KR20220112017A - Composition for anti-wrinkle effect comprising fermented product of Momordica charantia or compound isolated from therefrom as effective component - Google Patents

Abstract

The present invention relates to a composition for ameliorating skin wrinkles comprising, as an active ingredient, a fermented product of Momordica charantia L. or a compound isolated therefrom. The fermented product of Momordica charantia L. of the present invention or a novel compound isolated therefrom has an excellent inhibitory ability against matrix metalloproteinase-1 (MMP-1) as well as an excellent procollagen Type-1 synthesis ability, and thus can be effectively used as a raw material for cosmetics, foods, and external skin preparations for ameliorating skin wrinkles.

Description

Composition for anti-wrinkle effect comprising fermented product of Momordica charantia or compound isolated from therefrom as effective component

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

The skin mainly protects the human body from external physical damage and chemicals, prevents bacteria, fungi, and viruses from invading the skin, and acts as a protective barrier to prevent moisture loss. Looking at the structure of the skin, it is composed of three layers: the epidermis, the dermis, and the subcutaneous fat.

Recently, as exposure to strong ultraviolet rays increases due to climate change or environmental pollution, moisture loss and skin aging of the skin are accelerated. Skin aging can be divided into intrinsic physiological aging, which is naturally induced by the deterioration of physiological functions of a living body with increasing age, and exogenous photoaging, which is progressed by long-term UV exposure. The clinical features of intrinsic skin aging are thinning of the skin and decreased elasticity, and histological findings are that the thickness of the epidermis and dermis is thinned, blood vessels are reduced, and the number of fibroblasts in the dermis is reduced. In addition, the clinical features of extrinsic skin aging, photoaging, are rough skin, loss of elasticity, irregular pigmentation, and increased deep wrinkles.

Various causes of wrinkles have been reported. First, when the skin is exposed to excessive ultraviolet rays, collagen degradation, which is a component of the skin, is promoted, so that the skin loses elasticity and wrinkles are formed. In addition, when the skin becomes too dry due to excessive temperature change, humidity drop, or wind, the skin function as a barrier to the outside decreases and wrinkles may occur. In addition, when the skin is exposed to reactive oxygen species or free radicals, lipid peroxide is generated by oxidative action, and as a result, skin constituent proteins such as collagen are deformed or reduced, thereby generating wrinkles. Various cosmetic compositions are being studied to suppress the occurrence of wrinkles due to these external and internal factors.

On the other hand, Korea Patent Publication No. 2015-0106070 discloses 'a skin condition improvement product containing bitter melon extract as an active ingredient'. There is no description about the composition for improving wrinkles.

The present invention has been derived from the above needs, and the present inventors have prepared a fermented gourd fermented by adding a filtrate of a pulverized mushroom to a bitter gourd hot water extract and a chemical separated from a fraction of the fermented bitter gourd (Formulas 1 to 3) By confirming that the MMP (Matrix metalloproteinase)-1 inhibitory ability and the type 1 pro-collagen synthesis ability 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 preventing or improving skin wrinkles.

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.

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 preventing or improving skin wrinkles.

Since the fermented bitter melon or the novel compound isolated therefrom of the present invention has excellent MMP-1 inhibitory ability and type 1 procollagen synthesis ability, it is expected to be usefully utilized as a raw material for cosmetics, food and external preparations for skin wrinkle improvement. It is expected.

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). EGCG (positive control): (-)-epigallocatechin gallate.
Figure 3 shows human skin fibroblasts (CCD-986sk) after UVB irradiation, hot water extract of bitter gourd, fermented bitter melon or fermented bitter melon fraction (n-Hexane layer, EtOAc layer, n-BuOH layer, H ₂ O layer) treatment This is the result of confirming the MMP-1 inhibitory activity. Nor: UV non-irradiated group (no sample treatment). Cont: UV irradiation group (no sample treatment), EGCG (positive control group): (-)-epigallocatechin gallate.
Figure 4 is after UVB irradiation on human skin fibroblasts (CCD-986sk) treated with hot water extract of bitter gourd, fermented bitter melon or fractions of bitter melon fermented (n-Hexane layer, EtOAc layer, n-BuOH layer, H ₂ O layer) This is the result of confirming the level of type 1 procollagen synthesis.
Figure 5 is APM-1 (Formula 1), APM-2 (Formula 2) or APM-4 (Formula 3) separated from the ethyl acetate fraction (EtOA) of bitter gourd fermentation after UVB irradiation on CCD-986sk by treatment with a compound This is the result of confirming the level of type 1 procollagen synthesis.
6 is a result of confirming the expression level of MMP-1 and type 1 procollagen by treating the APM-2 compound after UVB irradiation on CCD-986sk. Nor: UV non-irradiated group (no sample treatment). Cont: UV irradiation group (no sample treatment), EGCG (positive control group): (-)-epigallocatechin gallate.

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; as an active ingredient, it provides a cosmetic composition for preventing or improving skin wrinkles.

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 gourd, a fraction thereof, or a compound isolated therefrom of the present invention has excellent MMP (Matrix metalloproteinase)-1 inhibitory ability and type 1 pro-collagen synthesizing ability, and thus is effective in improving skin wrinkles.

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 , a salt formed when coordinated with tris(hydroxymethyl)-aminomethane or 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 fatty 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 comprising at least one compound selected from Formulas 1 to 3; as an active ingredient, it provides a composition for external application for skin for preventing or improving skin damage caused by UV irradiation.

In the composition for external application for skin 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.

In the composition for external application for skin of the present invention, the 'skin damage caused by ultraviolet irradiation' refers to a phenomenon in which wrinkles are generated or skin elasticity is reduced when repeatedly or long-term exposure to ultraviolet rays of sunlight.

In the composition for external application for skin of the present invention, the fermented bitter gourd or a compound isolated therefrom inhibits MMP-1, a collagen degrading enzyme increased by UV irradiation, and the sum of type 1 procollagen reduced by UV irradiation Since it can increase performance, it has an effect of preventing the generation of wrinkles or deterioration of skin elasticity caused by repeated or long-term exposure to UV rays.

The composition for external application for skin of the present invention may be applied to the human body in various ways or forms, but preferably, it may be prepared in the form of an external preparation for skin that can be applied topically to the skin. For example, powders, ointments, gels, creams, liniments, lotions, liquids, or aerosols may be used, but the present invention is not limited thereto.

In the case of formulation for external use for skin, it can be prepared by appropriately mixing additives, etc. in addition to the composition of the present invention. The additives are commonly used in the art, and can be formulated within a range that does not impair the purpose and effect 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 health functional food composition for preventing or improving skin wrinkles, 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.

The health functional food composition of the present invention contains, as an active ingredient, the compound of the present invention, which has excellent MMP-1 inhibitory ability and type 1 procollagen synthesizing ability, or fermented bitter gourd containing the compound, and is effective in improving skin wrinkles. .

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 wrinkle improvement or skin moisturizing enhancement effect is significantly increased compared to using only cosmetics. It has the advantage of being able to see a more effective skin wrinkle improvement and moisturizing enhancement 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 ingestion 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 it is prepared as a drink, a natural carbohydrate or flavoring agent may be included as an additional ingredient 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.

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, 17.4 g of n-BuOH fraction and 540.9 g of H ₂ O fraction were obtained.

Then, it has excellent MMP-1 inhibitory activity and type 1 procollagen synthesis 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. Cytotoxicity measurement

CCD-986sk cell line (Human skin fibroblast cells) purchased from ATCC (American Type Culture Collection) is DMEM (Dulbeccos's Modified Eagle's Medium) supplemented with 10% FBS (fetal bovine serum) and 1% penicillin/streptomycin (100 U/ml). , Gibco, USA) using 37 °C, 5% CO ₂ Adapted to an incubator and subcultured. After dispensing the cultured CCD-986sk cells into the play, the sample (a fraction of bitter gourd extract, fermented bitter melon, or a compound isolated from the fermented bitter melon) was treated and cultured, followed by treatment with MTT solution (5 mg/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). No cytotoxicity was found

Example 4. Measurement of MMP-1 inhibitory activity

The content of MMP-1 protein present in the culture medium of human skin fibroblasts (CCD-986sk) irradiated with UVB (20 mJ/cm ² ) was measured using an ELISA kit (R&D Systems, USA). The standard solution of the MMP-1 kit was diluted 1/2 to 6.25-100 ng/ml, 100 μl of the standard solution and 100 μl of the sample were dispensed into a micro plate of the MMP-1 ELISA kit, and horseradish peroxidase (HRP) was administered. 100 μl of the conjugate was added and incubated at 37° C. for 2 hours. Each well was repeatedly washed 5 times with buffer solution to remove unbound antigen, and 100 μl of chromogenic tetramethyl benzidine was added and reacted for 30 minutes. Then, 100 μl of stop solution was added, and absorbance was measured at 450 nm after 15 minutes. As a positive control, (-)-EGCG (epigallocatechin gallate) was used.

First, as a result of measuring the MMP-1 inhibitory ability of the bitter gourd fermented product and its fractions, the MMP-1 inhibitory ability of the 100 µg/ml-treated bitter gourd hot-water extract group was about 14%, whereas the MMP-1 The inhibitory ability of -1 was about 22%, and it was confirmed that the MMP-1 inhibitory activity was increased when the hot water extract of bitter gourd was reacted with an enzyme. In addition, it was confirmed that the MMP-1 inhibitory ability of about 36% was shown in the 100 μg/ml treatment group of the EtOAc fraction among the fractions of the fermented bitter gourd (see FIG. 3 and Table 2). Through this, it was expected that compared to the hot water extract of bitter gourd, the fermented bitter melon or a fraction of the fermented bitter melon could reduce the production of MMP-1 and suppress collagen degradation.

MMP-1 inhibitory activity Samples MMP-1 inhibition (%) ^a 25 μg/mL 50 μg/mL 100 μg/mL Yeoju hot water extract 7.7±1.5 9.7±2.1 14.3±2.2 fermented bitter melon 9.0±2.0 16.5±1.8 22.9±1.4 n-Hexane fraction 7.3±2.0 10.7±1.5 14.7±2.4 EtOAc fraction 13.3±0.9 20.8±1.1 36.5±2.0 n-BuOH fraction 0.1±0.1 1.3±2.7 3.5±2.0 H ₂ O fraction 0.1±0.1 1.0±2.5 1.5±3.0 (-)-EGCG ^b 57.5±0.7 64.1±1.3 74.3±1.3

a: IC50 values were determined by regression analysis and expressed as mean ± SD of three replicates.

b: positive control.

Example 5. Measurement of Type 1 Procollagen Synthesis Rate

Human skin fibroblasts (CCD-986sk) irradiated with UVB (20 mJ/cm ² ) were dispensed at a concentration of 5 × 10 ⁴ cells/well in a 24-well-plate and cultured for 24 hours, followed by 2 with PBS (Phosphate bufferes sailne). Washed twice and serum-free culture was added. After 24 hours, the sample was treated and cultured for 48 hours, and the culture medium was collected to measure the amount of type 1 procollagen synthesis. The degree of collagen synthesis in the cell culture was measured using a procollagen type I C-peptide assay kit (Takara Bio Inc., Japan). As a positive control, (-)-EGCG (epigallocatechin gallate) was used.

First, as a result of measuring the amount of type 1 procollagen synthesis of fermented bitter melon and its fractions, the amount of procollagen synthesis in the UVB-irradiated group (cont, untreated) was significantly reduced compared to the non-UVB-irradiated group (Nor, untreated). However, after UVB irradiation, the experimental group treated with fermented bitter melon or its n-Hexane fraction, EtOAc fraction, n-BuOH, and H ₂ 0 fraction showed an increase in the amount of type 1 procollagen synthesis compared to the UVB irradiation group. In particular, it was confirmed that the EtOAc fraction among the fractions of fermented bitter gourd produced type 1 procollagen at a level similar to that of the positive control group (EGCG) at all concentrations of 25 μg/ml, 50 μg/ml, or 100 μg/ml (Fig. 4). and Table 3).

Synthesis of type 1 procollagen Samples type I pro-collagen synthesis rate (%) 25 μg/mL 50 μg/mL 100 μg/mL Yeoju hot water extract 20.8±0.2 25.7±0.5 30.3±0.6 fermented bitter melon 38.7±0.5 52.3±0.8 76.2±1.0 n-Hexane fraction 21.3±0.3 25.0±0.5 33.3±0.8 EtOAc fraction 52.8±0.4 70.7±0.8 82.3±1.2 n-BuOH fraction 38.7±0.5 42.6±0.4 55.3±0.8 H ₂ O fraction 43.4±0.7 53.2±1.0 62.3±0.6 (-)-EGCG 65.3±1.0 73.5±1.2 85.6±1.3

In addition, the amount of synthesis of type 1 procollagen of APM-1 (Formula 1), APM-2 (Formula 2) or APM-4 (Formula 3) isolated from the ethyl acetate fraction of fermented bitter gourd, which had the best type 1 inhibitory ability As a result of measuring, the amount of procollagen synthesis was significantly reduced in the UVB-irradiated group (cont, untreated) compared to the non-UVB-irradiated group (Nor, untreated), but after UVB irradiation, APM-1, APM-2 or APM-4 The experimental group treated with the compound significantly increased the amount of procollagen type 1 synthesis compared to the UVB irradiation group, and in particular, it was confirmed that the APM-1 or APM-2 compound synthesized type 1 procollagen at a level similar to that of the positive control group ( Fig. 5 and Table 4).

Synthesis of type 1 procollagen Samples type I pro-collagen synthesis rate (%) 10 μM 20 μM 30 μM APM-1 52.8±0.5 90.6±0.8 96.7±1.1 APM-2 38.7±0.3 81.1±1.2 93.4±1.2 APM-4 20.5±0.3 29.2±0.3 34.3±0.7 (-)-EGCG 48.3±0.7 59.9±0.8 64.3±0.8

In addition, as a result of confirming the expression levels of MMP-1 and type 1 procollagen of the APM-2 compound isolated from the EtOAc fraction of the fermented bitter melon by western blot, the MMP-1 expression level increased by UVB irradiation was APM- 2 was decreased, and it was confirmed that the expression level of type 1 procollagen decreased by UVB irradiation was increased by treatment with APM-2 (FIG. 6).

Through this, compared to the bitter gourd hot water extract, the fermented bitter melon, the fraction of the fermented bitter melon, or the compounds of Formulas 1 to 3 isolated therefrom, are the primary component of the extracellular matrix that provides structural support in the skin dermis, type 1 pro It was expected to be effective in improving skin wrinkles and enhancing elasticity by promoting the synthesis of collagen.

Claims (4)

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 cosmetic composition for preventing or improving skin wrinkles, comprising as an active ingredient.
[Formula 1]

[Formula 2]

[Formula 3]

[Claim 2] The cosmetic composition for preventing or improving skin wrinkles according to claim 1, wherein the fermented bitter melon extract is fermented with a 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 composition for external application for skin for preventing or improving skin damage caused by ultraviolet irradiation, comprising as an active ingredient a fermented bitter melon comprising at least one compound selected from the following Chemical Formulas 1 to 3.
[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 melon comprising at least one compound selected from the following Chemical Formulas 1 to 3; A health functional food composition for preventing or improving skin wrinkles, containing as an active ingredient.
[Formula 1]

[Formula 2]

[Formula 3]

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