JP4280007B2 - Screening method for skin tightening substances - Google Patents
Screening method for skin tightening substances Download PDFInfo
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- JP4280007B2 JP4280007B2 JP2001275879A JP2001275879A JP4280007B2 JP 4280007 B2 JP4280007 B2 JP 4280007B2 JP 2001275879 A JP2001275879 A JP 2001275879A JP 2001275879 A JP2001275879 A JP 2001275879A JP 4280007 B2 JP4280007 B2 JP 4280007B2
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- albumin
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- tannic acid
- skin tightening
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Description
【０００１】
【発明の属する技術分野】
本発明は、皮膚引き締め物質のスクリーニング方法に関する。さらに詳しくは、動物の細胞中に含まれる可溶性タンパク質であるアルブミンに対する皮膚引き締め物質の反応性の良否をアルブミンに対するタンニン酸の反応性との比較のもとに表示する皮膚引き締め物質のスクリーニング方法に関する。
【０００２】
【従来の技術】
皮膚は、常に外界の刺激にさらされており、ハリ、弾力を喪失し易い。このような皮膚を引き締める物質の開発に対する要求は根強いものがある。しかしながら、皮膚を引き締める物質を探索するにあたって、どのようにしてスクリーニングすべきかについては、明瞭な指針はない。例えば特開平１１−２６３７１８号公報は、ヒト皮膚細胞のインテグリン発現物質を有効成分とする皮膚たるみ改善・引き締め剤を開示している。同公報は各サンプルを上腕内側に塗布し、数週間後の皮膚弾性値あるいは皮膚の引き締まり感を官能的に評価している。特開平１１−２６９０５７号公報は、プルーン果肉から採取された蛋白凝集性物質を有効成分とする皮膚収斂剤を開示し、皮膚収斂剤としての効果を蛋白凝集力試験により行っている。この試験法は、試料溶液とアルブミン水溶液とを反応させた後、濁度を測定し、蛋白凝集力（さらには皮膚収斂作用）の指標としている。特開２０００−３０２６６６号公報は、硫酸化グリコシド誘導体又はその塩を含有する皮膚引き締め剤を開示し、皮膚引き締め剤としての効果をコラーゲンゲル引き締め促進能、皮膚の弾力性変化あるいは皮膚の引き締まり感を官能的に評価している。引き締まり感を官能的に評価する方法は、漠然とした方法であり、個人により差が出るおそれがある。アルブミン水溶液の濁度を測定する方法はある濃度を越えると濁度が一定となり、測定できなくなる欠点がある。コラーゲンゲル引き締め能測定法は真皮モデルである線維芽細胞包理コラーゲンゲル等の使用が必要であり、その測定法は繁雑である。
【０００３】
【発明が解決しようとする課題】
本発明者らは、皮膚引き締め物質を探索し、より簡便にかつ客観的にスクリーニングする方法を探求し、開発することに成功した。
【０００４】
【課題を解決するための手段】
本発明は、皮膚引き締め物質及びタンニン酸をそれぞれアルブミンと反応させ、アルブミンが半量に減少するときの皮膚引き締め物質の濃度とアルブミンを半量に減少するときのタンニン酸濃度とを測定し、アルブミンを半量に減少するときの皮膚引き締め物質濃度に対するアルブミンを半量に減少するときのタンニン酸濃度との比率として表示することを特徴とする皮膚引き締め物質のスクリーニング方法を提供する。
【０００５】
【発明の実施の形態】
本発明で使用するアルブミンとしては、卵アルブミン、血清アルブミン（serum albumin）、乳アルブミン（lacta albumin）等のいずれでもよい。例えば、市販のALBUMIN,BOVINE Fraction V Minimum 96%（シグマ社製）を挙げることができる。
【０００６】
本発明で使用するタンニン酸としては、例えばタンニン酸特級（小宗化学社製）を挙げることができる。試薬によっては塩を含むものがある。このような試薬の場合には、塩の存在によりタンニン酸とアルブミンとの反応により生ずる沈殿が生成しなくなるため、脱塩処理が必要である。
【０００７】
タンニン酸は収斂作用を持つものとして一般に知られており、アルブミンは動物組織中に広く存在しており、アルブミンに対するタンニン酸の凝集力測定値として皮膚引き締め物質のアルブミン凝集力測定値を表示することは、皮膚引き締め物質の一般的評価方法として有用である。
本発明において、皮膚引き締め物質のアルブミン量を半減させるときの濃度の測定は次のようにして行う。
【０００８】
エタノール水溶液で数種の水準に希釈した皮膚引き締め物質とアルブミン水溶液とを反応させ、ついで反応により生じた沈殿を濾過除去する。除去後のアルブミン水溶液中の未反応アルブミンを測定する。このとき皮膚引き締め物質の希釈度は、アルブミンの未反応量が初期値の５０％を挟むように選択し、アルブミン量が半減するときの皮膚引き締め物質の濃度を求める。
【０００９】
ついでエタノール水溶液で数種の水準に希釈したタンニン酸とアルブミン水溶液とを反応させ、上記と同様に操作して、アルブミンの未反応量が初期値の５０％となるときのタンニン酸濃度を求める。
【００１０】
皮膚引き締め物質濃度に対するタンニン酸濃度は次のようにして表示される。
【００１１】
X = B/A × 100 (%)
Xはアルブミン量を半減させたときの皮膚引き締め物質の濃度Aに対するアルブミン量を半減させたときのタンニン酸濃度Bとの比率（％）で表示した値である。
【００１２】
【実施例】
以下の各例において、特にことわりのない限り％は重量％を意味する。なお下記実施例において、高速液体クロマトグラフィの測定条件は次の通りであった。
【００１３】
使用機器： ウォーターズ社製
カラム： スーパーローズ６（ファーマシアバイオテク社製）
溶離液： CH3CN 1 : 0.05M CH3COONH4 pH 6.5
流量： ０．４ ml/min.
検出器： 996 Photodiode array、検出波長200 nm - 600 nm (278 nm)
注入量： ２００μl。
【００１４】
実施例１
１０％エタノールを用いて０％、０．１％、０．２％及び０．３％濃度となるように希釈した柿渋エキスパウダーMF（丸善製薬社製）４gに１％ウシ血清アルブミン水溶液１gを添加し、３０℃で１時間反応させた。反応した沈殿をアクロLC(German Science)で濾過後、高速液体クロマトグラフィ(HPLC)により未反応のアルブミンを測定した。測定結果を図１に示す。さらに１０％エタノールを用いて０％、０．０１％、０．０２％及び０．０３％濃度となるように希釈したタンニン酸について、上記と同様にしてウシ血清アルブミン水溶液と反応させ、未反応アルブミンを測定した。測定結果を図２に示す。アルブミンが半量反応したときの柿渋エキスパウダーMFの濃度（IC50）は０．１１％、アルブミンが半量反応したときのタンニン酸濃度は０．０２１％であった。これらより、柿渋エキスパウダーMFは１９．１％のタンニン酸に相当する引き締め効果のあることがわかった。
【００１５】
実施例２
１０％エタノールを用いて０％、０．０２％、０．０５％及び０．１％濃度となるように希釈したグラヴィノール（ブドウ種子抽出物、キッコーマン社製）４gに１％ウシ血清アルブミン水溶液１gを添加し、３０℃で１時間反応させた。反応した沈殿をアクロLC(German Science)で濾過後、HPLCにより未反応のアルブミンを測定した。測定結果を図３に示す。さらに１０％エタノールを用いて０％、０．０１％、０．０２％及び０．０３％濃度となるように希釈したタンニン酸について、上記と同様にしてウシ血清アルブミン水溶液と反応させ、未反応アルブミンを測定した。測定結果を図４に示す。アルブミンが半量反応したときのグラヴィノールの濃度（IC50）は０．０４１％、アルブミンが半量反応したときのタンニン酸濃度は０．０２４％であった。これらより、グラヴィノールは５８．５％のタンニン酸に相当する引き締め効果を有していることがわかった。
【００１６】
実施例３
１０％エタノールを用いて０％、０．８％、０．９％及び１．０％濃度となるように希釈したアップルフェノン（りんご抽出物、ニッカウヰスキー社製）４gに１％ウシ血清アルブミン水溶液１gを添加し、３０℃で１時間反応させた。反応した沈殿をアクロLC(German Science)で濾過後、HPLCにより未反応のアルブミンを測定した。測定結果を図５に示す。さらに１０％エタノールを用いて０％、０．０１％、０．０２％及び０．０３％濃度となるように希釈したタンニン酸について、上記と同様にしてウシ血清アルブミン水溶液と反応させ、未反応アルブミンを測定した。測定結果を図６に示す。アルブミンが半量反応したときのアップルフェノンの濃度（IC50）は０．９３５％、アルブミンが半量反応したときのタンニン酸濃度は０．０２８％であった。これらより、アップルフェノンは２．９９％のタンニン酸に相当する引き締め効果を有していることがわかった。
【００１７】
【発明の効果】
本発明によれば、各種皮膚引き締め物質の引き締め効果をタンニン酸濃度で表示比較することができる。またタンニン酸濃度に換算することにより客観的に評価することができ、その測定法もまた簡便である。本発明によれば、各種皮膚引き締め物質のスクリーニングを簡便かつ客観的に実施することができる。
【図面の簡単な説明】
【図１】 実施例１の柿渋エキスパウダーMFの測定結果を示す。
【図２】 実施例１のタンニン酸の測定結果を示す。
【図３】 実施例２のグラヴィノールの測定結果を示す。
【図４】 実施例２のタンニン酸の測定結果を示す。
【図５】 実施例３のアップルフェノンの測定結果を示す。
【図６】 実施例３のタンニン酸の測定結果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a screening method for skin tightening substances. More specifically, the present invention relates to a screening method for a skin tightening substance, which displays whether the reactivity of the skin tightening substance to albumin, which is a soluble protein contained in animal cells, is compared with the reactivity of tannic acid to albumin.
[0002]
[Prior art]
The skin is constantly exposed to external stimuli, and it tends to lose its elasticity and elasticity. There is a persistent demand for the development of substances that tighten the skin. However, there is no clear guidance on how to screen for substances that tighten the skin. For example, Japanese Patent Application Laid-Open No. 11-263718 discloses a skin sagging improving / tightening agent containing an integrin-expressing substance of human skin cells as an active ingredient. This publication applies each sample to the inner side of the upper arm and sensuously evaluates the skin elasticity value or skin tightness after several weeks. Japanese Patent Application Laid-Open No. 11-269057 discloses a skin astringent containing as an active ingredient a protein aggregating substance collected from prune pulp, and the effect as a skin astringent is performed by a protein agglutination test. In this test method, a sample solution and an aqueous albumin solution are reacted, and then the turbidity is measured, which is used as an index of protein aggregating power (and skin convergence effect). Japanese Patent Application Laid-Open No. 2000-302666 discloses a skin tightening agent containing a sulfated glycoside derivative or a salt thereof, and has an effect as a skin tightening agent to enhance collagen gel tightening ability, skin elasticity change or skin tightening feeling. It is evaluated sensuously. The method of sensuously evaluating the tightness is a vague method and may vary depending on the individual. The method for measuring the turbidity of an aqueous albumin solution has a drawback that the turbidity becomes constant when a certain concentration is exceeded, and measurement cannot be performed. Collagen gel tightening ability measurement method requires the use of fibroblast-embedded collagen gel or the like which is a dermis model, and the measurement method is complicated.
[0003]
[Problems to be solved by the invention]
The present inventors have succeeded in searching for and developing a method for screening for a skin tightening substance and screening more simply and objectively.
[0004]
[Means for Solving the Problems]
The present invention reacts each of a skin tightening substance and tannic acid with albumin, measures the concentration of the skin tightening substance when albumin is reduced to half, and the tannic acid concentration when albumin is reduced to half. The present invention provides a screening method for a skin tightening substance, characterized in that albumin is displayed as a ratio of the tannic acid concentration when reducing albumin to half the amount of the skin tightening substance concentration when decreasing to 50%.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
The albumin used in the present invention may be any of egg albumin, serum albumin, lactalbumin and the like. For example, commercially available ALBUMIN, BOVINE Fraction V Minimum 96% (manufactured by Sigma) can be mentioned.
[0006]
Examples of tannic acid used in the present invention include tannic acid special grade (manufactured by Kosou Kagaku Co.). Some reagents contain salts. In the case of such a reagent, since the precipitation which arises by reaction of a tannic acid and albumin will not produce | generate by presence of a salt, a desalting process is required.
[0007]
Tannic acid is generally known to have an astringent action, albumin is widely present in animal tissues, and the albumin cohesive force measurement value of the skin tightening substance should be displayed as the cohesive force measurement value of tannic acid against albumin Is useful as a general method for evaluating skin tightening substances.
In the present invention, the concentration when the amount of albumin of the skin tightening substance is halved is measured as follows.
[0008]
A skin tightening substance diluted to several levels with an aqueous ethanol solution is reacted with an aqueous albumin solution, and then the precipitate produced by the reaction is filtered off. Unreacted albumin in the aqueous albumin solution after removal is measured. At this time, the degree of dilution of the skin tightening substance is selected so that the unreacted amount of albumin sandwiches 50% of the initial value, and the concentration of the skin tightening substance when the amount of albumin is halved is obtained.
[0009]
Subsequently, tannic acid diluted to several levels with an aqueous ethanol solution is reacted with an aqueous albumin solution and operated in the same manner as above to determine the tannic acid concentration when the unreacted amount of albumin is 50% of the initial value.
[0010]
The tannic acid concentration relative to the skin tightening substance concentration is displayed as follows.
[0011]
X = B / A x 100 (%)
X is a value expressed as a ratio (%) to the tannic acid concentration B when the amount of albumin is halved with respect to the concentration A of the skin tightening substance when the amount of albumin is halved.
[0012]
【Example】
In the following examples, “%” means “% by weight” unless otherwise specified. In the following examples, the measurement conditions for high performance liquid chromatography were as follows.
[0013]
Equipment used: Waters column: Super Rose 6 (Pharmacia Biotech)
Eluent: CH 3 CN 1: 0.05M CH 3 COONH 4 pH 6.5
Flow rate: 0.4 ml / min.
Detector: 996 Photodiode array, detection wavelength 200 nm-600 nm (278 nm)
Injection volume: 200 μl.
[0014]
Example 1
1 g of 1% bovine serum albumin aqueous solution was added to 4 g of persimmon astringent extract powder MF (manufactured by Maruzen Pharmaceutical Co., Ltd.) diluted to a concentration of 0%, 0.1%, 0.2% and 0.3% with 10% ethanol. The mixture was added and reacted at 30 ° C. for 1 hour. The reacted precipitate was filtered through Acro LC (German Science), and unreacted albumin was measured by high performance liquid chromatography (HPLC). The measurement results are shown in FIG. Further, tannic acid diluted to a concentration of 0%, 0.01%, 0.02% and 0.03% with 10% ethanol is reacted with bovine serum albumin aqueous solution in the same manner as above, and unreacted. Albumin was measured. The measurement results are shown in FIG. The concentration (IC50) of persimmon astringent extract powder MF when albumin reacted in half was 0.11%, and the tannic acid concentration when albumin reacted in half was 0.021%. From these results, it was found that persimmon astringent extract powder MF has a tightening effect equivalent to 19.1% tannic acid.
[0015]
Example 2
1% bovine serum albumin aqueous solution in 4 g of gravinol (grape seed extract, manufactured by Kikkoman) diluted with 10% ethanol to a concentration of 0%, 0.02%, 0.05% and 0.1% 1 g was added and reacted at 30 ° C. for 1 hour. The reacted precipitate was filtered with Acro LC (German Science), and unreacted albumin was measured by HPLC. The measurement results are shown in FIG. Further, tannic acid diluted to a concentration of 0%, 0.01%, 0.02% and 0.03% with 10% ethanol is reacted with bovine serum albumin aqueous solution in the same manner as above, and unreacted. Albumin was measured. The measurement results are shown in FIG. The concentration of gravinol (IC50) when the half amount of albumin reacted was 0.041%, and the tannic acid concentration when the half amount of albumin reacted was 0.024%. From these, it was found that gravinol has a tightening effect corresponding to 58.5% tannic acid.
[0016]
Example 3
1 g of 1% bovine serum albumin aqueous solution is added to 4 g of applephenone (apple extract, manufactured by Nikka Whiskey Co., Ltd.) diluted to a concentration of 0%, 0.8%, 0.9% and 1.0% with 10% ethanol. Was added and reacted at 30 ° C. for 1 hour. The reacted precipitate was filtered with Acro LC (German Science), and unreacted albumin was measured by HPLC. The measurement results are shown in FIG. Further, tannic acid diluted to a concentration of 0%, 0.01%, 0.02% and 0.03% with 10% ethanol is reacted with bovine serum albumin aqueous solution in the same manner as above, and unreacted. Albumin was measured. The measurement results are shown in FIG. The concentration of applephenone (IC50) when the half amount of albumin reacted was 0.935%, and the tannic acid concentration when the half amount of albumin reacted was 0.028%. From these, it was found that applephenone has a tightening effect equivalent to 2.99% tannic acid.
[0017]
【The invention's effect】
According to the present invention, it is possible to display and compare the tightening effect of various skin tightening substances by the tannic acid concentration. Moreover, it can evaluate objectively by converting into a tannic acid density | concentration, The measuring method is also simple. According to the present invention, screening of various skin tightening substances can be carried out simply and objectively.
[Brief description of the drawings]
FIG. 1 shows the measurement results of persimmon astringent extract powder MF of Example 1.
2 shows the measurement results of tannic acid of Example 1. FIG.
FIG. 3 shows the measurement results of gravinol in Example 2.
4 shows the measurement results of tannic acid in Example 2. FIG.
FIG. 5 shows the measurement results of apple phenon of Example 3.
6 shows the measurement results of tannic acid in Example 3. FIG.
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2001275879A JP4280007B2 (en) | 2001-09-12 | 2001-09-12 | Screening method for skin tightening substances |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2001275879A JP4280007B2 (en) | 2001-09-12 | 2001-09-12 | Screening method for skin tightening substances |
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|---|---|
| JP2003083962A JP2003083962A (en) | 2003-03-19 |
| JP4280007B2 true JP4280007B2 (en) | 2009-06-17 |
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| KR101338210B1 (en) * | 2012-02-24 | 2013-12-06 | 주식회사 아이피어리스 | The evaluation method of astringent activity using protein quantification |
| CN103760271B (en) * | 2014-01-27 | 2015-12-09 | 余显维 | The detection method of brain heart clearing capsule |
| CN106706788B (en) * | 2016-12-26 | 2019-05-17 | 合肥智汇医药科技有限公司 | A kind of quantification and qualification method of flavonoids effective constituent in extractive from leaves of persimmon |
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