WO2018199231A1 - Placenta extract - Google Patents

Abstract

In the present invention, exosomes are extracted from placenta extracts and a method is established for producing swine placenta extracts, which contain a high level of exosomes. In the exosomes, multiple miRNAs having anti-aging effects were found. Exosomes containing these miRNAs can be used for: oral compositions such as tablets, supplements, and beverages having anti-aging effect; cosmetics such as lotions, cosmetic oils, and facial cleansing creams; and a fibroblast cell growth promoter.

Description

Placenta extract Cross reference

This application claims priority based on Japanese Patent Application No. 2017-089024 filed in Japan on April 28, 2017, and all the contents described in this application are incorporated herein by reference in their entirety. Incorporated. In addition, all the contents described in all patents, patent applications, and documents cited in the present application are incorporated herein by reference in their entirety.

The present invention relates to the provision of various compositions containing exosomes derived from placenta (oral compositions, cosmetic compositions, fibroblast proliferation promoters), placenta extracts containing exosomes, and methods for producing exosomes.

Placenta (placenta) extract is formulated in cosmetics and supplements for the purpose of beauty and health benefits (Patent Document 1). Placenta extracts contain amino acids, peptides, nucleic acids, etc., which have been thought to synergistically contribute to physiological functions, but elucidate the identification of functionally involved components and their absorption into the body. There are many things that are not done. Against this background, the identification of functional components in placenta extracts has been desired in the food and pharmaceutical markets.

Recently, cell secretions known as exosomes (extracellular endoplasmic reticulum) have attracted attention. In vivo, exosomes are secreted from cells derived from various organs. These exosomes contain physiologically active substances such as miRNA (microRNA), amino acids and peptides, and are known to function as transporters and to help crosstalk between organs like hormones (Non-patent Document 1). ). It has also been reported that exosomes are released from the placenta at the time of pregnancy and work on the crosstalk between the fetus and the mother (Non-patent Document 2).

It is also known that exosomes may be absorbed from the gastrointestinal tract because miRNA derived from food exosomes is detected in human serum (Non-patent Document 3). These food-derived exosomes have been reported to be absorbed and play a useful role in biological functions such as immune regulation, but there are many unclear points about the mechanism, and in particular, elucidation at the cellular level is not sufficient. There was not (nonpatent literature 4).

MiRNA is a small RNA consisting of 20 to 25 bases. It is known that thousands of miRNAs exist in living bodies such as humans. Although miRNA is RNA that is not translated into protein, expression of other genes can be directly regulated by binding partially complementary to messenger RNA (mRNA) sequences. One type of miRNA may be involved in the expression control of a plurality of genes, or the function may be exhibited by a combination of a plurality of miRNAs. That is, although miRNA may be able to function even if it is single, it cooperates with other kinds of miRNA to suppress gene expression of multiple mRNAs.

On the other hand, although many of the beauty effects and health effects of placenta extract are expected to have an anti-aging effect, the elucidation at the cellular level has not been sufficient. From this background, placenta extract has also been desired to be elucidated at the cellular level.

Aging at the cell level can be examined in vitro using cell proliferation ability and cell size as indicators. For example, it is known that fibroblasts present in animal support tissues have a proliferating ability that decreases with an increase in the number of culture passages as in the case of aging of living bodies. In addition, although the number of passages in which the growth ability is reduced differs depending on the animal species, human fibroblasts do not grow in about 80 passages (Non-patent Document 5). It is also known that the cell size increases simultaneously with the decrease in proliferation ability. This phenomenon is due to changes in the molecular structure of proteins in the cells accompanying aging and a decrease in the metabolic capacity of the cells. (Non-patent paper 6)

Living body aging is also considered to reflect cellular aging, and even in rapidly growing organs such as skin, liver, and bone, growth capacity and metabolic speed may decrease with aging. It was known.

JP 2013-34423 A

An object of the present invention is to find an active ingredient in a placenta extract whose active ingredient is unknown and a method for producing a placenta extract containing this active ingredient, and to provide a more active fractional ingredient.

In order to solve the above problems, the present inventors have extracted a exosome from a placenta extract and established a method for producing a porcine placenta extract having a high exosome and exosome content. Furthermore, 162 types of miRNAs described in Tables 1 to 3 are contained in the exosome, and these are identical to the base sequences of miRNAs contained in human-derived exosomes, of which 27 types (SEQ ID NO: 3, 8, 10, 18, 22, 29, 33, 34, 37, 39, 41, 51, 60, 61, 66, 71, 72, 75, 76, 77, 105, 108, 109, 138, 148, 157 and 159) were confirmed to decrease in senescent cells.
In addition, when placenta-derived exosomes and synthetic miRNAs having the same base sequence as miRNAs contained in the exosomes were added to human fibroblasts, cell proliferation was promoted and the cell area decreased. confirmed.

That is, the exosome according to one embodiment of the present invention has (i) a nucleic acid molecule having the base sequence represented by any one of SEQ ID NOs: 1 to 162, (ii) 90% or more identity with the base sequence of (i). Or (iii) at least one selected from the group consisting of nucleic acid molecules having a base sequence having a deletion, addition and / or substitution of one or two bases in the base sequence of (i) An exosome characterized in that it is an extract from mammalian placenta. However, when the nucleic acid molecule is RNA, T shown in each base sequence is U.

In a preferred embodiment of the exosome, at least one nucleic acid molecule is SEQ ID NO: 3, 8, 10, 18, 22, 29, 33, 34, 37, 39, 41, 51, 60, 61, 66, 71, 72. 75, 76, 77, 105, 108, 109, 138, 148, 157 and 159. However, the base sequence of each nucleic acid molecule has 90% or more identity with the base sequence, or has a deletion, addition and / or substitution of one or two bases in the base sequence. Also good.

In another preferred embodiment of the above exosome, at least one nucleic acid molecule contains a nucleic acid molecule having a base sequence represented by SEQ ID NOs: 16, 74 and 87. However, the base sequence of each nucleic acid molecule has 90% or more identity with the base sequence, or has a deletion, addition and / or substitution of one or two bases in the base sequence. Also good.

In another embodiment of the present invention, an oral composition containing the exosome or mir-125a, mir-26a and / or mir-30c miRNA as an active ingredient is provided.

In still another embodiment, a cosmetic composition containing the exosome or mir-125a, mir-26a and / or mir-30c miRNA as an active ingredient is provided.

Yet another embodiment is a fibroblast proliferation promoter containing the exosome or mir-125a, mir-26a and / or mir-30c miRNA as an active ingredient.

In another aspect of the present invention, a method for producing a placenta extract comprising extracting a placenta of a mammal with hot water, performing enzymatic degradation with a protease, and recovering a placenta extract containing a exosome having a spherical structure. Is provided.

In still another aspect, the present invention provides a method for suppressing cellular senescence, comprising administering the exosome, or mir-125a, mir-26a and / or mir-30c miRNA to mammalian cells.

According to the present invention, it is possible to provide placenta-derived exosomes and miRNAs contained in exosomes. It was also confirmed that fibroblasts proliferated after adding exosomes to human fibroblasts. Based on the above, by providing a composition containing exosomes derived from placenta orally or transdermally, the structure of the dermis is strengthened, and it is provided as a beauty and anti-aging application that improves skin tension, wrinkles, sagging, etc. Became possible. Further, when a synthetic miRNA having the same base sequence as the miRNA contained in the exosome is added, the same effect is seen and the same effect can be expected.

It is an electron micrograph of the exosome contained in the porcine placenta extract. It is a figure which shows the particle size analysis of the exosome contained in the pig placenta extract. It is the photograph which immunostained the human fibroblast (66PDL) at the time of exosome addition and non-addition. It is a figure which shows the cell number of the human fibroblast (66PDL) at the time of exosome addition and non-addition. It is a figure which shows the cell size of the human fibroblast (66PDL) at the time of exosome addition and non-addition. It is the photograph which immunostained the human fibroblast (83PDL) at the time of exosome addition and non-addition. It is a figure which shows the cell number of a human fibroblast (83PDL) at the time of exosome addition and non-addition. It is a figure which shows the cell size of a human fibroblast (83PDL) at the time of exosome addition and non-addition. It is the result of having evaluated the macrophage migration ability of the exosome of this invention by a transwell transfer assay.

In addition, the placenta delivered by the normal delivery of the pig (SPF pig) without a specific disease cause is used as the pig placenta used in the present invention. In addition, the exosome in the present invention is a spherical composition comprising a lipid, encapsulating peptides, proteins, nucleic acids and the like.

Examples of the extraction solvent for the porcine placenta extract used in the present invention include water, lower alcohols such as methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, and isobutanol or hydrous lower alcohols, propylene glycol, 1,3-butylene glycol, 1,2-butylene glycol, 1,4-butylene glycol, 1,5-pentanediol, 1,2-pentanediol, 1,3-pentanediol, 1,4-pentanediol, 1,3,5-pentanetriol 1 selected from glycerin, acids (hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, etc.) and alkalis (sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, etc.) that have been appropriately adjusted. Examples include seeds or a mixture of two or more, especially water Door is preferable. However, if it is not preferable to use a solvent depending on the application, use only water, or perform extraction with a highly volatile solvent that can be easily removed after extraction, and dissolve in water after removing the solvent. Such a method is also possible. Furthermore, it is preferable to add protease. As the protease to be used, protease A “Amano” SD, protease M “Amano” SD or protease P “Amano” 3SD, which is a food processing protease manufactured by Amano Enzyme, is preferable, but not limited thereto.

About the extraction method, the weight ratio of the solvent to the raw material or the extraction time can be arbitrarily set. The weight ratio of the solvent to the raw material can be arbitrarily set within the range of, for example, raw material: solvent 4: 1 to 1: 100, and a weight ratio of 1: 1 to 1:20 is particularly preferable.

The porcine placenta extract used in the present invention can be further subjected to a purification operation after solvent extraction. Examples of the purification operation include fractionation using chromatography, filter paper, membrane filter, ultrafiltration membrane, etc. Filtration, drying, pH adjustment, deodorization, decolorization, and long-term storage and storage can be exemplified, and these can be arbitrarily selected and combined treatments can be performed.

The exosome used in the present invention can be obtained at a high concentration by centrifuging the porcine placenta extract powder with water or from the porcine placenta extract by ultracentrifugation. Purification operations such as filtration can be performed before and after centrifugation.

Nucleic acid molecules are extracted from the exosomes obtained in this way, analyzed for nucleotide sequences using a next-generation sequencer, and searched for a database of human microRNAs. As a result, the nucleotide sequences shown in Tables 1 to 3 are found. I understood. That is, at least in pig placenta, there are nucleic acid molecules having the same sequence as these human microRNAs. A preferred nucleic acid molecule is microRNA, in which case T (thymine) in the sequence is U (uracil). In the present specification, the placenta refers to placental tissue that can be obtained from a mother after childbirth, and is an organ that is formed in the uterus of a pregnant animal and connects the mother and the fetus. Since the placenta of mammals is structurally and functionally similar, placenta other than pigs, such as sheep placenta, horse placenta, bovine placenta and human placenta, can also be used. Among these, pig placenta is preferable from the viewpoint of availability and safety.

In the present specification, microRNA (microRNA; miRNA) typically means what is called mature miRNA. A mature miRNA is an endogenous non-coding RNA of about 20 to 25 bases encoded on the genome. miRNA is first transcribed from a miRNA gene on genomic DNA as a primary transcript having a length of about several hundred to several thousand bases, and then processed to be a precursor miRNA having a hairpin structure of about 60 to 70 bases. It becomes. Thereafter, it moves from the nucleus into the cytoplasm and further undergoes processing to become a double-stranded mature miRNA of about 20-25 bases. It is known that double-stranded mature miRNAs function to inhibit the translation of target genes by forming a complex with a protein called RISC, with one strand forming a complex with a protein called RISC. . Therefore, in the present specification, “miRNA” includes not only endogenous single-stranded and double-stranded miRNAs but also synthetic nucleic acids having the same base sequence as these miRNAs. MiRNA also encompasses its precursors.

For example, “miR-125a” includes both the precursor and mature form of the microRNA, but is preferably a mature form, and examples of the mature form include miR-125a-5p (SEQ ID NO: 163) and miR-125a. -3p (SEQ ID NO: 166) is exemplified. “MiR-30c” includes miR-30c-5p (SEQ ID NO: 164) and miR-30c-3p (SEQ ID NO: 167) as mature forms. Similarly, “miR-26a” includes miR-26a-5p (SEQ ID NO: 165) and miR-26a-3p (SEQ ID NO: 168) as mature forms. The base sequences of these miRNAs are disclosed in, for example, the miRBase database created by Sanger Laboratories: http://microrna.sanger.ac.uk/.

In the present invention, it means any of the following nucleic acids in relation to the miRNAs shown in Tables 1 to 3.
(I) RNA having a base sequence represented by any of SEQ ID NOs: 1 to 162,
(Ii) RNA having 90% or more, preferably 95% or more identity with the base sequence represented by any of SEQ ID NOs: 1 to 162, or (iii) base sequence represented by any of SEQ ID NOs: 1 to 162 RNA having a nucleotide sequence having a deletion, addition and / or substitution of one or two bases. However, T (thymine) shown in each sequence of Tables 1 to 3 is U (uracil). In general, 7 bases starting from the 2nd to 7th bases from the 5 ′ end of the mature miRNA are called seed sequences. It is known that this sequence must be completely complementary to the sequence of the target mRNA, but the other bases do not necessarily have to be completely identical. Therefore, in the miRNA of the present invention, it is sufficient that the core sequences of the sequences shown in SEQ ID NOs: 1 to 162 are completely matched, and the 5 ′ side or 3 ′ side sequence thereof is a deletion of one or several bases. May have additions and / or substitutions. The term “several” includes 1 to 10, preferably 5 or less, and more preferably 1 to 3. The core sequence (seed sequence) can be identified by analyzing the target mRNA.

“Identity” refers to an optimal alignment when two nucleotide sequences are aligned using mathematical algorithms known in the art (preferably the algorithm uses one or the other of the sequences for optimal alignment). The percentage of identical nucleotide residues to all overlapping nucleotide residues) (which can be considered the introduction of gaps to both).
For example, nucleotide sequence identity is determined using the homology calculation algorithm NCBI (National Center for Biotechnology Information) BLAST-2 under the following conditions (gap open = 5 penalty; gap extension = 2 penalty; x_dropoff = 50; It can be calculated by aligning two nucleotide sequences with expected value = 10; filtering = ON).

The miRNA used in the present invention can be obtained by synthesizing as in the test conducted in the present invention, or by extracting and purifying miRNA having a similar base sequence. For example, it may be a chimeric nucleic acid in which a part of the bases in the miRNA base sequences shown in (i) to (iii) above are replaced with deoxyribonucleotides.

The exosome, miRNA, porcine placenta extract, and oral composition containing them used in practicing the present invention may be in any form such as liquid, solid, powder, paste, etc. You can choose.

The contents of the extract, exosome, and miRNA are not particularly limited as long as the effects of the present invention can be confirmed, but generally 0.0001 mg / g to 10 mg / g (the denominator is denominator) (Indicating the weight of the preparation). The range is preferably 0.001 mg / g to 10 mg / g, and most preferably 0.001 mg / g to 1 mg / g.

The exosome, miRNA, and fibroblast growth promoter in the present invention are preferably used in pharmaceutical preparations. As the pharmaceutical, either oral administration or parenteral administration can be employed. In administration, the active ingredient can be mixed with a solid or liquid nontoxic pharmaceutical carrier suitable for administration methods such as oral administration, rectal administration and injection, and administered in the form of a conventional pharmaceutical preparation. Examples of such preparations include solid preparations such as tablets, granules, powders and capsules, liquid preparations such as solutions, suspensions and emulsions, freeze-dried preparations, and the like. It can be prepared by conventional means. Examples of the non-toxic pharmaceutical carrier include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, Examples include albumin, water, and physiological saline. If necessary, conventional additives such as a stabilizer, a wetting agent, an emulsifier, a binder, and a tonicity agent can be appropriately added and used.

In addition, when exosomes and miRNA are blended in oral compositions, cosmetics, and fibroblast proliferation promoters, the components and additives exemplified below are arbitrarily selected and used as long as the effects of the present invention are not impaired. Various preparations can be produced, and the content in the preparation is not particularly limited, but a concentration range of 0.0001 to 50% is usually preferred.

For example, as an oral composition containing exosomes and miRNA, as it is or after adding various nutritional supplements, nourishing tonics, fatigue recovery, constitution improvement, etc., it is contained in foods and drinks, nutritional supplements, health supplements, It is eaten as a nutritionally-adjusted food, functional health food, food for specified health use, functional nutrition food, functional indication food, supplement, and food material. For example, after adding the above-mentioned appropriate auxiliaries, it may be used for edible by using conventional means, and forming into an edible form, for example, granular, granular, tablet, capsule, paste, etc. In addition, various foods such as processed marine products such as kamaboko and chikuwa, livestock products such as sausage and ham, Western confectionery, Japanese confectionery, raw noodles, Chinese noodles, boiled noodles, buckwheat noodles, sauce, soy sauce, sauce, Seasonings such as sugar, honey, powdered candy, syrup, etc., spices such as curry powder, mustard powder, pepper powder, jam, marmalade, chocolate spread, pickles, red vegetables, sprinkles, canned and bottled vegetables and fruits Dairy products such as processed vegetables and fruits, cheese, butter, yogurt, miso soup, soup, fruit juice, vegetable juice, whey beverage, soft drink, alcoholic beverages, liquid food, etc. Was added to the general preference foods and beverages can be used, also, pet food, can also be used for animals and plants for feed and the like. In addition, in food and beverage compositions, it has health benefits such as nutritional supplementation, fatigue recovery, tonicity, cell activation (cell aging prevention), taste improvement, color tone, aroma, glossiness, stabilization, thickening It can be used for such purposes. Furthermore, in addition to these, expecting various pharmaceutical effects of each of the raw materials known so far, and combining these, the enhancement of the intended effect of the present invention can be achieved. It is also possible to make a product that expects an effect.

For example, cosmetics containing exosomes and miRNA can be used in various skin external preparations (including preparations used for animals) in general, specifically, ampoules, capsules, pills, tablets, powders, Granules, solids, liquids, gels, bubbles, emulsions, sheets, mists, sprays, etc. in suitable forms for use 1) Pharmaceuticals, 2) Quasi-drugs, 3) Topical or systemic skin cosmetics (For example, lotion, milky lotion, cream, cosmetic liquid, ointment, lotion, oil, pack, etc., cosmetics for eyelashes, cosmetics for eyelashes, eyelash treatments, permanents for eyelashes, hair restorers for eyelashes, etc. Cosmetics for eyelashes, facial cleanser and skin cleanser, massage agent, cleansing agent, hair remover, hair remover, shaving treatment, after shave lotion, pre-show bronze, Makeup cosmetics such as wave cream, foundation, lipstick, blusher, eye shadow, eyeliner, mascara, perfume, nail polish, nail enamel, nail enamel remover, poultice, plaster, tape , Sheets, patches, aerosols, etc.), 4) medicinal and / or cosmetic preparations applied to the scalp / hair (eg shampoo, rinse, hair treatment, pre-hair treatment, permanent solution, Hair dyes, hair styling agents, hair styling agents, hair growth and hair nourishing agents, poultices, plaster agents, tape agents, sheet agents, patches, aerosol agents, etc.), 5) bathing agents used in bath water, 6) other , Anti-smell, deodorant, deodorant, antiperspirant, sanitary goods, sanitary cotton, wet tissue, toothpaste, mouth freshener, gargle, and the like.

The exosome, miRNA, and oral composition, cosmetic, and fibroblast growth promoter containing the exosome or miRNA of the present invention are within the range that does not impair the effects of the present invention, if necessary, in addition to the above essential components. Thus, various preparations can be produced by arbitrarily selecting and using the components and additives exemplified below, and the content in the preparation is not particularly limited, but the concentration range is usually 0.0001 to 50%. preferable.

Examples of polymers, thickeners and gelling agents include guar gum, locust bean gum, queens seed, carrageenan, galactan, arabic gum, tara gum, tamarind, fur celeran, karaya gum, troarooi, cara gum, tragacanth gum, alginic acid and sodium salt Salt, mannan; starch such as rice, corn, potato, wheat; xanthan gum, dextran, succinoglucan, curdlan, hyaluronic acid and its salt, xanthan gum, pullulan, gellan gum, chitin, chitosan, agar, gypsophila extract, chondroitin sulfate, Casein, collagen, gelatin, albumin; methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose and Salts such as sodium, methyl hydroxypropyl cellulose, cellulose sodium sulfate, cellulose dialkyldimethylammonium sulfate, crystalline cellulose, cellulose and other derivatives thereof; soluble starch, carboxymethyl starch, methyl hydroxypropyl starch, starch such as methyl starch -Based polymers, starch derivatives such as hydroxypropyltrimonium chloride starch, octenyl succinic acid corn starch aluminum; sodium alginate, alginic acid derivatives such as propylene glycol alginate; polyvinyl pidoridone (PVP), polyvinyl alcohol (PVA), vinyl pidoridone and vinyl alcohol Polymer, polyvinyl methyl ether; polyethylene glycol, polypropylene Glycol, polyoxyethylene / polyoxypropylene copolymer; amphoteric methacrylate copolymer such as (methacryloyloxyethylcarboxybetaine / alkyl methacrylate) copolymer, (acrylates / stearyl acrylate / ethylamine methacrylate oxide) copolymer; Dimethicone / vinyl dimethicone) crosspolymer, (alkyl acrylate / diacetone acrylamide) copolymer, (alkyl acrylate / diacetone acrylamide) copolymer AMP; polyvinyl acetate partially saponified product, maleic acid copolymer; vinyl pyrrolidone / dialkyl methacrylate Aminoalkyl copolymer; acrylic resin alkanolamine; polyester, water-dispersible polyester; polyacrylamide; polyethyl acrylate, etc. Acrylate ester copolymer, carboxyvinyl polymer, polyacrylic acid and its salts such as sodium salt, acrylic acid / methacrylic acid ester copolymer; acrylic acid / methacrylic acid alkyl copolymer; cation such as polyquaternium-10 Cellulose, diallyldimethylammonium chloride / acrylamide copolymer such as polyquaternium-7, acrylic acid / diallyldimethylammonium chloride copolymer such as polyquaternium-22, and acrylic acid / diallyldimethylammonium chloride / acrylamide copolymer such as polyquaternium-39 Acrylic acid / cationized methacrylic acid ester copolymer, acrylic acid / cationized methacrylic acid amide copolymer, polyquaternium-47 acrylic acid / methyl acrylate / salt Methacrylamidopropyltrimethylammonium copolymer, chloromethacrylic acid choline ester polymer; cationized polysaccharide such as cationized oligosaccharide, cationized dextran, guar hydroxypropyltrimonium chloride; polyethyleneimine; cation polymer; polyquaternium-51 2-methacryloyloxyethyl phosphorylcholine polymer and butyl methacrylate copolymer; acrylic resin emulsion, polyethyl acrylate emulsion, polyacryl alkyl ester emulsion, polyvinyl acetate resin emulsion, natural rubber latex, synthesis Polymer emulsions such as latex; nitrocellulose; polyurethanes and various copolymers; various silicones; acrylic-silicone graft copolymers Various silicone copolymers; various fluoropolymers; 12-hydroxystearic acid and its salts; dextrin fatty acid esters such as dextrin palmitate and dextrin myristate; silicic anhydride, fumed silica (ultrafine silicic anhydride) Aluminum magnesium silicate, magnesium magnesium silicate, metal soap, dialkyl phosphate metal salt, bentonite, hectorite, organically modified clay mineral, sucrose fatty acid ester, fructooligosaccharide fatty acid ester are preferable.

Antioxidants include α-lipoic acid, salts of α-lipoic acid, and derivatives of α-lipoic acid; tocopherol derivatives such as tocopherol (vitamin E), tocotrienol, tocopherol acetate; BHT, BHA; propyl gallate, etc. Gallic acid derivatives; vitamin C (ascorbic acid) and / or derivatives thereof; erythorbic acid and derivatives thereof; sulfites such as sodium sulfite; bisulfites such as sodium bisulfite; thiosulfates such as sodium thiosulfate; metabisulfite Salts: thiotaurine, hypotaurine; thioglycerol, thiourea, thioglycolic acid, cysteine hydrochloride are preferred. Preferred examples of the reducing agent include thioglycolic acid, cysteine, cysteamine and the like. Preferred examples of the oxidizing agent include aqueous hydrogen peroxide, ammonium persulfate, sodium bromate, and percarbonate.

Antiseptic and antibacterial agents include hydroxybenzoic acid and its salts or esters such as methylparaben, ethylparaben, propylparaben, butylparaben; salicylic acid; sodium benzoate; phenoxyethanol; 1,2-pentanediol, 1,2-hexane 1,2-diols such as diols; isothiazolinone derivatives such as methylchloroisothiazolinone and methylisothiazolinone; imidazolinium urea; dehydroacetic acid and its salts; phenols; halogenated bisphenols such as triclosan, acid amides , Quaternary ammonium salts; trichlorocarbanide, zinc pyrithione, benzalkonium chloride, benzethonium chloride, sorbic acid, chlorhexidine, chlorhexidine gluconate, halocarban, hexachlorophene, Nokichioru; phenol, isopropyl phenol, cresol, thymol, p-chlorophenol, phenylphenol, other phenols such as phenylphenol sodium; phenylethyl alcohol, photosensitive Motorui, be mentioned as antibacterial zeolite, silver ion.

Examples of chelating agents include edetate (ethylenediaminetetraacetate) such as EDTA, EDTA2Na, EDTA3Na, and EDTA4Na; hydroxyethylethylenediaminetriacetate such as HEDTA3Na; pentetate (diethylenetriaminepentaacetate); phytic acid; Phosphonic acid and its sodium salt; sodium oxalate; polyamino acids such as polyaspartic acid and polyglutamic acid; sodium polyphosphate, sodium metaphosphate, phosphoric acid; sodium citrate, citric acid, alanine, dihydroxyethylglycine Gluconic acid, ascorbic acid, succinic acid, and tartaric acid are preferable.

pH adjusters / acids / alkalis include citric acid, sodium citrate, lactic acid, sodium lactate, glycolic acid, succinic acid, acetic acid, sodium acetate, malic acid, tartaric acid, fumaric acid, phosphoric acid, hydrochloric acid, sulfuric acid, monoethanol Amine, diethanolamine, triethanolamine, isopropanolamine, triisopropanolamine, 2-amino-2-methyl-1,3-propanediol, 2-amino-2-hydroxymethyl-1,3-propanediol, arginine, hydroxylated Sodium, potassium hydroxide, aqueous ammonia, guanidine carbonate, and ammonium carbonate are preferable.

Examples of powders include mica, talc, kaolin, sericite, montmorillonite, kaolinite, mica, muscovite, phlogopite, synthetic mica, red mica, biotite, permiculite, magnesium carbonate, calcium carbonate, aluminum silicate, silica Barium acid, calcium silicate, magnesium silicate, strontium silicate, metal tungstate, magnesium, zeolite, barium sulfate, calcined calcium sulfate, calcium phosphate, fluorine apatite, hydroxyapatite, ceramic powder, bentonite, smectite, clay, mud, Metal soap (for example, zinc myristate, calcium palmitate, aluminum stearate), calcium carbonate, bengara, yellow iron oxide, black iron oxide, ultramarine, bitumen, carbon black, titanium oxide, fine particles and super Titanium oxide, zinc oxide, fine and ultrafine zinc oxide, alumina, silica, fumed silica (ultrafine silica), mica titanium, fish scale foil, boron nitride, photochromic pigment, synthetic fluorophlogopite, fine particle composite powder Body, gold, aluminum, etc., various sizes and shapes of inorganic powders, and these are treated with various surface treatment agents such as hydrogen silicone, cyclic hydrogen silicone, or other silane or titanium coupling agents Inorganic powders such as hydrophobized or hydrophilized powders; starch, cellulose, nylon powder, polyethylene powder, polymethyl methacrylate powder, polystyrene powder, copolymer resin powder of styrene and acrylic acid, polyester powder, Benzoguanamine resin powder, polyethylene terephthalate, polymethy Organic powders and surface-treated powders of various sizes and shapes, such as polyurethane powder, polyethylene terephthalate / aluminum / epoxy powder, urethane powder, silicone powder, Teflon (registered trademark) powder, organic-inorganic composite powder The body is preferred. Inorganic salts include sodium chloride-containing salts such as salt, common salt, rock salt, sea salt, natural salt; potassium chloride, aluminum chloride, calcium chloride, magnesium chloride, bittern, zinc chloride, ammonium chloride; sodium sulfate, aluminum sulfate, Aluminum sulfate / potassium sulfate (alum), aluminum sulfate / ammonium sulfate, barium sulfate, calcium sulfate, potassium sulfate, magnesium sulfate, zinc sulfate, iron sulfate, copper sulfate; sodium phosphates such as 1Na, 2Na and 3Na phosphates, phosphoric acid Potassium, calcium phosphates and magnesium phosphates are preferred.

Vitamins and their derivatives include vitamin A such as retinol, retinol acetate, retinol palmitate; thiamine hydrochloride, thiamine sulfate, riboflavin, riboflavin acetate, pyridoxine hydrochloride, pyridoxine dioctanoate, pyridoxine dipalmitate, Flavin adenine dinucleotide, cyanocobalamin, folic acid, nicotinic acid such as nicotinic acid amide / benzyl nicotinate, vitamin B group such as choline; vitamin C such as ascorbic acid and its sodium salt; vitamin D; α, Vitamin E such as β, γ, and δ-tocopherol; other vitamins such as pantothenic acid and biotin; ascorbic acid phosphates such as ascorbic acid phosphate sodium salt and ascorbic acid phosphate magnesium salt, Ascorbic acid fatty acid esters such as ascorbyl tetraisopalmitate, ascorbyl stearate, ascorbyl palmitate and ascorbyl dipalmitate, ascorbic acid alkyl ethers such as ascorbic acid ethyl ether, ascorbic acid glucosides such as ascorbic acid-2-glucoside and Ascorbic acid derivatives such as fatty acid esters and tocopheryl phosphate ascorbyl; vitamin derivatives such as tocopherol nicotinate, tocopherol acetate, tocopherol acetate, tocopherol linoleate, tocopherol phosphate, tocopherol phosphate etc., tocotrienol, and other various vitamin derivatives Is preferable.

Anti-inflammatory and anti-inflammatory agents include glycyrrhizic acid and its derivatives, glycyrrhetinic acid derivatives, salicylic acid derivatives, hinokitiol, guaiazulene, allantoin, indomethacin, zinc oxide, hydrocortisone acetate, prednisone, diphedramine hydrochloride, chlorpheniramine maleate; peach leaf extract A plant extract such as a koji leaf extract is preferable.

Preferred examples of hormones include estradiol, estrone, ethinyl estradiol, cortisone, hydrocortisone, prednisone and the like.

As plant / animal / microbe extracts, Iris extract, Ashitaba extract, Asunaro extract, Asparagus extract, Avocado extract, Achacha extract, Almond extract, Altea extract, Arnica extract, Aloe extract, Apricot extract, Ginkgo biloba extract, Ginkgo biloba extract , Incheon extract, Fennel extract, Turmeric extract, Oolong tea extract, Peony extract, Ages extract, Echinacea leaf extract, Enmeo extract, Ogon extract, Oat extract, Auren extract, Barley extract, Panax ginseng extract, Hypericum extract, Odori extract , Dutch mustard extract, orange extract, dried seawater, seaweed extract, oyster leaf extract, oyster extract, hydrolyzed elastin, hydrolyzed wheat powder, water content Silk, Cascon extract, Chamomile extract, Oil-soluble chamomile extract, Carrot extract, Kawara mugi extract, Oat extract, Calcade extract, Licorice extract, Oil-soluble Licorice extract, Kiwi extract, Kiou extract, Cyprid extract, Quina extract, Cucumber extract, Kiri leaf extract, Guanosine, guava extract, cucumber extract, gardenia extract, kumazasa extract, clara extract, walnut extract, chestnut extract, grapefruit extract, clematis extract, black rice extract, brown sugar extract, black vinegar, chlorella extract, mulberry extract, gentian extract, Gentian pepper extract, tea Extract, yeast extract, kokuboku extract, coffee extract, burdock extract, rice extract, rice fermented extract, rice bran fermented extract, rice germ oil, comfrey extract Collagen, Cowberry extract, Saishin extract, Psycho extract, Saitai extract, Saffron extract, Salvia extract, Salmon extract, Sasa extract, Hawthorn extract, Sansha extract, Salamander extract, Shiitake extract, Giant extract, Shikon extract, Perilla extract, Linden extract, Shimosukeso Extract, jatoba extract, peonies extract, ginger cucumber extract, ginger root extract, white birch extract, white jellyfish extract, horsetail extract, stevia extract, stevia fermented product, west river willow extract, horse chestnut extract, hawthorn extract, elderberry extract, yarrow extract , Mint extract, sage extract, mallow extract, nematode extract, assembly extract, Sakuhakuhi extract, Diou extract, da Izu extract, Tissou extract, thyme extract, dandelion extract, lichen extract, tea extract, clove extract, chigaya extract, chimpi extract, tea tree oil, tea tea extract, red pepper extract, toukiki extract, toukisenka extract, tonin extract, spruce extract, dokudami extract, tomato Extract, natto extract, carrot extract, garlic extract, wild rose extract, hibiscus extract, bacmond extract, lotus extract, parsley extract, birch extract, honey, hamamelis extract, parietalia extract, hyacinth extract, bisabolol, cypress extract, bifidobacteria extract, Biwa extract, dandelion extract, burdock extract, bukkuri extract, butcher bloom extract, grape extract, grape seed extract, propolis, loofah extract Safflower extract, peppermint extract, body extract, button extract, hop extract, squid extract, pine extract, maroonnier extract, citrus extract, sorghum extract, melissa extract, mozuku extract, peach extract, cornflower extract, eucalyptus extract, yukinoshita extract, yuzu extract, lily extract , Yokuinin extract, Artemisia extract, Lavender extract, Green tea extract, Egg shell membrane extract, Apple extract, Rooibos tea extract, Ganoderma extract, Lettuce extract, Lemon extract, Forsythia extract, Forsythia extract, Rose extract, Rosemary extract, Roman chamomile extract, Extracts such as royal jelly extract and bitumen extract are preferred.

As pigments / colorants / dyes / pigments, brown 201, black 401, purple 201, purple 401, blue 1, blue 2, blue 201, blue 202, blue 203, blue 204 , Blue 205, Blue 403, Blue 404, Green 201, Green 202, Green 204, Green 205, Green 3, Green 401, Green 402, Red 102, Red 104-1 Red 105-1, Red 106, Red 2, Red 201, Red 202, Red 203, Red 204, Red 205, Red 206, Red 207, Red 208, Red 213 Red 214, Red 215, Red 218, Red 219, Red 220, Red 221, Red 223, Red 225, Red 226, Red 227, Red 228, Red 23 No.-1, Red 230-2, Red 231, Red 232, Red 3, Red 401, Red 404, Red 405, Red 501, Red 502, Red 503, Red 504, Red 505, Red 506, Orange 201, Orange 203, Orange 204, Orange 205, Orange 206, Orange 207, Orange 401, Orange 402, Orange 403, Yellow 201, Yellow 202 -1, Yellow 202-2, Yellow 203, Yellow 204, Yellow 205, Yellow 4, Yellow 401, Yellow 402, Yellow 403-1, Yellow 404, Yellow 405, Yellow 406 No., yellow 407, legal dyes such as yellow No. 5; other acidic dyes such as Acid Red 14; Aryanor Sienna Brown, Arianor Madder Red, Ar Base dyes such as anorSteel Blue and Arianor Straw Yellow; Nitro dyes such as HC Blue 2 and Basic Blue 26; disperse dyes; inorganic white pigments such as titanium dioxide and zinc oxide; inorganic red pigments such as iron oxide (Bengara) and iron titanate; inorganics such as γ-iron oxide Brown pigments; inorganic yellow pigments such as yellow iron oxide and ocher; inorganic black pigments such as black iron oxide and low-order titanium oxide; inorganic purple pigments such as mango violet and cobalt violet; chromium oxide, chromium hydroxide, Chita Inorganic green pigments such as cobaltate; Inorganic blue pigments such as ultramarine and bitumen; Titanium oxide coated mica, titanium oxide coated bismuth oxychloride, titanium oxide coated talc, colored titanium oxide coated mica, bismuth oxychloride, fish scale foil, etc. Pearl pigments; metal powder pigments such as aluminum powder, copper powder, gold; surface-treated inorganic and metal powder pigments; red 201, red 202, red 204, red 205, red 220, red 226, red 228, Red 405, Orange 203, Orange 204, Yellow 205, Yellow 401, Blue 404, Red 3, Red 104, Red 106, Red 227, Red 230, Red 401 , Red 505, Orange 205, Yellow 4, Yellow 5, Yellow 202, Yellow 203, Green 3 Organic pigments such as zirconium, barium or aluminum lakes such as Blue No. 1; surface-treated organic pigments; anthraquinones such as astaxanthin and alizarin, anthocyanidins, β-carotene, catenal, capsanthin, chalcone, calsamine, quercetin, crocin, chlorophyll, Natural pigments and dyes such as naphthoquinones such as curcumin, cochineal, shikonin, bixin, flavones, betacyanidine, henna, hemoglobin, lycopene, riboflavin, rutin; p-phenylenediamine, toluene-2,5-diamine, o-, Oxidative dye intermediates and couplers such as m-, or p-aminophenol, m-phenylenediamine, 5-amino-2-methylphenol, resorcin, 1-naphthol, 2,6-diaminopyridine and their salts An auto-oxidizing dye such as indoline; and dihydroxyacetone are preferred.

In addition to these, ingredients described in the Japanese Pharmacopoeia, Pharmaceutical Additive Standards, Food Additives Official Standards, etc., and Japanese and foreign patent publications and patent publications whose international patent classification IPC belongs to the classification of A61K7 and A61K8 It is possible to contain known pharmaceutical ingredients, food ingredients, etc., such as ingredients described in the official gazette (including published and republished publications) in known combinations, blending ratios and blending amounts.

In the present specification, “administering” means administration of the above-mentioned composition at a dose effective for treatment. “Amount effective for treatment” means a dose that produces an effect of suppressing the aging of cells, and is preferably an amount that increases the vitality of mammalian cells and provides a cosmetic or health improvement effect. . The exact dosage will vary depending on the purpose of the treatment and can be ascertained by one skilled in the art using known techniques. Those skilled in the art need to make adjustments for systemic versus local delivery, age, weight, general health, sex, diet, time of administration, drug interaction, and severity of symptoms. This can be confirmed by routine experimentation.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Examples of production of exosomes, miRNA, and pig placenta extract used in the present invention are described in detail below, but the exosomes and pig placenta extract of the present invention are not limited to the following production examples.

(Production Example 1) Pig placenta extract 1 kg of frozen porcine placenta was added with 8 kg of water at 50 ° C, protease M (manufactured by Amano Enzyme) was added and stirred for 2 hours, and then heated at 90 ° C for 1 hour. Protease was inactivated. After cooling to 30 ° C., it was filtered to obtain 2 kg of porcine placenta extract.

(Production Example 2) Exosomes 2 kg of the pig placenta extract obtained in Production Example 1 was dried to obtain 400 g of a powdery pig placenta extract. Among them, 1 g of the extract was dissolved in 64 mL of water and filtered through a 0.22 μm filter. The filtrate was centrifuged at 2000 × g for 20 minutes, and the supernatant was collected. The supernatant was further centrifuged at 110,000 × g for 70 minutes to recover 46 μg of exosomes. Furthermore, 10 μg of exosome was diluted 100-fold with phosphate buffer, and the number of exosomes was confirmed using a nanoparticle / microparticle multianalyzer (qNano, manufactured by Meiwa Forsys). As a result, it was 1.07 × 10 ¹² cells / mL. It was.

(Test 1) Confirmation of exosome 1 g of placenta extract was dissolved in 1 mL of water and then filtered through a 0.22 μm filter to obtain a test sample. At room temperature, a 10-fold diluted sample sample was dropped onto a copper grid mesh with a carbon indicator film, and the sample was dispersed by allowing to stand for 10 seconds. A copper grid mesh with a carbon indicator film was moved onto the distilled water droplets, and was further allowed to stand at room temperature for 30 seconds for washing treatment. Negative staining was performed by moving a copper grid mesh with a carbon indicator film onto a 2% uranyl acetate aqueous solution droplet and allowing it to stand at room temperature for 10 seconds. The grid was dried and then photographed with a transmission electron microscope HITACHI H-7600 at an acceleration voltage of 100 kV.

(Test results)
As shown in FIG. 1, spherical compositions having a particle size of about 40 nm and about 300 nm were observed.

(Test 2) Confirmation of particle size of exosome 10 mg of placenta extract was dissolved in 1 mL of water and then filtered through a 0.22 μm filter to obtain a test sample. The test sample was further diluted 100 times, and the particle size was measured with a dynamic light scattering photometer (ZETASIZER NANO-ZS, manufactured by Malvern).

(Test results)
As shown in FIG. 2, it was confirmed that the particle sizes of 41.14 nm and 268.2 nm were large.

(Test 3) Confirmation of miRNA in exosome Using the exosome of Production Example 2, the encapsulated miRNA was extracted with exoRNeasy (Qiagen). The sequence of the miRNA was analyzed with a next-generation sequencer (Ion PGM sequencer, manufactured by Thermo Fisher Scientific).

(Test results)
As a result of collating the obtained sequence data with the human miRNA database, it was confirmed that the miRNAs shown in Tables 1 to 3 were included.

(Test 4) miRNA decreased by cellular senescence
The cells used were TIG-3 (human fibroblasts) (purchased from Geriatric Research Bank). MiRNA was extracted from TIG-3 at the 39th passage (hereinafter described as 39PDL) as young cells and TIG-3 at the 80th passage (hereinafter described as 80PDL) as senescent cells, using miRNeasy (manufactured by Qiagen). Each miRNA was subjected to comparative analysis using 3D-Gene (manufactured by TORAY) DNA microarray (human miRNA ver12.1).

(Test results)
Of the miRNAs contained in the exosomes shown in Tables 1 to 3, miRNAs that were decreased in senescent cells compared to young cells were shown as decreased * in senescent cells. In 80 PDL senescent cells, 27 types of miRNA were confirmed to be decreased. When cells are aged, these 27 types (SEQ ID NOs: 3, 8, 10, 18, 22, 29, 33, 34, 37, 39, 41, 51, 60, 61, 66, 71, 72, 75, 76, 77, 105, 108, 109, 138, 148, 157, 159) decreases, and the ability to control the expression of genes involved in the function of young cells is thought to decrease.

(Test 5) Evaluation when miRNA was added to fibroblasts TIG-3 of passage 74 was used as the cells. The following synthetic miRNAs (both manufactured by QIAGEN) corresponding to three miRNAs containing the amounts shown in Table 4 among the miRNAs derived from the placenta extract were each diluted to 10 nM, and transferred to cells using lipofectamine RNAmax. Erected.
hsa-miR-125a-5p: 5′-UCCCUGAGACCCUUUAACCUGUGA-3 ′ (SEQ ID NO: 163)
hsa-miR-30c-5p: 5'-UGUAAACAUCCUACACUCUCAGC-3 '(SEQ ID NO: 164)
hsa-miR-26a-5p: 5′-UUCAAGUAAUCCAGGAUAGGCU-3 ′ (SEQ ID NO: 165)

Transfection is performed twice every 24 hours, and after 48 hours after addition, the cells are fixed, and actin and cell nuclei are fluorescently stained using Alexa Fluor 488 Phalloidin (manufactured by Thermo Fisher). The size was analyzed. Both the number of nuclei and the cell size are shown by comparison values with cells transfected with a control synthetic Hsa-miRNA (QIAGEN).

(Test results)
A comparison of cell number and cell size is shown in Table 4. The increase in the number of cells and the decrease in the cell size were confirmed by the addition of hsa-miR-125a-5p, hsa-miR-30c-5p and hsa-miR-26a-5p. Since it is known that when cells age, cell growth stops and the shape of the cells becomes flattened, so these miRNAs are considered to have anti-aging activity.

(Test 6) Evaluation at the time of adding exosome to fibroblasts TIG3 (human-derived fibroblasts) (purchased from Geriatric Research Bank) was used as the cells. TIG3 was seeded in a 96-well plate, cultured in DMEM medium, passaged 66 (hereinafter referred to as 66PDL) and passage 83 (hereinafter described as 83PDL), and 66PDL and 83PDL cells were obtained in Production Example 2 The exosome solution diluted to 5.15 × 10 ³ particles / mL with PBS was added to 66PDL fibroblasts, and the exosome solution diluted to 5.15 × 10 ⁴ particles / mL was added to 83PDL fibroblasts.
Addition is performed twice every 24 hours, cells are fixed 48 hours after addition, and actin / nuclear cells are fluorescently stained using Alexa Fluor488 Phalloidin (Thermo Fisher), and the number and size of the stained cells are determined. Analyzed.

(Test results)
Fig. 3 shows a photograph after fluorescent staining of 66PDL, Fig. 4 shows the number of cells, Fig. 5 shows the cell size, Fig. 6 shows a photo after fluorescent staining of 83PDL, Fig. 7 shows the number of cells, and Fig. 8 shows the cell size. . From FIG. 3 to FIG. 8, it was confirmed that both 66PDL and 83PDL increased the number of cells and decreased the cell size by adding exosomes. It is known that when cells age, cell growth stops and the shape of the cells becomes flattened, so exosomes are considered to have anti-aging activity.

(Prescription example)
Although the example of a formulation of the exosome obtained by this invention and a pig placenta extract is shown below, this invention is not necessarily limited to these.

(Formulation Example 1) Tablets by mass%
1. Placenta extract obtained in Production Example 1 3.0
(Or exosome obtained in Production Example 2 0.003)
2. Lactose 30.0
3. Cornstarch 58.0
4). Crystalline cellulose 7.0
5). Polyvinylpyrrolidone 3.0

(Formulation example 2) Supplement mass%
1. Placenta extract obtained in Production Example 1 10.0
(Or exosome obtained in Production Example 2 0.01)
2. Hesperidin 80.0
3. Starch 10.0
4). Perfume

(Prescription Example 3) Gummy Mass%
1. Reduced water tank 40.0
2. Granulated sugar 20.0
3. Glucose 18.0
4). Placenta extract obtained in Production Example 1 2.0
(Or exosome obtained in Production Example 2 0.002)
5). Water 10.0
6). Gelatin 9.0
7). Citric acid 0.9
8). Dye 0.1
9. Perfume

(Formulation example 4) Chewing gum mass%
1. Gum base 20.0
2. Sugar 15.0
3. Isomaltose 20.0
4). Palatinose 10.0
5). Xylitol 10.0
6). Corn syrup 13.0
7). Placenta extract obtained in Production Example 1 2.0
(Or exosome obtained in Production Example 2 0.002)
8). Minamata 10.0
9. Perfume

(Formulation example 5) Beverage mass%
1. Fructose dextrose liquid sugar 40.0
2. Grapefruit juice 40.0
3. Placenta extract obtained in Production Example 1 2.0
(Or exosome obtained in Production Example 2 0.002)
4). Yoyokan Juice 2.0
5). Sodium ascorbate 1.0
6). Perfume appropriate amount 7. Residue with 100 purified water

(Formulation example 6) Vegetable juice beverage
1. Commercial vegetable juice 99.0
2. Placenta extract obtained in Production Example 1.0
(Or exosome obtained in Production Example 2 0.001)

(Formulation example 7) Tablets by mass%
1. Cornstarch 30.0
2. Carboxymethylcellulose calcium 20.0
3. Microcrystalline cellulose 38.0
4). Placenta extract obtained in Production Example 1 2.0
(Or exosome obtained in Production Example 2 0.002)
5). Polyvinylpyrrolidone 6.0
6). Talc 3.0
7). Sodium ascorbate 1.0
8). Perfume

(Formulation example 8) Confectionery mass%
1. Powdered sugar 88.0
2. Lactose 2.0
3. Citric acid 2.0
4). Gelatin 1% aqueous solution 4.0
5). Placenta extract obtained in Production Example 1.0
(Or exosome obtained in Production Example 2 0.001)
6). Sodium ascorbate 2.0
7). Sucrose fatty acid ester 1.0
8). Perfume

(Formulation Example 9) Milk Mass%
1. Squalane 5.0
2. Olive oil 5.0
3. Jojoba oil 5.0
4). Cetyl alcohol 1.5
5). Glycerol monostearate 2.0
6). Polyoxyethylene (20) cetyl ether 3.0
7). Polyoxyethylene (20) soorbitan monooleate 2.0
8.1,3-Butylene glycol 1.0
9. Glycerin 2.0
10. Placenta extract obtained in Production Example 1.0
(Or exosome obtained in Production Example 2 0.001)
11. Preservative (paraoxybenzoic acid ester) Appropriate amount 12. Fragrance Appropriate amount

(Formulation Example 10) Cosmetic Oil Mass%
1. Liquid paraffin 30.0
2. Squalane 20.0
3. Olive oil 20.0
4). Isopropyl palmitate 10.0
5). Hamasge Whole Hot Water Extract 3.0
6). Placenta extract obtained in Production Example 1.0
(Or exosome obtained in Production Example 2 0.001)
7). Dingji Hot Water Extract 1.0
8). Grapefruit fruit or leaf hot water extract 3.0
9. Tochu Nakaba Hot Water Extract 3.0
10. Olive oil 1.0
11. Shea fat 1.0
12 Butylhydroxyanisole 0.1
13. Vitamin E derivative 0.1
14 Preservative (benzalkonium chloride) Perfume (rose water) Residue with 100 purified water

(Formulation Example 11) Flexible lotion mass%
1. Glycerin 5.0
2. 1,3-butylene glycol 5.0
3. Monolauric acid polyoxyethylene sorbitan (20E.O) 1.0
4). Ethanol 15.0
5). Placenta extract obtained in Production Example 1.0
(Or exosome obtained in Production Example 2 0.001)
6). Bodaiju flower or fruit 50% 1,3-butylene glycol extract 5.0
7). Yuzu fruit 30% ethanol extract 1.0
8). Hakobe Whole 30% Ethanol Extract 1.0
9. Carp stem or leaf 30% ethanol extract 1.0
10. Kiwi fruit 50% ethanol extract 1.0
11. Birch sap 1.0
12 Antibacterial (lactoferrin solution) appropriate amount13. Preservative (paraoxybenzoic acid ester) Perfume (Western light load water) Residue with 100 purified water

(Prescription Example 12) Pack mass%
1. Polyvinyl alcohol 15.0
2. Sodium carboxymethylcellulose 5.0
3. Propylene glycol 3.0
4). Ethanol 10.0
5). Placenta extract obtained in Production Example 1.0
(Or exosome obtained in Production Example 2 0.001)
6). Raspberry fruit 50% diglycerin extract 1.0
7). Dingyo 50% Diglycerin extract 1.0
8). Wenzhou citrus peel 50% 1,3-butylene glycol extract 1.0
9. Green tea leaf hot water extract 1.0
10. Momoha Hot Water Extract 2.0
11. Pig placenta hot water extract 1.0
12 Preservative (paraoxybenzoic acid ester) Perfume (rose water) Residue with 100 purified water

(Prescription Example 13) Essence liquid mass%
1. Polyvinyl alcohol 15.0
2. Xanthan gum 0.4
3. Hydroxyethyl cellulose 0.2
4). Carpoxyvinyl polymer 0.2
5). Potassium hydroxide 0.025
6.1,3-Butylene glycol 5.0
7). Placenta extract obtained in Production Example 1.0
(Or exosome obtained in Production Example 2 0.001)
8). Button rhizome or root bark 60% ethanol extract 1.0
9. Dipotassium glycyrrhizinate 0.2
10. Preservative (methyl paraoxybenzoate) Perfume (rose water) Appropriate amount 12. Residue with 100 purified water

(Prescription Example 14) Facial cleansing cream mass%
1. Myristic acid 25.0
2. Stearic acid 5.0
3. Tallow fatty acid 5.0
4). Propylene glycol 10.0
5). Potassium hydroxide 6.0
6). Palm oil fatty acid diethanolamide 6.0
7). Placenta extract obtained in Production Example 1.0
(Or exosome obtained in Production Example 2 0.001)
8). Apple fruit 20% 1,3-butylene glycol extract 2.0
9. Kawara mugwort leaves 20% 1,3-butylene glycol extract 2.0
10. Rosehip fruit 30% ethanol extract 1.0
11. Kakkon hot water extract 2.0
12 Glycyrrhizic acid 1.0
13. Apricot kernel 0.1
14 Preservative (salicylic acid) 0.1
15. Fragrance (today water) Appropriate amount 16. Residue with 100 purified water

(Prescription Example 15) Body soap mass%
1. Potassium laurate 15.0
2. Potassium myristate 5.0
3. Propylene glycol 5.0
4). Placenta extract obtained in Production Example 1.0
(Or exosome obtained in Production Example 2 0.001)
5). Oolong tea leaves 70% 1,3-butylene glycol extract 1.0
6). Toki rhizome 70% 2,3-butylene glycol extract 0.5
7). Wakame 70% 1,3-butylene glycol extract 0.5
8). Melilot flower or leaf 70% 1,3-butylene glycol extract 0.5
9. Aloe pulp 70% ethanol extract 2.0
10. Monoammonium glycyrrhizinate 0.1
11. Preservative (Undecylenic acid, phenol) Appropriate amount 12. Perfume (lavender water) Residue with 100 purified water

(Formulation Example 16) Sunscreen cosmetics (O / W type)
1. Oxybenzone 2.0
2. Octyl paramethoxycinnamate 5.0
3. Dihydroxymethoxybenzophenone 0.2
4). Squalane 10.0
5). Vaseline 5.0
6). Stearyl alcohol 3.0
7). Stearic acid 3.0
8). Glyceryl monostearate 2.0
9. Polyethyl acrylate 1.0
10.1,3-butylene glycol 6.0
11. Edetate disodium 0.1
12 Triethanolamine 1.0
13. Titanium dioxide 5.0
14 Placenta extract obtained in Production Example 1.0
(Or exosome obtained in Production Example 2 0.001)
15. White birch bark 70% dipropylene glycol extract 0.5
16. Cornflower leaf 50% propylene glycol extract 0.5
17. Kakkon 50% propylene glycol extract 0.5
18. Palm fruit 50% dipropylene glycol extract 0.5
19. Preservative (benzyl paraoxybenzoate) Perfume (jasmine water) Residue with 100 purified water

(Formulation example 17) Sunscreen cosmetics (oil type)
1. Liquid paraffin 70.0
2. Tetrahydroxybenzophenone 1.0
3. Cetyl octanoate 23.8
4). Placenta extract obtained in Production Example 1.0
(Or exosome obtained in Production Example 2 0.001)
5). Roman chamomile flower 10% 1,3-butylene glycol extract 1.0
6). Burdock root hot water extract 0.5
7). Horse chestnut whole plant 30% 1,3-butylene glycol extract 0.5
8). Mallow flower or leaf 50% propyl alcohol extract 0.5
9. Pearl barley 50% 1,3-butylene glycol extract 0.5
10. Aroma fruit 30% butanol extract 0.5
11. Glaze root or leaf hot water extract 0.5
12 Ethyl paraaminobenzoate 0.2
13. Antioxidant (butylhydroxytoluene) Appropriate amount14. Perfume (cornflower water)

(Formulation Example 18) Hair Liquid Mass%
1. Ethanol 29.0
2. Polyoxypropylene butyl ether phosphate 10.0
3. Polyoxypropylene monobutyl ether 5.0
4). Triethanolamine 1.0
5). Trimethylene glycol 5.0
6). Placenta extract obtained in Production Example 1.0
(Or exosome obtained in Production Example 2 0.001)
7). Assembly leaf or stem 50% 1,2-butylene glycol extract 1.0
8). Panax ginseng root hot water extract 1.0
9. Lemon fruit 50% 1,3-butylene glycol extract 0.5
10. Apple fruit 30% ethanol extract 1.0
11. Birch sap 1.0
12 Azulene 1.0
13. Antibacterial / preservative (paraben, benzalkonium chloride) Fragrance (orange water) Residue with 100 purified water

(Test 7) Evaluation of Macrophage Migration Ability by Transwell Migration Assay The action of the exosome prepared in Production Example 2 was examined by a transwell migration assay using RAW264.7 mouse macrophage-like cells. 750 μL of serum-containing medium was added to each well of the 24-well plate, and the insert was placed. RAW264.7 cells were suspended in a serum-free medium at 2 × 10 ⁶ cells / ml, and 100 μL each was seeded on the insert of each well. Each sample was added to the cells and cultured at 37 ° C. in the presence of 5% CO ₂ . After 24 hours, each sample was added again and further cultured at 37 ° C. in the presence of 5% CO ₂ . 48 hours after the first sample was added, the cells were stained with Giemsa, and the number of cells that migrated under the insert was measured and analyzed. For analysis of the number of cells, photographs of 5 fields were taken for each insert, and the total number was taken as the number of migrating cells. Samples were duplicated (n = 2).

The result is shown in FIG. The vertical axis in FIG. 9 indicates the number of migrated cells, and the horizontal axis indicates each sample used. “Untreated” means a sample using a phosphate buffer (PBS). “Reagent only” means a reagent of Total exosome isolation kit manufactured by Invitrogen Corporation used for purification of exosomes. “No treatment” indicates that the exosome solution obtained in Production Example 2 was used as it was. The “filter treatment” is obtained by treating the exosome solution obtained in Production Example 2 with a 0.22 μm filter. “Exosome removal” means the supernatant during purification of the kit, and is a fraction that does not contain exosomes. And “exosome” means a precipitate fraction during purification of a kit containing exosomes.

As shown in FIG. 9, when a sample containing exosomes was used with respect to control samples such as “untreated”, “reagent only”, and “exosome removal”, a significantly higher number of cell migration was detected, It was found that the exosome of the present invention has an activating effect on immune cells such as macrophages.

(Test 8) Wound healing assay
Exosomal macrophage migration ability prepared in Production Example 2 was examined by wound healing assay. In each well of the 6-well plate, RAW264.7 mouse macrophage-like cells were seeded so as to be confluent on the next day. After culturing at 37 ° C. in the presence of 5% CO ₂ for 24 hours, a 100% confluent cell surface is scratched with a yellow tip, washed with PBS, and then a new culture solution containing each sample. Was replaced. The cells were cultured in an environment of 37 ° C. and 5% CO ₂ , and 0 and 24 hours later photographs were taken to measure and analyze the width of the scratch portion. The width of the scratched scratch was measured from the photograph taken, and the ratio of how much was repaired in 24 hours was calculated. Based on the following formula, an average value and standard deviation in four experiments were calculated. Repair rate (%) = 100 − [(scratch width after 24 hours; μm) / (0 hour wound width; μm) × 100]

As a result, compared to the fraction containing no exosome as a sample, the repair rate was significantly higher when the purified fraction containing exosome was used, and the exosome of the present invention activated macrophage migration to recover the wound site. It was suggested to promote. From the above results, the exosome according to the present invention is also expected to be used as a macrophage migration promoter, immune cell activator or wound healing promoter.

According to the present invention, it has become possible to provide pig placenta-derived exosomes, miRNAs, and porcine placenta extracts having a high exosome content with high physiological activity. *

Claims (10)

1. (I) a nucleic acid molecule having the base sequence represented by any one of SEQ ID NOs: 1 to 162,
   (Ii) a nucleic acid molecule having 90% or more identity with the base sequence of (i), or (iii) deletion or addition and / or substitution of one or two bases in the base sequence of (i) A nucleic acid molecule having a base sequence,
   An exosome comprising at least one nucleic acid molecule selected from the group consisting of and an extract from a mammalian placenta. However, when the nucleic acid molecule is RNA, T shown in each base sequence is U.
2. Said at least one nucleic acid molecule is SEQ ID NO: 3, 8, 10, 18, 22, 29, 33, 34, 37, 39, 41, 51, 60, 61, 66, 71, 72, 75, 76, 77. 105, 108, 109, 138, 148, 157 and 159, The nucleic acid molecule of the base sequence shown by 159 is contained. However, the base sequence of each nucleic acid molecule has 90% or more identity with the base sequence, or has a deletion, addition and / or substitution of one or two bases in the base sequence. Also good.
3. The exosome according to claim 1, wherein the at least one nucleic acid molecule comprises a nucleic acid molecule having a base sequence represented by SEQ ID NOs: 16, 74 and 87. However, the base sequence of each nucleic acid molecule has 90% or more identity with the base sequence, or has a deletion, addition and / or substitution of one or two bases in the base sequence. Also good.
4. An oral composition containing the exosome according to any one of claims 1 to 3, or mir-125a, mir-26a and / or mir-30c miRNA as an active ingredient.
5. A cosmetic composition comprising the exosome according to any one of claims 1 to 3 or mir-125a, mir-26a and / or mir-30c miRNA as an active ingredient.
6. A fibroblast proliferation promoter comprising the exosome according to any one of claims 1 to 3 or mir-125a, mir-26a and / or mir-30 miRNA as an active ingredient.
7. A method for producing a placenta extract, comprising extracting mammalian placenta with hot water, performing enzymatic degradation with a protease, and collecting a placenta extract containing exosomes having a spherical structure.
8. The method for producing a placenta extract according to claim 7, further comprising a step of extracting the placenta extract with sterile water.
9. The method for producing a placenta extract according to claim 7 or 8, wherein the mammalian placenta is a pig placenta.
10. An exosome according to any one of claims 1 to 3, or a miRNA of mir-125a, mir-26a and / or mir-30c, is administered to a mammalian cell, thereby suppressing cell senescence Method.
    
    

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