KR101205031B1 - Improvement material for hair care and white hair preventive using soybean methanol - Google Patents

Abstract

The present invention relates to a hair growth prevention and hair growth prevention agent using soybean methanol to provide a functional material that is excellent for hair growth and prevent white hair,
Soybeans were dried at room temperature for 5 days, then pulverized, and 20 ~ 30% of water was added to methanol and immersed at 50 ℃ for 3 hours. Freeze-dried, and the freeze-dried sample was dissolved in 2% concentration in a solvent to obtain a liquid soy methanol extract, and the solvent, ethanol and water were mixed with the soy methanol extract to obtain hair growth and white hair prevention improving agent to obtain hair essence and The feature is that it can be used together.

Description

IMPROVEMENT MATERIAL FOR HAIR CARE AND WHITE HAIR PREVENTIVE USING SOYBEAN METHANOL}

The present invention relates to a preparation for improving hair growth and white hair prevention using soybean methanol, and more particularly, to improve hair growth while preventing hair loss or white hair caused by a lack of melanin pigment, thereby improving healthy hair. It is about the provision of improved sanctions that can be utilized.

The color of human hair or skin depends on the amount of melanin present in the hair or skin, and melanin is dopa- tion from tyrosine (DOPA) in pigmented cells (melanosites) in the hair follicles and epidermal basal layer. Biosynthesis by catalysis of tyrosinase via

Human skin color is determined by pigment components such as melanin and carotene, blood vessel distribution, and thickness of the stratum corneum. Among them, melanin is the most decisive factor. When abnormality occurs in melanogenesis, hyperpigmentation such as blemishes and freckles occurs.

Unlike skin, hair is known to have white hair or hair loss due to genetic factors or external factors such as stress.

The color of the hair appears in the hair when the melanocytes, the pigment cells in the hair roots, supply the pigments to the melanocytes in the hair follicles.

Existing patents related to white hair prevention include melanin synthesis promoter and external skin preparation (JP, Publication No. 2007-008888), dietary supplement composition for improving white hair and dietary supplement containing the same (KR, Publication No. 2005-0109345), melanin production promoter And a composition for promoting the production of melanin (JP, Publication No. 2004-345959), hair-healing aid based on Chinese herbal medicines (KR, Publication No. 2002-0095855), hair cosmetic composition for inhibiting the production of gray hair, containing dandelion extract (KR, publication) Most of them focus on substances that only increase melanocyte synthesis of melanocytes, such as number 2002-0068589).

In addition, the screening method of anti-harmonic substances was mainly limited to the method of measuring the activity of tyrosinase that increases melanin synthesis, quantifying the amount of melanin produced or visually determining the melanocyte activity. (JP, Publication No. 2002-212039 et al.).

A patent published last year (Japanese Patent Application, Publication No. 2006-028143) selected MITF as an indicator for screening anti-hairs and measured changes in the expression of MITF genes by real-time PCR. One of the changes in messenger RNA (mRNA) in melanocytes has a limitation in vitro screening method to find anti-white hair.

Another patent (Korean Patent Application, Publication No. 2006-0134302) conducted a mouse model and human application experiments to induce the white hair to evaluate the anti-white hair, but the effect of suppressing white hair is evaluated by summing the scores according to visual evaluation. There is a problem in that it cannot have objectivity.

Therefore, the present invention was invented to solve the above problems, soybeans were dried at room temperature for 5 days and then pulverized, and the sample was immersed in methanol for 20 to 30% and immersed for 3 hours at 50 ° C with the solution in a tube. After concentrating under reduced pressure at 45 ℃ and stored at low temperature, freeze-dried samples were lyophilized, and the freeze-dried sample was dissolved in a solvent at 2% concentration to obtain a liquid soy methanol extract, the solvent and ethanol and soybean methanol extract By mixing water and using it together with a hair essence, it is excellent in hair growth and can provide a functional agent which can prevent white hair.

The present invention improves melanin synthesis by using soy methanol extract with liquid essence to increase tyrosinase, which is excellent for white hair suppression (prevention) mechanism, in particular, prolongs the growth phase of hair follicles and degenerative and resting metabolic processes. It is an invention having various effects, such as being able to do it normally and helping hair growth.

1 is a graph showing the polyphenol and flavonoid content contained in soybean methanol of hair growth and white hair prevention improving agent using soybean methanol to which the technique of the present invention is applied.
Figure 2 is a graph showing the electron donating ability of hair growth and white hair prevention improving material using soybean methanol to which the technique of the present invention is applied.
Figure 3 is a graph showing the cytotoxicity test results of the hair growth and white hair prevention improving agent using soy methanol to which the technique of the present invention is applied.
Figure 4 is a graph showing the melanin content of hair growth and white hair prevention improving agent using soybean methanol to which the technique of the present invention is applied.
5 is a graph showing the active state of trocinases in the state of treating melanocytes of hair growth and leukemia prevention improving agent using soybean methanol to which the technique of the present invention is applied.
Figure 6 is a graph showing the yellow state of trosinase in the state treated with the cell extract of hair growth and white hair prevention improving agent using soybean methanol to which the technique of the present invention is applied.

Looking at the preferred extraction method of the present invention for achieving the above object with reference to the accompanying drawings as follows.

1 is a graph showing the polyphenols and flavonoid content contained in soybean methanol of hair growth and white hair prevention improving agent using soybean methanol to which the technique of the present invention is applied, and FIG. 2 is hair growth using soybean methanol to which the technique of the present invention is applied. And a graph showing the electron donating ability of the anti-hair growth improving agent, FIG. 3 is a graph showing the cytotoxicity test results of the hair growth and white hair prevention improving agent using soybean methanol to which the technique of the present invention is applied, and FIG. Figure 5 is a graph showing the melanin content of hair growth and white hair prevention improving agent using soybean methanol to which the technique is applied, Figure 5 is treated with melanin cells of hair growth and white hair prevention improving agent using soybean methanol to which the technique of the present invention is applied Figure 6 is a graph showing the active state of trocinase, Figure 6 is a hair growth using soy methanol to which the technique of the present invention is applied It will be described with the redundancy status of Trojan or when the scoring in a cell extract of the mother material to improve the prevention state as a graph showing FIG.

Soybean is a herbaceous plant (Glycine maxim), which is a herbaceous plant that begins in spring, which belongs to the legume (Leguminosae), and has been used for food for a long time. The effects and effects of soybeans are antioxidant, anti-mutant, osteoporosis and hyperlipidemia, Anti-obesity, anti-obesity and anti-cancer effects such as blood sugar and cholesterol control have been reported (Lee et al. 1998: Shin et al. 2003: Choi et al. 2004: Kang et al. 2004: Kim et al. 2005).

 In the present invention, soybean methanol containing pravonoids and polyphenols, which are known to be excellent in anti-oxidation, anti-aging, anti-aging and atherosclerosis, and excellent in preventing hair loss and white hair from viruses and stress. It is characterized in that the extract to be utilized as an agent for improving hair growth and white hair prevention.

Prevention of hair growth and white hair prevention using soy methanol.

Soybeans were dried at room temperature for about 5 days, then pulverized, and 20 ~ 30% of water was added to methanol, and the sample soaked at 50 ℃ for 3 hours was dispensed with the solution in a tube and concentrated under reduced pressure at 45 ℃. Store at 5 ~ -20 ℃ for refrigeration or freezing.

The cryopreserved sample is freeze-dried and the freeze-dried sample is dissolved in a solvent [ethanol: DMSO (1: 1)] at a concentration of 2% to obtain a liquid soy methanol extract.

A solvent, ethanol and water are mixed with the soybean methanol extract to obtain a hair growth and white hair prevention improving agent using a liquid essence type soy methanol extract.

The mixing ratio of the soybean methanol extract and the solvent, ethanol and water is 20-50% of the whole soybean methanol extract and the hair growth prevention agent, and the solvent has excellent characteristics similar to oils naturally found in hair. Jojoba oil used as a hair conditioner is mixed at a ratio of 1 to 5%, 100% ethanol 8 to 15% and water 10 to 35%.

In the present invention, soybean methanol extract 15ml + solvent Jojoba oil 1.5ml + 3.75ml ethanol + 9.75ml water was mixed to obtain a hair growth and white hair prevention improving agent using 30ml soybean methanol extract.

The yield of the sample was 6.6% as measured by freeze drying.

Hair growth and white hair prevention experiments were carried out using soybean methanol extract extracted from soybean used as a hair growth and prevention of hair growth as described above.

Experimental Example  One.

Total phenolic compound content.

Total phenolic compound content was colorimetrically determined by the Folin-Denis method (Folin and Denis 1912). After adding 1 ml of folin-reagent to 1 ml of the sample and allowing to stand for 3 minutes, 1 ml of 10% Na 2 CO 3 was mixed and left at room temperature for 1 hour to measure absorbance at 760 nm. The calibration curve was prepared using tannic acid.

The total polyphenol content of the soy methanol extract was 53.7 mg / g as in FIG.

Total flavonoid content was determined using the Davis method (AOAC 1995). 10 ml of di (ethylene glycol) reagent and 1 ml of 1N NaOH were added to 1 ml of the sample solution, mixed well, and reacted for 1 hour in a 37 ° C. water bath, followed by measurement of absorbance at 420 nm. The calibration curve was prepared using rutin.

The flavonoid content of soy methanol extract was 62.7 mg / g as in FIG.

Experimental Example  2.

Electron donating ability .

The electron donating ability of the soybean methanol extract was measured by the method of Blois (Blois 1958). The freeze-dried soybean methanol extract powder was prepared by dissolving it in DMSO at concentrations of 100, 500, and 1000 µg / ml, taking 1 ml into a test tube, and adding 4 ml of 4 × 10-4M DPPH solution for 10 seconds in a 60 ° C water bath. After shaking and standing at room temperature for 20 minutes, the absorbance was measured at 525 nm. The same experiment was performed by adding 1 ml of ethanol instead of the sample in the extract-free addition group, and showed the electron donating ability as the decrease ratio of absorbance to the extract addition group. As a positive control group, the synthetic antioxidant BHT was tested in the same manner, and the electron donating ability (%) was obtained by the following equation.

Electron donation is (%) = (1-absorbance of extract added / absorbent without extract) x 100

As shown in FIG. 2, the electron donating ability of the positive control group, BHT, showed a positive (+) positive-response relationship, and the BHT and soybean methanol extracts were 68.4% and 14.4% at 1000 ppm, respectively.

Experimental Example  3.

In Vitro  Cell line experiments.

(3-1) cell lines and cultures.

Melan-a cells, an immortalized cell line derived from C57BL / 6 mice, RPMI-1640, sold by Bennett (Cancer Research Center, London, England), containing 10% Fetal Bovine Serum (FBS), 1% PS (Penicillin / Steptomycin), and 200 nM TPA (12-O-tetradecanoylphorbol-13-acetate) The medium was incubated in an incubator at 37 ° C. and 5% CO 2.

(3-2) MTT assay

In MTT assay, MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide] reagent is absorbed into cells and then forms formazan by succinate dehydronase of mitochondria. Accumulation means the activity of mitochondria, broadly the activity of cells, and is a representative method for measuring the growth rate of cells.

Melan-a cells were dissolved in RPMI-1640 medium containing 10% FBS, 1% P / S, and 200 nM TPA, stabilized in 37 ° C., 5% CO 2 incubator for 72 hours, and used in the experiment.

Dissolve Melan-a cells in 96-well plates with the appropriate cell number (0.5 × 104 cells / well), incubate for 24 hours in a 37 ° C, 5% CO ₂ incubator, and then concentrate the soy methanol extracts (0,6.25, 12.5, 25). , 50, 100, 200) was added to each 200 and incubated for 48 hours in 37 ℃, 5% CO ₂ incubator.

The plate was centrifuged at 1000 rpm for 10 minutes, washed once with PBS, 200 μl of medium containing 0.5 mg / ml MTT, and incubated at 37 ° C. in a 5% CO ₂ incubator for 3 hours.

The plate was centrifuged at 1000 rpm for 10 minutes to allow the cells to sink to the bottom, discarded the medium, 200 μl of DMSO, dissolved in the plate shaker for 15 minutes, and the absorbance at 540 nm was measured with an ELISA reader. Cell growth rate was calculated by the following equation.

Cell growth rate (%) = (absorbance of sample added / absorbance of sample added) x 100

MTT assay was performed to confirm the cytotoxicity of Melan-a cells of the freeze-dried soybean methanol extract powder. As a result, the soy methanol extract was 80% at 3.125, 6.25, 12.5, 25, and 50 ppm as shown in FIG. The growth rate was higher, but the cytotoxicity was observed at 100 ppm (57.4%), and the maximum allowable concentration was 50 ppm.

Experimental Example  4.

Melanin Generation .

Melan-a cells were dissolved in RPMI-1640 medium containing 10% FBS, 1% P / S, and 200 nM TPA, stabilized in 37 ° C., 5% CO ₂ incubator for 72 hours, and used in the experiment.

Dissolve Melan-a cells in a 96-well plate with the appropriate cell number (2 × 104 cells / well), incubate for 24 hours in a 37 ° C, 5% CO ₂ incubator, and then extract the soybean methanol extract by concentration (0, 3.125, 6.25). , 12.5㎍ / ㎖), add 500μl each, add primary substance treatment, incubate for 72 hours at 37 ℃, 5% CO ₂ incubator, and incubate for 72 hours with secondary substance treatment. It was dissolved and the absorbance was measured at 490 nm. Melanin quantification was calculated by the following equation.

Melanin synthesis growth rate (%) = {(absorbance of sample added / no sample added

                        Absorbance) -1} × 100

In order to determine the melanin content, the amount of melanin was measured after treating soybean methanol extract in each concentration of Melan-a cells.

As a result, as shown in FIG. 4, the positive control group, IBMX, had a melanin content of 131.6% at 12.5 ppm and soy methanol extracts at 11.125%, 6.25, 12.5, and 25 ppm, respectively, at 116.1% and 131.0, respectively. %, 145.9%, and 151.2% were high.

(4-1) Intra - cellular tyrosinase activity assay .

Melan-a cells were dissolved in RPMI-1640 medium containing 10% FBS, 1% P / S, and 200 nM TPA, stabilized in 37 ° C., 5% CO ₂ incubator for 72 hours, and used in the experiment.

Dissolve the Melan-a cells in a round 60∮ cell culture dish with the appropriate cell count (4 × 105 cells / well), incubate for 24 hours in a 37 ° C, 5% CO ₂ incubator, and then extract the soybean methanol extract by concentration (0, 3.125). , 6.25, 12.5, 25㎍ / ㎖), add 5ml each, incubate for 72 hours at 37 ℃, 5% CO ₂ incubator, remove the medium, wash with PBS, and put on ice 1% triton X-100 200 μl of the solution was dissolved, and then transferred to an e-tube, and placed in ice, and left for 1 hour while vortexing at 10 minute intervals while being embedded in ice. Absorbance was measured with a protein assay solution (Bio Rad, USA) to calculate the same amount of protein.

After 100 μl of cell extract and 40 μg of protein were mixed, 100 μl of L-DOPA was added and placed in a 5 ° C. CO ₂ incubator at 37 ° C. for 10 hours at 490 nm. .

Synthetic growth rate (%) = {(absorbance of sample addition group / no sample addition group

                        Absorbance) -1} × 100

To determine how much soybean methanol extract increases the activity of tyrosinase, an enzyme that plays a key role in melanin synthesis, samples of melanocytes were treated to measure tyrosinase activity over time.

As shown in FIG. 5, the soy methanol extract increased tyrosinase activity by 175.0% and 190.0% at 6.25 and 12.5 ppm, respectively, compared to the normal group without treatment after 60 minutes as shown in FIG. 5, and the positive control IBMX at 152.5 at 15,5 ppm. Increased by%

(4-2) Cell - extracted tyrosinase activity assay .

Melan-a cells were dissolved in RPMI-1640 medium containing 10% FBS, 1% P / S, and 200 nM TPA, stabilized for 72 hours in a 37 ° C., 5% CO ₂ incubator and used for the experiment.

Dissolve Melan-a cells in a round 60 둥근 cell culture dish with appropriate cell number (4 × 105 cells / well), incubate for 72 hours at 37 ° C, 5% CO ₂ incubator, wash with PBS, and then wash with 1% triton X-100. After 200 μl of the solution was dissolved in an e-tube, and placed in ice, and left for about 1 hour while vortexed at 10 minute intervals in a sieve. Pick the supernatant centrifuged at 4 ℃ 14000rpm for 20 minutes, and extract 50µl of the supernatant, 49µl of 0.1M phosphate buffer (pH 6.8), and soybean methanol extract by concentration (0, 3.125, 6.25, 12.5, 25µg / ml). ) Was mixed with 1 μl and left for 1 hour. 100 μL of L-DOPA was added thereto and placed in a 37 ° C. 10% CO ₂ incubator, and the change in absorbance was measured at 490 nm for 10 hours at 10 minute intervals.

Synthetic growth rate (%) = [(absorbance of the sample addition group / absorbance of the sample addition group) -1] × 100

Soybean methanol extract was treated with the cell extract to measure tyrosianse activity over time.

As shown in FIG. 6, the soy methanol extract increased 112.5% and 110.2% tyrosinase activity at 12.5 and 6.25 ppm, respectively, compared to the normal group without treatment after 60 minutes as shown in FIG. 6, and the positive control IBMX was 127.2 at 12.5 ppm. % Increased.

Soybean methanol extract extracted by the technique of the present invention as described above, the main components of the hair and scalp is made of protein to protect the hair follicles from internal or external stress through transdermal coating and at the same time healthy from hair loss and white hair It can be used to help you get your hair done.

It contains two or more selected from the group consisting of jojoba oil and soybean methanol as active ingredients, and it can be used as a material that simultaneously promotes hair growth and suppresses white hair induction by removing waste matter, exfoliation, and nourishment of the scalp hair follicles.

Jojoba oil has been used as an excellent hair conditioner with properties similar to the oils found naturally in hair. In addition, Jojoba Oil oil, a natural plant extract, reduces water loss in the scalp, helps transdermal absorption of the soybean methanol extract and metabolism of the hair cycle, thus making hair healthy and smooth and helping to circulate unbalanced scalp. do.

Soy methanol extract promotes melanin synthesis to keep your hair healthy and the strong sulfated flavonoids relieve white hair and hair loss caused by aging and stress. It also helps to suppress apoptosis and increase the normal activity of the entire scalp.

Polyphenols act as antioxidants to protect cells and chemicals in the body, and they also help the normal hair growth cycle and activate cells that restore the function of the scalp hair follicles found only in the hair follicles.

Claims (2)

Soybeans were dried at room temperature for 5 days, pulverized, 20 ~ 30% of water was added to methanol, and the sample was immersed at 50 ℃ for 3 hours. The sample was dispensed with a solution in a tube and concentrated under reduced pressure at 45 ℃. Kept in;
Freeze dried samples;
The freeze-dried sample was dissolved in a solvent at a concentration of 2% to obtain a liquid soy methanol extract;
Hair growth and prevention of white hair using the soybean methanol extract, characterized in that the soybean methanol extract obtained by mixing a solvent, ethanol and water. The method of claim 1, wherein
20-50% of the soy methanol extract,
1 to 5% of crude jojoba oil in a solvent,
Ethanol (100%) 8-15%,
A hair growth and white hair prevention improving agent using soybean methanol, which is mixed at a rate of 10 to 35% of water.

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