KR20220112330A - Composition for improving skin whitening comprising fermented product of Momordica charantia or compound isolated therefrom as effective componenet - Google Patents

Abstract

The present invention relates to a composition for skin whitening containing a fermented product of Momordica charantia or a compound isolated therefrom as an active component. The fermented product of Momordica charantia or a novel compound isolated from the same has excellent tyrosinase inhibitory activity and melanin biosynthesis inhibition rate, so that it can be usefully used as a cosmetic or food for improving skin whitening or a therapeutic material for the treatment of melanin pigmentation disease.

Description

Composition for improving skin whitening comprising fermented product of Momordica charantia or compound isolated therefrom as effective componenet

The present invention relates to a skin whitening composition comprising a fermented bitter melon or a compound isolated therefrom as an active ingredient.

Melanin is a major factor that determines the color of hair and skin, and serves to protect skin cells from UV rays, but excessive melanin accumulation causes hyperpigmentation such as freckles or freckles. Melanin-forming cells present in the basal layer of the epidermis have a specific structure called melanosomes, where melanin is produced by the action of melanogenic enzymes. Melanin includes black and brown eumelanin and yellow and red pheomelanin, and the synthesis of melanin is tyrosine by tyrosinase and DOPA and dopa. It starts with conversion to DOPA quinone.

In addition, TRP-2 (tyrosinase-related protein 2) is dopa chrome (DOPA chrome) to 5,6-dihydroxyindole-2-carboxylic acid (5,6-dihydroxyindole-2-carboxylic acid), TRP-1 is Catalyzes the oxidation reaction with indole-5,6-quinone-2-carboxylic acid, tyrosinase and TRP-1 and TRP-2 for eumelanin synthesis is involved In addition, microphthalmia-associated transcription factor (MITF), which is well known as a regulator of melanin formation, is a transcription factor with a helix-loop-helixleucine zipper structure. It is well known to regulate proliferation and survival. Factors involved in melanin biosynthesis include α-MSH (α-melanocyte stimulating hormone), adrenocorticotropic hormone, and PGE2 (prostaglandine E2), and it has been reported to regulate melanin synthesis through cAMP. Therefore, studies related to the inhibition of the activity of major factors of melanin production in relation to skin whitening are being actively conducted.

Kojic acid, vitamin derivatives, arbutin, and dibutylated hydroxytoluene (BHT), which are currently used as synthetic antioxidants, have a whitening effect due to their low price and excellent antioxidant activity. and its consumption is regulated. Therefore, research to discover materials of functional ingredients from natural products is being actively conducted domestically and internationally in order to develop materials with biochemical efficacy due to the development of the research level and the increase in technology.

On the other hand, Korean Patent Application Laid-Open No. 2016-0016195 discloses 'An antioxidant or anti-aging composition comprising an ethyl acetate fraction fractionated from bitter melon extract as an active ingredient', and Korean Patent Publication No. 2015-0094832 discloses skin aging prevention, Although a 'cosmetic composition containing bitter melon fruit extract as an active ingredient and a method for producing the same,' which has antioxidant, skin wrinkle improvement, or skin whitening effects, the hot water extract of the present invention is fermented with a mushroom mycelium There is no description of 'a composition for skin whitening containing fermented bitter melon or a compound isolated therefrom' as an active ingredient.

The present invention has been derived from the above needs, and the present inventors have prepared a fermented bitter gourd fermented by adding a filtrate of a pulverized mushroom to a bitter gourd hot water extract and a compound isolated from the fraction of the fermented bitter gourd (Formulas 1 to 3). By confirming that the tyrosinase (tyrosinase) inhibitory activity and melanin biosynthesis inhibition rate were excellent, the present invention was completed.

In order to solve the above problems, the present invention provides a compound of Formula 1 or an acceptable salt thereof; a compound of formula 2 or an acceptable salt thereof; a compound of formula 3 or an acceptable salt thereof; Or a fermented bitter melon comprising at least one compound selected from Formulas 1 to 3; as an active ingredient, it provides a cosmetic composition for skin whitening.

In addition, the present invention relates to a compound of Formula 1 or an acceptable salt thereof; a compound of formula 2 or an acceptable salt thereof; a compound of formula 3 or an acceptable salt thereof; Or a fermented bitter melon comprising at least one compound selected from Formulas 1 to 3; as an active ingredient, it provides a health functional food composition for skin whitening.

In addition, the present invention relates to a compound of Formula 1 or an acceptable salt thereof; a compound of formula 2 or an acceptable salt thereof; a compound of formula 3 or an acceptable salt thereof; Or a fermented bitter melon containing one or more compounds selected from Formulas 1 to 3; provides a pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease containing as an active ingredient.

The fermented bitter melon or the novel compound isolated therefrom of the present invention has excellent tyrosinase inhibitory activity and melanin biosynthesis inhibition rate, so it is useful as a cosmetic or food for improving skin whitening or as a material for treatment of melanin pigmentation diseases. It is expected that it can be used.

1 is a result of High Performance Liquid Chromatography (HPLC) performed on an ethyl acetate fraction (EtOAc) of a bitter gourd hot water extract and a fermented bitter melon extract. Red arrow: APM-1 compound (t _R 11.6 min), APM-2 compound (t _R 9.2 min) and APM-4 compound (t _R 7.3 min) isolated in the EtOAc fraction.
2 is a hot water extract of bitter gourd, fermented bitter melon, fractions of fermented bitter melon (n-Hexane layer, EtOAc layer, n-BuOH layer, H ₂ O layer) or compounds isolated therefrom (APM-1, APM-2, APM It is the result of confirming the cytotoxicity of -4). Arbutin: positive control.
Figure 3A is a result of confirming the intracellular tyrosinase inhibitory activity of bitter gourd hot water extract, bitter gourd fermented product or fractions of bitter gourd fermented product (n-Hexane layer, EtOAc layer, n-BuOH layer, H ₂ O layer); Figure 3B is the result of confirming the intracellular tyrosinase inhibitory activity of APM-1 (Formula 1), APM-2 (Formula 2) or APM-4 (Formula 3) compounds isolated from the ethyl acetate fraction (EtOAc) of fermented bitter gourd. to be. Nor: UV non-irradiated group (no sample treatment). Cont: UV irradiation group (no sample treatment), α-MSH: α-Melanocyte-stimulating hormone, Arbutin (arbutin): positive control group.
Figure 4A is the result of confirming the inhibition of melanin biosynthesis in cells of the hot water extract of bitter melon, fermented bitter melon or fractions of fermented bitter melon (n-Hexane layer, EtOAc layer, n-BuOH layer, H ₂ O layer), Fig. 4B is fermentation of bitter melon It is a result of confirming the inhibition of melanin biosynthesis in cells of APM-1 (Formula 1), APM-2 (Formula 2) or APM-4 (Formula 3) compounds isolated from the ethyl acetate fraction of water (EtOAc). Nor: UV non-irradiated group (no sample treatment). Cont: UV irradiation group (no sample treatment), α-MSH: α-Melanocyte-stimulating hormone, Arbutin (arbutin): positive control group.
Figure 5 is melanoma cell line B16F10 cells involved in melanin synthesis after treatment with APM-2 (Formula 2) compound isolated from the ethyl acetate fraction (EtOAc) of fermented bitter gourd MITF (microphthalmia-associated transcription factor), TRP-1 (tyrosinase related protein 1), TRP2, and the result of confirming the expression levels of tyrosinase. Nor: UV non-irradiated group (no sample treatment). Cont: UV irradiation group (no sample treatment), α-MSH: α-Melanocyte-stimulating hormone, Arbutin (arbutin): positive control group.

In order to achieve the object of the present invention, the present invention provides a compound of Formula 1 or an acceptable salt thereof; A compound of Formula 2 or an acceptable salt thereof; A compound of Formula 3 or an acceptable salt thereof; Or a fermented bitter melon comprising at least one compound selected from the following Chemical Formulas 1 to 3; provides a cosmetic composition for skin whitening containing as an active ingredient.

In the cosmetic composition of the present invention, the compounds of Formulas 1 to 3 may be isolated from fermented bitter melon, preferably separated from the ethyl acetate fraction (Ethly acetate, EtOA) of fermented bitter melon. It is not limited, and it is possible to extract or synthesize it from other natural products.

In the cosmetic composition of the present invention, the compound represented by Formula 1 is a Monaspilosuslin compound having a molecular formula of C ₁₂ H ₈ O ₆ .

In the cosmetic composition of the present invention, the fermented bitter gourd may be a fermented bitter melon ( Agaricus bisporus ) mycelium, but is not limited thereto. The mushroom mycelium may refer to a filtrate obtained by filtering a mixture of a mushroom pulverized product and distilled water, and the filtrate contains polyphenol oxidase.

The fermented bitter melon extract of the present invention, a fraction thereof, or a compound isolated therefrom has excellent tyrosinase inhibitory activity and melanin biosynthesis inhibitory rate, and thus is effective in improving skin whitening.

When the fermented bitter gourd or its fraction of the present invention is treated at a concentration of 50 to 100 μg/ml, or the compound (Formulas 1 to 3) isolated from the fraction of the fermented bitter melon is treated at a concentration of 20 to 30 uM, intracellular tyrosinase Inhibitory activity, melanin biosynthesis inhibition rate, MITF (microphthalmia-associated transcription factor) involved in melanin synthesis, TRP-1 (tyrosinase related protein 1), TRP2 and the effect of reducing the expression level of Tyrosinase is excellent.

As used herein, the "acceptable salt thereof" refers to any salt of a compound of the present invention that is not toxic or unsuitable for pharmaceutical use while retaining the biological properties of the compound of the present invention. Such salts may be derived from, and include, various organic and inorganic counterions known in the art. Such salts include organic or inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, glutaric acid, Pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, chromic acid, cinnamic acid, mandelic acid, phthalic acid, Lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphoric acid, camphor Porsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfate, gluconic acid , acid addition salts formed with glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, cyclohexylsulfamic acid, quinic acid or muconic acid; or an acidic proton present in the parent compound is a metal ion (eg alkali metal ion, alkaline earth metal ion or aluminum ion), alkali metal or alkaline earth metal hydroxide (eg sodium, potassium, calcium, magnesium, aluminum, lithium, zinc) and barium hydroxide), ammonia, or an organic base such as an aliphatic, cycloaliphatic or aromatic organic amine such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanol Amine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine piperazine , tris(hydroxymethyl)-aminomethane or a salt formed when coordinated with tetramethylammonium hydroxide, and the like.

Also, examples of salts include sodium, potassium, calcium, magnesium, ammonium or tetraalkylammonium salts, and if the compound contains a basic functional group, salts with non-toxic organic or inorganic acids, such as hydrohalides (e.g., hydrohalides chloride or hydrobromide), sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, ru Bate, lactate, malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate, tartrate, citrate, benzoate, 3-(4-hydroxybenzoyl fumarate, tarta) late, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate mesylate), ethanesulfonate, 1,2-ethane -disulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, camphorsulfonate, 4- Methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate, gluconate, benzoate ate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylsulfamate, quinate or muconate salts and the like.

The cosmetic composition of the present invention is preferably in any one formulation selected from solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, foundation and spray, more preferably is skin, skin softener, skin toner, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, eye cream, moisture cream, hand cream, essence, nourishing essence, pack, cleansing foam, cleansing water, cleansing It may have any one formulation selected from cream, body lotion, body cleanser, soap and powder, but is not limited thereto. The cosmetic composition composed of each of these formulations may contain various carriers and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.

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

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

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

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

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

The cosmetic composition of the present invention may further contain excipients including fungicides, hydrotropes, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, and the like.

The present invention also provides a compound of Formula 1 or an acceptable salt thereof; a compound of Formula 2 or an acceptable salt thereof; a compound of Formula 3 or an acceptable salt thereof; Or a fermented bitter melon containing one or more compounds selected from Formulas 1 to 3; provides a health functional food composition for skin whitening containing as an active ingredient.

In the health functional food composition of the present invention, the compounds of Formulas 1 to 3 are isolated from the fraction of the fermented bitter melon, and the fermented bitter melon or the compound isolated therefrom is as described above.

As used herein, the term 'whitening' refers to suppressing, inhibiting or alleviating hyperpigmentation of the skin. Hyperpigmentation of the skin includes freckles, melasma, hyperpigmentation after UV exposure, hyperpigmentation after inflammation, age spots, brown spots or age spots, and the like.

The health functional food composition of the present invention contains the compound of the present invention having excellent tyrosinase inhibitory activity and melanin biosynthesis inhibitory rate or a fermented bitter melon containing the compound as an active ingredient, and thus is effective in improving skin whitening.

The health functional food composition of the present invention has the advantage of having a more excellent effect by ingesting it in the form of inner beauty food. The inner beauty is a food called 'eating cosmetics or beauty food'. It refers to a food that absorbs various ingredients good for the skin into the body and changes the skin constitution to make it healthy. Considering your lifestyle, you can choose and consume inner beauty food that suits you. When a cosmetic containing the above-described cosmetic composition and an inner beauty food containing a compound of the present invention or a fermented bitter melon containing the compound are mixed, the skin whitening improvement effect is significantly increased compared to using only cosmetics. It has the advantage of seeing a whitening improvement effect.

When the compound of the present invention or a fermented bitter melon containing the compound is used as a food additive, the compound of the present invention or a fermented bitter melon containing the compound may be added as it is or used together with other foods or food ingredients, It can be used suitably according to a conventional method. The mixed amount of the active ingredient may be appropriately used depending on the purpose of its use (prevention or improvement). In general, in the production of food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term intake for health purposes, the amount may be less than or equal to the above range.

There is no particular limitation on the type of the food. Examples of foods to which the health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea There are drinks, alcoholic beverages, vitamin complexes, etc., and includes all health foods in the ordinary sense. The beverage includes carbonated beverages, functional ionic beverages, juices (eg, apple, pear, grape, aloe, tangerine, peach, carrot, tomato juice, etc.), sikhye, and the like.

The functional food of the present invention includes ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrin, cyclodextrin, etc.) or sugar alcohols (eg, , xylitol, sorbitol, erythritol, etc.). As the flavoring agent, natural flavoring agents (eg, taumatine, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used. In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonated beverages It may further contain a carbonation agent, etc. used for

In addition to the health functional food composition, various nutrients, vitamins, inhibitors, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, It may further contain a carbonation agent, etc. used in carbonated beverages. The ratio of these added ingredients is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition of the present invention.

The present invention also provides a compound of Formula 1 or an acceptable salt thereof; a compound of Formula 2 or an acceptable salt thereof; a compound of Formula 3 or an acceptable salt thereof; Or a fermented bitter melon containing one or more compounds selected from Formulas 1 to 3; provides a pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease containing as an active ingredient.

In the pharmaceutical composition of the present invention, the compounds of Formulas 1 to 3 are isolated from the fraction of the fermented bitter melon, and the fermented bitter melon or the compound isolated therefrom is as described above.

As used herein, the term "melanin hyperpigmentation (hyperpigmentation)" refers to darkening or darkening compared to other areas due to excessive increase in melanin in a specific area of the skin or nails. The disease of melanin hyperpigmentation includes freckles, age spots, liver spots, melasma, brown or dark spots, solar pigmentation, cyanic melasma, hyperpigmentation after drug use, gravidic chloasma, or abrasions and post-inflammatory hyperpigmentation due to wounds including burns or dermatitis, and the like.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient, and such carriers are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, Calcium Phosphate, Alginate, Gelatin, Calcium Silicate, Microcrystalline Cellulose, Polyvinylpyrrolidone, Cellulose, Water, Syrup, Methyl Cellulose, Methylhydroxybenzoate, Propylhydroxybenzoate, Talc, Magnesium Stearate and Minerals oils and the like, but are not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. When adding the above ingredients, skin safety according to complex use, ease of formulation, and stability of active ingredients can be considered.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by topical application to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like. Considering that the pharmaceutical composition of the present invention is applied to treat or prevent melanin hyperpigmentation disease, it is preferable that it is applied topically to the skin.

The pharmaceutical composition of the present invention may be prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. It may be prepared by introduction into a dose container. At this time, the formulation may be prepared in any one formulation selected from injections, creams, patches, sprays, ointments, warning agents, lotions, liniments, pastas, and cataplasmas, and may additionally include a dispersant or stabilizer. have.

When the pharmaceutical composition of the present invention contains an effective amount of the compound (Formulas 1 to 3) or a fermented bitter gourd containing the compound, it is possible to provide a desirable skin whitening effect. In the present invention, the term 'effective amount' refers to an amount of a compound capable of inhibiting pigmentation or an amount of fermented bitter gourd containing the compound. The effective amount of the compound included in the composition of the present invention or the bitter gourd fermented product containing the compound will vary depending on the form in which the composition is commercialized, the method in which the compound or the bitter gourd fermented product containing the compound is applied to the skin, and the length of time it stays on the skin. . A preferred dosage of the compound of the present invention or a pharmaceutical composition comprising the fermented bitter gourd containing the compound as an active ingredient varies depending on the patient's condition and weight, the degree of disease, drug form, administration route and period, but can be determined by those skilled in the art. may be appropriately selected.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Preparation Example 1. Preparation of fermented bitter melon

4.5 kg of dried bitter gourd purchased from Dusonae Herb Co., Ltd. was immersed in 45 L of water, extracted at 90° C. for 3 hours, and concentrated under reduced pressure at low temperature to obtain 1.5 kg of hot water extract of bitter gourd. In addition, 40 g of Agaricus bisporus was mixed with 100 ml of distilled water for the enzymatic reaction of Yeoju hot water extract, crushed with a blender, and filtered with 5-layer gauze to prepare an enzyme source containing polyphenol oxidase. . To measure the polyphenol oxidase activity of the prepared enzyme source (60 ml), the content of benzotropolone produced after the oxidation reaction using pyrogallol as a substrate was measured at 360 nm with a UV spectrophotometer. The absorbance was measured, and an enzyme source with an absorbance value of 0.78 to 0.79 was used for the enzymatic reaction of the hot water extract of bitter melon. The absorbance was measured after putting 1.5 ml of the enzyme source in a quartz cell together with 1.5 ml of 200 mM sodium phosphate buffer (in 50 mM sodium phosphate buffer, pH 7.2) and inducing an oxidation reaction for 5 minutes. In the blank group, 1.5 ml of the substrate solution and 1.5 ml of distilled water were added (refer to Food Chemistry, 88, 2004, 69-77 and BBRC, 130(2), 1985, 633-639).

Then, 15 g of the hot water extract of bitter gourd was diluted in 40 ml of distilled water, put in a 500 ml beaker together with 60 ml of the enzyme source, and reacted at room temperature for 80 minutes using a stirrer, and then concentrated under reduced pressure to prepare a fermented bitter melon. .

Example 1. Isolation of compounds of formulas 1 to 3 from fermented bitter gourd

The enzyme reaction experiment of bitter gourd was repeated 36 times, and 750 g of the produced fermented bitter melon (MeOH) was diluted in 2 ℓ of 10% methanol (MeOH), and then n-hexane (hexane, 2 ℓ × 3 times), ethyl acetate (EtOAc, After fractionation and extraction using 2 ℓ × 3 times) and n-butanol (BuOH, 2 ℓ × 3 times), the fractions for each solvent were concentrated under reduced pressure and dried to dry 0.2 g of n-hexane fraction, 3.3 g of EtOAc fraction, 17.3 g of n-BuOH fraction and 540.9 g of H ₂ O fraction were obtained.

Then, it has excellent tyrosinase inhibitory activity and melanin biosynthesis inhibition rate. 2.3 g of the soluble part of the EtOAc fraction was subjected to column chromatography while increasing the MeOH content of H ₂ O/MeOH using a Toyopearl HW-40 column (2.5 cm id × 42 cm) to obtain EMC01-EMC06 small fractions. APM-1 (t _R 11.6 min, 19.7 mg) and APM-2 from 171.4 mg of small fraction EMC02 by performing column chromatography using YMC Pack ODS A-302 column (4.6 cm id × 150 mm, 5 μm) as a filler. (t _R 9.2 min, 16.3 mg) A compound was obtained, and APM-4 (t _R 7.3 min, 13.4 mg) was obtained from a small fraction EMC01 using the same method as above (FIG. 1). For mobile phase, 0.1% formic acid (solvent A) and acetonitrile (solvent B) were used, and gradient elution was analyzed with 100% solvent A for 30 min, 0% A; The material was analyzed with a solvent composition of 100% B, and the flow rate of the mobile phase was maintained at 1.0 mL/min to detect the compound at 280 nm (Table 1).

Example 2. Structural determination of compounds of formulas 1 to 3 isolated from fermented bitter gourd

The compound isolated from the ethyl acetate fraction of the fermented bitter melon was determined as the Monaspilosuslin compound represented by Formula 1 and the novel compounds represented by Formulas 2 and 3 through spectrochemical and structural analysis.

2-1. Formula 1 (Monaspilosuslin)

Compound 1 was obtained in the form of a brown amorphous powder, and the molecular weight was estimated by checking the base peak of m/z 323 [M+Na] ⁺ as a result of FABMS measurement. In the ¹ H NMR spectrum, signals corresponding to trans -olefins were measured at δ 7.85 (1H, d, J = 15.6 Hz, H-8) and 6.23 (1H, d, J = 15.6 Hz, H-7), and one A singlet proton corresponding to a canine double bond was observed at δ 5.77 (1H, s, H-10). Also one oxygenated methine δ 4.09 (1H, m, H-3), one hydroxymethyl group δ 3.70 (1H, d, J = 7.2 Hz, H-13), 3.63 (1H, d, J = 7.2 Hz, H -13), two methylene groups δ 2.00 (1H, m, H-2), 1.80 (1H, m, H-4), 1.78 (1H, m, H-2), 1.77 (1H, m, H- 4) was observed, and three methyl groups were observed at δ 1.96 (3H, s, H-14), 1.08 (3H, s, H-12), and 0.86 (3H, s, H-11). In ¹³ C NMR and HSQC spectra, one carbonyl group was observed at δ 173.3 (C-15), and signals such as trans -olefin, oxygenated methine, hydroxymethyl, and methyl were observed. As a result of 2D NMR measurement of ¹ H- ¹ H COSY, HMBC and NOESY, the structure of Compound 1 was determined as a monaspilosuslin compound, a sequiterpene compound isolated from rice fermented using Monascus pilosus microorganisms.

2-2. Formula 2 (New)

Compound 2 was obtained in the form of a yellow, amorphous powder, and showed maximum absorbance at 227 and 291 nm in the UV spectrum, suggesting that it is a γ-lactone skeleton. The molecular weight was estimated by confirming the base peak of m/z 143.0344 [M+H] ⁺ in the HRFABMS spectrum, and the molecular formula was confirmed as C ₆ H ₇ O ₄ . In the 1H NMR spectrum, a proton signal corresponding to one double bond was observed at δ 7.91 (1H, s, H-6), and one methyl group was observed at δ 2.27 (3H, s, H-7). In ¹³ C NMR and HSQC spectra, one carbonyl carbon signal was observed at δ 168.5 (C-2), and one methyl group was observed at δ 13.8 (C-7) along with five double-bonded carbon signals. In the key HMBC spectrum, H-6 showed correlation with C-4 and 5, and H-7 showed HMBC correlation with C-2, 3, and 4. As a result of the above results, Compound 2 was identified as a novel compound not previously reported, and the structure was determined as a tetronic acid derivative compound.

2-3. Formula 3 (New)

Compound 3 was also obtained in the form of a yellow amorphous powder, and showed maximum absorbance at 226 and 290 nm in the UV spectrum, suggesting that it is a γ-lactone skeleton. The molecular weight was estimated by confirming the base peak of m/z 1145.0499 [M+H] ⁺ in the HRFABMS spectrum, and the molecular formula was identified as C ₆ H ₉ O ₄ . In the ¹ H NMR spectrum, one oxygenated proton signal was observed at δ 4.18 (1H, dd, J = 9.6, 4.2 Hz, H-5), and one hydroxymethyl group was observed at δ 4.32 (1H, dd, J = 11.4, 4.2 Hz). , H-4), 4.09 (1H, dd, J = 11.4, 9.6 Hz, H-6), and one methyl group was observed at δ 1.98 (3H, s, H-7). In ¹³ C NMR and HSQC spectra, one carbonyl carbon signal was observed at δ 187.6 (C-2), two double-bonded carbon signals and one oxygenated carbon δ 67.5 (C-5), hydroxymethyl δ 71.4 (C-6) ) and one methyl group was observed at δ 14.8 (C-7). In the key HMBC spectrum, H-5 showed correlation with C-2, 4, and 6, and H-6 showed HMBC correlation with C-4, 5, and H-7 with C-2, 3, 4. . The absolute arrangement of the C-5 position was determined by comparison with the specific rotation value of the tetronic acid derivative. Compound 3 was measured as [α] _D : -13.9 ( c 0.1, MeOH). The absolute configuration of C-5 was confirmed as the S -configuration, and the structure of Compound 3 was determined as a novel compound that had not been previously reported.

Example 3. Confirmation of cytotoxicity

B16F10 cells, a melanoma cell line, were prepared using DMEM (Dulbeccos's Modified Eagle's Medium, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (100 U/ml) at 37°C, 5% CO ₂ Adapted to the incubator and subcultured. After dispensing the cultured B16F10 cells into a 96-well-plate (5×10 ⁴ cells/well), the samples (hot water extract of bitter melon, fermented bitter melon, fractions of fermented bitter melon or compounds isolated from the fraction) were treated with 0.02 ml of each concentration. After incubation for 4 hours, MTT solution (5 mg/ml) was treated with 0.02 ml and reacted. After the reaction was completed, absorbance was measured at 540 nm using an ELISA plate reader (Tecan Austria GmBH, Grodig, Austria).

As a result, both the fermented bitter melon, the fraction of the fermented bitter melon, or the compound isolated therefrom showed a high survival rate of 90% or more (Fig. 2). It was found that there was no cytotoxicity.

Example 3. Measurement of tyrosinase inhibitory activity in B16F10 cells

Intracellular tyrosinase inhibitory activity was measured using L-DOPA as a substrate according to the method of Hye-Jin Park et al . (J. Nat. Med., 2018, 72, 127-135). As a positive control, arbutin was used.

First, as a result of measuring the intracellular tyrosinase inhibitory activity of fermented bitter melon and its fractions, the α-MSH-treated group (Cont, untreated) compared with the α-MSH untreated group (Nor, untreated). Although the synase activity was increased, the experimental group treated with α-MSH after pretreatment with bitter gourd hot water extract, bitter gourd ferment or its n-Hexane fraction, EtOAc fraction, or n-BuOH fraction was compared to the α-MSH treated group with tyrosinase activity decreased. In particular, when fermented bitter melon or its n-Hexane fraction, EtOAc fraction, or n-BuOH fraction was treated at a concentration of 100 μg/ml, the tyrosinase activity was significantly reduced compared to the hot water extract of bitter melon, and EtOAc fraction 100 μg/ The intracellular tyrosinase inhibitory activity of the ml-treated group was about 48%, and the tyrosinase inhibitory activity was the best among the fractions of fermented bitter gourd ( FIG. 3A ).

In addition, as a result of measuring the intracellular tyrosinase inhibitory activity of APM-1 (Formula 1), APM-2 (Formula 2) or APM-4 (Formula 3) isolated from the ethyl acetate fraction of fermented bitter gourd, α-MSH The treatment group (Cont, sample untreated) had increased tyrosinase activity compared to the α-MSH untreated group (Nor, untreated), but after pretreatment with APM-1, APM-2 or APM-4 compounds, α -MSH-treated experimental group had decreased tyrosinase activity compared to α-MSH-treated group, and in particular, when APM-2 or APM-4 compound was treated at a concentration of 30 uM, tyrosinase of about 27% and 25%, respectively It was found that the tyrosinase inhibitory activity of the APM-2 or APM-4 compound was superior to that of the positive control group, compared to the tyrosinase inhibitory activity of the arbutin 100 uM treatment group was about 8%. could be (Fig. 3B).

Example 4. Measurement of inhibition of melanin biosynthesis in B16F10 cells

In order to measure the inhibition of intracellular melanin biosynthesis, B16F10 cells were aliquoted in a 12-well plate so as to become 1×10 ⁶ cells/ml, cultured for 24 hours, and then the sample was pretreated for 1 hour. Thereafter, each well was treated with α-MSH (1 nM) and cultured for 48 hours to induce melanin synthesis. After 48 hours, the medium was removed, washed with PBS, and centrifuged to obtain a cell pellet. 200 μl of a solution prepared so that DMSO was 10% (v/v) in 1N NaOH was added to the melanocyte pellet and dissolved at 60° C. for 1 hour, and absorbance was measured at 405 nm. The amount of melanin synthesis was expressed as a percentage (%) compared to the α-MSH alone treatment group. As a positive control, arbutin was used.

First, as a result of measuring the intracellular melanin biosynthesis inhibition rate of the fermented bitter melon and its fractions, the α-MSH treatment group (Cont, sample untreated) showed a higher melanin synthesis amount compared to the α-MSH untreated group (Nor, untreated). However, in the experimental group treated with α-MSH after pre-treatment with the fermented bitter melon or its EtOAc fraction or n-BuOH fraction, the melanin production decreased compared to the α-MSH treated group, and in particular, the fermented bitter melon or its EtOAc fraction, n When the -BuOH fraction was treated at a concentration of 100 μg/ml, it was confirmed that the amount of melanin production was significantly reduced compared to the hot water extract of bitter melon. The inhibition rate of melanin biosynthesis in the EtOAc fraction 100 μg/ml treated group of fermented bitter gourd was about 56%, the highest melanin biosynthesis inhibition rate among the fractions of fermented bitter gourd. In comparison, it was found that the EtOAc fraction of fermented bitter gourd had a better effect on inhibiting melanin biosynthesis than the positive control ( FIG. 4A ).

In addition, as a result of measuring the inhibition of intracellular melanin biosynthesis of APM-1 (Formula 1), APM-2 (Formula 2) or APM-4 (Formula 3) isolated from the ethyl acetate fraction of the fermented bitter melon, the α-MSH treatment group (Cont, untreated sample), the amount of melanin synthesis increased compared to the α-MSH untreated group (Nor, untreated), but treated with α-MSH after pretreatment with APM-1, APM-2 or APM-4 compounds One experimental group decreased melanin production compared to the α-MSH treated group, and in particular, when the APM-2 compound was treated at a concentration of 30 uM, the melanin biosynthesis inhibition rate was about 30%, and it was confirmed that the melanin biosynthesis inhibition rate was superior to that of the positive control group. (Fig. 4B).

Example 5. Measurement of the expression inhibitory activity of factors related to melanin biosynthesis

MITF (microphthalmia-associated transcription factor) is an important transcriptional regulator in the melanin synthesis process, and serves to promote the transcription of melanin synthetase enzymes TRP-1 (tyrosinase related protein 1), TRP2 and tyrosinase.

Western blot was performed to examine the effect of the APM-2 (Formula 2) compound of the present invention on the expression levels of MITF, TRP-1, TRP-2 and tyrosinase, which are factors related to whitening. Specifically, the B16F10 cell suspension was dispensed in a 100 mm culture vessel (2×10 ⁴ cells/well) and then cultured for 24 hours to stabilize the cells. After removing the medium, the medium was treated with α-MSH (1 nM/ml) and incubated for 48 hours in a medium treated with APM-2 compounds by concentration (10 uM, 20 uM), then the medium was removed again and washed twice with PBS. did. Dissolved with 100 μl of lysis buffer and centrifuged for 20 minutes at 4° C. and 12,000 rpm to obtain only the supernatant. The obtained supernatant was quantified by Bradford assay, and 20 μl of protein was loaded on 10% SDS-polyacrylamide gel and separated by electrophoresis. The separated protein was transferred to a PVDF membrane, reacted with a blocking buffer (5% skim milk in TBST) at room temperature for 1 hour, and then washed three times with TBST at 10-minute intervals. After washing, MITF (Santacruz, 1:1000), TRP-1 (Santacruz, 1:1000), TRP-2 (Santacruz, 1:1000), tyrosinase (Santacruz, 1:1000) or ß-actin (Santacruz 1: 1000) using the primary antibody of 4 ℃ overnight, then washed 3 times with TBST every 10 minutes again, and then at room temperature using a secondary antibody of mouse anti-rabbit IgG HRP or bovine anti-goat IgG HRP. The reaction was carried out for 2 hours. After the secondary antibody reaction was completed, washed 3 times, reacted with ECL (Amersham Pharmacia) solution in the dark, and the band was confirmed using the LAS4000 chemiluminescence detection system (Fuji).

As a result, the expression levels of MITF, TRP-1, TRP-2 and Tyrosinase were increased in the α-MSH-treated group (Cont, untreated) compared to the α-MSH untreated group (Nor, untreated), but 10 uM or It was confirmed that the expression levels of MITF, TRP-1, TRP-2 and Tyrosinase decreased when α-MSH was treated after pretreatment with APM-2 at a concentration of 20 uM compared to α-MSH alone treatment. In particular, it was confirmed that the expression levels of MITF, TRP-1, TRP-2 and Tyrosinase were inhibited by 36%, 57%, 43%, and 26%, respectively, when APM-2 at a concentration of 20 uM was treated (FIG. 5).

From the above results, the fermented bitter gourd, the fraction of the fermented bitter melon of the present invention, or the compounds of Formulas 1 to 3 isolated therefrom have excellent intracellular tyrosianase inhibitory activity and melanin biosynthesis inhibition rate, and the melanin synthetase TRP- 1, since it can inhibit the expression of TRP2 and Tyrosinase, it was expected to be effective in improving skin whitening.

Claims (5)

A compound of Formula 1 or an acceptable salt thereof; A compound of Formula 2 or an acceptable salt thereof; A compound of Formula 3 or an acceptable salt thereof; Or a fermented bitter melon containing one or more compounds selected from the following Chemical Formulas 1 to 3; A cosmetic composition for skin whitening containing as an active ingredient.
[Formula 1]

[Formula 2]

[Formula 3]

[Claim 3] The cosmetic composition for skin whitening according to claim 1, wherein the fermented bitter melon extract is fermented with hot water extract of bitter gourd ( Agaricus bisporus ) mycelium. A compound of Formula 1 or an acceptable salt thereof; A compound of Formula 2 or an acceptable salt thereof; A compound of Formula 3 or an acceptable salt thereof; Or a fermented bitter melon comprising at least one compound selected from the following Chemical Formulas 1 to 3; A health functional food composition for skin whitening containing as an active ingredient.
[Formula 1]

[Formula 2]

[Formula 3]

A compound of Formula 1 or an acceptable salt thereof; A compound of Formula 2 or an acceptable salt thereof; A compound of Formula 3 or an acceptable salt thereof; Or a fermented bitter gourd comprising at least one compound selected from the following Chemical Formulas 1 to 3; A pharmaceutical composition for preventing or treating melanin hyperpigmentation disease as an active ingredient.
[Formula 1]

[Formula 2]

[Formula 3]

According to claim 4, wherein the melanin hyperpigmentation disease is freckles, senile spots, liver spots, melasma, brown or dark spots, solar pigmentation, cyanic melasma, hyperpigmentation after drug use, gestational brown spots (gravidic) chloasma), or a pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease, characterized in that post-inflammatory hyperpigmentation due to wounds or dermatitis.

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