JP2022018719A - Agent for promoting differentiation of epidermal stem cell into epidermal keratinocyte - Google Patents
Agent for promoting differentiation of epidermal stem cell into epidermal keratinocyte Download PDFInfo
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- JP2022018719A JP2022018719A JP2020122039A JP2020122039A JP2022018719A JP 2022018719 A JP2022018719 A JP 2022018719A JP 2020122039 A JP2020122039 A JP 2020122039A JP 2020122039 A JP2020122039 A JP 2020122039A JP 2022018719 A JP2022018719 A JP 2022018719A
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- epidermal
- stem cells
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- epidermal stem
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Abstract
Description
本発明は、バラ科に属するニワウメ（Ｐｒｕｎｕｓ ｊａｐｏｎｉｃａ）の抽出物を含有することを特徴とする表皮幹細胞の表皮角化細胞への分化促進剤に関する。 The present invention relates to an agent for promoting the differentiation of epidermal stem cells into epidermal keratinized cells, which comprises an extract of Prunus japonica belonging to the family Rosaceae.
脊椎動物、特に哺乳動物の組織は、傷害もしくは疾患、又は加齢などに伴い細胞・臓器の損傷が起こった場合、再生系が働き、細胞・臓器の損傷を回復しようとする。この作用に、当該組織に備わる幹細胞が大きな役割を果たしている。幹細胞は、あらゆる細胞・臓器に分化する多能性を有しており、この性質により細胞・臓器の損傷部を補うことで回復に導くと考えられている。このような幹細胞を、生体外において培養し、目的の細胞に分化誘導させ、再生したい臓器や組織に類似した３次元的な構築を行うといった、次世代の技術である再生医療や再生美容に期待が集まっている。 In vertebrates, especially mammalian tissues, when cell / organ damage occurs due to injury or disease, or aging, the regenerative system works to recover the cell / organ damage. Stem cells in the tissue play a major role in this action. Stem cells have pluripotency to differentiate into all cells and organs, and it is thought that this property leads to recovery by compensating for damaged parts of cells and organs. Expectations for next-generation technologies such as regenerative medicine and regenerative beauty, such as culturing such stem cells in vitro, inducing differentiation into target cells, and performing three-dimensional construction similar to the organ or tissue to be regenerated. Are gathered.
哺乳動物の幹細胞研究において、近年、骨髄、肝臓、膵臓、脂肪、皮膚など、あらゆる臓器・組織に幹細胞が存在することが明らかにされ、各臓器・組織の再生および恒常性維持を司っていることがわかってきた（非特許文献１～４）。皮膚内における幹細胞は、表皮及び真皮に存在していると考えられており（非特許文献５，６）、それらは多分化能を示し、皮膚の細胞にも分化可能であると考えられる（非特許文献７）。 In recent years, research on stem cells in mammals has revealed that stem cells are present in all organs and tissues such as bone marrow, liver, pancreas, fat, and skin, and are responsible for the regeneration and maintenance of homeostasis of each organ and tissue. It has become clear (Non-Patent Documents 1 to 4). Stem cells in the skin are considered to be present in the epidermis and dermis (Non-Patent Documents 5 and 6), and they are considered to be pluripotent and capable of differentiating into skin cells (non-patent documents 5 and 6). Patent Document 7).
皮膚は、外部からの様々な環境因子（紫外線や乾燥等）に対するバリア機能において、重要な役割を果たしている。特に、表皮角化細胞は、皮膚の中でも最表面の層である表皮の大部分を占めており、それらの環境因子から体を守る重要な細胞である。表皮角化細胞は、表皮の最下層の基底層で分裂し、上層に向かって移動するとともに、分裂能を失って分化していき、有棘層、顆粒層を経て、最上層の角質層に到達して角質細胞に分化する。角質層は表皮角化細胞の最終分化産物であり、やがて皮膚より剥がれ落ちる。この表皮角化細胞の増殖と分化の過程はターンオーバーと呼ばれており、表皮幹細胞は、この表皮のターンオーバーに重要な役割を果たしていると考えられている（非特許文献８）。よって、ターンオーバーの正常化と、より強固な表皮バリア機能を発揮するためには、表皮幹細胞を最外層の角質層へと分化を導くことが重要である。 The skin plays an important role in the barrier function against various environmental factors (ultraviolet rays, dryness, etc.) from the outside. In particular, epidermal keratinocytes occupy most of the epidermis, which is the outermost layer of the skin, and are important cells that protect the body from these environmental factors. Epidermal keratinocytes divide in the basal layer, which is the lowest layer of the epidermis, move toward the upper layer, lose their ability to divide, and differentiate, passing through the stratum spinosum and the stratum granulosum to the uppermost stratum corneum. It reaches and differentiates into keratinocytes. The stratum corneum is the final differentiation product of epidermal keratinocytes and eventually peels off the skin. The process of proliferation and differentiation of epidermal keratinocytes is called turnover, and epidermal stem cells are considered to play an important role in this epidermal turnover (Non-Patent Document 8). Therefore, in order to normalize the turnover and exert a stronger epidermal barrier function, it is important to induce the differentiation of epidermal stem cells into the outermost stratum corneum.
ニワウメ（学名：Ｐｒｕｎｕｓ ｊａｐｏｎｉｃａ）は、バラ目バラ科スモモ属に属する落葉低木であり、生薬の郁李仁（イクリニン）はニワウメの種子を乾燥させたもので、緩下、利尿に効用があるとして用いられている。その他のニワウメの薬効としては自然保湿因子（ＮＭＦ）成分産生促進効果（特許文献１）及びペルオキシソーム増殖剤応答性受容体活性化効果（特許文献２）を有すること等が知られている。 Prunus japonica (scientific name: Prunus japonica) is a deciduous shrub belonging to the genus Plum of the Rosales family. ing. Other medicinal effects of Prunus japonica are known to have a natural moisturizing factor (NMF) component production promoting effect (Patent Document 1) and a peroxisome proliferating agent-responsive receptor activating effect (Patent Document 2).
安全で安定性に優れ、表皮幹細胞の表皮角化細胞への分化促進効果に優れた素材が望まれているが、未だ十分満足し得るものが提供されていないのが現状である。 There is a demand for a material that is safe, has excellent stability, and has an excellent effect of promoting the differentiation of epidermal stem cells into epidermal keratinocytes, but at present, a material that is sufficiently satisfactory has not been provided.
このような事情により、本発明者らは鋭意検討した結果、ニワウメの抽出物が優れた表皮幹細胞の表皮角化細胞への分化促進作用を有していることを見出した。さらに、その抽出物を含有する外用剤が、安全で安定であり、シミやくすみを改善する効果を有することを見出し、本発明を完成するに至った。 Under these circumstances, the present inventors have diligently studied and found that the extract of Prunus japonica has an excellent effect of promoting the differentiation of epidermal stem cells into epidermal keratinized cells. Furthermore, they have found that the external preparation containing the extract is safe and stable, and has an effect of improving spots and dullness, and have completed the present invention.
即ち、本発明は以下の発明を包含する。
（１）ニワウメの抽出物を有効成分として含有する、表皮幹細胞の表皮角化細胞への分化促進剤。
（２）（１）に記載の剤を含有する、表皮幹細胞の分化機能低下又は不全により、正常に表皮角化細胞が形成されないことに起因する疾患又は病態の治療、改善及び予防用皮膚外用剤。
（３）（１）に記載の剤を含有する、表皮幹細胞の分化機能低下又は不全により、正常に表皮角化細胞が形成されないことに起因する疾患又は病態の治療、改善及び予防用医薬品。
（４）（１）に記載の剤を含有する、表皮幹細胞の分化機能低下又は不全により、正常に表皮角化細胞が形成されないことに起因する疾患又は病態の治療、改善及び予防用食品。
（５）表皮幹細胞を、ニワウメの抽出物を含有する培地で培養する工程を含む、表皮幹細胞の表皮角化細胞への分化促進方法。
（６）表皮幹細胞を、ニワウメの抽出物を含有する培地で培養する工程を含む、表皮角化細胞の製造方法。
That is, the present invention includes the following inventions.
(1) An agent for promoting the differentiation of epidermal stem cells into epidermal keratinized cells, which contains an extract of Prunus japonica as an active ingredient.
(2) A skin external preparation for treating, improving and preventing a disease or pathological condition caused by the normal formation of epidermal keratinocytes due to a decrease or insufficiency of the differentiation function of epidermal stem cells containing the agent according to (1). ..
(3) A drug for treating, improving and preventing a disease or pathological condition caused by the normal formation of epidermal keratinocytes due to a decrease or insufficiency of the differentiation function of epidermal stem cells, which comprises the agent according to (1).
(4) A food for treating, improving and preventing a disease or pathological condition caused by the normal formation of epidermal keratinocytes due to a decrease or insufficiency of the differentiation function of epidermal stem cells, which comprises the agent according to (1).
(5) A method for promoting the differentiation of epidermal stem cells into epidermal keratinized cells, which comprises a step of culturing epidermal stem cells in a medium containing an extract of chicken horse.
(6) A method for producing epidermal keratinized cells, which comprises a step of culturing epidermal stem cells in a medium containing an extract of Prunus japonica.
本発明によれば、ニワウメの抽出物を有効成分として含有する表皮幹細胞の表皮角化細胞への分化促進剤が提供される。従って、本発明は、表皮幹細胞の分化機能低下又は不全により、正常に表皮角化細胞が形成されないことに起因する疾患又は病態の治療、改善及び予防に、有効である。 According to the present invention, there is provided an agent for promoting the differentiation of epidermal stem cells into epidermal keratinized cells, which contains an extract of Prunus japonica as an active ingredient. Therefore, the present invention is effective for the treatment, improvement and prevention of diseases or pathological conditions caused by the normal formation of epidermal keratinocytes due to the deterioration or deficiency of the differentiation function of epidermal stem cells.
本発明に用いるニワウメ（学名：Ｐｒｕｎｕｓ ｊａｐｏｎｉｃａ、別名：コニワザクラ、チョウコウイクリ）は、バラ目バラ科スモモ属に属する落葉低木であり、中国北部原産で、日本各地で観賞用に栽培されている。 Prunus japonica (scientific name: Prunus japonica, also known as Koniwazakura, Chokouikuri) used in the present invention is a deciduous shrub belonging to the genus Plum of the Rosales family, native to northern China, and cultivated for ornamental purposes in various parts of Japan.
本発明において、ニワウメの抽出物は、その花、果実、種子、幹、樹皮、根等の植物体の一部又は植物体全体、あるいはそれらの混合物の抽出物をいうが、本発明において抽出原料として使用する部位は、種子が好ましい。又、抽出には、植物体を生のまま使用しても良く、前処理として、乾燥、粉砕、細切等の処理を行っても良い。 In the present invention, the extract of Prunus japonica refers to an extract of a part of a plant such as flowers, fruits, seeds, stems, bark, roots, etc., the whole plant, or a mixture thereof. Seeds are preferred as the site to be used as. In addition, the plant may be used as it is for extraction, or may be dried, crushed, shredded or the like as a pretreatment.
抽出溶媒としては、例えば、水、低級アルコール類（メタノール、エタノール、１－プロパノール、２－プロパノール、１－ブタノール、２－ブタノール等）、液状多価アルコール類（１，３－ブチレングリコール、プロピレングリコール、グリセリン等）、ケトン類（アセトン、メチルエチルケトン等）、アセトニトリル、エステル類（酢酸エチル、酢酸ブチル等）、炭化水素類（ヘキサン、ヘプタン、流動パラフィン等）、エーテル類（エチルエーテル、テトラヒドロフラン、プロピルエーテル等）が挙げられる。好ましくは、水、低級アルコール及び液状多価アルコール等の極性溶媒が良く、特に好ましくは、水、エタノール、１，３－ブチレングリコール及びプロピレングリコールが良い。これらの溶媒は一種でも二種以上を混合して用いても良い。また、これらの溶媒に酸やアルカリを添加して、ｐＨを調整した溶媒を用いることもできる。また、抽出方法は、特に限定されないが、例えば、連続抽出や浸漬抽出が挙げられる。抽出温度は、加熱抽出であっても良いし、常温抽出や冷温抽出であっても良く、抽出物の使用用途や抽出溶媒等によって、適宜選択できる。 Examples of the extraction solvent include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol, etc.). , Glycerin, etc.), Ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.) Etc.). Polar solvents such as water, lower alcohols and liquid polyhydric alcohols are preferable, and water, ethanol, 1,3-butylene glycol and propylene glycol are particularly preferable. These solvents may be used alone or in admixture of two or more. Further, it is also possible to use a solvent whose pH is adjusted by adding an acid or an alkali to these solvents. The extraction method is not particularly limited, and examples thereof include continuous extraction and immersion extraction. The extraction temperature may be heat extraction, normal temperature extraction or cold temperature extraction, and may be appropriately selected depending on the intended use of the extract, the extraction solvent and the like.
本発明に用いる抽出物は、抽出した溶液のまま用いても良いが、必要に応じて、本発明の効果を奏する範囲で、濃縮（減圧濃縮、膜濃縮等による濃縮）、希釈、濾過、活性炭等による脱色や脱臭等の処理を行ってから用いても良い。更には、抽出した溶液を濃縮乾固、噴霧乾燥、凍結乾燥等の処理を行い、乾燥物として用いても良い。 The extract used in the present invention may be used as it is in the extracted solution, but if necessary, concentration (concentration by vacuum concentration, membrane concentration, etc.), dilution, filtration, activated carbon is performed within the range in which the effect of the present invention is exhibited. It may be used after performing treatments such as decolorization and deodorization. Further, the extracted solution may be subjected to treatments such as concentrated drying, spray drying, freeze-drying and the like, and used as a dried product.
このようにして得られたニワウメの抽出物は、生体レベル又は培養レベルで、表皮幹細胞の表皮角化細胞への分化促進剤として使用できる。本発明に係る表皮幹細胞の表皮角化細胞への分化促進剤は、表皮幹細胞を、表皮角化細胞へと効率的に分化させるための表皮幹細胞培養用培地添加剤、研究用試薬としても使用することができる。 The extract of Prunus japonica thus obtained can be used as an agent for promoting the differentiation of epidermal stem cells into epidermal keratinized cells at the biological level or the culture level. The agent for promoting the differentiation of epidermal stem cells into epidermal keratinocytes according to the present invention is also used as a medium additive for culturing epidermal stem cells and a research reagent for efficiently differentiating epidermal stem cells into epidermal keratinocytes. be able to.
本発明に係る表皮幹細胞の表皮角化細胞への分化促進剤を、ヒトを含めた哺乳動物の表皮幹細胞に適用することで、表皮幹細胞の表皮角化細胞への分化を促進することができる。 By applying the agent for promoting the differentiation of epidermal stem cells into epidermal keratinocytes according to the present invention to the epidermal stem cells of mammals including humans, the differentiation of epidermal stem cells into epidermal keratinocytes can be promoted.
本発明に係る表皮幹細胞の表皮角化細胞への分化促進剤は、有効成分であるニワウメの抽出物が優れた表皮幹細胞の表皮角化細胞への分化促進作用を有するので、表皮幹細胞の分化機能低下又は不全により、正常に表皮角化細胞が形成されないことに起因する疾患又は病態を治療、改善及び予防するのに有効である。ここで、その疾患又は病態としては、例えば、シミ、くすみ、熱傷、創傷、瘢痕、ケロイド、角質肥厚、毛穴のひらき、ニキビ痕、肌荒れ、乾燥肌、敏感肌、アトピー性皮膚炎、乾癬などが挙げられるが、これらに限定されない。 The agent for promoting the differentiation of epidermal stem cells into epidermal keratinized cells according to the present invention has an excellent effect of promoting the differentiation of epidermal stem cells into epidermal keratinized cells by an extract of chicken horse, which is an active ingredient. It is effective in treating, ameliorating and preventing diseases or pathological conditions caused by normal formation of epidermal keratinocytes due to deterioration or insufficiency. Here, the disease or pathological condition includes, for example, spots, dullness, burns, wounds, scars, keloids, keratin thickening, open pores, acne scars, rough skin, dry skin, sensitive skin, atopic dermatitis, psoriasis and the like. However, but not limited to these.
本発明はまた、表皮幹細胞を、ニワウメの抽出物を含有する培地で培養することで、表皮幹細胞の表皮角化細胞への分化を促進する方法に関する。換言すれば、本発明に係る方法は、表皮幹細胞を、ニワウメの抽出物を含有する培地で培養する工程を含む、表皮幹細胞の表皮角化細胞への分化促進方法、表皮角化細胞の製造方法ということができる。 The present invention also relates to a method for promoting the differentiation of epidermal stem cells into epidermal keratinized cells by culturing the epidermal stem cells in a medium containing an extract of chicken horse. In other words, the method according to the present invention comprises a step of culturing epidermal stem cells in a medium containing an extract of chicken seaweed, a method for promoting differentiation of epidermal stem cells into epidermal keratinocytes, and a method for producing epidermal keratinocytes. It can be said.
本発明の表皮幹細胞の表皮角化細胞への分化促進剤、皮膚外用剤、医薬品及び食品は、上記抽出物をそのまま使用しても良く、抽出物の効果を損なわない範囲内で、化粧品、医薬部外品、医薬品又は食品等に用いられる成分である油脂類、ロウ類、炭化水素類、脂肪酸類、アルコール類、エステル類、界面活性剤、金属石鹸、ｐＨ調整剤、防腐剤、香料、保湿剤、粉体、紫外線吸収剤、増粘剤、色素、酸化防止剤、美白剤、キレート剤、賦形剤、皮膜剤、甘味料、酸味料等の成分が含有されていても良い。 As the agent for promoting the differentiation of epidermal stem cells into epidermal keratinized cells, the external skin preparation, pharmaceuticals and foods of the present invention, the above extracts may be used as they are, and cosmetics and pharmaceuticals may be used as long as the effects of the extracts are not impaired. Oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH regulators, preservatives, fragrances, moisturizers, which are ingredients used in external products, pharmaceuticals, foods, etc. Ingredients such as agents, powders, ultraviolet absorbers, thickeners, pigments, antioxidants, whitening agents, chelating agents, excipients, film agents, sweeteners and acidulants may be contained.
本発明の表皮幹細胞の表皮角化細胞への分化促進剤、皮膚外用剤、医薬品及び食品の剤形としては、例えば、化粧水、クリーム、乳液、ゲル剤、エアゾール剤、エッセンス、パック、洗浄剤、浴用剤、ファンデーション、打粉、口紅、軟膏、パップ剤、錠菓、チョコレート、ガム、飴、飲料、散剤、顆粒剤、錠剤、糖衣錠剤、カプセル剤、シロップ剤、丸剤、懸濁剤、液剤、乳剤、坐剤、注射剤等が挙げられる。注射用製剤の場合は単位投与量アンプル又は多投与量容器の状態で提供される。 Dosage forms of the epidermal stem cells of the present invention into epidermal keratinized cells, external skin preparations, pharmaceuticals and foods include, for example, cosmetics, creams, emulsions, gels, aerosols, essences, packs and cleaning agents. , Baths, foundations, powders, lipsticks, ointments, poultices, tablets, chocolates, gums, candy, beverages, powders, granules, tablets, sugar-coated tablets, capsules, syrups, rounds, suspensions, liquids , Emulsions, suppositories, injections and the like. In the case of an injectable product, it is provided in the form of a unit dose ampoule or a multi-dose container.
外用の場合、本発明に用いる上記抽出物の含有量は、固形物に換算して０．０００１重量％以上が好ましく、０．００１～１０重量％がより好ましい。さらに、０．０１～５重量％が最も好ましい。０．０００１重量％未満では十分な効果は望みにくい。１０重量％を超えると、効果の増強は認められにくく不経済である。 In the case of external use, the content of the extract used in the present invention is preferably 0.0001% by weight or more, more preferably 0.001 to 10% by weight in terms of solid matter. Further, 0.01 to 5% by weight is most preferable. If it is less than 0.0001% by weight, it is difficult to expect a sufficient effect. If it exceeds 10% by weight, the enhancement of the effect is hardly recognized and it is uneconomical.
内用の場合、摂取量は年齢、体重、症状、治療効果、投与方法、処理時間等により異なる。通常、成人１人当たりの１日の摂取量としては、５ｍｇ以上が好ましく、１０ｍｇ～５ｇがより好ましい。さらに、２０ｍｇ～２ｇが最も好ましい。 For internal use, the intake varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, and the like. Usually, the daily intake per adult is preferably 5 mg or more, more preferably 10 mg to 5 g. Further, 20 mg to 2 g is most preferable.
上記の量はあくまで例示であって、組成物の種類や形態、一般的な使用量、効能・効果などを考慮して適宜設定・調整すればよい。 The above amounts are merely examples, and may be appropriately set and adjusted in consideration of the type and form of the composition, general usage amount, efficacy / effect, and the like.
本発明に係る方法において、表皮幹細胞から分化誘導して製造された表皮角化細胞は、一般的に体外で培養後、創傷部や組織を再生させたい部位に直接注射などで移植することが可能である。表皮幹細胞を培養する培地、また同時に用いる添加剤としては、特に限定はされず、表皮幹細胞の分化のために一般的に使用されている培地及び添加剤を用いればよい。 In the method according to the present invention, epidermal keratinized cells produced by inducing differentiation from epidermal stem cells can generally be cultured in vitro and then transplanted directly into a wound or a site where tissue is to be regenerated by direct injection or the like. Is. The medium for culturing the epidermal stem cells and the additives used at the same time are not particularly limited, and media and additives generally used for the differentiation of epidermal stem cells may be used.
具体的には、表皮幹細胞を培養する培地には、幹細胞の生存及び増殖に必要な成分（無機塩、炭水化物、ホルモン、必須アミノ酸、非必須アミノ酸、ビタミン、脂肪酸）を含む基本培地、例えば、Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（Ｄ－ＭＥＭ）、Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ（ＭＥＭ）、ＲＰＭＩ １６４０、Ｂａｓａｌ Ｍｅｄｉｕｍ Ｅａｇｌｅ（ＢＭＥ）、Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ：Ｎｕｔｒｉｅｎｔ Ｍｉｘｔｕｒｅ Ｆ－１２（Ｄ－ＭＥＭ／Ｆ－１２）、Ｇｌａｓｇｏｗ Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ（Ｇｌａｓｇｏｗ ＭＥＭ）、Ｈａｎｋ’ｓ ｂａｌａｎｃｅｄ ｓａｌｔ ｓｏｌｕｔｉｏｎ（ハンクス液）等が挙げられる。また、上記培地には、細胞の増殖速度を増大させるために、必要に応じて、塩基性線維芽細胞増殖因子（ｂＦＧＦ）、上皮細胞増殖因子（ＥＧＦ）等の増殖因子、腫瘍壊死因子（ＴＮＦ）、ビタミン類、インターロイキン類、インスリン、トランスフェリン、ヘパリン、ヘパラン硫酸、コラーゲン、ウシ血清アルブミン（ＢＳＡ）、フィブロネクチン、プロゲステロン、セレナイト、Ｂ２７－サプリメント、Ｎ２－サプリメント、ＩＴＳ－サプリメント等を添加してもよく、また、抗生物質（ペニシリン、ストレプトマイシン等）等を添加してもよい。さらに、本発明の分化促進剤とは別に、表皮幹細胞から表皮角化細胞への分化誘導因子を添加してもよい。その分化誘導因子としては、例えば、カルシウム塩、リゾフォスファチジン酸（ＬＰＡ）等が挙げられる。培地の各成分は、滅菌された状態で使用する。 Specifically, the medium for culturing epidermal stem cells is a basic medium containing components necessary for the survival and proliferation of stem cells (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids), for example, Dulvecco. 's Modified Eagle Medium (D-MEM), Minimum Essential Medium (MEM), RPMI 1640, Basic Medium Eagle (BME), Dulvecco's Modern EagleM Examples thereof include Glassgo Minimum Essential Medium (Grasgow MEM), Hank's balanced salt solution (Hanks solution), and the like. In addition, in order to increase the growth rate of cells, the above medium contains growth factors such as basic fibroblast growth factor (bFGF) and epithelial cell growth factor (EGF), and tumor necrosis factor (TNF), if necessary. ), Vitamin, interleukins, insulin, transferase, heparin, heparan sulfate, collagen, bovine serum albumin (BSA), fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement, etc. Often, antibiotics (penicillin, streptomycin, etc.) and the like may be added. Furthermore, apart from the differentiation-promoting agent of the present invention, a differentiation-inducing factor from epidermal stem cells to epidermal keratinized cells may be added. Examples of the differentiation-inducing factor include calcium salt, lysophosphatidic acid (LPA) and the like. Each component of the medium is used in a sterilized state.
また、上記以外には、１～２０重量％の含有率で血清が培地に含まれることが好ましい。しかしながら、血清はロットの違いにより成分が異なり、その効果にバラツキがあるため、ロットチェックを行った後に使用することが好ましい。 In addition to the above, it is preferable that serum is contained in the medium at a content of 1 to 20% by weight. However, since serum has different components depending on the lot and its effect varies, it is preferable to use it after performing a lot check.
上記の本発明に係る表皮幹細胞の分化促進剤あるいは本発明に係る方法に準じて、ニワウメの抽出物を、単独で、あるいは培地と別々に又は培地と混合し、表皮幹細胞の分化促進のための試薬キットとして提供することもできる。当該キットは、必要に応じて取扱い説明書等を含むことができる。あるいは、上記のニワウメの抽出物を培地と混合し、表皮幹細胞の分化促進用培地として提供することもできる。 According to the above-mentioned epidermal stem cell differentiation promoting agent according to the present invention or the method according to the present invention, an extract of chicken horse beetle can be used alone or separately from the medium or mixed with the medium to promote the differentiation of epidermal stem cells. It can also be provided as a reagent kit. The kit may include an instruction manual or the like, if necessary. Alternatively, the above extract of Prunus japonica can be mixed with a medium and provided as a medium for promoting differentiation of epidermal stem cells.
表皮幹細胞の培養に用いる培養器は、表皮幹細胞の培養が可能なものであれば特に限定されないが、例えば、フラスコ、シャーレ、ディッシュ、プレート、チャンバースライド、チューブ、トレイ、培養バッグ、ローラーボトルなどが挙げられる。培養器は、細胞非接着性であっても接着性であってもよく、目的に応じて適宜選択される。細胞接着性の培養器は、細胞との接着性を向上させる目的で、細胞外マトリックス等による細胞支持用基質などで処理したものを用いてもよい。細胞支持用基質としては、例えば、コラーゲン、ゼラチン、ポリ－Ｌ－リジン、ポリ－Ｄ－リジン、ラミニン、フィブロネクチンなどが挙げられる。 The incubator used for culturing epidermal stem cells is not particularly limited as long as it is capable of culturing epidermal stem cells, and examples thereof include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. Can be mentioned. The incubator may be cell non-adhesive or adhesive, and is appropriately selected depending on the intended purpose. As the cell adhesion incubator, those treated with a cell-supporting substrate or the like using an extracellular matrix or the like may be used for the purpose of improving the adhesion to cells. Examples of the cell-supporting substrate include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.
表皮幹細胞培養に使用される培地に対するニワウメの抽出物の添加濃度は、上述の本発明に係る表皮幹細胞の分化促進剤におけるニワウメの抽出物の含有量に準じて適宜決定することができるが、ニワウメの抽出物の乾燥物に換算して、例えば１～１００００μｇ／ｍＬ、好ましくは１０～５０００μｇ／ｍＬ、最も好ましくは５０～２０００μｇ／ｍＬの濃度が挙げられる。また、表皮幹細胞の培養期間中、これらの抽出物を定期的に培地に添加してもよい。 The concentration of the extract of Prunus japonica added to the medium used for culturing the epidermis stem cells can be appropriately determined according to the content of the extract of Prunus japonica in the above-mentioned agent for promoting the differentiation of the epidermis stem cells according to the present invention. In terms of the dried product of the extract, for example, a concentration of 1 to 10000 μg / mL, preferably 10 to 5000 μg / mL, and most preferably 50 to 2000 μg / mL can be mentioned. In addition, these extracts may be added to the medium on a regular basis during the culture period of epidermal stem cells.
表皮幹細胞の培養条件は、表皮幹細胞の培養に用いられる通常の条件に従えばよく、特別な制御は必要ではない。例えば、培養温度は、特に限定されるものではないが約３０～４０℃、好ましくは約３６～３７℃である。ＣＯ２ガス濃度は、例えば約１～１０体積％、好ましくは約２～５体積％である。なお、培地の交換は２～３日に１回行うことが好ましく、毎日行うことがより好ましい。前記培養条件は、表皮幹細胞が生存及び増殖可能な範囲で適宜変動させて設定することもできる。 The culture conditions for epidermal stem cells may follow the usual conditions used for culturing epidermal stem cells, and no special control is required. For example, the culture temperature is not particularly limited, but is about 30 to 40 ° C, preferably about 36 to 37 ° C. The CO 2 gas concentration is, for example, about 1 to 10% by volume, preferably about 2 to 5% by volume. The medium is preferably exchanged once every 2 to 3 days, and more preferably every day. The culture conditions can be appropriately varied and set within a range in which epidermal stem cells can survive and proliferate.
表皮幹細胞の表皮角化細胞への分化が促進されたか否かのｉｎ ｖｉｔｒｏでの判定は、当業者が通常行う方法によって行うことが可能であり、例えば、本発明に係る表皮幹細胞の表皮角化細胞への分化促進剤の非存在下で培養した表皮幹細胞と比較して、本発明に係る表皮幹細胞の分化促進剤の存在下で培養した該幹細胞において表皮角化細胞マーカー遺伝子の発現レベルがｍＲＮＡレベル又はタンパク質レベルで有意に高いか否かで評価することができる。表皮角化細胞マーカー遺伝子としては、例えば、ＬＯＲ（ロリクリン）、ＦＬＧ（フィラグリン）、ＩＶＬ（インボルクリン）、ＯＣＬＮ（オクルディン）、ＣＬＤＮ（クローディン）などが挙げられるが、これらに限定はされない。 In vitro determination of whether or not the differentiation of epidermal stem cells into epidermal keratinized cells can be performed by a method usually performed by those skilled in the art, for example, epidermal keratinization of epidermal stem cells according to the present invention. The expression level of the epidermal keratinization cell marker gene is mRNA in the stem cells cultured in the presence of the differentiation promoting agent of the epidermal stem cells according to the present invention, as compared with the epidermal stem cells cultured in the absence of the differentiation promoting agent into cells. It can be evaluated by whether it is significantly higher at the level or protein level. Examples of the epidermal keratinized cell marker gene include, but are not limited to, LOR (loricrin), FLG (filaggrin), IVL (volucrin), OCLU (occludin), and CLDN (claudin).
次に、実施例により本発明を更に具体的に説明するが、本発明はこれに限定されるものではない。なお、特に指定のない場合は、実施例に示す％とは重量％を示す。 Next, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto. Unless otherwise specified,% shown in Examples means% by weight.
（製造例１）ニワウメの熱水抽出物１の調製
ニワウメ（Ｐｒｕｎｕｓ ｊａｐｏｎｉｃａ）の種子の乾燥物１０ｇに２００ｍＬの水を加え、９５～１００℃で２時間抽出した。得られた抽出液を濾過し、その濾液を濃縮し、凍結乾燥してニワウメの熱水抽出物１を２．６ｇ得た。
(Production Example 1) Preparation of Hot Water Extract 1 of Prunus japonica 200 mL of water was added to 10 g of dried seeds of Prunus japonica, and the mixture was extracted at 95 to 100 ° C. for 2 hours. The obtained extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 2.6 g of hot water extract 1 of Prunus japonica.
（製造例２）ニワウメの５０％エタノール抽出物の調製
ニワウメ（Ｐｒｕｎｕｓ ｊａｐｏｎｉｃａ）の種子の乾燥物１０ｇを２００ｍＬの５０％エタノール水溶液に室温で７日間浸漬し、抽出を行った。得られた抽出液を濾過した後、エバポレーターで濃縮乾固してニワウメの５０％エタノール抽出物を１．７ｇ得た。
(Production Example 2) Preparation of 50% ethanol extract of Prunus japonica 10 g of dried seeds of Prunus japonica was immersed in 200 mL of a 50% ethanol aqueous solution at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness with an evaporator to obtain 1.7 g of a 50% ethanol extract of Prunus japonica.
（製造例３）ニワウメのエタノール抽出物の調製
ニワウメ（Ｐｒｕｎｕｓ ｊａｐｏｎｉｃａ）の種子の乾燥物１０ｇを２００ｍＬのエタノールに室温で７日間浸漬し、抽出を行った。得られた抽出液を濾過した後、エバポレーターで濃縮乾固してニワウメのエタノール抽出物を０．２ｇ得た。
(Production Example 3) Preparation of Ethanol Extract of Prunus japonica 10 g of dried seeds of Prunus japonica was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. After filtering the obtained extract, it was concentrated to dryness with an evaporator to obtain 0.2 g of an ethanol extract of Prunus japonica.
（製造例４）ニワウメの１，３－ブチレングリコール抽出物の調製
ニワウメ（Ｐｒｕｎｕｓ ｊａｐｏｎｉｃａ）の種子の乾燥物１０ｇを２００ｍＬの１，３－ブチレングリコールに室温で７日間浸漬し、抽出を行った。得られた抽出液を濾過して、ニワウメの１，３－ブチレングリコール抽出物を１９０ｇ得た。
(Production Example 4) Preparation of 1,3-butylene glycol extract of Prunus japonica 10 g of dried seeds of Prunus japonica was immersed in 200 mL of 1,3-butylene glycol at room temperature for 7 days for extraction. The obtained extract was filtered to obtain 190 g of 1,3-butylene glycol extract of Prunus japonica.
（製造例５）ニワウメの熱水抽出物２の調製
ニワウメ（Ｐｒｕｎｕｓ ｊａｐｏｎｉｃａ）の植物体全体の乾燥物１０ｇに２００ｍＬの水を加え、９５～１００℃で２時間抽出した。得られた抽出液を濾過し、その濾液を濃縮し、凍結乾燥してニワウメの熱水抽出物２を３．０ｇ得た。
(Production Example 5) Preparation of Hot Water Extract 2 of Prunus japonica 200 mL of water was added to 10 g of a dried product of the whole plant of Prunus japonica, and the mixture was extracted at 95 to 100 ° C. for 2 hours. The obtained extract was filtered, the filtrate was concentrated, and freeze-dried to obtain 3.0 g of hot water extract 2 of Prunus japonica.
（処方例１） 化粧水１
処方 含有量（％）
１．ニワウメの熱水抽出物１（製造例１） ２．０
２．１，３－ブチレングリコール ８．０
３．グリセリン ２．０
４．キサンタンガム ０．０２
５．クエン酸 ０．０１
６．クエン酸ナトリウム ０．１
７．エタノール ５．０
８．パラオキシ安息香酸メチル ０．１
９．ポリオキシエチレン硬化ヒマシ油（４０Ｅ．Ｏ．） ０．１
１０．香料 適量
１１．精製水にて全量を１００とする
［製造方法］成分１～６及び１１と、成分７～１０をそれぞれ均一に溶解し、両者を混合し濾過して製品とする。
(Prescription example 1) Toner 1
Prescription content (%)
1. 1. Prunus japonica hot water extract 1 (Production Example 1) 2.0
2.1,3-butylene glycol 8.0
3. 3. Glycerin 2.0
4. Xanthan gum 0.02
5. Citric acid 0.01
6. Sodium citrate 0.1
7. Ethanol 5.0
8. Methyl paraoxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40EO) 0.1
10. Appropriate amount of fragrance 11. [Manufacturing method] Ingredients 1 to 6 and 11 having a total amount of 100 in purified water and components 7 to 10 are uniformly dissolved, and both are mixed and filtered to obtain a product.
（比較処方例１） 従来の化粧水
処方例１において、ニワウメの熱水抽出物１（製造例１）を精製水に置き換えたものを、従来の化粧水とした。
(Comparative Prescription Example 1) Conventional Toner In Formulation Example 1, the hot water extract 1 of Prunus japonica (Production Example 1) was replaced with purified water, and the conventional lotion was used.
（処方例２） 化粧水２
処方例１において、ニワウメの熱水抽出物１（製造例１）をニワウメの熱水抽出物２（製造例５）に置き換えたものを、化粧水２とした。
(Prescription example 2) Toner 2
In Formulation Example 1, the hot water extract 1 of Prunus japonica (Production Example 1) was replaced with the hot water extract 2 of Prunus japonica (Production Example 5), which was referred to as lotion 2.
（処方例３） クリーム
処方 含有量（％）
１．ニワウメの５０％エタノール抽出物（製造例２） １．０
２．スクワラン ５．５
３．オリーブ油 ３．０
４．ステアリン酸 ２．０
５．ミツロウ ２．０
６．ミリスチン酸オクチルドデシル ３．５
７．ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．） ３．０
８．ベヘニルアルコール １．５
９．モノステアリン酸グリセリン ２．５
１０．香料 ０．１
１１．パラオキシ安息香酸メチル ０．２
１２．１，３－ブチレングリコール ８．５
１３．精製水にて全量を１００とする
［製造方法］成分２～９を加熱溶解して混合し、７０℃に保ち油相とする。成分１及び１１～１３を加熱溶解して混合し、７５℃に保ち水相とする。油相に水相を加えて乳化して、かき混ぜながら冷却し、４５℃で成分１０を加え、さらに３０℃まで冷却して製品とする。
(Prescription example 3) Cream prescription content (%)
1. 1. 50% ethanol extract of Prunus japonica (Production Example 2) 1.0
2. 2. Squalene 5.5
3. 3. Olive oil 3.0
4. Stearic acid 2.0
5. Beeswax 2.0
6. Octyldodecyl myristate 3.5
7. Polyoxyethylene cetyl ether (20EO) 3.0
8. Behenyl alcohol 1.5
9. Glycerin monostearate 2.5
10. Fragrance 0.1
11. Methyl paraoxybenzoate 0.2
12.1,3-Butylene Glycol 8.5
13. [Manufacturing method] Ingredients 2 to 9 having a total amount of 100 in purified water are heated and dissolved and mixed, and kept at 70 ° C. to prepare an oil phase. Ingredients 1 and 11 to 13 are heated, dissolved and mixed, and kept at 75 ° C. to prepare an aqueous phase. An aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring, the component 10 is added at 45 ° C, and the product is further cooled to 30 ° C to obtain a product.
（処方例４） 乳液
処方 含有量（％）
１．ニワウメのエタノール抽出物（製造例３） ０．０１
２．スクワラン ５．０
３．オリーブ油 ５．０
４．ホホバ油 ５．０
５．セタノール １．５
６．モノステアリン酸グリセリン ２．０
７．ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．） ３．０
８．ポリオキシエチレンソルビタンモノオレエート（２０Ｅ．Ｏ．） ２．０
９．香料 ０．１
１０．プロピレングリコール １．０
１１．グリセリン ２．０
１２．パラオキシ安息香酸メチル ０．２
１３．精製水にて全量を１００とする
［製造方法］成分１～８を加熱溶解して混合し、７０℃に保ち油相とする。成分１０～１３を加熱溶解して混合し、７５℃に保ち水相とする。油相に水相を加えて乳化して、かき混ぜながら冷却し、４５℃で成分９を加え、さらに３０℃まで冷却して製品とする。
(Prescription example 4) Emulsion prescription content (%)
1. 1. Ethanol extract of Prunus japonica (Production Example 3) 0.01
2. 2. Squalene 5.0
3. 3. Olive oil 5.0
4. Jojoba oil 5.0
5. Cetanol 1.5
6. Glycerin monostearate 2.0
7. Polyoxyethylene cetyl ether (20EO) 3.0
8. Polyoxyethylene sorbitan monooleate (20EO) 2.0
9. Fragrance 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12. Methyl paraoxybenzoate 0.2
13. [Manufacturing method] Ingredients 1 to 8 having a total amount of 100 in purified water are heated and dissolved and mixed, and kept at 70 ° C. to prepare an oil phase. Ingredients 10 to 13 are heated and dissolved, mixed, and kept at 75 ° C. to prepare an aqueous phase. An aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring, component 9 is added at 45 ° C, and the product is further cooled to 30 ° C to obtain a product.
（処方例５） ゲル剤
処方 含有量（％）
１．ニワウメの１，３－ブチレングリコール抽出物（製造例４） １．０
２．エタノール ５．０
３．パラオキシ安息香酸メチル ０．１
４．ポリオキシエチレン硬化ヒマシ油（６０Ｅ．Ｏ．） ０．１
５．香料 適量
６．１，３－ブチレングリコール ５．０
７．グリセリン ５．０
８．キサンタンガム ０．１
９．カルボキシビニルポリマー ０．２
１０．水酸化カリウム ０．２
１１．精製水にて全量を１００とする
［製造方法］成分２～５と、成分１及び６～１１をそれぞれ均一に溶解し、両者を混合して製品とする。
(Prescription example 5) Gel preparation Prescription content (%)
1. 1. Prunus japonica 1,3-butylene glycol extract (Production Example 4) 1.0
2. 2. Ethanol 5.0
3. 3. Methyl paraoxybenzoate 0.1
4. Polyoxyethylene hydrogenated castor oil (60EO) 0.1
5. Appropriate amount of fragrance 6.1,3-butylene glycol 5.0
7. Glycerin 5.0
8. Xanthan gum 0.1
9. Carboxyvinyl polymer 0.2
10. Potassium hydroxide 0.2
11. [Manufacturing method] Ingredients 2 to 5 and components 1 and 6 to 11 having a total amount of 100 in purified water are uniformly dissolved, and the two are mixed to obtain a product.
（処方例６） パック
処方 含有量（％）
１．ニワウメの熱水抽出物１（製造例１） １．０
２．ニワウメの１，３－ブチレングリコール抽出物（製造例４） ５．０
３．ポリビニルアルコール １２．０
４．エタノール ５．０
５．１，３－ブチレングリコール ８．０
６．パラオキシ安息香酸メチル ０．２
７．ポリオキシエチレン硬化ヒマシ油（２０Ｅ．Ｏ．） ０．５
８．クエン酸 ０．１
９．クエン酸ナトリウム ０．３
１０．香料 適量
１１．精製水にて全量を１００とする
［製造方法］成分１～１１を均一に溶解し製品とする。
(Prescription example 6) Pack prescription content (%)
1. 1. Prunus japonica hot water extract 1 (Production Example 1) 1.0
2. 2. Prunus japonica 1,3-butylene glycol extract (Production Example 4) 5.0
3. 3. Polyvinyl alcohol 12.0
4. Ethanol 5.0
5.1,3-butylene glycol 8.0
6. Methyl paraoxybenzoate 0.2
7. Polyoxyethylene hydrogenated castor oil (20EO) 0.5
8. Citric acid 0.1
9. Sodium citrate 0.3
10. Appropriate amount of fragrance 11. [Manufacturing method] Ingredients 1 to 11 having a total amount of 100 in purified water are uniformly dissolved to prepare a product.
（処方例７） ファンデーション
処方 含有量（％）
１．ニワウメの５０％エタノール抽出物（製造例２） １．０
２．ステアリン酸 ２．４
３．ポリオキシエチレンソルビタンモノステアレート（２０Ｅ．Ｏ．） １．０
４．ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．） ２．０
５．セタノール １．０
６．液状ラノリン ２．０
７．流動パラフィン ３．０
８．ミリスチン酸イソプロピル ６．５
９．カルボキシメチルセルロースナトリウム ０．１
１０．ベントナイト ０．５
１１．プロピレングリコール ４．０
１２．トリエタノールアミン １．１
１３．パラオキシ安息香酸メチル ０．２
１４．二酸化チタン ８．０
１５．タルク ４．０
１６．ベンガラ １．０
１７．黄酸化鉄 ２．０
１８．香料 適量
１９．精製水にて全量を１００とする
［製造方法］成分２～８を加熱溶解し、８０℃に保ち油相とする。成分１９に成分９をよく膨潤させ、続いて、成分１及び１０～１３を加えて均一に混合する。これに粉砕機で粉砕混合した成分１４～１７を加え、ホモミキサーで撹拌し７５℃に保ち水相とする。油相に水相をかき混ぜながら加え、乳化する。その後、冷却し、４５℃で成分１８を加え、かき混ぜながら３０℃まで冷却して製品とする。
(Prescription example 7) Foundation prescription content (%)
1. 1. 50% ethanol extract of Prunus japonica (Production Example 2) 1.0
2. 2. Stearic acid 2.4
3. 3. Polyoxyethylene sorbitan monostearate (20EO) 1.0
4. Polyoxyethylene cetyl ether (20EO) 2.0
5. Cetanol 1.0
6. Liquid lanolin 2.0
7. Liquid paraffin 3.0
8. Isopropyl myristate 6.5
9. Sodium Carboxymethyl Cellulose 0.1
10. Bentonite 0.5
11. Propylene glycol 4.0
12. Triethanolamine 1.1
13. Methyl paraoxybenzoate 0.2
14. Titanium dioxide 8.0
15. Talc 4.0
16. Bengala 1.0
17. Yellow iron oxide 2.0
18. Appropriate amount of fragrance 19. [Manufacturing method] Ingredients 2 to 8 having a total amount of 100 in purified water are dissolved by heating and kept at 80 ° C. to prepare an oil phase. Ingredient 9 is well swollen with ingredient 19, then ingredients 1 and 10-13 are added and mixed uniformly. Ingredients 14 to 17 pulverized and mixed by a pulverizer are added thereto, and the mixture is stirred with a homomixer and kept at 75 ° C. to prepare an aqueous phase. Add the aqueous phase to the oil phase while stirring to emulsify. Then, it is cooled, the component 18 is added at 45 ° C., and the product is cooled to 30 ° C. while stirring.
（処方例８） 浴用剤
処方 含有量（％）
１．ニワウメのエタノール抽出物（製造例３） １．０
２．炭酸水素ナトリウム ５０．０
３．黄色２０２号（１） 適量
４．香料 適量
５．硫酸ナトリウムにて全量を１００とする
［製造方法］成分１～５を均一に混合し製品とする。
(Prescription example 8) Prescription content for bath (%)
1. 1. Ethanol extract of Prunus japonica (Production Example 3) 1.0
2. 2. Sodium bicarbonate 50.0
3. 3. Yellow No. 202 (1) Appropriate amount 4. Appropriate amount of fragrance 5. [Manufacturing method] Ingredients 1 to 5 having a total amount of 100 with sodium sulfate are uniformly mixed to prepare a product.
（処方例９） 軟膏１
処方 含有量（％）
１．ニワウメの熱水抽出物１（製造例１） ５．０
２．ニワウメの１，３－ブチレングリコール抽出物（製造例４） １．０
３．ポリオキシエチレンセチルエーテル（３０Ｅ．Ｏ．） ２．０
４．モノステアリン酸グリセリン １０．０
５．流動パラフィン ５．０
６．セタノール ６．０
７．パラオキシ安息香酸メチル ０．１
８．プロピレングリコール １０．０
９．精製水にて全量を１００とする
［製造方法］成分３～６を加熱溶解して混合し、７０℃に保ち油相とする。成分１、２及び７～９を加熱溶解して混合し、７５℃に保ち水相とする。油相に水相を加えて乳化して、かき混ぜながら３０℃まで冷却して製品とする。
(Prescription example 9) Ointment 1
Prescription content (%)
1. 1. Prunus japonica hot water extract 1 (Production Example 1) 5.0
2. 2. Prunus japonica 1,3-butylene glycol extract (Production Example 4) 1.0
3. 3. Polyoxyethylene cetyl ether (30EO) 2.0
4. Glycerin monostearate 10.0
5. Liquid paraffin 5.0
6. Cetanol 6.0
7. Methyl paraoxybenzoate 0.1
8. Propylene glycol 10.0
9. [Manufacturing method] Ingredients 3 to 6 having a total amount of 100 in purified water are melted by heating and mixed, and kept at 70 ° C. to prepare an oil phase. Ingredients 1, 2 and 7 to 9 are heated, dissolved and mixed, and kept at 75 ° C. to prepare an aqueous phase. An aqueous phase is added to the oil phase to emulsify, and the product is cooled to 30 ° C. with stirring to obtain a product.
（処方例１０） 軟膏２
処方例９において、ニワウメの熱水抽出物１（製造例１）をニワウメの熱水抽出物２（製造例５）に置き換えたものを、軟膏２とした。
(Prescription example 10) Ointment 2
In Formulation Example 9, the hot water extract 1 of Prunus japonica (Production Example 1) was replaced with the hot water extract 2 of Prunus japonica (Production Example 5), and the ointment 2 was used.
（処方例１１） 散剤１
処方 含有量（％）
１．ニワウメの熱水抽出物（製造例１） １．０
２．乾燥コーンスターチ ３９．０
３．微結晶セルロース ６０．０
［製造方法］成分１～３を混合し、製品とする。
(Prescription example 11) Powder 1
Prescription content (%)
1. 1. Hot water extract of Prunus japonica (Production Example 1) 1.0
2. 2. Dried cornstarch 39.0
3. 3. Microcrystalline Cellulose 60.0
[Manufacturing method] Ingredients 1 to 3 are mixed to prepare a product.
（処方例１２） 散剤２
処方例１１において、ニワウメの熱水抽出物１（製造例１）をニワウメの熱水抽出物２（製造例５）に置き換えたものを、散剤２とした。
(Prescription example 12) Powder 2
In Formulation Example 11, the hot water extract 1 of Prunus japonica (Production Example 1) was replaced with the hot water extract 2 of Prunus japonica (Production Example 5), which was designated as powder 2.
（処方例１３） 錠剤
処方 含有量（％）
１．ニワウメのエタノール抽出物（製造例３） ５．０
２．乾燥コーンスターチ ２５．０
３．カルボキシメチルセルロースカルシウム ２０．０
４．微結晶セルロース ４０．０
５．ポリビニルピロリドン ７．０
６．タルク ３．０
［製造方法］成分１～４を混合し、次いで成分５の水溶液を結合剤として加えて顆粒成型する。成型した顆粒に成分６を加えて打錠する。１錠０．５２ｇとする。
(Prescription example 13) Tablet prescription content (%)
1. 1. Ethanol extract of Prunus japonica (Production Example 3) 5.0
2. 2. Dried cornstarch 25.0
3. 3. Carboxymethyl cellulose calcium 20.0
4. Microcrystalline Cellulose 40.0
5. Polyvinylpyrrolidone 7.0
6. Talc 3.0
[Manufacturing method] Ingredients 1 to 4 are mixed, and then an aqueous solution of ingredient 5 is added as a binder to form granules. Ingredient 6 is added to the molded granules and the mixture is locked. One tablet weighs 0.52 g.
（処方例１４） 錠菓
処方 含有量（％）
１．ニワウメのエタノール抽出物（製造例３） ２．０
２．乾燥コーンスターチ ４９．８
３．エリスリトール ４０．０
４．クエン酸 ５．０
５．ショ糖脂肪酸エステル ３．０
６．香料 ０．１
７．精製水にて全量を１００とする
［製造方法］成分１～４及び７を混合し、顆粒成型する。成型した顆粒に成分５及び６を加えて打錠する。１粒１．０ｇとする。
(Prescription example 14) Tablet confectionery prescription content (%)
1. 1. Ethanol extract of Prunus japonica (Production Example 3) 2.0
2. 2. Dried cornstarch 49.8
3. 3. Erythritol 40.0
4. Citric acid 5.0
5. Sucrose fatty acid ester 3.0
6. Fragrance 0.1
7. [Manufacturing method] Ingredients 1 to 4 and 7 having a total amount of 100 are mixed with purified water and granulated. Ingredients 5 and 6 are added to the molded granules and the mixture is locked. One grain is 1.0 g.
（処方例１５） 飲料１
処方 含有量（％）
１．ニワウメの熱水抽出物１（製造例１） ０．０５
２．ステビア ０．０５
３．リンゴ酸 ５．０
４．香料 ０．１
５．精製水にて全量を１００とする
［製造方法］成分１～３を少量の水に溶解する。次いで、成分４及び５を加えて混合する。
(Prescription example 15) Beverage 1
Prescription content (%)
1. 1. Hot water extract of Prunus japonica 1 (Production Example 1) 0.05
2. 2. Stevia 0.05
3. 3. Malic acid 5.0
4. Fragrance 0.1
5. [Manufacturing method] Ingredients 1 to 3 having a total amount of 100 in purified water are dissolved in a small amount of water. Then, components 4 and 5 are added and mixed.
（処方例１６） 飲料２
処方例１５において、ニワウメの熱水抽出物１（製造例１）をニワウメの熱水抽出物２（製造例５）に置き換えたものを、飲料２とした。
(Prescription example 16) Beverage 2
In Formulation Example 15, the hot water extract 1 of Prunus japonica (Production Example 1) was replaced with the hot water extract 2 of Prunus japonica (Production Example 5), and the beverage 2 was used.
次に、本発明の効果を詳細に説明するため、実験例を挙げる。 Next, in order to explain the effect of the present invention in detail, an experimental example will be given.
実験例１ 表皮幹細胞の表皮角化細胞への分化促進効果の評価
表皮幹細胞のモデルとして、ヒト不死化表皮細胞を用いた。ヒト不死化表皮細胞を、φ６０ｍｍ ｄｉｓｈに１×１０５個播種し、ＨｕＭｅｄｉａ ＫＧ－２培養液にて、３７℃、５体積％ＣＯ２条件下で培養した。培養を開始してから２４時間後に、各試料（終濃度は表１に記載）を添加したＨｕＭｅｄｉａ ＫＧ－２培養液にて７２時間培養した後、細胞を回収し、総ＲＮＡの抽出を行った。細胞からの総ＲＮＡの抽出はＲＮＡｉｓｏ Ｐｌｕｓ（タカラバイオ）を用いて行い、総ＲＮＡ量は分光光度計（Ｎａｎｏｄｒｏｐ）を用いて２６０ｎｍにおける吸光度により求めた。ｍＲＮＡ発現量の測定は、細胞から抽出した総ＲＮＡを基にしてリアルタイムＲＴ－ＰＣＲ法により行った。リアルタイムＲＴ－ＰＣＲ法には、ＰｒｉｍｅｒＳｃｒｉｐｔ ＲＴ Ｍａｓｔｅｒ Ｍｉｘ（タカラバイオ）及びＳＹＢＲ Ｓｅｌｅｃｔ Ｍａｓｔｅｒ Ｍｉｘ（ライフテクノロジーズ）を用いた。即ち、５００ｎｇの総ＲＮＡを逆転写反応後、ＰＣＲ反応（９５℃：１５秒間、６０℃：６０秒間、４０ｃｙｃｌｅｓ）を行った。その他の操作は定められた方法に従い、表皮角化細胞への分化マーカーであるインボルクリン（ＩＶＬ） ｍＲＮＡの発現量を、内部標準である１８ＳｒＲＮＡの発現量に対する割合として求めた。ＩＶＬ発現促進率は、コントロール（試料未添加）群のＩＶＬ ｍＲＮＡの発現量に対する試料添加群のＩＶＬ ｍＲＮＡの発現量の比率として算出した。尚、各遺伝子の発現量の測定に使用したプライマーは次の通りである。
Experimental Example 1 Evaluation of the effect of promoting the differentiation of epidermal stem cells into epidermal keratinized cells Human immortalized epidermal cells were used as a model of epidermal stem cells. Human immortalized epidermal cells were seeded in 1 × 10 5 cells in a φ60 mm dish and cultured in HuMedia KG-2 culture medium at 37 ° C. under 5% by volume CO 2 conditions. Twenty-four hours after the start of culturing, cells were collected and total RNA was extracted after culturing in HuMedia KG-2 culture medium to which each sample (final concentration is shown in Table 1) was added for 72 hours. .. Extraction of total RNA from cells was performed using RNAiso Plus (Takara Bio), and the total amount of RNA was determined by absorbance at 260 nm using a spectrophotometer (Nanodrop). The mRNA expression level was measured by the real-time RT-PCR method based on the total RNA extracted from the cells. For the real-time RT-PCR method, PrimerScript RT Master Mix (Takara Bio) and SYBR Select Master Mix (Life Technologies) were used. That is, after a reverse transcription reaction of 500 ng of total RNA, a PCR reaction (95 ° C: 15 seconds, 60 ° C: 60 seconds, 40 cycles) was performed. For other operations, the expression level of involucrin (IVL) mRNA, which is a marker for differentiation into epidermal keratinocytes, was determined as a ratio to the expression level of 18S rRNA, which is an internal standard, according to a predetermined method. The IVL expression promotion rate was calculated as the ratio of the expression level of IVL mRNA in the sample-added group to the expression level of IVL mRNA in the control (non-sampled) group. The primers used to measure the expression level of each gene are as follows.
ＩＶＬ用のプライマーセット
ＣＣＡＴＣＡＧＧＡＧＣＡＡＡＴＧＡＡＡＣＡＧ（配列番号１）
ＧＣＴＣＧＡＣＡＧＧＣＡＣＣＴＴＣＴＧ（配列番号２）
１８ＳｒＲＮＡ用のプライマーセット
ＴＧＣＡＣＣＡＣＣＡＡＣＴＧＣＴＴＡＧＣ（配列番号３）
ＴＣＴＴＣＴＧＧＧＴＧＧＣＡＧＴＧＡＴＧ（配列番号４）
Primer set for IVL CCATCAGGAGCAAAATTGAAACAG (SEQ ID NO: 1)
GCTCGACAGGGCACTTCTG (SEQ ID NO: 2)
Primer set for 18S rRNA TGCACCACCAACTGCTTAGC (SEQ ID NO: 3)
TCTTCTGGGTGGGCAGTGATG (SEQ ID NO: 4)
これらの実験結果を表１に示した。その結果、本発明のニワウメの抽出物には、優れたＩＶＬ発現促進効果（表皮幹細胞の表皮角化細胞への分化促進効果）が認められた。 The results of these experiments are shown in Table 1. As a result, the extract of Prunus japonica of the present invention was found to have an excellent effect of promoting IVL expression (effect of promoting differentiation of epidermal stem cells into epidermal keratinized cells).
実験例２ 使用試験
処方例１の化粧水１及び比較処方例１の従来の化粧水を用いて、シミ、くすみがある５人（２６～６６歳）を対象に１ヶ月間の使用試験を行った。使用後、シミ、くすみの程度をアンケートにより判定した。
Experimental Example 2 Use test Using the conventional lotion 1 of Prescription Example 1 and the conventional lotion of Comparative Prescription Example 1, a one-month use test was conducted on 5 people (26-66 years old) with stains and dullness. rice field. After use, the degree of stains and dullness was judged by a questionnaire.
その結果、本発明のニワウメの抽出物を含有する化粧水により、シミ、くすみが軽減した。尚、試験期間中、皮膚トラブルは１人もなく、安全性においても問題なかった。又、処方成分の劣化についても問題なかった。 As a result, the stain and dullness were alleviated by the lotion containing the extract of Prunus japonica of the present invention. During the test period, there was no skin trouble and there was no problem in terms of safety. In addition, there was no problem with deterioration of the formulated ingredients.
以上のことから、本発明で用いるニワウメの抽出物は、優れた表皮幹細胞の表皮角化細胞への分化促進作用を有し、安定性にも優れていた。よって、本発明で用いるニワウメの抽出物は、再生医療や再生美容を含む医療や美容の分野において利用でき、化粧品、医薬部外品、医薬品及び食品への応用が期待される。 From the above, the extract of Prunus japonica used in the present invention has an excellent effect of promoting the differentiation of epidermal stem cells into epidermal keratinized cells and is also excellent in stability. Therefore, the extract of Prunus japonica used in the present invention can be used in the fields of medicine and cosmetology including regenerative medicine and regenerative beauty, and is expected to be applied to cosmetics, quasi-drugs, pharmaceuticals and foods.
Claims (6)
A method for producing epidermal keratinized cells, which comprises a step of culturing epidermal stem cells in a medium containing an extract of Prunus japonica.
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