JP7266202B2 - Cosmetics and anti-inflammatory agents - Google Patents
Cosmetics and anti-inflammatory agents Download PDFInfo
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Description
本発明は、化粧料及び抗炎症剤に関する。 The present invention relates to cosmetics and anti-inflammatory agents.
アミノ酸配列ＧＲＬＫＲＭＧＥＲＬＫＲＫＩＱＫＷＩＲＷからなるポリペプチドが抗菌作用を有することが知られており、このポリペプチドを含有する抗菌剤、抗真菌剤または消毒剤、及び、熱傷、褥瘡、創傷、皮膚潰瘍、下肢潰瘍、糖尿病性潰瘍、閉塞性動脈疾患及び閉塞性動脈硬化症、蜂巣炎、急性リンパ管炎、リンパ節炎、丹毒、皮膚膿瘍、壊死性皮下感染、ブドウ球菌性熱傷様皮膚症候群（ＳＳＳＳ）、毛包炎、フルンケル（面疔）、化膿性汗腺炎、カルブンケル（癰）、感染性爪囲炎、紅色陰癬及び重症感染症（敗血症）から成る群から選択される疾患の予防、改善又は治療剤が提案されている（特許文献１）。 It is known that a polypeptide consisting of the amino acid sequence GRLKRMGERLKRKIQKWIRW has an antibacterial effect, and antibacterial agents, antifungal agents or disinfectants containing this polypeptide, burns, decubitus ulcers, wounds, skin ulcers, leg ulcers, diabetes mellitus. ulcer, obstructive arterial disease and arteriosclerosis obliterans, cellulitis, acute lymphangitis, lymphadenitis, erysipelas, skin abscess, necrotizing subcutaneous infection, staphylococcal scalded skin syndrome (SSSS), folliculitis , furuncle (comedone), hidradenitis suppurativa, carbuncle (carbuncle), infectious paronychia, erythema and severe infection (sepsis). (Patent Document 1).
新規な化粧料と抗炎症剤、肌荒れ防止剤、美白剤、にきび改善剤、コラーゲン産生促進剤、ヒアルロン酸産生促進剤、基底膜タンパク産生促進剤、基底膜タンパク分解抑制剤、シワ改善又は抑制剤、皮膚のたるみ・はり改善剤、フケ改善剤、育毛剤を提供することを課題とする。特に、化粧料としては、肌荒れ防止剤、美白剤、にきび改善剤、コラーゲン産生促進剤、ヒアルロン酸産生促進剤、基底膜タンパク産生促進剤、基底膜タンパク分解抑制剤、シワ改善又は抑制剤、皮膚のたるみ・はり改善剤等の皮膚化粧料、フケ改善剤、育毛剤等の毛髪化粧料を提供することを課題とする。 Novel cosmetics and anti-inflammatory agents, anti-rough skin agents, whitening agents, acne-improving agents, collagen production promoters, hyaluronic acid production promoters, basement membrane protein production promoters, basement membrane protein degradation inhibitors, wrinkle improvement or inhibitors , a skin sagging/tension improving agent, a dandruff improving agent, and a hair restorer. In particular, as cosmetics, rough skin preventing agents, whitening agents, acne improving agents, collagen production promoters, hyaluronic acid production promoters, basement membrane protein production promoters, basement membrane protein degradation inhibitors, wrinkle improving or suppressing agents, skin An object of the present invention is to provide a skin cosmetic such as an agent for improving skin sagging and tightness, a hair cosmetic such as an agent for improving dandruff, and a hair restorer.
本発明の課題を解決するための手段は、次のとおりである。
１．配列番号１に記載のアミノ酸配列からなるポリペプチド、またはそのアミノ酸配列に対し９５％以上の相同性を有するポリペプチドを含有する、化粧料。
２．配列番号１に記載のアミノ酸配列からなるポリペプチド、またはそのアミノ酸配列に対し９５％以上の相同性を有するポリペプチドを含有する、抗炎症剤。
３．配列番号１に記載のアミノ酸配列からなるポリペプチド、またはそのアミノ酸配列に対し９５％以上の相同性を有するポリペプチドを含有する、肌荒れ防止剤。
４．配列番号１に記載のアミノ酸配列からなるポリペプチド、またはそのアミノ酸配列に対し９５％以上の相同性を有するポリペプチドを含有する、美白剤。
５．配列番号１に記載のアミノ酸配列からなるポリペプチド、またはそのアミノ酸配列に対し９５％以上の相同性を有するポリペプチドを含有する、にきび改善剤。
６．配列番号１に記載のアミノ酸配列からなるポリペプチド、またはそのアミノ酸配列に対し９５％以上の相同性を有するポリペプチドを含有する、フケ改善剤。
７．配列番号１に記載のアミノ酸配列からなるポリペプチド、またはそのアミノ酸配列に対し９５％以上の相同性を有するポリペプチドを含有する、育毛剤。
８．配列番号１に記載のアミノ酸配列からなるポリペプチド、またはそのアミノ酸配列に対し９５％以上の相同性を有するポリペプチドを含有する、コラーゲン産生促進剤、ヒアルロン酸の産生促進剤、基底膜タンパクの産生促進剤、基底膜タンパクの分解抑制剤、シワ改善又は抑制剤、皮膚のたるみ・はり改善剤。
Means for solving the problems of the present invention are as follows.
1. A cosmetic comprising a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having 95% or more homology to the amino acid sequence.
2. An anti-inflammatory agent comprising a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having 95% or more homology to the amino acid sequence.
3. An agent for preventing rough skin, comprising a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having 95% or more homology to the amino acid sequence.
4. A whitening agent comprising a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having 95% or more homology to the amino acid sequence.
5. An acne-improving agent comprising a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having 95% or more homology to the amino acid sequence.
6. An anti-dandruff agent comprising a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having 95% or more homology to the amino acid sequence.
7. A hair restorer containing a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having 95% or more homology to the amino acid sequence.
8. A collagen production promoter, a hyaluronic acid production promoter, and a basement membrane protein production containing a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having 95% or more homology to the amino acid sequence Accelerator, basement membrane protein decomposition inhibitor, wrinkle improving or inhibiting agent, skin sagging/tension improving agent.
本発明のポリペプチドを用いることにより、化粧料、抗炎症剤、肌荒れ防止剤、美白剤、にきび改善剤、コラーゲン産生促進剤、ヒアルロン酸産生促進剤、基底膜タンパク産生促進剤、基底膜タンパク分解抑制剤、シワ改善又は抑制剤、皮膚のたるみ・はり改善剤、フケ改善剤、育毛剤を提供することができた。特に、化粧料としては、肌荒れ防止剤、美白剤、にきび改善剤、コラーゲン産生促進剤、ヒアルロン酸産生促進剤、基底膜タンパク産生促進剤、基底膜タンパク分解抑制剤、シワ改善又は抑制剤、皮膚のたるみ・はり改善剤等の皮膚化粧料、フケ改善剤、育毛剤等の毛髪化粧料を提供することができた。 By using the polypeptide of the present invention, cosmetics, anti-inflammatory agents, anti-rough skin agents, whitening agents, acne-improving agents, collagen production promoters, hyaluronic acid production promoters, basement membrane protein production promoters, and basement membrane proteolytic agents It was possible to provide an inhibitor, a wrinkle-improving or suppressing agent, an agent for improving sagging and tightness of the skin, an agent for improving dandruff, and a hair restorer. In particular, as cosmetics, rough skin preventing agents, whitening agents, acne improving agents, collagen production promoters, hyaluronic acid production promoters, basement membrane protein production promoters, basement membrane protein degradation inhibitors, wrinkle improving or suppressing agents, skin Skin cosmetics such as sagging and firmness improving agents, hair cosmetics such as dandruff improving agents and hair restorers could be provided.
本発明は、下記配列番号１に記載のアミノ酸配列からなるポリペプチド、またはそのアミノ酸配列に対し９５％以上の相同性を有するポリペプチドを使用することを特徴とする。
配列番号１：ＧＲＬＫＲＭＧＥＲＬＫＲＫＩＱＫＷＩＲＷ
The present invention is characterized by using a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 below, or a polypeptide having 95% or more homology to the amino acid sequence.
SEQ ID NO: 1: GRLKRMGERLKRKIQKWIRW
なお、本発明で使用するポリペプチドは、既知の抗菌活性物質であるが、抗炎症作用、肌荒れ防止作用、美白作用、にきび改善作用、コラーゲン産生促進作用、ヒアルロン酸産生促進作用、基底膜タンパク産生促進作用、基底膜タンパク分解抑制作用、シワ改善又は抑制作用、皮膚のたるみ・はり改善作用、フケ改善作用、育毛作用を有することは、これまでに知られていない新規な知見である。
そして、本発明は、このポリペプチドの抗炎症作用、肌荒れ防止作用、美白作用、にきび改善作用、コラーゲン産生促進作用、ヒアルロン酸産生促進作用、基底膜タンパク産生促進作用、基底膜タンパク分解抑制作用、シワ改善又は抑制作用、皮膚のたるみ・はり改善作用、フケ改善作用、育毛作用を利用した化粧料と抗炎症剤、肌荒れ防止剤、美白剤、にきび改善剤、コラーゲン産生促進剤、ヒアルロン酸産生促進剤、基底膜タンパク産生促進剤、基底膜タンパク分解抑制剤、シワ改善又は抑制剤、皮膚のたるみ・はり改善剤、フケ改善剤、育毛剤に関する。本発明の化粧料としては、にきび改善剤、コラーゲン産生促進剤、ヒアルロン酸産生促進剤、基底膜タンパク産生促進剤、基底膜タンパク分解抑制剤、シワ改善又は抑制剤、皮膚のたるみ・はり改善剤等の皮膚化粧料、フケ改善剤、育毛剤等の毛髪化粧料が挙げられる。
The polypeptide used in the present invention is a known antibacterial active substance, and has anti-inflammatory action, rough skin prevention action, whitening action, acne improvement action, collagen production promotion action, hyaluronic acid production promotion action, and basement membrane protein production. It is a new and hitherto unknown finding that it has stimulatory action, inhibitory action on basement membrane proteolysis, wrinkle-improving or inhibiting action, skin sagging/tension-improving action, dandruff-improving action, and hair-growing action.
Then, the present invention provides anti-inflammatory action, rough skin prevention action, whitening action, acne improvement action, collagen production promotion action, hyaluronic acid production promotion action, basement membrane protein production promotion action, basement membrane protein degradation inhibitory action, wrinkle-improving or suppressing action, skin sagging and firmness-improving action, dandruff-improving action, cosmetics using hair-growth action, anti-inflammatory agents, anti-roughness agents, whitening agents, acne-improving agents, collagen production promoters, and hyaluronic acid production promoters agent, basement membrane protein production promoter, basement membrane protein degradation inhibitor, wrinkle improving or suppressing agent, skin sagging and firmness improving agent, dandruff improving agent, and hair restorer. Cosmetics of the present invention include acne-improving agents, collagen production promoters, hyaluronic acid production promoters, basement membrane protein production promoters, basement membrane proteolytic inhibitors, wrinkle-improving or suppressing agents, and skin sagging and firmness-improving agents. and hair cosmetics such as anti-dandruff agents and hair restorers.
本発明で使用するポリペプチドは、アミノ末端（Ｎ末端）及び／又はカルボキシル末端（Ｃ末端）が修飾されていてもよい。
本発明のポリペプチドは、キャッピング構造が付加されていてもよい。ここで、キャッピング構造は、ポリペプチドのＮ末端及び／又はＣ末端に付加し、反応性を失わせる構造である。
本発明で使用するポリペプチドは、市販のペプチド合成機を用いた化学合成等の常法により製造することができる。また、末端修飾も、周知の方法により行うことができる。
Polypeptides used in the present invention may be modified at the amino-terminus (N-terminus) and/or the carboxyl-terminus (C-terminus).
A capping structure may be added to the polypeptide of the present invention. Here, the capping structure is a structure added to the N-terminus and/or C-terminus of the polypeptide to render it inactive.
Polypeptides used in the present invention can be produced by conventional methods such as chemical synthesis using a commercially available peptide synthesizer. Terminal modification can also be performed by a well-known method.
本発明で使用するポリペプチドは、抗炎症作用、肌荒れ防止作用、美白作用を有するので、抗炎症、肌荒れ防止、美白を目的とする医薬品、医薬部外品、皮膚化粧料、毛髪化粧料として用いることができる。
本発明で使用するポリペプチドは、抗炎症作用を有するので、抗炎症を目的とする医薬品、医薬部外品、皮膚化粧料、毛髪化粧料として用いることができる。
本発明で使用するポリペプチドは、肌荒れ防止作用、美白作用を有するので、肌荒れ防止、美白を目的とする医薬品、医薬部外品、皮膚化粧料、毛髪化粧料として用いることができる。
Since the polypeptide used in the present invention has anti-inflammatory, anti-roughness and whitening effects, it can be used as pharmaceuticals, quasi-drugs, skin cosmetics and hair cosmetics for the purpose of anti-inflammatory, anti-roughness and whitening. be able to.
Since the polypeptide used in the present invention has an anti-inflammatory effect, it can be used as an anti-inflammatory drug, quasi-drug, skin cosmetic, and hair cosmetic.
Since the polypeptide used in the present invention has rough skin prevention and skin whitening effects, it can be used as pharmaceuticals, quasi-drugs, skin cosmetics, and hair cosmetics for the purpose of rough skin prevention and skin whitening.
本発明の抗炎症剤を医薬品、医薬部外品、皮膚化粧料、毛髪化粧料として用いる場合は、皮膚外用剤、皮膚洗浄剤、毛髪洗浄剤、育毛剤、洗口剤、うがい薬、点眼剤、貼付剤、毛髪化粧料、メイクアップ化粧料等として用いることができる。
本発明の肌荒れ防止剤を医薬品、医薬部外品、皮膚化粧料、毛髪化粧料として用いる場合は、皮膚外用剤、皮膚洗浄剤、毛髪洗浄剤、育毛剤、毛髪化粧料、メイクアップ化粧料等として用いることができる。
本発明の美白剤を医薬品、医薬部外品、皮膚化粧料、毛髪化粧料として用いる場合は、皮膚外用剤、メイクアップ化粧料等として用いることができる。
When the anti-inflammatory agent of the present invention is used as a pharmaceutical, quasi-drug, skin cosmetic, or hair cosmetic, it can be used as an external skin preparation, skin cleanser, hair cleanser, hair restorer, mouthwash, gargle, eye drops. , patches, hair cosmetics, make-up cosmetics, and the like.
When the rough skin preventing agent of the present invention is used as a pharmaceutical, quasi-drug, skin cosmetic, or hair cosmetic, it can be used as an external skin preparation, skin cleanser, hair cleanser, hair restorer, hair cosmetic, make-up cosmetic, or the like. can be used as
When the whitening agent of the present invention is used as a pharmaceutical, quasi-drug, skin cosmetic, or hair cosmetic, it can be used as an external skin preparation, make-up cosmetic, or the like.
本発明のにきび改善剤、コラーゲン産生促進剤、ヒアルロン酸産生促進剤、基底膜タンパク産生促進剤、基底膜タンパク分解抑制剤、シワ改善又は抑制剤、皮膚のたるみ・はり改善剤、フケ改善剤、育毛剤を医薬品、医薬部外品、皮膚化粧料、毛髪化粧料として用いる場合は、皮膚外用剤、皮膚洗浄剤、毛髪洗浄剤、育毛剤、毛髪化粧料、メイクアップ化粧料として用いることができる。 Acne improving agent of the present invention, collagen production promoter, hyaluronic acid production promoter, basement membrane protein production promoter, basement membrane proteolytic inhibitor, wrinkle improving or suppressing agent, skin sagging and firmness improving agent, dandruff improving agent, When the hair restorer is used as a pharmaceutical, quasi-drug, skin cosmetic, or hair cosmetic, it can be used as a skin external preparation, skin cleanser, hair cleanser, hair restorer, hair cosmetic, or makeup cosmetic. .
以下、本発明を実施例に基づき説明する。
実施例において、ポリペプチドとして、ＧＲＬＫＲＭＧＥＲＬＫＲＫＩＱＫＷＩＲＷ－ａｍｉｄｅ（以下、ポリペプチド１という）を使用した。
EXAMPLES The present invention will be described below based on examples.
In the examples, GRLKRMGERLKRKIQKWIRW-amide (hereinafter referred to as polypeptide 1) was used as the polypeptide.
１．ホルボール １２－ミリスタート １３－アセタート（以下、ＰＭＡという。）による炎症に対する抗炎症効果
表皮角化細胞にＰＭＡ刺激により炎症を惹起させ、ポリペプチド１による２４時間後の炎症関連タンパクの変化を検討した。
1. Anti-inflammatory Effect on Inflammation by Phorbol 12-myristate 13-acetate (hereinafter referred to as PMA) Inflammation was induced in epidermal keratinocytes by PMA stimulation, and changes in inflammation-related proteins by polypeptide 1 after 24 hours were examined. .
１×１０５ｃｅｌｌｓ／ｗｅｌｌ／２ｍｌとなるように調整した正常ヒト新生児ケラチノサイト（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）（以下ＨＥＫｎとする）を、Ｈｕｍｅｄｉａ－ＫＧ２増殖添加剤セット（５００ｍｌ用）（倉敷紡績株式会社製）を添加したＥｐｉ－Ｌｉｆｅ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）（以下Ｅｐｉ－Ｌｉｆｅ・ＫＧ２培地という。）で、３７℃、５％ＣＯ２インキュベーター内でサブコンフルエントになるまで培養した。
培養後、Ｅｐｉ－Ｌｉｆｅ・ＫＧ２培地にＰＭＡを５０ｎｇ／ｍｌ、ポリペプチド１を０．００１％添加した培養液に培地交換し、３７℃、５％ＣＯ２インキュベーター内で培養した。なお、ポリペプチド１を添加していないものをｃｏｎｔｒｏｌとした。
２４時間培養後、培養上清を回収し、培養上清中のＴＮＦ－α、ＭＭＰ－９、ＮＧＡＬ、ＩＬ－８量をＥＬＩＳＡ ｋｉｔ（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）を用いて測定した。結果を図１～４に示す。
Normal human neonatal keratinocytes (Thermo Fisher Scientific) (hereinafter referred to as HEKn) adjusted to 1×10 5 cells/well/2 ml were added to Humedia-KG2 growth additive set (for 500 ml) (manufactured by Kurashiki Boseki Co., Ltd.). ) supplemented with Epi-Life (Thermo Fisher Scientific) (hereinafter referred to as Epi-Life KG2 medium), and cultured at 37° C. in a 5% CO 2 incubator until subconfluent.
After culturing, the medium was changed to Epi-Life·KG2 medium supplemented with 50 ng/ml of PMA and 0.001% of polypeptide 1, and cultured in a 37° C., 5% CO 2 incubator. In addition, the thing which did not add the polypeptide 1 was made into control.
After culturing for 24 hours, the culture supernatant was collected, and the amounts of TNF-α, MMP-9, NGAL and IL-8 in the culture supernatant were measured using an ELISA kit (R&D Systems). The results are shown in Figures 1-4.
（結果）
図１に示したように、ＰＭＡ刺激による炎症により、ＴＮＦ－αが３８ｐｇ／ｍｌ発現した。しかし、ポリペプチド１を０．００１％添加することにより、ＰＭＡを添加しても、ＴＮＦ－αの発現量は８ｐｇ／ｍｌと小さくなり、炎症が抑制できた。
図２に示したように、ＰＭＡ添加前はＭＭＰ－９の発現量が１７１ｐｇ／ｍｌであったが、ＰＭＡ添加後の炎症により、ＭＭＰ－９が１６５０ｐｇ／ｍｌと増大した。一方、ポリペプチド１を０．００１％添加することにより、ＰＭＡを添加してもＭＭＰ－９の発現量は２７７ｐｇ／ｍｌと、ＰＭＡ添加前と同程度となり、炎症が抑制できた。
図３に示したように、ＰＭＡ添加前はＮＧＡＬの発現量が６８１５ｐｇ／ｍｌであったが、ＰＭＡ添加後の炎症により、ＮＧＡＬが１２２３６ｐｇ／ｍｌと増大した。一方、ポリペプチド１を０．００１％添加することにより、ＰＭＡを添加してもＮＧＡＬの発現量は８３４０ｐｇ／ｍｌとＰＭＡ添加前と近い値となり、炎症が抑制できた。
図４に示したように、ＰＭＡ添加前はＩＬ－８の発現量が９４ｐｇ／ｍｌであったが、ＰＭＡ添加後の炎症により、ＩＬ－８が５１８０ｐｇ／ｍｌと増大した。一方、ポリペプチド１を０．００１％添加することにより、ＰＭＡを添加してもＩＬ－８の発現量は２７３６ｐｇ／ｍｌと小さくなり、炎症が抑制できた。
(result)
As shown in FIG. 1, PMA-stimulated inflammation resulted in TNF-α expression of 38 pg/ml. However, by adding 0.001% of polypeptide 1, the expression level of TNF-α decreased to 8 pg/ml even with the addition of PMA, and inflammation could be suppressed.
As shown in FIG. 2, the expression level of MMP-9 was 171 pg/ml before addition of PMA, but increased to 1650 pg/ml due to inflammation after addition of PMA. On the other hand, by adding 0.001% of polypeptide 1, the expression level of MMP-9 was 277 pg/ml even with the addition of PMA, which was about the same level as before the addition of PMA, and inflammation could be suppressed.
As shown in FIG. 3, the expression level of NGAL was 6815 pg/ml before addition of PMA, but increased to 12236 pg/ml due to inflammation after addition of PMA. On the other hand, when 0.001% of polypeptide 1 was added, the expression level of NGAL was 8340 pg/ml, a value close to that before the addition of PMA, even when PMA was added, and inflammation could be suppressed.
As shown in FIG. 4, the IL-8 expression level was 94 pg/ml before PMA addition, but increased to 5180 pg/ml due to inflammation after PMA addition. On the other hand, by adding 0.001% of polypeptide 1, the expression level of IL-8 decreased to 2736 pg/ml even with the addition of PMA, and inflammation could be suppressed.
２．Ｐ．ａｃｎｅｓ死菌による炎症に対する抗炎症効果
佐藤らの報告（Ｂｉｏｌ Ｐｈａｒｍ Ｂｕｌｌ．３４（２），２９５－２９９，（２０１１））を参考に、ニキビの原因菌であるＰ．ａｃｎｅｓの死菌を使用した炎症の系を用いて、表皮角化細胞での抗炎症作用を検討した。
ＮＧＡＬはニキビの部位にて増加するタンパクとして知られている（Ｂｒ Ｊ Ｄｅｒｍａｔｏｌ．１６５（２），３０２－３１０，（２０１１））。
表皮角化細胞にＰ．ａｃｎｅｓの死菌刺激により炎症を惹起させ、ポリペプチド１による２４時間後の炎症関連タンパクの変化を検討した。
2. P. Anti-inflammatory effect on inflammation caused by dead bacteria of P. acnes With reference to the report by Sato et al. An anti-inflammatory effect on epidermal keratinocytes was examined using an inflammation system using killed bacteria of P. acnes.
NGAL is known as a protein that increases in acne sites (Br J Dermatol. 165(2), 302-310, (2011)).
In epidermal keratinocytes, P. Inflammation was induced by stimulating killed bacteria of acnes, and changes in inflammation-related proteins by polypeptide 1 after 24 hours were examined.
アクネ菌としては、Ｐｒｏｐｉｏｎｉｂａｃｔｅｒｉｕｍ ａｃｎｅｓ（ＮＢＲＣ１０７６０５）を用いた。ＧＡＭ寒天培地を用いて３５℃、５日間嫌気培養した後、リン酸緩衝生理食塩水（ＰＢＳ）に分散し、菌数を測定した。菌数測定後、４％パラホルムアルデヒド／ＰＢＳ（－）で３０ｍｉｎ振とうしながら固定し、ＰＢＳ（－）で２回洗浄後、ＰＢＳ（－）に分散させてアクネ菌死菌溶液とした。
１×１０５ｃｅｌｌｓ／ｗｅｌｌ／２ｍｌとなるように調整したＨＥＫｎをＥｐｉ－Ｌｉｆｅ・ＫＧ２培地で３７℃、５％ＣＯ２インキュベーター内でサブコンフルエントになるまで培養した。
培養後、Ｅｐｉ－Ｌｉｆｅ・ＫＧ２培地にアクネ菌死菌溶液を１×１０８ｃｆｕ／ｍｌの濃度となるように、またポリペプチド１をそれぞれ０．０００１％、０．０００５％、０．００１％添加した培養液に培地交換し、３７℃、５％ＣＯ２インキュベーター内で培養した。なおポリペプチド１を添加していないものをｃｏｎｔｒｏｌとした。
２４時間培養後、培養上清を回収し、培養上清中のＭＭＰ－９、ＮＧＡＬ量をＥＬＩＳＡ ｋｉｔ（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）を用いて測定した。結果を図５、６に示す。
Propionibacterium acnes (NBRC107605) was used as acne bacteria. After anaerobic culture at 35° C. for 5 days using GAM agar medium, the cells were dispersed in phosphate-buffered saline (PBS) and counted. After measuring the number of bacteria, the cells were fixed with 4% paraformaldehyde/PBS(-) with shaking for 30 minutes, washed twice with PBS(-), and dispersed in PBS(-) to obtain a killed acne solution.
HEKn adjusted to 1×10 5 cells/well/2 ml was cultured in Epi-Life·KG2 medium at 37° C. in a 5% CO 2 incubator until subconfluent.
After culturing, P. acnes killed solution was added to Epi-Life KG2 medium to a concentration of 1×10 8 cfu/ml, and polypeptide 1 was added to 0.0001%, 0.0005%, and 0.001%, respectively. The medium was replaced with the added culture medium, and cultured in a 37°C, 5% CO 2 incubator. In addition, the thing which did not add the polypeptide 1 was made into control.
After culturing for 24 hours, the culture supernatant was collected, and the amounts of MMP-9 and NGAL in the culture supernatant were measured using an ELISA kit (R&D Systems). The results are shown in FIGS.
（結果）
図５に示したように、Ｐ．ａｃｎｅｓの死菌添加前はＭＭＰ－９の発現量が４２８ｐｇ／ｍｌであったが、Ｐ．ａｃｎｅｓの死菌添加後の炎症により、ＭＭＰ－９が１４３２ｐｇ／ｍｌと増大した。一方、ポリペプチド１を０．０００１％、０．０００５％、０．００１％添加することにより、Ｐ．ａｃｎｅｓの死菌を添加してもＭＭＰ－９の発現量はそれぞれ７０１ｐｇ／ｍｌ、４９２ｐｇ／ｍｌ、３８５ｐｇ／ｍｌと小さくなり、添加量に依存して、炎症を抑制した。特に、ポリペプチド１を０．００１％添加することにより、Ｐ．ａｃｎｅｓの死菌の添加前後でＭＭＰ－９の発現量が変わらず、炎症を抑制できた。
図６に示したように、Ｐ．ａｃｎｅｓの死菌添加前はＮＧＡＬの発現量が５１００ｐｇ／ｍｌであったが、Ｐ．ａｃｎｅｓの死菌添加後の炎症により、ＮＧＡＬが７４３９ｐｇ／ｍｌと増大した。一方、ポリペプチド１を０．０００１％、０．０００５％、０．００１％添加することにより、Ｐ．ａｃｎｅｓの死菌を添加してもＮＧＡＬの発現量はそれぞれ５８１５ｐｇ／ｍｌ、６２９０ｐｇ／ｍｌ、５２１８ｐｇ／ｍｌと小さくなり、炎症を抑制できた。特に、ポリペプチド１を０．００１％添加することにより、Ｐ．ａｃｎｅｓの死菌の添加前後でＮＧＡＬの発現量が変わらず、炎症を抑制できた。
すなわち、ポリペプチド１は、Ｐ．ａｃｎｅｓ死菌による炎症に対する抗炎症効果があり、にきび改善作用を有することが確認できた。
(result)
As shown in FIG. Before the addition of killed P. acnes, the expression level of MMP-9 was 428 pg/ml. MMP-9 increased to 1432 pg/ml due to inflammation after addition of killed P. acnes. On the other hand, by adding 0.0001%, 0.0005% and 0.001% of polypeptide 1, P. Even with the addition of killed bacteria of acnes, the expression levels of MMP-9 decreased to 701 pg/ml, 492 pg/ml and 385 pg/ml, respectively, and inflammation was suppressed depending on the amount added. In particular, the addition of 0.001% of polypeptide 1 significantly reduced P. The expression level of MMP-9 did not change before and after the addition of killed P. acnes, and inflammation could be suppressed.
As shown in FIG. Before the addition of killed P. acnes, the expression level of NGAL was 5100 pg/ml. Inflammation following the addition of killed acnes increased NGAL to 7439 pg/ml. On the other hand, by adding 0.0001%, 0.0005% and 0.001% of polypeptide 1, P. Even when killed bacteria of acnes were added, the expression levels of NGAL decreased to 5815 pg/ml, 6290 pg/ml and 5218 pg/ml, respectively, and inflammation could be suppressed. In particular, the addition of 0.001% of polypeptide 1 significantly reduced P. The expression level of NGAL did not change before and after the addition of killed P. acnes, and inflammation could be suppressed.
Thus, polypeptide 1 is P. It was confirmed that there is an anti-inflammatory effect against inflammation caused by dead acnes bacteria and that it has an acne-improving effect.
３．Ｍａｌａｓｓｅｚｉａ死菌による炎症に対する抗炎症効果
脂漏性皮膚炎やマラセチア毛包炎、フケの原因菌となるＭａｌａｓｓｅｚｉａ ｆｕｒｆｕｒの死菌を使用した炎症の系を用いて、表皮角化細胞での抗炎症作用を検討した。
ＩＬ－６、ＩＬ－８はマラセチア菌との共培養により増加するタンパクとして知られている。（ＦＦＭＳ Ｉｍｍｕｎｏｌ Ｍｅｄ Ｍｉｃｒｏｂｉｏｌ．５４，２０３－２１４，（２００８））
表皮角化細胞にＭａｌａｓｓｅｚｉａ死菌刺激により炎症を惹起させ、ポリペプチド１による２４時間後の炎症関連タンパクの変化を検討した。
3. Anti-inflammatory effect on inflammation caused by killed Malassezia bacteria Anti-inflammatory effect on epidermal keratinocytes using an inflammation system using killed bacteria of Malassezia furfur, which causes seborrheic dermatitis, Malassezia folliculitis, and dandruff It was investigated.
IL-6 and IL-8 are known to be proteins that are increased by co-cultivation with Malassezia. (FFMS Immunol Med Microbiol. 54, 203-214, (2008))
Inflammation was induced in epidermal keratinocytes by stimulation with killed Malassezia, and changes in inflammation-related proteins by polypeptide 1 after 24 hours were examined.
Ｍａｌａｓｓｅｚｉａ菌としては、Ｍａｌａｓｓｅｚｉａ ｆｕｒｆｕｒ（ＮＢＲＣ０６５６）を用いた。ＧＰＬＰ寒天培地を用いて３０℃、５日間培養した後、リン酸緩衝生理食塩水（ＰＢＳ）に分散し、菌数を測定した。菌数測定後、４％パラホルムアルデヒド／ＰＢＳ（－）で３０ｍｉｎ振とうしながら固定し、ＰＢＳ（－）で２回洗浄後、ＰＢＳ（－）に分散させてＭａｌａｓｓｅｚｉａ菌死菌溶液とした。
１×１０５ｃｅｌｌｓ／ｗｅｌｌ／２ｍｌとなるように調整したＨＥＫｎをＥｐｉ－Ｌｉｆｅ・ＫＧ２培地で３７℃、５％ＣＯ２インキュベーター内でサブコンフルエントになるまで培養した。
培養後、Ｅｐｉ－Ｌｉｆｅ・ＫＧ２培地にＭａｌａｓｓｅｚｉａ菌死菌溶液を１×１０７ｃｆｕ／ｍｌの濃度となるように、またポリペプチド１を０．０００２５％、０．０００５％、０．００１％添加した培養液に培地交換し、３７℃、５％ＣＯ２インキュベーター内で培養した。なおポリペプチド１を添加していないものをｃｏｎｔｒｏｌとした。
２４時間培養後、培養上清を回収し、培養上清中のＭＭＰ－９、ＮＧＡＬ、ＩＬ－６、ＩＬ－８量をＥＬＩＳＡ ｋｉｔ（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）を用いて測定した。結果を図７～１０に示す。
Malassezia furfur (NBRC0656) was used as the Malassezia bacterium. After culturing at 30° C. for 5 days using GPLP agar medium, the cells were dispersed in phosphate-buffered saline (PBS) and counted. After measuring the number of bacteria, the cells were fixed with 4% paraformaldehyde/PBS(-) with shaking for 30 minutes, washed twice with PBS(-), and dispersed in PBS(-) to obtain a Malassezia-killed cell solution.
HEKn adjusted to 1×10 5 cells/well/2 ml was cultured in Epi-Life·KG2 medium at 37° C. in a 5% CO 2 incubator until subconfluent.
After culturing, Malassezia-killed cell solution was added to Epi-Life KG2 medium at a concentration of 1×10 7 cfu/ml, and polypeptide 1 was added at 0.00025%, 0.0005% and 0.001%. The medium was exchanged with the culture solution obtained from the above method, and cultured in a 37° C., 5% CO 2 incubator. In addition, the thing which did not add the polypeptide 1 was made into control.
After culturing for 24 hours, the culture supernatant was collected, and the amounts of MMP-9, NGAL, IL-6 and IL-8 in the culture supernatant were measured using an ELISA kit (R&D Systems). The results are shown in Figures 7-10.
（結果）
図７に示したように、Ｍａｌａｓｓｅｚｉａの死菌添加前はＭＭＰ－９の発現量が８８ｐｇ／ｍｌであったが、Ｍａｌａｓｓｅｚｉａの死菌添加後の炎症により、ＭＭＰ－９が３５４ｐｇ／ｍｌと増大した。一方、ポリペプチド１を０．０００２５％、０．０００５％、０．００１％添加することにより、Ｍａｌａｓｓｅｚｉａの死菌を添加してもＭＭＰ－９の発現量はそれぞれ１６５ｐｇ／ｍｌ、１１０ｐｇ／ｍｌ、９５ｐｇ／ｍｌと小さくなり、添加量に依存して、炎症を抑制できた。特に、ポリペプチド１を０．０００５％、０．００１％添加することにより、Ｍａｌａｓｓｅｚｉａの死菌の添加前後でＭＭＰ－９の発現量がほぼ同じ値を示し、炎症が抑制された。
図８に示したように、Ｍａｌａｓｓｅｚｉａの死菌添加前はＮＧＡＬの発現量が４２８３ｐｇ／ｍｌであったが、Ｍａｌａｓｓｅｚｉａの死菌添加後の炎症により、ＮＧＡＬが５０７２ｐｇ／ｍｌと増大した。一方、ポリペプチド１を０．０００２５％、０．０００５％、０．００１％添加することにより、Ｍａｌａｓｓｅｚｉａの死菌を添加してもＮＧＡＬの発現量はそれぞれ４４２５ｐｇ／ｍｌ、４５８１ｐｇ／ｍｌ、３７２６ｐｇ／ｍｌと小さくなり、炎症を抑制できた。
図９に示したように、Ｍａｌａｓｓｅｚｉａの死菌添加後の炎症により、ＩＬ－６が１６ｐｇ／ｍｌと増大した。一方、ポリペプチド１を０．０００２５％、０．０００５％、０．００１％添加することにより、Ｍａｌａｓｓｅｚｉａの死菌を添加してもＩＬ－６の発現量は２ｐｇ／ｍｌ、１ｐｇ／ｍｌ、検出限界以下と小さく、添加量に依存して、炎症を抑制できた。
図１０に示したように、Ｍａｌａｓｓｅｚｉａの死菌添加前はＩＬ－８の発現量が１１９ｐｇ／ｍｌであったが、Ｍａｌａｓｓｅｚｉａの死菌添加後の炎症により、ＩＬ－８が４７０ｐｇ／ｍｌと増大した。一方、ポリペプチド１を０．０００２５％、０．０００５％、０．００１％添加することにより、Ｍａｌａｓｓｅｚｉａの死菌を添加してもＩＬ－８の発現量はそれぞれ１０２ｐｇ／ｍｌ、１１１ｐｇ／ｍｌ、８６ｐｇ／ｍｌと小さく、Ｍａｌａｓｓｅｚｉａの死菌添加前と同程度であり、炎症を抑制できた。
すなわち、ポリペプチド１は、Ｍａｌａｓｓｅｚｉａ ｆｕｒｆｕｒの死菌による炎症に対する抗炎症効果があり、フケの改善作用があることが確認できた。
(result)
As shown in FIG. 7, the expression level of MMP-9 was 88 pg / ml before the addition of killed Malassezia, but the inflammation after the addition of killed Malassezia increased MMP-9 to 354 pg / ml. . On the other hand, by adding 0.00025%, 0.0005%, and 0.001% of polypeptide 1, the expression levels of MMP-9 were 165 pg/ml, 110 pg/ml, respectively, even when killed Malassezia was added. It became as small as 95 pg/ml, and inflammation could be suppressed depending on the added amount. In particular, addition of 0.0005% and 0.001% of polypeptide 1 showed substantially the same expression level of MMP-9 before and after addition of killed Malassezia, and inflammation was suppressed.
As shown in FIG. 8, the expression level of NGAL was 4283 pg/ml before addition of killed Malassezia, but increased to 5072 pg/ml due to inflammation after addition of killed Malassezia. On the other hand, by adding 0.00025%, 0.0005%, and 0.001% polypeptide 1, the expression levels of NGAL were 4425 pg/ml, 4581 pg/ml, and 3726 pg/ml, respectively, even with the addition of killed Malassezia cells. ml, and the inflammation could be suppressed.
As shown in FIG. 9, IL-6 increased to 16 pg/ml due to inflammation after addition of killed Malassezia. On the other hand, by adding 0.00025%, 0.0005%, and 0.001% of polypeptide 1, the expression levels of IL-6 were 2 pg/ml and 1 pg/ml, even if killed Malassezia were added. It was small, below the limit, and could suppress inflammation depending on the amount added.
As shown in FIG. 10, the expression level of IL-8 was 119 pg / ml before the addition of killed Malassezia, but IL-8 increased to 470 pg / ml due to inflammation after the addition of killed Malassezia. . On the other hand, by adding 0.00025%, 0.0005%, and 0.001% of polypeptide 1, the expression levels of IL-8 were 102 pg/ml, 111 pg/ml, respectively, even with the addition of killed Malassezia. It was as small as 86 pg/ml, which was almost the same level as before the addition of killed Malassezia, and inflammation could be suppressed.
That is, it was confirmed that the polypeptide 1 has an anti-inflammatory effect against inflammation caused by dead bacteria of Malassezia furfur and has an improving effect on dandruff.
４．外毛根鞘細胞の増殖促進作用
毛髪の毛球から毛包の全長に存在している外毛根鞘細胞の増殖促進作用を検討した。
毛包外毛根鞘細胞 （Ｈａｉｒ Ｏｕｔｅｒ Ｒｏｏｔ Ｓｈｅａｔｈ Ｃｅｌｌｓ 以下「ＯＲＳ」、ＳｃｉｅｎＣｅｌｌ Ｒｅｓｅａｒｃｈ Ｌａｂｏｒａｔｏｒｉｅｓ）は、９６－ｗｅｌｌプレートに２×１０４ｃｅｌｌｓ／ｗｅｌｌで播種して、２４時間培養した。培養後、培地を除去して、ポリペプチド１を添加したポリペプチド１含有ＭＳＣＭ培地に替えた。
ポリペプチド１は、Ｍｅｓｅｎｃｈｙｍａｌ Ｓｔｅｍ Ｃｅｌｌ Ｍｅｄｉｕｍ培地（ＳｃｉｅｎＣｅｌｌ Ｒｅｓｅａｒｃｈ Ｌａｂｏｒａｔｏｒｉｅｓ）に付属の５％牛胎児血清を添加した培地（以下ＭＳＣＭ培地という。）で、０．０００３１２５、０．０００６２５、０．００１２５質量％となるよう希釈してポリペプチド１含有ＭＳＣＭ培地を調製した。
サンプル添加処理から２４時間後に培養上清を回収し、Ｃｅｌｌ Ｃｏｕｎｔｉｎｇ Ｋｉｔ（同仁化学研究所）を用いてＷＳＴ－１ ｃｅｌｌ ｐｒｏｌｉｆｅｒａｔｉｏｎ ａｓｓａｙにより細胞増殖率を算出した。結果を図１１に示す。
4. Effect of Promoting Proliferation of Outer Root Sheath Cells The effect of promoting the proliferation of outer root sheath cells present from the hair bulb to the entire length of the hair follicle was examined.
Hair Outer Root Sheath Cells (hereinafter “ORS”, ScienceCell Research Laboratories) were seeded in a 96-well plate at 2×10 4 cells/well and cultured for 24 hours. After culturing, the medium was removed and replaced with polypeptide 1-containing MSCM medium supplemented with polypeptide 1.
Polypeptide 1 was 0.0003125, 0.000625, 0.00125% by mass in a medium supplemented with 5% fetal bovine serum attached to Mesenchymal Stem Cell Medium (ScienCell Research Laboratories) (hereinafter referred to as MSCM medium). Polypeptide 1-containing MSCM medium was prepared by diluting the
Twenty-four hours after the sample addition treatment, the culture supernatant was collected, and the cell proliferation rate was calculated by WST-1 cell proliferation assay using a Cell Counting Kit (Dojindo Laboratories). The results are shown in FIG.
（結果）
図１１に示したように、ポリペプチド１を添加しなかった外毛根鞘細胞に比べて、ポリペプチド１を０．０００３１２５、０．０００６２５、０．００１２５質量％となるよう希釈したポリペプチド１含有ＭＳＣＭ培地を添加した外毛根鞘細胞は、それぞれ１０６．９２、１１４．３３、１１８．１８％と、増殖率が増大した。
すなわち、ポリペプチド１は、外毛根鞘細胞の増殖を促進し、育毛作用があることが確認できた。
(result)
As shown in FIG. 11, polypeptide 1 containing diluted polypeptide 1 to 0.0003125, 0.000625, 0.00125% by mass compared to outer root sheath cells to which polypeptide 1 was not added Outer root sheath cells to which the MSCM medium was added had increased proliferation rates of 106.92, 114.33 and 118.18%, respectively.
That is, it was confirmed that polypeptide 1 promotes proliferation of outer root sheath cells and has a hair growth effect.
５．コラーゲンとヒアルロン酸の産生促進作用
線維芽細胞に対するヒトプロコラーゲンＩ型Ｃ末端ペプチドとヒアルロン酸の産生促進効果を検討した。
新生児由来正常ヒト線維芽細胞（Ｎｏｒｍａｌ ｈｕｍａｎ ｄｅｒｍａｌ ｆｉｂｒｏｂｌａｓｔｓ以下「ＮＨＤＦ」、ＬＯＮＺＡ）は、２４－ｗｅｌｌプレートに４×１０４ｃｅｌｌｓ／ｗｅｌｌで播種して、６日間培養した。培養後、培地を除去して、ポリペプチド１を添加したポリペプチド１含有ＤＭＥＭ培地に替えた。
ポリペプチド１は、０．５％牛胎児血清（株式会社ニチレイバイオサイエンス）、および１％（ｖ／ｖ）Ｐｅｎｉｃｉｌｌｉｎ－Ｓｔｒｅｐｔｏｍｙｃｉｎ（Ｓｉｇｍａ Ａｌｄｒｉｃｈ）含有ＤＭＥＭ（Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ Ｍｅｄｉｕｍ）培地（ＧＩＢＣＯ）（以下ＤＭＥＭ培地という。）で、０．０００１、０．００１質量％となるよう希釈してポリペプチド１含有ＤＭＥＭ培地を調製した。
サンプル添加処理から２４時間後に培養上清を回収し、培養上清中のヒトプロコラーゲンＩ型Ｃ末端ペプチド（タカラバイオ）とヒアルロン酸量（Ｒ＆Ｄ Ｓｙｓｔｅｍｓ）をそれぞれＥＬＩＳＡキットで測定した。また、細胞をＭＴＴ Ａｓｓａｙにより細胞生存率を測定した。ＥＬＩＳＡにて算出した結果をＭＴＴ Ａｓｓａｙにより算出した細胞生存率で割り返したものを結果として用いた。結果を図１２、１３に示す。
5. Effect of Promoting Production of Collagen and Hyaluronic Acid The effect of promoting the production of human procollagen type I C-terminal peptide and hyaluronic acid on fibroblasts was examined.
Neonatal-derived normal human fibroblasts (hereinafter “NHDF”, LONZA) were seeded in a 24-well plate at 4×10 4 cells/well and cultured for 6 days. After culturing, the medium was removed and replaced with polypeptide 1-containing DMEM medium supplemented with polypeptide 1.
Polypeptide 1, 0.5% fetal bovine serum (Nichirei Biosciences), and 1% (v / v) Penicillin-Streptomycin (Sigma Aldrich) containing DMEM (Dulbecco's Modified Eagle's Medium) medium (GIBCO ) (hereinafter referred to as DMEM medium) to prepare polypeptide 1-containing DMEM medium by diluting to 0.0001 and 0.001% by mass.
The culture supernatant was collected 24 hours after the sample addition treatment, and the amount of human procollagen type I C-terminal peptide (Takara Bio) and the amount of hyaluronic acid (R&D Systems) in the culture supernatant were each measured using an ELISA kit. In addition, the cells were measured for cell viability by MTT Assay. The results obtained by dividing the results calculated by ELISA by the cell viability calculated by MTT Assay were used as the results. The results are shown in FIGS.
（結果）
図１２に示したように、ポリペプチド１を添加しなかった線維芽細胞に比べて、ポリペプチド１を０．０００１、０．００１質量％となるよう希釈したポリペプチド１含有ＤＭＥＭ培地を添加した線維芽細胞は、ヒトプロコラーゲンＩ型Ｃ末端ペプチドの産生量が、それぞれ１１０．３７、１２４．２４％と増大した。
図１３に示したように、ポリペプチド１を添加しなかった線維芽細胞に比べて、ポリペプチド１を０．００１質量％となるよう希釈したポリペプチド１含有ＤＭＥＭ培地を添加した線維芽細胞は、ヒアルロン酸の産生量が、１３２．３１％に増大した。
すなわち、ポリペプチド１は、ヒトプロコラーゲンＩ型Ｃ末端ペプチド及びヒアルロン酸の産生量を増大し、シワ改善、シワ抑制、皮膚のたるみ・はり改善作用があることが確認できた。
(result)
As shown in FIG. 12, a DMEM medium containing polypeptide 1 diluted to 0.0001% by mass or 0.001% by mass of polypeptide 1 was added to fibroblasts to which polypeptide 1 was not added. Fibroblasts increased the production of human procollagen type I C-terminal peptide to 110.37% and 124.24%, respectively.
As shown in FIG. 13, compared to fibroblasts to which polypeptide 1 was not added, fibroblasts to which polypeptide 1-containing DMEM medium diluted to 0.001% by mass of polypeptide 1 was added were , the production of hyaluronic acid increased to 132.31%.
That is, it was confirmed that polypeptide 1 increases the production of human procollagen type I C-terminal peptide and hyaluronic acid, and has the effects of improving wrinkles, suppressing wrinkles, and improving skin sagging and firmness.
６．基底膜タンパクの産生促進及び分解抑制作用
ケラチノサイトに対する４型コラーゲン、ラミニン３３２、ＭＭＰ－２、ＭＭＰ－９の産生量を検討した。４型コラーゲンとラミニン３３２は表皮基底膜の構成成分であり、ＭＭＰ－２は４型コラーゲンとラミニン３３２の、ＭＭＰ－９は４型コラーゲンの分解酵素であることが知られている。
6. Effect of Promoting Production and Suppressing Degradation of Basement Membrane Protein The amounts of type 4 collagen, laminin 332, MMP-2 and MMP-9 produced by keratinocytes were examined. It is known that type 4 collagen and laminin 332 are constituents of the epidermal basement membrane, MMP-2 is an enzyme that degrades type 4 collagen and laminin 332, and MMP-9 is an enzyme that degrades type 4 collagen.
ＨＥＫｎを６－ｗｅｌｌプレートに１×１０５ｃｅｌｌｓ／ｗｅｌｌで播種して、Ｅｐｉ－Ｌｉｆｅ・ＫＧ２培地にてサブコンフルエントの状態まで培養した。培養後、培地を除去して、ポリペプチド１を添加したポリペプチド１含有Ｅｐｉ－Ｌｉｆｅ・ＫＧ２培地に替えた。
ポリペプチド１は、Ｅｐｉ－Ｌｉｆｅ・ＫＧ２培地で、０．０００１、０．００１質量％となるよう希釈してポリペプチド１含有Ｅｐｉ－Ｌｉｆｅ・ＫＧ２培地を調製した。
サンプル添加処理から２４時間後に培養上清を回収し、培養上清中のＭＭＰ－２、ＭＭＰ－９量をＥＬＩＳＡ ｋｉｔ（Ｒ＆Ｄ ｓｙｓｔｅｍｓ社）を用いて測定した。また細胞を１００μｌ Ｌａｅｍｍｌｉ ｂｕｆｆｅｒ（０．０９Ｍ Ｔｒｉｓ－ＨＣｌ（ｐＨ６．８）、３％ ＳＤＳ、１０．３％ ｇｌｙｃｅｒｏｌ）に溶解し、３０分間４℃にてシェイカー上で細胞を溶解させた。
次いで、４°Ｃで１５，０００×ｇの条件で１０分間遠心分離した。上清を４×ＳＤＳ－ｌｏａｄｉｎｇ ｂｕｆｆｅｒと混合し、９５°Ｃで３分間熱処理した後、ＳＤＳ－ＰＡＧＥにかけてタンパク質を分離した。ゲル内のタンパク質は、電気的にＰＶＤＦ膜に転写した。
HEKn was seeded on a 6-well plate at 1×10 5 cells/well and cultured in Epi-Life·KG2 medium until subconfluent. After culturing, the medium was removed and replaced with Epi-Life·KG2 medium containing polypeptide 1 supplemented with polypeptide 1.
Polypeptide 1 was diluted with Epi-Life·KG2 medium to 0.0001 and 0.001% by mass to prepare polypeptide 1-containing Epi-Life·KG2 medium.
The culture supernatant was collected 24 hours after the sample addition treatment, and the amounts of MMP-2 and MMP-9 in the culture supernatant were measured using an ELISA kit (R&D Systems). The cells were dissolved in 100 μl Laemmli buffer (0.09M Tris-HCl (pH 6.8), 3% SDS, 10.3% glycerol) and lysed on a shaker at 4° C. for 30 minutes.
Then, it was centrifuged at 4°C and 15,000 xg for 10 minutes. The supernatant was mixed with 4×SDS-loading buffer, heat-treated at 95° C. for 3 minutes, and subjected to SDS-PAGE to separate proteins. Proteins in the gel were electrically transferred to a PVDF membrane.
転写後、膜をＳｔａｒｔｉｎｇ Ｂｌｏｃｋ Ｂｌｏｃｋｉｎｇ Ｂｕｆｆｅｒ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）に浸し、約３０分間ブロッキングした。
次に、ＰＶＤＦ膜をＳｔａｒｔｉｎｇ Ｂｌｏｃｋ Ｂｌｏｃｋｉｎｇ Ｂｕｆｆｅｒで２０００倍希釈した一次抗体（Ａｎｔｉ－Ｃｏｌｌａｇｅｎ ＩＶ、Ａｂｃａｍ、およびＡｎｔｉ－Ｌａｍｉｎｉｎ ３３２、Ｍｉｌｌｉｐｏｒｅ）に浸し、４°Ｃで一晩反応させた。次いで、ＰＢＳＴ（ＰＢＳ、０．０５％ Ｔｗｅｅｎ ２０）を用いて３回洗浄後、１０，０００倍希釈した二次抗体（Ｇｏａｔ ａｎｔｉ－Ｒａｂｂｉｔ ＩｇＧ （Ｈ＋Ｌ） Ｓｅｃｏｎｄａｒｙ Ａｎｔｉｂｏｄｙ、ＨＲＰ、Ｐｏｌｙｃｌｏｎａｌ、 Ｉｎｖｉｔｒｏｇｅｎ Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ、およびＧｏａｔ ａｎｔｉ－Ｍｏｕｓｅ ＩｇＧ （Ｈ＋Ｌ） Ｓｅｃｏｎｄａｒｙ Ａｎｔｉｂｏｄｙ、ＨＲＰ、Ｐｏｌｙｃｌｏｎａｌ、 Ｉｎｖｉｔｒｏｇｅｎ Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）に浸し、室温で１時間反応させた。
ＰＢＳＴで３回洗浄した後、ＥＣＬ ｄｅｔｅｃｔｉｏｎ ｋｉｔ（ＧＥ ｈｅａｌｔｈｃａｒｅ）を用いて、化学発光検出装置（ＬＡＳ－４０００ ｍｉｎｉ、富士フィルム株式会社）でシグナルを検出した。
検出されたバンドのシグナル強度を解析（Ｍｕｌｔｉ Ｇａｕｇｅ、フジフィルム株式会社）し、コントロールに対する相対値を算出し４型コラーゲンおよびラミニン３３２の増加量を評価した。
結果を図１４、１５に示す。
After transfer, the membrane was soaked in Starting Block Blocking Buffer (Thermo Fisher Scientific) and blocked for about 30 minutes.
Next, the PVDF membrane was immersed in a primary antibody (Anti-Collagen IV, Abcam, and Anti-Laminin 332, Millipore) diluted 2000-fold with Starting Block Blocking Buffer, and allowed to react overnight at 4°C. Then, after washing three times with PBST (PBS, 0.05% Tween 20), 10,000-fold diluted secondary antibody (Goat anti-Rabbit IgG (H + L) Secondary Antibody, HRP, Polyclonal, Invitrogen Thermo Fisher Scientific , and Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP, Polyclonal, Invitrogen Thermo Fisher Scientific) and allowed to react at room temperature for 1 hour.
After washing with PBST three times, signals were detected with a chemiluminescence detector (LAS-4000 mini, Fuji Film Co., Ltd.) using an ECL detection kit (GE healthcare).
The signal intensity of the detected band was analyzed (Multi Gauge, Fuji Film Co., Ltd.), the relative value to the control was calculated, and the increased amount of type 4 collagen and laminin 332 was evaluated.
The results are shown in FIGS.
（結果）
図１４に示したように、ポリペプチド１を添加しなかったケラチノサイトに比べて、ポリペプチド１を０．０００１質量％となるよう希釈したポリペプチド１含有Ｅｐｉ－Ｌｉｆｅ・ＫＧ２培地を添加したケラチノサイトは、４型コラーゲンとラミニン３３２の産生量が、それぞれ１４４．７５、１２９．９６％増大した。
図１５に示したように、ポリペプチド１を添加しなかったケラチノサイトに比べて、ポリペプチド１を０．０００１、０．００１質量％となるよう希釈したポリペプチド１含有Ｅｐｉ－Ｌｉｆｅ・ＫＧ２培地を添加したケラチノサイトは、４型コラーゲンとラミニン３３２の分解酵素であるＭＭＰ－２の産生量が、それぞれ９６．０９、７１．８３％に抑制され、４型コラーゲンの分解酵素であるＭＭＰ－９の産生量が、それぞれ６４．８８、７４．３９％に抑制された。
すなわち、ポリペプチド１は、基底膜タンパクの産生を促進するとともに、基底膜分解酵素の産生を抑制し、シワの改善・抑制作用、皮膚のたるみ・はり改善作用があることが確認できた。
(result)
As shown in FIG. 14, compared to keratinocytes to which polypeptide 1 was not added, keratinocytes to which polypeptide 1-containing Epi-Life KG2 medium diluted to 0.0001% by mass of polypeptide 1 was added were , the production of type 4 collagen and laminin 332 increased by 144.75% and 129.96%, respectively.
As shown in FIG. 15, Epi-Life KG2 medium containing polypeptide 1 diluted to 0.0001% by mass and 0.001% by mass of polypeptide 1 was added to keratinocytes to which polypeptide 1 was not added. In the added keratinocytes, the production amounts of MMP-2, which is an enzyme that degrades type 4 collagen and laminin 332, are suppressed to 96.09% and 71.83%, respectively, and the production of MMP-9, which is an enzyme that degrades type 4 collagen. were suppressed to 64.88 and 74.39%, respectively.
That is, it was confirmed that polypeptide 1 promotes the production of basement membrane proteins, suppresses the production of basement membrane-degrading enzymes, and has wrinkle-improving/inhibiting effects and skin sagging/tension-improving effects.
処方例１ 皮膚外用剤
成分 配合量（重量％）
１．本発明で使用するポリペプチド ０．０００１
２．精製水 １
３．ワセリン 残余
処方例２ 皮膚洗浄料
成分 配合量（重量％）
１．本発明で使用するポリペプチド ０．００００１
２．ラウロイルグルタミン酸Ｎａ １５
３．ココイルグルタミン酸Ｋ １０
４．ココイルアスパラギン酸Ｎａ ５
５．ココイルメチルタウリンＮａ ２．４
６．スルホコハク酸ラウレス２Ｎａ ０．２
７．トリメチルグリシン ６
８．モノステアリン酸デカグリセリル ０．５
９．変性デンプン ０．４
１０．グリセリン ２５
１１．１，３－ブチレングリコール １４
１２．ココアンホ酢酸Ｎａ ３．９
１３．ポリクオタニウム－７ ０．１
１４．精製水 残余
Formulation example 1 External skin preparation components Blending amount (% by weight)
1. Polypeptide used in the present invention 0.0001
2. Purified water 1
3. Vaseline residue
Formulation example 2 Skin cleanser component Amount (% by weight)
1. Polypeptide used in the present invention 0.00001
2.
3. Cocoyl glutamic acid K10
4. Sodium cocoyl aspartate 5
5. Cocoyl methyl taurine Na 2.4
6. Disodium laureth sulfosuccinate 0.2
7.
8. Decaglyceryl monostearate 0.5
9. Modified starch 0.4
10.
11. 1,3-
12. Sodium cocoamphoacetate 3.9
13. Polyquaternium-7 0.1
14. purified water residue
処方例３ 毛髪洗浄料
成分 配合量（重量％）
１．本発明で使用するポリペプチド ０．０００５
２．ラウラミドプロピルヒドロキシスルタイン ５
３．ココイルアラニンＮａ ５
４．ココイルメチルアラニンＮａ ５
５．セテアレス－６０ミリスチルグリコール １
６．ペンチレングリコール １
７．ココイルグルタミン酸Ｎａ １
８．ポリクオタニウム－５１ ０．００１
９．（メタクリル酸グリセリルアミドエチル／メタクリルサンステアリル）コポリマー
０．００１
１０．ヒドロキシプロピルグアーヒドロキシプロピルトリモニウムクロリド
０．００１
１１．クエン酸 ０．２
１２．精製水 残余
処方例４ 育毛剤
成分 配合量（重量％）
１．本発明で使用するポリペプチド ０．０００００５
２．エタノール ５０
３．１，３－ブチレングリコール １０
４．センブリ抽出物 ０．５
５．パントテニルエチルエーテル ０．５
６．精製水 残余
Formulation Example 3 Hair cleansing component Amount (% by weight)
1. Polypeptide used in the present invention 0.0005
2. Lauramidopropyl hydroxysultaine 5
3. Cocoyl alanine Na 5
4. Cocoyl Methylalanine Na 5
5. ceteareth-60 myristyl glycol 1
6. Pentylene glycol 1
7. Sodium cocoyl glutamate 1
8. Polyquaternium-51 0.001
9. (glycerylamidoethyl methacrylate/methacrylsanstearyl) copolymer
0.001
10. Hydroxypropyl Guar Hydroxypropyltrimonium Chloride
0.001
11. Citric acid 0.2
12. purified water residue
Prescription Example 4 Hair restorer component Amount (% by weight)
1. Polypeptide used in the present invention 0.000005
2.
3. 1,3-
4. assembly extract 0.5
5. Pantothenyl ethyl ether 0.5
6. purified water residue
処方例５ 洗口剤
成分 配合量（重量％）
１．本発明で使用するポリペプチド ０．００１
２．マスティック １
３．ソルビトール １０
４．ポリグリセリン脂肪酸エステル １０
５．エタノール ２０
６．精製水 残余
処方例６ 点眼剤
成分 配合量（重量％）
１．本発明で使用するポリペプチド ０．０００１
２．ホウ酸 １．５
３．ホウ砂 ０．１
４．ポリソルベート８０ ０．５
５．ｌ－メントール ０．０１
６．ヒドロキシプロピルメチルセルロース ０．１
７．精製水 残余
処方例７ メイクアップ化粧料
成分 配合量（重量％）
１．本発明で使用するポリペプチド ０．００００１
２．シクロメチコン １０
３．ジメチコン ３０
４．ポリエーテル変性シリコーン ６
５．トリイソステアリン酸ポリグリセリル－２ １
６．シリコーン処理酸化チタン ８
７．シリコーン処理酸化鉄 ０．５
８．シリコーン処理酸化亜鉛 １
９．シリコーン処理群青 ２
１０．パルミチン酸デキストリン ０．５
１１．ポリメタクリル酸メチル ４
１２．シリカ ２
１３．１，３－ブチレングリコール ５
１４．精製水 残余
Formulation Example 5 Mouthwash component Amount (% by weight)
1. Polypeptide used in the present invention 0.001
2. Mastic 1
3.
4. Polyglycerol
5.
6. purified water residue
Formulation Example 6 Eye drop component Amount (% by weight)
1. Polypeptide used in the present invention 0.0001
2. Boric acid 1.5
3. Borax 0.1
4.
5. l-menthol 0.01
6. Hydroxypropyl methylcellulose 0.1
7. purified water residue
Formulation Example 7 Makeup cosmetic ingredients Blending amount (% by weight)
1. Polypeptide used in the present invention 0.00001
2.
3.
4. Polyether modified
5. Polyglyceryl-2 1 triisostearate
6.
7. Siliconized iron oxide 0.5
8. Siliconized zinc oxide 1
9. Silicone treated ultramarine blue 2
10. Dextrin palmitate 0.5
11. Polymethyl methacrylate 4
12. Silica 2
13.1,3-butylene glycol 5
14. purified water residue
Claims (6)
Priority Applications (1)
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|---|---|---|---|
| JP2022205114A JP2023052021A (en) | 2018-02-19 | 2022-12-22 | Anti-inflammatory agent and anti-skin irritation agent |
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|---|---|---|---|
| JP2018027412 | 2018-02-19 | ||
| JP2018027412 | 2018-02-19 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2022205114A Division JP2023052021A (en) | 2018-02-19 | 2022-12-22 | Anti-inflammatory agent and anti-skin irritation agent |
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| Publication Number | Publication Date |
|---|---|
| JP2019142835A JP2019142835A (en) | 2019-08-29 |
| JP7266202B2 true JP7266202B2 (en) | 2023-04-28 |
Family
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| JP2018198894A Active JP7266202B2 (en) | 2018-02-19 | 2018-10-23 | Cosmetics and anti-inflammatory agents |
| JP2022205114A Pending JP2023052021A (en) | 2018-02-19 | 2022-12-22 | Anti-inflammatory agent and anti-skin irritation agent |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2022205114A Pending JP2023052021A (en) | 2018-02-19 | 2022-12-22 | Anti-inflammatory agent and anti-skin irritation agent |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005053867A (en) | 2003-08-07 | 2005-03-03 | Showa Denko Kk | Dandruff-suppressing composition |
| JP2007204475A (en) | 2006-02-02 | 2007-08-16 | Omp Inc | Method for improving therapy of skin |
| WO2010101237A1 (en) | 2009-03-06 | 2010-09-10 | アンジェスＭｇ株式会社 | Polypeptides and antibacterial or antiseptic use of same |
| JP2012500257A (en) | 2008-08-19 | 2012-01-05 | セル バイオテク カンパニー リミテッド | Novel use of bacteriocin derived from Enterococcus faecalis SL-5 |
| JP2013129648A (en) | 2011-11-22 | 2013-07-04 | Anges Mg Inc | Skin care agent containing antimicrobial active polypeptide having collagen gel contractile activity, human skin fibroblast proliferative activity and hyaluronic acid-producing activity |
| JP2017002057A (en) | 2007-07-26 | 2017-01-05 | ルバンス セラピュティックス インク．Ｒｅｖａｎｃｅ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． | Antimicrobial peptide, composition, and method for using the same |
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2018
- 2018-10-23 JP JP2018198894A patent/JP7266202B2/en active Active
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- 2022-12-22 JP JP2022205114A patent/JP2023052021A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005053867A (en) | 2003-08-07 | 2005-03-03 | Showa Denko Kk | Dandruff-suppressing composition |
| JP2007204475A (en) | 2006-02-02 | 2007-08-16 | Omp Inc | Method for improving therapy of skin |
| JP2017002057A (en) | 2007-07-26 | 2017-01-05 | ルバンス セラピュティックス インク．Ｒｅｖａｎｃｅ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． | Antimicrobial peptide, composition, and method for using the same |
| JP2012500257A (en) | 2008-08-19 | 2012-01-05 | セル バイオテク カンパニー リミテッド | Novel use of bacteriocin derived from Enterococcus faecalis SL-5 |
| WO2010101237A1 (en) | 2009-03-06 | 2010-09-10 | アンジェスＭｇ株式会社 | Polypeptides and antibacterial or antiseptic use of same |
| JP2013129648A (en) | 2011-11-22 | 2013-07-04 | Anges Mg Inc | Skin care agent containing antimicrobial active polypeptide having collagen gel contractile activity, human skin fibroblast proliferative activity and hyaluronic acid-producing activity |
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| Publication number | Publication date |
|---|---|
| JP2019142835A (en) | 2019-08-29 |
| JP2023052021A (en) | 2023-04-11 |
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