DNA damage inhibitor

Abstract

To provide DNA damage inhibitors with excellent safety and efficacy. [Solution] The present invention provides a DNA damage inhibitor characterized by containing kukui tree seed extract, which is highly safe and effective. These DNA damage inhibitors effectively suppress DNA damage caused by DNA damage inducers such as benzo[a]pyrene. Therefore, by including kukui tree seed extract, it becomes possible to provide pharmaceuticals, quasi-drugs, cosmetics, foods, etc., that can effectively protect against ultraviolet rays, chemical substances, and the like.

Description

Technical Field

[0001] The present invention relates to a DNA damage inhibitor.

Background Art

[0002] DNA is a polymer in which a double strand composed of sugar, phosphate, and four types of bases forms a double helix. Although various genetic information such as development, growth, and reproduction is incorporated in DNA, it is known that DNA is damaged by various factors.

[0003] External factors of DNA damage are mainly classified into physical factors and chemical factors. As physical factors, ultraviolet rays contained in sunlight, ionizing radiation such as X-rays and γ-rays, etc. are known. Also, as chemical factors, it is known that mainly electrophilic chemical substances react with DNA, resulting in various structural changes in DNA. These DNA damages can trigger cancerization when they occur in somatic cells, and may affect the next generation when they occur in germ cells. Therefore, it is very important to evaluate the DNA damaging properties of test substances.

[0004] Several methods are known as methods for evaluating the DNA damaging properties of test substances. For example, the post-labeling method for detecting DNA adducts, the Rec assay using Bacillus subtilis, the unscheduled DNA synthesis test using the DNA repair process, the SOS test, etc. can be mentioned. Also, the comet assay for detecting DNA strand breaks using electrophoresis is known, and the in vivo comet assay using experimental animals has been test guideline-ized by the OECD (Organization for Economic Cooperation and Development). On the other hand, although the in vitro comet assay using cultured cells has not been test guideline-ized by the OECD, there have been many research reports so far.

[0005] The kukui tree (scientific name: Aleurites moluccanus), belonging to the genus Aleurites in the family Euphorbiaceae, is widely distributed, mainly in Southeast Asia. To date, antiviral agents containing active ingredients present in the kukui tree (Patent Document 1), hair growth agents characterized by being made from the kukui tree (Patent Document 2), MMP inhibitors containing kukui tree seed extracts, collagen production promoters, wrinkle improvers, hyaluronic acid production promoters, and cell proliferation promoters (all of which are in Patent Document 3) have been known. However, it has not been reported to date that kukui tree seeds have DNA damage inhibitory effects. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Special Publication No. 5-506847 [Patent Document 2] Japanese Patent Application Publication No. 11-286414 [Patent Document 3] Japanese Patent Publication No. 2022-34789 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The objective of this invention is to provide a DNA damage inhibitor with excellent safety and efficacy. [Means for solving the problem]

[0008] In other words, the present invention provides a DNA damage inhibitor characterized by containing an extract of the seeds of the kukui tree.

[0009] Furthermore, the present invention provides a pharmaceutical product, quasi-drug, cosmetic product, or food product characterized by containing the DNA damage inhibitor as an active ingredient. [Effects of the Invention]

[0010] The present invention makes it possible to provide a DNA damage inhibitor with excellent safety and efficacy. [Modes for carrying out the invention]

[0011] The kukui tree (Aleurites moluccanus) used in this invention is a deciduous tree native to Southeast Asia, belonging to the genus Aleurites in the family Euphorbiaceae, and grows to a height of 10 to 20 meters. Its dark green, oval leaves are arranged spirally, and its yellowish-green fruits are about 5 centimeters in diameter and grow in clusters of 3 to 6.

[0012] The "kukui tree seeds" used in this invention refer to the brown to black, lumpy mass inside the fruit, consisting of about two layers, excluding the pulp. Furthermore, the white to light brown, nut-like substance inside the mass is also considered a seed in this invention. These are also called kernels. This nut-like substance is also called a kukui nut or candle nut. Seeds can be obtained from the growing region or purchased commercially. For extraction, the seeds themselves may be extracted, or they may be dried, crushed, or finely chopped.

[0013] Examples of extraction solvents include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), and ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.). Preferably, polar solvents such as water, lower alcohols, and liquid polyhydric alcohols are used, and water is particularly preferred. These solvents may be used individually or in mixtures of two or more. In addition, a solvent with pH adjusted by adding an acid or alkali to the above extraction solvent may be used. There are no particular limitations on the amount of solvent used; for example, it should be 10 times or more, preferably 20 times or more, relative to the dry weight of the kukui tree seeds, but it is preferable to be 100 times or less for convenience in operations such as concentration or isolation after extraction. Furthermore, the extraction temperature and time can be appropriately selected depending on the type of solvent used, the pressure during extraction, etc.

[0014] The extraction method is not particularly limited and can be performed by methods such as heat extraction (e.g., 40-100°C), room temperature extraction (e.g., 15-40°C), cold extraction (e.g., 0-15°C), stirring extraction, or column extraction. When the solvent is water, heat extraction is preferred from the viewpoint of extraction efficiency.

[0015] The above extract may be used as is, but if necessary, it may be used after treatment such as concentration (concentration by vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation, etc., to the extent that the effects of the present invention are achieved. Furthermore, the extracted solution may be treated by concentration to dryness, spray drying, freeze-drying, etc., and used as a dried product.

[0016] The DNA damage inhibitor in this invention contains a seed extract of the kukui tree and suppresses DNA damage by protecting cells from the induction of DNA damage by ultraviolet light, ionizing radiation, chemical substances, etc. The types of DNA damage are not particularly limited and include, for example, oxidation, hydrolysis, pyrimidine dimer formation, interstrand crosslinking, single-strand breaks, double-strand breaks, etc.

[0017] The evaluation method for DNA damage inhibitors in the present invention is not particularly limited and can include, for example, in vitro evaluation methods using bacteria or cultured cells, or in vivo evaluation methods using experimental animals. Examples of in vitro evaluation methods include post-labeling, Rec assay, SOS test, irregular DNA synthesis test, and comet assay. Examples of in vivo evaluation methods include post-labeling, irregular DNA synthesis test, and comet assay.

[0018] The target cells of the DNA damage inhibitor in the present invention are not particularly limited, but examples include epidermal keratinocytes, dermal fibroblasts, pigment cells, hepatocytes, renal cells, germ cells, and immune cells.

[0019] The DNA damage inhibitor in the present invention may use the above extract as is, or it may contain ingredients used in pharmaceuticals, quasi-drugs, cosmetics, or foods, such as oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, humectants, powders, UV absorbers, thickeners, pigments, antioxidants, whitening agents, chelating agents, excipients, film-forming agents, sweeteners, and acidulants, to the extent that the effect of the extract is not impaired.

[0020] The present invention can be used in any of pharmaceuticals, quasi-drugs, cosmetics, and foods. Examples of its dosage forms include, for example, lotions, creams, milky lotions, gel agents, aerosol agents, essences, packs, detergents, bath agents, foundations, face powders, lipsticks, ointments, patches, lozenges, capsule agents, chocolates, gums, candies, beverages, powders, granules, tablets, sugar-coated tablets, syrup agents, pills, suspensions, liquid agents, emulsions, suppositories, injection solutions, and the like.

[0021] In the case of external use, the content of the above extract used in the present invention is preferably 0.0001% by weight or more, more preferably 0.001 - 10% by weight, in terms of solids. Further, 0.01 - 5% by weight is most preferable. If it is less than 0.0001% by weight, it is difficult to expect sufficient effects. If it exceeds 10% by weight, it is difficult to recognize an enhancement of the effect and it is uneconomical.

[0022] In the case of internal use, the intake amount varies depending on age, body weight, symptoms, treatment effect, administration method, treatment time, etc. Usually, as the daily intake amount per adult, 5 mg or more is preferable, 10 mg - 5 g is more preferable. Further, 20 mg - 2 g is most preferable.

[0023] Next, in order to explain the present invention in detail, production examples, experimental examples, and formulation examples of the extract used in the present invention are given as examples, but the present invention is not limited thereto. Unless otherwise specified, the % shown in the examples indicates % by weight.

Examples

[0024] (Production Example 1) Preparation of hot water extract of kukinochi seeds 200 mL of water was added to 10 g of the dried product of kukinochi seeds, and extraction was carried out at 95 - 100°C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.1 g of the hot water extract of kukinochi seeds.

[0025] (Production Example 2) Preparation of 50% ethanol extract of kukinochi seeds 10 g of dried kukui tree seeds were immersed in 200 mL of 50% ethanol aqueous solution at room temperature for 7 days to extract the extract. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 0.2 g of a 50% ethanol extract of kukui tree seeds.

[0026] (Manufacturing Example 3) Preparation of ethanol extract from kukui tree seeds 10 g of dried kukui tree seeds were immersed in 200 mL of ethanol at room temperature for 7 days for extraction. After filtering the resulting extract, it was concentrated to dryness using an evaporator to obtain 1.7 g of ethanol extract of kukui tree seeds.

[0027] (Manufacturing Example 4) Preparation of 1,3-butylene glycol extract from kukui tree seeds 10 g of dried kukui tree seeds were immersed in 200 mL of 1,3-butylene glycol at room temperature for 7 days for extraction. The resulting extract was filtered to obtain 192 g of 1,3-butylene glycol extract of kukui tree seeds.

[0028] Next, to explain the effects of the present invention in detail, experimental examples will be given. [Examples]

[0029] Experimental Example: DNA Damage Suppression by Comet Assay TK6 (human lymphoblast-derived) cells were seeded in 24-well plates and cultured overnight. Subsequently, DNA damage inducers and hot water extracts of kukui seeds were added. Specifically, in the case of metabolic activation, 2.5 μg / mL of benzo[a]pyrene and hot water extracts of kukui seeds at various concentrations were added in the presence of rat liver homogenate (S9), and the cells were cultured for 2 hours. In the case of no metabolic activation, 1,000 μg / mL of ethyl methanesulfonate and hot water extracts of kukui seeds at various concentrations were added in the absence of S9, and the cells were cultured for 2 hours. TK6 cells were harvested, suspended in phosphate-buffered saline (PBS), mixed with 0.5% low-melting-point agarose, dropped onto glass slides, and left on ice to prepare specimens. The specimens were left in a cooled lysis buffer (100 mM EDTA, 2.5 M sodium chloride, 10 mM Tris-base, 1% Triton X-100, 10% DMSO, pH 10.0) for at least one hour. Then, the specimens were left in a cooled electrophoresis buffer (300 mM sodium hydroxide, 1 mM EDTA) for 20 minutes, followed by electrophoresis for 20 minutes. After neutralizing the specimens with a neutralization solution (0.4 M Tris-base, pH 7.5), they were dehydrated with ethanol. After staining the specimens with propidium iodide, the cells were observed under a fluorescence microscope.

[0030] (Judgment method) Each cell in a single field of view was classified according to the following criteria, and the degree of DNA damage was evaluated by calculating a weighted average score (Comet Score). • Type 1: Undamaged nucleus without a tail (1 item) • Type 2: Two nuclei with small DNA fragments • Type 3: Nucleus with a thin tail (3 points) • Type 4: A nucleus with a clear tail (4 points) ·Type5:Nuclear 5 points

[0031] [Table 1]

[0032] [Table 2]

[0033] Table 1 shows that the DNA damage inducer benzo[a]pyrene resulted in an increase in the comet score compared to the negative control. On the other hand, when a hot water extract of kukui tree seeds was added simultaneously, a decrease in the comet score was observed depending on the concentration of the kukui tree seed extract.

[0034] Table 2 shows that ethyl methanesulfonate, a DNA damage inducer, resulted in an increase in the comet score compared to the negative control. On the other hand, when a hot water extract of kukui tree seeds was added simultaneously, a decrease in the comet score was observed, depending on the concentration of the kukui tree seed extract. [Examples]

[0035] (Example prescription 1) Lotion Formula Content (%) 1. Hot water extract of kukui tree seeds (Production Example 1) 2.0 2,1,3-Butylene glycol 8.0 3. Glycerin 2.0 4. Xanthan gum 0.02 5. Citric acid 0.01 6. Sodium citrate 0.1 7. Ethanol 5.0 8. Methyl parahydroxybenzoate 0.1 9. Polyoxyethylene hydrogenated castor oil (40 E.O.) 0.1 10.Fragrance (appropriate amount) 11. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Components 1-6 and 11 and components 7-10 are uniformly dissolved, mixed together, and filtered to obtain the product.

[0036] (Prescription example 2) Cream Formula Content (%) 1. 50% ethanol extract of kukui tree seeds (production example 2) 1.0 2. Squalane 5.5 3. Olive oil 3.0 4. Stearic acid 2.0 5. Beeswax 2.0 6. Octyldodecyl myristate 3.5 7. Polyoxyethylene cetyl ether (20 E.O.) 3.0 8. Behenyl alcohol 1.5 9. Glyceryl monostearate 2.5 10.Fragrance 0.1 11. Methyl parahydroxybenzoate 0.2 12.1,3-Butylene glycol 8.5 13. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Heat and dissolve components 2-9, mix, and maintain at 70°C to form the oil phase. Heat and dissolve components 1 and 11-13, mix, and maintain at 75°C to form the aqueous phase. Add the aqueous phase to the oil phase and emulsify, then cool while stirring. Add component 10 at 45°C, and further cool to 30°C to obtain the final product.

[0037] (Example prescription 3) Emulsion Formula Content (%) 1. Ethanol extract of kukui tree seeds (production example 3) 0.01 2. Squalane 5.0 3. Olive oil 5.0 4. Jojoba oil 5.0 5. Cetanol 1.5 6. Glyceryl monostearate 2.0 7. Polyoxyethylene cetyl ether (20 E.O.) 3.0 8. Polyoxyethylene sorbitan monooleate (20E.O.) 2.0 9.Fragrance 0.1 10. Propylene glycol 1.0 11. Glycerin 2.0 12. Methyl parahydroxybenzoate 0.2 13. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Heat and dissolve components 1-8, mix, and maintain at 70°C to form the oil phase. Heat and dissolve components 10-13, mix, and maintain at 75°C to form the aqueous phase. Add the aqueous phase to the oil phase and emulsify, then cool while stirring. At 45°C, add component 9, and cool further to 30°C to obtain the final product.

[0038] (Prescription example 4) Gel Formula Content (%) 1. Kukui tree seeds 1,3-Butylene glycol extract (Production Example 4) 1.0 2. Ethanol 5.0 3. Methyl parahydroxybenzoate 0.1 4. Polyoxyethylene hydrogenated castor oil (60 E.O.) 0.1 5.Fragrance (appropriate amount) 6.1,3-Butylene glycol 5.0 7. Glycerin 5.0 8. Xanthan gum 0.1 9. Carboxyvinyl polymer 0.2 10. Potassium hydroxide 0.2 11. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Dissolve components 2-5 and components 1 and 6-11 uniformly, then mix them together to obtain the product.

[0039] (Prescription example 5) pack Formula Content (%) 1. Hot water extract of kukui tree seeds (Production Example 1) 1.0 2. Kukui tree seeds 1,3-Butylene glycol extract (Production Example 4) 5.0 3. Polyvinyl alcohol 12.0 4. Ethanol 5.0 5.1,3-Butylene glycol 8.0 6. Methyl parahydroxybenzoate 0.2 7. Polyoxyethylene hydrogenated castor oil (20 E.O.) 0.5 8. Citric acid 0.1 9. Sodium citrate 0.3 10.Fragrance (appropriate amount) 11. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Dissolve ingredients 1-11 uniformly to form the product.

[0040] (Prescription example 6) Foundation Formula Content (%) 1. 50% ethanol extract of kukui tree seeds (production example 2) 1.0 2. Stearic acid 2.4 3. Polyoxyethylene sorbitan monostearate (20E.O.) 1.0 4. Polyoxyethylene cetyl ether (20 E.O.) 2.0 5. Cetanol 1.0 6. Liquid lanolin 2.0 7. Liquid paraffin 3.0 8. Isopropyl myristate 6.5 9. Sodium carboxymethylcellulose 0.1 10. Bentonite 0.5 11. Propylene glycol 4.0 12. Triethanolamine 1.1 13. Methyl parahydroxybenzoate 0.2 14. Titanium dioxide 8.0 15. Talc 4.0 16. Bengara 1.0 17. Yellow iron oxide 2.0 18.Fragrance (appropriate amount) 19. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Heat and dissolve components 2-8, maintain at 80°C to form the oil phase. Swell component 9 thoroughly in component 19, then add components 1 and 10-13 and mix uniformly. Add components 14-17, which have been crushed and mixed in a pulverizer, and stir with a homomixer, maintaining at 75°C to form the aqueous phase. Add the aqueous phase to the oil phase while stirring and emulsify. Then, cool, add component 18 at 45°C, and cool to 30°C while stirring to obtain the product.

[0041] (Prescription example 7) Bath additive Formula Content (%) 1. Ethanol extract of kukui tree seeds (Production Example 3) 1.0 2. Sodium bicarbonate 50.0 3. Yellow No. 202 (1) appropriate amount 4.Fragrance (appropriate amount) 5. Add sodium sulfate to bring the total volume to 100. [Manufacturing Method] Mix ingredients 1-5 uniformly to form the product.

[0042] (Prescription example 8) Ointment Formula Content (%) 1. Hot water extract of kukui tree seeds (Production Example 1) 5.0 2. Kukui tree seeds 1,3-Butylene glycol extract (Production Example 4) 1.0 3. Polyoxyethylene cetyl ether (30 E.O.) 2.0 4. Glyceryl monostearate 10.0 5. Liquid paraffin 5.0 6. Cetanol 6.0 7. Methyl parahydroxybenzoate 0.1 8. Propylene glycol 10.0 9. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Heat and dissolve components 3-6, mix, and maintain at 70°C to form the oil phase. Heat and dissolve components 1, 2 and 7-9, mix, and maintain at 75°C to form the aqueous phase. Add the aqueous phase to the oil phase and emulsify, then cool to 30°C while stirring to obtain the final product.

[0043] (Prescription example 9) Powder Formula Content (%) 1. Hot water extract of kukui tree seeds (Production Example 1) 1.0 2. Dried corn starch 39.0 3. Microcrystalline cellulose 60.0 [Manufacturing method] Mix ingredients 1-3 and prepare as a powder.

[0044] (Prescription example 10) Tablets Formula Content (%) 1. Ethanol extract of kukui tree seeds (Production Example 3) 5.0 2. Dried corn starch 25.0 3. Carboxymethylcellulose calcium 20.0 4. Microcrystalline cellulose 40.0 5. Polyvinylpyrrolidone 7.0 6. Talc 3.0 [Manufacturing Method] Mix ingredients 1-4, then add an aqueous solution of ingredient 5 as a binder and form into granules. Add ingredient 6 to the formed granules and compress into tablets. Each tablet should weigh 0.52g.

[0045] (Prescription example 11) Tablet confectionery Formula Content (%) 1. Ethanol extract of kukui tree seeds (Production Example 3) 2.0 2. Dried cornstarch 49.8 3. Erythritol 40.0 4. Citric acid 5.0 5. Sucrose fatty acid ester 3.0 6.Fragrance 0.1 7.Purified water 0.1 [Manufacturing Method] Mix ingredients 1-4 and 7 and form into granules. Add ingredients 5 and 6 to the formed granules and compress into tablets. Each tablet should weigh 1.0g.

[0046] (Prescription example 12) Beverages Formula Content (%) 1. Hot water extract of kukui tree seeds (Production Example 1) 0.05 2. Stevia 0.05 3. Malic acid 5.0 4.Fragrance 0.1 5. Dilute with purified water to make a total volume of 100. [Manufacturing Method] Dissolve ingredients 2 and 3 in a small amount of water. Then add ingredients 1, 4 and 5 and mix. [Industrial applicability]

[0047] According to the present invention, kukui tree seed extract has a DNA damage inhibitory effect with excellent safety and efficacy, and can therefore be used to prevent aging such as age spots and wrinkles caused by DNA damage.

Claims

1. A DNA damage inhibitor characterized by containing kukui tree seed extract.

2. A pharmaceutical product, quasi-drug, cosmetic, or food product characterized by containing the DNA damage inhibitor described in claim 1 as an active ingredient.

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