JP2014141450A - Elastin production promoter - Google Patents
Elastin production promoter Download PDFInfo
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Abstract
Description
本発明は、ペプチド分子等を含有するエラスチン産生促進剤に関する。 The present invention relates to an elastin production promoter containing a peptide molecule or the like.
皮膚の老化は、細胞外マトリックスの合成とマトリックスメタロプロテアーゼなどによる分解との間のバランスの不均衡により生ずる。皮膚老化は加齢、乾燥、酸化、物理的刺激や、紫外線などの外的ストレスが要因とされている。組織学的な現象としては、真皮中のコラーゲン、エラスチン、グルコサミノグリカンなどの細胞外マトリックス成分の変化が挙げられ、これらの質的および量的な現象が皮膚老化において重要であると考えられる。また、紫外線照射時には、真皮内の線維芽細胞のエラスチン合成が抑制され、それに伴ってシワができやすくなる。よって、紫外線照射時にエラスチン合成を促進することができれば、シワを抑制することも可能であると考えられる。 Skin aging is caused by an imbalance in the balance between extracellular matrix synthesis and degradation by matrix metalloproteases and the like. Skin aging is caused by aging, drying, oxidation, physical stimulation, and external stress such as ultraviolet rays. Histological phenomena include changes in extracellular matrix components such as collagen, elastin, and glucosaminoglycans in the dermis, and these qualitative and quantitative phenomena are considered to be important in skin aging. . In addition, when irradiated with ultraviolet light, elastin synthesis in fibroblasts in the dermis is suppressed, and wrinkles are easily generated accordingly. Therefore, if elastin synthesis can be promoted during ultraviolet irradiation, wrinkles can be suppressed.
特許文献１には、２種類以上のムコ多糖、コラーゲンおよび／もしくはエラスチンまたはその部分分解物、コエンザイムＱ１０を有効成分とする経口用皮膚老化予防または改善剤が記載されている。しかし、これらの成分から３つの成分を除いた比較例２（コラーゲンペプチドを含む）では、肌の弾力性、水分量、肌明度の改善効果がほとんど認められないことが分かると記載されている。
非特許文献１〜５には、コラーゲンペプチドによって皮膚の水分量が増加することが記載されている。しかし、水分量増加はエラスチン産生促進とは無関係である。また、いかなるペプチドが水分量増加作用を示すかも不明である。
非特許文献６および７には、Ｐｒｏ−Ｈｙｐ等が線維芽細胞の増殖促進作用を有し、またヒアルロン酸の産生を促進することが記載されている。しかし、これらのコラーゲンペプチドによって、エラスチン遺伝子のｍＲＮＡはほぼ変化が無かったことが記載されている。
Patent Document 1 describes an oral skin aging preventive or ameliorating agent comprising two or more kinds of mucopolysaccharides, collagen and / or elastin or a partially decomposed product thereof, and coenzyme Q10 as an active ingredient. However, it is described that in Comparative Example 2 (including the collagen peptide) in which three components are removed from these components, it can be seen that almost no effect of improving skin elasticity, moisture content, and skin brightness is observed.
Non-patent documents 1 to 5 describe that the moisture content of the skin is increased by the collagen peptide. However, the increase in water content is not related to the promotion of elastin production. In addition, it is unclear which peptide has an effect of increasing water content.
Non-Patent Documents 6 and 7 describe that Pro-Hyp and the like have a fibroblast proliferation promoting action and promote hyaluronic acid production. However, it is described that the mRNA of the elastin gene was not substantially changed by these collagen peptides.
本発明が解決しようとする課題は、皮膚のシワおよびたるみを予防および改善することができる、従来技術よりも優れたエラスチン産生促進剤を提供することにある。 The problem to be solved by the present invention is to provide an elastin production promoter superior to the prior art, which can prevent and improve skin wrinkles and sagging.
本発明者らは、鋭意検討した結果、ペプチド分子であるＧｌｕ−Ｈｙｐ−Ｇｌｙ、Ｓｅｒ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ−Ｇｌｙ、Ｇｌｕ−Ｈｙｐ、Ｌｅｕ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ−Ｇｌｙ、Ｌｅｕ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ、Ｐｒｏ−ＡｌａおよびＨｙｐ−Ｇｌｙならびにこれらの組み合わせに、優れたエラスチンの産生促進作用があることを見出して、本発明を完成させるに至った。即ち本発明は、以下の通りである。
［１］ Ｇｌｕ−Ｈｙｐ−Ｇｌｙ、Ｓｅｒ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ−Ｇｌｙ、Ｇｌｕ−Ｈｙｐ、Ｌｅｕ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ−Ｇｌｙ、Ｌｅｕ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ、Ｐｒｏ−ＡｌａおよびＨｙｐ−Ｇｌｙからなる群から選択されるペプチドまたはその薬学上許容される塩を含有する、エラスチン産生促進剤。
［２］ Ｇｌｕ−Ｈｙｐ−Ｇｌｙ、Ｓｅｒ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ−Ｇｌｙ、Ｇｌｕ−ＨｙｐおよびＬｅｕ−Ｈｙｐ−Ｇｌｙからなる群から選択されるペプチドまたはその薬学上許容される塩を含有する、［１］記載のエラスチン産生促進剤。
［３］ Ｇｌｕ−Ｈｙｐ−Ｇｌｙ、Ｓｅｒ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ−Ｇｌｙ、Ｇｌｕ−Ｈｙｐ、Ｌｅｕ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ−Ｇｌｙ、Ｌｅｕ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ、Ｐｒｏ−ＡｌａおよびＨｙｐ−Ｇｌｙからなる群から選択される２以上のペプチドまたはそれらの薬学上許容される塩を含有する、［１］記載のエラスチン産生促進剤。
［４］ Ｇｌｕ−Ｈｙｐ−Ｇｌｙ、Ｓｅｒ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ−Ｇｌｙまたはその薬学上許容される塩と、Ｇｌｕ−Ｈｙｐ、Ｌｅｕ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ−Ｇｌｙ、Ｌｅｕ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ、Ｐｒｏ−ＡｌａおよびＨｙｐ−Ｇｌｙからなる群から選択されるペプチドまたはその薬学上許容される塩とを含有する、［３］記載のエラスチン産生促進剤。
［５］ 美肌化粧料として用いられる、［１］〜［４］のいずれか記載のエラスチン産生促進剤。
［６］ Ｇｌｕ−Ｈｙｐ−Ｇｌｙ、Ｓｅｒ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ−Ｇｌｙ、Ｇｌｕ−Ｈｙｐ、Ｌｅｕ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ−Ｇｌｙ、Ｌｅｕ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ、Ｐｒｏ−ＡｌａおよびＨｙｐ−Ｇｌｙからなる群から選択されるペプチドまたはその薬学上許容される塩を含有する、健康食品。
As a result of intensive studies, the present inventors have found that peptide molecules such as Glu-Hyp-Gly, Ser-Hyp-Gly, Ala-Hyp-Gly, Glu-Hyp, Leu-Hyp-Gly, Ala-Hyp, Pro-Hyp -Gly, Leu-Hyp, Pro-Hyp, Pro-Ala and Hyp-Gly and combinations thereof were found to have an excellent elastin production promoting effect, and the present invention was completed. That is, the present invention is as follows.
[1] Glu-Hyp-Gly, Ser-Hyp-Gly, Ala-Hyp-Gly, Glu-Hyp, Leu-Hyp-Gly, Ala-Hyp, Pro-Hyp-Gly, Leu-Hyp, Pro-Hyp, Pro -An elastin production promoter containing a peptide selected from the group consisting of Ala and Hyp-Gly or a pharmaceutically acceptable salt thereof.
[2] containing a peptide selected from the group consisting of Glu-Hyp-Gly, Ser-Hyp-Gly, Ala-Hyp-Gly, Glu-Hyp and Leu-Hyp-Gly, or a pharmaceutically acceptable salt thereof, [1] The elastin production promoter according to [1].
[3] Glu-Hyp-Gly, Ser-Hyp-Gly, Ala-Hyp-Gly, Glu-Hyp, Leu-Hyp-Gly, Ala-Hyp, Pro-Hyp-Gly, Leu-Hyp, Pro-Hyp, Pro -The elastin production promoter according to [1], comprising two or more peptides selected from the group consisting of Ala and Hyp-Gly or a pharmaceutically acceptable salt thereof.
[4] Glu-Hyp-Gly, Ser-Hyp-Gly, Ala-Hyp-Gly or a pharmaceutically acceptable salt thereof, Glu-Hyp, Leu-Hyp-Gly, Ala-Hyp, Pro-Hyp-Gly, The elastin production promoter according to [3], comprising a peptide selected from the group consisting of Leu-Hyp, Pro-Hyp, Pro-Ala and Hyp-Gly or a pharmaceutically acceptable salt thereof.
[5] The elastin production promoter according to any one of [1] to [4], which is used as a skin care cosmetic.
[6] Glu-Hyp-Gly, Ser-Hyp-Gly, Ala-Hyp-Gly, Glu-Hyp, Leu-Hyp-Gly, Ala-Hyp, Pro-Hyp-Gly, Leu-Hyp, Pro-Hyp, Pro A health food containing a peptide selected from the group consisting of Ala and Hyp-Gly or a pharmaceutically acceptable salt thereof.
本発明によって、Ｇｌｕ−Ｈｙｐ−Ｇｌｙ、Ｓｅｒ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ−Ｇｌｙ、Ｇｌｕ−Ｈｙｐ、Ｌｅｕ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ−Ｇｌｙ、Ｌｅｕ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ、Ｐｒｏ−ＡｌａおよびＨｙｐ−Ｇｌｙのペプチド分子を含有する、従来技術よりも優れたエラスチン産生促進剤を提供することができる。これらのペプチド分子によって、エラスチン産生が促進され、皮膚のシワおよびたるみを予防および改善することができる。 According to the present invention, Glu-Hyp-Gly, Ser-Hyp-Gly, Ala-Hyp-Gly, Glu-Hyp, Leu-Hyp-Gly, Ala-Hyp, Pro-Hyp-Gly, Leu-Hyp, Pro-Hyp, It is possible to provide an elastin production promoter that is superior to the prior art and contains peptide molecules of Pro-Ala and Hyp-Gly. These peptide molecules promote elastin production and can prevent and ameliorate skin wrinkles and sagging.
以下、本発明を詳細に説明する。
１．ペプチド
本発明に用いられるペプチドは、Ｇｌｕ−Ｈｙｐ−Ｇｌｙ、Ｓｅｒ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ−Ｇｌｙ、Ｇｌｕ−Ｈｙｐ、Ｌｅｕ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ−Ｇｌｙ、Ｌｅｕ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ、Ｐｒｏ−ＡｌａおよびＨｙｐ−Ｇｌｙであり、本ペプチドは薬学上許容される塩とすることができる。好ましいペプチドとして、Ｇｌｕ−Ｈｙｐ−Ｇｌｙ、Ｓｅｒ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ−Ｇｌｙ、Ｇｌｕ−Ｈｙｐ、Ｌｅｕ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ−ＧｌｙおよびＬｅｕ−Ｈｙｐが挙げられ、より好ましくはＧｌｕ−Ｈｙｐ−Ｇｌｙ、Ｓｅｒ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ−Ｇｌｙ、Ｇｌｕ−ＨｙｐおよびＬｅｕ−Ｈｙｐ−Ｇｌｙが挙げられ、特に好ましくはＧｌｕ−Ｈｙｐ−Ｇｌｙ、Ｓｅｒ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ−Ｇｌｙが挙げられる。また、本ペプチドは、２つ以上を組み合わせて用いることも好ましく、特にＧｌｕ−Ｈｙｐ−Ｇｌｙ、Ｓｅｒ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ−Ｇｌｙまたはその薬学上許容される塩と、Ｇｌｕ−Ｈｙｐ、Ｌｅｕ−Ｈｙｐ−Ｇｌｙ、Ａｌａ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ−Ｇｌｙ、Ｌｅｕ−Ｈｙｐ、Ｐｒｏ−Ｈｙｐ、Ｐｒｏ−ＡｌａおよびＨｙｐ−Ｇｌｙからなる群から選択されるペプチドまたはその薬学上許容される塩とを組み合わせことが好ましい。
「薬学上許容される塩」としては、例えば、塩酸塩、硫酸塩、リン酸塩等の無機酸塩、メタンスルホン酸塩、ベンゼンスルホン酸塩、コハク酸塩、シュウ酸塩等の有機酸塩、ナトリウム塩、カリウム塩、カルシウム塩等の無機塩基塩、トリエチルアンモニウム塩等の有機塩基塩等が挙げられる。
Hereinafter, the present invention will be described in detail.
1. Peptides used in the present invention are Glu-Hyp-Gly, Ser-Hyp-Gly, Ala-Hyp-Gly, Glu-Hyp, Leu-Hyp-Gly, Ala-Hyp, Pro-Hyp-Gly, Leu-Hyp. Pro-Hyp, Pro-Ala and Hyp-Gly, and the peptide can be a pharmaceutically acceptable salt. Preferred peptides include Glu-Hyp-Gly, Ser-Hyp-Gly, Ala-Hyp-Gly, Glu-Hyp, Leu-Hyp-Gly, Ala-Hyp, Pro-Hyp-Gly and Leu-Hyp, and more Preferred are Glu-Hyp-Gly, Ser-Hyp-Gly, Ala-Hyp-Gly, Glu-Hyp and Leu-Hyp-Gly, particularly preferably Glu-Hyp-Gly, Ser-Hyp-Gly, Ala- Hyp-Gly is mentioned. In addition, it is also preferable to use a combination of two or more of the present peptides, in particular, Glu-Hyp-Gly, Ser-Hyp-Gly, Ala-Hyp-Gly or a pharmaceutically acceptable salt thereof, and Glu-Hyp, Leu. -Combining a peptide selected from the group consisting of Hyp-Gly, Ala-Hyp, Pro-Hyp-Gly, Leu-Hyp, Pro-Hyp, Pro-Ala and Hyp-Gly, or a pharmaceutically acceptable salt thereof. Is preferred.
Examples of the “pharmaceutically acceptable salt” include inorganic acid salts such as hydrochloride, sulfate, and phosphate, and organic acid salts such as methanesulfonate, benzenesulfonate, succinate, and oxalate. Inorganic base salts such as sodium salt, potassium salt and calcium salt, and organic base salts such as triethylammonium salt.
本ペプチドは、例えば「固相合成法」および「液相合成法」（例えば、特開２００３−１８３２９８）等で合成することができる。なお、固相合成法の場合はさらにＦｍｏｃ法とＢｏｃ法の方法が知られており、本ペプチドはいずれの方法で合成してもよい。固相合成法の例を、以下に具体的に説明する。表面をアミノ基で修飾した直径０．１ｍｍ程度のポリスチレン高分子ゲルのビーズを固相として用い、縮合剤としてジイソプロピルカルボジイミドを用いる。まず、Ｃ末のアミノ酸のアミノ基をＦｍｏｃ基またはＢｏｃ基で保護して、上記ポリスチレン高分子ゲルのアミノ基とペプチド結合を形成させる。固相を溶媒でよく洗い、残存する試薬、アミノ酸を洗浄除去し、その後、固相に結合しているアミノ酸のアミノ基の保護基を除去する。続いて、アミノ基を保護したアミノ酸を用いて、順次、同様の反応を繰り返すことで、固相上でペプチドを合成する。最後に、固相をトリフルオロ酢酸で温浸させることで、ペプチドを固相から切り離すことで、ペプチドを合成することができる。
本ペプチドは、ゼラチンにエンド型プロテアーゼおよびエキソ型プロテアーゼの２種以上を組み合わせて加水分解することによっても製造することができる。また、上記加水分解をしたペプチド混合物自体またはこれを部分精製した混合物をエラスチン産生促進剤として用いることもできる。
This peptide can be synthesized by, for example, “solid phase synthesis method” and “liquid phase synthesis method” (for example, JP-A No. 2003-183298). In the case of the solid phase synthesis method, methods of the Fmoc method and the Boc method are further known, and this peptide may be synthesized by any method. An example of the solid phase synthesis method will be specifically described below. A polystyrene polymer gel bead having a diameter of about 0.1 mm whose surface is modified with an amino group is used as a solid phase, and diisopropylcarbodiimide is used as a condensing agent. First, the amino group of the C-terminal amino acid is protected with an Fmoc group or a Boc group to form a peptide bond with the amino group of the polystyrene polymer gel. The solid phase is thoroughly washed with a solvent, the remaining reagent and amino acid are washed away, and then the amino group protecting group of the amino acid bonded to the solid phase is removed. Subsequently, a peptide is synthesized on a solid phase by sequentially repeating the same reaction using an amino acid with an amino group protected. Finally, the peptide can be synthesized by digesting the solid phase with trifluoroacetic acid to separate the peptide from the solid phase.
This peptide can also be produced by hydrolyzing gelatin with a combination of two or more of endo-type protease and exo-type protease. The hydrolyzed peptide mixture itself or a partially purified mixture thereof can also be used as an elastin production promoter.
本発明において、本ペプチドは化学修飾されていても良い。化学修飾はアミノ酸単位で行われうるが、例えば、ヒドロキシプロリンの水酸基、Ｎ末アミノ酸のアミノ基およびＣ末アミノ酸のカルボキシル基が挙げられる。このような化学修飾によって、弱酸性から中性で溶解可能にでき、後述する他の有効成分との相溶性向上なども可能となる。
具体的には、ヒドロキシプロリンの水酸基の化学修飾としては、例えばＯ−アセチル化等が挙げられる。Ｎ末アミノ酸のアミノ基の化学修飾としては、例えばポリペプチジル化、スクシニル化、マレイル化、アセチル化、脱アミノ化、ベンゾイル化、アルキルスルホニル化、アリルスルホニル化、ジニトロフェニル化、トリニトロフェニル化、カルバミル化、フェニルカルバミル化、チオール化等が挙げられる。Ｃ末アミノ酸のカルボキシル基の化学修飾としては、例えばエステル化、アミド化等が挙げられる。さらに、本ペプチドをカチオン化する場合は、エチレンジアミン化、スペルミン化などを行うことができる。
In the present invention, the peptide may be chemically modified. Chemical modification can be performed in amino acid units, and examples thereof include a hydroxyl group of hydroxyproline, an amino group of an N-terminal amino acid, and a carboxyl group of a C-terminal amino acid. By such chemical modification, it is possible to dissolve from weakly acidic to neutral, and to improve compatibility with other active ingredients described later.
Specifically, examples of the chemical modification of the hydroxyl group of hydroxyproline include O-acetylation. Examples of chemical modification of the amino group of the N-terminal amino acid include polypeptidylation, succinylation, maleylation, acetylation, deamination, benzoylation, alkylsulfonylation, allylsulfonylation, dinitrophenylation, trinitrophenylation, Examples thereof include carbamylation, phenylcarbamylation, and thiolation. Examples of the chemical modification of the carboxyl group of the C-terminal amino acid include esterification and amidation. Furthermore, when the present peptide is cationized, it can be subjected to ethylenediamine formation, spermination or the like.
化学修飾の具体的手段や処理条件は、通常のペプチドの化学修飾技術が適用される。例えば、ヒドロキシプロリンの水酸基のＯ−アセチル化は水溶媒中または非水溶媒中で無水酢酸を作用させることなどによって行うことができる。例えば、Ｃ末アミノ酸のカルボキシル基のエステル化はメタノールへの懸濁後に乾燥塩化水素ガスを通気することなどによって行うことができ、そのアミド化はカルボジイミドなどを作用させることによって行うことができる。さらに、化学修飾のその他の具体例として、特公昭６２−４４５２２号公報や特公平５−７９０４６号公報等に記載の化学修飾技術が適用できる。 As specific means and processing conditions for chemical modification, ordinary peptide chemical modification techniques are applied. For example, O-acetylation of the hydroxyl group of hydroxyproline can be performed by reacting acetic anhydride in an aqueous solvent or a non-aqueous solvent. For example, esterification of the carboxyl group of the C-terminal amino acid can be performed by passing dry hydrogen chloride gas after suspension in methanol, and amidation can be performed by acting carbodiimide or the like. Furthermore, as other specific examples of chemical modification, the chemical modification techniques described in Japanese Patent Publication No. Sho 62-44522 and Japanese Patent Publication No. 5-79046 can be applied.
２．エラスチン産生促進剤
本ペプチド等は、後述の評価試験に記載の通り、エラスチン発現促進作用を有する。従って、本ペプチド等は、エラスチンの産生を促進して、皮膚のシワおよびたるみを予防および改善することができる。また、美容のための化粧料として用いることもできる。
本発明のエラスチン産生促進剤は、経口的に又は非経口的に種々の形態で投与することができる。その形態としては、経口的に投与する場合は、例えば、錠剤、顆粒剤、カプセル剤、粉剤、液剤、懸濁製剤、乳化製剤等が挙げられ、または飲食品に混合することもできる。非経口的に投与する場合は、例えば、皮膚への塗布、注射剤、経皮剤、坐剤、点鼻剤及び吸入剤等が挙げられる。好ましくは、錠剤、顆粒剤、カプセル剤、皮膚への直接塗布する液剤、軟膏、クリーム剤、パップ剤等が挙げられる。なお、本ペプチドは、消化管でアミノ酸への分解もほとんど起こらず、腸管で迅速に吸収されるため、経口投与による摂取が好適である。本ペプチドは食事または飲料に混ぜて摂取させることも好ましい。
2. Elastin production promoter This peptide and the like have an elastin expression promoting action as described in the evaluation test described later. Therefore, this peptide etc. can promote the production of elastin and prevent and improve skin wrinkles and sagging. It can also be used as a cosmetic for cosmetic purposes.
The elastin production promoter of the present invention can be administered orally or parenterally in various forms. As the form, when administered orally, a tablet, a granule, a capsule, a powder, a liquid agent, a suspension formulation, an emulsion formulation etc. are mentioned, for example, It can also mix with food-drinks. In the case of parenteral administration, for example, application to skin, injection, transdermal agent, suppository, nasal drop, inhalant and the like can be mentioned. Preferable examples include tablets, granules, capsules, liquids directly applied to the skin, ointments, creams, poultices and the like. In addition, since this peptide hardly decomposes into amino acids in the gastrointestinal tract and is rapidly absorbed in the intestinal tract, it is preferably taken by oral administration. It is also preferable to ingest the peptide by mixing it with food or beverages.
本ペプチドの投与量は、対象の状態や体重、化合物の種類、投与経路等によって異なるが、成人１日当たり、経口投与の場合は、例えば、約０．１〜１０００ｍｇ、好ましくは約１〜５００ｍｇ、より好ましくは約１０〜２００ｍｇが挙げられ、皮膚に直接投与する場合は、例えば、約０．０００１〜９０重量％、好ましくは約０．００１〜５０重量％、より好ましくは約０．０１〜１０重量％が挙げられる。その他の形態の製剤は、これらの投与量を参考にして適宜決めることができる。これら製剤は、１日１〜数回に分けて投与するか、または１〜数日に１回投与することができる。
本発明のエラスチン産生促進剤は、本発明の効果を害しない範囲で、適宜他の有効成分や製剤用の成分を含有させても良い。他の有効成分として、例えばヒアルロン酸等が挙げられる。他の有効成分の配合量としては、各々の作用に応じて適宜、変更することができる。
The dose of this peptide varies depending on the condition and body weight of the subject, the type of compound, the route of administration, etc., but in the case of oral administration per day for adults, for example, about 0.1 to 1000 mg, preferably about 1 to 500 mg, More preferably, about 10 to 200 mg can be mentioned. When administered directly to the skin, for example, about 0.0001 to 90% by weight, preferably about 0.001 to 50% by weight, more preferably about 0.01 to 10%. % By weight. Formulations in other forms can be appropriately determined with reference to these dosages. These preparations can be administered divided into 1 to several times a day, or once to several days.
The elastin production promoter of the present invention may contain other active ingredients and ingredients for preparation as appropriate as long as the effects of the present invention are not impaired. Examples of other active ingredients include hyaluronic acid. The blending amount of other active ingredients can be appropriately changed according to each action.
医薬製剤に製剤化する際に用いる薬学上許容される担体としては、希釈剤、結合剤（シロップ、アラビアゴム、ゼラチン、ソルビット、トラガカント、ポリビニルピロリドン）、賦形剤（乳糖、ショ糖、コーンスターチ、リン酸カリウム、ソルビット、グリシン）、滑沢剤（ステアリン酸マグネシウム、タルク、ポリエチレングリコール、シリカ）、崩壊剤（バレイショデンプン）および湿潤剤（ラウリル硫酸ナトリウム）等を挙げることができる。本医薬製剤は、従来公知の方法に従って、本ペプチド、他の有効成分、薬学上許容される担体等を混合して製造することができる。 Pharmaceutically acceptable carriers used in formulating pharmaceutical preparations include diluents, binders (syrup, gum arabic, gelatin, sorbit, tragacanth, polyvinylpyrrolidone), excipients (lactose, sucrose, corn starch, Examples include potassium phosphate, sorbit, glycine), lubricants (magnesium stearate, talc, polyethylene glycol, silica), disintegrants (potato starch), wetting agents (sodium lauryl sulfate), and the like. The pharmaceutical preparation can be produced by mixing the peptide, other active ingredients, a pharmaceutically acceptable carrier and the like according to a conventionally known method.
３．健康食品
本ペプチド等は、天然コラーゲンに由来するペプチドであるため、日常的に摂取または塗布しても極めて安全である。そこで、本ペプチド等の優れたエラスチン産生促進作用を利用した、本ペプチド等を含有する飲食品、すなわち健康食品等としても有用である。本発明の健康食品における本ペプチド等は、利用する効果に応じて適宜、含有量を変更して用いることができる。
3. Health foods The present peptides and the like are peptides derived from natural collagen, so they are extremely safe even if they are taken or applied on a daily basis. Therefore, it is also useful as a food or drink containing the peptide or the like, that is, a health food or the like using the excellent elastin production promoting action of the peptide or the like. The peptide or the like in the health food of the present invention can be used by changing the content as appropriate according to the effect to be used.
以下、本発明を実施例、比較例、評価試験によりさらに詳細に説明するが、本発明はこれらに何ら限定されるものではない。
実施例１〜１１
前記のペプチド固相合成法を用いて、以下のペプチドを合成した。
（実施例１）Ｇｌｕ−Ｈｙｐ−Ｇｌｙ（ＥＯＧ）
（実施例２）Ｓｅｒ−Ｈｙｐ−Ｇｌｙ（ＳＯＧ）
（実施例３）Ａｌａ−Ｈｙｐ−Ｇｌｙ（ＡＯＧ）
（実施例４）Ｇｌｕ−Ｈｙｐ（ＥＯ）
（実施例５）Ｌｅｕ−Ｈｙｐ−Ｇｌｙ（ＬＯＧ）
（実施例６）Ａｌａ−Ｈｙｐ（ＡＯ）
（実施例７）Ｐｒｏ−Ｈｙｐ−Ｇｌｙ（ＰＯＧ）
（実施例８）Ｌｅｕ−Ｈｙｐ（ＬＯ）
（実施例９）Ｐｒｏ−Ｈｙｐ（ＰＯ）
（実施例１０）Ｐｒｏ−Ａｌａ（ＰＡ）
（実施例１１）Ｈｙｐ−Ｇｌｙ（ＯＧ）
EXAMPLES Hereinafter, although an Example, a comparative example, and an evaluation test demonstrate this invention further in detail, this invention is not limited to these at all.
Examples 1-11
The following peptides were synthesized using the peptide solid phase synthesis method described above.
(Example 1) Glu-Hyp-Gly (EOG)
(Example 2) Ser-Hyp-Gly (SOG)
(Example 3) Ala-Hyp-Gly (AOG)
(Example 4) Glu-Hyp (EO)
(Example 5) Leu-Hyp-Gly (LOG)
(Example 6) Ala-Hyp (AO)
(Example 7) Pro-Hyp-Gly (POG)
(Example 8) Leu-Hyp (LO)
(Example 9) Pro-Hyp (PO)
(Example 10) Pro-Ala (PA)
(Example 11) Hyp-Gly (OG)
実施例１２
コラーゲンペプチドＨＤＬ−１２ＳＰ（新田ゼラチン製）。
ＬＣ−ＭＳ／ＭＳで分析したところ、本コラーゲンペプチドには以下のペプチドがそれぞれ含まれていた。
ＥＯＧ：３０８．１ｐｐｍ，ＳＯＧ：３５１．２ｐｐｍ，ＡＯＧ：３３０．７ｐｐｍ，ＥＯ：５７．９ｐｐｍ，ＬＯＧ：２２３．４ｐｐｍ，ＧＰＯ：７５．５ｐｐｍ，ＰＯ：１４３．６ｐｐｍ，ＯＧ：５８１０．４ｐｐｍ，ＰＡ：１４９８．６ｐｐｍ，ＡＯ：６２．９ｐｐｍ。
Example 12
Collagen peptide HDL-12SP (made by Nitta Gelatin).
When analyzed by LC-MS / MS, the collagen peptides contained the following peptides, respectively.
EOG: 308.1 ppm, SOG: 351.2 ppm, AOG: 330.7 ppm, EO: 57.9 ppm, LOG: 223.4 ppm, GPO: 75.5 ppm, PO: 143.6 ppm, OG: 58.10.4 ppm, PA: 1498.6 ppm, AO: 62.9 ppm.
比較例１
前記のペプチド固相合成法を用いて、Ｇｌｙ−Ｐｒｏ−Ｈｙｐ（ＧＰＯ）を合成した。
比較例２
デキストリン（ＴＫ−１６、松谷化学社製）。
Comparative Example 1
Gly-Pro-Hyp (GPO) was synthesized using the peptide solid phase synthesis method.
Comparative Example 2
Dextrin (TK-16, manufactured by Matsutani Chemical Co.).
比較例３
コラーゲンペプチドＨＤＬ−５０ＳＰ（新田ゼラチン製）。
ＬＣ−ＭＳ／ＭＳで分析したところ、本コラーゲンペプチドには以下のペプチドがそれぞれ含まれていた。
ＡＯＧ：０．１ｐｐｍ，ＥＯ：０．１ｐｐｍ，ＬＯＧ：３．８ｐｐｍ，ＧＰＯ：０．１ｐｐｍ，ＰＯ：８．３ｐｐｍ，ＯＧ：１１．３ｐｐｍ，ＰＡ：３６．７ｐｐｍ，ＡＯ：６．０ｐｐｍ。
Comparative Example 3
Collagen peptide HDL-50SP (made by Nitta Gelatin).
When analyzed by LC-MS / MS, the collagen peptides contained the following peptides, respectively.
AOG: 0.1 ppm, EO: 0.1 ppm, LOG: 3.8 ppm, GPO: 0.1 ppm, PO: 8.3 ppm, OG: 11.3 ppm, PA: 36.7 ppm, AO: 6.0 ppm.
試験例１
エラスチン発現促進試験
ヒト正常皮膚線維芽細胞ＮＨＤＦ（ＮＢ）を用いた。１０％ＦＢＳ含有ＤＭＥＭ／Ｆ１２で前培養し、４×１０４細胞／ｍｌ×１０ｍｌ（４×１０５細胞／皿）で３日培養した。細胞がサブコンフルエントになっていることを確認後、ＰＢＳ１０ｍｌに置き換えた。ＵＶ照射区はクロストロリンカーでＵＶＢランプを用い、照射量が２．５ｍＪ／ｃｍ２（３５００ｕＷ／ｃｍ２×７秒）になるように蓋を開け、照射した。その後、ＵＶ照射区およびＵＶ非照射区を共に、試験培地９．５ｍｌ（ＤＭＥＭ／Ｆ１２培地）に置き換えた。終濃度０．００５、０．０５、０．５または５ｍＭとなるように試験培地に可溶化したサンプルを各々０．５ｍｌ添加し、６時間反応させた。コントロールには試験培地のみを１０ｍｌ添加した。細胞より全ＲＮＡを抽出し、逆転写を行いリアルタイムＰＣＲにかけた。リアルタイムＰＣＲでは標的遺伝子としてエラスチン（Hs00355783_m1）を測定した。補正遺伝子はＧＡＰＤＨで行った。計算は検量線法を用い、プライマー＆プローブはTaqMan Gene ExpressionのFAM色素を用いた。また、ＵＶ非照射区およびＵＶ照射区のそれぞれのコントロール値を１．０とした。ＵＶ非照射区コントロールを１．０とした場合、ＵＶ照射区コントロール値は０．３２であり、ＵＶ照射することによってエラスチン産生は非常に抑制される。
Test example 1
Elastin expression promotion test Human normal skin fibroblast NHDF (NB) was used. The cells were precultured in DMEM / F12 containing 10% FBS, and cultured at 4 × 10 4 cells / ml × 10 ml (4 × 10 5 cells / dish) for 3 days. After confirming that the cells were subconfluent, the cells were replaced with 10 ml of PBS. In the UV irradiation section, a UVB lamp was used as a cross trolinker, and the lid was opened so that the irradiation amount was 2.5 mJ / cm 2 (3500 uW / cm 2 × 7 seconds). Thereafter, both the UV irradiation group and the UV non-irradiation group were replaced with 9.5 ml of the test medium (DMEM / F12 medium). 0.5 ml of each sample solubilized in the test medium to a final concentration of 0.005, 0.05, 0.5 or 5 mM was added and reacted for 6 hours. For control, 10 ml of test medium alone was added. Total RNA was extracted from the cells, reverse transcribed and subjected to real-time PCR. In real-time PCR, elastin (Hs00355783_m1) was measured as a target gene. The correction gene was GAPDH. The standard curve method was used for the calculation, and TaqMan Gene Expression FAM dye was used as the primer and probe. Moreover, each control value of UV non-irradiation section and UV irradiation section was set to 1.0. When the UV non-irradiation control is 1.0, the UV irradiation control value is 0.32, and elastin production is greatly suppressed by UV irradiation.
実施例１〜１１および比較例１のペプチドについて、エラスチン発現促進作用をＵＶ照射区とＵＶ非照射区に分けて測定した。表１にＵＶ非照射区について、表２にＵＶ照射区についての結果を示す。＊、＊＊および＊＊＊は、Paired-t-testにおいて、コントロールに対してそれぞれP<0.05、P<0.01、P<0.001で有意であることを示す。
また、試験培地９．５ｍｌに対し、各々の終濃度の合計が０．１ｍＭとなるように実施例１のＥＯＧ、実施例２のＳＯＧまたは実施例３のＡＯＧと他のペプチドとを１：１で混合したものを０．５ｍｌ添加した。コントロールには試験培地のみを１０ｍｌ添加した。ＵＶ非照射区とＵＶ照射区についてのエラスチン発現促進作用を試験を行った。＊、＊＊および＊＊＊は、Paired-t-testにおいて、コントロールに対してそれぞれP<0.05、P<0.01、P<0.001で有意であることを示す。
表１および３から分かる通り、本発明のペプチドはＵＶ非照射区においてエラスチン発現促進作用を有している。また、表２および３では、ＵＶ照射という過酷な条件にも関わらず、コントロールより有意にエラスチン発現を促進しており、ＵＶ照射時にも非常に有効である。なかでも実施例１〜８のペプチドが高い作用を有しており、特に実施例１〜５のペプチドが好ましく、実施例１〜３のペプチドが最も好ましい。
さらに、実施例１〜３のペプチドと、その他の実施例１〜１１のペプチドとを組み合わせることで、エラスタチン発現促進作用が相乗的に向上する。
As can be seen from Tables 1 and 3, the peptide of the present invention has an elastin expression promoting effect in the UV non-irradiated section. In Tables 2 and 3, despite the harsh conditions of UV irradiation, the expression of elastin is significantly promoted compared to the control, and it is very effective during UV irradiation. Especially, the peptide of Examples 1-8 has a high effect | action, The peptide of Examples 1-5 is especially preferable, and the peptide of Examples 1-3 is the most preferable.
Furthermore, the elastatin expression promoting action is synergistically improved by combining the peptides of Examples 1 to 3 and the other peptides of Examples 1 to 11.
試験例２
臨床試験
試験食品として実施例１２、比較例２および３を用いて、臨床試験を行った。被験者は、乾燥肌、肌荒れに自覚を持つ健康な女性８５人に対して、実施例１２摂取群、比較例２摂取群、比較例３摂取群に無作為に割付し、実施例１２群２８人、比較例２群２８人、比較例３群２９人で二重盲検試験を行った。患者は３３歳〜５５歳であった。各試験食品を５ｇ摂取を８週間連続摂取した。評価方法は、専門医による診断により頬のたるみを評価した。また、Skin Surface Analyzer（商標）により目じりシワ数を、Cutometer（商標）により目じりの肌弾力（Ｒ２）を測定し、評価した。評価は０週目、４週目、８週目で行った。
Test example 2
A clinical test was conducted using Example 12, Comparative Examples 2 and 3 as clinical test test foods. The subjects were randomly assigned to the intake group of Example 12, the intake group of Comparative Example 2, and the intake group of Comparative Example 3 to 85 healthy women who were aware of dry skin and rough skin, and 28 people in the Example 12 group. A double-blind test was conducted with 28 people in Comparative Example 2 group and 29 people in Comparative Example 3 group. Patients were between 33 and 55 years old. Each test food was ingested 5 g for 8 weeks. The evaluation method evaluated cheek sagging by diagnosis by a specialist. Further, the number of eyes wrinkles was measured with Skin Surface Analyzer ™, and the skin elasticity (R2) of eyes was measured with Cutometer ™ and evaluated. Evaluation was performed at 0th week, 4th week, and 8th week.
頬たるみの試験結果を以下に示す。＊はTwo-way-ANOVAにおいて、比較例２に対してP<0.05で有意であることを示す。
目尻しわ数の試験結果を以下に示す。＊はTwo-way-ANOVAにおいて、比較例２に対してP<0.05で有意であることを示す。
弾力性（目じり）の試験結果を以下に示す。＊はTwo-way-ANOVAにおいて、比較例２に対してP<0.05で有意であることを示す。
以上の通り、特殊な酵素で加水分解させたＥＯＧ、ＳＯＧ、ＡＯＧ等が多いペプチド混合物（実施例１２）は、デキストリン（比較例２）および従来から知られている通常の酵素で加水分解したペプチド混合物（比較例３）と比べて、頬たるみ、目尻しわおよび弾力性（目じり）において、有意な有効性を示した。 As described above, the peptide mixture (Example 12) rich in EOG, SOG, AOG and the like hydrolyzed with a special enzyme is a peptide hydrolyzed with dextrin (Comparative Example 2) and a conventionally known ordinary enzyme. Compared with the mixture (Comparative Example 3), it showed significant effectiveness in cheek sagging, wrinkles on the outer corners of the eyes, and elasticity (eye contact).
本発明によって、ペプチド分子を含有する、従来技術よりも優れたエラスチン産生促進剤を提供することができる。 According to the present invention, it is possible to provide an elastin production promoter containing peptide molecules, which is superior to the prior art.
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