KR20200028783A - A cosmetic composition for improving wrinkles containing an enzyme-treated soybean embryo extract as an active ingredient and a process for producing the same. - Google Patents

Abstract

The present invention relates to a cosmetic composition for preventing skin aging and wrinkle amelioration. More specifically, disclosed is a method for manufacturing a cosmetic composition which is excellent in the prevention of skin aging and amelioration of wrinkles while being applicable to skin by removing fermentation smell due to microbial fermentation through the bioconversion of a soybean germ extract using an enzyme. To this end, the method for manufacturing a cosmetic composition for ameliorating wrinkles comprising an enzyme-treated soybean germ extract as an active ingredient comprises the following steps; a) adding a phosphate buffer to the soybean germ extract; b) adding an enzyme to the soybean germ extract to which the buffer is added; and c) mixing the soybean germ extract to which the enzyme is added at 35 to 40°C at 100 to 200 rpm for 3 to 27 hours.

Description

A cosmetic composition for improving wrinkles containing an enzyme-treated soybean embryo extract as an active ingredient and a process for producing the same.}

The present invention relates to a cosmetic composition and a method for manufacturing the same, and more particularly, to a cosmetic composition for preventing skin aging and improving wrinkles and a method for manufacturing the same.

Causes of skin aging such as wrinkle formation and elasticity in the skin, endogenous factors such as photoaging and age increase and menopause caused by ultraviolet rays, skeletal and muscle shape, genetic factors caused by muscles' continuous relaxation and contraction, and environmental pollution. It can be classified as environmental factors due to substances and stress. Skin aging caused by these various factors includes wrinkles and skin elasticity decreases due to decreased keratinocytes and fibroblast division, decreased collagen synthesis, increased MMP production and increased melanin signaling. , Spots, freckles and blotch increase. In particular, the hormonal balance is broken around the forties, and the ability to regenerate cells, the ability to synthesize collagen, and the ability to produce skin lipids, which play an important role in moisturizing skin, is also significantly reduced. In addition, during the first 5 years after menopause, collagen decreases by about 30%, and the abnormal entanglement of these collagen fibers increases, resulting in a significant decrease in skin elasticity, hyaluronic acid responsible for moisture in the skin, and glycosaminoglyc, a type of glycoprotein. It has been reported that the amount of cells produced is significantly reduced.

As described above, various methods are applied to improve the skin aging phenomenon of women due to photoaging and natural aging, and typical methods include applying AHA, retinoic acid, retinol and vitamins. However, there are many controversies over skin irritation and clinical efficacy. In addition, hormone replacement therapy alleviates the symptoms of menopause, but if estrogen is used directly, the possibility of causing many side effects, such as breast cancer and cervical cancer, and female hormone itself cannot be legally used in cosmetics, other countries such as Europe Estrogen-like plant ingredients with few side effects are used to relieve menopausal symptoms.

In addition, the most actively researched field for improving skin aging these days is research using plant extracts that have a similar function to biological female hormones. It has been found that most plants have the above-mentioned substances, and especially contains a lot of soybeans, red clover, etc., since they are structurally similar to estrogen and have similar effects on living bodies, they are collectively referred to as 'Phytoestrogen' It is called this.

In particular, the isoflavone and lignan lines are widely studied among various phytoestrogens. However, there have been few studies on the skin of the above components, and so far, it has not been widely used as an external preparation for skin.

On the other hand, Patent Publication No. 10-2018-0057330 discloses a composition used for preventing hair loss by inoculating microorganisms with soybean germ extract, but there is a problem that fermentation odor occurs when using microorganisms.

Therefore, the present invention was devised to solve the above problems, and in the cosmetic composition for preventing skin aging and improving wrinkles, it is possible to remove fermentation odor caused by microbial fermentation and apply to the skin, but also has anti-aging and wrinkle improvement effects. It is intended to provide an excellent cosmetic composition.

In addition, as a cosmetic composition having an excellent effect of preventing skin aging and improving wrinkles, an object of the present invention is to provide an optimal method of preparing a cosmetic composition comprising an enzyme-treated soybean germ extract as an active ingredient.

In order to solve the above problems, the present invention, a) adding a phosphate buffer to the soybean embryo extract; b) adding an enzyme to the soybean germ extract to which the buffer is added; And c) mixing the enzyme-added soybean germ extract at 35-40 ° C. for 100-200 rpm for 3-27 hours; It provides a method for preparing a cosmetic composition for improving wrinkles comprising an enzyme-treated soybean germ extract containing as an active ingredient.

In addition, the extract provides a method for preparing a cosmetic composition for improving wrinkles, characterized in that it is extracted with any one solvent selected from the group consisting of water, alcohol having 1 to 6 carbon atoms, hexane, ethyl acetate and mixed solvents thereof.

In addition, the enzyme provides a cosmetic composition for wrinkle improvement, characterized in that the pectinase.

In addition, the soybean embryo extract provides a cosmetic composition for improving wrinkles, characterized in that 0.01 to 10% by weight based on the total weight of the cosmetic composition.

In addition, it provides a cosmetic composition for improving wrinkles prepared using the above manufacturing method.

According to the present invention, by subjecting the soybean germ extract to enzymatic treatment under specific conditions to bioconvert glycosides to non-glycosides, inhibit fermentation odors that occurred during conventional microbial fermentation and convert glycosides to non-glycosides, improving skin penetration and aging the skin It is possible to provide a cosmetic composition comprising an enzyme-treated soybean germ extract having an excellent effect in preventing and improving wrinkles as an active ingredient.

In addition, as a method for preparing a cosmetic composition having excellent skin anti-aging and wrinkle improvement effects, it is possible to provide an optimal method for preparing a cosmetic composition containing an enzyme-treated soybean germ extract as an active ingredient under specific conditions.

Figure 1 (A) is a quantitative graph of Daidzin measured using HPLC of the soybean germ extract prepared in Preparation Example,
Figure 1 (B) is a quantitative graph of Genistin measured using HPLC of the soybean embryo extract prepared in Preparation Example,
Figure 1 (C) is a quantitative graph of Daidzein measured using HPLC of the soybean embryo extract prepared in Preparation Example,
1 (D) is a quantitative graph of Genistein measured using HPLC of soybean germ extract prepared in Preparation Example,
2 is a quantitative graph of isoflavones of soybean embryo extract measured using HPLC,
3 (A) is a graph showing the survival rate of RAW 264.7 cells of the enzyme-treated soybean embryo extract prepared in Example 1,
Figure 3 (B) is a graph showing the survival rate of B16F10 cells of the enzyme-treated soybean embryo extract prepared in Example 1,
3 (C) is a graph showing the survival rate of HDF cells of the enzyme-treated soybean embryo extract prepared in Example 1,
4 is a graph showing the elastase inhibitory activity of oleanolic acid,
5 is a graph showing the elastase inhibitory activity of the enzyme-treated soybean germ extract prepared in Example 1,
6 is a graph showing the melanin production inhibitory ability of the enzyme-treated soybean germ extract prepared in Example 1,
7 is a graph showing the anti-aging effect of the enzyme-treated soybean germ extract prepared in Example 1,
8 is a graph showing the skin moisture content improvement degree of the composition prepared in the application example,
9 is a graph showing the skin roughness improvement degree of the composition prepared in the application example,
10 is a graph showing the measurement of the degree of wrinkle improvement around the eye area of the composition prepared in the application example.

Hereinafter, the present invention will be described in detail through preferred embodiments. Prior to this, the terms or words used in the present specification and claims should not be construed as being limited to ordinary or lexical meanings, and the inventor appropriately explains the concept of terms to explain his or her invention in the best way. Based on the principle that it can be defined, it should be interpreted as meanings and concepts consistent with the technical spirit of the present invention. Therefore, the configuration of the embodiments described herein is only one of the most preferred embodiments of the present invention, and does not represent all of the technical spirit of the present invention, and various equivalents and modification examples that can replace them at the time of this application It should be understood that there may be. Also, in the specification, when a part “includes” a certain component, it means that the component may further include other components, not exclude other components, unless specifically stated to the contrary.

The present inventors in the cosmetic composition containing an enzyme-treated soybean germ extract that prevents skin aging and improves wrinkles as an active ingredient, removes the fermentation odor that occurred during microbial treatment of the soybean germ extract, and contains it in the soybean germ extract As a result of repeated studies to increase the skin penetration power by converting the glycosides into non-glycosides, when processing enzymes in soybean germ extract under specific conditions, it is possible to convert the glycosides to non-glycosides without using microorganisms, resulting in fermentation As it does not occur, as the glycoside contained in the soybean germ extract is converted into a non-glycoside, the skin penetration power of the soybean germ extract has been improved to discover that it has excellent skin aging prevention and wrinkle improvement effects and has led to the present invention.

Therefore, the present inventors a) adding a phosphate buffer to the soybean embryo extract; b) adding an enzyme to the soybean germ extract to which the buffer is added; And c) mixing the enzyme-added soybean germ extract at 35-40 ° C. for 100-200 rpm for 3-27 hours; Disclosed is a method for preparing a cosmetic composition for wrinkle improvement, comprising an enzyme-treated soybean germ extract comprising as an active ingredient.

The soybean embryo of step a) contains more isoflavones than soybeans, and the isoflavones are phytochemicals that are naturally present in soybeans and are a type of "phytoestrogen". The isoflavones contained in soybeans are also called phytoestrogens because they are structurally similar to the female hormone estrogen and have a similar biological action. The phytoestrogens have so far been found to provide potential alternative therapies for hormone-dependent diseases, including cancer, menopause syndrome, cardiovascular disease and osteoporosis. Chemically, soy isoflavones refer to soybeans, especially flavonoid-based compounds contained in soybeans (bean eyes).

In addition, the isoflavone is a compound having a 3-phenylchrome skeleton, the most well-known isoflavones are genistein, daidzein, formononetin, biochanin A, It is coumestrol, and there are 12 isoflavones in soybeans. The isoflavones of the soybeans are divided into glycosides and aglycones according to their chemical structure. The non-glycosides are three types: genistein, daidzein, and glycitein. , As a glycoside, daidzin and genistin, 7-O-glucosides, in which carbohydrate is attached to carbon 7 of daidzein and genistein, respectively, and carbohydrate to carbon 6 Combined forms of 6'-O-acetylglucosides include 6'-O-acetyl daidzin and 6'-O-acetylgenistin, and 6'-O-malonylglucosides 6'-O-malonyl daidzin and 6'-O-malonyl genistin There is this.

In addition, 7'-O-glucosides, 6'-O-acetylglucosides, and 6'-O-malonylglucosides, which are sugar-coupled glycosides, are converted into non-glycosides, which can be called free isoflavones that have been separated by enzymes. do.

In particular, the non-glycosylated form of removing sugar has much better skin absorbing power than the glycoside, which is a form generally associated with sugar.

<12 types of soy isoflavones of soybeans>

In the present invention, the soybean embryo extract of step a) may be extracted with any one solvent selected from the group consisting of alcohols having 1 to 6 carbon atoms, hexane, ethyl acetate and mixed solvents thereof. The extract of the present invention may be preferably extracted with a solvent selected from water, an alcohol having 1 to 6 carbons and a mixed solvent thereof, or may be a lower alcohol aqueous solution having 1 to 6 carbons. The concentration of the aqueous alcohol solution may be 10 to 100% (v / v), preferably 70 to 100% (v / v). The extraction solvent of the present invention may most preferably be an aqueous ethanol solution of 95% (v / v).

The extraction method in the present invention can be used without limitation as long as it is known in the art, for example, there are methods such as cold immersion, hot water extraction, ultrasonic extraction, reflux cooling extraction, but are not limited thereto.

The supernatant can be filtered with a filter paper (Whatman NO.2) after centrifugation to separate only the extract from the extract prepared by the above method, and the combined filtrate after extraction as above is concentrated under reduced pressure at 40 to 60 ° C in the extract. After removing the remaining solvent, and azeotropically concentrating 2-3 times with water of 25-50 times the weight of the concentrated concentrate under reduced pressure, the same amount of water is added again to homogeneously suspend it, and the final extract may be further purified or frozen. It may be dried by a method such as drying to remove the solvent to solidify or may be prepared in powder form.

In step a), the buffer is a solution in which the hydrogen ion concentration index (pH) hardly changes even with the addition of a strong acid or a strong base. It is used for the purpose of maintaining a constant pH in the present invention. As it is preferably used sodium phosphate buffer (pH 7.0).

In addition, the amount of the phosphate buffer added may be 50 to 250 parts by weight, and preferably 50 to 150 parts by weight, compared to 100 parts by weight of the soybean germ extract.

The enzyme added in step b) generally acts to increase the reaction rate in addition to the reactants in the chemical reaction, and the chemical reaction occurring in the organism is accelerated by the catalyst, especially the role of the catalyst in the organism It is called enzymes and consists of proteins.

In the present invention, the enzyme can convert the glycoside contained in the soybean germ extract to the non-glycoside. The non-glycoside has a higher skin penetration power than the glycoside, and thus can be well absorbed by the skin, and has an excellent effect on preventing skin aging and improving wrinkles.

In the present invention, as the enzyme, pectinase may be preferably used, and pectinase may be added to 1-100 µl compared to 1 mg of the soybean embryo extract, preferably 5-50 µl may be added, , Most preferably 5-15 μl may be added.

The pectinase is an enzyme that hydrolyzes pectin, and in the form of action of the enzyme, it is decomposed sequentially in the end group pectinase that produces oligosaccharides by randomly decomposing from the inside of α-1,4 bonds and end groups. It can be classified as an exo-type pectinase producing monosaccharides, the former being liquefied, and the latter being glycated.

In addition, the pectinase bioconverts the isoflavone glycoside contained in the soybean germ extract into an isoflavone non-glycoside in the mixing step described later, so that the isoflavone penetrates the skin better.

In addition, when the amount of the enzyme added in step b) is less than 1 µl, the efficiency of converting the glycoside contained in the soybean germ extract into a non-glycoside may decrease, and when the amount of the addition exceeds 100 µl, The conversion efficiency to non-glycosides may be the same and may not be economical.

In the step c), the enzyme-treated soybean embryo extract is preferably mixed at 35 to 40 ° C, preferably at 36 to 38 ° C, at 100 to 200 rpm, preferably at 130 to 170 rpm, for 3 to 27 hours, preferably 5 to 15 hours.

When the mixture is mixed, the glycoside contained in the soybean embryo extract is bioconverted to a non-glycoside, and when the mixing temperature is less than 35 ° C or exceeds 40 ° C, the activity of the enzyme is lowered so that the amount of the glycoside converted to the non-glycoside is It can be very reduced.

In addition, when the mixing time is less than 3 hours, a large amount of glycosides may not be converted into non-glycosides, so that the amount of non-glycosides may decrease, and when it exceeds 27 hours, the converted non-glycosides may be converted into glycosides again. The amount of glycosides may decrease.

In the present invention, the soybean embryo extract may be 0.01 to 10% by weight relative to the entire composition, preferably 0.05 to 5% by weight, and most preferably 0.1 to 3% by weight.

If the soybean germ extract is less than 0.01% by weight compared to the total composition, the effect of preventing skin aging and improving wrinkles is negligible, and when it exceeds 10% by weight, the effect is the same, which is not economical.

In another aspect, the present invention provides a cosmetic composition for improving wrinkles manufactured using the above-described manufacturing method.

The cosmetic composition containing the enzyme-treated soybean germ extract according to the present invention as an active ingredient may preferably be formulated into a cosmetic composition for preventing skin wrinkles, enhancing skin elasticity, and moisturizing the skin. The cosmetic composition of the present invention is not particularly limited in its formulation, and may be, for example, a cosmetic composition having a formulation of softening makeup, nutritional makeup, massage cream, nutrition cream, pack, gel, or skin adhesion type cosmetic, It may also be a transdermal dosage form such as lotion, ointment, gel, cream, patch or spray.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Manufacturing example  : Soybean germ extract

Pour 500 g of distilled water into 200 g of soybean germ (Cosmos Co., Ltd.) and boil until the water is almost gone. 2 g of 70 (v / v)% ethanol is added to 200 g of boiled soybean embryos, and extracted at room temperature for 3 days. After extraction, to separate only the extract, centrifugation is performed at 8000 rpm for 20 minutes, and then the supernatant is filtered with filter paper (Whatman NO.2). After filtration, vacuum decompression is performed to remove ethanol. After vacuum decompression, freeze-drying was performed to obtain a powdered isoflavone-enriched soybean embryo extract.

Example 1

After dissolving 5 mg of isoflavone-enriched soybean germ extract obtained in Preparation Example in 5 ml of sodium phosphate 100 mM (pH7.0), 50 µl of pectinase (Novozyme, Pectinex Ultra SP-L 10,000 PECTU / ml) was added and 37 ℃ At 150 rpm for 7 hours, the mixture was bioconverted to prepare an enzyme-treated soybean embryo extract.

Example 2

Soybean embryo extract was prepared in the same manner as in Example 1, except that the reaction time in Example 1 was changed to 5 hours.

Example 3

Soybean embryo extract was prepared in the same manner as in Example 1, except that the reaction time in Example 1 was changed to 24 hours.

Comparative example  One

Soybean embryo extract was prepared in the same manner as in Example 1, except that the reaction time in Example 1 was changed to 0 hours.

Comparative example  2

Soybean embryo extract was prepared in the same manner as in Example 1, except that the reaction time in Example 1 was changed to 1 hour.

Comparative example  3

Soybean embryo extract was prepared in the same manner as in Example 1, except that the mixing temperature in Example 1 was changed to 30 ° C.

Comparative example  4

Soybean embryo extract was prepared in the same manner as in Example 1, except that the mixing temperature in Example 1 was changed to 45 ° C.

Comparative example  5

Soybean embryo extract was prepared in the same manner as in Example 1, except that the mixing temperature in Example 1 was changed to 50 ° C.

Comparative example  6

Soybean embryo extract was prepared in the same manner as in Example 1, except that the enzyme was not used in Example 1.

Application example

After freeze-drying the soybean germ extract prepared in Example 1, a gel-type composition was prepared by adding 1% by weight relative to the total composition.

Experimental Example 1

After taking a sample to confirm the isoflavone content of the soybean embryo extract prepared in the above preparation example, after dissolving 1 mg in 1 ml of dimethyl sulfoxide (DMSO), PVDF SYRINGE FILTER (13mm, 0.2㎛, 100units / Whatman Cat No .. 6779 1302) was filtered to prepare HPLC samples. The isoflavone content of the sample prepared according to the following experimental method was measured. The results are shown in Table 1, FIGS. 1 and 2, FIG. 1 (A) is a quantitative graph of Daidzin measured using HPLC, and FIG. 1 (B) shows Genistin measured using HPLC Quantitative graph, Figure 1 (C) is a quantitative graph of Daidzein measured using HPLC, Figure 1 (D) is a quantitative graph of Genistein measured using HPLC, Figure 2 is measured using HPLC This is a quantitative graph of isoflavones from one soybean embryo extract.

<Experiment Method>

For the analysis of the sample, an Agilent Eclipse XDB-C18 column (5 µm, 4.6 × 250 mm) was used using a YL9100 HPLC system from Younglyn (Korea), and the column temperature was maintained at 25 ° C. As the mobile phase, distilled water containing 2% by weight acetic acid (HPLC grade, solvent A) and 100% by weight acetonitrile (HPLC grade, solvent B) were used. After starting solvent A at 95% by weight and lowering it to 45.4% by weight for 21 minutes. It is maintained at 45.5% by weight for 10 minutes and then raised to 95% by weight for 5 minutes. The sample injection volume was 5 µl, the flow rate of the mobile phase was 1 ml / min, and absorbance was measured at a wavelength of 262 nm. As a standard for isoflavone analysis, daidzin, genistin, daidzein, and genistein were used, and all of the standards were Sigma (Sigma Chemical, USA) products.

Referring to Table 1, FIGS. 1 and 2, the contents of the isoflavones Daidzin, Genistin, Daidzein, and Genistein can be seen, and the dividend chains Daidzin (8.12% by weight) and Genistin (4.8% by weight) are non-dividend chains Daidzein (2.07). Weight%) and Genistein (0.69% by weight).

Experimental Example 2

In order to measure the isoflavone content of the enzyme-treated soybean germ extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 6, each sample was taken, dissolved in 1 ml of dimethyl sulfoxide (DMSO) 1 mg each, and then PVDF SYRINGE An HPLC sample was prepared by filtering with a filter (13mm, 0.2㎛, 100units / Whatman Cat No. 6779 1302). The isoflavone content of each sample was measured in the same manner as in Experimental Example 1, and the results are shown in Table 2.

As a standard for isoflavone analysis, daidzin, genistin, daidzein, and genistein were used, and all of the standards were Sigma (Sigma Chemical, USA) products.

Referring to Table 2, when the enzyme pectidase was added to the soybean embryo extract and mixed at 37 ° C for a specific time (7, 5, 24 hours) (Examples 1 to 3), the glycosides Daidzin and Genistin were It can be seen that the amounts converted to Daidzein and Genistein, which are non-dividends, are very large, but when the mixing time (0, 1 hour) is different (Comparative Example 1,2), the amount of the glycosides converted to non-glycoside is Example 1 It can be seen that it is lower than when compared to the third. In addition, even when the temperature (30 ° C, 45 ° C, 50 ° C) is different (Comparative Examples 3, 4, 5), it was found that the amount of glycoside conversion to non-glycoside is lower than that of Examples 1 to 3. You can. And, when the enzyme is not used (Comparative Example 6), it can be seen that the glycoside is hardly converted into the non-glycoside.

Experimental Example 3

In order to measure the cytotoxicity of the enzyme-treated soybean embryo extract prepared in Example 1, an experiment was conducted as follows, and the results are shown in FIG. 3. Figure 3 (A) is a graph showing the survival rate of RAW 264.7 cells, Figure 3 (B) is a graph showing the survival rate of B16F10 cells, Figure 3 (C) is a graph showing the survival rate of HDF cells.

<Experiment Method>

After adjusting the concentrations of RAW 264.7 cells, B16F10 cells, and HDF (human dermal fibroblast) cells pre-sold by the Korea Cell Line Bank (KCLB) to 3 × 10⁴ cells / well, the cells were dispensed into 96-well plates for 24 hours at 37 ° C., 5 Incubate in weight% CO ₂ incubator. After preparing samples at 5, 10, 20, 50 and 100 µg / ml, the culture solution is removed. After removal, the enzyme-treated soybean embryo extract prepared in Example 1 was replaced with a dissolved culture medium, and then cultured in a 37 ° C, 5%, CO ₂ incubator for 48 hours. After preparing a 1% by weight NR serum-free medium using a Neutral red (NR) solution, replace it with an existing culture medium, incubate for 3 hours, and then replace with a 10% by weight formaldehyde cell fixation solution to fix the cells for 20 minutes. 100 μl of NR desorb solution (1% glacial acetic acid, 49% ethanol, 50% distilled water) was dispensed to extract NR, and absorbance was measured at 540 nm with a Microplate reader (BIO-TEK, Synergy-HT, USA).

Referring to Figure 3, the enzyme-treated soybean germ extract prepared in Example 1 can be seen that the cell viability of more than 90% at all concentrations in each cell, in the case of HDF cells, cell viability as the concentration increases You can see that it increases. Therefore, in the case of the enzyme-treated soybean embryo extract of Example 1, since there was little cytotoxicity in each cell, it can be understood as safe when used on the skin.

Experimental Example 4

In order to measure the Elastase inhibitory activity of the enzyme-treated soybean germ extract and oleanolic acid (Sigma Aldrich) prepared in Example 1, experiments were carried out in the following experimental method, and the results were analyzed by the following analytical method. 3, 4 and 4 and 5. Table 3 is a table showing the elastase inhibitory activity of oleanolic acid, Table 4 is a table showing the elastase inhibitory activity of the sample of Example 1, Figure 4 is a table showing the elastase inhibitory activity of oleanolic acid, Figure 5 is an example Table 1 shows the elastase inhibitory activity of the sample.

<Experiment Method>

Each test substance was prepared for each concentration, and 20 µl was taken into an epp.tube, and 100 µl of 1.6 mM N-succinyl- (Ala) 3-p-nitroanilide and 20 µl of 0.4 U / ml porcine pancreas elastase solution as a substrate, 10 µM tris- After adding 60 μl of HCl buffer (pH 8.0) one after another to react at room temperature for 5 minutes, absorbance was measured at 410 nm to determine the elastase inhibition rate.

Elastase inhibition (%) = 100- (absorbance in the sample-added group / absorbance in the non-added group) × 100

<Analysis method>

Statistical significance of all experimental results was confirmed by using the t-test (paired t-test) function of Microsoft Office Excel 2007, and it was judged to be statistically significant when the p value was less than 0.05.

Referring to Tables 3 and 4, FIGS. 4 and 5, a test result confirming the elastase inhibitory activity using oleanolic acid known to have antioxidant activity as a positive control, was concentration-dependent in the test concentration range, and was statistically significant (p <0.05). It showed a level of elastase inhibitory activity (IC ₅₀ : 12.06 µg / ml), which confirmed that there was no problem in this test procedure.

Based on this, the enzyme-treated soybean embryo extract prepared in Example 1 could not confirm IC ₅₀ , but was concentration-dependent in the concentration range of 12.5 to 800 μg / ml, and was statistically significant (p <0.05). It was confirmed that it exhibits elastase inhibitory activity. In particular, at a concentration of 800 μg / ml, elastase inhibitory activity of 38.72% was shown.

Experimental Example 5

In order to measure the ability of the enzyme-treated soybean germ extract prepared in Example 1 to inhibit melanocyte production, the experiment was conducted three times, and the results are shown in FIG. 6 (* p <0.05, ** p <0.01). , *** p <0.001). All experimental results were expressed as mean ± standard deviation (Mean ± SD), and statistical processing was analyzed using SPSS Window Version 17.0 (SPSS Inc., Illinois, USA), and the significance test was conducted by Student's t-test. It was determined that there was a statistically significant difference when the P value was less than 0.05.

<Experiment Method>

In order to measure the change in absorbance according to the relative degree of biosynthesis of melanin pigment in cells, B16F10 cells were cultured in a 96-well plate at a concentration of 2 × 10³ cells / well for 24 hours at 37 ° C. and incubated in a 5% by weight CO ₂ incubator. In order to induce melanin biosynthesis, the enzyme-treated soybean germ extract prepared in Example 1 was added at a concentration of 5, 10, 20, 50, and 100 µg / ml, respectively, to a culture solution containing 5% by weight of serum and 10 nM of α-MSH. After replacing with the existing culture medium and incubated in a 5% by weight CO ₂ incubator at 37 ° C. for 72 hours, absorbance was measured at 405 nm. As a positive control, arbutin (Arbutin, Sigma Aldrich) was treated at concentrations of 100, 200, and 300 µg / ml to measure absorbance in the same manner, and the enzyme-treated soybean embryo extract prepared in Example 1 was added as a control (con). Absorbance was measured in the same manner using the uncultured medium.

α-MSH induces melanin pigment synthesis by increasing tyrosinase mRNA transcription and tyrosinase synthesis and activity in melanocytes in the skin, so measuring the degree of biosynthesis of melanin by adding α-MSH confirms the effect on the sample's anti-melanin reaction Did

Referring to FIG. 6, as a result of the experiment, arbutin treated as a positive control significantly inhibited relative melanin biosynthesis at a concentration of 200 μg / ml by 24.78%, and the enzyme-treated soybean embryo extract of Example 1 was significantly melanin-dependent in concentration. When biosynthesis was inhibited and the concentration was 100 μg / ml, the melanin biosynthesis inhibition rate was 26.09%.

Experimental Example 6

In order to measure the ability to inhibit MMP-1 expression of the enzyme-treated soybean germ extract prepared in Example 1, experiments were conducted three times according to the following experimental method, and the results are shown in FIG. 7 (* p <0.05, ** p <0.01, *** p <0.001). The experimental results were analyzed in the same manner as in Experimental Example 5.

<Experiment Method>

HDF cells were incubated in a 96 well plate at a concentration of 3 x 10 mm 3 cell / well and incubated in a 5% by weight CO ₂ incubator at 37 ° C. for 24 hours. After adding the enzyme-treated soybean germ extract prepared in Example 1 at concentrations of 5, 10, 20, 50, and 100 μg / ml to the new culture medium, respectively, and replacing them with the existing culture solution, UVB (ultraviolet rays) (100 mJ / cm²) was added. After irradiation for a minute, incubation was carried out for 24 hours at 37 ° C in a 5% by weight CO ₂ incubator. 100 µl of the culture supernatant and 100 µl of the coating buffer were mixed, transferred to a new 96-well plate, and stored frozen for 24 hours. After coating, after washing with PBS-T (0.05% by weight Tween-20 in PBS), 100 μl of blocking buffer (0.1% by weight BSA in PBS) is added and reacted at 37 ° C. for 1 hour. After blocking, washed with PBS-T, treated with 50 μl of primary antibody (anti-MMP-1 mouse antibody, Sigma, diluted 1000-fold) and reacted at 37 ° C. for 1 hour. After washing with PBS-T, alkaline phosphatase-conjugated secondary antibody (anti-mouse IgG antibody, Sigma, 4000-fold dilution) was treated with 50 µl each and then left at 37 ° C. for 1 hour. After washing with PBS-T, 200 μl of p-nitrophenyl phosphate (9.7 wt% diethanolamine buffer, 0.5 mM MgCl ₂ , pH 9.8) was treated, reacted at 37 ° C. for 1 hour in the dark, and absorbance was measured at 405 nm. Absorbance was measured in the same manner using a culture solution to which the enzyme-treated soy embryo extract was not added as a control (con).

MMP-1, also known as collagenase, is primarily expressed during physiological and pathological tissue remodeling in vivo. It has been reported that UV is induced by stimulating the MMP-1 Promoter region.

Referring to FIG. 7, the experimental results showed that the expression level of MMP-1 in the fibroblast culture medium was increased by UVB, and the expression level of MMP-1 was significantly increased by 84.32%, 76.26%, 64.29%, 58.67% and 49.36% depending on the extract concentration. It was decreased and it was confirmed that the MMP-1 expression level recovered to the control level. Therefore, it can be seen that the enzyme-treated soybean embryo extract of Example 1 has an anti-aging effect.

Experimental Example 7

In order to measure the skin moisture content of the composition prepared in the above application example, a total of three skin moisture contents were measured while self-using the composition on the face once a day for 4 weeks to 31 adults and men and women, and indoors to obtain accurate data Moisture content was measured using a Corneometer CM 825 (Courage & Khazaka, Germany) instrument prepared at a constant time in an indoor environment condition maintaining a temperature of 22 ~ 24 ℃ and an indoor humidity of 40-60%. It is shown in FIG. 8. At the time of measurement, the lower part of both cheekbones was repeatedly measured three times. The measurement results were analyzed in the same manner as in Experimental Example 5.

Referring to Table 5 and FIG. 8, the moisture content of the stratum corneum layer increased to a significant level from 2 weeks after the test in both regions, and 16.59% and 25.63% improved after 2 weeks and 4 weeks, respectively, in the left region and in the right region After 2 and 4 weeks, the improvement was 19.98% and 29.30%, respectively.

Experimental Example  8

In order to measure the improvement of the skin roughness of the composition prepared in the above application example, 31 adult men and women were evaluated by using the composition on their face once a day for 4 weeks in the evening, to evaluate the degree of skin roughness 3 times in order to obtain accurate data Moisture content was measured using an Antera 3D (Miravex, Ireland) device prepared at a constant time in an indoor environment that maintains an indoor temperature of 22 ~ 24 ℃ and an indoor humidity of 40-60%, and the results are shown in Tables 6 and 9 below. It is shown in. At the time of measurement, the entire cheek was repeatedly measured three times. The measurement results were analyzed in the same manner as in Experimental Example 5.

Referring to Tables 6 and 9, in the experiment results, the skin roughness index decreased significantly after 2 weeks of testing in both regions, and 9.71% and 16.26% improved after 2 weeks and 4 weeks, respectively, in the left region and in the right region In the case of 2 weeks and 4 weeks, the improvement was 7.71% and 15.36%, respectively.

Experimental Example  9

In order to measure the improvement of the eye wrinkles of the composition prepared in the above application example, the skin roughness of the skin was evaluated 3 times while using the composition on the face once a day for 4 weeks to 31 adults and men and women to obtain accurate data. The moisture content was measured using an Antera 3D (Miravex, Ireland) device prepared at a constant time in an indoor environment condition that maintains an indoor temperature of 22 to 24 ° C and an indoor humidity of 40 to 60%, and the results are shown in Tables 7 and 10 below. It is shown in. At the time of measurement, the sides of both eyes were repeatedly measured three times. The measurement results were analyzed in the same manner as in Experimental Example 5.

Referring to Tables 7 and 10, the results of the experiment showed that in the case of the right eye area, the wrinkle index decreased to a significant level from 2 weeks after the test, and in the case of the left eye area, the wrinkle index decreased although it was not significant. . In the case of left eye wrinkles, the improvement was 4.64% and 10.13%, respectively, after 2 and 4 weeks, and in the case of right eye wrinkles, 3.28% and 7.58%, respectively, after 2 and 4 weeks.

Experimental Example  10

Satisfaction was evaluated for the subjective skin improvement effect of 31 subjects who self-used the composition prepared in the preparation example once a day in the evening, and the results are shown in Table 8 below.

Referring to Table 8 above, it can be seen that most of the response rate is 80% or more, and it can be seen that most of the subjects judged that the skin condition was improved.

As described above, by controlling the mixing conditions of the enzyme and soybean embryo extract and enzyme according to the present invention, bio-conversion of a large amount of isoflavone glycoside contained in soybean embryo extract to non-glycoside prevents skin aging suitable for cosmetic compositions and improves wrinkles , It is possible to provide a cosmetic composition comprising an enzyme-treated soybean germ extract showing an effect of improving skin moisturizing and skin roughness as an active ingredient and a method for manufacturing the same.

It has been described in detail with reference to the production examples, experimental examples and drawings of the present invention. The description of the present invention is for illustration only, and those skilled in the art to which the present invention pertains will understand that it is possible to easily modify to other specific forms without changing the technical spirit or essential features of the present invention.

Accordingly, the scope of the present invention is indicated by the claims, which will be described later, rather than by the detailed description, and all the modified or modified forms derived from the meaning, scope, and equivalent concepts of the claims are included in the scope of the present invention. Should be interpreted.

Claims (5)

a) adding phosphate buffer to the soybean embryo extract;
b) adding an enzyme to the soybean germ extract to which the buffer is added; And
c) mixing the enzyme added soybean germ extract at 35-40 ° C. for 100-200 rpm for 3-27 hours;
Method for preparing a cosmetic composition for improving wrinkles comprising an enzyme-treated soybean germ extract comprising as an active ingredient. According to claim 1,
The extract is water, 1 to 6 carbon atoms, alcohol, hexane, ethyl acetate and a method for producing a cosmetic composition for improving wrinkles, characterized in that extracted with any one solvent selected from the group consisting of mixed solvents thereof. According to claim 1,
The enzyme is pectinase, characterized in that the cosmetic composition for wrinkle improvement. According to claim 1,
The soybean embryo extract is a cosmetic composition for improving wrinkles, characterized in that 0.01 to 10% by weight relative to the total weight of the cosmetic composition. A cosmetic composition for improving wrinkles prepared by using the method of any one of claims 1 to 4.

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