KR20230130259A - Composition for anti-wrinkle comprising methyl gallate oxidation reactant or hexahydroxydiphenic acid dimethyl ester isolated therefrom as effective component - Google Patents

Abstract

The present invention relates to a composition for anti-wrinkle comprising a methyl gallate oxidation reactant or hexahydroxydiphenic acid dimethyl ester isolated therefrom as an effective component. A methyl gallate oxidation reactant prepared by mixing methyl gallate and pear-derived oxidase source and then performing oxidation reaction, and hexahydroxydiphenic acid dimethyl ester isolated therefrom have excellent MMP-1 activity inhibition effects and type 1 procollagen synthesis inducing effects, to be expected to be useful as cosmetics, foods, and external skin agents for reducing skin wrinkles.

Description

Composition for improving skin wrinkles comprising methyl gallate oxidation reactant or HHDP dimethyl ester isolated therefrom as an effective ingredient {Composition for anti-wrinkle comprising methyl gallate oxidation reactant or hexahydroxydiphenic acid dimethyl ester isolated therefrom as effective component}

The present invention relates to a composition for improving skin wrinkles containing methyl gallate oxidation reactant or HHDP dimethyl ester isolated therefrom as an active ingredient.

Skin aging refers to both tangible and intangible changes that occur in the skin as one ages. Skin aging can be divided into intrinsic physiological aging, which is naturally caused by a decline in the body's physiological functions with age, and extrinsic photoaging, which occurs due to long-term exposure to ultraviolet rays. Clinical characteristics of intrinsic skin aging include thinning of the skin, decreased elasticity, thinning of the epidermis and dermis, decreased blood vessels, and decreased number of fibroblasts in the dermis. In addition, the clinical characteristics of photoaging, which is extrinsic skin aging, are that the skin becomes rough and loses elasticity, irregular pigmentation occurs, and deep wrinkles increase.

There are several reported causes of wrinkles. First, when the skin is exposed to excessive ultraviolet rays, the breakdown of collagen, a skin component, is accelerated, causing the skin to lose elasticity and wrinkles to form. In addition, when the skin becomes excessively dry due to excessive temperature changes, low humidity, wind, etc., the skin's function as a defensive barrier against the outside world deteriorates, causing wrinkles to form. And when the skin is exposed to reactive oxygen species or free radicals, lipid peroxidation is produced due to oxidation, which can deform skin constituent proteins such as collagen and create wrinkles. Various cosmetic compositions are being studied to suppress the occurrence of wrinkles caused by these external and internal factors. However, in the case of functional cosmetics containing chemical ingredients, there is a problem in that they cause skin irritation and are not suitable for sensitive skin, so interest is focused on functional cosmetics derived from natural plants that are less irritating to the skin and are eco-friendly.

Meanwhile, methyl gallate (MG) is methyl-3,4,5-trihydroxybenzoic acid, and its common name is gallicin. Studies on the physiological activity of methyl gallate have reported antiviral activity, antioxidant activity, antibacterial effect, and growth inhibitory effect on oral cancer cells.

Meanwhile, Korean Patent Publication No. 2014-0117053 discloses the skin wrinkle-improving effect of a composition for inhibiting the formation of advanced glycation end products containing an extract of Chrysanthemum agaris or methyl gallate or a salt thereof as an active ingredient, and Korean Patent No. 1897485 Although a composition for skin whitening and wrinkle improvement containing gallic acid glycoside as an active ingredient is disclosed, a composition for skin wrinkle improvement containing the methyl gallate oxidation product of the present invention or HHDP dimethyl ester separated therefrom as an active ingredient is disclosed. There is nothing written about it.

The present invention was developed in response to the above-mentioned needs, and the methyl gallate oxidation reaction product prepared by adding pear-derived oxidase to methyl gallate and the compound isolated therefrom, HHDP dimethyl ester, are MMP- in human skin fibroblasts. 1 The present invention was completed by confirming that the inhibition ability and type 1 procollagen synthesis ability were excellent.

In order to solve the above problems, the present invention provides a cosmetic composition for preventing or improving skin wrinkles containing methyl gallate oxidation reactant as an active ingredient.

Additionally, the present invention provides a health functional food composition for preventing or improving skin wrinkles containing methyl gallate oxidation reactant as an active ingredient.

In addition, the present invention provides a composition for topical skin application for preventing or improving skin damage caused by ultraviolet irradiation, containing methyl gallate oxidation reaction product as an active ingredient.

Additionally, the present invention provides a cosmetic composition for preventing or improving skin wrinkles containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient.

Additionally, the present invention provides a health functional food composition for preventing or improving skin wrinkles containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient.

Additionally, the present invention provides a composition for topical skin application for preventing or improving skin damage caused by ultraviolet irradiation, containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient.

The present invention relates to a composition for improving skin wrinkles containing methyl gallate oxidation reactant or HHDP dimethyl ester isolated therefrom as an active ingredient. After mixing methyl gallate and pear-derived oxidase source, oxidation The methyl gallate oxidation product prepared by reaction and the HHDP dimethyl ester isolated therefrom have excellent MMP-1 activity inhibition effects and type 1 procollagen synthesis induction effects, so they are raw materials for cosmetics, foods, and external skin preparations for improving skin wrinkles. It is expected that it will be useful.

Figure 1 shows the results of HPLC analysis of methyl gallate (A), methyl gallate oxidation reactant (B), and MGP-1 (C).
Figure 2 shows the results confirming the MMP-1 inhibitory ability in human skin fibroblasts (CCD-986sk) according to treatment with the methyl gallate oxidation reactant of the present invention. UVB irradiation promoted the expression of MMP-1. *, ** indicate a statistically significant decrease in MMP-1 expression of the methyl gallate oxidation reactant compared to methyl gallate, * is p < 0.05, ** is p < 0.01.
Figure 3 shows the results of confirming the type 1 procollagen synthesis rate in human skin fibroblasts (CCD-986sk) according to treatment with the methyl gallate oxidation reactant of the present invention. UVB irradiation inhibited procollagen synthesis. * means that the type 1 procollagen synthesis rate of the methyl gallate oxidation reactant was statistically significantly increased compared to methyl gallate, and * is p<0.05.
Figure 4 shows the results confirming the MMP-1 inhibition ability in human skin fibroblasts (CCD-986sk) according to MGP-1 (HHDP dimethyl ester) treatment of the present invention. UVB irradiation promoted the expression of MMP-1. MG is the methyl gallate treated group, and EGCG is the (-)-epigallocatechin gallate treated group used as a positive control. *, ** indicate a statistically significant decrease in MMP-1 expression of MGP-1 compared to methyl gallate, * is p < 0.05, ** is p < 0.01.
Figure 5 shows the results of confirming the type 1 procollagen synthesis rate in human skin fibroblasts (CCD-986sk) according to MGP-1 (HHDP dimethyl ester) treatment of the present invention. UVB irradiation inhibited procollagen synthesis. MG is the methyl gallate treated group, and EGCG is the (-)-epigallocatechin gallate treated group used as a positive control. * means that the type 1 procollagen synthesis rate of MGP-1 was statistically significantly increased compared to methyl gallate, and * is p<0.05.
Figure 6 shows the results of Western blot confirmation of MMP-1 and procollagen protein expression in human skin fibroblasts (CCD-986sk) according to MGP-1 (HHDP dimethyl ester) treatment of the present invention (A) and the results of quantification thereof. It is (B). UVB irradiation promoted the expression of MMP-1 and inhibited procollagen synthesis. EGCG is a (-)-epigallocatechin gallate treated group used as a positive control. *, ** indicate a statistically significant change in the protein expression level of MGP-1 compared to the group treated with UVB alone, * is p < 0.05, ** is p < 0.01.
Figure 7 shows the results of confirming the cytotoxicity of the methyl gallate oxidation reactant (A) and MGP-1 (HHDP dimethyl ester) (B) of the present invention in human skin fibroblasts (CCD-986sk). EGCG is a (-)-epigallocatechin gallate treated group used as a positive control.
Figure 8 shows the results of confirming the amount of MGP-1 produced according to the oxidation reaction time (A), methyl gallate concentration (B), enzyme source concentration (C), and reaction temperature (D).

In order to achieve the object of the present invention, the present invention provides a cosmetic composition for preventing or improving skin wrinkles containing methyl gallate oxidation reactant as an active ingredient.

The methyl gallate oxidation reactant is

1) mixing pear pulp and water, pulverizing, and then filtering to obtain pear-derived oxidase source as a filtrate; and

2) Mixing the pear-derived oxidase source obtained in step 1) with methyl gallate dissolved in ethanol, followed by oxidation reaction while stirring at 10 to 50°C for 20 to 200 minutes. can,

preferably

1) mixing 200g of pear pulp with 100-400mL of water, grinding, and then filtering to obtain pear-derived oxidase source as a filtrate; and

2) Mix the pear-derived oxidase source obtained in step 1) above with the lysate containing 100 to 400 mg of methyl gallate dissolved in 10 mL of ethanol, then stir for 110 to 140 minutes at 20 to 40°C for oxidation reaction. It may be manufactured including the step of:

more preferably

1) mixing 200g of pear pulp and 200mL of water, pulverizing, and then filtering to obtain pear-derived oxidase source as a filtrate; and

2) mixing the pear-derived oxidase source obtained in step 1) with a lysate of 200 mg of methyl gallate dissolved in 10 mL of ethanol, followed by oxidation reaction while stirring at 30°C for 120 minutes; Although manufactured, it is not limited to this.

In one embodiment of the present invention, the methyl gallate is converted to HHDP dimethyl ester through an oxidation reaction with a pear-derived oxidase source.

The methyl gallate oxidation reactant preferably includes HHDP dimethyl ester (hexahydroxydiphenic acid dimethyl ester) represented by the following formula (1), but is not limited thereto.

In one embodiment of the present invention, the methyl gallate oxidation reactant has excellent MMP-1 inhibition ability and type 1 procollagen synthesis ability and is effective in preventing or improving skin wrinkles.

In the cosmetic composition according to one embodiment of the present invention, the cosmetic composition for improving skin wrinkles is cream, softening lotion, nourishing lotion, pack, essence, hair tonic, shampoo, rinse, hair conditioner, hair treatment, gel, skin. Lotion, skin softener, skin toner, astringent, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, hand cream, foundation, nutritional essence, sunscreen, soap, cleansing foam, cleansing lotion, cleansing cream, It may have any one formulation selected from the group consisting of body lotion and body cleanser, but is not limited thereto. A cosmetic composition composed of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these components can be easily selected by a person skilled in the art.

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

When the formulation of the cosmetic composition of the present invention is powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the cosmetic composition is a spray, chlorofluorohydride may be used as a carrier ingredient. It may contain 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. These include 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, the carrier component includes water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

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

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

Additionally, the present invention provides a health functional food composition for preventing or improving skin wrinkles containing methyl gallate oxidation reactant as an active ingredient.

The health functional food composition of the present invention contains as an active ingredient the methyl gallate oxidation product of the present invention, which has excellent MMP-1 inhibition ability and type 1 procollagen synthesis ability, and is effective in preventing or improving skin wrinkles.

The health functional food composition of the present invention has the advantage of having more excellent effects when consumed in the form of inner beauty food. The inner beauty is a food called 'edible cosmetics or beauty food'. It refers to foods that change the skin constitution to a healthy one by absorbing various ingredients that are good for the skin into the body. Just like choosing cosmetics that suit your skin type, it is important to check your skin condition and health. Considering your lifestyle, you can choose and consume inner beauty foods that suit each individual. When cosmetics containing the above-mentioned cosmetic composition are mixed with inner beauty food containing the compound of the present invention, the effect of improving skin wrinkles is significantly increased compared to using only cosmetics, and can be more effective in preventing or improving skin wrinkles.

When using the health functional food composition of the present invention as a food additive, the health functional food composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The mixed amount of active ingredients can be appropriately used depending on the purpose of use (prevention or improvement). Generally, when manufacturing a 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 materials. However, in the case of long-term intake for health purposes, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There are no particular restrictions on the types of health functional foods. Examples of foods to which the health functional food composition can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, and tea. There are drinks, alcoholic beverages, and vitamin complexes, and it includes all health foods in the conventional sense.

Additionally, the health functional food composition of the present invention can be manufactured into food, especially functional food. The functional food of the present invention includes ingredients commonly added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when manufacturing a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredient. The natural carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrins, cyclodextrins, etc.), or sugar alcohols (e.g., For example, xylitol, sorbitol, erythritol, etc.) is preferable. The flavoring agent may be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the above 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 regulators, stabilizers, preservatives, glycerin, alcohol, carbonic acid. It may further contain carbonating agents used in beverages. The ratio of the ingredients 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 product composition of the present invention.

In addition, the present invention provides a composition for topical skin application for preventing or improving skin damage caused by ultraviolet irradiation, containing methyl gallate oxidation reaction product as an active ingredient.

In the composition for topical skin application of the present invention, the term 'skin damage caused by ultraviolet ray irradiation' refers to the phenomenon of wrinkle formation or decrease in skin elasticity upon repeated or long-term exposure to ultraviolet rays of the sun.

In the composition for topical skin application of the present invention, the compound can inhibit the activity of MMP-1 increased by ultraviolet irradiation and increase the ability to synthesize type 1 procollagen, which has been reduced by ultraviolet irradiation, so it can be repeatedly exposed to ultraviolet rays. It is effective in preventing the formation of wrinkles or loss of skin elasticity caused by long-term exposure.

The composition of the present invention can be applied to the human body in various ways or forms, but is preferably prepared in the form of an external skin preparation that can be applied topically to the skin. Examples include powder, ointment, gel, cream, liniment, lotion, liquid, or aerosol, but are not limited thereto.

When formulating the above skin external application, it can be prepared by appropriately mixing additives in addition to the composition of the present invention. The above additives are commonly used in the art and can be mixed within the range that does not impair the purpose and effect of the present invention.

Additionally, the present invention provides a cosmetic composition for preventing or improving skin wrinkles containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient.

The HHDP dimethyl ester is characterized as being separated from the methyl gallate oxidation reaction product prepared by adding pear-derived oxidase source to methyl gallate, but is not limited to this, and can be isolated or synthesized from other natural products. .

As used herein, the term “acceptable salt thereof” refers to any salt of a compound of the invention that retains the biological properties of the compound of the invention and is not toxic or unsuitable for pharmaceutical use. Such salts may be derived from and include a variety of organic and inorganic counterions known in the art. These 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, chric acid, cinnamic acid, mandelic acid, phthalic acid, Lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzene sulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphoric acid, camphor acid. Forsulfonic acid, 4-methylbicyclo [2.2.2]-oct-2-en-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, etc.; or the acidic proton present in the parent compound is a metal ion (e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion), an alkali metal, or an alkaline earth metal hydroxide (e.g., sodium, potassium, calcium, magnesium, aluminum, lithium, zinc). and barium hydroxide), ammonia, or substituted with an organic base such as aliphatic, cycloaliphatic or aromatic organic amines 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.

Additionally, examples of salts include sodium, potassium, calcium, magnesium, ammonium, or tetraalkylammonium salts, and when the compound contains a basic functional group, salts with non-toxic organic acids or inorganic acids, such as hydrohalides (e.g., hydrohalides). chloride or hydrobromide), sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, lutein Bates, lactate, malonate, succinate, sorbate, ascorbate, maleate, maleate, fumarate, tartarate, citrate, benzoate, 3-(4-hydroxybenzoyl fumarate, tartarate) 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-en-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethyl acetate, tert-butylacetate, lauryl sulfate, gluconate, benzoate. It may include ate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylsulfamate, quinate, or muconate salt.

Additionally, the present invention provides a health functional food composition for preventing or improving skin wrinkles containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient.

Additionally, the present invention provides a composition for topical skin application for preventing or improving skin damage caused by ultraviolet irradiation, containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be explained in more detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited thereto.

Preparation Example 1. Preparation of methyl gallate oxidation reactant

1) Manufacturing of pear-derived oxidative enzyme source

An enzyme source derived from Korean pear was prepared and used to prepare methyl gallate oxidation reactant. In October 2020, 200 mL of distilled water was added to 200 g of pear flesh harvested in Yeongcheon-si, Gyeongsangbuk-do, pulverized in a blender, and filtered using 5-layer gauze to prepare pear-derived oxidase source as a filtrate. The polyphenol oxidase activity of the pear-derived oxidase source was determined by measuring the amount of benzotropolone produced after the oxidation reaction using pyrogallol as a substrate by measuring the absorbance at 360 nm using a UV spectrometer. was measured and analyzed. 1.5 mL of the enzyme source and 1.5 mL of 200mM pyrogallol (in 50 mM sodium phosphate buffer, pH 7.2) were mixed and placed in a quartz cell to induce an oxidation reaction for 5 minutes. Then, the absorbance was measured, and OD ₃₆₀ = 0.71 to 0.75. The phosphorus oxidase source was then used to prepare the oxide. The blank group was measured by adding 1.5 mL of substrate solution and 1.5 mL of distilled water.

2) Preparation of methyl gallate oxidation reactant

Dilute 100 mg of methyl gallate with a purity of 99% or higher in 10 mL of 100% (v/v) ethanol and place it in a 1 L beaker along with 300 mL of the pear-derived oxidase source (OD ₃₆₀ = 0.71-0.75) prepared above. , reaction was performed at room temperature (20 ± 5°C) using a stirrer for 120 minutes. Afterwards, it was dried using a low-temperature vacuum concentrator, and the above method was repeated a total of 20 times to obtain methyl gallate oxidation product (1.6 g).

Example 1. Separation and identification of newly generated compounds from methyl gallate oxidation reactants

1) Separation of newly generated compounds from methyl gallate oxidation reactants

To confirm changes in the components of the methyl gallate oxidation reactant prepared in Preparation Example 1, HPLC was performed. HPLC analysis conditions are as disclosed in Table 1 below. 0.1% (v/v) formic acid (HCOOH) was used as mobile phase solvent A, acetonitrile (MeCN) was used as solvent B, and gradient elution was performed using 100% solvent A. Starting with the final 100% solvent B, the compound was detected at 280 nm while maintaining a mobile phase flow rate of 1.0 mL/min for 30 minutes.

As a result of HPCL, as shown in FIGS. 1A and 1B, it was confirmed that a newly generated peak that was not detected in methyl gallate was present in the methyl gallate oxidation reaction product.

parameter condition device Shimadzu LC20AD detector Shimadzu SPD-M20A photodiode-array detector(PDA) wavelength 280nm column YMC-Pack ODS A-302 column (4.6mm i.d.x150mm) oven temperature 40℃ flow rate 1.0mL/min injection volume 5㎕ mobile phase Time (minutes) Solvent A: 0.1% HCOOH Solvent B: MeCN 0 100 0 10 80 20 15 50 50 20 30 70 25 0 100 30 0 100-End

The new product was separated and purified using column chromatography. The methyl gallate oxidation reactant was filled with Toyoperal HW-40 gel to a height of 40 cm in a column with a diameter of 2.5 cm, and then tightly packed using MeOH. Afterwards, the methyl gallate oxidation product (1.5 g) was dissolved in 5 mL of MeOH, and the solution was adsorbed on the column, and MeOH was eluted to obtain subfrations KM01 to KM06. Six subfractions were confirmed to have peaks generated through HPLC analysis. Among them, newly generated peaks were detected in subfractions KM03 and KM06, and it was confirmed that they were relatively abundant. Accordingly, column chromatography was performed on KM03 using YMC ODS gel as a filler, and MGP-1, a newly produced compound, was separated and purified. 545 mg of MGP-1 was obtained. The purity of the isolated single material was confirmed through HPLC analysis (Figure 1C).

The structure of the purified single substance was identified through UV, NMR, and FABMS spectrum measurements.

2) Identification of a newly generated compound (MGP-1) from methyl gallate oxidation reactant

The isolated compound MGP-1 was obtained in the form of a brown amorphous powder. As a result of ESIMS spectrum measurement, the base peak of m/z 389 [M+Na] ⁺ was confirmed to estimate the molecular weight of the compound, and the molecular formula was C ₁₆ . It was confirmed as H ₁₄ O ₁₀ . As a result of measuring the ¹ H NMR spectrum, a signal corresponding to an aromatic proton was observed at δ 7.08 (2H, s, H-2, 2'), and no signal corresponding to the other aromatic region was observed. Additionally, a methoxy group corresponding to δ 3.50 (6H, s, 7/7'-OCH ₃ ) was observed, confirming the unique signal of methyl gallate.

^{13 Through} C-NMR and HSQC spectrum analysis, a signal corresponding to carbonyl carbon was observed at δ 168.4 (C-7, 7'), and a carbon signal corresponding to 12 aromatic regions was observed, indicating that it is a dimeric compound of methyl gallate. It was estimated. In particular, the signal corresponding to C-6 and 6', δ 109.4, was observed, confirming that it was a C - C bond.

2D NMR was measured to confirm the exact binding of the functional group. In the HMBC spectrum, H-2/2' showed an HMBC correlation up to C-1, 3, 4, 6, and 7, and the methoxy group showed a correlation up to C-7, indicating that the methoxy group was bound to C-7. was confirmed. As a result of NMR and ESIMS spectra, the structure of MGP-1 was determined to be HHDP dimethyl ester in which methyl gallate is dimerized through a C - C bond.

To prove the absolute configuration corresponding to the biphenyl of compound MGP-1, the CD spectrum was measured, and a positive Cotton effect was shown around 225 nm and a negative Cotton effect was shown around 250 nm, proving the S -configuration.

As a result of the above results, MGP-1 is a compound in which methyl gallate is dimerized by polyphenol oxidase. The structure was determined as ( S )-HHDP dimethyl ester (Formula 1), and it was confirmed to be the main compound of the oxidation reactant.

Example 2. Wrinkle improvement effect of methyl gallate oxidation reactant

1) Measurement of MMP-1 inhibitory activity

The MMP-1 inhibitory activity of the methyl gallate oxidation reactant of the present invention was measured in human skin fibroblasts (CCD-986sk) irradiated with ultraviolet rays.

Human skin fibroblasts (CCD-986sk) purchased from ATCC (American Type Culture Collection) were cultured in DMEM (Dulbeccos's Modified Eagle's Medium) supplemented with 10% FBS (fetal bovine serum) and 1% penicillin/streptomycin (100 U/mL). was cultured in an incubator at 37°C and 5% CO ₂ .

After culture, human skin fibroblasts were inoculated into a 24-well plate at a concentration of 5×10 ⁴ cells/well and cultured for 24 hours. After washing the cells twice with PBS (phosphate buffer saline), serum-free culture medium was added and cultured for 24 hours. Afterwards, 20 mJ/cm ² of ultraviolet rays (UVB) was pretreated, 50 to 200 ㎍/mL of methyl gallate or methyl gallate oxidation reactant was added, and cultured for 48 hours. Then, the culture medium was collected and used to measure MMP-1 protein content. used. The MMP-1 standard solution from the MMP-1 ELISA kit (R&D Systems, Minneapolis, MN, USA) was diluted 1/2 to 6.25~100ng/mL, and 100㎕ of the standard solution and 100㎕ of the sample were placed on the plate of the MMP-1 ELISA kit. After adding 100㎕ of horseradish peroxidase conjugate, the mixture was incubated at 37°C for 2 hours. Each well was repeatedly washed with a buffer solution five times to remove unbound antigen, and then 100 μl of chromogenic tetramethyl benzidine was added and reacted for 30 minutes. Then, 100 μl of stop reagent was added, and after 15 minutes, the absorbance was measured at 450 nm using an ELISA reader (Tecan Austria GmBH, Grodig, Austria).

As a result, as shown in Figure 2, the content of MMP-1 increased by UVB irradiation was reduced by treatment with methyl gallate or methyl gallate oxidation reactant, and the reduction effect of methyl gallate oxidation reactant was greater than that of methyl gallate. It was remarkable.

2) Measurement of type 1 procollagen synthesis ability

The ability of the methyl gallate oxidation reactant of the present invention to synthesize type 1 procollagen was measured in human skin fibroblasts (CCD-986sk) irradiated with ultraviolet rays. Human skin fibroblasts were inoculated into a 24-well plate at a concentration of 5×10 ⁴ cells/well and cultured for 24 hours. After washing the cells twice with PBS (phosphate buffer saline), serum-free culture medium was added and cultured for 24 hours. Afterwards, 20 mJ/cm ² of ultraviolet rays (UVB) was pretreated, 50 to 200 ㎍/mL of methyl gallate or methyl gallate oxidation reactant was added, and after culturing for 48 hours, the culture medium was collected to determine the amount of type 1 procollagen synthesis. It was used for measurement. The degree of procollagen synthesis in cell culture was measured using a type 1 procollagen C-peptide assay kit (Takara Bio Inc., Japan).

As a result, as shown in Figure 3, it was confirmed that the type 1 procollagen synthesis ability, which was reduced by UVB, increased in a concentration-dependent manner by treatment of the sample, and the type 1 procollagen synthesis ability of the methyl gallate oxidation reactant was methyl gallate. It was superior to .

Example 3. Compound MGP-1 [( S )-HHDP dimethyl ester] wrinkle improvement effect

1) Measurement of MMP-1 inhibitory activity

The experiment was conducted in the same manner as described in Example 2, except that MGP-1, a compound isolated from the methyl gallate oxidation product, was used as a sample for the experiment to confirm MMP-1 inhibitory activity.

As a result, as shown in Figure 4, the expression of MMP-1 increased by UVB was decreased by treatment of the sample, and the MMP-1 inhibitory activity of MGP-1 was more significant than that of methyl gallate (MG).

2) Measurement of type 1 procollagen synthesis ability

The experiment was conducted in the same manner as the method disclosed in Example 2, but the ability to synthesize type 1 procollagen was measured using MGP-1, a compound isolated from the methyl gallate oxidation reaction product, as a sample of the experiment. did.

As a result, as shown in Figure 5, it was confirmed that the type 1 procollagen synthesis ability, which was reduced by UVB, increased by treatment of the sample, and the type 1 procollagen synthesis ability of MGP-1 was superior to that of methyl gallate.

3) MMP-1 and type 1 procollagen Western blot

The protein expression levels of MMP-1 and type 1 procollagen were confirmed through Western blot experiments.

Human skin fibroblasts (CCD-986sk) were diluted to 2×10 ⁴ cells/mL, dispensed into a 100 mm culture dish, and cultured for 24 hours to stabilize the cells. Afterwards, the medium was removed, MGP-1 or positive control was pretreated for 1 hour, then α-MSH (10nM/mL) was treated, and cultured for 48 hours. After incubation, the medium was removed and washed twice with PBS, 100 ㎕ of lysis buffer was added to dissolve, and then centrifuged at 4°C and 12,000 rpm for 20 minutes to obtain the supernatant. The protein content of the supernatant was quantified using the Bradford assay, and the same amount of protein for each experimental sample was loaded into 10% SDS-polyacryamide and then electrophoresed to separate the proteins by size. The separated protein was transferred to a PVDF membrane using a transfer device and then reacted in blocking buffer [5% (w/v) skim milk] for 1 hour at room temperature. After reaction, the reaction was washed three times at 10-minute intervals using TBST, and then reacted with MMP-1 or procollagen primary antibody at 4°C for 16 hours. Afterwards, it was washed again with TBST a total of 3 times at 10-minute intervals, and the secondary antibody corresponding to the primary antibody was reacted at room temperature for 2 hours. After the reaction, the cells were washed three times at 10-minute intervals using TBST, reacted with ECL (Amersham Pharmacia, England) solution in the dark, and used a LAS4000 chemiluminescence detection system (Fuji, Tokyo, Japan). After confirming the band, the density was measured.

As a result, as shown in Figure 6, the expression of MMP-1 increased by UVB irradiation was decreased by MGP-1 treatment, and the expression of procollagen decreased by UVB irradiation was increased by MGP-1 treatment.

Example 4. Cytotoxicity measurement

Human skin fibroblasts (CCD-986sk) were distributed at 5 × 10 ⁴ cells/well in a 96-well plate for cell culture and cultured for 48 hours, and then each sample was treated according to concentration and cultured for 24 hours. Afterwards, 5 mg/mL of MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide) reagent was added to each well, and after 4 hours of incubation, the supernatant was removed and 150 ㎕ of DMSO was added. (dimethyl sulfoxide) was added to dissolve the formazan crystals, and the absorbance was measured at 540 nm using a microplate reader. For cytotoxicity, the relative cell survival rate of the methyl gallate oxidation product of the present invention and the compound MGP-1 isolated therefrom was calculated by taking the survival rate of the group treated with α-MSH alone as 100%.

As a result, as shown in Figure 7, it was confirmed that the methyl gallate oxidation reactant of the present invention and the compound MGP-1 isolated therefrom did not exhibit cytotoxicity in human skin fibroblasts.

Example 5. Establishment of optimal conditions for manufacturing method of methyl gallate oxidation reactant to enhance MGP-1 content

Through the above examples, MGP-1 separated from the methyl gallate oxidation reaction product prepared by the production method of the present invention has an increased wrinkle improvement effect compared to methyl gallate, so to establish an optimal production method to significantly increase its content. did.

To establish optimal conditions, the oxidation reaction time, enzyme (pear-derived oxidase source) concentration, substrate (methyl gallate) concentration, and reaction temperature were adjusted to confirm the content of MGP-1.

The method for preparing the methyl gallate oxidation reactant in Preparation Example 1 was the same, but the oxidation reaction time was adjusted from 20 to 180 minutes. As a result, as the reaction time increased, as shown in Figure 8A, the MGP-1 content increased until 120. There was little change in content after 1 minute. Therefore, the optimal reaction time was set at 120 minutes.

Next, changes in MGP-1 content depending on the content of methyl gallate used as a substrate were confirmed. The method for preparing the methyl gallate oxidation reactant in Preparation Example 1 was the same, but the methyl gallate concentration was prepared at 1.25, 5, 10, 20, 40, 80, and 160 mg/mL, and then the oxidation reaction was performed. As a result, it was confirmed that the largest amount of MGP-1 was produced in the oxidation reaction product using methyl gallate at a concentration of 20 mg/mL, as shown in Figure 8B. Therefore, the optimal substrate concentration was set at 20 mg/mL.

Next, the amount of MGP-1 production according to the concentration of enzyme source was confirmed. The method for preparing the methyl gallate oxidation reactant in Preparation Example 1 was the same, but the reaction was performed for 120 minutes under the optimal conditions, the methyl gallate concentration was fixed at 20 mg/mL, and the pear flesh was added during the production of the pear-derived oxidase source. The concentration of the enzyme source was changed by adjusting the amount. Pear flesh was prepared by adding 12.5, 25, 50, 100, 200, and 400 g to 100 mL of water, and the polyphenol oxidase activity of each enzyme source was confirmed to be 0.65, 0.68, 0.74, 0.73, 0.72, and 0.73. . As a result of comparing the amount of MGP-1 produced according to the concentration of the enzyme source, as shown in Figure 8C, the amount of MGP-1 produced increased as the concentration of the enzyme source increased, and even when the concentration of 100 g/100 mL or more was added, the amount of MGP-1 was maintained. There was little change in production volume. Therefore, the optimal enzyme source concentration was set at 100g/100mL.

The amount of MGP-1 produced was confirmed according to the oxidation reaction temperature. The methyl gallate oxidation reaction product was prepared in the same manner as in Preparation Example 1, but the reaction time, substrate concentration, and enzyme source concentration confirmed as the optimal conditions were used. The oxidation reaction temperature was set at 10, 20, 30, 50, 70, and 90°C, and as a result of checking the amount of MGP-1 produced according to the reaction temperature, the amount of MGP-1 produced was the highest when the reaction was performed at 30°C as shown in Figure 8D. There were a lot.

When combining the above results, the optimal oxidation conditions are disclosed in Table 2 below.

condition result Optimal oxidation reaction time 120 minutes optimal substrate concentration 20mg/mL (based on ethanol) optimal enzyme concentration 100g/100mL (based on water) temperature 30℃

Claims (9)

A cosmetic composition for preventing or improving skin wrinkles containing methyl gallate oxidation reaction product as an active ingredient. The method of claim 1, wherein the methyl gallate oxidation reactant is
1) mixing 200g of pear pulp with 100-400mL of water, grinding, and then filtering to obtain pear-derived oxidase source as a filtrate; and
2) Mix the pear-derived oxidase source obtained in step 1) above with the lysate containing 100 to 400 mg of methyl gallate dissolved in 10 mL of ethanol, then stir for 110 to 140 minutes at 20 to 40°C for oxidation reaction. A cosmetic composition for preventing or improving skin wrinkles, comprising: The cosmetic composition for preventing or improving skin wrinkles according to claim 1, wherein the methyl gallate oxidation reactant includes HHDP dimethyl ester (hexahydroxydiphenic acid dimethyl ester) represented by the following formula (1).
[Formula 1]
A health functional food composition for preventing or improving skin wrinkles containing methyl gallate oxidation reactant as an active ingredient. An external skin composition for preventing or improving skin damage caused by ultraviolet irradiation, containing methyl gallate oxidation reaction product as an active ingredient. A cosmetic composition for preventing or improving skin wrinkles containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient. The cosmetic composition for preventing or improving skin wrinkles according to claim 6, wherein the HHDP dimethyl ester is separated from the methyl gallate oxidation reaction product. A health functional food composition for preventing or improving skin wrinkles containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient. An external skin composition for preventing or improving skin damage caused by ultraviolet irradiation, containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient.