Method for extracting nucleic acid, composition, and cosmetic composition

Abstract

To provide a method for extracting nucleic acids from microalgae.SOLUTION: A method for extracting nucleic acids includes the steps for preparing microalgae, extracting nucleic acids from the microalgae, and purifying the nucleic acids extracted in the nucleic acid extracting step. In the nucleic acid extracting step, a solution containing microalgae is subjected to heating and / or shaking. Preferably, the microalgae are Euglena. Also preferably, the nucleic acids include β-D-glucosyl-hydroxymethyl uracil.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] The present invention relates to a method for extracting nucleic acid, a composition, and a cosmetic composition. [Background technology]

[0002] Nucleic acid is a general term for DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), and is made up of base pairs consisting of adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U), as well as sugar and phosphate.

[0003] Nucleic acids have the following usefulness, and there is a demand for a stable supply of nucleic acid raw materials. Nucleic acid units are natural materials that not only provide nucleic acid medicines and nucleic acid analysis methods, but can also be used as raw materials for cosmetics, supplements, and biodegradable plastics.

[0004] Guanine (G) is an antioxidant. Guanine (G) and adenine (A) absorb ultraviolet rays (UVA (which causes sun tan) and UVB (which causes sun burn). Thymine (T) and cytosine (C) absorb ultraviolet rays (UVB, which causes sun burn). Adenine (A) is the "currency" of energy for living organisms. Guanine (G) is also required for energy metabolism. The nucleic acid units can also provide biodegradable fibers.

[0005] The following problems are exemplified with regard to the supply of nucleic acids. Safety and security: Anything that comes into contact with human hands or is ingested should be supplied using safe ingredients and safe methods, and there is a need to establish purification methods that utilize ethanol, salt, and kitchen detergent ingredients that do not place a burden on nucleic acids. Bulk supply sources: There is a need to utilize bulk supply sources while keeping in mind the 3Rs (Reduce, Reuse, Recycle) without destroying nature or using large amounts of energy. Security of nucleic acid resources: There is a need to establish a stable method of obtaining nucleic acid resources without relying on foreign countries.

[0006] On the other hand, microalgae such as Euglena (genus name: Euglena, Japanese name: Midorimushi) are attracting attention as promising biological resources for use as food, feed, fuel, etc. Patent Document 1 describes a method for producing sphingolipids using Euglena as a raw material. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2019-099528 A Summary of the Invention [Problem to be solved by the invention]

[0008] In the algae industry, commercial production of dried algae is 15,000 t per year, with the potential to extract 450 t of DNA each year. There has been a demand for a method for efficiently extracting nucleic acids from algae. The present invention has been made in view of the above problems, and an object of the present invention is to provide a method for extracting nucleic acid. Another object of the present invention is to provide a composition containing nucleic acid of a microalga. Another object of the present invention is to provide a cosmetic composition containing nucleic acid from microalgae. [Means for solving the problem]

[0009] According to the present invention, the above problem is solved by a method for extracting nucleic acid, which includes the steps of preparing microalgae, extracting nucleic acid from the microalgae, and purifying the nucleic acid extracted in the nucleic acid extraction step, in which the nucleic acid extraction step involves heating and / or shaking a solution containing the microalgae. In this case, the microalgae may be Euglena. In this case, the nucleic acid may contain β-D-glucosyl-hydroxymethyluracil.

[0010] Furthermore, according to the present invention, the above-mentioned problems are solved by a composition containing nucleic acid of a microalga and selected from a cosmetic composition, a pharmaceutical composition, and a food composition. In this case, the microalgae may be Euglena. In this case, the nucleic acid may contain β-D-glucosyl-hydroxymethyluracil. In this case, the composition is preferably a cosmetic composition. In this case, the cosmetic composition is preferably for use as a sunscreen.

[0011] Moreover, according to the present invention, the above-mentioned problems are solved by a cosmetic composition for sunscreen use, which contains nucleic acid from microalgae as an active ingredient. In this case, the microalgae may be Euglena. In this case, the nucleic acid may contain β-D-glucosyl-hydroxymethyluracil. Effect of the Invention

[0012] According to the present invention, a method for extracting nucleic acid can be provided. Furthermore, according to the present invention, a composition containing nucleic acid of a microalga can be provided. Furthermore, according to the present invention, a cosmetic composition containing nucleic acid of microalgae can be provided. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a flow diagram showing a method for extracting nucleic acid. [Diagram 2] FIG. 1 is a diagram showing the basic procedure for extracting nucleic acids from Euglena. [Diagram 3] This is a photograph of a sample that was left standing at 4°C for one week. [Figure 4] (Left) A photograph of a sample that was heated in a microwave oven and then centrifuged. (Right) A photograph showing the results of measuring the absorbance of the supernatant. [Diagram 5] (Left) Photograph of a sample that was heated to 60°C and then centrifuged. (Right) Photograph of a sample that was heated to 60°C and then left to stand at 4°C for two days. [Figure 6] (Right) Photograph of a sample shaken at room temperature for 2 hours. (Left) Photograph of a sample shaken at room temperature for 2 hours and then left to stand at 4°C for 2 days. [Figure 7] FIG. 13 is a diagram showing experimental results. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, an embodiment of the present invention will be described with reference to FIGS. The present embodiment relates to a method for extracting nucleic acid, a composition containing nucleic acid from microalgae, and a cosmetic composition containing nucleic acid from microalgae.

[0015] [Microalgae] In this embodiment, the term "microalgae" refers to minute algae with a body length of several μm to several hundred μm, the existence of which cannot be recognized individually by the naked eye. The algae bodies of microalgae that can be used as the microalgae in this embodiment are not particularly limited, and the algae bodies of known microalgae can be used, and may be either prokaryotic or eukaryotic.

[0016] Examples of microalgae include Euglenida, Chlorophyta such as Chlorella, Dunaliella, and Botryococcus, Cyanobacteria such as Spirulina and Star Jelly, Labyrinthulea such as Aurantiochytrium, Glaucophyta, Rhodophyta, and Chlorarachniophyte. Examples of microalgae that can be classified into the following classes include microalgae: rachniophyta, cryptophyta, Phaeophyta, haptophyta such as Pleurochrysis and Emiliania, heterokontophyta, dinoflagellates, chromerida, primitive red algae, diatoms, coccolithophorids, dinoflagellates, euphthalmophytes, and goldenrods. The taxonomic group to which the microalgae belong may not be determined, and it is preferable if it has been shown by molecular phylogeny that the microalgae belong to these taxa or are closely related to them.

[0017] <Euglena> In this embodiment, "Euglena" includes microorganisms taxonomically classified in the genus Euglena, variants thereof, and closely related species in the family Euglenaceae. Here, the genus Euglena refers to a group of organisms belonging to the Excavata, Euglenozoa phylum, Euglenophyceae, Euglenales, and Euglenaceae family among eukaryotes.

[0018] Specific species in the genus Euglena include Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proxima, Euglena spirogyra, and Euglena viridis. As the Euglena, Euglena gracilis, in particular Euglena gracilis Z strain, can be used. In addition, other Euglena species such as Euglena gracilis Z mutant SM-ZK strain (chloroplast-deficient strain) and E. gracilis var. bacillaris, gene mutants such as chloroplast mutants of these species, and Astasia longa and other Euglena species may also be used.

[0019] The genus Euglena is widely distributed in freshwater such as ponds and marshes, and may be isolated from these for use, or any species of the genus Euglena that has already been isolated may be used. The genus Euglena includes all mutant strains thereof, including those obtained by genetic methods such as recombination, transduction, transformation, and the like.

[0020] (Euglena algae) In this embodiment, live Euglena cells separated by centrifugation, filtration, sedimentation, or the like can be used as they are as the Euglena algae bodies. Live Euglena cells can be used as they are after harvesting from the culture tank, but it is preferable to wash them with water or physiological saline. Alternatively, they may be used in the form of a dispersion in which the Euglena algae bodies are dispersed in a liquid such as water. In this embodiment, it is preferable to use dried Euglena algae bodies obtained by freeze-drying or spray-drying live Euglena cells as the Euglena algae bodies. Furthermore, mechanically treated algae bodies obtained by subjecting living Euglena cells to mechanical treatment such as ultrasonic irradiation or homogenization may be used as the Euglena algae bodies. Furthermore, the mechanically treated product may be dried to be used as the Euglena algae bodies.

[0021] <Green algae> In this embodiment, "green algae" refers to unicellular or multicellular organisms in the division Chlorophyta, and examples of such organisms include those that perform photosynthesis using chlorophyll. In this embodiment, the term "green algae" includes all microorganisms classified as green algae in the zoological and botanical classification, their varieties, and their mutant species. Specifically, these include the Class Trebouxiophyceae, Class Prasinophyceae, Class Trebouxiophyceae, Class Ulvophyceae, Class Pedinophyceae, Class Pleurastrophyceae, etc., which are subclassifications of the phylum Chlorophyta. Among these, it is preferable to use Chlorella, which is classified into multiple phyla as Chlorophyceae or Trebouxiophyceae, and it is also possible to use Haematococcus or Chlamydomonas, which are classified into Chlorophyceae. In this embodiment, green algae include all mutant strains thereof, including those obtained by genetic methods such as recombination, transduction, transformation, and the like.

[0022] (chlorella) In this embodiment, "Chlorella (including Parachlorella)" refers to a microorganism that is a freshwater unicellular green alga mainly belonging to the Chlorophyta division, Chlorophyceae class, and Chlorella genus, and that has chlorophyll in its cells and appears green. Chlorella includes all plants classified in the genus Chlorella, their varieties, and their variants. The genus Chlorella is a plant classified in the order Chlorococcales and family Oocystaceae. Examples of microbial species of the genus Chlorella include Prenoidosa, Erpsoidea, Vulgaris, Regilaris, Sorokiniana, etc. Among these, it is preferable to use the Vulgaris species, which is widely used in research.

[0023] (Cockomixa) In this embodiment, "Coccomyxa" refers to a group of microalgae belonging to the genus Coccomyxa in the family Chlorococcaceae. Examples of Coccomyxa include, but are not limited to, Coccomyxa minor and Coccomyxa gloeobotrydiformis.

[0024] (Haematococcus) In this embodiment, "Haematococcus" refers to a group of microalgae belonging to the genus Haematococcus in the class Trebouxiophyceae. Examples of Haematococcus include, but are not limited to, Haematococcus pluvialis, Haematococcus lacustris, Haematococcus capensis, Haematococcus droebakensi, and Haematococcus zimbabwiensis.

[0025] (Chlamydomonas) In this embodiment, "Chlamydomonas" refers to a group of microalgae belonging to the genus Chlamydomonas in the class Trebouxiophyceae. Examples of Chlamydomonas include, but are not limited to, Chlamydomonas asymmetrica, Chlamydomonas debaryana, Chlamydomonas moewusii, Chlamydomonas nivalis, and Chlamydomonas reinhardtii.

[0026] <Blue-green algae> In this embodiment, "cyanobacteria" refers to bacteria belonging to the phylum Cyanobacteria, a group of eubacteria also known as cyanobacteria, characterized by producing oxygen through photosynthesis. There are single-celled floating ones, ones that form a cluster of a small number of cells, ones with a structure in which cells are arranged in a filamentous form, and the like. Although there is no particular limitation, single-celled ones are preferred. In this embodiment, the term "cyanobacteria" includes all microorganisms classified as cyanobacteria in the zoological and botanical classification, their varieties, and their mutant species. Examples of cyanobacteria include, but are not limited to, those in the genera listed below. For example, the genera Synechocystis, Microcystis, Arthrospira, Cyanothece, Alcaligenes, Anabaena, Synechococcus, Thermosynechococcus, Gloeobacter, Acaryochloris, Nostoc, Trichodesmium, Prochloron, and Prochlorococcus can be mentioned. In this embodiment, the cyanobacteria include all mutant strains thereof, including those obtained by genetic methods such as recombination, transduction, transformation, and the like.

[0027] (Spirulina) In this embodiment, "spirulina" refers to a group of microalgae belonging to the genus Arthrospira of the order Oscillatory, or the genus Spirulina of the family Oscillatory, order Nostocaceae, of the order Cyanobacteria. Examples of spirulina include, but are not limited to, Spirulina platensis, Spirulina maxima, Spirulina geitleri, Spirulina siamese, Spirulina major, Spirulina subsalsa, Spirulina princeps, Spirulina laxissima, Spirulina curta, and Spirulina spirulinoides.

[0028] <Labyrinthula> In this embodiment, the term "Labyrinthulea" includes all microorganisms classified into Labyrinthulea in the classification of zoology or botany, their variants, and their mutant species. Examples of Labyrinthula include the genera Labyrinthula, Althornia, Aplanochytrium, Japonochytrium, Labyrinthuloides, Schizochytrium, Thraustochytrium, Oblongichytrium, Parietichytrium, Ulkenia, and Aurantiochytrium. Preferred are microorganisms belonging to the genus Aurantiochytrium, and particularly preferred is Aurantiochytrium limacinum.

[0029] <True eye drop algae> In this embodiment, "Eustigmatophyte" refers to a unicellular organism belonging to the Eustigmatophyceae class of the Heterokontophyta. The term "Euonymus unicolor" includes all mutants thereof, including those obtained by genetic techniques such as recombination, transduction, transformation, and the like. As the true eyespot algae, algae of the genus Nannochloropsis in the order Eustigmatales can be used.

[0030] [Nucleic acid] In this embodiment, "nucleic acid" refers to microalgae's DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), or derivatives thereof (methylated, oxidized, dimerized), modified forms (thiophosphates, thiolated, phosphorylated, aminated, biotinylated, fluorescently labeled, etc.), and artificially created mimics of DNA and RNA (primers, including non-natural nucleic acid bases, etc.).

[0031] The nucleic acid in this embodiment may contain β-D-glucosyl-hydroxymethyluracil (β-D-Glucosyl-hydroxymethyluracil, CAS registration number: 53910-89-7, also known as β-D-Glucopyranosyloxymethyluracil). β-D-Glucosyl-hydroxymethyluracil is a special modified base called base J. It is synthesized by modification of the thymine base in DNA and is present in the nuclear DNA of animal flagellates and Euglena, but not in other protozoans or metazoans.

[0032] [Nucleic acid extraction method] The nucleic acid extraction method of this embodiment includes a step of preparing microalgae (step S1), a step of extracting nucleic acid from the microalgae (step S2), and a step of purifying the nucleic acid extracted in the nucleic acid extraction step (step S3).In the nucleic acid extraction step, a solution containing the microalgae is heated and / or shaken. Each step will be described below with reference to FIG.

[0033] (Preparing microalgae) In the step of preparing microalgae, microalgae from which nucleic acids are to be extracted are prepared (step S1). As the raw material microalgae, it is preferable to use dried algae bodies from the viewpoint of separating and purifying nucleic acids, but it is not limited thereto. In this embodiment, it is preferable to use dried algae bodies obtained by subjecting live cells of microalgae to a freeze-drying process or a spray-drying process as the raw material.

[0034] Alternatively, live cells of microalgae collected by centrifugation, filtration, sedimentation, or the like after cultivation can be used as the raw material. Live cells of microalgae can be used as they are after harvesting from the culture tank, but it is preferable to wash them with water or physiological saline. Alternatively, they can be used in the form of a dispersion in which the algae bodies of the microalgae are dispersed in a liquid such as a culture medium or water. Furthermore, the raw material may be a mechanically treated product of algae bodies obtained by subjecting microalgal cells to ultrasonic irradiation treatment or mechanical treatment such as homogenization. Also, the raw material may be a dried product of the mechanically treated product, which is then subjected to a drying treatment.

[0035] (Step of extracting nucleic acid) In the step of extracting nucleic acid, a fraction containing nucleic acid is separated from the microalgae prepared in the step of preparing microalgae (step S2). In the step of extracting nucleic acids, the solution containing the microalgae can be heated and / or shaken to efficiently extract the nucleic acids.

[0036] The grinding liquid (extraction liquid) used for extraction may be appropriately selected depending on the type of microalgae. The disruption solution may contain, for example, a protease or a surfactant, which can disrupt barriers such as cell walls, cell membranes, and nuclear membranes under relatively mild conditions, facilitating the extraction of nucleic acids.

[0037] The proteolytic enzyme is not particularly limited as long as it has the ability to degrade proteins to the extent that it can destroy cell walls, cell membranes, and nuclear membranes (in the case of eukaryotes). Examples of the proteolytic enzyme include serine proteases such as protease K, trypsin, chymotrypsin, subtilisin, aspartic acid proteases such as pepsin and cathepsin D, and cysteine ​​proteases such as papain, cathepsin, caspase, and calpain. One or more of these may be used.

[0038] The surfactant is not particularly limited as long as it contributes to the destruction of cell walls, cell membranes, and nuclear membranes. As shown in the examples, when the microalgae is Euglena, a solution containing kitchen detergent and sodium chloride can be used as the disruption liquid without using a protease.

[0039] Heating is performed by heating the solution of the microalgae and the disrupted liquid with a heating means. As the heating means, a general-purpose heater or a high-frequency heating device can be used, but there is no particular limitation. The heating temperature may be any temperature higher than room temperature (25° C.), and is preferably 30° C. or higher, more preferably 40° C. or higher, even more preferably 50° C. or higher, and particularly preferably 60° C. or higher. From the viewpoint of suppressing boiling or evaporation of the solution and decomposition or denaturation of nucleic acid, the upper limit of the heating temperature is less than 100° C., preferably 90° C. or lower, more preferably 80° C. or lower, and even more preferably 70° C. or lower. The heating time may be any time sufficient to extract nucleic acids from the microalgae, and may be adjusted appropriately depending on the amount of microalgae and the amount of solution, for example, 1 minute or more, 10 minutes or more, 30 minutes or more, or 1 hour or more. When using a high-frequency heating device, the microwave output and heating time may be adjusted appropriately depending on the amount of solution to be heated.

[0040] The shaking is performed by shaking a solution of the microalgae and the disruption liquid. At this time, it is also possible to use a shaking means such as a shaker or agitator. The shaking time is not particularly limited as long as it is sufficient to extract nucleic acid from the microalgae, but is, for example, 30 minutes or more, preferably 1 hour or more, and more preferably 2 hours or more. The temperature at which shaking is performed is not particularly limited, but may be room temperature (25°C), or may be combined with heating to a temperature higher than room temperature (25°C), for example, 30°C or higher, 40°C or higher, 50°C or higher, or 60°C or higher.

[0041] When a culture solution containing cultured microalgae is prepared in the step of preparing microalgae, the algae bodies of the microalgae may be collected by known methods such as centrifugation of the culture solution or simple sedimentation, membrane filtration, etc., and the algae bodies may be washed and then dried by known drying methods (vacuum freeze drying, spray drying, heated vacuum drying, etc.) to be used in the separation step. The microalgae prepared in the step of preparing microalgae may also be crushed by known crushing methods such as an ultrasonic crusher.

[0042] (Nucleic acid purification step) In the step of purifying nucleic acid, the nucleic acid extracted in the step of extracting nucleic acid is purified (step S3). Specifically, the nucleic acid is purified by removing substances other than the nucleic acid from the solution containing the nucleic acid obtained in the nucleic acid extraction step.

[0043] The method for purifying nucleic acid is not particularly limited as long as it can purify the target nucleic acid, but examples include centrifugation, decantation, layer separation, washing, and the use of an appropriate separation column. As in the method shown in the Examples, it is also possible to centrifuge the solution containing the nucleic acid obtained in the nucleic acid extraction step, recover the supernatant and the precipitate at the interface, add ethanol to cause precipitation, and wash the precipitated nucleic acid with a solvent such as ethanol.

[0044] [Application] The nucleic acid from microalgae obtained by the nucleic acid extraction method of this embodiment can be used for various purposes depending on its characteristics, etc. For example, the nucleic acids of microalgae can be used as cosmetic compositions, pharmaceutical compositions, food compositions, functional cosmetics such as sunscreens, and nutritional supplements. In addition, the nucleic acids of microalgae can be used as raw materials for biodegradable and recyclable bioplastics and for nucleic acid medicines such as mRNA vaccines. Furthermore, the nucleic acids of microalgae can also be applied to nucleic acid analysis methods (PCR) and the like.

[0045] The nucleic acid from microalgae obtained by the nucleic acid extraction method of this embodiment can be used as an active ingredient of an antioxidant or an ultraviolet absorber, as described below.

[0046] <Antioxidants> The antioxidant according to this embodiment is an antioxidant that contains nucleic acid of microalgae as an active ingredient. Since guanine (G) contained in nucleic acids has antioxidant properties, it is possible to use the nucleic acids of microalgae as an active ingredient in antioxidants.

[0047] <Ultraviolet absorbing agent> The ultraviolet absorbent according to this embodiment is an ultraviolet absorbent that contains nucleic acid of microalgae as an active ingredient. Guanine (G) and adenine (A) contained in nucleic acids have the effect of absorbing at least a part of ultraviolet A rays (UVA), which are ultraviolet rays with wavelengths of 320 to 400 nm. Thymine (T) and cytosine (C) contained in nucleic acids have the effect of absorbing at least a part of ultraviolet B rays (UVB), which are ultraviolet rays with wavelengths of 280 to 320 nm. Therefore, it is possible to use the nucleic acid of microalgae as an active ingredient of an ultraviolet absorbent. In this case, if vitamin B2 (riboflavin) is used in combination, it can cover the entire UVA wavelength range and has the effect of releasing the absorbed energy to guanine, which can then absorb light again (photosensitizer), so a photosensitizer that absorbs ultraviolet rays, such as riboflavin, can also be added.

[0048] [Composition] The antioxidants and UV absorbers containing the nucleic acid of the microalgae according to this embodiment as an active ingredient can be used as compositions such as cosmetic compositions, pharmaceutical compositions, and food compositions by adding various additives.

[0049] <Cosmetic composition> The nucleic acid of the microalgae of this embodiment can be suitably used in cosmetic compositions by utilizing its antioxidant effect. The nucleic acid of the microalgae of this embodiment has an ultraviolet absorbing effect, and therefore is preferably used as a cosmetic composition for sunscreen. The cosmetic composition can be applied to any type of cosmetic, for example, skin care cosmetics such as sunscreen cosmetics, lotions, milky lotions, creams, and serums, and makeup cosmetics such as foundations, concealers, makeup bases, lipsticks, blushers, eyeshadows, and eyeliners.

[0050] In addition to the nucleic acid of the microalgae, the cosmetic composition according to this embodiment can contain one or more freely selected components that can be used in general cosmetic compositions. For example, all additives that can be commonly used in the cosmetic field, such as base materials, preservatives, emulsifiers, colorants, antiseptics, surfactants, UV absorbers, antioxidants, moisturizers, UV absorbers, fragrances, antiseptic and antifungal agents, extender pigments, coloring pigments, alcohol, and water, may be contained.

[0051] In the cosmetic composition according to this embodiment, the content of the nucleic acid of the microalgae is not particularly limited and can be freely set according to the purpose.

[0052] <Pharmaceutical Composition> The nucleic acid of the microalgae of this embodiment can be used as a pharmaceutical composition. The pharmaceutical composition of the present embodiment is provided by blending a pharma- ceutical composition having an antioxidant effect or an ultraviolet absorbing effect with a pharma- ceutical carrier or additive in an amount sufficient to effectively exert the said effect. The pharmaceutical composition may be a drug or a quasi-drug. The pharmaceutical composition may be applied externally or internally, and may be used in the form of an oral preparation, an injection such as a subcutaneous injection, an intravenous injection, an intradermal injection, an intramuscular injection, and / or an intraperitoneal injection, a transmucosal application preparation, a transdermal application preparation, or the like. The dosage form of the pharmaceutical composition can be appropriately set depending on the form of application, and examples thereof include liquid preparations such as solutions and suspensions, semi-solid preparations such as ointments and gels, and solid preparations such as tablets, granules, capsules, powders, and dusting agents.

[0053] The pharmaceutical composition according to this embodiment can contain one or more pharma- ceutical acceptable additives selected freely. For example, when the pharmaceutical composition according to the present embodiment is applied to an oral preparation, it may contain all additives that can be commonly used in the field of pharmaceutical preparations, such as excipients, binders, disintegrants, surfactants, preservatives, colorants, flavorings, fragrances, stabilizers, antiseptics, antioxidants, etc. In addition, it may be made into a sustained-release preparation by utilizing a drug delivery system (DDS).

[0054] In addition to the nucleic acid of the microalgae, one or more other substances known to have antioxidant or ultraviolet absorbing properties can also be added to the pharmaceutical composition of this embodiment.

[0055] <Food composition> The nucleic acid of the microalgae of this embodiment can also be used in food products. In the food field, the food composition of this embodiment can provide a food composition having the antioxidant and UV absorbing effects by incorporating an effective amount of microalgal nucleic acid that can effectively exert the said effects as a food ingredient into various foods. It is also possible to provide food compositions by combining the nucleic acids of microalgae with foods for specified health uses, foods with nutrient function claims, foods with functional claims, foods for hospital patients, supplements, etc. Examples of the food composition include seasonings, processed meat products, processed agricultural products, beverages (soft drinks, alcoholic beverages, carbonated beverages, dairy beverages, fruit juice beverages, tea, coffee, nutritional drinks, etc.), powdered beverages (powdered juice, powdered soup, etc.), concentrated beverages, confectioneries (candy (throat lozenges), cookies, biscuits, gum, gummy candy, chocolate, etc.), bread, cereals, etc. In addition, in the case of foods for specified health uses, foods with nutrient functions, foods with functional claims, etc., the foods may be in the form of capsules, lozenges, syrup, granules, powder, etc.

[0056] Here, a "specified health food" is a food that contains functional ingredients that affect physiological functions, etc., and can be labeled as suitable for specific health uses with the permission of the Commissioner of the Consumer Affairs Agency. A food with nutrient functions is a food that is used to supplement nutritional components (vitamins, minerals), and displays the function of the nutritional components. In order to be sold as a food with nutrient functions, the amount of nutritional components contained in the recommended daily intake must be within the range of the set upper and lower limits, and in addition to displaying the nutritional function, it must also display a warning. Foods with functional claims are foods that display functionality based on scientific evidence at the responsibility of the business operator, and information on safety and functionality is submitted to the Commissioner of the Consumer Affairs Agency before sale.

[0057] In addition to the nucleic acid of the microalgae, the food composition according to the present embodiment can contain one or more components that can be used in general food compositions. For example, all additives that can be used in the food industry, such as various seasonings, preservatives, emulsifiers, stabilizers, flavorings, colorants, preservatives, and pH adjusters, can be added.

[0058] In addition to the nucleic acids of microalgae, one or more other substances known to have antioxidant or ultraviolet absorbing properties can also be added to the food composition of this embodiment. EXAMPLES

[0059] The present invention will be described in detail below with reference to specific examples, but the present invention is not limited to these. In the following examples, nucleic acids were extracted from Euglena, a type of microalgae. Euglena gracilis powder (Euglena algae, manufactured by Euglena Co., Ltd.) was used as Euglena.

[0060] <Extraction method> FIG. 2 is a diagram showing the basic procedure for extracting nucleic acids from Euglena. 10 ml of grinding liquid (1% kitchen detergent (Frosch (Asahi Kasei Home Products Co., Ltd.)), 3 M sodium chloride)) was added to 1 g of Euglena powder, and nucleic acid was extracted by heating, shaking, etc. After centrifugation, the supernatant and the precipitate at the interface were collected in a 50 ml tube and precipitated by adding ethanol, and the precipitated nucleic acid was washed with 70% ethanol. Absorbance measurements of the nucleic acid solutions were performed at 260 nm using a NanoDrop microspectrophotometer (Thermo Fisher Scientific). The yield of nucleic acid was calculated from the obtained wavelength, and the purity of nucleic acid was evaluated by the A260 / A280 ratio.

[0061] <Purification method> 1) The volume of the aqueous solution of nucleic acid was estimated. 2) 100% ethanol was added in an amount 2.5 times the amount of the nucleic acid aqueous solution. 3) Mix thoroughly until uniform. 4) Allow to stand at room temperature for 2 to 10 minutes. 5) Centrifuge at 13,000 rpm for 15 minutes at 4°C or room temperature. 6) The upper (aqueous) phase was decanted off. 7) 1 ml of 70% ethanol was added. 8) The contents of the tube were mixed gently. 9) Centrifuge at 13,000 rpm for 2 minutes at room temperature. 10) The upper (aqueous) phase was decanted and discarded. 11) Place the tube upside down on a paper towel to remove all moisture. 12) The precipitate was dried completely.

[0062] <Confirmation of nucleic acid purity> The absorption spectrum of the nucleic acid solution at 260 nm (A260) was confirmed using NanoDrop1000. DNA purity was assessed by estimating the absorbance ratio A260 / A280 (>1.8), and DNA purity due to protein and polysaccharide contamination was assessed by estimating the absorbance ratio A260 / A230 (2.0–2.2). When necessary, bands were observed by gel electrophoresis.

[0063] <How to calculate yield> On the premise that the nucleic acid concentration is 50 μg / ml when the absorbance at 260 nm is 1, when the absorbance is L, the concentration of the nucleic acid solution is = L × 50 [μg / ml]. The yield when 1 g of Euglena powder is used is 50 × L × c * [μg / 1g](c * was calculated using the dilution rate.

[0064] The conditions examined in the nucleic acid extraction process are shown below. <Standing at low temperature> Extraction procedure: Euglena powder (1 g as is or 0.5 g fine powder) was added to 10 ml of the crushed liquid cooled to 4°C and left to stand at a low temperature of 4°C for one week. FIG. 3 is a photograph of a sample that was left standing at 4° C. for one week.

[0065] <Microwave heating> 10 ml of the grinding liquid was added to 1 g of Euglena powder and allowed to stand at room temperature for 5 minutes. After shaking at room temperature for 30 minutes, the mixture was heated in a microwave oven (500 W, 60 seconds). The mixture was cooled to room temperature and allowed to stand at 4°C for 2 days or centrifuged (room temperature, 1000 rpm, 15 minutes). Figure 4 shows a photograph (left) of a sample that was heated in a microwave oven and then centrifuged, and a photograph (right) showing the results of measuring the absorbance of the supernatant. Accurate nucleic acid concentrations could not be obtained because the sample would suddenly boil after a few seconds of microwave heating. From the absorbance, it could be inferred that a higher yield would be obtained by heating in a microwave oven.

[0066] <Hot plate heating> Extraction procedure: 10 ml of the 60°C grinding liquid was added to 1 g of Euglena powder, and the mixture was left to stand at 60°C for 40 minutes, after which it was left to stand at 4°C for 2 days or centrifuged (room temperature, 10,000 rpm, 15 minutes). Figure 5 shows a photograph of a sample that was heated to 60°C and then centrifuged (left), and a photograph of a sample that was heated to 60°C and then left to stand at 4°C for two days (right).

[0067] <Shaker shaking> Extraction procedure: 10 ml of the grinding liquid was added to 1 g of Euglena powder, and the mixture was shaken at room temperature for 2 hours using a shaker, and then allowed to stand at 4°C for 2 days. FIG. 6 shows a photograph of a sample that had been shaken at room temperature for 2 hours (right) and a photograph of a sample that had been shaken at room temperature for 2 hours and then allowed to stand at 4° C. for 2 days (left).

[0068] <Experimental Results> The experimental results are shown in Figure 7. A comparison of samples No. 1 to 6 with No. 7 to 14 revealed that the extraction efficiency was significantly improved when heating or shaking was performed. In particular, under the conditions of samples No. 8 to 14, the yield was recorded to be 2 mg / g or more, which was significantly higher than the value of 0.08 mg / g for broccoli. By adding operations such as heating and shaking to the extraction process, a yield of 0.1 mg / g or more could be achieved. It was found that a white precipitate was formed at the boundary with the supernatant after standing at 4°C for 2 days. Heating in a microwave oven is expected to produce a high extraction effect. When extracting cells by disrupting them, contamination with proteins and polysaccharides is expected.

Claims

1. Providing microalgae; Extracting nucleic acids from the microalgae; A step of purifying the nucleic acid extracted in the step of extracting the nucleic acid is carried out, A method for extracting nucleic acid, wherein the step of extracting nucleic acid comprises heating and / or shaking a solution containing the microalgae.

2. The method for extracting nucleic acid according to claim 1 , wherein the microalgae is Euglena.

3. The method for extracting nucleic acid according to claim 2, wherein the nucleic acid contains β-D-glucosyl-hydroxymethyluracil.

4. Contains nucleic acid from microalgae, The composition is selected from a cosmetic composition, a pharmaceutical composition, and a food composition.

5. The composition according to claim 4 , wherein the microalgae is Euglena.

6. The composition of claim 5, wherein the nucleic acid comprises β-D-glucosyl-hydroxymethyluracil.

7. The composition according to any one of claims 4 to 6, which is a cosmetic composition.

8. The cosmetic composition according to claim 7, which is for use as a sunscreen.

9. A cosmetic composition for sunscreen use, comprising nucleic acid of microalgae as an active ingredient.

10. The cosmetic composition according to claim 9 , wherein the microalgae is Euglena.

11. The cosmetic composition according to claim 10, wherein the nucleic acid comprises β-D-glucosyl-hydroxymethyluracil.

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