KR20200058932A - Hair loss prevention and hair growth improving composition containing a bellflower extract or a bellflower fractio - Google Patents

Abstract

The present invention relates to a composition for preventing hair loss and improving hair growth, wherein the composition comprises any one fraction selected from an ethanol extract of bellflower root, a chloroform fraction of bellflower root, an ethylacetate fraction of bellflower root, a butanol fraction of bellflower root, and a water fraction of bellflower root. In addition, it can be seen that the composition for preventing hair loss and improving hair growth has no toxicity to dermal papilla cells and also has an effect of increasing cell growth and decreasing an amount of DHT production near 50%, thereby providing an excellent effect of improving hair growth. The composition of the present invention also comprises a natural material as an effective component so the composition is hypoallergenic and excellently safe to the skin, and thus can be mixed in a variety of hair cosmetics and used in various dosage forms.

Description

Hair loss prevention and hair growth improving composition containing a bellflower extract or a bellflower fractio}

The present invention relates to a hair loss prevention and hair growth improvement composition containing a bellflower extract or a bellflower fraction as an active ingredient, specifically, an anti-inflammatory effect, a dihydro-testosterone production inhibitory effect that causes hair loss, and a bellflower extract excellent in antibacterial activity effect Or it relates to a hair loss prevention and hair growth improvement composition containing a bellflower fraction as an active ingredient.

In addition to the purpose of protecting the hair and scalp of human body, the importance of hair loss is recognized in terms of aesthetics and sexuality, but the exact mechanism of hair loss is unknown, and environmental, genetic, and mental The physical rhythm is broken due to various causes such as physical and back, and the balance is not balanced, which greatly affects not only the scalp but also the hair follicle, leading to hair loss and hair loss.

With the rapidly growing hair loss population, the demand for scalp care hair loss prevention hair care products has increased significantly, and there have been many research and development related to this, but the material development is still insignificant. Until now, products approved for hair loss treatment include Finasteride and Minoxidil, but these two products have known side effects, and hair growth efficacy substances have not been properly developed. These studies have been continuously conducted so far as necessary.

As a prior art related to the prevention of hair loss using natural substances, for example, patent document 1 includes a complex extract of chrysanthemum, safflower, marigold, gold silver, bokbok and badger oil as an active ingredient, preventing hair loss and promoting hair growth The hair cosmetic composition for dragon hair is disclosed, and as a herbal composition for preventing hair loss and promoting hair growth in Patent Document 2: 20 to 25% by weight, Phellinus linteus extract, 20 to 25% by weight, golden extract 20 to 25% by weight, based on the total amount of the herbal composition 15 to 20% by weight of extract, 5 to 10% by weight of Cornus officinalis extract, 5 to 10% by weight of 고 extract, 5 to 10% by weight of ginseng extract, 5 to 10% by weight of succinate extract, 5 to 10% by weight of green tea leaf extract, extract of gold and silver coins 5 to 10% by weight, herbal extracts containing 5 to 10% by weight of at least one extract selected from silk extracts and hwangnyeon extracts and epidermis extracts; And with respect to the total amount of the herbal composition, genistein (genistein) 1 to 2% by weight and resveratrol (resveratrol) 1 to 2% by weight of polyphenols; including hair loss prevention and hair growth promoting herbal composition disclosed.

In addition, Patent Document 3, based on the total weight of the plant material, 6 to 12% by weight of ginseng, 10 to 15% by weight of black beans, 6 to 12% by weight of Eoseongcho, 6 to 12% by weight of wolfberry, 10 to 15% by weight of licorice, As a plant extract extracted from plant materials containing 6 to 12% by weight of dermis, 6 to 12% by weight of jujube, 6 to 12% by weight of cotyledon, 10 to 15% by weight of green tea leaves and 6 to 10% by weight of white sorghum, Disclosed is a composition for preventing hair loss and improving hair growth, comprising a plant extract that inhibits 5α-reductase expression of dermal papilla cells (HFDPC) and promotes β-catenin expression of human dermal papilla cells (HFDPC).

Applicants of the present invention are natural extracts of bellflower extract and fractions that show antioxidant and skin cell regeneration effects, anti-inflammatory effects that reduce NO and PGE ₂ production, and dihydro-testosterone production inhibitory effects that cause hair loss. Confirmed the possibility and completed the present invention.

Patent Document 1; Republic of Korea Registered Patent Publication No. 10-1009763 Patent Document 2; Republic of Korea Registered Patent Publication No. 10-1440046 Patent Document 3; Republic of Korea Registered Patent Publication No. 10-1758998

The problem to be solved in the present invention is to provide a hair loss prevention and hair growth improvement composition containing a bellflower extract or a bellflower fraction as an active ingredient, and more specifically, reducing antioxidant and skin cell regeneration effects, reducing NO and PGE ₂ production. It is an object of the present invention to provide a composition for preventing hair loss and improving hair growth, which contains an anti-inflammatory effect and a bellflower extract or a bellflower fraction having an excellent effect of inhibiting the production of dihydro-testosterone that causes hair loss.

As a solution for solving the problems according to the present invention, a hair loss prevention and hair growth improvement composition containing a bellflower extract or a bellflower fraction is a bellflower chloroform fraction, a bellflower ethyl acetate fraction, a bellflower butanol fraction and a bellflower water fraction obtained from a bellflower ethanol extract or a bellflower ethanol extract It consists of containing at least one bellflower fraction selected from the active ingredient.

One embodiment according to the hair loss prevention and hair growth improvement composition containing the bellflower extract or the bellflower fraction of the present invention is a bellflower chloroform obtained from a bellflower ethanol extract or a bellflower ethanol extract as an active ingredient relative to the total weight of the composition, a bellflower ethylacetate It consists of 0.1 to 80% by weight of at least one bellflower fraction selected from fractions, bellflower butanol fraction and bellflower water fraction.

Another embodiment according to the hair loss prevention and hair growth improvement composition containing a bellflower extract or a bellflower fraction as a solution of the problems of the present invention is a hair tonic, a hair conditioner, a hair essence, a hair lotion, and hair nutrition containing the composition of the present invention Lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nutrition cream, hair moisture cream, hair massage cream, hair wax, hair aerosol, hair pack, hair nutrition pack, hair soap, hair cleansing foam, hair oil, hair dryer , Hair preservation treatment agent, hair dye, hair wave agent, hair decolorant, hair gel, hair glaze, hair dresser, hair lacquer, hair moisturizer, hair mousse and hair spray.

There is an antioxidant effect of bellflower ethanol extract containing as an active ingredient in the hair loss prevention and hair growth improvement composition of the present invention, skin cell regeneration effect, anti-inflammatory effect to reduce NO and PGE ₂ production, and dihydro-testosterone production inhibitory effect that causes hair loss. In addition, the bellflower chloroform and ethyl acetate fraction show anti-hair loss and hair growth effects because they have antibacterial activity effects that inhibit the growth of dandruff bacteria and proliferation of dermal papilla cells, and have the advantage of being applied to hair care products.

1 is a diagram of DPPH radical scavenging activity and ABTS radical scavenging ability of the bellflower ethanol extract of the present invention
Figure 2 is a chart for the skin cell regeneration effect of the bellflower ethanol extract of the present invention
Figure 3 is a chart for PGE ₂ inhibitory activity of the bellflower ethanol extract of the present invention
Figure 4 is a chart for proliferation activity of dermal papilla cells of the bellflower ethanol extract of the present invention
Figure 5 is a chart for the amount of DHT production of bellflower ethanol extract and control of the present invention
6 is a diagram of DPPH radical scavenging activity and ABTS radical scavenging ability of bellflower fractions of the present invention
7 is a chart for skin cytotoxicity of bellflower fractions of the present invention
8 and 9 is a chart for the skin cell regeneration effect of the bellflower fractions of the present invention
10 is a diagram for RAW264.7 cytotoxicity of bellflower fractions and controls of the present invention
11 is a chart for PGE ₂ inhibitory activity of the bellflower chlorform and ethyl acetate fractions of the present invention and the control group
12 is a toxicity confirmation chart for dermal papilla cells of the bellflower fractions of the present invention
13 is a chart for the amount of DHT production of the control of bellflower fractions of the present invention

Hereinafter, the present invention will be described in more detail through specific contents for carrying out the present invention, <Examples> and <Test Examples>, but the embodiments of the present invention may be modified in various other forms, so that the present invention The scope is not limited to the embodiments described below.

The hair loss prevention and hair growth improvement composition containing the bellflower extract or bellflower fraction according to the present invention is a bellflower chloroform fraction obtained from an bellflower ethanol extract or a bellflower ethanol extract, an bellflower ethyl acetate fraction, a bellflower butanol fraction as an active ingredient relative to the total weight of the composition And 0.1 to 80% by weight of at least one bellflower fraction selected from bellflower water fractions.

The bellflower ethanol extract according to the present invention is extracted at 35 ° C using 70% (v / v) of ethanol as an extraction solvent.

The bellflower fractions are chloroform, ethyl acetate and butanol sequentially added to the above-mentioned bellflower ethanol extract, and separated by using the property that the layers are separated according to polarity differences, respectively, to thereby separate the bellflower chloroform fraction, the bellflower ethyl acetate fraction, the bellflower butanol fraction and the bellflower water fraction. To obtain

Hereinafter, the present invention will be described in detail through <Examples> and <Test Examples>, and the examples below are only intended to illustrate the present invention and do not limit the contents of the present invention.

<Example 1> Preparation of bellflower ethanol extract

A freshly picked 3-year-old Jangsaeng Doraji, purchased from Jiri Mountain's Moral Farm, was carefully selected, then shredded to dryness at 40 ° C for 24 hours, pulverized to form a powder.

70% ethanol was added at a volume ratio of 10 times to the bellflower powder, and the mixture was extracted twice by stirring at 35 ° C for 48 hours, and the resulting extract was filtered under reduced pressure (filter paper No. 2) and concentrated under reduced pressure (EYERA Rotary Evaporator N- After removing the extraction solvent with 1000), this concentrate is used as a sample, and the highest concentration is set to 200 mg / ml, and then an in vitro test is performed.

<Example 2> Separation of bellflower fractions

The ethanol extract obtained in <Example 1> was dissolved in distilled water using a separating funnel, and then left to stand by adding chloroform to separate the chloroform layer to obtain a bellflower chloroform fraction.

Ethyl acetate was added to the remaining solution to stand, and then the ethyl acetate layer was separated to obtain a bellflower ethyl acetate fraction, and the butanol layer was separated by adding butanol to the remaining solution from which the bellflower ethyl acetate fraction was separated to separate the bellflower butanol fraction. Obtained and finally the remaining bellflower water fraction was obtained.

After removing the solvent from each separated fraction by concentration under reduced pressure, the solvent extract is tested with a sample of the bellflower chloroform fraction, bellflower ethyl acetate fraction, bellflower butanol fraction, bellflower water fraction dissolved in DMSO as a sample.

A. Prevention of hair loss and improvement of hair growth efficacy of bellflower ethanol extract

<Test Example 1> antioxidant activity

The causes of hair loss are genetic and environmental factors, and environmental factors are closely related to free radicals. Reactive oxygen promotes aging through oxidation and reduces hair vitality. In addition, it binds to cholesterol and turns into lipid peroxide and sticks to the walls of blood vessels, narrowing the blood vessels of the scalp and clogging pores, reducing blood flow, making it difficult to supply nutrients to the hair roots. In addition, it damages the cell membrane of the cells constituting the hair follicles, causing cell death and causing scalp inflammation to cause hair loss. DPPH and ABTS Radical scavenging activity were measured to confirm the antioxidant activity of the bellflower ethanol extract.

 One). DPPH radical scavenging ability

  The DPPH assay method is described in Yoshida et al. (1989). 50 μl of deionized water is added to the control of a 96 well plate, and 5 μl of a sample diluted with deionized water is added to the test group as triples. Add 25 µl of ethanol to all wells, add 75 µl of DPPH (0.5 mM 1,1-diphenyl-2- picrylhydrazyl) solution and mix. After reacting at room temperature for 25 minutes for 30 minutes, absorbance was measured at 517 nm. As a solvent control group, the radical scavenging ability of the control group was expressed as a percentage as shown in [Calculation Formula 1] below, and the results are shown in [Table 1] and [Figure 1] below.

[Calculation formula 1]

 2). ABTS radical scavenging ability

  The ABTS assay method was measured by modifying the method of Re et al. (1999). That is, after mixing 5 ml of 7 mM ABTS and 88 μl of 140 mM potassium persulfate, the light is blocked for 16 hours at room temperature to form ABTS cations. The solution was then diluted with PBS so that the absorbance value at 414 nm was 1.5. After 190 µl of the prepared dilution solution and 10 µl of the sample were mixed and reacted for 6 minutes at room temperature, the absorbance was measured at 734 nm. As a solvent control group, the radical scavenging ability of the control group was expressed as a percentage as shown in [Calculation Formula 1] below, and the results are shown in [Table 1] and [Figure 1] below.

[Calculation formula 2]

Sample concentration (mg / ml) DPPH radical scavenging ability (%) ABTS radical scavenging capacity (%) One 13.3 ± 5.4 10.0 ± 1.9 10 31.5 ± 1.0 24.6 ± 2.5 50 57.5 ± 2.2 99.9 ± 0.2 100 90.0 ± 7.5 99.7 ± 0.2 200 90.3 ± 3.3 100.0 ± 0.4 * IC ₅₀ : DPPH 42.9mg / ml, ABTS 23.1mg / ml

As shown in [Table 1], the activity of Radical scavenging of ethanol extract increased concentration-dependently. DPPH radical scavenging activity of bellflower ethanol extract showed 57.5% of activity at a concentration of 50 mg / ml, and ABTS radical scavenging activity of 24.6% at a concentration of 10 mg / ml. ABTS radical scavenging ability was higher than DPPH radical scavenging ability.

<Test Example 2> Skin cell toxicity and cell regeneration efficacy evaluation test

 One). Cell lines and culture

  To examine the skin cell toxicity of the sample, the cell toxicity was measured through HaCaT cells, which are human-derived epidermal cells. That is, the HaCaT cell line cultured in the cell culture flask was passaged by removing the cells from the flask with a 10 ml pipette, and the cells were cultured with DMEM / LOW medium containing 10% FBS.

 2). Cytotoxicity test

HaCaT cells were dispensed at 1x10 ⁴ / well in a 96 well plate and incubated with 10% FBS medium for 24 hours, and the final concentration of each sample was 1, 10, 50, 100 and 200 μg / Prepared at a concentration of ㎖, treated with cells, and cultured for 24 hours.

To confirm cytotoxicity, absorbance was measured at 490 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA) using the Cell Titer 96 Aqueous One solution cell proliferation assay kit (Promega, WI, USA). It is shown in Table 2.

Sample name (µg / ml) Cell Viability (%) Control group 100 ± 5.2 Bellflower ethanol extract One 120.9 ± 7.3 ^* 10 117.4 ± 6.7 ^* 50 118.2 ± 7.8 ^* 100 110.3 ± 3.4 ^* 200 93.9 ± 4.6 *: p <0.05, compared with Control group

As shown in [Table 2], there was no toxicity of the bellflower ethanol extract at concentrations of 1, 10, 50, 100, and 200 μg / ml.

 3). Evaluation of skin cell regeneration effect

The HaCaT cell line was dispensed into a 96 well plate at a concentration of 2 × 10 ³ cells / ml, and cultured in a CO ₂ incubator controlled at 37 ° C., 95% humidity, and 5% CO ₂ for 24 hours. Each sample was treated with cells at appropriate concentrations within a range of concentrations of 1, 10, 50, 100, and 200 μg / ml in fresh medium, and then cultured for 1 and 2 days. Absorbance at 450 nm with a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA) using Cell Counting Kit-8 (CCK-8, Dojindo Mol. Tech., Rockville., MD, USA) to measure cell line viability. Was measured. The regeneration effect of the cells was expressed as the absorbance of the sample treatment group compared to the control group that did not process the sample, and the results are shown in [Table 3] and [Figure 2] below.

Sample name (µg / ml) Cell proliferation rate (%) 1 day 2 days Control group 100.0 ± 1.8 100.0 ± 6.3 Bellflower ethanol extract One 108.0 ± 2.4 ^* 113.8 ± 11.6 10 113.2 ± 5.7 ^* 131.2 ± 4.2 ^* 50 113.0 ± 2.6 ^* 125.8 ± 7.4 ^* 100 116.8 ± 2.8 ^* 127.6 ± 7.2 ^* 200 113.5 ± 3.3 ^* 143.7 ± 16.9 ^* *: p <0.05, compared with Control group

As shown in [Table 3], the 1-day incubation increased by 16.8% compared to the control group, the 2-day incubation increased by 25.8, 27.6% at 50, 100 μg / mL, and increased by 43.7% at 200 μg / mL. The concentration-dependent proliferative activity was confirmed.

<Test Example 3> anti-inflammatory efficacy test

 One). Cell culture

The cell line RAW264.7 used in the experiment was used for pre-sale from the Korea Cell Line Bank (KCLB, Seoul, Korea). Each cell line was cultured in a 10 cm well plate and passaged 2-3 times a week, and the medium was a growth medium containing 10% Fetal Bovine Serum (FBS, Lonza, Valais, Switzerland) medium. Cell lines were cultured in an incubator controlled at 95% humidity, 5% CO ₂ , and 37 ° C, and the medium was changed once every 2 days. At this time, a medium antibiotic (Penicillin streptomycin, Gibco, CA, USA) was used to suppress contamination or proliferation of microorganisms. When the cells were covered with 80% of the dish, they were washed with phosphated-buffered saline-EDTA (PBS-EDTA), followed by trypsin treatment, and the cells were cultured by changing the medium every 48 hours.

 2). Nitric Oxide production measurement

  The RAW 264.7 cell line was dispensed into 96 wells, and then cultured until the cells grew to about 80% or more of the bottom. To induce inflammation, lipopolysaccharide (LPS, Sigma-Aldrich, MO, USA) was treated at a concentration of 1 μg / ml.

To check the anti-inflammatory efficacy of the licorice extract for sample and contrast, dissolve in the medium in the cells and incubate for 24 hours so that the concentration of the sample and licorice extract is 1, 10, 50, 100, 200 μg / ml with LPS, NO production was measured using Griess reagent. Griess reagent was made by mixing 1% sulfanilamide and 0.1% naphthylethylene-diamine dihydrochloride in 2.5% phosphate solution. 50 μl of the supernatant of the cultured cells was added to a 96 well plate, 50 μl of Griess reagent was added thereto, and the mixture was reacted at room temperature for 15 minutes, and absorbance at 540 nm using a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA). Was measured. Measurement of anti-inflammatory efficacy is measured by measuring the amount of NO produced in the sample and licorice extract-treated groups along with LPS for the amount of NO produced in the group treated with LPS only by treating LPS in a macrophage cell line to determine whether the sample has an effect of inhibiting NO production. The results are shown in [Table 4] and [Figure 4] below.

Sample name (µg / ml) NO production (%) Control 11.6 ± 0.2 Control (+ LPS) 100.0 ± 1.2 Bellflower ethanol extract + LPS One 81.3 ± 8.1 ^* 10 67.0 ± 4.4 ^* 100 67.2 ± 4.9 ^* 200 65.7 ± 5.0 ^* Licorice extract 10 79.8 ± 4.2 ^* 50 68.9 ± 3.9 ^* 100 57.6 ± 5.6 ^* 200 46.3 ± 5.9 ^* *: p <0.05, compared with Control group

As shown in [Table 4], 10 μg / mL of bellflower ethanol extract showed 33% inhibition of production compared to the control group treated with LPS only, and showed a 34.3% inhibitory capacity at concentration of 200 μg / mL. The licorice extract with high anti-inflammatory activity showed a production inhibitory ability of 19.2% at a concentration of 10 µg / ml and a high production inhibitory ability of 53.7% at a concentration of 200 µg / ml. The bellflower ethanol extract shows higher activity at lower concentrations than licorice extract.

3). PGE ₂ production measurement

Measurement of PGE ₂ was used by purchasing the Prostaglandin E2 EIA Kit from Cayman Chemical Company (Ann Arbor, Michigan, USA), and the measurement method was quantified according to the manufacturer's analysis method. That is, RAW 264.7 cells were sampled and licorice extract together with LPS at a concentration of 0.01, 0.1, 1, 10, 50, 100, 200 μg / ml for 24 hours, and the cell culture supernatant was used for PGE ₂ measurement. 50 μl of each culture was loaded onto 96-well plates coated with goat anti-mouse. Here, 50 µl of primary antibody solution and 50 µl of PGE ₂ conjugate were added to react for 18 hours at 4 ° C., washed 5 times with Washing buffer, and then treated with 200 µl of Ellman's reagent and reacted with stirring for 60 to 90 minutes. Then, absorbance was measured at 405 to 420 nm, and the results are shown in [Table 5] and [Figure 3] below.

Sample name (µg / ml) Prostaglandin E ₂  Production amount (%) Control 34.6 ± 1.8 Control (+ LPS) 100.0 ± 5.0 Bellflower ethanol extract + LPS One 81.8 ± 0.8 ^* 10 88.8 ± 4.5 ^* 100 61.5 ± 3.9 ^* 200 59.4 ± 1.2 ^* Licorice extract 50 84.3 ± 2.1 ^* 100 67.2 ± 1.4 ^* *: p <0.05, compared with Control group

As shown in [Table 5], the amount of PGE ₂ produced in the bellflower ethanol extract was decreased by 18.2% and 11.2% at concentrations of 1 and 10 µg / ml compared to the LPS-treated group, and decreased by 40.6% at 200 µg / ml. Licorice extract was inhibited at 15.7% at 50µg / ml and 32.8% at a concentration of 100µg / ml. Inhibition of PGE ₂ production showed higher activity than licorice extract, and it was confirmed that the ethanol extract of bellflower ethanol extract had very high NO production inhibitory activity and PGE ₂ inhibitory activity, and had higher activity compared to licorice extract.

<Test Example 4> Proliferation efficacy test for dermal papilla cells

 Hair papilla cells (HFDPC) constitute hair follicles, and many studies have shown that proliferation and differentiation of hair papilla cells are primarily involved in the progression of hair growth cycle and hair formation. Active growth and differentiation of hair papillae occurs actively during the growth phase when hair is actively growing, hair growth is stopped, and these cells are killed during the degenerative, resting, and alopecia phases when hair loss occurs. Therefore, the proliferation of dermal papilla cells was measured to test the anti-hair loss effect of the bellflower ethanol extract.

 One). Cytotoxicity test

Cytotoxicity was confirmed by HDFPC cell, a papillary cell. That is, the HDFPC cell line cultured in the cell culture flask was passaged by removing the cells from the flask with a 10 ml pipette, and the cells were cultured with folicle dermal papilla cell growth medium containing 10% FBS. The HDFPC cell line was dispensed into a 96 well plate at a concentration of 1 × 10 ⁴ cells / well, and cultured in a CO ₂ incubator adjusted to 37 ° C., humidity 95%, and CO ₂ 5% for 24 hours. Samples were dissolved in fresh medium so that the final concentrations were 1, 10, 50, 100, and 200 µg / ml, treated with cell lines, and cultured for 24 hours. To measure the viability of the cell line, absorbance was measured at 490 nm using a microplate reader (Molecular Devices, VersaMax ELISA Microplate Reader, USA) using MTS reagent, and to confirm that toxicity was seen in papillary cells when treated with bellflower ethanol extract. After dividing the dermal papilla cells into 5x10 ³ / well, after 24 hours, the bellflower ethanol extract was treated with the concentration as shown in [Table 6] to confirm the toxicity of the cells. The survival rate of the cells was expressed as the absorbance of the sample treatment group compared to the control group not treated with the sample, and the results are shown in [Table 6] below.

Sample name (µg / ml) Cell Viability (%) Control group 100.0 ± 0.0 Bellflower ethanol extract One 108.3 ± 3.3 10 109.0 ± 3.1 50 101.7 ± 3.0 100 101.9 ± 3.7 200 85.2 ± 1.4 ^* *: p <0.05, compared with Control group

As shown in [Table 6], toxicity was confirmed at a concentration of 200 μg / ml of the bellflower ethanol extract.

 2). Cell regeneration efficacy test

  Based on the confirmed results of the papilloma cell toxicity test, treatment was performed on the papillary cells at a concentration of 1-100 μg / ml of bellflower ethanol extract, and then the proliferation process was examined until 3 days. As a result, as shown in [Table 7] below, Day 1 showed a high proliferation of 124.8% at 200 μg / ml, and showed a tendency to increase concentration-dependently. Decreased.

Sample name (µg / ml) Cell proliferation rate (%) 1 day 2 days 3 days Control group 100.0 ± 2.4 100.0 ± 6.2 100.0 ± 6.0 Bellflower ethanol extract One 100.3 ± 3.0 95.6 ± 1.2 107.4 ± 2.8 10 117.6 ± 7.0 ^* 104.1 ± 1.3 106.8 ± 2.4 100 122.6 ± 6.0 ^* 98.9 ± 3.4 102.8 ± 2.8 200 124.8 ± 6.1 ^* 96.4 ± 3.3 88.3 ± 5.3 ^* *: p <0.05, compared with Control group

<Test Example 5> Dihydrotestosterone production inhibitory activity test

By measuring the ability to inhibit the production of Dihydrotestosterone, which is known to cause hair loss, the efficacy of preventing the hair loss of the developed ingredients was measured. In the treatment of the sample used in the test, the liver was homogenized after PBS corresponding to twice the weight of the rat was added. After adding 50 μl of the test solution to 0.5 ml of homogenized liver, the mixture was reacted with stirring at 37 ° C. for 30 minutes. After completion of the reaction, 0.5 ml of extraction buffer (50 mM Na ₂ HPO _4, 0.25 M sucrose) was added and centrifuged at 4 ° C. and 1,500 g for 20 minutes to take a supernatant and proceed with a Dihydrotestosterone (DHT) kit. The DHT kit used IB59116 manufactured by Immuno-Biological Laboratories, Inc., and the results are shown in [Table 8] and [FIG. 5] below.

Sample name (µg / ml) Dihydrotestosterone (%) Control 100.0 ± 5.4 Bellflower ethanol extract One 78.3 ± 6.7 ^* 10 64.5 ± 7.3 ^* 50 76.8 ± 11.1 100 76.5 ± 14.0 Finasteride One 100.0 ± 0.9 10 68.5 ± 2.5 ^* 100 48.5 ± 2.8 ^* *: p <0.05, compared with Control group

As shown in [Table 8], the bellflower ethanol extract showed 23.5% inhibitory activity at a concentration of 100 μg / ml compared to the non-added control group, and Finasteride known to inhibit DHT production was 31.5 at a concentration of 10 μg / ml. % High activity inhibitory activity was confirmed.

Through the above <Test Example 1> to <Test Example 5> it was confirmed the hair loss prevention and improvement effect of the bellflower ethanol extract according to the present invention.

Below, in order to identify a substance having a hair loss prevention effect of a bellflower ethanol extract, the fraction obtained in Example 2 above, that is, a bellflower croroform fraction, a bellflower ethyl acetate fraction, a bellflower butanol fraction, and a bellflower water fraction as a sample The hair loss prevention effect was tested in the same manner as the test methods of <Test Example 1> to <Test Example 5>.

B. Prevention of hair loss and efficacy for improving hair growth of bellflower fraction

<Test Example 6> Antioxidant activity test of bellflower fractions

 In order to confirm the antioxidant activity of bellflower fractions, DPPH scavenging ability and ABTS Radical scavenging ability were measured in the same manner as in <Test Example 1>, and the results are shown in [Table 9] and [Table 10], respectively. Attached.

Sample concentration
(mg / ml) Sample name (%) Bellflower chloroform
Fraction Bellflower ethyl acetate fraction Bellflower
Butanol fraction Bellflower
Water fraction One 17.0 ± 3.0 11.5 ± 3.6 0.0 ± 2.3 5.1 ± 0.6 10 50.7 ± 7.3 48.0 ± 1.4 40.9 ± 4.1 8.9 ± 1.5 50 85.4 ± 6.6 77.2 ± 3.5 78.3 ± 3.2 26.8 ± 3.1 100 89.2 ± 4.3 94.4 ± 2.7 89.8 ± 1.0 41.2 ± 2.4 200 96.9 ± 2.7 96.8 ± 2.2 93.0 ± 0.3 51.9 ± 3.9 * IC ₅₀ : bellflower chloroform fraction 19.5mg / mL, bellflower ethyl acetate fraction 29.6mg / mL, bellflower butanol fraction 16.2mg / mL, bellflower water layer fraction 121.8mg / mL

Sample concentration
(mg / ml) Sample name (%) Bellflower chloroform
Fraction Bellflower ethyl acetate fraction Bellflower
Butanol fraction Bellflower
Water fraction One 32.0 ± 91.3 27.2 ± 0.7 19.5 ± 2.3 7.8 ± 1.2 10 91.3 ± 3.3 91.1 ± 2.5 90.0 ± 3.9 27.2 ± 1.3 50 100.0 ± 6.0 100.0 ± 3.3 100.0 ± 0.5 67.8 ± 2.3 100 97.6 ± 7.7 96.6 ± 5.6 100.0 ± 3.6 98.7 ± 1.4 200 95.3 ± 11.5 84.0 ± 5.9  93.4 ± 10.0 99.9 ± 0.2 * IC ₅₀ : bellflower chloroform fraction 3.7 mg / ml, bellflower ethyl acetate fraction 4.2mg / ml, bellflower butanol fraction 4.9mg / ml, bellflower water layer fraction 39.8mg / ml

As shown in [Table 9] and [Table 10], except for the bellflower water fraction, the remaining fractions showed superior DPPH scavenging effect as they were closer to the stock solution. Among them, the butanol fraction showed 50% or more scavenging ability at a lower concentration than other fractions (16.2 mg / ml), showing the most excellent effect among the four fractions.When comparing the IC ₅₀ values, the bellflower butanol fraction was followed by the bellflower chloroform , Ethyl acetate fractions were excellent in antioxidant effect.

Likewise, the ABTS scavenging ability was closer to the stock solution, and the radical scavenging ability of all fractions was superior. As a result of comparing the IC ₅₀ values, the bellflower chloroform and ethyl acetate fractions exhibiting the highest activity even at low concentrations showed the most excellent effect. As a result of comparing DPPH and ABTS activity with IC ₅₀ value, it was confirmed that ABTS radical scavenging ability was much superior to DPPH radical scavenging ability in all fractions.

<Test Example 7> Skin cell toxicity and regenerative effect test of bellflower fractions

 One). Skin cell toxicity test

  In the same manner as in <Test Example 2>, the skin cytotoxicity of the bellflower fractions was tested. In the cytotoxicity test, toxicity was observed at a high concentration of 50 μg / ml or more, and the toxicity was shown as a result of retesting with a minimum concentration of 0.001 to 10 μg / ml. The results are shown in Table 11 below and attached as [FIG. 7].

Sample name (㎍ / ml) Cell Viability (%) Control group 100 ± 5.2 Chloroform fraction 0.001 117.7 ± 4.9 ^* 0.005 109.3 ± 7.9 0.01 114.7 ± 10.3 ^* 0.1 102.2 ± 1.1 Ethyl acetate fraction 0.01 101.9 ± 13.9 0.1 110.0 ± 4.1 ^* One 107.3 ± 3.7 ^* 10 104.7 ± 2.3 ^* Butanol fraction 0.005 101.2 ± 7.8 0.01 100.9 ± 12.9 0.1 107.8 ± 2.6 ^* One 107.5 ± 1.8 ^* Water fraction 0.005 114.7 ± 5.1 ^* 0.01 101.8 ± 1.2 0.1 103.4 ± 7.5 One 98.6 ± 2.3 *: p <0.05, compared with Control group

 2). Skin cell regeneration effect test

  Based on the confirmed <Test Example 7> toxicity test results, the skin cell regeneration effect of bellflower fractions was tested and the results are shown in [Table 12] and [Figure 8] below.

Sample name (㎍ / ml) Cell proliferation rate (%) 1 day 2 days Control group Bellflower chloroform fraction 0.001 115.7 ± 3.5 ^* 108.2 ± 2.1 0.005 117.9 ± 1.9 ^* 115.6 ± 8.6 ^* 0.01 120.4 ± 4.0 ^* 105.6 ± 4.0 0.1 115.1 ± 2.6 ^* 105.3 ± 2.1 Bellflower ethyl acetate
Fraction 0.01 110.7 ± 5.7 ^* 107.8 ± 9.4 0.1 110.0 ± 2.7 ^* 116.5 ± 6.6 ^* One 108.5 ± 5.4 ^* 106.6 ± 9.6 10 108.5 ± 4.6 ^* 106.9 ± 3.9 Bellflower butanol fraction 0.005 113.6 ± 3.2 ^* 110.0 ± 6.2 0.01 121.1 ± 3.7 ^* 110.6 ± 2.2 0.1 125.7 ± 10.2 ^* 108.0 ± 2.3 One 131.4 ± 8.1 ^* 112.5 ± 7.3 Bellflower water fraction 0.005 110.8 ± 3.8 ^* 109.0 ± 8.6 0.01 119.3 ± 3.6 ^* 108.4 ± 3.9 0.1 116.2 ± 3.9 ^* 113.6 ± 5.7 ^* One 118.6 ± 9.3 ^* 106.7 ± 5.4 *: p <0.05, compared with Control group

As shown in [Table 12], all fractions had skin cell regeneration efficacy. In particular, the butanol fraction was found to have a proliferation efficiency of 25.7% at a concentration of 0.1 µg / ml and a high proliferation capacity of 31.4 at a concentration of 1 µg / ml. Chloroform and water fractions also showed proliferation rates close to 20%, but from day 2, proliferation rates decreased in all fractions compared to day 1. In the skin cell regeneration efficacy, the highest activity was found in the butanol fraction.

<Test Example 8> Anti-inflammatory efficacy test of bellflower fractions

 One). NO production inhibitory activity test

  In the same manner as in <Test Example 3>, the NO production inhibitory activity test of bellflower fractions was carried out at a concentration at which no toxicity appeared, and the results are shown in [Table 13] below.

Sample name (㎍ / ml) NO production (%) Control 11.6 ± 0.2 Control (+ LPS) 100.0 ± 1.2 Licorice extract + LPS 10 79.8 ± 4.2 ^* 50 68.9 ± 3.9 ^* 100 57.6 ± 5.6 ^* 200 46.3 ± 5.9 ^* Bellflower chloroform fraction One 81.5 ± 9.8 ^* 10 74.6 ± 3.6 ^* 50 61.5 ± 3.2 ^* 100 51.0 ± 4.2 ^* Bellflower ethyl acetate
Fraction One 79.2 ± 4.2 ^* 10 68.3 ± 3.0 ^* 100 57.0 ± 5.0 ^* 200 45.7 ± 5.6 ^* Bellflower butanol fraction 0.01 94.3 ± 4.5 0.1 92.9 ± 0.9 ^* One 88.5 ± 1.0 ^* 10 87.8 ± 2.6 ^* Bellflower water fraction One 91.3 ± 3.5 ^* 10 88.0 ± 2.2 ^* 50 90.5 ± 1.9 ^* 100 92.2 ± 1.7 ^* *: p <0.05, compared with Control group

As shown in [Table 13], the inhibitory activity for NO production was a chloroform, and the ethyl acetate fraction showed a tendency of concentration-dependent decrease in NO production. In the bellflower butanol and water fractions, the inhibitory effect of NO production was not higher than that of the chloroform fraction and the ethyl acetate fraction, but the activity was confirmed in all fractions. When compared to the licorice extract, which is known to have a high anti-inflammatory effect, the bellflower ethyl acetate fraction having the highest activity was found to have a similar effect of reducing NO production. The chloroform fraction also showed high production inhibitory activity similar to that of the ethyl acetate fraction.

2). PGE ₂ production inhibitory activity test

Based on the NO production inhibitory activity test results, the PGE ₂ production inhibitory effect was tested with a bellflower ethyl acetate fraction and a bellflower chloroform fraction having excellent NO production inhibitory effects, and the results are shown in [Table 14] and [FIG. 11] below.

Sample name (㎍ / ml) Prostaglandin E ₂  Inhibitory activity Control 34.6 ± 1.8 Control (+ LPS) 100.0 ± 5.0 Licorice extract + LPS 50 84.3 ± 2.1 ^* 100 67.2 ± 1.4 ^* Bellflower ethyl acetate fraction One 80.5 ± 1.6 ^* 10 67.8 ± 3.5 ^* 100 67.3 ± 2.0 ^* 200 63.2 ± 4.3 ^* Bellflower chloroform fraction One 74.3 ± 2.4 ^* 10 67.5 ± 2.2 ^* 50 76.3 ± 1.4 ^* 100 95.9 ± 0.9 *: p <0.05, compared with Control group

As shown in [Table 14], in the chloroform fraction, the amount of PGE ₂ production was changed at concentrations of 1, 10, and 50 μg / ml compared to the control group, but it was difficult to confirm the significance, and the bellflower ethyl acetate fraction had a concentration of 100 μg / ml licorice extract In 36.8% and 32.8%, it showed almost similar results to NO production inhibitory activity.

<Test Example 9> Antibacterial activity test of bellflower fractions

Dandruff is a phenomenon in which rice bran-shaped sclerosis occurs mainly due to the delamination of the stratum corneum from the scalp, and may be regarded as a minor symptom of psoriasis and seborrheic dermatitis. One of the causes of dandruff is the rapid reproduction of Malassezia furfur ( M. furfur ) fungi, which inhabit the skin for a long time and cause surface infection of the skin under certain conditions.Scalpitis, seborrheic dermatitis, eczema, dandruff, itching, hair loss, Since it causes acne, we observed the antibacterial activity effect of the bellflower extract against acne and dandruff.

 1) .Antibacterial test strain and culture

The strains used in this experiment were propionibacterium acnes ( P. acnes , acne fungus) and Malassezia furfur ( M. furfur , dandruff), which were distributed in the Microbial Resource Center KCTC (Korean Collection for Type Cultures) and used in the experiment. Reinforced Clostridial Medium shown in [Table A] below was used as the growth medium for P. acnes , and cultured using [Table B] shown below as M. furfur growth medium.

Table A

Table B

 2). Antibacterial activity measurement

To measure the antibacterial activity, a paper disc diffusion method of the sample was used using P. acnes and M. furfur strains. After taking a single colony of the cultured strain and inoculating it in a liquid medium, P. acnes and M. furfur were cultured at 37 ° C. for over 48 hours, and then 1000 μl was taken and uniformly plated on a solid medium. A sterilized disc paper (Toyo seisakusho, 8 mm) was prepared and inoculated with 25, 50 μl of each sample at a concentration of 200 μg / ml to measure antibacterial activity. As a result, a clear zone of 0.2 cm and 0.5 cm was formed when 25 μl of ethyl acetate fraction and chloroform fraction were treated among the fractions, as shown in [Figure 1] below, and when 0.8 μl treatment, 0.8 cm and 0.7 cm below. A clear zone was formed. Antibacterial activity against dandruff which is a cause of hair loss was confirmed by measuring high antibacterial activity in ethyl acetate and chloroforum fractions (in the picture below, in the photo of the antibacterial activity of dandruff of the bellflower fractions in Figure 1, 25 μl in the upper part and 50 μl in the lower part) to be).

[Figure 1]

 <Test Example 9> All cells of the bellflower fraction proliferation efficacy test

 One). Cytotoxicity test

  According to the same method as in <Test Example 4>, the bellflower fractions were treated with concentrations of 1, 10, 50, 100, and 200 µg / ml to confirm the proliferative effect on the dermal papilla cells, thereby confirming toxicity. Finasteride was used as a comparative control, and the results are shown in [Table 15] and [Figure 12] below.

Sample name (㎍ / ml) Cell Viability (%) Control group 100.0 ± 0.0 Bellflower chloroform
 Fraction One 106.4 ± 11.0 10 107.8 ± 1.8 ^* 50 60.4 ± 2.7 ^* 100 54.7 ± 2.1 ^* 200 54.0 ± 1.1 ^* Bellflower ethyl acetate fraction One 97.7 ± 1.2 10 87.4 ± 1.6 ^* 50 76.1 ± 7.7 ^* 100 76.1 ± 10.1 ^* 200 77.5 ± 3.3 ^* Bellflower butanol fraction One 100.2 ± 2.8 10 88.7 ± 1.9 ^* 50 62.8 ± 2.1 ^* 100 44.7 ± 0.6 ^* 200 43.4 ± 0.6 ^* Bellflower water fraction One 95.0 ± 3.1 10 101.4 ± 3.6 50 99.9 ± 2.9 100 103.4 ± 6.5 200 104.9 ± 3.5 Finasteride One 106.7 ± 1.3 ^* 10 105.3 ± 4.8 50 99.9 ± 3.6 100 100.9 ± 3.6 200 79.4 ± 4.7 ^* *: p <0.05, compared with Control group

As shown in [Table 15], toxicity was observed at most concentrations except for the bellflower water fraction and Finasteride.

 2). Hair papilla regeneration effect test

  In the results of the cytotoxicity test, the dermal papilla cell regeneration effect test was performed to test the dermal papilla cell regeneration activity only with a bellflower water fraction that did not show a toxic effect. Cell proliferation rate was measured.

As a result, as shown in [Table 16], the bellflower water fraction was found to have a significant cell proliferation capacity at a concentration of 50 μg / ml or more when cultured for 1 day, 2 days, 3 days, and 16.9% at a concentration of 200 μg / ml. Cell proliferation ability was shown. At the same concentration, 17.7% on day 2 and 18.2% on day 3 showed proliferative activity. It did not show a high rate of increase with increasing cell culture period.

Sample name (㎍ / ml) Breast papilla cell proliferation rate (%) 1 day 2 days 3 days Control 100.0 ± 0.5 100.0 ± 6.8 100.0 ± 4.8 Bellflower water fraction 10 103.3 ± 4.5 108.2 ± 5.7 107.2 ± 4.2 50 110.3 ± 2.3 ^* 109.3 ± 5.5 ^* 115.0 ± 5.6 ^* 100 112.9 ± 8.3 ^* 113.3 ± 5.6 ^* 115.8 ± 2.0 ^* 200 116.9 ± 6.6 ^* 117.7 ± 2.3 ^* 118.2 ± 3.8 ^* *: p <0.05, compared with Control group

<Test Example 10> Dihydrotestosterone production inhibitory activity test of bellflower fractions

 The effect of preventing the hair loss of the fraction was measured by measuring the inhibitory ability of Dihydrotestosterone, which is known to cause hair loss. The liver homogenate of the rat was treated with the homogenate of the rat homogenate containing a large amount of 5 alpha reductase as an enzyme solution and then cultured to measure the amount of dihydrotestosterone produced.

As shown in [Table 17], the ethyl acetate fraction showed inhibitory activity of about 20% at the concentrations of 50 µg / ml and 10 µg / ml, and the bellflower butanol fraction could not confirm the activity-dependent inhibitory activity.

As shown in [Table 18] below, the bellflower chloroform fraction showed a high reduction effect of 48.4% at a concentration of 100 µg / ml compared to the sample-untreated control group, and was the most effective among the fractions. Finasteride, which is known to inhibit DHT production, showed an inhibitory activity of 51.5% at a concentration of 100 μg / ml, showing almost the same effect as that of the bellflower chloroform fraction, thereby confirming the very high inhibitory effect of DIhydrotestosterone production.

Sample (µg / ml) Dihydrotestosterone (%) Control 109.1 ± 1.2 Bellflower ethyl acetate fraction One 127.6 ± 3.0 ^* 10 101.6 ± 1.2 ^* 50 83.9 ± 3.7 ^* 100 80.8 ± 5.7 ^* Bellflower butanol fraction One 71.3 ± 12.3 ^* 10 81.9 ± 8.7 ^* 50 76.1 ± 1.7 ^* 100 82.4 ± 3.2 ^* *: p <0.05, compared with Control group

Sample (µg / ml) Dihydrotestosterone (%) Control 100.0 ± 5.4 Bellflower chloroform fraction One 67.4 ± 2.4 ^* 10 63.3 ± 7.3 ^* 50 64.7 ± 3.7 ^* 100 51.6 ± 2.0 ^* Finasteride One 100.0 ± 0.9 10 68.5 ± 2.5 ^* 100 48.5 ± 2.8 ^* Liver (no reaction) 107.5 ± 1.5 *: p <0.05, compared with Control group

According to the results of the above <Test Examples>, the bellflower extract and fractions according to the present invention have an antioxidant effect and a skin cell regeneration effect, an anti-inflammatory effect that reduces NO and PGE ₂ production, and a dihydro-testosterone production inhibitory effect that causes hair loss. It showed that it is possible to confirm the potential as a natural extract to prevent hair loss, and the bellflower chloroform and ethyl acetate fraction had antibacterial activity effects to inhibit the growth of dandruff bacteria, confirmed the effect of proliferating the papillary cells, and reduced the amount of DHT production by 50% Since it can be seen that there is a bellflower extract and fractions according to the present invention can predict that the hair loss prevention and hair growth effect, and also predict that the bellflower extract and fractions of the present invention can be applied to the hair care product for hair loss prevention and hair growth Can be.

Claims (8)

A hair loss prevention and hair growth improvement composition comprising a bellflower extract or a bellflower fraction obtained from a bellflower extract as an active ingredient. The method according to claim 1, wherein the bellflower extract is a bellflower ethanol extract, the bellflower fraction is any one or more fractions selected from bellflower chlorform fraction, bellflower ethyl acetate fraction, bellflower butanol fraction and bellflower water fraction, preventing hair loss and improving hair growth. Composition. The method for preventing hair loss and improving hair growth according to claim 1 or 2, wherein the active ingredient contains 0.1 to 80% by weight based on the total weight of the composition. The method for preventing hair loss and improving hair growth according to claim 3, wherein the active ingredient has a DPPH radical scavenging ability. The method for preventing hair loss and improving hair growth according to claim 3, wherein the active ingredient has an ABTS radical scavenging ability. The method for preventing hair loss and improving hair growth according to claim 3, wherein the active ingredient has antioxidant activity. The method for preventing hair loss and improving hair growth according to claim 3, wherein the active ingredient has the ability to inhibit the production of nitrogen monoxide. The hair loss prevention and hair growth improvement composition according to claim 3 is hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nutrition cream, hair moisture cream, hair massage Cream, hair wax, hair aerosol, hair pack, hair nutrition pack, hair soap, hair cleansing foam, hair oil, hair drying agent, hair preservative, hair dye, hair wave agent, hair bleach, hair gel, hair glaze, hair dresser, Hair lacquer, hair moisturizer, hair mousse, and hair loss prevention and hair growth improvement composition, characterized in that made of any one of the formulation.

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