Damaged DNA repairing composition including GABA as active ingredient

Abstract

[Problem] To provide a composition for a supplement, medicine, food or drink, feed, or similar that is capable of repairing damaged DNA. [Solution] This damaged DNA repairing composition that is characterized by including GABA as an active ingredient can be used as a highly safe supplement, medicine, food or drink, or feed, for anti-aging or health maintenance.

Description

DNA damage repair composition containing GABA as an active ingredient

[0001] This invention relates to a technical field related to the repair of damaged DNA.

[0002] DNA, which carries genetic information, can be damaged by external factors such as radiation, ultraviolet rays, and chemical substances, as well as internal factors such as reactive oxygen species generated during intracellular metabolism and changes in intracellular pH. DNA damage inhibition, which prevents DNA damage from occurring in the first place, and DNA damage repair, which repairs damaged DNA, play important roles in cells protecting genetic information and maintaining normal function.

[0003] It has been reported that the ability to suppress DNA damage and repair damaged DNA declines with age, and that the accumulation of mutations due to DNA damage is a cause of some neurodegenerative diseases. Therefore, research and development of substances and materials that suppress or repair DNA damage is progressing in order to prevent DNA damage that is involved in the development of tumor cells and various aging symptoms, or to repair damaged DNA and prevent these diseases from occurring. For example, as DNA damage inhibitors to avoid or reduce factors that damage cellular DNA, such as oxidative stress, ultraviolet rays, and chemical substances, many have been reported, including plant extracts of the genera Hypnum, Hypnum, and Thuidium, which are useful as antioxidants with the ability to scavenge free radicals such as reactive oxygen species (Patent Document 1), plant extracts of the genus Gentiana, or extracts of the genus Loquat (Patent Document 3), as well as specific water-soluble kefir fractions (Patent Document 4).

[0004] The genomic DNA of cells is constantly being damaged by various internal and external factors. DNA damage includes various types of damage that affect the genomic DNA sequence, such as oxidation of bases, abnormal binding due to mismatches between bases, and DNA breaks. In particular, DNA double-strand breaks (DSBs) are serious damage in which both strands of DNA are broken. As DNA damage repair agents to restore already damaged DNA to its original normal state, UV-induced DNA damage repair agents containing goji berry extract as an active ingredient (Patent Document 5) and plant extracts of one or more plants from the genus Panax in the Araliaceae family or the genus Rumex in the grass family (Patent Document 6) have been reported.

[0005] On the other hand, gamma-aminobutyric acid (GABA) is a non-protein-constituting amino acid that is widely present in nature and is known as a neurotransmitter in the central nervous system of mammals. Following the revision of the food and drug classification in 2001, it became possible to use it as a food, and it is commercially available as a supplement in tablet or capsule form. A wide range of physiological activities have been reported, including tranquilizing effects and blood pressure lowering effects (Non-Patent Document 1), as well as effects that improve skin elasticity (Patent Document 7) and inhibit mammary gland tissue dysplasia (Patent Document 8). In addition, GABA has been reported to have antioxidant activity that suppresses hydrogen peroxide-induced DNA cleavage in human peripheral lymphocytes (Non-Patent Document 2).

[0006] Japanese Patent Publication No. 2006-193472, Japanese Patent Publication No. 2006-151831, Japanese Patent Publication No. 2014-118405, Japanese Patent Publication No. 2009-102397, Japanese Patent Publication No. 2012-211106, Japanese Patent Publication No. 2017-39680, Japanese Patent Publication No. 2018-30826, International Publication No. 2024 / 106501

[0007] Journal of Agricultural and Food Chemistry (2012) Vol.60, No.6, p.1586-1594

[0008] The object of the present invention is to provide a DNA damage repair composition containing a highly safe substance as an active ingredient. Another object of the present invention is to provide a supplement, pharmaceutical, food or beverage, or animal feed for repairing damaged DNA.

[0009] The inventors have discovered that GABA present in living organisms enhances the ability to repair damaged DNA, and based on this discovery, they have completed the present invention relating to the use of GABA in food, beverages, supplements, and pharmaceuticals.

[0010] The present invention relates to the following compositions (1) to (6), the use of (7) and (8), or the method of use of (9): (1) A composition for repairing damaged DNA, comprising GABA as an active ingredient. (2) The composition according to (1) above for the prevention or improvement of health disorders, including those caused by DNA double-strand breaks. (3) The composition according to (2) above, wherein the health disorder is a disease or a side effect of treatment. (4) The composition according to (1) above, which is an orally administered preparation, food or beverage, or pharmaceutical. (5) The composition according to (1) above, wherein the daily intake of GABA is 10 to 3000 mg. (6) The composition according to (1) above, wherein the damaged DNA is a DNA double-strand break. (7) The use of GABA in the manufacture of a damaged DNA repair agent. (8) The use of GABA as a damaged DNA repair agent. (9) A method of using GABA for the repair of damaged DNA.

[0011] According to the present invention, it is possible to provide a DNA damage repair composition containing highly safe GABA as an active ingredient that repairs DNA damage. Furthermore, it is possible to provide easily ingestible supplements, pharmaceuticals, foods and beverages, and animal feed for repairing damaged DNA. One phenomenon affected by damaged DNA is aging. Ingesting GABA may lead to anti-aging through the repair of damaged DNA.

[0012] Furthermore, acetaldehyde, which is excessively produced in the body by drinking alcohol, smoking, etc., is known to cause DNA damage. However, by ingesting GABA, it is expected to repair damaged DNA and reduce toxicity. Skin condition changes thought to be caused by ultraviolet rays such as spots and wrinkles are thought to be caused in part by ultraviolet rays reaching the inside of the skin, generating reactive oxygen species and causing DNA damage. However, by ingesting GABA, it is expected to promote the repair of damaged DNA by reactive oxygen species and lead to maintaining health.

[0013] The results of Example 1 are shown. After culturing human breast cancer-derived T-47D cells and removing the medium, 10 mM H ２ O ２ was added at 400 μL / well and exposed at room temperature for 15 minutes. Then H ２ O ２ was removed, and PBS was added, and PBS adjusted so that GABA was 1.1 μg / mL was added, and it was incubated at 37°C, in 5% CO ２ . PBS was used as a control. After treatment for 0, 1, 2, and 4 hours respectively, formaldehyde was added to a final concentration of 1% for immobilization, immunostained, observed with a microscope, and the number of DNA double-strand breaks was shown by measuring the signal of the marker for DNA double-strand breaks per cell. The results of Example 2 are shown. After culturing human breast cancer-derived T-47D cells and removing the medium, PBS was added, and PBS adjusted so that GABA was 1.1 μg / mL was added, and it was incubated at 37°C, in 5% CO ２ for 4 hours. Then, after exposing to 10 mM H ２ O ２ for 15 minutes, it was immobilized, immunostained, observed with a microscope, and the number of DNA double-strand breaks was shown by measuring the signal of the marker for DNA double-strand breaks per cell. Images of cell nuclei obtained in Example 3 are shown. The repair effect of damaged DNA by GABA was evaluated by the comet assay. The pre-treatment group, as well as the untreated group which is the experimental treatment group, the group untreated after H ２ O ２ treatment, and the group untreated after H ２ O ２Images of cell nuclei from the GABA-treated group after treatment, taken using a fluorescence microscope with a 10x objective lens and a total magnification of 100x. The total damaged DNA score obtained in Example 3 is shown. Pre-treatment group, experimental treatment group (untreated group), and H ２ O ２ After treatment, untreated group and H ２ O ２ This figure shows the total DNA damage score calculated for the shape of 100 randomly selected cell nuclei from each of the GABA-treated groups.

[0014] GABA, which is administered externally to repair damaged DNA in this invention, is an abbreviation for γ-aminobutyric acid. The abbreviation GABA, derived from the first letters of its English name, is commonly used. It is a type of amino acid that is widely found in plants and animals. GABA has the function of improving blood flow to the brain, increasing oxygen supply, and enhancing brain metabolism. GABA is present in the brain and spinal cord of living organisms and is known as a neurotransmitter of the central nervous system. It is used to activate blood flow in the diencephalon, enhance the metabolic function of brain cells, and alleviate autonomic nervous system dysfunction caused by stress. It is also known to have an effect on improving skin elasticity.

[0015] GABA in this invention is not particularly limited to GABA extracted from vegetables, fruits, grains, etc., GABA produced from fermented foods, or GABA produced from organic synthesis. The above vegetables, fruits, and grains refer to pumpkins, eggplants, tomatoes, cucumbers, rice, brown rice, malt, soybeans, etc., and fermented foods refer to fermented foods such as kimchi, pickles, fermented milk, and natto derived from lactic acid bacteria, yeast, and natto bacteria. GABA can be obtained by fermenting germinated rice, green tea, or rice bran with lactic acid bacteria, or by fermenting glutamic acid using lactic acid bacteria. Furthermore, it may be obtained by enzymatically converting glutamic acid and / or sodium glutamate using naturally occurring glutamate decarboxylase (GAD) as a raw material, or by isolating bacteria from fermented foods and preparing it in a culture medium.

[0016] These fermented solutions and extracted solutions may be appropriately dried by freeze-drying or spray-drying and powdered. Although not particularly limited, the GABA content is 0.1% to 30% in liquid form and 0.1% to 99% in powder form. The daily intake for humans is 1 to 3000 mg as GABA, and 10 mg to 3000 mg is preferred. More preferably, it is 20 mg to 1000 mg, and even more preferably 100 mg to 1000 mg. If it is less than 10 mg as GABA, the effect cannot be expected, and if it is 3000 mg or more, it becomes difficult to take a single dose.

[0017] The action of the composition for repairing damaged DNA containing GABA of the present invention can be confirmed by cell experiments using cultured cells. For example, after exposing cultured cells to hydrogen peroxide to cleave and damage double-stranded DNA, the repair effect of damaged DNA when GABA is added can be confirmed by measuring the signal of the marker for DNA double-strand breaks per cell by immunostaining.

[0018] DNA damage is known to cause gene mutations and is involved in cancer, lifestyle-related diseases, or various aging symptoms. Enhancing the ability to repair damaged DNA leads to anti-aging. Anti-aging, also called anti-aging, aims to extend the healthy life span. In 2019, the World Health Organization defined aging (functional decline due to aging, excluding dementia) as a disease in the 11th edition of the International Classification. The causes of cell aging are diverse, including damage to genomic DNA, daily inflammation, stress such as oxidative stress and lysosomal stress.

[0019] Also, maintaining good physical and mental conditions without getting sick is called maintaining health. The composition for repairing damaged DNA of the present invention can enhance the ability to repair damaged DNA including DNA double-strand breaks, so it can be used for maintaining health to prevent diseases such as cancer, cardiovascular disease, neurodegenerative disease, and diabetes caused by DNA damage, or for improving conditions caused by the side effects of treatment for diseases.

[0020] The GABA-containing DNA repair composition of the present invention may be in the form of a food or beverage rich in GABA. The form of the food or beverage is not particularly limited, but may be in the form of a powder, granules, capsules, or tablets. Other forms may include food ingredients, food additives, or syrups, suspensions, drinks, liquid foods, soft drinks, milk drinks, lactic acid bacteria drinks, functional seasonings, gel-like foods, puddings, yogurts, confectionery / cakes, breads, noodles, pasta, chocolates, candies, chewing gum, etc. Furthermore, the food or beverage is not limited to human consumption and includes feed for mammals such as dogs and cats kept as pets or livestock. In addition to ordinary food and beverages, the concept of food or beverage also includes beverages, so-called supplements and health foods, enteral nutrition foods, foods for special dietary uses, nutritional functional foods, and foods for specified health uses.

[0021] One embodiment of the DNA damage repair composition of the present invention is preferably used in at least one of the following applications: pharmaceuticals, food (supplements), and food additives. When used in pharmaceuticals, there are no particular limitations, but examples include oral formulations such as powders, granules, capsules, pills, and tablets. The effective dose of the pharmaceutical composition varies depending on the condition of the recipient (including age, physical condition, etc.) and dosage form, but the oral dose for a human (an adult weighing 60 kg) can be set within the range of the above-mentioned daily intake for humans.

[0022] Next, specific examples of the present invention will be described by the following embodiments, but the present invention is not limited to these embodiments.

[0023] [Example 1] <Cell test> Cultured cells were treated with hydrogen peroxide (H ２ O ２ The effect of adding GABA to DNA after it has been damaged by exposure to GABA (which causes double-strand breaks) was investigated.

[0024] Human breast cancer-derived T-47D cells were used as the cultured cells. T-47D cells were cultured in RPMI1640 medium, which was phenol red-free and contained 10% char-filtered fetal bovine serum. 400 μL / well was placed in an 8-well slide chamber at 37°C and 5% CO2. ２ The cultures were incubated under static conditions. After 3 days of incubation, the culture medium was removed and replaced with RPMI1640 medium without phenol red, and incubated for 2 days. The culture medium was removed and 10 mM H was added. ２ O ２ The solution was added at a concentration of 400 μL / well and exposed to room temperature for 15 minutes.

[0025] After that, H ２ O ２ Remove the GABA, and either GABA-free or GABA-containing at 1.1 μg / mL, at 37°C and 5% CO2. ２ Cells were incubated in PBS. The solvent was PBS, and after 0, 1, 2, or 4 hours, formaldehyde was added to a final concentration of 1% for fixation, followed by immunostaining. P-Histone H2A.X was used as the primary antibody, and Alexa Fluor 546 was used as the secondary antibody. 100 cells were observed per well, and the number of γH2A.X fluorescence signals (focuses) formed in each cell nucleus was measured as an indicator of the number of DNA double-strand breaks. Statistical analysis was performed using the Mann-Whitney U-test, and significance was tested after Bonferroni correction. The results are shown in Figure 1.

[0026] At 0h, H ２ O ２ Intracellular DNA double-strand breaks (DSBs) were observed. After 1 hour, no DSB repair was observed even with the addition of GABA, but after 2 hours, a tendency toward DSB repair was observed with GABA addition. After 4 hours, GABA addition significantly repaired DSBs compared to the control group. These results indicate that the addition of GABA has the effect of promoting DSB repair in cells.

[0027] [Example 2] <Cell Experiment> To confirm whether the reduction in DNA double-strand breaks (DSBs) observed in Example 1 is due to the inhibitory or repairing effect of GABA, the same cultured cells as in Example 1 were exposed to GABA 1.1 μg / mL for 4 hours, and then subjected to 10 mM H２ O ２ The samples were exposed to light for 15 minutes to allow them to fix, and the number of DSBs was counted by immunohistochemistry. The results are shown in Figure 2.

[0028] H after GABA exposure ２ O ２ No reduction in DSB was observed when exposed to [the substance]. When the results of Example 2 are compared with the results of Example 1, it is shown that the reduction in DSB by GABA in Example 1 is due to the DSB repair effect of GABA, not the DSB inhibitory effect.

[0029] [Example 3] <Cell Experiment> The repair of damaged DNA by GABA was evaluated using the comet assay. The experiment was performed three times independently, with a pre-treatment group and an experimental treatment group (untreated group, H2). ２ O ２ After treatment, untreated group and H ２ O ２ The GABA-treated group was compared after treatment. Human mammary cancer-derived T-47D cells were used as cultured cells. RPMI 1640 medium containing 10% fetal bovine serum and free of phenol red (hereinafter, RPMI 1640 medium free of phenol red is referred to as "Medium") was used as the culture medium, with a volume of 2 mL in a 35 mm culture dish at 37°C and 5% CO2. ２ The cells were cultured under static conditions. After 3 days of culture, the culture medium was removed and replaced with a medium that did not contain fetal bovine serum, and incubated for 1 day. This was designated as the pre-treatment group. Next, a portion of the pre-treatment group was subjected to the following primary and secondary treatments as the experimental treatment group. Specifically, as the primary treatment, after removing the culture medium from the pre-treatment group, PBS or H ２ O ２ Two mL of PBS dissolved in 10 mM was added, and the mixture was exposed at room temperature for 20 minutes. Next, as a secondary treatment, after removing the supernatant, either a medium solution without GABA or a medium solution with 1.1 μg / mL of GABA was added, and the mixture was exposed at 37°C and 5% CO2. ２ Incubate for 4 hours, then H ２ O ２ Treatment group (untreated group), H ２ O ２ The group that underwent post-processing was designated as the GABA-treated group.

[0030] Subsequently, adherent cells were detached from all groups using 0.05% trypsin. The cells were then divided into 2 × 10⁶ cells. ４ Cells were collected individually, and the supernatant was removed after centrifugation. 20 μL of 1% low-melt agarose was added and spread onto a glass slide. After refrigeration at 4°C to allow the agarose to solidify completely, lysis buffer was added to dissolve the cell membrane and nuclear membrane. The glass slides were immersed in an electrophoresis tank and electrophoresis was performed. TBE buffer was used as the buffer. After drying the glass slides, they were stained with DAPI and mounted on coverslips. Figure 3 shows images of the cell nuclei of each group taken using a fluorescence microscope with a 10x objective lens and a total magnification of 100x. Figure 4 shows the total damaged DNA score calculated from the shape of 100 randomly selected cell nuclei from each group, based on the following literature. Tukey's multiple comparison method was used to test for statistical significance. Zu-Chuan Weng, Yasuyuki Ogawa: Comet Assay: A Powerful Analytical Method for Detecting Genotoxicity, Journal of Occupational Safety and Health (2010) Vol. 3, No. 1, pp. 79-82.

[0031] <Results> H ２ O ２ DNA cleaved by this process moves within the agarose gel via electrophoresis. Observation with a fluorescence microscope reveals that the cleaved DNA strands move toward the anode, forming a tail. The comet assay is a method for evaluating damaged DNA using this tail. Figure 3-3 shows H ２ O ２ In the untreated group after treatment, tails appeared at a high frequency, as shown in Figure 3-4. ２ O ２ In the GABA-treated group after treatment, tails appeared at a moderate frequency. On the other hand, in the pre-treatment group shown in Figure 3-1 and the untreated group shown in Figure 3-2, tails appeared at a low frequency. Figure 4 shows the total damaged DNA score for each group. There was no difference in the total score between the pre-treatment group and the untreated group, indicating that the experimental procedure did not affect DNA damage. H was a positive target. ２ O ２ In the untreated group after treatment, the total value increased 3.5 times compared to the untreated group. ２ O ２ In the GABA-treated group after processing, the ratio was 2.3 times. ２O ２ The total value of the GABA-treated group after processing is H ２ O ２ The level after treatment was 0.7 times that of the untreated group, and this decrease was statistically significant. Therefore, the comet assay also confirmed that the addition of GABA has the effect of repairing damaged DNA.

Claims

1. A DNA repair composition containing GABA as an active ingredient.

2. The composition according to claim 1 for the prevention or improvement of health disorders, including those caused by DNA double-strand breaks.

3. The composition according to claim 2, wherein the health hazard is a disease or a side effect of treatment.

4. The composition according to claim 1, which is an orally administered preparation, food or beverage, or pharmaceutical product.

5. The composition according to claim 1, wherein the daily intake of GABA is 10 to 3000 mg.

6. The composition according to claim 1, wherein the damaged DNA is a DNA double-strand break.

7. Use of GABA in the manufacture of DNA damage repair agents.

8. Use of GABA as a DNA damage repair agent.

9. A method for using GABA to repair damaged DNA.

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