KR20240171393A - A composition for improving, preventing and treating of hair loss, promoting hair growth comprising Cannabidiol - Google Patents

Abstract

The present invention relates to a composition for promoting hair growth, promoting hair growth, improving, preventing or treating hair loss, and by containing cannabidiol as an active ingredient, the composition can improve, prevent or treat hair loss and not only has an excellent effect of promoting hair growth or hair growth, but also has no side effects as it is derived from a natural product, so it can be utilized in various ways as a health functional food composition, a pharmaceutical composition, a quasi-drug composition and a cosmetic composition for promoting hair growth, preventing or improving hair loss.

Description

{A composition for improving, preventing and treating of hair loss, promoting hair growth comprising Cannabidiol}

The present invention relates to a composition containing cannabidiol (CBD) as an active ingredient, which promotes hair growth or hair growth and improves, prevents, or treats hair loss.

In recent modern society, the number of people suffering from hair loss is rapidly increasing due to various direct and indirect factors such as genetic factors, stress, westernized eating habits, and aging. Hair loss symptoms are steadily increasing not only in men but also in young women.

The above hair loss occurs when the decrease and shrinkage of hair follicles increases, causing repeated hair growth and loss. The hair follicles contain hair roots, hair papillae, and hair matrix cells.

The above-mentioned papilla cells are located at the very bottom of the hair follicle and are known to play a very essential role in the formation and growth of hair follicles because they are closely related to capillaries. In addition, since papilla cells directly control the hair production cycle, they are mainly used in research related to hair loss. Currently, the drug treatments approved by the U.S. Food and Drug Administration (FDA) to treat hair loss are minoxidil, which induces hair growth, and finasteride, which delays hair loss by inhibiting the activity of dihydrotestosterone (DHT), which directly affects male pattern baldness. However, although these treatments are widely used domestically and internationally, various side effects such as dizziness, allergic dermatitis, decreased sexual function, and infertility have been reported.

Therefore, there is a need to develop a safe treatment that is effective and has fewer side effects compared to existing hair loss treatments.

Meanwhile, overseas, hemp is classified into hemp and marijuana based on the content of delta-9tetrahydrocannabinol (THC), a hallucinogenic ingredient contained in marijuana. Hemp has a THC content of 0.3% or less and is used for industrial purposes, while marijuana with a THC content of 5-20% or more is restricted for use. The number of compounds in marijuana known to date is approximately 400, most of which are cannabinoids, phenols, and terpenes. Of these, about 90 cannabinoids, known as the main effective ingredients of marijuana, have been identified.

Cannabidiol (CBD), known as the main active ingredient of cannabis, has high activity but does not have psychoactive effects like THC, and has been approved for medical use by the US FDA. Medicines containing cannabidiol (CBD) as the main active ingredient include sativex, cesamet, and marinol. In addition, pharmacological research on cannabidiol (CBD) is actively underway, and various research results have been reported, such as anti-inflammatory effects, liver damage protection effects, and anti-diabetic effects.

Republic of Korea Patent No. 2107715 Republic of Korea registered patent no. 1853634

The purpose of the present invention is to provide a health functional food composition containing cannabidiol (CBD) as an active ingredient, which promotes hair growth or hair growth and prevents or improves hair loss.

In addition, another object of the present invention is to provide a pharmaceutical composition containing cannabidiol (CBD) as an active ingredient, which promotes hair growth or hair growth and prevents or treats hair loss.

In addition, another object of the present invention is to provide a pharmaceutical composition containing cannabidiol (CBD) as an active ingredient, which promotes hair growth or hair growth and alleviates hair loss.

In addition, another object of the present invention is to provide a cosmetic composition containing cannabidiol (CBD) as an active ingredient, which promotes hair growth or hair growth and improves hair loss.

The health functional food composition for promoting hair growth, promoting hair growth, preventing or improving hair loss of the present invention to achieve the above-mentioned purpose may contain cannabidiol (CBD) as an active ingredient.

The above cannabidiol (CBD) can be used at a concentration of 0.1 to 8 μM.

In addition, the pharmaceutical composition of the present invention for promoting hair growth, promoting hair growth, preventing or treating hair loss to achieve the other purposes mentioned above may contain cannabidiol (CBD) as an active ingredient.

In addition, the pharmaceutical composition for promoting hair growth, promoting hair growth, or alleviating hair loss of the present invention to achieve another purpose described above may contain cannabidiol (CBD) as an active ingredient.

In addition, the cosmetic composition for promoting hair growth, promoting hair growth, or improving hair loss of the present invention to achieve another purpose described above may contain cannabidiol (CBD) as an active ingredient.

The composition for promoting hair growth, promoting hair growth, improving, preventing or treating hair loss of the present invention is non-toxic, promotes the growth of hair cells, and significantly increases the expression of a protein that promotes the growth of hair cells, as well as increases the expression of a protein that promotes the growth cycle of hair cells, and therefore has excellent effects of promoting hair growth, promoting hair growth, improving, preventing or treating hair loss, and is effective in the manufacture of competitive health functional food compositions, pharmaceutical compositions, quasi-drug compositions and cosmetic compositions.

Figure 1A is a graph showing the DPPH radical scavenging activity (IC50) according to the concentration of ascorbic acid (L-ascorbic acid) used as positive control group 1; Figure 1B is a graph showing the DPPH radical scavenging activity (IC50) according to the concentration of cannabidiol (CBD) of Example 1 of the present invention.
Figure 2A is a graph showing the ABTS radical scavenging activity (IC50) according to the concentration of ascorbic acid (L-ascorbic acid) used as positive control group 1; Figure 2B is a graph showing the ABTS radical scavenging activity (IC50) according to the concentration of cannabidiol (CBD) of Example 1 of the present invention.
Figure 3 is a graph showing the cell survival rate according to the concentration of cannabidiol (CBD) of Example 1 of the present invention.
FIG. 4 is a graph showing the mRNA expression of growth factors related to hair growth, specifically (A) FGF1, (B) FGF7, (C) VEGF, and (D) IGF, when treated with cannabidiol (CBD) of Example 1 of the present invention.
Figure 5A is a graph showing the mRNA expression of genes related to hair growth inhibition, specifically (A) AR and (B) TGFβ1, when treated with cannabidiol (CBD) of Example 1 of the present invention.
FIG. 6A is a graph showing a Western blot and its quantification for measuring p-AKT/AKT protein expression upon treatment with cannabidiol (CBD) of Example 1 of the present invention; FIG. 6B is a graph showing a Western blot and its quantification for measuring p-ERK/ERK protein expression upon treatment with cannabidiol (CBD) of Example 1 of the present invention.

The present invention relates to a composition containing cannabidiol (CBD) as an active ingredient, which promotes hair growth or hair growth and improves, prevents, or treats hair loss.

Hereinafter, the present invention will be described in detail.

The composition for promoting hair growth, promoting hair growth, improving hair loss, preventing or treating hair loss of the present invention contains cannabidiol (CBD) as an active ingredient.

The above cannabidiol (CBD) is obtained from cannabis and is a non-psychoactive cannabinoid that can be used for recreational and medical purposes.

The above cannabis ( Cannabis sativa L. ) is an annual plant of the Cannabaceae family that has been widely cultivated in tropical and temperate regions since 12,000 years ago, centered around Central Asia, and includes wild ginseng, and cannabis chemovars and their variants, including variants var. indica and var. kafiristanica, Cannabis sativa subspecies sativa, Cannabis sativa subspecies indica, Cannabis sativa subspecies ruderalis, and genetically hybridized, selfed, or hybridized plants thereof, containing various kinds of cannabinoid compounds known as medicinal and pharmaceutical ingredients.

The above cannabidiol (CBD) is a compound with a molecular weight of 314.464 g/mol and a chemical formula of C21H30O2, and the drug Epidiolex, which is a drug of the above cannabidiol, is approved by the U.S. Food and Drug Administration for the treatment of epilepsy. Long-term side effects of the drug include drowsiness, loss of appetite, diarrhea, fatigue, anxiety, lethargy, and sleep disorders.

In the present invention, cannabidiol (CBD) is used at a concentration of 0.1 to 8 μM, preferably 0.5 to 6 μM. If the concentration is below the lower limit, the effects of promoting hair growth, promoting hair growth, improving hair loss, preventing or treating the condition may be significantly reduced, and if it exceeds the upper limit, toxicity may be induced and verification of safety is required.

The term "promoting hair growth" of the present invention means promoting hair growth and ultimately increasing the proportion of hair in the growth phase among all hair. Therefore, the term can have the same meaning as "improving hair growth", as it suppresses hair loss caused by a decrease in the proportion of hair in the growth phase.

The term "hair growth promotion" of the present invention means not only the hair growth function that generates new hair, or the function of promoting hair growth, but also the function of promoting the delay from the growth phase to the regression phase, thickening the hair, improving the density of the hair, and making existing hair grow healthily.

The term "hair loss improvement" of the present invention refers to the action of preventing hair loss by weakening the regression phase of hair follicles and inducing the growth phase. Therefore, the term can have the same meaning as "hair loss alleviation", "hair loss prevention" and "hair loss treatment", as it suppresses hair loss caused by a decrease in the proportion of hair in the growth phase.

The term "prevention" in the present invention means all actions of the composition of the present invention to suppress or delay hair loss by weakening the regression phase of hair follicles and inducing the growth phase.

The term "treatment" in the present invention means all actions of the composition of the present invention to suppress or delay hair loss by weakening the regression phase of hair follicles and inducing the growth phase.

Meanwhile, the term 'containing as an effective ingredient' in this specification means including a sufficient amount of cannabidiol (CBD) to achieve the efficacy or activity. Since cannabidiol (CBD) is a natural product and does not cause side effects to the human body even when administered in excessive amounts, the quantitative upper limit of cannabidiol (CBD) included in the composition of the present invention can be selected and implemented within an appropriate range by those skilled in the art.

The term "cosmetic composition" of the present invention can be prepared in the form of general emulsified formulations and solubilized formulations. The emulsified formulations include nourishing toners, creams, essences, etc., and the solubilized formulations include flexible toners, etc. The cosmetic composition can be prepared in a formulation selected from the group consisting of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, ampoules, powder foundations, emulsion foundations, wax foundations, and sprays, but is not limited thereto. Specifically, the cosmetic composition can be prepared in the form of a hypoallergenic cosmetic skin protectant, flexible toner, nourishing toner, nourishing cream, massage cream, essence, eye cream, serum, cleansing cream, cleansing foam, cleansing water, pack, cream, essence, spray, or powder.

In addition, the cosmetic composition may additionally include one or more cosmetically acceptable carriers that are blended with general skin cosmetics, and may appropriately blend conventional ingredients such as oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., but is not limited thereto.

The cosmetically acceptable carrier included in the above cosmetic composition varies depending on the formulation.

When the formulation of the above cosmetic composition is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or mixtures thereof can be used as a carrier component.

When the formulation of the above cosmetic composition is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane/butane or dimethyl ether may be additionally included.

When the formulation of the above cosmetic composition is a solution or emulsion, a solvent, a solubilizer or an emulsifier is used as a carrier component, and for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil can be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan can be used.

When the formulation of the above cosmetic composition is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as carrier components.

When the formulation of the above cosmetic composition is soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. can be used as carrier components.

In addition, the present invention provides a pharmaceutical composition for promoting hair growth, preventing or treating hair loss, comprising cannabidiol (CBD) as an active ingredient.

In the present invention, the pharmaceutical composition may additionally contain a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable carrier" in the present invention refers to a carrier or diluent that does not stimulate a living organism and does not inhibit the hair growth promoting, hair loss preventing or therapeutic activity and properties of the composition of the present invention. In a composition formulated as a liquid solution, acceptable pharmaceutical carriers include sterile and biocompatible substances, such as saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed.

Additionally, the pharmaceutically acceptable carrier of the present invention may include a non-natural carrier.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount, wherein the term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention, and the effective dosage level can be determined according to the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the excretion rate, the treatment period, the drug used in combination with or concurrently with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered alone or in combination with a known pterygium treatment agent. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration.

In addition, the present invention provides a pharmaceutical composition for promoting hair growth or alleviating hair loss, which contains cannabidiol (CBD) as an active ingredient.

The term "quasi-drug" in the present invention means a product that is used for the purpose of diagnosing, treating, improving, alleviating, managing or preventing diseases of humans or animals, and has a milder effect than a pharmaceutical product. For example, according to the Pharmaceutical Affairs Act of the Republic of Korea, a quasi-drug is a product excluding products used for the purpose of pharmaceutical products, and includes products used for the treatment or prevention of diseases of humans or animals, products that have a mild effect on the human body or do not act directly, etc.

The pharmaceutical composition of the present invention can be manufactured in a form selected from the group consisting of a body cleanser, foam, soap, mask, ointment, cream, lotion, essence, and spray, but is not limited thereto.

When the composition containing cannabidiol (CBD) of the present invention is used as an over-the-counter drug additive, the composition containing cannabidiol (CBD) of the present invention may be added as is or used together with other over-the-counter drugs or over-the-counter drug ingredients, and may be used appropriately according to a conventional method.

In addition, the present invention provides a health functional food composition for promoting hair growth and preventing or improving hair loss, which contains cannabidiol (CBD) as an active ingredient.

In the present invention, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients having useful functionality for the human body.

Here, "functionality" means obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during the manufacturing process. In addition, the formulation of the health functional food can be manufactured without limitation as long as it is a formulation recognized as a health functional food.

The health functional food of the present invention can be manufactured in various forms of formulations, and unlike general drugs, it has the advantage of having no side effects that may occur with long-term use of drugs since it uses food as a raw material, and it is highly portable, so the health functional food of the present invention can be taken as a supplement to enhance the effects of promoting hair growth and alleviating hair loss.

The health functional food of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As a sweetener, a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame can be used.

In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination. The ratio of these additives is not particularly important, but is generally selected in the range of 1 to 100 parts by weight per 100 parts by weight of the health functional food of the present invention, but is not particularly limited thereto.

Hereinafter, preferred examples are presented to help understand the present invention, but the following examples are only illustrative of the present invention, and it will be apparent to those skilled in the art that various changes and modifications are possible within the scope and technical idea of the present invention, and it is natural that such changes and modifications fall within the scope of the appended patent claims.

Example 1.

Cannabidiol (CBD, Averix Bio, Wilson, NC, USA) obtained from hemp was used.

<Example> In vitro ( In vitro )

Cell culture

Human dermal papilla cells (HDPCs) were obtained from Promo cell (C-12071, Heidelberg, Germany) and used in the experiment. They were cultured in low glucose Dulbecco's modified eagle's medium (DMEM, Hyclone, Logan, Utah, USA) supplemented with 10% fetal bovine serum (FBS, Welgene, Gyeongsan, Korea) and 1% Antibiotic-Antimycotic (Gibco, MA, USA). The cells were cultured at 37°C and 5% CO2, and the medium was changed every two days. 0.25% trypsin-EDTA was used for subculture.

Statistical analysis

All experiments were repeated at least three times, and data were expressed as mean ± standard deviation (S.D.). One-way analysis of variance (ANOVA) test was performed on the mean using SPSS 26 (SPSS Inc, Armonk, NY, USA), and the least-significant difference (LSD) test was performed post hoc. Statistical significance for the difference in the mean values of each group was expressed at p<0.05, p<0.01, and p<0.001.

Test Example 1. Measurement of antioxidant activity

1-1. DPPH radical scavenging activity (%): The DPPH free radical scavenging method by Blosis was used as a method for measuring antioxidant activity. DPPH radical scavenging activity is a representative experimental method used to measure the amount of antioxidant substances contained in a sample. 100 μl of a sample of a certain concentration and 100 μl of a 60 μM DPPH solution were mixed, and reacted at room temperature in the dark for 30 minutes. The reaction solution was measured using the absorbance at 450 nm using a UV spectrophotometer (Infinite M200, Tecan, Salzburg, Austria), and the result was calculated. The amount of sample required to reduce the absorbance of the control group to which no sample was added by 50% was expressed as IC50.

1-2. ABTS radical scavenging activity (%): ABTS radical scavenging activity was measured to compare and evaluate antioxidant efficacy. 7 mM ABTS and 2.45 mM potassium persulfate were dissolved in distilled water, and reacted for 12 hours in the dark. The absorbance value of the reaction solution was corrected to 0.70±0.02 using ethanol at 415 nm. 5 μl of the sample was added to 95 μl of the ABTS solution, reacted for 15 minutes, and then the absorbance was measured at 415 nm.

Figure 1A is a graph showing the DPPH radical scavenging activity (IC50) according to the concentration of ascorbic acid (L-ascorbic acid) used as positive control group 1; Figure 1B is a graph showing the DPPH radical scavenging activity (IC50) according to the concentration of cannabidiol (CBD) of Example 1 of the present invention.

In addition, Fig. 2A is a graph showing the ABTS radical scavenging activity (IC50) according to the concentration of ascorbic acid (L-ascorbic acid) used as positive control group 1; Fig. 2B is a graph showing the ABTS radical scavenging activity (IC50) according to the concentration of cannabidiol (CBD) of Example 1 of the present invention.

Ascorbic acid (L-ascorbic acid), known as a representative antioxidant, was used as positive control group 1.

As shown in Figures 1A and 1B, the IC50 value of the positive control group 1, ascorbic acid (L-ascorbic acid), was confirmed to be 16.62±0.13 μM, and the IC50 value of cannabidiol (CBD) was confirmed to be 15.46±0.24 μM.

In addition, as shown in FIGS. 2A and 2B, the ABTS radical scavenging activity was confirmed to have an IC50 value of 19.32±0.57 μM for ascorbic acid (L-ascorbic acid), which is a positive control group 1, and an IC50 value of 13.90±0.06 μM for cannabidiol (CBD).

It is known that excessively generated active oxygen in the metabolic process of the human body has negative effects such as cell damage and inflammation. In addition, these active oxygens are known to be the main factor in causing hair loss by reducing hair production and causing cell damage.

Cannabidiol (CBD) has been confirmed to have excellent radical scavenging activity, and based on this, it can be expected to have an effect of preventing cell damage from active oxygen.

Test Example 2. Measurement of cell viability

To evaluate the cytotoxicity of cannabidiol (CBD) in Example 1, EZ-Cytox reagent, a water soluble tetrazolium salt-1 (WST-1) measurement method, was used. Dermal papilla cells were seeded in a 96-well plate at 4 × 10 ⁴ cells/well and adapted for 24 hours in a 37 °C, 5% CO2 incubator. Afterwards, cannabidiol (CBD) was treated at various concentrations. DMSO containing the sample dissolved in it was used as the control group, and minoxidil (MNXD) was used as positive control group 2 and incubated for another 24 hours. 10 μl of EZ-Cytox solution was added per well, incubated for 1 hour, and then the absorbance was measured at 450 nm using an ELISA plate reader.

Figure 3 is a graph showing the cell survival rate according to the concentration of cannabidiol (CBD) of Example 1 of the present invention.

Recently, research on hair papilla cells, which are closely related to hair growth and regeneration, has been actively conducted, and research on the biological mechanisms of hair papilla cell proliferation and death is on the rise.

As shown in Fig. 3, when cannabidiol (CBD) was treated at a concentration of 10 μM or higher, it was confirmed that cell viability decreased to less than 80%, indicating cytotoxicity, and it was confirmed that cell proliferation occurred at a concentration of cannabidiol (CBD) of 5 μM or lower.

Accordingly, considering the high proliferation rate of mammary papilla cells, cannabidiol (CBD) of Example 1 was used at a concentration of 5 μM or less in subsequent experiments.

Test Example 3. Measurement of hair growth-related gene expression_Real-time PCR

To evaluate the gene expression levels of key molecules related to cell growth, dermal papilla cells were cultured. The control group was treated with DMSO, the positive control group 2 was treated with minoxidil at a concentration of 10 μM, and cannabidiol (CBD) at concentrations of 1 μM, 2.5 μM, and 5 μM, respectively. After culturing for 24 h, the cells were washed three times with cold PBS, and RNA was isolated using a Nucleo spin RNA/Protein kit (MACHEREY-NAGEL, Düren, Germany). The extracted RNA was quantified, and cDNA was synthesized according to the manual using a High Capacity cDNA Reverse Transcription Kit (Applied biosystems, MA, USA). Primers include glyceraldehyde-3-phos-phate dehydrogenase (GAPDH; Hs0286624_g1), fibroblast growth factor 1 (FGF1; Hs01092738_m1), fibroblast growth factor 7 (FGF7; Hs00940253_m1), and vascular endothelial growth factor A (VEGF; Hs00900055_m1), insulin like growth fac-tor-1 (IGF-1; Hs01547656_m1), androgen receptor (AR; Hs00171172_m1), and transforming growth factor beta 1 (TGFβ1; Hs00998133_m1) were used, and mRNA expression level was measured using Luna Universal qPCR Master Mix (New England Biolabs, MA, USA) using the CFX-96 Real-time PCR System (Bio-Rad, The study was conducted in Hercules, CA, USA.

Figure 4 is a graph showing the mRNA expression of growth factors related to hair growth, specifically (A) FGF1, (B) FGF7, (C) VEGF, and (D) IGF, when treated with cannabidiol (CBD) of Example 1 of the present invention. *p<0.05, **p<0.01, ***p<0.001

To confirm the hair growth effect of cannabidiol (CBD), real-time PCR was used to evaluate hair growth-related genes. Fibroblast growth factors, which stimulate hair growth, induce the growth phase, and play a role in healing during the wound recovery process, are closely related to hair growth.

As shown in Figures 4A to 4D, the expression level of FGF1, a fibroblast growth factor in hair papilla cells, increased in the minoxidil treatment group, which is the positive control group 2, compared to the control group (CON), and the cannabidiol (CBD) treatment group showed a tendency for the expression level to increase at a concentration of 2.5 μM compared to the control group (CON), and it was confirmed that the expression levels in the 1 μM and 5 μM concentration treatment groups were significantly increased, respectively.

In addition, FGF7 is known as a biomarker that promotes the proliferation of hair germ and stromal cells and confirms a new hair cycle in hair papilla cells. As a result of measuring the expression level of FGF7, there was no significant difference between the minoxidil treatment group and the cannabidiol (CBD) 1 μM and 2.5 μM treatment groups compared to the control group (CON), but it was confirmed that there was a significant increase of 158.42% in the 5 μM treatment group.

In addition, it is known that hair papilla cells supply nutrients to hair follicles through capillaries. VEGF is emerging as an important factor in the growth process of hair follicles by expanding blood vessels, and research results have been reported in animal experiments that VEGF not only has the effect of creating blood vessels around hair follicles, but also increases hair size and promotes growth rate. As a result of measuring the expression of VEGF, there was no significant difference in the minoxidil treatment group compared to the control group (CON), and it was confirmed that the cannabidiol (CBD) treatment group showed an increasing trend, although not significant, at all concentrations.

In addition, IGF-1, which has a similar structure to insulin, is known to be a factor that helps hair growth by promoting the growth of hair follicle cells and preventing apoptosis of hair papilla cells. As a result of measuring the expression of IGF-1, it was confirmed that it increased in the minoxidil treatment group compared to the control group (CON), and the cannabidiol (CBD) treatment group showed an increasing trend although it was not significant at a concentration of 2.5 μM, and it was confirmed that it significantly increased in the cannabidiol (CBD) 1 μM and 5 μM treatment groups, respectively.

It was confirmed that treatment with cannabidiol (CBD) increased the growth regulatory factors FGF1, FGF7, IGF-1, and VEGF in hair papilla cells compared to the control group, and showed a higher increase rate than the minoxidil treatment group used as positive control group 2. Therefore, it is thought that cannabidiol (CBD) may play an important role in increasing hair growth.

Test Example 4. Gene Expression Related to Hair Growth Inhibition

To evaluate the level of gene expression related to hair growth inhibition, the same method as in Test Example 3 was used.

Figure 5A is a graph showing the mRNA expression of genes related to hair growth inhibition, specifically (A) AR and (B) TGFβ1, when treated with cannabidiol (CBD) of Example 1 of the present invention. *p<0.05, **p<0.01, ***p<0.001

Androgen receptor (AR) is known to reduce the size and number of hair follicles, promote secretion in the catagen phase of the hair growth cycle, and induce cell apoptosis; TGFβ1 is known to be a factor that affects hair loss by inhibiting hair growth and promoting the catagen phase in the cell cycle more than normal.

As shown in Figures 5A and 5B, the expression of AR was confirmed to decrease in the minoxidil treatment group (positive control group 2) compared to the control group (CON), and it was confirmed to significantly decrease in the cannabidiol (CBD) treatment group compared to the control group (CON).

In addition, the expression of TGFβ1 in mammary papilla cells was confirmed to be significantly reduced in the minoxidil treatment group compared to the control group (CON), and was confirmed to be significantly reduced in all cannabidiol (CBD) treatment groups compared to the control group (CON).

In the group treated with cannabidiol (CBD), factors involved in hair growth inhibition showed a high reduction rate, indicating that cannabidiol (CBD) is effective for hair growth.

Test Example 5. Measurement of protein expression related to hair growth regulation_Western blot

Human papilla cells were treated with cannabidiol (CBD) at various concentrations (1, 2.5, 5 μM) or 10 μM minoxidil for 24 h, washed twice with cold PBS, and proteins were extracted using a Nucleo spin RNA/Protein kit (MACHEREY-NAGEL, Düren, Germany). Proteins were separated by SDS-PAGE and transferred to a 0.2 μm Trans-Blot Turbo mini PVDF transfer pack (Bio-Rad Laboratories, Hercules, CA, USA). Nonspecific protein binding sites were blocked by reacting with Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% BSA for 1 h. The membrane was incubated overnight at 4 °C in a refrigerator with the primary antibody AKT (#4685s, Cell Signaling Technology, MA, USA), phospho-AKT (#4060s, Cell Signaling Technology, MA, USA), ERK (#9102s, Cell Signaling Technology, MA, USA), and phospho-ERK (#9101s, Cell Signaling Technology, MA, USA) and washed three times for 10 minutes with TBST (TBS containing 0.1% Tween 20). Then, the membrane was reacted with the secondary antibody Goat anti-rabbit IgG conjugates horseradish peroxidase solution at room temperature for 2 hours, and then washed three times for 5 minutes with TBST. β-actin (sc-47778, Santa Cruz Biotechnology, TX, USA) was used as the internal standard protein, and the expression of the target protein was measured using the ChemiDocTM MP Imaging System (Bio-rad, Hercules, CA, USA) with Clarity Max Western ECL Substrate luminescent reagent (Bio-rad, Hercules, CA, USA). The bands were quantified and displayed using Image J software (Version v1.32j, National Institute of Health).

FIG. 6A is a graph showing a Western blot and its quantification for measuring p-AKT/AKT protein expression upon treatment with cannabidiol (CBD) of Example 1 of the present invention; FIG. 6B is a graph showing a Western blot and its quantification for measuring p-ERK/ERK protein expression upon treatment with cannabidiol (CBD) of Example 1 of the present invention.

AKT and ERK are members of the mitogen activated protein kinase (MAPK) family and are regulated by growth factors and play a role in cell proliferation and various interactions within cells.

As shown in Figures 6A and 6B, the expression of p-AKT/AKT, a factor involved in the proliferation of breast papilla cells, was confirmed to have increased by 17.58% in the minoxidil-treated group, which is the positive control group 2, compared to the control group (CON), and was confirmed to have increased by 16.89%, 39.73%, and 45.39%, respectively, in the cannabidiol (CBD)-treated group compared to the control group (CON), depending on the concentration.

In addition, the expression of p-ERK/ERK was confirmed to increase by 38.05% in the minoxidil treatment group, which is the positive control group 2, compared to the control group (CON), and the cannabidiol (CBD) treatment group was confirmed to increase by 29.68%, 33.37%, and 72.45%, respectively, according to concentration compared to the control group (CON).

Activated AKT and ERK are deeply involved in cell proliferation and are known to induce proliferation of human papilla cells. Treatment with cannabidiol (CBD) in papilla cells showed a tendency to increase the protein expression levels of p-AKT/AKT and p-ERK/ERK, and it was confirmed that in the high concentration of cannabidiol (CBD) treatment group of 5 μM, each significantly increased compared to the control group.

Therefore, it was confirmed that cannabidiol (CBD) has a positive effect on the proliferation and growth of hair papilla cells by increasing the activation of both AKT and ERK in hair papilla cells. Based on these results, cannabidiol (CBD) can be used as a material that can help hair growth, and cannabidiol (CBD) can also be used to improve hair loss-related diseases.

Below, a formulation example of a composition containing the powder of the present invention is described, but the present invention is not intended to be limited thereto but is merely intended to be described specifically.

Preparation Example 1. Preparation of a mountain product

500 mg of cannabidiol (CBD) of Example 1

100 mg lactose

10 mg of talc

The above ingredients are mixed and filled into a sealed bag to produce a powdered preparation.

Preparation Example 2. Preparation of tablets

300 mg of cannabidiol (CBD) of Example 1

Corn starch 100 mg

100 mg lactose

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are manufactured by pressing them according to the usual method for manufacturing tablets.

Preparation Example 3. Preparation of capsules

200 mg of cannabidiol (CBD) of Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above ingredients are mixed according to the conventional capsule manufacturing method and filled into a gelatin capsule to manufacture a capsule.

Preparation Example 4. Preparation of injection

600 mg of cannabidiol (CBD) of Example 1

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Na ₂ HPO _4, 12H ₂ O 26 mg

According to the manufacturing method of conventional injections, it is manufactured with the above ingredient content per ampoule.

Preparation Example 5. Preparation of liquid preparation

4 g of cannabidiol (CBD) of Example 1

10 g of isoflavone

Mannitol 5 g

Appropriate amount of purified water

According to the usual method for manufacturing a liquid, each ingredient is dissolved in purified water, an appropriate amount of lemon flavor is added, the above ingredients are mixed, purified water is added, and the total amount is adjusted to 100 g by adding purified water, and then the liquid is filled into a brown bottle and sterilized.

Preparation Example 6. Preparation of granules

1,000 mg of cannabidiol (CBD) of Example 1

Vitamin mixture appropriate amount

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Appropriate amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium phosphate monobasic 15 mg

55 mg of dibasic calcium phosphate

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the above vitamin and mineral mixture is a preferred example of mixing ingredients relatively suitable for granules, but the mixing ratio may be arbitrarily modified, and granules may be manufactured by mixing the above ingredients according to a conventional method for manufacturing granules, and then used to manufacture a health functional food composition according to a conventional method.

Preparation Example 7. Manufacturing of functional beverages

1,000 mg of cannabidiol (CBD) of Example 1

1,000 mg of citric acid

100 g of oligosaccharide

2 g of plum concentrate

1 g of taurine

Add purified water to make a total of 900 mL

The above ingredients are mixed according to a conventional health beverage manufacturing method, stirred and heated at 85°C for about 1 hour, the resulting solution is filtered, placed in a sterilized 2 L container, sealed and sterilized, then refrigerated and used to manufacture the functional beverage composition of the present invention.

The above composition ratio is a preferred example of mixing ingredients suitable for relatively preferred beverages, but the mixing ratio may be arbitrarily modified according to regional and national preferences such as demand class, demand country, and intended use.

Preparation Example 8. Manufacturing of cosmetics in emulsion form

Cosmetics in emulsified formulations such as nutritious toners, creams, and essences, as well as cosmetics in soluble formulations such as flexible toners, were manufactured.

An emulsified cosmetic was manufactured with the composition described in Table 1 below. The manufacturing method is as follows.

1) A mixture of raw materials 1 to 9 was heated to 65 to 70°C.

2) 10 raw materials were added to the mixture of step 1).

3) The mixture of raw materials 11 to 13 was heated to 65 to 70°C and completely dissolved.

4) While going through the above step 3), the mixture of the above 2) was slowly added and emulsified at 8,000 rpm for 2 to 3 minutes.

5) Dissolve the raw material of 14 in a small amount of water, add it to the mixture of step 4), and emulsify for 2 more minutes.

6) After weighing each of the raw materials from 15 to 17, add them to the mixture of step 5) and emulsify for another 30 seconds.

7) The mixture of step 6) was emulsified, degassed, and cooled to 25 to 35°C to produce an emulsified cosmetic.

furtherance Emulsion type 1 Emulsion type 2 Emulsion type 3 1 Stearic acid 0.3 0.3 0.3 2 Stearyl alcohol 0.2 0.2 0.2 3 Glyceryl Monostearate 1.2 1.2 1.2 4 beeswax 0.4 0.4 0.4 5 Polyoxyethylene sorbitan
Monolauric acid ester 2.2 2.2 2.2 6 Methyl parahydroxybenzoate 0.1 0.1 0.1 7 Paraoxybenzoic acid profile 0.05 0.05 0.05 8 Cetyl ethylhexanoate 5 5 5 9 Triglycerides 2 2 2 10 Cyclomethicone 3 3 3 11 Distilled water ~ 100 ~ 100 ~ 100 12 Concentrated glycerin 5 5 5 13 Triethanolamine 0.15 0.15 0.15 14 polyacrylic acid polymer 0.12 0.12 0.12 15 pigment 0.0001 0.0001 0.0001 16 incense 0.10 0.10 0.10 17 Example 1 0.0001 1 10

Preparation Example 9. Preparation of cosmetics in the form of a solubilized formulation

A cosmetic in the form of a solubilized formulation was manufactured with the composition described in Table 2 below. The manufacturing method is as follows.

1) Add raw materials 2 to 6 to raw material 1 (purified water) and dissolve using a still mixer.

2) Add raw materials 8 to 11 to raw material 7 (alcohol) and completely dissolve.

3) The mixture of step 2) was slowly added to the mixture of step 1) to solubilize it.

furtherance Soluble formulation 1 Soluble formulation 2 Availability Formulation 3 1 Purified water ~ 100 ~ 100 ~ 100 2 Concentrated glycerin 3 3 3 3 1,3-Butylene glycol 2 2 2 4 EDTA-2Na 0.01 0.01 0.01 5 pigment 0.0001 0.0002 0.0002 6 Example 1 0.1 5 5 7 Alcohol (95%) 8 8 8 8 Methyl parahydroxybenzoate 0.1 0.1 0.1 9 polyoxyethylene
Hydrogenated diethyl ester 0.3 0.3 0.3 10 incense 0.15 0.15 0.15 11 Cyclomethicone - - 2

Claims (5)

A health functional food composition for promoting hair growth, promoting hair growth, preventing or improving hair loss, characterized by containing cannabidiol (CBD) as an active ingredient. A health functional food composition for promoting hair growth, promoting hair growth, preventing or improving hair loss, characterized in that in claim 1, the cannabidiol (CBD) is used at a concentration of 0.1 to 8 μM. A pharmaceutical composition for promoting hair growth, promoting hair growth, preventing or treating hair loss, characterized by containing cannabidiol (CBD) as an active ingredient. A pharmaceutical composition for promoting hair growth, promoting hair growth, or alleviating hair loss, characterized by containing cannabidiol (CBD) as an active ingredient. A cosmetic composition for promoting hair growth, promoting hair growth, or improving hair loss, characterized by containing cannabidiol (CBD) as an active ingredient.