KR102289135B1 - Herbal medicine mixed extract and uses therof - Google Patents

Abstract

The present invention relates to the use of a mixed plant extract comprising earl yam, Angelica asiatica and licorice, wherein the plant mixed extract containing earl yam, Angelica basil and licorice is prepared according to the present invention for antioxidant, anti-inflammatory, wound healing, whitening, and wrinkle improvement. and anti-aging effect, and in addition, it has a significant effect compared to Ssanghwa-tang extracted with other plants, so it can be used usefully. Because it does not exceed the mixing ratio and mixing amount of the components, it is very safe for human application and use.

Description

Herbal medicine mixed extract and its use {HERBAL MEDICINE MIXED EXTRACT AND USES THEROF}

The present invention relates to the anti-aging use of a plant mixture extract comprising ear pea, Angelica lily and licorice.

The skin is a very complex body organ that is structurally composed of an epidermal layer, a dermal layer, and a hypodermal layer. It mainly protects the body from external stimuli, prevents moisture loss, maintains body temperature, and prevents infection. It acts as a protective barrier, such as The epidermis contains keratinocytes in the outer layer of the skin, melanocytes between the epidermis and the dermis, and fibroblasts in the deeper layer, the dermis, to maintain the structure and function of the skin. do. Collagen and elastin, one of the main substances constituting the dermis, are substances that give strength and tension to the skin and affect skin elasticity. Collagen is synthesized by the action of fibroblasts and decomposed by collagenase, and elastin is decomposed by elastase. Moisture in the skin cells is lost, the action of collagenase increases and the cross-linked form of collagen decreases, leading to skin aging and the formation of skin wrinkles. This anti-wrinkle method promotes collagen biosynthesis to maintain skin elasticity, replenishes ceramide itself in the stratum corneum, and enhances the function of MMP-1, a collagenase that decomposes collagen by UV rays. There is a method of inhibiting the function of elastase, which decomposes elastin, an insoluble elastic fiber of connective tissue, and breaks the basal layer network structure to create wrinkles.

On the other hand, melanin is produced by melanin-producing cells as a defense action to suppress the generation of free radicals that cause cell damage in the skin. It plays an important role in reducing irritation to the skin. On the other hand, the production of melanin also causes melasma and freckles, which are major concerns in women's beauty. When the skin is excessively exposed to UV rays, keratinocytes increase the transcription of POMC (pro-opomelanocortin) gene in response to cell damage and stimulate the production of melanin, α-melanocyte stimulating hormone (α-MSH), TGF- Increases the production and secretion of beta, PGE2, etc. TGF-beta, PGE2, and α-MSH secreted from keratinocytes stimulate cell activation of melanocytes that synthesize melanin and synthesize melanin as part of a mechanism to protect the skin from external stress. It is transmitted to keratinocytes and the like through dendritic cells, etc.

However, since brown and black pigments generated from melanocytes have a negative effect on skin diseases as well as skin beauty, various attempts have been made to block melanin synthesis as part of research to obtain white skin with skin diseases in the cosmetic industry. (Tobin DJ, Shibahara S, Slominski A, Melanin pigmentation in mammalian skin and its hormonal regulation. J. Physiol Rev, 84(4), pp.1155-228, 2004). Representative methods for inhibiting this include a method of inhibiting tyrosinase, the main enzyme that makes L-DOPA in melanin formation, a method of binding the enzyme so that it does not work, and a method of inhibiting the oxidation process of L-DOPA in the melanin formation process. There is a way.

An increase in reactive oxygen species (ROS) caused by external stimuli may increase skin pigmentation and cause cell and tissue damage. Excessive ROS induces inflammation of the skin and suppresses immune function, thereby reducing skin regeneration and wound repair ability, and activating enzymes such as α-MSH (melanin stimulating hormone), related cytokines and tyrosinase to produce abnormal melanin production. (melanogenesis), resulting in skin aging and hyperpigmentation. It has been reported that antioxidants that inhibit ROS have an effect on skin damage caused by UV exposure, and in clinical studies, it was reported that wound wounds healed quickly in burn patients with high antioxidant properties. A wound is generally damaged to the dermis layer and the continuity of the skin structure is destroyed by the opening of the skin. Wound healing is when the wound site is restored through the inflammatory response, the formation of regenerated tissue, and the re-epithelialization stage. complex steps such as In particular, chronic wounds are mainly caused by a delayed inflammatory stage, but wounds caused by chronic diseases do not heal well and develop mainly in the form of ulcers. Extrinsic skin photoaging caused by UV exposure causes wrinkles, delayed wound healing, prolonged chronic inflammation, and localized pigmentation such as uneven spots on the skin. Aged, cultured cells become broad, flat, and vacuolised. There is a β-galactosidase activity that can be detected in senescent cells arrested at G1 of the cell cycle, i.e., the so-called senesence-associated beta-galactosidase (SA-β-gal) activity. Currently, SA-β-gal is used as a major biomarker of cellular senescence as a method to easily identify senescent cells.

Ssanghwa-tang is a traditional recipe recorded in Donguibogam and Bangyakhappyeon. Samultang, which consists of Paeonia lactiflora, Sukjihwang (Rehmannia glutinosa), Angelica gigas, Cnidiumofficinale, and Paeonia lactiflora, Astragalus membranaceus membranaceus. , cinnamon (Cinnamomum cassia), licorice (Glycyrrhiza glabra), ginger (health) (Zingiber officinale), and jujube (Zizyphus jujuba) are combined. Since ancient times, Ssanghwa-tang has been prescribed as a healing medicine when the mind and body are tired and the qi and blood are damaged. The effects of Ssanghwa-tang reported so far include anti-fatigue, improvement of liver function, central nervous system suppression, anti-pain and antispasmodic effect, promotion of sex hormone secretion, and improvement of osteoporosis. Although excellent efficacy of Ssanghwa-tang has been reported as described above, studies on skin diseases and skin protection have not been actively conducted.

Therefore, in this study, PAG (Paeonia lactiflora, Angelica gigas, Glycyrrhiza glabra) was prepared by selecting and extracting PAG (Paeonia lactiflora, Angelica gigas, Glycyrrhiza glabra) from among the components of Ssanghwatang, melanocytes, keratinocytes, and human dermal fibroblasts. And macrophages were used to confirm antioxidant, anti-inflammatory, whitening, wrinkle improvement, wound healing and anti-aging effects.

An object of the present invention is to provide an anti-aging cosmetic composition.

In addition, it is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease.

Another object of the present invention is to provide a pharmaceutical composition for external use on the skin.

It is also an object of the present invention to provide an anti-aging food composition.

In addition, it is an object of the present invention to provide a food composition for preventing or improving melanin hyperpigmentation.

In order to solve the above problems, the present invention provides an anti-aging cosmetic composition containing a plant mixed extract including ear pea, Angelica lily and licorice as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease containing a mixed extract of a plant consisting of ear buds, Angelica basil and licorice as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for external use on the skin containing as an active ingredient a plant mixed extract comprising ear peony, angelica and licorice.

In addition, the present invention provides an anti-aging food composition containing a plant mixed extract comprising ear peony, angelica and licorice.

In addition, the present invention provides a food composition for preventing or improving melanin hyperpigmentation containing a plant mixture extract consisting of earl yam, Angelica lily and licorice.

The plant mixed extract containing ear pea, Angelica basil and licorice produced according to the present invention has antioxidant, anti-inflammatory, wound healing, whitening, wrinkle improvement and anti-aging effects, and in addition, compared to Ssanghwa-tang extracted with other plants Because it shows a remarkable effect, it can be used usefully. In particular, the PAG, a mixture of three kinds of herbal medicines selected from Ssanghwa-tang, is very safe for human application and taking because the mixing ratio and mixing amount do not exceed the mixing ratio and mixing amount of the ingredients of Ssanghwa-tang. It has a characteristic that

1 is a diagram confirming the cytotoxicity of PAG and SHT to melanocytes B16F10.
2 is a diagram confirming the cytotoxicity of PAG and SHT to keratinocytes HaCaT.
3 is a diagram confirming the cytotoxicity of PAG and SHT to macrophage RAW 264.7.
Figure 4 is a diagram confirming the cytotoxicity of PAG and SHT to human dermal fibroblasts HDF.
5 is a diagram confirming the antioxidant effects of PAG and SHT through DPPH, Hydrogen peroxidase scavening, and reducing power.
6 is a view confirming the melanin synthesis inhibitory effect of PAG and SHT.
7 and 8 are diagrams confirming the tyrosinase activity inhibitory effect of PAG and SHT.
9 is a view confirming the effect of inhibiting the elastase activity of PAG.
10 is a view confirming the wound healing effect of PAG and SHT.
11 is a view confirming the anti-inflammatory effect of PAG and SHT.
12 is a diagram confirming the inhibitory effect of PAG and SHT on SA-β-gal activity.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary according to the intention of a user or operator, or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

Throughout this specification, '%' used to indicate the concentration of a specific substance is (w/w) % for solid/solid, (w/v) % for solid/liquid, and Liquid/liquid is (v/v) %.

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

In one aspect, the present invention relates to an anti-aging cosmetic composition containing as an active ingredient a plant mixed extract comprising ear pea, Angelica lily and licorice.

In one embodiment, the cosmetic composition of the present invention may be an anti-aging cosmetic composition having antioxidant activity, skin whitening activity, wrinkle improvement and wound healing activity.

In one embodiment, the cosmetic composition of the present invention may be a cosmetic composition for skin whitening that inhibits melanin production and inhibits tyrosinase activity.

In one embodiment, the cosmetic composition of the present invention may be a cosmetic composition for improving wrinkles that inhibits elastase activity.

In one embodiment, earl yew, Angelica basil and licorice may be mixed in a weight ratio of 10:4:3.

In one embodiment, Rehmannia glutinosa , Astragalus membranaceus , Cnidium officinale, Cinnamomum cassia , ginger (health; dried ginger) ( Zingiber officinale ) and jujube ( Zizyphus jujuba ) It may further include an extract.

As used herein, the term “extract” refers to an active ingredient separated from a natural product, that is, a substance exhibiting a desired activity. The extract can be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof, and includes a dry powder thereof or any form formulated using the extract. In addition, the extract includes those obtained by fractionating the extract that has undergone the extraction process. The extraction method of the extract is not particularly limited, and for example, it may be extracted by stirring extraction, shaking extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction. As the extraction solvent, a _{polar solvent such as water, a C 1} -C ₄ lower alcohol or a non-polar solvent such as hexane, chloroform, dichloromethane or ethyl acetate, or a mixture of two or more thereof may be used.

The extract of the present invention may be used as it is extracted from a natural product using a solvent, or a fermented product of the extract prepared by additionally including a fermentation process using an enzyme or microorganism to increase activity. The fermentation is to increase the biological activity or absorption rate of the extract of the present invention, and the effect on the skin such as anti-aging, whitening, wrinkle improvement, wound healing, antioxidant, anti-inflammatory, etc. of the extract confirmed in the present invention can be improved. have. The type of enzyme or microorganism used for fermentation of the extract is not particularly limited, and as long as it is possible to ferment a natural product as in the present invention, it may be selected and used without limitation.

The cosmetic composition of the present invention may be prepared including a cosmetically effective amount of the above-described active ingredients of the present invention and a cosmetically acceptable carrier.

As used herein, the term “cosmetic effective amount” refers to an amount sufficient to achieve the whitening, anti-wrinkle or anti-aging efficacy of the composition of the present invention described above.

The appearance of the cosmetic composition contains a cosmetically or dermatologically acceptable medium or base. It is in any dosage form suitable for topical application, for example solutions, gels, solids, kneaded dry products, emulsions obtained by dispersing the oily phase in an aqueous phase, suspensions, microemulsions, microcapsules, microgranules or, ionic (liposomes) and It may be provided in the form of a non-ionic vesicular dispersant, or in the form of a cream, skin, lotion, powder, ointment, spray or concealer stick. These compositions can be prepared according to conventional methods in the art. The composition according to the invention can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition according to an embodiment of the present invention is not particularly limited in its formulation, for example, softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing It can be formulated into cosmetics such as cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil, and body essence.

When the formulation of the cosmetic composition of the present invention is a paste, cream, or gel, animal fiber, vegetable fiber, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. This can be used.

When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydro It may contain a propellant such as carbon, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene fatty acid esters of glycol, 1,3-butylglycol oil, glycerol fatty esters, polyethylene glycol or sorbitan.

When the formulation of the cosmetic composition of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanth may be used.

When the formulation of the cosmetic composition of the present invention is surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate as carrier components , fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linoline derivative or ethoxylated glycerol fatty acid ester, etc. may be used.

The cosmetic composition of the present invention includes skin, lotion, cream, essence, pack, foundation, color cosmetics, sun cream, two-way cake, face powder, compact, makeup base, skin cover, eye shadow, lipstick, lip gloss, lip fix, eyebrow pencil It can be applied to cosmetics such as , lotion, and detergents such as shampoo and soap.

The cosmetic composition according to an embodiment of the present invention may further include functional additives and components included in general cosmetic compositions in addition to the extract containing the active ingredient. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high-molecular peptides, high-molecular polysaccharides, sphingolipids, and seaweed extract.

In the cosmetic composition of the present invention, a component contained in a general cosmetic composition may be further blended with the functional additive as needed. Other ingredients included include oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation An accelerator, a cooling agent, a limiting agent, purified water, etc. are mentioned.

In one aspect, the present invention relates to a pharmaceutical composition for external use on the skin containing a plant mixed extract comprising ear pea, Angelica lily and licorice as an active ingredient.

In one embodiment, the pharmaceutical composition for external application to the skin may have anti-inflammatory and wound healing activity.

In one embodiment, the pharmaceutical composition for external application to the skin may further include extracts of Sukhumidhuang, Astragalus, chrysanthemum, broiler broiler, ginger (health) and jujube.

In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease containing a mixed extract of a plant consisting of ear buds, Angelica basil and licorice as an active ingredient.

In one embodiment, the melanin hyperpigmentation disease is freckles, senile spots, liver spots, melasma, brown or dark spots, solar pigmentation, cyanic melasma, hyperpigmentation after drug use, gravidic chloasma ), or post-inflammatory hyperpigmentation due to wounds or dermatitis, including abrasions and burns.

The term "treatment" as used in the present invention means any action that improves or advantageously changes the symptoms of an inflammatory disease or its complications by administering the extract of the present invention or a composition comprising the same. Those of ordinary skill in the art to which the present invention pertains, with reference to the data presented in the Korean Medical Association, etc., know the exact criteria of the disease for which the composition of the present application is effective, and can determine the degree of improvement, improvement and treatment will be.

In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of inflammation by administration of the pharmaceutical composition according to the present invention.

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral formulations, topical formulations, suppositories, sterile injection solutions and sprays.

The therapeutically effective amount of the composition of the present invention may vary depending on several factors, for example, the administration method, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's Health status, type of cancer, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. Compounds described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

As used herein, the term “pharmaceutically acceptable” refers to exhibiting non-toxic properties to cells or humans exposed to the composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, stearic acid Calcium, sucrose, dextrose, sorbitol, talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

As used herein, the term "administration" means providing a predetermined substance to a patient by any suitable method, and parenteral administration (eg, intravenous, subcutaneous, intraperitoneal or topical administration according to a desired method) ) or oral administration, and the dosage varies according to the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.

The composition of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) or orally according to a desired method, and the dosage may vary depending on the patient's weight, age, sex, health status, The range varies depending on the diet, administration time, administration method, excretion rate, and the severity of the disease. The daily dose of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to divide and administer once to several times a day.

Liquid preparations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. and the like may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.

In one aspect, the present invention relates to an anti-aging food composition comprising a plant mixed extract comprising ear pea, Angelica basil and licorice.

In one embodiment, the food composition may further include Sukhumid Hwang, Astragalus, Chunkyung, broiler broiler, ginger (health) and jujube extract.

In one aspect, the present invention relates to a food composition for skin whitening and wrinkle improvement containing a plant mixed extract consisting of ear bud, angelica and licorice.

In one aspect, the present invention relates to a food composition for preventing or improving melanin hyperpigmentation containing a plant mixed extract consisting of ear buds, Angelica basil and licorice.

When the composition of the present invention is used as a food composition, the extract may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. In addition to the active ingredient, the composition may contain a food additive that is pharmaceutically acceptable, and the mixing amount of the active ingredient may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment).

The term "food supplement additive" used in the present invention refers to a component that can be added to food as an auxiliary, and is added to the manufacture of health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplement additive of the present invention.

The food composition of the present invention may include a health functional food. The term "health functional food" as used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful in the human body. Here, the term 'functionality' refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the ordinary technical field, and at the time of the preparation, it can be prepared by adding raw materials and components commonly added in the conventional technical field. In addition, if the formulation of the health functional food is also recognized as a health functional food, it can be prepared without limitation. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, and has excellent portability, and the present invention health functional food can be taken as an adjuvant to enhance the effect of anticancer drugs.

In addition, there is no limitation on the type of health food in which the composition of the present invention can be used. In addition, the composition comprising the extract of the present invention as an active ingredient can be prepared by mixing known additives with other suitable auxiliary ingredients that may be contained in health functional foods according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding the extract according to the present invention as a main component to juice, tea, jelly, juice, and the like.

Since the extract of the present invention is made from a natural plant as a raw material, even when used as a pharmaceutical composition or a food composition, side effects may be less than that of a general synthetic compound.

The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the contents of the present invention, and the present invention is not limited thereto.

Example 1. Preparation of mixed extracts of ear pea, Angelica and licorice

To develop as a functional material with anti-aging effects of wound healing, skin whitening, wrinkle improvement, antioxidant, and anti-inflammatory, it is used as a traditional prescription based on the traditional medicine Donguibogam and Bangyakhappyeon. A mixed extract was prepared by selecting three kinds of herbal medicines that are being used, namely, Paeonia lactiflora , Angelica gigas , and Licorice ( Glycyrrhiza glabra). Specifically, the Korean herbal medicine Semosan 3399 (417 Paengseongbuk-ro, Paengseong-eup, Pyeongtaek-si, Gyeonggi-do) was used for ear peony, angelica, and licorice. The purchased domestic herbal medicine, Earl Pole (300g), Angelica (120g) and Licorice (90g) were mixed and placed in a nonwoven fabric, and then washed 2-3 times. After immersion in water for 1 hour, 3L of drinking water is added, and the mixed extract (PAG) is recovered using a circulating non-pressure semi-automatic centrifugal herbal extractor ME-45 (Migang Co., Ltd., Busan, Korea) and converted into an extract (PAG). After use, the herbal medicine residues in the sieve were removed. The extract (PAG) was vacuum-packed continuously at 75 mL/pack using an automatic packaging machine (film adhesive) KD-101-A-20R (Kukje Industrial Co., Ltd., Seoul, Korea). One package of extract was stored in a deep freezer for 2 hours and then frozen, and then freeze-dried at -50°C and 50 mTorr using a freeze dryer TFD5503 (Ilshin Biobase, Gyeonggi-do, Korea), and this was used as a PAG sample ( 32.8 mg/mL). As shown in Table 1 below, the mixing ratio and mixing amount of the three selected oriental medicinal materials are Ssang-hwa-tang (Ssang-hwa-tang, SHT), which is traditionally widely used as medicine or tea. Since the mixing ratio and mixing amount of the constituent drugs are not exceeded, the PAG sample is safe for human application and consumption.

Example 2. Confirmation of flavonoid and phenol content of the mixed extract

The contents of flavonoids and phenols of the mixed extract (PAG) prepared in Example 1 were compared with those of Ssanghwa-tang (SHT). Specifically, the confirmation of the total flavonoid content is to make a total solution of 1 mL by mixing 300 μL of 95% ethanol, 20 μL of 10% aluminum chloride hexahydrate, 20 μL of 1M potassium acetate and 560 μL of distilled water in 100 μL of SHT or PAG, respectively, and then vortexing. After mixing and reacting at room temperature for 40 minutes, absorbance was measured at 415 nm using a Molecular Devices microplate reader (PerkinElmer, USA). Confirmation of the total flavonoid content of SHT and PAG was calculated using a standard curve using quercetin and expressed as QE/100mg. In addition, the total phenol content is calculated by mixing 400 µL of 2% sodium carbonate, 20 µL of 50% Folin-Ciocalteu reagent, and 560 µL of distilled water to 20 µL of each of SHT or PAG to make a total of 1 mL solution, vortex and mix for 30 minutes After the reaction, absorbance was measured at 750 nm using a Molecular Devices microplate reader (PerkinElmer, USA). The total phenol content of SHT and PAG was calculated using a standard curve using gallic acid and expressed as QE/100 mg.

As a result, it was found that the total flavonoid and total phenol content of Ssanghwa-tang (SHT), in which 6 other herbal medicines were added in addition to earl yam, angelica, and licorice, and the mixed extract (PAG) of the present invention, were found to be similar (Table 2).

Example 3. Confirmation of Cytotoxicity of Mixed Extracts

The cytotoxicity of the mixed extract PAG of the present invention was confirmed in keratinocytes HaCaT, melanocyte B16F10, human dermal fibroblast HDF and macrophage RAW 264.7, respectively. Specifically, DMEM containing penicillin (100 IU/mL), streptomycin (100 μg/mL), and 10% FBS (fetal bovine serum) after inoculation of HaCaT, B16F10, HDF and RAW 264.7 cell lines into the bottom of a culture dish (Dulbecco' Modifide Eagle' Medium) medium was added, maintained at 37°C, and cultured in an incubator containing 5% carbon dioxide. HaCaT, B16F10, HDF and RAW 264.7 cell lines were aliquoted into 24-well plates at a concentration of 5×10⁴ cells per well, and then cultured in a cell culture condition at 37° C. and 5% carbon dioxide incubator for 24 hours. Discard the medium, wash with 1×PBS, change to a fresh medium without 10% FBS, and treat test substance SHT or PAG at a certain concentration (0, 125, 250, 500, 1000, 2000, 3000 μg/mL) After incubation for 24 hours. After incubation, add 100 μL of MTT solution (0.5% 3-(4,5-dimethylthiazol-2-yl)-2,5 dephenyl-2H-tetrazolium bromide solution) to each well, and formazan Incubated for 4 hours until formation. After 4 hours, the culture medium was removed, and then 1000 μL of DMSO (dimethylsulfoxide) solution was added, shaken for 10 minutes, and then aliquoted into a 96-well plate and absorbance was measured at 540 nm with an ELISA reader.

As a result, neither PAG nor SHT was found to be toxic up to 2000 μg/mL in melanocytes (B16F10) and up to 2000 μg/mL in keratinocytes (HaCaT), confirming that PAG is safer for cytotoxicity. were (Figures 1 and 2). In addition, neither mixture was found to be cytotoxic in macrophages (RAW 264.7) and human dermal fibroblasts (HDF) ( FIGS. 3 and 4 ).

Example 4. Confirmation of the antioxidant effect of the mixed extract

4-1. Measurement of DPPH radical scavenging ability

In order to measure the antioxidant effect of the PAG mixed extract of the present invention, DPPH radical scavenging ability was measured and compared with SHT. Specifically, in a 96-well plate, add 100 µL of each concentration of SHT or PAG diluted with PBS (125-2000 µg/mL) to a 96-well plate, and 100 µL of 99% ethyl alcohol and 0.4 mM DPPH diluted with 99% ethyl alcohol. After adding 100 μL of (2,2-diphenyl-1-picrylhydrazyl) solution and reacting in the dark for 30 minutes, absorbance was measured at 517 nm using a Molecular Devices microplate reader (PerkinElmer, USA), and the following Equation 1 was used to represent the graph.

4-2. Measurement of hydrogen peroxide radical scavenging ability

In order to measure the antioxidant effect of the PAG mixed extract of the present invention, hydrogen peroxide (Hydrogen peroxide) radical scavenging ability was measured and compared with SHT. _{Specifically, 20 μL of SHT or PAG diluted with PBS and 1.0 MH 2} O ₂ were mixed in a 96-well plate for each concentration (125-2000 μg/mL) in a 96-well plate and reacted at 37° C. for 5 minutes, and then this was again 1.25 mM 30 μL of ABTS and 1 U/mL peroxidase were added, reacted at 37° C. for 10 minutes, and absorbance was measured at 405 nm with a Molecular Devices microplate reader (PerkinElmer, USA), and a graph was shown using Equation 2 below.

4-3. Measuring reducing power

In order to measure the antioxidant effect of the PAG mixed extract of the present invention, the reducing power was measured and compared with SHT. Specifically, SHT or PAG diluted with PBS and 0.2 M sodium phosphate buffer (pH 6.6) and 1% potassiumferricyanide (K ₃ Fe(CN) ₆ ) in a 96-well plate by concentration (125-2000 μg/mL) After reacting at 50 °C for 20 minutes with 250 μL of a mixture of 250 μL of 10% trichloroacetic acid (CCl ₃ COOH), centrifuge the reaction solution at 4000 rpm for 10 minutes, mix 500 μL of distilled water with 500 μL of the supernatant, FeCl ₃ ·H ₂ O) 100 μL was added to make a total solution of 1.1 mL, mixed by vortexing, and reacted at room temperature for 10 minutes. Absorbance was measured at 700 nm using a Molecular Devices microplate reader (PerkinElmer, USA).

As a result of measuring the above three types of antioxidant activity, both SHT and PAG showed ROS scavenging rates of reactive oxygen species in a concentration-dependent manner, and as a result of comparing _{the antioxidant efficacy of SHT and PAG with IC 50 values,} In both reducing power, PAG showed higher antioxidant efficacy at lower concentrations ( FIG. 5 ).

Example 5. Confirmation of the whitening effect of the mixed extract

5-1. Check melanin production

Intracellular melanin synthesis was artificially increased by α-melanocyte stimulating hormone (α-MSH), and the regulation of melanin synthesis by PAG treatment was confirmed. Specifically, B16F10 mouse melanoma cells (Korea Cell Line Bank, Seoul, Korea) were inoculated in a 6-well plate at 2×10 ⁵ cells/well, and penicillin (100 IU/mL), streptomycin (100 μg/mL) and 10% DMEM (Dulbecco'Modifide Eagle'Medium) medium containing FBS (fetal bovine serum) was added, and the cells were cultured under cell culture conditions (37°C, 5% CO ₂ ) for 24 hours. When the cells are attached, remove the medium, wash the cells with 1mL of PBS, and then treat the sample solution, SHT or PAG by concentration (125~1000 μg/mL), and treat the medium with 100 nM of melanogenesis-stimulating hormone (α-MSH). After allowing the melanogenesis process to be accelerated, cultured for 48 hours. After removing the medium and washing the cells with 1 mL of PBS, 1 mL of PBS was added again, and the cells were scraped with a scraper and centrifuged at 5000 rpm for 1 minute. The supernatant was removed by centrifugation, and the degree of intracellular pigmentation was visually measured by taking an image of the cell sediment pellet. In addition, 40 μL of protein lysis buffer (PRO-PREP™) was added to the cell precipitate and the cells were lysed by leaving them on ice for 30 minutes, followed by centrifugation at 4° C. at 15,000 rpm for 30 minutes, and the supernatant was tyrosinase ( tyrosinase) activity was used as an enzyme solution. The cell precipitate was recovered again, 250 μL of 1 N sodium hydroxide (NaOH) solution was added, and left to stand at 60° C. for 1 hour using a dry bath incubator MK200-2 (allsheng, China) to be suspended, then vortexed to 96- Transfer 70 μL per well to a well plate and measure the absorbance at 405 nm using a Molecular Devices microplate reader (PerkinElmer, USA) to determine the amount of melanin production, and compare with the results of the blank sample solution treated with the solvent in which the sample was dissolved. As a positive control, arbutin (100 μg/mL) was used.

As a result, the amount of melanin production increased by melanogenesis-stimulating hormone (α-MSH) had an inhibitory effect on melanogenesis in both groups treated with SHT or PAG, but at the same concentration, PAG more inhibited melanogenesis than SHT. And, unlike SHT, PAG significantly inhibited melanin production even in the lowest concentration 125 μg/mL treatment group used in the experiment. In addition, in the pigmentation image data, it was visually confirmed that the hyperpigmentation increased by α-MSH was reduced to the control level in the PAG 1000 μg/mL treatment group ( FIG. 6 ).

5-2. Confirmation of Tyrosinase Inhibitory Effect

Melanin production is oxidized by the enzyme tyrosinase. In the melanin production stage, tyrosinase oxidizes tyrosine to DOPA and plays an important role in forming eumelanin by generating DOPA chrome through successive steps. In the formation of melanin, the tyrosinase enzyme activity inhibitor can inhibit melanin synthesis by inhibiting the initial stage of melanin production, so the effect of PAG on tyrosinase activity was confirmed. Specifically, B16F10 mouse melanoma cells (Korea Cell Line Bank, Seoul, Korea) were inoculated in a 6-well plate at 2×10 ⁵ cells/well, penicillin (100 IU/mL), streptomycin (100 μg/mL) and 10 DMEM (Dulbecco'Modifide Eagle'Medium) medium containing % FBS (fetal bovine serum) was added, and the cells were cultured under cell culture conditions (37°C, 5% CO ₂ ) for 24 hours. When the cells are attached, remove the medium, wash the cells with 1mL of PBS, and then treat the sample solution, SHT or PAG by concentration (125~1000 μg/mL), and treat the medium with 100 nM of melanogenesis-stimulating hormone (α-MSH). After accelerating the process of melanin production, cultured for 48 hours. After removing the medium and washing the cells with 1 mL of PBS, 1 mL of PBS was added again, and the cells were scraped with a scraper and centrifuged at 5000 rpm for 1 minute. The supernatant was removed by centrifugation and a cell precipitate pellet was obtained. 40 μL of protein lysis buffer (PRO-PREP™) was added to the cell sediment pellet and the cells were lysed by standing on ice for 30 minutes, followed by centrifugation at 4° C. at 15,000 rpm for 30 minutes to recover the supernatant. The recovered supernatant was used as an enzyme solution for measuring activity. After quantifying the enzyme solution by the Bradford method, put 40 μg of this enzyme solution and 40 μL of 100 mM sodium phosphate buffer (pH 6.8) in a 96-well plate, and add 160 μL of 10 mM L-DOPA as a substrate. After dark reaction at 37°C for 1 hour, absorbance was measured at 475 nm using a Molecular Devices microplate reader (PerkinElmer, USA). As a positive control, arbutin (arbutin, 100 μg/mL) was used. In addition, the inhibitory effect of tyrosinase activity in the cell free system is to dissolve the sample SHT and PAG in PBS as a solvent, and set a concentration range that can confirm the inhibition of tyrosinase activity within a concentration range without cytotoxicity (125). ~1000 μg/mL) to measure the inhibition of tyrosinase activity. Specifically, in a test tube, 100 μL of 100 mM sodium phosphate buffer (pH 6.5), 20 μL of sample solutions SHT and PAG by concentration (125-1000 μg/mL), and mushroom tyrosinase solution (mushroom tyrosinase) , 110 U/mL) and 40 μL were added in this order. To this solution, add 40 μL of 1.5 mM tyrosine solution dissolved in 100 mM sodium phosphate buffer (pH 6.8), react at 25°C for 5 minutes, and then use a Molecular Devices microplate reader (PerkinElmer, USA) at 475 nm. to measure the absorbance. _{The sample concentration (IC 50} ) when the activity inhibition rate was 50% was calculated using an appropriate program. Instead of the sample solution, a solvent in which the sample was dissolved was used as a blank sample solution for correction.

As a result, the tyrosinase activity increased by melanogenesis-stimulating hormone (α-MSH) did not show a significant inhibitory effect by SHT treatment, but PAG showed significant tyrosinase activity from the 500 μg/mL treatment concentration. negatively inhibited (FIG. 7). This result is consistent with the result of the whitening effect that the pigmentation image increased by α-MSH of [FIG. 6] is greatly reduced from the PAG treatment concentration of 500 μg/mL. In addition, as a result of measuring the tyrosinase inhibitory activity of SHT and PAG in the cell free system, 50% maximum inhibitory concentration IC ₅₀ (the half maximal inhibitory concentration) When compared by values, PAG showed a better effect at a lower concentration than SHT ( FIG. 8 ).

Example 6. Confirmation of the wrinkle improvement effect of the mixed extract

In order to confirm the anti-wrinkle effect of the mixed extract PAG of the present invention, the inhibitory effect on elastase activity was compared with SHT. Specifically, after inoculating human dermal fibroblasts HDF human dermal fibroblasts (Cell Bio, Seoul, Korea) on the bottom of a culture dish, penicillin (100 IU/mL), streptomycin (100 μg/mL) and 10% FBS (fetal) DMEM (Dulbecco'Modifide Eagle'Medium) medium containing bovine serum) was added, maintained at 37°C, and cultured in an incubator containing _{5% CO 2 .} After washing the cultured cells with PBS, 0.1% triton X-100·0.2M Tris solution (PH 8.0) solution was added and dissolved. This solution was pipetted several times to homogenize the cells. After centrifuging the solution at 4° C. at 3000 rpm for 20 minutes, the supernatant was taken and used as an enzyme solution containing elastase. The elastase solution was quantified by the Bradford method, and an amount containing 100 μg of protein per well was put into 96 wells, and 0.2M Tris-HCl buffer (PH 8.0) was added to make 88 μL. After adding 10 μL of SHT and PAG, which are the sample solutions, to each well by concentration (125-1000 μg/mL), STANA (N-succinyl-tri-alanyl-p-nitroanilide, 50 mM) solution, which is an elastase substrate, was added to each well. 2 μL per well was added and incubated at 37°C. After 90 minutes, absorbance was measured at 405 nm using a Molecular Devices microplate reader (PerkinElmer, USA). As a positive control, phosphoramidon (PPR; 800 μM) was used.

As a result, both SHT and PAG had an effect of inhibiting elastase activity, but PAG exhibited a better elastase activity inhibitory effect ( FIG. 9 ). The elastin present in the dermal layer of the skin forms a network structure and plays an important role in maintaining the elasticity of the skin, and the amount of elastin in the dermis can be controlled by the activity of an enzyme called elastase. . When the skin is stimulated by ultraviolet rays and free radicals, the activity of elastase increases, decomposition of elastin occurs, the network structure sags, and wrinkles are formed. known to be active. Therefore, it is inferred that the PAG of the present invention can maintain elasticity and prevent skin sagging by maintaining the mechanical properties of the skin tissue by lowering the activity of elastase, an elastin degrading enzyme, which is one of the main causes of skin aging and wrinkles. can do.

Example 7. Confirmation of wound healing effect of mixed extract

In order to confirm the wound healing effect of the mixed extract PAG of the present invention, a scratch is made on the cell monolayer with a physical stimulus, and a certain interval is measured for each time to capture an image that causes the cells to move and close the scratch area under a microscope over time. By doing so, the degree of wound recovery or cell migration was measured. Specifically, HaCaT immortalized human keratinocyet cells (Korea Cell Line Bank, Seoul, Korea) were inoculated into a 6-well plate at ^{a concentration of 5 × 10 5} cells/well, and after 24 hours, the supernatant was removed and serum-free for 12 hours. free) medium was starvated. Each well was scratched using the end of a pipet tip of 200 μL, and then treated with SHT or PAG diluted by concentration (125~1000 μg/mL), and then microscopic camera Digiretina 16 Camera (TUCSEN, China) every 12 hours. was taken, and the captured image was measured by quantifying the degree of cell closure using the image J program.

Unlike SHT, the PAG of the present invention showed a significant cell migration effect at the concentrations of 500 μg/mL and 1000 μg/mL, showing an excellent wound healing effect ( FIG. 10 ).

Example 8. Confirmation of the anti-inflammatory effect of the mixed extract

COX-2 (cyclooxygenase-2) is a major enzyme in the biosynthesis of prostaglandin (PG: prostaglandin), an inflammation-inducing substance. is known In order to confirm the anti-inflammatory effect of the mixed extract PAG of the present invention, the mRNA expression inhibitory effect of COX-2 (cyclooxygenase-2), which is produced by immune cells such as macrophages and plays an important role in the inflammatory process, was confirmed. Specifically, RAW 264.7 mouse leukaemic monocyte macrophage cells (Korea Cell Line Bank, Seoul, Korea) were ^{aliquoted to 2×10 6} cells/well in a 6-well plate, and when cells were attached, SHT or PAG samples were treated by concentration (125~ 1000 μg/mL) was treated with Lipopolysaccharides (LPS) 1 μg/mL and incubated for 18 hours. As a positive control, Celecoxib (5 μM) was treated. After incubation, total intracellular mRNA was extracted according to the manual of the RNAiso Plus (Takara Biochemical Inc., Japan) sample, and cDNA was synthesized according to the manual using the cDNA Synthesis kit (Takara). cDNA was prepared by using a Real-Time PCR (polymerase chain reaction) (CFX Connect, Biorad) machine and the primer set in Table 3 below according to the sample ratio of the manual instructed by SYBR Premix Ex Taq II (Takara) to prepare COX-2 mRNA The expression level was measured.

As a result, both SHT and PAG showed an anti-inflammatory effect by significantly reducing the expression of COX-2 increased by LPS stimulation. shown (Fig. 11).

Example 9. Confirmation of the anti-aging effect of the mixed extract

As dermal fibroblasts age, they are arrested at G1 of the cell cycle, and the increase in SA-β-gal (senesence-associated beta-galactosidase) results in a positive reaction in staining, and senescence markers such as p16INK4A are measured. Therefore, the measurement of SA-β-gal activity and p16INK4A is used as a major biomarker of cellular senescence. Therefore, a Senescence Detection kit (BioVision, USA) was used to confirm the expression of SA-β-gal in dermal fibroblasts of the mixed extract PAG of the present invention, which was X-gal (5-Bromo-4-Chloro-3- Indolyl-β-D-Galactoside), an enzyme expressed only in senescent cells and tissues, is hydrolyzed and dyed blue around the nucleus of aging dermal fibroblasts. Specifically, HDF human dermal fibroblasts (Cell Bio Co., Ltd., Seoul, Korea) were ^{aliquoted to 1×10 5} cells/well in a 24-well plate and cultured for 24 hours. After 24 hours, the cultured medium was removed, and after 4 hours of culture with DMEM (Dulbecco'Modifide Eagle'Medium) medium without penicillin and FBS (fetal bovine serum), UVB (30mJ/cm2) was irradiated and SHT or PAG was applied. By concentration (125-1000 μg/mL), the cells were further cultured for 24 hours under cell culture conditions (37° C., 5% CO _{2 ).} After incubation for 24 hours, after washing with PBS, 0.5 mL of a fixative solution was added and reacted at room temperature for 12 minutes to immobilize. The immobilized cells were treated with 0.5 mL of a staining solution mixture (staining solution 470 μL, staining supplement 5 μL and 20 mg/mL X-gal in DMSO 25 μL) and reacted at 37° C. for 12 hours to stain the cells. The stained cells were photographed using an optical microscope camera Digiretina 16 Camera (TUCSEN, China), and the number of stained cells was measured by dividing the image into compartments, and the proportion of aged cells was compared with the control group to indicate the degree of cellular senescence. .

As a result, both SHT and PAG had an inhibitory effect on SA-β-gal activity, but PAG showed a superior SA-β-gal activity inhibitory effect ( FIG. 12 ).

Claims (14)

A pharmaceutical composition for the treatment of open wounds caused by physical scratching of the epidermis of the skin, containing a mixed extract consisting of ear buds, Angelica basil and licorice at a concentration of 500-1000 μg/mL. delete delete delete delete delete delete delete delete delete delete delete delete delete

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