JP6452064B2 - Yanagita dessprout extract and method for producing the same, enzyme activity inhibitor and anti-aging agent, cosmetic composition and functional food - Google Patents
Yanagita dessprout extract and method for producing the same, enzyme activity inhibitor and anti-aging agent, cosmetic composition and functional food Download PDFInfo
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- Coloring Foods And Improving Nutritive Qualities (AREA)
- Cosmetics (AREA)
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Description
本発明は、皮膚の老化の改善・防止する作用を有する新規な植物抽出物及びその応用品に関する。 The present invention relates to a novel plant extract having an action of improving / preventing skin aging and its applied product.
皮膚の水分保持性、柔軟性又は弾力性は、真皮に含まれるエラスチン、コラーゲン等の細胞外マトリックス成分に起因しているが、これらエラスチン、コラーゲンの減少又は分解が皮膚老化の原因の１つといわれている。
細胞外マトリックス成分のうち、エラスチン、コラーゲン及びゼラチン（変性コラーゲン）は、それぞれの分解酵素であるエラスターゼ、コラゲナーゼ及びゼラチナーゼにより分解される。そのため、エラスチン、コラーゲン及びゼラチンの分解酵素に関する合成量抑制もしくは酵素活性を阻害することにより皮膚の抗老化作用が期待できる。そこで、皮膚の老化防止やしわ防止作用等の抗老化作用を期待して、エラスチン、コラーゲン、ゼラチンの分解酵素の活性阻害剤を配合した皮膚外用剤や各種化粧料組成物、機能性食品が開発されている。
The moisture retention, flexibility or elasticity of the skin is attributed to extracellular matrix components such as elastin and collagen contained in the dermis. The decrease or degradation of these elastin and collagen is said to be one of the causes of skin aging. ing.
Of the extracellular matrix components, elastin, collagen and gelatin (denatured collagen) are degraded by elastase, collagenase and gelatinase, which are the respective degradation enzymes. Therefore, the anti-aging effect of the skin can be expected by suppressing the synthesis amount or inhibiting the enzyme activity relating to the degrading enzymes of elastin, collagen and gelatin. Therefore, in anticipation of anti-aging effects such as skin aging prevention and wrinkle prevention effects, skin external preparations and various cosmetic compositions and functional foods containing elastin, collagen and gelatin degrading enzyme activity inhibitors were developed. Has been.
エラスターゼ活性阻害剤として、例えば、特許文献１にはオオウメガサソウを有効成分とするエラスターゼ活性阻害剤及び該阻害剤を含む化粧料組成物が開示されている。また、コラゲナーゼ活性阻害剤として、例えば、特許文献２には、アセロラ種子の抽出物を有効成分とするコラゲナーゼ活性阻害剤及び該阻害剤を含む化粧料組成物が開示されている。また、ゼラチナーゼ活性阻害剤として、例えば、特許文献３にはリンゴの未熟果実の水・アルコール溶媒抽出物を有効成分とするゼラチナーゼ活性阻害剤及び該阻害剤を含む皮膚老化抑制剤が開示されている。 As an elastase activity inhibitor, for example, Patent Document 1 discloses an elastase activity inhibitor containing Euphorbia as an active ingredient and a cosmetic composition containing the inhibitor. Moreover, as a collagenase activity inhibitor, for example, Patent Document 2 discloses a collagenase activity inhibitor containing an extract of acerola seeds as an active ingredient and a cosmetic composition containing the inhibitor. Moreover, as a gelatinase activity inhibitor, for example, Patent Document 3 discloses a gelatinase activity inhibitor containing a water / alcohol solvent extract of an immature apple fruit as an active ingredient, and a skin aging inhibitor containing the inhibitor. .
より効果的に皮膚の老化を改善・抑制するためには、エラスチン及びコラーゲン両方、並びにゼラチンの分解を抑制することが望ましい。
非特許文献１にはヤナギタデの全草抽出物に関して、エラスターゼ活性阻害作用があることが報告されている。また、非特許文献１ではヤナギタデの全草抽出物にはコラゲナーゼ遺伝子発現抑制作用を有していることも報告されている。一方、特許文献４では、ヤナギタデの全草抽出物は、エラスターゼ活性阻害作用を有するが、コラゲナーゼ活性阻害作用を有さないと報告されている。
In order to more effectively improve / suppress skin aging, it is desirable to suppress the degradation of both elastin and collagen and gelatin.
Non-Patent Document 1 reports that an elastase activity inhibitory action is associated with a whole plant extract of Willow. Non-Patent Document 1 also reports that the whole plant extract of Willow radish has a collagenase gene expression inhibitory action. On the other hand, in Patent Document 4, it is reported that the whole plant extract of the willow tree has an elastase activity inhibitory action, but does not have a collagenase activity inhibitory action.
上記非特許文献１や特許文献４で報告されているように、ヤナギタデの全草抽出物には、エラスターゼ活性阻害作用及びコラゲナーゼ遺伝子発現抑制作用を有しているため、エラスターゼの酵素活性を阻害し、かつ、生成されるコラゲナーゼ量を減少させることができる。しかしながら、ヤナギタデの全草抽出物は、コラゲナーゼに対する酵素活性阻害作用を有さないため、新たに生成されるコラゲナーゼ量は減少するものの、既に存在しているコラゲナーゼに起因するコラーゲンの分解を抑制することができないという課題があった。また、上記文献を含め、ヤナギタデ由来成分のゼラチナーゼ活性阻害作用についての報告例はない。 As reported in Non-Patent Document 1 and Patent Document 4, the whole plant extract of Willow radish has an elastase activity inhibitory action and a collagenase gene expression inhibitory action, thereby inhibiting the enzyme activity of elastase. And the amount of collagenase produced can be reduced. However, since the whole plant extract of willow tree has no enzyme activity inhibitory action on collagenase, the amount of newly produced collagenase is reduced, but it suppresses the degradation of collagen caused by the existing collagenase. There was a problem that it was not possible. Moreover, there is no report example about the gelatinase activity inhibitory effect of the component derived from a willow tree including the said literature.
かかる状況下、本発明の目的は、エラスターゼ、コラゲナーゼ及びゼラチナーゼに対する酵素活性阻害作用を併せ持つヤナギタデ由来の抽出物、及びヤナギタデ由来成分を有効成分として含有する酵素活性阻害剤及び抗老化剤、並びに当該酵素活性阻害剤を配合した化粧料組成物ないし機能性食品を提供することである。 Under such circumstances, an object of the present invention is to extract from Yanagita that has an enzyme activity inhibitory action on elastase, collagenase and gelatinase, an enzyme activity inhibitor and anti-aging agent containing the component derived from Yanagita as an active ingredient, and the enzyme It is to provide a cosmetic composition or a functional food containing an activity inhibitor.
本発明者は、上記課題を解決すべく鋭意研究を重ねた結果、ヤナギタデのスプラウトにコラゲナーゼ活性阻害作用、エラスターゼ活性阻害作用及びゼラチナーゼ活性阻害作用を有する有効成分が含まれていることを見出した。そして、ヤナギタデスプラウトから活性の低下を抑制して、有効成分を抽出する方法を見出し、本発明を完成させた。 As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that an active ingredient having a collagenase activity inhibitory action, an elastase activity inhibitory action and a gelatinase activity inhibitory action is contained in the sprout of Yanagitade. And the method of suppressing an active fall from a willow desprout and extracting an active ingredient was discovered, and this invention was completed.
すなわち、本発明は、以下の発明に係るものである。
＜１＞ ヤナギタデ（Persicaria hydropiper）のスプラウトと抽出溶媒とを接触させ、前記ヤナギタデスプラウトに含有される成分を抽出して得られるヤナギタデスプラウト抽出物。
＜２＞ 前記抽出溶媒が、必須成分として１，３−ブチレングリコールを含有し、さらに水及びエタノールの少なくとも１種を含有する混合溶媒である前記＜１＞に記載のヤナギタデスプラウト抽出物。
＜３＞ 前記抽出溶媒における１，３−ブチレングリコールの割合が、２０体積％以上８０体積％以下である前記＜２＞に記載のヤナギタデスプラウト抽出物。
＜４＞ 前記抽出溶媒が、水及びエタノールの少なくとも１種である前記＜１＞に記載のヤナギタデスプラウト抽出物。
＜５＞ ヤナギタデスプラウト由来成分を有効成分として含有し、エラスターゼ、コラゲナーゼ及びゼラチナーゼから選択される少なくとも１種の酵素に対する活性阻害作用を有する酵素活性阻害剤。
＜６＞ エラスターゼ、コラゲナーゼ及びゼラチナーゼのすべての酵素に対する活性阻害作用を有する前記＜５＞に記載の酵素活性阻害剤。
＜７＞ ヤナギタデスプラウト由来成分が、前記＜１＞から＜４＞のいずれかに記載のヤナギタデスプラウト抽出物である前記＜５＞または＜６＞に記載の酵素活性阻害剤。
＜８＞ 前記＜５＞から＜７＞のいずれかに記載の酵素活性阻害剤を含有する抗老化剤。
＜９＞ 前記＜５＞から＜７＞のいずれかに記載の酵素活性阻害剤を配合してなる化粧料組成物。
＜１０＞ 前記＜５＞から＜７＞のいずれかに記載の酵素活性阻害剤を配合してなる機能性食品。
＜１１＞ ヤナギタデ（Persicaria hydropiper）のスプラウトと、１，３−ブチレングリコールと水及びエタノールの少なくとも１種とを含有する抽出溶媒とを接触させ、前記スプラウトに含有される成分を抽出するヤナギタデスプラウト抽出物の製造方法。
＜１２＞ 前記抽出溶媒における１，３−ブチレングリコールの割合が、２０体積％以上８０体積％以下である前記＜１１＞に記載のヤナギタデスプラウト抽出物の製造方法。
That is, the present invention relates to the following inventions.
<1> A Yanagita Desprout extract obtained by bringing a sprout of Persicaria hydropiper into contact with an extraction solvent and extracting components contained in the Yanagita Desprout.
<2> The willow desprout extract according to <1>, wherein the extraction solvent is a mixed solvent containing 1,3-butylene glycol as an essential component and further containing at least one of water and ethanol.
<3> The Yanagita dessprout extract according to <2>, wherein a ratio of 1,3-butylene glycol in the extraction solvent is 20% by volume or more and 80% by volume or less.
<4> The willow desprout extract according to <1>, wherein the extraction solvent is at least one of water and ethanol.
<5> An enzyme activity inhibitor having an activity inhibitory action against at least one enzyme selected from elastase, collagenase, and gelatinase, containing a component derived from willow desprout as an active ingredient.
<6> The enzyme activity inhibitor according to the above <5>, which has an activity inhibitory action on all enzymes of elastase, collagenase and gelatinase.
<7> The enzyme activity inhibitor according to <5> or <6>, in which the willow desprout-derived component is the willow red sprout extract according to any one of <1> to <4>.
<8> An anti-aging agent comprising the enzyme activity inhibitor according to any one of <5> to <7>.
<9> A cosmetic composition comprising the enzyme activity inhibitor according to any one of <5> to <7>.
<10> A functional food comprising the enzyme activity inhibitor according to any one of <5> to <7>.
<11> Yanagita Desprout Extract for Contacting Sprout of Persicaria hydropiper with an Extraction Solvent Containing 1,3-Butylene Glycol, Water and at least One of Ethanol to Extract Components Contained in the Sprout Manufacturing method.
<12> The method for producing a Yanagita desprout extract according to <11>, wherein the proportion of 1,3-butylene glycol in the extraction solvent is 20% by volume or more and 80% by volume or less.
本発明によれば、エラスターゼ、コラゲナーゼ及びゼラチナーゼに対する酵素活性阻害作用を有するヤナギタデスプラウト抽出物、及びヤナギタデスプラウト由来成分を有効成分として含有する酵素活性阻害剤及び抗老化剤、並びにヤナギタデスプラウト由来成分を配合した化粧料組成物及び機能性食品が提供される。 According to the present invention, the extract of the willow desprout having an enzyme activity inhibitory action on elastase, collagenase and gelatinase, the enzyme activity inhibitor and anti-aging agent containing the component derived from the spider sprout as active ingredients, and the component derived from the spider sprout Cosmetic compositions and functional foods are provided.
以下、本発明について例示物等を示して詳細に説明するが、本発明は以下の例示物等に限定されるものではなく、本発明の要旨を逸脱しない範囲において任意に変更して実施できる。なお、本明細書において、「〜」という表現を用いた場合、その前後の数値または物理値を含む意味で用いることとする。 Hereinafter, the present invention will be described in detail with reference to examples and the like, but the present invention is not limited to the following examples and the like, and can be arbitrarily modified and implemented without departing from the gist of the present invention. In addition, in this specification, when the expression “to” is used, it is used in a sense including numerical values or physical values before and after that.
＜１．ヤナギタデスプラウト抽出物＞
本発明のヤナギタデスプラウト抽出物（以下、「本発明の抽出物」と称す場合がある。）は、ヤナギタデ（Persicaria hydropiper）のスプラウトと抽出溶媒とを接触させ、前記スプラウトに含有される成分を抽出して得られる抽出物である。
<1. Yanagita Desprout Extract>
The willow desprout extract of the present invention (hereinafter sometimes referred to as “the extract of the present invention”) is obtained by bringing a sprout of a willow tree (Persicaria hydropiper) into contact with an extraction solvent to extract components contained in the sprout. It is the extract obtained by doing.
本発明のヤナギタデスプラウト抽出物は、エラスターゼ、コラゲナーゼ、及びゼラチナーゼに対する酵素活性阻害作用を有することに特徴がある。そのため、エラスターゼによるエラスチンの分解、コラゲナーゼによるコラーゲンの分解、ゼラチナーゼによるゼラチンの分解を抑制することができる。また、上述の通り、特許文献４や非特許文献１で開示されたヤナギタデの全草抽出物では、エラスターゼの活性阻害作用を有すが、コラゲナーゼの活性阻害作用は有さない。 The willow desprout extract of the present invention is characterized by having an enzyme activity inhibitory action on elastase, collagenase, and gelatinase. Therefore, degradation of elastin by elastase, degradation of collagen by collagenase, and degradation of gelatin by gelatinase can be suppressed. Moreover, as described above, the whole plant extract of Willow radish disclosed in Patent Document 4 and Non-Patent Document 1 has an elastase activity inhibitory action, but does not have a collagenase activity inhibitory action.
本発明の抽出物の原材料となるヤナギタデ（Persicaria hydropiper）はイヌタデ属植物であり、そのスプラウト（芽タデ）は、さしみのつま、タデ酢等として食用にされている。ヤナギタデスプラウトは、事前処理せずにそのまま溶媒抽出に使用することもできるが、通常、抽出効率を高めるため、スライス、粉砕、圧搾またはすりおろす等により、細粒物として使用する。 The willow tree (Persicaria hydropiper), which is the raw material of the extract of the present invention, is a plant of the genus Inodade, and its sprout (bud seed) is edible as sashimi-tsuma or vinegar. Yanagita desprout can be used for solvent extraction as it is without pretreatment, but is usually used as a fine-grained material by slicing, crushing, squeezing or grated in order to increase extraction efficiency.
本発明において、「抽出物」とは、抽出対象となるヤナギタデスプラウト、又はこれを必要に応じて乾燥、細切したものを溶媒抽出して、有効成分の含有率を高めた形態のものを総括した概念である。具体的にはヤナギタデスプラウトを抽出原料として得られる抽出液、該抽出液の希釈液若しくは濃縮液、又はこれらの粗精製物若しくは精製物のいずれもが含まれる。なお、抽出液を乾燥して得られる乾燥物も、抽出物に該当するものとする。 In the present invention, the “extract” is a generalized form of the extract of Yanagita desprouts to be extracted, or those that have been dried and shredded as necessary to increase the content of active ingredients. Concept. Specifically, an extract obtained by using Yanagita desprout as an extraction raw material, a diluted solution or a concentrated solution of the extract, or a crudely purified product or a purified product thereof is included. In addition, the dried material obtained by drying an extract liquid shall also correspond to an extract.
抽出溶媒としては、ヤナギタデスプラウトに含有されるコラゲナーゼ活性阻害作用、エラスターゼ活性阻害作用及びゼラチナーゼ活性阻害作用を発現する有効成分を抽出できるものであればよい。具体例としては、水、メタノール、エタノール、プロピルアルコール、イソプロピルアルコール等の炭素数１〜５の低級アルコール；アセトン、メチルエチルケトン等の低級脂肪族ケトン；１，３−ブチレングリコール、プロピレングリコール、グリセリン等の炭素数２〜５の多価アルコールなどが挙げられる。なお、抽出溶媒として使用し得る水には、精製水、イオン交換水、生理食塩水、リン酸緩衝液、リン酸緩衝生理食塩水等も含まれる。
これらの抽出溶媒は単独又はこれら２種以上の混合物として使用することができ、２種以上の溶媒の混合液を抽出溶媒として使用する場合、その混合比は適宜調整することができる。
Any extraction solvent may be used as long as it can extract an active ingredient that expresses collagenase activity inhibitory action, elastase activity inhibitory action, and gelatinase activity inhibitory action contained in the willow sprout. Specific examples include lower alcohols having 1 to 5 carbon atoms such as water, methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene glycol, propylene glycol, glycerin, and the like. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms. The water that can be used as the extraction solvent includes purified water, ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.
These extraction solvents can be used alone or as a mixture of two or more thereof. When a mixture of two or more solvents is used as the extraction solvent, the mixing ratio can be appropriately adjusted.
なお、抽出溶媒には、本発明の効果を損なわない範囲で添加剤を含んでいてもよい。例えば、ｐＨ調整剤、粘度調整剤などが挙げられる。 The extraction solvent may contain an additive as long as the effects of the present invention are not impaired. For example, a pH adjuster, a viscosity adjuster, etc. are mentioned.
本発明のヤナギタデスプラウト抽出物において、抽出溶媒が１，３−ブチレングリコールを必須溶媒とし、さらに水及びエタノールの少なくとも１種を含有する混合溶媒であることが好ましい。ヤナギタデのスプラウトと、１，３−ブチレングリコールと水及びエタノールの少なくとも１種とを含有する抽出溶媒とを接触させ、前記スプラウトに含有される成分を抽出する製造方法により得られたヤナギタデスプラウト抽出物は、他の溶媒で抽出した場合と比較して、エラスターゼ及びコラゲナーゼ及びゼラチナーゼのすべての酵素に対する優れた活性阻害作用を示す。 In the willow desprout extract of the present invention, the extraction solvent is preferably a mixed solvent containing 1,3-butylene glycol as an essential solvent and further containing at least one of water and ethanol. Yanagita sprout extract obtained by a production method of contacting a sprout of willow tree with an extraction solvent containing 1,3-butylene glycol and at least one of water and ethanol and extracting the components contained in the sprout Shows superior activity inhibitory action on all enzymes of elastase, collagenase and gelatinase, as compared with the case of extraction with other solvents.
なお、１，３−ブチレングリコールを単独で抽出溶媒として使用し、ヤナギタデスプラウト抽出物を得た場合、当該抽出物によるエラスターゼ、コラゲナーゼ及びゼラチナーゼに対する活性阻害作用は上記混合溶媒と比較して小さい。
このように抽出溶媒として１，３−ブチレングリコール単独でなく、水及びエタノールの少なくとも１種を含有する混合溶媒とすることにより、エラスターゼ、コラゲナーゼ及びゼラチナーゼに対する活性阻害作用が向上する理由については、現在のところ完全に明らかではないが、１，３−ブチレングリコールと、極性の異なる水やエタノールとの相乗作用により、ヤナギタデスプラウトから、エラスターゼ、コラゲナーゼ及びゼラチナーゼに対する活性阻害作用を有する成分が効率的に抽出されるものと推測される。
なお、水やエタノールは、ヤナギタデスプラウトと抽出溶媒とを接触させる際に、１，３−ブチレングリコールと共存する必要があり、１，３−ブチレングリコール単独で抽出したヤナギタデスプラウト抽出物に、水やエタノールを添加したり、水やエタノールにより抽出したヤナギタデスプラウト抽出物に、１，３−ブチレングリコールを添加しても優れたコラゲナーゼ活性阻害作用及びエラスターゼ活性阻害作用はほとんど示さない。
In addition, when 1,3-butylene glycol is used alone as an extraction solvent to obtain a Yanagita desprout extract, the activity-inhibiting action against elastase, collagenase and gelatinase by the extract is small compared to the above mixed solvent.
The reason why the activity inhibiting action on elastase, collagenase and gelatinase is improved by using a mixed solvent containing at least one of water and ethanol instead of 1,3-butylene glycol alone as the extraction solvent is as follows. However, it is not completely clear, but due to the synergistic action of 1,3-butylene glycol with water and ethanol having different polarities, components having an inhibitory action on elastase, collagenase and gelatinase are efficiently extracted from the willow moth sprout. Presumed to be.
In addition, water or ethanol needs to coexist with 1,3-butylene glycol when bringing the willow desprout into contact with the extraction solvent, and water or ethanol is added to the willow desprout extract extracted with 1,3-butylene glycol alone. Even if ethanol is added or 1,3-butylene glycol is added to a Yanagita dessprout extract extracted with water or ethanol, excellent collagenase activity inhibitory activity and elastase activity inhibitory activity are hardly exhibited.
また、水、エタノール、１，３−ブチレングリコールは、皮膚外用剤、化粧料組成物として適用可能である溶媒である。上記混合溶媒を使用する場合、水、エタノールのみでは粘度が低すぎる場合があるが、適度な粘度を有する１，３−ブチレングリコールにより、保湿効果も加わるため、皮膚外用剤、化粧料組成物として好適である。 Water, ethanol, and 1,3-butylene glycol are solvents that can be applied as an external preparation for skin and a cosmetic composition. In the case of using the above mixed solvent, the viscosity may be too low only with water and ethanol, but 1,3-butylene glycol having an appropriate viscosity also adds a moisturizing effect, so that the skin external preparation and cosmetic composition Is preferred.
エラスターゼ及びコラゲナーゼに対する、より優れた活性阻害作用を得るためには、抽出溶媒における１，３−ブチレングリコールの割合が、２０体積％以上８０体積％以下であることが好適であり、より好適には４０体積％以上８０体積％以下である。
なお、水とエタノールの割合は任意であるが、水とエタノールの合計を１００体積％としたときに、４０体積％以上６０体積％以下であることが好ましい。
In order to obtain a superior activity inhibitory action against elastase and collagenase, the proportion of 1,3-butylene glycol in the extraction solvent is preferably 20% by volume or more and 80% by volume or less, and more preferably It is 40 volume% or more and 80 volume% or less.
In addition, the ratio of water and ethanol is arbitrary, but when the total of water and ethanol is 100% by volume, it is preferably 40% by volume or more and 60% by volume or less.
また、同様にゼラチナーゼに対する活性阻害作用を得るためには、抽出溶媒における１，３−ブチレングリコールの割合が、２０体積％以上８０体積％以下であることが好適であり、より好適には４０体積％以上８０体積％以下である。
なお、水とエタノールの割合は任意であるが、水とエタノールの合計を１００体積％としたときに、４０体積％以上６０体積％以下であることが好ましい。
Similarly, in order to obtain an activity inhibiting action on gelatinase, the proportion of 1,3-butylene glycol in the extraction solvent is preferably 20% by volume or more and 80% by volume or less, and more preferably 40% by volume. % To 80% by volume.
In addition, the ratio of water and ethanol is arbitrary, but when the total of water and ethanol is 100% by volume, it is preferably 40% by volume or more and 60% by volume or less.
また、本発明の酵素活性阻害剤の使用形態として、経口摂取を想定する場合には、抽出溶媒が水及びエタノールの少なくとも１種であることが好ましい。この場合、水及びエタノールの割合は任意であるが、水とエタノールの合計を１００体積％としたときに、４０体積％以上６０体積％以下であることが好ましい。 Moreover, when assuming oral intake as a usage form of the enzyme activity inhibitor of the present invention, the extraction solvent is preferably at least one of water and ethanol. In this case, although the ratio of water and ethanol is arbitrary, when the sum total of water and ethanol is 100 volume%, it is preferable that they are 40 volume% or more and 60 volume% or less.
ヤナギタデスプラウトから抽出物を抽出する方法は特に限定されず、常法に従って行なわれる。例えば、ヤナギタデスプラウトと、抽出溶媒とを互いに充分に攪拌し混合後、ヤナギタデスプラウトから溶媒中に、有効成分が十分に抽出されるまで一定期間静置する。抽出時間は、抽出溶媒の種類や、ヤナギタデスプラウトと抽出溶媒との割合によっても変化するが、０．５〜３時間程度である。
また、抽出物に含まれる残渣を取り除くため、濾過や遠心分離を行ってもよい。また、得られた抽出液はそのまま利用してもよいが、常法に従って希釈、濃縮、乾燥、精製等の処理を施してもよい。
なお、この抽出操作は品質劣化を避けるために常温で行うのが好ましいが、抽出効率を上げるために加温状態にして行うことも可能である。また、必要に応じてヤナギタデスプラウト抽出物の割合を高めるため、減圧濃縮や凍結乾燥により溶媒除去してもよい。
The method for extracting the extract from the willow sprout is not particularly limited, and is performed according to a conventional method. For example, a willow desprout and an extraction solvent are sufficiently stirred and mixed with each other, and then allowed to stand for a certain period until the active ingredient is sufficiently extracted from the willow desprout into the solvent. Although extraction time changes also with the kind of extraction solvent, and the ratio of a willow desprout and an extraction solvent, it is about 0.5 to 3 hours.
Moreover, in order to remove the residue contained in the extract, filtration or centrifugation may be performed. Moreover, although the obtained extract may be used as it is, it may be subjected to treatments such as dilution, concentration, drying and purification according to a conventional method.
This extraction operation is preferably performed at room temperature in order to avoid quality deterioration, but can also be performed in a warmed state in order to increase extraction efficiency. Further, if necessary, the solvent may be removed by concentration under reduced pressure or lyophilization in order to increase the proportion of the willow sprout extract.
抽出溶媒は上述の通り、ヤナギタデスプラウトに含有される、エラスターゼ活性阻害作用、コラゲナーゼ活性阻害作用及びゼラチナーゼ活性阻害作用を発現する有効成分を抽出できるものであればよい。
好適なヤナギタデスプラウト抽出物の製造方法を例示すると、ヤナギタデのスプラウトと、１，３−ブチレングリコールと水及びエタノールの少なくとも１種とを含有する抽出溶媒とを接触させ、前記スプラウトに含有される成分を抽出する方法が挙げられる。１，３−ブチレングリコールと水及びエタノールの少なくとも１種とを含有する抽出溶媒を使用することにより、エラスターゼ、コラゲナーゼ及びゼラチナーゼに対する、より優れた阻害活性を有する抽出物を得ることができる。この製造方法の場合、抽出溶媒におけるブチレングリコールの割合が、２０体積％以上８０体積％以下が好適であり、より好適には４０体積％以上８０体積％以下である。
As described above, the extraction solvent is not particularly limited as long as it can extract an active ingredient that expresses elastase activity inhibitory activity, collagenase activity inhibitory activity, and gelatinase activity inhibitory activity, contained in the willow sprout.
An example of a suitable method for producing a willow desprout extract is as follows: a sprout of willow tide is brought into contact with an extraction solvent containing 1,3-butylene glycol, water and at least one of ethanol, and the components contained in the sprout The method of extracting is mentioned. By using an extraction solvent containing 1,3-butylene glycol and at least one of water and ethanol, an extract having more excellent inhibitory activity against elastase, collagenase and gelatinase can be obtained. In the case of this production method, the proportion of butylene glycol in the extraction solvent is preferably 20% by volume to 80% by volume, and more preferably 40% by volume to 80% by volume.
ヤナギタデスプラウトに対する抽出溶媒の量は特に制限はないが、通常、ヤナギタデスプラウトに対して重量比で１〜１００倍量程度である。抽出溶媒に、１，３−ブチレングリコールと水及びエタノールの少なくとも１種とを含有する抽出溶媒を使用する場合には、ヤナギタデスプラウトに対して重量比で５〜５０倍量であることが好ましい。 The amount of the extraction solvent with respect to the willow desprout is not particularly limited, but is usually about 1 to 100 times the weight ratio with respect to the willow desprout. When an extraction solvent containing 1,3-butylene glycol and at least one of water and ethanol is used as the extraction solvent, the amount is preferably 5 to 50 times by weight with respect to Yanagita desprout.
本発明のヤナギタデスプラウト抽出物は、エラスターゼ、コラゲナーゼ及びゼラチナーゼに対する阻害作用を併せ持つと共に、人体に対する毒性や刺激性が少ない。そのため、各種外用剤、服用剤、化粧料組成物、機能性食品等の用途に使用することができる。なお、本発明のヤナギタデスプラウト抽出物の酵素活性阻害作用は、ヒト由来のエラスターゼ、コラゲナーゼ及びゼラチナーゼに限定されるわけではない。そのため、直接的にヒトに使用する応用品のみならず、ヒト由来以外のエラスターゼ、コラゲナーゼ及びゼラチナーゼ阻害作用を利用した応用品にも使用できる。 The willow desprout extract of the present invention has an inhibitory action on elastase, collagenase and gelatinase, and is less toxic and irritating to the human body. Therefore, it can be used for various external preparations, medications, cosmetic compositions, functional foods and the like. In addition, the enzyme activity inhibitory action of the willow desprout extract of the present invention is not limited to human-derived elastase, collagenase and gelatinase. Therefore, it can be used not only for an application product directly used for humans but also for an application product utilizing elastase, collagenase and gelatinase inhibitory effects other than those derived from humans.
＜２．酵素活性阻害剤＞
本発明の酵素活性阻害剤は、ヤナギタデスプラウト由来成分を有効成分として含有し、エラスターゼ、コラゲナーゼ及びゼラチナーゼから選択される少なくとも１種の酵素に対する活性阻害作用を有する。すなわち、本発明の酵素活性阻害剤は、ヤナギタデスプラウト由来成分を有効成分として含有する、エラスターゼ活性阻害剤、コラゲナーゼ活性阻害剤、ゼラチナーゼ活性阻害剤として使用することができる。また、本発明の酵素活性阻害剤は、エラスターゼ、コラゲナーゼ及びゼラチナーゼのすべてに対する活性阻害作用を有する酵素活性阻害剤として使用することもできる。
<2. Enzyme activity inhibitor>
The enzyme activity inhibitor of the present invention contains a Yanagita desprout-derived component as an active ingredient, and has an activity inhibiting action on at least one enzyme selected from elastase, collagenase and gelatinase. That is, the enzyme activity inhibitor of the present invention can be used as an elastase activity inhibitor, a collagenase activity inhibitor, or a gelatinase activity inhibitor containing a Yanagita desprout-derived component as an active ingredient. Moreover, the enzyme activity inhibitor of this invention can also be used as an enzyme activity inhibitor which has the activity inhibitory effect with respect to all of elastase, collagenase, and gelatinase.
ここで、本発明の酵素活性阻害剤が含有する「ヤナギタデスプラウト由来成分」は、ヤナギタデスプラウト未加工品、加工品に含まれる成分のうち、エラスターゼ、コラゲナーゼ及びゼラチナーゼから選択される少なくとも１種の酵素に対する阻害作用を有する成分である。すなわち、本発明の酵素活性阻害剤に含有されるヤナギタデスプラウト由来成分は、エラスターゼ、コラゲナーゼ及びゼラチナーゼから選択される少なくとも１種の酵素に対する阻害作用を有する限り、その態様には任意であり、上述したヤナギタデスプラウト抽出物のみならず、未処理のヤナギタデスプラウト及びその細断物、ヤナギタデスプラウトの搾り汁やヤナギタデスプラウト乾燥物等のヤナギタデスプラウト未加工品、加工品の形態でもよい。 Here, the “Yanagita desprout-derived component” contained in the enzyme activity inhibitor of the present invention is at least one enzyme selected from elastase, collagenase, and gelatinase among components contained in unprocessed and processed products It is a component that has an inhibitory action on. That is, as long as the component derived from the Yanagita desprout contained in the enzyme activity inhibitor of the present invention has an inhibitory action on at least one enzyme selected from elastase, collagenase and gelatinase, the embodiment is optional and has been described above. It may be in the form of unprocessed and processed products such as unwashed willow-destroy sprout and its unprocessed willow-tasted sprout and its shredded products, squeezed juice of willow-toothed sprout and dried willow.
但し、本発明の酵素活性阻害剤におけるヤナギタデスプラウト由来成分として、エラスターゼ、コラゲナーゼ及びゼラチナーゼに対する阻害作用を有する有効成分の含有率が高く、これらの酵素に対する阻害作用に優れる点で、ヤナギタデスプラウト抽出物がより好適である。そのため、本発明の酵素活性阻害剤は、有効成分としてヤナギタデスプラウト抽出物を含有することが好ましい。
また、上述の通り、ヤナギタデスプラウト抽出物は、エラスターゼ、コラゲナーゼ及びゼラチナーゼに対する阻害作用を併せ持つため、これを有効成分として含有する、エラスターゼ、コラゲナーゼ及びゼラチナーゼのすべてに対する活性阻害作用を有する酵素活性阻害剤として好適に使用することができる。また、本発明の酵素活性阻害剤は、有効成分としてヤナギタデスプラウト抽出物を含有する場合でも、エラスターゼ活性阻害剤、コラゲナーゼ活性阻害剤、ゼラチナーゼ活性阻害剤としても使用することができる。
However, in the enzyme activity inhibitor of the present invention, as a component derived from the willow fly desprout, the content of active ingredients having an inhibitory action on elastase, collagenase and gelatinase is high, and the Yanagita desprout extract is superior in an inhibitory action on these enzymes. More preferred. Therefore, it is preferable that the enzyme activity inhibitor of the present invention contains a willow red sprout extract as an active ingredient.
In addition, as described above, the willow desprout extract has an inhibitory action on elastase, collagenase and gelatinase, and therefore contains this as an active ingredient, and as an enzyme activity inhibitor having an activity inhibitory action on all of elastase, collagenase and gelatinase. It can be preferably used. In addition, the enzyme activity inhibitor of the present invention can be used as an elastase activity inhibitor, collagenase activity inhibitor, or gelatinase activity inhibitor even when it contains a Yanagita desprout extract as an active ingredient.
本発明の酵素活性阻害剤におけるエラスターゼ活性阻害作用、コラゲナーゼ活性阻害作用及びゼラチナーゼ阻害作用は、有効成分であるヤナギタデスプラウト由来成分の量に依存し、エラスターゼ、コラゲナーゼ及びゼラチナーゼのいずれかの酵素の活性阻害率が３０％以上（好適には４０％以上）となる範囲で決定される。より好適には、エラスターゼ、コラゲナーゼ及びゼラチナーゼのすべての酵素に対する活性阻害率が４０％以上となる範囲で決定される。
なお、本発明の酵素活性阻害剤は、上述の通り、エラスターゼ活性阻害剤、コラゲナーゼ活性阻害剤、又はゼラチナーゼ活性阻害剤として用いることもできる。
本発明の酵素活性阻害剤の有効成分として、ヤナギタデスプラウト抽出物を使用されている場合、ヤナギタデスプラウト抽出物は、コラゲナーゼ活性阻害作用が高いため、コラゲナーゼ活性阻害剤として好適である。また、ヤナギタデスプラウト抽出物は、ゼラチナーゼ活性阻害作用が高いため、ゼラチナーゼ活性阻害剤として好適である。
The elastase activity inhibitory action, collagenase activity inhibitory action and gelatinase inhibitory action in the enzyme activity inhibitor of the present invention depend on the amount of the component derived from the Yanagita desprout which is an active ingredient, and the activity inhibition of any enzyme of elastase, collagenase and gelatinase The rate is determined within a range of 30% or more (preferably 40% or more). More preferably, the activity inhibition rate for all enzymes of elastase, collagenase and gelatinase is determined within a range of 40% or more.
In addition, the enzyme activity inhibitor of this invention can also be used as an elastase activity inhibitor, a collagenase activity inhibitor, or a gelatinase activity inhibitor as above-mentioned.
In the case where a Yanagita dessprout extract is used as an active ingredient of the enzyme activity inhibitor of the present invention, the Yanagita desprout extract is suitable as a collagenase activity inhibitor because of its high collagenase activity inhibitory action. In addition, the willow moth sprout extract is suitable as a gelatinase activity inhibitor because it has a high gelatinase activity inhibitory action.
本発明の酵素活性阻害剤の有効成分として、ヤナギタデスプラウト抽出物を使用されている場合、ヤナギタデスプラウト抽出物を、原液をそのまま用いても、濃縮して濃縮液として用いてもよく、原液あるいは濃縮液を希釈溶媒に溶解して使用してもよい。この希釈溶媒としては、酵素活性阻害作用を著しく損なわないものであれば特に限定されるものではなく、水、エチルアルコール、イソプロピルアルコール、グリセリン、プロピレングリコール、１，３−ブチレングリコール等を例示することができる。 In the case where a willow moth sprout extract is used as an active ingredient of the enzyme activity inhibitor of the present invention, the willow moth sprout extract may be used as a stock solution as it is, or may be concentrated and used as a concentrated solution. The solution may be used by dissolving in a diluting solvent. The diluting solvent is not particularly limited as long as it does not significantly impair the enzyme activity inhibiting action, and examples thereof include water, ethyl alcohol, isopropyl alcohol, glycerin, propylene glycol, 1,3-butylene glycol and the like. Can do.
本発明の酵素活性阻害剤の形態としては特に限定はないが、一般に皮膚に塗布する形の皮膚外用剤として用いられる場合には、液状やクリーム状である。この場合、酵素活性阻害剤は、必要に応じて、通常医薬品、医薬部外品に配合される、油性成分、可溶化剤、保湿剤、色素、乳化剤、増粘剤、香料等の任意の成分を含有することができる。また、本発明の効果を損なわない範囲で、他の薬効植物抽出物を添加してもよい。 The form of the enzyme activity inhibitor of the present invention is not particularly limited, but when used as a skin external preparation generally applied to the skin, it is liquid or creamy. In this case, the enzyme activity inhibitor is an optional component such as an oily component, a solubilizer, a moisturizer, a dye, an emulsifier, a thickener, a fragrance, etc. Can be contained. Moreover, you may add another medicinal plant extract in the range which does not impair the effect of this invention.
また、本発明の酵素活性阻害剤は、入浴剤、ボディーソープ、シャンプー等の入浴用組成物に配合して用いてもよい。剤型としては、一般に用いられる、水溶液、Ｗ／Ｏ型又はＯ／Ｗ型エマルション、適当な賦形剤等を用いて顆粒剤その他の粉末、錠剤等としてもよい。また、本発明の効果を損なわない範囲で、他の薬効植物抽出物を添加してもよい。 In addition, the enzyme activity inhibitor of the present invention may be used in a bath composition such as a bath agent, body soap, or shampoo. The dosage form may be a granule or other powder, tablet or the like using a commonly used aqueous solution, W / O type or O / W type emulsion, an appropriate excipient and the like. Moreover, you may add another medicinal plant extract in the range which does not impair the effect of this invention.
また、本発明の酵素活性阻害剤は経口用組成物とすることもできる。この場合、経口投与に利用される剤形としては、具体的には、固形製剤として、粉末剤、顆粒剤、錠剤、カプセル剤、トローチ等が挙げられる。また、液状製剤として内用液剤、外用液剤、懸濁剤、乳剤、シロップ剤等が例示され、これら剤形やその他の剤形が目的に応じて適宜選択される。また、必要に応じて、医薬品・医薬部外品・食品などに配合される任意の成分を含有することができる。また、本発明の効果を損なわない範囲で、他の薬効植物抽出物を添加してもよい。 Moreover, the enzyme activity inhibitor of this invention can also be used as an oral composition. In this case, specific examples of dosage forms used for oral administration include solid preparations, powders, granules, tablets, capsules, troches and the like. Examples of liquid preparations include internal liquids, external liquids, suspensions, emulsions, syrups and the like, and these and other dosage forms are appropriately selected according to the purpose. In addition, it can contain any component that is blended in pharmaceuticals, quasi-drugs, foods, and the like as necessary. Moreover, you may add another medicinal plant extract in the range which does not impair the effect of this invention.
（抗老化剤）
また、本発明の抗老化剤は上記本発明の酵素阻害剤を含有してなり、エラスターゼ、コラゲナーゼ、及びゼラチナーゼの少なくとも一つの酵素に対する活性阻害作用（好適にはエラスターゼ、コラゲナーゼ、及びゼラチナーゼのすべての酵素に対する活性阻害作用）に起因して抗老化作用を有する。
ここで、本発明における「抗老化作用」とは、皮膚のしわ、たるみなどを有効に防止・改善などを含む概念である。
本発明の抗老化剤は、上記本発明の酵素阻害剤をそのまま使用してもよく、従来の公知の抗老化剤に使用される任意の成分を含有していてもよい。
(Anti-aging agent)
The anti-aging agent of the present invention comprises the enzyme inhibitor of the present invention, and has an activity inhibitory action on at least one of elastase, collagenase, and gelatinase (preferably all of elastase, collagenase, and gelatinase). It has an anti-aging action due to the activity inhibition action on the enzyme).
Here, the “anti-aging effect” in the present invention is a concept including effectively preventing and improving skin wrinkles and sagging.
The anti-aging agent of the present invention may use the enzyme inhibitor of the present invention as it is, or may contain any component used for a conventionally known anti-aging agent.
本発明の抗老化剤の形態としては特に限定はないが、一般に皮膚に塗布する形の皮膚外用剤として用いられる場合には、液状やクリーム状である。この場合、抗老化剤は、必要に応じて、通常医薬品、医薬部外品に配合される、油性成分、可溶化剤、保湿剤、色素、乳化剤、増粘剤、香料等の任意の成分を含有することができる。また、本発明の効果を損なわない範囲で、他の薬効植物抽出物を添加してもよい。 The form of the anti-aging agent of the present invention is not particularly limited, but when used as a skin external preparation generally applied to the skin, it is liquid or creamy. In this case, the anti-aging agent is an optional component such as an oily component, a solubilizer, a moisturizer, a dye, an emulsifier, a thickener, and a fragrance, which are usually blended in pharmaceuticals and quasi drugs. Can be contained. Moreover, you may add another medicinal plant extract in the range which does not impair the effect of this invention.
また、本発明の抗老化剤は、入浴剤、ボディーソープ、シャンプー等の入浴用組成物に配合して用いてもよい。剤型としては、一般に用いられる、水溶液、Ｗ／Ｏ型又はＯ／Ｗ型エマルション、適当な賦形剤等を用いて顆粒剤その他の粉末、錠剤等としてもよい。また、本発明の効果を損なわない範囲で、他の薬効植物抽出物を添加してもよい。 Moreover, you may mix | blend and use the anti-aging agent of this invention for bathing compositions, such as a bath agent, a body soap, and a shampoo. The dosage form may be a granule or other powder, tablet or the like using a commonly used aqueous solution, W / O type or O / W type emulsion, an appropriate excipient and the like. Moreover, you may add another medicinal plant extract in the range which does not impair the effect of this invention.
また、本発明の抗老化剤は経口用組成物とすることもできる。この場合、経口投与に利用される剤形としては、具体的には、固形製剤として、粉末剤、顆粒剤、錠剤、カプセル剤、トローチ等が挙げられる。また、液状製剤として内用液剤、外用液剤、懸濁剤、乳剤、シロップ剤等が例示され、これら剤形やその他の剤形が目的に応じて適宜選択される。また、必要に応じて、医薬品・医薬部外品・食品などに配合される任意の成分を含有することができる。また、本発明の効果を損なわない範囲で、他の薬効植物抽出物を添加してもよい。 Moreover, the anti-aging agent of this invention can also be used as an oral composition. In this case, specific examples of dosage forms used for oral administration include solid preparations, powders, granules, tablets, capsules, troches and the like. Examples of liquid preparations include internal liquids, external liquids, suspensions, emulsions, syrups and the like, and these and other dosage forms are appropriately selected according to the purpose. In addition, it can contain any component that is blended in pharmaceuticals, quasi-drugs, foods, and the like as necessary. Moreover, you may add another medicinal plant extract in the range which does not impair the effect of this invention.
（化粧料組成物）
本発明の化粧料組成物は、上記本発明の酵素阻害剤を配合してなることを特徴とする。上述の通り、本発明の酵素阻害剤に含まれるヤナギタデスプラウト由来の有効成分（特にはヤナギタデスプラウト抽出物）は、エラスターゼ、コラゲナーゼ及びゼラチナーゼに対する阻害作用を併せ持ち、これらの酵素に対する活性阻害作用に起因する抗老化作用を有し、かつ、各種化粧料基材及び化粧料添加物に対する安定性も高いため、化粧料組成物に好適に使用することができる。
(Cosmetic composition)
The cosmetic composition of the present invention is characterized by blending the enzyme inhibitor of the present invention. As described above, the active ingredient derived from the willow fly sprout contained in the enzyme inhibitor of the present invention (particularly the extract of the willow fly sprout) has an inhibitory action on elastase, collagenase and gelatinase, and is attributable to the activity inhibitory action on these enzymes. Since it has an anti-aging action and has high stability to various cosmetic base materials and cosmetic additives, it can be suitably used in a cosmetic composition.
化粧料組成物への本発明の酵素阻害剤の配合割合は任意であるが、化粧料組成物が抗老化作用を十分に発揮する範囲で適宜選択される。本発明の酵素阻害剤の有効成分として、ヤナギタデスプラウト抽出物を使用されている場合、ヤナギタデスプラウト抽出物の化粧料組成物に対する割合は、ヤナギタデスプラウト抽出物における溶媒の割合や溶媒の種類等にもよるが、例えば、化粧料組成物の形態がジェルの場合は１〜５０重量％程度、形態が乳液の場合は１〜５０重量％程度、形態がクリームの場合は１〜５０重量％程度である。 The blending ratio of the enzyme inhibitor of the present invention to the cosmetic composition is arbitrary, but is appropriately selected within the range where the cosmetic composition sufficiently exhibits the anti-aging action. In the case where a willow moth sprout extract is used as an active ingredient of the enzyme inhibitor of the present invention, the proportion of the willow moth sprout extract to the cosmetic composition is also the ratio of the solvent or the kind of the solvent in the willow moth sprout extract. However, for example, when the form of the cosmetic composition is gel, it is about 1 to 50% by weight. When the form is emulsion, it is about 1 to 50% by weight. When the form is cream, it is about 1 to 50% by weight. .
本発明の化粧料組成物は、慣用の化粧料基材を適宜配合し、所望の剤型とすることができる。その形態は特に制限はないが、ジェル、乳液、クリーム等の形態が挙げられる。 The cosmetic composition of the present invention can be made into a desired dosage form by appropriately blending a conventional cosmetic base material. The form is not particularly limited, and examples thereof include gels, emulsions, and creams.
化粧料基材としては、目的とする剤型に合わせて、水、水溶性有機溶媒、油脂、ロウ、高級脂肪酸、高級アルコール、エステル類等の従来公知の化粧料基材が適宜選択される。
また、本発明の化粧料組成物には、本発明の効果を損なわない範囲で通常化粧料や皮膚外用医薬で使用される任意の成分を添加することができる。かかる任意成分の具体例としては、増粘・ゲル化剤、酸化防止剤、紫外線吸収剤、色素剤、金属封鎖剤、防腐剤、ｐＨ調整剤、香料、ミツロウ等が挙げられる。これら任意成分の配合割合は、その目的に応じて適宜選択して決定することができる。また、本発明の効果を損なわない範囲で、他の薬効植物抽出物を添加してもよい。
As the cosmetic base material, conventionally known cosmetic base materials such as water, water-soluble organic solvents, fats and oils, waxes, higher fatty acids, higher alcohols, and esters are appropriately selected according to the intended dosage form.
Moreover, in the cosmetic composition of this invention, the arbitrary components normally used by cosmetics and a skin external medicine can be added in the range which does not impair the effect of this invention. Specific examples of such optional components include thickening / gelling agents, antioxidants, ultraviolet absorbers, pigments, metal sequestering agents, preservatives, pH adjusters, fragrances, beeswax and the like. The blending ratio of these optional components can be selected and determined as appropriate according to the purpose. Moreover, you may add another medicinal plant extract in the range which does not impair the effect of this invention.
（機能性食品）
一方、日常的に飲食することで、本発明の酵素阻害剤を摂取したい場合には、本発明の酵素阻害剤を食品、飲料に含有させて機能性食品としてもよい。
ここでいう「機能性食品」とは、一般食品に加えて、健康食品、栄養補助食品、栄養機能食品、栄養保険食品等、健康の維持の目的で摂取する食品および／又は飲料を意味している。なお、機能性食品として製品化する場合には、食品に用いられる様々な添加剤、具体的には、着色料、保存料、増粘安定剤、酸化防止剤漂白剤、防菌防黴剤、酸味料、調味料、乳化剤、強化剤、製造用剤、香料等を添加していてもよい。
(Functional food)
On the other hand, when it is desired to ingest the enzyme inhibitor of the present invention by eating and drinking on a daily basis, the enzyme inhibitor of the present invention may be contained in foods and beverages to make a functional food.
The term “functional food” as used herein means foods and / or beverages ingested for the purpose of maintaining health, such as health foods, nutritional supplements, functional nutritional foods, nutrition insurance foods, in addition to general foods. Yes. In addition, when commercializing as a functional food, various additives used in food, specifically, coloring agents, preservatives, thickening stabilizers, antioxidant bleaching agents, antibacterial and antifungal agents, A sour agent, a seasoning, an emulsifier, a reinforcing agent, a manufacturing agent, a fragrance and the like may be added.
本発明の機能性食品の対象となる、食品、飲料は特に限定されるものではない。例えば、食品として、ソーセージ、ハム、魚介加工品、ゼリー、キャンディー、チューインガムなどの食品類が挙げられる。また、飲料としては、各種の茶類、清涼飲料水、酒類、栄養ドリンクなどが挙げられる。この中でも、茶、ゼリーであることが特に好ましい。
本発明の酵素阻害剤は、このような食品、飲料に添加することにより、簡易に経口摂取することができる。
The foods and beverages that are the targets of the functional food of the present invention are not particularly limited. Examples of food include foods such as sausage, ham, processed seafood, jelly, candy, and chewing gum. Examples of the beverage include various teas, soft drinks, alcoholic beverages, and energy drinks. Among these, tea and jelly are particularly preferable.
The enzyme inhibitor of the present invention can be easily taken orally by adding to such foods and beverages.
本発明の機能性食品に対する本発明の酵素阻害剤の配合量は、その機能性食品の種類によって異なり、エラスターゼ、コラゲナーゼ、及びゼラチナーゼの少なくとも一つの酵素に対する活性阻害作用（好適にはエラスターゼ、コラゲナーゼ、及びゼラチナーゼのすべての酵素に対する活性阻害作用）が発現するように適宜調整される。 The compounding amount of the enzyme inhibitor of the present invention with respect to the functional food of the present invention varies depending on the type of the functional food, and it inhibits the activity of at least one of elastase, collagenase, and gelatinase (preferably elastase, collagenase, And the activity inhibitory action of gelatinase on all enzymes) is appropriately adjusted.
以下、実施例により本発明を更に詳細に説明するが、本発明は、その要旨を変更しない限り以下の実施例に限定されるものではない。なお、以下において、「１，３−ブチレングリコール」を「ＢＧ」と記載する場合がある。 EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to a following example, unless the summary is changed. In the following, “1,3-butylene glycol” may be referred to as “BG”.
＜１．ヤナギタデスプラウトの調製＞
ヤナギタデ（「ＪＡ筑前あさくら」の紅たで部会から購入）のスプラウト（芽の部分）を乾燥して得られたヤナギタデスプラウトの乾燥物を、ミクロパウダー（ウェスト社製）で十分に粉砕し、ヤナギタデスプラウト乾燥粉砕物を得た。
<1. Preparation of Yanagita Desprout>
The dried sprout (bud part) of Yanagitade ("JA Chikuzen Asakuri" red sprout) was dried and thoroughly dried with Micropowder (West). Yanagita desprout dried pulverized product was obtained.
＜２．評価方法＞
（１）エラスターゼ活性阻害率の評価
エラスターゼ活性阻害作用の評価は、ブタ膵臓由来エラスターゼ及びヒト白血球由来エラスターゼの２種類のエラスターゼを使用して行った。
（１−１）ブタ膵臓由来エラスターゼ活性阻害率の評価
ブタ膵臓由来エラスターゼ（SIGMA-ALDRICH社製）および合成基質スクシニル-Ｌ-アラニル-Ｌ-アラニル-Ｌ-アラニン-ｐ-ニトロアニリド）（SIGMA-ALDRICH社製）を使用した。まず、基質をジメチルスルホキシドで０．０５Ｍに調製し、使用時に、０.２Ｍ Ｔｒｉｓ-ＨＣｌ緩衝液（ｐＨ８．０）で１０倍希釈した５ｍＭ溶液を使用した。酵素は０.２Ｍ Ｔｒｉｓ-ＨＣｌ緩衝液（ｐＨ８．０）で１０μｇ/ｍＬに調製した。基質５０μＬを酵素１００μＬおよび試料５０μＬとともに３７℃で８分間反応させ、その後、吸光度４０５ｎｍで測定した（Ａ）。さらに酵素を添加せずに緩衝液を加えて同様の操作と測定を行った（Ｂ）。同様の測定を、試料を添加せずに抽出溶媒を加えて実施した（Ｃ、Ｄ）。上記方法で吸光度を求め、下記式（１）により、エラスターゼ活性阻害率を算出した。
<2. Evaluation method>
(1) Evaluation of elastase activity inhibition rate The elastase activity inhibition action was evaluated using two types of elastases, porcine pancreatic elastase and human leukocyte-derived elastase.
(1-1) Evaluation of porcine pancreatic elastase activity inhibition rate Porcine pancreatic elastase (manufactured by SIGMA-ALDRICH) and synthetic substrate succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide) (SIGMA- ALDRICH) was used. First, the substrate was adjusted to 0.05 M with dimethyl sulfoxide, and at the time of use, a 5 mM solution diluted 10-fold with 0.2 M Tris-HCl buffer (pH 8.0) was used. The enzyme was adjusted to 10 μg / mL with 0.2 M Tris-HCl buffer (pH 8.0). 50 μL of the substrate was reacted with 100 μL of the enzyme and 50 μL of the sample at 37 ° C. for 8 minutes, and then the absorbance was measured at 405 nm (A). Further, a buffer solution was added without adding the enzyme, and the same operation and measurement were performed (B). The same measurement was performed by adding an extraction solvent without adding a sample (C, D). Absorbance was calculated | required with the said method, and the elastase activity inhibition rate was computed by following formula (1).
（１−２）ヒト白血球由来エラスターゼ活性阻害率の評価
上記「（１−１）ブタ膵臓由来エラスターゼ活性阻害率の評価」において、ブタ膵臓由来エラスターゼに代えて、ヒト白血球由来エラスターゼ（SIGMA-ALDRICH社製）を使用し、ヒト白血球由来エラスターゼ活性阻害率を評価した。
まず、基質であるスクシニル-Ｌ-アラニル-Ｌ-アラニル-Ｌ-アラニン-ｐ-ニトロアニリド）（SIGMA-ALDRICH社製）をジメチルスルホキシドで０．０５Ｍに調製し、使用時に、０.２Ｍ Ｔｒｉｓ-ＨＣｌ緩衝液（ｐＨ８．０）で１０倍希釈した５ｍＭ溶液を使用した。酵素は０.２Ｍ Ｔｒｉｓ-ＨＣｌ緩衝液（ｐＨ８．０）で１０μｇ/ｍＬに調製した。
基質５０μＬを酵素１００μＬおよび試料５０μＬとともに３７℃で６０分間反応させ、その後、吸光度４０５ｎｍで測定した（Ａ）。さらに酵素を添加せずに緩衝液を加えて同様の操作と測定を行った（Ｂ）。同様の測定を、試料を添加せずに抽出溶媒を加えて実施した（Ｃ、Ｄ）。上記方法で吸光度を求め、下記式（１）により、阻害率を算出した。
(1-2) Evaluation of human leukocyte-derived elastase activity inhibition rate In the above "(1-1) Evaluation of porcine pancreas-derived elastase activity inhibition rate", instead of porcine pancreas-derived elastase, human leukocyte-derived elastase (SIGMA-ALDRICH) Manufactured leukocytes) and the inhibition rate of human leukocyte-derived elastase activity was evaluated.
First, the substrate succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide) (manufactured by SIGMA-ALDRICH) was prepared to 0.05 M with dimethyl sulfoxide, and 0.2 M Tris- A 5 mM solution diluted 10-fold with HCl buffer (pH 8.0) was used. The enzyme was adjusted to 10 μg / mL with 0.2 M Tris-HCl buffer (pH 8.0).
50 μL of the substrate was reacted with 100 μL of the enzyme and 50 μL of the sample at 37 ° C. for 60 minutes, and then the absorbance was measured at 405 nm (A). Further, a buffer solution was added without adding the enzyme, and the same operation and measurement were performed (B). The same measurement was performed by adding an extraction solvent without adding a sample (C, D). Absorbance was calculated | required with the said method and the inhibition rate was computed by following formula (1).
エラスターゼ活性阻害率（％）＝[１−（Ａ−Ｂ）/（Ｃ−Ｄ）]×１００・・・（１）
（但し、Ａ：試料溶液の４０５ｎｍにおける吸光度、Ｂ：試料溶液ブランクの４０５ｎｍにおける吸光度、Ｃ：対照溶液の４０５ｎｍにおける吸光度、Ｄ：対照溶液ブランクの４０５ｎｍにおける吸光度である。）
Elastase activity inhibition rate (%) = [1− (A−B) / (C−D)] × 100 (1)
(However, A: Absorbance at 405 nm of the sample solution, B: Absorbance at 405 nm of the sample solution blank, C: Absorbance at 405 nm of the control solution, D: Absorbance at 405 nm of the control solution blank.)
（２）コラゲナーゼ活性阻害率の評価
〔試薬の調製〕
基質溶液：
Ｐｚペプチド(BACHEM社製)３．９ｍｇを、０．１Ｍトリス塩酸緩衝液(ｐＨ７．１、含２０ｍＭ塩化カルシウム)１０ｍＬに溶解して使用した(０．５ｍＭに相当)。
酵素溶液：コラゲナーゼ(TYPE IV、SIGMA-ALDRICH社製)５ｍｇを緩衝液１ｍＬに溶解させ２０μＬずつ分注し、−８０℃で保管した。使用時に緩衝液で５０倍に希釈して反応に用いた。
(2) Evaluation of collagenase activity inhibition rate [Preparation of reagents]
Substrate solution:
3.9 mg of Pz peptide (manufactured by BACHEM) was dissolved in 10 mL of 0.1 M Tris-HCl buffer (pH 7.1, containing 20 mM calcium chloride) and used (corresponding to 0.5 mM).
Enzyme solution: 5 mg of collagenase (TYPE IV, manufactured by SIGMA-ALDRICH) was dissolved in 1 mL of buffer solution, dispensed in 20 μL aliquots, and stored at −80 ° C. At the time of use, it was diluted 50 times with a buffer and used for the reaction.
〔コラゲナーゼ活性阻害作用測定法〕
これらの試料溶液５０μＬ、コラゲナーゼ溶液５０μＬ及び基質溶液４００μＬを混合し、３７℃で３０分間反応させた。次いで、２５ｍＭクエン酸溶液１ｍＬで反応を停止し、酢酸エチル５ｍＬで抽出した。遠心分離(１６００ｇ、１０分間)後、酢酸エチルを対照として、酢酸エチル層の波長３２０ｎｍにおける吸光度を測定した。対照には、試料溶液の代わりに各抽出溶媒を用い、また、それぞれのブランクとして、酵素溶液の代わりに超純水を加えて同様の操作を行った。これらの値からコラゲナーゼ活性阻害率を下記式（２）により算出した。
[Method for measuring collagenase activity inhibitory action]
These sample solutions 50 μL, collagenase solution 50 μL, and substrate solution 400 μL were mixed and reacted at 37 ° C. for 30 minutes. The reaction was then stopped with 1 mL of 25 mM citric acid solution and extracted with 5 mL of ethyl acetate. After centrifugation (1600 g, 10 minutes), the absorbance of the ethyl acetate layer at a wavelength of 320 nm was measured using ethyl acetate as a control. For the control, each extraction solvent was used in place of the sample solution, and as a blank, ultrapure water was added instead of the enzyme solution, and the same operation was performed. From these values, the collagenase activity inhibition rate was calculated by the following formula (2).
コラゲナーゼ活性阻害率(％)＝〔１−（Ａ−Ｂ）／（Ｃ−Ｄ）〕×１００・・・（２）
（但し、Ａ：試料溶液の３２０ｎｍにおける吸光度、Ｂ：試料溶液ブランクの３２０ｎｍにおける吸光度、Ｃ：対照溶液の３２０ｎｍにおける吸光度、Ｄ：対照溶液ブランクの３２０ｎｍにおける吸光度である。）
Collagenase activity inhibition rate (%) = [1- (AB) / (CD)] × 100 (2)
(However, A: Absorbance at 320 nm of the sample solution, B: Absorbance at 320 nm of the sample solution blank, C: Absorbance at 320 nm of the control solution, D: Absorbance at 320 nm of the control solution blank.)
（３）ゼラチナーゼ活性阻害率の評価
ゼラチナーゼ活性阻害作用は、ヒト線維肉腫細胞ＨＴ-１０８０（Cell No. JCRB9113）由来ゼラチナーゼ（ＭＭＰ−２、ＭＭＰ−９）およびゼラチン含有ＳＤＳ-ＰＡＳＥゲルを用いたゼラチンザイモグラフィにより評価した。HT-1080細胞を１０％ウシ胎児血清（ＦＢＳ）および１％非必須アミノ酸（ＮＥＡＡ）を含むMEM培地でサブコンフルエントになるまで培養した。MEM培地に交換し２４時間インキュベート後、遠心分離により培養上清を回収した。培養上清に２×SDS-PAGEサンプル緩衝液を等量混和し、ゼラチナーゼ溶液とした。０．２％ゼラチン含有１０％ ＳＤＳ-ＰＡＳＥゲルに、ゼラチナーゼ溶液を１レーンあたり１０μＬ入れ、室温で電気泳動を行った。レーンごとに切断し、ゲル洗浄溶液（２．５％ TritonX-100）で２回洗い、ゲル中のＳＤＳを洗い流した。切断したゲルを、あらかじめ試料を添加した反応溶液（１０ｍＭ Ｔｒｉｓ-ＨＣｌ、４０ｍＭ ＮａＣｌ、２ｍＭ ＣａＣｌ2）入りチューブに入れ、２４時間３７℃で反応を行った。対照には、試料のかわりに水を加えた。ＣＢＢ染色後、脱染したゲル画像をスキャナで取り込み画像解析ソフトImageJにより定量化した。得られた各バンドのピーク面積から下記式（３）によりゼラチナーゼ活性阻害率を求めた。
(3) Evaluation of gelatinase activity inhibition rate Gelatinase activity inhibition action was performed using gelatin derived from human fibrosarcoma cell HT-1080 (Cell No. JCRB9113) gelatinase (MMP-2, MMP-9) and gelatin-containing SDS-PASE gel. It was evaluated by zymography. HT-1080 cells were cultured in MEM medium containing 10% fetal bovine serum (FBS) and 1% non-essential amino acids (NEAA) until they became subconfluent. After replacing with MEM medium and incubating for 24 hours, the culture supernatant was collected by centrifugation. An equal volume of 2 × SDS-PAGE sample buffer was mixed with the culture supernatant to obtain a gelatinase solution. Gelatinase solution was placed in a 10% SDS-PASE gel containing 0.2% gelatin per lane, and electrophoresis was performed at room temperature. Each lane was cut and washed twice with a gel washing solution (2.5% Triton X-100) to wash away SDS in the gel. The cut gel was placed in a tube containing a reaction solution (10 mM Tris-HCl, 40 mM NaCl, 2 mM CaCl 2 ) to which a sample had been added in advance, and reacted at 37 ° C. for 24 hours. For the control, water was added instead of the sample. After CBB staining, the destained gel image was captured by a scanner and quantified by image analysis software ImageJ. The gelatinase activity inhibition rate was calculated from the peak area of each obtained band according to the following formula (3).
ゼラチナーゼ活性阻害率（％）＝１−Ａ/Ｂ・・・（３）
（但し、Ａ：試料溶液のピーク面積、Ｂ：対照溶液のピーク面積である。）
Gelatinase activity inhibition rate (%) = 1−A / B (3)
(However, A: peak area of the sample solution, B: peak area of the control solution.)
＜３．酵素活性阻害作用の評価＞
「評価１」：ブタ膵臓由来エラスターゼ及びコラゲナーゼ阻害作用の評価（単一溶媒）
抽出溶媒として、水、エタノール、１，３−ブチレングリコール（ＢＧ）のそれぞれ単種の溶媒を用いてヤナギタデスプラウト抽出物を製造し、エラスターゼ及びコラゲナーゼそれぞれの活性阻害率を評価した。
<3. Evaluation of enzyme activity inhibitory action>
"Evaluation 1": Evaluation of porcine pancreatic elastase and collagenase inhibitory action (single solvent)
As a solvent for extraction, a Yanagita desprout extract was produced using a single solvent of water, ethanol, and 1,3-butylene glycol (BG), and the activity inhibition rates of elastase and collagenase were evaluated.
実験例１（抽出溶媒：水）
ヤナギタデスプラウト１ｇ（乾燥重量）を、水１０ｍＬに浸漬し、２時間攪拌した。遠心分離後、上澄みをろ過して得られた抽出液（原液）を実験例１の抽出物とした。
実験例１の抽出物（原液）に所定量の純水を加えて適当な濃度に希釈して、上述の方法でブタ膵臓由来エラスターゼ及びコラゲナーゼの活性阻害率を評価した。実験例１の抽出物におけるエラスターゼ活性阻害率は、原液濃度５体積％で１１％であり、コラゲナーゼ活性阻害率は、原液濃度０．５体積％で３２％であった。結果を表１に示す。
Experimental Example 1 (Extraction solvent: water)
Yanagita dessprout 1 g (dry weight) was immersed in 10 mL of water and stirred for 2 hours. The extract obtained by filtering the supernatant after centrifugation (stock solution) was used as the extract of Experimental Example 1.
A predetermined amount of pure water was added to the extract (stock solution) of Experimental Example 1 to dilute to an appropriate concentration, and the activity inhibition rate of porcine pancreatic elastase and collagenase was evaluated by the method described above. The elastase activity inhibition rate in the extract of Experimental Example 1 was 11% at a stock solution concentration of 5% by volume, and the collagenase activity inhibition rate was 32% at a stock solution concentration of 0.5% by volume. The results are shown in Table 1.
実験例２（抽出溶媒：エタノール）
ヤナギタデスプラウト１ｇ（乾燥重量）を、エタノール１０ｍＬに浸漬し、２時間攪拌した。遠心分離後、上澄みをろ過して得られた抽出液（原液）を実験例２の抽出物とした。実験例２の抽出物（原液）に所定量の純水を加えて適当な濃度に希釈して、上述の方法でブタ膵臓由来エラスターゼ及びコラゲナーゼの活性阻害率を評価した。実験例２の抽出物におけるエラスターゼ活性阻害率は、原液濃度５体積％で０％であり、コラゲナーゼ活性阻害率は、原液濃度０．５体積％で４％であった。結果を表１に示す。
Experimental Example 2 (Extraction solvent: ethanol)
Yanagita desprout 1 g (dry weight) was immersed in 10 mL of ethanol and stirred for 2 hours. After centrifugation, the extract (stock solution) obtained by filtering the supernatant was used as the extract of Experimental Example 2. A predetermined amount of pure water was added to the extract (stock solution) of Experimental Example 2 to dilute to an appropriate concentration, and the activity inhibition rate of porcine pancreatic elastase and collagenase was evaluated by the method described above. The elastase activity inhibition rate in the extract of Experimental Example 2 was 0% at a stock solution concentration of 5% by volume, and the collagenase activity inhibition rate was 4% at a stock solution concentration of 0.5% by volume. The results are shown in Table 1.
実験例３（抽出溶媒：ＢＧ）
ヤナギタデスプラウト１ｇ（乾燥重量）を、ＢＧ１０ｍＬに浸漬し、２時間攪拌した。遠心分離後、上澄みをろ過して得られた抽出液（原液）を実験例３の抽出物とした。実験例３の抽出物（原液）に所定量の純水を加えて適当な濃度に希釈して、上述の方法でブタ膵臓由来エラスターゼ及びコラゲナーゼの活性阻害率を評価した。実験例３の抽出物におけるエラスターゼ活性阻害率は、原液濃度５体積％で０％であり、コラゲナーゼ活性阻害率は、原液濃度０．５体積％で２９％であった。結果を表１に示す。
Experimental Example 3 (Extraction solvent: BG)
1 g of Yanagita dessprout (dry weight) was immersed in 10 mL of BG and stirred for 2 hours. The extract obtained by filtering the supernatant after centrifugation (stock solution) was used as the extract of Experimental Example 3. A predetermined amount of pure water was added to the extract (stock solution) of Experimental Example 3 to dilute to an appropriate concentration, and the activity inhibition rates of porcine pancreatic elastase and collagenase were evaluated by the method described above. The elastase activity inhibition rate in the extract of Experimental Example 3 was 0% at a stock solution concentration of 5% by volume, and the collagenase activity inhibition rate was 29% at a stock solution concentration of 0.5% by volume. The results are shown in Table 1.
「評価２」：ブタ膵臓由来エラスターゼ及びコラゲナーゼ阻害作用の評価（混合溶媒）
抽出溶媒として、水、エタノール、１，３−ブチレングリコール（ＢＧ）の混合溶媒を用いてヤナギタデスプラウト抽出物を製造し、エラスターゼ（ブタ膵臓由来）、コラゲナーゼそれぞれの活性阻害率を評価した。
"Evaluation 2": Evaluation of porcine pancreatic elastase and collagenase inhibitory action (mixed solvent)
As the extraction solvent, a Yanagita desprout extract was produced using a mixed solvent of water, ethanol, and 1,3-butylene glycol (BG), and the activity inhibition rates of elastase (derived from porcine pancreas) and collagenase were evaluated.
実験例４（エタノール/水 混合溶媒）
ヤナギタデスプラウト１ｇ（乾燥重量）を、エタノールと水の混合溶媒１０ｍＬ（エタノール：２０〜８０体積％、水：８０〜２０体積％）に浸漬し、２時間攪拌した。遠心分離後、上澄みをろ過して得られた抽出液（原液）を実験例４の抽出物とした。実験例４の抽出物（原液）に所定量の純水を加えて適当な濃度に希釈して、上述の方法でエラスターゼ（ブタ膵臓由来）及びコラゲナーゼの活性阻害率を評価した。
図１にエタノール濃度とエラスターゼ活性阻害率の関係、図２にエタノール濃度とコラゲナーゼ活性阻害率の関係を示す。なお、活性評価における抽出物濃度（原液濃度）は、エラスターゼ活性阻害では５体積％であり、コラゲナーゼ活性阻害では２体積％である。
Experimental Example 4 (Ethanol / water mixed solvent)
1 g (dry weight) of willow moth sprout was immersed in 10 mL of a mixed solvent of ethanol and water (ethanol: 20 to 80% by volume, water: 80 to 20% by volume) and stirred for 2 hours. The extract obtained by filtering the supernatant after centrifugation (stock solution) was used as the extract of Experimental Example 4. A predetermined amount of pure water was added to the extract (stock solution) of Experimental Example 4 to dilute to an appropriate concentration, and elastase (derived from porcine pancreas) and collagenase activity inhibition rates were evaluated by the method described above.
FIG. 1 shows the relationship between ethanol concentration and elastase activity inhibition rate, and FIG. 2 shows the relationship between ethanol concentration and collagenase activity inhibition rate. In addition, the extract concentration (stock solution concentration) in activity evaluation is 5% by volume for elastase activity inhibition, and 2% by volume for collagenase activity inhibition.
実験例４の抽出物を使用したエラスターゼ活性阻害評価では、エタノール４０〜８０体積％でエラスターゼ活性阻害効果が認められた。また、コラゲナーゼ活性阻害評価では、２０〜８０体積％（特に２０〜６０体積％）で強いコラゲナーゼ活性阻害効果が認められた。 In the elastase activity inhibition evaluation using the extract of Experimental Example 4, an elastase activity inhibitory effect was observed at 40 to 80% by volume of ethanol. Moreover, in collagenase activity inhibition evaluation, the strong collagenase activity inhibitory effect was recognized by 20-80 volume% (especially 20-60 volume%).
実験例５（ＢＧ/水 混合溶媒）
ヤナギタデスプラウト１ｇ（乾燥重量）を、ＢＧと水の混合溶媒１０ｍＬ（ＢＧ：２０〜８０体積％、水：８０〜２０体積％）に浸漬し、２時間攪拌した。遠心分離後、上澄みをろ過して得られた抽出液（原液）を実験例５の抽出物とした。実験例５の抽出物（原液）に所定量の純水を加えて適当な濃度に希釈して、上述の方法でエラスターゼ（ブタ膵臓由来）及びコラゲナーゼの活性阻害率を評価した。
図３にＢＧ濃度とエラスターゼ活性阻害率の関係、図４にＢＧ濃度とコラゲナーゼ活性阻害率の関係を示す。なお、活性評価における抽出物濃度（原液濃度）は、エラスターゼ活性阻害では５体積％であり、コラゲナーゼ活性阻害では、０．２体積％である。
Experimental Example 5 (BG / water mixed solvent)
1 g (dry weight) of Willow moth sprout was immersed in 10 mL of a mixed solvent of BG and water (BG: 20 to 80% by volume, water: 80 to 20% by volume) and stirred for 2 hours. The extract obtained by filtering the supernatant after centrifugation (stock solution) was used as the extract of Experimental Example 5. A predetermined amount of pure water was added to the extract (stock solution) of Experimental Example 5 to dilute to an appropriate concentration, and elastase (derived from porcine pancreas) and collagenase activity inhibition rates were evaluated by the method described above.
FIG. 3 shows the relationship between the BG concentration and the elastase activity inhibition rate, and FIG. 4 shows the relationship between the BG concentration and the collagenase activity inhibition rate. The extract concentration (stock solution concentration) in the activity evaluation is 5% by volume for the elastase activity inhibition and 0.2% by volume for the collagenase activity inhibition.
実験例５の抽出物を使用したエラスターゼ活性阻害評価では、ＢＧ４０〜８０体積％で強いエラスターゼ活性阻害効果が認められた。またコラゲナーゼ活性阻害では２０〜８０体積％で強いコラゲナーゼ活性阻害効果が認められた。 In the elastase activity inhibition evaluation using the extract of Experimental Example 5, a strong elastase activity inhibitory effect was observed at BG 40-80% by volume. Further, in the inhibition of collagenase activity, a strong collagenase activity inhibitory effect was observed at 20 to 80% by volume.
「評価３」：ヒト白血球由来エラスターゼ阻害作用の評価
実験例６（抽出溶媒：水）
上述の実験例１と同様の方法で得られたヤナギタデスプラウト水抽出物（原液）を実験例６の抽出物とした。実験例６の抽出物（原液）に所定量の純水を加えて適当な濃度（原液濃度：１〜５０体積％）に希釈して得られた評価用の希釈溶液を使用し、上述の方法でヒト白血球由来エラスターゼの活性阻害率を評価した。表２に結果を示す。
“Evaluation 3”: Evaluation example 6 of human leukocyte-derived elastase inhibitory action (extraction solvent: water)
The Yanagita desprout water extract (stock solution) obtained by the same method as in Experimental Example 1 was used as the extract of Experimental Example 6. Using the diluted solution for evaluation obtained by adding a predetermined amount of pure water to the extract (stock solution) of Experimental Example 6 and diluting to an appropriate concentration (stock solution concentration: 1 to 50% by volume), the method described above Was used to evaluate the activity inhibition rate of human leukocyte-derived elastase. Table 2 shows the results.
実験例７（５０％ ＢＧ/水 混合溶媒）
ヤナギタデスプラウト１ｇ（乾燥重量）を、ＢＧと水の混合溶媒１０ｍＬ（ＢＧ：５０体積％、水：５０体積％）に浸漬し、２時間攪拌した。遠心分離後、上澄みをろ過して得られた抽出液（原液）を実験例７の抽出物とした。実験例７の抽出物（原液）に所定量の純水を加えて適当な濃度（原液濃度：１〜５０体積％）に希釈して得られた評価用の希釈溶液を使用し、上述の方法でヒト白血球由来エラスターゼの活性阻害率を評価した。表２に結果を示す。
Experimental Example 7 (50% BG / water mixed solvent)
1 g (dry weight) of Willow moth sprout was immersed in 10 mL of a mixed solvent of BG and water (BG: 50% by volume, water: 50% by volume) and stirred for 2 hours. After centrifugation, the extract (stock solution) obtained by filtering the supernatant was used as the extract of Experimental Example 7. Using the diluted solution for evaluation obtained by adding a predetermined amount of pure water to the extract (stock solution) of Experimental Example 7 and diluting to an appropriate concentration (stock solution concentration: 1 to 50% by volume), the method described above Was used to evaluate the activity inhibition rate of human leukocyte-derived elastase. Table 2 shows the results.
表２の結果から、ヤナギタデスプラウト水抽出物（実験例６）、ヤナギタデスプラウト５０％ＢＧ抽出物（実験例７）共に、ヒト白血球由来エラスターゼへの阻害活性を有していることが分かる。そして、表１、表２から、実験例１の抽出物（抽出溶媒：水）は、ブタ膵臓由来エラスターゼと比較して、ヒト白血球由来エラスターゼへの阻害活性に特に優れることが分かる。 From the results shown in Table 2, it can be seen that both the water extract of the Yanagita desprout (Experimental Example 6) and the extract of the willow fly desprout 50% BG (Experimental Example 7) have inhibitory activity on human leukocyte-derived elastase. From Tables 1 and 2, it can be seen that the extract of Example 1 (extraction solvent: water) is particularly excellent in inhibitory activity against human leukocyte-derived elastase as compared to porcine pancreatic-derived elastase.
「評価４」：ゼラチナーゼ阻害作用の評価
実験例８（抽出溶媒：水）
上述の実験例１と同様の方法で得られたヤナギタデスプラウト水抽出物（原液）を実験例８の抽出物とした。実験例８の抽出物（原液）に所定量の純水を加えて適当な濃度（原液濃度：１〜５０体積％）に希釈して得られた評価用の希釈溶液を使用し、上述の方法でゼラチナーゼ（ＭＭＰ−２，ＭＭＰ−９）の活性阻害率を評価した。表３に結果を示す。
"Evaluation 4": Evaluation experiment example 8 of gelatinase inhibitory action (extraction solvent: water)
The Yanagita desprout water extract (stock solution) obtained by the same method as in Experimental Example 1 was used as the extract of Experimental Example 8. Using the diluted solution for evaluation obtained by adding a predetermined amount of pure water to the extract (stock solution) of Experimental Example 8 and diluting to an appropriate concentration (stock solution concentration: 1 to 50% by volume), the method described above Thus, the activity inhibition rate of gelatinase (MMP-2, MMP-9) was evaluated. Table 3 shows the results.
実験例９（５０％ ＢＧ/水 混合溶媒）
ヤナギタデスプラウト１ｇ（乾燥重量）を、ＢＧと水の混合溶媒１０ｍＬ（ＢＧ：５０体積％、水：５０体積％）に浸漬し、２時間攪拌した。遠心分離後、上澄みをろ過して得られた抽出液（原液）を実験例９の抽出物とした。実験例９の抽出物（原液）に所定量の純水を加えて適当な濃度（原液濃度：１〜５０体積％）に希釈して得られた評価用の希釈溶液を使用し、上述の方法でゼラチナーゼ（ＭＭＰ−２，ＭＭＰ−９）の活性阻害率を評価した。表３に結果を示す。
Experimental Example 9 (50% BG / water mixed solvent)
1 g (dry weight) of Willow moth sprout was immersed in 10 mL of a mixed solvent of BG and water (BG: 50% by volume, water: 50% by volume) and stirred for 2 hours. The extract obtained by filtering the supernatant after centrifugation (stock solution) was used as the extract of Experimental Example 9. Using the diluted solution for evaluation obtained by adding a predetermined amount of pure water to the extract (stock solution) of Experimental Example 9 and diluting to an appropriate concentration (stock solution concentration: 1 to 50% by volume), the method described above Thus, the activity inhibition rate of gelatinase (MMP-2, MMP-9) was evaluated. Table 3 shows the results.
表３の結果から、ヤナギタデスプラウト水抽出物（実験例８）、ヤナギタデスプラウト５０％ＢＧ抽出物（実験例９）共に、２種（ＭＭＰ−２およびＭＭＰ−９）のゼラチナーゼへの、強い阻害活性を有していることがわかる。表１、図２、図３にしめすように、ゼラチナーゼと同様に活性中心に金属を配位するコラゲナーゼ（ＭＭＰ-１）に対する阻害活性を有していることから、金属イオンをキレートすることにより、酵素活性を抑制していると考えられる。 From the results of Table 3, strong inhibitory activity of two kinds (MMP-2 and MMP-9) on gelatinase in both the willow moth sprout water extract (Experimental Example 8) and the Willow moth sprout 50% BG extract (Experimental Example 9). It can be seen that As shown in Table 1, FIG. 2 and FIG. 3, since it has an inhibitory activity against collagenase (MMP-1) that coordinates a metal to the active center in the same manner as gelatinase, by chelating a metal ion, It is thought that the enzyme activity is suppressed.
本発明のヤナギタデスプラウト抽出物は、コラゲナーゼ及びエラスターゼに対する酵素活性阻害剤、並びに抗老化剤、化粧料組成物及び機能性食品として好適に適用することができる。 The willow desprout extract of the present invention can be suitably applied as an enzyme activity inhibitor for collagenase and elastase, as well as an anti-aging agent, a cosmetic composition and a functional food.
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