JP4731350B2 - Anti-aging agent, skin cosmetics and food and drink for beauty - Google Patents
Anti-aging agent, skin cosmetics and food and drink for beauty Download PDFInfo
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- JP4731350B2 JP4731350B2 JP2006040237A JP2006040237A JP4731350B2 JP 4731350 B2 JP4731350 B2 JP 4731350B2 JP 2006040237 A JP2006040237 A JP 2006040237A JP 2006040237 A JP2006040237 A JP 2006040237A JP 4731350 B2 JP4731350 B2 JP 4731350B2
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Description
本発明は、植物抽出物を含有するＩＶ型コラーゲン産生促進剤、ラミニン５産生促進剤、マトリックスメタロプロテアーゼ阻害剤、抗老化剤、皮膚化粧料及び美容用飲食品に関する。 The present invention relates to a type IV collagen production promoter, laminin 5 production promoter, matrix metalloproteinase inhibitor, anti-aging agent, skin cosmetic, and cosmetic food and drink containing plant extracts.
皮膚は角層、表皮、基底膜及び真皮から構成されている。基底膜は表皮真皮境界部に存在し、表皮と真皮を繋ぎ止めるだけでなく、皮膚機能の維持に重要な役割を果たしている（非特許文献１参照）。基底膜の主要骨格はＩＶ型コラーゲンからなる網目構造をしている。基底膜と表皮の境界に存在し、基底膜と表皮を繋ぎとめているのがラミニン５を主成分とする各種糖蛋白質である。若い皮膚においては、基底膜の働きにより表皮、真皮の相互作用が恒常性を保つことで水分保持、柔軟性、弾力性等が確保され、肌は外見的にも張りや艶があってみずみずしい状態に維持される。 The skin is composed of the stratum corneum, epidermis, basement membrane and dermis. The basement membrane is present at the epidermal dermis boundary, and plays an important role in maintaining skin function as well as connecting the epidermis and dermis (see Non-Patent Document 1). The main skeleton of the basement membrane has a network structure made of type IV collagen. Various glycoproteins mainly composed of laminin 5 exist at the boundary between the basement membrane and the epidermis and connect the basement membrane and the epidermis. In young skin, the interaction between the epidermis and dermis is kept constant by the action of the basement membrane, ensuring moisture retention, flexibility, elasticity, etc. Maintained.
ところが、紫外線の照射、空気の著しい乾燥、過度の皮膚洗浄等、ある種の外的因子の影響があったり、加齢が進んだりすると、基底膜の主要構成成分であるＩＶ型コラーゲンや、ラミニン５は分解・変質を起こし、基底膜構造が破壊される（非特許文献２参照）。その結果、皮膚は保湿機能や弾力性が低下し、角質は異常剥離を始めるから、肌は張りや艶を失い、荒れ、シワ等の老化症状を呈するようになる。このように、皮膚の老化に伴う変化、即ち、シワ、くすみ、きめの消失、弾力性の低下等には、基底膜成分の減少、基底膜の構造変化が関与している。 However, when there is an influence of certain external factors such as ultraviolet irradiation, excessive drying of the air, excessive skin washing, etc., or when aging progresses, type IV collagen, which is a main component of the basement membrane, and laminin 5 undergoes degradation and alteration, and the basement membrane structure is destroyed (see Non-Patent Document 2). As a result, the skin's moisturizing function and elasticity are lowered, and the keratin begins to exfoliate abnormally, so the skin loses its tension and gloss, and exhibits aging symptoms such as roughness and wrinkles. As described above, changes associated with skin aging, that is, wrinkles, dullness, disappearance of texture, decrease in elasticity, and the like, are associated with a decrease in basement membrane components and a change in basement membrane structure.
近年の研究により、この変化を誘導する因子として、マトリックスメタロプロテアーゼ（ＭＭＰｓ；Matrix metalloproteinases）の関与が指摘されている。このＭＭＰｓの中でも、ゼラチナーゼ群に属する酵素であるＭＭＰ−２及びＭＭＰ−９は、基底膜の主要構成成分であるＩＶ型コラーゲンやラミニン５を分解する酵素として知られているが、その発現及び活性は紫外線の照射により大きく増加し、紫外線による基底膜成分の減少、基底膜の構造変化の原因となり、皮膚におけるシワやたるみの形成等の大きな要因となることが明らかとなっている（非特許文献３参照）。 Recent studies have pointed out the involvement of matrix metalloproteinases (MMPs) as factors that induce this change. Among these MMPs, enzymes belonging to the gelatinase group, MMP-2 and MMP-9, are known as enzymes that degrade type IV collagen and laminin 5, which are the main components of the basement membrane. Is significantly increased by irradiation with ultraviolet rays, causing a decrease in basement membrane components and structural changes of the basement membrane due to ultraviolet rays, and it has been clarified that it becomes a major factor in the formation of wrinkles and sagging in the skin (non-patent literature). 3).
したがって、ＩＶ型コラーゲン及びラミニン５の産生を促進することによって基底膜構造の再構築を誘導し、またＭＭＰｓの活性を阻害することにより基底膜成分の減少、基底膜の構造変化を抑制し、皮膚機能を改善する物質の開発が望まれている。 Therefore, it promotes the production of type IV collagen and laminin 5 to induce remodeling of the basement membrane structure, and by inhibiting the activity of MMPs, it suppresses the decrease in basement membrane components and the basement membrane structure, Development of substances that improve function is desired.
ＩＶ型コラーゲン産生促進作用を有するものとしては、例えば、加水分解カゼイン、プレエキス、ブナの芽エキス、エリスリナエキス、可溶性卵殻膜、カッコンエキス及び西洋キズタエキス（特許文献１参照）、サポニン又はサポゲノール（特許文献２参照）等が知られている。 Examples of the type IV collagen production promoting action include hydrolyzed casein, pre-extract, beech bud extract, erythrina extract, soluble eggshell membrane, cuckoo extract and western kizuta extract (see Patent Document 1), saponin or sapogenol (patent) Document 2) is known.
また、ラミニン５産生促進作用を有するものとしては、例えば、カッコン抽出物、カンゾウ抽出物またはそのフラボノイド画分、ヒオウギ抽出物、ジタノキ抽出物、イボツヅラフジ抽出物、コロハ抽出物および乳清抽出物（特許文献３参照）オノニス抽出物、メリロート抽出物、モヤシ抽出物、アズキ抽出物（特許文献４参照）、大豆抽出物（特許文献５参照）等が知られている。 Moreover, as what has a laminin 5 production promotion effect | action, for example, a cuckoo extract, a licorice extract or its flavonoid fraction, a cypress extract, a cypress extract, a lobster extract, a Koroha extract and a whey extract ( (See Patent Document 3) Onionis extract, Merirot extract, bean extract, azuki bean extract (see Patent Document 4), soybean extract (see Patent Document 5), and the like are known.
さらに、マトリックスメタロプロテアーゼ阻害作用を有するものとしては、例えば、黒霊芝抽出物（特許文献６参照）、クリ渋皮発酵物からの抽出物（特許文献７参照）等が知られている。
本発明は、天然物の中からＩＶ型コラーゲン産生促進作用、ラミニン５産生促進作用、マトリックスメタロプロテアーゼ阻害作用を有する物質を見出し、それを有効成分として含有するＩＶ型コラーゲン産生促進剤、ラミニン５産生促進剤、マトリックスメタロプロテアーゼ阻害剤、抗老化剤、皮膚化粧料及び美容用飲食品を提供することを目的とする。 The present invention finds a substance having a type IV collagen production promoting action, laminin 5 production promoting action, matrix metalloprotease inhibitory action from natural products, and a type IV collagen production promoting agent laminin 5 production containing the substance as an active ingredient An object is to provide an accelerator, a matrix metalloproteinase inhibitor, an anti-aging agent, a skin cosmetic, and a cosmetic food and drink.
上記目的を達成するために、第１に、本発明は、クロバナツルアズキからの抽出物を有効成分として含有することを特徴とするＩＶ型コラーゲン産生促進剤、ラミニン５産生促進剤、マトリックスメタロプロテアーゼ阻害剤又は抗老化剤を提供する。 In order to achieve the above-mentioned object, first, the present invention comprises an extract from type IV azuki bean as an active ingredient, a type IV collagen production promoter, laminin 5 production promoter, matrix metalloproteinase Inhibitors or anti-aging agents are provided.
第２に、本発明は、クロバナツルアズキからの抽出物を配合したことを特徴とする皮膚化粧料及び美容用飲食品を提供する。 2ndly, this invention provides the skin cosmetics and the cosmetics food / beverage products characterized by mix | blending the extract from a black-breasted adzuki bean.
本発明のマトリックスメタロプロテアーゼ阻害剤においては、前記抽出物が、マトリックスメタロプロテアーゼ−２（ＭＭＰ−２）阻害作用及び／又はマトリックスメタロプロテアーゼ−９（ＭＭＰ−９）阻害作用を有することが好ましい。 In the matrix metalloprotease inhibitor of the present invention, the extract preferably has a matrix metalloproteinase-2 (MMP-2) inhibitory action and / or a matrix metalloproteinase-9 (MMP-9) inhibitory action.
本発明によれば、優れたＩＶ型コラーゲン産生促進作用、ラミニン５産生促進作用、マトリックスメタロプロテアーゼ阻害作用を有するクロバナツルアズキ抽出物を有効成分として含有するＩＶ型コラーゲン産生促進剤、ラミニン５産生促進剤、マトリックスメタロプロテアーゼ阻害剤及び抗老化剤、並びに当該抽出物を配合した皮膚化粧料及び美容用飲食品を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, the type IV collagen production promoter and laminin 5 production promotion which contain as an active ingredient the cerebral azuki bean extract which has the outstanding type IV collagen production promotion effect, laminin 5 production promotion effect, and a matrix metalloprotease inhibitory effect An agent, a matrix metalloprotease inhibitor and an anti-aging agent, and a skin cosmetic and a cosmetic food and drink containing the extract can be provided.
以下、本発明について詳細に説明する。
〔抗老化剤，ＩＶ型コラーゲン産生促進剤，ラミニン５産生促進剤，マトリックスメタロプロテアーゼ阻害剤〕
本発明の抗老化剤、ＩＶ型コラーゲン産生促進剤、ラミニン５産生促進剤又はマトリックスメタロプロテアーゼ阻害剤は、クロバナツルアズキからの抽出物を有効成分として含有する。
Hereinafter, the present invention will be described in detail.
[Anti-aging agent, type IV collagen production promoter, laminin 5 production promoter, matrix metalloprotease inhibitor]
The anti-aging agent, type IV collagen production promoter, laminin 5 production promoter, or matrix metalloprotease inhibitor of the present invention contains an extract from black beetle as an active ingredient.
ここで、本発明において「抽出物」には、クロバナツルアズキを抽出原料として得られる抽出液、当該抽出液の希釈液若しくは濃縮液、当該抽出液を乾燥して得られる乾燥物、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。 Here, in the present invention, the “extract” refers to an extract obtained using black bean azuki bean as an extraction raw material, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or these Either a crude product or a purified product is included.
本発明において使用する抽出原料は、クロバナツルアズキ（学名：Phaseolus atropurpureus）である。クロバナツルアズキ（Phaseolus atropurpureus）は、本州、九州、沖縄等に分布しているマメ科インゲンマメ属に属するつる性の多年草であり、これらの地域から容易に入手することができる。抽出原料として使用し得る部位としては、例えば、幹部、地上部、根部、全草等が挙げられるが、好ましくは幹部である。 The extraction raw material used in the present invention is black bean azuki (Scientific name: Phaseolus atropurpureus). Black-headed azuki bean (Phaseolus atropurpureus) is a perennial vine belonging to the genus of the leguminous kidney bean distributed in Honshu, Kyushu, Okinawa, etc., and can be easily obtained from these regions. Examples of the part that can be used as the extraction raw material include a trunk, an above-ground part, a root part, and a whole plant, and a trunk part is preferable.
クロバナツルアズキ抽出物に含まれるＩＶ型コラーゲン産生促進作用、ラミニン５産生促進作用又はマトリックスメタロプロテアーゼ阻害作用を有する物質の詳細は不明であるが、植物の抽出に一般に用いられている抽出方法によって、クロバナツルアズキからＩＶ型コラーゲン産生促進作用、ラミニン５産生促進作用又はマトリックスメタロプロテアーゼ阻害作用を有する抽出物を得ることができる。 The details of the substance having type IV collagen production promoting action, laminin 5 production promoting action or matrix metalloprotease inhibitory action contained in the black azuki bean extract is unknown, but depending on the extraction method generally used for plant extraction, An extract having a type IV collagen production promoting effect, a laminin 5 production promoting effect, or a matrix metalloprotease inhibitory effect can be obtained from the black beetle.
例えば、クロバナツルアズキを乾燥した後、そのまま又は粗砕機を用いて粉砕し、抽出溶媒による抽出に供することにより、ＩＶ型コラーゲン産生促進作用、ラミニン５産生促進作用又はマトリックスメタロプロテアーゼ阻害作用を有する抽出物を得ることができる。乾燥は天日で行ってもよいし、通常使用される乾燥機を用いて行ってもよい。また、ヘキサン等の非極性溶媒によって脱脂等の前処理を施してから抽出原料として使用してもよい。脱脂等の前処理を行うことにより、極性溶媒による抽出処理を効率よく行うことができる。 For example, after drying the blackwood azuki bean or pulverizing as it is or using a crusher, it is subjected to extraction with an extraction solvent, thereby extracting type IV collagen production promoting action, laminin 5 production promoting action or matrix metalloprotease inhibitory action You can get things. Drying may be performed in the sun or using a commonly used dryer. Moreover, after performing pretreatment, such as degreasing, with a nonpolar solvent such as hexane, it may be used as an extraction raw material. By performing pretreatment such as degreasing, extraction with a polar solvent can be performed efficiently.
抽出溶媒としては、極性溶媒を使用するのが好ましく、例えば、水、親水性有機溶媒等が挙げられ、これらを単独で又は２種以上を組み合わせて、室温又は溶媒の沸点以下の温度で使用することが好ましい。 As the extraction solvent, it is preferable to use a polar solvent, and examples thereof include water and hydrophilic organic solvents. These are used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. It is preferable.
抽出溶媒として使用し得る水としては、純水、水道水、井戸水、鉱泉水、鉱水、温泉水、湧水、淡水等のほか、これらに各種処理を施したものが含まれる。水に施す処理としては、例えば、精製、加熱、殺菌、濾過、イオン交換、浸透圧調整、緩衝化等が含まれる。したがって、本発明において抽出溶媒として使用し得る水には、精製水、熱水、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。 Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, and those subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline, and the like.
抽出溶媒として使用し得る親水性有機溶媒としては、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の低級脂肪族アルコール；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の多価アルコール等が挙げられる。 Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycol, propylene glycol and glycerin.
２種以上の極性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。例えば、水と低級脂肪族アルコールとの混合液を使用する場合には、水１０質量部に対して低級脂肪族アルコール１〜９０質量部を混合することが好ましく、水と低級脂肪族ケトンとの混合液を使用する場合には、水１０質量部に対して低級脂肪族ケトン１〜４０質量部を混合することが好ましく、水と多価アルコールとの混合液を使用する場合には、水１０質量部に対して多価アルコール１０〜９０質量部を混合することが好ましい。 When using the liquid mixture of 2 or more types of polar solvents as an extraction solvent, the mixing ratio can be adjusted suitably. For example, when using a liquid mixture of water and a lower aliphatic alcohol, it is preferable to mix 1 to 90 parts by weight of a lower aliphatic alcohol with respect to 10 parts by weight of water. When using a mixed solution, it is preferable to mix 1 to 40 parts by mass of a lower aliphatic ketone with 10 parts by mass of water, and when using a mixed solution of water and a polyhydric alcohol, water 10 It is preferable to mix 10 to 90 parts by mass of polyhydric alcohol with respect to parts by mass.
抽出処理は、抽出原料に含まれる可溶性成分を抽出溶媒に溶出させ得る限り特に限定はされず、常法に従って行うことができる。例えば、抽出原料の５〜１５倍量（質量比）の抽出溶媒に、抽出原料を浸漬し、常温又は還流加熱下で可溶性成分を溶出させた後、濾過して抽出残渣を除去することにより抽出液を得ることができる。得られた抽出液は、該抽出液の希釈液若しくは濃縮液、該抽出液の乾燥物、又はこれらの粗精製物若しくは精製物を得るために、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。 The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be performed according to a conventional method. For example, the extraction raw material is immersed in 5 to 15 times the extraction solvent (mass ratio) of the extraction raw material, the soluble components are eluted at room temperature or under reflux, and then filtered to remove the extraction residue. A liquid can be obtained. The obtained extract is diluted, concentrated, dried, purified, etc. according to a conventional method in order to obtain a diluted or concentrated solution of the extract, a dried product of the extract, or a crude purified product or a purified product thereof. Processing may be performed.
精製は、例えば、活性炭処理、吸着樹脂処理、イオン交換樹脂処理等により行うことができる。得られた抽出液はそのままでもＩＶ型コラーゲン産生促進剤、ラミニン５産生促進剤又はマトリックスメタロプロテアーゼ阻害剤の有効成分として使用することができるが、濃縮液又は乾燥物としたものの方が使用しやすい。 Purification can be performed by, for example, activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, or the like. The obtained extract can be used as it is as an active ingredient of a type IV collagen production promoter, laminin 5 production promoter or matrix metalloprotease inhibitor, but a concentrated solution or a dried product is easier to use. .
クロバナツルアズキ抽出物は、特有の匂いと味とを有しているため、その生理活性の低下を招かない範囲で脱色、脱臭等を目的とする精製を行うことも可能であるが、皮膚化粧料等に配合する場合には大量に使用するものではないから、未精製のままでも実用上支障はない。 Since the black azuki bean extract has a unique smell and taste, it can be purified for the purpose of decolorization, deodorization, etc. within a range that does not reduce its physiological activity. Since it is not used in a large amount when blended into a material, there is no practical problem even if it is not purified.
以上のようにして得られるクロバナツルアズキ抽出物は、抗老化作用、ＩＶ型コラーゲン産生促進作用、ラミニン５産生促進作用又はマトリックスメタロプロテアーゼ阻害作用を有しているため、それらの作用を利用して抗老化剤、ＩＶ型コラーゲン産生促進剤、ラミニン５産生促進剤又はマトリックスメタロプロテアーゼ阻害剤の有効成分として用いることができる。 Since the extract of Azuki beetle obtained as described above has an anti-aging action, a type IV collagen production promoting action, a laminin 5 production promoting action or a matrix metalloprotease inhibitory action, It can be used as an active ingredient of an anti-aging agent, type IV collagen production promoter, laminin 5 production promoter or matrix metalloprotease inhibitor.
本発明の抗老化剤、ＩＶ型コラーゲン産生促進剤、ラミニン５産生促進剤又はマトリックスメタロプロテアーゼ阻害剤は、クロバナツルアズキからの抽出物のみからなるものでもよいし、クロバナツルアズキからの抽出物を製剤化したものでもよい。 The anti-aging agent, type IV collagen production promoter, laminin 5 production promoter, or matrix metalloprotease inhibitor of the present invention may be composed only of an extract from black-spotted azuki bean, or an extract from black-spotted red bean is used. It may be formulated.
クロバナツルアズキからの抽出物は、デキストリン、シクロデキストリン等の薬学的に許容し得るキャリアーその他任意の助剤を用いて、常法に従い、粉末状、顆粒状、錠剤状、液状等の任意の剤形に製剤化して提供することができ、他の組成物（例えば、後述する皮膚化粧料、美容用飲食品等）に配合して使用できるほか軟膏剤、外用液剤、貼付剤等として使用することができる。この際、助剤としては、例えば、賦形剤、結合剤、崩壊剤、滑沢剤、安定剤、矯臭剤等を用いることができる。 The extract from black-spotted azuki bean is prepared by using a pharmaceutically acceptable carrier such as dextrin and cyclodextrin and other optional auxiliaries according to a conventional method and in any form such as powder, granules, tablets, and liquids. It can be provided after being formulated into a form, and can be used by blending with other compositions (for example, skin cosmetics and cosmetic foods and beverages described later), as well as ointments, liquids for external use, patches, etc. Can do. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent and the like can be used.
なお、本発明の抗老化剤、ＩＶ型コラーゲン産生促進剤、ラミニン５産生促進剤又はマトリックスメタロプロテアーゼ阻害剤は、必要に応じてＩＶ型コラーゲン産生促進作用、ラミニン５産生促進作用又はマトリックスメタロプロテアーゼ阻害作用を有する他の天然抽出物等を、クロバナツルアズキからの抽出物とともに配合して有効成分として用いることができる。 The anti-aging agent, type IV collagen production promoter, laminin 5 production promoter or matrix metalloprotease inhibitor of the present invention is an IV collagen production promoting action, laminin 5 production promoting action or matrix metalloprotease inhibition as required. Other natural extracts or the like having an action can be blended together with an extract from black-spotted azuki bean and used as an active ingredient.
本発明の抗老化剤は、クロバナツルアズキからの抽出物が有するＩＶ型コラーゲン産生促進作用を通じて、ＩＶ型コラーゲンの産生を促進することができるとともに、クロバナツルアズキからの抽出物が有するラミニン５産生促進作用を通じて、ラミニン５の産生を促進することができる。また、本発明の抗老化剤は、クロバナツルアズキからの抽出物が有するマトリックスメタロプロテアーゼ阻害作用を通じて、マトリックスメタロプロテアーゼの活性を阻害することができ、特にマトリックスメタロプロテアーゼ−２及び／又はマトリックスメタロプロテアーゼ−９の活性を阻害することができる。これにより、基底膜構造の再構築を誘導し、皮膚の老化症状を予防・改善することができる。ただし、本発明の抗老化剤は、これらの用途以外にもＩＶ型コラーゲン産生促進作用、ラミニン５産生促進作用又はマトリックスメタロプロテアーゼ阻害作用を発揮することに意義のあるすべての用途に用いることができる。 The anti-aging agent of the present invention can promote the production of type IV collagen through the action of promoting the production of type IV collagen possessed by the extract from black azuki bean, and the production of laminin 5 possessed by the extract from black azuki bean Through the promoting action, the production of laminin 5 can be promoted. In addition, the anti-aging agent of the present invention can inhibit the activity of matrix metalloprotease through the matrix metalloprotease inhibitory action possessed by the extract from black-spotted azuki bean, in particular, matrix metalloprotease-2 and / or matrix metalloprotease. -9 activity can be inhibited. Thereby, reconstruction of the basement membrane structure can be induced, and skin aging symptoms can be prevented and improved. However, in addition to these uses, the anti-aging agent of the present invention can be used for all uses that are meaningful for exerting a type IV collagen production promoting effect, a laminin 5 production promoting effect, or a matrix metalloprotease inhibitory effect. .
本発明のＩＶ型コラーゲン産生促進剤は、クロバナツルアズキからの抽出物が有するＩＶ型コラーゲン産生促進作用を通じて、ＩＶ型コラーゲンの産生を促進することができ、これにより、基底膜構造の再構築を誘導し、皮膚の老化症状を予防・改善することができる。ただし、本発明のＩＶ型コラーゲン産生促進剤は、これらの用途以外にもＩＶ型コラーゲン産生促進作用を発揮することに意義のあるすべての用途に用いることができる。 The type IV collagen production-promoting agent of the present invention can promote the production of type IV collagen through the action of promoting the production of type IV collagen possessed by the extract from black-spotted azuki bean, thereby reconstructing the basement membrane structure. It can induce and prevent or improve skin aging symptoms. However, the type IV collagen production-promoting agent of the present invention can be used for all purposes that are meaningful for exerting the effect of promoting type IV collagen production in addition to these uses.
本発明のラミニン５産生促進剤は、クロバナツルアズキからの抽出物が有するラミニン５産生促進作用を通じて、ラミニン５の産生を促進することができる。これにより、基底膜構造の再構築を誘導し、皮膚の老化症状を予防・改善することができる。ただし、本発明のラミニン５産生促進剤は、これらの用途以外にもラミニン５産生促進作用を発揮することに意義のあるすべての用途に用いることができる。 The laminin 5 production promoter of the present invention can promote the production of laminin 5 through the laminin 5 production promoting action possessed by the extract from the blackwood azuki bean. Thereby, restructuring of the basement membrane structure can be induced, and skin aging symptoms can be prevented and improved. However, the laminin 5 production promoter of the present invention can be used for all purposes that are meaningful for exerting a laminin 5 production promoting action in addition to these uses.
本発明のマトリックスメタロプロテアーゼ阻害剤は、クロバナツルアズキからの抽出物が有するマトリックスメタロプロテアーゼ阻害作用を通じて、マトリックスメタロプロテアーゼの活性を阻害することができ、特に、マトリックスメタロプロテアーゼ−２及び／又はマトリックスメタロプロテアーゼ−９の活性を阻害することができる。これにより、基底膜構造の再構築を誘導し、皮膚の老化症状を予防・改善することができる。ただし、本発明のマトリックスメタロプロテアーゼ阻害剤は、これらの用途以外にもマトリックスメタロプロテアーゼ阻害作用を発揮することに意義のあるすべての用途に用いることができる。 The matrix metalloprotease inhibitor of the present invention can inhibit the activity of matrix metalloprotease through the matrix metalloprotease inhibitory action possessed by the extract from black-spotted red bean, and in particular, matrix metalloprotease-2 and / or matrix metalloproteinase. The activity of protease-9 can be inhibited. Thereby, reconstruction of the basement membrane structure can be induced, and skin aging symptoms can be prevented and improved. However, the matrix metalloprotease inhibitor of the present invention can be used for all uses other than these uses that are meaningful for exhibiting a matrix metalloprotease inhibitory action.
なお、クロバナツルアズキからの抽出物は、マトリックスメタロプロテアーゼ（ＭＭＰｓ）のうちのマトリックスメタロプロテアーゼ−２（ＭＭＰ−２）及び／又はマトリックスメタロプロテアーゼ−９（ＭＭＰ−９）の活性を阻害する作用を有するため、それらの作用を利用してマトリックスメタロプロテアーゼ−２（ＭＭＰ−２）阻害剤又はマトリックスメタロプロテアーゼ−９（ＭＭＰ−９）阻害剤の有効成分として用いることができる。 In addition, the extract from the black azuki bean has an action of inhibiting the activity of matrix metalloproteinase-2 (MMP-2) and / or matrix metalloprotease-9 (MMP-9) among matrix metalloproteinases (MMPs). Therefore, it can be used as an active ingredient of a matrix metalloproteinase-2 (MMP-2) inhibitor or a matrix metalloproteinase-9 (MMP-9) inhibitor by utilizing these actions.
〔皮膚化粧料〕
本発明の皮膚化粧料は、クロバナツルアズキからの抽出物を配合したものである。クロバナツルアズキからの抽出物は、ＩＶ型コラーゲン産生促進作用、ラミニン５産生促進作用及びマトリックスメタロプロテアーゼ阻害作用を有しており、皮膚に適用した場合の使用感と安全性とに優れているため、皮膚化粧料に配合するのに好適である。この場合、クロバナツルアズキからの抽出物をそのまま配合してもよいし、クロバナツルアズキからの抽出物より製剤化した抗老化剤、ＩＶ型コラーゲン産生促進剤、ラミニン５産生促進剤又はマトリックスメタロプロテアーゼ阻害剤を配合してもよい。クロバナツルアズキからの抽出物、又は上記抗老化剤、上記ＩＶ型コラーゲン産生促進剤、上記ラミニン５産生促進剤若しくは上記マトリックスメタロプロテアーゼ阻害剤を皮膚化粧料に配合することによって、皮膚化粧料にＩＶ型コラーゲン産生促進作用、ラミニン５産生促進作用、マトリックスメタロプロテアーゼ阻害作用を付与することができる。
[Skin cosmetic]
The skin cosmetic of the present invention is a blend of an extract from black-spotted azuki bean. Since the extract from the black azuki bean has a type IV collagen production promoting action, a laminin 5 production promoting action and a matrix metalloprotease inhibitory action, it has excellent usability and safety when applied to the skin. Suitable for blending into skin cosmetics. In this case, an extract from black-spotted azuki bean may be blended as it is, or an anti-aging agent, a type IV collagen production promoter, a laminin 5 production-promoting agent, or a matrix metalloproteinase formulated from an extract from black-spotted red bean You may mix | blend an inhibitor. By adding an extract from black-spotted azuki bean or the anti-aging agent, the type IV collagen production promoter, the laminin 5 production promoter or the matrix metalloprotease inhibitor to the skin cosmetic, Type collagen production promoting action, laminin 5 production promoting action, matrix metalloprotease inhibitory action can be imparted.
クロバナツルアズキからの抽出物を配合し得る皮膚化粧料としては、特に限定されるものではなく、例えば、軟膏、クリーム、乳液、ローション、パック、ファンデーション、リップクリーム、入浴剤、ヘアートニック、ヘアーローション、石鹸、ボディシャンプー等が挙げられる。 There are no particular limitations on the skin cosmetics that can be blended with an extract from black-spotted azuki, such as ointments, creams, emulsions, lotions, packs, foundations, lip balms, bath preparations, hair arts, hair lotions. , Soap, body shampoo and the like.
クロバナツルアズキからの抽出物を皮膚化粧料に配合する場合、その配合量は、皮膚化粧料の種類に応じて適宜調整することができるが、好適な配合率は標準的な抽出物に換算して約０．０００１〜１０質量％であり、特に好適な配合率は標準的な抽出物に換算して約０．００５〜５質量％である。 In the case where an extract from black-spotted adzuki bean is blended in skin cosmetics, the blending amount can be adjusted as appropriate according to the type of skin cosmetic, but a suitable blending ratio is converted to a standard extract. About 0.0001 to 10% by mass, and a particularly suitable blending ratio is about 0.005 to 5% by mass in terms of a standard extract.
本発明の皮膚化粧料は、クロバナツルアズキからの抽出物が有するＩＶ型コラーゲン産生促進作用、ラミニン５産生促進作用及びマトリックスメタロプロテアーゼ阻害作用を妨げない限り、通常の皮膚化粧料の製造に用いられる主剤、助剤又はその他の成分、例えば、収斂剤、殺菌、抗菌剤、美白剤、紫外線吸収剤、保湿剤、細胞賦活剤、抗老化剤、消炎・抗アレルギー剤、抗酸化・活性酸素除去剤、油脂類、ロウ類、炭化水素類、脂肪酸類、アルコール類、エステル類、界面活性剤、香料等を併用することができる。このように併用することで、より一般性のある製品となり、また、併用された他の有効成分との間の相乗作用が通常期待される以上の優れた効果をもたらすことがある。 The skin cosmetic of the present invention can be used for the production of ordinary skin cosmetics as long as it does not interfere with the type IV collagen production promoting action, laminin 5 production promoting action and matrix metalloprotease inhibitory action of the extract from black-spotted azuki bean. Main agent, auxiliaries or other ingredients such as astringents, bactericides, antibacterial agents, whitening agents, UV absorbers, moisturizers, cell activators, anti-aging agents, anti-inflammatory / anti-allergic agents, antioxidant / reactive oxygen scavengers , Oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, perfumes and the like can be used in combination. When used in combination, it becomes a more general product, and a synergistic effect with other active ingredients used in combination may lead to an excellent effect that is more than normally expected.
〔美容用飲食品〕
クロバナツルアズキからの抽出物は、ＩＶ型コラーゲン産生促進作用、ラミニン５産生促進作用及びマトリックスメタロプロテアーゼ阻害作用を有しており、消化管で消化されるようなものではないことが確認されており、安全性にも優れているため、美容用飲食品に配合するのに好適である。この場合に、クロバナツルアズキからの抽出物をそのまま配合してもよいし、クロバナツルアズキからの抽出物より製剤化した抗老化剤、ＩＶ型コラーゲン産生促進剤、ラミニン５産生促進剤又はマトリックスメタロプロテアーゼ阻害剤を配合してもよい。
[Beauty food and drink]
It has been confirmed that the extract from the black bean has a type IV collagen production promoting action, a laminin 5 production promoting action and a matrix metalloprotease inhibitory action and is not digested in the digestive tract. Since it is excellent in safety, it is suitable for blending into cosmetic foods and drinks. In this case, an extract from black-spotted azuki bean may be blended as it is, or an anti-aging agent, a type IV collagen production promoter, a laminin 5 production-promoting agent, or a matrix metallo, formulated from an extract from black-spotted red bean A protease inhibitor may be blended.
ここで、「美容用飲食品」とは、美肌を図ることを目的とした飲食物、又は肌荒れ、皮膚の老化及びこれらに伴って生じる各種皮膚疾患を予防・改善することを目的とした飲食物を意味する。 Here, “beauty food and drink” refers to food and drink intended for beautifying the skin, or food and drink intended to prevent and improve rough skin, aging of the skin and various skin diseases associated therewith. Means.
クロバナツルアズキからの抽出物、又は上記抗老化剤、上記ＩＶ型コラーゲン産生促進剤、上記ラミニン５産生促進剤若しくは上記マトリックスメタロプロテアーゼ阻害剤を美容用飲食品に配合する場合、それらにおける有効成分の配合量は、使用目的、症状、性別等を考慮して適宜変更することができるが、添加対象飲食品の一般的な摂取量を考慮して、成人１日あたりのクロバナツルアズキからの抽出物の摂取量が約１〜１０００ｍｇになるようにするのが好ましい。 In the case where an extract from black azuki bean or the anti-aging agent, the type IV collagen production promoter, the laminin 5 production promoter or the matrix metalloprotease inhibitor is added to a cosmetic food or drink, The compounding amount can be appropriately changed in consideration of the purpose of use, symptoms, sex, etc., but taking into consideration the general intake of foods and drinks to be added, an extract from the black bean per adult per day It is preferable that the intake of be about 1 to 1000 mg.
本発明の美容用飲食品は、クロバナツルアズキからの抽出物をその活性を妨げないような任意の飲食品に配合したものであってもよいし、クロバナツルアズキからの抽出物を主成分とする栄養補助食品であってもよい。 The cosmetic food and drink of the present invention may be a blend of an extract from black-spotted azuki bean in any food and drink that does not interfere with its activity, and an extract from black-spotted red bean as a main component. It may be a dietary supplement.
本発明の美容用飲食品を製造する際には、例えば、デキストリン、デンプン等の糖類；ゼラチン、大豆タンパク、トウモロコシタンパク等のタンパク質；アラニン、グルタミン、イソロイシン等のアミノ酸類；セルロース、アラビアゴム等の多糖類；大豆油、中鎖脂肪酸トリグリセリド等の油脂類などの任意の助剤を添加して任意の形状の飲食品にすることができる。 When producing the cosmetic food and drink of the present invention, for example, sugars such as dextrin and starch; proteins such as gelatin, soybean protein and corn protein; amino acids such as alanine, glutamine and isoleucine; cellulose and gum arabic Polysaccharides: Foods and drinks of any shape can be made by adding arbitrary auxiliaries such as oils and fats such as soybean oil and medium chain fatty acid triglycerides.
クロバナツルアズキからの抽出物を配合し得る美容用飲食品は特に限定されないが、その具体例としては、清涼飲料、炭酸飲料、栄養飲料、果実飲料、乳酸飲料等の飲料（これらの飲料の濃縮原液及び調整用粉末を含む）；アイスクリーム、アイスシャーベット、かき氷等の冷菓；そば、うどん、はるさめ、ぎょうざの皮、しゅうまいの皮、中華麺、即席麺等の麺類；飴、チューインガム、キャンディー、ガム、チョコレート、錠菓、スナック菓子、ビスケット、ゼリー、ジャム、クリーム、焼き菓子等の菓子類；かまぼこ、ハム、ソーセージ等の水産・畜産加工食品；加工乳、発酵乳等の乳製品；サラダ油、てんぷら油、マーガリン、マヨネーズ、ショートニング、ホイップクリーム、ドレッシング等の油脂及び油脂加工食品；ソース、たれ等の調味料；スープ、シチュー、サラダ、惣菜、漬物；その他種々の形態の健康・栄養補助食品；錠剤、カプセル剤、ドリンク剤などが挙げられ、これらの飲食品にクロバナツルアズキからの抽出物を配合するときに、通常用いられる補助的な原料や添加物を併用することができる。 Beauty foods and drinks that can be blended with an extract from black-spotted adzuki beans are not particularly limited, and specific examples include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks (concentration of these drinks) Ice cream, ice sherbet, shaved ice, etc .; noodles such as buckwheat, udon, harsame, gyoza skin, sushi mai, Chinese noodles, instant noodles; candy, chewing gum, candy, gum , Chocolate, tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, baked confectionery, etc .; fish and fishery processed foods such as kamaboko, ham, sausage; dairy products such as processed milk, fermented milk; salad oil, tempura oil , Margarine, mayonnaise, shortening, whipped cream, dressing and other fats and oils processed foods; sauces Seasonings such as soup, stew, salad, side dish, pickles; various other forms of health and nutritional supplements; tablets, capsules, drinks, etc. When blending, auxiliary materials and additives usually used can be used in combination.
なお、本発明の抗老化剤、ＩＶ型コラーゲン産生促進剤、ラミニン５産生促進剤、マトリックスメタロプロテアーゼ阻害剤、皮膚化粧料又は美容用飲食品は、ヒトに対して好適に適用されるものであるが、それぞれの作用効果が奏される限り、ヒト以外の動物に対して適用することもできる。 The anti-aging agent, type IV collagen production promoter, laminin 5 production promoter, matrix metalloprotease inhibitor, skin cosmetic or cosmetic food or beverage of the present invention is suitably applied to humans. However, as long as each effect is exhibited, it can also be applied to animals other than humans.
以下、製造例、試験例及び配合例を示し、本発明を具体的に説明するが、本発明は下記の各例に何ら制限されるものではない。 Hereinafter, although a manufacture example, a test example, and a compounding example are shown and this invention is demonstrated concretely, this invention is not restrict | limited to each following example at all.
〔製造例１〕クロバナツルアズキ抽出物の製造
細切りにしたクロバナツルアズキの幹部の乾燥物２００ｇに抽出溶媒２０００ｍＬを加え、還流抽出器で８０℃にて３時間加熱抽出し、熱時濾過した。得られた抽出液を減圧下に濃縮し、乾燥してクロバナツルアズキ抽出物を得た。抽出溶媒として、水、５０質量％エタノール（水とエタノールとの質量比＝１：１）、エタノールを用いたときの各抽出物の収率を表１に示す。
[Production Example 1] Manufacture of black-spotted azuki bean extract To 200 g of finely-sliced dried black-spotted red bean trunk, 2000 mL of extraction solvent was added, and the mixture was heated and extracted at 80 ° C for 3 hours with a reflux extractor and filtered while hot. The obtained extract was concentrated under reduced pressure and dried to obtain a black bean extract. Table 1 shows the yield of each extract when water, 50 mass% ethanol (mass ratio of water to ethanol = 1: 1), and ethanol were used as the extraction solvent.
［表１］
試 料 抽出溶媒 抽出物収率（％）
１ 水 ２０．５
２ ５０％エタノール １４．３
３ エタノール ８．４
[Table 1]
Sample extraction solvent extract yield (%)
1 Water 20.5
2 50% ethanol 14.3
3 Ethanol 8.4
〔試験例１〕ＩＶ型コラーゲン産生促進作用試験
製造例１で得られたクロバナツルアズキ抽出物（試料１〜３）について、以下のようにしてＩＶ型コラーゲン産生促進作用を試験した。
[Test Example 1] Type IV Collagen Production Promoting Action Test The black-spotted azuki bean extract obtained in Production Example 1 (Samples 1 to 3) was tested for type IV collagen production promoting action as follows.
ヒト線維芽細胞を９６穴プレートに播種し、３７℃、５％ＣＯ２−９５％ａｉｒの条件下にて、試料（試料１〜３）添加培地（試料濃度：４００μｇ／ｍＬ）で７２時間培養した後、上清１００μＬをエライザプレートに移し換え４℃、一晩でプレートに吸着させたのち、溶液を捨て、０．０５％Ｔｗｅｅｎ−２０を含むリン酸生理緩衝液（ＰＢＳ−Ｔ）にて洗浄を行った。その後、１％のウシ血清アルブミンを含むリン酸生理緩衝液で、ブロッキング操作を行った。溶液を捨て、０．０５％Ｔｗｅｅｎ−２０を含むリン酸生理緩衝液（ＰＢＳ−Ｔ）にて、洗浄を行い、抗ヒトコラーゲンタイプＩＶ抗体（ウサギＩｇＧ，ノボテック社製）を反応させた。溶液を捨て、０．０５％Ｔｗｅｅｎ−２０を含むリン酸生理緩衝液（ＰＢＳ−Ｔ）にて、洗浄を行い、ＨＲＰ標識抗ウサギＩｇＧ抗体と反応させたのち、同様の洗浄操作を行い、発色反応を行った。 Human fibroblasts are seeded in a 96-well plate and cultured in a medium (sample concentration: 400 μg / mL) for 72 hours under conditions of 37 ° C. and 5% CO 2 -95% air. After that, 100 μL of the supernatant was transferred to an ELISA plate and adsorbed on the plate overnight at 4 ° C., then the solution was discarded, and a phosphate physiological buffer solution (PBS-T) containing 0.05% Tween-20 was used. Washing was performed. Thereafter, a blocking operation was performed with a phosphate physiological buffer containing 1% bovine serum albumin. The solution was discarded, washed with a phosphate physiological buffer solution (PBS-T) containing 0.05% Tween-20, and reacted with an anti-human collagen type IV antibody (rabbit IgG, manufactured by Novotech). Discard the solution, wash with phosphate physiological buffer (PBS-T) containing 0.05% Tween-20, and react with HRP-labeled anti-rabbit IgG antibody. Reaction was performed.
ＩＶ型コラーゲン産生促進率（％）は、標準品を用いて上記ＥＬＩＳＡを行い、検量線を作成し、試料無添加時のＩＶ型コラーゲン産生量を１００％として算出した。各試料のＩＶ型コラーゲン産生促進率（％）を表２に示す。 The type IV collagen production promotion rate (%) was calculated by conducting the above ELISA using a standard product, creating a calibration curve, and assuming that the amount of type IV collagen produced when no sample was added was 100%. Table 2 shows the type IV collagen production promotion rate (%) of each sample.
[表２]
試 料 ＩＶ型コラーゲン産生促進率（％）
１ １４８
２ １６３
３ １５５
[Table 2]
Sample IV type collagen production promotion rate (%)
1 148
2 163
3 155
表２に示されるように、クロバナツルアズキ抽出物（試料１〜３）は、優れたＩＶ型コラーゲン産生促進作用を有することが確認された。 As shown in Table 2, it was confirmed that the black azuki bean extract (samples 1 to 3) has an excellent type IV collagen production promoting action.
〔試験例２〕ラミニン５産生促進作用試験
製造例１で得られたクロバナツルアズキ抽出物（試料１〜３）について、以下のようにしてラミニン５産生促進作用を試験した。
[Test Example 2] Laminin 5 production promoting effect test The black and white azuki bean extract (Samples 1 to 3) obtained in Production Example 1 was tested for laminin 5 production promoting action as follows.
正常ヒト表皮角化細胞を２４穴プレートに播種し、３７℃、５％ＣＯ２-９５％ａｉｒの条件下にて、試料添加培地（試料濃度：５０μｇ／ｍＬ）で２４時間培養した後、上清１００μＬをエライザプレートに移し換え、４℃、一晩でプレートに吸着させた。その後、溶液を捨て、０．０５％Ｔｗｅｅｎ−２０を含むリン酸生理緩衝液（ＰＢＳ−Ｔ）にて洗浄を行った。その後、１％ウシ血清アルブミンを含むリン酸生理緩衝液で、ブロッキング操作を行った。溶液を捨て、０．０５％Ｔｗｅｅｎ−２０を含むリン酸生理緩衝液（ＰＢＳ−Ｔ）にて洗浄を行い、抗ヒトラミニン５抗体（マウスＩｇＧ，ケミコン社製）を反応させた。溶液を捨て、０．０５％Ｔｗｅｅｎ−２０を含むリン酸生理緩衝液（ＰＢＳ−Ｔ）にて洗浄を行い、ビオチン標識抗マウスＩｇＧ（アマシャムバイオサイエンス社製）を反応させた。溶液を捨て、０．０５％Ｔｗｅｅｎ−２０を含むリン酸緩衝液（ＰＢＳ−Ｔ）にて洗浄を行い、ストレプトアビジン−ペルオキシダーゼ複合体（カルビオケム社製）と反応させた後、同様の洗浄操作を行い、発色反応を行った。 Normal human epidermal keratinocytes are seeded in a 24-well plate and cultured in a sample-added medium (sample concentration: 50 μg / mL) at 37 ° C. and 5% CO 2 -95% air for 24 hours. 100 μL of the clean was transferred to an ELISA plate and adsorbed on the plate at 4 ° C. overnight. Thereafter, the solution was discarded and washed with a phosphate physiological buffer solution (PBS-T) containing 0.05% Tween-20. Thereafter, a blocking operation was performed with a phosphate physiological buffer containing 1% bovine serum albumin. The solution was discarded, washed with a phosphate physiological buffer solution (PBS-T) containing 0.05% Tween-20, and reacted with anti-human laminin 5 antibody (mouse IgG, manufactured by Chemicon). The solution was discarded and washed with a phosphate physiological buffer solution (PBS-T) containing 0.05% Tween-20 to react with biotin-labeled anti-mouse IgG (Amersham Bioscience). After discarding the solution, washing with a phosphate buffer solution (PBS-T) containing 0.05% Tween-20 and reacting with a streptavidin-peroxidase complex (Calbiochem), the same washing operation was performed. The color reaction was performed.
ラミニン５産生促進率は（％）、試料無添加時における吸光度を１００％として算出した。各試料のラミニン５産生促進率（％）を表３に示す。 The laminin 5 production promotion rate was calculated as (%), and the absorbance when no sample was added was calculated as 100%. Table 3 shows the laminin 5 production promotion rate (%) of each sample.
[表３]
試 料 ラミニン５産生促進率（％）
１ １３８
２ １４５
３ １４０
[Table 3]
Sample laminin 5 production promotion rate (%)
1 138
2 145
3 140
表３に示される結果より、クロバナツルアズキ抽出物（試料１〜３）は、優れたラミニン５産生促進作用を有することが確認された。 From the results shown in Table 3, it was confirmed that the black azuki bean extract (samples 1 to 3) has an excellent laminin 5 production promoting action.
〔試験例３〕ＭＭＰ−２活性阻害作用試験
（１）ＭＭＰ−２の調製
ヒトＭＭＰ−２のｃＤＮＡ（Collier. I.E. et al.，「J. Biol. Chem.」，1998年，第263巻第14号，p.6579-6587）について、５’ＰＣＲプライマー（配列番号１）及び３’ＰＣＲプライマー（配列番号２）を用いて、ＭＭＰ−２の３．３ｋｂ ｃＤＮＡ断片を含むｐＳＧ−ＧｅｌＡを鋳型としてＰＣＲ反応を行った。得られた３０Ａｌａから４７４Ｖａｌまでをコードする１．３ｋｂＰＣＲ断片をＢａｍ ＨＩ／Ｓａｌ Ｉ部位にクローン化した。なお、ｐＴＨ−７２発現ベクターは、タンデムリピートのＴ７プロモーターの下流に、リボソーム結合部位、翻訳開始コドン、マルチクローニング部位、ヘキサヒスチジンタグをコードする配列、および翻訳終止コドンを含んでいる。
Test Example 3 MMP-2 Activity Inhibitory Action Test (1) Preparation of MMP-2 Human MMP-2 cDNA (Collier. IE et al., “J. Biol. Chem.”, 1998, Vol. 263, Vol. P. 6579-6587), using 5 ′ PCR primer (SEQ ID NO: 1) and 3 ′ PCR primer (SEQ ID NO: 2) as a template for pSG-GelA containing a 3.3 kb cDNA fragment of MMP-2 As a result, PCR reaction was performed. The resulting 1.3 kb PCR fragment encoding 30 Ala to 474 Val was cloned into the Bam HI / Sal I site. The pTH-72 expression vector includes a ribosome binding site, a translation initiation codon, a multicloning site, a sequence encoding a hexahistidine tag, and a translation stop codon downstream of the T7 promoter of tandem repeat.
ヒトＭＭＰ−２は、Ｉｔｏｈらの方法（Itoh，M. et al.，「J. Biol. Chem.」，1996年，第119巻，p.667-673）により発現した。また、精製及び巻き戻し（以下「リフォールディング」という。）は、公知の方法（西村義文 大野茂雄 監修，「タンパク実験プロトコール 細胞工学別冊 実験プロトコールシリーズ２ 構造解析編」，1997年）に準じて行った。 Human MMP-2 was expressed by the method of Itoh et al. (Itoh, M. et al., “J. Biol. Chem.”, 1996, Vol. 119, p. 667-673). In addition, purification and rewinding (hereinafter referred to as “refolding”) is performed according to a known method (supervised by Yoshifumi Nishimura, Shigeo Ohno, “Protein Experiment Protocol Cell Engineering Separate Volume Experiment Protocol Series 2 Structural Analysis”, 1997). It was.
すなわち、ＭＭＰ−２のｃＤＮＡを含む発現ベクター（ｐＴＨ−ＭＭＰ−２）を、大腸菌ＢＬ２１（ＤＥ３）株にトランスフェクトし、ＩＰＴＧで発現誘導した。発現タンパクは、Ｎｉ−ＮＴＡ樹脂（Quiagen INC.社製）を用いてアフィニティー精製後、リフォールディングを行い、酢酸４−アミノフェニル水銀と３７℃、６０分間反応を行うことで、活性型へ移行させた後、ＥＤＴＡを加えた。これを酵素標本とし、蛍光性ペプチド基質（ＭＯＣＡｃ／ＤＮＰペプチド）切断活性反応を行い、下記の条件にて高速液体クロマトグラフィーによる生成物のピーク面積を測定し、これを酵素活性の指標とした。 That is, an expression vector (pTH-MMP-2) containing MMP-2 cDNA was transfected into Escherichia coli BL21 (DE3) strain, and expression was induced with IPTG. The expressed protein is affinity-purified using Ni-NTA resin (manufactured by Quiagen INC.), Refolded, and reacted with 4-aminophenylmercuric acetate at 37 ° C for 60 minutes to shift to the active form. After that, EDTA was added. Using this as an enzyme sample, a fluorescent peptide substrate (MOCAc / DNP peptide) cleavage activity reaction was carried out, and the peak area of the product was measured by high performance liquid chromatography under the following conditions, and this was used as an index of enzyme activity.
＜高速液体クロマトグラフィー条件＞
製品名：ＬＣ−８０２０（東ソー社製）
固定相：Wakosil 5C18（和光純薬工業社製）
溶離液：３０％アセトニトリル＋０．１％ＴＨＦ
流速：１．０ｍＬ／ｍｉｎ
検出：励起波長３２５ｎｍ，蛍光波長４１０ｎｍ
検出器：８２０−ＦＰ（日本分光社製）
<High performance liquid chromatography conditions>
Product name: LC-8020 (manufactured by Tosoh Corporation)
Stationary phase: Wakosil 5C 18 (Wako Pure Chemical Industries, Ltd.)
Eluent: 30% acetonitrile + 0.1% THF
Flow rate: 1.0 mL / min
Detection: excitation wavelength 325 nm, fluorescence wavelength 410 nm
Detector: 820-FP (manufactured by JASCO)
（２）ＭＭＰ−２阻害活性の測定
試料（試料１〜３）を蒸留水に溶解させて（試料濃度：４．０ｍｇ／ｍＬ）、懸濁物を除くため、当該試料溶液をろ過した。ＭＭＰ−２阻害活性の測定は、４０μＬの活性型ＭＭＰ−２、２０μＬの試料溶液、２０μＬのアッセイバッファー（５００ｍＭのトリス−塩酸緩衝液(ｐＨ７．５)，１．５Ｍの塩化ナトリウム，１００ｍＭの塩化カルシウム，５００μＭの硫酸亜鉛，３０ｍＭのアジ化ナトリウム，０．０５％のＢｒｉｊ３５）を３７℃で１５分間プレインキュベーションした後、４．１６μＭのＭＯＣＡｃ／ＤＮＰペプチド１２０μＬを添加し、３７℃で２時間反応させた後、ＥＤＴＡ１０μＬ（２００ｍＭ）を添加した。反応液中の生成物について高速液体クロマトグラフィー分析によるピーク面積を測定した。試料溶液の代わりに蒸留水を加えた反応液の生成物についても同様に高速液体クロマトグラフィー分析によるピーク面積を測定し、下記の式に基づいて、ＭＭＰ−２阻害率（％）を求めた。
(2) Measurement of MMP-2 inhibitory activity Samples (samples 1 to 3) were dissolved in distilled water (sample concentration: 4.0 mg / mL), and the sample solution was filtered to remove the suspension. MMP-2 inhibitory activity was measured using 40 μL of active MMP-2, 20 μL of sample solution, 20 μL of assay buffer (500 mM Tris-HCl buffer (pH 7.5), 1.5 M sodium chloride, 100 mM chloride). Calcium, 500 μM zinc sulfate, 30 mM sodium azide, 0.05% Brij 35) was preincubated for 15 minutes at 37 ° C., and then added with 120 μL of 4.16 μM MOCAc / DNP peptide and reacted at 37 ° C. for 2 hours. Then, 10 μL (200 mM) EDTA was added. The peak area by the high performance liquid chromatography analysis was measured about the product in a reaction liquid. The peak area by high performance liquid chromatography analysis was similarly measured for the product of the reaction solution to which distilled water was added instead of the sample solution, and the MMP-2 inhibition rate (%) was determined based on the following formula.
ＭＭＰ−２阻害率(％)＝(Ａ−Ｂ)／Ｃ×１００
式中、Ａは「試料溶液添加時のピーク面積」を表し、Ｂは「ブランクのピーク面積」を表し、Ｃは「コントロールのピーク面積」を表す。
結果を表４に示す。
MMP-2 inhibition rate (%) = (A−B) / C × 100
In the formula, A represents “peak area when sample solution is added”, B represents “blank peak area”, and C represents “control peak area”.
The results are shown in Table 4.
［表４］
試 料 ＭＭＰ−２阻害率（％）
１ １１
２ １５
３ １３
[Table 4]
Sample MMP-2 inhibition rate (%)
1 11
2 15
3 13
表４に示すように、クロバナツルアズキからの抽出物は、優れたＭＭＰ−２活性阻害作用を有することが確認された。 As shown in Table 4, it was confirmed that the extract from black-spotted azuki bean has an excellent MMP-2 activity inhibitory action.
〔試験例４〕ＭＭＰ−９阻害作用試験
製造例１にて得られたクロバナツルアズキ抽出物（試料１〜３）について、以下のようにしてＭＭＰ−９阻害作用を試験した。
[Test Example 4] MMP-9 Inhibitory Action Test The black-spotted azuki bean extract (Samples 1 to 3) obtained in Production Example 1 was tested for MMP-9 inhibitory action as follows.
（１）一本鎖ＤＮＡの調製
正常ヒト表皮角化細胞を３５ｍｍシャーレに播種し、３７℃、５％ＣＯ２−９５％ａｉｒの条件下にて、２４時間培養した。培養終了後、リン酸生理緩衝液１ｍＬで洗浄し、ハンクス液１ｍＬに交換した。これにＴＯＲＥＸ ＦＬ２０ＳＥ−３０／ＤＭＲを４灯装着した紫外線照射装置を光源として紫外線ＵＶ−Ｂを５０ｍＪ／ｃｍ２照射した。照射後直ちに試料添加培地（試料濃度：１０μｇ／ｍＬ）に交換し、２４時間培養した。培養終了後、細胞を１ｍＬのＲＮＡ抽出用試薬（トリゾール，インビトロジェン社製）で溶解し、クロロホルムを２００μＬ添加した後、遠心（１２０００回転，４℃，１５分間）にて上層ＲＮＡ層を単離し、さらにイソプロパノールで濃縮した。濃縮沈殿させた総ＲＮＡをＴＥ溶液（１０ｍＭのトリス−塩酸／１ｍＭのＥＤＴＡ，ｐＨ８．０）に溶解して総ＲＮＡ標品とし、ＰＣＲ装置（TaKaRa PCR Themal Cycler MP，タカラバイオ社製）及びリアルタイムＰＣＲキット（TaKaRa ExScript RT reagent Kit，RR035A，タカラバイオ社製）を用いて、ＭＭＰ−９のｍＲＮＡ発現量を測定するために鋳型に使用する一本鎖ＤＮＡを合成した。また、試料を添加せず紫外線を照射せずに培養した細胞、及び試料を添加せず紫外線を照射して培養した細胞についても、同様に総ＲＮＡを調製し、一本鎖ＤＮＡを合成した。
(1) Preparation of single-stranded DNA Normal human epidermal keratinocytes were seeded in a 35 mm petri dish and cultured under conditions of 37 ° C. and 5% CO 2 -95% air for 24 hours. After completion of the culture, it was washed with 1 mL of a phosphate physiological buffer and replaced with 1 mL of Hanks' solution. An ultraviolet ray UV-B was irradiated at 50 mJ / cm 2 using an ultraviolet ray irradiation device equipped with four TOREX FL20SE-30 / DMR as a light source. Immediately after irradiation, the medium was replaced with a sample-added medium (sample concentration: 10 μg / mL) and cultured for 24 hours. After completion of the culture, the cells were lysed with 1 mL of RNA extraction reagent (Trizol, manufactured by Invitrogen), 200 μL of chloroform was added, and then the upper RNA layer was isolated by centrifugation (12,000 rpm, 4 ° C., 15 minutes) The mixture was further concentrated with isopropanol. Concentrated and precipitated total RNA is dissolved in TE solution (10 mM Tris-HCl / 1 mM EDTA, pH 8.0) to obtain a total RNA preparation, PCR apparatus (TaKaRa PCR Themal Cycler MP, manufactured by Takara Bio Inc.) and real time Using a PCR kit (TaKaRa ExScript RT reagent Kit, RR035A, manufactured by Takara Bio Inc.), a single-stranded DNA used as a template for measuring the expression level of MMP-9 mRNA was synthesized. In addition, total RNA was similarly prepared for cells cultured without adding a sample and irradiated with ultraviolet light, and cells cultured with no sample added and irradiated with ultraviolet light, and single-stranded DNA was synthesized.
（２）サイバーグリーン法を用いたリアルタイム−ＰＣＲ反応
ＭＭＰ−９遺伝子増幅用プライマーとして、センスプライマー（配列番号３）及びアンチセンスプライマー（配列番号４）を作製した。また、Ｇ３ＰＤＨ遺伝子増幅用プライマーとして、センスプライマー（配列番号５）及びアンチセンスプライマー（配列番号６）を作製した。
(2) Real-time-PCR reaction using the cyber green method A sense primer (SEQ ID NO: 3) and an antisense primer (SEQ ID NO: 4) were prepared as primers for MMP-9 gene amplification. In addition, a sense primer (SEQ ID NO: 5) and an antisense primer (SEQ ID NO: 6) were prepared as primers for G3PDH gene amplification.
上述したようにしてそれぞれ培養した細胞から調製した一本鎖ＤＮＡ及び検量線作成用一本鎖ＤＮＡ溶液を用いて、リアルタイムＰＣＲ装置（Real Time PCR System Smart Cycler II，Cepheid社製）及びリアルタイムＰＣＲキット（SYBR Premix Ex Taq，RR041A，タカラバイオ社製）にてリアルタイムＰＣＲ反応を行った。なお、検量線作成用一本鎖ＤＮＡ溶液は、原液濃度の相対値を便宜的に「１０００００」とし、以降１０倍希釈を繰り返して、濃度値「１０００００」、「１００００」、「１００」及び「１０」の５段階の希釈系列とした。反応は、９５℃で１０秒間保温した後、９５℃で５秒間、５７℃で２０秒間の反応を４５サイクル繰り返し、１サイクルごとにサイバーグリーン色素の発光量を測定した。 Real-time PCR device (Real Time PCR System Smart Cycler II, manufactured by Cepheid) and real-time PCR kit using single-stranded DNA prepared from cells cultured as described above and single-stranded DNA solution for preparing calibration curve (SYBR Premix Ex Taq, RR041A, manufactured by Takara Bio Inc.) was used for real-time PCR reaction. In the single-strand DNA solution for preparing a calibration curve, the relative value of the stock solution is set to “100,000” for convenience, and 10-fold dilution is repeated thereafter to obtain concentration values “100,000”, “10000”, “100” and “100”. 10 ”was a five-stage dilution series. The reaction was held at 95 ° C. for 10 seconds, followed by 45 cycles of reaction at 95 ° C. for 5 seconds and 57 ° C. for 20 seconds, and the amount of luminescence of the cyber green dye was measured for each cycle.
（３）解析
サイクルごとのサイバーグリーン色素の発光量から、ＭＭＰ−９及びＧ３ＰＤＨのそれぞれをコードするＤＮＡ断片の増幅曲線を作成した。検量線作成用一本鎖ＤＮＡ溶液の希釈系列の増幅曲線から横軸に濃度、縦軸に増幅曲線の２次導関数が最大となるサイクル数をとった検量線を作成した。各発現定量用サンプルについては、増幅曲線の２次導関数が最大となるサイクル数を検量線上にプロットし、相対的な発現量を算出した。ＭＭＰ−９の発現量は、同一サンプルにおけるＧ３ＰＤＨの発現量の値で補正を行った後、さらに試料を添加せず紫外線を照射せずに培養した細胞から調製した一本鎖ＤＮＡを用いて算出したＭＭＰ−９の発現量の補正値を１００としたときの、試料を添加せずに紫外線を照射して培養した細胞から調製した一本鎖ＤＮＡを用いて算出したＭＭＰ−９の発現量の補正値、及び試料を添加し紫外線を照射して培養した細胞から調整した一本鎖ＤＮＡを用いて算出したＭＭＰ−９の発現量の補正値を算出した。得られた補正値から下記の式に基づいてＭＭＰ−９のｍＲＮＡ発現抑制率（％）を算出した。
(3) Analysis Amplification curves of DNA fragments encoding each of MMP-9 and G3PDH were prepared from the amount of luminescence of the cyber green dye for each cycle. A calibration curve was prepared from the amplification curve of a dilution series of a single-stranded DNA solution for preparing a calibration curve, with the horizontal axis representing the concentration and the vertical axis representing the number of cycles that maximized the second derivative of the amplification curve. For each expression quantification sample, the number of cycles that maximized the second derivative of the amplification curve was plotted on a calibration curve, and the relative expression level was calculated. The expression level of MMP-9 was calculated using single-stranded DNA prepared from cells cultured after correction with the value of the expression level of G3PDH in the same sample and without addition of a sample and without irradiation with ultraviolet rays. Of the expression level of MMP-9 calculated using single-stranded DNA prepared from cells cultured by irradiating with ultraviolet rays without adding a sample when the corrected value of the expression level of MMP-9 is 100 The correction value and the correction value of the expression level of MMP-9 calculated using single-stranded DNA prepared from cells cultured by adding the sample and irradiating with ultraviolet rays were calculated. Based on the obtained correction value, the mRNA expression suppression rate (%) of MMP-9 was calculated based on the following formula.
ｍＲＮＡ発現抑制率(%)＝{(Ａ−Ｂ)−(Ａ−Ｃ)}／(Ａ−Ｂ)×１００
式中、Ａは「試料無添加・紫外線非照射の補正値」を表し、Ｂは「試料無添加・紫外線照射の補正値」を表し、Ｃは「試料添加・紫外線照射の補正値」を表す。
結果を表５に示す。
mRNA expression suppression rate (%) = {(A−B) − (A−C)} / (A−B) × 100
In the formula, A represents “correction value of no sample addition / ultraviolet irradiation”, B represents “correction value of no sample addition / ultraviolet irradiation”, and C represents “correction value of sample addition / ultraviolet irradiation”. .
The results are shown in Table 5.
［表５］
試 料 ｍＲＮＡ発現抑制率（％）
１ ７８
２ ８７
３ ８３
[Table 5]
Sample mRNA expression suppression rate (%)
1 78
2 87
3 83
表５に示すように、クロバナツルアズキ抽出物は、ＭＭＰ−９のｍＲＮＡの発現を抑制可能であることが確認された。このことから、クロバナツルアズキ抽出物は、優れたＭＭＰ−９阻害作用を有することが推認される。 As shown in Table 5, it was confirmed that the black azuki bean extract can suppress the expression of MMP-9 mRNA. From this, it is presumed that the black-spotted azuki bean extract has an excellent MMP-9 inhibitory action.
〔配合例１〕
下記組成の乳液を常法により製造した。
クロバナツルアズキ幹部５０質量％エタノール抽出物（製造例１）
０．０１ｇ
ホホバオイル ４．０ｇ
モモ葉エキス ０．１ｇ
オリーブオイル ２．０ｇ
スクワラン ２．０ｇ
セタノール ２．０ｇ
モノステアリン酸グリセリル ２．０ｇ
ポリオキシエチレンセチルエーテル（20E.O.） ２．５ｇ
オレイン酸ポリオキシエチレンソルビタン（20E.O.） ２．０ｇ
グリチルレチン酸ステアリル ０．１ｇ
サルビアエキス ０．１ｇ
加水分解コンコンキオリン ０．１ｇ
１，３−ブチレングリコール ３．０ｇ
ヒノキチオール ０．１５ｇ
香料 ０．０５ｇ
精製水 残部（全量を１００ｇとする）
[Formulation Example 1]
An emulsion having the following composition was produced by a conventional method.
Black-headed Azuki stem 50% by mass ethanol extract (Production Example 1)
0.01g
Jojoba oil 4.0g
Peach leaf extract 0.1g
Olive oil 2.0g
Squalane 2.0g
Cetanol 2.0g
Glyceryl monostearate 2.0g
Polyoxyethylene cetyl ether (20E.O.) 2.5g
Oleic acid polyoxyethylene sorbitan (20E.O.) 2.0g
Stearyl glycyrrhetinate 0.1g
Salvia extract 0.1g
Hydrolyzed Conconchiolin 0.1g
1,3-butylene glycol 3.0 g
Hinokitiol 0.15g
Fragrance 0.05g
Purified water remainder (total amount is 100 g)
〔配合例２〕
下記組成のクリームを常法により製造した。
クロバナツルアズキ幹部５０質量％エタノール抽出物（製造例１）
０．０５ｇ
アロエエキス ０．１ｇ
ニンジンエキス ０．１ｇ
センブリエキス ０．１ｇ
流動パラフィン ５．０ｇ
サラシミツロウ ４．０ｇ
スクワラン １０．０ｇ
セタノール ３．０ｇ
ラノリン ２．０ｇ
ステアリン酸 １．０ｇ
オレイン酸ポリオキシエチレンソルビタン（20E.O.） １．５ｇ
モノステアリン酸グリセリル ３．０ｇ
油溶性甘草エキス ０．１ｇ
１，３−ブチレングリコール ６．０ｇ
パラオキシ安息香酸メチル １．５ｇ
香料 ０．１ｇ
精製水 残部（全量を１００ｇとする）
[Formulation Example 2]
A cream having the following composition was produced by a conventional method.
Black-headed Azuki stem 50% by mass ethanol extract (Production Example 1)
0.05g
Aloe extract 0.1g
Carrot extract 0.1g
Assembly extract 0.1g
Liquid paraffin 5.0g
Salami beeswax 4.0g
Squalane 10.0g
Cetanol 3.0g
Lanolin 2.0g
Stearic acid 1.0g
Oleic acid polyoxyethylene sorbitan (20E.O.) 1.5g
3.0 g glyceryl monostearate
Oil soluble licorice extract 0.1g
1,3-butylene glycol 6.0 g
1.5 g of methyl paraoxybenzoate
Fragrance 0.1g
Purified water remainder (total amount is 100 g)
〔配合例３〕
下記組成のクリームを常法により製造した。
クロバナツルアズキ幹部５０質量％エタノール抽出物（製造例１）
０．０５ｇ
エイジツエキス ０．１ｇ
シャクヤクエキス ０．１ｇ
ワレモコウエキス ０．１ｇ
モノステアリン酸ポリエチレングリコール ２．０ｇ
自己乳化型モノステアリン酸グリセリン ５．０ｇ
ステアリン酸 ２．０ｇ
セタノール ２．０ｇ
スクワラン １２．０ｇ
マカダミアナッツ油 ３．０ｇ
メチルポリシロキサン ０．２ｇ
香料 ０．０１ｇ
防腐剤（パラオキシ安息香酸メチル） ０．１５ｇ
甘草エキス ０．１ｇ
１，３−ブチレングリコール ７．０ｇ
キサンタンガム ０．２ｇ
精製水 残部（全量を１００ｇとする）
[Composition Example 3]
A cream having the following composition was produced by a conventional method.
Black-headed Azuki stem 50% by mass ethanol extract (Production Example 1)
0.05g
Ages extract 0.1g
Peonies extract 0.1g
Walnut extract 0.1g
Polyethylene glycol monostearate 2.0g
Self-emulsifying glyceryl monostearate 5.0 g
Stearic acid 2.0g
Cetanol 2.0g
Squalane 12.0g
Macadamia nut oil 3.0g
0.2g of methylpolysiloxane
Fragrance 0.01g
Preservative (Methyl paraoxybenzoate) 0.15g
Licorice extract 0.1g
1,3-butylene glycol 7.0 g
Xanthan gum 0.2g
Purified water remainder (total amount is 100 g)
〔配合例４〕
下記組成の美容液を常法により製造した。
クロバナツルアズキ幹部５０質量％エタノール抽出物（製造例１）
０．０１ｇ
カミツレエキス ０．１ｇ
シソエキス ０．１ｇ
シラカバエキス ０．１ｇ
キサンタンガム ０．３ｇ
ヒドロキシエチルセルロース ０．１ｇ
カルボキシビニルポリマー ０．１ｇ
１，３−ブチレングリコール ４．０ｇ
グリチルリチン酸ジカリウム ０．１ｇ
グリセリン ２．０ｇ
水酸化カリウム ０．２５ｇ
香料 ０．０１ｇ
防腐剤（パラオキシ安息香酸メチル） ０．１５ｇ
エタノール ２．０ｇ
精製水 残部（全量を１００ｇとする）
[Formulation Example 4]
A serum having the following composition was produced by a conventional method.
Black-headed Azuki stem 50% by mass ethanol extract (Production Example 1)
0.01g
Chamomile extract 0.1g
Perilla extract 0.1g
Birch extract 0.1g
Xanthan gum 0.3g
Hydroxyethylcellulose 0.1g
Carboxyvinyl polymer 0.1g
1,3-butylene glycol 4.0 g
0.1g dipotassium glycyrrhizinate
Glycerin 2.0g
Potassium hydroxide 0.25g
Fragrance 0.01g
Preservative (Methyl paraoxybenzoate) 0.15g
Ethanol 2.0g
Purified water remainder (total amount is 100 g)
〔配合例５〕
下記組成の美容液を常法により製造した。
クロバナツルアズキ幹部５０質量％エタノール抽出物（製造例１）
０．０５ｇ
クワエキス ０．１ｇ
リョクチャエキス ０．１ｇ
カルボキシビニルポリマー ０．２ｇ
キサンタンガム ０．２ｇ
ヒアルロン酸ナトリウム ０．１ｇ
ポリエチレングリコール ３．０ｇ
グリセリン ６．０ｇ
１，３−ブチレングリコール ３．０ｇ
グリチルリチン酸ジカリウム ０．１ｇ
防腐剤（パラオキシ安息香酸メチル） ０．１５ｇ
Ｌ−アルギニン ０．１５ｇ
精製水 残部（全量を１００ｇとする）
[Formulation Example 5]
A serum having the following composition was produced by a conventional method.
Black-headed Azuki stem 50% by mass ethanol extract (Production Example 1)
0.05g
Mulberry extract 0.1g
Ryokucha Extract 0.1g
Carboxyvinyl polymer 0.2g
Xanthan gum 0.2g
Sodium hyaluronate 0.1g
Polyethylene glycol 3.0g
Glycerin 6.0g
1,3-butylene glycol 3.0 g
0.1g dipotassium glycyrrhizinate
Preservative (Methyl paraoxybenzoate) 0.15g
L-Arginine 0.15g
Purified water remainder (total amount is 100 g)
〔配合例６〕
下記組成のパックを常法により製造した。
クロバナツルアズキ幹部５０質量％エタノール抽出物（製造例１）
０．０５ｇ
ヨクイニンエキス ０．１ｇ
ホップエキス ０．１ｇ
ポリビニルアルコール １５．０ｇ
ポリエチレングリコール ３．０ｇ
プロピレングリコール ７．０ｇ
エタノール １０．０ｇ
パラオキシ安息香酸メチル ０．０５ｇ
グリチルリチン酸ジカリウム ０．１ｇ
香料 ０．０５ｇ
精製水 残部（全量を１００ｇとする）
[Composition Example 6]
A pack having the following composition was produced by a conventional method.
Black-headed Azuki stem 50% by mass ethanol extract (Production Example 1)
0.05g
Yokuinin extract 0.1g
Hop extract 0.1g
Polyvinyl alcohol 15.0g
Polyethylene glycol 3.0g
Propylene glycol 7.0g
Ethanol 10.0g
0.05 g of methyl paraoxybenzoate
0.1g dipotassium glycyrrhizinate
Fragrance 0.05g
Purified water remainder (total amount is 100 g)
〔配合例７〕
下記組成の洗浄用化粧水を常法により製造した。
クロバナツルアズキ幹部５０質量％エタノール抽出物（製造例１）
０．０５ｇ
ラベンダーエキス ０．１ｇ
ハマメリスエキス ０．１ｇ
メリッサエキス ０．１ｇ
１，３−ブチレングリコール ４．０ｇ
グリセリン ４．０ｇ
ジプロピレングリコール ２．０ｇ
ポリオキシエチレンポリオキシプロピレン
デシルテトラデシルエーテル ０．７ｇ
エタノール ３．０ｇ
グリチルリチン酸ジカリウム ０．１ｇ
香料 ０．００１ｇ
防腐剤（パラオキシ安息香酸メチル） ０．１５ｇ
精製水 残部（全量を１００ｇとする）
[Formulation Example 7]
A cleaning lotion having the following composition was produced by a conventional method.
Black-headed Azuki stem 50% by mass ethanol extract (Production Example 1)
0.05g
Lavender extract 0.1g
Hamamelis extract 0.1g
Melissa extract 0.1g
1,3-butylene glycol 4.0 g
Glycerin 4.0g
Dipropylene glycol 2.0g
Polyoxyethylene polyoxypropylene
Decyl tetradecyl ether 0.7g
Ethanol 3.0g
0.1g dipotassium glycyrrhizinate
Fragrance 0.001g
Preservative (Methyl paraoxybenzoate) 0.15g
Purified water remainder (total amount is 100 g)
〔配合例８〕
下記組成の乳液を常法により製造した。
クロバナツルアズキ幹部５０質量％エタノール抽出物（製造例１）
０．０５ｇ
ローズマリーエキス ０．１ｇ
モノステアリン酸ポリオキシエチレンソルビタン １．０ｇ
テトラオレイン酸ポリオキシエチレンソルビット １．５ｇ
親油型モノステアリン酸グリセリン １．０ｇ
ステアリン酸 ０．５ｇ
ベヘニルアルコール １．５ｇ
パルミチン酸セチル ０．５ｇ
スクワラン １０．０ｇ
メチルポリシロキサン ０．５ｇ
香料 適量
１，３−ブチレングリコール ７．０ｇ
グリチルレチン酸ステアリル ０．１ｇ
キサンタンガム ０．１ｇ
精製水 残部（全量を１００ｇとする）
[Formulation Example 8]
An emulsion having the following composition was produced by a conventional method.
Black-headed Azuki stem 50% by mass ethanol extract (Production Example 1)
0.05g
Rosemary extract 0.1g
1.0 g polyoxyethylene sorbitan monostearate
Tetraoleic acid polyoxyethylene sorbit 1.5g
Lipophilic glyceryl monostearate 1.0g
Stearic acid 0.5g
Behenyl alcohol 1.5g
Cetyl palmitate 0.5g
Squalane 10.0g
Methyl polysiloxane 0.5g
Perfume appropriate amount 1,3-butylene glycol 7.0g
Stearyl glycyrrhetinate 0.1g
Xanthan gum 0.1g
Purified water remainder (total amount is 100 g)
本発明のＩＶ型コラーゲン産生促進剤、ラミニン５産生促進剤、マトリックスメタロプロテアーゼ阻害剤、抗老化剤、皮膚化粧料又は美容用飲食品は、皮膚の老化症状の予防・改善に有用である。 The type IV collagen production promoter, laminin 5 production promoter, matrix metalloprotease inhibitor, anti-aging agent, skin cosmetic, or cosmetic food or drink of the present invention is useful for the prevention and improvement of skin aging symptoms.
Claims (5)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2006040237A JP4731350B2 (en) | 2006-02-17 | 2006-02-17 | Anti-aging agent, skin cosmetics and food and drink for beauty |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2006040237A JP4731350B2 (en) | 2006-02-17 | 2006-02-17 | Anti-aging agent, skin cosmetics and food and drink for beauty |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2007217352A JP2007217352A (en) | 2007-08-30 |
| JP4731350B2 true JP4731350B2 (en) | 2011-07-20 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2006040237A Active JP4731350B2 (en) | 2006-02-17 | 2006-02-17 | Anti-aging agent, skin cosmetics and food and drink for beauty |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4731350B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5008168B2 (en) * | 2006-02-21 | 2012-08-22 | 丸善製薬株式会社 | Anti-aging agent and skin cosmetics |
| JP2009298723A (en) * | 2008-06-12 | 2009-12-24 | Maruzen Pharmaceut Co Ltd | Type iv collagen production promoter |
| CN102548571B (en) | 2009-06-12 | 2014-08-27 | 基因雷克斯制药有限公司 | Compositions and methods for prevention and treatment of brain diseases and conditions |
| JP6161953B2 (en) * | 2013-05-23 | 2017-07-12 | 日本メナード化粧品株式会社 | Hair restorer, muscle activator and energy production promoter |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0925225A (en) * | 1995-05-09 | 1997-01-28 | Sansho Seiyaku Co Ltd | Skin external agent and production of active component for the agent |
| JP2001122764A (en) * | 1999-10-21 | 2001-05-08 | Koreana Cosmetics Co Ltd | Skin cosmetic composition |
| JP2002154919A (en) * | 2000-11-14 | 2002-05-28 | Pola Chem Ind Inc | Elongation inhibitor of dendrite of melanocyte and cosmetic comprising the same |
| JP2002179529A (en) * | 2000-12-13 | 2002-06-26 | Pola Chem Ind Inc | Composition for bleaching |
| JP2002265324A (en) * | 2001-03-07 | 2002-09-18 | Ichimaru Pharcos Co Ltd | Cosmetic composition containing moisture retention plant extract |
| JP2002265343A (en) * | 2001-03-07 | 2002-09-18 | Ichimaru Pharcos Co Ltd | Cosmetic composition |
| JP2003201208A (en) * | 2001-12-28 | 2003-07-18 | Ichimaru Pharcos Co Ltd | Cosmetic composition |
| JP2005060334A (en) * | 2003-08-19 | 2005-03-10 | Okinawa Pref Gov | Anti-obesity agent having lipase inhibiting activity and antioxidation activity |
-
2006
- 2006-02-17 JP JP2006040237A patent/JP4731350B2/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0925225A (en) * | 1995-05-09 | 1997-01-28 | Sansho Seiyaku Co Ltd | Skin external agent and production of active component for the agent |
| JP2001122764A (en) * | 1999-10-21 | 2001-05-08 | Koreana Cosmetics Co Ltd | Skin cosmetic composition |
| JP2002154919A (en) * | 2000-11-14 | 2002-05-28 | Pola Chem Ind Inc | Elongation inhibitor of dendrite of melanocyte and cosmetic comprising the same |
| JP2002179529A (en) * | 2000-12-13 | 2002-06-26 | Pola Chem Ind Inc | Composition for bleaching |
| JP2002265324A (en) * | 2001-03-07 | 2002-09-18 | Ichimaru Pharcos Co Ltd | Cosmetic composition containing moisture retention plant extract |
| JP2002265343A (en) * | 2001-03-07 | 2002-09-18 | Ichimaru Pharcos Co Ltd | Cosmetic composition |
| JP2003201208A (en) * | 2001-12-28 | 2003-07-18 | Ichimaru Pharcos Co Ltd | Cosmetic composition |
| JP2005060334A (en) * | 2003-08-19 | 2005-03-10 | Okinawa Pref Gov | Anti-obesity agent having lipase inhibiting activity and antioxidation activity |
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| Publication number | Publication date |
|---|---|
| JP2007217352A (en) | 2007-08-30 |
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