Drug and health food for suppressing gene mutation

Abstract

To provide a drug and a health food for suppressing gene mutations using nucleic acids and RNA.SOLUTION: The present invention provides a drug and a health food comprising a yeast extract and, in addition to the yeast extract, one or more components derived from a plant, fish, and a dairy product as active ingredients, and particularly containing RNA as an active ingredient. The drug and the health food do not inhibit DNA damage, but promote an action of repairing damaged DNA or promote an action of killing cells containing damaged DNA to suppress gene mutations.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a drug and a health food for suppressing gene mutations. Specifically, it relates to a drug and a health food for suppressing gene mutations, which contain, in addition to yeast extract, components derived from plants, fish, and dairy products as active ingredients, and particularly contain RNA as an active ingredient. Note that health foods include beverages.

Background Art

[0002] Gene mutations are a cause of various diseases including cancer. Therefore, suppressing gene mutations is considered to lead to the prevention of many diseases. Each cell that makes up a living organism has a nucleus, and DNA is present in the nucleus. DNA is easily damaged by environmental factors such as ultraviolet rays, reactive oxygen species, and chemical substances. For example, when DNA is irradiated with ultraviolet rays, two adjacent bases (for example, thymine-thymine) dimerize, resulting in DNA damage. When there is DNA damage, replication errors are likely to occur at that part during DNA replication, leading to gene mutations. As another example, when the chemical substance ethylnitrosourea (ENU: N-Nitroso-N-ethylurea) enters the body, ENU acts directly on DNA and alkylates it, inducing DNA damage and causing gene mutations. However, DNA damage does not necessarily result in gene mutations. The living body is equipped with a DNA damage repair mechanism, and DNA damage sites are repaired by the action of DNA repair enzymes, etc. before DNA replication. It is said that tens of thousands of DNA damages occur per cell per day in humans, but most of them are repaired and do not lead to gene mutations. When DNA damage exceeds the repair capacity, repair cannot be completed in time, DNA replication occurs leaving DNA damage, and it is considered that gene mutations occur.

[0003] So far, there have been several reports on the suppression of DNA damage by the intake of foods and the like. However, in these studies, DNA damage has been targeted, and it is unclear whether the suppression of such DNA damage leads to the suppression of gene mutations. In studies related to nucleic acids, there are reports in in vitro tests (Non-Patent Document 1) that the addition of high-molecular DNA promotes the repair of DNA damage after ultraviolet irradiation, and reports in in vivo tests (Non-Patent Document 2) that ribonucleotide intake suppresses DNA damage in mice caused by cyclophosphamide administration. In addition, there is a report in an in vitro test (Patent Document 1) that the addition of low-molecular nucleoproteins (DNA and proteins) suppresses oxidative damage to DNA. However, also in these studies, as described above, DNA damage has been targeted, and it is unclear whether their action leads to the suppression of gene mutations.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] The present inventors have made efforts to elucidate the mechanism of gene mutation suppression. And an object of the present invention is to provide a drug and a health food for suppressing gene mutations. The inventors of the present invention have found that yeast extract and components derived from plants, fish, and dairy products (hereinafter also referred to as yeast extract, etc.) exhibit an effect of suppressing gene mutations, and in particular, RNA contained in the yeast extract, etc. exhibits an effect of suppressing gene mutations. Based on this, the present invention has completed a drug and a health food for suppressing gene mutations.

Means for Solving the Problems

[0007] The ENU has strong mutagenicity even when compared with other mutagenic compounds and is known to induce gene mutations in various organs throughout the body. Therefore, the inventors of the present invention selected intraperitoneal administration of ENU as a method for inducing gene mutations and used a test method called the Pig-a assay, which can examine the frequency of gene mutations from a small amount of peripheral blood collected from mice, etc., to verify the inhibitory effect of ingestion of yeast extract, etc. (RNA contained in the yeast extract) on gene mutations. And the inventors of the present invention have found an inhibitory effect of RNA ingestion on gene mutations from the results of the test method called the Pig-a assay.

[0008] Furthermore, the point of this test is that RNA contained in yeast extract, etc. was ingested in its original RNA form (the form originally present in food). The report of Non-Patent Document 2 clarified that ingestion of monoribonucleotides suppresses DNA damage in mice (protection against damage), but usually, in food, it exists not in the form of monomers but in the form of RNA (polymer, polymer form). The inventors of the present invention have found that by ingesting in the form of RNA this time, the effect of the mutagenic compound changes depending on the amount of RNA in the diet of mice, and have found an inhibitory effect on gene mutations in a form close to the RNA ingested in our daily life.

[0009] The present invention has been completed based on the above findings. That is, one aspect of the present invention is 1. A drug containing yeast extract, which contains RNA contained in the yeast extract as an active ingredient, and suppresses gene mutations. 2. A drug that further contains one or more components derived from plants, fish, and dairy products in addition to the yeast extract, and contains RNA as an active ingredient, the drug for suppressing gene mutations according to 1. 3. The drug for suppressing gene mutations according to 1 or 2, wherein the RNA promotes the action of repairing damaged DNA or promotes the action of killing cells containing damaged DNA. 4. The drug for suppressing gene mutations according to any one of 1 to 3, which contains 0.08 to 20% by mass of the RNA. 5. The drug for suppressing gene mutations according to any one of 1 to 4, wherein the RNA contained in the yeast extract is RNA extracted from Torula yeast. 6. A health food containing a yeast extract, which contains RNA contained in the yeast extract as an active ingredient, a health food for suppressing gene mutations. 7. A health food that further contains one or more components derived from plants, fish, and dairy products in addition to the yeast extract, and contains RNA as an active ingredient, the health food according to 6. 8. The health food according to 6 or 7, wherein the RNA promotes the action of repairing damaged DNA or promotes the action of killing cells containing damaged DNA. 9. The health food according to any one of 6 to 8, which contains 0.08 to 20% by mass of the RNA. 10. The health food according to any one of 6 to 9, wherein the RNA contained in the yeast extract is RNA extracted from Torula yeast. Relates to.

[0010] Furthermore, another aspect of the present invention relates to a method for suppressing gene mutations, characterized by oral administration to the drug for suppressing gene mutations and the health food, and further relates to the use for suppressing gene mutations, targeting the drug for suppressing gene mutations and the health food.

Effects of the Invention

[0011] The present invention provides a drug and a health food that highly suppress gene mutations with very high safety and almost no side effects. In addition, the drug and the health food for suppressing gene mutations of the present invention can suppress gene mutations by promoting the action of repairing DNA when it is damaged or promoting the action of killing cells containing damaged DNA.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0013] The present invention will be described in more detail below. Nucleic acids are a general term for DNA (deoxyribonucleic acid) that retains genetic information and RNA (ribonucleic acid) that transmits genetic information and synthesizes proteins according to the information held by DNA. They not only play an important role in cell growth and proliferation but are also very important substances in maintaining life activities. One of the food components described in the present invention is highly contained in the nucleic acid of Torula yeast. Among foods, Torula yeast contains particularly a large amount of nucleic acid. Torula yeast is a yeast that has been recognized as safe for food use by the US Food and Drug Administration (FDA), and its cells are produced using sugars such as pulp waste liquid and molasses. The nucleic acid (RNA) extracted from the cells is used as a health food. In the present invention, the effect of Torula yeast extract on gene mutations was examined, and in addition to yeast extracts other than Torula yeast, the effects of active ingredients contained in components derived from plants, fish, and dairy products, particularly RNA, on gene mutations were also examined.

[0014] Gene mutations are caused by damage to genes by ultraviolet rays, reactive oxygen species, chemical substances in the environment, etc. As a result, abnormal proteins are expressed, which are considered to cause various diseases. Regarding cancer, the accumulation of gene mutations is thought to induce carcinogenesis. In particular, since it is considered that cancer occurs when a mutation occurs in a gene that regulates cell division, it is considered important to suppress DNA damage and gene mutations for maintaining health. Organisms have several defense mechanisms to suppress gene mutations. One is the DNA damage repair reaction, and the second is the apoptosis reaction dependent on mismatch repair proteins. In addition, autophagy may be involved in the repair of DNA damage.

[0015] The drugs and health foods for suppressing gene mutations in one aspect of the present invention use, but are not limited to, components derived from plants such as yeast extracts, corn, wheat, soybeans, and rice, as well as components derived from fish and dairy products. Regarding yeast, there are, for example, brewer's yeast, Torula yeast, milk yeast, and baker's yeast, and extracts (especially RNA) from these yeasts can be used. The active ingredient in the drug and health products for suppressing gene mutations of the present invention is a yeast extract or the like, and in particular, RNA is considered. Hereinafter, when referred to as a "gene mutation suppressing substance", it represents a yeast extract or the like.

[0016] The administration forms of the drug for suppressing gene mutations of the present invention include parenteral administration by injection (subcutaneous, intravenous, intramuscular, intraperitoneal injection), ointment, suppository, aerosol, etc., or oral administration by tablets, capsules, granules, pills, syrups, solutions, emulsions, suspensions, etc. The drug for suppressing gene mutations of the present invention contains RNA of the gene mutation suppressing substance in an amount of about 0.01 to 99.5% by mass, preferably about 0.05 to 50% by mass, and more preferably about 0.08 to 20% by mass based on the mass of the total composition.

[0017] In addition to the gene mutation inhibitor, which is an active ingredient, the agent for suppressing gene mutations of the present invention can also contain other pharmaceutically or veterinarily active compounds. The clinical dosage of the gene mutation inhibitor contained in the agent for suppressing gene mutations of the present invention varies depending on age, body weight, patient sensitivity, degree of symptoms, etc. The dosage of the RNA of the gene mutation inhibitor contained in the agent for suppressing gene mutations or health food is usually 0.01% by mass or more, preferably 0.05% by mass or more, more preferably 0.08% by mass or more, based on the mass of the meal taken at one time. However, amounts outside the above range can also be used if necessary.

[0018] The gene mutation inhibitor of the present invention is formulated for administration by conventional means in the pharmaceutical industry. That is, tablets, capsules, granules, and pills for oral administration are prepared using excipients such as sucrose, lactose, glucose, starch, mannitol; binders such as hydroxypropyl cellulose, syrup, gum arabic, gelatin, sorbitol, tragacanth, methyl cellulose, polyvinyl pyrrolidone; disintegrants such as starch, carboxymethyl cellulose or its calcium salt, microcrystalline cellulose, polyethylene glycol; lubricants such as talc, magnesium stearate or calcium, silica; wetting agents such as sodium laurylate, glycerol, etc.

[0019] Injections, solutions, emulsions, suspensions, syrups, and aerosols are prepared using solvents for the active ingredient such as water, ethyl alcohol, isopropyl alcohol, propylene glycol, 1,3-butylene glycol, polyethylene glycol; surfactants such as sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene ether of hydrogenated castor oil, lecithin; suspending agents such as carboxymethyl sodium salt, cellulose derivatives such as methyl cellulose, natural gums such as tragacanth, gum arabic; preservatives such as esters of paraoxybenzoic acid, benzalkonium chloride, sorbate, etc. For ointments, which are transdermal preparations, for example, white petrolatum, liquid paraffin, higher alcohols, macrogol ointment, hydrophilic ointment, aqueous gel base, etc. are used. Suppositories are prepared using, for example, cacao butter, polyethylene glycol, lanolin, fatty acid triglyceride, coconut oil, polysorbate, etc.

[0020] Formulation examples of the agent for suppressing gene mutation of the present invention are shown below. Formulation Example 1 Tablets RNA in the gene mutation suppressing substance 1 - 30 g Lactose 200 - 300 g Microcrystalline cellulose 500 - 700 g Corn starch 300 - 400 g Hydroxypropyl cellulose 50 - 200 g CMC - Ca 100 - 200 g Magnesium stearate 10 - 60 g Total amount 1,500 g or more (*) After mixing the above components by a conventional method, 10,000 sugar-coated tablets are manufactured. (*) Depending on the proportion of RNA in the gene mutation suppressing substance (yeast extract), the total amount increases. The same applies to the "total amount" below. Formulation Example 2 Capsules RNA in the gene mutation suppressing substance 1 - 30 g Lactose 300 - 500 g Microcrystalline cellulose 500 - 1200 g Magnesium stearate 10 - 100 g Total amount 1,500 g or more After mixing the above components by a conventional method, they are filled into gelatin capsules to manufacture 10,000 capsules. Formulation Example 3 Soft capsules RNA in the gene mutation suppressing substance 2 - 40 g PEG400 400 - 500 g Saturated fatty acid triglyceride 1000 - 2000 g Hakka oil 0.2 - 2 g Polysorbate 80 5 - 20 g Total amount 2,000 g or more After mixing the above components, fill No. 3 soft gelatin capsules by a conventional method to produce 10,000 soft capsules of the soft capsule preparation. Formulation Example 4 Ointment RNA in the gene mutation inhibitor 0.1 - 3 g Liquid paraffin 5 - 20 g Cetanol 10 - 30 g White petrolatum 60 - 80 g Ethylparaben 0.05 - 3 g l-menthol 0.2 - 2 g Total amount 100.0 g or more Mix the above components by a conventional method to obtain an ointment. Formulation Example 5 Suppository RNA in the gene mutation inhibitor 1 - 30 g Witepsol H15* 400 - 600 g Witepsol W35* 500 - 600 g Polysorbate 80 0.5 - 21 g Total amount 1,000 g or more “*: Trademark name of triglyceride-based compound, Witepsol = Witepsol (registered trademark)” Melt and mix the above components by a conventional method, pour them into a suppository container, cool and solidify to produce 1,000 suppositories. Formulation Example 6 Injection RNA in the gene mutation inhibitor 1 - 15 mg Distilled water for injection 5 mL Dissolve and use at the time of use.

[0021] The present invention also relates to a health food containing a gene mutation inhibitor. The active ingredient in the health food of the present invention is a gene mutation inhibitor, that is, yeast extract or the like, particularly RNA. As a health food of the present invention, for example, it is preferably implemented as a health food having a gene mutation inhibitory effect. Further, it may be mixed with various components such as known sweeteners, acidulants, vitamins, etc. to make a product that suits the user's taste. For example, it can be provided in the form of tablets, capsules, drinks, jellies, dairy products such as yogurt, seasonings, processed foods, supplements, desserts, confectioneries, etc. The manufacturing process of these health foods is not particularly limited. For example, during the processing of health foods, the target health food can be manufactured by adding the above-mentioned sweeteners, etc. by appropriate means. The gene mutation inhibitory substance can be blended in the range of about 1 mg to 20 g or 0.08 g to 20 g per 100 g of food.

[0022] Specific substances that can be added to the health food of the present invention include, but are not limited to, the following. As the white-roe extract, after removing the skin, fibers, blood vessels, etc. from the white roe, it is purified to remove oil, and then subjected to enzymatic decomposition treatment with nuclease and protease to produce water-soluble nucleoprotein. As the white roe, for example, white roe of salmon, trout, sardine, cod, etc. can be used. Examples of collagen include porcine collagen peptide, fish collagen peptide (including gelatin), and collagen-containing mineral complex. The above-mentioned collagen can be used alone or as a mixture of two or more kinds.

[0023] Chondroitin is a kind of glycosaminoglycan (mucopolysaccharide), and chondroitin and its derivatives and their salts having a structure in which sulfuric acid is bound to a sugar chain in which a disaccharide of D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) repeats as a basic structure are mentioned.

[0024] As the hyaluronic acid, it is a kind of proteoglycan, and hyaluronic acids and their salts which have a basic structure of a structure in which disaccharide units in which the 1-position of β-D-glucuronic acid and the 3-position of β-D-N-acetyl-glucosamine are bonded are linked, and low-molecular-weight hyaluronic acids or hyaluronic acid degradation products obtained by subjecting such hyaluronic acids to enzymatic treatment using hyaluronidase or the like, or heat and pressure treatment can be mentioned. Specific hyaluronic acids that can be added to health foods include chicken comb extracts and the like.

[0025] Arginine is not particularly limited as long as it can be added in a form that can be used in foods, and examples include arginine alone and a form in which an arginine molecule and an acid molecule are bonded. Magnesium carbonate is also a medical pharmaceutical, and is not particularly limited as long as it can be added in a form that can be used in foods. As the magnesium salt, magnesium oxide, magnesium chloride, etc. may be added instead of magnesium carbonate or a part thereof. Zinc is not particularly limited as long as it can be added in a form that can be used in foods, and can be administered in forms such as zinc gluconate, zinc sulfate, edible zinc yeast, etc.

[0026] The vitamins are not particularly limited as long as they are vitamins or their derivatives, or their salts that can exhibit the effects of the present invention. For example, vitamin C (ascorbic acid), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B6 (pyridoxine), vitamin B12 (cobalamin), folic acid (vitamin B9), niacin (vitamin B3), calcium pantothenate, etc. can be mentioned.

[0027] As other components, sweeteners such as fructose, glucose, liquid sugar, refined sugar, rare sugar-containing syrup, erythritol and sucralose, fruit juices such as pineapple juice, preservatives such as sodium benzoate coloring agents such as caramel pigment, emulsifiers (for example, derived from soybeans), flavors, acidulants, etc. can be mentioned. When the health food of the present invention contains these other components, appropriate amounts of each component can be added.

[0028] Examples of the components of the health food of the present invention are shown below. Health Food Example 1 Beverage (amount per 720 mL) RNA in the gene mutation inhibitor 0.6 g - 18 g White fish extract 4000 - 4500 mg Collagen 60 - 90 g Chondroitin 100 - 200 mg Hyaluronic acid 50 - 80 mg Arginine 1500 - 2000 mg Magnesium carbonate 1000 - 2000 mg Zinc 30 - 40 mg Folic acid 2 - 4 mg Niacin 100 - 200 mg Vitamin C 3000 - 4000 mg Vitamin B1 13 - 14 mg Vitamin B2 14 - 15 mg Vitamin B6 15 - 16 mg Vitamin B12 25 - 27 mg Calcium pantothenate 70 - 90 mg Other added ingredients Appropriate amount (Other added ingredients: fructose glucose liquid sugar / refined sugar / rare sugar-containing syrup / erythritol / pineapple juice, etc.) Health Food Example 2 Jelly (amount per 15 g) RNA in the gene mutation inhibitor 10 - 300 mg White fish extract 100 - 200 mg Zinc 1 - 2 mg Vitamin C 60 - 90 mg Vitamin B1 0.7 - 0.9 mg Vitamin B2 1.0 - 1.2 mg Vitamin B6 0.8 - 1.0 mg Other added ingredients Appropriate amount (Other added ingredients: collagen / chondroitin / hyaluronic acid / sweetener / vitamin B12 / fruit puree, etc.) Health Food Example 3 Capsule-Type Supplement (Amount per 12 Capsules) RNA in Gene Mutation Suppressant: 80 - 2400 mg White-Grain Extract: 500 - 700 mg Arginine: 400 - 600 mg Zinc: 6 - 9 mg Vitamin C: 400 - 600 mg Copper: 0.4 - 0.6 mg Other Additive Ingredients: Appropriate Amount (Other Additive Ingredients: Brewer's Yeast / Icho Leaf Extract / Deodorized Garlic Extract / Pig Plantar Extract / Dextrin / Selenium / Gelatin / Sucrose Fatty Acid Ester / Sodium Ferrous Citrate / Calcium Pantothenate / Vitamin B1 / Vitamin B2 / Vitamin B12 / Vit amin A / Vitamin D / Folic Acid, etc.)

Examples

[0029] Hereinafter, one aspect of the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples. The mice and samples used in the following examples are as follows. 1. Mouse Rearing Male C57BL / 6J mice were introduced at 5 weeks of age, divided into 3 groups considering the average body weight, and fed an experimental diet after acclimation. ENU was administered intraperitoneally on the 15th day of feeding the experimental diet. The experimental diet was continuously fed after the administration of ENU. The food intake from the 8th day to the 10th day of feeding the experimental diet and the food intake from the 20th day to the 22nd day after the administration of ENU were measured. The body weight was measured once a week starting from the beginning of feeding the experimental diet. Blood was collected from the tail vein on the 8th, 15th, 22nd, 29th, and 43rd days after the administration of ENU and used for the Pig-a assay.

[0030] 2. Materials and Methods 2-1. Experimental Animals and Their Rearing Environment Male C57BL / 6J mice were purchased at 5 weeks of age from CLEA Japan, Inc. and maintained in the barrier breeding room of the Utsunomiya Office of the Special Immunology Research Institute, Inc.

[0031] 2-2. Grouping The mice were weighed at the time of introduction and grouped to achieve an average weight. The types and numbers of experimental diets fed to each group were as follows. Group 1: Low nucleic acid diet intake group, 8 mice Group 2: 0.6% RNA diet intake group, 8 mice Group 3: Normal diet for mice (CRF-1) intake group, 8 mice The grouping and the weights at the time of introduction are shown in Table 1.

Table 1

[0032] 2-3. Diet The low nucleic acid diet and the 0.6% RNA diet were prepared by Nippon Kairea Co., Ltd. The unopened diet was stored at 4°C, and the opened diet was stored at room temperature in the breeding room. The composition of the low nucleic acid diet is as shown in Table 2 below. The 0.6% RNA diet was prepared by subtracting the mass percentage of the added yeast extract from the mass percentage of corn starch in the composition shown in Table 2. The yeast extract used in the 0.6% RNA diet was derived from Torula yeast and was provided by Fordies Co., Ltd. The 0.6% RNA diet used a yeast extract containing 70% RNA. The commercially available normal diet for mice CRF-1 was purchased from Oriental Yeast Co., Ltd. and used after autoclaving. Table 3 shows the data obtained from the homepage of Oriental Yeast Co., Ltd. in October 2020. CRF-1 uses brewer's yeast, corn, wheat (bran), defatted soybeans, soybeans, oil-degummed rice bran, alfalfa, fish meal, and skim milk powder as raw materials. Although the blending ratios of these raw materials and RNA are not disclosed, as shown in Table 4, RNA is contained in CRF-1. This RNA is considered to be derived from each raw material. During the domestication period, all groups were fed the normal diet CRF-1.

Table 2

Table 3

[0033] 2-4. Measurement of the amount of nucleic acid in feed The nucleic acid (DNA, RNA) contents in the low-nucleic acid feed, 0.6% RNA feed, and normal feed were measured by the method described in Patent 6660994 (Japanese Patent Application No. 2018-219366, Japanese Unexamined Patent Application Publication No. 2020-085623). Specifically, after crushing the solid feed with a crusher and pulverizing it with a mortar and pestle, it was dissolved in a phosphate buffer solution with pH = 6.0, and a sample solution was prepared by performing protease treatment, protease deactivation treatment, and nuclease treatment, and analysis by HPLC was performed according to the method described in Patent 6660994 (Japanese Patent Application No. 2018-219366, Japanese Unexamined Patent Application Publication No. 2020-085623). The amounts in terms of total deoxynucleotide and total ribonucleotide in the low-nucleic acid feed, 0.6% RNA feed, and normal feed are shown in Table 4.

Table 4

[0034] 2-5. Preparation and administration of ENU Ethylnitrosourea (ENU) reagent (N8509-5G, N-Nitroso-N-ethylurea Bulk package, purity 56%) from SIGMA was used. ENU was dissolved in PBS (phosphate buffered saline) and intraperitoneally administered at 67.2 mg / kg body weight.

[0035] 2-6. Measurement of feed intake On the day before measuring the food intake, the bedding was changed and the weight of the feeder containing the experimental diet was measured. Approximately 24 hours later, the weight of the feeder containing the experimental diet and the weight of the experimental diet that had fallen onto the bedding were measured. The food intake per cage was calculated from the difference between the sum of these two weights and the weight of the feeder the previous day. The food intake per animal was calculated by dividing the food intake per cage by the number of animals housed. The same procedure was carried out continuously for 3 days. Table 5 shows the results of measuring the food intake for 3 days starting from the 7th day of feeding the experimental diet.

Table 5

Table 6

[0036] 2 - 7. Body weight measurement Figure 1 shows the results of measuring the body weight of the mice at the time of introduction and once a week after starting the feeding of the experimental diet, between 1 pm and 2 pm. There was no difference between the low nucleic acid diet group and the 0.6% RNA diet group. The normal diet group had a lower body weight compared to the others. This is presumably due to the differences in the basic composition of the experimental diets.

[0037] 3. Pig - a assay test method 3 - 1 Principle The Pig-a assay is a method capable of analyzing gene mutations using a small amount of peripheral blood and a flow cytometer. When a mutation occurs in the Pig-a (Phosphatidylinositol glycan anchor biosynthesis, class A) gene, the cell loses its GPI anchor. The GPI anchor serves to connect various proteins to the cell membrane surface. Normally, proteins such as CD24, which are GPI-anchor-binding proteins, are presented on the surface of red blood cell membranes. However, CD24 protein is not presented on red blood cells in which a Pig-a gene mutation has occurred. By utilizing this, red blood cells are stained with a fluorescently labeled antibody against the CD24 protein, and the mutation frequency of the Pig-a gene can be measured by counting the number of red blood cells with and without the CD24 protein presented on the cell membrane surface using a flow cytometer.

[0038] 3-2 Blood Sampling The mouse was placed in a fixator, and its tail was disinfected with 70% alcohol. A 23G injection needle was inserted into the tail vein to cause bleeding, and 4 μL of blood was immediately collected with a pipette and mixed with 1 μL of EDTA. Blood sampling can also be performed by incising a part of the tail vein with a razor blade to cause bleeding.

[0039] 3-3 Blood Sample Preparation and Staining Before staining, 100 μL of physiological saline containing 1% fetal bovine serum, 3 μL of PE / Cy7 anti-mouse TER-119 / Erythroid Cells (BioLegend), and 2 μL of FITC anti-mouse CD24 (BioLegend) were mixed to prepare an antibody staining solution. 1 μL of the collected blood sample was added to 150 μL of physiological saline containing serum, mixed well, and then centrifuged at 1000×g for 5 minutes at 4°C. After removing the supernatant, it was suspended in 100 μL of the antibody staining solution and left to stand in the dark at 4°C for 40 minutes for staining. After stirring the stained blood sample, it was centrifuged at 1000×g for 5 minutes at 4°C. The supernatant was removed and suspended in 500 μL of physiological saline containing serum. Note that the sample can be used for measurement within 24 hours after staining and should be stored in a cold and dark place until immediately before measurement.

[0040] Measurement by 3-4 flow cytometer The stained blood samples were measured using a flow cytometer, Gallios (Beckman Coulter) or FACS CantoII (BD FACS). Figure 2 shows an example of the results of the Pig-a assay in which blood 43 days after ENU administration was measured using a flow cytometer. First, gate P1 was set for a single cell population on a plot of forward scatter light and side scatter light (an operation to select a specific cell population in the sample as the analysis target on the data) (Figure 2-A). Further, gate P2 was set for a population of TER-119-positive cells, which are red blood cell markers, from within the single cell population (gate P1), using the fluorescence of PE / Cy7 labeled with an anti-TER-119 antibody as an index (Figure 2-B). Next, gate P3 was set for the CD24-negative portion among the red blood cell population (gate P2), using the fluorescence of FITC labeled with an anti-CD24 antibody as an index (Figure 2-C). The number of CD24-negative cells in the red blood cell population (gate P2) was counted, and the Pig-a gene mutation frequency was calculated by dividing the number of CD24-negative cells (= the number of cells within gate P3, that is, the number of Pig-a gene mutant red blood cells) by the number of TER-119-positive cells (= the number of cells within gate P2, that is, the total number of red blood cells measured). Note that each sample was measured so that the number of red blood cells (TER-119-positive cells) was approximately 1 million. In the Pig-a assay using mouse blood, as shown in Figure 2-C, more CD24-negative red blood cells (gate P3) were observed in the blood of mice administered with the mutagen ENU compared to the blood of mice administered with PBS as a negative control (data not shown), and induction of gene mutation could be detected.

[0041] 4. Results of Pig-a assay Figure 3 shows a graph of the gene mutation frequency measured over time by the Pig-a assay in mice fed a low nucleic acid diet, mice fed a 0.6% RNA diet, and mice fed a normal diet after administration of ENU. In mice fed a low-nucleic acid diet, mice fed a 0.6% RNA diet, and mice fed a normal diet, no difference in the Pig-a gene mutation frequency was observed 8 days or 15 days after ENU administration. Subsequently, when breeding continued with each experimental diet, the gene mutation frequency tended to decrease in mice fed a 0.6% RNA diet and mice fed a normal diet compared to mice fed a low-nucleic acid diet 22 days or 29 days after ENU administration. On the 43rd day after ENU administration, the gene mutation frequency significantly decreased in mice fed a 0.6% RNA diet and mice fed a normal diet compared to mice fed a low-nucleic acid diet. Regarding the results of this test, if the intake of yeast extract, etc. is a defensive effect against DNA damage itself, a difference would appear in the initial stage after ENU administration between the group that ingested yeast extract, etc. and the non-ingested group. However, gene mutations occurred to the same extent in both groups 8 - 15 days after ENU administration. From this, it is judged that the point of action of yeast extract, etc. is not a defensive effect against DNA damage. Therefore, it is considered that the intake of yeast extract, etc. (especially RNA) does not have a defensive effect against DNA damage itself, but promotes DNA repair, thereby suppressing gene mutations. On the other hand, when significant DNA damage occurs, in order to prevent the accumulation of gene mutations, the cells are removed by cell death, etc. It is considered that the intake of yeast extract, etc. (especially RNA) can prevent the final accumulation of gene mutations even under conditions with strong DNA damage exceeding the repair capacity.

Claims

Claim 1 A drug containing yeast extract, which is a drug for suppressing gene mutations and contains RNA contained in the yeast extract as an active ingredient. Claim 2 A drug according to claim 1, which further contains one or more of plant, fish and dairy product-derived components in addition to the yeast extract, and contains RNA as an active ingredient, for suppressing gene mutations. Claim 3 The drug for suppressing gene mutations according to claim 1 or claim 2, wherein the RNA promotes the action of repairing damaged DNA or promotes the action of killing cells containing damaged DNA. Claim 4 The drug for suppressing gene mutations according to any one of claims 1 to 3, which contains 0.08 to 20% by mass of the RNA. Claim 5 The drug for suppressing gene mutations according to any one of claims 1 to 4, wherein the RNA contained in the yeast extract is RNA extracted from Torula yeast. Claim 6 A health food containing yeast extract, which is a health food for suppressing gene mutations and contains RNA contained in the yeast extract as an active ingredient. Claim 7 A health food according to claim 6, which further contains one or more of plant, fish and dairy product-derived components in addition to the yeast extract, and contains RNA as an active ingredient, for suppressing gene mutations. Claim 8 The health food for suppressing gene mutations according to claim 6 or claim 7, wherein the RNA promotes the action of repairing damaged DNA or promotes the action of killing cells containing damaged DNA. Claim 9 The health food according to any one of claims 6 to 8, which contains 0.08 to 20% by mass of the RNA. Claim 10 The health food according to any one of claims 6 to 9, wherein the RNA contained in the yeast extract is RNA extracted from Torula yeast.

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