JP2023178803A - Epidermal stem cell activator - Google Patents
Epidermal stem cell activator Download PDFInfo
- Publication number
- JP2023178803A JP2023178803A JP2022091715A JP2022091715A JP2023178803A JP 2023178803 A JP2023178803 A JP 2023178803A JP 2022091715 A JP2022091715 A JP 2022091715A JP 2022091715 A JP2022091715 A JP 2022091715A JP 2023178803 A JP2023178803 A JP 2023178803A
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- Prior art keywords
- epidermal stem
- royal jelly
- salt
- acid
- stem cell
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Abstract
Description
本発明は、表皮幹細胞活性化剤に関する。 The present invention relates to an epidermal stem cell activator.
皮膚は、表皮、真皮、及び皮下組織からなり、主に外部からの傷害及び経皮水分喪失に対するバリア機能を有している。最も外側の表皮は、さらに外側から角質層、顆粒層、有棘層、基底層の４つの層からなり、主に表皮角化細胞から構成される。基底層には表皮幹細胞が存在し、表皮幹細胞は表皮角化細胞の前駆細胞に分化したのち、有棘層、顆粒層を形成する細胞へと分化し、最終的には角質層を形成する細胞へと分化する。加齢に伴い、表皮幹細胞の機能が低下し、それが皮膚の菲薄化、皮膚のターンオーバーの遅れ、及び皮膚のバリア機能低下に繋がることが報告されている（非特許文献１～３）。 The skin consists of the epidermis, dermis, and subcutaneous tissue, and mainly has a barrier function against external damage and transepidermal water loss. The outermost epidermis consists of four layers, from the outside: the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale, and is mainly composed of epidermal keratinocytes. Epidermal stem cells exist in the basal layer, and after the epidermal stem cells differentiate into precursor cells of epidermal keratinocytes, they differentiate into cells that form the stratum spinosum and stratum granulosum, and finally cells that form the stratum corneum. differentiate into It has been reported that with aging, the function of epidermal stem cells decreases, which leads to skin thinning, delayed skin turnover, and decreased skin barrier function (Non-Patent Documents 1 to 3).
特許文献１にマンネンタケ胞子の超臨界抽出物が、表皮幹細胞の表皮角化細胞への分化を促進できることが記載されている。しかしながら、引用文献１には、表皮幹細胞を活性化することについて開示されていない。 Patent Document 1 describes that a supercritical extract of C. spores can promote the differentiation of epidermal stem cells into epidermal keratinocytes. However, Cited Document 1 does not disclose activation of epidermal stem cells.
本発明は、新規な表皮幹細胞活性化剤を提供し、特に皮膚のターンオーバーの低下、皮膚の老化、及び／又は皮膚の菲薄化の抑制に有効な表皮幹細胞活性化剤を提供することを目的とする。 An object of the present invention is to provide a novel epidermal stem cell activator, and particularly to provide an epidermal stem cell activator that is effective in reducing skin turnover, inhibiting skin aging, and/or skin thinning. shall be.
本発明者らが、上記目的を達成すべく鋭意研究の結果、ローヤルゼリー及びローヤルゼリーに含まれるいくつかの脂肪酸が表皮幹細胞を活性化でき、それによって、表皮幹細胞が幹細胞性を維持したまま増殖性を向上させることができ、また、皮膚のターンオーバーの低下抑制、皮膚の老化抑制、皮膚の菲薄化の抑制等ができることを見出し、本発明の完成に至った。 As a result of intensive research aimed at achieving the above objective, the present inventors found that royal jelly and some fatty acids contained in royal jelly can activate epidermal stem cells, thereby allowing epidermal stem cells to proliferate while maintaining their stemness. The present inventors have discovered that it is possible to improve skin turnover, suppress skin turnover, suppress skin aging, suppress skin thinning, etc., and have completed the present invention.
すなわち、本発明は、例えば以下の発明に関する。
［１］ローヤルゼリー、１０－ヒドロキシ－２－デセン酸若しくはその塩、１０－ヒドロキシデカン酸若しくはその塩、デセン二酸若しくはその塩、及び、セバシン酸若しくはその塩からなる群より選択される少なくとも一つを有効成分として含有する、表皮幹細胞活性化剤。
［２］皮膚のターンオーバーの低下、皮膚の老化、及び皮膚の菲薄化のうち少なくとも一つを抑制する、［１］に記載の表皮幹細胞活性化剤。
［３］［１］又は［２］に記載の表皮幹細胞活性化剤を含む、化粧品、食品組成物、又は医薬組成物。
That is, the present invention relates to, for example, the following inventions.
[1] At least one selected from the group consisting of royal jelly, 10-hydroxy-2-decenoic acid or a salt thereof, 10-hydroxydecanoic acid or a salt thereof, decenedioic acid or a salt thereof, and sebacic acid or a salt thereof An epidermal stem cell activator containing as an active ingredient.
[2] The epidermal stem cell activator according to [1], which suppresses at least one of a decrease in skin turnover, skin aging, and skin thinning.
[3] A cosmetic, food composition, or pharmaceutical composition comprising the epidermal stem cell activator according to [1] or [2].
本発明によれば、新規な表皮幹細胞活性化剤、皮膚のターンオーバーの低下抑制、皮膚の老化抑制、皮膚の菲薄化抑制に用いることができる表皮幹細胞活性化剤を提供できる。 According to the present invention, it is possible to provide a novel epidermal stem cell activator, an epidermal stem cell activator that can be used to suppress decline in skin turnover, suppress skin aging, and suppress skin thinning.
以下、本発明を実施するための形態について詳細に説明する、ただし、本発明は以下の実施形態に限定されるものではない。 EMBODIMENT OF THE INVENTION Hereinafter, the form for implementing this invention is demonstrated in detail, However, this invention is not limited to the following embodiment.
本実施形態に係る表皮幹細胞活性化剤は、ローヤルゼリー、１０－ヒドロキシ－２－デセン酸若しくはその塩、１０－ヒドロキシデカン酸若しくはその塩、デセン二酸若しくはその塩、及び、セバシン酸若しくはその塩からなる群より選択される少なくとも一つを有効成分として含有する。 The epidermal stem cell activator according to the present embodiment is made of royal jelly, 10-hydroxy-2-decenoic acid or a salt thereof, 10-hydroxydecanoic acid or a salt thereof, decenedioic acid or a salt thereof, and sebacic acid or a salt thereof. Contains at least one selected from the group consisting of: as an active ingredient.
ローヤルゼリーは、蜜蜂のうち日齢３～１２日の働き蜂が下咽頭腺及び大腮腺から分泌する分泌物を混合して作る乳白色のゼリー状物質である。ローヤルゼリー中の主な生理活性成分としては、例えば、ローヤルゼリーに特有な１０－ヒドロキシ－２－デセン酸、１０－ヒドロキシデカン酸等の有機酸類をはじめ、タンパク質、アミノ酸、ペプチド、脂質、糖類、ビタミンＢ類、葉酸、ニコチン酸、パントテン酸等のビタミン類、各種ミネラル類等が挙げられる。 Royal jelly is a milky white jelly-like substance produced by mixing the secretions secreted from the hypopharyngeal glands and magnus glands of worker bees that are 3 to 12 days old. The main physiologically active ingredients in royal jelly include organic acids such as 10-hydroxy-2-decenoic acid and 10-hydroxydecanoic acid, which are unique to royal jelly, as well as proteins, amino acids, peptides, lipids, sugars, and vitamin B. Examples include vitamins such as folic acid, nicotinic acid, and pantothenic acid, and various minerals.
本明細書においてローヤルゼリーは、例えば生ローヤルゼリーであってもよく、生ローヤルゼリーに処理を施したローヤルゼリー処理物であってもよい。 In this specification, the royal jelly may be, for example, raw royal jelly or a processed royal jelly obtained by processing raw royal jelly.
生ローヤルゼリーは、例えば、常法に従い養蜂産品として入手することができる。ローヤルゼリーの産地は、制限されず、日本、中国、ブラジル、ヨーロッパ諸国、オセアニア諸国、アメリカ等のいずれであってもよい。生ローヤルゼリーは、採取したローヤルゼリーを凍結保存した凍結ローヤルゼリーを含む。 Raw royal jelly can be obtained, for example, as a beekeeping product according to conventional methods. The origin of royal jelly is not limited and may be Japan, China, Brazil, European countries, Oceania countries, America, etc. Fresh royal jelly includes frozen royal jelly obtained by freezing and preserving collected royal jelly.
ローヤルゼリー処理物としては、生ローヤルゼリーを濃縮又は希釈したローヤルゼリー濃縮物又は希釈物、生ローヤルゼリーを乾燥させて粉末化したローヤルゼリー粉末、ローヤルゼリーをエタノール等の有機溶媒で抽出したローヤルゼリーエタノール抽出物等のローヤルゼリー有機溶媒抽出物、ローヤルゼリーをタンパク質分解酵素で処理した酵素分解ローヤルゼリー等が挙げられる。ローヤルゼリー処理物は複数の処理が施されたものであってもよい。ローヤルゼリーは酵素分解及び粉末化された酵素分解ローヤルゼリー粉末であってもよい。ローヤルゼリー処理物としては、酵素処理ローヤルゼリーであることが好ましく、酵素処理ローヤルゼリーの粉末であることがより好ましい。 Processed royal jelly products include royal jelly concentrate or dilution obtained by concentrating or diluting raw royal jelly, royal jelly powder obtained by drying and powdering raw royal jelly, and royal jelly organic products such as royal jelly ethanol extract obtained by extracting royal jelly with an organic solvent such as ethanol. Examples include solvent extracts, enzymatically decomposed royal jelly obtained by treating royal jelly with proteolytic enzymes, and the like. The processed royal jelly may be one that has been subjected to a plurality of treatments. The royal jelly may be an enzymatically decomposed royal jelly powder that has been enzymatically decomposed and powdered. The royal jelly treated product is preferably enzyme-treated royal jelly, and more preferably enzyme-treated royal jelly powder.
ローヤルゼリー濃縮物は、例えば、生ローヤルゼリーから水分を除去することにより得ることができる。ローヤルゼリー希釈物は、例えば、生ローヤルゼリーに水分を添加することにより得ることができる。 Royal jelly concentrate can be obtained, for example, by removing water from raw royal jelly. A diluted royal jelly can be obtained, for example, by adding water to raw royal jelly.
ローヤルゼリー粉末は、例えば、凍結乾燥及び噴霧乾燥等の本技術分野における公知の方法により生ローヤルゼリーを粉末化することにより得ることができる。乾燥方法としては、通風乾燥や天日乾燥等の自然乾燥、電気等で加熱して乾燥させる強制乾燥、凍結乾燥等、一般食品加工で採用される公知のいずれの方法を使用することができる。好ましくは、凍結乾燥である。なお、乾燥時間は特に制限されず、通風や天日乾燥等の自然乾燥の場合は、約３日程度、電気等で加熱して強制乾燥させる場合は、５０℃程度で１～３日程度を挙げることができる。通常、水分含量が１０質量％以下、好ましくは５質量％以下になるように乾燥させることが好ましい。なお、通風や天日乾燥等の自然乾燥の場合のように水分含量を１０質量％以下にすることが難しい場合は、その後、凍結乾燥機にかけてさらに水分を下げる処理を行ってもよい。また、凍結乾燥又は噴霧乾燥後に粉砕機（例えば、ピンミル、ハンマーミル、ボールミル、ジェットミル）により粉砕してローヤルゼリー粉末を得てもよい。 Royal jelly powder can be obtained, for example, by pulverizing raw royal jelly by methods known in the art, such as freeze drying and spray drying. As a drying method, any known method employed in general food processing can be used, such as natural drying such as ventilation drying and sun drying, forced drying that is heated and dried with electricity, and freeze drying. Preferably, it is freeze-dried. There are no particular restrictions on the drying time; in the case of natural drying such as ventilation or sun drying, it takes about 3 days, and in the case of forced drying by heating with electricity, it takes about 1 to 3 days at about 50℃. can be mentioned. Usually, it is preferable to dry so that the moisture content is 10% by mass or less, preferably 5% by mass or less. Note that if it is difficult to reduce the moisture content to 10% by mass or less, such as in the case of natural drying such as ventilation or sun drying, the moisture content may be further reduced by using a freeze dryer. Alternatively, after freeze-drying or spray-drying, the royal jelly powder may be obtained by pulverizing with a pulverizer (eg, pin mill, hammer mill, ball mill, jet mill).
ローヤルゼリー有機溶媒抽出物は、例えば、エタノール、メタノール、プロパノール、アセトン等の有機溶媒を溶媒として生ローヤルゼリー又はローヤルゼリー粉末を抽出することで得ることができる。抽出時間は、原料として用いられる生ローヤルゼリーの形態、溶媒の種類及び量、抽出の際の温度及び攪拌条件等に応じて適宜設定することができる。抽出後、ろ過、遠心分離等により固形分を除去してもよい。また、抽出された溶液をそのまま用いてもよいし、当該溶液から溶媒を除去して、濃縮液又は粉末として用いてもよい。ローヤルゼリー有機溶媒抽出物としては、ローヤルゼリーエタノール抽出物であることが好ましい。 Royal jelly organic solvent extract can be obtained, for example, by extracting raw royal jelly or royal jelly powder using an organic solvent such as ethanol, methanol, propanol, or acetone as a solvent. The extraction time can be appropriately set depending on the form of raw royal jelly used as a raw material, the type and amount of solvent, the temperature during extraction, stirring conditions, etc. After extraction, solid content may be removed by filtration, centrifugation, etc. Further, the extracted solution may be used as it is, or the solvent may be removed from the solution and used as a concentrated liquid or powder. The royal jelly organic solvent extract is preferably a royal jelly ethanol extract.
酵素分解ローヤルゼリーは、例えば、生ローヤルゼリー又はローヤルゼリー粉末をタンパク質分解酵素で処理することで得ることができる。タンパク質分解酵素としては、例えば、エンドペプチダーゼ作用を有する酵素、エキソペプチダーゼ作用を有する酵素、エンドペプチダーゼ作用とエキソペプチダーゼ作用の両方を有する酵素からなる群より選択されることが好ましい。特に、エンドペプチダーゼ作用とエキソペプチダーゼ作用の両方を同時に有するペプチダーゼであることが好ましい。かかるペプチダーゼを使用した酵素処理によれば、一段階酵素処理でタンパク質を低分子化することができるので、操作が簡便であるとともに、ローヤルゼリーに含まれる有用成分の生理活性の消失及び大幅な低減を防止することができるという利点がある。 Enzyme-decomposed royal jelly can be obtained, for example, by treating raw royal jelly or royal jelly powder with a proteolytic enzyme. The proteolytic enzyme is preferably selected from the group consisting of, for example, an enzyme having endopeptidase action, an enzyme having exopeptidase action, and an enzyme having both endopeptidase action and exopeptidase action. In particular, a peptidase having both endopeptidase action and exopeptidase action is preferred. Enzyme treatment using such peptidase allows proteins to be reduced in molecular weight in one step, making the operation simple and eliminating or significantly reducing the physiological activity of useful components contained in royal jelly. This has the advantage of being preventable.
タンパク質分解酵素は、その由来は特に制限されず、動物、植物、及び微生物（細菌、ウィルス、真菌類（カビ、酵母、キノコ等）、藻類等）に由来するペプチダーゼを広く使用できる。 The origin of the proteolytic enzyme is not particularly limited, and peptidases derived from animals, plants, and microorganisms (bacteria, viruses, fungi (molds, yeast, mushrooms, etc.), algae, etc.) can be widely used.
「エキソペプチダーゼ」は、「アミノペプチダーゼ」と「カルボキシペプチダーゼ」に分類される。また、ペプチダーゼは、至適ｐＨによって、それぞれ酸性、中性、アルカリ性という用語を各酵素につけることがあり、例えば、「酸性エキソペプチダーゼ」、「中性アミノペプチダーゼ」、「アルカリ性エンドペプチダーゼ」のように記載することもある。 "Exopeptidase" is classified into "aminopeptidase" and "carboxypeptidase". In addition, peptidases are sometimes referred to as acidic, neutral, or alkaline depending on their optimum pH, such as "acid exopeptidase," "neutral aminopeptidase," and "alkaline endopeptidase." It may also be stated.
少なくともエンドペプチダーゼ活性を有するタンパク質分解酵素としては、動物由来(例えば、トリプシン、キモトリプシン等)、植物由来(例えば、パパイン等）、微生物由来(例えば、乳酸菌、酵母、カビ、枯草菌、放線菌等)のエンドペプチダーゼ等が挙げられる。 Proteolytic enzymes having at least endopeptidase activity are derived from animals (e.g., trypsin, chymotrypsin, etc.), plants (e.g., papain, etc.), and microorganisms (e.g., lactic acid bacteria, yeast, molds, Bacillus subtilis, actinomycetes, etc.). Examples include endopeptidases and the like.
少なくともエキソペプチダーゼ活性を有するタンパク質分解酵素としては、カルボキシペプチダーゼ、アミノペプチダーゼ、微生物由来(例えば、乳酸菌、アスペルギルス属菌、リゾープス属菌等)のエキソペプチダーゼ、エンドペプチダーゼ活性も併せて有するパンクレアチン、ペプシン等が挙げられる。 Proteolytic enzymes having at least exopeptidase activity include carboxypeptidase, aminopeptidase, exopeptidase derived from microorganisms (e.g., lactic acid bacteria, Aspergillus spp., Rhizopus spp.), pancreatin, pepsin, etc. that also have endopeptidase activity. can be mentioned.
エキソペプチダーゼ活性とエンドペプチダーゼ活性の両方を有する酵素の好ましい例としては、ストレプトマイセス・グリセウス（Ｓｔｒｅｐｔｏｍｙｃｅｓ ｇｒｉｓｅｕｓ）産生ペプチダーゼ（商品名：アクチナーゼＡＳ）、アスペルギルス・オリゼー（Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ）産生ペプチダーゼ（商品名：プロテアーゼＡ、フレーバーザイム、プロテアックス、スミチームＬＰ－Ｇ）、アスペルギルス・メレウス（Ａｓｐｅｒｇｉｌｌｕｓ ｍｅｌｌｅｕｓ）産生ペプチダーゼ（商品名：プロテアーゼＰ）を挙げることができる。 Preferred examples of enzymes having both exopeptidase and endopeptidase activities include peptidase produced by Streptomyces griseus (trade name: actinase AS), and peptidase produced by Aspergillus oryzae (trade name: Protease A, Flavorzyme, Proteax, Sumiteam LP-G), and peptidase produced by Aspergillus melleus (trade name: Protease P) can be mentioned.
また、エキソペプチダーゼ活性を有する酵素の好ましい例としては、アスペルギルス・オリゼー（Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ）産生ペプチダーゼ（商品名：ウマミザイムＧ、Ｐｒｏｍｏｄ １９２Ｐ、Ｐｒｏｍｏｄ １９４Ｐ、スミチームＦＬＡＰ）、アスペルギルス・ソーエ（Ａｓｐｅｒｇｉｌｌｕｓ ｓｏｊａｅ）産生ペプチダーゼ（商品名：Ｓｔｅｒｎｚｙｍｅ Ｂ１５０２４）、アスペルギルス属産生ペプチダーゼ（商品名：コクラーゼＰ）、リゾプス・オリゼー（Ｒｈｉｚｏｐｕｓ ｏｒｙｚａｅ）産生ペプチダーゼ（商品名：ペプチダーゼＲ）を挙げることができる。 Further, preferable examples of enzymes having exopeptidase activity include peptidase produced by Aspergillus oryzae (trade name: Umamizyme G, Promod 192P, Promod 194P, Sumyzyme FLAP), Aspergillus soe. jae) produced peptidase ( Examples include peptidase produced by Aspergillus (trade name: Sternzyme B15024), peptidase produced by Rhizopus oryzae (trade name: Peptidase R).
さらに、エンドペプチダーゼ活性を有する酵素の好ましい例としては、バチルス・サブチリス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）産生ペプチダーゼ（商品名：オリエンターゼ２２ＢＦ、ヌクレイシン）、バチルス・リシェニフォルミス（Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ）産生ペプチダーゼ（商品名：アルカラーゼ）、バチルス・ステアロサーモフィラス（Ｂａｃｉｌｌｕｓ ｓｔｅａｒｏｔｈｅｒｍｏｐｈｉｌｕｓ）産生ペプチダーゼ（商品名：プロテアーゼＳ）、バチルス・アミロリケファシエンス（Ｂａｃｉｌｌｕｓ ａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ）産生ペプチダーゼ（商品名：ニュートラーゼ）、バチルス属産生ペプチダーゼ（商品名：プロタメックス）を挙げることができる。 Further, preferred examples of enzymes having endopeptidase activity include peptidase produced by Bacillus subtilis (trade name: Orientase 22BF, Nucleisin), and peptidase produced by Bacillus licheniformis (trade name: Alcalase), Bacillus stearothermophilus-produced peptidase (product name: Protease S), Bacillus amyloliquefaciens-produced peptidase (product name: Neutrase), Bacillus-produced peptidase Putidase (product Name: Protamex).
生ローヤルゼリーをタンパク質分解酵素で処理する際の反応条件（タンパク質分解酵素の使用量、反応時の温度、ｐＨ、反応時間等）は、使用するタンパク質分解酵素の種類等に応じて、適宜設定すればよい。 The reaction conditions (amount of proteolytic enzyme used, temperature during reaction, pH, reaction time, etc.) when treating raw royal jelly with proteolytic enzymes can be set appropriately depending on the type of proteolytic enzyme used, etc. good.
ローヤルゼリーは、市販されているものを用いてもよい。市販されているローヤルゼリーの具体例としては、例えば、酵素分解ローヤルゼリーキング（株式会社山田養蜂場製）等が挙げられる。 Commercially available royal jelly may be used. Specific examples of commercially available royal jelly include enzymatically decomposed royal jelly king (manufactured by Yamada Bee Farm Co., Ltd.).
上述したように、１０－ヒドロキシ－２－デセン酸（１０Ｈ２ＤＡ）及び１０－ヒドロキシデカン酸（１０ＨＤＡ）は、いずれもローヤルゼリーに含まれる脂肪酸の１種である。また、これらは体内で代謝されることにより、デセン二酸（２－ＤＡ）又はセバシン酸（ＳＡ）となる。デセン二酸は、２－デセン二酸であってもよく、１０Ｈ２ＤＡ及び２－ＤＡは、ｃｉｓであってもｔｒａｎｓであってもよい。１０Ｈ２ＤＡ、１０ＨＤＡ、ＤＡ及びＳＡは、合成品であってもよく、例えばローヤルゼリー由来等の天然由来のものであってもよい。ローヤルゼリー由来の１０Ｈ２ＤＡ、及び１０ＨＤＡは、例えば、ローヤルゼリーから有機溶媒等により抽出精製して得ることができる。 As mentioned above, 10-hydroxy-2-decenoic acid (10H2DA) and 10-hydroxydecanoic acid (10HDA) are both types of fatty acids contained in royal jelly. Further, these are metabolized in the body to become decenedioic acid (2-DA) or sebacic acid (SA). Decenedioic acid may be 2-decenedioic acid, and 10H2DA and 2-DA may be cis or trans. 10H2DA, 10HDA, DA and SA may be synthetic products or may be naturally derived products such as those derived from royal jelly. 10H2DA and 10HDA derived from royal jelly can be obtained, for example, by extracting and purifying royal jelly using an organic solvent or the like.
１０Ｈ２ＤＡ、１０ＨＤＡ、２－ＤＡ又はＳＡの塩としては、特に制限されないが、例えば、ナトリウム塩、カリウム塩等のアルカリ金属塩；カルシウム塩、マグネシウム塩等のアルカリ土類金属塩；アンモニウム塩；トリメチルアミン塩、トリエチルアミン塩、シクロヘキシルアミン塩、エタノールアミン塩、ジエタノールアミン塩、トリエタノールアミン塩、プロカイン塩等の脂肪族アミン塩；Ｎ，Ｎ－ジベンジルエチレンジアミン等のアラルキルアミン塩；ピリジン塩、ピコリン塩、キノリン塩、イソキノリン塩等の複素環芳香族アミン塩；テトラメチルアンモニウム塩、テトラエチルアンモニウム塩、ベンジルトリメチルアンモニウム塩、ベンジルトリブチルアンモニウム塩、メチルトリオクチルアンモニウム塩、テトラブチルアンモニウム塩等の第４級アンモニウム塩；アルギニン塩、リジン塩、ヒスチジン塩、アルギニン塩、アスパラギン酸塩、グルタミン酸塩等のアミノ酸塩；塩酸塩、硫酸塩、硝酸塩、リン酸塩、炭酸塩、炭酸水素塩、過塩素酸塩等の無機酸塩；酢酸塩、プロピオン酸塩、乳酸塩、マレイン酸塩、フマル酸塩、酒石酸塩、リンゴ酸塩、クエン酸塩、アスコルビン酸塩等の有機酸塩；メタンスルホン酸塩、イセチオン酸塩、ベンゼンスルホン酸塩、ｐ－トルエンスルホン酸塩等のスルホン酸塩等が挙げられる。 Salts of 10H2DA, 10HDA, 2-DA or SA are not particularly limited, but include, for example, alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; trimethylamine salts. , aliphatic amine salts such as triethylamine salt, cyclohexylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, procaine salt; aralkylamine salt such as N,N-dibenzylethylenediamine; pyridine salt, picoline salt, quinoline salt , heterocyclic aromatic amine salts such as isoquinoline salt; quaternary ammonium salts such as tetramethylammonium salt, tetraethylammonium salt, benzyltrimethylammonium salt, benzyltributylammonium salt, methyltrioctylammonium salt, tetrabutylammonium salt; arginine Amino acid salts such as salts, lysine salts, histidine salts, arginine salts, aspartates, glutamates; inorganic acid salts such as hydrochlorides, sulfates, nitrates, phosphates, carbonates, hydrogen carbonates, perchlorates, etc. ; Organic acid salts such as acetate, propionate, lactate, maleate, fumarate, tartrate, malate, citrate, ascorbate; methanesulfonate, isethionate, benzenesulfone Examples include acid salts, sulfonate salts such as p-toluenesulfonate, and the like.
本実施形態に係る表皮幹細胞活性化剤は、ローヤルゼリー、１０－ヒドロキシ－２－デセン酸若しくはその塩、１０－ヒドロキシデカン酸若しくはその塩、デセン二酸若しくはその塩、及び、セバシン酸若しくはその塩からなる群より選択される少なくとも一つを有効成分として含有するため、表皮幹細胞を活性化させる作用を有する。「表皮幹細胞を活性化させる」とは、表皮幹細胞の幹細胞性を維持しつつ、増殖性を向上させることを意味する。また、「幹細胞性」とは、自己増殖能を有しかつ分化多能性を有することを意味する。つまり、本実施形態に係る表皮幹細胞活性化剤は、表皮幹細胞の幹細胞性を維持しつつ、増殖性を向上させるための組成物とも言い換えられる。 The epidermal stem cell activator according to the present embodiment is made of royal jelly, 10-hydroxy-2-decenoic acid or a salt thereof, 10-hydroxydecanoic acid or a salt thereof, decenedioic acid or a salt thereof, and sebacic acid or a salt thereof. Since it contains at least one selected from the group consisting of as an active ingredient, it has the effect of activating epidermal stem cells. "Activating epidermal stem cells" means improving proliferation while maintaining the stemness of epidermal stem cells. Moreover, "stem cell nature" means having self-propagation ability and having pluripotency. In other words, the epidermal stem cell activator according to the present embodiment can be said to be a composition for improving the proliferative property of epidermal stem cells while maintaining their stemness.
表皮幹細胞の幹細胞性は未分化マーカーの発現によって確認することができ、増殖性は細胞増殖性マーカーの発現によって確認することができる。未分化性マーカーとしては、例えば、ｐ６３等が挙げられる。細胞増殖性マーカーとしては、Ｋｉ６７、ＰＣＮＡ等が挙げられる。 The stemness of epidermal stem cells can be confirmed by the expression of undifferentiated markers, and the proliferation ability of epidermal stem cells can be confirmed by the expression of cell proliferation markers. Examples of undifferentiated markers include p63. Examples of cell proliferation markers include Ki67 and PCNA.
表皮幹細胞の活性化は、例えば、表皮幹細胞に対して本実施形態に係る表皮幹細胞活性化剤を有効量使用した場合、表皮幹細胞活性化剤を使用しない場合と比べて、未分化性マーカー及び細胞増殖性マーカーを発現する表皮幹細胞数が、１０％以上の増加であればよく、２０％以上、３０％以上、５０％以上、７０％以上、又は１００％以上の増加であることが好ましい。 For example, when an effective amount of the epidermal stem cell activator according to the present embodiment is used for epidermal stem cells, the activation of epidermal stem cells is more likely to occur when the undifferentiated markers and cells are The number of epidermal stem cells expressing proliferative markers may increase by 10% or more, preferably by 20% or more, 30% or more, 50% or more, 70% or more, or 100% or more.
本実施形態に係る表皮幹細胞活性化剤は、上記有効成分によって表皮幹細胞を活性化させることにより、皮膚のターンオーバーの低下の抑制作用、皮膚の老化の抑制作用、及び／又は皮膚の菲薄化の抑制作用を有する。 The epidermal stem cell activator according to the present embodiment activates epidermal stem cells with the above-mentioned active ingredient, thereby inhibiting a decrease in skin turnover, inhibiting skin aging, and/or inhibiting skin thinning. Has an inhibitory effect.
皮膚のターンオーバーは、表皮のターンオーバーを意味し、生体の恒常性の維持に重要である一方で、加齢又は肌ストレス等により表皮のターンオーバーが乱れると、肌の菲薄化、色素沈着、くすみ、肌のトーン、及び小じわ等の皮膚老化症状の原因となる。より具体的には、表皮のターンオーバーは、表皮幹細胞の増殖と分化のバランス、さらに表皮角化細胞の増殖により維持されている。加齢又は肌ストレスは、表皮幹細胞及び表皮角化細胞の老化を誘発し、表皮のターンオーバーを低下させると考えられている。すなわち、本実施形態に係る表皮幹細胞活性化剤は、表皮幹細胞を活性化することで、表皮のターンオーバーの低下を抑制し、上記の皮膚老化症状の改善が期待できる。表皮のターンオーバーの低下抑制作用は、例えば、三次元皮膚モデルの基底層における表皮幹細胞の数及び増殖能、さらにそれらに伴う表皮の厚みの評価、又は平面培養系における表皮角化細胞の細胞増殖能等で評価できる。 Skin turnover refers to the turnover of the epidermis and is important for maintaining homeostasis of the body. However, when the turnover of the epidermis is disrupted due to aging or skin stress, skin thinning, pigmentation, It causes skin aging symptoms such as dullness, skin tone, and fine lines. More specifically, epidermal turnover is maintained by the balance between proliferation and differentiation of epidermal stem cells, as well as by the proliferation of epidermal keratinocytes. Aging or skin stress is thought to induce senescence of epidermal stem cells and epidermal keratinocytes and reduce epidermal turnover. That is, the epidermal stem cell activator according to the present embodiment suppresses a decrease in epidermal turnover by activating epidermal stem cells, and can be expected to improve the above-mentioned skin aging symptoms. The effect of suppressing the decrease in epidermal turnover can be evaluated, for example, by evaluating the number and proliferation ability of epidermal stem cells in the basal layer of a three-dimensional skin model, as well as the thickness of the epidermis associated with these, or the cell proliferation of epidermal keratinocytes in a flat culture system. It can be evaluated based on ability etc.
皮膚の菲薄化抑制作用は、例えば、表皮幹細胞に本実施形態に係る表皮幹細胞活性化剤を有効量使用した場合、表皮幹細胞活性化剤を使用しない場合と比べて、表皮の厚みが１０％以上の増加であればよく、２０％以上、３０％以上、５０％以上、７０％以上、又は１００％以上の増加であることが好ましい。表皮の厚みは、特に制限されないが、基底層から角質層までの厚みであってもよいし、基底層から顆粒層までの厚みであってもよい。 The effect of suppressing skin thinning is, for example, when an effective amount of the epidermal stem cell activator according to the present embodiment is used for epidermal stem cells, the thickness of the epidermis is 10% or more compared to when no epidermal stem cell activator is used. It is sufficient that the increase is 20% or more, 30% or more, 50% or more, 70% or more, or 100% or more. The thickness of the epidermis is not particularly limited, but may be the thickness from the basal layer to the stratum corneum, or the thickness from the basal layer to the granular layer.
皮膚の老化は、表皮における細胞（表皮幹細胞及び表皮角化細胞）の老化を意味し、細胞老化とは、加齢、ＤＮＡ損傷又はストレス等により誘導され、細胞が安定的に細胞増殖を停止した状態であることを指す。老化細胞の特徴としては形態学的な変化、代謝変化、遺伝子発現変化等様々である。表皮では、表皮幹細胞の老化又は表皮角化細胞の老化が、加齢による肌表現型を誘発する要因の一つであると考えられる。すなわち、本実施形態に係る表皮幹細胞活性化剤は、表皮幹細胞を活性化させることで、皮膚の老化を抑制することが期待できる。皮膚の老化の抑制作用は、例えば表皮における細胞倍加時間、細胞周期の変化、或いは、表皮における細胞における老化により特異的に増加するＳＡβガラクトシダーゼ活性、ｐ１６、ｐ２１遺伝子発現、又は老化により低下するテロメア長、若しくはラミン発現等の複数の指標で評価できる。 Skin aging refers to the aging of cells in the epidermis (epidermal stem cells and epidermal keratinocytes), and cellular senescence is induced by aging, DNA damage, stress, etc., and cells have stably stopped cell proliferation. Refers to a state. Senescent cells have various characteristics such as morphological changes, metabolic changes, and gene expression changes. In the epidermis, senescence of epidermal stem cells or senescence of epidermal keratinocytes is considered to be one of the factors that induce age-related skin phenotypes. That is, the epidermal stem cell activator according to this embodiment can be expected to suppress skin aging by activating epidermal stem cells. The effect of suppressing skin aging is, for example, due to cell doubling time in the epidermis, changes in the cell cycle, SAβ galactosidase activity, p16, p21 gene expression, which increases specifically with aging in cells in the epidermis, or telomere length, which decreases with aging. , or can be evaluated using multiple indicators such as lamin expression.
本実施形態に係る表皮幹細胞活性化剤における上記有効成分の含有量は、表皮幹細胞活性化剤全量に対して乾燥固形分換算で０．１質量以上１００質量％以下であってよく、０．１質量％以上、１質量％以上、３質量％以上、３．５質量％以上、５質量％以上、７質量％以上、１０質量％以上、１５質量％以上、２０質量％以上、２５質量％以上、３０質量％以上、３５質量％以上、４０質量％以上、４５質量％以上、５０質量％以上、５５質量％以上、６０質量％以上、６５質量％以上、７０質量％以上、７５質量％以上、８０質量％以上、８５質量％以上、９０質量％以上、９３質量％以上、９５質量％以上、９８質量％以上、９９質量％以上、又は１００質量％であってもよい。また、本実施形態に係る表皮幹細胞活性化剤における上記有効成分の含有量は、表皮幹細胞活性剤全量に対して乾燥固形分換算で１００質量％以下、９９質量％以下、９８質量％以下、９５質量％以下、９３質量％以下、９０質量％以下、８５質量％以下、８０質量％以下、７５質量％以下、７０質量％以下、６５質量％以下、６０質量％以下、５５質量％以下、５０質量％以下、４５質量％以下、４０質量％以下、３５質量％以下、３０質量％以下、２５質量％以下、２０質量％以下、１５質量％以下、１０質量％以下、８質量％以下、５質量％以下、３質量％以下、又は１質量％以下であってもよい。 The content of the active ingredient in the epidermal stem cell activator according to the present embodiment may be 0.1% by mass or more and 100% by mass or less in terms of dry solid content based on the total amount of the epidermal stem cell activator, and may be 0.1% by mass or more and 100% by mass or less Mass% or more, 1 mass% or more, 3 mass% or more, 3.5 mass% or more, 5 mass% or more, 7 mass% or more, 10 mass% or more, 15 mass% or more, 20 mass% or more, 25 mass% or more , 30% by mass or more, 35% by mass or more, 40% by mass or more, 45% by mass or more, 50% by mass or more, 55% by mass or more, 60% by mass or more, 65% by mass or more, 70% by mass or more, 75% by mass or more , 80% by mass or more, 85% by mass or more, 90% by mass or more, 93% by mass or more, 95% by mass or more, 98% by mass or more, 99% by mass or more, or 100% by mass. Further, the content of the above-mentioned active ingredient in the epidermal stem cell activator according to the present embodiment is 100% by mass or less, 99% by mass or less, 98% by mass or less, 95% by mass or less in terms of dry solid content based on the total amount of the epidermal stem cell activator. mass% or less, 93 mass% or less, 90 mass% or less, 85 mass% or less, 80 mass% or less, 75 mass% or less, 70 mass% or less, 65 mass% or less, 60 mass% or less, 55 mass% or less, 50 mass% or less, 45 mass% or less, 40 mass% or less, 35 mass% or less, 30 mass% or less, 25 mass% or less, 20 mass% or less, 15 mass% or less, 10 mass% or less, 8 mass% or less, 5 It may be less than or equal to 3% by mass, or less than or equal to 1% by mass.
本実施形態に係る表皮幹細胞活性化剤における上記含有量は、有効成分としてローヤルゼリー、１０－ヒドロキシ－２－デセン酸若しくはその塩、１０－ヒドロキシデカン酸若しくはその塩、デセン二酸若しくはその塩、及び、セバシン酸若しくはその塩からなる群より２つ以上が選択された場合、それぞれの成分の含有量を適宜に調節してもよい。 The above content in the epidermal stem cell activator according to the present embodiment includes royal jelly, 10-hydroxy-2-decenoic acid or a salt thereof, 10-hydroxydecanoic acid or a salt thereof, decenedioic acid or a salt thereof, and , sebacic acid, or a salt thereof, the content of each component may be adjusted as appropriate.
本実施形態の表皮幹細胞活性化剤は、例えば、実験的に表皮幹細胞を活性化させる場合等に使用することができ、また、体内での表皮幹細胞の活性化に使用するためにヒトに投与することもできる。本実施形態の表皮幹細胞活性化剤は、経口投与でもよく、非経口投与でもよい。経口投与は経腸投与を含み、非経口投与は局所投与を含み、特に経皮投与を含む。 The epidermal stem cell activator of this embodiment can be used, for example, to experimentally activate epidermal stem cells, and can also be administered to humans for use in activating epidermal stem cells in the body. You can also do that. The epidermal stem cell activator of this embodiment may be administered orally or parenterally. Oral administration includes enteral administration, and parenteral administration includes topical administration, especially transdermal administration.
本実施形態の表皮幹細胞活性化剤が経口投与される場合の投与量は、有効成分、組成物の形態及び適用方法・適用量によって異なり得るが、例えば、有効成分がローヤルゼリーである場合、体重６０ｋｇの成人に一日当たり、乾燥固形分換算で１０ｍｇ～３００００ｍｇのローヤルゼリーであればよく、好ましくは１００ｍｇ～２００００ｍｇ、１５０ｍｇ～１５０００ｍｇ、６００ｍｇ～１２０００ｍｇ、１２００ｍｇ～１００００ｍｇ、又は２４００ｍｇ～８０００ｍｇのローヤルゼリーである。 The dose when the epidermal stem cell activator of this embodiment is orally administered may vary depending on the active ingredient, the form of the composition, and the method and amount of application; for example, if the active ingredient is royal jelly, The amount of royal jelly required for an adult per day is 10 mg to 30,000 mg in terms of dry solid content, preferably 100 mg to 20,000 mg, 150 mg to 15,000 mg, 600 mg to 12,000 mg, 1,200 mg to 10,000 mg, or 2,400 mg to 8,000 mg.
また、有効成分が、１０－ヒドロキシ－２－デセン酸又はその塩である場合、投与量が体重６０ｋｇの成人に一日当たり、１ｍｇ～２００ｍｇの１０－ヒドロキシ－２－デセン酸又はその塩であればよく、好ましくは１０ｍｇ～１００ｍｇの１０－ヒドロキシ－２－デセン酸又はその塩である。有効成分が、１０－ヒドロキシデカン酸又はその塩である場合、投与量が体重６０ｋｇの成人に一日当たり、０．１ｍｇ～５０ｍｇの１０－ヒドロキシデカン酸又はその塩であればよく、好ましくは１ｍｇ～２０ｍｇの１０－ヒドロキシデカン酸又はその塩である。有効成分が、デセン二酸又はその塩である場合、投与量が体重６０ｋｇの成人に一日当たり、０．１ｍｇ～３０ｍｇのデセン二酸又はその塩であればよく、好ましくは１ｍｇ～１５ｍｇのデセン二酸又はその塩である。有効成分が、セバシン酸又はその塩である場合、投与量が体重６０ｋｇの成人に一日当たり、０．１ｍｇ～２０ｍｇのセバシン酸又はその塩であればよく、好ましくは０．５ｍｇ～１５ｍｇのセバシン酸又はその塩である。 In addition, when the active ingredient is 10-hydroxy-2-decenoic acid or its salt, the dosage is 1 mg to 200 mg of 10-hydroxy-2-decenoic acid or its salt per day for an adult weighing 60 kg. Often, preferably 10 mg to 100 mg of 10-hydroxy-2-decenoic acid or a salt thereof. When the active ingredient is 10-hydroxydecanoic acid or a salt thereof, the dosage may be 0.1 mg to 50 mg of 10-hydroxydecanoic acid or a salt thereof, preferably 1 mg to 50 mg per day for an adult weighing 60 kg. 20 mg of 10-hydroxydecanoic acid or its salt. When the active ingredient is decenedioic acid or a salt thereof, the dosage may be 0.1 mg to 30 mg, preferably 1 mg to 15 mg of decenedioic acid or a salt thereof, per day for an adult weighing 60 kg. It is an acid or its salt. When the active ingredient is sebacic acid or a salt thereof, the dosage may be 0.1 mg to 20 mg of sebacic acid or a salt thereof, preferably 0.5 mg to 15 mg of sebacic acid per day for an adult weighing 60 kg. Or its salt.
当該投与量は、摂取する人の健康状態、投与方法、有効成分の種類及び他の剤との組み合わせ等の因子に応じて、適宜増減することができる。本実施形態に係る表皮幹細胞活性化剤は、一日当たりの有効投与量が上述した範囲内にあれば、一日一回投与されてもよいし、一日二回、一日三回等、複数回に分けて投与されてもよい。本実施形態の表皮幹細胞活性化剤は、投与してすぐ効果が得られるが、１～４週間又は１か月以上、６か月以上、１年以上の継続的な投与は、効果をより持続できるため、好ましい。 The dosage can be increased or decreased as appropriate depending on factors such as the health condition of the person taking it, the method of administration, the type of active ingredient, and the combination with other agents. The epidermal stem cell activator according to the present embodiment may be administered once a day, twice a day, three times a day, etc., as long as the effective daily dose is within the above-mentioned range. It may be administered in divided doses. The epidermal stem cell activating agent of this embodiment can be effective immediately after administration, but continuous administration for 1 to 4 weeks, 1 month or more, 6 months or more, or 1 year or more will make the effect more sustained. This is preferable because it can be done.
本実施形態の表皮幹細胞活性化剤が非経口投与される場合の投与量は、有効成分、適用する部位及び適用範囲によって異なり得るが、例えば、有効成分がローヤルゼリーである場合、皮膚への適用量が、乾燥固形分換算で０．０１ｍｇ～５０ｍｇの有効成分であればよく、好ましくは０．０２ｍｇ～４０ｍｇであればよく、０．０２ｍｇ～３５ｍｇ、又は、０．０２５ｍｇ～３０ｍｇの有効成分である。 When the epidermal stem cell activator of this embodiment is administered parenterally, the dose may vary depending on the active ingredient, the site of application, and the scope of application. For example, if the active ingredient is royal jelly, the dose applied to the skin The active ingredient may be 0.01 mg to 50 mg, preferably 0.02 mg to 40 mg, or 0.02 mg to 35 mg, or 0.025 mg to 30 mg in terms of dry solid content. .
また、有効成分が、１０－ヒドロキシ－２－デセン酸又はその塩である場合、皮膚への適用量が、０．００１ｍｇ～０．３ｍｇの有効成分であればよく、好ましくは０．０１ｍｇ～０．１ｍｇの有効成分である。有効成分が１０－ヒドロキシデカン酸又はその塩である場合、皮膚への適用量が、０．０００１ｍｇ～０．０８ｍｇの有効成分であればよく、好ましくは０．００１ｍｇ～０．０６ｍｇの有効成分である。有効成分がデセン二酸又はその塩である場合、皮膚への適用量が、０．０００１ｍｇ～０．０５ｍｇの有効成分であればよく、好ましくは０．００１ｍｇ～０．０４ｍｇの有効成分である。有効成分がセバシン酸又はその塩である場合、皮膚への適用量が、０．０００１ｍｇ～０．０３ｍｇの有効成分であればよく、好ましくは０．００１ｍｇ～０．０２ｍｇの有効成分である。 Further, when the active ingredient is 10-hydroxy-2-decenoic acid or its salt, the amount applied to the skin may be 0.001 mg to 0.3 mg, preferably 0.01 mg to 0. .1mg of active ingredient. When the active ingredient is 10-hydroxydecanoic acid or a salt thereof, the amount applied to the skin may be 0.0001 mg to 0.08 mg, preferably 0.001 mg to 0.06 mg. be. When the active ingredient is decenedioic acid or a salt thereof, the amount applied to the skin may be 0.0001 mg to 0.05 mg, preferably 0.001 mg to 0.04 mg. When the active ingredient is sebacic acid or a salt thereof, the amount applied to the skin may be 0.0001 mg to 0.03 mg, preferably 0.001 mg to 0.02 mg.
本発明の一実施形態は、上記実施形態の表皮幹細胞活性化剤を有効成分として含む組成物を提供し、該組成物は経口組成であっても、局所用組成物であってもよく、特に上記実施形態の表皮幹細胞活性化剤を有効成分として含む、化粧品、食品組成物又は医薬組成物を提供する。本実施形態の表皮幹細胞活性剤、又は、化粧品、食品組成物若しくは医薬組成物をヒトに適用し、ヒトの表皮幹細胞に作用させることによって、表皮幹細胞を活性化することで、皮膚のターンオーバーの低下を抑制する効果、皮膚の老化を抑制する効果、及び／又は皮膚の菲薄化を抑制する効果も得られる。それによって、美肌効果、皮膚のアンチエイジング効果を得ることができる。 One embodiment of the present invention provides a composition comprising the epidermal stem cell activator of the above embodiment as an active ingredient, and the composition may be an oral composition or a topical composition, particularly Cosmetics, food compositions, or pharmaceutical compositions containing the epidermal stem cell activator of the above embodiment as an active ingredient are provided. The epidermal stem cell activator of this embodiment, or the cosmetic, food composition, or pharmaceutical composition is applied to humans and acts on human epidermal stem cells, thereby activating the epidermal stem cells and thereby reducing skin turnover. The effect of suppressing skin deterioration, the effect of suppressing skin aging, and/or the effect of suppressing skin thinning can also be obtained. As a result, skin beautification and skin anti-aging effects can be obtained.
本実施形態の表皮幹細胞活性剤を含む化粧品、食品組成物又は医薬組成物における上記有効成分の含有量は特に限定されず、上記表皮幹細胞活性化剤の経口投与又は非経口投与の投与量を達成できる有効量であればよい。 The content of the above-mentioned active ingredient in the cosmetic, food composition, or pharmaceutical composition containing the epidermal stem cell activator of this embodiment is not particularly limited, and the dose that achieves the oral or parenteral administration of the above-mentioned epidermal stem cell activator is not particularly limited. Any effective amount is sufficient.
本実施形態に係る表皮幹細胞活性化剤、又は表皮幹細胞活性化剤を含む化粧品、食品組成物若しくは医薬組成物は、上記有効成分に加えて、他の成分をさらに含んでいてもよい。他の成分としては、例えば、薬学的に許容される成分（例えば、賦形剤、結合材、滑沢剤、崩壊剤、乳化剤、界面活性剤、基剤、溶解補助剤、懸濁化剤）、食品として許容される成分（例えば、ミネラル類、ビタミン類、フラボノイド類、キノン類、ポリフェノール類、アミノ酸、核酸、必須脂肪酸、清涼剤、結合剤、甘味料、崩壊剤、滑沢剤、着色料、香料、安定化剤、防腐剤、徐放調整剤、界面活性剤、溶解剤、湿潤剤）、化粧品として許容される成分（例えば、美白剤、保湿剤、酸化防止剤、油性成分、紫外線吸収剤、界面活性剤、増粘剤、アルコール類、粉末成分、色材、水性成分、水、各種皮膚栄養剤）を挙げることができる。 The epidermal stem cell activator according to this embodiment, or the cosmetic, food composition, or pharmaceutical composition containing the epidermal stem cell activator may further contain other ingredients in addition to the above-mentioned active ingredients. Other ingredients include, for example, pharmaceutically acceptable ingredients (e.g., excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizing agents, suspending agents). , ingredients acceptable as foods (e.g. minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants) , fragrances, stabilizers, preservatives, sustained release modifiers, surfactants, solubilizers, humectants), cosmetically acceptable ingredients (e.g. whitening agents, humectants, antioxidants, oily ingredients, ultraviolet absorbers) agents, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, and various skin nutrients).
本実施形態に係る表皮幹細胞活性化剤、又は表皮幹細胞活性化剤を含む食品組成物若しくは医薬組成物は、固体、液体、ペースト等のいずれの形状であってもよく、錠剤（素錠、糖衣錠、発泡錠、フィルムコート錠、チュアブル錠、トローチ剤等を含む）、カプセル剤、丸剤、粉末剤（散剤）、細粒剤、顆粒剤、液剤、懸濁液、乳濁液、シロップ、ペースト、注射剤（使用時に、蒸留水又はアミノ酸輸液若しくは電解質輸液等の輸液に配合して液剤として調製する場合を含む）、塗布剤若しくは貼付剤等の局所用剤形等の剤形であってもよい。これらの各種製剤は、例えば、有効成分と、必要に応じて他の成分とを混合して上記剤形に成形することによって調製することができる。 The epidermal stem cell activator according to the present embodiment, or the food composition or pharmaceutical composition containing the epidermal stem cell activator may be in any form such as solid, liquid, paste, etc. (including effervescent tablets, film-coated tablets, chewable tablets, lozenges, etc.), capsules, pills, powders, fine granules, granules, liquids, suspensions, emulsions, syrups, and pastes. , even if it is in the form of an injection (including when prepared as a liquid by mixing with distilled water or an infusion such as an amino acid infusion or an electrolyte infusion at the time of use), or a topical dosage form such as a liniment or patch. good. These various preparations can be prepared, for example, by mixing the active ingredient and, if necessary, other ingredients, and molding the mixture into the above-mentioned dosage form.
食品組成物としては、食品の３次機能（体調調節機能）が強調された食品であることが好ましい。食品の３次機能が強調された食品としては、例えば、健康食品、機能性表示食品、栄養機能食品、栄養補助食品、サプリメント及び特定保健用食品を挙げることができる。 The food composition is preferably a food that emphasizes the tertiary function (physical condition regulating function) of the food. Foods with emphasized tertiary functions include, for example, health foods, foods with functional claims, nutritionally functional foods, nutritional supplements, supplements, and foods for specified health uses.
食品組成物の形態は特に限定されず、例えば、飲料類（コーヒー、ジュース、茶飲料等の清涼飲料、乳飲料、乳酸菌飲料、ヨーグルト飲料、炭酸飲料等）；スプレッド類（カスタードクリーム等）；ペースト類（フルーツペースト等）；洋菓子類（チョコレート、ドーナツ、パイ、シュークリーム、ガム、ゼリー、キャンデー、クッキー、ケーキ、プリン等）；和菓子類（大福、餅、饅頭、カステラ、あんみつ、羊羹等）；氷菓類（アイスクリーム、アイスキャンデー、シャーベット等）；食品類（カレー、牛丼、雑炊、味噌汁、スープ、ミートソース、パスタ、漬物、ジャム等）；調味料類（ドレッシング、ふりかけ、旨味調味料、スープの素等）であってもよい。 The form of the food composition is not particularly limited, and includes, for example, beverages (coffee, juice, tea drinks, and other soft drinks, milk drinks, lactic acid bacteria drinks, yogurt drinks, carbonated drinks, etc.); spreads (custard cream, etc.); and pastes. (fruit paste, etc.); Western sweets (chocolate, donuts, pies, cream puffs, gum, jelly, candy, cookies, cakes, pudding, etc.); Japanese sweets (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); (Ice cream, popsicles, sherbet, etc.); Foods (curry, gyudon, rice porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); Seasonings (dressing, furikake, umami seasonings, soup seasonings, etc.); (element etc.) may be used.
化粧品の剤形は、例えば、可溶化系、乳化系、粉末系、油液系、ゲル系、軟膏系、エアゾール系、水－油２層系、水－油－粉末３層系等であってよい。化粧品としては、例えば、洗顔料、化粧水、乳液、クリーム、ジェル、エッセンス、美容液、パック、マスク、ミスト、ＵＶ予防化粧品等の基礎化粧品、ファンデーション、口紅、頬紅、アイシャドウ、イライナー、マスカラ等のメークアップ化粧品、洗顔料、マッサージ用剤、クレンジング用剤、アフターシェーブローション、プレシェーブローション、シェービングクリーム、ボディソープ、石けん、シャンプー、リンス、へアートリートメント、整髪料、へアートニック剤、ヘアミスト、ヘアフォーム、ヘアリキッド、ヘアジェル、ヘアスプレー、育毛剤、制汗剤、入浴剤、マウスリンス、口腔化粧品、歯磨剤、ハンドクリーム、ハンドソープ等であってよい。 Dosage forms for cosmetics include, for example, solubilized systems, emulsified systems, powder systems, oil liquid systems, gel systems, ointment systems, aerosol systems, water-oil two-layer systems, water-oil-powder three-layer systems, etc. good. Examples of cosmetics include facial cleansers, lotions, emulsions, creams, gels, essences, serums, packs, masks, mist, basic cosmetics such as UV prevention cosmetics, foundations, lipsticks, blushers, eye shadows, eye liners, mascara, etc. Make-up cosmetics, facial cleansers, massage agents, cleansing agents, aftershave lotions, pre-shave lotions, shaving creams, body soaps, soaps, shampoos, conditioners, hair treatments, hair conditioners, hair tonic agents, hair mist, hair foam. , hair liquid, hair gel, hair spray, hair restorer, antiperspirant, bath additive, mouth rinse, oral cosmetics, toothpaste, hand cream, hand soap, etc.
以下、本発明を実施例に基づいてより具体的に説明する。ただし、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail based on Examples. However, the present invention is not limited to the following examples.
（実験例１：三次元培養系を用いた生ローヤルゼリー及び酵素分解ローヤルゼリーによる表皮幹細胞活性化能の検討）
［１．ヒト表皮角化細胞の三次元培養］
表皮幹細胞を含む、ヒト表皮角化細胞（ＨＰＥＫｐ、Ｊｕｖｅｎｉｌｅ Ｐｏｏｌｅｄ、セルンテック社製）を、ＣｎＴ－Ｐｒｉｍｅ、Ｅｐｉｔｈｅｌｉａｌ Ｃｕｌｔｕｒｅ Ｍｅｄｉｕｍ（セルンテック社製）を用い、３７℃、５％ＣＯ２下でサブコンフルエントに達するまで培養を行った。培養したＨＰＥＫｐをＰＢＳで１回洗浄し、アキュターゼ（登録商標、イノベーティブセルテクノロジーズ社製）で処理することで、細胞を剥離した。剥離されたＨＰＥＫｐをＣｎＴ－Ｐｒｉｍｅ培地に懸濁し、遠心分離（１，１００ｒｐｍ、３ｍｉｎ）により回収した。回収したＨＰＥＫｐの細胞数をカウントし、ＣｎＴ－Ｐｒｉｍｅ培地に懸濁し、三次元培養に用いた。
(Experimental example 1: Examination of epidermal stem cell activation ability by raw royal jelly and enzymatically decomposed royal jelly using a three-dimensional culture system)
[1. Three-dimensional culture of human epidermal keratinocytes]
Human epidermal keratinocytes (HPEKp, Juvenile Pooled, manufactured by Cerntech), including epidermal stem cells, are grown to subconfluence at 37°C and 5% CO2 using CnT-Prime, Epithelial Culture Medium (manufactured by Cerntech). Culture was carried out until. The cultured HPEKp was washed once with PBS and treated with Accutase (registered trademark, manufactured by Innovative Cell Technologies) to detach the cells. The detached HPEKp was suspended in CnT-Prime medium and collected by centrifugation (1,100 rpm, 3 min). The number of recovered HPEKp cells was counted, suspended in CnT-Prime medium, and used for three-dimensional culture.
６０ｍｍ培養シャーレに、三次元培養用のカルチャーインサート（Ｍｉｌｌｉｃｅｌｌ－ＰＣＦ ｃｅｌｌ ｃｕｌｔｕｒｅ ｉｎｓｅｒｔ、フィルター孔径：０．４μｍ、フィルター直径：１２ｍｍ、メルクミリポア社製）を置き、インサートあたり、２×１０５細胞／４００μＬとなるように調整した細胞懸濁液をインサート内部に播種し、インサート外部にはＣｎＴ－Ｐｒｉｍｅ培地を添加して３日間培養した。培養３日後にインサート内外の培地を三次元培養用培地であるＣｎＴ－ＰＲ－３Ｄ培地（セルンテック社製）に置き換えた。さらに１日後に、インサート内の培地のみを除き、インサート外の培地を交換することで気層液層培養を開始した。インサート外の培地交換の際、生ローヤルゼリー（ＮＲＪ、山田養蜂場社製）、又は酵素分解ローヤルゼリー（ＥＲＪ、山田養蜂場社製）を添加したＣｎＴ－ＰＲ－３Ｄ培地を用いた。具体的には、ＮＲＪ及びＥＲＪは、０．４５μｍフィルター（ミリポア社製）、さらに０．２μｍフィルター（ミリポア社製）を通して滅菌を行ったのち、１００又は４００μｇ／ｍＬとなるようにＣｎＴ－ＰＲ－３Ｄ培地に添加し、該培地をインサート外に添加した。培養２、５、９、１１、及び１３日後にそれぞれ培地交換を実施し、培養終了まで継続してＮＲＪ又はＥＲＪを添加した培地で培養した。 A culture insert for three-dimensional culture (Millicell-PCF cell culture insert, filter pore size: 0.4 μm, filter diameter: 12 mm, manufactured by Merck Millipore) was placed in a 60 mm culture dish, and 2 × 10 5 cells/400 μL per insert. A cell suspension prepared as follows was seeded inside the insert, and CnT-Prime medium was added to the outside of the insert and cultured for 3 days. After 3 days of culture, the medium inside and outside the insert was replaced with CnT-PR-3D medium (manufactured by Cerntech), which is a medium for three-dimensional culture. After one day, only the medium inside the insert was removed and the medium outside the insert was replaced to start air-layer culture. When replacing the medium outside the insert, a CnT-PR-3D medium supplemented with fresh royal jelly (NRJ, manufactured by Yamada Bee Farm Co., Ltd.) or enzymatically degraded royal jelly (ERJ, manufactured by Yamada Bee Farm Co., Ltd.) was used. Specifically, NRJ and ERJ are sterilized through a 0.45 μm filter (manufactured by Millipore) and then a 0.2 μm filter (manufactured by Millipore), and then CnT-PR- 3D medium and the medium was added outside the insert. The medium was replaced after 2, 5, 9, 11, and 13 days of culture, and the cells were cultured in a medium supplemented with NRJ or ERJ until the end of culture.
［２．細胞固定］
インサートを回収し、上記１．で得られた三次元培養表皮を４％パラホルムアルデヒド（ＰＦＡ）固定液につけ細胞固定を行った後、ＰＢＳで洗い、１０％ショ糖含有ＰＢＳ溶液に浸漬し、シェーカーで浸透処理を行った。続いて、２０％ショ糖含有ＰＢＳ溶液に置き換えさらに浸透処理を行った。その後、さらに新鮮な２０％ショ糖含有ＰＢＳ溶液に置き換え４℃で一晩浸透処理した。インサート内の膜を回収しＯＣＴコンパウンド（サクラファインテックジャパン社製）に浸漬させたのち、凍結切片作製用の包埋皿クリオモルド１号に垂直に置き、液体窒素の上で固め、液体窒素を用いて包埋した。
[2. Cell fixation]
Collect the insert and proceed as described in 1. The three-dimensionally cultured epidermis obtained was immersed in a 4% paraformaldehyde (PFA) fixative to fix cells, then washed with PBS, immersed in a PBS solution containing 10% sucrose, and permeated with a shaker. Subsequently, the solution was replaced with a PBS solution containing 20% sucrose and further permeation treatment was performed. Thereafter, the solution was replaced with a fresh 20% sucrose-containing PBS solution and the permeation treatment was carried out at 4°C overnight. After collecting the membrane inside the insert and immersing it in OCT compound (manufactured by Sakura Finetech Japan), it was placed vertically in an embedding dish Cryomold No. 1 for frozen section preparation, solidified on liquid nitrogen, and then immersed in OCT compound (manufactured by Sakura Finetech Japan). It was embedded.
［３．表皮凍結切片作成と染色、画像取得］
クリオスタット（ライカ社製）を用い、上記２．で固定及び包埋した三次元培養表皮から１０μｍ厚の表皮凍結切片を作成し、スライドガラスに貼り付け、ヘマトキシリン・エオジン染色（ＨＥ染色）及び免疫染色を行った。
[3. Epidermal frozen section preparation, staining, and image acquisition]
Using a cryostat (manufactured by Leica), perform the above 2. A 10 μm thick frozen section of the epidermis was prepared from the three-dimensionally cultured epidermis fixed and embedded in , and was pasted on a glass slide, followed by hematoxylin and eosin staining (HE staining) and immunostaining.
ＨＥ染色は、１０μｍ厚の凍結切片を作成し、水道水でＯＣＴコンパウンドを洗い流した後、マイヤー・ヘマトキシリン染色液につけて５分間核を染色した。その後、流水で１０分間洗い、色出しを行った。０．１％エオシンＹ染色液で２分間細胞質を染色した後、７０％、８０％、１００％エタノールで段階的に脱水し、レモゾール（富士フイルム和光純薬社製）に１分間、３回つけて透徹を行った後、エンテランニュー（メルクミリポア社製）で封入した。オールインワン蛍光顕微鏡（キーエンス社製）を用いて、ＨＥ染色した表皮凍結切片を観察し、代表的な画像を図１に示した。また、三次元培養表皮の厚さ（基底層から顆粒層までの厚さ）を測定し、図２に示した。取得した画像の８０～１１０地点をランダムに選定し、イメージＪソフトウェアで計測し、その平均値を算出した。 For HE staining, frozen sections with a thickness of 10 μm were prepared, and after washing off the OCT compound with tap water, they were immersed in Mayer hematoxylin staining solution to stain the nucleus for 5 minutes. Thereafter, it was washed with running water for 10 minutes and colored. After staining the cytoplasm with 0.1% eosin Y staining solution for 2 minutes, it was dehydrated in stages with 70%, 80%, and 100% ethanol, and soaked in Lemosol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) three times for 1 minute. After clearing, the cells were sealed with Entellinu (manufactured by Merck Millipore). HE-stained epidermal cryosections were observed using an all-in-one fluorescence microscope (manufactured by Keyence Corporation), and representative images are shown in FIG. 1. In addition, the thickness of the three-dimensionally cultured epidermis (thickness from the basal layer to the granular layer) was measured and shown in FIG. 2. 80 to 110 points of the acquired images were randomly selected and measured using Image J software, and the average value was calculated.
免疫染色は、三次元培養表皮幹細胞マーカー（未分化性マーカー）ｐ６３、細胞増殖性マーカーＫｉ６７、及び、表皮前駆細胞マーカーＫｒｔ１４に対する抗体を用いて行った。まず、スライドガラス上の表皮凍結切片をＰＢＳＭＴ（２％スキムミルク、０．１％Ｔｒｉｔｏｎ－Ｘ１００含有ＰＢＳ）で１時間ブロッキングしたのち、１次抗体として１：５００に希釈したウサギ抗Ｋｉ６７抗体（ＮＢ１１０－８９７１９、Ｎｏｖｕｓ Ｂｉｏｌｏｇｉｃａｌｓ社製）、１：２００に希釈したマウス抗ｐ６３抗体（ａｂ７３５、ａｂｃａｍ社製）、及び１：１０００に希釈したニワトリ抗Ｋ１４抗体（９０６００４、ＢｉｏＬｅｇｅｎｄ社製）を添加し、ＰＢＳＴ（０．１％Ｔｒｉｔｏｎ－Ｘ１００含有ＰＢＳ）中、４℃で一晩インキュベーションした。次に、ＰＢＳＴで洗浄し、２次抗体としての、ＰＢＳＴ中にそれぞれ１：１０００に希釈した、抗ウサギＩｇＧ（Ａ２１２０６、ライフテクノロジーズ社製）、ＡＦ６４７標識抗マウスＩｇＧ（Ａ３１５７１、ライフテクノロジーズ社製）、及びＣｙ３標識抗ニワトリＩｇＹ（７０３－１６５－１５５、ジャクソンイムノリサーチ社製）を添加し、室温で１時間反応させた。さらに、ＰＢＳＴで洗浄し、Ｐｒｏｌｏｎｇ Ａｎｔｉｆａｄｅ Ｋｉｔ ｗｉｔｈ ＤＡＰＩ（Ｐ３６９３１、Ｉｎｖｉｔｒｏｇｅｎ社製）の褪色防止用封入剤を添加し、表皮凍結切片をカバーガラスで封入した。ＬＳＭ ９８０共焦点顕微鏡（ＺＥＩＳＳ社製）を用い、免疫染色した表皮凍結切片を観察し、代表的な画像を図３に示す。なお、図３中のＭｅｒｇｅは、ｐ６３、Ｋｉ６７、及びＫｒｔ１４の三つの蛍光を重ねた画像である。 Immunostaining was performed using antibodies against the three-dimensional cultured epidermal stem cell marker (undifferentiated marker) p63, the cell proliferation marker Ki67, and the epidermal progenitor cell marker Krt14. First, a cryosection of the epidermis on a slide glass was blocked with PBSMT (PBS containing 2% skim milk, 0.1% Triton-X100) for 1 hour, and then rabbit anti-Ki67 antibody (NB110- 89719, manufactured by Novus Biologicals), a mouse anti-p63 antibody (ab735, manufactured by ABCAM) diluted at 1:200, and a chicken anti-K14 antibody (906004, manufactured by BioLegend) diluted at 1:1000, and PBST ( The cells were incubated overnight at 4°C in PBS containing 0.1% Triton-X100. Next, it was washed with PBST, and the secondary antibodies were anti-rabbit IgG (A21206, manufactured by Life Technologies) and AF647-labeled anti-mouse IgG (A31571, manufactured by Life Technologies), each diluted 1:1000 in PBST. , and Cy3-labeled anti-chicken IgY (703-165-155, manufactured by Jackson ImmunoResearch) were added and allowed to react at room temperature for 1 hour. Furthermore, it was washed with PBST, a mounting medium for preventing fading of Prolong Antifade Kit with DAPI (P36931, manufactured by Invitrogen) was added, and the epidermal frozen section was mounted with a cover glass. The immunostained frozen sections of the epidermis were observed using an LSM 980 confocal microscope (manufactured by ZEISS), and representative images are shown in FIG. 3. Note that Merge in FIG. 3 is an image in which three fluorescences of p63, Ki67, and Krt14 are superimposed.
図１及び２に示されるように、ＮＲＪ又はＥＲＪを添加して培養した場合、無添加の場合に比べて、三次元培養表皮の厚さが有意に増加したことが確認された。また、ＥＲＪ及びＮＲＪを添加して培養した三次元培養表皮の細胞は密に詰まっているが、無添加の三次元培養表皮では、細胞がまばらにしか存在していなかった。 As shown in FIGS. 1 and 2, when cultured with the addition of NRJ or ERJ, it was confirmed that the thickness of the three-dimensional cultured epidermis significantly increased compared to the case without addition. Furthermore, cells in the three-dimensionally cultured epidermis cultured with the addition of ERJ and NRJ were densely packed, whereas cells were only sparsely present in the three-dimensionally cultured epidermis without any additives.
図３に示されるように、ＮＲＪ又はＥＲＪを添加して培養した三次元培養表皮のいずれの場合においても、無添加の場合に比べてｐ６３、Ｋｉ６７陽性細胞が増加したことが確認できた。Ｍｅｒｇｅにおける明るい領域はＫｒｔ１４陽性領域であり、ＮＲＪ又はＥＲＪを添加して培養した三次元培養表皮のいずれの場合においても、無添加の場合に比べてＫｒｔ１４陽性細胞が増加したことが確認できた。そして、ＮＲＪ又はＥＲＪを添加して培養した三次元培養表皮のいずれの場合においても、ｐ６３、Ｋｉ６７及びＫｒｔ１４の全てのマーカーを発現する細胞が、無添加の場合に比べて増加したことが確認された。 As shown in FIG. 3, it was confirmed that p63- and Ki67-positive cells increased in both three-dimensionally cultured epidermis cultured with the addition of NRJ or ERJ, compared to the case without the addition of NRJ or ERJ. The bright areas in Merge are Krt14-positive areas, and it was confirmed that Krt14-positive cells increased in both cases of three-dimensionally cultured epidermis cultured with the addition of NRJ or ERJ compared to the case without addition. In both cases of three-dimensionally cultured epidermis cultured with the addition of NRJ or ERJ, it was confirmed that the number of cells expressing all markers, p63, Ki67, and Krt14, increased compared to the case without the addition of NRJ or ERJ. Ta.
上記結果から、ＮＲＪ又はＥＲＪを添加して、表皮幹細胞を含むヒト表皮角化細胞を培養したことにより、三次元培養表皮の厚みが増し、また、表皮幹細胞の幹細胞性を維持したまま増殖性が向上したことがわかった。これらのことから、ＮＲＪ又はＥＲＪが表皮幹細胞を活性化でき、それによって、皮膚のターンオーバーの低下を抑制し、皮膚の菲薄化を抑制できることが確認できた。 From the above results, by adding NRJ or ERJ and culturing human epidermal keratinocytes including epidermal stem cells, the thickness of the three-dimensionally cultured epidermis increases, and the proliferative property of epidermal stem cells is increased while maintaining their stemness. I found that I had improved. From these results, it was confirmed that NRJ or ERJ can activate epidermal stem cells, thereby suppressing a decrease in skin turnover and suppressing skin thinning.
（実験例２：三次元培養系を用いたローヤルゼリーに含まれる脂肪酸による表皮幹細胞活性化能の検討）
ＨＰＥＫｐ培養時に、ＮＲＪ又はＥＲＪの代わりに、これらのローヤルゼリーに含まれる脂肪酸である、１０－ヒドロキシ－２－デセン酸（１０Ｈ２ＤＡ、Ｈａｎｇｚｈｏｕ Ｅａｓｔｂｉｏｐｈａｒｍ社製）、１０－ヒドロキシデカン酸（１０ＨＤＡ、Ｃｏｍｂｉ－Ｂｌｏｃｋｓ社製）、デセン二酸（２－ＤＡ、Ｓｕｎｄｉａ ＭｅｄｉＴｅｃｈ社製）、又はセバシン酸（ＳＡ、Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製）を培地全量に対して８０μＭとなるようにインサート外の培地に添加すること以外は実験例１と同様にして、行った。なお、１０Ｈ２ＤＡ、１０ＨＤＡ、２－ＤＡ、ＳＡはＤＭＳＯに溶解後、三次元培養用培地に溶解した。ＨＥ染色の結果を図４に、三次元培養表皮の厚みを図５に、免疫染色の結果を図６にそれぞれ示す。
(Experimental example 2: Examination of the ability of fatty acids contained in royal jelly to activate epidermal stem cells using a three-dimensional culture system)
During HPEKp culture, 10-hydroxy-2-decenoic acid (10H2DA, manufactured by Hangzhou Eastbiopharm) and 10-hydroxydecanoic acid (10HDA, Combi-Blocks), which are fatty acids contained in these royal jelly, were used instead of NRJ or ERJ. (manufactured by Sundia MediTech), decenedioic acid (2-DA, manufactured by Sundia MediTech), or sebacic acid (SA, manufactured by Sigma-Aldrich) to the medium outside the insert at a concentration of 80 μM relative to the total volume of the medium. It was carried out in the same manner as in Experimental Example 1. Note that 10H2DA, 10HDA, 2-DA, and SA were dissolved in DMSO and then dissolved in a three-dimensional culture medium. The results of HE staining are shown in FIG. 4, the thickness of the three-dimensional cultured epidermis is shown in FIG. 5, and the results of immunostaining are shown in FIG. 6.
図４及び５に示されるように、１０Ｈ２ＤＡ、１０ＨＤＡ、２－ＤＡ又はＳＡを添加して培養した場合、無添加の場合に比べて三次元培養表皮の厚みが有意に増加したことが確認された。また、三次元培養表皮の厚みの増加は、１０Ｈ２ＤＡ、１０ＨＤＡ、２－ＤＡ、ＳＡの順に大きく、１０ＨＤＡと２－ＤＡとでは、三次元培養表皮の厚みの増加はほぼ同程度であった。 As shown in Figures 4 and 5, it was confirmed that when cultured with the addition of 10H2DA, 10HDA, 2-DA, or SA, the thickness of the three-dimensional cultured epidermis significantly increased compared to the case without addition. . Furthermore, the increase in the thickness of the three-dimensionally cultured epidermis was larger in the order of 10H2DA, 10HDA, 2-DA, and SA, and the increase in the thickness of the three-dimensionally cultured epidermis was approximately the same between 10HDA and 2-DA.
図６に示されるように、実験例１と同様、１０Ｈ２ＤＡ、１０ＨＤＡ、２－ＤＡ、又はＳＡを添加して培養した三次元培養表皮のいずれの場合においても、無添加の場合と比較して、ｐ６３、Ｋｉ６７、Ｋｒｔ１４陽性細胞が増加したことが確認できた。 As shown in FIG. 6, as in Experimental Example 1, in any case of three-dimensionally cultured epidermis cultured with the addition of 10H2DA, 10HDA, 2-DA, or SA, compared to the case without the addition of It was confirmed that p63, Ki67, and Krt14 positive cells increased.
上記結果から、１０Ｈ２ＤＡ、１０ＨＤＡ、２－ＤＡ、又はＳＡを添加して、表皮幹細胞を含むヒト表皮角化細胞を培養したことにより、三次元培養表皮の厚みが増し、また、表皮幹細胞の幹細胞性を維持したまま増殖性が向上したことがわかった。これらのことから、１０Ｈ２ＤＡ、１０ＨＤＡ、２－ＤＡ又はＳＡが表皮幹細胞を活性化でき、それによって皮膚のターンオーバーの低下を抑制し、皮膚の菲薄化を抑制できることが確認できた。 From the above results, by adding 10H2DA, 10HDA, 2-DA, or SA to culture human epidermal keratinocytes including epidermal stem cells, the thickness of the three-dimensionally cultured epidermis increases, and the stemness of epidermal stem cells increases. It was found that the proliferation ability was improved while maintaining the From these results, it was confirmed that 10H2DA, 10HDA, 2-DA, or SA can activate epidermal stem cells, thereby suppressing a decrease in skin turnover and suppressing skin thinning.
（実験例３：複製老化系を用いた生ローヤルゼリー及び酵素分解ローヤルゼリーによる抗皮膚老化能の評価）
実施例１と同様に、表皮幹細胞を含む、ヒト表皮角化細胞（ＨＰＥＫｐ、Ｊｕｖｅｎｉｌｅ Ｐｏｏｌｅｄ、セルンテック社製）をサブコンフルエントに達するまで培養し、剥離した。剥離されたＨＰＥＫｐをＣｎＴ－Ｐｒｉｍｅ培地に懸濁し、遠心分離（１，１００ｒｐｍ、３ｍｉｎ）により回収した。回収したＨＰＥＫｐを、５×１０３細胞／ｃｍ２となるように調製して、ＣｎＴ－Ｐｒｉｍｅ培地の入った新しいシャーレに播種し培養した。培養の際、ＮＲＪ（山田養蜂場社製）、又はＥＲＪ（山田養蜂場社製）を添加したＣｎＴ－Ｐｒｉｍｅ培地を用いた。具体的には、ＮＲＪ及びＥＲＪは、０．４５μｍのフィルターに通し、さらに、０．２μｍフィルターに通して滅菌を行ったのち、１００又は１０μｇ／ｍＬとなるようにＣｎＴ－Ｐｒｉｍｅ培地に添加した。以降培養終了まで継続してＮＲＪ及びＥＲＪを添加した培地で培養した。また、ＨＰＥＫｐの抗皮膚老化能の評価は細胞の分裂回数の累積値（Ｐｏｐｕｌａｔｉｏｎ ｄｏｕｂｌｉｎｇ ｌｅｖｅｌ、ＰＤＬ）を時間軸にプロットした曲線で評価した。すなわち、各継代回数内の培養において培養終了時の細胞数を培養開始時の細胞数で割り、さらに対数をとることで分裂回数を求め、各継代時の分裂回数の累積値（ＰＤＬ）を時間軸にプロットして評価した。なお、ＨＰＥＫｐの継代数が３の時点から評価を開始した。その結果を図７に示す。
(Experimental Example 3: Evaluation of anti-skin aging ability of raw royal jelly and enzymatically decomposed royal jelly using a replicative aging system)
In the same manner as in Example 1, human epidermal keratinocytes (HPEKp, Juvenile Pooled, manufactured by Cerntech), including epidermal stem cells, were cultured until they reached subconfluence and then detached. The detached HPEKp was suspended in CnT-Prime medium and collected by centrifugation (1,100 rpm, 3 min). The recovered HPEKp was adjusted to 5×10 3 cells/cm 2 , seeded in a new petri dish containing CnT-Prime medium, and cultured. During culture, CnT-Prime medium supplemented with NRJ (manufactured by Yamada Bee Farm Co., Ltd.) or ERJ (manufactured by Yamada Bee Farm Co., Ltd.) was used. Specifically, NRJ and ERJ were passed through a 0.45 μm filter and then sterilized through a 0.2 μm filter, and then added to the CnT-Prime medium at a concentration of 100 or 10 μg/mL. Thereafter, the cells were continuously cultured in a medium supplemented with NRJ and ERJ until the end of the culture. Furthermore, the anti-skin aging ability of HPEKp was evaluated using a curve in which the cumulative number of cell divisions (Population doubling level, PDL) was plotted on the time axis. In other words, in culture within each passage number, the number of cells at the end of culture is divided by the number of cells at the start of culture, and the number of divisions is calculated by taking the logarithm, and the cumulative value (PDL) of the number of divisions at each passage is calculated. was evaluated by plotting it on the time axis. Note that the evaluation was started when the passage number of HPEKp was 3. The results are shown in FIG.
図７に示されるように、無添加で培養した場合、ＨＰＥＫｐのＰＤＬは、ある一定の日数が経過すると増加しなくなった。この結果から、細胞は老化とともに、分裂回数が減っていくことがわかる。一方、ＮＲＪ又はＥＲＪを添加して培養したいずれの場合も、無添加の場合と比較してＰＤＬがより高く、３０日後であってもＰＤＬは増加傾向にあった。 As shown in FIG. 7, when cultured without additives, the PDL of HPEKp stopped increasing after a certain number of days. These results show that as cells age, the number of cell divisions decreases. On the other hand, in both cases of culture with addition of NRJ or ERJ, PDL was higher than in the case of no addition, and PDL tended to increase even after 30 days.
この結果及び実験例１の結果から、ＮＲＪ又はＥＲＪが表皮幹細胞を活性化でき、それによって、皮膚の老化を抑制できることが確認できた。
From this result and the results of Experimental Example 1, it was confirmed that NRJ or ERJ can activate epidermal stem cells, thereby suppressing skin aging.
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