JPH11349436A - Collagenase inhibitor and skin agent containing the same and used for external use - Google Patents
Collagenase inhibitor and skin agent containing the same and used for external useInfo
- Publication number
- JPH11349436A JPH11349436A JP10172217A JP17221798A JPH11349436A JP H11349436 A JPH11349436 A JP H11349436A JP 10172217 A JP10172217 A JP 10172217A JP 17221798 A JP17221798 A JP 17221798A JP H11349436 A JPH11349436 A JP H11349436A
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- Prior art keywords
- ethanol
- collagenase
- skin
- collagenase inhibitor
- fraction
- Prior art date
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- Medicines Containing Plant Substances (AREA)
Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は、主として間葉系細
胞により産生される間質系のコラゲナーゼに対し、特異
的に作用するコラゲナーゼ阻害剤、及びこれを含有して
成る、皮膚の老化症状の防止,改善に有効な皮膚外用剤
に関する。さらに詳しくは、ユーカリノキ又はその近縁
植物の50容量%エタノールによる抽出物より、精製分
画して得た分画物を含有して成るコラゲナーゼ阻害剤、
さらにこれを含有して成る皮膚外用剤に関する。[0001] The present invention relates to a collagenase inhibitor which acts specifically on stromal collagenase produced mainly by mesenchymal cells, and a skin aging condition comprising the same, which comprises the same. It relates to an external preparation for skin that is effective for prevention and improvement. More specifically, a collagenase inhibitor comprising a fraction obtained by purifying and fractionating from a 50% by volume ethanol extract of Eucalyptus or a related plant thereof,
Further, the present invention relates to a skin external preparation containing the same.
【0002】[0002]
【従来の技術】近年、マトリックス線維の分解を促進す
るマトリックスメタロプロテアーゼ(MMP)が種々の
病態に関与していることが明らかになってきた。MMP
の中でも、線維芽細胞等の間葉系細胞や、炎症部位に存
在する好酸球等により産生される間質系のコラゲナーゼ
が、がん細胞の転移や潰瘍形成、歯周炎やう歯形成等に
関与することが報告されている。また前記コラゲナーゼ
は、皮膚真皮のコラーゲン線維を分解し、皮膚弾性の低
下やしわの発生にも関与している。これらコラゲナーゼ
の産生が、紫外線やアレルゲン等の外部刺激により誘導
されるサイトカインによって促進されることも知られて
いる。2. Description of the Related Art In recent years, it has become clear that matrix metalloprotease (MMP), which promotes degradation of matrix fibers, is involved in various disease states. MMP
Among them, mesenchymal cells such as fibroblasts, and stromal collagenase produced by eosinophils and the like present at the site of inflammation are involved in metastasis and ulceration of cancer cells, periodontitis and caries formation, etc. Has been reported to be involved. In addition, the collagenase degrades collagen fibers in the dermis of the skin, and is also involved in reducing skin elasticity and generating wrinkles. It is also known that production of these collagenases is promoted by cytokines induced by external stimuli such as ultraviolet rays and allergens.
【0003】それゆえ、前記のようなコラゲナーゼの活
性を阻害するコラゲナーゼ阻害剤のスクリーニングも盛
んに行われてきた。たとえば、アシルフェニルグリシン
誘導体(特開平7−101925)、ザクロ実,レモン
バーム実及び葉,グアバ,ハマメリスといった植物の抽
出物(特開平7−196526,同7−291873,
同8−283133)、ポリポレン酸及びこれを含有す
るホウロクタケ抽出物(特開平9−40552)、ジカ
ルボン酸(特開平9−124472)、アスペルギルス
属微生物の産生物質(特開平9−241287)などが
開示されている。[0003] Therefore, screening for a collagenase inhibitor that inhibits the activity of collagenase as described above has been actively conducted. For example, acylphenylglycine derivatives (JP-A-7-101925), plant extracts such as pomegranate seeds, lemon balm seeds and leaves, guava and hamamelis (JP-A-7-195526, and 7-291873).
No. 8-283133), polypolenic acid and holo mushroom extract containing the same (JP-A-9-40552), dicarboxylic acid (JP-A-9-124472), substances produced by microorganisms belonging to the genus Aspergillus (JP-A-9-241287), and the like. Have been.
【0004】しかしながら、これまでに得られたコラゲ
ナーゼ阻害剤の中には、細菌性のコラゲナーゼに対し強
い阻害活性を示し、間質系のコラゲナーゼに対する特異
性の低いものや、阻害活性が十分でないもの、安定性に
欠けるものも存在していた。[0004] However, among the collagenase inhibitors obtained so far, those having a strong inhibitory activity against bacterial collagenase, having low specificity for stromal collagenase, or those having insufficient inhibitory activity. Some lacked stability.
【0005】[0005]
【発明が解決しようとする課題】そこで本発明において
は、線維芽細胞等の間葉系細胞や好酸球等により産生さ
れる間質系のコラゲナーゼに対し特異的に阻害活性を示
し、炎症や創傷、がんの転移、潰瘍形成、皮膚の老化
等、コラゲナーゼの関与する病態を改善し、或いは防止
し得るコラゲナーゼ阻害剤を得、さらには皮膚弾性の低
下やしわの形成といった皮膚老化症状の防止及び改善に
有効な皮膚外用剤を得ることを目的とした。Accordingly, in the present invention, the present invention specifically exhibits an inhibitory activity against stromal collagenase produced by mesenchymal cells such as fibroblasts, eosinophils, etc., and exhibits inflammation and Obtain collagenase inhibitors that can improve or prevent collagenase-related pathologies such as wounds, cancer metastasis, ulceration, and skin aging, and prevent skin aging symptoms such as reduced skin elasticity and wrinkles And to obtain a skin external preparation effective for improvement.
【0006】[0006]
【課題を解決するための手段】上記の課題を解決するた
め、本発明者らは安定性及び安全性が高く、しかも高活
性のコラゲナーゼ阻害剤のスクリーニングを行い、すで
に、ユーカリノキ,サルビア,ドクダミ,ボタン及びシ
ャクヤク又はこれらの近縁植物の抽出物に優れたコラゲ
ナーゼ阻害活性を見いだし、これを含有するコラゲナー
ゼ阻害剤について開示した(特願平9−33498
1)。今回、前記植物抽出物の中で、ユーカリノキ又は
その近縁植物の50容量%エタノールによる抽出物をさ
らに分画することにより、特に高いコラゲナーゼ阻害活
性を示す画分を得ることができ、これを含有させること
によって、本発明を完成するに至った。Means for Solving the Problems To solve the above-mentioned problems, the present inventors screened a collagenase inhibitor having high stability and safety and high activity, and have already screened Eucalyptus, Salvia, Dokudami, An excellent collagenase inhibitory activity was found in extracts of buttons and peonies or their closely related plants, and a collagenase inhibitor containing the same was disclosed (Japanese Patent Application No. 9-33498).
1). This time, among the plant extracts, a fraction exhibiting particularly high collagenase inhibitory activity can be obtained by further fractionating an extract of Eucalyptus or a closely related plant thereof with 50% by volume of ethanol. As a result, the present invention has been completed.
【0007】すなわち本発明においては、ユーカリノキ
(Eucalyptus globulus Labillardiere)又はその近縁
植物(Eucalyptus polybractea R.T.Baker,Eucalyptus
dives Schauer)の50容量%エタノールによる抽出物
を、スチレンポリマー製の吸着ゲルクロマトグラフィー
にてエタノール−水混合溶媒で分画し、50容量%エタ
ノール溶出画分を分取し、次いでシリカゲルクロマトグ
ラフィーにかけ、クロロホルム−メタノール−水混合溶
媒で溶出した後に、50容量%エタノールにより溶出さ
れる画分を分取して、これをそのまま、或いは溶媒,基
剤等に含有させてコラゲナーゼ阻害剤とする。そしてさ
らに、このコラゲナーゼ阻害剤を外用剤基剤に含有させ
て、皮膚外用剤とする。That is, in the present invention, Eucalyptus globulus Labillardiere or a closely related plant thereof ( Eucalyptus polybractea RTBaker, Eucalyptus)
dives Schauer) was fractionated with 50% ethanol by a mixed solvent of ethanol and water by adsorption gel chromatography made of styrene polymer, and the fraction eluted with 50% ethanol by ethanol was then subjected to silica gel chromatography. After elution with a mixed solvent of chloroform-methanol-water, a fraction eluted with 50% by volume of ethanol is collected and used as it is or as a collagenase inhibitor as it is contained in a solvent, a base or the like. Further, this collagenase inhibitor is contained in an external preparation base to prepare a skin external preparation.
【0008】なおユーカリノキ抽出物は、従来より化粧
料や皮膚外用剤に配合されており、Porphyromonas ging
ivalisにより産生されたコラゲナーゼを抑制する作用が
知られている(特開平8−109118)。しかしこの
植物の抽出物より、間質系コラゲナーゼに対し、特異的
且つ高い阻害作用を有する画分を得たという報告はまだ
なされていない。[0008] Note that eucalyptus extract is formulated in cosmetics and skin external agents conventionally, Porphyromonas ging
effect of inhibiting collagenase produced is known by Ivalis (JP 8-109118). However, there has been no report that a fraction having a specific and high inhibitory effect on stromal collagenase was obtained from the plant extract.
【0009】[0009]
【発明の実施の形態】本発明のコラゲナーゼ阻害剤を得
る際、出発材料として用いるユーカリノキ(Eucalyptus
globulus Labillardiere)は、生薬ユーカリ油の基原
植物として用いられるオーストラリア原産のフトモモ科
に属する常緑高木である。その近縁植物(Eucalyptus p
olybractea R.T.Baker,Eucalyptus dives Schauer)も
用いることができる。抽出には、前記植物の花,茎,
葉,根の各部位及び全木を用いることができる。本発明
においては、これら植物の50容量%エタノールによる
抽出物を用いる。DESCRIPTION OF THE PREFERRED EMBODIMENTS In obtaining the collagenase inhibitor of the present invention, Eucalyptus used as a starting material
globulus Labillardiere) is an evergreen tree belonging to the family Myrtaceae native to Australia and used as a base plant of crude drug eucalyptus oil. Related plants ( Eucalyptus p
olybractea RTBaker, Eucalyptus dives Schauer) can also be used. For extraction, flowers, stems,
Leaves, roots and whole trees can be used. In the present invention, an extract of these plants with 50% by volume of ethanol is used.
【0010】上記植物は生のまま抽出操作に供しても良
いが、抽出効率を考えると、細切,乾燥,粉砕等の処理
を行った後抽出を行うことが好ましい。抽出は、抽出溶
媒である50容量%エタノールに浸漬して行う。抽出効
率を上げるため攪拌を行ったり、抽出溶媒中でホモジナ
イズすることもできる。抽出温度としては5℃〜50℃
程度が適切である。抽出時間は、4時間〜10日間程度
である。The above plants may be subjected to an extraction operation as they are, but in consideration of the extraction efficiency, it is preferable to carry out the extraction after performing a treatment such as shredding, drying and pulverization. The extraction is performed by immersing in 50% by volume ethanol as an extraction solvent. Stirring may be performed to increase the extraction efficiency, or homogenization may be performed in an extraction solvent. 5 ℃ -50 ℃ as extraction temperature
The degree is appropriate. The extraction time is about 4 hours to 10 days.
【0011】ユーカリノキ又はその近縁植物の50容量
%エタノール抽出物は、まずスチレンポリマー製の吸着
ゲルクロマトグラフィーにて、エタノール−水混合溶媒
で分画し、50容量%エタノールで溶出される画分を得
る。スチレンポリマー製の吸着ゲルとしては、MCI
GEL(三菱化学株式会社製)等が好ましく使用でき
る。前記画分をさらにシリカゲルクロマトグラフィーに
かけ、クロロホルム−メタノール−水混合溶媒で溶出
し、この混合溶媒では溶出されないシリカゲル吸着物質
を50容量%エタノールで溶出し、目的とする画分を得
る。なお、前記吸着ゲルクロマトグラフィー,シリカゲ
ルクロマトグラフィーは、カラムクロマトグラフィーと
して行うのが適切である。A 50% by volume ethanol extract of Eucalyptus or a closely related plant thereof is first fractionated by an adsorption gel chromatography made of styrene polymer with a mixed solvent of ethanol and water, and a fraction eluted with 50% by volume of ethanol Get. As an adsorption gel made of styrene polymer, MCI
GEL (manufactured by Mitsubishi Chemical Corporation) or the like can be preferably used. The fraction is further subjected to silica gel chromatography, and eluted with a mixed solvent of chloroform-methanol-water. A silica gel adsorbed substance that is not eluted with this mixed solvent is eluted with 50% by volume of ethanol to obtain a desired fraction. The adsorption gel chromatography and the silica gel chromatography are suitably performed as column chromatography.
【0012】上記ユーカリノキ等の50容量%エタノー
ルによる抽出物より得られる分画物は、そのままでもコ
ラゲナーゼ阻害剤として利用できるが、水,エタノール
等の極性溶媒に溶解又は懸濁したり、乳剤,クリーム,
ゲル,軟膏等の基剤に分散してコラゲナーゼ阻害剤とす
ることもできる。The fraction obtained from the 50% by volume ethanol extract of Eucalyptus or the like can be used as it is as a collagenase inhibitor, but it can be dissolved or suspended in a polar solvent such as water or ethanol, or can be used as an emulsion, cream,
Collagenase inhibitors can also be dispersed in a base such as gel or ointment.
【0013】さらに本発明に係るコラゲナーゼ阻害剤に
は、活性酸素消去剤,抗炎症剤,抗がん剤,美白剤,皮
膚細胞賦活剤,殺菌剤等の生理活性成分の他、油類,界
面活性剤,保湿剤,紫外線吸収剤,粉体,香料,防腐剤
等、一般的な医薬品及び化粧料用原料をも含有させるこ
とができる。The collagenase inhibitor according to the present invention includes physiologically active components such as active oxygen scavengers, anti-inflammatory agents, anticancer agents, whitening agents, skin cell activators, bactericides, oils, and interface agents. Raw materials for general pharmaceuticals and cosmetics, such as activators, humectants, ultraviolet absorbers, powders, fragrances, preservatives, etc., can also be included.
【0014】また本発明においては、上記のようにして
得たコラゲナーゼ阻害剤を外用剤基剤に含有させて皮膚
外用剤とする。皮膚外用剤への配合量としては、0.0
001〜5.0重量%程度が適切である。In the present invention, the collagenase inhibitor obtained as described above is contained in an external preparation base to prepare an external preparation for skin. The amount added to the skin external preparation was 0.0
About 001 to 5.0% by weight is appropriate.
【0015】なお本発明に係る皮膚外用剤は、ローショ
ン剤,乳剤,ゲル剤,クリーム剤,軟膏等の剤型で提供
することができ、さらに化粧水,乳液,クリーム,パッ
ク等の皮膚化粧料、メイクアップベースローション,メ
イクアップベースクリーム,乳液状又はクリーム状或い
は軟膏型のファンデーション,アイカラー,チークカラ
ーといったメイクアップ化粧料、ハンドクリーム,レッ
グクリーム,ボディローション等の身体用化粧料などと
しても提供することができる。また、本発明に係るコラ
ゲナーゼ阻害剤の他、油類,界面活性剤,保湿剤,紫外
線吸収剤,顔料,香料,防腐剤等の一般的な医薬品及び
化粧料用原料や、活性酸素消去剤,抗炎症剤,美白剤,
皮膚細胞賦活剤等の生理活性成分も含有させることがで
きる。The external preparation for skin according to the present invention can be provided in the form of lotions, emulsions, gels, creams, ointments and the like, and further, skin cosmetics such as lotions, emulsions, creams, packs, etc. , Makeup base lotion, makeup base cream, emulsion or creamy or ointment type foundation, makeup cosmetics such as eye color, cheek color, body cosmetics such as hand cream, leg cream, body lotion, etc. Can be provided. Further, in addition to the collagenase inhibitor according to the present invention, general pharmaceutical and cosmetic raw materials such as oils, surfactants, humectants, ultraviolet absorbers, pigments, fragrances, and preservatives, active oxygen scavengers, Anti-inflammatory, whitening,
Physiologically active ingredients such as skin cell activators can also be included.
【0016】[0016]
【実施例】さらに本発明の特徴について、実施例により
詳細に説明する。EXAMPLES Further, the features of the present invention will be described in detail with reference to examples.
【0017】[実施例1]ユーカリノキ(Eucalyptus g
lobulus Labillardiere)の葉の乾燥粉末500gを、
50容量%エタノール水溶液1,000mlに浸漬し、
25℃にて7日間静置した。その後植物粉末をろ別除去
し、ろ液をMCI GEL(三菱化学株式会社製)カラ
ムクロマトグラフィーにかけ、エタノール−水混合溶媒
で分画し、50容量%エタノールにより溶出される画分
を分取した。次いでこの画分をシリカゲル(和光純薬工
業株式会社製)カラムクトマトグラフィーにかけ、クロ
ロホルム−メタノール−水混合溶媒(容量比=7:3:
0.5)にて溶出し、この混合溶媒で溶出されないカラ
ム吸着物質について、50容量%エタノールにより溶出
させて得られた画分をコラゲナーゼ阻害剤とした。Example 1 Eucalyptus g
lobulus Labillardiere) 500 g of dry powder of leaves
Immerse in 1,000 ml of 50% by volume ethanol aqueous solution,
It was allowed to stand at 25 ° C. for 7 days. Thereafter, the plant powder was removed by filtration, and the filtrate was subjected to MCI GEL (manufactured by Mitsubishi Chemical Corporation) column chromatography, fractionated with a mixed solvent of ethanol and water, and fractions eluted with 50% by volume of ethanol were fractionated. . Next, this fraction was subjected to column chromatography on silica gel (manufactured by Wako Pure Chemical Industries, Ltd.), and a chloroform-methanol-water mixed solvent (volume ratio = 7: 3:
A fraction obtained by eluting with 50% by volume of the column adsorbed material eluted in 0.5) and not eluted with this mixed solvent was used as a collagenase inhibitor.
【0018】実施例1のコラゲナーゼ阻害剤を、3容量
%塩酸−メタノール中で環流して加水分解したところ、
水溶性の糖を含む固形物と、メタノール可溶性の固形物
が得られ、実施例1の主成分が配糖体であることが示さ
れた。またシリカゲル薄層クロマトグラフィーにて定性
分析を行ったところ、実施例1の主成分はポリフェノー
ル化合物であったが、フラボノイド骨格を有するもので
はないことが確認された。なお、前記加水分解物のう
ち、メタノール可溶性画分においても高いコラゲナーゼ
阻害活性を認め、実施例1の主成分のアグリコンもコラ
ゲナーゼ阻害作用を有することが示された。When the collagenase inhibitor of Example 1 was hydrolyzed by refluxing in 3% by volume hydrochloric acid-methanol,
A solid containing a water-soluble saccharide and a solid soluble in methanol were obtained, indicating that the main component of Example 1 was a glycoside. In addition, qualitative analysis was performed by silica gel thin layer chromatography, and it was confirmed that the main component of Example 1 was a polyphenol compound, but did not have a flavonoid skeleton. Among the hydrolysates, a high collagenase inhibitory activity was also observed in the methanol-soluble fraction, indicating that the aglycone as the main component of Example 1 also has a collagenase inhibitory activity.
【0019】上記の実施例1について、出発材料とした
50容量%エタノール抽出物、及び実施例1のメタノー
ル可溶性加水分解物とともに、コラゲナーゼに対する阻
害作用を比較評価した。コラゲナーゼとしては、精製I
型コラゲナーゼ(0.25ユニット/ml)及びヒト線
維芽細胞由来コラゲナーゼを用いた。コラゲナーゼ活性
は、コラゲナーゼを0.25mg/mlのフルオレセイ
ンイソチオシアネート(FITC)で標識したI型コラ
ーゲンと、トリス(ヒドロキシメチル)アミノメタン−塩
酸緩衝液(pH7.5)中35℃で2時間インキュベー
トし、次いで反応液に40mMのο-フェナントロリン
5μlを添加し、35℃で30分間処理した後、エタノ
ール沈殿法により分解されたコラーゲン含有上清を得、
この上清中に含有されるコラーゲンの蛍光強度を蛍光分
光光度計により、励起波長495nm,蛍光波長520
nmで測定して求めた。本発明に係る実施例1等のコラ
ゲナーゼ阻害作用は、各試料25μg/mlの存在下
に、上記各コラゲナーゼを前記標識コラーゲンと作用さ
せて酵素活性を測定し、(1)式によりコラゲナーゼ阻
害率を算出して表した。結果は表1に示した。For Example 1 described above, a 50% ethanol extract as a starting material and the methanol-soluble hydrolyzate of Example 1 were used to compare and evaluate the inhibitory effect on collagenase. Purified I as collagenase
Type collagenase (0.25 units / ml) and human fibroblast-derived collagenase were used. Collagenase activity was determined by incubating collagenase with type I collagen labeled with 0.25 mg / ml fluorescein isothiocyanate (FITC) in a tris (hydroxymethyl) aminomethane-hydrochloric acid buffer (pH 7.5) at 35 ° C. for 2 hours. Then, 5 μl of 40 mM o-phenanthroline was added to the reaction solution, and the mixture was treated at 35 ° C. for 30 minutes to obtain a collagen-containing supernatant decomposed by an ethanol precipitation method.
The fluorescence intensity of the collagen contained in the supernatant was measured with a fluorescence spectrophotometer at an excitation wavelength of 495 nm and a fluorescence wavelength of 520.
It was determined by measuring in nm. The collagenase inhibitory activity of Example 1 and the like according to the present invention is determined by measuring the enzyme activity by allowing each of the above collagenases to act on the above-mentioned collagen in the presence of 25 μg / ml of each sample. Calculated and expressed. The results are shown in Table 1.
【数1】 (Equation 1)
【0020】なお、ヒト線維芽細胞由来コラゲナーゼと
しては、ヒト線維芽細胞を、1ウェル当たり5×104
個となるように48穴マイクロプレートに播種し、24
時間後にインターロイキン-1α(IL-1α)及び表皮成
長因子(EGF)各10ng/mlを添加したダルベッ
コ修正基礎栄養培地(DMEM)に交換し、さらに37
℃で48時間培養した後、培地を採取し、採取した培地
にトリプシンを0.04mg/mlとなるように添加し
て、37℃で30分間反応させ、上清中のコラゲナーゼ
を活性化したものを用いた。As the human fibroblast-derived collagenase, human fibroblasts were used at 5 × 10 4 cells / well.
Seeded into a 48-well microplate so that
After time, the medium was replaced with Dulbecco's modified basal nutrient medium (DMEM) supplemented with 10 ng / ml of each of interleukin-1α (IL-1α) and epidermal growth factor (EGF).
After culturing at 48 ° C. for 48 hours, the medium was collected, trypsin was added to the collected medium to a concentration of 0.04 mg / ml, and the mixture was reacted at 37 ° C. for 30 minutes to activate collagenase in the supernatant. Was used.
【0021】[0021]
【表1】 表1より明らかなように、本発明の実施例1は、精製I
型コラゲナーゼ及びヒト線維芽細胞由来コラゲナーゼに
対し、ともに99%と、ほぼ完全に活性を阻害してい
た。一方、出発材料である50容量%エタノール抽出物
では、各コラゲナーゼに対し90%の阻害作用が見られ
た。また、実施例1のメタノール可溶性加水分解物にお
いても、各コラゲナーゼに対し99%という高い阻害作
用が認められていた。[Table 1] As is evident from Table 1, Example 1 of the present invention
Both types of collagenase and human fibroblast-derived collagenase almost completely inhibited the activity, at 99%. On the other hand, in the 50% by volume ethanol extract as a starting material, a 90% inhibitory effect on each collagenase was observed. In addition, the methanol-soluble hydrolyzate of Example 1 also showed a high inhibitory effect of 99% on each collagenase.
【0022】続いて、本発明の実施例1について熱及び
光に対する安定性を評価した。実施例を100℃で10
分間熱処理した場合、及び3カ月間露光保存した場合の
それぞれについて、25μg/ml添加時のコラゲナー
ゼ阻害率を測定し、未処理の場合のコラゲナーゼ阻害率
と比較して表2に示した。コラゲナーゼとしては、I型
コラゲナーゼ標品を用いた。Subsequently, the stability to heat and light of Example 1 of the present invention was evaluated. Example 10 at 100 ° C.
The collagenase inhibitory rate when 25 μg / ml was added was measured for each of the cases of heat treatment for 3 minutes and exposure and storage for 3 months, and the results are shown in Table 2 in comparison with the collagenase inhibitory rates of the untreated cases. As collagenase, a type I collagenase preparation was used.
【0023】[0023]
【表2】 表2より明らかなように、本発明の実施例1は熱及び光
に対し非常に良好な安定性を示し、100℃で10分間
の熱処理を行った場合には98%、3カ月間の露光保存
を行った場合には95%のコラゲナーゼ阻害活性を保持
していた。[Table 2] As is clear from Table 2, Example 1 of the present invention shows very good stability to heat and light, and 98% when heat-treated at 100 ° C. for 10 minutes, and exposure for 3 months. When stored, it retained 95% collagenase inhibitory activity.
【0024】また、本発明の実施例1について、培養ヒ
ト線維芽細胞に対する細胞毒性を評価した。ヒト由来線
維芽細胞を、1ウェル当たり2.0×104個となるよ
うに96穴マイクロプレートに播種し、24時間後に、
実施例1を1.0mg/ml〜100.0mg/ml含
有する1.0容量%牛胎仔血清添加DMEMにて37℃
で24時間さらに培養して、生細胞数を計測して細胞生
存率を求め、50%致死濃度(LD50)を算出した。そ
の結果、実施例1のLD50値は100.0mg/mlよ
り大きい値となっており、試験した濃度では細胞毒性は
認められなかった。Further, Example 1 of the present invention was evaluated for cytotoxicity to cultured human fibroblasts. Human-derived fibroblasts were seeded in a 96-well microplate at 2.0 × 10 4 cells / well, and 24 hours later,
Example 1 at 1.0 mg / ml to 100.0 mg / ml in DMEM supplemented with 1.0% by volume fetal calf serum at 37 ° C.
The cells were further cultured for 24 hours, and the number of viable cells was counted to determine the cell viability, and the 50% lethal concentration (LD 50 ) was calculated. As a result, the LD 50 value of Example 1 was greater than 100.0 mg / ml, and no cytotoxicity was observed at the concentrations tested.
【0025】続いて、本発明に係る皮膚外用剤について
の実施例の処方を示す。Next, the formulations of the examples of the external preparation for skin according to the present invention are shown.
【0026】 [実施例2] 皮膚用ローション剤 (1)エタノール 10.0(重量%) (2)ヒドロキシエチルセルロース 1.0 (3)コラゲナーゼ阻害剤(実施例1) 0.1 (4)パラオキシ安息香酸メチル 0.1 (5)精製水 88.8 製法:(1)〜(4)を順次(5)に添加し、均一に溶解する。[Example 2] Lotion for skin (1) Ethanol 10.0 (wt%) (2) Hydroxyethylcellulose 1.0 (3) Collagenase inhibitor (Example 1) 0.1 (4) Paraoxybenzoate Methyl acid 0.1 (5) Purified water 88.8 Production method: (1) to (4) are sequentially added to (5) and uniformly dissolved.
【0027】 [実施例3] 皮膚用乳剤 (1)ステアリン酸 0.2(重量%) (2)セタノール 1.5 (3)ワセリン 3.0 (4)流動パラフィン 7.0 (5)ポリオキシエチレン(10E.O.)モノオレイン酸 1.5 エステル (6)酢酸トコフェロール 1.0 (7)グリセリン 5.0 (8)パラオキシ安息香酸メチル 0.1 (9)トリエタノールアミン 1.0 (10)精製水 79.5 (11)コラゲナーゼ阻害剤(実施例1) 0.2 製法:(1)〜(6)の油相成分を混合,加熱して均一に溶解
し、70℃に保つ。一方、(7)〜(10)の水相成分を混
合,加熱して均一とし、70℃とする。この水相成分に
前記油相成分を攪拌しながら徐々に添加して乳化し、冷
却した後40℃にて(11)を添加,混合する。Example 3 Skin Emulsion (1) Stearic acid 0.2 (% by weight) (2) Cetanol 1.5 (3) Vaseline 3.0 (4) Liquid paraffin 7.0 (5) Polyoxy Ethylene (10E.O.) monooleic acid 1.5 ester (6) Tocopherol acetate 1.0 (7) Glycerin 5.0 (8) Methyl parahydroxybenzoate 0.1 (9) Triethanolamine 1.0 (10 ) Purified water 79.5 (11) Collagenase inhibitor (Example 1) 0.2 Production method: The oil phase components (1) to (6) are mixed, heated and uniformly dissolved, and kept at 70 ° C. On the other hand, the aqueous phase components (7) to (10) are mixed and heated to be uniform, and the temperature is set to 70 ° C. The oil phase component is gradually added to the aqueous phase component while stirring to emulsify, and after cooling, (11) is added and mixed at 40 ° C.
【0028】 [実施例4] 皮膚用ゲル剤 (1)ジプロピレングリコール 10.0(重量%) (2)カルボキシビニルポリマー 0.5 (3)水酸化カリウム 0.1 (4)パラオキシ安息香酸メチル 0.1 (5)精製水 88.8 (6)コラゲナーゼ阻害剤(実施例1) 0.5 製法:(5)に(2)を均一に溶解した後、(1)に(4)を溶解し
て添加し、次いで(3)を加えて増粘させ、(6)を添加,混
合する。Example 4 Skin Gel (1) Dipropylene glycol 10.0 (% by weight) (2) Carboxyvinyl polymer 0.5 (3) Potassium hydroxide 0.1 (4) Methyl paraoxybenzoate 0.1 (5) Purified water 88.8 (6) Collagenase inhibitor (Example 1) 0.5 Production method: After uniformly dissolving (2) in (5), dissolving (4) in (1) Then, (3) is added to increase the viscosity, and (6) is added and mixed.
【0029】 [実施例5] 皮膚用クリーム (1)ミツロウ 6.0(重量%) (2)セタノール 5.0 (3)還元ラノリン 8.0 (4)スクワラン 27.5 (5)グリセリル脂肪酸エステル 4.0 (6)親油型グリセリルモノステアリン酸エステル 2.0 (7)ポリオキシエチレン(20E.O.)ソルビタン 5.0 モノラウリン酸エステル (8)プロピレングリコール 5.0 (9)パラオキシ安息香酸メチル 0.1 (10)精製水 36.4 (11)コラゲナーゼ阻害剤(実施例1) 1.0 製法:(1)〜(7)の油相成分を混合,溶解して75℃に加
熱する。一方、(8)〜(10)の水相成分を混合,溶解して
75℃に加熱する。次いで、上記水相成分に油相成分を
添加して予備乳化した後、ホモミキサーにて均一に乳化
し、冷却後40℃にて(11)を添加,混合する。Example 5 Skin Cream (1) Beeswax 6.0 (% by weight) (2) Cetanol 5.0 (3) Reduced Lanolin 8.0 (4) Squalane 27.5 (5) Glyceryl fatty acid ester 4.0 (6) Lipophilic glyceryl monostearate 2.0 (7) Polyoxyethylene (20E.O.) sorbitan 5.0 Monolaurate (8) Propylene glycol 5.0 (9) Paraoxybenzoic acid Methyl 0.1 (10) Purified water 36.4 (11) Collagenase inhibitor (Example 1) 1.0 Production method: Mix and dissolve oil phase components (1) to (7) and heat to 75 ° C . On the other hand, the aqueous phase components (8) to (10) are mixed and dissolved, and heated to 75 ° C. Next, the oil phase component is added to the water phase component and pre-emulsified, and then uniformly emulsified by a homomixer. After cooling, (11) is added and mixed at 40 ° C.
【0030】 [実施例6] 水中油型乳剤性軟膏 (1)白色ワセリン 25.0(重量%) (2)ステアリルアルコール 25.0 (3)グリセリン 12.0 (4)ラウリル硫酸ナトリウム 1.0 (5)パラオキシ安息香酸メチル 0.1 (6)精製水 36.3 (7)コラゲナーゼ阻害剤(実施例1) 0.6 製法:(1)〜(4)の油相成分を混合,溶解して均一とし、
75℃に加熱する。一方、(5)を(6)に溶解して75℃に
加熱し、これに前記油相成分を添加して乳化し、冷却後
40℃にて(7)を添加,混合する。Example 6 Oil-in-water emulsion ointment (1) White petrolatum 25.0 (% by weight) (2) Stearyl alcohol 25.0 (3) Glycerin 12.0 (4) Sodium lauryl sulfate 1.0 (5) Methyl paraoxybenzoate 0.1 (6) Purified water 36.3 (7) Collagenase inhibitor (Example 1) 0.6 Production method: Mix and dissolve oil phase components (1) to (4) And uniform
Heat to 75 ° C. On the other hand, (5) is dissolved in (6) and heated to 75 ° C., the oil phase component is added thereto to emulsify, and after cooling, (7) is added and mixed at 40 ° C.
【0031】 [実施例7] 化粧水 (1)エタノール 10.00(重量%) (2)1,3-ブチレングリコール 5.00 (3)コラゲナーゼ阻害剤(実施例1) 0.01 (4)香料 0.10 (5)精製水 84.89 製法:(1)〜(4)を順次(5)に添加して均一に混合,溶解
する。Example 7 Toner (1) Ethanol 10.00 (% by weight) (2) 1,3-butylene glycol 5.00 (3) Collagenase inhibitor (Example 1) 0.01 (4) Fragrance 0.10 (5) Purified water 84.89 Production method: (1) to (4) are sequentially added to (5), and uniformly mixed and dissolved.
【0032】 [実施例8] 美容液 (1)グリセリン 2.00(重量%) (2)1,3-ブチレングリコール 3.00 (3)ポリオキシエチレン(25E.O.)オレイルエーテル 0.50 (4)コラゲナーゼ阻害剤(実施例1) 0.05 (5)エタノール 15.00 (6)パラオキシ安息香酸メチル 0.10 (7)香料 0.10 (8)精製水 79.25 製法:(6),(7)を(5)に溶解し、これを(1)〜(4)ととも
に(8)に添加して均一に混合,溶解する。Example 8 Serum (1) Glycerin 2.00 (% by weight) (2) 1,3-butylene glycol 3.00 (3) Polyoxyethylene (25E.O.) oleyl ether 0.50 (4) Collagenase inhibitor (Example 1) 0.05 (5) Ethanol 15.00 (6) Methyl paraoxybenzoate 0.10 (7) Fragrance 0.10 (8) Purified water 79.25 Production method: (6) ) And (7) are dissolved in (5), and this is added to (8) together with (1) to (4) and uniformly mixed and dissolved.
【0033】 [実施例9] エモリエントクリーム(油中水型) (1)流動パラフィン 30.00(重量%) (2)マイクロクリスタリンワックス 2.00 (3)ワセリン 5.00 (4)ジグリセリルジオレイン酸エステル 5.00 (5)L-グルタミン酸ナトリウム 1.60 (6)L-セリン 0.40 (7)プロピレングリコール 3.00 (8)パラオキシ安息香酸メチル 0.10 (9)精製水 52.75 (10)香料 0.10 (11)コラゲナーゼ阻害剤(実施例1) 0.05 製法:(5),(6)を(9)の一部に溶解して50℃とし、5
0℃に加熱した(4)に攪拌しながら徐々に添加する。こ
れをあらかじめ混合し70℃に加熱溶解した(1)〜(3)に
均一に分散し、これに(7),(8)を(9)の残部に溶解して
70℃に加熱したものを攪拌しながら添加し、ホモミキ
サーにて乳化する。冷却後、40℃にて(10),(11)を添
加,混合する。[Example 9] Emollient cream (water-in-oil type) (1) Liquid paraffin 30.00 (wt%) (2) Microcrystalline wax 2.00 (3) Vaseline 5.00 (4) Diglyceryl geo Leic acid ester 5.00 (5) Sodium L-glutamate 1.60 (6) L-serine 0.40 (7) Propylene glycol 3.00 (8) Methyl parahydroxybenzoate 0.10 (9) Purified water 52. 75 (10) Fragrance 0.10 (11) Collagenase inhibitor (Example 1) 0.05 Production method: (5) and (6) are dissolved in a part of (9) to 50 ° C.
Add slowly to (4) heated to 0 ° C. with stirring. This was previously mixed and uniformly dispersed in (1) to (3), which were heated and dissolved at 70 ° C., and (7) and (8) were dissolved in the remainder of (9) and heated to 70 ° C. Add while stirring and emulsify with a homomixer. After cooling, (10) and (11) are added and mixed at 40 ° C.
【0034】 [実施例10] メイクアップベースクリーム (1)ステアリン酸 12.00(重量%) (2)セタノール 2.00 (3)グリセリルトリ2-エチルヘキサン酸エステル 2.50 (4)自己乳化型グリセリルモノステアリン酸エステル 2.00 (5)プロピレングリコール 10.00 (6)水酸化カリウム 0.30 (7)精製水 69.58 (8)酸化チタン 1.00 (9)ベンガラ 0.10 (10)黄酸化鉄 0.40 (11)香料 0.10 (12)コラゲナーゼ阻害剤(実施例1) 0.02 製法:(1)〜(4)の油相成分を混合し、75℃に加熱して
均一とする。一方(5)〜(7)の水相成分を混合し、75℃
に加熱,溶解して均一とし、これに(8)〜(10)の顔料を
添加し、ホモミキサーにて均一に分散させる。この水相
成分に前記油相成分を添加し、ホモミキサーにて乳化し
た後冷却し、40℃にて(11),(12)を添加,混合する。Example 10 Makeup Base Cream (1) Stearic acid 12.00 (% by weight) (2) Cetanol 2.00 (3) Glyceryl tri-2-ethylhexanoate 2.50 (4) Self-emulsification Type glyceryl monostearate 2.00 (5) Propylene glycol 10.00 (6) Potassium hydroxide 0.30 (7) Purified water 69.58 (8) Titanium oxide 1.00 (9) Bengala 0.10 ( 10) Yellow iron oxide 0.40 (11) Fragrance 0.10 (12) Collagenase inhibitor (Example 1) 0.02 Production method: Mix oil phase components (1) to (4) and heat to 75 ° C And make it uniform. On the other hand, mix the aqueous phase components (5) to (7)
The mixture is heated and dissolved to make the mixture uniform, and the pigments (8) to (10) are added to the mixture and uniformly dispersed by a homomixer. The oil phase component is added to the water phase component, emulsified by a homomixer, cooled, and (11) and (12) are added and mixed at 40 ° C.
【0035】 [実施例11] 乳液状ファンデーション (1)ステアリン酸 2.00(重量%) (2)スクワラン 5.00 (3)ミリスチン酸オクチルドデシル 5.00 (4)セタノール 1.00 (5)デカグリセリルモノイソパルミチン酸エステル 9.00 (6)1,3-ブチレングリコール 6.00 (7)水酸化カリウム 0.10 (8)パラオキシ安息香酸メチル 0.10 (9)精製水 53.40 (10)酸化チタン 9.00 (11)タルク 7.40 (12)ベンガラ 0.50 (13)黄酸化鉄 1.10 (14)黒酸化鉄 0.10 (15)香料 0.15 (16)コラゲナーゼ阻害剤(実施例1) 0.15 製法:(1)〜(5)の油相成分を混合し、75℃に加熱して
均一とする。一方(6)〜(9)の水相成分を混合し、75℃
に加熱,溶解して均一とし、これに(10)〜(14)の顔料を
添加し、ホモミキサーにて均一に分散させる。この水相
成分に前記油相成分を添加し、ホモミキサーにて均一に
乳化した後冷却し、40℃にて(15),(16)を添加,混合
する。Example 11 Emulsion Foundation (1) Stearic acid 2.00 (% by weight) (2) Squalane 5.00 (3) Octyldodecyl myristate 5.00 (4) Cetanol 1.00 (5) Decaglyceryl monoisopalmitate 9.00 (6) 1,3-butylene glycol 6.00 (7) Potassium hydroxide 0.10 (8) Methyl parahydroxybenzoate 0.10 (9) Purified water 53.40 ( 10) Titanium oxide 9.00 (11) Talc 7.40 (12) Bengala 0.50 (13) Yellow iron oxide 1.10 (14) Black iron oxide 0.10 (15) Fragrance 0.15 (16) Collagenase Inhibitor (Example 1) 0.15 Production method: The oil phase components (1) to (5) are mixed and heated to 75 ° C. to make uniform. On the other hand, the aqueous phase components of (6) to (9) were mixed and
The mixture is heated and dissolved to make the mixture uniform, and the pigments (10) to (14) are added to the mixture and uniformly dispersed by a homomixer. The oil phase component is added to the water phase component, and the mixture is uniformly emulsified by a homomixer, then cooled, and (15) and (16) are added and mixed at 40 ° C.
【0036】 [実施例12] ハンドクリーム (1)セタノール 4.0(重量%) (2)ワセリン 2.0 (3)流動パラフィン 10.0 (4)グリセリルモノステアリン酸エステル 1.5 (5)ポリオキシエチレン(60E.O.)グリセリル 2.5 イソステアリン酸エステル (6)酢酸トコフェロール 0.5 (7)グリセリン 20.0 (8)パラオキシ安息香酸メチル 0.1 (9)精製水 59.2 (10)コラゲナーゼ阻害剤(実施例1) 0.2 製法:(1)〜(6)の油相成分を混合,溶解して75℃に加
熱する。一方、(7)〜(9)の水相成分を混合,溶解して7
5℃に加熱する。次いで、上記水相成分に油相成分を添
加して予備乳化した後、ホモミキサーにて均一に乳化し
て冷却し、40℃にて(10)を添加,混合する。Example 12 Hand Cream (1) Cetanol 4.0 (% by weight) (2) Vaseline 2.0 (3) Liquid paraffin 10.0 (4) Glyceryl monostearate 1.5 (5) Polyoxyethylene (60E.O.) glyceryl 2.5 isostearate (6) Tocopherol acetate 0.5 (7) Glycerin 20.0 (8) Methyl paraoxybenzoate 0.1 (9) Purified water 59.2 ( 10) Collagenase inhibitor (Example 1) 0.2 Production method: Mix and dissolve the oil phase components (1) to (6) and heat to 75 ° C. On the other hand, the aqueous phase components (7) to (9)
Heat to 5 ° C. Next, the oil phase component is added to the water phase component and pre-emulsified, then uniformly emulsified and cooled with a homomixer, and (10) is added and mixed at 40 ° C.
【0037】上記本発明の実施例のうち実施例2〜実施
例6について、紫外線によるしわの発生に対する防止効
果を評価した。なお、各実施例において実施例1のコラ
ゲナーゼ阻害剤を50容量%エタノールに代替したもの
を比較例2〜比較例6とした。しわ発生防止効果は、ヘ
アレスマウス5匹を1群とし、各群について実施例及び
比較例をそれぞれ1日1回背部に塗布し、1J/cm2
/週の長波長紫外線(UVA)を50週間照射し、ヘア
レスマウスにおけるしわの発生状況を観察し、表3に示
す判定基準に従って点数化して行った。この際、精製水
のみを塗布した群を対照とした。結果は各群の平均値を
算出し、UVA照射日数との関係により表4に示した。With respect to Examples 2 to 6 among the above Examples of the present invention, the effect of preventing wrinkles caused by ultraviolet rays was evaluated. In each of the Examples, Comparative Example 2 to Comparative Example 6 were obtained by replacing the collagenase inhibitor of Example 1 with 50% by volume of ethanol. The effect of preventing wrinkles was determined by applying 5 hairless mice to one group, applying Examples and Comparative Examples to the back once a day for each group, and applying 1 J / cm 2
/ Week long-wave ultraviolet light (UVA) was applied for 50 weeks, the occurrence of wrinkles in hairless mice was observed, and scored according to the criteria shown in Table 3. At this time, a group to which only purified water was applied was used as a control. The results were calculated as the average value of each group, and are shown in Table 4 in relation to the number of UVA irradiation days.
【表3】 [Table 3]
【0038】[0038]
【表4】 表4に示されるように、対照群においては、UVA照射
日数が40週を超える頃には形成されたしわの深さは中
程度にまで達し、50週後には深いしわの発生が認めら
れていた。これに対し、本発明の実施例塗布群では、い
ずれにおいても50週後に微小〜軽微なしわが認められ
た程度で、しわの発生は顕著に抑制されていた。一方比
較例塗布群では、酢酸トコフェロールを含有する比較例
3塗布群で、発生したしわの程度の軽減が若干認められ
た他は、有意なしわの発生防止或いは軽減は認められな
かった。[Table 4] As shown in Table 4, in the control group, the depth of wrinkles formed reached a moderate level when the number of UVA irradiation days exceeded 40 weeks, and the occurrence of deep wrinkles was observed after 50 weeks. Was. On the other hand, in each of the groups to which the examples of the present invention were applied, the occurrence of wrinkles was remarkably suppressed to the extent that slight to slight wrinkles were observed after 50 weeks in each case. On the other hand, in the group to which the comparative example was applied, in the group to which the comparative example 3 containing tocopherol acetate was applied, the occurrence or occurrence of wrinkles was not significantly prevented or reduced except that the degree of wrinkles was slightly reduced.
【0039】続いて、本発明の実施例2〜実施例6につ
いて、抗炎症作用及び創傷治癒促進効果を評価した。背
部皮膚に人工的に炎症又は創傷を形成した1群5匹のマ
ウスを用い、各群において、実施例及び比較例をそれぞ
れ0.5gずつ1日2回、炎症部位又は創傷部位に7日
間塗布し、7日目に各部位の状態を観察した。抗炎症作
用については「有効」,「やや有効」,「無効」、創傷
治癒促進効果については「完全治癒」,「ほぼ治癒」,
「治癒不完全」の3段階でそれぞれ評価し、各評価を得
たマウスの数にて表5に示した。Subsequently, with respect to Examples 2 to 6 of the present invention, the anti-inflammatory effect and the effect of promoting wound healing were evaluated. Using a group of 5 mice with artificial inflammation or wound on the back skin, in each group, apply 0.5 g of each of Examples and Comparative Examples twice a day to an inflamed or wounded site for 7 days. Then, on day 7, the condition of each part was observed. "Effective", "Slightly effective", "Ineffective" for anti-inflammatory effect, "Complete healing", "Almost healing" for wound healing promoting effect,
The results were evaluated in three stages of “incomplete healing”, and the results are shown in Table 5 in terms of the number of mice that obtained each evaluation.
【0040】[0040]
【表5】 表5より明らかなように、抗炎症作用については、本発
明の実施例塗布群ではいずれにおいても無効と評価され
たマウスは見られず、2例以上のマウスにおいて有効な
抗炎症作用が認められていた。また創傷治癒促進効果に
ついても、本発明の実施例塗布群では創傷治癒の不完全
なマウスはいずれにおいれも認められておらず、2例以
上のマウスで完全な治癒を認めていた。これに対し比較
例塗布群では、比較例3及び比較例5塗布群でやや有効
な抗炎症作用の認められたマウスが各1例見られた他
は、何らかの抗炎症作用の認められたマウスは見られな
かった。また、比較例3塗布群で創傷がほぼ治癒したマ
ウスが1例見られた他は、いずれの塗布群においてもマ
ウスの創傷治癒は不完全であった。[Table 5] As apparent from Table 5, no anti-inflammatory effect was observed in any of the mice to which the present invention was applied in the group to which the present invention was applied, and an effective anti-inflammatory effect was observed in two or more mice. I was Regarding the effect of promoting wound healing, none of the mice to which the wound healing was incomplete were observed in the group to which the Examples of the present invention were applied, and complete healing was recognized in two or more mice. On the other hand, in the group to which the comparative example was applied, one mouse each having a slightly effective anti-inflammatory effect was observed in each of the groups to which the comparative example 3 and the comparative example 5 were applied. I couldn't see it. In addition, the wound healing of the mice was incomplete in any of the application groups, except for one mouse in which the wound was almost healed in the group to which Comparative Example 3 was applied.
【0041】次に、本発明の実施例2〜実施例12につ
いて、6カ月間の実使用試験を行った。パネラーとして
は、しわや皮膚弾性の低下といった皮膚の老化症状を顕
著に呈する40才〜60才代の女性を用い、1群20名
とした。この使用試験においても、上記と同様に、各実
施例において配合した実施例1のコラゲナーゼ阻害剤
を、50容量%エタノールに代替したものを比較例2〜
比較例12とした。使用試験は、皮膚老化症状を呈する
パネラーの各群に、実施例及び比較例のそれぞれをブラ
インドにて使用させて行った。使用試験開始前及び使用
試験終了後に皮膚の状態を観察し、しわ及び皮膚弾性の
各改善状況について「改善」,「やや改善」,「変化な
し」の3段階にて評価した。なお、しわの程度について
は写真撮影及びレプリカ採取により、皮膚弾性について
はキュートメーターにより測定して評価した。結果は、
各評価を得たパネラー数にて表6に示した。Next, a practical use test for 6 months was performed on Examples 2 to 12 of the present invention. As the panelists, women in their 40s to 60s who exhibited remarkable skin aging symptoms such as wrinkles and decreased skin elasticity were used, and one group consisted of 20 women. In this use test, the collagenase inhibitor of Example 1 blended in each Example was replaced with 50% by volume of ethanol in Comparative Examples 2 to 3 in the same manner as described above.
Comparative Example 12 was made. The use test was conducted by blindly using each of the examples and comparative examples for each group of panelists exhibiting skin aging symptoms. Before the start of the use test and after the end of the use test, the condition of the skin was observed, and the wrinkle and skin elasticity were evaluated in three stages of "improvement", "slight improvement", and "no change". The degree of wrinkles was evaluated by photographing and replica sampling, and the skin elasticity was measured and evaluated by a cute meter. Result is,
Table 6 shows the number of panelists who obtained each evaluation.
【0042】[0042]
【表6】 表6に示されるように、しわの改善状況については、本
発明の実施例使用群ではすべてにおいて改善傾向が認め
られていた。特に、実施例2〜実施例6,及び実施例1
2使用群では、50%以上のパネラーにおいて明確な改
善を認めていた。皮膚弾性の改善状況については、実施
例使用群ではすべて改善傾向が認められており、35%
以上のパネラーで明確な改善を認めていた。これに対
し、比較例使用群ではしわ及び皮膚弾性ともに明確な改
善を認めたパネラーは見られず、しわについては70%
以上、皮膚弾性については65%以上のパネラーで症状
の改善を認めなかった。[Table 6] As shown in Table 6, the improvement of the wrinkles was observed in all the groups using the examples of the present invention. In particular, Examples 2 to 6, and Example 1
In the two-use group, a clear improvement was recognized in 50% or more of the panelists. Regarding the state of improvement of skin elasticity, the improvement tendency was observed in all the groups using the examples, and 35%
The above panelists have clearly seen improvement. On the other hand, in the group using the comparative example, no panelists who recognized a clear improvement in both wrinkles and skin elasticity were observed, and the wrinkles were 70%.
As described above, regarding skin elasticity, 65% or more of panelists did not improve symptoms.
【0043】なお、本発明の実施例2〜実施例12につ
いては、上記の使用試験期間中に含有成分の析出,分
離,凝集、変色,変臭といった状態変化は全く見られな
かった。また、各実施例使用群において、皮膚刺激性反
応や皮膚感作性反応を示したパネラーも存在しなかっ
た。In Examples 2 to 12 of the present invention, no change in state such as precipitation, separation, agglomeration, discoloration, or odor of the contained components was observed at all during the above use test period. In addition, in each group used in Examples, there was no paneler showing a skin irritating reaction or a skin sensitizing reaction.
【0044】[0044]
【発明の効果】以上詳述したように、本発明により、線
維芽細胞等の間葉系細胞や好酸球等により産生される間
質系のコラゲナーゼに対し、特異的且つ高い阻害活性を
示すコラゲナーゼ阻害剤を得ることができた。本発明に
係るコラゲナーゼ阻害剤は、炎症や創傷、がんの転移、
潰瘍形成、皮膚の老化等、コラゲナーゼの関与する病態
の改善、或いは防止に有用である。As described in detail above, the present invention exhibits a specific and high inhibitory activity against stromal collagenase produced by mesenchymal cells such as fibroblasts and eosinophils. A collagenase inhibitor could be obtained. Collagenase inhibitors according to the present invention, inflammation and wounds, cancer metastasis,
It is useful for improving or preventing pathologies involving collagenase such as ulceration and skin aging.
【0045】また本発明により、紫外線等の種々の外来
刺激や、加齢に伴う真皮線維芽細胞におけるコラーゲン
の分解の進行を抑制して、マトリックス線維の破壊を防
止することにより、しわや皮膚弾性の低下といった皮膚
の老化症状を有効に防止或いは改善し、抗炎症作用及び
創傷治癒促進作用にも優れる皮膚外用剤を得ることがで
きた。According to the present invention, wrinkles and skin elasticity are prevented by suppressing the progress of collagen degradation in dermal fibroblasts due to various external stimuli such as ultraviolet rays and aging, and preventing the destruction of matrix fibers. A skin external preparation which effectively prevents or ameliorates the aging symptoms of the skin, such as a decrease in skin loss, and which is also excellent in the anti-inflammatory action and the wound healing promoting action, was obtained.
Claims (2)
billardiere)又はその近縁植物(Eucalyptus polybrac
tea R.T.Baker,Eucalyptus dives Schauer)の50容
量%エタノールによる抽出物を、スチレンポリマー製の
吸着ゲルクロマトグラフィーにてエタノール−水混合溶
媒で分画して、50容量%エタノール溶出画分を分取
し、次いでシリカゲルクロマトグラフィーにかけ、クロ
ロホルム−メタノール−水混合溶媒で溶出した後に、5
0容量%エタノールにより溶出して得られる画分を含有
して成る、コラゲナーゼ阻害剤。[Claim 1] Eucalyptus globulus La
billardiere) or its closely related plant ( Eucalyptus polybrac)
tea RTBaker, Eucalyptus dives Schauer) with 50% by volume of ethanol was fractionated with an ethanol-water mixed solvent by styrene polymer adsorption gel chromatography, and a 50% by volume ethanol eluted fraction was collected. Then, the mixture was subjected to silica gel chromatography, eluted with a mixed solvent of chloroform-methanol-water, and
A collagenase inhibitor comprising a fraction obtained by elution with 0% by volume of ethanol.
含有して成る、皮膚外用剤。2. An external preparation for skin comprising the collagenase inhibitor according to claim 1.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP10172217A JPH11349436A (en) | 1998-06-03 | 1998-06-03 | Collagenase inhibitor and skin agent containing the same and used for external use |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP10172217A JPH11349436A (en) | 1998-06-03 | 1998-06-03 | Collagenase inhibitor and skin agent containing the same and used for external use |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH11349436A true JPH11349436A (en) | 1999-12-21 |
Family
ID=15937773
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP10172217A Pending JPH11349436A (en) | 1998-06-03 | 1998-06-03 | Collagenase inhibitor and skin agent containing the same and used for external use |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH11349436A (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000051562A1 (en) * | 1999-03-03 | 2000-09-08 | Shiseido Company, Ltd. | Matrix metalloprotease inhibitor and utilization thereof |
JP2002138013A (en) * | 2000-10-27 | 2002-05-14 | Pias Arise Kk | Hne-and-acrolein formation inhibiting and hne-and- acrolein decomposition promoting agent, and skin aging care preparation for external use |
JP2003002819A (en) * | 2001-06-22 | 2003-01-08 | Naris Cosmetics Co Ltd | Skin care composition |
JP2003201214A (en) * | 2001-10-23 | 2003-07-18 | Shiseido Co Ltd | Matrix metalloprotease activity inhibitor |
EP1180371A4 (en) * | 2000-03-27 | 2004-04-14 | Shiseido Co Ltd | Agents promoting the formation of skin basement membrane, agents promoting the formation of artificial skin and process for producing artificial skin |
JP2007291042A (en) * | 2006-04-27 | 2007-11-08 | Kao Corp | Vegf production promoter |
JP2010163459A (en) * | 2010-04-12 | 2010-07-29 | Geol Kagaku Kk | Mushroom-originated composition |
KR101877047B1 (en) * | 2016-04-22 | 2018-07-10 | (주)에스디생명공학 | Composition for preventing or improving skin wrinkle comprising Eucalyptus globulus extract treated by enzyme as active ingredient |
-
1998
- 1998-06-03 JP JP10172217A patent/JPH11349436A/en active Pending
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000051562A1 (en) * | 1999-03-03 | 2000-09-08 | Shiseido Company, Ltd. | Matrix metalloprotease inhibitor and utilization thereof |
EP1180371A4 (en) * | 2000-03-27 | 2004-04-14 | Shiseido Co Ltd | Agents promoting the formation of skin basement membrane, agents promoting the formation of artificial skin and process for producing artificial skin |
US7645595B2 (en) | 2000-03-27 | 2010-01-12 | Shiseido Company, Ltd. | Method of production of artificial skin |
JP2002138013A (en) * | 2000-10-27 | 2002-05-14 | Pias Arise Kk | Hne-and-acrolein formation inhibiting and hne-and- acrolein decomposition promoting agent, and skin aging care preparation for external use |
JP2003002819A (en) * | 2001-06-22 | 2003-01-08 | Naris Cosmetics Co Ltd | Skin care composition |
JP2003201214A (en) * | 2001-10-23 | 2003-07-18 | Shiseido Co Ltd | Matrix metalloprotease activity inhibitor |
JP2007291042A (en) * | 2006-04-27 | 2007-11-08 | Kao Corp | Vegf production promoter |
JP2010163459A (en) * | 2010-04-12 | 2010-07-29 | Geol Kagaku Kk | Mushroom-originated composition |
KR101877047B1 (en) * | 2016-04-22 | 2018-07-10 | (주)에스디생명공학 | Composition for preventing or improving skin wrinkle comprising Eucalyptus globulus extract treated by enzyme as active ingredient |
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