JP2022159441A - Skin condition change inhibitor and screening method therefor - Google Patents
Skin condition change inhibitor and screening method therefor Download PDFInfo
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- JP2022159441A JP2022159441A JP2022129725A JP2022129725A JP2022159441A JP 2022159441 A JP2022159441 A JP 2022159441A JP 2022129725 A JP2022129725 A JP 2022129725A JP 2022129725 A JP2022129725 A JP 2022129725A JP 2022159441 A JP2022159441 A JP 2022159441A
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Abstract
Description
本発明は、日々変化する肌状態を安定に保つための皮膚外用剤の有効成分、及びそのスクリーニング方法に関する。 TECHNICAL FIELD The present invention relates to an active ingredient of an external skin preparation for stabilizing the skin condition, which changes daily, and a screening method therefor.
美しい肌、健康的な肌に対する需要は高い。人の肌状態は年単位で大きく変化していくため、加齢により増加する、シミ、シワなどに対する原因や対策等のスキンケアに関する種々の情報はメディアから多く提供され、化粧品、健康食品、美容家電、エステティックなど、様々なスキンケア市場が存在する。また、思春期から若齢成人にかけて悩みの多い、ニキビ、肌荒れに対するスキンケアも多く提案されている。
一方、ある日にはごわついていた肌の触感が翌日にはすべすべだと感じる、週の途中でニキビが現れてまた消える、メイクのりの良い日と悪い日があるなど、程度の差こそあれ日常生活において肌状態の良い日と悪い日が存在することを多くの人が感じている。短期的な肌状態の変化に関しては、多くの人が日常的に好不調を気にしているにも関わらず、美容方法や生活習慣の改善方法についての情報が提供されるのみで、有効な解決手段が存在していなかった。
There is a high demand for beautiful and healthy skin. Since people's skin condition changes greatly from year to year, various information on skin care such as causes and countermeasures for age spots, wrinkles, etc., which increase with age, is provided by the media, and is widely used in cosmetics, health foods, and beauty appliances. There are various skin care markets such as , esthetics, etc. In addition, there are many proposals for skin care for pimples and rough skin, which are common concerns from puberty to young adults.
On the other hand, the texture of the skin that was stiff one day feels smooth the next day, pimples appear and disappear again in the middle of the week, there are days when makeup is good and days when makeup is good, and so on. Many people feel that there are good days and bad days in their lives. With regard to short-term changes in skin condition, although many people are concerned about their condition on a daily basis, simply providing information on beauty methods and ways to improve lifestyle habits is an effective solution. the means did not exist.
日間で変動する肌状態としては、乾燥感、しっとり感、ゴワツキ、凹凸感などが挙げられる。これらは皮膚再外層に存在する角層の、水分蒸散を抑制するバリア機能、天然保湿因子や油分の放出、剥離状態や層数、過角化による毛穴や脂腺の詰まりなどに大きく影響される肌状態であることが推測されている。
これらの肌状態の日間での変動は、一般に、仕事の忙しさや子育てなど私生活の負荷が比較的長期的に続いている状態で、さらにストレスを感じた後に実感することが多い。
Examples of skin conditions that change from day to day include dryness, moistness, stiffness, unevenness, and the like. These are greatly affected by the barrier function of the stratum corneum, which exists in the outermost layer of the skin, to suppress water evaporation, the release of natural moisturizing factors and oil, the peeling state and number of layers, and the clogging of pores and sebaceous glands due to hyperkeratosis. It is presumed that it is a skin condition.
These day-to-day variations in skin condition are generally felt after feeling stress in a state where the burden of personal life, such as busy work and child-rearing, continues for a relatively long period of time.
ストレスを感じた際には、全身性ホルモンであるコルチゾールが増加することが知られている(非特許文献1)。コルチゾールは、表皮角化細胞に対する過剰な角化を誘導する作用(非特許文献2)や表皮角化細胞のヒアルロン酸合成を抑制する作用(非特許文献3)を有することが知られていて、その作用を緩和して肌状態を緩和させることが提案されている(特許文献1)。一方で、コルチゾールは抗炎症作用を有することも知られており、肌荒れを抑制するために皮膚外用剤に配合されてきた。
また、コルチゾールは、細胞内で11βヒドロキシステロイドデヒドロゲナーゼタイプ2(11β-HSD2)により不活性型のコルチゾンに変換されることで、その影響が一過性のものとなるが、11βヒドロキシステロイドデヒドロゲナーゼタイプ1(11β-HSD1)により、再活性化されコルチゾールとなることで、局所での濃度が高まることが知られている(非特許文献8)。
また、長期的に負荷が続いている人においては、全身性の炎症性サイトカインであるTNFαやIL1-βが増加することが知られている(非特許文献4、非特許文献5)。これらの炎症性サイトカインは、表皮角化細胞の11β-HSD1の遺伝子(HSD11B1)の発現を増加させることが知られている(非特許文献6、非特許文献7)。さらには、11β-HSD1活性が抑制されると、表皮が角化ではなく増殖方向に変化したり(非特許文献9)、脂腺細胞の脂質合成が抑えられたりする(非特許文献10)ことも報告されている。
It is known that cortisol, which is a systemic hormone, increases when stress is felt (Non-Patent Document 1). Cortisol is known to have the effect of inducing excessive keratinization of epidermal keratinocytes (Non-Patent Document 2) and the effect of suppressing hyaluronic acid synthesis of epidermal keratinocytes (Non-Patent Document 3). It has been proposed to alleviate the effects of these agents to alleviate skin conditions (Patent Document 1). On the other hand, cortisol is also known to have an anti-inflammatory effect, and has been incorporated into external preparations for skin to suppress rough skin.
In addition, cortisol is converted into inactive cortisone by 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) in cells, resulting in a transient effect, but 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is known to reactivate cortisol and increase its local concentration (Non-Patent Document 8).
In addition, it is known that TNFα and IL1-β, which are systemic inflammatory cytokines, are increased in people who are under long-term stress (Non-Patent
本発明は、肌状態の日間変動を抑制する成分をスクリーニングする方法を提供することを課題とする。また、肌状態変動抑制用の組成物を提供することを課題とする。 An object of the present invention is to provide a method of screening for an ingredient that suppresses daily variations in skin condition. Another object of the present invention is to provide a composition for suppressing skin condition fluctuations.
本発明者らは、ストレス等で日間変動する肌状態を改善するために鋭意研究したところ、TNFαやIL1-β存在下で表皮角化細胞における11β-HSD1活性を抑制することにより、コルチゾールの再活性化を抑制することができ、ストレスの影響を一過性に留めることができることを見出した。コルチゾールは全身性のホルモンで、従来局所での不活性化又は再活性化に着目した例はなかった。これらの知見に基づき、TNFα又はIL1-βの存在下で表皮角化細胞における11β-HSD1活性を抑制し得る成分が、肌状態の日間変動を抑制する成分となり得ることに想到し、本発明を完成するに至った。 The present inventors have conducted intensive research to improve skin conditions that fluctuate daily due to stress, etc., and have found that cortisol reactivation can be achieved by suppressing 11β-HSD1 activity in epidermal keratinocytes in the presence of TNFα and IL1-β. It was found that the activation can be suppressed and the effects of stress can be limited to a temporary effect. Cortisol is a systemic hormone, and so far there have been no studies focused on its local inactivation or reactivation. Based on these findings, it was conceived that a component capable of suppressing 11β-HSD1 activity in epidermal keratinocytes in the presence of TNFα or IL1-β could be a component that suppresses daily fluctuations in skin condition, and the present invention was developed. Completed.
すなわち、本発明は以下の通りである。
[1]TNFα又はIL1-βの存在下での表皮角化細胞における11βヒドロキシステロイドデヒドロゲナーゼタイプ1(11β-HSD1)の活性を指標とする、肌状態変動抑制剤のスクリーニング方法。
[2]前記11β-HSD1の活性が、11β-HSD1をコードする遺伝子(HSD11B1)の発現量であり、
TNFα又はIL1-βの存在下で被験物質を表皮角化細胞に添加する工程、及び
被験物質を添加した表皮角化細胞におけるHSD11B1の発現量が、TNFα又はIL1-βの存在下で被験物質を添加しなかった表皮角化細胞におけるHSD11B1の発現量と比較して小さくなる被験物質を選択する工程、
を含む、[1]に記載のスクリーニング方法。
[3]HSD11B1の発現量が20%以上小さくなる被験物質を選択する、[2]に記載のスクリーニング方法。
[4]さらに、TNFα又はIL1-βの存在下での表皮角化細胞における11βヒドロ
キシステロイドデヒドロゲナーゼタイプ2(11β-HSD2)の活性を指標とする、[1]~[3]のいずれかに記載のスクリーニング方法。
[5]前記11β-HSD1の活性が、11β-HSD1をコードする遺伝子(HSD11B1)の発現量であり、
前記11β-HSD2の活性が、11β-HSD2をコードする遺伝子(HSD11B2)の発現量であり、
TNFα又はIL1-βの存在下で被験物質を表皮角化細胞に添加する工程、及び
被験物質を添加した表皮角化細胞におけるHSD11B1の発現量が、TNFα又はIL1-βの存在下で被験物質を添加しなかった表皮角化細胞におけるHSD11B1の発現量と比較して小さくなり、かつ
被験物質を添加した表皮角化細胞におけるHSD11B2の発現量が、TNFα又はIL1-βの存在下で被験物質を添加しなかった表皮角化細胞におけるHSD11B2の発現量と比較して変化しない被験物質、を選択する工程、
含む、[4]に記載のスクリーニング方法。
[6]HSD11B1の発現量が20%以上小さくなり、かつ
HSD11B2の発現量が20%以上小さくならない被験物質を選択する、[5]に記載のスクリーニング方法。
[7][1]~[6]のいずれかに記載のスクリーニング方法を行う工程、及び
前記工程により選択された物質を含有させる工程、
を含む、組成物の設計方法。
[8]前記組成物が、肌状態変動抑制用の皮膚外用剤である、[7]に記載の設計方法。[9]アサイヤシ果実抽出物、加水分解エラスチン、加水分解ゴマタンパク、シュードアルテロモナス発酵物抽出物、タモギタケ抽出物、チョウセンゴミシ果実抽出物からなる群より選択される1種または2種以上を含有する、肌状態変動抑制用組成物。
[10]シュードアルテロモナス発酵物抽出物を含有する、[9]に記載の組成物。
[11]さらにセンブリ抽出物を含有する、[10]に記載の組成物。
[12]皮膚外用剤である、[9]~[11]のいずれかに記載の組成物。
[13]化粧料または医薬部外品である、[12]に記載の組成物。
That is, the present invention is as follows.
[1] A screening method for a skin condition fluctuation inhibitor, wherein the activity of 11β hydroxysteroid dehydrogenase type 1 (11β-HSD1) in epidermal keratinocytes in the presence of TNFα or IL1-β is used as an indicator.
[2] the activity of 11β-HSD1 is the expression level of the gene (HSD11B1) encoding 11β-HSD1;
A step of adding a test substance to epidermal keratinocytes in the presence of TNFα or IL1-β; selecting a test substance that reduces the expression level of HSD11B1 in the epidermal keratinocytes that were not added;
The screening method according to [1], comprising
[3] The screening method of [2], wherein a test substance that reduces the expression level of HSD11B1 by 20% or more is selected.
[4] Any one of [1] to [3], wherein the activity of 11β hydroxysteroid dehydrogenase type 2 (11β-HSD2) in epidermal keratinocytes in the presence of TNFα or IL1-β is used as an indicator. screening method.
[5] the activity of 11β-HSD1 is the expression level of the gene (HSD11B1) encoding 11β-HSD1;
The activity of 11β-HSD2 is the expression level of the gene (HSD11B2) encoding 11β-HSD2,
A step of adding a test substance to epidermal keratinocytes in the presence of TNFα or IL1-β; The expression level of HSD11B1 in epidermal keratinocytes to which no test substance was added is lower than the expression level of HSD11B1, and the expression level of HSD11B2 in epidermal keratinocytes to which the test substance was added decreased in the presence of TNFα or IL1-β when the test substance was added. selecting a test substance that does not change in comparison with the expression level of HSD11B2 in epidermal keratinocytes without
The screening method of [4], comprising
[6] The screening method of [5], wherein the test substance is selected that reduces the expression level of HSD11B1 by 20% or more and does not reduce the expression level of HSD11B2 by 20% or more.
[7] The step of performing the screening method according to any one of [1] to [6], and the step of containing the substance selected by the above steps,
A method of designing a composition, comprising:
[8] The designing method according to [7], wherein the composition is an external skin preparation for suppressing skin condition fluctuations. [9] Contains one or more selected from the group consisting of acai palm fruit extract, hydrolyzed elastin, hydrolyzed sesame protein, pseudoalteromonas fermented product extract, oyster mushroom extract, and schizandra fruit extract A composition for suppressing skin condition fluctuations.
[10] The composition according to [9], which contains a Pseudoalteromonas ferment extract.
[11] The composition of [10], further comprising a japonica japonicum extract.
[12] The composition according to any one of [9] to [11], which is an external preparation for skin.
[13] The composition of [12], which is a cosmetic or quasi-drug.
本発明により、表皮角化細胞における11β-HSD1の活性を指標とする、肌状態変動抑制剤のスクリーニング方法が提供される。また、本発明により、肌状態の日間変動の抑制に有効な組成物が提供される。 INDUSTRIAL APPLICABILITY The present invention provides a screening method for a skin condition fluctuation inhibitor, using the activity of 11β-HSD1 in epidermal keratinocytes as an indicator. In addition, the present invention provides a composition that is effective in suppressing daily fluctuations in skin condition.
本発明のスクリーニング方法は、TNFα又はIL1-βの存在下での表皮角化細胞(ケラチノサイト)における11βヒドロキシステロイドデヒドロゲナーゼタイプ1(11
β-HSD1)の活性を指標とする。11β-HSD1は、細胞内で不活性化されたコルチゾール(コルチゾン)を、再活性化する酵素である。
本発明のスクリーニング方法は、さらに、TNFα又はIL1-βの存在下での表皮角化細胞における11βヒドロキシステロイドデヒドロゲナーゼタイプ2(11β-HSD2)の活性をも指標とすることが好ましい。11β-HSD2は、細胞内でコルチゾールをコルチゾンに変換することで不活性化する酵素である。
The screening method of the present invention is a 11β hydroxysteroid dehydrogenase type 1 (11
The activity of β-HSD1) is used as an index. 11β-HSD1 is an enzyme that reactivates intracellularly inactivated cortisol (cortisone).
The screening method of the present invention preferably also uses the activity of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) in epidermal keratinocytes in the presence of TNFα or IL1-β as an indicator. 11β-HSD2 is an enzyme that inactivates cortisol by converting it to cortisone inside cells.
本発明の方法では、ストレス状態(異常時)において増加するTNFαやIL1-βの存在下でスクリーニングを行うことにより、通常はこれらのサイトカインにより増加する11β-HSD1の活性を抑制する、すなわち平常時に戻す物質を探索することができる。なお、11β-HSD2は、該サイトカイン存在下では特に変動しないことが、本発明者らにより分かっている。
本発明の方法により選択される物質は、ストレス状態で一時的に増加したコルチゾールが、表皮角化細胞で役割を終えてコルチゾンに変換された後に、11β-HSD1により再活性化して持続的にコルチゾールが維持されるのを抑制する作用を有するものである。コルチゾール量を抑えることにより、ストレスの肌状態への影響を一過性にとどめることができる。また、本発明の方法により選択される物質は、好ましくは11β-HSD2の活性には影響を与えない、具体的にはコルチゾールの不活性化を抑制しない作用を有するものである。
In the method of the present invention, screening is performed in the presence of TNFα and IL1-β, which increase under stress (abnormal state), thereby suppressing the activity of 11β-HSD1, which is normally increased by these cytokines. You can search for substances to return. The present inventors have found that 11β-HSD2 does not change in the presence of the cytokine.
The substance selected by the method of the present invention is one in which cortisol, which temporarily increases under stress, finishes its role in epidermal keratinocytes and is converted to cortisone, and then reactivated by 11β-HSD1 to sustain cortisol. It has the effect of suppressing the maintenance of By suppressing the amount of cortisol, the effects of stress on the skin condition can be limited to a temporary period. In addition, the substance selected by the method of the present invention preferably does not affect the activity of 11β-HSD2, specifically, does not suppress the inactivation of cortisol.
なお、11β-HSD1及び11β-HSD2のアミノ酸配列、並びにこれらをコードする遺伝子のDNA塩基配列は公知であり、GenBank等に開示されている。 The amino acid sequences of 11β-HSD1 and 11β-HSD2 and the DNA sequences of genes encoding them are known and disclosed in GenBank and the like.
本発明において、前記酵素(11β-HSD1及び11β-HSD2)の活性とは、好ましくはこれらの酵素を構成するタンパク質をコードする遺伝子又は前記タンパク質の発現量により表される。
あるいは、これらの酵素の反応活性の測定値であってもよく、例えば11β-HSD1活性についてはコルチゾンをコルチゾールに変換する活性を、11β-HSD2活性についてはコルチゾールをコルチゾンに変換する活性を、それぞれ任意の方法で測定した値であってもよい。
In the present invention, the activity of the enzymes (11β-HSD1 and 11β-HSD2) is preferably represented by genes encoding proteins constituting these enzymes or expression levels of the proteins.
Alternatively, it may be a measurement value of the reaction activity of these enzymes, for example, the activity of converting cortisone to cortisol for 11β-HSD1 activity, and the activity of converting cortisol to cortisone for 11β-HSD2 activity. It may be a value measured by the method of
本発明のスクリーニング方法は、通常は、TNFα又はIL1-βの存在下で被験物質を表皮角化細胞に添加する工程、及び被験物質を添加した表皮角化細胞におけるHSD11B1の発現量が、TNFα又はIL1-βの存在下で被験物質を添加しなかった表皮角化細胞におけるHSD11B1の発現量と比較して小さくなる被験物質を選択する工程を含む。また、好ましくは、被験物質を添加した表皮角化細胞におけるHSD11B2の発現量が、TNFα又はIL1-βの存在下で被験物質を添加しなかった表皮角化細胞におけるHSD11B2の発現量と比較して変化しない被験物質を選択する工程を含む。
本発明においては、HSD11B1遺伝子の発現抑制と肌状態変動抑制とが相関する。そのため、被験物質を細胞に添加することで、HSD11B1遺伝子の発現抑制が生じた被験物質は、肌状態変動抑制作用を有する成分として選択できる。
また、HSD11B2遺伝子の発現抑制と肌状態変動亢進とが相関する。そのため、被験物質を細胞に添加することで、HSD11B2遺伝子の発現抑制が生じた被験物質は、肌状態変動抑制作用を有する成分として選択することを控えた方がよい。
The screening method of the present invention usually comprises a step of adding a test substance to epidermal keratinocytes in the presence of TNFα or IL1-β, and the expression level of HSD11B1 in the epidermal keratinocytes to which the test substance was added is determined by TNFα or A step of selecting a test substance that reduces the HSD11B1 expression level in the presence of IL1-β compared to epidermal keratinocytes to which no test substance was added. Also, preferably, the expression level of HSD11B2 in epidermal keratinocytes to which the test substance was added is compared with the expression level of HSD11B2 in epidermal keratinocytes to which no test substance was added in the presence of TNFα or IL1-β. A step of selecting test substances that do not change is included.
In the present invention, suppression of HSD11B1 gene expression correlates with suppression of skin condition fluctuations. Therefore, a test substance that suppresses the expression of the HSD11B1 gene by adding the test substance to cells can be selected as a component that has an effect of suppressing skin condition fluctuations.
In addition, suppression of HSD11B2 gene expression correlates with increased variation in skin condition. Therefore, it is better to refrain from selecting a test substance that suppresses the expression of the HSD11B2 gene by adding the test substance to the cells as a component that suppresses skin condition fluctuations.
本発明のスクリーニング方法において被験物質を表皮角化細胞に添加する工程を行う際のTNFα又はIL1-βの存在量は、1~100ng/mLとすることが好ましい。これは、スクリーニング系としてストレス状態(異常時)を模倣するための条件である。なお、非ストレス状態(通常時)のTNFα又はIL1-βの存在量は、一般に10pg/mL未満である。 In the screening method of the present invention, the amount of TNFα or IL1-β present in the step of adding a test substance to epidermal keratinocytes is preferably 1 to 100 ng/mL. This is a condition for imitating a stress state (abnormal state) as a screening system. The amount of TNFα or IL1-β present in a non-stressed state (normal time) is generally less than 10 pg/mL.
本発明のスクリーニング方法において、選択される被験物質は、被験物質を添加した表皮角化細胞におけるHSD11B1の発現量が、TNFα又はIL1-βの存在下で被験物質を添加しなかった表皮角化細胞におけるHSD11B1の発現量と比較して小さければよく、好ましくは被験物質添加後12または24時間経過後に20%以上小さく、より好ましくは30%以上小さく、さらに好ましくは40%以上小さいものである。この範囲の発現減少であれば、表現形にも影響を与えることができ、かかる減少を引き起こす物質は十分に肌状態変動抑制効果を示し得ると考えられる。
また、本発明のスクリーニング方法において、選択される被験物質は、被験物質を添加した表皮角化細胞におけるHSD11B2の発現量が、TNFα又はIL1-βの存在下で被験物質を添加しなかった表皮角化細胞におけるHSD11B2の発現量と比較して変化しないものが好ましく、より好ましくは被験物質添加後12または24時間経過後に20%以上小さくならない、より好ましくは15%以上小さくならない、さらに好ましくは10%以上小さくならないものである。また、HSD11B2の発現量は、被験物質非添加の場合より大きくなってもよいが(発現亢進)、好ましくは20%以上大きくならない、より好ましくは15%以上大きくならない、さらに好ましくは10%以上大きくならないものである。この範囲の発現変化であれば、コルチゾールの不活性化に与える影響が緩和なものとなるので、HSD11B1遺伝子の発現抑制効果を打ち消すことがないと考えられる。
In the screening method of the present invention, the test substance to be selected is that the expression level of HSD11B1 in the epidermal keratinocytes to which the test substance was added is equal to that of the epidermal keratinocytes to which the test substance was not added in the presence of TNFα or IL1-β. preferably 20% or more, more preferably 30% or more, still more preferably 40% or more 12 or 24 hours after addition of the test substance. A decrease in expression within this range can also affect phenotypes, and substances that cause such a decrease are considered to be capable of sufficiently exhibiting an effect of suppressing skin condition fluctuations.
In addition, in the screening method of the present invention, the test substance to be selected is such that the expression level of HSD11B2 in the epidermal keratinocytes to which the test substance has been added is equal to It is preferable that the expression level of HSD11B2 does not change in comparison with the expression level of HSD11B2 in transformed cells, more preferably it does not decrease by 20% or more 12 or 24 hours after the addition of the test substance, more preferably it does not decrease by 15% or more, still more preferably 10%. It should not be smaller than this. In addition, the expression level of HSD11B2 may be greater than when the test substance is not added (enhanced expression), but preferably not greater than 20%, more preferably not greater than 15%, still more preferably not greater than 10%. It should not be. If the change in expression falls within this range, the effect on the inactivation of cortisol is moderated, so it is considered that the effect of suppressing the expression of the HSD11B1 gene is not canceled.
HSD11B1遺伝子又はHSD11B2遺伝子の発現量は、任意の方法を用いて測定することができる。例えば、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、既知の複数のメラノサイト活性化因子の制御因子の遺伝子の塩基配列はそれぞれ公開されており、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。
また、例えば、前記遺伝子によりコードされるタンパク質の細胞内存在量を、常法により定量的に測定して、前記遺伝子の発現量としてもよい。
The expression level of the HSD11B1 gene or HSD11B2 gene 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. Nucleotide sequences of genes of regulatory factors of a plurality of known melanocyte activating factors have been published, and those skilled in the art can appropriately design primers and subject them to PCR.
Alternatively, for example, the intracellular abundance of the protein encoded by the gene may be quantitatively measured by a conventional method and used as the expression level of the gene.
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(微生物を含む)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。
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、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。
A test substance targeted by the screening method of the present invention may be a pure substance, an extract derived from animals or plants (including microorganisms), or a mixture thereof.
Animal and plant-derived extracts refer not only to the extracts themselves derived from animals or plants, but also to collectively include extract fractions, purified fractions, extracts or fractions, and solvent-removed purified products. The plant-derived extracts include wild or grown plants, extracts using those sold as raw materials of herbal medicines, commercially available extracts, and the like.
In the extraction operation, in addition to using the whole plant part, parts such as the plant body, the ground part, the rhizome part, the tree trunk, the leaf part, the stem part, the flower spike, and the flower bud can be used. It is preferable to cut to improve the extraction efficiency. Extraction solvents include water, alcohols such as ethanol, isopropyl alcohol and butanol, polyhydric alcohols such as 1,3-butanediol and polypropylene glycol, ketones such as acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran. One or more selected from polar solvents such as polar solvents can be exemplified as suitable. As a specific extraction method, for example, 1 to 30 parts by mass of a solvent is added to 1 mass of the part used for extraction of the plant body or the like or its dry matter, and at room temperature for several days, at a temperature near the boiling point. If possible, a method of immersing for several hours, cooling to room temperature, optionally removing insoluble matter and/or solvent, and fractionating and purifying by column chromatography or the like can be mentioned.
本明細書において、「肌状態」とは、特に限定されないが、肌の乾燥感(かさつき、保湿不足)、しっとり感(水分量、油分量)、ゴワツキ感(肌理)、凹凸感、柔らかさ、ハリ・弾力、肌色などを言い、主観的又は客観的なものを問わない。
また、「肌状態の変動」とは、特に限定されないが、肌状態が日間で好調になったり不
調になったりする変化をいう。
また、「肌状態の変動の抑制」とは、特に限定されないが、肌状態が日間で好調になったり不調になったりする変化の幅を小さくすることをいう。
したがって、本発明のスクリーニング方法により選択された肌状態変動抑制剤は、肌状態変動抑制用の組成物の有効成分として好適に配合することができる。
In the present specification, "skin condition" is not particularly limited, but skin dryness (dryness, lack of moisture retention), moistness (moisture content, oil content), rough feeling (texture), unevenness, softness. , firmness/elasticity, skin color, etc., subjective or objective.
In addition, "variation in skin condition" is not particularly limited, but refers to a change in skin condition between good and bad days.
In addition, "suppression of fluctuations in skin condition" is not particularly limited, but refers to reducing the range of changes in skin condition between good and bad over the course of the day.
Therefore, the skin condition fluctuation suppressing agent selected by the screening method of the present invention can be suitably blended as an active ingredient of a composition for suppressing skin condition fluctuation.
すなわち、本明細書は、本発明のスクリーニング方法を行い、それにより選択された物質(肌状態変動抑制剤)を含有させる工程を含む、組成物の設計方法も提供する。そして、かかる組成物は、経皮投与される組成物(例えば、化粧料、医薬部外品、医薬品等の皮膚外用剤)の態様とすることができ、肌状態変動抑制用の組成物とすることが好ましい。
また、組成物の剤型は特に限定されない。例えば、化粧料として設計される場合の剤型は、通常知られているローション剤型、乳液剤型、エッセンス剤型、クリーム剤型、粉体含有剤型等が挙げられる。
That is, the present specification also provides a method of designing a composition, which includes the step of carrying out the screening method of the present invention and containing a substance (skin condition fluctuation suppressing agent) selected thereby. Such a composition can be in the form of a transdermally administered composition (e.g., cosmetics, quasi-drugs, external preparations for skin such as pharmaceuticals), and is used as a composition for suppressing skin condition fluctuations. is preferred.
Moreover, the dosage form of the composition is not particularly limited. For example, formulations designed as cosmetics include commonly known lotion formulations, emulsion formulations, essence formulations, cream formulations, powder-containing formulations, and the like.
本発明のスクリーニング方法により選択された肌状態変動抑制剤を含有する組成物を設計する際、肌状態変動抑制剤の含有量(配合量)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0.001質量%以上であり、通常80質量%以下、好ましくは30質量%以下、より好ましくは10質量%以下である。上記範囲とすることで、好適に肌状態変動抑制効果を奏する組成物とすることができる。
また、該組成物に含有させる肌状態変動抑制剤の種類は、1種類のみでなく2種類以上であってもよい。
When designing a composition containing the skin condition fluctuation inhibitor selected by the screening method of the present invention, the content (blended amount) of the skin condition fluctuation inhibitor is usually 0.00001% by mass or more, preferably 0. 0001% by mass or more, more preferably 0.001% by mass or more, and usually 80% by mass or less, preferably 30% by mass or less, more preferably 10% by mass or less. By setting the content within the above range, the composition can be suitably produced to suppress skin condition fluctuations.
Moreover, the number of skin condition fluctuation suppressing agents to be contained in the composition may be one or two or more.
本発明の肌状態変動抑制剤としては、アサイヤシ果実抽出物、加水分解エラスチン、加水分解ゴマタンパク、シュードアルテロモナス発酵物抽出物、タモギタケ抽出物、チョウセンゴミシ果実抽出物が好ましく挙げられ、これらの1種または2種以上を肌状態変動抑制用組成物に配合することができる。また、これらのうちシュードアルテロモナス発酵物抽出物が特に好ましい。さらにシュードアルテロモナス発酵物抽出物とセンブリ抽出物とを組み合わせて肌状態変動抑制用組成物に配合することが好ましい。 Preferred examples of the skin condition fluctuation suppressing agent of the present invention include acai palm fruit extract, hydrolyzed elastin, hydrolyzed sesame protein, pseudoalteromonas fermented product extract, oyster mushroom extract, and schizandra fruit extract. One or more of them can be blended in the composition for suppressing skin condition fluctuations. Among these, the Pseudoalteromonas fermented product extract is particularly preferred. Furthermore, it is preferable to combine the Pseudoalteromonas fermented product extract and the assembly extract and incorporate them into the composition for suppressing skin condition fluctuations.
本発明のスクリーニング方法により選択された肌状態変動抑制剤を含有する組成物を設計する際は、前述した態様や用途に応じて、適宜必要な成分を配合するよう、設計してよい。
例えば、化粧料組成物として設計する場合は、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、その剤型や用途についても、何ら限定されない。以下、主に化粧料に適用される場合に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。
When designing a composition containing the skin condition fluctuation suppressing agent selected by the screening method of the present invention, it may be designed so as to appropriately incorporate necessary components according to the above-described aspects and applications.
For example, when it is designed as a cosmetic composition, it is possible to incorporate a wide range of ingredients that are commonly used in cosmetics, and there are no restrictions on the dosage form or application. Hereinafter, components that can be contained in cosmetics when mainly applied to cosmetics will be described.
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、本発明のスクリーニング方法により選択された肌状態変動抑制剤と重複して配合してもよい。
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、エラグ酸、コウジ酸、リノール酸、ニコチン酸アミド、5,5’-ジプロピルビフェニル-2,2’-ジオール、5’-アデニル酸二ナトリウム、トラネキサム酸セチル、4-メトキシサリチル酸カリウム塩、ハイドロキノン、パントテン酸等が挙げられる。
化粧料における美白成分の含有量は、通常0.01~30質量%であり、0.1~10質量%が好ましく、0.3~5質量%がより好ましい。
Active ingredients include whitening ingredients, wrinkle-improving ingredients, anti-inflammatory ingredients, extracts derived from animals and plants, and the like. In addition, it may be mixed with the skin condition fluctuation inhibitor selected by the screening method of the present invention.
The whitening component is not particularly limited as long as it is commonly used in cosmetics. For example, 4-n-butylresorcinol, ascorbic acid glucoside, 3-O-ethylascorbic acid, tranexamic acid, arbutin, ellagic acid, kojic acid, linoleic acid, nicotinamide, 5,5′-dipropylbiphenyl-2, 2'-diol, disodium 5'-adenylate, cetyl tranexamate, potassium 4-methoxysalicylate, hydroquinone, pantothenic acid and the like.
The content of the whitening component in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, more preferably 0.3 to 5% by mass.
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、三フッ化イソプロピルオキソプロピルアミノカルボニルピロリジンカルボニル
メチルプロピルアミノカルボニルベンゾイルアミノ酢酸ナトリウム、ニコチン酸アミド、ビタミンA又はその誘導体(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、酢酸レチノール等)、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 wrinkle-improving component is not particularly limited as long as it is commonly used in cosmetics. For example, sodium trifluoride isopropyloxopropylaminocarbonylpyrrolidinecarbonylmethylpropylaminocarbonylbenzoylaminoacetate, nicotinamide, vitamin A or derivatives thereof (retinol, retinal, retinoic acid, tretinoin, isotretinoin, tocopherol retinoate, retinol palmitate , retinol acetate, etc.), benzyl ursolic acid, ursolic acid phosphate, betulinic acid benzyl ester, and benzylic acid phosphate.
The content of the wrinkle-improving component in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, more preferably 1 to 5% by mass.
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、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、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、マヨナラエキス、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。
化粧料中における動植物由来抽出物の含有量は、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。
食品中における動植物由来抽出物の含有量は、通常0.01~80質量%であり、0.1~50質量%が好ましく、1~30質量%がより好ましい。
The extract derived from animals and plants is not particularly limited as long as it is generally used in pharmaceuticals, cosmetics, foods, and the like. For example, akebi extract, asunaro extract, asparagus extract, avocado extract, amacha extract, almond extract, arnica extract, aloe extract, aronia extract, apricot extract, ginkgo biloba extract, Indian mushroom extract, fennel extract, udo extract, ageitsu extract, eleuthero extract , Trillium extract, Scutellaria root extract, Phellodendron extract, Coptis extract, Panax ginseng extract, Hypericum extract, Dead nettle extract, Orange extract, Kakyoku extract, Kakon extract, Chamomilla extract, Carrot extract, Kawara mugwort extract, Kiwi extract, Cucumber extract, Guava extract, Kujin extract, gardenia extract, kumazasa extract, clara extract, walnut extract, grapefruit extract, black rice extract, chlorella extract, mulberry extract, turmeric extract, gentian extract, gentian extract, gennoshoko extract, black tea extract, burdock extract, rice extract, fermented rice extract , rice bran fermented extract, rice germ oil, lingonberry extract, salvia extract, soapwort extract, sasa extract, hawthorn extract, sansha extract, Japanese pepper extract, shiitake extract, rhododendron extract, rhizome extract, perilla extract, linden extract, meadowsweet extract, peony extract, pepper Kyou extract, calamus root extract, white birch extract, horsetail extract, stevia extract, fermented stevia, ivy extract, hawthorn extract, elderberry extract, yarrow extract, mint extract, sage extract, mallow extract, cnidium extract, assembly Extract, Sohakuhi extract, Rheum extract, Soybean extract, Taisou extract, Thyme extract, Dandelion extract, Tea extract, Clove extract, Chimp extract, Tencha extract, Capsicum extract, Angelica extract, Angelica extract, Calendula officinalis extract, Tonin extract, Spruce extract, Houttuynia cordata extract, Tomato extract , natto extract, carrot extract, garlic extract, wild rose extract, hibiscus extract, bakumondou extract, lotus extract, parsley extract, birch extract, hamamelis extract, cypress extract, cypress extract, loquat extract, coltsfoot extract, coltsfoot extract, bukuryo extract, butcher bloom extract, grape extract, grape seed extract, luffa extract, safflower extract, peppermint extract, bodaiju extract, Botan extract, hop extract, pine extract, mayonara extract, horse chestnut extract, skunk cabbage extract, soapberry extract, melissa extract, mozuku extract, peach extract, cornflower extract, eucalyptus extract, saxifrage extract, yuzu extract, lily extract, yokuinin extract, mugwort extract, lavender extract, green tea Extracts such as apple extract, rooibos tea extract, litchi extract, lettuce extract, lemon extract, forsythia extract, astragalus extract, rose extract, rosemary extract, Roman chamomile extract, royal jelly extract, burnet extract are preferred.
The content of the animal or plant-derived extract in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, more preferably 1 to 5% by mass.
The content of the animal or plant-derived extract in the food is usually 0.01 to 80% by mass, preferably 0.1 to 50% by mass, more preferably 1 to 30% by mass.
抗炎症成分としては、アラントイン、イソプロピルメチルフェノール、クラリノン、グラブリジン、サリチル酸、トコフェロール酢酸エステル、トコフェロールニコチン酸エステル、トラネキサム酸、ニコチン酸アミド、パントテン酸、パントテニルアルコール等が挙げられ、好ましくは、サリチル酸及びその誘導体、トラネキサム酸及びその誘導体、パ
ントテニルアルコール並びにパントテ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 anti-inflammatory ingredients include allantoin, isopropylmethylphenol, clarinone, glabridin, salicylic acid, tocopherol acetate, tocopherol nicotinate, tranexamic acid, nicotinamide, pantothenic acid, pantothenyl alcohol, etc. Salicylic acid and pantothenyl alcohol are preferred. derivatives thereof, tranexamic acid and its derivatives, pantothenyl alcohol and pantothenic acid and its salts.
The content of the anti-inflammatory ingredient in the cosmetic is usually 0.01 to 30% by mass, preferably 0.1 to 10% by mass, more preferably 1 to 5% by mass.
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、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、イソノナン酸2-エチルヘキシル、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ン酸コレステリル、ジ-2-エチルヘキサン酸エチレングリコール、ジペンタエリスリトール脂肪酸エステル、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ル、トリ-2-エチルヘキサン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-エチルヘキサン酸ペンタンエリスリトール、トリ-2-エチルヘキサン酸グリセリル、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。
Polar oil, volatile hydrocarbon oil, etc. are mentioned as an oily component.
Polar oils include synthetic ester oils such as isopropyl myristate, cetyl octanoate, octyldodecyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, decyl oleate, hexyldecyl dimethyloctanoate, and lactic acid. Cetyl, myristyl lactate, lanolin acetate, 2-ethylhexyl isononanoate, isocetyl stearate, isocetyl isostearate, cholesteryl 12-hydroxystearate, ethylene glycol di-2-ethylhexanoate, dipentaerythritol fatty acid ester, monoisostearate N- Alkyl glycol, neopentyl glycol dicaprate, diisostearyl malate, glyceryl di-2-heptylundecanoate, trimethylolpropane tri-2-ethylhexanoate, trimethylolpropane triisostearate, pentane tetra-2-ethylhexanoate Mention may be made of erythritol, glyceryl tri-2-ethylhexanoate, trimethylolpropane triisostearate.
さらに、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ル、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、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。 In addition, cetyl 2-ethylhexanoate, 2-ethylhexyl palmitate, glyceryl trimyristate, tri-2-heptyl undecanoic acid glyceride, castor oil fatty acid methyl ester, oleic oil, cetostearyl alcohol, acetoglyceride, palmitic acid 2 -heptylundecyl, diisobutyl adipate, N-lauroyl-L-glutamic acid 2-octyldodecyl ester, di-2-heptylundecyl adipate, ethyl laurate, di-2-ethylhexyl sebacate, 2-hexyl myristate Decyl, 2-hexyldecyl palmitate, 2-hexyldecyl adipate, diisopropyl sebacate, 2-ethylhexyl succinate, ethyl acetate, butyl acetate, amyl acetate, triethyl citrate, octyl methoxycinnamate and the like.
また、天然油として、アボガド油、ツバキ油、タートル油、マカデミアナッツ油、トウモロコシ油、ミンク油、オリーブ油、ナタネ油、卵黄油、ゴマ油、パーシック油、小麦胚芽油、サザンカ油、ヒマシ油、アマニ油、サフラワー油、綿実油、エノ油、大豆油、落花生油、茶実油、カヤ油、コメヌカ油、シナギリ油、日本キリ油、ホホバ油、胚芽油、トリグリセリン、トリオクタン酸グリセリル、トリイソパルミチン酸グリセリル等が挙げられる。 In addition, as natural oils, avocado oil, camellia oil, turtle oil, macadamia nut oil, corn oil, mink oil, olive oil, rapeseed oil, egg yolk oil, sesame oil, persic oil, wheat germ oil, sasanqua oil, castor oil, linseed oil, Safflower oil, cottonseed oil, perilla oil, soybean oil, peanut oil, tea seed oil, kaya oil, rice bran oil, sinagiri oil, Japanese pungent oil, jojoba oil, germ oil, triglycerin, glyceryl trioctanoate, glyceryl triisopalmitate etc.
揮発性炭化水素油としては、イソドデカン、イソヘキサデカン等が挙げられる。 Volatile hydrocarbon oils include isododecane, isohexadecane, 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) 、グリセリン脂肪酸エステル類(モノステアリン酸グリセリル等)、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(登録商標)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, anionic surfactants such as alkyl sulfate triethanolamine ether, stearyltrimethylammonium chloride, benzalkonium chloride, laurylamine oxide. cationic surfactants such as betaine surfactants (alkylbetaine, amidobetaine, sulfobetaine, etc.), imidazoline amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-
Sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acid esters (glyceryl monostearate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hydrogenated castor oil derivatives, glycerin alkyl Ether, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbit fatty acid esters (POE-sorbit monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoiso stearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE 2-octyldodecyl ether, etc.), POE alkylphenyl ethers (POE nonylphenyl ether, etc.), Pluronic (registered trademark) ) types, POE/POP alkyl ethers (POE/POP 2-decyltetradecyl ether, etc.), tetronics, POE castor oil/hydrogenated castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), sucrose fatty acid esters , nonionic surfactants such as alkyl glucosides, 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-ブチレングリコール、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-butylene glycol, 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、グアーガム、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, caroninic acid, chitin, chitosan, carboxymethyl Chitin, agar, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, alkyl-modified carboxyvinyl polymer, sodium polyacrylate, polyethylene glycol, bentonite and the like.
粉体類としては、表面を処理されていてもよい、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よい、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よい、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よい赤色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 the powders, powders such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, anhydrous silicic acid (silica), aluminum oxide, and barium sulfate, which may be surface-treated, may be used. Inorganic pigments such as red iron oxide, yellow iron oxide, black iron oxide, cobalt oxide, ultramarine blue, Prussian blue, titanium oxide, and zinc oxide that may be treated, mica titanium that may be treated, fish phosphorus foil, Pearl agents such as bismuth oxychloride, optionally laked Red No. 202, Red No. 228, Red No. 226, Yellow No. 4, Blue No. 404, Yellow No. 5, Red No. 505, Red No. 230, Red No. 223 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 and other organic pigments, polyethylene powder, polymethyl methacrylate, nylon powder, and organic powders such as organopolysiloxane elastomers.
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 UV absorbers include para-aminobenzoic 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.
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, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples as long as the gist thereof is not exceeded.
<実施例1>
培養ヒト成人表皮角化細胞(NHEK、クラボウ)を増殖培地とともに24穴マルチウェルプレートに播種した後、培地を被験物質または溶媒対照を含む培地に交換し、24時
間培養した。その後、表1に示す各被験物質(各0.25容量%)と10ng/mLのTNFα(R&D SYSTEMS社)を含む培地に交換し24時間培養した後に培地を除き、QIAshredder、RNeasy Mini Kit、QIACube(いずれもQIAGEN)を用いてRNAを精製し、Superscript VILO cDNA Synthesis Kit(Thermo Fisher Scientific)にてcDNA化した。cDNAから、QuantiTect Primer Assay(QIAGEN)を用いてリアルタイムPCRを行い、HSD11B1及びHSD11B2の各mRNA量を測定した(n=3)。内在性コントロールとして、HSD11B1にはRPL27を、HSD11B2にはGAPDHのmRNA量を測定し、内在性コントロールのmRNA量に対する比率を解析し、被験物質の値と被験物質を含まずに溶媒のみを培地に添加した溶媒対照との間で、HSD11B1及びHSD11B2の遺伝子発現量を比較した。なお、プライマーは表2に記載のもの(QIAGEN社)を用いた。
溶媒対照の平均値と比較して20%以上のHSD11B1遺伝子発現の減少を示す場合に、該被験試料はHSD11B1遺伝子発現抑制作用を有すると判定することとした。また、溶媒対照の平均値と比較して20%以上のHSD11B2遺伝子発現の減少を示す場合に、該被験試料はHSD11B2遺伝子発現抑制作用を有すると判定することとした。
<Example 1>
Cultured human adult epidermal keratinocytes (NHEK, Kurabo Industries) were seeded in a 24-well multiwell plate with a growth medium, then the medium was replaced with a medium containing the test substance or solvent control and cultured for 24 hours. Thereafter, the medium was replaced with a medium containing each test substance (0.25% by volume each) shown in Table 1 and 10 ng/mL TNFα (R&D SYSTEMS), and after culturing for 24 hours, the medium was removed, and QIAshredder, RNeasy Mini Kit, and QIACube (both from QIAGEN), and converted to cDNA using Superscript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time PCR was performed from the cDNA using QuantiTect Primer Assay (QIAGEN) to measure the amount of each mRNA of HSD11B1 and HSD11B2 (n=3). As endogenous controls, the mRNA levels of RPL27 for HSD11B1 and GAPDH for HSD11B2 were measured, and the ratio to the endogenous control mRNA was analyzed. Gene expression levels of HSD11B1 and HSD11B2 were compared between the added solvent control. The primers listed in Table 2 (QIAGEN) were used.
When the HSD11B1 gene expression decreased by 20% or more compared to the mean value of the solvent control, the test sample was judged to have an HSD11B1 gene expression-suppressing effect. In addition, when the HSD11B2 gene expression decreased by 20% or more compared to the mean value of the solvent control, the test sample was determined to have an HSD11B2 gene expression-suppressing effect.
被験物質を添加したNHEKにおけるHSD11B1遺伝子の発現量の解析結果を図1に示す。溶媒対照での発現量を100としたときの相対値で示し、低値であるほど、HSD11B1遺伝子の発現が抑制されたことを表す。これまでHSD11B1遺伝子の発現抑制効果の知られていなかった、シュードアルテロモナス発酵物抽出物、加水分解エラスチン、加水分解ゴマタンパク、アサイヤシ果実抽出物、タモギタケ抽出物、チョウセンゴミシ果実抽出物、及びシュードアルテロモナス発酵物抽出物に、HSD11B1遺伝子発現抑制作用が認められた。さらに、加水分解ゴマタンパク、及びシュードアルテロモナス発酵物抽出物には、有意なHSD11B1遺伝子発現抑制作用を認められた。
被験物質を添加したNHEKにおけるHSD11B2遺伝子の発現量の解析結果を図2に示す。溶媒対照での発現量を100としたときの相対値で示し、低値であるほど、HSD11B2遺伝子の発現が抑制されたことを表す。HSD11B1の遺伝子発現抑制作用が認められたシュードアルテロモナス発酵物抽出物、加水分解エラスチン、加水分解ゴマタンパク、アサイヤシ果実抽出物、タモギタケ抽出物、及びチョウセンゴミシ果実抽出物には、いずれもNHEKにおけるHSD11B2遺伝子の発現に対する抑制作用は認められなかった。
FIG. 1 shows the analysis results of the expression level of the HSD11B1 gene in NHEK to which the test substance was added. The expression level in the solvent control is shown as a relative value to 100, and the lower the value, the more the expression of the HSD11B1 gene is suppressed. Pseudoalteromonas fermented product extract, hydrolyzed elastin, hydrolyzed sesame protein, acai palm fruit extract, Pleurotus cornucopiae extract, Schisandra fruit extract, and pseudo, which had no previously known effect of suppressing the expression of the HSD11B1 gene. The Alteromonas fermented product extract was found to have an HSD11B1 gene expression-suppressing effect. Furthermore, the hydrolyzed sesame protein and the Pseudoalteromonas fermented product extract were found to have a significant HSD11B1 gene expression suppressing effect.
FIG. 2 shows the analysis results of the expression level of the HSD11B2 gene in NHEK to which the test substance was added. The expression level in the solvent control is shown as a relative value when the expression level is 100, and the lower the value, the more the HSD11B2 gene expression is suppressed. The Pseudoalteromonas fermented product extract, hydrolyzed elastin, hydrolyzed sesame protein, acai palm fruit extract, Pleurotus cornucopiae extract, and Schisandra schisandra fruit extract, all of which have been found to have the gene expression-suppressing effect of HSD11B1, have No inhibitory action was observed on the expression of the HSD11B2 gene.
<実施例2>
以下の手順で、HSD11B1遺伝子発現の抑制効果を有し、かつHSD11B2遺伝子の発現を抑制しない物質が、表皮角化細胞におけるコルチゾールの再活性化を抑制し得ることを確認した。
NHEKを増殖培地とともに96穴マルチウェルプレートに播種した後、シュードアルテロモナス発酵物抽出物(0.25容量%)又は溶媒対照を含む培地に交換し24時間培養した。その後、各被験物質とTNFαと1μMのコルチゾール(Sigma-Aldrich社)を含む培地に交換し、24時間培養した。その後、再度コルチゾールを含まない各被験物質とTNFαのみを加えた培地に交換し、さらに培養した。24時間後の培養系に含まれるコルチゾール量をDetectX CORTISOL Enzyme Immunoassay Kit (ARBOR assays社)にて測定した。該コルチゾール量を、細胞に使用されて不活性化されたコルチゾール変換体であるコルチゾンが、
活性型コルチゾールに再生された量であるとして評価した。
シュードアルテロモナス発酵物抽出物を添加したNHEKでは、溶媒対照よりもコルチゾール量が有意に低く、コルチゾンのコルチゾールへの再活性化が抑制されたことが確認された。
<Example 2>
By the following procedure, it was confirmed that a substance that has an effect of suppressing HSD11B1 gene expression but does not suppress HSD11B2 gene expression can suppress reactivation of cortisol in epidermal keratinocytes.
NHEKs were seeded in 96-well multiwell plates with growth medium, then changed to medium containing Pseudoalteromonas fermentate extract (0.25% by volume) or solvent control and cultured for 24 hours. Thereafter, the medium was replaced with a medium containing each test substance, TNFα and 1 μM cortisol (Sigma-Aldrich) and cultured for 24 hours. After that, the medium was replaced with a medium containing only each test substance not containing cortisol and TNFα, and the cells were further cultured. After 24 hours, the amount of cortisol contained in the culture system was measured using DetectX CORTISOL Enzyme Immunoassay Kit (ARBOR assays). Cortisone, an inactivated cortisol convertor, is used by cells to
It was evaluated as the amount regenerated to active cortisol.
The NHEK supplemented with the Pseudoalteromonas ferment extract had significantly lower cortisol levels than the solvent control, confirming that the reactivation of cortisone to cortisol was suppressed.
<実施例3>
以下の手順で、センブリ抽出物が、シュードアルテロモナス発酵物抽出物の表皮角化細胞におけるコルチゾールの再活性化抑制効果をより高めることを確認した。
被験物質として、シュードアルテロモナス発酵物抽出物(0.25容量%)、シュードアルテロモナス発酵物抽出物とセンブリ抽出物(香栄興業株式会社)の混合物(各0.25容量%)、センブリ抽出物(0.25容量%)を用い、実施例2と同様の方法で活性型コルチゾールの再生量を評価した。
結果を図3に示す。低値を示すほど、コルチゾールが再活性化されていないことを表す。シュードアルテロモナス発酵物抽出物単独では、溶媒対照よりも細胞培養中のコルチゾール量が有意に低いことから、HSD11B1によるコルチゾンのコルチゾールへの再活性化が抑制されたことが分かる。また、センブリ抽出物単独では、コルチゾール量に影響を及ぼさなかった。一方、シュードアルテロモナス発酵物抽出物とセンブリ抽出物との混合物を添加した場合は、シュードアルテロモナス発酵物抽出物単独よりもコルチゾール再活性化が抑制されることが認められた。これにより、シュードアルテロモナス発酵物抽出物の表皮角化細胞におけるコルチゾールの再活性化抑制作用が、センブリ抽出物により増強されたことが推測された。
<Example 3>
It was confirmed by the following procedure that the assembly extract enhances the cortisol reactivation inhibitory effect of the Pseudoalteromonas fermented product extract in epidermal keratinocytes.
As test substances, Pseudoalteromonas fermented product extract (0.25% by volume), a mixture of Pseudoalteromonas fermented product extract and assembly extract (Koei Kogyo Co., Ltd.) (each 0.25% by volume), Using the assembly extract (0.25% by volume), the amount of activated cortisol regenerated was evaluated in the same manner as in Example 2.
The results are shown in FIG. A lower value indicates less reactivation of cortisol. The Pseudoalteromonas fermentate extract alone inhibited the reactivation of cortisone to cortisol by HSD11B1, as the amount of cortisol in the cell culture was significantly lower than the solvent control. Also, jersey extract alone had no effect on cortisol levels. On the other hand, when the mixture of the Pseudoalteromonas fermentate extract and the assembly extract was added, it was found that cortisol reactivation was suppressed more than the Pseudoalteromonas fermentate extract alone. From this, it was speculated that the cortisol reactivation inhibitory action of the Pseudoalteromonas fermented product extract in epidermal keratinocytes was enhanced by the assembly extract.
<実施例4>
表3に示す処方で、水中油型の乳液を調製した。すなわち、成分A及び成分Bを80℃でそれぞれ加温して混合し、撹拌下でAにBを徐々に加え、撹拌冷却して乳液を得た。
<Example 4>
An oil-in-water emulsion was prepared according to the formulation shown in Table 3. That is, component A and component B were heated at 80° C. and mixed, B was gradually added to A with stirring, and the mixture was stirred and cooled to obtain a milky lotion.
<実施例5>
実施例4で調製した試験製剤及び比較製剤を、被験者(各群5名、計10名)に5日間顔面に朝晩2回使用してもらい、肌状態4項目、すなわち経皮水分蒸散量(TEWL)、角層水分量、皮膚表面の粗さ、及び隆起部の体積を毎日同時刻に測定した。TEWLはVapometer SWL5001JT(キーストンサイエンティフィック)を用いて、角層の水分量はCorneometer CM825(Courage+Khazaka)を用いて、皮膚表面の粗さと隆起部の体積はANTERA 3D(Miravex)を用いて、それぞれ測定した。上記肌状態4項目の各日間変動率は、下記式に従い、すなわち各測定日の測定値と1日目の測定値との差の絶対値の、1日目の測定値に対する割合として算出した。
肌状態の変動率(%)={(各日の測定値-1日目の測定値)/1日目の測定値}×100
各項目の測定値の各群の平均値及び誤差を、図4~図7に示す。各図において低値を示すほど1日目からの変化が少ないことを表す。比較製剤と比較してシュードアルテロモナス発酵物抽出物及びセンブリ抽出物を含む試験製剤では、全ての測定項目で概ね変動率が小さく、特に、5日目には全項目で変動率が小さかった。このことから、試験製剤は肌状態の日間変動の抑制に有効であり、連続で使用することにより、より効果を発揮することがわかる。
<Example 5>
Subjects (5 subjects in each group, 10 subjects in total) were asked to use the test formulation and comparative formulation prepared in Example 4 on the face twice in the morning and evening for 5 days. ), stratum corneum water content, skin surface roughness, and ridge volume were measured at the same time every day. TEWL was measured using a Vapometer SWL5001JT (Keystone Scientific), the water content of the stratum corneum was measured using a Corneometer CM825 (Courage + Khazaka), and the roughness of the skin surface and the volume of ridges were measured using an ANTERA 3D (Miravex). did. The daily variation rate of each of the four skin condition items was calculated according to the following formula, that is, as a ratio of the absolute value of the difference between the measured value on each measurement day and the measured value on the first day to the measured value on the first day.
Variation rate of skin condition (%) = {(measured value of each day - measured value of 1st day) / measured value of 1st day} x 100
The average values and errors of each group of measured values for each item are shown in FIGS. 4 to 7. FIG. In each figure, the lower the value, the smaller the change from the first day. Compared to the comparative formulation, the test formulation containing the Pseudoalteromonas fermentate extract and the assembly extract showed generally lower variability in all measured items, especially on
本発明により、肌状態の日間変動を抑制し得る物質のスクリーニング方法が提供される。これにより、肌状態変動抑制用組成物等の設計・製造に有用となり得る素材の探索が可能になるので、産業上有用である。 INDUSTRIAL APPLICABILITY The present invention provides a screening method for substances capable of suppressing daily fluctuations in skin condition. This makes it possible to search for materials that can be useful in the design and production of compositions for suppressing skin condition fluctuations, etc., which is industrially useful.
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