JPH0959166A - Epithelial cell proliferation promoter - Google Patents

Epithelial cell proliferation promoter

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Publication number
JPH0959166A
JPH0959166A JP23068295A JP23068295A JPH0959166A JP H0959166 A JPH0959166 A JP H0959166A JP 23068295 A JP23068295 A JP 23068295A JP 23068295 A JP23068295 A JP 23068295A JP H0959166 A JPH0959166 A JP H0959166A
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glycoside
skin
water
soybeans
epithelial cell
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JP23068295A
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JP3302535B2 (en )
Inventor
Masaru Matsuura
Akio Obata
Minoru Saito
Kouichirou Tobe
Nobuyuki Yamatsugu
明雄 小幡
信幸 山次
光一朗 戸辺
實 斉藤
勝 松浦
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Kikkoman Corp
Noda Sangyo Kagaku Kenkyusho
キッコーマン株式会社
財団法人野田産業科学研究所
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Abstract

PROBLEM TO BE SOLVED: To obtain an epithelial cell proliferation promoter, a skin cosmetic, a hair tonic, a skin preparation for external use, etc., having multiplying effect on epithelial cell, comprising malonylisoflavone glycoside as an active ingredient. SOLUTION: This epithelial cell proliferation promoter comprises malonylisoflavone glycoside as an active ingredient. The glycoside is obtained from soybeans or an extracted solution of soybeans with water and, for example, malonyldaizin etc., are preferably used. The glycoside, for example, is obtained by immersing peeled soybeans in water adjusted to pH7.5 to pH9.0 with caustic soda, etc., at 45-65 deg.C for 2-4 hours, removing the soybeans to give the immersion water as an extracted solution with water, removing protein from the extracted solution by an ultrafilter membrane to give a filtrate, bringing the filtrate into contact with an adsorbent and eluting the glycoside from the adsorbed material by using an aqueous solution of an alcohol or an alkali aqueous solution of an alcohol.

Description

【発明の詳細な説明】 DETAILED DESCRIPTION OF THE INVENTION

【0001】 [0001]

【産業上の利用分野】本発明はマロニルイソフラボン配糖体を上皮細胞増殖因子として含有させた、新規な上皮細胞増殖促進剤ないし皮膚用剤に関するものである。 The present invention relates to malonylisoflavone glycoside was contained as epidermal growth factor, it relates to a novel epithelial cell growth-promoting agent or dermatological agents.

【0002】 [0002]

【従来の技術及び課題】最近皮膚の細胞成長因子、例えば上皮成長因子(EGF)、線維芽細胞成長因子(FG BACKGROUND OF THE INVENTION Recently cell growth factors of the skin, such as epidermal growth factor (EGF), fibroblast growth factor (FG
F)等が発見され、これらの成長因子を利用して、皮膚の老化防止を目的とする皮膚化粧料や、皮膚の創傷治癒期間の短縮を目的とする皮膚外用剤が開発されている。 F) and the like are found, by taking advantage of these growth factors, skin cosmetics and for the purpose of anti-aging of the skin, skin external agents have been developed for the purpose of reduction of wound healing period of the skin.
例えばグルコサミノグリカンと線維芽細胞成長因子とを配合した皮膚化粧料(特開平4−187613)、α− For example glycosaminoglycans and fibroblast growth factor and skin cosmetics containing (JP 4-187613), α-
ヒドロキシ酢酸を配合した皮膚化粧料及び皮膚外用剤(特開平5−112422)、カバノキ科植物の抽出エキスを含有させた皮膚老化防止化粧料(特開平6−26 Formulated with hydroxyacetic acid skin cosmetic and external preparation for skin (JP-A 5-112422), skin anti-aging cosmetic composition which contains the extract of Betulaceae plants (JP-A-6-26
3627)等を挙げることができる。 3627), and the like.

【0003】一方大豆のイソフラボン化合物は、エステロゲン作用、抗酸化作用、抗溶血作用、抗コレステロール作用、抗脂血作用、抗菌作用等、種々の生理作用を有することが知られており、これらの生理作用を利用した皮膚化粧料等に関する特許出願もなされており、例えば大豆イソフラボン化合物含有美白化粧料(特公昭60− Meanwhile isoflavone compounds soybean, estrogen action, antioxidant action, anti-hemolytic activity, anti-cholesterol effect, antilipidemic activity, antibacterial action, etc., are known to have various physiological effects, these physiological have been made patent applications relating to skin cosmetics utilizing the action, for example, soy isoflavone compounds containing whitening cosmetic (JP-B-60-
19885)、大豆サポニン含有化粧料組成物(特開昭59−106419)、大豆胚軸の水抽出物を有効成分とする保湿剤(特開昭63−243013)等が知られている。 19,885), soybean saponin-containing cosmetic composition (JP 59-106419), a humectant and water extract of the active ingredient of soybean hypocotyl (JP 63-243013) and the like are known. また最近になって、大豆中にはマロニルダイジン及びマロニルゲニスチン等のマロニルイソフラボン配糖体の存在が確認され、これらが大豆中のイソフラボン化合物の主成分であることが判ってきた。 Also recently, the presence of malonyl isoflavone glycosides such malonyldaidzin and malonylgenistin during soybeans was confirmed, it has been found that these are the main component of isoflavone compounds in the soy. このマロニルイソフラボン配糖体は水に溶け易く、またそれ自身抗酸化作用があり、またその構造の類似性から上記したような薬理効果が期待されている。 The malonyl isoflavone glycosides more soluble in water, also has its own antioxidant, also pharmacological effects as mentioned above are expected from the similarity of its structure.

【0004】本発明者等は先に、大豆の水抽出液そのものが皮膚外用剤として効果のあることを見い出し特許出願した(特願平6−305697)が、さらに検討を進めた結果、大豆の水抽出液中にはマロニルイソフラボン配糖体が含有されており、抽出液を吸着剤に接触させ、、次いでアルコール水溶液で溶出させることによりマロニルイソフラボン配糖体が簡単に取得できるという知見を得、特許出願した(特願平7−112705)。 [0004] The present inventors have previously water extract itself soybeans were patent application found that is effective as a skin external preparation (Japanese Patent Application No. 6-305697) is a result of further studying, the soybean the aqueous extract are contained in malonyl isoflavone glycoside, extract into contact with the adsorbent to obtain a finding that malonyl isoflavone glycosides can be obtained simply by ,, then eluted with aqueous alcohol, and patent application (Japanese Patent application No. 7-112705).
そして更に、このマロニルイソフラボン配糖体が上皮細胞の増殖に効果のあることを見い出した。 And further found that the malonyl isoflavone glycoside is effective in proliferation of epithelial cells.

【0005】 [0005]

【課題を解決するための手段】本発明はこのような知見に基づきなされたものであって、上皮細胞増殖因子としてマロニルイソフラボン配糖体を含有させた皮膚用剤である。 SUMMARY OF THE INVENTION The present invention was made based on this finding, a dermatological agent which contains the malonylisoflavone glycoside as epidermal growth factor. 以下本発明を具体的に説明する。 Specifically describe the present invention following.

【0006】<マロニルイソフラボン配糖体の取得>大豆中のマロニルダイジン [0006] <Acquisition of malonyl isoflavone glycoside> malonyldaidzin in soy

【化1】 [Formula 1] マロニルゲニスチン Malonylgenistin

【化2】 ## STR2 ## 等のマロニルイソフラボン配糖体の含量は、大豆の種類、収穫時期等によってことなるが、文献{J.Agric.Fo The content of malonyl isoflavone glycoside etc, the type of soybean, varies depending harvest time and the like, the literature {J.Agric.Fo
od Chem.,42,1674(1994)}によれば表1の通りである。 od Chem., as shown in Table 1 according to 42,1674 (1994)}.
なお含量は大豆1gあたりのマイクログラムである。 The content is micrograms per soy 1g. 表1 大豆(年度) マロニルダイジン マロニルゲニスチン アメリカ種 Vinton81(1989) 410 958 Vinton81(1990) 300 743 Vinton81(1991) 237 545 Pioneer9111(1989) 690 1756 Pioneer9202(1989) 630 1705 Prize(1989) 709 1342 HP204(1989) 345 915 LS301(1989) 752 1558 KL72(1989) 198 1042 Strayer2233(1989) 385 883 日本種 Keburi(1991) 562 1232 Keburi(1992) 322 670 Kuro daizu(1991) 375 1187 Kuro daizu(1992) 222 717 Raiden(1991) 407 1191 Raiden(1992) 242 723 Table 1 Soybean (FY) malonyldaidzin malonylgenistin American species Vinton81 (1989) 410 958 Vinton81 (1990) 300 743 Vinton81 (1991) 237 545 Pioneer9111 (1989) 690 1756 Pioneer9202 (1989) 630 1705 Prize (1989) 709 1342 HP204 ( 1989) 345 915 LS301 (1989) 752 1558 KL72 (1989) 198 1042 Strayer2233 (1989) 385 883 Japan species Keburi (1991) 562 1232 Keburi (1992) 322 670 Kuro daizu (1991) 375 1187 Kuro daizu (1992) 222 717 Raiden (1991) 407 1191 Raiden (1992) 242 723

【0007】大豆からマロニルイソフラボン配糖体を製造する方法としては特開平3-170495号に記載されているように、粉砕大豆をアルコール抽出したのち、その抽出物を数段の水不混和性有機溶媒で抽出処理して製造する方法が知られているが製造方法の容易性、収率の点から以下に記載の如く、大豆の水抽出液から取得する方法が好ましい。 [0007] As a method for producing a malonyl isoflavone glycosides from soy is described in JP-A-3-170495, After the milled soybeans were alcohol extraction, water-immiscible organic several stages that extract ease of ways are known a method of manufacturing extraction process to a solvent, as described below in terms of yield, how to get from the water extract of soybeans is preferred.

【0008】すなわちマロニルイソフラボン配糖体を得るための原料は、丸大豆、脱皮大豆、脱脂大豆等であり、これらの水抽出液が好適に用いられる。 Namely material for obtaining malonylisoflavone glycosides, whole soybeans, dehulled soybeans are defatted soybean, these water extract is preferably used. 例えば丸大豆、脱皮大豆あるいは脱脂大豆を20〜80℃の水に1 For example 1 whole soybeans, dehulled soybeans, or defatted soybean 20 to 80 ° C. Water
〜30時間浸漬して得られる抽出液であるが、中でも脱皮大豆や脱脂大豆が好適である。 An extract obtained by immersing 30 hours, with preference given dehulled soybeans and defatted soybeans. もちろん、醤油、味噌、豆腐、納豆、豆乳、或いはもやしの製造に際して生ずる大豆の浸漬廃液も有効に利用できる。 Of course, soy sauce, miso, tofu, natto, soy milk, or dipping waste soybean arising during sprouts produced can also be effectively utilized. また大豆煮汁や木綿豆腐製造において生じるホエーも利用可能である。 The whey produced in soybean broth and tofu manufacturing also available. そして大豆の水抽出液を得るのに好ましい態様としては、脱皮大豆を、カセイソーダ等によりpH10.0以下、好ましくは7.5〜9.0に調整した45〜65℃ And as a preferred embodiment for obtaining a water extract of soybeans, dehulled soybeans, pH 10.0 or less by sodium hydroxide or the like, 45 to 65 ° C. preferably adjusted to 7.5 to 9.0
の水に2〜4時間浸漬し、浸漬大豆を除いて得られる浸漬水を水抽出液とする。 The soaked 2-4 hours in water, the soaking water obtained by removing soaked soybean and water extraction liquid.

【0009】この場合、浸漬水のpHを10.0以上にするとマロニルイソフラボン配糖体の収率が低下する。 [0009] In this case, the yield of malonyl isoflavone glycosides decreases when the pH of the soaking water above 10.0. これはマロニルイソフラボン配糖体が分解し、イソフラボン配糖体となるためである。 This is because the malonyl isoflavone glycoside is decomposed, the isoflavone glycoside. また大豆成分の抽出率を上げるためには、浸漬温度は高いほうが有利であるが、7 In order to increase the extraction rate of the soybean component is soaking temperature is advantageously the higher, 7
0℃以上にするとマロニルイソフラボン配糖体が分解しはじめる。 When the 0 ℃ than malonyl isoflavone glycoside begins to decompose. したがって抽出率と分解率とを考慮すると4 Thus extraction rate and considering the decomposition rate 4
5〜65℃が適当である。 5~65 ℃ is appropriate.

【0010】また脱脂大豆を使用する場合の脱脂大豆は低変性脱脂大豆が好適であり、これを直接水に浸漬し、 Further defatted soybean for using defatted soybean is preferably low-denatured defatted soybean which was directly immersed in water,
抽出するか、低変性脱脂大豆の粉砕物を水又はアルカリ水で抽出し、不溶残渣を除去したのち抽出液を塩酸でpH To be extracted, the pulverized low-denatured defatted soybean was extracted with water or alkaline water, pH of the extract solution with hydrochloric acid after removing the insoluble residue
4.3付近で酸沈して得られる分離大豆蛋白を除いた液、すなわち大豆ホエーでもよい。 Liquid excluding the isolated soybean protein obtained by the acid precipitation in the vicinity of 4.3, i.e., may be a soy whey.

【0011】このような大豆の抽出液を必要により限外濾過膜を用いて蛋白質を除去した濾液、あるいは上記大豆ホエーのように塩酸でpH4.3程度に調整し、抽出液中に溶解している蛋白質を沈澱させ、その上澄液を吸着剤に接触させる。 [0011] Such soybean extract filtrate to remove protein using an ultrafiltration membrane by requiring or adjusted to about pH4.3 with hydrochloric acid as described above soybean whey, is dissolved in the extract precipitating proteins are, contacting the supernatant to the adsorbent. 上記濾液あるいは上澄液は、そのまま吸着剤と接触させる方法と、濾液あるいは上澄液をカセイソーダでpH8.0程度に調整した後接触させる方法とがある。 The filtrate or supernatant, and a method for directly contacting with the adsorbent, and a method of contacting After adjusting the filtrate or supernatant to approximately pH8.0 with sodium hydroxide.

【0012】前者の場合は吸着剤に対する吸着量が増大し、後者の場合はマロニルイソフラボン配糖体とイソフラボン配糖体及びアグリコンの分離が容易であるという利点がある。 [0012] In the former case increases the adsorption amount with respect to the adsorbent, the latter case is advantageous in that malonylisoflavone glycoside and isoflavone glycosides and aglycon separation is easy. いずれの場合でも、使用する吸着剤は、例えば合成吸着剤、活性炭、アルミナ等であり、具体的にはダイヤイオンHP-20(三菱化学製)、精製白鷺活性炭(武田薬品工業製)、活性アルミナ(和光純薬製)等を挙げることができる。 In either case, the adsorption agent used is, for example, a synthetic adsorbent, activated carbon, an alumina or the like, in particular Diaion HP-20 (Mitsubishi Chemical), purified Shirasagi activated carbon (manufactured by Takeda Chemical Industries, Ltd.), activated alumina it can be mentioned (manufactured by Wako pure Chemical Industries, Ltd.), and the like. 接触はバッチ法、カラム法等一般的方法でよく、例えば、吸着剤を充填したカラムに抽出液を通過させることにより行うことができ、こうすることにより抽出液中のマロニルイソフラボン配糖体の殆どが吸着剤に吸着される。 Contact a batch process may be a column method such as a general method, for example, can be carried out by passing the extract to a column filled with adsorbent, most malonyl isoflavone glycoside in the extraction solution by doing so There is adsorbed by the adsorbent.

【0013】次いで吸着剤に吸着したマロニルイソフラボン配糖体をアルコール水溶液又はアルカリ性アルコール水溶液を用いて、マロニルイソフラボン配糖体を溶出させる。 [0013] Then the malonylisoflavone glycoside adsorbed on the adsorbent using the aqueous alcohol solution or an alkaline alcohol solution, eluting the malonylisoflavone glycoside. 得られた溶液は減圧濃縮し、あるいは減圧濃縮後、凍結乾燥してマロニルイソフラボン配糖体の乾燥粉末を得る。 The resulting solution was concentrated under reduced pressure, or after concentration under reduced pressure, to obtain a dry powder of malonyl isoflavone glycosides and lyophilized.

【0014】上記濃縮液あるいは乾燥粉末はマロニルダイジン及びマロニルゲニスチンの混合物であり、これを分別して取得する場合には、逆相クロマトグラフィーにより分取することができる。 [0014] The concentrated solution or dry powder is a mixture of malonyldaidzin and malonylgenistin, the case of obtaining by fractionating this can be fractionated by reverse phase chromatography. 例えばODS樹脂(山村化学製)を充填したカラムに濃縮液を通液し、マロニルイソフラボン配糖体を吸着させ、アルコール水溶液で溶出してマロニルダイジン、マロニルゲニスチンを分画し、 For example was passed through the concentrated solution to a column filled with ODS resin (manufactured by Yamamura Chemical), adsorbed the malonylisoflavone glycoside, fractionated malonyldaidzin and eluted with an aqueous alcohol solution, the malonylgenistin min,
これらの画分を減圧濃縮後、凍結乾燥してマロニルダイジン及びマロニルゲニスチンの乾燥粉末を得る。 After concentration under reduced pressure of these fractions, to obtain a dry powder of malonyldaidzin and malonylgenistin lyophilized.

【0015】なお、マロニルダイジン及びマロニルゲニスチンを効率よく得るためには以下の方法によることが好ましい。 [0015] It is preferable according to the following method in order to efficiently obtain malonyldaidzin and malonylgenistin. すなわち大豆の抽出液を吸着剤と接触させるまでは上記と同様であるが、溶出をアルコール水溶液の濃度を変えて順次行い、大まかにマロニルダイジンとマロニルゲニスチンを分別しこれらの溶出液をODSカラムで精製するのである。 Namely an extract of soybeans to be contacted with the adsorbent is as defined above, elute sequentially performed by changing the concentration of the alcohol aqueous solution was fractionated roughly malonyldaidzin and malonylgenistin of the eluate ODS column than is purified. このようにして大豆の抽出液より簡単にマロニルイソフラボン配糖体、あるいはマロニルダイジン及びマロニルゲニスチンを分別して得ることができる。 In this way easily malonyl isoflavone glycoside from the extract of soybean, or can be obtained by fractionating malonyldaidzin and malonylgenistin.

【0016】このようにして得られたマロニルイソフラボン配糖体は、上皮細胞を増殖させる効果を有し、これを通常用いられる各種基剤、例えば油分、界面活性剤、 [0016] The thus obtained malonyl isoflavone glycoside has the effect of proliferating epithelial cells, which normally various bases to be used include oil, surfactant,
保湿剤、アルコール、増粘剤、香料、酸化防止剤、キレート剤、色素、防腐剤等に配合することにより、上皮細胞増殖促進剤、皮膚化粧剤、育毛剤、皮膚外用剤、抗炎症剤等の皮膚用剤とすることができる。 Moisturizers, alcohols, thickeners, perfumes, antioxidants, chelating agents, dyes, by blending the antiseptic agent and the like, epithelial cell growth promoting agents, skin cosmetic agents, hair growth agents, skin external agents, anti-inflammatory agents such as It may be of a skin agent. マロニルダイジン、マロニルゲニスチンの配合量は、皮膚外用剤、皮膚化粧剤、抗炎症剤いずれの場合でも0.001〜100 Malonyl daidzin, the amount of malonyl genistin, skin external preparation, dermocosmetic agent, in either case an anti-inflammatory agent 0.001
0ppmである。 It is 0ppm. また他の上皮細胞増殖因子との併用もできる。 In addition it is also used in combination with other epidermal growth factor.

【0017】次にマロニルイソフラボン配糖体の上皮細胞増殖効果について検討した結果を示す。 [0017] Next, the results of investigation of the epidermal growth effect of malonyl isoflavone glycosides. なお使用した細胞は、米国白人皮膚由来の正常ヒト皮膚上皮細胞(No Incidentally cells used were American Caucasian skin from normal human skin epithelial cells (No
rmal human epidermal keratinocyte:NHEK)(倉敷紡績株式会社扱い)である。 rmal human epidermal keratinocyte: NHEK) is (Kurabo Industries Ltd. to handle).

【0018】使用培地:改変MCDB153培地(倉敷紡績製)にヒト組み換え型上皮成長因子(hEGF)をO. [0018] The use medium: modified MCDB153 medium (Kurabo Industries, Ltd.) to O. human recombinant epidermal growth factor (hEGF) 1ng/ml、インスリンを5μg/ml、ハイドロコーチゾンを0.5μg/ml、抗菌剤としてゲンタマイシンを50μg/ml、アンフォテンシンを0.0 1 ng / ml, insulin 5 [mu] g / ml, hydrocortisone a 0.5 [mu] g / ml, gentamicin 50 [mu] g / ml as an antimicrobial agent, Ann follower Tianjin 0.0
5μg/mlを添加し、表皮角化細胞増殖用培地(K− Was added 5 [mu] g / ml, epidermal keratinocytes growth medium (K-
DM)とした。 It was DM).

【0019】培養方法:正常ヒト皮膚上皮細胞(NHEK) [0019] The culture method: normal human skin epithelial cells (NHEK)
を上記K−DM培地に5000cells/mlとなるように懸濁し、48wellプレート(住友ベークライト製) Was suspended so that 5000 cells / ml in the K-DM medium, 48-well plate (manufactured by Sumitomo Bakelite)
に0.3ml/wellとなるように接種し、これを5 Inoculated so as to 0.3ml / well in, this 5
%CO 2と95%空気の雰囲気下、37℃、湿度100 % CO 2 and an atmosphere of 95% air, 37 ° C., a humidity of 100
%の培養機内で4時間培養する。 Cultured for 4 hours at% of culture-flight. 培養後、表2に示す試料液を10μl添加し、更に72時間培養後、K−DM After culturing, adding 10μl of the sample solutions shown in Table 2, after further 72 h culture, K-DM
培地を除去し、新たに新鮮なK−DM培地を0.3ml The medium was removed, new fresh K-DM medium 0.3ml
/wellと試料を再添加し、72時間培養し、細胞の増殖を測定した。 / Well and the sample re-added, and cultured for 72 hours, cell proliferation was measured.

【0020】細胞増殖の測定:和光純薬工業製のCel [0020] measurement of cell proliferation: manufactured by Wako Pure Chemical Industries, Ltd. Cel
l Counting Kit{(WST−1:2-(4-I l Counting Kit {(WST-1: 2- (4-I
odophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl- odophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl-
2H-tetrazolium,monosodium salt}を使用し、発色反応させ、分光光度計で波長***での吸光度(OD)を測定した。 2H-tetrazolium, using monosodium salt}, by coloring reaction was measured absorbance (OD) at a wavelength *** a spectrophotometer. 本測定方法は吸光度と細胞数は直線的な比例関係にある。 This measurement method absorbance and cell number are in a linear proportional relationship. なお比較2〜4は水に難溶性であるので溶解限度の濃度で実験を行なった。 Incidentally comparison 2-4 Experiments were performed at a concentration of solubility limit since it is poorly soluble in water.

【0021】以上の結果を表2に示す。 [0021] The above results are shown in Table 2. 表2 試料 濃度(ppm) OD値 有意差検定 マロニルダイジン 3×10 -5 0.371 * (本発明) 3×10 -4 0.358 * 3×10 -3 0.377 * 3×10 -2 0.397 * 3×10 -1 0.367 3 0.321 3×10 0.312 マロニルゲニスチン 3×10 -5 0.414 * (本発明) 3×10 -4 0.418 ** 3×10 -3 0.408 * 3×10 -2 0.347 3×10 -1 0.334 3 0.314 3×10 0.328 ダイジン 3×10 -5 0.300 (比較1) 3×10 -4 0.313 3×10 -3 0.293 3×10 -2 0.298 3×10 -1 0.315 3 0.306 3×10 0.312 ゲニスチン 3×10 -5 0.350 (比較2) 3×10 -4 0.330 3×10 -3 0.341 3×10 -2 0.327 3×10 -1 0.32 Table 2 Sample Concentration (ppm) OD value significance test malonyldaidzin 3 × 10 -5 0.371 * (invention) 3 × 10 -4 0.358 * 3 × 10 -3 0.377 * 3 × 10 -2 0.397 * 3 × 10 -1 0.367 3 0.321 3 × 10 0.312 malonylgenistin 3 × 10 -5 0.414 * (invention) 3 × 10 -4 0.418 ** 3 × 10 -3 0.408 * 3 × 10 -2 0.347 3 × 10 -1 0.334 3 0.314 3 × 10 0.328 daidzin 3 × 10 -5 0.300 (Comparative 1) 3 × 10 -4 0.313 3 × 10 -3 0.293 3 × 10 -2 0.298 3 × 10 -1 0.315 3 0.306 3 × 10 0.312 genistin 3 × 10 -5 0.350 (Comparative 2) 3 × 10 -4 0.330 3 × 10 -3 0.341 3 × 10 -2 0.327 3 × 10 -1 0.32 3 0.320 ダイゼイン 3×10 -5 0.312 (比較3) 3×10 -4 0.306 3×10 -3 0.293 1×10 -2 0.284 2×10 -2 0.246 3×10 -2 0.209 ゲニステイン 3×10 -5 0.343 (比較4) 3×10 -4 0.302 3×10 -3 0.329 1×10 -2 0.318 2×10 -2 0.198 3×10 -2 0.071 水(対照) 0.315 濃度(ppm):細胞培養液中濃度を示す OD値:5回の繰り返し試験の平均値である。 3 0.320 daidzein 3 × 10 -5 0.312 (Comparative 3) 3 × 10 -4 0.306 3 × 10 -3 0.293 1 × 10 -2 0.284 2 × 10 -2 0.246 3 × 10 -2 0.209 genistein 3 × 10 -5 0.343 (Comparative 4) 3 × 10 -4 0.302 3 × 10 -3 0.329 1 × 10 -2 0.318 2 × 10 -2 0 .198 3 × 10 -2 0.071 water (control) 0.315 concentration (ppm): OD value represents the concentration in the cell culture medium: 5 times the mean value of repeated tests. 有意差検定 *:5%危険率で有意差有り **:1%危険率で有意差有り また水を用いたとき(対照)の増殖活性を1とし、それぞれの試料の増殖活性を図1に示した。 Significance test *: significant difference at 5% risk factor There **: significant difference of 1% risk rate there also when using water as a 1 proliferative activity (control), the proliferative activity of each sample in Figure 1 Indicated.

【0022】表2及び図1から明らかなように、マロニルイソフラボン配糖体であるマロニルダイジン、マロニルゲニスチンを3×10 -5 〜3×10 -1 ppm添加した試料はヒト上皮細胞増殖効果を示し、特にマロニルダイジンは添加量3×10 -5 〜3×10 -2 ppm、マロニルゲニスチンは3×10 -5 〜3×10 -3 ppmの範囲で、 [0022] As is clear from Table 2 and Figure 1, malonyldaidzin a malonyl isoflavone glycoside, sample was added malonylgenistin 3 × 10 -5 ~3 × 10 -1 ppm indicates human epidermal growth effect particularly malonyldaidzin addition amount 3 × 10 -5 ~3 × 10 -2 ppm, malonylgenistin in the range of 3 × 10 -5 ~3 × 10 -3 ppm,
有意に効果のあることが認められた。 It has been found that significant an effect.

【0023】また生理活性作用があるとされるダイジン、ゲニスチン、ダイゼイン、ゲニステイン(比較1〜 Further daidzin which is to be physiological activity, genistin, daidzein, genistein (Comparison 1
4)においては、ダイジン、ゲニスチンは効果が認められたが有意な効果でなく、またダイゼイン、ゲニステインではほとんど効果が認められず、高い濃度では阻害が認められた。 In 4), daidzin, genistin effect is not but significant effect was observed, also daidzein, not observed little effect genistein, inhibition was observed at high concentrations.

【0024】 [0024]

【実施例】以下に実施例を示す。 [Example] the following examples are set forth. 実施例1 脱皮大豆の温水抽出液20Lを、塩酸でpH4.0に調整し2時間放置後、濾過助剤(ラジオライト#500、昭和化学製)を添加し、ブフナーロートで吸引濾過した。 Hot water extract 20L of Example 1 dehulled soybeans and left for 2 hours and adjusted to pH4.0 with hydrochloric acid, was added filter aid (Radiolite # 500, Showa Chemical Co., Ltd.), and suction filtered through a Buchner funnel.
この濾液を活性炭(精製白鷺クロマト用、武田薬品工業製)を充填したカラム(5×22cm、430ml)に1. 1 The filtrate activated carbon (purified Shirasagi for chromatography, Takeda Chemical Industries, Ltd.) packed column (5 × 22 cm, 430 ml) to.
5L/hrの流速で通液させてマロニルイソフラボン配糖体を吸着させた後、1%アンモニア水3Lでカラムを洗浄した。 After adsorption of the malonylisoflavone glycosides liquid permeation is at a flow rate of 5L / hr, the column was washed with 1% aqueous ammonia 3L. 次いで1%アンモニア含有50%エタノール水溶液5Lで溶出し、得られた溶出液を減圧下、50℃で濃縮し、マロニルダイジン1.23g、マロニルゲニスチン1.05gを含有する濃縮液500mlを得た。 Then eluted with 1% ammonia-containing 50% aqueous ethanol 5L, under reduced pressure and the resulting eluate was concentrated at 50 ° C., malonyldaidzin 1.23 g, to obtain a concentrated liquid 500ml containing malonylgenistin 1.05 g. この濃縮液を2NのカセイソーダでpH8.0としODS樹脂充填カラム(4×24cm、300ml)に流速30ml/min. The concentrate was brought to pH8.0 with sodium hydroxide of 2N ODS resin packed column (4 × 24cm, 300ml) in a flow rate of 30 ml / min.
で通液し、蒸留水0.5Lで洗浄後、2%、5%及び1 In and passing liquid, washed with distilled water 0.5 L, 2%, 5% and 1
0 %エタノール水溶液各2Lで溶出を行い、5%エタノール水溶液の溶出区分よりマロニルダイジン画分を、1 Performed eluting with 0% aqueous ethanol each 2L, the malonyldaidzin fractions from elution Segment 5% aqueous ethanol solution, 1
0%エタノール水溶液の溶出区分よりマロニルゲニスチン画分を得、それぞれを減圧下、50℃で減圧濃縮した後、凍結乾燥し、マロニルダイジンをナトリウム塩として652mg、マロニルゲニスチンをナトリウム塩として412mg得た。 Give malonylgenistin fractions from elution Segment 0% ethanol aqueous solution to vacuum concentration, respectively under reduced pressure, at 50 ° C., and lyophilized to give 412 mg 652 mg, the malonylgenistin as the sodium salt as the sodium salt malonyl daidzin. こうして得られたマロニルダイジンナトリウム塩又はマロニルゲニスチンナトリウム塩を上皮細胞増殖因子として以下の皮膚用剤を調整した。 Thus obtained Malo Niruda discoidin sodium or malonylgenistin sodium salt was adjusted following dermatological as epidermal growth factor.

【0025】<皮膚化粧剤> マロニルダイジン−Na 1 マロニルゲニスチン−Na 1 エタノール 80 グリセリン 45 ポリオキシエチレン硬化ヒマシ油 5 香料 1 精製水 867 これらを混合し皮膚化粧剤とした。 [0025] it was <dermocosmetic agent> malonyldaidzin -Na 1 malonylgenistin -Na 1 ethanol 80 Glycerin 45 Polyoxyethylene hardened castor oil 5 Perfume 1 Purified water 867 by mixing these skin cosmetic.

【0026】<皮膚外用剤>親水軟膏基剤(小堺製薬製)1kgにマロニルダイジン−Na及びマロニルゲニスチン−Naを各200mgずつ精製水200mlに溶解後混和して皮膚外用剤とした。 [0026] <endermic liniment> was hydrophilic ointment base (Kosakai Seiyaku) 1 kg in malonyldaidzin -Na and malonylgenistin -Na by mixing dissolved in purified water 200ml by each 200mg external preparation for skin.

【0027】<育毛剤>8%エタノール溶液1lにマロニルダイジン−Na及びマロニルゲニスチン−Naを各2mg溶解して育毛剤とした。 [0027] The malonyldaidzin -Na and malonylgenistin -Na to <hair restorer> 8% ethanol solution 1l was each 2mg dissolved to hair tonic.

【0028】<使用例1>上記した<皮膚化粧剤>を5 [0028] The <Example 1> above <skin cosmetic agent> 5
0代の女性20名に1ヵ月間使用させ、アンケート調査したところ、15名のパネルから皮膚のはりに効果があり、内10名はしわの防止に効果があるという回答を得た。 0 generations to use one month to women 20 people, was questionnaire survey, there is effect from the 15 panelists on the beams of the skin, internal 10 people got the answer that there is an effect in the prevention of wrinkles. また残りの5名も肌がしっとりとしたとの回答があった。 The rest of the five also had responded with the skin is moist and.

【0029】<使用例2>肌あれ症(乾燥肌)の20〜 [0029] 20 of the <Example 2> skin any diseases (dry skin)
40代の女性10名に1ヵ月間、右手に上記、〈皮膚外用剤>を、左手にはコントロールとしてマロニル配糖体無添加の軟膏を、毎日就寝前に塗布してもらい、その後左右の手の肌あれ症状の状態を観察してもらったところ、10名とも右手の方がしっとりと滑らかとなり、肌あれの治癒効果を認めた。 1 month to 10 women in their 40s, above on the right, the <external preparation for skin>, on the left the ointment of malonyl glycosides additive-free as a control, asked to applied before going to bed every day, then left and right hands of rough skin was asked to observe the state of the symptoms, those of the right hand becomes moist and smooth least 10 people, showed a skin any of the healing effect.

【0030】<使用例3>上記〈育毛剤〉を50代の男性10名に1ヵ月間、洗髪後頭皮に使用してもらったところ6名が髪に艶が出、抜け毛が減ったとの効果を認めた。 [0030] The effect of the <Example 3> above 1-month <hair tonic> to 10 males in their 50s, 6 people was asked to use the shampoo after the scalp gloss to the hair out, hair loss has decreased the admitted.

【0031】 [0031]

【発明の効果】本発明はマロニルイソフラボン配糖体が上皮細胞増殖因子として作用し、上皮細胞増殖促進に効果があるという知見に基づくものであり、これにより皮膚の老化防止、肌あれ改善等の皮膚化粧剤、抗炎症剤、 According to the present invention acts malonyl isoflavone glycoside as the epidermal growth factor is based on the finding that there is an effect on the epithelial cell growth promoting, thereby anti-aging of the skin, the improvement there be skin skin cosmetic agent, an anti-inflammatory agent,
創傷治療剤等として有用な皮膚外用剤等を提供することができる。 It is possible to provide a useful skin external preparation such as a wound therapeutic agent.

【図面の簡単な説明】 BRIEF DESCRIPTION OF THE DRAWINGS

【図1】正常ヒト皮膚上皮細胞に対する、各試料の増殖活性を示す。 [1] on normal human skin epithelial cells, indicating the proliferative activity of each sample.

───────────────────────────────────────────────────── フロントページの続き (72)発明者 小幡 明雄 千葉県野田市野田339番地 キッコーマン 株式会社内 (72)発明者 山次 信幸 千葉県野田市野田339番地 キッコーマン 株式会社内 (72)発明者 戸辺 光一朗 千葉県野田市野田339番地 キッコーマン 株式会社内 ────────────────────────────────────────────────── ─── of the front page continued (72) inventor Akio Obata Chiba Prefecture Noda Noda 339 address Kikkoman within Co., Ltd. (72) inventor mountain following Nobuyuki Chiba Prefecture Noda Noda 339 address Kikkoman within Co., Ltd. (72) inventor Tobe light Ichiro Chiba Prefecture Noda Noda 339 address Kikkoman within Co., Ltd.

Claims (4)

    【特許請求の範囲】 [The claims]
  1. 【請求項1】 マロニルイソフラボン配糖体を有効成分とする上皮細胞増殖促進剤。 1. A epidermal growth promoting agent comprising as an active ingredient malonylisoflavone glycoside.
  2. 【請求項2】 マロニルイソフラボン配糖体を含有させた皮膚用剤。 2. A dermatological agent is contained malonylisoflavone glycoside.
  3. 【請求項3】 マロニルイソフラボン配糖体が、大豆又は大豆の水抽出液から取得したものである請求項1又は2記載の皮膚用剤。 3. malonylisoflavone glycosides, dermatological agent according to claim 1 or 2, wherein is obtained from the aqueous extract of soybean or soybean.
  4. 【請求項4】 皮膚用剤が皮膚化粧剤、育毛剤、皮膚外用剤、抗炎症剤のいずれかである請求項2記載の皮膚用剤。 4. A dermatological skin cosmetic agent, hair tonic, skin external agents, skin agent according to claim 2, wherein either the anti-inflammatory agent.
JP23068295A 1995-08-17 1995-08-17 Epithelial cell growth-promoting agent Expired - Fee Related JP3302535B2 (en)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6093411A (en) * 1998-03-16 2000-07-25 The Procter & Gamble Company Compositions for regulating skin appearance
US6479054B1 (en) 1998-09-21 2002-11-12 Showa Sangyo Co., Ltd. Process for obtaining genistin-rich isoflavone composition
EP1074240A3 (en) * 1999-07-27 2003-01-29 JOHNSON &amp; JOHNSON CONSUMER PRODUCTS, INC. Reducing hair growth, hair follicle and hair shaft size and hair pigmentation
US7112344B2 (en) 2003-08-11 2006-09-26 I-Hung Chu Vapor fraction from seeds of Glycine max (L.)Merr. and composition thereof
JP2007008908A (en) * 2005-07-04 2007-01-18 Takahito Tokuyama Cell proliferation enhancer and cell-repairing agent using cereals except rice or pulse crops as raw material
US7282226B2 (en) 2003-08-11 2007-10-16 I-Hung Chu Vapor fraction from seeds of Glycine max (L.) Merr. and composition thereof
JP2009242324A (en) * 2008-03-31 2009-10-22 Naris Cosmetics Co Ltd Keratinocyte proliferation promoter
JP2010150203A (en) * 2008-12-25 2010-07-08 Lion Corp Hair growing agent composition
US7897144B2 (en) 2001-02-28 2011-03-01 Johnson & Johnson Comsumer Companies, Inc. Compositions containing legume products
US8093293B2 (en) 1998-07-06 2012-01-10 Johnson & Johnson Consumer Companies, Inc. Methods for treating skin conditions
US8106094B2 (en) 1998-07-06 2012-01-31 Johnson & Johnson Consumer Companies, Inc. Compositions and methods for treating skin conditions
US8431550B2 (en) 2000-10-27 2013-04-30 Johnson & Johnson Consumer Companies, Inc. Topical anti-cancer compositions and methods of use thereof

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6093411A (en) * 1998-03-16 2000-07-25 The Procter & Gamble Company Compositions for regulating skin appearance
US8106094B2 (en) 1998-07-06 2012-01-31 Johnson & Johnson Consumer Companies, Inc. Compositions and methods for treating skin conditions
US8093293B2 (en) 1998-07-06 2012-01-10 Johnson & Johnson Consumer Companies, Inc. Methods for treating skin conditions
US6479054B1 (en) 1998-09-21 2002-11-12 Showa Sangyo Co., Ltd. Process for obtaining genistin-rich isoflavone composition
EP1074240A3 (en) * 1999-07-27 2003-01-29 JOHNSON &amp; JOHNSON CONSUMER PRODUCTS, INC. Reducing hair growth, hair follicle and hair shaft size and hair pigmentation
US7985404B1 (en) * 1999-07-27 2011-07-26 Johnson & Johnson Consumer Companies, Inc. Reducing hair growth, hair follicle and hair shaft size and hair pigmentation
US8431550B2 (en) 2000-10-27 2013-04-30 Johnson & Johnson Consumer Companies, Inc. Topical anti-cancer compositions and methods of use thereof
US7897144B2 (en) 2001-02-28 2011-03-01 Johnson & Johnson Comsumer Companies, Inc. Compositions containing legume products
US7282226B2 (en) 2003-08-11 2007-10-16 I-Hung Chu Vapor fraction from seeds of Glycine max (L.) Merr. and composition thereof
US7112344B2 (en) 2003-08-11 2006-09-26 I-Hung Chu Vapor fraction from seeds of Glycine max (L.)Merr. and composition thereof
JP2007008908A (en) * 2005-07-04 2007-01-18 Takahito Tokuyama Cell proliferation enhancer and cell-repairing agent using cereals except rice or pulse crops as raw material
JP2009242324A (en) * 2008-03-31 2009-10-22 Naris Cosmetics Co Ltd Keratinocyte proliferation promoter
JP2010150203A (en) * 2008-12-25 2010-07-08 Lion Corp Hair growing agent composition

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