Composition for promoting hair growth or preventing, treating, or alleviating hair loss, comprising strain of genus janthinobacterium or violacein

Abstract

The present invention relates to a composition for promoting hair growth or preventing or treating hair loss, the composition comprising a microorganism of the genus Janthinobacterium or violacein. The microorganism of the genus Janthinobacterium or violacein according to one aspect or a solvate or salt thereof has the effects of increasing the sphere size of dermal papilla cells, inducing hair follicle growth, inducing the differentiation or proliferation of hair follicle stem cells, and reducing hair follicle degeneration, and thus has the effect of promoting hair growth or treating hair loss.

Description

Composition for promoting hair growth or preventing, treating or improving hair loss, comprising a strain of the genus Xantinobacterium or violacein

[0001] The present invention relates to a composition for promoting hair growth or preventing or treating hair loss, comprising a strain of the genus Janthinobacterium sp. or violacein.

[0002] Hair loss is a condition in which hair falls out from the scalp tissue along with itching due to the constriction of the scalp capillaries caused by various factors such as genetic, environmental, and psychological factors. It is one of the concerns of modern people. The causes of hair loss are excessive mental stress, mental overwork, and an instant food lifestyle, which lead to poor blood circulation in the scalp, malnutrition of hair root cells, and a decline in the function of the endocrine system. This significantly reduces the number of hair root cells growing compared to the number of hair root cells dying, resulting in premature hair loss even during the growth phase. Furthermore, inhibition of the growth of dermal papilla cells, which are involved in hair follicle development and hair growth, is known to cause a decrease in the size of the dermal papilla region within the hair follicle, which can lead to a decrease in hair thickness and hair loss.

[0003] Medications used to prevent hair loss include androgen regulators like finasteride and chemical scalp preparations like minoxidil. However, these all contain chemical ingredients, leading to resistance among users to long-term use. Therefore, there is a pressing need for hair loss prevention or treatment products that are safe for the human body.

[0004] One object of the present invention is to provide a composition for promoting hair growth or preventing, treating or improving hair loss, comprising a microorganism of the genus Janthinobacterium, a dried product, a lysate, a lysate, a culture medium, an extract thereof or a fraction of an extract thereof; or violacein, a hydrate, a solvate or a salt thereof.

[0005] Another object of the present invention is to provide a pharmaceutical composition for promoting hair growth or preventing, treating or improving hair loss, comprising the above composition.

[0006] Another object of the present invention is to provide a food composition for promoting hair growth or preventing or improving hair loss, including the composition.

[0007] Another object of the present invention is to provide a cosmetic composition for promoting hair growth or preventing or improving hair loss, including the composition.

[0008] Another object of the present invention is to provide a method for promoting hair growth, preventing hair loss, treating hair loss, or improving hair loss, which comprises administering to a subject a composition comprising a microorganism of the genus Janthinobacterium, a dried product, a lysate, a lysate, a culture medium, an extract thereof, or a fraction of an extract thereof; or violacein, a hydrate, a solvate, or a salt thereof.

[0009] Another object of the present invention is to provide a use of a microorganism of the genus Janthinobacterium, a dried product, a lysate, a lysate, a culture medium, an extract thereof, or a fraction of an extract thereof; or violacein, a hydrate, a solvate, or a salt thereof, or a composition comprising the same, for promoting hair growth or preventing, treating, or improving hair loss.

[0010] Another object of the present invention is to provide a use of a composition comprising a microorganism of the genus Janthinobacterium, a dried product, a lysate, a lysate, a culture medium, an extract thereof, or a fraction of an extract thereof; or violacein, a hydrate, a solvate, or a salt thereof, for the manufacture of a pharmaceutical product for promoting hair growth or preventing, treating, or improving hair loss.

[0011] One aspect is a composition for promoting hair growth or preventing, treating or improving hair loss, comprising a microorganism of the genus Janthinobacterium, a dried product, a lysate, a lysate, a culture medium, an extract thereof or a fraction of an extract thereof; or violacein, a hydrate, a solvate or a salt thereof.

[0012] In one specific example, the microorganism of the genus Zantinobacterium may be, but is not limited to, Zantinobacterium sp. SUN098 deposited under the deposit number KCTC 15091BP.

[0013] The term “crusted material” in this specification may mean a product obtained by crushing the cell wall of the strain itself by chemical or physical force.

[0014] The term "culture medium" in this specification may be used interchangeably with "culture supernatant", "conditioned medium" or "conditioned medium", and may mean the entire medium including the strain, its metabolites, extra nutrients, etc., obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that the strain of the genus Xantinobacterium can grow and survive in vitro. The medium may be selected from known liquid media or solid media, and may be, for example, but is not limited to, R2A agar medium, TSA agar medium, ISP liquid medium, GSS liquid medium, BL agar medium, ISP agar medium, Bennett's agar medium, NA agar medium, M3 medium, or LB medium.

[0015] The above culture solution may include the culture itself, a concentrate thereof, or a lyophilized product obtained by culturing the strain, or a culture supernatant, a concentrate thereof, or a lyophilized product obtained by removing the strain from the culture.

[0016] Meanwhile, the liquid from which the bacterial cells have been removed from the culture medium is called a "supernatant," and can be obtained by leaving the culture still for a certain period of time and taking only the liquid from the upper layer excluding the part that has settled to the lower layer, removing the bacterial cells through filtration, or centrifuging the culture to remove the sediment at the bottom and taking only the liquid at the upper layer.

[0017] The term "culture solution extract" in this specification means an extract obtained from the culture solution or a concentrate thereof, and may include an extract, a diluted or concentrated extract, a dried product obtained by drying the extract, or a controlled or purified product thereof, or a fraction obtained by fractionating the same.

[0018] The above extract or fraction may be, but is not limited to, a dried substance, a crushed substance, a dissolved substance, or a culture solution of a microorganism extracted or fractionated with hexane, chloroform, ethyl acetate, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

[0019] The alcohol having 1 to 6 carbon atoms may be methanol, ethanol, propanol, isopropanol, 1,3-propanediol, butanol, pentanol, hexanol, etc.

[0020] In one specific example, the microorganism of the genus Zantinobacterium, the dried product, crushed product, lysate, culture medium of the microorganism, the extract thereof or the fraction of the extract may contain violacein, a hydrate, a solvate or a salt thereof.

[0021] The above violacein is a purple pigment, named “3-(2-(5-Hydroxyindol-3-yl)-5-oxo-2-pyrrolin-4-ylidene)-2-indolinone”, may have a molecular formula of C20H13N3O3, and may be Cas No. 548-54-9, UNII-QJH0DSQ3SG, and / or BRN 0049923.

[0022] The above violacein may be obtained by extraction and isolation from natural products and / or strains, or may be manufactured by conventional organic synthesis methods, but is not limited thereto. The above violacein may be isolated from a microorganism of the genus Zantinobacterium, for example, Zantinobacterium sp. SUN098.

[0023] The above violacein may include a solvate or salt thereof (e.g., a pharmaceutically acceptable salt). It may also include all possible hydrates that can be prepared therefrom, and all possible stereoisomers.

[0024] The term "solvate" as used herein means a higher-order compound formed between molecules or ions of a solute and molecules or ions of a solvent.

[0025] The above solvate may include both stoichiometric solvates and non-stoichiometric solvates.

[0026] The term "salt" or "pharmaceutically acceptable salt" used herein refers to a salt that can be used pharmaceutically among salts, which are substances in which cations and anions are bound by electrostatic attraction. The salt can typically be a metal salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, etc. For example, the metal salt can be an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), an aluminum salt, etc.; the salt with an organic base can be a salt with triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine, etc.; the salt with an inorganic acid can be a salt with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.; salts with basic amino acids include salts with arginine, lysine, ornithine, etc.; salts with acidic amino acids include salts with aspartic acid, glutamic acid, etc., but are not limited thereto.

[0027] The term "hydrate" in this specification refers to a compound to which water is bound, and is a broad concept that includes inclusion compounds in which there is no chemical bonding between water and the compound.

[0028] The above violacein may include a violacein derivative.

[0029] The above violacein derivative may be at least one selected from the group consisting of deoxyviolacein, proviolacein, and oxyviolacein.

[0030] In this specification, the term "alopecia" means a phenomenon in which hair falls out from the scalp or a condition in which hair becomes thicker or thinner, "prevention or improvement of hair loss" means preventing and suppressing hair loss as described above, and "promotion of hair growth" or "promotion of hair growth" means not only promoting the creation of new hair but also helping existing hair grow healthily.

[0031] The above hair loss may be one or more selected from among Alopecia Areata, Androgenetic Alopecia, Telogen Effluvium, Traumatic Alopecia, Trichotillomania, Pressure Alopecia, Anagen Alopecia, Pityriasis Gravis, Alopecia Syphlltiac, Alopecia Seborrhecia, Symptomatic Alopecia, Cicatricial Alopecia, and Congenital Alopecia, but is not limited thereto.

[0032] In one specific example, the composition may cause one or more of an increase in the sphere size of hair papilla cells, induction of hair follicle growth, induction of hair growth, induction of differentiation or proliferation of hair follicle stem cells, and reduction of hair follicle regression.

[0033] The composition may increase the expression of a gene associated with hair follicle growth or hair growth. The gene may be one or more selected from the group consisting of, but not limited to, ALPL, LEF1, and WNT5A.

[0034] The composition may increase the expression of a gene that induces differentiation or proliferation of hair follicle stem cells. The gene may be one or more selected from the group consisting of FGF7, FGF10, and NOG, but is not limited thereto.

[0035] The composition may increase GSK3β / β-catenin signaling associated with hair growth.

[0036]

[0037] Another aspect is a pharmaceutical composition for promoting hair growth or preventing, treating or improving hair loss comprising the above composition.

[0038] The composition is as described above.

[0039] The pharmaceutical composition may additionally comprise a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a glidant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium dihydrogen phosphate anhydrous, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

[0040] When formulating the above pharmaceutical composition, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include Witepsol, macrogol, Tween 61, cacao butter, liurin butter, and glycerogelatin.

[0041] The pharmaceutical composition may contain carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins or other stabilizers to increase stability or absorbability.

[0042] The pharmaceutical composition may be formulated as an oral or parenteral dosage form. The oral dosage form may be a granule, powder, liquid, tablet, capsule, dry syrup, or a combination thereof. The parenteral dosage form may be an injection.

[0043] The pharmaceutical composition may be administered by any method known in the art. The administration may be administered directly to the subject by any means, including intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. The administration may be systemic or local.

[0044] The above administration may include, but is not limited to, application to the scalp.

[0045] The above administration is 0.00001 mg to 1,000 mg of the composition according to one specific example per subject per day, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, Or it may be administered 10 mg to 25 mg. However, the dosage may be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage by considering these factors. The frequency of administration can be once a day or twice or more within the range of clinically acceptable side effects, and the administration site can be administered in one or more sites, and the total number of administration days can be administered daily or at intervals of 2 to 5 days, from 1 day to 30 days for a single treatment. If necessary, the same treatment can be repeated after an appropriate period. For animals other than humans, the same dosage as for humans per kg can be administered, or the above dosage can be converted into an amount based on the volume ratio (e.g., average value) of the organs (heart, etc.) of the target animal and humans.

[0046] The above pharmaceutical composition may be administered in combination with different compositions for promoting hair growth or preventing or treating hair loss.

[0047] As used herein, the terms "combination therapy," "combination administration," or "in combination" refer to any form of simultaneous or concurrent treatment using at least two separate therapeutic agents. The components of the combination therapy may be administered simultaneously, sequentially, or in any order. The components may be administered in different dosages, at different frequencies, or via different routes, as appropriate.

[0048] The term "administered simultaneously" as used herein is not particularly limited and means that the components of the combination therapy are administered substantially simultaneously, for example, as a mixture or in an immediately subsequent sequence.

[0049] The term "administered sequentially" as used herein is not particularly limited and means that the components of the combination therapy are administered not simultaneously, but one after another or in clusters with a specific time interval between administrations. The time intervals may be the same or different between the administrations of each of the components of the combination therapy, and may be selected, for example, from the range of 2 minutes to 96 hours, 1 day to 7 days, or 1 week, 2 weeks, or 3 weeks. Typically, the time interval between administrations may range from several minutes to several hours, for example, from 2 minutes to 72 hours, from 30 minutes to 24 hours, or from 1 to 12 hours. Additional examples include time intervals ranging from 24 to 96 hours, from 12 to 36 hours, from 8 to 24 hours, and from 6 to 12 hours.

[0050]

[0051] Another aspect provides a food composition for promoting hair growth or preventing or improving hair loss, comprising the composition.

[0052] The composition is as described above.

[0053] The term "improvement" herein may mean any action that at least reduces a parameter associated with the condition being treated, for example, the severity of a symptom.

[0054] The food composition may include ingredients commonly added during food manufacturing. For example, the composition may include proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be used, but are not limited thereto.

[0055] In addition to the above, the food composition according to the aspect may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0056] The above food composition may include a health functional food, particularly in the form of a tablet, capsule, powder, granule, liquid, or pill. In addition, the food may be in the form of a beverage, powdered beverage, solid, chewing gum, tea, vitamin complex, or food additive.

[0057] The above health functional food refers to any food manufactured using nutrients that are easily deficient in daily meals or raw materials or ingredients (hereinafter referred to as “functional raw materials”) that have functions useful to the human body, and helps maintain health or prevent and / or improve certain diseases or symptoms. There are no special restrictions on the final product form.

[0058] The above health functional food may be provided mixed with a conventionally known health functional food for preventing or improving hair loss, or with another existing health functional food. If the health functional food includes another health functional food with cancer prevention or improvement effects, it is important to mix the two in an amount that achieves maximum effect with minimal side effects, as can be readily determined by those skilled in the art.

[0059]

[0060] Another aspect is a cosmetic composition for promoting hair growth or preventing or improving hair loss, comprising the composition.

[0061] The composition is as described above.

[0062] In one specific example, the cosmetic composition may be prepared in one or more formulations selected from the group consisting of serum, toner, essence, paste, mask pack, patch, gel, cream, lotion, nourishing lotion, nourishing cream, moisturizing cream, massage cream, powder, soap, cleanser, oil, foundation, makeup base, wax, and spray.

[0063] The above cosmetic composition may further comprise any conventional cosmetic ingredient selected from additional ingredients commonly used in cosmetics, such as thickeners, dispersants, fragrances, fillers, preservatives, antiseptics, neutralizers, sweeteners, vitamins, free radical scavengers, metal ion sequestrants, functional ingredients, and mixtures thereof. A person skilled in the art can select any additional ingredient and / or its amount such that the beneficial properties of the composition according to the present disclosure are not adversely affected or substantially not affected by the anticipated addition.

[0064] When the above cosmetic composition is a surfactant-containing cleansing formulation, it may further include, as a carrier component, an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyl taurate, a sarcosinate, a fatty acid amide ether sulfate, an alkylamidobetaine, an aliphatic alcohol, a fatty acid glyceride, a fatty acid diethanolamide, a vegetable oil, a linolenic derivative, or an ethoxylated glycerol fatty acid ester.

[0065] When the cosmetic composition according to the present invention is in the form of a cream or gel, it may further include animal oil, vegetable oil, wax, paraffin, starch, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide as a carrier component.

[0066] When the above cosmetic composition is in the form of a solution or emulsion, it may further include a solvent, solvating agent or emulsifying agent, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, propylene glycol, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0067] If the above cosmetic composition is in the form of a suspension formulation, it may further include a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth as a carrier component.

[0068] If the above cosmetic composition is in the form of a powder or spray formulation, it may further include lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder as a carrier component, and particularly if it is in the form of a spray formulation, it may further include a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether.

[0069] The above cosmetic composition may be applied alone or in combination, or may be applied in combination with other cosmetic compositions other than those of the present invention. Furthermore, the cosmetic composition according to the present invention may be used according to conventional methods, and the frequency of application may vary depending on the user's skin condition or preference.

[0070]

[0071] Another aspect is a method for promoting hair growth, preventing hair loss, treating hair loss, or improving hair loss, comprising administering to a subject a composition comprising a microorganism of the genus Janthinobacterium, a dried product, a lysate, a lysate, a culture medium, an extract thereof, or a fraction of an extract thereof; or violacein, a hydrate, a solvate, or a salt thereof.

[0072] The composition and administration are as described above.

[0073] In this specification, “subject” means a subject in need of treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0074]

[0075] Another aspect is a use of a microorganism of the genus Janthinobacterium, a dried product, a lysate, a lysate, a culture medium, an extract thereof, or a fraction of an extract thereof; or violacein, a hydrate, a solvate, or a salt thereof, or a composition comprising the same, for promoting hair growth or preventing, treating, or improving hair loss.

[0076]

[0077] Another aspect is the use of a composition comprising a microorganism of the genus Janthinobacterium, a dried product, a lysate, a lysate, a culture broth, an extract thereof or a fraction of an extract thereof; or violacein, a hydrate, a solvate or a salt thereof, for the manufacture of a medicament for the treatment of hair loss.

[0078] Depending on the aspect, microorganisms of the genus Xantinobacterium or violacein have the effect of increasing the sphere size of hair papilla cells, inducing hair follicle growth, inducing differentiation or proliferation of hair follicle stem cells, and reducing hair follicle regression, thereby promoting hair growth or preventing or treating hair loss.

[0079] Figure 1 is a graph confirming the cytotoxicity of the SUN098 strain extract.

[0080] Figure 2 shows the sphere size of mammary papilla cells according to treatment with SUN098 strain extract.

[0081] Figure 3 shows the degree of sphere formation in mammary papilla cells according to treatment with SUN098 strain extract.

[0082] Figure 4 shows changes in the expression of signature genes related to hair growth according to treatment with SUN098 strain extract in 2D and 3D cultures of hair papilla cells.

[0083] Figure 5 is a graph confirming the cytotoxicity of violacein.

[0084] Figure 6 shows the sphere size of mammary papilla cells according to violacein treatment.

[0085] Figure 7 shows the degree of sphere formation in mammary papilla cells according to violacein treatment.

[0086] Figure 8 shows changes in the expression of signature genes related to hair growth according to violacein treatment in 2D and 3D cultures of hair papilla cells.

[0087] Figure 9 shows the activity of β-Catenin according to violacein treatment.

[0088] Figure 10 shows the stability of β-Catenin according to treatment with violacein at different concentrations.

[0089] Figure 11 shows the stability of β-Catenin according to the treatment time of violacein.

[0090] Figure 12 shows β-Catenin activity in the cytoplasm and nucleus according to treatment with violacein.

[0091] Figure 13 shows the expression of hair-inductive genes following treatment with violacein.

[0092] Figure 14 shows the expression of β-Catenin upper signal according to concentration-dependent violacein treatment.

[0093] Figure 15 shows the expression of β-Catenin upper signals according to the treatment time of violacein.

[0094] Figure 16 shows the mechanism of action of GSK3β / β-Catenin according to concentration-dependent violacein treatment.

[0095] Figure 17 shows the mechanism of action of GSK3β / β-Catenin according to the treatment time of violacein.

[0096] Figure 18 compares the stability of β-Catenin according to treatment with violacein and GSK3β inhibitor in 3D culture.

[0097] Figure 19 is a graph evaluating the degree of TCF / LEF promoter activation by treatment with violacein and positive controls (lithium chloride and minoxidil).

[0098] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0099]

[0100] Example 1. Confirmation of hair growth efficacy of extract of Janthinobacterium sp. SUN098 strain.

[0101] 1.1. Preparation of extract of Janthinobacterium sp. SUN098 strain

[0102] The extract of Janthinobacterium sp. SUN098 strain, deposited under accession number KCTC 15091BP, was prepared as follows. Specifically, the culture broth of the SUN098 strain was mixed with ethyl acetate in a 1:1 (v / v) ratio, sonicated (water-bath sonicator) for 20 minutes to disrupt the cells, and centrifuged at 13,000 rpm for 10 minutes. The supernatant was concentrated under reduced pressure to powder the sample, and diluted with each solvent to an appropriate concentration for activity testing.

[0103]

[0104] 1.2. Confirmation of cytotoxicity of SUN098 strain extract

[0105] The cytotoxicity of the Janthinobacterium sp. SUN098 strain extract was examined in human dermal papilla cells (HDPCs), which regulate hair follicle growth and regression. Specifically, 2,000 cells / well of HDPCs were seeded in a 96-well plate and cultured for 24 hours under 5% CO2 and 37°C conditions. Afterwards, the SUN098 strain extract was treated to the HDPCs at various concentrations and further cultured for 48 or 72 hours. Cytotoxicity was tested using the WST-1 assay, and the results are shown in Fig. 1.

[0106] As shown in Fig. 1, no toxicity was observed up to 20 μM when treated with the SUN098 strain extract, but inhibition of cell proliferation was observed when treated with a concentration of 50 μM or higher. Therefore, SUN098 strain extract at a concentration of 20 μM or lower was used in subsequent experiments.

[0107]

[0108] 1.3. Confirmation of mammary gland spheroid size according to treatment with SUN098 strain extract

[0109] Dermal papilla cells exist in the form of spheroids in the dermal papilla region involved in the growth of hair follicles, and the size of the dermal papilla spheroids is known to be proportional to the survival rate of the hair follicles, the degree of growth, and the diameter of the hair. When the SUN098 strain extract was treated to dermal papilla cells, the effect on hair growth was confirmed by observing the change in spheroid size. Specifically, 5,000 cells / well of dermal papilla cells were seeded in a 96-well round bottom ultra-low attachment plate and cultured for 24 hours under 5% CO2, 37℃ conditions. Afterwards, 10 μM or 20 μM of the SUN098 strain extract, which was not confirmed to be toxic in two-dimensional culture, was treated to the dermal papilla spheroids for 72 hours to confirm the size, and the results are shown in Fig. 2.

[0110] As shown in Fig. 2, it was confirmed that the sphere size of hair follicle cells increased in a concentration-dependent manner when treated with the SUN098 strain extract. Based on this, it was expected that the SUN098 strain extract would have hair growth efficacy.

[0111] Additionally, we attempted to confirm the hair growth efficacy of the SUN098 strain extract by culturing multiple spheroids rather than single spheroids. Specifically, 2 × 10 5 The cells were inoculated at a density of 10 cells / plate and cultured at 5% CO2, 37°C for 24 hours. Afterwards, the SUN098 strain extract was treated to the mammary papilla spheroids for 72 hours, and the size of multiple spheroids was measured.

[0112] As shown in Fig. 3, it was confirmed that the sphere size of the hair papilla cells increased as the treatment concentration of the SUN098 strain extract increased. In addition, it was confirmed that the number of hair papilla cells with a size of 50 μm or more increased in a concentration-dependent manner with the SUN098 strain. In a three-dimensional culture of hair papilla cells, it was confirmed that treatment with the SUN098 strain increased not only cell growth but also aggregative properties.

[0113]

[0114] 1.4. Confirmation of hair growth-related gene expression by SUN098 strain treatment

[0115] We confirmed that the SUN098 strain extract induced the growth of hair papilla cells, and confirmed the expression of signature genes related to hair growth. Specifically, hair papilla cells were cultured in two- or three-dimensional (2D) or three-dimensional (3D) plates at a density of 5 × 10 5 cells / dish or 2×10 5 The cells were seeded at a density of 10 cells / plate and cultured at 37°C in 5% CO2 for 24 hours. Subsequently, the SUN098 strain extract was treated with the hair papilla cells for 24 hours, and the treated cells were collected and subjected to reverse transcription polymerase chain reaction (RT-PCR). Through RT-PCR, the expression levels of genes known to be associated with hair growth were confirmed, and gene expression in two-dimensional or three-dimensionally cultured hair papilla cells was confirmed. The results are shown in Fig. 4.

[0116] As shown in Fig. 4, treatment with the SUN098 strain extract increased the expression of ALPL, LEF1, and WNT5A, which are known to be expressed more in the dermal papilla of anagen hair follicles. In addition, treatment with the SUN098 strain increased the expression of FGF7, FGF10, and NOG, which are known to be important signaling substances that induce differentiation and proliferation of hair follicle stem cells.

[0117] Therefore, the expression of all eight signature genes in two-dimensional or three-dimensional cultured hair papilla cells increased by treatment with the SUN098 strain extract, confirming the hair growth efficacy of the SUN098 strain extract.

[0118]

[0119] Example 2. Confirmation of the hair growth efficacy of violacein.

[0120] 2.1. Confirmation of cytotoxicity of violacein

[0121] To determine whether violacein is cytotoxic to human papilla cells, we treated the cells. Specifically, 2,000 cells / well of human papilla cells were seeded in 96-well plates and cultured for 24 hours under 5% CO2 and 37°C. The cells were then treated with violacein at various concentrations and further cultured for 48 or 72 hours. Cytotoxicity was tested using the WST-1 assay, and the results are shown in Figure 5.

[0122] As shown in Figure 5, no toxicity was observed up to 200 nM when violacein was treated, but inhibition of cell proliferation was confirmed when a concentration of 500 nM or higher was treated.

[0123]

[0124] 2.2. Confirmation of the size of mammary papilla spheroids according to violacein treatment

[0125] When treating peritoneal papilla cells with violacein, we aimed to determine the efficacy of peritoneal papilla cells for hair growth by observing changes in spheroid size. Specifically, peritoneal papilla cells were seeded at 5,000 cells / well in 96-well round-bottom ultra-low attachment plates and cultured for 24 hours under 5% CO2 and 37°C. Subsequently, peritoneal papilla spheroids were treated with various concentrations of violacein for 72 hours to determine their size, and the results are shown in Fig. 6.

[0126] As shown in Fig. 6, it was confirmed that the sphere size of mammary papilla cells increased in a concentration-dependent manner when treated with violacein. In addition, the spheroid size increased up to a concentration of 200 nM, at which cell proliferation inhibition was not observed, and it was confirmed that the size decreased at concentrations of 500 nM or higher.

[0127] Additionally, we attempted to confirm the hair growth efficacy of violacein by culturing multiple spheroids rather than single spheroids. Specifically, 2 × 10 5 Cells / plate were seeded at a density of 10 cells / plate and then cultured at 5% CO2, 37°C for 24 hours. Afterwards, the mammary papilla spheroids were treated with violacein at various concentrations for 72 hours, and the size of multiple spheroids was measured.

[0128] As shown in Figure 7, the sphere size of the mammary papilla cells increased in a concentration-dependent manner when violacein was treated, and it was confirmed that the growth of the mammary papilla cells decreased due to cytotoxicity at a concentration of 500 nM or higher.

[0129] From the above results, the efficacy of violacein in increasing hair follicle cell growth and aggregative property was confirmed, and thus its hair growth efficacy was expected.

[0130]

[0131] 2.3. Confirmation of hair growth-related gene expression by violacein treatment

[0132] We confirmed that violacein induces the growth of hair papilla cells and confirmed the expression of signature genes related to hair growth. Specifically, hair papilla cells were cultured in two- or three-dimensional (2D) or three-dimensional (3D) plates at a density of 5 × 10 5 cells / dish or 2 × 10 5 Cells were seeded at a density of 10 cells / plate and cultured at 37°C in 5% CO2 for 24 hours. RT-PCR was then performed using 2D and 3D cultured dermal papilla cells, and after treatment with 200 nM violacein for 24 hours, the expression of genes associated with hair growth was confirmed. The results are shown in Figure 8.

[0133] As shown in Fig. 8, when violacein was treated in two-dimensional cultured cells, the expression of signature genes was confirmed to increase, confirming that the increased expression of these genes induces hair follicle growth and differentiation and proliferation of hair follicle stem cells. Furthermore, in a three-dimensional culture condition similar to the biological environment, gene expression was confirmed to increase, similar to two-dimensional cultured cells, confirming the hair growth efficacy of violacein.

[0134]

[0135] 2.4. Confirmation of β-Catenin activity following violacein treatment

[0136] We confirmed that violacein increases the spheroids of hair papilla cells and sought to determine whether it increases the activity of β-catenin, which is known as one of the important biomarkers for hair growth. Specifically, 293T cells were seeded in a 6-well plate at a density of 3 × 10 5The cells were seeded at a density of 10 cells / well and cultured at 5% CO2 and 37°C for 24 hours. Subsequently, violacein was treated at various concentrations for 24 hours, and the treated cells were collected and the activity of β-catenin was confirmed using a TOP-Flash luciferase reporter assay, which is shown in Figure 9.

[0137] As shown in Figure 9, it was confirmed that β-Catenin activity increased in a concentration-dependent manner when treated with violacein. Therefore, it was expected that violacein could induce hair growth by increasing β-Catenin, a key signal that induces differentiation and proliferation of hair follicle stem cells.

[0138]

[0139] Example 3. Confirmation of the hair growth signaling mechanism of violacein.

[0140] 3.1. Confirmation of β-Catenin Stability Following Treatment with Violacein

[0141] β-Catenin is known to be continuously degraded, so we wanted to check whether β-Catenin is maintained stably when treated with violacein. To check whether the protein stability of β-Catenin increases in actual dermal papilla cells, we treated them with violacein at various concentrations. Specifically, dermal papilla cells were seeded at 5 × 10 in a 100 mm dish. 5 The cells were seeded at a density of 10 cells / dish and cultured at 5% CO2 and 37°C for 24 hours. Subsequently, violacein was treated at various concentrations for 12 hours, and the treated cells were collected and the activity of β-Catenin was confirmed through Western blotting. The results are shown in Figure 10.

[0142] As shown in Fig. 10, it was confirmed that the expression of p-β-Catenin (S33 / S37 / T41), a degradation form of β-Catenin, decreased as the treatment concentration of violacein increased. Since a decrease in p-β-Catenin means an increase in the stability of β-Catenin, it was confirmed that the stability of β-Catenin increased by treatment with violacein.

[0143] Additionally, the stability of β-Catenin was examined by treating the cells with 200 nM violacein, the most effective concentration, over time. Furthermore, the transcriptional activity of β-Catenin following violacein treatment was confirmed through nuclear and cytoplasmic fractions. The results are presented in Figures 11 and 12.

[0144] As shown in Figure 11, it was confirmed that as the treatment time of violacein increased, the expression of p-β-Catenin decreased and the expression of β-Catenin increased.

[0145] As shown in Figure 12, it was confirmed that β-Catenin expression in the nucleus increased when violacein was treated.

[0146] From the above results, it was confirmed that violacein not only increases the stability of β-Catenin, but also enters the nucleus and enhances its activity as a transcription factor.

[0147]

[0148] 3.2. Confirmation of hair-inductive gene expression following violacein treatment

[0149] β-Catenin is known to induce hair follicle growth by increasing the expression of various hair growth-related signature genes in dermal papilla cells. Therefore, we wanted to determine whether treatment with violacein, which increases β-catenin, increases the expression of genes that induce hair follicle growth. Specifically, dermal papilla cells were seeded at 5 × 10 in a 100 mm dish. 5 The cells were seeded at a density of 10 cells / dish and cultured at 5% CO2 and 37°C for 24 hours. Subsequently, violacein was treated at various concentrations for 12 hours, and the treated cells were collected and the expression level of proteins that induce hair follicle growth was confirmed through Western blotting. The results are shown in Figure 13.

[0150] As shown in Figure 13, it was confirmed that the expression of ALP, VEGF, and FGF2 increased when violacein was treated.

[0151]

[0152] 3.3. Confirmation of expression of β-Catenin upstream genes

[0153] Experiments were conducted to identify the upstream regulatory proteins that induce the increase in β-catenin by violacein. Specifically, 5 × 10 breast papilla cells were seeded in a 100 mm dish. 5 Cells were seeded at a density of 10 cells / dish and cultured at 37°C in 5% CO2 for 24 hours. Subsequently, cells were treated with violacein at various concentrations for 12 hours, and the treated cells were collected for Western blotting. Protein expression analysis confirmed the expression of GSK3β and p-GSK3β (S9, T390), known as key upstream regulators of β-catenin. The results are shown in Figures 14 and 15.

[0154] As shown in Fig. 14, it was confirmed that the expression of p-GSK3β increased in a concentration-dependent manner when violacein was treated. GSK3β is one of the phosphorylation enzymes, and the activity of the enzyme is regulated by phosphorylation, and phosphorylation of serine 9 and threonine 390 of GSK3β (p-GSK3β) is known to inhibit the activity of GSK3β. In the case of GSK3β, it induces the phosphorylation of β-Catenin, thereby increasing the expression of p-β-catenin (S33 / S37 / T41) and inducing the degradation of β-Catenin. However, when p-GSK3β (S9, T390) increases, the phosphorylation of β-Catenin is inhibited, which stops the degradation of β-Catenin and induces the stabilization of β-Catenin, thereby inducing the transcriptional activity of β-Catenin. Therefore, treatment with violacein was confirmed to enhance β-Catenin signaling through an increase in p-GSK3β (S9, T390).

[0155] In addition, as shown in Fig. 15, when violacein was treated over time, an increase in p-GSK3β (S9, T390) was observed, confirming that β-Catenin was increased.

[0156]

[0157] 3.4. Confirmation of expression of GSK3β / β-Catenin upstream genes

[0158] Experiments were performed to identify the upstream regulators that induce the increase in intracellular GSK3β / β-Catenin signaling by violacein. Specifically, 5 × 10 5Cells were seeded at a density of 10 cells / dish and cultured at 37°C in 5% CO2 for 24 hours. Subsequently, violacein was treated at various concentrations for 12 hours, and the treated cells were collected and subjected to Western blotting. Through protein expression analysis, the expression of p-PKA, p-ERK, and p-AKT, known as kinases targeting GSK3β (S9), was confirmed. The expression of p-p38, known as kinase targeting GSK3β (T390), was also confirmed. The results are shown in Figures 16 and 17.

[0159] As shown in Figure 16, it was confirmed that the expression of genes that induce an increase in GSK3β (S9) and GSK3β (T390) expression increased. In addition, as shown in Figure 17, it was confirmed that the expression of the above regulators increased even when violacein was treated over time.

[0160] From the above results, it was confirmed that violacein increases phosphorylation of S9 and T390 residues of GSK3β through signals such as PKA, AKT, p38, or ERK.

[0161]

[0162] Example 4. Comparison of β-Catenin Expression by Violacein and GSK3β Inhibitors

[0163] We confirmed that violacein regulates GSK3β, known as an upstream gene of β-catenin, and compared it with the treatment results of CHIR99021, known as a GSK3β inhibitor, to confirm the hair growth efficacy. Specifically, in order to culture hair papilla cells in three dimensions, 2×10 cells were cultured in a 6-well flat bottom ultra-low attachment plate. 5The cells were seeded at a density of 10 cells / plate and cultured at 5% CO2, 37°C for 24 hours. The three-dimensionally cultured dermal papilla cells were then treated with 200 nM violacein or 2.5 μM CHIR99021 for 48 hours, and the protein expression level of β-catenin was confirmed by Western blotting. The results are shown in Figure 18.

[0164] As shown in Figure 18, when CHIR99021, a GSK3β inhibitor, was treated, it was confirmed that the expression of β-Catenin, a downstream gene of GSK3β, increased and the expression of p-βCatenin decreased. When violacein was treated, it was confirmed that, similar to the GSK3β inhibitor, the expression of β-Catenin increased and the expression of p-βCatenin decreased.

[0165] From the above results, the inhibitory effect of violacein on GSK3β activity and the effect of increasing β-Catenin expression through this were reconfirmed.

[0166]

[0167] Example 5. Evaluation of the efficacy of violacein on increasing TCF / LEF transcription factor activity.

[0168] TCF / LEF promoter activation evaluation (TOPFlash luciferase reporter assay) The difference in hair growth between patients with hair loss and normal individuals is closely related to the activity of the WNT / β-catenin signaling pathway in dermal papilla cells.

[0169] Activation of β-catenin induces activation of the TCF / LEF transcription factor, thereby increasing the expression of target genes related to hair growth. Therefore, we aimed to confirm the activation of the TCF / LEF transcription factor according to the presence or absence of violacein treatment using a luciferase reporter assay.

[0170] Specifically, to quantitatively evaluate the degree of intracellular β-Catenin transcriptional activity of violacein, a TOPFlash luciferase reporter assay was performed. First, 293T cells transfected with p-SV-β-galactosidase plasmid (Promega) and TCF / LEF response element-driven luciferase reporter plasmid (Addgene) vectors were treated with violacein at concentrations of 0.1 and 0.2 μM for 24 hours, and then the luminescence value was measured using the luciferase reporter assay. Lithium chloride (LiCl) 5 mM and minoxidil (MNX) 10 μM were used as positive controls. The results of the efficacy evaluation test for the ability of violacein to increase TCF / LEF transcription factor activity are shown in Figure 19.

[0171] As shown in Fig. 19, it was confirmed that luciferase activity increased in a concentration-dependent manner when treated with nanomole violacein. In particular, it was confirmed that luciferase activity increased statistically significantly by 43.01 ± 0.40% and 68.80 ± 0.60%, respectively, compared to the control group at concentrations of 0.1 and 0.2 μM. It was confirmed that luciferase activity increased by 64.62 ± 1.61% and 34.39 ± 0.16%, respectively, compared to the control group in the positive control group with 5 mM LiCl and 10 μM MNX. In addition, 100 nM and 200 nM violacein showed statistically significantly (p<0.001) higher luciferase activity levels than the positive control group, 10 μM MNX, confirming that violacein promotes the transcriptional activity of β-Catenin.

[0172] In summary, through the efficacy evaluation test on the ability of the above violacein to increase TCF / LEF transcription factor activity, it was confirmed that violacein treatment and Minoxidil (10 μM to 2 μg / mL) treatment could induce the activation of intracellular TCF / LEF transcription factor in a concentration-dependent manner, and it was confirmed that violacein could induce a higher effect at a lower concentration (100 to 200 nM) compared to the activation of TCF / LEF transcription factor by Minoxidil.

[0173] [Accession number]

[0174] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0175] Accession number: KCTC15091BP

[0176] Date of acceptance: 20220921

[0177]

Claims

1. A composition for promoting hair growth or preventing, treating or improving hair loss, comprising microorganisms of the genus Janthinobacterium, dried products, fragments, lysates, culture solutions, extracts thereof or fractions of extracts thereof; or violacein, a hydrate, a solvate or a salt thereof.

2. In the first paragraph, the extract or fraction is a composition obtained by extracting or fractionating a dried substance, a fragment, a dissolved substance, or a culture medium of a microorganism with hexane, chloroform, ethyl acetate, an alcohol having 1 to 6 carbon atoms, or a mixed solvent thereof.

3. A composition according to claim 1, wherein the microorganism of the genus Janthinobacterium, the dried product, the crushed product, the dissolved product, the culture medium, the extract thereof or the fraction of the extract contains violacein, a hydrate, a solvate or a salt thereof.

4. A composition according to claim 1, wherein the violacein is isolated from a natural product or strain, or manufactured by an organic synthetic method.

5. A composition according to claim 4, wherein the violacein is isolated from a microorganism of the genus Zantinobacterium.

6. A composition in paragraph 5, wherein the violacein is isolated from Xantinobacterium sp. SUN098 deposited under the deposit number KCTC 15091BP.

7. In the fourth paragraph, a composition comprising a violacein derivative.

8. A composition in claim 7, wherein the violacein derivative comprises at least one selected from the group consisting of deoxyviolacein, proviolacein, and oxyviolacein.

9. A composition according to claim 1, wherein the hair loss is at least one selected from the group consisting of Alopecia Areata, Androgenetic Alopecia, Telogen Effluvium, Traumatic, Trichotillomania or Pressure Alopecia, Papillomas, Alopecia Syphlltiac, Alopecia Seborrhecia, Symptomatic Alopecia, Non-scarring Alopecia, Cicatricial Alopecia and Congenital Alopecia.

10. A composition according to claim 1, which causes at least one selected from the group consisting of an increase in the sphere size of hair papilla cells, induction of hair follicle growth, induction of differentiation or proliferation of hair follicle stem cells, and reduction of hair follicle regression.

11. A food composition for promoting hair growth or preventing or improving hair loss, comprising a composition according to any one of claims 1 to 10.

12. A cosmetic composition for promoting hair growth or preventing or improving hair loss, comprising a composition according to any one of claims 1 to 10.

13. A method for promoting hair growth or preventing, treating or improving hair loss, comprising administering to a subject a composition comprising a microorganism of the genus Janthinobacterium, a dried product, a fragment, a lysate, a culture medium, an extract thereof or a fraction of an extract thereof; or violacein, a hydrate, a solvate or a salt thereof.

14. A method according to claim 13, wherein the microorganism of the genus Janthinobacterium is Janthinobacterium sp. SUN098 deposited under the deposit number KCTC 15091BP.

15. A method according to claim 13, wherein the violacein is isolated from a natural product or strain, or manufactured by an organic synthetic method.

16. A method according to claim 15, wherein the violacein is isolated from a microorganism of the genus Zantinobacterium.

17. A method in claim 16, wherein the violacein is isolated from Zantinobacterium sp. SUN098 deposited under the deposit number KCTC 15091BP.

18. A method in claim 15, wherein the violacein comprises a violacein derivative.

19. A method according to claim 18, wherein the violacein derivative is selected from the group consisting of deoxyviolacein, proviolacein, and oxyviolacein.

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