KR101080297B1 - Cosmetic composition comprising sophora japonica l.-derived stem cells for preventing or improving alopecia - Google Patents

Abstract

PURPOSE: A composition containing Sophora japonica L.-derived stem cells is provided to suppress 5-alpha-reductase and to prevent and treat alopecia. CONSTITUTION: A cosmetic composition for preventing or treating alopecia contains 2 x 10^3/kg-2 x 10^9/kg of Sophora japonica L.-derived stem cells. A method for preparing the stem cells comprises: a step of isolating leaves, stems, roots, or flowers of Sophora japonica L. and culturing in a medium to obtain callus; and a step of continuously performing sub-culture of the callus. The medium is MS(Murashige & Skoogs).

Description

Cosmetic composition for preventing or improving hair loss comprising stem cells derived from the painting tree {Cosmetic composition comprising Sophora japonica L.-derived stem cells for preventing or improving alopecia}

The present invention relates to a cosmetic composition for preventing or improving hair loss, including stem-derived stem cells. More specifically, the present invention has an effect of inhibiting 5 alpha reductase (5-alpha-reductase), which promotes the production of dihydrotestosterone, which is a hair loss-inducing hormone, is used for preventing and improving hair loss. It can be used as a composition.

In male expression, testosterone is involved in male impulse, skeletal muscle increase, male external genital development, scrotum growth, and spermatogenesis, and dihydrotestosterone is known to be involved in tissues such as acne, sebum increase, hair loss and prostatic hyperplasia. ( J. Invest . Dermatol . 1995; 105 (2): 209-14). 5-alpha-reductase is present in male hormone-reactive tissues such as sebaceous glands, hair follicles, prostate or epididymis and is a testosterone, a male hormone that is biosynthesized in the testes, and dihydrotestosterone, the active male hormone. , DHT) is a reductase involved in the conversion. It is known that NADPH is used as a coenzyme in this conversion process. Dehydrotestosterone secreted after puberty acts as a major causative agent of excessive sebum production in sebocytes, causing acne and male hair loss. In particular, many hairy cells provide a cause of hair loss because dihydrotestosterone binds to hormone receptors better than testosterone. Accordingly, studies are being actively conducted to develop a substance that inhibits the activity and production of 5-alpha-reductase, which promotes the production of dihydrotestosterone, and to use it as a hair regrowth or hair loss prevention agent. In particular, since male-type hair loss causes serious mental pain for men in the modern society, which emphasizes beauty, researches are being actively conducted to identify and improve the mechanism of hair loss.

Conventionally, female hormones such as estrogen have been used for the treatment of male hair loss. However, female hormones have been reported to cause skin inflammation and side effects due to hormone administration compared to its effects, and are currently discontinued or used in very small amounts. Recently, Merck in the United States has synthesized a finasteride (finasteride, proscar), which inhibits the action of 5 alpha-reductase to inhibit the production of dihydrotestosterone, and is commercially available as a hair growth agent for oral administration. However, this hair regrowth is expensive and produced for the application of the scalp is directly applied to the situation is less effective and other side effects of oral administration, such as the situation is limited to use.

In order to solve the problem of the skin, cosmetics using natural products have recently been developed to reduce skin irritation caused by various chemicals. Natural ingredients have less side effects on the skin, and as the consumer's response to cosmetics using natural ingredients has increased recently, the development value of cosmetics as a raw material for cosmetics is increasing.

Sophora japonica L. ) is a deciduous arborescent of the dicotyledonous plant Rosaceae, and its buds are called lumps or lumps. Flowers, berries, bark, stems, and roots are used mainly for arteriosclerosis, intestinal bleeding, uterine bleeding, gum inflammation, swelling, burns, hypertension, cerebral hemorrhage, stroke, and numbness of the hands and feet.

Stem cells are present in the plant's birthplace. Stem cells are undifferentiated cells that have the capacity to self-renewal and to differentiate into various types of tissues or organs. These plant stem cells are differentiated to their own personality and function to renew the tissues and organs of plants such as roots and stems. These plant stem cells are classified as pluripotent stem cells, which have the ability to form various organs through typical functions.

Therefore, the present inventors have studied the physiological mechanism of the stem cells, and as a result, it contains a large amount of an effective substance that effectively inhibits 5 alpha-reductase which causes hair loss by converting testosterone to dihydrotestosterone, thereby promoting excellent hair growth. The present invention was completed by finding an effect and preparing a cosmetic composition for preventing or improving hair loss containing the same.

An object of the present invention is to provide a cosmetic composition for preventing or improving hair loss containing stem cells derived stem cells.

The cosmetic composition for preventing or improving hair loss of the present invention may contain a stem-derived stem cell as an active ingredient.

In one embodiment, the composition may contain 2 x 10 ³ stems / kg to 2 x 10 ⁹ stems / kg stem cells.

In one embodiment, the stem cells may be included in 0.25-50% by weight of the composition.

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The cosmetic composition according to the present invention has an effect of preventing and improving hair loss by inhibiting the activity of 5-alpha-reductase, which contains stem wood-derived stem cells and promotes the production of dihydrotestosterone.

Figure 1 shows the inhibition rate of 5 alpha reductase activity according to the concentration of the cedar stem cells. In Figure 1, column 1 represents 1% of the stem cell, row 2 of the stem cell 0.5%, row 3 of the stem cell 0.25%, row 4 shows the radioactive results of the control group. In FIG. 1, the middle band indicated in each column is testosterone, and the band indicated at the top of the middle band represents dihydrotestosterone. The higher the concentration of stem cell treatment, the more the results show that the production of dihydrotestosterone.
Figure 2 shows the inhibition rate of tyrosinase activity according to the concentration of stem cells. In Figure 2, ◆ indicates stem cells, and ■ indicates the results during ascorbic acid treatment.
Figure 3 shows the cell activity according to the concentration of stem cells.
Figure 4 shows the antimicrobial activity according to the concentration of stem cells.

Hereinafter, the present invention will be described in more detail.

The present invention provides a cosmetic composition for preventing or improving hair loss containing stem cells derived stem cells.

The present inventors, in order to find out that the stem-derived stem cells are effective in preventing or improving hair loss, the stem cells were isolated and cultured from the fir-trees, and identified as stem cells through experiments of differentiation of stem cells. The stem cells were then found to inhibit the activity of the 5 alpha reductase, a hair loss-inducing hormone.

Stem cells derived from a painting tree are Sophora japonica L. ). Specifically, the stem-derived stem cells are separated from one or more selected from the group consisting of leaves, stems, roots and flowers of the painting tree, and then primary cultured in the callus-inducing medium to obtain callus. The callus obtained can be obtained by continuous secondary passage culture.

The stem-derived stem cells are preferably obtained from the leaves, stems or roots of the shrub. Callus induction medium may be used a conventional solid medium or liquid medium, and the components of the solid medium or liquid medium may be those known to those skilled in the art. For example, MS (Murashige & Skoogs) medium can be used. Induced callus can be obtained by cell subculture in a solid or liquid medium. Secondary passage culture may also use a conventional solid medium or liquid medium, for example MS (Murashige & Skoogs) medium.

The inventors confirmed that the stem cells through the differentiation capacity test for the cells prepared from the above.

In order to determine whether the stem cell derived from the cedar tree inhibits the activity of 5 alpha-reductase, the present inventors treated the stem cells with 5 alpha-reductase produced by the fibroblasts of the epidermis. As a result, it was confirmed that the conversion rate of testosterone to dihydrotestosterone was suppressed when the stem cells were treated. From this, it can be seen that stem cell derived stem cells have an effect of preventing or improving hair loss by inhibiting the activity of 5 alpha-reductase, which acts as a cause of male alopecia.

Painting tree-derived stem cells may be contained in the composition of the present invention from 2 x 10 ³ / kg to 2 x 10 ⁹ / kg. Preferably, it may be contained at 5 x 10 ³ pieces / kg to 1 x 10 ⁹ pieces / kg.

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Painting tree-derived stem cells may be included in 0.25-50% by weight of the composition of the present invention. Preferably 0.25-30% by weight, more preferably 0.25-1% by weight.

The composition of the present invention may include, in addition to stem cell derived stem cells, a conventional solvent that can be used in preparing the composition as the remaining amount of the composition. The solvent may include water, but is not limited thereto.

In addition to the stem-derived stem cells, the composition of the present invention may include a typical blending component hinokithiol, capsicum tink, tocopherol acetate, nico acid amide, nico acid benzine, pyrotonolamine, salicylic acid, L-mentone and the like.

The cosmetic composition for preventing or improving hair loss of the present invention can be used in various cosmetic or cosmetic preparations such as topical application agents such as ointments, creams, lotions, liquids, sprays, etc., in use for the purpose of preventing or improving hair loss. Preferably, it is effective to formulate into topical application preparations such as ointments, lotions, sprays and the like and apply them directly to the hair loss site. It can also be formulated into any formulation that can be applied to conventional scalp, such as shampoos, hair conditioners, hair lotions, liquid hair growth promoter types, and the like.

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

However, the following Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.

Example

(1) Isolation and culture of stem cell derived stem cells

The trunk, leaves and roots of the ash tree were used for callus induction. The sample was cut to about 10 cm in length, washed, and then sterilized with 0.5% sodium hypochloride solution containing 0.1% of Tween 20 for 20 minutes while stirring. Ultrasonic sonication was performed for 30 seconds during this period to remove micro marks on the surface of the biological sample. After washing once with sterile water, put it in 70% ethanol solution and sterilize the surface again for 1 minute, and then cut the sterilized and washed biological sample into a size of about 0.5 cm and place agar in MS (Murashige & Skoogs) medium. The culture was started by transferring the mixed solid culture medium. MS (Murashige & Skoogs) medium was the most suitable medium used for inducing callus cells. 0.5-1.0% of agar is added to the MS medium to be used as a solid medium. Phytohormone auxin is added as a growth regulator, but it is preferable to use 1-naphthalene acetic acid (NAA) instead of 2,4-D, which is seriously harmful to humans. In order to escape the detrimental controversy of the growth regulators, no growth regulators were added, and instead natural coconut milk was added with MS (NH 4 NO 3 (1650 mg / L), KNO 3 (1900 mg / L), CaCl 22 H 2 O (440 mg / L), MgSO 47 H 2 O). (370 mg / L), KH2PO4 (170 mg / L), KI (0.83 mg / L), H3BO3 (6.2 mg / L), MnSO44H2O (22.3 mg / L), ZnSO47H2O (8.6 mg / L), Na2MoO42H2O (0.25 mg / L), CuSO45H2O (0.025 mg / L), CoCl26H2O (0.025 mg / L), FeSO47H2O (27.8 mg / L), Na2EDTA2H2O (37.3 mg / L), Inositol (100 mg / L), Nicotinic acid (0.5 mg / L), Pyridoxine HCl (0.5 mg / L), Thiamine HCl (0.1 mg / L), Glycine (2 mg / L), Sucrose (3%)) and added to the medium for excellent cell growth, without growth regulator Medium was used.

All manipulations were performed aseptically on a sterile operating table, and callus was induced and grown from 2-3 weeks after the start of incubation at 25 ° C dark conditions. Callus was well-derived from various biological samples, and excellent ones were transferred to fresh medium every month and cultured to obtain a stem cell line derived from a cedar.

(2) Identification of stem cells

In addition to self renewal, traits that characterize plant stem cells include pluripotency. In order to induce differentiation of the stem cells, the cultures were cultured at 25 ± 1 ° C. under dark conditions of MS medium containing 10 mg / L of NAA, 2 mg / L of Kinetin, 6 mg / L of GA3, and 6% of sucrose. As a result, it was confirmed that the stem cells were differentiated into stems, leaves, and roots, respectively.

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Experimental Example  One: 5 alpha - Reductase  Activity inhibitory test

5 alpha-reductase activity inhibitory 5 alpha-reductase used in the experiment was used an enzyme produced by the epidermal fibroblasts.

Fibroblasts isolated from the skin cells were inoculated with 1 × 10 ⁴ cells per microplate hole and cultured. In each hole, 0.1.mμ.Ci of radiolabeled testosterone with ³ H (tritium) was added and cultured to determine whether the fibroblasts used it. The stem-derived stem cells were included at 0.25-1% concentration (wt%). A medium containing no stem cell derived from the cedar was used as a control.

After incubation for 24 hours, the supernatant was obtained, and steroids were obtained with 1 ml of an ethyl acetate-cyclohexane (1: 1) extractant. The steroid thus obtained was placed on a thin layer chromatography plate and developed with a chloroform / methanol mixture (98/2 (v / v)). Radioactivity of the points corresponding to testosterone and dihydrotestosterone was measured by using a densitometer (see FIG. 1), and the conversion rate was calculated. The results were compared with the control group and 5 alpha-reductase inhibitory ability according to Equation 1 below. Was evaluated. The same experiment was repeated twice and the results are shown in Table 1 below.

<Equation 1>

5 alpha-reductase inhibitory activity (%) = [A-B] / [A] X 100

(Wherein A represents the conversion rate of testosterone to dihydrotestosterone when no stem cell is added, and B represents the conversion rate of testosterone to dihydrotestosterone upon addition of stem cells).

   Painting Stem Cell Treatment Concentration 0.25% 0.5% One%  5 alpha-reductase inhibitory activity 45.8% 45.3% 65.2%

Experimental results As shown in Table 1, it can be seen that the stem-derived stem cells have a 5 alpha-reductase inhibitory effect. From this, the stem-derived stem cells have a hair loss prevention or improvement effect.

Experimental Example  2. Experiment of inhibitory effect on tyrosinase activity

Tyrosinase activity inhibition experiment was performed to investigate the degree of inhibitory activity of tyrosinase activity in the cedar stem cells. As tyrosinase activity inhibiting substrate, 10 mM L-DOPA dissolved in sodium phosphate buffer (pH 6.4) was used. The cedar stem cells were diluted using 0.1 M sodium phosphate buffer solution to the concentrations shown in Table 2 below. 40 μl of L-DOPA was added to 240 μl of the sample solution according to each concentration, and 20 μl of tyrosinase (500 U / ml) was added thereto, followed by reaction at 37 ° C. for 10 minutes, and then absorbance at 450 nm. Ascorbic acid at the same concentration was used as a positive control. Ascorbic acid is known to have tyrosinase inhibitory activity as a substance with whitening activity. Inhibition of tyrosinase activity was calculated according to the following formula 2, the results are shown in Table 2 and FIG.

<Equation 2>

Tyrosinase activity inhibition rate (%) = 100-[(b-b ') / a] X100

(In the above formula, a is the absorbance of only tyrosinase, b is the absorbance of tyrosinase and stem cells together, b ': the absorbance of not adding tyrosinase).

Example Positive control
(Concentration 100 µg / ml) Stem Cell Concentration (wt%) 6.25 12.5 25 50 100 Tyrosinase inhibition rate (%) 91.5 96.2 97.8 94.6 99.5 68.7

As shown in Table 2, it can be seen that the tyrosinase inhibitory ability of the cedar stem cells. At low concentrations, it showed an excellent effect on inhibiting tyrosinase activity and showed much higher inhibitory effect compared to ascorbic acid, a positive control group, indicating that the whitening effect of the stem cells is excellent.

Experimental Example  3. Cytotoxicity Test

Water soluble tetrazolium salt (WST) is a water-soluble tetrazolium salt that reacts with living cells to produce orange water-soluble formazan. Therefore, it is a method to measure the cytotoxicity to test substance by measuring this amount.

Toxic hardness for human keratinocytes (HaCat) was measured to determine whether the stem cells derived from the present invention of the present invention can exhibit a skin improvement effect without skin irritation. Human keratinocytes (ATCC CLS 300493, USA) were counted in 5 x 10 ³ cells / well in 96-well plates using an hemacytometer and then aliquoted. Time incubation. After culturing for 24 hours, the culture medium was removed, and 100-200 μl of the stem cells of the preparation of the present invention was added to each well at a concentration of 0-2 (w / v)%. It was. Thereafter, 10 μl of WST was added to each well, and the cells were further incubated for 4 hours under the same conditions. Thereafter, absorbance was measured at 420 nm with an Enzyme-Linked Immunosorbent Assay (ELISA) leader. Cell viability was expressed as a percentage based on the absorbance intensity of the control with pure water. DMEM (10% FBS) was used as a positive control and dimethyl sulfoxide (DMSO) was used as a negative control. The cell activity was calculated according to the following Equation 3, and the value when the value for the cell activity of the positive control group was viewed as 100 is shown in Table 3 and FIG. 3.

<Equation 3>

Cell activity (%) = [absorbance of stem cell treatment / absorbance of negative control] X 100

Example Negative Control
10% Stem Cell Concentration (wt%) 0.125 0.25 0.5 One 2 Cell activity (%) 106.4 102.8 99.9 88.9 85.5 16.5

As shown in Table 3, the compositions of the embodiments of the present invention can be seen that the cytotoxicity is significantly lower than the negative control at the treatment concentration, and the cell activity is lowered as the stem cell concentration is higher. These compositions can bring about skin cell regeneration effects.

Experimental Example  4. Antibacterial activity test by concentration

Diluting the stem-derived stem cells in the liquid medium in the range of 1-0.1% by weight, inoculating 50 μl of E. coli in each medium, and incubating for 24 hours with stirring. It calculated according to the following formula 4. As a control, only E. coli was inoculated into a liquid medium containing no stem cells. The results are shown in Table 4 and FIG.

<Equation 4>

% Of bacterial growth inhibition = [(absorbance of control group-absorbance of stem cells) / absorbance of control group] X 100

Experimental Example Stem Cell Concentration (wt%) 0.0625 0.125 0.25 0.5 One 2 Bacterial growth inhibition rate (%) 3.8 10.3 35.8 62.5 88.8 90.2

As shown in Table 4, as a result of the antimicrobial activity of the stem cells, it can be seen that the antimicrobial activity increases as the concentration increases.

Formulation Example 1 Hair Lotion with Hair Loss Prevention or Improvement

The hair lotion was prepared by the conventional method using the component of Table 5 below.

ingredient Content (% by weight) Example picture tree stem cell 1.0 Cetostearyl alcohol 2.0 Stearyl Triethyl Ammonium Chloride 2.0 Hydroxyethyl cellulose 0.5 Pyrotonolamine 0.1 Spices 0.5 Purified water Balance system 100.0

Formulation Example 2: Hair Lotion with Hair Loss Prevention or Improvement

Hair tonic was prepared in a conventional manner using the components shown in Table 6 below.

ingredient Content (% by weight) Example picture tree stem cell 1.0 ethanol 55.0 Castor oil 5.0 glycerin 3.0 Pyrotonamine 0.1 Spices, pigments 0.01 Purified water Balance system 100.0

Claims (5)

A cosmetic composition for preventing or improving hair loss containing stem cell derived stem cells.
The cosmetic composition for preventing or improving hair loss according to claim 1, wherein the composition contains 2 x 10 ³ stems / kg to 2 x 10 ⁹ stems / kg.
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