JP2023022363A - cytoprotective agent - Google Patents

Abstract

【Theme】
To provide a cytoprotective agent which is safe even when taken for a long period of time, and exhibits preventive or ameliorative effect on deterioration of mitochondrial function and protective effect on cells.
[Solution]
A cytoprotective agent comprising, as an active ingredient, a milt extract, preferably a fish milt hydrolyzate having a low molecular weight containing 50 to 100% of a fraction having a molecular weight of 5,000 or less. Such cytoprotective agents have a mitochondrial function-improving action in normal liver model cells and fatty liver model cells, and therefore reduce mitochondrial functions such as reduction in oxidative phosphorylation associated with mitochondrial dysfunction due to fatty liver, aging, aging, etc. Prevention or amelioration (treatment) of diseases or conditions (symptoms) caused by a decrease in
[Selection figure] None

Description

TECHNICAL FIELD The present invention relates to a cell-protecting agent, and more particularly to a cell-protecting agent containing milt extract as an active ingredient, and a cell-protecting food and drink containing the cell-protecting agent.

Salmon milt extract (hereinafter sometimes referred to as "SME") is a food ingredient rich in amino acids and nucleic acids, which has the effect of significantly reducing oxidative damage to genes. It was known to have (Patent Document 1). In order to search for new functionality of the foodstuff, the present inventors have been earnestly conducting research. In a previous study, a system for measuring the amount of oxidized low-density lipoprotein (LDL) was used in a carbon nanotube (CNT) electrode method, and it was reported that SME effectively suppresses oxidation of LDL (Patent Document 2).

In addition, when fatty liver model mice were ingested with SME, they reported that the expression of mitochondrial metabolism-related genes (PGC1, PPAR, CPT1, CRLS, Thioredoxin) increased compared to the SME non-ingestion group (non-patent Reference 1). In other words, it was suggested that SME may act as a mitochondrial metabolism promoting agent. However, its effects on mitochondrial respiration, the most important function of mitochondria, remained unclear. In other words, it is still insufficient to prove the mitochondrial function-improving action of food, and it is necessary to demonstrate further cell physiologically that food ingredients added to cells enhance mitochondrial function.

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 oxygen (O) through the actions of a series of enzymes (complexes I, II, III, IV) while utilizing NADH ₂ ⁺ and FADH ₂ generated by glycolysis and the TCA cycle. ₂ ) to form water (H ₂ O). The process of complexes I to IV is called an electron transfer system because electrons are transferred between proteins and coenzymes in the inner mitochondrial membrane. In the process of this electron transport chain, complexes I, III, and IV move protons (H ⁺ ) from the matrix side to the intermembrane space side. This results in a concentration gradient of H ⁺ across the mitochondrial inner membrane. The energy released in the electron transport chain is stored as an electrochemical potential.

In addition, since the inner membrane of mitochondria is impervious to ions, a potential difference DΨ called membrane potential is generated on both surfaces of the inner membrane. Three H ⁺ from the intermembrane space move to the matrix side through complex V, and one molecule of ATP is synthesized from H ⁺ , ADP and phosphate. These processes, called oxidative phosphorylation, are central to aerobic metabolism. The produced ATP is transported to the cytoplasm via the ADP-ATP translocator present in the mitochondrial inner membrane. The series of processes described above is called the respiratory chain.

Japanese Patent No. 3978716 Japanese Patent No. 6628071

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

It is becoming clear that the activation of mitochondrial function is deeply related to health promotion, for example, the relationship between mitochondria and various metabolic disorders represented by diabetes and aging. However, there are few reports on improvement of mitochondrial function by food. The reasons for this include the difficulty of scientifically proving the effects of food on mitochondrial function, and the fact that a system for sharing this method with society has not been established.

The present inventors have previously established a measurement system for mitochondrial function and applied it to clarify the effect of milt extract containing SME on mitochondrial function, especially on respiratory function, thereby promoting aging, aging, and mitochondria. Proposed pharmaceuticals and functional foods that have preventive or ameliorating (therapeutic) effects on diseases or conditions (symptoms) caused by decreased mitochondrial function such as decreased oxidative phosphorylation associated with dysfunction (Japanese Patent Application No. 2020-120617 Specification).

Here, nonalcoholic fatty liver disease (hereinafter sometimes referred to as “NAFLD” or “fatty liver”) is defined as “alcoholic liver disease without a clear drinking history. It has been reported that the prevalence of NAFLD in health checkup patients is about 40% in men and about 20% in women. About steatohepatitis and its treatment” http://jldf.jp/public/nafld.shtml). NAFLD is nonalcoholic fatty liver (NAFL), in which the pathology rarely progresses, and nonalcoholic steatohepatitis, which is an advanced form of NAFLD and has findings similar to alcoholic hepatitis on the liver tissue. NASH), a broad disease concept that includes liver cirrhosis (Id.).

Furthermore, the principle of treatment for NAFLD is to correct the underlying obesity, diabetes, dyslipidemia, and hypertension by improving lifestyle habits such as diet and exercise therapy. It is said that there is no fixed evaluation for treatment such as intervention and pharmacotherapy (ibid.). Therefore, there is a need for a new material that has no side effects, can be safely ingested on a daily basis, and is expected to have effects on fatty liver, such as inhibition of progression of pathological conditions.

Based on the results of the present inventors' previous work, the object of the present invention is to prepare a fatty liver model using cultured hepatocytes and to clarify the effect of SME on mitochondrial function. An object of the present invention is to provide pharmaceuticals and functional foods that exhibit a preventive or ameliorating (therapeutic) effect on the deterioration of mitochondrial function in fatty liver and a cytoprotective effect.

As a result of intensive studies to solve the above problems, the present inventors found that human liver-derived cell C3A (hereinafter sometimes referred to as "normal liver model cell") and mitochondrial function in fatty liver model cells using C3A. We found that the decrease was inhibited by SME, thereby exerting a cytoprotective effect, and that this protective effect was mediated through increased expression of complex I and complex III genes in the mitochondrial respiratory chain. The present invention has been accomplished based on such findings.

That is, the present invention is as follows.
(1) A cytoprotective agent containing a milt extract as an active ingredient, wherein the hydrolyzed fish milt is reduced in molecular weight so that the milt extract contains 50 to 100% of a fraction with a molecular weight of 5000 or less. A cytoprotective agent characterized by being a substance.
(2) The agent for cell protection according to (1), which protects cells in fatty liver.
(3) cytoprotection is based on improvement of mitochondrial function by enhancement of any selected from the group consisting of basal respiration, maximal respiration, reserve respiratory capacity and ATP production of mitochondria, or improvement of respiration not by mitochondria; The agent for cell protection according to (1) or (2).
(4) The cytoprotection according to any one of (1) to (3), wherein the cytoprotection is based on an increase in the expression level of one or both of complex I gene and complex III gene of the mitochondrial respiratory chain. agent.
(5) Cell-protecting food and drink containing the cell-protecting agent according to any one of (1) to (4).

As another embodiment of the present invention, an agent for promoting the expression of one or both of the complex I gene and the complex III gene of the mitochondrial respiratory chain, containing the milt extract as an active ingredient, and such an expression promoting agent. Food and drink consisting of can be mentioned.

As another embodiment of the present invention, a subject in need of cell protection (specifically, a subject in need of prevention or improvement [treatment] of a disease or condition caused by decreased mitochondrial function), A method for preventing or ameliorating (treating) a disease or condition caused by a decrease in mitochondrial function, or a method for preventing or improving (treatment) a disease or condition caused by a decrease in mitochondrial function, comprising the step of administering a milt extract Milt extract for use, use of milt extract in the manufacture of prophylactic or ameliorating (therapeutic) agents for diseases or conditions caused by decreased mitochondrial function, and use as a cytoprotective agent in fatty liver Mention may be made of the use of the milt extract, the milt extract for use in hepatocyte protection, and the use of the milt extract for the preparation of a cytoprotective agent.

In another embodiment of the present invention, cytoprotection includes improvement of mitochondrial function, preferably improvement of mitochondrial function in fatty liver. In another embodiment of the present invention, the milt extract includes, for example, cells containing, as an active ingredient, a fish milt hydrolyzate (decomposition product) obtained by treating fish milt with a nuclease and a protease. Protective agents may be mentioned.

Here, in the present invention, cytoprotection includes not only fatty liver but also cytoprotection for prevention or improvement (treatment) of diseases or conditions (symptoms) caused by decreased mitochondrial function.

According to the present invention, the milt extract has a cytoprotective effect, that is, a cytoprotective effect based on the improvement of mitochondrial function, and various diseases caused by reduced mitochondrial function such as fatty liver, metabolic abnormalities, and aging. Alternatively, it is expected that the prevention of the condition, the suppression or improvement (treatment) of the pathological condition, and the promotion of health can be carried out without side effects. The milt extract used in the present invention has no safety problems and can be freely prepared in the form of functional foods or pharmaceuticals. can be taken over a long period of time.

FIG. 3 is a diagram showing the molecular weight distribution of SME (salmon milt extract). In the figure, the horizontal axis is the retention time (minutes), and the vertical axis is the absorbance in the ultraviolet region (wavelength 260 nm (upper) or 280 nm (lower)). In the figure, the standard molecular weight of 12,000 is the elution position of cytochrome C. FIG. 3 is a diagram showing the molecular weight distribution of DNA-Na (high molecular weight DNA); In the figure, the horizontal axis is the retention time (minutes), and the vertical axis is the absorbance in the ultraviolet region (wavelength: 260 nm). The approximate molecular weight of 12,000 in the figure is the elution position of cytochrome C. FIG. 2 shows the results of cytotoxicity test (n=6). In the figure, SME indicates salmon milt extract, and DNA-Na indicates high-molecular-weight DNA (the same applies hereinafter). FIG. 3 shows the results of evaluation of mitochondrial copy number by real-time PCR (n=4). FIG. 4 shows the change in OCR profile upon sample addition (n=4). In the figure, arrows indicate the addition time of each reagent. It is a diagram showing the results of comparison (n=4) of each mitochondrial function item calculated from AUC of OCR (*P<0.05 vs. control, **P<0.01 vs. control, n.s. : not significant). The symbols in the figure have the following meanings. A, Maximal respiration; B, Basal respiration; C, Spare respiratory capacity; D, Proton leak; E, ATP production; F, Non -mitochondrial respiration FIG. 3 shows changes in cell viability and LDH activity by SME in a fatty liver model. In FIG. 7, the left figure (A) shows the cell viability of the GFIO group, which is a fatty liver model (GFIO), and the GFIO + SME group in which GFIO and SME were co-cultured, and the right figure (B) shows the LDH activity of the same group. indicates Each figure is expressed as a relative value with the control group (water) set to 1 (**P<0.01, ***P<0.0001, n.s.: not significant, n=6). FIG. 3 shows enhancement of lipid droplet formation in a fatty liver model. The upper figure (A) shows an image stained with Oil red O, where a is the control group, b is the GFIO group, and c is the GFIO+SME group. The lower figure (B) represents the absorbance value at 550 nm (***P<0.0001, n=6). FIG. 3 shows the effect of SME on improving mitochondrial function in a fatty liver model. Left panel (A) shows changes in the OCR profile, that is, changes in the OCR profile by SME in a fatty liver model. The right figure (B) is an explanation of the OCR profile, and the gray area or frame represents the area of items related to mitochondrial function (Maximal respiration, Basal respiration, Spare respiratory capacity, Proton leak: proton leak, ATP production: ATP production, non-mitochondrial respiration: respiration that does not depend on mitochondria). FIG. 4 shows changes in mitochondrial function by SME in a fatty liver model. The AUC of each mitochondrial function item was calculated from the OCR profile (Fig. 9A) of the fatty liver model group (GFIO) and the SME co-culture group (GFIO + SME), and each group was expressed as a relative value with the control group being 1 (* P<0.05, **P<0.01, ***P<0.001, n.s.: not significant, n=13-14). FIG. 3 shows changes in mitochondrial copy number in a fatty liver model. The mitochondrial copy numbers of the GFIO group and the GFIO+SME group were expressed as relative values with the control group being 1 (ns: not significant, n=6). FIG. 4 shows changes in expression levels of respiratory chain complex-related genes by SME in a fatty liver model. The gene expression levels of the GFIO group, which is a fatty liver model, and the GFIO + SME group, in which SME was co-cultured, were expressed as relative values with the control group being set to 1 (*P<0.05, **P<0.01, n.s.: not significant, n=6). NDUFS8: Complex I, SDHB: Complex II, UQCRC: Complex III, COX6B1: Complex IV, ATP5G1: Complex V. BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the outline|summary of the morphological observation method of the mitochondria in a fatty liver model. FIG. 3 shows the effect of SME on mitochondrial morphological changes in a fatty liver model. FIG. 3 shows the effect of SME on the expression levels of genes involved in mitochondrial fusion and fission in a fatty liver model.

Embodiments of the present invention will be described in detail below, but the following description is an example of the embodiments of the present invention, and the present invention is not limited to the following descriptions. In addition, when the expression "~" is used in this specification, it is used as an expression including numerical values or physical property values before and after it. In addition, the content (%) of active ingredients means percent by mass (wt%) unless otherwise specified.

The cytoprotective agent of the present invention contains milt extract as an active ingredient. In the present specification, the milt extract may be a hydrolyzate (decomposition product) of fish milt that has been reduced in molecular weight so as to contain 50 to 100% of a fraction with a molecular weight of 5000 or less, preferably protease or It is a hydrolyzate (enzymatic degradation product) by enzymatic treatment such as nuclease.

Here, as the fish milt, the milt (testis) of fish such as salmon, trout, and cod can be used because it contains a large amount of DNA and can be used effectively as a waste product from the processing of marine products. is preferred. Among these, salmon milt is particularly preferable from the viewpoint of availability of raw materials.

Although the method for preparing the milt extract is not particularly limited, for example, it can be prepared according to the methods described in JP-A-2003-325149, JP-A-2004-16143, JP-A-2016-204340, and the like. .

For example, after removing the skin, muscles, blood vessels, etc. from the above-mentioned fish soft roe as necessary, further refinement as necessary to remove oil and crush (hereinafter sometimes referred to as "pretreatment"). Those prepared by a method involving treatment with proteases and nucleases are preferred.

Although there is no particular limitation on the properties of the protease to be used, serine proteases such as trypsin are preferred. Serine proteases selectively hydrolyze peptide bonds at the carboxyl side of arginine and lysine, and are therefore suitable for hydrolyzing arginine-rich protamines.

The properties of the nuclease to be used are not particularly limited, but a nuclease that cleaves with a 5' phosphate group is preferred, and a certain degree of thermostability is preferred. These proteases and nucleases may be used by appropriately selecting commercially available products.

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 powder by spray drying or the like. Moreover, if necessary, it can be used after further purification.

Here, the aforementioned salmon milt extract (SME) is prepared by subjecting pretreated salmon milt to protease and nuclease treatment (enzymatic degradation) as described above.

The milt extract in the present invention includes a hydrolyzate of milt-derived DNA (hereinafter sometimes referred to as "DNA component") and a milt-derived protein hydrolyzate (hereinafter sometimes referred to as "protein component"), such as Protamine, hydrolysates thereof (proteins, peptides, amino acids), milt-derived polyamines, and the like are included.

The content of DNA components in the milt extract of the present invention is generally 1-80%, preferably 10-50%, more preferably 26-36%. The content of the DNA component is a value based on anion exchange chromatography (IC analysis).

Although the molecular weight and the like of the DNA component of the milt extract in the present invention are not particularly limited, a DNA component having a low molecular weight containing about 50 to 100% of the fraction having a molecular weight of 5,000 or less, preferably 3,000 or less, is preferred. The molecular weight distribution is a value based on gel permeation chromatography (hereinafter sometimes referred to as "GPC analysis") (absorbance at a wavelength of 260 nm).

The lower limit of the molecular weight of the active ingredient (molecular weight as DNA) is not particularly limited as described above. It is preferable to contain about 0 to 50% of a fraction having a molecular weight of 3,000 or more.

In the milt extract of the present invention, the content of protein components (total amino acids) is generally 10-80%, preferably 30-60%, more preferably 40-50%. The content of protein components (total amino acids) is a value measured by the method used in the Japanese Food Standard Composition Tables 2015 Edition (7th Edition) Amino Acid Composition Tables.

Although the molecular weight and the like of the protein component of the milt extract in the present invention are not particularly limited, protein components having a molecular weight of 5,000 or less, preferably 3,000 or less, containing about 50 to 100% of the protein component are preferred. The molecular weight distribution is a value based on the GPC analysis (absorbance at a wavelength of 280 nm).

From the action thereof, the agent for cell protection of the present invention is an agent for improving mitochondrial function of hepatocytes, an agent for activating basal respiration of mitochondria, an agent for activating maximal mitochondrial respiration, an agent for activating preliminary mitochondrial respiration, and mitochondrial ATP. It can also be called an agent for activating production and an agent for activating respiration that does not depend on mitochondria. "Activation" in these inventions can also be called "enhancement".

In addition, the cytoprotective agent of the present invention can also be called a mitochondrial respiratory chain complex I gene expression-enhancing agent and a mitochondrial respiratory chain complex III gene expression-enhancing agent.

In addition, the cytoprotective agent of the present invention can also be said to be an agent for suppressing mitochondrial fragmentation due to its action.

Furthermore, the cytoprotective agent for fatty liver according to the present invention exhibits cytoprotective effects not only for fatty liver, but also for various diseases and conditions caused by reduced mitochondrial function such as metabolic abnormalities and aging. It can also be called an agent.

In the present invention, mitochondrial respiration refers to the production of ATP by consuming oxygen in mitochondria. In the present invention, mitochondrial respiratory capacity refers to the ability of mitochondria to produce and/or be able to produce ATP. The mitochondrial respiratory capacity is specifically expressed by mitochondrial basal respiration, maximal respiration, preliminary respiratory capacity, and the like.

Moreover, the mitochondrial function in the present invention refers to supplying energy (ATP) to cells through the above mitochondrial respiration.

Here, a decrease in oxidative phosphorylation associated with aging, senescence, and mitochondrial dysfunction lowers respiratory reserve capacity, resulting in an increased risk of disease. Reduced respiratory reserve is associated with cardiovascular disease (Sansbury BE, et al., Chem-Biol Interact, 191:288-95, 2011), neurodegenerative diseases (Yadava N and Nicholls DG, J Neurosci, 27:7310-7). Ann NY Acad Sci, 1147:53-60, 2008) and smooth muscle cell death (Hill BG, et al., Biochim Biophys Acta, 1797:285-95, 2010). have been reported to be associated with

The present inventors have previously reported that in the assay system for searching for food ingredients that activate mitochondria, which the present inventors established, food ingredients were added to liver cultured cells (normal liver model cells), and the mitochondria of the cells function was evaluated. As a result, since the milt extract in the present invention has the effect of enhancing mitochondrial respiratory capacity (preliminary respiratory capacity, etc.), it is possible to activate oxidative phosphorylation (activate mitochondrial respiratory chain) and enhance mitochondrial function. Therefore, the milt extract, which is an active ingredient in the present invention, is effective in reducing oxidative phosphorylation associated with aging, aging and mitochondrial dysfunction, as well as heart disease, neurodegenerative disease, and smooth muscle cell death. It was speculated that it is effective for the prevention or improvement of such.

Based on these findings, the present inventors have found that using milt extract containing SME as an active ingredient, aging, aging, diseases caused by reduced mitochondrial function such as reduced oxidative phosphorylation associated with mitochondrial dysfunction, or We have proposed pharmaceuticals and functional foods that are effective in preventing or improving (therapeutic) conditions (symptoms) (Japanese Patent Application No. 2020-120617).

In the present invention, the effect of SME on hepatocyte function was evaluated by the above measurement system in a fatty liver model using cultured liver cells, as will be specifically shown in Examples described later. As a result, it was shown that SME exerts an effect of promoting (improving) lowered mitochondrial functions (maximal respiration, basal respiration, reserve respiratory capacity, ATP production) in a fatty liver model. SME also increased (improved) non-mitochondrial respiration.

Furthermore, when the gene expression levels of complexes IV of the mitochondrial respiratory chain complex were measured, it was found that SME suppressed the decrease in the expression level of complexes I and III genes in a fatty liver model. From this, SME induced the expression of the complex I and III genes of the mitochondrial respiratory chain complex, and as a result, suppressed the decline of mitochondrial functions such as maximal respiration, preliminary respiratory capacity, and basal respiration in the fatty liver model. Conceivable.

Furthermore, in an experiment using fluorescence staining of mitochondria, fragmentation of mitochondria was observed in fatty liver model cells, and addition of SME under these conditions suppressed fragmentation. In addition, since the expression of the gene (DRP1) associated with mitochondrial fission was significantly decreased in SME, it is considered that SME worked to suppress fission, that is, maintained the fusion state. This result agreed with the result of the fluorescence staining image.

Based on these results, in fatty liver model cells, SME not only acts to increase the expression of genes related to the mitochondrial electron transport chain, but also increases mitochondrial fusion through suppression of division (fragmentation). , which is thought to improve mitochondrial respiratory function.

Therefore, the milt extract of the present invention can be used not only in hepatocytes of fatty liver but also in the prevention or improvement (treatment) of diseases or conditions (symptoms) caused by decreased mitochondrial function (particularly respiratory capacity). . Here, the disease or condition (symptoms) that can be prevented or ameliorated is not particularly limited as long as it is a disease or condition (symptom) caused by a decrease in mitochondrial function (especially respiratory function). (including alcoholic fatty liver, nonalcoholic steatohepatitis, liver cirrhosis), various metabolic disorders (e.g., diabetes), aging, decreased oxidative phosphorylation associated with aging and mitochondrial dysfunction, heart disease, Examples include neurodegenerative diseases, smooth muscle cell death, and the like.

As used herein, the term "prevention or amelioration" of a disease or condition is used to include control, delay of progress, alleviation, prevention of onset, prevention of recurrence, suppression, etc. of a disease or condition.

The milt extract in the present invention is usually readily water-soluble and has excellent miscibility with ingredients that are usually added to foods, beverages and pharmaceuticals. Moreover, since it is a component contained in foods, it is excellent in safety. Therefore, a cytoprotective agent containing this as an active ingredient can be properly incorporated into a daily diet and ingested without difficulty.

The cytoprotective agent of the present invention is an agent that contains milt extract as an active ingredient of a cytoprotective agent whose use is limited to "used for protecting cells", and can be used alone or in foods, beverages or pharmaceuticals ( formulation). In addition, it can be contained in various compositions such as food, beverages and pharmaceuticals as an active ingredient of a cell protecting agent. Thereby, a food/beverage composition or a pharmaceutical composition of the cell protective agent can be obtained. The obtained food/drink composition or pharmaceutical composition for cell protection is useful for diseases or conditions (symptoms) caused by the above-mentioned deterioration of cell functions, such as fatty liver, aging, aging and oxidative stress associated with mitochondrial dysfunction. In addition to decreasing phosphorylation, it can be effectively used for prevention or improvement (treatment) of heart disease, neurodegenerative disease, smooth muscle cell death and the like.

In the present invention, the cytoprotective agent is not particularly limited as long as it is in an orally ingestible form such as a solution, suspension, emulsion, powder, or solid molding. Specific examples include powders, capsules, tablets, granules, powders, as well as forms used in ordinary foods such as beverages and foods, and pharmaceuticals. Moreover, as the form of the preparation, powders, capsules, tablets, granules, drinks, etc., which are easy to control the intake, can be preferably exemplified.

In the present invention, the milt extract may be used as it is as a cytoprotective agent, or may be used after being prepared into a desired formulation by a conventionally known method. For example, it can be mixed with various additives permitted for the production of pharmaceutical preparations and molded as a composition. Any additive may be added as long as it does not impair the effects of the present invention. stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, colorants, flavors, buffers, antioxidants, pH adjusters, acidulants and the like.

Examples of the excipient include lactose, starch, cellulose, maltitol, dextrin and the like. Examples of the surfactant include glycerin fatty acid ester, sucrose fatty acid ester, and the like. Examples of the coating agent include gelatin, pullulan, shellac, and zein. Examples of the oils and fats include wheat germ oil, rice germ oil, safflower oil, and the like. Examples of the waxes include beeswax, rice bran wax, and carnauba wax. Examples of sweeteners include sucrose, glucose, fructose, stevia, saccharin, and sucralose. Examples of the acidulant include citric acid, malic acid, gluconic acid and the like.

Examples of the crude drugs include Korean ginseng, American ginseng, Panax ginseng, Ganoderma lucidum, propolis, agaricus, blueberry, ginkgo biloba and extracts thereof. Examples of the vitamins include oil-soluble vitamins such as vitamins D and K, and water-soluble vitamins such as vitamins B1, B2, B6, B12, C, niacin, pantothenic acid, folic acid, and biotin.

Further, according to the present invention, there is provided a food, drink or a drug containing a cytoprotective agent containing milt extract as an active ingredient. Here, "comprising" means that it may contain a physiologically acceptable carrier according to the desired product form, and also contains other auxiliary ingredients that can be used in combination.

In the present invention, the food and drink is anything other than pharmaceuticals, and is not particularly limited as long as it is in a form that can be orally ingested as described above. Specifically, instant foods such as instant noodles, retort foods, canned foods, microwave foods, instant soups/miso soups, freeze-dried foods; soft drinks, fruit juice drinks, vegetable drinks, soy milk drinks, coffee drinks, tea Beverages such as beverages, powdered beverages, concentrated beverages, alcoholic beverages; Flour products such as bread, pasta, noodles, cake mixes, fried chicken powder, and bread crumbs; Caramel, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, Confectionery such as crackers, Japanese confectionery, dessert confectionery; Seasonings such as sauces, processed tomato seasonings, flavor seasonings, cooking mixes, sauces, dressings, soups, curry and stew ingredients; Processed oils and fats, butter, Fats and oils such as margarine and mayonnaise; Dairy products such as milk drinks, yogurts, lactic acid beverages, ice creams and creams; Processed marine products such as processed eggs, fish ham and sausages, fish paste products; Livestock hams and sausages, etc. processed livestock products; processed agricultural products such as canned agricultural products, jams and marmalades, pickles, boiled beans, cereals; and frozen foods.

Foods and drinks include health foods (e.g., functional foods, dietary supplements, health supplements, nutrient-enriched foods, nutrient-adjusting foods, supplements, etc.), foods with health claims (e.g., specified health foods, foods with nutrient function claims, Foods with functional claims, etc.), foods for special dietary uses (e.g., food for sick people, powdered milk for infants, powdered milk for pregnant or breastfeeding women, etc.) It also includes categories such as foods and drinks labeled as reducing, preventing, or improving the risk of diseases or conditions (symptoms) caused by a decrease or the like.

According to another aspect of the present invention, there may be provided a food or drink containing the cytoprotective agent, wherein the food or drink is labeled with a function of preventing or improving the disease or condition described above.

In the present invention, pharmaceuticals are prepared as oral preparations or parenteral preparations according to conventional methods, using additives that are acceptable for formulation. In the case of oral preparations, as mentioned above, there are no particular restrictions as long as they are in a form that can be taken orally. Parenteral formulations can be in the form of injections or suppositories. From the point of view of simplicity, an oral formulation is preferred. Additives acceptable for formulation include those described above.

In the present invention, the content of the active ingredient in the cytoprotective agent is difficult to uniformly define depending on the type and form of the formulation and the purpose of prevention or improvement. , the dry weight of milt extract is usually 20 to 2,000 mg, preferably 50 to 1,500 mg, and more preferably about 100 to 1,000 mg. The content in the formulation may be appropriately set in consideration of the daily intake so that the required daily intake of the active ingredient can be taken.

EXAMPLES 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.

[Example 1] Functional evaluation in normal liver model 1. Method 1.1. Reagents/equipment/consumables MEM (manufactured by gibco): Unless otherwise noted, MEM is 10% FBS at final concentration, 1% Penicillin-Streptomycin-Neomycin, 406.0 mg/mL GlutaMAX, 10.0 mg/L Phenol Red ( gibco).
・Extracellular flux analyzer XFp device (manufactured by Agilent Technologies)
・XFp Cell Culture Miniplate (manufactured by Agilent Technologies)
・XFp sensor cartridge (manufactured by Agilent Technologies)
・Calibration solution: XF Calibrant (pH 7.4) (manufactured by Agilent Technologies)

・XFp Cell Mito Stress Test Kit (manufactured by Agilent Technologies):
- Oligomycin (complex V inhibitor)
- Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (uncoupling agent: administration of this agent prevents H ⁺ generated in the electron transport chain from passing through ATP synthase (complex V) As a result, the inflowing H ⁺ reacts with O ₂ to form H ₂ O. In other words, oxygen is consumed to the maximum.)
- Rotenone/Antimycin A (Complex I inhibitor/Complex III inhibitor)

・Assay medium: composition when total 10 mL
- Seahorse XF DMEM Medium, pH 7.4 (Phenol Red (-), manufactured by Agilent Technologies: 9.8 mL
- 100x GlutaMAX (manufactured by gibco): 100 μL
- 100 mM Sodium Pryuvate (manufactured by gibco): 100 μL
- Glucose (manufactured by Nacalai Tesque Co., Ltd.): 0.01 g

・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 salmon milt extract from Nissei Bio. This product was prepared by pretreating salmon milt, followed by protease and nuclease hydrolysis, according to the description in Example 1 of JP-A-2004-16143. The results of GPC analysis of this product are shown in FIG. 1, 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).

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

As shown in FIG. 1, A ₂₈₀ /A ₂₆₀ =0.5789 (A ₂₈₀ is the absorbance at 280 nm, A ₂₆₀ is the absorbance at 260 nm), and salmon milt extract (SME) contains both nucleic acids and proteins (including peptides and amino acids). ). In addition, the DNA content of this product is 33.6%, and the protein component (total amino acid) content is 47.4%. In addition, DNA content and protein component content are the values based on the said analytical method, respectively.

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

Based on the above analysis results, the fractions of DNA-Na based on molecular weight are as follows.
Molecular weight fraction 2,000,000 to 669,000 (elution time 25.1 minutes to less than 30.3 minutes): 10.3%
Fractions with a molecular weight of 669,000 or less (elution time after 30.3 minutes): 89.4%
Based on the above, the fraction having a molecular weight of 2,000,000 or less in the DNA-Na used in Examples is 99.7%.

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 ).

1.3. Cytotoxicity Test C3A was seeded in a 96-well plate at 1.0×10 ⁴ cells/well and precultured in a CO ₂ incubator for 24 hours. Next, SME or DNA-Na was added to a final concentration of 0-200 μg/mL. After culturing for 21 hours, WST-1 reagent (manufactured by Dojindo Laboratories) was added at 100 μL/well, and after further 3 hours of culture, microplate reader (PerkinElmer, ARVO MX 1420 MULTILABEL COUNTER) was used to measure 450 nm. Absorbance was measured. Cell viability was calculated from the absorbance value for the control (0 μg/mL) (n=6 for each group).

1.4. Analysis of Mitochondrial Copy Number The mitochondrial number was evaluated as a relative value obtained by dividing the mitochondrial DNA copy number by the nuclear DNA copy number. The total DNA of the cells was collected, and each target gene (mitochondrial DNA, ND1 [NADH dehydrogenase subunit 1]; nuclear DNA, GAPDH) was quantified by real-time PCR to determine the ratio.

First, C3A was seeded in a 24-well plate at 2.0×10 ⁵ cells/well, cultured for 24 hours, the supernatant was discarded, 1 mL of MEM was added to each well, then water, SME ( 0-200 μg/mL) or DNA-Na (0-2 μg/mL) was added. After 24 hours, the cells were collected by trypsinization in order to extract DNA using the ArchivePure DNA Blood Kit (#2900257, 5 PRIME Inc.). The concentration of purified DNA was quantified by Nanodrop (manufactured by Invitrogen). Expression levels were measured using THUNDERBIRDSYBR qPCR Mix (manufactured by TOYOBO) and CFX Connect real-time PCR analysis system (manufactured by BIO RAD) and analyzed by the 2-(ΔΔCT) method. Comparison between groups was performed with the control group as 1. FIG. 4 is represented by mean±SD (n=4 for each group).

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)

1.5. Analysis of Mitochondrial Function Using Extracellular Flux Analyzer XFp C3A was seeded on XFp Cell Culture Miniplate (manufactured by Agilent Technologies) at 1.0×10 ⁴ cells/well and placed in a CO ₂ incubator (37° C., 5% CO ₂ ) for 24 hours. In parallel, 200 μL/well of the calibration solution was added to the XFp sensor cartridge and left in an incubator (37° C., no CO ₂ control) to hydrate the sensor. This time, hydration was started at the same timing as cell seeding, so the cells were allowed to stand for 48 hours. Hydration times ranged from 12-72 hours.

After the cells were precultured for 24 hours, sterilized water (control), SME stock solution (final concentration 200 μg/mL), or DNA-Na stock solution (final concentration 2 μg/mL) was added (n=4 for each group). These addition concentrations were set to the maximum concentrations at which no toxicity was observed. After that, it was cultured in a _CO2 incubator for 24 hours.

After 24 hours of stimulation, the supernatant in the wells of the XFp Cell Culture Miniplate was replaced with assay medium and allowed to stand in an incubator (37°C, no _CO2 control) for 1 hour. Meanwhile, the hydrated XFp sensor cartridge was calibrated using an extracellular flux analyzer XFp (Agilent Technologies). After completion of incubation of the XFp Cell Culture Miniplate, the oxygen consumption rate (OCR) was measured using an extracellular flux analyzer XFp. The final concentrations of reagents used in the measurement are as follows: Oligomycin (final concentration: 4 µM), FCCP (final concentration: 1 µM), Rotenone/Antimycin A (final concentration: 0.5 µM). Once the reagent was added, the mix-wait-measurement (2-0-3 minutes) process was repeated four times and set to add the next reagent.

The data were analyzed using the accompanying software, Wave ver2.3.0.19 (manufactured by Seahorse Bioscience). From the profile data, the area under the curve (AUC) was calculated for each functional item and compared between the groups. A one-way analysis of variance (one-way ANOVA) was used for statistical analysis, and a multi-group comparison test was performed by Tukey's multiple comparison test. The significance level was set at 5%.

2. Results 2.1. Cytotoxicity At the concentrations used this time, SME had no toxicity to hepatocytes (Fig. 3, left panel). On the other hand, DNA-Na showed no toxicity up to a final concentration of 2 μg/mL, but cytotoxicity was observed at a final concentration of 20 μg/mL or higher (*P<0.05 vs. control) (right figure in FIG. 3). .

2.2. Mitochondrial Number There was no significant change in the mitochondrial copy number in both the SME-added group and the DNA-Na-added group at concentrations at which cytotoxicity was not observed and at lower concentrations (Fig. 4).

2.3. Mitochondrial function In addition to the fact that no difference in the level of basal respiration was observed, the response to each reagent addition showed a typical profile, and no large errors were observed at each measurement point. Performance and reproducibility were good (Fig. 5).

Data on mitochondrial function were then calculated from the data in this profile (Fig. 6). A significant increase in maximal respiration and reserve respiratory capacity was observed in the SME-added group compared to the Control group and the 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 change was observed in any parameter compared to the control group.

3. Discussion As is clear from the above examples, this research was conducted to establish a screening method for foods that activate mitochondrial function, and as a result of the examination, we were able to determine the experimental conditions.
Moreover, from the results of this time, it was found that SME increases maximal respiration and preliminary respiratory capacity of mitochondria. In particular, respiratory reserve, calculated from maximal respiration, is considered an important aspect of mitochondrial function and is defined as the difference between basal ATP production and its maximal activity. When cells are stressed, their energy demand increases, requiring more ATP to maintain cellular function. Cells with a larger respiratory reserve can produce more ATP and overcome more stress, 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).

If a cell's spare respiratory capacity is not sufficient to supply the required amount of ATP, the cell risks being driven into senescence or cell death. Decreases in oxidative phosphorylation associated with aging, aging and mitochondrial dysfunction reduce respiratory reserve and consequently increase disease risk. Decreased respiratory reserve is associated with 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 (HillBG, et al., Biochim Biophys Acta, 1797:285-95, 2010). reported to do. If SME is a food that enhances respiratory reserve, SME may activate oxidative phosphorylation and enhance mitochondrial function (activate mitochondrial function).

Considering the possibility that nucleic acid contributes to this mitochondrial activation, the present inventors provided the DNA-Na group in Example 1 above, but the activating effect of DNA-Na was not observed. Alternatively, although not experimentally proven, it is speculated that the abundance of amino acids in SME may have contributed to the increase in these parameters.

[Example 2] Functional evaluation in fatty liver model cells 1. Method 1.1. Reagents/Equipment/Consumables The same as in 1.1 of Example 1 were used, except that DNA-Na (high molecular weight DNA) was not used.

1.2. Cell culture The cells were subcultured in the same manner as in 1.2 of Example 1.

1.3. Cytotoxicity Test C3A was seeded in a 96-well plate at 1.25×10 ⁴ cells/well and precultured in a CO ₂ incubator for 24 hours. Thereafter, the supernatant was replaced with FBS-free MEM (0% FBS, 1% Penicillin-Streptomycin-Neomycin, 406 mg/mL GlutaMAX, 10 mg/L Phenol Red [manufactured by gibco]) to starve the cells. Cultured for 24 hours in a _CO2 incubator.

Next, as a fatty liver model group (GFIO group), glucose, fructose, insulin and oleic acid were added to the final concentrations shown in Table 3. This concentration condition was referred to the past literature (Prins GH, et al., Nutrients, 11: 507, 2019). As an SME-added group (GFIO+SME group), an SME stock solution (final concentration: 200 μg/mL) was simultaneously added in addition to glucose, fructose, insulin and oleic acid. The same amount of sterilized water was added to the control group.

After culturing for 21 hours, 50 μL of the cell supernatant was collected in a 96-well plate and diluted 2-fold to prepare a sample for measuring lactate dehydrogenase (LDH) activity. After recovery, 100 μL/well of WST-1 reagent (manufactured by Dojindo Laboratories) was added, and after 3 hours of culture, absorbance at 450 nm was measured using a microplate reader (PerkinElmer, ARVO MX 1420 MULTILABEL COUNTER). was measured.

Cell viability was calculated from the absorbance value for the control (0 μg/mL) (n=6 for each group). The absorbance at 450 nm was measured, and the cell viability was calculated from the absorbance value for the control (0 μg/mL) (n=6 for each group).

LDH Cytotoxicity Detection Kit (manufactured by Takara Bio Inc.) was used to measure LDH activity, and cytotoxicity was measured. Absorbance at 490 nm was measured with a microplate reader (manufactured by PerkinElmer, ARVO MX 1420 MULTILABEL COUNTER) according to the package insert of the kit, and calculated from absorbance values for controls (n=6 for each group).

1.4. Observation of lipid droplets by Oil Red O staining C3A cells were seeded in a 24-well plate at 1.25 × 10 ⁵ cells/well, cultured for 24 hours, and the supernatant was added to FBS-free MEM (0% FBS , 1% Penicillin-Streptomycin-Neomycin, 406 mg/mL GlutaMAX, 10 mg/L Phenol Red [manufactured by gibco]) to starve the cells. After that, stimulation was given in the same manner as in the cytotoxicity test.

After 24 hours, the supernatant was replaced with 200 μL/well of a 10% neutral buffered formalin solution (manufactured by Wako Pure Chemical Industries, Ltd.) and allowed to stand for 10 minutes to fix the cells. After fixation, the supernatant was removed, and washing was performed twice with 200 μL/well of phosphate buffer (manufactured by Wako Pure Chemical Industries, Ltd.). After that, 200 μL/well of 60% isopropanol (manufactured by Kanto Kagaku Co., Ltd.) was added and allowed to stand for 10 minutes. The supernatant was exchanged with 200 μL/well of Oil Red O solution (3 mg/mL of Oil Red O [manufactured by Wako Pure Chemical Industries, Ltd.] dissolved in 60% isopropanol) and allowed to stand at room temperature for 20 minutes. . After that, 200 μL/well of 60% isopropanol was added and allowed to stand for 10 minutes. After removing the supernatant and washing twice with 200 μL/well of phosphate buffer, the cells were observed under a microscope (IX71, manufactured by OLYMPUS) and the images were saved.

After microscopic observation, the supernatant was replaced with 120 μL/well of 100% isopropanol (manufactured by Kanto Kagaku Co., Ltd.), followed by cell lysis and Oil Red O extraction. After collecting 100 μL of the supernatant in a 96-well plate, the absorbance at 550 nm was measured with a microplate reader (PerkinElmer, ARVO MX 1420 MULTILABEL COUNTER) (n=6 for each group).

1.5. Analysis of Mitochondrial Function Using Extracellular Flux Analyzer XFp C3A was seeded on XFp Cell Culture Miniplate (manufactured by Agilent Technologies) at 1.25×10 ⁴ cells/well and placed in a CO ₂ incubator (37° C., 5% CO ₂ ) for 24 hours. The supernatant was then replaced with FBS-free MEM (0% FBS, 1% Penicillin-Streptomycin-Neomycin, 406 mg/mL GlutaMAX, 10 mg/L Phenol Red [gibco]) to starve the cells. After that, it was cultured in a _CO2 incubator for 24 hours. Next, stimulants were added to the final concentrations shown in Table 3.

After stimulation for 24 hours, the supernatant in the wells of the XFp Cell Culture Miniplate was replaced with an assay medium and allowed to stand in an incubator (37°C, without _CO2 control) for 1 hour. Meanwhile, calibration of the 48-hour hydrated XFp sensor cartridge was performed using an extracellular flux analyzer XFp (manufactured by Agilent Technologies). After completing the incubation of the XFp Cell Culture Miniplate, the oxygen consumption rate (OCR) was measured using an extracellular flux analyzer XFp.

The final concentrations of the reagents used in the measurement are as follows: Oligomycin (final concentration: 2 µM), FCCP (final concentration: 1 µM), Rotenone/Antimycin A (final concentration: 0.5 µM). Once the reagent was added, the mix-wait-measurement (2-0-3 minutes) process was repeated four times and set to add the next reagent.

The data were analyzed using the accompanying software, Wave ver2.3.0.19 (manufactured by Seahorse Bioscience). From the profile data, the area under the curve (AUC) was calculated for each functional item and compared between the groups. FIG. 10 is represented by mean±SD (each group n=13-14).

1.6. Mitochondrial copy number analysis
Cells were stimulated under the same conditions as the observation of lipid droplets by Oil Red O staining.
Cells were harvested by trypsinization in order to extract DNA using the ArchivePure DNA Blood Kit (#2900257, 5 PRIME Inc.). The concentration of DNA purified with the kit was quantified by Nanodrop (manufactured by Invitrogen). The expression level was measured using THUNDERBIRD SYBR qPCR Mix (manufactured by TOYOBO) and CFX Connect real-time PCR analysis system (manufactured by BIO RAD) and analyzed by the 2-(ΔΔCT) method. The primers used are shown in Table 4. 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. FIG. 11 is expressed as mean±SD (n=4 for each group).

1.7. Analysis of Gene Expression Level of Mitochondrial Respiratory Chain Complex A stimulus was applied in the same manner as the mitochondrial copy number analysis. After 24 hours, they were recovered with Lysis Buffer (manufactured by Life Technologies). RNA was extracted using PureLink RNA Mini Kit (manufactured by Life Technologies), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (manufactured by TOYOBO). Gene expression levels were analyzed using the same real-time PCR analysis system (manufactured by BIO RAD) as described above.

Target genes are ND1: ubiquinone oxidoreductase core subunit S8 (NDUFS8), succinate dehydrogenase complex iron sulfur subunit B (SDHB), ubiquinol-cytochrome c reductase core protein 1 (UQCRC1), cytochrome oxidase subunit 6B1 (COX6B1), ATP synthase, H ⁺ transporting, mitochondrial fo complex, subunit C1 (subunit 9) (ATP5G1), and the expression level of each gene was corrected with the expression level of GAPDH. Comparison between groups was performed with the control group as 1, and the results were expressed as mean ± SD (n = 6 for each group). The primers used are shown in Table 4.

1.8. Statistical Analysis For statistical analysis, one-way analysis of variance (one-way, manufactured by ANOVA) was used, and multi-group comparison test was performed by Tukey's multiple comparison test. The significance level was set at 5%.

2. Results 2.1. Cell viability and cytotoxicity The cell viability of the GFIO group, which is a fatty liver model, decreased to 30.5% of that of the control group. Moreover, the cell viability in the GFIO+SME group was suppressed to 45.5% of that in the control group (Fig. 7A). In addition, no significant change in LDH activity was observed in each group (Fig. 7B).

2.2. Formation of Lipid Droplets In the GFIO group and GFIO+SME group, which are fatty liver models, oil red O-stained lipid droplets were significantly observed compared to the control group (FIG. 8A). When the amount of lipid droplets in each group was measured by absorbance, the absorbance was significantly higher in the GFIO group and the GFIO+SME group than in the control group (Fig. 8B). No change was observed in the amount of lipid droplets in the GFIO group and the GFIO+SME group.

2.3. Mitochondrial Function-Enhancing Effect of SME The OCR behavior measured this time had a typical profile and was under stable measurement conditions (FIG. 9). From this profile, items related to mitochondrial function were calculated (Fig. 10). In the GFIO group, the items of maximal respiration, basal respiration, reserve respiratory capacity, and ATP production were significantly lower than those in the control group. In addition, in the GFIO+SME group, the decreased maximal respiration, basal respiration, reserve respiratory capacity, and ATP production in the GFIO group were almost the same as in the control group. Respiration independent of mitochondria in the GFIO+SME group was significantly higher than in the control group and the GFIO group.

2.4. Effect of SME on Mitochondrial Copy Number No significant change was observed in the mitochondrial copy number in each of the control group, GFIO group and GFIO+SME group (FIG. 11).

2.5. Effect of SME on Expression Levels of Respiratory Chain Complex-Related Genes In the GFIO group, which is a fatty liver model, the expression level of NDUFS8 (complex I) was reduced by 79.1% of that in the control group. The expression level of NDUFS8 in the GFIO+SME group was 108.1% of that in the control group. In addition, the expression level of UQCRC (complex III) in the GFIO+SME group increased to 128.4% of that in the control group. As for other genes, no significant changes were observed in any group (Fig. 12).

3. Discussion According to Example 2, it was confirmed that the fatty liver model using cultured liver cells was under conditions in which mitochondrial function was lowered (FIG. 10). A previous study by Sakurai et al. (Sakurai T, et al., J Sci Food Agric, 99:1675-81, 2019 ) yielded consistent results. In the fatty liver model of this example, decreases in maximal respiration, basal respiration, reserve respiratory capacity, and ATP production were evident, and these decreases were found to be suppressed by the addition of SME (Fig. 10).

Therefore, it was shown that SME has the effect of enhancing mitochondrial function in the fatty liver model as well as in the normal liver model. Interestingly, in the fatty liver model of this example, the effect of SME extended to ATP production enhancement (Fig. 10).

In addition, non-mitochondrial respiration reflects the amount of oxygen consumed by enzymes such as NADPH oxidase and xanthine oxidase present on the cell surface (Lewis N, et al., Sci Rep 10: 3493, 2020; Manes C, et al. al., J Reprod Fertil 104: 69-75, 1995). SME addition increased non-mitochondrial respiration, suggesting the possibility that SME affects cellular functions other than mitochondria.

Mitochondrial maximal respiration and respiratory reserve reflect the cell's ability to overcome stresses such as oxidation and inflammation (Desler C, et al., J Aging Res 2012, Article ID 192503, 2012; Kramer AK , et al., Redox Biology 2, 206-210, 2014). Also, when cells are stressed, their energy demand increases and more ATP is required to maintain cell function (Desler C, et al., J Aging Res 2012, Article ID 192503, 2012; Yamamoto H, et al., Oxid Med Cell Longev 2016, Article ID 1735841, 2016).

Hepatocytes with impaired mitochondrial function have been reported to have reduced maximal respiration and reserve respiratory capacity (Zelickson RB, et al., Biochem Biophys Acta 1807: 1573-82, 2011). Mitochondria in the liver model were thought to be impaired in their normal function and reduced in their ability to produce the energy necessary to maintain cell function. Therefore, as shown in FIG. 7A, the decreased cell viability in the fatty liver model may be due to decreased mitochondrial function (Redegeld AMF, et al., Eur J Pharmacol 228: 229-36, 1992; Han D, et al., Biochem Pharmacol 67: 439-51, 2004).

On the other hand, the addition of SME suppressed the decrease in cell viability in the fatty liver model (Fig. 7A), suggesting the possibility that SME has a cytoprotective function. LDH activity, an index that increases due to cytopathy or cell death (Legrand C, et al., J. Biotechnol 25: 231-43, 1992; Ruch JR, et al., Toxicol Appl Pharmacol 100: 451-64, 1989) From the fact that no change was observed (Fig. 7B), it is considered that the toxicity of SME to cells is small.

In the fatty liver model cells used in this example, the amount of lipid droplets was significantly increased compared to the control group (Fig. 8), so it is considered that the conditions for preparing the fatty liver model of this example were appropriate. . In addition, no change was observed in the lipid droplet amount due to the co-culture of the fatty liver model and SME. Even in the fatty liver model mouse of the previous study, no change in the amount of triglyceride and cholesterol in the liver was observed due to SME intake (Sakurai T, et al., J Sci Food Agric, 99:1675-81, 2019). Since the results were similar to those of the example, it is possible that SME does not affect the formation of lipid droplets.

Changes in mitochondrial copy number were investigated to elucidate the mechanism by which SME suppressed the decline in mitochondrial function, but no significant change was observed in the three groups used in this example (Fig. 11). It has been reported that no change in mitochondrial copy number was observed in high-fat diet-induced obese rats (Claycombe JK, et al., J Nutr Biochem 31: 113-21, 2016), and this example is similar. This resulted in

Since no change in mitochondrial copy number was observed due to the addition of SME even in the normal liver model, it is highly likely that the mechanism by which SME improves mitochondrial function is not due to quantitative changes in mitochondria, but rather to qualitative changes. was taken.

The mitochondrial respiratory chain consists of five complexes (complexes IV), and hydrogen derived from NADH undergoes the action of a series of complexes to produce water (H ₂ O). This process involves the transfer of electrons between the mitochondrial inner membrane and coenzymes. At the same time, protons (H ⁺ ) are transported from the matrix side to the intermembrane space by complexes I, III and IV. ATP is synthesized when these protons flow into the matrix side via complex V (ATP synthase). These processes are called oxidative phosphorylation (Elston T, et al., Nature 391: 510-3, 1998; Noji H and Yoshida M, J Biol Chem 276: 1665-8, 2001).

When the gene expression levels of complexes IV were measured, the expression level of NDUFS8 (complex I) was significantly decreased in the fatty liver model compared to the control group. It became clear that this decrease was suppressed by the addition of SME (Fig. 12).

Complex I is thought to act as the rate-limiting enzyme for the entire oxidative phosphorylation (Santamarina BA, et al., J Nutr Biochem 26: 1348-56, 2015; Paradies G, et al., World J Gastroenter 20: 14205-18, 2014). It was also found that the addition of SME significantly increased the expression level of UQCRC (complex III) compared to the control group (Fig. 12). Complex III is central to the electron transport pathway of oxidative phosphorylation (Santamarina BA, et al., J Nutr Biochem 26, 1348-1356, 2015). This suggests that SME induced the expression of complexes I and III of the mitochondrial respiratory chain complex, and as a result, suppressed the decline of mitochondrial functions such as maximal respiration, preliminary respiratory capacity, and basal respiration in the fatty liver model. be done. These results revealed that SME exerts an effect on gene expression associated with the mitochondrial electron transport system.

The present inventors previously found and proposed that SME improves mitochondrial function in a normal liver model (Japanese Patent Application No. 2020-120617). In the present invention, it was further clarified that the decrease in mitochondrial function in a fatty liver model is suppressed by the addition of SME. It was also suggested that this effect might be mediated through increased expression of Complex I and Complex III in the mitochondrial respiratory chain.

From the above, it was clarified that SME has a mitochondrial function-improving action in human normal liver and fatty liver models, and a cytoprotective action based thereon.

[Example 3] Mitochondrial fusion and fission 1. Background It has been reported that the balance between mitochondrial fusion and fission affects mitochondrial respiratory function (Westermann B, Biochim Biophys Acta 1817, 1833-8, 2012). Mitochondrial fusion improves mitochondrial respiratory function. On the other hand, mitochondria that have been damaged by stress such as reactive oxygen species (ROS) divide by separating the damaged site, and are thought to contribute to maintaining normal function. Therefore, both fission and fusion mechanisms are necessary, and the balance between them is important. The effect of SME on mitochondrial morphological changes is unknown, and clarification of this will lead to further elucidation of the mechanism of mitochondrial hyperfunction.

As described above (Example 2), in the fatty liver model, maximal respiration, basal respiration, reserve respiratory capacity, and ATP production were clearly decreased, but the addition of SME improved them. Therefore, in this example, the effects of SME on mitochondrial morphological changes were investigated in a fatty liver model using cultured liver cells C3A.

2. Method 2.1. Fig. 13 shows an overview of the mitochondrial morphology observation method. First, a C3A cell suspension was prepared using MEM containing 10% fetal bovine serum (FBS), seeded in a 35 mm diameter glass bottom dish at 1.875×10 ⁵ cells/dish, and placed in a CO ₂ incubator. Pre-incubated for 24 hours. Thereafter, the supernatant was replaced with FBS-free MEM (0% FBS, 1% Penicillin-Streptomycin-Neomycin, 406 mg/mL GlutaMAX, 10 mg/L Phenol Red [manufactured by gibco]) to starve the cells. and cultured in a _CO2 incubator for 24 hours.

Sterilized water or GFIO or GFIO+SME was then added as in the stimulation conditions described above. After 24 hours, the cells were washed with PBS and then stained with Hoechst 33342 (manufactured by Dojindo Laboratories) for nuclear staining and MitoTracker Green FM (manufactured by Thermo Fisher Scientific) for mitochondria staining. After washing with PBS, the cells were filled with FluoroBrite DMEM (manufactured by Thermo Fisher Scientific) and images were acquired using a fluorescence microscope BZ-9000 (manufactured by Keyence). Images were analyzed with Image J and the mitochondrial perimeter was calculated in the pixel range of 1000-80000.

2.2. Expression level analysis of genes involved in mitochondrial fusion and fission Stimuli were applied in the same manner as for mitochondrial copy number analysis. After 24 hours, the cells were collected with Lysis Buffer (manufactured by Life Technologies). RNA was extracted using PureLink RNA Mini Kit (manufactured by Life Technologies), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (manufactured by TOYOBO).

Gene expression levels were analyzed using the same real-time PCR analysis system (manufactured by BIO RAD) as described above. The target genes are dynamin related protein 1 (DRP1) related to mitochondrial fission, optic atrophy 1 (OPA1) related to fusion and mitofusin 1/2 (MFN1/2), and the expression level of each gene is the expression level of GAPDH. corrected by Comparison between groups was performed with the control group as 1, and the results were expressed as mean ±SD (n=8 for each group). The primers used are shown in Table 5.

3. Statistical Analysis Kruskal-Wallis test was performed for the analysis of the perimeter of mitochondria, and Dunn's multiple comparison test was performed for multigroup comparison test. One-way analysis of variance (one-way ANOVA) was used for gene expression level analysis, and a multi-group comparison test was performed by Tukey's multiple comparison test. The significance level was set at 5%.

4. Results 4.1. Effect of SME on mitochondrial morphological changes The median mitochondrial circumference was 3.6, 3.0, and 3.5 μm in the control group, GFIO group, and GFIO+SME group, respectively. It was significantly higher (Fig. 14).

4.2. Effect of SME on the expression levels of genes involved in mitochondrial fusion and fission The gene DRP1 involved in mitochondrial fission showed the most significant change, and its expression was significantly increased in the GFIO group compared to the control group (P< 0.05), and the increase was significantly reduced in the GFIO+SME group (P<0.001) (FIG. 15).

While no significant difference was observed between the control group and the GFIO group, the expression of the gene OPA1 involved in mitochondrial fusion decreased in the GFIO+SME group compared to the GFIO group (P<0.05) (FIG. 15). Expression of MFN1 and MFN2 showed no significant difference between the GFIO group and the GFIO+SME group (FIG. 15).

5. Discussion In the extracellular flux analyzer experiment described above (Example 2), SME activated mitochondrial respiratory function. As one candidate for the activation mechanism, we considered the possibility of increasing the number of fused mitochondria by adding SME. In this experiment using fluorescence staining of mitochondria, fragmentation of mitochondria was observed in fatty liver model cells, and addition of SME under these conditions suppressed the fragmentation. The results are consistent with previous reports that fusion between mitochondria improves mitochondrial respiratory function (Westermann B, Biochim Biophys Acta 1817, 1833-8, 2012).

In addition, the fact that the expression of the gene DRP1 associated with mitochondrial fission decreased significantly in the GFIO+SME group suggests that SME worked to suppress the fission, that is, it is thought that the fusion state was maintained. This result agreed with the result of the fluorescence staining image.

Therefore, in fatty liver model cells, SME not only acts to increase the expression of genes related to the mitochondrial electron transport chain, but also improves mitochondrial respiratory function by increasing mitochondrial fusion through suppression of division. It was suggested that

Claims (5)

A cytoprotective agent containing a milt extract as an active ingredient, wherein the milt extract is a fish milt hydrolyzate degraded to contain 50 to 100% of a fraction with a molecular weight of 5000 or less. A cytoprotective agent characterized by: 2. The cytoprotective agent according to claim 1, which protects hepatocytes in fatty liver. The cytoprotection is based on either or both of mitochondrial function improvement by enhancement of any selected from the group consisting of mitochondrial basal respiration, maximal respiration, reserve respiratory capacity and ATP production, and improvement of non-mitochondrial respiration. The agent for cell protection according to claim 1 or 2, which is The agent for cell protection according to any one of claims 1 to 3, wherein the cell protection is based on an increase in the expression level of one or both of complex I gene and complex III gene of the mitochondrial respiratory chain. A cell-protecting food or drink comprising the cell-protecting agent according to any one of claims 1 to 4.

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