Mitochondrial function activator

Abstract

To provide a mitochondrial function activator that can be expected to prevent or improve (treat) diseases or conditions (symptoms) caused by decreased mitochondrial functions, such as decreased oxidative phosphorylation associated with aging, senescence, and mitochondrial dysfunction, by effectively activating mitochondrial functions.SOLUTION: A mitochondrial respiration activator comprises milt extract (hydrolyzed fish milt), preferably mitochondrial extract having a low molecular weight so as to contain 50 to 100% of a fraction having a molecular weight of 5000 or less. Such an activator has an activating effect on mitochondrial functions to prevent or improve (treat) diseases or conditions (symptoms) caused by decreased mitochondrial functions, such as decreased oxidative phosphorylation associated with aging, senescence, and mitochondrial dysfunction.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a mitochondrial function activator, and more specifically to a mitochondrial function activator containing a milt extract as an active ingredient, and a food or drink for activating mitochondrial function comprising said activator. [Background technology]

[0002] Salmon milt extract (hereinafter sometimes referred to as "SME") is a food ingredient that is rich in amino acids and nucleic acids, and it has been known to have the effect of significantly reducing oxidative damage to genes (Patent Document 1). The present inventors have been conducting intensive research to explore new functionalities of this food ingredient. In a past study, using a carbon nanotube (CNT) electrode method and a measurement system for the amount of oxidized low-density lipoprotein (LDL), it was reported that SME effectively suppresses the oxidation of LDL (Patent Document 2).

[0003] In addition, it was reported that when SME was administered to fatty liver model mice, the expression of mitochondrial metabolism-related genes (PGC1, PPAR, CPT1, CRLS, Thioredoxin) was increased compared to the group that did not receive SME (Non-Patent Document 1). In other words, it was suggested that SME may act as a mitochondrial metabolism promoter. However, its effect on mitochondrial respiration, the most important function of mitochondria, remains unknown. In other words, as a future task, it is still insufficient to prove that food has an effect of improving mitochondrial function, and it is necessary to further demonstrate by cell physiological methods that food ingredients added to cells enhance mitochondrial function.

[0004] Mitochondria are intracellular organelles that play a central role in energy (ATP) production. In the cristae of the inner mitochondrial membrane, hydrogen is converted to NADH2 produced by glycolysis and the TCA cycle. +Using FADH2 and FADH3, it reacts with oxygen (O2) through the action of a series of enzymes (complexes I, II, III, IV) to become water (H2O). The process of complexes I to IV is called the electron transport system because electrons are transferred between proteins and coenzymes in the inner mitochondrial membrane. In the process of this electron transport system, complexes I, III, and IV transport protons (H + As a result, H + A concentration gradient of 1000 keV is created. The energy released in the electron transport chain is stored as electrochemical potential.

[0005] In addition, because the inner mitochondrial membrane does not allow ions to pass through, a potential difference DΨ called the membrane potential is generated on both sides of the inner membrane. + moves to the matrix side through complex V, and H + One molecule of ATP is synthesized from ADP, phosphate, and ATP. This process is called oxidative phosphorylation and is the core of aerobic metabolism. The ATP produced is transported to the cytoplasm via the ADP-ATP translocator present in the inner mitochondrial membrane. The above series of processes is called the respiratory chain. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 3978716 [Patent Document 2] Patent No. 6628071 [Non-patent literature]

[0007] [Non-Patent Document 1] Sakurai T, et al., Dietary salmon milt extracts attenuate hepatosteatosis and liver dysfunction in diet-induced fatty liver model. J Sci Food Agric, 99:1675-81, 2019 Summary of the Invention [Problem to be solved by the invention]

[0008] It is becoming clear that activating mitochondrial function is deeply related to health promotion, for example, the relationship between mitochondria and various metabolic disorders such as diabetes and aging. However, there are few reports of food improving mitochondrial function. This is due to the methodological difficulties in scientifically proving the effects of food on mitochondrial function, and the lack of an established system for sharing such methods with society.

[0009] The objective of the present invention is to establish a mitochondrial function measurement system and use it to clarify the effect of a milt extract containing SME on mitochondrial function, particularly respiratory function, thereby providing pharmaceuticals and functional foods that have the effect of preventing or improving (treating) diseases or conditions (symptoms) caused by decreased mitochondrial function, such as decreased oxidative phosphorylation associated with aging and mitochondrial dysfunction. [Means for solving the problem]

[0010] As a result of intensive research to solve the above problems, the present inventors established a mitochondrial function measurement system using human liver-derived cells (C3A) and an extracellular flux analyzer XFp, and using this measurement system, found that SME (salmon milt extract), which is safe even when taken for a long period of time, has an effect of activating mitochondrial function, particularly on the maximum respiration and spare respiratory capacity of hepatocytes. The present invention was achieved based on such findings.

[0011] That is, the present invention is as follows. (1) A mitochondrial function activator containing a milt extract as an active ingredient, the milt extract being a hydrolysate of fish milt. (2) The mitochondrial function activator according to (1) above, wherein the milt extract is reduced in molecular weight so that it contains 50 to 100% of a fraction having a molecular weight of 5,000 or less. (3) A food or drink for activating mitochondrial function, comprising the mitochondrial function activator described in (1) or (2) above.

[0012] Other embodiments of the present invention include a method for preventing or ameliorating (treating) a disease or condition (symptom) caused by decreased mitochondrial function, comprising the step of administering a milt extract to a subject in need of such prevention or amelioration (treatment) of a disease or condition (symptom) caused by decreased mitochondrial function; a milt extract for use as an agent for preventing or ameliorating (treatment) a disease or condition (symptom) caused by decreased mitochondrial function; a milt extract for use in the prevention or amelioration (treatment) of a disease or condition (symptom) caused by decreased mitochondrial function; and use of the milt extract for the manufacture of an agent for preventing or ameliorating (treatment) a disease or condition (symptom) caused by decreased mitochondrial function. As another embodiment of the present invention, a mitochondrial function activator containing, as an active ingredient, a fish milt hydrolysate (decomposition product) obtained by treating fish milt with nuclease and protease can be mentioned as an example of a milt extract. Effect of the Invention

[0013] According to the present invention, it is expected that the milt extract has a mitochondrial function activating effect and can prevent or improve (treat) diseases caused by various metabolic disorders, aging, and decreased mitochondrial function, and promote health without side effects. The milt extract used in the present invention has no safety issues and can be freely prepared in the form of functional foods or pharmaceuticals, so it can be taken over a long period of time not only by healthy people but also by the elderly, the infirm, and those recovering from illness. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram showing a strategy (measurement system) for discovering food components that activate mitochondria. [Diagram 2] This is a diagram showing the molecular weight distribution of SME (salmon milt extract). The horizontal axis of the figure is retention time (min), and the vertical axis is absorbance in the ultraviolet region (wavelength 260 nm (upper row) or 280 nm (lower row)). In the figure, the molecular weight guideline of 12,000 is the elution position of cytochrome c. [Diagram 3] This is a diagram showing the molecular weight distribution of DNA-Na (high molecular weight DNA). The horizontal axis of the figure is retention time (min), and the vertical axis is absorbance in the ultraviolet region (wavelength 260 nm). The molecular weight guideline of 12,000 in the figure is the elution position of cytochrome C. [Figure 4] 1 shows the results of a cytotoxicity test (n=6), in which SME represents salmon milt extract, and DNA-Na represents high molecular weight DNA (hereinafter the same). [Diagram 5] FIG. 1 shows the results of evaluation of mitochondrial copy number by real-time PCR (n=4). [Figure 6] This figure shows the change in OCR profile upon addition of samples (n=4). In the figure, the arrows indicate the addition time of each reagent. [Figure 7] This is a schematic diagram for explaining a typical OCR profile (excerpted from the Prime Tech website). In the diagram, the value of each mitochondrial function item is calculated by its respective area. [Figure 8]This figure shows the results of comparing each mitochondrial function item calculated from the AUC of OCR (n=4). The symbols in the figure mean the following: A, Maximal respiration; B, Basal respiration; C, Spare respiratory capacity; D, Proton leak; E, ATP production; F, Non-mitochondrial respiration. *P<0.05 vs. control. **P<0.01 vs. control. ns, not significant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The following describes in detail an embodiment of the present invention, but the following description is an example of an embodiment of the present invention, and the present invention is not limited to the following description. In this specification, the expression "~" is used to include the numerical value or physical property value before and after it. In addition, the content (%) of the active ingredient means mass percent (wt%) unless otherwise specified.

[0016] The mitochondrial function activator of the present invention contains a milt extract as an active ingredient. As described below, the milt extract is a hydrolysate (decomposition product) of fish milt, preferably a hydrolysate (enzymatic decomposition product) obtained by enzymatic treatment with protease, nuclease, or the like, and preferably contains a specific molecular weight fraction.

[0017] Here, the milt of fish is preferably the milt (testes) of fish such as salmon, trout, and cod, from the viewpoints of containing a large amount of DNA and of effectively utilizing waste from fishery processing. Among these, salmon milt is particularly preferred from the viewpoint of raw material availability.

[0018] The method for preparing the milt extract is not particularly limited, but it can be prepared, for example, in accordance with the methods described in JP-A-2003-325149, JP-A-2004-16143, JP-A-2016-204340, etc.

[0019] For example, it is preferable to prepare the product by removing the skin, tendons, blood vessels, etc. from the above-mentioned fish milt as necessary, and then purifying it as necessary to remove oil and roughly crushing it (hereinafter sometimes referred to as "pretreatment"), followed by treatment with protease and nuclease.

[0020] Although there is no particular limitation on the nature of the protease used, a serine protease such as trypsin is preferred. Serine proteases selectively hydrolyze peptide bonds at the carboxyl side of arginine and lysine, and are therefore suitable for hydrolysis of protamines that contain a large amount of arginine.

[0021] The properties of the nuclease to be used are not particularly limited, but for example, a nuclease that cleaves leaving a phosphate group at the 5' end is preferred, and it is preferable that the nuclease has a certain degree of thermal stability. These proteases and nucleases may be appropriately selected from commercially available products.

[0022] These enzyme-treated products can be used as the milt extract in the present invention as they are, or, if necessary, in the form of a powder obtained by spray drying or the like. If necessary, they can also be further purified before use.

[0023] Here, the above-mentioned salmon milt extract (SME) is prepared by treating pretreated salmon milt with protease and nuclease (enzymatic hydrolysis) as described above.

[0024] The milt extract of the present invention includes hydrolysates of milt-derived DNA and hydrolysates of milt-derived proteins, such as protamine and its hydrolysates, and milt-derived polyamines.

[0025] The DNA content in the milt extract of the present invention is usually 1 to 80%, preferably 10 to 50%, and more preferably 26 to 36%.

[0026] The molecular weight of the milt extract in the present invention is not particularly limited, but it is preferable that the molecular weight of the DNA is reduced so that the fraction with a molecular weight of 5000 or less, preferably 3000 or less, accounts for about 50 to 100%. The molecular weight distribution is a value based on the following GPC analysis (absorbance at a wavelength of 260 nm).

[0027] As described above, the lower limit of the molecular weight of the active ingredient (molecular weight as DNA) is not particularly limited as long as the molecular weight is approximately 330 or more (number of bases is 1 or more), and it is preferable that the fraction with a molecular weight of 330 or more is 1 to 100%, and the fraction with a molecular weight of 3,000 or more is approximately 0 to 50%.

[0028] In the present invention, the molecular weight distribution can be measured by fractionating a sample based on its molecular weight using gel permeation chromatography (GPC) and then quantifying the molecular weight using a UV detector.

[0029] The mitochondrial function activator of the present invention can also be called a mitochondrial respiratory capacity activator, a mitochondrial spare respiratory capacity activator, a mitochondrial maximum respiratory capacity activator, etc., based on its action.

[0030] In the present invention, mitochondrial respiration refers to the production of ATP by consuming oxygen in mitochondria. In addition, in the present invention, mitochondrial respiratory capacity refers to the ability of mitochondria to produce ATP and / or the ability to produce ATP. Specifically, mitochondrial respiratory capacity is represented by basal respiration, maximum respiration, reserve respiratory capacity, etc. of mitochondria.

[0031] In the present invention, the mitochondrial function refers to the supply of energy (ATP) to cells through the above-mentioned mitochondrial respiration. On the other hand, in addition to the above-mentioned function, it is known that mitochondria also perform various metabolisms and regulations (for example, various metabolisms including synthesis of steroids and heme, regulation of intracellular concentrations of calcium and iron, regulation of cell cycle and apoptosis, transport of various substances, etc.), but these are not included in the "mitochondrial function" in the present invention.

[0032] FIG. 1 shows an outline of the strategy (measurement system) for searching for food ingredients that activate mitochondria, which was established by the present inventors and used in the present invention. In this measurement system, food ingredients are added to cultured liver cells, and the mitochondrial function of the cells is evaluated. An extracellular flux analyzer is used for the evaluation. If a food ingredient that activates mitochondria is discovered, the inventors will carry out expression level analysis of genes involved in mitochondrial metabolism, which they have established, lipid composition analysis using mass spectrometry or enzymatic methods, mitochondrial copy number analysis using real-time PCR, and cytotoxicity testing in order to elucidate the mechanism of activation. In the present invention, the details of the mitochondrial copy number analysis and mitochondrial function analysis will be specifically shown in the Examples described below.

[0033] Here, the decline in oxidative phosphorylation associated with aging, senescence, and mitochondrial dysfunction reduces the respiratory reserve, resulting in an increased risk of disease. A decline in the respiratory reserve has been reported to be associated with various diseases, including heart disease (Sansbury BE, et al., Chem-Biol Interact, 191:288-95, 2011), neurodegenerative diseases (Yadava N and Nicholls DG, J Neurosci, 27:7310-7, 2007; Nicholls DG. Ann NY Acad Sci, 1147:53-60, 2008), and smooth muscle cell death (Hill BG, et al., Biochim Biophys Acta, 1797:285-95, 2010).

[0034] As specifically shown in the Examples below, the milt extract of the present invention has the effect of enhancing mitochondrial respiratory capacity (spare respiratory capacity, etc.), and therefore may activate oxidative phosphorylation (activate the mitochondrial respiratory chain) and enhance mitochondrial function. Therefore, it is presumed that the active ingredient of the present invention is effective in preventing or improving not only the decrease in oxidative phosphorylation associated with aging, senescence, and mitochondrial dysfunction, but also heart disease, neurodegenerative disease, smooth muscle cell death, etc.

[0035] Therefore, the milt extract of the present invention can be used to prevent or improve (treat) a disease or condition (symptom) caused by a decrease in mitochondrial respiratory capacity. Here, the disease or condition (symptom) that can be prevented or improved is not particularly limited as long as it is a disease or condition (symptom) caused by a decrease in mitochondrial respiratory capacity, and examples thereof include a decrease in oxidative phosphorylation associated with aging, senescence, and mitochondrial dysfunction, as well as heart disease, neurodegenerative disease, and smooth muscle cell death.

[0036] In this specification, the term "prevention or amelioration" of a disease or condition is used to mean regulation, delay of progression, alleviation, prevention of onset, prevention of recurrence, suppression, and the like of a disease or condition.

[0037] The milt extract of the present invention is generally water-soluble and has excellent miscibility with ingredients that are usually added to foods, beverages, and medicines. In addition, since it is an ingredient contained in foods, it is also highly safe. Therefore, a mitochondrial function activator containing this as an active ingredient can be appropriately incorporated into daily dietary life and taken without difficulty and with peace of mind.

[0038] The mitochondrial function activator of the present invention is an agent containing a milt extract as an active ingredient of the mitochondrial function activator, the use of which is limited to "use for activating mitochondrial function", and can be used alone as a food or drink or a medicine (preparation). In addition, it can be contained as an active ingredient of the mitochondrial function activator in various compositions such as food or drink or medicine. In this way, a food or drink composition or a pharmaceutical composition for activating mitochondrial function can be obtained. The obtained food or drink composition or pharmaceutical composition for activating mitochondrial function can be effectively used for the prevention or improvement (treatment) of the above-mentioned diseases or conditions (symptoms) caused by the decrease in mitochondrial function, such as the decrease in oxidative phosphorylation associated with aging, senescence, and mitochondrial dysfunction, as well as heart disease, neurodegenerative disease, smooth muscle cell death, etc.

[0039] In the present invention, the mitochondrial function activator is not particularly limited as long as it is in a form that can be orally ingested, such as a solution, suspension, emulsion, powder, solid molding, etc. Specific examples include powders, capsules, tablets, granules, powders, as well as forms used in ordinary foods such as beverages and foods and pharmaceuticals. In addition, suitable examples of the formulation form include powders, capsules, tablets, granules, drinks, etc., which are easy to adjust the intake amount.

[0040] In the present invention, the milt extract may be used as it is as a mitochondrial function activator, or may be used after preparing a desired preparation by a conventional method. For example, it may be mixed with various additives permitted for preparation and molded into a composition. The additives may be any additives that are added within a range that does not impair the effects of the present invention, and examples of such additives include herbal medicines, vitamins, minerals, excipients, surfactants, coating agents, oils and fats, waxes, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, colorants, flavors, buffers, antioxidants, pH adjusters, acidulants, etc.

[0041] Examples of the excipients include lactose, starch, cellulose, maltitol, dextrin, etc. Examples of the surfactants include glycerin fatty acid esters, sucrose fatty acid esters, etc. Examples of the coating agents include gelatin, pullulan, shellac, zein, etc. Examples of the fats and oils include wheat germ oil, rice germ oil, safflower oil, etc. Examples of the waxes include beeswax, rice bran wax, carnauba wax, etc. Examples of the sweeteners include sucrose, glucose, fructose, stevia, saccharin, sucralose, etc. Examples of the acidulants include citric acid, malic acid, gluconic acid, etc.

[0042] Examples of the herbal medicines include Korean ginseng, American ginseng, Panax notoginseng, Ganoderma lucidum, propolis, agaricus, blueberries, ginkgo leaves and extracts thereof, etc. Examples of the vitamins include oil-soluble vitamins such as vitamin D and K, water-soluble vitamins such as vitamins B1, B2, B6, B12, C, niacin, pantothenic acid, folic acid, biotin, etc.

[0043] The present invention also provides a food, drink, or pharmaceutical product comprising a mitochondrial function activator having a milt extract as an active ingredient. Here, "comprises" means that the product may contain a physiologically acceptable carrier depending on the desired product form, and may also contain other auxiliary ingredients that can be used in combination.

[0044] In the present invention, the term "food and drink" refers to any food and drink other than pharmaceuticals, and is not particularly limited as long as it is in a form that can be orally ingested as described above. Specific examples of the food and drink include instant foods such as instant noodles, retort foods, canned foods, microwave foods, instant soups, miso soups, freeze-dried foods, and other instant foods; beverages such as soft drinks, fruit juice drinks, vegetable drinks, soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, and alcoholic drinks; flour products such as bread, pasta, noodles, cake mixes, fried chicken powder, and bread crumbs; confectioneries such as caramel, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, and dessert sweets; and soybeans. These include condiments such as seasonings, tomato processed seasonings, flavor seasonings, cooking mixes, sauces, dressings, soups, curry and stew bases; processed oils and fats, butter, margarine, mayonnaise, etc.; dairy products such as milk drinks, yogurt, lactic acid bacteria drinks, ice cream, cream, etc.; processed eggs, fish ham and sausages, fish paste products, etc.; processed livestock products such as meat ham and sausages; processed agricultural products such as canned agricultural goods, jams, marmalades, pickles, boiled beans, cereals, etc.; frozen foods, etc.

[0045] Food and beverages also include health foods (e.g., functional foods, nutritional supplements, health supplements, nutritionally fortified foods, nutritionally regulated foods, supplements, etc.), health functional foods (e.g., foods for specified health uses, nutritionally functional foods, foods with functional claims, etc.), foods for special dietary uses (e.g., foods for the sick, infant formula, milk powder for pregnant or lactating women, etc.), as well as foods and beverages labeled for risk reduction, prevention, or improvement of diseases or conditions (symptoms) caused by aging, decreased oxidative phosphorylation associated with mitochondrial dysfunction, etc.

[0046] According to another aspect of the present invention, there can be provided a food or beverage comprising the mitochondrial function activator, which is indicated as having the function of preventing or ameliorating a disease or condition that can be prevented or ameliorated by mitochondrial function activation.

[0047] In the present invention, a pharmaceutical product is prepared as an oral or parenteral formulation in accordance with a conventional method by combining additives acceptable for formulation. In the case of an oral formulation, as described above, there is no particular limitation as long as it is in a form that can be taken orally. In the case of a parenteral formulation, it can be in the form of an injection or suppository. From the viewpoint of simplicity, an oral formulation is preferable. Examples of additives acceptable for formulation include those similar to those described above.

[0048] In the present invention, the content of the active ingredient in the mitochondrial function activator is difficult to uniformly define due to the type and form of the preparation, the purpose of prevention or improvement, etc., but for example, it is usually about 20 to 2,000 mg, preferably 50 to 1,500 mg, and more preferably 100 to 1,000 mg per day for an adult (body weight 60 kg) in terms of dry weight of milt extract. The content in the preparation may be appropriately set in consideration of the daily intake so that the required daily intake of the active ingredient can be obtained. EXAMPLES

[0049] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0050] 1. Method 1.1. Reagents, equipment and consumables ·MEM (Gibco): Unless otherwise specified, the MEM contained, at final concentrations, 10% FBS, 1% Penicillin-Streptomycin-Neomycin, 406.0 mg / mL GlutaMAX, and 10.0 mg / L Phenol Red (Gibco). Extracellular flux analyzer XFp device (Agilent Technologies) XFp Cell Culture Miniplate (Agilent Technologies) XFp sensor cartridge (Agilent Technologies) Calibration solution: XF Calibrant (pH 7.4) (Agilent Technologies)

[0051] ·XFp Cell Mito Stress Test Kit (manufactured by Agilent Technologies): - Oligomycin (complex V inhibitor) - Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (uncoupler: administration of this agent inhibits the H produced in the electron transport chain) + leaks into the matrix without passing through ATP synthase (complex V). As a result, the inflow of H + reacts with O2 to form H2O. In other words, oxygen is consumed to the maximum.) - Rotenone / Antimycin A (Complex I inhibitor / Complex III inhibitor)

[0052] Assay medium: composition for a total of 10 mL - Seahorse XF DMEM Medium, pH7.4 (Phenol Red (-), manufactured by Agilent Technologies): 9.8mL - 100x GlutaMAX (Gibco): 100μL - 100 mM Sodium Pryuvate (Gibco): 100 μL - Glucose (Nacalai Tesque): 0.01g

[0053] ·SME (salmon milt extract) 10 mg of SME was dissolved in sterile water to prepare a 10 mg / mL SME stock solution. In this example, the SME used was a salmon milt extract manufactured by Nissei Bio Co., Ltd. This product was prepared by pretreating salmon milt and then hydrolyzing it with protease and nuclease according to the description in Example 1 of JP 2004-16143 A. The results of the GPC analysis of this product are shown in Figure 2, and the relationship between retention time and molecular weight is shown in Table 1. The molecular weight guideline of 12,000 was based on the retention time of cytochrome C (MW 12,400).

[0054] [Table 1]

[0055] As shown in Figure 2, A 280 / A 260 =0.5789(A 280 is the absorbance at 280 nm, A 260 is the absorbance at 260 nm), and it can be seen that salmon milt extract (SME) is a mixture of nucleic acids and proteins (including peptides and amino acids). The DNA content of this product is 33.6%.

[0056] From the above analysis results (absorbance at a wavelength of 260 nm), the fractions of the salmon milt extract based on molecular weight are as follows: Fraction with molecular weight of 12,000 to 5,000 (elution time 20 to 25 minutes): 0.3% Fraction with molecular weight of 5,000 or less (elution time 25 minutes or later): 99.6% From the above, the fraction with a molecular weight of 12,000 or less in the salmon milt extract used in the Examples accounts for 99.9%.

[0057] ·DNA-Na (high molecular weight DNA) 10 mg of DNA-Na was dissolved in sterile water to prepare a 10 mg / mL DNA-Na stock solution. The DNA-Na used above is a product of Nissei Bio Co., Ltd. This product is high molecular weight DNA prepared by treating salmon milt with a protein-degrading enzyme (protease) under conditions that do not degrade DNA, adding alcohol to the enzyme-treated solution, extracting and purifying the DNA as DNA-Na salt, and drying it. Note that in this preparation process, the hydrolyzate produced by the protease treatment is removed from the DNA-Na. The results of the GPC analysis of this product are shown in Figure 3, and the relationship between retention time and molecular weight is shown in Table 2. The molecular weight guideline of 12,000 is based on the retention time of cytochrome C (MW 12,400).

[0058] [Table 2]

[0059] From the above analytical results, the molecular weight fractions of DNA-Na are as follows: Molecular weight 2,000,000 to 669,000 fraction (elution time 25.1 to 30.3 minutes): 10.3% Fraction with molecular weight of 669,000 or less (elution time 30.3 minutes or later): 89.4% From the above, the fraction of DNA-Na used in the examples having a molecular weight of 2,000,000 or less was 99.7%.

[0060] 1.2.Cell culture A human liver-derived cell line (C3A) was subcultured in MEM (10% FBS, 1% Penicillin-Streptomycin-Neomycin) in a CO2 incubator (37°C, 5% CO2).

[0061] 1.3.Cytotoxicity test Add 1.0×10 C3A to a 96-well plate. 4The cells were seeded at 100 cells / well and pre-cultured in a CO2 incubator for 24 hours. SME or DNA-Na was then added to a final concentration of 0-200 μg / mL. After 21 hours of culture, 100 μL / well of WST-1 reagent (Dojindo Laboratories, Inc.) was added, and after further culture for 3 hours, the absorbance at 450 nm was measured using a microplate reader (PerkinElmer ARVO MX 1420 MULTILABEL COUNTER). The cell viability was calculated from the absorbance value relative to the control (0 μg / mL) (n=6 per group).

[0062] 1.4. Mitochondrial copy number analysis The number of mitochondria was evaluated as a relative value obtained by dividing the copy number of mitochondrial DNA by the copy number of nuclear DNA. Total DNA was collected from the cells, and the ratio of each target gene (mitochondrial DNA, ND1; nuclear DNA, GAPDH) was calculated by real-time PCR.

[0063] First, C3A was diluted to 2.0 × 10 5 After culturing for 24 hours, the supernatant was discarded, and 1 mL of MEM was added to each well, followed by addition of water, SME (0-200 μg / mL), or DNA-Na (0-2 μg / mL). After 24 hours, the cells were harvested by trypsinization in order to extract DNA using ArchivePure DNA Blood Kit (#2900257, 5 PRIME Inc.). The concentration of purified DNA was quantified using Nano drop (Invitrogen). Expression levels were measured using THUNDERBIRD SYBR qPCR Mix (TOYOBO) and CFX Connect real-time PCR analysis system (BIO RAD), and were analyzed by 20-dose ELISA. -(ΔΔCT) Analysis was performed using the method described above. Comparisons were made between groups, with the control group set as 1. Figure 5 shows the mean ± SD (n = 4 per group).

[0064] The primers used are as follows: ND1-forward (F), 5'-ATGGCCAACCTCCTACTCCT-3' (SEQ ID NO: 1) ND1-reverse (R), 5'-GCGGTGATGTAGAGGGTGAT-3' (SEQ ID NO: 2) GAPDH-F, 5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ ID NO: 3) GAPDH-R, 5'-GAAGATGGTGATGGGATTTC-3' (SEQ ID NO: 4)

[0065] 1.5. Analysis of mitochondrial function using the extracellular flux analyzer XFp C3A was placed in an XFp Cell Culture Miniplate (Agilent Technologies) at 1.0 × 10 4 The cells were seeded at 100 cells / well and pre-cultured in a CO2 incubator (37°C, 5% CO2) for 24 hours. In parallel, 200μL / well of calibration solution was added to the XFp sensor cartridge and the sensor was left in an incubator (37°C, no CO2 control) to hydrate. In this case, hydration was started at the same time as the cells were seeded, so the sensor was left in the incubator for 48 hours. The hydration time was between 12 and 72 hours.

[0066] After pre-incubation for 24 hours, sterile water (control), SME stock solution (final concentration 200μg / mL), or DNA-Na stock solution (final concentration 2μg / mL) was added (n=4 per group). The concentrations of these solutions were set to the maximum concentrations at which no toxicity was observed. Then, the cells were cultured for 24 hours in a CO2 incubator.

[0067] After 24 hours of stimulation, the supernatant in the wells of the XFp Cell Culture Miniplate was replaced with the assay medium and left to stand in an incubator (37°C, no CO2 control) for 1 hour. During this time, the calibration of the hydrated XFp sensor cartridge was performed using an extracellular flux analyzer XFp (manufactured by Agilent Technologies). After the incubation of the XFp Cell Culture Miniplate was completed, the oxygen consumption rate (OCR) was measured using the extracellular flux analyzer XFp. The final concentrations of the reagents used during the measurement were as follows: Oligomycin (final concentration: 4 μM), FCCP (final concentration: 1 μM), Rotenone / Antimycin A (final concentration: 0.5 μM). When the reagent was added, the mix-wait-measurement (2-0-3 min) process was repeated four times, and the next reagent was added.

[0068] The data was analyzed using the accompanying software, Wave ver2.3.0.19 (Seahorse Bioscience). From the profile data, the area under the curve (AUC) was calculated for each functional item and compared between groups. One-way ANOVA was used for statistical analysis, and multigroup comparison tests were performed using Tukey's multiple comparison test. The significance level was set at 5%.

[0069] 2.Results 2.1.Cytotoxicity At the concentrations used in this study, SME was not toxic to hepatocytes (Figure 4). On the other hand, DNA-Na was not toxic up to a final concentration of 2 μg / mL, but cytotoxicity was observed at final concentrations of 20 μg / mL or more (*P<0.05 vs. control).

[0070] 2.2. Mitochondrial number At concentrations where no cytotoxicity was observed or at concentrations lower than those, there was no significant change in mitochondrial copy number in either the SME- or DNA-Na-added groups (Figure 5).

[0071] 2.3.Mitochondrial function No differences in basal respiration levels were observed. In addition, the reactions upon addition of each reagent showed typical profiles, and no significant errors were observed at each measurement point, indicating that the stability and reproducibility of the measurements under these conditions were good (Figure 6).

[0072] Next, data on mitochondrial function was calculated from this profile data as explained in Figure 7 (Figure 8). In the SME-added group, a significant increase was observed in maximum respiration and spare respiratory capacity compared to the control group and DNA-Na-added group. No significant changes were observed in other parameters compared to the control group. In the DNA-Na-added group, no significant changes were observed in any of the parameters compared to the control group.

[0073] 3. Discussion As is clear from the above examples, this study was conducted to establish a screening method for foods that activate mitochondrial function, and as a result of the investigation, the experimental conditions were able to be determined. Our results also show that SME increases mitochondrial maximum and spare respiratory capacity. In particular, spare respiratory capacity calculated from maximum respiration is considered an important aspect of mitochondrial function and is defined as the difference between basal ATP production and its maximum activity. When cells are exposed to stress, their energy demand increases and more ATP is required to maintain cellular function. Cells with a larger spare respiratory capacity can generate more ATP and overcome more stresses, including oxidative stress (Yamamoto H, et al., Oxid Med Cell Longev, 2016:1735841, 2016; Desler C, et al., J Aging Res, 2012:Article ID 192503, 2012).

[0074] If a cell's respiratory reserve is insufficient to supply the required amount of ATP, the cell is at risk of senescence or cell death. The decline in oxidative phosphorylation associated with aging, senescence, and mitochondrial dysfunction reduces the respiratory reserve, thereby increasing the risk of disease. A decrease in the respiratory reserve has been reported to be associated with various diseases, such as heart disease (Sansbury BE, et al., Chem-Biol Interact, 191:288-95, 2011), neurodegenerative diseases (Yadava N and Nicholls DG, J Neurosci, 27:7310-7, 2007; Nicholls DG. Ann NY Acad Sci, 1147:53-60, 2008), and smooth muscle cell death (Hill BG, et al., Biochim Biophys Acta, 1797:285-95, 2010). If SME is a food that increases spare respiratory capacity, it is possible that SME may activate oxidative phosphorylation and enhance mitochondrial function (activate mitochondrial function).

[0075] The inventors considered the possibility that nucleic acids may contribute to the activation of mitochondria and provided the DNA-Na group in the above examples, but no activation effect was observed due to DNA-Na. In addition, although this has not been experimentally proven, it is speculated that the abundant amino acids in SME may have contributed to the increase in these parameters. From the above, it is unclear which components in SME affect mitochondrial function, but we believe that proving this is scientifically important and should be a topic for future study.

Claims

1. A method for producing an agent for activating maximum or spare mitochondrial respiration, comprising the steps of: The milt extract is a hydrolysis product of DNA and protein of salmon milt with nuclease and protease, and contains 50 to 100% of a fraction having a molecular weight of 5,000 or less. A method for producing the activator, comprising the steps of:

2. A method for producing a food or drink for activating maximum or spare respiratory capacity of mitochondria, comprising the method for producing the activator according to claim 1.

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