Composition for preventing hair loss or promoting hair growth comprising extract of above-ground part of carrots

Abstract

The present disclosure relates to a composition that is capable of preventing hair loss and promoting hair growth by comprising the extract of the above-ground part of carrots. The composition comprising the extract of the above-ground part of carrots of the present disclosure significantly increases the viability of dermal papilla cells and can inhibit the expression of 5a-reductase, thereby exhibiting excellent effects in preventing hair loss and promoting hair growth.

Claims

1. A composition for preventing hair loss or promoting hair growth, the composition comprising an extract of above-ground part of carrots.

2. The composition of claim 1, wherein the above-ground part of carrots is a leaf, a flower, a stem, a sprout, or a mixture thereof.

3. The composition of claim 1, wherein the extract of the above-ground part of carrots is extracted using water, C1 to C4 alcohol, or a mixture thereof as a solvent.

4. The composition of claim 1, wherein the extract of the above-ground part of carrots is contained in a concentration of 0.001% to 0.02% by weight.

5. The composition of claim 1, inhibits 5α-reductase.

6. A pharmaceutical composition for external use for skin, the composition comprising an extract of an above-ground part of carrots, wherein the above-ground part of carrots is extracted at room temperature by putting the above-ground part of carrots in a mixed solvent in which water and ethanol are mixed in a weight ratio of 1:9 to 5:5.

7. A cosmetic composition comprising an extract of an above-ground part of carrots, wherein the above-ground part of carrots is extracted at room temperature by putting the above-ground part of carrots in a mixed solvent in which water and ethanol are mixed in a weight ratio of 1:9 to 5:5.

Description

BEST MODE

[0019] Hereinafter, the present disclosure will be described in detail. Prior to this, the terms or words used in the present specification and claims should not be construed as being limited to conventional or dictionary meanings. An inventor should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that the concept of terms can be appropriately defined in order to describe his or her own invention in the best way. Therefore, since the configurations described in the embodiments described herein are only the most preferred embodiments of the present disclosure and do not represent all the technical ideas of the present disclosure, it should be understood that there may be various equivalents and modifications that may replace them at the time of the present application.

[0020] The composition for preventing hair loss or promoting hair growth of the present disclosure includes an extract of the above-ground part of carrots (Daucus carota).

[0021] The above-ground part of carrots may be leaves, flowers, stems, buds, or a mixture thereof, but the composition may preferably include leaves and stems extracts.

[0022] The extract of the above-ground part of carrots may be obtained by extracting the above-ground part of carrots by using water, C1 to C4 alcohol, or a mixture thereof as a solvent, but preferably, a mixture of water and ethanol as a solvent may be used. More preferably, the extract is preferable to use a mixed solvent in which water and ethanol are mixed in a weight ratio of 1:9 to 5:5 as a solvent.

[0023] The extract of the above-ground part of carrots may be obtained by extracting the above-ground part of carrots at room temperature, preferably, at a temperature range of 10° C. to 30° C., and more preferably, 15° C. to 25° C.

[0024] The extract of the above-ground part of carrots may be included in a concentration of 0.001% to 1% by weight in the composition, preferably 0.005% to 0.05% by weight, and most preferably, 0.01% to 0.02% by weight.

[0025] The composition of the present disclosure may significantly increase the viability of dermal papilla cells and inhibit the expression of 5α-reductase.

[0026] Human dermal papilla cells exist at the base of the follicle, and dermal papilla cells are responsible for hair growth and hair follicle cycle control by supplying oxygen and nutrition to the cells that make up the follicle. Therefore, when the proliferation of dermal papilla cells is promoted, hair becomes healthy, hair growth is promoted, and hair loss can be prevented. Because hair growth proceeds as the epithelial cells surrounding the dermal papilla divide to form a hair shaft, the dermal papilla cells play an important role in regulating the division of epithelial cells. In addition, in androgenetic alopecia, the site where male hormones act on hair follicles is also the dermal papilla, and dermal papilla cells play a very important role in hair growth.

[0027] In addition, 5α-reductase is known to have type 1 and type 2 isoenzymes. 5α-reductase-1 of type 1 is mainly distributed in a dermal papilla cell, sebaceous glands, and keratinocytes of epidermis and hair follicles, and 5α-reductase-2 of type 2 is mainly distributed in the prostate, genital skin, seminal vesicles, and epididymis. 5α-reductase is an enzyme that acts with testosterone to produce dihydrotestosterone (DHT), a major cause of hair loss, and the reduction of 5α-reductase-1 gene expression in human dermal papilla cells is known to be involved in hair loss prevention.

[0028] The composition is a pharmaceutical composition for external application to the skin and may be a cream, gel, patch, spray, ointment, plaster, lotion, liniment agent, paste, or cataplasma formulation, but is not limited thereto.

[0029] In addition, the composition is a cosmetic composition and may be a hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair conditioner, hair treatment, hair cream, hair nutrition cream, hair moisture cream, hair massage cream, hair wax, hair aerosol, hair pack, hair nourishment pack, hair soap, hair cleansing foam, hair oil, hair drying agent, hair preservative, hair dye, hair wave agent, hair bleach, hair gel, hair glaze, hairdressing agent, hair lacquer, hair moisturizer, hair mousse, or hair spray formulation, but is not limited thereto.

[0030] The composition may include ingredients commonly used in cosmetic compositions in addition to the extract of the above-ground part of carrots. For example, the composition may include adjuvants commonly used in the field of cosmetology or dermatology, such as fatty substance, organic solvent, solubilizer, thickener, gelling agent, emollient, antioxidant, suspending agent, stabilizer, foaming agent, fragrance, surfactant, water, an ionic or nonionic emulsifier, a filler, a sequestering agent, a chelating agent, a preservative, vitamins, a blocking agent, a wetting agent, essential oil, a dye, a pigment, a hydrophilic or lipophilic active agent, a lipid vesicle, or any other ingredients commonly used in cosmetics. The adjuvant is introduced in an amount generally used in the field of cosmetology or dermatology.

[0031] Hereinafter, examples and experimental examples will be described in detail to describe the present disclosure in detail. However, the embodiments according to the present disclosure may be modified in various other forms, and the scope of the present disclosure should not be construed as being limited to the embodiments described below. The embodiments of the present disclosure are provided to more completely explain the present disclosure to those of ordinary skilled in the art.

Example: Preparation of the Extract of the Above-Ground Part of Carrots

[0032] Carrot leaves were washed with distilled water, dried, and pulverized with a blender to obtain a fine powder sample. Next, a solvent in which ethanol and purified water were mixed at a weight ratio of 7:3 was added in an amount of about 20 times the volume of the fine powder sample and then extracted once at 25° C. After the extraction was performed, the resulting filtrate was filtered through a 400 mesh filter, concentrated using a decompression concentrator, and dissolved in purified water to a concentration of 5 mg/mL to prepare a stock solution. Then, the stock solution was diluted with purified water, and the working solution adjusted to individual concentration was used for the following test.

Test Example 1: Cell Culture

Human Follicle Dermal Papilla Cell Culture

[0033] Human follicle dermal papilla cells were purchased from PromoCell Co., and a human follicle dermal papilla cell growth medium containing fetal calf serum, bovine pituitary extract, basic fibroblast growth factor and insulin was used and cultured at 37° C. and 5% CO.sub.2 incubator.

MCTT HCE™ Culture

[0034] MCTT HCE™ is a human corneal model (organization) that reproduces the corneal epithelium using human corneal epithelial cells, and the tissue manufactured by Korea Bio-solution was purchased and used. The tissue placed on the agarose gel was transferred to a 24-well plate and pre-cultured at 37° C. and 5% CO.sub.2 incubator.

Test Example 2: Cytotoxicity Evaluation

MTT Assay

[0035] MTT assay is a representative method for measuring cell viability using the principle that MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) reacts with dehydrogenase of living cells to generate purple formazans.

[0036] In order to check the cytotoxicity, 1 × 10.sup.4 cells/mL of human dermal papilla cell were dispensed into a 48 well plate, and when the amount reached 80% or more, the extract of the above Example was treated with the concentration shown in Table 1 below and cultured for 24 hours. Then, 200 .Math.L of a 0.5 mg/mL solution of MTT diluted in the medium were added and cultured at 37° C. for 4 hours. After removing the solution, adding 200 .Math.L of DMSO, and transferring 100 .Math.L to a 96 well plate, absorbance was measured at 585 nm using a microplate reader. The average absorbance value for each sample group was obtained, and the cell viability was evaluated by comparing the average absorbance value with the absorbance value of the control group.

TABLE-US-00001 Division Cytotoxicity Treatment concentration (wt%) Cell viability (%) Untreated group - 100.000 Example 0.00005 100.496 0.0005 104.311 0.005 105.798 0.01 110.631 0.02 115.396 0.05 14.222

[0037] As shown in Table 1, it was confirmed that cytotoxicity did not appear when the extract of the above Example was treated at 0.02% by weight or less and that cell viability rapidly decreased due to strong cell toxicity when the concentration of the extract was more than 0.05% by weight.

Test Example 3: Dermal Papilla Cell Viability

[0038] When the cultured human dermal papilla cells were dispensed in a 48 well plate at 1 × 10.sup.4 cell/mL, and when the amount reached 80% or more, the extract of Example was treated at the concentrations shown in Table 2 below. The positive control group was treated with minoxidil at concentrations of 10 and 50 .Math.M. After culturing the cells for 24, 48, and 72 hours, 200 .Math.L of a 0.5 mg/mL solution of MTT diluted in the medium were added and cultured at 37° C. for 4 hours. After removing the solution, adding 200 .Math.L of DMSO, and transferring 100 .Math.L to a 96 well plate, absorbance was measured at 585 nm using a microplate reader. The average absorbance value for each sample group was obtained, and the cell viability was evaluated by comparing the average absorbance value with the absorbance value of the control group.

TABLE-US-00002 Division 24h 48h 72h Positive control group 10 .Math.M 104.056 107.385 115.000 50 .Math.M 107.407 116.168 124.815 Example 0% by weight 100.000 100.000 100.000 0.005% by weight 103.704.sup.# 109.980.sup.# 118.519.sup.# 0.01% by weight 116.931.sup.# 120.559.sup.# 128.519.sup.# 0.02% by weight 121.693.sup.# 125.749.sup.# 135.185.sup.#

[0039] As shown in Table 2, when the extract of Example was treated with 0.01 to 0.02% by weight, it was confirmed that the viability of human dermal papilla cells was superior to that of minoxidil, a positive control group was increased.

[0040] In addition, in order to confirm the effect according to the solvent composition, a water solvent (Comparative Example 1) and a mixed solvent (Comparative Example 2) in which water and ethanol were mixed in a weight ratio of 7:3 were used to prepare an extract of the above-ground part of carrots. After that, the viability test for human dermal papilla cell was performed in the same manner as above.

TABLE-US-00003 Purified water extraction (room temperature) Concentration (wt % ) 24H 48H 72H 0.005 90.585 93.923 91.023 0.01 94.047 95.844 91.377 0.02 100.142 96.198 90.574

TABLE-US-00004 Concentration (% by weight) Comparative Example 1 / Example Ratio (%) 24H 48H 72H 0.005 87.3 85.4 76.8 0.01 80.4 79.5 71.1 0.02 82.3 76.5 67.0

[0041] As shown in Table 3 above, when extracted with purified water, the viability of human dermal papilla cells is the same as 100% or less, so it can be confirmed that the extract of the above-ground part of carrots with purified water does not substantially improve hair loss.

[0042] In addition, the ratio (%) of Comparative Example ⅟Example of the 9 cells of Table 4 is, on average, about 78.5%. It was confirmed that the viability of human dermal papilla cells was significantly higher when extracted with 70% by weight of ethanol than when extracted with a water solvent.

TABLE-US-00005 30% by weight of ethanol extraction (room temperature) Concentration (% by weight) 24H 48H 72H 0.005 99.48 100.458 95.62 0.01 100.832 96.081 91.573 0.02 101.334 95.522 90.257

TABLE-US-00006 Concentration Comparative Example 2 / Example Ratio (%) (% by weight) 24H 48H 72H 0.005 95.9 91.3 80.7 0.01 86.2 79.7 71.3 0.02 83.3 76.0 66.8

[0043] The ratio (%) of Comparative Example 2/Example of the 9 cells of Table 6 was about 81.2% on average. It was confirmed that the viability of human dermal papilla cells was significantly higher when extracted with 70% by weight of ethanol than when extracted with 30% by weight ethanol.

[0044] Next, in order to confirm the effect according to the extraction temperature, the viability of the human dermal papilla cells was measured after obtaining the extract by heating it to 80° C. while using 70% by weight of ethanol (Comparative Example 3).

TABLE-US-00007 70% by weight of ethanol extraction (heated to 80° C.) Concentration (% by weight) 24H 48H 72H 0.005 108.195 111.45 102.377 0.01 111.175 112.723 106.796 0.02 113.063 112.265 116.618

TABLE-US-00008 Concentration (% by weight) Comparative Example 3 / Example Ratio (%) 24H 48H 72H 0.005 104.3 101.3 86.4 0.01 95.1 93.5 83.1 0.02 92.9 89.3 86.3

[0045] In the case of the present disclosure, as shown in Table 1, while the viability of human dermal papilla cell increases over time, the extracts obtained by heating in Comparative Example 3 of Table 7 showed a tendency to decrease over time. In addition, in Table 8 comparing the two results, it was confirmed that the heated extract showed only an effect of about 85% level after 72 hours when compared with the extract at room temperature.

[0046] Therefore, it was confirmed that the extract of the above Example could exhibit the effect of preventing hair loss and promoting hair growth by improving the viability of dermal papilla cells.

Test Example 4: Evaluation of Gene Expression of 5α-Reductase-1

Real-Time Polymerase Chain Reaction (qRT-PCR)

[0047] This test is a method to confirm the gene expression level of 5α-reductase-1 using a real-time polymerase chain reaction. RNA extracted from cells is used to make cDNA by reverse transcriptase and amplified through a polymerase chain reaction so that the expression level of the corresponding gene can be compared in real-time and relatively.

[0048] When the cultured human dermal papilla cells were dispensed in a 6-well plate at 2 × 10.sup.5 cell/mL, and the amount reached 90% or more, the extract of Example was treated in each well and cultured for 24 hours. Thereafter, cells were suspended by treatment with 0.05% Trypsin-EDTA (Gibco), and total RNA in the cells was extracted using an RNA extraction kit (RNeasy mini kit, Qiagen). cDNA was synthesized from the extracted total RNA using RNA to cDNA EcoDryTM Premix (Oligo dT) (Clontech). A real-time polymerase chain reaction was performed using the synthesized cDNA, Taqman Fast Advanced Master Mix (Applied Biosystems), and Taqman primer (5α-reductase-1: Hs00971645_g1, Applied Biosystems) of each target. For the relative quantitative analysis of each gene, GAPDH (Hs02786624_g1, Applied Biosystems), a housekeeping gene, was used.

[0049] During cDNA amplification, the amplification amount was checked in real-time to obtain a threshold cycle (CT), which is an intersection between the amplification curve and the threshold line, and a relative quantitative value (RQ) was obtained based on this value to confirm the relative mRNA expression of the target gene. The RQ was calculated by the following Formula.

[00001]${\text{RQ = 2}}^{-\text{ΔΔ}{\text{C}}_{\text{T}}}$

[00002]$\text{ΔΔ}{\text{C}}_{\text{T}}=\text{Δ}{\text{C}}_{\text{T}}\left(\text{treatment}\right)-\text{Δ}{\text{C}}_{\text{T}}\left(\text{control}\right)$

[00003]$\text{Δ}{\text{C}}_{\text{T}}={\text{C}}_{\text{T}}\left(\text{target gene}\right)-{\text{C}}_{\text{T}}\left(\text{housekeeping gene}\right)$

[0050] Treatment (test group): Example treatment [0051] Control (control group): negative control group [0052] target gene: 5α-reductase-1 [0053] housekeeping gene: GAPDH

TABLE-US-00009 Negative control group Example extract (0.005% by weight) 5α-reductase-1 gene expression level (RQ) Average 1.000 ± 0.062 0.672 ± 0.018 Significance probability (p-value) for negative control group - <0.05.sup.#

[0054] As shown in Table 9, it was confirmed that the extract of the above-ground part of carrots of Example, suppressed the expression of the 5α-reductase-1 gene (mRNA) compared to the negative control group.