JP6013115B2 - A skin care agent comprising an antimicrobial active polypeptide having collagen gel contracting activity, human skin fibroblast proliferation activity and hyaluronic acid production activity. - Google Patents
A skin care agent comprising an antimicrobial active polypeptide having collagen gel contracting activity, human skin fibroblast proliferation activity and hyaluronic acid production activity. Download PDFInfo
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- JP6013115B2 JP6013115B2 JP2012215415A JP2012215415A JP6013115B2 JP 6013115 B2 JP6013115 B2 JP 6013115B2 JP 2012215415 A JP2012215415 A JP 2012215415A JP 2012215415 A JP2012215415 A JP 2012215415A JP 6013115 B2 JP6013115 B2 JP 6013115B2
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
本発明は、優れたコラーゲンゲル収縮活性、ヒト皮膚線維芽細胞増殖活性およびヒアルロン酸産生活性を有する抗菌活性ポリペプチドを含有するスキンケア剤に関する。
The present invention relates to a skin care agent containing an antimicrobial active polypeptide having excellent collagen gel contractile activity, human skin fibroblast proliferation activity and hyaluronic acid producing activity.
皮膚は表皮、真皮、皮下組織の３層に主に分けられる。真皮は皮膚構造の維持に極めて重要であり、コラーゲンなどの繊維により強固かつ柔軟性のある真皮結合組織を形成している。コラーゲンなどの結合組織の線維を構成するタンパク質は、線維芽細胞により合成および分解されている。つまり結合組織の状態はコラーゲン等の線維と相互作用することで線維芽細胞によりコントロールされている。
The skin is mainly divided into three layers: epidermis, dermis, and subcutaneous tissue. The dermis is extremely important for maintaining the skin structure, and forms a strong and flexible dermis connective tissue with fibers such as collagen. Proteins that constitute connective tissue fibers such as collagen are synthesized and degraded by fibroblasts. That is, the state of connective tissue is controlled by fibroblasts by interacting with fibers such as collagen.
通常、線維芽細胞培養条件下で線維芽細胞をコラーゲンゲル中に埋包して培養するとコラーゲンゲルは収縮する。すなわちコラーゲンゲルの強度が増している。このコラーゲンゲルの収縮は、細胞数や培地中の血清量によっても異なり、ゲル中の線維芽細胞数が多いほど、培地中の血清量が多いほど顕著になる。しかし、線維芽細胞の存在しない培養下や浮遊性細胞を用いた培養下ではコラーゲンゲルの収縮は全く起こらず、強度も変化しない。このコラーゲンゲルの埋包培養は一般に真皮結合組織のモデルとされている（非特許文献１）。
Normally, when fibroblasts are embedded in a collagen gel and cultured under fibroblast culture conditions, the collagen gel contracts. That is, the strength of the collagen gel is increased. The contraction of the collagen gel varies depending on the number of cells and the amount of serum in the medium, and becomes more noticeable as the number of fibroblasts in the gel increases and the amount of serum in the medium increases. However, the collagen gel does not contract at all in culture without fibroblasts or culture using suspension cells, and the strength does not change. This embedding culture of collagen gel is generally regarded as a model of dermal connective tissue (Non-patent Document 1).
皮膚の老化に関しては、コラーゲンゲルの埋包培養実験において、老齢者由来の線維芽細胞埋包培養と若年者由来線維芽細胞埋包培養とを比較すると、前者の細胞においては後者の細胞よりコラーゲンゲルの収縮が低下していることが報告されており、老化によりゲルの収縮能が低下することが明らかとなっている（非特許文献２）。この状態は、コラーゲンゲルの収縮能が低下することで真皮結合組織を若いころのように収縮させて、引き締まった状態に維持することが困難になっていることを示している。実際に、老化に伴い頬や首筋、腕等の皮膚に若い頃にはあまり認められなかったタルミが生じることは周知の事実である。このタルミは皮膚の真皮結合組織が収縮力を失い、さらには強度や弾力性を失うことで生じると考えられる。従ってコラーゲンゲルの収縮能を高めることで、皮膚のしわやタルミを改善する効果を発揮すると報告されている（特許文献１および２）。
Regarding skin aging, in the collagen gel embedding culture experiment, comparing the fibroblast-embedded culture derived from the elderly with the fibroblast-embedded culture derived from the young, the former cells were more collagenous than the latter cells. It has been reported that the shrinkage of the gel is reduced, and it has been clarified that the shrinkage ability of the gel is lowered by aging (Non-patent Document 2). This state shows that it is difficult to keep the dermis connective tissue contracted like a young age and maintain a tightened state due to a decrease in the contractility of the collagen gel. In fact, it is a well-known fact that with the aging, tarmi, which was not recognized much when it was young, occurs in the skin such as the cheeks, neck and arms. This talmi is thought to occur when the dermal connective tissue of the skin loses its contractile force and further loses its strength and elasticity. Therefore, it has been reported that the effect of improving wrinkles and talmi of the skin is exhibited by increasing the contractility of the collagen gel (
コラーゲンゲル収縮能だけでなく、線維芽細胞増殖活性およびコラーゲン産生活性を有するものがいくつか報告がなされており、ポリペプチドとしてはＥＧＦやｂＦＧＦが知られている。またこれらと同等の活性を持つものとしてラクトフェリン（乳清）も知られており（特許文献３）、これらを有効成分としたスキンケア効果を持つ組成物が提供されている。しかし格段にスキンケア効果の優れた有効成分は見出されておらず、新しい有効成分の発見が求められている。
Some reports have not only collagen gel contractility but also fibroblast proliferation activity and collagen production activity, and EGF and bFGF are known as polypeptides. In addition, lactoferrin (whey) is also known as one having the same activity (Patent Document 3), and a composition having a skin care effect using these as active ingredients is provided. However, no active ingredient with much superior skin care effect has been found, and the discovery of a new active ingredient is required.
本発明の目的は、優れたコラーゲンゲル収縮活性、ヒト皮膚線維芽細胞増殖活性およびヒアルロン酸産生活性を有する抗菌活性ポリペプチドのスキンケア剤を提供することである。
An object of the present invention is to provide an antibacterial active polypeptide skin care agent having excellent collagen gel contraction activity, human skin fibroblast proliferation activity and hyaluronic acid production activity.
抗菌活性を持つペプチドに着目して、コラーゲン収縮活性があることを見出し、さらにヒト皮膚線維芽細胞増殖活性およびヒアルロン酸産生がペプチドにより増強されることを見出して、本発明に至った。
Focusing on peptides with antibacterial activity, they found that they have collagen contracting activity, and found that human skin fibroblast proliferation activity and hyaluronic acid production are enhanced by the peptides, leading to the present invention.
すなわち、本発明は、
（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有効成分とする、コラーゲンゲル収縮促進剤。
（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有効成分とする、線維芽細胞増殖促進剤。
（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有効成分とする、ヒアルロン酸産生促進剤。
（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有効成分とする、シワ改善・抑制剤。
（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有効成分とする、皮膚のたるみ・はり改善剤。
（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有効成分とする、皮膚引き締め剤。
に関する。
That is, the present invention
(1) A collagen gel contraction promoter comprising, as an active ingredient, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a polypeptide having 95% or more homology with the amino acid sequence.
(2) A fibroblast proliferation promoter comprising as an active ingredient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a polypeptide having a homology of 95% or more with respect to the amino acid sequence.
(3) A hyaluronic acid production promoter comprising as an active ingredient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a polypeptide having a homology of 95% or more with the amino acid sequence.
( 4 ) A wrinkle improving / suppressing agent comprising as an active ingredient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having a homology of 95% or more with the amino acid sequence.
( 5 ) A skin sagging / swelling improver comprising, as an active ingredient, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a polypeptide having 95% or more homology with the amino acid sequence.
( 6 ) A skin tightening agent comprising as an active ingredient a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or a polypeptide having a homology of 95% or more with respect to the amino acid sequence.
About.
本発明により、優れたヒト皮膚線維芽細胞増殖活性、ヒアルロン酸産生活性および収縮活性を有するポリペプチドが提供された。さらにこのポリペプチドを有効成分とする用途が提供された。
According to the present invention, a polypeptide having excellent human skin fibroblast proliferation activity, hyaluronic acid producing activity and contractile activity is provided. Furthermore, the use which uses this polypeptide as an active ingredient was provided.
本明細書において、「ポリペプチド」と「ペプチド」の用語は同義に用いられる。
In the present specification, the terms “polypeptide” and “peptide” are used synonymously.
本発明のスキンケア剤の有効成分であるポリペプチドは、市販のペプチド合成機を用いた化学合成等の定法により容易に製造することができる。
The polypeptide which is an active ingredient of the skin care agent of the present invention can be easily produced by a conventional method such as chemical synthesis using a commercially available peptide synthesizer.
なお、本発明のスキンケア剤の有効成分であるポリペプチドは、いずれも新規機能をもつ既知の抗菌活性物質である。
The polypeptides that are active ingredients of the skin care agent of the present invention are all known antibacterial active substances having a novel function.
本発明のポリペプチドは、優れた線維芽細胞増殖活性、ヒアルロン酸産生活性およびコラーゲンゲル収縮活性を有するので、皮膚細胞に対する細胞増殖活性および収縮活性を促すスキンケア剤として用いることができる。
Since the polypeptide of the present invention has excellent fibroblast proliferation activity, hyaluronic acid production activity and collagen gel contraction activity, it can be used as a skin care agent that promotes cell proliferation activity and contraction activity on skin cells.
スキンケア剤としての使用方法は公知のポリペプチド系のスキンケア剤と同様であり、ローション、クリーム、パック、ゲル、乳剤、バルサム、軟膏、液剤、粉剤、またはその他の局所的もしくは内部的に適用可能な形態で適用される。
The method of use as a skin care agent is the same as that for known polypeptide-based skin care agents, and can be applied to lotions, creams, packs, gels, emulsions, balsams, ointments, solutions, powders, or other topically or internally. Applied in form.
以下、本発明を実施例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。
Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
１．抗菌活性の測定
「最小発育阻止濃度（minimum
inhibitory concentration : MIC）を以下の通り測定した。MICは菌の発育が阻止される最小の薬剤濃度であり、抗菌剤の効力、菌の感受性の強さなどの指標として用いられ、その濃度以上であれば生育できないという濃度である。この測定方法は日本化学療法学会またはCLINICAL AND LABORATORY STANDARD INSTITUTE(CLSI)が標準法として定める方法に従って測定されるが、ここではCLSIが2007年1月に発行した「M100-S17/M7-A7」(Performance Standards for Antimicrobial
Susceptibility Testing : Seventeenth Informational Supplemen、Vol. 27 No.1)に準拠し、微量液体希釈法で測定した。すなわちマイクロタイタープレートあるいは試験管を用いて、薬剤感受性試験を行った。
（方法）
菌株にはアクネ菌（Propionibacterium acnes）、大腸菌（Escherichia
coli）、黄色ブドウ球菌（Staphylococcus
aureus）、緑膿菌（Pseudomonas
aeruginosa）を用いた。
各菌株を液体培地で4〜6時間培養した後、A600における吸光度を測定した。McFarland
#0.5にしたがって、ミューラー・ヒントン・ブロス(Mueller-Hinton
broth : MHB)で希釈した。各菌株を1.0×105 CFU/mL（最終濃度）程度になるように添加した。配列番号１に記載のポリペプチドを任意の濃度に調整し、さらにそこから段階希釈を行った。各濃度段階のペプチドを分注したマイクロタイタープレートあるいは試験管に各菌株を添加した。このプレートあるいは試験管を37℃で20時間培養し、各菌株の生育が阻止された最小の濃度を最小生育阻止濃度(MIC)とした。
1. Measurement of antibacterial activity "minimum growth inhibitory concentration
inhibitory concentration (MIC) was measured as follows. MIC is the minimum drug concentration at which fungal growth is prevented, and is used as an indicator of the efficacy of antibacterial agents and the strength of bacterial sensitivity. This measurement method is measured according to the method defined by the Japanese Society of Chemotherapy or CLINICAL AND LABORATORY STANDARD INSTITUTE (CLSI) as a standard method. Performance Standards for Antimicrobial
Susceptibility Testing: Based on Seventeenth Informational Supplemen, Vol. 27 No. 1), measured by a micro liquid dilution method. That is, a drug sensitivity test was performed using a microtiter plate or a test tube.
(Method)
The strains include Propionibacterium acnes, Escherichia
coli), Staphylococcus
aureus), Pseudomonas
aeruginosa).
After each strain was cultured for 4-6 hours in a liquid medium, the absorbance was measured at A 600. McFarland
According to # 0.5, Mueller-Hinton Bros
broth: MHB). Each strain was added to a concentration of about 1.0 × 10 5 CFU / mL (final concentration). The polypeptide described in SEQ ID NO: 1 was adjusted to an arbitrary concentration, and further serial dilution was performed therefrom. Each strain was added to a microtiter plate or test tube into which peptides at each concentration step were dispensed. This plate or test tube was cultured at 37 ° C. for 20 hours, and the minimum concentration at which growth of each strain was inhibited was defined as the minimum growth inhibition concentration (MIC).
結果を表１に示す。 The results are shown in Table 1.
（結果）
表１に示されるように、本発明のポリペプチドは、アクネ菌への抗菌活性を持ち、優れた抗菌活性を示した。
(result)
As shown in Table 1, the polypeptide of the present invention has antibacterial activity against acne bacteria and showed excellent antibacterial activity.
２．コラーゲンゲル収縮アッセイ（Floating
Matrix Contraction Assay(FMC)
（方法）
室温にて24ウェルプレートを2%BSA/PBSでコーティングし、37℃、5% CO2インキュベーター内で静置しておく。使用前にこのプレートをPBSで洗い、乾燥を防ぐためにPBSを添加しておく。使用時にPBSを吸引除去し、2.0×106 cells/mLとなるように調整した正常ヒト新生児包皮皮膚線維芽細胞（NHDF(NB)）とコラーゲン溶液との混合液500 μLを24ウェルプレートの各ウェルに播種した。37℃、5% CO2インキュベーター内で1時間培養後、培地で各濃度に調整した配列番号１に記載のポリペプチドを各ウェルに500 μLずつ添加した。無刺激には培地のみを各ウェルに500 μLずつ添加した。プレートを静かに揺らし、ゲルを浮遊させた後に37℃、5% CO2インキュベーター内に静置した。３日間培養後、コラーゲンゲルを撮影し、画像解析ソフトを用いて画像からコラーゲンゲルの面積を測定した。刺激開始時のゲル面積を191.0mm2（ウェル底の直径：15.6 mm2）とした。収縮率（％）は以下の計算式により算出した。
（計算式）
収縮率（%）＝（（無刺激時のゲル面積−ポリペプチド刺激時のゲル面積）／無刺激時のゲル面積）×100
2. Collagen gel contraction assay (Floating
Matrix Contraction Assay (FMC)
(Method)
A 24-well plate is coated with 2% BSA / PBS at room temperature and allowed to stand in a 37 ° C., 5% CO 2 incubator. Wash the plate with PBS before use and add PBS to prevent drying. PBS was removed by suction at the time of use, and 500 μL of a mixture of normal human neonatal foreskin skin fibroblasts (NHDF (NB)) and collagen solution adjusted to 2.0 × 10 6 cells / mL was added to each 24-well plate. Wells were seeded. After culturing at 37 ° C. in a 5% CO 2 incubator for 1 hour, 500 μL of the polypeptide of SEQ ID NO: 1 adjusted to each concentration with a medium was added to each well. For no stimulation, 500 μL of medium alone was added to each well. The plate was gently shaken to float the gel, and then placed in a 37 ° C., 5% CO 2 incubator. After culturing for 3 days, the collagen gel was photographed, and the area of the collagen gel was measured from the image using image analysis software. The gel area at the start of stimulation 191.0mm 2 (well bottom diameter: 15.6 mm 2) was. The shrinkage rate (%) was calculated by the following formula.
(a formula)
Shrinkage rate (%) = ((gel area at no stimulation−gel area at polypeptide stimulation) / gel area at no stimulation) × 100
結果を表２に示す。 The results are shown in Table 2.
（結果）
表２に示されるように、本発明のポリペプチドは、優れた収縮活性を示した。
(result)
As shown in Table 2, the polypeptide of the present invention showed excellent contractile activity.
３．線維芽細胞増殖活性の測定
（方法）
0.5×104
cells/well/100 μLとなるように調整した正常ヒト新生児包皮皮膚線維芽細胞（NHDF(NB)）を、1% 血清含有培地で37℃、5% CO2インキュベーター内で約3時間培養後、同じ培地で各濃度に調整した配列番号１に記載のポリペプチドを各ウェルに100 μLずつ添加した。無刺激群には培地のみを各ウェルに100 μLずつ添加した。さらに約48時間培養後、Cell Counting Kit(WST-1)（商品名、同仁化学）により蛍光測定装置マルチラベルカウンター Wallac 1420 ARVOsx（商品名、Perkin
Elmer）を用いて細胞増殖を測定した。波長450 nm，620 nmの吸光度により測定値のO.D.450 −O.D.620を求め、この値から細胞を含まない空ウェルのO.D.450 − O.D.620の平均値を引いたものをNet O.D.450 とした。細胞増殖活性は無刺激のNet O.D.450に対するペプチド添加時の割合で評価した。
3. Measurement of fibroblast proliferation activity (method)
0.5 × 10 4
Normal human neonatal foreskin skin fibroblasts (NHDF (NB)) adjusted to cells / well / 100 μL were cultured in a medium containing 1% serum at 37 ° C in a 5% CO 2 incubator for about 3 hours. 100 μL of the polypeptide described in SEQ ID NO: 1 adjusted to each concentration in the same medium was added to each well. In the unstimulated group, 100 μL of medium alone was added to each well. After further incubation for about 48 hours, Cell Counting Kit (WST-1) (trade name, Dojindo Chemical Co., Ltd.), fluorescence measuring device multi-label counter Wallac 1420 ARVOsx (trade name, Perkin
Elmer) was used to measure cell proliferation. Seeking OD 450 -OD 620 measured values by absorbance at a wavelength of 450 nm, 620 nm, OD 450 of empty wells without cells from this value - the minus the mean value of the OD 620 was Net OD 450. Cell proliferation activity was evaluated by the ratio at the time of peptide addition to unstimulated Net OD 450 .
結果を表３および図１に示す。 The results are shown in Table 3 and FIG.
（結果）
表３および図２に示されるように、本発明のポリペプチドは、優れた線維芽細胞増殖活性を示した。さらにポリペプチドの濃度依存的にその増殖活性は強まっていた。
(result)
As shown in Table 3 and FIG. 2, the polypeptide of the present invention showed excellent fibroblast proliferation activity. Furthermore, the proliferation activity increased in a polypeptide concentration-dependent manner.
４．ヒアルロン酸産生量の測定と同時に行った線維芽細胞増殖活性の測定
（方法）
2×104 cells/well/500 μLとなるように調整した正常ヒト新生児包皮皮膚線維芽細胞（NHDF(NB)）を1% FBS含有で増殖添加剤の含まれていないMedium106培地で37℃、5%CO2インキュベータ内で一晩培養した。同じ培地で各濃度に調整した配列番号１に記載のポリペプチドを500μLずつウェルに添加し、さらに37℃、5% CO2インキュベータ内で培養した。5日間培養後、培養上清を1.5mLチューブに回収し、15000rpm、4℃、5分間遠心後、上清を新しいチューブに回収した。この培養上清中のヒアルロン酸量をQnE Hyaluronic Acid (HA) ELISA Assay（商品名、Biotech Trading Partners, LLC）を用いて測定した。
また同時に線維芽細胞増殖率をCell
Counting Kit(WST-1)（商品名、同仁化学）を用いて測定した。培養上清を回収したウェルに、培地とWST-1を10：1で混合した液を500μLずつ加え、約2時間37℃、5% CO2インキュベータ内で静置した後、波長450 nm、620 nmの吸光度を測定した。各測定値のO.D.450
- O.D.620を求め、測定ウェルのO.D.450 - O.D.620の値から細胞を含まない空ウェルのO.D.450 - O.D.620の平均値を引いたものをNet O.D.450 とした。細胞増殖率は配列番号１に記載のポリペプチド無添加のNet O.D.450に対する配列番号１に記載のポリペプチド添加時の割合(%)で評価した。
4). Measurement of fibroblast proliferation activity performed simultaneously with measurement of hyaluronic acid production (method)
Normal human neonatal foreskin dermal fibroblasts (NHDF (NB)) adjusted to 2 × 10 4 cells / well / 500 μL at 37 ° C. in Medium 106 medium containing 1% FBS and no growth additives, The cells were cultured overnight in a 5% CO 2 incubator. 500 μL of the polypeptide described in SEQ ID NO: 1 adjusted to each concentration in the same medium was added to each well and further cultured in a 37 ° C., 5% CO 2 incubator. After culturing for 5 days, the culture supernatant was collected in a 1.5 mL tube, centrifuged at 15000 rpm at 4 ° C. for 5 minutes, and the supernatant was collected in a new tube. The amount of hyaluronic acid in the culture supernatant was measured using QnE Hyaluronic Acid (HA) ELISA Assay (trade name, Biotech Trading Partners, LLC).
At the same time, the fibroblast proliferation rate
Measurement was performed using Counting Kit (WST-1) (trade name, Dojin Chemical). To the well from which the culture supernatant was collected, 500 μL of a 10: 1 mixed medium and WST-1 was added and allowed to stand in a 5% CO 2 incubator for 2 hours at 37 ° C. The absorbance at nm was measured. OD 450 for each measurement
-OD 620 was determined, and Net OD 450 was obtained by subtracting the average value of OD 450 -OD 620 of empty wells without cells from the value of OD 450 -OD 620 of the measurement well. The cell growth rate was evaluated by the ratio (%) at the time of addition of the polypeptide described in SEQ ID NO: 1 to Net OD 450 without addition of the polypeptide described in SEQ ID NO: 1.
結果を表４および図２に示す。 The results are shown in Table 4 and FIG.
（結果）
表４および図２に示されるように、本発明のポリペプチドは、優れたヒアルロン酸産生を示した。さらにポリペプチドの濃度依存的にそのヒアルロン酸産生量は増加していた。
(result)
As shown in Table 4 and FIG. 2, the polypeptide of the present invention showed excellent hyaluronic acid production. Furthermore, the amount of hyaluronic acid produced increased depending on the concentration of the polypeptide.
Claims (6)
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