KR102630951B1 - Composition for anti-inflammation comprising 5-hydroxymaltol - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition containing 5-hydroxymaltol and a composition for preventing, improving or treating inflammatory diseases. According to the present invention, 5-hydroxymaltol is responsible for producing NO and expressing iNOS. , It has the effect of suppressing ROS accumulation and acting as an anti-inflammatory agent by regulating MAPK, NF-κB, and Nrf-2/HO-1 signaling, so it can be usefully used to prevent, improve, or treat inflammatory diseases.

Description

Anti-inflammatory composition comprising 5-hydroxymaltol {Composition for anti-inflammation comprising 5-hydroxymaltol}

The present invention relates to an anti-inflammatory composition containing 5-hydroxymaltol and a composition for preventing, improving or treating inflammatory diseases.

The innate or non-specific immune response serves as the first barrier against invasion by harmful substances and organisms in the body and involves macrophages and inflammatory biomolecules. Inflammation occurs in all human tissues and is a beneficial response to humans that exerts a protective effect against external pathogens and internal damage. However, excessive inflammation causes chronic diseases such as rheumatoid arthritis, atherosclerosis, and diabetes. Macrophages develop in tissues throughout the body and not only phagocytose foreign substances and dead cells, but also respond to various infectious signals. Macrophages possess toll-like receptors that serve as the primary host defense against infection. Lipopolysaccharide (LPS) is a major component of the cell wall of Gram-negative bacteria and induces strong cell signals from macrophages in the innate immune system. LPS activates macrophages by binding to toll-like receptor 4 (TLR 4), and activates the nuclear factor κB (NF-κB) pathway and three mitogen-activated protein kinases. protein kinase (MAPK) pathway, i.e., extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK)/stress-activated protein kinase (stress- It induces several intracellular signaling pathways, including activated protein kinases) and p38 MAPK. Macrophages are activated upon LPS treatment, causing excessive production of inflammatory mediators, cytokines, and reactive oxygen species (ROS). Nitric oxide (NO) induced by NO synthase is a proinflammatory mediator, causing oxidative stress and inflammation. In LPS-treated macrophages, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are overexpressed, promoting the pathogenesis of inflammatory diseases. Heme oxygenase 1 (HO-1) plays an important role in anti-inflammatory and iron homeostasis. Expression of HO-1 has been considered an adaptive (acquired) cellular response to inflammation and oxidative damage, and the nuclear factor erythroid 2-related factor 2, which plays an important role in cellular protection against inflammation and oxidative stress. It is known to be mediated by factor 2, Nrf2).

Meanwhile, beet (Beta vulgaris) is also called root vegetable, iris bean, and fire beet, and is native to southern Europe and North Africa along the Mediterranean coast. It is a popular crop that can be easily grown at home in foreign countries because it is relatively easy to cultivate and the entire plant can be used. In Korea, it is mainly produced in Icheon, Gyeonggi-do, Pyeongchang, Gangwon-do, and Jeju Island. The red beet root is similar to Ganghwa turnip in Korea. 8% of beets are made up of chlorine, which purifies the liver and is effective in skeletal formation and infant development. In addition, it contains a large amount of iron and vitamins, which helps produce red blood cells, purifies the blood, and improves blood circulation disorders. It is known to be good for menstrual irregularities or menopausal women. In addition, beets are known to not only prevent stomach damage and protect the stomach mucosa, but are also effective in aging, anemia, stress, vision recovery, cell replication, and detoxification.

The present inventors prepared beet fermentation products, isolated and purified compounds showing antioxidant activity, and found that the purified compounds can be applied as anti-inflammatory agents by regulating NF-κB, MAPK, and Nrf-2 signals. was confirmed and the present invention was completed.

Republic of Korea Patent Publication No. 10-2021-0094745

The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases.

Additionally, an object of the present invention is to provide a food composition and a cosmetic composition for preventing or improving inflammatory diseases.

Additionally, an object of the present invention is to provide an anti-inflammatory composition.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases containing 5-hydroxymaltol, a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient. do.

In addition, the present invention provides a food composition for preventing or improving inflammatory diseases containing 5-hydroxymaltol, a foodologically acceptable salt thereof, or a solvate thereof as an active ingredient.

Additionally, the present invention provides a cosmetic composition for preventing or improving inflammatory diseases containing 5-hydroxymaltol or a cosmetically acceptable salt or solvate thereof as an active ingredient.

In addition, the present invention provides an anti-inflammatory composition containing 5-hydroxymaltol, a salt thereof, or a solvate thereof as an active ingredient.

According to the present invention, 5-hydroxymaltol isolated from the beet fermentation product of the present invention inhibits NO production, iNOS expression, ROS accumulation, MAPK signaling activation, and NF-κB signaling activation in macrophages. In addition, since it has the effect of upregulating Nrf-2/HO-1 signaling, it can be usefully used as an anti-inflammatory agent and for the prevention, improvement or treatment of inflammatory diseases.

Figure 1 is a schematic diagram showing the process of separation and purification of an active compound (5-hydroxymaltol) from beet fermentation product prepared by fermentation of Lactobacillus rhamnosus GG strain.
Figure 2 is data showing the results of HPLC analysis of beet fermentation product prepared by fermentation of Lactobacillus rhamnosus GG strain and active compounds purified therefrom:
(A) HPLC chromatograms of unfermented beets (0 day) and beet ferments (5 days) (red boxes indicate new peaks); and
(B) HPLC chromatogram of isolated and purified 5-hydroxymaltol.
Figure 3 is a diagram showing the purification process using C-18 Prep TLC from a fraction of beet fermentation product prepared using Lactobacillus rhamnosus GG and the results of TLC plate analysis of the purified material.
Figure 4 is a diagram showing the chemical structure, antioxidant activity, and cytotoxicity analysis results of 5-hydroxymaltol derived from beet fermentation product prepared using Lactobacillus rhamnosus GG (value = mean ± SD; n =3; ** p<0.01; *** p<0.001):
(A) Molecular structure of 5-hydroxymaltol;
(B) DPPH radical scavenging activity of 5-hydroxymaltol; and
(C) RAW 264.7 macrophages were exposed to different concentrations of 5-hydroxymaltol for 1 day and cell proliferation (cell viability) was examined using the MTS assay.
Figure 5 is a graph showing the inhibitory effect of 5-hydroxymaltol on LPS-induced NO production and iNOS expression in RAW 264.7 cells (value = mean ± standard deviation; n = 3; * p <0.05; ** p <0.01;***p<0.001):
(A) Results of measuring NO production following treatment with LPS (1 μg/mL) and/or 5-hydroxymaltol (200, 500, and 1000 μM); and
(B) Results of measuring iNOS mRNA and protein levels following treatment with LPS (1 μg/mL) and/or 5-hydroxymaltol (200, 500, and 1000 μM).
Figure 6 is a graph showing the inhibitory effect of 5-hydroxymaltol on LPS-induced IL-1β and TNF-α production and IL-1β and TNF-α mRNA expression in RAW 264.7 cells (value = mean ± standard) Deviation; n=3; * p<0.05; ** p<0.01; *** p<0.001):
(A) Results of measuring the mRNA expression and production levels of IL-1β following treatment with LPS (1 μg/mL) and/or 5-hydroxymaltol (200, 500, and 1000 μM); and
(B) Results of measuring the mRNA expression and production levels of TNF-α according to treatment with LPS (1 μg/mL) and/or 5-hydroxymaltol (200, 500, and 1000 μM).
Figure 7 is a diagram showing the inhibitory effect of 5-hydroxymaltol on the nuclear translocation of NF-κB (p65) induced by LPS in RAW 264.7 cells (value = mean ± standard deviation; n = 3; * p <0.05;**p<0.01).
Figure 8 is a diagram showing the inhibitory effect of 5-hydroxymaltol on LPS-induced MAPK activation in RAW 264.7 cells (value = mean ± standard deviation; n = 3; * p < 0.05).
Figure 9 is a diagram showing the inhibitory effect of 5-hydroxymaltol on LPS-induced ROS accumulation in RAW 264.7 cells (value = mean ± standard deviation; n = 3; * p <0.05; *** p < 0.001 ).
Figure 10 is a diagram showing the effect of increasing Nrf2 and HO-1 protein levels by 5-hydroxymaltol in RAW 264.7 cells (value = mean ± standard deviation; n = 3; * p <0.05; ** p < 0.01) :
(A) Results of Western blot analysis of the expression levels of HO-1 and Nrf2 according to 5-hydroxymaltol treatment time for total protein; and
B) Results of immunoblot analysis of the expression level of Nrf2 in nuclear and cytoplasmic proteins depending on 5-hydroxymaltol treatment.
Figure 11 is a schematic diagram showing the mechanism of the anti-inflammatory and cytoprotective effects of 5-hydroxymaltol against LPS-induced inflammation.

Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

Throughout this specification, '%' used to indicate the concentration of a specific substance means (w/w) % for solid/solid, (w/v) % for solid/liquid, and Liquid/liquid is (v/v) %.

In one aspect, the present invention provides a pharmaceutical agent for the prevention or treatment of inflammatory diseases comprising 5-hydroxymaltol represented by the following formula (1) or a pharmaceutically acceptable salt or solvate thereof as an active ingredient: It relates to the composition:

5-hydroxymaltol of the present invention may be manufactured by synthesis, or may be purchased and used as a commercially available product, but is preferably extracted, separated, and purified from beet fermentation product. It may be obtained by doing so.

In one embodiment, the fermented beet product of the present invention includes the steps of (1) preparing a medium containing lactic acid bacteria at a concentration of 7×10 ⁵ CFU/ml; and (2) mixing beet powder with the medium at a concentration of 2% (w/v), culturing and fermenting at 25-35°C for 3-7 days, and then centrifuging to obtain a supernatant. It may be manufactured. The medium may be MRS medium, but is not limited thereto, and in addition to the above medium, various commercial media that can be used for culturing lactic acid bacteria can be used. The lactic acid bacteria may be Lactobacillus rhamnosus , and more preferably Lactobacillus rhamnosus GG.

In one embodiment, 5-hydroxymaltol of the present invention is prepared by: (a) preparing beet fermentation product; (b) preparing an extract by mixing the fermented beet product with an ethyl acetate (EtOAc) solvent at a volume ratio of 2:1 to 1:2; and (c) purifying 5-hydroxymaltol by performing at least one chromatography selected from the group consisting of column chromatography, preparative TLC, and preparative HPLC; separating from the beet fermentation product and It may be refined. In step (c), column chromatography, preparative TLC, and preparative HPLC may be performed sequentially to prepare each fraction and purify 5-hydroxymaltol therefrom. The column chromatography may be performed using one or more fillers selected from the group consisting of ODS, silica gel, and Sephadex, and preferably, ODS column chromatography, silica gel column chromatography, and Sephadex column chromatography. It may be performed sequentially. The preparative TLC and preparative HPLC may be performed using an ODS-coated glass plate and an ODS-filled column, respectively.

In one embodiment, the composition of the present invention can inhibit nitric oxide (NO) production or secretion and ROS production or accumulation, and can inhibit the expression or production of iNOS protein or gene.

In one embodiment, the composition of the present invention can inhibit the expression or production of pro-inflammatory cytokines, and in particular, can inhibit the expression or production of proteins or genes of TNF-α and IL-1β.

In one embodiment, the composition of the present invention is capable of inhibiting the NF-κB signaling pathway, inhibiting the activation and nuclear localization of NF-κB, and inhibiting the expression or production of the protein or gene of NF-κB p65. And it can increase the expression or production of IκB-α protein or gene.

In one embodiment, the composition of the present invention can inhibit the MAPK signaling pathway, inhibit MAPK activation, and particularly inhibit phosphorylation of ERK, p38, and JNK.

In one embodiment, the composition of the present invention can increase Nrf-2/HO-1 signaling, and in particular, can increase the expression or production of HO-1 and Nrf-2 proteins or genes.

In one embodiment, The inflammatory diseases include arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, dermatitis, allergy, atopy, periodontitis, gingivitis, and otitis media. , sore throat, arthritis, rheumatoid arthritis, tendonitis, tenosynovitis, degenerative nerve inflammation, and acute to chronic inflammatory diseases.

In one embodiment, the composition of the present invention may include 5-hydroxymaltol at a concentration of 0.01 to 5,000 μM, preferably 0.1 to 1,500 μM, more preferably 50 to 1,500 μM. It is more preferable to include it at a concentration of 50 to 1,100 μM.

In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of an inflammatory disease by administering the pharmaceutical composition according to the present invention, and "treatment" refers to any action that inhibits or delays the occurrence, spread, and recurrence of an inflammatory disease by administering the pharmaceutical composition according to the present invention. It refers to any action that improves or beneficially changes the symptoms of an inflammatory disease. Anyone with ordinary knowledge in the technical field to which the present invention pertains can refer to the data presented by the Korean Medical Association, etc. to know the exact criteria for diseases for which our composition is effective and to determine the degree of improvement, improvement, and treatment. will be.

The term "therapeutically effective amount" used in combination with an active ingredient in the present invention refers to an amount effective in preventing or treating inflammatory diseases, and the therapeutically effective amount of the composition of the present invention is determined by several factors, such as the method of administration. , may vary depending on the target area, patient condition, etc. Therefore, when used in the human body, the dosage must be determined as appropriate by considering both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. These considerations in determining an effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceutically effective amount" refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's Factors including health status, type of inflammatory disease, cause of inflammatory disease, severity, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, treatment period, combination or drugs used simultaneously, and It may be determined based on other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition of the present invention may contain a carrier, diluent, excipient, or a combination of two or more commonly used in biological products. As used in the present invention, the term “pharmaceutically acceptable” means that the composition exhibits non-toxic properties to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compounds described in, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group including oral formulations, topical formulations, suppositories, sterile injectable solutions, and sprays, with oral or injectable formulations being more preferable.

As used in the present invention, the term "administration" means providing a predetermined substance to an individual or patient by any appropriate method, and is administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally) according to the desired method. Alternatively, it can be applied topically as an injection formulation) or orally administered, and the dosage range varies depending on the patient's weight, age, gender, health status, diet, administration time, administration method, excretion rate, and severity of the disease. Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are used. etc. may be included together. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc. The pharmaceutical composition of the present invention may be administered by any device capable of transporting the active agent to target cells. Preferred administration methods and formulations include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection. Injections include aqueous solvents such as physiological saline solution and Ringer's solution, non-aqueous solvents such as vegetable oil, higher fatty acid esters (e.g., ethyl oleate, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). It can be manufactured using stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulphite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH adjustment, and agents to prevent microbial growth. It may contain pharmaceutical carriers such as preservatives (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

As used in the present invention, the term "individual" refers to monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits, including humans who have or may develop the inflammatory disease. Or, it refers to all animals including guinea pigs, and the diseases can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to the subject. The pharmaceutical composition of the present invention can be administered in combination with existing therapeutic agents.

The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, white sugar, dextrose, sorbitol, and talc can be used. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The pharmaceutical composition of the present invention can also be provided in the form of an external preparation containing 5-hydroxymaltol as an active ingredient. When the pharmaceutical composition for preventing or treating inflammatory diseases of the present invention is used as an external skin agent, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, and foaming agents are added. agent), fragrance, surfactant, water, ionic emulsifier, non-ionic emulsifier, filler, metal ion sequestrant, chelating agent, preservative, vitamin, blocking agent, wetting agent, essential oil, dye, pigment, hydrophilic activator, lipophilic It may contain adjuvants commonly used in the field of dermatology, such as active agents or lipid vesicles or any other ingredients commonly used in topical skin preparations. Additionally, the ingredients may be introduced in amounts commonly used in the field of dermatology.

When the pharmaceutical composition for preventing or treating inflammatory diseases of the present invention is provided as an external skin preparation, it may be in the form of an ointment, patch, gel, cream, or spray, but is not limited thereto.

In one aspect, the present invention provides a food composition for preventing or improving inflammatory diseases comprising 5-hydroxymaltol represented by the following formula (1) or a foodologically acceptable salt or solvate thereof as an active ingredient: It's about:

[Formula 1]

.

In addition to containing 5-hydroxymaltol, which is an active ingredient, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional food compositions.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements, etc. There is.

In addition, the food composition contains, in addition to the extract as an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid, and salts thereof. , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. In the present invention, 'health functional food composition' refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food, and refers to food that has It means taking it for the purpose of controlling nutrients or obtaining useful health effects such as physiological effects. The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions of the Food Additives Code and General Test Methods approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards. Items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations. For example, health functional foods in the form of tablets are prepared by granulating a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, and other additives in a conventional manner, adding a lubricant, etc., and compression molding, or The mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary. Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of the active ingredient of the present invention mixed with additives such as excipients, and soft capsules can be prepared by mixing the active ingredient of the present invention with additives such as excipients. It can be manufactured by filling the mixture with a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary. The health functional food in the form of a ring can be prepared by molding a mixture of the active ingredient of the present invention, excipients, binders, disintegrants, etc., using a known method. If necessary, it can be coated with white sugar or other coating agents. Alternatively, the surface can be coated with substances such as starch or talc. Health functional food in the form of granules can be manufactured into granules by mixing a mixture of excipients, binders, disintegrants, etc. of the active ingredients of the present invention by a known method, and may contain flavoring agents, flavoring agents, etc., if necessary. You can.

The health functional food composition according to the present invention can be manufactured in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled foods, jam, mamalade, etc.), fish, meat and their processed foods (e.g. ham, sausages, etc.) corned beef, etc.), bread and noodles (e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine , can be produced by adding 5-hydroxymaltol of the present invention to vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., soybean paste, soy sauce, sauce, etc.). In addition, nutritional supplements are not limited to this, but can be prepared by adding 5-hydroxymaltol of the present invention to capsules, tablets, pills, etc. In addition, the health functional food is not limited to this, but for example, the 5-hydroxymaltol itself of the present invention can be manufactured in the form of tea, juice, and drink and liquefied, granulated, encapsulated, and used for drinking (health beverage). It can be powdered and consumed. Additionally, in order to use 5-hydroxymaltol of the present invention in the form of a food additive, it can be prepared and used in the form of powder or concentrate. Additionally, it can be prepared in the form of a composition by mixing 5-hydroxymaltol of the present invention with known active ingredients known to be effective in preventing or improving inflammatory diseases.

When using the 5-hydroxymaltol of the present invention as a health drink, the health drink composition may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional drink. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.

In addition, 5-hydroxymaltol of the present invention can be contained as an active ingredient in a food composition for preventing or improving inflammatory diseases, and the amount is not particularly limited to an amount effective to achieve the effect of preventing or improving inflammatory diseases, but the total It is preferably 0.01 to 100% by weight based on the total weight of the composition. The health functional food composition of the present invention can be prepared by mixing 5-hydroxymaltol with other active ingredients known to be effective in inflammatory diseases.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, and preservatives. , may contain glycerin, alcohol, or carbonating agent. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In one aspect, the present invention provides a cosmetic composition for preventing or improving inflammatory diseases comprising 5-hydroxymaltol represented by the following formula (1) or a cosmetically acceptable salt or solvate thereof as an active ingredient: It's about:

[Formula 1]

.

The “cosmetic composition” of the present invention can be prepared by containing a cosmetically effective amount of the 5-hydroxymaltol of the present invention described above and a cosmetically acceptable carrier.

In the present invention, the term “cosmetically effective amount” refers to an amount sufficient to achieve the inflammatory disease prevention or improvement effect of the composition of the present invention described above.

The appearance of the cosmetic composition contains a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, such as solutions, gels, solids, pasty anhydrous products, emulsions obtained by dispersing the oil phase in the water phase, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) and It may be provided in the form of a non-ionic vesicular dispersion, or in the form of a cream, skin, lotion, powder, ointment, spray or concealer stick. These compositions can be prepared according to conventional methods in the art. The composition according to the invention can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition according to an embodiment of the present invention is not particularly limited in its formulation, and includes, for example, softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, and cleansing lotion. It can be formulated into cosmetics such as cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil, and body essence.

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

When the formulation of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the cosmetic composition is a spray, chlorofluorohydride may be used additionally. 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, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, and sarcosinate are used as carrier ingredients. , fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linoline derivative, or ethoxylated glycerol fatty acid ester can be used.

The cosmetic composition of the present invention includes skin, lotion, cream, essence, pack, foundation, color cosmetics, sunscreen, two-way cake, face powder, compact, makeup base, skin cover, eye shadow, lipstick, lip gloss, lip fix, and eyebrow pencil. , can be applied to cosmetics such as lotion and detergents such as shampoo and soap.

The cosmetic composition according to an embodiment of the present invention may further include functional additives and components included in general cosmetic compositions in addition to the 5-hydroxymaltol. The functional additive may include ingredients selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids, and seaweed extract.

In addition to the above-mentioned functional additives, the cosmetic composition of the present invention may also contain components included in general cosmetic compositions, if necessary. Other ingredients included include oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, disinfectants, antioxidants, plant extracts, pH adjusters, alcohol, pigments, fragrances, and blood circulation agents. Examples include accelerators, cooling agents, limiting agents, and purified water.

In one aspect, the present invention relates to an anti-inflammatory composition comprising 5-hydroxymaltol represented by Formula 1 above or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.

In one embodiment, the anti-inflammatory composition may be one or more selected from the group consisting of pharmaceutical compositions, cosmetic compositions, and food compositions.

In the present invention, “anti-inflammatory” may be used interchangeably with “inhibiting or improving inflammation,” and may mean any action that alleviates the inflammatory response.

The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

Example 1. Separation and purification of 5-hydroxymaltol from beet fermentation product

1-1. Preparation of fermented beets

Beet powder from Jeju was obtained, washed with distilled water, and ground with a blender (Shinil, Seoul, Korea) to prepare beet powder. The strain for fermentation, Lactobacillus rhamnosus GG (KCTC 3237), was purchased and used from the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC, Seoul, Korea). Stock cultures were stored at -80°C in Man, Rogosa, and Sharpe (MRS) medium (Difco, Detroit, USA) containing 20% (v/v) glycerol.

Lactobacillus rhamnosus GG strain was inoculated into MRS medium at a concentration of 7×10 ⁵ CFU/ml and pre-cultured for 1 day at 180 rpm and 30°C. The beet powder (1kg) was cultured with MRS medium (50L) containing Lactobacillus rhamnosus GG strain at 180 rpm and 30°C for 5 days, and the fermented medium was centrifuged to produce a supernatant, i.e., beet fermentation product. (50L) was obtained.

The fractionation and purification process for isolating the active compound from the beet fermentation product is shown in Figure 1, and the specific method is as described below.

Meanwhile, as a result of performing HPLC analysis on the beets before fermentation (0 day) and the beets after fermentation (5 days), a peak that was not visible in the HPLC spectrum of unfermented beets appeared in the HPLC spectrum of fermented beets at about 7 minutes, indicating fermentation. It was confirmed that a new material was created (Figure 2A).

1-2. Preparation of EA extract from beet fermentation

An EA extract of the beet fermented product was obtained by mixing and extracting 50L of the beet fermented product obtained by the above method with 50L of ethyl acetate (EA). After concentrating the EA extract, it was solubilized with methanol and used in the following examples.

1-3. Primary fractionation using ODS chromatography

The EA extract solubilized with methanol was loaded on an RP-18 manual column (ODS C-18 open column, 10 × 14 cm), and ODS (octadecyl silica) chromatography was performed on 0% methanol (water), 10% Fractionated with methanol and 20% methanol. As a result of examining the antioxidant activity of the three primary fractions using DPPH (diphenyl-picrylhydrazyl) scavenging activity analysis, the 0% methanol fraction showed an antioxidant effect and was used as a sample for the secondary fraction.

1-4. Secondary fractionation using silica gel chromatography

The 0% methanol fraction was concentrated, then loaded on a column (30 × 450 mm) filled with silica gel 60 (0.035-0.2 mm) resin (MERCK, Darmstadt, Germany) and chloroform:methanol (10:1, Four fractions (fractions 1, 2, 3, and 4) were obtained through silica gel chromatography using v/v) as an elution solvent. As a result of examining the antioxidant activity of the four fractions, fraction No. 2 showed an antioxidant effect, and it was concentrated and used as a sample for the third fractionation.

1-5. Third fractionation using Sephadex G-10

Fraction No. 2 was loaded onto a column (3 × 45 cm) filled with Sephadex G-10 (Sephadex G-10 ^TM , 40-120 μm) powder (Cytiva, Marlborough, MA, USA) gel and water was used as an elution solvent. Sephadex gel chromatography was performed using. From this, two fractions (fraction No. 2-1 and fraction No. 2-2) were separated and their antioxidant activity was examined. As a result, the second fraction (fraction No. 2-2) showed an antioxidant effect, and it was concentrated to form the fourth fraction. It was used as a sample for fractionation.

1-6. Fourth fractionation using ODS C18 prep TLC

The fraction 2-2 was spotted on a preparative TLC glass plate coated with ODS C-18 (Silica gel 60 RP-18 F ₂₅₄ plate, 10×20 cm; Sigma, Burlington, IA, USA) and methanol was added to the TLC chamber. :Water (1:1, v/v) was used as a developing solvent to allow the material to be separated in the form of a band. After the development was completed, the glass plate was taken out of the chamber and the fluorescence was analyzed with a UV detector (UV254nm), and it was confirmed that the material was fractionated into two bands (material 1 and material 2) (Figure 3). Each gel containing the fractionated band portion was separated by scraping from the glass plate using a knife, and each was collected in a 50 mL conical tube. Next, 20 mL of methanol was added to each gel containing the separated materials, the mixture was vortexed, and centrifuged to purify material No. 1 and material No. 2. As a result of examining the antioxidant activity of each substance, substance No. 1 showed excellent antioxidant effect, and HPLC was performed for final separation and purification of the substance.

1-7. Purification of active compounds using Prep HPLC

Material No. 1 obtained through the ODS C18 prep TLC was injected into preparative high-performance liquid chromatography (preparative HPLC). HPLC analysis was performed using Shimadzu HPLC (Shimadzu, Tokyo, Japan). HPLC separation was performed using an RP-18 (10 × 250 mm) column, and the separated material was sieved through a 0.2 μm filter. At this time, the injection volume was 0.5 mL, the flow rate was 2.5 mL/min, the column temperature at 220 and 254 nm was room temperature, the mobile phase consisted of water (solvent A) and acetonitrile (solvent B), and in the case of gradient elution, the solvent B was initially set at 0%, increased to 5% at 20 minutes, and increased to 100% at 30 minutes. The peak of the final isolated and purified active compound was displayed at 36 minutes (Figure 2B).

1-8. Structural analysis of effective compounds

Finally, in order to analyze the structure of the separated and purified effective compound, the structure was identified through NMR (nuclear magnetic resonance) analysis, and the molecular weight was measured with an ESI-Mass spectrometer, confirming that the molecular weight was 143 g/mol. Through this, it was confirmed that the substance purified from the beet fermentation product of the present invention was 5-hydroxymaltol (Figure 4A).

1-9. Analysis of the antioxidant effect of 5-hydroxymaltol and its effect on macrophage proliferation

The antioxidant effect of 5-hydroxymaltol was investigated through DPPH scavenging activity analysis. First, 5-hydroxymaltol (0, 100, 200, 400, 500μM) or 10mM N-acetyl-L-cysteine (NAC) was dispensed into each well of a 96-well plate, and 200μM DPPH solution (4mM stock, methanol ) were mixed and incubated for 30 minutes. Absorbance at OD ₅₁₇ was measured using a VersaMax ELISA (enzyme-linked immunosorbent assay) reader (Molecular Devices, San Jose, CA, USA), and DPPH scavenging activity was calculated using the formula:

(Here, Sample is the absorbance of the sample, Control is the absorbance of the control group treated with only DPPH, and Blank is the absorbance of the sample not treated with DPPH.)

As a result of the DPPH scavenging activity analysis, 5-hydroxymaltol showed a strong antioxidant effect in a concentration-dependent manner. In particular, it increased to 42.5% at a high concentration of 500 μM, showing an antioxidant effect similar to that of NAC, a positive control (Figure 4B).

In addition, to confirm the effect of 5-hydroxymaltol on cell proliferation of the macrophage cell line RAW 264.7, RAW 264.7 macrophages were obtained from the Korea Cell Line Bank (Seoul, Korea) and MTS analysis was performed. Macrophages were grown in high-glucose DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal calf serum (Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (Hyclone) in a 5% CO ₂ atmosphere and at 37°C. cultured under conditions. RAW 264.7 cells (3×10 ⁵ cells/mL) were seeded in 96-well plates and cultured for 1 day, then incubated with increasing 5-hydroxymaltol concentrations (0, 50, 100, 200, 400, 500, 750, and 1000 μM) for 1 day. Using CellTiter 96 AQueous One Solution (Promega, Madison, WI, USA) according to the manufacturer's protocol, mix with the culture medium treated with 5-hydroxymaltol at a ratio of 1:5 (solution: culture medium, v/v). Afterwards, 100 μL of the mixture was placed in a 96-well plate and reacted at 37°C for 2 hours. Absorbance was measured using a VersaMax ELISA reader at OD ₄₉₀ , and cell viability was investigated from this.

As a result of culturing RAW 264.7 macrophages with various concentrations of 5-hydroxymaltol (up to 1000 μM), treatment with 5-hydroxymaltol at a high concentration of 1000 μM did not have any effect on cell proliferation (Figure 4C).

Example 2. Inhibitory effect of 5-hydroxymaltol on NO production and iNOS expression

2-1. Analysis of NO production inhibition effect

Nitric oxide (NO) is a biological mediator produced in LPS-stimulated macrophages. To evaluate the inhibitory effect of 5-hydroxymaltol on NO production induced by LPS (lipopolysaccharide from E. coli O111:B4; InvivoGen, San Diego, CA, USA) in RAW 264.7 cells, with or without LPS. NO assay was performed by treating RAW 264.7 macrophages with 5-hydroxymaltol (200, 500, and 1000 μM) for 24 hours.

Specifically, RAW 264.7 cells (3×10 ⁵ cells/mL) were cultured in a 6-well plate and incubated for 1 day. Cells were treated with 5-hydroxymaltol (0 and 500 μM) without LPS treatment or with various concentrations of 5-hydroxymaltol (0, 200, 500, and 1000 μM) for 24 h before treatment with LPS (1 μg/mL). did. The amount of NO production in the upper culture medium was measured using the NO Plus Detection kit (iNtRON Biotechnology, Gyeonggi-do, Korea). 100 μL of culture medium or nitrite standard was dispensed into each well of a 96-well plate, and 50 μL of N1 buffer (sulfanilamide in buffer) was added to induce a preliminary reaction. The 96-well plate was then incubated for 20 min and the mixture was reacted with 50 μL of N2 buffer (naphthyl-ethylenediamine in buffer). After incubating the mixture for 10 minutes, NO production was measured by the absorbance value at OD ₅₆₀ nm using a VersaMax ELISA reader.

As a result, LPS stimulation induced a 51-fold increase in NO concentration, while cells treated with 200, 500, and 1000 μM 5-hydroxymaltol showed a decrease of 34%, 41%, and 54%, respectively, compared to the LPS-treated control group ( Figure 5A). Therefore, NO production is induced by LPS, and 5-hydroxymaltol appears to reduce NO production induced by LPS.

2-2. Analysis of iNOS protein expression inhibition effect

The protein level of inducible NO synthase (iNOS) was evaluated using Western blot analysis. Macrophages treated or untreated with 5-hydroxymaltol were treated with LPS (1 μg/mL) for 24 hours, then protein lysates were separated and subjected to Western blot analysis.

In addition, total RNA was isolated from RAW 264.7 macrophages treated or not with 5-hydroxymaltol and then stimulated with LPS (1 μg/mL) for 1 day, and subjected to reverse transcription-quantitative polymerase chain reaction (RT). The mRNA level of iNOS was examined through -qPCR). Total RNA was purified using the TaKaRa MiniBEST RNA Extraction Kit (TaKaRa, Kyoto, Japan) and RT-qPCR using RNA-direct™ SYBR® Green Realtime qPCR Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer's protocol. was carried out. RT-qPCR mixture contains 10 μL RNA-direct SYBR Green Realtime qPCR Master Mix, 1 μL 50mM Mn(OAc) ₂ , 2 μL template RNA (100 ng/μL), 2 μL specific primer-F (10 ng/μL), 2 μL It contained specific primer-R (10 ng/μL) and 3 μL of nuclease-free H ₂ O. Specific primers for iNOS used in the RT-PCR were purchased from Bioneer (Daejeon, Korea).

As a result, as shown in Figure 5B, iNOS protein expression was increased in RAW 264.7 cells stimulated with LPS, while 5-hydroxylmaltol highly suppressed this. Additionally, analysis of the transcript level of iNOS in RAW 264.7 cells showed that LPS increased the iNOS transcript level, while 5-hydroxylmaltol decreased the increased iNOS transcript level (Figure 5B). LPS treatment increased the messenger RNA (mRNA) and protein levels of iNOS by 21- and 14-fold, respectively, whereas 5-hydroxymaltol (1000 μM) decreased the mRNA and protein levels of iNOS by 46%, respectively, compared to the LPS-treated control group. and decreased by 61%.

Therefore, it was confirmed that 5-hydroxymaltol can inhibit NO production through down-regulation of iNOS.

Example 3. Inhibitory effect of 5-hydroxymaltol on production and expression of TNF-α and IL-1β

LPS-activated macrophages produce proinflammatory cytokines and growth factors. Evaluating the effect of 5-hydroxymaltol on increased protein and transcript levels of TNF-α and IL-1β in LPS-stimulated RAW 264.7 cells using enzyme-linked immunosorbent assay (ELISA) and RT-qPCR analysis. did.

First, for ELISA analysis, RAW 264.7 macrophages were cultured in a 6-well plate. Attached cells were pretreated with various concentrations of 5-hydroxymaltol (0, 200, 500, and 1000 μM) for 24 h and stimulated with LPS (1 μg/mL) for 24 h. Measurements of TNF-α and IL-1β were assessed in the supernatants. The amount of IL-1β was measured and quantified using the IL-1β Mouse ELISA Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol, and the amount was measured and quantified using the ELISA MAX™ kit (BioLegend, San Diego, CA, USA). The amount of TNF-α was measured and quantified.

Meanwhile, for RT-qPCR analysis, RAW 264.7 cells were cultured in 6-well plates containing 5-hydroxymaltol (1000 μM), treated with LPS (1 μg/mL) for 1 day, and then incubated with TaKaRa according to the manufacturer's protocol. Total RNA was purified using the MiniBEST RNA Extraction Kit (TaKaRa, Kyoto, Japan). RT-qPCR analysis was performed in the same manner as Example 2-2. Specific primers for TNF-α and IL-1β used in the RT-PCR were purchased from Bioneer (Daejeon, Korea).

The results showed that in RAW 264.7 cells, LPS treatment increased the mRNA and protein levels of IL-1β by 101-fold and 1.6-fold, respectively, whereas 5-hydroxymaltol (1000 μM) increased the mRNA and protein levels by 61% and 23%, respectively, compared to LPS-treated controls. down-regulated (Figure 6A). LPS treatment increased the mRNA and protein levels of TNF-α by 5.9- and 11.9-fold, respectively, compared to the untreated control, while 5-hydroxymaltol (1000 μM) increased the mRNA and protein levels of TNF-α compared to the LPS-treated control. downregulated by 42% and 43%, respectively (Figure 6B). These results indicate that 5-hydroxymaltol inhibits IL-1β and TNF-α cytokine production through downregulation of IL-1β and TNF-α genes.

Additionally, LPS treatment significantly increased the production of TNF-α and IL-1β in RAW 264.7 cells, whereas 5-hydroxymaltol treatment decreased LPS-stimulated TNF-α and IL-1β production in a concentration-dependent manner. LPS treatment increased the transcript levels of TNF-α and IL-1β genes, whereas 5-hydroxymaltol pretreatment downregulated LPS-stimulated TNF-α and IL-1β gene expression (Figure 6A, 6B).

From these results, it was confirmed that 5-hydroxymaltol reduced the immune response of RAW 264.7 cells by suppressing LPS-stimulated cytokine production.

Example 4. Inhibitory effect of 5-hydroxymaltol on LPS-stimulated NF-κB activation

Nuclear translocation of NF-κB is known to increase the expression of inflammation-related genes in macrophages. Therefore, immunoblot analysis was performed to determine whether 5-hydroxymaltol could inhibit nuclear translocation of NF-κB.

RAW 264.7 macrophages were pretreated with 5-hydroxymaltol for 1 h and stimulated with LPS (1 μg/mL) for 30 min. Macrophage cells were harvested and lysed using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA). Each nuclear and cytoplasmic fraction sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred to an Immobilin-FL polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). ) ordered. After blocking with Odyssey® blocking buffer (LICOR, Lincoln, NE, USA) for 1 h, the blots were incubated with primary antibodies overnight at 4°C. Blots were washed and then incubated with diluted IRDye 680- and IRDye 800-labeled secondary antibodies for 60 minutes using Odyssey blocking buffer containing 0.2% Tween-20/0.01% sodium dodecyl sulfate. Protein bands were detected with an Odyssey CLx imaging machine (LI-COR). Primary antibodies against p65 were obtained from Cell Signaling Technology (Beverly, MA, USA), and antibodies against lamin B and β-actin were from Santa Cruz Biotechnology, Inc (Dallas, TX, USA). ) was purchased and used. Lamin B and β-actin were used as internal controls for nuclear and cytoplasmic fractions to quantify their relative expression levels.

The nuclear translocation of NF-κB in LPS-treated cells was increased 8.6-fold compared to the control group, while 5-hydroxymaltol downregulated nuclear p65 protein by 73% compared to the LPS-treated control group. In addition, as a result of examining the cytosolic inhibitor κB (IκB), a regulator of NF-κB, 5-hydroxymaltol pretreatment blocked the downregulation of IκB-α in LPS-treated cells, compared to the LPS-treated control group. Compared to , cytoplasmic IκB increased twofold. These results suggest that LPS treatment induces nuclear translocation of NF-κB(p65) with downregulation of IκB-α, whereas 5-hydroxymaltol pretreatment inhibits nuclear translocation of NF-κB(p65) in LPS-treated cells. and can block downregulation of IκB-α (Figure 7).

Therefore, it was confirmed that 5-hydroxymaltol can act as an inhibitor of LPS-induced NF-κB activation in RAW 264.7 macrophages.

Example 5. Inhibitory effect of 5-hydroxymaltol on LPS-stimulated MAPK activation

MAPKs are known to regulate cell function, proliferation, gene expression, cell survival, and cell death. The effect of 5-hydroxymaltol on LPS-stimulated phosphorylation and activation of MAPK in RAW 264.7 cells was evaluated using immunoblot analysis.

RAW 264.7 cells were incubated with 1000 μM 5-hydroxymaltol for 1 hour and then treated with LPS (1 μg/mL) for 30 minutes. The total protein extract was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblot analysis was performed in the same manner as in Example 4 above. Primary antibodies against pp38, JNK, pJNK, ERK, and pERK were obtained from Cell Signaling Technology (Beverly, MA, USA), and antibodies for p38 and β-actin were from Santa Cruz Biotechnology, Inc (Dallas, TX, USA). Purchased and used from . β-Actin was used as an internal control for total protein to quantify relative expression levels.

While LPS treatment induced the phosphorylation levels of ERK, p38, and JNK by 4.8-fold, 3-fold, and 1.9-fold, respectively, compared to the control group, 5-hydroxymaltol decreased the phosphorylation levels of ERK, p38, and JNK compared to the LPS-treated control group. were found to be downregulated by 69%, 73%, and 59%, respectively (Figure 8).

Therefore, it was confirmed that 5-hydroxymaltol can inhibit LPS-induced phosphorylation of ERK, p38, and JNK, thereby inhibiting MAPK signaling, thereby suppressing the inflammatory response of LPS-induced RAW 264.7 cells.

Example 6. Inhibitory effect of 5-hydroxymaltol on LPS-induced ROS production

LPS-stimulated macrophages induce reactive oxygen species (ROS) accumulation and oxidative stress. Therefore, the effect of 5-hydroxymaltol on cellular ROS accumulation in LPS-induced RAW 264.7 cells was analyzed using CellROX Green Dye.

ROS were measured using the CellROX Green probe (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's protocol. RAW 264.7 cells (2×10 ⁶ cells/10 mL) were cultured on plates and incubated for 1 day. Macrophages were pretreated with 5-hydroxymaltol (1000 μM) or NAC (10 mM) for 1 hour and treated with LPS (1 μg/mL) for 30 minutes. CellROX Green Dye was then applied to the cells, and the cells were incubated at 37°C for 10 minutes. The medium was discarded, and the cells were washed with 1×phosphate-buffered saline. Stained macrophages were visualized at 10× magnification using a fluorescence microscope (Lionheart, Biotek, VT, USA). The level of fluorescence signal was quantified relative to the control group (untreated group).

Figure 9 shows that LPS treatment increased the cell density of CellROX Green, while 5-hydroxymaltol and N-acetyl cysteine (NAC) increased the ROS content of cells induced by LPS, i.e., decreased ROS production. . Fluorescence intensity increased 13.6-fold in LPS-treated cells compared to the untreated control, and decreased 87% in 5-hydroxymaltol-treated cells compared to the LPS-treated control.

Therefore, it was confirmed that 5-hydroxymaltol exhibits a strong ROS scavenging effect in RAW 264.7 cells.

Example 7. Effect of 5-hydroxymaltol on increasing protein expression of HO-1 and Nrf2

Nrf2 and HO-1 signaling pathways are antioxidant systems that reduce oxidative stress in animal models. The present inventors investigated whether 5-hydroxymaltol is involved in the Nrf2 and HO-1 signaling pathways.

First, RAW 264.7 macrophages were incubated with 1000 μM 5-hydroxymaltol, and total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western blot using Nrf2 and HO-1 antibodies. Protein expression levels were analyzed through . β-actin was used as an internal control, from which the relative expression level was quantified.

Additionally, RAW 264.7 macrophages were treated with 500 μM 5-hydroxymaltol for 12 hours, then nuclear and cytoplasmic fractions were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then Nrf2 antibody was used. Immunoblot analysis was then performed in the same manner as in Example 4. Lamin B and β-actin were used as internal controls for nuclear and cytoplasmic fractions to quantify relative expression levels.

Primary antibodies against Nrf2 and HO-1 were obtained from Cell Signaling Technology (Beverly, MA, USA), and antibodies against lamin B and β-actin were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX, USA). did.

Western blot analysis showed that 5-hydroxymaltol treatment increased the protein expression of HO-1 and Nrf2 by 3.9-fold and 3.8-fold, respectively, by 12 hours, and that 5-hydroxymaltol treatment increased the protein expression of Nrf2 and HO-1 in a time-dependent manner. It was shown to increase protein levels (Figure 10A). Immunoblot analysis of cytoplasmic and nuclear fractions for Nrf2 protein in RAW 264.7 cells showed that nuclear Nrf2 protein level was significantly increased by 1.9-fold by treatment with 5-hydroxymaltol (Figure 10B).

Therefore, it was confirmed that 5-hydroxymaltol has the effect of increasing the Nrf2/HO-1 signaling pathway.

Overall, through the above examples of the present invention, 5-hydroxymaltol isolated from the beet fermentation product of the present invention inhibits NO production and iNOS expression, ROS accumulation, MAPK signaling activation, and NF-κB nuclear translocation. In addition to inhibiting, it exerts an anti-inflammatory effect on RAW 264.7 cells by upregulating Nrf-2/HO-1 signaling (Figure 11), through which 5-hydroxymaltol exerts an anti-inflammatory effect and prevents inflammatory diseases. It was confirmed that it can be useful for treatment.

Claims (10)

Anti-inflammatory pharmaceutical composition comprising 5-hydroxymaltol or a pharmaceutically acceptable salt or solvate thereof represented by the following formula (1) as an active ingredient:
[Formula 1]
. The pharmaceutical composition according to claim 1, which inhibits nitric oxide (NO) production or secretion and ROS production or accumulation. The pharmaceutical composition according to claim 1, which inhibits the expression or production of pro-inflammatory cytokines. The pharmaceutical composition according to claim 1, which inhibits the NF-κB signaling pathway. The pharmaceutical composition according to claim 1, which inhibits the MAPK signaling pathway. The pharmaceutical composition of claim 1, which increases Nrf-2/HO-1 signaling. delete An anti-inflammatory food composition comprising 5-hydroxymaltol represented by the following formula (1) or a foodologically acceptable salt or solvate thereof as an active ingredient:
[Formula 1]
. An anti-inflammatory cosmetic composition comprising 5-hydroxymaltol or a cosmetically acceptable salt or solvate thereof represented by the following formula (1) as an active ingredient:
[Formula 1]
.

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