Antibacterial and antioxidant composition containing grape callus culture solution as an active ingredient

Abstract

This invention provides antioxidant and antibacterial compositions containing active ingredients derived from plants. [Solution] The present invention relates to an antibacterial and antioxidant composition containing grape callus culture solution containing epsilon(ε)-Viniferin, delta(δ)-Viniferin, and resveratrol as active ingredients. Specifically, the grape callus, its culture solution, and its extract of the present invention are non-cytotoxic, exhibit significant antioxidant and antibacterial effects against acne bacteria, and have particularly remarkable antioxidant and antibacterial effects compared to pure viniferin. Therefore, the grape callus, its culture solution, and its extract can be usefully used as active ingredients in antioxidant and antibacterial compositions.

Description

Technical Field

[0001] The present invention relates to a composition for antibacterial and antioxidant use containing a grape callus culture solution containing epsilon(ε)-Viniferin, delta(δ)-Viniferin, resveratrol, etc. as an active ingredient.

Background Art

[0002] In the process of the worldwide conversion from an industrial-centered society to a service-centered society, human health has become the biggest topic. The rapid expansion of the industrial society based on petroleum substances has brought convenience to humans, but the sequelae of rapid development have not only polluted the living environment but also caused a situation that harms human health itself. In contrast, there is a growing interest and demand for environmentally friendly substances centered on products close to daily life.

[0003] Oxygen is an essential element for maintaining life and is used to produce energy in the body through the process of respiration. However, oxygen does not only give us energy. Oxygen produces harmful oxygen called reactive oxygen species in the process of combining with food in the body, and this reactive oxygen species acts as a cause of diseases in our bodies. Recently, the medical community has named reactive oxygen species as the main culprit of human aging. Therefore, the development of natural product-derived antioxidants that can protect the human body from the toxicity of reactive oxygen species is being steadily carried out.

[0004] Acne is a skin condition that occurs in most people of all ages, forming pustules or cysts. Acne develops due to a combination of factors, including excessive sebum secretion, hormonal imbalances, abnormal keratinization within hair follicles, and the proliferation and inflammatory response of Propionibacterium acnes. Propionibacterium acnes, the bacterium that causes acne, is an anaerobic skin flora that proliferates within hair follicles. Lipase hydrolyzes triglycerides in sebum, producing free fatty acids. This stimulates the hair follicles, generating chemotactic factors that trigger an inflammatory response. While antibiotics are the most common treatment for acne caused by Propionibacterium acnes infection, their effectiveness can decrease due to antibiotic resistance. Therefore, research into herbal substances that do not cause side effects or resistance in acne treatment is actively underway.

[0005] On the other hand, many studies have attempted to produce a variety of useful substances (e.g., secondary metabolites) derived from plants using biotechnology. Among these, the most effective production method is plant cell culture. This method can effectively produce secondary metabolites through the use of culture media, culture environment, and various elicitor treatments. Furthermore, it is commercially important because it allows for the continuous and stable mass production of useful substances derived from plants without being subject to the constraints of cultivation or the natural environment (highly toxic pesticides, heavy metal contamination, seasonal influence, climatic conditions, etc.).

[0006] Furthermore, as related prior art, Registered Korean Patent No. 10-1253374 (Cosmetic Composition for Improving Acne-Prone Skin) utilizes kimchi lactic acid bacteria fermentation extract and soybean fermentation extract as a cosmetic composition for improving acne-prone skin, and Registered Korean Patent No. 10-0879032 (Antibacterial, Antioxidant, and Anti-inflammatory Inhibitor with Natural Antibacterial and Anti-inflammatory Effects and Cosmetic Composition Containing the Same) provides an antibacterial cosmetic composition using a mixed extract of the herbal medicines licorice, rhizome, rhizome, astragalus, coptis japonica, sophora, rhizome, and gromwell. However, the antibacterial and antioxidant effects of grape callus against acne bacteria are not known.

[0007] Therefore, the inventors have worked to develop a method for producing large quantities of useful plant-derived substances and to develop functional cosmetics, health functional foods, and pharmaceutical materials using these substances. As a result, the grape callus culture solution of the present invention exhibits significant antioxidant and antibacterial effects, and in particular, shows remarkable antioxidant and antibacterial effects compared to pure viniferin. Thus, the present invention was completed by demonstrating that the grape callus culture solution can be usefully used as a natural product-derived antioxidant and antibacterial pharmaceutical composition, cosmetic composition, and health food. [Overview of the project] [Problems that the invention aims to solve]

[0008] The object of the present invention is to provide an antioxidant and antibacterial composition containing one or more selected from the group consisting of grape callus, crushed callus, callus culture solution, and callus extract as an active ingredient.

[0009] Another object of the present invention is to provide a method for preventing, improving, or treating oxidative stress, comprising the step of administering to an individual a composition containing, as an active ingredient, one or more selected from the group consisting of grape callus, callus crushed material, callus culture solution, and callus extract in a pharmaceutically effective amount.

[0010] Another object of the present invention is to provide a method for preventing, improving, or treating a bacterial infection, comprising the step of administering to an individual a composition containing, as an active ingredient, one or more selected from the group consisting of grape callus, callus crushed material, callus culture solution, and callus extract in a pharmaceutically effective amount.

[0011] Another object of the present invention is to provide a method for preventing, improving, or treating acne, comprising the step of administering to an individual a composition containing, as an active ingredient, one or more selected from the group consisting of grape callus, callus crushed material, callus culture solution, and callus extract in a pharmaceutically effective amount.

[0012] Another object of the present invention is to provide uses for compositions containing one or more selected from the group consisting of grape callus, callus crushed material, callus culture solution, and callus extract as active ingredients, for use as compositions for the prevention, improvement, or treatment of oxidative stress.

[0013] Another object of the present invention is to provide uses for compositions containing one or more selected from the group consisting of grape callus, callus crushed material, callus culture solution, and callus extract as active ingredients, for use as compositions for the prevention, improvement, or treatment of bacterial infections.

[0014] Another object of the present invention is to provide uses for compositions containing one or more selected from the group consisting of grape callus, callus crushed material, callus culture medium, and callus extract as active ingredients for use as pharmaceutical compositions for the prevention or treatment of acne.

[0015] Furthermore, another object of the present invention is to provide uses for compositions containing one or more selected from the group consisting of grape callus, crushed callus, callus culture solution, and callus extract as active ingredients, for use as cosmetic compositions for preventing and improving acne. [Means for solving the problem]

[0016] To achieve the objectives of the present invention, the present invention provides an antioxidant and antibacterial pharmaceutical composition containing one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract as an active ingredient.

[0017] Furthermore, the present invention provides a pharmaceutical composition for the prevention and treatment of acne, containing one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract as an active ingredient.

[0018] The present invention also provides an antioxidant and antibacterial cosmetic composition containing, as an active ingredient, any one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract.

[0019] The present invention also provides an acne prevention and improvement cosmetic composition containing, as an active ingredient, any one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract.

[0020] The present invention also provides a health food for antioxidant and antibacterial effects containing, as an active ingredient, any one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract.

[0021] The present invention also provides a method for preventing or improving oxidative stress, which includes administering to an individual a composition containing, as an active ingredient, any one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract in a pharmaceutically effective amount.

[0022] The present invention also provides a method for treating oxidative stress, which includes administering to an individual a composition containing, as an active ingredient, any one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract in a pharmaceutically effective amount.

[0023] Another object of the present invention is to provide a method for preventing or improving bacterial infection, which includes administering to an individual a composition containing, as an active ingredient, any one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract in a pharmaceutically effective amount.

[0024] Another object of the present invention is to provide a method for treating bacterial infection, which includes administering to an individual a composition containing, as an active ingredient, any one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract in a pharmaceutically effective amount.

[0025] The present invention also provides a method for preventing or improving acne, which includes administering to an individual a composition containing as an active ingredient any one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract.

[0026] The present invention also provides a method for treating acne, which includes administering to an individual a composition containing as an active ingredient any one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract.

[0027] The present invention also provides the use of a composition containing as an active ingredient any one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract for use as a composition for preventing, improving or treating oxidative stress.

[0028] The present invention also provides the use of a composition containing as an active ingredient any one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract for use as a composition for preventing, improving or treating bacterial infection.

[0029] The present invention also provides the use of a composition containing as an active ingredient any one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract for use as a pharmaceutical composition for preventing or treating acne.

[0030] In addition, the present invention provides the use of a composition containing as an active ingredient any one or more selected from the group consisting of grape callus, callus crushed product, callus culture solution, and callus extract for use as a cosmetic composition for preventing and improving acne.

Advantages of the Invention

[0031] The grape callus, its culture medium, and its extract of the present invention are non-cytotoxic, exhibit significant antioxidant and antibacterial effects against acne bacteria, and have particularly remarkable antioxidant and antibacterial effects compared to pure vinylin. Therefore, the grape callus, its culture medium, and its extract can be usefully used as active ingredients in antioxidant and antibacterial compositions. [Brief explanation of the drawing]

[0032] [Figure 1] Figure 1 shows the cytotoxicity of vinylin and grape callus culture medium (sample name A) in MNT-1 cells. To compare with vinylin, grape callus culture medium (sample name A) was diluted according to the epsilon vinylin content standard and treated in wells. [Figure 2] Figure 2 shows the cytotoxicity of vinylin and grape callus culture medium (sample name A) in Raw264.7 cells. To compare with vinylin, grape callus culture medium (sample name A) was diluted according to the epsilon vinylin content standard and treated in wells. [Figure 3] Figure 3 shows the results of DPPH analysis to confirm the antioxidant effects of grape callus culture solution (sample name A) and grape callus culture solution extract (sample name a). [Figure 4] Figure 4 shows the minimum concentration at which grape callus culture solutions (sample names A and C) inhibited the growth of three types of P. acnes after 72 hours of treatment. [Figure 5] Figure 5 shows the minimum concentration required to suppress the growth of three types of *P. acne* after 72 hours of treatment with grape callus culture solution (sample names A and C). [Figure 6] Figure 6 shows the minimum concentration required to suppress the growth of three types of grape callus culture solution (sample names A and C) after 48 hours of treatment. [Figure 7] Figure 7 shows the antibacterial effect of a grape callus culture extract (sample name a) confirmed using a disc diffusion test. Best mode for carrying out the invention

[0033] The present invention will be described in detail below.

[0034] The present invention provides an antioxidant and antibacterial composition containing one or more selected from the group consisting of grape callus, crushed callus, callus culture solution, and callus extract as an active ingredient.

[0035] In this invention, the callus is an amorphous tissue or cell mass that has lost the ability to undergo normal organ formation or tissue differentiation, is mostly cellular, and is a special tissue or cell mass that is formed when tissue cut from a plant is cultured in a tissue culture medium containing a growth regulator, when a plant is wounded, or when a growth regulator is applied to a wound on a plant.

[0036] Furthermore, the aforementioned crushed product refers to a cell lysate obtained by crushing cells using a chemical method with a common surfactant (detergent) or a physical method with a French press or the like, and the method can be manufactured in accordance with the usual methods published in the industry, and the method is not particularly limited.

[0037] Furthermore, the culture medium refers to the remaining culture medium (condition medium) after the process of culturing cells in a conventional manner published in the industry, and after removing the pellet using physical methods such as centrifugation and filter mesh filtration. The method of production can be in accordance with a conventional method published in the industry, and the method is not particularly limited.

[0038] The aforementioned extract means an extract obtained by conventional methods known to the art, either of the callus itself, callus powder, callus crushed material, or callus culture medium, which can be produced by conventional methods published to the art and are not particularly limited. Examples include cold maceration, hot water extraction, ultrasonic extraction, and reflux quenching. Commonly available solvents can be used as extraction solvents, such as water, anhydrous or aqueous lower alcohols with 1-4 carbon atoms, acetone, ethyl acetate, butyl acetate, and 1,3-butylene glycol.

[0039] On the other hand, the grape callus and its culture medium are preferably manufactured as described below, but are not limited thereto.

[0040] The step of inducing callus from grape tissue; and

[0041] The step of culturing the induced grape callus with the addition of an activity inducer and a solubilizer.

[0042] The grapes in step 1) above are one or more parts selected from the group consisting of grape flowers, leaves, stems, branches, fruits and roots, more specifically preferably grape flowers, and the grape varieties include, but are not limited to, Shimizu, Benitan, Tamnara, Koro, Kurotama, Kurohoshi, Mizutama, Tunuri, Tamukumchu, Hong Alam, Narsha, Hong Sodam, Alden, Campbell Early, Delaware, Duchess, Golden Muscat, Himrod Seedless, Schuyler, Beniizu, Kyoho, Pione, Super Hamburg, Tano Red, Muscat Hamburg and Seibel 9110.

[0043] In step 1) above, induction of Caroline is carried out by culturing plant tissue or its sections in a tissue culture medium. Various basic plant culture media can be used as the tissue culture medium, such as MS (Murashige and Skoog, 1962) medium, N6 (Chu et al., 1975), B5 (Gamborg et al., 1968), NN (Nitsch and Nitsch, 1967) medium, and White medium. Furthermore, it is desirable to use a tissue culture medium to which plant growth regulators have been added. The aforementioned plant growth regulator can utilize substances known to the industry, but preferably 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), dicamba (2-methoxy-3,6-dichlorobenzoic acid), IAA (indole-3-acetic acid), IBA (indole-3-butyric acid), MCPA (2-methyl-4-chlorophenoxyacetic acid), NAA (1-naphthylacetic acid), NOA (2-naphthyloxyacetic acid), and picloram (4-amino-2,5,6-trichloropicolinic acid). The culture medium may also contain oxins such as acid; cytokinins such as BAP (6-benzylaminopurine), 2iP (N6-(2-isopentyl)adenine), Kinetin (6-furfurylaminopurine), Thidiazuron (1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea), and Zeatin (4-hydroxy-3-methyl-trans-2-butenylaminopurine) and their derivatives. In addition, alkaloids, terpenoids, carotenoids, phenolic compounds, and natural extracts that act on cell differentiation and proliferation may be added to the tissue culture medium.IAA, NAA, 2,4-D, and kinetin can be added more preferably, and even more preferably, 0.05 to 0.2 mg / L of IAA, 0.05 to 0.2 mg / L of NAA, 1 to 2 mg / L of 2,4-D, and 0.2 to 0.3 mg / L of kinetin can be added, but are not limited thereto.

[0044] In step 2) above, the activity inducer may include methyl jasmonic acid, SA, or prazelin 22, and more preferably methyl jasmonic acid, but is not limited thereto. The solubilizer may be cyclodextrin, methyl cyclodextrin, or stevioside, and more preferably stevioside, but is not limited thereto. The methyl jasmonic acid and stevioside may be added at concentrations of 50-350 μM and 40-60 mM, respectively, and more preferably at concentrations of 100 μM and 50 mM, respectively, but is not limited thereto.

[0045] Furthermore, the aforementioned composition contains Propionibacterium acnes, Staphylococcus epidermis, Staphylococcus aureus, Staphylococcus saprophyticus, Pseudomonas aeruginosa, Enterococcus faecalis, Actinomyces baumannii, Bacillus cereus, Bacillus subtilis subsp. spizenii, Klebsiella pneumonia, and Klebsiella oxytoka. It is desirable that the composition has antibacterial activity against one or more bacteria from the group consisting of *Oxitoca* and *Micrococcus luteus*, and in particular exhibits significant bacteriostatic and bactericidal activity against *Propionibacterium acnes*, therefore, it is desirable that the composition be used as a pharmaceutical composition for the prevention and treatment of acne.

[0046] Furthermore, the present invention provides for the use of antioxidant and antibacterial compositions containing one or more selected from the group consisting of grape callus, callus crushed material, callus culture solution, and callus extract as active ingredients, for use as pharmaceutical compositions for the prevention or treatment of acne.

[0047] In specific examples of the present invention, after inducing grape callus, the inventors prepared the culture medium and its extract, and then confirmed the cytotoxicity. The results showed that epsilon-viniferin was not cytotoxic only at a concentration of 20 μg / ml, but the grape callus culture medium did not show cytotoxicity even when it contained 100 μg / ml of epsilon-viniferin (see Figures 1 and 2).

[0048] Furthermore, the inventors conducted DPPH analysis to confirm the antioxidant effect of grape callus culture solution. The results showed that epsilonviniferin and deltaviniferin at 500 μg / ml eliminated approximately 25% of radicals compared to the control group. However, in the case of the grape callus culture solution (sample name A) and grape callus culture solution extract (sample name a) of the present invention, it was confirmed that more than 60% of radicals were eliminated at 500 μg / ml (based on epsilonviniferin content) (see Figure 3).

[0049] Furthermore, the inventors confirmed the antibacterial effect of grape callus culture solution and found that the grape callus culture solution of the present invention exhibits significant bactericidal and bacteriostatic effects against acne bacteria, and in particular shows a remarkable antibacterial effect compared to pure vinylin (see Figures 4 to 7).

[0050] Therefore, the grape callus, its culture medium, and its extract of the present invention are non-cytotoxic, exhibit significant antioxidant effects, and show remarkable antibacterial effects against acne bacteria, making them useful as active ingredients in antioxidant and antibacterial compositions.

[0051] The pharmaceutical composition according to the present invention may contain a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulation and include, but are not limited to, carbohydrate compounds (e.g., lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, etc.), acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, acacia gum, vegetable oils (e.g., corn oil, cottonseed oil, soy milk, olive oil, coconut oil), polyethylene glycol, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0052] The pharmaceutical compositions of the present invention may additionally contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the components mentioned above. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0053] The pharmaceutical composition of the present invention can be administered by various routes, such as orally or parenterally, and all methods of administration are foreseeable, for example, by oral, rectal, intravenous, intramuscular, or subcutaneous injection. In this case, transdermal administration is preferred as the parenteral route, and among these, topical application is the most preferred.

[0054] The appropriate dosage of the pharmaceutical composition of the present invention can be formulated in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, administration route, excretion rate, and response sensitivity. The oral dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg / kg (body weight) for adults. In the case of topical preparations, an amount of 1.0 to 3.0 ml can be applied once to five times a day for a period of one month or more for adults. However, the above dosage is not limited to the range of the present invention.

[0055] The pharmaceutical compositions of the present invention can be manufactured in unit volume form or by being contained in multi-volume containers by formulation using pharmaceutically acceptable carriers and / or excipients by a method readily available to a person with ordinary skill in the art to which the invention pertains. The dosage form may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of an X agent, acid agent, powder, granule, purified or capsule, and may additionally contain dispersants or stabilizers.

[0056] Furthermore, the present invention provides an antioxidant and antibacterial cosmetic composition containing one or more selected from the group consisting of grape callus, crushed callus, callus culture solution, and callus extract as an active ingredient.

[0057] Furthermore, the present invention provides applications for compositions containing one or more selected from the group consisting of grape callus, crushed callus, callus culture solution, and callus extract as active ingredients, for use as cosmetic compositions for preventing and improving acne.

[0058] The aforementioned Grapevine, callus fragments, callus culture solution, and callus extract, their effects, and their role in treating acne are the same as described for the antioxidant and antibacterial compositions. Specific details will be based on the above description, and only the components specific to the cosmetic composition will be described below.

[0059] The cosmetic composition according to the present invention may additionally contain ingredients commonly used in cosmetic compositions, in addition to the active ingredient. For example, it may include common auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and antifungals, and carriers.

[0060] The cosmetic composition of the present invention can be manufactured in any formulation commonly produced in the industry, for example, in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a nourishing cream, astringent lotion, softening lotion, lotion, essence, nourishing gel, or massage cream.

[0061] When the formulation of the cosmetic composition is a paste, cream, or gel, animal oils, vegetable oils, waxes, paraffin, starch, tracand, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide can be used as the carrier component.

[0062] When the formulation of the cosmetic composition is in the form of a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier component, and in particular in the case of a spray, propellants such as chlorohydrocarbon, propane / boutane, or dimethyl ether may be additionally included.

[0063] When the formulation of the cosmetic composition is a solution or emulsion, a solvent, solubilizer, or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or sorbitan fatty acid ester is used.

[0064] When the formulation of the cosmetic composition is a suspension, the carrier component can be a liquid diluent such as water, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester, or microcrystalline cellulose, aluminum methhydroxyl, bentonite, aga, or tracant.

[0065] When the formulation of the cosmetic composition is a surfactant-containing cleanser, the carrier component can be aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinate monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamide betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

[0066] In addition, the present invention provides an antioxidant and antibacterial health food containing one or more selected from the group consisting of grape callus, crushed callus, callus culture solution, and callus extract as an active ingredient.

[0067] The grape callus, crushed callus, callus culture solution, and callus extract, and their effects, are the same as those described for the antioxidant and antibacterial compositions. Specific details will be based on the above information, and only the components specific to the health food composition will be described below.

[0068] The health food according to the present invention may contain ingredients that are normally added during food manufacturing, in addition to the active ingredient. For example, it may contain proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. Examples of carbohydrates mentioned above include monosaccharides, such as glucose and fructose; disaccharides, such as maltodextrin, sucrose, and oligosaccharides; and polysaccharides, such as common sugars like dextrin and cyclodextrin, and sugar alcohols like xylitol, sorbitol, and erythritol. Natural flavorings [thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavorings (sakarin, aspartum, etc.)] may be used as flavorings.

[0069] The amount of active ingredients mixed can be appropriately determined depending on the intended use. Generally, the amount of active ingredients in health foods can be added at a rate of 0.01 to 15 times the total weight of the food, and in health beverage compositions, it can be added at a rate of 0.02 to 5 g, preferably 0.3 to 1 g, per 100 ml. However, in the case of long-term intake for health and hygiene purposes, or for health regulation purposes, the above amounts are below the above range and there are no safety issues, so the active ingredients can be used in amounts above the above range.

[0070] When the health food of the present invention is manufactured as a health beverage composition, in addition to the active ingredients of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, etc. may be added.

[0071] Furthermore, in addition to the components mentioned above, the health food may contain various nutrients, vitamins, electrolytes, flavorings, colorings, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. Additionally, the health food of the present invention may contain fruit pulp for the production of natural fruit juices, beverages, and vegetable drinks. These components can be used independently or in combination.

[0072] Furthermore, the present invention provides a method for preventing or improving oxidative stress, comprising the step of administering to an individual a composition containing, as an active ingredient, one or more selected from the group consisting of grape callus, callus crushed material, callus culture solution, and callus extract in a pharmaceutically effective amount.

[0073] Furthermore, the present invention provides a method for treating oxidative stress, comprising the step of administering to an individual a composition containing, as an active ingredient, one or more selected from the group consisting of grape callus, callus crushed material, callus culture solution, and callus extract in a pharmaceutically effective amount.

[0074] Another object of the present invention is to provide a method for preventing or improving bacterial infection, comprising the step of administering to an individual a composition containing, as an active ingredient, one or more selected from the group consisting of grape callus, callus crushed material, callus culture solution, and callus extract in a pharmaceutically effective amount.

[0075] Another object of the present invention is to provide a method for treating a bacterial infection, comprising the step of administering to an individual a composition containing, as an active ingredient, one or more selected from the group consisting of grape callus, callus crushed material, callus culture solution, and callus extract in a pharmaceutically effective amount.

[0076] Furthermore, the present invention provides a method for preventing or improving acne, comprising the step of administering to an individual a composition containing, as an active ingredient, one or more selected from the group consisting of grape callus, callus crushed material, callus culture solution, and callus extract in a pharmaceutically effective amount.

[0077] Furthermore, the present invention provides a method for treating acne, comprising the step of administering to an individual a composition containing, as an active ingredient, one or more selected from the group consisting of grape callus, callus crushed material, callus culture solution, and callus extract in a pharmaceutically effective amount.

[0078] The grape callus, crushed callus, callus culture solution, callus extract, and acne are the same as described for the antioxidant and antibacterial compositions, and specific descriptions are based on the above.

[0079] On the other hand, the antioxidant and antibacterial composition produced by the present invention is non-cytotoxic, exhibits significant antioxidant and antibacterial effects against acne bacteria, and has been confirmed to have particularly remarkable antioxidant and antibacterial effects compared to pure vinylin. Therefore, the antioxidant and antibacterial composition of the present invention can be usefully used for the prevention, improvement, or treatment of acne.

[0080] The present invention will be described in detail below with reference to examples and experimental examples.

[0081] However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

[0082] <Example 1> Preparation of grape callus culture solution

[0083] <1-1> Grape callus induction

[0084] We used the grape variety Campbell (Vitis vinifera L. cv Campbell Early) to induce callus from grape tissue, which was then propagated.

[0085] Specifically, grape blossoms were cut, washed with running water, and then immersed in a 70% ethanol solution for 1 minute for surface sterilization. Surface sterilization was performed by placing the grape blossom tissue in a glass bottle on a sterile workbench and immersing it in a commercial Lux (approximately 1% sodium hypochlorite) solution for 15 minutes, followed by three washes with sterile distilled water. After surface sterilization, the tissue was used for culture after removing any remaining moisture using sterile filter paper. After surface sterilization, the grape blossom tissue was cut using a scalpel, and each tissue (petals, stamens, and rhizomes) was incised into the culture medium. The culture medium used for grape callus induction is shown in [Table 1], and the medium used contained 0.1 mg / L IAA, 0.1 mg / L NAA, 1.5 mg / L 2,4-D, and 0.25 mg / L kinetin as growth regulators. Cancer culture was performed under constant temperature conditions of 25°C. The white callus formed after culturing was subjected to seasonal culture in a solid medium of the same composition approximately every four weeks.

[0086] [Table 1]

[0087] <1-2> Grape Callus Culture Solution Recovery

[0088] Grape callus derived from the above example <1-1> and stabilized was cultured and the grape callus culture solution was collected.

[0089] Specifically, the grape callus derived and stabilized from Example <1-1> described above was inoculated into three flasks at a rate of 10 g each into 100 ml of MS1D (MS 4.4 g, sucrose 30 g, MES 0.5 g, myo-inositol 0.1 g, thiamine HCl 0.4 mg, 2,4-D 1 mg / L) liquid medium, which is called MS (Murashige & Skoog) medium containing 1 mg / L 2,4-D. The cultures were then incubated at 25°C and 90 rpm. After culturing for 14 days, the grown callus was sterilized and transferred to a dry 3 L bioreactor, to which 1.2 L of MS1D liquid medium was added. After culturing for 7 days, the sample was treated with 100 μM MeJA (methyl jasmonate) and 50 mM stevioside together. Subsequently, the sample was filtered through a filter mesh to separate the callus from the callus culture medium, thereby obtaining the grape callus culture medium (sample name A).

[0090] In addition, the content of epsilonviniferin, deltaviniferin, and resveratrol present in the prepared grape callose culture medium is measured and the results are shown in Table 2.

[0091] On the other hand, the content of epsilon-viniferin, delta-viniferin, and resveratrol was determined by adding the same volume of ethyl acetate to 20 ml of the aforementioned grape keras culture solution (sample name A) and mixing thoroughly. The ethyl acetate layer was then collected and the ethyl acetate was removed using an evaporator. Finally, after dissolving in 1 ml of ethanol and diluting to an appropriate volume, the content of epsilon-viniferin, delta-viniferin, and resveratrol was analyzed by HPLC.

[0092] <1-3> Preparation of grape callus culture extract

[0093] The grape callus culture solution (sample name A) obtained in the above example <1-2> was extracted as ethyl acetate to produce an extract of grape callus culture solution (sample name a).

[0094] Specifically, 20 ml of the grape callus culture solution (sample name A) obtained in the above example <1-2> was mixed with the same volume of ethyl acetate, and the ethyl acetate layer was collected and removed using an evaporator. Finally, it was dissolved in 1 ml of ethanol to prepare an extract of the callus culture solution (sample name a).

[0095] In addition, the content of epsilonviniferin, deltaviniferin, and resveratrol in the callus culture extract (sample name a) was measured using the same method as described in <1-2> above, and the results are shown in Table 2 below.

[0096] <1-4> Preparation of Sample C

[0097] In the above example <1-2>, the callus was inoculated into a flask and cultured for 14 days to grow. Instead of a bioreactor, the callus was subcultured into a pre-sterilized and dried flask, and 300 ml of MS1D liquid medium was added. After culturing for 7 days, the sample was treated with 100 μM MeJA (methyl jasmonate) and 50 mM stevioside together. After 5 days of culture, the sample was filtered through a filter to separate the callus from the callus culture solution, thereby obtaining grape callus culture solution (named sample C).

[0098] [Table 2]

[0099] <1-5> Isolation and Purification of Delta-Biniferin

[0100] The grape callus culture solution prepared in <1-2> above was partitioned and extracted into water and ethyl acetate, and the resulting ethyl acetate layer was concentrated under reduced pressure. The obtained concentrate (15 g) was separated in one step using an MPLC instrument on a reversed-phase column (10 × 30 cm). At this time, water-acetonitrile was used as the eluent, and the water-acetonitrile mixed solution was eluted by lowering the polarity from 100:1 to 100% acetonitrile to obtain seven fractions (AG). Fraction C, which contained a large amount of the target compound viniferin (trance-d-viniferin, chemical formula 1 below), was concentrated to obtain a concentrate of 1.3 g. The obtained concentrate of 1.3 g was subjected to reversed-phase column chromatography using a recycling LC instrument under water-acetonitrile (2:2) mixed solvent conditions to obtain 160 mg of viniferin with a purity of 80% or higher. The obtained fraction was eluted with 95% methanol using gel chromatography (Sephadex LH-20) to obtain 12 mg of 98% pure viniferin. The structure of the obtained compound is as follows: 1 H-NMR, 13 The findings were investigated using 1C-NMR, 2D-NMR, DEPT, and mass spectrometry.

[0101] trans-δ-Viniferin: 1H-NMR (400MHz, Acetone-d6) δ7.45 (1H, dd, J=8.5, 2.0Hz, H-14b), 7.27 (1H, s, H-10b), 7 .25(2H, d, J=8.5Hz, H-2(6)a), 7.07(1H, d, J=16.5Hz, H-7b), 6.92(1H, d, J=16.5Hz, H-8b), 6.88(1H, d, J=8.1H z, H-13b), 6.86(2H, d, J=8.5Hz, H-3(5)a), 6.55(2H, d, J=2.0Hz, H-2(6)b), 6.29(1H, t, J=2.0Hz, 12a), 6.27(1 H, t, J=2.0Hz, H-4b), 6.21(2H, d, J=2.0, H-10(14)a), 5.47(1H, d, J=8.0Hz, H-7a), 4.48(1H, d, J=8.0Hz, H-8a); 13C-NMR (100MHz, Acetone-d6) δ159.7(C-12b), 158.8(C-11(13)a), 158.6(C-3(5)b), 157.5(C-4 a), 144.3(C-9a), 139.8(C-1b), 131.6(C-11b), 131.2(C-1a), 130.8(C-9b), 128.2(C-7b), 127 .7(C-14b), 127.6(C-2(6)a), 126.3(C-8b), 123.0(C-10b), 115.2(C-3(5)a), 109.2(C-13b), 106.4(C-10(14)a), 104.7(C-2(6)b), 101.8(C-4b), 101.4(C-12a), 93.1(C-7a), 56.9(C-8a).

[0102] [ka]

[0103] <1-6> Isolation and Purification of Epsilon Viniferin

[0104] The grape callus culture solution prepared in <1-2> above was partitioned and extracted into water and ethyl acetate, and the resulting ethyl acetate layer was concentrated under reduced pressure. The obtained concentrate (10 g) was subjected to one-step separation using an MPLC instrument on a reversed-phase column (10 × 30 cm). At this time, water-acetonitrile was used as the eluent, and the water-acetonitrile mixed solution was eluted by decreasing the polarity from 70:1 to 100% acetonitrile to obtain five fractions (AE). Fraction B, which contained a large amount of the target compound viniferin (trans-delta-viniferin, chemical formula 2 below), was concentrated to obtain a concentrate of 1.5 g. The obtained concentrate of 1.5 g was subjected to reversed-phase column chromatography using a recycling LC instrument under water-acetonitrile (2:1) mixed solvent conditions to obtain 100 mg of viniferin with a purity of 85% or higher. The obtained fraction was eluted with 95% methanol using gel chromatography (Sephadex LH-20) to obtain 30 mg of 98% pure viniferin. The structure of the obtained compound is as follows: 1 H-NMR, 13The findings were investigated using 1C-NMR, 2D-NMR, DEPT, and mass spectrometry.

[0105] trans-ε-viniferin 1 H-NMR (400MHz, MeOD) δ7.14 (2H, d, J=8.0Hz, H-2(6)a), 7.03 (2H, d, J=8.0Hz, H-2(6)b) , 6.80(1H, d, J=13.6Hz, H-7b), 6.78(2H, d, J=8.0Hz, H-3(5)a), 6.65(2H, d, J=8.0Hz, H- 3(5)b), 6.56(1H, d, J=13.6Hz), 6.28(1H, brs, H-12b), 6.21(1H, brs, H-12a), 6.18(2H, brs, H-10a, 14a), 5.37(1H, d, J=8.0Hz, H-7a), 4.35(1H, d, J=4.0Hz, H-8a);13C-NMR(10 0MHz, MeOD) δ161.3(C-11b), 158.6(C-11a, 13a), 158.3(C-13b), 157.1(C-4a), 156.9( C-4b), 146.0(C-9a), 135.6(C-9b), 132.5(C-1a), 129.1(C-1b, 7b), 127.5(C-2(6)b), 1 27.0(C-2(6)a), 122.4(C-8b), 118.8(C-10b), 115.1(C-3(5)b), 115.1(C-3(5)a), 106. 3(C-14a, 10a), 103.1(C-14b), 101.0(C-12a), 95.6(C-12b), 93.5(C-7a), 56.9(C-8a).

[0106] [ka]

[0107] <Experimental Example 1> Confirmation of cytotoxicity of grape callus culture solution

[0108] To confirm the presence or absence of cytotoxicity of grape callus culture, epsilon-viniferin, and delta-viniferin, an MTT assay was performed using MNT-1 and Raw264.7 cells.

[0109] Specifically, Raw264.7 (murine macrophage) was cultured in DMEM (High glucose) medium supplemented with 10% FBS and 1% g gentamicin at 37°C and 5% CO2, while MNT-1 (human melanoma cell) was cultured in DMEM / MEM (High glucose) medium supplemented with 20% FBS and 1% gentamicin at 37°C and 5% CO2. Subsequently, MNT-1 (10⁴ cells / well) and Raw264.7 (2 x 10⁴ cells / well) were cultured in 96 wells each, and after diluting the samples with the medium, they were treated in each well. After 24 hours, the wells were treated with MTT solution at a concentration of 100 μg / mL and reacted in an incubator at 37°C and 5% CO2 for 1 hour. After that, all the culture medium was removed, 100 μl of acid-isopropanol was added to each well, and the absorbance was measured using an ELISA reader at 570 nm. Additionally, the samples were diluted according to the epsilon-viniferin content standard and treated in wells for comparison with vinyliferin.

[0110] As a result, as shown in Figures 1 and 2, epsilonviniferin showed no cytotoxicity only at a concentration of 20 μg / ml, but grape callus culture medium (sample name A) did not show cytotoxicity even when containing 100 μg / ml of epsilonviniferin (Figures 1 and 2).

[0111] <Experimental Example 2> Confirmation of the antioxidant effect of grape callus culture solution

[0112] DPPH analysis was performed to confirm the antioxidant effect of grape callus culture solution.

[0113] Specifically, a 0.2 mM DPPH (1,1-Diphenyl-2-picryhydrazyl) solution was prepared using 80% methanol. Then, 180 μl of DPPH and 20 μl of the sample were thoroughly mixed and reacted at room temperature for 10 minutes. The absorbance was then measured at 517 nm using an ELISA reader. For comparison with vinylin, the sample was diluted according to the epsilon vinylin content standard and treated in wells. As a control, the sample was used instead of the sample, with the DPPH reagent added to the sample dilution solvent. Ascorbic acid was treated as a positive control group.

[0114] As a result, as shown in Figure 3, epsilonviniferin and deltaviniferin at 500 μg / ml resulted in approximately 25% reduction of radicals compared to the control group. However, in the case of the grape callus culture solution (sample name A) and grape callus culture solution extract (sample name a) of the present invention, it was confirmed that more than 60% of radicals were reduced at 500 μg / ml (based on epsilonviniferin content) (Figure 3).

[0115] <Experimental Example 3> Confirmation of the antibacterial effect of grape callus culture solution

[0116] <3-1> Culturing of acne bacteria

[0117] To confirm the antibacterial effect of Grapecorn culture solution on acne bacteria, Propionibacterium acnes (KCTC3314, KCTC3320, KCTC5012) were cultured in RCM (Reinforced Clostridial Medium) at 37°C under Anarobic conditions.

[0118] <3-2> Confirmation of MIC (minimum inhibitory concentration) against acne bacteria

[0119] MIC is an index used to measure the bacteriostatic characteristics of antimicrobial substances. After diluting the grape carose culture solution of the present invention to concentrations between 500 nM and 3.91 nM, the minimum concentration that inhibited the growth of three types of *P. acnes* was identified.

[0120] Specifically, the three types of *P. acnes* cultured in <3-1> above were cultured in 5 ml of RCM liquid medium under analogy conditions for 72 hours in a 37°C incubator. In addition, eight master plates were prepared by diluting each of the samples by half in DW, starting from the highest concentration, in 96-well plates. Subsequently, 50 μl of each diluted sample was transferred to other 96-well plates. The three types of *P. acnes* were diluted to 1 x 10⁶ cells / 50 μl (in 2x RCM), and 50 μl of the bacteria (1 x 10⁶ cells) was transferred to each of the 96-well plates containing the samples.

[0121] To measure the MIC, the 0-hour OD600nm was measured using a microplate reader. Subsequently, the 96-well plate was incubated under analog conditions at 37°C for 72 hours. After that, the 72-hour OD600nm was measured again using a microplate reader. After subtracting the 0-hour OD value from the 72-hour OD value, the growth of three types of p.acnes, corresponding to the control and sample concentrations, was compared, and antibacterial activity was confirmed through OD value comparison.

[0122] As a result, as shown in Figure 4, grape callus culture solution (sample name A) inhibited the growth of *P. acnes* 3320, 3314, and 5012 at average concentrations of 151.72 nM, 176.02 nM, and 117.45 nM, respectively. Grape callus culture solution with a high δ-viniferin content (sample name C) inhibited the growth of *P. acnes* 3320, 3314, and 5012 at average concentrations of 88.43 nM and 133.19 nM, respectively. On the other hand, pure ε-viniferin and pure δ-viniferin did not show growth inhibition against the three *P. acnes* species.

[0123] Therefore, it was confirmed that the grape callus culture solution of the present invention exhibits significantly greater probiotic activity compared to pure vinylin.

[0124] <3-3> MBC (minimum bactericidal concentration) analysis of acne bacteria

[0125] MBC, as an index for measuring the bactericidal characteristics of antimicrobial substances, diluted the grape callus culture solution of the present invention to concentrations between 500 nM and 3.91 nM, and then confirmed the minimum concentration that inhibited the growth of three types of P. acnes.

[0126] Specifically, the minimum sterilization concentration was measured using the plate on which the MIC described in <3-1> above was measured.

[0127] As a result, as shown in Figure 5, when the 72-hour sterilization of antimicrobial substances was measured via MBC, it was confirmed that grape callus culture solution (sample name A) showed the highest growth inhibitory effect against P. acnes 3320, 3314, and 5012 at concentrations of 125 nM, respectively. Furthermore, it was confirmed that grape callus culture solution (sample name C) showed the greatest growth inhibition at concentrations of 125 nM against P. acnes 3320 and 5012, and at 62.5 nM against P. acnes 3314.

[0128] Furthermore, as shown in Figure 6, the 48-hour sterilization of antimicrobial substances was measured via MBC. The results showed that grape callus culture solution (sample name A) showed the highest growth inhibitory effect against P. acnes 3320 and 3314 at 125 nM, and growth inhibition against P. acnes 5012 was confirmed at 62.5 nM. Additionally, grape callus culture solution (sample name C) showed the most significant growth inhibition at concentrations of 62.5 nM against P. acnes 3320 and 5012, and at 125 nM against P. acnes 3314.

[0129] In addition, we confirmed that pure ε-viniferin and pure δ-viniferin did not inhibit the growth of the three P.acnes species at both 48-hour and 72-hour intervals.

[0130] Therefore, it was confirmed that the grape callus culture solution of the present invention exhibits significant bactericidal activity compared to pure vinylin.

[0131] <3-4> Confirmation of antibacterial effect by disc diffusion test

[0132] The disc diffusion test is a method for testing the sensitivity of bacteria to antimicrobial agents. If an antimicrobial effect is detected, a clear zone is formed around the disc paper, and the degree of clear zone formation can be used to measure the antimicrobial activity.

[0133] Specifically, P.acnes 3320 was cultured in 5 ml of RCM liquid medium under Anarobic conditions for 72 hours in a 37°C incubator. Subsequently, 5 x 10 5 ~1x10 6 After wetting a cotton swab with the diluted bacteria, it was spread evenly onto RCM agar medium. A disc paper was placed on an agar plate, immersed in the undiluted sample, and then incubated under analog-lowering conditions for 72 hours at 37°C incubator. The size of the clear zone was then measured using a vernier caliper.

[0134] As a result, as shown in Figure 7 and Table 3, when the grape callus culture extract (sample name a) was treated with 5 μg and 10 μg of ε-viniferin, antibacterial activity against two species of *P. acnes* (*P. acnes* 3320 and 5012) was observed. Specifically, an 8 mm clear zone appeared in the control group treated with 50% EtOH, confirming that the grape callus culture extract (sample name a) of the present invention exhibits antibacterial activity against *Propionibacterium acnes*.

[0135] [Table 3] [Industrial applicability]

[0136] The present invention relates to an antibacterial and antioxidant composition containing grape callus culture solution containing epsilon(ε)-Viniferin, delta(δ)-Viniferin, and resveratrol as active ingredients. Specifically, the grape callus, its culture solution, and its extract of the present invention are non-cytotoxic, exhibit significant antioxidant and antibacterial effects against acne bacteria, and have the advantage of having particularly remarkable antioxidant and antibacterial effects compared to pure viniferin, making them useful for various applications.

Claims

1. An antimicrobial pharmaceutical composition containing grape callus culture solution or an extract of grape callus culture solution as an active ingredient.

2. The antimicrobial pharmaceutically active composition according to claim 1, characterized in that the grape callus is derived from one or more parts selected from the group consisting of grape flowers, leaves, stems, branches, fruits, and roots.

3. The antimicrobial pharmaceutically active composition according to claim 1, characterized in that the composition has antimicrobial activity against Propionibacterium acnes.

4. The antimicrobial pharmaceutical composition according to claim 1, characterized in that the composition has bacteriostatic and bactericidal activity.

5. A pharmaceutical composition for the prevention and treatment of acne, containing grape callus culture solution or an extract of grape callus culture solution as an active ingredient.

6. The pharmaceutical composition for preventing and treating acne according to claim 5, characterized in that the composition has antibacterial activity against Propionibacterium acnes.

7. The pharmaceutical composition for the prevention and treatment of acne according to claim 5, characterized in that the composition has bacteriostatic and bactericidal activity against Propionibacterium acnes.

8. An antibacterial cosmetic composition containing grape callus culture solution or an extract of grape callus culture solution as an active ingredient.

9. A cosmetic composition for preventing and improving acne, containing grape callus culture solution or an extract of grape callus culture solution as an active ingredient.

10. A composition for the prevention, improvement, or treatment of bacterial infection, comprising grape callus culture solution or an extract of grape callus culture solution as an active ingredient.

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