KR102668908B1 - Composition for Improving Hair Health Using Mixture of Powder of Mastic Gum and an Extract of Pepermint - Google Patents

Abstract

The present invention promotes the proliferation of dermal papilla cells and enhances the expression of VEGF, IGF-1, and β-catenin, which are related to hair growth and hair growth promotion. Furthermore, it promotes hair growth in animal models, improves hair density, and improves hair growth ratio. Disclosed is a composition for improving hair health using a complex of mastic gum powder and peppermint extract, which has the activity of increasing hair health.

Description

Composition for Improving Hair Health Using Mixture of Powder of Mastic Gum and an Extract of Peppermint}

The present invention relates to a composition for improving hair health, particularly a composition for promoting hair growth or inhibiting hair loss, using a complex of mastic gum powder and peppermint extract.

Hair is formed from hair follicles. There are about 5 million hair follicles in an adult, distributed throughout the entire human skin except for the palms and soles of the feet. There are 1 million on the head, and 100,000 of these are on the scalp. (Bulletin of Food Technology 2009, 22(4):651-663). Scalp hair accounts for about 20% of human hair, and has the function of protecting the head from shock by cushioning the head in addition to the physiological functions of hair such as evaporation of sweat and regulating body temperature, and is suitable for modern people's tendency to value appearance. It also has an important social function in creating an individual's personality and image (Clin Dermatol 2001, 19(2):161-166).

The exact cause of hair loss is not fully known to date, but it is due to inhibition of proliferation or decreased function of dermal papilla cells related to hair cycle regulation, abnormalization of the hair cycle due to the action of male hormones, and decreased blood flow to the scalp. Abnormal changes in the hair cycle, drugs such as anticancer drugs, mental stress, physical stimulation, and environmental pollution are being discussed (J Invest Dermatol, 1999, 113: 873-877; Mol Cell Endocrinol, 2002 198:89-95; J Dermatol. Sci. 2011, 62: 154-159).

Dermal papilla cells, a type of specialized fibroblast within the hair bulb, interact with various types of epithelial cells within the hair follicle and play an important role in hair follicle formation, hair regeneration, and hair growth. It is known that the growth, proliferation, and inhibition of apoptosis of dermal papilla cells are important targets for the development of drugs that maintain the hair growth phase and suppress hair loss (Physiol. Rev. 81: 449-494; J. Dermatol Sci. 2011, 62: 154-159; FASEB J. 2008, 22: 1725-1736.

IGF-1 acts to promote hair growth by promoting the growth of dermal papilla cells and inhibiting apoptosis of dermal papilla cells. IGF-1 is a substance produced in the dermal papilla and is a representative growth factor that helps hair growth by promoting the differentiation of keratinocytes in hair follicles (Ann Dermatol. 2012;24(1):26-31).

VEGF (Vascular endothelial growth factor) is an angiogenesis factor that is known to enhance hair growth and differentiation of dermal papilla cells by improving blood circulation by growing the vascular endothelium. It is an indicator factor used to suppress hair loss or screen hair growth-inducing substances. (Korean J Dermatol. 1999;37(1):23-30; Exp Cell Res. 2012 Aug 15;318(14):1633-40).

Cell proliferation is closely related to the progression of the cell cycle, which consists of G0/G1, S, and G2/M phases, and this cell cycle progression is influenced by Wnt/β-catenin signaling, which leads to hair growth. Wnt/β-catenin signaling also plays an important role in hair regeneration (Dev. Biol. 2005, 185: 82-91; Mol. Cell. Biol. 2005, 25: 9063-9072; J. Immunol. 2001, 166 : 4713-4720).

Currently, the most commonly used drugs for hair loss treatment are substances that inhibit the activity of type 2 5α-reductase, including Minoxidil preparations (see U.S. Patents Nos. 4,139,619 and 4,596,812) and finasteride (U.S. Patent Nos. Examples include No. 5547957, No. 5760046 or Republic of Korea Patent No. 375083.

Hair loss treatment requires long-term treatment, so minimizing side effects is key. Accordingly, efforts are being made to discover new hair growth ingredients or materials from natural products that have relatively few side effects on the human body.

Accordingly, the present invention discloses the hair growth promoting and hair loss inhibiting activities of a complex of mastic gum ( Pistacia lentiscus ) powder and peppermint ( Mentha piperita ) extract.

The purpose of the present invention is to provide a composition for promoting hair growth or inhibiting hair loss using a complex of mastic gum powder and peppermint extract.

Other or specific purposes of the present invention will be presented below.

In the present invention, as confirmed in the examples and experimental examples below, the complex of mastic gum powder and peppermint extract promotes the proliferation of dermal papilla cells and promotes hair growth and VEGF, IGF-1, and β-catenin related to hair growth promotion. It was completed by confirming that it enhances expression and further promotes hair growth in animal models, improves hair density, and increases the rate of hair growth.

The present invention is provided based on the results of these experiments, and the composition for promoting hair growth or inhibiting hair loss of the present invention is characterized in that it contains a complex of mastic gum powder and peppermint extract as active ingredients.

In this specification, “pemmermint extract” refers to extracting the leaves, branches, fruits, seeds, or mixtures of peppermint, which are the target of extraction, with water, lower alcohols with 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, and acetone. , hexane, ether, chloroform, ethyl acetate, butylacetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or mixed solvents thereof. It refers to an extract obtained by using a supercritical extraction solvent such as carbon dioxide or pentane, or a fraction obtained by fractionating the extract. The extraction method is cold immersion, reflux, and heating considering the polarity, degree of extraction, and degree of preservation of the active substance. , ultrasonic radiation, supercritical extraction, etc. can be applied. In the case of a fractionated extract, the extract is suspended in a specific solvent and then mixed with a solvent of different polarity to form a fraction obtained by adsorbing the crude extract onto a column filled with silica gel and then mixed with a hydrophobic solvent, a hydrophilic solvent, or a mixture of these. It is meant to include fractions obtained using the mobile phase. In addition, the meaning of the extract includes concentrated liquid extract or solid extract from which the extraction solvent has been removed by methods such as freeze-drying, vacuum drying, hot air drying, and spray drying. Preferably, it refers to an extract obtained using water, ethanol, or a mixed solvent thereof as an extraction solvent, and more preferably 30 to 80% (v/v) ethanol extract.

In addition, in this specification, the meaning of “active ingredient” refers to an ingredient that can exhibit the desired activity alone or in combination with a carrier that is not active on its own.

In addition, in this specification, the term "hair loss" should be understood as including all symptoms classified as hair loss in the art, regardless of the direct or indirect cause of its occurrence. Specifically, it includes all symptoms of hair loss due to poor blood circulation, excessive secretion of sebum due to excessive production of dihydrotestosterone, deterioration of scalp function due to peroxides or bacteria, aging, genetic factors, stress, and their combined effects. .

The composition of the present invention may be included in any amount (effective amount) depending on the use, formulation, purpose of formulation, etc., as long as the active ingredient can exhibit hair growth promoting or hair loss inhibiting activity. The typical effective amount is based on the total weight of the composition. It will be determined within the range of 0.001% by weight to 50.0% by weight. Here, “effective amount” means that when the composition of the present invention is administered to mammals, preferably humans, to which it is applied during the administration period recommended by medical experts, etc., it can exhibit the intended functional and pharmacological effects, such as hair growth promotion. It refers to the amount of active ingredient contained in the composition of the invention. Such effective amounts can be determined experimentally within the scope of the ordinary ability of those skilled in the art.

In addition to the active ingredients, the composition of the present invention has already been tested for safety in the art in order to increase or reinforce the effect of promoting hair growth or inhibiting hair loss, or to improve the convenience of taking or ingesting through the addition of similar activities such as blood pressure regulating activity. and may further include any compounds or natural extracts known to have the corresponding activity. These compounds or extracts include compounds, extracts, and pharmaceuticals listed in compendial documents such as the Pharmacopoeia of each country (in Korea, the “Korean Pharmacopoeia”) and the Code of Health Functional Foods of each country (in Korea, it is the “Standards and Specifications for Health Functional Foods” notified by the Ministry of Food and Drug Safety). The laws of each country that govern the manufacture and sale of compounds or extracts and health functional foods that have received product approval in accordance with the laws of each country (in Korea, this is the Pharmaceutical Affairs Act) (in Korea, it is the “Health Functional Foods Act”) 」) includes compounds or extracts whose functionality has been individually recognized.

For example, dexpanthenol, biotin, L-menthol, zinc pyrithione, etc., which are ingredients with hair loss symptom alleviating functionality listed in the Ministry of Food and Drug Safety Notification “Functional Cosmetic Standards and Test Methods” under the Korean Cosmetics Act, are listed in the Korea Health Functional Food Act. GABA-containing powder derived from L-glutamic acid, katsuobushi oligo peptide, Natto bacteria culture powder, Somoktae peptide complex, salmon peptide, Olive leaf extract, sardine peptide, casein hydrolyzate, coenzyme Q10, grape seed enzymatically digested extract powder, Haitai oligopeptide, etc. may be examples of these compounds or extracts.

In a specific embodiment, the composition of the present invention can be considered a food composition.

The food composition of the present invention can be manufactured in any form, for example, beverages such as tea, juice, carbonated beverages, and electrolyte drinks, processed oils such as milk and yogurt, gums, rice cakes, Korean snacks, bread, snacks, noodles, etc. It can be manufactured into health functional food preparations such as foods, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, etc.

In addition, the food composition of the present invention can have any product classification in terms of legal and functional classification as long as it complies with the enforcement laws and regulations at the time of manufacture and distribution. For example, it is a health functional food according to the Korean 「Act on Health Functional Foods」, or confectionery, beans, tea, and beverages according to each food type according to the food code of the Korean 「Food Sanitation Act」 (Ministry of Food and Drug Safety Notification 「Food Standards and Specifications」) , special purpose food, etc.

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to, mixed with, or infiltrated into food when manufacturing, processing, or preserving food. Since they are consumed daily and for a long period of time with food, their safety must be guaranteed. In the food additive code of each country that regulates the manufacturing and distribution of food (in Korea, it is the Food Sanitation Act), food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korea Food Additive Code (Ministry of Food and Drug Safety Notification “Food Additive Standards and Specifications”), food additives are classified into chemical synthetics, natural additives, and mixed preparations in terms of composition. These food additives are classified into sweeteners and flavors in terms of function. It is classified into preservatives, emulsifiers, acidulants, thickeners, etc.

Sweeteners are used to impart an appropriate sweetness to foods, and both natural and synthetic ones can be used in the food composition of the present invention. Preferably, a natural sweetener is used, and natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents are used to improve taste or aroma, and both natural and synthetic ones can be used. Preferably, natural products are used. When using natural products, they can serve the purpose of enhancing nutrition in addition to flavor. Natural flavoring agents may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, coriander leaves, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. You can also use things obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo nuts. Natural flavoring agents may be liquid concentrates or solid extracts. In some cases, synthetic flavoring agents may be used, and as synthetic flavoring agents, esters, alcohols, aldehydes, terpenes, etc. may be used.

Preservatives include calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc., and emulsifiers include acacia gum, carboxymethyl cellulose, xanthan gum, and pectin. etc. may be used, and as acidulants, acidic acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, etc. may be used. In addition to improving taste, acidulants may be added to ensure that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms. As a thickening agent, a suspending agent, settling agent, gel forming agent, bulking agent, etc. may be used.

In addition to the food additives described above, the food composition of the present invention may contain bioactive substances or minerals known in the art and whose safety is guaranteed as food additives for the purpose of supplementing and reinforcing functionality and nutrition.

Such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, etc., and minerals include calcium preparations such as calcium citrate and magnesium stearate. Magnesium preparations such as iron citrate, iron preparations such as chromium chloride, potassium iodine, selenium, germanium, vanadium, zinc, etc. are included.

The food composition of the present invention may contain the above-described food additives in an appropriate amount to achieve the purpose of addition depending on the product type.

Regarding other food additives that can be included in the food composition of the present invention, each country's food code or food additive code can be referred to.

The composition of the present invention may be considered a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier in addition to the active ingredient and can be prepared into an oral formulation or a parenteral formulation depending on the route of administration by a conventional method known in the art. Here, the route of administration may be any suitable route, including topical route, oral route, intravenous route, intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and for specifics, reference can be made to the pharmacopoeia of each country, including the Korean Pharmacopoeia.

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared as powder, granules, tablets, pills, sugar-coated tablets, capsules, solutions, gels, syrups, suspensions, and wafers along with a suitable carrier according to methods known in the art. It can be manufactured in a dosage form such as: Examples of suitable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as corn starch, potato starch, and wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Cellulose such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease. Serol, etc. can be mentioned. When formulating, appropriate binders, lubricants, disintegrants, colorants, diluents, etc. can be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferrite, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, and wax, and as a lubricant, oleic acid. Examples include sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium and calcium salts, and polydethylene glycol. Disintegrants include starch and methyl cellulose. , agar, bentonite, xanthan gum, starch, alginic acid or its sodium salt, etc. Additionally, diluents include lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories along with a suitable carrier according to methods known in the art. When formulated as an injection, an aqueous isotonic solution or suspension can be used as a suitable carrier. Specifically, an isotonic solution such as PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, or 5% dextrose can be used. . When formulated for transdermal administration, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and aerol preparations. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide. When formulated as a suppository, the carrier is Wethepsol ( witepsol), Tween 61, polyethylene glycols, cocoa fat, laurel paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc. can be used.

Regarding the specific formulation of pharmaceutical compositions, it is known in the art, and references can be made to, for example, Remington's Pharmaceutical Sciences (19th ed., 1995). The above documents are considered a part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, gender, age, patient's severity, and administration route. It may be in the /kg range. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the invention in any respect.

In another specific aspect, the composition of the present invention can be considered a cosmetic composition.

Even if the composition of the present invention is identified as a cosmetic composition, the cosmetic composition may be classified as an arbitrary product in terms of its use and by law, and is specifically used for purposes such as alleviating hair loss, promoting hair growth, improving skin troubles, and improving atopic dermatitis. It can be functional cosmetics, non-functional general cosmetics, etc. The product form can take any product form, specifically solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, shampoo, surfactant-containing cleansing, oil, powder foundation, emulsion foundation. It can take the form of products such as wax foundation, spray, etc. Specific product forms may include softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray, or powder.

In addition to the active ingredient, the cosmetic composition of the present invention may contain ingredients commonly used in cosmetic compositions, such as stabilizers, solubilizers, surfactants, vitamins, conventional auxiliaries such as pigments and flavorings, and carriers.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier ingredient. You can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent is used as a carrier component, specifically water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, fatty acid ester of sorbitan, etc. can be used.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, and microcrystals. Cellulose, aluminum metahydroxide, bentonite, agar, etc. can be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.

The cosmetic composition of the present invention can be manufactured according to a cosmetic composition manufacturing method commonly used in the art, except that it contains active ingredients that exhibit hair growth promoting activity, etc.

As described above, according to the present invention, a composition for promoting hair growth or inhibiting hair loss using a complex of mastic gum powder and peppermint extract can be provided.

The composition of the present invention can be commercialized into foods, drugs, hair cosmetics, etc.

Figures 1 and 2 show the results of evaluating the degree of proliferation of dermal papilla cells.
Figure 3 shows the results of antioxidant activity evaluation.
Figure 4 shows the Western blot results of VEGF, IGF-1, and β-catenin.
Figure 5 shows the results of evaluating hair growth index in an animal model.
Figure 6 shows the results of hair density evaluation in an animal model.
Figure 7 shows the evaluation results of hair growth phase ratio in an animal model.

Hereinafter, the present invention will be described with reference to examples and experimental examples. However, the scope of the present invention is not limited to these examples and experimental examples.

<Example> Preparation of a composite of mastic gum powder and peppermint extract

Mastic gum powder was homogenized by putting mastic gum in a grinder, pulverizing it, and then sieving it using an AMKCO mechanical Sieve.

Peppermint extract was obtained by adding 4 L of 50% alcohol to 200 g of dried peppermint and extracting at 50°C for 6 hours. Afterwards, the filtered extract was concentrated under reduced pressure below 50°C and then freeze-dried to prepare a powdery extract.

The peppermint extract was mixed with mastic gum powder at a weight ratio of 7:3 (mastic gum being 7 weight ratio) to prepare a composite of mastic gum powder and peppermint extract (FHH-MG).

<Experimental Example> Evaluation of hair loss inhibition or hair growth promotion efficacy

1. In vitro efficacy evaluation

1.1 Cell proliferation rate evaluation

This test method is performed to confirm the appropriate concentration that is not toxic to cells when treating cells with the test product. This is a method of measuring the amount of living cells using WST (water soluble tetrazolium salts) and is used for cytotoxicity evaluation, cell viability, and cell division ability. WST is a highly sensitive water soluble tetrazolium salt that reacts with intracellular dehydrogenase to produce formazan. Therefore, the more living cells there are, the more formazan is produced, resulting in a higher absorbance, and this can be used to determine the degree of cell toxicity or the degree of cell proliferation promotion.

Human hair papilla cells (Human Follicle Dermal Papilla Cell, PromoCell) are grown in Human Follicle Dermal Papilla Cell Growth Medium (PromoCell) with Fetal Calf Serum, Bovine Pituitary Extract, Basic Fibroblast Growth Factor, and Insulin at 37°C and 5% CO2 conditions. was cultured.

The cultured cells are divided into ⁵ Afterwards, WST substrate solution (CCK-8, Dojindo) was added to the medium and incubated at 37°C for 2 hours, and the absorbance (OD) was measured at 450 nm using a microplate reader (VARIOSKAN LUX, Thermo Fisher Scientific). Based on the measured OD value, the cell proliferation rate was calculated as a percentage compared to the sample untreated group.

The results are the same as Figures 1 and 2. Figure 1 shows the results when each single substance and the complex (FHH-MG) of the mastic gum and peppermint extract of the example were treated at 200 ppm and cultured for 48 and 72 hours, and Figure 2 shows the results of the complex (FHH-MG) at 62.5 , These are the results when treated with 125 and 250 ppm and cultured for 24, 48, and 72 hours.

Referring to Figure 1, it can be seen that the cell proliferation rate when cultured for 48 and 72 hours promotes the proliferation of dermal papilla cells more when treated with the composite (FHH-MG) than with the mastic gum and peppermint extract single substances. there is.

Referring to Figure 2, it can be seen that the complex (FHH-MG) promotes the proliferation of dermal papilla cells compared to the control group in proportion to the treatment concentration and culture time.

Minoxidil, used as a positive control in Figures 1 and 2, also promoted proliferation of dermal papilla cells compared to the control group.

1.2 Antioxidant efficacy evaluation

1.2.1 SOD activity evaluation

This test used Marklun's method (Eur. J. Biochem., 47: 469-474, 1974.) to measure superoxide dismutase (SOD) activity by observing inhibition of pyrogallol autoxidation.

SOD is an antioxidant enzyme that is known to protect the living body from free radicals by converting oxygen radicals harmful to cells into hydrogen peroxide and then converting them into harmless water and oxygen molecules by catalase.

Cultured human hair papilla cells _were divided _into ⁵ and cultured again for 24 hours. Afterwards, the culture medium was taken and centrifuged at 2,000 xg for 10 minutes, and only the supernatant excluding cell debris was collected and used for testing.

The SOD kit (BIOMAX) was used for SOD activity. The stock solution was used as the culture medium, the test was performed according to the enclosed test method, and the OD value was measured using a microplate reader (VARIOSKAN LUX, Thermo Fisher Scientific). Based on the measured OD value, SOD activity was calculated according to the method suggested by the manufacturer.

The results are shown in Figure 3. Referring to Figure 3, it was observed that the SOD activity of hair papilla cells, which was reduced by H ₂ O ₂ , increased in a concentration-dependent manner compared to the control group according to treatment with the complex (FHH-MG). In addition, it was confirmed that when the complex (FHH-MG) was treated at 125 and 250 ppm, it increased more than the positive control group, Minoxidil.

1.2.2 CAT activity evaluation

This test used Aebi's method (Methods in enzymology. Academic press. 105: 121-126. 1984.), which checks the activity of catalase (CAT) by measuring the absorbance that decreases as hydrogen peroxide decomposes.

CAT is a representative antioxidant enzyme, and is involved in the reaction that decomposes hydrogen peroxide (two molecules of H2O2) generated by SOD into one molecule of oxygen and two molecules of water and the decomposition of reactive oxygen species (ROS). It is known that

Cultured human hair papilla cells _were divided _into ⁵ and cultured again for 24 hours. Afterwards, the culture medium was taken and centrifuged at 2,000 xg for 10 minutes, and only the supernatant excluding cell debris was collected and used for testing.

CAT kit (BIOMAX) was used for CAT activity. The stock solution was used as the culture medium, the test was performed according to the enclosed test method, and the OD value was measured using a microplate reader (VARIOSKAN LUX, Thermo Fisher Scientific). Based on the measured OD value, CAT activity was calculated according to the method suggested by the manufacturer.

The results are shown together in Figure 3.

Referring to Figure 3, it was observed that the CAT activity of hair papilla cells, which had been reduced by H ₂ O ₂ , increased in a concentration-dependent manner compared to the control group following treatment with the complex (FHH-MG). In addition, it was confirmed that when the complex (FHH-MG) was treated, it increased more than the positive control group, Minoxidil.

1.3 Evaluation of hair growth stimulating factor protein production amount

This test is a test method that quantifies the production of hair growth stimulating factors, such as VEGF, IGF-1, and β-catenin proteins, through reaction with enzyme-labeled antibodies. The production of VEGF, IGF-1, and β-catenin proteins for the test products was measured in human hair papilla cells.

The cultured human hair papilla cells were divided into 5 x 10 ⁴ cells and dispensed into a 6-well plate. When the cells were cultured at more than 80%, the test product was added to each well and cultured for 24 hours. Afterwards, the culture medium was taken and centrifuged at 2,000 xg for 10 minutes, and only the supernatant excluding cell debris was collected and used for testing.

The Human VEGF ELISA kit (abcam) was used to quantify VEGF protein, the Human IGF-1 ELISA kit (abcam) was used to quantify IGF-1 protein, and the Human β-catenin ELISA kit (MyBioSource) was used to quantify β-catenin protein. was used. The stock solution was used as the culture medium, and the test was performed according to the test method suggested by the manufacturer. The OD value was measured using a microplate reader (VARIOSKAN LUX, Thermo Fisher Scientific), and the measured value was entered into the regression equation obtained from the standard curve. Protein concentration was calculated by substituting and multiplying by the dilution factor.

The results are shown in Figure 4.

Referring to Figure 4, it was observed that the IGF-1 protein increased in a concentration-dependent manner compared to the control group following treatment with the complex (FHH-MG), and increased more than the positive control group, Minoxidil. In addition, VEGF and β-catenin proteins increased compared to the control group following treatment with the complex (FHH-MG), and were observed to increase more than the positive control group, Minoxidil.

1.4. Statistical analysis

Statistical analysis was verified using the IBM SPSS statistics 25.0 program, and the significance of the experimental and control groups was confirmed with a hypothesized mean difference of 5% ( p <0.05).

After the normality test, significance was confirmed through an independent sample T-test (parametric method) depending on whether normality was satisfied.

2. In vivo efficacy evaluation

2.1 Animal rearing and experiment composition

Five-week-old male C57BL/6 mice were purchased from Orient Bio Co., Ltd. and underwent an acclimation period for one week. Experimental animals are maintained under breeding conditions with a temperature of 24°C ± 0.5°C, humidity of 55% to 65%, and light and dark automatically adjusted in a 12-hour cycle, with free feeding of laboratory animal feed. All experimental procedures and procedures are approved by the Institutional Animal Care and Use Committee of Yonsei University Health System (approval number: 2023-0017) and are performed in compliance with ethical regulations in the SPF area of Yonsei University Avison Biomedical Research Center (ABMRC).

The negative control group uses distilled water, which is the solvent in which the sample was dissolved, and the positive control group uses Minoxidil (Sigma, US), which is used as a hair loss treatment. After an acclimation period, the mouse is anesthetized by inhalation with isoflurane, and then the hair on the back of the mouse is first removed using an animal clipper. Apply the hair remover to the area where the hair was removed, leave it on for 3 minutes, and then remove the fine hairs remaining on the skin. After 24 hours of skin stabilization after hair removal, 6-week-old mice with pink telogen body hair on the removed dorsal side were weighed, divided into 5 groups of 6, and treated with the test product “FHH-MG (100, 150, 200 mg/kg) orally administer 100 μl 5 times a week. Weight is measured once a week to check the effect of administration on weight change. 7 and 21 days after the experiment began, they were anesthetized with CO ₂ and sacrificed.

2.2 Hair growth index measurement

For 3 weeks after the start of the experiment, photos are taken once a week using a scanner (Aura X Pro; CZUR) to evaluate hair growth through hair growth index measurement. Changes in hair growth on the removed back area of the mouse were observed with the naked eye to determine 0-19% (1-1.5 points), 20-39% (2-2.5 points), 40-59% (3 points) depending on the degree of hair growth. -3.5 points), 60-79% (4-4.5 points), 80-100% (5 points). The average value of the judgment scores evaluated by experienced researchers through a blind test is calculated.

The results are shown in Figure 5. Referring to Figure 5, it can be seen that administration of the complex (FHH-MG) increases the hair growth index compared to the control group from 7 to 14 days. Additionally, Minoxidil also increased the hair growth index compared to the control group.

2.3 Hair density measurement

To measure hair density according to the number of hair follicles, skin tissue from mice administered orally for 7 days was extracted, fixed in 10% formalin for more than 24 hours, and paraffin blocks and paraffin embedded slides were made. The deparaffinized tissue was stained with a hematoxylin (104302; merck) solution for the nucleus and then washed in running water. The cytoplasm was stained with an eosin (230251; sigma) solution and then washed with water. The completely dehydrated tissue was then washed using a mounting solution. Fix it. Using an optical microscope (BX43F; OLYMPUS), the number of hair follicles in the dermis and subcutaneous tissue is measured at 400x magnification.

The results are shown in Figure 6. Referring to Figure 6, it can be seen that administration of the complex (FHH-MG) increases hair density compared to the control group. Additionally, Minoxidil also increased hair density compared to the control group.

2.4 Growth rate measurement

To measure the growth phase ratio according to the number of hair follicles, skin tissue from mice administered orally for 7 days was extracted, fixed in 10% formalin for more than 24 hours, and paraffin blocks and paraffin embedded slides were made. The deparaffinized tissue was stained with a hematoxylin (104302; merck) solution for the nucleus and then washed in running water. The cytoplasm was stained with an eosin (230251; sigma) solution and then washed with water. The completely dehydrated tissue was then washed using a mounting solution. Fix it. Using an optical microscope (BX43F; OLYMPUS), measure the ratio of the number of subcutaneous hair follicles to the total number of hair follicles in the tissue magnified at 400x magnification.

The results are shown in Figure 7. Referring to Figure 7, it can be seen that administration of the complex (FHH-MG) increases the growth phase ratio compared to the control group. Additionally, Minoxidil also increased the growth rate compared to the control group.

2.5 Statistical analysis

Statistical analysis is verified using the IBM SPSS statistics 25.0 program, and the significance of the test group and control group is confirmed with a hypothesized mean difference of 5% (p<0.05).

After the normality test, significance is checked through an independent sample T-test (parametric method) depending on whether normality is satisfied.

Claims (6)

A food composition for improving hair health containing a complex of mastic gum powder and peppermint extract as active ingredients,
The composite is a composite of mastic gum powder and peppermint extract in a weight ratio of 7:3 (mastic gum powder is 7),
The peppermint extract is a mixed solvent extract of water and ethanol,
A composition wherein the hair health improvement is promoting hair growth or inhibiting hair loss.
According to paragraph 1,
A composition characterized in that the peppermint extract is a 50% ethanol extract.
A pharmaceutical composition for promoting hair growth or suppressing hair loss comprising a complex of mastic gum powder and peppermint extract as an active ingredient,
The composite is a composite of mastic gum powder and peppermint extract in a weight ratio of 7:3 (mastic gum powder is 7),
A composition in which the peppermint extract is a mixed solvent extract of water and ethanol.
According to clause 3,
A composition characterized in that the peppermint extract is a 50% ethanol extract.
A cosmetic composition for promoting hair growth or suppressing hair loss comprising a complex of mastic gum powder and peppermint extract as active ingredients,
The composite is a composite of mastic gum powder and peppermint extract in a weight ratio of 7:3 (mastic gum powder is 7),
A composition in which the peppermint extract is a mixed solvent extract of water and ethanol.
According to clause 5,
A composition characterized in that the peppermint extract is a 50% ethanol extract.