JP3302535B2 - Epithelial cell growth promoter - Google Patents

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 skin preparation 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 have been discovered, and a skin cosmetic for preventing skin aging and a skin external preparation for shortening the wound healing period of skin have been developed using these growth factors.
For example, skin cosmetics containing glucosaminoglycan and fibroblast growth factor (JP-A-4-187613), α-
Skin cosmetics and skin external preparations containing hydroxyacetic acid (JP-A-5-112422), skin aging preventive cosmetics containing extracts of birch plants (JP-A-6-26)
3627) and the like.

[0003] On the other hand, isoflavone compounds of soybean are known to have various physiological actions such as estrogen action, antioxidant action, antihemolytic action, anticholesterol action, antilipidemic action and antibacterial action. Patent applications have been filed for skin cosmetics and the like utilizing the action, for example, whitening cosmetics containing soy isoflavone compounds (Japanese Patent Publication No.
19885), a soybean saponin-containing cosmetic composition (JP-A-59-106419), a humectant containing an aqueous extract of soybean hypocotyl as an active ingredient (JP-A-63-2443013), and the like are known. Also, recently, the presence of malonyl isoflavone glycosides such as malonyl daidzin and malonyl genistin in soybeans has been confirmed, and it has been found that these are the main components of the isoflavone compounds in soybeans. 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 have previously found that an aqueous extract of soybean itself is effective as an external preparation for the skin, and filed a patent application (Japanese Patent Application No. 6-305697). The water extract contains malonyl isoflavone glycosides, and the knowledge that malonyl isoflavone glycosides can be easily obtained by contacting the extract with an adsorbent and then eluting with an aqueous alcohol solution, A patent application was filed (Japanese Patent Application No. 7-112705).
Further, they have found that this malonyl isoflavone glycoside is effective for the proliferation of epithelial cells.

[0005]

The present invention has been made based on such findings, and is a skin preparation 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 Malonylgenistin

Embedded image The content of malonyl isoflavone glycosides, etc. depends on the type of soybeans, harvest time, etc.
According to od Chem., 42, 1674 (1994), Table 1 shows the results.
The content is microgram per 1 g of soybean. Table 1 Soybean (FY) Malonyl daidzin Malonilgenistin 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 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

[0007] As a method for producing malonyl isoflavone glycoside from soybean, as described in JP-A-3-170495, ground soybeans are extracted with alcohol, and the extract is then subjected to several stages of water-immiscible organic compounds. Although a method of producing by extraction with a solvent is known, a method of obtaining from an aqueous extract of soybean as described below is preferable from the viewpoint of easiness of the production method and yield.

That is, raw materials for obtaining malonyl isoflavone glycosides include whole soybeans, dehulled soybeans, defatted soybeans and the like, and an aqueous extract thereof is preferably used. For example, whole soybeans, dehulled soybeans or defatted soybeans are added to water at 20-80 ° C for 1
It is an extract obtained by immersing for up to 30 hours, and among them, dehulled soybeans and defatted soybeans are preferred. Needless to say, soybean immersion waste liquid generated during the production of soy sauce, miso, tofu, natto, soy milk, or sprouts can also be used effectively. Whey produced in the production of soy broth or cotton tofu can also be used. As a preferred embodiment for obtaining an aqueous extract of soybeans, a dehulled soybean is adjusted to a pH of 10.0 or less, preferably 7.5 to 9.0 with caustic soda or the like, at 45 to 65 ° C.
Immersed in water for 2 to 4 hours, and the immersion water obtained by removing the immersed soybeans is used as a water extract.

In this case, when the pH of the immersion water is set to 10.0 or more, the yield of malonyl isoflavone glycoside decreases. This is because malonyl isoflavone glycoside is decomposed into isoflavone glycoside. In order to increase the extraction rate of the soybean component, it is advantageous that the immersion temperature is higher.
At 0 ° C. or higher, malonyl isoflavone glycosides begin to decompose. Therefore, considering the extraction rate and decomposition rate, 4
5-65 ° C is suitable.

When defatted soybeans are used, low-denatured defatted soybeans are suitable as the defatted soybeans.
Extract or extract the ground material of low-denatured defatted soybeans with water or alkaline water, remove the insoluble residue, and extract the extract with hydrochloric acid.
A solution from which isolated soybean protein obtained by acid precipitation near 4.3 is removed, that is, soybean whey may be used.

If necessary, such a soybean extract is subjected to filtration using an ultrafiltration membrane to remove proteins, or the pH is adjusted to about 4.3 with hydrochloric acid as in the case of the above-mentioned soybean whey, and dissolved in the extract. The protein is precipitated and the supernatant is contacted with the adsorbent. The filtrate or the supernatant may be brought into contact with the adsorbent as it is, or the filtrate or the supernatant may be adjusted to about pH 8.0 with caustic soda and then brought into contact.

In the former case, the amount of adsorption to the adsorbent is increased, and in the latter case, the malonyl isoflavone glycoside, the isoflavone glycoside and the aglycone are easily separated. In any case, the adsorbent used is, for example, a synthetic adsorbent, activated carbon, alumina, etc. Specifically, Diaion HP-20 (manufactured by Mitsubishi Chemical), purified Shirasagi activated carbon (manufactured by Takeda Pharmaceutical), activated alumina (Made by Wako Pure Chemical Industries). The contact may be performed by a general method such as a batch method or a column method.For example, the contact can be performed by passing the extract through a column filled with an adsorbent, whereby most of the malonyl isoflavone glycoside in the extract can be obtained. Is adsorbed on the adsorbent.

Next, the malonyl isoflavone glycoside adsorbed on the adsorbent is eluted using an aqueous alcohol solution or an aqueous alkaline alcohol solution. The resulting 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-mentioned 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, the concentrated solution is passed through a column filled with an ODS resin (manufactured by Yamamura Chemical) to adsorb malonyl isoflavone glycoside, and eluted with an aqueous alcohol solution to fractionate malonyl daidin and malonyl genistin,
These fractions are concentrated under reduced pressure and freeze-dried to obtain a dry powder of malonyl daidzin and malonyl genistin.

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

The malonyl isoflavone glycoside thus obtained has an effect of proliferating epithelial cells, and is used in combination with various commonly used bases such as oils, surfactants,
Formulated with humectants, alcohols, thickeners, fragrances, antioxidants, chelating agents, pigments, preservatives, etc., to promote epithelial cell growth, skin cosmetics, hair growth agents, skin external preparations, anti-inflammatory agents, etc. Skin preparation. The amounts of malonyl daidzin and malonyl genistin are 0.001 to 100 in the case of 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 studies on the effect of malonyl isoflavone glycoside on proliferating epithelial cells will be described. The cells used were normal human skin epithelial cells (No.
rmal human epidermal keratinocyte (NHEK) (Kurashiki Spinning Co., Ltd.).

Medium used: Human modified epidermal growth factor (hEGF) was added to a modified MCDB153 medium (Kurashiki Boseki Co., Ltd.). 1 ng / ml, insulin 5 μg / ml, hydrocortisone 0.5 μg / ml, gentamicin 50 μg / ml as an antibacterial agent, and amphotensin 0.0
5 μg / ml was added, and the medium for growing epidermal keratinocytes (K-
DM).

Culture method: Normal human skin epithelial cells (NHEK)
Is suspended in the above K-DM medium at a concentration of 5000 cells / ml, and is 48-well plate (manufactured by Sumitomo Bakelite).
Is inoculated at a concentration of 0.3 ml / well.
% CO ₂ and 95% air atmosphere, 37 ° C, humidity 100
Incubate for 4 hours in a% incubator. After the culture, 10 μl of the sample solution shown in Table 2 was added, and after further culturing for 72 hours, K-DM was added.
Remove the medium and add fresh 0.3 ml K-DM medium.
/ Well and the sample were added again, cultured for 72 hours, and the proliferation of the 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-
A color reaction was performed using 2H-tetrazolium, monosodium salt 反 応, and the absorbance (OD) at a wavelength of *** was measured with a spectrophotometer. In this measurement method, the absorbance and the number of cells have a linear proportional relationship. Since Comparative Examples 2 to 4 are hardly soluble in water, the experiments were performed at a concentration of the solubility limit.

Table 2 shows the above results. Table 2 Sample concentration (ppm) OD value Significance test Malonyldidine 3 × 10 ⁻⁵ 0.371 * (in the present 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 * (the present 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 Daizin 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 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): Indicates the concentration in the cell culture solution OD value: Average value of 5 repetition tests. Significance test *: Significant difference at 5% risk ratio **: Significant difference at 1% risk ratio In addition, the proliferation activity of water (control) was set to 1, and the proliferation activity of each sample was shown in FIG. Indicated.

As is clear from Table 2 and FIG. 1, the samples to which malonyl isoflavone glycoside malonyl daidzin and malonyl genistin were added in an amount of 3 × 10 ^{-5 to} 3 × 10 ^-1 ppm showed a human epithelial cell proliferation effect. In particular, malonyl daidzin is added in the range of 3 × 10 ^{−5 to} 3 × 10 ⁻² ppm, and malonylgenistin is in the range of 3 × 10 ^{−5 to} 3 × 10 ⁻³ ppm.
A significant effect was observed.

In addition, daidzin, genistin, daidzein, and genistein which are considered to have a physiologically active action (comparative 1 to
In 4), the effects of daidzin and genistin were recognized but not significant, and the effects of daidzein and genistein were hardly observed, and inhibition was observed at a high concentration.

[0024]

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

<Skin Cosmetic> Malonyldaidine-Na 1 Malonylgenistin-Na 1 Ethanol 80 Glycerin 45 Polyoxyethylene hydrogenated castor oil 5 Fragrance 1 Purified water 867 These were mixed to prepare a skin cosmetic.

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

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

<Use example 1> The above <Skin cosmetic>
Twenty women in their 0s were allowed to use them for one month, and a questionnaire survey showed that 15 of the panel responded that they were effective for skin abrasion, and 10 of them were effective for preventing wrinkles. The other five respondents also stated that their skin was moist.

<Use Example 2> 20 to 20 of rough skin (dry skin)
For 10 months, 10 women in their 40s applied the above <External Skin Preparation> to the right hand and ointment without malonyl glycoside as a control to the left hand every day before going to bed for 1 month. As a result, the right hand became smoother and smoother, and the healing effect of the rough skin was recognized.

<Use Example 3> Ten men in their 50s used the above <hair restorer> on their scalp after washing their hair for one month, and six of them showed gloss and reduced hair loss. Admitted.

[0031]

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 proliferation, thereby preventing skin aging and improving skin roughness. Skin cosmetics, anti-inflammatory agents,
An external preparation for skin and the like useful as a wound treatment agent and the like can be provided.

[Brief description of the drawings]

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

──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Nobuyuki Yamaji 339 Noda, Noda City, Chiba Prefecture Kikkoman Corporation (72) Inventor Koichiro Tobe 339 Noda Noda City, Chiba Prefecture Examiner, Kikkoman Corporation Tsurumi Hideki (56) Reference JP-A-3-170495 (JP, A) ACS Symposium Series, 1992, no. 507, pp. 98-113 ACS Symposium Series, 1994, no. 546, pp. 330- 339 (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 35/78 A61K 7/00 A61K 31/70 BIOSIS (DIALOG) CA (STN) MEDLINE (STN)

Claims (1)

(57) [Claims] 1. An epithelial cell growth promoter comprising malonyl isoflavone glycoside as an active ingredient.