KR102353335B1 - Inhibitor compostion for TLR4-MD2 comprising fraction of ethanol extract of Hibiscus Syriacus - Google Patents

Abstract

In the present invention, a fraction of an ethanol extract of Mugunghwa or a flavonoid compound isolated therefrom binds to TLR4-MD and inhibits the activity of TLR4-MD, inhibits LPS-induced activation of TLR4-MD, and inhibits and prevents inflammation . Specifically, it inhibited NO production induced by LPS and 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 and It was confirmed that it has an anti-inflammatory effect by reducing teratogenesis. Therefore, it is possible to use the fraction of the ethanol extract of Mugunghwa for the prevention, improvement or treatment of inflammatory diseases, and/or for alleviating skin inflammation.

Description

TLR4-MD2 inhibitor composition comprising fraction of ethanol extract of Mugunghwa {Inhibitor composition 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 an ethanol extract of Mugunghwa or a flavonoid isolated therefrom. It can be used as a composition.

The inflammatory response is promoted by oxidative stress that occurs in the body. Oxidative stress causes changes in nucleic acid (DNA), the main component of genes in cells, protein, the main component of muscle, and fat, which is the main component of cell membranes, or causes cell activity to decrease. This causes problems in the body's function itself, which not only causes apoptosis, but also increases the gene expression of specific cells that cause degenerative diseases, thereby initiating or exacerbating the inflammatory response.

One of the endotoxins, lipopolysaccharide (LPS), increases the expression of inflammatory cytokines such as TNF-α, IL-6, and IL-1β in Raw 264.7 macrophages, nitric oxide (NO), prostaglandin E ₂ (prostaglandin E ₂ , PGE ₂ ) and other inflammatory mediators are secreted. In the inflammatory state, COX-2 and NO synthetase (NOS) are induced to generate excess PGE ₂ , NO, etc., 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.

Toll-like receptor (TLR), a receptor of such LPS, has been found to be closely related to oxidative stress. Toll-like receptor (TLR) is a membrane protein that plays a central role in the innate immune response, and is highly expressed in intravascular sentinel cells such as macrophages and dendritic cells. do. Among them, TLR2 or TLR4 mainly detects lipoprotein 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, which induces the pathology of various diseases and is involved in carcinogenesis. However, it is known that there is no endogenous enzyme that can directly inactivate the generated reactive oxygen species and NO, ONOO ^{- action in vivo.}

Toll-like receptor 4 (TLR4) is the first receptor identified in the TLR family. Innate immune signaling amplified through MyD88 (myeloid differentiation 88)-dependent and MyD88-independent signaling processes. to activate Due to the role of TLR4, interest in research to utilize TLR4 as a target for treating various immune diseases is increasing. LPS recognized through accessory molecules such as lipopolysaccharide (LPS)-binding protein (LBP), cluster of differentiation 14 (CD 14) and myeloid differentiation factor 2 (MD2) activate TLR4. Activated TLR4 induces early activation and nuclear translocation of NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) through Myd88-dependent signaling, and mitogen-activated protein kinases (MAPKs) induce activation. 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 effective techniques for inflammatory signaling pathways by inhibiting TLR4-MD2 activation.

Republic of Korea 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 an 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, including 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 alleviating skin inflammation comprising the TLR4-MD2 inhibitor.

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

In the present invention, "Mugunghwa (Hibiscus syriacus)" belongs to the deciduous shrub of the mallow family of the dicotyledonous plants. The height reaches 3-4m, and the young branches have many hairs, but they gradually disappear. Mugunghwa is easy to cultivate in the garden and can be propagated by seeds, but since it is propagated by cuttings, it is easy to maintain without changing its characteristics. The leaves are alternate phyllotaxis, egg-shaped, and usually split into three, and the edges have serrations. The outer skin of Mugunghwa is peeled and used as a raw material for paper, and dried and used as medicine. The young leaves are edible, and the flowers and leaves can be consumed as tea.

In the present invention, "extract" refers to an extract obtained by extraction treatment, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a prepared 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 by

In the present invention, drying of the extract may be carried out by a known method in the range in which useful components from the collected sample are not destroyed, for example, by a method of freeze-drying in a vacuum state. In addition, the crushing or pulverization may be pulverized by crushing or pulverizing to the extent that useful components of the sample can be sufficiently extracted in the subsequent extraction process. The drying, crushing, or pulverizing processes may be performed in reverse order or repeated if necessary.

In the extraction of the present invention, the extraction method is not particularly limited, and extraction may be performed 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 can 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.

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

In the present invention, the term “fraction” refers to a result obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various components.

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), followed by fractionation using alcohol having 1 (C1) to 4 (C4) carbon atoms, ethyl acetate, n-hexane, or a mixed solvent thereof can be

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

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

In addition, as a result of analyzing the flavonoid component in the fraction of the ethanol extract of Mugunghwa, cyanidin-3-O-galactoside, cyanidin-3-O-glucoside (Cyanidin-3-O) -glucoside), orientin-7-O-glucoside (glucoside Orientin-7-O-glucoside), cyanidin-3,5-O-diglucoside (Cyanidin-3,5-O-diglucoside), isoorien Tin-4'-O-glucoside (Isoorientin-4'-O-glucoside), Isovitexin-4'-O-glucoside (Isovitexin-4'-O-glucoside), Vitexin-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-glucopinoside (Apigenin-8-C-β-D-glucopyranoside), isovitexin-2"-O-rhamnoside (Isovitexin-2"-O-rhamnoside), Apigenin-6-C-β-D-glucopinoside, apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside Sid (Apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside), Kaempferol-O-glucoside derivative, Kaempferol-7-O-glucoside (Kaempferol-7-O-glucoside), kaempferol-3-O-glucoside (Kaempferol-3-O-glucoside), apigenin-7-O-glucoside, and kaempferol It was confirmed that 17 kinds of flavonoid compounds of roll-3-(6''-acetylglucoside) (Kaempferol-3-(6''-acetylglucoside)) were included.

In the example of the present invention, the binding relationship between 17 kinds of flavonoids isolated from the ethanol extract of Mugunghwa and TLR4-MD2, which is known to be involved in the inflammatory reaction, was confirmed. The double kaempferol-O-glucoside derivative (Kaempferol-O-glucoside derivative) could not be 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, isoorientin- 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-glucopinoside (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.

Also cyanidin-3-O-galactoside, apigenin-6-C-β-D-glucopinoside, apigenin-6-C-β-D-glucopyranoside Genin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside), kaempferol-7- O-glucoside (Kaempferol-7-O-glucoside) and kaempferol-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 (Isovitexin-4'-O-glucoside) and kaempferol-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 form hydrogen bonds with MD2 and TLR4. confirmed not to have

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

Cyanidin-3-O-glucoside Cyanidin-3,5-O-diglucoside (Cyanidin-3,5-O-) contained in the fraction of the ethanol extract of Mugunghwa of the present invention diglucoside), isoorientin-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-glucopinoside (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- Glucopinoside (Apigenin-6-C-β-D-glucopyranoside), apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6-C-glucoside-7) -(6"-O-acetyl)-glucoside), kaempferol-7-O-glucoside (Kaempferol-7-O-glucoside) and kaempferol-3-O-glucoside (Kaempferol-3-O-glucoside) has a hydrogen bond with MD2, orientin-7-O-glucoside (glucoside Orientin-7-O-glucoside) and kaempferol-3-(6''-acetylglucoside) (Kaempferol-3-(6') It was confirmed that '-acetylglucoside)) formed a hydrogen bond with TLR4 to bind to TLR4-MD2, and the fraction of 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.

The fraction of the ethanol extract of Mugunghwa of the present invention is 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), isoorientin-4'- O-glucoside (Isoorientin-4'-O-glucoside), isovitexin-4'-O-glucoside (Isovitexin-4'-O-glucoside), vitexin-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-glucopinoside (Apigenin-8-C-β-D-glucopyranoside), isovitexin-2"-O-rhamnoside (Isovitexin-2"-O-rhamnoside), apigenin-6- C-β-D-glucopinoside (Apigenin-6-C-β-D-glucopyranoside), apigenin-6-C-glucoside-7-(6″-O-acetyl)-glucoside (Apigenin-6 -C-glucoside-7-(6"-O-acetyl)-glucoside), Kaempferol-O-glucoside derivative, Kaempferol-7-O-glucoside O-glucoside), kaempferol-3-O-glucoside (Kaempferol-3-O-glucoside), apigenin-7-O-glucoside (Apigenin-7-O-glucoside) and kaempferol-3-(6) It may include a flavonoid selected from the group consisting of ''-acetylglucoside) (Kaempferol-3-(6''-acetylglucoside)).

Specifically, the flavonoid is cyanidin-3-O-glucoside that forms hydrogen bonds with both MD2 and TLR4 (Cyanidin-3-O-glucoside, Cyanidin-3,5-O-diglucoside (Cyanidin-3,5) -O-diglucoside), isoorientin-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-glucopinoside (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-glucopinoside (Apigenin-6-C-β-D) -glucopyranoside), apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside), kaempferol-7-O-glucoside (Kaempferol-7-O-glucoside) and kaempferol-3-O-glucoside (Kaempferol-3-O-glucoside); and

Orientin-7-O-glucoside and kaempferol-3-(6''-acetylglucoside) forming a hydrogen bond with TLR4 (Kaempferol-3-(6''-) acetylglucoside)) may be selected.

In addition, Apigenin-7-O-glucoside, which has the strongest binding strength with TLR4-MD2, forms hydrogen bonds with LYS122 amino acids of MD2 at 2.205 A and 3.098 A, and SER127 amino acids of MD2 It was confirmed that hydrogen bonding with 2.844 A and 2.873A with SER441 amino acid of TLR4 was confirmed. Specifically, the flavonoid may be apigenin-7-O-glucoside (Apigenin-7-O-glucoside). .

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

TLR4-MD2 is activated and 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 TLR4-MD2 activation.

Compounds with strong binding affinity to TLR4-MD2 inhibit or inhibit the activity of TLR4-MD2 by binding to TLR4-MD2, thus blocking the TLR4 signaling pathway induced by LPS, resulting in decreased 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 best.

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

In addition, in the zebrafish model of inflammatory sepsis with LPS, it was confirmed that the fraction of ethanol extract of Mugunghwa sharply reduced the mortality caused by LPS. %, but it was confirmed that the malformation rate was reduced by the treatment of the fraction (PS) of the ethanol extract of Mugunghwa.

That is, it was confirmed that the fraction of the ethanol extract of Mugunghwa has excellent inhibitory and inhibitory effects on the inflammatory reaction known to be induced by TLR4-MD2. It can be used in pharmaceuticals, food, and quasi-drugs for the purpose of being used as a cosmetic composition for alleviating 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 and the ethanol extract of Mugunghwa is the same as described above.

In the present invention, “inflammatory disease” refers to TNF-α (tumor necrosis factor-alpha), IL-12 (interleukin- 12), IL-6 (interleukin-6), prostaglandin, or nitric oxide (NO) refers to a disease caused by an inflammatory substance.

In the present invention, the inflammatory disease is 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 that inhibits or delays 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 that improves or beneficially changes the symptoms of the inflammatory disease 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 manufacture of pharmaceutical compositions other than the active ingredient. At this time, the fraction of the ethanol extract of Mugunghwa included in the composition is not particularly limited thereto, but may include 0.001 wt% to 99 wt%, preferably 0.01 wt% to 50 wt%, 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, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-drying agents and suppositories It may have a dosage form, and may be oral or parenteral various dosage forms. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin. It can be prepared by mixing and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may also be used. Liquid formulations for oral administration include suspensions, internal 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 sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level includes the subject type and severity, age, sex, and disease type. , drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, and 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, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those 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 an inflammatory disease, and any one is applicable. For example, any of non-human animals such as monkey, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, pig, goat, human, bird and fish may be used, and the pharmaceutical composition may be administered parenterally or subcutaneously. , intraperitoneally, intrapulmonary and intranasally, and for local treatment, if necessary, by any 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 drug form, 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 dural or intracerebrovascular injection, but is not limited thereto.

A suitable total daily amount can be determined by treatment within the scope of sound medical judgment, and is 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 It can be administered in divided doses from once a day to several times a day.

In another aspect of the present invention, there is provided a quasi-drug composition for preventing or improving inflammatory diseases comprising the TLR4-MD2 inhibitor.

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

In the present invention, "improvement" refers to any action that improves or benefits the symptoms of suspected inflammatory disease and onset individuals by using a composition comprising a fraction of an ethanol extract of Mugunghwa as an active ingredient.

In the present invention, “quasi-drugs” refer to textiles, rubber products, or similar products used for the purpose of treating, alleviating, treating or preventing diseases of humans or animals, and have weak or no direct action on the human body, and are devices or machines Products that are not sterilized or similar, or products that are used for sterilization, insecticide and similar purposes to prevent infection It refers to items that are not heavy instruments, machines, or devices, and items used for the purpose of pharmacologically affecting the structure and function of humans or animals, excluding those that are not instruments, machines, or devices. include

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 can be added as it is or used together with other quasi-drug components, and can be used appropriately according to a conventional method. can The mixing amount of the active ingredient may be appropriately determined according to the purpose of use.

The external preparation for skin is not particularly limited thereto, but is 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, shampoo, toothpaste, air freshener gel, cleaning gel, disinfectant cleaner, detergent, shower foam, mouthwash, hand wash or mask. .

In another aspect of the present invention, there is provided a food composition for preventing or improving inflammatory diseases comprising the TLR4-MD2 inhibitor.

The description of the TLR4-MD2 inhibitor, inflammatory disease, prevention, and improvement in the present invention is the same 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, chewing gum, tea, vitamin complexes, and health supplements.

In addition to the fraction of the ethanol extract of Mugunghwa, other ingredients may be added to the food composition, and the type is not particularly limited. For example, it may contain, as an additional ingredient, various herbal extracts, food-logically acceptable food supplements or natural carbohydrates, such as conventional food, but is not limited thereto.

As used herein, the term "food supplement additive" refers to a component that can be supplementally added to food, and is added to manufacture health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplement additive of the present invention.

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. Hygin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used.

The food composition of the present invention may include a health functional food. The term "health functional food" used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful in the human body. Here, the term "functionality" refers to obtaining a useful effect for health purposes such as regulating nutrients or physiological action with respect to 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 components commonly added in the art. In addition, unlike general drugs, there are no side effects that may occur when taking the drug for a long time by using food as a raw material, and it can be excellent in portability.

The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, the fraction of the Mugunghwa ethanol extract of the present invention in the manufacture of food 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 health and hygiene purposes or for health control, the above amount may be used 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 includes all health functional foods in the ordinary sense.

In another aspect of the present invention, there is provided a feed additive composition for preventing or improving inflammatory diseases comprising the TLR4-MD2 inhibitor.

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

As used herein, the term “feed additive” refers to a substance added in a trace amount to a 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, an emulsifier, a preservative, etc. added to prevent quality deterioration, and an amino acid agent, a vitamin agent, an enzyme agent, a probiotic agent, a flavor agent, and a non-protein nitrogen added to increase the efficacy It may further include a compound, a silicate agent, a buffer, a colorant, an extractant, an oligosaccharide, and the like, and may further include a feed mixture, etc., but is not limited thereto.

In another aspect of the present invention, there is provided a cosmetic composition for alleviating skin inflammation comprising the TLR4-MD2 inhibitor.

The description of the TLR4-MD2 inhibitor in the present invention is the same as described above.

In the present invention, skin inflammation alleviation may be to relieve inflammatory skin degeneration caused by an inflammatory stimulus.

In the present invention, the skin inflammation refers to any skin inflammatory disease accompanied by 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, warts, epidermal nevus, Sebaceous nevus, seborrheic keratosis, acanthosis nigricans, actinic keratosis, dermatofibroma, psoriasis, chronic eczema, tuberculosis or deep mycosis, infectious granulomatous granulomatous cell tumor, acute contact dermatitis with hereditary lichenosis and spongiosis as the main symptoms, pneumonitis It may be eczema, herpes cold, vesicular hyposensitization, vesicular dermatophytosis, but is not limited thereto.

The cosmetic composition of the present invention may include, without limitation, commonly acceptable ingredients in addition to the active ingredients, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers. have.

The cosmetic composition according to the present invention includes solutions, external ointments, creams, foams, nourishing lotions, softening lotions, packs, soft water, emulsions, makeup bases, essences, soaps, liquid detergents, bathing agents, sunscreen creams, sun oils, suspensions, Emulsion, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, may be prepared in one or more formulations selected from the group consisting of patches and sprays, It is not limited.

In addition, the cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and conventional ingredients include, for example, oil, water, surfactant, humectant, lower alcohol, A thickener, a chelating agent, a colorant, a preservative, a fragrance, etc. may be appropriately mixed, but the present invention is 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, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or Mixtures thereof may 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, solubilizer or emulsifier 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, fatty acid ester of polyethylene glycol or 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, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters; Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.

When the formulation of the present invention is soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionate, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugar, etc. may be used as carrier components. can

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcositate, fatty acid amide as carrier components 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 ethanol extract of Mugunghwa binds to TLR4-MD and inhibits the activity of TLR4-MD, inhibits LPS-induced activation of TLR4-MD, and inhibits and prevents inflammatory responses. Specifically, it inhibited NO production induced by LPS and 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 and It was confirmed that it has an anti-inflammatory effect by reducing teratogenesis. Therefore, it is possible to use the fraction of the ethanol extract of Mugunghwa for the prevention, improvement or treatment of inflammatory diseases, and/or for alleviating skin inflammation.

1 is a result of analyzing the flavonoids contained in the fraction of the ethanol extract of Mugunghwa.
FIG. 2 shows chemical formulas of 16 compounds except for a kaempferol-O-glucoside derivative (No. 13) among 17 flavonoids of Table 3. FIG.
Figure 3 is the result of analyzing the photo and MTT of cells according to the process flow of the ethanol extract of Mugunghwa.
4 is a flow cytometric analysis result of cells according to the treatment concentration of the fraction of the ethanol extract of Mugunghwa.
5 is an analysis of the NO production inhibitory effect induced by the LPS treatment of the fraction of the ethanol extract of Mugunghwa.
6 is an analysis of the inhibitory effect of _{PGE 2} expression induced by LPS treatment of a fraction of an ethanol extract of Mugunghwa.
7 is a result of analyzing the expression of COX-2, 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, iNOS protein according to the treatment concentration of the fraction 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 mRNA expression levels of TNF-α, IL-6, and IL-12 according to the treatment concentration of a fraction of an 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 ethanol extract of Mugunghwa in zebrafish.
15 is an analysis of the malformation inhibitory effect according to the fraction treatment of the ethanol extract of Mugunghwa in zebrafish.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

<Materials and Methods>

1. Preparation of anthocyanin fractions from Mugunghwa

Mugunghwa ( Hibiscus syriacus ) used Pulsae and Paektanshim grown at the National Academy of Sciences (Suwon, South Korea) between July and August 2017. The sandalwood core of Mugunghwa or the petals of Mugunghwa firebird were freeze-dried for 3 days and stored at a temperature below -20℃ until extraction.

After pulverizing 1.5 kg of petals, they were extracted three times with 40.0 L ethanol at room temperature (25° C.) for 48 hours, followed by filtration. 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.

Mugunghwa anthocyanin fraction was filtered with 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 chromatography solvent.

2. Flavonoid Analysis (MS Spectral Analysis)

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

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 (Phase A: water containing 0.1% formic acid, phase B) : acetonitrile with 0.1% formic acid) was eluted using a chromatographic gradient. The optimized linear gradient is shown in Table 1.

hour Phase B 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 Premier™; Waters Corp.) was operated in negative ion mode under the following conditions to perform the analysis.

capillary voltage 2.3 kV

cone voltage, 50V

Source temperature, 110 ℃

desolvation temperature, 350 °C

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

3. MTT analysis

MTT assay was performed to determine 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, the 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 μg/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 the 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 the number of apoptotic cells. Raw 264.7 cells ^{were inoculated at a density of 1×10 5} /ml and cultured overnight, and a fraction (PS) of ethanol extract of Mugunghwa at a series of concentrations (0, 50, 100, 200, 400, 800 and 1000㎍/㎖) treated and incubated for 24 hours.

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

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

5. NO Production Analysis

RAW 264.7 cells were seeded in a 24-well plate at ^{1×10 5 cells/ml and cultured.} The 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 1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 5% phosphoric acid.

An equal volume of Griess reagent was mixed with each sample and reacted at room temperature for 15 minutes. Then, the absorbance was measured at 540 nm using a microplate reader. The concentration of nitrite was calculated using a 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 the M-MLV reverse transcriptase kit (Promega, Madison, Wis.).

Quantitative real-time PCR was performed to analyze the gene expression changes 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

Cell proteins were isolated using a pro-prep protein extraction solution (iNtRON Biotechnology). The concentration of the isolated protein was measured by Bradford analysis (Bio-Rad, Hercules, CA), and the protein was separated by electrophoresis on 10% SDS-PAGE. The isolated protein was transferred to a PVDF membrane (Millipore), the membrane was blocked with 5% skim milk in TBS-T (1 x TBS, 0.1 % Tween-20, pH 7.4) at room temperature for 12 h, and then with primary antibody overnight at 4 °C. cultured. After washing the unreacted primary antibody, it was reacted with the secondary antibody bound to horseradish peroxidase (HRP) at room temperature for 2 hours, and protein expression was confirmed using a chemiluminescent reagent (Millipore). In this case, 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 at a concentration of 0 to 800 μg/ml or/and 500 ng/ml of LPS. _{Cell-free culture supernatant 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- 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 acids and active hydrogen bond lengths were predicted.

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

After treatment with 0 to 200 μg/ml and 2 hours of concentration of zebrafish lava in zebrafish lava on the 3rd day of hatching for 2 hours, 2 μl (500ng/ml) of LPS was injected into the yolk part of lava with a micro-injector. After injection, the survival rate was measured for 48 hours.

<Result>

1. Analysis of anthocyanin fractions from Mugunghwa

Anthocyanins contained in the anthocyanin fraction of Mugunghwa were analyzed using the UPLC system. As shown in Table 3, 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), isoorientin-4'-O-glucoside (Isoorientin-4'-O-glucoside), isovitexin-4'-O-glucoside, vitexin-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-glucopinoside (Apigenin-8-C-β-D-glucopyranoside), isovitexin-2"-O-rhamnoside (Isovitexin-2"-O-rhamnoside), apigenin-6-C-β- D-glucopinoside (Apigenin-6-C-β-D-glucopyranoside), apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6-C-glucoside) -7-(6"-O-acetyl)-glucoside), Kaempferol-O-glucoside derivative, Kaempferol-7-O-glucoside , kaempferol-3-O-glucoside, apigenin-7-O-glucoside and kaempferol-3-(6''-acetyl) Glucoside) (Kaempferol-3-(6''-acetylglucoside)) was confirmed to contain 17 types of compounds (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 assay

In order 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 μg/ml, and MTT analysis Toxicity was analyzed. As a result, it was confirmed that there was no cytotoxicity up to a concentration of 800 μg/ml, and it was confirmed that there was some cytotoxicity at 1000 μg/ml ( FIG. 3 ).

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

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

3. Check the anti-inflammatory effect

3.1 NO and PGE ₂ expression regulation of

Next, in order to confirm the anti-inflammatory effect of the fractions of the ethanol extract of Mugunghwa, the inflammatory factors, NO production and changes in the expression _{of PGE 2 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 by LPS (500ng/ml), and as the treatment concentration of the ethanol extract of Mugunghwa was increased, it was confirmed that the NO production induced by LPS was inhibited (FIG. 5). ).

In addition _{, as a result of ELISA analysis of PGE 2} protein expression, it was confirmed that, similarly, as the treatment concentration of the ethanol extract of Mugunghwa increased, _{the protein expression of PGE 2} induced by LPS was inhibited (FIG. 6).

3.2 Regulation of inflammatory regulators iNOS and COX-2 expression

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), which regulates the expression of PGE2, by RT_PCR and Western blot, the treatment concentration of the ethanol extract of Mugunghwa As the increase in iNOS or COX2 mRNA expression induced by LPS decreased (FIG. 7), it was confirmed that the expression of these proteins also decreased (FIG. 8).

4. Inhibition of inflammatory cytokine expression

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

As a result of the analysis, it was confirmed that mRNA or protein expression of inflammatory cytokines IL-6, IL-12, and TNF-α increased during LPS treatment. In cells treated with both LPS and ethanol extract of Mugunghwa, the IL-6 , It was confirmed that the mRNA or protein expression of IL-12, TNF-α was 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 had an anti-inflammatory effect by suppressing the expression of inflammatory cytokines.

5. TLR4-MD2 Binding Assay

After confirming the anti-inflammatory effect of the ethanol extract of Mugunghwa extract, the binding relationship between 17 kinds of flavonoid compounds isolated from the ethanol extract of Mugunghwa and TLR4-MD2 was investigated to determine whether they are involved in the inhibition of TLR4-MD2, which is involved in the induction of inflammation. analyzed.

Among them, 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, isoorientin- 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-glucopinoside (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 (Cyanidin-3-O-galactoside), apigenin-6-C-β-D-glucopinoside (Apigenin-6-C-β-D-glucopyranoside), Apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside), kaempferol-7 -O-glucoside (Kaempferol-7-O-glucoside) and kaempferol-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 kaempferol-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 hydrogen bond with MD2 and TLR4. It was confirmed that it does not have (Table 4).

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

Compounds with strong binding affinity to TLR4-MD2 inhibit or inhibit the activity of TLR4-MD2 by binding to TLR4-MD2, thus blocking the TLR4 signaling pathway induced by LPS, resulting in decreased 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 best.

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

In Table 4, 1COG, Cyanidin-3-O-galactoside; 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 is 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 after 48 hours. On the other hand, the fraction (PS) of the ethanol extract of Mugunghwa at 50 μg/ml sharply reduced the mortality due to LPS, but it was confirmed that about 5% of them died. When the fraction (PS) of the ethanol extract of Mugunghwa was treated at 100 μg/ml and 200 μg/ml, apoptosis induced by LPS was completely blocked.

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

<110> Jeju National University Industry-Academic Cooperation Foundation <120> Inhibitor composition 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)

As a pharmaceutical composition for preventing or treating inflammatory diseases comprising a fraction of ethanol extract of Mugunghwa,
The fraction is
Cyanidin-3-O-galactoside (Cyanidin-3-O-galactoside), cyanidin-3-O-glucoside (Cyanidin-3-O-glucoside), orientin-7-O-glucoside (glucoside) Orientin-7-O-glucoside), cyanidin-3,5-O-diglucoside (Cyanidin-3,5-O-diglucoside), isoorientin-4'-O-glucoside (Isoorientin-4'- O-glucoside), isovitexin-4'-O-glucoside (Isovitexin-4'-O-glucoside), vitexin-4'-O-glucoside-2''-O-rhamnoside (Vitexin-4) '-O-glucoside-2''-O-rhamnoside), apigenin-8-C-β-D-glucopinoside (Apigenin-8-C-β-D-glucopyranoside), apigenin-6-C- β-D-glucopinoside (Apigenin-6-C-β-D-glucopyranoside), apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6-C) -glucoside-7-(6"-O-acetyl)-glucoside), kaempferol-7-O-glucoside (Kaempferol-7-O-glucoside), kaempferol-3-O-glucoside (Kaempferol-3- O-glucoside), apigenin-7-O-glucoside and kaempferol-3-(6''-acetylglucoside) (Kaempferol-3-(6''-acetylglucoside) ) is a flavonoid compound composed of
The flavonoid compound is
A pharmaceutical composition comprising hydrogen bonding to TLR4 or MD2 of TLR4-MD2 to inhibit the activity of TLR4-MD2. delete delete delete delete As a quasi-drug composition for the prevention or improvement of inflammatory diseases comprising a fraction of the ethanol extract of Mugunghwa,
The fraction is
Cyanidin-3-O-galactoside (Cyanidin-3-O-galactoside), cyanidin-3-O-glucoside (Cyanidin-3-O-glucoside), orientin-7-O-glucoside (glucoside) Orientin-7-O-glucoside), cyanidin-3,5-O-diglucoside (Cyanidin-3,5-O-diglucoside), isoorientin-4'-O-glucoside (Isoorientin-4'- O-glucoside), isovitexin-4'-O-glucoside (Isovitexin-4'-O-glucoside), vitexin-4'-O-glucoside-2''-O-rhamnoside (Vitexin-4) '-O-glucoside-2''-O-rhamnoside), apigenin-8-C-β-D-glucopinoside (Apigenin-8-C-β-D-glucopyranoside), apigenin-6-C- β-D-glucopinoside (Apigenin-6-C-β-D-glucopyranoside), apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6-C) -glucoside-7-(6"-O-acetyl)-glucoside), kaempferol-7-O-glucoside (Kaempferol-7-O-glucoside), kaempferol-3-O-glucoside (Kaempferol-3- O-glucoside), apigenin-7-O-glucoside and kaempferol-3-(6''-acetylglucoside) (Kaempferol-3-(6''-acetylglucoside) ) is a flavonoid compound composed of
The flavonoid compound is
A quasi-drug composition, characterized in that it inhibits the activity of TLR4-MD2 by hydrogen bonding with TLR4 or MD2 of TLR4-MD2. As a food composition for preventing or improving inflammatory diseases comprising a fraction of an ethanol extract of Mugunghwa,
The fraction is
Cyanidin-3-O-galactoside (Cyanidin-3-O-galactoside), cyanidin-3-O-glucoside (Cyanidin-3-O-glucoside), orientin-7-O-glucoside (glucoside) Orientin-7-O-glucoside), cyanidin-3,5-O-diglucoside (Cyanidin-3,5-O-diglucoside), isoorientin-4'-O-glucoside (Isoorientin-4'- O-glucoside), isovitexin-4'-O-glucoside (Isovitexin-4'-O-glucoside), vitexin-4'-O-glucoside-2''-O-rhamnoside (Vitexin-4) '-O-glucoside-2''-O-rhamnoside), apigenin-8-C-β-D-glucopinoside (Apigenin-8-C-β-D-glucopyranoside), apigenin-6-C- β-D-glucopinoside (Apigenin-6-C-β-D-glucopyranoside), apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6-C) -glucoside-7-(6"-O-acetyl)-glucoside), kaempferol-7-O-glucoside (Kaempferol-7-O-glucoside), kaempferol-3-O-glucoside (Kaempferol-3- O-glucoside), apigenin-7-O-glucoside and kaempferol-3-(6''-acetylglucoside) (Kaempferol-3-(6''-acetylglucoside) ) is a flavonoid compound composed of
The flavonoid compound is
A food composition, characterized in that it inhibits the activity of TLR4-MD2 by hydrogen bonding with TLR4 or MD2 of TLR4-MD2. A cosmetic composition for alleviating skin inflammation comprising a fraction of an ethanol extract of Mugunghwa,
The fraction is
Cyanidin-3-O-galactoside (Cyanidin-3-O-galactoside), cyanidin-3-O-glucoside (Cyanidin-3-O-glucoside), orientin-7-O-glucoside (glucoside) Orientin-7-O-glucoside), cyanidin-3,5-O-diglucoside (Cyanidin-3,5-O-diglucoside), isoorientin-4'-O-glucoside (Isoorientin-4'- O-glucoside), isovitexin-4'-O-glucoside (Isovitexin-4'-O-glucoside), vitexin-4'-O-glucoside-2''-O-rhamnoside (Vitexin-4) '-O-glucoside-2''-O-rhamnoside), apigenin-8-C-β-D-glucopinoside (Apigenin-8-C-β-D-glucopyranoside), apigenin-6-C- β-D-glucopinoside (Apigenin-6-C-β-D-glucopyranoside), apigenin-6-C-glucoside-7-(6"-O-acetyl)-glucoside (Apigenin-6-C) -glucoside-7-(6"-O-acetyl)-glucoside), kaempferol-7-O-glucoside (Kaempferol-7-O-glucoside), kaempferol-3-O-glucoside (Kaempferol-3- O-glucoside), apigenin-7-O-glucoside and kaempferol-3-(6''-acetylglucoside) (Kaempferol-3-(6''-acetylglucoside) ) is a flavonoid compound composed of
The flavonoid compound is
A cosmetic composition comprising hydrogen bonding to TLR4 or MD2 of TLR4-MD2 to inhibit the activity of TLR4-MD2.

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