KR20220149352A - Composition for preventing or treating autoimmune disease comprising compound isolated from Paeonia lactiflora extract as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, treating, and alleviating autoimmune diseases containing a compound represented by chemical formula 1 isolated from a Perilla frutescens extract and a pharmaceutically acceptable salt thereof as active components. The composition containing the compound or a salt thereof as an active component can be used for preventing, alleviating, or treating autoimmune diseases.

Description

A composition for preventing and treating autoimmune diseases comprising a compound isolated from perilla leaf as an active ingredient {Composition for preventing or treating autoimmune disease comprising compound isolated from Paeonia lactiflora extract as effective component}

It relates to a composition for preventing and treating autoimmune diseases comprising a compound isolated from perilla leaves, Perilla frutescens var. acuta as an active ingredient, and more specifically, comprising a compound isolated from perilla leaf extract as an active ingredient And it relates to a composition that can be used for the prevention, improvement or treatment of autoimmune diseases by inhibiting the differentiation into Th17 cells and the production of IL-17.

According to the expression of specific cytokines and transcription factors, CD4 ⁺ T cells are Th1 (T helper type1), Th2 (T helper type2), CD4 ⁺ CD25 ⁺ immunoregulatory T cells (regulatory T cells) and Th17 cells (T helper 17 cells). ) can be divided into subtypes such as Among them, IL-17-producing Th17 cells have been found to play a direct role in the pathogenesis of inflammation and autoimmunity, and their role in host defense against pathogens or inducing abnormal immune responses through animal models and human cell studies. As the major etiological roles of IL-17 and Th17 cells in inflammation and autoimmune diseases have been identified, their inhibition and regulation have been proposed as important strategies for disease treatment, and inhibition of Th17 cell line cytokines, neutralization and specific transcription factors Potential target treatment methods through inhibition control are being studied.

Rheumatoid arthritis is an autoimmune disease that affects various tissues and organs in the body. As a symptom, it causes severe pain in joints such as knuckles and causes abnormalities in various organs such as muscles, lungs, heart, skin, blood vessels, nerves and eyes in addition to joints. Although the above symptoms do not appear significantly, it gradually progresses with time and develops into a disease that causes joint damage and functional abnormalities.

Crohn's disease is a disease in which lesions such as ulcers occur discontinuously in any part of the digestive tract from the mouth to the anus.

In autoimmune diseases such as rheumatoid arthritis and Crohn's disease, an increase in the expression level of IL-17 according to the differentiation of TH17 has been reported as one of the causes. In fact, IL-17 enhances granulopoiesis by promoting the expression of G-CSF and GM-CSF, and has the effect of promoting the formation of germinal centers and the production of autoantibodies. In this regard, in patients with rheumatoid arthritis, an inflammatory autoimmune disease, IL-17 is detected high in serum, and IL-17A induces the expression of IL-1β and IL-6 in the synovial cells of rheumatoid arthritis patients. In addition, as a major function of IL-17, it is known that it inhibits matrix production in chondrocytes and osteoblasts, causing joint damage and leading to a lack of tissue regeneration.

Autoimmune diseases, such as rheumatoid arthritis and Crohn's disease, have not yet been treated or prevented, and many patients suffer. Therefore, there is an urgent need to develop a preventive and therapeutic agent for autoimmune diseases that is effective and does not cause side effects while being safe.

The researchers of the present invention were developing a preventive and therapeutic agent for autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and Crohn's disease. The present invention has been completed by finding that it can be applied.

Korean Patent Registration No. 10-1970457

One aspect relates to a pharmaceutical composition for preventing or treating autoimmune diseases, comprising as an active ingredient the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof isolated from a perilla leaf extract.

Another aspect relates to a method of preparing a pharmaceutical composition for preventing or treating the autoimmune disease.

Another aspect relates to a food composition for preventing or improving autoimmune diseases, comprising as an active ingredient the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof isolated from the perilla leaf extract.

Another aspect relates to a method for preventing, treating, or improving an autoimmune disease using the composition.

(1) A pharmaceutical composition for preventing or treating autoimmune diseases, comprising as an active ingredient a compound represented by the following formula (1) or a salt thereof isolated from the perilla leaf extract:

[Formula 1]

.

.

(2) The extraction solvent of the perilla leaf extract is water, C ₁ to C ₄ alcohol, or a mixture thereof. A pharmaceutical composition for preventing or treating autoimmune diseases.

(3) the compound is isolated by adding one or more solvents selected from the group consisting of water, C ₁ to C ₅ linear or branched alcohol, hexane, dichloromethane, and ethyl acetate to the perilla leaf extract. A pharmaceutical composition for preventing or treating a disease.

(4) The compound is a pharmaceutical composition for preventing or treating autoimmune diseases, wherein the compound is isolated by adding one or more solvents selected from the group consisting of hexane, dichloromethane, ethyl acetate, butanol and water to the perilla leaf extract.

(5) The compound is a pharmaceutical composition for preventing or treating autoimmune diseases, wherein the compound is separated by sequentially adding hexane, dichloromethane, ethyl acetate, butanol and water to the ethanol extract of perilla leaf.

(6) The separation is a pharmaceutical composition for preventing or treating autoimmune diseases by separating the dichloromethane layer obtained by fractionation with dichloromethane through silica gel column chromatography.

(7) Silica gel column chromatography method is a pharmaceutical composition for preventing or treating autoimmune diseases that is separated through a gradient method of a solvent.

(8) The salt is a physiologically acceptable salt with an inorganic acid, an organic acid, an inorganic base, or an autoimmune disease prevention or treatment pharmaceutical composition.

(9) the inorganic acid is at least one selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, and the organic acid is citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid ), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, one or more selected from the group consisting of glutamic acid and aspartic acid A pharmaceutical composition for preventing or treating immune diseases.

(10) The pharmaceutical composition for preventing or treating autoimmune diseases further comprising a diluent or a carrier.

(11) A pharmaceutical composition for preventing or treating autoimmune diseases that is formulated as an oral formulation or parenteral formulation.

(12) The oral preparations are tablets, capsules, pills, powders, granules, suspensions or syrups, and the parenteral preparations are creams, lotions, ointments, liquids, gels, cataplasmas, patches, and aerosols. , Liquid extract, elixir, acupuncture, sachet, or injection is a pharmaceutical composition for preventing or treating autoimmune diseases.

(13) extracting the perilla leaves with 50% (v/v) to 100% (v/v) ethanol;

fractionating the ethanol extract obtained from the above step with 30% (v/v) to 99% (v/v) dichloromethane; and

Separating the dichloromethane fraction obtained from the above step by silica gel column chromatography to obtain the compound of Formula 1 of claim 1;

(14) A food composition for the prevention or treatment of autoimmune diseases, comprising as an active ingredient a compound represented by the following formula (1) or a salt thereof isolated from the perilla leaf extract:

[Formula 1]

.

.

The compound represented by Formula 1 or a salt thereof isolated from the perilla leaf extract according to an aspect inhibits the production of IL-17, and more specifically, inhibits the polarization of Th17 cells to effectively inhibit the expression of IL-17. can be suppressed Accordingly, the composition comprising the compound or a salt thereof as an active ingredient can be used for preventing, improving, or treating autoimmune diseases.

1 shows the solvent fractionation process (scheme) of perilla leaf extract.
Figure 2A shows the cell viability (%) according to the toxicity evaluation of each ethanol extract of perilla, cheonnyeoncho fruit, cheonnyeoncho stem, and gujeolcho, and FIG. The IL-17A expression rate (%) of the extract is shown.
Fig. 3A shows the cell viability (%) according to the toxicity evaluation of the pericidal fraction, and Fig. 3B shows the IL-17A expression rate (%) of the pericidal fraction.
4A shows the cell viability (%) according to the toxicity evaluation of the perilla dichloromethane (DCM) fraction, and FIG. 4B shows the IL-17A expression rate (%) of the DCM fraction.
5 shows the IL-17A expression rate (%) of each sub-fraction of perilla dichloromethane (DCM).
6 shows the TLC results of the PFD4-2 subfraction.
7A shows the ¹ H-NMR spectrum (400 MHz, CD ₃ OD) result of PFD4-2-5-6.
7B shows the ¹³ C-NMR spectrum (100 MHz, CD ₃ OD) result of PFD4-2-5-6.
Fig. 7C shows the structure of tomentic acid, a compound isolated as an active ingredient from a dichloromethane fraction of perilla leaves.
8 shows the separation process of tomentic acid from the dichloromethane fraction of perilla leaf ethanol extract.
FIG. 9 shows the IL-17A expression rate (%) measured using PFD 4-2-5-6 (tomentic acid) and rosmarinic acid isolated from perilla leaflets.
FIG. 10 shows measurements of perilla leaf ethanol extract, PFD 4-2-5-6 (tomentic acid) isolated from perilla leaf, tomentic acid standard, and PFD 2-3-2-2 (ursolic acid). IL-17A expression rate (%) is shown.
11 shows the treatment process of perilla leaf extract and tomentic acid isolated therefrom in a DSS-induced inflammatory enteritis model.
12A and 12B show the results of measuring changes in the length of the intestine after treatment with perilla leaf extract, tomentic acid, and mesalazine in an animal model of DSS-induced inflammatory enteritis.
13A and 13B show histological changes in the intestine in an animal model of DSS-induced inflammatory enteritis.
14A, 14B, and 14C show the expression rate (%) of CD4 ⁺ IL-17 ⁺ cells in the spleen (Spleen), mesenteric lymph nodes (MLN), and lamina propria leukocytes (LPL), respectively.

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

All technical terms used in the present invention, unless otherwise defined, have the same meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are hereby incorporated by reference in their entirety.

One aspect provides a pharmaceutical composition for preventing or treating autoimmune diseases, comprising as an active ingredient a compound represented by Formula 1 or a salt thereof isolated from a perilla extract.

[Formula 1]

As used herein, the term "autoimmune disease" refers to a disease that attacks one's own cells due to an abnormality in the immune system or causes an abnormal reaction in an autologous organ. For example, rheumatoid arthritis, Crohn's disease, progressive systemic sclerosis (Scleroderma), systemic lupus erythematosus (lupus), insulin-dependent childhood diabetes mellitus caused by pancreatic cell antibody, multiple sclerosis, autoimmune hemolytic anemia ( Autoimmune hemolytic anemia), Myasthenia gravis, Grave's disease or inflammatory bowel disease.

In one embodiment, the autoimmune disease is inflammatory bowel disease. Inflammatory bowel disease, such as Crohn's disease and ulcerative colitis, is a chronic inflammatory autoimmune disease caused by excessive inflammation of the mucous membrane.

As used herein, the term “extraction” refers to separating the components of a herbal medicine for incorporation into a pharmaceutical composition. However, depending on the specific content of each step, it may be harmful in the sense of the first step of separating the active ingredient from the herbal medicine.

As used herein, "Perilla leaves, Perilla frutescens var. acuta " refers to the leaves of perilla frutescens var. acuta KUDO., an annual herb belonging to the family Lamiaceae.

The extraction solvent of the perilla leaf extract may be water, an organic solvent, or a mixture thereof. The organic solvent may be, for example, a C ₁ to C ₄ alcohol, a polar solvent such as ethyl acetate or acetone, or a non-polar solvent such as ether, chloroform, benzene, hexane or dichloromethane, or a mixed solvent thereof.

The alcohol may be 1 to 100% (v/v). Alternatively, the alcohol may be provided by diluting the alcohol with dilution water such as 1 to 99% (v/v) of the alcohol in water.

In one embodiment, the extraction solvent of the perilla leaf extract is water, C ₁ to C ₄ alcohol, or a mixture thereof. In one embodiment, the extraction solvent may be ethanol.

Ethanol according to an embodiment is a preferred extraction solvent for extracting tomentic acid in a high content from the perilla leaf extract. For example, ethanol extraction is more preferable since thomentic acid is hardly included during water extraction. In addition, during methanol extraction, ethanol extraction is more preferable for use as a pharmaceutical composition or a food composition because there is toxicity due to methanol.

For sufficient and effective extraction of the active ingredient, the extraction solvent may be 1 to 30 times by volume, preferably 2 to 10 times by volume, based on the weight of perilla leaf. The term “fold by volume” means a multiple of the volume of solvent compared to the weight of the perilla leaf, that is, a multiple corresponding to ml per gram.

The extraction temperature of the extract may be carried out at 4 ℃ to 120 ℃. In addition, the extraction time may be 12 hours to 120 hours. If the extraction temperature is lower than 4 ℃, the extraction may be difficult, and if it exceeds 120 ℃, the extract may be denatured. If the extraction time is less than 12 hours, some of the active ingredients may not be extracted, and if it exceeds 120 hours, impurities other than the active ingredients may be extracted together, which is not preferable. Alternatively, the extraction temperature may be room temperature.

If necessary, the extraction process may be repeated 1 to 10 times, for example, 2 to 5 times.

The perilla leaf extract may be prepared according to a conventional method for producing a plant extract, and specifically may be a cold extraction method, a warm needle extraction method, a heat extraction method, an ultrasonic extraction method, etc. can

The perilla leaf extract can be extracted with an extraction solvent, the perilla leaves are removed, filtered through filter paper, and the filtrate is concentrated using a vacuum rotary concentrator or vacuum dryer, and then stored at room temperature.

Fractionation may be added to obtain a better active active substance or to remove impurities. The fraction means a means for separating by adding a fractionation solvent to the extract. The fraction following the extraction is obtained by adding a fractionation solvent to the perilla leaf extract, for example, water, C ₁ to C ₅ linear or branched alcohol, hexane, dichloromethane ethyl acetate, chloroform, acetone. , may be obtained by fractionation by adding one or more fractionation solvents selected from the group consisting of ether and benzene.

When two or more types of fractionation solvents are used, the fractions may be individual solvent fractions obtained by performing solvent fractionation using two or more types of fractionation solvents simultaneously or sequentially. For example, the fraction may be each solvent fraction obtained by performing solvent fractionation in the order of n-hexane, dichloromethane, ethyl acetate, butanol and water.

The fractionation solvent may be, for example, a 10% (v/v) to 99% (v/v) aqueous solution or a solvent having a concentration of 100% (v/v).

In one embodiment, the compound of Formula 1 is a perilla leaf extract by using one or more organic solvents or water selected from the group consisting of hexane, dichloromethane, ethyl acetate, butanol and chloroform, simultaneously or sequentially performing a fractionation process further It can be obtained as a fraction by carrying out.

In one embodiment, the compound is isolated by adding one or more solvents selected from the group consisting of water, C ₁ to C ₅ linear or branched alcohol, hexane, dichloromethane, and ethyl acetate to the perilla leaf extract.

In one embodiment, the compound is isolated by adding one or more solvents selected from the group consisting of hexane, dichloromethane, ethyl acetate, butanol and water to the perilla leaf extract.

In one embodiment, the compound may be isolated by sequentially adding hexane, dichloromethane, and ethyl acetate to the ethanol extract of perilla leaf. For example, the compound can be isolated from the dichloromethane (DCM) fraction of ethanol extract of perilla leaves.

The fractionation temperature may be carried out at 4 ℃ to 120 ℃. Alternatively, the fractionation temperature may be room temperature.

If necessary, filtration, concentration, and freeze-drying methods known in the art may be additionally used. The dried product or concentrate of the extract and/or fraction used in the present invention means dried or concentrated by a known method as described above.

If necessary, the fractionation process may be repeated 1 to 10 times, for example, 2 to 5 times.

If necessary, a suspension process of the extract may be performed before the fractionation process.

The fractionated extract obtained by performing the fractionation process may be concentrated using a vacuum rotary concentrator or a vacuum dryer, and then stored at room temperature.

In one embodiment, in the separation, the dichloromethane layer obtained by fractionation with dichloromethane may be separated through silica gel column chromatography.

In one embodiment, the silica gel column chromatography method can be separated through a solvent gradient method (gradient method).

In one embodiment, the salt may be a physiologically acceptable salt with an inorganic acid, an organic acid, an inorganic base or an organic base.

In one embodiment, the inorganic acid is at least one selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, and the organic acid is citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid ( fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid or aspartic acid may be at least one selected from the group consisting of have.

The salt may be a pharmaceutically acceptable salt or a food acceptable salt. The term "pharmaceutically acceptable salt" or "food acceptable salt" refers to generally safe, non-toxic, not biologically or otherwise unsuitable for use in the manufacture of pharmaceutical and food pharmaceutical compositions. .

The compound of Formula 1 may be synthesized in vitro or extracted from crude drugs. When extracted from crude drugs, it can be extracted separately from perilla leaves.

The compound of Formula 1 may be referred to as tormentic acid.

The tomentic acid compound represented by Formula 1 or a salt thereof may decrease the expression of Th17-related cytokines and transcription factors, IL-17A, by participating in the regulation of cell differentiation.

Th17 cells are an essential T cell subset that accumulates on mucosal surfaces and protects the host from bacterial and fungal infections. In addition to its protective immune function, it also serves as an important effector of autoimmune inflammation. Th17 cells are characterized by secretion of IL-17A, IL-17F, IL-21, and IL-22 cytokines. Unregulated or inappropriate activation of Th17 cells plays an important role in several inflammatory pathologies.

The tomentic acid compound represented by Formula 1 or a salt thereof inhibits the production of IL-17, and more specifically, inhibits the polarization of Th17, thereby effectively inhibiting the expression of IL-17. Accordingly, the composition comprising the compound or a salt thereof as an active ingredient can be used for preventing, improving, or treating autoimmune diseases.

As used herein, the term “T helper 17 cells (Th17, Th17 cells, T helper 17 cells)” refers to a group that produces interleukin 17 (IL-17) among pre-inflammatory helper T cells. They are associated with regulatory T cells and are associated with signals that differentiate Th17s and thus inhibit actual regulatory T cell differentiation. Th17s is developmentally distinct from Th1 and Th2. Th17 cells play an important role in maintaining the mucosal barrier and contribute to clearing pathogens from mucosal surfaces, but are also associated with autoimmune and inflammatory diseases. Depletion of Th17 cells on the mucosal surface is also associated with chronic inflammation and microbial metastasis.

The compound represented by Formula 1 or a pharmaceutically acceptable salt thereof isolated from the perilla leaf extract provides excellent use for preventing, improving or treating autoimmune diseases.

The compound of Formula 1 in the pharmaceutical composition may be provided as a derivative, isomer, pharmaceutically acceptable salt or solvate thereof.

The derivative refers to a compound obtained by substituting a part of the structure of the compound with another atom or group of atoms. The term "substitution" refers to introduced instead of a hydrogen atom when a derivative is formed by substituting one or more hydrogen atoms in an organic compound with another atomic group, and "substituent" refers to an introduced atomic group. The substituent is, for example, a hydroxyl group, a halogen atom (eg, fluorine (F), chlorine (Cl), bromine (Br), and iodine (I)), a C ₂ to C ₁₀ alkenyl group, a C ₂ to C ₁₀ alkynyl group. , C ₁ to C ₁₀ heteroalkyl group, C ₆ to C ₁₀ aryl group, C ₆ to C ₁₀ heteroaryl group, C ₁ to C ₁₀ alkoxy, nitro group, cyano group, amino group, carboxyl group or a salt thereof, sulfonyl group, It may be a sulfamoyl group, a sulfonic acid group or a salt thereof, phosphoric acid or a salt thereof, or a combination thereof.

The isomers may be structural isomers or stereoisomers.

The solvate refers to a compound solvated in an organic or inorganic solvent. The solvate is, for example, a hydrate.

In one embodiment, the pharmaceutical composition may further include a diluent or carrier. The diluent or carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be exemplified by microcrystalline cellulose, lactose, or low-substituted hydroxycellulose, but is not limited thereto. The disintegrant may be exemplified by sodium starch glycolate, or anhydrous calcium monohydrogen phosphate, but is not limited thereto. The binder may be exemplified by polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, or hydroxypropyl cellulose, but is not limited thereto. The lubricant may be exemplified by magnesium stearate, silicon dioxide, or talc, but is not limited thereto.

The diluent or carrier may be appropriately selected, for example, in the range of 0.001 to 90 parts by weight per 100 parts by weight of the composition.

In one embodiment, it may be formulated as an oral preparation or a parenteral preparation.

In one embodiment, the oral formulation may be exemplified as a tablet, capsule, pill, powder, liquid, dry syrup, granule, suspending agent or syrup, but is not limited thereto. The parenteral formulations include creams, lotions, ointments, liquids, gels, cataplasmas, patches, aerosols, fluid extracts, elixirs, needles, sachets, injections, skin emulsifiers, skin suspensions, transdermal delivery patches, It may be exemplified as a drug-containing bandage, but is not limited thereto.

Administration of the composition may be administered by a method known in the art. For example, it can be administered directly to a subject by any means, such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. . The administration may be systemically or locally.

In another aspect, extracting the perilla leaves with 50% (v/v) to 100% (v/v) ethanol;

fractionating the ethanol extract obtained from the above step with 30% (v/v) to 99% (v/v) dichloromethane; and

It provides a method for preparing a pharmaceutical composition for preventing or treating autoimmune diseases, including; obtaining the compound of Formula 1 by separating the dichloromethane fraction obtained from the above step by silica gel column chromatography.

Another aspect provides a food composition for preventing or treating autoimmune diseases, comprising the compound represented by Formula 1 or a salt thereof isolated from the perilla leaf extract as an active ingredient.

The composition for food may be used, for example, as a pharmaceutical, supplement, health food or functional food for a patient suffering from an autoimmune disease or a person who may suffer from an autoimmune disease. There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include various dairy products such as milk and yogurt, beverages, tea, drinks, alcoholic beverages and vitamin complexes, and snacks such as bread, chocolate, candy, and confectionery. It includes all foods in the sense of phosphorus.

The food composition may be provided as a supplement or a health functional food.

Another aspect provides a method for preventing, improving or treating an autoimmune disease comprising the compound represented by Formula 1 or a salt thereof isolated from the perilla leaf extract as an active ingredient.

As used herein, the term "prevention" refers to inhibiting the development of a disease or disorder in an individual who has not been diagnosed with the disease or disease, but is prone to the disease or disease.

As used herein, the term “treatment” refers to (a) inhibiting the development of a disease or disorder in a subject, (b) alleviating the disease or disorder, and (c) eliminating the disease or disorder.

As used herein, the term “improvement” refers to any action in which a disease or symptom of a disease is improved in an individual.

As used herein, the term "individual" refers to monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, chimpanzees, etc. means a mammal of

As used herein, the term "included as an active ingredient" means to include an amount sufficient to achieve the efficacy or activity of the compound represented by Formula 1, a derivative thereof, or a mixture thereof isolated from the perilla leaf extract. For example, the content of the active ingredient in the pharmaceutical composition for preventing or treating autoimmune diseases may be 0.001% to 99.9% by weight, 0.1% to 99% by weight, or 1% to 50% by weight, but is limited thereto. It is not, and it can be used by appropriately adjusting the content to a desired amount according to the mode of use and the method of use of the composition.

In one embodiment, the compound represented by Formula 1, a derivative thereof, or a mixture thereof isolated from the perilla leaf extract may be included in an amount of 1 to 80% by weight based on the total weight of the pharmaceutical composition, and may be a solvate or pharmaceutical composition. It may also be provided in the form of a commercially acceptable salt.

In one embodiment, the compound represented by Formula 1, a derivative thereof, or a mixture thereof isolated from the perilla leaf extract may be included in an amount of 1 to 80% by weight based on the total weight of the food composition, and may be a solvate or a food ingredient. It may also be provided in the form of an acceptable salt.

In addition, the composition may additionally include a known therapeutic agent for autoimmune diseases in addition to the active ingredient.

In the composition, the compound represented by Formula 1, a derivative thereof, or a mixture thereof may be included in an effective amount pharmaceutically or foodologically. For example, the compound represented by Formula 1, a derivative thereof, or a mixture thereof may be present in a concentration of 0.001 to 1000 nM in the composition. For example, in the composition, 5 nM, 10 nM, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 75 nM, 100 nM, or these values may be present in a range having an upper limit or a lower limit. For example, it may be present in a concentration of 1 to 10 M in the composition.

Or in the composition, the compound represented by Formula 1, a derivative thereof, or a mixture thereof is 0.001 to 200 μg/mL, for example, 0.5, 1, 2, 4, 5, 7, 8, 10, 15, 20, 30 , 50, 100, 200 μg/mL or a range having the upper or lower limit thereof, such as 0.5 to 30 μg/mL, 1 to 20 μg/mL, 4 to 15 μg/mL, 7 to 10 μg/mL, 0.1 to 100 μg /mL.

Alternatively, the compound represented by Formula 1, a derivative thereof, or a mixture thereof in the composition may be included in an amount of 0.1 μg/mL or more.

For example, in the composition, the compound represented by Formula 1, a derivative thereof, or a mixture thereof may contain all, 1/2, or 1/3 of the daily dose required for the prevention, treatment, or improvement of autoimmune diseases. may be included in an amount.

or 0.001 to 100 μg/mL, for example, 0.5, 1, 2, 4, 5, 7, 8, 10, 15, 20, 30 μg/mL of perilla leaf ethanol extract or the dichloromethane fraction isolated therefrom in the composition mL or a range using these as upper or lower limits, such as 0.5 to 30 µg/mL, 1 to 20 µg/mL, 4 to 15 µg/mL, and 7 to 10 µg/mL.

Alternatively, the ethanol extract of perilla leaf or the dichloromethane fraction separated therefrom in the composition may be included in an amount of 0.1 μg/mL or more.

The composition may be prepared in a unit dose form by formulating using a pharmaceutically or food acceptable additive or carrier, or may be prepared by internalizing in a multi-dose container.

A suitable dosage of the composition varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient, usually skilled in the art. A physician or the like can readily determine and prescribe an effective dosage for the desired treatment, prevention or improvement. According to one embodiment, the daily dose of the composition may be 0.001 to 10 g / kg.

For example, the dosage of the pharmaceutical composition for the treatment of autoimmune diseases may be determined in consideration of the absorption of the active ingredient, the inactivation rate, and the drug used in combination, and 0.1 mg/kg (body weight) based on the daily active ingredient. ) to 500 mg/kg (body weight), 0.1 mg/kg (body weight) to 400 mg/kg (body weight), or 1 mg/kg (body weight) to 300 mg/kg (body weight), and may be administered once or It can be administered in several divided doses, but is not limited thereto.

For example, an ethanol extract of perilla leaf, a dichloromethane fraction thereof, or a compound represented by Formula 1 isolated therefrom, a derivative thereof, or a mixture thereof is 10 to 100 mg/kg (body weight), 20 to 80 mg/kg (body weight), or 25 to 50 mg/kg (body weight).

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of perilla ethanol extract

6 kg of perilla leaves (from Korea, Yeongcheon) were purchased and extracted with 100% ethanol. 650 g of ethanol extract were obtained. This process was repeated 3 times, and the concentrate was stored at 4°C until use.

Example 2. Preparation of fractions from perilla leaf ethanol extract

The perilla leaf ethanol extract prepared in Example 1 was fractionated with an immiscible solvent using the difference in polarity. Specifically, the ethanol extract was fractionated by polarity using n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc), butanol and water to obtain a hexane layer, a DCM layer, an EtOAc layer, a butanol layer and a water layer. This process was repeated 3 times, and the concentrate was stored at 4°C until use.

1 shows the solvent fractionation process (scheme) of perilla leaf extract.

More specifically, FIG. 1 shows a solvent fractionation process using n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc), butanol and water of an ethanol extract of perilla leaf.

Comparative Example 1. Preparation of ethanol extracts of cheonnyeoncho fruit, cheonnyeoncho stem, gujeolcho, and green tea

Each ethanol extract was obtained from the fruit of cheonnyeoncho, the stem of cheonnyeoncho, the gujeolcho, and green tea in the same manner as the method for preparing the ethanol extract of perilla leaf described in Example 1.

Test Example 1. Evaluation of toxicity of ethanol extracts prepared in Example 1 and Comparative Example 1 and measurement of IL-17A expression level

Toxicity evaluation and IL-17A expression levels were measured using the ethanol extract of perilla leaf obtained in Example 1 and the ethanol extracts of perilla leaf obtained in Comparative Example 1, stem, gujeolcho, and green tea.

(1) Toxicity evaluation (cell activity measurement)

After removing the pancreas from B6 mice at 6 weeks of age, using CD4-beads, only CD4 ⁺ T cells were isolated by MACS positive separation, 5 x 10 ⁵ cells were seeded in a 96-multi well plate, and cultured in a 5% CO ₂ incubator. The cultured cells were treated with each ethanol extract (2, 5, 10 μg/mL ) of perilla leaf, chrysanthemum stem, and gujeolcho, and then cultured for 1 day, then added MTT and cultured at 37°C for 4 hours. did. After dissolving formazan by adding 100 ul of 0.04N HCl in isopropanol, absorbance was measured at 570 nm using a microplate reader (Emax, Molecular Devices) to measure cell activity. The control group was the DMSO-treated group.

Figure 2A shows the cell viability (%) according to the toxicity evaluation of each ethanol extract of perilla leaf, fruit of cheonnyeoncho, stem of ch.

More specifically, Figure 2A shows the toxicity evaluation results measured by varying the concentration of each ethanol extract to 2, 5, 10 ㎍ / mL.

As shown in Figure 2A, each ethanol extract did not show cytotoxicity.

(2) IL-17A expression rate (%) measurement (IL-17A inhibitory effect measurement)

After removing the pancreas from B6 mice at 6 weeks of age, only CD4 ⁺ T cells were isolated by MACS positive separation using CD4-beads. -1.2 x 10 ⁶ cells were seeded in a multi-well plate and treated with IL-6 and TGF-β at concentrations of 25 ng/ml and 2.5 ng/ml, respectively, for differentiation into Th17. The cultured cells were treated with each ethanol extract (5, 10 μg/mL) of perilla leaf, chrysanthemum fruit, chrysanthemum stem, gujeolcho, and green tea, and then cultured for 1-3 days, followed by CD4-APC and IL-17A-PE After staining with an intracellular staining kit, IL-17A expression levels were measured by FACS. The control group was the DMSO-treated group.

Figure 2B shows the IL-17A expression rate (%) of each ethanol extract of perilla, cheonnyeoncho fruit, cheonnyeoncho stem, gujeolcho, and green tea.

More specifically, Figure 2B shows the IL-17A expression rate (%) measured by varying the concentration of each of the ethanol extracts at 5 and 10 μg/mL.

As shown in FIG. 2B , the ethanol extract of perilla leaves effectively inhibited IL-17A expression at concentrations of 5 and 10 μg/mL.

Test Example 2. Evaluation of toxicity of the perilla leaf fraction prepared in Example 2 and measurement of IL-17A expression level

A hexane fraction (HEX FR.), a dichloromethane fraction (DCM FR.), an ethyl acetate fraction (EA FR.), a butanol fraction (BuOH FR.), a water fraction (Water FR) obtained from the ethanol extract of perilla leaf in Example 2 .) was used to evaluate toxicity and measure the expression level of IL-17A.

(1) Toxicity evaluation (cell activity measurement)

After removing the pancreas from B6 mice at 6 weeks of age, using CD4-beads, only CD4 ⁺ T cells were isolated by MACS positive separation, 5 x 10 ⁵ cells were seeded in a 96-multi well plate, and cultured in a 5% CO ₂ incubator. The cultured cells were treated with hexane, dichloromethane, ethin acetate, butanol, and water fractions of perilla leaf ethanol extract at concentrations of 10, 20, 50, and 100 μg/mL, respectively, and then cultured for 1 day and then added with MTT. Incubated at ℃ for 4 hours. After dissolving formazan by adding 100 ul of 0.04N HCl in isopropanol, absorbance was measured at 570 nm using a microplate reader (Emax, Molecular Devices) to measure cell activity. The control group was the DMSO-treated group.

Figure 3A shows the toxicity evaluation of perilla hexane fraction (HEX FR.), dichloromethane fraction (DCM FR.), ethyl acetate fraction (EA FR.), butanol fraction (BuOH FR.), water fraction (Water FR.) Cell viability (%) is shown.

More specifically, FIG. 3A shows the results of toxicity evaluation measured by varying concentrations of each of the perilla fractions at 10, 20, 50, and 100 μg/mL.

(2) IL-17A expression rate (%) measurement (IL-17A inhibitory effect measurement)

After removing the pancreas from B6 mice at 6 weeks of age, only CD4 ⁺ T cells were isolated by MACS positive separation using CD4-beads. -1.2 x 10 ⁶ cells were seeded in a multi-well plate and treated with IL-6 and TGF-β at concentrations of 25 ng/ml and 2.5 ng/ml, respectively, for differentiation into Th17. The cultured cells were treated with hexane, dichloromethane, ethinacetate, butanol, and water fractions of the ethanol extract of perilla leaf at concentrations of 10, 20, 50, and 100 μg/mL, respectively, and then cultured for 1-3 days, followed by CD4-APC After staining with IL-17A-PE intracellular staining kit, the expression level of IL-17A was measured by FACS. The control group was the DMSO-treated group.

Figure 3B shows IL-17A of perilla hexane fraction (HEX FR.), dichloromethane fraction (DCM FR.), ethyl acetate fraction (EA FR.), butanol fraction (BuOH FR.), water fraction (Water FR.) Expression rate (%) is shown.

More specifically, FIG. 3B shows the IL-17A expression rate (%) measured by varying concentrations of 10, 20, 50, and 100 μg/mL of each perilla fraction.

As shown in FIG. 3B , the perilla dichloromethane fraction particularly effectively inhibited IL-17A expression.

Test Example 3. Toxicity evaluation and IL-17A expression level of dichloromethane fraction from perilla leaf prepared in Example 2

In Example 2, the dichloromethane fraction (DCM FR.) obtained from the ethanol extract of perilla leaf was used at different concentrations to evaluate toxicity and measure the expression level of IL-17A.

(1) Toxicity evaluation (cell activity measurement)

The cultured cells were treated with the dichloromethane fraction obtained from the ethanol extract of perilla leaflet at concentrations of 1, 5 and 10 μg/mL, and measured in the same manner as in the toxicity evaluation of Test Example 2.

Figure 4A shows the cell viability (%) according to the toxicity assessment of the perilla dichloromethane (DCM) fraction.

More specifically, FIG. 4A shows the toxicity evaluation results measured using the control (100% perilla ethanol extract, 10 μg/mL) and the perilla dichloromethane fraction (DCM FR.) at concentrations of 1, 5 and 10 μg/mL. indicates.

As shown in FIG. 4A , the perilla dichloromethane fraction showed no cytotoxicity at concentrations of 1, 5, and 10 μg/mL.

(2) IL-17A expression rate (%) measurement (IL-17A inhibitory effect measurement)

The cultured cells were treated with the dichloromethane fraction obtained from the perilla ethanol extract at concentrations of 1, 5, and 10 μg/mL, and measured in the same manner as in Test Example 2 for measuring the IL-17A expression rate (%).

The case where differentiation was maximally induced by adding only IL-6 and TGF-β was designated as Th17.

FIG. 4B shows the results of measuring the expression level of IL-17A measured using the control group, Th17, 10 μg/mL of perilla ethanol extract (positive control), and perilla dichloromethane fraction (DCM FR.).

More specifically, FIG. 4B shows the results of measuring the expression level of IL-17A measured by varying concentrations of the perilla dichloromethane fraction (DCM FR.) at 0.1, 1, 5, and 10 μg/mL.

As shown in FIG. 4B , the dichloromethane fraction of perilla leaves effectively inhibited IL-17A expression at concentrations of 0.1 μg/mL or more.

Example 3. Separation of perilla dichloromethane sub-fractions

The active ingredient was separated from the dichloromethane fraction obtained from the ethanol extract of perilla leaf in the method of Example 2 using a bioassay-guided isolation method. First, Silica Open Column Chromatography (chloroform : methanol = 100 : 1 ~ 50 : 1 ~ 20 : 1 ~ 10 : 1 ~ 5: 5 ~ 0 : 1) was performed on the perilla dichloromethane fraction into 7 fractions. divided, and the activity of each fraction was confirmed.

5 shows the IL-17A expression rate (%) of each sub-fraction of perilla dichloromethane (DCM).

As a result, fraction 4 (PFD4) having high IL-17 expression inhibition activity was selected.

Example 4. Separation of active ingredient from perilla dichloromethane fraction

(1) Separation of active ingredient from perilla DCM fraction

The active ingredient was separated by selecting fraction 4 (PFD4) of the dichloromethane fraction of perilla leaf obtained in the method of Example 3.

PFD4 was obtained from six subfractions obtained by applying open column chromatography (chloroform: methanol = 100: 1 to 20: 1 to 1: 1 to 0: 1) using silica gel as a stationary phase. Silica Open Column Chromatography (chloroform: methanol = 100: 1 to 0: 1) was performed by selecting the PFD4-2 layer with the highest amount among them to obtain 7 sub-fractions, and among them, PFD4-2- Silica Open Column Chromatography (chloroform : acetone = 4.5 : 1 ~ 3 : 1 ~ 1 : 1 ~ 0 : 1, methanol 100%) was performed on the 5th layer (232 mg). As a result, fraction 6 (4-2) -5-6) was obtained as an active ingredient.

6 shows the TLC results of the PFD4-2 subfraction.

(2) Identification of the active ingredient isolated from the perilla DCM fraction

7A shows ¹ H-NMR spectrum (400 MHz, CD ₃ OD) of PFD4-2-5-6.

7B shows the ¹³ C-NMR spectrum (100 MHz, CD ₃ OD) of PFD4-2-5-6.

The ¹ H-NMR and ¹³ C-NMR results of the isolated active ingredient, Compound 1, were compared with the literature, and the compound structure was identified as tomentic acid, respectively.

Compound 1 - White powder; C ₃₀ H ₄₈ O ₅ MS m/z 489 [M+H] ⁺ ; ¹ H NMR (500 MHz, Pyridine- d5 ): δ 5.52 (1H, brs, H-12), 4.04 (1H, ddd, J =10.5, 9.5, 4.5 Hz, H-2), 3.32 (1H, d, J =9.5 Hz, H-3), 3.06 (1H, ddd, J =13.5, 12.5, 4.5 Hz, H-16), 2.98 (1H, s, H-18), 1.65 (3H, s, Me-27) ), 1.37 (3H, s, Me-29), 1.20 (3H, s, Me-23), 1.05 (3H, d, J =6.0 Hz, Me-30), 1.04 (3H, s, Me-26) , 1.01 (3H, s, Me-24), 0.93 (3H, s, Me-25); ¹³ C NMR (125 MHz, Pyridine- d5 ): δ 180.4 (C-28), 144.6 (C-13), 127.7 (C-12), 83.6 (C-3), 72.4 (C-19), 68.3 ( C-2), 55.7 (C-5), 54.3 (C-18), 48.5 (C-17), 48.1 (C-9), 48.0 (C-1), 42.6 (C-20), 42.1 (C) -14), 40.1 (C-8), 39.8 (C-4), 38.5 (C-10, C-22), 33.2 (C-7), 29.2 (C-23), 29.1 (C-15), 26.8 (C-29), 26.7 (C-21), 26.1 (C-16), 24.6 (C-27), 24.5 (C-11), 18.9 (C-6), 17.5 (C-24), 17.1 (C-26), 16.7 (C-25), 16.4 (C-30).

Fig. 7C shows the structure of tomentic acid, a compound isolated as an active ingredient from a dichloromethane fraction of perilla leaves.

8 shows the separation process of tomentic acid from the dichloromethane fraction of perilla leaf ethanol extract.

Test Example 4. Evaluation of the Th17 inhibitory effect

PFD 4-2-5-6 (tormentic acid), PFD 2-3-2-2 (ursolic acid) isolated from perilla leaves, tormentic acid purchased as a standard product, and rosmarinic acid purchased separately acid) to measure the Th17 inhibitory effect.

Cells differentiated with IL-6 and TGF-β were treated with PFD 4-2-5-6, PFD 2-3-2-2, standard product tomentic acid or rosmarinic acid, and then IL-17A expression was measured to differentiate Th17. The degree of inhibition was evaluated.

In FIGS. 9 and 10 , Th0 refers to a group not treated with IL-6 and TGF-β for differentiation. In addition, in FIGS. 9 and 10, Th17 refers to a group that maximizes differentiation by adding only IL-6 and TGF-β.

9 shows the IL-17A expression rate (%) measured using PFD 4-2-5-6 (tomic acid) and rosmarinic acid isolated from perilla leaflets. The effect of rosmarinic acid, which occupies the most content in the DCM layer of perilla leaf, was measured together to be used as an index component for extract standardization.

More specifically, FIG. 9 shows IL-17A expression rates measured by using rosmarinic acid purchased from Sigma at concentrations of 1, 5, and 10 μg/mL, respectively, separately from PFD 4-2-5-6 (tomentic acid) isolated from perilla leaflets. (%) is shown.

From Fig. 9, it was confirmed that tomentic acid and rosmarinic acid isolated from perilla leaf inhibit IL-17A expression, thereby confirming the Th17 inhibitory effect.

10 shows an ethanol extract of perilla leaves, PFD 4-2-5-6 isolated from perilla leaves (tomentic acid), tomentic acid standard (tomentic acid, purchased from Core Sciences), and PFD 2-3-2- The expression rate (%) of Th17A measured using 2 (ursolic acid) is shown.

More specifically, FIG. 10 shows ethanol extracts of perilla leaves at 5, 10, 20, and 50 µg/mL, PFD 4-2-5-6 (tomenic acid) isolated from perilla leaves at 1, 2, and 5 µg/mL; IL measured using PFD 2-3-2-2 (ursolic acid) isolated from perilla leaflets at 1, 2, 5 μg/mL tormentic acid standard, and 0.5, 1, 2 μg/mL -17A expression rate (%) is shown.

From FIG. 10, it was confirmed that tomentic acid (PFD 4-2-5-6) isolated from the ethanol extract of perilla leaf inhibited IL-17A expression, thereby confirming the Th17 inhibitory effect.

PFD 4-2-5-6 (tomentic acid) isolated from perilla leaf showed significant inhibitory effect and suppressed IL-17A expression at 10 μg/mL and at 5 and 10 μg/mL of tomentic acid purchased as standard. It was confirmed that PFD 4-2-5-6 isolated from perilla leaf and tomentic acid purchased as a standard product showed the same level of efficacy as the scale for the ratio was not significantly different.

Test Example 5. Determination of the effect of tomentic acid isolated from perilla lobules in dextran sulfate sodium (DSS)-induced inflammatory enteritis model

Seven-week-old C57BL/6 (male) mice were purchased from Orient Bio and acclimatized in SPF condition ((IACUC number: #202000120). To induce intestinal inflammation, drinking water containing 2.5% DSS was provided for 7 days, and water Consumption was checked daily (tested by dividing 6 animals into 6 groups).

The vehicle (vehicle, corn oil) treated group was used as a negative control. In addition, a group treated with only DSS (DSS-induced and no drug) was used as a modality control group.

DSS and perilla ethanol extract 50 mg/kg/day treatment group, DSS and tomentoic acid 25 mg/kg/day treatment group, DSS and tomento acid 50 mg/kg/day treatment group, DSS and mesalazine (50 mg/kg/day p.o.) The treatment group was used as the test group.

After 7 days, water containing 2.5% DSS was replaced with water, and after 1 day (day 8), the animals were sacrificed, and the spleen, mesenteric lymph nodes (MLN) and intestines were extracted and tested.

11 shows the treatment process of vehicle, perilla ethanol extract, tomentic acid isolated from perilla extract, and mesalazine in a DSS-induced inflammatory enteritis model.

12A and 12B show the results of measuring changes in the length of the intestine after treatment with perilla ethanol extract, tomentic acid, and mesalazine in an animal model of DSS-induced inflammatory enteritis.

12A is a vehicle-treated group (Control), DSS-induced and drug-untreated group (DSS), DSS and perilla ethanol extract 50 mg/kg/day treated group (P.F extracts 50 mg/kg), DSS and tomentoic acid 25 mg /kg/day treatment group (Tormentic acid 25 mg/kg), DSS and tomentoic acid 50 mg/kg/day treatment group (Tormentic acid 50 mg/kg), DSS and mesalazine 50 mg/kg/day The results of measuring changes in the length of the intestine after treatment with the treatment group (Mesalazine 50 mg/kg) are shown. In addition, FIG. 12B is a graphical representation of this.

The more severe the intestinal inflammation, the shorter the length of the intestine, and the closer to the control, the better the intestinal inflammation.

As shown in FIGS. 12A and 12B , the DSS-treated group (DSS) significantly reduced the length of the intestine compared to the vehicle-treated group (Control). The decrease in intestinal length was significantly suppressed.

In addition, as shown in FIGS. 12A and 12B , tomentic acid isolated from the perilla leaf extract significantly inhibited the decrease in the length of the intestine due to inflammatory enteritis induced by DSS.

13A and 13B show histological changes in the intestine in an animal model of DSS-induced inflammatory enteritis.

More specifically, FIG. 13A shows the results of measuring histological photographs of the colon in mice by H&E staining. It can be observed that the mucosa layer of the DSS-treated group is destroyed compared to the vehicle-treated group (Control). On the other hand, a slight edema is observed in the group treated with tomentic acid 25 mg/kg, but DSS The destruction of the mucosal layer was significantly improved compared to the treatment group.

In addition, Figure 13B is a numerical representation of the severity of the pathological condition of the stained tissue.

13A and 13B show that tomentic acid isolated from perilla lobules significantly inhibited DSS-induced histological changes in the intestine.

It also shows that the histological changes of the intestine induced by DSS in the 25 mg/kg or 50 mg/kg treatment group of tomentic acid are more inhibited than in the mesalazine 50 mg/kg treatment group.

Test Example 6. Cytokine expression inhibition evaluation

14A, 14B, and 14C show the expression rate (%) of CD4 ⁺ IL-17 ⁺ cells isolated from the spleen, mesenteric lymph nodes (MLN), and lamina propria leukocytes (LPL) of experimental mice, respectively. ) is indicated.

That is, after separating CD4 ⁺ T cells from the spleen, MLN, and LPL of mice, respectively, stimulation with PMA, ionomycin, and Golgi-plug for 4 hours, CD4 ⁺ IL-17A ⁺ was analyzed by flow cytometry. it will be analyzed

From Figures 14A, 14B, and 14C, it is judged that the improvement effect of DSS-induced inflammatory enteritis is due to inhibition of the expression of cytokines such as IL-17.

statistical analysis

All data and figures are expressed as mean ± standard deviation.

This study is the result of a research conducted as a regional cooperation area industry promotion project supported by the Ministry of Trade, Industry and Energy and the Korea Institute for Advancement of Technology (P0004697).

Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (14)

A pharmaceutical composition for preventing or treating autoimmune diseases, comprising as an active ingredient a compound represented by the following formula (1) or a salt thereof isolated from the perilla leaf extract:
[Formula 1]

. The pharmaceutical composition for preventing or treating autoimmune diseases according to claim 1, wherein the extraction solvent of the perilla leaf extract is water, C ₁ to C ₄ alcohol, or a mixture thereof. The method according to claim 1, wherein the compound is separated by adding one or more solvents selected from the group consisting of water, C ₁ to C ₅ linear or branched alcohol, hexane, dichloromethane, and ethyl acetate to the perilla leaf extract. A pharmaceutical composition for preventing or treating autoimmune diseases. The pharmaceutical composition for preventing or treating autoimmune diseases according to claim 1, wherein the compound is isolated by adding one or more solvents selected from the group consisting of hexane, dichloromethane, ethyl acetate, butanol and water to the perilla leaf extract. The pharmaceutical composition for preventing or treating autoimmune diseases according to claim 1, wherein the compound is isolated by sequentially adding hexane, dichloromethane, ethyl acetate, butanol and water to the ethanol extract of perilla leaf. The pharmaceutical composition for preventing or treating autoimmune diseases according to claim 5, wherein the separation is performed by separating the dichloromethane layer obtained by fractionation with dichloromethane through silica gel column chromatography. The pharmaceutical composition for preventing or treating autoimmune diseases according to claim 6, wherein silica gel column chromatography is separated through a gradient method of a solvent. The pharmaceutical composition for preventing or treating autoimmune diseases according to claim 1, wherein the salt is a physiologically acceptable salt with an inorganic acid, an organic acid, an inorganic base, or an organic base. The method according to claim 1, wherein the inorganic acid is at least one selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, and the organic acid is citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid ( fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, at least one selected from the group consisting of glutamic acid and aspartic acid A pharmaceutical composition for preventing or treating phosphorus autoimmune diseases. The pharmaceutical composition for preventing or treating autoimmune diseases according to claim 1, wherein the pharmaceutical composition comprises a diluent or a carrier. The pharmaceutical composition for preventing or treating autoimmune diseases according to claim 1, which is formulated as an oral formulation or a parenteral formulation. The method according to claim 10, wherein the oral formulation is a tablet, capsule, pill, powder, granule, suspension or syrup, and the parenteral formulation is a cream, lotion, ointment, liquid, gel, cataplasma, patch, A pharmaceutical composition for preventing or treating autoimmune diseases, which is an aerosol, fluid extract, elixir, acupuncture, sachet, or injection. extracting the perilla leaves with 50% (v/v) to 100% (v/v) ethanol;
fractionating the ethanol extract obtained from the above step with 30% (v/v) to 99% (v/v) dichloromethane; and
Separating the dichloromethane fraction obtained from the above step by silica gel column chromatography to obtain the compound of Formula 1 of claim 1;
A method for preparing a pharmaceutical composition for preventing or treating autoimmune diseases according to claim 1. A food composition for preventing or treating autoimmune diseases, comprising as an active ingredient a compound represented by the following formula (1) or a salt thereof isolated from the perilla leaf extract:
[Formula 1]

.

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