WO2020190072A1 - Composition for promoting skin wound healing or skin regeneration, containing (r)-ginsenoside rg3 as active ingredient - Google Patents

Abstract

The present invention relates to a composition for promoting skin wound healing or skin regeneration, containing, as an active ingredient, (R)-ginsenoside Rg3 or a pharmaceutically acceptable salt thereof. (R)-ginsenoside Rg3 increases the mobility of and infiltration into cells, is noncytotoxic, and forms new tissues and reduces scarring at a wound site, thereby being very effective for skin wound healing and skin regeneration. Therefore, (R)-ginsenoside Rg3 or a pharmaceutically acceptable salt thereof can be effectively used as a pharmaceutical or cosmetic composition for the promotion of skin wound healing and skin regeneration.

Description

(R)-Ginsenoside RG3 as an active ingredient for healing skin wounds or promoting skin regeneration

The present invention relates to a composition for promoting skin wound healing or skin regeneration comprising (R)-ginsenoside Rg3 as an active ingredient, and more specifically, R-form ginsenoside Rg3 (ginsenoside Rg3 ( 20R)) or a pharmaceutically/cosmetically acceptable salt thereof as an active ingredient, to a pharmaceutical or cosmetic composition for promoting skin wound healing or skin regeneration.

The skin acts as a barrier to protect the body and is the body's primary line of defense against diseases, and the epidermis, which is present on the outermost wall of the skin, provides a barrier against microbial invasion.

Skin damage, depending on the severity, can occur as a result of burns, cuts, abrasions, and ulcers. If the skin damage is not recovered, the skin barrier function is lost and the skin tissue is exposed to microbial infection, which can further damage the skin. Although recent research on wound healing has been further subdivided into cell biology and molecular biology, and clinical applications for promoting wound healing are becoming more extensive, there are many questions about the cellular biology and molecular biological mechanisms for skin wound healing. . Wound healing is the tissue's response to injury and is a process of tissue repair that consists of a combination of several cell types, including keratinocytes, fibroblasts, endothelial cells, macrophages, and platelets. This process includes cell proliferation and migration rates, collagen deposition and remodeling, wound contraction, and angiogenesis.

Skin wound healing is a complex process in which the skin or other organs or tissues repair themselves after the wound. In normal skin, the epidermis (the outermost layer) and the dermis (the inner or deep layer) form a protective barrier against the external environment. Once the protective barrier is broken, the normal physiological process of wound healing begins immediately. However, the injured person's health, age, diseases such as diabetes, the presence of foreign bodies or necrotic tissues can affect the speed of skin wound healing. If the speed of healing of skin wounds is slow, there is a risk of secondary infection through the wound site. In addition, in some wounds, incomplete control of wound dissipation leads to excessive scar formation, which leaves scar tissue functionally and cosmetically inferior. Therefore, if a wound occurs, it is important to heal the wound quickly and without side effects.

Currently, commercialized products used to heal skin wounds include Fucidine of Dongwha Pharm, Inc. and Madekasol of Dongkuk Pharm. However, studies have been reported that Staphylococcus aureus , which is resistant to fucidic acid, the main component of fucidin, is increasing (Arch Dis Child 2004;89:74-44). In addition, side effects such as allergic reactions and rashes have been reported steadily when using fucidin and madekasol, so the need for research on new pharmaceuticals is emerging.

As a patent document on wound healing, Korean Patent Registration No. 10-1899818 discloses a wound healing composition containing trakeloside as an active ingredient, and Korean Patent No. 10-1838156 discloses a round leaf Ipomoea Quamoclit extract. A wound healing composition containing as an active ingredient has been reported.

On the other hand, ginseng is a well-known herbal medicine in Asia, and various pharmacological and therapeutic effects have been reported on the central nervous system, endocrine system, immune system, cardiovascular system, and aging through a number of experimental and clinical studies (Kitts et al., 2000; Nam, 2002; Liu and Xiao, 1992). The main active ingredient of ginseng is triterpene glycoside, called ginsenoside (G). Ginsenosides have been reported to have various effects, including anti-fatigue and anti-stress effects, immunomodulation, antioxidant, anti-inflammatory, neuronal protection, and anti-tumor effects (Attele et al., 1999; Park et al., 2003; Shibata, 2001; Leung and Wong, 2010).

In particular, among ginsenosides, ginsenoside Rg3 (G-Rg3) is the most studied component after G-Rg1 and G-Rb1. It suppresses tumors (Shinkai et al., 1996), inhibits angiogenesis and anti-tumor metastasis (Mochizuki). et al., 1995; Liu et al., 2009), anticancer (Yun et al., 2001; Li et al., 2005), liver protection (Lee et al., 2005), neuroprotection (Tian et al., 2005), immunomodulation (Wang and Meng, 1999), vasodilation (Kim et al., 2003), antidiabetic and insulin secretion promotion (Kang et al., 2010; Park et al, 2008), antioxidant activity (Lee et al. al., 2008a) and various pharmacological effects have been reported (Cheng et al., 2008).

G-Rg3 has a molecular formula of C ₄₂ H ₇₂ O ₁₃ and a molecular weight of 784, and there is a pair of stereoisomers, according to the position of the hydroxyl group attached to the C-20 position of the non-sugar of the formula 1 below. It is classified into an epimer of 20(R)-Rg3 and 20(S)-Rg3 of Formula 2 (Shoji, 1990; Lee BH and Nah SY, 2007). This 20(S)/20(R)-Rg3 is produced by selective attack of hydroxyl groups after removal of sugar residues attached to C-20 (Kang et al., 2006).

The isomers have the same molecular weight, but exhibit different physical and biological properties due to structural properties from each other (Lee and Nah, 2007; Ni et al., 2005; Bae et al., 2002). The 20(S)-Rg3 isomer is more water-soluble and has a higher bioavailability than the 20(R)-Rg3 isomer (Chang et al., 2009), and the 20(S)-Rg3 isomer has a melting point of 248-250. °C (dec.), and 20(R)-Rg3 differs from 299-302 °C (dec.) (Bae et al., 2002). These have different pharmacological activity, so their utility is different, and it has been reported that the pharmacological activity of 20(S)-Rg3, which is generally more water-soluble, is strong. 20(S)-Rg3 has stronger antioxidant activity than 20(R)-Rg3 (Kang et al., 2007; Lee et al., 2008c), and is a more efficient regulator of potential-dependent Ca ²⁺ , K ⁺ or Na ⁺ pathways. (Kang et al., 2005), and acted as an antagonist of the stronger serotonin type 3 receptor F (5-HT(3A) receptor) (Lee et al., 2007). In addition, 20(S)-Rg3 showed stronger cytotoxicity than 20(R)-Rg3, and was reported to have anti-cancer effects (Popovich and Kitts, 2002; Qi et al., 2010b). In promoting immune response, 20(R)-Rg3 was stronger than that of 20(S)-Rg3 (Wei et al., 2012).

These findings suggest that the three-dimensional structure of the hydroxyl group of C-20 influences the biological and pharmacological effects of ginsenosides (Yue et al., 2006; Qi et al., 2010b).

Therefore, the inventors of the present invention were conducting a more in-depth study on the activity of ginsenoside Rg3, whereas the S-form of ginsenoside Rg3 did not show any effect on skin wound healing or skin regeneration, whereas the R-form of ginsenoside Rg3 By discovering the fact that it exhibits an effect of (R)-ginsenoside Rg3, a new use of skin wound healing and skin regeneration promotion was discovered, and the present invention was completed.

Accordingly, a technical problem to be solved in the present invention is to provide a material having a skin wound healing and skin regeneration effect, and a skin wound healing and skin regeneration method using the material.

In order to solve the above technical problem, the present invention provides a pharmaceutical composition for healing skin wounds or promoting skin regeneration comprising (R)-ginsenoside Rg3 of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. to provide:

Formula 1

In addition, the present invention provides a cosmetic composition for healing skin wounds or promoting skin regeneration comprising (R)-ginsenoside Rg3 of Formula 1 or a cosmetically acceptable salt thereof as an active ingredient:

The (R)-ginsenoside Rg3 (ginsenoside Rg3(20R) or 20(R)-ginsenoside Rg3) is extracted and separated from ginseng or red ginseng using a method known in the art for extraction and separation. It can be obtained or chemically synthesized and used.

(R)-ginsenoside Rg3 represented by Chemical Formula 1 of the present invention can be used in the form of a pharmaceutically/cosmetically acceptable salt, and as a salt, a pharmaceutically/cosmetically acceptable free acid Acid addition salts formed by are useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous or phosphorous acid, and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , Sebacate, fumarate, maleate, butin-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, me Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, Maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention is prepared in a conventional manner, for example, (R)-ginsenoside Rg3 is dissolved in an excess acid aqueous solution, and the salt is dissolved in a water-miscible organic solvent such as methanol, ethanol, acetone or It can be prepared by precipitation using acetonitrile. Further, the mixture may be dried by evaporating a solvent or an excess of acid, or may be prepared by suction filtration of the precipitated salt.

In addition, bases can be used to make pharmaceutically/chemically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

According to one embodiment of the present invention, the (R)-ginsenoside Rg3 increases cell mobility and invasive ability of colon cancer cell lines (Caco2) and keratinocytes (HaCaT), does not exhibit cytotoxicity, and It is very effective for skin wound healing and skin regeneration by forming new tissues in the area and reducing scars. On the other hand, (S)-ginsenoside Rg3, an isomer of (R)-ginsenoside Rg3, has little or no effect on cell mobility and invasiveness of colon cancer cell lines (Caco2) and keratinocytes (HaCaT). have.

In the present invention, "wound" refers to damage to humans resulting from a disorder or disease in which tissue is cut, torn, broken, burned, or traumatized, or causing such damage.

In the present invention, "healing" is the acceleration or acceleration of the time from the occurrence of the wound to the time the wound is closed (complete wound contraction).

In the present invention, “skin regeneration” is a process of tissue recovery from damage, chemotaxis, cell differentiation and replication, synthesis of matrix proteins, blood vessel neovascularization and reconstruction of wounds. It is a complex biological process involving the back. One of the representative substances that are generated early in the skin regeneration process and regulate the subsequent processes is growth factor, which has a strong influence over the entire skin regeneration process while controlling the growth, differentiation, and metabolism of cells.

The “cell infiltration” generally refers to a phenomenon in which cells migrating in a tissue gather at one site. According to a specific embodiment of the present invention, cell infiltration by treatment with (R)-ginsenoside Rg3 on the wound site This occurs and the skin wound can be healed by proliferation of cells along with the formation of neovascularization.

Types of wounds to which the composition for skin wound healing or skin regeneration of the present invention can be applied are, but are not limited to, cuts and lacerations, surgical incisions or wounds, punctures, abrasions (graze), scratches, pressure wounds , Abrasion, friction wounds (e.g. diaper rash, friction blisters), pressure sore ulcers (e.g. bedsores or bulges), thermal wounds (directly or through conduction, convection or radiation) and sources of heat and electricity From burns), chemical wounds (such as acid or alkali burns), or pathogenic infections (such as viral, bacterial or fungal infections), including open or intact boils, skin rashes, blemishes, and acne, ulcers, chronic wounds (diabetes) -Related injuries, such as lower leg and foot ulcers, venous leg ulcers and bedsores).

In the present invention, the "active ingredient" refers to a component that exhibits a desired activity by itself or can exhibit activity together with a carrier that is not active by itself.

(R)-ginsenoside Rg3 of the present invention stimulates neurological activation of healing, and subsequent inflammatory activity related to cell growth and proliferation. Accordingly, the composition of the present invention can be used to stimulate epithelial cell proliferation and bottom layer keratinocytes for wound healing, especially skin wound healing purposes. These wounds can be shallow or deep and include damage to the dermis and epidermis of the skin.

The (R)-ginsenoside Rg3 of the present invention heals wounds at a normal rate or promotes the healing of anastomosis and other wounds caused by surgery in humans with poor healing.

(R)-ginsenoside Rg3 of the present invention is a condition that induces normal individuals and abnormal wound healing, such as uremia, malnutrition, vitamin deficiency, obesity, infection, immunosuppression and systemic treatment using steroids, radiation therapy, and Surgical wounds, resection wounds, severe wounds associated with damage to the dermis and epidermis, soft tissue damage such as muscle rupture, ocular tissue wounds, tooth tissue wounds, oral wounds, in individuals with complications related to anti-neoplastic drugs and antimetabolites, For wound healing of wounds, including wounds and ulcers of the gastrointestinal mucosa, diabetic ulcers, skin ulcers, elbow ulcers, arterial ulcers, venous congestive ulcers, and heat-induced burns, exposure to extremely high or cryogenic temperatures, or exposure to chemicals. It is clinically useful for stimulation. The composition may also promote healing of wounds associated with ischemic and ischemic injuries, such as chronic venous inferior ulcers, caused by impairment and/or insufficiency of venous circulatory system reflux; Promoting skin repair following skin loss; Increase in epidermal tension and epidermal thickness; And for increasing adhesion of skin grafts to the wound layer and promoting re-epithelialization from the wound layer.

(R)-ginsenoside Rg3 or a pharmaceutically/cosmetically acceptable salt thereof contained in the composition for promoting skin wound healing or skin regeneration of the present invention is 0.001 to 10% by weight, preferably based on the total weight of the composition. It may be included in an amount of 0.001 to 5% by weight. If the content is less than 0.001% by weight, the effect of promoting skin wound healing and skin regeneration, which is the target effect of the present invention, cannot be obtained, and when it exceeds 10% by weight, the increase in the effect according to the increase in the content is not proportional. It may be inefficient and there is a problem in that the stability of the formulation is not secured.

According to one embodiment of the present invention, the present invention provides a pharmaceutical composition comprising (R)-ginsenoside Rg3 or a pharmaceutically acceptable salt thereof as an active ingredient.

When the composition of the present invention is used as a pharmaceutical composition, it may further contain a pharmaceutically acceptable carrier in addition to (R)-ginsenoside Rg3 or a pharmaceutically acceptable salt thereof as an active ingredient. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and carbohydrate compounds (eg, lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, etc.) , Gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, gum arabic, vegetable oil (e.g. corn oil, cotton seed oil , Soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, etc., but are not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (latest edition), Mach Publishing Company, Easton, PA.

A preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 to 100 mg/kg on an adult basis. In addition, in the case of an external preparation, it is recommended to apply once to 5 times a day in an amount of 1.0 to 3.0 ml based on an adult and continue for more than 1 month. The above dosage does not limit the scope of the present invention.

Formulations of the pharmaceutical composition of the present invention include oral dosage forms such as powders, granules, tablets, capsules, emulsions, syrups, aerosols, external preparations such as ointments and creams, and sterile injectable solutions according to conventional methods. It can be formulated and used in any form suitable for the formulation.

When the pharmaceutical composition for skin wound healing or skin regeneration of the present invention is provided as an external preparation for skin, it may be a formulation such as an ointment, patch, gel, cream or spray, but is not limited thereto.

In the case of parenteral administration, in one embodiment, (R)-ginsenoside Rg3 is used as a pharmaceutically acceptable carrier, i.e., in unit dose injectable form (solution, suspension, or emulsion) at the desired degree of purity. It is non-toxic to the subject at the dosage and concentration and is generally formulated by mixing with ones compatible with the other ingredients of the formulation.

In the case of topical administration, formulations such as ointments, creams, and gels can be used at the dosages described above for the composition. Suitable formulations known in the art can be found from Remington's Pharmaceutical Sciences (latest edition), Mach Publishing Company, Easton, PA.

Ointments are generally (1) oily bases, i.e. those consisting of fixed oils or hydrocarbons, such as white petrolatum or mineral oils, or (2) absorbent bases, i.e. any anhydrous substances or substances capable of absorbing water, such as anhydrous. It is prepared using lanolin. Typically, after preparation of the substrate, whether oily or absorbent, the active ingredient ((R)-ginsenoside Rg3) is added in an amount that provides the desired concentration.

The cream is an oil/water emulsion. They typically consist of an oil phase (internal phase) comprising fixed oils, hydrocarbons and the like, such as wax, petrolatum, mineral oil, and the like, and an aqueous phase (continuous phase) comprising water and optional water-soluble substances such as additional salts. The two phases are emulsifiers such as surface active agents such as sodium lauryl sulfate; It is stabilized by the use of hydrophilic colloids such as acacia colloidal clay, veggies and the like. In the formation of the emulsion, the active ingredient ((R)-ginsenoside Rg3) is usually added in an amount to achieve the desired concentration.

Gels include oily substrates, water, or emulsion-suspension substrates, such as substrates selected from those described above. To the substrate is added a gelling agent that forms a matrix in the substrate and increases its viscosity. Examples of the gelling agent include hydroxypropyl cellulose, acrylic acid polymer, and the like. Typically, the active ingredient ((R)-ginsenoside Rg3) is added to the formulation at the desired concentration at the time prior to addition of the gelling agent.

The compositions of the present invention also contemplate wound care dressings or bandages comprising a therapeutically effective amount of (R)-ginsenoside Rg3 or a pharmaceutically acceptable salt thereof. Wound care bandages or dressings may include an outer fabric support, preferably an elastomeric fabric support; And an inner pad, wherein the inner pad preferably comprises an outer membrane surface made from a film-forming material, and a therapeutically effective amount of R)-ginsenoside Rg3, or a pharmaceutically acceptable salt thereof, is added to the composition. It is introduced into the matrix at the dosages described above. The pad may be integral with the outer fabric support or may be separate.

According to one embodiment of the present invention, the present invention provides a cosmetic composition comprising (R)-ginsenoside Rg3 or a cosmetically acceptable salt thereof as an active ingredient.

In addition to (R)-ginsenoside Rg3 or a cosmetically acceptable salt thereof as an active ingredient, the cosmetic composition according to one embodiment of the present invention includes ingredients commonly added to the cosmetic composition, such as antioxidants, stabilizers, solubilizers. , Conventional auxiliaries such as vitamins, pigments and flavors, and carriers may be further added.

The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , Oil, powder foundation, emulsion foundation, wax foundation, spray, etc. may be formulated, but is not limited thereto. In more detail, it may be prepared in the form of a nourishing cream, astringent lotion, a soft lotion, lotion, essence, a nourishing gel or a massage cream.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide are used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, toss, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether.

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

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspending agents such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, crystallites Sex cellulose, aluminum metahydroxide, bentonite, agar or tracanth gum, and the like can be used.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used.

The pharmaceutical or cosmetic composition containing (R)-ginsenoside Rg3 or a pharmaceutically acceptable salt thereof of the present invention as an active ingredient is administered to the wound site of the individual, subcutaneous administration or topical administration by application, etc. There is an advantage in that it can achieve the purpose of skin wound healing and skin regeneration by forming new tissues and reducing scars.

In one specific embodiment, the present invention administers a composition comprising (R)-ginsenoside Rg3 or a pharmaceutically/cosmetically acceptable salt thereof as an active ingredient to an individual to heal skin wounds and regenerate the skin. It provides a method of making and its use.

In the present invention, the term "subject" refers to monkeys, cattle, horses, pigs, sheep, dogs, cats, rats, mice, including humans with conditions or diseases in which symptoms can be improved by administering the composition of the present invention, It means a mammal such as a chimpanzee.

The administration is oral or parenteral administration, preferably parenteral administration, more preferably topical administration by application, and the dosage thereof is the formulation method, the mode of administration, the patient's age, weight, sex, pathological condition, etc. It can be easily changed and used by those of ordinary skill in the art depending on the factors of.

On the other hand, (R)-ginsenoside Rg3 of the present invention or its pharmacologically/cosmetically acceptable salt has no skin irritation and cytotoxicity, and is excellent in human stability, so there are almost no side effects to the human body, so it is safe even when used for a long time. Because it can be used, it can be safely used in medicines or cosmetics.

(R)-ginsenoside Rg3 according to the present invention increases the cell mobility and invasive ability of Caco2 cells (Colon Epithelial Colorectal adenocarcin noma cells, colon adenocarcinoma cells) and HaCaT (Human keratinocyte, keratinocyte line), and increases cytotoxicity. It does not appear, and it is very effective for skin wound healing and skin regeneration by forming new tissue at the wound site and reducing scarring. Accordingly, (R)-ginsenoside Rg3 or a pharmaceutically acceptable salt thereof may be usefully used as a pharmaceutical or cosmetic composition for healing skin wounds and promoting skin regeneration.

1 is a result of measuring the cytotoxicity of (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 to Caco2 cells.

2 is a result of measuring the mobility of HaCaT cells treated with (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3.

3 is a graph showing the results of observing the invasion of HaCaT cells treated with (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 under a microscope and quantifying them.

4 is a graph showing the results of observing the invasion of Caco2 cells treated with (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 under a microscope and quantifying them.

5 is a photograph of visual observation of a reduction in incision area as an incision healing effect through animal experiments of (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3, and a graph quantifying it.

Hereinafter, examples, etc. will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Example 1> Confirmation of cytotoxicity of (R)-ginsenoside Rg3

Cytotoxicity of (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 in Caco2 cells (Colon Epithelial Colorectal adenocarcin noma cells, colon adenocarcinoma cells) and HaCaT cells was confirmed through MTT assay.

MTT (3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a pale yellow substrate that produces dark blue formazan in living cells and does not react in dead cells. The amount of cup produced is used to measure the number of living cells.

Specifically, Caco2 cells and HaCaT cells were prepared in DMEM (Dulbecco's Modification of Eagle's Medium) medium containing 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin under conditions of 37° C. and 5% CO _2. Cultured in.

MTT (3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent was dissolved in PBS (phosphate buffered saline, pH 7.4) at a concentration of 1 mg/ml and then 0.2㎛ syringe filter ) Was used after filtration, and (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 were each diluted with PBS and used. The cultured cells were dispensed in a 24 well-plate at a concentration of 2×10 ⁴ cells/well, cultured for 24 hours in a cell incubator at 37° C. in the presence of 5% CO ₂ , and cell adhesion was confirmed. Each of the wells was treated with (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 at different concentrations (12.5, 25, 50 and 100 μM), and then cultured again for 48 hours. After the treatment of (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3, MTT solution (1 mg/ml in PBS) was added in an amount equal to 1/10 of the total volume of the medium, and 37°C In the CO ₂ incubator was reacted for 3 hours. After removing all of the culture medium, 100 μl of DMSO (dimethyl sulfoxide) was added to each well, and formazan formed in the cells was dissolved, and the absorbance was measured with an ELISA meter at 540 nm. The degree of proliferation activity of cells was calculated according to Equation 1 below based on the absorbance intensity of the control group using pure water and expressed as a percentage. As a control, cells treated with DMSO were used instead of the sample. The results are shown in Table 1 and Fig. 1 below.

Table 1 shows the IC ₅₀ values of (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 for Caco2 and HaCaT cells, and FIG. 1 shows (R)-ginsenoside Rg3 and and (S) -This is the result of measuring the relative cell viability of Ginsenoside Rg3 against Caco2 and HaCaT cells. As shown here, (S)-ginsenoside Rg3 has IC ₅₀ values for Caco2 and HaCaT cells, respectively, 59.1uM and 83.1uM, whereas (R)-ginsenoside Rg3 has IC ₅₀ values for Caco2 and HaCaT cells. These were 262.5 uM and 172.4 uM, respectively, confirming that (R)-ginsenoside Rg3 had a higher cell viability at the same concentration and no cytotoxicity compared to (S)-ginsenoside Rg3.

Compounds	IC 50 (uM)
	Caco2	HaCaT
20(R)-Rg3	262.5	172.4
20(S)-Rg3	59.1	83.1

<Example 2> Measurement of cell mobility of (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3

Cellular mobility of HaCaT cells by (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 was measured.

Specifically, the degree of cell migration was measured using a transwell chamber (Corning Costerm Corning, NY, USA) with a polycarbonate having a pore size of 8 μm. First, HaCaT cells were dispensed to the upper part of the chamber so that it became 2.4 × 10 ⁶ cells/well. A culture medium containing 10% FBS was dispensed into the lower layer of the chamber. Then, the HaCaT cells were each treated with (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 at concentrations that do not show cytotoxicity, and then cultured for 24 hours. After the cultured cells were fixed and stained using a Diff Quick kit (sysmex, Kobe, Japan), the number of infiltrated cells was counted using an optical microscope. As a control, HaCaT treated with DMSO was used. The experiment was repeated three times, and the values were expressed as mean±standard deviation. In addition, significance was verified by one-way analysis of variance (One-way ANOVA, Tukey's HSD) at p<0.05 significance level, and SPSS 17.0 (Chicago, USA) statistical program was used. The results are shown in FIG. 2.

FIG. 2 is a graph illustrating microscopic observation and quantification of cell mobility of keratinocytes (HaCaT) treated with (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3, respectively. As shown here, in HaCaT cells treated with (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3, cell mobility increased compared to the control, but only (R)-ginsenoside Rg3 increased in concentration. As a result, it was found that (R)-ginsenoside Rg3 specifically increased cell migration by increasing cell mobility.

<Example 3> Measurement of cell invasive ability of (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3

Cell invasive ability of Caco2 and HaCaT by (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 was measured.

Specifically, the degree of invasion of cells was measured using a transwell chamber (Corning Costerm Corning, NY, USA) with a filter coated with polycarbonate and 1% gelatin having a pore size of 8 μm. First, Caco2 and HaCaT cells were dispensed into the upper part of the chamber so that it became 2.4 × 10 ⁶ cells/well. In the lower layer of the chamber, a culture medium containing 0.2% BSA and 1 μg/mL fibronectin for Caco2 cells, and a culture medium containing 10% FBS for HaCaT cells was dispensed. Then, the Caco2 and HaCaT cells were each treated with (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 at concentrations that do not show cytotoxicity, and then cultured for 24 hours. After the cultured cells were fixed and stained using a Diff Quick kit (sysmex, Kobe, Japan), the number of infiltrated cells was counted using an optical microscope. As a control, DMSO-treated Caco2 and HaCaT were used. The experiment was repeated three times, and the values were expressed as mean±standard deviation. In addition, significance was verified by one-way analysis of variance (One-way ANOVA, Tukey's HSD) at p<0.05 significance level, and SPSS 17.0 (Chicago, USA) statistical program was used. The results are shown in FIGS. 3 and 4.

3 and 4 show microscopic observation and quantification of the invasion of colon adenocarcinoma cell lines (Caco2) and keratinocytes (HaCaT) treated with (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 at each concentration. It is a graph. As shown here, the number of infiltrated cells in Caco2 and HaCaT cells treated with (R)-ginsenoside Rg3 increased compared to the control.In particular, (R)-ginsenoside Rg3 infiltrated with increasing concentration. It was confirmed that the number of cells was increased proportionally. On the other hand, in Caco2 cells treated with (S)-ginsenoside Rg3, the number of infiltrated cells decreased compared to the control, so that (S)-ginsenoside Rg3 did not affect or slightly decreased in invasion of cells. there was.

<Example 4> (R)-Ginsenoside Rg3 and (S)-Ginsenoside Rg3 In a wound-induced animal model, visually observed skin wound healing effect

The skin wound healing ability of (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 was measured by the following method.

After anesthetizing a 6-week-old Balb/C mouse, the hair was removed and four wounds were made on the skin using a 5 mm punch. Two wounds were randomly assigned per head, using (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 as a treatment group and DMSO as a control group.

Dilute 10 mM of (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 each to 1/100 in a viscous glycerol/PBS solution made by mixing 100% glycerol and PBS solution in a ratio of 1:1. The final concentration was prepared to be 0.1 mM. This was applied once daily for 15 days in an amount of about 0.02 mg/kg per total weight of mice to the incision site of the animals in the writing treatment group. Only DMSO was treated as a control group.

In order to observe the progress of incision healing in the mice, the incision site was photographed at a certain distance for each group using a digital camera every day after incision induction, and the changes in the wound were visually observed.

5 is a photograph of visual observation of reduction in incision area as an incision healing effect of (R)-ginsenoside Rg3 and (S)-ginsenoside Rg3 through animal experiments, and a graph quantifying the degree of reduction in skin wounds. . As shown here, the wound area of the (R)-ginsenoside Rg3 treatment group showed a tendency to be significantly reduced during the treatment period compared to the (S)-ginsenoside Rg3 treatment group. Specifically, from day 1 of the wound, the wound size began to decrease compared to the control and (S)-ginsenoside Rg3, and on the 7th day, the wound size was significantly smaller than that of the control group and (S)-ginsenoside Rg3. I could confirm that it was healing.

Claims (4)

1. A pharmaceutical composition for healing skin wounds or promoting skin regeneration comprising (R)-ginsenoside Rg3 of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

   Formula 1

   
2. The method of claim 1,

   The pharmaceutical composition for promoting skin wound healing or skin regeneration, characterized in that the composition is an ointment, patch, gel, cream or spray.
3. A cosmetic composition for healing skin wounds or promoting skin regeneration comprising (R)-ginsenoside Rg3 of Formula 1 or a cosmetically acceptable salt thereof as an active ingredient.

   Formula 1

   
4. A method for curing skin wounds of an individual and promoting skin regeneration, comprising administering to the individual a composition comprising the (R)-ginsenoside Rg3 of Formula 1 or an acceptable salt thereof as an active ingredient.

   Formula 1

   

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