WO2023113151A1 - Composition for alleviating, preventing or treating inflammatory diseases - Google Patents

Abstract

The present invention relates to a composition for alleviating, preventing or treating inflammatory diseases and, specifically, to a composition for alleviating, preventing or treating inflammatory diseases, comprising estafiatin as an active ingredient.

Description

Composition for improving, preventing or treating inflammatory diseases

This patent application claims priority to Korean Patent Application No. 10-2021-0179758 filed with the Korean Intellectual Property Office on December 15, 2021, the disclosure of which is incorporated herein by reference.

The present invention relates to a composition for improving, preventing or treating inflammatory diseases, and specifically, to a composition for improving, preventing or treating inflammatory diseases comprising estafiatin as an active ingredient.

The immune system in the living body maintains homeostasis by secreting various inflammatory factors including cytokines in response to the invasion of external pathogens or abnormal cellular reactions in the living body. When this homeostatic balance is disrupted and an inflammatory response is excessively induced, an inflammatory disease occurs.

Such inflammatory diseases include infectious inflammatory diseases such as pneumonia, periodontitis, hepatitis, and sepsis, and autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. In addition, there are inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, metabolic diseases such as obesity, diabetes, and atherosclerosis, and neurodegenerative diseases such as Alzheimer's and Parkinson's diseases.

Pattern recognition receptors (PRRs) present in the cell membrane and cytoplasm recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), and NF- Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), IL through activation of inflammatory transcription factors such as κB and MAP-Kinase It increases the expression of various inflammatory proteins including -1β inducible nitric oxide synthase (iNOS).

On the other hand, unlike most inflammatory proteins, some inflammatory proteins including IL-1β and IL-18 are released to the outside of cells by a specific immune signaling mechanism called inflammasome.

In addition, in various diseases such as metabolic diseases, neurodegenerative diseases, and autoimmune diseases, it has been confirmed that the NLRP3 inflammasome is involved in the onset and prognosis of diseases more specifically than the general inflammatory activation mechanism. Accordingly, the NLRP3 inflammasome has emerged as a key control factor for various inflammatory-mediated diseases, and research and development of new drugs based on NLRP3 inflammasome inhibition is actively progressing worldwide.

On the other hand, studies on physiologically active substances capable of preventing or suppressing diseases from natural products are increasing. Research is actively under way to find out.

Artemisia scoparia is a perennial plant belonging to the Asteraceae family and is an edible and medicinal halophyte that grows on sandy beaches. Estafiatin isolated from artemisia extract is known as a typical sesquiterpene lactone, and sesquiterpene lactone is mostly found in Asteraceae plants.

Although various compounds of the sesquiterpene lactone series have been scientifically proven to have anti-obesity, anti-cancer, and antioxidant effects, studies on their effects on inflammatory diseases have not been known, and treatment in patients is limited. There are many, so the development of new drugs to complement them is continuously required.

The present inventors have made intensive research efforts to develop a composition for improving, preventing or treating inflammatory diseases. As a result, the present invention was completed by identifying that estafiatin has an effect on inflammatory diseases.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising estapiatin as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving inflammatory diseases comprising estapiatin as an active ingredient.

Another object of the present invention is to provide a use of estapiatin for improving, preventing or treating inflammatory diseases.

Another object of the present invention is to provide a method for treating inflammatory diseases using estapiatin.

The present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases containing estafiatin as an active ingredient, a food composition for preventing or improving inflammatory diseases containing estafiatin as an active ingredient, and improving inflammatory diseases by estafiatin. , preventive or therapeutic use, and methods for treating inflammatory diseases using estapiatin.

Hereinafter, the present invention will be described in more detail.

One example of the present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases comprising estafiatin as an active ingredient.

As used herein, the term "estapiatin" is a compound with a molecular formula of C ₁₅ H ₁₈ O ₃ and has a molar mass of 246.3 g/mol, and the structural formula is shown in Structural Formula I below:

[Structural formula I]

In the present invention, the inflammatory disease may be at least one selected from the group consisting of infectious inflammatory disease, autoimmune disease, inflammatory bowel disease and gout, but is not limited thereto.

In the present invention, the infectious inflammatory disease may be at least one selected from the group consisting of pneumonia, gastritis, meningitis, periodontitis, hepatitis, sepsis, bronchitis, otitis media, sinusitis, sore throat and osteomyelitis, but is not limited thereto.

In the present invention, the autoimmune disease may be at least one selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, systemic sclerosis, vasculitis, dermatomyositis, and polymyositis, but is not limited thereto.

In the present invention, the inflammatory bowel disease may be at least one selected from the group consisting of ulcerative colitis, ulcerative duodenitis, Crohn's disease, enteritis, and ischemic colitis, but is not limited thereto.

The pharmaceutical composition of the present invention may contain a pharmaceutically effective amount of the extract of Artemisia.

As used herein, the term "comprising as an active ingredient" means containing an amount sufficient to achieve the efficacy or activity of the artemisia extract.

The term "pharmaceutically effective amount" in the present specification means an amount sufficient to achieve the efficacy or activity of the above-mentioned artemisia extract.

The composition of the present invention may further include adjuvants such as pharmaceutically suitable and physiologically acceptable carriers, excipients, and diluents in addition to the extract of Artemisia.

Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate , cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose ( It can be prepared by mixing sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Oral preparations include suspensions, solutions for oral use, emulsions, syrups, ointments, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, transdermal preparations, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.

As preparations for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The pharmaceutical composition according to the present invention may be administered to mammals including humans by various routes. The administration method may be any method commonly used, and may be administered by, for example, oral, dermal, intravenous, intramuscular, subcutaneous, etc. routes, preferably orally.

For example, the composition of the present invention may be in the form of a tablet containing starch or lactose, or in the form of a capsule either alone or containing an excipient, or in the form of an elixir or suspension containing a flavoring or coloring chemical. It may be administered orally, buccally or sublingually. Such liquid formulations may be prepared by suspending agents (e.g. methylcellulose, semi-synthetic glycerides such as witepsol or mixtures of apricot kernel oil and PEG-6 esters or PEG-8 and caprylic/capric acid). It can be formulated with pharmaceutically acceptable excipients such as mixtures of glycerides).

The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity, A ordinarily skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis.

In the embodiment of applying the composition of the present invention to humans, the composition of the present invention may be administered alone, but is generally mixed with a pharmaceutical carrier selected in consideration of the administration method and standard pharmaceutical practice. can be administered.

The dosage of the extract-containing composition of the present invention may vary depending on the patient's age, weight, sex, dosage form, state of health and degree of disease, and may be administered once or several times a day at regular time intervals according to the judgment of a doctor or pharmacist. Split doses may also be administered.

In the present invention, the pharmaceutical composition has an amount of artemisia extract per 1 kg of body weight of 5 to 500 mg/day, 5 to 400 mg/day, 5 to 300 mg/day, 5 to 200 mg/day, 5 to 100 mg/day. day, 10 to 500 mg/day, 10 to 400 mg/day, 10 to 300 mg/day, 10 to 200 mg/day, 10 to 100 mg/day, 20 to 500 mg/day, 20 to 400 mg/day , 20 to 300 mg/day, 20 to 200 mg/day, 20 to 100 mg/day, 50 to 500 mg/day, 50 to 400 mg/day, 50 to 300 mg/day or 50 to 200 mg/day, For example, it may be administered in the range of 50 to 100 mg/day.

If the daily dose of the artemisia extract-containing composition of the present invention is less than the above dose, a significant effect cannot be obtained, and if it exceeds the dose, it is not only uneconomical but also out of the range of the usual dose, so there is a concern that undesirable side effects may occur. Since it can occur, it is good to set it as the said range.

As used herein, the term "treatment" refers to all activities in which symptoms of an inflammatory disease are improved or cured by administration of the composition according to the present invention.

As used herein, the term "prevention" refers to any action that suppresses or delays the symptoms of an inflammatory disease by administering the composition according to the present invention.

In the present invention, estapiatin may be derived from a wormwood extract, but is not limited thereto.

In the present specification, the term "Mugwort" is a perennial plant belonging to the genus Artemisia of the Asteraceae family ( Compositae ), and is a perennial plant that grows in sandy seashores and resembles Artemisia, but has a different smell. The stem of wormwood is tree-like and does not die in winter, but the stem of wormwood dries out completely in winter. Mugwort is a rare herb that is rarely used in oriental medicine.

In the present invention, the mugwort may be one or more selected from the group consisting of leaves, stems, fruits, roots, and flowers, and may be, for example, leaves, but is not limited thereto.

In the present invention, the artemisia extract may be a crude extract obtained by extracting artemisia artemisia with a polar solvent, a non-polar solvent, or a mixture thereof.

In the present invention, the polar solvent may be at least one selected from the group consisting of water, alcohol, acetic acid, dimethyl formamide (DMFO), and dimethyl sulfoxide (DMSO), but is not limited thereto.

In the present invention, the alcohol is a straight chain or branched alcohol having 1 to 4 carbon atoms, for example, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxy It may be ethanol, ethylene glycol or a mixture thereof, but is not limited thereto.

In the present invention, in the case of a mixture of solvent water and alcohol, 10% or more to less than 100% (v / v), 20% or more to less than 100% (v / v), 30% or more to 100% (v / v) Less than, 40% or more to less than 100% (v/v), 50% or more to less than 100% (v/v), 50% or more to less than 95% (v/v), 50% or more to 92% (v/v) v) less than, 60% or more to less than 100% (v/v), 60% or more to less than 95% (v/v), 60% or more to less than 92% (v/v), 70% or more to 100% ( v/v), greater than or equal to 70% to less than 95% (v/v), greater than or equal to 70% to less than 92% (v/v), greater than or equal to 80% to less than 100% (v/v), greater than or equal to 80% to 95 It may be an aqueous solution of less than % (v/v), 80% or more to less than 92% of a straight-chain or branched alcohol having 1 to 4 carbon atoms, for example, 90% (v/v), but is not limited thereto no.

In the present invention, the fractions are a normal hexane fraction obtained by solvent fractionation of a crude extract extracted with a solvent with normal hexane, a chloroform fraction obtained by solvent fractionation with chloroform for an aqueous solution fraction remaining after solvent fractionation of a crude extract extracted with a solvent with normal hexane, and a chloroform fraction obtained with solvent The aqueous fraction remaining after solvent fractionation of the extracted crude extract with normal hexane, the ethyl acetate fraction obtained by solvent fractionation with ethyl acetate, the aqueous fraction remaining after solvent fractionation with chloroform, and the aqueous fraction remaining after solvent fractionation of the crude extract extracted with normal hexane It may be one or more fractions selected from the group consisting of a saturated butanol fraction obtained by solvent fractionation with saturated butanol in the aqueous fraction remaining after solvent fractionation with ethyl acetate in the aqueous fraction remaining after solvent fractionation with chloroform, and a mixture thereof. It is not limited.

The extract of artemisia contained in the composition of the present invention is a natural plant material and has no cytotoxicity.

Another example of the present invention relates to a food composition for preventing or improving inflammatory diseases comprising estapiatin as an active ingredient.

In the present invention, the inflammatory disease may be at least one selected from the group consisting of infectious inflammatory disease, autoimmune disease, inflammatory bowel disease and gout, but is not limited thereto.

In the present invention, the infectious inflammatory disease may be at least one selected from the group consisting of pneumonia, periodontitis, hepatitis and sepsis, but is not limited thereto.

In the present invention, the autoimmune disease may be at least one selected from the group consisting of rheumatoid arthritis and multiple sclerosis, but is not limited thereto.

In the present invention, the inflammatory bowel disease may be at least one selected from the group consisting of ulcerative colitis, ulcerative duodenitis, Crohn's disease, enteritis, and ischemic colitis, but is not limited thereto.

When the food composition of the present invention is used as a food additive, the food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. In general, when preparing food or beverage, the food composition of the present invention may be added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material.

There is no particular limitation on the type of food. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages, vitamin complexes, and the like, and includes all foods in a conventional sense.

The beverage may contain various flavoring agents or natural carbohydrates as additional ingredients. The aforementioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. . The ratio of the natural carbohydrates may be appropriately determined by a person skilled in the art.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , carbonation agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain fruit flesh for the production of natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives can also be appropriately selected by those skilled in the art.

In the food composition, the contents overlapping with the pharmaceutical composition are omitted in consideration of the complexity of the present specification.

As used herein, the term "improvement" refers to all activities in which symptoms of inflammatory diseases are improved or cured by administration of the composition according to the present invention.

Another example of the present invention relates to a method for preparing an extract of Artemisia.

In the present specification, the term "extract" has a meaning commonly used as a crude extract in the art, but also includes fractions obtained by additional fractionation of the extract in a broad sense. In other words, the extract of Wispswort includes not only those obtained by using an extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods are also included in the extract of artemisia of the present invention. Also, the extract of artemisia may be in the form of a solution, concentrate or powder.

The extraction method used in the present invention may be any method commonly used, and may be, for example, cold brewing, hot water extraction, ultrasonic extraction, or reflux cooling extraction, but is not limited thereto.

The manufacturing process of the artemisia extract according to the present invention in more detail is as follows:

Room temperature extraction is performed with an extraction solvent of about 8 to 50 times the weight of the mugwort powder. After extraction, filter and collect the filtrate. The extraction temperature is not particularly limited, but is preferably 15 to 110°C, preferably 20 to 90°C.

The extraction process may be repeated once or several times, and in one preferred example of the present invention, a method of re-extracting again after the first extraction may be adopted, which means that even if the extract is effectively filtered in the case of mass production, its water content Since this is high, loss occurs, and the extraction efficiency drops only with the first extraction, so this is to be prevented. In addition, as a result of verifying the extraction efficiency at each stage, it was found that about 80 to 90% of the total extraction amount was extracted by the secondary extraction.

In the present invention, the extraction step may be reflux extraction twice, preferably 8 to 12 times by volume. When filtering after extraction and repeating the previously obtained residue, the obtained residue is again combined with the extraction solvent, about 5 to 15 times the volume, and the filtrate, and concentrated under reduced pressure to prepare an extract. Although the extraction efficiency can be increased by mixing the filtrate obtained after the second extraction and each extraction, the extract of the present invention is not limited to the number of extractions.

In the present invention, if the amount of the solvent used in preparing the artemisia extract is too small, stirring becomes difficult and the solubility of the extract decreases, resulting in a decrease in extraction efficiency. Since it is not economical and problems in handling may occur, the amount of the solvent used is preferably within the above range.

The filtered extract obtained in this way is about 10 to 30 times, preferably 15 to 25 times, more preferably about 15 to 25 times the total amount of the concentrate in order to adjust the remaining lower alcohol content so that it is suitable for use as a pharmaceutical and food raw material. It can be azeotropically concentrated 1 to 5 times, preferably 2 to 3 times with 20 weight times of water, and then homogeneously suspended by adding the same amount of water again, and then freeze-dried to prepare a powdery artemisia extract.

The present invention relates to a composition for improving, preventing or treating inflammatory diseases, and specifically, to a composition for improving, preventing or treating inflammatory diseases comprising estafiatin as an active ingredient.

1 is a structural formula of estafiatin.

Figure 2 is a graph of the results of measuring cell viability by evaluating cytotoxicity after treating mouse macrophages with estafiatin according to an embodiment of the present invention.

Figure 3a is a graph of the result of measuring the gene expression level of the inflammatory cytokine IL-6 after treating mouse macrophages stimulated with LPS with estapiatin according to an embodiment of the present invention.

Figure 3b is a graph of the result of measuring the gene expression level of the inflammatory cytokine TNF-α after treating mouse macrophages stimulated with LPS with estapiatin according to an embodiment of the present invention.

Figure 3c is a graph of the result of measuring the gene expression level of iNOS after treating mouse macrophages stimulated with LPS with estapiatin according to an embodiment of the present invention.

Figure 4a is a graph of the result of measuring the protein expression level of the inflammatory cytokine IL-6 after treating mouse macrophages stimulated with LPS with estapiatin according to an embodiment of the present invention.

Figure 4b is a graph of the result of measuring the protein expression level of the inflammatory cytokine TNF-α after treating mouse macrophages stimulated with LPS with estapiatin according to an embodiment of the present invention.

Figure 5 is a Western blot result of measuring the iNOS protein expression level after treating mouse macrophages stimulated with LPS with estapiatin according to an embodiment of the present invention.

Figure 6 is a Western blot showing the results of measuring phosphorylation of inflammatory transcription factors (p65, p38, JNK, ERK) after treating mouse macrophages stimulated with LPS with estapiatin according to an embodiment of the present invention. .

Figure 7a is a graph showing the results of measuring the NLRP3 inflammasome inhibitory effect (IL-1β release) after treating estapiatin to NLRP3 inflammasome induced by LPS+ATP according to an embodiment of the present invention.

Figure 7b is a graph showing the results of measuring the NLRP3 inflammasome inhibitory effect (IL-1β release) after treating estapiatin to NLRP3 inflammasome induced by LPS+Nigericin according to an embodiment of the present invention.

Figure 7c is a graph showing the results of measuring the NLRP3 inflammasome inhibitory effect (IL-1β release) after treating estapiatin to NLRP3 inflammasomes induced by LPS+MSU according to an embodiment of the present invention.

Figure 8a is a NLRP3 inflammasome inhibitory effect (caspase 1, IL-1β and β-actin protein production) after treatment with estapiatin to NLRP3 inflammasome induced by LPS + ATP according to an embodiment of the present invention. is the result of measuring

Figure 8b is a NLRP3 inflammasome inhibitory effect (caspase 1, IL-1β and β-actin protein production) after treatment with estapiatin to NLRP3 inflammasome induced by LPS + Nigericin according to an embodiment of the present invention. is the result of measuring

Figure 8c is a NLRP3 inflammasome inhibitory effect (caspase 1, IL-1β and β-actin protein production amount) after treatment with estapiatin to NLRP3 inflammasome induced by LPS + MSU according to an embodiment of the present invention. is the result of measuring

Figure 9a shows the effect of inhibiting the production of inflammatory cytokine IL-6 in peritoneal fluid after administration of estapiatin to an LPS-induced mouse septic shock animal model according to an embodiment of the present invention. It is a graph of the result of measuring .

Figure 9b shows the effect of inhibiting the production of the inflammatory cytokine TNF-α in the peritoneal fluid after administration of estapiatin to an LPS-induced mouse septic shock animal model according to an embodiment of the present invention. It is a graph of the result of measuring .

Figure 9c shows the effect of inhibiting the production of the inflammatory cytokine IL-1β in the peritoneal fluid after administration of estapiatin to an LPS-induced mouse septic shock animal model according to an embodiment of the present invention. It is a graph of the result of measuring .

Figure 9d shows the effect of inhibiting the production of inflammatory cytokine CXCL1 in peritoneal fluid after administration of estapiatin to an LPS-induced mouse septic shock animal model according to an embodiment of the present invention. This is the resulting graph.

Figure 9e is a measurement of the inhibitory effect on the production of the inflammatory cytokine CXCL2 in peritoneal fluid after administration of estapiatin to an LPS-induced mouse septic shock animal model according to an embodiment of the present invention. This is the resulting graph.

Figure 10a is a measurement of the inhibitory effect on the production of the inflammatory cytokine IL-6 in serum after administration of estapiatin to an LPS-induced mouse septic shock animal model according to an embodiment of the present invention. This is the resulting graph.

Figure 10b is a measurement of the inhibitory effect on the production of the inflammatory cytokine TNF-α in serum after administration of estapiatin to an LPS-induced mouse septic shock animal model according to an embodiment of the present invention. This is the resulting graph.

Figure 10c is a measurement of the inhibitory effect on the production of the inflammatory cytokine IL-1β in serum after estafiatin was administered to an LPS-induced mouse septic shock animal model according to an embodiment of the present invention. This is the resulting graph.

Figure 10d is a result of measuring the inhibitory effect on the production of inflammatory cytokine CXCL1 in serum after administering estapiatin to an LPS-induced mouse septic shock animal model according to an embodiment of the present invention. it's a graph

Figure 10e is a result of measuring the effect of inhibiting the production of inflammatory cytokine CXCL2 in serum after administering estapiatin to an LPS-induced mouse septic shock animal model according to an embodiment of the present invention. it's a graph

11 is a graph showing the result of measuring the lethality improvement effect after administering estapiatin to an LPS-induced mouse septic shock animal model according to an embodiment of the present invention.

A pharmaceutical composition for preventing or treating inflammatory diseases comprising estapiatin as an active ingredient.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Throughout this specification, unless otherwise specified, "%" used to indicate the concentration of a particular substance is (weight/weight) % for solids/solids, (weight/volume) % for solids/liquids, and liquid/liquid is (volume/volume) %.

Preparation Example 1. Preparation of ethanol aqueous solution extract

140 g of the powder of the aerial part of Artemisia was added with 2.2 L of 95% (V/V) ethanol, pulverized with a homogenizer, extracted at room temperature for 24 hours, and filtered with a filter paper (No. 2, Whatman) to obtain an aqueous ethanol solution. got In addition, 1 L of 95% (V/V) ethanol was added to the residue, followed by extraction and filtration again in the same manner as above. The obtained ethanol aqueous solution extraction filtrate was concentrated under reduced pressure at 45° C. using a vacuum concentrator equipped with an aspirator (A-3S, Eyela, Tokyo, Japan) to prepare an ethanol aqueous solution extract.

Preparation Example 2. Isolation of estapiatin

The extract of the aqueous solution of artemisia sagebrush obtained in Preparation Example 1 was suspended in double distilled water (1.2 L), and then solvent fractionation was repeated three times with normal hexane (1.2 L) to obtain a normal hexane fraction, and then chloroform (CHCl ₃ , 1.2 L) was subjected to repeated solvent fractionation three times to obtain a chloroform fraction. Subsequently, the aqueous fraction was subjected to repeated solvent fractionation with ethyl acetate (EtOAc, 1.2 L) three times to obtain an ethyl acetate fraction, and the aqueous fraction was subjected to repeated solvent fractionation with saturated butanol (BuOH, 0.9 L) three times to obtain a saturated butanol fraction. . Each obtained fraction was concentrated under reduced pressure at 45° C. using a vacuum concentrator to prepare a normal hexane fraction, a chloroform fraction, an ethyl acetate fraction, and a hydrosaturated butanol fraction.

The ethyl acetate fraction (13.74 g) was purified using MPLC equipped with a silica gel column. That is, the ethyl acetate fraction (13.74 g) was adsorbed onto silica gel, filled in a column, and then connected to a silica gel column (SNAP KP-Sil 340 g, Biotage, Seongnam, Korea). Thereafter, chloroform/methanol = 9:1, 4:6, and 0:10 (v/v) solvent systems were eluted and fractionated by a step-wise elution method (3,400 mL for each step, 100 mL/min).

Fraction A (6.37 g, chloroform/methanol = 9:1, v/v) obtained from the silica gel column chromatography was re-purified using MPLC equipped with a silica gel column. That is, in the same manner as above, fraction A was adsorbed onto silica gel, filled in a column, and then connected to a silica gel column for compound separation (ZIP KP-Sil 120 g, Biotage, Seongnam, Korea). Subsequently, the gradient elution method (50 mL) with normal hexane / ethyl acetate = 90:10 (v / v) for the first 10 minutes and normal hexane / ethyl acetate = 0:100 (v / v) from 10 to 40 minutes /min) to obtain fraction A7 (1.14 g, retention value 23-25 min) containing estapiatin.

Experimental Example 1. Structure confirmation of estapiatin isolated from ethanol aqueous solution extract

The structure of estapiatin, a compound isolated in Preparation Example 2, could be confirmed through MS and NMR analysis. From the HR-ESI-MS (positive) spectrum of estapiatin, a molecular weight ion peak m/z 269.1254 [M+Na] ⁺ was observed, and it was found that the molecular weight of this compound was 246 (C ₁₅ H ₁₈ O ₃ ) .

From ¹ H-NMR and ¹³ C-NMR spectra, this compound was suggested to be a guaiadienolide-based compound. That is, in the ¹ H-NMR (500 MHz, CD ₃ OD) spectrum, 4 proton signals attributed to 2 sp ² methylenes [δ 6.21 (1H, d, J = 3.6 Hz, H-13a), 5.48 (1H, d, J = 3.6 Hz, H-13b), 4.95 (1H, br. s, H-14a), 4.86 (1H, br. s, H-14b)] and five methine proton signals [ δ 2.97 (1H, dt, J = 11.0, 8.0 Hz, H-1), 3.37 (1H, s, H-3), 2.31 (1H, dd, J = 11.0, 8.5 Hz, H-5), 4.08 ( 1H, dd, J = 11.0, 8.5 Hz, H-6), 2.87 (1H, m, H-7)] were observed. In addition, 5 types of proton signals attributed to 3 types of methylene groups [δ 2.06 (1H, dt, J = 11.0, 8.0 Hz, H-2a), 2.06 (1H, dd, J = 14.0, 7.0 Hz, H -2b), 2.26 (2H, m, H-8), 2.21 (1H, m, H-9a), 1.53 (1H, m, H-9b)] and one methine proton signal [δ 1.62 (3H , s, H-15)] was observed. In ^{the 13} C-NMR (125 MHz) spectrum, one carbonyl carbon signal [δ 169.7 (C-12)] and three quaternary signals [δ 65.8 (C-4), 146.1 (C-10), 139.5 (C-11)] were observed, which was consistent with the results of ESI-MS and ¹ H-NMR spectrum analysis. From the above results, this compound was structurally interpreted as estapiatin, a guaianolide-based compound.

Table 1 below shows ¹ H- (500 MHz) and ¹³ C-NMR (125 MHz) data (CD ₃ OD) of estapiatin, and its structure is shown in FIG. 1 .

location	δH (rel. Int., Multi., J in Hz)	δC
One	2.97 (1H, td, 11.0, 8.0)	44.8
2a	2.06 (1H, dd, 14.0, 7.0)	33.1
2b	1.81 (1H, dd, 3.5, 1.0)
3	3.37 (1H, s)	63.3
4	-	65.8
5	2.31 (1H, dd, 11.0, 8.5)	50.8
6	4.08 (1H, dd, 11.0, 8.5)	80.5
7	2.87 (1H, m)	44.1
8	2.26 (2H, m)	28.6
9a	2.21 (1H, m)	29.2
9b	1.53 (1H, m)
10	-	146.1
11	-	139.5
12	-	169.7
13a	6.21 (1H, d, 3.5)	120.2
13b	5.48 (1H, d, 3.5)
14a	4.95 (1H, br.s)	115.3
14b	4.86 (1H, br. d, 2)
15	1.62 (3H, s)	18.6

Experimental Example 2: Inhibitory effect of estapiatin on inflammatory response

Bone marrow was isolated from the femur and tibia of 8- to 12-week-old C57BL/6J mice using a 26 g syringe. The isolated bone marrow was cultured in a 150° cell culture plate in IMDM medium, 30% L929 cell supernatant, 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% non-essential amino acids. After differentiation into macrophages for 6 days in a medium composed of (non-essential amino acid) and 1% sodium pyruvate, they were used in the experiment.

2-1. Cytotoxicity assessment

Differentiated macrophages were seeded in 200 μL at a concentration of 1×10 ⁶ cells/ml in a 48-well plate. Then, it was stabilized for 12 hours at 37°C and 5% CO ₂ conditions. After removing the supernatant of the cultured cells, 200 μl of estafiatin was diluted to a concentration of 0.5, 1, 2, 4, 8 or 16 μM in a serum-free medium and treated at 37°C, 5% CO ₂ conditions. Incubated for 24 hours. Thereafter, the supernatant of the cultured cells was removed, and 200 μL of an MTT solution having a concentration of 400 μg/m was treated, followed by reaction at 7°C and 5% CO ₂ for 4 hours. Then, the reaction solution was removed, and 200 μl of DMSO was added to measure the absorbance at 570 nm. Based on the cell viability of cells not treated with anything (100%), the viability of cells treated with the sample was calculated and shown in FIG. 2 and Table 2.

Cytotoxicity assessment
Estapiatin (μM)	-	0.5	One	2	4	8	16
cell viability (%)	100	98.27	97.61	101.3	93.5	79.84	76.2

As can be seen in Figure 2 and Table 2, it was confirmed that the cell viability was maintained at 93% or more when mouse bone marrow-derived macrophages were treated with estapiatin at a concentration of 4 μM or less. That is, it was found that no cytotoxicity was observed when estapiatin was treated at a concentration of 4 μM or less.

2-2. Gene expression level measurement

Differentiated macrophages were seeded at a concentration of 1×10 ⁶ cells/mL in 1 ml each in a 12-well plate. Then, it was stabilized for 12 hours at 37°C and 5% CO ₂ conditions. After removing the supernatant of the cultured cells, estapiatin was diluted to a concentration of 4 μM in a serum-free medium and treated with 900 ul each. After 2 hours, 100 ul of LPS was added at a concentration of 1 μg/ml, followed by incubation at 37° C. and 5% CO ₂ for 0, 3, 6 or 12 hours. After removing the supernatant of the cultured cells, the cells attached to the plate were homogenized with trizol. After RNA was extracted from each sample, the expression levels of inflammatory cytokines and iNOS genes were quantified by qPCR, and are shown in FIGS. 3a to 3c and Table 3.

Inflammatory cytokine and iNOS gene expression levels
incubation time (hour)	0	3	3	6	6	12	12
LPS (ng/ml)	-	100	100	100	100	100	100
Estapiatin (μM)	-	-	4	-	4	-	4
IL-6 expression level	One	8899	1809	13692	1073	7501	692.8
TNF-α expression level	One	173.9	160.9	147.4	97.59	111	60.91
iNOS expression level	One	421.6	23.67	1006	123.5	848.3	59.82

As can be seen in Figures 3a to 3c and Table 3, when mouse bone marrow-derived macrophages were treated with LPS, it was confirmed that the gene expression of IL-6, TNF-α, and iNOS was suppressed over time. When LPS and estapiatin were treated together, the gene expression of IL-6, TNF-α, and iNOS by LPS was further suppressed. That is, it was found that estapiatin can be usefully used as an anti-inflammatory composition.

2-3. Protein expression level measurement

Differentiated macrophages were seeded in 200ul each in a 48-well plate at a concentration of 1x10 ⁶ cells/mL. Then, it was stabilized for 12 hours at 37°C and 5% CO ₂ condition. After removing the supernatant of the cultured cells, estafiatin was diluted in a serum-free medium at a concentration of 1, 2 or 4 μM and treated with 180 ul each. After 2 hours, 20 ul of LPS was added at a concentration of 1 μg/ml, followed by incubation at 37° C. and 5% CO ₂ conditions for 24 hours. After taking a sample of the supernatant of the cultured cells, the amount of inflammatory cytokines was measured through an enzyme-linked immunosorbent assay (ELISA) technique and shown in FIGS. 4a and 4b and Table 4.

Production of inflammatory cytokines (IL-6, TNF-α)
LPS (ng/ml)	-	100	100	100	100
Estapiatin (μM)	-	-	One	2	4
IL-6 (pg/ml)		6682	4693	1875	0
TNF-α (pg/ml)		7259	5607	2732	0

As can be seen in Figures 4a, 4b and Table 4, it was confirmed that the production of IL-6 and TNF-α by LPS was inhibited in a concentration-dependent manner when mouse bone marrow-derived macrophages were treated with estapiatin. In particular, the production of IL-6 and TNF-α was completely suppressed when estapiatin was treated at 4 μM. That is, it was found that estapiatin can be usefully used as an anti-inflammatory composition.

2-4. Measurement of iNOS and β-actin protein production

Differentiated macrophages were seeded in 1 ml each in a 12-well plate at a concentration of 1x10 ⁶ cells/ml. Then, it was stabilized for 12 hours at 37°C and 5% CO ₂ condition. After removing the supernatant of the cultured cells, 900 ul each of estapiatin was diluted to a concentration of 2.5, 5 or 10 μM in a serum-free medium. After 2 hours, 100 ul of LPS was added at a concentration of 1 μg/ml, followed by incubation at 37° C. and 5% CO ₂ conditions for 24 hours. After removing the supernatant of the cultured cells, the cells attached to the plate were homogenized using NP40 cell lysis buffer. Protein was extracted from each sample. The amount of iNOS and β-actin protein production was measured by western blot and shown in FIG. 5 .

As can be seen in Figure 5, it was confirmed that iNOS protein production by LPS was inhibited in a concentration-dependent manner when mouse bone marrow-derived macrophages were treated with estapiatin. That is, it was found that estapiatin can be usefully used as an anti-inflammatory composition.

2-5. Measurement of phosphorylation of inflammatory transcription factors

Differentiated macrophages were seeded in 1 ml each in a 12-well plate at a concentration of 1x10 ⁶ cells/ml. Then, it was stabilized for 12 hours at 37°C and 5% CO ₂ conditions. After removing the supernatant of the cultured cells, estapiatin was diluted to a concentration of 4 μM in a serum-free medium and treated with 900 ul each. After 2 hours, 100 ul of LPS was added at a concentration of 1 μg/ml, followed by incubation at 37°C and 5% CO ₂ conditions for 0, 15, 30, or 60 minutes, respectively. After removing the supernatant of the cultured cells, the cells attached to the plate were homogenized using NP40 cell lysis buffer containing a phosphatase inhibitor. After extracting proteins from each sample, the amount of each protein produced was measured by Western blotting and is shown in FIG. 6 .

As can be seen in Figure 6, it was confirmed that phosphorylation of inflammatory transcription factors by LPS was inhibited when mouse bone marrow-derived macrophages were treated with estapiatin. That is, it was found that estapiatin can be usefully used as an anti-inflammatory composition.

Experimental Example 3: Measurement of NLRP3 inflammasome inhibitory effect of estapiatin

3-1. Measurement of IL-1β release

Differentiated macrophages were seeded in 200 μL at a concentration of 1×10 ⁶ cells/ml in a 48-well plate. Then, it was stabilized for 12 hours at 37°C and 5% CO ₂ conditions. After removing the supernatant of the cultured cells, 180 ul of LPS was treated in a serum-free medium at a concentration of 100 ng/ml and primed for 5 hours. Then, estapiatin was diluted to a concentration of 10, 20 or 40 μM and added in 20 ul portions (working at a concentration of 1, 2 or 4 μM). After 1 hour, ATP (20 ul at a concentration of 10 mM and incubated for 30 minutes), Nigericin (Nigericin, 20 ul at a concentration of 25 μM and cultured for 30 minutes), MSU (20 ul at a concentration of 2 mg/ml and then cultured for 4 hours) ) were treated respectively. After collecting cultured cell supernatant samples, the amount of inflammatory cytokines was measured by ELISA technique and shown in FIGS. 7a to 7c and Tables 5 to 7.

IL-1β release by ATP
LPS (ng/ml)	-	100	100	100	100	100
ATP (mM)	-	-	One	One	One	One
Estapiatin (μM)	-	-	-	One	2	4
IL-1β (pg/ml)	0	0	3328	1394	307	0

Nigericin-induced IL-1β release
LPS (ng/ml)	-	100	100	100	100	100
Nigericin (μM)	-	-	2.5	2.5	2.5	2.5
Estapiatin (μM)	-	-	-	One	2	4
IL-1β (pg/ml)	0	0	8153	4758	2928	888.5

IL-1β release by MSU
LPS (ng/ml)	-	100	100	100	100	100
MSU (μg/ml)	-	-	200	200	200	200
Estapiatin (μM)	-	-	-	One	2	4
IL-1β (pg/ml)	0	0	7906	5948	1397	0

As can be seen in Figures 7a to 7c and Tables 5 to 7, estapiatin inhibits cleavage and IL-1β maturation of caspase 1 increased by ATP, nigericin, and MSU. It was confirmed that the inhibition was concentration-dependent. In particular, it was found that 4 μM of estapiatin effectively inhibited caspase 1 cleavage and IL-1β maturation.

3-2. Measurement of caspase 1, IL-1β and β-actin protein production

Differentiated macrophages were seeded at 1 ml each in a 12-well plate at a concentration of 1x10 ⁶ cells/ml. Then, it was stabilized for 12 hours at 37°C and 5% CO ₂ conditions. After removing the supernatant of the cultured cells, 450 ul of LPS was treated in a serum-free medium at a concentration of 100 ng/ml and primed for 5 hours. Estapiatin was diluted to a concentration of 10, 20 or 40 µM and added in 50 ul each (1. 2 or 4 µM concentration). After 1 hour, ATP (50 ul at a concentration of 10 mM and incubated for 30 minutes), Nigericin (50 ul at a concentration of 25 μM and then cultured for 30 minutes), MSU (50 ul at a concentration of 2 mg / ml and then cultured for 4 hours) each was treated. The adherent cells and culture medium were homogenized together with Triton X-100 cell lysis buffer. After protein was extracted from each sample, the amounts of caspase 1, IL-1β, and β-actin protein produced were measured by western blotting, and are shown in FIGS. 8a to 8c.

As can be seen in Figures 8a to 8c, estapiatin inhibited the cleavage of caspase 1 and IL-1β maturation increased by ATP, nigericin, and MSU in a concentration-dependent manner.

Experimental Example 4: Verification of anti-inflammatory efficacy of estapiatin

4-1. Measurement of inhibitory effect on inflammatory cytokine production

200 μl of LPS (2.5 mg/kg) was intraperitoneally administered to 8-week-old female C57BL/6 mice to induce septic shock. Immediately thereafter, 200 ul of 1.25 or 2.5 mg/kg of estapiatin was administered intraperitoneally. In the control group, the same dose of saline was administered intraperitoneally. Blood was collected from the orbital venous plexus 4 hours after administration. After euthanasia, 1 ml of PBS was intraperitoneally administered, and intraperitoneal exudate was collected. The intraperitoneal exudate and coagulated blood were centrifuged at 15000 RPM for 10 minutes to separate only the supernatant. The amount of inflammatory cytokines in each sample was measured by ELISA and shown in FIGS. 9a to 10e and Tables 8 to 9.

Inflammatory cytokines in serum
LPS (mg/kg)	-	2.5	2.5	2.5
Estapiatin (mg/kg)	-	-	1.25	2.5
IL-6 (pg/ml)	0	88663	67310	47245
TNF-α (pg/ml)	0	742.8	589.8	204.7
IL-1β (pg/ml)	459.5	772.1	609.4	543.6
CXCL1 (pg/ml)	0	97143	64244	38472
CXCL2 (pg/ml)	232.4	24146	23708	11805

Inflammatory cytokines in peritoneal fluid
LPS (mg/kg)	-	2.5	2.5	2.5
Estapiatin (mg/kg)	-	-	1.25	2.5
IL-6 (pg/ml)	0	29848	6521	6527
TNF-α (pg/ml)	0	0	0	0
IL-1β (pg/ml)	0	91.72	2.808	36.1
CXCL1 (pg/ml)	0	74303	70685	51835
CXCL2 (pg/ml)	10.84	3162	2804	1408

As can be seen in Figures 9a to 10e and Tables 8 to 9, it was confirmed that the production of inflammatory cytokines by LPS was inhibited in a concentration-dependent manner in mice administered with estapiatin. That is, it was found that estapiatin can be usefully used as an anti-inflammatory composition.

4-2. Measurement of lethality improvement effect

200 μl of LPS (25 mg/kg) was intraperitoneally administered to 8-week-old female C57BL/6 mice to induce septic shock. Immediately thereafter, 200 ul of estapiatin at 1.25 or 2.5 mg/kg was administered intraperitoneally. In the control group, the same dose of saline was administered intraperitoneally. Then, the survival rate of mice was checked every 4 hours and shown in FIG. 11 .

As can be seen in Figure 11, it was confirmed that mortality by LPS was improved in a concentration-dependent manner in mice administered with estapiatin. That is, it was found that estapiatin can be usefully used as an anti-inflammatory composition.

The present invention relates to a composition for improving, preventing or treating inflammatory diseases, and specifically, to a composition for improving, preventing or treating inflammatory diseases comprising estafiatin as an active ingredient.

Claims (18)

1. A pharmaceutical composition for preventing or treating inflammatory diseases comprising estapiatin represented by the following structural formula I as an active ingredient:

   [Structural formula I]

   

   .
2. According to claim 1, wherein the inflammatory disease is an infectious inflammatory disease, an autoimmune disease, inflammatory bowel disease and at least one member selected from the group consisting of gout, inflammatory disease prevention or treatment for the pharmaceutical composition.
3. The method of claim 2, wherein the infectious inflammatory disease is at least one selected from the group consisting of pneumonia, gastritis, meningitis, periodontitis, hepatitis, sepsis, bronchitis, otitis media, sinusitis, sore throat and osteomyelitis, an agent for preventing or treating inflammatory diseases academic composition.
4. The agent for preventing or treating inflammatory diseases according to claim 2, wherein the autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, systemic sclerosis, vasculitis, dermatomyositis, and polymyositis. academic composition.
5. The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 2, wherein the inflammatory bowel disease is at least one selected from the group consisting of ulcerative colitis, ulcerative duodenitis, Crohn's disease, enteritis and ischemic colitis.
6. According to claim 1, wherein the estafiatin is wormwood ( Artemisia scoparia ) Of the extract derived from, inflammatory disease prevention or treatment for the pharmaceutical composition.
7. [Claim 7] The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 6, wherein the extract of artemisia is a crude extract extracted with a solvent consisting of water, methanol, ethanol or a combination thereof.
8. According to claim 7, wherein the artemisia extract is obtained by fractionating the crude extract with one or more solvents selected from the group consisting of normal hexane, chloroform, ethyl acetate and saturated butanol, inflammatory disease prevention or treatment pharmaceutical composition.
9. According to claim 6, wherein the Artemisia is at least one selected from the group consisting of leaves, stems, fruits, roots and flowers of Artemisia, a pharmaceutical composition for preventing or treating inflammatory diseases.
10. A food composition for preventing or improving inflammatory diseases comprising estapiatin represented by the following structural formula I as an active ingredient:

    [Structural Formula I]

    

    .
11. 11. The method of claim 10, wherein the inflammatory disease is an infectious inflammatory disease, an autoimmune disease, inflammatory bowel disease, and at least one selected from the group consisting of gout, inflammatory disease prevention or improvement food composition.
12. The food for preventing or improving inflammatory diseases according to claim 11, wherein the infectious inflammatory disease is at least one selected from the group consisting of pneumonia, gastritis, meningitis, periodontitis, hepatitis, sepsis, bronchitis, otitis media, sinusitis, sore throat and osteomyelitis. composition.
13. The food for preventing or improving inflammatory diseases according to claim 11, wherein the autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, systemic sclerosis, vasculitis, dermatomyositis, and polymyositis. composition.
14. The food composition for preventing or improving inflammatory diseases according to claim 11, wherein the inflammatory bowel disease is at least one selected from the group consisting of ulcerative colitis, ulcerative duodenitis, Crohn's disease, enteritis and ischemic colitis.
15. [Claim 11] The method of claim 10, wherein the estafiatin is wormwood ( Artemisia scoparia ) Of the extract derived from, inflammatory disease prevention or improvement food composition.
16. [Claim 16] The food composition for preventing or improving inflammatory diseases according to claim 15, wherein the extract of artemisia is a crude extract extracted with a solvent consisting of water, methanol, ethanol or a combination thereof.
17. 17. The method of claim 16, wherein the artemisia extract is obtained by fractionating the crude extract with one or more solvents selected from the group consisting of normal hexane, chloroform, ethyl acetate and saturated butanol, inflammatory disease preventing or improving food composition.
18. [Claim 16] The food composition for preventing or improving inflammatory diseases according to claim 15, wherein the sagebrush is at least one selected from the group consisting of leaves, stems, fruits, roots and flowers of sagebrush.

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