JP6317993B2 - Screening method for skin improvers - Google Patents
Screening method for skin improvers Download PDFInfo
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- JP6317993B2 JP6317993B2 JP2014096389A JP2014096389A JP6317993B2 JP 6317993 B2 JP6317993 B2 JP 6317993B2 JP 2014096389 A JP2014096389 A JP 2014096389A JP 2014096389 A JP2014096389 A JP 2014096389A JP 6317993 B2 JP6317993 B2 JP 6317993B2
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- extract
- skin
- improving agent
- nk1r
- test substance
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Plant Substances (AREA)
Description
本発明は、肌改善剤のスクリーニング方法、並びに該スクリーニングにより肌改善剤として選択された物質を含有する組成物及びその製造方法に関する。 The present invention relates to a method for screening a skin improving agent, a composition containing a substance selected as a skin improving agent by the screening, and a method for producing the same.
年を経るごとに肌の弾力性や張りは徐々に失われていき、また、色素沈着を起こし、くすみの増加による色調変化も生じる。肌の老化による肌状態の悪化は、人に与える印象を大きく変えるため、肌の改善を期待し、様々な皮膚外用剤、とりわけ、化粧料への関心は高く、様々な研究が盛んに行われている。
例えば、皮膚の老化を防止し、張りや艶を維持するために、女性ホルモンの一種であるエストロゲンに着目し、エストロゲン様作用剤を含む美容用食品や皮膚化粧料が提案されている(特許文献1)。
また、ストレスが肌の老化を促進することが経験的に知られている。すなわち、生理現象のみならず、物理的ストレスや心理的ストレスにより、つや、透明感、張りなどの肌状態が変化することが経験的に知られている。それに対し、例えば、皮膚が受ける酸化的ストレスに着目し、透明感の向上についての研究がなされている(特許文献2)。他にも、機能性食品や化粧品などによる肌状態改善も提案されている(特許文献3)。
心理的ストレスにより皮膚内のサブスタンスP(以下、SPという。)が増加することが知られている(非特許文献1)。SPは皮脂腺を増大させ皮脂分泌を促進することが知られており、これによる過剰な皮脂分泌の抑制が試みられている(特許文献4)。SPについては、脱毛症との関係についても研究がなされている(特許文献5)。しかしながら、SPが具体的に肌の状態にどのような影響を及ぼすかについては知られていない。
一方、肌の弾力、ハリには真皮のコラーゲンが重要であることが知られており、コラーゲンに着目した研究は精力的に行われている。コラーゲンは複数のタイプがあり、I型コ
ラーゲンとIII型コラーゲンが知られているが、I型コラーゲンを中心とした研究がほとんどであり、III型コラーゲンに関する報告はほとんどない。III型コラーゲンは胎児の時期に最も皮膚に存在しており、加齢に伴い減少することが知られている(非特許文献2)。また、III型コラーゲンは、I型と比較して、それ自身の透明性が高いことが知られている(
非特許文献3)。しかしながら、III型コラーゲンが肌の透明感や柔らかさに関与していることは知られていなかった。
As the years go by, the elasticity and tension of the skin is gradually lost, and pigmentation occurs and color changes occur due to increased dullness. Deterioration of the skin condition due to aging of the skin greatly changes the impression given to humans, so it is expected to improve the skin, and interest in various skin external preparations, especially cosmetics, is high, and various studies are actively conducted. ing.
For example, in order to prevent skin aging and maintain tension and gloss, attention has been focused on estrogen, a type of female hormone, and cosmetic foods and skin cosmetics containing estrogenic agents have been proposed (Patent Documents) 1).
It is also empirically known that stress promotes skin aging. That is, it is empirically known that not only physiological phenomena but also skin conditions such as gloss, transparency, and tension change due to physical stress and psychological stress. On the other hand, for example, paying attention to the oxidative stress that the skin receives, research has been conducted on improving transparency (Patent Document 2). In addition, improvement of the skin condition by functional foods and cosmetics has been proposed (Patent Document 3).
It is known that substance P in the skin (hereinafter referred to as SP) increases due to psychological stress (Non-patent Document 1). It is known that SP increases sebaceous glands and promotes sebum secretion, and attempts have been made to suppress excessive sebum secretion (Patent Document 4). Regarding SP, research has also been conducted on the relationship with alopecia (Patent Document 5). However, it is not known how SP specifically affects the skin condition.
On the other hand, it is known that collagen of the dermis is important for the elasticity and firmness of the skin, and research focusing on collagen has been conducted energetically. There are several types of collagen, and type I collagen and type III collagen are known. However, most studies center on type I collagen, and there are few reports on type III collagen. It is known that type III collagen is most present in the skin during the fetal period and decreases with age (Non-patent Document 2). In addition, type III collagen is known to have high transparency as compared with type I (
Non-patent document 3). However, it has not been known that type III collagen is involved in skin transparency and softness.
本発明は、肌の透明感、柔らかさを改善する肌改善剤のスクリーニング方法を提供することを課題とする。 This invention makes it a subject to provide the screening method of the skin improving agent which improves the transparency and softness of skin.
本発明者は、肌の透明感、柔らかさを改善するための方法について研究を進め、心理ストレスにより分泌されるSPがIII型コラーゲン産生を阻害することを見出した。すなわ
ち、真皮線維芽細胞中のSPが主要受容体であるNK1R(以下、NK1Rという。)へ結合することが、III型コラーゲンの産生を阻害し、肌の透明感、柔らかさに影響すると
いう知見を得た。
そして、当該知見をもとに、真皮線維芽細胞中のNK1Rの発現量を指標とすることで、肌の透明感や柔らかさの改善効果を有する物質をスクリーニングできることに想到した。
The inventor conducted research on methods for improving the transparency and softness of the skin, and found that SP secreted by psychological stress inhibits type III collagen production. That is, the finding that SP in dermal fibroblasts binds to NK1R (hereinafter referred to as NK1R), which is the main receptor, inhibits the production of type III collagen and affects the transparency and softness of the skin. Got.
And based on the said knowledge, it came to the idea that the substance which has the effect of improving the transparency and softness of the skin can be screened by using the expression level of NK1R in dermal fibroblasts as an index.
すなわち、本発明は以下の通りである。
[1] 真皮線維芽細胞のNK1R量を指標とする肌の透明感、柔らかさを改善する肌改善剤のスクリーニング方法であって、
被験物質を添加しない真皮線維芽細胞のNK1R量(コントロール)、及び被験物質を添加して培養した真皮線維芽細胞のNK1R量を得る測定ステップ、前記ステップで得られたNK1R量を比較し、コントロールと比較してNK1R量が減少した被験物質を肌改善剤として選択する選択ステップ、を含む、スクリーニング方法。
[2] 前記選択ステップは、被験物質を添加して培養した真皮線維芽細胞のNK1R量がコントロールのNK1R量の80%以下である場合に、前記被験物質を肌改善剤として選択する、[1]に記載のスクリーニング方法。
[3] 真皮線維芽細胞のIII型コラーゲン量を指標とする肌の透明感、柔らかさを改善
する肌改善剤のスクリーニング方法であって、
SPを添加し被験物質を添加しない真皮線維芽細胞のIII型コラーゲン量(ネガティブ
コントロール)、及びSPを添加し被験物質を添加して培養した真皮線維芽細胞のIII型
コラーゲン量を得る測定ステップ、
前記ステップで得られたIII型コラーゲン量を比較し、ネガティブコントロールと比較
してIII型コラーゲン量が増加した被験物質を肌改善剤として選択する選択ステップ、を
含む、スクリーニング方法。
[4] 前記測定ステップは、SP及び被験物質を添加しない真皮線維芽細胞のIII型コ
ラーゲン量(コントロール)を測定するステップをさらに含み、前記選択ステップは、以下の式から算出された値が10%以上ある場合に、前記被験物質を肌改善剤として選択する、[3]に記載のスクリーニング方法。
[(α−α1)/(α0−α1)]*100 [%] 式(1)
ここで、αはSP及び被験物質を添加して培養した真皮線維芽細胞のIII型コラーゲン量
、α1はネガティブコントロール、α0はコントロールである。
[5] [1]〜[4]のいずれかに記載の方法により肌改善剤として選択された物質を
含有してなる組成物。
[6] 前記物質がハス胚芽エキスである、[5]に記載の組成物。
[7] 皮膚外用剤である、[5]又は[6]に記載の組成物。
[8] 化粧料(ただし、医薬部外品を含む)である、[5]〜[7]のいずれかに記載の組成物。
[9] 肌の透明感及び/又は肌の柔らかさを改善する肌改善用である、[6]〜[8]のいずれかに記載の組成物。
[10] [1]〜[4]のいずれかに記載の方法により肌改善剤として選択された物質を組成物に配合するステップを含むことを特徴とする組成物の製造方法。
That is, the present invention is as follows.
[1] A screening method for a skin improving agent that improves skin transparency and softness using the NK1R amount of dermal fibroblasts as an index,
NK1R amount of dermal fibroblasts without addition of test substance (control) and measurement step to obtain NK1R amount of dermal fibroblasts cultured with addition of test substance, NK1R amount obtained in the previous step is compared, control And a selection step of selecting, as a skin improving agent, a test substance having a reduced amount of NK1R compared to the screening method.
[2] In the selection step, when the NK1R amount of the dermal fibroblasts cultured with the test substance added is 80% or less of the NK1R amount of the control, the test substance is selected as a skin improving agent. ] The screening method of description.
[3] A screening method for a skin improving agent that improves skin transparency and softness using the amount of type III collagen of dermal fibroblasts as an index,
Measurement step of obtaining the amount of type III collagen of dermal fibroblasts to which SP is added and no test substance is added (negative control), and the amount of type III collagen of dermal fibroblasts cultured by adding SP and adding the test substance,
A screening method comprising the step of comparing the amount of type III collagen obtained in the above step and selecting a test substance having an increased amount of type III collagen as compared with a negative control as a skin improving agent.
[4] The measurement step further includes a step of measuring a type III collagen amount (control) of dermal fibroblasts to which SP and a test substance are not added, and the selection step has a value calculated from the following equation of 10: The screening method according to [3], wherein the test substance is selected as a skin ameliorating agent when there is at least%.
[(Α−α 1 ) / (α 0 −α 1 )] * 100 [%] Formula (1)
Here, α is the amount of type III collagen in dermal fibroblasts cultured with SP and a test substance added, α 1 is a negative control, and α 0 is a control.
[5] A composition comprising a substance selected as a skin improving agent by the method according to any one of [1] to [4].
[6] The composition according to [5], wherein the substance is a lotus germ extract.
[7] The composition according to [5] or [6], which is an external preparation for skin.
[8] The composition according to any one of [5] to [7], which is a cosmetic (including a quasi-drug).
[9] The composition according to any one of [6] to [8], which is for skin improvement that improves skin transparency and / or skin softness.
[10] A method for producing a composition, comprising a step of incorporating a substance selected as a skin improving agent into the composition by the method according to any one of [1] to [4].
本発明により、肌の透明感、柔らかさを改善する肌改善剤のスクリーニング方法が提供される。 According to the present invention, there is provided a screening method for a skin improving agent that improves skin transparency and softness.
心理的ストレスにより、皮膚中のSP量は増加する。また、真皮線維芽細胞には、SPの受容体であるNK1Rが発現している。本発明のスクリーニング方法の第一の実施態様は、真皮線維芽細胞のNK1R量を指標とする肌改善剤のスクリーニング方法である。
具体的には、被験物質を添加しない真皮線維芽細胞(コントロール)のNK1R量、及び被験物質を添加して培養した真皮線維芽細胞のNK1R量を得る測定ステップ、前記ステップで得られたNK1R量を比較し、コントロールと比較してNK1R量が減少した被験物質を肌改善剤として選択する選択ステップ、を含む、スクリーニング方法である。
The amount of SP in the skin increases due to psychological stress. In addition, NK1R, which is a receptor for SP, is expressed in dermal fibroblasts. The first embodiment of the screening method of the present invention is a screening method for a skin improving agent using the amount of NK1R of dermal fibroblasts as an index.
Specifically, the measurement step of obtaining the NK1R amount of dermal fibroblasts (control) to which no test substance is added and the NK1R amount of dermal fibroblasts cultured with the addition of the test substance, the NK1R amount obtained in the above step And a selection step of selecting a test substance having a reduced NK1R amount as compared with a control as a skin improving agent.
以前から、シワの形成に関連するI型コラーゲンに着目した研究は盛んになされている。本発明者はNK1RとSPの結合により、III型コラーゲンの産生が阻害されることを
新たに見出した。III型コラーゲン産生を阻害するNK1Rを減少させることで、I型コ
ラーゲンが関連するシワ形成抑制とは異なり、肌の透明感、柔らかさが改善されることに想到した。すなわち、被験物質を添加したときの真皮線維芽細胞中のNK1R量を測定し、その変動量から、被験物質の肌改善作用を推測できることを見出した。さらに、真皮線維芽細胞のNK1R量を指標とするスクリーニング方法により、肌の透明感、柔らかさに特に効果を発揮する肌改善剤を提供し、当該肌改善剤は、従来知られているI型コラーゲンが有するシワ形成抑制効果に加え、透明感、柔らかさを改善する効果を有することに想到した。
Researches focusing on type I collagen related to the formation of wrinkles have been extensively conducted. The inventor has newly found that the production of type III collagen is inhibited by the binding of NK1R and SP. By reducing NK1R, which inhibits type III collagen production, it was conceived that, unlike wrinkle formation inhibition associated with type I collagen, the transparency and softness of the skin were improved. That is, the amount of NK1R in dermal fibroblasts when a test substance was added was measured, and it was found that the skin improvement effect of the test substance can be estimated from the amount of change. Furthermore, by a screening method using the amount of NK1R of dermal fibroblasts as an indicator, a skin improving agent that exhibits a particularly effective effect on the transparency and softness of the skin is provided. The skin improving agent is a conventionally known type I In addition to the wrinkle formation inhibitory effect of collagen, it has been conceived that it has an effect of improving transparency and softness.
本発明のスクリーニング方法の第一の実施態様における手順の一例を以下に挙げるが、本発明の趣旨を逸脱しない限り以下の内容に限定されるものではなく、適宜変更して実施することができる。 An example of the procedure in the first embodiment of the screening method of the present invention is given below, but is not limited to the following contents without departing from the gist of the present invention, and can be implemented with appropriate modifications.
<測定ステップ>
NK1Rを構成するタンパク質をコードするmRNAの発現量は、任意の方法を用いて測定することができる。例えば、当該遺伝子の配列に特異的に結合する配列を有するDNA断片をプライマーとして用いてPCRを行い、定量的な検出を行う。なお、NK1RをコードするmRNAの遺伝子配列は公開されており、当業者は適宜プライマーを設計してPCRに供することができる。
<Measurement step>
The expression level of mRNA encoding the protein constituting NK1R can be measured using any method. For example, PCR is performed using a DNA fragment having a sequence that specifically binds to the sequence of the gene as a primer, and quantitative detection is performed. In addition, the gene sequence of mRNA encoding NK1R has been made public, and those skilled in the art can design primers appropriately and use them for PCR.
まず、コントロールとして被験物質を添加しない真皮線維芽細胞において、該細胞からmRNAを抽出し、指標とするNK1Rをコードする遺伝子の発現量を、該遺伝子を特異
的に検出するプライマーを用いてq−PCRを行い、定量的に測定する。また、真皮線維芽細胞に被験物質を添加し、37℃で約3〜72時間培養する。その後、該細胞からmRNAを抽出し、指標とするNK1Rをコードする遺伝子の発現量を、該遺伝子を特異的に検出するプライマーを用いてq−PCRを行い、定量的に測定する。被験物質を添加した真皮線維芽細胞における該遺伝子の発現量が、被験物質を添加しなかった該細胞における該遺伝子の発現量(コントロール)に対して減少した場合、好ましくはコントロールに対して80%以下、より好ましくは70%以下となった場合に、前記被験物質は肌改善剤として選択される。該選択された物質は、肌の透明感、柔らかさを改善する作用を有し、該肌改善剤は、化粧料、医薬部外品、食品、医薬品に配合することができるが、化粧料に配合されることが好ましい。
First, in dermal fibroblasts to which no test substance is added as a control, mRNA is extracted from the cells, and the expression level of the gene encoding NK1R used as an index is determined using a primer that specifically detects the gene q − Perform PCR and measure quantitatively. In addition, a test substance is added to dermal fibroblasts and cultured at 37 ° C. for about 3 to 72 hours. Thereafter, mRNA is extracted from the cells, and the expression level of the gene encoding NK1R as an index is quantitatively measured by q-PCR using a primer that specifically detects the gene. When the expression level of the gene in dermal fibroblasts to which the test substance is added is reduced relative to the expression level (control) of the gene in the cells to which the test substance is not added, preferably 80% of the control Hereinafter, the test substance is selected as a skin improving agent when it becomes 70% or less. The selected substance has the effect of improving the transparency and softness of the skin, and the skin improving agent can be added to cosmetics, quasi drugs, foods, and pharmaceuticals. It is preferable to mix.
本発明のスクリーニング方法の第二の実施態様は、真皮線維芽細胞のIII型コラーゲン
量を指標とする肌改善剤のスクリーニング方法である。
具体的には、SPを添加し被験物質を添加しない真皮線維芽細胞のIII型コラーゲン量
(ネガティブコントロール)、及びSPを添加し被験物質を添加して培養した真皮線維芽細胞のIII型コラーゲン量を得る測定ステップ、前記ステップで得られたIII型コラーゲン量を比較し、ネガティブコントロールと比較してIII型コラーゲン量が増加した被験物質
を肌改善剤として選択する選択ステップ、を含む、スクリーニング方法である。
The second embodiment of the screening method of the present invention is a screening method for a skin improving agent using the amount of type III collagen in dermal fibroblasts as an index.
Specifically, the amount of type III collagen in dermal fibroblasts added with SP and no test substance (negative control), and the amount of type III collagen in dermal fibroblasts cultured with addition of SP and added test substance A screening method comprising: a measurement step for obtaining a test substance that compares the amount of type III collagen obtained in the above step, and that selects a test substance having an increased amount of type III collagen as compared with a negative control as a skin improving agent. is there.
本発明のスクリーニング方法の第二の実施態様における手順の一例を以下に挙げるが、本発明の趣旨を逸脱しない限り以下の内容に限定されるものではなく、適宜変更して実施することができる。 An example of the procedure in the second embodiment of the screening method of the present invention is given below. However, the procedure is not limited to the following contents without departing from the gist of the present invention, and can be implemented with appropriate modifications.
まず、被験物質及びSPを添加しない真皮線維芽細胞における、指標とするIII型コラ
ーゲンの量をELISA法にて定量的に測定する(コントロール)。また、真皮線維芽細胞にSPを添加し、37℃で6〜72時間培養し、該細胞における、指標とするIII型コ
ラーゲンの量をELISA法にて定量的に測定する(ネガティブコントロール)。さらに、真皮線維芽細胞にSPおよび被験物質を添加し、37℃で6〜72時間培養し、該細胞における、指標とするIII型コラーゲン量をELISA法にて定量的に測定する。被験物
質を添加しなかった該細胞におけるIII型コラーゲンの量がネガティブコントロールに対
して増加した場合、好ましくは以下の式(1)で算出された値が、10%以上、より好ましくは15%以上となった場合に、前記被験物質は肌改善剤として選択される。
[(α−α1)/(α0−α1)]*100 [%] 式(1)
ここで、αはSP及び被験物質を添加した時の真皮線維芽細胞のIII型コラーゲン量、
α1はネガティブコントロール、α0はコントロールである。
該選択された物質は、肌の透明感、柔らかさを改善する作用を有し、該肌改善剤は、化粧料、医薬部外品、食品、医薬品に配合することができるが、化粧料に配合されることが好ましい。
First, the amount of type III collagen as an index in dermal fibroblasts to which no test substance and SP are added is quantitatively measured by ELISA (control). Further, SP is added to dermal fibroblasts, cultured at 37 ° C. for 6 to 72 hours, and the amount of type III collagen as an index in the cells is quantitatively measured by ELISA (negative control). Furthermore, SP and a test substance are added to dermal fibroblasts, cultured at 37 ° C. for 6 to 72 hours, and the amount of type III collagen as an index in the cells is quantitatively measured by ELISA. When the amount of type III collagen in the cells to which no test substance is added is increased relative to the negative control, the value calculated by the following formula (1) is preferably 10% or more, more preferably 15% or more The test substance is selected as a skin-improving agent.
[(Α−α 1 ) / (α 0 −α 1 )] * 100 [%] Formula (1)
Where α is the amount of type III collagen in the dermal fibroblast when SP and the test substance are added,
α 1 is a negative control and α 0 is a control.
The selected substance has the effect of improving the transparency and softness of the skin, and the skin improving agent can be added to cosmetics, quasi drugs, foods, and pharmaceuticals. It is preferable to mix.
<真皮線維芽細胞>
本発明において、スクリーニングに用いる細胞の種類は、真皮線維芽細胞である。例えば、ヒト、サル、マウス、ラット、モルモット、ウサギ、ネコ、イヌ、ウマ、ウシ、ヒツジ、ヤギ、ブタ等の哺乳動物の真皮線維芽細胞を用いることができる。好ましくは、ヒト真皮線維芽細胞である。市販の真皮線維芽細胞としては、正常ヒト皮膚線維芽細胞(NHDF)(倉敷紡績株式会社製)を使用することができる。真皮線維芽細胞の培養の条件としては、通常の培養条件、例えば市販のダルベッコ改変イーグル培地(DMEM)を用いる他、本発明のスクリーニング方法の実行を妨げない培養条件であれば、特段の限定なく適用することができる。
<Dermal fibroblasts>
In the present invention, the type of cells used for screening is dermal fibroblasts. For example, dermal fibroblasts of mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats and pigs can be used. Preferably, it is a human dermal fibroblast. As human dermal fibroblasts, normal human dermal fibroblasts (NHDF) (manufactured by Kurashiki Boseki Co., Ltd.) can be used. The conditions for the cultivation of dermal fibroblasts, normal culture conditions, e.g., except for using a commercially available Dulbecco's modified Eagle medium (DMEM), as long as the culture conditions that do not interfere with the execution of the screening methods of the present invention, no particular limitation Can be applied without.
<被験物質>
本発明のスクリーニング方法が対象とする被験物質は、純物質、動植物由来の抽出物、又はそれらの混合物等のいずれであってもよい。
動植物由来の抽出物は、動物又は植物由来の抽出物自体のみならず、抽出物の画分、精製した画分、抽出物乃至は画分、精製物の溶媒除去物の総称を意味するものとし、植物由来の抽出物は、自生若しくは生育された植物、漢方生薬原料等として販売されるものを用いた抽出物、市販されている抽出物等が挙げられる。中でも、化粧料としたときの透明性、柔らかさの観点から、ハス胚芽エキスが好ましい。
抽出操作は、植物部位の全草を用いるほか、植物体、地上部、根茎部、木幹部、葉部、茎部、花穂、花蕾等の部位を使用することできるが、予めこれらを粉砕あるいは細切して抽出効率を向上させることが好ましい。抽出溶媒としては、水、メタノール、エタノール、イソプロピルアルコール、ブタノールなどのアルコール類、1,3−ブタンジオール、ポリプロピレングリコールなどの多価アルコール類、アセトン、メチルエチルケトンなどのケトン類、ジエチルエーテル、テトラヒドロフランなどのエーテル類等の極性溶媒から選択される1種乃至は2種以上が好適なものとして例示することができる。具体的な抽出方法としては、例えば、植物体等の抽出に用いる部位乃至はその乾燥物1質量に対して、溶媒を1〜30質量部加え、室温であれば数日間、沸点付近の温度であれば数時間浸漬し、室温まで冷却した後、所望により不溶物及び/又は溶媒除去し、カラムクロマトグラフィー等で分画精製する方法が挙げられる。
本発明においては、動植物由来の極性溶媒による抽出物をろ過後、カラムクロマトグラフィーにて分画し、有効成分濃度を高め、前記スクリーニング方法にて、より効果の高い画分を選択し肌改善剤として用いることが好ましい。
<Test substance>
The test substance targeted by the screening method of the present invention may be any of a pure substance, an extract derived from animals and plants, or a mixture thereof.
The extracts derived from animals and plants mean not only animal or plant-derived extracts themselves, but also generic names of extract fractions, purified fractions, extracts or fractions, and solvent-removed products of purified products. Examples of plant-derived extracts include native or grown plants, extracts using products sold as herbal medicine ingredients, and commercially available extracts. Among these, a lotus germ extract is preferable from the viewpoint of transparency and softness when it is used as a cosmetic.
For the extraction operation, the whole plant part can be used, and other parts such as the plant body, the above-ground part, the rhizome part, the tree trunk part, the leaf part, the stem part, the flower ear, and the flower bud can be used. It is preferable to improve the extraction efficiency by cutting. Examples of the extraction solvent include water, alcohols such as methanol, ethanol, isopropyl alcohol, and butanol, polyhydric alcohols such as 1,3-butanediol and polypropylene glycol, ketones such as acetone and methyl ethyl ketone, diethyl ether, and tetrahydrofuran. One or two or more selected from polar solvents such as ethers can be exemplified as preferable ones. As a specific extraction method, for example, 1 to 30 parts by mass of a solvent is added to 1 part by mass of a plant or the like used for extraction of a plant or the like. For example, after immersion for several hours and cooling to room temperature, the insoluble matter and / or solvent may be removed if desired, followed by fractional purification by column chromatography or the like.
In the present invention, after extracting an extract from a polar solvent derived from animals and plants, fractionating by column chromatography, increasing the active ingredient concentration, and selecting the more effective fraction by the screening method, the skin improving agent It is preferable to use as.
<肌改善剤として選択された物質を含有する組成物>
本発明のスクリーニング方法により肌改善剤として選択された物質は、任意の調製方法により組成物に含有させることができる。
組成物としては、化粧料、医薬部外品、医薬品、食品などが好適に例示でき、日常的に使用できることから、化粧料、医薬部外品がより好ましい。その投与経路としては、特に限定されるものではないが、肌の透明感、柔らかさの改善効果を発揮するために皮膚への貯留性、標的部位への到達効率等を考慮し、経皮投与を採用して皮膚外用剤とすることが好ましい。
<Composition containing a substance selected as a skin improving agent>
The substance selected as the skin improving agent by the screening method of the present invention can be contained in the composition by any preparation method.
As the composition, cosmetics, quasi-drugs, pharmaceuticals, foods and the like can be suitably exemplified, and cosmetics and quasi-drugs are more preferable because they can be used on a daily basis. The route of administration is not particularly limited, but it is transdermally administered in consideration of the retention on the skin, the efficiency of reaching the target site, etc. in order to exert the effect of improving skin transparency and softness. Is preferably used as a skin external preparation.
本発明の組成物中における、肌改善剤の含有量(配合量)は、皮膚外用剤として用いる場合は、通常、0.00001質量%以上、好ましくは0.0001質量%以上、より好ましくは0.01質量%以上であり、通常10質量%以下、好ましくは5質量%以下である。食品中における動植物抽出物の含有量は、通常、0.01質量%以上、好ましくは0.1質量%以上であり、より好ましくは1質量%以上であり、通常80質量%以下であり、好ましくは50質量%以下であり、より好ましくは30質量%以下である。肌改善剤の含有量(配合量)が少なすぎると所望の効果が得られにくい場合があり、多すぎると効果が頭打ちになるばかりか組成物の処方の自由度を損なう場合があるからである。
また、組成物に含有させる肌改善剤の種類は、1種類のみでなく2種類以上であってもよい。
When used as a skin external preparation, the content (blending amount) of the skin improving agent in the composition of the present invention is usually 0.00001% by mass or more, preferably 0.0001% by mass or more, more preferably 0. 0.01 mass% or more, usually 10 mass% or less, preferably 5 mass% or less. The content of the animal or plant extract in the food is usually 0.01% by mass or more, preferably 0.1% by mass or more, more preferably 1% by mass or more, and usually 80% by mass or less, preferably Is 50% by mass or less, more preferably 30% by mass or less. If the content (blending amount) of the skin-improving agent is too small, the desired effect may be difficult to obtain. If it is too much, the effect will reach its peak, and the degree of freedom of formulation of the composition may be impaired. .
Moreover, the kind of skin improvement agent contained in a composition may be not only one type but two or more types.
組成物の製造に際しては、化粧料、医薬部外品、医薬品、食品などの製剤化で通常使用される成分を、それぞれの用途に応じて、適宜含有させることができる。 In the production of the composition, components usually used in the preparation of cosmetics, quasi-drugs, pharmaceuticals, foods, and the like can be appropriately contained according to each application.
化粧料に適用される場合、通常化粧料に使用される成分を広く配合することが可能であり、また、その剤形や用途についても、何ら限定されない。以下、化粧料に適用される場合、化粧料中に含有させることができる成分について説明する。例えば、炭化水素類、エステル類、トリグリセライド類、脂肪酸、高級アルコール等の油性成分、アニオン界面活性剤類、両性界面活性剤類、カチオン界面活性剤類、非イオン界面活性剤類等の界面活性
剤、多価アルコール類、増粘・ゲル化剤、酸化防止剤、紫外線吸収剤、色剤、防腐剤、粉体等を任意に配合することができる。有効成分としては、美白成分、シワ改善成分、抗炎症成分、動植物由来の抽出物等が挙げられる。なお、上記説明した本発明の実施態様に係る肌改善剤と併用してもよい。
化粧料における美白成分の含有量は、通常0.001〜10質量%であり、0.01〜10質量%が好ましく、0.1〜5質量%がより好ましい。
When applied to cosmetics, it is possible to broadly blend the components normally used in cosmetics, and the dosage form and use are not limited at all. Hereinafter, the components that can be contained in the cosmetic when applied to the cosmetic will be described. For example, surfactants such as oils such as hydrocarbons, esters, triglycerides, fatty acids, higher alcohols, anionic surfactants, amphoteric surfactants, cationic surfactants, nonionic surfactants Polyhydric alcohols, thickening / gelling agents, antioxidants, ultraviolet absorbers, colorants, preservatives, powders, and the like can be arbitrarily blended. Examples of active ingredients include whitening ingredients, wrinkle-improving ingredients, anti-inflammatory ingredients, animal and plant extracts. In addition, you may use together with the skin improvement agent which concerns on the embodiment of this invention demonstrated above.
Content of the whitening component in cosmetics is 0.001-10 mass% normally, 0.01-10 mass% is preferable and 0.1-5 mass% is more preferable.
美白成分としては、一般的に化粧料に用いられているものであれば特に限定はない。例えば、4−n−ブチルレゾルシノール、アスコルビン酸グルコシド、3−О−エチルアスコルビン酸、トラネキサム酸、アルブチン、1−トリフェニルメチルピペリジン、1−トリフェニルメチルピロリジン、2−(トリフェニルメチルオキシ)エタノール、2−(トリフェニルメチルアミノ)エタノール、2−(トリフェニルメチルオキシ)エチルアミン、トリフェニルメチルアミン、トリフェニルメタノール、トリフェニルメタン及びアミノジフェニルメタン、N−(p−トルイル)システイン酸、N−(p−メトキシベンゾイル)システイン酸等が挙げられる。更にその他の美白成分として、N−ベンゾイル−セリン、N−(p−メチルベンゾイル)セリン、N−(p−エチルベンゾイル)セリン、N−(p−メトキシベンゾイル)セリン、N−(p−フルオロベンゾイル)セリン、N−(p−トリフルオロメチルベンゾイル)セリン、N−(2−ナフトイル)セリン、N−(4−フェニルベンゾイル)セリン、N−(p−メチルベンゾイル)セリン メチルエステル、N−(p−メチルベンゾイル)セリン エチルエステル、N−(2−ナフトイル)セリン メチルエステル、N−ベンゾイル−O−メチルセリン、N−(p−メチルベンゾイル)−O−メチルセリン、N−(p−メチルベンゾイル)−O−アセチルセリン、N−(2−ナフトイル)−O−メチルセリン等があげられる。
これらの美白成分は、既に市販されているものもあれば、合成により入手することもできる。例えば、3−О−エチルアスコルビン酸は、特開平8−134055号公報に記載の公知の方法で合成することが出来る。市販品(日本精化製「VCエチル」)もあるので、これらを入手して使用することが可能である。1−トリフェニルメチルピペリジン、1−トリフェニルメチルピロリジン、2−(トリフェニルメチルオキシ)エタノール、2−(トリフェニルメチルアミノ)エタノール、2−(トリフェニルメチルオキシ)エチルアミン、トリフェニルメチルアミン、トリフェニルメタノール、トリフェニルメタン、アミノジフェニルメタンは特許文献WO2010/074052号パンフレットに、N−(o−トルオイル)システイン酸、N−(m−トルオイル)システイン酸、N−(p−トルオイル)システイン酸、N−(p−メトキシベンゾイル)システイン酸、N−(4−フェニルベンゾイル)システイン酸、N−(p−トルオイル)ホモシステイン酸、はWO2011/087006号パンフレットに、N−ベンゾイル−セリン、N−(p−メチルベンゾイル)セリン、N−(p−エチルベンゾイル)セリン、N−(p−メトキシベンゾイル)セリン、N−(p−フルオロベンゾイル)セリン、N−(p−トリフルオロメチルベンゾイル)セリン、N−(2−ナフトイル)セリン、N−(4−フェニルベンゾイル)セリン、N−(p−メチルベンゾイル)セリン メチルエステル、N−(p−メチルベンゾイル)セリン エチルエステル、N−(2−ナフトイル)セリン メチルエステル、N−ベンゾイル−O−メチルセリン、N−(p−メチルベンゾイル)−O−メチルセリン、N−(p−メチルベンゾイル)−O−アセチルセリン、N−(2−ナフトイル)−O−メチルセリン等はWO2011/074643号パンフレットに、それぞれその合成方法が公開されているので、該開示に従い合成することができる。
化粧料における美白成分の含有量は、通常0.001〜10質量%であり、0.01〜10質量%が好ましく、0.1〜5質量%がより好ましい。
The whitening component is not particularly limited as long as it is generally used in cosmetics. For example, 4-n-butylresorcinol, ascorbic acid glucoside, 3-O-ethylascorbic acid, tranexamic acid, arbutin, 1-triphenylmethylpiperidine, 1-triphenylmethylpyrrolidine, 2- (triphenylmethyloxy) ethanol, 2- (triphenylmethylamino) ethanol, 2- (triphenylmethyloxy) ethylamine, triphenylmethylamine, triphenylmethanol, triphenylmethane and aminodiphenylmethane, N- (p-toluyl) cysteic acid, N- (p -Methoxybenzoyl) cysteic acid and the like. Furthermore, as other whitening components, N-benzoyl-serine, N- (p-methylbenzoyl) serine, N- (p-ethylbenzoyl) serine, N- (p-methoxybenzoyl) serine, N- (p-fluorobenzoyl) ) Serine, N- (p-trifluoromethylbenzoyl) serine, N- (2-naphthoyl) serine, N- (4-phenylbenzoyl) serine, N- (p-methylbenzoyl) serine methyl ester, N- (p -Methylbenzoyl) serine ethyl ester, N- (2-naphthoyl) serine methyl ester, N-benzoyl-O-methylserine, N- (p-methylbenzoyl) -O-methylserine, N- (p-methylbenzoyl) -O -Acetylserine, N- (2-naphthoyl) -O-methylserine and the like.
Some of these whitening components are already on the market, or they can be obtained by synthesis. For example, 3-O-ethylascorbic acid can be synthesized by a known method described in JP-A-8-134055. There are also commercially available products (“VC ethyl” manufactured by Nippon Seika Co., Ltd.), and these can be obtained and used. 1-triphenylmethylpiperidine, 1-triphenylmethylpyrrolidine, 2- (triphenylmethyloxy) ethanol, 2- (triphenylmethylamino) ethanol, 2- (triphenylmethyloxy) ethylamine, triphenylmethylamine, triphenyl Phenylmethanol, triphenylmethane, and aminodiphenylmethane are disclosed in pamphlet of WO2010 / 074052, in which N- (o-toluoyl) cysteic acid, N- (m-toluoyl) cysteic acid, N- (p-toluoyl) cysteic acid, N -(P-methoxybenzoyl) cysteic acid, N- (4-phenylbenzoyl) cysteic acid and N- (p-toluoyl) homocysteic acid are disclosed in WO2011 / 088706, N-benzoyl-serine, N- (p −Me Rubenzoyl) serine, N- (p-ethylbenzoyl) serine, N- (p-methoxybenzoyl) serine, N- (p-fluorobenzoyl) serine, N- (p-trifluoromethylbenzoyl) serine, N- ( 2-naphthoyl) serine, N- (4-phenylbenzoyl) serine, N- (p-methylbenzoyl) serine methyl ester, N- (p-methylbenzoyl) serine ethyl ester, N- (2-naphthoyl) serine methyl ester N-benzoyl-O-methylserine, N- (p-methylbenzoyl) -O-methylserine, N- (p-methylbenzoyl) -O-acetylserine, N- (2-naphthoyl) -O-methylserine, etc. Since the methods for synthesizing them are disclosed in each pamphlet It is possible to follow synthesis.
Content of the whitening component in cosmetics is 0.001-10 mass% normally, 0.01-10 mass% is preferable and 0.1-5 mass% is more preferable.
本発明の組成物には、本発明に係るスクリーニング方法で選択された肌改善剤に加え、更にシワ改善成分を含有することができる。シワ改善成分としては、一般的に化粧料に用いられているものであれば特に限定はない。例えば、ビタミンA又はその誘導体であるレ
チノール、レチナール、レチノイン酸、トレチノイン、イソトレチノイン、レチノイン酸トコフェロール、パルミチン酸レチノール、酢酸レチノールや、ウルソール酸ベンジルエステル、ウルソール酸リン酸エステル、ベツリン酸ベンジルエステル、ベンジル酸リン酸エステルが挙げられる。化粧料におけるシワ改善成分の含有量は、通常0.01〜30質量%であり、0.1〜10質量%が好ましく、1〜5質量%がより好ましい。
The composition of the present invention may further contain a wrinkle improving component in addition to the skin improving agent selected by the screening method according to the present invention. The wrinkle improving component is not particularly limited as long as it is generally used in cosmetics. For example, vitamin A or its derivatives retinol, retinal, retinoic acid, tretinoin, isotretinoin, retinoic acid tocopherol, retinol palmitate, retinol acetate, ursolic acid benzyl ester, ursolic acid phosphate ester, betulinic acid benzyl ester, benzyl Acid phosphate ester is mentioned. Content of the wrinkle improvement component in cosmetics is 0.01-30 mass% normally, 0.1-10 mass% is preferable and 1-5 mass% is more preferable.
動植物由来の抽出物としては、一般的に医薬品、化粧料、食品等に用いられているものであれば特に限定はない。例えば、アケビエキス、アスナロエキス、アスパラガスエキス、アボカドエキス、アマチャエキス、アーモンドエキス、アルニカエキス、アロエエキス、アロニアエキス、アンズエキス、イチョウエキス、インドキノエキス、ウイキョウエキス、ウドエキス、エイジツエキス、エゾウコギエキス、エンメイソウエキス、オウゴンエキス、オウバクエキス、オウレンエキス、オタネニンジンエキス、オトギリソウエキス、オドリコソウエキス、オレンジエキス、カキョクエキス、カッコンエキス、カモミラエキス、カロットエキス、カワラヨモギエキス、カンゾウエキス、キウイエキス、キューカンバーエキス、グアバエキス、クジンエキス、クチナシエキス、クマザサエキス、クララエキス、クルミエキス、グレープフルーツエキス、黒米エキス、クロレラエキス、クワエキス、ケイケットウエキス、ゲットウヨウエキス、ゲンチアナエキス、ゲンノショウコエキス、紅茶エキス、ゴボウエキス、コメエキス、コメ発酵エキス、コメヌカ発酵エキス、コメ胚芽油、コケモモエキス、サルビアエキス、サボンソウエキス、ササエキス、サンザシエキス、サンシャエキス、サンショウエキス、シイタケエキス、ジオウエキス、シコンエキス、シソエキス、シナノキエキス、シモツケソウエキス、シャクヤクエキス、ショウキョウエキス、ショウブ根エキス、シラカバエキス、スギナエキス、ステビアエキス、ステビア発酵物、セイヨウキズタエキス、セイヨウサンザシエキス、セイヨウニワトコエキス、セイヨウノコギリソウエキス、セイヨウハッカエキス、セージエキス、ゼニアオイエキス、センキュウエキス、センブリエキス、ソウハクヒエキス、ダイオウエキス、ダイズエキス、タイソウエキス、タイムエキス、タンポポエキス、茶エキス、チョウジエキス、チンピエキス、甜茶エキス、トウガラシエキス、トウキエキス、トウキンセンカエキス、トウニンエキス、トウヒエキス、ドクダミエキス、トマトエキス、納豆エキス、ニンジンエキス、ニンニクエキス、ノバラエキス、ハイビスカスエキス、バクモンドウエキス、ハスエキス、ハス胚芽エキス、パセリエキス、バーチエキス、ハマメリスエキス、ヒキオコシエキス、ヒノキエキス、ビワエキス、フキタンポポエキス、フキノトウエキス、ブクリョウエキス、ブッチャーブルームエキス、ブドウエキス、ブドウ種子エキス、ヘチマエキス、ベニバナエキス、ペパーミントエキス、ボダイジュエキス、ボタンエキス、ホップエキス、マツエキス、マロニエエキス、ミズバショウエキス、ムクロジエキス、メリッサエキス、モズクエキス、モモエキス、ヤグルマギクエキス、ユーカリエキス、ユキノシタエキス、ユズエキス、ユリエキス、ヨクイニンエキス、ヨモギエキス、ラベンダーエキス、緑茶エキス、リンゴエキス、ルイボス茶エキス、レイシエキス、レタスエキス、レモンエキス、レンギョウエキス、レンゲソウエキス、ローズエキス、ローズマリーエキス、ローマカミツレエキス、ローヤルゼリーエキス、ワレモコウエキス等のエキスが好ましいものとして挙げられる。なお、本発明のスクリーニング方法において得られた肌改善剤が植物エキスであった場合、上記植物リスティングされた植物エキスと重畳的に含有させてもよい。化粧料中における動植物由来抽出物の含有量は、通常0.0001質量%以上であり、0.001質量%以上が好ましく、0.01質量%以上がより好ましい。また、通常30質量%以下であり、10質量%以下が好ましく、5質量%以下がより好ましい。
食品中における動植物抽出物の含有量は、通常0.001質量%であり、0.01質量%以上が好ましく、1質量%以上がより好ましい。また、通常80質量%以下であり、50質量%以下が好ましい。
The extracts derived from animals and plants are not particularly limited as long as they are generally used for pharmaceuticals, cosmetics, foods and the like. For example, akebi extract, asunaro extract, asparagus extract, avocado extract, achacha extract, almond extract, arnica extract, aloe extract, aronia extract, apricot extract, ginkgo biloba extract, Indian mushroom extract, fennel extract, udo extract, ages extract, sorghum extract , Enmezo extract, Ogon extract, Oat extract, Auren extract, Panax ginseng extract, Hypericum extract, Adrianthus extract, Orange extract, Oyster extract, Cuckoo extract, Chamomile extract, Carrot extract, Kawara mugwort extract, Licorice extract, Kiwi extract, Cucumber extract, Guava extract, Kujin extract, Gardenia extract, Kumazasa extract, Clara extract, Walnut extract, Grapefruit extract, Black rice , Chlorella extract, mulberry extract, caquette extract, gentian extract, gentian extract, Gennosho extract, tea extract, burdock extract, rice extract, fermented rice extract, rice fermented extract, rice germ oil, bilberry extract, salvia extract, bonito extract , Sasa extract, Hawthorn extract, Sansha extract, Salamander extract, Shiitake extract, Giant extract, Shikon extract, Perilla extract, Linden extract, Citrus extract, Peonies extract, Pepper extract, Ginger root extract, Birch extract, Japanese cedar extract, Stevia extract, Stevia fermentation , Kizuta extract, Hawthorn extract, Elderberry extract, Achillea millefolium extract, Pepper extract, Sage extract, Mallow Kiss, Senkyu Extract, Sembura Extract, Sakuhakuhi Extract, Daiou Extract, Soybean Extract, Taiso Extract, Thyme Extract, Dandelion Extract, Tea Extract, Clove Extract, Chimpi Extract, Green Tea Extract, Capsicum Extract, Toki Extract, Tokinsenka Extract, Tonin Extract, Spruce Extract , Dokudami extract, tomato extract, natto extract, carrot extract, garlic extract, wild rose extract, hibiscus extract, bacmond extract, lotus extract, lotus germ extract, parsley extract, birch extract, hamamelis extract, cypress extract, hinoki extract, loquat extract, fukidan popo Extract, Fukinoto extract, Bukuryo extract, Butcher bloom extract, Grape extract, Grape seed extract, Loofah extract, Safflower extract, Pepper Mint extract, body extract, button extract, hop extract, pine extract, maroni extract, mizuba sho extract, mukuroji extract, melissa extract, mozuku extract, peach extract, cornflower extract, eucalyptus extract, yukinoshita extract, yuzu extract, lily extract, yokoinin extract, mugwort extract, Preference is given to extracts such as lavender extract, green tea extract, apple extract, rooibos tea extract, litchi extract, lettuce extract, lemon extract, forsythia extract, forsythia extract, rose extract, rosemary extract, roman chamomile extract, royal jelly extract, and bitumen extract As mentioned. In addition, when the skin improving agent obtained in the screening method of the present invention is a plant extract, it may be contained in a superimposed manner with the plant extract listed above. The content of the animal or plant-derived extract in the cosmetic is usually 0.0001% by mass or more, preferably 0.001% by mass or more, and more preferably 0.01% by mass or more. Moreover, it is 30 mass% or less normally, 10 mass% or less is preferable and 5 mass% or less is more preferable.
The content of the animal or plant extract in the food is usually 0.001% by mass, preferably 0.01% by mass or more, and more preferably 1% by mass or more. Moreover, it is 80 mass% or less normally, and 50 mass% or less is preferable.
抗炎症成分としては、クラリノン、グラブリジン、グリチルリチン酸、グリチルレチン酸、パントテニルアルコール等が挙げられ、好ましくは、グリチルリチン酸及びその塩、
グリチルレチン酸アルキル及びその塩、並びに、グリチルレチン酸及びその塩である。
化粧料中における抗炎症成分の含有量は、通常0.01〜30質量%であり、0.1〜10質量%が好ましく、1〜5質量%がより好ましい。
Examples of the anti-inflammatory component include clarinone, glabrizine, glycyrrhizic acid, glycyrrhetinic acid, pantothenyl alcohol, etc., preferably glycyrrhizic acid and salts thereof,
These are alkyl glycyrrhetic acid and its salt, and glycyrrhetic acid and its salt.
Content of the anti-inflammatory component in cosmetics is 0.01-30 mass% normally, 0.1-10 mass% is preferable and 1-5 mass% is more preferable.
油性成分としては、極性油、揮発性炭化水素油等が挙げられる。
極性油としては、合成エステル油として、ミリスチン酸イソプロピル、オクタン酸セチル、ミリスチン酸オクチルドデシル、パルミチン酸イソプロピル、ステアリン酸ブチル、ラウリン酸ヘキシル、ミリスチン酸ミリスチル、オレイン酸デシル、オレイン酸オクチルドデシル、ジメチルオクタン酸ヘキシルデシル、乳酸セチル、乳酸ミリスチル、酢酸ラノリン、ステアリン酸イソセチル、イソステアリン酸イソセチル、12−ヒドロキシステアリル酸コレステリル、ジ−2−エチルヘキシル酸エチレングリコール、モノイソステアリン酸N−アルキルグリコール、ジカプリン酸ネオペンチルグリコール、リンゴ酸ジイソステアリル、ジ−2−ヘプチルウンデカン酸グリセリン、トリ−2−エチルヘキシル酸トリメチロールプロパン、トリイソステアリン酸トリメチロールプロパン、テトラ−2−エチルヘキシル酸ペンタンエリスリトール、トリ−2−エチルヘキシル酸グリセリン、トリイソステアリン酸トリメチロールプロパンを挙げることができる。
Examples of the oil component include polar oil and volatile hydrocarbon oil.
Polar oils include synthetic ester oils such as isopropyl myristate, cetyl octanoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, octyldodecyl oleate, dimethyloctane Hexyldecyl acid, cetyl lactate, myristyl lactate, lanolin acetate, isocetyl stearate, isocetyl isostearate, cholesteryl 12-hydroxystearylate, ethylene glycol di-2-ethylhexylate, N-alkyl glycol monoisostearate, neopentyl glycol dicaprate , Diisostearyl malate, glycerin di-2-heptylundecanoate, trimethylolpropane tri-2-ethylhexylate, triiso Stearate trimethylolpropane, tetra-2-ethylhexyl acid pentane erythritol, tri-2-ethylhexyl glyceryl, may be mentioned trimethylolpropane triisostearate.
更に、セチル2−エチルヘキサノエート、2−エチルヘキシルパルミテート、トリミリスチン酸グリセリン、トリ−2−ヘプチルウンデカン酸グリセライド、ヒマシ油脂肪酸メチルエステル、オレイン酸オイル、セトステアリルアルコール、オレイルアルコール、ステアリルアルコール、オクチルドデカノール、アセトグリセライド、パルミチン酸2−ヘプチルウンデシル、アジピン酸ジイソブチル、N−ラウロイル−L−グルタミン酸−2−オクチルドデシルエステル、アジピン酸ジ−2−ヘプチルウンデシル、エチルラウレート、セバチン酸ジ−2−エチルヘキシル、ミリスチン酸2−ヘキシルデシル、パルミチン酸2−ヘキシルデシル、アジピン酸2−ヘキシルデシル、セバチン酸ジイソプロピル、コハク酸2−エチルヘキシル、酢酸エチル、酢酸ブチル、酢酸アミル、クエン酸トリエチル、オクチル メトキシシンナメート等も挙げられる。 Furthermore, cetyl 2-ethylhexanoate, 2-ethylhexyl palmitate, glyceryl trimyristate, glyceride tri-2-heptylundecanoate, castor oil fatty acid methyl ester, oleic acid oil, cetostearyl alcohol, oleyl alcohol, stearyl alcohol, Octyldodecanol, acetoglyceride, 2-heptylundecyl palmitate, diisobutyl adipate, N-lauroyl-L-glutamic acid-2-octyldodecyl ester, di-2-heptylundecyl adipate, ethyl laurate, sebacate diacid 2-ethylhexyl, 2-hexyldecyl myristate, 2-hexyldecyl palmitate, 2-hexyldecyl adipate, diisopropyl sebacate, 2-ethylhexyl succinate, acetic acid Chill, butyl acetate, amyl acetate, triethyl citrate, may also be mentioned octyl methoxycinnamate and the like.
また、天然油として、アボカド油、アマニ油、エノ油、オリーブ油、カヤ油、牛脂、ゴマ油、小麦胚芽油、コメヌカ油、サザンカ油、サフラワー油、スクワラン、大豆油、茶実油、ツバキ油、シナギリ油、タートル油、ナタネ油、トウモロコシ油、胚芽油、パーシック油、ヒマシ油、ホホバ油、日本キリ油、マカデミアナッツ油、ミンク油、綿実油、椰子油、落花生油、卵黄油、カルナウバワックス、トリグリセリン、トリオクタン酸グリセリン、トリイソパルミチン酸グリセリン等が挙げられる。 Natural oils include avocado oil, flaxseed oil, eno oil, olive oil, kayak oil, beef tallow, sesame oil, wheat germ oil, rice bran oil, sasanqua oil, safflower oil, squalane, soybean oil, tea seed oil, camellia oil, Cinnagi oil, turtle oil, rapeseed oil, corn oil, germ oil, persic oil, castor oil, jojoba oil, Japanese kiri oil, macadamia nut oil, mink oil, cottonseed oil, coconut oil, peanut oil, egg yolk oil, carnauba wax, bird Examples include glycerin, glycerin trioctanoate, and glycerin triisopalmitate.
炭化水素油としては、例えば、直鎖状又は分岐状の炭化水素油が挙げられ、揮発性の炭化水素油であっても不揮発性の炭化水素油であってもよい。炭化水素油の具体例としては、合成スクワラン、植物性スクワラン、スクワレン、流動イソパラフィン、軽質イソパラフィン、水添ポリイソブテン、イソドデカン、ステアリン酸、軽質流動イソパラフィン、イソヘキサデカン、流動パラフィン、プリスタン、α−オレフィンオリゴマー、オゾケライト、セレシン、パラフィン、パラフィンワックス、ポリエチレンワックス、ポリエチレン・ポリプロピレンワックス、(エチレン/プロピレン/スチレン)コポリマー、(ブチレン/プロピレン/スチレン)コポリマー、ポリイソブチレン、マイクロクリスタリンワックス、ワセリン等が挙げられる。 Examples of the hydrocarbon oil include linear or branched hydrocarbon oils, which may be volatile hydrocarbon oils or non-volatile hydrocarbon oils. Specific examples of hydrocarbon oils include synthetic squalane, vegetable squalane, squalene, liquid isoparaffin, light isoparaffin, hydrogenated polyisobutene, isododecane, stearic acid, light liquid isoparaffin, isohexadecane, liquid paraffin, pristane, α-olefin oligomer, Examples include ozokerite, ceresin, paraffin, paraffin wax, polyethylene wax, polyethylene / polypropylene wax, (ethylene / propylene / styrene) copolymer, (butylene / propylene / styrene) copolymer, polyisobutylene, microcrystalline wax, petrolatum and the like.
界面活性剤としては、脂肪酸セッケン(ラウリン酸ナトリウム、パルミチン酸ナトリウム等)、ラウリル硫酸カリウム、アルキル硫酸トリエタノールアミンエーテル、スルホコハク酸エステルやポリオキシエチレンアルキル硫酸ナトリウム等のアニオン界面活性剤類、塩化ステアリルトリメチルアンモニウム、塩化ベンザルコニウム、ラウリルアミンオキサイド、ジアルキルアンモニウム塩等のカチオン界面活性剤類、ベタイン系界面活性剤(
アルキルベタイン、アミドベタイン、スルホベタイン等)、イミダゾリン系両性界面活性剤(2−ココイル−2−イミダゾリニウムヒドロキサイド−1−カルボキシエチロキシ2ナトリウム塩等)、アシルメチルタウリン等の両性界面活性剤類、ソルビタン脂肪酸エステル類(ソルビタンモノステアレート、セスキオレイン酸ソルビタン等) 、グリセリン
脂肪酸類(モノステアリン酸グリセリン等)、プロピレングリコール脂肪酸エステル類(モノステアリン酸プロピレングリコール等)、硬化ヒマシ油誘導体、グリセリンアルキルエーテル、POEソルビタン脂肪酸エステル類(POEソルビタンモノオレエート、モノステアリン酸ポリオキシエチレンソルビタン等)、POEソルビット脂肪酸エステル類(POE−ソルビットモノラウレート等)、POEグリセリン脂肪酸エステル類(POE−グリセリンモノイソステアレート等)、POE脂肪酸エステル類(ポリエチレングリコールモノオレート、POEジステアレート等) 、POEアルキルエーテル類(POE2−
オクチルドデシルエーテル等)、POEアルキルフェニルエーテル類(POEノニルフェニルエーテル等)、プルロニック型類、POE・POPアルキルエーテル類(POE・POP2−デシルテトラデシルエーテル等)、テトロニック類、POEヒマシ油・硬化ヒマシ油誘導体(POEヒマシ油、POE硬化ヒマシ油等)、ショ糖脂肪酸エステル、アルキルグルコシド等の非イオン界面活性剤類、等が挙げられる。
Surfactants include fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, alkylsulfuric triethanolamine ether, sulfosuccinic acid esters, polyoxyethylene alkyl sodium sulfate and other anionic surfactants, stearyl chloride Cationic surfactants such as trimethylammonium, benzalkonium chloride, laurylamine oxide, dialkylammonium salts, betaine surfactants (
Amphoteric surfactants such as alkylbetaines, amidebetaines, sulfobetaines, etc.), imidazoline-based amphoteric surfactants (such as 2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt), and acylmethyltaurines , Sorbitan fatty acid esters (such as sorbitan monostearate, sorbitan sesquioleate), glycerin fatty acids (such as glyceryl monostearate), propylene glycol fatty acid esters (such as propylene glycol monostearate), hardened castor oil derivatives, glycerin Alkyl ethers, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbite fatty acid esters (POE-sorbit monolaure) POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE2-
Octyldodecyl ether, etc.), POE alkylphenyl ethers (POE nonylphenyl ether, etc.), Pluronic types, POE / POP alkyl ethers (POE / POP2-decyltetradecyl ether, etc.), Tetronics, POE castor oil / cured Castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), nonionic surfactants such as sucrose fatty acid ester, alkyl glucoside, and the like.
多価アルコールとしては、ポリエチレングリコール、グリセリン、1,3−ブタンジオール、エリスリトール、ソルビトール、キシリトール、マルチトール、プロピレングリコール、ジプロピレングリコール、ジグリセリン、イソプレングリコール、1,2−ペンタンジオール、2,4−ヘキシレングリコール、1,2−ヘキサンジオール、1,2−オクタンジオール等が挙げられる。 Polyhydric alcohols include polyethylene glycol, glycerin, 1,3-butanediol, erythritol, sorbitol, xylitol, maltitol, propylene glycol, dipropylene glycol, diglycerin, isoprene glycol, 1,2-pentanediol, 2,4 -Hexylene glycol, 1,2-hexanediol, 1,2-octanediol and the like.
増粘剤としては、グアガム、クインスシード、カラギーナン、ガラクタン、アラビアガム、ペクチン、マンナン、デンプン、キサンタンガム、カードラン、メチルセルロース、ヒドロキシエチルセルロース、カルボキシメチルセルロース、メチルヒドロキシプロピルセルロース、コンドロイチン硫酸、デルマタン硫酸、グリコーゲン、ヘパラン硫酸、ヒアルロン酸、ヒアルロン酸ナトリウム、トラガントガム、ケラタン硫酸、コンドロイチン、ムコイチン硫酸、ヒドロキシエチルグアガム、カルボキシメチルグアガム、デキストラン、ケラト硫酸、ローカストビーンガム、サクシノグルカン、カロニン酸,キチン、キトサン、カルボキシメチルキチン、寒天、ポリビニルアルコール、ポリビニルピロリドン、カルボキシビニルポリマー、アルキル変性カルボキシビニルポリマー、ポリアクリル酸ナトリウム、ポリエチレングリコール、ベントナイト等が挙げられる。 Thickeners include guar gum, quince seed, carrageenan, galactan, gum arabic, pectin, mannan, starch, xanthan gum, curdlan, methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, methylhydroxypropylcellulose, chondroitin sulfate, dermatan sulfate, glycogen, Heparan sulfate, hyaluronic acid, sodium hyaluronate, tragacanth gum, keratan sulfate, chondroitin, mucoitin sulfate, hydroxyethyl guar gum, carboxymethyl guar gum, dextran, kerato sulfate, locust bean gum, succinoglucan, caronic acid, chitin, chitosan, carboxymethyl Chitin, agar, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, al Le-modified carboxyvinyl polymers, sodium polyacrylate, polyethylene glycol, and bentonite.
粉体類としては、表面を処理されていても良い、マイカ、タルク、カオリン、合成雲母、炭酸カルシウム、炭酸マグネシウム、無水ケイ酸(シリカ)、酸化アルミニウム、硫酸バリウム等の粉体類、表面を処理されていても良い、ベンガラ、黄酸化鉄、黒酸化鉄、酸化コバルト、群青、紺青、酸化チタン、酸化亜鉛の無機顔料類、表面を処理されていても良い、雲母チタン、魚燐箔、オキシ塩化ビスマス等のパール剤類、レーキ化されていても良い赤色202号、赤色228号、赤色226号、黄色4号、青色404号、黄色5号、赤色505号、赤色230号、赤色223号、橙色201号、赤色213号、黄色204号、黄色203号、青色1号、緑色201号、紫色201号、赤色204号等の有機色素類、ポリエチレン末、ポリメタクリル酸メチル、ナイロン粉末、オルガノポリシロキサンエラストマー等の有機粉体類、が挙げられる。 As powders, the surface may be treated, mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, silicic anhydride (silica), aluminum oxide, barium sulfate, etc. May be treated, bengara, yellow iron oxide, black iron oxide, cobalt oxide, ultramarine, bitumen, titanium oxide, zinc oxide inorganic pigments, surface may be treated, titanium mica, fish phosphorus foil, Pearl agent such as bismuth oxychloride, red 202, red 228, red 226, yellow 4, blue 404, yellow 5, red 505, red 230, red 223 which may be raked No., Orange No. 201, Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No. 201, Purple No. 201, Red No. 204, organic dyes, polyethylene powder, polymeta Methyl acrylic acid, nylon powder, organic powders such as organopolysiloxane elastomers, and the like.
紫外線吸収剤としては、パラアミノ安息香酸系紫外線吸収剤、アントラニル酸系紫外線吸収剤、サリチル酸系紫外線吸収剤、桂皮酸系紫外線吸収剤、ベンゾフェノン系紫外線吸収剤、糖系紫外線吸収剤、2−(2'−ヒドロキシ−5'−t−オクチルフェニル)ベンゾ
トリアゾール、4−メトキシ−4'−t−ブチルジベンゾイルメタン等の紫外線吸収剤類
、等が挙げられる。
Examples of the UV absorber include paraaminobenzoic acid UV absorbers, anthranilic acid UV absorbers, salicylic acid UV absorbers, cinnamic acid UV absorbers, benzophenone UV absorbers, sugar UV absorbers, 2- (2 UV absorbers such as'-hydroxy-5'-t-octylphenyl) benzotriazole, 4-methoxy-4'-t-butyldibenzoylmethane, and the like.
また、化粧料として適用される場合の剤型は、通常知られているローション剤形、乳液剤形、エッセンス剤形、クリーム剤形、粉体含有剤形の何れをも取ることが出来る。 Moreover, the dosage form in the case of applying as cosmetics can take any of the conventionally known lotion dosage forms, emulsion dosage forms, essence dosage forms, cream dosage forms, and powder-containing dosage forms.
組成物の製造は、本発明に係るスクリーニング方法により肌改善剤として選択された物質を組成物に配合するステップを含み、常法に従ってこれらの成分を配合することにより、行うことができる。また、肌改善剤として選択された物質は、精製などの処理を行ってから、組成物に配合してもよい。 Manufacture of a composition can be performed by including the step which mix | blends the substance selected as a skin improving agent with the screening method which concerns on this invention in a composition, and mix | blends these components in accordance with a conventional method. Moreover, the substance selected as the skin improving agent may be blended into the composition after undergoing a treatment such as purification.
本発明のスクリーニング方法により肌改善剤として選択される物質を含有する組成物は、肌の透明感、柔らかさの改善の用途に好適に用いることができる。
本発明のスクリーニング方法により肌改善剤と選択された物質は、心理ストレスに起因すると推察されるSP及び/又はNK1Rの形成を抑制すること、さらには、III型コラ
ーゲン産生を阻害する、SPとNK1Rとの結合を拮抗、若しくは遮断し得る作用を有すると考えられ、新しいメカニズムによるものとなりうる。
The composition containing a substance selected as a skin improving agent by the screening method of the present invention can be suitably used for the purpose of improving skin transparency and softness.
The substance selected as a skin improving agent by the screening method of the present invention suppresses the formation of SP and / or NK1R presumed to be caused by psychological stress, and further inhibits the production of type III collagen, SP and NK1R It is thought that it has an action capable of antagonizing or blocking the binding to, and may be due to a new mechanism.
以下、本発明を実施例により更に詳細に説明するが、本発明は、その要旨を超えない限り、以下の実施例に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention still in detail, this invention is not limited to a following example, unless the summary is exceeded.
<実施例1>
正常ヒト真皮線維芽細胞を24穴プレートに5.0×104cells/well播種し
、10%ウシ胎児血清(FBS)含有ダルベッコ改変イーグル培地(DMEM、SIGMA ALDRICH社)にて37℃、5%CO2条件下で48時間培養した後、培地を0.1%ウシ血清アルブミン(BSA)含有DMEMに交換し、さらに24時間培養した。培地交換24時間後、細胞をリン酸緩衝食塩液(PBS)にて洗浄し、製造例1に示す精製ハス胚芽エキスを溶解させた培地に培地交換し、更に6時間培養した。培養終了後、細胞を回収し、RNeasy Mini Kit(QIAGEN社)を用いてトータルRNAを抽出し、得られたトータルRNAからSuperScript VILO cDNA
Synthesis Kit(invitrogen社)を用いてcDNAを合成した。合成したcDNAをテンプレートとしてQuantiFast SYBR Green
PCR Kit(QIAGEN社)を用いてリアルタイムPCRを行い、検量線法によりNK1R mRNA発現量を相対定量した。このとき、18S rRNAを内在性コントロールとし、初期遺伝子量を補正した。
コントロールとしては、被験物質を溶解させた培地に培地交換をせずに培養した以外は上記と同様にして、NK1R mRNA発現量を相対定量した。
被験物質であるハス胚芽エキス添加培養におけるNK1RのmRNA発現量と、被験物質を添加しなかったコントロールの発現量とを比較し、その変化を確認した。また、市販のハス胚芽エキス(ハス胚芽エキスパウダーMF 丸善製薬製)についても同様にNK1RのmRNA発現量を測定した。結果を表1に示す。
<Example 1>
Normal human dermal fibroblasts were seeded in a 24-well plate at 5.0 × 10 4 cells / well, and 10% fetal bovine serum (FBS) -containing Dulbecco's modified Eagle medium (DMEM, SIGMA ALDRICH) at 37 ° C., 5% After culturing for 48 hours under CO 2 conditions, the medium was replaced with 0.1% bovine serum albumin (BSA) -containing DMEM and further cultured for 24 hours. After 24 hours of medium replacement, the cells were washed with phosphate buffered saline (PBS), replaced with a medium in which the purified lotus germ extract shown in Production Example 1 was dissolved, and further cultured for 6 hours. After completion of the culture, the cells are collected, total RNA is extracted using RNeasy Mini Kit (QIAGEN), and SuperScript VILO cDNA is obtained from the obtained total RNA.
CDNA was synthesized using Synthesis Kit (Invitrogen). QuantFast SYBR Green using the synthesized cDNA as a template
Real-time PCR was performed using PCR Kit (QIAGEN), and the expression level of NK1R mRNA was relatively quantified by a calibration curve method. At this time, 18S rRNA was used as an endogenous control, and the initial gene dosage was corrected.
As a control, the expression level of NK1R mRNA was relatively quantified in the same manner as described above, except that the culture was performed without replacing the medium in a medium in which the test substance was dissolved.
The expression level of NK1R mRNA in the culture containing a lotus germ extract as a test substance was compared with the expression level of a control to which no test substance was added, and the change was confirmed. Further, the mRNA expression level of NK1R was measured in the same manner for a commercially available lotus germ extract (lotus germ extract powder MF Maruzen Pharmaceutical). The results are shown in Table 1.
<製造例1: 精製ハス胚芽エキス>
以下の手順に従い、精製ハス胚芽エキスを調製した。即ち、ハス胚芽エキスをメタノールにて抽出し、ろ過後、溶媒を留去した。酢酸エチルと水を同量ずつ加え、液体−液体分配を実施後、水層のみを取り出し、ダイヤイオンHP20(三菱化学株式会社製)にて、非吸着画分を採取し、精製ハス胚芽エキスとした。
<Production Example 1: Purified lotus germ extract>
Purified lotus germ extract was prepared according to the following procedure. That is, the lotus germ extract was extracted with methanol, and after filtration, the solvent was distilled off. Add the same amount of ethyl acetate and water, perform liquid-liquid partitioning, take out only the aqueous layer, collect the non-adsorbed fraction with Diaion HP20 (manufactured by Mitsubishi Chemical Corporation), and extract the purified lotus germ extract and did.
表1の結果より、精製ハス胚芽エキス及び市販のハス胚芽エキスのNKIR量はコントロールに対し、それぞれ56.4%、72.6%であり、2つのハス胚芽エキスはNK1RのmRNA発現量を80%以下に抑制し、特に、精製ハス胚芽エキスは市販のハス胚芽エキスより高いNK1RのmRNA発現抑制効果を有することがわかる。
上記の結果より、これらのハス胚芽エキスを肌改善剤として選択し、以下の試験例に使用した。
From the results in Table 1, the NKIR levels of the purified lotus germ extract and the commercially available lotus germ extract are 56.4% and 72.6%, respectively, and the two lotus germ extracts have an NK1R mRNA expression level of 80%. In particular, it can be seen that the purified lotus germ extract has a higher NK1R mRNA expression inhibitory effect than the commercially available lotus germ extract.
From the above results, these lotus germ extracts were selected as skin improvers and used in the following test examples.
<実施例2>
正常ヒト真皮線維芽細胞を24穴プレートに5.0×104cells/well播種し
、10%FBS含有DMEM培地にて37℃、5%CO2条件下で培養した後、培地を0.1%ウシBSA含有DMEMに交換し、さらに培養した。培地交換24時間後、SPのみを添加(ネガティブコントロール)、あるいは、SPおよびハス胚芽エキスを添加し、さらに6時間培養した。培養終了後、培養上清中のIII型コラーゲン量をELISA法に
より確認した(α、α1)。
コントロールとしては、SPおよびハス胚芽エキスを添加せずに培養した以外は上記と同様にして、III型コラーゲン量を確認した。コントロール(α0)を1としてそれぞれ
の値を表した結果を表2に示す。
この結果は、SP添加による真皮線維芽細胞中のIII型コラーゲン量の減少を、ハス胚
芽エキスが抑制しており、ハス胚芽エキスが優れた肌改善作用を有する肌改善剤となることを示している。特に精製ハス胚芽エキスは市販のハス胚芽エキスより顕著な効果を示した。
真皮中に存在するコラーゲンの1種であるIII型コラーゲンは、I型コラーゲンと比較
して、ゲルにした場合の透明性及び柔軟性が高い。心理的ストレスにより肌状態が悪化するような状況においては特に、本発明のスクリーニング方法により肌改善剤として選択された物質を含有する化粧料を使用することにより、III型コラーゲンが増加し、透明感、
柔らかさのある肌への改善効果が期待できる。
<Example 2>
Normal human dermal fibroblasts were seeded in a 24-well plate at 5.0 × 10 4 cells / well, cultured in DMEM medium containing 10% FBS under conditions of 37 ° C. and 5% CO 2. The medium was replaced with DMEM containing% bovine BSA and further cultured. After 24 hours of medium exchange, only SP was added (negative control), or SP and lotus germ extract were added, and further cultured for 6 hours. After completion of the culture, the amount of type III collagen in the culture supernatant was confirmed by ELISA (α, α 1 ).
As a control, the amount of type III collagen was confirmed in the same manner as described above except that the cells were cultured without adding SP and lotus germ extract. Table 2 shows the results of representing each value with control (α 0 ) being 1.
This result shows that the lotus germ extract suppresses the decrease in the amount of type III collagen in the dermal fibroblasts due to the addition of SP, and the lotus germ extract becomes a skin improvement agent having an excellent skin improving action. Yes. In particular, the purified lotus germ extract showed a remarkable effect over the commercially available lotus germ extract.
Type III collagen, which is one type of collagen present in the dermis, is more transparent and flexible when gelled than type I collagen. Especially in the situation where the skin condition deteriorates due to psychological stress, the use of a cosmetic containing a substance selected as a skin improving agent by the screening method of the present invention increases the type III collagen, resulting in transparency. ,
Expected to improve soft skin.
<製造例2: 本発明の肌改善剤を含有する皮膚外用剤の製造(ローション)>
以下の手順に従い、本発明の肌改善剤のスクリーニング方法により選択された肌改善剤を含有する組成物(皮膚外用剤)を調製した。
即ち、表3の処方成分(B)を50℃にて加熱溶解し、処方成分(A)を撹拌しながら、加熱溶解した(B)を添加し、本発明の組成物(皮膚外用剤1)を得た。
<Manufacture example 2: Manufacture (lotion) of the external preparation for skin containing the skin improvement agent of this invention>
In accordance with the following procedure, a composition (skin external preparation) containing the skin improving agent selected by the screening method for the skin improving agent of the present invention was prepared.
That is, the formulation component (B) in Table 3 was dissolved by heating at 50 ° C., and the formulation component (A) was heated and dissolved while stirring, and the composition of the present invention (skin external preparation 1) Got.
<製造例3: 本発明の肌改善剤を含有する皮膚外用剤の製造(クリーム)>
以下の手順に従い、本発明の肌改善剤のスクリーニング方法により選択された肌改善剤を含有する組成物(皮膚外用剤)を調製した。
即ち、表4の処方成分(A)及び(B)を70℃にて、それぞれ加熱溶解し、処方成分(A)に(B)を撹拌しながら加え、室温まで冷却し、本発明の組成物(皮膚外用剤2)を得た。
<Manufacture example 3: Manufacture (cream) of the skin external preparation containing the skin improving agent of this invention>
In accordance with the following procedure, a composition (skin external preparation) containing the skin improving agent selected by the screening method for the skin improving agent of the present invention was prepared.
That is, the formulation components (A) and (B) in Table 4 were each dissolved by heating at 70 ° C., and (B) was added to the formulation component (A) with stirring, and the mixture was cooled to room temperature. (Skin external preparation 2) was obtained.
<試験例1:肌改善の評価>
上記実施例1、2より選択された肌改善剤を含む皮膚外用剤1(ローション)を、製造例1に従って調製した。
平均年齢42.5歳の被験者17名(女性、年齢層36〜49歳)に、皮膚外用剤1を1日2回、連日3か月間使用してもらい、塗布前、3か月間塗布後の肌の明るさ、黄味、弾力及び肌の柔らかさをそれぞれ測定した。
<Test Example 1: Evaluation of skin improvement>
A skin external preparation 1 (lotion) containing a skin improving agent selected from Examples 1 and 2 was prepared according to Production Example 1.
17 subjects (female, age group 36-49 years) with an average age of 42.5 years used skin external preparation 1 twice a day for 3 consecutive days, before application, after application for 3 months Skin brightness, yellowness, elasticity and skin softness were measured respectively.
<試験例1-1:肌の透明感の評価>
頬部を色彩色差計(コニカミノルタ株式会社社製 CR-400)にて、肌の明るさL*
とくすみに関連する黄味b*を3回測定し、平均値を取得し、肌の透明感の評価とした。結果を図1、2に示す。図1、2から、本発明のスクリーニング方法により選択された肌改善剤含有ローションを3か月間塗布後には、肌の明度が高くなり、黄味が顕著に低下したことがわかる。これらの結果は本発明のスクリーニング方法により選択された肌改善剤含有ローションを用いて、肌の透明感が改善されたことを示すものである。
<Test Example 1-1: Evaluation of skin transparency>
Skin cheeks with color difference meter (CR-400 manufactured by Konica Minolta Co., Ltd.), skin brightness L *
Yellowness b * related to dullness was measured three times, an average value was obtained, and the skin transparency was evaluated. The results are shown in FIGS. 1 and 2, it can be seen that after applying the skin-improving agent-containing lotion selected by the screening method of the present invention for 3 months, the lightness of the skin was increased and the yellowness was significantly reduced. These results indicate that the skin transparency was improved using the skin-improving agent-containing lotion selected by the screening method of the present invention.
<試験例1-2:肌の弾力の評価>
肌の弾力としては、頬部の弾力を皮膚粘弾性測定装置(Courage+Khazaka社製 cutometer MPA580)を用いて測定した。吸引圧400mbarにて2秒間吸引、2秒間開放の1サイクルで得られた波形データから、戻り率(Ur/Uf:メーカパラメータR7)を取得した。結果を図3に示す。Ur/Ufは、弾力性部分を完全な波形(弾力性100%)と比較した値であって、この値が高い程、肌の弾力が高いことを示す。図3から、本発明のスクリーニング方法により選択された肌改善剤含有ローションを3か月間塗布後には肌の弾力が改善されたことがわかる。
<Test Example 1-2: Evaluation of skin elasticity>
As the elasticity of the skin, the elasticity of the cheeks was measured using a skin viscoelasticity measuring apparatus (Courage + Khazaka cutometer MPA580). The return rate (Ur / Uf: manufacturer parameter R7) was obtained from the waveform data obtained in one cycle of suction for 2 seconds at a suction pressure of 400 mbar and release for 2 seconds. The results are shown in FIG. Ur / Uf is a value obtained by comparing the elastic portion with a complete waveform (elasticity 100%), and the higher this value, the higher the elasticity of the skin. FIG. 3 shows that the skin elasticity was improved after applying the skin-improving agent-containing lotion selected by the screening method of the present invention for 3 months.
<試験例1-3:肌の柔らかさの評価>
肌の柔らかさとして、頬部を触覚センサシステム(株式会社アクシム製 ビーナストロン)にて、往路の2g、5g荷重時の周波数変化(Δf)を測定した。結果を図4、5に示す。縦軸の周波数変化Δf[Hz]は、値が大きいほど、肌が柔らかいということを示している。図4、5から、本発明のスクリーニング方法により選択された肌改善剤含有ローションを3か月間塗布後には、2g荷重時、5g荷重時それぞれにおいて、Δfは塗布前に比較して高くなり、肌の柔らかさが改善されたことがわかる。
<Test Example 1-3: Evaluation of skin softness>
As the softness of the skin, the change in frequency (Δf) at the time of 2 g and 5 g loads on the outward path was measured using a tactile sensor system (Venustron manufactured by Axim Co., Ltd.). The results are shown in FIGS. The frequency change Δf [Hz] on the vertical axis indicates that the larger the value, the softer the skin. 4 and 5, after applying the skin-improving agent-containing lotion selected by the screening method of the present invention for 3 months, Δf is higher at 2 g load and at 5 g load than before application, It can be seen that the softness of is improved.
本発明のスクリーニング法により、新たな作用機序に基づく肌の透明感、柔らかさを改善する肌改善剤として有効な成分を探索することができる、スクリーニング方法が提供され、また、本発明により新たなアプローチによる化粧料が提供されるため、産業上非常に有用である。 The screening method of the present invention provides a screening method capable of searching for an effective component as a skin improving agent for improving skin transparency and softness based on a new mechanism of action. This is very useful in the industry because cosmetics are provided by a simple approach.
Claims (4)
被験物質を添加しない真皮線維芽細胞のNK1Rの発現量(コントロール)、及び被験物質を添加して培養した真皮線維芽細胞のNK1Rの発現量を得る測定ステップ、前記ステップで得られたNK1Rの発現量を比較し、コントロールと比較してNK1Rの発現量が減少した被験物質を肌改善剤として選択する選択ステップ、を含む、スクリーニング方法。 A screening method for a skin improving agent that improves skin transparency and softness using the expression level of NK1R in dermal fibroblasts as an index,
Expression of NK1R dermal fibroblasts without the addition of test substance (control), and measuring step of obtaining expression of NK1R dermal fibroblasts cultured by adding the test substance, expression of NK1R obtained in the step A selection method comprising comparing a quantity and selecting a test substance having a reduced expression level of NK1R as a skin improving agent as compared with a control.
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