KR20230024227A - Uses of fractions of spinach extract comprising novel compounds for improving skin condition - Google Patents

Abstract

The present invention relates to a use of a fraction containing a novel compound isolated from spinach for improving skin condition, wherein the compound is represented by formulas 1 to 6. More specifically, the present invention relates to a use of a novel medicagenic acid glycoside compound isolated from spinach and a fraction containing the same for skin whitening, wrinkle alleviation, antioxidant activity, skin inflammation prevention, and skin aging prevention. The fraction of spinach extract containing five novel glycoside compounds and celosin I alleviate inflammatory responses in skin cells and exhibits excellent skin whitening, skin wrinkle alleviation, antioxidant, skin aging alleviation, and moisturizing effects, and thus can be very useful for ameliorating skin conditions, including inflammatory skin diseases.

Description

Uses of fractions of spinach extract comprising novel compounds for improving skin condition

The present invention relates to the use of a fraction containing a novel compound isolated from spinach to improve skin conditions, and more particularly, to a novel medicagenic acid glycoside compound isolated from spinach and a fraction containing the same for skin whitening and wrinkles. Improvement, antioxidant, prevention of skin inflammation and prevention of skin aging.

The skin is stimulated by various pollutants such as environmental pollution and automobile exhaust, as well as retinol and lactic acid, which are widely used as raw materials for cosmetics, and the occurrence of skin diseases such as aging, skin inflammation such as atopy or acne, and wrinkles is increasing. . Specifically, hormonal imbalances intensify due to various pollutants, reducing the synthesis ability of fibroblasts in the skin or reducing the immune function, and retinol and lactic acid, which are effective for wrinkle improvement and regeneration, are decomposed by ultraviolet rays and have practical efficacy This causes skin damage caused by ultraviolet rays.

The main cause of skin diseases such as skin damage and aging is caused by the inflammatory reaction in the body, and nitric oxide (NO), a kind of reactive radical species in the inflammatory reaction in the body, It is known to play an important role in various physiological and pathological processes. Nitric oxide is a very unstable compound and is produced through the activation of inducible nitric oxide synthase (iNOS), an enzyme that produces nitric oxide (NO), by an inflammatory substance such as lipopolysaccharide (LPS). In addition, it is known that various types of cytokines or chemokines are involved in the process of infiltrating inflammatory cells into lesions, and receptors related thereto are expressed in various cells such as inflammatory cells or keratinocytes.

In order to improve or prevent such skin damage and aging, non-steroidal antioxidants such as benzidamine, intomethacin, ibuprofen, etc., or dexamethasone, hydrocortisone, etc. Skin wrinkles and skin inflammatory diseases were improved by using steroid-based antioxidants. In addition, substances having inhibitory activity against derosinase such as hydroquinone, ascorbic acid, kojic acid, and glutathione are used to treat skin whitening or skin hyperpigmentation. improved. However, since the above chemicals cause side effects when used excessively and have problems with stability and safety, it is necessary to develop new substances that are safe and have excellent skin condition improvement effects with few side effects.

Accordingly, the present inventors have conducted intensive research to develop a new substance derived from natural products capable of exhibiting anti-inflammatory effects, inhibiting melanin production, improving skin wrinkles, and exhibiting antioxidant effects, and as a result, a novel substance isolated from spinach extract was found. The present invention was completed by finding that a fraction containing 5 kinds of glycoside compounds and celosin I exhibited such an effect.

Accordingly, an object of the present invention is a pharmaceutical, cosmetic, food, water for skin whitening, skin wrinkle improvement, skin moisturizing, skin aging improvement, antioxidation and skin inflammation improvement containing compounds represented by Formulas 1 to 6 as active ingredients. To provide a medical or feed composition:

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

Another object of the present invention is to provide a composition for skin whitening, skin wrinkle improvement, skin moisturizing, skin aging improvement, antioxidant and skin inflammation improvement comprising a spinach fraction prepared according to a method comprising the following steps as an active ingredient:

(a) extracting spinach with a solvent selected from the group consisting of water, organic solvents, subcritical fluids, supercritical fluids, and mixtures thereof; and

(b) obtaining fractions containing the compounds of Formulas 1 to 6 by fractionating the spinach extract by chromatography.

is to do

In order to achieve the above object of the present invention, the present invention provides a pharmaceutical preparation for skin whitening, skin wrinkle improvement, skin moisturizing, skin aging improvement, antioxidation and skin inflammation improvement comprising compounds represented by Formulas 1 to 6 as active ingredients. , cosmetic, food, veterinary or feed compositions are provided:

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

In order to achieve another object of the present invention, the present invention is skin whitening, skin wrinkle improvement, skin moisturizing, skin aging improvement, antioxidant and skin inflammation improvement comprising spinach fraction prepared according to a method comprising the following steps as an active ingredient A composition is provided for:

(a) extracting spinach with a solvent selected from the group consisting of water, organic solvents, subcritical fluids, supercritical fluids, and mixtures thereof; and

(b) obtaining fractions containing the compounds of Formulas 1 to 6 by fractionating the spinach extract by chromatography.

Hereinafter, the present invention will be described in detail.

The present invention relates to skin whitening, skin wrinkle improvement, skin moisturizing, skin aging improvement, A pharmaceutical, cosmetic, food, veterinary or feed composition for antioxidation and improvement of skin inflammation is provided:

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

.

.

In one embodiment of the present invention, the present inventors isolated novel glycosides of medicagenic acid represented by Chemical Formulas 1 to 6 from spinach extracts. The novel glycosides represented by Chemical Formulas 1 to 6 have been confirmed to exhibit significantly improved skin condition improvement efficacy (skin whitening, skin wrinkle improvement, skin moisturizing, skin aging improvement, antioxidant and skin inflammation improvement) compared to medicagen acid, which It was also confirmed that the fraction of the spinach extract containing spinach extract exhibited significantly improved skin condition improvement efficacy (skin whitening, skin wrinkle improvement, skin moisturizing, skin aging improvement, antioxidation and skin inflammation improvement) compared to the spinach extract.

In the present invention, the term "pharmaceutically acceptable" means that it can be approved by the government or a regulatory agency equivalent thereto to be used in animals, more specifically in humans, by avoiding significant toxic effects when used in normal pharmaceutical dosages, or It means approved or recognized as listed in a pharmacopeia or listed in other general pharmacopeias.

In the present invention, the term "pharmaceutically acceptable salt" means a salt that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. The salt may be useful as an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts are salts of inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. These pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulphate, sulphite, 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, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, Toxybenzoates, phthalates, terephthalates, benzenesulfonates, toluenesulfonates, chlorobenzenesulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, hydroxybutyrates, glycolates, but is not limited to maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

In the present invention, the term "isomer" refers to optical isomers (eg, essentially pure enantiomers, essentially pure diastereomers or mixtures thereof) as well as conformational isomers ( Includes conformation isomers (i.e. isomers that differ only in the angle of one or more chemical bonds), position isomers (particularly tautomers) or geometric isomers (e.g. cis-trans isomers) can do.

In the present invention, the term "essentially pure", when used in relation to, for example, enantiomers or diastereoisomers, contains about 90% or more, preferably about 95%, of a specific compound exemplified by the enantiomer or diastereomer. or more, more preferably about 97% or more or about 98% or more, even more preferably about 99% or more, even more preferably about 99.5% or more (w / w).

In the present invention, the term "hydrate" means a compound to which water is bound, and may mean a broad concept including an inclusion compound having no chemical binding force between water and the compound.

In the present invention, the term “solvate” may refer to a higher order compound formed between solute molecules or ions and solvent molecules or ions.

In the present invention, the glycoside compounds represented by Chemical Formulas 1 to 6 may be extracted from spinach.

Specifically, the glycoside compound represented by Chemical Formulas 1 to 6 may be obtained by: (a) extracting spinach with a solvent selected from the group consisting of water, an organic solvent, a subcritical fluid, a supercritical fluid, and mixtures thereof; (b) obtaining fractions by fractionating the spinach extract by chromatography; and (c) isolating the compounds of Formulas 1 to 6 according to claim 1 from the fractions.

In the present invention, the spinach (Spinacia oleracea L.) is an annual or biennial herb of the dicotyledonous plant Centrioleaceae, and the spinach includes stems, flowers, roots, shoots, outposts, or mixtures thereof.

In the present invention, step (a) is a step of preparing a spinach extract by a known natural product extraction method. In the step (a), extraction may be performed with one or more solvents selected from the group consisting of solvents selected from the group consisting of water, organic solvents, subcritical fluids, supercritical fluids, and mixtures thereof. The organic solvent having 1 to 6 carbon atoms is alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride chloride), hexane, cyclohexane, and petroleum ether, but may be selected from the group consisting of, but is not limited thereto. When the extraction solvent in step (a) is a mixed solvent of water and organic solvent, water and organic solvent may be mixed in a ratio of 90:10 to 10:90, specifically 90:10, 80:20, It can be mixed in a ratio of 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 or 10:90.

In the present invention, step (b) is a step of separating fractions containing the novel glycosides of Chemical Formulas 1 to 6 from the spinach extract prepared in step (a).

In the present invention, the type of the chromatography method is not particularly limited, and any method used in the art for separating or purifying a desired component may be used without limitation. Preferably, the chromatography method may be column chromatography, more preferably medium pressure liquid chromatography (MPLC), high performance liquid chromatography (HPLC), or ultra high performance liquid chromatography (UHPLC).

Chromatography in step (b) may be performed 1 to 5 times in consideration of yield and purity, but is not limited thereto.

In step (b), when the spinach extract is chromatographically fractionated, water, an organic solvent, or a mixed solvent thereof may be used as a mobile phase or a developing solvent, and preferably a mixed solvent of water and a non-polar solvent may be used.

The type of the non-polar solvent is not particularly limited, but may be preferably methanol or acetonitrile, and most preferably methanol.

In the step (b), the chromatography method may be performed by sequentially developing water and a non-polar solvent as a mobile phase according to a concentration gradient, wherein the ratio of the water and the non-polar solvent is sequentially from 100:0 to 0:100 Chromatography can be performed while changing. Specifically, 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 water and a non-polar solvent as the mobile phase and a chromatographic method may be performed using a ratio of 0:100.

In the step (b) of the present invention, when fractionation of the spinach extract is carried out by sequentially developing water and a non-polar solvent as a mobile phase according to a concentration gradient, in a 20 to 100% (v / v) non-polar solvent aqueous solution, preferably Preferably, it may be characterized by obtaining a fraction that is fractionated in a 30 to 100% (v / v) aqueous solution of a non-polar solvent.

According to one embodiment of the present invention, the novel glycoside compounds of Chemical Formulas 1 to 6 are obtained by chromatographic fractionation of spinach extract using a mixed solvent of water and methanol, 30 to 100% (v / v) of methanol It was confirmed that it was contained in the aqueous fraction.

In the present invention, step (c) is a step of separating desired new glycosides of Formulas 1 to 6 from the fraction of the spinach extract obtained in step (b).

According to one embodiment of the present invention, the novel glycosides of Chemical Formulas 1 to 6 were confirmed to have excellent anti-inflammatory activity in skin cells. Therefore, it can be used for preventing or improving inflammatory skin diseases and skin inflammation.

The inflammatory skin disease or skin inflammation may be selected from the group consisting of atopic dermatitis, acne, psoriasis, seborrheic dermatitis, contact dermatitis, systemic lupus erythematosus, allergic skin disease, acne, lupus erythematosus and urticaria, but is limited thereto It is not.

According to another embodiment of the present invention, it was confirmed that the novel glycosides of Chemical Formulas 1 to 6 can be used for skin whitening because of their excellent activity of inhibiting melanin accumulation in skin cells.

In the present invention, the "skin aging" refers to aging due to aging (chronoaging) or aging due to exposure to sunlight (photoaging), extreme climatic conditions, exposure to environmental factors such as cigarette smoke, fine dust, and chemical pollutants It means all the changes that the skin experiences. Skin changes due to aging include all recognizable changes, such as external and visible changes through touch or the like, or histological changes in skin tissue. Preferably, the skin aging may include oxidation (ie, accumulation of active oxygen), skin wrinkles due to collagen reduction, skin pigmentation, skin inflammation, skin fibrosis, and skin dryness. In the present invention, the skin aging can be caused by various factors, and the composition of the present invention can help anti-aging of the skin through various mechanisms such as antioxidant, skin wrinkle improvement, moisturizing, whitening or anti-inflammatory.

In the present invention, the "photo-aging" refers to a phenomenon in which the skin is aged by exposure to sunlight (ultraviolet rays), and when exposed to the sun (ultraviolet rays) for a long period of time, deep wrinkles are formed on the face and neck, In addition, the skin becomes dry and the skin surface becomes rough or causes pigmentation such as spots and freckles.

In the present invention, the "improvement of skin wrinkles" means maintaining or enhancing the ability related to wrinkles and elasticity of the skin. Collagen, which is a collagen fiber in the dermal layer of the skin, and elastin, an elastic fiber, are major proteins that play such a role and govern skin elasticity, and the biosynthesis of collagen is influenced internally and externally by the skin. Specifically, in skin cells, when cell activity is reduced due to natural aging, collagen fibers are reduced, or as an external factor, excessive irradiation of ultraviolet rays or active oxygen generated by stress reacts with a thiol group (thiol: -SH) of a protein. This inhibits the activity of target proteins or increases the expression of decomposing enzymes such as collagen and elastin, thereby increasing wrinkles and reducing elasticity of the skin, leading to skin aging.

In the present invention, the "antioxidation" means to scavenge reactive oxygen species (ROS) that cause oxidative damage in the human body. Reactive oxygen species are generated in large quantities when irradiated with ultraviolet rays and damage DNA, RNA, proteins, cell membranes, and cell structures, and are considered one of the main causes of aging.

The pharmaceutical composition according to the present invention contains a compound represented by Formulas 1 to 6, a pharmaceutically acceptable salt thereof, an isomer thereof, a hydrate thereof, or a solvate thereof alone, or one or more pharmaceutically acceptable carriers or excipients. Or it may further contain a diluent. In the above, "pharmaceutically acceptable" is a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans and does not usually cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions. says

A pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like. In addition, the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol, and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, and the like in addition to the above components. Other pharmaceutically acceptable carriers and formulations may be referenced as described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration can be

The pharmaceutical composition of the present invention may be formulated into a preparation for oral administration or parenteral administration according to the administration route as described above.

In the case of preparations for oral administration, the composition of the present invention may be formulated into powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, etc. using a method known in the art. can For example, preparations for oral use may be obtained by combining the active ingredient with a solid excipient, which is then milled and, after adding suitable auxiliaries, processed into a mixture of granules to obtain tablets or dragees. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose, and the like, fillers such as gelatin, polyvinylpyrrolidone, and the like may be included. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant, if desired. Furthermore, the pharmaceutical composition of the present invention may further include an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, and a preservative.

In the case of preparations for parenteral administration, they may be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995, which is a generally known formula for all pharmaceutical chemistry.

The total effective amount of the composition of the present invention can be administered to the patient in a single dose or by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 1,000 mg, most preferably 0.1 μg to 500 mg per kg of patient body weight per day. However, the dose of the pharmaceutical composition is determined by considering various factors such as the formulation method, administration route, and number of treatments as well as the patient's age, weight, health condition, sex, severity of disease, diet, and excretion rate. Therefore, considering this point, those skilled in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as it exhibits the effects of the present invention.

Furthermore, the pharmaceutical composition of the present invention can be administered in parallel with a known substance having an effect of preventing, improving and treating inflammatory skin diseases and the like.

Furthermore, it can be preferably formulated according to each disease or component by using an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

In the present invention, the cosmetic composition can be prepared in any formulation that is conventionally prepared, for example, a solution, emulsion, suspension, paste, cream, lotion, gel, powder, spray, surfactant-containing cleansing, oil , Soap, liquid cleanser, bath additive, wax foundation, makeup base, essence, toner, foam, pack, softening water, sunscreen cream or sun oil, etc. may be formulated, but is not limited thereto.

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

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

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

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

When the formulation of the present invention is a surfactant-containing cleanser, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, 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 cosmetic composition of the present invention may further include conventional adjuvants and carriers such as commonly used antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances, and the like. For example, the cosmetic composition may further include auxiliary ingredients such as glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate, and allantoin.

The food composition of the present invention includes all types of functional food, health functional food, nutritional supplement, health food and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, a composition containing the compounds of Chemical Formulas 1 to 6 may be prepared in the form of tea, juice, or drink to be consumed, or granulated, encapsulated, or powdered to be consumed. In addition, it can be prepared in the form of a composition by mixing the compounds of Formulas 1 to 6 of the present invention with known active ingredients known to have anti-wrinkle, anti-aging, skin elasticity enhancement and skin moisturizing effects.

In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled fruit, jam, marmalade, etc.), fish, meat and their processed foods (e.g. ham, sausage corned beef). etc.), breads and noodles (e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable It can be prepared by adding the compounds of Formulas 1 to 6 to proteins, retort foods, frozen foods, various seasonings (eg, soybean paste, soy sauce, sauce, etc.).

Specifically, the present invention relates to a health functional food comprising a compound represented by Chemical Formulas 1 to 6, a pharmaceutically acceptable salt thereof, an isomer thereof, a hydrate thereof or a solvate thereof, and a food pharmaceutically acceptable carrier or additive. functional food). The health functional food defined in the present invention has sufficiently established functionality and safety for the human body newly defined through the Health Functional Food Act amended in 2008, and thus has the health function specified in the Food and Drug Administration Notice No. 2008-72 of the Food and Drug Administration. It means that it is a health functional food listed in the regulations on recognition of functional food ingredients.

These health functional foods may be formulated in the form of conventional health functional foods known in the art. The health supplement may be prepared into, for example, powders, granules, tablets, pills, capsules, suspensions, emulsions, syrups, precipitates, liquids, extracts, gum, tea, jelly, or beverages. Any carrier or additive known to be usable in the art for the preparation of the formulation to be prepared may be used as the pharmaceutically acceptable carrier or additive.

The health functional food of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts , organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like.

In addition, various flavoring agents or natural carbohydrates may be further contained. Examples of the natural carbohydrate include common sugars such as monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), polysaccharides (eg, dextrin, cyclodextrin, etc.) and xylitol, sorbitol, erythritol, etc. of sugar alcohols may be contained. In addition, natural flavoring agents (eg, thaumatin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be included as flavoring agents.

In addition, in order to use the compounds of Formulas 1 to 6 of the present invention in the form of food additives, they may be prepared and used in the form of powder or concentrate.

In the food composition of the present invention, the preferred content of the compound represented by Formulas 1 to 6, a pharmaceutically acceptable salt thereof, an isomer thereof, a hydrate thereof, or a solvate thereof is 0.0001 to 90% by weight, preferably 0.0001 to 90% by weight, based on the total weight of the food composition. Preferably it may be contained in the range of 0.01 to 5% by weight.

The veterinary composition of the present invention may further include appropriate excipients and diluents according to conventional methods. The excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose , polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben , propylparaben and mineral oil.

The veterinary composition according to the present invention may further include a filler, an anti-coagulant, a lubricant, a wetting agent, a spice, an emulsifier, a preservative, etc. It can be formulated using methods well known in the art to provide sustained or delayed release, and the dosage form can be powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules. , It may be in the form of suppositories, sterile injection solutions, sterile external preparations, and the like.

The veterinary composition according to the present invention may vary depending on the age, sex, and weight of the animal, but may be administered in an amount of 0.1 to 100 mg/kg once to several times a day, and the dosage is the route of administration, the severity of the disease, It may increase or decrease according to gender, weight, age, etc. Accordingly, the dosage is not intended to limit the scope of the present invention in any way.

In addition to the composition according to the present invention, the 'feed composition' may use food ingredients and food additives described in the Food Additive Code that can be used as food described in the food standards and specifications ('Food Code') as an active ingredient, Even if they are not food ingredients or food additives that can be used as food additives, raw materials that fall under the scope of single feed in Attached Table 1 of 'Standards and Specifications for Feed, etc.' and raw materials that fall under the scope of supplementary feed under Attached Table 2 can be used.

The 'feed composition' may be an extractant in supplementary feed according to 'standards and specifications for feed, etc.', and may be a formulated feed containing the supplementary feed.

In the case of preparing the feed composition, the content of the compound represented by Formulas 1 to 6 is not particularly limited as long as the content shows improvement in skin condition, but, for example, 0.1 to 99% by weight, 0.5 to 95% by weight, 1 to 10% by weight. 90% by weight, 2 to 80% by weight, 3 to 70% by weight, 4 to 60% by weight, may be included in 5 to 50% by weight.

The compound of the above formula, which is an active ingredient in the feed composition, varies depending on the condition, body weight, presence or absence or degree and period of disease of the consuming animal, but may be appropriately selected by a person skilled in the art. For example, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, still more preferably 20 to 800 mg, and most preferably 50 to 500 mg based on the daily dose. There is, and the number of administrations need not be particularly limited, but can be adjusted by a person skilled in the art within the range of three times a day to once a week. In the case of long-term intake for the purpose of health and hygiene or health control, it may be less than the above range.

The present invention also provides a composition for skin whitening, skin wrinkle improvement, skin moisturizing, skin aging improvement, antioxidant and skin inflammation improvement comprising a spinach fraction prepared according to a method comprising the following steps as an active ingredient:

(a) extracting spinach with a solvent selected from the group consisting of water, organic solvents, subcritical fluids, supercritical fluids, and mixtures thereof; and

(b) obtaining fractions containing the compounds of Formulas 1 to 6 by fractionating the spinach extract by chromatography.

In the present invention, a detailed description of steps (a) and (b) may refer to the above.

A fraction of spinach extract containing 5 new glycoside compounds and celosin I alleviates inflammatory reactions in skin cells, and exhibits excellent skin whitening, skin wrinkle improvement, antioxidant, skin aging improvement and moisturizing effects. Therefore, it can be very usefully used for improving skin conditions including inflammatory skin diseases.

1 is a view showing the results of UPLC analysis of spinach extract.
Figure 2 shows the manufacturing process of the effective fraction (Fr.2) containing 5 new substances and celosin I compound from spinach extract.
Figure 3 is a view showing the results of CAD chromatogram analysis of spinach extract, fraction 1 (Fr.1) and fraction 2 (Fr.2).
4 is a diagram showing the results of UPLC-QTOF-MS analysis of 5 new substances (SOA1, SOA2, SOA4, SOA5, SOA6) and celosin I compound (SOA 3) isolated from spinach extract.
Figure 5 is a view showing the results of in vitro experiments evaluating the whitening efficacy of spinach extract, fraction 1 (SO Fr.1), fraction 2 (SO Fr.2), and five new compound glycosides.
Figure 6 is a result of evaluating the cytotoxicity and whitening efficacy of fraction 2 (SO Fr.2) of spinach extract in B16F10 cells.
7 is a view showing the results of an in vitro experiment evaluating the skin inflammation improvement efficacy of spinach extract, fraction 1 (SO Fr.1), fraction 2 (SO Fr.2), and five kinds of new compound glycosides.
8 is a result of evaluating the tyrosinase inhibitory activity of spinach fraction 2 (Fr.2) by Western blotting.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1: Preparation of spinach extract and component profile analysis

After extracting 100 kg of spinach at 50 degrees Celsius for 90 minutes using 18-fold 30% alcohol, it was filtered/concentrated.

Components of the prepared spinach extract were analyzed by UPLC.

Specifically, for UPLC analysis, the spinach extract was filtered once with a 0.25 mm membrane filter for UPLC. After mounting a column (Waters BEH C18 column, 2.1 Х 100 mm, 1.7 μm) on a UPLC instrument (Waters UPLC-Q-TOF), each filtered fraction was loaded in an amount of 0.3 μl.

At this time, the solvent used in the UPLC analysis was acetonitrile + 0.1% formic acid / water + 0.1% formic acid [10: 90 -> 100: 0 (v / v)], and the elution rate was 0.4 ml / min. . The degree of separation of the substances separated from the UPLC was confirmed by chromatography using MS (Mass spectrometry) and CAD (Charged Aerosol Detector) as detectors.

The UPLC-QTOF-MS analysis conditions and results are shown in FIG. 1 and Table 1.

As can be seen in Figure 1, 5 new substances (SOA1, SOA2, SOA4, SOA5, SOA6) and celosin I compound (SOA 3) were identified from the spinach extract through UPLC analysis.

Example 2: Fractionation of Spinach Alcohol Extract

Effective fractions and novel compounds were separated from the extract obtained in Example 1 in the following manner.

Specifically, spinach extract (20 g) was loaded with a column (YMC-DAD-50-700S (50 x 700 mm, 10 μm)) in an MPLC instrument (YMC LAB-300), and then the extract was loaded. At this time, as a solvent methanol/water [10:90 -> 100:0 (v/v)] was used, the elution rate was 100 ml/min, and a small fraction (SO Fr. 1, Fr.2) was obtained (FIG. 2).

Specifically, the effective fraction SO Fr.2 (16.5 g) was loaded with a column (Waters X-bridge, 19 x 250 mm, 5 μm) in an MPLC instrument (YMC Lc-Forte/R), and then the effective fraction was loaded. At this time, acetonitrile + 0.1% formic acid / water + 0.1% formic acid [15:85 -> 100:0 (v / v)] was used as the solvent, the elution rate was 11 ml / min, UV 210, 254, Repeatedly performed at a wavelength of 280 nm, compounds with novel structures represented by Chemical Formulas 1, 2, 3 and 4 having the following physical properties SO peak 1 (SOA1, 238.6 mg), 2 (SOA2, 170.2 mg), 3 (SOA3, 39.4 mg) and 4 (SOA4, 24.2 mg) were obtained (FIGS. 3 and 4).

Example 3: Structural analysis of 5 new materials

The molecular weight and molecular formula of the oleanane-type triterpene saponin compound obtained in Example 2 were determined using a high-resolution Qtof-MS mass spectrometer (Vion IMS-Qtof-MS (Waters, USA). In addition, nuclear magnetic resonance (NMR) Determine molecular structure using ¹ H-NMR, ¹³ C-NMR and 2D NMR (COSY, HSQC, HMBC, ROESY) spectroscopic data through analysis (Bruker Avance-800 MHz, Bruker Avance-900 MHz Bruker, Germany) did

As a result of comparative analysis of the instrumental analysis results with those of published literature, it was confirmed that the oleanane-type triterpene saponins represented by Formulas 1 to 6 below. The detailed analysis results are as follows.

In compound 1 , a molecular ion [M+H] ⁺ of m/z 1133.5383 was observed from the HR-Qtof-MS spectrum, and through this, the molecular formula of C ₅₄ H ₈₄ O ₂₅ was determined. Through MS cleavage pattern analysis, a cleavage ion at m/z 971 due to loss of glucose (162 Da) and molecular ions at m/z 825, 679 due to loss of pentose rhamnose and fucose (146 Da) were observed. . In addition, the molecular ion of sapogenin, m/z 503, was detected by the loss of glucuronic acid (176 Da), which is identical to the molecular ion value of medicagenic acid. In ^{the 1} H NMR spectrum, 8 methyl protons ( δ _H 1.89, 1.64, 1.45, 1.40, 1.19, 1.03, 0.79, 0.77), 1 olefin proton ( δ _H 5.33) and 4 anomeric protons ( δ _H 6.20, 5.91 , 5.13, 5.06), and two oxygen-adjacent methine protons ( δ _H 4.74, 4.63) were observed. In ^{the 13} C NMR spectrum, three carbonyl carbons ( δ _C 180.9, 176.6, 172.7), a pair of olefin carbons ( δ _C 143.8, 122.4), and four enemeric carbons ( δ _C 106.2, 105.2, 101.1, 94.5) and 1 oxygen-substituted methylene carbon ( δ _C 62.4) and 18 methine carbons ( δ _C 85.7, 84.5, 78.1, 77.9, 77.2, 76.9, 75.9, 75.8, 74.4, 74.1, 72.8, 72.6, 72.1, 71.9, 71.3, 71.2, 70.0, 68.2) were confirmed. As a result of confirmation through double 2D NMR, a signal was observed in which the methyl proton peak [ δ _H 1.89 (3H, s, H ₃ -24)] was adjacent to the carbonyl peak ( δ _C 180.9), and these NMR spectral patterns and Medicagenic acid ( medicagenic acid, 2β,3β-dihydroxyolean-12-ene-23,28-dioic acid) as an aglycone was confirmed to be an oleanane-type saponin compound. In addition, δ _H 5.13 (1H, d, J = 8.1 Hz,, H-1′, GlcA) peak was δ _C 85.7 (C-3) among the four enomeric protons through 2D NMR (COSY, HSQC, HMBC). , and it was confirmed that the δ _H 5.91 (1H, d, J = 8.0 Hz, H-1′′, Fuc) peak was linked to the δ _C 176.6 (C-28) carbonyl group. Additionally, the δ _H 6.20 (1H, d, J = 1.8 Hz, H-1′′′, Rham) peak is connected to the 2-carbon position of fucose ( δ _C 74.1, C-2′′), and δ _H It was confirmed that the 5.06 (1H, d, J = 7.2 Hz, H-1′′′′, Glu) peak was bound to the carbon at position 4 ( δ _C 84.5 C-4′′′) of rhamnose. Combining the previous literature and the above results, compound 1 is 3- O -β-d-glucuronopyranosyl-2β,3β,-dihydroxyolean-12-ene-23,28-dioic acid-28- O -β-d-glucopyranosyl- It was identified as (1→4) -α -l-rhamnopyranosyl-(1→2)-β-d-fucopyranoside and named as spinasaponin C.

In compound 2 , a molecular ion [M+H] ⁺ of m/z 1265.5810 was observed from the HR-Qtof-MS spectrum, and the molecular formula of C ₅₉ H ₉₂ O ₂₉ was determined. Through MS cleavage pattern analysis, the molecular ion at m/z 1133 was detected due to the loss of xylose (132 Da), a pentose sugar, and the cleavage ion at m/z 971 due to the loss of glucose (162 Da) and rhamnose, a pentose sugar. Molecular ions at m/z 825, 679 due to the loss of fucose (146 Da) were observed. In addition, the molecular ion of sapogenin, m/z 503, was detected by the loss of glucuronic acid (176 Da), which is identical to the molecular ion value of medicagenic acid. As a result of comparative analysis of the 1D and 2D NMR spectra and the prior literature, compound 2 showed a similar spectrum when compared to compound 1 , and it was confirmed that a pentose sugar, xylose sugar, was additionally linked to compound 1 . Through 2D NMR data, the enomeric proton of xylose sugar ( δ _H 5.30, 1H, d, J = 7.2 Hz, H-1′′, Xyl) is linked to C-3′ of glucuronic acid ( δ _C 86.1, C-3', GlcA) was confirmed. Therefore, by combining the previous literature and the above results, compound 2 is 3- O -β-d-xylopyranosyl-(1→3)-β-d-glucuronopyranosyl-2β,3β,-dihydroxyolean-12-ene-23,28- It was identified as dioic acid-28- O -β-d-glucopyranosyl-(1→4) -α -l-rhamnopyranosyl-(1→2)-β-d-fucopyranoside and named as spinasaponin D. did

In compound 3 , the molecular ion [M+H] ⁺ of m/z 1103.5301 was observed from the HR-Qtof-MS spectrum, and the molecular formula of C ₅₃ H ₈₂ O ₂₄ was determined. Through MS cleavage pattern analysis, a molecular ion of m/z 971 was detected due to the loss of pentose sugar xylose (132 Da), and m/z 825 due to the loss of pentose rhamnose and fucose (146 Da). 679 molecular ions were observed. In addition, the molecular ion of sapogenin, m/z 503, was detected by the loss of glucuronic acid (176 Da), which is identical to the molecular ion value of medicagenic acid. As a result of comparative analysis of the 1D and 2D NMR spectra and the preceding documents, compound 3 showed a similar spectrum when compared to compound 1 , and it was found that the glucose sugar position of compound 1 was substituted with xylose sugar, which is a pentose sugar. Confirmed. Through 2D NMR data, the enomeric proton of xylose sugar ( δ _H 5.03, 1H, d, J = 7.2 Hz, H-1′′′′, Xyl) is linked to C -4′′′ of rhamnose ( δ H 5.03, 1H, d, J = 7.2 Hz, H-1′′′′, Xyl) _C 85.8, C-4′′′, Rham) was confirmed. Combining the above results, compound 3 is 3- O -β-d-glucuronopyranosyl-2β,3β,-dihydroxyolean reported in the previous literature (J. Asian Nat. Prod. Res., 2014, 16, 240-247). -12-ene-23,28-dioic acid-28- O -β-d-xylopyranosyl-(1→4)- α -l-rhamnopyranosyl-(1→2)-β-d-fucopyranoside (Celosin I) compound It was confirmed that

In compound 4 , the molecular ion [M+H] ⁺ of m/z 1235.5725 was observed from the HR-Qtof-MS spectrum, and the molecular formula of C ₅₈ H ₉₀ O ₂₈ was determined. Through MS cleavage pattern analysis, a molecular ion of m/z 1103 was detected due to the loss of xylose (132 Da), a pentose sugar. Molecular ions at m/z 957, 825, and 679 were observed. In addition, the molecular ion of sapogenin, m/z 503, was detected by the loss of glucuronic acid (176 Da), which is identical to the molecular ion value of medicagenic acid. As a result of comparative analysis of 1D and 2D NMR spectra and the preceding literature, compound 4 showed a similar spectrum when compared to compound 2 , and it was found that the glucose sugar position of compound 2 was substituted with xylose sugar, which is a pentose sugar. Confirmed. Through 2D NMR data, the additional enomeric proton of xylose sugar ( δH _4.94 , 1H, d, J = 7.2 Hz, H-1′′′′′, Xyl-II) is C-4′′′ of rhamnose ′ ( δ _C 84.6, C-3′′′′, Rham). Therefore, by combining the previous literature and the above results, compound 4 is 3- O -β-d-xylopyranosyl-(1→3)-β-d-glucuronopyranosyl-2β,3β,-dihydroxyolean-12-ene-23,28- It was identified as dioic acid-28- O -β-d-xylopyranosyl-(1→4)- α -l-rhamnopyranosyl-(1→2)-β-d-fucopyranoside and named as spinasaponin E. did β

In compound 5 , the molecular ion [M+H] ⁺ of m/z 1175.5487 was observed from the HR-Qtof-MS spectrum, and the molecular formula of C ₅₆ H ₈₆ O ₂₆ was determined. Through MS cleavage pattern analysis, a cleavage ion at m/z 1013 due to loss of glucose (162 Da) and a molecular ion at m/z 867 due to loss of rhamnose (146 Da), a pentose sugar, were observed. In addition, the molecular ion of m/z 679 due to the loss of acetylated fucose (188 Da) and the molecular ion of m/z 503 of sapogenin were detected due to the loss of glucuronic acid (176 Da). It is the same as the molecular ion value. As a result of comparative analysis of 1D and 2D NMR spectra and the preceding literature, compound 5 showed a similar spectrum when compared to compound 1 , and compound 5 had an acetyl group added to the backbone of compound 1 ( δ _H 4.94 3H, s , OAc-CH ₃ ; δ _C 171.3, OAc, 20.6, OAc-CH ₃ ). Through 2D NMR data, it was confirmed that the acetyl group was substituted at position 4 of the fucose sugar ( δ _C 74.5, C-4′′, Fuc), and the enomeric proton of the acetylated fucose sugar was ( δ _H 5.95 , 1H, d, J = 8.1 Hz, H-1′′, Fuc) It was confirmed that it was linked to the carbonyl group ( δ _C 176.6, C-28) of meticazenic. Therefore, by combining the previous literature and the above results, compound 5 is 3- O -β-d-glucuronopyranosyl-2β,3β,-dihydroxyolean-12-ene-23,28-dioic acid-28- O -β-d-glucopyranosyl It was identified as -(1→4)- α -l-rhamnopyranosyl-(1→2)-4- O -acetyl-β-d-fucopyranoside and named as spinasaponin F.

In compound 6 , a molecular ion [M+H] ⁺ of m/z 1307.5915 was observed from the HR-Qtof-MS spectrum, and the molecular formula of C ₆₁ H ₉₄ O ₃₀ was determined. Through MS cleavage pattern analysis, the molecular ion at m/z 1175 was detected due to the loss of xylose (132 Da), a pentose sugar, and the cleavage ion at m/z 1013 due to glucose loss (162 Da) and the A molecular ion of m/z 867 was observed due to the loss (146 Da), and a molecular ion of m/z 679 was detected due to the loss of acetylated fucose (188 Da). In addition, the molecular ion of sapogenin, m/z 503, was detected by the loss of glucuronic acid (176 Da), which is identical to the molecular ion value of medicagenic acid. As a result of comparative analysis of the 1D and 2D NMR spectra and the prior literature, compound 6 showed a similar spectrum when compared to compound 5 , and it was confirmed that a pentose sugar, xylose sugar, was additionally linked to compound 5 . Through 2D NMR data, the enomeric proton of xylose sugar ( δ _H 5.15, 1H, d, J = 7.2 Hz, H-1′′, Xyl) is linked to C-3′ of glucuronic acid ( δ _C 85.6, C-3', GlcA) was confirmed. Therefore, by combining the previous literature and the above results, compound 6 is 3- O -β-d-xylopyranosyl-(1→3)-β-d-glucuronopyranosyl-2β,3β,-dihydroxyolean-12-ene-23,28- It was identified as dioic acid-28- O -β-d-glucopyranosyl-(1→4)- α -l-rhamnopyranosyl-(1→2)-4- O -acetyl-β-d-fucopyranoside, spinasaponin G (spinasaponin G).

Compound 1 (SOA1) 3- O -β-d-glucuronopyranosyl-2β,3β,-dihydroxyolean-12-ene-23,28-dioic acid-28- O -β-d-glucopyranosyl-(1→4)- α -l-rhamnopyranosyl-(1→2)-β-d-fucopyranoside (Spinasaponin C)

[Formula 1]

1) Properties: Amorphous white crystals

2) Molecular weight: 1133.24

3) Molecular formula: C ₅₄ H ₈₄ O ₂₅

4) ¹ H-NMR (Pyridine- d ₅ , 900 MHz) δ _H 6.20 (1H, d, J = 1.8 Hz, H-1′′′, Rham), 5.91 (1H, d, J = 8.1 Hz, H -1′′, Fuc), 5.33 (1H, s, H-12), 5.13 (1H, d, J = 8.1 Hz, H-1′, GlcA), 5.06 (1H, d, J = 7.2 Hz, H -1′′′′, Glu), 4.74 (1H, d, J = 2.7 Hz, H-2), 4.63 (1H, d, J = 2.7 Hz, H-3), 3.02 (1H, dd, J = 2.7 Hz, H -3) 13.5, 3.6 Hz, H-18), 2.21 (1H, d, J = 12.6 Hz, H-1a), 1.89 (3H, s, H ₃ -24), 1.64 (3H, d, J = 6.3 Hz, H ₃ -6′′′, Rham), 1.45 (3H, s, H ₃ -25), 1.40 (3H, d, J = 6.3 Hz, H ₃ -6′′, Fuc), 1.19 (3H, s, H ₃ -27), 1.03 (3H, s, H ₃ -26), 0.79 (3H, s, H ₃ -30), 0.77 (3H, s, H ₃ -29); ¹³ C-NMR (Pyridine- d ₅ , 225 MHz) δ _C 44.1 (C-1), 70.0 (C-2), 85.7 (C-3), 52.6 (C-4), 52.3 (C-5), 20.9 (C-6), 32.7 (C-7), 40.1 (C-8), 48.4 (C-9), 36.5 (C-10), 23.7 (C-11), 122.4 (C-12), 143.8 (C-13), 42.0 (C-14), 28.0 (C-15), 23.1 (C-16), 46.8 (C-17), 41.8 (C-18), 46.0 (C-19), 30.4 ( C-20), 33.6 (C-21), 32.0 (C-22), 180.9 (C-23), 13.8 (C-24), 16.6 (C-25), 17.2 (C-26), 25.8 (C -27), 176.6 (C-28), 32.8 (C-29), 23.5 (C-30), 105.2 (C-1′, GlcA), 74.4 (C-2′, GlcA), 77.2 (C-3) ′, GlcA), 72.8 (C-4′, GlcA), 76.9 (C-5′, GlcA), 172.7 (C-6′, GlcA), 94.5 (C-1′′, Fuc), 74.1 (C- 2′′, Fuc), 75.8 (C-3′′, Fuc), 72.6 (C-4′′, Fuc), 72.1 (C-5′′, Fuc), 16.5 (C-6′′, Fuc) , 101.1 (C-1′′′, Rham), 71.2 (C-2′′′, Rham), 71.9 (C-3′′′, Rham), 84.5 (C-4′′′, Rham), 68.2 (C-5′′′, Rham), 18.3 (C-6′′′, Rham), 106.2 (C-1′′′′, Glu), 75.9 (C-2′′′′, Glu), 78.1 (C-3′′′′, Glu), 71.3 (C-4′′′′, Glu), 77.9 (C-5′′′′, Glu), 62.4 (C-6′′′′, Glu) .

Compound 2 (SOA2) 3- O -β-d-xylopyranosyl-(1→3)-β-d-glucuronopyranosyl-2β,3β,-dihydroxyolean-12-ene-23,28-dioic acid-28- O -β-d-glucopyranosyl-(1→4)- α -l-rhamnopyranosyl-(1→2)-β-d-fucopyranoside (Spinasaponin D)

[Formula 2 ]

1) Properties: Amorphous white crystals

2) Molecular weight: 1265.36

3) Molecular formula: C ₅₉ H ₉₂ O ₂₉

4) ^1H -NMR (Pyridine- d ₅ , 800 MHz) δH _6.36 (1H, s, H-1′′′′, Rham), 6.04 (1H, d, J = 8.0 Hz, H-1′′ ′, Fuc), 5.46 (1H, brs, H-12), 5.30 (1H, d, J = 7.2 Hz, H-1′′, Xyl), 5.28 (1H, d, J = 8.0 Hz, H- 1′, GlcA), 5.17 (1H, d, J = 8.0 Hz, H-1′′′′′, Glu), 4.84 (1H, brs, H-2), 4.77 (1H, brs, H- 3), 3.12 (1H, d, J = 13.6, 3.2 Hz, H-18), 2.26 (1H, d, J = 12.0 Hz, H-1a), 2.01 (3H, s, H ₃ -24), 1.70 (3H, d, J = 5.6 Hz, H ₃ -6′′′, Rham), 1.58 (3H, s, H ₃ -25), 1.48 (3H, d, J = 6.4 Hz, H ₃ -6′ ′′, Fuc), 1.26 (3H, s, H ₃ -27), 1.14 (3H, s, H ₃ -26), 0.90 (3H, s, H ₃ -30), 0.85 (3H, s, H ₃ -29); ¹³ C-NMR (Pyridine- d ₅ , 200 MHz) δ _C 44.9 (C-1), 71.0 (C-2), 86.8 (C-3), 53.3 (C-4), 53.1 (C-5), 21.6 (C-6), 33.4 (C-7), 40.8 (C-8), 49.0 (C-9), 37.3 (C-10), 24.3 (C-11), 123.1 (C-12), 144.3 (C-13), 42.7 (C-14), 28.6 (C-15), 23.8 (C-16), 47.3 (C-17), 42.5 (C-18), 46.6 (C-19), 31.1 ( C-20), 34.3 (C-21), 32.7 (C-22), 180.9 (C-23), 14.5 (C-24), 17.3 (C-25), 17.8 (C-26), 26.5 (C -27), 177.0 (C-28), 33.5 (C-29), 24.2 (C-30), 106.3 (C-1′, GlcA), 74.4 (C-2′, GlcA), 86.1 (C-3) ′, GlcA), 71.8 (C-4′, GlcA), 77.0 (C-5′, GlcA), 170.6 (C-6′, GlcA), 106.5 (C-1′′, Xyl), 75.8 (C- 2′′, Xyl), 78.4 (C-3′′, Xyl), 71.8 (C-4′′, Xyl), 67.7 (C-5′′, Xyl), 95.1 (C-1′′′, Fuc ), 74.7 (C-2′′′, Fuc), 76.8 (C-3′′′, Fuc), 73.5 (C-4′′′, Fuc), 72.7 (C-5′′′, Fuc), 17.2 (C-6′′′, Fuc), 101.8 (C-1′′′′, Rham), 72.1 (C-2′′′′, Rham), 72.8 (C-3′′′′, Rham) , 85.8 (C-4′′′′, Rham), 68.9 (C-5′′′′, Rham), 19.0 (C-6′′′′, Rham), 107.4 (C-1′′′”′ , Glu), 76.8 (C-2′′′′′, Glu), 79.2 (C-3′′′′′, Glu), 72.2 (C-4′′′′′, Glu), 78.8 (C- 5′′′′′, Glu), 63.3 (C-6′′′′′, Glu).

Compound 3 (SOA3) 3- O -β-d-glucuronopyranosyl-2β,3β,-dihydroxyolean-12-ene-23,28-dioic acid-28- O -β-d-xylopyranosyl-(1→4)- α -l-rhamnopyranosyl-(1→2)-β-d-fucopyranoside (Celosin I)

[Formula 3 ]

1) Properties: Amorphous white crystals

2) Molecular weight: 1103.22

3) Molecular formula: C ₅₃ H ₈₂ O ₂₄

4) ¹ H-NMR (Pyridine- d ₅ , 900 MHz) δ _H 6.41 (1H, br s, H-1′′′, Rham), 6.04 (1H, d, J = 8.1 Hz, H-1′′ , Fuc), 5.41 (1H, s, H-12), 5.22 (1H, br s, H-1′, GlcA), 5.03 (1H, d, J = 7.2 Hz, H-1′′′′, Xyl ), 4.85 (1H, m, H-2), 4.75 (1H, m, H-3), 3.12 (1H, d, J = 12.6 Hz, H-18), 2.31 (1H, br s, H-1a ), 2.00 (3H, s, H ₃ -24), 1.70 (3H, d, J = 6.3 Hz, H ₃ -6′′′, Rham), 1.57 (3H, s, H ₃ -25), 1.48 ( 3H, d, J = 5.4 Hz, H ₃ -6′′, Fuc), 1.26 (3H, s, H ₃ -27), 1.14 (3H, s, H ₃ -26), 0.89 (3H, s, H ₃ -30), 0.84 (3H, s, H ₃ -29); ¹³ C-NMR (Pyridine- d ₅ , 225 MHz) δ _C 44.9 (C-1), 70.7 (C-2), 86.7 (C-3), 53.3 (C-4), 52.9 (C-5), 21.6 (C-6), 33.4 (C-7), 40.8 (C-8), 49.0 (C-9), 37.2 (C-10), 24.3 (C-11), 123.1 (C-12), 144.3 (C-13), 42.7 (C-14), 28.6 (C-15), 23.7 (C-16), 47.4 (C-17), 42.4 (C-18), 46.6 (C-19), 31.1 ( C-20), 34.3 (C-21), 32.7 (C-22), 181.0 (C-23), 14.6 (C-24), 17.3 (C-25), 17.8 (C-26), 26.4 (C -27), 177.0 (C-28), 33.5 (C-29), 24.1 (C-30), 106.0 (C-1′, GlcA), 75.3 (C-2′, GlcA), 78.2 (C-3) ′, GlcA), 73.7 (C-4′, GlcA), 77.6 (C-5′, GlcA), 173.0 (C-6′, GlcA), 95.1 (C-1′′, Fuc), 74.4 (C- 2′′, Fuc), 77.0 (C-3′′, Fuc), 73.5 (C-4′′, Fuc), 72.7 (C-5′′, Fuc), 17.3 (C-6′′, Fuc) , 101.7 (C-1′′′, Rham), 71.3 (C-2′′′, Rham), 72.9 (C-3′′′, Rham), 85.8 (C-4′′′, Rham), 68.6 (C-5′′′, Rham), 18.9 (C-6′′′, Rham), 108.0 (C-1′′′′, Xyl), 76.6 (C-2′′′′, Xyl), 79.2 (C-3′′′′, Xyl), 72.2 (C-4′′′′, Xyl), 67.9 (C-5′′′′, Xyl).

Compound 4 (SOA4) 3- O -β-d-xylopyranosyl-(1→3)-β-d-glucuronopyranosyl-2β,3β,-dihydroxyolean-12-ene-23,28-dioic acid-28- O -β-d-xylopyranosyl-(1→4)- α -l-rhamnopyranosyl-(1→2)-β-d-fucopyranoside (Spinasaponin E)

[Formula 4 ]

1) Properties: Amorphous white crystals

2) Molecular weight: 1235.33

3) Molecular formula: C ₅₈ H ₉₀ O ₂₈

4) ¹ H-NMR (Pyridine- d ₅ , 900 MHz) δ _H 6.25 (1H, s, H-1′′′′, Rham), 5.91 (1H, d, J = 8.1 Hz, H-1′′ ′, Fuc), 5.34 (1H, brs, H-12), 5.16 (1H, d, J = 8.1 Hz, H-1′, GlcA), 5.15 (1H, d , J = 8.1 Hz, H-1 ′′, Xyl-I), 4.94 (1H, d, J = 7.2 Hz, H-1′′′′′, Xyl-II), 4.72 (1H, brs, H-2), 4.65 (1H, d , J = 3.6 Hz, H-3), 3.02 (1H, dd, J = 13.5, 3.6 Hz, H-18), 2.18 (1H, d, J = 12.6 Hz, H-1a), 1.88 (3H, s , H ₃ -24), 1.60 (3H, d, J = 6.3 Hz, H ₃ -6′′′′, Rham), 1.44 (3H, s, H ₃ -25), 1.40 (3H, d, J = 6.3 Hz, H ₃ -6′′′, Fuc), 1.19 (3H, s, H ₃ -27), 1.03 (3H, s, H ₃ -26), 0.78 (3H, s, H ₃ -30), 0.76 (3H, s, H ₃ -29); ¹³ C-NMR (Pyridine- d ₅ , 200 MHz) δ _C 44.1 (C-1), 70.1 (C-2), 85.7 (C-3), 52.6 (C-4), 52.4 (C-5), 20.9 (C-6), 32.7 (C-7), 40.1 (C-8), 48.4 (C-9), 36.5 (C-10), 23.7 (C-11), 122.4 (C-12), 143.8 (C-13), 42.1 (C-14), 28.0 (C-15), 23.0 (C-16), 46.8 (C-17), 41.8 (C-18), 46.0 (C-19), 30.4 ( C-20), 33.6 (C-21), 32.0 (C-22), 180.7 (C-23), 13.8 (C-24), 16.6 (C-25), 17.1 (C-26), 25.8 (C -27), 176.6 (C-28), 32.8 (C-29), 23.5 (C-30), 104.9 (C-1′, GlcA), 73.6 (C-2′, GlcA), 85.5 (C-3) ′, GlcA), 71.0 (C-4′, GlcA), 76.4 (C-5′, GlcA), 173.5 (C-6′, GlcA), 105.5 (C-1′′, Xyl-I), 74.8 ( C-2′′, Xyl-I), 77.3 (C-3′′, Xyl-I), 70.4 (C-4′′, Xyl-I), 66.7 (C-5′′, Xyl-I), 94.5 (C-1′′′, Fuc), 73.8 (C-2′′′, Fuc), 76.0 (C-3′′′, Fuc), 72.6 (C-4′′′, Fuc), 72.1 ( C-5′′′, Fuc), 16.6 (C-6′′′, Fuc), 101.0 (C-1′′′′, Rham), 71.2 (C-2′′′′, Rham), 71.9 ( C-3′′′′, Rham), 84.6 (C-4′′′′, Rham), 68.0 (C-5′′′′, Rham), 18.2 (C-6′′′′, Rham), 107.0 (C-1′′′′′, Xyl-II), 75.7 (C-2′′′′′, Xyl-II), 78.0 (C-3′′′′′, Xyl-II), 70.4 ( C-4′′′′′, Xyl-II), 67.0 (C-5′′′′′, Xyl-II).

compound 5 (SOA5) 3- O -β-d-glucuronopyranosyl-2β,3β,-dihydroxyolean-12-ene-23,28-dioic acid-28- O -β-d-glucopyranosyl-(1→4)- α -l-rhamnopyranosyl-(1→2)-4- O -acetyl-β-d-fucopyranoside (Spinasaponin F)

[Formula 5 ]

1) Properties: Amorphous white crystals

2) Molecular weight: 1175.28

3) Molecular formula: C ₅₆ H ₈₆ O ₂₆

4) ¹ H-NMR (Pyridine- d ₅ , 900 MHz) δ _H 6.13 (1H, s, H-1′′′, Rham), 5.95 (1H, d, J = 8.1 Hz, H-1′′, Fuc), 5.35 (1H, s, H-12), 5.14 (1H, d, J = 7.2 Hz, H-1′, GlcA), 5.07 (1H, d, J = 8.1 Hz, H-1′′′ ′, Glu), 4.74 (1H, brs, H-2), 4.64 (1H, brs, H-3), 3.02 (1H, dd, J = 13.5, 3.6 Hz, H-18), 2.22 (1H , d, J = 12.6 Hz, H-1a), 1.95 (3H, s, OAc-CH ₃ ), 1.90 (3H, s, H ₃ -24), 1.72 (3H, d, J = 6.3 Hz, H ₃ -6′′′, Rham), 1.46 (3H, s, H ₃ -25), 1.20 (3H, s, H ₃ -27), 1.16 (3H, d, J = 6.3 Hz, H ₃ -6′′ , Fuc), 1.02 (3H, s, H ₃ -26), 0.78 (3H, s, H ₃ -30), 0.77 (3H, s, H ₃ -29); ¹³ C-NMR (Pyridine- d ₅ , 225 MHz) δ _C 44.1 (C-1), 70.0 (C-2), 85.7 (C-3), 52.6 (C-4), 52.3 (C-5), 20.9 (C-6), 32.8 (C-7), 40.1 (C-8), 48.4 (C-9), 36.5 (C-10), 23.7 (C-11), 122.4 (C-12), 143.8 (C-13), 42.1 (C-14), 27.9 (C-15), 23.1 (C-16), 46.8 (C-17), 41.7 (C-18), 46.0 (C-19), 30.4 ( C-20), 33.6 (C-21), 32.1 (C-22), 180.9 (C-23), 13.8 (C-24), 16.7 (C-25), 17.2 (C-26), 25.8 (C -27), 176.6 (C-28), 32.8 (C-29), 23.5 (C-30), 105.2 (C-1′, GlcA), 74.4 (C-2′, GlcA), 77.1 (C-3) ′, GlcA), 72.8 (C-4′, GlcA), 77.1 (C-5′, GlcA), 172.0 (C-6′, GlcA), 94.3 (C-1′′, Fuc), 74.4 (C- 2′′, Fuc), 73.3 (C-3′′, Fuc), 74.5 (C-4′′, Fuc), 70.2 (C-5′′, Fuc), 16.2 (C-6′′, Fuc) , 171.3 (OAc), 20.6 (OAc-CH ₃ ), 101.5 (C-1′′′, Rham), 71.1 (C-2′′′, Rham), 71.8 (C-3′′′, Rham), 84.4 (C-4′′′, Rham), 68.4 (C-5′′′, Rham), 18.5 (C-6′′′, Rham), 106.2 (C-1′′′′, Glu), 75.8 (C-2′′′′, Glu), 78.1 (C-3′′′′, Glu), 71.3 (C-4′′′′, Glu), 78.1 (C-5′′′′, Glu) , 62.4 (C-6′′′′, Glu).

Compound 6 (SOA6) 3- O -β-d-xylopyranosyl-(1→3)-β-d-glucuronopyranosyl-2β,3β,-dihydroxyolean-12-ene-23,28-dioic acid-28- O -β-d-glucopyranosyl-(1→4)- α -l-rhamnopyranosyl-(1→2)-4- O -acetyl-β-d-fucopyranoside (Spinasaponin G)

[Formula 6 ]

1) Properties: Amorphous white crystals

2) Molecular weight: 1307.39

3) Molecular formula: C ₆₁ H ₉₄ O ₃₀

4) ¹ H-NMR (Pyridine- d ₅ , 900 MHz) δ _H 6.13 (1H, s, H-1′′′′, Rham), 5.94 (1H, d, J = 8.1 Hz, H-1′′ ′, Fuc), 5.34 (1H, s, H-12), 5.15 (1H, d, J = 7.2 Hz, H-1′′, Xyl), 5.16 (1H, d , J = 7.2 Hz, H-1 ′, GlcA), 5.06 (1H, d, J = 8.1 Hz, H-1′′′′′, Glu), 4.72 (1H, br s, H-2), 4.65 (1H, br s, H-3 ), 3.02 (1H, dd, J = 13.5, 2.7 Hz, H-18), 2.19 (1H, d, J = 12.6 Hz, H-1a), 1.94 (3H, s, OAc- _CH3 ), 1.89 ( 3H, s, H ₃ -24), 1.72 (3H, d, J = 6.3 Hz, H ₃ -6′′′′, Rham), 1.46 (3H, s, H ₃ -25), 1.20 (3H, s , H ₃ -27), 1.16 (3H, d, J = 6.3 Hz, H ₃ -6′′′, Fuc), 1.02 (3H, s, H ₃ -26), 0.81 (3H, s, H ₃ - 30), 0.77 (3H, s, H ₃ -29); ¹³ C-NMR (Pyridine- d ₅ , 225 MHz) δ _C 44.1 (C-1), 70.1 (C-2), 85.7 (C-3), 52.6 (C-4), 52.4 (C-5), 20.9 (C-6), 32.7 (C-7), 40.1 (C-8), 48.4 (C-9), 36.5 (C-10), 23.7 (C-11), 122.4 (C-12), 143.8 (C-13), 42.1 (C-14), 27.9 (C-15), 23.2 (C-16), 46.8 (C-17), 41.7 (C-18), 46.0 (C-19), 30.4 ( C-20), 33.6 (C-21), 32.0 (C-22), 180.7 (C-23), 13.8 (C-24), 16.6 (C-25), 17.2 (C-26), 25.8 (C -27), 176.6 (C-28), 32.8 (C-29), 23.5 (C-30), 105.0 (C-1′, GlcA), 73.6 (C-2′, GlcA), 85.6 (C-3) ′, GlcA), 73.3 (C-4′, GlcA), 75.7 (C-5′, GlcA), 173.9 (C-6′, GlcA), 105.5 (C-1′′, Xyl), 74.8 (C- 2′′, Xyl), 77.3 (C-3′′, Xyl), 70.4 (C-4′′, Xyl), 66.7 (C-5′′, Xyl), 94.3 (C-1′′′, Fuc ), 74.4 (C-2′′′, Fuc), 73.6 (C-3′′′, Fuc), 74.5 (C-4′′′, Fuc), 70.2 (C-5′′′, Fuc), 16.2 (C-6′′′, Fuc), 171.2 (OAc), 20.6 (OAc-CH ₃ ), 101.5 (C-1′′′′, Rham), 71.1 (C-2′′′′, Rham) , 71.8 (C-3′′′′, Rham), 84.4 (C-4′′′′, Rham), 68.4 (C-5′′′′, Rham), 18.4 (C-6′′′′, Rham), 106.2 (C-1′′′′′, Glu), 75.8 (C-2′′′′′, Glu), 78.0 (C-3′′′′′, Glu), 71.2 (C-4 ′′′′ ′, Glu), 77.9 (C-5′′′′′, Glu), 62.3 (C-6′′′′′, Glu).

Example 4: Cytotoxicity and whitening efficacy of spinach extracts and fractions in Melan A cells

Measurement of melan A cell proliferation by drugs for spinach extracts and fractions was performed using RPMI 1640 medium (Hyclon, USA) supplemented with 10% Fetal Bovine Serum (6Х10) to culture Melan A, a mouse melanocyte. After suspension at a concentration of ⁴ cells/ml, 100 μl of the suspension was inoculated into a 96-well plate, and the cells were cultured for 24 hours. After 24 hours, the culture medium was removed and cultured for 24 hours by replacing the medium with 200 nM of PMA (Sigma aldrich) in RPMI medium containing 10% FBS to induce melanin biosynthesis. In order to confirm the effect of the drug during melanin production, the medium was cultured for 2 days after treatment with the drug when the medium was exchanged. As described in the CCK-8 (Dojindo) kit for counting cells, 10 μl of the CCK-8 solution was mixed with 90 μl of the medium, and 100 μl per well was added. After reacting for at least 30 minutes to 1 hour, 450 Absorbance was measured in nm. Cell viability was calculated according to Equation 1 with the negative control treated with 0.1% DMSO as 100%, and the results are shown in the figure below.

[Equation 1]

Heating blok에서 15분간 멜라닌을 용출시켰으며 멜라닌 함량을 ELISA를 이용하여 405 nm에서 흡광도를 측정하였다. 멜라닌 생성량은 대조군을 100%로 하여 나타내었고, 그 결과를 도 5에 나타내었다.Measurement of melanin production in Melan A cells by drugs for spinach extracts, fractions and novel compounds was performed using RPMI 1640 medium (Hyclone, USA) was used to suspend the suspension at a concentration of 1Х10 ⁶ cells/ml, inoculate 1 ml each into a 12-well-plate, and culture the cells for 24 hours. After 24 hours, the culture medium was removed and cultured for 24 hours by replacing the medium with 200 nM of PMA (Sigma aldrich) in RPMI medium containing 10% FBS to induce melanin biosynthesis. In order to confirm the effect of the drug during melanin production, the medium was cultured for 2 days after treatment with the drug when exchanging the medium. After inducing melanin production, in order to measure melanin accumulation, the culture medium was removed, washed with PBS (Phosphate buffered saline, Hyclon), and treated with trypsin-EDTA to recover the cells into micro tubes. The recovered cells were added with 200 μl of 2N NaOH and

Melanin was eluted from the heating block for 15 minutes, and the absorbance at 405 nm was measured for the melanin content using ELISA. The amount of melanin produced was shown as 100% in the control group, and the results are shown in FIG. 5 .

Example 5: Cytotoxicity and whitening efficacy of spinach extracts, fractions and 5 new compounds in B16F10 cells

In the present invention, in order to evaluate the cytotoxicity of the spinach extract, the effective fraction, and the novel compounds prepared in Example 2, B16F10 cells were treated at concentrations of 0, 5, 10, 25, 50, and 100 μg/mL to determine the cell viability through cell viability. Cytotoxicity was evaluated. Cell viability was analyzed using the WTS kit (ez-cytox, Dogen). B16F10 cells were dispensed in a 96 well plate at a concentration of 1 x 10 ⁴ cell, cultured for 24 hours, and each spinach extract was treated by concentration and cultured for 24 hours. After incubation, the medium was removed, 90 μL of the medium and 10 μL of the WTS solution were added, and after 1 hour, the absorbance was measured at 450 nm using a microplate reader. As a result, as shown in Figure 6, it was confirmed that the spinach extract prepared in the present invention did not show cytotoxicity in the concentration range of 100 μg / mL or less.

에서 가열하여 세포 내의 melanin을 확보하였다. E-tube에서 96 well plate로 옮긴 후 microplate reader기를 사용하여 405 nm에서 흡광도를 측정하였다. 양성대조군으로는 200 μM PTU(phenylthiourea)를 사용하였다.In order to examine the effect of spinach extract, effective fractions, and novel compounds on melanin biosynthesis, B16F10 cells were treated at the same concentration as the cytotoxic concentration, and the intracellular melanin content was measured. B16F10 cells were divided into 2 x 10 ⁴ cells per well in a 24 well plate, and after 24 hours, the culture medium was exchanged with a medium containing spinach extract with 200 μM of IBMX by concentration, followed by culture for 72 hours. After 72 hours, the medium was removed, washed three times with DPBS, treated with trypsin, and the cells were collected in an e-tube. The recovered cells were incubated at 70 °C for 60 minutes after adding 100 μL of 1N NaOH.

was heated to secure intracellular melanin. After transferring from the E-tube to a 96 well plate, the absorbance was measured at 405 nm using a microplate reader. As a positive control, 200 μM PTU (phenylthiourea) was used.

The results for this are shown in FIG. 6 .

Example 6: Skin inflammation improvement efficacy of spinach extracts, fractions and 5 new compounds

Cytotoxicity tests were conducted on mouse macrophage RAW 264.7 cells (American Type culture collection) for spinach extracts, fractions and novel compounds. RAW 264.7 cells were cultured in DMEM medium (HyClone) supplemented with 10% fetal bovine serum (Gibco) and antibiotics (Penicillin 100 units/ml + 100 μg/ml) at a concentration of 1x10 ⁵ cells/ml in a 96 well plate at 100 μl. Inoculated and cultured for 16 hours. Then, after culturing in serum-free DMEM medium for 16 hours, spinach extracts and fractions were treated at concentrations of 5, 10, and 20 μg/ml. After 2 hours of reaction, LPS (Sigma) was added at a concentration of 100 ng/ml and cultured for 24 hours. As described in the CCK-8 kit (Dojindo), 10 μl of the CCK-8 solution and 90 μl of the medium were mixed, added to the cells, reacted for 1 hour, and absorbance was measured at 450 nm.

Inhibition of the production of TNF-α and IL-6 induced by LPS in RAW264.7 mouse macrophages was evaluated by spinach extracts and fractions. RAW 264.7 cells were inoculated into a 96 well plate by 100 μl at a concentration of 1×10 ⁵ cells/ml in DMEM medium supplemented with 10% fetal bovine serum. After culturing for 16 hours, the medium was replaced with serum-free DMEM medium and cultured for 16 hours. Spinach extracts and fractions were treated at concentrations of 5, 10, and 20 μg/ml for 2 hours, then LPS (Sigma) was added at a concentration of 100 ng/ml and cultured for 6 hours. After recovering 100 μl of the supernatant, the experiment was conducted by the method described in the mouse TNF ELISA set (BD) and mouse IL-6 ELISA set (BD) instructions.

Inhibition of nitric oxide (NO) production induced by LPS in mouse macrophage RAW264.7 was evaluated by spinach extracts and fractions. For cell preparation, mouse macrophage RAW264.7 cells were dispensed into a 96 well plate at a concentration of 1x10 ⁶ cells/ml in a DMEM medium supplemented with 10% fetal bovine serum, and adhered for 16 hours. It was replaced with an untreated DMEM medium and cultured for 3 hours. Spinach extracts and fractions were treated with 5, 10, and 20 μg/ml for 2 hours at each concentration, and then treated with LPS (Sigma) at a concentration of 100 ng/ml and cultured for 24 hours. After taking 100 μl of the cell supernatant and transferring it to a 96 well plate, Griess reagent ° solution, which is a mixture of N-(1-naphthyl) ethylenediamine dihydrochloride (Sigma) and sterile water, and Sulfanilamide (Sigma), Phosphoric acid (Sigma) and sterile water are mixed. Griess reagent ± solution was mixed 1: 1 and added by 100 μl. After 10 minutes of reaction, absorbance was measured at 540 nm.

The results of this are shown in FIG. 7 .

Example 7: Verification of anti-whitening efficacy through inhibition of tyrosinase production by spinach fraction (Fr.2)

In order to evaluate the tyrosinase production inhibitory activity of the spinach effective fraction (SO Fr.2) according to the present invention, the following experiment was performed.

In order to examine the inhibitory activity of spinach fractions on tyrosinase production of the present invention, B16F10 melanoma cells were added to 0.8 × 10 ⁵ cells in 2 ml each in a 6-well plate and cultured for 24 hours in a 37°C, CO ₂ incubator. After culturing, the medium was removed, and the spinach fraction was treated with the normal culture medium and the medium containing IBMX 200 νM at concentrations of 10, 25, and 50 νg/ml, and cultured for 72 hours under the same culture conditions. After culturing for 72 hours, a tyrosine-EDTA solution was added to recover the cells in each plate, the cells were separated, collected in a microtube, and centrifuged to remove the supernatant. After washing the separated pellet with PBS and centrifuging, the pellet was treated with PRO-PREP TM Protein Extract Solution (Intron, Inc.) and reacted at -20 ° C for 15 minutes. After centrifugation, the supernatant was transferred to a tube and stored at -20 °C. The protein purified by the above method was subjected to electrophoresis using 12% SDS-PAGE and transferred to a PVDF membrane. After going through a blocking process for 1 hour in a Tris buffer solution containing 5% skim milk, it was reacted with a tyrosinase antibody. After the reaction, anti-mouse IgG-HRP-conjugated antibody was added, reacted for a certain period of time using ECL Western blotting Substrate (Thermo), and then developed. As a result, tyrosinase expression was suppressed from the spinach fraction at a concentration of 10 νg/ml. Therefore, it was confirmed that the expression of enzymes involved in the melanin production step was suppressed in the spinach fraction.

A fraction of spinach extract containing 5 new glycoside compounds and celosin I alleviates inflammatory reactions in skin cells, and exhibits excellent skin whitening, skin wrinkle improvement, antioxidant, skin aging improvement and moisturizing effects. Therefore, it can be used very usefully for the purpose of improving skin conditions, including inflammatory skin diseases, and thus has high industrial applicability.

Claims (7)

A composition for skin whitening, skin wrinkle improvement, skin moisturizing, skin aging improvement, antioxidation and skin inflammation improvement comprising a compound represented by Formulas 1 to 6 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

The composition according to claim 1, wherein the compounds represented by Formulas 1 to 6 are isolated from spinach.
The composition according to claim 1, wherein the composition is a pharmaceutical, cosmetic, food, veterinary or feed composition.
A composition for skin whitening, skin wrinkle improvement, skin moisturizing, skin aging improvement, antioxidant and skin inflammation improvement comprising a spinach fraction prepared according to a method comprising the following steps as an active ingredient:
(a) extracting spinach with a solvent selected from the group consisting of water, organic solvents, subcritical fluids, supercritical fluids, and mixtures thereof; and
(b) obtaining fractions containing the compounds of Formulas 1 to 6 by fractionating the spinach extract by chromatography.
[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

The method of claim 4, wherein the organic solvent in step (a) is alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, or ethyl acetate. ), a composition characterized in that it is selected from the group consisting of methylene chloride, hexane, cyclohexane and petroleum ether.
The composition according to claim 4, wherein the chromatography in step (b) is performed by sequentially developing water and a non-polar solvent as a mobile phase according to a concentration gradient.
[Claim 7] The composition according to claim 6, wherein in step (b), a fraction to be fractionated in an aqueous solution of 20 to 100% (v/v) of a non-polar solvent is obtained.

Images