KR102498763B1 - 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 fermented bitter gourd extract or a compound isolated therefrom as an active ingredient. Since it is excellent, 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 composition for skin whitening containing fermented bitter melon or a compound isolated therefrom as an active ingredient.

Melanin is a major factor determining the color of hair and skin. It serves to protect skin cells from ultraviolet rays, but accumulation of excessive melanin causes hyperpigmentation such as freckles or melasma. Melanin-forming cells present in the basal layer of the epidermal layer have specific structures called melanosomes, where melanin is produced due to the action of melanogenic enzymes. Melanin consists of black and brown eumelanin and yellow and red pheomelanin. It starts with conversion to quinone (DOPA quinone).

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

Kojic acid, vitamin derivatives, arbutin, and BHT (dibutylated hydroxytoluene), which are currently used as synthetic antioxidants, are cheap and have excellent antioxidant activity, resulting in whitening effects. It is being used and its usage is regulated. Therefore, in order to develop materials with biochemical efficacy due to the development of research level and increase in technology, research is being actively conducted to discover materials of functional ingredients from natural products at home and abroad.

On the other hand, Korean Patent Publication No. 2016-0016195 discloses 'an antioxidant or anti-aging composition containing an ethyl acetate fraction fractionated from bitter gourd extract as an active ingredient', and Korean Patent Publication No. 2015-0094832 discloses anti-aging, 'Cosmetic composition containing bitter gourd fruit extract as an active ingredient and manufacturing method of bitter gourd fruit extract therefor' having antioxidation, skin wrinkle improvement or skin whitening effect is disclosed, There is no description about 'a composition for skin whitening containing fermented bitter gourd extract or a compound isolated therefrom as an active ingredient'.

The present invention has been derived from the above needs, and the inventors have found that the fermented gourd extract fermented by adding the filtrate of button mushroom pulverized to the hot water extract of gourd extract and the compounds (Formulas 1 to 3) isolated from the fractions of the fermentation gourd gourd The present invention was completed by confirming that the tyrosinase inhibitory activity and the melanin biosynthesis inhibitory rate were excellent.

In order to solve the above problems, the present invention is 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 fermented bitter gourd containing at least one compound selected from Formulas 1 to 3; provides a cosmetic composition for skin whitening containing as an active ingredient.

In addition, the present invention is 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 fermented bitter gourd containing at least one compound selected from Formulas 1 to 3; provides a health functional food composition for skin whitening containing as an active ingredient.

In addition, the present invention is 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 fermented bitter gourd containing at least one compound selected from Formulas 1 to 3; provides a pharmaceutical composition for preventing or treating hypermelanin hyperpigmentation disease containing as an active ingredient.

Since the fermented bitter gourd extract of the present invention or the novel compound isolated therefrom has excellent tyrosinase inhibitory activity and melanin biosynthesis inhibitory rate, it is useful as a cosmetic or food for improving skin whitening or a therapeutic material for the treatment of melanin pigmentation disease. It is hoped that it can be used.

1 is a result of HPLC (High Performance Liquid Chromatography) performed on the hot water extract of bitter melon and the ethyl acetate fraction (EtOAc) of fermented bitter gourd extract. Red arrows: 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 from the EtOAc fraction.
2 is a hot water extract of bitter gourd extract, fermented bitter gourd extract, fermented bitter gourd extract fractions (n-Hexane layer, EtOAc layer, n-BuOH layer, H ₂ O layer) or compounds separated therefrom (APM-1, APM-2, APM This 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 extract, fermented bitter gourd extract, or fractions of fermented bitter gourd bitter gourd (n-Hexane layer, EtOAc layer, n-BuOH layer, H ₂ O layer), Figure 3B is a 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 melon. am. Nor: UV non-irradiation group (sample untreated). Cont: UV irradiation group (sample untreated), α-MSH: melanocyte-stimulating hormone, Arbutin: positive control.
Figure 4A is a result of confirming the intracellular melanin biosynthesis inhibition rate of bitter gourd extract, fermented bitter gourd extract or fermented bitter gourd extract fractions (n-Hexane layer, EtOAc layer, n-BuOH layer, H ₂ O layer), and Fig. 4B is bitter gourd fermentation This is the result of confirming the intracellular melanin biosynthesis inhibition rate of APM-1 (Formula 1), APM-2 (Formula 2) or APM-4 (Formula 3) compounds isolated from the ethyl acetate fraction (EtOAc) of water. Nor: UV non-irradiation group (sample untreated). Cont: UV irradiation group (sample untreated), α-MSH: melanocyte-stimulating hormone, Arbutin: positive control.
Figure 5 is a melanoma cell line, B16F10 cells, MITF (microphthalmia-associated transcription factor), TRP-1 involved in melanin synthesis after treatment with the APM-2 (Formula 2) compound isolated from the ethyl acetate fraction (EtOAc) of fermented bitter melanoma. This is the result of confirming the expression levels of (tyrosinase related protein 1), TRP2 and Tyrosinase. Nor: UV non-irradiation group (sample untreated). Cont: UV irradiation group (sample untreated), α-MSH: melanocyte-stimulating hormone, Arbutin: positive control.

In order to achieve the object of the present invention, the present invention is a compound of Formula 1 or an acceptable salt thereof; a compound of Formula 2 below or an acceptable salt thereof; a compound of Formula 3 below or an acceptable salt thereof; Or fermented bitter gourd containing one or more compounds selected from the following 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 isolated from the ethyl acetate fraction (Ethly acetate, EtOA) of fermented bitter gourd. It is not limited, and it is possible to extract or synthesize 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 melon may be fermented hot water extract of bitter melon ( Agaricus bisporus ) mycelium, but is not limited thereto. The mushroom mycelium may mean a filtrate obtained by filtering a mixture of mushroom pulverized material and distilled water, and the filtrate contains polyphenol oxidase.

The fermented bitter gourd 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.

Intracellular tyrosinase when the fermented product of bitter melon or a fraction thereof of the present invention is treated at a concentration of 50 to 100 μg/ml, or the compounds (Formulas 1 to 3) isolated from the fermented bitter gourd extract fraction are treated at a concentration of 20 to 30 uM. It is excellent in reducing the expression level of inhibitory activity, melanin biosynthesis inhibition rate, MITF (microphthalmia-associated transcription factor), TRP-1 (tyrosinase related protein 1), TRP2 and Tyrosinase involved in melanin synthesis.

As used herein, the above "acceptable salt thereof" refers to any salt of the compound of the present invention that is not toxic or unsuitable for pharmaceutical use while maintaining the biological properties of the compound of the present invention. Such salts may be derived from and include a variety of 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, cricic 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, camphoric acid Forsulfonic 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 (e.g. alkali metal ion, alkaline earth metal ion or aluminum ion), alkali metal or alkaline earth metal hydroxide (e.g. sodium, potassium, calcium, magnesium, aluminum, lithium, zinc) and barium hydroxide), substituted with ammonia, or organic bases such as aliphatic, cycloaliphatic or aromatic organic amines such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanol Amines, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine piperazine , tris(hydroxymethyl)-aminomethane or tetramethylammonium hydroxide and a salt formed when coordinately bonded, and the like.

In addition, examples of the salt include sodium, potassium, calcium, magnesium, ammonium, or tetraalkylammonium salts, and when the compound contains a basic functional group, a salt with a non-toxic organic acid or inorganic acid, such as a hydrohalide (eg, hydrohalide) chloride or hydrobromide), sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, lutein Bait, Lactate, Malonate, Succinate, Sorbate, Ascorbate, Maleate, Maleate, Fumarate, Tartarate, Citrate, Benzoate, 3-(4-Hydroxybenzoylfumarate, Tarta rate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate mesylate), ethanesulfonate, 1,2-ethane -Disulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besilate), 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, benzo ate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylsulfamate, quinate or muconate salt, and the like.

The cosmetic composition of the present invention is preferably any one formulation selected from solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, foundations and sprays, more preferably skin, skin softener, skin toner, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, eye cream, moisture cream, hand cream, essence, nutrient 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 dosage forms may contain various carriers and additives necessary and appropriate for formulation of the dosage form, 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. can be used.

When the formulation of the cosmetic composition 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 chlorofluorohydro propellants 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, such as 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, 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, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like may be used.

When the formulation of the cosmetic composition of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyl taurate, and 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.

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

The present invention also relates to a compound of Formula 1 or an acceptable salt thereof; the compound of Formula 2 or an acceptable salt thereof; the compound of Formula 3 or an acceptable salt thereof; Or fermented bitter gourd containing at least one compound 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 fractions of fermented bitter gourd extract, and the fermented bitter gourd extract or compounds isolated therefrom are as described above.

As used herein, the term 'whitening' refers to inhibiting, inhibiting, or alleviating hyperpigmentation of the skin. Hyperpigmentation of the skin includes freckles, melasma, hyperpigmentation after ultraviolet exposure, hyperpigmentation after inflammation, senile lentigines, brown spots or age spots.

The health functional food composition of the present invention contains, as an active ingredient, the compound of the present invention having excellent tyrosinase inhibitory activity and melanin biosynthesis inhibitory rate, or a fermented product of bitter gourd containing the compound, and 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 refers to food referred to as 'eating cosmetics or beauty food', which absorbs various ingredients good for the skin into the body and changes the skin constitution to a healthy one. You can select and consume the inner beauty food that suits your individual lifestyle considering your lifestyle. When cosmetics containing the above-mentioned cosmetic composition and inner beauty food containing the compound of the present invention or fermented bitter gourd containing the compound are mixed, the skin whitening improvement effect is significantly increased compared to using only cosmetics, resulting in more effective skin care. It has the advantage of seeing the whitening improvement effect.

When the compound of the present invention or fermented bitter gourd containing the compound is used as a food additive, the compound of the present invention or fermented bitter gourd containing the compound may be added as it is or used together with other foods or food ingredients, It can be used appropriately according to a conventional method. Mixed amounts of active ingredients can be appropriately used depending on the purpose of use (prevention or improvement). In general, when preparing 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 the above range.

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

The functional food of the present invention includes components commonly added during food preparation, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared as a drink, natural carbohydrates or flavors may be included as additional ingredients in addition to active ingredients. The natural carbohydrates are monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrins, cyclodextrins, etc.) or sugar alcohols (eg, maltose, sucrose, etc.) , xylitol, sorbitol, erythritol, etc.) are preferred. As the flavoring agent, natural flavoring agents (eg, thaumatin, 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, flavors, colorants, 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 used for

In addition to the health functional food composition, various nutrients, vitamins, inhibitors, flavoring agents, colorants, 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 carbonating agent used in carbonated beverages. The ratio of the components to be added 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 relates to a compound of Formula 1 or an acceptable salt thereof; the compound of Formula 2 or an acceptable salt thereof; the compound of Formula 3 or an acceptable salt thereof; Or fermented bitter gourd containing one or more compounds selected from Formulas 1 to 3; provides a pharmaceutical composition for preventing or treating hypermelanin 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 fractions of fermented bitter melon, and the fermented bitter gourd extract or compounds isolated therefrom are as described above.

In the present specification, the term “melanin hyperpigmentation” means that certain parts of the skin or nails or nails become black or dark compared to other parts due to an excessive increase in melanin. The melanin hyperpigmentation disease is freckles, senile spots, liver spots, melasma, brown or sunspots, solar pigment spots, cyanic melasma, hyperpigmentation after drug use, pregnancy brown spots (gravidic chloasma), or scratches and hyperpigmentation after wounds including burns or inflammation due to dermatitis; and the like, but are not limited thereto.

The pharmaceutical composition of the present invention may include pharmaceutically acceptable carriers in addition to active ingredients, and these carriers are commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, 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 components, skin safety according to combined use, ease of formulation, and stability of active ingredients may be considered.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can 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 for the treatment or prevention of melanin hyperpigmentation disease, it is preferably applied topically to the skin.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier or excipient according to a method that can be easily performed by those skilled in the art, or It can be prepared by incorporating into a dose container. At this time, the formulation may be prepared as any one formulation selected from injections, creams, patches, sprays, ointments, warnings, lotions, liniments, pastas, and cataplasmas, and may additionally include a dispersing agent or a stabilizer. there is.

The pharmaceutical composition of the present invention can provide a desirable effect of improving skin whitening when containing an effective amount of a compound (Formulas 1 to 3) or fermented bitter gourd containing the compound. In the present invention, 'effective amount' means the amount of a compound capable of suppressing pigmentation or fermented bitter gourd containing the compound. The effective amount of the compound included in the composition of the present invention or the fermented bitter gourd containing the compound will vary depending on the form in which the composition is commercialized, how the compound or fermented bitter gourd containing the compound is applied to the skin, and how long it stays on the skin. . A preferred dosage of the compound of the present invention or a pharmaceutical composition containing fermented bitter gourd containing the compound as an active ingredient varies depending on the condition and body weight of the patient, the severity of the disease, the type of drug, the route of administration and the duration, but it is determined by those skilled in the art. can be selected appropriately.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate 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 Dusonaeyakcho Co., Ltd. was immersed in 45 L of water, extracted at 90 ° C. for 3 hours, and then concentrated under reduced pressure to obtain 1.5 kg of hot water extract of bitter gourd. In addition, for the enzymatic reaction of the hot water extract of Yeoju, 40 g of button mushrooms ( Agaricus bisporus ) was mixed with 100 ml of distilled water, pulverized with a mixer, and filtered through 5 layers of gauze to prepare an enzyme source containing polyphenol oxidase. . In order 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 having an absorbance value of 0.78 to 0.79 was used for the enzymatic reaction of the hot water extract of Momordica charantia. The absorbance was measured after putting 1.5 ml of the enzyme source together with 1.5 ml of 200 mM pyrogallol (in 50 mM sodium phosphate buffer, pH 7.2) into a quartz cell and inducing an oxidation reaction for 5 minutes. and 1.5 ml of distilled water (refer to Food Chemistry, 88, 2004, 69-77 and BBRC, 130(2), 1985, 633-639).

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

Example 1. Isolation of Compounds of Chemical Formulas 1 to 3 from Fermented Gochujang

The enzyme reaction experiment of bitter gourd was repeated 36 times, and 750 g of the fermented bitter gourd produced was diluted in 2 ℓ of 10% methanol (MeOH), and then n-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 obtain 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 portion of the EtOAc fraction was subjected to column chromatography using a Toyopearl HW-40 column (2.5 cm id × 42 cm) while increasing the MeOH content of H ₂ O/MeOH to obtain EMC01 to EMC06 subfractions. Column chromatography using a YMC Pack ODS A-302 column (4.6 cm id × 150 mm, 5 μm) as a filler was performed to obtain APM-1 (t _R 11.6 min, 19.7 mg) and APM-2 from 171.4 mg of the small fraction EMC02. (t _R 9.2 min, 16.3 mg) was obtained, and APM-4 (t _R 7.3 min, 13.4 mg) was obtained from the small fraction EMC01 using the same method as above (FIG. 1). As the mobile phase, 0.1% formic acid (solvent A) and acetonitrile (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 mash

The compounds isolated from the ethyl acetate fraction of the fermentation product of bitter melon were determined to be Monaspilosuslin represented by Formula 1 and 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 confirming 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). A singlet proton corresponding to two double bonds 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 ^{the 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, compound 1 was structured as monaspilosuslin, a sequiterpene compound isolated from fermented rice using Monascus pilosus microorganisms.

2-2. Formula 2 (New)

Compound 2 was obtained in the form of a yellow amorphous powder, and showed maximum absorbances of 227 and 291 nm in the UV spectrum, suggesting that it was 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 the 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 bond carbon signals. In the Key HMBC spectrum, H-6 showed correlations with C-4 and 5, and H-7 showed HMBC correlations with C-2, 3 and 4. As a result of the above, compound 2 was identified as a novel compound not previously reported, and its 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 absorbances of 226 and 290 nm in the UV spectrum, suggesting that it was a γ-lactone skeleton. The molecular weight was estimated by checking the base peak of m/z 1145.0499 [M+H] ⁺ in the HRFABMS spectrum, and the molecular formula was confirmed 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 ^{the 13} C NMR and HSQC spectra, one carbonyl carbon signal was observed at δ 187.6 (C-2), two double bond carbon signals, one oxygenated carbon δ 67.5 (C-5), and 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 a correlation with C-2, 4, and 6, and H-6 showed a correlation with C-4, 5, and H-7 with C-2, 3, and 4. . The absolute arrangement of the C-5 position was determined by comparison with the specific rotation values of tetronic acid derivatives. Compound 3 was determined as [α] _D : -13.9 ( c 0.1, MeOH). The absolute configuration of C-5 was confirmed by the S -configuration, and the structure of Compound 3 was determined as a novel compound not previously reported.

Example 3. Confirmation of cytotoxicity

Melanoma cell line B16F10 cells 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 and 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), 0.02 ml of sample (hot water extract of bitter gourd extract, fermented bitter gourd extract, fermented bitter gourd extract, fractions or compounds isolated from fractions) was treated at each concentration. After incubation for 4 hours, 0.02 ml of MTT solution (5 mg/ml) was treated 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, all of the fermented bitter gourd extract, the fermented bitter gourd extract fractions, or the compounds isolated from them showed a high survival rate of 90% or more (FIG. 2). No cytotoxicity was found.

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). Arbutin was used as a positive control.

First, as a result of measuring the intracellular tyrosinase inhibitory activity of fermented bitter gourd extract and its fractions, the α-MSH treated group (Cont, sample untreated) was compared to the α-MSH untreated group (Nor, sample not treated) as tyrosinase. Although the synase activity was increased, the experimental group treated with α-MSH after pre-treatment of hot water extract, fermented product of M. charantia or its n-Hexane fraction, EtOAc fraction, or n-BuOH fraction treated with α-MSH compared to the α-MSH treatment group. activity decreased. In particular, when the fermented bitter gourd extract 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 gourd extract. The intracellular tyrosinase inhibitory activity of the ㎖ treatment group was about 48%, and the tyrosinase inhibitory activity was the best among the fractions of fermented bitter melon (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 melon, α-MSH The treated group (Cont, sample untreated) showed increased tyrosinase activity compared to the α-MSH untreated group (Nor, sample untreated), but after pretreatment with APM-1, APM-2 or APM-4 compound, α -The experimental group treated with MSH showed a decrease in tyrosinase activity compared to the α-MSH treated group. In particular, when the APM-2 or APM-4 compound was treated at a concentration of 30 uM, the tyrosinase activity was reduced by 27% and 25%, respectively. tyrosinase inhibitory activity, and compared to the tyrosinase inhibitory activity of the arbutin 100 uM treated group of about 8%, it was found that the tyrosinase inhibitory activity of the APM-2 or APM-4 compound was better than that of the positive control group. could (Fig. 3B).

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

In order to measure the intracellular melanin biosynthesis inhibition rate, B16F10 cells were dispensed in a 12-well plate to be 1×10 ⁶ cells/ml, incubated for 24 hours, and then samples were 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 cell pellets. 200 μl of a solution of 10% (v/v) DMSO in 1N NaOH was added to the melanocyte pellet, 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. Arbutin was used as a positive control.

First, as a result of measuring the inhibition of intracellular melanin biosynthesis of fermented bitter gourd extract and its fractions, the α-MSH treated group (Cont, sample untreated) showed a higher melanin synthesis than the α-MSH untreated group (Nor, sample untreated). However, the experimental group treated with α-MSH after pre-treatment of fermented bitter gourd or its EtOAc fraction and n-BuOH fraction showed a decrease in melanin production compared to the α-MSH treated group, especially fermented bitter gourd extract 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 produced was significantly reduced compared to the hot water extract of Bitter gourd. The melanin biosynthesis inhibition rate of the 100 μg/ml treatment group of the EtOAc fraction of the fermentation of bitter melon was about 56%, which was the highest among the fractions of the fermented bitter gourd melanoma. When compared, it was found that the EtOAc fraction of fermented bitter gourd extract was more effective in inhibiting melanin biosynthesis than the positive control group (FIG. 4A).

In addition, as a result of measuring the intracellular melanin biosynthesis inhibition rate 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 treated group (Cont, sample untreated) increased the amount of melanin synthesis compared to the α-MSH untreated group (Nor, sample untreated), but α-MSH was treated after pretreatment with APM-1, APM-2 or APM-4 compound One experimental group showed a decrease in melanin production compared to the α-MSH treatment 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%, confirming that the melanin biosynthesis inhibition rate was superior to that of the positive control group. (FIG. 4B).

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

MITF (microphthalmia-associated transcription factor) is an important transcriptional regulator in the melanin synthesis process, and plays a role in promoting the transcription of melanin synthesizing enzymes TRP-1 (tyrosinase related protein 1), TRP2 and tyrosinase.

Western blotting 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 whitening-related factors. Specifically, the B16F10 cell suspension was dispensed into a 100 mm culture vessel (2×10 ⁴ cells/well) and cultured for 24 hours to stabilize the cells. After removing the medium, it was treated with α-MSH (1 nM/ml) and cultured for 48 hours with the medium treated with APM-2 compound at different concentrations (10 uM, 20 uM), then the medium was removed again and washed twice with PBS. did It was 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 a 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) for 1 hour at room temperature, and 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) at 4 ° C overnight using the primary antibody, washed three times with TBST at 10-minute intervals, and at room temperature using the secondary antibody of mouse anti-rabbit IgG HRP or bovine anti-goat IgG HRP. Reacted for 2 hours. After the secondary antibody reaction was completed, the mixture was washed three 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 increased in the α-MSH treated group (Cont, no sample treatment) compared to the α-MSH untreated group (Nor, no sample treatment), but 10 uM or When α-MSH was treated after pretreatment with 20 μM of APM-2, it was confirmed that the expression levels of MITF, TRP-1, TRP-2, and Tyrosinase were reduced compared to α-MSH alone. In particular, it was confirmed that the treatment of APM-2 at a concentration of 20 uM inhibited the expression levels of MITF, TRP-1, TRP-2, and Tyrosinase by 36%, 57%, 43%, and 26%, respectively (FIG. 5).

Through the above results, the fermented bitter gourd extract of the present invention, the fermented bitter gourd extract fraction, or the compounds of Formulas 1 to 3 isolated therefrom have excellent intracellular tyrosyanase inhibitory activity and melanin biosynthesis inhibition rate, and the melanin synthase TRP- 1, TRP2 and Tyrosinase expression can be inhibited, so it was expected to be effective in improving skin whitening.

Claims (5)

a compound of Formula 1 below or an acceptable salt thereof; a compound of Formula 2 below or an acceptable salt thereof; and a compound of Formula 3 or an acceptable salt thereof; A cosmetic composition for skin whitening containing at least one compound selected from among active ingredients.
[Formula 1]

[Formula 2]

[Formula 3]

The cosmetic composition for skin whitening according to claim 1, wherein the compounds of Formulas 1 to 3 are isolated from fermented bitter melon. a compound of Formula 1 below or an acceptable salt thereof; a compound of Formula 2 below or an acceptable salt thereof; and a compound of Formula 3 or an acceptable salt thereof; Health functional food composition for skin whitening containing at least one compound selected from among as an active ingredient.
[Formula 1]

[Formula 2]

[Formula 3]

a compound of Formula 1 below or an acceptable salt thereof; a compound of Formula 2 below or an acceptable salt thereof; and a compound of Formula 3 or an acceptable salt thereof; A pharmaceutical composition for the prevention or treatment of hypermelanin hyperpigmentation disease containing at least one compound selected from among as an active ingredient.
[Formula 1]

[Formula 2]

[Formula 3]

The method of claim 4, wherein the melanin hyperpigmentation disease is freckles, senile spots, liver spots, melasma, brown or sunspots, solar pigment spots, cyanic melasma, hyperpigmentation after drug use, pregnancy brown spots (gravidic spots) chloasma), or a pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease characterized by hyperpigmentation after inflammation due to wounds or dermatitis.

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