KR20230130258A - Manufacturing method of methyl gallate oxidation reactant containing hexahydroxydiphenic acid dimethyl ester - Google Patents

Abstract

The present invention relates to a method for producing a methyl gallate oxidation product containing HHDP dimethyl ester, and a composition for skin whitening containing the methyl gallate oxidation product prepared by the above production method or a compound isolated therefrom as an active ingredient. The methyl gallate oxidation reactant prepared by mixing methyl gallate and a pear-derived oxidase source and then performing an oxidation reaction includes an HHDP dimethyl ester compound, and the methyl gallate oxidation reactant and the HHDP dimethyl ester separated therefrom. Since its tyrosinase inhibitory activity and melanin synthesis inhibitory activity are superior to methyl gallate, it can be usefully used as a functional cosmetic for skin whitening, a health functional food for preventing or improving melanin hyperpigmentation disease, or a treatment thereof.

Description

Manufacturing method of methyl gallate oxidation reactant containing hexahydroxydiphenic acid dimethyl ester}

The present invention relates to a method for producing a methyl gallate oxidation product containing HHDP dimethyl ester, and a composition for skin whitening containing the methyl gallate oxidation product prepared by the above production method or a compound isolated therefrom as an active ingredient.

Melanocytes in the basal layer of the epidermis produce melanin due to various irritating factors in the skin, and when this melanin reaches the stratum corneum and causes hyperpigmentation, the skin appears dark. Factors involved in melanin synthesis include follicular hormone, progesterone, pigment cell-stimulating hormone, ultraviolet rays, stress, and heredity.

Skin whitening refers to the process of quickly discharging unnecessary melanin pigment and restoring the original transparency of the person's skin. Substances registered as whitening ingredients with the Food and Drug Administration include mulberry extract, arbutin, ethyl ascorbyl ether, oil-soluble licorice extract, ascorbyl glucoside, niacinamide, alpha-bisabolol, and ascorbyl tetraisopalmitate. Whitening cosmetics are made into lotions, liquids, creams, mask packs, etc.

Mulberry extract, arbutin, alpha-bisabolol, and oil-soluble licorice extract inhibit the activation of the tyrosinase enzyme involved in melanin synthesis, and ascorbyl glucoside, ethyl ascorbyl ether, and ascorbyl tetraisopalmitate inhibit the tyrosinase enzyme. It inhibits the oxidation of stimulated tyrosine, and niacinamide inhibits the transition of already produced melanin from melanocytes to keratinocytes, preventing the final step of melanin entering actual skin cells.

A representative substance used for medical purposes other than cosmetics is hydroquinone, which is prescribed as a medicine and is mainly used as a skin ointment. In Korea, to purchase an ointment containing more than 4% of this substance, you must have a doctor's prescription, and 2% ointment can be purchased at a pharmacy without a prescription. Unlike whitening cosmetics, hydroquinone ointment used for medical purposes directly destroys skin cells containing melanin and helps new skin cells grow.

However, the above substances have the problem of narrow product application scope, such as the effect of inhibiting tyrosinase activity only at relatively high concentrations or the effect does not last long due to low stability, and hydroquinone has skin irritation and stability issues. Its use in cosmetics is limited.

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. 1999-0086660 discloses a whitening cosmetic composition containing methyl gallate, and Korean Patent No. 0907685 contains phenolic compounds (including methyl gallate) isolated from Nogeun as an active ingredient. Although a cosmetic composition for skin whitening has been disclosed, the method for producing a methyl gallate oxidation reaction product containing HHDP dimethyl ester of the present invention, the methyl gallate oxidation reaction product prepared by the above production method, or a compound isolated therefrom as an active ingredient. There is no description of the skin whitening composition it contains.

The present invention was developed in response to the above-mentioned needs, and provides a method for preparing a methyl gallate oxidation product containing HHDP dimethyl ester by adding a pear-derived oxidase to methyl gallate, and the methyl gallate oxidation product The present invention was completed by confirming that HHDP dimethyl ester, a compound isolated therefrom, had superior tyrosinase inhibitory activity and melanin synthesis inhibitory activity in melanocytes compared to methyl gallate.

In order to solve the above problem, the present invention provides a method for producing a methyl gallate oxidation product comprising mixing methyl gallate and a pear-derived oxidase source and then performing an oxidation reaction.

Additionally, the present invention provides a methyl gallate oxidation reactant containing HHDP dimethyl ester prepared by the above production method.

Additionally, the present invention provides a cosmetic composition for skin whitening containing a methyl gallate oxidation product containing the HHDP dimethyl ester as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving melanin hyperpigmentation disease, which contains a methyl gallate oxidation product containing the HHDP dimethyl ester as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating melanin hyperpigmentation disease, which contains a methyl gallate oxidation product containing the HHDP dimethyl ester as an active ingredient.

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

In addition, the present invention provides a health functional food composition for preventing or improving melanin hyperpigmentation disease containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating melanin hyperpigmentation disease containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient.

The present invention relates to a method for producing a methyl gallate oxidation product containing HHDP dimethyl ester, and a composition for skin whitening containing the methyl gallate oxidation product prepared by the above production method or a compound isolated therefrom as an active ingredient. The methyl gallate oxidation reactant prepared by mixing methyl gallate and a pear-derived oxidase source and then performing an oxidation reaction includes an HHDP dimethyl ester compound, and the methyl gallate oxidation reactant and the HHDP dimethyl ester separated therefrom. Since its tyrosinase inhibitory activity and melanin synthesis inhibitory activity are superior to methyl gallate, it can be usefully used as a composition for skin whitening and prevention, improvement or treatment of melanin hyperpigmentation disease.

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 showing tyrosinase activity in melanocytes (B16F10) following treatment with the methyl gallate oxidation reactant of the present invention. Tyrosinase activity was induced by treatment with α-MSH. *, ** indicate a statistically significant decrease in tyrosinase activity compared to the methyl gallate treated group, * is p<0.05, ** is p<0.01.
Figure 3 shows the results showing the amount of melanin synthesis in cells (B16F10) following treatment with the methyl gallate oxidation reactant of the present invention. Melanin synthesis was induced by treatment with α-MSH. *, ** indicate a statistically significant decrease in melanin synthesis compared to the methyl gallate treatment group, * is p < 0.05, ** is p < 0.01.
Figure 4 shows the results showing tyrosinase activity in melanocytes (B16F10) according to MGP-1 (HHDP dimethyl ester) treatment of the present invention. Tyrosinase activity was induced by treatment with α-MSH. MG is the methyl gallate treatment group, and arbutin is the positive control group.
Figure 5 is a result showing the amount of melanin synthesis in melanocytes (B16F10) according to MGP-1 (HHDP dimethyl ester) treatment of the present invention. Melanin synthesis was induced by treatment with α-MSH. MG is the methyl gallate treatment group, and arbutin is the positive control group. *, ** indicate a statistically significant decrease in melanin synthesis compared to the methyl gallate treatment group, * is p < 0.05, ** is p < 0.01.
Figure 6 shows the results of Western blot confirmation of protein expression levels of factors related to whitening efficacy according to MGP-1 (HHDP dimethyl ester) treatment of the present invention (A) and the results of quantification (B). Arbutin is a positive control. *, ** indicate a statistically significant decrease in protein expression compared to the α-MSH treatment group alone, * is p < 0.05, ** is p < 0.01.
Figure 7 shows the results showing the cytotoxicity of the methyl gallate oxidation reactant of the present invention and MGP-1 (HHDP dimethyl ester) in melanocytes (B16F10).
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 method for producing a methyl gallate oxidation product comprising mixing methyl gallate and a pear-derived oxidase source and then performing an oxidation reaction.

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

In one embodiment of the present invention, the pear-derived oxidase source includes pear-derived polyphenol oxidase.

In one embodiment of the present invention, the manufacturing method is specifically

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;

more specifically

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 include a step of doing so,

Even more specifically

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 stirring at 30°C for 120 minutes to cause an oxidation reaction; It may be possible, but it is not limited to this.

Additionally, the present invention provides a methyl gallate oxidation reactant containing HHDP dimethyl ester prepared by the above production method.

Methyl gallate has the characteristic of being converted to HHDP dimethyl ester through an oxidation reaction with pear-derived oxidase source.

Additionally, the present invention provides a cosmetic composition for skin whitening containing a methyl gallate oxidation product including HHDP dimethyl ester as an active ingredient.

The methyl gallate oxidation reaction product is effective in skin whitening due to its excellent ability to inhibit tyrosinase activity and melanin synthesis.

In the cosmetic composition according to one embodiment of the present invention, the cosmetic composition for skin whitening 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, body. It may have any one formulation selected from the group consisting of 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. .

In addition, the present invention provides a health functional food composition for preventing or improving melanin hyperpigmentation disease, which contains a methyl gallate oxidation product containing the HHDP dimethyl ester as an active ingredient.

The health functional food composition of the present invention contains the compound of the present invention, which has excellent tyrosinase activity inhibition ability and melanin synthesis inhibition ability, as an active ingredient, and is effective in preventing or improving melanin hyperpigmentation disease.

As used herein, the term “hyperpigmentation” refers to the excessive increase of melanin in a specific area of the skin or hands or toenails, making it darker or darker than other areas. The melanin hyperpigmentation disease includes freckles, senile spots, liver spots, freckles, brown or black spots, solar pigment spots, cyanic melasma, hyperpigmentation after drug use, hyperpigmentation after inflammation due to wounds or dermatitis, or Including, but not limited to, gravidic chloasma.

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 skin whitening effect is significantly increased compared to using only cosmetics, making it more effective for melanin hyperpigmentation disease.

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 pharmaceutical composition for preventing or treating melanin hyperpigmentation disease, which contains a methyl gallate oxidation product containing the HHDP dimethyl ester as an active ingredient.

The “melanin hyperpigmentation disease” is the same as described above.

The pharmaceutical composition of the present invention contains as an active ingredient a methyl gallate oxidation product containing HHDP dimethyl ester of the present invention, which has excellent ability to inhibit tyrosinase activity and melanin synthesis, and is effective in preventing or treating melanin hyperpigmentation disease. There is.

The pharmaceutical composition of the present invention may further include appropriate carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions. The pharmaceutical dosage form of the composition according to the present invention can be used alone or in combination with other pharmaceutically active compounds, as well as in appropriate combinations.

The preferred dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. You can.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered topically on the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

Additionally, the present invention provides a cosmetic composition for skin whitening 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 product prepared by the production method of the present invention, but is not limited thereto, and can be separated 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.

In addition, the present invention provides a health functional food composition for preventing or improving melanin hyperpigmentation disease containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating melanin hyperpigmentation disease containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in more detail through preparation examples and examples. However, the following preparation examples and 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. 1.5mL of the enzyme source and 1.5mL of 200mM pyrogallol diluted in 50mM 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~ An oxidase source of 0.75 was used for subsequent oxide production. 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. Whitening effect of methyl gallate oxidation reactant

1) Measurement of tyrosinase inhibitory activity

In order to evaluate the whitening efficacy of the methyl gallate oxidation product prepared by the production method of the present invention, the inhibitory activity of tyrosinase, an enzyme involved in melanin synthesis, was measured. Mouse-derived melanocytes (B16F10) were cultured in DMEM (Dulbeco's modified) supplemented with 10% (v/v) FBS (fetal bovine serum) and 1% (v/v) penicillin/streptomycin (100U/mL). Using eagle'medium) medium, the cells were adapted to an incubator at 37°C and 5% CO ₂ and subcultured. Thereafter, the stabilized cells were inoculated into a 100 mm culture dish at a density of 2×10 ⁶ cells/dish and cultured for 24 hours. Samples of Preparation Example 1 were prepared at different concentrations, added at 2 mL each, and pretreated for 1 hour. Afterwards, each well was treated with 1 nM α-MSH for 48 hours to induce tyrosinase activity. After 48 hours, the medium was removed, washed with PBS, and 200 ㎕ of lysis buffer was added to the cells, and the cells were collected and lysed on ice for 30 minutes. Afterwards, the supernatant obtained by centrifugation was treated with 100mM of sodium phosphate (NaH ₂ PO ₄ ) and reacted at 37°C for 5 minutes, then 100mM of catechol solution was added and measured at 405nm. Absorbance was measured.

As a result, as shown in Figure 2, the methyl gallate oxidation reactant of the present invention inhibited the tyrosinase activity increased by α-MSH in a concentration-dependent manner and had superior tyrosinase inhibitory activity compared to methyl gallate.

2) Measurement of melanin synthesis inhibition activity

To evaluate the whitening efficacy of the methyl gallate oxidation reactant of the present invention, the melanin synthesis inhibitory activity was measured. Mouse-derived melanocytes (B16F10) were inoculated into a 12-well plate at 1×10 ⁶ cells/ml and cultured for 24 hours, and then pretreated with the oxidation reaction product of Preparation Example 1 for 1 hour at each concentration. Afterwards, each well was treated with 1 nM α-MSH for 48 hours to induce melanin synthesis. After 48 hours, the medium was removed, washed with PBS, and centrifuged to obtain a cell pellet. 200 ㎕ of a solution prepared with 10% (v/v) DMSO in 1N NaOH was added to the precipitated melanocytes and dissolved at 60°C for 1 hour, and the absorbance was measured at 405 nm. The amount of melanin synthesis was expressed as a percentage (%) compared to the α-MSH treatment group alone.

As a result, as shown in Figure 3, the methyl gallate oxidation reactant of the present invention inhibited the synthesis of melanin increased by α-MSH in a concentration-dependent manner, and the melanin synthesis inhibitory activity of the methyl gallate oxidation reactant compared to methyl gallate This was remarkable.

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

1) Measurement of tyrosinase inhibitory activity

The experiment was conducted in the same manner as that 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 tyrosinase inhibitory activity. Arbutin was used as a positive control group.

As a result, as shown in Figure 4, the tyrosinase inhibitory activity of MGP-1 was more significant than that of methyl gallate, and the effect was also superior to that of arbutin, a positive control.

2) Measurement of melanin synthesis inhibition 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 melanin synthesis inhibitory activity. Arbutin was used as a positive control group.

As a result, as shown in Figure 5, the melanin synthesis inhibitory activity of MGP-1 was more significant than that of methyl gallate, and the effect was also superior to that of arbutin, a positive control.

3) Measurement of protein expression level related to whitening

To examine the expression levels of whitening-related factors such as MITF (microphthalmia transcription factor), TRP-1 (dihydroxy indole carboxylic acid oxidase), TRP-2 (DOPA Chrome tautomerase), and tyrosinase protein, B16F10 cells were grown at 2×10 ^cells / After diluted to mL, it was 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, washing was performed a total of 3 times at 10-minute intervals using TBST, and then washed with MITF (Santacruz, 1:1000), TRP-1 (Santacruz, 1:1000), TRP-2 (Santacruz, 1:1000), and tyrosine. Sinase (Santacruz, 1:1000) and β-actin (Santacruz, 1:1000) primary antibodies were reacted at 4°C for 16 hours. Afterwards, the secondary antibodies of mouse anti-rabbit IgG HRP or bovine anti-goat IgG HRP were diluted 1:1000 and incubated at room temperature for a total of three times at 10-minute intervals using TBST. It was reacted 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, it was confirmed that the protein expression of tyrosinase, MITF, TRP-1, and TRP-2, which was increased by α-MSH treatment, was significantly reduced by MGP-1 treatment.

Example 4. Cytotoxicity assay

The cytotoxicity of methyl gallate oxidation reactant and MGP-1 was confirmed through MTT analysis. MTT analysis is performed by dehydrogenation to convert the yellow water-soluble substrate MTT tetrazolium into the blue-violet water-insoluble MTT formazan (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium bromide], the concentration of MTT formazan was measured using an ELISA plate reader (Tecan Austria GmBH, Grodig, Austria) at 540 nm.

Mouse-derived melanocytes (B16F10) were dispensed into a 96-well plate for cell culture at 5 × 10 ⁴ cells/well and cultured for 24 hours. Samples were prepared by concentration, 0.02 mL was added, and pretreated for 1 hour. α-MSH was added and cultured in an incubator at 37°C and 5% CO ₂ for 24 hours. The control group was cultured under the same conditions by adding the same amount of distilled water as the sample. 0.02 mL of MTT solution prepared at a concentration of 5 mg/mL was added here, and cultured for 4 hours. The culture medium was removed, 0.15 mL of DMSO was added to each well, and the mixture was incubated at room temperature for 30 minutes, and the absorbance was measured at 540 nm with an ELISA 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 melanocytes.

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 whitening effect compared to methyl gallate, so an attempt was made to establish a production method that maximizes the MGP-1 content. .

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

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 (12)

A method of producing a methyl gallate oxidation reactant comprising mixing methyl gallate and a pear-derived oxidase source and then subjecting the oxidation reaction. The method of claim 1, wherein the methyl gallate oxidation reactant includes HHDP dimethyl ester (hexahydroxydiphenic acid dimethyl ester) represented by the following formula (1).
[Formula 1]
The method of claim 1, wherein the manufacturing method 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 method for producing a methyl gallate oxidation reactant comprising the step of: A methyl gallate oxidation reactant comprising HHDP dimethyl ester prepared by the production method of any one of claims 1 to 3. A cosmetic composition for skin whitening containing a methyl gallate oxidation product containing the HHDP dimethyl ester of claim 4 as an active ingredient. A health functional food composition for preventing or improving melanin hyperpigmentation disease, comprising a methyl gallate oxidation product containing the HHDP dimethyl ester of claim 4 as an active ingredient. The method of claim 6, wherein the melanin hyperpigmentation disease is caused by freckles, senile spots, liver spots, freckles, brown or dark spots, solar pigment spots, cyanic melasma, hyperpigmentation after drug use, wounds, or dermatitis. A health functional food composition for preventing or improving melanin hyperpigmentation disease, characterized by post-inflammatory hyperpigmentation or gravidic chloasma. A pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease, comprising as an active ingredient a methyl gallate oxidation product containing the HHDP dimethyl ester of claim 4. A cosmetic composition for skin whitening containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient. The cosmetic composition for skin whitening according to claim 9, wherein the HHDP dimethyl ester is separated from the methyl gallate oxidation product of claim 4. A health functional food composition for preventing or improving melanin hyperpigmentation disease containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient. A pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease containing HHDP dimethyl ester or an acceptable salt thereof as an active ingredient.