KR20210041401A - Inhibitor compostion for TLR4-MD2 comprising fraction of ethanol extract of Hibiscus Syriacus - Google Patents

Abstract

Provided in the present invention is a fraction of an ethanol extract of Hibiscus syriacus or a flavonoid compound isolated therefrom, which binds to TLR4-MD and thus inhibits the activity of TLR4-MD, inhibits LPS-induced activation of TLR4-MD, and inhibits and prevents inflammatory responses. Specifically, it was confirmed that the fraction of the ethanol extract of Hibiscus syriacus or the flavonoid compound isolated therefrom inhibits the NO production induced by LPS, and effectively inhibits the expression of iNOS and COX-2 proteins and pro-inflammatory cytokines TNF-α, IL-6, and IL-12, and reduces a survival rate and teratogenicity in a zebrafish sepsis model, thereby providing an anti-inflammatory effect. Thus, the fraction of the ethanol extract of Hibiscus syriacus can be utilized for preventing, ameliorating or treating inflammatory diseases and/or for alleviating skin inflammation.

Description

{Inhibitor compostion for TLR4-MD2 comprising fraction of ethanol extract of Hibiscus Syriacus}

The present invention relates to a composition for inhibiting TLR4-MD2 comprising a fraction of the ethanol extract of Mugunghwa or flavonoids isolated therefrom, wherein the fraction of the ethanol extract of Mugunghwa inhibits the activation of TLR4-MD2 to prevent, improve and treat inflammatory diseases It can be used as a composition.

Inflammatory reaction is promoted by oxidative stress generated in the body. Due to oxidative stress, nucleic acids (DNA), which are the main components of genes in cells, proteins, which are the main components of muscle, fat, which are the main components of cell membranes, are transformed or the activity of the cells decreases. Is a problem with the body's function itself, which initiates or worsens the inflammatory response by increasing the gene expression of specific cells that cause apoptosis as well as degenerative diseases.

Lipopolysaccharide (LPS), one of the endotoxins, increases the expression of inflammatory cytokines such as TNF-α, IL-6, and IL-1β in Raw 264.7 macrophages, and nitric oxide (NO) and prostaglandins It secretes inflammation mediating substances such as _{E 2} (prostaglandin E ₂ , PGE _{2 ).} In the inflammatory state, COX-2 and NO synthase (NOS) are induced to _{produce excessive amounts of PGE 2} and NO, and various inflammatory diseases are promoted. In particular, NOS, an enzyme that produces NO, and COX, an enzyme that mediates the biosynthesis of various prostaglandins (PGs), are known as important mediators for regulating the inflammatory response.

The LPS receptor, Toll-like receptor (TLR), has been found to have a close relationship with oxidative stress. Toll-like receptor (TLR) is a membrane protein that plays a central role in the innate immune response, and it is frequently expressed in sentinel cells in blood vessels such as macrophages and dendritic cells. do. Among them, TLR2 or TLR4 mainly detects lipoproteins in the outer cell membrane/cell wall of bacteria, and plays a role in inducing ROS (reactive oxygen species) that causes oxidative stress. In the case of TLR4, even when macrophages are stimulated by LPS stimulation, various inflammatory factors are generated due to NF-κB activation, leading to pathologies of various diseases and involved in carcinogenesis. However, it is known that there are no endogenous enzymes that can directly inactivate the action of generated reactive oxygen species and NO, ONOO ^{-in vivo.}

Toll-like receptor 4 (TLR4) is the first receptor identified in the TLR family, and innate immune signaling amplified through MyD88 (myeloid differentiation 88)-dependent signaling processes and MyD88-independent signaling processes. Activates. Due to the role of TLR4, there is a growing interest in research for utilizing TLR4 as a target for treating various immune diseases. LPS, recognized through accessory molecules such as lipopolysaccharide (LPS)-binding protein (LBP), cluster of differentiation 14 (CD 14) and myeloid differentiation factor 2 (MD2), activates TLR4. Activated TLR4 induces initial activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and migration to the nucleus through a Myd88-dependent signaling process, and mitogen-activated protein kinases (MAPKs) Induces activation. The activation of NFκB and MAPKs induces inflammation by secreting inflammatory cytokines such as tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), and interleukin 6 (IL-6).

Therefore, TLR4-MD2 can be a target for treating inflammatory diseases, and there is a need for research on a technology that effectively inhibits the inflammatory signaling pathway by activation of TLR4-MD2.

Korean Patent Registration No. 10-1976415

One object of the present invention is to provide a TLR4-MD2 (Toll-like receptor 4-myeloid differentiation factor 2) inhibitor composition comprising a fraction of the ethanol extract of Mugunghwa or a flavonoid compound isolated therefrom.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising the TLR4-MD2 (Toll-like receptor 4-myeloid differentiation factor 2) inhibitor as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for preventing or improving inflammatory diseases comprising the TLR4-MD2 (Toll-like receptor 4-myeloid differentiation factor 2) inhibitor.

Another object of the present invention is to provide a food composition for preventing or improving inflammatory diseases comprising the TLR4-MD2 inhibitor.

Another object of the present invention is to provide a cosmetic composition for relieving skin inflammation comprising the TLR4-MD2 inhibitor.

In order to achieve the above object, the present invention provides a TLR4-MD2 (Toll-like receptor 4-myeloid differentiation factor 2) inhibitor composition comprising a fraction of the ethanol extract of Mugunghwa or a flavonoid compound isolated therefrom. .

In the present invention, "Mugunghwa (Hibiscus syriacus)" belongs to the deciduous shrub of the dicotyledonous plant Malvaceae Malvaceae. The height reaches 3-4m, and there are many hairs on the young branches, but they gradually disappear. Mugunghwa is easy to cultivate in a garden and can be propagated with seeds, but since it is propagated by cuttings, it is easy to maintain without altering its traits. Leaves are alternate, egg-shaped, usually divided into three, and there are saw teeth on the edge. The skin of the hibiscus is peeled and used as a raw material for paper, and dried and used as a medicine. Young leaves are edible, and flowers and leaves can be drunk as tea.

In the present invention, "extract" refers to an extract obtained by an extraction treatment, a diluted solution or a concentrate of the extract, a dried product obtained by drying the extract, a preparation or purified product of the extract, or a mixture thereof, the extract itself and the extract. It includes extracts of all formulations that can be formed.

In the present invention, drying of the extract may be performed by a known method within a range in which useful components are not destroyed from the collected sample, for example, it may be performed by a method of freeze-drying in a vacuum state. Further, the crushing or pulverization may be pulverized or pulverized to a degree that sufficient components of the sample can be sufficiently extracted during the subsequent extraction process. The drying and crushing or pulverizing process may be performed in reverse order or repeatedly performed as necessary.

In the extraction of the present invention, the method of extracting is not particularly limited, and may be extracted according to a method commonly used in the art.

In the present invention, the Mugunghwa extract can be obtained by an extraction process using water, an organic solvent, or a mixed solvent thereof. Specifically, it may be obtained by using an alcohol _{having 1 (C 1} ) to 4 (C ₄ ) carbon atoms such as water, methanol, ethanol, and butanol, or a mixed solvent thereof. In addition, the alcohol may be ethanol.

In the example of the present invention, ethanol was added to the lyophilized petals of Mugunghwa, extracted for 48 hours at 10°C, and filtered to obtain an ethanol extract of Mugunghwa.

In the present invention, the term "fraction" means a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents.

The fractionation method for obtaining the fraction in the present invention is not particularly limited, and may be performed according to a method commonly used in the art. A non-limiting example of the fractionation method may be a fraction separated by chromatography on an ethanol extract of Mugunghwa.

In addition, the fraction is obtained by suspending the ethanol extract of Mugunghwa in distilled water (water), and then fractionating using an alcohol having 1 (C1) to 4 (C4) carbon atoms, ethyl acetate, n-hexane, or a mixed solvent thereof. Can be.

In the example of the present invention, a fraction having a high anthocyanin content obtained by fractionating the ethanol extract of Mugunghwa using a Diaion® HP-20 column was separated.

Accordingly, the fraction of the ethanol extract of Mugunghwa of the present invention may be a fraction obtained by separating the ethanol extract of Mugunghwa with a Diaion® HP-20 column, and specifically, may be a fraction having a high anthocyanin content.

In addition, as a result of analyzing flavonoid components in the fractions of the ethanol extract of Mugunghwa, cyanidin-3-O-galactoside, cyanidin-3-O-glucoside (Cyanidin-3-O -glucoside), Orientin-7-O-glucoside, Cyanidin-3,5-O-diglucoside, Isooriene Tin-4'-O-glucoside (Isoorientin-4'-O-glucoside), isovitexin-4'-O-glucoside (Isovitexin-4'-O-glucoside), vitaxin-4'-O- Glucoside-2''-O-rhamnoside (Vitexin-4'-O-glucoside-2''-O-rhamnoside), isovitexin-7-O-glucoside, Apigenin-8-C-β-D-glucopyranoside, isovitexin-2"-O-rhamnoside, Apigenin-6-C-β-D-glucopyranoside, apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside Seed (Apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside), Kaempferol-O-glucoside derivative, Camperol-7-O-glucoside (Kaempferol-7-O-glucoside), campferol-3-O-glucoside, apigenin-7-O-glucoside, and camphor It was confirmed to contain 17 kinds of flavonoid compounds such as rol-3-(6''-acetylglucoside) (Kaempferol-3-(6''-acetylglucoside)).

In an example of the present invention, the binding relationship between 17 flavonoids isolated from the fractions of the ethanol extract of Mugunghwa and TLR4-MD2, which is known to be involved in the inflammatory reaction, was confirmed. Of these, the Kaempferol-O-glucoside derivative was not analyzed because the same structure was not confirmed in Pubchem.

As a result of the analysis, cyanidin-3-O-glucoside, cyanidin-3,5-O-diglucoside, isorientin- 4'-O-glucoside (Isoorientin-4'-O-glucoside), vitexin-4'-O-glucoside-2``-O-rhamnoside (Vitexin-4'-O-glucoside-2'') -O-rhamnoside), apigenin-8-C-β-D-glucopynoside (Apigenin-8-C-β-D-glucopyranoside) and apigenin-7-O-glucoside (Apigenin-7-O- glucoside) was confirmed to have hydrogen bonds with both MD2 and TLR4.

In addition, cyanidin-3-O-galactoside, apigenin-6-C-β-D-glucopynoside (Apigenin-6-C-β-D-glucopyranoside), api Genin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside), camphorol-7- O-glucoside (Kaempferol-7-O-glucoside) and camperol-3-O-glucoside (Kaempferol-3-O-glucoside) have a hydrogen bond with MD2, and orientin-7-O-glucoside ( Orientin-7-O-glucoside), isovitexin-4'-O-glucoside and camperol-3-(6''-acetylglucoside) (Kaempferol-3- (6''-acetylglucoside)) was confirmed to have a hydrogen bond with TLR4.

On the other hand, isovitexin-7-O-glucoside and isovitexin-2"-O-rhamnoside are hydrogen bonded to MD2 and TLR4. It was confirmed that it did not have

Among them, the docking score of apigenin-7-O-glucoside was -8.4, which showed the best binding power with TLR4-MD2. Specifically, apigenin-7-O-glucoside forms a hydrogen bond with the LYS122 amino acid of MD2 and 2.205 A and 3.098 A, and has a hydrogen bond with the SER127 amino acid of MD2 and 2.844 A. Confirmed. In addition, it was confirmed that it has a hydrogen bond with SER441 amino acid of TLR4 and 2.873 A.

Cyanidin--3-O-glucoside contained in the fractions of the ethanol extract of Mugunghwa of the present invention Cyanidin-3,5-O-diglucoside (Cyanidin-3,5-O- diglucoside), isorientin-4'-O-glucoside, vitexin-4'-O-glucoside-2''-O-rhamnoside (Vitexin-4'- O-glucoside-2''-O-rhamnoside), apigenin-8-C-β-D-glucopynoside (Apigenin-8-C-β-D-glucopyranoside) and apigenin-7-O-glucoside (Apigenin-7-O-glucoside) forms hydrogen bonds with both MD2 and TLR4, and cyanidin-3-O-galactoside, apigenin-6-C-β-D- Glucopynoside (Apigenin-6-C-β-D-glucopyranoside), apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6-C-glucoside-7) -(6"-O-acetyl)-glucoside), campferol-7-O-glucoside (Kaempferol-7-O-glucoside) and campferol-3-O-glucoside (Kaempferol-3-O-glucoside) Has a hydrogen bond with MD2, and orientin-7-O-glucoside and campferol-3-(6''-acetylglucoside) (Kaempferol-3-(6' '-acetylglucoside)) formed a hydrogen bond with TLR4 to bind to TLR4-MD2, and the fraction of the ethanol extract of Mugunghwa containing the flavonoid may inhibit or inhibit TLR4-MD2 activity.

Therefore, the TLR4-MD2 inhibitor of the present invention includes a fraction of the ethanol extract of Mugunghwa.

The TLR4-MD2 inhibitor includes a flavonoid compound that inhibits or inhibits the activity of TLR4-MD2 by hydrogen bonding to MD2 and TLR4.

Fractions of the ethanol extract of Mugunghwa of the present invention include Cyanidin-3-O-galactoside, Cyanidin-3-O-glucoside, orien Tin-7-O-glucoside (glucoside Orientin-7-O-glucoside), Cyanidin-3,5-O-diglucoside (Cyanidin-3,5-O-diglucoside), isorientin-4'- O-glucoside (Isoorientin-4'-O-glucoside), isovitexin-4'-O-glucoside (Isovitexin-4'-O-glucoside), vitaxin-4'-O-glucoside-2' '-O-rhamnoside (Vitexin-4'-O-glucoside-2``-O-rhamnoside), isovitexin-7-O-glucoside (Isovitexin-7-O-glucoside), apigenin-8- C-β-D-glucopynoside (Apigenin-8-C-β-D-glucopyranoside), isovitexin-2"-O-rhamnoside (Isovitexin-2"-O-rhamnoside), apigenin-6- C-β-D-glucopynoside (Apigenin-6-C-β-D-glucopyranoside), apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6 -C-glucoside-7-(6"-O-acetyl)-glucoside), campferol-O-glucoside derivative, campferol-7-O-glucoside) O-glucoside), campferol-3-O-glucoside, apigenin-7-O-glucoside and campferol-3-(6 It may contain flavonoids selected from the group consisting of''-acetylglucoside) (Kaempferol-3-(6''-acetylglucoside)).

The flavonoids are specifically, Cyanidin-3-O-glucoside Cyanidin-3-O-glucoside Cyanidin-3,5-O-diglucoside (Cyanidin-3,5) which forms hydrogen bonds with both MD2 and TLR4. -O-diglucoside), isorientin-4'-O-glucoside, vitexin-4'-O-glucoside-2''-O-rhamnoside (Vitexin- 4'-O-glucoside-2''-O-rhamnoside), apigenin-8-C-β-D-glucopynoside (Apigenin-8-C-β-D-glucopyranoside) and apigenin-7-O -Glucoside (Apigenin-7-O-glucoside);

Cyanidin-3-O-galactoside having a hydrogen bond with MD2, apigenin-6-C-β-D-glucopynoside (Apigenin-6-C-β-D) -glucopyranoside), apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside), Campferol-7-O-glucoside and campferol-3-O-glucoside; And

Orientin-7-O-glucoside forming a hydrogen bond with TLR4 and camphorol-3-(6``-acetylglucoside) (Kaempferol-3-(6``- acetylglucoside)).

In addition, Apigenin-7-O-glucoside, which has the strongest binding strength with TLR4-MD2, forms a hydrogen bond with LYS122 amino acid of MD2 and 2.205 A and 3.098 A, and SER127 amino acid of MD2 It was confirmed that hydrogen bonds were made with 2.844 A and 2.873 A with SER441 amino acid of TLR4, and the flavonoid may be specifically apigenin-7-O-glucoside. .

In addition, the apigenin-7-O-glucoside forms a hydrogen bond with LYS122 amino acid of MD2 and 2.205 A and 3.098 A, and hydrogen bonds with SER127 amino acid of MD2 and 2.844 A. , It may be characterized by forming a hydrogen bond with SER441 amino acid of TLR4 and 2.873 A.

When activated, TLR4-MD2 is involved in the inflammatory response. Inhibition of TLR4-MD2 can inhibit the inflammatory response. Therefore, the TLR4-MD2 inhibitor of the present invention can be used for prevention, improvement, and treatment of inflammatory diseases induced by activation of TLR4-MD2.

Compounds with strong binding power to TLR4-MD2 bind to TLR4-MD2 and inhibit or inhibit the activity of TLR4-MD2, thus blocking the TLR4 signaling pathway induced by LPS, causing a decrease in the activation of NFκB and MAPKs. It can inhibit the occurrence or progression of inflammation by reducing the secretion of inflammatory cytokines, NO and ROS.

Therefore, the TLR4-MD2 inhibitory activity of apigenin-7-O-glucoside, which has the best binding ability with TLR4-MD2, may be the most excellent.

In an example of the present invention, it was confirmed that the fraction of the ethanol extract of Mugunghwa inhibited the generation of NO induced by LPS and inhibited _{the expression of prostaglandin E 2} (prostaglandin E ₂ , PGE _{2 ).} In addition, it was confirmed that the expression of the inflammatory regulator iNOS or COX-2 was inhibited in a concentration-dependent manner, and the expression of the inflammatory cytokines IL-12, IL-6, and TNF-α was also decreased in a concentration-dependent manner.

In addition, in the zebrafish model for inflammatory sepsis with LPS, it was confirmed that the fraction of the ethanol extract of Mugunghwa dramatically reduced the mortality rate due to LPS, and as a result of malformaion analysis, the malformation rate in zebrafish was about 60. %, but it was confirmed that the malformation rate was decreased by the treatment of the fraction (PS) of the ethanol extract of Mugunghwa.

In other words, it was confirmed that the fraction of the ethanol extract of Mugunghwa has excellent inhibitory and inhibitory effects on the inflammatory response known to be induced in TLR4-MD2, and the fraction of the ethanol extract of Mugunghwa improves, prevents, and treats inflammatory diseases caused by TLR4-MD2. It can be used in pharmaceuticals, foods, and quasi-drugs for the purpose of, and it can be used as a cosmetic composition for relieving skin inflammation.

In another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating inflammatory diseases comprising the TLR4-MD2 inhibitor as an active ingredient.

In the present invention, the description of the TLR4-MD2 inhibitor, the ethanol extract of Mugunghwa is as described above.

In the present invention, "inflammatory diseases" are TNF-α (tumor necrosis factor-alpha), IL-12 (interleukin-alpha) secreted by immune cells such as macrophages by excessively promoting the human immune system due to harmful stimulation such as inflammation inducing factors. 12), IL-6 (interleukin-6), prostaglandin (prostagladin), or nitric oxide (nitric oxide) refers to a disease caused by an inflammatory substance such as.

In the present invention, the inflammatory disease is any one selected from the group consisting of any one selected from the group consisting of asthma, bronchitis, sepsis, arthritis, hepatitis, rheumatoid arthritis, osteoarthritis, ulcerative colitis, myocarditis, multiple sclerosis, and viral infection. Can be Specifically, it may be sepsis.

In the present invention, "prevention" means any action of inhibiting or delaying the onset of the inflammatory disease by administration of the composition according to the present invention.

In the present invention, "treatment" refers to any action in which the symptoms of the inflammatory disease are improved or advantageously changed by administration of the composition according to the present invention.

The pharmaceutical composition of the present invention may further include an appropriate carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions other than the active ingredient. At this time, the fraction of the ethanol extract of Mugunghwa contained in the composition is not particularly limited thereto, but may include 0.001% by weight to 99% by weight, preferably 0.01% by weight to 50% by weight, based on the total weight of the composition.

The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried agents, and suppositories. It may have a dosage form, and may be a variety of oral or parenteral dosage forms. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin. It can be prepared by mixing and the like. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. In the present invention, the term "pharmacologically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the individual, age, sex, and type of disease. , Drug activity, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors, and can be easily determined by a person skilled in the art.

The pharmaceutical composition of the present invention is not particularly limited as long as it is an individual for the purpose of preventing or treating inflammatory diseases, and any can be applied. For example, non-human animals such as monkeys, dogs, cats, rabbits, mormons, rats, mice, cows, sheep, pigs, goats, etc., humans, birds and fish, etc. can be used, and the pharmaceutical composition may be parenterally or subcutaneously , Intraperitoneal, intrapulmonary and intranasal administration, and for local treatment, if necessary, may be administered by a suitable method including intralesional administration. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the individual, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. For example, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or cerebrovascular injection, but is not limited thereto.

Suitable total daily use amount can be determined by treatment within the range of correct medical judgment, and generally in an amount of 0.001 to 1000 mg/kg, preferably 0.05 to 200 mg/kg, more preferably 0.1 to 100 mg/kg The amount of can be divided into once to several times a day to administer.

Another aspect of the present invention provides a quasi-drug composition for preventing or improving inflammatory diseases comprising the TLR4-MD2 inhibitor.

In the present invention, the description of the TLR4-MD2 inhibitor, inflammatory disease and prevention is as described above.

In the present invention, "improvement" refers to any action in which symptoms of an individual with suspicion of an inflammatory disease and an onset of inflammatory disease are improved or benefited by using a composition containing a fraction of the ethanol extract of Mugunghwa as an active ingredient.

In the present invention, “quasi-drugs” are fibers, rubber products or similar materials used for the purpose of treating, alleviating, treating or preventing diseases of humans or animals. A product corresponding to one of the preparations used for sterilization, insecticide, and similar purposes to prevent infection, and for the purpose of diagnosing, treating, alleviating, treating or preventing diseases of humans or animals. Means non-instruments, machines, or devices, and those used for the purpose of pharmacologically affecting the structure and function of humans or animals, excluding those that are not devices, machines, or devices. Skin external preparations and personal hygiene products are also used. Includes.

When the fraction of the ethanol extract of Mugunghwa of the present invention is added to a quasi-drug composition for the purpose of preventing or improving inflammatory diseases, the extract or fraction may be added as it is or used with other quasi-drug components, and used appropriately according to a conventional method. I can. The mixing amount of the active ingredient can be appropriately determined according to the intended use.

The external preparation for skin is not particularly limited thereto, but may be preferably prepared and used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel.

The personal hygiene product is not particularly limited thereto, but preferably includes soap, wet tissue, tissue paper, shampoo, toothpaste, air freshener gel, cleaning gel, disinfectant cleaner, detergent, shower foam, mouthwash, hand wash, or mask. .

Another aspect of the present invention provides a food composition for preventing or improving inflammatory diseases comprising the TLR4-MD2 inhibitor.

In the present invention, the description of the TLR4-MD2 inhibitor, inflammatory disease, prevention, and improvement is as described above.

The food composition of the present invention may include the form of pills, powders, granules, needles, tablets, capsules or liquids, and there is no particular limitation on the type of food to which the fraction of the ethanol extract of Mugunghwa of the present invention can be added, For example, there are various beverages, gums, teas, vitamin complexes, and dietary supplements.

In addition to the fraction of the ethanol extract of Mugunghwa, other components may be added to the food composition, and the kind is not particularly limited. For example, as an additional component, various herbal extracts, food additives, or natural carbohydrates that are acceptable for food may be included as an additional component, but are not limited thereto.

In the present invention, the term "food supplementary additive" refers to a component that can be added auxiliary to food, and is added to the manufacture of health functional foods of each formulation, and can be appropriately selected and used by those skilled in the art. Examples of food additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. are included, but the types of the food additives of the present invention are not limited by the above examples.

Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; And polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (such as taumatin), stevia extract (rebaudioside A, glysir) Hibiscus) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used advantageously.

The food composition of the present invention may contain health functional food. The term "health functional food" as used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body. Here, the term "functionality" refers to obtaining useful effects for health purposes such as controlling nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and ingredients commonly added in the art. In addition, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material, and it may be excellent in portability.

The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food, the fraction of the ethanol extract of Mugunghwa of the present invention may be added in an amount of 1 to 50% by weight, preferably 5 to 10% by weight of the raw material composition, but is not limited thereto. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be used even below the above range.

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and all health functional foods in the usual sense are included.

Another aspect of the present invention provides a feed additive composition for preventing or improving inflammatory diseases comprising the TLR4-MD2 inhibitor.

In the present invention, the description of the TLR4-MD2 inhibitor, inflammatory disease, prevention and improvement is as described above.

In the present invention, the term "feed additive" is a generic term for a substance added in trace amounts to feed for nutritional or specific purposes, and in the present invention, it means a substance added for the purpose of preventing or improving inflammatory diseases. Here, an animal is a concept including livestock and pets.

The feed additive of the present invention may further include a binder, emulsifier, preservative, etc. added to prevent quality degradation, and amino acid, vitamin, enzyme, probiotic, flavoring, non-protein nitrogen added to increase utility A compound, a silicate, a buffer, a colorant, an extractant, an oligosaccharide, etc. may be additionally included, and in addition, a feed mixture may be additionally included, but the present invention is not limited thereto.

Another aspect of the present invention provides a cosmetic composition for relieving skin inflammation comprising the TLR4-MD2 inhibitor.

In the present invention, the description of the TLR4-MD2 inhibitor is as described above.

Relieving skin inflammation in the present invention may be relieving inflammatory skin degeneration caused by irritation causing inflammation.

In the present invention, the skin inflammation refers to all skin inflammatory diseases accompanying an inflammatory reaction occurring in the skin. More specifically, it may be any one selected from the group consisting of atopic dermatitis, contact dermatitis, seborrhea, and acne. Sebaceous nevus, seborrheic keratosis, melanocytosis, actinic keratosis, cutaneous fibroids, psoriasis, chronic eczema, tuberculosis or deep mycosis, infectious granulomatous granulocyte tumors, acute contact dermatitis with lichen planus and spongy spongiasis It may be eczema, herpes zoster, hydrophobic pseudosensitization, or hydrophobic dermatophytes, but is not limited thereto.

The cosmetic composition of the present invention may include, without limitation, ingredients that are generally permitted in addition to the active ingredients, and may include conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and a carrier. have.

The cosmetic composition according to the present invention is a solution, external ointment, cream, foam, nutrient lotion, softening lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid cleaning agent, bathing agent, sunscreen cream, sun oil, suspension, Emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, can be prepared in one or more formulations selected from the group consisting of patches and sprays, but It is not limited.

In addition, the cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers blended in general skin cosmetics, and as common ingredients, for example, oil, water, surfactants, moisturizers, lower alcohols, A thickener, a chelating agent, a colorant, a preservative, a fragrance, and the like may be appropriately blended, but are not limited thereto. The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation.

When the formulation of the present invention is an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or Mixtures of these can be used.

When the formulation of the present invention is a powder, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fatty ester, polyethylene glycol or fatty acid ester of sorbitan may be used. have.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, micro Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.

When the formulation of the present invention is a soap, an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolyzate, isethionate, a lanolin derivative, an aliphatic alcohol, vegetable oil, glycerol, sugar, etc. may be used as a carrier component. I can.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcositate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters may be used.

In the present invention, the fraction of the ethanol extract of Mugunghwa binds to TLR4-MD, inhibits the activity of TLR4-MD, inhibits the activation of TLR4-MD induced by LPS, and inhibits and prevents inflammatory reactions. Specifically, it inhibited the production of NO induced by LPS, effectively inhibited the expression of iNOS and COX-2 proteins and pro-inflammatory cytokines TNF-α, IL-6, and IL-12, and the survival rate of the sepsis zebrafish model and It was confirmed that it has an anti-inflammatory effect by reducing teratogenicity. Therefore, the fraction of the ethanol extract of Mugunghwa can be used for prevention, improvement or treatment of inflammatory diseases or/and for alleviating skin inflammation.

1 is a result of analyzing flavonoids contained in a fraction of the ethanol extract of Mugunghwa.
FIG. 2 shows the chemical formulas of 16 types of compounds excluding the camperol-O-glucoside derivative (No. 13) among the 17 flavonoids in Table 3. FIG.
3 is a result of analyzing the photographs and MTT of cells according to the treatment dynamics of the fractions of the ethanol extract of Mugunghwa.
4 is a result of flow cytometric analysis of cells according to the treatment concentration of a fraction of the ethanol extract of Mugunghwa.
5 is an analysis of the inhibitory effect of NO generation induced by LPS treatment of a fraction of the ethanol extract of Mugunghwa.
6 is an analysis of the effect of inhibiting _{PGE 2} expression induced by LPS treatment of a fraction of the ethanol extract of Mugunghwa.
7 is a result of analyzing the expression of COX-2 and iNOS mRNA according to the treatment concentration of the fraction of the ethanol extract of Mugunghwa.
8 is a result of analyzing the expression of COX-2 and iNOS proteins according to the treatment concentration of the fractions of the ethanol extract of Mugunghwa.
9 is a result of ELISA analysis of the protein expression level of TNF-α according to the treatment concentration of the fraction of the ethanol extract of Mugunghwa.
10 is a result of ELISA analysis of the protein expression level of IL-6 according to the treatment concentration of the fraction of the ethanol extract of Mugunghwa.
11 is a result of ELISA analysis of the protein expression level of IL-12 according to the treatment concentration of the fraction of the ethanol extract of Mugunghwa.
12 is a result of RT-PCR analysis of the mRNA expression levels of TNF-α, IL-6, and IL-12 according to the treatment concentration of the fractions of the ethanol extract of Mugunghwa.
Figure 13 shows the binding site of apigenin-7-O-glucoside (Apigenin-7-O-glucoside) and TLR4-MD2.
14 is a result of analyzing the change in survival rate according to the fraction treatment of the ethanol extract of Mugunghwa in zebrafish.
15 is an analysis of the effect of inhibiting the formation of malformation according to the treatment of fractions of the ethanol extract of Mugunghwa in zebrafish.

Hereinafter, the present invention will be described in more detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

<Materials and methods>

1. Preparation of Mugunghwa anthocyanin fraction

Hibiscus syriacus was used as a firebird (Pulsae) and sandalwood (Paektanshim) grown in the National Institute of Mountain Science (Suwon, Korea) between July and August 2017. The petals of Mugunghwa Sandalwood or Mugunghwa Firebird were freeze-dried for 3 days and stored at a temperature of -20℃ or less until extraction.

After crushing 1.5 kg of petals, it was extracted three times with 40.0 L ethanol for 48 hours at room temperature (25° C.), and then filtered. The filtrate was freeze-dried after removing the solvent at 30° C. or lower using a rotary evaporator.

The extract was adsorbed using Diaion® HP-20 (Mitsubishi Chemical Company, Japan) and eluted with alcohol to prepare a rich anthocyanin fraction. fraction, PS) was prepared.

The anthocyanin fraction of Mugunghwa was filtered through a 0.2 mm polytetrafluoroethylene (PTFE) filter, and UPLC-QTOF-MS and biological activity analysis were performed.

EP grade was used for the extraction solution, and LC-MS grade was used for the chromatographic solvent.

2. Flavonoid analysis (MS spectrum analysis)

Samples were separated by performing chromatography using a binary solvent delivery system, an automatic sampler, and a UPLC system equipped with a UV detector (Waters Corp., Milford, MA, USA).

Aliquots of 3.0 µl of each sample were injected into a BEH C18 column (2.1 × 100 mm, 1.7 µM) at a flow rate of 0.4 ml/min and two mobile phases (A phase: water containing 0.1% formic acid, B phase). : Acetonitrile containing 0.1% formic acid) was eluted using a chromatographic gradient. The optimized linear gradient is shown in Table 1.

time B phase concentration (%, v/v) 0 minutes One% 0 to 1.0 minutes 1-5% 1.0 to 10.0 minutes 5-30% 10.0 to 17.0 minutes 30-60% 17.0 to 17.1 minutes 60 100% 17.1 to 19.0 minutes 100% 19.0 to 20 minutes 10%

A quadrupole time-of-flight mass spectrometer (Q-Tof PremierTM; Waters Corp.) was analyzed by operating in negative ion mode under the following conditions.

Capillary voltage 2.3 kV

Cone voltage, 50V

Source temperature, 110 ℃

Desolvation temperature, 350 ℃

Full scan data and MS/MS spectra were collected using MassLynx software (Waters Corp.).

3. MTT analysis

MTT analysis was performed to measure the relative viability of cells. First, Raw 264.7 cells ^{were inoculated at a density of 1×10 5} /ml, and cultured at 37°C for 12 hours. Next, a fraction (PS) of the ethanol extract of Mugunghwa was treated at a series of concentrations (0, 0.1, 0.5, 1, 25, 50, 100, 200, 400, 800 or 1000㎍/mL) and cultured for 24 hours. After incubation, MTT solution (0.5 mg/ml) was added to each well and reacted for 45 minutes.

Formazan crystals formed from cells were dissolved by adding a DMSO solvent, and absorbance was measured at 540 nm using a microplate reader (Thermo Electron Corporation).

4. Flow cytometry

Flow cytometry was performed using the Muse® Count & Viability Assay Kit (Millipore, Billerica, MA) to measure the total number of cells, cell viability, and apoptotic cell population. Raw 264.7 cells ^{were inoculated at a density of 1×10 5} / ㎖ and cultured overnight, and a fraction (PS) of the ethanol extract of Mugunghwa at a series concentration (0, 50, 100, 200, 400, 800 and 1000 ㎍ / ㎖) Treated and incubated for 24 hours.

The cells were recovered, washed with cold PBS, reacted for 5 minutes with Muse® Count & Viability Assay Kit, and analyzed using Muse® cellcycler.

Apoptosis induction control was _{treated with H 2} O ₂ (100 μM).

5. NO generation analysis

RAW 264.7 cells were inoculated into a 24-well plate at ^{1×10 5 cells/ml and cultured.} Fractions of the ethanol extract of Mugunghwa were treated with 0 to 800 µg/ml or/and 500 ng/ml of LPS for 24 hours. Thereafter, the culture supernatant was recovered and the amount of NO production was analyzed using Griess reagent.

Griess reagent was prepared by mixing 5% phosphoric acid with 1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride.

Each sample was mixed with the same volume of Griess reagent, and reacted at room temperature for 15 minutes. Thereafter, absorbance was measured at 540 nm using a microplate reader. The concentration of nitrite was calculated using the standard concentration of sodium nitrite.

6. RNA isolation and quantification (RT-PCR)

Total RNA was isolated from Raw 264.7 cells using Easy Blue (iNtRON Biotechnology, Seongnam, Korea). 1 μg of the isolated RNA was reverse transcribed using an M-MLV reverse transcriptase kit (Promega, Madison, WI).

Quantitative real-time PCR was performed to analyze changes in gene expression of iNOS, COX-2, TNF-α, IL-6 and IL-12p35. GAPDH was used as an internal control.

The primers used are shown in Table 2.

Gene Forward primers (5'→3') Reverse primers (5'→3') iNOS CCT CCT CCA CCC TAC CAA GT
(SEQ ID NO: 1) CAC CCA AAG TGC TTC AGT CA
(SEQ ID NO: 2) COX-2 TGC TGT ACC AGC AGT GGC AA
(SEQ ID NO:3) GCA GCC ATT TCC TTC TCT CC
(SEQ ID NO: 4) TNF-α ATG AGC ACA GAA AGC ATG AT
(SEQ ID NO: 5) TAC AGG CTT GTC ACT GA AT
(SEQ ID NO: 6) IL-6 AAG TGC ATC ATC GTT GTT TTC A
(SEQ ID NO: 7) GAG GAT ACC ACT CCC AAC AG
(SEQ ID NO: 8) IL-12p35 AAG ACA TCA CAC GGG ACCC AA
(SEQ ID NO: 9) GAG GAT ACC ACT TCC CAA CAG
(SEQ ID NO: 10) GAPDH AGG TCG GTG TGA ACG GAT TTG
(SEQ ID NO: 11) TGT AGA CCA TGT AGT TGA GGT CA
(SEQ ID NO: 12)

7. Western Blot Analysis

Proteins of the cells were isolated using a pro-prep protein extraction solution (iNtRON Biotechnology). The concentration of the separated protein was measured by Bradford analysis (Bio-Rad, Hercules, CA), and the protein was separated by electrophoresis on 10% SDS-PAGE. Transfer the separated protein to a PVDF membrane (Millipore), block the membrane with 5% skim milk in TBS-T (1 x TBS, 0.1% Tween-20, pH 7.4) at room temperature for 12 hours, then overnight with primary antibody at 4°C. Cultured. After washing the unreacted primary antibody, it was reacted with the secondary antibody to which HRP (horseradish peroxidase) was bound at room temperature for 2 hours, and protein expression was confirmed using a chemiluminescent reagent (Millipore). At this time, β-actin was used as an internal control.

8. Enzyme-linked immunosorbent assay (ELISA)

RAW 264.7 macrophages were treated with a fraction of the ethanol extract of Mugunghwa or/and 500 ng/ml LPS at a concentration of 0 to 800 µg/ml. _{The culture supernatant without cells was recovered, and the concentration of PGE 2} , TNF-α, IL-6 or IL-12 was measured using an ELISA kit (R&D Systems, Minneapolis, MN).

9. Molecular Docking

TLR4-MD2 (PDB ID: 3FX1) was obtained from the RCSB protein database bank (PDB, http://www.rcsb.org), and SMILES (simplified molecular-input line-) of 17 compounds isolated from the anthocyanin fraction of Mugunghwa. entry system) was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov). The molecular docking score was calculated using Mcule and Autodock vina. Four docking structures (Pose) were calculated, representative images were displayed using UCSF Chimera, and amino acid and active hydrogen bond lengths were predicted.

10. Analysis of the inhibitory effect of LPS-induced inflammatory sepsis in zebrafish

After incubating the zebrafish laba on the third day of hatching, the Mugunghwa fraction (PS) was treated at a concentration of 0 to 200 µg/ml for 2 hours, and then 2µl (500 ng/ml) of LPS was added to the yolk part of the laba by using a microinjector. After injection using, the survival rate was measured for 48 hours.

<Result>

1. Analysis of the anthocyanin fraction of Mugunghwa

Anthocyanins contained in the anthocyanin fraction of Mugunghwa were analyzed using the UPLC system. As shown in Table 3 as a result of analysis, cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, orientin-7- O-glucoside (glucoside Orientin-7-O-glucoside), cyanidin-3,5-O-diglucoside (Cyanidin-3,5-O-diglucoside), isorientin-4'-O-glucoside (Isoorientin-4'-O-glucoside), isovitexin-4'-O-glucoside, vitaxin-4'-O-glucoside-2''-O- Rhamnoside (Vitexin-4'-O-glucoside-2``-O-rhamnoside), isovitexin-7-O-glucoside, apigenin-8-C-β- D-glucopynoside (Apigenin-8-C-β-D-glucopyranoside), isovitexin-2"-O-rhamnoside (Isovitexin-2"-O-rhamnoside), apigenin-6-C-β- D-glucopynoside (Apigenin-6-C-β-D-glucopyranoside), apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6-C-glucoside) -7-(6"-O-acetyl)-glucoside), campferol-O-glucoside derivative, campferol-7-O-glucoside , Campferol-3-O-glucoside, apigenin-7-O-glucoside and campferol-3-(6''-acetyl It was confirmed that glucoside) (Kaempferol-3-(6''-acetylglucoside)) compound (17 types) was included (FIGS. 1 and 2).

NO. I.D. Molecular
formula Retention time
(min) Calculated ion
(m/z) Fragments PubChem CID One Cyanidin-3-O-galactoside C ₁₂ H ₂₁ O ₁₁ ⁺ 4.34 449.1084 259, 287, 421 441699 2 Cyanidin-3-O-glucoside C ₂₁ H ₂₁ O ₁₁ ⁺ 4.43 449.1084 259,287. 421 44256715 3 Orientin-7-O-glucoside C ₂₇ H ₃₀ O ₁₆ 4.61 609.1456 327, 357, 447 44257973 4 Cyanidin-3,5-O-diglucoside C ₂₇ H ₃₁ O ₁₆ ⁺ 4.87 611.1612 259, 287, 449 44256718 5 Isoorientinm-4'-O-glucoside C ₂₇ H ₃₀ O ₁₆ 5.21 609.1456 193, 285, 299, 327, 357, 447 44257975 6 Isovitexin-4'-O-glucoside C ₂₇ H ₃₀ O ₁₅ 5.32 593.1506 116, 447 154105 7 Vitexin-4'-O-glucoside-2''-O-rhamnoside C ₃₃ H ₄₀ O ₁₉ 5.43 739.2086 431, 447, 593 44257755 8 Isovitexin-7-O-glucoside (saponarin) C ₂₇ H ₃₀ O ₁₅ 5.54 593.1506 283, 311, 431 441381 9 Apigenin-8-C-β-D-glucopyranoside (Vitexin) C ₂₁ H ₂₀ O ₁₀ 6.02 431.0987 283, 311 5280441 10 Isovitexin-2"-O-rhamnoside C ₂₇ H ₃₀ O ₁₄ 6.20 577.1557 293, 311, 431 44257672 11 Apigenin-6-C-β-D-glucopyranoside (Isovitexin) C ₂₁ H ₂₀ O ₁₀ 6.28 431.0978 283, 311, 341 162350 12 Apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside C ₂₉ H ₃₂ O ₁₆ 6.50 635.1671 431 44257840 13 Kaempferol-O-glucoside derivative C ₃₁ H ₃₄ O ₁₈ 6.94 693.1612 227, 255, 284, 300, 311 N.F. 14 Kaempferol-7-O-glucoside C ₂₁ H ₂₀ O ₁₁ 7.23 447.0927 227, 255, 285 10095180 15 Kaempferol-3-O-glucoside C ₂₁ H ₂₀ O ₁₁ 7.45 447.0927 151, 257, 285 44258798 16 Apigenin-7-O-glucoside C ₂₁ H ₂₀ O ₁₁ 7.53 431.0978 268, 271 44257792 17 Kaempferol-3-(6``-acetylglucoside) C ₂₃ H ₂₂ O ₁₂ 7.85 489.1033 227,255, 284, 429 44258855

2. Cytotoxicity analysis

To analyze the cytotoxicity of the fractions of the ethanol extract of Mugunghwa, RAW 264.7 macrophages were treated with fractions of 0, 0.1, 0.5, 1, 25, 50, 100, 200, 400, 800 and 1000㎍/㎖, and analyzed by MTT. Toxicity was analyzed. As a result, it was confirmed that there was no cytotoxicity up to the concentration of 800µg/ml, and it was confirmed that there was some cytotoxicity at 1000µg/ml (Fig. 3).

Next, fractions of the ethanol extract of Mugunghwa were treated with 0, 50, 100, 200, 400, 800 and 1000㎍/㎖, and cell viability, total cell number, and dead cell number were analyzed through flow cytometry. At this time, the control group was treated with _{H 2} O _2.

Fractions of the ethanol extract of Mugunghwa did not show cytotoxicity for 24 hours even at a concentration of 800 µg/ml or less, but it was confirmed that the survival rate of 1000 µg/ml decreased and the total number of cells was also decreased. In addition, it was confirmed that the number of apoptotic cells increased at a concentration of 800µg/ml or more. Therefore, in subsequent experiments, a concentration of 800 μg/ml or less was used, which does not show cytotoxicity (Fig. 4).

3. Checking the effect of inhibiting inflammation

3.1 NO and PGE ₂ Regulation of expression of

Next, in order to confirm the inflammation inhibitory effect of the fractions of the ethanol extract of Mugunghwa, the changes in the expression _{of NO and PGE 2 as inflammatory factors were analyzed.}

As a result of NO production analysis, it was confirmed that only the fraction of the ethanol extract of Mugunghwa did not affect NO production in cells treated with 800 µg/ml. On the other hand, it was confirmed that NO production was increased in the cells induced inflammation with LPS (500 ng/ml), and it was confirmed that NO production induced by LPS was inhibited as the treatment concentration of the fraction of the ethanol extract of Mugunghwa increased (Fig. 5 ).

Further analysis of the PGE ₂ protein expression ELISA, similarly as the ethanol extract of rose of Sharon fraction concentration is increased as in the protein expression of PGE ₂ induced by LPS was confirmed inhibited (FIG. 6).

3.2 Inflammation regulator iNOS and COX-2 expression regulation

In addition, as a result of analyzing the mRNA and protein expression of inducible nitric oxide synthase (iNOS) involved in NO production and cyclooxygenase-2 (COX2) that regulates the expression of PGE2 by RT_PCR and Western blot, the treatment concentration of the fraction of the ethanol extract of Mugunghwa As was increased, the expression of iNOS or COX2 mRNA induced by LPS decreased (FIG. 7), and it was confirmed that the expression of these proteins decreased (FIG. 8).

4. Inhibition of the expression of inflammatory cytokines

Next, the effect of inhibiting the expression of inflammatory cytokines IL-6, IL-12, and TNF-α of the fractions of the ethanol extract of Mugunghwa was confirmed through RT-PCR and ELISA analysis.

As a result of the analysis, it was confirmed that the mRNA or protein expression of the inflammatory cytokines IL-6, IL-12, and TNF-α was increased during LPS treatment, and the IL-6 in cells treated with both LPS and Mugunghwa ethanol extract fractions , IL-12, TNF-α mRNA or protein expression was confirmed to be significantly reduced compared to the cells treated with LPS alone (Figs. 9 to 12).

That is, it was confirmed that the fraction of the ethanol extract of Mugunghwa suppressed the expression of inflammatory cytokines and had an anti-inflammatory effect.

5. TLR4-MD2 binding assay

The anti-inflammatory effect of the fractions of the ethanol extract of Mugunghwa was confirmed.To determine whether they are involved in the inhibition of TLR4-MD2, which is involved in inducing inflammation, the binding relationship between the 17 flavonoid compounds isolated from the fractions of the ethanol extract of Mugunghwa and TLR4-MD2 was investigated. Analyzed.

Among them, the Kaempferol-O-glucoside derivative was not analyzed because the same structure was not confirmed in Pubchem.

As a result of the analysis, cyanidin-3-O-glucoside, cyanidin-3,5-O-diglucoside, isorientin- 4'-O-glucoside (Isoorientin-4'-O-glucoside), vitexin-4'-O-glucoside-2``-O-rhamnoside (Vitexin-4'-O-glucoside-2'') -O-rhamnoside), apigenin-8-C-β-D-glucopynoside (Apigenin-8-C-β-D-glucopyranoside) and apigenin-7-O-glucoside (Apigenin-7-O- glucoside) was confirmed to have hydrogen bonds with both MD2 and TLR4.

In addition, cyanidin-3-O-galactoside, apigenin-6-C-β-D-glucopynoside (Apigenin-6-C-β-D-glucopyranoside), Apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside, camphorol-7 -O-glucoside (Kaempferol-7-O-glucoside) and camperol-3-O-glucoside (Kaempferol-3-O-glucoside) have a hydrogen bond with MD2, orientin-7-O-glucoside (Orientin-7-O-glucoside), isovitexin-4'-O-glucoside, and camphorol-3-(6''-acetylglucoside) (Kaempferol-3 -(6``-acetylglucoside)) was confirmed to have a hydrogen bond with TLR4.

On the other hand, isovitexin-7-O-glucoside and isovitexin-2"-O-rhamnoside (Isovitexin-2"-O-rhamnoside) are hydrogen bonded to MD2 and TLR4. It was confirmed that it did not have (Table 4).

Among them, the docking score of apigenin-7-O-glucoside was -8.4, which showed the best binding power with TLR4-MD2. Specifically, apigenin-7-O-glucoside forms a hydrogen bond with the LYS122 amino acid of MD2 and 2.205 A and 3.098 A, and has a hydrogen bond with the SER127 amino acid of MD2 and 2.844 A. Confirmed. In addition, it was confirmed that it has a hydrogen bond with SER441 amino acid of TLR4 and 2.873 A (FIG. 13).

Compounds with strong binding power to TLR4-MD2 bind to TLR4-MD2 and inhibit or inhibit the activity of TLR4-MD2, thus blocking the TLR4 signaling pathway induced by LPS, causing a decrease in the activation of NFκB and MAPKs. It can inhibit the occurrence or progression of inflammation by reducing the secretion of inflammatory cytokines, NO and ROS.

Therefore, it is judged that the TLR4-MD2 inhibitory activity of apigenin-7-O-glucoside, which has the best binding ability with TLR4-MD2, is the most excellent.

Binding to MD2 TLR4 Name Docking score

)H-bond distance (

) Binding A.A. H-bond distance (

) Binding A.A. 1COG -6.8 2.032 LYS125 3.396 LYS125 3.223 HIS155 2COG -7.3 2.145 ASP29 2.409 GLU422 2.463 ASP29 3.214 LYS125 2.807 PHE126 3OOG -6.4 2.082 GLU422 2.980 SER455 4COD -6.6 2.950 GLY123 2.471 GLU422 3.300 LYS125 2.879 HIS155 5IOG -6.4 2.839 LYS55 2.704 SER445 2.975 LYS128 6IOG -6.3 3.040 SER455 7VGR -7.4 3.364 LYS125 2.149 SER445 3.326 PHE126 2.894 ARG447 3.416 ARG447 8IOG -6.7 N.D. N.D. N.D. N.D. 9ACG -6.9 3.261 LYS125 2.149 GLU422 2.860 HIS155 10IR -6.8 N.D. N.D. N.D. N.D. 11AG -7.2 1.953 LYS55 12AG -7.2 3.259 LYS125 3.267 LYS125 2.081 PHE126 3.003 HIS155 13KG ⁵ 14KG -7.3 3.525 LYS125 15KG -6.2 3.348 LYS125 2.879 HIS155 16AG -8.4 2.205 LYS122 2.873 SER441 3.098 LYS122 2.844 SER127 17KA -6.1 2.975 ARG447

1COG, Cyanidin-3-O-galactoside in Table 4; 2COG, Cyanidin-3-O-glucoside; 3OOG, Orientin-7-O-glucoside; 4COD, Cyanidin-3,5-O-diglucoside; 5IOG, Isoorientinm-4'-O-glucoside; 6IOG, Isovitexin-4'-O-glucoside; 7VGR, Vitexin-4'-O-glucoside-2"-O-rhamnoside; 8IOG, Isovitexin-7-O-glucoside (saponarin); 9ACG, Apigenin-8-C-β-D-glucopyranoside (Vitexin); 10IR, Isovitexin-2"-O-rhamnoside; 11AG, Apigenin-6-C-β-D-glucopyranoside (Isovitexin); 12AG, Apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside ; 13KG, Kaempferol-O-glucoside derivative; 14KG, Kaempferol-7-O-glucoside; 15KG, Kaempferol-3-O-glucoside; 16AG, Apigenin-7-O-glucoside; 17KA, Kaempferol-3-(6''-acetylglucoside).

H-bond is a hydrogen bond, A.A. Indicates amino acids, and N.D. indicates that no H-bond was detected.

6. Analysis of the inhibitory effect of LPS-induced inflammatory sepsis in zebrafish

When LPS was injected into the zebrafish, about 60% of the zebrafish died 48 hours later. On the other hand, the fraction (PS) of the ethanol extract of Mugunghwa of 50㎍/㎖ rapidly reduced the mortality rate due to LPS, but it was confirmed that about 5% died. When the fractions (PS) of the ethanol extract of 100 µg/ml and 200 µg/ml were treated, the death induced by LPS was completely blocked.

In addition, as a result of malformation analysis, it was confirmed that malformaion increased to about 60% in zebrafish due to LPS. The deformity was reduced to 40% by the fraction (PS) treatment of the ethanol extract of Mugunghwa at 50 µg/ml, and the deformity rate decreased to the same level as that of the LPS-free group at a concentration of 100 µg/ml or more (FIG. 15).

<110> Jeju National University Industry-Academic Cooperation Foundation <120> Inhibitor compostion for TLR4-MD2 comprising fraction of ethanol extract of Hibiscus Syriacus <130> DP20190216 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS forward primer <400> 1 cctcctccac cctaccaagt 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> iNOS reverse primer <400> 2 cacccaaagt gcttcagtca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2 forward primer <400> 3 tgctgtacca gcagtggcaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COX-2 reverse primer <400> 4 gcagccattt ccttctctcc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha forward primer <400> 5 atgagcacag aaagcatgat 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha reverse primer <400> 6 tacaggcttg tcactgaat 19 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL-6 forward primer <400> 7 aagtgcatca tcgttgtttt ca 22 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6 reverse primer <400> 8 gaggatacca ctcccaacag 20 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-12p35 forward primer <400> 9 aagacatcac acgggaccca a 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-12p35 reverse primer <400> 10 gaggatacca cttcccaaca g 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH forward primer <400> 11 aggtcggtgt gaacggattt g 21 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH reverse primer <400> 12 tgtagaccat gtagttgagg tca 23

Claims (8)

TLR4-MD2 (Toll-like receptor 4-myeloid differentiation factor 2) inhibitor composition comprising a fraction of the ethanol extract of Mugunghwa or a flavonoid compound isolated therefrom. In claim 1,
The fractions are cyanidin-3-O-galactoside, cyanidin-3-O-glucoside, orientin-7-O-glucoside Seed (glucoside Orientin-7-O-glucoside), Cyanidin-3,5-O-diglucoside, Isoorientin-4'-O-glucoside (Isoorientin- 4'-O-glucoside), isovitexin-4'-O-glucoside, vitaxin-4'-O-glucoside-2''-O-rhamnoside ( Vitexin-4'-O-glucoside-2''-O-rhamnoside), isovitexin-7-O-glucoside, apigenin-8-C-β-D-glucoside Pinoside (Apigenin-8-C-β-D-glucopyranoside), isovitexin-2"-O-rhamnoside (Isovitexin-2"-O-rhamnoside), apigenin-6-C-β-D-glucose Pinoside (Apigenin-6-C-β-D-glucopyranoside), apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6-C-glucoside-7- (6"-O-acetyl)-glucoside), campferol-O-glucoside derivative), campferol-7-O-glucoside (Kaempferol-7-O-glucoside), camphorol -3-O-glucoside (Kaempferol-3-O-glucoside), apigenin-7-O-glucoside, camphorol-3-(6''-acetylglucoside) (Kaempferol-3-(6''-acetylglucoside)) that contains a flavonoid selected from the group consisting of, TLR4-MD2 inhibitor composition. In claim 2,
The flavonoid is apigenin-7-O-glucoside (Apigenin-7-O-glucoside) will, TLR4-MD2 inhibitor composition. In claim 3, the apigenin-7-O-glucoside forms a hydrogen bond with LYS122 amino acid of MD2 2.205 A and 3.098 A, and hydrogen bonds with SER127 amino acid of MD2 2.844 A To achieve, TLR4-MD2 inhibitor composition, characterized in that the hydrogen bond with SER441 amino acid of TLR4 and 2.873 A. A pharmaceutical composition for preventing or treating inflammatory diseases comprising the TLR4-MD2 inhibitor of claim 1 as an active ingredient. A quasi-drug composition for preventing or improving inflammatory diseases comprising the TLR4-MD2 inhibitor of claim 1. A food composition for preventing or improving inflammatory diseases comprising the TLR4-MD2 inhibitor of claim 1. A cosmetic composition for relieving skin inflammation comprising the TLR4-MD2 inhibitor of claim 1.

Images