Method for producing tissue culture of Dogtooth violet having antioxidant activity and antioxidant

Abstract

To provide methods for producing tissue cultures containing functional ingredients of Erythronium japonicum efficiently in order to stably supply and effectively use Erythronium japonicum as a functional material.SOLUTION: Provided are a method for producing a tissue culture of Erythronium japonicum having antioxidant activity comprising: continuously proliferating a cell mass containing an adventitious bud primordium of Erythronium japonicum; culturing the proliferated cell mass to form adventitious buds; dividing the obtained adventitious buds and transplanting them into a hormone-free medium, elongating the green leaf blades from the adventitious buds, and inducing anthocyanin in the leaf blades; and collecting green leaf blades or anthocyanin-induced leaf blades, and an antioxidant comprising the tissue culture of Erythronium japonicum obtained by the method.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a tissue culture of Dogtooth Violet having antioxidant activity, and an antioxidant containing said tissue culture. [Background technology]

[0002] It takes a long time, 7 to 8 years, for the dogtooth violet (Erythronium japonicum Decne.) to grow from seed to flower. The dogtooth violet seeds are highly dormant, and after about 5 months at room temperature (20 to 25°C), they will not germinate unless they are exposed to temperatures below 10°C, a unique feature not found in other plants. Once germinated, they grow thin leaves and form bulbs at their base. The bulbs grow little by little every year, and when they reach a size where they can develop two leaves, they bloom and form seeds, but it is said that bulbs only grow and rarely divide in nature. Therefore, there is no way to multiply dogtooth violet in a short period of time, and since the colonies gradually decrease when the bulbs are harvested, most of the existing dogtooth violet colonies are protected areas and are not easily accessible.

[0003] Extracts from the leaves and whole plant of Katakuri have been reported to have useful functions such as skin cell activation, antioxidant, whitening, and anti-inflammatory effects (Patent Document 1), tumor necrosis factor (TNF) α production inhibitory effect (Patent Document 2), and nitric oxide (NO) production inhibitory effect (Patent Document 3), and are expected to be used in medicines and cosmetics for the treatment and prevention of autoimmune diseases, neurodegenerative diseases, skin diseases, etc. However, because Katakuri has special characteristics that are not found in ordinary plants as described above, it is not possible to collect stems and leaves used as extraction materials from Katakuri's native plants, and artificial cultivation is also difficult. Therefore, in order to effectively use Katakuri as a functional material, a method for stably supplying its tissue culture in a short period of time was desired. Therefore, the present inventors have established a culture system that can be stably grown by tissue culture using Katakuri seeds (Patent Application No. 2020-007521).

[0004] To date, tissue cultures have been used to stably produce large amounts of functional components contained in plants, and callus from night-blooming cereus (Patent Document 4) and rose (Patent Document 5) have been reported. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2009-143849 A [Patent Document 2] JP 2018-135328 A [Patent Document 3] JP 2018-135294 A [Patent Document 4] Patent No. 6779627 [Patent Document 5] Patent No. 5577443 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for efficiently producing a tissue culture containing functional components of Dogtooth Violet, in order to stably supply and effectively utilize Dogtooth Violet as a functional material. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above problems, and as a result of investigating the antioxidant activity of proliferated cell masses produced by tissue culture of Dogtooth Violet and materials at various growth stages differentiated therefrom, they have found that the greened leaf blades elongated from adventitious buds formed from proliferated cell masses have antioxidant activity superior to that of proliferated cell masses, and that leaf blades in which anthocyanins have been induced can be efficiently harvested by treating the adventitious buds with weak light for 24 hours during their formation, and that the anthocyanin-induced leaf blades have antioxidant activity equivalent to that of an exogenous plant (naturally grown), thereby completing the present invention. Up to now, there have been no reports on the antioxidant activity of Dogtooth Violet cultures, and in studies on antioxidant activity using cultured plant tissues, there have been no reports that callus has low antioxidant activity and leaf blades have high antioxidant activity.

[0008] That is, the present invention includes the following inventions. [1] The following steps: (1) A step of continuously growing a cell mass containing adventitious shoot primordia of Dogtooth Violet (Erythronium japonicum Decne.), (2) culturing the cell mass grown in step (1) at 6 to 18°C ​​for 3 to 6 months in the dark or under 12-hour illumination to form adventitious buds; (3) dividing the adventitious buds formed in step (2), transplanting them into a hormone-free 1 / 2 MS medium, and culturing them at 16 to 20°C under illumination for 12 to 16 hours to elongate the greened leaf blades; (4) harvesting the greened leaf blades within one month from the start of the culture in step (3); A method for producing a tissue culture of Dogtooth Violet having antioxidant activity, comprising: [2] The method according to [1], wherein during the culture in the step (2), the cell mass is treated at 6 to 16°C under weak light for 24 hours for 3 to 4 weeks to induce anthocyanin. [3] The method according to [2], wherein the illuminance of the 24-hour low light illumination is 300 to 1000 Lux. [4] A tissue culture of Dogtooth Violet obtained by the method described in any one of [1] to [3]. [5] The tissue culture of Dogtooth Violet according to [4], wherein the antioxidant activity is 120 to 300 μmol TE / g (dry matter). [6] An antioxidant comprising the tissue culture of Dogtooth Violet or its extract as described in [4] or [5] as an active ingredient. Effect of the Invention

[0009] According to the present invention, a method for efficiently producing a tissue culture of Dogtooth Violet having antioxidant activity is provided. The tissue culture of Dogtooth Violet of the present invention has a high ORAC value and is therefore effective in improving and preventing various diseases and pathologies associated with reactive oxygen species. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows the morphology of cell clusters containing adventitious shoot primordia propagated by tissue culture from very immature seeds of Dogtooth Violet. [Diagram 2] FIG. 2 shows the morphology of leaf primordia and leaf blades from adventitious buds formed from cell clusters containing adventitious bud primordia of Dogtooth violet (FIG. 1). [Diagram 3] FIG. 3 shows the morphology of the leaf of a wild dogtooth violet. [Figure 4] FIG. 4 shows the morphology of red cell clusters (with anthocyanin induction) containing adventitious buds formed from cell clusters containing adventitious bud primordia of Dogtooth violet (FIG. 1). [Diagram 5] FIG. 5 shows the results of measuring the absorbance of the red pigment accumulated in the adventitious buds and leaf blades of Dogtooth Violet. [Figure 6] FIG. 6 shows the morphology of leaf blades elongated from adventitious buds formed from cell clusters containing adventitious bud primordia of Dogtooth violet. [Figure 7] FIG. 7 shows the morphology of leaf blades (with anthocyanin induction) elongated from adventitious buds formed from cell clusters containing adventitious bud primordia of Dogtooth violet. [Figure 8] FIG. 8 shows a comparison of the ORAC values ​​per dry weight of wild-grown Dogtooth Violet leaves and Dogtooth Violet tissue cultures at each stage. [Figure 9] FIG. 9 shows the morphology of broad leaf blades sprouted from bulbs formed during cultivation of Dogtooth Violets. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 1. Method for producing tissue culture of Dogtooth Violets The method for producing a tissue culture of Dogtooth Violet of the present invention comprises the following steps: (1) A step of continuously growing a cell mass containing adventitious shoot primordia of Dogtooth Violet (Erythronium japonicum Decne.), (2) culturing the cell mass grown in step (1) at 6 to 18°C ​​for 3 to 6 months in the dark or under 12-hour illumination to form adventitious buds; (3) dividing the adventitious buds formed in step (2), transplanting them into a hormone-free 1 / 2 MS medium, and culturing them at 16 to 20°C under illumination for 12 to 16 hours to elongate the greened leaf blades; (4) harvesting the greened leaf blades within one month from the start of the culture in step (3). Each step will be described below.

[0012] Process (1): In step (1), cell masses containing adventitious shoot primordia of Dogtooth Violet (Erythronium japonicum Decne.) are continuously grown.

[0013] In this step, the medium for growing the cell mass containing adventitious shoot primordia may be a basic medium commonly used in plant tissue culture, such as MS medium (Murashige-Skoog medium), LS medium (Linsmeier-Skoog medium), Gamborg's medium, White's medium, Tulecke's medium, or Nitsch & Nitsch's medium to which sugars such as sucrose, glucose, fructose, or maltose have been added. In addition, media modified by replacing some of the medium components of the basic medium with similar components, adding vitamins (thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, calcium pantothenate, vitamin B12, biotin, para-aminobenzoic acid, folic acid, etc.), amino acids (glycine, glutamine, glutamic acid, aspartic acid, lysine, etc.), or changing the concentration of sugars such as sucrose, may be used.

[0014] Auxins, cytokinins, and gibberellins are added to the above-mentioned basic medium as plant hormones. Examples of auxins include naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxyacetic acid (MCPA), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), with NAA being preferred. Examples of cytokinins include benzyladenine (BA), zeatin, kinetin, 6-(γ,γ-dimethylalamino)purine (2iP), and N-(2-chloro-4-pyrido)-N'-phenylurea (CPPU), with BA being preferred. Examples of gibberellins include gibberellin A1 (GA1), gibberellin A3 (GA3), gibberellin A4 (GA4), gibberellin A7 (GA7), etc., with GA3 being preferred.

[0015] In the present invention, the plant hormone contained in the proliferation medium for the cell mass containing adventitious shoot primordia may be one type, but it is preferable to combine two or more types. In the case of one type, gibberellins are preferable, and in the case of two or more types, a combination of gibberellins and auxins, or a combination of gibberellins, auxins and cytokinins is preferable.

[0016] The range of the content of plant hormone in the medium varies depending on the type of plant hormone, but from the viewpoint of promoting the proliferation of cell masses containing adventitious shoot primordia, a total of 1 to 10 mg / L is preferable, 1 to 5 mg / L is more preferable, and 2 to 4 mg / L is even more preferable. For example, the content of auxins in the medium is 0.1 to 1.0 mg / L, the content of cytokinins is 0.5 to 2.0 mg / L, and the content of gibberellins is 0.2 to 1.0 mg / L. A particularly preferred combination of plant hormones is a combination of NAA, BA, and GA3, with the content of NAA being 1.0 mg / L, the content of BA being 0.5 to 2.0 mg / L, and the content of GA3 being 0.2 to 1.0 mg / L.

[0017] More specifically, ME4 medium (based on 1 / 2MS medium, to which 30 g / L of sucrose and 2 g / L of gellan gum as a support have been added, and which has been supplemented with 1.0 mg / L of naphthalene acetic acid (NAA), 2.0 mg / L of benzyladenine (BA), and 0.2 mg / L of gibberellin A3 (GA3)) is preferred.

[0018] The culture temperature in step (1) may be 6 to 18° C., preferably 12 to 16° C., and more preferably 14 to 16° C. The culture period can be appropriately adjusted depending on the culture temperature, but is preferably 2 to 3 months. Culture is carried out several times continuously under the same conditions.

[0019] The form of the medium for growing cell clusters containing adventitious shoot primordia is preferably a solid medium, and the sterilized ultra-immature seeds are placed on the medium and cultured. For the solid medium, agar, agarose, gellan gum, etc. are used as a gelling agent for the medium. The medium can be solidified by a general method such as pouring a gelling agent dissolved in water into a basal medium and treating under heating and pressure in an autoclave. The amount of gelling agent added may be an amount sufficient to obtain a sufficiently solidified state as a medium, and varies depending on the type, but for example, 0.8 to 1.2% for agar, 0.1 to 1.0% for agarose, and 0.1 to 0.4% for gellan gum are preferable. The pH of the medium for growing cell clusters containing adventitious shoot primordia is preferably in the range of 4.5 to 7.0, which is suitable for cell cluster growth.

[0020] As the culture vessel, a flat plastic petri dish is usually used, but a test tube, an Erlenmeyer flask, a wide-mouth bottle, or the like can also be used.

[0021] The culture in step (1) is carried out in a dark place. In this specification, the term "dark place" is synonymous with the dark conditions usually used in plant tissue culture, and does not necessarily mean a completely dark place. For example, even if the culture is temporarily exposed to normal light conditions during observation, it is considered to be a dark place. In addition, the method of setting the dark conditions is not particularly limited, but examples thereof include a method of turning off the lights in the culture room, a method of sealing a container containing the culture in a light-shielding container, or a method of wrapping the container in aluminum foil or the like.

[0022] Process (2): In step (2), the cell mass grown in step (1) is cultured in the dark or under 12-hour illumination, preferably in the dark, to form adventitious shoots. The culture in step (2) is performed at 6 to 18°C ​​for 3 to 6 months, which is longer than the growth cycle period in step (1), and the other conditions (type of basal medium, form of medium, culture vessel) are the same as those in step (1) and may be as described above. This step is characterized by culturing under the same conditions for a longer period, unlike the cycle in which the grown cells are continuously maintained and grown.

[0023] During the culture in step (2), the cell masses can be placed at 6 to 16°C under weak light (300 to 1000 Lux) for 24 hours for 3 to 4 weeks, thereby forming adventitious buds in which anthocyanins are induced.

[0024] Process (3): In step (3), the adventitious buds formed in step (2) are divided and transplanted into hormone-free 1 / 2 MS medium, and cultured at 16 to 20°C, preferably 18°C, under illumination for 12 to 16 hours to allow the greened leaf blades to elongate. The basal medium used here is preferably 1 / 2 MS medium, which is a medium in which the inorganic salts contained in MS medium are diluted by half. Division is performed so that, for example, about 3 to 4 buds are included. The illuminance of the 12 to 16 hours of illumination is preferably 4500 to 10000 Lux. The culture in this step is preferably carried out for about one month, and is not preferable if it is carried out for more than one month because it will cause aging.

[0025] Process (4): In step (4), the green leaf blades are harvested within one month from the start of the culture in step (3). In the present invention, the fresh weight of the leaf blades that can be harvested through the above steps is about 0.03 to 0.3 g. The length of the leaf blades is approximately 3 cm or more.

[0026] 2. Antioxidants The antioxidant of the present invention contains as an active ingredient a leaf blade (hereinafter referred to as "Dogtooth violet tissue culture") that has been differentiated and greened from a cell mass containing adventitious bud primordia of Dogtooth violet obtained by the manufacturing method in 1., or an extract thereof.

[0027] The tissue culture of Dogtooth violet, which is the active ingredient of the present invention, has an ORAC (Oxygen Radical Absorbance Capacity) value, which is an index of antioxidant activity, of 120 to 300 μmol TE / g (dry matter). Here, the ORAC value can be calculated by converting the antioxidant power of a material into the amount of the antioxidant Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). The ORAC method is a method for measuring the decomposition process of fluorescein (FL) caused by peroxy radicals generated by AAPHz (2,2'-azo-bis(2-amidinopropane)dihydrochloride). If the sample has antioxidant power, the decomposition rate of fluorescein will be slow, and the antioxidant power converted into the standard substance can be calculated by comparing the sample with Trolox, which is a standard substance.

[0028] The Dogtooth violet tissue culture can be washed with water and used as is, or after shredding, drying, crushing, etc., in the form of a slurry, fine grains, granules, or powder. For ease of handling when used in a product, however, it is preferable to subject it to extraction after the above treatment and use it as an extract.

[0029] The extraction method for obtaining the Katakuri tissue culture is not particularly limited, and may be, for example, a heated extraction method, or a room temperature or cold extraction method. Examples of the solvent used for extraction 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.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.). Among these solvents, water, lower alcohols, and liquid polyhydric alcohols are preferred, and water and ethanol are more preferred. These solvents may be used alone or in combination of two or more, and for example, an aqueous ethanol solution of 30 to 70 v / v% may be used. In addition, a solvent whose pH has been adjusted by adding an acid or alkali to the above extraction solvent may be used.

[0030] The amount of the solvent used is not particularly limited, and may be, for example, 10 times or more, preferably 20 times or more, relative to the dry weight of the above-mentioned Katakuri tissue culture, but is preferably 100 times or less for the convenience of operations in the case of concentration or isolation after extraction. The extraction temperature and time depend on the type of solvent used, and may be, for example, 10 to 100°C, preferably 30 to 90°C, and 30 minutes to 24 hours, preferably 1 to 10 hours.

[0031] The extraction can be carried out by immersing the tissue culture of Dogtooth violet in the above-mentioned extraction solvent and leaving it to stand or stirring it. Alternatively, the tissue culture may be disrupted in the extraction solvent using a bead-type disrupter or homogenized in the extraction solvent.

[0032] The extraction temperature and time can be appropriately determined depending on the method of pretreatment of the Katakuri tissue culture, the type of extraction solvent, the extraction method, etc. For example, when extraction is performed by immersion in an extraction solvent containing a lower alcohol, extraction is usually performed at 2 to 30° C. for 1 to 14 days, and preferably at 25 to 30° C. for 2 to 7 days. After extraction, the extract is collected according to a standard method, for example, by filtration, centrifugation, etc.

[0033] The extract may be used as it is in the form of an extracted solution, but if necessary, it may be used after being subjected to treatments such as concentration (using an organic solvent, vacuum concentration, membrane concentration, etc.), dilution, filtration, decolorization with activated carbon, etc., deodorization, ethanol precipitation, etc., within the scope that does not affect the effect of the extract. Furthermore, the extracted solution may be subjected to treatments such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.

[0034] The antioxidant according to the present invention has the function of suppressing the production of "active oxygen" because the extract of the Katakuri tissue culture, which is the active ingredient, contains anthocyanin, a type of antioxidant polyphenol. Therefore, the antioxidant according to the present invention is effective in treating, improving, and preventing diseases and pathologies caused by active oxygen. Examples of diseases and pathologies caused by active oxygen include, but are not limited to, arteriosclerosis, cancer, skin aging (age spots, wrinkles, etc.), skin diseases (atopic dermatitis), dementia, eye diseases (cataracts, retinitis pigmentosa, age-related macular degeneration, etc.), brain and nervous system diseases (Parkinson's disease, Alzheimer's disease), respiratory system diseases (bronchitis, etc.), circulatory system diseases (ischemic arrhythmia, myocardial infarction, cerebral infarction, hypertension, etc.), digestive system diseases (gastric ulcer, colitis, fatty liver, etc.), and diabetes.

[0035] When the antioxidant of the present invention is administered to a living body, it can be administered as it is, but it can also be provided by being incorporated into various compositions such as cosmetics, quasi-drugs, pharmaceuticals, food and beverages, etc. together with appropriate additives within a range that does not impair the effects of the present invention.

[0036] When the antioxidant of the present invention is incorporated into cosmetics or quasi-drugs, the dosage form may be any of an aqueous solution, a solubilized solution, an emulsion, a powder, a powder dispersion, an oil solution, a gel, an ointment, an aerosol, a water-oil two-layer system, or a water-oil-powder three-layer system. The cosmetics or quasi-drugs may be produced by appropriately blending various ingredients, additives, bases, etc. that are usually used in skin external compositions together with the antioxidant of the present invention according to their types, in accordance with a method known in the art. The form may be any of a liquid, emulsion, cream, gel, paste, spray, etc. Examples of ingredients contained in compositions for external use on the skin include oils and fats (olive oil, coconut oil, jojoba oil, castor oil, etc.), waxes (lanolin, beeswax, carnauba wax, etc.), hydrocarbons (liquid paraffin, squalene, petrolatum, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, etc.), higher alcohols (myristyl alcohol, cetanol, stearyl alcohol, etc.), esters (isopropyl myristate, isopropyl palmitate, cetyl octanoate, etc.), organic acids (citric acid, lactic acid, α-hydroxyacetic acid, pyrrolidone carboxylic acid, etc.), sugars (maltitol, sorbitol, xylobiose, N-acetyl-D-glucosamine, etc.), proteins and protein hydrolysates, amino acids and their salts, vitamins, plant and animal extracts, various surfactants, moisturizers, UV absorbers, antioxidants, stabilizers, preservatives, disinfectants, fragrances, etc.

[0037] Examples of types of cosmetics and quasi-drugs include lotions, milky lotions, gels, beauty serums, general creams, sunscreen creams, packs, masks, facial cleansers, cosmetic soaps, foundations, face powders, bath additives, body lotions, body shampoos, hair shampoos, hair conditioners, and hair growth agents.

[0038] When the antioxidant of the present invention is incorporated into a pharmaceutical product, it can be mixed with pharmacologically and pharmacy-acceptable additives and formulated into various preparations in a dosage form suitable for application to the affected area. As pharmacologically and pharmacy-acceptable additives, a formulation base material or carrier, excipient, diluent, binder, lubricant, coating agent, disintegrant or disintegration aid, stabilizer, preservative, antiseptic, bulking agent, dispersant, wetting agent, buffer, solubilizer or solubilization aid, isotonicity agent, pH regulator, propellant, colorant, sweetener, flavoring agent, flavoring agent, flavoring agent, etc., appropriately selected according to the dosage form and application, may be added appropriately to prepare various dosage forms that can be administered orally or parenterally, systemically or locally, by various known methods. When the pharmaceutical product is provided in each of the above forms, it can be manufactured by a method commonly used by those skilled in the art, for example, the method shown in each article of the General Provisions for Preparations [2] of the Japanese Pharmacopoeia.

[0039] The form of the pharmaceutical product is not particularly limited, and examples thereof include oral preparations such as tablets, sugar-coated tablets, capsules, lozenges, granules, powders, liquids, pills, emulsions, syrups, suspensions, and elixirs, and parenteral preparations such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, and intraperitoneal injections), drips, suppositories, ointments, lotions, eye drops, sprays, transdermal absorption agents, transmucosal absorption agents, and patches. In addition, the pharmaceutical product may be a dried product that is redissolved when used, and in the case of an injection preparation, it is provided in the form of a unit-dose ampule or a multi-dose container.

[0040] When the antioxidant of the present invention is used as a pharmaceutical for treating, improving, and preventing the above-mentioned skin-related diseases and damages, the form suitable for use is an external preparation, for example, an ointment, cream, gel, liquid, patch (poultice, plaster), foam, spray, aerosol, etc. An ointment refers to a homogeneous semi-solid external preparation, including an oleaginous ointment, an emulsion ointment, and a water-soluble ointment. A gel refers to an external preparation in which a water-insoluble component hydrate compound is suspended in an aqueous liquid. A liquid refers to a liquid external preparation, including a lotion, a suspension, an emulsion, a liniment, etc.

[0041] The pharmaceutical functions as a preventive drug for suppressing the onset of the above-mentioned diseases and pathological conditions caused by active oxygen, and / or as a therapeutic drug for improving the condition to a normal state. The active ingredient of the pharmaceutical of the present invention is derived from a natural product and is commonly used, and therefore has a high level of safety. Therefore, when used as a pharmaceutical for treating, improving, and preventing the above-mentioned diseases, it can be orally or parenterally administered in a wide range of dosages to mammals such as humans, mice, rats, rabbits, dogs, and cats.

[0042] The content of the antioxidant in the above-mentioned cosmetics, pharmaceuticals, and quasi-drugs is not particularly limited, but is preferably 0.001 to 30% by weight, more preferably 0.01 to 10% by weight, calculated as the dry solid content of the extract of the Katakuri tissue culture relative to the total weight of the preparation (composition). The above amounts are merely examples, and may be appropriately set and adjusted in consideration of the type and form of the composition, the general amount used, efficacy, and effects. In addition, the method of adding the active ingredient in the formulation may be added in advance or during the production process, and may be appropriately selected in consideration of workability.

[0043] The antioxidant of the present invention can also be incorporated into food and drink. In the present invention, the term "food and drink" refers to not only general food and drink, but also foods that can be taken for the purpose of maintaining or improving health other than pharmaceuticals, such as health foods, functional foods, health functional foods, or special purpose foods for preventing or improving diseases caused by active oxygen. Health foods include foods provided under the names of dietary supplements, dietary supplements, supplements, etc. Health functional foods are defined by the Food Sanitation Act or the Health Promotion Act, and include foods for specified health uses and foods with nutritional functions that can display specific health effects, functions of nutritional ingredients, reduction of disease risk, etc., as well as functional foods that can display the contents of the scientific evidence-based functionality notified to the Commissioner of the Consumer Affairs Agency. Special purpose foods also include foods for sick people, foods for the elderly, foods for infants, foods for pregnant women, etc. that display that they are suitable for specific subjects or patients with specific diseases. Here, the display of specific health effects, functions of nutritional ingredients, etc. attached to foods and drink can be displayed on product containers, packaging, instructions, package inserts, etc., product flyers and pamphlets, product advertisements such as newspapers and magazines, etc.

[0044] The form of the food or drink may be any form suitable for consumption, such as solid, liquid, granule, powder, capsule, cream, or paste. In particular, in the case of the above-mentioned health foods, etc., the form is preferably, for example, tablet, round, capsule, powder, granule, fine granule, lozenge, liquid (including syrup, milk, and suspension).

[0045] Types of food and beverages include, but are not limited to, bread, noodles, confectionery, dairy products, processed seafood and livestock foods, oils and fats and processed oils and fats, seasonings, various beverages (soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks, dairy drinks, etc.), and concentrated concentrates and powders for adjusting such beverages.

[0046] Food and drink may be appropriately mixed with additives that are commonly used depending on the type of food and drink. Any additives that are permitted under the Food Sanitation Act can be used as additives, and examples of such additives include sweeteners such as glucose, sucrose, fructose, isomerized liquid sugar, aspartame, and stevia; acidulants such as citric acid, malic acid, and tartaric acid; excipients such as dextrin and starch; binders, diluents, flavorings, colorants, buffers, thickeners, gelling agents, stabilizers, preservatives, emulsifiers, dispersants, suspending agents, and preservatives.

[0047] In the case of general foods and beverages, they can be produced by adding an extract of the Katakuri tissue culture to the normal production process of the foods and beverages. In the case of health foods, they can be produced in accordance with the above-mentioned method for producing pharmaceuticals. For example, tablet-shaped supplements can be produced by adding and mixing an extract of the Katakuri tissue culture with additives such as excipients, and molding the mixture by applying pressure with a tablet press or the like.

[0048] The amount of extract of Dogtooth Violet tissue culture in food or beverages may be any amount sufficient to exert an antioxidant effect, and may be appropriately determined taking into consideration the general intake amount of the target food or beverage, the form of the food or beverage, efficacy and effects, taste, preference, cost, etc. EXAMPLES

[0049] The present invention will be specifically described below by showing examples, but the present invention is not limited to these examples.

[0050] (Reference Example) Cultivation of cell masses containing adventitious bud primordium tissue of Dogtooth violet Very immature seeds of Dogtooth violet (Erythronium japonicum Decne.) (ovules approximately 3 mm long and 1 mm wide, aseptically removed from 1.5-2 cm ovaries approximately one week after cleistodes) were cultured in somatic embryo induction medium (a 1 / 2 MS medium base supplemented with 30 g / L sucrose, 2 g / L gellan gum as a support, 0.1 mg / L naphthalene acetic acid (NAA), and 1 mg / L gibberellin A3 (GA3)) at 20°C for 5 months in the dark to induce somatic embryo tissue. The induced somatic embryo tissue was cultured in ME4 medium (based on 1 / 2MS medium, with 30 g / L sucrose, 2 g / L gellan gum as a support, 1 mg / L naphthalene acetic acid (NAA), 2 mg / L benzyladenine (BA), and 0.2 mg / L gibberellin A3 (GA3)) at 16°C for 4 to 5 months in the dark to obtain tissue containing adventitious shoot primordia of a size that can be divided (referred to as "adventitious shoot primordium tissue"). The formed adventitious shoot primordium tissue was divided, and the resulting divided mass was placed on ME4 medium (same as above) in a petri dish and cultured at 16°C for 3 months in the dark to proliferate the divided mass (adventitious shoot primordium tissue) (Figure 1).

[0051] Example 1: Evaluation of antioxidant capacity (ORAC value) of Dogtooth Violets (1) Test 1 The antioxidant capacity of tissue cultures of Dogtooth Violet (Erythronium japonicum Decne.) was evaluated. Adventitious shoot primordia-containing cell masses (Fig. 1), leaf primordia elongated from adventitious shoots (Fig. 2), and wild Dogtooth Violet leaves (Fig. 3) were each washed with water and freeze-dried for 70 hours, and then crushed to a particle size of 500 μm or less to produce freeze-dried powder.

[0052] To 1 g of each of the freeze-dried powders, 10 mL of a mixture of hexane and dichloromethane (1:1 (v / v)) was added, stirred at room temperature for 30 seconds, centrifuged at 3000 rpm for 10 minutes, and the supernatant was removed. 10 mL of a mixture of hexane and dichloromethane (1:1 (v / v)) was added again to the precipitate, and the above operation was repeated. Next, 10 mL of MWA solution (methanol 90: water 9.5: acetic acid 0.5 (v / v)) was added to the precipitate, and ultrasonic treatment was performed at 37 ° C for 5 minutes. After leaving it at room temperature for 10 minutes, it was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected. 10 mL of MWA solution (methanol 90: water 9.5: acetic acid 0.5 (v / v)) was added again to the precipitate, and the above operation was repeated. The collected supernatants were mixed and the volume was adjusted to 25 mL, and used as samples for measuring antioxidant activity. Antioxidant capacity was measured by the ORAC (Oxygen Radical Absorbance Capacity) method. The ORAC value was expressed as the equivalent amount of Trolox (TE: Trolox Equivalent), an antioxidant standard substance. The antioxidant content of each sample was compared relative to the antioxidant content of wild dogtooth violets leaves, which was set at 100%. The results are shown in Table 1.

[0053] [Table 1]

[0054] As shown in Table 1, the antioxidant activity of the cell mass containing adventitious bud primordia was low, but antioxidant activity was observed in the leaf primordia that extended from the adventitious buds, suggesting that if the buds are cultured and propagated in the direction of elongation, they may be used as functional materials.

[0055] (2) Test 2 The cell masses containing adventitious shoot primordia were transplanted into ME4 modified medium (1 / 2MS, BA 2mg / L, IAA 1mg / L, GA 30.2mg / L, sucrose 60g / L, gellan gum 2g / L) and cultured at 16°C under 24-hour lighting (illumination 10,000 Lux) for one month, resulting in cell masses with a large red color (Figure 4).

[0056] The cell masses containing adventitious bud primordia were cultured in ME4 medium (1 / 2MS, BA 2mg / L, NAA 1mg / L, GA 30.2mg / L, sucrose 30g / L, gellan gum 2g / L) at 14℃ for 3 months in the dark to form adventitious buds. The adventitious buds were then divided into several buds and transplanted into hormone-free 1 / 2MS medium (1 / 2MS0 medium: 1 / 2MS, sucrose 20g / L, gellan gum 2g / L) and cultured at 18℃ for 1 month under 16-hour lighting to elongate the leaf blades (GL) (Figure 6).

[0057] During the cultivation process using the above method, a few percent of the leaves showed red color (anthocyanin accumulation) (AL) (Figure 7). This red pigment was identified as anthocyanin by absorbance measurement (Figure 5).

[0058] The obtained samples were freeze-dried and powdered in the same manner as in Test 1, and the antioxidant capacity was measured. The antioxidant content of each sample was compared relative to the antioxidant content in the anthocyanin-induced leaf blade (AL), which was set at 100%. The results are shown in Table 2. A comparison of the ORAC values ​​of each material obtained in Tests 1 and 2 is shown in Figure 8.

[0059] [Table 2]

[0060] As shown in Table 2 and FIG. 8, it was confirmed that the leaf blades (GL) elongated from adventitious buds, especially the leaf blades (AL) in which anthocyanin was induced, are effective as functional materials.

[0061] (Example 2) Fresh weight per leaf blade used in antioxidant activity analysis The leaf blades used in the antioxidant activity analysis in Example 1 were roughly classified into three types, and the fresh weights due to differences in leaf blade morphology were investigated. The classifications were broad leaf blades sprouted from bulbs (Figure 9), leaf blades elongated from adventitious buds (Figure 6), and thin, needle-like single leaves resembling those sprouted from seeds. Several of these were grouped together and the fresh weights were measured to determine the average fresh weight per leaf. The results of measuring the fresh weights of the various leaf blades are shown in Table 3.

[0062] [Table 3]

[0063] As shown in Table 3, the fresh weight of a thin single leaf was 0.04 g, whereas the fresh weight of a broad leaf sprouted from a bulb and a leaf blade elongated from an adventitious bud was 0.10 g and 0.09 g, respectively, more than twice as much.

[0064] From the above, it was found that leaf blades should be elongated not from thin simple leaves, but from leaves that have already formed bulbs or from large adventitious buds.

[0065] (Example 3) Number of adventitious shoots formed by cell clusters containing adventitious shoot primordia Of the materials cultured in ME4 medium (1 / 2MS, BA 2mg / L, NAA 1mg / L, GA 30.2mg / L, sucrose 30g / L, gellan gum 2g / L) at 14℃ for 3 months in the dark, 5 petri dishes (4 cells per dish) in which many adventitious buds had developed were selected and the number of adventitious buds was measured. Note that there are individual differences among proliferating cell clumps, and some cell clumps do not easily develop adventitious bud primordia, so cell clumps that are easy to transition to adventitious buds were used as materials. The number of adventitious buds in proliferating cell clumps is shown in Table 4.

[0066] [Table 4]

[0067] As shown in Table 4, the average number of adventitious buds per cell cluster ranged from 3.20 to 4.75, with the average value being 3.94±0.66.

[0068] (Example 4) Conditions for obtaining antioxidant materials through tissue culture of Dogtooth Violets (1) Adventitious shoot formation from cell clusters containing adventitious shoot primordium tissue ME4 medium (1 / 2MS, BA 2mg / L, NAA 1mg / L, GA 30.2mg / L, sucrose 30g / L, gellan gum 2g / L) and BA1 medium (1 / 2MS, BA 1mg / L, sucrose 20g / L, gellan gum 2g / L) were used as adventitious bud formation media, and the cell masses containing adventitious bud primordium tissue obtained in the reference example were cultured under various conditions shown in Table 5-1 below to form adventitious buds.

[0069] [Table 5-1]

[0070] (2) Leaf blade elongation from adventitious buds The adventitious shoots A and B obtained in (1) were cultured under the conditions shown in Table 5-2 below (culture period: 1 month), and leaf blades were allowed to elongate from the adventitious shoots and examined for the presence or absence of greening.

[0071] [Table 5-2]

[0072] As shown in Table 5-2, in the absence of transplantation, neither adventitious bud A nor adventitious bud B greened, and remained yellow with little growth. On the other hand, after dividing the adventitious buds including several buds, they were transplanted to hormone-free 1 / 2MS medium (1 / 2MS0 medium: 1 / 2MS, sucrose 20g / L, gellan gum 2g / L) and cultured at 18°C ​​under 16 hours of light. Movement was observed from the first day, and after one week, more than half of the adventitious buds greened. After about one month of culture, the leaf blades were able to grow to their original size. On the other hand, when the adventitious buds were transplanted to high sucrose 1 / 2MS0 medium (the sucrose concentration of 1 / 2MS0 medium was changed to 60g / L) and cultured, they hardly greened or grew. In addition, the longer the culture period of the test materials was, the more adventitious bud primordia transitioned to adventitious buds.

[0073] From the above results, tissue culture of Dogtooth Violet, which is used as an antioxidant material, could be prepared by culturing cell masses containing adventitious bud primordia at 6-18℃ for 3-6 months in the dark or under 12-hour lighting to form adventitious buds, dividing the adventitious buds into several pieces, transplanting them to hormone-free 1 / 2MS medium, culturing them for about 1 month under 18℃ and 16-hour lighting, and harvesting the leaf blades. In addition, adventitious buds could be more efficiently prepared from cell masses by culturing them in the dark at 14-16℃ for about 4 months.

[0074] (Example 5) Anthocyanin accumulation in adventitious shoots The incidence rate of leaf blades containing anthocyanin with high antioxidant activity was only a few percent under normal conditions of 18°C ​​and 12-16 hour lighting. Therefore, to determine the conditions under which anthocyanin can be stably induced, adventitious shoots were cultured under the conditions in Table 6 below, and an investigation was conducted into the presence or absence of anthocyanin accumulation as well as the greening of the adventitious shoots. The illuminance of the 24 hour lighting was weak light of 300-1000 Lux, and the illuminance of the 12 hour or 16 hour lighting was 4500-10000 Lux.

[0075] [Table 6]

[0076] As shown in Table 6, anthocyanin accumulation was observed in some plants under 12- or 16-hour illumination at 18°C, but was not stable. On the other hand, stable anthocyanin accumulation was observed when the plants were cultivated for about three weeks under 24-hour weak light at 6°C or 16°C, and at the same time, adventitious buds turned green. Note that the number of plants in which anthocyanin accumulation was observed was not 100%, but rather in a range of 50-80% (average 75.5%). However, without treatment under 24-hour weak light, the incidence of leaf blades containing anthocyanin was low at 5-7%.

[0077] (Example 6) Incidence of anthocyanin-induced leaf blade (AL) Cultivated under low light for 24 hours, the temperature conditions (6℃, 16℃, 18℃) were changed and the incidence of leaf blades containing anthocyanin (AL) was investigated after 1, 2 and 3 weeks. The materials used were those that had been cultivated in the dark for 5 months on ME4 medium (same as above) at 6℃. The results are shown in Table 7.

[0078] [Table 7]

[0079] As shown in Table 7, the incidence of leaf blades in which anthocyanins were induced varied depending on the temperature, being highest at 16°C, at approximately 74% in the third week. This was followed by 48% at 6°C in the third week, and at 18°C, where even in the third week it was only 3.7%. From the above, it was confirmed that the most favorable conditions for inducing anthocyanins in the leaf blades were 16°C and 24-hour weak light.

[0080] (Example 7) Fresh weight of leaf blades grown under optimal conditions (GL) and anthocyanin-induced leaf blades (AL) The fresh weight of 10 individuals of each of the leaf blades (GL) and anthocyanin-induced leaf blades (AL) cultivated under the optimal conditions thus far identified was measured. The results are shown in Table 8.

[0081] [Table 8]

[0082] As shown in Table 8, the fresh weight of the leaf blade (GL) was 0.16g on average. On the other hand, the anthocyanin-induced leaf blade (AL) was 0.09g, so the leaf blade (GL) was heavier. However, the weight was generally close to the target weight (about 0.1g). [Industrial Applicability]

[0083] The present invention can be used in the fields of production of cosmetics, pharmaceuticals, food and beverages, etc., having antioxidant effects.

Claims

1. The following steps: (1) A step of continuously growing a cell mass containing adventitious shoot primordia of Dogtooth Violet (Erythronium japonicum Decne.); (2) culturing the cell mass grown in step (1) at 6 to 18°C ​​for 3 to 6 months in the dark or under 12-hour illumination to form adventitious buds; (3) dividing the adventitious shoots formed in step (2), transplanting them into a hormone-free ½ MS medium, and culturing them at 16 to 20° C. under illumination for 12 to 16 hours to elongate the greened leaf blades; (4) harvesting the greened leaf blades within one month from the start of the culture in step (3); A method for producing a tissue culture of Dogtooth Violet having antioxidant activity, comprising:

2. The method according to claim 1, wherein during the culture in the step (2), the cell mass is treated at 6 to 16° C. under weak light for 24 hours for 3 to 4 weeks to induce anthocyanin.

3. The method according to claim 2, wherein the illuminance of the 24-hour low-light illumination is 300 to 1000 Lux.

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