KR102676679B1 - Composition comprising Graviola callus lysate extract as an active ingredient having skin whitening, antioxidant, skin condition improvement and hair loss inhibitory effects - Google Patents

Abstract

The present invention relates to a composition containing graviola callus soluble extract as an active ingredient that has skin whitening, antioxidant, skin condition improvement, and hair loss inhibition effects.
The graviola callus dissolved extract according to the present invention inhibits melanin biosynthesis or promotes cell proliferation and has effects such as skin whitening, skin regeneration, wound improvement, and hair loss inhibition, and can be used as a material for cosmetics and medicine. .

Description

Composition comprising Graviola callus lysate extract as an active ingredient having skin whitening, antioxidant, skin condition improvement and hair loss inhibitory effects}

It relates to a composition containing a dissolved extract derived from Graviola callus as an active ingredient that has skin whitening, antioxidant, skin condition improvement, and hair loss prevention effects.

In modern society, with the improvement of living standards and changes in the social atmosphere that emphasizes individual value, interest in beauty is focused regardless of age and gender, and research on aging prevention is actively underway.

Skin is one of the important tissues of the human body that first comes into contact with the external environment, protects the human body, and has biochemical and physical functions. Today's cosmetics are products that are used directly on human skin, so they must not only be safe for the skin, but also have biological functions that protect the skin, clean it, moisturize it, keep it supple, and maintain its physiological functions. The skin is an organ that is relatively vulnerable to external stimuli as it is easily exposed and affected by external stimuli, and is also one of the organs that is considered important in an individual's pursuit of external aesthetics in social life. Skin aging can be classified into intrinsic aging and extrinsic aging. Aging that occurs due to physiological changes over time is called intrinsic aging, and external stimulation such as heat or ultraviolet rays causes the skin to become thinner, lose elasticity, or lose melanin. Aging in which skin pigmentation diseases such as freckles, freckles, and age spots increase due to increased production reactions can be referred to as extrinsic aging.

In addition, in line with the social phenomenon of population aging, the development of functional cosmetics that delay aging is steadily increasing. Among them, natural materials with whitening functionality are highly preferred by consumers. In the basal layer of the skin, there is a pigment called melanin, which is a critical element of human skin color. This pigment is a phenol-based polymer that is a complex of black pigment and protein. In melanin biosynthesis, tyrosinase, a specific enzyme present in melanocytes, converts tyrosine into dopa (L-DOPA; L-3,4-dihydroxyl-L-phenylalanine) and dopaquinone. It is involved in oxidation to produce the final product, indole-5,6-dihydroquinone, which is melanin. If this synthesis of melanin occurs excessively within the skin, skin darkening, dark spots, freckles, etc. may occur, and in severe cases, it may even cause skin cancer. In order to prevent this excessive pigmentation phenomenon and achieve a whitening effect, the production of melanin and the enzymes involved, tyrosinase, and products such as dopa and dopaquinone must be reduced.

Meanwhile, human aging can occur not only in the skin but also in the hair. Hair loss causes great mental pain to those affected, but there is no clear way to treat it, so most hair loss patients are giving up treatment. Hair has its own hair cycle that goes through a growth phase, a catagen phase, a telogen phase, and then a growth phase, so it always maintains a constant number of hairs. However, due to genetics, aging, stress, irregular lifestyle habits, hormonal abnormalities, etc., the hair papilla at the hair root becomes smaller or the hair cycle becomes shorter, causing hair loss.

In this situation, recognizing the need for in-depth research to effectively solve phenomena such as freckles, freckles, and hair loss, which can become not only aesthetic but also serious social problems, the present inventors have developed skin whitening, skin condition improvement, and hair loss prevention effects. We developed a graviola (Soursop) callus dissolved extract that is excellent and can be used safely without any side effects on the skin.

Graviola is called by the scientific name of soursop ( Annona muricata L.) and is a plant of the Annonaceae family, well known as graviola or soursop. It has been used as an edible food in tropical regions since ancient times, and is also widely used in folk remedies for high blood pressure, nerve sedatives, asthma, cough, and headaches. In the past, attempts and research have been conducted to use Graviola leaf extract as a cosmetic material, but there has been no commercialization using Graviola callus.

Republic of Korea Patent Publication No. 10-2017-0048624

The present invention aims to solve the above-described problems and other problems associated therewith.

The purpose of the present invention is to provide a cosmetic composition for skin whitening, improving skin condition, or preventing or improving hair loss, containing graviola callus dissolved extract as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for preventing, improving, or treating skin disease or hair loss, containing the dissolved extract of Graviola callus as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing, improving, or treating skin disease or hair loss, containing graviola callus soluble extract as an active ingredient.

Another object of the present invention is to provide a method for producing a composition for whitening skin, improving skin condition, or preventing or improving hair loss, including the step of obtaining a graviola callus dissolved extract from graviola.

The technical problem to be achieved according to the technical idea of the invention disclosed in this specification is not limited to the problem to solve the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Additionally, the scope of the present application cannot be considered limited by the specific description described below.

In one aspect for achieving the above object, the present invention provides a composition for skin whitening, improving skin condition, or preventing, improving, or treating hair loss, containing a dissolving extract of Graviola callus as an active ingredient. Specifically, the present invention develops a naturally derived graviola callus soluble extract that has little skin irritation and is safe for human skin cells, and has skin improving effects such as skin whitening, skin aging prevention, skin regeneration, and hair loss inhibition effects. Provided is a composition containing graviola callus dissolved extract as an active ingredient.

In another aspect for achieving the above object, the present invention provides a cosmetic composition for skin whitening, improving skin condition, or preventing or improving hair loss, containing the dissolved extract of Graviola callus as an active ingredient.

In the present invention, 'Graviola ( Annona muricata Linn.)' is a member of the Magnolia family, Popunia family, cultivated in tropical regions such as South America, North America, the Philippines, and Indonesia. The leaves are oval, 7-20 cm long, and 2-5 cm wide. have It is reported to be rich in major components such as flavonoids, isoquinoline, alkaloids, and annonaceous acetogenins.

In the present invention, 'callus' is a cell mass derived from plant tissue. When the plant tissue is wounded, the cells recover their ability to divide, block the wound, and become enlarged. It is subcultured in a new medium. By doing so, it can permanently divide and proliferate. These cell masses are amorphous tissues or cell masses that have lost the ability to produce normal organ formation or tissue differentiation, and are mostly composed of podocytes. In a broad sense, they also include plant tumor tissues caused by infections such as Agrobacterium. . In other words, when cells in a plant proliferate, their direction is immediately determined and they are regularly assembled into not only specific tissues but also organs. However, when the undifferentiated cell mass formed through tissue culture is stimulated from the outside with various plant growth hormones, the control maintained until then is released, and the cells are interpreted to proliferate at will. When the cell mass obtained by dedifferentiation is placed in a liquid medium with the same composition and cultured with shaking, the cells separate and continue to proliferate in suspension. These cultured cells can permanently divide and proliferate by subculturing them in new media.

In the present invention, 'callus' may be derived from the tissue of the plant graviola.

In the present invention, 'extract' refers to a liquid component obtained by immersing the desired material, such as a Graviola callus culture, in various solvents and then extracting it at room temperature or at a heated state for a certain period of time, and removing the solvent from the liquid component. It refers to the results such as the obtained solid content. In addition, it can be comprehensively interpreted to include all diluted solutions of the above results, concentrated solutions thereof, crude preparations, purified products, dissolved extracts, etc.

The extract can be obtained by extraction with water or various organic solvents, but is not particularly limited thereto, and is preferably extracted with water, alcohol with 1 to 4 carbon atoms, or a mixed solvent thereof. In addition, the method for obtaining the extract is not particularly limited thereto, but is preferably a hydrothermal extraction method in which the dried product or processed product is immersed in the solvent and extracted at a temperature of 90 to 120 ° C., at a room temperature of 10 to 25 ° C. Methods such as cold needle extraction, heat extraction by heating to 40 to 100°C, ultrasonic extraction by applying ultrasonic waves, and reflux extraction using a reflux cooler can be used. Preferably, extraction is done using hot water extraction. It may be, and more preferably, it may be extracted using a cyclic hot water extraction method.

As an example, the dissolved extract can be obtained by a cyclic hot water extraction method. Specifically, the extraction solvent is preferably added at 1 to 400 times the weight for extraction, the reduced pressure evaporation temperature is preferably 90 to 120°C, but is not limited thereto, and the condensation extraction temperature is preferably 4 to 15°C. Without limitation, the extraction time is preferably 46 to 50 hours, but is not limited thereto. Drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, and more preferably freeze drying, but is not limited to these.

Alternatively, it may be prepared by extraction using water, an organic solvent, or a mixed solvent thereof. The extracted liquid can be used directly or after being concentrated and/or dried.

In the present invention, the extract can be concentrated or diluted, a distillate of the extract can be used, or the extract can be freeze-dried again and used in powder form.

The graviola callus dissolved extract of the present invention is not toxic to skin cells, and the present invention can provide a cosmetic composition containing the graviola callus dissolved extract for skin whitening, improving skin condition, or preventing or improving hair loss.

In the present invention, the 'skin whitening' not only brightens the skin tone by inhibiting the synthesis of melanin pigment, but also improves hyperpigmentation of the skin caused by ultraviolet rays, hormones, or genetics. Here, hyperpigmentation of the skin may include, but is not limited to, freckles, freckles, senile lentigo, brown spots, or age spots.

In the present invention, the 'skin condition improvement' may include skin regeneration, skin aging prevention, or wound improvement, but is not limited thereto.

In the present invention, the 'improvement' refers to all actions that improve or benefit the skin condition by using a composition containing the dissolved graviola callus extract according to the present invention.

The term 'skin regeneration' in the present invention refers to the recovery process of skin tissue against damage caused by external and internal causes. Damage due to external causes may include ultraviolet rays, external contaminants, wounds, trauma, etc., and damage due to internal causes may include stress, etc., but are not limited thereto.

The term 'skin aging prevention' in the present invention refers to both natural (endogenous) aging that occurs due to physiological changes in the body over time and preventing or delaying photoaging that occurs in areas exposed to sunlight.

Skin aging of the present invention may be photoaging caused by UV-B ultraviolet rays, but is not limited thereto. Specifically, photoaging of skin cells may occur due to light with a wavelength of 280 nm to 40 nm included in sunlight. In particular, damage to skin or fibers may occur due to irradiation of ultraviolet rays such as UV-B, which has a wavelength in the range of 280 nm to 320 nm, and a blackening of the skin may occur. When UV-B is irradiated, the accumulation of active oxygen (e.g. H ₂ O ₂ ) and free radicals within skin cells is promoted, and the radicals stimulate the intracellular signaling system to oxidize biomolecules such as DNA, proteins, and lipids. It can cause stress, which can cause damage to skin tissue. When oxidative stress in skin cells increases, stimulation occurs in keratinocytes in the epidermis or fibroblasts in the dermis, and through a series of intracellular signaling processes, the expression of genes such as MMP (matrix metalloproteinase), a collagen-degrading enzyme, increases. This can cause a decrease in collagen, which is a major component of the skin and accounts for 90% of the dermis and protects the skin from external stimulation or force by providing strength and tension to the skin, resulting in skin aging. Wrinkles may form.

The term 'wound' in the present invention refers to a disruption in the normal continuity of the skin structure due to physical damage to the skin, specifically contusion or bruise, laceration, avulsion, and penetrating wound. (penetrated wound), non-healing traumatic wound, destruction of tissue by irradiation, abrasion, bone gangrene, gun-shot wound, cut, burn, frostbite, skin ulcer, dry skin, keratosis skin, Cracks, tears, dermatitis, pain due to dermatophytosis, wounds such as surgical wounds, vascular disease wounds, corneal wounds, bedsores, flat sores, conditions related to diabetes and poor circulation, chronic ulcers, suture sites after plastic surgery, spinal injury wounds , refers comprehensively to damage to parts of an entity, such as gynecological wounds, chemical wounds, and acne.

The improvement of the skin condition of the present invention may be due to cell proliferation promoting activity, and may specifically promote the proliferation of human keratinocytes or fibroblasts, but is not limited thereto.

In the present invention, 'hair loss' refers to a phenomenon in which hair completely falls out of the scalp, and refers to loss of hair growth and partial or complete loss of hair.

The composition of the present invention may prevent or improve hair loss, and may promote hair growth or hair growth. The term 'hair growth' refers to hair growing from the scalp, and the term 'hair growth' refers to hair becoming longer and thicker and is used in the same sense as 'hair', another term used in the art.

The 'prevention' refers to all actions that inhibit or delay hair loss by administering or applying the composition of the present invention to an individual, and the 'improvement' refers to improving hair loss or hair condition by using the composition of the present invention. It means all actions that are beneficial.

The composition of the present invention may reduce the expression level of hair loss inducing factors, and specifically may reduce the gene expression level of SRD5A1 or DKK-1, which are hair loss inducing factors. Additionally, the composition of the present invention may increase the expression level of factors that induce hair follicle growth, and specifically may increase the gene expression level of β-catenin.

The composition of the present invention may inhibit melanin biosynthesis, and preferably may inhibit the oxidation activity of tyrosine or L-dopa, a melanin precursor, but is not limited thereto.

The composition of the present invention may enhance the antioxidant effect, but is not limited thereto. The term 'antioxidant' refers to the action of suppressing oxidation. The human body maintains a balance between prooxidants and antioxidants, but due to various factors, this balance becomes unbalanced and oxidation occurs. If it leans towards promotion, oxidative stress is induced, causing potential cell damage and pathological diseases. Reactive oxygen species (ROS), which are the direct cause of this oxidative stress, are unstable and highly reactive, easily reacting with various biological substances, and attacking macromolecules in the body, causing irreversible damage to cells and tissues, mutations, and mutations. It causes cytotoxicity and carcinogenesis. Reactive nitrogen species (RNS), such as NO, HNO ₂ , and ONOO ^- , are produced in large quantities due to the immune response of macrophages, neutrophils, and other immune cells during inflammatory reactions, and ROS is also produced at this time. The above-described free radicals oxidize and destroy cells in the body, thereby exposing them to various diseases. In addition, the antioxidant refers to the function of suppressing oxidation of cells by highly reactive free radicals or reactive oxygen species (ROS) due to oxidative stress caused by intracellular metabolism or the influence of ultraviolet rays. This includes removing free radicals or reactive oxygen species, thereby reducing damage to cells.

The composition of the present invention may contain the graviola callus dissolved extract in various amounts as long as it has the effect of skin whitening, skin regeneration, skin aging prevention or wound improvement, and hair loss prevention or improvement. Specifically, the graviola callus extract The dissolved extract may be contained in an amount of 0.1 to 10% by weight based on the total weight of the composition. This ratio is only an exemplary range, and it is obvious to those skilled in the art that even if it is 20% or 30% or more, it has excellent skin whitening, skin condition improvement effects, and hair loss prevention or improvement effects.

In addition to the graviola callus dissolved extract, the cosmetic composition according to the present invention contains various ingredients commonly used in cosmetic compositions, such as water-soluble ingredients, powder ingredients, oils, and interfaces, as necessary, within the range that does not reduce the effect of the present invention. It can be composed of activators, moisturizers, viscosity modifiers, preservatives, antioxidants, fragrances, pigments, etc.

Non-limiting examples of the surfactants that can be used include anionic surfactants, cationic surfactants, nonionic surfactants, and amphoteric surfactants. More specifically, the anionic surfactant includes alkylbenzene sulfonate, polyoxyalkylene alkyl sulfuric acid ester salt, alkyl sulfuric acid ester salt, olefin sulfonate, alkyl phosphate, polyoxyalkylene alkyl ether phosphate, and dialkyl sulfosuccine. Salts, fatty acid salts, etc. may be mentioned, and nonionic surfactants include polyoxyethylene alkyl ether, polyoxyethylene fatty acid ester, polyhydric alcohol fatty acid partial ester, polyoxyethylene polyhydric alcohol fatty acid partial ester, polyglycerol fatty acid ester, and polyoxyethylene fatty acid ester. Examples include ethylene hydrogenated castor oil derivatives and fatty acid diethanolamide. In addition, cationic surfactants include tertiary aliphatic amine salts, alkyltrimethylammonium halides, dialkyldimethylammonium halides, etc., and examples of amphoteric surfactants include amide betaine type, imidazolinium betaine type, and sulfobetaine type. etc. can be mentioned.

Examples of the moisturizing agent include glycerin, propylene glycol, 1,3-butylene glycol, dipropylene glycol, and sorbitol. Examples of the preservative include benzoic acid, dehydroacetic acid, paraoxybenzoic acid ester (methyl paraoxybenzoate, butyl paraoxybenzoate, etc.), phenoxyethanol, etc. In addition, the antioxidants include ascorbic acid, BHA, etc., and in addition, ultraviolet absorbers, anti-inflammatory agents, fresheners, etc. can be added.

The cosmetic composition of the present invention includes solution, external ointment, cream, foam, nutritional lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid cleanser, bath agent, sunscreen cream, sun oil, suspension, and emulsion. It can be prepared in a formulation selected from the group consisting of liquid, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch and spray. no.

The cosmetic composition of the present invention may further include one or more carriers acceptable in cosmetic formulations, and common ingredients include, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, Preservatives, fragrances, etc. may be appropriately mixed, but are not limited thereto. Here, 'acceptable carrier in cosmetic preparations' means a compound or composition that is already known and used or a compound or composition that will be developed in the future that can be included in cosmetic preparations and has no toxicity, instability or irritation beyond what the human body can adapt to when contacted with the skin. It says something that doesn't exist. The carrier may be included in the composition of the present invention in an amount of about 1% by weight to about 99.99% by weight based on the total weight thereof, preferably about 90% by weight to about 99.99% by weight of the weight of the composition. However, since the ratio varies depending on the formulation in which the composition of the present invention is manufactured, its specific application area (face, neck, etc.), and its preferred application amount, the ratio does not limit the scope of the present invention in any respect. It should not be understood as

In the cosmetic formulation included in the cosmetic composition of the present invention, acceptable carriers vary depending on the formulation of the cosmetic composition.

When the formulation of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. are used as carrier ingredients. may be used, but is not limited thereto. These may be used alone or in combination of two or more types.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silcate, polyamide powder, etc. may be used as carrier ingredients, and especially in the case of a spray, chlorofluorohydride may be additionally used. It may include propellants such as locarbon, propane/butane, or dimethyl ether, but is not limited thereto, and these may be used alone or in a mixture of two or more types.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butyl glycol oil, etc. can be used, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan. may be used, but is not limited thereto, and may be used alone or in a mixture of two or more types.

When the formulation of the present invention is a suspension, the carrier components include water, liquid diluents such as ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and miso. Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used, but are not limited thereto, and they may be used alone or in a mixture of two or more types.

When the formulation of the present invention is soap, alkali metal salts of fatty acids, hemiester salts of fatty acids, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. are used as carrier components. It may be, but is not limited to, these may be used alone or in combination of two or more types.

In one aspect for achieving the above object, the present invention provides a quasi-drug composition for preventing, improving, or treating skin disease or hair loss, containing a soluble extract of Graviola callus as an active ingredient.

The ‘graviola’, ‘callus’, ‘extract’ and ‘composition’ are as described above.

The term 'skin disease' of the present invention may be selected from the group consisting of skin wounds, skin scars, and skin pigmentation, but is not limited thereto.

Non-limiting examples of the terms 'skin wound' and 'skin scar' of the present invention include abrasions, scars caused by abrasions, etc.

The term 'skin pigmentation' of the present invention refers to all diseases caused by skin pigmentation due to increased production of melanin. Non-limiting examples of the skin pigmentation disease include melasma, freckles, spots, solar pigmentation, pigmentation after drug use, pigmentation after inflammation, and pregnancy-related pigmentation.

The term 'hair loss' in the present invention refers to hair growth defects and partial or complete loss of hair, and includes alopecia areata, androgenetic alopecia, tinea capitis, and hypotrichosis. , hereditary hypotrichosis simplex, circumscribed alopecia, alopecia congenitalis, alopecia pubis, alopecia seborrheica, alopecia senilis, alopecia totalis. Totalis), alopecia universalis, or telogen effluvium, etc. may be included, but are not limited thereto.

The term 'prevention' of the present invention refers to any act of suppressing or delaying skin diseases such as skin wounds, skin scars, and skin pigmentation by administering or applying the graviola callus dissolved extract of the present invention to an individual, and hair loss. It means any action that suppresses or delays.

The term 'improvement' of the present invention refers to all actions that improve or benefit skin diseases such as skin wounds, skin scars, and skin pigmentation by using the graviola callus dissolved extract of the present invention, and hair loss or hair condition. It means any action that improves or becomes beneficial.

The term 'treatment' of the present invention refers to any action that improves, alleviates, or benefits skin diseases such as skin wounds, skin scars, and skin pigmentation by administering or applying the graviola callus dissolved extract of the present invention to an individual, It refers to all actions that improve, alleviate, or benefit hair loss.

In the present invention, 'quasi-drugs' are articles used for the purpose of diagnosing, treating, alleviating, treating or preventing diseases in humans or animals, but are not instruments, machines, or devices, and are not intended to have a pharmacological effect on the structure and function of humans or animals. It refers to products used for the purpose, excluding those that are not instruments, machines, or devices. One specific example may include preparations for internal use, but is not limited thereto, and the formulation method, dosage, usage method, and components of quasi-drugs etc. can be appropriately selected from conventional techniques known in the technical field.

In addition to the above ingredients, the quasi-drug composition of the present invention may further include pharmaceutically acceptable carriers, excipients, or diluents as needed. The pharmaceutically acceptable carrier, excipient, or diluent is not limited as long as it does not impair the effect of the present invention, and includes, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc. It can be included.

In one aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing, improving, or treating skin disease or hair loss, containing graviola callus soluble extract as an active ingredient.

The ‘graviola’, ‘callus’, ‘extract’, ‘skin disease’, ‘hair loss’, ‘prevention’, ‘improvement’, ‘treatment’ and ‘composition’ are the same as described above.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc.

Carriers for parenteral administration may also include water, suitable oils, saline solutions, aqueous glucose and glycols, and the like. Additionally, stabilizers and preservatives may be additionally included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.

The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally, and parenteral administration methods include intravenous, intramuscular, intraarterial, intracentral, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, and intranasal. , may be administered enterally, topically, sublingually or intrarectally, and preferably centrally administered, but is not limited thereto.

The pharmaceutical composition of the present invention can be formulated into a preparation for oral administration or parenteral administration according to the administration route described above. When formulated, one or more buffers (e.g. saline or phosphate buffered saline (PBS)), antioxidants, bacteriostatic agents, chelating agents (e.g. EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g. For example, aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrating agents, surfactants, diluents or excipients may be used.

Solid preparations for oral administration include tablets, pills, powders, granules, solutions, gels, syrups, slurries, suspensions or capsules, etc. These solid preparations include the pharmaceutical composition of the present invention and at least one excipient, e.g. For example, starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol and maltitol. It can be prepared by mixing cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropylmethyl-cellulose, or gelatin. For example, tablets or dragees can be obtained by combining the active ingredient with solid excipients, grinding them, adding suitable auxiliaries, and processing them into a granule mixture.

In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water or liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, or preservatives may be included. there is. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be additionally included. .

When administered parenterally, the pharmaceutical composition of the present invention can be formulated with a suitable parenteral carrier in the form of injections, transdermal administration, and nasal inhalation according to methods known in the art. The above injections must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. For injections, examples of suitable carriers may be, but are limited to, solvents or dispersions including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), mixtures thereof, and/or vegetable oils. That is not the case. More preferably, suitable carriers include Hanks' solution, Ringer's solution, PBS containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. . In order to protect the injection from microbial contamination, it may additionally contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Additionally, in most cases, the injection may additionally contain an isotonic agent such as sugar or sodium chloride.

In the case of transdermal administration, forms such as ointments, creams, lotions, gels, external solutions, paste preparations, linear preparations, and aerol preparations are included. Here, 'transdermal administration' means administering a pharmaceutical composition topically to the skin so that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.

For inhalation administration, the compositions used according to the invention may be packaged in pressurized packs or using a suitable propellant, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. For pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. Formulations for parenteral administration are described in Remington's Pharmaceutical Science, 15th Edition, 1975 Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a text generally known in all pharmaceutical chemistry.

In the pharmaceutical composition of the present invention, the amount of the active compound in the unit dose formulation may vary, and specifically, it may be adjusted to about 0.01 mg to about 1 g per time based on an average human weighing 70 kg. However, the administered dose may vary depending on the requirements of humans and mammals, the severity of the disease being treated, and the final composition of the compound used. It falls to those skilled in the art to determine the appropriate dosage for a particular situation.

The pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, or methods using biological response modifiers.

In one aspect for achieving the above object, the present invention provides a method for producing a composition for whitening skin, improving skin condition, or preventing or improving hair loss, including the step of obtaining a graviola callus dissolved extract from graviola.

The ‘graviola’, ‘callus’, ‘extract’, ‘skin whitening’, ‘skin condition improvement’, ‘hair loss’, ‘prevention’, ‘improvement’ and ‘composition’ are the same as described above.

In the present invention, the step of obtaining the Graviola callus dissolved extract from the Graviola includes producing a Graviola callus culture by inducing and culturing the Graviola callus from the Graviola plant tissue, and producing Graviola callus from the Graviola callus culture. It may include preparing a callus dissolved extract. The graviola plant tissue can be obtained by collecting leaves, roots, stems, etc. of graviola.

In the step of preparing the graviola callus culture, the step may include collecting graviola plants and sterilizing them, and the sterilization may be performed if there is a sterilization method commonly used in the plant cell induction process in the technical field. It can be used without restrictions, but specifically, graviola leaves, roots, stems, etc. are collected, washed in running water, sterilized with 70% ethanol (EtOH) solution for 30 to 60 seconds, and then 2% with a small amount of Tween20 added. It may be sterilized by immersing it in a sodium hypochlorite solution for 7 to 15 minutes.

In addition, the step of preparing the graviola callus culture involves inducing graviola callus in a callus induction medium containing 6-Benzylaminopurine (BA) and/or Picloram, “ It may be a “callus induction step,” and is preferably a medium containing 1 to 20 μM of picloram and 0.1 to 1 μM of 6-benzylaminopurine (BA), but is not limited thereto. As an example, this medium is a basic MS medium containing the picloram and 6-BA. Specifically, based on 1L of the basic MS medium, trace elements include CoCl ₂ ·6H ₂ O 0.11uM, CuSO ₄ ·5H ₂ O 0.10 uM, FeNaEDTA 100uM, H ₃ BO ₃ 100.27uM, KI 5.00uM, MnSO _{4.H 2} O 100uM, Na ₂ MoO _{4.2H 2} O 1.03uM, ZnSO ₄ .7H ₂ O 29.91 uM, macro elements include CaCl ₂ 2.99mM, KH ₂ PO ₄ 1.25mM, KNO ₃ 18.79mM, MgSO ₄ It contains 1.50mM, NH ₄ NO ₃ 20.31mM, and vitamins include Glycine 26.64uM, myo-Inositol 554.94 uM, Nicotinic acid 4.06 uM, Pyridoxine HCl 2.43 uM, and Thiamine HCl 0.30 uM.

This callus induction step can be specifically performed by cutting and wounding leaves, stems, roots, etc., placing them on callus induction medium, and culturing them in the dark at 26 to 28°C.

In the present invention, the step of preparing the graviola callus culture may include a “callus stabilization step” of culturing the graviola callus in a solid medium and then additionally culturing it in a liquid medium. Specifically, the induced graviola callus can be selected and stabilized on a solid medium, and then, in order to grow callus, 0.1 to 1 μM of BA and 1 to 20 μM of Picloram are added to the MS medium. It may include culturing using a medium containing this.

Thereafter, it may include a "callus mass culture step" of culturing in a liquid medium to mass-cultivate the graviola callus obtained by growing on a solid medium. The liquid medium is placed in a 500 ml Erlenmeyer flask and the cultured solid culture medium is added. It may include the step of cultivating a sufficient amount of callus by inoculating and repeatedly culturing graviola callus. The liquid culture may be carried out at 22 to 26°C and may be cultured at 50 to 150 rpm.

In the step of preparing the graviola callus culture, an appropriate medium can be selected to specifically induce and cultivate graviola callus.

In the present invention, in the step of preparing the Graviola callus dissolved extract, the step of drying the obtained Graviola callus culture to prepare a Graviola callus dried material and mixing the dried material with purified water to prepare a dissolved extract may be included. You can. Specifically, the Graviola callus dried product can be prepared by washing the Graviola callus culture 3 to 5 times with purified water and then freeze-drying it at -80 to -100°C, and the freeze-dried Graviola callus is placed in purified water at a temperature of 90 to 90 degrees Celsius. A hydrothermal distillate can be obtained by evaporating under reduced pressure at a high temperature of 120°C and heating cyclically for 46 to 50 hours. Thereafter, the graviola callus dissolved extract can be prepared by rapid cooling through a cold water cooling pipe and condensation extraction. The condensation extraction may be carried out at 4 to 15°C, but is not limited thereto.

Additionally, the composition of the present invention can be prepared as a composition in an appropriate form by the known methods mentioned above, depending on the purpose.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention should not be construed as limited by these examples.

The graviola callus dissolved extract according to the present invention inhibits melanin biosynthesis or promotes cell proliferation and has antioxidant effects, and has effects such as skin whitening, skin regeneration, wound improvement, and hair loss inhibition, and is used in cosmetics, pharmaceuticals, etc. It can be used as a material.

Figure 1 is a diagram showing graviola callus cultured in the present invention.
Figure 2 shows the results of confirming the cell stability and cell proliferation effects of the graviola callus lysate extract of the present invention on human keratinocytes.
Figure 3 shows the results of confirming the cell stability and cell proliferation effects of the graviola callus lysate extract of the present invention on human fibroblasts.
Figure 4 shows the results of confirming the skin regenerative ability of the graviola callus dissolved extract of the present invention.
Figures 5 and 6 show the results of confirming the antioxidant activity of the graviola callus dissolved extract of the present invention.
Figure 7 shows the results confirming the melanin biosynthesis inhibitory effect of the graviola callus dissolved extract of the present invention.
Figure 8 shows the results of confirming the effect of the graviola callus dissolved extract of the present invention on inhibiting the oxidation activity of L-DOPA, a melanin precursor.
Figure 9 shows the results of confirming the effect of the graviola callus dissolved extract of the present invention on inhibiting the oxidation activity of tyrosine, a melanin precursor.
Figure 10 shows the results of confirming the cell stability effect of the graviola callus lysate extract of the present invention on human dermal papilla cells.
Figure 11 shows the results of confirming the effect of the graviola callus dissolved extract of the present invention on the expression level of SRD5A1.
Figure 12 shows the results of confirming the effect of the graviola callus dissolved extract of the present invention on the expression level of DKK-1.
Figure 13 shows the results of confirming the effect of the graviola callus soluble extract of the present invention on the expression level of β-catenin.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Graviola callus induction and culture

1.1. Plant material preparation

Graviola plant tissues such as young Graviola leaves, stems, and roots are collected, washed in running water, and then sterilized for 30 to 60 seconds using a 70% EtOH (Merck, cat.603-002-00-5) solution. , Sterilize the sterilized graviola plant tissue by immersing it in a 2% sodium hypochlorite solution with a small amount of Tween20 added for 7 to 15 minutes. Wash the sterilized graviola plant tissue five times repeatedly with sterilized distilled water to remove all remaining sterilizing solution. did. Graviola callus was induced using sterilized tissues as follows.

1.2. Exploration of graviola callus induction and growth conditions

Disinfected and washed graviola plant tissue samples were each cut to a certain size, wounded, placed on callus induction medium, and cultured in the dark at a temperature range of 26 to 28°C. For callus induction, 1 to 20 μM of Picloram (Duchefa, Cat. P0914.0005), 6-Benzylaminopurine (BA, Duchefa Cat.B0904.0001) media containing 0.1 to 1 μM were used.

1.3. Graviola callus stabilization and culture

The induced graviola callus was stabilized after a good callus selection process on solid medium. Afterwards, for growth, the basic MS medium was cultured using a medium containing 1 and 10 μM of BA and Picloram, respectively, which are mainly used for plant callus growth. The formed graviola callus was subcultured every two weeks and maintained in good culture (Figure 1).

1.4. Graviola callus liquid culture

For mass culture of Graviola callus obtained by growing on a solid medium, the culture was switched to a liquid medium culture stage. For this purpose, 100 ml of MS liquid medium containing hormones of the same composition as the solid medium was placed in a 500 ml Erlenmeyer flask, and three well-cultured solid culture plates (plates) were inoculated with Graviola callus and cultured at 25°C and 100 rpm.

1.5. Manufacture of Graviola callus dissolved extract

Using the Graviola callus culture obtained in liquid culture, a Graviola callus dissolved extract was prepared. At this time, it was manufactured through a cyclic hydrothermal distillation extraction method to minimize the loss and destruction of biologically active substances and increase the preservation and acquisition of active ingredients. The Graviola callus culture was washed five times with purified water and then freeze-dried at -80°C to obtain dried Graviola callus. After adding 2 L of purified water to 5 g of freeze-dried graviola callus, it was evaporated under reduced pressure at a high temperature of 98℃ and heated cyclically for more than 48 hours to obtain a hydrothermal distillate. This was rapidly cooled through a cold water cooling pipe (cooling coil maintained at 15℃). Condensation and extraction were performed. In order to conduct experiments based on accurate concentrations, the extract was freeze-dried again and the Graviola callus dissolved extract powder was used in the following tests. Graviola leaf extract was also prepared in the same way and used for comparative testing.

Example 2. Safety test on human keratinocytes of graviola callus lysate extract

In order to confirm whether the graviola callus lysate extract of the present invention can show improvement effects without skin irritation even when used for a long period of time, the presence or absence of cytotoxicity to human keratinocytes was tested. At this time, graviola leaf extract was also tested as a comparative test group.

2.1. Human keratinocyte culture

Prior to the safety test, the keratinocyte cell line HaCaT, a human skin cell, was first cultured in an incubator at 37°C and 5% CO ₂ . When the degree of culture was equivalent to 85 to 90% of the area of the culture vessel, cells were detached and counted by trypsin treatment, and subculture was performed at 5Х10 ³ cells/cm ² .

DMEM (Dulbecco's Modified Eagle Medium, GIBCO) medium supplemented with 10% FBS (GIBCO), 100 U/ml penicillin, and 100 μg/ml streptomycin was used to culture the cells.

2.2. Safety test on human keratinocytes

Human keratinocytes were counted in a 24-well plate at 5Х10 ³ cells/well using a hemacytometer and then dispensed. When cultured in DMEM containing 10% FBS for 48 hours and reached ~60% of the surface area of the culture vessel, it was replaced with FBS-free DMEM containing graviola callus soluble extract or graviola leaf extract and cultured for an additional 24 hours. After incubation, 50 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, SigmaM5655) solution (2.5 mg/ml) was added and cultured for an additional 3 hours. Afterwards, all cell culture was discarded, 200 μl of DMSO (dimethyl sulfoxide, Sigma D2650) was added to each well, and after stirring, 100 μl was taken into 96 wells and the absorbance was measured at 570 nm using ELISA (Enzyme-Linked Immunosorbent Assay). did. The degree of cytotoxicity was expressed as a percentage based on the absorption intensity of the control group (untreated group) using pure water.

The graviola callus dissolved extract of the present invention had no cytotoxicity at all concentrations and showed a higher degree of cell proliferation than the untreated group (Figure 2). The group treated with a concentration of 125 μg/ml of graviola callus lysate extract showed the highest cell proliferation rate, approximately 1.2 times higher than the untreated group. On the other hand, graviola leaf extract showed cell death at a low concentration of 62.5 μg/ml, and a cell survival rate of 55% was confirmed at the highest concentration tested, 500 μg/ml. Therefore, it was confirmed that Graviola callus and its extract do not cause skin irritation due to cytotoxicity even when used for a long period of time compared to Graviola leaf extract, and at the same time induce the proliferation of human keratinocytes.

Example 3. Cell safety evaluation of graviola callus lysate extract on human fibroblasts

3.1. Human fibroblast culture

Prior to the safety test, the fibroblast (keratinocyte) cell line CCD-986sk, a human skin cell, was cultured in an incubator at 37°C and 5% CO ₂ . When the degree of culture was equivalent to 85 to 90% of the area of the culture vessel, cells were detached and counted by trypsin treatment, and subculture was performed at 5Х10 ³ cells/cm ² . For cell culture, DMEM (Dulbecco's Modified Eagle Medium, GIBCO) medium supplemented with 10% FBS (GIBCO), 100 U/ml penicillin, and 100 μg/ml streptomycin was used.

3.2. Safety and cell proliferation effects of Graviola callus lysate extract on human fibroblasts

First, human fibroblasts were counted in a 24-well plate at 5Х10 ³ cells/well using a hemacytometer and then dispensed. When cultured in DMEM containing 10% FBS for 48 hours and reached 50% of the surface area of the culture vessel, it was replaced with FBS-free DMEM containing graviola callus soluble extract or graviola leaf extract and cultured for an additional 24 hours. Graviola leaf extract was obtained in the same manner as the Graviola callus dissolved extract and was used in the experiment. After incubation, 50 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, SigmaM5655) solution (2.5 mg/ml) was added and cultured for an additional 3 hours. Afterwards, all cell culture was discarded, 200 μl of DMSO was added to each well, and after stirring, 100 μl was taken into 96 wells and the absorbance was measured at 570 nm using ELISA (Enzyme-Linked Immunosorbent Assay). The degree of cytotoxicity was expressed as a percentage based on the absorbance intensity of the control group using pure water (extract treatment concentration 0 μg/ml).

As a result of the cytotoxicity test on human fibroblasts of the graviola callus dissolved extract and the graviola leaf extract of the present invention, it was confirmed that the graviola leaf extract was cytotoxic by appearing similar to the control group or lowering the survival rate, and graviola callus The dissolved extract significantly promoted cell proliferation compared to the untreated group and the Graviola leaf extract. In particular, a cell survival rate of 168% was confirmed at the highest tested concentration of 1000 μg/ml (Figure 3), compared to the Graviola leaf extract. It was confirmed that cell safety and cell proliferation effect were high.

Example 4. Analysis of skin regeneration effect of graviola callus dissolved extract

The degree of wound regeneration was investigated as one of the main physiological activity mechanisms of cosmetic compositions for skin regeneration and skin soothing. The skin regenerative activity of the graviola callus dissolved extract of the present invention was evaluated as follows. The human keratinocyte cell line HaCaT was distributed at a rate of 1Х10 ⁵ cells/well in a 6 well plate and cultured in 10% FBS/DMEM medium for 24 hours to form a keratinocyte monolayer, then the culture medium was removed and wound. A cell-free zone (scratch wound) was formed in each well using a maker. Afterwards, each well was washed three times with 1 ml of phosphate buffer (pH 7.4), and 3 ml of graviola callus lysate extract, stepwise diluted with FBS-free DMEM culture medium, was added to each well and cultured in a CO ₂ incubator. The regenerative capacity (healing capacity) of the graviola callus dissolved extract was measured by setting the control group treated with only FBS-free DMEM medium containing no extract as 100. Regenerative capacity was measured by staining cells and comparing the wound area with the no-added group through a microscope.

As a result of the experiment, when treated with graviola callus lysate extract, the regenerative activity of skin cells significantly increased compared to the no-added group (Figure 4). In addition, it was confirmed that at the highest test concentration of 500 μg/ml, the wound regeneration effect increased by about 91% compared to the no additive group.

Example 5. Confirmation of antioxidant effect of graviola callus dissolved extract

To evaluate the antioxidant activity of the graviola callus dissolved extract, the free radical scavenging effect of the sample was measured using DPPH (2,2-diphehylpictyl-hydrazyl). Graviola callus lysate extract was diluted with phosphate buffer (pH 7.4) to prepare the test concentration. 100 μL of 0.2 mM DPPH methanol solution was added to 100 μL of each sample solution and reacted at room temperature for 30 minutes. Afterwards, 100 μL of methanol was added to 100 μL of each sample solution as a blank, and the DPPH methanol solution was used as a negative untreated group. The absorbance was measured at 520 nm, and the DPPH inhibition rate (antioxidation rate) was calculated using the formula below. As a comparison test group for the graviola callus dissolved extract of the present invention, the graviola leaf extract was tested in the same manner. Additionally, vitamin C (VitC), known to have excellent antioxidant properties, was used as a positive control.

[Equation]

DPPH inhibition rate (%) = 100 - [(Absorbance of sample - Absorbance of each sample blank)/Absorbance of negative untreated group Х 100]

As a result of the experiment, as shown in Figure 5, the antioxidant capacity increased in a concentration-dependent manner as the treatment concentration of the graviola callus dissolved extract treatment increased, and the highest treatment concentration, 1,000 μg/ml, showed a high antioxidant rate that increased by 94% compared to the no addition group. showed. In addition, as shown in Figure 6, when compared to the Graviola leaf extract, the antioxidant rate was more than 30% higher at a concentration of 1,000 μg/ml, and the Graviola callus dissolved extract of the present invention had an antioxidant effect compared to the Graviola leaf extract. It was confirmed that it was even better.

Example 6. Confirmation of melanin synthesis inhibition ability of graviola callus dissolved extract

To confirm the whitening activity of the graviola callus lysate extract, it was treated with B16F10 cells, which are skin cells that form melanin, and the amount of melanin biosynthesis produced by the cells during culture was compared and evaluated. In more detail, B16F10 cells were distributed at 1Х10 ⁴ cells/well in a 96 well plate, and treated with freeze-dried callus extract at a concentration of 62.5 μg/ml in the culture medium to 500 μg/ml. After the sample was treated with an appropriate concentration, it was cultured for an additional 72 hours to induce inhibition of melanin biosynthesis. After additional incubation, the supernatant from each well was aspirated, washed with PBS, and 150 μL of 1N NaOH was added to each well. The melanin formed in the cells was completely dissolved by leaving it at 65°C for 45 minutes, and then 100 μL of the supernatant from each well was taken and the absorbance was measured at 405 nm. The whitening activity was evaluated by expressing the relative reduction in melanin synthesis as a percentage based on the melanin synthesis induction group treated with α-MSH (alpha-Melanocyte Simulating Hormone).

[Equation]

Melanin synthesis inhibition rate (%) =

100-{(reaction absorbance of each sample solution/reaction absorbance of negative control) Х 100}

As a result of the experiment, as shown in Table 1 below, compared to the no addition group, treatment with the graviola callus dissolved extract showed a melanin biosynthesis inhibition effect of 10 to 65% or more within the tested concentration, confirming the whitening effect (Table 1, Figure 7).

Treatment group No additives α-MSH
Addition group α-MSH +
62.5(μg/ml) α-MSH +
125(μg/ml) α-MSH +
250(μg/ml) α-MSH +
500(μg/ml) melanin
Synthesis rate (%) 24.77 100 89.60 64.82 55.83 34.38

Example 7. Confirmation of the inhibitory effect of L-DOPA oxidation activity of graviola callus dissolved extract

The freeze-dried graviola callus lysate extract was diluted with sodium phosphate buffer (0.1 M, pH 6.8) to prepare the final concentration of the entire reaction solution from 250 μg/ml to 1,000 μg/ml. As a test method, 850ul of sodium phosphate buffer (0.1 M, pH 7.0), 50ul of callus sample diluted to the appropriate concentration, and 50ul of mushroom tyrosinase solution were sequentially added to the test tube and reacted at 37°C for 6 minutes. 50ul of 0.06mM L-DOPA (L-3,4-dihydroxyphenylalanine, Sigma D9628-25G) solution was added to this solution and reacted at 37°C for 1 minute. After the reaction was completed, absorbance was measured at 475 nm using an ELISA reader. As a blank sample solution, 0.1M phosphate buffer solution (pH 7.0) was added instead of the sample solution.

As shown in Table 2 below, the graviola callus dissolved extract showed oxidation inhibition ability against the L-DOPA substrate even at low concentrations. In particular, it showed an inhibitory activity of close to 50% at a concentration of 1,000 μg/ml (Figure 8).

Graviola callus dissolved extract treatment concentration (μg/ml) Inhibition ability (%) No additives 0 250 24.99 500 36.35 1000 49.41

Example 8. Confirmation of the inhibitory effect of tyrosine oxidation activity of graviola callus dissolved extract

The graviola callus dissolved extract was diluted with sodium phosphate buffer (0.1 M, pH 6.8) to prepare the final concentration of the entire reaction solution from 250 μg/ml to 1,000 μg/ml. 290 μL of sodium phosphate buffer, 100 μL of prepared sample, 10 mM, and 100 μL of substrate were added to a 2 mL tube and preincubated at 37°C for 5 minutes. Afterwards, 10 μL of 2,500 unit/mL tyrosinase solution was added and reacted at 37°C for 10 minutes. After completion of the reaction, the absorbance was measured at 475 nm, and 0.1M phosphate buffer solution (pH 7.0) was used as a blank sample solution instead of the sample solution.

As a result, as shown in Table 3 below, the graviola callus dissolved extract showed inhibitory ability against the tyrosine substrate even at low concentrations. In particular, it showed an inhibitory activity of close to 48% at a concentration of 1,000 μg/ml (Figure 9).

Graviola callus dissolved extract treatment concentration (μg/ml) Inhibition ability (%) No additives 0 250 19.98 500 33.49 1000 47.92

Example 9. Confirmation of cell safety of Graviola callus lysate extract on dermal papilla cells

9.1. Human dermal papilla cell culture

Prior to the safety test, human dermal papilla cells HFDPC were first cultured in an incubator at 37°C and 5% CO ₂ . When the degree of culture was equivalent to 85 to 90% of the area of the culture vessel, cells were detached and counted by trypsin treatment, and subculture was performed at 5Х10 ³ cells/cm ² . For cell culture, DMEM (Dulbecco's Modified Eagle Medium, GIBCO) medium supplemented with 10% FBS (GIBCO), 100 U/ml penicillin, and 100 μg/ml streptomycin was used.

9.2. Confirmation of safety and cell proliferation effects of graviola callus lysate extract on human dermal papilla cells

First, human dermal papilla cells were counted and dispensed into a 24-well plate at 5Х10 ³ cells/well using a hemacytometer. When cultured in DMEM containing 10% FBS for 48 hours and reached 50% of the surface area of the culture vessel, it was replaced with FBS-free DMEM containing graviola callus soluble extract or graviola leaf extract and cultured for an additional 24 hours. After incubation, 50 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, SigmaM5655) solution (2.5 mg/ml) was added and cultured for an additional 3 hours. Afterwards, all cell culture was discarded, 200 μl of DMSO was added to each well, and after stirring, 100 μl was taken into 96 wells, and the absorbance was measured at 570 nm using ELISA (Enzyme-Linked Immunosorbent Assay). The degree of cytotoxicity was expressed as a percentage based on the absorbance intensity of the control group using pure water (extract treatment concentration 0 μg/ml).

As a result of the cytotoxicity test on human dermal papilla cells of the graviola callus dissolved extract and graviola leaf extract of the present invention, the graviola leaf extract showed a cell survival rate of 85.7% at 62.5 μg/ml compared to the untreated group, and the highest concentration was 1000 μg. /ml showed a low cell survival rate of 51.8%. On the other hand, the graviola callus lysed extract did not show serious cytotoxicity in human dermal papilla cells like the graviola leaf extract, and a cell survival rate of 101% was confirmed at the highest tested concentration of 1000 μg/ml (FIG. 10).

Example 10. Confirmation of hair loss inhibition effect of graviola callus dissolved extract

To test the hair loss inhibition effect of the graviola callus lysate extract, the expression levels of hair loss inducing factors and hair follicle growth cycle inducing factors in dermal papilla cells were evaluated at the mRNA level.

10.1 Culture and sample processing of human dermal papilla cells

The cultured cells were equally counted at 1Х10 ⁴ cells/well in a 6-well plate using a hemacytometer and then dispensed. The cells were cultured in DMEM containing 10% FBS for 24 hours, and when the culture vessel reached 60 to 70% of the surface area, they were replaced with FBS-free DMEM containing the graviola callus lysate extract according to the present invention and cultured for 24 hours. .

10.2 RT-PCR for hair loss inhibition and inducing factors

After culturing, the solution was dissolved using QIAzol Lysis Reagent (QIAGEN, 79306, USA), then 0.2 mL chloroform (Duksan, 67-66-3, Korea) was added and left at room temperature. Centrifugation was carried out at 12,000 rpm and 4°C for 20 min to separate the supernatant containing protein from the supernatant containing mRNA. 0.5 mL isopropanol (Merck, 109634, Germany) was added to the supernatant and left at room temperature for 10 minutes. RNA was precipitated by centrifugation at 12,000 rpm and 4°C. After washing with 75% ethanol, the ethanol was removed and dried at room temperature. The extracted RNA was dissolved in RNase Free dH ₂ O, mixed with 5ХPrimeScript RT Master Mix, and reacted at 37°C for 10 min, 85°C for 5 seconds to synthesize cDNA. Expression of hair-related DKK-1, β-catenin, and SRD5A1 genes was confirmed through RT-PCR using the synthesized cDNA. The primer sequences used are as shown in Table 4 below.

Primer name order sequence number DKK-1 F TGA TGA GTA CTG CGC TAG TC One R CTC CTA TGC TTG GTA CAC AC 2 β-catenin F ATG ACT CGA GCT CAG AGG 3 R ATT GCA CGT GTG GCA AGT TC 4 SRD5A1 F ACT GCA TCC TCC TGG CCA TGT TC 5 R GGC ATA GCC ACA CCA CTC CAT GA 6

As a result, it was confirmed that the gene expression levels of SRD5A1 and DKK-1, which are one of the factors causing hair loss, were decreased in the group treated with the graviola callus dissolved extract. The expression level of the SRD5A1 gene was significantly reduced to 46.76 and 45.56% at treatment concentrations of 750 μg/ml and 1000 μg/ml, respectively, compared to the untreated group (Figure 11), and the expression level of the DKK-1 gene was 500 μg/ml, At treatment concentrations of 750 μg/ml and 1000 μg/ml, the expression levels were reduced by 83.43, 67.76, and 74.38%, respectively, compared to the untreated group (Figure 12).

In addition, the expression level of the β-catenin gene, which is one of the growth phase inducers of hair follicles and has a positive effect on the hair growth cycle, increased by 114.45 and 112.05% at treatment concentrations of 125 μg/ml and 250 μg/ml, respectively. It was seen (Figure 13). Based on the above results, it is expected that treatment with graviola callus lysate extract will have a positive effect on suppressing hair loss by decreasing the expression level of hair loss-causing factors and increasing the expression level of genes that induce hair follicle growth.

As above, specific parts of the content of the present invention have been described in detail, and those skilled in the art will understand that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

A cosmetic composition for skin whitening or improving skin condition containing graviola callus dissolved extract as an active ingredient. According to paragraph 1,
A cosmetic composition wherein the skin condition improvement is skin regeneration or wound improvement. According to paragraph 1,
The composition is a cosmetic composition that inhibits melanin biosynthesis. According to paragraph 3,
The composition is a cosmetic composition that inhibits tyrosine (Tyrosine) or L-dopa (L-dopa) oxidation activity. According to paragraph 1,
The composition is a cosmetic composition that promotes antioxidant activity. delete delete A quasi-drug composition for preventing, improving or treating skin diseases containing graviola callus dissolved extract as an active ingredient. According to clause 8,
A quasi-drug composition, wherein the skin disease is at least one selected from the group consisting of skin wounds, skin scars, and skin pigmentation. A pharmaceutical composition for preventing, improving or treating skin diseases containing graviola callus soluble extract as an active ingredient. According to clause 10,
A pharmaceutical composition, wherein the skin disease is at least one selected from the group consisting of skin wounds, skin scars, and skin pigmentation. delete A method for producing a composition for skin whitening or improving skin condition, comprising the step of obtaining a graviola callus dissolved extract from graviola.