JPH0959166A - Epithelial cell proliferation promoter - Google Patents

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

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel epithelial cell growth promoter or dermatological agent containing malonyl isoflavone glycoside as an epidermal growth factor.

[0002]

2. Description of the Related Art Recently, skin cell growth factors such as epidermal growth factor (EGF) and fibroblast growth factor (FG) have been developed.
F) and the like were discovered, and by utilizing these growth factors, skin cosmetics for the purpose of preventing skin aging and skin external preparations for the purpose of shortening the skin wound healing period have been developed.
For example, skin cosmetics containing glucosaminoglycan and fibroblast growth factor (JP-A-4-187613), α-
Skin cosmetics and external preparations for skin containing hydroxyacetic acid (JP-A-5-112422), and skin anti-aging cosmetics containing an extract of birch plants (JP-A-6-26).
3627) and the like.

On the other hand, isoflavone compounds of soybean are known to have various physiological actions such as an estrogen action, an antioxidant action, an antihemolytic action, an anticholesterol action, an antilipemic action and an antibacterial action. A patent application relating to skin cosmetics and the like utilizing the action has been made, for example, a whitening cosmetic containing a soy isoflavone compound (Japanese Patent Publication No. 60-
19885), a soybean saponin-containing cosmetic composition (JP-A-59-106419), a moisturizer containing an aqueous extract of soybean hypocotyl as an active ingredient (JP-A-63-243013), and the like are known. Moreover, the presence of malonyl isoflavone glycosides such as malonyl daidzin and malonyl genistin has been recently confirmed in soybean, and it has been found that these are the main components of the isoflavone compound in soybean. This malonyl isoflavone glycoside is easily soluble in water, has an antioxidant effect by itself, and is expected to have the above-mentioned pharmacological effects due to its structural similarity.

The present inventors previously found that the water extract of soybean itself was effective as an external preparation for skin, and filed a patent application (Japanese Patent Application No. 6-305697). Malonyl isoflavone glycosides are contained in the water extract, and the finding that malonyl isoflavone glycosides can be easily obtained by contacting the extract with an adsorbent and then eluting with an aqueous alcohol solution, We applied for a patent (Japanese Patent Application No. 7-112705).
Furthermore, they have found that this malonyl isoflavone glycoside has an effect on the proliferation of epithelial cells.

[0005]

The present invention has been made based on such findings, and is a dermatological agent containing malonyl isoflavone glycoside as an epidermal growth factor. Hereinafter, the present invention will be described specifically.

<Acquisition of Malonyl Isoflavone Glycoside> Malonyl daidzin in soybean

Embedded image Malonyl genistin

Embedded image The content of malonyl isoflavone glycosides, etc., varies depending on the type of soybean, the harvest time, etc., but the literature {J. Agric.
od Chem., 42, 1674 (1994)}.
The content is microgram per 1 g of soybean. Table 1 Soybean (FY) Malonyldaidine Malonylgenistin American Varieties 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 Japanese 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

As a method for producing malonyl isoflavone glycosides from soybeans, as described in JP-A-3-170495, ground soybeans are subjected to alcohol extraction, and then the extract is subjected to several stages of water-immiscible organic compounds. Although a method of producing by extracting with a solvent is known, a method of obtaining from a water extract of soybean is preferable as described below from the viewpoint of ease of production and yield.

That is, the raw materials for obtaining malonyl isoflavone glycosides are whole soybeans, dehulled soybeans, defatted soybeans, etc., and their water extracts are preferably used. For example, add whole soybeans, dehulled soybeans or defatted soybeans to water at 20-80 ° C.
It is an extract obtained by soaking for about 30 hours, and among them, dehulled soybeans and defatted soybeans are preferable. Of course, soy sauce, miso, tofu, natto, soy milk, or soybean dipping waste liquid generated during the production of sprouts can also be effectively used. Also, whey produced in soybean juice and cotton tofu production can be used. And as a preferred embodiment for obtaining the water extract of soybean, the dehulled soybean is adjusted to pH 10.0 or less by caustic soda or the like, preferably 45 to 65 ° C., which is adjusted to 7.5 to 9.0.
The soaked water obtained by removing the soaked soybeans is used as a water extract.

In this case, the yield of malonyl isoflavone glycoside decreases when the pH of the immersion water is set to 10.0 or higher. This is because malonyl isoflavone glycosides are decomposed into isoflavone glycosides. In order to increase the extraction rate of soybean ingredients, it is advantageous to use a higher soaking temperature.
Malonyl isoflavone glycosides begin to decompose at temperatures above 0 ° C. Therefore, considering the extraction rate and the decomposition rate, 4
A temperature of 5 to 65 ° C is suitable.

When defatted soybeans are used, the defatted soybeans are preferably low-denaturation defatted soybeans, which are directly immersed in water,
Extract or pulverize low-denaturing defatted soybeans with water or alkaline water to remove insoluble residues, and then add the extract to pH with hydrochloric acid.
A liquid obtained by removing precipitated soybean protein obtained by acid precipitation in the vicinity of 4.3, that is, soybean whey may be used.

If necessary, the soybean extract is filtered to remove protein using an ultrafiltration membrane, or the soybean whey is adjusted to about pH 4.3 with hydrochloric acid and dissolved in the extract. The proteins present are allowed to settle and the supernatant is contacted with the adsorbent. The above-mentioned filtrate or supernatant may be contacted with the adsorbent as it is, or the filtrate or supernatant may be adjusted to pH 8.0 with caustic soda and then contacted.

In the former case, the amount of adsorption to the adsorbent is increased, and in the latter case, there is an advantage that the malonyl isoflavone glycoside, the isoflavone glycoside and the aglycone can be easily separated. In any case, the adsorbent used is, for example, a synthetic adsorbent, activated carbon, alumina or the like, and specifically, Diaion HP-20 (manufactured by Mitsubishi Chemical), purified Shirasagi activated carbon (manufactured by Takeda Chemical Industries), activated alumina. (Manufactured by Wako Pure Chemical Industries, Ltd.) and the like. The contact may be carried out by a general method such as a batch method or a column method. For example, the extraction solution may be passed through a column packed with an adsorbent, whereby almost all malonyl isoflavone glycosides in the extraction solution may be contacted. Are adsorbed by the adsorbent.

Then, the malonyl isoflavone glycoside adsorbed on the adsorbent is eluted with an aqueous alcohol solution or an alkaline alcohol aqueous solution. The obtained solution is concentrated under reduced pressure, or concentrated under reduced pressure and then freeze-dried to obtain a dry powder of malonyl isoflavone glycoside.

The above concentrated liquid or dry powder is a mixture of malonyl daidzin and malonyl genistin, and when it is obtained by fractionation, it can be fractionated by reverse phase chromatography. For example, a concentrated liquid is passed through a column filled with ODS resin (manufactured by Yamamura Chemical Co., Ltd.) to adsorb malonyl isoflavone glycoside, and eluted with an aqueous alcohol solution to fractionate malonyl daidzin and malonyl genistin,
These fractions are concentrated under reduced pressure and then freeze-dried to obtain a dry powder of malonyldaididine and malonylgenistin.

In order to efficiently obtain malonyl daidzin and malonyl genistin, the following method is preferable. That is, the above is the same as above until the soybean extract is brought into contact with the adsorbent, but the elution is sequentially performed by changing the concentration of the alcohol aqueous solution, and the malonyldaididine and the malonylgenistin are roughly separated and these eluates are separated by an ODS column. Purify. In this way, malonyl isoflavone glycosides or malonyl daidzin and malonyl genistin can be easily separated and obtained from the soybean extract.

The malonyl isoflavone glycoside thus obtained has the effect of proliferating epithelial cells, and various bases usually used such as oil, surfactant,
By blending with moisturizers, alcohols, thickeners, fragrances, antioxidants, chelating agents, pigments, preservatives, etc., epithelial cell growth promoters, skin cosmetics, hair growth agents, skin external preparations, anti-inflammatory agents, etc. It can be used as a skin preparation. The amount of malonyl daidzin and malonyl genistin used is 0.001 to 100 for any of the skin external preparations, skin cosmetics, and anti-inflammatory agents.
It is 0 ppm. It can also be used in combination with other epidermal growth factors.

Next, the results of examining the epithelial cell growth effect of malonyl isoflavone glycoside will be shown. The cells used were normal human skin epithelial cells (No.
rmal human epidermal keratinocyte (NHEK) (handled by Kurashiki Spinning Co., Ltd.).

Medium used: Modified MCDB153 medium (manufactured by Kurashiki Spinning Co., Ltd.) was supplemented with human recombinant epidermal growth factor (hEGF). 1 ng / ml, insulin 5 μg / ml, hydrocortisone 0.5 μg / ml, gentamicin 50 μg / ml as antibacterial agent, amphotensin 0.0
5 μg / ml was added to the medium for growing epidermal keratinocytes (K-
DM).

Culture method: Normal human skin epithelial cells (NHEK)
Suspended in the above K-DM medium at 5000 cells / ml, and a 48-well plate (Sumitomo Bakelite)
To 0.3 ml / well, and inoculate this to 5
% CO ₂ and 95% air atmosphere, 37 ℃, humidity 100
% Incubator for 4 hours. After culturing, 10 μl of the sample solution shown in Table 2 was added, and after culturing for 72 hours, K-DM was added.
Remove the medium and add 0.3 ml of fresh K-DM medium.
/ Well and the sample were re-added and cultured for 72 hours, and the proliferation of cells was measured.

Measurement of cell proliferation: Cel manufactured by Wako Pure Chemical Industries
l Counting Kit {(WST-1: 2- (4-I
odophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl-
2H-tetrazolium, monosodium salt} was used to cause color reaction, and the absorbance (OD) at a wavelength *** was measured with a spectrophotometer. In this measuring method, the absorbance and the number of cells are linearly proportional. Since Comparatives 2 to 4 are sparingly soluble in water, experiments were conducted at a concentration at the solubility limit.

The above results are shown in Table 2. Table 2 Sample concentration (ppm) OD value Significant difference test Malonyl daidzin 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 Malonyl genistin 3 × 10 ⁻⁵ 0.414 * (invention) 3 × 10 ⁻⁴ 0.418 ** 3 × 10 ^-3 0.408 * 3 x 10 ^-2 0.347 3 x 10 ^-1 0.334 3 0.314 3 x 10 0.328 Daihatsu 3 x 10 ^-5 0.300 (Comparison 1) 3 x 10 ^-4 0.313 3x10 ^-3 0.293 3x10 ^-2 0.298 3x10 ^-1 0.315 3 0.306 3x10 0.312 Genistin 3x10 ^-5 0.350 (Comparison 2) ^{3 × 10 -4 0.330 3 × 10} -3 0.341 3 × 10 -2 0.327 3 × 10 -1 0.32 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 X10 ^-2 0.209 genistein 3x10 ^-5 0.343 (comparative 4) 3x10 ^-4 0.302 3x10 ^-3 0.329 1x10 ^-2 0.318 2x10 ^-2 0 198 3 × 10 ^-2 0.071 Water (control) 0.315 Concentration (ppm): Indicates concentration in cell culture solution OD value: Average value of 5 repeated tests. Significant difference test *: Significant difference at 5% risk rate **: Significant difference at 1% risk rate Moreover, the proliferative activity when water was used (control) was set to 1, and the proliferative activity of each sample is shown in FIG. Indicated.

As is apparent from Table 2 and FIG. 1, the samples containing malonyldaididine and malonylgenistin, which are malonyl isoflavone glycosides, added at 3 × 10 ^{-5 to} 3 × 10 ^-1 ppm show human epithelial cell proliferation effect. particularly malonyldaidzin addition amount ^{3 × 10 -5 ~3 × 10 -2} ppm, malonylgenistin in the range of ^{3 × 10 -5 ~3 × 10 -3} ppm,
It was confirmed to be significantly effective.

Also, daidzin, genistin, daidzein, genistein (Comparative 1
In 4), the effects of daidzin and genistin were recognized, but the effects were not significant, and the effects of daidzein and genistein were hardly observed, and inhibition was observed at high concentrations.

[0024]

Examples are shown below. Example 1 20 L of the hot water extract of dehulled soybeans was adjusted to pH 4.0 with hydrochloric acid and allowed to stand for 2 hours, then a filter aid (Radiolite # 500, Showa Kagaku) was added, and suction filtration was carried out using a Buchner funnel.
This filtrate was applied to a column (5 × 22 cm, 430 ml) packed with activated carbon (for purified Shirasagi chromatography, manufactured by Takeda Pharmaceutical Co., Ltd.).
The solution was passed at a flow rate of 5 L / hr to adsorb malonyl isoflavone glycoside, and then the column was washed with 3 L of 1% ammonia water. Then, elution was carried out with 5 L of a 50% aqueous ethanol solution containing 1% ammonia, and the obtained eluate was concentrated at 50 ° C. under reduced pressure to obtain 500 ml of a concentrated solution containing 1.23 g of malonyldaididine and 1.05 g of malonylgenistin. The concentrated solution was adjusted to pH 8.0 with 2N caustic soda, and the flow rate was 30 ml / min in an ODS resin packed column (4 × 24 cm, 300 ml).
After washing with 0.5 L of distilled water, 2%, 5% and 1
Elute with 2 L each of 0% ethanol aqueous solution, and extract the malonyldaidine fraction from the elution fraction of 5% ethanol aqueous solution to 1 L.
Malonylgenistin fractions were obtained from the elution fraction of a 0% ethanol aqueous solution, concentrated under reduced pressure at 50 ° C. under reduced pressure, and then freeze-dried to obtain 652 mg of malonyldaidinidine as a sodium salt and 412 mg of malonylgenistin as a sodium salt. Using the malonyl daidzin sodium salt or malonyl genistin sodium salt thus obtained as an epidermal growth factor, the following skin preparations were prepared.

<Skin cosmetic agent> Malonyl daidzin-Na 1 Malonyl genistin-Na 1 Ethanol 80 Glycerin 45 Polyoxyethylene hydrogenated castor oil 5 Perfume 1 Purified water 867 These were mixed to prepare a skin cosmetic agent.

<External skin preparation> Malonyldaididine-Na and malonylgenistin-Na (200 mg each) were dissolved in 1 kg of a hydrophilic ointment base (manufactured by Kosakai Pharmaceutical Co., Ltd.) in 200 ml of purified water and mixed to prepare an external skin preparation.

<Hair Growth Agent> Malonyl daidzin-Na and malonyl genistin-Na (2 mg each) were dissolved in 1 liter of an 8% ethanol solution to prepare a hair growth agent.

<Use Example 1> 5% of the above <skin cosmetic agent>
When 20 women in their 0's were allowed to use it for 1 month and a questionnaire survey was conducted, a panel of 15 people answered that it was effective for skin scalp and 10 of them were effective in preventing wrinkles. The remaining 5 respondents also said that their skin was moisturized.

<Use Example 2> 20 for skin roughness (dry skin)
10 females in their 40s were asked to apply the above-mentioned <skin external preparation> to their right hands for one month, and to their left hands a malonyl glycoside-free ointment as a control every day before bedtime, and then on their left and right hands. As a result of observing the condition of skin roughening, the 10 hands showed that the right hand became smoother and smoother, and the healing effect of the skin roughening was recognized.

<Use Example 3> When 10 males in their 50's were used on the scalp after washing the hair for 1 month with the above <Hair growing agent>, the effect was that 6 of them had shiny hair and reduced hair loss. Admitted.

[0031]

INDUSTRIAL APPLICABILITY The present invention is based on the finding that malonyl isoflavone glycoside acts as an epithelial cell growth factor and is effective in promoting epithelial cell growth, whereby it is possible to prevent skin aging, improve skin roughness, etc. Skin cosmetics, anti-inflammatory agents,
It is possible to provide a skin external preparation and the like useful as a wound treatment agent and the like.

[Brief description of drawings]

FIG. 1 shows the proliferative activity of each sample on normal human skin epithelial cells.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Akio Obata 339 Noda, Noda, Chiba Prefecture, Kikkoman Corporation (72) Inventor Nobuyuki Yamaji 339 Noda, Noda, Chiba Prefecture, Kikkoman Corporation (72) Inventor, Tobe Koichiro 339 Noda, Noda City, Chiba Prefecture Kikkoman Corporation

Claims (4)

[Claims] 1. An epithelial cell growth promoter containing malonyl isoflavone glycoside as an active ingredient. 2. A skin agent containing malonyl isoflavone glycoside. 3. The dermatological agent according to claim 1 or 2, wherein the malonyl isoflavone glycoside is obtained from soybean or an aqueous extract of soybean. 4. The dermatological agent according to claim 2, which is one of a skin cosmetic agent, a hair-growth agent, a skin external agent and an anti-inflammatory agent.