer cell-damaging effects (in vitro) and antitumor effects (in vivo).

[0056] (Beverages containing ultrafine bubble-containing solutions) The beverage containing the ultrafine bubble-containing solution according to the present invention is characterized by containing an ultrafine bubble-containing solution in which at least one of carbon dioxide and hydrogen is contained within the ultrafine bubbles. Because the ultrafine bubble-containing beverage has ROS scavenging ability, it can have a wide range of effects, such as maintaining and improving health and preventing diseases through ROS scavenging.

[0057] (Pharmaceuticals containing ultrafine bubble-containing solutions) The pharmaceutical product comprising an ultrafine bubble-containing solution according to the present invention is characterized by comprising an ultrafine bubble-containing solution in which at least one of carbon dioxide and hydrogen is contained within the ultrafine bubbles. Because the ultrafine bubble-containing pharmaceutical product has ROS scavenging ability, cytotoxic effect on cancer cells, and antitumor effect on cancer-bearing animals, it can be used as a preventive and therapeutic agent for diseases caused by reactive oxygen species, as a preventive and therapeutic agent for any disease selected from the group consisting of geriatric diseases and cancer, and as a preventive and therapeutic agent for other diseases.

[0058] (Other uses of ultrafine bubble-containing solutions) The ultrafine bubble-containing solution according to the present invention, which contains at least one of carbon dioxide and hydrogen gases inside the ultrafine bubbles, can be used in a wide range of fields, including food, cosmetics, medicine, agriculture, and detergents. [Examples]

[0059] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0060] (1) Preparation of an ultrafine bubble-containing solution according to the present invention Experimental Example 1: Preparation of a solution containing ultrafine bubble carbon dioxide according to the present invention The aforementioned solution containing ultrafine bubble carbon dioxide (aqueous solution) was prepared by passing carbon dioxide (CO2, 99.5%) from argon gas in a pressurized tank through porous ceramics in a beaker containing 200 mL of water at a rate of 0.4 L / min for 30 minutes, while stirring with a magnetic stirrer as shown in Figure 1 (the aforementioned porous ceramics passage method) (hereinafter also referred to as "containing solution CO2"). The ultrafine bubble carbon dioxide in the containing solution is also referred to as "UFB / CO2" below.

[0061] Experimental Example 2: Preparation of a solution containing ultrafine bubble hydrogen according to the present invention The ultrafine bubble hydrogen was generated by electrolysis (the fine bubble production electrolysis method) using a double-bottle hydrogen generator (manufactured by Woo, Gas & Water Double Hydrogen Bottle®) as shown in Figure 2 (hereinafter also referred to as "UFB / EH2"). The solution (aqueous solution) containing the ultrafine bubble hydrogen was prepared by adding it to 200 mL of water for 30 minutes (hereinafter also referred to as "containing solution EH2"). The concentration of the ultrafine bubble hydrogen produced by electrolysis was 0.15% by volume and was quantified using a specific gravity bottle.

[0062] Experimental Example 3: Preparation of an ultrafine bubble carbon dioxide and hydrogen mixed solution according to the present invention The ultrafine bubble carbon dioxide and hydrogen mixed solution is prepared by blowing carbon dioxide (CO2, 99.5%) from argon gas from a pressurized tank into the ultrafine bubble hydrogen-containing solution (prepared in item (2) above) at a rate of 0.4 L / min for 30 minutes while stirring with a magnetic stirrer, as described in item (1) above (hereinafter also referred to as "mixed solution CO2 / EH2"). Alternatively, the ultrafine bubble carbon dioxide and hydrogen mixed solution is prepared by performing electrolysis on the ultrafine bubble carbon dioxide-containing solution (prepared in item (1) above) for 30 minutes, as described in item (2) above (hereinafter also referred to as "mixed solution EH2 / CO2").

[0063] (2) Measurement of ultrafine bubbles using the IFA measuring device in the present invention In the present invention, the ultrafine bubbles were measured in water using the IFA measuring device shown in Figure 3, according to the IFA method described in Japanese Patent Application Publication No. 2016-109453, "Method and apparatus for measuring particle size distribution of fine bubbles" (see Figures 4 and 5).

[0064] (2) Antioxidant capacity of the ultrafine bubble-containing solution in the present invention Experimental Example 4: Generation of Reactive Oxygen Species (ROS) Hydroxyl radicals (OH·) were generated by the decomposition of 0.1 w / t% hydrogen peroxide (H₂O₂) under 5s ultraviolet radiation from a UV radiator (UV LIGHTSOURCE, SUPERCURE-203S). A mixture of 90 μL of 0.2 w / t% hydrogen peroxide and 20 μL of 100 mM G-CYPMPO was transferred to a disposable borosilicate ESR cell, and the ESR spectrum of the adduct between the hydroxyl radical and G-CYPMPO was analyzed.

[0065] Superoxide anion radical (·O2 -The compound was generated in a hypoxanthine / xanthine (HX / XO) system. A mixture of 10.970 units / mL of XO, 20 μL of 20 mM HX, and 20 μL of 100 mM G-CYPMPO was transferred to a disposable borosilicate ESR cell, and the ESR spectrum of the adduct between the hydroxyl radical and G-CYPMPO was analyzed.

[0066] Experimental Example 5: Measurement of reactive oxygen species by ESR A novel radical trapper, 2-(5,5-dimethyl-2-oxo-2-l5-[1,3,2]dioxaphosphinan-2-yl)-2-methyl-3,4-dihydro-2H-pyrro-line N-oxide, G-CYPMPO®, was used to capture free radicals of reactive oxygen species. 25 mg of G-CYPMPO® (100 mL) was dissolved in 2 mL of ultrapure water.

[0067] The ESR spectra of spin-trapping adducts were recorded using an ESR spectrometer (JEOL JES-TE25X). Typical ESR measurement conditions were as follows: Microwave power: 4mW, microwave frequency: 9.2GHz, magnetic field: 328.0mT, field sweep with: ±7.5mT, field modulation: 0.16mT, sweep time: 1 minute, 0.003663mT / point, total 4096 points, ESR measurement was performed at room temperature.

[0068] Kohri's ESR spin trap method, a representative technique, was used for data analysis. The peak-to-peak intensity of selected ESR lines of the free radical adducts was tracked in the presence and absence of antioxidants.

[0069] In the presence of a spin trapping agent (SP) and an antioxidant (AO), the following free radicals (R) undergo a wrapping reaction. R+SP→R Velocity constant of the added material: tk sp (1) R + AO → Product rate constant: k AO (2) If I0 and I are the ESR peak heights in the presence of ST alone and ST+AO, respectively, then the amount of product in equation (2) is I0-I. Therefore, I0 / I-1 was calculated to quantify the free radical scavenging capacity.

[0070] To quantify free radical scavenging capacity, the oxidizing agent species is mixed with the free radical generating system several hours before ESR measurement. If I0 and I are the highest ESR peaks in ST alone and in the presence of ST + oxidizing agent, respectively, then the amount of free radical generating system oxidized by the oxidizing agent species is I0-I. Therefore, I0 / I-1 was calculated to quantify the free radical scavenging capacity.

[0071] Example 1: Antioxidant capacity of the ultrafine bubble carbon dioxide-containing solution according to the present invention The free radical scavenging ability of a solution containing ultrafine bubble carbon dioxide (the porous ceramic carbon dioxide UFB) was investigated by ESR measurement. The I0 / I-1 values ​​obtained from the ESR spectrum of the G-CYPMPO® adduct of the ultrafine bubble-containing solution are shown in Figure 6.

[0072] As expected, the ultrafine bubble-containing solution was found to capture hydroxyl radicals (OH·), but contrary to expectations, the ultrafine bubble carbon dioxide-containing solution was found to capture hydroxyl radicals (OH·) most powerfully. At the same time, the ultrafine bubble carbon dioxide-containing solution also captured superoxide anion radicals (·O2). - The concentration was increased. On the other hand, the solution containing ultrafine bubble hydrogen (the electrolytic hydrogen UFB) had almost no hydroxyl radical (OH·) scavenging ability. Figure 6 shows the strong hydroxyl radical (OH·) scavenging ability, i.e., antioxidant ability, of the solution containing ultrafine bubble carbon dioxide at 6 hours and 24 hours.

[0073] Example 2: Antioxidant capacity of the solution containing ultrafine bubble hydrogen according to the present invention The free radical scavenging ability of solutions containing ultrafine bubble hydrogen (the aforementioned electrolytic hydrogen UFB) was investigated by ESR measurement. The I0 / I-1 values ​​obtained from the ESR spectrum of the G-CYPMPO® adduct of the ultrafine bubble-containing solution are shown in Figure 7.

[0074] The solution containing ultrafine bubble hydrogen (the electrolytic hydrogen UFB) exhibits good superoxide anion radicals (·O2 - ) showed. On the other hand, the solution containing ultrafine bubble carbon dioxide (the porous ceramic carbon dioxide UFB) showed superoxide anion radical (·O2 - ) showed no such thing as superoxide anion radicals (·O2) in the ultrafine bubble carbon dioxide solution at 6 hours and 24 hours. Figure 7 shows good superoxide anion radical (·O2) in the ultrafine bubble carbon dioxide solution. - ) This demonstrates scavenging ability, i.e., antioxidant capacity.

[0075] (3) Cancer cell damaging effect of the ultrafine bubble-containing solution in the present invention Example 3: Cytotoxic effect of ultrafine bubble hydrogen (the electrolytic hydrogen UFB) on cancer cells (in vitro) To investigate the damaging effect of the ultrafine bubble-containing solution in this invention on cancer cells, cell viability was measured in vitro. Cell viability was measured using the Cell Coefficient Kit-8 (CCK-8, manufactured by Dojindo) according to the protocol provided with the product.

[0076] The rat cancerous micosa cell line (RGK1) was established by exposing rat gastric mucosal cells to 1-methyl-3-nitro-1-cotrosoguanidine. RGK1 cells were cultured in Dulbecco-modified Eagle / F12 medium (Sigma-Aldrich) de-glutamine. Mouse Lewis lung carcinoma (LLC) cells were obtained from the RIKEN Cell Bank. LLC cells were cultured in RPMI1640 medium (Wako Pure Chemical Industries). The culture medium contained 10% heat-inactivated fetal bovine serum (BioWest) and 1% penicillin / streptomycin (Wako Pure Chemical Industries). Cells were cultured in a 37°C incubator under a 5% carbon dioxide (air) atmosphere.

[0077] The cancer cells were cultured in a medium containing electrolyzed hydrogen (UFB) and in a medium without it, and the results of the cell viability are shown in Figure 9. In the medium containing electrolyzed hydrogen (UFB), the cancer cell viability decreased to 80% with a significant difference. From this, it was found that the solution containing ultrafine bubbles of hydrogen had a cytotoxic effect on cancer cells (Figure 9).

[0078] Example 4: Antitumor effect (in vivo) of ultrafine bubble hydrogen (the electrolyzed hydrogen UFB) on Lewis lung cancer-bearing 57BL / 6J mice. Eight-week-old male mice were used in the in vivo assay. The mice were obtained from SLC Corporation. The animal protocol was approved by the Standing Committee on Animal Science, University of Tsukuba. LLC cells (2 × 10⁶) 6 The substance was administered subcutaneously to the flank of mice. After tumor transplantation, mice were allowed to freely drink water or the electrolyzed hydrogen UFB. After tumor transplantation, tumor size was measured on days 13, 16, 19, and 22. The mice were euthanized on day 22, and the tumor volume was calculated. The tumor volume was calculated using the following formula. Volume (mm³) = Length × Width 2 ×0.5

[0079] Figure 10 shows the time course of tumor size in tumor-bearing mice. The tumor size in the electrolyzed hydrogen filtration (UFB) group was smaller than that of the control group (water), and a significant difference was observed 19 days after tumor transplantation. This result indicates that electrolyzed hydrogen filtration inhibits tumor growth.

[0080] Based on the above in vitro and in vivo experiments, the antitumor effect of the ultrafine bubble-containing solution (solution containing electrolytic hydrogen UFB) according to the present invention was confirmed. [Explanation of symbols]

[0081] 310 Electric Balance 320 Platinum 330 Sample Solution 340 Particle Behavior Section 341 Hemisphere 342 flat plate 343 Piezo Stage

Claims

1. An aqueous solution containing ultrafine bubbles, comprising both ultrafine bubbles containing carbon dioxide and ultrafine bubbles containing hydrogen, wherein the ultrafine bubbles are stable for 2 to 3 months under closed conditions.

2. The aqueous solution containing ultrafine bubbles according to claim 1, wherein the 50% average particle size of the ultrafine bubbles containing carbon dioxide is in the range of 10 nm to 200 nm.

3. The aqueous solution containing ultrafine bubbles according to claim 1 or 2, wherein the 50% average particle size of the ultrafine bubbles containing hydrogen is in the range of 10 nm to 200 nm.

4. An aqueous solution containing ultrafine bubbles according to any one of claims 1 to 3, wherein the content of ultrafine bubbles containing carbon dioxide is in the range of 100 million bubbles / mL to 10 billion bubbles / mL.

5. An aqueous solution containing ultrafine bubbles according to any one of claims 1 to 4, wherein the content of the hydrogen-containing ultrafine bubbles is in the range of 0.1 billion bubbles / mL to 50 billion bubbles / mL.

6. An aqueous solution containing ultrafine bubbles according to any one of claims 1 to 5, wherein the ultrafine bubbles containing carbon dioxide are generated by the passage of carbon dioxide through porous ceramics.

7. An aqueous solution containing ultrafine bubbles according to any one of claims 1 to 6, wherein the ultrafine bubbles containing hydrogen are generated by a double-bottle hydrogen generator.

8. A method for producing an aqueous solution containing ultrafine bubbles according to any one of claims 1 to 7, wherein the ultrafine bubbles containing hydrogen are generated by a double-bottle hydrogen generator, and the aqueous solution contains both ultrafine bubbles containing carbon dioxide and ultrafine bubbles containing hydrogen.

9. A method for producing an aqueous solution containing ultrafine bubbles according to any one of claims 1 to 8, wherein the ultrafine bubbles containing carbon dioxide are generated by the passage of carbon dioxide through porous ceramics, and the aqueous solution contains both ultrafine bubbles containing carbon dioxide and ultrafine bubbles containing hydrogen.

10. A solution containing an ultrafine bubble containing hydrogen, comprising a prophylaxis for cancer or tumor A drug that prevents or treats, Here, the ultrafine bubbles containing hydrogen were generated by electrolysis using a double-bottle hydrogen generator (manufactured by Woo, Gas & Water Double Hydrogen Bottle®). The 50% average particle size of the hydrogen-containing ultrafine bubbles is in the range of 10 nm to 200 nm, the content is in the range of 0.1 billion bubbles / mL to 50 billion bubbles / mL, and the concentration is 0.15% by volume as determined by quantitative analysis using a specific gravity bottle. The aforementioned ultrafine bubbles containing hydrogen remain stable for one month when open. A preventive or therapeutic agent for cancer or tumors, comprising an aqueous solution containing ultrafine bubbles.

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