Energy production method by dissociation reaction of molecular hydrogen, Anti-aging method, fuel cell, and intercellular interaction promoter

Abstract

The present invention provides a method for extracting energy on the basis of the dissociation mechanism of molecular hydrogen generated by the hydrogenase activity of mitochondria, wherein a superoxide and molecular hydrogen overcome an energy barrier and undergo a propagation reaction, and dissociation energy is generated from the dissociation of molecular hydrogen into electrons and protons, or electrical energy is produced from moving electrons and protons.

Description

Energy production method by dissociation reaction of hydrogen molecules, anti-aging method, fuel cell and cell interaction promoter

[0001] The present invention relates to a method for producing energy by dissociation reaction of hydrogen molecules, an anti-aging method, a fuel cell, an agent for promoting cell-cell interaction, and the like.

[0002] In recent years, the use of hydrogen has become widespread as part of efforts to achieve carbon neutrality, and it is well known that fuel cells, which use hydrogen as an energy source, play a major role in the hydrogen society.

[0003] The basic mechanism of fuel cells is to extract energy by dissociating hydrogen molecules into electrons and protons. However, in order to dissociate hydrogen molecules into electrons and protons under normal temperature and pressure, it is necessary to overcome the activation energy barrier of the hydrogen molecules. Therefore, until now, catalysts mainly using platinum have been necessary.

[0004] On the other hand, in relation to this point, since ancient times when there was no oxygen on Earth, primitive life forms have extracted energy from hydrogen molecules by dissociating them into electrons and protons using hydrogenases ([Ni-Fe], [Fe-Fe]), in which transition metal elements such as nickel and iron are coordinated to the active center of enzymes as catalysts.

[0005] From ancient times, more than 2 billion years ago, when there was almost no oxygen in the atmosphere, the evolution of the photosynthetic apparatus by cyanobacteria released large amounts of oxygen molecules, resulting in a drastic change in the Earth's environment to an oxidized state. As the amount of hydrogen in the atmosphere became very small, high concentrations of hydrogen came to exist only in anaerobic environments with low oxygen concentrations, such as soil and the intestines of animals, including humans.

[0006] This is because anaerobic microorganisms depend on anaerobic metabolism for their energy production, and require bidirectional hydrogenases that, for example, dissociate hydrogen molecules as an electron donor source to extract electrons and protons, or, when ferredoxin, which has a lower redox potential, is used as an electron source, use protons as an electron acceptor to generate hydrogen molecules.

[0007] Furthermore, rodents that are raised in sterile conditions to prevent these anaerobic metabolic microorganisms from coexisting in their intestinal flora do not acquire normal immune education and immune tolerance, resulting in the development of severe autoimmune diseases.

[0008] This suggests that hydrogen molecules may regulate the energy metabolism of immune cells, i.e., the flow of electrons and protons, in the mitochondria of animal intestinal epithelial cells and immune cells infiltrating the same area.

[0009] As described above, it was previously thought that catalysts such as platinum or hydrogenase were essential to overcome the energy barrier involved in dissociating hydrogen under normal temperature and pressure conditions.

[0010] Incidentally, hydrogen is known to have the effect of improving energy metabolism (vital function improving effect) in the mitochondria of multicellular organisms such as humans, animals, and plants that do not have hydrogenase.

[0011] The administration of gaseous hydrogen molecules is one way to incorporate molecular hydrogen into all cells, including single-celled organisms. For example, the most common way to ingest this gaseous hydrogen is to drink water or other biocompatible liquids containing molecular hydrogen. However, other methods include inhaling gas containing molecular hydrogen, or intravenous infusion or transdermal absorption, which allow hydrogen to be directly delivered to the bloodstream using a liquid containing molecular hydrogen.

[0012] This function of hydrogen to improve vital functions has been attracting increasing attention in recent years, and is becoming essential for the health of humans and pets in particular. However, most of the vital functions of hydrogen have been explained so far as a scavenger function, i.e., electron donation to highly reactive oxygen species such as hydroxyl radicals and peroxynitrite (e.g., Non-Patent Documents 1 and 2). To this extent, only a part of the vital functions of hydrogen molecules has been explained, which has consequently limited the application of hydrogen molecules and / or impaired an important aspect of understanding their vital functions.

[0013] For example, hydroxyl radicals are not the cause of energy metabolism, but rather are the result or by-product of abnormal energy metabolism. They have a short half-life of nanoseconds and are toxic because they instantly oxidize surrounding biological molecules. Therefore, even if they are detoxified, it will not improve energy metabolism disorders such as diabetes.

[0014] Specifically, even if hydroxyl radicals, a type of reactive oxygen species, were generated inside a cell, which is extremely densely packed with countless proteins, nucleic acids, lipids, and other reactive small molecules, their half-life is on the order of nanoseconds, so chemical reaction theory makes it clear that the possibility of them encountering and reacting with hydrogen molecules supplied from outside is extremely low.

[0015] Hydroxy radicals are toxic because they are extremely reactive, and there is no way that the administered hydrogen would exist (wait) without reacting with the densely packed biological molecules that surround it until it reaches the site where the hydroxyl radical is generated and collides with a hydrogen molecule.

[0016] Therefore, although the scavenging function of hydrogen molecules against hydroxyl radicals is a conceivable reaction in a test tube, it does not occur in vivo.

[0017] Although hydroxyl radicals react with hydrogen molecules in a test tube, the reaction rate is more than an order of magnitude slower than the rate at which hydroxyl radicals react with their precursor, nearby superoxide.

[0018] So, regarding the unresolved question of how hydrogen acts on mitochondria to improve vital functions, the present inventors have discovered that complex I (including its potential and ubiquinone: coenzyme Q10, which functions as a proton carrier; the same applies hereinafter) is part of the mitochondrial respiratory chain and is thought to be a descendant of hydrogenase, an enzyme that dissociates hydrogen molecules, and plays the role of hydrogenase in mitochondria, becoming the starting point for improving vital functions. Hereinafter, this role and some of the phenomena that result from it will be referred to as hydrogenase activity.

[0019] The quinone molecule plays a central role in this reaction, acting as a carrier of electrons and protons in electron transfer in complexes I and III and participating in the catalytic reaction. The intermediate is a highly reactive semiquinone radical.

[0020] Semiquinone radicals are produced during reverse electron transport (RET), for example, during immune cell activation such as inflammation or during post-ischemic reperfusion injury, or remain in a radical state, serving as a central electron donor source for oxygen molecules in generating damagingly excessive superoxide.

[0021] Since hydrogen molecules can improve this dangerous condition, it is thought that the reaction between semiquinone radicals and hydrogen molecules is not limited to the enzymatic function of complex I or III, but also occurs non-enzymatically, suppressing the excessive production of superoxide.

[0022] Ishihara, G. , Kawamoto, K. , Komori, N. , & Ishibashi, T. Molecular hydrogen suppresses superoxide generation in the mitochondrial complex and reduced mitochondrial membrane potential. Biochem Biophys Res Commun. 522, 965-970 (2020). Current Pharmaceutical Design, 2019, 25, 946-955

[0023] The present invention aims to elucidate, through the inventor's demonstration experiments, the mechanism by which hydrogen molecules dissociate during hydrogenase activity in mitochondria, provide a method for extracting energy from this hydrogen molecule dissociation reaction, and demonstrate the industrial applicability of hydrogen molecules, water containing hydrogen molecules, gas, etc., based on this mechanism.

[0024] Another object of the present invention is to provide a novel intercellular interaction promoter for industrial use in fields such as medicine, healthcare, the health industry, agriculture, livestock farming, and fisheries, in which hydrogen molecules improve vital functions.

[0025] To solve the above problems, the present invention has the following configurations: [1] A method for producing energy through the dissociation reaction of hydrogen molecules, comprising: causing a propagation reaction between superoxide and hydrogen molecules across an energy barrier, and generating dissociation energy and / or electrical energy due to the transfer of electrons or protons when the hydrogen molecules dissociate into electrons and protons; [2] A method for producing energy through the dissociation reaction of hydrogen molecules, comprising: causing a propagation reaction between a quinone intermediate and hydrogen molecules across an energy barrier, and generating dissociation energy and / or electrical energy due to the transfer of electrons or protons when the hydrogen molecules dissociate into electrons and protons; [3] A method for producing energy through the dissociation reaction of hydrogen molecules, comprising: introducing hydrogen molecules into a reaction system containing superoxide and a quinone intermediate, causing a propagation reaction between the superoxide and / or the quinone intermediate and the hydrogen molecules across an energy barrier, and generating dissociation energy and / or electrical energy due to the transfer of electrons or protons when the hydrogen molecules dissociate into electrons and protons. [4] An anti-aging method characterized by carrying out the dissociation reaction of hydrogen molecules described in any one of [1] to [3] inside mitochondria. [5] The anti-aging method described in [4], in which hydrogen gas or a mixed gas containing hydrogen gas is inhaled or absorbed transdermally to introduce the hydrogen molecules into mitochondria. [6] The anti-aging method described in [4], in which physiological water or other hydrogen liquid containing or dissolved in hydrogen is orally ingested, absorbed transdermally, injected, or administered intravenously to introduce the hydrogen molecules into mitochondria. [7] A fuel cell characterized by circulating electrons extracted during the dissociation reaction of hydrogen molecules described in any one of [1] to [3] through a circuit. [8] A hydrogen-containing intercellular interaction promoter, which causes cells, including unicellular organisms, to secrete, liberate, or release cellular components or metabolic substances that exhibit intercellular interactions, including mitochondria, by acting on them with a component containing hydrogen molecules. [9] A hydrogen-containing intercellular interaction promoter, which causes mitochondria to secrete, liberate, or release cellular components or metabolic substances that exhibit intercellular interactions.

[10] The intercellular interaction promoter described in [8] or [9], which has hypoxia-inducing ability.

[11] A blood coagulation inhibitor comprising the cell-cell interaction promoter according to any one of [8] to

[10] .

[12] A mitochondrial releaser comprising the cell-cell interaction promoter according to any one of [8] to

[10] .

[13] Mitochondria or a mitochondrial preparation released by the method according to

[12] .

[14] A plant activator comprising the cell-cell interaction promoter according to any one of [8] to

[10] .

[15] A method for extracellularly growing mitochondria using hydrogen in the presence of living cells.

[16] A method for growing mitochondria in the presence of dead cells or cellular components using hydrogen, and providing the mitochondria or mitochondrial preparation according to

[13] for the normalization or activation of cells.

[17] A cell activator obtained by removing water from the mitochondria or mitochondrial preparation according to

[13] , or comprising a mitochondria-containing component containing a water-retaining substance such as trehalose and a small number of water molecules.

[18] A method for identifying a candidate drug having different efficacy due to a bioenergetic shift in the effector cells and the control cells, by administering a candidate drug such as a candidate molecule, protein, peptide, nucleic acid molecule, or gaseous molecule expected to have a medicinal effect to effector cells that have been treated with the cell-cell interaction promoter described in any one of [8] to

[10] and control cells that have not been treated with the agent,

[19] A method for enhancing the function of an intercellular interaction promoter, by using a candidate drug having different efficacy found by the method described in

[18] in combination with the intercellular interaction promoter described in any one of [8] to

[10] .

[0026] According to the present invention, instead of dissociating hydrogen molecules by lowering the activation energy of the hydrogen molecules using a catalyst as in the past, it is now possible to extract energy by dissociating hydrogen molecules into electrons and protons through a quantum reaction such as the quantum tunneling effect, and therefore this invention has extremely high utility value and potential for development in the energy industry.

[0027] Furthermore, by reacting superoxide or semiquinone radicals, which have been confirmed in mitochondria, with hydrogen molecules (including the reaction of hydrogen molecules with semiquinone radicals, which are generated when one electron is transferred from superoxide to a quinone molecule), hydrogenase activity is generated, causing the hydrogen molecules to dissociate, thereby improving vital functions. Furthermore, in the reactions between oxygen molecules and semiquinone radicals, and between superoxide and quinone, which are in a state of dynamic equilibrium with each other, hydrogen molecules are intervened to dissociate the hydrogen molecules, allowing the superoxide and / or semiquinone radicals to react with the hydrogen molecules, stabilizing the amount of superoxide generated and achieving an anti-aging effect.

[0028] Furthermore, by using electrons extracted by a quantum reaction mechanism including the quantum tunneling effect of hydrogen molecules and superoxide, it is possible to provide fuel cells and the like without using expensive catalysts.

[0029] Furthermore, in the fields of medicine, healthcare, the health industry, agriculture, livestock farming, and fisheries, hydrogen molecules can provide novel cell-cell interaction promoters that improve vital functions, which in humans and animals can achieve the effects of promoting the repair and regeneration of tissues, including nerve cells or tissues, such as promoting monocyte differentiation to promote tissue repair, promoting the conversion of inflammatory macrophages to repair macrophages, or removing inflammation from vascular endothelial cells to promote repair, and in plants can achieve the effects of activating dormant cells to promote germination, activating leaf veins, and promoting plant growth.

[0030] Graph showing the amount of superoxide quantified by MPEC when ubiquinone was 0 μM in Example 1. Graph showing the amount of superoxide quantified by MPEC when ubiquinone was 10 μM in Example 1. Graph showing the amount of superoxide quantified by MPEC when ubiquinone was 50 μM in Example 1. Bar graph showing the amount of NADH and ATP released from mitochondria quantified by HPLC in Example 2. Photograph of the results of applying the hydrogen-containing intercellular interaction promoter of the present invention to the soil of a pot in which finger limes were planted in Example 3. Bar graph showing the ratio of ATP to ADP quantified by HPLC in Example 4. Ratios of ATP to ADP ATP / ADP and NADH / NAD quantified by HPLC in Example 4. ＋ Line graph showing bioenergetic shift due to ratio change

[0031] (Method for producing energy by dissociation reaction of hydrogen molecules) In the present invention, the method for producing energy by dissociation reaction of hydrogen molecules extracts energy (dissociation energy and / or electrical energy due to the movement of electrons or protons) by dissociating hydrogen molecules into electrons and protons.

[0032] In this regard, hydrogen molecules are extremely stable molecules, and dissociating hydrogen molecules into electrons and protons requires a reaction that exceeds the energy barrier required for dissociation, that is, activation energy.

[0033] The dissociation reaction of hydrogen molecules is closely related to the energy industry and the life activities of living organisms. For example, it is widely known that platinum is used as a catalyst in fuel cells, and hydrogenase is used as a catalyst in the anaerobic metabolism of living organisms.

[0034] However, although mitochondria do not contain hydrogenase, they exhibit hydrogenase activity, which dissociates hydrogen molecules into electrons and protons.

[0035] The inventors of the present application conducted a demonstration experiment (Example 1) to investigate why mitochondria, which do not have hydrogenase, are able to dissociate hydrogen molecules into electrons and protons, and as a result of extensive research, they discovered the mechanism behind this.

[0036] Specifically, it was found that the reaction of hydrogen molecules with superoxide or semiquinone radicals (quinone intermediates) confirmed in mitochondria can induce hydrogenase activity and dissociate hydrogen molecules, thereby improving vital functions. In this case, the reaction involves the reaction of hydrogen molecules with semiquinone radicals, which are generated by the transfer of one electron from superoxide to a quinone molecule.

[0037] Furthermore, in the reactions between oxygen molecules and semiquinone radicals, and between superoxide and quinone, which are in a state of mutual dynamic equilibrium (a reaction system in which superoxide and quinone intermediates are mixed), hydrogen molecules are intervened to dissociate the reactions, causing the superoxide and / or semiquinone radicals to react with the hydrogen molecules, stabilizing the amount of superoxide generated and achieving an anti-aging effect.

[0038] Specifically, they conducted experiments to determine how hydrogen molecules intervene in the interaction (dynamic equilibrium) between superoxide and semiquinone radicals in mitochondria, and as a result, they found that superoxide and / or semiquinone radicals interact with hydrogen molecules, and a dissociation reaction occurs across the activation energy barrier via the quantum tunneling effect (a propagation reaction that reacts by propagation, wraparound, penetration, etc. across an energy barrier).

[0039] Furthermore, it was found that this dissociation reaction occurs between superoxide and hydrogen molecules even in the absence of quinone or semiquinone radicals.

[0040] As a result, in an environment of normal temperature and pressure, either or both of superoxide and / or semiquinone radicals undergo a dissociation reaction with hydrogen molecules, liberating electrons and protons or transferring electrons to oxygen molecules, quinones, or semiquinones, thereby producing energy.

[0041] Regarding the quantum propagation reaction (quantum tunneling effect) shown in Example 1, it has become clear from the data obtained by measuring the amount of superoxide that a reversal phenomenon occurs. Therefore, it is thought that Marcus theory can be applied to the optimum concentration of hydrogen molecules to be reacted with the respective concentrations of superoxide and semiquinone radical, or with respect to the concentration in the interaction.

[0042] (Anti-aging method using dissociation reaction of hydrogen molecules) Furthermore, the anti-aging method of the present invention, which applies the above mechanism, involves hydrogen molecules in the radical reaction that occurs between superoxide and semiquinone radicals in mitochondria (a reaction system in which superoxide and quinone intermediates are mixed), causing a quantum propagation reaction (quantum tunneling effect) between superoxide and / or semiquinone radicals and hydrogen molecules, thereby stabilizing the amount of superoxide generated.

[0043] In mitochondria, electrons are transferred between superoxide and quinone (ubiquinone: coenzyme Q10 in humans). This interaction occurs at a reaction rate constant (K,M). －１ S －１ ) is 1 x 10 ８ ~1x10 ９ This is a dynamic equilibrium state of extremely fast reaction, and superoxide and semiquinone radicals coexist.

[0044] In the present invention, hydrogen molecules are actively incorporated into the radical reaction in the presence or coexistence of superoxide and semiquinone radicals, thereby causing dissociation of the hydrogen molecules as shown in Example 1.

[0045] This suppresses the explosive generation of superoxide and prevents superoxide bursts. Furthermore, from the results of Example 1, conversely, when the amount of superoxide is small, hydrogen molecules can increase and stabilize the amount of superoxide generated.

[0046] Incidentally, it is known that the production of low levels of superoxide has a positive effect on the lifespan of living organisms (reference: Understanding Coenzyme Q. Physiol Rev. 2024 May 9; 104: 1533-1610).

[0047] In this regard, the above-mentioned literature has shown that some mice and nematodes lacking the ability to synthesize quinones have longer lifespans than normal individuals, and it has been described that in these individuals in which an extended lifespan has been confirmed, a slight increase in superoxide occurs, i.e., the RAS gene group may sense a slight increase in superoxide and recombine a wide range of gene expression switches to control metabolism and adjust the redox balance, thereby contributing to the extension of lifespan. According to this, in the case of superoxide at an excessively low concentration, it is understood that the redox balance is maintained at a level that contributes to life extension by stabilizing superoxide or increasing its generation.

[0048] Therefore, the present invention, which allows hydrogen molecules to intervene in radical reactions in the presence or coexistence of superoxide and semiquinone radicals, is thought to generate low levels of superoxide when the amount of superoxide is low, thereby creating the extended lifespan observed in some mice and nematodes, and as a result, to achieve an anti-aging effect.

[0049] Furthermore, the biological function-improving effect of hydrogen on mitochondria has traditionally been suggested by many researchers to be due to its selective reduction of specific reactive oxygen species, particularly hydroxyl radicals, and this effect has been observed in basic research and animal experiments. Therefore, hydrogen has been thought to be effective in anti-aging by neutralizing reactive oxygen species (ROS) through its antioxidant properties (scavenging function against reactive oxygen).

[0050] However, from the perspective of chemical reaction theory, even if hydroxyl radicals are generated inside cells, which are extremely densely confined with countless proteins, nucleic acids, lipids, and other reactive small molecules, their half-life is on the order of nanoseconds, making it extremely unlikely that they would react with hydrogen molecules supplied from outside. In other words, the idea that hydrogen molecules are scavenger for hydroxyl radicals is not true in vivo, and is a reaction that can only be thought of in a test tube.

[0051] However, while it has been confirmed that taking hydrogen into the body has medical benefits, there are still many unknowns about the specific mechanisms behind this.

[0052] The present inventors have previously discovered that complex I (including ubiquinone (coenzyme Q10), which is an electron and proton carrier for complex I and is considered to be a descendant of hydrogenase, an enzyme that dissociates hydrogen molecules), is a part of the mitochondrial respiratory chain, and that this complex I activates hydrogenase activity in mitochondria, dissociating hydrogen molecules and thereby becoming the starting point for various life function-improving effects (for example, rectification in the electron transport chain). In other words, the life function-improving effects are obtained by hydrogenase activity occurring in mitochondria.

[0053] The present invention actively incorporates hydrogen molecules, allowing them to intervene in radical reactions in mitochondria in the presence or coexistence of superoxide and semiquinone radicals, thereby dissociating the hydrogen molecules into electrons and protons, thereby enabling the expression and / or enhancement of hydrogenase activity.

[0054] Therefore, according to the anti-aging method of the present invention, anti-aging effects can be expected from both the effect described in the above-mentioned literature of generating superoxide at low levels and the effect of improving vital functions by expressing and / or enhancing hydrogenase activity.

[0055] Any method can be used to introduce hydrogen molecules into mitochondria, including, for example, inhaling or transdermal absorption of gaseous hydrogen or a mixed gas containing hydrogen gas, orally ingesting a hydrogen solution containing dissolved hydrogen molecules, injecting or infusing physiological water containing hydrogen molecules, or transdermal absorption by contacting the skin with (refluxed) hydrogen water.

[0056] (Fuel cell based on dissociation of hydrogen molecules) Furthermore, the fuel cell of the present invention dissociates hydrogen molecules by inducing a quantum propagation reaction (quantum tunneling effect) in the reaction between hydrogen molecules and superoxide or semiquinone radicals, and can generate electrical energy by circulating the extracted electrons (or protons) in a circuit.

[0057] Conventional fuel cells were thought to require a catalyst to overcome the activation energy barrier in order to cause the dissociation of hydrogen molecules under normal temperature and pressure conditions, and in fact, platinum catalysts were mainly used.

[0058] Fuel cells will play a major role in the hydrogen society in which hydrogen use has become widespread, as part of efforts to achieve carbon neutrality in recent years. However, the platinum used in the catalyst is highly rare and extremely expensive to produce, hindering their widespread adoption.

[0059] As shown in Example 1, the inventors of the present invention have succeeded in extracting electrons from hydrogen molecules by a method of dissociating hydrogen molecules into electrons and protons through a quantum mechanical reaction without the need for a catalyst such as a metal, that is, by overcoming an energy barrier through the quantum tunneling effect without the intervention of a catalytic reaction.

[0060] The electrons (or protons) extracted by dissociation of these hydrogen molecules can be used as electrical energy by flowing them through a circuit, just like in conventional fuel cells.

[0061] The superoxide used in the fuel cell of the present invention may be generated in any manner. For example, KO ２ It is thought that hydrogen can be generated by reacting oxygen with a superoxide salt of an alkali metal, such as potassium superoxide. The simplest way to obtain hydrogen is by electrolyzing water, but if the present invention is to be used in a fuel cell vehicle, it is also thought that hydrogen can be obtained industrially in large quantities from a supply facility such as a hydrogen station that uses solar cells containing perovskite, which are installed for conventional hydrogen supply.

[0062] The process of generating electrical energy involves the intervention of hydrogen molecules in a radical reaction in the presence or coexistence of superoxide and semiquinone radicals, and as shown in Example 1, the hydrogen molecules are dissociated into electrons and protons through a quantum propagation reaction. The electrons dissociated from the hydrogen molecules travel through an external circuit and are converted into electrical energy. The protons dissociated from the hydrogen molecules, i.e., hydrogen ions, pass through the electrolyte membrane as in conventional fuel cells and react with oxygen to generate water.

[0063] As described above, the inventors of the present application discovered through the demonstration experiment shown in Example 1 that hydrogen molecules are dissociated into electrons and protons by a quantum mechanical reaction rather than a catalytic reaction with superoxide and / or semiquinone radicals, and this mechanism makes it possible to extract the energy (kinetic energy and potential energy) possessed by the electrons, and the energy possessed by the electrons extracted by this dissociation can be converted into electrical energy to form a fuel cell.

[0064] Furthermore, in mitochondria, hydrogen molecules are dissociated into electrons and protons, which results in the expression and / or enhancement of hydrogenase activity, and the stabilization of superoxide due to the dissociation of hydrogen molecules, which can improve vital functions and provide anti-aging effects.

[0065] (Hydrogen-containing cell-cell interaction promoter) Next, various effects originating from hydrogenase activity, which is enhanced by dissociation of hydrogen molecules in mitochondria, will be described using a drug or activator called a hydrogen-containing cell-cell interaction promoter.

[0066] The hydrogen-containing intercellular interaction promoter of the present invention is an intercellular interaction promoter containing hydrogen for secreting, liberating, or releasing active ingredients, including mitochondria, from cells. Note that the present invention is not intended to act on the target cells themselves that are to be activated, but rather to indirectly act from cells other than the target cells, using cellular components or metabolic substances that have intercellular interactions that enable the activation of the target cells.

[0067] According to the present invention, it is possible to obtain the benefits of components that have intercellular interactions, including mitochondria, without the need to artificially extract mitochondria from cells by disrupting the cells. For example, by taking the present invention during, before, or after daily muscle stress through exercise, mitochondria are secreted, liberated, or released from muscle cells, thereby promoting the self-renewal or rejuvenation of cells and tissues throughout the body other than the cells from which the mitochondria originate, including stem cells that need to be activated.

[0068] For example, release of mitochondrial components from osteoblasts in an environment without hydrogen molecules has been reported (see Mitochondrial fragmentation and donut formation enhance mitochondrial secretion to promote osteogenesis. Cell Metab. 2023 Feb 7; 35(2): 345-360.e7. doi: 10.1016 / j.cmet.2023.01.003.), but the mechanism by which cells containing mitochondria, including yeast, fungi, and platelets, actively secrete mitochondria through the action of components containing hydrogen molecules in order to stimulate, repair, or activate other cells is not known.

[0069] Furthermore, the present invention can also provide an intercellular interaction promoter that contains hydrogen and has hypoxia-inducing ability, which induces hypoxia in target cells that secrete an active ingredient that has intercellular interaction including mitochondria.

[0070] For example, when this hydrogen-containing intercellular interaction promoter contains ultra-high concentrations of hydrogen exceeding a saturation concentration of 1.6 ppm, the oxygen concentration normally contained in a solution that comes into contact with the air at atmospheric pressure can be reduced to 50% or less. As a result, the mitochondria of the target cells that secrete the active ingredient having intercellular interaction, including mitochondria, also enter a state of hypoxia, causing reverse electron transport (RET) in the electron transport chain, which results in the inhibition of ATP synthesis by FoF1-ATPase and a decrease in ATP production.

[0071] In this state, the presence of hydrogen molecules promotes ATP synthesis despite the RET state, or reverses the rotation of FoF1-ATPase in a hypoxic and RET state, inhibiting ATP degradation and maintaining or increasing ATP concentration or slowing ATP consumption (see Examples). This is a reversible reaction desirable for mitochondrial stability, and depending on the cell or mitochondria, it may result in a reverse reaction. This is due to mitochondrial hydrogenase activity, and proton reduction may occur in the target mitochondria, generating hydrogen molecules. The induction of hydrogenase activity, which not only dissociates hydrogen molecules but also generates hydrogen molecules from protons, is associated with the mechanism by which the cell-cell interaction promoter of the present invention, which contains hydrogen and induces hypoxia in the target cells, secretes an active ingredient with cell-cell interaction properties including mitochondria.

[0072] The components used in the intercellular interaction promoter containing hydrogen and having hypoxia-inducing ability may be not only ultra-high concentration hydrogen-containing water exceeding the saturation concentration of hydrogen molecules in the water, but also substances containing chemical hypoxia, such as cobalt chloride (CoCl2), or enzymes that induce hypoxia.

[0073] (Blood coagulation inhibitor) The cell-cell interaction promoter of the present invention can also be used as a blood coagulation inhibitor. In the case of blood cells including platelets, aggregation is inhibited by applying the hydrogen-containing cell-cell interaction promoter of the present invention to the cells immediately after blood collection to release cell-cell interaction components including mitochondria. Depending on the time of hydrogen action, the released cell-cell interaction components including mitochondria may not contain hydrogen after the action of hydrogen.

[0074] The same applies when the present invention is applied to fat cells, bone marrow cells, stem cells, etc. collected from a living body. ＋ It may also contain precursors, vitamins B and K (including menaquinone: MK7 found in natto and phylloquinone: vitamin K1 found in plants), vitamins such as vitamins C and E, sugar chains including trehalose, quinones, and superoxides such as KO2.

[0075] The cell-cell interaction promoter of the present invention can exert different beneficial effects than platelet-rich plasma (PRP). Furthermore, in the case of, for example, therapeutic cultured cells or microbial cells, the contents and functions of the cell-cell interaction promoter may differ depending on the time for which hydrogen molecules are allowed to act. The promoter has different uses depending on whether the time is a short period of time, such as a few minutes to a few tens of minutes, within 1-2 hours, within 24 hours, or within 72 hours. In the case of microorganisms and animals, including humans, the method of the present invention contributes to the health of individuals with intestines, for example, in the intestinal environment.

[0076] (Mitochondria-releasing agent and mitochondria (preparation)) The cell-cell interaction promoter of the present invention reduces DNA damage in free mitochondria by releasing, secreting, or liberating mitochondria from blood cells including platelets or other stem cells and adipocytes, and also changes or partially amplifies the copy number of free mitochondrial DNA, thereby enabling qualitative, quantitative, and temporal changes in RNA, peptides, and proteins expressed from mitochondrial DNA.

[0077] This is because the released mitochondria can suppress cell degeneration and damage, including aging, of the target cells that affect cell-to-cell interactions, make the target cells fresh or young, or activate their metabolism, thereby protecting or enhancing the function of the target cells.

[0078] In this case, the cells that release mitochondria may be damaged, injured, or dead cells, such as muscle cells or fat cells damaged by anaerobic exercise. Muscle cell overload and damage caused by exercise can be considered a type of physiological anti-aging that releases mitochondria from muscles, and the intercellular interaction promoter of the present invention complements and enhances the benefits of this physiological release and release of mitochondria. In these cases, the effects of the hydrogen-containing intercellular interaction promoter of the present invention can be confirmed not only by suppressing oxidative damage and damage to mitochondrial DNA and subsequent mutation, but also by observing changes in blood muscle damage markers that increase following anaerobic or overload exercise in animals and humans.

[0079] The mitochondria may be mitochondria exposed to hydrogen, mitochondrial particles themselves contained in cells exposed to hydrogen, or mitochondrial components such as respiratory complexes (including supercomplexes such as CI+2xCIII and CI+2xCIII+CIV) and mitochondrial DNA (a structure containing mitochondrial DNA-binding proteins). Examples of mitochondria include mitochondrial donor cells, i.e., cells capable of transferring mitochondria to other cells, including fat cells, stem cells, blood cells, vascular endothelial cells, and other cells whose mitochondrial function has been enhanced or activated by hydrogen's uncoupling effect on the mitochondrial inner membrane potential difference or the rectification effect of, for example, RET in the electron transport system.

[0080] Furthermore, the mitochondria (preparation) includes providing a mitochondrial preparation for use in normalizing or activating cells by using hydrogen to proliferate mitochondria extracellularly in the presence of living cells, or by using hydrogen to proliferate mitochondria in the presence of dead cells or cell components.

[0081] Furthermore, a cell activator can be prepared by removing water from mitochondria (preparations) extracted from cells, blood, or cultured cell culture medium, or by adjusting or culturing them using the above methods, or by using a cell activator consisting of a mitochondria-containing component containing a water-retaining substance such as trehalose and a small amount of water molecules, and administered orally, by injection into the blood, subcutaneously, or intramuscularly, or by direct administration from a blood vessel to a target tissue using a catheter, or by administration into the brain or spinal cord, or subarachnoidally or subdurally, or by spraying into the respiratory tract and allowing it to be absorbed through the respiratory tract mucosa or alveoli, thereby activating cells or enhancing the effect of an anticancer drug on cancer cells.

[0082] Furthermore, in culturing or preparing target cells, multiple types of mixed gases containing hydrogen, oxygen, carbon dioxide, nitrogen, and the like are used to repeatedly select and cull cells. This allows for the preparation of cells that are rich in excellent mitochondrial function (functional mitochondrial) and exhibit mitochondrial donor ability under conditions such as normoxia or hypoxia in an in vivo environment, or in the presence of high concentrations of oxygen exceeding atmospheric oxygen concentrations, or in the presence of nitric oxide, carbon monoxide, carbon dioxide, high concentrations of carbon dioxide, helium, or the like, or in the presence of nanobubbles of these gas molecules. This method can be used to improve the capabilities of mesenchymal stem cells, including those derived from adipose tissue or bone marrow, nerve cells, or white blood cells such as monocytes, macrophages, or lymphocytes, and platelets, making them suitable for biotherapy, and these cells can be used for treatment.

[0083] Furthermore, soluble cytosolic-hydrogenase or (O2-sensitive or O2-resistant) membrane-bound hydrogenase can be expressed in mammalian cells, including human cells, or plant cells, and the cells can be cultured in the presence of hydrogen or in an anaerobic environment. Mitochondria can then be extracted from the culture medium or cells and used as a promoter of cell-cell interaction.

[0084] Furthermore, the mitochondrial respiratory complex (including the supercomplex), membrane fraction, or DNA (mitochondrial DNA), or a mixture thereof, can be used as a cell activator for promoting cell-to-cell interactions to activate and normalize senescent cells, dysfunctional cells, etc. For example, senescent cells can be senolyzed or reprogrammed and rejuvenated.

[0085] Furthermore, not only during the process of adjusting mitochondria (preparation), but also in the presence of hydrogen gas or a solution containing dissolved hydrogen molecules, the mitochondria (preparation) can be made to act on cells or living organisms.

[0086] The method can also be used to act on adipocytes, for example, by converting white adipocytes, which store lipids intracellularly, into brown or beige adipocytes that produce heat rather than ATP by lowering the membrane potential through mitochondrial uncoupling, thereby improving conditions such as metabolic syndrome (browning white adipocytes). The method is characterized by using molecules composed of hydrogen gas or a gas mixture containing hydrogen gas as an adipocyte metabolic activator. For example, excised white adipocytes can be browned or beige-colored using the method of the present invention and then returned to the living body, improving the energy metabolism of the entire individual and providing a lifestyle-related disease treatment that alleviates diabetes, hyperlipidemia, arteriosclerosis, and cardiovascular disorders.

[0087] Furthermore, it improves the body's lipid metabolism and provides a method for preventing the aggravation of diseases such as COVID-19, which are exacerbated by abnormalities in lipid metabolism, vascular endothelial cells, and the blood coagulation system.

[0088] Furthermore, it can be used as a metabolic activator that promotes lipid oxidation or lipid synthesis in adipocytes, characterized by being composed of water containing hydrogen molecules. Furthermore, these contents also contribute to the field of drug discovery, characterized by administering candidate drugs such as candidate molecules, proteins, peptides, nucleic acid molecules, or gaseous molecules that are expected to have medicinal effects to affected cells that have been treated with an intercellular interaction promoter and control cells that have not been treated, and finding candidate drugs that have different medicinal effects due to the bioenergetic shift in the affected cells and the control cells.

[0089] Furthermore, the present invention includes mitochondria in which the electron transport chain is strengthened by the action of hydrogen molecules to enhance the uptake of quinones into respiratory complexes I, III, IV, and V, particularly into the supercomplex consisting of these complexes, and into the quinone pool in the mitochondrial inner membrane.Similarly, the present invention also includes mitochondria in which the electron transport chain is strengthened by reducing cytochrome C or Fe-S (iron-sulfur) clusters in respiratory complexes III-IV, or the heme or porphyrin contained therein.

[0090] (Plant Activator) The cell-cell interaction promoter of the present invention can be used as an activator for plant cells. For example, when acting on soil microorganisms, a substance containing molecular hydrogen is applied to isolated and cultured bacteria, including anaerobic bacteria such as actinomycetes, to induce a bioenergetic shift in these cells, promoting cell-cell interactions. This can stimulate plant germination and growth, and revitalize plants that are dying or struggling to grow (Example 3). By isolating soil microorganisms and increasing the proportion of a certain population while applying the cell-cell interaction promoter of the present invention, effects specific to each cell type can be imparted to plant cells.

[0091] Even when acting on the plant itself, by acting on a part of the target plant, the hydrogen-containing intercellular interaction promoter of the present invention can activate parts that are not in direct contact with the plant, or indirectly activate the cells of other nearby plant individuals, or provide a method for attracting insects necessary for pollination.The hydrogen-containing intercellular interaction promoter itself exerts a pheromone effect, attracting insects, etc.

[0092] Furthermore, when the present invention is applied to, for example, plant seeds themselves, yeasts and fungi (mushrooms) that coexist with a wide variety of bacteria in the soil, and soil insects, nematodes, and pill bugs, even those that are inherently harmful to plants, the action of the present invention induces a bioenergetic shift in these cells, promoting intercellular interactions. In addition to plants, the present invention can also be applied to mushrooms in the soil where mycelia spread, or to the cells of other species that interact with them, thereby increasing the yield of cultivated mushrooms. Mushrooms are organisms similar to yeast, and since the bioenergetic shift described in this research example is induced in yeast mitochondria, the present invention can also be applied to fungus. For example, when cultivating mushrooms such as shiitake mushrooms using logs, electrostatic stimulation such as lightning strikes can be effective, and hydrogen, as an electron donor, has the same effect as electrostatic stimulation such as lightning strikes.

[0093] By applying the hydrogen-containing cell-cell interaction promoter to plants, yeast, and bacteria to convert or stimulate the metabolism of the cells, it is possible to promote the growth, germination (including seed germination of F1 varieties), flowering, and fruiting of plants, or to increase the sugar content and nutrient content of vitamins and other nutrients in fruits and harvested products.

[0094] In addition to the application of the hydrogen-containing intercellular interaction promoter, non-mammalian organisms such as plants (including cells and protoplast units), mushrooms (including fungi and filamentous fungi), yeast, and bacteria (including archaea that produce substances useful to humans and the environment, and which may be anaerobic or aerobic) can be grown or cultured in the presence of gaseous or water-dissolved hydrogen molecules to activate or alter their energy metabolism, thereby promoting plant growth and germination, or increasing the sugar content and vitamin and other nutrient content of fruits and harvested products. In other words, not only administration of hydrogen molecules alone but also addition of the hydrogen-containing intercellular interaction promoter can optimize the energy efficiency of each cell type in the bioenergy shift, alter metabolism, and produce useful phenotypes.

[0095] By applying the hydrogen-containing cell-cell interaction promoter to plants and optimizing (and often increasing) the electron transport and energy efficiency in, for example, photosystem I or II, it becomes possible to help plants secure the electron energy necessary for survival and growth, even when they have access to only a small amount of water.

[0096] This method aids plant growth in areas where plant ecosystems have been weakened by deforestation, such as deserts, and leads to greening. It is known that even when light rain does not return sufficient water to the earth, a certain amount of water vapor is maintained in the air. Even a slight increase in the efficiency of photosystem I or II can promote plant adaptation to water-deficient environments and their subsequent evolution. Conversely, this method can generate electricity by releasing electrons from plants and surrounding (including symbiotic) microorganisms in the soil or while they are waiting.

[0097] This includes optimizing electron transport and energy efficiency by supplying electrons and protons to the heme of the chlorophyll dimer and the oxygen-evolving complex when the oxygen-evolving complex (a cluster consisting of four manganese and one calcium) that is the active center of photosystem II is insufficient in extracting four electrons from water. This optimization of energy efficiency by hydrogen molecules in photosystem II optimizes (often increases) the efficiency of NADPH and ATP supply to the electron transport chain in photosystem I, making the electron transport chain to photosystem I more efficient and helping to fix carbon dioxide into organic matter. This, in turn, leads to a reduction in carbon dioxide emissions.

[0098] As a result, not only does it improve plant growth efficiency, but it also increases the sugar content, acidity, and other nutrient content of fruits. Also, some plants synthesize vitamin K1 (phylloquinone), which can be used as a supplement together with quinones or alone.

[0099] The method for administering the hydrogen-containing intercellular interaction promoter does not necessarily require continuous supply. For example, when promoting seed or seedling germination, the agent can be administered temporarily at a timing based on changes in the relevant genes of the cells, or at a timing synchronized or shifted with the irradiation of light of various wavelengths, or intermittently at regular or pre-programmed intervals. This can also serve as a substitute for the action of static electricity, such as that caused by lightning strikes.

[0100] Similarly, when hydrogen gas is added to the air in, for example, a plant growth chamber or a greenhouse, it can be added continuously or intermittently at preprogrammed intervals. The term "program" here refers to adjusting the electron transport system, i.e., energy metabolism, for the vital reactions required for photosynthesis, such as genes, metabolic products, water content, and light absorption efficiency at the cellular or individual plant level, and is linked to the state of the plant, so it does not necessarily refer to a single program.

[0101] (Other) The hydrogen-containing intercellular interaction promoter of the present invention uses hydrogen molecules generated by metal oxidation of water to alter the energy metabolism of soil microorganisms (including unicellular organisms with energy converters, such as bacteria, yeast, fungi, and archaea) and act on plant cells. When used as an energy metabolism converter containing hydrogen molecules to stimulate plant growth, hydrogen can be generated using hydrogen-generating metals, including alkali metals or amphoteric metals such as cobalt, nickel, iron, manganese, or aluminum, which are useful for the growth of plant cells, fungi (including filamentous fungi), mushrooms, and protoplasts of both fungi, yeast, and bacteria (including archaea). Metal nutrients that generate hydrogen molecules can be provided simultaneously with the generated hydrogen, and the culture medium and its manufacturing method can be used. Both of these applications involve the bioenergy shift of the present invention.

[0102] The hydrogen-generating metal used in this method is embedded in, for example, nonwoven fabric or a mesh bag used for tea bags, or placed directly in a bottle made of metal, glass, or plastic. Water, acidic water (in the case of aluminum, this includes reaction with basic compounds such as calcium hydroxide, which is amphoteric), or an aqueous solution containing an organic solvent containing alcohol or phosphorus is added, and the metal reacts with water molecules in the sealed container to generate hydrogen gas.

[0103] The hydrogen molecule concentration in the container is preferably as high as possible, for example, a high concentration exceeding the hydrogen saturation concentration of 1.6 ppm.

[0104] Instead of or in addition to generating hydrogen gas in a container, high-pressure hydrogen gas or liquid hydrogen used in fuel cells may be added to the culture solution containing nutrient metals. In this method, industrial hydrogen gas or liquid hydrogen is ultra-high pressure, so it must be diluted with water in stages, and a high-concentration hydrogen-filled hydrogen water container is used for dilution with water.

[0105] Just before use, the hydrogen concentration is adjusted in this hydrogen gas concentration dilution container to a concentration 100 to 200 times higher than, for example, a 1 to 20 ppm hydrogen concentration, and ultra-high concentration hydrogen water, approximately 1 / 100 to 1 / 200 of the required culture solution, is added to the culture solution. It can also be used for drinking water containing only hydrogen. In this case, a filter is used in the high-concentration hydrogen water container at the stage of adding it to the drinking water to prevent impurities other than hydrogen and water from being contained in the drinking water. The hydrogen molecules used may also be in a gaseous state, for example, in a nanobubble state, which is bubbled into the water or culture solution. Furthermore, nanobubble hydrogen can also be generated by depressurizing the container containing the high-concentration hydrogen water, and these can be supplied simultaneously.

[0106] The hydrogen-containing intercellular interaction promoter may be added with superoxides such as KO (potassium superoxide), or quinones such as ubiquinone, menaquinone, phylloquinone, or quinone rings, and reacted with water molecules in a sealed state to generate hydrogen gas, thereby forming a mixed solution with high concentration hydrogen. This may provide the above-mentioned mixed culture solution and the same culture solution containing metals useful for the growth of plant cells, mushrooms including fungi (including filamentous fungi), and protoplasts of both, yeast, and bacteria (including archaea).

[0107] Superoxides such as KO2 are known to generate superoxide when dissolved in water. This method can donate electrons to quinones such as Q10, and in the generation of hydrogen molecules, a culture medium containing not only quinols important for cellular energy metabolism but also quinone intermediates containing semiquinone radicals is obtained. NaO2 or LiO2 may also be used as the superoxide. This method is included in the energy production method of the present invention.

[0108] Furthermore, when using this superoxide, by adding organic acids such as formic acid or acetic acid, or acids such as hydrochloric acid or sulfuric acid to the culture solution in advance, not only can the pH be adjusted, but protons in the solution generate hydrogen gas from metals, and these organic acids and sulfides themselves affect the metabolism of cells, thereby improving the intended function of the culture solution.

[0109] In the above-mentioned hydrogen-containing intercellular interaction promoter, the generator or culture solution can be irradiated with, for example, ultraviolet light, infrared light, or visible light including light of 550 to 700 nm to photoexcite photoexcitable molecules such as metals, porphyrins, and quinones, thereby converting photosensitive metabolic activators such as quinones, porphyrins, and anthocyanins into different forms, thereby increasing the repertoire of culture solutions.

[0110] Light irradiation may be performed on the culture medium after the reaction with the generator. This method also includes adding a molecule containing isoprene, such as that contained in Q10, to the culture medium. This method also includes adding an HO decomposition enzyme such as catalase or an antioxidant such as glutathione (GSH or GSSG), or conversely, adding HO as an oxidizing agent.

[0111] The hydrogen-containing intercellular interaction promoter can be used as a cell metabolism activation culture medium by adding compounds containing a pyrrole ring, such as respiratory complexes and supercomplexes contained in mitochondria, mitochondrial DNA (structures containing mitochondrial DNA-binding proteins, etc.), chlorophyll, thylakoids (including electron transport complexes), hemes, urobilinogen (including urobilin), and porphyrins containing hemes coordinated with transition metals such as iron atoms, to the hydrogen-containing intercellular interaction promoter. Both mitochondria and thylakoids may be included, or any combination of the above compounds may be used.

[0112] The hydrogen-containing intercellular interaction promoter may be added with or without a hydrogen-generating metal-containing activator, such as xanthine or hypoxanthine, NMN, NR, or NADP. ＋ , NADPH, NAD ＋Alternatively, metabolic substrates such as NADH, FAD, FADH2, carbohydrates such as glucose and trehalose, and lipids such as triglycerides, triglycerol, cholesterols, phospholipids, and isoprene may be added. These contents also contribute to the field of drug discovery, which is characterized by administering a candidate drug, such as a candidate molecule, protein, peptide, nucleic acid molecule, or gaseous molecule, that is expected to have a medicinal effect to affected cells that have been treated with an intercellular interaction promoter and control cells that have not been treated with the promoter, and finding a candidate drug that has different medicinal effects due to the bioenergetic shift in the affected cells and the control cells.

[0113] The hydrogen-containing intercellular interaction promoter can be applied to yeasts that ferment raw materials when producing alcoholic beverages such as wine, sake, shochu, and whiskey. Fermented beverages, including wine, sake, and beer, have been essential to human life since ancient times. However, in recent years, the diversification of alcoholic beverages has led to an expansion of the industry toward more sophisticated beverages. Preference is determined by taste factors, such as what consumers perceive as "delicious," and health-consciousness, such as what consumers perceive as healthy. Among taste factors, acidity and sugar content are particularly important. Furthermore, the type and amount of nutrients, including amino acids and other vitamins, play a significant role in the fermentation process, as do the metabolism of microorganisms such as yeast. In this case, the hydrogen-containing intercellular interaction promoter can be applied to barrels used during the brewing process or the aging process after brewing.

[0114] The hydrogen-containing intercellular interaction promoter of the present invention is expected to have a biological effect similar to that of a supplement in a sense for those who are health-conscious. In this sense, the metabolic products that are decomposed or secreted or released by microorganisms such as yeast during the fermentation process are important, and the yeast also interacts with surrounding bacteria and their components, wood such as in barrels, and components containing lignin and cellulose, thereby diversifying the fermentation metabolites or receiving various metabolic stimuli from microorganisms including bacteria containing hydrogenase. By acting together with the hydrogen-containing intercellular interaction promoter, the yeast can further diversify the fermentation process and contribute to the fermentation industry.

[0115] By administering or allowing the hydrogen-containing cell-cell interaction promoter and related energy metabolites of the present invention to act on microorganisms such as yeast, it is possible to obtain microbial fermentation and decomposition metabolites that could not be obtained by conventional brewing methods, and it is expected that fermented foods that were previously obtained will be produced more efficiently, or will have a better taste and nutrient balance.

[0116] In yeast and other fungi, mitochondria are responsible for energy metabolism, and the invention of patent [JP WO2019 / 225669] induces mitochondrial hydrogenase activity and rectifies the electron transport chain, optimizing cellular metabolism and changing and streamlining fermentation metabolic functions.

[0117] The timing of adding the additives of the present invention, including molecular hydrogen, to yeast can be adjusted not only under conditions in which the additives are constantly present, but also intermittently, or by programming the timing related to the cellular energy metabolism and gene expression, to use an action program tailored to the taste and nutrients of the target fermentation product. The program here refers to adjustments made from the perspective of cellular energy metabolism, such as genes or metabolic products, water content, and light absorption efficiency at the cellular or microbial population level, and is linked to the state of the microbial cells and fermentation products, and does not necessarily refer to a single program.

[0118] The administration of the hydrogen-containing cell-cell interaction promoter is applicable to, for example, fungi such as yeast that ferment raw materials, and coexisting bacteria including archaea and Bacillus subtilis, when producing not only wine, sake, beer, and other alcoholic beverages, but also fermented foods such as miso, cheese, and natto. Archaea and bacteria have respiratory chain complexes, equivalent to the mitochondrial electron transport system, incorporated into their bacterial membranes, and many fungi express hydrogenases that use hydrogen molecules or protons as their original substrates.

[0119] The activation of hydrogenase itself and the subsequent production of useful metabolic products are also goals of administering hydrogen-containing cell-cell interaction promoters. Similar to the induction of hydrogenase activity in mitochondria and the rectification of the electron transport chain by hydrogen molecules, hydrogen molecules also optimize the metabolism of these archaeal and bacterial cells, leading to changes and increased efficiency in the fermentation metabolic function. Furthermore, as mentioned above, some bacteria, particularly Bacillus subtilis, synthesize vitamin K2 (menaquinone: MK7), which can be used as a supplement either together with quinones or alone.

[0120] When nematodes or cells are used as sensors for metabolites released by mammalian or plant cells, a hydrogen-containing intercellular interaction promoter can be used to induce metabolic changes in the sensor organism or cell, thereby educating and enhancing its sensor function. This is also a method for improving the function of olfaction, a chemical sensor found in almost all living organisms, from microorganisms and insects to humans. For example, cats can detect hydrogen through their sense of smell, suggesting that hydrogen molecular dissociation in olfactory molecules or sensors affects quantum coherence and decoherence, providing a method for improving quantum chemical sensors. As mentioned above, this provides valuable application value as a beneficial pheromone.

[0121] Hydrogen-containing cell-cell interaction promoters can induce metabolic changes in cells or organisms, including humans, plants, and microorganisms, thereby extending the cell's lifespan or increasing the number of cell divisions. This is possible by improving the balance of the electron transport chain and its closely related energy metabolic substances, which are present in almost all living organisms, from microorganisms and insects to humans, and by inducing the associated epigenetic reprogramming. Here too, the hydrogen molecule dissociation reaction and the resulting electrons and protons are thought to affect quantum coherence and decoherence, providing a quantum method for improving energy efficiency. When a dissociation reaction of hydrogen molecules occurs in the hydrogenase of microorganisms or in the mitochondria of eukaryotic cells (when hydrogenase activity is activated), the hydrogen molecules, which are boson particles, may in some cases be in a quantum-physical identical state, i.e., a state similar to Bose-Einstein condensation, in relation to the amino acid residues or metal atoms coordinated around them at the site of the dissociation reaction, i.e., the active center of the enzyme, or to the central metal of a porphyrin containing an iron-sulfur cluster (Fe-S cluster) or a pyrrole group involved in electron transfer, or to a porphyrin molecule not coordinated with a metal. When a hydrogen molecule dissociates in this state, it is converted into a plurality of fermions, i.e., protons and electrons, which cannot be in a quantum-physical identical state. This transition to a plurality of fermions with energy levels different from those of the boson particle state results in differences in energy levels and distribution at the position of the active center or during the reaction time. It is thought that this energy difference may change or promote the hydrogen molecule dissociation reaction in the hydrogenase or mitochondria. As will be described in detail later, Marcus theory can be applied to the hydrogen molecule dissociation reaction within cells, making it possible to apply the quantum tunneling effect to life.

[0122] Furthermore, the intercellular interaction promoter of the present invention can be used in a test system or kit to measure changes in the amount or concentration of mitochondria in body fluids or blood secreted or released from mitochondria-containing cells, such as striated muscle, smooth muscle, and platelets in the body, to understand the amount of free mitochondria in the blood (blood concentration and total amount in the blood) due to diurnal variation, exercise, physical and psychological stress, etc., and to optimally activate the function of mitochondrial release, secretion, or freedom accordingly.

[0123] In addition, a test system or kit using an intercellular interaction promoter may be used to confirm the mitochondrial concentration in the blood after application of the intercellular interaction promoter, and may also be used to optimize the function of mitochondrial release, secretion, or liberation.

[0124] Furthermore, a test system or kit using an intercellular interaction promoter could be used to observe the sequence of mitochondrial DNA contained in body fluids, blood, and urine, detect mutations, modifications, duplications, deletions, etc., and optimize the release, secretion, or liberation of mitochondria.

[0125] Furthermore, the mitochondrial DNA contained in the body fluids, blood, and urine to be observed may be mitochondrial DNA released by using an intercellular interaction promoter.

[0126] As described above, the test system or kit records fluctuations in the amount of free mitochondria due to stress on the subject's mind and body, and can be used to adjust the intensity and timing of exercise, as well as to avoid damage caused by mental stress, overwork, excessive exercise, etc. Furthermore, the use of an intercellular interaction promoter can reduce damage caused by mental stress, overwork, excessive exercise, etc.

[0127] As described above, the cell-cell interaction promoter of the present invention secretes, liberates, or releases cellular components or metabolites that have cell-cell interactions, including mitochondria, through the action of components containing hydrogen molecules.

[0128] That is, the cell-cell interaction promoter is composed of gaseous hydrogen molecules (hydrogen gas), a mixed gas containing hydrogen molecules, or water containing hydrogen gas, or any of these containing other active ingredients.

[0129] In the case of hydrogen gas or a mixed gas containing hydrogen gas, hydrogen gas can be administered to a subject (such as a human, plant, or microorganism, or a cell) using a conventionally known hydrogen gas supply device. Specifically, when the subject is a human or animal, administration is performed by inhalation of hydrogen molecular-containing gas or transdermal absorption using a conventionally known medical hydrogen gas (hydrogen mixed gas) supply device. A conventionally known medical hydrogen gas (hydrogen mixed gas) supply device is, for example, a hydrogen gas generator from Enoa. In the case of a mixed gas, the gases contained other than hydrogen are not particularly limited as long as they are not harmful to the living body, and examples include oxygen and air. Furthermore, when administering hydrogen gas or a mixed gas containing hydrogen gas by inhalation or transdermal absorption, it is possible to perform the treatment in a store equipped with a hydrogen gas supply device, or to purchase or lease the machine itself and perform the treatment yourself.

[0130] Water containing hydrogen gas (hydrogen water) can be administered to living organisms by drinking it as is. Water containing hydrogen gas may also contain conventionally known components, such as salts and nutrients, that are not toxic when administered to living organisms. For oral intake of hydrogen water, producers may produce hydrogen water using a hydrogen water generator and sell it in bottles, or individuals may purchase or lease a generator and generate and consume the water themselves. Furthermore, producers may produce and sell hydrogen powder that can be processed into a powder and dissolved in water.

[0131] The hydrogen water may contain nanobubbled hydrogen, and may be produced by applying ultrasonic waves or electromagnetic waves to the nanobubble hydrogen-containing water, or by heating the water to burst the bubbles or further break them down into smaller bubbles.

[0132] Other possible administration methods include intravenous drip infusion or transdermal absorption of nanobubble hydrogen gas or a liquid containing hydrogen molecules. (Example 1)

[0133] The inventors conducted the following experiments to investigate how hydrogen molecules improve the life-threatening condition caused by the excessive generation of superoxide by semiquinone radicals during immune cell activation due to inflammation or reverse electron transport (RET) during post-ischemic reperfusion injury, that is, to clarify the mechanism of the medical effects of hydrogen molecules when the mitochondrial electron transport system is in the RET state (e.g., dissociating hydrogen molecules and rectifying the flow of electrons). The inventors investigated how hydrogen molecules affect the dynamic equilibrium between superoxide and quinone, or between oxygen molecules and semiquinone radicals in solution, or the entire reaction system.

[0134] The dynamic equilibrium between superoxide and quinone, or oxygen molecules and semiquinone radicals, refers to the state of equilibrium in which superoxide and quinone in a solution repeatedly change to oxygen and semiquinone radicals through an oxidation-reduction reaction. ・－ , Q.H. － , Q.H. ・ In this case, reactions can occur not only between oxygen molecules but also between superoxide and semiquinone radicals, which are also included in this reaction system.

[0135] In this example, the reaction of hypoxanthine / xanthine oxidase in solution was used to investigate what reaction occurs between the generated superoxide and ubiquinone (coenzyme Q10) and / or hydrogen molecules.

[0136] In Figures 1 to 3, superoxide was generated by a reaction of hypoxanthine / xanthine oxidase at a concentration of 2 μM and 0.01 units / ml, and the amount was measured by the fluorescence count of MPEC (2-Methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo pyrazin-3: a reagent that specifically quantifies superoxide by utilizing a luminescent reaction with superoxide). The box plots in each figure show the difference in the amount of superoxide depending on the hydrogen molecule concentration. Each box shows the distribution and fluctuation of the amount of superoxide at each concentration of hydrogen molecules.

[0137] Figure 1 shows the results for 0 μM ubiquinone, 0 μM hydrogen molecules (left box), 0.2 μM hydrogen molecules (center box), and 0.8 μM hydrogen molecules (right box). The results show that when superoxide was generated by a reaction of 2 μM hypoxanthine / xanthine oxidase and 0.01 units / ml, in the absence of ubiquinone, an increase in superoxide was observed when the hydrogen molecules were 0.2 μM, but conversely, a decrease in superoxide was observed when the hydrogen molecules were 0 μM and 0.8 μM.

[0138] Figure 2 shows the results for 10 μM ubiquinone and 0 μM (left box), 0.2 μM (center box), and 0.8 μM (right box) hydrogen molecules. The results show that when superoxide was generated by a reaction of 2 μM hypoxanthine / xanthine oxidase at 0.01 units / ml, in the presence of 10 μM ubiquinone, an increase in superoxide was observed at 0 μM and 0.8 μM hydrogen molecules, but conversely, a decrease in superoxide was observed at 0.2 μM hydrogen molecules.

[0139] Figure 3 shows the results for 50 μM ubiquinone and 0 μM (left box), 0.2 μM (center box), and 0.8 μM (right box) hydrogen molecules. The results show that when superoxide was generated by a reaction with 2 μM hypoxanthine / xanthine oxidase at 0.01 units / ml, in the presence of 50 μM ubiquinone, an increase in superoxide was observed at 0 μM and 0.8 μM hydrogen molecules, whereas a decrease in superoxide was observed at 0.2 μM hydrogen molecules.

[0140] In both cases, it was found that hydrogen molecules intervened in the reactions of superoxide or semiquinone radicals, causing changes in the amount or stability of superoxide generated. In other words, hydrogen molecules interacted with the dynamic equilibrium between superoxide and quinone, or between oxygen molecules and semiquinone radicals, or with the entire reaction system, dissociating into electrons and protons in the process.

[0141] For example, the amount of superoxide produced in this reaction is reversed when the quinone concentration is 0 μM, 10 μM, and 50 μM. Even under the same quinone concentration, the amount of superoxide produced is reversed when the hydrogen concentration is 0.2 μM, and when it is 0 μM and 0.8 μM. In other words, it can be inferred that the interaction in the entire reaction system is not due to a typical catalytic reaction, but rather to the quantum tunneling effect, which can be explained by Marcus theory, and that the reaction proceeds by overcoming the activation energy barrier required for dissociation.

[0142] For the experiments shown in Figures 1 to 3, the average values ​​and SDs of four measurements were calculated, and two-dimensional analysis of variance was performed on the main effect (radical reactions between superoxide and semiquinone radicals and hydrogen molecules, respectively) and the interaction effect (interaction in the dynamic equilibrium reaction system between superoxide and quinone, or oxygen molecules and semiquinone radicals). The P value indicating a significant difference in the interaction of the experiments shown in Figures 1 to 3 was p = 2.38 × 10 －１７ This indicates that the observed effect is statistically highly significant and is not a chance response. (Example 2)

[0143] 4 is a bar graph showing the quantitative analysis of NADH (black) and ATP (white) released from mitochondria using HPLC (liquid chromatography). Yeast cells in a culture medium were treated with a hydrogen-containing intercellular interaction promoter, and the resulting yeast was then briefly coexisted with another yeast (control yeast). Mitochondria extracted from the target yeast were analyzed by HPLC (liquid chromatography). When mitochondria were subjected to the addition of, for example, (dimethyl)succinic acid or CoCl2, or under hypoxic conditions with an oxygen concentration of 50% or less, and observed under RET (reverse electron transport) conditions (the two leftmost bars in the graph), ATP synthesis (open bars) was reduced. However, when the hydrogen-containing intercellular interaction promoter of the present invention was applied (right arrow in the graph), ATP synthesis increased approximately two-fold (open bars on the right side of the graph). During this RET induction, NADH (black bar in the figure), the initial metabolite of the electron transport chain, increases with the increase in reactive oxygen species (ROS) such as superoxide. ＋ It was previously observed that NADH was retrosynthesized from ATP, but when the hydrogen-containing intercellular interaction promoter of the present invention was applied (right-pointing arrow in the figure), NADH was reduced (consumed), and the mitochondrial membrane potential also changed by several percent. These results indicate that when the hydrogen-containing intercellular interaction promoter of the present invention was applied, the electron transport system actually shifted from an ATP-reduced state caused by hypoxia to an ATP-increasing state. This indicates that the hydrogen-containing intercellular interaction promoter induced a so-called bioenergetic shift in the target yeast mitochondria, which changes the composition ratio of NADH and ATP, molecules essential for cellular energy. Figure 4 shows an analysis of data measured after inducing RET, such as hypoxia, in mitochondria derived from the target yeast. A similar bioenergetic shift was also observed in mitochondria extracted from the target yeast after RET was induced. A similar bioenergetic shift was also induced when the hydrogen-containing cell-cell interaction promoter of the present invention was directly applied to mitochondria extracted from yeast (Example 3).

[0144] Figure 5 shows photographs of the results of applying the hydrogen-containing intercellular interaction promoter of the present invention to the soil of a potted finger lime. The top panel shows approximately 2 ml of water containing the hydrogen-containing intercellular interaction promoter of the present invention, added once daily for seven days; the top right panel shows the results one month later. The bottom left panel shows the same results as the top panel, where water containing the hydrogen-containing intercellular interaction promoter of the present invention was added once daily to the soil, excluding the plant parts, for seven days; the bottom right panel shows the results one month later. Applying the hydrogen-containing intercellular interaction promoter to the soil showed at least twice the growth rate compared to the control case where only water was added. In this case, the agent was not applied to at least the above-ground parts of the plant, demonstrating enhanced secretion or release of active ingredients, including or not including mitochondria, which are involved in intercellular interactions, in soil microorganisms. Similar effects were observed not only in finger limes but also in seedlings of apple berries, tomatoes, strawberries, and other plants, including strawberries that were beginning to wither. New shoots were also observed to emerge from olive seedlings that were thought to have withered. (Example 4)

[0145] 6 is a bar graph showing the ratio of ATP to ADP (ATP / ADP) quantified by HPLC, which indicates the ATP synthesis ability as a function of mitochondrial cells. The two pairs in the left column are data obtained from mitochondrial cells not treated with metformin, and the two pairs in the right column are data obtained from mitochondrial cells treated with metformin. The decrease in the two right columns compared to the two left columns indicates the inhibition of ATP synthesis, which is the inhibitory effect of metformin on complex I. The open bars represent data when the intercellular interaction promoters described in claims 1 to 3 were not applied, and the black bars (H2 +) represent data when the intercellular interaction promoters described in claims 1 to 3 were applied.When the intercellular interaction promoters of the present invention were applied, even in the presence of metformin, the ATP / ADP ratio increased by approximately 76% due to the intercellular interaction promoters of the present invention, and some of the ATP synthesis inhibited by metformin was maintained.

[0146] That is, under conditions where the diabetes drug metformin reduces mitochondrial ATP synthesis through the inhibition of complex I, the bioenergetic shift was triggered by the cell-cell interaction promoter containing H of the present invention, and it was observed that the drug caused changes in energy metabolism. In this case, it was shown that ATP synthesis can be rescued to some extent by the bioenergetic shift, which may reduce side effects caused by overdose, and it also provides the possibility of identifying the potential of test drugs with unknown efficacy as candidate drugs from changes in ATP synthesis ability, which is directly linked to energy metabolism. As shown in Figure 7, methotrexate (MTX), used as a control for metformin, further reduced ATP synthesis by hydrogen (Figure 7b). This indicates that the mechanism of action of MTX is not only the inhibition of folate metabolism as previously thought, but also that, like metformin, it is an inhibitor of complex I. It was also confirmed that hydrogen enhances the action of low-dose MTX, which is used as an antirheumatic drug, and acts as an adjuvant. Furthermore, MTX also reduced the NADH / NAD+ ratio (Figure 7a), suggesting that MTX acted directly on complex I or the electron transport chain. Furthermore, hydrogen molecules inhibited this effect, indicating that MTX affected the action of MTX in mitochondria via the bioenergetic shift.

[0147] In other words, a repertoire of candidate molecules, proteins, peptides, nucleic acid molecules, gaseous molecules, etc. (hereinafter collectively referred to as candidate drugs) expected to have pharmacological effects was tested on diseased cells, experimental animals, normal cells, healthy organisms, or plants. The subjects were divided into two groups: one group in which a bioenergetic shift was induced by the application of hydrogen molecules for a short period of several minutes, or for a longer period, or in some cases for more than a day, and the other group in which no hydrogen molecules were applied. The candidate drug was then applied to each group under the same conditions, and the difference in pharmacological effect between the two groups was demonstrated. In other words, the bioenergetic shift induced by the cell-cell interaction promoter of the present invention was shown to change the drug's effect. By measuring the magnitude and timing of this change, the timing of administration of the cell-cell interaction promoter of the present invention can be improved, thereby optimizing the application of the cell-cell interaction promoter of the present invention. At the same time, when administering a drug with unknown efficacy or during the drug screening stage of drug discovery, by using the intercellular interaction promoter of the present invention, it is possible to identify previously unclear drug effects and optimize the timing of administering conventional drugs, and by using the intercellular interaction promoter of the present invention in combination with the resulting drug, it is possible to find better applications for the intercellular interaction promoter of the present invention, or to identify drugs that are effective through the bioenergetic shift, thereby contributing to drug discovery.

[0148] The present invention, based on the inventor's discovery that hydrogen molecules can be dissociated into electrons and protons through the quantum tunneling effect (quantum propagation reaction), allows the energy of electrons extracted from hydrogen molecules to be utilized in various energy industries, thereby contributing to the revitalization of the energy industry. Furthermore, the inventor has discovered that the mechanism by which hydrogen molecules act on mitochondria in living organisms is rooted in the dissociation of hydrogen molecules and the accompanying supply of electrons or protons, or the supply of energy. This hydrogen molecular dissociation reaction can improve various vital functions and provide anti-aging effects, contributing to the promotion of health in organisms that have mitochondria. Furthermore, by applying the method for dissociating hydrogen molecules into electrons and protons of the present invention to fuel cells, fuel cells can be produced more cheaply than before, contributing to the development of industries that use fuel cells.

[0149] The present invention also provides a method for promoting cell-cell interactions, including self-renewal or rejuvenation, by secreting components that promote cell-cell interactions, including mitochondria, from nucleated cells, including yeast, fungi, nematodes, insects, and animal cells. Specifically, the present invention provides a method for promoting self-renewal or rejuvenation of cells other than those from which the mitochondria originate, by increasing the number of circulating mitochondria in animal cells, for example, without artificially disrupting the cells that secrete, liberate, or release mitochondria. Furthermore, numerous reports on the improvement of disease and health conditions through the administration of molecular hydrogen have not only demonstrated that molecular hydrogen acts directly on target cells suffering from pathological conditions, hypofunction, or dysfunction, but also that the addition of the hydrogen-containing cell-cell interaction promoter of the present invention to immune cells or various stem cells at the mitochondrial, cellular, tissue, or individual levels of multicellular organisms may provide even greater benefits. In plants and microorganisms, the present invention acts on cells including bacteria, fungi, insects, and the like, other than target plants and microorganisms including yeast, to secrete, liberate, or release electron transport system components that act as energy converters that generate a proton concentration gradient across membranes, such as mitochondria or hydrogenase or complex I, and then acts on target plants or microorganisms, thereby providing a cellular energy state activation technology that enables plant growth promotion and microbial metabolic conversion. Furthermore, by converting the cellular energy state according to the present invention, i.e., by preparing cells in a state in which a bioenergetic shift has occurred, and administering a drug that affects metabolism together with the cells before the state conversion, the change in effect between the two states can be used as an indicator, contributing to the strengthening of combined use of the cell-cell interaction promoter of the present invention and also contributing to new drug discovery.

Claims

1. A method for energy production by dissociation reaction of hydrogen molecules, characterized in that superoxide and hydrogen molecules are caused to undergo a propagation reaction across an energy barrier, and when the hydrogen molecules dissociate into electrons and protons, dissociation energy and / or electrical energy due to the movement of electrons or protons is generated.

2. A method for energy production by dissociation reaction of hydrogen molecules, characterized in that a quinone intermediate and hydrogen molecules are caused to undergo a propagation reaction across an energy barrier, and when the hydrogen molecules dissociate into electrons and protons, dissociation energy and / or electrical energy due to the movement of electrons or protons is generated.

3. A method for energy production by dissociation reaction of hydrogen molecules, characterized in that hydrogen molecules are introduced into a reaction system in which superoxide and a quinone intermediate coexist, and the superoxide and / or the quinone intermediate and the hydrogen molecules are caused to undergo a propagation reaction across an energy barrier, and when the hydrogen molecules dissociate into electrons and protons, dissociation energy and / or electrical energy due to the movement of electrons or protons is generated.

4. An anti-aging method, characterized in that the dissociation reaction of hydrogen molecules according to any one of claims 1 to 3 is carried out inside mitochondria.

5. The anti-aging method according to claim 4, wherein the hydrogen molecules are taken into the mitochondria by inhaling hydrogen gas or a mixed gas containing hydrogen gas or by percutaneous absorption.

6. The anti-aging method according to claim 4, wherein the hydrogen molecules are taken into the mitochondria by oral ingestion, percutaneous absorption, injection or drip infusion of physiological saline or other hydrogen-containing liquids containing or dissolved with hydrogen.

7. A fuel cell, characterized in that electrons taken out during the dissociation reaction of hydrogen molecules according to any one of claims 1 to 3 are circulated in a circuit.

8. A cell interaction promoter containing hydrogen, which acts on cells including single-celled organisms with a component containing hydrogen molecules to secrete, release or discharge cell components or metabolites having cell-cell interactions including mitochondria.

9. A cell interaction promoter containing hydrogen, which acts on mitochondria to secrete, release or discharge cell components or metabolites having cell-cell interactions.

10. The cell interaction promoter according to claim 8 or 9, which has the ability to induce hypoxia.

11. An anticoagulant containing the cell interaction promoter according to any one of claims 8 to 10.

12. A mitochondrial releasing agent comprising the intercellular interaction promoter according to any one of claims 8 to 10.

13. Mitochondria or a mitochondrial preparation released by the method according to claim 12.

14. A plant activator comprising the intercellular interaction promoter according to any one of claims 8 to 10.

15. A method for growing mitochondria extracellularly using hydrogen in the presence of living cells.

16. A method of providing the mitochondria or mitochondrial preparation according to claim 13 for cell normalization or activation by growing mitochondria using hydrogen in the presence of dead cells or cell components.

17. A cell activator comprising a mitochondrial-containing component obtained by removing moisture from the mitochondria or mitochondrial preparation according to claim 13, or comprising a water-retaining substance such as trehalose and a small number of water molecules.

18. A method of administering a candidate drug such as a candidate molecule, protein, peptide, nucleic acid molecule or gaseous molecule for which a drug effect is expected to an effector cell on which the intercellular interaction promoter according to any one of claims 8 to 10 has been allowed to act and a control cell on which it has not been allowed to act, and finding a candidate drug for which the drug effect due to a bioenergetic shift is different between the effector cell and the control cell.

19. A method for enhancing the function of an intercellular interaction promoter, which is carried out by using in combination a candidate drug having a different drug effect found by the method according to claim 18 and the intercellular interaction promoter according to any one of claims 8 to 10.

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