JP2003083962A - Method for screening skin tightening substance - Google Patents
Method for screening skin tightening substanceInfo
- Publication number
- JP2003083962A JP2003083962A JP2001275879A JP2001275879A JP2003083962A JP 2003083962 A JP2003083962 A JP 2003083962A JP 2001275879 A JP2001275879 A JP 2001275879A JP 2001275879 A JP2001275879 A JP 2001275879A JP 2003083962 A JP2003083962 A JP 2003083962A
- Authority
- JP
- Japan
- Prior art keywords
- albumin
- concentration
- skin tightening
- tannic acid
- substance
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- Cosmetics (AREA)
- Medicines Containing Plant Substances (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【０００１】[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。TECHNICAL FIELD The present invention relates to a method for screening a skin tightening substance. More specifically, the present invention relates to a method for screening a skin tightening substance, which displays whether the reactivity of the skin tightening substance for albumin, which is a soluble protein contained in animal cells, is compared with the reactivity of tannic acid for albumin.
【０００２】[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１１−２６３７１
８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。2. Description of the Related Art The skin is constantly exposed to external stimuli and tends to lose its elasticity and elasticity. There is a strong demand for the development of such skin tightening substances. However, there is no clear guide on how to screen for a skin tightening substance. For example, JP-A-11-26371
Japanese Unexamined Patent Publication No. 8 discloses a skin sagging improving and tightening agent containing an integrin-expressing substance of human skin cells as an active ingredient. In this publication, each sample is applied to the inside of the upper arm, and the elasticity of the skin or the firmness of the skin after several weeks is evaluated organoleptically. Japanese Unexamined Patent Publication No. 11-269057 discloses a skin astringent containing a protein aggregating substance collected from prune pulp as an active ingredient, and its effect as a skin astringent is tested by a protein aggregating force test. This test method, after reacting a sample solution and an aqueous solution of albumin, measuring the turbidity,
It is used as an index of protein cohesion (and also skin astringent action). Japanese Unexamined Patent Publication No. 2000-302666 discloses a skin tightening agent containing a sulfated glycoside derivative or a salt thereof, and the effect as a skin tightening agent is to improve collagen gel tightening promoting ability, skin elasticity change or skin tightening feeling. It is evaluated sensually. The method of sensually evaluating the firmness is a vague method, and there is a possibility that individual differences will occur. The method of measuring the turbidity of an aqueous albumin solution has a drawback that the turbidity becomes constant when the concentration exceeds a certain level, and the measurement becomes impossible. The collagen gel tightening ability measurement method requires the use of a fibroblast-encapsulated collagen gel, which is a dermis model, and the measurement method is complicated.
【０００３】[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。DISCLOSURE OF THE INVENTION The present inventors have succeeded in searching for and developing a more convenient and objective screening method for a skin tightening substance.
【０００４】[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。The present invention comprises reacting a skin tightening substance and tannic acid with albumin,
When the concentration of the skin tightening substance when albumin is reduced to half and the concentration of tannic acid when albumin is reduced to half are measured, and when the albumin to the concentration of skin tightening substance when reducing albumin to half is reduced to half The method for screening a skin tightening substance is characterized by displaying it as a ratio with the tannic acid concentration of.
【０００５】[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 albumi
n）、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 Minimu
m 96%（シグマ社製）を挙げることができる。BEST MODE FOR CARRYING OUT THE INVENTION Albumin used in the present invention includes egg albumin and serum albumin (serum albumi
n), milk albumin (lacta albumin) or the like. For example, commercial ALBUMIN, BOVINE Fraction V Minimu
m 96% (manufactured by Sigma) can be mentioned.
【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。Examples of tannic acid used in the present invention include tannic acid special grade (manufactured by Kosou Chemical Co., Ltd.). Some reagents contain salts. In the case of such a reagent, a desalting treatment is necessary because the presence of a salt prevents the precipitation caused by the reaction between tannic acid and albumin.
【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。[0007] Tannic acid is generally known to have an astringent action, and albumin is widely present in animal tissues. As a cohesive force measurement value of tannic acid for albumin, an albumin cohesive force measurement value of a skin tightening substance is used. The labeling is useful as a general evaluation method for skin tightening substances. In the present invention, the concentration of the skin tightening substance when the amount of albumin is halved is measured as follows.
【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。A skin tightening substance diluted to several levels with an aqueous solution of ethanol is reacted with an aqueous solution of albumin, and then the precipitate generated by the reaction is removed by filtration. The unreacted albumin in the aqueous albumin solution after removal is measured. At this time, the dilution degree of the skin tightening substance is selected so that the unreacted amount of albumin is 50% of the initial value, and the concentration of the skin tightening substance when the amount of albumin is halved is determined.
【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。Then, tannic acid diluted to several levels with an aqueous ethanol solution is reacted with an aqueous albumin solution, and the same operation is performed as described above, and the tannic acid concentration when the unreacted amount of albumin reaches 50% of the initial value. Ask for.
【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。The tannic acid concentration relative to the skin tightening substance concentration is displayed as follows.
【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。X = B / A × 100 (%) X is expressed as a ratio (%) of the tannic acid concentration B when the albumin amount is halved to the skin tightening substance concentration A when the albumin amount is halved. It is the value.
【００１２】[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。EXAMPLES In the following examples,% means% by weight unless otherwise specified. In the following examples, the measurement conditions of high performance liquid chromatography were as follows.
【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。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】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。Example 1 1% per 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. 1 g of bovine serum albumin aqueous solution was added and reacted at 30 ° C. for 1 hour. After the reacted precipitate was filtered through Acro LC (German Science), unreacted albumin was measured by high performance liquid chromatography (HPLC). The measurement results are shown in FIG. Further 0%, 0.01%, 0.02% and 0.03 with 10% ethanol.
Tannic acid diluted to have a concentration of% was reacted with a 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 the albumin reacted half amount was 0.11%, and the tannic acid concentration when the albumin reacted half amount was 0.021%. From these, it was found that persimmon astringent extract powder MF had a tightening effect equivalent to 19.1% tannic acid.
【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 Scienc
e)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（IC5
0）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。Example 2 0%, 0.02%, 0.05 with 10% ethanol
1% to 4 g of Gravinol (grape seed extract, Kikkoman) diluted to a concentration of 0.1% and 0.1%
1 g of bovine serum albumin aqueous solution was added and reacted at 30 ° C. for 1 hour. The reacted precipitate was mixed with Acro LC (German Scienc
After filtration with e), unreacted albumin was measured by HPLC. The measurement result is shown in FIG. Tannic acid diluted with 10% ethanol to a concentration of 0%, 0.01%, 0.02%, and 0.03% was reacted with a bovine serum albumin aqueous solution in the same manner as above, and unreacted. Albumin was measured. The measurement results are shown in FIG. Gravinol concentration (IC5
0) was 0.041%, and the tannic acid concentration when half the amount of albumin reacted was 0.024%. From these,
Gravinol was found to have a tightening effect equivalent to 58.5% tannic acid.
【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。Example 3 1 to 4 g of applephenone (apple extract, Nikka Whiskey) diluted to a concentration of 0%, 0.8%, 0.9% and 1.0% with 10% ethanol. % Bovine serum albumin aqueous solution (1 g) was added and reacted at 30 ° C. for 1 hour. The reacted precipitate is acro LC (German Science)
After filtration with, the unreacted albumin was measured by HPLC.
The measurement result is shown in FIG. Tannic acid diluted with 10% ethanol to a concentration of 0%, 0.01%, 0.02%, and 0.03% was reacted with a bovine serum albumin aqueous solution in the same manner as above, and unreacted. Albumin was measured. The measurement result is shown in FIG. Applephenone concentration when half the amount of albumin reacted (IC50)
Was 0.935%, and the concentration of tannic acid when albumin had half reacted was 0.028%. From these, it was found that applephenone has a tightening effect equivalent to 2.99% tannic acid.
【００１７】[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。According to the present invention, the tightening effect of various skin tightening substances can be displayed and compared by the tannic acid concentration. Further, it can be objectively evaluated by converting it into a tannic acid concentration, and its measuring method is also simple. According to the present invention, various skin tightening substances can be screened easily and objectively.
【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。FIG. 1 shows the measurement results of persimmon astringent extract powder MF of Example 1.
【図２】 実施例１のタンニン酸の測定結果を示す。2 shows the measurement results of tannic acid in Example 1. FIG.
【図３】 実施例２のグラヴィノールの測定結果を示
す。FIG. 3 shows the measurement results of gravinol of Example 2.
【図４】 実施例２のタンニン酸の測定結果を示す。FIG. 4 shows the measurement results of tannic acid of Example 2.
【図５】 実施例３のアップルフェノンの測定結果を示
す。5 shows the measurement results of apple phenone of Example 3. FIG.
【図６】 実施例３のタンニン酸の測定結果を示す。FIG. 6 shows the measurement results of tannic acid of Example 3.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ａ６１Ｋ 35/78 Ａ６１Ｋ 35/78 Ｈ Ａ６１Ｐ 17/16 Ａ６１Ｐ 17/16 Ｇ０１Ｎ 33/15 Ｇ０１Ｎ 33/15 Ｚ Ｆターム(参考） 2G045 AA40 DA38 FB02 FB06 FB20 JA01 4C083 AA111 CC02 EE11 4C088 AB24 AB51 AB56 AC04 BA08 NA20 ZA89 ─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. 7 Identification code FI theme code (reference) A61K 35/78 A61K 35/78 H A61P 17/16 A61P 17/16 G01N 33/15 G01N 33/15 Z F Term (reference) 2G045 AA40 DA38 FB02 FB06 FB20 JA01 4C083 AA111 CC02 EE11 4C088 AB24 AB51 AB56 AC04 BA08 NA20 ZA89
Claims (2)
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。1. A skin tightening substance and tannic acid are respectively reacted with albumin, and the concentration of the skin tightening substance when albumin is reduced by half and the tannic acid concentration when albumin is reduced by half are measured. The method for screening a skin tightening substance is characterized by displaying the ratio of the concentration of albumin to the concentration of the skin tightening substance when the amount of tannin is reduced by half, and the concentration of tannic acid when the amount of albumin is reduced by half.
ラフィで測定することを特徴とする請求項１記載のスク
リーニング方法。2. The screening method according to claim 1, wherein the albumin concentration is measured by high performance liquid chromatography.
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 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2003083962A true JP2003083962A (en) | 2003-03-19 |
| JP4280007B2 JP4280007B2 (en) | 2009-06-17 |
Family
ID=19100667
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2001275879A Expired - Fee Related JP4280007B2 (en) | 2001-09-12 | 2001-09-12 | Screening method for skin tightening substances |
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| Country | Link |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101338210B1 (en) * | 2012-02-24 | 2013-12-06 | 주식회사 아이피어리스 | The evaluation method of astringent activity using protein quantification |
| CN103760271A (en) * | 2014-01-27 | 2014-04-30 | 余显维 | Detection method for Naoxinqing capsules |
| CN106706788A (en) * | 2016-12-26 | 2017-05-24 | 合肥智汇医药科技有限公司 | Qualitative and quantitative analysis method for flavonoid effective components in persimmon leaf extract |
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2001
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101338210B1 (en) * | 2012-02-24 | 2013-12-06 | 주식회사 아이피어리스 | The evaluation method of astringent activity using protein quantification |
| CN103760271A (en) * | 2014-01-27 | 2014-04-30 | 余显维 | Detection method for Naoxinqing capsules |
| CN106706788A (en) * | 2016-12-26 | 2017-05-24 | 合肥智汇医药科技有限公司 | Qualitative and quantitative analysis method for flavonoid effective components in persimmon leaf extract |
| 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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| JP4280007B2 (en) | 2009-06-17 |
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